Click on an contribution to preview the abstract content.
Connexin hemichannels and pannexin channels as drug targets in liver toxicity and disease (#13)
M. Vinken1, B. Cogliati2
1 Vrije Universiteit Brussel, In Vitro Toxicology and Dermato-Cosmetology, Brussels, Belgium
Because of its unique function in the body, it is not surprising that the liver is a frequent target in toxicity and disease. Liver pathology is typically associated with cell death and inflammation. Connexin (Cx) hemichannels and pannexin (Panx) channels have emerged as key players in a plethora of tissues in both processes over the past few years. In this research, it was investigated whether this also holds true for hemichannels consisting of Cx32 and Cx43 as well as Panx1 channels in the liver. Focus was hereby put on acute liver failure, non-alcoholic steatohepatitis (NASH) and liver fibrosis. It was found that inhibition of Cx32 and Cx43 hemichannels by using TAT-Gap24 and TAT-Gap19 peptides, respectively, in acetaminophen-induced acute liver failure counteracts inflammation, while their co-administration leads to diminished liver damage in mice. Likewise, mice with diet-induced NASH treated with TAT-Gap19 or TAT-Gap24 peptides display less inflammation and steatosis. Along the same line, TAT-Gap19 peptide reduces scar formation in carbon tetrachloride-evoked liver fibrosis in mice. Furthermore, inhibition of Panx1 channels by 10Panx1 peptide or genetic ablation of Panx1 alleviates liver damage, inflammation and oxidative stress in acetaminophen-intoxicated mice. Similar results are seen upon inducing NASH in whole body Panx1 knock-out mice, while these genetically modified animals respond in distinct ways to different experimental triggers of liver fibrosis. Collectively, these results suggest that Cx32 and Cx43 hemichannels as well as Panx1 channels may represent promising pharmacological targets for the treatment of acute and chronic liver toxicity and disease.
Keywords: liver, connexin, pannexin, hemichannel
Fine and ultrafine particles issued from oil fuels and second-generation biofuels combustion: a comparative study of the physico-chemical and in vitro toxicological characteristics (#33)
A. T. Juarez Facio1, M. Malleter1, C. Rüger2, J. - M. Vaugeois1, J. Yon3, C. Monteil1
1 ABTE - EA 4651, Rouen, France
Air pollution is a serious worldwide issue due to its health impacts. A correlation between air pollution and the development of respiratory and cardiovascular diseases has been demonstrated by numerous scientific studies. Particulate matter (PM) has drawn more attention by different aspects such as successive particulate air pollution episodes, physicochemical characteristics, and its pathological consequences. Depending on its size, PM is deposited at different levels of the respiratory system: coarse particles (PM10) in upper airways, fine particles (PM2,5) in the lower airways and ultrafine particles (PM0,1) until the deeper airways (alveoli). In addition to size, the composition of particles leads to different toxicological responses. PM is constituted by organic, inorganic, and biological compounds that can alter several biological activities. In outside environment, the main source of PM comes from combustion of fossil fuels and biomass. Biofuels seem to be an alternative in particle pollution control, although new methods to evaluate health effects from particles, and especially ultrafine particles, are required to support biofuels development. Interestingly, in vitro toxicology approaches such as primary cultures of lung cells grown at the air-liquid interface depict a situation close to physiological conditions and allow estimating the toxicity of combustion particles. The present project evaluates the biological effects of fine and ultrafine particles produced during oil fuels and biofuels combustion. We develop an innovative protocol of primary lung cells to fine and ultrafine particles exposure in which generation, characterization and particle exposition are done simultaneously under controlled conditions. During exposure, particle generation is done by a miniCAST adapted to liquid fuels, cell particle exposure is made by a Vitrocell® system and aerosol particles is sampled for chemical analysis. This approach will lead us to investigate the influence of the physicochemical composition, on the response patterns after particles exposure.
Keywords: Air-liquide Interface, biofuels, fine and ultrafine particles, Vitrocell ® exposure system, in vitro toxicology
How similar among different toxicogenomics study designs for Liver? (#58)
1 FDA/NCTR, Bioinformatics and Biostatistics, Jefferson, Arkansas, United States of America
Toxicogenomics (TGx) is an important tool to gain an enhanced understanding of toxicity at the molecular level. A broad ranges of TGx study design has been reported, some based on the existing animal models (e.g., one-day short-term assay or repeated dosing for 28 days) and the other applied in vitro systems (e.g., cell lines from rat, humans and cancer). A question is naturally rasied: how similar among different TGx study designs? In fact, this question can be asked in many different ways: (1) is a one-day in vivo short-term assay able to replace the 28-day standard and expensive toxicological assay? (2) are some biological processes more conservative across different preclinical testing systems than others? (3) do these preclinical testing systems have the similar resolution in differentiating drugs by their therapeutic uses? (4) Is it possible for in vitro to in vivo extrapolation? And (5) can genomic profiles from a cancel line predict drug-induced liver injury? In this presentation, these questions will be explored using several large genomics datasets including Open Toxicogenomics Project-Genomics Assisted Toxicity Evaluation System (TG-GATEs) and L1000 for assesing drug-induced livery injury.
Keywords: bioinformatics, toxicogenomics, study design, DILI, in vitro
Characterization of a human liver spheroid model comprised of HepaRG™ and hepatic stellate cells (#92)
D. Bovard1, E. Guedj1, A. Sewer1, A. Iskandar1, K. Luettich1, S. Frentzel1, J. Hoeng1
1 Philip Morris Internationale, Science & Innovation, Neuchâtel, Neuchâtel, Switzerland
In vitro hepatotoxicity assessment of Philip Morris International’s Reduced-Risk Products requires an in vitro liver model that closely mimics the human liver. Therefore, we have developed and characterized a spheroid model composed of HepaRG™ and human hepatic stellate cells for its morphology, metabolic capacity, viability, and functionality. Spheroids with HepaRG™ cells and stellate cells (mixed-cell) were compared for six weeks with spheroids composed only of HepaRG™ cells (HepaRG™-only).
Based on the spheroid aspects and staining of different cell types, both spheroid models had similar morphologies. ATP content and LDH secretion remained stable over the six weeks and were comparable between the two models. Given the essential function of the liver in metabolizing xenobiotics, the activities of CYP1A1/1B1, 1A2, and 3A4 were evaluated weekly, and the expression of phase 1 metabolism-associated genes was profiled. The metabolic capacity (based on the CYP inducibility after stimulation) was conserved at any time point tested; no differences were observed between the two models. Secretion of albumin, a marker of hepatic function, was slightly higher during the first two weeks in the mixed-cell model than in the HepaRG™-only model. Procollagen 1α1 levels in the medium surrounding the spheroids were greater in the mixed-cell spheroids regardless of treatment with TGFβ1, a known inducer of collagen production. A whole-human transcriptome analysis consistently showed an upregulation of genes associated with collagen secretion and an enrichment in senescence and apoptosis pathways in the mixed-cell spheroids when compared with HepaRG™-only spheroids.
In summary, the spheroid model composed of HepaRG™ and stellate cells demonstrated stable viability, functionality, and metabolic capacity over six weeks. The increased procollagen 1 secretion that was associated with an upregulation of genes involved in the secretion of collagen suggested that the cells were already fibrotic (or activated) at week 2 after the spheroid preparation. While the reason for stellate cell activation remains unclear, a non-adapted coculture medium could explain the results obtained. Given their fibrotic phenotype, the mixed-cell spheroid model needs to be optimized before being used for toxicological assessment.
Keywords: Liver, 3D models, hepaRG, human hepatic stellate cells, spheroid
Correlation between cytochrome P450 enzyme induction and up-regulation of oxidative stress mediators by the pyrethroid insecticide lambda-cyhalothrin in rat liver (#104)
M. A. Martínez1, I. Ares1, J. L. Rodríguez1, M. Martínez1, B. Lopez-Torres1, A. Anadón1, M. R. Martínez-Larrañaga1
1 Universidad Complutense de Madrid, Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Madrid, Spain
Although cytochrome P450 (CYP) enzymes normally generate metabolites with diminished biologic activity and represent a defense for detoxifying the ROS entities O2• and O22•, there are numerous examples where these enzymes catalyze the metabolic activation of chemically inert agents to electrophiles. This study aimed (i) to examine if lambda-cyhalothrin interacts with microsomal CYP system and (ii) to analyze whether oxidative stress, proinflammatory and apoptosis mechanisms should be also co-affected by this pyrethroid. All experimental procedures were conducted with ethic requirements and authorized by the Institutional Animal Care and Use Committee of our University. It was evaluated in the liver of male Wistar rats following oral pyrethroid exposure (4 and 8 mg/kg bw in corn oil, 6 days): (1) CYP isoform activities (CYP1A1, CYP2B1/2, CYP3A1/2, CYP2A1, CYP2C11 and CYP2B1). (2) Oxidative stress markers (ROS, and enzymatic antioxidant activities). (3) Gene expression of proinflammatory (NFκB, IL-1β), oxidative stress and apoptosis (Nrf2, p53, caspase-3, Bax) mediators. Quantitative real-time PCR assays for rat CYP1A1, CYP1A2, CYP2A1, CYP2B1/2, CYP2E1, CYP3A1/2, and CYP4A1 mRNA were also performed. The results demonstrated: (1) Lambda-cyhalothrin exposure produced significant increase in CYP3A1/2, CYP2A1 and CYP2B1 activities. (2) Hepatic CYP1A1, CYP1A2, CYP2A1, CYP2E1, CYP3A1 and CYP3A2 gene expressions increased significantly in both groups treated with lambda-cyhalothrin. The major significant increase of mRNA levels of CYP isoforms was observed in CYP2B1 (1463 % and 961 %) and CYP2B2 (604 % and 501 %). Finally, IL-1β, NFκB, Nrf2, p53, Casp-3, and Bax mRNA levels were also significantly increased by lambda-cyhalothrin exposure. In conclusion, the present study demonstrates, in liver microsomes from rats treated orally with lambda-cyhalothrin, an induction of CYP1A, CYP2E, CYP2B and CYP3A subfamilies; results confirmed analyzing gene expression by real-time PCR. Our study also provides links between inflammation, oxidative stress, NFκB activation and CYP regulation in lambda-cyhalothrin toxicity. Work supported by Project Ref. S2013/ABI-2728 from Comunidad de Madrid, and Project Ref. RTA2015-00010-C03-03 from Ministerio de Economía, Industria y Competitividad, Spain.
Keywords: Lambda-cyhalothrin, CYP enzymes, Gene expression, Pathways, Rat liver
Effects of 2-mercaptobenzimidazole and its methyl derivatives on liver drug- metabolizing enzyme system after repeated oral administration in rats (#111)
A. Miyajima-Tabata1, K. Sakemi-Hoshikawa2, M. Usami2, 3, K. Mitsunaga4, T. Irie2, Y. Ohno2, 5, M. Sunouchi2
1 National Institute of Health Sciences, Division of Medical Devices, Kawasaki, Japan
Purpose: 2-Mercaptobenzimidazole (MBI) and its methyl derivative 4(5)-MeMBI (1:1 mixture of 4-MeMBI and 5-MeMBI) are widely used as rubber antioxidants. MBI was strongly thyrotoxic due to the thioureylene structure in repeated oral administration toxicity studies, causing marked enlargement of the thyroid, and decreased thyroid hormones. On the other hand, MeMBIs (4-MeMBI, 5-MeMBI, and 4(5)-MeMBI) were also thyrotoxic but to a lesser degree with smaller or no changes in thyroid hormones. In this study, we examined and compared the effects of MBI and MeMBIs on the drug-metabolizing activity in rat liver microsomes, since the differences in their thyrotoxicity seem to depend on their toxicokinetic profiles.
Methods: MBI (0.3 mmol/kg/day), 4-MeMBI (0.6), 5-MeMBI (0.6), and 4(5)-MeMBI (0.6, 1.2) were administrated orally once a day to male Wistar rats for 8 days. Microsomal pellets were obtained from rat liver and the contents of cytochrome P450 (CYP), and cytochrome b5 (CYB5) were measured. The activities of NADPH-cytochrome P450 reductase (POR), 7-ethoxycoumarin O-deethylation (ECOD), 7-ethoxyresorufin O-deethylation (EROD), and 7-pentoxyresorufin O-depentylation (PROD) were determined, and the amounts of microsomal CYPs were determined semi-quantified by western blot analysis.
Results and discussion: MBI and MeMBIs increased the weight of liver and thyroid; MBI was most potent, and there was no additive or synergistic effect between 4-MeMBI and 5-MeMBI. MBI decreased the CYP content, and the activities of POR and ECOD, but increased the PROD activity, suggesting overall inhibition of the drug-metabolizing activity with simultaneous induction of CYP2B activity. In contrast, 4-MeMBI, 5-MeMBI, and 4(5)-MeMBI increased the contents of CYP and CYB5, and the activities of ECOD, EROD, and PROD, indicating that MeMBIs mostly induce CYP activity. 5-MeMBI and 4(5)-MeMBI appeared inhibitory for CYPs 2C11 and 2C13. There was no additive or synergistic effect, but was counteraction, between 4-MeMBI and 5-MeMBI. These effects on the liver drug-metabolizing system seem to be related to the toxicological differences between MBI and MeMBIs.
Keywords: benzimidazole, rat, drug metabolizing enzyme
Dosing corrected for species differences in toxicokinetics using PBPK modelling predicts equivalent reactive metabolite burden following acetaminophen overdose (#118)
D. Reddyhoff1, R. Sison-Young5, L. Livoti5, G. Vermeil De Conchard3, Y. Parmentier4, R. J. Weaver2, K. Park5, C. Fisher1, I. Gardner1, I. Copple5
1 Certara UK Limited, Simcyp Division, Sheffield, United Kingdom
Species differences in metabolic pathways, arising from cofactor turnover and differences in drug metabolizing enzyme affinity and expression, and physiology effect the toxicokinetics of compounds. Differences in phase I and II metabolism of acetaminophen (APAP) between rat and mouse affect production of the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI), which is primarily detoxified through conjugation with the anti-oxidant glutathione (GSH). However, excess production of NAPQI results in depletion of GSH and subsequent formation of protein adducts, oxidative-stress and liver injury.
Based on literature and publically accessible data repositories, PBPK models were established describing the disposition of APAP in both species. Specifically, these models incorporated sulphation, glucuronidation and CYP-mediated hydroxylation of APAP. Using these models, oral equivalent doses (OEDs) of 300 mg/kg and 1000 mg/kg were predicted for mouse and rat, respectively. OEDs were defined as the oral APAP dose resulting in the same hepatic NAPQI burden in both species. These doses were subsequently used in a preclinical single dose study in both species. Following overnight withdrawal of food, animals were dosed via oral gavage and sacrificed at 0.5, 1, 3, 6, 9 and 24h after dosing (n = 7-14 per timepoint). APAP and conjugated metabolite concentrations in plasma (APAP-GSH, APAP-CYS, APAP-NAC), as well as hepatic GSH concentrations were determined at each timepoint. Total hepatic NAPQI was then calculated from the AUC of all conjugated APAP metabolites in mouse via mass-balance analysis; assuming no protein binding, active secretion or re-absorption, and corrected for biliary clearance. A second approach assumed 1:1 stoichiometry between GSH and NAPQI, and calculated total hepatic NAPQI burden from total hepatic GSH depletion in both rat and mouse. Our experimental results show a mouse:rat total hepatic NAPQI burden ratio, based on GSH depletion, was 1.4. The ratio calculated based on mass balance in mouse and GSH depletion in rat was 0.64. Thus, PBPK predicted OEDs resulted in cross-species total hepatic NAPQI burden within 1.6 fold. Biomarker results show the mouse is intrinsically more susceptible to hepatic injury following APAP overdose. Despite the similar hepatic NAPQI burden at the chosen doses, mean mouse ALT and AST levels increased by 67 and 44-fold, respectively, rat levels only increased 3.5 and 7-fold, respectively.
Preclinical study design informed by PBPK modelling and simulation facilitates robust cross-species comparison of intrinsic susceptibility to toxicological hazard. Such an approach can reduce the number of animals required, help to refine study design, and inform on species selection for safety testing to facilitate extrapolation of findings to humans as part of the risk assessment of compounds.
This project has received funding from the IMI2 programme under grant agreement No 116030.
Keywords: PBPK, in vivo, toxicokinetics, acetaminophen
Comparable findings in the rat liver with a long-acting glucagon receptor agonist, SAR438544, and an 8-hour infusion of glucagon (#160)
A. Bube1, C. Hunger2, L. Müller3, S. Nellen2, S. Ramusovic4, A. Dudda5, T. Kißner1
1 Sanofi, Preclinical Safety, Frankfurt/Main, Hesse, Germany
This study was funded by Sanofi.
Background and Aims: Glucagon agonism using synthetic receptor agonists has potential for weight loss therapy. For further characterization of the safety profile of SAR438544, a long-acting glucagon receptor agonist, its effects on selected liver findings were assessed in rats following subcutaneous (SC) injection and were compared to effects of exposure profile matched (short acting) glucagon. Exposure profile machting was achieved by 8-h continuous intravenous (IV) infusion of (short acting) glucagon.
Materials and methods: Low (1 mg/kg/day) and high (10 mg/kg/day) doses of SAR438544 or low (0.146 mg/day) and high (1.46 mg/day) doses of (short acting) glucagon were administered daily for 7 days by SC injection or 8-h continuous intravenous (IV) infusion, respectively. Mortality, clinical signs, body weight, clinical chemistry, and anatomic pathology were recorded in the main group of rats. In a toxicokinetics satellite group blood was collected on Days 1 and 7 of dosing.
Results: Daily SC administration of long-acting glucagon or IV infusion of (short acting) glucagon were well tolerated with limited clinical observations. No deaths or consistent trends in body weight or food consumption were noted with either compound. Toxicokinetic profiles were generally similar. Both compounds resulted in findings in the liver including increased organ weight and glycogen accumulation.
Conclusion: The incidence and severity of liver findings in rats dosed daily with long-acting glucagon were consistent with the effects of daily dosed and exposure profile matched (short acting) glucagon.
Keywords: Liver, Glucagon
Relation between DMSO application and selected cytochromes P450 in developing liver (#183)
L. Luptakova1, S. Dvorcakova1, E. Petrovová2
1 University of Veterinary Medicine and Pharmacy in Kosice, Department of Biology and Genetics, Košice, Slovakia
Living organisms are exposed to a number of structurally different chemicals in their natural environment every day. In addition to the substances of natural origin, they also receive a large amount of synthetic foreign substances - xenobiotics. Xenobiotics cannot be used by organism, they are potentially harmful and therefore should be excluded from the body as soon as possible. The study evaluates the effect of dimethyl sulfoxide (DMSO) on the developing chicken liver after its application on the 4th embryonic day (ED4) in application dose 5, 10, 15, 20, 25, 30, 35 a 50 µl per egg. The liver was removed on ED9 for the gene expression analysis of selected cytochrome P450 genes (CYP1A5, CYP3A37 and CYP3A4).
Cytochrome activity and their gene expression is a major determinant of drug efficacy and toxicity, thereby determining the therapeutic outcome. In monitoring the activity of detoxifying enzymes in various poultry species great differences in enzyme kinetics were found. CYP3A37 and CYP3A4 isoforms belong to the cytochrome CYP3A group, mainly found in the liver and intestine. Their expression can be induced by a wide variety of compounds such as antibiotics, glucocorticoids or pesticides. DMSO has been shown to increase the expression of cytochrome family enzymes, especially the CYP3A family. The increased expression of the CYP1A5 gene after DMSO administration may be related to the fact that DMSO is capable of activating the aryl hydrocarbon receptor (AhR), which functions as a ligand-activated transcription factor regulating gene expression. AhR is also referred to as the regulator of xenobiotics metabolising enzymes. DMSO exposure activates AhR and induces AhR translocation to the nucleus and its binding to the target gene promoter, resulting in increased gene expression. When the ligand binds to the nuclear receptor, it causes nuclear receptor activation and subsequently it binds to the DNA promoter and induces gene transcription initiation.
In our study, gene expression in developing liver tissue isoforms of both cytochrome P450 families (CYP1A and CYP3A) was increased by DMSO administration. It could be concluded that gene expression increased in proportion to an increased application dose. The statistically significance increase of gene expression of isoforms CYP1A5 and CYP3A37 has been noticed in the application doses 15, 20, 25, 30, 35 and 50 ul. The isoform CYP3A4 has the similar results except the application dose 15 ul. Generally, the highest induction of gene expression by DMSO has been noticed for the isoform CYP1A5 in application dose 50 ul (8.83 times), followed by CYP3A37 (5.67 times). The lowest of gene expression induction has been noticed for isoform CYP3A4 (3.37 times).
This work was realized within the project of Ministry of Education VEGA No.1/0050/19.
Keywords: DMSO, gene expression, cytochrome P450, chick embryo, liver
Disruption of liver gene expression and ultrasonic vocalization of infant mouse offspring perinatally exposed to 2,3,7,8-tetrabromodibenzofuran (#212)
E. Kimura1, 2, G. Suzuki3, N. Uramaru4, F. Maekawa1
1 National Institute for Environmental Studies, Center for Health and Environmental Risk Research, Tsukuba, Japan
Exposure to chlorinated dioxins/furans that activate aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, has been reported to induce a variety of toxicities, such as tumorigenesis and cognitive impairments, in humans and laboratory animals. We have shown that in utero and lactational exposure to 2,3,7,8-tetrachlorodibezo-p-dioxin induces expression of AhR-target genes in the liver and suppressed ultrasonic vocalization (USV) of infant mouse offspring. Since certain brominated analogs are also able to activate AhR, it is reasonable to consider that such brominated congeners have toxicities relevant to chlorinated congeners. In the present study, we studied effects of perinatal exposure to 2,3,7,8-tetrabromodibenzofuran (TBDF) on gene expression and USV in the mouse. Pregnant C57BL/6J mice were orally administered TBDF at a dose of 0, 9, or 45 μg/kg b.w. on gestational day 12.5 (hereafter, named as the control, TBDF-9, and TBDF-45 groups, respectively). USVs of offspring on postnatal days 3–9 were measured for 1 min in sound-attenuate chamber, and the USV frequencies of 60–100 kHz were analyzed. Liver and brain tissues on postnatal day 5 were collected, and mRNA expression between the control and TBDF-45 groups was analyzed by gene microarray. In addition, the expression of the top four mRNAs that significantly increased or decreased was confirmed by quantitative PCR. As a result, total USV duration in the TBDF-45 group, but not the TBDF-9 group, was significantly lower than that in the control group. Gene microarray revealed significant changes in expression of 1,181 genes in the liver between the control and TBDF-45 groups. Among them, Cyp1a1, Cyp1a2, Fmo3, and Pnliprp1 mRNAs were significantly increased and Tff3, Ocstamp, Kcnk16, and Lgals2 mRNAs were significantly decreased in the TBDF-9 and -45 groups compared to those in the control group. On the other hand, no significant difference in expression of Cyp1a1 and Tff3 mRNAs was observed in the brain between the control and TBDF-45 groups. Our findings show gene expression changes in the liver, but not the brain, of the TBDF-exposed mouse offspring, which suggests that suppressed USV might be partly caused by impairment of peripheral tissues, including the liver.
Keywords: Brominated dioxins/furans, Developmental toxicity, Ultrasonic vocalization, Liver, Brain
Assessment of drug hepatotoxicity in 3D InSight™ Liver Microtissues with expanded panel of cytotoxicity markers (AST, LDH and ATP). (#245)
A. Pajak1, R. Class2, B. Twomey2, A. Kiessling2, A. Gorecka1, M. Kijanska1
1 InSphero AG, Schlieren, Zürich, Switzerland
3D InSight™ Human Liver Microtissues consisting of primary liver cells are an attractive tool for in vitro drug safety assessment due to preservation of liver specific functions and metabolic activity over a long culturing time. 3D InSight™ Human Liver Microtissues were previously shown to outperform 2D primary hepatocytes in prediction of hepatotoxicants causing drug-induced liver injury (DILI), based on cell viability assay.
In this study, we established a method to assess a clinically relevant marker for liver damage - aspartate aminotransferase (AST), and correlate AST levels with other cell viability and cytotoxicity markers (leakage of lactate dehydrogenase (LDH) and intracellular ATP). 3D InSight Human Liver Microtissues were exposed to a set of reference compounds with known DILI grade for 7 days. Measurement of all 3 markers was performed for a single microtissue, with LDH and AST measurements multiplexed in the same microtissue supernatant.
Cell viability profiles were able to discriminate between structurally related compounds manifesting different clinical toxicity grade, such as Troglitazone/Rosiglitazone and Tolcapone/Entacapone. Importantly, profiling of ATP levels together with AST and LDH leakage signal for DILI+ reference compounds showed either a strong correlation between decrease of ATP levels and increase of AST and LDH leakage signal or decrease of ATP levels with concomitant mild or no release of AST and LDH. Reduction of ATP levels with moderate-to-no leakage signal from LDH and AST can be an indicator of metabolic stress rather than necrosis as a cause of toxicity. Together, these results 1) prove feasibility of profiling of clinically relevant marker (AST) in human liver microtissues 2) expand cytotoxicity marker panel that can be measured in a single microtissue to ATP, AST and LDH 3) show application of complementary profiles of different toxicity markers in in vitro assessment of hepatotoxic compounds and their potential mechanism of toxicity.
We suggest this method as a promising approach for safety assessment in drug development with a direct correlation to DILI markers used in clinical trials.
Keywords: 3D liver tissues, in vitro toxicology, cytotoxicity markers, hepatotoxic compounds
The method of spheroid formation for 3D cultures of primary hepatocytes influences hepatocellular functions and hepatotoxicity (#248)
J. Moer1, D. Runge2, A. Ullrich3
1 PRIMACYT Cell Culture Technology GmbH, Schwerin, Mecklenburg-Western Pomerania, Germany
Primary hepatocytes of human and animal origin are the gold standard for all pharmacological-toxicological studies in drug development. They play a major role in eco-toxicological evaluation as well. Three-dimensional (3D) cultures became more popular in recent years since they might mimic the in-vivo cell morphology, polarity and cell-cell interactions better than traditional two-dimensional (2D) cultures. Here, two types of cell culture plates were used to generate 3D cultures with primary hepatocytes: the GravityPLUS Hanging Drop System with subsequent culture in Gravity TRAP plates in comparison to U-bottom ULA (ultra-low attachment) plates with cell repellent surfaces. Standard 2D cultures were performed as well.
Hepatocellular detoxification functions like urea release and CYP450 activity as well as the response to the hepatotoxin Diclofenac were analysed in these culture systems. The results were normalized to the corresponding volume of culture medium or to protein content.
The secretion of urea was improved and maintained at higher levels in U-bottom ULA plates compared to the Hanging Drop System. CYP1A activity was better inducible by ß-Naphthoflavone in U-bottom plates than in the Hanging Drop System at all 3 tested cell numbers. Basal Cytochrome P450 activities were higher in U-bottom plates and showed a better inducibility in these plates compared to the Gravity TRAP plates.
Diclofenac, a known and well-described hepatotoxic compound, did show similar effects on hepatocytes with regard to the ATP content in both 3D culture systems. Beside this, the decrease of ATP content due to Diclofenac treatment was higher in 2D culture than in the 3D culture systems.
In summary, our results indicate that major differences may exist between different 3D culture systems and in comparison to standard 2D culture methods. These differences may lead to different and conflicting results in the assessment of drug toxicity and drug-drug interaction.
Keywords: 3D culture, spheroid, hepatocytes hepatotoxicity, liver
Development and characterisation of 3D liver models to investigate drug toxicity (#252)
M. F. Kaluthantrige Don1, 2, 3, K. O'Holleran2, A. West3, M. Huch1, 2
1 University of Cambridge, Gurdon institute, Cambridge, United Kingdom
Drug induced liver injury (DILI) is a leading safety problem for the pharmaceutical industry and healthcare providers. Yet, many drugs are withdrawn from the market as causing human hepatotoxicity. Hence, identification of liver predictive models to maximize the amount of drug-candidate information is critical during preclinical stages of drug discovery. Work conducted by Meritxell Huch has shown that by isolating adult stem cells from a liver biopsy and culturing them in artificial ECM matrix and medium supplemented with specific growth factors, these cells naturally self-organize in fully functioning 3D liver structures defined as “organoids”. However, using the liver organoid as model to detect hepatotoxicity is currently an open question. To address this, initial focus was directed towards the differentiation of liver organoids into hepatocytes like cells as these are the main epithelial cells involved in drug metabolism. I have developed a protocol for the differentiation of liver organoids in a stem cell like state into organoids expressing hepatocyte cells, characterised by a higher expression of hepatocyte markers like ALBUMIN, and CYP3A4 genes. Transmission electron microscopy analysis showed cellular polarization marked by formation of bile canaliculi like structures in differentiated liver organoids. This led to the analysis of the transport activity of efflux transporters located at the apical membrane of hepatocytes. Immunostaining analysis showed expression of the bile salt export pump (BSEP), a bile canalicular transporter involved in secretion of bile and xenobiotics, but also an important target for drug toxicity. A transport assay using fluorescein diacetate confirmed the functional activity of BSEP to transport its substrate into the bile canaliculus. In summary, I have validated a method to differentiated organoids into mature hepatocytes and preliminarily investigated the structural functionality of this novel 3D model. Future work will aim to further characterise the metabolic activity and then investigate the toxicological predictivity of the model towards known human hepatotoxins.
Huch, M. et al. In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration. Nature 494, 247–50 (2013).
Keywords: Liver, Toxicology, Organoid, in vitro, DILI
Hepatic IGF signalling is dysregulated by in utero exposure to maternal smoking (#318)
C. Talia1, P. Filis1, U. Soffientini5, B. Lucendo-Villarin2, A. Douglas1, D. Hay4, S. Shaw1, J. Iredale4, M. Swortwood7, M. Huestis3, M. Bellingham5, L. Connolly6, P. O’Shaughnessy5, P. Fowler1
1 University of Aberdeen, Institute of Medical Sciences , Aberdeen, United Kingdom
Background: Insulin-like growth factors (IGF-1 and IGF-2), acting through their receptors (IGF1R and IGF2R), are major regulators of fetal growth. IGF bioavailability is regulated by high-affinity binding proteins (IGFBPs). Fetal expression of IGFBPs is tissue-specific with high abundance in the liver. Small for gestational age (SGA) newborns exhibit reduced IGF and increased circulating IGFBP. Maternal smoking during pregnancy is associated with intrauterine growth restriction, but molecular mechanisms remain unclear. Our aim was to evaluate whether in utero exposure to cigarette smoke (containing more than 7000 pollutants) could affect the IGF axis in the human fetal liver.
Methods: 80 human fetal livers from elective terminations of normal pregnancies (12-19 gestation week), were collected (NHS Grampian Research Ethics Committees, REC 04/S0802/21). RNA was extracted and the Illumina NextSeq platform produced 76 bp single end RNA sequencing reads. These reads were quality controlled, aligned to the human reference genome and quantified at gene regions. Significant differentially expressed genes were identified using a generalised linear model with a three-way interaction model between fetal sex, fetal age and maternal smoking status.
Results: IGFBP1 and IGF2BP2 were significantly upregulated in the older male (>17 gestation week) smoke-exposed fetuses. The developmental increase in IGFBP4 and IGFBP7 expression was significantly accelerated only in males exposed to smoke. IGF2R decreased throughout gestation only in male fetuses not exposed to cigarette smoke.
Conclusions: The significant changes in key elements of the IGF axis indicate a general dysregulation of IGF signalling within the male fetal liver, with a striking sex-difference. Interestingly, in epidemiological studies, males exposed to maternal smoking are more susceptible to growth restriction. Increased transcript levels of IGFBPs may decrease IGF bioavailability, ultimately resulting in reduced IGF signalling and potentially impairing fetal growth. Investigation of protein and gene expression of IGF pathway members in the placenta and other fetal tissues will be performed.
Funding: European Union’s Horizon 2020 Marie Skłodowska-Curie grant agreement No.722634; MRC MR/L010011/1
Keywords: human fetus, fetal liver, maternal smoking, endocrine disruptors, prenatal toxicology
Grayscale differential box counting as a measure of complexity of liver texture in common carp (Cyprinus carpio) sub-chronically exposed to perfluorooctanoic acid (PFOA) (#321)
M. Manera1, B. Sayyaf Dezfuli2, G. Castaldelli2, C. Martino3, L. Giari2
1 University of Teramo, Faculty of Biosciences, Food and Environmental Technologies, Teramo, Italy
Perfluorooctanoic acid (PFOA), a perfluorinated alkylated substance (PFAS), poses a worldwide concern for its wide distribution, bioaccumulation in food webs, long half-life in organisms, and potential toxic, carcinogenic and endocrine disrupting effects on animals. Fish are excellent candidates in aquatic biomonitoring programs and toxicologic testing, frequently focusing on liver due to its pivotal role in the health of the whole organism and its highly sensitivity to contaminants. Histopathology can be useful in evaluating toxicological effects on fish health and texture analysis can represent an objective, replicable diagnostic tool, potentially free from operator-dependent bias. In the present survey, liver histological texture was comparatively assessed in specimens of common carp (Cyprinus carpio) sub-chronically exposed to PFOA. Twenty specimens were exposed to two PFOA dosages (10 exposed to 200 ng l-1, 10 exposed to 2 mg l-1) for 56 days and compared to other 10 unexposed fish. Grayscale differential box counting (fractal dimension and lacunarity) was evaluated on representative pictures taken from liver histological sections. Differential box counting was implemented by converting two-dimensional grayscale images into pseudo three-dimensional information. Hence, fractal dimension and lacunarity acted as a measure of the complexity and of the heterogeneity of the grayscale levels distribution, respectively. Redundancy Analysis (RDA) was performed on the obtained numerical data in order to summarize the part of grayscale differential box counting variation that is explained by the following biometric/experimental variables: PFOA liver concentration, liver mass, proliferating cell nuclear antigen (PCNA) positive nuclei, after removing the effects of fish total length. The t-values of the regression coefficients of liver PFOA concentration and of liver mass with both fractal dimension and lacunarity, and of PCNA positive nuclei with lacunarity, showed values larger than 2, while the t-value of the regression of PCNA positive nuclei with fractal dimension appeared to be close to 2. Considering the selected biometric/experimental variables, liver PFOA concentration correlated with PCNA positive nuclei but did not correlate with liver mass, whereas PCNA positive nuclei correlated with liver mass. Interestingly, fractal dimension contributed better than lacunarity in treatment groups ordination. Recently, fractal analysis has been adopted to estimate the complexity loss associated with pathological changes. In the present survey, contrary as expected, liver texture modification related to liver PFOA concentration increase was associated with a significant complexity increase, related to reversible changes (hydropic degeneration), possibly acting as an initially adaptive strategy, rather than representing mere degeneration, to cope with PFOA challenge. The possible occurrence of a hormetic response should be further investigated.
Keywords: toxicologic pathology, hydropic degeneration, cellular pathology, hormesis
Pluripotent stem cells differentiation towards definitive endoderm. (#354)
1 University of Helsinki, Faculty of Pharmacy, Helsinki, Finland
Hepatocyte-like cells generated from induced pluripotent stem cells (iPSC) represent a promising tool as a human liver cell model for different applications including drug toxicity screening. The main complexity of this approach is that obtaining of mature functional hepatocytes remains challenging therefore require detailed study of every step of the cells differentiation. Current research is dedicated to comparison of different methods of obtaining of definitive endoderm (DE) – the first stage of stem cells differentiation towards hepatic lineage. Aim of this study is to develop an effective method of hPSC differentiation to cells of DE in 2D conditions. We differentiated two cell lines using six conditions, contained of growth factors (Activin A (AA), Wnt3a), or small molecules (sodium butyrate (NaB), IDE1). We checked three NaB, which were purchased from three different suppliers to test the affection of the product purity on cell viability. Change of cell morphology showed which condition facilitated faster cell differentiation and how different conditions influence cell viability. At four time points (day 0, 1, 4 and 6), we measured relative mRNA expression of gene markers for pluripotency, DE, hepatic, mesendoderm and ectoderm. We found that cells had different sensitivity to NaB obtained from different companies and one of them was excluded due to massive cell death. We observed effective DE formation with AA alone, with the combination of AA and Wnt-3A, and with the combination of AA and NaB obtained from two companies. NaB leads to the fastest differentiation, but it is toxic for cells, which restricts it’s usage for obtaining of big amount of DE cells. Hierarchical cluster analysis showed similarities between different conditions and allowed us to divide them in two groups, based on affection on the gene expression; none of them included IDE1. Immunofluorescent method confirmed effectiveness on protein level of four conditions for DE formation. We showed that the IDE-1 at the tested concentrations is ineffective for DE formation. In conclusion, we obtained an effective protocol for obtaining DE cells for the further differentiation towards hepatic lineage.
Keywords: iPSC, ESC, definitive endoderm
Versatile pro-fluorescent and fluorescent coumarin derivatives as substrates for different types of xenobiotic metabolizing enzymes (#355)
R. O. Juvonen1, J. Huuskonen2, O. Pentikäinen3, M. Finel4, H. Raunio1
1 University of Eastern Finland, School of Pharmacy, Faculty of Health Sciences, Kuopio, Finland
Detailed knowledge of xenobiotic metabolizing pathways is essential for understanding toxicity of substances and for evaluation of their health risks. For small-molecule drugs, cytochrome P450 (CYP) enzymes catalyze most functionalizing reactions, with glucuronosyltransferases (UGT) and sulfotransferases (ST) mediating most conjugation reactions. 7-hydroxycoumarin and its substituents are usually strongly fluorescent, whereas the parent coumarins or their ether derivatives such as glucuronides are non-fluorescent. We have established novel metabolism reactions for CYPs, UGTs, STs and catechol-O-methyltransferase (COMT) using coumarin or its derivatives as probe substrates. In these reactions, coumarin derivatives are oxidized to corresponding fluorescent 7-hydroxycoumarins by different CYPs, and 7-hydroxycoumarins are conjugated to non-fluorescent metabolites. The change in fluorescence can be determined in simple and sensitive assays in a multiwell plate format by either kinetic or end-point measurements. Coumarin substituted with different types of phenyls at position 3 or with chlorine, methoxy or methyl at position 6 are oxidized to fluorescent 7-hydroxycoumarins by human CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2C19 or CYP3A4 enzymes. Similar 7-hydroxycoumarins are glucuronidated or sulfonated by microsomal UGTs or sulfonated by cytosolic STs to non-fluorescent conjugates. The weakly fluorescent 6, 7-dihydroxcoumarin was methylated by cytosolic and microsomal COMT to strongly fluorescent 6-methoxy-7-hydroxycoumarin. The substrates were used to detect CYP and conjugating enzyme activities in the liver and intestine in humans and various preclinical animal species. Some of the substrates are selective for individual human CYP and UGT enzymes, and have facilitated evaluation of hepatic vs extrahepatic enzyme activities. In drug development, these new probe substrates can be used to study inhibitory potential of drug candidates towards specific enzymes in a high throughput format.
Keywords: xenobiotic, metabolism, liver, assay
CYP1A2 Enzyme Activity and Protein Abundance In Normal And Diseased Pediatric Livers (#356)
M. Czerwinski1, B. Ewy1, A. Kats2, M. Pritchard3, 4, S. Tague5, B. Ogilvie1
1 Sekisui XenoTech LLC, Scientific Consulting, Kansas City, Kansas, United States of America
CYP1A2 is a drug-metabolizing enzyme whose expression begins between birth and 4 weeks of age, and gradually increases to about half of the adult levels by 6 years of age. The enzyme constitutes 4 - 16% of the hepatic CYP pool, and is a major determinant of the biotransformation of ~9% of clinically used drugs. Interestingly, CYP1A2 activity decreases in adults with non-alcoholic fatty liver disease (NAFLD). Considering the increase in the number of medications given to children, as well as the rise in childhood obesity and NAFLD, this study aimed to determine whether donor age and health status influence CYP1A2 abundance, lobular localization and enzyme activity. Pediatric liver microsomes and a corresponding tissue microarray (TMA) were our test system. The TMA contained 25 tissues from donors aged 3 months to 18 years old and 5 adult controls. The donor demographics and health data were collected from interviews with next of kin and from hospital records. Three consecutively cut arrays were stained with hematoxylin and eosin (H&E) and Gömöri trichrome for a pathologist’s determination of liver disease status or with anti-CYP1A2 Ab. Liver microsomes were prepared and CYP1A2 phenacetin O-dealkylase activity assayed according to published methods. CYP1A2 protein was detected in all tissues, except in one 4-month old, and it increased with age. In tissues judged to be normal or having minimal pathological findings, CYP1A2 protein was located in zone 3 and 2 hepatocytes. In 20 samples that were free of significant necrosis and NAFLD, the abundance of CYP1A2 enzyme correlated with the donor age (R2 = 0.28). In microsomes prepared from these tissues, CYP1A2 enzyme activity was independent of donor age (R2 = 0.01). A diffuse localization, associated with reduced CYP1A2 level, was seen in tissues affected by necrosis and ischemia. Pediatric NAFLD was associated with diminished CYP1A2 staining (3 donors, 10 - 14 years old, steatosis 50 - 80%, BMI 32.2 - 32.5) paralleling what is seen in adults. In conclusion, the pediatric liver TMA but not microsomes, was a useful tool to elucidate the ontogeny of CYP1A2 protein in healthy livers. Better preservation of immunoreactive CYP1A2 protein than its enzymatic activity in our samples may reflect the priority given to organ transplantation over utilization of donor tissue for research.
Keywords: Cytochrome P450, Pediatric drug metabolism, Liver tissue microarray
Extracellular vesicles are involved in polycyclic aromatic hydrocarbon hepatotoxicity (#427)
N. van Meteren1, D. Gobart1, I. Gallais1, E. Le Ferrec1, D. Lagadic-Gossmann1, O. Sergent1
1 Univ Rennes, Inserm, EHESP, Irset , Institut de recherche en santé, environnement et travail - UMR_S 1085, Rennes, France
Purpose Polycyclic aromatic hydrocarbons (PAHs) are environmental pollutants that can be found in cigarette smoke and contaminated food, the main exposure for non-smokers, and that are considered for some of them as hepatotoxicants. Extracellular vesicles (EVs) are membrane-surrounded nanostructures released by cells into the extracellular environmenta that are now recognized as major actors of intercellular communication and in this context, as pathogenic mediators in several liver diseasesb. Regarding xenobiotic liver injury, EVs emerge as potential actors of drug induced liver injuryc, yet nothing is known concerning toxicant-associated liver diseases. We previously demonstrated that three PAHs i.e benzo(a)pyrene (BP), dibenzo(a,h)anthracene (DBA) or pyrene (PYR), were able not only to increase the EV release by primary rat and WIF-B9 hepatocytes but also to modify EV composition. Therefore, the aim of this work was to study the impact of hepatocyte-derived EVs on target hepatocytes.
Methods WIF-B9 and primary rat hepatocytes were treated by 100 nM BP, DBA or PYR. PAHs were selected based upon their various concentrations in common food and their various affinities for the AhR (Aryl hydrocarbon Receptor) as AhR mediates most of the biological effects of several PAHs by leading to the production of reactive oxygen species and metabolitesd. Then, EVs were isolated from extracellular medium by differential ultracentrifugation and put in contact with non-treated hepatocytes.
Results EVs released from PAH-treated hepatocytes (PAH-EVs) were more cytotoxic than control EVs, as they were able of causing an increase in apoptosis of target hepatocytes by activation of caspases. The triggering of apoptosis was dependent on an EV uptake by endocytosis. In line with this, PAH-EVs contained more pro-apoptotic components. In addition, greater levels of pro-oxidative components were found in PAH-EVs and PAH-EVs were capable of generating an oxidative stress in target hepatocytes. Finally, PAH-EVs were demonstrated to be able to reach the lysosomal compartment. As the expression of the iron storage protein, ferritin, was higher in PAH-EVs, it could be suggested that Fenton and Haber-Weiss reaction occurred in lysosomes leading to the production of the powerful oxidative species, hydroxyl radical. Thus, a lysosome membrane oxidative damage may explain the lysosome membrane permeabilization (LMP) found with PAH-EVs, that ultimately caused target hepatocyte death.
Conclusion PAH-EVs are implicated in apoptosis of target hepatocytes suggesting a possible involvement of extracellular vesicles in PAH-induced liver injury.
a Kowal et al. Curr. Opin. Cell Biol. 29, 116–125 (2014)
b Hirsova et al. Hepatology 64, 2219–2233 (2016)
c Holman et al. Toxicol. Sci. 151, 365–375 (2016)
d Collin et al. Free Radic. Biol. Med. 72, 11–22 (2014)
Keywords: Polycyclic aromatic hydrocarbon, Apoptosis, Extracellular vesicle, Hepatocyte, Cell communication
A new strategy for exploring the role of hyperthermia in MDMA-induced toxicity in primary mouse hepatic cells using GC-MS-based metabolomics (#452)
A. M. Araújo1, M. Enea1, E. Fernandes2, M. D. L. Bastos1, F. D. Carvalho1, P. Guedes de Pinho1, M. Carvalho1, 3
1 UCIBIO, REQUIMTE, Laboratory of Toxicology, Faculty of Pharmacy, University of Porto, Porto, Portugal
Hyperthermia is a life-threatening consequence of 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) abuse. In this work, a metabolomics approach based on gas chromatography-mass spectrometry was used to investigate the hepatic metabolic changes caused by MDMA under normothermic and hyperthermic conditions. For this purpose, freshly isolated mouse hepatocytes were exposed to three concentrations of MDMA (corresponding to LC01, LC10and LC30, as assessed by the MTT assay) for 24 h, at 37.0 °C or 40.5 °C, and the alterations on the intracellular metabolome were evaluated.The results obtained by the multivariate analysis showed that metabolic patterns of MDMA exposed cells are separated from control in a concentration-dependent manner, both in normothermic and hyperthermic conditions. Normothermic data revealed a significant alteration (p<0.05) in the levels of malate, fumarate, 2-oxoglutarate, lactate, phosphoric acid, ornithine, glutamate, cysteine, aspartate, myo-inositol, gluconic acid, among others. This panel of discriminant metabolites are mostly involved in energetic metabolism, amino acids metabolism, urea cycle, ammonia recycling, fatty acid metabolism and antioxidant defenses. These metabolic disturbances were significantly more pronounced at 40.5 °C. Importantly, our resultsalso demonstrated that hyperthermia per se induces significant alterations in levels of metabolites involved in the TCA cycle, amino acids metabolism and urea cycle. Taken together, these findings indicate that MDMA triggers significant metabolic alterations on hepatic cells and that these effects are clearly exacerbated under hyperthermic conditions, emphasizing the potential increased risks of MDMA abuse owing to the thermogenic action of the drug.
A.M.A. and M.E. thank Fundação para a Ciência e Tecnologia (FCT) for their PhD fellowships (SFRH/BD/107708/2015 and PQ/BD/109634/2015, respectively).This work was financed by national funds from FCT/MEC (UID/Multi/04378/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI/01/0145/FEDER/007728).M.C. acknowledges FCT through the UID/MULTI/04546/2019 project.
Keywords: Hyperthermia, MDMA, Primary mouse hepatocytes, Hepatotoxicity, Metabolomics
Proteomic Analysis Reveals Perfluoro-(3,5,7,9-tetraoxadecanoic) acid (PFO4DA) Induced Hepatotoxicity via Activation of PPARs on Male Mice (#476)
J. Dai1, H. Guo1, N. Sheng1
1 Institute of Zoology, Chinese Academy of Sciences, Key Laboratory of Animal Ecology and Conservation Biology, Beijing, China
Introduction: Perfluoro-(3,5,7,9-tetraoxadecanoic) acid (PFO4DA), an alternative to perfluorooctanoic acid (PFOA), has been detected in aqueous samples. However, its potential toxicities remain unclear.
Methods: Male mice were exposed to 0, 0.4, 2, or 10 mg/kg/d PFO4DA for 28 d. We measured triglyceride (TG) and total cholesterol (TCHO) content in serum and liver. Differentially expressed proteins (DEPs) between the control and 10 mg/kg/d groups were identified using isobaric tags for relative and absolute quantification (iTRAQ). Bioinformatics analysis were used to identify the networks between key proteins and DEPs and enrich the regarding GO terms and KEGG pathways. Western blotting was performed to verify iTRAQ results and analyze the expression levels of key proteins and nuclear receptors.
Results: Compared to the control group, changes of liver injury index and serum lipid content in 0.4 and 2 mg/kg/d PFO4DA groups did not exceed 20%. After 10 mg/kg/d PFO4DA exposure, relative liver weight and liver injury index significantly increased, indicating the occurrence of acute liver injury in mice. In serum, we observed no change in TG and TCHO content, but in liver there were significantly decreased TG and TCHO levels. Exposure to 10 mg/kg/d of PFO4DA led to 198 differentially expressed liver genes (56 down-regulated, 142 up-regulated), mainly involved in fatty acid metabolism, oxidation-reduction process and transport. Metabolic pathways, fatty acid degradation, peroxisome, and PPAR signal pathway were enriched, highlighting the stimulation of lipid metabolism in mice liver. The significantly increased PPARα and the downstream proteins after 10 mg/kg/d PFO4DA exposure might be mainly responsible for the decreased lipid content in liver.
Conclusion: This study concluded that PFO4DA exposure could cause hepatotoxicity, and decrease lipid content in mice liver. PPAR pathway activation in the mice liver may contribute to the observed toxic effects. As it has been detected in water samples with much higher concentrations than PFOA, efforts to remove or at least decrease its occurrence in drinking water should be made urgently.
1. Strynar, M.; Dagnino, S.; McMahen, R.; Liang, S.; Lindstrom, A.; Andersen, E.; McMillan, L.; Thurman, M.; Ferrer, I.; Ball, C. Identification of Novel Perfluoroalkyl Ether Carboxylic Acids (PFECAs) and Sulfonic Acids (PFESAs) in Natural Waters Using Accurate Mass Time-of-Flight Mass Spectrometry (TOFMS). Environ Sci Technol 2015, 49 (19), 11622-30.
Keywords: Perfluoropolyether Carboxylic Acids, Proteomic Analysis, PPAR pathways, Hepatotoxicity
Study of long term culture condition of hepatocytes for chronic toxicity test (#502)
S. Horiuchi1, Y. Kuroda1, R. Fujii1, S. - R. Kim1, S. Ishida1
1 National Institute of Health Sciences, Pharmacology, KAWASAKI, Japan
Purpose: At the early stage of drug development, drug-induced liver injury (DILI) is evaluated by in vitro assay using hepatocytes. However, hepatocytes are difficult to culture for a long time while maintaining their functions. Therefore, they are not suitable for evaluation of chronic toxicity. To solve this point, sandwich culture, 3D-bioreactor, and spheroid culture have been developed. By using such culture system, hepatocyte functions can be maintained for several weeks. However, these have not been widely used because they are complicated or require specific culture material and devices. Therefore, we examined simple method for long term culture of hepatocytes.
Keywords: human iPS cell-derived hepatocytes, human cryo-preserved hepatocytes, long term culture, bile canaliculi, drug metabolism
CYP-dependent destruction of hepatic sinusoidal endothelial cells and induction of cholestasis by the hepatotoxic pyrrolizidine alkaloid senecionine (#548)
S. Hessel-Pras1, A. Braeuning1, A. Adawy2, G. Guenther2, A. - M. Enge1, J. Ebmeyer1, C. J. Henderson3, J. G. Hengstler2, A. Lampen1, R. Reif2
1 German Federal Institute for Risk Assessemnt, Food Safety, Berlin, Germany
Pyrrolizidine alkaloids (PA) are phytotoxins that may cause severe liver damage. However, molecular mechanisms of PA hepatotoxicity are not well understood. Therefore, we investigated metabolism-dependent development of PA hepatotoxicity in vivo, using an acutely toxic dose of senecionine in mice. Analysis of the liver was performed by intravital two-photon microscopy, histology and clinical chemistry.
Pericentral liver sinusoidal endothelial cell (LSEC) necrosis was observed together with elevated sinusoidal marker proteins in the serum of senecionine-treated mice and increased platelet aggregation. In vitro experiments showed no cytotoxicity to the freshly isolated non-parenchymal cell fraction (predominantly LSECs) up to 500 µM senecionine. However, metabolic activation of senecionine by preincubation with primary mouse hepatocytes increased the cytotoxicity to cultivated LSECs. CYP-dependent bioactivation was confirmed in CYP reductase-deficient mice in vivo. Analysis of hepatic bile salt transport by intravital two-photon imaging revealed a delayed uptake of a fluorescent bile salt analogue from the hepatic sinusoids into hepatocytes and delayed elimination. This was accompanied by mRNA downregulation of hepatic bile salt transporters.
In conclusion, toxic metabolites are generated by hepatic CYPs during intoxication with senecionine that destroys LSECs in the pericentral region of the liver lobules. Together with the observation of compromised bile transport the results explain the observed cholestasis and the clinical symptoms of veno-occlusive disease due to platelet aggregation and LSEC destruction after PA intoxication.
Keywords: pyrrolizidine alkaloids, senecionine, hepatotoxicity, two-photon microscopy
Special aspects of the hepatotoxic action of tetrachloromethane in rats of different ages (#567)
T. A. Sinitskaya1, V. N. Rakitskii1, S. V. Skupnevskii1
1 Federal Scientific Center of Hygiene named after F.F. Erisman, Administration, Mytischchi, Russian Federation
Age-related changes cause shifts in the physiological and biochemical functions of the organism, the consequences of which may result the sensibility to the toxic effects of chemical agents.
Goal of the research was studying the special aspects in mechanism of carbon tetrachloride-induced hepatotoxicity in rats of different ages.
Materials and methods. The studies carried out on young rats (3 months) and older (18 months) male rats of Wistar line, divided into control and experimental groups. Liver pathology formed by daily per os of a 25% oil solution exposure of carbon tetrachloride for 4 days at 0.2 ml / 100 g weight. In the blood were determined standard kits to study clinical diagnostic parameters: ALAT, ASAT, hydroperoxide (HP) and malonic dialdehyde (MDA). Statistical processing of results - according to the criterion of Student's test.
Results. By comparison of different age’sanimals reactions with each other showed, that the hepatotoxic effect of CCl4 in age rats is higher. This manifested itself by a relative ("Experience / Control") increase in the activities of hepatospecific enzymes - ALATelderly = 10.42 (p <0.01), ALATyoung = 5.46 (p <0.001); ASATelderly = 6.1 (p <0.001), ASAT young = 3.46 (p <0.001). The de Ritis coefficient for the compared groups was: RdReld. = 0.65 (p <0.05), RdRyoung = 0.69 (p <0.001). The concentration of lipid peroxidation markers, reflecting the degree of toxic effects of CCl4 on the organism, revealed that the conversion of xenobiotics in older rats was reduced relatively young (HPeld. = 3.16 ± 0.13 µmol / l; HPyoung = 4.59 ± 0.12; MDAeld. = 38.29 ± 1.39 µmol / l, MDAyoung = 41.8 ± 0.96).
Consequently, despite of the relative decrease in the activity of the cytochrome system in older rats, the toxic effect of CCl4 in them is more pronounced, which may be based on the weakening of clearance and more longer contact of the body with toxic metabolites.
Keywords: ageing toxicity; age-related changes; CCl4 – induced liver injury
Human non-parenchymal cells protect against acetaminophen hepatotoxicity in a co-culture spheroid model (#568)
C. C. Bell1, L. C. Andersson1, R. Sargeant2, J. W. Dear3, D. P. Williams2, M. Söderberg1
1 AstraZeneca, Clinical Pharmacology and Safety Science, Gothenburg, Sweden
In addition to hepatocytes, the liver comprises a variety of non-parenchymal cells (NPCs) such as stellate, Kupffer and liver sinusoidal endothelial cells, which all have specialized functions. Incorporating NPCs into in vitro models may therefore provide a more physiologically relevant platform for studies of liver injury and/or disease.
In this study, cryopreserved primary human hepatocytes and mixed NPCs were co-cultured in 3D spheroids. The presence of each cell type was confirmed through immunohistochemistry for cellular markers such as CD68 (Kupffer cells) and CD31 (endothelial cells). Although faint staining for a-SMA and COL1A1 (markers of activated stellate cells) was observed in untreated co-culture spheroids, this increased significantly upon TGF-b treatment, indicating that the culture conditions were suitable for maintaining stellate cells in a quiescent state.
Interestingly, the addition of NPCs protected the spheroids from acetaminophen-induced toxicity, an effect which has previously been reported in animal models (Ju et al. 2002). NPC-containing spheroids were less sensitive when considering all readouts examined (depletion of ATP and glutathione, and miR-122 release), particularly after repeated dosing. This effect was observed with multiple NPC donors. Despite all spheroids containing the same number of hepatocytes, mRNA expression of CYP1A2, CYP2E1 and CYP3A4 (enzymes responsible for the bioactivation of APAP) was lower in co-cultures and may therefore have contributed to the protective effect observed.
To understand whether the introduction of NPCs increased the physiological relevance of the model, the expression of a panel of miRNAs associated with APAP-toxicity in patients (Vliegenthart et al. 2015) was compared between mono- and co-cultures. Of the six miRNAs analysed, only miR-122 was readily detected in cell culture media following APAP treatment, reflecting the high levels present in the liver. In addition, increased cellular expression of miRNAs implicated in inflammation (miR-155) and liver regeneration (miR-382) was observed in co-culture spheroids.
This work highlights the importance of multiple cell types in the liver’s response to toxic insult and suggests that the presence of non-parenchymal cells can significantly impact upon toxicity mechanisms.
Ju, C., Reilly, T. P., Bourdi, M., Radonovich, M. F., Brady, J. N., George, J. W., & Pohl, L. R. (2002). Protective Role of Kupffer Cells in Acetaminophen-Induced Hepatic Injury in Mice, Chemical Research in Toxicology, (15), 1504–1513.
Vliegenthart, A. D. B., Shaffer, J. M., Clarke, J. I., Peeters, L. E. J., & Caporali, A. (2015). Comprehensive microRNA profiling in acetaminophen toxicity identifies novel circulating biomarkers for human liver and kidney injury. Scientific Reports, (5:15501), 1–13.
Keywords: Hepatoxicity, In vitro models, miRNA, 3D
A Retrospective Analysis of Hepatocyte Hypertrophy in Repeated Dose Rat Studies (#640)
L. Pan1, T. Zhou1, J. Zhao1, S. McPherson1
1 WuXi AppTec (Suzhou) Co., Ltd., Suzhou, China
Hepatocyte hypertrophy is generally considered an adaptive change of the liver which reflects drug metabolism and hepatic enzyme induction, which sometime results in decreased systemic exposure. This is more often observed in nonclinical studies as high doses are administered to animals to investigate the toxicity of the test article. Extensive research has been conducted especially in rodents to address the significance of this change relative to drug metabolism and safety evaluation. The purpose of this investigation is to analyze the hepatocyte hypertrophy observed in rat studies conducted at the facility and provide references as background data. It was also intended to analyze if there are any relationships between hepatocyte hypertrophy and accumulation index of systemic exposure. A retrospective analysis was performed on approximately 150 repeated dose rat studies of 4-week to 26-week duration tested with non-biologics. The animals used on studies were Sprague Dawley and Wistar rats from BioLASCO Taiwan Co., Ltd., Sprague Dawley rats from Vital River Laboratory Animal Technology Co., Ltd. Beijing, and Wistar Han rats from Charles River Laboratories, USA. The results showed that hepatocyte hypertrophy was noted in approximately 14% studies and represents approximately 14% of total compounds tested. The hepatocyte hypertrophy was in centrilobular or diffuse pattern, of minimal to moderate severity, and was accompanied with increased relative liver weight (to body weight) by 12% to 98% relative to the concurrent control. There were no associated changes in ALT (Alanine Aminotransferase) or AST (Aspartate Aminotransferase) or the increases were of low magnitude and were not considered adverse. In 12 of the 21 studies, hepatocyte hypertrophy was the only test article-related change observed in the liver. Other liver changes included hepatocellular vacuolation and/or necrosis. The presence of hepatocyte hypertrophy does not appear to result in meaningful differences of accumulation index of systemic exposure. However, when this was observed along with follicular hypertrophy in the thyroid glands, which is another indicator of enzyme induction, these tend to be associated more with decreased systemic exposure and greater magnitude of liver weight increase. The hepatocyte hypertrophy was reversible in all studies analyzed expect that in 2 studies it was still noted as a minimal or mild change in one animal following a 2-week recovery or only in the high dose group following a 4-week recovery. For two compounds that both the IND enabling and longer term studies were conducted at WuXi, hepatocyte hypertrophy was noted in both the 4-week and 13- or 16‑week studies. Among the 19 compounds that resulted in hepatocyte hypertrophy in rat studies, only three compounds also had similar reversible changes in the non-rodent species. In most studies, the hepatocyte hypertrophy by itself was not considered an adverse change
Toxicol Pathol. 2010 Aug;38(5):776-95. Hepatic enzyme induction: histopathology. Maronpot RR1, Yoshizawa K, Nyska A, Harada T, Flake G, Mueller G, Singh B, Ward JM.
Toxicol Pathol. 2012 Oct;40(7):971-94. Liver hypertrophy: a review of adaptive (adverse and non-adverse) changes--conclusions from the 3rd International ESTP Expert Workshop. Hall AP1, Elcombe CR, Foster JR, Harada T, Kaufmann W, Knippel A, Küttler K, Malarkey DE, Maronpot RR, Nishikawa A, Nolte T, Schulte A, Strauss V, York MJ.
Keywords: Hepatocyte Hypertrophy, Rat, Repeated Dose
Development of high-throughput assays for the screening of drug-induced mitotoxicity (Glu/Gal assay) and membrane potential integrity (Mito-ID assay): workflow and software for efficient end-to-end accurate data delivery. (#657)
K. G. de Waepenaert1, B. Van Dijck1, D. Peeters2, N. Mesens1
1 Johnson and Johnson, MIT/PDMS/D&MS, Beerse, Belgium
Drug-induced hepatic injury is the most common reason cited for withdrawal of an approved drug and impaired mitochondrial function is increasingly implicated in this etiology. To shift early on in deselection of molecules with mitochondrial liabilities during the drug development process, the operational in vitro screening screenings assays needed to be tuned into high-throughput assay formats. We demonstrate that by implementation of modern HTS infrastructure and sophisticated data analysis software packages, throughput, efficiency, reproducibility and reliability could be optimized, essential for a robust fully integration of mitochondrial toxicity data compliant with the drug discovery data warehouse. The use of Echo 555 spotted plates, Genedata Screener, 3DX enabled the development of an end-to-end platform that was used to screen ~10,000 compounds in dose response and can be used in hit-to-lead compound DILI characterization in a streamlined and modern pharma research workflow.
Keywords: High Throughput / Mitotoxicity
Effects of antipsychotic drugs on mitochondrial bioenergetics in vitro. (#674)
A. Rosell-Hidalgo1, A. L. Moore1, T. Ghafourian1
1 University of Sussex, Department of Biochemistry and Biomedicine, Brighton, United Kingdom
Mitochondria are the cellular organelles that generate 95% of the energy needed for a cell to remain viable. It has long been recognized that mitochondrial dysfunction can be the result of drug-induced toxicities and a major mechanism of hepatotoxicity and cardiotoxicity . In fact, drug-induced liver injury is a major cause of safety-related drug-marketing withdrawals . The structural and functional characteristics of mitochondria provide a number of off-targets for some pharmaceutical drugs that can ultimately lead to cellular bioenergetic deficit, increased free radical production, alterations in cell signalling pathways and even cell death. The aim of this study was to identify detailed mechanisms involved in the toxicity of three antipsychotic drugs: chlorpromazine, haloperidol and olanzapine. Here we investigated the in vitro effects of these neuroleptic drugs on bioenergetic functions of isolated rat liver mitochondria using high-resolution respirometry in combination with simultaneous evaluation of membrane potential. O2 fluxes determined in the presence of 12.5 mM succinate and 1 μM rotenone supporting complex II-linked respiration showed that respiration was highly inhibited by chlorpromazine whereas olanzapine and haloperidol showed slight effects. Furthermore, complete dissipation of mitochondrial membrane potential was observed in the presence of 200 μM chlorpromazine. MTT assays revealed the effects of these antipsychotics on the viability of HepG2 cells. Results showed that both chlorpromazine and haloperidol were cytotoxic at 15 μM (80% viability) and 50 μM (40% viability), respectively. Interestingly, 50 μM olanzapine, the highest concentration tested, showed no cytotoxic effects on HepG2 cells. On the contrary, it increased proliferation with respect to the control (140% viability). The relative potencies of these therapeutic agents as inhibitors of mitochondrial function are in accordance with the known risk of adverse effects. Our data agree with reports indicating that olanzapine, an atypical antipsychotic, is a safer drug than the typical antipsychotics chlorpromazine and haloperidol .
1. Wallace, K.B. and A.A. Starkov, Mitochondrial Targets of Drug Toxicity. Annual Review of Pharmacology and Toxicology, 2000. 40(1): p. 353-388.
2. Sanuki, Y., et al., A rapid mitochondrial toxicity assay utilizing rapidly changing cell energy metabolism. J Toxicol Sci, 2017. 42(3): p. 349-358.
3. Modica-Napolitano, J.S., et al., Differential effects of typical and atypical neuroleptics on mitochondrial function in vitro. Arch Pharm Res, 2003. 26(11): p. 951-9.
Keywords: Mitochondria, HepG2, toxicity, drug-induced liver injury, high-resolution respirometry
PXR activation dissociates hepatosteatosis from insulin resistance in obese mice (#691)
O. Kummu1, M. Karpale1, J. Rysä2, J. Hukkanen3, J. Hakkola1
1 University of Oulu , Research Unit of Biomedicine, Pharmacology and Toxicology and Medical Research Center Oulu, Oulu, Finland
Pregnane X receptor (PXR; NR1I2) is a xenobiotic sensing nuclear receptor identified as a regulator of glucose and lipid metabolism. Many drugs and environmental chemicals activate PXR causing impaired glucose tolerance and liver steatosis, which precede type 2 diabetes. Still, the significance of PXR in energy metabolism and in the onset of metabolic syndrome remains unclear. We aimed to investigate how PXR activation affects metabolic health in obese mice.
C57BL/6N mice were fed high-fat diet for 15 weeks and then treated with PXR activator (pregnenolone-16α-carbonitrile, PCN, 50mg/kg) for 4 days. PCN treatment dramatically potentiated hepatosteatosis in obese mice. Despite the drastic steatotic effect, PCN treatment improved glucose tolerance and HOMA-IR index, and a trend towards better insulin sensitivity was seen in insulin tolerance test. Hepatic gluconeogenesis was not affected in pyruvate tolerance test although gluconeogenic genes PEPCK1 and G6P were repressed.
A gene expression profiling study was performed to identify glucose responsive genes that may be modified by PXR activation in mouse liver. IGF-binding protein 2 (IGFBP-2) was the top hit among the glucose repressed, but PCN upregulated candidate genes. IGFBP-2 has been previously reported to improve glucose homeostasis through IGF-dependent and independent mechanisms. The upregulation of IGFBP-2 was confirmed with qPCR in PCN-treated mouse liver, while PXR knockout abolished the effect. PXR activation increased plasma soluble IGFBP-2 in chow-fed and high-fat diet-fed mice, and also in humans.
In summary, PXR-activation greatly potentiated high-fat diet-induced hepatosteatosis. Despite the aggravation of hepatosteatosis, PXR activation improved glucose tolerance and insulin sensitivity. This was associated with increased liver and circulating IGFBP-2, which may provide a mechanistic explanation for dissociation of hepatosteatosis from insulin resistance.
1.Hakkola J, Rysä J, Hukkanen J. Regulation of hepatic energy metabolism by the nuclear receptor PXR. Biochim Biophys Acta. 2016;1859(9):1072-1082.
2. Rysä J, Buler M, Savolainen MJ, Ruskoaho H, Hakkola J, Hukkanen J. Pregnane X receptor agonists impair postprandial glucose tolerance. Clin Pharmacol Ther. 2013;93(6):556-563.
3.Hassani-Nezhad-Gashti F, Rysä J, Kummu O, Näpänkangas J, Buler M, Karpale M, Hukkanen J, Hakkola J. Activation of nuclear receptor PXR impairs glucose tolerance and dysregulates GLUT2 expression and subcellular localization in liver. Biochem Pharmacol. 2018;148:253-264.
4. Zhou J, Zhai Y, Mu Y, Gong H, Uppal H, Toma D, Ren S, Evans RM, Xie W. A novel pregnane X receptor-mediated and sterol regulatoryelement-binding protein-independent lipogenic pathway. J Biol Chem. 2006;281(21):15013-15020.
5. He J, Gao J, Xu M, Ren S, Stefanovic-Racic M, O'Doherty RM, Xie W. PXR ablation alleviates diet-induced and genetic obesity and insulin resistance in mice. Diabetes. 2013;62(6):1876-1887.
6.Russo VC, Azar WJ, Yau SW, Sabin MA, Werther GA. IGFBP-2: The dark horse in metabolism and cancer. Cytokine Growth Factor Rev. 2015;26(3):329-346.
7. Wittenbecher C, Ouni M, Kuxhaus O, Jähnert M, Gottmann P, Teichmann A, Meidtner K, Kriebel J, Grallert H, Pischon T, Boeing H, Schulze MB, Schurmann A. Insulin-like growth factor binding protein 2 (IGFBP-2) and the risk of developing type 2 diabetes. Diabetes. 2019;68:188-197.
Keywords: xenobiotic, nuclear receptor PXR, type 2 diabetes, hepatosteatosis, glucose metabolism
Effects of α-amanitin in HepG2 cells are not prevented by drugs used in Amanita phalloides intoxications (#706)
D. F. Rodrigues1, V. M. Costa1, M. D. L. Bastos1, F. D. Carvalho1
1 UCBIO-REQUIMTE, Faculty of Pharmacy, University of Porto, Laboratory of Toxicology, Porto, Portugal
Amatoxins, specially α-amanitin, are responsible for the major deleterious effects of Amanita phalloides mushrooms. However, until now, there is no clinical effective procedure or antidote for Amanita phalloides intoxications. The liver is a major target of α-amanitin toxicity, thus it is crucial to identify the mechanisms of α-amanitin hepatotoxicity and search for effective antidotes. The aim of this study was to evaluate the feasibility of HepG2 cells for this purpose.
α-Amanitin cytotoxicity was evaluated in HepG2 cells by MTT reduction and neutral red uptake assays, following exposure at different concentrations (0.1-20 µM) for 24 or 48 h. The effect of α-amanitin in nascent RNA synthesis, in total and reduced glutathione (GSH) levels, in mitochondrial membrane potential (MMP) and in ATP levels was assessed following exposure for 24h. Additionally, the influence of 1 µM oligomycin, an ATP synthesis inhibitor, and of 25 µM buthionine sulfoximine (BSO), an inhibitor of gamma-glutamyl-cysteine synthase, was evaluated towards the effects of α-amanitin following exposure 24h or 48h. Lastly, the influence of previously identified antidotes (1 mM N-acetylcysteine, 10 µM silibinin and 0.5 mM benzylpenicillin) but poorly effective for amatoxin-intoxications was evaluated towards the cytotoxic effects of α-amanitin 48h after exposure.
α-Amanitin caused a concentration- and time-dependent mitochondrial and lysosomal dysfunction. Additionally, α-amanitin produced a significant decrease in nascent RNA synthesis. While this amatoxin did not induce changes in MMP, it caused a significant increase in intracellular ATP levels, which was not prevent by incubation with oligomycin. α-Amanitin provoked a significant increase in total and reduced GSH levels that was abolished by pre-incubation with BSO. Notwithstanding, BSO provided partial protection towards the cytotoxic effects of α-amanitin. None of the clinically used antidotes conferred protection against α-amanitin cytotoxicity.
HepG2 cells have proven to be an interesting model for evaluating the mechanisms of α-amanitin hepatotoxicity. Nonetheless, lack of protection of the previously described antidotes for amatoxin poisoning towards α-amanitin cytotoxicity highlights the importance of the development of better antidotal strategies.
Acknowledgements: VMC thanks FCT for grant (SFRH/BPD/110001/2015). Work supported by FEDER funds through the Operational Programme for Competitiveness Factors – COMPETE and by national funds by FCT (PTDC/DTP-FTO/4973/2014– POCI-01-0145-FEDER- 016545).
Keywords: α-Amanitin, HepG2.
Computable biological network models for mechanistic 21st Century Toxicology (#757)
M. Talikka1, E. Scotti1, H. Yepiskoposyan1, J. Szostak1, M. C. Peitsch1, J. Hoeng1
1 PMI R&D, Philip Morris Products S.A., Quai Jeanrenaud 5, Neuchatel, Switzerland
Systems toxicology approaches with extensive molecular measurements complement apical endpoints in various areas of research, and new methods are needed to interpret these rich data and derive new hypotheses. Causal biological network models, scripted in the Biological Expression Language, facilitate the assembly of available biological knowledge in a structured format and, owing to their computability, offer mechanistic interpretation of molecular data in a well-defined biological context. The network model consists of biological entities (nodes) and relationships between the nodes (edges). Information regarding gene expression regulation by some of the nodes in the network backbone is employed to build a second, scorable layer to the network model. This layer is used to infer the activity of the backbone nodes from transcriptomic data, and the impact on the network as a whole can be assessed using the network perturbation amplitude algorithm.
Previously, we have constructed a suite of causal biological network models for the three phases of xenobiotic metabolism to better understand how toxicants are metabolized in the liver. In this work, we introduce a network model that was built to describe signaling pathways that contribute to biological processes involved in liver steatosis, involving hypoxia inducible factor 1 and 2a, sterol regulatory element binding protein, hepatocyte nuclear factors 1 and 4, and various nuclear receptors and molecules essential for lipid metabolism. Each statement (network edge) extracted from scientific literature was extensively annotated to trace back the source (PMID) as well as the biological context (i.e., species, tissue/cell type, and disease state).
