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Effect of L-Glutamic acid and N-acetyl cysteine on carbon tetrachloride-induced oxidative stress in rats (#14)
1 Institute of Animal Biology, Lviv, Ukraine
Carbon tetrachloride (CCl4) is one of the most widely used toxicant. CCl4-induced toxicity depending on dose and duration of exposure covers a variety of effects. The liver is especially sensitive to CCl4 since it contains a large amount of the enzymes that change the form of the chemical. The kidney and brain and other tissues of the body are also sensitive to CCl4. L-Glutamic acid (L-Glu) and N-acetyl cysteine (NAC) are antioxidants. Antioxidants play a critical role against CCl4 intoxication by scavenging active oxygen and free radicals and neutralizing lipid peroxides. These amino acids are necessary for the synthesis of key molecules, such as glutathione, which involved in process of xenobiotics detoxification in reaction of conjugation with glutathione.
The present study was carried out to evaluate the antioxidant effects of L-Glu and NAC on CCl4- induced oxidative stress in rats. Experiments were conducted on albino Wistar rats (males) weighing 200-220g. The duration of experimental period was 24 hours. CCl4 (3ml/kg) administrated intraperitoneally to all experimental groups of rats. After that rats from the second and third experimental groups intraperitoneally received an aqueous solution of L-Glu and NAC. Rats of the control group were administered by the appropriate amount of saline. Activity of some antioxidant enzymes, intensity of peroxidation processes, some biochemical indexes in various tissues and blood of rats was studied.
Activity of antioxidant enzymes decreased and level of lipid peroxidation expressed by lipid hydroperoxides significantly decreased (p≤0.05) in group of rats when CCl4 only was administered. L-Glu and NAC treatment was found to increase antioxidant enzymes activity(P<0.05) and decreased lipid hydroperoxides level. There was a difference between the CCl4 and CCl4 +L-Glu, CCl4 +NAC groups in others studied indexes.
The results obtained in this study show the protective action of L-Glu and NAC in carbon tetrachloride-induced oxidative stress in rats.
Keywords: Carbon tetrachloride, oxidative stress, antioxidants, L-Glutamic acid, N-acetyl cysteine
Mother’s residency (urban vs. rural) significantly influences newborns’ sex hormone levels, IL-6 and micronucleus frequency (#44)
A. Fucic1, M. Starcevic2, N. Sindicic Dessardo2, D. Batinic2, S. Kralik2, D. Plavec3, J. Krasic4, N. Sincic4, D. Loncarevic2
1 Institute for Medical Research and Occupational Health, Zagreb, Croatia
The association of newborn health risks due to the mother’s exposure to urban pollution has been investigated for decades but comparison of health risks with newborns whose mothers spent their pregnancy in agricultural areas is very limited. The purpose of this study was to compare for the first time IL-6, testosterone (T) and estrogen (E) levels, their ratio (E/T) and genome damage by micronucleus assay (MN) and nuclear bridge (NB) frequency between newborns born from mothers with urban or agricultural residency in order to assess the possible environmental endocrine effects and interaction between biomarkers pointing at different types of health risks. Fifty full-term newborns of both sexes, whose mothers were healthy and not occupationally exposed to any known carcinogen, were analyzed. All of the mothers filled in a questionnaire on life style, diet and residency. Multivariate analyses for dependent variables were done using generalized linear/nonlinear regression models using all effects models. Results showed significantly higher levels of E and E/T ratio in newborns of mothers from agricultural than from those born by mothers with urban residency. A lower level of E and E/T ratio was measured in newborns of mothers, who drank coffee every day, smoked and didn’t eat fish. Testosterone was significantly higher in boys of mothers with agricultural residency than from mothers with urban residency. Residence had no impact on difference in MN frequency. IL-6 levels were higher in newborns of mothers with agricultural residency but also in those who lived close to the highway. NB levels were significantly associated with E and E/T ratio levels. A significant association between E levels and IL-6 and between E and T levels was found. Our results for the first time show a significant impact of mother’s agricultural residency on sex hormones and IL-6 levels. Future research should focus on sex-specific effects of herbicide/insecticides on newborns’ immunological and endocrine status. Increased incidence of cancer and chronic diseases in agricultural areas may have origin in transplacental exposure. Acknowlegement: supported by European Regional Development Fund, Operational Programme Competitiveness and Cohesion KK.01.1.1.01.0008
Keywords: micronucleus assay, transplacental expousure; estrogen; interleukin; urban; rural
Assessment of Mitochondrial Function in Peripheral Blood Mononuclear Cells and Platelets as Potential Surrogates for Systemic Mitochondrial Perturbation. (#78)
J. Armitage1, D. Grimsditch1, G. Brunori1, S. Pearce1, R. Buckley1, A. Williams1, J. Lyon1, S. Gresham1
1 GlaxoSmithKline, Investigative Safety/Mitochondrial Toxicity, Ware, United Kingdom
Bioenergetic mitochondrial assessments are limited in in-vivo toxicity testing due to conflicting tissue requirements for mitochondrial isolation and pathology driving the need for bespoke investigative studies. Standard pre-clinical toxicity testing also relies on the use of animals which are generally young and metabolically healthy; as such they are relatively insensitive to compound-mediated mitochondrial perturbation, often remaining asymptomatic. This contrasts with patients whose metabolic capacity is often impaired by several factors; where a mild insult could result in a severe clinical effect. There are no easily accessible biomarkers to monitor compound effects on mitochondria in-vivo from a toxicology or pharmacology perspective. To compound this calcium loading and seahorse assays indicate that some in-vivo mitochondrial inhibition does not persist (or leaves an adapted phenotype) following mitochondrial isolation confusing interpretation (Broom et al 2015). To bridge this gap an exploratory study in healthy rats was conducted to assess the use of peripheral blood mononuclear cells (PBMCs) or platelets as a non-invasive method for detecting systemic mitochondrial perturbation by measuring oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) in these isolated cells. Rats were dosed with a known electron transport chain (ETC) inhibitor or a mitochondrial uncoupler (both GSK compounds terminated for mitochondrial toxicity). The compounds provide discrete (clinically relevant) mechanisms of mitochondrial toxicity resulting in differing effects on OCR in mitochondria. ETC inhibition causes: drop in OCR and ATP production, adaptive shift to glycolysis, increased reactive oxygen species (ROS), drop in body temperature (BT) and (as no alternate route to ATP production exists) when glucose and glycogen are exhausted mortality. In contrast, uncoupling dissipates the mitochondrial membrane potential (∆Ψ) by shuttling protons from the inner membrane space to the matrix causing: Maximal ETC activity in a futile effort to recover ∆Ψ, increase in OCR, drop in ATP production, shift to glycolysis and an increase in BT. As the ETC is still functional and uses all available substrates, an adequate dose results in prolonged hyperthermia and death before any substrate limitations. As circulatory cells are known to have altered energetics following activation by immune stimuli and thus potentially in response to tissue damage (Kramer et al 2014) a comparator study was performed where cells were isolated from naïve rat blood and exposed to the two compounds to explore this effect and allow comparison to the in-vivo results. These studies serve as a proof of concept regarding our ability to detect mitochondrial changes in blood cell populations; that could provide a non-invasive route to assessing mitochondrial function in routine in-vivo toxicity studies for new chemical entities where mitochondrial function is of interest.
Keywords: mitochondrial function, mitochondrial toxicity testing, electron transport chain inhibition, mitochondrial uncoupling
Intra-erythrocyte chromium as an indicator of exposure to hexavalent chromium: in-vivo evaluation. (#159)
J. Devoy1, F. Cosnier1, E. Bonfanti1, G. Antoine1, H. Nunge1, M. J. Decret1, L. Douteau1, M. Lorcin1, S. Sebillaud1, S. Grossmann1, S. Michaux1, S. Viton1, C. Seidel1, L. Gate1
1 INRS, Toxicology Department, Vandoeuvre les Nancy, France
Thousands of employees are potentially exposed to hexavalent chromium (Cr6) which is carcinogenic to humans.
There is currently no Cr6 specific biological exposure marker. Although considered the most reliable biomarkers, the blood and urinary chromium concentrations are not specific for Cr6 exposures. A previous in vitro study conducted on human blood samples has demonstrated that intra erythrocyte chromium (CrIE) is a specific indicator of the Cr6 exposure. However, due to insufficient data, this assay cannot be proposed to the hygienists in routine.
This work aims to provide additional in vivo data in rat (useful for the improvement of PBPK models and the extrapolation across species for use in risk assessment) regarding the comparative kinetics of incorporation and elimination of Cr6 in erythrocytes, plasma and urine.
Male Sprague-Dawley rats were iv injected with Cr6 and/or Cr3 solutions made from ammonium dichromate ((NH4)2Cr2O7) or chromium chloride (CrCl3) dissolved in saline. Three doses of Cr6 (corresponding to 0.13, 1.31 and 2.62 mg of Cr6 per kg of rat), one of Cr3 (corresponding to 1.31 mg Cr3 / kg rat) and one mixture Cr6/Cr3 (each at 1.31 mg / kg rat) were thus administered. Control groups were administered with saline solution only. Blood and urine were collected at different time points (until 48 h and day 90 for urine and blood, respectively).
Erythrocytes selectively incorporate Cr6 at the expense of Cr3 and Cr3 has no effect on Cr6 incorporation into erythrocytes. The Cr6 incorporation into the erythrocytes is rapid (less than 10 min to reach the maximum) and the Cr remains trapped in the erythrocytes for a few days (quite stable for 2 days and 62% of the initial concentration in CrIE after 5 days). In addition, CrIE concentration is proportional to the amount of Cr injected. By way of comparison, the CrU concentration reaches a maximum 6 h after injection then returning to the threshold level in less than 24 h.
These results confirm the relevance of CrIE as a specific indicator of recent or older exposures to Cr6. Since the life expectancy of human erythrocytes is longer than those of rat (120 days / 60 days), a higher accumulation capacity and a slower elimination can be expected in human. Samples taken at the beginning and end of the workshift week could allow a good evaluation of the recent exposure to Cr6.
Keywords: erythrocyte, hexavalent, chromium, biomonitoring
Cell-free, circulating microRNAs reflect air pollution-induced environmental health risks (#163)
J. Krauskopf1, K. van Veldhoven2, 3, M. Chadeau-Hyam2, R. Vermeulen4, G. Carrasco-Turigas5, 6, M. Nieuwenhuijsen5, 6, P. Vineis2, T. M. de Kok1, J. C. S. Kleinjans1
1 Maastricht University, Toxicogenomics, Maastricht, Netherlands
The WHO estimated that worldwide more than 80% of the people in cities are exposed to air pollution levels that exceed the air quality limits. A large source of air pollution originates from traffic emission which consists of a complex mixture of compounds that contributes to the pathogenesis of many diseases. In search of an early diagnostic biomarker for improved environmental health risk assessment, recent studies have shown that certain microRNAs (miRNAs) are responsive to exposure to traffic-related air pollution (TRAP).
Here, we present a genome-wide analysis of cell-free, circulating miRNAs (cimiRNAs) in a human healthy population exposed to different levels of TRAP. The cross-over study included blood sampling from 24 volunteers after 2 hours of resting or intermittently cycling at high and low TRAP exposure sites (4 scenarios per volunteer) in Barcelona, Spain. Real-time exposure of particulate matter (PM10, PM2.5, UFP and black carbon), nitrogen oxides (NO, NO2) and carbon oxides (CO, CO2) were measured during each intervention. Furthermore, next-generation sequencing analysis was used to quantify global cimiRNA levels across all subjects.
Associations between TRAP levels and cimiRNA levels were evaluated using multivariate normal models (False discovery rate <= 0.1). We identified 8 cimiRNAs to be associated with the mixture of TRAP and 27 cimiRNAs that were specifically associated with the individual pollutants NO, NO2, CO, CO2, BC and UFP. We did not find significant associations between cimiRNA levels and PM10 or PM2.5.
Bioinformatics analysis revealed potential molecular mechanisms by which these cimiRNAs can target complex regulatory networks that are implicated in the development of major air pollution-induced diseases. These networks included among others the hub genes TP53, VEGFA, IL6 and PTEN which have known roles in the pathogenesis of diseases such as lung cancer, asthma as well as multiple cardiovascular and neurodegenerative diseases. Further mechanistic studies are needed to confirm the regulatory roles of these cimiRNAs; however, this study presents a new avenue through which TRAP potentially induces human health effects. Furthermore, it provides novel evidence for the potential of global cimiRNA profiles to be used in biomarker based environmental health-risk assessment.
Keywords: microRNAs, health-risk assessment, air pollution, particulate matter, traffic pollution
Alcohol induced changes in the serum and placental metabolome during pregnancy (#184)
O. Kärkkäinen1, A. Lehikoinen2, J. Repo1, M. Lehtonen1, S. Auriola1, S. Heinonen3, K. Vähäkangas1, K. Hanhineva1
1 University of Eastern Finland, Kuopio, Finland
Alcohol use during pregnancy is the leading preventable cause of developmental disability in children. Understanding the changes in the metabolome due to alcohol during pregnancy will enable to identify sensitive biomarkers of prenatal alcohol exposure and to find possible novel targets for treatment. Clinical value is obvious, since fetal alcohol spectrum disorders are under diagnosed conditions worldwide and no treatment options exist for prevention or alleviation of the symptoms.
We have analyzed first trimester serum samples from alcohol users (n=19), drug users (n=24), tobacco smokers (n=40) and controls (n=55), and placental samples from alcohol exposed (n=6) and control (n=6) pregnancies using untargeted liquid chromatography mass spectrometry based metabolomics. Samples were collected during routine clinical visits and used after an informed consent was gained from the mothers.
Increased levels of glutamate and decreased levels of glutamine and serotonin were associated with alcohol use during pregnancy in the first trimester serum samples. Furthermore, we found that in the placental tissue, alcohol use during pregnancy was associated with altered phospholipid levels. Especially the levels of phosphatidylethanolamines were increased in the placentas by alcohol.
These results give insight to the pathological processes caused by prenatal alcohol exposure, especially in the placenta. Furthermore, these results show that metabolomics can be used to pursue biomarkers of alcohol exposure during pregnancy. Especially placenta seems to be very interesting tissue for this purpose.
Keywords: alcohol, metabolomics, pregnancy, placenta, metabolome
Predictive toxicogenomics space modeling serves effectively to sensitive biomarker-based read across from capturing toxic mode-of-action of lowest-observable effect levels (#214)
P. J. Kohonen1, 2, P. Nymark1, 2, V. Hongisto2, R. Grafström1, 2
1 Karolinska Institutet, Institute of Environmental Medicine, Stockholm, Sweden
The predictive toxicogenomics space (PTGS) concept was previously developed for predicting cellular toxicity and organ pathology from analyzing transcriptomics data generated in cell culture experiments (Kohonen et al., Nat Commun, 2017). Directed initially towards human drug-induced liver injury, the 14 gene component-based PTGS proved to effectively capture pathological states from analyzing human and rat hepatocyte data. Our extended work provides now a standardized PTGS-based biomarker analysis protocol that couples compound grouping and read-across with defining first the lowest-observable effect levels and toxic mode-of-action (MoA) to component or gene level. Scoring of microarray or RNA-seq data applying the U.S. EPA BMD Express 2.2 software identify initially points of departure (POD) biomarkers that are up to 100-fold more sensitive than previously identified biomarkers. The POD data in turn serves to connect the lowest observable toxic effect levels of agents under study to chemicals and drug molecules present in the Connectivity Map or the Library of Integrated Network-based Cellular Signatures perturbation classes. The novel PTGS-based protocol permits ab initio toxicity prediction to biomarker level of any agent coupled to potency, MoA and biological read-across data. Coupling of the biomarker data to key events in adverse outcome pathways is then a further dimension. Overall, quantitative PoD-focused biomarker discovery is bound to increase the applicability of in vitro and in silico-based data modeling for replacement of animal experiments in toxicity testing.
Keywords: toxicogenomics, DILI, mode-of-action, benchmark dose, read-accross
Assessing Aflatoxin B1 exposure in humans by measuring Aflatoxin M1 in urine (#267)
R. Ortiz-Martinez1, M. C. De Luna-Lopez1, A. G. Valdivia-Flores1, T. Quezada-Tristan1
1 UNiversidad Autonoma de Aguascalientes, Centro de Ciencias Agropecuarias, Aguascalientes, Mexico
In humans, as well as in several other species; toxicology uses biomarkers as predictive tools to detect early exposure or damage caused by chronic exposure.
A biomarker is a measurable variation in the cellular or biochemical components, processes, structures or functions in a biological system or samples; these changes are induced by xenobiotics
After the ingestion of AFB1 (AFB1) by human or animals, a metabolite called aflatoxin M1 (AM1) is produced and subsequently eliminated through urine and milk. In fact, AFM1 is a linear biomarker to estimate AFB1 exposure.
The main objective of the study was to assess AFM1 concentration in urine of a group of volunteers and relate it to previous AFB1 exposure.
A convenience sampling (non-probabilistic sampling), was used in Aguascalientes, Mexico. In the study both female and male individuals (≥18 years old) were included. The participants in the research signed informed consent.
The samples were analyzed in accordance to the directions specified on a commercial ELISA kit (detection range: 0–4.0 ng/mL; specificity and sensitivity: 100%).
The study was carried out over a 2-year period. During this time, AFM1 determination was performed in 660 urine samples.
Main results indicate that 46% male and 54% female were included in the study. The average age of volunteers was 20 year-old. Sixty percent of the samples had detectable levels of AFM1. The concentration of AFM1 in urine samples ranged from 0.15 to 0.40 ng/mL. The highest AFM1 concentrations were found on the > 45 years-old volunteer group.
The results indicated an unnoticed AFB1 exposure that may cause subsequent damages, even cancer.
Gupta R, 2014. Biomarkers in Toxicology. Academic Press, Elsevier. San Diego, CA. USA.
Atongbiik M, Opoku N, Kweku F. 2017. Aflatoxin contamination in cereals and legumes to reconsider usage as complementary food ingredients for Ghanaian infants: A review. Journal of Nutrition & Intermediary Metabolism. 10:1-7.
Gurbay A, Sabuncuoglu SA, Girgin G, Sahin G, Yigit S, Yurdakok M, Tekinalp G, 2010. Exposure of newborns to aflatoxin M1 and B1 from mothers breast milk in Ankara, Turkey.Food Chem Toxicol. 48(1):314-9.
Jager AV, Tonin FG, Souto PCMC, Privatti RT and Oliveira CA. 2014. Determination of Urinary Biomarkers for Assessment of Short-Term Human Exposure to Aflatoxins in São Paulo, Brazil. Toxins 2014, 6, 1996-2007.
Keywords: biomarkers, AFB1, AFM1
Regioselective synthesis of neoeriocitrin dihydrochalcone from naringin dihydrochalcone by Bacillus megaterium CYP102A1 and its effects on human cytochrome P450 activities (#273)
H. T. H. Nguyen1, C. - H. Yun1
1 Chonnam National University, Biological Sciences and Biotechnology, Gwangju, Republic of Korea
Naringin dihydrochalcone (naringin DC) is well-known as an artificial sweetener with a strong antioxidant activity, that has potential applications in food and pharmaceutical fields. It is originally derived from the flavonoid naringin which occurs naturally in citrus fruits, especially in grapefruit. Naringin DC is a glycoside of phloretin which shows an inhibitory effect on active transport of glucose into cells by SGLT1 and SGLT2. It was suggested that naringin DC might be a potential therapeutic agent for the treatment of AD against multiple targets that include Aβ pathology, neuroinflammation and neurogenesis. A large set of natural compounds and their metabolites are known to effect on the catalytic activities of human cytochrome P450 enzyme, which are the major metabolizing enzymes. In this study, we have tried to find an enzymatic strategy for the efficient synthesis of potentially valuable metabolites from naringin DC. Effects of the naringin DC and its metabolites on P450 activities were studied. A set of Bacillus megaterium CYP102A1 variants was used to find efficient regioselective hydroxylases toward naringin DC. Human liver microsomes and recombinant human P450s were used to make metabolites of naringin DC. We found three highly active CYP102A1 variants to hydroxylate naringin DC among wild type (CYP102A1) and its 60 variants. Highly active variants produced one major metabolite and its chemical structure was determined by LC/MS and NMR. The major metabolite is neoeriocitrin dihydrochalcone (neoeriocitrin DC), which has a catechol structure of naringin DC. Inhibitory effects of the naringin DC and its metabolites on human P450 catalyzed reaction were observed. The synthesis of neoeriocitrin DC from naringin DC has been achieved by using biocatalytic strategy of CYP102A1 enzyme with highly efficient yields. At present, as neoeriocitrin DC is not commercially available, its biological functions have not been studied. This result suggests that neoeriocitrin DC can be used for further biological studies at the levels of cells and animals. Consumption of the naringin DC should be considered as a factor for the drug-drug interactions as the naringin DC show inhibitory effects P450 activities. Here, we reported an efficient synthesis of neoeriocitrin DC from naringin DC by using CYP102A1 and inhibitory effects of naringin DC on P450 activities were shown.
Keywords: Naringin dihydrochalcone, CYP102A1
Results from the Norwegian human biomonitoring study in the EuroMix project: Exposure to the pesticides boscalid and imazalil from the diet in Norway (#282)
H. Dirven1, F. Sonnet1, A. K. Sakhi2, C. Thomsen2, T. Husøy1
1 Norwegian Institute of Public Health, Department of Toxicology and Risk Assessment, Oslo, Norway
Background: The fungicides boscalid and imazalil were among the most frequently detected pesticides in the residues monitoring programs 2013-2017 in Norway.
The aim of the present study was to estimate the daily intake of these two pesticides and compare with measured concentrations in 24 h urine samples.
Methods: A human biomonitoring study was performed to study the exposure to chemicals present in food and personal care products (PCPs). In two 24 h periods two-three weeks apart, 144 participants (100 women and 44 men) kept detailed weighted food diaries and PCP diaries and collected all urine excreted. Individual-specific consumption data from both 24 h periods were used to estimate boscalid and imazalil exposure deterministically. A sensitive ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS-MS) method was developed to measure the boscalid metabolite 2-chloro-N-(4’-chloro-5-hydroxybiphenyl-2-yl)nicotinamide (M510F01) and imazalil in the 24 h urine pools collected at Day 1.
Results: Overall, the estimated dietary exposure of boscalid and imazalil was comparable between males and females. In the lower bound exposure scenarios, the estimated dietary exposure of boscalid ranged from 0-0.9 µg/kg bw/day and the estimated exposure of imazalil ranged from 0-0.81 µg/kg bw/day.
In 99 % of the samples M510F01was detected in concentrations from 0.04-15.03 ng/ml. There was a statistically significant difference between genders (P<0.0001) with a median concentration of 0.98 ng/ml for females, and 0.46 ng/ml for males. Imazalil was detected in 1 % of the samples. One of the reasons for the low detection of imazalil in urine samples could be the choice of the biomarker. Comparisons with estimated exposure levels for both boscalid and imazalil will be presented.
Conclusion: Widespread human exposure to the fungicide boscalid as measured by one of its metabolites in urine samples was observed.
Keywords: Pesticides, exposure, diet, biomonitoring, Boscalid
Dietary exposure to phthalates in the European population from infants to the elderly (#309)
C. Cascio1, K. Volk2, L. Castle3, C. Tlustos4, F. Poças5, D. Arcella1
1 European Food Safety Authority (EFSA) , Evidence Management Unit (DATA), Parma, Italy
Exposure assessment is one of the four pillars of chemical risk assessment carried out in EFSA. Exposure assessment methodologies can differ from one field to the other and this is of relevance when considering chemicals that are ubiquitous (such as phthalates) in different matrices and can contribute to an aggregate exposure. Several options are available to carry out exposure assessment, starting from crude conservative estimates following a tiered approach to refined exposure assessments based on individual food consumption data. EFSA selects the best approach on a case by case basis to guarantee the protection of EU citizens. Recently, EFSA received a mandate to update its 2005 risk assessments of five phthalates (1-5) which are authorised in the EU for use in plastic food contact materials: di-butylphthalate (DBP), butylbenzylphthalate (BBP), di(2-ethylhexyl)phthalate (DEHP), di-isononylphthalate (DINP) and diisodecylphthalate (DIDP). Dietary exposure (mean and 95th percentile) was estimated for different age groups from infants to the very elderly across 22 European countries by combining literature occurrence data with individual consumption data from the EFSA Comprehensive Food Consumption Database. Exposure estimates were assessed for the 5 phthalates individually and also as a group since some of them were placed into a Cumulative Assessment Group on the basis of co-exposure and due to sharing a common mode of action for toxicity. Data and methodology adopted to assess chronic dietary exposure to the named phthalates will be presented along with key results (6). A comparison of results with reported exposure estimates obtained using other methodologies (such as biomonitoring and total diet studies) and the uncertainties related to the approach used will also be discussed
Keywords: Exposure Assessment, Diet, Risk Assessment, Phthalates
Hallmarks of ageing are interconnected in placental tissue and influenced by particulate air pollution exposure during pregnancy (#316)
B. Janssen1, D. Martens1, W. Lefebvre2, C. Vanpoucke3, K. Vrijens1, T. Nawrot1, 4
1 Hasselt University, Centre for Environmental Sciences, Diepenbeek, Belgium
Observations from experimental studies have put forth a “core axis of ageing” involving telomeres, mitochondria, tumour suppressor gene p53 (TP53), and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PPARGC1A). In this study, we hypothesized that these hallmarks of ageing in placental tissue are interconnected and influenced by early-life ambient air pollution exposure during pregnancy.
In 680 newborns of the ongoing ENVIRONAGE birth cohort, we measured protein levels of TP53 and PPARGC1A in cord plasma and telomere length and mitochondrial DNA (mtDNA) content in placental tissue. Daily ambient particulate matter with a diameter less than 2.5 μm (PM2.5) was calculated for each participant’s home address using a spatial-temporal interpolation model in combination with a dispersion model. The associations between prenatal PM2.5 exposure and specific hallmarks of ageing were analysed with linear regression models, while accounting for covariates and potential confounders.
PM2.5 exposure averaged (SD) 13.5 µg/m3 (2.5) over the entire pregnancy period. A 5-μg/m3 increment in PM2.5 exposure during the 3rd trimester was associated with 13.2% (95%CI, −19.3% to −6.7%) shorter placental telomere length, 11.2% (95% CI: -4.1 to -17.7) lower placental mtDNA content, and 7.4% (95% CI: 2.1 to 13.0%) higher TP53 protein levels. Telomere length and mtDNA content were linked [a 10% shorter telomere length was associated with a 4.8% (95%CI: 3.6 to 6.1%) lower mtDNA content], and we observed a negative trend between TP53 protein levels and telomere length (p = 0.08). PPARGC1A protein levels were not associated with mtDNA content.
Prenatal air pollution is associated between candidate hallmarks of ageing (telomeres, mitochondria, TP53) in placental tissue. This is the first observational study demonstrating some degree of interconnectedness between master regulators of the molecular circuit linking PM-induced telomere damage and compromised mitochondrial biogenesis.
Keywords: particulate air pollution, placental tissue, mitochondria, telomeres, ageing
Selection process of relevant quantity data for the safety assessment of cosmetic products (#323)
A. Bernard1, F. Alby1, I. Laffont1, T. Cazard1, M. - P. Gomez-Berrada1, P. - J. Ferret1
1 Pierre-Fabre Dermo-Cosmetique, Safety assessment and cosmetovigilance department, Toulouse, France
For a few years, papers on consumption of cosmetic products have been increasingly present in the scientific literature. Thus, the original problem of lack of data is gradually being replaced by choosing the most relevant data to be used for exposure assessment. The aim of this work was to develop a method to select quantity data of cosmetic products applied by consumers, to be used in the Margin of Safety calculation.
The method was based on a scoring of published studies. First, each study was analyzed according to 10 parameters defined as follows: 4 parameters assessing the study in its entirety (year, duration of exposure, statistical method, data homogeneity), 4 parameters assessing the data collection method (supervision, weighting of the product, instruction of use, personal product) and 2 parameters assessing the panel (size and geographical area of the population). Depending on its relevance level, each parameter was given a score of 10, 100 or 1000. Then scores obtained were weighted according to the importance of each parameter in order to choose the most realistic amount data. Different weighting factors were used, from 1 for the most important parameter to 9 for the less important. Finally, the overall score of the study was calculated by adding all the weighted scores.
Because no reference guideline is currently available for cosmetic products intended for babies, we used this method to determine the most relevant quantities to be used in safety assessment. Thanks to a previous work (Ficheux et al., 2019) 8 studies were identified. As a result, this process led to the selection of the most relevant quantity data specific to European babies for 5 categories of products and specific to Asian babies for 3 categories of products. This method is going to be applied to other cosmetics such as sunscreen products.
This process allows the selection of the most relevant amount data based on recent consumption studies for specific populations. It can be useful as a new tool to choose more realistic data, especially when the daily amount proposed by the Scientific Committee on Consumer Safety (SCCS, 2018) is not representative of specific population as it is the case for babies.
Ficheux et al., 2019. Consumption and exposure to finished cosmetic products: A systematic review. Food Chem Toxicol 124, 280-299.
SCCS, 2018. The SCCS Notes of Guidance for the Testing of Cosmetic Ingredients and their Safety Evaluation, 10th revision. SCCS Notes of Guidance.
Keywords: Quantity data, selection method, cosmetic product, safety assessment
Effects of sterigmatocystin on antioxidative enzymes and expression of Hsps in male Wistar rats (#328)
M. Peraica1, D. Rašić1, A. Hulina2, V. Micek3, D. Jakšić4, L. Rumora2, M. Šegvić Klarić4
1 Institute for Medical Research and Occupational Health, Toxicology Unit, Zagreb, Croatia
Sterigmatocystin (STC), a precursor of aflatoxin B1, is mycotoxin which International Agency for Research on Cancer evaluated as Group 2B. STC was isolated in 1954 from Aspergillus versicolor and afterwards from other Aspergillus species producing it as aflatoxin B1 precursor or as final mycotoxin. Due to its structural similarity to carcinogen AFB1, STC carcinogenic potential was studied much more than its toxicity. The aim of this study was to evaluate the effect of STC on antioxidative enzymes and heat shock proteins (Hsp 70 and Hsp27) as parameters of oxidative stress. Male Wistar rats (N=5 per group) were treated with single oral STC doses of 1/16, 1/8, and 1/4 of LD50 (10, 20 and 40 mg kg-1 b.w.). Control group was treated with corn oil which was used as STC vehicle. Catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity in plasma, kidneys and liver were measured on a plate reader using commercial kits. Expressions of heat shock proteins Hsp70 i Hsp27 were measured in kidneys and liver using Western blot methods. CAT activity did not change in organs of treated animals. GPx activity was significantly lower in liver of rats treated with 1/8 and 1/4 LD50. Activity of SOD was significantly increased in kidneys of rats given 1/16 and 1/4 LD50. Expression of Hsp70 was significantly increased in liver samples of rats treated with 1/16 LD50; in kidneys the same dose augmented expression of Hsp70 but without significant difference to control group. Highest STC dose returned Hsp70 expression to control values. Expression of Hsp27 in liver and kidneys was not significantly changed at any STC dose. Taken together our findings suggest that acute oral exposure to STC causes oxidative stress both in liver and kidneys.
This work has been fully supported by the Croatian Science Foundation under the project MycotoxA (HRZZ-IP-09-2014-5982).
Keywords: catalase, glutathione peroxidase, heat shock proteins, superoxide dismutase
Combinatorial Effects of Pesticides on Toxicologically Relevant Liver Proteins in HepaRG Cells (#339)
F. F. Schmidt1, 3, A. Steinhilber3, A. Mentz4, J. Kalinowski4, D. Lichtenstein2, P. Marx-Stoelting2, A. Braeuning2, A. Lampen2, T. Joos1, O. Pötz3, 1
1 Natural and Medical Sciences Institute at the University of Tübingen, Protein Analytics, Reutlingen, Baden-Württemberg, Germany
Based on the steady increase of the world’s population, the protection of plants and crops is essential. To yield sufficient food supplies, pesticides and biocides are widely used in agriculture. Everyday new substances get market approval and therefore the development of novel in vitro methods for the detection of potential cumulative effects are required, since the reduction of animal testing is worth striving for.
On the basis of mRNA expression analysis, a selection of important toxicologically relevant liver proteins was made and analyzed with mass spectrometry- (MS) based immunoassays to investigate potential mixture effects of pesticides. The workflow includes a tryptic proteolysis to yield proteotypic peptides of each analyte and an immune enrichment by use of Triple X Proteomics-(TXP) antibodies, which recognize short C-terminal epitopes. The analysis was performed in targeted parallel reaction monitoring (PRM) mode on an ultra-high performance liquid chromatography- mass spectrometry (UHPLC-MS) device. Quantification of the target analytes was done by use of stable isotopically labeled standard peptides. This project focused amongst others on toxicologically relevant proteins like cytochrome P450 enzymes (CYPs, phase I), UDP-glucuronosyltransferases (UGTs, phase II), as well as transporters (phase 0 & III).
As a well-established human hepatocyte system, HepaRG cells were used for the investigation of single and combinatorial effects of pesticides and biocides. 27 proteins were analyzed quantitatively in cells with 30 different single pesticides after 24 hours of treatment. For instance, induction effects were observed for CYP1A1, CYP1A2, CYP3A4, TNFRSF12A and S100P. Based on these results, substances were grouped according to their protein expression profile similarities in very weak, weak, moderate and very strong correlation (Pearson). Four mixtures were generated and HepaRG cells were treated for 24, 48 and 72 hours and analyzed afterwards. Combinatorial effects (additive effects) were observed for several analytes after mixture treatment.
Weiss, F., et al. Scientific Reports, 2015.
Marx-Stoelting, P., et al. Archives of Toxicology, 2017.
Weiss, F., et al. Drug Metabolism and Disposition, 2018.
Keywords: Pesticide mixtures, Combinatorial effects, Biomarkers, Mass spectrometry-based immunoassays, Proteomics
GvHD: Non-Clinical Findings in the Development of CAR-T cells Projects (#346)
G. Di Gallo1, M. Magistrelli1, G. Pennella1, L. Mancini1, M. Russo1, E. Giannotti1, C. I. Bernardi1
1 Accelera Srl, Nerviano - Milan, Italy
Activation of the immune cell response targeting specific antigens and gene therapies are currently amongst the innovative and most promising frontiers for the care of hematological malignancies. In this context, ex-vivo gene modified autologous or allogeneic cells obtained and manufactured from human Peripheral Blood Mononuclear Cells (PBMC) demonstrate significant response in the treatment of different leukemia.
One of the major clinical complication (and limitation) of human cells administration in cancer patients is Graft versus Host Disease (GvHD) and different strategies have been developed to decrease this adverse reaction. The preclinical program should therefore take into careful consideration the different claims to properly evaluate the effects observed in preclinical species.
Based on the peculiarity of this therapeutic approach, customized preclinical safety programs have to be properly designed to at least identify the bio-distribution of the therapeutic cells, their persistency, and any adverse effects of cell administration. Particularly, the effect of lymphoid cells, presenting different modifications or obtained through different culture programs, to the host organism need to be investigated taking into account the different endpoints, while managing technical limitations of the animal models. The reaction of the exogenous cells versus the host environment and their relevance and predictivity for the clinical use has to be taken into account.
The experimental model and study plans used with different CAR-T cells projects and the effects observed with unmodified or mock cultured cells are presented, including bio-distribution and persistence evaluated through different bioassays (example flow cytometry, PCR technologies, immunohistochemistry, etc).
Clinical signs and histopathological findings from animals receiving unmodified cells and suggestive of immunological reactions following treatment with different cells preparations will be reported and compared to indicative changes observed in GvHD in models of animal disease.
Keywords: CAR-T cells, GvHD, Cell therapy
Diesel exhaust particle-altered inflammatory gene expression in alveolar macrophage cells relevant for lung toxicity (#373)
D. I. Kim1, M. - K. Song1, 2, H. - S. Yang1, K. Lee1, 2
1 Korea Institute of Toxicology, National Center for Efficacy evaluation for Respiratory disease product, Jeongeup, Republic of Korea
Many epidemiological and animal studies have shown that particulate matter 2.5 (PM2.5) is associated with lung injury via induces the production of inflammatory cytokines, the generation of reactive oxygen species, and alteration in macrophage polarization. However, studies on the relationship between PM2.5 and the inflammatory response in alveolar macrophages are still unclear.
In this study, we used gene expression profiling and gene ontology (GO) analysis to investigate whether diesel exhaust particle (DEP), one of main PM2.5 occurred from motor vehicles in urban enhances the inflammatory response through increasing the expression of cytokines and chemokines in alveolar macrophage (AM) cells.
The gene expression profiles in murine AM (MH-S) cells following 3 hrs exposure to 100 μg/ml DEP were investigated using RNA-Seq analyses. A combination of fold change ≥1.5 and p value <0.05 was used to define differentially expressed genes (DEGs). Overrepresentation of gene ontology terms representing biological processes and signaling pathway analysis was performed with bioinformatics tools (ExDEGA software, Ingenuity Pathway Analysis(IPA) and DAVID functional annotation tool).
The expression of 192 and 79 genes was up- and down-regulated >1.5-fold (p <0.05), respectively, after DEP exposure. GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of these genes revealed significant enrichment in several key biological processes related to inflammatory response, TNF signaling pathway and cellular response to IFN-gamma. Genes related to these functions, such as CIITA, CCL2, CSF1, CXCL2, ANXA1, CX3CL1, TLR5, NLRP3 and AGER have the ability to elicit local and systemic inflammatory response. Also, through a comparative toxicogenomics database (CTD) analysis, we determined that 17 genes are linked to respiratory tract disease.
DEP exposure modulates expression of cytokines and chemokines in alveolar macophages important in the development of lung injury. This suggests that alveolar macrophage-mediated inflammation may contributes to PM2.5-induced lung damage and that the inflammatory genes expressed should be studied in detail, thereby laying the groundwork for development of novel therapeutic targets for PM2.5-induced lung injury.
* This work was supported by Korea Institute of Toxicology (KK1905-02).
Keywords: diesel exhaust particle, gene expression, RNA sequencing, gene ontology, inflammation
Multiplex miRNA Profiling for Biomarker Discovery and Verification Studies Using the FirePlex® Platform (#406)
M. Tackett1, B. Heinrich1, I. Diwan1, G. Tejada1, C. Rafferty1, E. Atabakhsh1, D. Pregibon1
1 Abcam Inc, Cambridge, Massachusetts, United States of America
We have developed the FirePlex® Technology Platform to address the need for rapid and sensitive biomarker quantitation. Utilizing patented FirePlex hydrogel particles and a three-region encoding design, FirePlex assays allow for true, in-well multiplexing, providing flexible and customizable analyte quantification.
To facilitate miRNA biomarker discovery studies, we offer our standard FirePlex miRNA assays, for quantitation of 5-400 miRNA targets per sample and data acquisition on standard flow cytometers. For miRNA screening studies requiring faster workflows, we offer our high-throughput miRNA assays (miRNA-HT). The high-throughput assays allow for quantitation of 5-36 miRNA targets per sample and assay readout rapidly conducted on high-content imagers.
FirePlex miRNA assay combines particle-based multiplexing with single step RT-PCR signal amplification using universal primers. Thus, these assays leverage PCR sensitivity while eliminating the need for separate reverse-transcription reactions and mitigating amplification biases introduced by target-specific qPCR. Assy sensitivity is ~1000 miRNA copies per sample, with a linear dynamic range of ~5 logs. Assays can be performed without the need for RNA purification, making the FirePlex ideally suited for profiling in serum, plasma, exosomes, cell culture supernatants, urine, and directly from FFPE and tissues. The ability to multiplex targets in each well eliminates the need to split valuable samples into multiple reactions. Results are displayed and interpreted using the integrated, free-of-charge FirePlex Analysis Workbench.
Panels are available for biomarker discovery studies, as well as for specific research areas of interest. We also provide the option to design fully customizable miRNA panels for any sequence, from any species, at no additional cost.
Here we present the data from several studies investigating circulating miRNA profiles, as well as miRNA profiles obtained directly from FFPE tissues, using the FirePlex miRNA Assay Panels. Together, this novel combination of bioinformatics tools and multiplexed, high-sensitivity assays enables rapid discovery and verification of miRNA biomarker signatures from biofluid samples.
Keywords: biomarker, miRNA, quantitation, multiplex, profiling
Exposure of pregnant women to body moisturizer and anti-stretchmark care (#407)
T. Nguyen1, M. - P. Gomez-Berrada1, A. Bernard1, A. Rielland2, M. Bellec2, D. De Javel2, P. - J. Ferret1
1 Pierre Fabre Dermo Cosmetics, Safety Assessment Department, Toulouse, France
The European Regulation (EC) N°1223/2009 on cosmetic products defines pregnant women as a vulnerable consumer group (EU, 2009). Thus, a specific risk assessment with accurate exposure data is required. However, exposure values from the Scientific Committee on Consumer Safety guidelines (SCCS, 2018) do not consider pregnant women and little information is available in the literature. The aim of this study was to obtain consumption and real-life exposure values of two personal care products commonly used by pregnant women, body moisturizer and anti-stretchmark care.
The study was conducted on 43 French pregnant women enrolled thanks to a previous study on usage patterns of personal care products. The mean age of the subjects was 31 years old, 47.7% of them were at their 2nd trimester and 50% at their 3rd at the inclusion. The participants used their own product, either a body moisturizer or anti-stretchmark care or both, over a 3-week period according to their personal habits. To assess the exposure, products were weighed with a precise balance at the beginning and end of the study and the pregnant women were asked about their weight. Furthermore, the subjects recorded each application and the body areas where the products were applied in a follow-up form. Distribution data were generated with @Risk software.
Among the subjects, 24 were users of body moisturizer and 35 of anti-stretchmark care. 16 subjects used both products. The 90th percentile of daily frequency of use, amount and exposure were 1.46 use/day, 5.37 g/day and 84.63 mg/kg(bw)/day for body moisturizer and 1.83 use/day, 3.97 g/day and 60.91 mg/kg(bw)/day for anti-stretchmark care. The women mostly applied the body moisturizer on their legs (80% of the users), thighs (68%) and arms (52%) and the anti-stretchmark care on their belly (100%) and chest (57%).
This study provides actual exposure values and describes the consumption behavior of pregnant women for body moisturizer and anti-stretchmark care which could serve as a basis for the risk assessment.
EU, 2009. Regulation (EC) n°1223/2009 of the European Parliament and of the Council of 30th November 2009 on cosmetic products.
SCCS, 2018. The SCCS notes of guidance for the testing of cosmetic ingredients and their safety evaluation, 10th revision, SCCS/1602/18.
Keywords: pregnant women, cosmetic product, exposure, risk assessment
Optimization of a 5-Fluorouracil-Induced Intestinal Injury Model in Mice to Construct a Multi-Scale Predictive Model of Drug-Induced Intestinal Toxicity (#413)
F. Jardi1, M. Van Heerden1, L. Vereyken2, T. Erkens1, D. F. Rodrigues3, J. C. S. Kleinjans3, T. M. de Kok3, S. Ferreria4, I. Gardner4, C. Fisher4, M. Pritchard5, A. Lynch6, D. Sevin6, K. Beattie6, C. Pin7, L. Lammens1, S. de Jonghe1
1 Janssen, Toxicology, Pathology & LAM, Beerse, Belgium
Pharmaceutical industry faces an urgent need to improve early safety evaluation of new drug candidates. Computational systems toxicology could help identifying compounds with an acceptable safety profile in the clinic. However, its implementation within the drug discovery and development pipeline is still in its infancy. Our goal within the IMI project Translational Quantitative Systems Toxicology is to build a multi-scale predictive model of drug-induced gastrointestinal (GI) toxicity. To this end, we require new experimentation in clinically relevant animal models of GI toxicity - meeting both functional and phenotypic endpoints - that includes drug-response multi-omics profiles to identify the mechanisms underlying intestinal toxicity. Here, we describe a mouse model of 5-fluorouracil (5-FU)-induced GI injury that captures the pathophysiologic dynamics of the epithelium following exposure to chemotherapy. C57BL/6J male mice were treated with 5-FU at 20 or 50 mg/kg BID by IV bolus injection via a jugular vein catheter for 6, 24 or 96 h. The healing phase of the process was assessed in animals euthanized after a 48-h recovery period. Mice treated with 50 but not 20 mg/kg showed a progressive loss of body weight that reached 15% on day 4 and persisted after cessation of treatment. Also on day 4, 5-FU triggered a dose-dependent increase in diarrhea score, which was normalized after recovery. Histological evaluation demonstrated mucosal atrophy of the intestines in high-dose treated animals on day 4, with shortening of both villi height (32%) and crypt depth (7%), reduced crypt density (16%), and the presence of granulocytic infiltrates. Plasma citrulline levels were accordingly reduced. Despite a strong regenerative crypt hyperplasia, villus atrophy persisted in recovery animals. Regarding the impact of 5-FU on crypt cell death and proliferation, there was an increase in apoptosis at 6 h that peaked at 24 h, whereas inhibition of mitotic figures was first evident at 24 h but persisted until day 4. Ongoing omics analysis will shed light on the cell cycle signaling pathways affected by 5-FU. The biological processes identified will be integrated with our previously established computational model of epithelial cell dynamics to improve its predictivity for clinically relevant intestinal toxicity.
Keywords: Translational Quantitative Systems Toxicology, gastrointestinal toxicity, animal models, -omics analysis
A randomised, controlled study to evaluate the effects of switching from cigarette smoking to using a Tobacco Heating Product on Biomarkers of Exposure to cigarette smoke toxicants in healthy subjects (#419)
N. Gale1, M. McEwan1, G. Hardie1, J. Ebajemito1, O. M. Camacho1, F. Lowe1, C. J. Proctor1
1 British American Tobacco (Investments) Ltd, Research and Development, Southampton, United Kingdom
Preclinical assessments and 5-day clinical studies have shown that toxicant emissions are lower and associated exposure is reduced when using the glo tobacco heating product (THP) compared to smoking conventional cigarettes. However, it is unclear if these reductions are sustained.
This study aimed to test the hypothesis that reductions in toxicant exposure observed in confined studies over 5 days are sustained over a longer period of 90 days in an ambulatory setting. Biomarkers of exposure (BoE) to cigarette smoke toxicants when smokers switch to using glo compared with smokers who continue to smoke were assessed.
This novel study, conducted in the UK (ISRCTN81075760), was approved by a local Research Ethics Committee and run in accordance with ICH-GCP. Subjects were of either gender and aged 23–55 years. Regular smokers were randomly allocated to either continue to smoke their own brand cigarettes (CTS) or switch to using glo for one year. A separate smoking cessation group consisted of regular smokers intending to quit who were provided with assistance to do so (NRT/varenicline/counselling). The final group were participants who have never smoked. For the 90-day exposure segment of this study, subjects attended a Screening Visit plus a total of 4 non-residential clinic visits. Urinary and breath BoE endpoints were assessed at days 1, 30, 60 and 90. Safety evaluations included adverse events, vital signs, clinical laboratory evaluations, physical examinations, electrocardiography, and spirometry.
The results show significant and substantial reductions in the levels of BoE in the smoking cessation group, as well as in the glo group compared to the CTS arm.
This study demonstrated that when smokers switched from smoking combustible cigarettes to using glo, their exposure to smoke toxicants decreased. This confirms that these reductions are sustained for at least 90 days in an ambulatory setting and suggests that glo has the potential to be a reduced exposure and/or reduced risk tobacco product. Further research is required to confirm whether these exposure reductions translate to reductions in smoking-related health risks. The continuation of this clinical study will examine changes in health effect indicators in subjects switching to glo for a period of one year.
Keywords: Biomarkers, Tobacco Heating Product, Clinical Assessment, Smoking, Tobacco Harm Reduction
Changes in the mouse fecal microbiome upon cigarette smoke exposure and effect reversal upon switching to a potential RRP or cessation (#428)
J. Battey1, J. Szostak1, B. Phillips2, C. Teng2, C. K. Tung2, W. T. Lim2, Y. S. Yeo2, S. Ouadi1, K. Baumer1, J. Thomas1, J. Martinis1, N. Sierro1, N. V. Ivanov1, P. Vanscheeuwijck1, M. C. Peitsch1, J. Hoeng1
1 PMI R&D, Philip Morris Products S.A., Neuchâtel, Switzerland
Cigarette smoking causes adverse health effects that may occur shortly after smoking initiation and lead to the development of inflammation and cardiorespiratory disease progression. The microbiome is susceptible to the influence of environmental factors, such as smoking, and recent studies have indicated microbiome alterations in smokers. To reduce the risk of smoking-related diseases, Philip Morris International is developing potentially Reduced-Risk Products (RRPs) to which adult smokers can switch instead of continuing to smoke cigarettes.
Here, groups of mice were exposed to either cigarette smoke (CS) from a reference cigarette; aerosol from two RRPs, the Carbon Heated Tobacco Product (CHTP) 1.2 and the Tobacco Heating System (THS) 2.2; or fresh air (Sham) over the course of six months. Two groups were exposed to CS over three months and switched to either CHTP 1.2 or Sham for the remaining three months. Fecal samples were collected from these groups of mice and subjected to next-generation sequencing-based microbiomics analysis in order to identify microbial taxa whose relative abundance is altered in response to aerosol exposure and changes in aerosol exposure. We identify taxa that are increased in abundance upon CS exposure, such as certain Bacteroides and Akkermansia.species, as well as species that are reduced in relative abundance upon CS exposure, such as certain Lactobacillus species. After two months of switching from CS to CHTP 1.2 or to Sham exposure, one of the Lactobacillus species depleted by CS is increased significantly in both groups. These microbial changes could be important for understanding the effects of CS and of switching to RRPs on gut function and its relevance to disease via the microbiome.
Keywords: Microbiome, next generation sequencing
Prediction of interethnic differences in acetylcholinesterase inhibition upon chlorpyrifos exposure (#443)
S. Zhao1, L. Kamelia1, R. Boonpawa2, S. Wesseling1, B. Spenkelink1, I. M. C. M. Rietjens1
1 Wageningen University and research, Toxicology, Wageningen, Netherlands
This work was funded by a Grant from the China Scholarship Council (No. 201707720063 to (ZHAOSHENSHENG)
Chlorpyrifos (CPF) is an organophosphate (OP) insecticide. The exposure to CPF has been associated with acetylcholinesterase (AChE) inhibition in human red blood cell (RBC). RBC AChE inhibition has been used as indicator to define points of departure for risk assessment of CPF. The current study aimed at investigating interethnic differences in in vivo CPF exposure-related RBC AChE inhibition between the Chinese and Caucasian population. This was done by using physiologically based kinetic (PBK) models defined for both the Chinese or Caucasian population together with a reverse dosimetry approach to quantitatively convert concentration-response curves for RBC AChE inhibition to in vivo dose-response curves for these two populations. By doing so, the potential neurological risks for two targeted populations upon CPF exposure could be defined. The predicted in vivo dose-response curves for both populations revealed that CPF is 4- to 7- fold less toxic to Chinese than Caucasian as a result of interethnic differences in biotransformation. The average Chinese population appeared to be 4.6-fold slower in CPF bioactivation from CPF to Chlorpyrifos-oxon (CPO), 2.8-times more efficient in detoxcification from CPO to 3,5,6-trichloro-2-pyridinol (TCPy) and 2-times less efficient in detoxification from CPF to TCPy as compared to the average Caucasian popultaion, which could be partly explained by racial variation in the frequency of single-nucleotide polymorphisms (SNPs) for key enzymes involved. Collectively, these results highlight interethnic differences in CPF bioactivation and detoxification that may affect the ultimate risk and indicate that the newly developed PBK models for CPF coupled with reverse dosimetry are capable of predicting in vivo toxicokinetic of CPF and capturing possible interethnic differences in bioactivation and detoxification between the Chinese and Caucasian population.
Keywords: chlorpyrifos, interethnic difference, acetylcholinesterase inhibition, reverse dosimetry PBK modeling, toxicokinetic
Association between heavy metals in umbilical cord serum and DNA methylation of cord tissues in human (#446)
S. Nishizawa-Jotaki1, K. Sakurai1, A. Eguchi1, H. Tanabe1, M. Watanabe1, E. Todaka1, C. Mori1
1 Chiba Univesity, Center of Priventive Medical Sciennces, Chiab City, Japan
Prenatal exposures to heavy metals are known to be associated with fetal development and adverse outcomes in later life, in which DNA methylations are currently considered as one of the possible mechanisms . Whereas there might be a sex-specific association between exposure to heavy metals and DNA methylation , little is confirmed about the fact and the details.
The purpose of this study was to investigate the relationship between prenatal exposure to heavy metals and DNA methylation in offspring.
In a birth cohort study in Chiba (C-MACH), concentrations of heavy metals, mercury (Hg), manganese (Mn) and selenium (Se) in the Umbilical cord serum (UCS), and DNA methylation status in the Umbilical cord tissue (UCT) (a part of fetus) using a methylation array analysis, were examined and their association was analyzed by Spearman correlation adjusted by a false discovery rate in each sex of offspring.
Total 67 pregnant women who gave birth to 27 males and 40 females were participated in the end . Our previous study suggests that UCT is useful as an alternative surrogate for studying environmental effects on DNA methylation in human fetuses, compensating UC blood cells .
Only one locus was correlated to the concentrations of Hg in males and ten[sj5] loci were correlated to the concentrations of Se also in males, while no correlation was observed at any loci in females. There was no correlation between the concentrations of Mn and DNA methylation in either sex. The locus correlated to Hg concentration was on intron of gene body of HDHD1 gene on chromosome X and was a binding site for zinc finger protein CTCF (CCCTC-binding factor).
 Relton, C.L., et al. Int. J. Epidemiol. 2015
Keywords: prenatal exposure, heavy metals, DNA methylation
Chromosome damage in humans: from a group level indicator of genotoxic effects and cancer risk to an individual biomarker (#455)
H. Norppa1, K. Aimonen1, G. Vales1, H. K. Lindberg1, S. Suhonen1, M. Hartikainen1, J. Catalán1, 2
1 Finnish Institute of Occupational Health, Work Environment, TYÖTERVEYSLAITOS Helsinki, Finland
Cytogenetic biomarkers have for decades been used for assessing the genotoxic effects of human exposure to genotoxic carcinogens. A high frequency of chromosome aberrations in peripheral lymphocytes has been associated with an increased risk of cancer, and a similar relationship has also been found for lymphocyte micronuclei. Cytogenetic biomarkers have mostly been evaluated at the group level. This is reasonable, as cells with chromosomal aberrations or micronuclei are rare and their manual analysis is subjective and usually based on relatively low numbers of cells. A more extensive analysis has been time-consuming and expensive. However, the application of automated techniques is rapidly changing this scheme, as the number of cells scored can substantially be increased, while the time spent with the analyses and their expenses and the subjectivity are reduced. The most promising approach is offered by the reticulocyte micronucleus assay – the human equivalent of the rodent peripheral blood micronucleus test. As micronucleated reticulocytes are rapidly removed from blood circulation by the human spleen, micronuclei do not accumulate in normocytes in long-term exposure as they do in mice. The known time window from micronucleus induction in the bone marrow to the appearance of micronucleated reticulocytes in blood and their eventual disappearance from circulation makes it possible to apply the assay for following-up of genotoxic effects and for intervention studies. Due to the improved accuracy of the analysis, reticulocyte micronuclei may become an individual biomarker of the effects of genotoxic carcinogens and cancer risk.
Keywords: Chromosome, cancer risk, genotoxic effect, micronucleus, reticulocyte
Updating strategies for nonnegative matrix factorization to integrate cross omics layers (#498)
T. Kuijpers1, J. C. S. Kleinjans1, D. Jennen1
1 Maastricht University, Department of Toxicogenomics, Grow School for Oncology and Developmental Biology, Maastricht, Netherlands
The ongoing development of high-throughput technologies has generated large and complex data sets of different omics layers, such as mRNA, methylation and protein expression. It is believed that the integration of these different layers should lead to a more complete understanding of cellular events. However, this integration step is not trivial, due to the different distributions and dimensions in each layer, and therefore an appropriate computational method has to be selected. Previous studies have shown the promising results of detecting clusters and features by applying Nonnegative Matrix Factorization (NMF). Here, we propose a multi-layer NMF with a prior knowledge integration workflow to detect both inter and intra relationships in all layers of omics information.
The original NMF method by Lee and Seung decomposes one layer of information into a feature matrix W and a coefficient matrix H, by applying an update rule for both W and H. First, to take multiple omics layers into account, a new set of update rules has to be defined. Therefore, we introduce an update rule for H based on the omics layers Wi (i equals the number of omics layers). This will result in a clustering coefficient matrix H built from all omics layers and thus can be used to relate the different biological entities.
Second, the optimization problem for NMF is not necessarily convex and multiple local minima can be identified. Here, we hypothesize that initializing H with prior knowledge, a local minimum can be found associated with the features and clusters representing the different phenotypic endpoints or experimental conditions. This prior knowledge can contain information about exposure concentrations, compound information but also sample characteristics or disease development.
The proposed multi-layer semi-unsupervised NMF workflow gives valuable information about sample clustering and features. The workflow has been evaluated with toy data, but also with gene expression and CpG methylation values from the NCI60 tumor cell line database. With the integration of those two platforms by our workflow, it becomes possible to obtain the relationships between CpG and gene data for different biological clusters. Future development of the workflow to handle time series data could allow for dynamic cross omics analysis.
Keywords: omics integration, nonnegative matrix factorization, feature detection
Using Human Biomonitoring for the Risk Assessment of Polycyclic Aromatic Hydrocarbons in Occupational Exposures (#541)
S. Viegas1, B. C. Gomes2, H. Louro2, M. J. Silva2, A. S. Joksić5, T. Santonen6
1 ESTeSL-IPL, Environmental Health, Lisboa, Portugal
Background and Purpose: The Human Biomonitoring Initiative (HBM4EU) is a joint effort of 28 countries, the European Environment Agency and the European Commission, co-funded under Horizon 2020. HBM4EU is generating evidence of the current exposure of European citizens to chemicals and the possible health effects in order to assess the associated risks and support policy making towards human health protection. Polycyclic aromatic hydrocarbons (PAH) were considered one of the 1st priority substance groups to be addressed. In the scope of this project, the present work aimed to evaluate the added value of human biomonitoring (HBM) for the PAH risk assessment process, in the case of occupational exposure.
Methods: An extensive literature search was performed to identify scientific papers published between 2008 and 2018 that included air monitoring and HBM data in several occupational settings based in Europe. Among them, those papers presenting urinary 1-hydroxypyrene (1-OHP) quantification - the most common exposure biomarker of pyrene and a surrogate for exposure to PAHs mixtures - were selected. Based on the 1-OHP values the excess lifetime cancer risk (ELCR) for workers, concerning lung cancer, was estimated following the ECHA recent approach (https://echa.europa.eu/fi/applying-for-authorisation/evaluating-applications). ELCR values calculated using air and HBM data were compared.
Results: Based on the criteria described, only 7 out of 28 papers were considered for ELCR estimation. Overall, high ELCR values were estimated (several values higher than 10-4). Moreover, for some studies (3 out of 7) the ELCR estimation using HBM data yielded values higher than those estimated from air monitoring data. This might indicate that, for those specific workplaces, transdermal absorption or even hand-mouth exposure can have an important role in the total exposure to PAH and that the HBM data allows a more accurate PAH exposure assessment. Nevertheless, these findings should be interpreted with caution, since ELCR estimates from air monitoring data are based on Benzo[a]pyrene (BaP) concentrations while HBM-based ELCR determination uses urinary 1-OHP concentration that reflects exposure not only to BaP but to all PAHs, irrespectively of sources or routes of exposure.
This work claims attention for two main aspects, namely: i) the exposure levels are still high in some occupational settings and ii) there is a need for developing new occupational studies, applying a set of exposure biomarkers or a more specific biomarker for BaP exposure, which would allow a better ELCR estimation for exposed workers.
The authors are grateful to HBM4EU project. The HBM4EU project has received funding from the European Union's Horizon 2020 research and innovation program under grant agreement No 733032.
RAC-Committee for Risk Assessment (2018a). Note on reference dose-response relationship for the carcinogenicity of pitch, coal tar, high temperature and on PBT and vPvB properties. Helsinki Available at: https://echa.europa.eu/documents/10162/13637/ctpht_rac_note_en.pdf/a184ee42-0642-7454-2d18-63324688e13d [Accessed February 11, 2019].
Keywords: Polycyclic aromatic hydrocarbons, occupational exposure, human biomonitoring, risk assessment
Toxicity assessment caused by the insecticide methamidophos in bullfrog's tadpoles (#546)
L. I. Paulin1, A. C. Razo2, R. D. C. Guzmán3, N. I. Romero4
1 Instituto Politécnico Nacional, ENCB, Ciudad de México, Mexico
SECRETARÍA DE INVESTIGACIÓN Y POSGRADO (SIP20170116)
In Mexico, currently are sold various plagicides prohibited in other countries, thus it is important to determine its toxicity and how it affects to humans and animals. Methamidosphos is one of the above mentioned pesticides and belongs to a group of organophosphates that are characterized by causing neurological damage and alterations in various defense mechanisms. The purpose of this study was to assess the acute effects (median lethal concentration, LC50) as well as neurotoxic damage and the evaluation of oxidative stress markers in a non-lethal concentration.
It has been described that organophosphorus pesticides inhibit the enzyme's acetylcholinesterase activity, responsible of hydrolyzing acetylcholine, neurotransmitter of varied synapses, mainly in neuromotor plates and are precursors of increasing free radicals: O2•-, HO• and peróxidos:H2O2.
The excessive use of methamidophos on the agricultural fields close to acquifers, represents a level of risk to amphibian species, that is why the toxicity is evaluated in bullfrog's tadpoles, an animal with gastronomic importance, it is able to thrive in aquatic and terrestrial environments.
The results were, CL50 of methamidophos during the 48 h of exposure was 1.55 g/L. The methamidophos non-lethal concentration 0.155 g/L was used at 48 hours of exposure so as the acetylcholinesterase (AChE) inhibitory response, total protein levels and the antioxidant response that includes: the enzymes superoxide dismutase activity (SOD), catalase (CAT), glutathione peroxidase (GPx) and lipid hydroperoxidation (LPO) during 6, 12, 24 and 48 hours of exposure in bullfrog tadpoles.
It was established a drop in total proteins, in the entire period of exposure; acetilcholinesterase inhibition was demonstrated through the period of exposure. When assessing enzymatic activity, SOD increased significantly during the 48 hours compared to the control; on the other hand, the CAT had the highest peak at 12 h, being below the value control, it subsequently decreased; GPx showed no changes during the exposure, however, it was lower compared to the control; regarding to lipid hydroperoxidation, an increase was observed from 6 h until the end of the exposure time.
Conclusion: At sublethal concentration of 0.155 g/L methamidophos, oxidative stress and neurotoxic damage are generated in bullfrog tadpoles.
Ighodaro, O. y Akinloye, O. (2017). First line antioxidants-superoxide dismutase (SOD), catalase (CAT) and glutathione peroxide dismutase (GPx): Their fundamental role in the entire antioxidant defence grid.Alexandria Journal of Medicine, Vol. 30, 30-30.
Garrido, O., Meza, S. E., Anguiano, V., y Chamorro, G. (2014).Adaptation of Lorke's method to determine and compare ED50 values:The cases of tow anticonvulsants drugs. Journal of Pharmacological and Toxicological Methods, 66-69.
McCord, J. (2008).Superoxide dismutase, Lipid Peroxidation, and bell-shaped dose response curves. Dose-Response, 6(3), 223–238.http://doi.org/10.2203/dose-response.08-012.McCord
Medellín, L. y R. A. (2000). Rana catesbeiana. Vertebrados superiores exóticos en México: diversidad, distribución y efectos potenciales.Instituto de Ecología, Universidad Nacional Autónoma de México. Bases de datos SNIB-CONABIO. México. D.F. (pp. 1-6). Recuperado el 20 de noviembre del 2017 de:http://www.conabio.gob.mx/conocimiento/exoticas/fichaexoticas/Ranacatesbeiana00.pdf
Keywords: methamidophos, oxidative stress, neurotoxic damage, bullfrog tadpoles
Regucalcin expression profiles in formalin-fixed parafﬁn-embedded (FFPE) samples: histological and molecular assessments for detection of sex steroid illicit administration. (#579)
A. Benedetto1, E. Biasibetti1, C. Beltramo2, S. Peletto2, E. Bozzetta1, M. Pezzolato1
1 Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, C.I.B.A. - National Reference Center for biological screening of anabolic substances in food producing animals, Turin, Italy
The use of sex steroids in food producing animals is forbidden within the EU. Illicit growth promoters’ cocktails are known to determine, together with increased meat production, several perturbations in different tissue biomarkers, which can be exploited to setup novel diagnostic tools (Benedetto et al., 2018; Pezzolato et al., 2016).