When used in combination with transcriptomic data from relevant studies, the model provides mechanistic understanding and quantitative impact assessment on toxicant effects in the liver. These efforts are the beginning of the development of a suite of biological network models that can be used in the context of 21st Century Toxicology for a mechanistic and quantitative understanding of how toxic substances impact the biological system.
Keywords: systems toxicology, liver steatosis, biological network model, biological expression language
Comparison of 2D and 3D Cell-based Models Using Human Chemical Derived Hepatic Progenitors to Predict Drug-induced Liver Injury (#758)
S. Na1, 2, J. - Y. Kim1, S. Han1, A. - R. Lee1, J. Jeong3, D. Choi3, S. Hong4, S. - H. Kim4, S. Lee1, S. - J. Park1, G. Yoo1, M. H. Yoo1, D. - O. Kim1, K. - S. Moon1, 2
1 Korea Institute of Toxicology, Daejeon, Republic of Korea
Drug-induced liver injury (DILI) is one of the major cause of decline in new drug approval and withdrawal from the drug market. During drug development process, two-dimensional (2D) culture systems have been used to detect drug efficacy and toxicity. However, 2D systems have limitations to reflect the complexity of the liver microenvironment. In this study, we established 2D and 3D culture models using human chemically derived hepatic progenitors (hCdHs) which were reprogrammed from human primary hepatocytes, and also evaluated hepatotoxicity using traditional DILI drugs: diclofenac sodium (DF) and acetaminophen (APAP). For determining drug-induced liver injury, we analyzed multiple hepatotoxicity-related parameters: albumin, lactate dehydrogenase (LDH), alanine aminotransferase (ALT), glutamate dehydrogenase (GDH), malate dehydrogenase (MDH) and major cytochrome P450 levels (CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4). After DF treatment, both 2D and 3D culture models showed increases of ALT and LDH levels in a dose-dependent manner but 3D models displayed approximately 15-fold higher compared to 2D models at the highest concentration (1000 μM). APAP has been known metabolized by CYP1A2 (contributed 30%–56%) and CYP2E1 (contributed 30%–78%). After APAP treatment, CYP1A2 and CYP2E1 mRNA levels were elevated in 3D models. Our current study presented alterations in hepatotoxicity-related parameters in both 2D and 3D hCdHs culture systems after treatment of two well-known DILI drugs. Further investigations on hepatotoxicity in hCdHs culture systems may provide valuable insights into new appropriate model for evaluation of DILI.
Keywords: 2D and 3D cell-based models, hCdHs, drug-induced liver injury
Transcriptomic profiling of compound treated human liver spheroids to investigate the underlying mechanisms of drug induced liver injury observed in the clinic (#778)
M. Steemans1, A. De Bondt2, F. Van Goethem1, J. Van Houdt2, L. Lammens1, H. Goehlmann2, A. De Smedt1, M. Otieno3
1 Janssen Research & Development, Nonclinical Safety - Mechanistic & Investigative Toxicology, Beerse, Belgium
Drug-induced liver injury (DILI) is the most common cause for acute liver failure in the USA and Europe and is the leading cause of attrition of compounds in drug development. Although insights have been gained into the underlying mechanisms of idiosyncratic DILI, the prediction of human liver toxicity is still problematic. In this study, 3 compounds were selected which were active on the same therapeutic target and stopped in clinical development. Compound A and B induced clinical liver toxicity, while compound C did not.
To assess the underlying mechanisms of the clinical adverse effects, a mechanistic in vitro study was set-up to explore the transcriptomic profiles induced by these 3 compounds. In this proof-of-concept approach, different human liver models were used. Here, we will focus on the study performed with the 3D InSightTM Human Liver Microtissue model from InSphero.
Concentration selection was based on the clinical plasma concentrations (Cmax) and on the cytotoxicity profiles obtained in a range finder study. In total 336 RNA samples were isolated and microarray expression analysis was performed to assess the transcriptional response from >20,000 well-annotated genes, both after a 1- and 12-day exposure period. Differentially expressed genes and pathways were compared. In addition, the role of oxidative stress - cytotoxicity in the presence and absence of BSO - was assessed as possible initiator of hepatocellular necrosis.
This proof-of-concept study showed that the BSO-assay and transcriptional profiling in 3D human spheroids can discriminate between the 3 compounds in a way which is consistent with the observed clinical liver toxicities. Although this approach requires further validation (e.g. testing of high-quality annotated DILI reference compounds), it may eventually become a valuable prioritization screening tool in the selection process of safe NME candidates.
Keywords: DILI, Human Liver Spheroids, Transcriptomics
(S-)Metolachlor – Human Relevance Framework Assessment of Liver Tumour Induction in Female Rats (#782)
D. E. Cowie1, L. Brierley1, R. A. Currie1, R. Green1
1 Syngenta Ltd, Human Safety, Bracknell, United Kingdom
A number of dose- and time-related key and associative events have been identified that characterise the hypothesised MOA for (S-)metolachlor-induced liver tumours in female rats. A non-genotoxic mode of action (MOA), initiated via activation of the constitutive androstane receptor (CAR) was demonstrated using in vitro and in vivo mechanistic studies. CAR activation was demonstrated using a CAR transactivation assay. In mechanistic studies of up to 60 days duration, (S-)metolachlor, at the tumorigenic dose of 3000ppm, increased hepatic PROD & BROD activity, liver weight , smooth endoplasmic reticulum proliferation and centrilobular hypertrophy. A key event in the CAR-mediated MOA, increased cell proliferation was maximal after 1-3 days of treatment, but not at later time points (≥7 days exposure). In contrast, the non tumourigenic dose levels (≤300 ppm) produced only minor changes in a small subset of the measured liver parameters.
Keywords: Mode of Action, Liver, Carcinogenesis, Regulatory Toxicology
An optimized process for medium chain fatty acid profiling or quantitation in complex matrices using derivatization and LC-MSMS analysis (#819)
E. Andres1, P. Pujuguet2, A. Obry1, A. Huyard1, S. Shakir1, A. Montjardet2, C. Dini1
1 Oroxcell, Romainville, France
Free fatty acids (FFAs) are key molecules namely implicated in cell signaling and metabolism. The variation of the fatty acids profiles in liver cells are therefore relevant biomarkers of various toxicological effects, thus, fatty acid profiling may become a relevant endpoint for liver pathology evaluation.
Profiling and quantitation of these molecules is a powerful tool for the analysis of these molecules, however, their poor ionization efficiency under electrospray ionization mass spectrometry represent a clear limitation to the development of assays for such analysis.
In the present study, we describe the development of an optimized process for the profiling and quantification of free fatty acids by derivatization in complex matrixes such as liver cell homogenates.
Medium-chain fatty acids Pelargonic acid (C9), Capric acid (C10, Undecylic acid (C11), Lauric acid (C12), Tridecylic acid (13) and Myristic acid (C14) were analyzed using the same analysis method with clear separation and relative quantitation. Furthermore, absolute quantitation was achieved by comparison of the signal against a standard down to 50 ng/ml.
The same methodology can be anticipated as applicable to lower or higher free fatty acids chain length leading to cutting-edge technology for free fatty acids profiling in complex matrixes.
Keywords: Free fatty acids, liver, analysis, LC-MS/MS
Rifampicin induces the bone form of alkaline phosphatase (ALP) in humans (#848)
H. N. Abdelfattah1, P. Lehenkari2, J. Hukkanen3, J. Hakkola1
1 University of Oulu, Research Unit of Biomedicine and Medical Research Center Oulu, Oulu, Finland
Nuclear receptor Pregnane X receptor (PXR) is a xenobiotic-sensing nuclear receptor that regulates expression of drug metabolizing enzymes and drug transporters mainly in the liver and intestine1. Recently, PXR activation has been shown to have affect also other tissues including bone, but these effects are still poorly understood1,2. PXR knockout mice display osteopenia and reduced bone formation2.
We performed a clinical trial on healthy volunteers to discover novel functions of PXR activation. Rifampicin, a well establish ligand for human PXR, was used as a study compound. The design of the study was randomized, single-blind, placebo-controlled and cross-over. Rifampicin 600 mg a day or placebo was dosed on each arm for a week. Rifampicin induced alkaline phosphatase (ALP) blood level in healthy volunteers. Further analysis indicated that this represent the bone form of ALP.
To investigate the mechanism(s) involved, we used human osteoblast lineage differentiated from bone marrow-derived mesenchymal stromal cells. The differentiated cells were treated with different concentration of two PXR ligands i.e., rifampicin and hyperforin. Both compounds induced the mRNA level of bone biomarker genes (ALP, MGP, OPN, OPG). We also measured the ALP activity from the treated cells, and it was significantly increased. PXR expression was detected in the cells, however, the expression was very low compared with the human liver.
To further investigate potential role of PXR in the observed ALP induction by rifampicin, we treated mice and rats with a rodent PXR ligand PCN. However, PCN did not increase plasma ALP indicating that the rifampicin effect in humans is either species specific, or alternatively is not mediated by PXR.
In conclusion, we showed that rifampicin treatment induces the bone form ALP in the plasma of human volunteers. Further studies are required to establish the mechanism more precisely.
1 Hakkola, J., Rysä, J., & Hukkanen, J. (2016). Regulation of hepatic energy metabolism by the nuclear receptor PXR doi://doi.org/10.1016/j.bbagrm.2016.03.012.
2 Azuma, K., Casey, S. C., Ito, M., Urano, T., Horie, K., Ouchi, Y., . . . Inoue, S. (2010). Pregnane X receptor knockout mice display osteopenia with reduced bone formation and enhanced bone resorption. Journal of Endocrinology, 207(3), 257-263. doi:10.1677/JOE-10-0208.
Keywords: Rifampicin, Bone homeostasis, Pregnane X receptor (PXR), Alkaline phosphtase, PXR
Study of the hepatic metabolic effects induced by PFOA exposure using a multiplatform metabolomics approach (#860)
J. Villaret-Cazadamont1, N. Butin2, 3, C. Canlet1, 3, M. Tremblay-Franco1, 3, R. Gautier1, 3, F. Bellvert2, 3, F. Jourdan1, 3, D. Zalko1, N. Cabaton1, N. Poupin1
1 Toxalim (Research Centre in Food Toxicology), Université de Toulouse, INRA, ENVT, INP-Purpan, UPS, Toulouse, France
Perfluorialkylated substances (PFAS) are used for a wide range of industrial applications, including the manufacturing of anti-adhesive cookware coatings and waterproof clothing textiles. PFOA (perfluorooctanoic acid), one of the most studied PFAS, has raised major concerns regarding public health over the last years. Although its use is decreasing in the industry, consumers are still exposed to PFOA, which is extremely persistent and bioaccumulates in the environment. Dietary intake is reported as the main source of human exposure, especially through seafood and freshwater. Epidemiological surveys and in vivo studies in rodents suggest that PFAS can induce metabolic effects, especially on lipid metabolism, and might be involved in hepatotoxicity. However, the metabolic pathways affected by these compounds and the underlying mechanisms for the observed alterations remain to be characterized.
In this study, we aim at assessing the metabolic effects of acute and sub-chronic exposures to different concentrations of PFOA, focusing on the liver as the main metabolic target. We performed in vitro studies using the human hepatic cell line HepaRG, which constitutes a particularly relevant model for studying the long-term effects of low doses of xenobiotics and is recommended by OECD, as well as the American Tox 21 program. HepaRG cells were exposed to three concentrations of PFOA (0.001 µM, 0.1 µM and 10 µM) and during two exposure durations (24h and 7 days). Both extracellular and intracellular samples were collected to have access to the intracellular metabolic content and extracellular fluxes (intake and secretion of metabolites by cells). Three complementary approaches (untargeted NMR, targeted LC-HRMS and IC-HRMS) were performed on all samples to increase the coverage of the metabolome. The first results show a discrimination between exposed and non-exposed cells, as well as between the different doses of exposure, with distinct metabolomic patterns, suggesting the involvement of specific and distinct metabolic pathways. Further analysis of these fingerprints, in the context of the human genome-scale metabolic network, will allow getting a more comprehensive picture of the metabolic modulations induced by PFAS and improving the understanding of their Modes of Action at different doses.
Keywords: metabolomics, perfluorinated compounds, human hepatic cells, metabolic effects
What can an animal tell the toxicologist? Concordance of toxic effects to liver and kidney between rats and humans. (#865)
W. Zobl1, F. Moradi Afrapoli1, S. E. Escher1
1 Fraunhofer Institute for Toxicology and Experimental Medicine, Hannover, Germany
Aim: This study was designed to test the hypothesis that rat models allow an accurate prediction of liver and kidney toxicity in humans. Specifically, we investigated the predictivity of common liver and kidney toxicity related endpoints from subchronic and chronic rat studies in relation to liver and kidney toxicity found in clinical trials.
Background: Many drug discovery and development (DDD) projects face severe adverse effects of the active ingredient as late as in the clinical phase or even in the post-marketing phase . In many of these cases liver or kidney toxicity plays a major role. Previously, the power of animal tests to predict clinical trial outcomes has already been elucidated for large sets of substances [2,3]. However, analyses did not differentiate between different types of animal studies. Another obstacle in previous studies was the lack of reliable data on predicted negative, so a lack of detailed knowledge about the study scopes of the evaluated animal studies. Therefore the present study can facilitate animal model and lead selection during DDD as well as uncertainty assessment in chemical risk assessments.
Methods: Observations related to liver and kidney toxicity in subchronic and chronic preclinical safety studies in rats from the eTOX and RepDose databases have been curated. In total they cover more than 100 unique chemical entities. Based on a standardized effect terminology these observations were compared to clinical trial outcomes reported in the PharmaPendium database for the same substances.
Expected results: The predictivity of liver and kidney effect-related preclinical endpoints for related clinical trial outcomes is presented in terms of accuracy as well as positive and negative likelihood ratio (LR+/-). Conclusions will be drawn on strengths and weaknesses of different animal test parameters as markers of liver and kidney toxicity in humans.
1. Waring MJ, Arrowsmith J, Leach AR, Leeson PD, Mandrell S, Owen RM, et al. An analysis of the attrition of drug candidates from four major pharmaceutical companies. Nat Rev Drug Discov. 2015;14(7):475-86.
Keywords: Predictivity of animal models, Uncertainty and Risk assessment, Liver toxicology, Translational analysis, Drug development
Development of MS-based immunoassays for quantification of drug-induced liver injury candidate biomarkers across species (#897)
V. Anselm1, C. Sommersdorf1, A. Steinhilber1, W. Naboulsi1, F. Schmidt2, H. Hammer1, A. Tausch1, H. Planatscher1, T. Joos2, O. Pötz1
1 Signatope GmbH, Reutlingen, Germany
Drug-induced liver injury (DILI) shows various stages from mild to severe liver injury of which an adequate prognosis for liver damage is particularly difficult to determine. Currently, the golden standard to diagnose DILI is determining alanine transaminase and bilirubin levels, however, these markers lack of specificity and capability for an early onset diagnosis. Thus, DILI is one of the major reasons for withdrawals of approved drugs. Moreover, idiosyncratic factors prevent an effective safety toxicity assessment so far.
Within the recent years a set of proteins was identified by large consortia as the Safer and Faster Evidence-based Translation Consortium (SAFE-T) and the Predictive Safety Consortium as potential biomarkers to indicate DILI and provide insight into DILI mechanism.
Here, we aimed on establishing immunoaffinity mass spectrometry (MS)-based assays to accurately quantify these potential DILI biomarkers in plasma or serum of patients and animal models. In our approach, plasma or serum proteins are proteolytically cleaved and a unique peptide per candidate is captured by antibody-based immune precipitation. The peptides are then quantified by parallel reaction monitoring mass spectrometry in reference to a corresponding stable isotopic synthetic labelled peptide. Targeting conserved sequences in proteotypic peptides enabled assay development for several species with antibodies comprising the same epitope.
Results from assay development addressing DILI biomarkers across species such as rodents, canines, and humans will be presented and discussed. Protein biomarker levels are compared among the stated species.
Quantification of candidate biomarkers for DILI in animal models corresponding to the human biomarkers may facilitate drug development by identification of harmful drugs rather in preclinical studies than after drug approval.
Keywords: drug-induced liver injury, mass spectrometry-based immuno assays, plasma proteins, biomarker, animal models
Changes in bile acid profiles induced by cholestatic drugs in HepaRG hepatocytes cultured in bile acid-enriched medium (#899)
A. Guillouzo1, A. Burban1, A. Sharanek1, L. Humbert2, E. Gauliard2, C. Guguen-Guillouzo1, D. Rainteau2
1 University of Rennes 1, Inserm 1241 Numecan, Rennes, France
The two primary bile acids (BAs), cholic acid (CA) and chenodeoxycholic acid (CDCA), are synthesized in the liver, and their secretion into bile is facilitated by conjugation to taurine (T) or glycine (G). In the gut they are deconjugated and dehydroxylated to form secondary BAs, mainly lithocholic acid (LCA) and deoxycholic acid (DCA). Intrahepatic cholestatic diseases of various etiologies are characterized by accumulation of BAs in the liver. Many drugs can induce cholestasis in humans but limited information exists on associated-changes in serum and liver BA profiles. The aim of this work was to analyze changes in BA profiles induced by various cholestatic and non-cholestatic compounds in HepaRG hepatocytes cultured in presence of a cocktail of nine major BAs either at physiologic (1x) or 60-fold higher (60x) concentrations for 24h, following 24h pre-incubation with the BAs only. BAs were measured by HPLC-MS/MS. Whatever the condition, no marked effects on BA profiles were observed with the non-cholestatic drugs compared to untreated cultures. In the presence of 1xBAs the main changes were observed with major cholestatic drugs, such as cyclosporine A, troglitazone, bosentan, fasudil and chlorpromazine; they were typified by increased (CDCA) or detectable (LCA and DCA) amounts in both supernatants and cell layers (intracellular + bile canaliculi). With 60xBAs, CDCA, CA and DCA conjugates and sulfated LCA were decreased while their unconjugated forms were increased, in supernatants. Most conjugates, including sulfated LCA (LCA-S3 and TLCA-S3), and strikingly unconjugated CDCA, LCA, DCA and CA were markedly increased in cell layers. These data demonstrate that in presence of exogenous BAs cholestatic drugs can alter in vitro BA profiles in both supernatants and cell layers, particularly by causing preferential cellular accumulation of unconjugated toxic hydrophobic BAs.
Keywords: Cholestasis, bile acids, drugs, HepaRG hepatocytes, bile acid profile
Lipidomic analysis of PLHC-1 topminnow liver cells exposed to bisphenol F and bisphenol A diglycidyl ether (#29)
C. Porte1, E. Pérez-Albaladejo1, A. Solís1, I. Bani1
1 IDAEA -CSIC, Environmental Chemistry, Barcelona, Spain
Plasticizers are widespread environmental contaminants that have been described as obesogens in terrestrial vertebrates. However, there are currently no equivalent in-vitro methods to investigate their mode of action in fish. This work explores the use of PLHC-1 cells as an alternative model to assess the alteration of hepatic lipids after 24 h of exposure to bisphenol F (BPF) and a chlorinated derivative of bisphenol A diglycidyl ether (BADGE·2HCl). PLHC-1 lipid extracts were analyzed by flow injection coupled to high resolution mass spectrometry (FIA-ESI(+/-)-Orbitrap-Exactive). The analysis of the intracellular concentration of the chemicals revealed the highest bioconcentration of BADGE·2HCl, which in turn induced a significant depletion of triacylglycerides (TGs) in PLHC-1 cells at internal concentrations close to those described in the liver of marine mammals. Exposure to BPF induced the generation of reactive oxygen species and a lipidic profile characterized by (a) a significant decrease in phosphatidylcholine (PC)- and phosphatidylethanolamine (PE)-plasmalogens, which are the lipids more sensitive to oxidative damage, and (b) hydrolysis of TGs, particularly of those enriched in polyunsaturated fatty acids. Changes in the lipidic profile occurred at concentrations well below the cytotoxic effect of the chemicals, and provided evidence of the different modes of action of BPF and BADGE·2HCl. Overall, the use of topminnow liver cells in lipidomic studies is a powerful tool to evaluate the bioconcentration and metabolic/ lipidic responses to plastic additives in fish.
Keywords: plastic additives; liver cells; toxicity; bioconcentration; lipidome
Cadmium telluride quantum dots induced the histopathological changes of livers and kidneys in mice via elevating hydroxyl radicals and decreasing antioxidant capacities (#34)
P. Huang1, J. Wang1, M. Yang1, J. Li2
1 Capital Medical University, Department of Toxicology and Sanitary Chemistry, School of Public Health., Beijing, China
Although quantum dot (QD)-induced toxicity occurs due to free radicals, generation of oxidative stress mediated by ROS formation is considered an important mechanism. However, free-radical mechanisms are essentially difficult to elucidate at the molecular level because most biologically relevant free radicals are highly reactive and short-lived, making them difficult to directly detect, especially in vivo. Antioxidants play an important role in preventing or, in most cases, limiting the damage caused by ROS. Healthy people and animals possess many endogenous antioxidative substances that scavenge free radicals in vivo to maintain the redox balance and genome integrity. The antioxidant capacity of an organism is highly important but seldom studied. In this study, male ICR mice were administered a single intravenous dose (1.5 µmol/kg) of CdTe QDs, and liver and kidney function and morphology were subsequently examined at 1, 7, 14, and 28 days. Furthermore, ⋅OH production in the tissue was quantified by trapping ⋅OH with salicylic acid (SA) as 2,3-dihydroxybenzoic acid (DHBA) and detecting it using a high-performance liquid chromatography fluorescence method. The antioxidant capacities of the liver and kidneys were investigated using the EPR spin tapping technique. We found that the QD-induced histopathological changes were time-dependent with elevated ⋅OH and decreased antioxidant capacity, and could recover after a period of time. The ⋅OH exhibited delayed effects in terms of histopathological abnormalities. QD-induced antioxidant efficiency reduction was time dependent with GSH decrease. These experimental results offer new information on QD toxicity in vivo. Specifically, CdTe QDs can elevate •OH and deplete GSH to reduce the elimination ability of liver and kidneys for •OH and •O2-, thus inducing oxidative damage to tissues.
Keywords: quantum dot; antioxidative capacity; hydroxyl radical; superoxide radical; spin-trapping
In vivo toxicological evaluation of natural repellent in nanotechnological matrix (#45)
N. Andreo-Filho1, C. Sales1, C. Higushi1, I. Haridass2, W. Sanchez2, P. Lopes1, J. Grice2, M. Roberts2, 3, V. R. Leite-Silva1, 2
1 UNIFESP- Universidade Federal de Sao Paulo, 1Instituto de Ciências Ambientais Químicas e Farmacêuticas, Diadema, Brazil
It is well known that millions of people around the world are affected every year by diseases transmitted by several mosquitoes, among them Aedes aegypti, which transmits diseases such as dengue, chikungunya and Zika. Vector control and personal protection, such as the use of repellent, are important to minimize the onset of disease. For this reason, safer alternatives with an effective and lasting impact against various insects is necessary. Many essential oils have been labelled with repellent properties, such as citronella oil, in addition to their terpene alcohols. The encapsulation of citronella oil and / or association with essential oils or vegetable oils, is expected to increase repellent action time and decrease its characteristic odor. Lipid nanoparticulate systems (LNS) are underexplored and are highly promising for the delivery of bioactive substances. In order to optimize their properties and application, it was proposed in this project to carry out safety studies for solid colloidal carriers of lipid base associated with vegetable oils. The Fluorescence Lifetime Imaging Microscopy (FLIM) method, which is predominantly used to evaluate the metabolic state of the tissue in response to a change in the microenvironment by monitoring changes in the fluorescence lifetime of endogenous fluorophores such as NAD(P)H and FAD. Metabolic changes in tissues can be measured as changes in NAD(P)H or FAD fluorescence lifetime, and / or as changes in the redox ratio or proportion of free and to protein-bound NAD(P)H (α1/α2). FLIM images were acquired with the DermaInspect Multiphoton Microscope (JenLab GmbH) equipped with a TCSPC830 detection module (Becker & Hickl GmbH). In vivo experiments were conducted on three participants. We found that there were no significant changes in NAD(P)H lifetime and α1/α2 ratio in the viable epidermis in vivo following topical application of LNS formulations containing Citronella Oil. This suggests that LNS formulations are safe for application for use on human skin and do not cause any measurable changes in the metabolic activity of the viable epidermis.
Keywords: repellent, citronella, solid colloidal carriers, Fluorescence Lifetime Imaging Microscopy.
Evaluation of the effect of cellulose nanofibers on skin irritation using a 3D in vitro reconstructed human epidermis model (#64)
K. Fujita1, S. Obara1, J. Maru1, S. Endoh1, Y. Kitano2
1 National Institute of Advanced Industrial Science and Technology (AIST), Research Institute of Science for Safety and Sustainability (RISS), Tsukuba, Japan
Cellulose nanofibers (CNFs) are new nanomaterials with a potential to be used in various applications. However, to accelerate the practical use of CNFs in society, hazard assessment of CNFs needs to be performed. With regard to dermal exposure to chemical substances, evaluation of permeability of chemical substances and the consequent skin irritation is an important part of hazard assessment. For this, tests using animal skin have been conducted so far. However, for animal protection in Europe, animal testing for safety evaluation of cosmetics and their raw materials has been banned. In recent years, as a substitute for animal skin testing, three-dimensional (3D) in vitro reconstructed human epidermis (RHE) models have been gaining popularity. Unlike animal skin, human cells do not pose interspecies difference challenges. In addition, there are fewer variations between batches, leading to high reproducibility across test results. In view of these circumstances, our research project was aimed at developing a method for testing skin penetration of CNFs, aiming at support of voluntary safety management of business operators. There have been no reports of studies on dermal toxicity and skin permeability of CNFs using a 3D RHE model. Thus, we propose that an appropriate skin penetration test is required, as CNF has an intermediate property between gel and sol, and its viscosity changes with time and shear stress (thixotropy). CNF demonstrates diverse physical properties, such as fiber diameter, fiber length, morphology, functional group, and impurities, depending upon the type of raw material and the method of chemical treatment/defibration treatment used. Therefore, TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl radical)-oxidized CNFs (TOCN) were selected as a test material from representative CNFs developed in Japan. In this study, we analyzed CNF sample preparation conditions, and employed an in vitro skin irritation test using two 3D RHE models (EpiDerm™ (EPI-200SIT) and SkinEthicTM RHE), according to the OECD test guideline for the Testing of Chemicals 439. This study was supported by the New Energy and Industrial Technology Development Organization (NEDO), Japan.
Keywords: Cellulose nanofibers, Skin irritation, 3D RHE model, TEMPO
Amorphous silica nanoparticles trigger human dendritic cell maturation in vitro and provoke CD4 + T Cell proliferation (#136)
A. Feray1, M. Hullo1, N. Szely1, F. - X. Legrand2, E. Brun3, E. Guillet1, S. Barillet1, M. Pallardy1, A. Biola-Vidamment1
1 University Paris-Sud, INSERM UMR 996, Chatenay-Malabry, France
Danger signals activate dendritic cells (DCs) stimulating both the innate and adaptive immune responses. DCs could sense nanomaterials, considered as NAMPs (nanoparticles-associated molecular patterns) and undergo a maturation process enabling them to migrate to regional lymph nodes and to activate naive T-lymphocytes. Amorphous silica nanoparticles (aSNPs) are widely used in dietary supplements, biomedical applications, cosmetics or construction materials. General population is probably more exposed than initially anticipated and occupational exposure, particularly via the inhalation route, should be better evaluated. Indeed, despite generally presented as highly biocompatible as compared to their crystalline counterparts, aSNPs could contribute to or exacerbate the onset of allergic airway disease.
The aim of this work was to evaluate the effects of aSNPs on human DCs in vitro. Human monocyte-derived DCs were exposed for 16 hours to final concentrations of 12,5 and 25 µg/ml of fumed silica nanoparticles. We measured cell viability, phenotypical changes, cytokines production and allogenic CD4+ T cells proliferation upon NP treatment. Endotoxin levels were unlikely to have any effect on DCs since no activity was found in the media.
Results showed that the aSNP significantly upregulated the CD86 costimulatory molecule, as well as the CD83 maturation marker and the CXCR4 chemokine receptor surface expressions. Secretions of inflammatory cytokines such as IL-1b, IL-6, IL-8 or TNF‑a were significantly enhanced in a dose-dependent manner in the DC culture supernatants. To evaluate whether aSNPs could induce DC to become functionally mature, we assessed their capacity to activate allogeneic T cells. Results showed that the increase in T-lymphocytes proliferation in presence of aSNP-treated moDCs was statistically significant for all tested DC/T ratios compared to uncharged DCs. Moreover, analysis of the co-culture supernatants for the production of T cell-derived cytokines showed a significant increase of IL-9 and IL-17A and F, and an upregulation of IL-5, consistent with the pro-inflammatory phenotype of DCs described above.
Taken together, these results suggest that aSNPs are able to induce functional DCs maturation and could act as adjuvants of the immune system.
Keywords: amorphous silica nanoparticles, dendritic cells, danger signal, immunotoxicology, T-cells
Evaluation of DNA damage in the rat lung after inhalation exposure to TiO2 and SiO2 nanoparticles (#143)
F. Brandão1, 2, C. Costa1, 2, M. J. Bessa1, 2, A. Haase3, S. Fraga1, 2, J. P. Teixeira1, 2
1 Universidade do Porto, EPIUnit-Instituto de Saúde Pública da Universidade do Porto, Porto, Portugal
Titanium dioxide (TiO2 NPs) and silica (SiO2 NPs) nanoparticles are widely used for several applications, increasing the concern about the possible risks they may pose to human health. Inhalation is a major route of exposure for these nanoparticles. This study aimed at evaluating the potential DNA damage in the lungs of male Sprague Dawley rats(n=5/per group) exposed for 5 days, 6h/day by whole-body inhalation to aerosolised TiO2 NPs (0.5 to 10 mg/m3) and SiO2 NPs (0.5 to 5 mg/m3) at 5 days(nonrecovery group) and 21 days(recovery group) after the initial exposure. Rats administered (i.p.) with methyl methanesulfonate (MMS) at a dose of 100 mg/kg were used as positive controls (n=3). Lung cells were isolated by mechanical disruption and primary and oxidative DNA damage assessed by the alkaline and FPG-modified Comet assay versions, respectively. At least 100 cells/tissue (50 in each replicate gel) were scored using the Comet Assay IV software (Perceptive Instruments, Suffolk, UK) and the mean of the percentage of DNA in the comet tail (% tail intensity) was used as DNA damage descriptor.
Exposure to all tested TiO2 NPs doses did not induce significant primary DNA damage in the lung tissue in the nonrecovery (5.60±1.84 vs 5.47±2.03 % tail intensity;10 mg/m3) and recovery animal groups (9.81±4.51 vs 8.06±1.93; 10 mg/m3) compared to the respective controls. Similar findings were observed in the lung of aerosolised SiO2 NPs exposed rats either in the nonrecovery (14.87±3.19 vs 15.84±2.47 % tail intensity; 5 mg/m3) or recovery group (8.87±1.41 vs 6.36±1.17 % tail intensity; 5 mg/m3) compared to the controls. In addition, exposure to both types of NPs did not cause a significant increase in lung oxidative DNA damage in both groups. As expected, DNA damage in the lung of MMS-injected animals showed a significant increase in the % tail intensity (74.50±4.28) compared with the control group.
Our data suggest that inhalation exposure to the tested doses of TiO2 NPs and SiO2 NPs do not affect DNA integrity in the rat lung. Nevertheless, further research should be conducted, namely the evaluation of other genotoxicity endpoints to support these findings.
This work was supported by the Portuguese Foundation for Science and Technology (FCT) through the ERA-NET SIINN project NanoToxClass (ERA-SIINN/0001/2013). FB and MJB are recipients of FCT PhD scholarships (SFRH/BD/101060/2014 and SFRH/BD/120646/2016). The authors would also like to acknowledge the contribution of COST Action hCOMET (CA15132).
Keywords: in vivo, rat, inhalation, nanoparticles, genotoxicity
Co-delivery of pemetrexed and quercetin with multi-walled carbon nanotubes displayed synergic effects in pancreatic cancer cells (#147)
M. Balas1, M. A. Badea1, D. Ionita2, M. Prodana2, A. Dinischiotu1
1 University of Bucharest, Faculty of Biology, Department of Biochemistry and Molecular Biology, Bucharest, Romania
Combination of conventional and natural chemotherapeutics was proved to offer synergistic anticancer efficacy, sometimes minimizing adverse effects. In this study, we aimed to investigate in vitro, the cytotoxic activity of pemetrexed (PMX) and quercetin (QCN) delivered separately or simultaneously in multi-walled carbon nanotubes (MWCNTs) and to compare their anticancer potential.
Carboxyl-modified MWCNTs were mixed with QCN, PMX, and respectively QCN-PMX solution. The samples were characterized by FT-IR spectroscopy, DLS, zeta potential and drug release profile. Different doses (between 3.125 – 50 µg/mL MWCNTs, 2.5 – 40 µg/mL PMX and respectively 0.25 – 4 µg/mL QCN) of the obtained nanoformulations (MWCNT-PMX, MWCNT-QCN and MWCNT-PMX-QCN) were tested on two human cell lines: MDA-MB-231 (breast cancer cells) and PANC-1 (pancreatic tumor cells). Biological effects were assessed after 24 h exposure analyzing cell viability, cell morphology, cellular uptake and internalization into lysosomes as well as reactive oxygen species (ROS) production.
The results indicated higher stability of MWCNT-PMX-QCN nanoformulation compared with the other samples and smaller size of MWCNTs when conjugated with drugs (369 - 460 nm) compared with free MWCNTs (approx. 700 nm) as a result of electrostatic charges and lower aggregation. In vitro results showed higher cytotoxic effects of MWCNT-PMX-QCN nanoformulation compared with the single-drug ones in pancreatic cells. In breast cancer cells the cytotoxic effects were less pronounced and quite similar between the three nanoformulations. The MWCNT-PMX-QCN nanoformulation induced significant alterations of pancreatic cell morphology and a decrease of cell viability under 60% at a dose of 25 µg/mL MWCNTs – 20 µg/mL PMX - 2 µg/mL QCN. The evaluation of drug release revealed also the highest percent (approx. 42%) for MWCNT-PMX-QCN sample at pH 7.4 after 24 h. In accordance with these observations, a significant increase of lysosome number (by max. 2.2 fold compared to single-drug samples) and an elevation of the intracellular ROS level (by max. 1.6 fold) at doses over 25 µg/mL MWCNTs - 20 µg/mL PMX - 2 µg/mL QCN were also noticed after 24 h for the same sample confirming the cellular internalization and co-oxidant drug activity in pancreatic cancer cells.
We concluded that the combination of PMX and QCN exhibits synergic effects on pancreatic cancer cells but not in breast cancer cells, showing a superior therapeutic efficacy compared with single-drug nanoformulations by expressing higher stability, internalization rate, and oxidant activity.
Keywords: Pemetrexed, Quercetin, MWCNTs, PANC-1 cells, reactive oxygen species
Therapeutic effects of oxidized single-walled carbon nanotubes loaded with cisplatin on breast cancer multicellular tumor spheroids (#161)
M. A. Badea1, M. Balas1, M. Prodana2, D. Ionita2, A. Dinischiotu1
1 University of Bucharest, Faculty of Biology, Department of Biochemistry and Molecular Biology, Bucharest, Romania
The aim of this study was to evaluate the anti-tumoral efficiency of oxidized single-walled carbon nanotubes (SWCNT-COOH) loaded with cisplatin (CDDP) on breast cancer multicellular tumor spheroids (MCTSs).
SWCNTs were functionalized by acid treatment, resulting SWCNT-COOH. Further, SWCNT-COOH were mixed with dimethylformamide and CDDP to obtain the nanocomposite SWCNT-COOH-CDDP. The efficiency of drug encapsulation was checked by ICP-MS and it was found that the encapsulated CDDP is 192.82 μg/mL.
Breast cancer MCTSs were generated from MDA-MB-231 cells in a medium with 2.5% Matrigel. The toxicity of SWCNT-COOH-CDDP and free components was tested at doses of 1 µg/mL SWCNT-COOH/0.6 µg/mL CDDP and 4 µg/mL SWCNT-COOH/2.52 µg/mL CDDP after 24 and 48 h of incubation. Optical microscopy was used to analyze the morphology of treated and untreated MCTSs, while the presence of lysosomal vesicles was observed by fluorescence microscopy. The proliferative capacity of breast cancer MCTSs was assessed by evaluating the protein expression of proliferating cell nuclear antigen (PCNA) by immunoblotting. The protein expression of cathepsin B and phosphatidylinositol 3-kinase (PI3K) was also analyzed. The evolution of invasive potential of MCTSs was monitored after the embedding of MCTSs in a matrix composed of basement membrane proteins.
The results revealed that after 48 h of incubation with 4 µg/mL SWCNT-COOH-CDDP the dimensions of MCTSs decreased and their spherical morphology was altered probably due to the detachment of cells from the proliferative layer. However, the expression of PCNA remained constant after treatment, suggesting that nanocomposite SWCNT-COOH-CDDP did not affect the proliferative capacity of MCTSs. An increase in the lysosomes number in presence of SWCNT-COOH-CDDP was observed in correlation with the rise of cathepsin B expression, after 24 h of exposure, indicating that their uptake occurred by an endocytic pathway. An up-regulation followed by a down-regulation of PI3K expression after 24h respectively 48h was noticed. Moreover, after 48 h of incubation, the invasive potential of breast cancer MCTSs was significantly inhibited in the presence of 4 µg/mL SWCNT-COOH-CDDP in comparison with 2.52 µg/mL free CDDP.
We concluded that nanocomposite SWCNT-COOH-CDDP showed high efficiency in the transport of CDDP in the breast cancer MCTSs and anti-tumoral activity by inhibiting their invasive potential and initiating probably cell death.
Keywords: breast cancer spheroids, carbon nanotubes, cisplatin, invasivity
Silica nanoparticles induce inflammatory response by interfering with cell autophagy (#173)
M. Yang1, J. Duan1, P. Huang1, Z. Sun1
1 China Capital Medical University, Public Health, Beijing, China
Objective: To investigate the mechanisms of inflammation caused by Silica nanoparticles.
Methods: RAW264.7 mouse macrophage cells were cultured and randomly divided into 5 groups: negative control group, positive control group (LPS10ng / ml exposure 24 hours), silica nanoparticle exposure groups (5μg / ml, 10μg / ml, 20μg / ml , 40ug / ml ), the exposure time was 24h, the morphological changes of the cells were observed under microscope and the activity of lactate dehydrogenase (LDH) in macrophages, intracellular activity (ROS) concentration was used to detect the oxidative damage of the cells. The cytotoxicity of silica nanoparticles was determined by CCK-8 cell proliferation-toxicity method. The inflammatory responses were tested by ELISA and qPCR methods. The relationship between cell autophagy and cell inflammatory response was detected by Western Blot.
Results: Compared with the negative control group, the cell density was decreased and the irregularly morphological changes were observed. The data of the oxidative damage test showed that in the the exposed groups ROS and LDH activities were higher than negative control group, even in the 40ug / ml exposure group, the activities were higher than the positive control group (P <0.05). Compared with the negative control group, the levels of TNFa in the exposed groups were higher than those in the negative control (P <0.05). The expression at transcription level of the NLRP3, Caspase-1, TNF-α, IL-1β, IL-6 and IL-18 showed an increased trend in the exposed groups. Meanwhile the protein levels of NLRP3, IL-1β, NF-κB, P-NF-κB, Caspase-1, LC3, P62 in the exposed groups were higher than the negative control group. Dose-dependent relationships existed for all tests.
Conclusion: Silica nanoparticles induced oxidative stress and cytotoxicity to the RAW264.7 macrophages, then caused the inflammatory response which may associate with the interference of cell autophagy.
Keywords: silica nanoparticles, inflammation, autophagy
In vitro toxicity of model ZnO-Ag nanoparticles in human lymphocytes and hemocytes of mussel Mytilus galloprovincialis (#185)
I. Efthymiou1, G. Kalamaras2, K. Koukouvini1, E. Mouzourakis3, Y. Georgiou3, S. Dailianis2, Y. Deligiannakis3, D. Vlastos1
1 University of Patras, Department of Environmental and Natural Resources Management, Agrinio, Greece
The present study investigates the effects of ZnO-Ag nanoparticles (ZnO-Ag NPs) on two in vitro biological models, i.e. human lymphocytes and mussel hemocytes, using a battery of bioassays, commonly linked to cytotoxic and genotoxic/mutagenic, as well as cytotoxic and oxidative effects, respectively. In this regard, different concentrations of ZnO-Ag NPs manufactured through Flame Spray Pyrolysis were tested in cultured human lymphocytes (0.5, 5, 10 and 20 μg mL-1) via the Cytokinesis-Block micronucleus (CBMN) assay and in primary cultures of Mytilus galloprovincialis hemocytes (0.1, 0.5 and 1 μg mL-1), using cytotoxic (i.e. neutral red retention time/NRRT assay) and oxidative (determination of superoxide anions, nitric oxide and lipid peroxides) stress indices for determining NPs cytotoxic, oxidative and genotoxic potential in any case. The obtained results were also compared with relevant data derived from bulk metal ions Zn+2 and Ag+. According to the latter, none of the ZnO-Ag NPs concentrations exhibited genotoxicity in human lymphocytes, while the Cytokinesis block proliferation index (CBPI), used for the assessment of cytotoxicity, showed ZnO-Ag NPs cytotoxic potential, similar to the results in the case of bulk metal ions Zn+2 and Ag+. As far as mussel hemocytes are concerned, the results demonstrated a significant increase of cell death after treatment with ZnO-Ag NPs, with maximum values of cell death at concentration 1 μg mL-1. A significant increase of O2·- and MDA was shown, compared to those values observed in control cells in each case, whereas a statistically significant decrease of NO was demonstrated. The comparative study of cytotoxic and oxidative effects of ZnO-Ag NPs, ZnCl2 and AgNO3 demonstrates the cytotoxic nature of NPs compared with the bulk metal ions Zn+2 and Αg+ (ZnO-Ag NPs > AgNO3 > ZnCl2), as well as NPs oxidative potential in the mussel hemocytes.
Keywords: toxicity, nanoparticles, mussels, human lymphocytes
Biological effects of molybdenum(IV) sulfide in the form of nano- and microparticles after intratracheal instillation in rat (#186)
Z. Sobańska1, M. Szparaga1, K. Domeradzka1, K. Sitarek1, R. Świercz1, L. Zapór2, J. Gromadzinska1, W. Wąsowicz1, J. Grobelny3, E. Tomaszewska3, G. Celichowski3, J. Roszak1, M. Stępnik1
1 Nofer Institute of Occupational Medicine, Łódź, Poland
Considering application of molybdenum(IV) sulfide (MoS2) in the nanosize form as a lubricant and scarcity of data on its biological effect in vivo with contradictory results in vitro, the study was undertaken to characterize its activity after short- and long-term exposure by intratracheal instillation.
Prepared nano- and microparticles (bulk MoS2 from US Research Nanomaterials, Inc.) were disk-shaped (97x8.5 nm by TEM) and plates (1.92x0.273 µm by TEM), respectively. Sprague Dawley rats were treated via intratracheal instillation with micro- and nanosized MoS2 at 1.5 or 5 mg/kg, using single exposure (analysis after 1 and 7 days) or multiple exposures (7 exposures every 2 weeks with analysis after 90 days). The following parameters were assessed: blood hematology, biochemistry (albumin and total protein concentration, triglycerides, urea, total cholesterol and HDL, uric acid, ASPAT, ALAT, GSH-Px activity), cytotoxic effects in bronchoalveolar lavage (BAL), comet assay on blood leukocytes, histopathological evaluation.
No acute effect was observed 1 or 7 days after single exposure of the animals to MoS2 in both forms. No clinical signs of systemic toxicity were noticed after multiple exposures. Some hematological and biochemical changes were observed, however no uniform pattern of toxic effects was evident.
After 90 days histopathological analysis revealed inflammatory changes in lungs in all animals treated with nano- and microform. Index of histopathological changes (range: 0-4 points) reached on average 1.33 and 1.67 for nanoform and 1.33 and 2.83 for microform (dose 1.5 and 5 mg/kg bw, respectively). In control group small inflammatory lesions in lungs were observed in 5/12 animals (index 0.42). Comet assay showed no significant DNA damage in blood lymphocytes in the exposed groups.
In conclusion, repeated intratracheal exposure to micro- and nanosized MoS2 can lead to inflammatory changes in the rat respiratory system, slightly stronger for the microform.
Supported by the IV stage of the programme ,,Improvement of safety and work conditions" (2017-2019) by the Ministry of Science and Higher Education/the National Centre for Research and Development. Coordinator: Central Institute for Labour Protection – National Research Institute"
Keywords: MOLYBDENUM(IV) SULFIDE, MICROPARTICLES, NANOPARTICLES
Assessment of reactive oxygen species in tobacco (Nicotiana tabacum L.) seedlings exposed to silver nanoparticles (#268)
A. - M. Domijan1, R. Biba2, S. Babić3, P. Cvjetko2, M. Tkalec2, B. Balen2
1 University of Zagreb, Faculty of Pharmacy and Biochemistry, Department of Pharmaceutical Botany, Zagreb, Croatia
Silver nanoparticles (AgNPs) have wide application in many consumer products due to their unique chemical and physical features enhancing well known antibacterial and antifungal properties of silver. Increase in AgNPs production has raised many concerns about their possible toxicity, and many studies have already indicated that they induce oxidative stress through enhanced production of reactive oxygen species (ROS). Rapid accumulation of ROS and imbalance in their generation and scavenging could result in severe damage of proteins, lipids and DNA. Since many factors control the ROS metabolism, detecting ROS accurately is challenging. The aim of this study was to develop fast and reliable method for quantitative determination of ROS in the plant tissue and to test the method in AgNP-treated tobacco (Nicotiana tabacum L.) seedlings. Three weeks old seedlings were treated with 25, 50, 75, 100 and 150 μM polyvinylpyrrolidone (PVP)-coated AgNPs for 7 days. Cytosolic ROS level in the plant extracts was assessed with fluorescent probe dihydroethidium (DHE) that specifically detects superoxide radical, and for quantification of fluorescence microplate reader wavelengths were set at 520 nm for excitation and 600 nm for emission. To optimize the method, optimal incubation time of the reaction was tested by monitoring fluorescence immediately after addition of DHE, and after 5 and 15 minutes of incubation. The linearity of the method was tested by measuring fluorescence of several dilutions of the extracts. Testing the incubation time for the samples showed that incubation could lead to false results and should be omitted. Moreover, measuring several dilutions of the samples confirmed linearity of the method thus proving that this method could be used for ROS quantification in the plant extracts. Finally, obtained results showed dose-dependent increase of ROS in AgNP-PVP treated tobacco seedlings indicating that oxidative stress is involved in toxicity of AgNPs towards plants.
Keywords: AgNP, tobacco seedlings, oxidative stress, ROS
Mechanism of toxicity of amorphous silica nanoparticles in lung epithelial cells and macrophages (#289)
S. Diabaté1, S. Fritsch-Decker1, C. Marquardt1, R. Leibe1, C. Weiss1
1 Karlsruhe Institute of Technology, Institute of Toxicology and Genetic, Eggenstein-Leopoldshafen, Baden-Württemberg, Germany
Synthetic amorphous silica nanoparticles (SAS NPs) are the most abundant nanomaterials and widely used in industry and in consumer products. As mechanisms of toxicity are still insufficiently understood for silica NPs, we studied the effects of two different types of nanosilica (colloidal and pyrogenic) in human lung epithelial cells (A549) and in murine RAW264.7 macrophages. Both silica NPs dose-dependently induced membrane leakage and cell death in the absence of serum without obvious involvement of reactive oxygen species. Interestingly, at low concentrations nanosilica triggered autophagy, evidenced by morphological and biochemical hallmarks such as autophagolysosomes or increased levels of LC3-II, which serves to protect cells from cytotoxicity.
According to the oxidative stress model, also nanosilica elicits an, albeit modest, anti-oxidative response as well as pronounced pro-inflammatory reactions and cytotoxicity in macrophages. Interestingly however, these three tiers of toxicity seem to operate separately of each other for nanosilica. Specifically, impeding the anti-oxidative response by scavenging of reactive oxygen species does not prevent the pro-inflammatory and cytotoxic response. Furthermore, blocking the pro-inflammatory response does not impair cell death.
We further investigated the impact of the protein corona on the biological activity of colloidal and pyrogenic nanosilica. Adsorption of serum proteins to the nanosilica surface suppressed cytotoxicity as well as inflammation in both cell lines. Cytotoxicity precedes the onset of pro-inflammatory gene expression and cytokine release as exemplified for IL-8 in A549 cells and TNF-alpha in RAW264.7 macrophages. Formation of a protein corona not only inhibited cellular toxicity, but also the pro-inflammatory response.
As hazard assessment has been guided by the prevailing assumption of a dose-dependent coupling of sequential tiers of toxicity, identification of critical physico-chemical parameters to assist the safe-by-design concept should be enabled by simply monitoring one of the toxicity read-outs. Our results indicate a more complex scenario in the case of nanosilica, which triggers independent pleiotropic effects possibly also related to different material properties and primary cellular targets.
Keywords: amorphous silica nanoparticles, cell death, inflammation, lung epithelial cells, macrophages
Effects of silver nanoparticles and silver nitrate on photosynthesis and photosynthesis-related proteins in tobacco (Nicotiana tabacum) – a comparative study (#305)
M. Tkalec1, P. Peharec-Štefanić1, R. Biba1, P. Cvjetko1, S. Šikić2, B. Balen1
1 University of Zagreb, Department of Biology/Faculty of Science, Zagreb, Croatia
The small size of nanoparticles (NPs), with dimensions between 1 and 100 nm, results in unique chemical and physical characteristics, which is why they are being implemented in various consumer products. Therefore, one of the important concerns is the potential detrimental impact of NPs on environment. Among different types of available nanomaterials, the most frequently applied are silver nanoparticles (AgNPs) due to antimicrobial properties of silver. As plants are the vital part of ecosystem and the first component of the food chain, the investigation of NPs phytotoxic effects is of particular interest. In this study, we examined the effects of citrate-coated AgNPs and its bulk form (AgNO3) on photosynthesis and leaf proteome of tobacco plants exposed to 100 µM AgNPs and AgNO3 for 7 days. Silver accumulation in leaf tissue was determined by ICP-MS. Changes in photosynthesis were evaluated by measuring the chlorophyll fluorescence parameters, while content of photosynthetic pigments was analysed by HPLC. Two-dimensional gel electrophoresis (2-DE) and MALDI mass spectrometry were employed to reveal the changes in protein expression. Both types of treatments resulted with the similarly increased Ag uptake. Significantly decreased photochemical quenching and increased concentration of violaxantin was recorded after exposure to AgNPs. On the contrary, treatments with AgNO3 did not significantly influence fluorescence parameters, although they induced a negative effect on majority of photosynthetic pigments. Identified proteins with differential expression were found to be mostly photosynthesis-related and down-regulated, although almost half of the proteins exhibited different expression level between AgNPs and AgNO3 exposure. Obtained results indicate that the AgNPs effects observed in tobacco leaves are not simply due to the release of silver ions and can be correlated with distinct impact of silver nanoparticle form.
Keywords: chlorophyll fluorescence, nanosilver, photosynthetic pigments, proteomics
Surface modification of halloysite nanotubes increases surface area and airway toxicity in mice (#338)
K. S. Hougaard1, 2, K. K. Barfod1, K. M. Bendtsen1, T. Berthing1, J. Koivisto1, S. Poulsen1, E. Segal3, A. Holländer4, K. A. Jensen1, U. Vogel1, 5
1 National Research Centre for the Working Environment, Danish Nanosafety Centre, Copenhagen Ø., Denmark
Halloysite [Al2Si2O5(OH)2] is an abundant natural clay mineral that can occur as nanotubes of rolled-up aluminum silica sheets (HNTs). HNT size and structure depend on the formation conditions and therefore vary between locations of origin. Diameters are typically <100 nm, with length to diameter ratios up to 200. More than 50 000 metric tons of HNTs are mined annually. Due to the hollow nanostructure that allows for loading and subsequent release of compounds applications are wide, e.g. HNTs may be loaded with antimicrobial agents (natural essential oils) and subsequently incorporated into polymers to form packaging materials that may increase food shelf life. Considering their high production volume and physico-chemical characteristics, where HNTs have the characteristics of poorly soluble high aspect ratio nanomaterials (HARN), knowledge about their toxicity is highly warranted (Koivisto et al. 2018).
We assessed the toxicological response to HNTs following airway exposure, an evident occupational exposure route of concern during mining, processing and handling of HNTs. Both a pristine (NaturalNano, NN) and a HNT modified by surface chemical etching by sulfuric acid (NNEtched) were assessed. BET surface area was 4-5 times higher for the NNEtched compared to the pristine HNT. First, the potential cytotoxicity of the two HNTs was screened in vitro in MutaTMMouse lung epithelial cells. Then adult female C57BL/6J BomTac mice were intratracheally instilled once with 6, 18 or 54 µg HNTs in isoflurane anesthesia and compared to vehicle controls and 162 µg Carbon black Printex 90 (positive inflammation control) on day 1, 3, and 28 after instillation. Lung inflammation was determined by the cellular composition of bronchoalveolar lavage (BAL) fluid, acute phase response by Saa mRNA levels (real-time quantitative PCR) in lung and liver tissue, and genotoxicity was analyzed by the alkaline comet assay as DNA strand breaks in BAL cells, lung and liver tissue. None of the HNTs were cytotoxic, affected mouse body weight or induced genotoxicity. At the highest dose level, NNEtched increased neutrophil influx and Saa3 mRNA levels in lung at all assessed time points. At day 1 post-exposure, the induced inflammation correlated well with the deposited particle surface area, indicating that the increase in BET surface area due to etching could explain the difference in the inflammatory potential of the studied HNTs. This is consistent with observations for other nanosized particles.
EUs Horizon 2020 research and innovation program (grant #720815) supported the Nanopack project (www.nanopack.eu). The abstract reflects views of the authors only, the EU Commission is not responsible for any use hereof.
Koivisto AJ, Bluhme AB, Kling KI, Fonseca AS, Redant E, Andrade F, et al. Occupational exposure during handling and loading of halloysite nanotubes – A case study of counting nanofibers. NanoImpact. 2018;10:153-60.
Keywords: halloysite nanotubes, nanoparticles, inflammation, acute phase response, surface area
Surface chemistry can drive the safer applications of cadmium-based quantum dots related to sex-specific neurodevelopmental adverse outcomes (#364)
D. Leme2, Y. Suh1, S. Hong1, T. Workman1, M. Smith1, W. Griffith1, E. M. Faustman1
1 University of Washington, Institute for Risk Analysis and Risk Communication, Department of Environmental and Occupational Health Sciences, Seattle, Washington, United States of America
Engineered nanomaterials (ENMs) are widely applied to a large number of market sectors that impact the lives of billions of people. However, the ability to fully characterize the risks associated with human exposures to ENMs has been limited. Cadmium-selenium containing quantum dots (QDs) have unique optical properties that favor clinical applications. Therefore, with the growing use of QDs in medical applications, there is an increased probability QDs interact with sensitive human receptors. Consequently, there is a need to investigate associated human health risks, specifically those related to neurodevelopmental disorders. In vitro human neural progenitor cell (hNPC) lines that originate from different sexes are promising in vitro models to evaluate potential sex-specific chemical effects on neurodevelopment. Cytotoxicity of two CdSe/ZnS QDs of differing surface chemistries (ITK™: carboxyl functional group; Qtracker®: polyethylene glycol with no reactive functional group) was quantified by LDH assay on proliferating and differentiating hNPCs from male (NSC-H14) and female (hNP1™) donors. Cadmium chloride was used as control for cytotoxic effects related to Cd2+ release. Exposures (days in vitro 1, 24 h) to ITK at five different doses (2.5-40 nM) demonstrated a significant dose-dependent decrease in viability of both cell lines during the proliferation stage. Significant response difference to ITK between cells from male (NSC-H14) versus female (hNP1) origin was also observed, and hNPCs of male origin were more sensitive to ITK than cells of female origin. Qtracker, at the same dose range as ITK, did not induce cytotoxicity in the proliferation or differentiation stages of either hNPC line. Cd (2.5-40 nM) induced a significant dose-response increase in viability of differentiating hNP1. Our results indicate that the main contributing factor of the QDs cytotoxicity is not from Cd ion release. In addition, we find the surface coating of QDs affects the cytotoxicity of these ENMs on hNPCs, suggesting that PEGylation can be a strategy for the safer application of QDs in clinical settings. Our findings also demonstrate the need to further evaluate sex-specific neurodevelopmental effects of ENMs.
Financial support: This project is supported by the EPA (RD 83573801, RD 83451401) and the NIEHS (5P01ES009601, P30ES007033, T32ES015459). The views expressed in this paper are those of the authors and do not necessarily reflect the views of the U.S. EPA.
Keywords: nanotoxicology, developmental neurotoxicity, in vitro testing, human progenitor cell lines, sex-specific effects
Cobalt-impregnated tungsten nanoparticles and cobalt ions trigger toxicity in differentiating neuronal cells: potential link to parkinsonian neurodegeneration (#367)
G. S. Gupta1, A. Gliga1, J. Hedberg2, A. Serra3, 4, D. Greco3, 4, I. O. Wallinder2, B. Fadeel1
1 KAROLINSKA INSTITUTET, Institute of Environmental Medicine, Division of Molecular Toxicology, Stockholm, Sweden
Tire studs, with pins made of cobalt-doped tungsten carbide (WC-Co), are used in many countries during winter to improve the gripping power on icy roads. During their use, cobalt and tungsten based nanoparticles (NPs) may be released. Recent material flow analysis studies performed in the MISTRA Environmental Nanosafety program have shown that there is a high dissipation rate of tungsten in tire studs and that there is presently no functional recycling of the tungsten (Furberg et al., 2019). In the present study, tungsten and cobalt based NPs were studied to understand their potential neurotoxicity using a retinoic acid (RA) differentiated human neuroblastoma cell line. CoCl2 was also included as ionic control. Differentiated SH-SY5Y cells displayed characteristics of dopaminergic as well as cholinergic neurons. The hydrodynamic size of NPs was between 400–600 nm in MilliQ water. It was also observed that these NPs sediment within 1 h of dispersion in cell culture medium. The zeta potential values were negative and were reduced in cell culture medium. ICP-MS analysis revealed that WC-Co and CO NPs released approximately 77% and 96% Co ions, respectively, in cell culture medium after 24 h. TEM showed that the NPs were internalized in SH-SY5Y cells and triggered ultrastructural changes. Co NPs were specifically internalized in cells through endosome formation and the NPs caused mitochondrial damage. Co NPs were found the most toxic when compared to WC and WC-Co NPs as determined by the Alamar Blue assay and the toxicity was paralleled by the toxicity of the cobalt salt. We also observed that differentiating cells were more sensitive to Co NPs than undifferentiated and differentiated cells. Furthermore, calcium overload and oxidative stress were shown to play a role cell death. Real-time PCR analysis of cholinergic and dopaminergic markers in RA differentiated SH-SY5Y cells revealed overexpression of markers of cholinergic neurons after exposure to Co NPs and Co ions indicative of a relative decline in dopaminergic neurons. Finally, in silico analysis of publically available transcriptomics data (Serra et al., 2019) predicted a significant connection between WC-Co NPs, the parkinsonogenic chemical, MPTP and L-dopa, which ameliorates symptoms in patients with Parkinson’s disease. Overall, this study provides initial evidence of the neurodegenerative potential of Co-based NPs in human systems.
Supported by the Swedish Foundation for Strategic Environmental Research-MISTRA.
Keywords: Neurotoxicity, Nanomaterials
Neuro- & biochemical- toxicity of silver nanoparticles and silver nitrate in soil to Aporrectodea calginosa earthworms (#379)
R. Gooneratne1, N. Saleeb1, A. Laschin1, B. Robinson1, J. Cavanagh2, J. Ross1
1 Lincoln University, New Zealand, Agriculture & Life Sciences, Christchurch, New Zealand
Silver nanoparticles (AgNPs) now widely used in many industry applications discharged via the land application of sewage sludge (Meier et al. 2016) interact with soil biota, including earthworms. Because there are no regulations on discharge limits, improper discharge of waste from these industries can lead to environmental contamination and damage to ecosystem organisms (Kühnel and Nickei 2014). In this study Aporrectodea caliginosa earthworms were exposed to 0 (control), 0.3, 3, 30, 300 and 0 (control), 0.03, 0.3, 3, 10 mg/kg of AgNPs and AgNO3 in soil respectively for 4 weeks and select biochemical and neurotoxicity studies were conducted. The lipid peroxidation (a measure of thiobarbituric acid reactive substances; TBARS) and activities of antioxidant enzymes (catalase, glutathione peroxidase, superoxide dismutase, glutathione S transferase, lipid peroxidation), and nerve conduction velocity (NCV) of the medial giant fibers (MGF) using a novel non-invasive electrophysiological technique were measured in earthworms at 1, 2, 3, and 4 weeks. The TBARS and antioxidant enzyme activities were elevated by both AgNO3 and AgNPs and this was most evident in earthworms at 4 weeks > 3 > 2 > 1. In neurotoxicity studies, MGF NCV progressively decreased in A. caliginosa exposed to both AgNPs and AgNO3. Biochemical toxicity was > neurotoxicity. The findings highlight oxidative stress and neurotoxic effects of Ag compounds on earthworms and the importance of government authorities to have legislations in place to prevent excessive soil contamination by AgNPs produced by the expanding nanoparticle industry.
Kühnel D, Nickei C. (2014). The OECD meeting on ecotoxicology and environmental fate – Towards the development of improved OECD guidelines for the testing of nanomaterials. Sci Total Environ 472:347–353.
Meier C, Voegelin A, Pradas del Real A, Sarret G, Mueller CR, Kaegi R. (2016). Transformation of silver nanoparticles in sewage sludge during incineration Environ Sci Technol 50:3503–3510. DOI: 10.1021/acs.est.5b04804.
Keywords: silver nanoparticles, silver nitrate, earthworm, antioxidant enzymes, neurotoxicity
TiO2 NM 105 response obtained on three different rat models, vitro, ALI, and vivo. (#380)
O. Joubert1, Z. Doumandji1, M. Lovera-Leroux1, R. Hocquel1, T. Krebs4, O. Schmid3, L. Ferrari1, L. Gaté2, L. Chézeau2, V. Zhernovkov5, B. Rihn1
1 Université de Lorraine, Institut Jean Lamour UMR CNRS 7198, Nancy, France
Due to the growing use of nanomaterials in various industrial processes, the number of workers potentially exposed is increasing even though the toxicological properties of these compounds are not completely known. Since nanoparticles (NP) may become aerosolized, inhalation represents the main route of occupational exposure, and the first tissues to be exposed are therefore those of the respiratory system.
Keywords: nanoparticles, toxicology, transcriptomic, alternative model, air lung interface
High-throughput hazard-based scoring, ranking and grouping of engineered nanomaterials (#386)
V. Hongisto1, P. Nymark1, 2, P. J. Kohonen1, 2, J. Hattara1, R. Grafström1, 2
1 Misvik Biology, Toxicology, Turku, Finland
Traditional safety testing is costly and labor-intensive, and does not suffice for keeping pace with steadily increasing innovations that involve engineered nanomaterials (ENMs). High throughput screening (HTS) technology offers opportunity to partly tackle the problem, as it allows for rapid and cost-effective in vitro model-based definition of inherent toxicological properties and priority ranking of tested agents for further study. We report a NANOSOLUTIONS FP7 EU-funded HTS-driven case study of 31 ENMs covering nine core structures functionalized by carboxylation, amination/ammoniation and pegylation. Cellular ATP content, cell number changes, apoptosis frequency, DNA damage and nucleic acid oxidative stress markers were assessed for up to 72h in the human lung epithelial cell line BEAS-2B in conditions with or without 10% fetal bovine serum, involving the generation of almost 105 data points. The results were fused into a comprehensive, multi-time point and multi-endpoint inclusive toxicity score for rank ordering of the ENMs, including versus reference agents with known toxicity profiles. The ENMs were finally grouped related to score or individual assays applying the US-EPA ToxPi Toxicological Prioritization Index tool. The integrated analysis demonstrated unique toxicity profiles of the respective ENMs, including variable activity relative the end points and/or by showing different dose-dependencies early or later with time. Serum influenced the results by slightly enhancing, having no effect or markedly decreasing the toxicity. Ammoniation coupled to higher toxicity than the other functionalized groups. The scoring analysis clustered the ENMs into several distinguishable groups likely reflecting inherent physiochemical reactivity among other properties. We believe our study to effectively advocate for the general usefulness of HTS technology for safety assessment studies. This technology overall promises to aid the integration of safety testing measures during innovation of ENMs.
Keywords: High throughput screening, Nanomaterial, in vitro, Scoring, Ranking
Orally administered SiO2 nanoparticles differing in their specific surface area did not induce local or systemic toxicity (#399)
J. Cabellos1, G. Vales2, H. K. Lindberg2, K. A. Jensen3, R. Alturi3, J. Catalán2, 4, S. Vázquez-Campos1, G. Janer1
1 Leitat Technological Center, Barcelona, Spain
Understanding how the physicochemical properties of nanomaterials influence toxicity is critical to support their grouping and avoid case by case testing. Little attention has been placed on the role of specific surface area on oral toxicity. In this study, four amorphous methyl-coated SiO2 NPs consisting of two target sizes (100 and 300 nm), and two types of porosity (non-porous and highly porous particles) were used. As a result, their specific surface areas ranged from 10 to 844 m2/g. Female Swiss mice were administered by oral gavage for 5 consecutive days. Two SiO2 NP dose levels (100 and 1000 mg/kg b.w.) were tested for each of the four materials. All dispersions were characterized by TEM and Nanoparticle tracking analysis. Porous material dispersions tended to form agglomerates and were rather unstable. Animals were sacrificed one day after the last administration or after a three-week recovery period. No relevant toxicological effects were induced by any of the SiO2 NPs, as evaluated by body weight, gross pathology, relative organ weights (liver, spleen, kidneys), hematology, blood biochemistry (AST, ALT, creatinine and total protein concentration), genotoxicity (Comet assay in jejunum cells and micronucleus test in peripheral blood erythrocytes), liver and small intestine histopathology, and intestinal inflammation (cytokine determinations in jejunum mucosa and submucosa). The presence of SiO2 NP in the intestine was evaluated by a hyperspectral imaging microscopy system (CytoViva) using histological samples of jejunum tissue. A high intra-group variability in the percentage of pixels showing a SiO2 NP spectral signature was found. The highest percentages of positive pixels per sample were recorded in NP-treated animals, but no statistically significant differences in the mean percentages were observed either among the four NPs treatments, or with the control group. (Funded by EU H2020 caLIBRAte project, Grant Agreement No. 686239).
Keywords: silica nanoparticles, specific surface area, in vivo, oral toxicity, intestine
Impact of silver nanoparticles on physiological parameters of tobacco seedlings (#409)
R. Biba1, P. Cvjetko1, M. Tkalec1, P. Peharec-Štefanić1, A. - M. Domijan2, S. Šikić3, D. M. Lyons4, S. Babić5, B. Balen1
1 Faculty of Science, University of Zagreb, Department of Biology, Zagreb, Croatia
Silver nanoparticles (AgNPs) are among the most commonly applied nanomaterials due to their powerful antibacterial and antimicrobial properties that are being exploited in a number of consumer products. As a consequence, AgNPs are expected to enter natural ecosystems where they can undergo biodegradation or bio-accumulate in the food chain and act as a potential environmental hazard. Plants, as primary producers, are very likely to be influenced. Bioaccumulation of AgNPs is dependent on the characteristics of the particles, with mainly their size and surface coating determining their mobility and transport in the environment. In this study, we compared the effects of two differently coated AgNPs [polyvinylpyrrolidone (AgNP-PVP) and cetyltrimethylammonium bromide (AgNP-CTAB)], applied in three concentrations (25, 50 and 100 μM), on photosynthesis and oxidative stress parameters of tobacco (Nicotiana tabacum) seedlings. Silver uptake in the plant tissue was determined with inductively coupled plasma mass spectrometry (ICP-MS). Chlorophyll fluorescence parameters were measured by fluorimeter using a saturation pulse method. Dihydroethidium test was used to determine the content of reactive oxygen species (ROS). Ascorbate peroxidase (APX), pyrogallol peroxidase (PPX), superoxide dismutase (SOD) and catalase (CAT) activities were spectrophotometrically measured. Although both types of AgNPs significantly increased the silver content in the plant tissue, the highest amount of Ag was detected in 100 μM AgNP-CTAB treatment. Both AgNPs induced ROS formation that was again much more pronounced with AgNP-CTAB. Changes in antioxidant enzymes activities were also observed. AgNP-PVP significantly decreased PPX and APX activity in all tested concentrations, but no changes were detected in CAT and SOD activity. AgNP-CTAB decreased PPX and SOD activity, and increased CAT activity at the highest concentration. Both AgNP-PVP and AgNP-CTAB lowered most of the chlorophyll fluorescence parameters, indicating a negative influence on the photosynthetic apparatus. Even though both types of AgNPs caused phytotoxic effects on tobacco seedlings, these results indicate that the degree of damage correlates with the surface coating used for AgNPs stabilization.
Keywords: silver nanoparticles, Nicotiana tabacum, reactive oxygen species, fluorescence, antioxidant enzymes
A metabolomic study of the effect of gold nanostars vs gold nanospheres in Wistar rats after a single-dose intravenous administration. (#426)
M. Enea1, 2, A. M. Araújo1, P. Guedes de Pinho1, E. Pereira2, M. D. L. Bastos1, H. F. Carmo1
1 University of Porto, Biological Science, Porto, Portugal
The physical and chemical properties of gold nanoparticles (AuNPs) such as shape, influence their optical and biological effects. Regarding their biological effects, the existing studies fail in consistency and one of the causes is the low toxicological profile of AuNPs as well as the lack of sensitive methods to detect differences among different types of AuNPs. For this reason, innovative and sensitive approaches such as metabolomics are much needed.
The current work aimed at using a metabolomic approach to compare the effect of gold nanospheres vs.gold nanostars (of similar dimeter ~40-48 and coated with 11-mercaptoundecanoic acid) 24h after i.v. administration to Wistar rats (1.33*1011AuNPs/Kg). A gas chromatography-mass spectrometry (GC-MS)-based metabolomic study was performed to investigate the metabolomic changes in the liver and spleen of the AuNPs-exposed animals.
Multivariate analysis showed that the metabolic pattern of the liver from animals exposed to gold nanospheres discriminate from the liver exposed to gold nanostars. The spheres produced a significant increase in intracellular metabolites such as palmitic, oleic, and 5,8,11- eicosatrienoic acids while the nanostars increased dimethylglycine, uracil and reduced inosine, uridine, L-lysine, and phosphoric acid. The discriminated metabolites are associated to the biosynthesis of fatty acids, the metabolism of arachidonic acid, pyrimidine and purine, biotin and glycine as well as to the synthesis of aminoacids. Regarding the spleen, the multivariate analysis did not discriminate significantly between the effect of spheres and stars, but some of the discriminated metabolites are involved in the same pathways as in the case of the liver (biosynthesis of fatty acids, pyrimidine and purine metabolism).
Using a metabolomic approach we were able to obtain different metabolic profiles for gold nanospheres vs gold nanostars in the liver. This proves that metabolomics is a very useful tool for the study of the effect of gold nanoparticles and should be taken into consideration as a highly sensitive alternative for comparison studies between different types of AuNPs.
This work was supported by FEDER (POCI/01/0145/FEDER/007728, POCI-01-0145-FEDER-029584, POCI/01/0145/FEDER/007265 ) and by national funds (FCT, Fundação para a Ciência e Tecnologia) through UID/MULTI/04378/2013, UID/QUI/50006/2013 andthe grants PD/BD/109634/2015 (PDQS) and SFRH/BD/107708/2015. To all financing sources the authors are greatly indebted.
Keywords: gold nanostars, gold nanospheres, in vivo, metabolomics
Effects of titanium dioxide nanoparticles on T98G human glioblastoma cells (#450)
E. Fuster1, H. Candela1, J. Estévez1, E. Vilanova1, M. A. Sogorb1
1 Universidad Miguel Hernández de Elche, Instituto de bioingeniería, Elche, Spain
The effects of a set of TiO2 nanoparticles (NPs) (Z potential = +22.8±0.8 mV; mean size determined by transmission electronic microscopy (TEM) = 18±5 nm and mean size after 72 hours in cell culture media determined by dynamic light scattering = 12 nm) on T98G human glioblastoma cells were characterised.
TiO2 NPs were incorporated into the cells after 72 hours of exposure, as was verified by optic microscopy and by light scattering flow cytometry. TEM confirmed these results showing that NPs remain in the cytoplasm grouped in clusters and causing, apparently, autophagy.
RNAseq experiment showed that the exposure of T98G cells to TiO2 NPs in non-cytotoxic conditions (20 µg/ml during 72 hours) caused dysregulation in the expression of 1025 genes. The results of the RNAseq were further partly validated in independent experiments using a set of 5 genes.
The analysis of the ontology of the genes with altered expression determined that the more overrepresented biological processes among the differentially expressed genes were blood vessel endothelial cell proliferation involved in sprouting angiogenesis and negative regulation of fibroblast grow factor receptor signalling pathway together with other biological processes related to angiogenesis and vasculogénesis as positive regulation of vascular endothelial growth factor production, positive regulation of angiogenesis and blood vessel development.
Up to 6 different biological processes associated to mechanisms of inflammation were altered among the differentially expressed genes. The overexpression of the pro-inflammatory interleukins 6 and 8 were also verified by immunological methodologies.