Recently, it has been shown that sex steroids induce a reduction of regucalcin (RGN) expression in calve testis (Cucuzza et al., 2017). This effect can be detected by monitoring the expression of RGN at different levels (mRNA, proteins).
The aim of this work was to compare RGN mRNA levels with protein perturbations detected by immunohistochemistry (IHC) in testis.
FFPE testis samples of calves treated with nandrolone (n = 10; 50 mg / head / week, four injections), 17β-estradiol (n = 10; 5 mg / head / week, four injections), and a cocktail of the two hormones (n = 10; 5 + 50mg / head / week, four injections) were analyzed by RT-qPCR with specific RGN assay and by IHC with rabbit anti-RGN polyclonal antibody (Sigma).
Gene expression data were analyzed using GenEx software for relative quantification (ΔΔCt). The IHC was evaluated by pixel analysis (NIS-Elements 4.5) for the quantification of the percentage of positive staining area.
Relative quantification (RQ) results demonstrated that androgens induce a 2.85 fold reduction (RQ mean 0.35, range 0.21-0.58, p<0.05), estrogens induce a 4.16 fold reduction (RQ mean 0.24, range 0.17-0.34, p<0.01) and the association of the two hormones induces a 11.1 fold reduction (RQ mean=0.09, range 0.05-0.14, p<0.01) of RGN mRNA levels compared to untreated animals (RQ mean=1, range 0.74-1.34).The IHC showed a significant reduction (p<0.0001) in RGN expression in all treatments (nandrolone, 17β-estradiol and their association) compared to the control group.
The good correlation between RT-qPCR and IHC applied to FFPE testis samples confirms RGN as a useful biomarker to detect illegal administration of sex steroid hormones in veal calves.
Benedetto, A., Pezzolato, M., Peletto, S., Beltramo C., Bozzetta, E. 2018. Up‐regulation of progesterone receptor in formalin fixed sex accessory glands: towards the validation of a screening method for illicit growth promoters abuse in veal calves. J. Vet.Pharmacol. Ther. 41(S1):118-119. https://doi.org/10.1111/jvp.12654.
Cucuzza, L. S., Biolatti, B., Divari, S., Pregel, P., Scaglione, F. E., Sereno, A., & Cannizzo, F. T., 2017. Development and Application of a Screening Method of Absolute Quantitative PCR To Detect the Abuse of Sex Steroid Hormone Administration in Male Bovines. J. Agric. Food Chem. 65(23), 4866-4874. https://doi.org/10.1021/acs.jafc.7b00852.
Pezzolato, M., Botta, M., Baioni, E., Richelmi, G. B., Pitardi, D., Varello, K., Caramelli, M., & Bozzetta, E., 2016. Confirmation of the progesterone receptor as an efficient marker of treatment with 17 beta-estradiol in veal calves. Food Addit. Contam. Part A Chem. Anal. Control Expo. Risk Assess. 33(1), 60-65. https://doi.org/10.1080/19440049.2015.1107918.
Keywords: Biomarkers, Growth promoters, Immunohistochemistry, qPCR, FFPE samples
Comparison of the Tyrosinaemic Potential from Exposure to HPPD Inhibitors of Herbicidal & Medicinal Use (#596)
M. Provan1, J. Botham2, P. Botham3, M. Frericks4, J. - C. Garcin5, G. Semino-Beninel6, J. Zimmermann7
1 Regulatory Science Associates, Inverkip, United Kingdom
The class of chemicals known to be inhibitors of 4-hydroxyphenylpyruvate (HPPD) can cause ocular toxicity in rats. The mechanism has been clearly defined and key is a sustained elevation of plasma tyrosine (tyrosinaemia) above a threshold due to blockage of tyrosine catabolism. This communication addresses the human relevance of this mechanism of action (MOA) for dietary exposure to herbicidal HPPD inhibitors.
One HPPD inhibitor (nitisinone) is recommended as the medication of choice for the treatment of Hereditary Tyrosinaemia Type I (HT-1), a life-threatening inborn error of tyrosine catabolism, and is administered from birth. It is a highly potent HPPD inhibitor, designed under continuous administration to block the tyrosine catabolic pathway completely, and induces a moderate tyrosinaemia. Today, the dose level and frequency of administration have also been clearly defined by medical agencies; however clinical trial information on induced tyrosinaemia is available from the early development period.
HPPD inhibitors have also been developed as herbicides in a variety of crops, such as corn, cereals or rice and for one of these compounds (mesotrione) human data on plasma tyrosine concentrations following oral dosing are available. Herbicidal HPPD inhibitors have a lower potency of inhibition of HPPD than nitisinone (based on toxicodynamic and toxicokinetic differences), and produce less marked tyrosinaemia at equivalent dose levels.
A comparison of the tyrosinaemic potential in humans of the two HPPD inhibitors via medicinal use and from dietary exposure to herbicide residues has been made. The medical use of nitisinone induces a moderate tyrosinaemia through continuous daily exposure which, in 5% of patients, can cause reversible ocular toxicity that can be managed by dietary adjustment of tyrosine/phenylalanine. Although some of the low potency agrochemical HPPD inhibitors are also capable of causing ocular toxicity in rats, dietary exposure to residues is negligible and is thus highly unlikely to cause any tyrosinemia and hence ocular toxicity in humans.
This work was supported and funded by the European Crop Protection Association.
Keywords: HPPD exposure nitisinone mesotrione tyrosinaemia
Exploring the effect of anticancer drugs doxorubicin and mitoxantrone on cardiac mitochondrial plasticity using a proteomic approach (#641)
S. R. Brandão1, 2, A. R. Mendes2, F. D. Carvalho2, M. D. L. Bastos2, R. Ferreira1, V. M. Costa2
1 University of Aveiro, Department of Chemistry, Aveiro, Portugal
The current anticancer therapies have increased the number of cancer survivors, although the inherent cardiac side effects of some drugs are also increasing among survivors[1,2]. The cardiotoxicity of doxorubicin (DOX) and mitoxantrone (MTX) may be linked to the cardiac aging process, although the molecular modulation is not understood so far. So, our goal was to study the effects of DOX and MTX in the molecular mechanisms harbored in the heart of adult male CD-1 mice (3 months) and compare them with old CD-1 mice (18 months). All animals were injected with 6 intraperitoneal administrations twice a week for three weeks: control mice received saline solution and DOX- and MTX-treated mice received a total cumulative dose of 9 mg/kg and 6 mg/kg, respectively. During the entire experimental period, animal welfare was assessed daily, and mice were euthanized one week after the last injection. The experiments were performed with the approval of the Portuguese National Authority for Animal Health (General Directory of Veterinary Medicine) (reference number 0421/000/000/2016). After excising, aliquots of whole cardiac tissue homogenate and enriched mitochondrial fractions were prepared and analyzed by immunoblot and enzymatic assays. Enriched mitochondrial fractions were characterized by mass spectrometry-based proteomics (GeLC-MS/MS). Data highlighted a decrease on mitochondrial density for both DOX- and MTX-treated and aged animals, as assessed by citrate synthase activity. Additionally, DOX treatment led to an increase in the ETFDH-to-ATP synthase ratio. GeLC-MS/MS analysis of enriched mitochondrial fractions resulted in the identification of 693 different proteins, assigned to the biological processes “small molecule metabolic process”, “oxidation-reduction process” and “carboxylic acid metabolic process”, according to String v10.5. From the PLS statistical analysis, no proteome signature could be associated to each group, although the drugs induced down-regulation of branched-chain amino acid metabolism and fatty acid beta-oxidation with no clear connection with the cardiac aging process. Taken together, our data points to a modulation of mitochondrial plasticity induced by the anticancer drugs DOX and MTX. Indeed, the decrease on mitochondrial density may be associated to mitochondrial adaptations, such as metabolic shift to fatty acid beta-oxidation. Thus, more than alterations noticed in isolated cardiac mitochondria, these drugs seem to modulate mitochondria biogenesis.
This work was supported by FEDER funds through the Operational Programme for Competitiveness Factors–COMPETE and by national funds by Fundação da Ciência e a Tecnologia (FCT) within the project “PTDC/DTP-FTO/1489/2014 – POCI-01-0145-FEDER-016537”. SRB, ARM and VMC acknowledge FCT for their grants (SFRH/BD/138202/2018, SFRH/BD/129359/2017 and SFRH/BPD/110001/2015).
 Hrynchak, I.; Sousa, E.; Pinto, M.; Costa, V. M. The Importance of Drug Metabolites Synthesis: The Case-Study of Cardiotoxic Anticancer Drugs. Drug Metab. Rev. 2017, 49 (2), 158–96.
 Colombo, A.; Sandri, M. T.; Salvatici, M.; Cipolla, C. M.; Cardinale, D. Cardiac Complications of Chemotherapy: Role of Biomarkers. Curr. Treat. Options Cardiovasc. Med. 2014, 16 (6), 313.
 Senkus, E.; Jassem, J. Cardiovascular Effects of Systemic Cancer Treatment. Cancer Treat. Rev. 2011, 37 (4), 300–11.
 Szklarczyk, D.; Franceschini, A.; Wyder, S.; Forslund, K.; Heller, D.; Huerta-Cepas, J.; Simonovic, M.; Roth, A.; Santos, A.; Tsafou, K. P. STRING V10: Protein–Protein Interaction Networks, Integrated over the Tree of Life. Nucleic Acids Res. 2015, 43 (D1), D447–52.
Keywords: anticancer drugs, proteomics, mitochondria, cardiotoxicology, aging
Perfluorinated compounds in women of reproductive age exposed to contaminated drinking water in the Veneto Region, Italy (#701)
A. Abballe1, A. M. Ingelido1, E. Dellatte1, N. Iacovella1, V. Marra1, S. Valentini1, E. De Felip1
1 Istituto Superiore di Sanità, Department Environment and Health , Rome, Italy
Per- and polyfluoroalkyl substances (PFASs) have been widely produced and used for many years as water repellants and protective coatings in industrial and domestic products and due to their use and persistence to degradation they are widespread around the globe. Humans are generally exposed to low levels of these chemicals principally through diet, but consumption of drinking water can be an important source of exposure in communities living in areas where PFASs have contaminated water supplies. A well-known example is represented by the water contamination that occurred in Ohio and West Virginia and was investigated starting from 2006 by the C8 Project. Such project included the biomonitoring of PFAS in 69,030 subjects from six contaminated water districts.
A major episode of PFAS water contamination occurred in Veneto, a region in the North-Est of Italy. The contamination, identified in 2013, was originated from a chemical plant that has been producing PFASs in the area for decades. Contamination had affected also drinking water where the presence of several PFASs had been detected. On this evidence, a human biomonitoring study was carried out.
The study included a group of women of reproductive age, a population group which raised major concerns for the local sanitary authorities and the population because of the possible PFAS effects on maternal health and on foetal growth and development. PFAS concentrations were measured in a group of 121 exposed women (E) of reproductive age (20-40 years old), and in a group of 80 women (NE) from the same region living in areas not exposed to contaminated drinking water. Serum samples were analyzed for PFBA, PFPeA, PFHxA, PFHpA, PFOA, PFNA, PFDA, PFUdA, PFDoA, PFBS, PFHxS, and PFOS. About 250 μL of serum were spiked with labelled internal standards. Extraction was performed with acetonitrile, reduced and transferred to an autosampler vial to undergo instrumental analysis. Instrumental analysis was carried out by HPLC interfaced with a triple quadrupole mass spectrometer operated in the electrospray negative mode.
Mann-Whitney and Spearman tests were used to assess differences between groups in the concentrations of serum contaminants and correlations with determinants of exposure. The characteristics of study participants (age, body mass index (BMI), residence area) together with information on lifestyle in relation to water use and consumption were considered in the evaluation of results.
Serum concentrations of most of the analyzed contaminants were significantly higher in the E group, years of residence in the municipalities and BMI appear to be the most important determinants of exposure.
PFOA levels assessed in the contaminated areas resulted to be in the concentration range found to be associated with pregnancy induced hypertension in the C8 study, and much higher than the range of levels associated with adverse effects on birth weight and development in published studies.
Keywords: perfluorinated compounds, human exposure, drinking water, women of reproductive age, human serum
Development of a dietary-PTU model of gradual thyroid disruption (hypothyroidism) in the mouse (#703)
L. Claustre1, 2, C. Viguié2, C. Layssol1, A. Bury1, L. Mialon1, N. Bourgès-Abella1, M. Kolf-Clauw1
1 Toulouse Veterinary School, CREFRE, Université de Toulouse, INSERM, UPS, ENVT, Toulouse cedex3, France
Recommendations for the evaluation of thyroid disruption are very scarce and limited. It can include summary evaluation of histological structure of the gland and/or thyroid hormone plasma concentrations . We aim at determining which one of histological modification or hormonal concentrations (TH and/or TSH) is the more sensitive biomarker of thyroid disruption.
Our approach was to develop a model of gradual thyroid disruption (hypo) in mouse to analyze the relationship between circulating TH /TSH concentrations and quantitative parameters characterizing the thyroid architecture. The goal of the current study as a first step was to identify treatment conditions associated with major modification of both hormone levels and thyroid structure and low dose inducing more moderate modification to determine the range of PTU doses that could be used to produce a full scale dose-response relationship.
Materials and methods
Swiss adult male mice were allocated to 3 groups (n=4 each), a control fed with a standard iodine concentration diet (0.5 ppm) and two groups PTU-treated animals fed with iodine deficient diet (0.03-0.05 ppm), supplemented by PTU (10 and 1000 ppm). Animals were observed daily, blood was collected on days 0, 14 and at the end of a 28-day treatment. The thyroid were sampled at the end of the treatment. Thyroid histological structure and morphometric measurements (thyroid follicular density, colloid area, epithelial surface using NIH’s ImageJ software) were analyzed on hematoxylin-eosin and PAS stained sections respectively. The mean Activation Index (AI), expressed by the epithelial volume/colloid volume ratio was calculated for each group.
Major functional, macroscopic (enlargement) and histological changes were observed in PTU treated groups without clear clinical signs of hypothyroidism or changes in bodyweights. In both treated groups, thyroid parenchyma was modified with diffuse and/or focal follicular hyperplasia associated with epithelial hypertrophy of moderate (10 ppm) to severe intensity (1000 ppm). At the low dose, at day14, TT4 was decreased by 23% and fell below the assay limit of quantification (5 ng/ml) by the end of the treatment. In the high PTU dose, TT4 was already much lower than in the low dose at day 14 and below assay detection limits for most of the animals. Mean AI of the 10 ppm group was three-fold higher than in control. In the highest PTU dose group, the thyroid histological organization was so modified that is was not possible to determine an AI. From this results we identified a range of PTU doses from 1 to 100 ppm in iodine-deficient mice as a way to obtain different degrees of hypothyroidism to model the relationship between the two main types of parameters used to characterize thyroid disruption.
Keywords: thyroid disruption, mouse, activation index, dietary-PTU model
Activation of keratinocytes in response to multi-exposure of a cosmetic sensitizer in a reconstructed epidermis (#717)
R. Vallion1, 2, C. Raffalli1, A. Jaracz-Ros1, C. Callego1, P. - J. Ferret2, G. Schlecht-Louf1, M. Pallardy1, F. Bachelerie1, S. Kerdine-Römer1
1 Université Paris-Saclay, UMR996 - Inflammation Chimiokines et Immunopathologie, INSERM, Fac. de pharmacie - Univ.Paris-Sud, Châtenay-Malabry, France
Keratinocytes (KCs) are the main component of the epidermis, an epithelium in continuous self-renewal. The four distinct layers are characterized by the differentiation status of keratinocytes: the undifferentiated basal layer, the stratum spinosum, the stratum granulosum differentiated additional and the stratum corneum with dead corneocytes. During their maturation process, KCs move from the basal to the upper layer and orchestrate immune responses if microbes and molecules enter the stratum corneum due to mechanical or pathological skin barrier defects.
In certain diseases such as allergic contact dermatitis (ACD), KCs play a key role since they are the first cells to encounter the contact sensitizer (CS) in the skin. KCs contain enzymes that have metabolic activity to transform prohaptenes into biologically active haptenes, facilitating protein binding to form the antigenic complex. In addition, by expressing chemotactic factors and inflammatory cytokines when exposed to CS, KCs could initiate the immune response.
In this study, we investigate how repeated exposure to CS influences the process of epidermal differentiation. To answer this question, a 3D skin model composed of KCs (NIKS cell line) grown on a matrix of collagen and primary human fibroblasts was used. All along the differentiation time, the skin model was exposed to low concentrations (0.1 mM & 0.25 mM) of cinnamaldehyde (CinA), a well-known electrophilic compound. At the end of the differentiation, the 3D skin model was analyzed by immunohistochemistry, western blot and RT-qPCR. A biochip was also carried out to highlight new canonical pathways in order to propose new genes of interest.
Our results show that repeated exposure to CinA induces a slight increase in skin thickness and a lower percentage of apoptotic cells. An induction of filaggrin expression is measured in response to a chronic exposure to CinA. In addition, the transcription factor Nrf2 is activated and antioxidant genes such as ho-1 and nqo-1 are also induced. Preliminary results from the microarray show a high degree of segregation between groups and the analysis is currently under study.
This work shows that a low concentration of CS can modify the epidermis and seems relevant for cosmetic products often used with low doses of sensitizing molecules.
Keywords: Contact sensitizer, epidermis differentiation
Roles of Nrf2 protein in environmental chemicals' toxicity: Toxicogenomics data mining (#721)
K. Baralić1, K. Živančević1, B. Antonijević1, Z. Bulat1, M. Ćurčić1, E. Antonijević1, D. Javorac1, V. Matović1, D. Đukić-Ćosić1
1 University of Belgrade, Faculty of Pharmacy, Belgrade, Serbia
Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a protein encoded by NFE2L2 gene. It has a role in antioxidant proteins expression regulation, especially those that protect against the oxidative damage induced by injury and inflammation. Nrf2 might have an important role in oxidative stress and toxicity defense, while its activation is being used as biomarker of chemical damage. The aim of our study was to explore the influence of environmental chemicals on NFE2L2 gene using the toxicogenomics approach. Comparative Toxicogenomics Database (CTD; http://ctd.mdibl.org) was the main data mining tool for our analysis. Set Analyzer CTD tool listed 783 chemicals that interact with NFE2L2. Top environmental chemical influences for NFE2L2 gene were: sulforafan, sodium arsenite, tetrachlorodibenzodioxin, tobacco smoke, 2-tert-butylhydroquinone, resvertarol, paraquat, quercetin and cadmium chloride. SetAnalyser CTD tool listed 2,321 diseases connected with NFE2L2 gene. For the top 10 curated diseases (fatty liver, hepatomegaly, acute kidney injury, hyperglycemia, liver neoplasms, hepatocellular carcinoma, skin neoplasms, pulmonary fibrosis, gastrointestinal diseases and non-alcoholic fatty liver disease) NFE2L2 gene played a role in the ethyology and might be used as a biomarker. This can be connected with exposure to some chemicals. For example, paraquat, herbicide which causes severe pulmonary fibrosis, decreases the activity of NFE2L2 protein and expression of NFE2L2 mRNA. Tobacco smoke decreases the expression of NFE2L2 protein as well. However, it is important to consider the role of NFE2L2 gene as a therapeutic target in the treatment of some diseases. This gene has been listed in the CTD as a possible therapeutic target for cardiovascular diseases (heart failure and vascular system injuries). Sulforaphane and resvertarol, which increase activity of NFE2L2 protein and expression of NFE2L2 mRNA, might be used as a prevention of cardiovascular diseases. These substances also inhibit the reaction of other chemicals that decrease the expression of NFE2L2 protein. These results provide a basis for further in vitro and in vivo investigation of the molecular mechanisms behind Nrf2 role in environmental chemical’s toxicity (project 46009III).
Keywords: NFE2L2, toxicogenomics, The Comparative Toxicogenomics Database, sulforaphane, resvertarol
BMD Analysis of In Vitro and In Vivo Whole Transcriptome TempO-Seq Dose Response Data (#735)
M. Raghunathan1, M. Babic1, J. Yeakley1, B. Seligmann1
1 BioSpyder Technologies, Inc., Carslbad, California, United States of America
Gene expression (Gex) dose response measurements with Benchmark Dose (BMD) statistical analysis provide a sensitive and data-rich basis for chemical risk assessments and determination of human health guidance values. Unfortunately, this approach has been hampered by high costs and complexity of RNA-seq technologies and the low reproducibility of such assays. A targeted expression profiling assay with efficient NGS-based readout, TempO-Seq®, facilitates BMD analysis by drastically reducing cost per sample, simplifying high-throughput workflows, and providing the reproducibility required for quality dose response data. The assay proceeds directly from cell lysates without RNA extraction or reverse transcription; consistency of results is high without input normalization; and data analysis is fully automated (requiring no bioinformatics expertise). Furthermore, the assay allows for proportional attenuation of highly expressed targets, and its high tolerance to RNA damage and degradation allows Gex measurement of fixed archival tissue samples. We used TempO-Seq to compare dose response results from rat cell lines treated with fenofibrate in vitro vs. FFPE tissue from rats treated in vivo. Data is fine-grained enough to allow step-by-step analysis of cellular responses, with the expression activity associated with the PPARa agonist mode of action of fenofibrate increasing with dose and time. Both in vivo and in vitro BMD analysis shows dose and time-dependent activation of the expected processes (lipoprotein lipase activity, fatty-acid beta oxidation, triglyceride biosynthesis). Cellular amide metabolic processes, proposed as a mechanism of action for fibrate drugs, are detected as the second most sensitive pathway to fenofibrate treatment. We extended this dataset with analyses of cells and rats treated with β-estradiol, N-(2-Fluorenyl) acetamide, phenobarbital, 5,6-benzoflavone, and amiodarone, showing that cell line treatment provides a reasonable model for in vivo effects. BMD analysis of TempO-Seq dose response data permits the ranking of pathways that describe the mode of action of drugs, the comparison of in vivo to in vitro validates the utility of in vitro assays, and this approach will ultimately permit cross species analysis (e.g. animal in vivo to human in vitro).
Trejo C, Babić M, Imler E et al. Extraction-free whole transcriptome gene expression analysis of FFPE sections and histology-directed subareas of tissue. PLoS ONE. 2019;14(2):e0212031. doi:10.1371/journal.pone.0212031
Yeakley J, Shepard P, Goyena D, VanSteenhouse H, McComb J, Seligmann B. A trichostatin A expression signature identified by TempO-Seq targeted whole transcriptome profiling. PLoS ONE. 2017;12(5):e0178302. doi:10.1371/journal.pone.0178302
You B, Hour M, Chen L, Luo S, Hsu P, Lee H. Fenofibrate induces human hepatoma Hep3B cells apoptosis and necroptosis through inhibition of thioesterase domain of fatty acid synthase. Sci Rep. 2019;9(1). doi:10.1038/s41598-019-39778-y
Finger JH, Smith CM, Hayamizu TF, McCright IJ, Xu J, Law M, Shaw DR, Baldarelli RM, Beal JS, Blodgett O, Campbell JW, Corbani LE, Lewis JR, Forthofer KL, Frost PJ, Giannatto SC, Hutchins LN, Miers DB, Motenko H, Stone KR, Eppig JT, Kadin JA, Richardson JE, Ringwald M. 2017. The mouse Gene Expression Database (GXD): 2017 update. Nucleic Acids Res. 2017 Jan. 4;45 (D1): D730-D736.
Yang L, Allen B, Thomas R. BMDExpress: a software tool for the benchmark dose analyses of genomic data. BMC Genomics. 2007;8(1):387. doi:10.1186/1471-2164-8-387
Farrell E, Chen Y, Barazanji M, Jeffries K, Cameroamortegui F, Merkler D. Primary fatty acid amide metabolism: conversion of fatty acids and an ethanolamine in N18TG2 and SCP cells1,[S]. Journal of Lipid Research. 53: 247–256. doi: 10.1194/jlr.M018606
Keywords: Gene Expression, transcriptomics, BMD, dose response, FFPE
Development, testing, parameterisation and calibration of a human PBPK model for the plasticiser, Hexamoll® DINCH using in silico, in-vitro and human bio-monitoring data (#793)
G. Loizou1, K. McNally1
1 HSE Science and Research Centre, Buxton, United Kingdom
A physiologically based pharmacokinetic (PBPK) model for Hexamoll® DINCH (diisononyl-cyclohexane-1, 2-dicarboxylate) was developed to interpret the biokinetics in humans after single oral doses. The model was parameterised with in vitro and in silico derived parameters and uncertainty and sensitivity analysis was used during the model development process to assess structure, biological plausibility and behaviour prior to simulation and analysis of human biological monitoring (HBM) data. The model provided good simulations of the urinary excretion (Curine) of two metabolites; cyclohexane-1, 2-dicarboxylic acid mono hydroxyisononyl ester (OH-MINCH) and cyclohexane-1, 2-dicarboxylic acid mono carboxyisononyl ester (cx-MINCH) from the biotransformation of mono-isononyl-cyclohexane-1, 2-dicarboxylate (MINCH), the monoester metabolite of DINCH. However, good simulations could be obtained, with and without, a lymphatic compartment. Selection of an appropriate model structure was informed by sensitivity analysis which could identify and quantify the contribution to variability in Curine by parameters, such as, the fraction of oral dose (FracDose) that directly entered the lymphatic compartment via the lacteals in the gut and therefore by-passed the liver and the fraction of MINCH bio-transformed to cx-MINCH and OH-MINCH (FracMetabcx, FracMetabOH). By constraining FracDose, FracMetabcx and FracMetabOH within biologically plausible limits the presence of a lymphatic compartment was deemed an important model structure. Furthermore, the use of sensitivity analysis is important in the evaluation of uncertainty around in silico derived parameters. By quantifying their impact on model output sufficient confidence in the use of a model should be afforded. This type of approach could expand the use of PBPK models since parameterisation with in silico techniques allows for rapid model development. This in turn could assist in reducing the use of animals in toxicological evaluations by enhancing the utility of “read across” techniques.
Keywords: PBPK, in silico, in vitro, phthalate, human biological monitoring
Identification of urinary metabolites of Diethylamino Hydroxybenzoyl Hexyl Benzoate (Uvinul A plus) using microsomes and electrochemistry – application in exposure assessment study following dermal application (#816)
P. A. Dąbrowska1, B. Wielgomas1
1 Medical University of Gdańsk, Toxicology Department, Faculty of Pharmacy, Gdańsk, Poland
Uvinul A Plus (DHHB, Diethylamino Hydroxybenzoyl Hexyl Benzoate) is used in personal care products as an effective UV filter. It is considered to be safe and penetrate poorly through human skin. Human metabolic pathways of DHHB are not described yet, thus no validated biomarker is available for the assessment of internal dose following oral, dermal or combined exposure. In this work we applied electrochemical reactor and human liver microsomes (HLM) to simulate and study first phase metabolism and select potential candidate which may serve later as urinary biomarker of exposure. Finally, study on urinary elimination of metabolites following controlled dermal exposure was performed on 6 volunteers. Application amount of commercial personal care product containing 3% of Uvinul A Plus was 1 mg/cm2. The approximate applied dose of DHHB was 30 µg/cm2.
Several oxidative metabolites corresponding to hydroxylated products and mono- and di-N-dealkylated products were generated by both electrochemical reactor and HLM. Additionally, product of hydrolysis 2-(4-(diethylamino)-2-hydroxybenzoyl)benzoic acid (DHBA) was detected in HLM incubations but as expected, it was not observed in electrochemistry (EC) experiments. The range of tentatively identified metabolites generated by HLM and EC allowed to develop targeted LC-MS/MS method for their determination in human urine. DHBA was quantified in the samples using authentic standard.
Among human DHHB metabolites 2-(4-(diethylamino)-2-hydroxybenzoyl)benzoic acid (DHBA), was eliminated in urine in the highest amount and was not present in the pre-exposure samples. Thus DHBA may be considered as a potential urinary biomarker of exposure to DHHB. The study provides first experimental data on DHHB human skin penetration and suggest human biotransformation pathways. It also presents methodology employing electrochemistry for better characterization of possible biotransformation products.
Keywords: biomarkers, metabolism, electrochemistry, Uvinul A Plus, human
Emergent role of epigenetic biomarkers for pesticides exposure among farmworkers in Meknes (Morocco) (#840)
A. Menouni1, 2, R. C. Duca2, I. Berni1, D. Akroute1, L. Godderis2, S. El Jaafari1
1 Moulay Ismail University, Cluster of Competence Environment & Health, MEKNES, Morocco
Background: Pesticide exposure has been associated with acute and chronic adverse health effects. Current evidence supports that epigenetics may mediate these effects. DNA methylation (DNAm) is one of the broadly investigated epigenetic alteration. Therefore, to date, only limited human data is linking pesticide exposure to global DNAm alterations. The aim of the study was to characterize pesticides exposure in farmworkers and pesticides applicators and investigate whether DNA methylation patterns were related to pesticides exposure level.
Keywords: Pesticides, epigenetics, DNA methylation, Occupational exposure, Oxidative stress
Prenatal exposure to parabens and triclosan and assessment of possible health impacts (#867)
V. Karzi1, 2, I. Katsikantami1, 2, M. Tzatzarakis1, E. Vakonaki1, E. Iatrou1, A. Stavroulaki1, 2, P. Xezonaki3, S. Sifakis3, 4, A. Rizos2, A. Tsatsakis1
1 University of Crete, Laboratory of Toxicology and Forensic Sciences, Medical School, Heraklion, Crete, Greece
Background: Parabens (PBs) and triclosan (TCS) are antimicrobial agents widely used in personal care products such as deodorants, shampoos and shower gels, mouth pastes and washes, cosmetics, etc., making exposure to them inevitable. Problems in reproductive and respiratory system, thyroid gland’s dysfunction and cancer are the most frequently reported health problems.
Keywords: parabens, triclosan, biomonitoring, urine, pregnant women
Assessment of Drinking Water Chlorination By-products in View of Multiroute Exposure (#924)
A. Drazdova1, V. Girina1, V. Buraya1, A. Firago1
1 Scientific-Practical Center for Hygiene, Minsk, Belarus
Most chemical disinfection methods are accompanied by the formation of a huge number of disinfection byproducts (DBPs) in treated drinking water (DW) through reaction of the chemical disinfectant with naturally occurring inorganic and organic matter in the source water. In some cases number of them exceed 500. A number of DBPs cancerogenic or cause target-organ toxicity (including repro- and developmental toxicity). As a result population are chronically low-level exposed to a very large number of DBPs. At the dose levels tested in laboratory experiments, a number of these DBPs were either carcinogenic or caused target-organ toxicity, including reproductive/developmental toxicity. These dose levels are high compared with the low levels found in water. In our previous research the algorithm for evaluation integrated toxicity of complex mixtures of DBPs in DW [http://rspch.by/Docs/instr-015-1118] was developed and tested. It recommended for use on the stage of substantiation of the choice of DW disinfection method which will pose the lowest risks to public health.
For routine surveillance of DW classic approach are used – confirmation of compliance with national standards on DBPs. In Belarus 6 DBPs of chlorination have hygienic standards and routinely monitored. At the same time last scientific data and estimates have allowed to strengthen the regulation of volatile chlorination by-products (THM) in developed countries: for chloroform 0.06 mg/l with the total content of the priority 4 THM 0.1 mg/l. In Belarus the guideline value for chloroform is 0.2 mg/l (3.3 times less stringent). It based only on per oral intake of DW, while THM are volatile and express hazard also through inhalation and percutaneous exposure while bathing, showering and housework. For revision of guideline values for THM in DW the research is needed.
The purpose of this ongoing research is to conduct complex health risk assessment of DBPs mixtures in DW considering multiple routes of exposure.
In research we use 3 methods: 1) assessment of complex exposure with priority THMs based on results of laboratory assessment of THMs levels in DW and accounting of the specific contribution of 3 routes of exposure with all household DW use; 2) assessment of internal doses of THM due to the complex intake of THM from DW at home on the basis biomonitoring data of THM levels in blood and urine (biomarkers of the exposure) of the population living in areas exposed to the harmful factor (Minsk); 3) the study of the genetic polymorphism effect of enzymes involved in the metabolism of THM (CYP450, GSTT), determination of sensitive groups of the population to the exposure to THM (by genetic sensitivity biomarkers).
Provisional results show the added value of biomonitoring data for complex risk assessment, they characterize of internal exposure with THM. The results of research will be used for revision of guideline values for THM in DW.
Keywords: drinking water, |multiroute exposure, biomarkers, disinfection byproducts, health risk assessment
Risk assessment of traditional alcoholic beverages (#925)
M. Kokkinakis1, 3, I. Tsakiris2, M. N. Tzatzarakis1, A. Alegkakis1, E. K. Vakonaki1, A. Kokkinaki1, A. M. Tsatsakis1
1 University of Crete , Laboratory of Toxicology, Medical School, Heraklion, Crete , Greece
Purpose: Traditional Greek spirit beverages, tsipouro and tsikoudia, are very popular, consumed both bottled and in buck quantities, while many locals especially in agricultural areas are distilling their own fermented grape pomaces for private consumption. We analyzed distillates for the identification of chemical compounds produced during the primary metabolism of the fermentation process and a risk assessment approach was implemented in order to evaluate the magnitude of human risk.
Materials and methods: Totally, 56 drinks from the Greek market were collected, stored at -20oC and analyzed using either clinical chemistry analyzer or by gas chromatography coupled with flame ionization detector (GC-FID) and gas chromatography coupled with mass spectrometry detector (GC-MS).
Results and discussion: The concentration of cancinogenic compounds (ethanol, acetaldehyde), higher alcohols (isobutanol, isoamyl alcohol), esters (ethyl acetate) and methanol were measured in order to estimate the potential cancer risk and the dietary intake of the other compounds. European Food Safety Authority (EFSA) margin of exposure (MOE) was used for cancer risk characterization, while the no-observed-adverse-effect-level (NOAEL), the oral reference dose (RfD) and data from the Integrated Risk Information System (IRIS) were used in order to make estimates of the health risk assessment of the other compounds, in terms of the Health Risk Index (HRI).
Conclusion: The margin of exposure approach (MOE) for carcinogenic compounds, such as ethanol and acetaldehyde, was found to be less than 500 (mean value) well below to 10,000 as suggested by EFSA for public concern. Contradictory, the risk assessment of non carcinogenic compounds, such as alcohols, aldehydes and esters, identified a specific compound, the isobutanol, with health risk index (HRI) greater than 1, making those spirits possible of inducing health side effects (nausea, dizziness, headache and stupor) in case of huge consumption.
Keywords: alcoholic beverages, risk assessment, ethanol, acetaldehyde, isobutanol
Biodistribution of the new psychoactive stimulant 3,4-dimethylmethcathinone (3,4-DMMC) in Wistar rats assessed by gas chromatography-mass spectrometry (GC-MS) (#926)
D. Rouxinol1, D. C. Dias da Silva1, C. Teixeira1, A. C. Faria1, J. P. Silva1, F. D. Carvalho1, M. D. L. Bastos1, H. F. Carmo1
1 Faculty of Pharmacy, University of Porto, Biological Sciences, Porto, Portugal
3,4-Dimethylmethcathinone (3,4-DMMC) is a new psychoactive stimulant belonging to the first group of synthetic cathinones detected via the EU Early Warning System in 2010. As the pharmacokinetics of this drug is still unknown, the aim of this study was to validate a GC-MS methodology for the quantification of 3,4-DMMC in biological matrices and further apply it to the in vivo study of the drug biodistribution profile in Wistar rats.
Keywords: Dimethylmethcathinone, Gas Chromatography-Mass Spectrometry (GC-MS), Biodistribution
Effect of polystyrene nanoplastics on the polychaete Hediste diversicolor: A multibiomarker approach (#937)
M. S. S. L. Silva1, M. Oliveira1, P. Valente1, D. López1, E. Figueira1, M. Martins2, A. Pires1
1 University of Aveiro, Department of Biology, Aveiro, Portugal
Plastics became emergent pollutants all over the globe. Their increased production and persistence in the environment raise concerns about their impact on marine life. Polystyrene (PS) is one of the most produced plastic polymers, used in a large number of single-use/ low reuse products and is among the most frequently reported in the aquatic environment. Once in the marine environment, these plastics, like many others, will slowly break down into increasingly smaller particles, becoming more available for biota and threatening organisms both in the water column and sediments, as they tend to gradually sink to the ocean floor.
Polychaetes usually are the most abundant group in marine ecosystems and support much of the diversity at higher trophic levels. As benthic organisms, they are not only exposed to waterborne contaminants but also to contaminants present in the sediments. Thus, this study aims to assess the effects of PS on biochemical endpoints associated with oxidative status and energy metabolism, behaviour and regenerative capacity.
Hediste diversicolor specimens were collected in a reference site in Ria de Aveiro lagoon (Portugal), and after acclimatization they were exposed, for 28 days, to five different concentrations of 100 nm PS particles (0.0; 0.005; 0.05; 0.5; 5.0; 50.0 mg/L).
The results showed that burrowing activity of the organisms exposed to 0.005; 0.05 and 0.5 mg/L was significantly affected, with organisms taking more time to bury. The regeneration capacity, typical of these organisms, was not significantly different among tested concentrations, but a slight decrease was observed in exposed organisms.
PS demonstrated the ability to affect biochemical endpoints of the tested polychaetes. Overall, the antioxidant enzymes glutathione peroxidase (0.005 and 0.05 mg/L) and catalase (0.05 to 50 mg/L PS and the enzymes of phase II of biotransformation glutathione-S-transferases (0.05 to 50 mg/L PS were sensitive to PS exposure, displaying decreased activities. In contrast, an increase of electron transport was observed in organisms exposed at 0.05 to 50 mg/L of PS. Protein oxidation was reported in organisms exposed at 0.05 to 50 mg/L of PS.
Overall, the results highlight that PS induces alterations in the studied polychaetes, which may present potential impacts at the population level.
Keywords: Regeneration capacity, effects, sediments, nanoplastics
Biomarkers of exposure to estrogen-derived reactive metabolites: mass spectrometry-based methodologies to identify protein covalent adducts (#944)
C. Charneira1, S. A. Pereira2, A. M. M. Antunes1
1 Centro de Química Estrutural, Instituto Superior técnico, Ulisboa, Lisboa, Portugal
Estrone (E1) undergoes a CYP450-catalysed hydroxylation at position C-16, yielding 16-α-hydroxyestrone (16-α-OHE1) that is a reactive metabolite with the ability of covalently modifying the lysine residues of proteins, involving the formation of a Schiff base. This intermediate can be subsequently stabilized by two distinct mechanisms: via reductive stabilization, yielding the α-hydroxyamine adduct or via Heyns rearrangement, yielding a stable ketoamine adduct.
Upregulated levels of 16-α-OHE1 were identified in autoimmune  and in pulmonary hypertension patients  and the formation of 16-α-OHE1-derived protein covalent adducts is thought to have a role in the onset of some of these pathologies . Therefore, the development of analytical methodologies capable of unequivocally identifying and quantifying these adducts is a relevant pursuit.
We report herein the development of highresolution mass spectrometry-based methodologies for the identification of 16-α-OHE1 covalent adducts formed with the blood proteins hemoglobin and human serum albumin. The methodologies developed will be crucial towards the evaluation 16-α-OHE1-derived protein adducts as biomarkers of exposure to estrogen-derived reactive metabolites and as diagnosis tools of diseases more prevalent in women.
Funding: We thank Fundação para a Ciência e a Tecnologia (FCT), Portugal, for financial support through project UID/QUI/00100/2013 and iNOVA4Health-UID/Multi/04462/2013, as well as for the doctoral fellowship SFRH/BD/102846/2014 to C.C.
 Weidler C, Härle P, Schedel J, Schmidt M, Schölmerich J, Straub RH. J Rheumatol. 2004;31:489-94.
 Docherty CK, Harvey KY, Mair KM, Griffin S, Denver N, MacLean MR. Adv Exp Med Biol. 2018;1065:511-528.
 Dieker J, Berden JH, Bakker M, Briand JP, Muller S, Voll R, Sjöwall C, Herrmann M, Hilbrands LB, van der Vlag J. PLoS One. 2016, 25;11:e0165373
Keywords: 16-α-hydroxyestrone, protein covalent adducts, mass spectrometry, biomarkers of exposure.
DNA methylation patterns associated with seric metals concentration. Accessing effects of pollutants on human epigenetic modifications. (#955)
N. Y. Noronha1, M. Pinhel1, C. Nicoletti1, B. Affonso1, C. F. C. Brandão1, J. S. Marchini1, W. A. da Silva Jr.1, F. Barbosa Jr.2, C. B. Nonino1
1 University of São Paulo, School of Medicine, Ribeirão Preto, Brazil
Anthropogenic activities increase the exposure to metals and the major sources are drinking water and contaminated food. Despite knowledge about toxic potential of these compounds as well as its implication in non-transmissible chronic diseases, it is not yet established how it contributes to the aetiology and progression of these diseases. Large-scale genomic studies allow thousands of regions to be evaluated simultaneously and can provide a global approach for clinical studies. This study aims to analyze the effect of metals seric concentration on DNA methylation for further inferences regarding health problems related to environmental exposures. This is especially important because recently some environmental disasters occurred in Brazil, increasing exposure to toxic metals. DNA were extracted from women (n=42) and used to 450k beadchip methylation analysis, results are represented in beta values format which varies from 0 to 1. Serum was used to metals determination using ICP-MS, 15 metals were evaluated (Mg, Cu, Zn, Mo, Li, Rb, Sr, Se, Mn, Ni, Co, Cd, As, Al, Hg). Bioinformatics analysis were based on the Champ package pipeline. Singular Value Decomposition Analysis (SVD) was used to find the correlation of principal components and biological factors, bumphunter algorithm to find the metals-related Differentially Methylated Regions (DMRs), and linear regression models to find Differentially Methylated Positions (DMPs). Results: SVD analysis revealed that Hg, Al, Mo, As, Cd, Mn, Ni and Co contribute significantly on global DNA methylation. The number of significant DMRs for each metal were: Mo:21, Mn:17, Ni:18, Co:82, Cd:25, Al:56, Hg:32, and DMPs were Mo: 234, Mn:377, Ni: 271, Co:2, Cd, 1095, Al:245, Hg:567 (p<0.05). Conclusion: Seric metals concentration was related to DNA methylation beta values, and this may lead to pathway disruption due promoting changes in gene expression. This is an important topic in the field of chronic and non-communicable diseases which are the most important causes of death worldwide. This kind of study is important to support decisions regarding safety and legislations about metals usage and environment.
Keywords: metals, DNA methylation, epigenome, exposure
Biomonitoring of phthalate metabolites in urine from pregnant women in Crete, Greece (#963)
I. Katsikantami1, 2, V. Karzi1, 2, M. Tzatzarakis1, S. Sifakis3, 4, P. Xezonaki3, A. Rizos2, A. Tsatsakis1
1 Univerisity of Crete, Laboratory of Toxicology and Forensic Sciences, School of Medicine, Heraklion, Greece
Introduction: Exposure of pregnant women to phthalates was evaluated from the biomonitoring of six phthalate metabolites (MEHP, MEHHP, MEOHP, MiBP, MnBP, MBzP) in maternal urine.
Methods: A total of 100 urine samples were collected. Metabolites were deconjugated with enzymatic hydrolysis and extracted with 2 ml ethyl acetate for 20 min for three times. A solid phase extraction (SPE) procedure was applied to the organic extracts as a further cleanup process. The final extract was evaporated to dryness and reconstituted in methanol prior to instrumental analysis with LC-MS.
Results & Discussion: Positive samples were from 27% (MEHP) to 54% (MiBP) at median concentrations 17.9, 4.9, 41.5, 28.1, 46.7 and 6.1 ng/ml for MEHHP, MEOHP, MiBP, MnBP, MBzP and MEHP, respectively. MEHHP and MEOHP which are the oxidative metabolites of DEHP were significantly correlated with each other (rs=0.92, p<0.001) and also MnBP with MEOHP (rs=0.41, p=0.02) and MiBP (rs=0.95, p<0.001) and MBzP with MiBP (rs=0.70, p<0.001) and MnBP (rs=0.63, p=0.002), indicating their common sources of exposure. MEHP was significantly associated with frequent use of plastics for food storage (p=0.026). No significant associations came up for head circumference, birth weight and length, allergies, respiratory problems or other abnormalities.
Conclusion: Phthalate metabolites were detected in urine from pregnant women indicating their acute exposure to the pollutants during pregnancy. Use of plastics for food storage was proved to be a source of exposure to the compounds. It was found that acute maternal exposure had no effects for the infant development and health.
Keywords: phthalate metabolites, urine, pregnant women, biomonitoring
Selective citation in scientific literature on the human health effects of bisphenol A (#966)
M. Urlings1, B. Duyx1, G. Swaen1, L. Bouter2, 3, M. Zeegers1, 4
1 Maastricht University, NUTRIM School of Nutrition and Translational Research in Metabolism, Maastricht, Netherlands
Introduction: Bisphenol A is highly debated and studied in relation to a variety of health outcomes. This large variation in the literature makes BPA a topic that is prone to selective use of literature, in order to underpin one’s own findings and opinion. Over time selective use of literature, by means of citations can lead to skewed knowledge development and a biased scientific consensus. In this study we assess which factors drive citation and whether this results in the overrepresentation of harmful health effects of BPA.
Methods: A citation network analysis was performed to test various determinants of citation. A systematic search identified all relevant publications on human health effect of BPA. Data were extracted on potential determinants of selective citation, such as study outcome, study design, sample size, journal impact factor, authority of the author, self-citation and funding source. We applied random effect logistic regression to assess whether these determinants influence the likelihood of citation.
Results: 169 Publications on BPA were identified, with 12,432 potential citation pathways of which 808 citations occurred. Positive studies have a 1.5 times greater chance of being cited compared to negative studies. Additionally, authority of the author and self-citation are consistently found to be positively associated with the likelihood of being cited. Overall, the network seems to be highly influenced by two highly cited publications, whereas 60 out of 169 publications received no citations.
Conclusion: In the literature on BPA, citation is mostly driven by positive study outcome and author-related factors, such as high authority within the network. Interpreting the impact of these factors and the big influence of a few highly cited publications, it can be questioned to which extent the knowledge development in human literature on BPA is actually evidence-based.
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Keywords: Questionable Research Practise, selective citation, citation analysis, methodology, bisphenol A
Target Safety Assessments: Evaluation of the Toxicological Risk of Targeting FRS (Phenylalanyl-tRNA Synthetase) in the Treatment of Malaria (#16)
J. Barber1, D. Baud2, P. Willis2, C. Sadler1, R. Roberts1, 3
1 ApconiX, Alderley Edge, United Kingdom
Phenylalanyl-tRNA synthetase (FRS) is a highly conserved enzyme that catalyzes the ligation of phenylalanine to its cognate transfer tRNA during protein synthesis. Due to its vital role as part of the translational machinery, FRS has been identified as a potential target to treat the malaria parasite. However, since target-related toxicity accounts for >50% of all drug project failures, it is vital to understand the potential unintended consequences of target modulation in non-plasmodium (mammalian) species to assist in the determination of the required plasmodium/human safety ratio. We conducted a comprehensive in silico target safety review to understand the role of FRS in normal physiology as a basis for evaluation of the potential toxicity of FRS inhibitors. Based on published literature, it is clear that eukaryotic cells harbour two different types of FRS: the heterotetrameric cytosolic alpha (FRSA) and beta forms, and the monomeric mitochondrial forms. Pathogenic variants in FRS2 (encoding the human mitochondrial FRS) have been associated with phenotypes ranging from spastic paraplegia to fatal infantile Alpers encephalopathy. FRSA knockout mice are homozygous lethal. Heterozygote phenotypes include abnormal bone morphology, decreased bone mineral density, decreased circulating chloride and sodium levels, impaired glucose tolerance and increased total body fat amount. Based on these observations, we predict that potential target organs of toxicity caused by inhibition of FRS could include bone, immune system, kidney, liver, muscle, and the nervous system. Specifically, there may be a risk of abnormal bone development, perturbed glucose metabolism, immunosuppression, nephrotoxicity, reduced liver function, myopathy and an increased risk of epilepsy. Based on this toxicological profile, inhibition of host FRS could be a serious limitation; therefore, the specificity and selectivity of compounds will be a key for their success. However, a single genomic copy of mitochondrial FRS is targeted to the parasite mitochondria and is exclusive to malaria parasites within the apicomplexan phyla, hence drug targeting of FRS presents a unique opportunity to potentially target malarial FRS specifically. Nonetheless, it would be sensible to conduct an early rodent investigative study looking at in life effects and potential target organs to help identify whether the risks our in silico analysis has identified actually occur in vivo with inhibitors of FRS.
Keywords: Target safety assessment, drug safety, discovery toxicology, investigative toxicology, drug project toxicology
Blood level measurement of urea and creatinine in methamphetamine abuser patients (#85)
A. Ebadollahinatanzi1, G. Arabrahmatipour2
1 Agricultural Research, Education and Extension Organization (AREEO), Department of Medicinal Plants, Imam Khomeini Higher Education Center, Karaj, Iran (Islamic Republic of)
Purpose: The methamphetamine (C10H15N), known as the "Crystal", is a psychoactive substance and a stimulant for the nerves. Due to its psychomotor stimulating features such as increased alertness, euphoria and mood elevation, the drug can be greatly abused. The chronic methamphetamine abuse may cause damage in some organs including brain, heart, and liver of human. The study was aimed to measure the blood levels of urea and creatinine as kidney function indices in methamphetamine abuser and to predict urea dependent drug toxicity.
Methods: The specimens included 52 serums of people who had more than one year of history of drug addiction and consumed between 1-2 grams of crystal per day. They were tested for serum urea and creatinine levels. Experiments were performed in fasting mode using a quantitative diagnostic kit. Normal values for this method for urea and creatinine were 20-43 and 0.8-1.4 mg/dL, respectively.
Results: The mean age of patients was 29.61 ± 5.39 years. The mean values obtained from serum urea were found to be 17.98 ± 3.49 mg/dL. The highest and lowest values of urea were 23 and 10 mg/dL, respectively. The mean serum creatinine level was 0.90 ± 0.13 mg/dL and the highest and lowest of that were obtained as 1.10 and 0.60 mg/dL, respectively.
The study concludes; the creatinine levels in methamphetamine abusers are not significantly differed in comparison with normal population, whilst the blood urea level of these patients can be decreased due to disorders in protein synthesis which accompanied by methamphetamine toxicity.
Keywords: Methamphetamine abuser, Urea, Creatinine
Determination of the most susceptibility of bacteria to antimicrobial agents in endophthalmitis (#86)
G. Arabrahmatipour1, A. Ebadollahinatanzi2
1 Tehran University of Medical Sciences, Farabi Hospital Laboratory, Tehran, Iran (Islamic Republic of)
Purpose: Bacterial endophthalmitis is a rare and serious complication that may occur as a result of eye surgery. In this disease, the role of timely diagnosis and treatment as well as the use of appropriate antibiotics to prevent blindness is very important. The aim of this study was to laboratory survey for determination of the most antimicrobial susceptibility of pathogens producing bacterial endophthalmitis.
Methods: The samples were culture positive isolates of vitreous (n=101) from patients who had been referred, due to bacterial endophthalmitis, to Farabi Ophthalmology Hospital of Tehran University of Medical Sciences in 2013. Determination of the maximum susceptibility to antibiotics in the common bacterial pathogens agent of the disease was performed on the basis of laboratory standards and antibiotic disc diffusion method. The antibiotics disc were included of Cefazolin(CZ), Ceftazidime(CAZ), Chloramphenicol(C), Amikacin(AN), Ciprofloxacin(CP), Trimethoprim(SXT), Gentamycin(GM), Vancomycin(VA), Oxacillin(OX), Imipenem(IMP).
Results: Our results showed that, in this disease, the most common gram positive and negative bacteria are Staphylococcus epidermidis (35.58%) and Pseudomonas aeruginosa (12.5%), respectively. Among gram-positive species, Staphylococcus epidermidis was found to be most susceptible (100 %) to CZ antibiotic. Whilst for gram-negative bacteria, Pseudomonas aeruginosa was shown to be most susceptible (100%) to CP, GM, IMP antimicrobial agents and also (91.67 %) to AN antibiotic. In the light of this study physicians would be able to have a predictable susceptibility pattern for treatment of bacterial endophthalmitis.
Keywords: Antimicrobial agents, Susceptibility, Endophthalmitis
Predicting the need for hospitalization of intoxicated patients: a pilot study (#91)
C. C. Hunault1, L. Hondebrink1, S. J. Rietjens1, D. Dekker1, 2, I. de Vries1, D. W. de Lange1, 3
1 University Medical Center Utrecht, Dutch Poisons Information Center, Utrecht, Netherlands
Intoxicated patients are frequently admitted to the Emergency Department (ED) whereas hospital admission is not always necessary. No predictive models exist that could improve ED triage of these patients. In a pilot study, we aimed at identifying potential predictors for developing such a model.
We conducted a prospective cohort study of ED presentations involving intoxications in a Dutch University Hospital during a 1.5-year period (January 2015 – July 2016). The primary outcome was “necessary hospitalization”. This outcome was determined in retrospect by selecting patients according to their Poisoning Severity Score (moderate & severe categories) and/or the need for treatment on the ward. Potential predictors were covariates available in the first hours following ED presentation, including vital signs and findings based on clinical examination, ECG and laboratory analysis. After multiple imputation of the missing values, selection and prediction optimization were achieved using an Elastic Net regularization. The predictive performance was evaluated by using a cross-validation approach.
417 ED presentations were included for analysis. In 190 cases (45.6 %), hospitalization appeared necessary in retrospect. The strongest risk factors for a necessary hospitalization (factors with OR >1) were: ingestion of at least one modified-release preparation, hypotension, and pH <7.37. Normal glucose and pH values were strong protective factors (with OR <1). The expected severity, based on the reported exposures (mg product / kg bodyweight) and our Poisons Center’s comprehensive toxicological database, was also predictive. AUCs for predicting a necessary hospitalization were on average 0.70, depending on the included predictors. The calibration plots showed a good fit of the data.
This pilot study identified predictors of necessary hospitalization of intoxicated patients. Our findings should be confirmed in a study including a larger number of patients.
Keywords: Intoxication, Emergency Department, predictive model, pilot study, Poisoning Severity
The carbonylation pattern in type 2 diabetes using capillary electrophoresis (#142)
C. Purdel1, I. Dima-Adam1, G. Moldoveanu3, D. Margina2
1 UMF Carol Davila, Toxicology, Bucharest, Romania
Purpose: Type 2 diabetes like many other chronic diseases, is strongly related to oxidative stress. One of the biochemical consequences of oxidative stress is the carbonylation of the proteins. The aim was to find a pattern in the electrophoregrams of carbonyl proteins from patients with diabetes.
Materials and Methods: Samples from patients with type 2 diabetes were evaluated using derivatization with 2,4-dinitrophenylhydrazine and analysed using a capillary electrophoresis (CE) method. Electrophoretic separation was performed using an aqueous electrolyte system: 20 mM borate buffer with pH 9.0 and 15% w/w dextran 70. Injection was performed in the hydrodynamic mode at 0.5 psi for 10s, and the applied voltage was - 25 kV. The detection was performed at 370 nm, 365 and 214 nm.
Results: CE method offers qualitative information about the carbonylated species through electropherograms and the peak characteristics. Based on the retention time (RT), the peaks resulted from samples from patients with type 2 diabetes were grouped within 10 groups. We determined the average number of peaks for every patient serum sample, found in each RT group. This is an index showing the fragmentation degree which leads to the formation of more carbonylated species that have very close molecular mass. More than one third of the samples exhibited peaks in half of the groups. A suitable measurement as an indicator of a protein carbonylation pattern was the percentage of patients whose serum samples issued peaks in a certain RT group. 75% of the samples had carbonyls with RT inside group with lower RT values, meaning that in type 2 diabetes mellitus, carbonylation most frequently occurs on smaller fractions. We also observed a specific fragmentation dynamic.
Conclusion: A preliminary pattern of protein carbonylation associated to type 2 diabetes mellitus was issued. Further studies are needed to elucidate the chronology of protein chain lysis and carbonylation.
Keywords: carbonyl proteins, diabetes, electrophoresis
Spectrum of acute drug toxicity during the most popular house and techno party in the world (#189)
K. Slankamenac1, D. Müller2, A. Herzog1, H. Kupferschmidt3, A. von Eckardstein2, D. Keller1
1 University Hospital Zurich, Emergency Department, Zurich, Switzerland
Background: Since 1991, the Street Parade, world’s most popular house and techno parade in Zurich, is still a mecca for ravers. One Saturday in every August, about one million visitors celebrate this initially peaceful event which stands for love, freedom and tolerance. However, extensive drug abuse has also been commonly seen. The prevalence of acute drug toxicity (ADT) due to novel psychoactive substances (NPS) during the Street Parade is unknown. Therefore, the aim was to investigate the drug spectrum of acute intoxicated patients from the Street Parade presenting in the Emergency Department (ED).
Methods: We investigated consecutively urine samples of acute intoxicated patients who participated at the Street Parade and presented in a Swiss tertiary care ED in 2017 and 2018. The endpoints were the analysis of the drug spectrum and assessment of the prevalence of ADT by NPS. Samples were analyzed by a screening method using liquid chromatography coupled to high-resolution mass spectrometry. Substances were identified by their theoretical exact mass and by comparing acquired tandem mass spectrometry (MS/MS) to library spectra.
Results: In total, we analyzed 47 urine samples. Ten patients presented with symptoms of ADT but only a wide spectrum of different medications was detected. In 20 patients (42.5%), alcohol without any other drug was identified. Finally, 17 intoxicated patients (36.2%) consumed drugs plus alcohol. The three leading drugs were cocaine (21.3%), 3,4-methylenedioxymethamphetamine (MDMA) (19.1%) and tetrahydrocannabinol (THC) (17.0%) followed by methamphetamine (8.5%), methylphenidate (6.4%) and 2.1% for each lysergic acid diethylamide and amphetamine. Furthermore, one patient (2.1%) showed an abuse of NPS (methylon) in combination with alcohol, cocaine and MDMA. An overdose of methamphetamine occurred in five patients in 2018 whereas no overdose of methamphetamine was detected in 2017.
Conclusion: Cocaine, MDMA and THC in combination with alcohol are the most prevalent drugs in Street Parade patients whereas NPSs are still rare. Methamphetamine intoxications seem to increase. Thus, future preventive strategies need to sensitize the rave scene about the drug spectrum and possible health consequences.
Keywords: acute drug toxicity, novel psychoactive substances, drug spectrum, prevalence
Prevalence of clinical intoxications: a study of drug intoxications profile in an emergency department of a Portuguese hospital (#238)
P. Ferreira1, C. Fonseca1, 2, E. Gallardo3, A. R. T. S. Araujo1, 2, 4
1 Polytechnic Institute of Guarda, School of Health Sciences, Guarda, Portugal
Human intoxication processes have been one of the most serious public health problems due to the lack of control and prevention of intoxication associated to easy access of the population to a high number of substances with a high degree of toxicity. Acute intoxications represent one of many causes of admission to hospital emergency services. The profile of clinical intoxications in Portugal is not well established and therefore its assessment is of utmost importance to help healthcare professionals to respond more efficiently and adequately to intoxications episodes.
This work describes the retrospective and descriptive analysis of the adult patients who were classified as eventually intoxicated (overdose or poisoning) by the Manchester Triage System at the time of entry into the emergency department of the Hospital da Senhora da Oliveira in Guimarães city in the period of January 1, 2017 to May 31, 2018.
Over the studied period, of the 837 possible intoxications cases observed, 221 patients were seen with a drug-related intoxication and 492 with alcohol-related intoxication.
Of the drug-related intoxications studied, 78.7% involved female individuals, whereas 21.3% were male. The average age was approximately 44 years old. Most of these intoxications were voluntary (96.8%), and 54.8% of those without suicidal ideation. In 99.5% of the episodes, the administration route was oral. The majority of patients had mono-intoxication (84.6%) and drug and alcohol intoxication accounted for 10.6%. The pharmacological group more frequently mentioned were the anxiolytics, hypnotics and sedatives (54.8%), followed by (42.1%) and antiepileptics and anticonvulsants (15.4%). The average number of drugs involved in intoxications was 2. Intoxicated individuals received mostly gastrointestinal decontamination treatment, such as gastric lavage (67.0%) and activated charcoal (58.5%). The antidotes were given in 20.21% of the intoxications, where flumazenil represented 87% followed by acetylcysteine (13%). Most exposure patients (67.5%) were admitted.
This work contributed to the documentation and identification of the occurrence of clinical intoxications in Portugal and highlight the need of the improvement in the prevention and education in this field.
Keywords: Clinical intoxications, emergency service, profile intoxications, Portugal, toxic agents
Nutritional modulation of environmental toxicity and implications in inflammatory diseases (#253)
B. Hennig1, M. Petriello1
1 University of Kentucky, Lexington, Kentucky, United States of America
Exposure to environmental pollutants is associated with the development of many diseases through multiple mechanisms including the induction of chronic inflammation. Many organic pollutants are persistent and express high stability and ubiquity in the environment. For example, coplanar polychlorinated biphenyls (PCBs), which act as an agonist of the aryl hydrocarbon receptor, exert toxic effects on the endothelium and associated vasculature. Atherosclerosis, a chronic inflammatory disease initiated by vascular endothelial cell dysfunction, remains the leading cause of death worldwide. Furthermore, PCB-induced toxicity has been linked to increased expression of pro-inflammatory caveolin-1, the major structural protein in caveolae membrane domains. Caveolae are particularly abundant in endothelial cells, where they play a major role in the regulation of vesicular trafficking and signal transduction. PCBs are also known to affect the cellular redox status, which may initiate antioxidant responses through nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signaling. Our data show that PCB toxicity is modulated by cross-talk between caveolae and Nrf2 signaling. Cav-1 silencing (siRNA treatment) increased levels of Nrf2-ARE transcriptional binding, resulting in higher mRNA levels of the antioxidant genes glutathione s-transferase and NADPH dehydrogenase quinone-1 in both vehicle and PCB-treated systems. Nutrition may function as a modulator of vulnerability to environmental insults. Increasing evidence suggests that diets high in plant-derived bioactive food components (e.g., polyphenols) and omega-3 lipids are associated with a reduced risk of chronic inflammatory diseases such as atherosclerosis. Current data suggest that endothelial cell dysfunction and inflammatory events induced by exposure to persistent environmental pollutants such as coplanar PCBs can be downregulated by polyphenols, such as flavonoids, as well as by omega-3 PUFAs. Our data suggest that PCB-induced inflammation is a trigger of cardiovascular disease risks and that dietary polyphenols and omega-3 lipids exhibit anti-inflammatory protection via caveolae and cytosolic Nrf2 signaling.
(Supported in part by NIEHS/NIH grant P42ES007380 and the Kentucky Agricultural Experiment Station)
Keywords: environmental toxicity, Atherosclerosis, inflammation
Toxicity / adverse effect predictions based on computational toxicology techniques and large-scale databases (#277)
1 Meiji Pharmaceutical University, Medical Molecular Informatics, Tokyo, Japan
Understanding the features of chemical structures related to the adverse effects of drugs is useful for identifying potential toxicities/adverse effects of new drugs and chemical products. This can be based on the limited information available from post-marketing surveillance, assessment of the potential toxicities of metabolites and illegal drugs with unclear characteristics, screening of lead compounds at the drug discovery stage, and identification of leads for the discovery of new pharmacological mechanisms. This present study developed techniques used in computational toxicology such as quantitative structure-activity (toxicity) relationship (QSAR / QSTR) analysis to investigate the content of large-scale spontaneous report databases of adverse effects such as FDA Adverse Event Reporting System (FAERS; JAPIC-AERS) and Japanese Adverse Drug Event Report database (JADER). Furthermore, volcano plotting, a new visualization method for clarifying the relationships between drugs and adverse effects via comprehensive analyses, will be introduced. These analyses may produce a great amount of data that can be applied to drug repositioning.