In conclusion, TiO2 NPs are able to cause alterations in glia cells that seriously affect their role in maintenance of the nervous system and can eventually conduct to neurotoxicity.
ACKNOWLEDGMENT: This work was supported by Ramón Areces Foundation (grant CIVP18A3939).
Keywords: nanotoxicology, in vitro toxicology, neurotoxicology, glia, titanium dioxide nanoparticles
Precision-Cut Liver Slices as a promising ex vivo model for nanosafety studies (#481)
R. Bartucci1, C. Åberg1, Y. L. Boersma1, P. Olinga1, A. Salvati1
1 University of Groningen (RUG), Groningen Research Institute of Pharmacy (GRIP), Groningen, Netherlands
Significant efforts within the nanosafety community are currently focused in the implementation of novel advanced models for in vitro testing. Ideal models should mimic as much as possible the complexity of the in-vivo environments in which nanomaterials have been found to accumulate. Within this context, Precision-Cut Tissue Slices (PCTS) represent an interesting ex-vivo model already validated and extensively used for toxicological studies and drug metabolism¹: the maintained cellular complexity of the tissue and the possibility of preparing tissue slices from diverse species, including from human tissue, and different organs, are only few of the many advantages of using this model. The use of PCTS can also significantly contribute to the reduction of in-vivo studies in accordance with the 3Rs – replacement, reduction and refinement.
Within this context we aimed to test whether PCTS can be used as an ex vivo model for nanosafety assessment. We have focused as a first step on Precision Cut Liver Slices (PCLS): most in vivo studies show in fact that once NPs reach the blood stream, they mainly end up in the liver and – within this organ – often accumulate in Kupffer macrophages. Then we have used fluorescently labeled carboxylate polystyrene (PS-COOH) NPs and amino-modified polystyrene (PS-NH2) as model nanoparticles whose behavior and effects at cellular level have already been extensively characterized.2 In this way, we could determine how in vivo accumulation within the liver and in vitro effects at cellular level of these well characterized nanoparticles translate in the ex vivo model.
Thus, NPs have been added to PCLS in relevant biological media containing serum where a protein corona forms on their surface³. NP uptake and distribution in the PCLS have been explored by using confocal fluorescence imaging, in order to determine whether the NPs enter cells and in which cell types they accumulate over time. Eventual effects on the tissue slices have been assessed by histological analysis and by measuring the ATP content, caspase 3/7 activity and TUNEL assay.
Upon exposure to increasing doses of PS-NH2, toxic effects have been observed in the tissue, including activation of apoptosis, resembling overall what has been shown in in-vitro studies with the same NPs. Single cell analysis has been used to determine NP impact in the cells in which they accumulate within the tissue. Despite the large adsorption of NPs on the outer cell layer, uptake of NPs in the tissue was clearly visible inside the section and immune-staining has been used to determine the cell types in which NPs accumulated over time.
Overall, the results suggest that NP behavior in the PCLS can resemble several aspects of what observed in vivo and in vitro. Additional results obtained in lung and in human liver are also presented.
Keywords: Nanosafety, 3 R's, ex-vivo model, macrophages
Effect of carbon nanotubes on pulmonary surfactant (#504)
D. Kondej1, T. R. Sosnowski2
1 Central Institute for Labour Protection – National Research Institute, Department of Chemical, Aerosol and Biological Hazards, Warsaw, Poland
Purpose: Carbon nanotubes (CNTs) are an important group of nanomaterials (NMs) and due to their outstanding physical and chemical properties have a wide range of applications. It is estimated that CNTs constitute almost 30% value of the total NM market. By 2023, the global CNT market is expected to reach almost 4,000 tonnes, an annual increase of 12.8%. The potential CNT applications indicate the need for a detailed analysis of the impact of carbon nanomaterials on biological membranes. The aim of this study was to evaluate the influence of carbon nanotubes on the rheological properties of the air/liquid interface with the pulmonary surfactant (PS), which is the first barrier separating the inhaled air in pulmonary alveoli from the lung tissue.
Materials and methods: The experiments were carried out with dynamic pendant drop (DPD) method using PAT-1M tensiometer (Sinterface Technologies, Germany). The animal-derived preparation Survanta (Abbott Laboratories, France), intended for treatment of Respiratory Distress Syndrome (RDS) in newborn premature infants, was used as a model PS. The tests were conducted at 36.6 ± 0.2 °C for different CNT concentrations (ranging up to 1 mg/ml) with constant concentration of PS solution (2.5 mg phospholipids/ml). The droplet was oscillated at 10% surface area changes with various frequencies (0.1-0.5 Hz) what corresponded to a range of breathing patterns (2 s – 10 s per inspiration-expiration cycle).
Results: It was found that carbon nanotubes studied influence the dynamics of changes in surface tension, loss angle, surface elasticity and surface viscosity of oscillated air-liquid interface. An increase in the concentration of carbon nanotubes causes an increase in surface elasticity and a decrease in loss angle. An increase in the oscillation frequency of the air/liquid interface with PS results in an increase in surface elasticity and a decrease in loss angle and surface viscosity of the air/liquid interface contacted with the tested CNTs.
Conclusion: The change in rheological parameters of the interface indicates a disturbance of viscoelastic properties of the pulmonary surfactant in the presence of the carbon nanotubes.
Acknowledgment: This paper has been based on the results of the fourth stage of the National Programme “Improvement of safety and working conditions” partly supported in 2017–2019 by the Ministry of Science and Higher Education/National Centre for Research and Development. CIOP-PIB is the Programme’s main co-ordinator.
1. De Volder M.F.L., Tawfick S.H., Baughman R.H., Hart A.J. (2013). Carbon nanotubes: present and future commercial applications. Science, 339(6119), 535-539.
2. Carbon Nanotube Market 2018-2019. Yano Research Institute Ltd., Tokyo, Japan, 2018.
3. Kondej D., Sosnowski T.R. (2019). Interactions of carbon nanotubes and carbon nanohorns with a model membrane layer and lung surfactant in vitro. Journal of Nanomaterials, vol. 2019, Article ID 9457683, 10 pages. DOI: 10.1155/2019/9457683
4. Gehr P. (2018). Interaction of nanoparticles with biological systems. Colloids and Surfaces B: Biointerfaces, 172, 395-399.
Keywords: carbon nanotubes, pulmonary surfactant, rheological properties
Effect of surface charge on the genotoxic potential of nanomaterials: hazard classification. (#527)
G. Vales1, S. Suhonen1, K. Siivola1, J. Catalán1, K. Savolainen1, H. Norppa1
1 Finnish Institute of Occupational Health, Work Environment, Helsinki, Finland
A main challenge in nanotoxicology is to understand, how the physicochemical properties of an engineered nanomaterial (ENM) determine its hazard potential. Particle functionalization may influence the nature and extent of the ENM-biological structures interaction, thereby modulating the potential consequences of ENM exposure. Here, we studied the cytotoxic and genotoxic potential of nine sets of ENMs: silver nanoparticles (NPs), copper NPs, gold NPs (5-nm and 20-nm core sizes), titanium dioxide NPs, titanium dioxide nanorods, multi-walled carbon nanotubes, nanodiamonds, and quantum dots. Each ENM set comprised of a core particle with different surface functionalizations: -COOH (negative charge), -NH2 (positive charge) and –PEG (polyethylene glycol; neutral charge). Pristine core particles were also included for ENMs with adequate dispersibility. Human bronchial epithelial BEAS-2B cells were exposed to the ENMs at doses chosen based on cytotoxicity (cell counts, dead cells excluded by trypan blue). For a protype of a nanosafety classifier, we devised a point-based classification system for the hazard potential of the ENMs. For cytotoxicity (24- and 48-h exposure), points were given according to the dose when IC50 was reached. For genotoxicity (comet assay, 24 h; micronucleus assay, 48 h), points were given for a positive result, efficiency (lowest dose giving a statistically significant increase) and effectivity (maximum fold-effect in comparison with control). Four categories were established depending on the amount of points: non-(cyto/geno)toxic, weak equivocal, equivocal and (cyto/geno)toxic. Our results showed that the cytotoxicity of all ENMs (except the carbon nanomaterials) was enhanced by the NH2-functionalization, while the effect of functionalization on genotoxicity depended on the ENM and genotoxicity endpoint. Treatment time affected the cytotoxicity categorization. Differences were observed in genotoxicity categorization based on DNA damage and micronuclei. In conclusion, functionalization affected both the cytotoxicity and genotoxicity of the ENMs. ENM hazard categorization based on a single cell system is restricted to that system and depends on the experimental details.
Keywords: in vitro, nanoparticles, classification, hazard, surface charge
Bioavailability improvement of a monoamine oxidase-B inhibitor using PEGylated PCL-based nanoparticles (#534)
M. Pinto1, C. Fernandes1, E. Gil-Martins2, R. Silva2, S. Benfeito1, F. Cagide1, F. Borges1, F. Remião2
1 CIQUP - Centro de Investigação em Química, Departmento de Química e Bioquímica, Faculdade de Ciências, Universidade do Porto, Porto, Portugal
Parkinson’s disease is a neurodegenerative disorder, which has as current pharmacological treatment the administration of
In this context, the use of PEGylated nanoparticles (NPs) as drug delivery systems has been reported as an interesting tool to avoid drug toxicity and to increase the stealth capacity of drug candidates to surpass biological barriers. Thus, in the present work, a novel potent, selective and reversible IMAO-B (
Acknowledgements: "The work was supported by UID/MULTI/04378/2019 with funding from FCT/MCTES through national funds.” M. Pinto, C. Fernandes, S.
Keywords: Parkinson disease, chromone, monoamine oxidase B inhibitor, PEGylated nanoparticles, intestinal and brain permeability
Effects of double-walled carbon nanotubes on the early phase of respiratory syncytial virus infection in mice. (#595)
W. Watanabe1, A. Miyauchi4, T. Akashi2, A. Hirose3, H. Yoshida4, M. Kurokawa4
1 Kyushu University of Health and Welfare, Graduate School of Health Sciences, Nobeoka, Japan
Double-walled carbon nanotubes (DWCNTs) are important materials in the fields of nanotechnology. Effects of DWCNTs on the pneumonia in respiratory syncytial virus (RSV)-infected mice were already reported by our research group at EuroTox 2018. The aim of this study was to evaluate effects of DWCNTs on primary immunity responding to RSV infection in mice.
DWNT-1 (1 μm in length) and DWNT-15 (15 μm in length) were used in this study. Female (6 weeks old) BALB/c mice were intranasally exposed to DWCNTs (0-0.125 mg/kg) on days 1, 3 and 5 before RSV infection under anesthesia. These mice were intranasally infected with 3.5 x105 PFU of RSV under anesthesia.
On day 1 post-infection, the levels of TNF-α, a proinflammatory cytokine, in the bronchoalveolar lavage fluids (BALF) of RSV-infected mice were significantly suppressed due to DWNT-1-exposure (0.125 mg/kg) compared with the control, but not DWNT-15-exposure. Histopathological analysis for lung tissues showed that increase of infiltration of the inflammatory cells around artery and hypertrophy of the epithelial cells due to DWCNTs treatment compared with the control. Especially, ingestion of the carbon tubes in alveolar macrophages was confirmed in DWNT-1-treated mice.
Thus, exposure to DWNT-1 might affect the function of alveolar macrophages/monocytes in an early phase of infection, resulting in the exacerbation of the pneumonia in RSV-infected mice.
Keywords: DWCNTs, pneumonia
Tissue distribution of silver chalcogenide quantum dots in mouse model (#603)
J. - Z. Zhang1, H. Tang2, X. - Z. Chen1, W. - S. Xi1, Q. Su1, A. Cao1, H. Wang1
1 Shanghai University, Institute of Nanochemistry and Nanobiology, Shanghai, China
Silver chalcogenide quantum dots (QDs), including Ag2S, Ag2Se and Ag2Te, hold great promise in fields of bioimaging, biosensor, photocatalysis, optoelectronics and thermoelectric materials, because of their bright near-infrared (NIR) emission, Cd-free composition and high stability. Especially, the emission peak of silver chalcogenide QDs is within the second NIR window, and thus these QDs exhibit great potentials in high resolution bioimaging with deep penetration. But, their safety issues are still not well addressed, which may impede their development and applications. In this study, we synthesized polyethylene glycol coated silver chalcogenide QDs, and measured and compared their biodistribution in mice after a single intravenous injection. Three kinds of QDs had the same size (5-6 nm) and surface properties (polyethylene glycol), and good dispersibility and stability in physiological solutions. After mice were intravenously injected with QDs at a dose of 8 μmol/kg body weight (silver equivalent), their blood kinetics and tissue distribution profiles were obtained by measuring the Ag contents in tissues at different time points. All three kinds of QDs were quickly removed from blood. Among them, Ag2Te displayed the longest half-life in blood, while Ag2Se displayed the lowest half-life. All three QDs mainly accumulated in liver and spleen, and then eliminated along with time. Compared to gradual decrease of Ag2S and Ag2Te, Ag2Se decreased markedly in first 7 days and reached to background levels at day 14. The stability of silver chalcogenide and the different properties of chalcogenide ions in biosystems may contribute to the different biological behaviour of three QDs.
Keywords: silver chalcogenide, nanoparticle, distribution, in vivo
Discovery of an inhibitor of multiwall carbon nanotubes-stimulated IL-1β secretion via inflammasome activation (#613)
T. Nishimaki-Mogami1, H. Cui1, K. Soga1, R. Adachi1, N. Tamehiro1, A. Hachisuka1, K. Kondo1, A. Hirose1
1 National Institute of Health Sciences, Kawasaki, Japan
The NLRP3 inflammasome-mediated IL-1β production is implicated in the pathogenesis of various chronic inflammatory diseases. We have previously shown that multiwall carbon nanotubes (MWCNT) of certain length and needle-like shape can elicit robust NLRP3-inflammasome activation and IL-1β secretion in macrophages. In this study, we explored inhibitors of inflammasome-mediated IL-1β production. PMA-differentiated THP-1 macrophages pretreated with/without chemicals were stimulated with either MWCNT or ATP. Among chemicals tested, K-NK104 caused marked reduction in MWCNT-induced IL-1β production. This reduction was accompanied by decreased release of mature IL-1β and active caspase-1 fragment, while cellular levels of IL-1β precursor, procaspase-1, and NLRP3 were unaffected. Flow cytometry analysis showed that MWCNT uptake by cells was repressed by this compound. In contrast, extracellular ATP-elicited IL-1β production was unimpaired, suggesting that the NLRP3 inflammasome and its downstream pathways were unaffected by this compound. We conclude that K-NK104 inhibits MWCNT-stimulated inflammasome activation and the consequent IL-1β production by repressing internalization of MWCNT into cells. The target of K-NK104 during this process is now under investigation.
Keywords: carbon nanotubes, IL-1beta, inflammasomes, NLRP3
Toxicity assessment of engineered and airborne ceramic nanoparticles on a human 3D bronchial epithelium (#644)
M. J. Bessa1, 2, F. Brandão1, 2, A. Salmatonidis3, A. Vulpoi4, M. Viana3, F. R. Cassee5, S. Fraga1, 2, J. P. Teixeira1, 2
1 Instituto Nacional de Saúde Doutor Ricardo Jorge, Saúde Ambiental, Porto, Portugal
Nanotechnology has been contributing to major advances in the ceramic industry. Many nanoscale powders are currently used for production of ceramic materials. At the same time, some processes used in the manufacture of ceramic products have a high potential for nanoparticle (NP) release into the workplace environment that raise occupational health concerns. Therefore, there is an urgent need for assessing the toxicity of these intentionally used or unintentionally generated NPs. This study aimed to investigate the in vitro toxicity of engineered (ENPs) and airborne ceramic NPs in a 3D human bronchial epithelial model (MucilAir™) under air-liquid interface conditions.
Two commonly used ENPs, zirconium (ZrO2) and antimony-tin oxide (Sb2O3•SnO2) and the particulate matter (PM) < 2.5 µm and ultrafine (UFP) < 0.2 µm fractions of airborne NPs released during High Velocity Oxy-Fuel (HVOF) spraying were tested. MucilAir™ cultures were exposed three consecutive days (E1, E2, E3) to different doses of NPs aerosols in a Vitrocell® Cloud 12 system. Cytotoxicity was evaluated by the lactate dehydrogenase (LDH) release (24h after each exposure) and WST-1 metabolization (24 h after the last exposure) assays. Primary and oxidative DNA damage were evaluated in cultures collected 24h after the last exposure by the alkaline and FPG-modified comet assay versions, respectively.
A significant increase in LDH leakage was observed in bronchial cultures exposed to aerosolised ZrO2 NPs (5.6 µg/cm2 per exposure) 24h after E1 and E2, while for antimony-tin oxide NP-exposed cultures (11.0 µg/cm2) this effect was only detected 24h after E1. No significant changes upon cell viability were detected 24h after E3 in both ENP-exposed cultures. Moreover, no significant alterations of primary and oxidative DNA damage levels were detected following exposure to the ENPs aerosols comparing with control cultures. The chemical analysis revealed that airborne HVOF-generated NPs were mainly constituted by WC, CrC and Ni. Exposure to the aerosolised PM 2.5 fraction failed to affect plasma membrane integrity of the MucilAir™ cultures as evaluated by the LDH release. However, 24h after the last exposure a significant decrease in cellular metabolic activity in human bronchial epithelium exposed cultures compared to the control cultures was observed, as assessed by the WST-1 assay. On the other hand, cultures exposed to the lowest tested dose of the aerosolised UFP fraction exhibited a significant increase in LDH release only visible at 24 h after the first exposure, whereas no changes in the metabolic activity were detected. On the other hand, only human bronchial cultures exposed to the highest tested dose of the UFP fraction aerosol exhibited a significant increase of oxidative DNA damage comparing with control cultures. Thus, our findings highlight the potential health risks associated with exposure to engineered and unintentional NP emissions derived from ceramic industry processes.
This work was supported by the Portuguese Foundation for Science and Technology (FCT) through the CERASAFE project (SIINN/0004/2014). MJB and FB are recipients of FCT PhD scholarships (SFRH/BD/120646/2016 and SFRH/BD/101060/2014). The authors would also like to acknowledge the contribution of COST Action hCOMET (CA15132).
Keywords: cytotoxicity, genotoxicity, nanoparticle aerosol, human upper airway epithelium, air-liquid interface
Agglomeration state of titanium-di-oxide (TiO2) nanomaterials influences the toxicity/biological responses in human bronchial epithelial cells at the air-liquid interface (#646)
S. Murugadoss1, S. Diabaté2, S. Mülhopt5, H. - R. Paur5, F. Brassinne4, J. Mast4, L. Godderis3, C. Weiss2, P. Hoet1
1 Unit for Environment and Health, Katholieke Universiteit Leuven (KU Leuven), Leuven, Belgium
Agglomeration of nanomaterials (NMs) is a ubiquitous phenomenon and its dynamic behaviour throughout their life cycle poses a great challenge in assessing its impact on human health. While agglomerates are of prime importance in occupational exposure scenarios, their toxicological relevance remain poorly understood1,2. Therefore, the aim of this study was to compare the toxicity/biological responses induced by either agglomerates or individual/unbound particles. Two different sized titania particles, nano-TiO2 (primary size 17 nm) and a sub-micron TiO2 (117 nm) were selected for this study. Stable stock dispersions of non-agglomerated particles (median feret min size, 34 and 120 nm) and their respective agglomerates (137 and 309 nm) were prepared using a modified protocol published previously3. These dispersions were aerosolized and subsequently administered to human bronchial epithelial cell cultures (16HBE14o-) at the air-liquid interface4,5, a procedure which is more realistic in terms of inhalation exposure. The cells were exposed to different doses of TiO2 aerosols using electrostatic deposition. At the end of 4-hour exposure, the effects on cell membrane integrity (LDH release), metabolic activity (WST-1 reduction) and oxidative stress (glutathione depletion) were evaluated. Significant effects were observed only for nano-TiO2. Non-agglomerated particles (34 nm) induced a dose dependent increase of LDH. Further, they decreased metabolic activity and glutathione levels at the highest dose tested. In contrast to unbound particles, agglomerates of nano-TiO2 did not induce adverse effects although the deposited mass was similar. Similarly, exposure of cells to comparable doses of sub-micron TiO2, either in the form of primary or agglomerated particles, also did not provoke toxicity. These results suggest that the agglomeration state of TiO2 nanomaterials influences the toxicity/biological responses at the air-liquid interface, depending on the primary particle size. In addition to acute cellular toxicity, other end points such as genotoxicity and altered gene expression are currently investigated.
1. Bruinink A, Wang J, Wick P (2015) Effect of particle agglomeration in nanotoxicology. Arch Toxicol 89:659–675. doi: 10.1007/s00204-015-1460-6
2. Mitrano DM, Motellier S, Clavaguera S, Nowack B (2015) Review of nanomaterial aging and transformations through the life cycle of nano-enhanced products. Environ Int 77:132–147. doi: 10.1016/j.envint.2015.01.013
3. Guiot C, Spalla O (2013) Stabilization of TiO2 nanoparticles in complex medium through a pH adjustment protocol. Environ Sci Technol 47:1057–1064. doi: 10.1021/es3040736
4. Panas A, Comouth A, Saathoff H, et al (2014) Silica nanoparticles are less toxic to human lung cells when deposited at the air-liquid interface compared to conventional submerged exposure. Beilstein J Nanotechnol 5:1590–1602. doi: 10.3762/bjnano.5.171
5. Mülhopt S., Dilger M., Diabaté S., Schlager C., Krebs T., Zimmermann R., Buters J., Oeder S., Wäscher T., Weiss C. and Paur HR. (2016). Toxicity testing of combustion aerosols at the air-liquid interface with a self-1 contained and easy-to-use exposure system. Journal of Aerosol Science. 96, 38-55.
Keywords: TiO2 nanomaterials, agglomerates, air-liquid interface, human lung cells, toxicity
Clearance of multi-walled carbon nanotubes in rat lungs after intratracheal instillation: a comparison of different instillation devices (#654)
M. Hojo1, A. Maeno1, Y. Sakamoto1, A. Onuki1, Y. Hasegawa1, K. Yuzawa1, Y. Kubo1, A. Nagasawa1, M. Ohnishi2, Y. Goto2, T. Suzuki1, A. Inomata1, T. Moriyasu1, A. Hirose3, D. Nakae4
1 Tokyo Metropolitan Institute of Public Health, Tokyo, Japan
Background: An intratracheal instillation test has been used as an alternative exposure method to the inhalation. Quantifying the clearance of test materials is essential, especially for the evaluation of long-biopersistent fibers. We here examined the time course of histological changes and the lung burden after a single administration of multi-walled carbon nanotube (MWCNT).
Keywords: MWCNT, lung burden, inflammation, intratracheal instillation
Prospects for the use of alternative methods for testing the safety of nanomaterials in the Republic of Belarus (#658)
1 Republican unitary enterprise «Scientific practical centre of hygiene» , Ministry of Health, Minsk, Belarus
The number of names of nanomaterials and the volume of their application in various fields of science, medicine, energy, industry is growing rapidly not only throughout the world, but in the Republic of Belarus too. The speed of nanotechnology development is ahead of the development of methods for assessing their safety and regulatory documents. Currently in the republic there is no register of nanomaterials. The need to study the potential toxic properties of nanomaterials in products and in the air of the working area is not fixed by law, technical normative legal acts regulating circulation in the market for products containing nanomaterials are not developed.
The aim of this work was to develop methodological approaches for screening safety assessment of nanomaterials on cell cultures.
The objects of research were nanoscale particles of different chemical nature (carbon nanotubes, nanoparticles based on metals and metal salts, etc.), both in suspension and fixed on the carrier.
The main damaging properties of nanomaterials are based on their ability to penetrate and accumulate inside the cell, disrupting the functioning of the internal systems, causing oxidative stress, as well as, when penetrating the cell nucleus, the ability to induce mutations. Thus, for preliminary screening testing of the safety of nanomaterials, it is most appropriate to use a sequential testing scheme based on alternative methods using cell cultures of different origin. On the basis of the main routes of entry of nano-sized particles into the body, cell cultures of similar specifications (A549, CaCo2, skin-muscle embryonic fibroblasts) were selected for research. The developed testing scheme includes a number of methods for determining the general toxic effect, mutagenicity, the ability to assess cell membrane damage and the method of assessing cell membrane damage. method for studying the induction of reactive oxygen species using fluorescein diacetate staining, cytogenetic analysis under a microscope and cytofluorimetric methods).
The study allowed us to develop a specific algorithm for screening assessment of nanomaterials. But the issues of proper sample preparation, as well as a comparative assessment of the number of tested nanoparticles, remained unresolved. We cannot adequately compare the concentrations of large molecular particles (for example, carbon nanotubes, which practically do not penetrate cell membranes, including due to the formation of agglomerates) and low molecular size (for example, silver nanoparticles). Thus, the question of the quantitative determination of nanoparticles for hygienic rationing nanoparticles in products and in the air of the working zone is still open.
Keywords: nanomaterials safety, cytotoxicity, mutagenicity, alternative testing nanomaterials
Skin irritation potential of graphene based materials (#720)
M. Pelin1, M. Garrido2, C. Martín3, S. Sosa1, L. Fusco2, E. Vázquez3, M. Prato2, A. Tubaro1
1 University of Trieste, Dept. Life Sciences, Trieste, Italy
Graphene based materials (GBMs) are innovative 2D nanomaterials obtained by graphite exfoliation. Their unique physicochemical properties stay at the basis of the multiple potential GBMs applications, ranging from electronics to biomedicine. However, little is known about their negative impact on human health, especially after skin contact, which represents one of the major exposure routes to GBMs for humans, especially in occupational settings.
Hence, this study was aimed at investigating skin irritation properties of a panel of GBMs: a few layer graphene (FLG), exfoliated by ball milling of graphite, a FLG exfoliated using sodium dodecyl sulfate (FLG-SDS) or sodium dodecylbenzenesulfonate (FLG-SDBS), a CVD-graphene monolayer disk, obtained by chemical vapor deposition, a graphene oxide (GO) and a reduced GO (rGO). In compliance with the 3Rs principle, skin irritation was assessed using the SkinEthic™ Reconstructed human Epidermis (RhE) model, following the skin Irritation Test-42bis (42 min exposure to GBMs followed by 42 h post-exposure without the materials), a test compliant with the Organization for Economic Co-operation and Development (OECD) Test Guideline (TG) 439.
In general, none of the materials reduced RhE viability at levels lower than those predicting skin irritation (≤50%), suggesting no skin irritant properties for the tested GBMs. This result, obtained following the OECD TG 439, was further confirmed by the absence of cytokines (IL-1α, IL-6 and IL-8) release by GBMs-treated RhE and by no observed histological alterations.
On the whole, these results demonstrate, for the first time, that GBMs do not seem to induce skin irritation after a single acute exposure.
This study was supported by the European Commission funded Graphene Flagship (grant agreement no. 696656, and 785219).
Keywords: graphene based materials, skin irritation, OECD, reconstructed human epidermis, nanotoxicology
Assessment of potential toxicity of new synthesized nanofibers in pulmonary cells in vitro (#743)
J. Bacova1, P. Majtnerova1, L. Hromadko2, J. M. Macak2, T. Rousar1
1 University of Pardubice, Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, Pardubice, Czech Republic
Introduction: The development and production of new nanomaterials have been increasing in recent years. Various technologies enable the synthesis of unique nanomaterials, including nanofibers. The highest risk of toxicity effect of nanomaterials is emerging in workplaces and environment. Respiratory tract is the primary and the most frequent possibility of entering nanomaterials into the human body, therefore it is important to test the pulmonary toxicity.
Aim: Nanofibers are among the nanomaterials whose cytotoxicity is generally not so much tested comparing with other nanomaterials. Our aim was to determine and to characterize the potential pulmonary toxicity of several different inorganic nanofibers, produced by electrospinning and centrifugal spinning, compared to commercially available nanoparticles using human lung cell line in vitro.
Methods: We tested newly synthesized nanofibers to compare their biological effects with commercial TiO2 nanoparticles. The human lung carcinoma epithelial cells (= A549 cell line) were seeded into well plates. After seeding, the cells were exposed to solutions of nanofibers or nanoparticles at concentrations of 0-100 μg.ml–1 for up to 24 h. Cell damage after treatment was assessed by cell viability tests and microscopic analysis.
Results and conclusion: The results showed that TiO2 nanoparticles did not induce any decrease in cell viability after 1 and 4 h. After 24 h, 100 μg.ml‑1 TiO2 nanoparticles showed significant reduction of cell viability by 10% in comparison with control cells. On the other hand, after 1, 4 and 24 h, any changes in cell viability were not found in A549 cells treated with nanofibers in comparison with control cells. Our results were also supported by microscopic analysis showing no visible cell damage after incubation with nanofibers. In general, commercial TiO2 nanoparticles are more toxic than tested nanofibers of similar composition.
Acknowledgement: This study was supported by OP RDE project with acronym NANOBIO, reg. n. CZ.02.1.01/0.0/0.0/17_048/0007421.
Keywords: A549, nanofibers, nanoparticles, pulmonary toxicity.
Evaluation of the impact of TiO2 and SiO2 nanofibers on the neuronal cells. (#748)
J. Handl1, J. Capek1, J. Bacova1, L. Hromadko2, J. M. Macak2, T. Rousar1
1 University of Pardubice, Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, Pardubice, Czech Republic
Introduction: Nanomaterials, such as nanoparticles or nanofibers, have a large potential for use in a number of industry sectors. A number of nanomaterials have been used and new types of structures with improved properties are being produced. In particular, inorganic nanofibers produced by electrospinning possess interesting prospects (porous structure, high surface area and breathability, tunable surface reactivity, dimensions). However, their impact on biological systems has not been well characterized yet. Generally, nanomaterials can have potential hepatotoxic and nephrotoxic, but also neurotoxic effects. The consequences of the presence of nanomaterials in the body are not fully understood. Therefore it is necessary to conduct neurotoxicity studies of these materials.
Aim: In vitro testing of inorganic electrospun nanofibers, comparing their effect with nanoparticles in neuronal cells.
Methods: The effect of inorganic nanofibers (TiO2 and SiO2) was tested comparing with TiO2 P25 nanoparticles in human neuroblastoma SH-SY5Y cell line. The SH-SY5Y cells were seeded into 96-well plates at density of 2.5×104 cells/well and incubated in Dulbecco’s Modified Eagle’s Medium/Nutrient Mixture F-12 containing tested nanomaterials at concentrations up to 100 µg/ml. The cells were incubated up to 24 hours. The cell viability was tested and evaluated by the WST-1 test spectrophotometrically.
Results and Conclusion: We found that none of the tested nanofibers caused a decrease in cell viability after 1 to 6 hours incubation period. After 24 hours of incubation, we found that TiO2 P25 nanoparticles caused a decrease in cell viability below 80% at concentration of 100 µg/ml. Nanofibers, on the other hand, did not induce changes in cell viability. The results on no significant cell damage were also supported by fluorescence microscopic analysis.
Acknowledgement: This work was supported by OP RDE project „Strengthening interdisciplinary cooperation in research of nanomaterials and their effects on living organisms“, reg. n. CZ.02.1.01/0.0/0.0/17_048/0007421.
Keywords: Neurotoxicity, Nanofibers, SH-SY5Y cell line
Multiparametric platform for safety testing of nanoparticles based on a 3D liver tissue model (#764)
J. Fleddermann1, J. Susewind2, S. Kiefer1, A. Kraegeloh1
1 INM - Leibniz Institute for New Materials, Nano Cell Interactions, Saarbrücken, Saarland, Germany
Synthetic nanoparticles (NPs) offer numerous applications in technical fields and biomedicine. Evaluating their potential hazards at an early stage will provide standards for production processes and preclinical development of NPs. To assess the hazards emanating from NPs we developed a multiparametric platform based on a 3D liver tissue model. The liver plays an important role in the safety assessment of nanoparticles, as it decomposes many drugs and metabolites as well as is a relevant target organ of NPs after entering in the blood circulation. The 3D liver tissue model features two key advantages; 1) It allows the combined measurement of classical, functional, and morphology markers. 2) In contrast to commonly used 2D cell culture, nanoparticle penetration into the tissue can be studied. Knowledge of NP distribution in liver tissue is of relevance for understanding NP induced mechanisms in nanosafety as well as for their delivery efficiency in biomedical applications.
The test platform was established addressing different aspects: 1) characterization of spheroids, 2) NP induced cytotoxicity, 3) hepatocyte function, and 4) NP penetration. Liver microtissues were prepared by 3D cell culture of human hepatocarcinoma HepG2 cells using the hanging-drop method. Microscopy analysis revealed 400 µm sized spheroids with uniform cell arrangement and without holes. The cells expressed a liver specific bile canaliculi related protein (MRP-2). NPs with relevance for biomedical and cosmetic applications owning different element composition (SiO2, TiO2, Ag) were tested. Cytotoxicity of the NPs was studied addressing cell viability and oxidative stress. Only Ag NPs caused decrease in cell viability. As a functional marker the gene expression of an important liver-specific CYP P450 enzyme for metabolism of xenobiotics (Cyp1A2) was analyzed. In presence of all tested NPs the basal Cyp1A2 gene expression was not affected. In contrast menadione-induced Cyp1A2 gene expression was altered in presence of TiO2 and Ag NPs. A combination of modern imaging techniques allowed us to locate the NPs inside the spheroids and to study NP penetration into tissue. NP penetration was shown to be limited to about 20µm from the spheroid border.
The combination of various markers with 3D cell culture technology, microscopy and PCR-based analysis of the gene expression pattern of cells in an integrated test platform will provide an important contribution to NP risk assessment.
Keywords: nanosafety, 3D spheroids, cytotoxicity, penetration
The Ecotoxicity of Zinc Oxide Nanoparticles in Sunscreen FormulationsThe Ecotoxicity of Zinc Oxide Nanoparticles in Sunscreen Formulations (#805)
Q. Chang1, C. Fu2, J. Duan2, M. Sun1, Z. Xie1, M. Yang2, M. Wu2, X. Deng1
1 Shanghai University, Institute of Nanochemistry and Nanobiology, Shanghai, China
As zinc oxide nanoparticles (ZnO NPs) are effective sun blocking agents and increasingly being used in sunscreen products, however, they will ultimately be released into the environment, and therefore it is very important to investigate the biological effects on aquatic organism. In order to study the toxicity of ZnO nanoparticles in sunscreen to zebrafish embryos, five different groups were set up: (1) sunscreen containing ZnO nanoparticles in (SN-groups); (2) pure ZnO nanoparticles (PN-groups); (3) sunscreen containing ZnCl2 (SI-groups); (4) sunscreen containing all accessories but no ZnO (SA-group); and (5) Zebrafish culture medium (Control). The Zn concentrations in SN-, PN-, SI-groups were 0.1, 1, 10, 20, 50 mg/L. We assessed the acute toxicity and oxidative stress to zebrafish embryos. For the acute toxicity, the suspensions and solutions in each group did not significantly affect the toxicological endpoints in low concentrations groups (0.1, 1 mg/L). In the 10 mg/L concentration groups, SI-10 group had the strongest interference to zebrafish embryos, but in the 20 and 50 mg/L concentrations groups, PN-groups had the strongest interference with zebrafish embryos, followed by SN-groups, and finally SI-groups. Since the sedimentation of ZnO NPs has been increased in a time- and concentration-dependent manner, and the sedimentation of PN-groups was higher than that of SN-groups after exposure for 12 h in the 20 and 50 mg/L concentrations groups, sedimentation is a very important factor for the acute toxicity of ZnO NPs to zebrafish embryos. On the one hand, the LC50 of SN-, PN-, SI-group at 120 hours post fertilization (hpf) were 22.9, 14.898 and 34.73 mg/L, respectively, the zinc ions released by the SN- and PN-groups were less than 10 mg/L. On the other hand, the activity of CAT was significantly reduced in PN-10 group, which should be caused by oxidative stress. So zinc ions released did not play a major role in the ecological toxicity of ZnO nanoparticles to zebrafish embryos. The sunscreen accessories (SA) could alleviate the ZnO nanoparticles toxicity to zebrafish embryos by inhibiting the dissolution of zinc ions and reducing the deposition of ZnO nanoparticles, which provides a good idea to relieve the side effects of ZnO nanomaterials to the aquatic environment. This work was supported by the National Natural Science Foundation of China (21371118, 41573116, 21611130174).
Keywords: ZnO nanoparticles, Zebrafish embryos, Sunscreen, Toxicity
In vitro cytotoxicity and genotoxicity evaluation of five different nanoforms of manganese iron oxide (#859)
R. B. Azevedo1, F. Penteado1, M. L. Fascineli1, S. Suhonen2, P. Cácerez-Vélez1, K. Aimonen2, H. Norppa2, J. Catalán2, 3
1 University of Brasília, Institute of Biology-Department of Genetic and Morphology, Brasília, Brazil
Manganese iron oxide nanoparticles (MnFe2O4 NPs) are extensively used nanomaterials due to their unique magnetic and electric characteristics, such as superparamagnetism. However, their potential benefits may be accompanied by human health hazards and risks during exposure. In this study, five nanoforms of MnFe2O4 (primary particle diameter 5 nm) synthesized by two different methods, coprecipitation (Cp) or thermal decomposition (Td) and coated with citrate (Cit) or meso-2,3-dimercaptossuccinic acid (Dmsa) were toxicologically assessed in human bronchial epithelial BEAS 2B cells. Bare Cp-synthesized NPs were also analysed. Cytotoxicity was measured by luminometric detection of ATP at 6, 24 and 48 hours. DNA damage was assessed by single cell gel electrophoresis (comet) assay, and chromosome damage was assessed by the cytokinesis-block micronucleus (MN) assay. Based on the cytotoxicity results, BEAS 2B cells were exposed to 25.6- 256 µg.mL of MnFe2O4 NPs for 6 and 24 h in the comet assay, and to 25.6- 256 µg.mL for 48 h in the MN assay. All NPs were uptaken by BEAS 2B cells within 6 hours of treatment, as verified by transmission eletronic microscopy. Dynamic light scattering (DLS)-based characterization of NPs in the ultrapure water showed slightly bigger hydrodynamic size (Hs) for NPs synthesised by thermal decomposition (110±4nm for MnFe2O4-TdCit, 170±5nm for MnFe2O4-TdDmsa) than by coprecipitation (101±7nm for MnFe2O4-Cp, 74±3 nm for MnFe2O4-CpCit, and 102±4 nm for MnFe2O4-CpDmsa). None of the MnFe2O4 NPs did cause any statistically or toxicologically significant cytotoxic or genotoxic effect (p>0.05) in the concentration range up to 153.6 µg.mL- and up to 48 hours of exposure, when analyzed by ANOVA - 1 Way. This is the first in vitro comparative research to address the genotoxicity of manganese iron oxide nanoparticles, with the merit of a comparative factorial analysis, which pointed out that cytotoxicity is dictated by concentration, time and coating; in the other hand, regarding the genotoxicity end points, the time of exposure was a statistically and biologically significant factor for the damage to the deoxyribonucleic acid caused by both citrate-coated nanoparticles, independently of the route synthesis, when analyzed by ANOVA – 2 and 4 Way.
(Funded by EU FP7 People-Pirses-BRASINOEU, Grant Agreement 319816); INCT Nanobiotechnology (CNPq-Brazil, 573880/2008-5).
Keywords: In vitro genotoxicity, Cytotoxicity, Manganese iron oxide nanoparticles., coating.
Toxicological evaluation of textiles coated with antibacterial metal oxide nanoparticles by 2D and 3D in vitro skin model (#864)
R. D. Bengalli1, A. Colantuoni1, P. Mantecca1, L. Fiandra1
1 University of Milan-Bicocca, Earth and Environmental Sciences, Milano, Italy
Metal oxide (MeO) nanoparticles (NPs), such as copper (CuO) and zinc (ZnO) oxides, thanks to their antibacterial properties, are among the most eligible nanomaterials for the coating of textiles. Nevertheless, there is a general concern about their safety for human health. Within the EU funded H2020 project “PROTECT”, the skin toxicity of textiles coated with antibacterial CuO and ZnO nanoparticles was assessed by different in vitro approaches.
CuO and ZnO NPs were produced by sonochemical process and characterized by TEM and DLS. Textiles, coated with ZnO and CuO NPs, were subjected to extraction procedure (ISO 10993-12), including 72 hours incubation in artificial sweat solution (AS, pH 4.7 and 6.3) at 37°C. The stability of the NPs at the different AS pHs, so as the release of NPs and/or ions from the textiles after the extraction procedure in AS, was evaluated through CPS and ICP-OES techniques. For the Skin Corrosion test, Epiderm™ 3D in vitro skin models (Mattek) were exposed to different concentration of CuO and ZnO in water, according to the OECD TG.431 protocol, while for the Skin Irritation test, the 3D models were exposed to the textile extracts, according to ISO/TC 194/WG 8 for Medical Devices. Balb/3T3 fibroblasts were also used to understand the mechanisms of action of the cytotoxicity trigged by CuO and ZnO NPs. Cell viability, inflammatory mediators release, morphological changes and wound healing process (scratch assay) were investigated.
Data from the Corrosion test showed that CuO and ZnO NPs resulted non-corrosive up to 1000 ppm in water, according to the Globally Harmonized System (GHS) adopted by the OECD. For the Skin Irritation test, a significant reduction of tissue cells viability was induced by both metal oxide NPs extracted from textiles in AS pH 4.7, likely due to the high content of free Cu and Zn ions released in these conditions and detected by ICP-OES. At higher pH, the effects were observed at lower extent, due to the less solubility of NPs at these experimental conditions. Experiments on fibroblasts showed that ZnO NPs strongly affected cell viability and IL-8 release starting from the dose of 20 ppm. Moreover, the release of IL-8 resulted dose-dependent after the exposure to CuO NPs. Microscopy analyses showed that MeO NPs changed fibroblasts morphology and their ability to migrate during the wound healing process. All together, these data highlight that coated textiles seem to be safe on intact skin models, unless NPs dissolution in acid AS occurs. Nevertheless, further experiments are needed in order to understand the MeO NPs toxicity in case of wounded skin, as suggested by data on fibroblasts. In conclusion, NPs physical and chemical properties and appropriate in vitro tools are determinant parameters in order to assess NPs safety.
Acknowledgements: This work was supported by EU funded H2020-720851 PROTECT project.
Keywords: metal oxide nanoparticles, in vitro skin models, cytotoxicity, OECD tests
Neuro-inflammation after inhalation exposure to aerosol mixtures of alumina nanoparticles / hydrogen chloride gas in rats (#874)
S. Dekali1, D. Saurat1, A. Boyard1, S. De Araujo1, C. Frédéric1, A. Bourgois1, S. François1
1 French Armed Forces Biomedical Research Institute (IRBA), Emerging technological riks research unit, Brétigny-sur-Orge, France
Alumina nanoparticles (Al2O3NPs) have a wide range of applications in several industrial fields such as electronics or energy. These NPs can also be found as pollutants of interest in mixtures containing high amount of hydrogen chloride (HCl) gas after burning of solid composite propellants used for missile or rocket in defence or aerospace fields. The main route of exposure to these mixtures is inhalation but neurotoxicity could occur in addition to pulmonary effects. These co-exposures may represent a health risk for workers. Due to a lack of data related to potential neurotoxic effects, the aim of this study was to investigate brain toxicity.
In order to study cytotoxic effects using several pollutants concentrations, we performed first series of experiments using mouse microvascular endothelial cells (bEnd.3). Cells were exposed to Al2O3particles (primary sizes 13 and 500 nm), HCl or Al2O3particles / HCl mixtures during 24h. Treatment with HCl induced concentration-dependant decrease of cell viability, cell index and depletion of reduced glutathione. Incubations with Al2O3NPs (13 and 500 nm) mixed or not with HCl markedly decreased cell index, but only 500 nm Al2O3NPs ±HCl induced depletion of reduced glutathione.
To assess neurotoxicity, Wistar rats were exposed during 4 hours by nose-only inhalation to filtered air (controls), Al2O3NPs (primary sizes 13 and 500 nm), HCl gas (5 ppm) or Al2O3NPs / HCl gas mixtures. 24 hours post-exposure, aluminum biodistribution was quantified by Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) and different analytes were quantified in brain homogenates using ELISA (VEGF, IL-1β, IP-10, TNF-α, Fractalkine, IFN-γ, RANTES). While aluminum was undetectable in brains, significant increases of IL-1βand IP-10 were quantified after exposure to 500 nm Al2O3particles±HCl.
These first results show that only exposure to 500 nm Al2O3particles±HCl, but not to 13 nm Al2O3NPs, could induce indirect neuroinflammation after inhalation. Additional research is needed to identify mediators inducing this inflammation, but in vitro results suggest that a mechanism of oxidative stress generated on microvascular endothelial cells of the blood-brain barrier is probably involved.
Keywords: neurotoxicity, Inhalation, inflammation, alumina nanoparticles, hydrogen chloride gas
In vitro effects of ZnO and CuO NPs in mixture with DEP: different nano-bio-interactions affect viability and colony forming efficiency of A549 cells. (#928)
A. Zerboni1, T. Catelani2, P. Mantecca1
1 University of Milano-Bicocca, Department of Earth and Environmental Sciences, Milano, Italy
Most of the atmospheric ultrafine particles (UFPs) in urban areas derive from combustion sources, especially diesel exhaust particles (DEP), but also from non-exhaust sources or from the unintentional release of engineered nanoparticles (NPs) during production and use. Since the environmental exposure to DEP and NPs occur simultaneously, it is necessary to consider their possible interactive effects in biological system. Commercially available (cZnO, cCuO < 50 nm) from Sigma-Aldrich) and sonochemically synthesized ZnO and CuO NPs (sZnO, sCuO) from Bar-Ilan University, were used alone or in combination with standard DEP (NIST 2975) to expose A549 cells.
After 24-72h exposure to increasing metal oxide NPs concentrations (10-20 μg/ml), with and without DEP at 100 μg/ml, MTT test and Colony Forming Efficiency Assay (CFE) were performed to assess the cytotoxicity. The NP mixtures were characterized by DLS and TEM, while the NP dissolution in cell medium was measured by ICP-OES. In parallel to the cytotoxicity studies, morphological analyses on NP-cell interactions were performed by light and transmission electron microscopy.
The results suggest that the presence of DEP introduced new physico-chemical interactions able to increase the cytotoxicity of cZnO, but to decrease that of sZnO. For CuO NPs, the presence of DEP significantly reduced the cytotoxicity of cCuO and only slightly that of sCuO. This is probably due to different interferences with the metal oxide NP surface and/or to the modulation of ions release.
The results from CFE were coherent with those from MTT. On the basis of the morphology and cell density, four well distinguishable colony types were identified. Cytostatic effects and changes in colony morphology were observed especially after exposure to CuO and DEP+CuO NPs.
TEM analyses revealed that both ZnO and CuO NPs, as well as their mixture with DEP, were abundantly internalized in A549 cells, especially in the endo-lysosomal compartments and multilamellar bodies. We are performing additional investigations to discriminate the modality of nano-bio-interactions of CuO and ZnO in presence of DEP and to analyse cell-cell adhesion molecules and epithelial-to-mesenchymal transition mechanisms.
Acknowledgements: EU Horizon 2020 project PROTECT (grant agreement No 720851)
Keywords: mixture toxicity, zinc oxide, copper oxide, Diesel exhaust particles, lung cells
Oxidative stress in microbes after exposure to iron nanoparticles: analysis of aldehydes as oxidative damage products of lipids and proteins (#942)
T. Cajthaml1, 2, J. Semerád1, 2, J. Filip3
1 Institute of Microbiology of the Czech Academy of Sciences, Prague 4, Czech Republic
Nanoremediation represents a new branch of remediation that employs various nanomaterials to decontaminate polluted matrices of the environment. Despite the large spectrum nanomaterials used, nanoscale zero-valent iron (nZVI) and its related materials represent the most commonly used agents in environmental remediation practice. Application of nZVI and full-scale cleanup operations have been successfully demonstrated in the decontamination of various organic and inorganic pollutants. However, there is still a lack of suitable and standardized tests enabling estimation of ecotoxicity of these new nanomaterials and especially during the remediation attempt. It is of noteworthy, that classical ecotoxicity tests are not useable due to special features of the nanomaterials or interaction with the assays. One of the most important mechanisms of nanoparticle toxicity is oxidative stress. Oxidative stress is established when an imbalance of reactive oxygen species (ROS) occurs. The presence of elevated ROS concentrations or their insufficient catabolism can cause interaction with biomolecules including proteins, carbohydrates, lipids, and nucleic acids, leading to the formation of toxic and mutagenic products.
This research was funded by the Competence Center TE01020218 of the Technology Agency of the Czech Republic.
Keywords: nanoscale zero-valent iron, oxidative stress, lipid peroxidation, aldehydes, malondialdehyde
Antimicrobial Properties and Cytotoxicity of Polymeric Nanocomposites in TK6 lymphoblastoid cells (#970)
S. Ramzan1, A. Alshareef1, P. Parmar1, D. North1, S. Nadurata1, R. Mudhiar1, N. Singh1, Z. Ahmad1
1 De Montfort, Leicester, United Kingdom
Metallic nanocomposites have potential in a variety of applications such as, antibacterial formulations to improve the efficacy of antimicrobial agents, specific cell targeted drug delivery and for use as an anti-cancer agent. Although, the potential benefits of metallic nanocomposites are considerable, there is a distinct need to identify any potential cellular damage associated with these nanocomposites. The aim of this study was to characterize metallic nanocomposites, determine the release of drug from synthesised formulations, and assess antimicrobial effects and their potential in vitro cytotoxic effects.
TK6 lymphoblastoid-B cells were exposed for 24 hours to five different metallic composites made up of a polymer Poly (D,L-lactide-co-glycolide) used at 5%w/v, a drug amoxicillin 5% w/w, metallic nanoparticles used at 5% w/w including, silver of differing shapes (hexagonal, spherical and nanowires) gold and copper nanoparticles. The fully synthesised formulations were used at various concentrations (0µg/ml-10µg/ml). Physico-chemical characterization on all composites was performed. Drug release of the antibiotic amoxicillin was performed to assess the release of drug based on differences in shape and type of metal used. Antimicrobial efficacy was determined using the disk diffusion method on two bacteria, E.coli and S.aureus. MTT viability assay was used to determine cytotoxicity.
The physiochemical characterisation showed the metallic nanoparticles to be within 1-250nm and the metallic nanocomposites to be between 600nm-1.5μm. Drug release studies showed differential results based on the shape of particles, with the silver hexagonal formulation having the maximum %age release. Antimicrobial studies showed that the hexagonal formulation exhibited the strongest antimicrobial effect. The cytotoxicity showed that the copper composites were the most cytotoxic followed by spherical and hexagonal polymeric nanocomposites with gold being the least toxic.
Interestingly, there was a correlation between release of drug from the nanocomposites and the antimicrobial responses. As metal based composites find their use in as antibacterial
agents in humans, these findings are important in guiding the fabrication and biocompatibility of metallic composites.
Keywords: MTT assay, cytotoxicity testing, drug release, antibacterial agent, metallic nanocomposites
Improved aerosol generation method and newly designed whole body rodent inhalation apparatus for the testing of nanomaterials (#973)
J. Kanno1, 2, 3, Y. Taquahashi2, A. Hirose3
1 Japan Bioassay Research Center, Japan Organization of Occupational Health and Safety, Hadano, Kanagawa, Japan
Inhalation study is a gold standard for the assessment of respiratory toxicity of the nanomaterials (NM). To generate well-dispersed aerosol, we developed the “Taquann” dispersion method and a “direct injection” whole body inhalation system for the dispersed sample (designated as “Taquann System” J Toxicol Sci. 2013). The Mitsui MWNT-7 was used to evaluate the performance. Taquann method removes the aggregate/agglomerates effectively and enrich the well-dispersed single fibers without changing the length and width distribution. The method is based on two concepts; liquid-phase fine filtration and critical point drying to avoid re-aggregation by surface tension. The bulk sample of MWNT-7 was suspended and dispersed in tert-butyl alcohol (TBA), filtered by 25 micrometer mesh to remove aggregates/agglomerates, snap-frozen by liquid nitrogen, and vacuumed, mimicking the process of critical point drying (a method used for scanning electron microscope sample preparation). Aliquots of dry dispersed MWCNT were loaded in tube-shaped original cartridges, and the dispersed sample in the cartridges are periodically (every few minutes in most cases) injected to a newly designed inhalation chamber system by the compressed air to maintain a certain range of aerosol concentration. The length distribution of the MWNT recovered from the lungs of the exposed mice was similar to the fibers in original sample and aerosol in the chamber. Now the system is shown to work for Nikkiso MWCNT, three different makes of nano TiOs, potassium titanate whiskers, nano-sized agglomerates of double wall CNT and carbohydrate polymer. The advantages of the Taquann System include relatively cheap small scale equipment compared to traditional whole body inhalation chamber systems, low running cost, high flexibility for various types of NM samples, low chance of scattering of sample due to closed procedures after making TBA suspension, and low loss rate of sample. To date, we have developed four models of inhalation exposure systems (ver.1.0, 2.0, 2.5 and 3.0). The latest model of the Taquann system is now equipped with computer-controlled automatic cartridge loader/injector that can keep the aerosol concentration for up to 6 hours. The capacity of the chamber is for 25 mice or 12 rats per chamber. With respect to MWNT-7, 80 % of samples loaded into a cartridge were aerosolized and mass concentration was reached up to 6 mg per cubic meters. These improvements made long-term studies possible for testing chronic effects of NMs. (Supported by Grants from MHLW, Japan.)
Keywords: Whole Body Inhalation, Dispersion method, nanomaterial
Identifying cobalt neurotoxicity targets in vivo through RNA-Sequencing (#19)
S. Gómez-Arnaiz1, R. Tate2, S. Laovitthayanggoon1, C. Henderson1, M. H. Grant1
1 University of Strathclyde, Department of Biomedical Engineering, Glasgow, United Kingdom
Implantation of Metal-on-Metal (MoM) hip implants is a common orthopaedic procedure, with an estimated one million patients worldwide. These prostheses have come under recent scrutiny with reported long-term systemic toxicities in multiple organ systems. Cobalt (Co) is suspected of causing this systemic poisoning as a consequence of the progressive wear of Co alloy MoM implants eluting metallic particles and ions into the bloodstream. However, the pathological consequences of Co accumulation, particularly in the brain, are difficult to interpret and its mechanisms of toxicity in neural cells remain obscure. Here we investigate Co neurotoxicity using an in vivo rat model.
Cobalt content in neural tissue was measured by inductively coupled plasma mass spectrometry (ICP-MS) and RNA-Seq data were obtained from the prefrontal cortex and cerebellum of Sprague Dawley rats dosed for 28 days with daily i.p. injections of 1 mg/kg Body Weight of CoCl2 (treatment groups) or dH2O (controls).
After treatment, Co content was significantly increased in the prefrontal cortex, cerebellum, and hippocampus (p<0.01), and Co levels in blood (27.14±2.70μg/l) were within those of MoM patients. The number of Differentially Expressed Genes (DEGs) (p<0.05) in the prefrontal cortex (3564 up-regulated, 2694 down) and cerebellum (2037 up, 1568 down) indicates a transcriptional response to Co accumulation from the clinically relevant doses of Co used. Transcript molecular classification showed a common metal homeostasis dysregulation in prefrontal cortex and cerebellum, since DEGs from the most populated functional group transcribe for metal ion binding proteins for zinc, calcium, and magnesium. Ion channels and transporters that handle calcium were also dysregulated. Finally, the gene expression of zinc binding phosphodiesterase and carbonic anhydrase families was significantly altered, suggesting that Co may modulate cyclic nucleotide signalling and pH balance, respectively.
Our research reveals that cobalt has neurotoxic effects via accumulation in neural tissue and interference with the transcriptional regulation of different metal ion binding systems. These findings may point towards novel therapeutic targets for patients with cobalt poisoning.
Keywords: Cobalt (Co) neurotoxicity, metal-on-metal (MoM) hip prosthesis, arthroprosthetic cobaltism, RNA-sequencing (RNA-seq), metal dyshomeostasis
Exposure to flame retardant tris (2-butoxyethyl) phosphate induces memory deficit and neuroinflammatory responses in a mouse model of allergic asthma (#54)
T. - T. Win-Shwe1, R. Yanagisawa1, E. Koike1, H. Takano2
1 National Inst for Envir Studies, Tsukuba, Japan
Tris (2-butoxyethyl) phosphate (TBEP) is a phosphate ester and used as a flame retardant in various household appliances. However, its potential health effects including neurotoxicity and immunotoxicity are not known clearly. In this study, we aimed to examine the effects of dietary exposure of TBEP on novel object recognition ability and inflammatory markers in the brain of a mouse model of allergic asthma. Tolerable daily intake (TDI) of TBEP 2 μg/ kg/day was set as high dose and 1/10, 1/100 from high dose were set as medium and low doses. Five-week-old male C3H/HeJ mice were fed a chow diet containing three doses of TBEP (1.67, 16.7 or 167 μg/kg/day) and ovalbumin (OVA) was given intratracheally every other week from 5 to 11-week-old. At 11 weeks of age, a novel object recognition test was conducted 2 hours after final instillation of OVA. Then the hippocampi were collected to detect neurological and immunological biomarkers using real-time RT-PC method. Mast cell was examined by toluidine blue staining and immunohistochemical staining methods. In addition, microglia activation was investigated by ionized calcium-binding adapter molecule (Iba)-1 immunoreactivity using immunohistochemical analysis. Regarding novel object recognition test, no significant changes of discrimination ability between novel and familiar objects were observed in the control and low or medium-dose TBEP-exposed allergic asthmatic mice. However, impaired discrimination ability was observed in high-dose TBEP-exposed allergic asthmatic mice compared with the control. The mRNA expression levels of memory function-related genes such as the N-methyl-D aspartate (NMDA) receptor subunits NR1 and NR2B, and inflammatory markers interleukin (IL)-1 β, tumor necrosis factor (TNF)-α, oxidative stress marker heme oxygenase (HO)1 were significantly increased in the hippocampus of high-dose TBEP-exposed allergic asthmatic mice. Moreover, mast cells and microglia activation were remarkable in high-dose TBEP-exposed allergic asthmatic mice. Our results indicate the possibility that childhood to young adulthood exposure to a phosphate flame retardant TBEP impaired memory function accompanied with alteration of memory function-related genes and inflammatory markers expression in the hippocampus of allergic asthmatic mice.
Keywords: Tris (2-butoxyethyl) phosphate (TBEP), novel object recognition test, inflammatory biomarkers, hippocampus, allergic asthmatic mouse
Brain-derived Neurotrophic Factor Protects against Acrylamide-induced Neuronal and Synaptic Injury via the TrkB-MAPK-Erk1/2 Pathway (#88)
X. Chen1, J. Xiao1, P. Cao1, Z. Li1, Y. Zhang1, W. Cai1, W. Gao2, B. Li1
1 National Institute of Occupational Health and Poison Control, Chinese Center for Disease Control and Prevention, Beijing, China
In this study, we explored the neuroprotective effects of brain-derived neurotrophic factor (BDNF) in the human neuroblastoma cell line (NB-1 cells) after exposure to a potent neurotoxin, acrylamide (ACR). NB-1 cells, NB-1 cells co-cultured with Schwann cells (SCs), and a negative control group were exposed to increasing concentrations of ACR. Cytotoxicity and cell viability were determined by a -(4, 5-dimethylthiazol-2-yl)-3,5-diphenyl tetrazolium bromide assay. Protein and partial mRNA expression of BDNF, tropomyosin receptor kinase B (TrkB), mitogen-activated protein kinase-extracellular signal-regulated kinases (MAPK-Erk), and Synapsin I were tested by western blotting and reverse transcription polymerase chain reaction. Expression changes after the application of exogenous BDNF to NB-1 cells were also examined. To determine the mechanisms underlying neuronal damage repair, TrkB was blocked with the K252a inhibitor. ACR decreased cell viability in a dose- and time-dependent manner and damaged synapses, as evidenced by a decrease in Synapsin I expression. After ACR exposure, neurons initiated a self-protection mechanism, in which the levels of Synapsin I and BDNF were increased. This mechanism could be strengthened by downstream activation of the TrkB/MAPK/Erk1/2 pathway in the co-culture condition. The application of exogenous BDNF led to increased TrkB, MAPK-Erk, and Synapsin I levels. Thus, we have demonstrated that ACR is a neurotoxin, SCs may play a protective role via the BDNF-TrkB-MAPK-Erk1/2 signaling pathway, and exogenous BDNF can exert neuroprotective effects that can surpass those of SCs. Therefore, BDNF could potentially reverse ACR-induced neuronal damage and could be useful in the prevention and treatment of other neurodegenerative diseases.
Keywords: Acrylamide; Synapsin I; Brain-derived neurotrophic factor; Tropomyosin receptor kinase B; Mitogen-activated protein kinase-extracellular signal-regulated kinases 1/2
Gene expression changes induced by Type II piretroids exposure in human neuroblastoma SH-SY5Y cells (#102)
A. Anadón1, I. Ares1, J. L. Rodríguez1, M. Martínez1, B. Lopez-Torres1, M. R. Martínez-Larrañaga1, M. A. Martínez1
1 Universidad Complutense de Madrid, Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Madrid, Spain
Recent studies, using human neuroblastoma SH-SY5Y cells, indicate that the Type II pyrethroid insecticides alpha-cypermethrin and cyfluthrin affect the expression of genes involved in proinflammatory, apoptosis and oxidative stress pathways. In this study we have initiated a test of the hypothesis that the pyrethroid-induced alterations that could occur during neural development may be responsible for the behavior and cognitive impairment observed for these pyrethroid in adult life. To study the molecular changes taking place during embryogenesis and neurodevelopment as a consequence of pyrethroid exposure we used a neuronal cell line, SH-SY5Y cells. This in vitro model allows us to determine whether changes that take place in vivo during gestational pyrethroid-exposure occur also in pyrethroid-treated neurons in vitro. In the present study, mRNA levels of the following general neuronal markers were examined: (i) tubulin beta 3 (TUBB3) and growth-associated protein-43 (GAP43) both implicated in neurite and axon growth; (ii) neurofilament triplet H protein (NEFH) involved in cytoarchitecture organization; (iii) growth-associated protein 43 (GAP43), which is expressed at high levels during development and stressed by nerve injury adult motor-neurons; (iv) calcium/calmodulin-dependent protein kinase II (CAMK2), alpha (CAMK2A), and beta (CAMK2B) isoforms, both essential for learning and memory formation. We used quantitative real-time RT-PCR to measure the expression of these neural markers. We found that TUBB3, GAP43, NEFH, CAMK2A and CAMK2B genes were upregulated by both pyrethroids. We assume that this counter-regulation may serve as an endogenous protective function. These data suggest that pyrethroid exposure during early life may produce irreversible neuronal dysfunction and reorganization that last into adult life. These data may contribute to clarify the role of early exposure to industrial chemicals such as pyrethroids in neurodegenerative diseases. This work was supported by Project (ALIBIRD-CM Program) Ref. S2013/ABI-2728 from Comunidad de Madrid, and Project Ref. RTA2015-00010-C03-03 from Ministerio de Economía, Industria y Competitividad, Spain.
Keywords: Pyrethroids, Neurotoxicity, Neurodevelopmental markers, SH-SY5Y cells
Neurotoxicity assessment of silver nanoparticle using human iPS cells (#107)
Y. Kanda1, S. Yamada1, 2
1 National Institute of Health Sciences (NIHS), Division of Pharmacology, Kawasaki, Japan
Developmental neurotoxicity (DNT) has been assessed using experimental animals. Due to complexity of human brain development and species differences, alternative in vitro testing using human cells, such as human iPS cells (iPSCs), has been expected in terms of cost, time-consuming and high throughput. Here we examine the effects of a well-known nanomaterial silver nanoparticles (AgNPs) using iPSCs. Despite their extensive use as anti-bacterial and anti-viral agents in broad consumer products, such as cosmetics and textiles, AgNPs have been concerned various types of cytotoxicity, including DNT. We have focused on neural differentiation process in iPSCs as a possible endpoint of DNT in vitro. Exposure to AgNPs reduced the expression of several marker genes, such as a neurogenesis marker OTX2 in the neural induction from iPSCs. Since neural differentiation requires ATP as a source of energy, we examined the intracellular ATP content. AgNPs decreased intracellular ATP levels in iPSCs. To understand the mechanisms by which AgNPs suppressed ATP production, we further examined the effects of AgNPs on mitochondrial dynamics. AgNPs induced mitochondrial fragmentation and reduced the level of mitochondrial fusion protein mitofusin 1 (Mfn1) in iPSCs. Moreover, knockdown of Mfn1 in iPSCs inhibited neural induction via OTX2 downregulation. Taken together, these data suggest that AgNPs induce cytotoxicity via Mfn1-mediated mitochondrial dysfunction in iPSCs. Thus, neural differentiation capability using iPSCs can be used for evaluation of compounds with developmental neurotoxicity.
Keywords: Neurotoxicity; iPS cell; Nanomaterial
Pin1 is inactivated by environmental pollutant cobalt and contributes to neurodegenerative damage (#137)
F. Zheng1, 2, 3, Y. Li1, 2, F. Zhang1, 2, C. Zheng1, 2, Z. Luo1, 2, P. Cai2, 3, 4, W. Shao1, 2, 3, Z. Guo2, 3, Z. Min5, S. Wu2, 3, 6, K. P. Lu5, 7, H. Li2, 3, 1
1 Fujian medical university, Department of Preventive Medicine, School of public health, Fuzhou, China
There is emerging evidence that the aberrant expression of prolyl cis-trans isomerase (Pin1) may underlie the pathogenesis of neurodegenerative diseases. Despite its clinical significance, the molecular mechanisms of Pin1 and age-related effects remain unclear in the environmental toxin-induced neurodegeneration.
Upon the various environmental toxicants tested, only cobalt caused a significant and dose-dependent decrease in Pin1 protein levels and increase in the inactive form of Pin1 levels in human brain glioma (H4) cells, demonstrated by Western blot and immunofluorescent microscopy. Accompanied with Pin1 inactivation, CoCl2 induced neural cell damages including cell-cycle arrest and upregulation of apoptosis rates dose-dependently examined by flow cytometry, Hoechst and Annexin V/PI staining and Western blot of HIF1α and CASPASE 9 protein levels. In addition, CoCl2 resulted in the upregulation of phosphorylated Tau (P-Tau) and disturbance of cis and trans P-Tau. Thus, we constructed Pin1 knockdown cell lines by lentivirus transfection, followed by exposure to CoCl2 for 24 and 36 h. CoCl2 exaggerated the loss of cell viability, hypoxia and neurodegenerative damages in Pin1 knockdown cells. The similar pattern of CoCl2 was found in Pin1 overexpression cell lines. In vitro study indicates that CoCl2 functions alongside with Pin1. To verify the above findings and include age-effect in vivo, 2-month-old and 12-month-old of C57BL/6J mice and the corresponding Pin1 knockout (KO) C57BL/6J mice were used. In accordance with in vitro studies, in wildtype mice, Pin1 was significantly downregulated by CoCl2 in the hippocampus and cortex. The content of Co2+ in blood, cerebral cortex and hippocampus of CoCl2-treated groups were significantly higher than that of control group, accompanied by metal ion disturbance. Neural damage was also found by immunohistochemical examinations, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labelling (TUNEL) assay and Western blots. Additionally, neurodegenerative indicators such as Tau, P-Tau, cis-/trans-P-Tau, APP and Aβ1-42 levels were significantly upregulated. As predicted, the loss of learning, memory and spatial exploration abilities were found in response to CoCl2. Those damages were more predominant in the 12-month-old mice and Pin1 KO mice, compared with 2-month-old mice and wildtype mice, individually. By comparing WT mice with Pin1 KO mice, CoCl2 exposure strengthened the severity of neurodegenerative damage related to Pin1 downregulation, which became even more severe when aging.
In conclusion, CoCl2 triggers neurodegenerative damages associated with Pin1 downregulation, age-dependently both in vivo and in vitro. Our findings indicate that cobalt could be one of the environmental toxicants that trigger neurodegenerative diseases associated with Pin1.
The work was supported by the Joint Funds for the innovation of science and Technology, Fujian province (no. 2017Y9105).
Keywords: Pin1, Neurodegeneration, CoCl2, Aging, P-tau
Glutathione depletion and p38 activation trigger production of pro-inflammatory citokines in microglia exposed to mercury (II) (#190)
V. Branco1, R. Guerreiro1, T. Eanes1, T. Caetano1, C. Carvalho1
1 Faculty of Pharmacy University of Lisbon, Research Institute for Medicines (iMed.ULisboa), Lisbon, Portugal
Mercury (Hg) is widely known by its neurotoxicity albeit immunotoxicity occurs at lower exposure levels. Since microglia cells are the major representatives of the immune system in the CNS, we hypothesize that Hg compounds disrupt microglia homeostasis by interfering with redox regulation of signalling pathways. Thus, this work aims to study in microglia cells the effect of exposure to Hg2+on p50, p65 and p38 nuclear translocation and activation considering the interaction of Hg with the glutathione system.
N9 mouse microglia cells were used as the experimental model and, following exposure to Hg2+, were analysed for viability (MTT assay), GSH activity (DTNB assay), ROS production (DHCF assay), nuclear translocation of p38, p50, p65 (Western blot). Activation of p38 was assessed by measuring its phosphorylation by Western blot. Transcription of genes associated with pro-inflammatory activation of microglia (e.g.IL1-ß; TNF-α) was evaluated by qRT-PCR. LPS exposure (300 ng/mL; 24h) was used as a positive control for microglia activation. Pre-exposure to N-Acetylcysteine (NAC; 2.5 mM 24 h) and a specific p38 inhibitor (SB 239063; 10 µM 4 h) were used to modulate relevant pathways. All experiments were independently replicated at least 3 times.
Following 24 h of exposure to different concentrations of Hg2+ the EC50for a reduction in viability was 42.1 ± 3.7 µM. However, subsequent experiments showed that after exposure for 24 h to just 5 µM of Hg2+ there was a general increase in ROS levels (»40%) which was accompanied by a very significant depletion (»90%) of GSH.
Upon 6 h of exposure to Hg2+(5 µM), nuclear translocation of p50 was decreased whereas p65’s was increased. Most importantly, Hg2+induced a very significant accumulation of p38 in the nucleus (50% higher than in control), which was accompanied by an increase in its phosphorylation.
Likewise, after exposure to Hg2+, transcript levels of both IL1-ß and TNF-α were increased by 50% relatively to control. However, pre-exposure to NAC – which caused a 60% increase in basal GSH levels – reduced transcription of both cytokines by Hg2+back to control levels. Similarly, pre-exposure of N9 cells to the p38 inhibitor SB 239063 hindered activation of cytokine transcription by Hg2+.
These results show that disruption of redox signalling by Hg2+causes activation of inflammatory pathways leading to production of IL1-ß and TNF-α at exposure levels much below cytotoxic concentrations. GSH depletion and p38 activation are major events contributing to enhance cytokine production. This is of significance since it shows that activation of microglia at sub-cytotoxic exposure levels may result in the production of pro-inflammatory factors which may enhance cytotoxicity for neighbouring cells (e.g. neurons).
This work was supported by Project PTDC/MED-FAR/31136/2017 and by iMed.ULisboa through project UID/DTP/04138/2013 both funded by Fundação para a Ciência e Tecnologia, Portugal (FCT; www.fct.pt).
Keywords: mercury, microglia, p38, glutathione, NF-kB
Analysis of brain transcriptome in MPTP-lesioned adult zebrafish: insights into innate immune-related genes (#271)
B. Chen1, J. - P. Zhang1
1 Chinese Academy of Medical Sciences & Peking Union Medical College, Institute of Medicinal Biotechnology , BEIJING, China
Background and Objective: Parkinson's disease (PD) is a common age-related neurodegenerative disease. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin, has been used to model PD in multiple model organism. Zebrafish is an alternative vertebrate model for the study of neurotoxicity. The objective of this study is to understand the biomechanism of MPTP induced neurotoxicity in zebrafish.
Methods and Results: Adult zebrafish received an intraperitoneal injection of MPTP. The phenotype and role of genes and proteins related to neurotoxicity were tested by using zebrafish swimming behavior, Realtime-PCR, immunofluorescence and transcriptome analysis approaches. MPTP-lesioned adult zebrafish showed the significant decreases in average speed (75%), average acceleration (71%), mobility rate (22%), distance of activity (64%) and exploration rate (54%). The levels of tyrosine hydroxylase gene and protein were significantly decreased in MPTP group, which represented dopaminergic neuron loss. We performed comparative transcriptomics of the brain from wildtype and MPTP-lesioned zebrafish to identify transcriptional signatures involved in the mechanism of MPTP induced neurotoxicity. MPTP treatment caused alteration of gene expression patten, a total of 863 differently expressed genes (fold change ≥2, P < 0.05) were identified by RNA-seq. Application of KEGG enrichment algorithms revealed a number of key biological processes perturbed by MPTP. Particularly, MPTP differentially activated biological processes associated with innate immune-related pathways including Toll-like receptor signaling pathway, NOD-like receptor signaling pathway and Cytokine-cytokine receptor interaction, with significant upregulation of tlr1, tlr3, irf3, irf7, il21r, il7r genes compared to the wildtype brain.
Conclusions: Our work provides a convenient tool for neurotoxicity study and uses an innovative approach to indicate the potential roles of innate immune in the development of MPTP-induced zebrafish PD model.
Keywords: Neurotoxicity, Zebrafish, Neurodegeneration, RNA-seq, MPTP
Phenyl valerate esterase activity of human acetylcholinesterase (#307)
J. Estévez1, M. Terol1, M. A. Sogorb1, E. Vilanova1
1 University Miguel Hernández of Elche, Institute of Bioengineering, Elche, Spain
The toxicity of organophosphorus compounds (OPs) cannot be explained only by action on acetylcholinesterase or neuropathy target esterase (NTE). A fraction of the membrane bound phenylvalerate esterase activity (PVase) is associated to NTE, the key initiating molecular event in the OP-induced delayed neuropathy (OPIDN). An enzymatic fraction in chicken brain soluble PVase has been reported to be due to a butyrylcholinesterase protein. We showed that human butyrylcholinesterase (hBuChE) shows PVase activity and that the substrates acethylthiocholine and phenyl valerate showed competition in their activities but with a interaction different to the competitive model of substrates.
In addition, we have observed that human acetylcholinesterase has also phenyl valerate esterase activity, but with lower activity than human butyrylcholinesterase. The level of phenylvalerate esterase activity in cholinesterases depends on the species and the type of cholinesterase.
This work shows that the kinetic interactions between phenyl valerate and acetylthiocholine in human acetylcholinesterase are different to the competitive model of substrates according to the Michaelis-Menten reaction. In addition, PVase activity is enhanced in presence of low acetylthiocholine concentration. The approach introduced in this work suggests that other site could be involved in the interaction with phenyl valerate.
Keywords: human acetylcholinesterase, Phenyl valerate, Inhibition kinetics
Comparison between the cytotoxic effects of pure cylindrospermopsin and containing and non-containing cylindrospermopsin-extracts in the neuronal SH-SY5Y cell line (#387)
M. G. Hinojosa1, A. I. Prieto1, D. Gutiérrez-Praena1, V. Vasconcelos2, 3, A. M. Cameán-Fernández1, A. Jos1
1 University of Sevilla, Toxicology, Sevilla, Spain
Cylindrospermopsin (CYN) is one of the main common cyanotoxins produced by several genera of cyanobacteria. Due to its capacity of causing damage at different levels in the organism is considered a cytotoxin. Among its effects, neurotoxicity is one of the less clear due to the lack of studies. However, some studies demonstrate its neurotoxic potential in vitro and in vivo. The aim of the present study was to compare the cytotoxic effects between pure CYN and extracts from a producer and a non-producer cyanobacterial cultures in the SH-SY5Y human neuroblastoma cell line. For this purpose, cells were exposed to 0-10 µg/mL CYN, 0-10 µg/mL CYN from an Chrysosporum ovalisporum culture extract (CYN+), and the equivalent volume of extract of a non-producer Cylindrospermopsis raciborskii (CYN-) culture. The cytotoxicity assays performed were the MTS tetrazolium salt reduction (MTS), the neutral red uptake (NR), and the total protein content (PC) assays. The results provided a more sensitive response in the MTS assay in all cases, obtaining EC50 values after 24 hours of exposure of 0.866 ± 0.131, 1.111 ± 0.325, and 5.658 ± 1.180 µg/mL after exposure to CYN, CYN+ and CYN-, respectively, and 0.322 ± 0.081, 0.691 ± 0.165 and 5.164 ± 1.620 µg/mL, respectively, after 48 hours. Thus, it can be concluded that pure CYN exerts higher cytotoxicity in this cell line compared to the CYN+ extract from C. ovalisporum. Furthermore, it is important to highlight that some compounds different to CYN present in the extracts from non-producer (CYN-) could promote cytotoxic damage by themselves, playing a role in the cell-damaging potential of cyanobacterial cultures.
Acknowledgments: Spanish Ministerio de Economía y Competitividad (AGL2015-64558-R, MINECO/FEDER, UE), Junta de Andalucía for the contract of Maria Gracia Hinojosa (USE-16667), and Centro de Investigación, Tecnología e Innovación from the University of Sevilla.
Keywords: cylindrospermopsin, Chrysosporum ovalisporum, Cylindrospermopsis raciborskii, neurotoxicity, SH-SY5Y cell line
Behavioral effects of cypermethrin, lambdacyhalothrin, and betacyfluthrin. (#470)
M. Konopelko1, B. Nieradko-Iwanicka1
1 Medical University of Lublin, Chair and Department of Hygiene, Lublin, Poland
Pyrethroids are among the most commonly used insecticides. Their main mode of action is blocking voltage dependent sodium channels in neuron membranes.
The aim of the study was to compare behavioral effects after exposure to cypermethrin (CYP), lambdacyhalothrin (LCH), and betacyfluthrin (BCF) in a model of subacute poisoning in mice.
A total of 64 mice were divided into 8 groups of 8 animals:
Animas were tested in a Y maze on day 1 and 7 in order to measure their spontaneous locomotor activity and fresh spatial memory. They were also trained in passive avoidance (PA) on day1 and examined on day 2 and day7. The PA task is regarded as a measure of long-term memory retention.
The mean time (±SD) of memory retention in PA on day 2 was: for CYP females 165.7±40.3s, CYP males 180±0.0s, for LCH females 100.3±85.2s (p<0.05 vs controls), for LCH males 153.5±54.4s, for BCF females and males 180±0.0s. On day 7 the mean time of memory retention in PA was: for CYP females 98.38±68.8s, CYP males 152.2±31.8s, for LCH females 95.13±90.7s, for males 95.75±74.9s, for BCF females and males 180±0.0s.
Measuring locomotor activity in a Y-maze on day 1 CYP females had 45.38±12.74 arm entries, CYP males 36.75±6.96, LCH females 53.88±13.59, LCH males 34.75±7.29, BCF females 27.25±10 ( p<0.05 vs controls) males 29.38±9.88. On day 7: CYP females 34.5±19.73, CYP males 32.75±10.54, LCH females 46.63±23.22, LCH males 35.13±3.6, BCF females 29.5±10.66 (p<0.05 vs controls), BCF males 21.12±10.58 (p<0,05 vs controls).
The number of logical alternation in the Y maze on day 1 was: for CYP females 62.29±8.48, CYP males 69.43±9.85, LCH females 60.81±15.06, LCH males 60.98±16.72, BCF females 64.84±16.72, BCF males 62.14±15.28. On day 7 the numbers of logical alternation was for CYP females 59,54±10,95, CYP males 62.75±10.96, LCH females 54.16±9.33, LCH males 68.34±11.52, BCF females 65.48±18.07, BCF males 65.14±11.84.
To sum up: subacute poisoning with LCH significantly decreases memory retention in females on day 2. BCF reduces locomotor activity in females on day 1 and in males and females on day 7. LCH is the least neurotoxic of the three pyrethroids.
Keywords: cypermethrin, lambdacyhalothrin, betacyfluthrin, memory, motor activity
Integration of PBPK with ROS SB model for PFOS induced Neurotoxicity (#497)
D. Deepika1, R. P. Sharma1, M. Schuhmacher1, V. Kumar1
1 Universitat Rovira i Virgili, Department of Chemical Engineering, Tarragona, Spain
PFOS is one of the most abundant
Keywords: Neurotoxicity, PFOS, PBPK, ROS, systems biology
Fusarium mycotoxins alter neuronal network activity in surviving rat brain slices (#515)
V. Bódi1, V. Csikós1, 2, P. Varró1, A. Dobolyi1, 2, I. Világi1
1 Institute of Biology, Eötvös Loránd University, Department of Physiology and Neurobiology, Budapest, Hungary
Mycotoxins are toxic secondary metabolites produced by microscopic fungi often infecting agricultural crops. The most common Fusarium toxins – fumonisin B1 (FB1), deoxynivalenol (DON) and zearalenone (ZEA) – are structurally stable and able to enter into food chain. Consumption of grain containing toxin, whose amount exceeds tolerable daily intake (TDI) may cause adverse health effects. FB1 is known as a de novo sphingolipid biosynthesis depressor, DON is a powerful protein synthesis inhibitor, while ZEA can interact with estrogen receptors. Recent data suggest that these toxins may also have acute effects on neural cell cultures as well. Thus, we decided to examine with electrophysiological techniques whether they alter neuronal network activities after acute treatment.
In order to carry out ex vivo experiments, rat brain slices were incubated for 30 minutes in artificial cerebro-spinal fluid (ACSF), which contained each Fusarium toxin (FB1, DON or ZEA) in different concentrations (10, 50 and 100 µM). After the pre-treatment, electrically evoked field potentials in the hippocampus and seizure-like events in the neocortex were investigated. In addition, neuronal activation pattern was studied after intraperitoneal injection of FB1 (7.5 mg/kg bw), DON (1 mg/kg bw) or ZEA (5 mg/kg bw), by counting c-Fos positive cells.
Basic excitability in the hippocampus was increased by FB1, but long-term potentiation (LTP) was not altered, while DON and ZEA inhibited both excitability and LTP. Seizure-like events in the neocortex were not altered by FB1, but DON delayed the appearance of bursts, and ZEA increased the frequency of events. The number of activated neurons increased mostly in the nucleus accumbens, but we can see similar tendencies in other studied brain areas as well.
Based on our results, each Fusarium toxin has different acute effects on neuronal networks following direct exposure of rat brain slices to toxin-containing ACSF. However, after intraperitoneal injection, the toxins may not penetrate sufficiently through the blood-brain barrier to yield effective concentration required for significantly increased neuronal activation. Therefore, activation was detected only in few brain regions.
Supported by National Research, Development and Innovation Fund, grant number: NVKP_16-1-2016-0016.
F. Wu, JD. Groopman, JJ. Pestka (2014): Public Health Impacts of Foodborne Mycotoxins, Annu. Rev. Food Sci. Technol. 5:351–72
JF. Wentzel, MJ. Lombard, LH. Du Plessis, L. Zandberg (2018): Evaluation of the cytotoxic properties, gene expression profiles and secondary signalling responses of cultured cells exposed to fumonisin B1, deoxynivalenol and zearalenone mycotoxins, Arch Toxicol 91:2265–2282
Keywords: mycotoxin, electrophysiology, c-fos, brain slice, rat
Effect of Fusarium mycotoxins on behavior and neuronal network activity after subchronic exposure in rat (#517)
P. Varró1, V. Bódi1, V. Csikós1, 2, M. Pethő1, T. Hajnik1, I. Sebestyén3, A. Dobolyi1, 2, I. Világi1
1 Eötvös Loránd University, Department of Physiology and Neurobiology / Institute of Biology, Budapest, Hungary
Mycotoxins are toxic secondary metabolites produced by microscopic fungi; the most common Fusarium toxins – fumonisin B1 (FB1), deoxynivalenol (DON) and zearalenone (ZEA) – may contaminate the food chain. FB1 is known for inhibiting de novo sphingolipid biosynthesis, DON effectively decreases protein synthesis inhibitor while ZEA can interact with estrogen receptors. There is increasing evidence that these substances may affect nervous system function as well.
In the present study, neuronal effects of Fusarium toxins were studied in subacute toxicological experiments. Rats of both sexes were treated for 28 days via gavage with FB1 (50 and 500 µg/kg), DON (20 and 200 µg/kg) or ZEA (20 and 200 µg/kg).
Previous experiments indicated that the toxins cause changes activity of neurons in brain areas implicated in anxiety- and depression-related behaviors. For this reason, specific behavioral tests were performed on the treated rats. DON decreased open arm entry number in the elevated plus maze test, indicating increased anxiety level. ZEA increased the time spent with immobility in the forced swim test, which is characteristic of depression-like behavior.
Sleep-wake activity pattern was studied with chronic EEG recording. None of the mycotoxins caused any significant change in the length and circadian distribution of wakefulness, but ZEA slightly decreased the time spent with light sleep. Neuronal network activity on the microcircuit level was studied on rat brain slices of hippocampus and neocortex with the means of field potential recording; excitability, plasticity and seizure susceptibility were examined. FB1 and DON slightly increased excitability in both areas, while ZEA modified seizure-like event pattern in male rats. The higher dose of ZEA also inhibited the development of long-term potentiation in hippocampal slices.
To summarize, the three Fusarium toxins exert different effects on the nervous system, from the microcircuit to the behavioral level; severity of alterations may depend on the dose, sex and exact experimental endpoint.
Supported by National Research, Development and Innovation Fund, grant number: NVKP 16-1-2016-0016.
S. Marin, AJ. Ramos, G. Cano-Sancho, V. Sanchis (2013): Mycotoxins: Occurrence, toxicology, and exposure assessment, Food and Chem. Tox. 60:218-37
F. Wu, JD. Groopman, JJ. Pestka (2014): Public Health Impacts of Foodborne Mycotoxins, Annu. Rev. Food Sci. Technol. 5:351–72
MS. Bonnet, J. Roux, L. Mounien, M. Dallaporta, Js. Troadec (2012): Advances in Deoxynivalenol Toxicity Mechanisms: The Brain as a Target, Toxins 4: 1120-1138
Keywords: mycotoxin, electrophysiology, behavior, brain slice, rat
Deoxynivalenol affects neuronal activity and impairs motivational behavior in mothers (#529)
V. Csikós1, 2, P. Varró2, L. Barcsai2, V. Bódi2, I. Világi2, A. Dobolyi1, 2
1 Eötvös Loránd University and the Hungarian Academy of Sciences, Budapest, Hungary, MTA-ELTE Laboratory of Molecular and Systems Neurobiology, Department of Physiology and Neurobiology, BUDAPEST, Hungary
Deoxynivalenol (DON) or vomitoxin, is a trichothecene mycotoxin produced by Fusarium graminearum and culmorum. Mycotoxins or secondary metabolic products of mould fungi, are micro-pollutants, which may affect human and animal health. The neuronal and behavioural actions of DON were analyzed in the present study. To address which neurons can be affected by DON, the neuronal activation pattern following intraperitoneal injection of DON (1 mg/kg) was investigated in adult male rats while control animals received physiological saline solution. Neuronal activity was assessed by c-Fos immunohistochemistry. DON induced significant c-Fos activation in only a few brain regions, including the accumbens nucleus, the medial prefrontal cortex and the ventral tegmental area. Further double labeling studies suggested that in the accumbens nucleus, a subpopulation of medium spiny neurons may be activated by DON treatment. The activation pattern suggested that DON influenced the reward system of the brain. To study the behavioural relevance of this activation, we examined the effect of DON on a special goal-directed behaviour, the pup-carrying behaviour in mother rats. Pup retrieval latencies were increased by DON administration, and DON-treated mother rats spent less time with nursing, suggesting reduced maternal motivation. Consistently with the behavioural inhibition, electrophysiological recording on rat brain slices indicated that in vitro field responses evoked by electrical stimulation also decreased in the nucleus accumbens as a result of DON pretreatment.
Keywords: mycotoxin, deoxynivalenol, c-Fos, maternal behaviour, rat
Screening of various neural induced hiPSCs (hiNPCs) for the use in (developmental) neurotoxicity assays (#539)
J. Hartmann1, M. Pahl1, J. Klose1, L. Nimtz1, U. Hübenthal1, J. Tigges1, E. Fritsche1, 2
1 Leibniz Research Institute for Environmental Medicine, Modern Risk Assessment and Sphere Biology, Düsseldorf, North Rhine-Westphalia, Germany
For most chemicals in use today, the (developmental) neurotoxic ((D)NT) potential is unknown. This data gap needs to be filled, as the exposure to these chemicals might contribute to neurological diseases in children and adults. Animal experiments are currently used for the testing of (D)NT endpoints, but they are very time-consuming, expensive and the results are often difficult to interpret due to high variation of endpoints. In addition, there are known species differences between rodents and man. Therefore, there is a need for alternative testing methods to assess the (D)NT potential of chemicals. One approach is to use human neural cell-based in vitro-systems to analyze effects on developmental neurotoxicity endpoints like proliferation, differentiation and migration of neural progenitor cells (NPC) as well as for acute neurotoxicity by assessing the influence of substances on a functional neuronal network. To avoid ethical concerns and to ensure enough cell material for medium to high throughput testing, we use human induced pluripotent stem cells (hiPSCs) instead of primary human cells and differentiate them into the neural lineage. Thereby we generate 3D cell aggregates called neurospheres (hiNPCs).
For standardized substance testing it is essential that the neural induction (NI) is highly reproducible and quality criteria need to be defined and established. Therefore we neurally induce different iPS-lines and compare them by monitoring their differentiation status via FACS and qPCR analysis. In addition we plate them on an extracellular matrix to differentiate them into neurons and astrocytes and analyze their radial migration by immunocytochemistry and high content image analysis. To test their ability to form functional neuronal networks, we measure their electrophysiological activity on microelectrode arrays (MEAs). With these results, we will be able to define quality criteria which have to be met by the initial NI culture as well as the time frame in which the cells can be used for (developmental) neurotoxicity assessment.
Keywords: hiPSCs, neurotoxicity, developmental neurotoxicity (DNT), neural induction, microelectrode arrays (MEAs)
Biocompatibility evaluation of medical devices coming into contact with brain tissue (#572)
S. Schmid1, D. Bouard2, A. - L. Leoni1, K. Weber3
1 BSL BIOSERVICE Scientific Laboratories Munich GmbH, Planegg, Bavaria, Germany
The evaluation of local effects in brain tissue is a requirement according to ISO 10993-6: 2016 "Biological evaluation of medical devices - Part 6: Tests for local effects after implantation" for assessing the biological response of the neural tissue to an implanted material. Depending on its clinical use, the material can either be implanted in the brain parenchyma or be placed directly on the parenchymal surface in rats or rabbits. The evaluation must consist in the comparison of the biological response of the test item with the response of a control sample (medical devices with well-known clinical acceptability and biocompatibility characteristics). The local effects of the material to be tested are evaluated at the macroscopic and microscopic level, after the animals have been evaluated clinically.
Intracerebral implantation was performed in rats under general anaesthesia and strict aseptic conditions. In one group, a solid test item (rod shape) was inserted intracerebrally in one hemisphere caudally from the bregma while in one further group, other animals received a liquid test item, which was applied topically on the parenchymal surface of brain. For periods of 1 or 4 weeks, the animals were observed for general clinical findings and detailed behaviour. At the end of the implantation period the animals were necropsied and the brain was stored in 4% neutral-buffered formaldehyde.
Implantation sites were processed to microscopic slides and stained with haematoxylin and eosin, Fluoro-Jade® C (degenerating neurons) and Glial Fibrillary Acidic Protein (GFAP, astrocyte biomarker). Histopathological findings with the solid test item consisted of reactive macrophages, swollen astrocytes, reactive microglia cells, neuronal necrosis, neuronophagia and satellitosis, degenerating neurons, hemorrhage, hemosiderin, and ependymal reaction. The glial reactions formed a gliosis and were present in all affected sections. Implantation sites with fluid item caused focal meningeal fibrosis or lymphocytic infiltration, and in one case, a focal submeningeal gliosis with reactive astrocytes was noted. Overall, the intracerebral implantation of the solid material showed a higher irritative potential than the implantation of the liquid material on parenchymal surface due to the traumatic impact of implantation.
ISO 10993-6: 2016 "Biological evaluation of medical devices - Part 6: Tests for local effects after implantation"
Keywords: intracerebral implantation, neural tissue, local effects
Quantitative evaluation of the Key Events Relationships (KERs) resulting in impairment of learning and memory abilities (OECD AOP13) to support regulatory decision-making (#612)
D. Pamies1, H. de Castro Abrantes2, C. Nunes1, M. Bandarabadi1, D. Tavel1, M. Tafti1, J. - Y. Chatton2, M. - G. Zurich1
1 University of Lausanne, Department of physiology, Lausanne, Switzerland
Exposures to environmental chemicals during early life are suspected to contribute to the increasing incidence of neurodevelopmental disorders in children, such as lowered IQ, learning disabilities, attention deficit hyperactivity disorder (ADHD) and autism. This rise has major societal and financial consequences. There is undoubtedly a substantial genetic component to these disorders . However, findings from neuropathology, brain gene expression, twin and sibling concordance/recurrence risk analyses, all suggest that environmental influences (e.g. chemicals and drugs) in the prenatal period also impact the risk of developing these disorders. The adverse outcome pathway (AOP) concept is a revolutionary advance in toxicological science, based on the application of mechanistic information. An AOP represents the existing knowledge concerning the causal links between a molecular initiating event (MIE) and the cascade of key events (KEs) that lead to a specific adverse outcome of regulatory concern . Well-developed AOPs are expected to guide the identification of experimental testing and non-testing approaches to support regulatory decision-making . However, AOPs are currently a theoretical concept, and activities within regulatory, industry and academic institutions, are still trying to determine how the use of AOPs can support regulatory decision-making. Quantitative metrics obtained in human physiologically relevant vitro models coupled to in silico modelling to construct quantitative AOP (qAOP) and followed by in vitro - in vivo extrapolations (IVIVE) are key to set exposure thresholds and allow the use of AOPs in risk assessment. Therefore, we are currently using the human 3D iPSC-derived brain system (BrainSpheres) developed by the authors  to study already endorsed developmental brain AOPs. Several KEs of AOP13 "Chronic binding of antagonist to N-methyl-D-aspartate receptors (NMDARs) during brain development induces impairment of learning and memory abilities" are quantitatively evaluated after exposure to MIE triggers, and then a qAOP will be constructed to model the effects of xenobiotics within the BrainSpheres. Calcium signalling and multielectrode array have confirmed the presence of functional NMDR in BrainSpheres, and have also allowed to establish a dose-response of NMDAR inhibition after Lead exposure, finding the IC50 around 50 µM. The ultimate goal of this work is to increase the potential of the application of AOPs for regulatory decision-making and improve our capability to predict human toxic exposure thresholds without the need for animal testing.
1. Grandjean, P. and P.J. Landrigan. Lancet, 2006. 368(9553): p. 2167-78.
2. Villeneuve, D.L., et al. Toxicol Sci, 2014. 142(2): p. 312-20.
3. Bal-Price, A., et al. Crit Rev Toxicol, 2015. 45(1): p. 83-91.
4. Pamies, D., et al. ALTEX, 2017. 34(3): p. 362-376.
Keywords: 3D culture, myelination, AOP, MEA, 3Rs
Could Ochratoxin A be a possible etiological factor of Parkinson´s disease? (#620)
A. Vettorazzi1, 2, M. Izco3, A. Lopez de Cerain1, 2, L. Alvarez-Erviti3
1 University of Navarra, Pharmacology and Toxicology, Pamplona, Spain
Parkinson’s disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra and the presence of Lewy bodies which contain aggregates of alpha-synuclein (α-syn). There is increasing evidence that the transmission of the pathology between neurons plays a central role in disease progression. The neurodegenerative process might start in the enteric nervous system and spread via the vagus nerve to the lower brainstem, a process that precedes degeneration of the dopaminergic neurons.
People living in a rural area might be at significantly increased risk of getting PD due to exposure to potential neurotoxins present in the environment. Mycotoxins are a group of natural-occurring fungal secondary metabolites contaminating a huge variety of crops. Very few studies have been specifically designed to evaluate the neurotoxic effects of mycotoxins. However, one of the most relevant mycotoxins in terms of genotoxicity/carcinogenicity, ochratoxin A (OTA), has shown some neurotoxic effects in vivo.
The aim of the present project was to determine the effect and mechanisms of OTA on α-syn in vitro in both intestinal and neuroblastoma cell lines.
For that purpose, i) Caco-2 cells were transfected with a plasmid to express wild-type (WT) and α-syn and ii) a stable SH-SY5Y cell line overexpressing WT α-syn was selected. Cytotoxicity assay (Celltiter Blue assay) was carried out to select a range of subtoxic concentrations. A range of 25 to 500 nM OTA was selected for the subsequent evaluation of α-syn (Western blot) after 72h of exposure. The lower doses of OTA (25, 50 nM) had no effect upon α-syn however doses between 75 to 200 nM increased significantly the intracellular α-syn levels. Moreover, proteins (hsc70 and LAMP-2A) involved in chaperone-mediated autophagy, a pathway known to degrade α-syn were evaluated. Although no significant changes were observed in hsc70 a clear downregulation of LAMP-2A was observed at concentration 100 nM. Decreased LAMP-2A levels in cell lines reduced chaperone-mediated autophagy activity and increased the half-life of α-syn.
Our results provide preliminary data to understand the potential neurodegenerative effect of OTA as well as contribute with an experimental system able to detect other long-term effect neurotoxins.
Keywords: Ochratoxin, Parkinson, neurotoxicity, alpha-synuclein, mycotoxin
Establishment of in vitro assays for regulatory developmental neurotoxicity testing (#677)
K. Bartmann1, S. Masjosthusmann1, L. - C. Stürzl1, T. Waldmann2, M. Leist2, E. Fritsche1
1 IUF - Leibniz Research Institute for Environmental Medicine, Düsseldorf, Germany
The activation of key neurodevelopmental processes (KNDP) is an essential requirement for brain development. It is assumed that at least one of these processes is impaired during chemical exposure, leading to developmental neurotoxicity (DNT). To assess this DNT hazard, animal-free new approach methods (NAM) have been developed, which model certain KNDP in vitro. To allow conclusion on the DNT hazard of hits the information from a screen assay has to be combined with additional data and with information on the test method.
Based on a set of selected pesticides we are performing primary hit testing with different KNDP methods. Primary fetal human neural progenitor cells (NPC), growing in 3D cell aggregates called neurospheres, cover the KNDP NPC proliferation, migration and differentiation into neural effector cells (astrocytes, neurons and oligodendrocytes) as well as neuronal morphology. A further test method models neurite outgrowth of immature human dorsal root ganglia cells. To improve the readiness and highlight robustness and reliability of the test methods we are performing an exchange of these methods between two laboratories. Additionally, as a hit confirmation, human induced pluripotent stem cell (hiPSC)-derived iNPC are being differentiated into neurons and astroglia cells. A second hit confirmation for the neurite outgrowth assay of dorsal root ganglia cells is being performed with immortalized primary central neurons from an 8-week old mesencephalon to observe effects on the neurite outgrowth. Beside data from tests for structural impairment, we aim to establish the neuronal network formation (NNF) assay based on human iPSCs to test neuronal network activity on 24-well microelectrode arrays (MEA). Previous data show that some pesticides as rotenone affect more than one endpoint while others are likely to be specific for only one of the tests (e.g. disruption of oligodendrocyte maturation or the neural network function).
We will present first results of this study according to the experimental progress.
Keywords: developmental neurotoxicity, new approach methods
Synthetic cannabinoids 5F-PB22 and THJ-2201 promote in vitro CB1 receptor-dependent neuronal differentiation at in vivo-relevant concentrations (#688)
J. Alexandre1, R. Malheiro1, D. C. Dias da Silva1, H. F. Carmo1, F. D. Carvalho1, J. P. Silva1
1 Faculty of Pharmacy, University of Porto, UCIBIO-REQUIMTE, Porto, Portugal
The widespread recreational use of Synthetic Cannabinoids (SCs), a vast group of new psychoactive substances that activate cannabinoid receptors (CB1R, CB2R) with stronger potency than THC (the main psychoactive substance of cannabis), represents a major public health issue. SC use during and before pregnancy is especially alarming due to the possible onset of neurodevelopment disorders in the offspring. In the present study, the role of 2 commonly abused SCs, 5F-PB22 and THJ-2201, on in vitro neuronal differentiation and proliferation was evaluated in NG108-15 neuroblastoma cells.
Cells were differentiated for 3 days in serum-starved (1% FBS) culture medium supplemented with forskolin and retinoic acid, and exposed to SCs at non-toxic, in vivo-relevant concentrations (≤ 1 nM), either in acute (single addition at day 0) or repeated (one addition every 24 h) exposure settings. Differentiation ratios (number of neurites per total cell number), total neurite length and neuronal marker expression (e.g. β3-tubulin, p73) were assessed. Cell proliferation was followed up to 72 h (Sulforhodamine B assay).
5F-PB22 and THJ-2201 only impaired metabolic activity, measured by the MTT assay, at high concentrations (EC50 of 779 μM and 299 μM, respectively). Exposure of NG108-15 cells to 1 pM - 1 nM of either SC raised differentiation ratios (near 2-fold) and total neurite length, compared to vehicle-treated cells. Regulation of such processes depended on CB1R activation, as its inhibition with the selective antagonist SR141716A abrogated SC-induced effects. Interestingly, repeated 5F-PB22 exposure was required to reach effects similar to a single THJ-2201 dose. Different neuronal marker expression levels (higher 5F-PB22-induced β3-tubulin and p73 expression, compared to THJ-2201) suggest that neuronal maturation state varied between the tested SCs. Of note, none of the 2 SCs affected cell proliferation.
Overall, we report first-hand the CB1R-mediated enhancement of neurodifferentiation by 5F-PB22 and THJ-2201 at concentrations below 1 nM. Still, further research is required to identify the underlying action mechanisms and the consequences for neurodevelopment in vivo.
This work was supported by UCIBIO, via FCT/MCTES UID/Multi/04378/2019 funds, and by FEDER (POCI/01/0145/FEDER/029584 and POCI/01/0145/FEDER/007728) under the framework of QREN (NORTE-01-0145-FEDER-000024) and FCT/MCTES (PTDC/SAU-TOX/29584/2017) funds.
Keywords: New Psychoactive Substances, Developmental Neurotoxicology, Neuronal Development, Cannabinoid receptors, Endocannabinoid system
Neurotoxicity evaluation of acute pesticide exposure on Human progenitor neural stem cells (#689)
A. U. K. M. W. Wijesekara Mudiyanselage1, W. G. Carter1
1 University of Nottingham, Division of Medical Sciences & Graduate Entry Medicine, Derby, United Kingdom
Neurodevelopment in humans is susceptible to damage through exposure to toxic chemicals including pesticides. Pesticide-induced neurotoxicity was investigated using the human neural progenitor cells. Both undifferentiated and differentiated cells were challenged with either of two commonly employed organophosphorus pesticides, Azamethiphos-oxon or Chlorpyrifos-oxon, or the carbamate pesticide, Aldicarb, over a concentration range of 0.3-300 μM for 24 hrs. Cellular metabolic activities and cell viability were assessed using MTT, LDH, and ATP assays. Reactive oxygen species (ROS) generated in response to pesticide exposures were quantified using a dichlorofluorescein diacetate assay. Cellular protein modification and damage was also investigated using gel electrophoresis, and Western blotting. Cell metabolic activity and viability decreased in a pesticide concentration-dependent fashion. The inhibitor concentration producing 50% cell death (IC50) in undifferentiated cells was 12.01±1.128 μM, 12.16±1.982 μM and 14.3±2.393 μM for Azamethiphos-oxon, Chlorphyrifos-oxon and Aldicarb, respectively, whereas differentiated cells were more sensitive to pesticides with IC50s of 11.883±2.043 μM, 8.2475 ±0.8896 μM, and 13.88± 2.844 μM, respectively. Differentiated cells were also more susceptible to ROS damage than undifferentiated cells, with ROS damaged proteins increased in a concentration-dependent fashion. Collectively, our results demonstrate the vulnerability of neural stem cells to a toxic insult from a range of commonly encountered pesticides.
Keywords: Neurotoxicity, Pesticides, ReNCell CX
The increase in lipid peroxidation in the rat brain after acute exposure to Pb and/or Cd (#690)
D. Javorac1, A. Buha Djordjevic1, M. Anđelković1, K. Baralić1, E. Antonijević1, M. Ćurčić1, D. Đukić-Ćosić1, J. Kotur-Stevuljević2, B. Antonijević1, Z. Bulat1
1 University of Belgrade-Faculty of Pharmacy, Department of Toxicology "Akademik Danilo Soldatović", Belgrade, Serbia
Among metals lead (Pb) and cadmium (Cd) are leading industrial and environmental pollutants. It is well known that the nervous system is the main target organ of Pb toxicity. However, there is growing evidence that Cd as well can produce toxic effects associated with nervous system. Having in mind that one of the main mechanisms of metal toxicity is induction of prooxidants and antioxidants imbalance, the aim of the study was to investigate whether the combined acute treatment with Pb and Cd has more pronounced effects than intoxication with single toxic agent on the parameters of oxidative stress: malondialdehyde (MDA), total antioxidative status (TAS) and total oxidative status (TOS) in the rats brain.
Three experimental groups (6-8 Wistar rats) received single oral treatment of 150 mg Pb/kg b.w. (Pb group), 15 mg Cd/kg b.w. (Cd group), or mixture of these two doses (Pb+Cd group). Control group was untreated. All animals were sacrificed 24h after treatment, and brains were removed and homogenized for further analysis of MDA, TAS and TOS.
Co-treatment with Pb and Cd induced significant increase of MDA in rat brain compared to the controls, and both groups treated with only one metal. Interestingly, single treatment of Pb or Cd did not change MDA levels when compared to the control group. On the other hand, TAS levels were significantly increased in all treated groups, while no significant changes in TOS levels were observed
The results of the present study indicated that Pb and Cd mixture exhibited more pronounced toxic effect on lipid peroxidation status in rats brain when compared to Pb or Cd single treatment,. Further studies are needed to determine whether this observed exacerbation of oxidative stress in brain following mixture administration is the result of toxicokinetic or toxicodynamic interactions between these two toxic metals.
Keywords: mixture toxicity, neurotoxicity, oxidative stress, rats, lead and cadmium
Feasibility Studies for Prediction Models analysing Concentration Response Data from High Content Image Analyses (#745)
H. E. Keßel1, S. Masjosthusmann1, N. Förster1, A. Mosig1, E. Fritsche1
1 Leibniz Research Insitute for Environmental Medicine, Düsseldorf, Germany
Testing of chemicals for their potential to induce neurodevelopmental toxicity (DNT) in vitro is a stepwise process that starts with the in vitro experiments followed by data generation, data analyses and finally decision-making based on the in vitro findings. Each of these steps is crucial, highly dependent on the preceding one and has a lot of criteria to regard. Here we propose a data evaluation tool for effective, versatile and robust data evaluation, analyses and interpretation of in vitro concentration-response toxicity data. For DNT testing, we use primary human neural progenitor cells (NPC) that grow as neurospheres in suspension culture. After plating cells on the extracellular matrix protein laminin, they radially migrate out of the neurosphere and thereby differentiate into neurons and glia cells. For simultaneous analyses of the multiple endpoints in this in vitro model, i.e. neuronal and glia migration and differentiation, as well as neuronal morphology analyses, we engage the software ‘Omnisphero’ that was specifically designed in our lab for analysing data generated with High Content Image Analyses (HCA) of spheroid based neural models. The output of Omnisphero are concentration-response curves over multiple endpoints for each compound, making an automated evaluation of data necessary. Here, we present our efforts of creating an R based data evaluation workflow, where several fitting models are generated and compared, thus applying the optimal fit for each set of data. Based on the optimal fit, different prediction models are developed and compared to find the one that best represents the in vitro data and thus enables data interpretation.
The here proposed statistical workflow takes several statistical models for concentration-response fitting and analysis into account and thus enables more robust, informative and versatile concentration-response analysis.
Keywords: Developmental neurotoxicity, High-content image analysis, Neurospheres, Concentration-Response Curve, Benchmark Response
Repeated intravenous administrations of macrocyclic Gadolinium Based Contrast Agents in rats: evaluation of gadolinium retention in different organs. (#763)
S. Bussi1, A. Coppo1, R. Celeste1, A. Fanizzi1, A. Fringuello Mingo1, C. Botteron2, F. Maisano1, F. Tedoldi1, M. A. Kirchin1
1 Bracco Imaging SpA, Colleretto Giacosa, Italy
Gadolinium Based Contrast Agents (GBCAs) are paramagnetic agents used in magnetic resonance imaging (MRI) as they add relevant diagnostic information to the anatomical resolution of the MR images. Recent studies have found that tiny amounts of gadolinium can be retained in the brain and other tissues after cumulative administration of GBCAs. Using a healthy rat animal model, we compared the residual gadolinium levels in cerebrum, cerebellum, liver, kidney (right), skin, femur and blood after repeated exposure to four commercially-available macrocyclic GBCAs.
Sixty-five male Sprague-Dawley rats were divided into four treated groups (ProHance®, Dotarem®, Clariscan™, Gadovist®, n=15) and one control group (saline, n=5). Exposed animals received 20 GBCA administrations (four per week for five weeks) at 0.6 mmol/kg, a dose corresponding to a clinical dose of 0.1 mmol/kg based on the extrapolation factor set for rats in FDA Human Equivalent Dose (HED) guidance. After a wash-out period of 28-days, animals were sacrificed and tissues harvested for gadolinium (Gd) determination. Gd content was determined by Inductively Coupled Plasma Mass-Spectrometry. Since GBCAs are mainly excreted through the kidneys, we also dissected and processed to slides the left kidney for histopathologic evaluation.
Significantly (p≤0.005; all evaluations) lower levels of Gd were noted with ProHance than with Dotarem, Clariscan or Gadovist in all soft tissues. Significantly (p≤0.01) higher Gd levels were noted with Gadovist in the femur compared to all other GBCAs. No GBCA-induced macroscopic or microscopic findings were noted in the left kidney.
In conclusion, our findings reveal considerably lower retained levels of Gd in brain and soft body tissues of rats at 28 days after the cumulative administration of a total ProHance dose of 12 mmol/kg, than after equivalent cumulative doses of Dotarem, Clariscan and Gadovist administered under identical conditions. Moreover, no evidence of GBCA-induced renal changes was observed histopathologically at the tested cumulative dose.
Keywords: neurotoxicology ; regulatory toxicology, Gadolinium Based Contrast Agents, hystopathology, kidney
Effect of uranium on multipotency of neural stem cells in a primary neurosphere culture model (#795)
A. Becquet1, C. Gloaguen1, K. Tack1, C. Ibanez1
1 Institute of Radioprotection and Nuclear Safety, Laboratory of Experimental Radiotoxicology and Radiobiology, Fontenay aux Roses, France
Uranium exposure situations are diverse and originate from its natural presence in the environment, and from its use in specific professional activities in relation with the nuclear industry (extraction, nuclear fuel cycles, and dismantling operations). Uranium internal contamination can occur via ingestion of contaminated food and drinking water or via inhalation of particulate aerosols containing uranium dust. This latest situation is the main cause of contamination in nuclear occupational activities. These contaminations raise concern in terms of potential consequences on human health. They appear to have negative impact on the brain as experimental studies have shown that uranium exposure via ingestion or inhalation can lead to cognitive impairments in rats. Neurogenesis disruption has been proposed to underlie these effects. To address this question, we used in vitro neurosphere primary cultures from rat embryo’s telencephalon at embryonic day 13. We studied uranium impact on multipotency of neural stem cell within a range of concentrations (10, 50, 100 µM) versus control over 7 days of contamination. Our results show a significant effect on cell survival via a decrease of the absolute number of all cell types: neurons, astrocytes and mature oligodendrocytes at 50 and 100 µM. Among cells surviving after 7 days of contamination, analysis of apoptotic gene expression tend to suggest an adaptive response via Bax/Bcl2 balance in favour of cell survival at 100 µM condition, that will need further investigations. In this condition (100 µM), neurons exhibit an aborted morphology with a reduction of the axon and dendrite length correlated with a significant decrease of gene expression GAP43 known to be involved in dendritic arborization development. Regarding gliogenesis, uranium seems to have a direct action on the maintenance of a population of glial progenitors Olig2 positive, linked with a significant increase of NeuroG3 gene expression at 100 µM. All together, these results suggest that uranium exerts a specific action on late cell maturation phases rather than on early determination stages.
Keywords: neurotoxicity, stem cell, multipotency, metals, uranium
Doxorubicin and mitoxantrone effects on the brain of differently aged mice: an in vivo chemobrain study (#873)
A. Dias-Carvalho1, A. Reis-Mendes1, M. Duarte-Araújo2, R. Guedes1, S. Gonçalves-Monteiro3, F. D. Carvalho1, M. D. L. Bastos1, J. P. Capela4, V. M. Costa1
1 UCIBIO, REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Porto, Portugal, Porto, Portugal
Chemobrain is the designation given to chemotherapy-induced cognitive dysfunction. Doxorubicin (DOX) and mitoxantrone (MTX) are two widely use chemotherapeutic agents with a broad spectrum of activity against cancer; however, their toxicity has been shown to occur in several organs. Still, their neurological effects are largely unknown. This work aimed to evaluate the redox and energetic status in the brain of differently aged CD-1 mice [juvenile (4 weeks), adults (3 months) and old (18-20 months)] after exposure to clinically relevant doses of DOX and MTX. Animals received biweekly intraperitoneal administrations, for 3 weeks. All age groups received a total cumulative dose of 6 mg/kg MTX or a total cumulative dose of 18 mg/kg DOX, except the old group that received a total cumulative dose of 9 mg/kg DOX. The control groups received same number of saline injections. Throughout the protocol, animal well-being, as well as body weight and food and water consumption were determined. Mice were euthanized one week (adults and old animals) or seventeen days (juvenile) after the last drug injection. To evaluate the brain’s oxidative stress, total glutathione (GSHt), reduced glutathione (GSH) and oxidized glutathione (GSSG) levels were determined, as well as the GSH/GSSG ratio. To evaluate the brain’s energetic status, ATP levels were measured. In adult and juvenile mice, DOX (18 mg/kg) caused weight decrease after the last injection. In fact, as early as day 10, these DOX groups revealed lower food intake than their respective controls. Brain levels of GSHt, GSH and GSH/GSSG ratio were decreased in DOX adults, but DOX infant brains had no changes in these parameters. Nonetheless, DOX (18 mg/kg) increased brain ATP levels in juvenile mice. MTX did not cause significant changes in the brain glutathione levels or ATP levels in any of the groups tested. This data suggest that DOX significantly impairs the redox status of the adult brain while increasing ATP in juvenile mice, and DOX neurotoxicity requires further research.
ARM and VMC acknowledge FCT for their grants: SFRH/BD/129359/2017 and SFRH/BPD/110001/2015, respectively). This work was supported by FEDER funds [Operational Programme for Competitiveness Factors – COMPETE and by FCT within the project “PTDC/DTP-FTO/1489/2014 – POCI-01-0145-FEDER-016537”]
Simó M, et al. Neuroscience & Biobehavioral Reviews. 2013;37(8):1311-21.
Keywords: Doxorubicin, mitoxantrone, chemobrain, glutathione, chemotherapy
The Unfinished Symphony: Neurotoxicity potential and mitochondrial-mediated mechanisms of synthetic cathinones in dopaminergic human neuronal SH-SY5Y cells (#906)
H. S. Leong1, 2, M. Philp1, M. Simone2, P. K. Witting2, S. Fu1
1 University of Technology Sydney, School of Mathematical and Physical Sciences, NSW, Australia
Synthetic cathinones (SCs) emerged as a popular “legal” alternatives to the “traditional” psychostimulant drugs in recent years. Increasing reported hospital admissions leading to fatalities has raised concerns. However, the precise mechanism of drug toxicity is unclear. This paucity of understanding raises an interest to investigate in-vitro neurotoxicity and mechanistic pathways. SCs-induced mitochondrial dysfunction has been hypothesized to be a crucial factor in the onset of neurotoxicity. For this reason, butylone and pentylone were supplemented in the human neuronal cell line, SH-SY5Y to evaluate the neurotoxicity potential and potency using trypan blue and lactate dehydrogenase assays over the dose range 1 to 10 mM. Cells were cultured in commercial DMEM/F12 media and plated at a optimal density when cell confluency reached 70-80 %. Cells were then differentiated to a neuronal phenotype using 10 μM retinoic acid (RA) in the media for 3 days, followed by a mixture of 10 μM RA and 81 nM 12-O-tetradecanoylphorbol-13-acetate in the media for another 3 days. To define the mechanisms underlying neurotoxicity, measurements included: markers of oxidative stress, mitochondrial bioenergetics impairment, intracellular calcium (Ca2+) and cell death pathways were evaluated at two doses for each drug tested, EC15 (butylone 4.67 and pentylone 3.05 mM) and EC40 (butylone 5.91 and pentylone 4.06 mM), together with EC15 (2.60 mM) and EC40 (3.43 mM) of popular synthetic cathinone 3,4-methylenedioxypyrovalerone (MDPV). After 24 h of exposure, both butylone and pentylone exhibited a dose-dependent cytotoxicity, characterized by significant (p<0.0001 vs control) production of reactive oxygen species, decreased mitochondrial respiration, depletion of adenosine triphosphate contents and increased intracellular Ca2+ concentrations. Activation of caspases 3, and 7 indicated that these synthetic cathinones induced neurotoxicity primarily via the intrinsic apoptotic pathway. These data provide important insight into the calcium involvement in mitochondrial pathophysiology mechanism, ultimately identifying potentially novel therapeutic targets in treating acute-neurological complications or at least ameliorate the deleterious consequences arising from the illicit use of butylone and pentylone.
Keywords: Synthetic cathinones, neurotoxicity, SH-SY5Y
Are PON and GST polymorphisms associated with advanced oxidation protein products in pesticide-exposed subjects? (#112)
C. Fenga1, M. Teodoro1, G. Briguglio1, I. Polito1, F. Giambò1, D. Caccamo1, C. Costa2
1 University of Messina, Department of Biomedical, Odontoiatric , Morphological and Functional Images-Occupational Medicine Section, Messina, Italy
Recent studies have suggested oxidative stress as one of the mechanisms for the adverse health effects of pesticides . Oxidation generates several molecules, such as advanced oxidation protein products (AOPP), which could represent useful biomarkers of oxidative stress.
Glutathione S-tranferases (GSTs) and PON family genes are enzymes involved in the detoxification of xenobiotics, sharing antioxidant effect; genetic differences in expression and activity of these enzymes are often due to polymorphic alleles. These polymorphisms alter enzyme activity and consequently susceptibility towards many toxic compounds. The present study was aimed to assess the contribution of genetic polymorphisms of pesticide-metabolizing enzymes on AOPP production as a biomarker of oxidative stress.
45 healthy Caucasian males (age 42.08±12.78) working as greenhouse farmers were enrolled. Genomic DNA was isolated from peripheral blood lymphocytes. Genotyping of the PON2 S331C and GSTM1 polymorphisms was performed by PCR.
The serum concentrations of AOPP were determined by a microplate absorbance reader, as previously described .
Data were analyzed using Kruskaal-Wallis test followed by Dunn’s post hoc test using Prism version 6.01 (GraphPad software, La Jolla, USA).
Subjects were exposed to a mixture of pesticides with prevalent use of chlorpyrifos. No infectious or inflammatory diseases and no drug use was reported in the subjects in the three months preceding the survey. The majority of subjects had an adequate intake of food rich in antioxidants, did not smoke and did not abuse alcohol. All subjects used personal protective equipment.
Increased AOPP levels were observed in the subjects with S331CG and S331GG (2.25±1.54 and 2.21±1.22 nmol/ml respectively, mean±SD) mutated genotype, compared with WT subjects (1.32±0.814 nmol/ml). A similar trend was observed in subjects with deleted GSDTM1 genotype, compared with WT (2.01±1.40 and 1.48±0.99 nmol/ml respectively).
The results of the present study indicate that measurement of AOPP levels may provide a useful biomarker for the oxidative effect of chronic pesticide exposure, and polymorphic genes encoding PON2 and GSTM1 can be genetic determinants of pesticide toxicity.
Keywords: Pesticide; polymorphism; oxidative stress
Low-dose exposure to lead and neurobehavioral effects (#113)
C. Costa1, E. Micali2, M. Teodoro3, G. Briguglio3, I. Polito3, G. Nutile3, A. Alibrandi4, C. Fenga3
1 University of Messina, Department of Clinical and Experimental Medicine, Messina, Italy
Exposure to inorganic lead (Pb) in the environmental and occupational settings continues to be a serious public health problem. At high exposure levels, lead is known to cause encephalopathy, kidney damage, anaemia and toxicity to the reproductive system. This survey was conducted to evaluate the association between occupational exposure to low-dose Pb and mood states using biological markers and a validated and standardized test [1,2].
Thirtysix male workers, employed in a battery recycling plant and participating to an health surveillance program, were enrolled for the present study.
Biomarkers of exposure and effect (PbB, blood lead; ZPP, Zn protoporphirin) and a neuropsychological test (POMS, Profile of Mood States) were evaluated in the exposed workers compared to 36 age-matched control subjects.
Mean PbB level resulted 56.7±13.9 μg/dL in exposed workers and 15.5±1.6 μg/dL in control subjects; ZPP was 53.9±23.6 and 23.5±1.4 µg/dL in workers and controls respectively.
Environmental assessment of workplace lead levels was over the threshold limit value of 0.05 mg/m3 set by the American Conference of Governmental Industrial Hygienists (ACGIH).
The values of tension, depression, aggressiveness, tiredness and confusion resulted higher in the exposed workers than in controls. An inverse trend was found for the vigour, that resulted higher for the controls. In addition, Poisson regression test performed on single psychoemotional factors, has allowed to evidence a significant influence of Pb e ZPP levels on tension, anxiety and depression.
Authors found that blood lead levels considered borderline for occupational exposure in workers currently exposed to low-dose lead were associated with tension, anxiety, hostility and depression.
Therefore, neurobehavioral effects may occur also at concentrations several orders of magnitude below the clinical threshold for acute lead poisoning.
Keywords: Lead, Neurotoxicity, Neuriobehavioral effects
Glucorticoids: different approaches in PDE and OEL evaluation, but similar values. (#154)
E. Gillio Tos1, M. D. Rodda1, L. Brunasso Cattarello1, V. Bortolot1, A. Conto1
1 CHEMSAFE SRL, PHARMA BUSINNESS UNIT, Colleretto Giacosa (TO), Italy
The approach followed for setting PDE limits is the same outlined in ICH Q3C consensus guideline on residual solvents, in ICH Q3D consensus guideline on elemental impurities and in the EMA guideline EMA/CHMP/CVMP/SWP/169430/2012. The Permitted Daily Exposure (PDE) value for APIs is based on scientific evaluation of all available toxicological and pharmacological data, taking into account several different adjustment factors (Safety Factors or Uncertainty Factors) and the specific absorption/bioavailability of the compound under evaluation according to the selected Point of Departure (PoD). The same approach has been used for the calculation of the OEL values.
Glucocorticoids are a group of drugs with various anti-inflammatory and immunosuppressant as well as metabolic and endocrine effects. Systemic glucocorticoids are used for hormone replacement therapy (e.g., in Addison disease), for acute or chronic inflammatory diseases (e.g., rheumatoid arthritis), and for immunosuppression (e.g., after organ transplants). Local glucocorticoids are used to treat conditions like dermatoses, asthma, and anterior uveitis.
An extensive literature search has been carried out on fourteen glucocorticoids in order to find data useful to their PDE and OEL derivations.
Different criteria have been used for the selection of the Point of Departure (POD) in each calculation: NOAEL (the highest tested dose at which no “critical” effect is observed) or LOAEL (the lowest-observed-adverse-effect level) if no NOAEL is obtained. For some compounds, toxicological profile is poorly characterized and there were no useful quali-quantitative data to be used to allow a NOAEL or LOAEL determination, so the PDE and the OEL values for these four compounds were based on the lowest recommended daily dose (MED).
Although different approaches have been used, the results obtained are very similar. Indeed, the resulting calculated PDE and OEL values for each glucocorticoids range from 0.4 to 2 µg/day and from 0.1 to 0.8 µg/m3, respectively. After the OEL determination, all compounds need the same containment strategy.
Keywords: PDE/OEL evaluation, Glucocorticoids
The effects of umbilical cord Mesenchymal stem cells on the pulmonary fibrosis in silicosis rats (#215)
Y. Sha1, Y. Xie1, Z. Li1
1 Shenzhen Prevention and Treatment Center for Occupational Diseases,, Institute for Occupational Health Science, , Shenzhen, China
Objective To explore the effects of the umbilical cord mesenchymal stem cells(UC-MSCs) on the pulmonary fibrosis in silicosis rats.. Methods SPF male Sprague Dawley rats were randomly divided into control group，silica model group and UC-MSCs treatment group with 12 rats each group． SiO2 intra-tracheal injection (0.5ml of 50mg/mL/rat) were applied to silica model group and UC-MSCs treatment groups. After that UC-MSCs treatment group received 1 mL UC-MSCs suspension (3 × 106 cells/mL) by tail vein injection on the 29th , 36th , 43th and 50th day after exposure to the first silica suspension. On the 60th and 75th day after exposure to silica suspension, all animals were examed for pulmonary CT. Then the rats were euthanized on 75the day after the first exposure to silica．Lung’s histopathological examination of the rats from all the groups were carried out. The content of hydroxyproline in lungs,TGF-β1and IL-6 in serum were examined. The microRNA expression profiles were identified by Illumia sequencing. Results The lung’s histopathological examination showed a lot of inflammatory cell aggregation and collagen fiber deposition in silica model group, while in the UC-MSCs treatment group, there were less inflammatory cells and collagen fiber. The rats from silica model groups had higher HYP, TGF-β1and IL-6 than the rats from UC-MSCs treatment group and control group. For CT examination, different-sized granular high-density shadows or reticular fibrous shadows were found diffusely distributed in the lungs of the rats in silica model group. Lung field of rats in UC-MSCs treatment group were less high density shadows, and more clear. Lung fields of rats in the control group were clear and no obvious high-density shadow. 7 miRNAs were found to be up-regulated, whereas 19 miRNAs were found to be down-regulated in UC-MSCs treated group compared with the silicosis group. In the UC-MSCs treated group,the expression of miR-449a/c , miR-133a/b, miR-34c, miR-384, miR-135a increased, and were related to changes in mRNA for regulating the fibrosis process and macrophage function. The changes of microRNAs were verified by real time QPCR. Conclusion: UC-MSCs can alleviate the pulmonary fibrosis in silica model rats through microRNAs to regulating fiblosis preoces and macrophage function,and UC-MSCs can also down-regulate the level of TGF-β1 and IL-6 to decrease fibrosis.
Keywords: UC-MSCs, pneumosilicosis, transforming growth factor β1, silicosis, microRNAs
Use of the local lymph node assay:5-bromo-2-deoxyuridine flow cytometry method to predict the skin sensitization potential of PHMG, PGH, TRICLOSAN and mixtures of these compounds with the excipient propylene glycol (#217)
H. Kim1, R. Gautam2, S. Joo2, S. Yang2, M. Acharya2, A. Maharjan2, J. Jo2, C. Kim2, Y. Heo2
1 The Catholic University of Korea, College of Medicine, Dept. Preventive Medicine, Seoul, Republic of Korea
In commercial biocidal products such as polyhexamethylene guanidine (PHMG), oligo (2-(2-ethoxy) ethoxyethyl guanidine chloride (PGH), 2,4,4’-trichloro-2’-hydroxydiphenyl ether (triclosan) often serves as an antimicrobial agent, and the excipient propylene glycol (PG) is used to dissolve the active ingredients. The skin sensitization (SS) potentials of each of these substances are still being debated. Moreover, mixtures of PHMG, PGH or triclosan with PG have not been evaluated for SS potency. The Local lymph node assay: 5-bromo-2-deoxyuridine-Flow Cytometry Method (LLNA: BrdU-FCM), which was developed and validated by Korean Scientists, and adopted as OECD TG 442B at 2018, served to address these issues. All the experimental procedures were undertaken following the OECD TG. Test concentrations without systemic toxicities were determined as followings through 2-stage pre-screening tests: PHMG: 5, 10, 25%; PGH and triclosan: 2.5, 5, 10%; PG: 25, 50, 100%. The stimulation index (SI) versus AOO vehicle (acetone:olive oil=4:1) was dose-dependently increased to 0.99±0.13 for 5%, 1.62±0.36 for 10%, 4.43±0.76 for 25% for PHMG, 1.62±0.23 for 2.5%, 2.43±0.16 for 5%, 15.00±1.91 for 10% for PGH, and 1.10±0.09 for 2.5%, 2.40±0.50 for 5%, 6.19±0.57 for 10% for triclosan. Since the SI ≥ 2.7 is considered skin sensitization positive, these three test substances were predicted as skin sensitizer, but PG was predicted as a non-sensitizer (0.92±0.19 for 25%, 1.48±0.37 for 50%, 1.16±0.18 for 100%). Concerning a broad mixture ratio in manufacturing companies, the mixture ratios were decided as 1:4, 4:1, 9:1 weight/volume for PHMG, PGH, triclosan versus PG. Mixtures of PHMG, PGH, triclosan with PG were all positive in terms of SS potential but SS potency was mitigated as the proportion of PG increased. Since humans can be occupationally or environmentally exposed to mixtures of excipients with active ingredients such as biocides, the present study may give insights into further investigations of the SS potentials of various chemical mixtures. [supported by Korea National Research Foundation, Project no. 2017R1D1A3B03032723].
Keywords: local lymph node assay:5-bromo-2-deoxyuridine flow cytometry method, Skin sensitization alternative method, polyhexamethylene guanidine, oligo (2-(2-ethoxy) ethoxyethyl guanidine chloride, TCS
Occupational lung cancer risk caused by CrVI assessed using human biomonitoring data (#262)
S. Mahiout1, M. Kiilunen1, T. Santonen1
1 Finnish Institute of Occupational Health (FIOH), Helsinki, Finland
Hexavalent chromium (CrVI) compounds are known lung carcinogens, and their use is subject to authorisation under REACH. However, occupational exposure to CrVI remains a relevant concern, as CrVI compounds are still widely used in authorised industrial applications, mostly due to their superiority in producing hard and corrosion tolerant coatings. CrVI fumes are also formed, e.g., in manufacturing and welding of stainless steel. Such process-generated fumes are not covered by REACH.
We calculated lung cancer risks due to occupational CrVI exposure based on human biomonitoring (HBM) data. As cumulative exposure is essential in CrVI-related cancer incidence increase, data covering a ~40-year period (1980–2016) were used, originating from a database of the Finnish Institute of Occupational Health (FIOH). It consists of the urinary Cr (U-Cr) samples sent to FIOH for exposure monitoring by occupational health care units. Published equations were used to convert the data (p95) into corresponding air levels and to calculate lung cancer risks.
The measured U-Cr levels decreased substantially over the 40‑y period. One of the highest measured U-Cr levels was in welders: 0.77 µmol/l in the 1980s (n=3232), down to 0.13 µmol/l in the 2010s (n=5348). The estimated corresponding CrVI air levels were 10–19 and 2–3 µg/m3 in the 1980s and 2010s, respectively. The 40-y cumulative exposure was estimated as 216–384 µg/m3, and the resulting attributive risk (AR; lung cancer) 27–40%. However, the estimates for welders include uncertainties: 1) the correlation equations are based on plating activities, 2) U-Cr reflects total Cr exposure and welders are also exposed to CrIII, 3) HBM reflects exposure also via other than the inhalatory route, which are not particularly relevant to lung cancer. For platers, the measured U-Cr levels in the 1980s and 2010s were 0.46 µmol/l (n=771) and 0.12 µmol/l (n=3631), respectively, corresponding to air levels of 6–11 and 2–3 µg/m3. The cumulative 40-y exposure level was calculated as 162–282 µg/m3, resulting to an AR of 22–33%.
Even though the use of HBM data may in some cases result in overestimation of risk, it provides a useful tool for assessing risks of adverse health effects, as it reflects actual and total internal dose via all exposure routes.
Keywords: human biomonitoring, hexavalent chromium, lung cancer, risk assessment
Safety assessment of copper nanoparticles developed for printable electronics (#303)
P. Taxell1, H. Lindberg1, T. Kanerva1, J. Catalán1, A. Säämänen1, A. - K. Viitanen1
1 Finnish Institute of Occupational Health, Helsinki, Finland
Unique electrical properties of copper nanoparticles (Cu NPs) are being utilized in the development of conductive inks for printable electronics. Here, the safety of workers developing Cu NPs coated with polyvinyl pyrrolidone (PVP) in laboratory scale was assessed based on published toxicological data on Cu NPs and exposure measurements by on-line measurement of particle number concentration and size distribution (size range 6 nm-10 µm).
The toxicity data available on Cu NPs considers mainly the oxidized form of Cu (CuO). CuO NPs possess stronger toxic potential in vitro as compared to micron-sized CuO or soluble copper compounds, most likely explained by the efficient uptake of NPs, followed by intracellular release of copper ions. The few inhalation studies on CuO NPs in mice and rats suggest that inhaled CuO NPs can induce inflammatory responses and histological changes in rodent lungs. Intratracheal instillation of CuO NPs has been shown to induce oxidative stress, inflammation and even neoplastic lesions in rats. Based on sparse in vitro data on PVP-coated CuO NPs, the coating is not expected to increase the toxicity of Cu NPs.
Exposure measurements were carried out in four different work tasks involving Cu NPs: synthesis of Cu NPs, handling of Cu NP powder, precursor mixing and cleaning procedure of the sample. A significant increase in the particle number concentration was detected only inside the fume hood when Cu NPs were handled as dry powder. However, no simultaneous increase was detected in the breathing zone of the workers, indicating an effective capture of the released Cu NPs by the fume hood.
Although the current knowledge about the health hazards of Cu NPs is limited, the available data indicate that inhalation exposure to Cu NPs may cause pulmonary toxicity. In this case, workers’ exposure was considered negligible due to small scale process combined with effective use of control measures. Thus, the health risk was assessed to be low. The recommendations given included ensuring flawless and efficient operation of the fume hoods, changing the form of the material from powder to liquid or paste, when applicable, wearing a fit tested respirator (FFP3) in reactor cleaning and maintenance, and compiling a procedure for accidental situations.
Funded by EU H2020 NECOMADA project, Grant Agreement No. 720897.
Keywords: copper nanoparticles, occupational exposure, risk assessment, printable electronics
Two years of DNA damage monitoring in males and females occupationally exposed to nanoparticles (#440)
A. Rossnerova1, D. Pelclova2, V. Zdimal3, F. Elzeinova1, H. Margaryan1, I. Chvojkova1, J. Topinka1, J. Schwarz3, J. Ondracek3, M. Kostejn3, M. Komarc4, 5, S. Vlckova2, Z. Fenclova2, L. Lischkova2, S. Dvorackova6, P. Rossner1
1 Institute of Experimental Medicine of the Czech Academy of Sciences, Department of Genetic Toxicology and Nanotoxicology, Prague, Czech Republic
Due to the increase of nanomaterials (NM) application in many areas of human life during the last decades, assessment of genotoxicity of NM and nanoparticles (NP) is one of the main objectives of genetic toxicology. Despite this fact, human cytogenetic studies focused on micronuclei (MN) formation following the exposure to NP are still rare. Moreover, no relevant information on possible differences in sensitivity of males and females to NP exposure is available.
In this study we analyzed 4x (in September 2016 and 2017; pre-shift and post-shift each year) samples in a group of workers (both genders), working long time in nanocomposites research, and matched controls. Detail aerosol exposure monitoring of particulate matter (PM) including nano-sized fractions was completed during working shift in sampling days. The micronucleus assay using Pan-Centromeric Chromosome Paint was applied to recognize, beside the frequency of total MN in binucleated cells (BNC), also other types of chromosomal damage (losses and breaks), including the centromere positive (CEN+) and centromere negative (CEN-) micronuclei. Moreover, whole-chromosome painting for autosome #1 and both gonosomes (X and Y) were applied with the aim to identify the particular structural and numerical chromosomal aberrations.
Obtained results showed consistently: (i) differences in the risk of exposure to NP related to individual working processes; (ii) differences in chemical composition of nano-fraction; (iii) possible adaptation to chronic exposure of NP (total MN); (iv) acute exposure (2.5 h) could be a reason for the CEN+ MN increase; (v) females seem to be more sensitive to chromosomal losses. Additional data suggested increased frequency of numerical aberrations in gonosomes.
Supported by the Grant Agency of the Czech Republic #18-02079S and the Ministry of Education Youth and Sports Czech Republic #LO1508.
Keywords: DNA damage, Micronuclei, Nanoparticles, Occupational exposure
Influence of Genetic Variance on Biomarker Levels After Occupational Exposure to 1,6-Hexamethylene Diisocyanate (HDI) Monomer and HDI Isocyanurate (#458)
L. W. Taylor1, J. E. French2, Z. G. Robbins1, J. C. Boyer1, L. A. Nylander-French1
1 University of North Carolina, Department of Environmental Sciences and Engineering, Chapel Hill, North Carolina, United States of America
Isocyanates are a leading cause of occupational asthma globally, in which 5 – 15% of exposed workers develop isocyanate-induced asthma. However, very little is known about the mechanism of isocyanate-caused skin and respiratory sensitization. In order to close part of the knowledge gap, we investigated the influence of genetics in conjunction with skin and inhalation exposures to 1,6-hexamethylene diisocyanate (HDI) monomer its trimer HDI isocyanurate on plasma and urine biomarker levels of 1,6-diaminohexane (HDA) and trisaminohexyl isocyanurate (TAHI) in a population of 33 workers who spray automobiles with isocyanate-containing polyurethane paints. Linear mixed model analyses indicated that HDI monomer and isocyanurate skin and inhalation exposures are both important modifiers of HDA and TAHI biomarker levels, respectively. Therefore, in order to assess how genetics impacts biomarker levels, we used genome-wide single nucleotide polymorphism (SNP) microarray data (Affymetrix 6.0), a false discovery rate <0.10, and both skin and inhalation exposure levels as covariates in this model. Seven SNPs were significantly associated with HDA levels in plasma, five SNPs were associated with HDA in urine, eight SNPs were associated with TAHI levels in urine, while no SNPs reached significance for TAHI in plasma. Furthermore, the heterozygous genotype and homozygous minor allele genotype for these 20 SNPs were associated with an average of 10 – 16-fold higher biomarker levels compared to the homozygous major allele. To evaluate the potential biological pathways impacted by these SNPs, NCBI gene database was used to determine the genes proximal to each of the significant SNPs and then those genes were input into GeneMANIA and DAVID bioinformatics databases to infer gene-ontology based predicted network associations. The predicted molecular functions included transcription regulation, calcium ion transport, and TGF-β signaling. Our results demonstrate that genetics is an important modifier of biomarker levels following occupational exposure to HDI. In future studies, these SNPs can be used to study isocyanate toxicokinetics and to identify individuals who are susceptible to developing isocyanate-induced asthma.
Keywords: Occupational Exposure Assessment, Genetics, Biomarkers, Isocyanates, Gene-environment Interactions
Case report of the rapid successful treatment of methemoglobinemia caused by occupational exposure to aniline (#460)
S. Sarmanaev1, 2, N. Bondarenko2, I. Kryijevskikh3, I. Akhmetov1, R. Tuktarova4
1 FRCC PCM FMBA Russia, Moscow, Russian Federation
Purpose: Aniline is a colorless aromatic liquid that is widely used in the manufacturing of synthetic dyes. Occupational non-oral exposure this chemical has led to fast absorbed by all other routes and induces methemoglobinemia.
Keywords: occupational exposure, aniline, poisoning, methemoglobin, antidotes
Risk assessment for an aniline derivative ortho-toluidine by using human biomonitoring data and bioequivalent method in the HBM4EU project (#609)
P. Huuskonen1, B. Schaddelee-Scholten2, H. Buist2, J. Westerhout2, T. Santonen1
1 1 Finnish Institute of Occupational Health (FIOH), Työterveyslaitos, Finland
Ortho-toluidine (CAS 95-53-4) is an aniline derivative which is considered to be an animal and human carcinogen, and may cause methemoglobinemia. o-Toluidine is used as a curing agent in epoxy resins and as intermediate in producing herbicides, dyes, and rubber chemicals. It is listed in the candidate list of substances of very high concern for authorization under REACH. A risk assessment (RA) was performed in the HBM4EU project for o-toluidine by utilizing human biomonitoring (HBM) data since the possible health risks should be monitored, especially for workers.
After hazard characterization and exposure assessment, a literature search was conducted on studies concerning o-toluidine HBM data. The biomonitoring equivalent (BE) methodology was used for the comparison to estimate the urinary levels corresponding to the external intake levels. For the RA, the results of the BE method were compared to the available HBM studies and occupational exposure levels.
The existing cancer RA resulted in a Benchmark Dose causing 10% urinary bladder tumour incidence above background level (BMD10) of 42.2 mg/kg bw/day in rats, corresponding to an inhaled dose scaled to humans of 210 mg/m3 at occupational exposure. Converted to mg/bw/working day this BMD10 corresponds to 30 mg/kg/day. Using the BE method, this level corresponds to a urinary level of 1000 mg/L by assuming a 70-kg bw, a 1.5 L/day urinary volume and 75% excretion.
In conclusion, by applying the BE methodology and based on HBM studies, the workers exposed to o-toluidine have a cancer risk of 1:20 000 in the worst-case scenario (0.5 mg/L in urine). The exposure levels calculated based on HBM data were below the binding occupational exposure level (BOELV, 0.44 mg/m3) set under the EU Carcinogens and Mutagens Directive. However, results should be considered carefully due to uncertainties and the limited number of HBM data. There is clearly a need for further HBM studies and data on the biokinetics of o-toluidine exposure.
Keywords: Aniline, HBM4EU, ortho-toluidine, biomonitoring
Occupational exposure to monoclonal antibodies in Portuguese health units: are there reasons for concern? (#652)
A. M. Costa-Veiga1, 2, S. Viegas1, 2
1 H&TRC- Health & Technology Research Center, ESTeSL- Escola Superior de Tecnologia da Saúde, Instituto Politécnico de Lisboa, Lisboa, Portugal
Background The use of monoclonal antibodies (MABs) has been increasing in healthcare, namely, in the treatment of malignant and non-malignant diseases. These molecules are widely used in monotherapy and in chemotherapy cycles along with cytotoxic drugs. Currently the use is also increasing in veterinary practice. Thus, a considerable number of health professionals (e.g. pharmacists and pharmacy technicians, nurses, veterinarians, physicians and other) can be exposed to this new therapeutic class.
Aim To give an overview of the main guidelines to safe handling of MABs worldwide; To estimate the occupational risk of handling MABs in three Portuguese health institutions.
Methods A detailed literature search on B-on “all for all”, PubMed (includes Medline) and Web of Science was carried out using various combinations of corresponding descriptors and free text terms such as “antibodies, monoclonal", "occupational exposure", "safe handling", “management” and "risk assessment". A direct observation of the workplaces and tasks implicating the handle of the MABs in a general hospital, an ophthalmic clinic and in a veterinary hospital was also performed. This allow understanding the medication pathway at each health institution, to describe the most used monoclonal antibodies and the location (ward/pharmacy) where they are prepared or administered, critical moments where exposure can occur and recommend new procedures if needed.
Results Healthcare workers involved in the preparation or administration of MABs should be aware of the potential occupational exposure risks. Recommendations to allocate the preparation and administration of MABs according to their toxicity profile should be performed taking into account the literature evidence.
The authors are grateful to Polytechnic Institute of Lisbon for funding the project ONCOAMB Ambulatory oncology therapy: effects on Public Health and environment. IPL/2017/OncoAmb/ESTeSL.
Bauters T, Vandenbroucke J. Development of a flowchart for risk assessment and allocation of preparation of monoclonal antibodies. J Oncol Pharm Pract 2019; doi.org/10.1177/1078155217743095.
King J, et al. A review of the evidence for occupational exposure risks to novel anticancer agents – A focus on monoclonal antibodies. J Oncol Pharm Pract 2016; 22:1. doi.org/10.1177/1078155214550729.
Keywords: monoclonal antibodies, occupational exposure, safe handling, management
Evaluation of inflammatory biomarkers in agate grinding workers in Iran (#679)
E. Rafiei manesh1, M. Soukhtanloo1, H. Esmaily1, F. Ahmadi1
1 Mashhad university of medical Sciences, mashhad, Iran (Islamic Republic of)
Objective: Silicosis is a chronic progressive and life threatening occupational disease which is usually recognized at later stages of the disease. The aim of this study was to evaluate the levels of LDH (Lactate Dehydrogenase), CA125, HS-CRP (high sensitive C-reactive protein), MDA, SOD, MMP9 (Matrix Metalloproteinase, MMP2 and Copper as early biomarkers for silicosis.
Results: A total of 55 male workers were included in this study. The mean age of participants was 40.12±9.56 years and the mean of employment duration was18.27±12.54years which were statistically different between the groups. 145 (8 %) of all the individuals were current smokers. According to the analysis, there were significant differences according to spirometric parameters and plasma levels of MMP2, MMP9, HS-CRP and CA125 between 3 groups (P ≤ 0.05). In patients with silicosis all the spirometric parameters were lower compared to the other 2 groups. MMP2, MMP9, serum hs-CRP and CA125 concentrations were significantly higher in cases compared with controls. Significant correlations were also observed between values of HS-CRP and CA125 and spirometric parameters.
Keywords: Silicosis, Biomarkes, Agate industry
Experimental Study of Toxicity and Derive Occupational Exposure Limit to 6-chlorohexan-1-ol (#685)
V. M. Vasilkevich1, S. Sychik1, E. Fedorenko1, A. Drozdova1
1 Republican unitary enterprise "Scientific Practical Centre of Hygiene", Minsk, Belarus
In chemical and petrochemical production (for example, the manufacture of polyurethane foams, sealants, adhesives), 6-chlorohexan-1-ol is used, which can be released into the air during the production process and affect workers. According to the ECHA (European Chemicals Agency) 6-chlorohexan-1-ol database (CAS No. 2009-83-8) is a substance that can be harmful if swallowed and inhaled, can cause eye irritation, can cause mutations in the Ames test, does not cause sensitization and skin irritation, there is no information about the toxicity of the substance with repeated exposure. The purpose of the research was the study of the toxicity and hazard of 6-chlorohexan-1-ol in subacute and subchronic experiments on rats to substantiate the hygienic standard in the air of the working area for professional use of the substance.
In the subacute experiment (intragastric administration to rats for 14 days), the threshold of acute action (Limac – 420 mg/kg) was set to change behavioral parameters (p˂0.05 against the background of control). In a subchronic experiment (60 days), rats were intragastrically administered with 0,1 DL50 of 6-chlorohexan-1-ol – 375 mg/kg. The animals were found to have changes in morpho-functional parameters (p˂0,05 against control): body weight, neutrophil and lymphocyte content in the blood, glutathione system (SH-groups, reduced glutathione and glutatintransferase), cellular and humoral immunity (blood granulocytes, lysozyme and antimicrobial blood activity). The revealed changes were functional (reversible) and disappeared after 30 days of the recovery period. The experiment performed allows us to conclude that the subchronic dose-monotonic administration of 6-chlorohexan-1-ol to rats leads to activation of the mechanisms of antioxidant and nonspecific immune defense, which are of an adaptive nature. Using the logarithmic equations that take into account the experimental data and the physicochemical properties of the substance (molecular weight and volatility), an estimated occupational exposure limit of 6-chlorohexan-1-ol in the working area air is calculated – 8 mg/m3.