Keywords: adverse effect, FAERS, JADER, QSAR, computational toxicology
Transcriptomic approach to improve the understanding of 5- fluorouracil (5-FU) induced intestinal toxicity in vitro and in vivo (#348)
D. F. Rodrigues1, T. de Souza1, L. Coyle2, Y. Schrooders1, F. Jardi3, S. de Jonghe3, M. van Heerden3, L. Lammens3, D. Jennen1, J. C. Kleinjans1, T. M. de Kok1
1 Maastricht University, Department of Toxicogenomics, Maastricht, Netherlands
5-fluorouracil (5-FU) is a classical cytotoxic agent widely used in cancer therapy that has been associated with adverse drug reactions (ADRs) in several organs, including the Gastrointestinal (GI) tract. 5-FU has shown to induce acute toxicity in small and large intestines, supported by patients’ reports of diarrhoea, nausea and abdominal pain that often lead to interruption of cancer treatments, impairing patients’ quality of life and survival to the disease. Nevertheless, the understanding of the molecular mechanisms underlying 5-FU toxicity and how these relate to the ADRs experienced by patients is limited. In this study, we aim to expand our knowledge by establishing 5-FU induced transcriptomic responses and cytotoxicity in different models. In vitro human intestinal organoids, derived either from colon or small intestine (SI), were exposed to 0, 10, 100, 1000 µM of 5-FU. The in vivo study consisted in exposing mice to 0, 20 and 50 mg/kg of 5-FU. Both in vitro and in vivo exposures are based on PBPK model calculations considering the doses recommended to cancer patients. Following the in vitro exposure, cell viability and apoptosis were assessed as functional endpoints. Moreover, gene expression profiles of non-exposed versus exposed samples were also assessed for both models. Transcriptomics was measured by performing RNA sequencing, after which the most affected biological pathways and respective differentially expressed genes (DEGs) were evaluated. Cell cycle, DNA damage/repair, p53 signalling, mitochondrial ATP synthesis, metabolism and apoptosis were amongst the most altered pathways unveiled by the in vitro assays, demonstrating time and dose effects, particularly in colon organoids. In addition, comparison of the functional and transcriptomic outcomes is evaluated between both in vitro and in vivo experiments. In further studies, the molecular responses will be used to build a multi-scale predictive model of drug-induced intestinal toxicity. Taken together, this study provides insight into possible toxicity mechanisms as well as the in vitro to in vivo translation of results generated in organoids. Moreover, it potentially leads to a step towards the improvement of the quantitative systems toxicology (QST) in predicting 5-FU effects in intestines.
Keywords: 5-FU, intestinal toxicity, organoids, transcriptomics
Metabolomics evaluation of urine from PCa patients by GC-MS and NMR spectroscopy (#444)
A. R. Lima1, J. Pinto1, C. Jerónimo2, 3, R. Henrique2, 3, 4, M. D. L. Bastos1, M. Carvalho1, 5, P. Guedes de Pinho1
1 UCIBIO/REQUIMTE, Faculty of Pharmacy, University of Porto, Department of Biological Sciences, Laboratory of Toxicology,, Porto, Portugal
Prostate cancer (PCa) is one of the most common types of cancer in men. In this work, 41 PCa and 42 non-cancer (control) urine samples were analyzed by GC-MS (direct injection after derivatization) and 1H NMR spectroscopy in order to obtain a comprehensive PCa metabolic signature. Multivariate statistical analysis was used to evaluate the ability of the GC-MS and 1H NMR urinary metabolic profiles to distinguish PCa from controls. The created discriminant models were further validated using an external validation set (n= 18 PCa and n= 18 controls). The GC-MS model presented a sensitivity of 94%, a specificity of 84% and an accuracy of 92%, whereas the 1H NMR model presented a sensitivity of 78%, a specificity of 94% and an accuracy of 86%. In GC-MS approach we disclosed 15 metabolites significantly altered in PCa (including 3 unidentified compounds) and in 1H NMR approach we revealed 12 metabolites significantly altered in PCa (including 3 unidentified compounds). Among them, 12 metabolites were found over-expressed in PCa cases, namely sarcosine, propylene glycol, oxalic acid, threose and threitol (identified through GC-MS), and leucine, valine, 2-hydroxyvalerate, 2-hydroxyisobutyrate, pyruvate, acetone and hydroxyacetone (identified through 1H NMR), while 9 metabolites were down-expressed, comprising gluconic acid, arabitol, fucitol, ribitol, mannitol, glucose and myo-inositol (identified through GC-MS) and 2-furoylglycine and trigonelline (identified through 1H NMR). To the best of our knowledge, this is the first study reporting significant alterations in the levels of propylene glycol, oxalic acid, threose, threitol, hydroxyacetone, fucitol, mannitol, 2-furoylglycine and trigonelline in PCa biological samples. Based on these results, we were able to associate PCa metabolic signature to the dysregulation in 14 biochemical pathways, being the majority of these pathways associated with amino acids and energetic metabolism. So, our results prove the potential of GC-MS and 1H NMR metabolic signatures for discrimination of PCa patients from control subjects and towards a better understanding of the metabolic dysregulations associated with PCa progression and development.
A.R.L thanks Fundação para a Ciência e Tecnologia (FCT), Portugal, for her PhD grant (SFRH/BD/123012/2016). 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: Prostate cancer, Metabolomics, Gas chromatography–mass spectrometry, Proton nuclear magnetic resonance, Urine
The role of exosomes from human MSC 3D cultures in wound healing (#580)
S. P. Camões1, J. S. Rodrigues1, M. Gaspar1, S. Simões1, R. Ferreira2, A. Barros3, R. Vitorino2, N. G. Oliveira1, J. M. Santos4, J. P. Miranda1
1 Faculty of Pharmacy, Universidade de Lisboa, Research Institute for Medicines, Lisbon, Portugal
Exposure to toxic agents frequently leads to cutaneous toxicity, which often induces skin irritation and impairs the healing process. The current available therapeutic options often fail to promote full tissue regeneration, and therefore novel strategies to develop effective therapies for improving the healing process are needed. In this context, mesenchymal stem cells (MSCs) gained relevance due to their role in tissue regeneration via paracrine mechanisms. Recently, the secretion of exosomes has been suggested as a dominant mechanism by which MSCs exert their healing function. Our study aimed at evaluating the role of exosomes, derived from umbilical cord matrix-derived MSCs primed by 3D culturing, on cutaneous wound healing using in vivo methodologies paired with integrative proteomic analysis. As such, the whole secretome was obtained by collecting and concentrating the culture media conditioned by MSCs in 2D (CM2D) and 3D (CM3D). Exosomes were isolated from CM2D (Exo2D) and CM3D (Exo3D) by size exclusion chromatography. Size distribution of the isolated exosomes (135 ± 54 nm and 265 ± 37 nm for Exo2D and Exo3D, respectively) pointed out the influence of the culture system in its morphology, however without compromising the presence of CD9 and CD81 exosomal surface markers. Moreover, proteomic analysis of the isolated exosomes revealed that 3D conditions lead to higher protein diversity than the 2D environment. Indeed, Exo3D show 18 specific proteins, some of which are involved in cell chemotaxis, division and proliferation. Accordingly, the evaluation of the effect of Exo3D/2D and CM3D/2D in skin regeneration using an in vivo rat wound-splinting model suggested a significantly higher therapeutic potential of exosomes over the MSC secretome. Macroscopic observations show that Exo-treated wounds exhibited accelerated wound closure when compared to control wounds. Accordingly, histological examination revealed that Exo3D-treated wounds show an improvement in the healing profile, by promoting wound margin closure and complete tissue regeneration with hair re-growth. Overall, the results suggest that 3D MSCs-derived exosomes promoted wound healing, granting their potential new role as active players in cell-free-based therapies for different pathological or toxicological contexts. Moreover, omics approaches may help on the identification of new markers involved in the healing process and ultimately improve therapeutic outcomes.
The work was financially supported by Fundação para a Ciência e a Tecnologia (FCT) through TUBITAK/003/2014, PTDC/MED-TOX/29183/2017,UID/DTP/04138/2013, PD/BD/114280/2016 to S.P.C. and IF/00286/2015 to R.V; Universidade de Lisboa through BD2017/ULisboa and COST Actions CA16113 and CA16119.
Keywords: Human neonatal mesenchymal stem cells, exosomes, 3D cultures, wound healing
Manganese in the diets of infants and young children: A review of manganese in the diets of infants and children by the UK Committee on Toxicity. (#584)
F. Hill1, B. Doerr1, R. Acheampong2, J. Shavila2, D. Gott1
1 Food Standards Agency, Chemical 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)
Manganese is an essential micronutrient in the human diet, but high chronic exposures have been associated with a range of neurological signs and symptoms which combine, in severe cases, to cause a Parkinson-type syndrome called manganism. It is unclear whether children are more sensitive than adults but there is a large body of literature linking high manganese exposure, primarily measured using biomarkers such as concentrations in hair, tooth or blood, with neurodevelopmental effects in children such as IQ decrements and attention deficit hyperactivity disorder. Humans may be exposed to manganese found naturally in the environment and from industrial processes. In the UK, manganese exposure in workers, and the general public, from industrial activity is minimal, but there are uncertainties over the impact of dietary manganese on the neurological development of infants and young children.
The UK Committee on Toxicity have reviewed manganese exposure of UK infants and young children using data from the analysis of food samples and two dietary surveys: the national diet and nutrition survey, and the diet and nutrition survey of infants and young children. They have compared these exposures with current health-based guidance values, primarily that set by the WHO in their Guidelines for Drinking Water Quality. The Committee found that estimated exposures from the diet exceed current health-based guidance values for manganese in nearly all age groups. There is considerable uncertainty on the degree to which manganese in the diet is absorbed in the gastro-intestinal tract, and there are some inconsistencies in the data on adverse effects, such as contradictory sex-related differences and the nature of the dose-response relationship. There are also uncertainties in the exposure assessment. Therefore, the health risk from manganese in the diets of infants and young children is unknown. The aim of this poster is to highlight the limitations in the database on manganese and identify further research to help close these data and knowledge gaps.
Keywords: Manganese, Absorption, diet, neurological, infants and young children
Poisons and poisonings by snakes of medical importance in Angola (#601)
P. R. Oliveira1, M. D. L. Bastos2, D. V. Tambourgi3
1 Centro Nacional de Investigação Científica, Ministério do Ensino Superior Ciência e Tecnologia e Inovação, Luanda, Angola
Snakebite is considered a neglected tropical disease responsible for high morbidity and mortality in Asia and sub-Saharan Africa. In Angola the scenario is unknown.
The objectives of this study were: to evaluate the performance of health professionals towards to snakebite poisonings in four Angolan regions; ii) to biochemically analyse the venoms of the snakes collected in these regions; iii) to evaluate the immunogenicity of the venoms and produce the experimental antivenom serum.
A three-step methodological design was carried out. First-step - a prospective cross-sectional descriptive observational field study including 151 health professionals; Second-step - Biochemical characterization of the venoms of captured snakes, including: i) protein content of the venoms and characterization of their electrophoretic profiles; ii) determination of the glycosylation profile of the venom proteins; iii) evaluation of the proteolytic activity; iv) detection of the phospholipase activity; and v) evaluation of the hyaluronidase activity in a sample of venoms of eigth snakes. In the third step - the evaluation of the immunogenic potential of Angolan snake venoms in a murine animal model and detection of the antigenic components against the murine sera.
The results of the present investigation allowed to conclude that: 1 - there is a low level of knowledge of health professionals in the diagnosis, evaluation and therapy of Angolan snakebites; 2 - the clinical manifestations of Angolan snakebites can be local, systemic, ophthalmological and neurological; 3 - the venoms of snakes involved in ophidian accidents, have a remarkable intraspecies biochemical variability, related to the sex of the animals and their regions of origin. 4 - Venoms of snakes of medical importance in Angola are immunogenic. Viperid venoms (B. arietans, B. gabonica) were more immunogenic than the elapidic venom (N. nigricollis) and the murine sera produced recognized a considerable number of venom components.
Kasturiratne, A., Wickremasinghe, A. R., Silva, N. De, Gunawardena, N. K., de Silva, N., Gunawardena, N. K., … de Silva, H. J. (2008). The Global Burden of Snakebite: A Literature Analysis and Modelling Based on Regional Estimates of Envenoming and Deaths. PLoS Medicine, 5(11), e218–e218. https://doi.org/10.1371/journal.pmed.0050218
P.R.S.Oliveira et al. (2018) Snake venoms from Angola: Intra-specific variations and immunogenicity. Toxicon, 148 ( February),85-94. https://doi.org/10.1016/j.toxicon.2018.04.013
Slagboom, J., Kool, J., Harrison, R. A., & Casewell, N. R. (2017). Haemotoxic snake venoms : their functional activity , impact on snakebite victims and pharmaceutical promise. British Journal of Haematology, 177(February), 947–959. https://doi.org/10.1111/bjh.14591
Keywords: Knowledge of health professionals, Angola, snakevenom, biochemical profile, murine serum
A fatal case related to heroin injection (#678)
C. Jing1, Z. Y. Feng1, W. R. Hua1, W. A. Hua1, Z. Bo1, L. J. Yi1
1 Insitute of Forensic Science, Ministry of Public Security, forensic toxicoly, Beij, China
Objectives: To report an accidental death caused by injection of heroin. A 23-old man, who was very strong, was found dead after heroin injection for half an hour in a car. The death scene investigation showed evidence of acute intoxication with previous doses of heroin consumption after an abstinence period time.
1. Meissner M, Recker S, Reiter A, Friedrich HJ, Oehmichen M. Fatal versus non-fatal heroin "overdose”: blood morphine concentrations with fatal outcome in comparison to those of intoxicated drivers. Forensic Sci Int 2002,130:49~54.
Keywords: heroin death, Autopsy, Morphine/codeine
Olanzapine induced hepatotoxicity is investigated by individual susceptibility and metabolomics (#704)
B. Karahalil1, A. Elkama1, M. Ak2, E. Nemutlu3, N. İlik1
1 Gazi University, Department of Toxicology / Faculty of Pharmacy, Ankara, Turkey
Hepatotoxicity is one of the deleterious effects of antipsychotic drugs. Hepatic function is monitored by serum aminotransferase levels. However, serum aminotransferases may not be liver-specific and sensitive. Alpha-glutathione S-transferase (α-GST) may be liver specific due to having greater cytosolic concentration, shorter half-life and smaller molecular weight than aminotransferases. GST enzymes catalyze the biotransformation and detoxification reactions of many drugs. Single nucleotide polymorphisms on GSTs can change the enzyme activities and therefore in drug response. Antipsychotic drugs and psychotic disorders can change metabolomics and are related to individual susceptibility. We aimed to investigate whether α-GST can be a better indicator of hepatotoxicity rather than others and whether the polymorphisms on GST enzymes have an effect on hepatotoxicity among individuals. Blood samples were taken from 30 patients, who have psychotic disorders, treated with olanzapine at 3 different time periods: T1, before medication; T2, 10 days after medication and T3, 3 months after medication. GSTT1, M1 and P1 genotyping was performed by PCR-RFLP. Serum α-GST enzyme activities were measured by ELISA. We observed statistically significant increase in α-GST enzyme activity (p=0,047) and alanine aminotransferase (ALT) levels (p=0,006) in T2 compared to those in T1. However, the percentage increase in ALT between T1 and T2 was greater than that in α-GST. We did not find any significant association between α-GST enzyme activities and GSTs variations. Schizophrenia-specific metabolomics pattern was observed and furthermore, tryptophan levels were high as we expected.
Keywords: antipsychotic drug induced hepatotoxicity, serum α-GST activity, polymorphisms, PCR-RFLP, metabolomics
Screening and Regulatory approaches to risk assess in vitro chemical mediated changes in thyroid function. (#785)
1 Concept Life Sciences, Chapel-en-le-Frith, United Kingdom
The hypothalamic-pituitary-thyroid axis (HPT axis) is conserved across vertebrate evolution. Perturbation of thyroid hormone homeostasis (THH) can lead to adverse effects in thyroid function affecting growth, metabolism and cognitive function. In utero, appropriate thyroid hormone concentrations are absolutely required for normal nervous system development. Chemical disruption of THH can occur via a number of mechanisms, including increased hepatic thyroid hormone clearance, inhibition of iodide transport into the thyroid (sodium/iodide symporter), inhibition of iodide oxidation (thyroid peroxidase) and inhibition of thyroid hormone deiodination (deiodinases). To understand the effect of chemicals on these functions the following assays are utilised. 1. in vitro primary hepatic thyroid hormone metabolism (multiple species), 2. thyroid peroxidase inhibition (multiple species), 3. deiodinase inhibition (rat and human), 4. sodium/iodide symporter inhibition (rat). Rat sodium iodide symporter inhibition and rat and human deiodinase 1,2 and 3 inhibition assays are currently being validated. Here we report the validation of in vitro rat, dog, pig and human TPO inhibition and in vitro primary hepatocyte metabolism of thyroxine (T4). In concurrence with the literature, TPO inhibition by 6-propyl-2-thiouracil (PTU) shows broad sensitivity across the species tested. in rat (IC50 2.2 µM ) Dog (IC50 17.7 µM ) pig (IC50 7.6 µM) and human (IC50 50.9 µM ). T4 metabolism by primary cultures of human and rat hepatocytes show a consistent dose response induction (approximately 2 fold over vehicle control) in response to reference item administration. This suite of assays will be used to generate data in support of the current requirements for endocrine disruption hazard assessment. Alternatively, we are currently adapting our platform of assays (standalone Regulatory platform (GLP)) to a high throughput format to allow these in vitro assays to be used to generate data to study chemical endocrine disruption data early in your discovery program.
Keywords: thyroid function, Screening, in vitro chemical mediated, thyroid hormone homeostasis, THH
Using of liquid mass spectrometry for detecting testosterone in blood plasma (#803)
T. Yevtushenko1, M. Prodanchuk1, A. Grinko1, V. Mikhailov1, N. Shepelskaya1, Y. Kolyanchuk1, O. Kravchuk1
1 L.I. Medved's Research center of preventive toxicology, food and chemical safetyMinistry of health of Ukraine (State enterprise), Analytical laboratory, Kyiv, Ukraine
The purpose of our work is the development of the method for testosterone determination in blood plasma of rats that allows to identify the threat of reproductive toxicity of lambda - cyhalothrin. It is well-known that the determination of testosterone is very important for the estimation of various diseases. At the same time, the application of liquid mass spectrometry in the practice of laboratory diagnosis can largely solve controversial interpretations, regarding the significance of diagnostic of the results of the definition of testosterone using various enzyme immune sets. Also, liquid mass spectrometry provides a number of indisputable advantages in relation to the enzyme immune method, which allows more accurately differentiate male hypogonadism state.
The proposed method of determination of testosterone is based on liquid extraction of biological objects, purification of extracts on Strata NH2 cartridges (55μm, 70A), Phenomenex and chromatographic separation on a reverse-phase column with using a liquid chromatograph Shimadzu LC-30A in a gradient moving phase, detection and quantitative analysis with using mass detector LCMS-8050. Based on experimental data, the metrological characteristics were obtained and are the following: the detection limit (LOQ) at 0.01 ng / ml, coincidence (Sr) - 0.15%, intra-laboratory reproducibility (SR) - 0.25%, extended vagueness (U) (at P = 0.95) - 0.5%. It was shown that the using of liquid mass spectrometry makes it possible to determine the testosterone and establish common reference intervals for laboratory diagnosis.
The influence of lambda – cyhalothrin doses of 0.3 mg/kg, 3 mg/kg and 10 mg/kg on the level of testosterone in the blood plasma of rats was established with a high degree of reliability (P = 0.01) and discussed.
Thus, the developed method provided for the study of antiandrogenic activity of the lambda-cyhalothrin test sample. This method allows to determine the level of testosterone in plasma and to establish a violation of spermatogenesis.
Keywords: antiandrogenic activity, testosterone, development of method
Development of a new chromatographic screening method for the determination triazole metabolites in raturine using High-resolution Hybrid LC-Orbitrap. (#815)
P. Aleinov1, M. Prodanchuk1, O. Kravchuk1, A. Grinko1, O. Kuznecova1
1 L.I. Medved's Research center of preventive toxicology, food and chemical safetyMinistry of health of Ukraine (State enterprise), Analytical laboratory, Kyiv, Ukraine
Pesticide exposure is typically done based on residue data from food monitoring (raw commodities) and food consumption databases. Biomonitoring is an alternative and can bring added value for chemical risk assessment. Upon uptake, most pesticides are rapidly metabolized and excreted. Therefore, urine analysis typically comes down to measurement of pesticide metabolites as biomarkers of exposure. Major bottlenecks in biomonitoring of of pesticides:
The main strategy of this work is development of screening method for triazole urinary metabolites using LC-HRMS. We used triazole pesticides as the most common fungicides. The method was applied to analysis of rat’s urine samples. Various pesticide-biomarkers were identified. Metabolites were detected through non-targeted analysis followed by both suspect screening analysis of samples before and after exposure. The most selective and sensitive metabolites will be used in developing quantification method. Development of dedicated targeted methods is our next main step.
Keywords: triazole metabolites, biomarkers
Activation of xenobiotic-sensing nuclear receptor PXR increases blood pressure and stimulates plasma renin activity (#827)
F. Hassani-Nezhad-Gashti1, J. Hakkola1, J. Hukkanen2
1 University of Oulu , Research Unit of Biomedicine, Pharmacology and Toxicology , Oulu , Finland
Metabolic syndrome involves several related conditions exposing to diabetes and cardiovascular disease. The main features are obesity, impaired fasting glucose, elevated blood pressure and dyslipidemia.
Pregnane X receptor (PXR) is a nuclear receptor that was originally identified to regulate drug metabolizing enzymes and drug transporters, i.e. mechanisms involved in the detoxification of xenobiotics. Subsequently, this xenobiotic receptor has been recognized to possess much broader regulatory functions, PXR has been shown to promote several components of the metabolic syndrome including dyslipidemia and impaired glucose tolerance. In the current study we addressed the question if PXR activation affects blood pressure.
We conducted a clinical trial on healthy volunteers to study the effects of rifampicin (well establish ligand for human PXR) on blood pressure and performed ambulatory 24-hour blood pressure monitoring. The design of the study was randomised, single-blind with blinded study personnel, placebo-controlled and cross-over. Rifampicin 600 mg a day or placebo was dosed on each arm for a week and the 24-hour blood pressure was monitored at the end of each arm. Rifampicin induced both the systolic and the diastolic blood pressure and also the heart rate. Furthermore, after rifampicin treatment the plasma renin level was increased.
Since PXR expression is mainly limited to liver and intestine, we hypothesized that 4β-hydroxycholesterol (4βHOC), an LXR ligand, known to be significantly increased in human circulation after treatment with PXR agonists, could mediate the increase in plasma renin level. To test this hypothesis, we established a stable expression of LXRα in Calu-6 cells, which constitutively express renin. Treatment of these cells with 4βHOC increased renin expression. However, relatively high 4βHOC concentration was required for induction. In summary, these results establish blood pressure elevation via renin activity as a novel function regulated by PXR.
Keywords: PXR, LXR, Renin, Blood pressure
Severity use of drugs of abuse and cell aging (#872)
E. K. Vakonaki1, M. N. Tzatzarakis1, P. Fragkiadaki1, D. Nathena1, V. Karzi1, K. Kanaki1, E. M. A. Renieri1, E. Iatrou1, M. E. Flamourakis2, A. Alegkakis1, A. M. Tsatsakis1
1 University of Crete, Laboratory of Toxicology, Heraklion, Greece
Introduction: Telomeres are repeated 5′-TTAGGG-3′ sequences at the end of chromosomes, which maintain genomic stability. It is known that drugs of abuse can provoke shortening of telomeric ends. The aim of our study is to evaluate if the shortening depends on the severity of usage and the type of drug.
Methods: Blood samples were collected from sixteen opiates and/or cannabis abusers. Metaphase spread leukocytes were isolated from peripheral blood telomeres length were measured by 3D Quantitative Fluorescence in situ Hybridization procedures with a (C3TA2)3 PNA probe. The severity of cannabis abuse was calculated as cigars x week and the severity of opiates abuse as gr x week, medium heavy (<5 cigars per week) and hard heavy abuse (>5 cigars per week) were used to describe the extent of abuse.
Results: Cannabis use anged from 7 to 26 years and weekly consumed cigarettes varied between 1 to 200 cigs. Opiates use ranged from 2 to 26 years and the consumption of heroin ranged from 1 to 39 gr/week. Negative correlation was observed (r=-0.564, p=0.045) of 1st quartile of short telomere length and the severity of opiates abuse. Additionally, tendency of association (p<0.100) was found between opiates abuse and 3rd quartile of telomere length. In an alternative way of analysis, using categorical opiate abuse (<5 vs >5 gram/week), resulted to significant difference at measured median Short Telomeres Lenght with p=0.035.
Conclusion: A possible effect of opiate heavy abuse on telomere length was observed, while abuse effect of cannabis comparison was questionable.
Keywords: Telomeres, Cannabis, Opiates, cell aging
Detection of Colchicine from Biological Samples of Two Death by QuEChERs-UPLC-MS/MS (#902)
H. Jian1, W. Fanglin1, L. Yujing1, R. Xinxin1, Z. Shihao2
1 Institute of Forensic Science, Ministry of Public Security, Beijing, China
Background: In recent years, the number of cases involving murder and suicide using clinical drugs is climbing. Colchicine which is used mainly for the treatment and prevention of gout is widely used in China.
Keywords: QuEChERS, LC-MS/MS, colchicine, biological samples
Xanthones as potential P-glycoprotein modulators at the intestinal barrier: in vitro and ex vivo studies. (#922)
V. L. S. Silva1, R. Silva1, E. Gil-Martins1, E. Sousa2, 3, D. Resende2, 3, F. Remião1, C. Rocha-Pereira1
1 FFUP, UCIBIO/REQUIMTE, Department of Biological Sciences, Laboratory of Toxicology, Faculty of Pharmacy, University of Porto, Porto, Portugal
P-glycoprotein (P-gp) is a membrane efflux pump belonging to the ATP-binding cassette (ABC) transporter superfamily, with a polarized expression in barrier and excretory tissues . Due to its wide range of substrates and to its large efflux capacity, P-gp has an outstanding impact in the pharmaco/toxicokinetics of xenobiotics . This mechanism is particularly important at the intestinal level, significantly reducing the intestinal absorption of xenobiotics, limiting their access to the target organs, thus resulting in a decrease in their toxicity . Consequently, P-gp induction and/or activation has been proposed as a therapeutic strategy in intoxication scenarios . In fact, several xanthones derivatives have been shown to protect cells against toxic xenobiotics by increasing P-gp expression and activity [5, 6].
The aim of the present study was to investigate the potential effect of 6 newly synthetized xanthones (X1, X2, X5, X6, X12, X16) on P-gp expression/activity. In vitro studies were performed in SW480 cells and some xanthones were able to significantly increase P-gp expression [X1, X2, X6 and X12 significantly increased P-gp expression to 113%, 119%, 139% and 122%, respectively, when compared to control cells (100%)] and activity [X1, X5, X6 and X12 significantly increased P-gp activity to 130%, 133%, 113% and 119%, respectively, when compared to control cells (100%)], 24 hours after exposure, as observed by flow cytometry and spectrophotometry, respectively. Additionally, in a short incubation period of 90 minutes almost all the xanthones significantly and immediately increased P-gp activity [X1, X2, X5, X6 and X12 significantly increased P-g activity to 121%, 120%, 128%, 126% and 135%, respectively, when compared to control cells (100%)], thereby behaving as P-gp activators given the short period of exposure. Furthermore, the protection afforded by these xanthones against the cytotoxicity induced by mitoxantrone (MTX, 10 μM), a toxic P-gp substrate, was evaluated by the MTT reduction assay. However, the xanthones failed to protect against MTX-induced cytotoxicity. Nevertheless, the most promising compound, X12, was tested for its ability to increase P-gp activity ex vivo, using rat everted intestinal sacs and rhodamine123 (RHO123) as a fluorescent substrate (300 μM). A significant increase in RHO123 efflux was observed in the presence of X12, an effect selectively blocked by zosuquidar (10 μM), a third-generation P-gp inhibitor. Therefore, the obtained results demonstrated P-gp involvement in the increased RHO123 efflux, confirming the in vitro results concerning the X12 P-gp activation potential.
Taken together, the obtained in vitro and ex vivo results suggested the P-gp activation potential of some of the tested xanthones and highlighted a potential source of new P-gp inducers and activators, disclosing new perspectives in the therapeutics of P-gp substrates-induced intoxications.
1. Sharom, F.J., The P-glycoprotein multidrug transporter. Essays Biochem, 2011. 50(1): p. 161-78.
2. Gameiro, M., et al., Cellular Models and In Vitro Assays for the Screening of modulators of P-gp, MRP1 and BCRP. Molecules, 2017. 22(4).
3. Estudante, M., et al., Intestinal drug transporters: an overview. Adv Drug Deliv Rev, 2013. 65(10): p. 1340-56.
4. Silva, R., et al., Modulation of P-glycoprotein efflux pump: induction and activation as a therapeutic strategy. Pharmacol Ther, 2015. 149: p. 1-123.
5. Silva, R., et al., Induction and activation of P-glycoprotein by dihydroxylated xanthones protect against the cytotoxicity of the P-glycoprotein substrate paraquat. Arch Toxicol, 2014. 88(4): p. 937-51.
6. Martins, E., et al., Newly Synthesized Oxygenated Xanthones as Potential P-Glycoprotein Activators: In Vitro, Ex Vivo, and In Silico Studies. Molecules, 2019. 24(4).
This work received financial support from the European Union (FEDER funds) through the Program PT2020 (project NORTE-01-0145-FEDER-000024). This work is included in and supported by TOX-OER (Learning Toxicology through Open Educational Resources) Project (https://toxoer.com/) that was funded by the European Commission and co-funded by the Erasmus+ Programme of the European Union.
Keywords: P-glycoprotein, Induction, Activation, Xanthones, Intoxications
Poisons centre enquiries relating to synthetic cannabinoids receptor agonists (SCRAs) in the UK, 2009-2018. (#927)
I. M. Al-Banaa1, 2, A. Blain3, S. Rushton4, S. Thomas1
1 Newcastle University, Health Protection Research Unit for Chemical and Radiation Threats and Hazards, Newcastle Upon Tyne, United Kingdom
The misuse of new psychoactive substances (NPS), in particular SCRA, has recently been an important public health issue because of their potential to cause serious clinical effects. These previously uncontrolled drugs of misuse have sometimes been perceived as safe alternatives to cannabis. SCRAs include a diverse group of compounds with various chemical structures that bind to CB1 and CB2 receptors with different affinities resulting in different potencies and toxicological profiles. There is limited information available on the incidence and changes in time of SCRA-related toxicity, therefore the present study evaluated this using poisons centre enquiry data collected in the UK over the last decade.
Clinical enquiries to the UK National Poison Information Service (NPIS) were reviewed retrospectively to ascertain the incidence of reported NPS–related toxicity from January 2009 to December 2018. NPS were defined as drugs of misuse that were not legally controlled in the UK prior to 2009. SCRA related enquires included the use of those branded products likely to contain a SCRA.
Over the 10 years of the study, 4158 episodes involving NPS toxicity were reported to the NPIS, of which 2510(60%) involved a SCRA or SCRA-containing products. Of those exposed, 67% were 30 years of age or younger, 75% were male, 23% female and in 2% sex was not recorded. The median age of the users with known age 24 years (range: 10 -78). The incidence of SCRA-related enquiries increased between 2009 and 2015, but has subsequently decreased significantly (P=0.0041) and this decline has continued since.
In conclusion, up to 2015 SCRA-related toxicity was becoming increasingly common,, especially in younger people and males, but has since declined in frequency. While the introduction a generic drug control law based on psychoactivity (The UK Psychoactive Substances Act, May 2016) may have made a contribution, other factors are also likely to be important, including the use of pre-existing legislation to restrict sales via headshops and websites.
Keywords: synthetic cannabinoids receptor agonists
P-glycoprotein modulation by xanthonic derivatives: a strategy to fight Alzheimer’s disease (#940)
E. Gil-Martins1, D. J. Barbosa2, E. Sousa3, 4, D. Resende3, 4, F. Remião1, R. Silva1
1 UCIBIO - REQUIMTE, Department of Biological Sciences, Laboratory of Toxicology, Faculty of Pharmacy of the University of Porto , Porto, Portugal
This work received financial support from the European Union (FEDER funds) through the Program PT2020 (project NORTE-01-0145-FEDER-000024). This work is included in and supported by TOX-OER (Learning Toxicology through Open Educational Resources) Project (https://toxoer.com/) that was funded by the European Commission and co-funded by the Erasmus+ Programme of the European Union.
P-glycoprotein (P-gp) is the best characterized member of the ATP-binding cassette (ABC) transporters superfamily. Regardless its contribution to the multidrug resistance (MDR) in neoplastic cells, this ATP-dependent efflux pump was also found to be constitutively expressed in the apical surface of normal human epithelial tissues. Noteworthy, its great efflux capacity of a wide diversity of substrates, and its cellular polarized expression in several excretory and barrier tissues [e.g. Blood-Brain Barrier (BBB)] make this protein vital in the defense of susceptible organs, by limiting the absorption and distribution of either toxic xenobiotics (e.g. colchicine) or endogenous substrates [e.g. amyloid beta (Aβ) peptide] . For this reason, P-gp can be faced as a potential disease-modifying pathway when positively modulated (activated and/or induced), in several pathologies/diseases, including in Alzheimer's disease (AD), where one of the main pathological factors to be considered is the accumulation of Aβ peptide, a P-gp substrate .
Xanthonic derivatives have been previously reported to act as P-gp modulators . Accordingly, the key goal of this work was to evaluate the induction or/and activation potential of six newly synthetized xanthonic derivatives in the in vitro model of the human BBB, the hCMEC/D3 cell line . Furthermore, the neuroprotective effect of the most promising xanthone against the Aβ-induced cytotoxicity was also assessed.
The newly synthetized xanthonic derivatives demonstrated to interact with P-gp, leading to an increase in P-gp expression and transport activity 24 hours after the incubation, and to an immediate increase in P-gp activity after a short incubation period of 90 minutes, indicating not only P-gp induction, as well as a direct pump activation. Additionally, one xanthone significantly protected hCMEC/D3 cells against the cytotoxic effect induced by the Aβ peptide, being this neuroprotective effect selectively blocked by zosuquidar, a third-generation P-gp inhibitor, thus confirming P-gp involvement in the observed neuroprotection.
Therefore, P-gp induction/activation, by increasing the efflux of Aβ peptide, can be faced as a potential prevention/treatment therapeutic approach in AD.
1. Gameiro, M., et al., Cellular Models and In Vitro Assays for the Screening of modulators of P-gp, MRP1 and BCRP. Molecules, 2017. 22(4).
2. Querfurth, H.W. and F.M. LaFerla, Alzheimer's disease. N Engl J Med, 2010. 362(4): p. 329-44.
3. Martins, E., et al., Newly Synthesized Oxygenated Xanthones as Potential P-Glycoprotein Activators: In Vitro, Ex Vivo, and In Silico Studies. Molecules, 2019. 24(4).
4. Poller, B., et al., The human brain endothelial cell line hCMEC/D3 as a human blood-brain barrier model for drug transport studies. J Neurochem, 2008. 107(5): p. 1358-68.
Keywords: P-glycoprotein, Induction/Activation, Xanthones, Amyloid beta peptide, Alzheimer’s disease
Increased consumption of seaweed in human nutrition: a new source of exposure for toxic element? (#65)
S. Rubini1, S. Menotta2, E. Ferlizza2, 9, G. Fedrizzi2, G. Isani3, D. Gigliotti1, M. N. Losio4, F. Spinardi5, S. Barbieri6, S. Molesini7, S. Sciabica8, S. Manfredini8
1 Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia Romagna, Sezione di Ferrara, Ferrara, Italy
Italian Ministry of Health
The use of seaweed for human consumption dates back to ancient times in many regions of Asia and Africa. Western countries begun to introduce algae in their diet since the 1980s and the quantities of products imported each year are continuously increasing in the EU. Seaweed are rich in proteins, carbohydrates, vitamins and trace elements with undoubted beneficial effects for human health. However, due to the environment of collection and poor sanitizing treatments to which seaweed are subjected before consumption, they can be a potential source of intoxication and/or food poisoning. The ability of seaweed to accumulate iodine and heavy metals, arsenic in particular, which may constitute a danger to consumer health, is also well known. The increase in consumption was followed by an increase in attention by the European Food Safety Agency (EFSA), which recommends the collection of information on both production /import data and potential risks to human health. Seaweed for human consumption belong to three large groups: brown algae (Phaeophyceae), red algae (Rhodophyceae) and green algae (Chlorophyceae). The algae are mostly collected in nature, in coastal areas close to the coast, where can be found the greater concentration of polluting factors of different nature. In this study, 100 samples of algae were examined, including 51 Phaeophyceae, 33 Rhodophyceae, 12 Chlorophyceae and 4 mixed preparations. Evaluations of iodine and heavy metals were conducted using ICP-MS technique, with interesting results. The concentration of lead and cadmium did not differ from that of other food products. With regard to the total and inorganic content of arsenic and total iodine, values were higher than those indicated in the EU Commission Recommendation (EU) 2018/464 of 19 March 2018 on the monitoring of metals and iodine in seaweed, halophytes and seaweed products. Recommendation 2018/464 specified that the ingestion of seaweed products, in particular dried products, might lead to a dangerously high intake if these products contain more than 20 mg of iodine per kg of dry matter and/or 2 mg per Kg of total arsenic. This research was funded by the Italian Ministry of Health (PRC 2016017).
Keywords: seaweed, iodine, arsenic, heavy metals
Plant Protection Products: an ecotoxicological assessment of active substances and associated metabolites (#108)
A. Domingues1, S. Sousa1, B. Calçada1
1 ASCENZA, Physical Chemical Department, Setúbal, Portugal
Quantitative Structure Activity-Relationships (QSARs), commonly known as (Q)SARs, are computational models used to predict physicochemical, biological and environmental fate properties of compounds simply based on their chemical structure. The importance of these software’s is already accepted by the scientific community since they follow the 3Rs principle - Refinement, Reduction and Replacement, related with the use of animals for testing purposes. Moreover, these predictions are becoming more accurate with the continuous development of more relevant, reliable and adequate (Q)SAR models.
In this study, the software OECD (Q)SAR Toolbox was used to perform an ecotoxicological assessment of 21 active substances (registered by Ascenza Agro S.A.) and 48 of their metabolites. The main goal of this study was to investigate the ecotoxicological trend of metabolites compared with their active substances. Results show that 77% of the metabolites are equivalent or less toxic than their parent compounds, and 45% of these metabolites share their parental toxicophore. Overall, it was not possible to establish a connection between an active moiety and the presence of an ECOSAR alert.
Keywords: ecotoxicity, active substances, metabolites, toxicophore
Embryotoxicity of selected organic UV filters on zebrafish (Danio rerio) (#149)
J. Blahova1, L. Plhalova1, J. Cahova1, C. Cocilova2, C. Faggio2, Z. Svobodova1
1 University of Veterinary and Pharmaceutical Sciences Brno, 1Department of Animal Protection, Welfare and Behaviour, Brno, Czech Republic
Organic UV filters are able to absorb ultraviolet radiation and are extensively used in many cosmetics products. Over the past decade, their application has increased steadily as a consequence of growing concerns of negative effect of UV radiation.
Although UV filters are present in the aquatic environment at comparatively low concentrations, these levels are biologically relevant and pose a significant growing risk for aquatic organisms. The aim of this study was to assess the acute embryotoxicity of selected two organic UV filters (2-ethylhexyl 4-methoxycinnamate EHMC, phenylbenzimidazole sulfonic acid PBSA).
As a model organism we used zebrafish (Danio rerio), which belong to one of the model fish organisms commonly used in toxicity tests to evaluate negative effects of various chemicals, which are occurring in the aquatic ecosystem. Toxic effects were studied using evaluation of lethal endpoints, development disorder, and other sublethal endpoints such as hatching rate, formation of somites, and development of eyes, spontaneous movement, heartbeat, blood circulation, pigmentation, or edema at 24, 48, 72, and 96 hours post fertilization. The embryonal toxicity test was performed through the modified method of Fish Embryo Acute Toxicity (FET) Test (OECD guideline 236). Newly fertilized zebrafish eggs were exposed to various concentrations of a single substances and their mixtures as well. In our experiment, we focused especially on testing low environmentally relevant concentrations of organic UV filters, which are usually found in surface water. Moreover higher concentrations were tested in order to reveal if the effects on exposed organism might be dose dependent. Our results showed that higher concentrations cause mortality and changes in development.
This research was supported by project IGA VFU 226/2019/FVHE.
Keywords: embryonal toxicity test, hatching, mortality, personal care products, aquatic organism
POPs in muscles of farmed deer (#229)
M. Warenik-Bany1, S. Maszewski1, W. Pietron1, M. Pajurek1, J. Piskorska-Pliszczynska1
1 NATIONAL VETERINARY RESEARCH INSTITUTE , Department of Radiobiology, PUŁAWY, Poland
Human activity results in the release of many toxic substances into the environment. One of the particularly toxic groups is persistent organic pollutants (POPs), such as chlorinated and brominated dioxins, polychlorinated biphenyls and polybrominated diphenyl ethers. Scientific research indicates the relationship between the POP environment contamination and toxicity to both animals and humans. Specific toxic effects to humans can include reproductive disorders, disruption of the immune system, damage to the nervous systems, and developmental and carcinogenic effects. Wild deer accumulate a high concentration of
Keywords: chlorinated and brominated dioxins, PCBs, PBDE, deer
Study of the impact of gold nanoparticles on representative of aquatic ecosystem (#237)
D. Hlávková1, P. Palíková1, H. Čaloudová2, B. Havelková1, M. Beklová1
1 University of Veterinary and Pharmaceutical Sciences Brno, Department of Ecology and diseases of Game, fish and bees, Brno, Czech Republic
The study was supported by the Internal Grant Agency of the University of Veterinary and Pharmaceutical Sciences Brno (No. 217/2019/FVHE).
Nanoparticles (NPs) have diverse applications in electronics, medical devices and cosmetics. With their increasing production and growing usage, a rise of concentrations of NPs in the environment is expected. Therefore, investigating the potential aquatic toxicity of nanomaterials has become an important issue.
To better understand the potential ecotoxicological impact of gold nanoparticles (AuNPs) released into freshwater environment, the Daphnia magna 48-h immobilization test was used. The experiment was carried out on the basis of OECD guideline 202 (ČSN EN ISO 6341). The toxicities of three suspensions of AuNPs (PVP 11.5 d.nm, PVP 15.2 d.nm, citrate 1.1 d.nm) and ionic form of gold were assessed. The particle suspensions used in the toxicity tests were characterized by Transmission Electron Microscopy and by Dynamic Light Scattering. Concentrations were chosen on the basis of the range finding test. The swimming behavior of D. magna and visible uptake of AuNPs were investigated and compared as well.
D. magna showed the highest sensitivity to AuCl3 (48hEC50 = 0.591 mg.l-1) and citrate form of gold (48hEC50 = 78.919 mg.l-1). All the gold species in this study caused abnormal swimming by the D. magna. The gold nanoparticles were ingested by the D. magna and accumulated in the gut.
The ecotoxicity of AuNPs varies considerably according to the particle sizes. The smaller sized NPs were more toxic compare to larger ones. These organisms are good bioindicators for assessing the acute toxicity of environmental contaminants.
References are available within the author.
Keywords: ecotoxicology, Daphnia magna, gold
Reproductive and developmental toxicity of tebuconazole to Caenorhabditis elegans (#272)
Q. Lu1, Y. Bu2, R. Liu1
1 Southeast University, Key Laboratory of Environmental Medicine Engineering, Ministry of Education, School of Public Health, Nanjing, China
Tebuconazole (TEB) is a triazole internal absorption fungicide, widely used in agriculture, and there are a large number of residues in crops, water and soil. In this study, the alternative model Caenorhabditis elegans (C. elegans) was used to estimate the reproductive and developmental toxicity of TEB at five concentrations [0 control (M9 solution), 0.01μg/L, 0.1μg/L, 1μg/L and 10μg/L] according to the LC50 test results (1.87mg/L). In order to determine the reproductive toxicity of TEB, L4 C. elegans larvae was exposed to TEB for 24, 48 and 72h. After 24h exposure, a significant decrease in brood size was detected in nematodes exposed to 10ug/L TEB(P<0.01) and showed dose-dependent reduction . After 72h exposure, the brood size in each exposed group reduced dose-dependently compared with the control group (P < 0.01).However, there was no difference in generation time between control and the exposed. Moreover, after 24h exposure, the fluorescence intensity of distal-tip cells (DTCs) in each exposure group significantly decreased compared with the control (P<0.01) and showed a dose-dependent reduction. After 72h exposure, the fluorescence intensity of DTCs decreased significantly compared with the 24h and 48h exposure group, which showed a significant dose-dependently reduction. Meanwhile, the oocyte numbers were time-and dose-dependently reduced when exposed to TEB (P<0.01). In the developmental toxicity assessment, parent generation (P0) was exposed to TEB for 24h using L4 C.elegans larvae, but F1 generation was not exposed to TEB. The body length of parent and F1 generation was dose-dependently reduced (P<0.01). And the length of F1 generation was shorten than that of parent. Meanwhile, the body width of P0 showed dose-dependently decreased with the increasing of TEB exposure, while the body width of F1 showed dose-dependently increased. The body width of F1 generation is wider than that of its parent generation at the same dose level. The results show that TEB can produce reproductive toxicity with reduced brood size, oocyte number and fluorescence intensity of DTCs and intergenerational developmental-toxicity with shortened body length and biphasic body width. Brood size, oocyte number, DTCs, body length and body width can be sensitive indicators of reproductive and developmental toxicity.
(This work was supported by National Natural Science Foundation of China grant (No.81872579) and Fundamental Research Fund of Central Public Welfare Research Institutions in 2019.)
1.Yang YF , Chen PJ , Liao HC . Chemosphere, 2016, 150:615-623.
2. Moon J, Kwak JI, Kim SW, et al. Environ Pollut, 2017, 220:46-52.
3. Sancho E, Villarroel MJ, Ferrando MD. Ecotoxicol Environ Saf, 2016, 124(67):10-17.
4. Zhou J, Zhang J, Li F, et al. J Hazard Mater, 2016, 308:294-302.
Keywords: Tebuconazole, Reproductive toxicity, Developmental toxicity, risk assessment, Caenorhabditis elegans
Comparison of the ecotoxicological effects of PPCPs on Artemia franciscana and Aliivibrio fischeri in automated and manual systems (#388)
G. Mascilongo1, S. Bodini2, F. Di Giacinto1, M. Berti1, E. Moscetta2, D. Zezza1, P. Moscetta2, N. Ferri1
1 Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise , Acquatic and Terrestrial Ecosystems, Teramo, Italy
Pharmaceuticals and personal care products (PPCPs) represent a group of chemicals, that includes human and veterinary drugs, disinfectants and fragrances used in household substances and personal care (e.g. body cleaning product, sunscreens). The U.S. Environmental Protection Agency (EPA), has categorized PPCPs as emerging organic pollutants; few data are available to define the impact on the environment and human health, such as endocrine-disrupting effects, teratogenicity and carcinogenicity. Previous studies have classified PPCPs as pseudo-persistent, because these chemicals are continuously introduced in the ecosystem. Coastal areas and aquatic environments are the most threatened. The interest on the adverse effects of these substances on the environment has increased in the last years, as well as the development of innovative early warning systems able to detect their presence. The purpose of this study was to compare the ecotoxicological effects of several PPCPs on two different organisms: Artemia franciscana, a brine shrimp, and Aliivibrio fischeri (NRRL-B-11177), a luminescent bacterium. Aiming at this, ten ingredients normally used in sunscreens were tested, including conservation agents, solvents, surfactants and chelators. A. franciscana-based ARTOXKIT M (Microbiotests, Belgium) was used as a preliminary assay to measure the mortality effect at 24 h (APAT-IRSA 8060). The fully automated analyzer Easychem® Tox Lab and the manual system Microtox® M500 were used to evaluate the inhibitory effects on the bacterial light emission at 5, 15 and 30 minutes (EC50) (ISO 11348-3:2009). A Wilcoxon signed-rank test was performed to evaluate EC50 variations associated with PPCPs tested in different systems. Despite shortest time of contact, A. fischeri resulted to be more sensitive to PCBBs than A. franciscana. For all the tested chemicals, the EC50 values of A. fischeri, were observed to be lower than A. franciscana mortality concentration ranges. Importantly, it was observed that automated and manual methods for performing the A. fischeri test gave comparable results at 15 and 30 minutes (p > 0.05).
APAT IRSA-CNR. Manuali e Linee Guida 29/2003. Metodi analitici per le acque. 8000 - METODI ECOTOSSICOLOGICI. 8060 - Metodo di valutazione della tossicità acuta con Artemia sp. vol.3,1043-1050.
Bodini et al. 2018. Evaluation of a novel automated water analyzer for continuous monitoring of toxicity and chemical parameters in municipal water supply. Ecotoxicology and Environmental Safety,157, 335-342.
ISO 11348-3:2009. Water quality. Determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri (Luminescent bacteria test). Part 3: Method using freeze-dried bacteria.
Mascilongo et al. 2018. Comparison of the ecotoxicological effects of PPCPs on different luminescent bacteria in automated and manual systems. 2nd International Conference on Bioresources, Energy, Environment, and Materials Technology, Korea.
Keywords: PPCPs, Sunscreen, Bioassay, Toxicity
A novel sensor for behavioural toxicity testing with freshwater and marine bivalves: preliminary results (#412)
F. Di Giacinto1, L. Carbone2, G. Mascilongo1, M. Berti1, N. Ferri1
1 Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise , Terrestrial and aquatic ecosystems, Teramo, Italy
Various types of sensors have been developed to detect valve gape of molluscs as behavioural endpoint of toxicity assays. Electromagnetic inductance instruments, video monitoring techniques, Hall sensors, optical fibre, laser detector and other mechanical systems have been investigated as detection technologies of valve movements. This study aimed to develop and test a novel distance measuring system, based on an infrared proximity sensor and an open source hardware platform. It is able to measure gapes remotely, with high resolution (>0.1 mm) and without any disturbing attachments on different sizes of shells (height > 2 mm). The valve-gaping behaviour of freshwater and marine adult bivalves (Anodonta cygnea, Unio mancus, Sinanodonta woodiana, Pisidium casertanum, Chamelea gallina, Venus verrucosa, Callista chione, Ruditapes philippinarum) have been registered every 30 seconds by using the novel sensor. Apart from P. casertanum, all molluscs have been exposed for 72 hours at 0,49 mg/L of hexavalent chromium [Cr(VI)], as reference toxicant in ecotoxicological studies. Five ranges of valve gapes (VG) have been established for the frequency calculation of results: VG ≤ 20%, 21–40%, 41–60%, 61–80% and ≥ 81%. Both in control and exposed mollusc groups, the average amount of time percentage spent in each VG range has been evaluated per species. A descriptive analysis using the average values has been conducted. Overall, Cr(VI) produced a general reduction of VGs in all tested species. For A. cygnea, V. verrucosa and R. philippinarum, there were little changes between exposed molluscs and controls. On the other hand, the behaviours were particularly different for S. woodiana and C. gallina. For both species, the exposed groups spent more time in the VG ≤20%, while the controls respectively in VG=61–80% and VG ≥ 81%. Concerning P. casertanum, two individuals were observed directly on the field for 12 days. They showed dissimilar behaviours, one individual registered a flapping graph with prevalence in VG ≤ 20%. The pattern of the other one was almost always fixed at VG ≥ 81% without any valve movements, probably due to the presence of seven juveniles ready to be released. Generally, each species showed behavioural rhythm of valve movements to be investigated. In addition to VG that is a sensitive parameter to Cr(VI), more registrations and analyses are necessary to elaborate other behavioural parameters of tested species useful for increasingly sensitive toxicity testing.
Keywords: Freshwater and marine bivalves, Valve movement behaviour, Infrared proximity sensor, Hexavalent chromium.
Perchlorate toxicity in organisms from different trophic levels (#424)
R. L. Acevedo Barrios1, C. Sabater Marco2, J. Olivero Verbel3
1 Technological University of Bolívar, Faculty of Basic Sciences, Cartagena, Colombia
Perchlorate (ClO4-) is an emerging inorganic pollutant widely distributed in the environment, derived from natural and anthropogenic sources. It is considered a potent endocrine disruptor that affect the iodine fixation by the thyroid gland, impacting metabolism, reproduction and development in the biota. However, there are few reports of its ecotoxicological impact on wildlife. The objective of this work was to evaluate the adverse effects of perchlorate exposure on different models, HEK, N2a and 3T3 cell lines, Vibrio fischeri, Pseudokirchneriella subcapitata, Daphnia magna and Eisenia fetida. Perchlorate exhibited similar toxicity against tested cell lines, with LC50 values of 19, 15 and 19 mM for HEK, N2a and 3T3, respectively. In V. fischeri the toxicity, measured as reduction of bioluminescence, was considerably lower (EC50= 715 mM). The growth of the freshwater algae P. subcapitata was impaired by perchlorate with an LC50 value of 72 mM, and the toxic response on D. magna was greater (LC50= 5 mM). Finally, in the earthworm E. fetida, perchlorate induced avoidance behavior, weight loss, decrease egg production and hatchling, as well as morphological and histopathological effects, such as malformations, dwarfism and necrosis, displaying an LC50 of 56 mM in soil. In conclusion, exposure to perchlorate has a significant impact on the survival, development and reproduction of organisms from different trophic levels.
Acevedo-Barrios, R., Bertel-Sevilla, A., Alonso-Molina, J., & Olivero-Verbel, J. (2019). Perchlorate-Reducing Bacteria from Hypersaline Soils of the Colombian Caribbean. International Journal of Microbiology, 2019.
Acevedo-Barrios R, Bertel-Sevilla A, Alonso-Molina J, Olivero-Verbel J (2016) Perchlorate tolerant bacteria from saline environments at the Caribbean region of Colombia. Toxicol Lett (259):S103. https://doi. org/10.1016/j.toxlet.2016.07.257
Chen HX, Ding MH, Liu Q, Peng KL (2014) Change of iodine load and thyroid homeostasis induced by ammonium perchlorate in rats. J Huazhong Univ Sci Technol (Med Sci) 34 (5):672-678. doi:10.1007/s11596-014-1335-8
Duan Q, Wang T, Zhang N, Perera V, Liang X, Abeysekera IR, Yao X (2016) Propylthiouracil, Perchlorate, and Thyroid-Stimulating Hormone Modulate High Concentrations of Iodide Instigated Mitochondrial Superoxide Production in the Thyroids of Metallothionein I/II Knockout Mice. Endocrinol Metab 31 (1):174-184. doi:https://doi.org/10.3803/EnM.2016.31.1.174
Schmidt F, Schnurr S, Wolf R, Braunbeck T (2012) Effects of the anti-thyroidal compound potassium-perchlorate on the thyroid system of the zebrafish. Aquat Toxicol 109:47-58. doi:10.1016/j.aquatox.2011.11.004.
Vigliotta G, Motta O, Guarino F, Iannece P, Proto A (2010) Assessment of perchlorate-reducing bacteria in a highly polluted river. Int J Hyg Environ Health 213 (6):437-443. doi:10.1016/j.ijheh.2010.08.001.
Keywords: Biological models, biota, ecotoxicology, endocrine disruption, environmental risk
A combined in vitro/risk assessment approach to identifying aquatic environmental risks of cosmetic products: A case study of UV filters (#516)
I. R. D. Souza2, J. Costa2, A. D. P. M. Canavez1, C. A. Brohem1, M. Lorecini1, D. M. Leme2
1 Grupo Boticario, Center for Safety and Efficacy Assessment, Sao Jose dos Pinhais, Brazil
Personal care products, such as sunscreen UV filters, can profound impact aquatic ecosystems, raising concerns about their sustainability. Therefore, enhancing and promoting eco-friendly products in cosmetic industry requires robust methods for assessing the environmental impacts. In vitro screening assays have been proposed as key components of a future testing paradigm for mechanism-base regulatory ecotoxicology; and improvements in predicting ecotoxicity risks of products can be achieved by combining cell-based models (in vitro) with risk assessment tools. Grupo Boticário has developed an environmental risk assessment tool, named IARA™, which integrates data of bioaccumulation, biodegradation, acute toxicity and PEC/PNEC ratio, providing risk quotients for ranking cosmetic ingredients according to their levels of environmental risk (high-moderate-low). However, the quality of toxicity data available poses a challenge to the reliability of risk assessment. Aimed to improve the accuracy in predicting environmental risks of cosmetics ingredients, we have developed a combined in vitro/risk assessment approach based on in vitro fish cytotoxicity testing (MTT assay, ZFL cell line – Danio rerio normal liver) and IARA™ matrix. Cytotoxicity data of eight UV filters (ethylhexyl methoxycinnamate, homosalate, diethylamino hydroxybenzoyl hexyl benzoate, titanium dioxide, phenylbenzimidazole sulfonic acid, ethylhexyl salicylate, zinc oxide, polysilicone-15) were used to verify the efficacy of our approach in ranking the aquatic toxicity risks of sunscreen UV filters. High to low cytotoxicity was observed to ZFL cells exposed (96-well plates, monolayer cells, 24 hours) to all tested UV filters (0.01-100 g/L). Applying these in vitro cytotoxicity data into IARA™ matrix improved the prediction of environmental risks of UV filters compared to risks estimated by database data-driven IARA™, demonstrating the relevance of controlling the quality of toxicity data. In summary, the combined in vitro/risk assessment approach herein proposed can drive the safer aquatic environmental use of sunscreens, framing them into the sustainable context.
Financial support: CAPES, Grupo Boticário
Keywords: ecotoxicology, sustainable cosmetic products, fish toxicity, in vitro testing, ZFL cell line
Effect of the electrochemical advanced oxidation process on the ecotoxicity of a solution composed of norfloxacin in presence of sodium sulphate (#542)
M. T. Montañés1, M. García-Gabaldón1, L. Roca-Pérez1, J. J. Giner-Sanz1, J. Mora1, V. Pérez-Herranz1
1 Universitat Politècnica de València, Ingeniería Química y Nuclear, València, Spain
The presence of antibiotic compounds in surface waters is an emerging environmental issue since many of these substances are not biodegradable, toxic and capable of accumulating in aquatic organisms. In this sense, numerous researches have promoted the advanced electrochemical oxidation as a promising alternative technique to treat wastewaters containing toxic and refractory organic pollutants.
Ecotoxicological bioassays based on Lactuca Sativa seeds and bioluminescent bacterium have been carried out to analyse the ecotoxicity of a contaminated solution composed of norfloxacin (NOR), an antibiotic widely used, in sodium sulphate solution, as supporting electrolyte before and after the treatment by an electrochemical advanced oxidation process. The effect of some process variables (pH, anode material, reactor configuration, applied current, supporting electrolyte concentration...) on the toxicity evolution is evaluated.
The toxicity limit of the effluents contaminated with norfloxacin in the presence of sodium sulphate has been determined using statistical tools. The EC50 value obtained with Lactuca sativa seeds for norfloxacin is 336 mg·L-1 and this value decreases if sodium sulphate is added to the solution. However no synergy is observed.
All the samples treated by electrochemical oxidation are more toxic than the starting solutions. This is mainly due to the formation of persulphates due to the oxidation of the sulphates present in the solution. Since the boron-doped diamond anode (BDD) is able to produce more persulphates than the ceramic anodes, samples treated using the BDD anode present higher toxicity values.
Authors are very grateful to the Ministerio de Economía y Competitividad (Project CTQ2015-65202-C2-1-R) for their economic support.
Keywords: Electrochemical oxidation, Lactuca sativa, Norfloxacin, Sodium sulfate, Vibrio fischeri
Evaluation of the toxic effects of livestock drinking water by in vitro studies (#581)
F. Pennisi1, D. R. Francese1, M. Pezzolato1, K. Varello1, D. Sulla1, M. Prearo1, M. Messina1, M. C. Abete1, D. Meloni1, E. Bozzetta1
1 Istituto Zooprofilattico Sperimentale del Piemonte Liguria e Valle d'Aosta, Torino, Italy
Introduction: Water is a necessary component of human and animal life, but often the role of water in livestock feeding is underestimated. In European context the drinking water quality standards for human consumption are regulated by the Directive 98/83 EC, while in the zootechnical sector no specific rules concerning the qualitative and health characteristics of water are defined.
Materials and Methods: For sampling activity of drinking water, six sites with different characteristics, have been selected throughout the Piedmont region (north-western Italy). The samples were analysed to assess:
Results: Results show no positivity for microcystin, cylindrospermopsin, viral and bacterial agents as expected in drinking water.
Discussion: Results show the healthiness of the livestock drinking water tested and demonstrate the low risk for food producing animals and indirectly the poor risks of contamination for final consumers.
Brand W., de Jongh C. M., van der Linden S. C., Mennes W., Puijker L. M., van Leeuwen C. J., Van Wezel A.P., Schriks M., Heringa, M. B. (2013). Trigger values for investigation of hormonal activity in drinking water and its sources using CALUX bioassays. Environment international, 55, 109-118.
Vandermarken T., Croes K., Van Langenhove K., Boonen I., Servais P., Garcia-Armisen T., Brion N., Denison M. S., Goeyens L., Elskens, M. (2018). Endocrine activity in an urban river system and the biodegradation of estrogen-like endocrine disrupting chemicals through a bio-analytical approach using DRE-and ERE-CALUX bioassays. Chemosphere, 201, 540-549.
Keywords: drinking water, ER CALUX, livestock
Аssessing the impact of the lead waste on environmental objects (#637)
V. Ioda1, O. Boris1, T. Gomolko1
1 The Republican Unitary Enterprise "Scientific and Practical Center of Hygiene" , Minsk, Belarus
Production wastes consider as the toxic factor of the habitat forming the risk of chemical pollution of water, the soil and air and, as a result, having potential toxicity for live organisms including humans.
The purpose of the work is to determine the degree of toxicity of lead sludge for biological objects of the environment.
Methods. Learned lead sludge contain cadmium, lead and arsenic. Studies of Ecotoxicity of lead sludge on Tetrahymena pyriformis W. were carried out in acute, subacute and chronic experiments. The toxicity of lead sludge in the Eisenia foetida test model was studied in an acute experiment. Phytotoxicity of lead sludge was studied on seeds of higher plants.
Results. The toxicity on Tetrahymena pyriformis is caused by the impact on processes of cellular division and growth among generations leading to decrease in population decline. Observed decrease in vital activity of populations of Tetrahymena pyriformis during the whole period of observation, death of organisms, decrease in stability of the cell membranes of ciliates to the adverse effects of the environment. No mutagenic activity.
After a seven-day exposure of lead sludge in the test model of Eisenia foetida, the mean lethal concentration LC50 = 15,36 (7,39 – 31,93) g/kg was established. Changes in the behavioral reactions of animals in the form of reduced motor activity, reducing the rate being burying in the ground.
Test results on phytotoxicity. Noted the effect of inhibition of the development of the roots of seedlings of cucumbers equal to 40,31%, radish – 32,28%, oats – 56,02%, which exceeds the threshold of phytotoxicity, equal to 20 %, for each crop of seed, and indicates the presence of phytotoxic action of waste. The most sensitive were oat seeds – the average effective dilution (ER50) was 1,8.
Thus, it is possible to characterize the lead sludge waste as having a significant adverse effect on the biotic elements of the environment.