Keywords: 6-chlorohexan-1-ol, toxicity, occupational exposure limit
The Genetics of Occupational Asthma Development among Workers Exposed to Diisocyanates: A Systematic Review with Meta-Analysis (#731)
L. W. Taylor1, E. J. Price1, C. Poole2, L. A. Nylander-French1
1 University of North Carolina at Chapel Hill, Department of Environmental Sciences and Engineering, Gillings School of Global Public Health, Chapel Hill, North Carolina, United States of America
Diisocyanates are a widely used class of chemicals that pose an occupational safety concern for workers in the spray-paint and spray-foam insulation industries. Epidemiologic studies indicate that 5-15% of workers exposed to diisocyanates develop diisocyanate-induced occupational asthma (diisocyanate asthma, DA). Because only a subset of workers develops this disease, genetic susceptibility may play a role in the development of DA. As such, many researchers have studied genetic markers that may increase workers’ susceptibility for DA. The purpose of this systematic review was to compile the results on genetic susceptibility markers for DA and to meta-analyze the results for the most commonly studied genes. Three databases (Embase, Pubmed, and Scopus) were searched and 166 non-duplicate publications were identified, of which 24 relevant occupational studies were included in this review. The genome-wide association studies and candidate-gene studies on DA susceptibility identified single nucleotide polymorphisms (SNPs) within 71 different genes. Multiple studies reported on SNPs within 17 genes and, thus, those genes were included in meta-analysis: CDH17, CTNNA3, GSTM1, GSTM3, GSTP1, GSTT1, HLA-A, HLA-B, HLA-C, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRB1, NAT1, NAT2, TNF-α, and ZBTB16. These 17 genes code for proteins that are involved in many processes including xenobiotic presentation, lymphocyte and T-cell activation, response to oxidative stress, cell-cell adhesions, and interaction with histone deacetylase. Knowledge about the genetic markers that impact susceptibility to developing asthma after exposure to diisocyanates could help to determine the etiology of the disease and to identify more effective ways to protect workers’ health.
Keywords: Occupational asthma, Genetic Susceptibility, Diisocyanate Exposure, Systematic Review, Meta-Analysis
Health risk associated with delta-aminolevulinic acid dehydratase (ALAD) gene polymorphism (rs1800435C/G) in Bulgarian workers from battery recycling industry (#742)
H. Dimbarev1, D. Dimbareva1, T. P. Georgieva1, R. Georgieva1, T. I. Panev1, T. P. Kuneva2
1 National Center of Public Health and Analyses, Sofia, Bulgaria
According to data from the Association of Bulgarian Association of Metallurgical Industry in Bulgaria for the period 2012 - 2016 the production of primary and secondary lead including the production of batteries in Bulgaria has increased almost double.
A number of recent studies (WHO, IARC Monographs, 2010) found that chronic exposure to lead leads to lead poisoning at a higher frequency than expected. This implies reviewing the data and conducting new studies to assess the health effects of chronic low-exposure exposure.
Questions arise - what indicators are being explored; are they suitable? Are new strategies and biomarkers needed? Is there a traceability of results in dynamics; what are the risk reduction measures that are sustainable?
The aim of the present study is to determine the allelic frequency and distribution of individual haplotypes among Bulgarian population based on ALAD gene polymorphism (rs1800435C/G) and possibility to use as a prediction biomarker for prevention of lead intoxication.
The subject of this study is 80 workers from Bulgarian battery recycling industry, professionally exposed to lead. The following biomarkers was measured: Hematological parameters; blood lead content, delta aminolevulinic acid (DALA) levels in urine and rs1800435C/G polymorphism distribution.
All persons have signed informed consent for conducting the research.
The results of the molecular-genetic analysis demonstrates that 23% of the study population is heterozygous for ALAD-2. According to the literature, there is an increased risk for the health of these persons. Our results show trends in higher blood lead concentrations and DALA levels and decreased hemoglobin levels and white blood cells count in ALAD-2 heterozygous subjects.
The present study is first for the Bulgarian population. Tendencies of dependence between the lead of the excretion of lead and the genotype distribution are established. It is difficult to decide which genotype is genotype "at risk" because the results show that each genotype is susceptible to one or more adverse effects than others. Genetic polymorphism seems to have a strong impact on lead absorption and bioaccumulation, but its role in affecting the neurotoxicity of lead is still unclear.
1. IARC MONOGRAPHS ON THE EVALUATION OF CARCINOGENIC RISKS TO HUMANS, volume 98 ( 2010)
2. A. Moreira, et all Genotyping an ALAD Polymorphism with Real-Time PCR in Two Populations from the Iberian Peninsula, Biochem Genet DOI 10.1007/s10528-012-9500-x, Springer Science+Business Media, LLC, 2012
3 da Cunha Martins A et all. Effects of Lead Exposure and Genetic Polymorphisms on ALAD and GPx Activities in Brazilian Battery Workers, J Toxicol Environ Health A. 2015;78(16):1073-8
Keywords: ALAD -2 genotype, prediction biomarker, lead, occupational exposure
Serum Metabolomics of Occupational Noise Exposure workers in China (#762)
J. Ji1, L. Miao1, L. Wan1, R. Sun1, J. Zhang1, L. Yin1, Y. Pu1
1 Sotheast University, School of Public Health, Key Laboratory of Environmental Medicine Engineering of Ministry of Education, Nanjing, China
Occupational noise exposure has become a major public health problem in China. Previous studies have suggested that noise exposure is associated with hearing loss, psychiatric disorder and cardiovascular disease. In the present study, we sought to explore the hazards of occupational noise exposure and screen tentative metabolic markers from the perspective of serum metabolomics. The 1:1:1 matched case-control design was used in this study, 100 cases of occupational noise exposure workers (50 cases with noise-induced hearing loss while 50 cases without) were compared with 50 healthy workers by physical examination data like gender, age, smoking and drinking history. The average length of occupational noise exposure was 8.37±7.58 years in NIHL group, and 8.54±7.37 years in non-NIHL group. The noise intensity in the workplace was 80dB(A)-85dB(A). A nontargeted metabolomics approach based on UPLC-QTOF-MS was performed on serum samples to identify differentially expressed metabolites. The results showed significant differences in serum metabolic profile between exposure workers and healthy workers. The metabolite that was upregulated in both exposure groups is Oleamide. Eight metabolites including arachidonic acid, L-kynurenine, docosahexaenoic acid, dihomolinoleic acid, L-methionine, 13-HOTE, 6,10,14-Trimethyl-5,9,13-pentadecatrien-2-one and sphinganine 1-phosphate were downregulated in both exposure groups. Researches have proved that arachidonic acid metabolites metabolized by CYP450 in kidney are altered in diabetes, hepatorenal syndrome, and in various models of hypertension. The L-kynurenine is associated with nervous system disorders. Oleamide is being studied as a potential medical treatment for mood and sleep disorders. Our findings suggest that occupational noise exposure can cause disturbances in tryptophan metabolism, glycine and serine metabolism, and lipid metabolism pathway which are related to the autonomic nervous system and endocrine system. In conclusion, the present study provides new insights into the health effect caused by occupational noise exposure, and the hygienic significance of the abnormal metabolites will be investigated in further studies.
Keywords: Occupational noise exposure, Serum metabolomics
2,4-Dimethylaniline may contribute to the occurrence of bladder cancer among workers in a chemical factory (#813)
R. Wang1, T. Toyooka1, Y. Qi1, 2, Y. Yanagiba1, M. Suda1
1 Japan National Instiute of Occupational Safety and Health, Division of Industrial Toxicology and Health Effects Research, Kawasaki, Japan
Ten cases of bladder cancer was reported recently among workers in a chemical factory in Fukui of Japan, and several aromatic amines have been used for nearly thirty years there. All of the ten cases of cancer had the records of exposure to o-toluidine, a known human carcinogen classified into group I chemical by the International Agency for Research on Cancer, and nine cases were also exposed to 2,4-dimethylaniline (2,4-DMA). Information about the genotoxicity and carcinogenesis of 2,4-DMA is limited and inconsistent. The International Agency for Research on Cancer classifies 2,4-DMA as a Group 3 chemical, indicating no evidence of carcinogenicity to humans. Our study aimed to investigate whether and how 2,4-DMA is of genotoxic effects and its possible contribution to the occurrence of occupational bladder cancer. Methods: human urothelial (1T1) and hepatocyte (WRL-68) cells were treated with 2,4-DMA at different concentrations for 1-24 hr, and phosphorylated histone H2AX (γ-H2AX), a marker of DNA double strand breaks, was detected by western blot and immunofluorescence staining. To explore the mechanism underlying the genotoxic effects, reactive oxygen species (ROS) production following 2,4-DMA exposure and the mediation of CYP2E1 were evaluated. Results: it was showed that 2,4-DMA induced γ-H2AX in a dose-dependent way in both cell lines, and this effect was even comparable to o-toluidine. The double-strand breaks formed in 1T1 cells after 2,4-DMA treatment was confirmed by the biased sinusoidal field gel electrophoresis. In the mechanistic investigations, we found that 2,4-DMA induced intracellular ROS, an effect clearly attenuated by disulfiram, a strong inhibitor of CYP2E1. Furthermore, CYP2E1 inhibitors and the antioxidant, NAC, also attenuated γ-H2AX generation following exposure to 2,4-DMA. Conclusions: our results suggest that 2,4-DMA can strongly induce γ-H2AX via ROS produced by CYP2E1-mediated metabolism. Exposure to 2,4-DMA over a long period of time may have contributed to the development of bladder cancer. Our results suggested the necessity of re-assessment on the carcinogenicity of 2,4-DMA.
Keywords: 4-dimethylaniline, γ-H2AX, genotoxicity, bladder cancer
Frequency of GSTP1 and GSTM1 Null Genotype in Batik Textile Worker in Yogyakarta, Indonesia (#825)
D. A. A. Nugrahaningsih1, P. Hastuti2, S. Hartini3
1 Universitas Gadjah mada, Pharmacology and Therapy, DI Yogyakarta, Indonesia
Background: Glutathione S-transferases (GSTs) is composed of multiple isoenzymes. Some of the isoenzymes of GSTs are GSTP1 and GSTM1. Both GSTP1 and GSTM1 is known to have an important role in detoxifying various xenobiotics, including textile production related toxicant, some of which are suspected to be hazardous towards human health. Yogyakarta is one of the most productive batik textile business. Therefore many people, especially women, are work in batik textile factory in which they are exposed to potentially hazardous substance related to batik textile production.
Aim of work: To find the susceptibility of batik textile worker towards hazardous substance in batik textile production related to GSTM1 and GSTP1 polymorphism.
Conclusion: We demonstrated that GSTM1 null genotype or GSTT1 null genotypes are low in batik textile worker in Yogyakarta, Indonesia.
Keywords: GSTP1, GSTM1, batik textile occupational
CamkII Beta might protect the toxicity induced by benzene in G6PD deficient cells (#830)
H. Zhang1, T. Wang1, K. Wang1, B. Wang1, R. Sun1, L. Yin1, Y. Pu1, J. Zhang1
1 Southeast University, Key Laboratory of Environmental Medicine Engineering of Ministry of Education, School of Public Health,, Nanjing, China
Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common genetic disease, which affects nearly 400 million people worldwide. Calcium/calmodulin-dependent protein kinase type II beta chain(CAMK2B ) is a member of the serine/threonine protein kinase family and belongs to the Ca2+/calmodulin-dependent protein kinase subfamily. Here, the mRNA expression of CAMKIIB of G6PD deficient mice (G6pdxa-m1Neu mice) was found 41.8 times that of normal C3H mice in bone marrow cells. The mRNA expression of CAMK2B in G6PD low-expression mice and normal mice decreased by half after exposure to 150 mg/ (kg · d) benzene by subcutaneous exposure, but the mRNA expression level of CAMK2B in G6PD low-expression mouse was still 38 times that of normal mice. In order to investigate the role of camk2B in high expression of g6pd deficiency, K562 cells were treated with KN93, a chemical inhibitor of CAMK2B, to construct low expression CAMK2B cells. The proliferation rate of low-expression CAMK2B cells decreased and the apoptosis rate increased significantly compared with control cells. The generation of the mitochondrial ATP and the membrane potential was reduced in CAMK2B low expression cells. Then, K562 cells with low expression of CAMK2B and normal K562 cells were treated with 1,4-Benzoquinone. The toxicity of cell proliferation, apoptosis and the mitochondrial damage was increased in CAMK2B low expression cells with 1,4-BQ treatment. Therefore,a high expression of Camk2B may be a defect in balancing the low expression of G6PD and protect the toxicity induced by benzene in G6PD deficient mice. For further study, we hope to identify the mechanism of how G6PD deficient regulate a high expression in CAMK2B, that might be contribute a new insight for G6PD deficient genetic disorder. (This work was supported by the National Natural Science Foundation of China (Grants no. 81573120, 81730087)).
Keywords: G6PD deficiency, benzene, CAMK2B
Risk assessment of occupational exposure to DINP, DIDP and DPHP in plastics sector (#845)
T. M. Santonen1, S. Mahiout1, S. P. Porras1
1 Finnish Institute of Occupational Health, Työterveyslaitos, Finland
Background: The use of DiNP, DiDP and DPHP has increased in plastic product manufacturing to substitute older phthalates like DEHP. DiNP has shown anti-androgenic effects but with much lower potency than e.g. DEHP, whereas DiDP and DPHP have not clearly shown these effects. No occupational exposure limits (OELs) or biological limit values (BLVs) have been set for DiNP, DiDP or DPHP. Only few human biomonitoring (HBM) data exists on the exposure of workers to these phthalates, including the data from our own biomonitoring study in plastics workers.
Methods: For the risk assessment (RA) of DiNP, DiDP and DPHP, we used our own HBM data from Finnish plastics workers complemented with published data. DNELs calculated by ECHA were used for DiNP and DiDP RA after the adjustment for occupational exposure. For DPHP, a DNEL based on the published BMDL10 level for thyroid effects was calculated. One compartment model based methodology was applied to calculate biomonitoring equivalents (BEs) for these DNELs, and measured biomarker levels were compared to the BEs to calculate risk characterization ratios (RCRs).
Results and conlusion: Using the BE approach based on the DNEL values, the calculated BE for occupational population was 1.0 mg/L for both cx-MiNP (metabolite of DiNP) and cx-MiDP (metabolite of DIDP). The BE estimated for OH-MPHP (metabolite of DPHP) was 0.6 mg/L. RCRs were all well below 1, being the highest for DiNP (RCR=0.3 in worst case scenario). For DiDP and DPHP, RCRs were below 0.1, indicating a very low risk. Even though the BE approach used here is quite rough and gives only an estimate on the level corresponding the external DNEL value, in many cases it can be considered sufficient for RA. Current data on occupational exposure to these three phthalates is, however, very limited, and therefore exposure assessment should be viewed with caution. As these phthalates are being widely used to replace the already restricted phthalates, more occupational exposure data is needed.
Keywords: phthalates, biomonitoring equivalents, occupational exposure, risk assessment
Comparative assessment of reconstituted human airway epithelium 3D models derived from large and small airway epithelial cells exposed to whole cigarette smoke (#37)
K. Matsumura1, T. Kurachi1, S. Ishikawa1, S. Ito1
1 JAPAN TOBACCO INC., Scientific Product Assessment Center, Yokohama, Japan
Purpose: Based on the field of injury concept, cigarette smoke (CS) is thought to elicit common molecular changes throughout the respiratory tract. The recapitulation and investigation of this concept with in vitro observations might help understand the mechanism of action further. In this study, we used two different reconstituted human airway epithelium 3D models derived from large and small airway epithelial cells in a CS exposure test to investigate their potential and limitations for use in a field of injury concept in vitro study.
Methods: MucilAir and SmallAir from the same donor were individually exposed to 1R6F reference CS using the Vitrocell exposure system. Cultures were exposed to 1R6F smoke at three different concentrations prepared by controlling the dilution flow (1, 2, and 4 L/min). Cultures exposed to air were used as controls. Four repeated exposures were performed per day with a 5-min exposure and 60-min interval between exposures. Cultures at 4, 24, 48, and 72 h post-exposure timepoints were subjected to the following analyses: cytotoxicity, tissue integrity, histology, cytokine secretion, and microarray.
Results: Although there was a concentration-dependent increase in cytotoxicity, destruction of pseudostratified morphology and decreased tissue integrity were observed in MucilAir and SmallAir exposed to 1R6F smoke. SmallAir was more vulnerable to tissue damage than MucilAir. Cytokine release was increased in both models exposed to 1R6F smoke at all timepoints, and cytokine levels were higher in MucilAir than in SmallAir. The highest number of differentially expressed genes (DEGs) was observed 24 h post-exposure to 1R6F smoke at all concentrations in MucilAir, while the increased number of DEGs lasted for 72 h post-exposure at 1 and 2 L/min concentrations in SmallAir. These DEGs were similar in both models, and were predicted to be related to oxidative stress and inflammatory response.
Conclusion: CS-inducible biological effects on large and small airways were similar, but vulnerability and time-dependent reactivity were different. These findings provide informative insights into the CS effect aligned with the airway field of injury concept.
Keywords: Cigarette smoke, Whole-smoke exposure, Field of injury, Respiratory toxicity, Transcriptome
Effect of cigarette smoke extract on the functional expression of P-glycoprotein in human lung-derived A549/P-gp cells (#49)
M. Takano1, S. Higa1, Y. Furuichi1, R. Yumoto1
1 Hiroshima University, Graduate School of Biomedical & Health Sciences, Department of Pharmaceutics and Therapeutics, Hiroshima, Japan
[Purpose] The lung is the organ directly exposed to cigarette smoke in smokers, and cigarette smoking is known to affect various functional proteins in the lung. The alveolar epithelium is comprised of two morphologically and functionally different cell types, squamous type I cells and cuboidal type II cells. Using rat primary cultured alveolar epithelial cells, we previously reported that P-glycoprotein (P-gp; ABCB1) was expressed in type I-like cells, but not in type II cells, and that cigarette smoke extract (CSE) directly inhibited P-gp activity in alveolar epithelial cells. However, in order to understand the change in alveolar P-gp activity in smokers, the effect of long-term treatment with CSE should be examined. For this purpose, we have established A549/P-gp cell line naturally and stably expressing P-gp, because P-gp expression in native A549, an alveolar epithelial cell line derived from human lung, was negligible. In this study, we examined the effect of long-term treatment with CSE on P-gp expression and function using A549/P-gp cells.
[Methods] Expression of MDR1 mRNA and P-gp protein was measured by real-time PCR analysis and western blotting, respectively. P-gp activity in A549/P-gp cells was measured by uptake experiments using rhodamine 123 as a substrate. Intracellular reactive oxygen species (ROS) level was estimated by flow cytometry using dihydroethidium as a fluorescence probe.
[Results] A549/P-gp cells were pretreated with various concentrations of CSE for 96 hours, and P-gp activity was measured in the absence of CSE. CSE treatment suppressed P-gp activity in a concentration-dependent manner. MDR1 mRNA expression and P-gp protein level were also suppressed by the long-term treatment with CSE. Intracellular ROS level was increased by CSE treatment, which was suppressed by α-tocopherol. In addition, CSE-induced suppression of P-gp activity was also attenuated by co-treatment with α-tocopherol. In conclusion, long-term treatment of A549/P-gp cells with CSE suppressed P-gp activity as well as its expression in alveolar epithelial cells, and ROS may be involved in the suppression of P-gp by CSE. The role of intracellular signaling pathways including MAPK pathways in CSE-induced suppression of P-gp will also be discussed.
Keywords: Alveolar epithelial cells, Cigarette smoke extract, P-Glycoprotein, Reactive oxygen species, MAPK pathways
Dose-dependent cytotoxicity assessment of nitrogen dioxide following pure or compounded exposures through the air liquid interface: in vitro (#79)
P. Bin1, T. Yu1, S. X. - F. Adamson2, 3
1 National Institute of Occupational Health and Poison Control，Chinese Center for Disease Control and Prevention, Key Laboratory of Chemical Safety and Health, Beijing, China
As a major air pollutant from vehicle emission, nitrogen dioxide (NO2) is associated with various respiratory diseases. However, few studies investigate the dose-dependent effect of NO2 following pure or compounded exposure using the newly developed air-liquid interface (ALI) exposure system with airway epithelial cells to simulate the real life airway inhalation exposure route. This study aimed to investigate the dose-dependent cytotoxicity in human A549 cells that exposed to pure NO2 or gasoline engine exhausts (marked for compounded exposure of NO2) using the ALI exposure system. Following exposure to pure or compounded NO2 through ALI at a flow rate of 15 ml/min/well for 1 h, the cell relative viability (CRV) of A549 cells was analyzed using MTT assay. The benchmark dose (BMD) and limit of benchmark dose (BMDL) were calculated to evaluate the cytotoxicity of NO2 according the benchmark dose software developed by the U.S Environmental Protection Agency. Our results revealed that the CRV of A549 cells was significant decreased along with the increased concentration of NO2 in both pure and compounded exposure circumstances (p<0.05). The BMD and BMDL of NO2-induced cytotoxicity estimated by the best fitting model were 4.40 mg/m3 and 2.74 mg/m3 for pure exposure, 2.83 mg/m3 and 1.96 mg/m3 for compounded exposure, respectively. Taken together, our findings clearly show an increased dose-dependent cytotoxicity in A549 cells following the ALI exposure to NO2 from pure exposure to compounded exposure, which provides basic data for evaluating the toxic effect of NO2 through inhalation. (Funding Support: National Natural Science Foundation of China (No.81472955))
Keywords: Gasoline engine exhausts, Nitrogen dioxide, Air-liquid interface, A549 cells, Benchmark dose
Expression of receptors for adhesion molecules in monocytes exposed to urban particulate matter is independent of size and composition of the particles. (#176)
E. Alfaro-Moreno1, R. Quintana-Belmares2, A. Montiel-Davalos2, A. Gustafsson1, J. Miranda5, R. Lopez-Marure3, I. Rosas-Perez4
1 Örebro University, Man-Technology-Environment Research Center, Örebro, Sweden
Exposure to urban particulate matter has been related to increases in mortality and visits to emergency rooms. Among the many described effects, local and systemic outcomes have been reported. Endothelial dysfunction and activation of monocytes are among the systemic effects of inhaled particles. In this study, we did evaluate urban particulate matter with aerodynamic sizes of 10 (PM10) and 2.5 (PM2.5) μm collected from November 2012 to May 2013 in the central region of Mexico City, a place with high population density and heavy traffic. The particles were collected using a high volume sampler on cellulose nitrate membranes and mechanically dislodged from the membranes1. The recovered particles were characterized for their content of metals, total carbon, organic carbon, elemental carbon, polycyclic aromatic hydrocarbons (PAHs), phthalates, endotoxins, and size distribution. Human monocytes were exposed to 0.001, 0.003, 0.01, 0.03, 0.3, 3.0 and 30 μg/mL of the different particles and we did evaluate the expression of early (sLex, PSGL-1) and late (LFA-1, VLA-4, αV-β3) receptors for adhesion molecules by means of flow cytometry. Unexposed cultures were used as negative controls and cultures exposed to 10 ng/mL of TNFα were used as positive controls.
In the following table we show the ranges of concentrations for metals, total carbon, PAHs and endotoxins for PM10 and PM2.5:
1) Alfaro-Moreno E, et al. Induction of IL-6 and inhibition of IL-8 secretion in the human airway cell line Calu-3 by urban particulate matter collected with a modified method of PM sampling. Environmental Research 2009; 109: 528-535
Keywords: PM10, PM2.5, monocytes, adhesion molecules
E-cigarettes induce lower biological responses than conventional cigarettes: A comparison of in vitro toxicity following repeated whole aerosol exposure to human bronchial tissue for 4 weeks (#180)
L. Czekala1, R. Wieczorek2, E. Trelles Sticken2, L. Simms1, L. M. Bode2, M. Stevenson1
1 Imperial Brands PLC, Scientific Research and Harm Reduction, Bristol, United Kingdom
Numerous public health bodies and governments worldwide have indicated that e-cigarettes have a central role to play in combustible tobacco harm reduction. With the increasing popularity of the Next Generation Nicotine Delivery Products, it is important to assess their potential biological impact in a robust, advanced, human-relevant biological systems, more closely modelling human exposure scenario.
This study compared the in vitro toxicological responses of a 3D organotypic model of the human airway epithelia (MucilAir™, Epithelix) following repeated exposures to either myblu™ aerosol (Tobacco flavour e-liquid 1.6% [w/w] nicotine) or Kentucky Reference Cigarette (3R4F) smoke. MucilAir™ tissues were repeatedly exposed at the air liquid interface (ALI) for 4 weeks to either 30, 60 or 90 puffs of aerosol/smoke using Imperial Brands’ Smoke Aerosol Exposure In Vitro System (SAEIVS). The smoke/aerosol was generated using the Health Canada Intense smoking regime for 3R4F (55mL/2s/30s) and the CORESTA Recommended Method N°81 (55mL/3s/30s) for myblu™. Cigarette smoke was diluted with filtered air (1:17) whilst myblu™ aerosol was applied undiluted. Endpoints measured included Cilia Beat Frequency (CBF) and Cilia Active Area (CAA), cytotoxicity (LDH), and Transepithelial Electrical Resistance (TEER). Inflammatory markers (IL-1β; IL-6; IL-8; TNF-α; MMP-1, 3, 9) secreted into the culture media were measured using MESO Scale QuickPlex™. Tissue histology was assessed using H&E/Alcian Blue immunostaining. Fox-J1 and MUC-5-AC were used to stain for ciliated cells and mucin, respectively. The nicotine dosimetry was performed to assess smoke/vapour delivery to the in vitro model.
Data demonstrates a dose response to diluted cigarette smoke in various endpoints assessed, including changes to tissue morphology, significant increase in inflammatory markers, CBF & CAA at all doses tested. myblu™ aerosol did not induce such changes, even at highest dose, when tested undiluted for 4 weeks.
The results from various functional and mechanistic endpoints assessed, adds to the weight-of-evidence approach to substantiate the harm reduction potential of e-cigarettes for adult smokers. The study also shows that the in vitro 3D organotypic lung model is a sensitive and robust tool for the assessment of lung toxicity.
Keywords: E-cigarette, Respiratory, 3D lung models, In vitro toxicology, Lung
Toxicity of combustion-derived particles emitted from different biomass sources in human bronchial epithelial cells (#246)
S. Marchetti1, J. A. Holme2, P. Mantecca1, A. Colombo1, J. Øvrevik2, S. Mollerup3
1 University of Milano-Bicocca, POLARIS Research Centre, Department of Earth and Environmental Sciences, Milano, Italy
This work was supported by Research Council of Norway, through the Better Health programs (grants n° 260381).
Biomass burning is recognized as a main source of air pollutants. Combustion-derived particles (CDPs) have been linked to several respiratory diseases, including lung cancer. In the present study we investigated effects of CDPs originating from different sources on epithelial-to-mesenchymal transition (EMT), a crucial step in the carcinogenic process. The aim of the study was to characterize and compare the relative role of the particle core versus extractable organic compounds.
CDPs (PM10) were collected from a stove fueled with pellet, charcoal or wood, respectively, and chemically characterized. Human bronchial epithelial cells (HBEC3-KT) were exposed to 2.5 μg/cm2 of whole PM, organic extracts and washed particles. The endpoints measured included cell viability, inflammatory responses, and cell migration.
CDPs showed different chemical compositions: pellet PM was enriched in metals, while charcoal and wood ones have higher PAHs content.
The results showed that CDPs differentially modulated cell viability and proliferation, and induced alterations in cell migration. Interestingly, our data revealed that the effects induced by the particles and by the adsorbed chemicals depended on the PM source; whereas exposure to washed pellet and wood PMs in general gave less response than whole particles and organic extracts, responses induced by washed charcoal were higher than from pristine particles. Additional studies on the expression of genes involved in these processes will provide additional information on the toxicological mechanisms.
In conclusion, the present study suggests that specific components attached to the particles could be responsible for the diverse effects observed following exposure to pellet and wood PMs; whereas with regard to charcoal, the PM as such appeared more toxic. The study highlights the importance of studying CDPs from different biomass sources and that more targeted strategies should be implemented to reduce the biological impact caused by the emission of biomass-propelled heating systems and to prevent hazardous health effects.
This work was supported by Research Council of Norway, through the Better Health programs (grants n° 260381).
Keywords: Biomass, EMT, in vitro exposure, lung carcinogenesis
The pulmonary damages induced by Polyhexamethyleneguanidine phosphate (PHMG-p) are irreversible (#371)
H. - S. Yang1, M. Kang1, K. Lee1
1 Koreal Institute of Toxicology, National Center for Efficacy evaluation for Respiratory disease product, Jeongeup, Republic of Korea
Polyhexamethyleneguanidine phosphate (PHMG-p) was used as a disinfectant to prevent contamination or growth of bacteria. The causal relationship was revealed between PHMG-p inhalation exposure and induction of pulmonary fibrosis. However, little is known about recovery of PHMG-p induced damage, especially lung damage. To determine recovery possibility of PHMG-p-induced damage, we evaluated change of damage in recovery period. Male specific-pathogen-free Sprague Dawley rats aged 7-8 weeks were nose-only exposed of aerosolized PHMG-p. PHMG-p aerosol was generated by constant atomizer and clean air was added to adjust the target concentration. The rats were exposed for 6 hours/day, 5 day/week for 4 weeks. Hematological change, organ weight and histopathologic change were examined. PHMG-p exposed rat showed decrease in body weight and food intake, hematological alteration, organ weight change, and histopathologic change were observed. The change of food intake, hematological alteration and organ weight except lung weight were returned to normal range in recovery period. However, PHMG-p-induced pulmonary damage included increase of lung weight and severity of microscopic findings in lung were irreversible during recovery period.
* This study was funded by the Korea Ministry of Environment (MOE) as ‘‘the Environmental Health Action Program(2017001360002).'
Keywords: Inhalation toxicology, polyhexamethyleneguanidine phosphate
Increased Throughput and Cryopreservation of Precision-Cut Lung Slices Extend the Utility of Human-Relevant, 3-Dimensional Pulmonary Test Systems (#500)
B. Gilbert1, P. Desai1, K. Amin2, D. Sheehan1, N. Castro1, H. Behrsing1
1 Institute for In Vitro Sciences, Inc., Gaithersburg, Maryland, United States of America
Human-relevant, in vitro/ex vivo assays are considered an ethical and economically viable manner by which to screen the thousands of chemicals requiring hazard assessment. Of the 3-dimensional models, human precision-cut lung slices (PCLS) are often considered the most physiologically relevant pulmonary test system, but lower throughput and difficulties in cryopreservation have hampered PCLS use. We have modified a tissue slicer to accommodate 3 tissue cores for simultaneous slicing. Increased slice production was quantified using agarose and tissue cores in the slicer. To evaluate cryopreservation of PCLS, we have tested 5 cryopreservation formulations using PCLS (frozen on the day of slicing, or after overnight culture). Thawed slice viability in each of the groups was assessed with the WST-8 viability assay, prior to fixation and histological evaluation. The slicer modification resulted in 2.8-fold and 2.4-fold more slices from agarose cores, and lung cores, respectively. Cryopreservation efforts indicated freezing after slicing yields better average viability (48-73% of fresh, non-frozen control) than culturing overnight and freezing (13-54% of control) when assessing health over 4 days, post-thaw. Cryopreservation buffers containing University of Wisconsin preservation solution preserved viability the best (54%-90% of non-frozen control). Histological findings concurred with WST-8 viability results and indicated the retention of healthy lung tissue features (>75% of normal), post-thaw. The increased PCLS production indicates larger (or multiple) studies can be initiated from one donor lung. The promising cryopreservation results suggest slices can be banked and utilized at a later date, potentially even allowing the same donor’s tissue to be used repeatedly.
Keywords: in vitro, respiratory, Precision-Cut Lung Slices, cryopreservation, pulmonary
Excipients for orally inhaled drug products - How can cell culture models facilitate drug development and replace animal experiments? (#510)
J. K. Metz1, 2, L. Scharnowske1, F. Hans1, K. Knoth1, S. Schnur1, H. Zimmer3, M. Limberger4, H. Groß1, C. - M. Lehr5, 2, M. Hittinger1
1 PharmBioTec GmbH, Drug Delivery, Saarbrücken, Saarland, Germany
Cellular-based in vitro models of the respiratory tract support data for safety and efficacy testing of orally inhaled drug products . Recent studies consider more complex co-culture systems, physiological relevant air-liquid conditions and aerosol deposition. However, there are still no guidelines (f. e. OECD, FDA) considering cellular based in vitro tools of the lungs. A possible explanation is a missing in vitro - in vivo correlation, which proves relevance for replacing animal tests or even predicting human based data. In this study we compared in vitro cytotoxicity data of pharmaceutical excipients (IC50) with their related FDA approved concentration and their GHS classification. Calu-3, A549 and hAELVi cells were used as simple monolayers addressing conducting airways and the alveolar space. A standardized MTT assay was applied to determine a dose-response curve for IC50 calculation. In order to predict the GHS classification, excipients were tested up to 10 mg/mL (1%) and ranked according to the in vitro hazard classification from Sauer et al . Some FDA parenteral and pulmonal approved excipients were above 10 mg/mL and further studies were performed to determine the IC50 when possible. For some compounds, the solubility of a compound was reached before an IC50 was measured. Furthermore, we investigated inflammatory responses of selected compounds on differentiated THP-1 macrophages (dTHP-1) focussing on the release of TNF-α and Interleukin-8. The cellular models were able to predict some in vivo aspects, but the immune response data generated by dTHP-1 show – so far – high variability between experiments. Data on cytotoxicity outlined a suitable working range for formulation development which is in accordance with FDA and GHS data. Our future work will focus on combining different in vitro assays in order to establish an in vitro test strategy reducing animal experiments.
Acknowledgements: The presented data were collected from two projects. The BMBF project AeroSafe (031L0128C) with the aim to set up a test strategy for orally inhaled compounds and the ZIM project NanOK with the aim develop a new pulmonary drug formulation.
1. Hittinger M, Schneider-daum N, Lehr C-M. Cell and tissue-based in vitro models for improving the development of oral inhalation drug products. Eur. J. Pharm. Biopharm. 2017;118:73–8.
2. Sauer UG, Vogel S, Hess A, Kolle SN, Ma-Hock L, van Ravenzwaay B, et al. In vivo-in vitro comparison of acute respiratory tract toxicity using human 3D airway epithelial models and human A549 and murine 3T3 monolayer cell systems. Toxicol. Vitr. Elsevier Ltd; 2013;27:174–90.
Keywords: respiratory safety, pulmonal in vitro in vivo correlation, 3R principle
Study on the test of the inhalation exposure of sodium dichloroisocyanurate (NaDCC) aerosols for the inhalation toxicity testing (#525)
Y. - H. Kim1, 2, Y. - J. An3, 4, S. Jo1, S. - H. Lee1, S. - J. Choi1
1 Korea Institute of Toxicology, Jeonbuk Department of Inhalation Research, Jeongeup, Republic of Korea
Sodium dichloroisocyanurate (NaDCC) is one form of chlorine used for disinfection or biocide.1 The NaDCC is used to disinfect water and is now widely available for household water treatment.2,3 The inhalation safety data (i.e., inhalation toxicity data) of NaDCC are needed because NaDCC can be inhaled depending on its use conditions and approaches. To test the inhalation toxicity, the inhalation exposure methods to expose the target materials to experimental animals are preferentially established considering the reactivity, stability, and concentration levels of target materials.4,5 In this study, the test for development of inhalation exposure method for inhalation toxicity testing of NaDCC aerosols were conducted: (1) stability test of NaDCC solution concentration, (2) confirmation of maximum exposure concentration of NaDCC aerosols, (3) comparison of chlorine concentrations between NaDCC solution and NaDCC aerosols, and (4) validation of sampling method for collection of NaDCC aerosols. The concentrations of free available chlorine in 15% NaDCC solutions were maintained for six hours after preparation of the NaDCC solution: (1) mean concentrations and of free chlorine (0, 0.5, 1, 3, and 6 hours after preparation of NaDCC solution, n=5) = 9.87 ± 0.33% (distilled water (DW) solvent) and 9.69 ± 0.29% (tap water (TW) solvent) and (2) mean mass fraction of free chlorine in NaDCC solutions (0, 0.5, 1, 3, and 6 hours after preparation of NaDCC solution, n=5) = 65.8 ± 2.17% (DW) and 64.6 1.95% (TW). The maximum exposure concentration of NaDCC aerosols were 0.069 ± 0.011 mg/L of nose-only inhalation chamber and 1.61 ± 0.05 mg/L of whole-body chamber, respectively. The maximum exposure concentrations are higher than the LC50 value of NaDCC reported by the US EPA (Sprague Dawley Rat; LC50 <1.17 mg/L, >0.27mg/L; four hours exposure). The mass fraction of free chlorine in NaDCC aerosols collected by glass fiber filter averaged 51.1 ± 2.81% and the mass fraction values were similar, regardless of solvent types (DW vs. TW) and chamber types (whole-body chamber vs. nose-only inhalation chamber). The combined free chlorine were detected from the NaDCC aerosols at mean 4.53 ± 0.67%. The combined chlorine can be made by the steps of NaDCC aerosolization and filter sampling. It is expected that the reliable inhalation toxicity data for disinfectants are obtained by conducting the test for the establishment of the optimal inhalation exposure method presented in this study.
*This work was supported by the Korea Institute of Toxicology, Republic of Korea [KK-1904].
(1) Clasen, T.; Edmondson, P. Int J Hyg Environ Health 2006, 209(2), 173-181.
(2) Pinto, G.; Rohrig, B. J. Chem. Educ. 2003, 80(1), 41-44.
(3) Tyan, K.; Kang, J.; Jin, K.; Kyle, A.M. Am. J. Infect. Control 2018, 46(11), 1254-1261.
(4) Shim, H.E.; Lee, J.Y.; Lee, C.H.; Mushtaq, S.; Song, H.Y.; Song, L.; Choi, S.-J.; Lee, K.; Jeon, J. Chemosphere 2018, 207, 649-654.
(5) Ahn, K.; ensor, D.; Shama, M.; Ostraat, M.; Ramsden, J.; Kanno, J.; Ghazikhansari, M.; Lazos, R.; Guumian, M.; Cassee, F.R.; De Jong, W.H.; Jeon, K.; Yu, I.J. Toxicol. Open Access 2017, 3(2), 1000127.
Keywords: sodium dichloroisocyanurate (NaDCC), free available chlorine, inhalation toxicity, inhalation exposure
Functionalization of carbon nanotubes change their toxicity mechanisms induced in alveolar macrophages (#585)
S. Nahle1, R. Safar2, Z. Manel Doumandji1, J. Ghanbaja1, B. Rihn1, O. Joubert1, L. Ferrari1
1 Jean Lamour Lamour, Institute, Nancy, France
Functionalized multiwall carbon nanotubes (MWCNTs) have become the focus of increased research interest, particularly in their application as tools in areas such as the biomedical field. Despite the benefits associated with functionalization of the MWCNTs, particularly in overcoming issues relating to solubility, several studies have demonstrated that these functionalized nanoparticles display toxicity. For this study, three well-characterized MWCNTs of similar diameter and length but varying function groups were investigated: a pristine non-functionalized MWCNT (NM403), an anionic MWCNT with a carboxyl group (NRCWE-042) and finally a cationic MWCNT with an amino group (NRCWE-049). This study proposes that the MWCNT toxicity mechanisms are charge dependent and employed cytotoxicity assays, transcriptomics and proteomics to assess the toxicity of the three different MWCNTs using the NR8383 rat alveolar macrophage cell line as an in vitro model. The study findings indicated that all three MWCNTs altered ribosomal protein translation, cytoskeleton arrangement and induced inflammation. Additionally, functionalization of the MWCNTs was also shown to alter normal signaling pathways, providing evidence of dysregulation of the mTOR signaling pathway in conjunction with increased Lamtor gene expression. Furthermore, the type of functionalization was also shown to be important, with the cationic MWCNT activating the transcription factor EB and inducing cell death via autophagy while the anionic MWCNT altering eukaryotic translation initiation factor 4 (EIF4) and phosphoprotein 70 ribosomal protein S6 kinase (p70S6K) signaling pathway as well as upregulation Tlr2 gene expression. This study also provides evidence that lysosomal stress could be considered as a biomarker of effect to cationic carbon nanotubes. Considering all of the study findings, it can be concluded that functionalization of CNTs is directly linked to their observed toxicity in macrophages.
This work has received funding from the European Union’s Horizon 2020 research (SmartNanotox project).
Keywords: multiwall carbon nanotubes, functionalizing, alveolar macrophages, omics, In vitro toxicity
Comparison of toxicity of Oligo(2-(2-ethoxy)ethoxyethyl guanidinium chloride and Polyhexamethylene-guanidine phosphate in mice (#592)
J. Song1, M. Yang1, J. - H. Hwang1, S. - C. Han1, K. Lee1, 2
1 Korea Institute of Toxicology, Jeonbuk Department of Inhalation Research, Jeongeup, Republic of Korea
Oligo(2-(2-ethoxy)ethoxyethyl guanidinium chloride (PGH) and Polyhexamethylene-guanidine phosphate (PHMG) are polymeric biocides with a guanidine group. They are causative agents of the tragic humidifier disinfectant incident, which had caused death of over 179 people in Korea. The aim of this study was to assess and compare the toxic effects of PGH and PHMG when they are directly exposed to the lungs. To assess the toxicity of PGH and PHMG, 0.0125%, 0.0325%, and 0.0625% PGH or PHMG were instilled into the lungs of mice and the mice were necropsied at day 7 and day 14. Body weights, cytokine production, and histopathological examination were performed and T cell subset distribution was evaluated by flow cytometry assay. The body weights of the intermediate- and high-dose PGH or PHMG groups reduced right after instillation. The mice of the PGH group gained weight from day 4 onward, whereas the body weight of the PHMG-P groups did not recovered. Lung weights were significantly increased in the intermediate- and high-dose PGH or PHMG groups. Thymic atrophy was detected in the intermediate- and high-dose PHMG groups. Both PGH and PHMG-P induced immune cells infiltration, atrophy/necrosis of bronchial epithelium in the lungs. Lung fibrosis was observed only in the PHMG groups. Interestingly, the inflammatory changes were weakened in the PGH group at day 14, but exacerbated in the PHMG group, though the inflammation grades of the low-and intermediate-dose PGH group were higher than those of the PHMG group at day 7. Production of proinflammatory cytokines were increased at day 7 and then restored to baseline levels except IL-1β in the PGH group, whereas the PHMG group showed increasing cytokine levels at days 7 and 14. Because thymic atropy was detected in PHMG group, we studied the subset distribution of CD4+CD8-, CD4-CD8+, CD4+CD8+ in the thymus of both groups. The percentage and total numbers of CD4+/CD8+ cells were markedly suppressed in the PHMG group, whereas there were no changes in the PGH group. T cells are known to play an important role in limiting the innate immune responses. Aberrant T cells development might leads to an inappropriate resolution of inflammation with fibrotic changes in the PHMG group. PGH and PHMG leads to pulmonary inflammation when they were exposed to the lung. And Exposure of PHMG to the lungs showed more severe adverse effect than that of PGH.
Guarda G, Dostert C, Staehli F, Cabalzar K, Castillo R, Tardivel A, Schneider P, Tschopp J (2009) T cells dampen innate immune responses through inhibition of NLRP1 and NLRP3 inflammasomes. Nature 460:269-73
Izbicki G, Segel MJ, Christensen TG, Conner MW, Breuer R (2002) Time course of bleomycin-induced lung fibrosis. Int J Exp Pathol. 83, 111-119.
Kolb M, Margetts PJ, Anthony, DC, Pitossi F, Gauldie J (2001) Transient expression of IL-1beta induces acute lung injury and chronic repair leading to pulmonary fibrosis. J Clin Invest. 107, 1529-1536.
Korea Centers for Disease Cotnrol and Prevention. Interim report of epidemiological investigation on lung injury with unknown cause in Korea. Public Health Weekly Report KCDC 2011, 4, 817-832 (In Korean).
McDonnell G and Russell AD (1999) Antiseptics and disinfectants: activity, action, and resistance. Clin Microbiol Rev. 12, 147-179.
Mishra BB, Rathinam VA, Martens GW, Martinot AJ, Kornfeld H, Fitzgerald KA, Sassetti CM (2013) Nitric oxide controls the immunopathology of tuberculosis by inhibiting NLRP3 inflammasome-dependent processing of IL-1beta. Nat Immunol 14:52-60
Song JA, Park HJ, Yang MJ, Jung KJ, Yang HS, Song CW, Lee K (2014) Polyhexamethyleneguanidine phosphate induces severe lung inflammation, fibrosis, and thymic atrophy. Food Chem Toxicol 69:267-75
Keywords: Oligo(2-(2-ethoxy)ethoxyethyl guanidinium chloride, Polyhexamethylene-guanidine phosphate, pulmonary toxicity
Optimization and validation of VITROCELL® 24/48 in vitro inhalation exposure system ready for testing petroleum-derived substancesOptimization and validation of VITROCELL® 24/48 in vitro inhalation exposure system ready for testing petroleum-derived substances (#608)
S. Verstraelen1, J. Van Laer1, A. Jacobs1, K. Hollanders1, R. Brabers1, S. Remy1, L. Geerts1, M. Spruyt1, H. Witters1, L. Deferme2, E. Frijns1
1 VITO NV (Flemish Institute for Technological Research), Unit HEALTH, Mol, Belgium
There is an increasing demand to implement in vitro alternatives to in vivo experimentation in different research areas to comply with the 3R concept. As the inhalation route is one of the most relevant routes of exposure, alternatives approaches would need realistic lung cell models (e.g. 3D), realistic inhalation exposure systems (i.e. air-liquid interface (ALI)), and proper dosimetry techniques to increase the predictive ability of in vitro cell models and therefore accelerate the shift from in vivo towards in vitro testing. The ultimate goal of the ‘PETRALI’ project is to develop an alternative method for in vivo inhalation testing of petroleum-derived substances. This is preceded by (i) development and validation of a generation facility to obtain vapors from oil derivatives; (ii) optimization and validation of VITROCELL® 24/48 exposure system for negative control (clean air), positive control (nitrogen dioxide), and ethylbenzene (EB) testing. VITROCELL® 24/48 is designed to perform a dose-response profile in one run. Up to 6 dilutions with 6 inserts can be used for exposure to compounds and 6 inserts in the same system are used for negative and positive control exposure, respectively. A generation facility was successfully developed at VITO to volatilize a single compound (EB) and complex substances (gasoline). Experiments with VITROCELL® 24/48 were performed to test different humidification systems, trumpet heights, flows, and cell models (A549, Calu-3, MucilAirTM) to find the most optimal settings. Quality control charts for cell viability of negative and positive control exposures were established according to expectations. Generation up to 33000 mg/m3 EB gave no effect on A549 cell viability. Chemical analysis showed very low deposition (<1%) of EB. Generation of up to 50000 mg/m3 resulted in 53% cell viability compared to clean air. Additional endpoints, such as inflammation and oxidative stress, will be evaluated in A549 as well. An overview of the optimization and validation of VITROCELL® 24/48 using EB will be shown and further evaluation will define when the system is ready for testing petroleum-derived substances, e.g. gasoline.
 Scrucca, L. (2004). qcc: an R package for quality control charting and statistical process control. R News 4/1, 11-17.
Keywords: air-liquid interface, petroleum-derived substance, in vitro inhalation testing
AhR knockout alters formation of prostaglandins in a human model of alveolar epithelial type II cells (#622)
G. Vazquez-Gomez1, J. Neca2, M. Karasova1, J. Mathews3, M. Machala2, J. Vondracek1
1 Insitute of Biophysics CAS, Department of Cytokinetics, Brno, Czech Republic
Type II alveolar epithelial (AEII) cells play a major role in the maintenance of lung homeostasis, as they produce surfactant, serve as stem/progenitor cell population, and, in conjunction with immune cells, contribute to regulation of immune response and inflammation within lung tissue. Formation of prostaglandins, which are known as important inflammatory regulators, has been shown to have a major impact on lung anti-microbial defense and tissue damage. Several studies support the hypothesis that the aryl hydrocarbon receptor (AhR), transcription factor that mediates cellular responses to airborne pollutants, such as polycyclic aromatic hydrocarbons (PAHs), could play an indirect role in regulation of expression/activity of major inducible enzyme(s) involved prostaglandin production, such as cyclooxygenase-2. However, the role of AhR in prostaglandin production has not been largely explored in the context of human AEII cells. Therefore, in the present study, we employed both wild-type and AhR knockout human adenocarcinoma A549 cells as a surrogate model for human AEII cells, in order to comprehensively evaluate the functional role of AhR in regulation of prostaglandin synthesis (and other eicosanoids). The LC/MS/MS analysis of eicosanoids in cell culture media, derived from A549 cells, revealed that loss of AhR was associated with a minor increase of arachidonic acid levels and some of its lipoxygenase-derived metabolites. In contrast, the AhR knockout led to a striking increase in the levels of some prostaglandins, such as PGE2 (and its metabolite, 13, 14 dihydro-15-keto-PGE2) and PGF2alpha. Levels of several other prostaglandins, such as PGD2, PGA2 and PGF2beta were also increased in A549 AhR knockout cell medium, albeit to a lower extent. Importantly, when A549 cells were treated with a model inflammatory cytokine, tumor necrosis factor-alpha, this led to a massive up-regulation of PGE2, PGF2alpha, PGD2 and PGA2 production in AhR knockout cells, which was up to several orders of magnitude higher than in wild type A549 cells. The production of PGE2 (as well additional prostaglandins identified in the present study), known to play primarily protective roles in the context of lung injury, could be thus significantly altered by deregulation of AhR activity in human AEII cells. Therefore, we are currently exploring the impact of AhR knockout (and model environmental AhR ligands, such as PAHs and their mixtures) on principal enzymes involved in eicosanoid synthesis, or other inflammatory mediators in A549 cells, as well as in additional AEII cell models. [This study was supported by the Czech Science Foundation, grant No. 18-00145S.]
Keywords: type II pneumocytes, A549 cells, AhR, prostaglandin, inflammation
Safety assessment of compounds – an in chemico/in vitro test strategy for inhalable substances from chemical, consumer goods and pharmaceutical industry (#683)
B. Blömeke1, U. Bock1, J. Lichter1, A. Buth1, J. K. Metz2, M. Hittinger2, H. Groß2, B. Birk3, L. Ma-Hock3, N. L. Krutz4, C. - M. Lehr2, R. Landsiedel3
1 Trier University, Environmental Toxicology, Trier, Rhineland-Palatinate, Germany
The goal of our joint research project (BMBF 031L0128A-D) is to reduce the number of animal experiments needed for safety assessment by establishing a standardized in chemico/in vitro test strategy. Different and complex respiratory cell culture based models with different endpoints of often unknown relevance for human safety assessments have been described. However, a set of predictive markers to identify the human respiratory toxicity potential of a broad variety of inhalable substances still needs to be determined. Based on available in vivo data of chosen test materials and mechanistic knowledge, our project focuses on inflammatory effects, modulation of epithelial barrier function and activation of the innate immune system. Our objective is to establish the simplest but appropriate test strategy. Starting from the first line of defense in the alveolar region, we are studying human macrophages (differentiated THP-1, blood-derived macrophages and alveolar macrophages) followed by coculture of macrophages (differentiated THP-1) with alveolar epithelial cells hAELVi (human Alveolar Epithelial Lentivirus immortalized), and consequently further target cells. We include validated approaches for analyzing protein reactivity. Established protocols will be tested with representative compounds consisting of chemicals, pharmaceuticals and nanomaterials. A significant part of the work is dedicated to the optimization and standardization of research protocols and their performance in different laboratories in order to come to a robust protocol for routine assessment of the compound-induced effects. The standardized protocol and data gained here should benefit investigators and considerably contribute to reduction of the currently high number of so far indispensable animal experiments.
Acknowledgement: Funded by German Federal Ministry of Education and Research; Acronym: AeroSafe, 031L0128A-D, 2017-2020
Keywords: respiratory toxicity, alveolar type I cell, macrophages
Cadmium and Lead mixture and lung cancer development: Toxicogenomic data mining approach (#726)
K. S. Živančević1, K. Baralić1, D. Jovanović1, A. Jovanović1, B. Antonijević1, Z. Bulat1, M. Ćurčić1, A. Buha Djordjevic1, D. Javorac1, V. Matović1, D. Đukić-Ćosić1
1 University of Belgrade , Faculty of Pharmacy, Belgrade, Serbia
Lung cancer is one of the biggest problems in pulmology and the leading cause of cancer mortality worldwide. Recently, there has been an increasing attention for the influence of environmental pollutants in the etiopathogenesis of this multifactorial disease. Among them, metals, such as cadmium (Cd) and lead (Pb), are considered of great significance, both because of their toxicity and increasing human exposure.
The aim of this in silico study was to analyze the individual and combined effects of cadmium and lead on the expression and activity of genes associated with the development of lung cancer by using the toxicogenomic data mining approach.
The Comparative Toxicogenomic Database (CTD) and its tools (Batch Query, MyVenn, VennViewer and Set Analyzer) were used to obtain the information about the interactions of investigated metals with genes/proteins associated with lung cancer development. Data on the function of genes were obtained from the GeneCards database, while GeneMania prediction server revealed detailed gene interactions.
Cadmium interacted with a total of 2645 and lead with a total of 3058 genes, of which 109 (Cd), and 70 (Pb) were associated with the development of lung cancer (Batch Query). MyVenn CTD tool revealed that lead and cadmium interacted with 48 common genes (additively/synergistically with 21 and antagonistically with 7 genes). The most important are the following genes: AKT1, CRP, FAS, GPX1, GSTP1, GSTT1, HMOX1, IL10, IL1B, JUN, KRAS, MAPK1, MAPK3, NOS2, TNF and TP53. They participate in 189 different metabolic pathways associated with the development of lung cancer (Set Analyzer), including MAPK signaling pathway and TNF signal pathway that affect a wide range of biological processes, such as cell growth, adhesion, transcription, translation, cytoskeletal redistribution, cell proliferation, differentiation, apoptosis. GeneMania server revealed that most of these genes were in co-expression (54.28%) and physical interaction (22.70%).
These results, confirming both individual and combined effect of Cd and Pb on genes important for lung cancer development, could be considered the basis for further in vitro and in vivo investigation in order to clarify the mechanisms of the development of this disease. Additionally, identified genes/proteins could serve as potential biomarkers and could be included for assessment of mixture toxicity of investigated metals in future (project: III 46009).
University of Belgrade, Faculty of Pharmacy
Keywords: pulmonary toxicity, metals, genes, carcinogenicity, biomarkers
Development of immunocompetent human airway epithelial models with macrophages for inhalation toxicity evaluation of airborne substances (#866)
X. - Y. Huang1, B. Boda1, I. Fureraj1, I. Larafa1, C. Mas2, S. Huang1, S. Constant1
1 Epithelix, Plan-les-Ouates, Switzerland
In addition to its barrier function, airway epithelia plays also a key role in immune responses in respiratory system. Among the immune cells, airway macrophages are particularly important and active for eliminating inhaled airborne particles such as allergens as well as microbes. Their activation must be tightly regulated by both soluble factors in the lumen of the airways and through cell-cell interactions. To a better understanding of the complex crosstalk between the epithelial cells and macrophages, in vitro immunocompetent models are developed based on three dimensional (3D) fully differentiated human airway epithelia cultured at air-liquid interface, co-cultured with human fibroblasts and surrogates of airway macrophages.
We report herein a simple and transposable procedure for the long term co-culture of MucilAir™ or SmallAir™ with fibroblasts and THP-1 derived M0-like macrophages. Using serum free ImmunAir™ culture medium, co-cultures were successfully maintained and functional for two weeks incorporating 5 cells types (basal, ciliated, goblet or club cells, fibroblasts and M0 macrophages).
The phenotypic identification of successively differentiated macrophages were performed by quantitative PCR of ten cellular markers. The co-culture was exposed to different stimuli and both cell types responded to these challenges. Repeated apical exposure to TNF-a and LPS increased the release of interleukin 8, in a dose dependent manner. Effect of respiratory irritants and pollens applied topically will be presented.
These data suggest that this new generation of immunocompetent airway human models may be useful for inhalation toxicity evaluation of airborne substances.
Keywords: inhalation toxicity, MucilAir, SmallAir, Macrophages, airway epithelium
Cytotoxic effect of real-time gasoline engine emissions exposure on BEAS-2B cells and MucilAirTM (#968)
T. Cervena1, 2, V. Beranek3, M. Vojtisek3, P. Rossner1
1 Institute of Experimental Medicine CAS, Department of Genetic Toxicology and Nanotoxicology, Prague, Czech Republic
Road traffic is a major cause of air pollution-related adverse health effects despite technological improvements and a considerable decrease in type approval limits. In vitro toxicity tests of emissions are usually done by the treatment of model cell lines with extractable organic matter (EOM) under submerged conditions. The aim of our study was to focus on more realistic exposure conditions such as air-liquid interface (ALI) and physical interaction of solid particles and gaseous pollutants with cells. This study reports on an in-house developed air-liquid interface exposure system for direct exposure of lung cell cultures to conditioned exhaust fumes. This approach involved a proportional sampling of exhaust using a partial flow dilution tunnel, conditioning of the sample to a stable temperature, humidity and CO2 content. Parallel exposure to exhaust fumes and control air was achieved using 4 separate exposure boxes (2 control and 2 exposed). For this study, we used human lung cell line BEAS-2B grown at the air-liquid interface and 3D lung tissue model MucilAirTM (Epithelix Sarl, Geneva). To investigate the adverse effects, a typical direct injection spark ignition petrol engine was mounted on an engine dynamometer and operated according to the World Harmonized Light Duty Vehicle Test Cycle (WLTC). BEAS-2B cells were maintained in our exposure device with 0.2 l/min of filtered humidified air supplemented with approximately 5% of CO2 for >10h with no visible changes. Based on the preliminary data, two exposure periods were selected: one-day and repeated 5-days exposure. The cytotoxicity measured as lactate dehydrogenase (LDH) release into media showed a time-dependent increase in BEAS-2B cells but not in MucilAirTM. Changes in sample morphology we observed after 5-days exposure in MucilAirTM as slower cilia beating frequency. In conclusion, our exposure device is able to maintain cell culture under ALI conditions and it is possible to use it for a wide variety of exposure schemes. This work was supported by the grant of the Czech Science Foundation (18-04719S).
Keywords: ALI, 3D model, gasoline emissions
Efficient Creation of Electronic SEND Datasets between CRO - Establishment of the Global SEND Alliance (G-SEND) - (#17)
T. Anzai1, S. Horikawa3, M. Wasko2
1 Showa University, School of Medicine, Tokyo, Japan
The Standard for Exchange of Nonclinical Data (SEND), adopted by the US Food and Drug Administration (FDA), is a set of regulations for digitalization and standardization of nonclinical study data; thus, related organizations have begun implementing processes in support of SEND. SEND provides electronic data standards created by the Clinical Data Interchange Standards Consortium (CDISC), and CDISC also collaborates in the implementation of SEND. Furthermore, the Pharmaceutical Users Software Exchange (PhUSE), which includes members of the US FDA, has conducted various activities to promote realistic and effective methods to implement SEND. As we surveyed in 2018, there is a significant variation in the efficiency and quality of SEND data implementation across pharmaceutical companies and contractors (CROs) globally. To address this problem, the Global SEND Alliance (G-SEND) was established in August 2018 to facilitate the coordination and standardization of SEND datasets across CROs in Asia. This presentaion reports the first method for organizationally and jointly creating consistent SEND datasets between CROs using G-SEND.
Keywords: SEND, G-SEND, CDISC, PhUSE, FDA
Promoting the uptake of alternatives to animal testing through the development of eLearning tools (#27)
E. Hill1, J. van Luijk2, R. de Vries2, A. Ulrey1, K. Tsaioun3, R. Pearse4, C. Eskes5, M. Ritskes-Hoitinga1
1 Institute for In Vitro Sciences, Gaithersburg, Maryland, United States of America
In order to further promote the implementation of Directive 2010/63/EU, the European Commission issued calls for a number of related projects last year. One of these projects is aimed at facilitating the uptake of non-animal alternatives by developing two e-learning modules. The contract for this project was awarded to a consortium consisting of SYRCLE, the Swiss 3R Competence Centre, lnstitute for ln Vitro Sciences, Pharma Launcher and Ecorys UK. This consortium will develop two modules, i.e., one e-learning module focused on searching for existing non-animal alternatives (including systematic reviews) and one module targeted at researchers who want to develop reliable and relevant non-animal alternatives for regulatory use taking into account Good In Vitro Method Practices (GIVIMP). The quality of the developed modules will be assessed by external review groups. The learning outcomes will be presented as well as the design of the assignments through which these outcomes will be realised.
Keywords: e-learning, GIVIMP, alternatives, regulatory, in vitro
Toxicological assessment of flavored e-liquids in Sprague-Dawley rats in an OECD sub-chronic inhalation study complemented by systems toxicology endpoints (#35)
J. Ho1, D. Sciuscio2, K. Ulrike2, B. Phillips1, B. Titz2, P. Leroy2, G. Vuillaume2, M. Talikka2, S. Lebrun2, W. Xia1, Y. Xiang2, A. Elamin2, K. Trivedi2, N. Ivanov2, J. Hoeng2, M. Peitsch2, P. Vanscheeuwijck2
1 Philip Morris International Research Laboratories Pte. Ltd., Singapore, Singapore
Flavor substances are an important element that is commonly added to e-liquids for sensory pleasure, but relatively few studies have been performed to evaluate their toxicity via inhalation exposure. The toxicity of a mixture of flavor substances in an e-liquid was characterized in a 90-day inhalation study according to the OECD 413 Testing Guideline, using 28 flavor group representatives (FGR) selected by grouping 179 flavors into 28 distinct groups based on chemical structure, where substances predicted to show the highest potential toxicological effect from each group were chosen as FGR. Sprague-Dawley rats were exposed for six hours/day, five days/week for at least 13 weeks to aerosols of vehicle control, e-liquid (propylene glycol [PG], vegetable glycerin [VG], and nicotine), e-liquid with three concentrations of FGR mixture, or PG/VG with medium concentration of FGR mixture. The target test atmosphere concentrations of nicotine, PG, and VG were 23 µg/L, 1520 µg/L and 1890 µg/L, respectively. The concentrations of the 28 flavors were derived from current maximum levels used in products. The results indicated that inhalation of the flavored e-liquid caused very minimal local and systemic toxic effects. No significant changes were detected in the number of inflammatory cells and inflammatory markers in the bronchoalveolar lavage fluid of rats in all groups, indicating limited pulmonary inflammation. The systemic effects related to exposure to the FGR mixture were limited and mainly nicotine-mediated, including changes in hematology, blood chemistry, and organ weights. There were minimal histopathological findings noted, but some findings, such as laryngeal squamous metaplasia, were seen in some rats of all groups, including vehicle control. Macro- and microscopic findings in spleen, adrenal, and thymus were considered due to procedure-related stress. The FGR mixture added to the e-liquid did not induce a measurable biological response on the transcriptome level, as seen from nose, lung, and liver samples in the current study, except a nicotine effect on metabolic processes. In summary, the results revealed findings mainly associated with nicotine exposure and limited synergistic effects caused by flavors.
Keywords: Flavor substances, e-liquid, inhalation toxicity, OECD TG413, systems toxicology
Aluminium salts in vaccines: From ancient concepts to current knowledge (#66)
G. Crépeaux1, J. - D. Masson1, F. - J. Authier1, R. Gherardi1
1 Inserm, IRMB, BNMS E10, Créteil, France
Aluminum (Al) salts Al oxy-hydroxide (AlOOH, Alhydrogel®) and Al hydroxyphosphate (AlOHPO4, Adju-Phos®) are particulate compounds widely used as immunologic adjuvants in about 60% of human vaccines. Today the exact degree of safety of Al-containing vaccines is still the subject of persistent disagreement and the WHO even notes that “adjuvant safety is an important and neglected field”. Concerns linked to the use of Al particles emerged 20 years ago following recognition of their causative role in the so-called macrophagic myofasciitis (MMF) lesion detected in patients with myalgic encephalomyelitis/chronic fatigue syndrome, revealing an unexpectedly long-lasting biopersistence of Al within immune cells. Currently growing worries concern the potential role played by Al-adjuvant exposure in a large scale of diseases, among them chronic fatigue syndrome, Gulf war syndrome or autism spectrum disorders. In this field, the Autoimmune (Autoinflammatory) Syndrome Induced by Adjuvants" (ASIA) has been delineated.
Keywords: Vaccine adjuvant, Aluminum, Vaccine safety, toxicokinetics
180-day toxicological research of GM soybean line MON87701×MON89788: The results of morphological examination (#69)
N. S. Nikitin1
1 Federal State Budgetary Scientific Institution “Federal Research Centre of Nutrition, Biotechnology and Food Safety”, the laboratory of biotechnologies safety assessment and novel food sources , Moscow, Russian Federation
As a part of the state registration of GM soybean line MON87701×MON89788 in the Russian Federation, a comprehensive safety assessment was conducted, which included chronic toxicological research on rats in vivo. This article presents the results of morphological examination of internal organs of rats that consumed GM soybean for 180 days.
During this study organs of 32 Wistar rats were examined (16 in the control and test groups each). The rats were subjected to euthanasia by decapitation. Microscopic examination of internal organs was conducted at the time of autopsy: both groups showed no pathological changes and anatomically correct organ structure of typical sizes and forms, chest cavity and abdominal cavity position of organs was in the norm, the capsules, mucous membranes and serous membranes were moist, smooth and shiny, without focal changes, of a tightly elastic consistency, lymph nodes were not enlarged, lungs were airy to the touch, freely lying in the pleural cavity, pleurodiaphragmatic adhesions were absent.
Thymus, heart, lungs, liver, kidneys, adrenals, spleen, small intestine, testicles, prostate gland were histologically investigated. Organs were fixed in 10% formalin solution, preparations stained with hematoxiline-eosin and van Gieson's stain. Histological preparations were assessed in light microscope AxioImager Zl. Morphometry was performed with AxioVision 4.8.
Histological structure of the investigated organs showed no deviations. No pathological changes in tissue structure or endemic hemorrhage have been detected. Morphometric analysis of the liver, kidneys, spleen structure showed no differences between the groups: for control and test groups the average values of diameters of the renal glomeruli, the renal proximal tubules, the lumen of renal proximal tubules were 96,32±1,07 and 95,25±1,14 µm; 29,01±0,39 and 28,16±0,44 µm, 17,23±0,56 and 16,95±0,57 µm, respectively; the diameters of the white pulp of the spleen were 288,11±4,1 and 284,46±3,95 µm; the diameters of the hepatocytes were 13,85±0,43 and 13,04±0,34 µm.
Results of the morphological examination, together with hematological and biochemical examination, diagnosis of antioxidant status and monooxygenase system enzymes' activity in the liver, did not reveal any toxic effect of GM soybean line MON87701×MON89788 in comparison with its traditional counterpart.
This work was supported by the Ministry of Science and Higher Education of the Russian Federation (research project No. 0529-2019-0056).
Keywords: morphology, GMO, soybean, toxicology
Results of Preclinical and Clinical Safety Studies Of The Novel Adenoviral Gene Therapy (#94)
N. V. Eremina1, 2, V. Kazey1, A. Zhanataev2, A. D. Durnev2
1 Panacela Labs LLC, Moscow, Russian Federation
Mobilan is a recombinant bicistronic non-replicating adenoviral immunotherapeutic drug that directs expression of human toll-like receptor 5 (hTLR5) and its specific agonistic ligand, 502s, which is a recombinant form of the natural TLR5 ligand, flagellin. Mechanism of action involves transduction of tumor cells with Mobilan, which leads to constitutive autocrine stimulation of TLR5 pathway. This results in strong induction of the innate immune system with subsequent development of adaptive anti-tumor responses. Mobilan has been engineered for immunotherapy of prostate cancer and designed to be intratumorally injected into both lobes of the prostate gland.
A standard regulatory set of preclinical safety studies of this drug included toxicity studies with a single (acute toxicity; outbred white mice and rats; i.v. and i.m. injections) and multiple (chronic toxicity; outbred white rats and Chinchilla rabbits; i.m. injections; 109, 1010 and 1011 virus particles (v.p.) per dose), mutagenic activity (mouse bone marrow chromosomal aberration test; DNA-comet assay), immunotoxicity, allergenicity, and reproductive toxicity. Standard in vivo test systems were used and Mobilan was injected intramuscularly in two doses (109 and 1010 v.p. per dose) except for the cases specified above.
The results of toxicological studies did not reveal any factors that impede the clinical trials of Mobilan, however, it is necessary to consider its potential risks for patients with bleeding disorders or suffering from chronic inflammatory diseases. The maximum dose (1010 v.p./dose) without the observed adverse effect (NOAEL) was determined. Since standard approaches to interspecific dose recalculation are not applicable to such drugs, the NOAEL defined for animals with a safety factor of at least 10 (109 v.p./dose) was used in clinical trials as a starting dose.
Key safety results of Phase I clinical trials in patients with local non-metastatic prostate cancer demonstrated that favorable tolerability was observed in patients administered with Mobilan in several dose levels (109 – 1011 v.p. per ml per intraprostatic injection). Two reversible SAEs possibly related to Mobilan were documented: severe pollakiuria with leukocytosis and elevated C-reactive protein level in patient administered with 109 v.p. and acute prostatitis in patient administered with 3·109 v.p.
Keywords: gene therapy, immunotherapy, Mobilan, regulatory toxicology
R-ODAF: an omics data analysis framework for regulatory application (#105)
M. Verheijen1, W. Tong2, L. Shi3, T. Gant4, B. Seligman5, F. Caiment1
1 Maastricht University, Toxicogenomics, Maastricht, Netherlands
High throughput technologies to analyze biological molecules (genes, protein, metabolites ….) are collectively known as “omics”. The use of omics technologies for toxicology research and scientific publications is expanding. However, till date no omics data has been used to support a chemical regulatory application.
Regulatory agencies report that the “truth” of toxicity is difficult to assess when using omics data, mainly because of 2 issues:
In order to address these issues, a consensus on an omics analysis framework (ODAF) for regulatory application needs to be achieved. The purpose of the current research is to test and develop a regulatory ODAF (R-ODAF) proposal for the toxicogenomics community with the ambition to enable the regulatory bodies to consider omics as a relevant data type to support compound submissions.
Because transcriptomics data is by far the most abundantly available in toxicogenomics, the CEFIC LRI-C4 project focusses on generating a standardized procedure for the analysis of transcriptomics data, obtained using the three major platforms: microarrays, RNA sequencing and the new TempO-Seq® technology.
Keywords: Regulatory toxicology, Transcriptomics, Bioinformatics, omics analysis framework
A systematic review of the Monocyte Activation Test: How much proof is good enough? (#116)
J. Hochmuth1, A. Ménache2
1 Animal Rights, Ghent, Belgium
Pyrogenic contamination of parenteral pharmaceuticals is considered a serious public health risk and can result in symptoms ranging from mild reactions (e.g. fever) to septic shock and death. Therefore, for injectable formulations, pyrogen testing is mandatory during the routine quality control of injectable products by regulatory agencies, as well as during the manufacturing process.
Over the last 70 years, various pyrogen testing methods have been introduced, namely: in the 1940s, the Rabbit Pyrogen Test (RPT), which is an in vivo test that measures the fever reaction as an endpoint; in the 1970s, the Limulus Amoebocyte Lysate (LAL) test (also referred to as the bacterial endotoxin test), which is an animal-based in vitro test that uses the haemolymph of the horseshoe crab and only represents a partial replacement of the rabbit test, as it solely detects endotoxin; and in 1995, the Monocyte Activation Test (MAT), which is a non-animal based in vitro pyrogen test that represents a full replacement of the rabbit test.
Article 12 of the Directive 2010/63/EU specifies that an alternative to animal testing should be used whenever such method prevails. The MAT was validated as a replacement for the RPT and the LAL by the EU Reference Laboratory for alternatives to animal testing (ECVAM) back in 2000 and adopted by the European Pharmacopoeia in 2013. We conducted a systematic review comparing the performance of the MAT against that of the two widely used animal-based pyrogen testing methods. From a scientific perspective, the results clearly demonstrate that the MAT does not have the limitations of the animal-based tests, thus outperforming the latter. The RPT fails to detect human-specific pyrogens and the LAL does not detect non-endotoxin pyrogens.
We are certain that the MAT represents an extraordinary opportunity to safeguard public health and simultaneously end the suffering of 400,000 rabbits worldwide per year used in the RPT and that it could contribute to the conservation of the critically endangered 450 million-year-old horseshoe crab used in the LAL and the birds up the food chain that depend on them. We strongly urge the biomedical and pharmaceutical industries to adhere to article 12 of the Directive 2010/63/EU and make it a priority to replace the animal-based methods with the in vitro alternative.