Keywords: biotesting, ecotoxicity
Ecological risk assessment of pesticides in groundwater in Saiss plain of Morocco (#744)
I. Berni1, A. Menouni1, 2, I. El Ghazi1, R. C. Duca2, M. - P. Kestemont3, L. Godderis2, S. El Jaafari1
1 Moulay Ismail University, Cluster of Competency “Environment and Health”, Meknès, Morocco
In this study a pesticide monitoring survey was took place in 21 wells of Saiss plain in Morocco in September 2017 and January 2018 to explore 4 different scenarios of risk. For this purpose, passive samplers were deployed between 14 to 20 days in 21 traditional wells from the study area and 28 pesticide compounds were analyzed including fungicides, insecticides, herbicides and their metabolites. The Scenarios 1 and 2 were used to depict the risk of failing to meet the good groundwater chemical status as defined by WHO (The measured environmental concentrations (i.e. the mean MECs) of each pesticide per sampling site over the survey period were used to assess the first scenario and the max MECs were used to assess the second scenario). The Scenarios 3 and 4 were used to assess for the first time the ecological risk in groundwater bodies, defined as the likelihood of hazard to the groundwater communities stably residing in the 21 wells that may be affected by pesticide contamination (the mean MECs (Scenario 3) and the max MECs (Scenario 4)). The ecological risk was assessed through a new procedure called GERAp (Groundwater Ecological Risk Assessment due to pesticides). The main results of this study highlighted that: 1) the Scenario 1 provided information of little use for risk managers; 2) chlorothalonil, dicofol, chlorpyrifos methyl, bifenthrine were the compounds most frequently detected using the highest concentrations measured in the monitoring period; 3) a high ecological risk were found in 6 wells of Saiss plain due to 13 insecticides (scenario 3); 4) some pesticides that were banned in Morocco should be kept monitored in the next surveys because they showed a persistent occurrence in some wells such as DDE, DDT and endosulfan; 5) DDE, Diazinon and Permethrine are expected to damage groundwater communities at concentrations that are lower than the present legal limits (scenario 4).
Keywords: Pesticide, Saiss plain, passive sampler, ecological risk, groundwater
Comparing approaches to acute fish toxicity testing across sectors and regions to identify opportunities to advance the 3Rs (#863)
N. Gellatly1, N. Burden1, F. Sewell1
1 NC3Rs, London, United Kingdom
Submitted on behalf of the NC3Rs ecotoxicology working group.
The acute fish toxicity test has been a mainstay of ecotoxicology for over 30 years. The aim of the test is to establish the concentration of test material which causes the death of 50% of the exposed fish (LC50). As such, there is the potential for significant suffering over the 96 hours of the test. Assessment of the potential for acute fish toxicity is a core requirement under many regulatory frameworks across the world. Whilst in many sectors and regions there is still a requirement to generate in vivo test data, alternative appraoches to assess this endpoint are increasingly being accepted. The UK’s National Centre for the Replacement, Refinement and Reduction of Animals in Research (NC3Rs) ecotoxicology working group includes experts from government agencies, academia and a range of industries. The working group has compared approaches accepted across sectors and regions to identify differences and where there is scope to further apply the 3Rs.
Following this comparison, the working group have identified a number of aspects of acute fish toxicity testing requirements where there may be potential for refinement, reduction or replacement of in vivo studies, and where approaches taken in one region or sector could be more widely adopted. These include when it may be necessary to generate data in multiple species or for products as well as individual active ingredients or whether data can be extrapolated to reduce the number of studies conducted, exposure considerations (for example whether chronic toxicity data may be more relevant) and use of alternative approaches such as quantitative structure-activity relationship models. These factors will be discussed and opportunities for sharing experience in assessing acute toxicity across sectors, regions and species (including fish, birds and mammals) highlighted.
Keywords: 3Rs, ecotoxicology, regulatory, acute toxicity, fish
Pharma pollution as a selective pressure (#959)
M. Rodda1, E. Gillio Tos1, L. Brunasso Cattarello1, V. Bortolot1, A. Conto1
1 Chemsafe Srl, Parella, Italy
Pollution may act as a selective pressure. A great importance to define how pollution could drive selective pressure should be given to the kind of pollution: acute or chronic pollution. When the toxicological “death dilemma” is applied to selection it is possible to conjecture: i. if all individuals in a population share the same threshold above which death occurs stochastically, acute pollution can not produce significant selection into a population; ii. if the tolerance threshold is distributed among the individuals in a population and its exceedance leads to certain death, acute pollution can cause genetic drift of alleles. Moreover, chronic pollution may reduce the number of genetic variants in a population and select only those could give a high fitness in individuals.
One other aspect to take into consideration could be the generation time of the species considered, due to high generation turnover could fix faster the genetic variants than species have low turnover.
Between 1900 and 2000, the increase in world population was three times greater than during the entire previous history of humanity, an increase from 1.5 to 6.1 billion in just 100 years. In order to satisfy the therapeutic needs of the human population, the increasing production of pharmaceuticals and the subsequent presence of pharmaceuticals in the environment are unavoidable. Patient use of medicines is the principal pathway by which pharmaceuticals (prescription and over the counter) find their way into the aquatic environment. Typically, a fraction of the medicines taken by patients is excreted and enters waterways. To a lesser extent, pharmaceuticals can enter the environment through improper disposal of medicines and from manufacturing discharges.
The selective pressure of chronic exposure to pollutants into environment can positively select, for example, more efficient detoxification pathways.
In the last years particular concerns have been expressed on compounds that mimic hormones and can disrupt reproduction and development in animals. It can be hypothesized that the pressure of chronic exposure to exogenous hormones into environment could lead, for example, to the reduction of endogenous production of specific hormones or reduction of specific membrane/nuclear receptors. But what happen if the exogenous stimulus, i.e. pollution from medicinal hormones, decrease abruptly? Taking into account the short time period and the physiological importance of the endocrine system compared to the detoxification pathways, paradoxically it can be hypothesized that the sudden loss of this selective pressure can be considered itself a selective pressure opposed to the previous one. Such selective pressure will be directly proportional to the selective pressure placed by pollution and could cause effects comparable to those of acute pollution.
In conclusion, the measures aimed at remedying situations of chronic pollution should be carefully evaluated and modulated over time.
Keywords: Pollution, selective pressure, death dilemma
PM2.5 exposure impairs sperm quality through testicular damage dependent on NALP3 inflammasome in mouse (#18)
1 Hebei Medical University, Toxicology, Shijiazhuang, China
Exposure to ambient fine particular matter (PM2.5) has been clearly associated with male reproductive disorders. However, very limited toxicological studies were carried out to investigate the potential mechanism underlying the PM2.5-induced sperm quality decline. In the present study, we established a real time whole-body PM2.5 exposure mouse model to investigate the effects of PM2.5 on sperm quality and its potential mechanisms. Sixty male C57BL/6 mice were randomly subjected to three groups: filtered air group, unfiltered air group and concentrated air group. Half of the mice from each group were sacrificed for study when the exposure duration accumulated to 8 weeks and the rest of the mice were sacrificed when exposed for 16 weeks. Our results suggested that PM2.5 exposure could induce significant increase in circulating white blood cells and inflammation in lungs. PM2.5 exposure induced apparently DNA damages and histopathologic changes in testis. There was significantly decreased sperm density of mice, which was paralleled with the down-regulated testosterone levels in testes tissue of mice after exposure to PM2.5 for 16 weeks. The numbers of motile sperms were decreased and sperms with abnormal morphology were increased after PM2.5 exposure in a time-depended and dose-depended manner. PM2.5 exposure significantly increased the expression of the major components of the NACHT, LRR and PYD domains-containing protein3 (NALP3) inflammasome, accompanied by the increased expression of miR-183/96/182 targeting FOXO1 in testes. The present data demonstrated that sperm quality decline induced by PM2.5 could be partly explained by the inflammatory reaction in testis which was as a consequence of systemic inflammation. The molecular mechanism was depended on the activation of NALP3 inflammasome accompanied by miR-183/96/182 targeting FOXO1 in testes.
Keywords: ambient fine particular matter (PM2.5); sperm quality; NALP3 inflammasome; FOXO1; male reproductive system.
A case report of unknowing ingestion of Brugmansia Suaveolens Leaves presenting with Delirium in Sri Lanka (#31)
K. P. Jayawickreme1, K. V. Janaka1, S. Subasinghe1
1 Sri Jayawardenepura General Hospital, Medical Unit, Sri Jayawardenepura Kotte, Sri Lanka
Self Poisoning carries a high mortality and morbidity in Sri Lanka, and has a case fatality ratio of 9% but is under-evaluated. Poisoning cases of Datura or Brugmansia, which come under the Solanacea family, in other countries were almost always due to ingestion of seeds.It contains alkaloids like scopolamine, atropine and hyoscyamine which cause an anticholinergic toxindrome by blocking the muscarinic acetylcholine receptors of the nervous system. There have been a very few reported cases of accidental ingestion of Brugmansia seeds among children, but hardly any reported cases of Brugmansia leaf poisoning among adults in Sri Lanka.
A 60 year old female presented with acute delirium, and agitation. She had ingested a kanji drink made from leaves from her garden prior to the onset of symptoms, until which she was previously well. She had urinary retention, mydriasis, and sinus tachycardia. She was managed supportively with activated charcoal, hydration, and 1.5mg IV Midazolam to calm the patient. The delirium completely resolved within 15 hours . Her CT brain was normal and urine for toxicology was negative for illicit drug substances. After regaining consciousness she admitted that she made the kanji drink containing an unknown plants leaves from her garden, which we identified as Brugmansia suaveolens, with the help of a native medicine physician and specialist in botany. she did not require the antidote physostigmine and recovered fully.
Although seeds are the most toxic plant part in most cases of Brugmansia poisoning, leaves also have a significant degree of toxicity. It is important that medical professionals promptly recognize the features of anticholinergic syndrome and have a high index to suspect Brugmansia poisoning and start prompt treatment to improve outcome. Further research can be recommended on the degree of awareness of toxicity of toxic plants in the Sri Lankan community, and measures must be taken to improve awareness among the general public of toxic effects of plants and recognizing such plants by their appearance in order to prevent toxicities and fatalities
Keywords: Solanacea, Brugmansia suaveolens, Datura, Atropine, Anticholinergic syndrome
Establishment of an animal model of allergic inflammation caused by atmospheric dust (#36)
A. Onodera1, M. Nagaoka1, Y. Meguri1, A. Takemura1, Y. Kawai1
1 Kobe Gakuin University, Faculty of Pharmaceutical Sciences, Kobe, Japan
Purpose: Atmospheric dust is strongly suspected of contributing to upper respiratory allergy symptoms and has been called the Japanese national disease. However, to the best of our knowledge, there has been no replicate study on the atmospheric dust-specific immune responses. The establishment of an animal model is the current bottleneck in transitioning from epidemiological studies to basic studies. Therefore, our study aimed to investigate the atmospheric dust-specific inflammatory responses in the footpad of adjuvant-sensitised mice by measuring cytokine/chemokine production.
Materials and methods: Atmospheric dust was collected on the filters of a central ventilation system in a building in the centre of the Beijing city (CRM No. 28: Japan National Institute for Environmental Studies). Male BALB/c mice were used in all experiments. 500 µg of atmospheric dust was gently mixed with 50 µl of PBS and 2% of Tween 80. This mixture was emulsified using an equal volume of complete Freund's adjuvant (CFA) or incomplete FA (IFA). 100 µl of this emulsion was subcutaneously injected at the base of the tail with CFA and IFA, on days 0 and 14, respectively. Control mice were injected with only CFA or IFA. On day 28, 100 µg of atmospheric dust in 10 µl of PBS and 2% Tween 80 was subcutaneously injected into the footpad, and footpad swelling was measured using a dial gauge calliper at 24, 48 and 72 h after injection. Cytokine/chemokine mRNA production was then measured by real-time polymerase chain reaction, and the classification and ratio of lymphocytes was determined for the isolated footpad and nearby popliteal lymph node by flow cytometric data.
Results: At 24 h after subcutaneous injections, injection of atmospheric dust alone in mice footpads with atmospheric dust sensitisation caused them to be enlarged to approximately 1.3 times the size of those without atmospheric sensitisation. We also confirmed increases in cytokine mRNA expression of at least seven types in the footpads and increases in CD3+, CD4+ or CD8+ T lymphocytes and CD3− and CD19+ B lymphocytes. These results strongly suggested that atmospheric dust provokes an allergic immune response.
Keywords: inflammation, atmospheric dust, lymphocytes
Biological response modulation of human reconstituted airway epithelium repeatedly exposed to PM0.3-2.5 (#40)
S. Achard1, E. Seurat1, S. Achawi1, A. - L. Schang1, A. Verdin2, F. Cazier3, D. Courcot2
1 Faculty of Pharmacy - Paris Descartes University, Inserm 1153 HERA tem (Health Environmental Risk Assessment), Paris, France
Over the past decades, air pollution has risen dramatically worldwide. Many epidemiological studies have shown associations between respiratory and cardiovascular diseases and airborne pollutants, including particulate matter (PM) playing a major role in the human health disorders.
In order to understand the PM’s effects on the biological activity, the present study evaluates the impact of two types of fine particles (PM0.3-2.5) with similar aerodynamic diameter but different chemical compositions (polycyclic aromatic hydrocarbons, heavy metals …): PMtraf from traffic zone and PMind from industrial zone.
To mimic the real human exposure to PM0.3-2.5 an innovative 3D in vitro model was used: a human airway epithelium reconstituted, nasal origin, co-cultured with human airway fibroblast (MucilAir™-HF, Epithelix®).
The epithelia were exposed to PM at 45 or 90µg.cm2 twice a week for two consecutive weeks. 48h after each exposure, the culture medium on basal side was collected and inflammatory response was assessed by ELISA assay. At the end of each week, the membrane integrity was evaluated using the TEER (transepithelial electrical resistance) measurement and part of the epithelia was sacrificed for histological analysis and gene expression assessment. RNA extraction was conducted in order to analyse the PM’s influence on inflammatory and oxidative stress gene expression by RTqPCR.
Our results showed no loss of integrity whatever the PM tested, while a significant increase of the inflammatory response appears. IL-8, IL-6 and GM-CSF secretions were higher after PMtraf exposure compared to PMind, probably due to its chemical composition. In addition, the cytokine secretions were the highest during the first week of exposure, certainly due to the adaptation capacity of the 3D-model. Besides, some modulation of gene expression appears after PM exposure: increase of inflammatory biomarkers (IL-8, IL-6 and GM-CSF) and decrease of oxidative stress biomarkers (NOS and SOD).
The major outcome of this study is the demonstration that, 1/the origin of particles, influencing their chemical composition, acts in the development of respiratory disorders, and 2/the reconstituted human epithelium is adapted to evaluate the impact of repeated exposures to environmental pollutants.
Keywords: human reconstituted airway epithelium, Fine particulate matter, repeated exposures, chemical characterization
Toxicity of nonylphenol and nonylphenol ethoxylate in Caenorhabditis elegans (#48)
A. C. De la parra Guerra1, S. Stürzenbaum2, J. Olivero Verbel1
1 University of Cartagena, Environmental and Computational Chemistry Group, Cartagena, Colombia
Nonylphenol (NP) and its ethoxylated isomers (NPEOs, e.g. NP-9) are compounds used as raw materials for many industrial processes. These chemicals and their metabolites are commonly found in environmental matrices. The objective of this work was to evaluate physiological and neurotoxic effects of NP and NP-9 in Caenorhabditis elegans. Lethality and locomotion were assessed in larval stage L4, whereas growth was carried using the L1 stage of the wild strain N2, exposing worms to different concentrations of NP and NP-9. GFP transgenic strains mtl-2, gst-1, gpx-4, gpx-6, sod-4, hsp-70 and hsp-4 were employed to evaluate the activation of signaling pathways related to cellular stress, whereas RT-qPCR was utilized to measure mRNA expression for different genes associated with neurotoxicity (unc-30, unc-25, unc-49, dop-3, dat-1, mgl-1, eat-4, glt-3, glt-6) and oxidative stress (mtl-1 and mtl-2). The nematode lethality was concentration-dependent, with 24h-LC50 values of 122 and 3215 μM for NP and NP-9, is the value of the LC50 for NP-9 respectively. At non-lethal concentrations, locomotion and body length were significantly reduced by both xenobiotics, although NP was always more potent. GFP activity suggests NP and NP-9 activate ROS-mediated pathways, and in the case of glutathione peroxidase, the concentration-response curve for NP was bimodal, a typical endocrine disruption response. Nonylphenol significantly inhibited the transcription of several genes related to neurotoxicity, such as GABA, glutamate and dopamine; whereas NP-9 down-regulated GABA, glutamate and stress oxidative-related genes, although dopamine and glutamate displayed a non-monotonic concentration-response curve. In summary, NP and NP-9 induced neurotoxic responses in C. elegans through mechanisms that involve ROS and disturbances of the GABA, glutamate and dopamine pathways, effects observed at environmentally-relevant concentrations. Colciencias-UniCartagena (Convocation No. 727 of 2015 Res. 513, July 2015).
Araujo FG, Bauerfeldt GF, Cid YP (2018) Nonylphenol: Properties, legislation, toxicity and determination. Anais da Academia Brasileira de Ciências 90(2): 1903-1918. Doi: http://dx.doi.org/10.1590/0001-3765201720170023
Calafat AM, Kuklenyik Z, Reidy JA, Caudill SP, Ekong J, Needham LL (2005) Urinary concentrations of bisphenol A and 4-nonylphenol in a human reference population. Environmental Health Perspectives 113(4): 391-395. http://doi.org/10.1289/ehp.7534
Cao X, Wang X, Chen H, Li H, Tariq M, Wang C, Liu Y (2019) Neurotoxicity of nonylphenol exposure on Caenorhabditis elegans induced by reactive oxidative species and disturbance synthesis of serotonin. Environmental Pollution 244: 947-957. Doi: https://doi.org/10.1016/j.envpol.2018.09.140
García M, Tejeda L, Olivero-Verbel J (2018) Toxicity of atrazine-and glyphosate-based formulations on Caenorhabditis elegans. Ecotoxicology and Environmental Safety 156: 216-222. https://doi.org/10.1016/j.ecoenv.2018.02.075
Soares A, Guieysse B, Jefferson B, Cartmell E, Lester JN (2008) Nonylphenol in the environment: a critical review on occurrence, fate, toxicity and treatment in wastewaters. Environment international 34(7): 1033-1049. Doi: https://doi.org/10.1016/j.envint.2008.01.004
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Keywords: endocrine disruption, lethality, gene expression, ROS, neurotoxicity
The association of exposure to fine airborne particulate matter with cardiovascular diseases in Beirut Lebanon (#50)
N. K. Zgheib1, 2, A. Imad3, H. Ismaeel4, 2, K. Badr5, 2, N. Saliba6, 2, I. Lakkis3
1 American University Of Beirut, Department of Pharmacology and Toxicology, Faculty of Medicine, Beirut, Lebanon
Ambient air pollution represents a worldwide environmental risk factor for cardiovascular diseases (CVD). In Lebanon, it was estimated that annual median concentrations of PM2.5 exceed the recommended levels set by the WHO air quality guidelines. One study reported a potential for association between exposure to ambient air pollution and CVD in Lebanon. However, and due to the lack of air monitoring stations, the investigators used individual questionnaires about outdoor air pollution as proxy instead of quantitative air pollutant measurements. No studies in Lebanon applied a modeled spatial distribution of air pollutants to investigate this association. This study hence aimed to determine the relationship between the level of exposure to airborne particulate matter and coronary artery disease (CAD) in the city of Beirut using a modeled spatial distribution of PM2.5.
This study builds on a cohort of subjects living in the city of Beirut who were recruited between March 2014 and December 2017 under the Vascular Medicine Program of the American University of Beirut Medical Center. Data were collected for demographics, smoking habits, comorbidities, and exact place of residence. In addition, the coronaries of all participants were visualized by cardiac catheterization. In parallel, a spatial distribution for a representative meteorological situation in Beirut was obtained by simulating the transport of PM2.5, based on an accurate emissions inventory of the diesel generators, using the physically-based mesoscale and micro-scale dispersion model system GRAMM-GRAL. The modeled distribution of PM2.5 was used to determine the level of PM2.5 exposure at the participants’ place of residence.
Preliminary results from 340 subjects revealed that obstructive CAD (defined as 50% or more obstruction in at least one of the coronaries) was significantly associated with the levels of PM2.5 among smokers (OR 1.052, 95% CI (1.016–1.089)) per 1 µg.m-3 rise in PM2.5 concentration. These data suggest that ambient air pollution represents an additive risk to smoking on CVD. Further analysis is ongoing to include comorbidities and associations with cardiac index. This knowledge is a starting point for assessing the impact of PM2.5 on CVD and potentially driving public health interventions to reduce air pollution.
Keywords: CVD, Air pollution
Environmental pollution: a 3D skin model to assess protective properties of cosmetic ingredients (#55)
F. Richard1, T. Creusot1, A. Josseaume1, A. Thelu1, L. Beaudequin1, M. Floreani1, S. Catoire1, H. Ficheux1
1 THOR Personal Care, Toxicology, Compiegne, France
Ambient air pollution has become a major risk factor of health damage, as world population exposure increases. Consequences of pollutants on skin physiology are extensively reviewed, however some mechanisms of penetration and action remain unclear.
Our previous publication (Quantin et al., 2018) showed an impact of pollutants on inflammation and induction of cytochromes P450 through the AhR pathway in keratinocytes. This study aims at identifying exposure scenarios that mimic real conditions, resulting in an increase of particles absorption. We determined cell viability and biomarkers of skin exposed to pollution in a 3D skin model to highlight the potential protective capacities of skincare ingredients.
A 3D reconstructed human epidermis (RhE) VitroDerm (VD), internally validated and used in routine for skin irritation assessment, was exposed to a standardized mixture of pollutants, Urban Dust SRM 1649b for 48 hours at reactive doses. To validate the use of VD in the specific context of pollution, several endpoints identified in an internal review of the literature were investigated: cell viability by MTT assay and skin barrier function through histological staining of filaggrin. Then, cells were exposed to different conditions to determine a potential enhancing effect in Urban Dust absorption. Finally, protective effect of cosmetic ingredients was assessed.
The VD model showed promising response to pollution as we observed a decrease of cell viability after exposure to pollutants and a change in the identified biomarkers. Our model allows to assess the enhancing capacity of environmental factors, therefore increasing pollutants skin passage. These experimental conditions previously determined were used to observe the effect of pollution on skin barrier function through filaggrin expression as well as the efficacy of protective ingredients with the VD model.
In conclusion, pollution impacts skin physiology at different levels, altering skin barrier function through the expression of filaggrin. Moreover, pollutants passage can be enhanced by some environmental conditions. Further studies will allow us to determine whether exposure to other external factors, such as ultraviolet (UV) or cigarette smoke, could impact the skin barrier function.
P. Quantin, S. Catoire, A. Thélu, A. Patatian, H. Ficheux, 2018. The Toxicologist. Abstract and Poster #3056.
Keywords: particulate matter, percutaneous absorption, skin, pollution, in vitro testing
Effect of ferulic acid on airway damage and the change of TGF-β1/Smads signaling pathway induced by atmospheric PM2.5 in asthmatic rats (#57)
X. Zhao1, Y. Wang1, W. Lao1, Y. Zhou1
1 Beijing Union University, Research Institute for Science and Technology of Functional food, Beijing , China
Purpose: To explore the protective effect of Ferulic Acid ( FA) on the respiratory tract injury and the change of TGF-β1/Smads signaling pathway induced by PM2.5 in asthmatic rats.
Method: SD rats were randomly divided into the following seven groups (n = 8 for each group): control group, model (OVA, ovalbumin) group, PM2.5 (OVA+6.0 mg/kgPM2.5) group, FA (19.4) group (OVA+6.0 mg/kg PM2.5+19.4mg/kg FA), FA (38.8) group (OVA+6.0 mg/kg PM2.5+38.8mg/kg FA), FA (77.6) group (OVA+6.0 mg/kg PM2.5+77.6mg/kg FA) and positive control group (OVA+6.0 mg/kg PM2.5+dexamethasone). OVA-sensitized rats were used to build the asthmatic rat models. Rats were exposed to OVA for sensitization and challenge, and rats in the control group were sensitized and challenged only using saline. From the first day of the experiment, different treatments were given to rats with gavage capacity of 5ml/kg for 28 days continuously. At 29th day, rats were hocused with 5mg/kg chloral hydrate, exposed to PM2.5 by tracheal instillation for three times, then ultrasonic atomizing inhalation for 30 min for 5 consecutive days, The femoral artery blood and the lung tissues of the rats were collected respectively. The activities of superoxide dismutase(SOD), methane dicarboxylic aldehyde(MDA) in serum were detected by colorimetric method, The contents of TGF-β1, Smad3, Smad2 and Smad7 in lung tissue were detected by ELISA. The pathology of lung tissues was measured by HE staining.
Results: Compared with PM2.5 group, in the FA (19.4) group and FA(38.8) group, the contents of MDA in serum were significantly decreased (p<0.05) and the contents of TGF-β1, Smad2 and Smad3 in lung tissue were obviously decreased (p<0.01 ) and SOD activity in serum and Smad7 content in lung tissue were increased (p<0.01 or p<0.05).The contents of MDA were significantly decreased at the FA (38.8) and FA (77.6) group. The positive group and the FA intervention group could improve the bronchial injury by reduction of inflammatory cell infiltration and mucus secretion on pathological sections.
Conclusion: A different concentrations of FA could effectively inhibit the effects of PM2.5 on respiratory tract injuries in asthmatic rats. These protective effects may be achieved by inhibiting oxidative injuries and regulating TGF-β1/smads signaling pathway.
Keywords: asthmatic rat, particulate matter（PM2.5）, ferulic acid（FA）, respiratory injury, TGF-β1/Smads signaling pathway
Modelling of uptake, depuration and bioconcentration of arsenic, zinc and copper mixtures in juvenile milkfish (Chanos chanos) (#68)
M. - C. Lin1, Y. - M. Yeh1
1 Nanhua University, General Education Center, Dalin, Chiayi, Taiwan
This study investigates the uptake, depuration and bioconcentration of arsenic (As), zinc (Zn) and copper (Cu) in juvenile milkfish, Chanos chanos. A 14-day exposure experiment was conducted to assess the time-integrated uptake and depuration of As, Zn and Cu by juvenile milkfish during exposure to each of these three chemicals and in various combinations, As-Zn, As-Cu and Zn-Cu. These three chemicals were chosen for the experiments, because they are found in culture ponds of juvenile milkfish in the blackfoot disease (BFD) area, southwest Taiwan. The uptake rate constant (k1) and depuration rate constant (k2) as well as the bioconcentration factor (BCF) of juvenile milkfish were analyzed based on a simple toxicokinetic model. The k1 values for As, Zn and Cu in milkfish exposed to single chemicals were 846.49 ml g-1 d-1, 682.32 ml g-1 d-1 and 13.08 ml g-1 d-1, respectively, while k2 values were 4.27 d-1, 2.02 d-1 and 1.24 d-1, respectively. The k1 and k2 values of the chemicals accumulated in milkfish were As > Zn > Cu when the fish were exposed to single chemicals. It indicates that uptake and depuration of As by milkfish occur more rapidly than the other two chemicals. The values of BCF of As, Zn and Cu were 198.42, 337.92 and 10.52, respectively. The results demonstrate that milkfish exhibited a greater ability for Zn accumulation than As and Cu. The decrease of k1 (from 846.49 ml g-1 d-1 to 361.51 ml g-1 d-1), k2 (from 4.27 d-1 to 3.23 d-1) and BCF (from 198.42 to 112.08) of As accumulation in milkfish was observed when Zn was added into the As stock. The values of k1 and BCF were decreased (from 846.49 ml g-1 d-1 to 842.69 ml g-1 d-1 for k1 and from 198.42 to 161.94 for BCF, respectively), while the value of k2 was increased (from 4.27 d-1 to 5.20 d-1) when the As stock was in combination with Cu additive. The k1, k2 and BCF values of Zn in milkfish were enhanced (from 682.32 ml g-1 d-1 to 841.93 ml g-1 d-1 for k1, from 2.02 d-1 to 2.26 d-1 for k2 and from 337.92 to 373.34 for BCF, respectively) when the fish were exposed to As-Zn mixture, while the BCF value was reduced (from 337.92 to 301.84) with higher values of k1 (from 682.32 ml g-1 d-1 to 771.39 ml g-1 d-1 ) and k2 (from 2.02 d-1 to 2.56 d-1) when exposed to Zn-Cu mixture. When milkfish were exposed to As-Cu mixture and Zn-Cu mixture, the values of k1 of Cu in milkfish were reduced (from 13.08 ml g-1 d-1 to 8.94 ml g-1 d-1 for As additive and from 13.08 ml g-1 d-1 to 7.05 ml g-1 d-1 for Zn additive, respectively) and the values of BCF also decreased (from 10.52 to 8.01 for As additive and from 10.52 to 5.49 for Zn additive, respectively), while the value of k2 for As additive was relatively lower (from 1.24 d-1 to 1.12 d-1) and that for Zn additive was relatively higher (from 1.24 d-1 to 1.28 d-1). The BCFs for the binary mixtures are lower than those for the single chemicals, which suggests that there is inhibition of one chemical accumulation by the other ones.
Keywords: Bioconcentration, Milkfish, Modelling, Toxicokinetics
Mercury speciation of preserved historical sludge to estimate risks from sludge entrapped under the reclaimed area of Minamata Bay, Japan (#74)
M. Sakamoto1, T. Itai2, K. Marumoto1, A. Matsuyama1
1 National Institute for Minamata Disease, Minamata, Japan
A large amount of methylmercury (MeHg) was directly discharged into Minamata Bay and created the Minamata disease. Then, sludge with high levels of mercury (exceeding 25μg/g on dry basis) was entrapped under reclaimed land of the bay. The objective of this study was to obtain data on potential MeHg pollution risks possibly caused by sludge leakage from reclaimed land in Minamata Bay, Japan by analyzing preserved sludge as well as current sediment samples and to answer concerns from residents. In this study, we performed a survey of Hg concentration and speciation in preserved sludge samples collected before the start of the dredging project (i.e., 35-50 years ago) and compared those results to sediment collected recently (July 29 to June 1st 2015) in Minamata Bay. The total mercury (THg) on wet basis was 0.18 μg/g in the control (n = 1), 6.1 μg/g (range: 0.83–12.2) for current sediments (n = 5), and 241 μg/g (range: 22.4–3620) for the preserved sludge (n = 4). MeHg was 0.71 ng/g, 3.7 ng/g (range: 1.71–8.5), and 108 ng/g (range: 7.8–503) for the control, current bay sediments, and preserved sludge, and MeHg percentage was 0.41%, 0.12% (range: 0.051–0.21), and 0.03% (range: 0.014–0.049), respectively. For all samples, the %MeHg decreased exponentially with increases in the THg concentration. An X-ray absorption fine structure analysis suggested that the main chemical form of the preserved sludge is β-mercury sulfide (β-HgS) and our data showed that the extractability of THg to the seawater was much lower than that of MeHg. Results indicate that, although MeHg was directly discharged from the company into Minamata Bay during the Minamata epidemic and that the preserved sludge had extremely high THg concentrations, we can estimate that, in the unlikely event of sludge leakage from reclaimed land, the risk of MeHg from the reclaimed land into Minamata Bay is low and far below the levels which caused the Minamata epidemic.
Keywords: methylmercury, Minamata Bay, sediment, HgS
Toxicity of diuron metabolites in human cells (#101)
A. M. Mohammed1, M. Huovinen1, K. Vähäkangas1
1 University of Eastern Finland, School of Pharmacy/Toxicology, Faculty of Health Sciences, Kuopio, Finland
Diuron is a phenylurea herbicide which is commonly used across the globe. It is known to be toxic to aquatic organisms and animals such as rats. Diuron is metabolized to equally or more toxic compounds, N-(3,4-dichlorophenyl)urea (DCPU), N-(3,4-dichlorophenyl)-3-methylurea (DCPMU) and 3,4-dichloroaniline (DCA). In the literature, metabolism of diuron has been linked with the development of urothelial cancer in rats. According to our earlier study, diuron is toxic and possibly genotoxic to human cells. In this current study, we wanted to pursue the effects of these diuron metabolites on cell viability, cell proliferation and ROS production. Studies were carried out using human cancer cell lines: BeWo (placental choriocarcinoma), MCF-7 (breast adenocarcinoma) and CaCo-2 (colon adenocarcinoma). According to our results, all the metabolites reduced the viability in all studied cell lines with BeWo cells being the most sensitive. Relative cell counts (indicating cell proliferation) were statistically significantly reduced by DCPMU in CaCo-2 cells and DCA in MCF-7 cells. DCPU slightly reduced the relative cell counts in all the cell lines. ROS production was statistically significantly increased in the case of DCA in all the cells and DCPMU in BeWo and MCF-7 cells. DCPU statistically significantly increased the production of ROS in BeWo cells. Experiments for potential effects of DCPU on ROS production in MCF-7 cells are yet to be carried out. In conclusion, our data indicates that diuron metabolites are cytotoxic probably through induction of ROS production. Sensitivity of BeWo cells (representing human placenta) to diuron metabolites implicates for possible fetal toxicity.
Keywords: cytotoxicity, ROS, DCA, DCPMU, DCPU
Distribution of cyfluthrin in brain regions, induction of dopamine depletion and up-regulation of oxidative stress and inflammation markers (#103)
I. Ares1, J. L. Rodríguez1, M. Martínez1, B. Lopez-Torres1, M. R. Martínez-Larrañaga1, A. Anadón1, M. A. Martínez1
1 Universidad Complutense de Madrid, Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Madrid, Spain
Taking into account that several classes of pesticide exposure causing lesions in dopaminergic neurons, the widely usage of cyfluthrin could be a serious public health concern. The present study aimed (i) to explore cyfluthrin oral absorption and its distribution in CNS and elimination in male Wistar rats after single oral dose of 20 mg/kg bw in order to determine in plasma and brain regions (hypothalamus, striatum, hippocampus and frontal cortex) toxicokinetics parameters. Cyfluthrin concentrations in plasma and brain were quantified by LC/MS; (ii) to determine dopamine (DA) and its metabolites levels after cyfluthrin treatment (20 mg/kg bw, orally 6 days); and (iii) to investigate also in these brain regions the expression of genes linked to apoptosis (Bax, Bcl2, casp-3), oxidative stress (Gpx1, Nrf2, Sod2, Cox2), proinflammation (Cox2, Il-1β, Il-6, NF-κB, TNFa) and DA metabolism (TH, DT, rD1, rD2, MAOA, MAOB) by Real-Time PCR. All experimental procedures involving animals were conducted in accordance with the ethics requirements and authorized by the Institutional Animal Care and Use Committee of the Complutense University of Madrid. Our results demonstrated: (i) Cyfluthrin crosses the blood-brain barrier. Plasma and nervous tissue kinetics showed an extensive oral absorption of cyfluthrin and a slow elimination (T1/2β range 17-23 h). The ratio AUC(0-24) tissue/AUC(0-24) plasma for cyfluthrin was 3.17 for hypothalamus. (ii) Cyfluthrin modulates the level of neurotransmitters, cyfluthrin produced a loss of DA and their metabolites contents in striatum (-81%), hypothalamus (-69%), prefrontal cortex (-55%) and hypoccampus (-53%) with respect to control. (iii) Of the genes examined, in hypothalamus tissue, changes in mRNA levels (fold change >1.5) were observed on cyfluthrin-upregulated Gpx1, Cox2, Il-1β, NF-κB, TNFa, rD1 and MAOB genes. These results imply cyfluthrin as a dopamine neurotoxin and a possible environmental risk factor for 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: Cyfluthrin, plasma and brain tissue toxicokinetics, neurotoxicity, dopamine neurodegeneration
Effect of prenatal treatment with Valproic acid on offspring investigated by phase contrast X-ray CT (#114)
T. T. Lwin1, A. Yoneyama2, T. T. Win Shwe3, H. Watanabe3, K. Hyodo4, T. Takeda1
1 Kitasato University, School of Allied Health Sciences, Sagmihara, Japan
Prenatal exposure of antiepileptic drug Valproic acid (VPA) increases the risk of having offspring with autism spectrum disorder (ASD). Neuroimaging studies play an important role for insights into changes in the brain structure of ASD. However, due to the small absorption differences between soft tissues of brain, conventional imaging techniques cannot depict internal structural of brain without contrast agent. Thus, imaging system with high spatial resolution is required. Now, phase-contrast X-ray CT with less than 36μm spatial resolution depicted soft tissue structural changes in various animal model diseases. Here, brains of VPA-exposed offspring were imaged by phase-contrast X-ray CT.
Autism rat was created by exposure of rat fetuses to valproic acid (600 mg/kg) on the 12.5th day of gestation (VPA). Normal Control rat was created using normal saline at the same condition. 13 weeks old 6 VPA and 3 normal rat’s brains were used in this study. Rat’s brains were extracted under anesthesia and fixed with 10% formalin for imaging. A two-crystal X-ray interferometer-based phase-contrast X-ray imaging system was used for the observations with an X-ray CCD camera with a 2560 x 2100 pixel sensor of pixel size: 6.5 x 6.5 μm2 and 17.8-keV synchrotron radiation. After imaging, Hematoxylin-eosin (H&E) staining was performed to examine abnormal histopathological structures.
Results and Conclusion
High spatial resolution phase-contrast X-ray CT without contrast agent clearly depicted the anatomical structures of brain, cortex, thalamus, corpus callosum, hippocampus, and amygdale depending on different densities. Mild to moderate expansion of ventricle was found in VPA group. In statistical analysis, increased density of hippocampus, thalamus and amygdale were observed in VPA group compared to control group. The significantly increased density was shown in hippocampus especially in dentate gyrus. This increased density might be associated with various neurodegenerative processes leading to ASD. H&E stain also revealed decreased neuronal size and increased cell packing density in dentate gyrus that is consistent with increased density.
This study demonstrated that phase contrast X-ray CT enabled to detect the fine inner structural changes in brains of VPA-exposed offspring.
Keywords: Valproic acid, Rat, autism, phase-contrast X-ray CT
The toxic effects and underlying mechanims of PM2.5-induced cardiomyocytes apoptosis and cardiac dysfunction (#172)
X. Yang1, M. Man1, P. Huang1, J. Duan1, Z. Sun1
1 Capital Medical University, Department of Toxicology and Sanitary Chemistry, School of Public Health, Beijing, China
Background: Although the strongly causal associations were between fine particulate matter (PM2.5) and cardiovascular disease, the toxic effect and potential mechanism of PM2.5 on heart was poorly understood. This study was aimed to investigate the toxic effects and involving mechanisms of PM2.5-induced cardiomyocytes apoptosis and cardiac dysfunction.
Methods and Results: In vitro, PM2.5 markedly augmented cardiotoxicity including oxidative damage and apoptosis in cardiomyocytes AC16 as well as epigenetic alteration. The cell viability was decreased while the levels of LDH release, ROS generation and MDA were increased in a dose-dependent manner induced by PM2.5. Followed by the activities of SOD and GSH-Px were declined. Mitochondria damage and apoptosis induced by PM2.5 was observed with the protein levels of Caspase-3, Caspase-9 and Bax were up-regulated while the anti-apoptotic protein, Bcl-2 was down-regulated. DNA methylation profiling revealed a significant gene-ADRB2 was involved in the cardiac relative GO and KEGG pathways. Mechanistic study showed the role of ADRB2 hypermethylation in inhibitng the β2AR and the downstream pathways, such as PI3K/Akt and p53 pathway in PM2.5-treated AC16. The transgenic cell lines showed over-expression of ADRB2 weakened the PM2.5-induced cardiomyocytes apoptosis in opposite way, but was augmented by PI3K inhibitor. The aboved cardiac toxicity induced by PM2.5 was also consistent with in vivo study using animal model via echocardiography, TUNEL staining, ultrastructural and histopathological evaluation.
Conclusions: Our results demonstrated that ADRB2 hypermethylation and mitochondria-mediated apoptosis pathway played a critical role in PM2.5-induced cardiac dysfunction.
Keywords: PM2.5; DNA methylation; Mitochondria damage; Apoptosis; Cardiac dysfunction
Cigarette smoke extract produces superoxide in aqueous media by reacting with bicarbonate (#204)
H. Jeong1, J. - M. Park1, Y. - S. Seo1, S. - J. Choi2, M. - Y. Lee1
1 Dongguk University, College of Pharmacy, Goyang, Republic of Korea
Cigarette smoke (CS) contains free radicals, reactive oxygen species (ROS) and other pro-oxidants. In addition, CS is capable of stimulating cells or tissues to generate ROS by activating cellular ROS sources. Hence, oxidative stress plays a large role in toxicity of smoking. Here, we found CS to generate superoxide in cell-free, aqueous solution and characterized chemical reaction producing superoxide. CS was generated from the mainstream smoke of 3R4F reference cigarettes and vapor-phase cigarette smoke extract (CSE) was prepared by passing CS through an impinger containing phosphate-buffered saline (PBS). CSE was added to biocompatible aqueous solution such as water, Dulbecco’s modified Eagle media (DMEM), Hank’s balanced salt solution (HBSS), PBS or blood plasma, and superoxide was measured using water-soluble tetrazolium salt-1 (WST-1). CSE produced superoxide only in DMEM and HBSS. In the experiments using aqueous solution containing each component of HBSS, bicarbonate (HCO3-) was proved to be responsible for superoxide production. Detection of superoxide by WST-1 was abolished by superoxide dismutase or a superoxide scavenger TEMPOL, but not by catalase or a hydroxyl radical scavenger mannitol. Chemical species detected by WST-1 was confirmed again to be superoxide using electron paramagnetic resonance spectroscopy. Considering the superoxide-producing chemical reaction between peroxy acid and bicarbonate, peroxy acids in CSE were assumed to be a culprit for superoxide generation. Indeed, by pretreating CSE with peroxidase to remove peroxy acids, superoxide generation in bicarbonate solution was reduced significantly. In addition to CSE, tar-phase of CS, so-called cigarette smoke condensate, also produced superoxide in the presence of bicarbonate. Taken together, CS can generate superoxide in aqueous media containing bicarbonate, and the substrates of peroxidase such as peroxy acids, at least in part, participate in such chemical reaction. These results suggest that bicarbonate may be a critical determinant of oxidative toxicity of CS in biological environments and the experimental conditions such as bicarbonate concentration in aqueous media should be carefully considered in in vitro toxicity study of CS.
Keywords: cigarette smoke, superoxide, bicarbonate
Chemical characterization of industrial- and road traffic-influenced fine particles (PM0.3-2.5) and impact on xenobiotic metabolizing enzymes gene expression in human reconstituted airway epithelium (#205)
A. Verdin1, S. Achard4, G. Tremolet1, E. Seurat4, D. Dewaele2, L. Courcot3, D. Courcot1, F. Cazier4
1 Université du Littoral Côte d'Opale (U.L.C.O.), Unité de Chimie Environnementale et Interactions sur le Vivant (U.C.E.I.V.), DUNKERQUE, France
Atmospheric fine particulate matter (PM2.5) was recently classified as carcinogenic to humans (Group 1) by the International Agency for Research on Cancer (IARC). Despite the relationship already established by epidemiological studies between PM exposure and the onset of cardiorespiratory diseases, the physiopathological mechanisms responsible for these diseases remain poorly understood.
For a better understanding of the PM’s health impact, the present study aims to evaluate the impact of two samples of fine particles (PM0.3-2.5) differing by their emission sources (traffic vs industry) and thus by their chemical composition (heavy metals, paraffins, polycyclic aromatic hydrocarbons …).
The chemical characterization showed that PM collected under industrial influence (Ind-PM) were 8-folds more concentrated in PAHs and 1.5-folds in metals than PM under traffic influence (Traf-PM), while Traf-PM were 2-fold enriched in paraffins. However, metals composition was clearly influenced by industrial emissions in Ind-PM (Al, Ca, Co, Fe, Mg, Mn, Ni, V et Zn…) and by vehicles and traffic emissions Traf-PM (Ba, Cu, Mo, Sn…).
Then, for biological assessment and to mimic the real human exposure to fine particles, an innovative 3D in vitro model was used: a human airway epithelium reconstituted, nasal origin, co-cultured with human airway fibroblast (MucilAir™-HF, Epithelix®). Epithelia were exposed to PM at 45 or 90µg/cm2 twice a week for two consecutive weeks. At the end of each week, part of the epithelia was sacrificed for the evaluation of xenobiotic metabolizing enzymes gene expression by RTqPCR. A significant dose-dependent induction of CYP1A1, CYP1B1 and HMOX genes expression was observed after exposure to Ind-PM and to a lesser extent to Traf-PM. Such an induction could be responsible for the formation of PAHs-reactive metabolites that could lead to an increase in DNA damages by adduct formation. Thus, a genotoxicity study will be led to assess the potential impact of these two samples on genome.
Keywords: chemical characterization, fine particles, metabolic gene expression, human reconstituted airway epithelium, repeated exposures
Cord blood acrylamide levels and birth size, and interactions with genetic variants in acrylamide-metabolizing genes (#258)
J. Hogervorst1, T. Nawrot1, 2
1 Hasselt University, Centre for Environmental Sciences, Diepenbeek, Belgium
To date, 3 epidemiological studies have consistently shown an inverse association between prenatal acrylamide exposure and birth size. According to the Developmental Origins of Health and Disease hypothesis, suboptimal prenatal development likely predisposes to inferior health throughout life.
We investigated the association between acrylamide and glycidamide to hemoglobin adducts in cord blood and birth size, and the interaction between acrylamide and polymorphisms in acrylamide-metabolising genes. Through this, we aimed to probe the causality of the inverse relationship between acrylamide and fetal growth.
In 443 newborns of the ENVIRONAGE (ENVIRonmental influence ON AGEing in early life) birth cohort, we investigated the association between prenatal acrylamide exposure (acrylamide and glycidamide to hemoglobin adduct levels (AA-Hb and GA-Hb, respectively) in cord blood) and birth weight, length and head circumference.
Furthermore, we studied interaction with single nucleotide polymorphism (SNPs) in CYP2E1 (rs2480258, rs915906 and rs11101888), EPHX1 (rs1051740) and GSTP1 (rs1695 and rs1138272).
We used multiple linear regression for the statistical analyses.
The effect estimate for a 10 pmol/g hemoglobin increase in AA-Hb was -40 grams (95% CI: -71,-9; p: 0.01), for birth weight, -0.17 centimeters (95% CI: -0.31, -0.03; p: 0.02) for length, and -0.13 centimeters (95% CI: -0.24, -0.01; p: 0.03) for head circumference. For GA-Hb, the effect estimates were -53 (95% CI: -90, -0.16; p: 0.005), -0.24 (95% CI: -0.41, -0.08; p: 0.004) and -0.11 (95% CI: -0.25, 0.03; p: 0.11), respectively. The associations were similar or stronger in newborns of non-smoking mothers.
There was no statistically significant interaction between acrylamide exposure and the studied genetic variations. However, there were stronger inverse associations with birth weight and head circumference among newborns with homozygous wildtypes alleles for the CYP2E1 SNPS and with variant alleles for a GSTP1 SNP (rs1138272), especially in children of mothers who did not smoke during pregnancy.
We observed an inverse association between prenatal dietary acrylamide exposure and prenatal growth. In addition, the interaction pattern (although not statistically significant) with SNPs in CYP2E1 is an indication for the causality of this association. Larger other studies are needed to corroborate this finding.
Given the consistent results of the good quality epidemiological studies that were performed to study the link between acrylamide and birth size, and the data on possible interaction with SNPs in CYP2E1, suggesting causality, preventative measures leading to reduced exposure of pregnant women to acrylamide are urgently called for.
Keywords: Cord blood acrylamide, birth weight, birth length, head circumference, SNPs in acrylamide-metabolizing genes
Health risks associated with cigarette sidestream smoke inhalation (#269)
L. - A. Li1, H. - J. Liu1, H. - L. Lee2
1 National Health Research Institutes, National Institute of Environmental Health Sciences, Zhunan, Miaoli, Taiwan
Cigarette sidestream smoke particulate matter (CSSP) is a common source of indoor air pollutants for nonsmokers. We measure the contents of metals and polycyclic aromatic hydrocarbons (PAHs) in CSSP emitted from Long Life cigarettes, a leading brand in Taiwan. 29 metals and 17 PAHs are found in CSSP. CSSP-bound metals may increase the chance of developing cancer by 9.27~20.93 x 10-6 and the hazard quotient for non-cancer toxicity by 0.496~0.286 when a Long Life cigarette is smoked in a 60-m3 poorly ventilated room. In contrast to Western cigarettes, cadmium is the primary toxic metal present in Long Life CSSP and accounts for more than 90% and 80% of metal-associated cancer and non-cancer risk, respectively. PAHs that are carcinogenic and probably carcinogenic to human comprise about one fifth of the total PAH mass. Carcinogenic potency is equivalent to 144 ng benzo[a]pyrene (BaP) per cigarette. When smoking occurs in a 60-m3 room, CSSP-bound PAHs increase cancer risk by a 1.44 x 10-6 chance per cigarette. In addition, the concentration of PAHs in the room is equivalent to 2.4 x 10-6 mg/m3 BaP, which is above the reference concentration for developmental toxicity recommended by US Environmental Protection Agency. High concentrations of CSSP are cytotoxic. Elevation of AhR expression in lung cells can attenuate CSSP-induced ROS generation and cytotoxicity. However, metals and PAHs are not the causes for cytotoxicity and have no effect on AhR activity.
Keywords: sidestream smoke, metals, polycyclic aromatic hydrocarbons, cancer risk, non-cancer toxicity
Convolution neural networks for training the unbalanced toxicity assessment data and analyzing chemical functional group (#298)
Y. O. Lee1, Y. Kim2
1 KIST Europe, Smart Convergence Group, Saarbrücken, Saarland, Germany
Deep learning models with the capability of the automated feature extraction have been developed and outperformed the traditional statistical models in the toxicity prediction recently. However, there is little consideration of data imbalance between ‘active’ and ‘inactive’ chemicals in the dataset, and efficient extraction of the activated substructure of the chemicals. In this study, the methods of training the models in the data-imbalanced conditions and interpreting the chemical substructures highly related to the toxicity from the model are proposed with convolution neural networks.
First, a convolution neural network is designed to predict the binary outcome of chemicals; ‘active’ or ‘inactive’ to toxicity in the given ligands. Second, a hybrid method of oversampling of active chemicals and down-sampling of inactive chemicals is utilized for training the model in the imbalanced data conditions in order to prevent overfitting. Third, the activated substructures are extracted by tracing back of convolution layers’ output and filters. For efficient extraction, filters of each convolution layer in the model are designed with the recent SMILES representation methods.
The experimental results with TOX21 datasets showed that (i) the proposed training method improves the prediction accuracy of chemicals’ activity to toxicity by 10% of AUC, (ii) the extracted substructures from analyzing convolution layer’s filters is validated with chemical functional group from the literature reviews. These results have potentials to in-Silico toxicity analysis for the prediction of unknown chemical’s toxicity effects and for finding the substructures for toxic ligand binding. Testing the unknown chemicals for screening the toxicity assessment and validation of a new substructure founded as toxicity biomarker will be conducted as future works.
Keywords: Toxicity prediction, QSAR, Deep Learning
Explanation of estrogenic activity in waste water treatment plant effluents (#330)
T. Černá1, 2, J. Semerád1, 2, T. Cajthaml1, 2
1 Institute of Microbiology of the CAS, v. v. i., Prague, Czech Republic
There are many concerns about the effects of endocrine disruptors (EDs) on the environment and human health. Especially aquatic ecosystems are highly affected by waste water treatment plant (WWTP) effluents as a secondary source of pollution. Hormonal activity of a sample can be revealed by using two approaches: analytical methods combined with prediction modelling and ecotoxicological assays. Nowadays, with the development of highly sensitive analytical methods, a large spectrum of EDs is being quantified in all matrices. Detected concentrations can then be used for the calculation of the total hormonal activity of the sample on the basis of the compounds respective potentials. Correspondingly, with the second approach, certain hormonal activity of the whole sample can be determined by ecotoxicological assays. Discrepancies between results obtained from both approaches are plenteously described in the literature.
By comparing our results for more than 20 samples of WWTP effluents we tried to explain these differences. Estrogenic activity was observed in more than one third of the samples (0.66±0.06 – 4.27±0.63 ng/L of 17β-estradiol equivalent). Most frequently detected analytes were bisphenol A, irgasan and estrone. Due to additive effect of EDs, we focused on determining the limits of detection of selected EDs by a newly developed liquid‑chromatography method. Afterwards, we determined the estrogenic activity (EC50) of every single substance employing the yeast Saccharomyces cerevisiae with human estrogenic receptor. Based on the potency of the compounds we then expressed the estrogenic activity of a mixture as 17β-estradiol equivalent (EEQ) using the quantified limits of detection. No sample exceeded this calculated value (2.48 ng/mL EEQ). Our results showed that the selected analytes explained the majority of the total estrogenic activity in the examined effluents.
Acknowledge: This research was supported by the Technology Agency of the Czech Republic (TA ČR TE01020218) and Grant Agency of Charles University (GA UK 1202219, Charles Univ, Fac Sci).
Keywords: estrogenic activity, effluents, effective concentration
Analysis of the biological effects of Persistant Organic Pollutants (POPs) on human leukocyte cell lines and peripheral blood mononuclear cells (#334)
S. M. Vidali1, 2, P. Georgiadis1, D. Stefanos2, D. Vlastos3
1 National Hellenic Research Foundation, , Institute of Biology, Medicinal Chemistry and Biotechnology, Athens, Greece
Despite the ban on the manufacturing and application of polychlorinated biphenyls (PCBs), their environmental persistence as well as their ability to bioaccumulate in tissues of living organisms (Persistent Organic Polutants, POPs) remain a great concern. Within the context of the European EnviroGenomarkers program (www.envirogenomarkers.net) the effect of POPs, on genome-wide expression as well as CpG methylation of leukocyte DNA of healthy volunteers was studied. The epigenetic exposure profile showed extensive and highly statistically significant overlaps with published profiles associated with the risk of future B-cell chronic lymphocytic leukemia (CLL) as well as with clinical CLL, suggesting an etiological link between exposure and CLL. A thorough toxicogenomic study of human leukocyte exposure to POPs in vitro is therefore necessary to validate the results of the population study and elucidate the role of POPs in haematological cancers. Therefore, cytotoxicity (by the MTT assay) and genotoxicity (double or single strand breaks using the COMET assay) of three selected POPs (HCB, PCB118 and PCB153) in human leukocyte cell lines (Jurkat and U937) as well as in Peripheral Blood Mononuclear Cells (PBMCs) were examined. Increased cytotoxicity was observed, regardless the cell line and incubation period used, only at concentrations greater than 50 μM, whereas, no notable genotoxicity was apparent at all incubation periods and concentrations. Only PCB153 induced a marginal but disputable, in terms of biological relevance, increase in DNA damage. However, using the LuMA assay, a dose-dependent global DNA hypomethylation was observed in PBMCs treated with both PCB118 and PCB153 at concentrations as low as 5 μΜ. The epigenetic in vitro CpG modulation observed might provide a causal association between POPs exposure and haematological cancers.
Keywords: EnviroGenomarkers, CLL, Persistent Organic Pollutants, epigenetics, methylation
Assessment of Roundup® cardiotoxicity on guinea-pig isolated Langendorff perfused heart and human Induced Pluripotent Stem cells derived cardiomyocytes (#336)
R. Printemps1, S. Guilbot1, H. Didier1, M. Le Grand1
1 PhysioStim, Lautrec, France
Although pesticides are known to come with benefits for agriculture, there is a lot of controversy surrounding the use of these substances because of suspected hazardous health effects. Roundup®, one of the most popularized pesticide, is composed of Glyphosate, its active ingredient, and adjuvants. The purpose of this work was to examine the effects of Roundup® exposition on heart function using isolated guinea-pig (GP) perfused heart and human Induced Pluripotent Stem cells derived cardiomyocytes (hiPSC-CM) models.
Haemodynamic and ECG parameters were recorded on isolated GP heart using the Langendorff method (3 doses of Roundup®, acute exposition) while contractility (impedance) and electrophysiology (MEA) of up to 9 doses were recorded in Cor.4U hiPSC-CM using the xCELLigence RTCA cardio ECR platform (acute and chronic exposition, up to 24h).
On isolated GP heart experiments, Roundup® increased ECG parameters and decreased heart rate only at 100 µM. An atrio-ventricular block occurred in 1 of 5 preparations. A concentration-dependent decrease of contractile function was observed at 10 and 100 µM.
Acute and chronic exposition to Roundup® also induced modification of hiPSC-CM electrophysiology and contractile function. At the top concentration of 1 mM, hiPSC-CM stopped beating and Cell Index was rapidly and drastically affected, corresponding to cardiomyocytes death.
Both models were able to detect cardiotoxicity of Roundup® mainly characterized by a massive decrease of the heart’s contractile function and troubles in electrical conduction. The effects observed in man after Roundup® intoxication, as reported in the literature (moderate ~360 µM to severe intoxication ~5 mM), were consistent with those observed in these in vitro-ex vivo models. These predictive and sensitive assays can be useful for cardiotoxicity assessment of pesticides and to a larger extent to compounds other than medicines developed by pharmaceutical industries.
Keywords: cardiotoxicity, Human cardiomyocytes, pesticides
Toxicity and degradability of widely used personal care products (#340)
L. Linhartová1, 2, K. Michalíková1, 2, M. Ezechiáš1, T. Cajthaml1, 2
1 Institute of Microbiology of the CAS, v. v. i., Prague, Czech Republic
Endocrine disrupting compounds (EDCs) belong among widely discussed and studied pollutants in last decade. Well known synthetic and massively used estrogens 17α-ethynylestradiol and bisphenol A are commonly detected in the environment and the methods to eliminate these compounds are studied extensively. Nevertheless, not only studied compounds contribute to the hormonal activity in the environment. We have focused on personal care products (PCPs) daily used as antimicrobial compounds in dental hygiene. Hormonal activity of octenidine - OCT, hexadecylpyridinium - HDP, menthol - MEN, eucalyptol - EUC, limonene - LIM, thymol - THM, sanguinarine - SAN, hexetidine - HEX, chlorhexidine - CHX was examined by the yeast Saccharomyces cerevisiae with human estrogenic receptor (ER)/androgenic receptor (AR) tests and the human cell line tests CXCL12-T47D and AIZ-AR for confirmation of estrogenic and androgenic activity, respectively. None of the tested compounds were identified as agonists of steroid receptor, but five of the tested compounds (OCT, HDP, CHX, THM and MEN) were able to inhibit estrogenic and/or androgenic pathway. The Schild analysis was used to identify direct binding on the receptor. Only THM and MEN were determined as antagonists of steroid receptors. The mechanism of antagonistic property of OCT, HDP and CHX requires further studies. Since the consumption of these antimicrobial agents is immense, a degradation study was also performed. In vivo and in vitro experiments were carried out with a representative of white-rot fungi, very potent degraders of persistent organic pollutants. Irpex lacteus and extracellular enzyme manganese-dependent peroxidase were used for degradation tests. In vivo experiments showed that I. lacteus was able to degrade more than 70% of CHX and OCT in 14 days. In vitro experiments showed 52%, 30% and 27% degradation of CHX, OCT and HDP, respectively.
This research was funded by the Competence Center TE01020218 of the Technology Agency of the Czech Republic.
Keywords: Personal care products, Endocrine disrupting compounds, Biodegradability, White rot fungi
The novel mathematical model for the quantitative analysis of antagonist-receptor interactions (#342)
M. Ezechiáš1, T. Cajthaml1, 2
1 Institute of Microbiology of the CAS, v.v.i., Laboratory of Environmental Biotechnology, Prague, Czech Republic
The quantitative analysis of drug-receptor interactions developed by Schild and Cheng-Prusoff is widely used for the assessment of antagonist effects. These methods are derived from the Gaddum equation. However, the Gaddum equation is derived for the simple law of mass action and does not consider that the compounds can have different slopes of their curves. This means that the slope parameters (Hill coefficients) of dose-response curves are always treated to be equal 1. Simplification in the Gaddum equation often leads to an inaccurate estimation of the equilibrium dissociation constants of the competitive antagonists, which is the key characteristic of the receptor ligands. In our previous study, we described the development and validation of a new mathematical model for mixture toxicity. Using this model, we derived a novel form of the Gaddum equation which contains the original hill coefficient of the agonist. Standardized in vitro yeast reporter gene assay (BMAEREluc/ERα) has been used for the validation of the proposed model and several known estrogen antagonists have been measured by Schild and Cheng-Prusoff method. Our mathematical model significantly reduces the differences in values calculated by the Cheng-Prusoff and Schild methods and yields more accurate estimations of antagonist affinity. This novel form of the Gaddum equation could improve hazard identification and dose-response assessment of chemical compounds.
Ezechias, M., Cajthaml, T., 2016. Novel full logistic model for estimation of the estrogenic activity of chemical mixtures. Toxicology 359, 58-70.
Cheng, Y., Prusoff, W.H., 1973. Relationship between inhibition constant (K1) and concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic-reaction. Biochem. Pharmacol. 22, 3099-3108.
Schild, H.O., 1949. Pax and competitive drug antagonism. Brit. J. Pharm. Chemoth. 4, 277-280.
Keywords: Receptor theory, Antagonists, Equilibrium constant, Gaddum equation
Cellular response and extracellular vesicles characterization of human macrophages exposed to PM2.5 (#345)
P. Martin1, A. Héliot1, G. Trémolet1, Y. Landkocz1, D. Dewaele2, F. Cazier2, F. Ledoux1, D. Courcot1
1 Université du Littoral Côte d'Opale, Unité de Chimie Environnementale et Interactions sur le Vivant, Dunkerque, France
Exposure to atmospheric fine Particulate Matter (PM) is one of the major environmental causes involved in the development of inflammatory lung diseases, such as chronic obstructive pulmonary disease (COPD) or asthma. When PM is penetrating in the pulmonary system, alveolar macrophages represent the first line of defense, in particular by triggering a pro-inflammatory response, and also by their ability to recruit infiltrating macrophages from the bone marrow. The aim of this study was to evaluate the toxicological and inflammatory responses of infiltrating macrophages after exposure to PM. Extracellular vesicles (EVs) production has been evaluated following their exposure to PM2.5. Finally, the ability of these EVs to convey information from PM exposed macrophages to pulmonary epithelial cells was evaluated.
Undifferentiated infiltrating macrophages respond to fine particles exposure in a conventional manner, as their exposure to PM2.5 induced the expression of EMXs such as CYP1A1 and CYP1B1, the enzymes involved in oxidative stress SOD2, NQO1 and HMOX as well as pro-inflammatory cytokines in a dose-dependent manner. Exposure to PM also induced a greater release of EVs in a dose-dependent manner. In addition, the produced EVs were able to induce a pro-inflammatory phenotype on pulmonary epithelial cells, with the induction of the release of IL6 and TNFa proinflammatory cytokines. These results suggest that infiltrating macrophages participate in the pro-inflammatory response induced by PM2.5 exposure and that EVs could be involved in this mechanism.
Keywords: Infiltrating macrophages, extracellular vesicles, PM2.5, lung epithelial cells
The influence of the Cucurbitaceae and their selected plant secondary metabolites on structurally related phenoxy acid herbicides removal and phytotoxicity mitigation (#347)
E. Mierzejewska1, W. Toloczko2, M. Tankiewicz3, M. Urbaniak1, 4, 5
1 University of Lodz, Department of Applied Ecology, Lodz, Poland
The aim was to evaluate the effects of Cucurbitaceae (zucchini and cucumber) and their selected plant secondary metabolites (PSMs: ferulic and syringic acids) on 1) removal rate of phenoxy acid herbicides (2,4-D and MCPA) with chemical structures resembling those of PSMs, and 2) phytotoxicity mitigation. The former was measured using chromatographic and molecular methods enabling to determine the removal rate of studied herbicides and presence of bacterial tfdA genes responsible for its degradation; and the latter using Phytotoxkit test. The research aimed also to determine the 3) influence of 2,4-D and MCPA, as well as artificial PSMs application, on cucurbits condition measured as changes in their morphometric and physiological parameters.
The obtained results demonstrated that although the removal of 2,4-D reached 100% irrespectively of PSMs spiking or not, the phytotoxicity remained very high. In contrast, in variants with MCPA+PSMs the herbicide removal was 99%, whereas in sample without PSMs was almost two-fold lower. Also phytotoxicity was found to be lower in samples spiked with MCPA+PSMs.