Keywords: regulatory toxicology, in vitro, Monocyte Activation Test, Limulus Amoebocyte Lysate, Rabbit Pyrogen Test
Cannabinoid toxicity: Computational assessment of (eco)toxic effects (#141)
K. Venko1, M. Novič1
1 National Institute of Chemistry, Theory Department/ Laboratory for Cheminformatic, Ljubljana, Slovenia
The significant increase of cannabinoid application for the therapeutic and recreational purposes raises question, how safe is their uncritical use by majority of consumers. Especially, the new synthetic cannabinoids are continuously produced as designer drugs, but their toxic effects are even not evaluated. The time and cost consuming chemical risk assessment of new designer drugs of course is not concern of illegal market. Fortunately, at least fast and costless in silico safety profiling by publically or commercially available models can be performed. We made it for 120 natural and synthetic cannabinoids. When quantitative structure-activity relationship (QSAR) models are used for regulation purposes it is recommended to run as much as possible of available QSAR models for the endpoint of interest, since agreement among predictions generated from several independent QSAR models increases the confidence on the predictions (ECHA, Practical Guide - How to use and report (Q)SARs 3.1). For prediction of various toxic endpoints of health and environment many validated QSAR models are publically available. By using several of them from VEGA, TEST and QSAR toolbox we evaluated cannabinoid toxicity. Independent and consensus predictions were performed including toxic health effect like mutagenicity, carcinogenicity, developmental toxicity, skin sensitization, endocrine disruption and hepatotoxicity. Cannabinoids were detected also in wastewater, thus the evaluation of their eco-toxic effects is of interest too. Therefore, properties of cannabinoid bioaccumulation, degradability and toxicity towards fish, water flea and algae were determined. In general all cannabinoids were estimated as developmental toxicants. Majority of them are also endocrine disruptors and potential carcinogens. In environment they are ready degradable. For natural cannabinoids much more reliable predictions can be done, while majority of synthetic cannabinoids unfortunately failed out of applicability domain of models. The compendium of predictive QSAR models used in this study can be implemented for preliminary chemical safety profiling of practically any other substance of interest.
Keywords: cannabinoids, QSAR, in silico models, toxicity, virtual safety profiling
Assessment of endocrine disruption potential of ozone using the ECHA/EFSA guidance document on identifying endocrine-disrupting chemicals: Experiences gained and challenges faced (#151)
J. Choi1, J. Kurzke1, W. Mune1, P. Janz1, T. Sendor1, T. Rücker1
1 Ramboll Environment & Health GmbH, Munich, Bavaria, Germany
Biocidal and plant protection products to be used in the European Union are required to undergo an assessment for endocrine disruption (ED) potential pursuant to the Biocidal Products Regulation (EU 528/2012) and the Plant Protection Products Regulation (EC 1107/2009), respectively. A guidance document (GD) on the identification of endocrine-disrupting chemicals (EDCs) was published by the European Chemicals Agency (ECHA) and European Food Safety Authority (EFSA) and serves as a tool for applicants to identify EDCs using a defined set of ED criteria.
Ozone is currently regulated as a biocidal active substance; therefore, an assessment of ozone for ED potential is required. This task was undertaken by following the key steps laid out in the ECHA/EFSA GD: gather all relevant information, evaluate relevance/reliability, assemble and assess the lines of evidence using an Excel template prepared by ECHA/EFSA and draw conclusion with the extended option of conducting a mode of action (MoA) analysis.
There are indications of ozone triggering endocrine activity, such as altered hormone levels, that are mediated by the oestrogen, androgen, thyroid or steroidogenic (EATS) modalities, but there is limited evidence of EATS-mediated endocrine adverse effects. On the other hand, there are indications of ozone exposure leading to non-EATS-related activities and effects such as altered stress hormone responses and impaired metabolism. To gain a better understanding of these non-EATS activities and effects, a MoA analysis of ozone was performed. The effects of ozone are primarily mediated by local effects in the respiratory tract (e.g. local inflammation) due to its strong oxidising properties, which subsequently leads to oxidative stress that can subsequently trigger altered hormonal or metabolic responses. Overall, for both mammalian and environmental species, there is no biologically plausible link between endocrine activity and adverse effects observed from ozone. Therefore, ozone does not meet the ED criteria with respect to human health or environment relevance.
The ECHA/EFSA GD provides a clear approach of performing the ED assessment of chemicals; however, challenges were faced during the data compilation and assembling the lines of evidence. Recommendations are made on how to deal with these challenges.
Keywords: endocrine disruption, ozone, biocides, echa, efsa
Food derived from genetically modified animals: Formation of safety assessment system and new approaches to toxicological research (#157)
V. A. Tutelyan1, N. Tyshko1, E. Sadykova1
1 Federal State Budgetary Scientific Institution “Federal Research Centre of Nutrition, Biotechnology and Food Safety”, Moscow, Russian Federation
The intensive development of animal genetic engineering and entering of genetically modified (GM) animal-derived food at the food market has led to necessity of safety assessment system development, as well as to harmonization of Russian and international regulatory and methodical documents. According to the requirements of the European Union a full-scale veterinary examination of GM animals is considered to be the start point of safety assessment. The next stage of assessment involves the comprehensive studies aimed at the establishing of substantial equivalence of animal-derived GM food. In general, the set of studies is very similar to the approach used for GM plants, with the exception of only the first stage of the veterinary examination.
The Russian system of GM plants safety assessment includes the execution of scale investigations, such as general toxicological research and the study of specific types of toxicity. Such approach provides with the most complete and reliable information on potential reprotoxic, genotoxic, immunotoxic etc. effects of GM organism, as well as enables to reveal possible unintended effects of modification. At the same time the necessity of creation of animal-derived GM food safety assessment system inspired us to update of toxicological methods set. One of the strategic points of further development we believe the involvement of new knowledge in the toxicological studies (for example, advances in the oncology which allow not only to detect tumors at an early stage, but also to predict the risk of developing tumors, can be used in the study of mutagenesis and carcinogenesis). The search of sensitive biomarkers that respond to adverse effects is a constant and important aspect of scientific work that would not lose its relevance in the coming decades. Also an important research direction is the search for new models that will increase the research informativity: first, the development of models with traditionally used laboratory animals (e.g. models of adaptive potential reducing which allow to decompensate the adaptation processes of healthy organism and to identify the effects of negative impact); second, the use of new biological objects that facilitate extrapolation to humans (here can be possible the range from cell cultures and individual organs to GM organisms and synthetic biology-derived organisms, which are similar in their biochemical, physiological, pathological reactions with the humans); third, the possibility of use computer simulation within toxicological studies.
Thus, the further improvement of methodical approaches in the safety assessment of novel food determines the necessity of efforts integration not only medical and biological scientists, but also specialists in the field of mathematical analysis, computer science, analytical chemistry and other areas.
This work was supported by the Ministry of Science and Higher Education of the Russian Federation (research project No. 0529-2018-0113).
Keywords: genetically modified animal-derived, GM food safety assessment, biomarkers, toxicological research of novel food
Risk for human health from five phthalates used in plastic food contact materials (FCM): a cumulative risk assessment by the European Food Safety Authority (EFSA) (#162)
K. Volk1, J. Cara-Carmona1, D. Wölfle2, I. Waalkens-Berendsen2, D. Gott3, 4, F. Pantazi1, A. F. Castoldi1, L. Castle2
1 European Food Safety Authority (EFSA), Parma, Italy
EFSA has updated its 2005 risk assessments for five phthalates which are authorised for use in plastic FCM and may possibly migrate into food: di-butylphthalate (DBP), butyl-benzyl-phthalate (BBP), bis(2-ethylhexyl)phthalate (DEHP), di-isononylphthalate (DINP) and di-isodecylphthalate (DIDP). EFSA reconfirmed the same critical effects and individual Tolerable Daily Intakes (TDIs; mg/kg bw per day) that it had established in 2005 for such phthalates, i.e. reproductive effects for DBP (0.01), BBP (0.5), DEHP (0.05), and liver effects for DINP and DIDP (0.15 each). The possibility of a cumulative risk for humans deriving from co-exposure to phthalates causing similar effects was investigated, since consumers may simultaneously be exposed to several phthalates from the diet and other sources. A common mode of action, i.e. reduction in fetal testosterone levels, was considered plausible for DBP, BBP and DEHP. DINP also affected fetal testosterone levels even though liver is recognised as its main toxicity target. DIDP did not affect fetal testosterone levels. Thus, a group-TDI was proposed for DBP, BBP, DEHP and DINP by using the Relative Potency Factor (RPF) approach. DEHP was chosen as the index compound due to its most robust dataset and the group-TDI was set to 0.05 mg/kg bw per day, expressed as DEHP equivalents. The RPFs were calculated as the ratio between the TDI of the index compound and the individual TDIs for DBP (5.0) and BBP (0.1). For DINP, an additional assessment factor was introduced to cover for the 3-fold lower NOAEL for liver effects compared to that for reproductive effects and the resulting RPF for DINP was 0.3. For the four grouped-phthalates, an aggregated potency-adjusted dietary exposure (expressed as DEHP equivalents by applying the RPFs) was estimated to contribute to up to 23% of the group-TDI in the worst-case scenario. For DIDP, dietary exposure was estimated to be 1,500-fold below its individual TDI. This draft assessment is under public consultation until 14 April 2019, and will be revised afterwards based on the comments received.
Keywords: Chemical mixtures, Cumulative risk assessment, Regulatory toxicology, Food safety, Reproductive toxicity
Benchmark dose uncertainty as a possible indicator of the biological relevance of toxicological endpoint (#177)
M. Zinovieva1, P. Zhminko1, N. Nedopytanska1, M. Prodanchuk1
1 L.I.Medved's Research Center of Preventive Toxicology, Food and Chemical Safety Ministry of Health, Ukraine, Kyiv, Ukraine
Benchmark dose (BMD) analysis sometimes results in high uncertainty of BMD interval (upper/lower limit ratio, BMDU/L) associated with predefined effect size. Data sets resulted in high BMD uncertainty are qualified as low quality ones. We suppose that the level of BMD uncertainty may reflect also toxicological relevance of endpoint. Previously toxicity/safety profile assessment of novel central acetylcholinesterase reactivator S-XX was performed. S-XX was administered (i.m.) at doses 0-10-50-100-200 mg/kg to rats females (n=6). Red blood endpoints found to be affected. Different NOAELs were established for HCT (10 mg/kg), RBC (50 mg/kg), HGB (100mg/kg), MCH (100mg/kg). Statistical deviations unrelated to dose were found for MCHC.
Purpose. To analyze uncertainty levels of BMD determined for the number of hematological endpoints within one study.
Methods. BMD-analysis performed using PROAST66.24, critical effect size 10%.
Results. In contrast to NOAELs, BMDLs for HGB, RBC, and HCT were found to be similar (from 12 to 16mg/kg) and lowest between other red blood parameters. BMDU/L were similar (5.5-6.5) as well, and low (<10), reflecting acceptable data quality. BMDLs for other hematological endpoints – MCH, and MCHC were 81, and 224 mg/kg, as well BMDU/Ls were substantially higher – 89, and 49, indicating lower relevance of these endpoints.
Conclusion. Despite different NOAELs established for hematological endpoints, BMD methodology allowed distinguishing set of endpoints with similar BMD with low uncertainty. These endpoints might be considered as the most relevant hematological endpoints of the particular study.
Keywords: BMD, uncertainty, biological relevance
Sources of Uncertainty in the Threshold of Toxicological Concern approach (#182)
P. Bellion1, H. Hoellnagel2, H. Buist3, S. Gadhia4, J. P. Gosling5, D. Lovell6, S. Melching-Kollmuss7, A. Worth8, E. Rito9
1 DSM Nutritional Products, Kaiseraugst, Switzerland
The probabilistic approach using the genotoxicity and non-cancer (Cramer class) Thresholds of Toxicological Concern (TTC) is often perceived as accepting a higher risk than traditional, substance-specific risk assessments. However, robust scientific activities to describe the sources of uncertainty within the TTC approach have not yet been conducted or published. An ILSI Europe Expert Group was formed to examine how much uncertainty may be associated with the application of the TTC approach as compared to a substance-specific risk assessment, thus developing scientific knowledge about the sources of uncertainty being specific to the TTC. The initial phase of the project focuses on qualitative description and ranking of the identified sources of uncertainty, with a subsequent quantitative assessment.
Uncertainties addressing the development of the TTC approach include, but are not limited to, the variability of animal studies, the accurate use of uncertainty (“safety”) factors, choice of the point of departure (NO(A)EL, BMDL, TD50), overall database quality and data distribution, and the choice of the 5th percentile for threshold selection.
Potential uncertainties that stem from the practical application of the TTC approach are: chemical space covered by the reference database, excluded substance groups, uncertainties associated with the use of in silico prediction vs. experimental data, the applicability of one TTC value to cover different toxicological endpoints (repeated dose toxicity, DART, endocrine disruption, immunotoxicity, etc.), and the influence of Cramer class misclassification.
The level of uncertainty was found to be similar for some factors, irrespective if the risk assessment is based on TTC or substance-specific data. Examples include the inherent uncertainty and variability of animal studies or the accuracy of assays employed to assess the mutagenicity of the substance.
Keywords: TTC, uncertainty assessment, risk assessment, ILSI Europe
Similarity assessment of peroxisome proliferators based on intracellular metabolomics in HepG2 cells (#198)
H. G. Kamp1, S. Sperber1, B. Birk1, V. Haake2, T. Walk2, B. van Ravenzwaay1
1 BASF SE, Experimental Toxicology and Ecology, Ludwigshafen, Germany
BASF and metanomics established the database MetaMap®Tox containing the plasma metabolome of more than 800 compounds derived 28-day studies in rats. In 2016, we have published a case study on the value of such in vivo metabolomics data for read-across within a group of phenoxy carboxylic acid herbicides (van Ravenzwaay et al., 2016). In this case study, we identified 2,4-DP as the best out of two potential source compounds to predict the 90day-toxicity of the target compound MCPP. Over the last few years, a highly stable and reproducible liver in vitro model was established, in which the intracellular metabolome of HepG2 cells can be specifically altered through treatment with different hepatotoxicants. Within the EU-funded Horizon 2020 project EU-ToxRisk, we have now analysed the intracellular metabolome of HepG2 cells treated with different classes of peroxisome proliferators (phenoxy carboxylic acid herbicides, pharmacologically active peroxisome proliferators, DEHP and MEHP). The metabolome consisted of 236 unique metabolites, thereof 35 amino acids and derivatives, 11 carbohydrates and related compounds, 54 lipids, 14 energy metabolites, 6 nucleobases, 14 vitamins and cofactors as well as other miscellaneous or unknown metabolites. Most of the treatments resulted in clear changes of the intracellular metabolome in HepG2 cells at at least the highest sub-cytotoxic concentration used. In a multivariate statistical approach (PCA) clear separations from the control treatments along the first principal component were seen for the herbicides, pharmacologically active PPARalpha agonists as well as for MEHP. Furthermore, within the group of herbicides, the results show that for MCPP, the most similar treatment is 2,4-DP, whereas MCPA and 2,4-D are less similar. This result is in line with the outcome of the abovementioned in vivo case study.
Keywords: metabolomics, HepG2, in vitro, peroxisome proliferation, read across
Impurities in cosmetic products : which are the most common, and how to assess them in a cosmetic safety report ? (#202)
A. Chelle1, A. Perdriat-Loucano1, V. Levelut1, A. Nalin1
1 Eurofins | Evic Product Testing France , Aix-en-Provence, France
While assessing the safety of cosmetic product, an important part of the approach is to evaluate whether the cosmetic product contains or not substances that have not been intentionally added to the formulation. These substances, also commonly called impurities, are unintended substances which can appear as traces in the finished product. To guarantee the consumer safety, a safe level should be established for each of impurity. When no safe level has been established by the cosmetic Regulation, it has to be determined on a case-by-case analysis. This safe level is then compared to the exposure of the consumer from the finished product, to determine if there is a risk for health. However, information concerning the exact content of impurities in cosmetic products is often very poor. The aim of this study was to perform a wide review of different categories of cosmetic products on the market, in order to determine the major impurities and their occurrence. Then, an approach to determine their safe level was proposed.
Keywords: Cosmetics, Impurities, Traces, Risk assessment
Read-across approach using molecular descriptors for the prediction of rat repeated-dose toxicity (#230)
Y. Kitsunai1, J. - I. Takeshita2, 1, M. Watanabe1, T. Hosaka1, R. Shizu1, T. Sasaki1, K. Yoshinari1
1 University of Shizuoka, School of Pharmaceutical Sciences, Shizuoka, Japan
[Introduction] Read-across is an approach to predict the toxicity of untested substances, based on the similarity in the chemical structures and/or other characteristics of substances with existing toxicity information. However, since current read-across approaches are subjective, expert-driven methods in terms of the similarity judgment, there are concerns on objectivity and/or reproducibility. In this study, we tested a possible use of molecular descriptors to judge chemical similarity for read-across approach of repeated-dose toxicity (RDT) prediction. [Methods] The results of rat 28-day RDT and 42-day combined RDT and reproductive/developmental toxicity tests (458 substances and 432 endpoints (EPs)) were obtained from the toxicity database HESS (NITE, Japan). Liver function/injury-related EPs were divided into 6 groups, and anemia- and kidney injury-associated EPs were grouped, respectively, and a total of 8 groups were used. Molecular descriptors were calculated using Dragon 7 (Talete) and Euclidean distances between substances were calculated with the normalized descriptors. As verification substances, 20 substances were randomly selected and their EPs (8 groups) were compared with those of top 10 neighboring substances [Results and Discussion] We tested 4 descriptor sets: A) calculable 2385 descriptors, excluding constant values, B) 101 functional group-related descriptors, C) molecular weight-, hydrophobicity- and polar surface area-related 5 descriptors, D) extended-connectivity fingerprints (ECFP1024, maximum diameter of 4). Neighboring substances of each verification substance were different depending on the descriptor set used, although similar results were obtained with sets A, C and D for certain substances. Their relative distances between verification and neighboring substances varied for each descriptor set. The toxicity similarity with neighbors was also different depending on the descriptor set and verification substances. The overall accordance was higher for substances with low toxicity (e.g. negative for all 8 EP groups) than those with multiple EPs. These results suggest that the molecular descriptors can be used for read-across of RDT prediction although the selection of appropriate descriptors and objective/statistical determination of neighboring substances are needed.
Keywords: Read-across, repeated-dose toxicity, alternative method, computational toxicology, molecular descriptor
Investigating human cytochrome P450-related variability using PBK models for chemical risk assessment (#263)
K. Darney1, A. Gkrillas2, E. Testai3, F. Buratti3, E. Di Consiglio3, L. Turco3, S. Vichi3, N. Kramer4, E. Kasteel4, L. Lautz1, C. Béchaux1, J. - L. Dorne5
1 French Agency for Food, Environmental and Occupational Health & Safety (ANSES), Risk Assessment Department, Maisons-Alfort, France
A 100-fold default uncertainty factor (UF) has been applied for over 60 years to account for interspecies differences and human variability in safe levels of threshold toxicants in non-cancer risk assessment. Pathway-related UFs quantifying human variability in a range of metabolic pathways have been proposed as intermediate options between default UFs and chemical-specific adjustment factors. Physiologically based kinetic (PBK) models are well suited to model pathway-related UFs as they simulate human variability in chemical disposition.
Here, we propose a methodology for human risk assessment based on in vitro systems and PBK models that includes variability. Techniques are now available to determine isoform specific metabolism and kinetics parameters for chemicals using in vitro human cell systems. Human variability distributions for metabolic pathways estimated from previous work can be used to estimate lognormal distribution for clearance of chemicals. These distributions are used in a PBK model with Markov-Chain Monte Carlo and physiological parameters taken from the online software PopGen. Other parameters specific to the studied compound such as partition coefficients are estimated using quantitative structure–activity relationship (QSAR).
Three different case studies have been realized: 1) the application of the methodology to a data rich chemical, chlorpyrifos, in order to validate the methodology by comparing the results with data from literature; 2) an illustration of the utility of using variability distributions for metabolism in the human risk assessment of a data poor compound, phosmet 3) and triflumuron.
The proposed methodology avoids the use of default UFs which are both overly and not conservative enough.
The views in this publication do not necessarily represent those of EFSA, Anses, ISS and are the authors only.
Keywords: PBK modelling, human risk assessment, toxicokinetics, CYPs-related variability
Assessment of the specificity of tyrosine kinase inhibitors in relation to their cardiovascular toxicity, cutaneous toxicity and hepatotoxicity in cancer treatment (#295)
P. Nortier1, 2, M. Burbank1, G. Guyader1
1 French National Agency for Medicines and Health Products Safety (ANSM), Oncology, Saint Denis , France
Cancers remain the leading cause of death in France. The availability of new and more effective treatments with acceptable tolerance is still essential to improve patient survival. In drug development, anti-cancer drugs represent 60% of the drugs on the market in Europe. Chemotherapy was the first treatment in development and was largely used these last decades. However new therapeutics including immunotherapy, antibody drug-conjugate, tyrosine kinase inhibitors (TKI) have emerged because of chemotherapy’s side effects and its low remission rate. Targeted therapies such as TKI are less harmful and more effective than chemotherapy treatments because their action is specific on the tumor process.
Tyrosine kinase enzymes activate proteins involved in cell proliferation, survival, migration, differentiation, angiogenesis... Their inhibitors block these enzymes and in doing so, the tumor growth. They can be divided into multi-kinase or single-kinase inhibitors and are related to potential toxicity, resistance mechanisms, pharmacokinetics, selectivity and tumor environment.
On-targets and off-targets effects related to cardiotoxicity, cutaneous toxicity, and hepatotoxicity are the most commonly emerging toxicities seen with the TKI. In the well-known marketed TKI, sumatinib has been associated to cardiotoxicity and both erlotinib and gefitinib have been associated to cutaneous toxicity and hepatotoxicity.
We present here a review of the different TKI families on the market and in clinical development in France. We will discuss their specificities of action in light of their on-target and off-target effects. We will focus particularly on cardiotoxicity, cutaneous toxicity and hepatotoxicity. Finally, we will compare the toxic effects observed in both non-clinical and clinical development in order to predict the side effects observed in Human. Data from French clinical trial authorization as well marketing Authorization application will be analyzed. We will suggest new approaches optimization of non-clinical models to improve side effects detection in Human.
Keywords: tyrosine kinase inhibitor, oncology, cardiovascular toxicity, cutaneous toxicity, hepatotoxicity
“Hypoallergenic” cosmetic products : regulatory review and scientific approach – A practical case (#302)
V. Levelut1, A. Nalin1, A. Nanu2, C. Lidon1
1 Eurofins | Evic Product Testing France , Aix-en-Provence, France
Cosmetic products are regulated by two main pieces of legislation, applicable throughout the entire European Community: Regulation (EC) No 1223/2009 of the European Parliament and of the Council on cosmetic products and Commission Regulation (EU) No 655/2013 laying down common criteria for the justification of claims used in relation to cosmetic products. Implementation rules for cosmetic claim Regulation have not been clear from the beginning, so discussions followed and other documents were drafted. On 3rd July 2017 the sub-working group on claims released the updated Technical document on cosmetic claims, which should become applicable to all Member States as of 1st July 2019.
Concerning the specific type of claim “hypoallergenic”, Annex IV of the technical document provides a better definition of the term, and offers additional recommendations for supporting this claim. Evidence that a cosmetic product has a very low allergenic potential should be based on scientifically robust and statistically reliable data, coming both from the substances and the finished product. In this study, a practical approach was tested and implemented for the safety assessment of “hypoallergenic” products, in compliance with the latest revision of the Technical document on cosmetic claims.
Keywords: Cosmetics, Claim, Hypoallergenic, Sensitization, Regulation
EFSA Safety Assessment of Food Additives: Data and Methodology Used for the Assessment of Dietary Exposure for Different European Countries and Population Groups (#310)
P. Gergelova1, S. Ioannidou1, D. Arcella1, A. Tard2, P. E. Boon3, O. Lindtner4, C. Tlustos5, J. - C. Leblanc6
1 European Food Safety Authority (EFSA), Risk Assessment and Scientific Assistance (RASA) Department /Evidence Management Unit, Parma, Italy
Purpose: To assess chronic dietary exposure to food additives in different European countries and population groups.
Methods: The European Food Safety Authority’s (EFSA) Panel on Food Additives and Flavourings (FAF) estimates chronic dietary exposure to food additives with the purpose of re-evaluating food additives that were previously authorized in Europe. For this, EFSA uses concentration values (usage and/or analytical occurrence data) reported by food industry and European countries. These are combined, at individual level, with national food consumption data from the EFSA Comprehensive European Food Consumption Database including data from 33 dietary surveys from 19 European countries and considering six different population groups (infants, toddlers, children, adolescents, adults and the elderly). Dietary exposure is assessed based on two different sets of data: (a) Maximum permitted levels (MPLs) of use set down in the EU legislation (defined as regulatory maximum level exposure assessment scenario) and (b) usage levels and/or analytical occurrence data (defined as refined exposure assessment scenario). The refined exposure assessment scenario is sub-divided into the brand-loyal consumer scenario and the non-brand-loyal consumer scenario. Additional exposure scenarios considering consumers of specific food (e.g. food supplements) are also estimated, as appropriate.
Results: Since 2014, this methodology has been applied in more than 60 food additive exposure assessments conducted as part of scientific opinions of the EFSA FAF Panel (previously Panel on Food Additives and Nutrient Sources added to Food (ANS)). For example, under the non-brand-loyal scenario, the highest 95th percentile of exposure to silicates (E 552-553) and the second highest 95th percentile of exposure to quillaia (E 999) was estimated in toddlers up to 27.3 and 0.8 mg/kg body weight/day, respectively. The estimates under the brand-loyal scenario in toddlers resulted in exposures of 65.0 and 1.0 mg/kg body weight/day, respectively. For the regulatory maximum level exposure assessment scenario, the 95th percentile of exposure to silicates (E 552-553) was estimated in toddlers up to 72.9 and 9.6 mg/kg body weight/day, respectively.
Conclusions: Detailed and up-to-date information on food additive concentration values (usage and/or analytical occurrence data) and food consumption data enables the assessment of chronic dietary exposure to food additives to more realistic levels.
EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS), 2018. Scientific opinion on the re-evaluation of calcium silicate (E 552), magnesium silicate (E 553a(i)), magnesium trisilicate (E 553a(ii)) and talc (E 553b) as food additives. EFSA Journal 2018;16(8):5375, 50 pp. https://doi.org/10.2903/j.efsa.2018.5375
EFSA FAF Panel (EFSA Panel on Food Additives and Flavourings), 2019. Scientific Opinion on the re-evaluation of Quillaia extract (E 999) as a food additive and safety of the proposed extension of use. EFSA Journal 2019;17(3):5622, 50 pp. https://doi.org/10.2903/j.efsa.2019.5622
Keywords: food additives, dietary exposure assessment, silicates, quillaia extract
Hierarchical Bayesian meta-analysis of human variability in PON1 metabolism for the refinement of uncertainty factors in chemical risk assessment (#317)
L. Lautz1, K. Darney1, C. Bechaux1, F. Buratti2, E. Di Consiglio2, L. Turco2, S. Vichi2, E. Testai2, E. Kasteel3, N. Kramer3, J. - L. Dorne4
1 French Agency for Food, Environmental and Occupational Health & Safety (ANSES), Risk Assessment, Paris, France
Human paraoxonase (PON) exhibits a broad substrate specificity and a range of important activities, including drug metabolism, hydrolyzation of a number of organophosphorus compounds as well as the oxon metabolites of organophosphorothionates, more toxic than the parent, including insecticides and nerve agents. PON1 activity was polymorphically distributed in human populations and the frequency of the low activity phenotype varied among populations of different ethnic origins.
Here, inter-individual differences in PON1 activity have been investigated through a systematic review. All data were extracted in a structured database and meta-analyses were performed using a hierarchical Bayesian model in the R freeware to derive parameter, route and ethnic-specific variability distributions for PON1 activity. Two different approaches were applied. 1) First, non-genotyped data were meta-analysed in order to provide a distribution of PON1 activity. 2) Derivation of genotype-specific variability distributions using fast metabolizer as reference group to compare with other polymorphism. Reference group was respectively PON1*192 RR, PON1*55 RR and PON1-108 CC.
Overall, subgroup-specific distributions for PON1-variability provided the basis to derive PON1-related uncertainty factors (UF) to cover 95th or 99th percentiles of the population and were compared with the human default toxicokinetic UF (3.16). The results indicated that differences in activity related to PON1*192 are much higher than differences related to PON1*55 and PON1-108. The PON1-related UFs in healthy adults were within the default toxicokinetic UF except for the slow metabolizers PON1*192 QQ and PON1*55MM. From these results, an uncertainty factor of eight would be needed to protect 95% of the slow metabolizers and 10 to cover 99%.
These distributions allow to: 1) apply PON1-related UFs in the risk assessment process for compounds for which in vitro PON1metabolism evidence are available without the need for animal data; 2) integrate PON1-related variability distributions with in vitro metabolism data into physiologically based kinetic (PBK) models for quantitative in vitro in vivo extrapolation (QIVIVE); 3) estimate UFs in the risk assessment process using variability distributions on metabolism.
The views in this publication do not necessarily represent those of EFSA, Anses, ISS and are the authors only.
Keywords: human variability, toxicokinetics, uncertainty factor, PON1-related uncertainty factor, chemical risk assessment
Non-dietary risk assessment of secondary metabolites of micro-organisms in plant protection products (#319)
W. Pfau1, E. Hinarejos Esteve2, I. Aragao1, M. Borja2
1 GAB Consulting, Department of Toxicology, Stade, Germany
Plant protection products containing micro-organisms (MPCP) like bacteria or fungi as active substances (MPCA) are regarded as safe for both the environment and human health. Recently, secondary metabolites (SM) formed by these micro-organisms have come into focus and it is required that a formal non-dietary risk assessment is conducted. The use of the relevant EFSA model (EFSA, 2014) in conjunction with the threshold of toxicological concern (TTC) concept (EFSA, 2016) has been proposed (OECD, 2018). For SM of unknown toxicity, a QSAR evaluation for genotoxicity is required.
Here we describe generic preliminary first tier estimations which demonstrate a safe use in high or low crops considering a reasonable application rate of 1 kg MPCP per ha/day. The application rate of the secondary metabolite is calculated with its concentration and considered in the EFSA model together with appropriate default values.
Operator exposure depends on the formulation type (liquid, granular or powder) and crop type (high or low crops). MPCA containing SM at concentrations of < 0.2 to 1000 ppm or < 2 to 2000 ppm are predicted to be safe for operators in high and low crops, respectively. When additional personal protective equipment such as protective gloves (and face mask) during mixing/loading are considered safe concentration range from < 100 ppm to < 10000 ppm.
For workers estimated exposure depends on crop type and tasks and are predicted to be safe at SM concentrations of < 1000 ppm (< 5000 ppm with gloves) for re-entry in vineyards or orchards and < 5000 ppm (< 10000 ppm with gloves) in arable crops or vegetables.
First tier estimations predict a safe exposure level for child residents up to 100 ppm upon application in high crops and < 1000 ppm in low crops. However, the model overpredicts resident inhalation exposure for low application rates and a higher tier refinement is possible.
These safe concentrations of secondary metabolites in the MPCA apply to non-genotoxic metabolites of low volatility and give only a generic orientation. A formal and detailed risk assessment considering the level of SM in the MPCP and the intended uses is required.
EFSA (2014) EFSA Journal 2014;12(10):3874
EFSA (2016) EFSA supporting publication 2016:EN-1000
Keywords: Microbial plant protection, regulatory toxicology, non-dietary risk assessment, secondary metabolites
Assessment of Bisphenol AF as an Endocrine Disruptor (#401)
L. Escrivá1, A. Hanberg1, J. Zilliacus1, A. Beronius1
1 Karolinska Institutet, Institute of Environmental Medicine, Stockholm, Sweden
Identification of Endocrine Disruptors (ED) is the first step to minimize human and environmental exposure. Scientific criteria and guidance for ED assessment have recently been established for pesticides in the EU1. Bisphenol A (BPA) is a widely used chemical classified as toxic for reproduction and identified as ED under REACH. Its potential adverse effects have resulted in restrictions for certain uses and increased use of BPA-analogs as safer alternatives. However, the potential toxicity of most of them are still unknown. Bisphenol AF (BPAF) is a structural BPA-analog with greater estrogen and anti-androgen activity in several in vitro studies2.
The aim of this work was to assess the ED properties of BPAF for human health by applying the recently established EU criteria and guidance.
A systematic literature review was performed by a non-targeted search (CAS, chemical name and synonyms) in WOS, Pubmed and Scopus databases. Title and abstract screening using RAYYAN (rayyan.qcri.org) and full text selection was performed. All relevant information was extracted and systematically reported. Reliability and relevance of data was assessed using SciRAP (www.scirap.org). Data was synthesized into lines of evidence for endocrine activity and adversity, respectively, and weight of evidence evaluation was performed.
Ninety-six of 456 identified studies were selected based on title and abstract and 72 were finally included in the dossier after full text analysis. Relevant extracted information included 461 parameters evaluated in mammals, fish and several cell lines. Lines of evidence for endocrine activity showed predominance of estrogenic mechanisms in vitro (activation of estrogen receptors, cell proliferation) and in vivo (estradiol and testosterone levels). Adverse effects included gonads histopathology, alterations of prostate, testes, seminal vesicle, mammary gland, and disturbance of the estrus cycle, indicating estrogenic and anti-androgenic effects.
There is strong evidence that BPAF has endocrine activity and causes endocrine-related adverse effects based on the EU criteria. A mode of action analysis is required to demonstrate the biological link between the endocrine activity and adversity. This study illustrates the application of the EU criteria and guidance for ED assessment for a non-pesticide.
Acknowledgements: This research was supported by the European Food Safety Authority - EFSA (EU-FORA 2018).
1. ECHA / EFSA 2018. Guidance for the identification of endocrine disruptors in the context of Regulations (EU) No 528/2012 and (EC) No 1107/2009.
2. DTU 2017. Danish Center on Endocrine Disrupters. List of Endocrine Disrupting Chemicals - Final report.
Keywords: bisphenol AF, endocrine disruptor, risk assessment
The Use of in silico Models for the Prediction of Mutagenicity (#404)
R. Middlemiss1, I. Crooks1, J. Lopez-Belmonte1, L. Nielson1, C. Meredith1
1 British American Tobacco, Southampton, United Kingdom
In silico methods have been gaining recognition and relevance across different industries. They are used to predict the potential toxicity of chemicals and an important advantage of these tools is their capability to provide an immediate and accurate estimate of potential toxicity hazards. This feeds into chemical prioritisation and early-stage risk assessments, giving a reliable indication of any further biological testing requirements. The hazard identification step within toxicological risk assessment often begins with using (quantitative) structure-activity relationship (Q)SAR models and in the case of mutagenicity, Ames activity is investigated. The International Council for Harmonisation (ICH) M7 guidance for the assessment and control of mutagenic impurities in pharmaceutical products recommends the application of in silico prediction techniques as part of the hazard identification and risk assessment strategy. The guideline advises the use of two complementary in silico models. We have explored the use of an expert rule-based system (Derek Nexus) and a statistical-based system (Leadscope Model Applier) for the prediction of mutagenic potential. Twenty-five compounds were investigated covering ECHA harmonised and self-notified mutagens, and mutagens and non-mutagens identified from a literature search. These were analysed using the (Q)SAR models to evaluate their sensitivity and specificity for the endpoint of mutagenicity against public data. The sensitivity of both programmes individually was 100%. The specificity when using one programme alone was 73% and this was increased to 91% when two in silico models were combined. This demonstrates that by combining complementary models, the number of false positive predictions can be reduced and increases the confidence in predictions when used in combination.
Keywords: Mutagenicity, in silico
Six-month repeated dose toxicity of subcutaneously administered BM41, a novel Allergen Immunotherapy candidate, in Wistar rats (#415)
P. P. Chrusciel1, U. - M. Jaakkola1, L. Linko1, L. Aglas2, F. Ferreira-Briza2, F. Stolz3, L. Jongejan4, R. van Ree4, E. Yatkin1
1 University of Turku, Central Animal Laboratory, Turku, Finland
Birch pollen allergy is one of the most common respiratory disease in Europe. “BM4SIT – Innovations for Allergy” (www.bm4sit.eu) is an EU-funded project evaluating the efficacy of a novel Allergen Immunotherapy (AIT) candidate for the treatment of birch pollen allergy. A vaccine based on a hypoallergenic variant of Bet v 1, the major birch pollen allergen, called BM41 is designed to reduce allergic side effects and be more effective in the modulation of the allergic towards an anti-inflammatory immune response.
This repeated dose toxicity study was designed 1) to provide information on the major toxic effects of BM41 and 2) to indicate possible target organs and 3) to provide an estimate of the no-observed-adverse-effect level (NOAEL) of exposure after bi-weekly subcutaneous administration over a period of six months in Wistar rats. The study was performed according to OECD principles of Good Laboratory Practice (GLP) using 90 adult Wistar rats. The animals were allocated into three treatment groups to receive either Placebo only, Low dose of BM41 (20 µg) or High dose of BM41 (40 µg). For the adjuvant alhydrogel was used. Clinical signs, morbidity and mortality, body weights, water and food consumption were monitored during the experimental period. Blood, urine and tissue samples were collected at the end of the study for hematology, clinical chemistry, immunological and coagulation tests, urinalysis and for histopathological evaluation. Animals from the Main groups were sacrificed one week after the last dose (study week 24) while Recovery groups were kept for six more weeks (up to study week 30) after the treatment period for observation of reversibility or persistence of any toxic effects.
No animals in moribund state or having significant toxic symptoms were found and no mortality was recorded. Observed microscopic findings were either considered similar in Placebo and test item treated animals, and thus not considered related to treatment with BM41, incidental or within background changes seen microscopically in rats of this age.
This study demonstrates that 20 µg and 40 µg of BM41 did not cause significant effects on vital signs and did not produce toxicologically significant adverse effects. The dose 40 µg BM41/0,5 mg Alum/0.9 % NaCl in 250 µl reflects a no-observed-adverse-effect level (NOAEL) of exposure.
Keywords: BM41, repeated dose toxicity, Wistar rat, GLP, allergy
Nonclinical development of products intended for treatment of damaged skin (#420)
J. Løgsted1, T. Starostka1, A. Makin1
1 Citoxlab, Lille Skensved, Denmark
For pharmaceutical products intended for dermal application, the minipig is an ideal animal model due to the close similarity of the skin of this species to human skin. Under normal conditions, the skin constitutes a relatively profound barrier to the external surroundings. However, in some human skin diseases, superficial abrasions and sores develop, potentially increasing systemic absorption of substances intended for topical skin use. This issue is not covered when using healthy animals with intact skin in regulatory toxicity testing of products intended for dermal application. For such studies, development of a model for repeated administration on damaged skin is becoming relevant and we have used the Göttingen minipig in regulatory toxicity testing of dermal products, to address this issue. The model includes an initial wound healing phase. Depending on the duration of the study, on completion of the healing phase of the wounds, a different route of administration, for example subcutaneous dosing to give good systemic exposure to the Test Item, can be considered. This model has been accepted by the regulatory authorities. Surgically applied wounds are used in the study design as these can be inflicted in a very precise and reproducible way. It is not considered ethical to wound the animals repeatedly, therefore the initial wound healing phase being combined with a second dose route if necessary. This meets requirements for longer duration non-clinical studies and maximises systemic exposure of the test compound. All routine guideline requirements for evaluation of systemic toxicity in non-rodent species are integrated into the study design. We conclude that the combination of a wound healing study with a second dosing route constitutes a valid method for testing test compounds intended for use on damaged skin in humans. This poster presents different study design options that have been used in regulatory studies and based on the study outcomes, evaluates their appropriateness.
Keywords: Dermal toxicology, Wound healing, Minipigs
Critical review of the human database used for performance evaluation of defined approaches to skin sensitisation testing (#431)
M. Herzler1, J. Gordon2, H. - S. Ko3, J. Matheson2, J. Strickland4, H. - J. Thierse1, J. Truax5, N. Kleinstreuer4
1 German Federal Institute for Risk Assessment (BfR), Berlin, Germany
An estimated 15-20% of the general population suffer from contact allergy (Peiser et al., 2012). Human predictive patch tests (HPPT) have been employed to explore the sensitising properties of chemicals for decades, e.g. the Human Maximisation Test (Kligman 1966) or the Human Repeated Insult Patch Test (Politano and Api, 2008). HPPT data were used in the validation of the Local Lymph Node Assay (ICCVAM 1999) and have been integrated with other information to compare the relative sensitising potencies of different chemicals (Api et al., 2017; Basketter et al., 2014). In 2018, an OECD expert group began characterising the performance of published Defined Approaches (DAs) to skin sensitisation testing and assessment (OECD, 2016). Under this activity, the authors of this presentation curated a large HPPT dataset and analysed its suitability to serve as a reference point for DA performance in terms of classifying sensitisers using the potency categories provided by the Globally Harmonized System of Classification and Labelling of Chemicals (GHS). HPPT data reliability and inherent variability were critically reviewed to determine the potential role of these data in binary hazard classification and potency sub-categorisation, using HPPT study reports previously collated at NICEATM and later reviewed for quality and extended with additional data by BfR. Results from this activity are presented, with a more comprehensive review forthcoming. The final curated HPPT database will be available to the public to facilitate additional activities such as screening or modelling.
Api A.M. et al. (2017), Dermatitis 28(5), 299-307. DOI: 10.1097/der.0000000000000304
Basketter D.A. et al. (2014), Dermatitis 25(1), 11-21. DOI: 10.1097/der.0000000000000003
ICCVAM (1999), NIH Publication No. 99-4494
Kligman A.M. (1966), Journal of Investigative Dermatology 47(5), 393-409. DOI: 10.1038/jid.1966.160OECD (2017), DOI: 10.1787/9789264279285-en
OECD (2016), ENV/JM/MONO(2016)29/ANN1
Peiser M. et al. (2012), Cellular and Molecular Life Sciences 69(5), 763-781. DOI: 10.1007/s00018-011-0846-8
Politano V.T. and Api A.M. (2008), Regulatory Toxicology and Pharmacology 52(1), 35-38. DOI: 10.1016/j.yrtph.2007.11.004
Keywords: defined approaches to testing and assessment, skin sensitisation, human patch test database, regulatory review, classification and labelling
The use of dosimetric modeling in the derivation of acute inhalable DNELs for nickel metal and nickel compounds (#436)
M. Taylor1, S. Seilkop2, A. Oller1
1 NiPERA, Inc., Durham, North Carolina, United States of America
Under REACH legislation in the European Union, acute inhalation Derived No Effect Levels (DNELs) for local and systemic effects are required for substances that are classified for acute toxicity by inhalation or that cause local adverse effects. These DNELs are compared to worker exposure levels for the characterization of risk and to guide the selection of risk management measures. For nickel compounds, acute systemic DNELs can be based on the results of single-exposure mortality studies in rats. However, for local effects, there are no acute studies where lung toxicity was examined in any detail. Instead, acute local DNELs are based on lung inflammation associated with 16 or 28 day repeated exposure studies to nickel metal or nickel compounds. These rodent inhalation studies utilize exposure to nickel particulates in the respirable size range (MMAD ≤ 4 μm). By contrast, workers are usually exposed to more complex aerosols made up of larger inhalable particles (MMAD ≤ 100 μm), with fewer workers exposed to respirable-sized particles exclusively. Therefore, both inhalable and respirable DNELs were derived for systemic and local effects for comparison to workplace exposures. Dosimetric modeling estimates of interspecies differences in deposition (MPPD v2.1) were used to calculate human equivalent concentrations (HECs) based on pulmonary particle deposition (for systemic effects) or retention (for local effects) in rats. This modeling also allowed the incorporation of respirable or inhalable workplace particle size ranges in the calculations. Once HECs for nickel that were equivalent to points of departure from the animal studies were calculated, assessment factors were applied for remaining toxicokinetic and toxicodynamic differences. In addition, nickel-specific duration of exposure adjustments were incorporated into the local-effects DNEL calculations. The dosimetrically derived inhalable and respirable DNELs are more appropriate for worker risk characterization under REACH than those based solely on animal exposure concentration and particle size distributions.
Keywords: occupational risk assessment, DNEL, REACH, Dosiometric modeling
Proposal for a Selection of Priority Biocide Mixtures in Consumer Products: Screening the Potential Synergistic Toxicity on Pulmonary Fibrosis (#448)
J. Kim1, Y. Lee2, Y. - Y. Lim1, H. Keum1, H. Kim1, S. - I. Shin1
1 Korea Research Insitute of Chemical Technology (KRICT), Chemical Safety Research Center, Daejeon, Republic of Korea
Different biocidal products have been broadly developed for industrial, professional, and consumer uses to restrain any unwanted harmful organism. Biocides can be also added as additives into other substances for keeping chemical products and articles from biological influx and contamination. In European Union (EU), a new biocidal product regulation (BPR) applied from 2013 for enhancing the authorization process of biocides. In a similar way, a new Korean biocides regulation, entitled “Consumer Chemical Products and Biocides Safety Act (also known as K-BPR)”, recently entered into force in South Korea in January 2019. Korea Ministry of Environment very recently initiated R&D programs, “The Environmental Health Action Program” and “Technology Program for Establishing Biocide Safety Management”, for supporting the implementation of the K-BPR.
These regulations make chemical risk assessors consider the mixture toxicity between active substances, or between active substances and other additives in the authorization process of biocidal products, if any. This is due to the fact that biocides may trigger the mixture toxicity effects on human health, and the environment if they interact with other biocides or substances at the same time and place due to their mixture toxicity (also known as cocktail effect or combined toxicity). Under those regulations, an additive toxicity approach based on concentration addition model has been frequently recommended as a default method to evaluate the toxicity of mixtures when there is no evidence on toxicological interactions among mixture components. However, available data on such interactions in mixtures still lack. Combined inhalation exposures to biocide mixtures by consumer products can occur mainly using sprays and powders. Combined inhalation exposures to airborne toxicants may also cause pulmonary or even systemic inflammation.
Therefore, the objectives of this study were i) to conduct a meta-study for investigating possible combinations of biocides in consumer products based on the EU and Korea chemical databases on consumer products, and ii) to screen biocide mixtures which may cause potential synergistic toxicity on pulmonary fibrosis using a scoring and ranking system. This study highlights a priority list of biocide mixtures that need to be assessed as a priority by toxicity testing to identify their synergistic toxicity on pulmonary fibrosis.
Keywords: synergistic toxicity, pulmonary fibrosis, combined exposure, scoring and ranking system, biocide
Computational Toxicology @ German Toxicology Society (#461)
J. vom Brocke1, M. Frericks2, S. E. Escher3, L. T. Anger4
1 ECHA, Helsinki, Finland
German Toxicology Society
The working group „Computational Toxicology“ was founded in 2018 as a specialty section of the German toxicological society (GT). Its main purpose as an expert platform in this emerging field of toxicology is the advancement of computational methods and their application in regulatory risk assessment. We aim to facilitate research and training by enhancing the communication of its members, contributing to symposia, meetings, and hosting or supporting educational events. Relevant tools and methods include QSAR, read-across, building and analysing toxicological databases, PBTK and metabolism predictions.
General assemblies are held during the annual meetings of the German Society for Experimental and Clinical Pharmacology and Toxicology (DGPT). At the annual meeting in February 2018, the specialty section held its first symposium on computational toxicology. Contributing to continuous education, an "Advanced Course in Computational Pharmacology and Toxicology" was offered jointly with the Clinical Pharmacology (DGKliPha) subdivision of the DGPT.
Members have been actively engaged in international consortia sharing toxicity data, such as the IMI eTOX/eTRANSAFE, and contribute to the "in silico toxicology protocols" consortium which aims to standardise in silico tool use and their interpretation. Furthermore, members are engaged as lecturers for Computational Toxicology at universities and in education courses offered by the German Toxicology Society ("Fachtoxikologe GT") for certification as European Registered Toxicologist.
The specialty section currently has 27 members from 15 institutions in Germany, Finland, Switzerland, and the USA. At Eurotox 2019 we ask you and other interested members of academia, industry and authorities to get in touch and discuss future collaboration. E-mail us at: email@example.com
Keywords: Computational Toxicology, qsar, read-across, pbtk, regulatory risk assessment
Assessment of priority tobacco additives per the requirements of the EU Tobacco Products Directive (2014/40/EU) (#477)
R. Stabbert1, M. McEwan2, M. Ashley2, A. Clarke3, S. Coburn2, J. Collard2, I. Crooks2, M. Esposito1, J. Freiesleben4, D. Ghosh5, L. Giles6, G. Jaccard1, H. - K. Kim7, S. Larroque6, T. Lindegaard8, R. Lutz5, A. Manson9, J. Martinez6, J. Miller6, J. Murphy2, C. - H. Park7, T. Paschke6, G. Pollner10, C. Proctor2, E. Roemer11, M. Scharfe12, H. - O. Sohn7, D. Tafin Djoko1, P. Vlachos13, I. Vincze14, D. Wigotzki15, D. Yuki6, L. Simms3
1 Philip Morris Products S.A., PMI Research & Development, Neuchâtel, Switzerland
Non-clinical and clinical assessments were performed to fulfill the regulatory requirement per Art. 6 (2) of the EU Tobacco Products Directive 2014/40/EU, under which Member States shall require manufacturers and importers of cigarettes and roll-your-own tobacco containing an additive that is included in the priority list established by Commission Implementing Decision (EU) 2016/787 to carry out comprehensive studies. The results of smoke chemistry (39 World Health Organizations smoke emissions), in vitro toxicology, and clinical studies performed by independent Contract Research Organizations are presented. Minor changes in smoke chemistry parameters were observed when comparing emissions from test cigarettes containing priority additives with emissions from additive-free reference cigarettes, and only two of the additives (sorbitol and guar gum) tested led to significant increases in a limited number of smoke constituents. These changes were not observed when sorbitol or guar gum were tested in a mixture with other priority additives. None of the priority additives resulted in increases in in vitro toxicity in the Ames, micronucleus, and neutral red uptake assays. In the clinical study, two distinct endpoints were investigated, namely measuring plasma nicotine pharmacokinetics as a measure of nicotine uptake and analyses of changes in smoker puffing behavior as a measure of cigarette smoke inhalation. This clinical study indicated that the inclusion of none of the priority additives, either as single additive or as part of a chemical mixture, facilitated nicotine uptake. Furthermore, the data did not suggest that differences in the inhalation pattern of cigarette smoke of any of the priority additives tested occurred when compared with the additive-free reference cigarette.
European Union, Directive 2014/40/EU of the European Parliament and Council, On the approximation of the laws, regulations and administrative provisions of the Member States concerning the manufacture, presentation and sale of tobacco and related products and repealing Directive 2001/37/EC. In: Council, E. P. a., (Ed.), Directive 2014/40/EU, 2014.
European Union, Priority list of additives contained in cigarettes and roll-your-own tobacco subject to enhanced reporting obligations In: Commision, E., (Ed.), Decision (EU) 2016/787, 2016.
Keywords: tobacco additives, smoke chemistry, in vitro, smoking behavior, nicotine absorption
Read-across approach, based on a combined use of five in silico tools, predicts practically identical true compound toxicity (#488)
S. Heinz1, A. Granitzny1, A. Mol2, S. Nakagawa3, A. Fuchs1, R. Fautz1
1 Kao Germany GmbH, Safety & Toxicology, Darmstadt, Germany
The cosmetic industry has faced challenges in recent years regarding insufficient data for the safety assessment of new raw materials due to the animal testing ban. A commonly used method for filling data gaps is read-across. However, uncertainty remains, which has an impact on the reliability and acceptance of this approach. To improve this, we investigated five in silico tools, ToxRead, AMBIT, COSMOS, ChemTunes.ToxGPS and OECD Toolbox, to establish a relevant next generation safety assessment (NGSA) strategy based on a combined use of these tools. We hypothesize this strategy can raise confidence in predictions and lower the uncertainty. With this objective, we conducted case studies of 12 cosmetic ingredients with full toxicological profiles to determine if the read-across outcome, based on our novel NGSA, predicts the toxicity correctly. The combination of these five tools identified sufficient, relevant analogues with toxicological data, and allowed a successful read-across and prediction of the toxicity of the target compounds. For example, 14 relevant analogues were found for cinnamyl alcohol, a known skin sensitizer. 71% of these showed at least one positive experimental or QSAR-based result for sensitization. This correctly predicted the sensitizing potential of the target. Regarding systemic toxicity, the lowest point of departure, a NOAEL of 53 mg/kg bw/d, was covered by the lowest NOAEL found among the identified analogues (53.4 mg/kg bw/d). For genotoxicity, 35.7% of the analogues were negative and 35.7% had positive experimental results; 28.6% had no data. However, the hazard prediction feature of ChemTunes.ToxGPS predicted a genotoxic potential for 57% of all analogues. As a worst-case scenario, we predicted the target to be genotoxic. This is in line with the experimental results of cinnamyl alcohol, which shows mainly negative results but also an alert for genotoxicity.
Based on our results, we began developing a decision tree to provide guidance on when to use all five tools or when a limited number of tools could be sufficient (e.g. new material with no prevalence vs. new material with high prevalence).
In conclusion, our investigations indicate that a combined use of the in silico tools presented leads to a reliable and sufficiently conservative safety assessment approach for cosmetic compounds.
Keywords: read-across, uncertainty, in silico tools, case studies, safety assessment
A QSAR and Read-Across Methodology for Genotoxicity Endpoints to Support Registration of Agrochemicals in Europe (#493)
L. Brierley1, E. Booth1, E. Lessmann1, K. Bridgwood1, D. Parr-Dobrzanski1
1 Syngenta Ltd, Human Safety, Bracknell, United Kingdom
As part of the registration of agrochemicals in Europe QSAR and read-across may be used to assess the genotoxicity potential of metabolites and impurities reducing the reliance on in vitro and in vivo data generation.
QSAR and read-across usage have been embedded in the pharmaceutical industry ( e.g. ICH M7 guidelines) and as part of REACH requirements for a number of years. In the agrochemical industry, the acceptance of the use of these methods has been limited in a regulatory context. However, with the EFSA publication of Definition of Residue (not yet adopted) and Impurities guidance there has been a rapid expansion into QSAR/read-across processes to address these endpoints.
A transparent QSAR and read-across methodology based on the current guidance has been developed to support metabolites and impurities of agrochemical active ingredients. Three QSAR systems (DEREK Nexus, CAESAR and the OECD QSAR Toolbox) are used in concert and chemical grouping based on QSAR alerts and structural similarity/chemical reactivity with respect to genotoxic endpoints proposed. Additionally, metabolites with existing genotoxicity data are identified based on similarity and included in the assessment as representative compounds in the grouping approach.
Based on the output, additional genotoxicity testing may be proposed to support the grouping approach.
A degree of regulatory conservatism is taken into account when proposing a chemical grouping strategy based on the QSAR and read-across assessment.
An example of the QSAR and read-across approach will be demonstrated.
This approach can be transferred to support various endpoints where an understanding of the genotoxic potential is required for multiple chemical entities, e.g metabolites or impurities, while still maintaining a robust scientific methodology.
The aim is to use the methodology to support active ingredient submissions in Europe and beyond where a QSAR and read-across assessment is required.
Keywords: QSAR, read-across, regulatory toxicology, agrochemicals, genotoxicity
Incorporation of rabbit suitability as a test species in a Framework to evaluate an adequate adaption for PNDT 2nd species information requirement under REACH (#511)
N. Synhaeve1, J. E. Foreman2
1 ExxonMobil Petroleum and Chemical BVBA, Machelen, Belgium
Testing for pre-natal developmental toxicity (PNDT) in two species is an Annex X requirement under REACH. The preferred species are rat and rabbit. Due to the known sensitivity of rabbits to gastro intestinal (GI) imbalances ECHA initiated a project to investigate the impact of rabbit GI toxicity in PNDT studies. In this investigation ECHA found rabbits were more sensitive than rats for REACH substances with respect to lower maternal mLOAEL, more frequent GI toxicity, abortions, and mortality. Of interest this differs from investigations of PNDT studies conducted with pharmaceuticals where no species sensitivities were identified (Theunissen et al.), which suggests there could be a fundamental difference in the utility of rabbits for PNDT testing of REACH substances perhaps due to specific physical chemical properties not shared by pharmaceuticals. In particular ECHA identified that substances classified for skin irritation or corrosion, or substances with low water solubility (<1 mg/l) led to GI effects more frequently in rabbits. In their discussion ECHA recommended conducting a dose range finding (DRF) study on substances with these properties to conclude on the suitability of rabbits as test species. In accordance with ECHA’s recommendation a PNDT DRF study was conducted on a REACH registered UVCB substance where greater than 90% of the constituents had water solubility lower than 1 mg/l. Consistent with the ECHA evaluation our study suggests that the rabbit is not a suitable test species for the tested substance. The water solubility criteria has been included into a framework we developed to assess objectively our substances for the 2nd species information requirement under REACH. The framework has established a decision logic for considerations on whether the general adaptation possibilities of Annex XI of the REACH Regulation are adequate to generate the necessary information as required by ECHA. For this substance it has been determined that, in accordance with Annex XI section 3, an exposure based waiving adaptation is also adequate. The framework, results of the DRF study, and exposure based waiving adaption are presented here.
Keywords: developmental toxicity, rabbit, REACH, framework
Applying pathway-oriented thinking to problem formulation for planning a Systematic Review: a case study with aluminium-containing antiperspirants and female breast cancer risk (#518)
N. Roth1, M. F. Wilks1
1 University of Basel, Swiss Centre for Applied Human Toxicology, Basel, Switzerland
The use of evidence-based methods for evaluating human health risks from environmental chemical exposures is still in its infancy. Case-studies showing how Systematic Review principles and methods can be translated to the chemical risk assessment context are needed for advancing best practices in evidence-based toxicology. We have developed a stepwise approach to Problem Formulation, using aluminium-containing antiperspirants (Al-AP) and female breast cancer risk as a case-study. Regulatory bodies have concluded, albeit with high uncertainty, that Al-AP are not a risk factor for female breast cancer, however the existing evidence has not been systematically reviewed and critically evaluated. Since this is a broad consumer health topic with direct relevance to regulatory decision-making, our aim was to explore how evidence mapping and pathway-oriented thinking can be applied to Problem Formulation to support planning, scoping, and framing primary and secondary PECO (Population, Exposure, Control, Outcome) questions in the broader context of health risk assessment. We mapped the grey (regulatory toxicology) literature to identify the conceptual boundaries, breadth and depth of analysis, research and regulatory activities, and major knowledge gaps and research needs; as well as to evaluate the feasibility and value to conduct a Systematic Review on the topic. A conceptual model (analytical framework) was developed that maps and causally links key research questions, working hypotheses, routes of exposure, pathways of toxicity, and primary and secondary health outcomes, based on a three-level hierarchy integrating the various dimensions of a health risk assessment: risk (first level), hazard and exposure (second level), and mechanistic and biokinetic (third level) related information. The model can be used in a transparent, objective and iterative manner, as a dynamic and central tool to lay out the methodological foundation of a Systematic Review on the topic.
Keywords: evidence-based toxicology, problem formulation, systematic review, evidence map, grey literature
How to develop the best strategy to meet the reproductive toxicity information requirements within the EU REACH regulation (#566)
S. Bergeret1, M. Bilau1, S. Jacobs1
1 Arcadis Belgium nv/sa, Product Stewardship Solutions , Brussels, Belgium
Prenatal developmental toxicity studies in one or two species and/or an extended one-generation reproductive toxicity study (EOGRTS, since March 2015) are imposed on EU REACH registrants at the higher tonnage levels (>100 tonnes per year). Selecting the most appropriate strategy to meet these information requirements is critical for the registrant as it may significantly impact the budget needed as well as the timeline for dossier completion. The uncertainties related to the outcome of these higher tiered tests may be challenging in terms of data interpretation and decisions on appropriate risk management measures. The elaboration of the testing strategy starts with the evaluation of all relevant existing information and especially repeated dose toxicity and reproductive toxicity data. When further in vivo testing is required, refinement of complex study designs is required for animal welfare reasons. More specifically the design of the modular EOGRTS needs to be well-defined, referring to the premating exposure duration, dose selection, and potential additional cohorts for assessment of F2 generation, neurotoxicity or immunotoxicity. The refined study design elaborated for the test to be performed should be described in a testing proposal submitted by the Lead Registrant, together with considerations for alternative testing methods. Authorities can request reproductive toxicity testing combined with a subchronic toxicity study, either in parallel or in a tiered approach. Stepwise testing would facilitate optimized study designs by intermittent data interpretation. A comprehensive analysis of publicly available information on ongoing dossier evaluations and decision-making processes will be presented. Generating additional information of this magnitude also depends on collaboration with experienced testing facilities offering sufficient capacities. Extension of imposed timelines might need to be considered in light of limited capacity at the testing facilities.
Strategies for endpoint coverage will be presented and challenges for impacted registrants with substances in the Annex IX and/or Annex X tonnage band are highlighted.
Keywords: reproductive toxicity, REACH, EOGRTS, prenatal development toxicity
Assessment Strategy for the Identification of Endocrine Disruptors under the Biocidal Products and Plant Protection Products Regulations (#573)
N. Andersson1, M. Arena2, D. Auteri2, S. Barmaz2, E. Grignard3, A. Kienzler3, P. Lepper1, A. M. Lostia2, S. Munn3, J. M. Parra Morte2, F. Pellizzato1, J. Tarazona2, A. Terron2, S. van der Linden1
1 European Chemicals Agency, Helsinki, Finland
In 2018, the new scientific criteria for the determination of endocrine disrupting (ED) properties became applicable to the Biocidal Products Regulation (BPR) (EU) No. 528/2012 and the Plant Protection Products Regulation (PPPR) (EC) No. 1107/2009. In the same year, ECHA and EFSA have jointly published the Guidance document  on how to identify endocrine disruptors in accordance with these criteria.
According to the ED criteria, which are limited to hazard identification, a substance shall be considered as having ED properties: if it shows an adverse effect, if it shows endocrine activity, and if there is a biologically plausible link between the adverse effect and the endocrine activity (i.e. it has an endocrine mode of action). The criteria require a weight of evidence approach, taking into account all available information: this includes a systematic review of the scientific literature. Separate conclusions are required on whether the criteria are met with respect to humans and non‐target organisms.
The current data requirements under the BPR and PPPR contain more mammalian studies that may be informative on ED properties than studies on other taxonomic groups. Thus, in line with the general principle to avoid unnecessary animal testing, the assessment strategy in the guidance recommends to strive for a conclusion on the ED properties with regard to humans first, followed by a conclusion on mammals as non‐target organisms based on the same data set. Only when the ED criteria are not met for mammals as non‐target organisms, there will be a need to proceed to other taxonomic groups. Depending on the available data, additional data might need to be generated.
The guidance document addresses the necessary steps to establish whether the ED criteria are met. It describes the gathering and evaluation of all relevant information for the ED assessment, how to conduct a mode of action analysis (including assessing essentiality, consistency and specificity), and when and how to apply a weight of evidence approach. To facilitate the assessment, parameters have been assigned to different groups, depending on whether they provide information on endocrine activity, on adversity or both. First experiences with the practical application of the ED Guidance, including the template for data collection, are discussed.
 Guidance for the identification of endocrine disruptors in the context of Regulations (EU) No 528/2012 and (EC) No 1107/2009 https://doi.org/10.2903/j.efsa.2018.5311
Keywords: pesticides, biocides, endocrine disruptors
A review of the toxicological information available for Dicyclopentadiene (DCPD) pertinent to its assessment to potentially cause endocrine disruption (#615)
T. Petry1, N. Aygun Kocabas2, B. Mani3, E. Rushton4, M. Rooseboom5, N. Synhaeve6, G. Martin7
1 ToxMinds BVBA, Brussels, Belgium
Dicyclopentadiene (CAS No. 77-73-6), abbreviated as DCPD, is an olefinic hydrocarbon which is manufactured and imported into the European Union in quantities greater than 1,000 tons per year. Concerns related to foetotoxic effects observed in reproductive toxicity studies at high doses led the REACH registrants to self-classify DCPD as a Category 2 reproductive toxicant under the EU CLP Regulation. These also led to a review of DCPD by the French Agency for Food, Environmental and Occupational Health & Safety (ANSES) under the European Chemical Agency (ECHA)’s Community Rolling Action Plan (CoRAP).
To elucidate whether the observed developmental effects may be triggered by an endocrine mode of action, CEFIC’s Lower Olefins Sector Group (LOSG), composed of the main DCPD interested petrochemical companies, formed an ad-hoc toxicology expert working group to review the scientific evidence for this hypothesis. The LOSG group followed OECD (2018) and EFSA/ECHA (2018) principles for the assessment of endocrine disrupting properties by identifying, collating and assessing the existing information pertaining to the potential endocrine activity and adversity of DCPD. Existing in vitro and in vivo information was complemented with additional structure activity modelling using ECHA-recommended (Q)SAR software tools. Lines of evidence were then assembled and assessed following a weight of evidence approach.
The objective of this poster is to present and discuss the data underlying the outcome of this review. Overall, taking the information from (Q)SAR, mechanistic in vitro and OECD conceptual framework level 4 and 5 in vivo studies into account, lines of evidence for endocrine-mediated adversity could not be established. Hence, the weight of evidence supports the conclusion that DCPD does not cause developmental toxicity via an endocrine mode of action.
OECD (2018). OECD conceptual framework for testing and assessment of ED chemicals. http://www.oecd.org/env/ehs/testing/oecdworkrelatedtoendocrinedisrupters.htm.
EFSA/ECHA (2018). Guidance for the identification of endocrine disruptors in the context of Regulations (EU) No 528/2012 and (EC) No 1107/2009. https://echa.europa.eu/documents/10162/23036412/bpr_guidance_identif_ed_en.pdf/1a4d2811-3faa-fe61-1de2-3cbce8fd4d95.
Keywords: Endocrine disruption, Regulatory Toxicology
Can diet-induced obesity and food restriction separate body weight-related from drug-related findings in rats following treatment with an anti-obesity compound? (#621)
F. Bolze1, J. M. Rojas1, I. Thorup1, L. W. Andersen1, M. Skydsgaard1, H. K. Offenberg1, J. T. Jensen2, G. Jeppesen2, I. Sjögren1, C. M. Dalsgaard1, Y. Tingle3, J. J. Fels1, P. S. Galle1, K. S. Nielsen1, M. Dalgaard1
1 Novo Nordisk A/S, Research & Development, Maaloev, Denmark
Preclinical safety evaluation of anti-obesity drug candidates is a challenge, since marked body weight loss in normal weight rodents has detrimental effects on various organ systems. We hypothesize that the observed pathological changes in normal weight, healthy, chow fed, young rats, treated with a potent anti-obesity drug are attributed to secondary effects related to drug-induced weight-loss rather than being a direct effect of drug toxicity per se. It was therefore anticipated that 1) obese rats would develop less pathological findings compared to normal weight animals, due to excess energy storage and 2) that food restriction (FR) and drug dosing would result in a similar pattern of findings when inducing a comparable body weight loss.
To test this hypothesis, male and female Sprague-Dawley rats (n=12-16/sex/group) were fed ad libitum either a standard control chow diet or a 45 kJ% high fat diet (HFD) for 23 weeks to achieve diet-induced obesity (DIO). Subsequently, the animals underwent either FR or treatment with daily subcutaneous doses of an anti-obesity drug candidate for 4 weeks to induce a 20% body weight loss.
As previously shown, HFD resulted in only a modest additional weight gain in comparison to chow-feeding (Rojas et al.) and DIO did not prevent the development of histological changes in drug-treated rats. Notably, some histopathological changes (e.g. in testis and kidneys) were exclusively detected in drug-treated DIO males, suggesting a higher susceptibility of DIO rats to develop findings in certain organ systems. Apart from thymus atrophy and increased macrophage infiltration in females, drug-treatment and FR shared only a few identical findings related to elevated fuel mobilization in response to energy deprivation such as reduced adipocyte size. Specific findings (e.g. in pancreas, salivary and Brunner’s glands) were additionally found in chow as well as HFD fed drug-treated rats, indicating that a direct effect of the drug was accountable for these changes.
In conclusion, FR seems successful in differentiating body weight-related from drug-related findings, whereas DIO did not prevent findings induced by the anti-obesity drug. Factors such as age, degree of obesity, difference in nutritional constituents, stress level and mode of weight loss may affect the outcome of the study.
Rojas JM, Bolze F, Thorup I, Nowak J, Dalsgaard CM, Skydsgaard M, Berthelsen LO, Keane KA, Søeborg H, Sjögren I, Jensen JT, Fels JJ, Offenberg HK, Andersen LW, Dalgaard M. (2018). The Effect of Diet-induced Obesity on Toxicological Parameters in the Polygenic Sprague-Dawley Rat Model. Toxicological Pathology; 46(7):777-798. doi: 10.1177/0192623318803557.
Keywords: Spraque Dawley Rat, preclinical safety assessment, diet-induced obesity, food restriction, anti-obesity drug candidate
Tobacco and tobacco products test results before and after the implementation of the 2014/40 EU tobacco directive (#623)
I. Vidic Strac1, N. Dimitrov1, B. Damianic1, D. Brlek Gorski1, B. Vučić1, L. Hrnjkaš1
1 Croatian Institute of Public Health, Divison for Environmental Health, Zagreb, Croatia
In 2016 and 2017 tests were carried out on randomly bought tobacco and tobacco related products for the purpose of screening the market situation before and after the implementation of the 2014/40 EU Tobacco Products Directive. In this research test results for cigarettes, e-cigarettes and e-liquids were compared. The tests were carried out in accordance with EU Directives (2001/37/EC and 2014/40/EU) and requirements of national legislation (Ordinance on Health Safety of Consumer Items (OG 125/2009).
Results: Before and after implementation of Tobacco Products Directive mold was found in cigarette samples. Results also showed that organochlorinated pesticides in tobacco, content of lead and arsenic, as well as product declarations were in compliance with the requirements of the legislations. Carbon monoxide, nicotine and tar content in smoke condensate in samples from 2017 were within limits of compliance. In cigarete samples from 2016 elevated content of carbon monoxide in the smoke condensate were found. Cigarete samples from 2016 also had 5% of the tar content higher than the declared value. In samples from 2016 and 2017 lead and cadmium were found in mouthpieces. Lead concentrations in 30% of tested mouthpieces from 2016 were above MAC values according to the requirements of Commission Regulation (EU) No. 836/2012 amending Annex XVII to Regulation (EC) No 1907/2006 (REACH).It was found that acrylic copolymer mouthpiece measured 10 m/m% of lead. Tests were repeated in 2017, when two other samples with unacceptable lead content were found. Those samples were also acrylate-based materials (polyacrylamide, styrene-acrylate copolymer applied to a stainless steel base). In 2016 there were no legal restrictions regarding the concentration of nicotine of 20mg/mL in e-liquid. That was one of the reason why in 50% of the tested samples nicotine concentration were above this value and highest measured concentration was 28.9mg/mL. In samples tested in 2017, the nicotine concentration in all samples was compliant with the 2014/40/EU Directive. In addition, the labelling verification according to the Directive was conducted, as well as screening for selected allergens in products and comparison with the declared ingredients (in 2017). Only 25% of the samples complied with all the requirements.
Conclusion: Comparison of the test results proved a positive effect of the implementation of the new Tobacco Products Directive has had on the tobacco industry and tobacco related products. Since there is a wide variety of products available on the market and the variety of manufacturers, an uneven interpretation of labelling requirements has been noted. Hazard identification, monitoring of the market and smoking prevalence has to be part of health prevention, especially for the young population for whom tobacco products are banned but still very attractive.
Ordinance on Health Safety of Consumer Items (OG 125/2009)
Directive 2014/40/EU of the European Parliament and of the Council of 3 April 2014on the approximation of the laws, regulations and administrative provisions of the Member States concerning the manufacture, presentation and sale of tobacco and related products and repealing Directive 2001/37/EC
Directive 2001/37/EC of the European Parliament and of the Council of 5 June 2001 on the approximation of the laws, regulations and administrative provisions of the Member States concerning the manufacture, presentation and sale of tobacco products
Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH)
Keywords: tobacco, related products, implementation, screening of the market
Systematic evaluation of in vitro data for hazard and risk assessment – development of the SciRAP tool (#626)
A. Beronius1, J. Zilliacus1
1 Karolinska Institutet, Institute of Environmental Medicine, Stockholm, Sweden
There is currently a rapid development of in vitro methods as alternatives to animal studies for investigating health effects of chemical exposure, driven by stakeholder needs, academic research interests and increased regulatory focus on the 3R concept1,2. In vitro data also provide valuable mechanistic information to hazard and risk assessment of chemicals, for example in the assessment of endocrine disruptors according to new EU regulations3. However, tools for systematic evaluation of the reliability and relevance of in vitro data have been lacking. Here we present the development of a tool for evaluating in vitro data on the Science in Risk Assessment and Policy (SciRAP) on-line platform. Criteria for evaluating reliability, addressing aspects of both reporting and methodological quality, as well as relevance of in vitro studies were developed based primarily on requirements and recommendations in OECD test guidelines and corresponding guidance documents. This first version of the criteria is now available on the SciRAP platform (www.scirap.org) and is currently being tested for completeness and practical use by experts in the field of in vitro testing and health risk assessment. The output of a study evaluation using the SciRAP method is a colour profile, which provides a transparent overview of how the evaluator judged each criterion4. These colour profiles can be used as basis for evidence integration in hazard and risk assessment. Ongoing studies, using the SciRAP tool for evaluating in vivo studies, demonstrate how the SciRAP method can be adjusted for judging risk of bias domains when applying a systematic review approach. Future studies are planned to illustrate how evaluations of in vitro studies using the SciRAP tool can be used to integrate mechanistic data in hazard and risk assessment.
Keywords: reliability and relevance, risk assessment, in vitro data, Science in Risk Assessment and Policy (SciRAP), systematic review methodology
EDC-MixRisk: Novel Whole Mixture Approach to Improve Risk Assessment of EDC-Mixtures (#628)
E. P. Drakvik1, J. Rüegg1, A. Bergman1
1 Karolinska Institutet, Institute of Environmental Medicine, Stockholm, Sweden
On behalf of the EDC-MixRisk Consortium
Endocrine disrupting chemicals (EDCs) are linked to serious health problems such as diabetes, obesity, neurodevelopmental disorders and reproductive problems. We are exposed on a daily basis to a cocktail of EDCs that potentially interact and amplify each other’s effects. EDC-MixRisk, a H2020 project, has studied the effects of prenatal exposure to mixtures of potential EDCs on the development and health of children. The objectives of the project were i) Identification of mixtures of EDCs that are associated with multiple adverse health outcomes; ii) Identification of molecular mechanisms and pathways underlying these associations and iii) Development of methods for risk assessment of EDC-mixtures.
EDC-MixRisk has developed a novel, integrated approach which is grounded on interdisciplinary collaboration, including epidemiology, experimental biology and regulatory toxicology. Three health domains were addressed: 1) growth and metabolism, 2) neurodevelopment and 3) sexual development. By using whole mixture approach and epidemiology data from the Swedish pregnancy cohort SELMA, relevant EDC mixtures associated with adverse health outcomes in humans were identified. Then, reference mixtures were created to mimic real-life internal exposures, and these mixtures were tested in various cell and animal models. The experimental data were used to establish new methods and strategies for mixture risk assessment in order to complement current approaches and to better address environmental exposures.
The epidemiological analysis showed that prenatal exposure to mixtures of EDCs was associated with various effects in children's health and development, some effects being sex specific. The mixtures, tested in the variety of experimental models, affected hormone-regulated and disease-relevant outcomes at concentrations found in the pregnant women. By applying this novel whole-mixture approach, we found a higher number of children at risk compared to estimates by current methods based on a single compound assessment. The results call for a comprehensive and harmonized approach across policy and regulatory silos to tackle combined exposures to hazardous chemical mixtures, as current regulations seem to systematically underestimate health risks associated with co-exposures to EDCs or potential EDCs.
Birgersson L, Borbely G, Caporale N, Germain P-L, Leemans M, Rendel F, et al. From Cohorts to Molecules: Adverse Impacts of Endocrine Disrupting Mixtures. bioRxiv. 2017. doi: 10.1101/206664.
Gennings, C., Shu, H., Rudén, C., Öberg, M., Lindh, C., Kiviranta, H., Bornehag, C-G. (2018):
Keywords: EDCs, mixtures, epidemiology, risk assessment
Feasibility study for the applicability of the ECHA/EFSA guidance for the identification of endocrine disruptors. The example of α – cypermethrin (#631)
C. Rovida1, 3, F. Panza2, M. Locatelli3
1 Konstanz University, CAAT-Europe, Konstanz, Germany
Regulation 528/2012 on biocide products and Regulation 1107/2009 on plant protection products ask that the new approved active substances should not present endocrine disruptor activity. To support applicants ECHA and EFSA together have prepared and published a detailed guidance on how to collect and present data to demonstrate the presence or absence of endocrine disruptor activity. In order to understand the applicability of the ECHA/EFSA guidance, available data for an already approved substance was used to test the procedure proposed by ECHA/EFSA and understand if a conclusion on endocrine disruptor activity was possible. For this aim, α – cypermethrin was selected as it is already approved as both pesticide and biocide. This substance is also in the list of substances requiring additional testing according to the EPA (Environmental Protection Agency) EDSP (Endocrine Disruptor Screening Program) program.
The data that were analysed included the studies that are present in the RAR (Renewal Assessment Report) for the approval as pesticide with the addition of new studies that were performed after the publication of the RAR. All these data were inserted in the Excel table contained in Annex E of the guideline and analysed following the provided instruction.
Even disregarding minor problems for example related to adapt the template to accept in vitro studies, some other identified limitations are:
Conclusion is that additional endpoints should be included in the template, with more emphasis to in vitro tests, whose development and application should be encouraged to reduce the number of new in vivo studies and increase toxicological predictivity.
ECHA and EFSA (2018) with the technical support of the Joint Research Centre (JRC). Guidance for the identification of endocrine disruptors in the context of Regulations (EU) No 528/2012 and (EC) No 1107/2009. EFSA Journal;16(6):5311, 135 pp. https://doi.org/10.2903/j.efsa.2018.5311. ECHA-18-G-01-EN.
OECD (2018), Revised Guidance Document 150 on Standardised Test Guidelines for Evaluating Chemicals for Endocrine Disruption, OECD Series on Testing and Assessment, OECD Publishing, Paris. https://doi.org/10.1787/9789264304741-en
Draft Renewal Assessment Report prepared according to Regulation (EC) 1107/2009 Alpha-Cypermethrine
Keywords: endocrine disruptors, biocides, pesticides, risk assessment, ECHA/EFSA guidance
SweNanoSafe – a national platform promoting safe handling of nanomaterials (#639)
A. Hanberg1, M. Beckman1, R. Karlsson1, K. Midander1, A. C. Lagerkvist1, B. Fadeel1, M. Berglund1
1 Karolinska Institutet, Institute of Environmental Medicine, Stockholm, Sweden
On behalf of the Swedish National Platform for Nanosafety, SweNanoSafe.
The increased production and use of engineered nanomaterials (ENMs) in consumer and industrial products has raised environmental, health and safety concerns. There is a need for more knowledge on the properties and potential risks of ENMs as well as requirements for protective measures throughout their life cycle. To promote the safe handling of nanomaterials, the Swedish National Platform for Nanosafety was established in 2016. The platform is an assignment from the Swedish Government with the aim to, in cooperation with various stakeholders, ensure knowledge building and exchange on environmental, health and safety issues of nanomaterials. The aim is also to increase knowledge on hindrances to safe handling of nanomaterials and on how these hindrances can be addressed. The platform consists of a Steering Committee, a Project Team, a Cooperation Council, an Expert Panel and a web-based forum to facilitate knowledge transfer (www.swenanosafe.se). The Cooperation Council gathers representatives from various stakeholders i.e., authorities, industry, NGO’s and academia. The Expert Panel and the recently established research network provides expertise from different disciplines within the field of nanosafety. Safety and sustainability aspects of nanomaterials concern their whole life cycle, such as synthesis, development, production, use and management of waste. Currently, through a range of activities, information needs and knowledge gaps, together with other hindrances to the safe handling of nanomaterials, are being identified within the areas of regulations and guidance, research and development, education, as well as knowledge and information exchange. Actions to overcome various hindrances to the safe handling of nanomaterials will also be proposed by the platform, thereby promoting a coordinated approach to issues of nanosafety in Sweden.
Keywords: nanosafety, nanomaterial, national platform, sustainability, non-toxic environment
Impact of the new ERA guidance on the conduction of Pharmacokinetic ant Toxicity studies (#642)
R. A. Wess1
1 Innovative Environmental Services (IES) Ltd, Witterswil, Solothurn, Switzerland
In November 2018 the EMA issued a new guideline draft prescribing the testing course for the Environmental Risk Assessment (ERA) and the involvement of data from the preclinical studies. An analysis of the recent and draft guidances is shown and discussed.
An impact of the guideline update will be to affect the study design of preclinical studies due to the phase-out of the Predicted Environmental Concentration (PEC) refinement on the basis of marketing data.
The ERA is a requirement for the registration of a Human Medicinal Product (HMP) as module 1.6 in the (electronic) Common Technical Document ((e) CTD) format. Although a dossier can be rejected if the ERA lacks, the outcome of it cannot be a reason for denial of the Market Authorisation Application (MAA). A leaflet warning however, may be a consequence of an unfavourable ERA. Therefore the conduct of specific environmental fate and effect studies is often considered rather late, but it must be commenced more than one year before submission in order to present a complete dossier. Authorities often do not consider some unfinished studies as an incomplete dossier and normally grant an extension, provided a letter of commitment had been signed.
Accordingly, the ERA and its requirements are more or less out of mind during the toxicity and pharmacokinetic studies necessary for registration of a HMP. These studies always played a certain role in the ERA in that they had to be considered in the CMR (Carcinogenicity, Mutagenicity and toxicity to Reproduction) assessment and thus the applicability of the environmental action limit. As this is only a question arising for Active Pharmaceutical Ingredients (API) with a maximum daily dose below 2 mg, the impact of the toxicity studies can in all other cases be neglected. Much more important for the avoidance of an environmental leaflet warning (with negative marketing impact) is the recalculation of the PEC, which used to be possible on the basis of marketing data once the base set of data was determined. This option was key to avoid a leaflet waring but is now phased out according to the new draft guidance update.
In consequence the calculation of a Factor of excretion (F excreta) is the only remaining option for PEC mitigation, but it depends of API and metabolite quantification in both, the faeces and urine, which is thus of significantly increased importance.
EMEA European Medicines Agency Committee for Medicinal Products for Human Use (CHMP) (2006).
EMA European Medicines Agency Committee for Medicinal Products for Human Use (CHMP) (2018).
Keywords: Environmental Risk Asessment, Pharmacokinetic studies, ERA, EMA guidance draft, preclinical studies
Hazard assessment of hydrazine, a possible migration contaminant from drinking water apparatus (#653)
M. Matsumoto1, T. Igarashi1, K. Inoue1, T. Yamada1, A. Hirose1
1 National Institute of Health Scinences, Division of Risk Assessment, Kawasaki, Japan
The Drinking Water Quality Standards for lifetime exposure of contaminants have been established for 51 items under the Japanese Water Supply Act (JWSA). In addition, non-legally binding target values are also notified for “Complementary Items” and some of “Items for Further Study” that can be detected in drinking water or water sources. Chemical contaminants can be leached to drinking water from the water supply system, but such chemical contaminants were not well evaluated. Therefore, we searched chemicals that can be migrated from drinking water apparatus to identify and evaluate hazard of migration contaminants in drinking water. Firstly, we made a list of chemical items that used in drinking water apparatus by reference to the Japan Water Works Association (JWWA) publications. Twenty-five items out of ca. 150 items appeared in JWWA publications were found to be used in materials directory contact to drinking water, and 18 out of 25 items were already evaluated under JWSA. However, seven items recognized as “Items for Further Study” were lack of information for their toxicity and detected levels, and the target values were not yet established. Therefore, we subsequently conducted screening assessment of these seven items using publicly available risk assessment reports to identify their hazard. As a result, the lowest health-based value (Tolerable Daily Intake: TDI or Virtually Safe Dose: VSD) was provisionally obtained for hydrazine from seven items we evaluated. Then, we decided to further evaluate hydrazine to derive a health-based target value in drinking water because of high toxicity potential, high water solubility and a wide range of industrial use. The health-based target value of hydrazine in drinking water was calculated to be 0.005 mg/L with body weight (50 kg for adults) and drinking water intake (2L/day) by using the VSD at 10-5 risk of 2.1 x 10-4 mg/kg/day, which is based on hepatocellular adenomas and carcinomas in rats in a two-year drinking water study (OECD TG 451). Our health-based target value will be useful to identify a possible risk of hydrazine intake via drinking water. ACKNOWLEGMENT: This study was supported by a Health and Labour Sciences Research Grant (H28-Kenki-Ippan-005) from the Ministry of Health, Labour and Welfare, Japan.
Keywords: drinking water quality standard, migration contaminants, hydrazine
What is the risk of drinking water downstream from sites polluted with polycyclic aromatic hydrocarbons (PAHs)? Comparative toxicity of oxygenated polycyclic aromatic compounds (O-PACs) to associated PAHs. (#665)
M. Bisson1, E. Granier1, J. Michel1
1 INERIS, DRC, VERNEUIL en HALATTE, France
Introduction: In industrialized countries, a lot of PAH-contaminated sites can be identified. PAH high toxicity has already been demonstrated. However, other polycyclic aromatic compounds (PACs) can be found at these sites and may therefore contribute to the risk for humans and the environment such as oxygenated PACs (O-PACs). O-PACs are emitted from the same sources as PAHs and can be formed by oxidation of the parent PAHs. They show a higher mobility and persistence in soils than PAH, and thus, a possible risk for human by drinking groundwater. In order to better assess the health risk associated to PAH-contaminated sites (former coking plants, gasworks or wood preservation facilities), 11 O-PACs were selected for their frequency of occurrence in groundwater and structural diversity.
Method: A literature review summarizing existing data was performed on various toxicological endpoints for all 11 O-PACs. All results were gathered and analyzed in order to compare their toxicity to the associated PAH with the most similar structure. Since O-PACs are not extensively described in the databases, results were completed with (Q)SAR predictions and Threshold Toxicological Concern (TTC) safety assessment.
Results: 3 of these compounds were already investigated. Anthraquinone (ANTQ), dibenzofuran (DBF) and 9H-fluorenone (9HF) were compared to their respectively associated PAH : anthracene, acenaphthene and fluorene. In the overall toxicity comparison of ANTQ to its parent compound anthracene, ANTQ seams to represent a greater danger based on a more important carcinogenicity. On the other hand, DBF and 9HF present the same level of toxicity on every studied endpoint compared to acenaphthene and fluorene.
Conclusion: This preliminary work demonstrated that O-PACs present at least the same level of toxicity than their associated PAH, suggesting that a follow-up of those molecules could be implemented for groundwater in order to assess its quality.
Keywords: oxygenated polycyclic aromatic compound, polycyclic aromatic hydrocarbon, (Q)SAR, TTC, drinking water
The role of chemical analysis in supporting the European Union’s ban on characterising flavors in tobacco products (#705)
C. Vardavas1, M. N. Tzatzarakis1, C. N. Kyriakos1, A. Vardavas1, C. Girvalaki1, I. Lagou1, E. I. Iatrou1, P. D. Stivaktakis1, A. M. Tsatsakis1
1 University of Crete, Laboratory of Toxicology, Medical School, Heraklion, Greece
Introduction: In light of the evidence of flavoured tobacco products facilitating initiation of tobacco consumption and affecting consumption patterns, the European Union (EU) Tobacco Products Directive (TPD) requires Members States (MS) to prohibit the placing on the market of tobacco products with a characterising flavour, specifically boxed cigarettes and roll-your-own tobacco.
Methods: The objective of the EUREST-FLAVOURS project is to support the European Commission in the specification of the methodology to support the decision on whether a tobacco product has a characterising flavour.
Results: The approach for specifying the methodology for whether a tobacco product imparts a characterising flavour is based on a comparison of the smelling properties of test products with those of reference products through sensory analysis, complemented by a chemical assessment of the product composition through chemical analyses.
Conclusions: The EUREST-FLAVOURS project is developing clear science-based decision criteria that a tobacco product has a characterising flavour. Chemical analysis will contribute to supporting evidence that a tobacco product contains flavour compounds in order to support the EU TPD ban on characterising flavours.
Funding: The EUREST-FLAVOURS Project takes place with the financial support of the European Commission Single framework Contract Chafea/2016/Health/36.
Disclaimer: The content of this working document represents the views of the EUREST-FLAVOUR Consortium and is its sole responsibility; it can in no way be taken to reflect the views of the European Commission and/or Chafea or any other body of the European Union.
Keywords: flavors, tobacco products, sensory and chemical analysis, tobacco products directive (TPD)
Analysis of level 1 and 2 of the OECD Guidance Document 150 for Evaluating Chemicals for Endocrine Disruption and applicability in the EU (#709)
F. Panza1, C. Rovida2, M. Locatelli3
1 University of Milan, Drug science, Scienze e sicurezza chimico-tossicologiche dell'ambiente, Milano, Italy
OECD Guidance Document (GD) 150 for Evaluating Chemicals for Endocrine Disruption describes 5 levels with increasing complexity for the definition of the endocrine disruptor activity of chemicals.
Level 1 regulates Existing data and existing or new non-test information, including modelling programs to collect existing information and perform the preliminary assessment of the substance. Scope of level 2 is in vitro assays providing data to elucidate selected endocrine mechanism(s)/pathway(s). This step is very important to demonstrate the endocrine disruptor mechanism at the basis of an adverse effect, with special attention to the species-specific mechanisms.
With the focus on human toxicology and the EU market, α-cypermethrin was taken as case study to test the procedure described for the Level 1 and 2 in order to define opportunities and limits of the approach.
Analysis of α-cypermethrin was performed using the list of databases present in Annex D of the ECHA/EFSA guidance for the identification of endocrine disruptors. More than a hundred studies was retrieved, but in many cases the exact tested isomers was not specified and in general pubic available studies do not report enough details for the definition of endocrine disruptor activity. Appendix D reports also very interesting modelling programs that could provide the useful link between the chemical structure and a possible concern. Use and consultation of the programs is often cumbersome, requesting special expertise.
Regarding level 2, there are already some validated in vitro methods and many others are well advanced in the acceptability for the elucidation of specific AOP (Adverse Outcome Pathways) offering a tremendous opportunity for the demonstration of a possible ED activity. The applicability of level 2 requires the availability of CROs (Contract Reasearch Laboratories) to execute the experiments. The authors performed a detailed search of any possible lab that may offer the service. In spite of the efforts only 20 labs were found eligible for in vitro testing. An enquiry was sent to all of them, with reply from 16 and only 6 confirmed the possibility to offer the service for in vitro testing to assess endocrine disruptor properties. Two of them are also developing new systems for the assessment of thyroid disfunction, which has still no official OECD guidelines. The average cost to perform the whole set of tests is about 25,000€ per substance.
Conclusion is that in vitro tests for the assessment of ED properties is a useful opportunity but needs stimulus for wider applicability.
OECD (2018), Revised Guidance Document 150 on Standardised Test Guidelines for Evaluating Chemicals for Endocrine Disruption, OECD Series on Testing and Assessment, OECD Publishing, Paris. https://doi.org/10.1787/9789264304741-en
ECHA and EFSA (2018) with the technical support of the Joint Research Centre (JRC). Guidance for the identification of endocrine disruptors in the context of Regulations (EU) No 528/2012 and (EC) No 1107/2009. EFSA Journal;16(6):5311, 135 pp. https://doi.org/10.2903/j.efsa.2018.5311. ECHA-18-G-01-EN.
Draft Renewal Assessment Report prepared according to Regulation (EC) 1107/2009 Alpha-Cypermethrine
Keywords: endocrine disruptors, biocides, pesticides, risk assessment, in vitro testing
Comparison of single, paired and group housing effects on cardiovascular parameters and body temperature in telemetered cynomolgus monkeys (#716)
P. Singh1, A. I. El Amrani1, S. Loriot1, F. El Amrani-Callens1, M. E. Duclos1, R. Forster1
1 Citoxlab France, Evreux, France
Animal research in the European Union (EU) is regulated under Directive 2010/63/EU (protection of animals used for scientific purposes). This directive clearly indicates that animals, except those which are naturally solitary, should be socially housed in stable groups of compatible individuals. In the present investigation, we compared three housing conditions (single, paired and group housing) in four well-acclimated male telemeter-implanted cynomolgus monkeys. Body temperature (BT) and cardiovascular parameters, including heart rate (HR), arterial blood pressure [systolic (SAP), diastolic (DAP) and mean arterial pressure (MAP)] and ECG parameters (PQ interval, QRS complex and QT interval) were continuously recorded by telemetry over a period of 19 hours (from 16:00 to 11:00). The animals were housed in ETS-123 compliant cages and data were recorded first under group housing and then under single and paired housing conditions using a cross-over design. When compared to single housing conditions, paired housing had no significant effect on cardiovascular parameters, but the group housing configuration led to significant decreases in HR from 19:30 to 6:00 [maximum effect (Emax) at 21:00: 106 ±9 vs. 139 ±4 beats/min, p<0.001], DAP from 18:00 to 3:00 (Emax at 21:00: 64 ±3 vs. 87 ±6 mmHg, p<0.001), MAP from 19:00 to 1:00 (Emax at 21:00: 83 ±3 vs. 106 ±7 mmHg, p<0.001) and increases in QT interval (Emax at 00:00: 297 ±15 vs. 247 ±3 ms, p<0.01). There were no statistically significant changes between single and group housed animals in SAP or QT corrected for changes in HR according to the Bazett (QTcB) formula or the individual QT correction method (QTca). In group housed animals, there was a statistically significant increase in body temperature from 16:00 to 17:30 and from 7:00 to 8:00, reaching an Emax at 16:30 (38.8 ±0 vs. 38.2 ±0 °C, p<0.01). Based on quantitative cardiovascular parameters, the present preliminary findings suggest a benefit of group housing conditions over single or paired housing in cynomolgus monkeys. Paired housing conditions had no benefit over the single housing environment under our experimental conditions. These preliminary findings support the use of group housing in studies of cardiovascular safety assessment.
Keywords: Single versus group housing, NHP housing conditions, Cardiovascular telemetry
In silico acute toxicity protocols and models (#747)
G. J. Myatt1, D. Bower1, K. Cross1, C. Johnson1, D. P. Quigley1, R. Tice2, C. Zwickl3
1 Leadscope, Columbus, Ohio, United States of America
In silico toxicology is an important alternative approach to animal testing that provides a fast and inexpensive prediction of toxicity. While computational approaches can quickly calculate a prediction, the process of selecting and acquiring models, performing an expert review, integrating experimental data and model results, and documenting conclusions and uncertainties can be time-consuming and difficult to reproduce. It is also challenging to defend the results, primarily due to a lack of published procedures for performing an in silico assessment. To support the development of such protocols, a 60-member international cross-industry consortium has been assembled including representatives from international regulatory agencies and government research laboratories in the United States, Canada, Japan and Europe, as well as large companies from various industrial sectors (e.g., pharmaceutical, food, cosmetics, agrochemicals), academic groups and other stakeholders. The protocols ensure that any in silico assessments are performed in a consistent, repeatable, well-documented and defendable manner so as to support their broader acceptance. To support the implementation of the acute toxicity protocol, a series of in silico models to predict acute toxicity were developed that are based on GHS categories from acute rat oral toxicity studies. A battery of structural fragment-based models and alerts were used to predict these categories. The overall predictive accuracy is 74% and is based on a predicting the correct GHS category or an adjacent more conservative category.
Keywords: In silico, protocols, acute toxicity
New TTC database compilation to support thresholds of toxicological concern in the risk assessment of antimicrobials beyond Cramer Classes (#750)
A. Mostrag1, C. Yang1, 2, M. Cheeseman3, J. Rathman1, 5, N. Skoulis3, V. Vitcheva1, M. T. Cronin4
1 MN-AM, Columbus, Ohio, United States of America
Threshold of Toxicological Concern (TTC) is an alternative method applied in the risk/safety assessment for substances whose exposure is very low and when appropriate data are not available. The aim of this work was to expand the original Munro TTC dataset through integration of existing public data sources to extend TTC approach to antimicrobials. Global antimicrobial inventory was defined based on records from US EPA (319), EFSA (170), and ECHA (240) spanning the chemical types of disinfectants, antimicrobial, biocides, and preservatives. The expanded database includes over 1600 chemicals and data from several well-established datasets, e.g., COSMOS TTC, MUNRO, EFSA, EPA IRIS and ToxRefDB. Strict study inclusion criteria (e.g., study type/duration, route of exposure, species, number of doses) have been applied. Approximately 85% of the AM inventory is Cramer Class III, which can be considered simplistic to apply 90 mg/day for most of the antimicrobials (AMs). Instead of using Cramer Decision Tree, AM category concept was developed to bin the compounds structurally, which then were further delineated to sub-categories according to their potency. This large database increases the robustness of the chemical domains already covered by the Munro dataset and enables performing chemoinformatics analysis to go beyond the Cramer decision tree. In this study, a set of AM chemotypes based on ToxPrint chemotypes is identified to develop categories, taking into account the physical and biological properties that are related more directly to toxicity. Potency categories of antimicrobial chemotypes are then developed by correlating with NO(A)EL values. The possibility of grouping chemicals into potency categories using the chemotypes is then validated against the full dataset. Using these AM categories, several use cases such as caffeine, organophosphate, iodo-2-propynl butylcarbamate, etidronic acid, and ZnPTO were demonstrated to set up frame work for potential thresholds. This new method intends to reduce the need for chronic animal testing of active antimicrobial ingredients in premarket reviews while reduce animal testing of metabolites or impurities.
Keywords: Threshold of Toxicological Concern (TTC), ToxPrints, chemotypes, potency category, antimicrobials
Tyrosinaemia: Factors Affecting Production & Excretion of HPPA During Inhibition of HPPD (#755)
C. Strupp1, M. Provan2, J. Botham3, G. Semino-Beninel4, J. Zimmermann5, P. Botham3, M. Frericks7, J. - C. Garcin4
1 Gowan Crop Protection Ltd., Reading, United Kingdom
Tyrosineaminotranferase (TAT) is the first and rate limiting enzyme of tyrosine catabolism and when 4-hydroxyphenylpyruvate dioxygenase (HPPD) is inhibited the amount of 4-hydroxyphenylpyruvate (HPPA) increases, is then removed to the general circulation and transported to the kidney where it is actively transported to urine with related metabolites, collectively known as phenolic acids.
The extent of the steady-state tyrosinaemia induced upon inhibition of HPPD in mice and rats has been shown to be inversely correlated to the hepatic activity of (TAT) in that species. This correlation extrapolates well to humans where the hepatic activity of TAT and the extent of the tyrosinaemia in healthy humans has been reported to be similar to mice.
The activity of TAT in rabbit and dog have also been reported. The activity of TAT in the rabbit is similar to that of the female rat consistent with the extent of the tyrosinaemia in each species. Unlike female rat, the rabbit does not suffer ocular effects, despite ocular exposure to tyrosine, suggesting a further defence mechanism is active within the eye. The activity of hepatic TAT in the dog is significantly higher than in mice which should indicate a tyrosinaemia less pronounced than that in mice. . However, the degree of tyrosinaemia in the dog is greater than 1,000nmol/ml, the threshold for ocular toxicity to be expressed, the characteristic, tyrosine-mediated ocular lesion of the dog has been reported with different inhibitors of HPPD.
From this information, the dog clearly contradicts the association of TAT activity with the maximal extent of tyrosinaemia once HPPD is inhibited in rats, mice, rabbits and humans. The present study examines the kinetic variables, during the inhibition of HPPD, that may influence the disposition of HPPA following its production from tyrosine. This in turn allows definition of the relative contribution to the development and extent of tyrosinaemia across species of hepatic TAT activity in production of HPPA, versus those factors that control the removal of HPPA from systemic circulation. This work extends our understanding of the mechanism that controls tyrosine-mediated ocular toxicity in laboratory animal species and the consequence for humans.
Keywords: plant protection, HPPD, mode of action, tyrosinaemia
GHS “Serious Eye Damage” mixture classification: predictive capacity of the calculation method versus test data (#770)
D. Byrne1, R. Scazzola1, P. Botham3, P. Todd2, G. Boeije1
1 A.I.S.E. (International Association for Soaps, Detergents and Maintenance Products), Brussels, Belgium
Purpose: Under UN GHS (and thus also EU CLP) the eye hazard classification of a chemical mixture is primarily to be based on appropriate data for that mixture or (via bridging) data for similar mixtures. If no such data are available, the calculation method (based on additivity) shall be used. Whereas this is intended as a last resort in a tiered approach, to avoid animal testing and due to limited availability of validated in-vitro tests, it is nevertheless frequently used in some product categories. The current review assesses how well the calculation method can reproduce data-based classification for actual mixtures, as reported in the literature.
Keywords: GHS/CLP, eye classification, calculation, Draize
Analysis of mycotoxins and toxic elements in laboratory animals feed (#787)
L. Radko1, L. Panasiuk1, M. Durkalec1, P. Jedzinak1, A. Nawrocka1, S. Stypuła-Trębas1, A. Posyniak1
1 National Veterinary Research Institute, Department of Pharmacology and Toxicology, Pulawy, Poland
Purpose: Laboratory animals are the most widely models used in experiments in toxicological research. The lack of a standardized diet for laboratory animals can have profound effects on their health and can lead to less reproducible research outcomes [1, 2]. The laboratory feeds are commonly used by lab animal breeders and researchers and could be a potential source of toxic compounds and elements. Dietary toxicants such as mycotoxins and toxic elements are important to measure because these are ubiquitous contaminants [3, 4, 5]. The presence of mycotoxins in European feed has been reported worldwide for decades. Moreover, their co-occurrence in a feed is an important problem affecting animal health due to their multidirectional toxicity . Toxic elements are a group of such compounds, that can accumulate in the body leading to developmental abnormalities, reduced growth, and increased rates of mortality.
Methods: Forty samples of feed for laboratory animals (mouse, rat, hamster, guinea pig, rabbit, zebrafish) were collected from breeders in Poland. The samples came from domestic and foreign manufacturers. The concentrations of mycotoxins (aflatoxins, deoxynivalenol, ochratoxin A, zearalenone and enniatins) were analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). Toxic elements (arsenic, cadmium, lead, and mercury) were determined using inductively-coupled plasma mass spectrometry (ICP-MS) and atomic absorption (AAS) methods.
Results: Mycotoxins and toxic elements were detected in all study samples of feed for laboratory animals. The most frequently detected (95%) in lab feed were enniatins at the concentration ranging from 1-2025 µg/kg. Zearalenone (4-218 µg/kg) and deoxynivalenol (100-500 µg/kg) were quantified in 76% and 56% samples, respectively. Only in 3 samples, ochratoxin A occurred (0.1-4.7 µg/kg). Arsenic and cadmium in concentrations 50-100 µg/kg were detectable in 44% and 49% samples of feed, respectively. Lead (50-100 µg/kg) and mercury (1-5 µg/kg) were detected in 58% and 59%, respectively.
Conclusion: This results demonstrated that the occurrence of mycotoxins and toxic elements in feed for laboratory animals are significant. Chronic consumption of these diets can be considered as a risk for animals health. Consequently, lead to obtaining false study results, increased the number of animals used in experiments and greater difficulty in extrapolating outcomes to humans. Efforts directed at analytical control of laboratory animal feed will improve the reliability of toxicity tests in biomedical research and regulatory toxicology.
Funding source: This research was conducted within the statutory activity of the National Veterinary Research Institute in Pulawy, Poland. Congress cost was covered by KNOW (Leading National Research Centre) Scientific Consortium „Healthy Animal - Safe Food", the decision of the Ministry of Science and Higher Education No. 05-1/KNOW2/2015.
Keywords: laboratory animals, feed, mycotoxins, toxic elements
Comparison of two commercially available systems proposed for oral administration of capsules in rats (#797)
G. Chevalier1, D. Papineau1, A. Cirio2, Y. Lambert1, G. Repérant1, P. Singh1
1 Citoxlab France, Evreux, France
Two commercially available systems for the oral administration of capsules to rats were tested to select the most appropriate device for use: a flexible tube (Instech Laboratories) and a stainless steel dosing applicator (Torpac Inc.). Ten female Sprague-Dawley rats (304-338g) were allocated to 2 groups and received empty standard size-9 gelatine capsules once daily for 4 days by the oral route using one of the two administration systems. On Day 5, the rats received a coated placebo size-9 capsule (filled with Avicel PH-102) by the same method as on Days 1-4. The animals were habituated to the administration procedure using a 3mm diameter plastic gavage tube (on 3 successive days). Air or liquid (water or corn oil) was administered to facilitate movement of the capsule into the stomach. With the Torpac rigid tube, it was difficult to insert a sufficient length of tube into the esophagus. It was also difficult to withdraw the tube from the esophagus, and the capsule had to be carefully placed into the tube to ensure that the direction (thick side first) was correct. The animals in this group showed some signs of distress, such as vocalization, and loud breathing was noted in 2/5 animals one hour after administration on Days 4 and 5. With the Instech flexible tube, flushing with oil or water to eject the capsule was at times difficult due to pressure at the junction between the tube and the syringe, which had a tendency to come apart when the syringe plunger was pushed (therefore a screw syringe was used). The capsule had to be carefully placed into the tube with the appropriate orientation (thin side of empty capsules first and thick side of the coated capsules first) for administration to work properly. Insertion of the flexible tube was easy, and similar to our currently used technique for oral gavage administrations. The animals did not show signs of distress at handling or after administration using the Instech tube method. No regurgitation was observed in either group. Body weight change was not impacted by either administration method, and there were no macroscopic lesions in the digestive tract. In conclusion, using the Instech procedure and flushing with water or corn oil via a screw syringe is considered to be an appropriate method for use in local tolerance studies on the rat gastrointestinal tract.
Keywords: Oral administration in rats, dosing systems, capsule administration in rats
Intravitreal drugs: how define safety limits for high concern impurities (#810)
C. Landolfi1, E. Fabris1, C. Bartella1, L. Durando1
1 Angelini S.p.A., RR&D, S. Palomba - Pomezia (RM), Italy
Intravitreal drug administrations have become an efficient approach to deliver drugs at therapeutic levels. The advantage of this route of administration is an immediate and increased therapeutic effect at the intended site. The intravitreal injection, in fact, is used to administer active ingredients directly into the posterior chamber of the eye, to assure a direct pharmacological effect of the drug and to rapidly reach and maintain pharmacological concentrations. On the other hands, the intravitreal injection route presents several unique challenges. The eye is an extremely sensitive organ, there is a limited collection of excipients acceptable for intravitreal injection compared with other delivery routes. As intravitreal injection is an invasive route, therefore there is always a small but significant risk of infection with each new injection. Moreover, considering the very low doses and volume (less than 0.10 mL per eye), in the setting limits of actual or potential high concern impurities, such as genotoxic or sensitizing impurities as well as elemental impurities, a non-standard approach should be followed to assure safety levels of contaminants in the site of administration.
The relevant guidelines are ICH M7 and ICH Q3D for genotoxic and elemental impurities respectively. Moreover, considering the very sensitive route of administration, a safety assessment of potential or actual sensitizing impurities should be performed, even if not mandatory and formally required by international guidance.
The purpose of the present work was to describe the pragmatic approach employed in Angelini to set specific safety limits of such impurities in intravitreal drugs.
Potential genotoxic impurities were evaluated following the principles outlined by the ICH M7 guideline. However specific safety factors were adopted in defining appropriate safety limits.
Since the ICH Q3D guideline does not provide PDEs for elemental impurities for the intravitreal route, a case-by-case approach was followed to define appropriate limits considering the doses/exposure and the expected local effects.
In addition, a safety evaluation was performed to highlight the sensitizing potential of impurities and specific limits have been set.
Keywords: Genotoxic Impurities, Elemental impurities, Intravitreal route
Novel Methods for Estimating NOAEL Confidence Bounds and Optimising Similarity Measures for Read-Across Workflows (#811)
C. Yang1, 2, M. T. Cronin3, D. Ebbrell3, J. W. Firman3, T. Kawamoto4, J. C. Madden3, T. Magdziarz1, A. Mostrag5, J. F. Rathman5, 2, E. H. Theophilus6
1 MN-AM, Nürnberg, Bavaria, Germany
Read-across of toxicological information to fill data gaps relies on the efficient identification of analogues associated with high quality data. Analogues are evaluated based on chemical similarity to target and reliability of the study data. Molecular fingerprints have proved to be a key means of identifying structurally similar compounds when applied in similarity measures, e.g., the Tanimoto coefficient. However, current molecular fingerprints are somewhat limited in terms of their mechanistic basis. This study evaluated similarity measures based on various common molecular fingerprints (including Morgan, FeatMorgan, RDKit Topological, MACCS Keys, ToxPrint chemotypes). The performance of the various approaches to determine molecular similarity was assessed in a systematic manner by evaluating the quality of the analogues. Criteria were developed to compare types of fingerprints with regard to: coverage and diversity; information density; consistency of local neighbours; differentiating power between similar and dissimilar compounds; and similarity thresholds. In read-across for repeated-dose toxicity endpoints, the estimation of NOAEL ranges of a target molecule is desired based on study results of analogues. The suitability of the read-across depends on analogue quality and reliability of the study data available for the analogue. Overall molecular fingerprints representing more mechanistic basis. e.g. ToxPrint chemotypes, tend to result higher quality analogues. Subsequently higher quality analogues with reliable study data are in general expected to have lower uncertainty in their NOAEL values. To assess these concepts, a dataset of 900 structures with systemic NOAEL values from repeated-dose toxicity studies was curated from various public sources (e.g., COSMOS DB). Distributions of NOAEL differences for each pair in the dataset were established, and lower and upper confidence bounds of NOAEL values for a target were estimated based on analogues within a given range of similarities to the target. This novel method allows estimation of confidence intervals on the NOAEL value of the target based on well-qualified toxicity data and chemical similarity. This rigorous approach expands the applicability of analogue-based read-across estimations for repeated-dose toxicity.
Keywords: Read Across, NOAEL/LOAEL, Uncertainty, Analog quality, Molecular fingerprints
Predictive Capacity of the iSafeRat EICM: Eye Irritation/Corrosion Prediction Model (QSAR) (#812)
C. Charmeau-Genevois1, M. Delannoy1, J. M. Arbona2, M. Duplaa1, P. Thomas1
1 KREATiS, l'Isle d'Abeau, France
Currently, there are no in vitro nor in silico methods to replace the in vivo Draize method1,2,3 to classify eye irritation UN GHS Category 2 (Cat. 2) substances. IENn the chemical regulatory field in vitro methods (e.g.: BCOP1, ICE2) cover Cat. 1 (serious eye damage) and chemicals not requiring classification for eye irritation/corrosion (NC) with a “no prediction can be made range”. RhCE3 in vitro methods are not able to distinguish Cat. 1 and Cat. 2. iSafeRat EICM4,5 aims to predict both irritation and corrosion potency of chemicals aiming to fill the data gap Cat. 2 for the chemicals in its applicability domain (AD), replacing animal testing.
Herein we compare iSafeRat EICM’s predictive capacity to that of in vitro methods as stated in the OECD guidelines1,2,3 assuring the same positive criteria was used for comparison.
Compared to in vivo6 fully validated data, classified according to the UN GHS classification system, iSafeRat EICM has a prediction accuracy of 90% (including training and external validation data sets and Cat. 1, 2 and NC substances) within its AD. While the accuracy of in vitro test methods ranges between 69-84%. The iSafeRat EICM has 98% specificity (in vitro: 63-100%), 90% sensitivity (in vitro: 63-100%), 2% false positives (in vitro: 4-69%) and 10% false negatives (in vitro: 0-37%).
iSafeRat EICM’s predictive capacity is comparable to the highest performing in vitro models within its AD. Furthermore, it can accurately predict Cat. 2, which cannot be identified at all using in vitro methods.
 OECD TG437 (2017)
 OECD TG438 (2018)
 OECD TG492 (2018)
 iSafeRat EICM, formerly known as iSafeRabbit (Winner of 2015 NC3Rs CRACK-IT QSARs Mix Challenge - https://www.crackit.org.uk/challenge-19-qsars-mix)
 Delannoy, M. et al. Toxicology Letters: “iSafeRabbit QSAR to predict skin and eye irritation potency of organic chemicals”, 295:S99. EUROTOX (2018) Brussels, Belgium.
 OECD TG405 (2012)
Keywords: Eye, irritation, QSAR, in silico, model
Identification and quantification of fragrance allergens in aromas for e-cigarettes (#814)
A. Pawelec1, B. Wielgomas1
1 University of Gdansk, Department of Toxicology, Gdansk, Poland
Electronic cigarettes have been gaining popularity in recent years, although they have been available on the market for over a decade. However, to this day it has not been possible to determine a coherent, supported by scientific research statement about the impact on health of the use of these devices , .
An electronic cigarette is a device whose operating principle is based on heating a special solution (so-called e-liquid) and creating an aerosol that is inhaled by the user. The traditional e-liquid consists of three ingredients: a base - a mixture of propylene glycol and glycerol in varying ratios, nicotine and aroma, which is a mixture of fragrances and flavors compositions, giving the e-liquid a pleasant taste during "vaping". In addition to commercially available ready-made e-liquids, individual components can be easily bought, which allow to create a customized mixture by the user himself.
Aromas (fragrances and flavors compositions), consist of organic compounds (most often aldehydes, alcohols, esters and/or terpenes) of synthetic or natural origin, usually in the form of multicomponent mixtures. In contrast to ready-made e-liquids already containing nicotine, aromas alone are not covered by legal regulations and are not subject to any control system in Poland. Therefore, there is no obligation for manufacturer to specify ingredients on the packaging.
Fragrances are one of the most sensitizing groups of compounds added to cosmetics or food products . In aromas used for preparing e-liquids, these substances are present in high concentrations - they can cause respiratory or contact allergic reactions, which the user, due to the lack of specified composition on the packaging, may not be aware of.
The aim of the work was a qualitative and quantitative analysis of fragrance allergens in aromas used to preparation of e-liquids. The analyzes were carried out using gas chromatography with a flame ionization detector (GC-FID) and gas chromatography mass spectrometry (GC-MS). 40 commercially available aromas with different flavors were analyzed. The results of the research show the presence of fragrance allergens in the majority of the analyzed aromas.
 K. Kadimisetty et al., ACS Sensors 2 (2017) 670–678.
 L. Shahab et al., Ann. Intern. Med. 166 (2017) 390–400.
 T. Hamilton, G. C. et al., Skin Therapy Lett., vol. 16, no. 4 (2011) 1–4.
Keywords: allergens, e-cigarettes, gas chromatography
CLARITY-BPA Study: Analysis for Non-Monotonic Dose-Responses (#821)
C. Beevers1, M. Badding2, L. Barraj3, A. Williams2, C. Scrafford3, R. Reiss2
1 Exponent International, Harrogate, United Kingdom
A recently published study sponsored by the European Food Safety Authority (EFSA) described a methodology for evaluating non-monotonic dose responses (NMDR) by assessing study findings according to 6 checkpoints. The publication (Varret, 2018, Toxicol. Appl. Pharmacol. 339:10) suggests researchers consider a meta-analysis of available data when a finding fulfills at least 5 of the 6 checkpoints. This methodology was applied to the results of a large U.S. government-sponsored 2-year bisphenol A (BPA) rat study. This BPA study, called the Consortium Linking Academic and Regulatory Insights on Bisphenol A Toxicity (CLARITY-BPA) study, was a collaborative effort between the U.S. Food and Drug Administration (FDA), the National Toxicology Program (NTP), the National Institute for Environmental Health Sciences (NIEHS), and 14 academic scientists. It was designed to address some of the lingering toxicological issues associated with BPA, including its possible role in endocrine disruption and the potential to induce NMDR, by combining standard guideline-compliant research practices (the Core study) with innovative studies conducted by academics (Grantee studies). Within the Core study, rats were exposed to BPA at doses of 2.5, 25, 250, 2,500, and 25,000 μg/kg/day by oral gavage. Treatment and clinical endpoints were examined throughout the 2-year study period. The evaluation presented herein provides additional analyses of statistically significant findings beyond those conducted by the researchers. In the Core study, only 2 of the statistically significant findings met at least 5 of the 6 checkpoint requirements for NMDR. These were clinical chemistry changes in serum: an increase in percent basophils and decreased total bile acids. However, further evaluation showed these 2 findings to not be biologically relevant. In conclusion, this analysis found little evidence for NMDR or biologically relevant changes associated with BPA treatment.
Keywords: Bisphenol A, non-monotonic dose-response
Prediction of adverse effects in preclinical subchronic studies by analysis of adverse effects from shorter-term studies using e.g. the RepDose database. (#823)
F. Moradi Afrapoli1, M. Wehr1, A. Bitsch1, S. E. Escher1
1 Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM) , in silico Toxicology, Chemical Risk Assessment, Hannover, Germany
Preclinical animal toxicity studies aim to identify the chemicals short- and long-term functional and morphologic adverse effects. In the interest of reduction of de novo animal testing, we here explore the relationship between the occurrence of short-term effects (subacute treatment period) and adverse effects in longer-term studies (subchronic treatment).
For this approach we used the high quality data from the databases (DB) RepDose (www.fraunhofer-repdose.de) enriched with complementary studies from ToxRef DB (US EPA) and Hess DB (NEDO). This results in a dataset of 37.766 adverse effects from ~2000 chemicals in 970 subacute and 2.360 subchronic studies. The analysis was restricted to 277 compounds, which had at least one subacute and subchronic study. For ~70 compounds the adverse events were reported in same species (Rat) and in a same administration route (dietary) in both long- and short-term studies. Reported adverse events in short-term studies in Rat and dietary were applied as a diagnostic criteria for longer term events.
The investigations were carried out by Bayesian analyses based on the calculation of positive and negative likelihood ratio in KNIME Analytics platform. The sensitivity and specificity of each test were used for determining the diagnostic power of the tests and the diagnostic power was used to identify the connection between subacute and subchronic apical findings.
The investigation showed that many adverse effects in short-term studies can predict adverse outcomes in longer exposure. The realtion of toxic effects in most frequently affected organs such as liver and kidney and clinical chemistry parameters are shown.
- M Clark. Prediction of clinical risks by analysis of preclinical and clinical adverse events. biomedical informatics 2015
- M Clark, T Steger-Hartmann. A big data approach to the concordance of the toxicity of pharmaceuticals in animals and humans. Regulatory Toxicology and Pharmacology 2018
Keywords: repeated dose toxicity, likelihood ratio, adverse events, Predictivity of short-term animal studies, RepDose
Towards an automated workflow for adverse outcome pathway hypothesis: the use case of non-genotoxic-induced hepatocellular carcinoma (#842)
T. Doktorova1, T. Exner1, B. Hardy1, T. Mohoric1, N. Oki1
1 Edelweiss Connect GmbH, Basel, Basel-Stadt, Switzerland
The Adverse Outcome Pathway (AOP) concept as a tool for gathering and linking of information at different levels of biological organization has been largely accepted by regulatory bodies and its usability has been recognized by scientists and regulators. The process of AOP generation, however, is still done manually by experts screening through evidences and extracting probable associations. To facilitate this process and increase the reliability of the findings, we have developed an automated workflow for AOP hypothesis generation.
In brief, high-throughput screening, gene expression, in vivo and disease data for chemicals was gathered from ToxCast and the Comparative Toxicogenomics Database (CTD), and subjected to frequent itemset mining to look for relationships between genes, pathways and diseases that co-occur across datasets by using the chemicals as the aggregating variable for the analysis. This was supplemented by pathway mapping using Reactome to fill in gaps and identify events occurring at the cellular/tissue levels. Furthermore, in vivo data from TG-Gates (using several time-points and dose levels) was integrated to finally derive a gene, pathway, biochemical/hematological, histopathological and disease information network from which specific disease sub-networks can be queried.
To test the workflow, non-genotoxic-induced hepatocellular carcinoma (HCC) was selected. The first module of frequent itemset mining yielded over 200 genes (from ToxCast and CTD) belonging to approximately 20 major pathways. These were further refined by the inclusion of the TG-Gates module which resulted in the identification of several non-genotoxic-specific HCC-connected biomarker genes, biochemical parameters and histopathological findings repeatedly deregulated among dose levels and time-points.
With this study, we proved that computational predicted constructs could support the process of AOP development by using pre-existing knowledge in a fast and unbiased manner.
Keywords: Adverse Outcome Pathway, computational predictions, Hepatocellular carcinoma, non-genotoxic carcinogens, automation
A new in silico method to predict with high probability the absence of potential for endocrine disruption (#852)
P. Thomas1, C. Charmeau-Genevois2, F. J. Bauer2
1 CEHTRA, Isle d'Abeau, France
Endocrine disruption (ED) potential of substances is of high concern for human health and environment, as reflected in the updated chemical regulations. Since 2018, Biocide and Pesticide Regulations require examination of ED potential of the active substance and co-formulants, while the definition of ED and ECHA/EFSA guidelines to assess whether substances meet the endocrine criteria were published in 20181. Under pressure to reduce animal testing and given the complication and cost of studies to determine ED properties, in silico methods may be advantageously used. However, screening models are inaccurate and insufficient considering the gravity of the subject.
We have designed an in silico battery to predict with high probability the absence of ED potential for a substance (Non EDC) meaning that the substance does not meet the criteria of the best understood ED modes of action (MoAs), i.e. related to estrogenic, androgenic, thyroidal and steroidogenic (EATS) modalities as described in EFSA/ECHA guidance1. Our approach comprises 3 steps:
Finally, a consensus of the predictions is obtained via the 3 steps. No alerts for ED MoA means high certainty that the substance does not act with the well understood ED MoAs. If alerts occur, literature searches or further testing to assess the ED properties of the substance is advised. The results of our in silico assessment will help orient testing by providing clues of which biological target will likely be disrupted.
(1) European Chemicals Agency (ECHA) and European Food Safety Authority (EFSA) with support from the Joint Research Centre (JRC). Guidance for the identification of endocrine disruptors in the context of Regulations (EU) No 528/2012 and (EC) No 1107/2009 (Pre-publication version; June 2018)
(2) Ding, D.; Xu, L.; Fang, H.; Hong, H.; Perkins, R.; Harris, S.; Bearden, E. D.; Shi, L.; Tong, W. The EDKB: An Established Knowledge Base for Endocrine Disrupting Chemicals. BMC Bioinformatics 2010, 11 (Suppl 6), S5. https://doi.org/10.1186/1471-2105-11-S6-S5.
Keywords: endocrine disruption, in silico, high certainty, structure-activity relationship
Can the battery of in vitro and in silico methods resolve current deadlocks with skin sensitisation? (#889)
A. Sharma1, F. Sahigara2, C. Chesne3, F. J. Bauer2, P. Thomas4, C. C. Genevois2
1 eurosafe, safety assessment , saint gregoire, France
In the context of 3R principles to minimise animal testing, several in vitro chemistry-based (DPRA, GSH reactivity) and cell-based methods (MUSST, hCLAT, Keratinosens) have been developed and validated to identify potential skin sensitising chemicals according to the OECD guidelines. However, their application to evaluate the skin potency is still not feasible. Besides, these in vitro methods are only able to cover specific events of the skin sensitisation AOP and metabolism is not always into account. Moreover, no formal decision tree is yet adopted on how to combine the results from in chemico and in vitro methods. More recent methods such as SENS-IS and GARD (currently under OECD validation) have encompassed the limitations of monolayer culture model allowing a better assessment of the sensitisation potency of chemicals.
In silico approaches including read-across and (Q)SAR models are also gaining acceptance within various regulatory frameworks provided they are scientifically valid and respect the recommended OECD principles. In practice, no single (Q)SAR model is currently capable to conclude on the final sensitisation potential of chemicals, however a battery of QSAR predictions including models capable to cover metabolism and mechanisms of action can further assist the classical in vitro assessment. This is especially true when the battery results from in vitro studies are inconclusive.
This work will discuss various scenarios in which in silico methods can be complementary to the in vitro assessment to reach final conclusions. This will be illustrated by a case study of Trioctanoin for which no clear conclusions were possible from existing toxicological profile about its safe use as a cosmetic ingredient. Existing experimental studies on this compound and its read-across analogues suggests a negative skin sensitisation potential, although metabolism may have not been taken into account. To get further evidence, we performed an in silico evaluation on Trioctanoin as well as its potential metabolites generated using metabolism simulators and mechanism of action tools. Neither the parent compound, nor any of its metabolites were predicted as skin sensitisers. Based on the combined results from in silico and in vitro studies, we concluded that the skin sensitisation potential of Trioctanion was negative.
OECD (2015), Test No. 442C: In Chemico Skin Sensitisation : Direct Peptide Reactivity Assay (DPRA), OECD Guidelines for the Testing of Chemicals, Section 4, Éditions OCDE, Paris, https://doi.org/10.1787/9789264229709-en.
OECD (2018), Test No. 442E: In Vitro Skin Sensitisation : In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation, OECD Guidelines for the Testing of Chemicals, Section 4, Éditions OCDE, Paris, https://doi.org/10.1787/9789264264359-en.
OECD (2018), Test No. 442D: In Vitro Skin Sensitisation : ARE-Nrf2 Luciferase Test Method, OECD Guidelines for the Testing of Chemicals, Section 4, Éditions OCDE, Paris, https://doi.org/10.1787/9789264229822-en.
Cottrez F, SENS-IS, a 3D reconstituted epidermis based model for quantifying chemical sensitization potency: Reproducibility and predictivity results from an inter-laboratory study, Toxicol in vitro, June 2015.
Zeller K. S., Forreryd A., Lindberg T., Gradin R., Chawade A., and Lindstedt M., The GARD platform for potency assessment of skin sensitizing chemicals. ALTEX Online first published April 12, 2017, version 2 https://doi.org/10.14573/altex.1701101
ECHA (2016). Practical Guide - How to use and report (Q)SARs 3.1Practical Guide – How to use and report (Q)SARs. DOI: 10.2823/81818
Bauer, F.J., Thomas, P.C., Fouchard, S.Y., Neunlist, S.J.M., 2018a. A new classification algorithm based on mechanisms of action. Comput. Toxicol. 5, 8–15. https://doi.org/10.1016/j.comtox.2017.11.001
Bauer, F.J., Thomas, P.C., Fouchard, S.Y., Neunlist, S.J.M., 2018b. High-accuracy prediction of mechanisms of action using structural alerts. Comput. Toxicol. 7, 36–45. https://doi.org/10.1016/j.comtox.2018.06.004
Keywords: Skin Sensitisation, metabolism, in vitro, QSAR, SENS-IS
Benchmark Dose Modeling for Hematologic Effects of Occupational Benzene Exposure (#938)
C. M. North1, M. Rooseboom2, A. Dalzell3
1 ExxonMobil Biomedical Sciences, Inc, Toxicology & Environmental Sciences, Annandale, New Jersey, United States of America
Benzene is a primary constituent of petrochemical feedstocks used to manufacture other products, and can be present in some petroleum streams. It may cause a specific target organ toxicity to the bone marrow, resulting in effects ranging from subclinical (decreased blood cell counts) to severe (acute myeloid leukemia, aplastic anemia). In the context of developing a Derived No Effect Level it was proposed that utilizing subclinical blood effects as a point of departure would protect exposed individuals from both decreased blood cell counts as well as more serious manifestations of toxicity. Benzene has been reported to affect multiple blood count parameters, while most consistently reported are changes to neutrophil (granulocyte) counts. An innovative approach could combine a benchmark dose approach to blood effects for protection from later, more serious effects. Applying both literature- and effect size-based approaches, we identified a 22% decrease in neutrophil count as a conservative benchmark response. Multiple statistical models in PROAST 65.2 appear to suitably fit the results from Qu, et. al., (2003) with a mean value of 12 ppm (BMD) and lower and upper confidence interval values of 1.7 and 44.2 from the Hill model (similar to Exponential 3 model). Given that multiple models provided suitable fits a Bayesian Model Averaging approach could be applied using several approaches.
Qu Q, Shore R, Li G, Jin X, Chen LC, Cohen B, Melikian AA, Eastmond D, Rappaport S, Li H, Rupa D. Validation and evaluation of biomarkers in workers exposed to benzene in China. Research report (Health Effects Institute). 2003 Jun(115):1-72.
Keywords: benzene, hematotoxicity, dose response modeling, neutrophils, occupational
Evaluation of Sexual Maturity in the RasH2 Mouse Model (#943)
G. Quesseveur1, E. Drevon-Gaillot1, H. Voute1, M. Aujoulat1, M. Sillon1
1 Charles River, Scientific Operations, Lyon, France
The RasH2 transgenic mouse is one of the mouse models accepted by regulatory agencies as an alternative model to carcinogenicity studies. The RasH2 mouse contains multiple copies of the human c-Ha-ras proto-oncogene. This mouse model is one of the most sensitive to both genotoxic and non-genotoxic carcinogens.
Age of the animals is one of the most important parameter for designing toxicology studies and sexual maturity process is critical to distinguish juvenile from mature animals. Despite its increasing use in carcinogenicity studies, little is known about the sexual maturity profile of the RasH2 mouse model. The aim of this study was to evaluate the sexual maturity onset for both RasH2 males and females between 6 and 9 weeks old of age, using histopathological examination of the reproductive organs and sperm analysis.
Histopathological evaluation showed that testicular maturity was already present in 6 weeks old male mice. Consistent with this finding, we did not observe any differences in sperm count between 6, 7, 8 and 9 weeks old animals, suggesting that spermatozoid production is fully efficient at 6 weeks of age in RasH2 males. However, analysis of sperm motility and morphology revealed a significantly lower population of progressive spermatozoids and a lower proportion of normal spermatozoids in up to six out of ten 6 weeks old males, when compared with older animals. Altogether these results demonstrate that RasH2 males are considered to be sexually mature from 6 weeks of age, on the basis of histological and sperm count data, with evidence of functional sexual maturity from
Keywords: sexual maturity, RasH2 mice
EuroMix handbook for mixture risk assessment (#962)
J. Zilliacus1, A. Beronius1, A. Hanberg1, M. Luijten2, H. van der Voet3, J. van Klaveren2
1 Karolinska Institutet, Institute of Environmental Medicine, Stockholm, Sweden
Focus on risks to human health from combined exposure to multiple substances (“chemical mixtures”) has increased in the last couple of decades. There has been a rise in awareness and concern in the community, especially concerning unintentional environmental exposure to unknown chemical mixtures. EuroMix Horizon2020 project has developed methodology and tools for mixture risk assessment and provides a handbook for mixture risk assessment. The handbook is consistent with and expands upon the recent documents on mixture risk assessment published by OECD and EFSA.
The handbook contains concise descriptions of the EuroMix methodology and tools with reference to the EuroMix toolbox. The EuroMix toolbox is a web-based platform where toxicity and exposure data can be uploaded and mixture risk assessment can be performed. Annexes in the handbook provide detailed information or useful templates. Illustrative examples are also included as annexes.
The EuroMix methodology is component-based, tiered and very flexible, enabling assessment of both data-rich and data-poor substances. Substances are grouped based on toxicological considerations in assessment groups. Grouping based on other characteristics can also be applied in the EuroMix toolbox. Toxicity and exposure information for each substance in the assessment group is used for estimation of the combined risk using the dose-addition hypothesis and relative potency factors approach. The concept of adverse outcome pathways forms the basis for the toxicological considerations for grouping as well as for the identification of endpoints that can be measured or predicted to derive toxicity data and relative potency factors. The adverse outcome pathway approach supports the use of in vitro data in a tiered testing strategy. In silico modelling can be used for grouping and for setting test priorities. The dietary exposure assessment of mixtures is based on probabilistic methodology considering the individual consumption and concentration data and allowing estimation of different percentiles of exposure to the mixture. The EuroMix handbook and toolbox provide practical support to apply the OECD and EFSA guidance on mixture risk assessment.
Keywords: mixtures, risk assessment, adverse outcome pathway, probabilistic exposure assessment, relative potency factors
Role of Kinetically Derived Maximum Dose (KMD) in Top-Dose Selection for Chronic Repeated Dose Toxicity Studies (#972)
J. Domoradzki1, M. Corvaro2, C. Terry1
1 Corteva Agriscience, Indianapolis, Indiana, United States of America
Based on the importance of toxicokinetic data in understanding systemic exposure, OECD Health Testing Guidance includes and emphasizes that toxicokinetic data can be used to improve selection of doses for repeated dose mammalian toxicity studies. The KMD approach selects a dose-range more relevant for risk assessment purposes. Doses based on toxicokinetic data are quantitatively relevant to real-world human exposures as compared to testing at the limit dose. Since use of the Kinetically-Derived Maximum Dose (KMD) approach can result in test doses lower than those associated with the long-standing conventional Maximum Tolerated Dose (MTD) dose selection approach, challenges have been raised that this potentially compromises identification of health hazards used in regulatory classification and labeling of chemicals. Presentation of case studies associated with KMD vs MTD dose selection strategies will illustrate the following: 1) Testing at KMD selected dose levels offers appropriate protection of human health, particularly when knowledge of human exposures is rapidly expanding.; 2) KMD is consistent with current knowledge of dose-dependent transitions of toxicity responses.; 3) KMD evaluations can be retroactively applied to previous classification/labeling/risk assessments based on data from MTD testing.; 4) KMD approach testing honors commitments to reducing animal testing and minimizing animal stress.; and 5) The opportunity to remove inter-and intraspecies uncertainty factors exists with knowledge of systemic dose. Some of the chemicals highlighted will be Sulfoxaflor (route selection), 2,4-D (saturated renal clearance and toxicity), Arylex (pharmacodynamic response), ethyl benzene (post-hoc study analysis), acetaminophen (saturation of metabolic conjugation pathways), and ethyl tertiary butyl ether, afidopyropen (mode of action). These examples will illustrate the importance of understanding systemic dose and toxicokinetics of a chemical and its metabolites in top-dose selection, study interpretation and human relevance.
Keywords: kinetically derived maximum dose, maximum tolerated dose, repeated dose toxicity, toxicokinetics, dose proportinal kinetics
The effect of subacute poisoning with fenpropathrin on TNF alpha and interleukin 1 beta in mice kidneys (#139)
B. Nieradko-Iwanicka1, M. Jaremek2
1 Medical University of Lublin, Chair and Department of Hygiene, Lublin, Poland
Pyrethroids are insecticides of mainly neurotoxic properties. They are divided into 2 types. Fenpropathrin (FEN) has features of Type I and Type II pyrethroids. There are data that pyrethroids apart from neurotoxic properties, can be also nephrotoxic and immunotoxic.
The aim of the study was to assess the influence of fenpropathrin on kidney function and concentration of proinflammatory cytokines: TNF alpha and interleukin 1 beta in mice kidneys.
16 female mice were divided into two groups: control and the group receiving FEN at the dose of 11.9mg/kg ip for 28 consecutive days. On day 29 blood samples were obtained to measure serum creatinine concentration. The animals were sacrificed, and kidneys were obtained in order to measure TNF alpha and interleukin 1 beta in mice kidneys with use of ELISA assay.
The concentration of creatinine was (mean ± SD) in controls 0.2±0.0mg/dl, in the group exposed to FEN 0.225 ± 0.046mg/dl. TNF alpha concentration in the kidneys of controls was 6.154 ± 1.597 pg/ml and in the group intoxicated with FEN it was 6.318 ± 1.012pg/ml. Interleukin 1 beta concentration in the kidneys of controls was 4.67±1.154pg/ml while in the group intoxicated with FEN 27.983 ±26.382pg/ml (p<0.05).
In conclusion: FEN affects kidney function and increases the concentration of proinflammatory interleukin 1 beta in mice kidneys, which supports the hypothesis about nephrotoxic and immunotoxic properties of this compound.
Keywords: pyrethroid, fenpropathrin, inetrleukin1 beta, tumor necrosis factor, renal toxicity
Protective effects of Dendropanax Morbifera against cisplatin-induced nephrotoxicity without blocking chemotherapeutic efficacy in animal models (#228)
J. H. Park1, J. S. Kim1, J. S. Lim1, J. Y. Son1, K. S. Kim1, H. S. Kim1, J. H. Kwak1
1 Sungkyunkwan University, School of Pharmacy, Suwon, Republic of Korea
Cisplatin is a widely used chemotherapeutic agent for the treatment of a broad-spectrum of solid tumors. However, its clinical use is limited by occurs acute kidney injury (AKI) in many patients. Despite intensive research, there is no successful protective therapy against cisplatin-induced AKI. The aim of the present study was to investigate the renoprotective effects of dendropanax morbifera (DM) on cisplatin-induced AKI and which can be effectively targeted during cisplatin chemotherapy. In the experimental design, four groups of male Sprague-Dawley rats; Control (vehicle); cisplatin (6 mg/kg, i.p.); DM (25 mg/kg, oral) for 5 days; and DM (25 mg/kg, oral) 2 h before cisplatin injection were used. In the present study, injection of cisplatin resulted in reduction of body weight, increased blood urea nitrogen (BUN) and creatinine and pro-inflammatory cytokine levels including IL-6 and TNF-α along with alteration in normal histological architecture of kidney. Urinary excretion of protein-based nephrotoxicity biomarkers such as selenium-binding protein 1 (SBP1), kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), and tissue inhibitor of metalloproteinase-1 (TIMP-1) also increased in the cisplatin-treated group. On the contrary, DM significantly protected cisplatin-induced nephrotoxicity which was evident by significant reduction of renal injury biomarkers (BUN, creatinine, and KIM-1, NGAL, and SBP1). DM treatment markedly reduced cisplatin-induced oxidative stress in the kidney by increasing endogenous antioxidants activities (SOD and catalase). Further, DM treatment also reduced the levels of pro-inflammatory cytokines. In particular, protective effect of DM was clearly observed in histopathological examination wherein, kidneys from DM treatment markedly reduced cisplatin-induced severe kidney damages in the proximal tubules. In tumor xenograft model, DM did not affect cisplatin-mediated anticancer activity in transfected colon cancer cells, but enhanced the chemotherapeutic activity of cisplatin as well as exhibited protective effects on cisplatin-induced AKI. Taken together, these results demonstrate a protective role of DM in cisplatin-induced nephrotoxicity and support as a reliable strategy used for renoprotective agent during cisplatin-based cancer therapy.
Keywords: Dendropanax morbifera, Renal toxicity, Urinary biomarker, Chemotherapy, Xenograft
A mechanistic model incorporating IVIVE to quantify a proposed AOP on the nephrotoxicity of NSAIDs (#365)
J. Pletz1, T. Allen2, J. Madden1, M. T. Cronin1, S. Webb2
1 Liverpool John Moores University, School of Pharmacy and Biomolecular Sciences, Liverpool, United Kingdom
Non-steroidal anti-inflammatory drugs (NSAIDs) are recognised as nephrotoxicants that change intraglomerular haemodynamics and produce an excess of reactive oxygen species and inflammatory changes in the kidney. Adverse outcome pathways (AOPs) have been proposed for the nephrotoxicity of NSAIDs. One AOP is initiated by NSAIDs interacting with organic anion transporters located in the basolateral membrane of proximal tubular cells. There, these substances subsequently accumulate and uncouple or inhibit mitochondrial oxidative phosphorylation which may lead to acute tubular necrosis and acute renal failure. Mechanistic models enable the mathematical description of kinetic processes in defined compartments and hence a better understanding of the concentrations reached in the cell. The purpose of this study was to develop a mechanistic model of the kidney and run it using specific parameters for salicylic acid (SA) to investigate whether a quantitative relationship may be established between the therapeutic doses of SA and toxicity events in proximal tubular cells. The model was parameterised with physiologically based and, when available, kinetic data for SA and related compounds. In vitro transporter data were scaled to total kidney tissue level using an in vitro to in vivo extrapolation (IVIVE) approach. At 2.20 mM, the upper bound of therapeutic SA blood concentration reaching the kidney, concentrations predicted for the proximal tubular cell compartments were between 0.755 and 0.775 mM. The results indicated that at a blood concentration of 2.20 mM the molecular initiating event of adversely effecting mitochondrial oxidative phosphorylation of proximal tubular cells is triggered. At SA concentrations as low as 0.4 mM, permeability transition is observed in rat kidney mitochondria which is triggered by the substance’s interaction with the respiratory chain and associated with necrotic cell death. Also, the results showed that the mechanistic kidney model adequately predicts concentrations reached in various parts of the kidney. Validation of the model with additional datasets is necessary to assess the specificity of results.
Needs, C.J. & Brooks, P.M. Clin Pharmacokinet (1985) 10: 164. https://doi.org/10.2165/00003088-198510020-00004
Al-Nasser, I.A. Toxicology Letters (1999) 105: 1. https://doi.org/10.1016/S0378-4274(98)00373-7
Keywords: mechanistic modelling, nephrotoxicity, IVIVE, computational toxicology, quantitative AOP
Overexpression of organic anion transporters in HEK293 reveals high affinity for Aristolochic acid 1 (#368)
H. Bastek1, G. Mucic1, T. Zubel2, A. Mangerich2, S. Beneke1, D. Dietrich1
1 University of Konstanz, Human and Environmental Toxicology, Konstanz, Baden-Württemberg, Germany
The herbal derived toxin Aristolochic acid I (AAI), used in traditional medicines and found in contaminated grain products, is considered to be the major cause of Balkan endemic and Chinese herb nephropathy, both associated with renal fibrosis and upper urothelial cancer. Although the carcinogenic potential is attributed to AAI DNA adduct formation, the nephrotoxic mechanism is still under debate. Renal fibrosis is presumed to result from continuously sustained proximal tubular epithelial cell (PTEC) cytotoxicity. Organic anion transporters (OAT), specifically OAT1, OAT3 at the basolateral and OAT4 at the luminal side of human PTEC, are assumed to be important for reaching cellular AAI concentrations critical for PTEC viability.
Thus, the aim of this project was to determine the relative affinity of AAI to OAT1 and OAT3 in comparison to known substrates. HEK293 cells lacking endogenous expression of these transporters were stably transfected with OAT1-, OAT3- or control-eGFP constructs. Confocal microscopy verified localization of OAT1- and OAT3-eGFP to the cytoplasmic membrane, whereas control-eGFP cells demonstrated a ubiquitous intracellular eGFP signal. Western Blot analysis additionally confirmed OAT1 and OAT3 expression with a predicted size of about 120 kDa. Functionality of the transporters was confirmed via the concentration- and time-dependent uptake of radioactive labeled estrone sulfate and fluorescent 6-carboxyfluorescein (6-CF). Competitive inhibition of 6-CF transport with other OAT substrates showed variable affinity of the substrates for OAT-1 and OAT-3, i.e. para-aminohippuric acid (IC50: 118 µM and 440 µM), probenecid (IC50: 53 µM and 5 µM), and estrone sulfate (IC50: 428 µM and 5 µM). In contrast to the latter, AAI competed with 6-CF uptake the strongest resulting in relative IC50values of 1.9 and 1.2 µM for OAT1 and OAT3, respectively.
The demonstrated high affinity of AAI for OAT1 and OAT3 strongly suggests that observed PTEC-cytotoxicity stems from AAI (presumably plasma albumin bound) import available from the basal vasculature. OAT4 mediated AAI transport is currently under investigation and will elucidate the contribution of OAT4 for AAI loading from the primary urine, or conversely the evasion of AAI from the cells.
Xue, X., Gong, L.-K., Maeda, K., Luan, Y., Qi, X.-M., Sugiyama, Y., Ren, J., 2011. role of organic anion transporters 1 and 3 in kidney accumulation and toxicity of aristolochic acid I. Mol. Pharm. 8, 2183–2192.
Dickman, K.G., Sweet, D.H., Bonala, R., Ray, T., Wu, A., 2011. Physiological and Molecular Characterization of Aristolochic Acid Transport by the Kidney. J. Pharmacol. Exp. Ther. 338, 588–597.
Keywords: Aristolochic acid, Organic anion transporter, HEK293, nephrotoxicity
Protective effect of SIRT-1 inhibitor, EX527, against high fat diet-induced nephrotoxicity (#392)
A. Kundu1, J. H. Park1, J. S. Kim1, J. S. Lim1, H. S. Kim1
1 Sungkyunkwan University, School of Pharmacy, Suwon, Republic of Korea
Diabetes nephropathy (DN) is the leading cause of chronic kidney diseases in patients starting transplantation or renal replacement therapy. Previous study indicated that a selective SIRT1 inhibitor exhibits multiple biological functions including antidiabetic potentiality. The aim of this study was to investigate the protective mechanisms of EX527 on high fat-diet (HFD)-induced nephrotoxicity in ZDF rats. The development of DN is clearly observed followed by 60% fat diet for 21 weeks. The changes of body and kidney weights were significantly increased in HFD rats. Total cholesterol, triglyceride, LDL, blood urea nitrogen (BUN), creatinine levels were significantly increased in HFD induced diabetic rats. However, these biochemical parameters were significantly reduced in HFD rats followed by the treatment with EX527. In histopathological analysis, EX527 protected HFD-induced severe kidney injury damage. Urinary excretion of micro albumin and 4-hydroxyproline levels were significantly decreased in HFD rats by EX-527 treatment. Furthermore, urinary secretion of protein biomarkers (KIM-1, NGAL, SBP-1, and vimentin) associated with nephrotoxicity were dramatically reduced in HFD rats by EX-527 treatment. In particular, HFD-induced abnormal levels of oxidative stress molecules (MDA, SOD, Catalase, and GSH) and proliflammatory cytokines were significantly restored after treatment with EX527. Kidney fibrosis biomarkers (α-SMA, TGF-β, vimentin, α-tubulin, fibronectin and collagen-1) was restored significantly followed by the treatment of EX527. The down-regulation of SIRT-1, SIRT-3 and SIRT-4 were noticed in HFD-induced rats, whereas SIRT-3 expression was up-regulated followed by the treatment with EX-527. In conclusion, this study strongly suggests EX527 exerts a protective effect against HFD-induced DN via ameliorating oxidative stress and inflammation.
Keywords: Nephrotoxicity, EX527, Biomarkers, High fat diet, Oxidative stress
Applying immunoaffinity-proteomics to validate and identify drug-induced kidney injury biomarkers in Cynomolgus monkey’s urine (#659)
W. Naboulsi1, 2, H. Planatscher1, 2, J. - C. Gautier3, X. Zhou4, T. Joos1, 2, O. Pötz1, 2
1 Signatope GmbH, Reutlingen, Germany
Drug-induced kidney injury (DIKI) is still one of the major reasons for failure in drug development. This two-phase study was conducted in cynomolgus to evaluate the potential usefulness of novel biomarkers of nephrotoxicity.
First, in a 10-day (D) dose-range finding study, groups of 3 Cynomolgus males received the nephrotoxic antibiotic gentamicin, at dose-levels of 10, 25, or 50mg/kg/day for 10days. Urine samples were collected on different days. Minimal to mild proximal tubular injury was histologically confirmed at 10mg/kg/day while moderate to severe injury was observed at 25 and 50mg /kg/day, respectively. Several kidney safety biomarkers Osteopontin (SPP1), Cystatin-C, Clusterin (CLU), Retinol Binding protein 4 (RBP4), Alpha-1-microglobulin and Neutrophil gelatinase-associated lipocalin (NGAL) were quantified in the urine samples via a peptide-centric mass spectrometry-based immunoassay panel (IP-LC/MS). In the IP-LC/MS assay, targeted peptides representing the targeted biomarkers are enriched by antibodies which recognize a short epitope motif (TXP-antibodies). As results, SPP1, CLU and RBP4 were best to reflect the nephrotoxicity in the monkey’s urine.