The results show also that simultaneous application of studied herbicides and PSMs contributes to increased detection of herbicide degradative genes. Samples spiked with herbicides+PSMs demonstrated a mean two-fold increase in detection of tfdA genes in comparison to those amended only with herbicides. Such an increase in tfdA genes detection demonstrated that PSMs can enhance the biodegradation of structurally similar phenoxy acid herbicides. 16S rRNA gene sequence analysis revealed high homology to soil bacteria Rhodoferax saidenbachensis, Burkholderia spp.and Cupriavidus spp. commonly known to be involved in biodegradation processes of phenoxy acid herbicides.
As far as the studied herbicides are used to selectively control the growth of dicotyledonous weeds, its application can also affect the condition of cultivated plants. Obtained results demonstrated that application of these herbicides influences on cucurbits condition measured as lower plant biomass, leaves surface area and length, and chlorophyll content. Simultaneous application of both phenoxy acid herbicides+PSMs, alleviated this inhibitory effects, however this positive influence was observed only in the case of zucchini variants.
Acknowledgements: This work was supported by the University of Lodz Student Research Grant “Plant Secondary Metabolites as stimulators of bacterial degradation of 2,4-D and MCPA” and The European Structural and Investment Funds, OP RDE-funded project 'CHEMFELLS4UCTP' (No. CZ.02.2.69/0.0/0.0/17_050/0008485)”
Keywords: biodegradation, phenoxy herbicides, plant secondary metabollites, cucurbits, phytotoxicity
Toxic effects on Caenorhabditis elegans from sedimented dust in an urban area of northern Colombia (#433)
J. A. Osorio Martinez1, A. C. De la parra Guerra1, M. Duran Izquierdo1, J. de la Rosa2, J. Olivero Verbel1
1 University of Cartagena, Environmental and Computational Chemistry Group, Cartagena, Colombia
Sedimented dust is a heterogeneous mixture whose potential sources are soil erosion, atmospheric deposition, and anthropogenic activities. It is the dominant fraction of air pollutants in urban areas carrying toxic substances such as heavy metals, pesticides, and polycyclic aromatic hydrocarbons, among other chemicals. The objective of this work was to evaluate the toxic effects of sedimented dust extracts from an urban area crossed by a railway line at northern Colombia on Caenorhabditis elegans. Urban dust samples were collected in 21 points at the municipality of Aracataca (Magdalena) Colombia, a location influenced by coal transport on trains, intensive agriculture, and some urban traffic. A reference sample was taken 3 Km northeast of the urban area. Aqueous extracts (K medium) were obtained from dust particles (< 75 μm), and synchronized nematodes (L1 and L4) were exposed to the extracts evaluating lethality, growth, locomotion, reproduction, as well as gene expression with transgenic GFP strains: mtl-2 and sod-4. ICP-MS analysis was performed at points with low, medium and high lethality. Lethality varied between 0.88% and 60.2%, and surprisingly, nematode size increased in 85.7% of the samples, whereas locomotion was inhibited between 0.77% and 46.1%. Some extracts promoted egg hatching. The mtl-2 and sod-4 expression increased moderately in most samples, suggesting metal exposure. Trace elements concentrations (ppm) in analyzed samples increased in the order Ba > Zn > Sr > Pb > B > Rb > Ce. Growth and locomotion showed a negative association with Zr and Rb concentrations, respectively. In short, urban dust extracts impacted physiological parameters in C. elegans, such as survival, growth, and locomotion, modulating gene expression related to metal exposure and oxidative stress. Colciencias-Unicartagena, 785-2017.
Anbalagan, C., Lafayette, I., Antoniou-Kourounioti, M., Gutierrez, C., Martin, J. R., Chowdhuri, D. K., & De Pomerai, D. I. (2013). Use of transgenic GFP reporter strains of the nematode Caenorhabditis elegans to investigate the patterns of stress responses induced by pesticides and by organic extracts from agricultural soils. Ecotoxicology, 22(1), 72–85. https://doi.org/10.1007/s10646-012-1004-2
Tang, Zhenwu, Miao Chai, Jiali Cheng, Jing Jin, Yufei Yang, Zhiqiang Nie, Qifei Huang, and Yanhua Li. (2017). Contamination and Health Risks of Heavy Metals in Street Dust from a Coal-Mining City in Eastern China. Ecotoxicology and Environmental Safety 138 (April): 83–91. https://doi.org/10.1016/J.ECOENV.2016.11.003
Tejeda-Benitez, L., Flegal, R., Odigie, K., & Olivero-Verbel, J. (2016). Pollution by metals and toxicity assessment using Caenorhabditis elegans in sediments from the Magdalena River, Colombia. Environmental Pollution, 212, 238–250. https://doi.org/10.1016/J.ENVPOL.2016.01.057
Valotto, G., Zannoni, D., Rampazzo, G., Visin, F., Formenton, G., & Gasparello, A. (2018). Characterization and preliminary risk assessment of road dust collected in Venice airport (Italy). Journal of Geochemical Exploration, 190, 142–153. https://doi.org/10.1016/J.GEXPLO.2018.03.005
Keywords: urban dust, toxic effects, nematodes, gene expression.
Toxicity of organic matter originated from Microcystis aeruginosa (#468)
S. Šilhavecká1, 2, T. Cajthaml1, 2, J. Načeradská3, M. Pivokonský3
1 Charles University, Institute for Environmental Studies, Faculty of Science, Prague 2, Czech Republic
Nowadays, the eutrophication of surface water results in a massive increase in algal and cyanobacterial growth associated with the occurrence of water blooms. Due to the decomposition of the biomass, the concentrations of algal organic matter (AOM) including cyanobacterial toxins also increase in water reservoirs which are frequently used as a source of drinking water. The main problem arises during the drinking water treatment processes because the majority of dissolved organic compounds serve as precursors for the formation of potentially toxic disinfection by-products (DBPs) in drinking water. The mentioned compounds (e.g. cyanotoxins, DBPs) are currently considered to be a threat to drinking water quality due to their adverse effect on human health.
A method employing liquid chromatography with tandem mass detection (LC-MS/MS) has been developed and optimized for the determination of selected types of cyanobacterial toxins such as microcystins (MCs), anatoxin, cylindrospermopsin, nodularin. The fully optimized method has been used for the detection of cyanotoxins in a sample containing dissolved organic carbon (DOC) of cyanobacterium Microcystis aeruginosa. Cyanotoxins detected in the sample were: anatoxin (0.02 µg/mg of DOC), MC-RR (0.78 µg/mg of DOC), MC-YR (0.22 µg/mg of DOC), MC-LR (0.74 µg/mg of DOC), MC-LY (0.02 µg/mg of DOC) MC-LW (0.69 µg/mg of DOC) and MC-LF (0.02 µg/mg of DOC).
Moreover, toxic properties of the studied AOM-containing sample have also been established using Thamnotoxkit F with the crustacean Thamnocephalus platyurus for which LC50 was determined at 20.2 mg/l of DOC. Furthermore, IC50 for root growth inhibition of plant Lepidium sativum was established (180.3 mg/L of DOC). The growth of other tested organisms (Saccharomyces cerevisiae, Bacillus subtilis, Escherichia coli) was not affected by the exposure to organic matter originated from M. aeruginosa and the growth of alga Desmodesmus subspicatus was even stimulated.
This study was funded by project GA18-14445S of the Czech Grant Agency of the Czech Republic.
Keywords: cyanobacteria, organic matter, toxins
Aromatase activity in the presence of penconazole and essential metals (#491)
J. Jaklová Dytrtová1, 2, M. Jakl3
1 Institute of Organic Chemistry and Biochemistry of the CAS, Prague, Czech Republic
Triazoles are agrochemicals or pharmaceuticals used for protection of crop or skin against fungi or mildew. The mechanism of their action lies in the inhibition of sterols biosynthesis . It is known that essential metals significantly influence the behaviour of azoles in the manner of (i) complexes creation with them , (ii) changes of redox behaviour  and (iii) degradation pathways . It is not clear how the cocktail effect of other present biological active chemicals modify the inhibitory effect of azoles (penconazole) to aromatase. We provide the experiments in the gas phase to clarify the reaction mechanisms modifications as an effect of essential metals presence.
The research was supported by by GA CR (project No. 18-01710S).
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Keywords: azoles, fungicides, cocktail effect, gas phase experiments, ESI-MS
Migration of pesticide of the derivative class of phenoxyacetic acids in soil-water system (#496)
V. N. Rakitskii1, T. Sinitskaya1, N. Fedorova1, I. Gromova1, L. Goryacheva1
1 Federal Scientific Center of Hygiene named after F.F. Erisman, Administration, Mytischchi, Russian Federation
Introduction: Study of the migration of phenoxyacetic acid class pesticide in soil water system.
Keywords: herbicide, migration, soil, water
Newborn telomere length and the prenatal exposome: findings from the ENVIRONAGE birth cohort (#536)
D. S. Martens1, T. S. Nawrot1, 2
1 Hasselt University, Centre for Environmental Sciences, Diepenbeek, Belgium
Background:. The exposome encompasses all exposures over an entire life as from conception onward. Telomere length (TL) is marker of biological ageing and TL at birth may predict disease susceptibility later in life. Telomeres can be considered as cellular memories of exposures to oxidative stress and inflammation, and therefore TL may be a proxy for assessing the exposome. We evaluated the potential of TL at birth as a proxy for the prenatal exposome.
Methods: In the ENVIRONAGE birth cohort, Flanders, Belgium, we measured cord blood TL in 1200 mother-newborn pairs using a qPCR method. We collected data on maternal external and internal factors of the exposome. We used linear regression models to associate maternal residential air pollution and traffic exposure, temperature exposure, maternal internal indicators of inflammation and diet, education, BMI, and lifestyle factors during pregnancy with cord blood TL. Models were adjusted for parental age, newborn sex, gestational age and month of delivery.
Results: We found that high particulate matter (PM2.5) and black carbon (BC) air pollution and temperature exposures during pregnancy were negatively associated with cord blood TL. Higher traffic density near the residential address was negatively associated with cord blood TL. Furthermore we found that increased maternal plasma insulin and homocysteine levels were associated with shorter TL, but no associations were observed for folate, and IL-6. Low maternal education and a high BMI independently were associated with shorter TL. Maternal lifestyle factors during pregnancy including, diet, physical activity and alcohol consumption did not strongly predict newborn TL.
Conclusion: Our results show that TL, already at birth, may capture some of the individual exposure factors of the prenatal exposome. However, to explore additive effects of multi-exposures from the exposome, an integrative assessment and an exposomics approach modeling strategy will be applied in the future to fully evaluate these multi-exposures in association with newborn TL.
Keywords: exposome, telomere biology, ageing, in utero life
Activation of NRF2 and AHR signaling pathways and autophagy by ambient fine and quasi-ultrafine particles in human bronchial epithelial BEAS-2B cells (#575)
G. Badran1, 2, 4, A. Verdin1, C. Grare2, I. Abbas4, F. Ledoux1, M. Roumié4, P. Genevray3, Y. Landkocz1, F. Cazier3, J. - M. Lo Guidice2, D. Courcot1, G. Garçon2
1 Univ. Littoral Côte d′Opale, Unité de Chimie Environnementale et Interactions sur le Vivant, UCEIV EA4492, FR CNRS 3417, Dunkerque, France
Particulate matter (PM), a major class of air pollutants, represents a heterogeneous complex of inorganic (e.g. metals, ions), organic (e.g. polycyclic aromatic hydrocarbons: PAH), and biological (e.g. pollen, fungi) components. PM have been widely studied, but the relationship between its chemical components and its toxicity is still unclear. In this work, we also sought to compare the toxicological effects of the organic and inorganic fractions of ambient PM2.5-0.3, on the one hand, and the organic fractions of ambient PM2.5-0.3 and PM0.3 in human bronchial epithelial lung BEAS-2B cells, on the other hand.
Physico-chemical characterizations of PM2.5-0.3 and PM0.3 were carried out using GC-MS for PAH, O and N-PAH, and n-alkane quantification, ICP-AES for major and trace elements quantification, ionic chromatography for ion quantification, X-ray diffraction for crystalline phase study, and SEM-EDX for morphology and elemental composition at particle scale. After 6 and 24h of exposure at low doses (3 and 12μg/cm2), comparisons were made between the toxicological effects of native PM2.5-0.3 to its organic extractable matter (OEM2.5-0.3), and non-extractable matter (NEM2.5-0.3), and between those of OEM0.3 and OEM2.5-0.3. Redox status and xenobiotic metabolizing enzymes were also studied by evaluating expression of several genes implicated in both these signaling pathways (AHR, ARNT, CYP1A1, CYP1B1, GSTA-4, and EPHX1; NRF2, NQO1, KEAP1, SOD and HMOX; RT-qPCR) and oxidative damage (carbonyl proteins, 8-isoprostane, and 8-OHdG; Elisa). Autophagy was evaluated through ATG5, LC3b, Beclin1, and PARKIN (Western-blot).
Concentrations of organic compounds (PAH, N and O-PAH, and n-alkane) were higher in PM0.3 than in PM2.5-0.3, thereby supporting the strong influence of combustion processes on PM0.3 emission. All the fractions were able to induce NRF2 and AHR signaling pathways, and autophagy. However, by comparing the different fractions derived from PM2.5-0.3, native particle lead to the highest activation of these underlying mechanisms. Finally, OEM0.3 was found to be the most toxic, probably due to its richness in organic compounds. Taken together, these results supported the toxicity of both inorganic and organic fractions of PM2.5-0.3, and showed the crucial role played by PM0.3 in PM toxicity.
Keywords: Fine and ultrafine particles, physico-chemical characterization, oxidative stress, metabolic activation, autophagy
Total arsenic and arsenic speciation of cereals and seaweeds in South Korea (#602)
K. - W. Lee1, S. Choi1, M. - H. Kim1
1 Korea University, Seoul, Republic of Korea
Arsenic (As) has different toxic effects on human depending on its chemical species with unique characteristics. Inorganic As (iAs), including arsenite (As (III)) and arsenate (As (V)) is more toxic than organic As and is known to cause cardiovascular disease and cancer. As an important dietary source of iAs exposure, there needs to monitor As species in various cereals. In addition, seaweed, which is rich in dietary fibers and minerals, is a popular type of seafood in South Korea, and it is known that the concentration of As in seaweed is relatively high. The annual consumption of seaweed in South Korean is close to 5 kg/person, and more than 50 kinds of seaweed are used as foods. In this study, total As (tAs) and six iAs species, including As (III), As (V), monomethylarsonic acid (MMA), dimethylarsinic acid (DMA), arsenobetaine (AsB), and arsenocholine (AsC)) were monitored in barley (n = 16), oats (n = 13), glutinous rice (n = 20), corn (n = 18), black rice (n = 12), wheat (n = 5), white rice (n = 20) and brown rice (n = 17) for cereal samples and in kelp (n = 33), laver (n = 25), sea mustard (n = 25), agar (n = 20), seaweed fulvescens (n = 17) and gulfweed (n = 12) for seaweed samples. For the present study, tAs was analyzed using inductively coupled plasma-mass spectrometry, ICP-MS, and 6 species of As were analyzed by HPLC coupled with ICP-MS. It was found that low level of As in corn, barley, oat and wheat samples, and relatively high level of As in rice were found, and there is a positive correlation between tAs and iAs concentration in cereals. In seaweed, there was no observative correlation between tAs and iAs concentration, and high level of tAs was detected in seaweeds, whereas level of iAs was quite low except for gulfweed. This study will be used to assess potential risk of As species from cereal and seaweed consumption.
Keywords: arsenic speciation, inorganic arsenic, cereal, seaweed, ICP-MS
A 28-day repeated oral dose toxicity study of 5-ethyl-2-methyl-2-oxido-1,3,2-dioxaphosphinan-5-yl)methyl methyl methylphosphonate in mice (#604)
S. Takasu1, M. Tohnai2, Y. Ishii1, A. Kijima1, K. Ogawa1, T. Umemura1, 2
1 National institute of health sciences, Division of pathology, Kawasaki-shi, Kanagawa-ken, Japan
5-ethyl-2-methyl-2-oxido-1,3,2-dioxaphosphinan-5-yl)methyl methyl methylphosphonate (PMMMP) is one of the novel phosphorus based-flame retardants. Because PMMMP is known to be an indoor contaminant, possible exposure of human to PMMMP has been concerned. However, there have been few reports in terms of safety assessment for PMMMP. In the present study, we investigated a repeated oral dose toxicity of PMMMP using mice. Six-week-old male CD-1 mice were treated with PMMMP (containing 20% bis[(5-ethyl-2-methyl-2-oxido-1,3,2-dioxaphosphorinan-5-yl)methyl] methyl methylphosphonate as a contaminant) by gavage at doses of 100, 300 or 1000 mg/kg/day for 28 days. The typical toxicological parameters were analyzed at necropsy. There were no treatment-related clinical sign and significant changes of body weight and food consumption. The serum phosphorus level was significantly increased in 300 and 1000 mg/kg/day PMMMP-treated groups, but there were no changes in serum calcium level in all PMMMP-treated groups. The absolute and relative adrenal weights were significantly increased in all PMMMP-treated groups except the relative weight at the low dose. Histopathological examination revealed no treatment-related changes in any organs. Therefore, we concluded that the increases in serum phosphorus level were due to the treatment with phosphorus agent, but not toxicological effects. Also, weight changes in adrenals might have no toxicological significance. Thus, there were no significant toxicological changes in any parameters in the present study. Hence, no adverse effect level of PMMMP under the present experimental condition in male mice was estimated to be greater than 1000 mg/kg/day.
Keywords: Repeated oral dose toxicity, Phosphorus based-flame retardants
Pesticide residues in peppermint, chamomile and bladder herbal teas sold in Estonia (#616)
K. Eha1, L. Parts1
1 Tallinn Health Care College, Medical Technology Education Centre, Tallinn, Estonia
Using medicinal plants for treating illness is one of the oldest method to cope with diseases. According to WHO currently about 80% of Worlds’ population is primarily relying on herbal remedies when falling ill. It is well known that there are many contaminants and residues in herbal remedies that may cause harm to consumer – such as pesticides. To avoid harm from these substances it is essential to control the quality of herbs prior consumption.
The aim of this study was to determine pesticide residues in commercially sold peppermint, chamomile and bladder tea herbs. All samples were bought from local pharmacies and supermarkets to include as many producers possible at given time. Samples were prepared with extraction with hexane followed by silica column cleaning. The chamomile samples in repeated analysis were prepared by standard method: Foods of plant origin - Determination of pesticide residues using GC-MS and/or LC-MS/MS following acetonitrile extraction/partitioning and clean-up by dispersive SPE - QuEChERS-method EN 15662. The analysis were carried out with Agilent Technologies 7890B gas chromatography, Agilent Technologies 5977A mass-selective detector, Agilent MassHunter Qualitative Analysis B.07.00 and Agilent MassHunter Quantitative Analysis B.07.00 programs. The pesticides were selected based on EU Pesticide Database and Statistics Estonia Database.
Results: Residues of 5 pesticides were detected in 8 samples of peppermint, 4 samples consisted pesticide amounts exceeding allowed limits. In chamomile residues of 6 pesticides were detected in 7 samples, 2 samples consisted residues in quantifiable amounts, none exceeded EU limits for pesticide residues. Bladder tea herbs contained more than 14 different pesticide residues, from which 4 were detected in quantifiable amounts.
Conclusion: The origin of pesticide residues in organically produced herbal teas is unclear, but the amounts are in trace levels and therefore pose no substantial risk on their own for consumers health. There is a risk for synergistic effect with such wide array of different pesticides depending on consumer habits. Some herbal teas sold in supermarkets exceeded EU limits for pesticides and should be avoided.
WHO guidelines on good herbal processing practices for herbal medicines (2018). WHO
WHO guidelines for assessingquality of herbal medicineswith reference to contaminantsand residues (2007). WHO
Ana Lozanoa, Łukasz Rajskia,b, Noelia Belmonte-Vallesa, Ana Uclésa, Samanta Uclésa, Milagros Mezcuaa, Amadeo R. Fernández-Alba. (2012). Pesticide analysis in teas and chamomile by liquid chromatography and gas chromatography tandem mass spectrometry using a modified QuEChERS method: Validation and pilot survey in real samples. Journal of Chromatography A: 1268 (2012) 109– 122.
Keywords: Herbal medicine, chamomile, peppermint, bladder tea, pesticide residue
The Fusarium mycotoxins effect on glutathione system in broiler chicken (#630)
S. Kulcsár1, 2, B. Kövesi1, 2, M. Mézes1, 2, K. M. Balogh1, 2, E. Zándoki2
1 Szent István University, Department of Animal Nutrition, Gödöllő, Hungary
In temperate climates Fusarium mycotoxins, including deoxynivalenol (DON), fumonisin B1 (FB1) and T-2 toxin are the most relevant contaminants of cereal grains and they very frequently co-occur. Combined exposure might cause additive, synergistic or antagonistic toxic effects, but little is known about the actual multi-mycotoxin risk, yet. It is well known that Fusarium mycotoxins provoke oxidative stress, which is neutralized by the glutathione system.
The aim of this study was to investigate the intracellular biochemical and gene expression changes in case of multi-mycotoxin exposure, with attention to certain elements of the glutathione system, which is thought to be a major defence at the onset of low‐level oxidative stress. In vivo study was performed in broiler chicken in a short-term (72 hours) feeding trial with low (T-2 toxin: 0.25mg/kg; DON: 5 mg/kg; FB1: 20 mg/kg) and high (T-2 toxin: 0.5mg/kg; DON: 10 mg/kg; FB1: 40 mg/kg) doses of multi-mycotoxin exposure. Liver samples were taken, in which some parameters of the glutatione system, reduced glutathione (GSH) concentration, glutathione peroxidase (GPx) activity and changes in the gene expression of glutathione peroxidase4 (GPx4), glutathione synthetase (GSS), and glutathione reductase (GSR) were measured.
The results revealed that multi-mycotoxin exposure caused significant differences in the amount and activity of the glutathione redox system as a function of the exposure time. GPx4 and GSR expression was decreased in the low dose group and GSS expression was elevated in high dose group on day 1 as compared to the control. The results showed how the Fusarium multi-mycotoxin exposure activated the antioxidant defence system as a consequence of low level oxidative stress by its individual effects.
The research was supported by the NVKP_16-1-2016-0016 and EFOP-3.6.3-VEKOP-16-2017-00008 co-financed by the European Union and the European Social Fund projects.
Keywords: Fusarium, multi-mycotoxin, glutathione system, poultry, oxidative stress
Food levels of 9 bisphenol analogues in Catalonia (Spain): Comparing canned vs non-canned foodstuffs (#668)
J. L. Domingo1, N. González1, S. Cunha2, C. Monteiro2, J. O. Fernandes2, M. Marquès1, M. Nadal1
1 Universitat Rovira i Virgili, Department of Basic Medical Science, Reus, Spain
Bisphenols (BPs) comprise a wide group of chemicals used in the manufacture of polycarbonate plastics and epoxy resins. Traces of BPs may be contained in a broad range of products, such as food containers, dental fillings, medical devices, toys, thermal paper, and surface coatings of can, among others. Although bisphenol A (BPA) has been the most largely used analogue, it is being gradually replaced by some alternatives, labelled as “BPA-free”, that could be less toxic. However, since the chemical structure of these analogues is very similar to that of BPA, their endocrine disrupting potential could be similar or even higher. Diet accounts for more than 99% of the exposure to BPs, being food containers a potential source of high importance. Unfortunately, data relative to food concentrations of not only BPA but especially the rest of BP analogues are still very scarce. Therefore, an accurate chemical analysis is needed to evaluate the potential exposure to these group of chemicals. This study was aimed at determining the levels of 9 bisphenol analogues (BPA, BPS, BPF, BPB, BPAF, BPZ, BPE, BPAP, and BPP) in 40 food samples obtained from a duplicate diet study. Samples were divided into 2 groups: 1) canned food; and 2) fresh food, packed in glass containers or other BP-free materials. Samples were homogenized, and BPs were determined combining QuEChERS extraction with a dispersive liquid-liquid microextraction (DLLME), followed by a gas chromatography coupled to mass spectrometry. BPA was present in all the canned foods, with levels ranging between 3.45 and 88 µg/kg. Canned chicken also showed traces of BPB (3.86 µg/kg). On the other hand, BPA was also found in 9 out of 26 non-canned foods, with mushrooms presenting the highest concentration of BPA (9.56 µg/kg). Oil samples, both in can and glass, contained measurable levels of BPB (1.25 and 0.85 µg/kg, respectively). Additionally, fresh chicken also had BPB (4.19 µg/kg). Finally, BPE could be also quantified in nuts and mushrooms in glass (mean levels: 12.35 and 2.40 µg/kg, respectively). Our results are very valuable to assess the dietary intake of these chemicals and to understand the role and the distribution of each analogue in the industry.
Keywords: Bisphenol, BPA, Food toxicology, Dietary exposure, Endocrine disruptor
Levels of Alternaria toxins in feed for farm animals – a preliminary study (#670)
P. Jedziniak1, L. Panasiuk1
1 National Veterinary Research Institute, Department of Pharmacology and Toxicology, Pulawy, Poland
Purpose: Among the mycotoxins, Alternaria toxins seem to be one of the most important group. This is about 70 compounds of secondary metabolites of Alternaria alternata which toxins as alternariol (AOH), alternariol monomethyl ether (AME), tenuazonic acid (TeA), and altertoxins (ATX) are described to induce harmful effects in animals, including fetotoxic and teratogenic effects. However, data on the sensitivity of farm animals are very limited and do not allow the estimation of tolerance levels for individual toxins and mixtures thereof. However the presence of those compound was reported in wheat, sorghum, and barley, and in oilseeds such as sunflower and rapeseed, tomato, apples, citrus fruits, olives and several other fruits and vegetables, the data about animal feed are scarce . The aim of this study was to survey the presence of six Alternaria toxins in a different kind of feed and feed material (227 samples of swine feed, poultry feed, silage and grains) with LC-MS/MS method.
Methods: Analytes (AOH, AME, TeA, ATX, altenuene ALT, tentoxin TEN) were extracted from feed samples (1g) with 4 ml of solvent extraction (ACN:H2O:HCOOH; 79:20:1 v:v:v) by 30 min of horizontal shaking. After extraction, 100 µl of supernatant was transferred to a plastic tube. After evaporation (N2, 40°C), the dry residue was dissolved in 100 µl of 50% MeOH and centrifuged (14 000 rpm, 30 min). Finally, the sample was transferred to orange vials and determined with LC-MS/MS technique (chromatograph Nexera X2 coupled with tandem mass spectrometer LCMS 8050, Shimadzu). The chromatographic separation was obtained using Kinetex C18 column (100×2.1 mm, 2.6, Phenomenex) and as a mobile phase 100 mg/l NH4(CO2)2 and MeOH (pH~7.8). The method was validated: limits of quantification (LOQ) ranged between 5.0 and 50 μg/kg; the repeatability and reproducibility (expressed as CV, %) were between 4.0-35% and recoveries were ranged from 65% to 150%.
Results: The results show a relatively high occurrence of Alternaria toxins in analysed feed samples. TeA, AOH and TEN were determined in 54-56% of all samples; ALT, ATX and AME in 9, 15 and 21% of all samples, respectively. Over 73% of the poultry and swine feed samples were contaminated with TeA in the range of 10-980 µg/kg. The feed materials (grains) contained TEN in the relatively low concentration (mean 8.5 µg/kg) but in 77% of surveyed samples. The AOH was determined in 62% of silages samples (mean 17.5 µg/kg).
Funded by KNOW (Leading National Research Centre) Scientific Consortium „Healthy Animal - Safe Food”, decision of Ministry of Science and Higher Education No. 05-1/KNOW2/2015
1. EFSA on Contaminants in the Food Chain (CONTAM); Scientific Opinion on the risks for animal and public health related to the presence of Alternaria toxins in feed and food. EFSA Journal 2011;9(10):2407. [97 pp.] doi:10.2903/j.efsa.2011.2407. Available online: www.efsa.europa.eu/efsajournal
Keywords: mycotoxins, alternaria toxins, feed, lc-ms/ms
Assessment of heart rate variability under exposure to GSM 900-MHz signal from Mobile phone in healthy young people. (#675)
B. Selmaoui1, 2, S. Andrinome1, 2, E. Stephan-Blanchard2, F. Telliez2
1 INERIS, Experimental Toxicology, Verneuil-en-Halatte, France
Purpose: Recently a National Toxicology Program has issued their technical reports (NTP TR 595 (1) and NTP TR 596 (2)). They found that high exposure to radio frequency radiation (RFR) used by cell phones was associated with clear evidence of tumors in the hearts of male rats. In this context and given the large number of mobile phone users worldwide, the present study was focused on the effect of RFR of mobile phone on the heart rate variability (HRV). The aim of the present study was to analyze, in healthy subjects at rest, the influence of exposure to GSM 900MHz on HRV parameters.
Participants and Methods: Twenty-six young healthy volunteers participated in the experiment. The volunteers were selected following a routine clinical examination. Inclusion criteria included regular sleep habits, no medication, no chronic disease or disability, no recent acute illness, no smoking, and no neurological or psychiatric illness. Participants having a history of cardiovascular disease were excluded. Volunteers participated into two ECG recording sessions. For each session, the participants were exposed for 26 min to sham or real GSM RF exposure. Exposure to RF EMF was performed by a commercial dual-band GSM mobile phone (Nokia 6650). The HRV was evaluated by both time domain and frequency domain analysis. Standard deviation of all R–R intervals (SDNN) and root-mean-square of successive differences (RMSSD) were measured in the time domain analysis of HRV. For frequency domain parameters, spectral analysis was performed by using fast-Fourier transform method. We analyzed the very low-frequency component (VLF), the low-frequency component (LF), and the high-frequency component (HF). The LF and HF powers were also converted into normalized units (LFnu, HFnu). Sympathovagal balance was expressed as the LF/HF ratio.
Results: Analysis of time domain HRV parameters showed that SDNN was significantly higher during the exposure session when compared to the control session.
Keywords: Electromagnetic field, Radiofrequency, HRV, GSM 900 MHz
Aluminum increases colorectal cancer cell metastasis through Smad2/3 signaling pathway (#687)
C. H. Jeong1, H. C. Kwon1, D. H. Kim1, S. G. Han1
1 Konkuk university, Food Science and Biotechnology of Animal Resources, Seoul, Republic of Korea
Aluminum (Al) is an abundant element found in environment and in foodstuffs. Human body, such as the digestive system is continuously exposed to Al. However, the effects of Al to the intestinal epithelium have rarely investigated. Particularly, the influence of Al in the metastasis of colorectal cancer cells has not been reported. Therefore, we investigated whether Al influences on the metastasis of human colorectal cell line, HT-29. Cells were treated with Al for acute (72 h, 1-4 mM) and chronic (30 weeks, 100-200 μM) exposure schemes. Results showed that cells treated with Al either acute or chronic exposures promoted migration and invasion of cells. The acute exposure of cells to Al decreased cell adhesion, whereas the chronic exposure increased cell adhesion. To further study the underlying mechanisms, expression of the genes and proteins associated with cancer metastasis were measured in cells. Acute exposure of cells to Al decreased both mRNA and protein levels of E-cadherin, while vimentin and snail were increased. Chronic exposure of cells to Al, however, did not alter expression of vimentin. Furthermore, nuclear translocation of Smad2/3 and gene expression of MMP-7 and 9 increased in cells treated with Al. These results indicate that activation of Smad2/3 is a key signaling pathway in cancer cell metastasis due to Al. It seems that the exposure of Al to the digestive track is a potential risk factor in the initiation of metastasis.
Keywords: Aluminum exposure, Metastasis, Digestive track, Colorectal cancer, Smad2/3
Developmental and toxicological joint effects of selected fungicide mixtures in zebrafish embryo (#741)
C. Venâncio1, 2, R. Vieira3, S. M. Monteiro3, 2, L. Félix4
1 Universidade de Trás-os-Montes e Alto Douro (UTAD), Departamento de Zootecnia, Escola de Ciências Agrárias e Veterinárias, Vila Real, Portugal
An increase in the use of pesticides to control pests that attack agricultural and wine crops (eg, mildew and powdery mildew) has been observed over the years due to climate changes. These compounds, once applied, are subsequently detected at residual levels in food and freshwater. In this sense, natural-based products have been studied as possible ecofriendly alternatives. However, there is the need to assess the impact that these compounds to aquatic ecosystems. As such, the objective of this study was to evaluate the toxicological effects of a mixture of commonly used synthetic fungicides or Mix 1 at environment relevant concentrations ( azoxystrobin (10 µg/mL), tebuconazole (5 µg/mL) and mancozeb (0.5 µg/mL)), a mixture of natural compounds or Mix 2 (Equisetum extract (6.25 µg/mL), Mimosa tenuiflora extract (8 µg/mL) and thymol (0.2 µg/mL)), with fungicidal properties, as well as to the mixture of both classes of fungicides or Mix 3 (all compound at previous concentrations descript) in the development of the zebrafish embryo.
Embryos in the blastula stage (~ 2h post-fertilization) were exposed for a period of 96h to the Mixes described before. During exposure, the mortality, spontaneous movements, heartbeat, hatching rate, malformations effects were evaluated. At the end of exposure, the activity of the enzymes superoxide dismutase, catalase, glutathione reductase, glutathione S-transferase, lactate dehydrogenase, acetylcholinesterase and carboxylesterase as well as the levels of the reduced and oxidized forms of glutathione, lipid peroxidation and reactive oxygen species were evaluated.
All the animals showed a normal development although exposure to the Mix 2 induced an increase in glutathione peroxidase activity. The exposure to the Mix 1 showed no differences relative to the control group. Similarly, no significant differences between the Mix 1 and Mix 2 were observed.
The results of this study show that exposure to the synthetic compounds at environmental relevant concentrations was not effective in inducing embryo-physiological alterations and oxidative-, neurotransmission- and energy-related changes on zebrafish embryo. On the other hand, and as expected, the results show that natural compounds potentially induce antioxidant activity. Overall, the results obtained deserve further studies.
Acknowledgement - Interact R&D project, operation number NORTE-01-0145-FEDER-000017, in its ISAC research line, co-financed by the ERDF through NORTE 2020 and CITAB projects by European Investment Funds by FEDER/COMPETE/POCI– Operacional Competitiveness and Internacionalization Programme, under Project POCI-01-0145-FEDER-006958 and National Funds by FCT - Portuguese Foundation for Science and Technology, under the project UID/AGR/04033/2019.
Keywords: Natural compounds, Ecotoxicology, Fungicides, Zebrafish, Embryonic development
Used of urtica dioica and capsicum frutescens for pathogens' management (#746)
S. Benani1, A. Menouni1, A. Bouchelta1
1 University of moulay ismail, Faculty of sciences, Meknes, Morocco
In Morocco, vicia faba is considered the most important legume for both food and feed. However, the ravages caused by pathogens remain hardly controlled. The objective of the present study is to evaluate the efficacy of Urtica dioica and Capsicum frutescens’ extracts in protecting broad beans against Bruchus rufimanus infestation. To this end, the research was conducted at the National Institute for Agricultural Research in Meknes (INRA) during the 2015-2016 crop year. The experimentation was conducted in pots with four replicates to evaluate the efficacy of aqueous extracts of urtica dioica and capsicum frutescens on five varieties of Vicia faba beans. The cultures were spread with the aqueous extract of these two plants from the beginning of flowering to maturity. The results of the study showed that the aqueous extract of c. frustescens at 100g/l dose, decreased the rate of infestation of beans by bruchids by 9.49% while for the other treatments, no decrease was recorded. In addition, an increase in yield was notified for the aqueous extract of u. dioica at the dose of 50g / l with a percentage of 1.86%.
BOUCHIKHI T. Z, KHELIL M. A, BENDAHOU M ET PUJADE-VILLAR J., 2010-2011. Lutte contre les trois bruches Acanthoscelides obtectus (Say, 1831), Bruchus rufimanus Boheman, 1833 et Callosobruchus maculatus (Fabricius, 1775) (Coleoptera : Chrysomelidae : Bruchinae) par les huiles essentielles extraites d’Origanum glandulosum (Lamiacées), Butll. Inst. Cat. Hist. Nat., 76: 177-186. BOUGHDAD A et LAUGE G, 1995 .Vicia faba seed infestation and losses due to bruchus rufimanus Boch.( Coleoptera : Bruchidae) in Morocco.Fabis news letter 36/37 . BOUGHDAD A et LAUGE G. Cycle biologique de Bruchus Rufimanus boch.(COLEOPTERA :BRUCHIDAE) sur vicia Faba Var.Minor L.(LEGUMINEUSE ) au Maroc, ANNP -4éme Conférence Internationale Sur Les Ravageurs En Agricultures ; MONTEPELLIER 6-7-8 JANVIER 1997. BOUGHDAD A et LAUGE G, 1997. Infestation des graines de Vicia Faba L dues à Bruchus Rufimanus Boch (Coleoptera, Bruchidae) au Maroc .Al AWAMIA 97 . DELAHAYE Julien ,2015.utilisation de l’ortie –urtica dioica Thèsepour le diplôme d’état de docteur en pharmacie .Université de ROUEN, UFR de médecine et pharmacie. FAO stat 2006. Pages web : www.faostat.fao.org Khadija BOURARACH, Mohamed SEKKAT, Driss LAMNAOUER 1994. Activité insecticides de quelques plantes médicinales du Maroc. Actes Inst .Agron . Vet (Maroc) 1994, Vol. 14(3):31-36. HAMAMI A S, 2014. Bio écologie et diapause reproductrice de la bruche de la fève ;Bruchus rufimanus ; dans deux parcelles de fève et fèverole dans la région Haizer (Bouira), mémoire de magister, Université Mouloud Mammeri de Tizi-Ouzou, Algérie. HOFFEMAN A., LABEYRIE V. et BALACHOWSKY, 1962.Famille des Bruchidae. Entomologie app. à l’agriculture 434-494, (1), BALACHOWSKY Ed., Masson publ., Paris,564p. Ministère de l’agriculture et de la pêche maritime ,Agriculture en chiffre 2015. Page web: www.agriculture.gov.ma. MEDJDOUB B., KHELIL M.A et HUIGNARD J. Bio écologie de la bruche de la fève (B.rufimanus), relations spatio-temporelles entre la bruche et sa plante hôte (Vicia faba) dans deux parcelles situées à deux altitudes différentes dans la région de Kabylie (ALGERIE). AFPP – Neuvième Conférence Internationale Sur Les Ravageurs En Agriculture Montpellier – 26 ET 27 OCTOBRE 2011. Mukondowa NSAMBU,Bahananga MUHIGWA, Kituta RUBABURA, Mashimango BAGALWA, and Sanvura BASHWIRA ,2014 Evaluation in vitro de l’activivté insecticides des alcaloides, saponines,terpenoides et steroides extraits de Capscicum frutescens L.( SOLANACEAE) contre Antestiopsis orbitalis ghesquierei ,insectes ravageurs des caféiers. International journal of innovation and applied studies,1231-1243p. Rémy MUKENDI, Patrick TSHLENGE, Constant KABWE et M.B. Théodore MUNYULI. Efficacité des plantes médicinales dans la lutte contre ootheca mutabilis sahlb. (chrysomelidae) en champ de niébé (vigna unguiculata Walp.) en RD du Congo. Lebanese Science Journal, Vol. 15, No. 1, 2014.
Keywords: Integreted control, vicia faba, urtica dioica, bruchus rufimanus, Capsicum frutescens
Copper exposure reduces the lifespan in Caenorhabditis elegans (#781)
Y. Zhang1, C. Zhao1, H. Zhang1, Y. Pu1, L. Yin1
1 Southeast University, School of Public Health, Key Laboratory of Environmental Medicine Engineering of Ministry of Education, Nanjing, China
Environmental pollution from heavy metals has proven to be a major global environmental problem. As one of the most widely heavy metals, copper exposure does cause harm to human health. This study conducted Caenorhabditis elegans (C. elegans) model to explore the effect of copper on lifespan. The synchronized L1 stage C. elegans worms were exposed to different concentrations of copper (0, 0.01, 0.1, 1 and 10 mg/L) for 48 h，respectively. To evaluate copper effect on lifespan in C. elegans, we performed lifespan assay, body length assay and brood size assays in worms after copper treated. AM141 worms were utilized for PolyQ aggregation assay to evaluate aging, and the number of poly(Q) aggregates was counted with the epifluorescence microscope. Aging process response genes including daf-2, age-1 and daf-16 were measured by quantitative RT-PCR (qRT-PCR). Fitness-related traits including developmental rate, brood size and lifespan were important in life history of C. elegans. Brood size was used to measure capacity of reproduction, our results showed that brood size under different copper concentrations (0, 0.01, 0.1, 1 and 10 mg/L) were 234.11 ± 16.00, 198.77 ± 7.51, 171.00 ± 35.34, 146.11 ± 24.36 and 128.63 ± 32.20 (P<0.05), respectively. What’s more, body length of C. elegans were 346.53 ± 29.21, 322.86 ± 38.20, 291.49 ± 32.35, 287.08 ± 36.50, 279.16 ± 35.14 µm (P<0.05), respectively. The result of lifespan assay showed that the longest longevity of each group were 23 days, 21 days, 20 days, 19 days, and 18 days (P<0.05), respectively. Besides, the average longevity was 21.00 ± 1.76, 18.70 ± 1.42, 18.30 ± 1.95, 17.40 ± 1.71 and 16.80 ± 1.48 days (P<0.05), respectively. Then, the number of poly(Q)40::YFP aggregates revealed that copper promoted PolyQ-YFP accumulation in the muscle cells of AM141 worms. The result of qRT-PCR showed copper could promote the expression of daf-2 and age-1, and inhibit the expression of daf-16. In conclusion, our findings suggest that copper could inhibit growth and development of worms, and suppress survival and lifespan of C. elegans.
Keywords: Copper, lifespan, Caenorhabditis elegans
Mercury-Induced Cellular Damage is Associated with Enhanced Mitochondrial DNA Damage, Oxidative Stress and Mitochondrial Dysfunction (#789)
S. B. S. Rao1, S. Das1, M. B. Joshi2
1 Manipal Academy of Higher Education, Manipal School of Life Sciences, Department of Radiation Biology & Toxicology, Manipal, India
Background: Alterations in mitochondrial function has been associated with several pathological conditions induced by exposure to environmental xenobiotics. Here we investigate mercury induced cellular toxicity and its associated changes in mitochondrial structure and function.
Methodology: Human dermal fibroblast cells were treated with mercuric chloride (HgCl2), changes in mitochondrial structure and function were assessed. DNA damage assessment was done by long-amplicon PCR and alterations in mitochondrial structure / mass by Mitotracker red / Nonyl-acridine orange dye staining. Functional changes were analyzed by detection of cytosolic/ mitochondrial ROS, mitochondrial membrane potential, activities of respiratory complexes, aconitase activity, ATP and mitochondrial GSH levels. Induction of mitochondrial biogenesis was measured by changes in expression of PGC1-α and Nrf1.
Results & Conclusion: HgCl2-treated human dermal fibroblast cells showed higher lesion frequency in mitochondrial DNA versus nuclear DNA indicating higher sensitivity of mitochondrial genome. Initial increase in mitochondrial ROS was accompanied by a gradual increase in cytosolic ROS levels and this was paralleled by loss of mitochondrial membrane potential along with decline in mitochondrial respiratory complex activity, depletion in MMP, aconitase activity and respiratory enzyme complexes along with lowering of ATP levels. Decrease in mitochondrial function was associated with enhanced mitochondrial fission. Elevated expression of Nrf1 and PGC-1α indicated the involvement of mitochondrial biogenesis during mercury intoxication that may contribute to maintenance of mitochondrial mass. We infer that mitochondrial biogenesis act in a harmonious way to maintain cellular integrity and prevent cell death after mercury exposure in fibroblast cells.
Acknowledgement: We thank Director, Manipal School of Life Sciences and MAHE for providing the research facilities as well as ICMR, Government of India for the financial support (Grant Number: 53/8/2013-BMS).
Keywords: Mercury, Mitochondrial DNA Damage, Oxidative Stress
Lithium, selenium, cobalt and other elements in scalp hair from a group of young Spanish adults (#836)
A. Peña-Fernández1, M. González-Muñoz2, S. Angulo3, M. C. Lobo-Bedmar4
1 De Montfort University, Leicester School of Allied Health Sciences, Leicester, United Kingdom
Human scalp hair has been suggested as an appropriate tissue to determine body burden of various metals although this raises controversy in the scientific community due to different factors influencing their content in this matrix. Lithium (Li), selenium (Se) and cobalt (Co) have been linked to mood and brain function, recent reviews have reported an inverse association for these metals in human scalp hair with suicide rates. As a pilot study, we have determined the levels of these metals, including arsenic (As), mercury (Hg) and lead (Pb), in scalp hair from 37 young adults (20 to 24 years-old; 28 female and 9 male) from different towns in the Community of Madrid (Spain). After appropriate pre-treatment of samples following previous methodologies, metals were monitored by ICP-MS. The limits of detection (ng/g) were: Li (1.98), Se (5.14), Co (0.75), As (2.0), Hg (1.0), Pb (1.0). The evaluated concentrations (median and percentiles provided in ng/g) were as follows: Li 5.44 (4.45, 7.47), Se 309.04 (255.47, 331.79), Co 7.53 (2.78-17.04). Concentrations for As (0.014), Hg (1.72) and Pb (0.64) were presented as arithmetic means in µg/g. Although our results are not reliable due to the differences in the number of participants by sex, levels of Se and Hg were significantly higher in males, which is in accordance with similar studies and could be related to the higher and significant intake of fish and shellfish previously reported in male participants in this group of population by our team. In general, the presence of these metals in the Spanish group’s hair were within those highlighted in different studies performed in healthy Caucasian young adult populations. However, levels of Li, Co and Se were much lower than reference ranges reported for a Japanese population, which although from a different ethnic background, might suggest that the intake of these essential elements could be compromised in the Spanish group. Our results, although preliminary, might indicate that the intake of Li, Co and Se should be carefully monitored in Spanish young adults, as these have been recognised as essential metals for optimal brain function.
Keywords: Lithium, selenium, cobalt, human hair, monitoring
Effect of the bread-making process on mycotoxin levels (#856)
M. B. Martín Moya1, G. Meca1, E. Ferrer1, H. Berrada1, M. Fernández Franzón1
1 University of Valencia, Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, University of Valencia , Valencia, Spain
Wheat is a cereal very susceptible to contamination by mycotoxigenic fungi. In this research, it is analyzed the stability of Aflatoxins B1, B2, G1, G1, zearalenone and enniatins A, A1, B and B1 during baking process of sliced bread. Therefore, sliced bread is made with previously contaminated flour with strains of fungi producers of these mycotoxins. Levels of these mycotoxins produced on sliced bread are analyzed with liquid chromatography coupled with a triple quadrupole mass spectrometry (LC-QqQ-MS), previously the extraction of nine mycotoxins was optimized with methanol. The highest reduction in mycotoxin levels was found in the first fermentation (first proof), while the lowest reduction was observed in the baking stage. The effect of baking in sliced bread was determined by four different temperatures and times: 180ºC (100 minutes), 200ºC (60 minutes), 220ºC (40 minutes) and 220ºC (15 minutes). The results showed that maximum reduction occurs during the fourth condition and further reduced mycotoxin level was obtained with zearalenone, while aflatoxins B2 and G1 present greater reduction than the B1 and G2 and the lower decrease in the mycotoxin level in sliced bread was observed in enniatins. The mycotoxin reduction during sliced bread making is minimum, so it is important that the presence of fungi and mycotoxins are controlled in raw material.
Acknowledge: This research was supported by Conselleria de Educación, Investigación, Cultura y Deporte (AICO/2018/199) and University of Valencia (Programa propio- Acciones Espec iales) UV-18-INV_AE18
Cano-Sancho, G., Sanchis, V., Ramos, A. J., & Marín, S. (2013). Effect of food processing on exposure assessment studies with mycotoxins. Food Additives & Contaminants: Part A, 30(5), 867-875.
Keywords: sliced bread, wheat, aflatoxin, zearalenone, enniatin and baking.
Association between blood lead, high sensitivity C-reactive protein and metabolic syndrome (#875)
W. - J. Choi1
1 Gachon University College of Medicine, Gil Medical Center, Dept of Occupational and Environmental Medicine, Incheon, Republic of Korea
Purpose: Environmental exposure to toxic heavy metal such as lead and systemic inflammation have been suggested as risk factors for cardiovascular disease. However, little is known about the association between environmental lead exposure, elevated inflammation marker and metabolic syndrome. The aim of this study was to investigate the association between blood lead, high sensitivity C-reactive protein (hsCRP) and metabolic syndrome.
1. Lead Exposure and Cardiovascular Disease: A Systematic Review. Navas-Acien A, Guallar E, Silbergeld EK, Rothenberg SJ. Environ Health Perspect. 2007;115(3):472-82.
Keywords: lead, hsCRP, metabolic syndrome
Proposal of next-generation system in big data era based on chemical data science --- Integrated toxicity research support system adapted to the new era --- (#891)
1 Insilico Data, Ltd.,, Narasino / Chiba, Japan
Introduction: Changes in toxicity research approach and computer environments.
Purpose: Current system features and limitations and problems.
Conclusion: In the latest toxicity evaluation research, an approach considering the toxicity development mechanism such as AOP is important. As a result, in addition to the conventional toxicity prediction software, coordination with a system having a toxicity mechanism analysis function is required.
Keywords: AOP, toxicity prediction, AI, bigdata, data science
First detection of Acanthamoeba spp. and Balamuthia mandrillaris in different water ecosystems in Leicestershire (UK) (#896)
U. Anjum1, A. Magnet2, M. C. Lobo-Bedmar3, A. Peña-Fernández1
1 De Montfort University, Leicester School of Allied Health Sciences, Leicester, United Kingdom
Acanthamoeba spp., Naegleria fowleri and Balamuthia mandrillaris can produce severe brain infections in immunocompetent and immunocompromised individuals. These free‐living amoebae (FLA) have a worldwide distribution. Despite the rarity of brain infections by these organisms in the United Kingdom (UK), generally linked to travelling exposures, the incidence of Acanthamoeba keratitis (AK) is significantly higher in the UK than in other European countries or the United States. However, to date, isolation of these pathogens in the UK is limited to Acanthamoeba spp., mainly in drinking water supplies. Three sets of 30 water samples were collected, according to the US Environmental Protection Agency (EPA) method 1623, from different open water systems in Leicestershire (UK) per season between March and November 2018 using a portable water pump connected to a foam filter module. Water samples were collected in the same locations each season from: 15 ponds (in public parks)/water reservoirs; 7 from the River Soar; 2 from a canalised section of the River Soar, Grand Union canal; 1 from the River Biam and a marina near the River Soar; 4 from lakes highly frequented for fishing or leisure (John Merricks', Kings Lear’s; Bennion Pools Fishing and Abbey park). Water samples were concentrated using the IDEXX® Filta Max system according to manufacturer's instructions and EPA method 1623; DNA was extracted using a FastDNA® Kit. Real-time PCR was used to detect these FLA according to previous methodologies. To our knowledge, these FLA were detected for the first time in 12/90 (13.3%) of the monitored samples in Leicestershire. N. fowleri was not detected in any sample; whereas Acanthamoeba spp. was detected in 11 water samples (12.2%) in all three seasons and environments monitored except the marina, which may suggest a wide environmental distribution of this pathogen in England. B. mandrillaris was found in John Merricks’ lake (1.1%) in Spring 2018, which is the first report of the presence of this pathogen in the UK. Our results highlight a potential risk for human health that should be carefully considered due to the high number of users of these water environments, particularly of the River Soar. Awareness of the presence of these pathogens and specific control measures should be provided to users of these open water systems.
Keywords: Acanthamoeba spp., Balamuthia mandrillaris, open water systems, Leicestershire, risks.
Exposure to TBBPA impedes vascular growth and disturbs metabolic pathways during early development in zebrafish (#932)
Y. Wei1, X. Zhong1, J. Kang1, J. Qiu1, W. Ke1
1 Sun Yat-sen University, Department of Toxicology, School of Public Health, Guangzhou, China
Tetrabromobisphenol A (TBBPA), a widely-used brominated flame retardant, has been applied in a good number of industrial and commercial products. Of TBBPA’s adverse health effects, impact on development is the primary concern. Epidemiological and animal studies have revealed an association between exposure to TBBPA and developmental problems. However, the effects and the underlying mechanisms of developmental defects resulting from TBBPA exposure are largely unknown. The vascular system which supplies oxygen and nutrients, maintains homeostasis and protects from toxic agents, is crucial for tissue development. Disruption of vascular development has been directly correlated with miscarriages, birth defects, maternal placental complications, and neurodevelopmental problems. In this study, we investigated the impacts of TBBPA on early vascular development using a zebrafish model. Zebrafish embryos were continuously exposed to waterborne TBBPA ranging from 0.5 to 300 μg/L starting from 2 hours post fertilization (hpf). Fluorescent images of vasculatures in kdrl:eGFP zebrafish were acquired using confocal microscope. Quantitative RT-PCR was applied to assess the mRNA levels. TBBPA-exposed zebrafish larvae did not exhibit significant difference in mortality, hatching rate, malformation and body length at 72 hpf. TBBPA exposure at 100 and 300 μg/L resulted in a delayed growth of common cardinal vein (CCV). The expression of genes related to angiogenesis and differentiation of endothelial cell, including Notch2, Hey2, Cdh5, Fli1a, Tal1, Rag1 and Npas4l, was suppressed in TBBPA-treated larvae. In addition, TBBPA exposure led to a reduction in the expression of Hmgcra and PPARγ, critical genes in lipolysis pathway. Strikingly, the enzymes responsible for glucose metabolism, including Hk1, Gk, Pk and Pepckc, dramatically decreased in zebrafish larvae exposed to TBBPA. The results reveal that developing vasculature in zebrafish is a sensitive target for TBBPA exposure. The findings indicate that TBBPA inhibits vascular development, and disturbs lipid and glucose metabolic pathway, which provide new insight into the mode of action of TBBPA upon developmental exposure.
Keywords: Brominated Flame Retardants, TBBPA, Developmental toxicity, Vascular growth, Mode of action
Effect of DEHP and DBP on steroidogenesis of adrenal gland in male Wistar rats (#934)
S. Ahmad1, S. Raisuddin1
1 Jamia Hamdard, Department of Medical Elementology & Toxicology, New Delhi, India
Adrenal gland is a less focused endocrine organ for the endocrine disrupting effect of endocrine disrupting chemicals (EDCs) leading to the neglected study of steroidogenesis as the target of EDCs. The effects of two extensively used phthalate esters viz. di-ethyl hexyl phthalate (DEHP) and di-butyl phthalate (DBP) on adrenal gland were observed in Wistar rats in the present study to check the susceptibility of adrenal gland and steroidogenesis in it against the exposure of these extensively used plasticizers which are well known EDCs. Wistar rats were divided into seven groups (n = 6) and received the treatment for fourteen days. Group I was control and received only corn oil which is used as vehicle. Group II, III and IV received daily dose of DEHP of 250 mg/kg-BW, 750 mg/kg-BW and 1500 mg/kg-BW respectively while group V, VI and VII received daily dose of DBP of 100 mg/kg-BW, 500 mg/kg-BW and 1000 mg/kg-BW respectively. The comparative microscopic study of histological slides of endocrine glands i.e. pituitary, pineal, thyroid, parathyroid, adrenal gland and testes revealed the susceptibility of adrenal gland towards the DEHP and DBP. Steroidogenesis was analyzed by molecular docking of DEHP and DBP with the enzyme proteins of involved in steroidogenesis using Maestro Schrodinger 9.4 software showing the potential of DEHP and DBP to inhibit these proteins comparable to the known inhibitors of these enzymes. The mRNA expression study of the enzymes of involved in the steroidogenesis i.e. StAR, 3β-HSD, CYP21A1, CYP1B1 and CYP11B2 on exposure to DEHP and DBP by real time PCR has also assessed the sensitivity of the steroidogenesis towards DEHP and DBP. The mRNA expressions of StAR and CYP1B1 were up-regulated in dose dependant manner on exposure to DEHP and DBP. The expression of CYP21A1 was slightly up-regulated on DBP exposure but in case of DEHP it was comparatively more up-regulated. It was vice-versa in case of 3β-HSD that mRNA expression was slightly up-regulated on DEHP exposure and comparatively more up-regulated on exposure to DBP. CYP11B2 was down regulated on exposure to both DEHP and DBP. The present study gives a unique approach to elucidate the novel mechanism of endocrine disruption by EDCs through the analysis of the sensitivity of adrenal steroidogenesis on exposure to DEHP and DBP.
Keywords: Glucocorticoid biosynthesis, endocrine glands, phthalate esters, molecular docking
Heavy Metal Evaluation in Rescue Dogs (#956)
M. M. Melo1, S. E. M. T. Branco1, A. G. Costa1, M. R. Lempeck1
1 Universidade Federal de Minas Gerais, Clínica e Cirurgia Veterinárias, Belo Horizonte, Brazil
Purpose: Increasing environmental pollution caused by heavy metals, which are released by industrial and mine activities, is an important worldwide problem (Allan, 1997). Concentrations of lead, cadmium, arsenic, and mercury are strongly influenced by this type of discharge and are most frequent in wastewater. In Brazil, the Brumadinho dam disruption, on January 25, 2019, resulted in one of the largest mine tailings disasters. The dam rupture released about 12 million cubic meters of tailings. Initially, three dogs were used to retrieve victims. These animals remained for two months at the accident site, having direct contact with the mud contaminated by toxic discharge. In this context, the objective was to analyze metals in their blood circulation.
ALLAN, R. Introduction: Mining and metals in the environmental. J. Geochem. Exploration, v. 58, p.95-100, 1997.
BRADBURY, M. W. B.; DEANE, R. Permeability of the blood-brain barrier to lead. Neurotoxicology v. 14, p. 131-136, 1993.
EXLEY, C. The toxicity of aluminium in humans. Morphologie , v.100, p. 51-55, 2016.
Keywords: toxic metals, poisoning, dog exposure to heavy metals, mine tailings
Evaluation of the effect of perfluorooctanesulfonate (PFOS) on DNA damage and highly reactive oxygen species generation in human peripheral blood mononuclear cells (in vitro study) (#967)
K. J. Mokra1, P. Sicińska1, M. Jarosiewicz1, B. Bukowska1, J. Michałowicz1
1 University of Lodz, Department of Environmental Pollution Biophysics, Faculty of Biology and Environmental Protection, Lodz, Poland
This work was financed by NCN institution (DEC-2018/02/X/NZ7/00188)
Introduction: Perfluorinated compounds (PFCs) are commonly, widely produced substances used in industry since 1950. Due to their chemical properties and extensive using in consumer products such as textiles, food or coating for cookware, they are persist in the environment and have been detected in wildlife and humans. One of the most widely used PFCs is perfluorooctanesulfonate (PFOS), which was included in the group of organic pollutants (POPs) by Stockholm Convention. Up to now, the mechanism of PFOS action on human peripheral blood cells (PBMCs) has been poorly investigated. Taking the above into consideration we have assessed the potential of PFOS to generate highly reactive oxygen species (ROS, mainly hydroxyl radical) and to induce DNA damage in PBMCs.
Material and methods: PBMCs were separated from buffy coats by a density gradient method using Histopaque. The final concentrations of the compounds were in the range from 0,02 to 100 µM. In order to detect DNA single strand-breaks (SSBs) comet assay was employed. Highly reactive oxygen species were analyzed by flow cytometry using 3’-(p-hydroxyphenyl)-fluorescein (HPF).
Results and conclusion: Flow cytometry analysis (3’-(p-hydroxyphenyl)-fluorescein staining) showed that PFOS increased the intracellular highly ROS (20-100 µM) and the increase was concentration-dependent. However, observed changes were not statistically significant. Interestingly, comet assay analysis showed that PFOS (0,5-100 µM) induced DNA damage and no statistically significant alterations in this parameter were found only in PBMCs treated with the lowest tested concentration (0,02 µM).
Collectively, obtained results suggest that DNA damage and highly ROS generation not occurs in PBMCs of general population. It may be also concluded that PFOS induced DNA damage in tested cells in the concentrations which may enter the human body as a result of occupational exposure.
Keywords: PFOS, PBMCs, reactive oxygen species, DNA damage
Environmental factors related with chronic kidney disease of unknown origin (CKDu) of Centro America: relationship of 28 element profile concentrations in drinking water with the prevalence in communities of Nicaragua. (#976)
A. J. Arias Ariel1, 2, R. A. Ruiz1, L. Blanco2, M. A. Sogorb1, E. Torres2, E. Vilanova1, E. Roque2
1 University Miguel Hernandez, Institute of Bioengineering, Elche, Spain
Mesoamerican nephropathy (MeN), also known as chronic kidney disease of unknown etiology (CKDu) is a public health problem in rural communities causing thousands of deaths mostly (but not exclusively) young male of the Centro America Pacific coast with extensive volcanic activity.
The relationship of ionic profile in drinking water with the prevalence of CKDu was evaluated in an ecologic epidemiological study studying the concentration of 28 elements by ICP-MS in rural communities in Nicaragua affected by high or medium prevalence of CKDu. Moreover a preliminary assessment of the biomonitoring of elements in hair has been also performed.
The mean values of some elements were higher that WHO and EU guideline. Some samples showed high K, Ca, Ni, As and Se. In the communities with high CKDu prevalence, significantly higher concentrations were found with Mg, Ca, Fe, Ba, Sr. On the contrary, Cu, Cr, As, Se, Cd were higher in areas of medium prevalence. The cases of high Mg and Ba over the guidelines were found only in areas of high prevalence. The special case or As: although median is below the guideline it concentration are considered higher than usual in most drinking waters, however is higher in the group of intermediate prevalence
By other side, the binary ratios of some elements as Ni/Na, showed predictive value. Although we cannot still stablish a cause-effect relationship, there are significant statistic correlations among elements. Two polarized groups are observed: one include Mg, Ca, Sr, Co and other AS, Ni, CD with positive correlations intragroup end negative intergroup.
Environmental factors (profiles of elements in drinking water) influence the prevalence of CKDu, which deserves to be evaluated in other affected regions.
Project partially funded by “Programa de Cooperación al Desarrollo de la UMH“, supported by “Generalitat Valenciana (Consellería de Transparencia, Responsabilidad Social, Participación y Cooperación)”.
Keywords: kidney, elements, heavy metals, water
Genotoxicological studies of novel food sources: alkaline comet assay (#61)
S. I. Shestakova1
1 Federal State Budgetary Scientific Institution “Federal Research Centre of Nutrition, Biotechnology and Food Safety”, Moscow, Russian Federation
The Russian system of complex biomedical research of genetically modified organisms (GMO) of plant origin includes general toxicological studies as well as specific types of toxicity studies, such as genotoxicological, reprotoxicological, immunotoxicological, allergological. The feature of this approach is the use of various experimental models to identify possible unintended effects of genetic modification.
According to a modern concept of mutagenesis the mechanism of chromosomal aberrations associates with the molecular disorders induction which lead to DNA helix break. Therefore the use of a two-level approach, that includes the assessment of DNA structure integrity by the Alkaline Comet Assay (OECD 489) and Mammalian Bone Marrow Chromosomal Aberration Test (OECD 475), is of high diagnostic value when studying the genotoxic GMO effect.
Long-term experience of DNA comet research in framework of GMO safety assessment allowed to form a database (historical control) of DNA damage levels in the liver, kidney, bone marrow and rectum of healthy adult C57Bl/6 male mice (age 70-90 days). In total more than 100 animals (of control groups), 400 organs and 40 000 cells were examined and analyzed. Based on the data, the ranges of physiological fluctuations of DNA fragmentation levels for bone marrow cells were determined as 7.49±0.38% (from 4.61 to 9.82), for liver as 7.22±0.31% (from 5.62 to 9.45), for kidney as 7.65±0.39% (from 5.15 to 9.54) and for rectum cells as 7.92±0.40% (from 4.63 to 10.00) .
The possibility of historical control use provides more correct interpretation of data, that are obtained in genotoxicological studies of new GMO within the State registration procedure; such approach is consistent with current trends in toxicological studies.
This work was supported by the Ministry of Science and Higher Education of the Russian Federation (research project No. 0529-2019-0056).