Based on the aforementioned results, we followed-up the lowest nephrotoxic dose of gentamicin (10 mg/ kg/ day, n= 6 or 4) for 10 days and a 2-week recovery to explore the efficiency and the sensitivity of the urinary biomarkers. Here, urinary RBP4 was mostly affected with 6 to 19 - fold higher in the treated monkeys versus controls depending on the day of treatment.
This indicate the applicability of the IP-LC/MS assay to detect changes in urine-based proximal tubular injury biomarkers in monkeys. By utilizing our short epitope motif enrichment strategy, the developed assay can be applied in dogs, human, mouse and rat.
Still, not many data about proteome changes in DIKI -monkeys is available. Therefore, we will conduct a toxicoproteomics study to identify novel protein biomarker candidates for monitoring and detecting early events in DIKI. For this, 50 different TXP-antibodies will be selected to fractionate digests of kidney tissue samples collected from the above-mentioned gentamicin low dose -study. The immunoprecipitated peptides will be analysed by high-resolution nLC mass spectrometry to quantify regulated proteins. Applying such approach, we would avoid conventional tryptic fragments appear in conventional bottom-up proteomic studies, by this we aim to maximize our knowledge regarding proteome changes in nephrotoxicity.
The experiment has been conducted in compliance with applicable regulations for tests on animal.
Keywords: Drug-induced kidney injury, Biomarker, mass spectrometry-based immunoassay, toxicoproteomics, Cynomolgus monkey
Intravenous glutamine infusion is not toxic in partially nephrectomized rats (#662)
S. Bartels1, M. K. Bothe1, R. Abele1, J. Harleman1, C. Meyer1, H. Topp1, M. Westphal1, J. Stover1
1 Fresenius Kabi Deutschland GmbH, Oberursel, Germany
Susanne Bartels and Melanie Bothe contributed equally to the poster.
Rationale: Intravenous glutamine infusion is contraindicated in patients with severe renal insufficiency (creatinine clearance <25 ml/min). Clinical trials, however, raised the question whether glutamine infusion is also safe in patients with mild or moderate kidney injury. To address this concern we performed a non-clinical trial in partly nephrectomized rats instead of healthy animals to qualify glutamine from a toxicological point of view.
Methods: 5/6 nephrectomized rats received continuous intravenous infusion of either Dipeptiven® (alanyl-glutamine) or saline for 9 consecutive days. Standard toxicological parameters including clinical chemistry were analysed.
Results: Rats infused with Dipeptiven® only showed transiently increased plasma urea and ALT levels on single occasions during the treatment period, while creatinine levels were unchanged.
Conclusions: This study provides evidence that Dipeptiven® infusion was not toxic in rats with moderate kidney injury and supports the safety of Dipeptiven® administration in this subgroup of human patients.
Keywords: Renal toxicology, animal model, glutamine
Nephrotoxicity of uranium after low-dose chronic exposure of Nrf2 KO mice (#796)
C. Poisson1, B. Murgues1, J. Stéfani1, O. Delissen1, L. Manens1, A. Ocadiz1, I. Dublineau1, Y. Guéguen1, 2
1 IRSN, PSE-SANTE/SESANE/LRTOX, Fontenay-aux-Roses, France
Uranium is a radioelement present in the environment naturally and also due to human activities. Therefore, exposure of the population to uranium mainly occurs at low dose through drinking water. The kidney is the main target organ of uranium. Biomarkers of U toxicity have been identified but the mechanisms involved in kidney response at low dose are still lacking [1, 2].
Pro/anti-oxidative equilibrium is a defense mechanism frequently involved in acute uranium toxicity. However, we have previously shown that a strengthening of this system was observed during chronic exposure to low doses of uranium . A study conducted on animals deficient in Nrf2 (KO), a transcription factor involved in the regulation of the antioxidant system was carried out, via an exposure for 4 months of male and female C57Bl/6N Nrf2 WT or KO mice. Drinking water contamination with uranium (between 1 and 160 mg.L-1) leads to an increased uranium tissue content in the kidneys, liver and bones of Nrf2 KO animals compared to WT. It is also higher in females for the 3 organs studied. It results in increased urinary levels of several biomarkers of renal tubular damage and inflammation in uranium-exposed animals (NGAL, OPN, β2-microglobulin and Cystatin C) that is also more pronounced in females. Although the protein levels of KIM-1 and Clusterin are not modified in urines, it appears that exposure to uranium could lead to renal tubular damage. Antioxidant enzymes expression are also modified following exposure to uranium, especially for the highest dose (160 mg/L), but without any notable difference between WT and KO. Overall, we show that the tissue accumulation of uranium is Nrf2- and sex-dependent; that biological disturbances are greater in Nrf2-KO animals indicating a role for Redox control, and that females would be more sensitive to the nephrotoxicity of uranium.
Keywords: uranium, Nrf2, biomarker, low-dose, kidney
Generation and characterisation of induced pluripotent stem cells- derived renal proximal tubular-like cells (#890)
V. Chandrasekaran1, R. Gupta2, F. Caiment2, J. C. S. Kleinjans2, P. Jennings1, A. Wilmes1
1 Vrije Universiteit Amsterdam, Amsterdam, Netherlands
The kidney plays a vital role in whole body homeostasis, via blood filtration, reabsorption of required substances and excretion of excess and waste substances. The proximal tubule region is the major workhorse of the nephron and is also one of the most susceptible regions to injury by xenobiotics. Thus the proximal tubule is an important tissue to assess in integrated testing chemical safety assessment approaches.
The main objective of this study was to explore the possibility of differentiating induced human Pluripotent Stem cells (iPSC) into cells representing a proximal tubule phenotype for application to chemical safety assessment and personalised medicine. iPSC cells were differentiated using a 2-step protocol employing specific small molecules and growth factors. Differentiation was characterised by following the expression of pluripotency markers, renal development markers and proximal tubular markers via immunofluorescence, western blot analysis and RNA sequencing. The data demonstrate a temporal transition from pluripotent tissue, to intermediate mesoderm, renal vesicles and finally to a renal phenotype. The last stage could be maintained for up to 10 days. RNA sequencing was cross referenced with the network biology platform CellNet, which confirmed a renal tissue type and absence of similarities to other organs in the database. The cells were positive for megalin, were sensitive to parathyroid hormone and insensitive to vasopressin which are all characteristic traits of the proximal tubule nephron region. This iPSC derived renal proximal tubular-like model will be further characterised with respect to phase I and phase II metabolism and xenobiotic transport capabilities.
Keywords: iPSC, proximal tubulus, renal toxicity
Investigation on chemical induced mitochondrial toxicity in human proximal tubular epithelial cells. (#933)
G. Carta1, P. Jennings1
1 Vrije Universiteit Amsterdam, Molecular and computational toxicology, Amsterdam, Netherlands
The proximal tubule performs constitutive reabsorption of water, amino acids, protein, glucose and ions which is driven by energy dependent Na-K-ATPase. The energy required for this process is generated through oxidative phosphorylation and beta oxidation of fatty acids in the mitochondria. Proximal tubule cells have a high content of mitochondria, which make them especially sensitive to compounds which can injure mitochondria or impair their function. Mitochondrial impairment is a frequent mode of toxicity, that is often identified only late in the drug development pipeline. Thus, there is a need to develop a preclinical screen to identify potential renal mitochondrial liabilities.
The human proximal tubular cell line RPTEC/TERT1 was exposed to 22 electron transport chain (ETC) complex inhibitors of complex I, complex II and complex III. Mitochondrial function was investigated by monitoring glycolysis (lactate production, extracellular acidification rates (ECAR)), mitochondrial membrane potential (MMP) and oxygen consumption rates (OCR, Seahorse Bioanalyser). Transcriptomic studies were also performed using TempO-Seq analysis.
Resazurin reduction in combination with lactate production, the JC-1 assay, the seahorse assay and the TempO-seq analysis performed well to detect mitochondrial liabilities and exhibited similar potency rankings. The data will be used to support the development of a renal quantitative Adverse Outcome Pathway for chemical induced mitochondrial renal diseases, such as Fanconi Syndrome.
“This project has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 681002”
Keywords: Mitochondria, MMP, Kidney, Glycolysis
GM stack soybean MON87701×MON89788 reproduction toxicity investigation (#63)
E. A. Baranov1, S. Shestakova1, E. Sadykova1, N. Tyshko1
1 Federal State Budgetary Scientific Institution “Federal Research Centre of Nutrition, Biotechnology and Food Safety”, Moscow, Russian Federation
The system of genetically modified (GM) organisms safety assessment in the Russian Federation within the framework of new GM lines state registration includes a large-scale toxicological studies. Since 2011, according to established researcher practice, the reproduction toxicity study of GMO (generative function, pre- and postnatal development) is one of the obligatory stages.
This publication presents the results of GM stack soybean MON87701×MON89788 evaluation in the in vivo reproduction toxicity experiment on Wistar rats. The animals were divided into two groups, fed with rodent diet with inclusion of GM soybean (‘test’ group) and non-GM near-isogenic counterpart (‘control’ group) soy varieties. The soy was included into the diet at maximum possible level (~44%) not causing nutritional imbalance or metabolic disturbance for the experimental animals. Rats were monitored for body weight, feed consumption, and general health. The assessment of reproductive system was focused on the generative (indices of mating) and endocrine gonads function of parent animals’ and on pre-/postnatal offspring's development. Prenatal development was assessed on 14-15 females of each group, that were euthanized on the 20th day of pregnancy (one day prior to the expected day of delivery). Postnatal offspring development was being assessed during the first month of pups’ life (29 and 28 litters in test and control group, respectively).
Analyses of reproductive function (mating efficiency level, ranges of serum estradiol, progesterone and testosterone), offspring prenatal development (number of ovarian corpora lutea, resorptions, implantation sites, number of live and dead fetuses, pre- and post-implantation losses), postnatal development (number of live and dead pups, dynamic of body weight and length, physical developmental parameters) revealed no biologically meaningful differences between test and control groups. All parameters did not exceed physiological range. The results of the reproduction toxicity assessment along with other biomedical research data indicate the safety of the GM MON87701×MON89788 soybean stack.
This work was supported by the Ministry of Science and Higher Education of the Russian Federation (research project No. 0529-2019-0056).
Keywords: soybean, GMO, reprotoxicity
Two-generation reproduction toxicity studies of novel food sources: chronobiologic features (#77)
E. O. Sadykova1
1 Federal State Budgetary Scientific Institution “Federal Research Centre of Nutrition, Biotechnology and Food Safety”, Moscow, Russian Federation
The procedure of new food sources safety assessment includes a two-generation reproduction toxicity study on laboratory animals. A duration of such studies determines the need of exogenous factors background effects standardization (fluctuations of atmospheric pressure, humidity, geomagnetic activity, etc.) during the experiment. Since the development of adaptation to these factors has been formed throughout the whole period of mammals evolution, the seasonal variability of some physiological and biochemical parameters cannot be mitigated even in the controlled laboratory conditions. Thus, when analyzing the results of the reproduction toxicity experiments it is necessary to take into account the chronobiologic features of laboratory animals.
This publication presents the results of research, that was pointed at investigation of seasonal factors influence on the reproductive system function of Wistar rats. The reproductive function in the autumn/winter seasons and spring/summer seasons was evaluated with the indices of mating, postnatal development of the offspring (number of live and dead pups, dynamic of body weight and length, physical developmental parameters).
All parameters did not exceed physiological range and did not form clearly traceable trends. The indices of mating were ~94% regardless of season of the year. The offspring born in the autumn/winter and spring/summer seasons showed the survival rate as 99.4% and 99.7%, and the males/females ratio in litter as 56/44 and 53/47, respectively. Analyses of body weight and length dynamic also revealed no biologically meaningful differences between groups.
Thus, the analysis of the obtained data did not reveal a correlation with seasonal factors. Values of all studied parameters did not fall outside the limits physiological norm and did not form obviously traced tendencies.
This work was supported by the Ministry of Science and Higher Education of the Russian Federation (research project No. 0529-2019-0056).
Keywords: reproduction toxicity studies, indices of mating, postnatal development, seasonal variability, novel food sources
Validation of a Novel Human Stem Cell-Based Gene Expression Assay for In Vitro DART Assessment (#324)
I. Brandsma1, P. Racz1, T. Zwetsloot1, S. Hartvelt1, G. Hendriks1
1 Toxys, Leiden, Netherlands
Testing for developmental and reproductive toxicology (DART) is a crucial part of the toxicological risk assessment. Today, DART mostly relies on animal testing, although alternative in vitro tests, such as embryonic stem cells based assays, are used. However, these in vitro assays often do not provide mechanistic insight and the results are difficult to translate to human risk due to inter-species differences.
To improve in vitro identification of developmental toxicants, we identified potential biomarkers in human induced pluripotent stem cells (hiPSC), marking different developmental stages from pluripotent stem cells to terminally differentiated cells. To test whether compounds affect development, first we optimised the differentiation protocols for hiPSC towards cardiomyocytes, hepatocytes and neural rosettes and confirmed the expression of selected biomarkers (OCT4, BMP4, MYH6, FOXA2, SOX17, AFP, ALB, PAX6) by qPCR. During differentiation, expression of the pluripotency marker OCT4 decreased, while expression increased for matured tissue markers MYH6 in cardiomyocytes, ALB and AFP in hepatocytes and Pax6 during neuronal rosette formation.
Next, we exposed differentiating hiPSC cells to 15 teratogenic and non-teratogenic compounds. We observed a marked downregulation of the cardiomyocyte-specific biomarker MYH6, hepatocyte-specific markers ALB and AFP and/or neural specific biomarker PAX6 during teratogenic compound treatment 5-FU, thalidomide, retinoic acid, diphenylhydantoin, bitertanol, triadimenol and methoxyacetic acid. The late differentiation markers were not affected after mono-butyl-phthalate treatment, but the early mesoderm specific marker BMP4 was down-regulated. Two potential teratogenic azole fungicides, fluconazole and carbendazim, did not reduce the expression of any of the biomarkers. Three out of five non-teratogenic compounds, acrylamide, dimethyl phthalate and saccharin, did not reduce the biomarker expression in either of the three differentiation protocols and were correctly identified as non-teratogenic
Following the differentiation program by using selected biomarkers allows the quantitative analyses of potential teratogen exposure and provides mechanistic insight into the potential teratogenic mode of action of compounds.
Keywords: DART, Developmental toxicity, Teratogens, Stem cells, Biomarkers
Copper nanoparticles alter cell viability and steroidogenic activity of gonadal cells (#344)
S. Scsukova1, A. Bujnakova Mlynarcikova1, F. Alonso2, A. Sirotkin3
1 Slovak Academy of Sciences, Institute of Experimental Endocrinology, Biomedical Research Center, Bratislava, Slovakia
With the rapid development and widespread use of nanoparticles (NPs) in many industrial and biomedical applications, the environmental and occupational exposure of humans and animals to NPs is dramatically increasing. The results of recent studies have reported that NPs may pose adverse effects on male and female reproductive health by altering normal testis and ovarian structure, spermatogenesis and sperm quality, oogenesis, follicle maturation and sex hormone levels. The present study aimed to investigate dose-dependent and time-course effects of copper (Cu) NPs of different size on viability and steroidogenic activity of ovarian granulosa (GCs) and testis Leydig cells in vitro.
The immortalized human GC line COV434, primary GCs isolated from porcine ovarian follicles (3–5 mm in diameter) and mouse somatic Leydig TM3 cells were cultured with Cu NPs of different size (1–50 nm; 0.001–1 µg/ml; 0.4–40 µM) under basal conditions or in the presence of gonadotropins (follicle-stimulating hormone, FSH or luteinizing hormone, LH; both 100 ng/ml) and/or androstenedione (100 nM) for different time periods (24, 48, and 72 h). Cell viability was assessed by MTT and CytoTox-ONE Homogenous Membrane Integrity (LDH) assays. Steroid hormone (progesterone, estradiol, and testosterone) levels in culture media were measured by radioimmunoassay commercial kits.
Treatment of human COV434 and porcine GCs, and mouse Leydig TM3 cells with tested Cu NPs induced a significant concentration- and time-dependent inhibition of cell viability. Exposure of human and porcine GCs, and Leydig cells to Cu NPs altered basal as well as stimulated progesterone and estradiol, and testosterone secretion, respectively by cells after 48 and 72 h of culture. The effects of Cu NPs were dependent on their size and the way of their preparation.
The obtained results indicate that disruption of gonadal cell functional state via NPs may affect steroidogenic output and thus perturb mammalian reproductive function. Possible mechanisms of Cu NPs adverse effects should be further elucidated.
This work was supported by the Slovak Research and Development Agency under the contract No APVV-15-0296 (acronym ENDONANOSAFE) and VEGA Grant 2/0187/17.
Keywords: nanoparticles, ovarian granulosa cells, Leydig cells, cell viability, steroids
Molecular mechanisms behind blood vessel formation in human in vitro cellular vasculogenesis and angiogenesis model and their connection with teratogenesis (#378)
1 Tampere University, FICAM, Tampere, Finland
Purpose: New blood vessels are formed by two distinct processes, vasculogenesis and angiogenesis. In this work, the molecular mechanisms behind vasculogenesis and angiogenesis was investigated in in vitro model based on co-culture of HUVEC and hASC cells. The model has been validated and standardized for routine use to study inhibitors of blood vessel formation (Toimela et al. 2016). Disturbances in blood vessel formation during embryonal development are one of the main routes leading to embryonal malformations and defects, also notified in OECD AOPs (Project 1.6).
Methods: hASC-HUVEC co-cultures were established and the cultures were stimulated for six days to form vascular structures. Total RNA samples were collected on day 0, day 1, day 3 and day 6 during the process vasculature was formed. The assay included positive and negative (test substance solvent i.e. 0.5 % DMSO) controls. The molecular mechanism by which valproic acid, a commonly prescribed drug and known teratogen, inhibit formation of vasculature were investigated using RNA-Seq with next-generation sequencing (NGS) analysis on the RNA samples. RNA-sequencing data was aligned to human genome using STAR aligner, gene expression levels were quantified with featureCounts program, and tested for differential expression between sample groups using DESeq2. Resulting lists of differentially expressed genes were utilized to identify pathways with altered expression using Ingenuity Pathway Analysis software and David functional annotation tool.
Results: The biological pathways behind formation of vasculature were identified. Further, genes and pathways associated to mechanism by which Valporic acid inhibit formation of vasculature were identified.
Toimela T, Huttala O, Sabell E, Mannerström M, Sarkanen JR, Ylikomi T, Heinonen T: Intra-Laboratory Validated Human Cell-Based In Vitro Vasculogenesis/Angiogenesis Test with Serum-Free Medium. Reproductive Toxicology. 2016. DOI 10.1016/j reprotox. 2016.11.015.
Keywords: vasculogenesis, teratogenesis, developmental toxicity, in vitro, tissue models
Hazard identification of pesticide reproductive toxicity. Different methodological approaches. (#410)
N. Shepelska1, Y. Kolianchuk1, M. Prodanchuk1
1 L.I. Medved’s Research Center of Preventive Toxicology, Food and Chemical Safety, Kyiv, Institute of experimental toxicology and biomedical researches, Kyiv, Ukraine
Results of own reproductive toxicity studies of five pesticides in gonadotoxic activity identification test-system were compared with manufacturing firm data obtained in test-systems of two and three generation reproduction toxicity studies in the rats.
For the comparative analysis, the compounds that had a toxic effect on the reproductive system in the test-system for identification gonadotoxic activity but not showing signs of systemic toxicity were selected: α-cypermethrin, mancoceb, metribuzin, pyrimifos-methyl, chloromequat chloride.
In the test-system for identification gonadotoxic activity, the ability to destructive effect on the testes and epididymis morphology and function of sex hormones was revealed in all studied pesticides. This ability characterized by a change in the testes and epididymis weight (α-cypermethrin, metribuzin, pyrimifos-methyl), deterioration of the sperm parameters (pyrimifos-methyl, chloromequat chloride), and violation of the periodicity and duration of the estrous cycle stages in females (α-cypermethrin, mancoceb, metribuzin).
When α-cypermethrin exposed to males, such changes as a decrease in conception and fertility index were noted; mancoceb alters sexual behavior in males, leading to an increase in the duration of the precoital interval when mated with untreated females; metribuzin and pyrimiphos-methyl, when exposed to males, induce an increase in intrauterine death of embryos and fetuses in untreated females.
The following LOAELs of test compounds is established: α-cypermethrin - 2,0 mg/kg/b.w., mancoceb – 25 mg/kg/b.w., metribuzin - when exposed to ♂♂ < 0,4 mg/kg/b.w., and to ♀♀- 7,5 mg/kg/b.w., pyrimifos-methyl - when exposed to ♂♂ 5,0 mg/kg/b.w., chloromequat chloride - 50,0 mg/kg/b.w.
In the test-system of 2- and 3-generations reproduction toxicity study, α-cypermethrin, mancoceb, metribuzin, pyrimifos-methyl, chloromequat chloride did not show reproductive toxicity.
The presence of endocrine-destructive potential in the studied pesticides is confirmed by the numerous results of independent studies.
The results obtained showed a higher sensitivity, informativity and diagnostic significance of the gonadotoxic activity identification methodology in comparison with the methodology of the 2- and 3- generation reproduction toxicity studies.
Keywords: reproductive toxicity, pesticides, methodological approaches, gonadotoxic activity identification test-system, 2- and 3-generations reproduction toxicity test-systems
Irreversibility of non-monotonic and monotonic dose-response curves of pesticide Lambda-Cyhalothrin antiandrogenic effect (#416)
N. Shepelska1, Y. Kolianchuk1, I. Rashkivska1, M. Prodanchuk1
1 L.I. Medved’s Research Center of Preventive Toxicology, Food and Chemical Safety, Kyiv, Institute of experimental toxicology and biomedical researches, Kyiv, Ukraine
Research methods: Lambda-cyhalothrin (LCT) 98.06% of purity was administered by oral gavage to three groups of animals in doses 0,3; 3,0 and 10 mg/kg of body weight for 11 weeks. After the end of the exposure period, part of the males was selected to study the parameters of sperm and blood serum testosterone levels, while the remaining males were used for a recovery period without exposure for one full cycle of spermatogenesis (70 days). Morpho-functional indicators of the gonad state and the level of total testosterone in the blood serum were studied in all males after exposure and recovery period.
Results: Tested LCT causes antiandrogenic effect which characterized by impaired of spermatogenesis and oligospermia, as well as a change in the testosterone content in the blood serum of experimental animals. Dose dependence of the severity of oligospermia and spermatozoa adynamia is linear in nature both before and after the recovery period, increasing markedly at the end of the recovery period. While the response level of testosterone to increase of the dose is non-monotonic. The most pronounced significant decrease in the level of testosterone is noted at the end of exposure at a dose of 3.0 mg/kg of body weight. When exposed to the minimum and maximum doses, there is a tendency for this parameter to decrease. After the recovery period, the minimum and maximum doses cause the tendency to increase in testosterone, while the middle dose of LCT, significantly induces a decrease in the content of this hormone.
Conclusions: The analysis of the qualitative and quantitative characteristics of the observed effects at the end of the exposure and recovery periods allows presuming that the tested LCT is irreversible xenoagonists of estrogenic receptors with an intermediate degree of activity, causing damage to Sertoli cells and the spermatogonial population of the germinative cells, depending on the dose level of exposure. The parameters characterizing the processes of spermatogenesis, and the testosterone content did not reach the control level during the recovery period; this indicates the irreversibility of the anti-androgenic effect for 10 weeks, and possibly the complete irreversibility of the observed effects.
Keywords: irreversibility, lambda-cygalothrin, anti-androgenic effect, non-monotonic and monotonic dose-response curve
Optimising the Design of Minipig Embryofetal Studies (#417)
A. Makin1, J. Løgsted1, S. Ellemann-Laursen1
1 Citoxlab, Lille Skensved, Denmark
Rats and rabbits are the routine species of choice for developmental toxicity (embryofetal development (EFD)) studies. If for any reason these species are found unsuitable (e.g. due to issues of metabolism) another species is chosen, and a commonly used non-rodent species is the minipig. In our laboratories, we have long experience with EFD studies in the Gottingen minipig. When working with non-standard species for studies of this nature, a reliable study design producing robust data is imperative, whilst taking into consideration the requirements of the guidelines. The ICH S5 and OECD 414 guidelines make clear the study designs. We have experience from more than 10 studies and the data generated from these studies enables us to continually review and refine the study designs to ensure reliable and consistent results. Consideration is given to factors such as efficient synchronisation of estrus with the use of Regumate® (altrenogest) to maximise mating success, and we have a pregnancy rate of close to 100%. In this way the required number of pregnant sows for the study can be accurately estimated, eliminating excess and contributing to Reduction in animal use. Further, this also allows precise scheduling of the number of sows mated per day according to the facility capacity to perform the caesarean sections on the required day of gestation. Our mating success and the litter sizes in our studies are superior to the published literature (for example, data from the animal breeder). This poster presents background control animal data for all of the fundamental litter-based parameters and demonstrates the robustness of the methods used.
Keywords: Embryofetal studies, Minipigs
The effects of perfluorooctanoic acid (PFOA) on fetal and adult rat testis (#462)
1 University of Turku, Research Centre for Integrative Physiology and Pharmacology, Institute of Biomedicine, Turku, Finland
Perfluorooctanoicacid (PFOA) is widely dispersed synthetic chemical and it accumulates in living organisms. Male reproductive disorders have increased and may have their origin in fetal life. This study was designed to investigate the effects of PFOA on fetal and adult rat testis in vitro. Fetal testes (ED 17.5) or seminiferous tubule segments (stage VII-VIII) were cultured in 4 different PFOA concentrations: DMSO only, PFOA 10, 50 and 100 µg/ml for 24 h. Afterwards, cAMP, progesterone, testosterone and StAR protein levels were measured from the fetal testes culture. Apoptotic fetal Sertoli (SC) and Leydig cells (LC) were detected by using immunofluorescence and TUNEL. Number of apoptotic adult testicular cells were detected from squash samples by immunohistochemistry using cleaved caspase-3. Flow cytometry analyze was made for adult testicular cells using vimentin and FxCycle. Present study shows that PFOA has effect on steroidogenesis; the levels of cAMP, progesterone and testosterone as well as the expression of StAR decreased significantly in PFOA 100 µg/ml. Apoptotic cells increased and PFOA affected different testicular cell populations significantly by decreasing the amount of diploid, proliferating, meiotic I and G2/M –phase cells in adult rat testis. PFOA did not affect fetal, proliferating or adult rat SCs. In addition, we detected an increased tendency of apoptotic fetal LCs but the difference was not significant.
Keywords: Endocrine disruption, male reproductive toxicology, steroidogenic pathway, Sertoli cells, Leydig cells
Activation of sigma-1, MT1 and MT3 receptors prevents pre- and postnatal disturbances in rat offspring induced by cigarette smoke and ethanol exposure (#487)
A. S. Solomina1, E. D. Shreder1, L. G. Kolik2, A. D. Durnev1
1 FSBI "Zakusov Institute of Pharmacology", Department of Drug Toxicology , Moscow, Russian Federation
Background. Prenatal maternal smoking as well as alcohol exposure can result in a range of physical, neuropatological and behavioral alterations [1,2]. Fabomotizole (afobazole) is an effective drug with the safety profile for treatment generalized anxiety disorder in Russia, it has pronounced cytoprotective, neuroprotective and antioxidative effect via activation sigma-1, MT1 and MT3 receptors . Recently it was shown a strong relationship between DNA damage in the embryo cells during fetal development and cognitive dysfunction in postnatal offspring in the streptozotocin-induced diabetes model prevented by fabomotizole . The aim of the present work is assessment of fabomotizole effects on developmental abnormalities of rat offspring after maternal ethanol or cigarette smoke exposure.
Methods. Pregnant outbred rats were administered ethanol (4.3 g/kg/day, 40% v., orally) from gestational gay 10 (GD10) to GD19. Exposure to cigarette smoke from 4 cigarettes with filter, containing 13 mg of tar and 1 mg nicotine, was performed throughout the pregnancy once a day for 20 minutes in the chambers 72 dm3. Fabomotizole (1 and 10 mg/kg, orally, daily) was administered 15 minutes prior to the ethanol intake or exposure in the “smoking” chambers. DNA damage in placenta and fetus cells was evaluated on GD13. Rates of embryo- and fetal development were measured on GD20. Parameters of postnatal development were assessed by the unconditional reflexes formation ("turning on the plane" and "avoiding the edge" tests) and muscle strength ("horizontal rope" test) on postnatal day 5 (PD5). The same animals were examined in the tests "T-shaped maze" and "Extrapolation disposal" to assess congnitive function on PD60.
Results. Cigarette smoke or ethanol exposure led to significant increase in DNA damage in placenta and embryo cells, morphological disturbances of fetuses, retardation of sensory-motor reflexes formation and muscle tone in PD5 offspring as well as cognitive disorders revealed in the "T-shaped maze" and "Extrapolation disposal" tests on PD60 (p<0.05). Prenatal fabomotizole diminished DNA damage in embryo and placental tissues to the level of naïve control, decreased the number of fetuses with abnormal internal organs and impaired ossification and prevented the changes in reflexes formation and muscle strength in the PD5 offspring as well as dose-dependently reduced the disturbances, accelerated adaptation in an unfamiliar environment, and reproduction of cognitive tasks in PD60.
Conclusion. Fabomotizole in the range of anxiolytic and neuroprotective doses via multitargeting action corrects developmental disturbances in rat offspring exposed to prenatal cigarette smoke or ethanol. Thus, fabomotizole is promising for further studies as means of warning of a delay physical development, learning and memory in offspring.
 Hall BJ, Cauley M, Burke DA, Kiany A, Slotkin TA., Levin ED. Cognitive and Behavioral Impairments Evoked by Low-Level Exposure to Tobacco Smoke Components: Comparison with Nicotine Alone. Toxicol Sci. 2016 Jun;151(2):236-44. doi: 10.1093/toxsci/kfw042. Epub 2016 Feb 26.
 Rouzer SK, Cole JM, Johnson JM, Varlinskaya EI, Diaz MR. Moderate Maternal Alcohol Exposure on Gestational Day 12 Impacts Anxiety-Like Behavior in Offspring. Front Behav Neurosci. 2017. 11:183. doi: 10.3389/fnbeh.2017.00183. eCollection 2017
 Voronin MV, Kadnikov IA. Contribution of Sigma-1 receptor to cytoprotective effect of afobazole. Pharmacol Res Perspect. 2016 Nov 7;4(6):e00273. doi: 10.1002/prp2.273. eCollection 2016 Dec.
 Zabrodina VV, Shreder OV, Shreder ED, Durnev AD. Effect of Afobazole and Betaine on Cognitive Disorders in the Offspring of Rats with Streptozotocin-Induced Diabetes and Their Relationship with DNA Damage. Bull Exp Biol Med. 2016 Jul;161(3):359-66. doi: 10.1007/s10517-016-3414-2. Epub 2016 Aug 9.
Keywords: fabomotizole, sigma receptor, DNA damage, pre- and postnatal development, rats
Recent findings on reproductive, developmental and systemic toxicity of propyl paraben show no evidence of endocrine activity (#523)
S. Fayyaz1, R. Kreiling1
1 Clariant Produkte Deutschland GmbH, Global toxicology & Ecotoxicology, Sulzbach am Taunus, Hesse, Germany
Alkyl esters of p-hydroxybenzoic acid (parabens) like methyl-, ethyl-, propyl- and butyl paraben are widely used as preservatives in cosmetics, food and pharmaceuticals. It is well known that parabens are rapidly hydrolyzed to nontoxic p-hydroxybenzoic acid, conjugated and excreted through urine resulting in a very low concentration of the parent compound in blood and urine. Although parabens explicitly fulfil all OECD and ECHA RAAF criteria for category formation, a proposal for respective grouping and read-across was rejected by ECHA and individual substance evaluations take place by various MSCAs.
Concerns about potential endocrine activity of parabens which are primarily based on in vitro findings indicate an increased endocrine activity of parabens with increasing chain length (methyl- < ethyl- < propyl- < butyl paraben). However, the “potency” remained several orders of magnitude below the activity of the natural endogenous estrogen 17ß-estradiol. Despite clear shortcomings and limitations in available in vivo data which is questioning a biological relevance of these in vitro findings, methyl-, ethyl- and propyl paraben have been selected to CoRAP as suspected endocrine disruptor and extensive sets of identical higher tier animal studies are required for all three parabens by ECHA. The data requirements comprise inter alia subchronic toxicity (OECD 408), developmental toxicity (OECD 414) and full EOGRTS (OECD 443).
The data presented here for propylparaben as example, clearly demonstrate that repeated oral exposure of rats did not result in any finding of toxicological relevance. The NOAELs in these studies were uniformly placed at 1000 mg/kg body weight per day. Even more important, the data do not support any biological meaningful endocrine activity of parabens.
Keywords: Paraben, endocrine disruptor, subchronic toxicity, developmental toxicity, reproduction toxicity
Safeguarding food safety: Rapid screening of phosphodiesterase 5 (PDE5) inhibitors as adulterants in selected food matrices using enzyme assay (#577)
A. Y. Mohd Yusop1, 2, L. Xiao1, S. Fu1
1 University of Technology Sydney, Centre for Forensic Science, Ultimo, Australia
The global incidence of adulterated food products warrants a faster detection method to address the food safety concern. This study developed an enzyme assay procedure to rapidly screen phosphodiesterase 5 (PDE5) inhibitors found as adulterants in selected food matrices promoted to improve male sexual performance.
The assay used a fluorescein amidite (FAM)-labelled cyclic guanosine monophosphate (cGMP) as a substrate for PDE5 enzyme activity, aided by the presence of phosphate binding beads on its fluorescence polarisation. First, the enzyme assay was validated using certified reference materials of sildenafil. A dose-response-inhibition curve was plotted using a non-linear fit of log inhibitor versus response at a concentration ranged from 1 x 10-4 to 1 μM. Next, three blank food matrices free from any analyte of interest were submitted to the developed assay procedure to verify the interference’s effect. The resulting values were utilised as thresholds for positive identification. The applicability of the developed procedure was then established using five samples suspected to be adulterated with PDE5 inhibitors obtained from Malaysia and Australia. Finally, the same samples were submitted to a liquid chromatography-quadrupole time of flight (LC-QTOF) analysis to confirm the adulterants’ identities.
The validation results showed that sildenafil inhibits the PDE5 enzyme, exhibiting a symmetrical sigmoidal shape curve with an IC50 of 4.3 x 10-3 μM, ensuring the robustness of the assay performance. The results also displayed an excellent enzyme-substrate activity which was deemed fit for application to potentially adulterated samples. The blank samples yielded the percentage of PDE5 enzyme inhibition as 18.2% for chewing gum, 6.6% for hard candy, and 13.8% for jelly. The real samples outcome returned a percentage range of inhibition from 77.3% to 100.6%, indicating the presence of PDE5 inhibitors in all products, in agreement with the confirmatory LC-QTOF analysis.
The procedure proposed in this study provides a rapid screening and straightforward data interpretation to make a quick preliminary decision in separating adulterated and non-adulterated food products. It would be gainful in tackling the problems of food safety, such as adulteration with PDE5 inhibitors, to protect public health.
Keywords: food safety, adulteration, PDE5 inhibitors, enzyme assay, rapid screening
Comparative evaluation of bisphenol A analogues in silico (#599)
M. Vasilyeva1, S. Sychyk1, I. Ilyukova1
1 Republican Unitary Enterprise , Minsk, Belarus
The growing concern about widely used bisphenol A (BPA) as a chemical that destroys the endocrine system and its possible effects on human health have prompted the exclusion of BPA from consumer products, often referred to as “BPA free”. Similarly, structured analogues of BPA are widely used, but much less is known about their potential toxicity or estrogenic activity. Therefore, it is necessary to evaluate such chemicals in order to determine safer ones.
The goal is to conduct a comparative assessment of BFA compounds in silico and identify safer substances for further testing in vitro.
Methods: in silico approach based on OECD QSAR Toolbox.
For the detection of bisphenols in the general list of potentially endocrine disruptive properties, an assessment was made based on data models based on the chemical structure of substances (QSAR-forecasts). Based on the mechanisms described for the properties of endocrine disruption of BPA, and as end points, measures were taken of the potential properties of endocrine disruption of each substance: α-agonism of the estrogen receptor and antagonism of the androgen receptor. It was predicted that out of 100 bisphenols, 75% of the substances are capable of activating the α-estrogen receptor and 65% of the substances are capable of inhibiting the signaling of the androgen receptor (antagonism). Overall, 90% had a positive prognosis for estrogen α-receptor activation or an androgen receptor antagonism (or both). Based on the analyzed literature data on the use of analogues of BPA, those chemicals that are more often encountered during production were selected for more detailed analysis and further in vitro testing.
Keywords: in silico, bisphenol A, estrogen receptor, analogues of BPA
Embryotoxicity of Sodium Valproate is correlated to the dysregulation of autophagy (#605)
Y. Ma1, J. Zhang1
1 Chinese Academy of Medical Sciences & Peking Union Medical College, Institute of Medicinal Biotechnology, Beijing, China
Valproic acid (VPA) has been clinically used as a traditional first-line antiepileptic drug through many anti-epilepsy drugs emerged, such as sodium phenytoin, phenobarbital, gabapentin, lamotrigine, zonisamide, etc. Epilepsy is a serious chronic and devastating neurologic disorder characterized by spontaneous, transient, recurrent and unprovoked seizures. Current drugs for the treatment of epilepsy include sodium phenytoin, phenobarbital, diazepam, valproic acid, etc. Recent studies showed VPA therapeutic roles on cancer and brain-injure diseases. However, VPA has potential teratogenicity, which is a non-ignorable risk for fetal development when a pregnant woman with epilepsy uses VPA. Therefore, it is important to clarify the teratogenic mechanism of anti-epilepsy drugs. Zebrafish is an ideal animal for studying embryonic development, medicinal toxicology and pharmacology in vivo. Autophagy is a host mechanism to maintain intracellular homeostasis and a defense mechanism against invasion by pathogenic microorganisms. Many studies have reported VPA could induce autophagy. In this work, we investigate the correlation between the VPA teratogenicity and autophagy mechanism. Zebrafish embryos were treated with VPA (50,100 and 200μg/ml) at three different administration periods (6-10 hpf, 10-24 hpf, 6-24 hpf), and observed under a microscope and collected for western blotting at 48 hpf. The results showed that zebrafish embryos deformed, including deficient cardiac development with pericardial cyst edema, invaginated yolk, short and bent body, small head and eyes and weak color or colorless of body surface. The severity of these deformed phenotypes dependent on the time length and embryonic periods exposed in VPA; the organogenesis is the most sensitive stage in zebrafish. We verified that in zebrafish VPA up-regulated the levels of autophagy marker LC3B-II protein and selective adaptor p62 protein and autophagy related ATG3, ATG5, ATG7 and ATG10 which participated in formation of two ubiquitination complexes for autophagy production. Meanwhile, VPA also activated the apoptosis pathway. These results indicate that the embryotoxicity of VPA probably is resulted from its induction of a deficient autophagy and apoptosis.
Keywords: Valproic acid (VPA); embryotoxicity; autophagy; apoptosis;
Extended One-Generation Reproductive Toxicity of Thiamethoxam in Rats (#699)
V. Malashetty1, U. Bhatnagar1, N. Rajesh1, M. S. Mulla1
1 Vimta Labs Limited, Pre-Clinical Division, Hyderabad, India
Purpose: Thiamethoxam, a second generation neonicotinoid insecticide was assessed for its systemic and reproductive toxicity, developmental neurotoxicity and immunotoxicity through an extended one generation toxicity study (EOGRTS, OECD No. 443).
Methods: Thiomethoxam was administered to groups of Wistar rats at doses of 15, 50 and 150 mg/kg/day orally. Treatment of males was initiated 2 weeks before cohabitation and continued for 70 consecutive days whereas treatment of females began 2 weeks before cohabitation and continued until weaning of the F1 offspring. In view of reducing the number of animals without compromising on parameters to be assessed, cohort 1A was covered in the parental generation. F1 offspring assigned to cohorts 2A, 2B and 3. Groups of cohort 2A animals were dosed for 70 consecutive days post weaning to assess adult developmental neurotoxicity. Brain histopathology was evaluated in weaned animals (Cohort 2B) for developmental neurotoxicity on lactation day 21. Animals of cohort 3 were treated for 56 days and assessed for developmental immunotoxicity.
Results: No systemic/reproductive toxicity or abnormal changes in fertility parameters (sperm parameters, mating index, pre-coital interval, gestation index, litter size, number of live pups and sex ratio) were observed in parental generation. In Cohort 2A, attainment of puberty was delayed by 4 days in males treated at 150 mg/kg. Examination of reproductive parameters indicated that there was no evidence of treatment related effects that would trigger the need of a second generation. Brain histomorphometry analysis revealed decrease in overall width of hippocampus in males treated at 150 mg/kg. A significant decrease in acoustic startle response (65 to 120 db), total and ambulatory counts at 50 and 150 mg/kg was also observed. In weaned animals (Cohort 2B), no treatment related gross or histopathological findings were observed in brain. Evaluation of developmental immunotoxicity (Cohort 3) of thiamethoxam, did not exhibit the ability to mount an antibody (IgM and/or IgG) response up to 150 mg/kg in a T-cell dependent antibody response functional assay.
Based on the findings, NOAEL of thiamethoxam was considered as 15 mg/kg for development & neurobehavior endpoints and 150 mg/kg for its reproductive & immunotoxic potential.
Keywords: Thiamethoxam, EOGRTS
Assessment of a framework to identify analogues for read-across: case study (#895)
A. Y. Caballero1, 2, C. Toma1, D. Gadaleta1, Y. Perez3, 4, E. Benfenati1
1 Mario Negri Institute for Pharmacological Research, Environmental Health Sciences, Milano, Italy
Read-across is an alternative method for filling data gaps based on an analogue or chemical category approach. OECD and ECHA developed the most important guidance for read-across, but a methodology is not well defined yet. This study intends to explore the performance of a framework for analogues identification in read-across, through a case of study to predict the human aromatase (CYP19A1) binding of 2-aminobenzothiazol. A set of in vitro CYP19A1 binding data was collected from Tox21 as candidates for read-across. An automated framework was developed to select most suitable analogues, based on the evaluation of structural, physical-chemical and biological similarities, and was implemented in KNIME. For the structural similarity assessment Tanimoto index was used as similarity measure. To evaluate the physical-chemical similarity relevant properties were determined. Mechanistic structural alerts for CYP19A1 binding and Rat liver S9 metabolism were used to explore biological similarity. The final list of analogues for read-across was defined by: 1) identifying the intersection between structural and physical-chemical similarities, 2) retrieving only compounds with structural alerts in common with the target and, 3) retrieving only compounds with common metabolites. In the end, two analogues were identified: 2-Amino-6-methoxybenzothiazole and 2-Amino-6-ethoxybenzothiazole. The activity for both analogues was in concordance with the experimental activity of the target confirming the real-life validity of the here presented framework. Deeper analysis must be performed to explore new cases. Different integration approaches, parameters, interactive threshold values, and uncertainties must be consider to refine the framework.
Patlewicz, G., Helman, G., Pradeep, P., & Shah, I. (2017). Navigating through the minefield of read-across tools: a review of in silico tools for grouping. Computational Toxicology, 3, 1-18.
OECD (2017), Guidance on Grouping of Chemicals, Second Edition, OECD Series on Testing and Assessment, No. 194, OECD Publishing, Paris, https://doi.org/10.1787/9789264274679-en.
Keywords: read-across, similarity, analogues, CYP19A, framework
Extended One Generation Reproductive Toxicity Study- EORGTS (OECD 443): How to successfully integrate additional parameters to meet specific regulatory and scientific requirements (#935)
P. Allingham1, P. Takawale1, R. Subramani1, S. Schiffner1, K. Stricker1, J. Gandorfer1, N. Kamps1, L. Richter1, K. Weber2, A. - L. Leoni1
1 BSL BIOSERVICE Scientific Laboratories Munich GmbH, Planegg, Germany
Driven by the motivation to reduce the number of animals used for safety evaluation of chemicals, the EORGTS was developed to allow systemic toxicity evaluation in rats covering a wide range of parameters in the fields of reproduction and developmental toxicity, neurotoxicity and immunotoxicity, including also the evaluation of endocrine disrupting properties of test materials. The OECD 443 guideline together with its corresponding guidance document No. 151 offer details on how to conduct such a complex study but at the same time calls upon the registrant to go beyond and include further parameters if deemed scientifically necessary.
We will present a study design which allows the inclusion of additional offspring in some existing or additional cohorts in order to meet specific regulatory requirement such as the assesment of learning and memory behaviour, which is not a standard requirement of the OECD 443 guideline, and discuss what are the key elements to ensure the success of such an evaluation.
In addition, we will present examples of specific stains that can be used on brain tissue in order to address more specifically potential neurotoxicity (Fluoro-Jade® C and Glial Fibrillary Acidic Protein (GFAP)) when the toxicity profile of similar compounds or literature suggest potential risk for neuronal degeneration. In other spare offspring, specific brain tissues (e.g. hippocampus) or other organs can also be sampled on various postnatal days before or at weaning in order to address dedicated anatomical regions and perform histology or molecular analysis.
Regulatory authorities might ask to demonstrate transfer of test compound to the pups in the milk during suckling. We will present the study designs and methods that can be used to include such evaluation in OECD 443 studies.
Despite the logistic challenge of this type of study, this poster should encourage to further drive refinement using molecular analysis, specific histological techniques and analytical methods to gather always more valuable information and finally develop new approaches for chemical risk evaluation.
Keywords: EORGTS, Endocrine Disruptors, Neurotoxicity, Immunotoxicity, Developmental and Reproductive toxicity (DART)
Effects of paroxetine on biochemical parameters and reproductive function in male rats. (#952)
R. Mosbah1, A. Chettoum2
1 University of M'Hame Bougara, Departement of Biology, Boumerdes, Algeria
Selective serotonin reuptake inhibitors (SSRI) are a class of molecules used in treating depression, anxiety,and mood disorders. Paroxetine (PRT) is one of the mostly prescribed antidepressant which has attracted great attention regarding its side effects in recent years. This study was planned to assess the adverse effects of PRT on the biochemical parameters and reproductive system. Fourteen male wistar rats were randomly allocated into two groups (7 rats or each): control and treated with PRT at dose of 5mg/kg.bw for two weeks. At the end of the experiment, blood was collected from retro orbital plexus for measuring the biochemical parameters, whereas the reproductive organs were removed for measuring semen quality and the histological investigations. Results showed that PRT induced significant changes in some biochemical parameters and alteration of semen quality including sperm count, spermatids number and sperm viability, motility and abnormalities. The histopathological examinations of testis and epididymis revealed an alteration of spermatogenesis, cellular disorganization and vacuolization, enlargement of interstitial space, shrinkage and degenerative changes in the epithelium of seminiferous and epididymal tubules with few to nil numbers of spermatozoa in their lumen. In conclusion, PRT treatment caused changes in some biochemical parameters and sperm profile as well as histopathologic effects of reproductive organs.
Keywords: Paroxetine, toxicity, biochemical parameters, reproduction function, rat
Teratological evaluation of Artichoke leaf extract in rats. (#965)
E. Ujhazy1, M. Farkas1, R. Koprdova1, M. Nagy2, P. Mucaji2, M. Mach1
1 Centre of Experimental Medicine of the Slovak Academy of Sciences, Institute of Experimental Pharmacology and Toxicology, Bratislava, Slovakia
Artichoke (Cynara cardunculus var scolymus) leaf extract have been studied intensively for its antioxidative, hepatoprotective and choleretic effects as well as lipid-lowering and anti-atherogenic activity with increased elimination of cholesterol and inhibition of hepatocellular de novo cholesterol biosynthesis. However there is a very little data about its toxicity in commercial preparations and no data is available about its effects on development. The aim of our study was to evaluate the possible teratogenic effect of the dry extract of artichoke leaves in Wistar rats. Intact females were treated, from gestation day (GD) 5 until GD15, with 0.0, 150, 400 or 1000 mg/kg body weight of extract of artichoke leaves. At GD20, a cesarean section was performed for evaluation of maternal and fetal parameters. Artichoke did not induce changes in food consumption, preimplantation or postimplantation losses, placental weight or biochemical profile. Experimental groups showed similar body weight gain during pregnancy. No reductions in fetal and placental weight were observed in experimental groups. The number of live pups per litter was not statistically significant. No fetal skeletal or visceral malformations were detected. Anogenital distance was not influenced. The results showed that the consumption of artichoke during pregnancy did not affect significantly either mother or fetus.
Supported by the grant VEGA 2/0166/16.
Keywords: Artichoke, Cynara cardunculus, teratology, rat
Effect of Selective Serotonin Reuptake Inhibitors on the Serotonin System and Junctional Protein in Human Placenta (#971)
L. Ok1, 2, A. - A. Hudon Thibeault1, C. Vaillancourt1
1 National Institute of Scientific Research , Armand Frappier Institute , Laval, Québec, Canada
Selective Serotonin Reuptake Inhibitors (SSRIs) are the most common pharmacological intervention for the treatment of antenatal depression. This type of antidepressant works by increasing the level of serotonin at the synaptic space by blocking the serotonin transporter (SERT; SLC6A4) on the post-synaptic neurons. The serotonin system, including SERT and monoamine oxidase A (MAOA), is expressed and functional in the placenta. Our group has shown that the exposure of human placenta to certain SSRIs affects the serotonin system and the trophoblast cell fusion and invasion. We hypothesized that the exposure of primary placental cells to the SSRIs alter the expression of genes involved in placental serotonergic system and the junctional proteins associated with trophoblast cells fusion. Thus, this study aims to assess the effects of most commonly used SSRIs in human trophoblasts. Primary villous trophoblasts were isolated from normal full-term human placentas and were exposed at two concentrations (0.3uM and 0.03uM) of fluoxetine, norfluoxetine, sertraline, venlafaxine, or citalopram for 24-h. The mRNA level of SLC6A4, MAOA, Connexin 43(GJA1), Tight junction protein-1(TJP-1) and Syncytin-1 (ERVW-1) were analysed by RT-qPCR. Overall, our preliminary data shows that all SSRIs tested tend to decrease the mRNA level of SLC6A4, MAOA, Connexin 43 and Zo-1 in primary trophoblasts, and were greater in the cells treated with the lower concentration (0.03uM) of Citalopram, Fluoxetine and Sertraline than the cells treated at the higher concentration (0.3uM). This preliminary study suggest that SSRI alters the mRNA expression of serotonin system and junctional proteins in human primary trophoblasts. This results need to be confirmed by further assessing the effect of SSRIs on proteins expression and placental function. The use of SSRIs during pregnancy poses adverse effect on the fetal development and may be associated with the pregnancy complications such as gestational hypertension. Thus pursuing the work is important to better understand the effect of SSRI on the placenta which is crucial for the maintenance of normal pregnancy and a healthy fetal development.
Keywords: depression, SSRI, pregnancy, placenta, serotonin
The toxicity of triptolide and mechanism involved (#43)
Z. Huang1, F. Shen1, J. Li1, J. Zhou1, W. Wang1, Y. Cheng1
1 Sun Yat-sen University, School of Pharmaceutical Sciences, Guangzhou, China
Triptolide (TP) is the main active ingredients in Chinese medicinal herb Tripterygium wilfordii Hook. F. (TWHF) that is widely used in China. The toxicity of TP is the main factor limiting its clinical application. Acute toxicity and repeated dose toxicity showed that the LD50 of TP is 0.743 mg/kg•bw in mice, and it caused injuries in heart, liver, gastrointestine, male reproductive system. Further studies suggested oxidative stress as the main mechanism of TP-induced organ injuries. Our studies indicated that Nrf2-ARE defense response was involved in the cardiotoxicity, nephrotoxicity, hepatoxicity. Moreover, inhibition of SIRT3 deacetylase, activation of GSK-3β (glycogen synthase kinase-3β) and increased p53 nuclear translocation also contributed to mitochondrial damage of cardiomyocytes. In liver, TP blocked rescue system by inhibiting Notch1 signaling and activating PTEN/Akt/ Txnip (thioredoxin interacting protein), TP disrupted PKD1 (protein kinase D1)/NF-κb/SOD2 (superoxide dismutase 2) signaling, both of which led to oxidative damage in hepatocytes. In addition, accumulating evidences show that TP has obvious toxicity in reproductive system. Our study indicated that TP induced mitochondrial damage and led to cytotoxicity in mice sertoli cells by inhibition of SIRT1 and increased AMPK (adenosine monophosphate activated protein kinase) phosphorylation, which influencing PGC-1α (peroxisome proliferator-activated receptor coactivator-1α) activity and led to the suppression of glycolysis and overactivity of fatty acid β-oxidation. In summary, TP possesses multiple pharmaceutical effects while accompany with a series of toxicities. Its promising potency in therapeutics promotes accumulating molecular research of toxicity, which is beneficial for developing the strategy in ameliorating its toxicity.
Keywords: Triptolide, Systemic toxicity, Molecular mechanism
‘Notch or Not’ - Mystery of an Unexpected Gastrointestinal Toxicity of a Gamma Secretase Modulator (#127)
G. Schmitt1, S. Badillo1, T. Bergauer1, C. Bertinetti1, M. Odin1, S. Roberts1, I. Wells1, E. Wolz1
1 F.Hoffmann-La Roche, Roche Pharmaceutical Research and Early Development, Basel, Switzerland
Gamma Secretase (GSEC) is a key enzyme in the metabolism of the transmembrane protein Amyloid precursor protein (APP). Proteolysis of APP generates beta amyloid (Aβ), whose amyloid fibrillar form is the primary component of amyloid plaques found in the brain of Alzheimer's disease (AD) patients. Therefore, GSEC is explored as a therapeutic target to decrease toxic Aβ formation in AD. GSEC also catalyzes the cleavage of Notch, receptors of a highly conserved cell signaling system with important regulatory function in cell differentiation. Initially developed inhibitors of GSEC showed safety profiles unacceptable for AD patients, mainly due to interaction with Notch signaling pathways. Modulators of GSEC can reduce Aβ formation via a conformational change of the binding site without inhibiting the enzymatic activity, thus entailing selectivity versus Notch and other GSEC substrates, and a higher likelihood for a beneficial safety profile. The preclinical development of a small molecule GSEC modulator included toxicity studies in mice and minipigs. While the 2-week dose-range finding studies in both species and the 4-week GLP toxicity study in mice were uneventful, there were unexpected histopathology findings in the minipig 4-week GLP toxicity study raising concern for Notch-signaling (predominantly goblet cell hyperplasia/metaplasia in small intestine). Since GSEC modulators are not supposed to inhibit Notch signaling and the rodent study did not indicate any of otherwise typical Notch-related side effects, a mechanistic workup of the minipig findings was undertaken, including transcriptome profiling of affected intestinal tissue. The principal affected genes were identified as downstream Notch, involved in enterocyte differentiation (e.g., ATOH1 and SPDEF increased). Yet, the overall phenotype (pathology and transcriptome) was clearly different from typical signatures of GSEC inhibitors (e.g., HES1 remained unchanged for the modulator). These results underpin principal differences between modulators and inhibitors of GSEC. Seemingly overlapping pathological effects need to be understood at a molecular level to guide an improved screening and profiling of back-up molecules.
Keywords: Gamma Secretase, Notch, goblet cell hyperplasia
Exposure to an aerosol generated by a novel electronic cigarette using MESH™ technology causes lower biological alterations than cigarette smoke on buccal organotypic epithelial cultures (#170)
F. Zanetti1, A. Iskandar1, A. Kondylis1, A. Sewer1, F. Martin1, S. Majeed1, L. Ortega Torres1, C. Nury1, C. Merg1, E. Guedj1, T. Schneider1, K. Trivedi1, S. Frentzel1, N. Ivanov1, M. Peitsch1, J. Hoeng1
1 Philip Morris Products S.A., PMI R&D, Neuchâtel, Neuchâtel, Switzerland
Electronic cigarettes (EC) are growing in popularity, although their impact on human health is still debated. It is therefore important to improve understanding of their effects, particularly in the context of tobacco harm reduction strategy. In this study, we investigated the impact of EC aerosol in comparison with that of cigarette smoke (CS) on human organotypic buccal epithelial cultures. Cultures were exposed to 112 puffs of undiluted aerosol generated from a variant of a novel EC device with MESH™ technology (Philip Morris International) or to diluted CS. Nine independent exposure repetitions were performed to ensure robust observations. A systems toxicology approach was applied to study the impact of exposure; a series of endpoints were analyzed, including histological modifications, global mRNA and miRNA expression, secreted miRNA and inflammatory mediator expression, and targeted and untargeted proteomic approaches. Histological evaluation showed minimal morphological changes in cultures exposed to undiluted EC aerosol but major damage in those exposed to CS. Lower alterations in mRNA, miRNA, and protein expression were detected in cultures exposed to EC aerosol than in those exposed to CS. The inflammatory mediators secreted following EC aerosol exposure were distinct from those following CS exposure: IL-1α secretion was enhanced following EC aerosol exposure, while IL-1β was highly induced following CS. The inflammatory mediators secreted following EC aerosol exposure were distinct from those following CS exposure: IL-1β secretion was highly induced following CS exposure, but IL-1α secretion was enhanced following EC aerosol exposure. RNAScope® technology was further used to localize the expression of IL1A gene, showing no difference in the expression and localization of IL1A in the EC aerosol-exposed cultures compared with the air-exposed control. Interestingly, increased apical expression of the IL1A gene was detected in the CS-exposed cultures. Overall, the results indicated that EC aerosol exposure did not elicit tissue damage in contrast to CS exposure at comparable (and higher) nicotine concentrations. Molecular changes were detected in the in vitro buccal epithelial cultures following EC aerosol exposure; however, the impact remained generally much lower than CS exposure.
Keywords: Systems Toxicology, Electronic Cigarette, 21st Century Toxicology, Air-Liquid Interface, Oral Organotypic Culture
Predicting systemic concentrations following topical application using Physiologically Based Kinetic modelling (#188)
I. Sorrell1, H. Li1, R. Cubberley1, R. Pendlington1, B. Nicol1, D. Sheffield1, J. Pickles1
1 Unilever, SEAC, Sharnbrook, United Kingdom
Physiologically Based Kinetic (PBK) models are routinely used to predict human kinetic profiles of compounds. Using PBK models it is possible to predict systemic concentrations of xenobiotic compounds from different routes of exposure. Combined with dose-response toxicology data from appropriate in vitro studies this has application in assessing risk of systemic toxicity. The purpose of this study was to assess the accuracy of a PBK modelling approach for predicting plasma concentration time-profiles of topically applied compounds when the input data was generated using non-animal methods. Prediction of maximum plasma concentration (Cmax) and area under the curve (AUC) were compared to the observed kinetics from published clinical studies.
Plasma profiles following topical application were predicted using Gastroplus 9.0 PBK software for six compounds (diclofenac, salicylic acid, coumarin, nicotine, caffeine and N,N-diethyl-m-toluamide), and compared with existing clinical kinetic data from topical application studies. Dermal absorption was determined from ex vivo human skin penetration studies. Hepatic clearance was determined using primary human hepatocyte suspension cultures. Plasma protein binding data was taken from the literature or generated using rapid equilibrium dialysis. In all cases experimental data was used from the literature where available but generated if not.
The calculated and clinically observed Cmax values were in good agreement (R2 = 0.81). The comparison between calculated and observed AUC∞ are of similar accuracy (R2 = 0.84).
The combination of in vitro data generation and in silico PBK modelling showed good accuracy for predicting human Cmax and AUC values within an order of magnitude for all six compounds. Given the small number of compounds and due to the lack of topical clinical studies reporting measured kinetics, additional data and approaches are required to improve the confidence in predicting systemic concentrations of topically applied compounds. However, the results of this study suggest that PBK modelling is a suitable approach for estimating the internal concentration of compounds applied to the skin.
Keywords: PBK modelling, systemic exposure, toxicokinetics
Risk assessment of methanol in consumer products (#278)
J. S. Kang1, Y. C. Kwon1, M. K. Kim1, B. M. Lee1
1 Sungkyunkwan University, College of Pharmacy, Suwon, Republic of Korea
This work was supported by a grant from Ministry of Food and Drug Safety (MFDS), 2018.
Methanol also called methyl alcohol with chemical formula CH3OH. Methanol is classified as an alcoholic substance such as ethanol and have similar properties. It is a colorless liquid and has combustibility, volatility and toxicity. However, methanol contains less carbon and hydrogen than ethanol, and the boiling point of methanol is lower than ethanol. Methanol is converted from the liver to formaldehyde which may be fatal to humans. In addition, methanol produces metabolic acidosis (nausea, headache), central nervous system (CNS) depression, ocular toxicity and even death. Because of these toxicities, the Ministry of Food and Drug Safety (MFDS) is regulating methanol that is rapidly absorbed into the body (skin, mouth and lung) via consumer products. MFDS consequently set the limit concentration of methanol at 0.2 percent and 0.002 percent, in cosmetic products and wet wipes, respectively. To carry out risk assessment, systemic exposure dosage (SED) was estimated to be 0.0016 mg/kg/day and 0.0006 mg/kg/day respectively in cosmetics and wet wipes, with a total SED is 0.01606 mg/kg bw/day for adults. Also, SED was estimated to be 0.064 mg/kg bw/day and 0.00024 mg/kg bw/day respectively, with a total SED is 0.06424 mg/kg bw/day for children. NOAEL of methanol was found to be 500 mg/kg bw/day in rats, but the modified NOAEL was estimated to be 415 mg/kg bw/day (500 mg/kg bw/day x 0.83) because of the oral bioavailability of 83 percent. The margin of safety (MOS) for methanol in cosmetics and wet wipes was calculated to be 25841 and 6460 based on 415 mg/kg bw/day (NOAEL) / 0.01606 mg/kg bw/day (SED) and 415 mg/kg bw/day (NOAEL) / 0.06424 (SED) mg/kg bw/day, respectively. These data suggest that methanol has no risk to human when it is exposed to 0.2% and 0.002% of the finished cosmetics products and wet wipes, confirming its safety.
Keywords: Methanol, risk assessment, NOAEL, SED, cosmetics, wet wipes
Keywords: risk assessment, NOAEL, SED, cosmetics, methanol
Next Generation Risk Assessment of Coumarin in Personal Care Products (#358)
M. T. Baltazar1, G. Reynolds1, S. Cable1, M. - Y. Lee1, M. Delagrange1, M. Cotter1, D. Tang1, J. Reynolds1, P. Kukic1, T. Cull1, C. Thorpe1, A. Middleton1
1 Unilever, SEAC, Bedford, United Kingdom
Next Generation Risk Assessment (NGRA) is defined as an exposure-led, hypothesis-driven risk assessment approach that integrates one or more new approach methodologies (NAMs) to ensure the safety of consumer products without the use of animal testing data. The International Cooperation on Cosmetics Regulation (ICCR) principles1 were applied to a hypothetical safety assessment of 0.5% coumarin in face cream or shampoo. For the purpose of evaluating the use of NAMs, existing animal and human data on coumarin were excluded. Exposure calculations using specific consumer habits data were used to build a physiologically based kinetic model for dermally applied coumarin. For the systemic toxicity assessment, a battery of in vitro NAMs were used to identify points of departure (PoDs) for a variety of biological effects such as genotoxicity (ToxTracker®), receptor-mediated and immunomodulatory effects (Eurofins Safety44TM screen and BioSeek® Profiling, respectively), and non-specific pathways/general bioactivity [ToxCast data, in vitro cell stress panel and high-throughput transcriptomics (HTTr)]. A novel statistical Bayesian approach was applied to both the cell stress panel, HTTr and Toxcast dose-response data. The PoDs from the in vitro assays identified as demonstrating a dose response were plotted against the calculated in vivo exposure (Cmax with associated uncertainty) in order to calculate a margin of safety (MoS). From these results, we concluded that coumarin is not genotoxic, does not bind to any of the 44 receptors or shows any immunomodulatory effects. The most sensitive PoD was the No-Observed-Transcriptional-Effect-Level (NOTEL) which ranged between 2.6-12.4 µM across different cell lines (MCF7, HepG2, HepaRG). The predicted Cmax values for face cream and shampoo were lower than the all PoDs. However, the lower predicted Cmax for shampoo (0.04 µM compared with 0.4 µM for face cream) results in a MoS that can be more confidently used to assure safety. Further refinements to the risk assessment are discussed. This case study demonstrates the value of integrating exposure science with computational modelling and in vitro bioactivity data that form the basis of non-animal safety assessments.
1. Dent et al., 2018. Principles underpinning the use of new methodologies in the risk assessment of cosmetic ingredients. Computational Toxicology; 7: 20-26.
Keywords: Next Generation Risk Assessment, New approach methodologies, Cosmetics risk assessment
Development of in vitro hepatotoxicity assessment system to predict the toxicological potential of cosmetic raw materials (#372)
S. Sekine1, T. Nukaga1, 2, M. Kawaguchi2, A. Takemura2, T. Susukida2, S. Oeda1, M. Hirota1, H. Kouzuki1, K. Ito2
1 Shiseido Co., Ltd., Raw Material and Safety Assessment Group, Safety and Analytics Research Center , Yokohama, Japan
Safety assessment system of cosmetic raw materials is required after animal test of cosmetic compounds was prohibited at 2009 in Europe. It is so important to construct that the proper assessment of systemic toxicity is needed. However, liver toxic data of human is absolutely lacked and animal test of cosmetic compounds is fully prohibited, it is difficult to examine the liver toxicity risk. Clinical information is abundant in pharmaceutical products compared to cosmetic material and some mechanisms of drug induced liver injury (DILI) are reported. The combination of mitochondrial toxicity and cholestasis is useful to predict DILI risk 1). In this study, we optimized the prediction system of DILI and applied this system to predict DILI risk of cosmetic raw materials. We constructed In vitro assay system using HepG2 and sandwich cultured human hepatocytes based on 1) the toxicity caused by mitochondria dysfunction, 2) cholestasis, 3) the inhibition of bile canaliculi formation, and 4) the accumulation of lipid droplet in 55 drugs (DILI classification; most concern 19 compounds, less concern 27 compounds and no concern 9 compounds). Next, we tested ANN analysis based on in vitro assay and optimized algorithmic program to predict liver toxicity in clinical. We preliminary applied these test systems and algorithmic program to cosmetic compounds.
From the inhibitory potency of drug in these four in vitro assays, the optimal algorithm was built by using artificial neural network (ANN) technology to give the highest accuracy of DILI concerns (overall accuracy: 73%). Although we excluded the assay of intrahepatic lipid accumulation from the final algorithm to avoid “overfitting” ,the accuracy of DILI concerns prediction in the algorithm applying the ANN (overall accuracy: 62%). Moreover, among 55 drugs, there was no false predictions ("Most concerned drugs" as "No concern" and “No concern drugs” as “Most concern”) in the final algorism with three in vitro assays. In our mechanism-integrated in vitro prediction method is useful approach to predict the risk of DILI of drug candidates.
In conclusion, the combination of these cell-based assay is useful to recognize drugs classified high DILI risk. In addition, the predictability of liver injury is improved by using algorithmic program.
1) Hepatology 2015. 60: 2015-2022
Keywords: Adverse outcome pathway, Cosmetics, Alternatives to Animal tests, Systemic Toxicity, Liver toxicity
The UK Committee on Toxicity: Review of chemicals in the diets of infants and children aged 0 to 5 years (#582)
B. Doerr1, C. Tsoulli1, D. Hedley1, F. Hill1, R. Acheampong2, J. Shavila2, D. Gott1
1 Food Standards Agency, Chemcical Risk Assessment Unit, London, United Kingdom
On behalf of the Food Standards Agency (FSA, Chemical Risk Assessment Unit) and the Committee on Toxicity of Chemicals in Food, Consumer Products and the Environment (COT)
As part of an ongoing review of scientific evidence that will inform the UK Government’s updated dietary recommendations for infants and young children up to 5 years, the Committee on Toxicity of Chemicals in Food, Consumer Products and the Environment (COT) are reviewing the risks of toxicity from chemicals in the diet of this population. Parallel work considering benefits of relevant chemicals is being undertaken by the Scientific Advisory Committee on Nutrition (SACN). The chemicals assessed in 2018 include alcohol, caffeine, food additives, legacy chemicals, soya phytoestrogen, vitamin A, trans fatty acids, perchlorate, chlorate, furan and methylfurans, chromium, selenium and zinc.
The toxicity of these chemicals was reviewed along with the basis of published health based guidance values (HBGVs) or other reference values. Where applicable, exposure assessments were undertaken using UK occurrence data, either from Total Diet Studies (TDS) or Food Standards Agency (FSA) surveys and UK consumption data from the Diet and Nutrition Survey of Infants and Young Children (DNSIYC) and the National Diet and Nutrition Survey (NDNS). Calculated dietary, including breastmilk, exposures for the chemicals were either compared to the respective HBGVs or were used to calculate the margin of exposure (MOE) for risk characterisation.
The COT refers to and confirms its previous evaluations for legacy chemicals, soya phytoestrogens, vitamin A and for caffeine and alcohol in pregnant and breastfeeding women. Additives and trans fatty acids were outside the remit of the COT, exposures of chromium, selenium and zinc are not of toxicological concern. The data collected by the FSA on perchlorate and chlorate has been submitted to and forms part of EFSAs evaluations. In agreement with EFSA, the COT concluded that while there are considerable uncertainties in the assessment there is potential concern from dietary exposure to chlorate and perchlorate in infants and young children. The exposures to furan and methylfurans are of potential toxicological concern, however, there are numerous uncertainties in the assessment and the COT acknowledges that its assessment is based on worst case assumptions. Efforts to reduce concentrations of furan (and methylfurans) in the diets of infants and young children should continue.
Keywords: Infant diet, chemical risk, COT
Estimation of Acceptable Ranges of Hematological Parameters in Wistar Rats for a better understanding of Adverse and Non-Adverse Effects of Test Substances in Toxicity Studies (#680)
M. de Kort1, K. Weber2, B. Wimmer3, P. Allingham1, K. Wilutzky1, A. - L. Leoni1
1 BSL BIOSERVICE Scientific Laboratories Munich GmbH , Planegg/Munich, Bavaria, Germany
The aim of the present study was to define acceptable ranges for hematological control parameters which are essential for the evaluation of health effects and the derived impact of different test substances in toxicological studies. It can be shown that the health status of control animals after study completion could have much higher deviations than expected. Due to the differences in health status it is important to define acceptable ranges to generate a better understanding of adverse and non-adverse effects of test substances.
After generating a set of historical control data of two Wistar rat strains (RccHanTM WIST and Crl:WI(Han)) from different breeders, the data sets were statistically analyzed using minitab. As far it was feasible in a first step, outliers were identified and afterwards both data sets were compared using t-test analysis.
It was noticed that in some cases outliers can affect the set of study control data thus the respective outliers were verified based on the available histopathological findings. Several of these outliers had corresponding histopathological findings such as pulmonary or sperm granuloma, and based on these were excluded from the control data set. Comparing both data sets it can be shown that also the different methods in blood sampling and anesthesia as well as the fact that the animals were derived from different breeders result in an offset between both hematological data sets.
It can be shown that even animals from control groups, which should be healthy, could have large differences in their current health status, and can alter, due to control group comparison, the whole study outcome. By excluding all the outliers a data set from animals with a presumably good status in health was generated. The acceptable ranges were defined as mean value ± 2 standard deviations. Values which were higher or lower than the defined acceptable range therefore can indicate adverse effects of test substance exposition.
Envigo Historical Control Data of Hematological Data in HsdRccHanTM: WIST, Wistar Hannover Rats. 2018. 1-49.
Blood biochemistry and hematological changes in rats after administration of a mixture of three anesthetic agents. The Journal of Veterinary Medical Science 2018 Feb; PMID: 29249748
The impact of different blood sampling methods on laboratory rats under different types of anaesthesia. Sept.2015 Laboratory Animals Ltd. Laboratory Animals (2006) 40, 261–274
Keywords: Historical Control Data, Hematology, Toxicity Studies, Wistar Strains, Acceptable Ranges
Rat myocardium contractility changes associated with a subchronic lead intoxication (#723)
S. Klinova1, B. Katsnelson1, Y. Protsenko2, O. Lookin2, 3, A. Balakin2, L. Nikitina2, O. Gerzen2, S. Nabiev2, I. Minigalieva1, L. Privalova1, L. Katsnelson2, 3
1 The Yekaterinburg Medical Research Center for Prophylaxis and Health Protection in Industrial Workers, Yekaterinburg, Russian Federation
Chronic lead intoxication in humans is known to induce arterial hypertension and thus can lead to some secondary disturbances of the heart function. However, no proof has yet been produced to show that this intoxication affects myocardium contractility. The purpose of this work is to investigate the effect of lead intoxication on myocardial contractility.
Male outbred rats with initial body mass about 295 g were injected intraperitoneally with lead acetate (12.5 mg of Pb per kg body mass) 3 times a week for 5 weeks; the controls were receiving injections of sterile distilled water of the same volume. Cardiotoxic effects on myocardial contractility were studied by mechanical activity analysis of right ventricular trabeculae and papillary muscles isolated preparations that were put to contract in isometric, isotonic and physiological regimes of the contraction-relaxation cycle. Myocardial contractile function was also studied at the molecular level by measuring the sliding velocity of reconstructed thin filaments over myosin using an in vitro motility assay.
Rats developed an explicit, even if moderate, lead intoxication characterized by typical hematological and other impairments. Subchronic lead intoxication caused myocardial preparations of varying lengths to respond by decreased time and speed parameters of the isometric contraction while maintaining its amplitude and by a decreased passive stiffness of the trabecules. Lead intoxication led to a decrease in the maximal rate of isotonic shortening for all afterloads in papillary muscles and a decrease in thin filaments sliding velocity in in vitro motility assay. The later can be explained by the established increase of slow myosin isoforms. Muscles of the same type collected from the lead-exposed rats displayed marked changes in most of the main characteristics of afterload contrac