Keywords: genotoxicity, GMO, Comet Assay
Safety assessment of genetically modified soybean: potential genotoxicity (#62)
N. V. Tyshko1, S. Shestakova1, E. Sadykova1, N. Nikitin1
1 Federal State Budgetary Scientific Institution “Federal Research Centre of Nutrition, Biotechnology and Food Safety”, Moscow, Russian Federation
The potential genotoxicity evaluation of genetically modified organisms includes the Bone Marrow Chromosomal Aberration Test (OECD 475) and the Alkaline Comet Assay (OECD 489) within in vivo experiment on C57Bl/6 mice. The two-level approach based on performing of DNA integrity assessment along with changes in chromosome structure detectable by microscopic examination of the metaphase stage of cell division, allows to register both DNA helix break and chromosomal aberrations in genomes.
The aim of this study was to investigate the possible genotoxic risk of the genetically modified (GM) fat-free soybean flakes containing GM line MON87701×MON89788. The 40 days experiment was performed on male mice of C57Bl/6 line with the initial bodyweight of 16-18 g. The animals were divided equally and randomly into two groups receiving soybean flakes from traditional (control group) and GM (exposure group) soybean flakes in the diet throughout the entire study.
The DNA structure integrity of the exposure group rats did not have significant differences from the control animals and averaged 7.65±0.19% and 7.73±0.20% in the bone marrow, 5.87±0.55% and 5.47±0.17% in the kidneys, 5.68 ± 0.21% and 5.62±0.30% in the liver, 5.61±0.20% and 5.91±0.25% in the rectum, respectively. The percentage of cells with structural chromosomal aberrations in the bone marrow of the exposure group did not have significant differences from control animals and averaged 2.0±0.4% and 2.1±0.4% of damaged metaphases, respectively.
Thus, genotoxicological research did not reveal any genotoxic effect of soybean flakes containing GM line MON87701×MON89788GM compared to the flakes produced from traditional soybean. The levels of DNA structure integrity and of chromosomal aberrations were similar in the control and exposure groups.
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, genotoxicity, Comet Assay, Chromosomal Aberration Test
Influence of genotoxic asarone isomers on DNA strand break repair mechanisms (#81)
L. Hermes1, K. Lausen1, S. Haupenthal1, M. Esselen1
1 University of Muenster, Institute of Food Chemistry, Muenster, North Rhine-Westphalia, Germany
α- und β-asarone are phenylpropanoids occurring in essential oils and rhizome of the plant species Acorus Calamus, which are used as food supplements, for tea or as flavoring compounds for sweets or alcoholic beverages. Both isomers are classified to be carcinogenic in rodents and are known for their genotoxic properties [1-3]. The mechanism of action underlying these genotoxic effects is not elucidated so far, which prevents an adequate risk assessment. Considering the toxic potential of the asarone isomers cytochrome P 450 monooxygenase-mediated oxidation seems to be a crucial step because the generated epoxide induces DNA adduct formation. This metabolic activation is postulated to be responsible for genotoxic and mutagenic effects of asarone isomers in vitro .
Data on persistence of DNA damage and the cellular response to these genotoxic effects are limited. Absent mutagenic effects in mammalian cells as well as a decrease of DNA adduct levels after 6 h suggest that DNA damage is recognized and signaling cascades are activated, which especially initiate DNA repair. Preliminary tests focusing on DNA repair showed that after 24 h DNA strand breaks were completely repaired. In a next step different key elements of DNA repair mechanisms were investigated, with a deeper focus on double strand break repair. An increase of phosphorylated histone H2AX, which is considered as marker for double strand breaks, was determined after short-time incubation up to 2 h. Furthermore, there are first indications that single strand break repair mechanisms are involved as well, demonstrated by a rapid repair of oxidative DNA damage in the Comet assay and via immunofluorescence of 8-Oxo-dG. To sum up, it can be said that activation of DNA repair mechanisms seems to play a crucial role in the cellular response to genotoxic asarone isomers or their respective metabolites. The potential contribution of base excision repair is currently under investigation.
 R. W. Wiseman, E. C. Miller, J. A. Miller and A. Liem, Cancer Res., 1987, 47(9), 2275.
Keywords: DNA damage response, DNA repair, Genotoxicity
Comparative study of Salmonella typhimurium tester strains TA1537, TA97 and TA97a in mutagenicity evaluation of tobacco products (#87)
Y. Sakai1, T. Ishii1, Y. Takahashi1, T. Hashizume1, T. Fukushima1
1 Japan Tobacco Inc., Scientific Product Assessment Center, R&D Group, Yokohama, Japan
The Ames test is widely employed to assess mutagenicity using bacteria. The Organisation for Economic Cooperation and Development (OECD) test guideline (TG) 471 states that at least five tester strains should be used for the Ames test, and Salmonella typhimurium TA1537, TA97 and TA97a can be used interchangeably. The Ames test has been conducted to examine the mutagenicity of tobacco products and to compare relative mutagenicity of cigarette smoke. It is well known that cigarette smoke shows clear mutagenic activity in some tester strains including TA1537, however, few studies have been reported using strains TA97 and TA97a. Thus, here, we compared strains TA97, TA97a and TA1537 in terms of their sensitivity to detect a mutagenic response and the discriminatory power to distinguish between different types of cigarette.
We selected four types of test cigarette (i.e., 3R4F, 1R6F, 100% single grade burley, and 100% single grade flue-cured) for use in this study. The cigarette smoke condensate (CSC) derived according to International Organization for Standardization smoking conditions was subjected to the Ames test. The assay was performed according to OECD TG471 in the presence and absence of metabolic activation by S9.
Regarding sensitivity, we compared the minimum dose where a significant increase in the revertant colonies was observed in a dose-dependent manner for each strain. In the presence of S9, all three strains showed clear, positive responses for all the CSCs. Especially a significant increase in revertant colonies was observed for TA97a at a lower dose than for the other two strains. In the absence of S9, TA97 showed a positive response to the CSCs, whereas the other two strains did not due to growth inhibition. Regarding the discriminatory power, in the presence of S9, the three strains provided consistent results in terms of the mutagenicity rank-order (i.e., burley > 3R4F ≈ 1R6F > flue-cured) and the ability to discriminate statistically dose responses found in the different CSCs.
We suggest that S. typhimurium strains TA97, TA97a and TA1537 have different sensitivities in detection of a positive mutagenic response, whereas the three strains are comparable in their ability to discriminate between different types of cigarette smoke. Further investigation is needed to understand the mechanism underlying the difference in sensitivity between the three strains in mutagenicity assessment of cigarettes.
Keywords: Ames test, mutagenicity, cigarette, Salmonella typhimurium, TA97
In situ detection of DNA double strand breaks by immunofluorescent γ-H2AX staining in mice exposed to multiwalled carbon nanotubes (#125)
K. Aimonen1, K. Huumonen1, 2, S. Savukoski1, H. Lindberg1, A. Schoonenberg3, K. Välimäki3, S. Libertini4, H. Wolff1, J. Catalán1, 5, H. Norppa1
1 Finnish Institute of Occupational Health, Helsinki, Finland
Phosphorylation of histone H2AX (γ-H2AX) at serine 139 is an acknowledged biomarker of DNA double strand breaks in cultured cells and tissue biopsies. However, γ-H2AX in situ staining has rarely been used to detect genotoxic effects of nanomaterials.
The application of immunofluorescent (IF) γ-H2AX staining on tissue samples has many advantages. The same paraffin embedded tissues can be used for both histopathology and γ-H2AX analysis, which allows implementing the γ-H2AX assay also on previously conducted in vivo studies. The detection of γ‑H2AX in situ enables the localization of the genotoxic effect in tissue-specific structures and even cell types. Analysis by microscopy can easily discriminate between cells with different levels of DNA damage and apoptotic cells.
The purpose of this study was to further evaluate the use of IF γ-H2AX staining for the genotoxicity assessment of nanomaterials in vivo. Groups of C57BL-6 female mice were exposed to three doses (10, 40 and 80 µg/mouse) of multiwalled carbon nanotubes (MWCNTs; Mitsui-7) by single pharyngeal aspiration. Lung samples for genotoxicity (Comet assay) and histopathological evaluation were collected 24 h and 28 d post-exposure and the results were compared to a negative control group. The IF γ-H2AX staining was performed on formalin-fixed paraffin-embedded lung samples after deparaffination and antigen retrieval by boiling. An autostainer was used for primary (rabbit monoclonal anti-gamma H2AX phospho-Ser139) and secondary (goat anti-rabbit IgG) antibody incubations and for tyramide amplification of the fluorescent signal (Alexa Fluor™ 488 Tyramide SuperBoost™ Kit; ThermoFisher Scientific) according to manufacturer’s instructions. Samples were counterstained with 4’,6-diamidino-2-phenylindole and digitized with 20x fluorescent scanning. Expression of γ-H2AX foci was analyzed using marker counter module of a digital microscope application. For each sample, all nuclei in four randomly selected annotations (200 µm x 200 µm) were classified as negative, weak positive (≤ 3 foci), positive (> 3 foci), or apoptotic (pan-stained nucleus).
The results showed a dose-dependent induction of γ-H2AX positivity 24 h post-exposure. 28 days later, the effect of the MWCNT exposure was lower, although the percentage of γ-H2AX positive nuclei remained elevated in the lungs of the exposed mice. These results were in line with comet assay data (% of DNA in tail) from the same animals. Hence, it has been shown that IF γ-H2AX staining can be used to complement the comet assay for monitoring DNA damage induced by nanomaterials in vivo.
[Supported by the Finnish Work Environment Fund]
Keywords: in vivo genotoxicity, γ-H2AX, MWCNT
Value of Cooperation of Industry & Regulatory Agencies Example: Cancerogenicity (#126)
G. H. Bode1, S. Wagner2
1 University of Goettingen, Germany , Institute for Pharmacology & Toxicology, Goettingen, Germany
Carcinogenicity studies are the most time-consuming, costly and resource intensive non-clinical investigations required for pharmaceuticals and chemicals. Therefore, the test strategies must be thoroughly analyzed to select the best options. The International Conferences on Harmonization (ICH) published first recommendations in 1997 (1), and later research data analyses (2) contributed to the optimization of the evaluation processes for bioassays.
Keywords: cancerogenicity, transgenic animals, need for assays
Possible threshold setting for aniline toxicity with transgenic Big Blue® Fischer344 rats. (#167)
S. Yoshida1, A. Hueser2, T. Amanuma1, J. Maeda1, K. Inagaki1, Y. Kitamura1, T. Masaki1, R. Young3, C. Beevers4, F. Weysser2, T. Toga1
1 Nihon Nohyaku Co., Ltd., Tokyo, Japan
Aniline, an environmental contaminant and residue/impurity in products, has been identified as a human health concern due to the occurrence of splenic tumors in rats. There are carcinogenic mechanisms proposed; genotoxicity via direct interaction with DNA (non-threshold), or an indirect, threshold-based mechanism via the accumulation of iron and the generation of oxidative stress after methemoglobin (MeHb) formation (see proposed AOP). We attempted to clarify whether aniline is mutagenic or not in transgenic rats. Toxicological mechanisms were also investigated concurrently.
Male Fischer 344 Big Blue® rats were given daily oral doses of 0, 25, 50 or 100 mg/kg/day aniline for 28 days. Mutant frequency was measured in liver, spleen and bone marrow at Day 31. Aniline concentration in plasma, MeHb in blood, erythrocyte (RBC), hemoglobin concentration (Hb), peripheral immature erythrocyte (reticulocyte: RETI) were also measured on day 3 or 4 of dosing and on completion of dosing (day 28 or 29). A portion of spleen was subjected also to histopathology.
Exposure to aniline was confirmed by dose-dependent plasma concentrations but there was no increase of mutant frequency in any tissues. Around day 4, MeHb was increased dose-dependently, while decreased Hb and increased RETI were limited to the top dose. At termination, decreased RBC and Hb and increased RETI were also observed from the lower doses as increase of MeHb became prominent. Iron deposition was confirmed in spleen by Prussian blue staining.
Overall, aniline is not considered to be a direct mutagen. After MeHb formation and subsequent erythrotoxicity, iron deposition in spleen and compensatory erythropoiesis in bone marrow (increase of RETI) was observed, which is in line with the proposed mechanism. A threshold-based risk assessment can be applied for aniline.
Keywords: Aniline, Genotoxicity, Transgenic rat, Big Blue®, Methemoglobin
Exposure to Dioxin Modulated Distinct Responses of Two Subtypes of Diffuse Large B-Cell Lymphoma Cells Determined by Computational Prediction and Gene Expression Profile (#187)
C. - Y. C. Chuang1, Y. Wang1, C. - Y. Li1, Y. - Y. Li1, Y. - K. Chen2
1 National Tsing Hua University, Biomedical Engineering and Environmental Sciences, Hsinchu, Taiwan
The incidence of non-Hodgkin lymphoma (HNL) has increased dramatically worldwide especially in developed countries. Environmental exposure to dioxin, have been implicated correlated with non-Hodgkin’s lymphoma (NHL) in epidemiological studies. Dioxin is a persistent organic compound containing polychlorinated biphenyl structure, and enable to activate aryl hydrocarbon receptor (AHR) and modulate inflammation in immune responses. Diffuse large B-cell lymphoma (DLBCL) is the most common type of NHL, and classified into two major biologically distinct molecular subtypes: germinal center B-cell (GCB) and activated B-cell (ABC). Patients with ABC DLBCL have been observed presenting substantially worse outcomes of treatment and poor prognosis. Thus, this study used the approaches of computational prediction and gene expression profiling to characterize the modulation of exposure to dioxin in distinct responses of two subtypes of DLBCL for investigating the potential genetically based indicators toward treatment of DLBCL.
This study firstly performed a computational method for the analysis of differentially expressed genes (DEGs) respectively in DLBCL tissues and in human cell lines exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; the most toxic dioxin) according to the microarray datasets from ArrayExpress. The results of the constructed gene-network showed that the biological pathways associated with DLBCL development included regulation of cell cycle phase transition, type I interferon production, Wnt signaling pathway, programmed cell death, and inflammasome-related genes. In the comparison of SU-DHL2 cells (ABC-like) with SU-DHL4 cells (GCB-like), TCDD treatment activated AhR with no obvious effect on NLRP3 and Caspase-1, which was leading to cell cycle arrest at the S to G2/M phase transition and reduce apoptosis in dose-dependent. Additionally, the distinct pathway in two subtypes of DLBCL cells presented ATF4-CREBBP-CYP1A1-TP53-CDKN1A-CTNNB1-RAC1-TLR4-NFkB-AKT1-GSK3 . The current study indicated that exposure to dioxin could reduce the sensitivity to inflammasome and cause cell proliferation underlying reduction of apoptosis potentially for lymphogenesis and drug resistance of treatment. These novel target genes for lymphomagenesis identified in this study can provide a reference for disease prevention and cancer treatment.
Keywords: Computational toxicology, Dioxins, Diffuse large B-cell lymphoma, Lymphogenesis
Mutagenicity assessment of a battery of compounds using a miniaturized-Ames test (#203)
J. Sanz Serrano1, D. Muruzabal1, A. Lopez de Cerain1, 2, A. Azqueta1, 2, A. Vettorazzi1, 2
1 University of Navarra, Pharmacology and Toxicology, Pamplona, Spain
The bacterial reverse mutation test (Ames test) is a robust test, with an internationally agreed protocol (OECD 471), that is included in several standard genotoxicity testing strategies for pharmaceuticals (ICH) or food additives/contaminants (EFSA).
Several miniaturized versions of the Ames test have been used scaling down the original approach, reducing not only material costs but also time.
The aim of this work was to assess the predictivity of a 6-well plate miniaturized-Ames test comparing the results with the standard Ames test and increasing historical data. For that purpose, OECD Test Guideline 471 was followed using 5 Salmonella typhimurium strains (TA97a, TA98, TA100, TA102, and TA1535) with and without metabolic activation.
For that purpose, genotoxic compounds with different mechanisms of action (4-nitroquinoline 1-oxide [4NQO], cisplatin, colchicine, etoposide, methyl methanesulfonate [MMS] and potassium bromate) and non genotoxic compounds (TritonX-100, fluometuron, D-mannitol, ethylenediaminetetraacetic acid and Tris(2-ethylhexyl)phosphate) were evaluated at different concentrations in the miniaturized 6-well plates version of the Ames test. Furthermore, commonly used positive controls for each strain were included in each assay at one concentration: 4-nitro-o-phenylenediamine (NPD), mitomycin C, sodium azide (NAAZ), 2-aminofluorene and 2-aminoanthracene.
As expected, MMS, cisplatin, 4NQO and etoposide were found to be positive at least in one or more strains with and/or without metabolic activation. Results from positive controls were within historical data. Thus, 100% (11/11) of concordance with the standard Ames test was found but with the advantage of using 10-fold less quantity of compound.
Financial support: Spanish Ministry of Economy and Competitiveness (BIOGENSA, AGL2015-70640-R). J.S. thanks the Asociación de Amigos de la Universidad de Navarra and the Government of Navarra for the pre-doctoral grants received.
Keywords: Ames, miniaturized, reverse mutation, mutagenicity, genotoxicity
Mode of action and human relevance for amisulbrom-induced rodent liver tumors (#231)
S. Hayashi1, K. Kusakari1, M. Kimura1, C. Hayakawa1, Y. Kuroda1, K. Takeuchi1, S. Furukawa1
1 Nissan Chemical Corporation, Toxicology & Environmental Science Department, Shiraoka-shi, Japan
Amisulbrom is a sulfonamide fungicide active ingredient and has been reviewed by Japan, EU, and US. In rodent carcinogenicity studies of amisulbrom, the incidences of hepatocellular adenomas increased in male/female rats and male mice. Amisulbrom has no genotoxic potential. In order to investigate the mode of action (MoA) and human relevance of amisulbrom-induced liver tumors in rodent, we conducted two experiments; 1) evaluating constitutive androstane receptor (CAR)-mediated mode of action using CAR KO rats, and 2) evaluating the effects on human hepatocyte using chimeric mice with humanized liver. In the experiment 1, Wild type (WT) and CAR KO rats received amisulbrom at 20,000 ppm or phenobarbital sodium salt (NaPB) at 500 ppm via the diet for 7 days. There were increased body weight relative liver weights, liver Cyp2b1/2b2 mRNA levels, and hepatocellular proliferation in the WT rats treated with amisulbrom or NaPB. In contrast, the treatment of the CAR KO rats with amisulbrom or NaPB did not cause these changes. In the experiment 2, chimeric mice with humanized liver (estimated more than 90% of the liver replaced with human hepatocytes) and severe combined immunodeficiency (SCID) mice, which were not transplanted human hepatocytes, received amisulbrom at 8,000 ppm via diet for 7 days. There were increased body weight relative liver weights, liver CYP2B6/Cyp2b10 mRNA levels and PROD activity in the chimeric mice and the SCID mice treated with amisulbrom. Increased hepatocellular proliferation was observed only in the SCID mice treated with amisulbrom, but not in the chimeric mice. These results suggest that amisulbrom has a potential for CAR activation in rodents and humans, and the amisulbrom-induced liver tumors in rodents is due to CAR activation and subsequent hepatocellular proliferation. In contrast, amisulbrom fails to induce proliferation of human hepatocytes in the chimeric mice with humanized liver. In conclusion, it is suggested that amisulbrom-induced liver tumors are mediated by CAR activation and the MoA for rodent liver tumors formation is not relevant to humans.
Keywords: Rodent liver tumors, Mode of action, human relevance, Constitutive androstane receptor, Humanize mice
Triorganotin derivatives: Time-dependent expression of Vimentin, Annexin A5 and selected nuclear receptors mRNA in MDA-MB-231 breast cancer cells (#249)
D. Macejova1, B. Mosna1, P. Bobal2, J. Otevrel2, J. Brtko1
1 Department of Endocrine Regulations and Psychofarmacology, Institute of Experimental Endocrinology BMC SAS, Bratislava, Slovakia
Trirganotin compounds are typical environmental contaminants that are used as biocides, agricultural fungicides, wood preservatives, and special paints for marine ships and endocrine disrupters (1, 2). A remarkable breakthrough in this field has been found that the triorganotin compounds are agonists of the RXR nuclear receptor subtypes (3). Vimentin plays a very important role in the process of metastasis and its expression is typical for neoplastic cells with metastatic properties. The observed protein is a key element regulating the expression of the EMT-related transcription factors and thus it is associated with the metastatic spread of cancer. In addition, overexpression of Vimentin indicates the aggressive and invasive type of breast cancer (4). Annexin 5 is the protein playing an anti-apoptotic role, promoting metastatic process and progression of breast cancer (5). In this study, in vitro the effects of triorganotin ligands of nuclear retinoid X receptors in human MDA-MB-231 breast cancer cells were analysed. The cells were exposed to tributyltin and triphenyltin derivatives (TBT-Cl, TPT-Cl, TBT-ITC, TPT-ITC), 9-cis retinoic acid (9cRA) (100 nM) and/or all-trans retinoic acid (ATRA) (1 µM) for 6, 12, 24 and 48 hours. Expression of mRNA genes for Vimentin, Annexin A5 and selected nuclear receptors was analyzed by semi-quantitative real-time PCR. ATRA, 9cRA and tributyltin derivatives alone or in combination with ATRA, was found to significantly induce the expression of Vimentin and Annexin A5 mRNA after 6 h. However, after 12 h and 24 h the expression was decreased and these effects were fully manifested after 48 hours of cultivation with the substances. In the case of RARbeta receptors, the action of triorganotin derivatives resulted in increased gene expression after all time ranges, with the combined effect with ATRA resulting in a synergistic enhancement of expression. Expression of RARgamma mRNA was found significantly increased after 6 hours, but after 24 and 48 hours the increase was diminished. After 6 hours after administration of retinoic acids and triorganotin derivatives, expression of RXRalpha was increased, but a decrease in expression was observed after 48 hours in cells treated with TBT-Cl and ATRA alone and with combination of ATRA with triorganotin derivatives. Given the role of vimentin in the epithelial-mesenchymal transition process and annexin A5 in the membrane repair process, the synergistic effect of triorganotin compounds, and the ATRA mediated by RXR/RAR heterodimer could represent a promising opportunity to inhibit metastasis of aggressive forms of hormone-resistant tumours. This project has been supported by the grants APVV-15-0372 and Vega 2/0171/17.
1. Hiromori et al. 2016 Jar cells. J. Steroid. Biochem. Mol. Biol. 155:190-198
Keywords: retinoic acid, retinoic acid receptors, Vimentin, Annexin A5, triroganotin derivatives
Genotoxicity, homocysteine, dietary micronutrients and MTHFR gene polymorphisms in psoriatic patients treated by Goeckerman regimen (#264)
P. Borsky1, M. Beranek2, 3, A. Malkova1, Z. Fiala1, J. Kremlacek4, K. Hamakova5, L. Zaloudkova2, T. Adamus6, V. Palicka2, L. Borska4
1 Faculty of Medicine in Hradec Kralove, Charles University, Institute of Hygiene and Preventive Medicine, Hradec Kralove, Czech Republic
Goeckerman regimen (GR) of psoriasis vulgaris is a therapeutic combination of crude coal tar application and ultraviolet irradiation. Both these agents could induce genotoxic effects. Insufficient amounts of micronutrients including vitamin B12 and folic acid lead to genomic instability and possibly intensified genotoxic effects of GT.
We examined DNA damage, serum homocysteine, vitamin B12, folic acid, and two polymorphisms (C677T and A1298C) in the MTHFR gene in patients with exacerbated psoriasis vulgaris treated by GR.
Study group consisted of thirty-five patients classified according to PASI score. Genotoxicity was evaluated by the number of micronucleated binucleated cells (MNBC). Serum homocysteine, vitamin B12 and folic acid were determined immunochemically. DNA analysis was performed via real-time PCR.
The median of PASI score decreased from 19.2 to 4.9, MNBC increased from 10 to 18 ‰ after GR (P < 0.001 in both cases). Homocysteine, vitamin B12 and folic acid were not changed significantly by the therapy. Correlations of MNBC with homocysteine and vitamin B12 before the regimen were observed. Hyperhomocysteinemia was an independent predictor of genotoxicity (OR 9.91; 95% CI, 2.09–55.67; P = 0.003). There was found a significantly higher MNBC In CC homozygous patients (A1298C polymorphism), than in AC heterozygotes and AA homozygotes.
Homocysteine is engaged in the pathogenesis of psoriasis vulgaris. Its serum levels correlating with MNBC enabled prediction of DNA damage induced by Goeckerman regimen. A potential link between the MTHFR C1298A polymorphism and genotoxic effects of this therapy was found. Both micronutrients status and homocysteine metabolic pathway contribute to genotoxicity of Goeckerman regimen.
Supported by the projects PROGRES Q40-09 and Q40-11 of Charles University in Prague, Faculty of Medicine in Hradec Kralove, Czech Republic.
Keywords: Goeckerman regimen, Genotoxicity, Homocysteine, Vitamin B12, MTHFR
Genotoxic effects of different technical products of dimethoate (#286)
N. A. Ilyushina1, O. Egorova1, G. Masaltsev1, N. Averianova1, V. Rakitskii1
1 FBES «Federal Scientific Center of Hygiene named after F.F. Erisman» of the Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing, Department of Genetic Toxicology, Mytishchi, Russian Federation
Technical products of generic pesticides may have dissimilar toxicological profiles due to different levels of impurities. Dimethoate is an organophosphate insecticide and acaricide. The data on its genotoxicity are controversial.
The aim of this study was to assess and compare genotoxic potentials of two technical grade active ingredients (TGAI) of dimethoate from different manufacturers.
The Ames test and mammalian erythrocyte micronucleus test were used for this study. It was shown that TGAIs of dimethoate induced reverse gene mutations in Salmonella typhimurium both in the presence and in the absence of S9 mix. However, the effect levels were different. TGAI1 shown statistically significant genotoxic effects only in two strains. At high concentration (5 mg/plate) fold-increase in the number of revertant colonies per plate relative to vehicle was 1.7/1.9 (TA-100/TA-102; -S9) and 1.7/2.0 (TA-100/TA-102; +S9). TGAI2 induced the higher levels of mutations in 3 strains: 2.4/2.7/2.8 (TA-97/TA-100/TA-102; -S9) and 2.2/3.3/2.3 (TA-97/TA-100/TA-102; +S9) at concentration 5 mg/plate and the effects were dose-dependent. TGAI1 was non-genotoxic in micronucleus test in vivo, whereas after administration of TGAI2 to CD-1 mice (oral gavage; three doses) we observed the statistically significant dose-dependent increase in the incidence of micronucleated polychromatic erythrocytes (mPCE) in bone marrow (2.1-fold relative to negative control at high dose). However, the incidence of mPCE was slightly beyond the upper Poisson-based 95% control limit for the historical negative control. 95%Wald confidence intervals for the mean of mPCE were [0.09;0.11] and [0.17; 0.30] for vehicle and dimethoate at high dose (60 mg/kg b.w.), respectively.
The observed difference between two technical products probably is due to the quality and quantity of impurities. Dimethoate may contain omethoate and isodimethoate as the relevant impurities. According to FAO specification, the maximum levels of these impurities must not exceed 2 g/kg and 3 g/kg, respectively. Genotoxicity of omethoate was shown in some research .
Therefore, our data indicate that dimethoate has a weak genotoxic potential and the different technical products of the same pesticide active ingredient may reveal dissimilar genotoxicity level.
1. Omethoate; Hazardous Substance Databank Number 6715. Available at: https://toxnet.nlm.nih.gov/cgi-bin/sis/search/a?dbs+hsdb:@term+@DOCNO+6715
Keywords: genotoxicity, pesticides, TGAIs, Ames test, micronucleus test
Cylindrospermopsin induces genotoxic damage in rats by the comet and micronucleus tests (#314)
L. Diez-Quijada Jiménez1, M. Llana-Ruiz-Cabello1, G. Catunescu2, M. Puerto1, A. Jos Gallego1, A. M. Cameán-Fernández1
1 Faculty of Pharmacy, University of Seville, Area of Toxicology, Seville, Spain
Nowadays, as a result of climate changes and water eutrophication an increase in the production of toxic cyanotoxins is happening. Cylindrospermopsin (CYN) is a cyanotoxin produced by different species with cytotoxic and hepatotoxic effects. Humans can be in contact with CYN by different routes, being the oral intake the main one. Among the toxic effects of CYN its genotoxicity is a keypoint. Previous studies pointed out that CYN is pro-genotoxic in vitro. Therefore, in accordance with the European Food Safety Authority (EFSA), it is needed to assess its genotoxicity in vivo. In this work, the genotoxic potential of CYN in Wistar rats was evaluated in liver, stomach and blood by the standard comet assay (OECD 489) and on bone marrow by the micronucleus test (MN, OECD 474). Moreover, the enzime-modified comet assay (Endonuclease III (Endo III) and Formamido pyrimidine glycosylase (FPG)) was used to assess oxidative DNA damage. Animals were exposed to CYN by oral gavage (7.5, 23.7, and 75 µg/kg body weight). Results for the standard comet assay showed no significant increase in DNA strand breaks at any dose assayed. However, after the post-treatment with Endo III a significant increase in the % of DNA in tail was observed in liver and blood cells exposed to the highest dose. Additionally, oxidative damage was observed in blood cells in presence of FPG after exposure to 23.7 and 75 µg/kg. Moreover, results obtained for the MN test showed CYN genotoxicity at any dose tested with increase in the % MN in immature erythrocytes. Therefore, these results show that CYN is genotoxic in vivo providing important data for a better risk assessment of this biotoxin.
Acknowledgments: This work was supported by the Spanish Ministry of Economy and Competitiveness for financing the project (AGL2015-64558-R, MINECO / FEDER, UE), and the grant FPI (BES-2016–078773) awarded to Leticia Díez-Quijada Jiménez. María Llana-Ruiz-Cabello also gratefully acknowledges Junta de Andalucía for her postdoctoral grant associated to AGR7252 project. Giorgiana M. Cătunescu also wants to grateful to EFSA the fund under the EU-FORA Programme allowing her contribution in the present work
Keywords: In vivo, Cylindrospermopsin, Genotoxicity, Micronucleus Test, Comet assay.
Genotoxic and genoprotective effects induced by a stilbene extract in HepG2 cells (#315)
C. Medrano Padial1, M. Puerto1, E. Cantos-Villar2, T. Richard3, A. M. Cameán-Fernández1, S. Pichardo1
1 Faculty of Pharmacy, University of Seville, Area of Toxicology, Seville, Spain
The addition of sulfur dioxide seems to be essential during several processes involved in winemaking and ageing wine. However, due to health concerns and a recent growing consumer interest in wines containing less additives, its use has been a field of discussion in wine industry. Stilbenes are candidates of great interest for this purpose, not only because of their antioxidant and antimicrobial activities, but also because they are naturally found in the grapevine. In the present study, the in vitro genotoxicity and genoprotective effects of an extract from grapevine shoot, with a stilbene richness of 86%, was investigated. The exposure concentrations were selected based on previous studies in which the EC50 was determined in HepG2 cells. The standard and the FPG-modified version of the comet assay, after 24 h or 48 h of exposure to the extract, did not show genotoxicity at any of the studied concentrations. In order to evaluate the protection ability of our extract against DNA induced damage, HepG2 were pretreated with Ro19-8022 and then exposed to the extract during 24 h or 48 h. There were significantly lower levels of DNA breaks compared with control in cells preincubated with 15.95 µg/mL and 31.90 µg/mL for 24 h and with 22.30 µg/mL for 48 h, indicating an enhanced antioxidant defense. Similarly, incubation of Ro19-8022-treated cells with our extract led to a concentration-related decreased in induced DNA damage. Significant changes respect the control were shown from 31.90 µg/mL and 11.15 µg/mL after 24 h and 48 h of exposure respectively. In summary, our extract showed no genotoxic effects at any concentration tested. In addition, the extract presents interesting antioxidant abilities in vitro. Therefore, considering its promising usefulness as additive in wines, further studies are required in order to confirm its suitability and safety for this purpose.
Acknowledgments: The authors thank the Ministerio de Economía, Industria y Competitividad and INIA for the financial support for this project (RTA2015-00005-C02-02). Moreover, we thank the CITIUS Biology Service (University of Seville) for the technical assistance offered.
Keywords: genotoxicity, comet assay, stilbene extract.
A proposed workflow, considering non-testing methods, to qualify non-genotoxic impurities (#423)
M. Fuart Gatnik1, S. Kovarich1, L. Ceriani1, R. Calabrese1, L. Broccardo1, L. Sartori1
1 S-IN Soluzioni Informatiche Srl, Computational Toxicology, Vicenza, Italy
A reflection paper has been recently published by EMA addressing open issues in the qualification approach of non-genotoxic impurities (NGI) in chemically synthesized pharmaceuticals according to the ICH Q3A/Q3B guidelines. As highlighted in the EMA document, little guidance is provided on which criteria and methods should be applied to qualify NGIs, and concerns are expressed from a scientific and 3R’s perspective on the current approach. When qualification of NGIs is required and data from the regular (non-)clinical development with the API batches is not considered sufficient, a recommendation to consider non-animal testing strategies, including both in silico and in vitro approaches, to evaluate the toxicity of individual NGIs, is discussed.
In the present poster we propose a workflow for the assessment of toxicity profile of individual NGIs based on non-animal testing strategies, with a particular emphasize on QSAR and read-across methodologies. A preliminary case-by-case analysis is performed considering the use and route of administration of the API as well as information on similar structures in order to define the set of endpoints to be investigated, and the most suitable in silico approaches to apply. The workflow can be represented as a decision tree, including the following key steps (each to be considered in a tier approach): 1) Evaluation of the applicability of the TTC approach for the target NGI. 2) Structural comparison with the API. 3a) NGI core structure is shared with the API: analysis of structural differences between the NGI and the API, including the assessment of any toxicologically relevant alert found in the NGI and not in the API. QSAR predictions are generated for selected endpoints to integrate alert analysis. 3b) NGI structure is not related to the API: QSAR predictions are generated for selected endpoints. 4) Search for structural analogues (including the API, if relevant) with experimental data on critical endpoints and/or endpoints not covered by the QSAR screening. 5) Evaluation of the feasibility of read-across. The proposed workflow offers the possibility to acquire impurity-specific data, also if testing is not feasible, and to decide on further in vitro testing, besides meeting 3R’s principles.
European Medicines Agency (2018). Reflection paper on the qualification of non-genotoxic impurities.
Keywords: non-genotoxic impurities, QSAR, read-across, non-testing, 3R’s
Estragole DNA adduct formation in different liver cell models (#435)
S. Yang1, S. Wesseling1, I. M. C. M. Rietjens1
1 Wageningen University, Toxicology, Wageningen , Netherlands
Estragole, one of the food-borne alkenylbenzenes, can naturally occur in a variety of herbs and spices such as sweet basil, fennel, star anise and essential oils. Estragole is of concern because of its genotoxicity and carcinogenicity, induced via DNA adduct formation after bioactivation (Miller et al.,1983). In previous in vitro studies, alkenylbenzene DNA adduct formation was studied upon exposure of HepG2 cells to 1'-hydroxy metabolites instead of to the parent compounds, because of the limited P450 enzyme activities present in the HepG2 cells (Jeurissen, et al., 2008; Alhusainy, et al. 2013). Use of 1'-OH estragole to induce DNA adduct formation may however result in levels of DNA adducts overwhelming DNA repair and thus will not provide a suitable in vitro model to study repair of alkenylbenzene DNA adducts. Therefore, this study was designed to define the most suitable cell model(s) to form a detectable level of DNA adducts upon exposure to the parent compound. HepG2 and HepaRG cells and primary rat hepatocytes were pretreated with or without CYP inducers and incubated with estragole or 1'-OH estragole. The DNA adduct formation upon exposure of the cells to both 1'-OH estragole or estragole increased in the order HepG2 cells < HepaRG cells < primary rat hepatocytes with levels upon exposure to estragole being 30 to 40 fold lower than upon exposure to 1'-OH estragole. The DNA adduct levels were not significantly affected by the CYP inducers.
It was concluded that non-induced HepaRG cells and primary hepatocytes exposed to estragole provide the most suitable in vitro models to study DNA adduct formation and subsequent repair.
Alhusainy et al. Inhibition of methyleugenol bioactivation by the herb-based constituent nevadensin and prediction of possible in vivo consequences using physiologically based kinetic modelling. Fd Chem Toxicol 59 (2013) 564-571.
Keywords: DNA adduct formation, CYP inducers
Rhus coriaria extract: protective effect against UVA irradiation in human dermal microvascular endothelial cells (HMEC-1) (#473)
L. Marabini1, G. Melzi1, F. Lolli2, S. Khalilpour2, M. Marinovich2
1 Università degli Studi di Milano, Department of Environmental Science and Policy, Milan, Italy
Rhus coriaria L. (sumac) is a plant traditionally used in some countries (Turkey, Iran) as an anti- microbial and anti-inflammatory agent in skin disorders (1). Its fruits are rich in tannins, anthocyanins, flavonoids and terpenoids and gallic acid derivatives (2). Recently, different polyphenol-enriched extracts have been proposed for the prevention of UV-mediated skin damage. The extract was obtained by ethanol maceration for 48h of dried fruits. Then, the mixture was filtered and taken to dryness under reduced pressure and freezed dried. The extract (mERC) has been characterized in its components gallotannins and flavonols. UV-A rays can penetrate up to the dermal layer also affecting the microvascular endothelial cells of the papillary dermis, determining inflammation and photoaging, associated with redox imbalance. Treatment with the extract (10 and 25 µg/ml, 1h in serum-free media) was followed by the exposure to UV-A (10-15-20 J/cm2) radiation in PBS on HMEC-1 cells. Cytotoxicity (MTT and apoptosis) and DNA damage (alkaline comet test, ɣH2AX foci formation) have been evaluated at T0 and T24 after exposure. The mechanisms of UV-A mediated DNA damage on this cell line have been also studied in order to highlight the ability of the extract components to interfere with oxidative damage (oxidized base formations, SBBs, apurinic sites), but also with the formation of cyclobutane pyrimidine dimers ( CPDs) by comet modified with enzymes and immunohistochemistry assays.
1) Bedi M.K. and Shenefelt P.D. (2002). Archives of Dermatology 138(2),232-242.
2) Shabbir A. .(,2012) J Anim Plant Sci 22(2). 505-512.
Keywords: UVA rays, genotoxicity, HMEC-1 cells, Rhus coriaria
UV-B damage: a potential molecular play of Vitis vinifera L. extract (#483)
F. Lolli1, L. Marabini2, G. Melzi2, S. Piazza1, M. Marinovich1
1 University of Milan, Department of Pharmacological and bimolecular Sciences, Milan, Italy
Ultraviolet component of sunlight UV-B (280-315 nm) is one of the major cause of skin damage. UV-B radiations have a low wavelength being absorbed almost completely by the epidermis; however, they interact directly with DNA causing molecular rearrangements. Photoaging and the development of skin cancer are of increasing relevance since lifestyle changes have led to an increase in the individual UV doses. Therefore, new prevention strategies have to be developed in order to reduce UV damage and delay photoaging process. In this context, different polyphenol-enriched botanicals have been proposed for the prevention of UV-mediated skin damage. Here it has been evaluated the antioxidant and DNA protective potential of an aqueous extracts of Vitis vinifera L., validated for the contents of anthocyanins, flavonoids and caffeic acid, against UV-B radiation in human keratinocyte (HaCaT) cell line. Treatment with the extract (100 µg/ml, 1h in serum-free media), was followed by the exposure to UV-B (20-30-40-80-160-320-640 mJ/cm2) radiation in PBS. The extract protects from the direct DNA damage induced by UV-B exposure even at doses where no oxidative damage was observed, as indicated by the alkaline comet and ɣH2AX tests, evaluated at T0 after the exposure. Moreover, it seems that the extract maintains the protective apoptotic pathway induced by UV-B. All these evidences led us to study the effect of the extract on DNA damage at molecular level, looking at the expression of genes involved in several pathways of DNA damage recognition, repair and apoptosis induction. Interestingly, the extract is able to modulate the expression of several genes involved both in damage signalling and NER (Nucleotide excision repair) pathway, mostly at 40 mJ/cm2 dose. The most important effect of the extract is on GADD45α especially at 40 and 80 mJ/cm2. This gene is pivotal in DNA repair pathway induced by UVR exposure, acting also on negative growth control. All together these findings suggest a potential interesting play of the extract also at the molecular level, modulating the expression of key genes involved in DNA damage signalling and repair.
Keywords: genotoxicology, dna repair, natural extracts, uv radiations
Difenoconazole – Liver Tumour Mode of Action and Human Relevance Assessment (#524)
R. A. Currie1, H. K. Bhandal1, R. C. Peffer2
1 Syngenta, Bracknell, United Kingdom
Dietary administration of CD-1 mice for up to 18 months with difenoconazole (DFZ) resulted in toxicity in excess of a maximum tolerated dose (MTD) from 2500 – 4500ppm. Gavage pharmacokinetic studies demonstrate that the systemic exposure of the doses used in this study are not dose-proportional and therefore these mice were dosed in excess of a kinetically-limited top dose. At these excessive doses, liver tumor incidence was statistically increased in males at 4500 ppm and both sexes at 2500 ppm. We show that DFZ causes activation of the constitutive androstane receptor (CAR), which leads to increased expression of CAR-responsive pro-proliferative and anti-apoptotic genes and transient increases in hepatocellular proliferation. Associative events that are mediated by CAR activation include increased CYP enzyme expression and activity (primarily CYP2B and CYP3A), hepatocellular hypertrophy, and increased liver weight. These key and associative events are absent in CAR-knockout mice, demonstrating their CAR-dependency. The prolonged CAR-dependent liver weight increase at doses of 2500 ppm and above, result after several months of dosing, in fatty change, bile stasis, and ultimately necrosis of hepatocytes and is clearly in excess of the MTD. This effect serves as a late-acting, modulatory factor for the CAR-dependent alteration in proliferation which is the initial and main driver for the carcinogenic process. Furthermore we demonstrate that DFZ is not an activator of human CAR in a transactivation reporter assay, under conditions where it is a highly effective activator of an equivalent mouse CAR reporter. In an in vitro study with human hepatocytes, moderate increases in CYP2B and CYP3A activity by DFZ indicate some potential for CAR activation in human liver cells. However, DFZ does not stimulate the key event of cell proliferation in human hepatocytes in vitro, whereas it does in mouse hepatocytes in vitro. This pattern of effects matches the known species differences that have been demonstrated for other CAR activators, and the weight of evidence indicates that it represents a qualitative difference in the established MOA for DFZ between mice and humans. consequently the data support the conclusion that DFZ does not pose a carcinogenic hazard to humans.
Keywords: carcinogenesis, liver toxicity, constitutive androstane receptor, mode of action, human relevance
A novel extension of the ToxTracker genotoxicity assay identifies aneugenic and clastogenic properties of chemicals. (#538)
I. Brandsma1, R. Derr1, N. Moelijker1, G. Hendriks1
1 Toxys, Leiden, Netherlands
ToxTracker® is a mammalian stem cell-based reporter assay that detects activation of specific cellular signalling pathways upon chemical exposure. ToxTracker contains six different GFP-tagged reporter cell lines that together allow the discrimination between induction of DNA damage, oxidative stress and/or protein damage in a single test. Genotoxicity is detected by the Bscl2-GFP reporter for promutagenic DNA lesions and DNA replication stress, and the Rtkn-GFP reporter for DNA double strand breaks.
Here we investigated whether the ToxTracker assay could be adapted to allow the discrimination between clastogenic and aneugenic compounds. We included a DNA stain in the ToxTracker assay for cell cycle analyses and the detection of aneuploidy. Clastogenic compounds can cause cell cycle arrest, but generally do not cause aneuploidy. Aneugenic compounds, on the other hand, cause a cell cycle arrest in G2/M phase and aneuploidy. To validate the assay, we tested 4 clastogenic, 7 aneugenic, 2 pro-mutagenic and 3 non-genotoxic compounds in ToxTracker ACE (aneugen clastogen evaluation). As expected, the clastogenic, and pro-genotoxic compounds (with metabolic activation) activated the genotoxicity reporters in ToxTracker, but did not cause aneuploidy. The aneugenic microtubule disruptors and aurora kinase inhibitors arrested the cells in G2/M phase and caused an increase in aneuploidy.
Together, the differential activation of the ToxTracker genotoxicity reporters, in combination with the cell cycle analysis and polyploidy detection, allows for rapid identification of clastogens and aneugens and can further discriminate between microtubule poisons and kinase inhibitors.
Keywords: ToxTracker, genotoxicity, mode-of-action, reporter assay
In vitro treatment of DMBA to murine mammary tissue-derived organoids induced adenocarcinomas/squamous cell carcinomas after their subcutaneous injection to nude mice (#571)
T. Imai1, 2, R. Masui2, R. Nakanishi1, Y. Machida1, 3, M. Ochiai2, M. Naruse1
1 National Cancer Center Research Institute, Central Animal Division, Tokyo, Japan
We previously reported that single administration of 7,12-dimethylbenz[a]anthracene (DMBA, 50 mg/kg body weight) by gavage induced mammary adenocarcinomas with or without adenosquamous characteristics with an incidence of over 70% after more than 13 weeks of DMBA administration in BALB/c-Trp53＋/－ mice. In addition, point mutations of Hras gene were frequently detected in the induced carcinomas. However, it could not be clarified whether the Hras mutation was an initial event or other molecular events by DMBA administration first occurred before the Hras mutations in the in vivo model. The purpose of the present study is to establishment of a simple model for evaluation of early molecular events of DMBA-induced mammary carcinogenesis, in which actual tumor development could be confirmed as its end point. We therefore here examined whether a short-term treatment of DMBA to normal mammary tissue-derived organoids of BALB/c-Trp53＋/－ mice in vitro would induce carcinomas after their subcutaneous injection to nude mice or not. [Materials and methods] Treatments with DMBA at concentrations of 0, 0.2 and 0.6 μM plus S9 mix to mammary organoids of BALB/c-Trp53＋/－ mice for 24 hours were repeated three times of passages of the organoids, followed by their subcutaneous injection to nude mice. [Results] The mammary organoids treated in vitro with 0.6 μM DMBA developed to adenocarcinomas and/or squamous cell carcinomas with the incidence of 4 of 4 but not in organoids treated with 0 and 0.2 μM DMBA (p=0.012) after 8 weeks of their subcutaneous injection to nude mice. [Conclusion and future plan] It was demonstrated that carcinogenic alterations of mouse mammary tissue-derived organoids treated with DMBA in vitro were induced in the nude mouse subcutis. We are now investigating whole exome sequence analyses of the DMBA-treaetd organoids before subcutaneous injection, to clarify the early genetic mutations in this carcinogenesis model.
Keywords: mammary, mouse, organoid, carcinoma, Trp53
Generating a Historical Control Database for the Comet Assay of Mouse Testes (#589)
M. Young1, K. Pant1, S. Bruce1, R. Kulkarni1, S. Springer1, M. Klug Laforce1
1 MilliporeSigma (BioReliance® Toxicology Testing Services), Rockville, Maryland, United States of America
The OECD comet assay test guideline (TG489, 2016) requires that prior to using any organ in the assay the laboratory should build a historical database to establish positive and negative control ranges and distributions for relevant tissues and species. Different tissues and different species, as well as different vehicles and routes of administrations, may give different negative control % tail DNA values. It is therefore important to establish negative control ranges for each tissue and species. To build a historical database for mouse testes, male Hsd:ICR (CD-1) mice were dosed with vehicle (saline) or the positive control methyl methanesulfonate (MMS) at 40 mg/kg/day via oral gavage once per day for three consecutive days. Initial experiments were performed with one, two, or three days of dose administration. The one and two day dose administration regimens did not result in a significant increase in DNA damage with MMS. The three day dose administration was successful. In order to build a database, five independent experiments, with two groups of five animals each, were conducted with saline and MMS. On study day 3, animals were euthanized, testes collected, single cell suspensions prepared, and cell suspensions processed for the comet assay, per the OECD Guideline. The comet assay results from these five experiments demonstrated that oral dosing of MMS for three days results in significant DNA damage in mouse testes (% tail DNA range: 7.38 to 9.03) and the response was significantly higher than the vehicle control (% tail DNA range: 0.55 to 1.25).
Keywords: Genetox, historical control, OECD, Comet assay
Exosome-mediated horizontal gene transfer: a possible new risk for genome editing (#614)
R. Ono1, Y. Yasuhiko1, K. - I. Aisaki1, S. Kitajima1, J. Kanno1, 2, Y. Hirabayashi3
1 National Institute of Health Sciences (NIHS), Division of Cellular and Molecular Toxicology, Center for Biological Safety and Research, Kawasaki, Japan
The CRISPR/Cas system allows the introduction of double strand breaks (DSBs) at particular loci in the genome. DSBs are subsequently repaired through non-homologous end joining (NHEJ), or homologous recombination (HR).
We showed that DSBs introduced into the mice zygote by the CRISPR/Cas system are repaired by the capture of unintentional sequences, including retrotransposons, mRNA, and CRISPR-Cas9 vector sequences. This DSB repair mechanism with the capture of unintentional sequences were partially mediated by reverse transcription (RT), because the captured sequences were apparently derived from RT-mediated spliced mRNAs (Ono et al. 2015).
Therefore, it is possible that unintentional insertions associated with DSB repair represent a potential risk for human genome editing gene therapies. To address this possibility, comprehensive sequencing of DSB sites was performedin vivo and in vitro (mouse) by deep sequencing of PCR products amplified with two primers across the target DSB site. Although most of the unintentional insertion sequences were derived from plasmid vectors, 0,27 % of the unintentional insertions were derived from bovine DNA fragments (Ono et al. 2019).
To determine the origin of bovine DNA fragments, we used goat serum, rabbit serum, and exosome-free FBS instead of FBS in the cell culture medium. Goat BovB, and rabbit LINE1 sequences were horizontally transferred to DSB sites by using goat and rabbit serum, respectively, however, almost no bovine DNA sequences were captured by using exosome-free FBS, suggesting that these horizontal gene transfers were mediated by exosomes.
We demonstrated that horizontal gene transfer assisted by CRISPR-Cas9 occurs in NIH-3T3 cells and mouse embryos, suggesting that exosome-mediated horizontal gene transfer is the driving force behind mammalian genome evolution. The findings of this study also highlight an emerging new risk for this leading-edge technology.
Ono R et al. Scientific reports, 2015, 5: 12281.
Ono R et al. Communications Biology, 2019,2: 57.
Keywords: genome editing, on-target risk, exosome
Mixture effects on BP-dependent AhR activation, metabolite and DNA adduct formation (#618)
L. Gödtke1, A. John2, A. Braeuning1, A. Seidel2, A. Lampen1, S. Hessel-Pras1
1 Federal Institute for Risk Assessment, Food Safety, Berlin, Berlin, Germany
The prototypical carcinogen benzo[a]pyrene (BP), but also other non-carcinogenic polycyclic aromatic hydrocarbons (PAH) like pyrene (PYR) and fluoranthene (FA), are frequently found as contaminants in the diet. To identify mixture effects of BP, PYR and FA in proportions as occurring in grilled meat, the activation of the nuclear receptors AHR and CAR and their target gene expression (CYP1A1, CYP2B6) were investigated in human HepaRG hepatocarcinoma cells. To also examine important downstream key events, BP metabolites and the BP-dependent formation of DNA adducts were investigated under the influence of mixtures.
In contrast to the well-known AHR agonist BP, PYR and FA activated AHR only weakly and binary/ternary mixtures were less efficient than BP alone. However, analysis of CYP1A1 gene expression showed synergistic effects after PAH co-exposure in HepaRG cells. By contrast, PYR and FA were strong CAR agonists, whereas BP was less potent. Mixtures containing BP caused a strong decrease of CAR transactivation in line with a lower CYP2B6 expression level. Analyzing BP metabolites and the BP-dependent formation of DNA adduct levels revealed higher levels of detoxified mutagenic BP-diol epoxide with simultaneous lower DNA adduct levels after treatment of HepaRG cells with binary and ternary BP mixtures with FA and PYR.
PAH mixtures can modulate the induction of gene expression which may result in higher detoxification or bioactivation of carcinogenic xenobiotics like BP itself. The study supports the general concept that for the risk assessment of complex mixtures, alterations of molecular key events such as the activation of multiple receptors must be taken into account.
Keywords: PAH, HepaRG, Benzoapyrene, DNA adducts, nuclear receptors
Preclinical safety assessment of aqueous fern extracts (#635)
L. Lauer1, I. Zilkowski1, J. Bertrams1, C. Turek1, M. B. Müller1, N. Mörbt1, P. Vögele1, F. C. Stintzing1
1 WALA Heilmittel GmbH, Bad Boll, Germany
Ferns and preparations thereof have been used in traditional medicine for a long time to treat a variety of ailments. However, there is also a hazard emerging from ferns, since poisonings have been reported in literature. Besides acute toxicity, mutagenicity is known for some fern species like Pteridium aquilinum (L.) Kuhn (P. aquilinum). Hence, a preclinical safety assessment is required for herbal medicines containing ferns. In the present work, the toxicological potential of two aqueous extracts from P. aquilinum and Dryopteris filix-mas (L.) Schott (D. filix-mas) ferns used as active substances in medicinal products was investigated. Both extracts were produced according to officially accepted pharmaceutical standards and evaluated in bacterial reverse mutation assays with and without exogenic metabolic activation following OECD standards. The aqueous extract of D. filix-mas was additionally tested in an in vitro cytotoxicity assay using mammalian cells performed in accordance with OECD and ISO standards. Moreover, the concentration of the mutagenic constituent ptaquiloside of P. aquilinum was determined in the aqueous P. aquilinum extract by a validated HPLC procedure. The aqueous extract of D. filix-mas exhibited cytotoxicity only at the highest concentration applied (50 mg/mL) and no evidence for mutagenicity was revealed. The aqueous extract of P. aquilinum was inconspicuous in the mutation assay with a ptaquiloside level below the limit of detection (0.1 mg/L). According to the results obtained, the two aqueous fern extracts do not exhibit any mutagenic potential. However, the cytotoxicity of the aqueous extract of D. filix-mas needs to be considered when used in herbal medicine at high concentrations. The data presented contribute to the preclinical safety assessment of aqueous fern extracts and herbal products derived therefrom.
Keywords: in vitro, mutagenicity, aqueous extract, fern
Characterization of enzymes oxidizing the tyrosine kinase inhibitor vandetanib and elucidation of the high efficiency of cytochrome P450 3A4 to generate N-desmethylvandetanib (#636)
R. Indra1, K. Vavrova1, P. Takacsova1, P. Pompach1, V. Martinek1, Z. Heger2, 3, V. Adam2, 3, V. M. Arlt4, M. Stiborova1
1 Department of Biochemistry, Faculty of Science, Charles University, Prague 2, Czech Republic
Metabolism affects the pharmacological efficiency of the tyrosine kinase inhibitor vandetanib. Here, we investigated the in vitro metabolism of vandetanib using (i) hepatic subcellular systems rich in drug-metabolizing enzymes (microsomes) isolated from livers of humans and several animal models, and (ii) human and/or rat recombinant biotransformation enzymes such as cytochromes P450 (CYPs) and flavin-containing monooxygenases (FMOs). In addition to the structural characterization of vandetanib metabolites, individual human and rat enzymes capable of oxidizing this drug were identified. Two vandetanib metabolites, N-desmethylvandetanib and vandetanib N-oxide, were formed in incubations with hepatic microsomes. The generation of N-desmethylvandetanib was attenuated by inhibitors of CYP3A and 2C subfamilies in both human and rat microsomes, while an inhibitor of CYP2D6 only decreased formation of this metabolite in human microsomes. The FMO inhibitor methimazol decreased the formation of vandetanib N-oxide in both rat and human microsomes. These results indicated that in the microsomal systems studied, CYP3A, 2C and/or 2D are mainly responsible for the formation of N-desmethylvandetanib and FMO1 and 3 mainly for the generation of vandetanib N-oxide. Human recombinant CYP3A4>>>2D6, 3A5, 1A1 and 2C8 oxidized vandetanib to N-desmethylvandetanib, while rat recombinant CYP2C11>>3A1>3A2>1A1>1A2>2D1>2D2 were effective in catalyzing this reaction. Cytochrome b5 which serves as electron donor to CYP enzymes influenced the CYP-catalyzed formation of N-desmethylvandetanib; CYP3A4 was most affected. Human CYP3A4 is not only the most efficient enzyme oxidizing vandetanib to N-desmethylvandetanib, but also most important due to its high expression in human liver. Molecular modeling indicated that binding of more than one molecule of vandetanib into the CYP3A4-active center can be responsible for the high efficiency of CYP3A4 N-demethylating vandetanib to N-desmethylvandetanib. Indeed, the CYP3A4-mediated reaction was allosterically modulated exhibiting kinetics of positive cooperativity, which corresponds well to the in-silico docking model, where two bound vandetanib molecules were found in the active center of CYP3A4.
Supported by GACR (grant 18-10251S).
Keywords: Vandetanib, Cytochrome P450
Migration and Invasion of human renal cancer cells are impaired upon treatment with thymoquinone (#638)
V. Keser1, J. G. Costa2, C. Jackson1, N. Saraiva2, N. Almeida2, S. P. Camões1, M. Castro1, J. P. Miranda1, A. S. Fernandes2, N. G. Oliveira1
1 Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, Lisboa, Portugal
Thymoquinone (TQ), 2-methyl-5-isopropyl-1,4-benzoquinone, is a monoterpene isolated from the oil of Nigella sativa seeds, also known as black seed. The cytotoxic effects of TQ have been reported in different cancer cell lines. Nevertheless, the impact of this bioactive compound on the metastatic properties of cancer cells should be further studied, particularly in the context of renal clear cell carcinoma. The aim of this work was to evaluate the effects of TQ on the migration and invasion of human renal cancer cells (786-O cells) resorting to complementary cell-based methodologies. Firstly, noncytotoxic concentrations of TQ were determined using the crystal violet (CV) assay. TQ significantly decreased the collective migration of 786-O cells, according to the wound healing assay, although no effect was observed in terms of chemotactic migration (transwell assay, using FBS as the chemoattractant). Furthermore, TQ significantly reduced the invasiveness potential of 786-O cells, when compared with non-treated cells, using a transwell cell invasion assay. Overall, these results suggest that TQ has an important potential to counteract renal cancer cell migration/invasion anticipating further studies to unveil the mechanisms involved.
Keywords: Renal Cancer, thymoquinone, migration, invasion
Uterine adenocarcinomas in isopyrazam-treated rats occur via a mode of action that has no relevance to humans (#656)
P. P. Parsons1, R. C. Peffer2, R. Richards-Doran2, D. E. Cowie2, R. A. Currie2, K. Lichti-Kaiser3, E. McInnes2, D. C. Wolf3, P. Sawhney-Coder4, R. J. Handa5, D. R. Grattan6, K. D. Yi3
1 Exponent International, Human Health, Harrogate, United Kingdom
Female rats treated with 3000ppm (232.8 mg/kg BW/day) isopyrazam (IZM) for 2-years had a greater incidence of uterine adenocarcinomas, lesser incidence of pituitary and mammary tumors, and ~40% reduction in body weight (BW) gain; tumor incidence was comparable to control at 500ppm(34.9 mg/kg bw/day).
For this investigative study, female rats were treated with 0, 500 and 3000ppm IZM (28 and 194 mg/kg BW/day) for up to 18 months. Decreased fat pad weights and reduced BW gain followed a dose-responsive pattern. The high dose group cycled regularly for a longer period of time and had a greater percentage of rats in persistent estrus from weeks 52 to 80 compared to control. Decreased fat pad weights, increased liver weights and reduced BW gain followed a dose-responsive pattern. Prolactin and leptin levels were significantly reduced at 3000 ppm and dopamine signaling to the pituitary was greater than controls at ≥26 weeks.
A mode of action (MoA) has been established that begins with decreased food utilization, fat pad weight and BW gain, and consequently decreased leptin levels which caused subsequent alteration in hypothalamic signaling resulting in an altered pattern of transition into reproductive senescence. The greater time spent in persistent estrus and exposure to circulating estrogens leads to an increase in uterine adenocarcinomas. Onset of senescence in rats is due to altered signaling within the hypothalamus, while menopause and reproductive senescence in humans is driven by depletion of a pre-defined number of primordial follicles in the ovaries with aging. The fundamental physiological difference in control of the reproductive cycle and the transition into reproductive senescence between rats and humans indicate that the established MoA leading to uterine tumor formation in rats is not relevant to humans
Keywords: isopyrazam, uterine adenocarcinoma, mammary fibroadenoma, mode of action, carcinogenesis
The study of the cytotoxicity of zoledronic acid in a chronic experiment (#684)
R. V. Bogdanov1, V. Y. Afonin1, P. N. Lepeshko1, E. V. Chernyshova1
1 Republican unitary enterprise «Scientific Practical Center of Hygiene», Laboratory of Industrial Toxicology, Minsk, Belarus
The aim of the research was to study the cytogenetic effects of zoledronic acid under conditions of chronic inhalation exposure for 4 months in white rats. Cyclophosphamide was used as a positive control. In the experiment, concentrations of drugs at the level of 0.10, 0.05 and 0.01 mg/m3 were studied. At the end of the experiment, cytogenetic damage to nuclear cells was taken into account in washes of the lungs using light microscopy.
In the experiment, it was found that after two months of zoledronic acid administration, cytogenetic disorders (micronuclei in mono and polynuclear cells, bridges) increase depending on the concentration, while the maximum concentration (0.10 mg/m3) is 2 times higher than the control indicators (p<0.05). In case of administration of cyclophosphamide, an increase in cytogenetic disorders is observed at all studied concentrations (p<0.05), when exposed to a concentration of 0.10 mg/m3, 4 times the control values. In the second month of exposure, a high level of cell death of various types (interphase and reproductive) is observed against the background of seasonal activation of proliferation.
In the group of animals treated with zoledronic acid, at the 4th month of the experiment, a decrease in the number of cells with cytogenetic damage was noted by 4-6 times to the control level, which is explained by a decrease in the intensity of proliferation during this observation period. When exposed to cyclophosphomide, the number of cells with cytogenetic damage also decreased by 2-3 times, but significantly exceeded the values in the control group.
2 months after cessation of exposure to drugs, compared with the control, a high level of cells with cytogenetic damage remained only at a concentration of 0.1 mg/m3 cyclofasfamide (p<0.05), while at a concentration of 0.01 mg/m3 of zoledronic acid an increase was observed damaged cells (p<0.05).
As a result of the experiment, it can be concluded that cellular cytogenetic disorders during chronic inhalation exposure to zoledronic acid and cyclophosphamide are the result of direct and distant mutagenic action, as well as the effects of seasonal and compensatory mechanisms of cell replacement repair.
Keywords: zoledronic acid, сyclophosphamide, cytogenetic effects.
Improving the predictive performance of in silico aromatic amine mutagenicity alerts through the analysis of proprietary data. (#697)
R. E. Tennant1
1 Lhasa Limited, Leeds, United Kingdom
Aromatic amines are frequently used as building blocks in the synthesis of pharmaceutical products. Unfortunately, metabolic activation to DNA-reactive species results in aromatic amines being potentially mutagenic, which is often demonstrated by a positive result in the Ames test. In silico tools can be utilised to predict the potential mutagenicity of a query structure; however, aromatic amines are notoriously difficult to develop alerts for due to a combination of their complex structure-activity relationships and poor reproducibility of experimental results. We describe how physicochemical property relationships can be incorporated within alerts to refine the specificity of in silico predictions.
Alerts for aromatic amines have been developed based principally on data from publicly available literature and refined further using proprietary data. Despite refinements, validation studies indicate that the alerts generate many false positive predictions. Analysis of the physicochemical properties for a public and proprietary data set has previously identified a number of parameters which appeared to be important for determining absolute mutagenic activity of aromatic amines in the Ames test.
Previous analysis led to an in silico model containing 4 structural alerts for aromatic amines with molecular size restrictions to exclude compounds with a heavy atom count of >25. For a proprietary data set of 651 compounds, the predictive performance of the model is: specificity = 87.7%, sensitivity = 63.0% and accuracy = 82.2%, compared with the unrestricted model: specificity = 84.6%, sensitivity = 63.7% and accuracy = 79.9%. In this study we have re-analysed the 4 alerts to determine whether maintaining a size restriction is appropriate following modifications to the scope of the alerts. It was found that lowering the heavy atom count to exclude compounds with a heavy atom count >24 further improved specificity to 88.3% and accuracy to 82.6% while maintaining sensitivity. Overall, incorporation of this restriction increases specificity by 3.7 percentage points, resulting in an additional 19 compounds predicted correctly as Ames negative. This increase in specificity comes at a cost of a concomitant reduction of 0.7 percentage points in sensitivity, creating 1 false negative compound. Overall, the accuracy of the model was increased by 2.7 percentage points.
Keywords: in silico, alerts, mutagenicity
Genotoxicity of Synthetic Pyrethroids in Bone Marrow of Mice Micronucleus Test and Fluctuation of Ames Assay (#714)
O. Kravchuk1, N. Nedopytanska1, T. Tkachuk1, V. Bubalo1, O. Tkachuk1, O. Zubko1, O. Kostik1
1 L.I.Medved Research Center of Preventive Toxicology, Food and Chemical Safety, Ministry of Health, Ukraine, Kyiv, Ukraine
Pyrethroids are analogues of natural pyrethrins, first isolated from plants of the genus Pyréthrum, a family of astroids known for their insecticidal properties. We know synthetic pyrethroids (SP) about 70 years. Nowadays, there are a large number of SP have different insecticidal activity, and also can be used in combination with other chemical compounds. Study of genotoxicity is a mandatory of the toxicological assessment to justifying their safe usage in Ukraine. The Mammalian Erythrocyte Micronucleus Test in vivo (MN) and Ames test are recognized as most sensitive, widely and frequently used tests to identify genotoxic features.
The aim of the studies was to research the genotoxicity of 5 SP via MN in vivo and Ames assay. The tests are conducted following the SOPs, comply with GLP.
The MN was used to study mutagenic activity of 5 SP: Cypermethrin 94.0% at doses 46.0, 9.2, 1.84 mg/kg body weight, 2 samples of Alpha-cypermethrin - 94.0 and 94.7% in doses of 20.0, 2.0, 0.2 mg/kg, and 2 samples of Lambda-cyhalothrin - 95.2 and 97.1% in doses of 5.0, 1.0, 0.2 mg/kg. Acclimatization took 5 days before dosing. One treatment by gavage. Exposure time - 24 hours. We counted micronuclei in polychromatophilic erythrocytes (MNPCE) of bone marrow of mice (OECD 474).
Study of mentioned above 5 SP in fluctuation Ames assay using S.typhimurium TA98, TA100 w/wo S9, preincubation was 90 min. Selection of concentrations in Ames test were based on preliminary experiment which was performed before the main test. In the absence of cytotoxicity and precipitation in preliminary experiment the following concentrations (2.5; 0.5; 0.1; 0.02; 0.004; 0.0008 mg/ml ) were defined for all 5 SP.
As a result: obtained experimental data of positive and negative controls were ranged with own laboratories historical control. The results of experimental studies indicate that Cypermethrin in doses from 46.0 to 1.84 mg/kg body weight, 2 samples of Alpha-cypermethrin in doses from 20.0 to 0.2 mg/kg, and 2 samples of Lambda-cyhalothrin in doses of 1.0 and 0.2 mg/kg did not reveal a significant increase in the level of MNPCE. Both samples of Lambda-cyhalothrin in doses: 5.0 mg/kg b.w. induces statistically significant excess of the spontaneous frequency of MNPCE (p≤0.05). Ames assay results showed statistically significant absence of the mutagenic effect.
Keywords: genotoxicity, Ames assay, micronucleus test, lambda-cyhalotrin, pyrethroids
DNA damage signaling and genotoxic effects induced by complex mixtures of PAHs generated by biomass burning air particulate matter in human lung cells (#733)
M. F. de Oliveira Galvão1, I. Sadiktsis2, S. R. Batistuzzo de Medeiros3, K. Dreij1
1 Karolinska Institutet, Unit of Biochemical Toxicology, Institute of Environmental Medicine, Stockholm, Sweden
Most research concerning the effects of air pollutants on human health focuses on urban centers and on the role of vehicular and industrial emissions as major sources of pollution. However, approximately 3 billion people world-wide are exposed to air pollution from biomass burning1. Herein, particulate matter (PM) emitted from artisanal cashew nut roasting, an important economic and social activity worldwide2,3, was investigated. This study focused on: i) chemical characterization of polycyclic aromatic hydrocarbons (PAHs) and their oxy-PAH derivatives; ii) time-dependent activation of DNA damage signaling and genotoxic effects, and iii) differential expression of genes involved in xenobiotic metabolism, inflammation, cell cycle arrest and DNA repair using A549 lung cells. Among the PAHs, chrysene, benzo[a]pyrene (B[a]P), benzo[b]fluoranthene, and benz[a]anthracene showed the highest concentrations (7.8-10 ng/m3), while among oxy-PAHs, benzanthrone and 9,10-anthraquinone were the most abundant. Testing of PM extracts was based on B[a]P equivalent doses (B[a]Peq). IC50 values for viability was 5.7 and 3.0 nM B[a]Peq at 24 h and 48 h, respectively. Based on this, all other experiments were conducted at doses up to 2 nM B[a]Peq. At these low doses, we observed a dose-dependent activation of DNA damage signaling (phosphorylation of Chk1) and genotoxicity (double strand breaks). In comparison, effects of B[a]P alone was observed at micromolar range. To our knowledge, no other study has demonstrated an activation of pChk1, a biomarker used to estimate the carcinogenic potency of PAHs in vitro4, in lung cells exposed to biomass burning extracts. Persistent increased gene expression of several important stress response mediators of xenobiotic metabolism (CYP1A1, CYP1B1), inflammation (IL-8, TNF-α), cell cycle arrest (CDKN1A), and DNA repair (DDB2) was also identified. In conclusion, our data show high potency of biomass burning PM to induce cellular stress including genotoxicity, and more potently so when compared to B[a]P alone. Our study provides new data that will help elucidate the mechanism of lung cancer development associated with biomass burning. In addition, the results of this study support the establishment of new guidelines for human health protection in regions strongly impacted by biomass burning.
1. WHO. Household air pollution and health. (2018). Available at: https://www.who.int/en/news-room/fact-sheets/detail/household-air-pollution-and-health. (Accessed: 14th December 2018)
2. de Oliveira Galvão, M. F. et al. Characterization of the particulate matter and relationship between buccal micronucleus and urinary 1-hydroxypyrene levels among cashew nut roasting workers. Environ. Pollut. 220, 659–671 (2017).
3. de Oliveira Galvão, M. F. et al. Cashew nut roasting: Chemical characterization of particulate matter and genotocixity analysis. Environ. Res. 131, 145–152 (2014).
4. Dreij, K. et al. Cancer Risk Assessment of Airborne PAHs Based on in Vitro Mixture Potency Factors. Environ. Sci. Technol. 51, 8805–8814 (2017).
Keywords: Cashew nut roasting, Air particulate matter, Polycyclic Aromatic Hydrocarbons, DNA damage response, Genotoxicity
Genotoxic Safety Evaluation of Termite mushroom Termitomyces Albuminosus (#768)
J. H. Yoon1, Y. S. Kim1, E. A. Kwon1, J. W. Yun1, J. S. Park1, B. C. Kang1, 2
1 Seoul National University Hospital, Department of Experimental Animal Research, Biomedical research institute, Seoul, Republic of Korea
Termitomyces albuminosus, also known as Termite mushroom, has pharmacological effects such promotion of neuronal growth and prevention of age-related diseases, leading to its use as functional food or supplements. Despite its increasing consumption, there is a lack of comprehensive information regarding its safety. In this study, we conducted three genotoxicity tests to evaluate the safety of Termitomyces albuminosus powder (TAP): Ames test (OECD TG 471) using 4 strains of Salmonella typhimurium and 1 strain of Escherichia coli and chromosome aberration test (OECD TG 473) using Chinese Hamster Lung fibroblast (CHL) cell line for evaluation of clastogenic potential, and In vivo micronucleus test using ICR mice (OECD TG 474). TAP did not induce reverse mutations in all the bacterial strains used in this study nor chromosomal aberrations in CHL fibroblasts at concentrations up to 5000 μg/plate regardless of S-9-mediated metabolic activation. In ICR mice, no changes were noted in clinical signs and body weight in the groups treated up to 3000 mg/kg of TAP. There was no statistically significant increase in the frequencies of micronucleated polychromatic erythrocytes at any TAP-treated groups compared with the negative control group (6.0±1.58 for TAP vs 5.2±0.84 for the vehicle control, p>0.05) and in the ratio of polychromatic erythrocyte to total erythrocytes (53.9 ± 6.48 % for TAP and 47.0 ± 5.24 % for the vehicle control, p>0.05). In conclusion, our data from the standard battery of genetotoxicity tests demonstrated that TAP has no genetotoxic potential under the conditions of this study. These findings, together with the recent subchronic toxicity test results, suggest that Termitomyces Albuminosus is a safe and nontoxic for human consumption when appropriately used.
Keywords: Termitomyces Albuminosus, Termite mushroom, genetic toxicity, Ames test, chromosome aberration test; micronucleus test
Development of a Biomolecular Data Computing environment for computer predicted adverse outcome pathways: benzene as a case study (#771)
S. Krishnan1, M. van Stee1, S. Ruiter1, J. Westerhout1, B. Schaddelee-Scholten1, R. Stierum1
1 TNO, Healthy Living, Zeist, Netherlands
Purpose: A Biomolecular Data Computing infrastructure (BDC) is being developed to enable a top down toxicoinformatics modelling approach that is data driven from external exposure to molecular pathways to health effects. Here, the application is demonstrated to propose adverse outcome pathways invoked by benzene, known to cause acute myeloid leukemia (AML).
Keywords: benzene, chromosome aberrations, adverse outcome pathways, computational toxicology, KNIME
Combined Pig-a, Micronucleus and Comet: An In Vivo Genotoxicity Assessment of Benzene and Cyproterone Acetate Using a Triple Endpoint Approach (#792)
D. A. Kidd1, L. Jordan1, R. Smith1, J. Whitwell1, J. Clements1
1 Covance Laboratories Ltd, Genetic Toxicology, Harrogate, United Kingdom
Cyproterone Acetate (CYP) is a synthetic sex steroid derived from hydroxyprogesterone and known carcinogen. Existing data (1, 2) have shown it induces Liver DNA adducts, gene mutation and tumours (at high dose levels only, predominantly female rats), negative for mouse micronucleus (single dose regimen), negative in vitro for HPRT, Ames and chromosome aberrations. Currently no in vivo gene mutation data exists for extended treatments.
Benzene (BZN) is a genotoxic carcinogen. Genotoxicity data indicate that BZN (including metabolites) are Ames negative but clastogenic and aneugenic, producing micronuclei, chromosomal aberrations, sister chromatid exchanges and DNA strand breaks (3).
Using a 28 day dosing regimen, female rats were treated with CYP (50, 100 & 200 mg/kg/day) and male rats treated with BZN (500, 1000 & 2000 mg/kg/day) and tested for induction of gene mutations (Pig-a assay), micronucleus (peripheral blood) and gross DNA damage in the Liver (Comet assay). Groups of 5 animals were dosed with vehicle, test article (see table). A mutagen control (ENU @ 20 mg/kg/day) was included. Blood was collected Days -1, 15, 29 (Pig-a) and Day 28 (micronucleus). Liver was sampled for Comet 3 hours post the last administration.
Results showed that CYP was negative for the two Pig-a endpoints (RBCs and RETs), positive for micronucleus and that the Comet endpoint was unscorable (due to high proportion of diffused cells). BZN was negative for the two Pig-a endpoints (RBCs and RETs), positive for micronucleus and weakly positive for the Comet endpoint. ENU was positive for all endpoints analysed.
The CYP pig-a mutation data was negative following dosing to 200 mg/kg/day (a dose that showed extensive toxicity to the liver). Concentrations selected for testing were based on published data for MN and mutation data from acute dosing studies (1) and a true MTD not achieved.
BZN a known genotoxic carcinogen produced the same profile as that for CYP, providing support for existing data that CYP may be a genotoxic carcinogen. Further testing at higher doses in the pig-a assay may be required to elucidate the true mutagenic profile.
1) Kasper P, Pharmacology & Toxicology, 2001, May;88(5), 223-31.
2) Rabe T et al., Drug Safety, 1996, January;14(1), 25-38.
3) Kitamoto S et al., Mutation Research. Genetic Toxicology and Mutagenesis, 2015, July;776-778, 137-43.
Keywords: Pig-a assay, in vivo micronucleus, in vivo Comet assay, Benzene, Cyproterone acetate
Use of (Q)SAR models to investigate potential CMR properties of e-liquid ingredients (#806)
D. Zarini1, S. Zucchi1, I. Trampolin2, A. Orro3, E. Ferri1
1 Trusticert srl, Milan, Italy
Electronic cigarettes (e-cigs) are designed to heat and aerosolized mixtures of propylene glycol, glycerol, flavorings, humectants and, optionally, nicotine. Unlike cigarettes, the process involves no tobacco and no combustion; however, the inhalation and exhalation of vapour is reminiscent of smoking.
In this context, the use of these devices, might play an important role in smoking cessation and reduction; however, there is still a lack of international consensus over the public health role of the e-cig.
Despite the large use of e-cigs, still few toxicological studies are available on the potential long term effects of inhaled of many characterizing flavors used in e-cig products.
For instance, the FDA GRAS (Generally Recognized As Safe) designation for some flavorings compounds and for propylene glycol, does not apply to inhalation, and currently, there are no controlled long-term studies of the effects of inhaling heated aerosolized mixture in humans.
Thus, there is legitimate concern over the health effects of chronically inhaling these substances and the lack of toxicological studies.
In this respect, the aim of this study was to determine potential Cancerogenic, Mutagenic and Reprotoxic (CMR) properties of several e-liquid ingredients by means of in silico methods.
With reference to our e-liquid ingredients and CMR effects, we first conducted an in depth screening, through the literature reviews; and we found experimental data gap for all the three categories.
Specifically, for the investigated e-liquid ingredients, we observed 35%, 85% and 70% of experimental data gap for Cancerogenicity, Mutagenicity and Reprotoxicity effects, respectively.
By following a battery approach, almost all data gaps were successfully filled using Quantitative Structure-Activity Relationship (Q)SAR methods. The predictions were performed using several open source software (VEGA, Toxtree, ToxRead and T.E.S.T.) and the results were combined to obtain the highest possible prediction accuracy (consensus approach).
This in silico study is a part of a broader integrated approach (literature research, in chemico, in vitro and computational analysis) specifically designed to assess the potential risk associated with characterizing flavors and e-liquid ingredients.
Keywords: Toxicity, QSAR, Electronic Cigarettes, chronic toxicity, Human health
Evaluation of the MultiFlow platform to supplement the in vitro micronucleus test with genotoxic Mode of Action-based information (#857)
F. Van Goethem1, K. De Vlieger1, S. Bryce2, J. Bemis2, N. Hall2, J. Van Gompel1, S. Dertinger2, A. De Smedt1
1 Janssen Pharmaceutica, Nonclinical Safety, Beerse, Belgium
The identification of genotoxic hazard is part of the routine test battery in pharmaceutical development. The objective is to identify direct DNA-damaging agents as well as those with different modes of action (MoA) like aneugens. The in vitro micronucleus (MN) test is routinely used to screen compounds for the induction of in vitro chromosomal damage. However, MN induction is not always indicative of a DNA reactive MoA, and determining the causal molecular pathway can be a relevant driver to mitigate the genotoxic flag. This MoA information is also highly useful when managing potential risk in supporting the Discovery teams.
Here we describe the evaluation of the MultiFlow (MF) platform, a DNA damage-based assay which multiplexes p53, γH2AX, phospho-histone H3, and polyploidization biomarkers into a single flow cytometric analysis. Human TK6 cells were exposed to 50 pharmaceutical internal test compounds (containing DNA reactive clastogens, tubulin poisons, kinase inhibitors, and non-genotoxicants) for 24 hr over a range of concentrations. Cell aliquots were removed at 4 and 24 hr for analysis and multiplexed response data were evaluated using 3 machine learning-based models which classified each compound as clastogenic, aneugenic or non-genotoxic. In addition, fold increases in biomarkers against global evaluation factors (GEFs) were also considered.
The obtained classifications were compared with the outcome of the in vitro MN test in TK6 cells. Of the 50 compounds tested, 24/26 and 18/24 showed comparable positive and negative outcomes respectively. The real value of the MF approach was demonstrated by the fact that the MoA-based classifications (signatures) correspond with the known profiles of the selected compounds. A detailed analysis of the MF responses often indicated atypical profiles for those compounds not inducing MN; observations that trigger additional work to explore deeper levels of information including aneugen molecular initiating events, discriminating primary vs. secondary DNA damaging agents, and the need for extended recovery designs in the MN test.
Overall, the obtained data show the strategic importance of understanding the underlying MoA driving MN induction, information which is needed to prioritize and select the most promising drug candidates in Discovery.
Keywords: Multiflow, genotoxicity, micronucleus
Validation of the in vivo comet assay in various organs of Wistar rats (#876)
C. Freitag1, M. Eberl2, H. Gehrke1
1 Eurofins BioPharma Product Testing Munich GmbH, In vitro Department of Pharmacology and Toxicology, Planegg, Bavaria, Germany
The in vivo alkaline Comet Assay (OECD Test Guideline 489) also known as single cell gel electrophoresis assay is a sensitive method for the detection of DNA single and double-strand breaks as well as alkali labile sites in different organ tissues. It can be performed as part of the standard testing battery for pharmaceuticals integrated in repeated dose studies or as a follow-up to a positive result in an in vitro clastogenicity test for industrial chemicals.
According to the validation trial from 2006-2012 coordinated by the Japanese Center for the Validation of Alternative Methods (JaCVAM), historic control data were only obtained for the two organs liver and stomach. As also different tissues can be of interest concerning the mode of action of substances, we established this technique 2017 in our lab and did an internal validation study investigating the dose-dependent effects of ethyl methanesulfonate (EMS) as positive control at three concentrations in several tissues like lung, glandular stomach, liver, small intestine (jejunum), colon, kidney and spleen.
The test groups consisted of 3 male rats. As negative control 0.9% physiological saline was applied and for the positive control 100, 200 and 300 mg EMS per kg body weight (bw) were selected as appropriate concentrations. 4 h after the application, the animals were sacrificed one by one and the organs of interest were removed. Each organ was processed independently by three people to investigate the inter-analyst variability. The single cells were embedded in agarose and lysed overnight to remove cell membranes. After an unwinding step of the DNA in a high alkaline solution, electrophoresis was performed. The slides were dried in ice-cold ethanol and stained with GelRed® solution. 50 cells per slide were evaluated from two different scorers. Overall, 504 slides were analysed obtained from 3 different analysts, which processed 7 organs and prepared two slides for each of it from 12 animals in total.
Keywords: Comet Assay, in vivo
Applicability of in-silico tools to predict mutagenic activity of pesticides (#885)
F. Frenzel1, K. Herrmann1, A. Holzwarth1, S. Rime1, B. Fischer1, P. Marx-Stoelting1, C. Kneuer1
1 German Federal Institute for Risk Assessment, Department of Pesticides Safety, Berlin, Berlin, Germany
In-silico studies based on computer-aided prediction models or (Q)SAR tools are increasingly submitted for the purpose of authorisation and/or approval of pesticides. For regulatory authorities, it is thus indispensable to develop a detailed understanding of the potential and the limitations of such in-silico tools. The correct output of (Q)SAR tools depends on model assumptions and on the underlying data. Whereas chemical structures of pharmaceuticals are already implemented in the training datasets, molecular structures of pesticides and their metabolites are thought to be underrepresented, raising questions about the regulatory readiness of the respective (Q)SAR tools.
Keywords: in silico, QSAR, mutagenicity, pesticides
Expression of γ-Glutamyltransferase in rats` hepatocytes after carbendazim exposure (#909)
V. Lisovska1, E. Bagley1, N. Nedopytanska1, O. Reshavska1
1 L.I.Medved`s Research Center of Preventive Toxicology, Food and Chemical Safety, Ministry of Health, Kyiv, Ukraine , Kyiv, Ukraine
It was shown that carbendazim (methyl 1H-benzimidazole-2-yl carbamate (CAS)) leads to pathological changes in the liver, including neoplastic. Disturbance of metabolic processes in cells occurring in the process of carcinogenesis revealed by changes of enzymes activity that can be considered as preneoplastic and tumor markers.
γ-Glutamyltransferase (γ-GGT) enzyme is one of the classical markers of preneoplastic changes in population of hepatocytes. According to data obtained in chronic experiments carbendazim classified as a carcinogen of the C group (a possible carcinogen for a human) that causes a hepatocarcinogenic effect.
The aim of this study was to investigate the expression of γ-GGT in hepatocytes of rats under the action of a generic 98 % carbendazim.
The experiment was done using the "NDEA-hepatectomy" model proposed by N. Ito et al. which is based on the initiation of hepatocytes by a single intraperitoneally injection of N-nitrozodiethylamine (NDEA) at a dose of 200 mg/kg associated with partial hepatectomy. The study was conducted on 75 male Wistar rats of 200 - 250 g b.w., 15 rats for each group: 1 – negative control, water; 2 - positive control, phenobarbital in the dose 37.5 mg/kg; 3, 4, and 5 - carbendazim at doses of 25, 75 and 300 mg/kg. The test substance was administered to animals by oral gavage for 8 weeks. After the end of the exposure period, all rats were sacrificed and liver slices are fixed in ice-cold acetone for examination of GGTase.
The results of the study showed that carbendazim in a dose of 25 mg/kg caused no effect; in a dose of 75 mg/kg - the number of γ-GGT positive foci significantly increased per slide unit area (cm2). Also, the tendency to increase of the area γ-GGT positive foci were noted (p = 0.06). At dose 300 mg/kg (1/5 LD50) the number and area of foci did not differ from the negative control, this may indicate the activation of compensatory mechanisms of body self-regulation. Consequently, the dynamics of the increase in the number of hepatocytes expressing γ-GGT is paradoxical in terms of the effect in small doses.
Keywords: Carbendazim, γ-Glutamyltransferase, preneoplastic markers, hepatocytes, rats
In vivo evaluation of glyphosate genotoxicity (#911)
V. Karzi1, P. Stivaktakis1, M. Tzatzarakis1, E. Vakonaki1, C. Chalkiadaki1, A. Kalliantasi1, P. Apalaki1, M. Panagiotopoulou1, I. Tsatsakis1, I. Fragkiadoulaki1, A. Tsatsakis1
1 University of Crete, Laboratory of Toxicology and Forensic Sciences, Medicine School, Heraklion, Greece
Background: Glyphosate is the active substance of the widely used herbicide Roundup®. Because of its ubiquity in the environment, humans get exposed to glyphosate daily in low or high doses, posing a real life risk for human health.
Keywords: glyphosate, long term exposure, genotoxicity, CBMN
Prognostic efficacy of cellular test-models in assessing the mutagenicity of chemicals (#954)
M. Anisovich1, N. Dudchik1, I. Ilyukova1
1 Republican unitary enterprise «Scientific practical centre of hygiene» , Ministry of Health, Minsk, Belarus
Research on the mutagenic properties of chemical compounds in cell cultures is a promising alternative to replace animal testing. But at this time there is a problem of insufficient reliability of the results. The mutagenic properties found in in vitro tests are not always confirmed in animal experiments.
Keywords: mutagenic properties, cell cultures, prognostic efficiency in vitro tests, cytofluorimetric methods
Cytoprotective autophagy protects cardiac cells from Dichlorvos-induced ER stress and necroptosis (#24)
I. Ben Salem1, 2, M. Boussabbeh3, 2, J. Pires Da Silva4, H. Bacha2, S. Abid2, C. Lemaire4
1 University of Sousse, Faculty of Medicine , Sousse, Tunisia
Dichlorvos (O,O-dimethyl-2,2-dichlorovinyl phosphate; DDVP) is an organophosphate pesticide (OP) that is widely used for the control of agriculture and animals pests. In the present study, we investigated the underlying mechanism of DDVP-induced toxicity in cardiac cells.
We show that 24h treatment with DDVP inhibits the growth of cells by inducing necroptosis. In fact, DDVP treatment upregulated RIP1 expression and chemical inhibition of RIP1 kinase activity by necrostatin-1 (Nec-1) protected the cells from death. After a short-time of treatment (6h) with DDVP, an increase in the level of Beclin-1 and LC3-II and an accumulation of the CytoID® autophagy detection probe were observed. In addition, inhibition of autophagy by chloroquine (CQ) significantly increases DDVP-induced necroptosis, suggesting that activation of autophagy is a cardioprotective mechanism against the toxicity induced by this OP.
We also demonstrate that inhibition (EX527) or knockdown (siRNA) of the deacetylase sirtuin 1 (SIRT1) significantly increases necroptosis induced by DDVP, whereas SIRT1 activation by resveratrol (RSV) greatly prevents the cytotoxic effects of this pesticide. When autophagy was inhibited by CQ, the protective effect of RSV against the cardiotoxicity of DDVP was not observed. Altogether, our results suggest that activation of SIRT1 protects cardiac cells from the toxicity of DDVP by an autophagy-dependent pathway.
Keywords: Dichlorvos, ER stress, Necroptosis, autophagy, SIRT1
Dried blood spots combined to an UPLC–MS/MS method for thesimultaneous determination of antihypertensive drugs in forensic toxicology (#25)
Y. Zhang1, J. Chang1, X. Wu1, L. Dong1
1 People's Republic of China - Institute of Forensic Science, Ministry of Public Security, Beijing, China
A method for the simultaneous determination of 4 antihypertensive drugs(Nimodipine, Valsartan, Arotinolol, Indapamide), using the dried blood spot (DBS) sampling technique combined with the UPLC–MS/MS technology was developed to study its applicability within the forensic toxicology. The DBS samples, prepared from a blood volume of 50 mL and using the bloodstain cards, were extracted with a methanol/acetonitrile mixture. The chromatographic separation was performed using an ACQUITY®HSS C18( 50 mm×2.1 mm, 1.8 μm ) and an acetonitrile/Water (0.1% formic acid) gradient. The detection was accomplished with a TQ Detector, operating in the ESI+ and MRM modes. The method was validated in terms of selectivity, matrix effect, extraction recovery (46%–106%), carryover, LOD and LOQ (0.2–0.8 ng/mL and 1–4 ng/mL, respectively), linearity (LOQ to 100 ng/mL), intraday and interday precision (2.6–13% and 4.1–14%, respectively), accuracy (2.3% to 4.8%) and dilution integrity. An eight months stability study at room temperature, 2–8 ◦C and−10 ◦C, was also performed, with the best results obtained at−10 ◦C.
The procedure was applied to 50 real samples. The results were compared with the methodologies routinely applied in the laboratory and the statistical analysis allowed to establish an acceptable correlation. This study permitted to determine that the DBS can represent an alternative or a complement to conventional analytical and sampling techniques, responding to some of the present issues concerning the different forensic toxicology applications
Keywords: dried blood spot, Antihypertensive drugs; Ultra performance liquid chromatography - tandem mass spectrometry
Assay of Staphylococcal Enterotoxin B by a QCM Biosensor (#26)
1 University of Defence, Faculty of Military Health Sciences, Hradec Kralove, Czech Republic
Staphylococcus aureus is a causative agent of infectious diseases and a producer of highly toxic proteins called Staphylococcal enterotoxins. In the current analytical praxis, presence of staphylococcal enterotoxins is assayed by standard immunochemical techniques or by instrumental methods based typically on the combination of chromatography and mass spectrometry. Despite availability of the current methods, they are not fully suitable for a simple, label-free, mode of use by an unskilled worker. In this study, a biosensor based on quartz crystal microbalance (QCM) sensor is performed as a tool for simple and reliable determination of Staphylococcal Enterotoxin B (SEB) which is one of the most important staphylococcal enterotoxins.
QCM sensors with 10 MHz basic frequency of oscillations and gold electrodes were used in this study for biosensor construction. The biosensor contained antibody against SEB immobilized through protein A on the QCM sensors and the interdigitated layer was stabilized by iron nanoparticles. The assay worked as a label-free and sample was applied without any pretreatment. Enzyme-Linked Immunosorbent Assay (ELISA) served for validation purpose.
The biosensors were tested for standard solution of SEB and calibration curve was prepared. In the assay, limit of detection 113 µg/ml was reached for a sample sized 50 µl. The assay by biosensors was compared to standard ELISA and correlation between the two methods was done resulting in coefficient of determination 0.993. Long term stability was also tested for an interval 90 days. In the end of the interval, the biosensors were able to provide approximately 85 % of the initial sensitivity.
In a conclusion, the biosensors for SEB assay appears as a simple but reliable tool to be performed in field conditions, protection against warfare use of toxins etc. The facts that the assay is cheap and label free without any necessity to apply any reagents are significant advantages.
A long-term organization development plan “Medical Aspects of Weapons of Mass Destruction” and Specific research funds (Faculty of Military Health Sciences, University of Defense, Czech Republic) is gratefully acknowledged.
Keywords: immunosensor, piezoelectric, quartz crystal microbalance, SEB, staphylococcal enterotoxin
Solid-phase microextraction as a universal tool for quantitative in vitro-to-in vivo extrapolation studies (#59)
L. Henneberger1, M. Mühlenbrink1, B. Escher1, 2
1 Helmholtz Centre for Environmental Research - UFZ, Cell Toxicology, Leipzig, Saxony, Germany
Reducing and eventually replacing animal tests by in vitro bioassays requires the quantitative extrapolation of effect data generated with in vitro test systems to whole organisms (quantitative in vitro-to-in vivo extrapolation, QIVIVE). QIVIVE models usually compare the nominal effect concentrations of the chemicals in the in vitro bioassays with total plasma concentrations in vivo. However, other dose metrics have been suggested that account for differences in bioavailability of the chemicals in vitro and in vivo due to different composition of e.g., cell culture media and human plasma. A better comparison is possible if freely dissolved concentrations in the assay medium (Cfree,medium) and in plasma (Cfree,plasma) are used. In this study we want to demonstrate that solid-phase microextraction (SPME), a widely used sample preparation technique, can support QIVIVE studies in many different aspects. SPME has been applied in previous studies to determine partitioning in diverse biological phases from bovine serum albumin and phospholipid liposomes to complex matrices like cell culture media and plasma. We demonstrated that SPME cannot only generate partitioning data that are required as input parameters for mass balance models used to predict Cfree,medium and Cfree,plasma but can also be used for the time-resolved experimental quantification of Cfree,medium in cell-based in vitro bioassays and to determine Cfree,plasma in plasma samples from different species. We found that Cfree,medium in in vitro test systems can be several orders of magnitude lower than the nominal concentration (Cnom) and was not necessarily linearly related to Cnom. In human plasma Cfree,plasma was lower than Cfree,medium at the same Cnom, which can be explained by the fact that human plasma has more proteins and lipids than commonly used cell culture media. By comparing Cfree,plasma determined in human and trout plasma we found similar values for neutral and basic chemicals, but differences of several orders of magnitude for several acidic chemicals. These results emphasise the need to account for bioavailability for successful QIVIVE and that SPME may be used as a universal experimental tool that improves our understanding on how chemicals distribute in vitro and in vivo.
Keywords: IVIVE, free concentration, protein binding, mass balance models, PBTK models
Generation of proliferating mouse hepatocytes (upcyte® mouse hepatocytes) (#60)
N. Nagy1, T. Evenburg1, S. Rohrmoser1, A. Noerenberg1, T. Johannssen1
1 upcyte technologies, Hamburg, Germany
The concern about the use of laboratory animals is increasing and leads to the support of alternative methods. Laboratory mice are frequently used for gene knockout studies in vivo. Additionally, isolated mouse cells are an appropriate tool for gene knockout studies on a cellular level. However, the use of primary mouse cells is hampered by e.g. short culture longevity, the limited quantity of cells that can be isolated from one mouse and the lack of proliferation capacity.
Since we have successfully generated several human upcyte® cells (e.g. upcyte® hepatocytes), the feasibility of the upcyte® technology on other species is of interest. Here, we show for the first time that the transduction of proliferation-inducing genes could extend the lifespan of primary mouse hepatocytes without losing their primary characteristics. For this purpose, primary mouse hepatocytes from three wildtype (WT) and three knockout (KO) C57BL/6 mice were isolated and subsequently transduced with upcyte® proliferation genes.
Murine hepatocytes were isolated from three wildtype (mouse donor number 16, 21 and 22) and three knockout (mouse donor number 17, 23 and 24) C57/BL6 mice using a two-step collagenase perfusion technique. Primary cells were transduced and cells were monitored for proliferating spots of hepatocytes.
After 13 days proliferating cells were visible, whereas only senescent cells where found in untransduced control wells. For all six mice proliferating upcyte® cells were found. All six upcyte® mouse hepatocytes were analysed for their morphology and for the expression of mouse hepatocyte marker proteins.
In conclusion, the upcyte® technology can be used to generate proliferating mouse hepatocytes from wildtype and knockout mice, while retaining their phenotype. The resulting cells called “upcyte® mouse hepatocytes” express hepatocyte markers such as CK8, CK18 and MSA. Thus, the upcyte® technology can contribute to the 3Rs concept and provide a suitable tool for knockout studies on a cellular level.
Keywords: in vitro toxicology, hepatocyte, liver, toxicology, mouse
Analysis of hepatotoxic mixture effects of pesticides in vitro (#89)
A. Braeuning1, D. Lichtenstein1, A. Mentz2, F. Schmidt3, J. Kalinowski2, O. Pötz4, 3, A. Lampen1
1 German Federal Institute for Risk Assessment, Food Safety, Berlin, Berlin, Germany
Consumers are exposed to mixtures of different contaminants and pesticide residues via the diet. This raises questions with respect to potential adverse health effects, especially when several compounds of a mixture exert toxicity by a similar mode of action. Efficient testing strategies for the nearly infinite number of combinations of chemicals are lacking. In addition, reduction of animal testing in toxicological risk assessment is a societal need. Consequently, it is important to establish in vitro tools to assess possible mixture effects.
A mixture testing strategy for different chemical groups of pesticides was developed by compiling an in vitro assay toolbox of different cellular hepatotoxic effect markers, together with PCR array-based expression analysis of hepatotoxicity-related transcripts and mass spectrometry-based determination of changes in protein expression. Human HepaRG liver cells were used as a metabolically competent in vitro system of human liver.
Following initial screening analyses with individual compounds, mRNA and protein hepatotoxicity marker patterns were determined for approximately 30 different pesticidal active compounds. Bioinformatic data evaluation was applied to deduce the hepatotoxic similarities as well as individual potencies of the compounds. The correlation between mRNA and protein marker deregulation and cellular triglyceride accumulation was established. Based on these results, effects of equipotent mixtures of compounds with similar or dissimilar modes of action were measured over a broad concentration range. In summary, we describe an in vitro toolbox for pesticidal active compounds in human cells for a testing of hepatotoxic mixture effects.
Keywords: hepatotoxicity, pesticides, in vitro testing, liver cells, mixture toxicity
Evaluation of primary human corneal epithelial cell lines from three different suppliers for use in in vitro mechanistic studies. (#95)
C. Taylor1, M. Burman1, M. Vidgeon-Hart1, P. McGill2
1 GlaxoSmithKline R&D, MSD, Ware, United Kingdom
An antibody drug conjugate (ADC), in development within GSK as an anti-cancer therapeutic, is currently undergoing clinical trials. Preliminary results have shown adverse ocular events involving the cornea, an effect reported with similar ADCs. Primary human corneal epithelial (PHCE) cells from three suppliers (ScienCell, MatTek and American Type Culture Collection (ATCC)) were evaluated as potential in vitro models to investigate the mechanism of toxicity.
Characterisation experiments were performed on the PHCE cells from each supplier, applying a range of endpoints: phenotypic markers for corneal epithelial and stem cells (by immunocytochemistry (ICC) and quantitative polymerase chain reaction (QPCR)), electron microscopy (EM) and assessment of doubling times. Optimisation/standardisation of culture conditions (e.g. seeding density, culture vessels, extracellular matrix (ECM)) was also conducted. To standardise laboratory methods, one dissociation method was optimised and seeding densities for sub-culturing were standardised to approximately 4000-6000 cells/cm2.
The characterisation studies confirmed the PHCE cells evaluated expressed a range of phenotypic markers, consistent with corneal epithelium markers reported in literature (e.g. positive expression via ICC of cytokeratin 3 and 19 and positive gene expression (QPCR) of Involucrin, Integrin a9, ABCG2 and p63). A 48-hour doubling time was defined for PHCEs from all suppliers. Culture on Thermanox coverslips coated with a range of ECMs (e.g. Matrigel, laminin, poly-l-lysine) showed no overt morphological differences, or changes in growth dynamics, when compared to those cultured on uncoated coverslips. ScienCell cultures sporadically developed a spindle-like phenotype (potentially batch related), which did not express any corneal epithelial markers, therefore further characterisation work with these cells was not pursued. Although the three suppliers recommended slightly different methodology, we are now using consistent culture conditions and have initiated the mechanistic work using the MatTek and ATCC-sourced PHCEs.
Keywords: Corneal, Epithelial, In vitro, Antibody-Drug-Conjugate
In Vitro Assays in a High-throughput Screening Platform – Strengths and Challenges (#97)
1 NCATS/NIH, Rockville, Maryland, United States of America
The US Tox21 (Toxicology in the 21st century) effort represents a paradigm shift in toxicity testing of chemical compounds from traditional in vivo animal tests to less expensive and higher throughput in vitro assays that are based on target-specific mechanisms and biological observations. To assess the toxicological effects of hundreds of thousands of the environmental chemicals, a quantitative high-throughput screening platform has been utilized to profile hundreds of thousands of environmental chemicals using a battery of in vitro cell-based assays. The millions of data points generated from the primary screening were made publicly accessible. These rich datasets provide researchers with opportunities for further data mining, understanding compound action, and prioritizing compound for further in-depth studies. One of the critical components of screening in toxicological research is data quality and reproducibility. This presentation will focus on assay optimization, validation, and screening performance. The screening technology and case study will be presented to illustrate the screening strengths and weaknesses. The challenge of developing in vitro assays with more physiological relevance will also be addressed and discussed.
Keywords: In vitro assays, Tox21
Two Read-across Case Studies Using IATA Approach Focused on Biological Similarity (#99)
M. OKamoto1, S. Nakagawa1, Y. Nukada1, O. Morita1
1 Kao Corporation, R&D - Core Technology - Safety Science Research, Akabane, Japan
Integrated Approaches to Testing and Assessment (IATA) is one of the appropriate approaches to establish reasonable read-across strategy. However, it is still unclear how the biological similarity considering adverse outcome pathway (AOP) affects the prediction of toxicity. Therefore, we verified the usefulness of considering the AOP-related biological similarity by using IATA-based read-across.
p-Alkylphenols and Chlorobenzenes were chosen as the target categories in this study. First, ADME and putative AOP for the category members were organized by in silico simulation or the published information. Next, AOP-related biological endpoint were examined by in chemico or in vitro tests. In case of p-Alkylphenols, chemicals were applied for GSH trapping test with metabolic reaction and cytotoxicity assay. In case of Chlorobenzenes, chemicals were applied for microarray to know the intracellular responses broadly. And then NO(A)ELs of target chemicals were decided by considering the similarity of biological responses.
Regarding p-Alkylphenols, reactive metabolite quinone methides (QMs)-induced hepatotoxicity is hypothesized as a major toxicological effect. As a result of the metabolite prediction by in silico tools, there was the difference of QMs production among the 4-position's complexity. Additionally, the p-Alkylphenols with complicated structure at 4-position produced lower amount of GSH adducts with metabolite and showed high cell viability. These results suggested that the potency of hepatotoxicity depended on the metabolic reaction of 4-alkyl structures in p-Alkylphenols, though all the members were structurally similar. Regarding Chlorobenzenes, parent chemicals and their metabolites were known to cause hepatotoxicity through several stress responses. As a result of microarray, a part of category chemicals induced similar biological responses including β-oxidation, mitochondrial disorder. in conclusion, it is suggested that AOP-based biological response could support prediction of the tendency and similarity of toxicity, which could not be inferred just from chemical/physical similarities.
Keywords: Read-across, IATA, AOP, similarity of biological response
Toxicological comparison of cigarette smoke and next generation product aerosol bubbled extracts using High Content Screening (#100)
E. Trelles Sticken1, R. Wieczorek1, L. M. Bode1, L. Simms2, M. Stevenson2
1 Reemtsma Cigarettenfabriken GmbH, An Imperial Brands PLC Company, BioToxLab, Hamburg, Hamburg, Germany
Smoking is a cause of serious disease in smokers. Tobacco-based and tobacco-free next generation products (NGPs) are understood to be a less harmful alternative to cigarettes, thereby creating a huge global public health opportunity if significant numbers of adult smokers fully switch.
The objective of this study was to compare the in vitro biological response of Phosphate Buffered Saline (PBS) which had either cigarette smoke or a selection of NGP aerosols bubbled through it. The in vitro response of each extract was determined in Normal Human Bronchial Epithelial cells using high content screening after 4 and 24 hours exposures. Products investigated were the Kentucky reference cigarette (3R4F), a tobacco heating product (THP), a hybrid product (HYB) and a myblu™ e-cigarette (Tobacco Flavour 1.6% Nicotine).
The 3R4F and THP were smoked using the Health Canada Intense method. HYB and myblu were vaped according to CORESTA Recommended Method N°81. The smoke and aerosols were bubbled through a series of impingers containing PBS. For every test day, fresh PBS solutions with 1.8 puffs/ml and 4 puffs/ml for the 3R4F and NGP samples respectively were produced. Chemical analysis of the 3R4F PBS solutions detected nicotine with an average of 86 ± 12µg/ml. The three NGP solutions contained nicotine levels from 70 ± 1µg/ml (HYB), over 150 ± 17µg/ml (THP) to 175 ± 17µg//ml (myblu).
The 3R4F bubbled PBS caused a significant dose dependent decrease in cell count and significantly altered y-H2AX, NfKB, p-c-Jun and cell count endpoints (at concentrations >1%). A partial overlap with endpoints induced by the THP solution was observed at concentrations considerably higher than 3R4F. By contrast, myblu and HYB extracts did not induce any significant activity in all the parameters tested at the maximum use concentration (10%).
This data suggests that the extracts from NGPs elicit little to no in vitro biological activity, even at higher exposure concentrations, compared to combustible cigarettes under the conditions tested.
Keywords: Next Generation Product, High content screening, Aerosol, E-Cigarette, in vitro
Evaluation of the biological effects of tobacco vapor and cigarette smoke using three-dimensional-reconstructed tissue from human airways (#106)
T. Kuarchi1, K. Yoshida1, S. Ishikawa1
1 Japan Tobacco Inc., Scientific Product Assessment Center, Yokohama, Kanagawa, Japan
The popularity of e-cigarettes and heated tobacco products is increasing worldwide. Our novel tobacco vapor product (NTV) is one of these heated tobacco products, and studies on NTV vapor have shown that their vapor has a greatly reduced level of several harmful and potentially harmful constituents (HPHCs) compared with that from cigarette smoke (CS), with approximately 99% reduction. The objective of this study was to evaluate the biological effects of NTV vapor on three-dimensional reconstructed human airway tissue in comparison with that elicited by CS. The tissue was primary human bronchial epithelial cells differentiated at an air–liquid interface and exposed to NTV vapor or CS from a 3R4F reference cigarette using a whole-smoke exposure system. The HPHC level in NTV vapor was much lower than that in CS, so the tissue was exposed to undiluted NTV vapor to achieve the highest concentration of exposure. Conversely, CS was diluted with clean air for exposure to match the concentration of total particulate matter in CS with the concentration of aerosol-collected mass in NTV vapor. The biological effects on the tissue were assessed by measurement of cytotoxicity (assays of adenylate kinase and lactate dehydrogenase), inflammation (interleukin-8 level) and tissue-barrier function (transepithelial electrical resistance) 48 h post-exposure. The tissue exposed to 45 puffs of CS showed significant changes in all analyzed endpoints compared with the air-exposure control. Significant changes were not observed in tissues exposed to ≤840 puffs of NTV vapor compared with the air-exposure control. The number of NTV vapor puffs required for induction of significant changes in the tissue was 1,120 puffs. This exposure condition (i.e., continuous exposure to ≥1,000 puffs of undiluted vapor) differs markedly from actual exposure conditions in humans, but enables detection of the biological effect of NTV vapor and its comparison with that of CS. Our results suggested that NTV vapor had reduced biological effects on a model of human airways in vitro. The number of puffs necessary for induction of a significant effect using NTV vapor was approximately 25-fold that required using CS.
Keywords: Tobacco vapor, Three-dimensional reconstructed human airway tissue, Whole-smoke exposure system, Cytotoxicity, Inflammation
Assessment of skin sensitising potential of agrochemical formulations using OECD accepted in vitro test methods (#117)
J. Ball1, H. Scott1, E. Smith1, S. Bennett2, W. Masinja3, 4, C. Elliott3, M. Tate1, M. Cumberbatch1
1 Gentronix, Alderley Edge, United Kingdom
Assessment of skin sensitising potential of plant protection products represents a critical component of the safety evaluation process for agrochemicals. Skin sensitisation assessment has traditionally used animal tests, but OECD accepted in vitro assays now exist: Direct Peptide Reactivity Assay (DPRA; OECD TG442C), KeratinoSensTM (OECD TG442D) and human Cell Line Activation Test (h-CLAT; OCD TG442E). However, these assays currently lack validation for their use in the evaluation of complex mixtures. As such their regulatory use for complex mixtures is currently not common place, with more evidence required to establish their suitability for mixture testing. Here we investigate the applicability of these 3 assays for evaluating skin sensitisation potential of 10 agrochemical formulations: 4 suspension concentrates [SC], 2 emulsifiable concentrates [EC], 2 flowable concentrates for seed treatment [FS], and 2 water dispersibles [WG]. Of these, 6 were previously positive in in vivo skin sensitisation tests. The testing approach calculated an average molecular weight (MW) for each formulation by considering the MWs and individual proportions of each component, resulting in average MWs for testing ranging from 177 to 2259 kDa. For each assay, the procedure for solvent selection and solubilisation was performed according to the respective DB-ALM protocols. For DPRA, 5/10 formulations (2 FS; 3 SC) were compatible and solubilised successfully in dimethyl sulphoxide (DMSO):acetonitrile (1:1) with concentrations ranging from 25mM to 100mM. For KeratinoSensTM, all formulations were soluble in DMSO, however, observed cytotoxicity resulted in testing of most formulations at dose ranges lower than OECD recommendations. Preliminary data from DPRA and/or KeratinoSensTM revealed a ‘sensitiser’ prediction that correlated with in vivo methods for 5/6 formulations. Confirming a negative result for the formulations with in vivo ‘non-sensitiser’ predictions was more problematic due to solubility/cytotoxicity profiles impacting assay performance to recommended guidelines, currently leading to their inconclusive interpretation. This is an ongoing study, with performance of all 3 OECD accepted assays being investigated, together with the hypothesis that individual co-formulants may be influencing assay results.
Keywords: Skin sensitisation, in vitro
Application of adverse outcome pathways and quantitative in vitro-in vivo extrapolation (QIVIVE) modelling for risk assessment based on in vitro data (#129)
A. Mally1, B. Birk2, S. Di Fiore3, B. Ellinger4, S. Jarzina1, P. Reiser1, F. Taverne5, N. Kramer5
1 University of Würzburg, Toxicology, Würzburg, Germany
The Adverse outcome pathway (AOP) framework has been adopted as a valuable tool to support transition of drug/chemical safety testing from animal studies to more mechanism-based in vitro endpoints. Systematic mapping of AOPs and identification of key events (KEs) for a given hazard endpoint may serve as a basis for the development of suitable in vitro assays that can be integrated with quantitative in vitro-in vivo extrapolation (QIVIVE) modelling to estimate risk based on in vitro data.
The aim of this work was to provide a proof-of-concept for this approach using kidney toxicity as an exemplary area of repeated dose systemic toxicity. AOPs for kidney injury due to (a) receptor mediated endocytosis leading to lysosomal overload and (b) inhibition of mitochondrial DNA polymerase g were mapped and critically evaluated in terms of biological plausibility, essentiality of KEs, dose-response and temporal concordance using both literature data and novel data generated by in vitro assays reflecting the KEs in each of the AOPs. Quantitative response-response relationships between KEs established using model stressors for each AOP (a: polymyxin antibiotics, b: acyclic nucleoside phosphonate antiviral drugs) were used to predict downstream KEs based on in vitro data reflecting early KEs. Finally, toxicokinetic models comprising a proximal tubule cell compartment were established and utilized to transform in vitro effect concentrations to a human equivalent dose. Model outcomes for selected nephrotoxic drugs were compared to human clinical data to assess the performance of the in vitro approach to risk assessment. Using polymyxin B as an example, we show that predicted in vivo effective doses are in the range of therapeutic doses known to be associated with a risk for nephrotoxicity. While these data support the overall approach of the project, there are yet limitations and sources of uncertainty that remain to be addressed in the future.
Keywords: AOP, QIVIVE, in vitro testing, risk assessment, nephrotoxicity
The kinetic Direct Peptide Reactivity Assay (kDPRA): An in chemico method to characterize the skin sensitization potency of chemicals (#131)
S. N. E. Kolle1, B. Wareing1, A. Natsch2, N. Alepée3, B. Birk1, T. Haupt2, E. Hill4, P. Kern5, L. Nardelli3, H. Raabe4, M. Rucki6, T. Ruwona4, C. Ryan8, S. Verkaart7, W. Westerink7, R. Landsiedel1
1 BASF SE, Experimental Toxicology and Ecology, Ludwigshafen am Rhein, Rhineland-Palatinate, Germany
While the skin sensitization hazard of substances can readily be identified using non-animal methods, the classification of potency into UN GHS sub-categories 1A and 1B remains challenging. The kinetic direct peptide reactivity assay (kDPRA) is a modification of the DPRA (OECD TG 442C) wherein the reaction kinetics of a test substance towards a synthetic cysteine-containing peptide is evaluated. For this purpose, several concentrations of the test substance are incubated with the synthetic peptide for several incubation times at 25°C. After the respective incubation time, the reaction is stopped by addition of the fluorescent dye monobromobimane (mBBr). The highly reactive and non-fluorescent mBBr rapidly reacts with unbound cysteine moieties of the model peptide to form a fluorescent complex. The remaining non-depleted peptide concentration is determined thereafter by fluorescence measurement at precisely defined time points. Kinetic rates of peptide depletion are then used to distinguish between two levels of skin sensitization potency, i.e. to discriminate between CLP/UN GHS sub-categories 1A and 1B. During an in house validation (Wareing et al., 2017) 35 of 38 substances with LLNA-based sensitizing potency were correctly assigned to the potency sub-categories, and the predictivity for 14 human data was similarly high. These results warranted the kDPRA for further validation. Here we present the results of a ring trial testing 24 blind-coded chemicals in seven labs. In parallel we present the extension of the kDPRA database to further assess the predictive capacity of the assay. Eventually the kDPRA should be used as a part of defined approach(es) with a quantitative data integration procedure for skin sensitization potency assessment.
 Upon the abstract submission deadline the ring trial was still in progress and the substance identities remained blind coded. Therefore no results could be presented in the abstract.
Keywords: skin sensitization, skin sensitizing potency, peptide reactivity, UN GHS categories, kinetic DPRA
Improving the prediction of hepatotoxicity: Impact of protein binding in the generation of in vivo relevant intracellular concentrations (#135)
K. R. Brouwer1, J. Jackson1, R. St. Claire III1, R. Laethem1
1 BIOIVT, ADME-TOX, Durham, North Carolina, United States of America
Purpose: The unbound intracellular concentration (ICC) is the driving force for processes that occur inside the hepatocyte, including metabolism, induction (metabolic and transporter), efflux based drug interactions, and hepatotoxicity. In sandwich-cultured hepatocytes, the intracellular milieu contains the components for drug binding, and in concert with hepatic uptake and efflux (basolateral and canicular) transporters, drug metabolizing enzymes, and key regulatory pathways allow generation of in vivo relevant unbound intracellular concentrations. Protein on the outside of the cell can also limit hepatic uptake of a drug. If hepatic uptake and intracellular concentration are dependent on the free concentration, then, parameters generated from experiments performed in the absence and presence of protein should be equal when normalized for protein binding.
Methods: Sandwich-cultured rat hepatocytes and B-CLEAR® technology were used to determine the ICC for 10 compounds (taurocholate, telmisartan, methotrexate, valsartan, DPDPE (1 and 10µM), digoxin, pitavastatin, rosuvastatin, and pravastatin) in the presence and absence of a physiological concentration of bovine serum albumin (4% BSA). IC50 values for inhibition of CYP2C9 and CYP3A4 metabolism by fluconazole and ketoconazole, respectively, were determined in the presence and absence of 4% BSA using Transporter Certified™ human hepatocytes in sandwich culture. The fraction unbound (fu) in 4% BSA was determined in a separate experiment using equilibrium dialysis. The extent of protein binding was used to normalize the values obtained in the absence of protein (Predicted value), and compared to the value obtained in the presence of protein (Observed value) and the fold change was calculated.
Results: The ICC was over predicted for valsartan (2.1X), and underpredicted for pitavastatin (3.2X), rosuvastatin (2.2X), and telmisartan (90X). For the other compounds evaluated (pravastatin, digoxin, DPDPE - 10µM, and taurocholate), predicted values were not different from the observed values. Normalization of the IC50 values for CYP2C9 by fluconazole were over predicted by 1.9X, while the IC50 values for inhibition of CYP3A4 by ketoconazole were under predicted by 257X. In vivo it is the rate limiting step - the slowest process that determines the overall rate. The lack of agreement between observed and predicted ICC values may be due to measurement of the extent and not the affinity of the protein binding. If the dissociation rate of the drug off of the protein is much greater than the uptake rate, protein binding may not be a limiting factor in drug uptake.
Conclusions: If active transport processes are involved in hepatic uptake, the slowest process will limit the hepatic uptake. Addition of physiologic protein concentrations to in vitro systems may improve predictions of intracellular drug exposure and effects.
Keywords: Hepatotoxicity, Intracellular Concentrations, Protein Binding, Transporters, In vivo Prediction
Safety Testing of Cosmetics for Eye Irritation In Vitro: Evaluation of Results From Over 40 Studies (#144)
E. H. Theophilus1, V. Rana1, D. Mihai1, N. Habeeb1, T. Suwa1, B. Yasso2, B. Varsho2, G. DeGeorge2
1 Shiseido, R&D, East Windsor, New Jersey, United States of America
Cosmetics safety assessment typically involves: 1) theoretical evaluation of ingredients and raw materials to determine local and systemic toxicity potential, and 2) testing to confirm local toxicity effects of final formulations. Interactions among ingredients are not easily predicted by single-ingredient theoretical evaluations and this is where safety testing is critical. Given the momentum regarding refinement, reduction, or replacement (3Rs) of animal-based tests with in vitro methods, it is important to define testing strategies that meet those requirements and provide confident safety assurance for each toxicity endpoint (e.g., eye irritation potential). The Chorioallantoic Membrane Vascular Assay (CAMVA) and Bovine Corneal Opacity and Permeability (BCOP) assays, when used in combination, have demonstrated to be relevant and reliable methods to predict eye irritation potential of cosmetics. These assays predict the possible effects of mixtures in the human eye fairly well partly because, together, they represent relevant eye areas (i.e., conjunctiva and cornea). In the BCOP assay, there are currently two suggested classifications at the low end of the eye irritation spectrum: 1) GHS (no category) and 2) Gautheron (mild irritant). Results from our 40+ test batteries have been used to establish a prediction model for safety assessment, which can be used to guide decision-making regarding cometics warning labels. According to our data analyses, a formulation could be confidently predicted as “practically non-irritating” to the human eye when the CAMVA RC50 is >70% and the BCOP in vitro irritancy score (IVIS) is ≤3. Comparing in vitro test results predictions with post-marketing surveillance analyses allows evaluation of the accuracy of the two-test battery prediction model, and confirms the effectiveness of safety evaluation recommendations for product market release to better ensure consumer safety.
Keywords: cosmetics, safety evaluation, eye irritation, in vitro
Donor-to-Donor Variability of Reconstructed Human Airway Tissues in Response to Cigarette Smoke (#168)
S. Mori1, K. Matsumura1, K. Ishimori1, S. Ishikawa1, S. Ito1
1 JAPAN TOBACCO INC. , Scientific Product Assessment Center, R&D Group, Yokohama, Japan
Reconstructed human airway (RHuA) tissue is considered a reliable in vitro model for inhalation toxicity testing with airborne materials such as cigarette smoke (CS) because of its resemblance to in vivo tissues. However, it potentially possesses donor specific characteristics related to variations of genotype, resulting in differences of responder responses to toxicants. Therefore, it is important to understand and biologically interpret such variabilities when assessing toxicities of interest. In this study, we exposed commercially available RHuA (MucilAir) derived from three different donors to an aqueous extract (AqE) of CS. Tissues were exposed to three different concentrations of CS-AqEs. Tissues incubated with fresh medium alone were used as untreated controls. After 24 h of exposure, cytotoxicity (secreted adenylate kinase; AK), ciliary functions (beating frequency and area), IL-8 secretion and global gene expression profiles were analyzed. AK secretion increased by CS-AqEs exposure differed among donors, suggesting that reactivity to toxicants was different. Consistent with this result, dose-response curves of IL-8 secretion and ciliary beating area differed although all assays used in this study showed some reaction to CS. Furthermore, microarray analysis revealed that genes that had statistically significant altered expression levels related to CS exposure only had a 13.6% overlap, implying that the other differentially expressed genes (DEGs) responded dependent upon the donor origin. However, canonical pathway analysis with the 13.6% of overlapping DEGs showed that these genes were related to oxidative stress responses including NRF2 mediated oxidative stress, a well-known biological event elicited by cigarette smoking. Taken together, donor dependent characteristics were observed in RHuA tissues in response to CS, but the key responses were well conserved. In conclusion, we demonstrated the potential of donor-to-donor variability in an advanced in vitro test using RHuA, which might cause misleading results if there are no consensus targeted endpoints or appropriate dose settings. Therefore, transcriptomic data will be a useful tool to complement these results and investigate the key biological effects of test substances that might highlight fit-for-purpose endpoints.
Keywords: organotypic culture, tobacco, transcriptome, inflammation, oxidative stress
hTERT Immortalized Adult Dermal Melanocytes: An in vitro Cell Model for the Study of Skin Pigmentation (#200)
J. L. Rodriguez1, C. Zou1, R. Menth1, M. Judge1
1 ATCC Cell Systems, Gaithersburg, Maryland, United States of America
Skin pigmentation is a complex process; melanocytes produce melanin and package it into melanosomes that are in turn exocytosed into the surrounding extracellular matrix. Numerous genes play roles in controlling pigmentation at various levels of melanin production. Mutations in these genes are characteristic of multiple skin disorders, including hyperpigmentation, hypopigmentation, and mixed hyper-/hypopigmentation. Additionally, extrinsic factors secreted by the surrounding resident cell types also regulate the melanin expression in adult melanocytes. Human primary cells can be a useful model for elucidating melanocyte biology. However, primary cells have their limitations such as donor variability and limited lifespan. Consequently, a need exists for a more robust human cell model system for the study of skin pigmentation.
In this study, we immortalized primary dermal melanocytes by expressing human telomerase reverse transcriptase (hTERT) in cells that were isolated from an adult donor. The immortalized primary melanocytes were cultured continuously for more than 40 population doublings without any signs of replicative senescence, yet retained melanin production. The immortalized primary melanocytes maintained a consistent expression of the melanocyte-specific marker TRP-1, and lacked expression of the fibroblast-specific marker TE7. In addition, we demonstrate the capability of these immortalized primary melanocytes to transfer melanosomes to keratinocytes, the ability to modulate melanogenesis with stimulators and inhibitors, and their capacity to incorporate into a functional 3D human dermal organotypic culture. Taken together, the hTERT immortalized primary melanocytes described here provide a versatile in vitro cell model for the study of melanin production and melanocyte:keratinocyte interactions in the dermal environment.
Keywords: hTERT, immortalized, primary, melanocyte
Screening of the cytotoxic, genotoxic, apoptotic and cell cycle effects of Rubus rosaefolius (Rosaceae) leaf extract on human HepG2/C3A cells. (#209)
E. L. Maistro1, A. P. Quadros2, L. Almeida1, I. Baraldi1, R. Niero3, M. Petreanu3, M. Mantovani4
1 São Paulo State University (UNESP), Speech and Hearing Therapy Department, Marilia, Brazil
The Rubus rosaefolius plant recently have been some of its therapeutic properties confirmed by scientific analysis, among them the analgesic, antimicrobial, antihypertensive, antioxidant, antiproliferative effects in tumor cells, diuretic, gastroprotective and antidepressant. Such confirmation makes this plant of great interest to the pharmaceutical industry. However, before the commercial exploitation of R. rosaefolius as a herbal remedy, it is necessary to carry out tests evaluating the biosafety of the use of this plant by humans, ascertaining if it is free of cellular and genetic toxicity. In view of the above context, this research aimed to analyse the action of R. rosaefolius leaf extract on human hepatoma (HepG2/C3A) cells, regarding its cytotoxic, genotoxic, apoptosis induction, and cell cycle effects. The cytotoxicity of the extract after 24, 48 and 72 h exposure in a concentration spectrum of 0.01 to 100 µg/mL was evaluated by the MTT test. Results showed absence of cytotoxic effect of the extract in MTT test on HepG2/C3A cells at all concentrations and exposure time tested. The in vitro comet assay showed an increase in the DNA damage (class 1) at 0.1 µg/mL concentration and above. Micronucleus test evidenced no clastogenic/aneugenic effects. Flow cytometry analysis showed significant increase in the number of the apoptotic cells at 10, 20 and 100 µg/mL concentrations, and interference of the extract in the cell cycle with increase number of the cells arrested in the S phase (only at 100 µg/mL). Under our experimental conditions, this preliminary in vitro biosafety evaluation showed that leaf extract of the medicinal plant R. rosaefolius presented some toxic effects on HepG2/C3A human cultured cells. Complementary studies are being performed to better determine the risk of this plant extract to the humam cells.
Financial Support: Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (Grant: 2017/24149-4).
Keywords: Genotoxic assessment, comet assay, flow cytometry, Micronucleus test, MTT assay
The THP-1 Toolbox: a new method that integrates the 4 key events of skin sensitization (#210)
E. Clouet1, 2, R. Béchara1, C. Raffalli1, M. - H. Damiens1, H. Groux3, M. Pallardy1, P. - J. Ferret2, S. Kerdine-Römer1
1 UMR996 - Inflammation, Chemokines and Immunopathology, INSERM, Univ Paris-Sud, Université Paris-Saclay, Châtenay-Malabry, France
Allergic contact dermatitis (ACD) is an adverse health effect that develops following repeated exposure to skin sensitizers. In the European Union, an animal testing ban has been applied under the Cosmetics Regulation, leading to development of reliably predictive non-animal methods. An adverse outcome pathways (AOP) for chemical induced skin sensitization has been already proposed in 2012 by OECD. AOPs outline causally linked key steps between a direct initiating event leading to an adverse health outcome. Four different key events (KE) have been identified and associated in the AOP for skin sensitization: (1) protein-binding reactions, reactivity, and metabolism, (2) epidermal inflammatory response, (3) DC activation and (4) T-cell proliferation. Different in vitro chemistry-based assays have been developed and allow the evaluation of sensitization hazards.
Since DC play a key role in the skin sensitization phase leading to the development of ACD, we propose to combine different tests covering all KE defined by AOP in a same cell line,the THP-1 cell, acting as a DC.
We decided to study the ROS production and GSH depletion as cellular oxidative stress for KE1, Nrf2 activation pathway and gene expressions for KE2, phenotype modifications as cell-surface markers and cytokines production for KE3, and T cell proliferation for KE4. All of those measurements were performed on the THP-1 cell-line, after exposure to a variety of chemicals, including irritants, non-sensitizers and allergens (pro/prehaptens).
Results showed early ROS production and reduction of intracellular glutathione are correlated with the potency of the chemicals such as cinnamaldehyde or methylisothiazolinone. Those chemicals as well as antioxidants specifically activate the Nrf2-Keap1 pathway, which were measured by western blot and a Nrf2 DNA-binding ELISA. They also strongly induced phenotype maturation of THP-1 cell-line with CD54 and CD86 expression at cell-surface and specific cytokine production such as IL-8, IL-18. All sensitizers were able to induce the T cell proliferation while non-sensitizers and irritants did not.
In the present study, we have demonstrated that the three main KE of skin sensitization AOP can be addressed in a same cell line as well as lymphocyte activation.
Keywords: THP-1, skin sensitization, Integrated AOP
Application of the Human Cell Line Activation Test to predict the skin sensitization potential of DDAC, PHMG, troclosan and propylene glycol (#219)
S. Yang1, R. Gautam1, A. Maharjan1, J. Jo1, C. Kim1, M. Acharya1, H. Kim2, Y. Heo1
1 Daegu Catholic University, College of Bio and Medical Sciences, Dept. Occupational Health, Gyeongsan-si, Republic of Korea
The Human Cell Line Activation Test (h-CLAT) is an alternative in vitro test method using dendritic cells for prediction of skin sensitization and adopted as OECD TG 442E. The h-CLAT method was used to predict skin sensitization potential of didecyldimethylammonium chloride (DDAC), polyhexamethylene guanidine (PHMG), 2,4,4’-trichloro-2’-hydroxydiphenyl ether (triclosan), which often serves as biocidal agents. Propylene glycol (PG) was also tested, which is used to dissolve the active ingredients. The skin sensitization (SS) potentials of each of these substances are still being debated. All the experimental procedures were undertaken following the OECD TG. Proficiency testing of the nine coded substances listed in OECD Test Guideline 442E correctly detected all sensitizers and non-sensitizers. On 3 independent runs, DDAC, PHMG and triclosan were predicted to be sensitizers (in 2 of 2 or 2 of 3 runs, the CD86 RFI is ≥150% and/or the CD54 RFI is ≥200% at any tested concentration). In terms of DDAC, the CD54 RFI % exceeded 200% in all 3 runs and the CD86 RFI % exceeded 150% in 1 run. Concerning on PHMG and triclosan, the CD54 RFI % exceeded 200% and the CD86 RFI % exceeded 150% in consecutive 2 runs for both substances. Meanwhile, PG was also classified into a sensitizer, in that the CD54 RFI % exceeded 200% in 2 runs. Since humans can be occupationally or environmentally exposed to those biocidal or excipient chemicals, the present study could help regulatory bodies in their assessment of the skin sensitization potency of DDAC, PHMG, triclosan and PG. [supported by Korea National Research Foundation, Project no. 2017R1D1A3B03032723].
Keywords: Human Cell Line Activation Test, Skin sensitization alternative method, didecyldimethylammonium chloride, polyhexamethylene guanidine, Triclosan
Investigation of the genotoxic potential of green smoothies in silico and in vitro (#220)
J. Reinhard1, M. Frericks1, T. Hofmann1, M. Speitling1, B. van Ravenzwaay1
1 BASF SE, Experimental Toxicology and Ecology, Ludwigshafen, Rhineland-Palatinate, Germany
Green smoothies contain raw green vegetables or herbs blended together with fruits. Due to their high amount of phytochemicals, they are advertised as a healthy addition to the normal diet. However, recent studies indicate that the excessive consumption of raw vegetables and leafy greens may not be as healthy as advertised and it is recommended to rotate the green ingredients in the smoothie to avoid toxic side effects. Moreover, blending of vegetables leads to a ruptured cell wall, thus a higher number and a different spectrum of phytochemicals are available for absorption. In this project, an in silico genotoxicity assessment, as well as a dietary risk assessment was performed on green smoothies. This included a database search for the phytochemical content and associated concentration values in common smoothie ingredients, followed by the prediction of their genotoxic potential by three different QSAR models. Thereby, endpoints regarding the Ames test, micronucleus test, and chromosome aberration were chosen. Compounds of toxicological concern were added to the dietary risk assessment and their concentration values in one smoothie were calculated and compared against the threshold of toxicological concern (TTC) for mutagenic compounds. HPLC-MS was performed to evaluate the number of phytochemicals in a sterile-filtered smoothie supernatant, showing the presence of thousands of compounds. In contrast to that, database search yielded a maximum of 384 known phytochemicals for a whole smoothie. Also, not for all phytochemicals a concentration value was available. QSAR analysis showed a genotoxic potential for multiple phytochemicals, but for several others a prediction was not possible due to missing data. Furthermore, the QSAR analysis showed high variations of the datasets between different models, resulting in different outcomes for the same endpoint. For a reliable dietary risk assessment too many data were missing. All in all, this project shows that there is need to define a safe exposure level also for plant-based compounds. For this, however, the databases for the chemical content in plants, as well as the datasets embedded in QSAR models do not provide sufficient information. This means, that more toxicological data are needed, and further research is necessary regarding the chemical content in plants.
Keywords: QSAR, phytochemicals
Vascularised cardiac tissue model for the assessment of efficacy and cardio toxicity (#240)
T. A. Tolvanen1, M. Toivanen1, T. Toimela1, T. Heinonen1
1 Tampere University / FICAM, Medicine and Health Technology, Tampere, Finland
Background: Cardiotoxic effects are among the most common reasons for discontinuation of development of a novel drug candidate at the Phase I or later. A commonly used test system is human induced pluripotent stem cell (hiPSC) derived cardiomyocyte (CM) monoculture. Monoculture lacks the 3D-tissue structure and phenotype of adult CMs and resemble closely the foetal one. As the microenvironment is essential for the full maturation of the cells, in our model hiPSC-CMs are cultured with human cellular vasculature. We hypothesized that using these more mature CM, would improve the predictability of the effective physiological concentration of a drug compared to monocultured CM.
Materials and methods: Vasculature was formed from Human adipose stromal cells (hASC) and human umbilical cord vascular endothelial cells (HUVEC) as described earlier. hiPSC-CM (iCell CM2, Cellular dynamics) were seeded on top of the vasculature. CM were let to mature for 8 days on top of the vasculature after which the cardiovascular model was used for chemical testing. For the assessment of the validity of our model in assessment of cardiac effects we used multi electrode array (MEA) measurements, and compared the published data obtained from monocultured CM. Set of 30 drugs with known effects on human heart functions and three negative controls were studied. This set of drugs included AR-agonists, AR-antagonist and various ion channel blockers.
Results and conclusion: The used negative controls did not significantly alter field potential duration or beat rate. The results with known drugs showed that majority of the effects in our model were obtained with concentrations closer to clinically observed Cmax values compared to monocultured CM. These data suggest that culturing CM together with vasculature increases the level of maturation and provides more physiologically relevant model for assessing the cardiac effects of different compounds.
Keywords: in vitro, cardio toxicity, tissue model
In vitro viability tests to evaluate Fe3O4NPs cytotoxicity in human mesenchymal stem cells (#243)
U. De Simone1, F. Caloni2, M. Roccio3, A. Spinillo3, M. A. Avanzini4, T. Coccini1
1 Laboratory of Clinical and Experimental Toxicology, Toxicology Unit, ICS Maugeri SpA-SB, IRCCS, Pavia, Italy
The emerging applications of superparamagnetic iron oxide nanoparticles (SPIONs), including magnetite (Fe3O4NPs), in life sciences and industrial and biomedical (diagnosis and therapy) fields, raise public health and scientific concerns over possible environmental and human health implications since Fe3O4NP toxicity is not yet fully understood.
In this study, in vitro tests, neutral red uptake (NRU), MTT tests and trypan blue assay (TB), were applied to evaluate cytotoxicity of Fe3O4NPs (1-300 mg/ml) after short-term exposure (24-48 h) using human mesenchymal stem cell derived from umbilical cord lining (CL-hMSCs), as an innovative and alternative cell model.
NRU results showed a concentration-dependent absorbance increase. Apparently, an enhancement of cell viability from 18 to 260% at 10-300 mg/ml Fe3O4NP was observed. Similar data were also obtained from MTT test (high cell viability) after NP exposure compared to control.
This absorbance enhancement was not supported by the evidence obtained with morphological analysis by phase-contrast microscopy. Cellular visual inspection, at both time points, showed cell density decrease and loss of the monolayer features at ≥ 50 μg/ml, and morphological alterations (large/flat cells, debris) at ≥150 μg/ml. Notably, the cell morphology changes paralleled with the results obtained with TB. The latter showed a cell death (35%) at ≥10 μg/ml, with maximum effect (65%) at 300 mg/ml Fe3O4NPs after 48 h.
Experiments carried out in a cell-free system confirmed that Fe3O4NPs interfered with the enzymatic activity of MTT and NRU assays (20-50% and 50-450% absorbance increase, respectively).
Altogether our data suggest that the agglomeration and settling of Fe3O4NPs in the specific medium used for CL-hMSC cultures, associated to the difficulty to remove them (by washing) from this cell type, appeared to be linked to light absorbance interference leading to overestimation (false) viability. On the contrary, in this culture conditions, TB seemed a suitable test to determine cell viability compared to MTT and NRU.
Grant from the Italian Ministries of Health, Research and Education
Keywords: Fe3O4NPs; stem cells; in vitro cytotoxicity; nanoparticles
Contraction properties of human in vitro cardiac tissue model (#255)
M. Toivanen1, T. Tolvanen1, J. Virtanen2, S. Tuukkanen2, I. Miinalainen3, L. Eklund4, T. Toimela1, T. Heinonen1
1 Tampere University, FICAM (Finnish Center for Alternative Methods), Faculty of Medicine and Health Technology, Tampere, Finland
Background: Cardiotoxicity is one of the major causes for drug attrition and withdrawal during drug development process and post approval. Contraction force is an essential part of heart function and drugs affecting the contraction of cardiomyocytes can potentially cause severe cardiac risks. Human induced pluripotent stem cell -derived cardiomyocytes can be used for cardiotoxicity testing in vitro. They are commonly used as a cardiomyocyte monoculture in which they, however, remain immature phenotype resembling more fetal than adult cardiomyocytes.
Materials and methods: In this study, contraction properties of vascularized cardiac tissue models were studied. The cardiac tissue models were constructed by first co-culturing human adipose stromal cells (hASCs) and human umbilical vein endothelial cells (HUVECs) to produce vascular-like networks and then seeding cardiomyocytes on top of the vascular structures. The contraction force of cardiac tissue models was measured using in-house developed piezoelectric cantilever sensor. The contracting structures of cardiomyocytes in the cardiac tissue models were characterized using electron microscopy techniques and immunofluorescence imaging and compared to cardiomyocytes cultured in monoculture.
Results: Contraction forces of 7.2 to 16.6 µN were measured from the cardiac tissue models. The cardiomyocytes in the cardiac tissue models had more mature morphology compared to cardiomyocytes in monocultures.
Keywords: in vitro, cardiac tissue model, contraction force
Identifying and characterising stress pathways of concern for consumer safety risk assessments (#256)
A. Middleton1, M. T. Baltazar1, P. Carmichael1, S. Hatherell1, H. Li1, B. Nicol1, J. Reynolds1, P. Russell1, S. Scott1, C. Westmoreland1, A. White1
1 Unilever, SEAC, Sharnbrook, United Kingdom
As recently outlined by the International Cooperation on Cosmetics Regulation (ICCR) , key principles of modern non-animal cosmetic safety risk assessments are that they should be exposure-led, hypothesis driven, use a tiered and iterative approach and adopt robust methods for which sources of uncertainty are characterised and documented. In particular, many compounds for which consumer safety risk assessments need to be conducted are not associated with specific toxicity modes of action, but rather exhibit non-specific toxicity leading to cell stress. In this work, a cellular stress panel is described, consisting of forty biomarkers representing nine stress pathways and cell health markers, including oxidative stress, mitochondrial toxicity and ER-stress, measured predominantly using high content imaging. To evaluate the panel, data were generated using HepG2 cells for fifteen compounds at typical human exposure levels. The compounds were selected either because they are known to either be toxic to humans at such levels (and therefore present a ‘high risk’ from a consumer safety perspective) and have a mode-of-action associated with cellular stress (e.g. doxorubicin, troglitazone, diclofenac), or compounds widely regarded as innocuous (i.e. ‘low risk’) to humans (e.g. caffeine, niacinamide and phenoxyethanol). A key metric here is the point-of-departure (POD), the lowest concentration at which biological responses can be detected within a given set of assays. For each compound, dose response data (eight concentration points) were generated for each biomarker at three timepoints. A Bayesian model was then developed to quantify the evidence that, for a given set of dose-response data, a biological response does occur, and if so, a credibility range for the estimated POD. These PODs are then compared to Cmax estimates calculated using physiologically-based kinetic models. Results from the panel indicate a clear differentiation between the ‘low-risk’ and ‘high-risk’ compounds at typical human exposure levels, with low risk ones triggering significantly fewer stress pathways, and at higher concentrations, than the high-risk compounds. Comparisons to analogous stress panel data generated using other cell models (HepaRG and NHEK) will also be presented. Overall, the results provide a strong indication that the panel could serve as an assay for identifying and characterising stress pathways of concern, as part of a weight of evidence-based risk assessment that follows the ICCR principles.
 Dent, Matthew, et al. "Principles underpinning the use of new methodologies in the risk assessment of cosmetic ingredients." Computational Toxicology 7 (2018): 20-26.
Keywords: new approach methodologies, Bayesian modelling, Dose response modelling, Cellular stress, Point of departure
Three-dimensional in vitro co-culture model of adipocytes and endothelial cells using magnetic levitation: toxicological evaluation of caffeine (#270)
P. S. Lopes1, A. Ueoka1, L. Fernandes1, B. Sufi2, W. Magalhães2, N. Andreo-Filho1, V. R. Leite-Silva1
1 Federal University of São Paulo, Pharmaceutical Sciences, Diadema, Brazil
To evaluate new active substances and formulations for the treatment of cellulite and adipocyte dysfunctions, conventional two-dimensional (2D) cell cultures are usually employed. However, 2D culture models do not mimic the complexity of the adipose tissue. The interactions between the cells and their microenvironment govern various processes, such as cell differentiation, proliferation, and gene expressions. In this context, we are proposing a three-dimension new levitation cell culture system based on magnetic particles to assess the toxicological aspects of caffeine, as a model drug of lipogenic activity. The goal of this study was to compare cytotoxicity in 2D cell culture models based on OECD/GD 129 and the 3D models. The 3T3-L1 preadipocyte cells and rat endothelial cells were cultured in DMEM 10% FBS. Three-dimensional levitation cell cultures were based on previously established methodology and were set up using 96-well Bio-Assembler™kit (Nano3D Biosciences™Inc.) consisting of nanoshuttle (NS) solution and a plate magnetic drive. 3D cultures levitated for 1 day were induced for adipogenic differentiation (0.5 mM isobutylmethyxanthine, 1µM dexamethasone, 1.7 mM insulin in DMEM 10% FBS) for 72 h. After this, the induction medium was replaced with DMEM 10% FBS containing 1.7mM insulin. Caffeine 7.0 mM was added after 72h and maintained for 8 days. The 2D cultures were not submitted to adipogenic differentiation. Both cultures – 2D and 3D - were evaluated using PrestoBlue™ viability dye. The results of cell viability for 2D cultures were 14.96 % for caffeine and 3.43 % for DMSO meanwhile for 3D were 13.01 % for caffeine and 8.7 % for DMSO. The major difference was observed only for the positive control, as the undifferentiated cells also did not present difference in the cell viability in both systems. In conclusion, it will be possible to come up with a 3D in vitro model to evaluated new adipogenic actives in research and development of new cosmetic products.
Keywords: in vitro model; safety; three dimensional cell culture (3D); cytotoxicity; cosmetic
Prediction of skin sensitization potency for risk assessment using noble biomarkers IL-1β and iNOS (#280)
M. K. Kim1, Y. C. Kwon1, J. S. Kang1, B. M. Lee1
1 Sungkyunkwan University, College of Pharmacy, Suwon, Gyeonggi-Do, Republic of Korea
This work was supported by a grant from Ministry of Food and Drug Safety (MFDS), 2018.
Biomarkers related to skin sensitization were analyzed in THP-1 human monocytic leukemia cells to predict skin sensitization potency for risk assessment, as an alternative animal tests. Cell viabilities of 90% (CV90) and 75% (CV75) were determined by WST-1 assay to establish the comparative conditions of 24 selected test materials. In addition, biomarkers related to skin sensitization were analyzed by western blotting under equivalent comparative conditions. In biomarker analyses, IL-1β, iNOS, IL-1β + iNOS, and THP-1 IL-1β + Raw 264.7 IL-1β were found to be suitable biomarkers for the prediction of skin sensitization potency following their classification as either skin sensitizers or non-sensitizers (accuracies of 91.7%, 87.5%, 83.3%, and 82.6%, respectively). In addition, a high positive correlation was found between these biomarkers and skin sensitization potency, with a correlation coefficient (R) of 0.7 or more (correlation coefficients of 0.77, 0.72, 0.70, and 0.84, respectively). Finally, the skin sensitization potency EC3 (%) was predicted using a biomarker correlation equation, with resulting prediction accuracy for the EC3 value (%) obtained from animal data was calculated as 83.3%, 79.2%, 79.2%, and 73.9%, respectively. These results suggest that biomarker analysis using IL-1β and iNOS in human THP-1 cells can be alternatively used to predict skin sensitization potency for risk assessment.
Keywords: IL-1β, iNOS, skin sensitization, risk assessment, biomarker
Mechanism-based alternative method for developmental toxicity testing in zebrafish embryos (#281)
R. Narumi1, J. Tasaki1, S. Liu1, N. Ikeda1, O. Morita1
1 Kao Corporation, Safety Science Research, Akabane, Ichikaimachi, Haga-gun, Tochigi, Japan
These days it is required to establish alternative testing methods for safety assessments. However, alternative methods for developmental toxicity tests have not been well developed because of its complicated toxicological responses. Zebrafish early embryos are non-protected animals (eg. Directive 2010/63/EU) and considered to be one of the promising models for screening of common birth defects owing to the conserved developmental program, low experimental costs, rapid development and transparency. Although conserved toxicity endpoints are necessary for accurate prediction of developmental toxicity in mammals, there is little information about cross-species conservation of teratogenic responses between mammals and fishes. We focused on 5 major targets of congenital birth defects (cranium, palate, nervous system, heart and musculoskeletal systems) and analyzed morphological, cellular and molecular responses to teratogens. In the present study, we investigated the conserved mechanisms of palate malformation between mammals and zebrafish.
Zebrafish embryos were exposed to 12 chemical compounds (valproic acid, warfarin, caffeine, imatinib, retinoic acid, salicylic acid, 5-fluorouracil, methotrexate, thalidomide, hydroxyurea, phenytoin and dexamethasone), which induce cleft palate in human or rodents. Palatal morphology and the number of proliferative cells and apoptotic cells were examined in zebrafish palate at 96 hpf using immunofluorescence staining and confocal microscopy. Also, we investigated the involvement of the canonical Wnt signaling pathway, which is one of the key contributors to orofacial clefts. Chemical rescue of the cleft palate were performed by simultaneous treatment with Wnt agonists (BIO, CHIR99021, and WAY262611) and specific teratogens (warfarin and valproic acid).
All 12 teratogens induced palatal defects in zebrafish embryos which showed decreased proliferation and increased apoptosis in the palate. These phenotypes were rescued at the cellular and molecular levels by the treatment with the Wnt agonists.
We showed the conserved responses to the teratogens between mammals and zebrafish: malformation of palate and regulation of proliferation/apoptosis via the Wnt signaling pathway. Thus, our results suggest that zebrafish early embryo assay would be a suitable model for assessing chemical-induced cleft palate as well as being a screening tool for prediction of cleft palate in mammals. We will confirm the key endpoints based on conserved molecular mechanisms by a comprehensive analysis as a next step for accurate prediction of teratogenicity in mammals.
Keywords: Alternative method, developmental toxicity, zebrafish, conserved mechanisms, teratogenicity
Data sharing on the INTERVALS platform and meta-analysis of in vitro toxicology assessment of diverse e-liquid and heat-not-burn products (#283)
S. Boue1, A. Stan1, J. Hoeng1, M. Peitsch1
1 Philip Morris Products S.A., Science and Innovation, Neuchatel, Switzerland
Extensive scientific studies are conducted to assess the relative risks of various candidate modified risk tobacco products compared with those of smoking cigarettes. As the scientific community conducts such assessments for diverse products and in a variety of laboratory models, knowledge on toxicity is spread across numerous scientific articles. We believe that by fostering the consolidation of data and knowledge gained from studies assessing novel tobacco/nicotine delivery products on a community platform, new hypotheses may be generated, and the weight of evidence may be increased. Therefore, we have created and are further developing INTERVALS (www.intervals.science), an online platform supporting independent, third-party collaboration by proactively sharing detailed protocols, tools, and data from assessment studies. Data files are accompanied by relevant information to foster reproducible research and encourage data reanalysis.
We will present a meta-analysis of in vitro toxicology assessment studies, including aerosol characterization, neutral red uptake assay, and mouse lymphoma assay, for various e-liquid and heat-not-burn platforms compared with the 3R4F reference cigarette. These studies have been conducted by multiple organizations using different methods and models. The content of the separate publications has been curated and included in INTERVALS in an interoperable format so that a meta-analysis of results can be performed.
The direct comparison of the platforms tested in separate studies with different study designs (e.g., different lists of chemicals quantified in the aerosols) makes it difficult to compare every single result across all individual studies. However, the overall result is consistent in that all of the studies included in this analysis demonstrate the reduction of harmful or potentially harmful chemicals and of toxicity assessed in vitro for the tested platforms compared with cigarettes. As the scientific community integrates more studies and datasets into INTERVALS, it will become easier to conduct such meta-analyses and review results obtained across institutions, models, and platforms.
Funding information: Philip Morris International is the sole source of funding and sponsor of this research and platform.
Keywords: Heat-not-burn, Meta-analysis
The molecular basis for a functional dermal barrier in two biotechnologically produced human skin equivalents: the Phenion® FT Skin model and the OS-REp model (#284)
K. R. Mewes1, L. Vierkotten1, A. Fischer1, M. Merkel1, N. Blasius1, C. Petrick1, G. Görtz1, T. Beyer1, D. Petersohn1
1 Henkel AG & Co. KGaA, Biophysics & Biological Research, Duesseldorf, North Rhine-Westphalia, Germany
Biotechnologically produced 3D skin equivalents are the state-of-the-art tools to study human skin physiology and pathology under standardized conditions in vitro and to replace animal experiments in the toxicological assessment of chemicals. In healthy human skin a functional and selective barrier, mainly located in the stratum corneum, discriminates between chemicals which penetrate the skin and subsequently reach the deeper tissue layers, or which remain on the tissue surface without any effect on the skin. Thus, lipid composition and structure of the dermal barrier are crucial for the access of chemicals into the skin and subsequently influence all downstream reactions, both in vivo and in 3D tissue models. The barrier lipid composition of 2 skin equivalents, the Phenion® Full-Thickness (FT) Skin Model and the Phenion Open Source Reconstructed Epidermis (OS-REp), was analyzed chromatographically. Ceramides, cholesterol and cholesterol derivatives, triglycerides and phosphatidyl choline were identified in all samples tested, although in slightly differing quantities. The lipid profiles of both 3D skin models closely matched the profile of native human foreskin tissue, the source for keratinocytes and fibroblasts which give rise to the tissue equivalents. Major enzymes of the epidermal lipid metabolism, e.g. ceramidases and serine palmitoyltransferase, were expressed in keratinocytes in monolayer culture and/or in the epidermis of the FT- and OS-REp models, as demonstrated by immunofluorescence and RT-PCR. Barrier integrity was analyzed by TEER value evaluation during the whole tissue culture period. The similarity of the lipid pattern in the 3D skin models with intact human skin, together with the presence of key enzymes of barrier lipid synthesis, provides strong evidence for a physiological barrier function. This is a key prerequisite for using the skin models in the toxicological assessment of substances, e. g. in in vitro skin irritation or corrosion tests or in dermal absorption studies. Thus, both the Phenion® FT Skin Model and the OS-REp model are well-suited to be used as in vitro surrogates for native human skin, or epidermis, respectively, in experiments which require a barrier function.
Keywords: skin model, barrier, lipid composition, alternative method, enzyme expression
Using 3D human liver microtissues to model NASH progression in vitro for drug discovery and safety testing (#291)
S. Ströbel1, J. Rupp1, K. Fiaschetti1, P. Guye1, A. Rea1, E. Thoma1, R. Kostadinova1
1 InSphero AG, Schlieren , Zürich, Switzerland
Non-alcoholic fatty liver disease (NAFLD) is the most prevalent type of liver disease and currently affects ~30% of the population. With progression to non-alcoholic steatohepatitis (NASH), this disease can eventually lead to liver cirrhosis and failure. To date, there are no approved drugs for NASH treatment and drug development has been impeded by the lack of predictive in vitro models reflecting the complex pathology of NASH. Here, we present a human in vitro NASH model based on 3D microtissue technology. Engineered to incorporate the primary human hepatocytes, hepatic stellate cells, Kupffer cells (KCs) and liver endothelial cells (LECs), this model includes all the liver cell types that play a crucial role in disease initiation and progression. Upon treatment with free fatty acids and LPS in diabetic medium these microtissues showed key physiological aspects of NASH. The lipotoxic NASH stimuli increased the lipid accumulation within the hepatocytes as well as the tissue secretion of pro-inflammatory markers, such as TNF-a, IL-6, IL-8, MCP-1, MIP-1a and IP-10 . Furthermore, lipotoxic stress stimuli increased the expression of pro-fibrotic markers such as collagen type I and III and the release of pro-collagen type I.
In summary, we present a human 3D NASH model that recapitulates key biological aspects of the NAFLD spectrum of diseases, including inflammation, steatosis and fibrosis. Compatible with high-throughput screening approaches, this model is a powerful tool for assessing efficacy of anti-NASH drugs.
Keywords: NASH, 3D liver co-culture, drug efficacy testing, high-throughtput drug screening
The mixture of persistent organic pollutants present in human follicular fluid stimulates the estradiol secretion by adult granulosa tumor spheroids via the classic and non-classic estrogen receptors. (#299)
A. Ptak1, J. Gogola1, M. Hoffmann1, S. Nimpsz1
1 Jagiellonian University in Cracow, Physiology and Toxicology of Reproduction, Cracow, Poland
Epidemiological studies have found that women have detectable levels of organic pollutants such as hexachlorobenzene (HCB), 2,2-dichlorodiphenyldichloroethylene (p,p’-DDE), polychlorinated biphenyl 153 (PCB153), perfluorooctanoate (PFOA), and perfluorooctane sulfonate (PFOS) in their follicular fluid [1,2]. Thus, these chemicals may act on ovarian tissue in a paracrine manner. Our goal was to elucidate the effects of the mixture of these compounds, similar to the profile found in human follicular fluid, on 17β-estradiol (E2) secretion by KGN cell spheroids, which represent adult granulosa tumor subtype.
In this study KGN cells (RBRC-RCB1154, Riken Cell Bank, Ibaraki, Japan; after approval from Drs. Yoshiro Nishi and Toshihiko Yanase) were cultured using a three-dimensional (3D) model to reflect tumor microenvironment. Spheroids were cultured in DMEM/F12 medium containing 10% FBS with the mixtures of the test compounds, as follows, Mix 1 (2 ng/ml PFOA, 8 ng/ml PFOS, 50 pg/ml HCB, 1 ng/ml p,p’-DDE, and 100 pg/ml PCB153), Mix 10 (10-times concentrate), and Mix 0.1 (10-times diluted compare with Mix 1) with testosterone (100nM) as a substrate. Secretion of E2 was determined by ELISA kits (DRG Instruments GmbH, Marburg, Germany) and the expression of aromatase was evaluated by real-time PCR (Hs00240671_m1, Applied Biosystems/ThermoFisher Scientific) and confirmed by western blot (ab39742, Abcam). In addition, caspase activity was detected using a Caspase-Glo® 3/7 assay kit (Promega, France). Statistical analysis was performed using one-way ANOVA (Tukey’s test, P<0.05).
We found that all of the mixtures stimulated E2 secretion and that this effect was independent of apoptosis. Moreover, a mixture of the five compounds does not affect aromatase expression. To investigate the mechanism by which the mixtures stimulate E2 secretion, we used pharmacological inhibitors and found that the mixtures acted through the classic estrogen receptors ERα and ERβ as well as the non-classical GPR30 pathway. Taken together, our results demonstrate for the first time that mixtures of persistent organic pollutants present in follicular fluids may stimulate E2 secretion through the classic and non-classic estrogen receptors pathways in granulosa tumor cells.
Acknowledgments: This study was funded by the National Science Centre, Poland (grant number 2016/21/B/NZ7/01080).
 Petro, E.M., Leroy, J.L., Covaci, A., Fransen, E., De Neubourg, D., Dirtu, A.C., De Pauw, I., Bols, P.E., 2012. Endocrine disrupting chemicals in human follicular fluid impair in vitro oocyte developmental competence. Hum. Reprod. 27, 1025e1033.
 Petro, E.M.L., D'Hollander, W., Covaci, A., Bervoets, L., Fransen, E., De Neubourg, D., De Pauw, I., Leroy, J.L.M.R., Jorssen, E.P.A., Bols, 2014. Perfluoroalkyl acid contamination of follicular fluid and its consequence for in vitro oocyte developmental competence. Sci. Total Environ. 496, 282e288.
Keywords: persistent organic pollutant, granulosa tumor, estradiol secretion
Characterization of a human proximal tubule epithelial cell/fibroblast transwell co-culture system (#313)
F. Piossek1, N. Schlichenmaier1, S. Beneke1, D. Dietrich1
1 University of Konstanz, Biology, Human and Environmental Toxicology, Konstanz, Baden-Württemberg, Germany
This project is supported by a collaborative research grant from Boehringer Ingelheim (#FP747/17) and the state Baden-Württemberg (KPK-InViTe).
Predicting compound mediated nephrotoxicity in humans is still problematic irrespective of recent advancements in in silico, in vitro and in vivo approaches. Moreover current systems are geared toward identification of single compound toxicity in a single cell type and not optimized to reflect the physiological reality in the patient e.g. polypharmacy of elderly patients with impaired renal function. Indeed, the majority of in vitro approaches address renal proximal tubule toxicity using primary human proximal tubule epithelial cells (hRPTEC), transformed hRPTEC or transformed animal RPTEC in 2D plastic dishes cultured at 21% O2 and high glucose (20 mM). While continuous comparability and quality of primary cells cannot be guaranteed, transformed cells present with reduced functionality due to their transformation. Moreover, primary and transformed renal epithelial cells will provide for falsified readings due to their 2D monoculture, abnormal microenvironment, i.e. missing physiological signaling crosstalk with other cell types, and hyperoxic (21% O2) and hyperglucose (20 mM) conditions.
To overcome the latter obstacles, we are developing a transwell-based co-culture system encompassing human hRPTEC/TERT1 and human fibroblasts (fHDF/TERT166), cultured at physiological glucose (5 mM). Culturing at routine non-physiological O2 (21%) levels were compared to physiological O2 (5%). Co-cultures were characterized with regard to gene expression (mRNA and protein level) and physiological functionality (transepithelial electrical resistance (TEER), lactate:glucose ratio, viability, vectorial anion and cation transport). To determine and to compare the sensitivity of the co-culture systems, single and repeated treatments with clinically relevant cisplatin concentrations were initiated.
Preliminary data at 21% O2 suggest a tight barrier, enabling active vectorial transport of +/- charged molecules in the co-culture systems. Single exposures to cisplatin at concentrations ≤ 10 µM had no impact on TEER whereas higher concentrations severely diminished TEER within 48h (50 µM) or 24h (100 µM) of exposure. Concurrent analyses of co-cultures at 5% O2, as well as repeated cisplatin treatments of co-cultures at 5% and 21% O2 are ongoing.
Keywords: nephrotoxicity, RPTEC/TERT1, transwell co-culture, physiological oxygen, cisplatin
Problem with incorrect classification of substances in terms of irritation or serious eye damage using Short Time Exposure test method (#320)
D. Krakowian1, A. Daniel-Wójcik1, D. Gądarowska1
1 Institute of Industrial Organic Chemistry, Branch Pszczyna, Toxicological studies, Pszczyna, Poland
Short time exposure (STE) test method is a cytotoxicity-based in vitro assay. After exposure to a test item, the cytotoxicity is quantitatively measured as the relative viability of SIRC ( Statens Seruminstitut Rabbit Cornea) cells. Decreased cell viability is used to predict potential adverse effects leading to ocular damage. The test items can be classified as chemicals inducing serious eye damage (category 1 in UN GHS) or as chemicals not requiring classification for eye irritation or serious eye damage (no category).
The study was performed according to OECD TG no. 491 (2018)  to confirm correct classification.
In this study 37 random substances were checked and the results were compared with the ECHA database . There were 3 independent runs with 3 repetitions each.
The confluent monolayer SIRC cells (ECACC 89090404) were treated with two concentrations of the test items (5 % and 0,05% w/w) for 5 minutes. After washing the test items with DPBS, the medium MEMα (ThermoFisher Scientific) with MTT (0,5 mg/ml, Merck) was added and cells were incubated for 2 hours (37 ± 1°C, 5 ± 1 % CO2, 90 ± 10 % RH). The extraction of formazan was performed with 0.04 N hydrochloric acid in isopropanol. Next, the absorbance was measured (FLUOStar Omega) at 570nm with reference wavelength (690 nm). The obtained cell viability is compared to the solvent control (saline or mineral oil) and used to estimate the potential eye hazard of the test chemical.
There were 18 correct (48.6 %) and 11 incorrect (29.7 %) classifications. 8 substances (21.6 %) were not classified ("no prediction can be made") and they require further studies. Importantly, in substances belong to category 1 (according to ECHA database) 3 test items (of 9) were classified as “no category”. What is more, 7 of 14 substances that cause eye irritation (category 2) were also incorrect classified as chemicals not requiring classification. This situation is very dangerous for health, because 27 % of substances were assigned to a safer category.
In conclusion, the STE test method needs to be changed to reduce the number of incorrect categorizations. To improve this method, we strongly recommend adding overnight post-incubation of cells before performing MTT test to exclude the delayed effect of the test item on the cells.
 Test No. 491: Short Time Exposure In Vitro Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage. OECD Guidelines for the Testing of Chemicals, Section 4: Health Effects.
Keywords: STE, eye irritation, eye damage, cell viability, human health
Integration of extracellular metabolomics and intracellular transcriptomics to unravel the mechanisms of 5-FU-induced gastrointestinal toxicity (#326)
T. Souza1, D. F. Rodrigues1, Y. Schrooders1, L. Coyle2, J. Vappiani3, D. Sevin3, D. Jennen1, J. C. S. Kleinjans1, T. M. de Kok1
1 Maastricht University, Toxicogenomics, Maastricht, Netherlands
5-fluorouracil (5-FU) is a chemotherapy agent whose use in the clinic has been hampered by reports of gastrointestinal (GI) adverse drug reactions (ADRs, i.e., mucositis, diarrhea). Despite that, there still exists a gap in the knowledge of the molecular mechanisms leading to toxicity in these target organs, and how these changes can be quantitatively linked to observed ADRs. Therefore, the aim of the current work was to investigate molecular-level alterations induced by 5-FU in cultures of human intestinal organoids derived from colon and small intestine (SI). For this, alterations in the transcriptome and metabolome in the media were quantified in organoids following exposure to three doses (10, 100 and 1000 mM) over a time course (24, 48 and 72h). Alterations at the transcriptome level were first evaluated by generating co-expression networks using the complete set for each organ. Analyses of the modules revealed large global effects induced 5-FU, and investigation of underlying cellular processes yielded a number of modules with equivalent biological responses across colon and SI experimental models. These processes comprised, for instance, cell cycle, mitochondrial-related processes such as the TCA cycle and electron transport chain, as well as transcription and translation-related processes. Pathways involved in inflammation such as TNFaand TGFbwere also found to be enriched across modules. In both cases, the eigengenes (vectors summarizing expression) for each dose/time point pair of such modules were found to vary in a dose-dependent manner, with the highest dose often showing the largest variation when compared to lower doses. To investigate the relationship between gene expression and extracellular metabolomics, correlation analyses were conducted between modules’ eigengenes and levels of metabolites measured in the media. A number of metabolites were significantly correlated to changes at the transcriptome level and an integrated pathway analysis using both transcriptome and metabolome revealed the alteration of metabolic pathways related to metabolism of nucleotides, in particular purines and pyrimidines, as well as urea cycle and metabolism of amino acids. In summary, the results from this work identified relevant co-expression networks embedded with relevant biological information that can be related to the toxic effects induced by 5-FU. Furthermore, integration with metabolomics highlighted specific sections of metabolic networks linking intracellular changes to external traits. Taken together, these findings may serve as basis for further investigations targeting the quantitative modeling of these pathways/networks in drug-induced GI toxicity.
Keywords: 5-FU, organoids, co-expression analysis, transcriptomics, metabolomics
The role of bile salts in cholestatic injury and fibrosis using a human 3D in vitro model (#331)
C. J. Messner1, 2, L. Mauch1, L. Suter-Dick2
1 University of Applied Sciences Northwestern Switzerland, Institute for Chemistry and Bioanalytics, Muttenz, Basel-Land, Switzerland
Bile formation and secretion is fundamental for the successful intestinal absorption of lipids and fat-soluble vitamins. On the other hand, a disrupted regulation of bile acid modulation and accumulation in the liver can contribute towards progressive liver damage and fibrosis. The molecular mechanisms of this process are still unclear; therefore a physiologically relevant human in vitro model would be beneficial to elucidate the role of bile in the onset of fibrosis. A multicellular, 3D liver microtissue comprising HepaRG, THP-1 and hTERT-HSC has been shown to recapitulate key fibrotic events elicited by known pro-fibrotics such as methotrexate. In this work, we aimed at utilising this 3D human in vitro model to investigate the role of bile salts in the progression of cholestatic injury and fibrosis. Furthermore, we investigated the direct effects bile salts may have on the three separate cell types using standard 2D monolayer culture.
We exposed both the 2D monocultures and 3D co-culture systems to bile salts treatment (50% cholic acid & 50% deoxycholic acid) for 7-14 days. Bile salts had a minimal effect on the 2D culture of both hTERT-HSC and differentiated THP-1. Treatment on 2D hTERT-HSC showed no cytotoxicity and no increase in activation markers (α-SMA, Collagens I and IV). The 2D differentiated THP-1 had a slight decrease in viability and no significant increase in TNF-α and TGF-β1 expression. Bile salts had a more pronounced effect on the 2D HepaRG, leading to a decrease in viability and increased miR-122, -192 and -34a release at 24 hours. In comparison in the 3D co-culture of the three cell types, there was an increase in expression of the key fibrotic markers α-SMA, Collagens I and IV, vimentin and the pro-inflammatory markers IL-6, TNF-α and TGF-β1. Exposure to bile salts also led to miRNA release (miR-122, -192 and -34a) and a decrease in albumin production, indicating hepatocellular damage in the 3D culture system. Finally, the expression of CYP7A1, a key regulatory cytochrome in bile acid synthesis, which is known to be inhibited by bile acid accumulation, was also decreased in the microtissue model.
In conclusion, we have demonstrated that the hepatocellular damage occurs in both 2D and 3D culture systems, suggesting that HepaRG are an appropriate cell type for studying bile-induced hepatocellular damage. This is also confirmed by the expected decrease in CYP7A1. We also demonstrated that the bile salt treatment does not result in activation of stellate cells or elicit an inflammatory response in the THP-1 in 2D monolayer culture, which suggests that the hepatocellular damage and the cellular interplay is required for eliciting a fibrotic response. These results suggest that the 3D multicellular microtissue model can recapitulate liver damage caused by bile acids and can therefore be used to elucidate the processes involved during the onset of fibrosis by cholestatic injury.
Keywords: cholestasis, microtissues, fibrosis, model, miRNA
Effects of Electrospun Nanofiber Curcumin on Bisphenol A Exposed Caco-2 Cells (#335)
Y. Turgut1, B. Yurdakok-Dikmen1, R. Uyar1, M. Birer2, F. Acarturk2, A. Filazi1
1 Ankara University, Faculty of Veterinary Medicine/ Department of Pharmacology and Toxicology, Ankara, Turkey
Purpose: Curcumin is the major polyphenolic compound of curcuminoids, extracted from Curcuma longa L. (turmeric). Curcumin gained increasing interest for its anti-inflammatory, anti-diabetic, anti-carcinogenic and anti-rheumatic properties with good tolerability and safety. However, several problems prevent marketing of curcumin as a drug such as the poor aqueous solubility, intense staining color, and extremely low oral bioavailability. In order to enhance the solubility, curcumin loaded polyvinylpyrrolidone (PVP) K90 nanofibers were prepared using electrospinning method and physicochemical properties of nanofibers were characterized. Bisphenol A (BPA), the major endocrine disruptor chemical, which stimulate estrogen receptors at very low concentrations, induce estrogen related carcinogenesis inducing proliferation in colon. Therefore, the aim of this study was to determine the effects of electrospun nanofiber curcumin on Bisphenol A treated human colorectal adenocarcinoma cells (Caco-2) in vitro.
Methods: Electrospinning solution ; consisted of PVP 12% and curcumin (10mg) was prepared in ethanol. The mixture was stirred for 2 h at room temperature to obtain homogeneous solution and used for electrospinning. Caco-2 cells (ATCC HTB-37,USA) were seeded at 80% confluence; where curcumin nanofiber at concentration of 2.7, 6.4 and 12,8 µg/ml were coincubated with BPA at 2nM-2µM. Following 24h coexposure, MTT assay along with standard trypan blue technique by JuLI Br Counting starter kit (NanoEnTek Inc, Seoul, South Korea) were used.
Results: BPA induced proliferation in the cells at 8 nM. Viability of the cells compared to untreated control against curcumin nanofibers were 67,64±1,06 for 2.7 µg/ml, 55.12±1.12 for 6.4 µg/ml, 50,88±3.03 for 12.8 µg/ml; while BPA at 8 nM were 85.97±8.11. A significant difference were observed for curcumin nanofibers compared to BPA only control (p<0.05); while between 6.4 and 12.8 µg/ml, no difference were observed (p>0.05). The current study supports the enhanced cytotoxic potential of curcumin nanofiber effective at 6.4 µg/ml concentration on Caco-2 colon cancer cells; where antiproliferative effects on cell proliferation induced by the environmental carcinogen Bisphenol A were found in vitro.
1) Alcigir ME,Dogan HO,Yurdakok-Dikmen B.,Dogan K., Vural SA, Yilmaz FM,Isgoren A.(2018) An Investigation of the Effects of Curcumin on the Changes in the Central Nervous System of the Rats Exposed to Arochlor 1254 in the Prenatal Period
2) Ruzgar G., Birer M., Tort S., Acarturk F. Studies on Improvement of Water-Solubility of Curcumin with Electrospun Nanofibers
3) Yurdakok-Dikmen B.,Alcigir ME., Dogan HO.,Dogan K.,Vural SA,Yilmaz FM.,Isgoren A.(2018)Effects of Curcumin in PCB Exposed F98 Glioma Cells
4) Xian-Yang Q,Tomokazu F,Linqing Y,Hiroko Z,Hiromi A,Qin Z,Jun Y,Hideko S. Effects of bisphenol A exposure on the proliferation and senescence of normal human mammary epithelial cells
Keywords: Bisphenol A, Curcumin, Electrospun nanofibers, In vitro toxicity, Caco-2
In vitro modelling of the GFB – characterization of a podocyte/endothelial cell co-culture system (#349)
N. Schlichenmaier1, F. Piossek1, S. Beneke1, D. Dietrich1
1 University of Konstanz, Human and Environmental Toxicology, Konstanz, Germany
The kidney is responsible for the excretion of xenobiotics and continuously exposed to drugs. The filtration units of the kidney, the glomeruli, are bundles of capillaries functioning as size-selective glomerular filtration barrier (GFB). The GFB results from the tight interaction of endothelial cells, forming the capillaries, and podocytes, specialized cells that cover the capillaries with interdigitating foot processes. Destruction of the GFB by nephrotoxins or disease, e.g. diabetes, results in glomerulosclerosis, proteinuria and end-stage renal disease. Consequently, to better understand and detect glomerulotoxicity in humans there is a need for a suitable in vitro model system. Obviously, the predictivity of in vitro model systems will be improved by mimicking the normal physiological environment as closely as possible. We addressed this issue by i.) co-cultivating podocytes and endothelial cells, thereby enabling cell-cell interactions and the development of an in vitro GBM, and ii.) cultivating the cells at physiological oxygen levels (10 %).
Cells (PODO/TERT256 & HUVEC/TERT2) were cultivated under 10% and 21% O2. Expression levels of podocyte markers were analyzed at the mRNA (RT-qPCR) and protein level (ICC). Barrier permeability was investigated using fluorescently labelled dextrans of different molecular sizes. Cytotoxicity of known glomerulotoxins was analyzed via LDH leakage.
PODO/TERT256, showing tight cell-cell confluency with elongated flat cells, expressed several podocyte specific markers and reacted to known glomerulotoxins when cultivated at 21% O2. Cultivation at 10% O2, resulted in characteristics typical of primary podocytes, i.e. individualized cells with a more rounded morphology and cytoplasmic protrusions (filipodia). This resembled a more in vivo like phenotype in relation to their functional tasks. Analysis of podocyte specific markers and sensitivity to toxins is ongoing. Concurrently, assessment of barrier permeability demonstrated the ability of PODO/TERT256 to form a size-selective filtration barrier. Preliminary results suggested an increased size-selectivity when PODO/TERT256 were co-cultivated with endothelial cells. Analysis of podocyte specific markers and sensitivity to toxins in the co-culture is ongoing.
Keywords: glomerulus, physiological oxygen, co-culture, nephrotoxicity
The development of a generic physiologically based kinetic model to predict in vivo endocrine activity in rats based on in vitro bioassays (#353)
M. Zhang1, B. van Ravenzwaay2, 1, I. M. C. M. Rietjens1
1 Wageningen University, Division of Toxicology, Wageningen, Netherlands
The development of non-animal based testing strategies of chemicals is important in current human safety testing. Many efforts focus on the development and standardization of in vitro models that provide concentration-response data. However, concentration-response data obtained from in vitro models are inadequate for human risk and safety assessment. In order to use these data for risk assessment purposes, the in vitro concentration-response data should be translated to in vivo dose-response data to obtain points of departure (PODs) to set safe human exposure levels. It has been proven that in vivo dose-response data can be predicted by in vitro concentration-response data using physiologically based kinetic (PBK) modelling-based reverse dosimetry, thus enabling the use of in vitro toxicity data for risk assessment and prioritization. Given the definition of the PBK model, it can be resource and time consuming to develop the model for the individual compound, efforts should be directed at the development of generic PBK models for large groups of compounds. The present study assessed the potential of the generic PBK model to predict the in vivo endocrine activities in rats for a series of compounds. PBK models for these compounds were developed using a generic approach and in vitro concentration-response data from the MCF-7/BOS proliferation assay and the yeast estrogen/androgen screening (YES/YAS) assay were translated into in vivo dose-response data. The benchmark dose (BMD) values derived from the predicted dose-response data were compared with the BMD values obtained from the in vivo uterotrophic assay or in vivo hershberger assay to evaluate the model predictions. The discrepancy in the ability of the in vitro assays to predict the in vivo toxicity may be related to the fact that the variation between the in vitro data of one compound obtained in the same assay could be up to 2 orders of magnitude in terms of EC50. Taken the large variation within the in vitro assay data into account, the predictions are reasonable. The current study indicates the feasibility of using the combination of in vitro toxicity data and a generic PBK model to predict in vivo endocrine activities for groups of endocrine disruptors. Further studies can expand the current approach for other in vivo endpoints.
Louisse J, Beekmann K, Rietjens IM (2016) Use of physiologically based kinetic modeling-based reverse dosimetry to predict in vivo toxicity from in vitro data. Chemical Research in Toxicology
Wetmore BA, Wambaugh JF, Ferguson SS, et al. (2011) Integration of dosimetry, exposure, and high-throughput screening data in chemical toxicity assessment. Toxicological Sciences 125(1):157-174
Zhang M, van Ravenzwaay B, Fabian E, Rietjens IM, Louisse J (2018) Towards a generic physiologically based kinetic model to predict in vivo uterotrophic responses in rats by reverse dosimetry of in vitro estrogenicity data. Archives of toxicology 92(3):1075-1088
Keywords: in vitro-in vivo extrapolation, physiologically based kinetic model, endocrine activity
Particles from different pyrotechnic smokes induced anti-oxidant and inflammatory responses in primary pulmonary cells after air-liquid interface exposure (#370)
C. Corbiere1, F. Cazier-Dennin2, M. Janona1, A. Ollivier1, V. Martin de Lagarde1, C. Logie1, J. - M. Vaugeois1, D. Dewaele3, F. Cazier3, C. Monteil1
1 University of Rouen Normandy, Health Department - ABTE-ToxEMAC, Rouen, France
This work was supported by ANR ASTRID project FUMITOX (project number ANR-15-ASTR-0023)
Smokes are widely used for military or civilian applications: obscuring, signaling, security or festivity. Smokes generate short-lived aerosol clouds which increase atmospheric particulate matters. However, there is a lack of data on the biological effects of such emissions. Moreover, there is a multiple type of smokes, characterized by various compositions and it is not excluded that they generate particles with different toxicological properties.
Consequently, the aim of this study was to develop an in vitro methodology using primary human pulmonary cells (SAEC) to assess toxicological effects of particles obtained from combustion of three different smokes: a red signaling smoke (F1) and two obscurant ones (F3 and F4). Cells were exposed at the Air-Liquid Interface, using a novel approach for preparation of standardized particle suspensions. Cytotoxicity (MTT), gene expression (RT-qPCR) and cytokine secretion (ELISA) were explored after 24h exposure.
Results show that particles did not induce cytotoxicity but altered genes expression that was dependent on particles type. Particles from F1 significantly induced superoxide dismutase 2 (SOD2), NADPH quinone oxidoreductase-1 (NQO-1) and heme oxygenase-1 (HO-1) expressions whereas particles from F3 moderately increased the expression of these genes. HO-1 expression was not modified after exposure with particles from F4 whereas SOD2 and NQO-1 expressions were significantly increased. In addition, particles from F3 and F4 decreased catalase expression. Concerning inflammatory response, particles from the 3 smokes induced IL-8 gene expression (F1>F2=F3). TNF-alpha expression was moderately induced after exposure of particles and only particles from F4 induced IL-6 expression and secretion.
These results showed that all particles types induce an anti-oxidant response as well as an inflammatory response. However, different response profiles were observed, which might depend on the different composition of particles. In conclusion, the methodology used in this study is applicable to the toxicological evaluation of particles produced by different smokes and obscurants and could be useful to assess human health risk.
Keywords: smokes particles, cytotoxicity, Air-Liquide Interface exposure, primary human pulmonary cells, antioxidant and inflammatory responses
Optimization of Spectrophotometric Direct peptide reactivity assay for skin sensitization (#374)
S. - A. Cho1, B. H. Kim2, S. An1
1 AMOREPACIFIC R&D UNIT, Safety and Microbiology Lab/ Safety & Regulatory Research Division, Yongin-si,, Republic of Korea
This research was supported by a grant (19182MFDS49) from Ministry of Food and Drug safety in 2019.
The chemical hapten bind to cellular protein, called haptenation, is considered essential process in skin sensitization. Thus, examination of reactivity of chemicals with peptides or proteins has been considered as candidate animal alternatives for identifying skin sensitization potential. Direct peptide reactivity assay monitored the reactivity of peptides with haptens by using chromatographic methods such as HPLC and adopted OECD TG. Globally, there have been many attempts to develop an easy and accurate in chemico tool with same KE (chemical-peptide reactivity) as DPRA. In previous study, we developed the convenient detecting method using spectrophotometric analysis for the monitoring peptide reactivity with haptens and identified the possibility as a new animal alternative. However, our model has a relatively low accuracy in the lysine peptide single predictive model (50% accuracy), so that some sensitizers couldn’t be predicted as sensitizer. Thus we performed the optimization studies to achieve higher accuracy of lysine peptide reactivity in this study. Lysine (Ac-RWAAKAA-COOH) was used as model peptides and these peptides were reacted with 23 chemicals (19 sensitizers, 4 non sensitizer) that is used as proficiency chemical in animal alternative studies of sensitization at various peptide-chemical reaction ratios. And non-reacted peptides were monitored by the fluorometer using fluorescamine as a detection reagent for free amine group, respectively. The condition of 1:20 peptide-chemical reaction ratio (Lysine peptide 100uM: chemical 2mM) and 15% depletion cut off –lysine model showed higher accuracy (above 80%) than previous our model. From these results, we were able to confirm the possibility of a lysine peptide reactivity assay as a single prediction model with high accuracy.
Cho, S.A., Jeong, Y.H., Kim, J.H., Kim, S., Cho, J.C., Heo, Y., Suh, K.D., Shin, K., An, S., 2014. Method for detecting the reactivity of chemicals towards peptides as an alternative test method for assessing skin sensitization potential. Toxicol Lett 225, 185-191.
Keywords: Skin sensitization, Spectrophotometric, Direct peptide reactivity, animal alternative, cosmetic
Toxicological risk assessment of pyrrolizidine alkaloids - Investigations of the hepatotoxic and genotoxic potential (#384)
L. Rutz1, L. Gao1, J. - H. Küpper2, D. Schrenk1
1 University of Kaiserslautern, Food Chemistry and Toxicology, Kaiserslautern, Rhineland-Palatinate, Germany
Background: Pyrrolizidine alkaloids (PAs) are a large group of natural toxins synthesized as secondary metabolites by different plant species. To date, approximately 600 different PAs are known1. PAs can be found as contaminants in foods like teas, herbs and honey2. They are generally considered acutely and chronically hepatotoxic, genotoxic and carcinogenic3.
 CONTAM (2011), EFSA Journal 9(11):2406.
Keywords: pyrrolizidine alkaloids, genotoxicity, structure-dependent cytotoxicity, micronucleus test, Alamar blue assay
Electrophysiological evaluation of LUHMES cells as model of human dopaminergic neurons (#402)
U. Kraushaar1, D. Loser1, 2, 3, T. Danker2, C. Moeller3, M. Leist4
1 NMI Natural and Medical Sciences Institute, Electrophysiology, Reutlingen, Germany
The loss of dopaminergic neurons in the substantia nigra plays an important role in the development of the Parkinson’s disease. The symptoms of this disease typically occur after around 80% of these neurons degenerated. This cell decay can be caused or promoted by genetic defects or environmental factors including chemical compounds like pesticides. For a proper testing of neurotoxic effects on these neurons as well as for the development of neuroprotective drugs, assays based on animal primary cells lack predictivity since the correlation between animal and human data is weak in some cases. Therefore, models based on human neuronal cells have the potential to overcome the limitations of animal models. One interesting neuronal cell line is the LUHMES (Lund human mesencephalic) line, which consists of immortalized fetal human mesencephalic precursor cells that can be differentiated into fully post-mitotic dopaminergic neurons within one week.
We currently investigate the electrophysiological properties of these neurons using manual and automated patch clamp as well as high-throughput calcium imaging for a functional characterization on both single cell and network level.
LUHMES neurons were capable to generate spontaneous and stimulated action potentials. The underlying Na+ channels were TTX-sensitive. Biophysical and pharmacological tests indicate the presence of the Nav 1.2 subtype.
Furthermore, we checked for the presence of neurotransmitter receptors and compared them to data obtained by mRNA analysis from these cells. We found that several key receptor subtypes were expressed functionally in the cells, including dopamine, serotonin and acetylcholine receptors. Next, we investigated whether the neurons were capable of forming functional neuronal networks using a high-throughput calcium imaging system. While at rest cells were quiescent, oscillatory network activitiy was visible in the presence of neurotransmitter receptor agonists like serotonin and norepinephrine as well as by modulating the extracellular K+ and Ca2+ concentration. These oscillations were sensitive to modulators like Haloperidol or the anticonvulsive drug Phenytoin dose dependently.
The results show that differentiated cells derived from LUHMES cells express electrophysiologically neuronal characteristics and form functional networks. The capability of using increased throughput techniques including automated patch clamp and HTS Ca imaging makes this cells attractive for neurotox experiments at industrial relevant scales.
Keywords: patch clamp, Ca imaging, dopaminergic neurons, compound test, LUHMES
New-Tiered Approach to In Vitro Predictive Toxicity Screening using retrospective analyses. (#403)
A. Marmugi1, B. Kiehr1, H. Ahrens2, J. - C. Garcin1, A. Becker1
1 BAYER SAS, Toxicology Profiling & Coordination, Crop Science, Sophia Antipolis, France
Background: Product safety is a major question to address during the agrochemical development process to ensure that products do not pose adverse effects to human. One of the greatest challenges is accurately predicting unanticipated adverse effects in animal toxicity studies that can result in late stage failure of promising new candidates. Early in vitro screening of new candidates is therefore essential to improve the selection process, as well as to minimize and refine animal use.
Objective: Our goal is to build a tiered toxicity screening toolbox with assays that allow a robust translation of in vitro toxicity data into meaningful prediction of potential in vivo effects.
Methods: We have incorporated a battery of assays that provides predictive indicators for endocrine disruption (ED), genotoxicity, carcinogenicity and developmental toxicity. Pharmacokinetic profiling is conducted in parallel using in vitro ADME assays and in vivo kinetics in a minimal number of animals. This combination gives a view of the potential cellular activity and exposure.
Results: The screening battery was tested on candidates from a promising chemical class. The approach was first evaluated through a retrospective analysis. We used a former candidate from the same class with late stage development failure, partly due to ED alerts observed in rodents. The experimental data (short-term and developmental toxicity study) were compared to the in vitro systems, to evaluate the predictiveness and accuracy of the in vitro data. We observed robust correlations, thus validating the screening approach for a comprehensive interpretation of data for new candidates. We showed that they display a much-improved toxicity profile compared to the reference compound with a remarkably lower bioavailability, a low ED risks based on safety margin and a lower teratogenicity index using zebrafish embryos.
Conclusions & Perspectives: Retrospective weight of evidence validated our new-tiered toxicity screening. Only the two safest candidates identified were promoted for further toxicity studies. Using the same conceptual approach, we are currently conducting data analysis of at least 100 fully developed molecules in order to learn about the correlation or gaps of our screening strategy.
Keywords: agrochemical product, in vitro screening, retrospective analysis, endocrine disruption, meaningful prediction of in vivo effects
Using the Real Architecture For 3D Tissue (3D RAFT™) System as a Versatile Tool to Build in vitro Models Relevant for Toxicity Testing (#414)
M. K. Bunger1, N. El-Andaloussi2, S. Schapermeier2, S. Busch2, T. D'Souza1, C. Schwartz2, J. Schroder2
1 Lonza, Walkersville, Inc, Morrisville, North Carolina, United States of America
Conventional in vitro assays are based on cells grown on two-dimensional (2D) substrates, which are not representative for the true in vivo cell environment. In tissue environments, cells interact with neighboring cells and with the extracellular matrix (ECM). Three-dimensional (3D) cell culture methods mimic these interactions and allow cells to grow in structures resembling more the in vivo environment.
The RAFT™ 3D Culture System uses a collagen matrix at physiologically relevant concentrations. Cells and neutralized collagen are mixed and dispensed into wells of standard cell culture plates or transwell inserts, and subsequently incubated at 37°C to allow the formation of a hydrogel. Specialized RAFT™ Absorbers are placed on top of the hydrogels. These absorbers gently remove abundant medium and compact the hydrogel to a layer approximately 100 μm thick. The cultures are then ready to use, but additional epithelial or endothelial cells may be added on top.
The resulting models provide valuable tools to investigate tissues in an in vivo-like micro-environment, potentially for use in pre-clinical efficacy and safety testing. This presentation focuses on skin, lung and liver models.
A full-thickness skin model was generated by embedding primary human dermal fibroblasts within the RAFT™ Collagen and seeding and differentiating human primary keratinocytes on top of the air-lifted cultures. Histological and immuno-histochemical evaluation confirmed the resemblance to native skin.
A RAFT™ 3D lung co-culture model containing normal or asthmatic bronchial epithelial and smooth muscle cells was compared to 2D cultures with respect to cell proliferation and morphology as well as growth factor and cytokine secretion.
We also demonstrate the feasibility of using the RAFT™ 3D System to create a robust model for the long-term maintenance of primary human liver cells. We compared the viability and morphology of primary human hepatocytes and the maintenance of Cytochrome P450 activity grown in the traditional Sandwich Model with that of cell cultured in the RAFT™ 3D System. Hepatocyte metabolism is stabilized in the RAFT™ 3D Cell Culture System for up to 17 days in culture, which enables long-term toxicity analysis using primary hepatocytes.
Keywords: 3D Cell Culture, In vitro toxicity testing, liver, lung, skin
Implementation of a mucus containing advanced in vitro model of the human intestinal barrier for a more predictive evaluation of food grade nanomaterials (#418)
C. Hempt1, 2, C. Hirsch1, P. Wick1, T. Buerki-Thurnherr1
1 Empa, Particles-Biology Interactions, St.Gallen, Switzerland
Nanotechnology provides many benefits to the food industry due to their versatile properties. Engineered nanomaterials (ENM) deliver for example new tastes, antimicrobial properties or improve the nutritional value of food (novel food). However, the impact of ENMs on the gut epithelium and their translocation through the intestinal barrier is still poorly investigated and understood. Mechanistic insights required for the safe design and use of ENMs in food applications can be obtained from advanced human in vitro models of the intestinal barrier that contain mucus and different cell types of the intestine (e.g. enterocytes, goblet cells and M cells). The mucus layer as a physical barrier is particularly important to achieve predictive results, however, it interferes with many conventional assays.
Keywords: advanced in vitro model, food safety, nanotoxicology, E551, SAS
In vitro toxic assessment of pyrotechnic red signaling smoke particles (#425)
C. Corbière1, M. Mekki1, C. Rozay1, F. Cazier2, D. Dewaele2, C. Logie1, J. - M. Vaugeois1, V. André3, C. Monteil1
1 University of Rouen Normandy, Health Department/ABTE-ToxEMAC, Rouen, France
This work was supported by the Direction Générale de l'Armement and the Regional Council of Haute-Normandie
Pyrotechnic smokes are widely used for military operations such as obscuring or signaling purposes as well as for civilian applications such as security or festivity. Smokes generate short-lived aerosol clouds which increase atmospheric particulate matters. Because of the recognized health risks for military and civilians exposed to old smokes, a variety of alternative pyrotechnic smokes have been developed. However, there is a lack of data on the biological effects of emissions produced by these alternative pyrotechnic smokes.
In this study, we examined the toxicity of particles obtained from combustion of a red signaling smoke (RSS) on human alveolar cells (A549), and studied the anti-oxidant and inflammatory responses by RT-qPCR. Cytotoxicity (MTT, trypan blue tests), cell cycle distribution and gene expressions were assessed after 24h and 48h exposure to particles collected by an impactor placed to the smoke source and suspended in the culture medium. In parallel, mutagenicity of organic extract prepared from RSS was evaluated using the bacterial Ames assay.
Particles significantly decreased cell viability (trypan blue) at 0.25 mg/mL whereas mitochondrial activity (MTT) was unaltered at this concentration. Exposure to 0.25 mg/mL of particles significantly increased cells in the sub-G1 phase and decreased cells in the G0/G1 phase. At this concentration, particles induced superoxide dismutase 2, heme oxygenase-1, NADPH quinone oxidoreductase-1 as well as IL-6 and IL-8 expressions. In parallel, Ames test showed significant response in Salmonella typhimurium tester strains TA98 + S9mix at 12.5 µg/plate (≈ 0.06 mg/mL), and in a larger extent at 2 µg/plate (≈ 0.009 mg/mL) in YG1041 + S9mix, a strain highly sensitive to aromatic amines.
Results showed that it is relevant to analyze multiple biomarkers to evaluate effect of particles. In this study, particles from RSS induced antioxidant and inflammatory responses as well as mutagenicity. These effects are likely due to the chemical composition of particles that contained numerous compounds (aromatic amines, quinones, naphthalene derivatives, azoic dye derivatives and metals). This study outlined the requirement of improving the knowledge of the toxicity of pyrotechnic mixtures like smoke particles in the context of protection of the human health.
Keywords: signaling smoke particles, cytotoxicity, mutagenicity, human alveolar cells, antioxidant and inflammatory responses
Quantification of seizurogenic activity with multiwell microeletrode array technology for proconvulsant risk assessment. (#434)
K. Gkatzis1, D. Millard1, H. Hayes1, A. Nicolini1, C. Arrowood1, J. Ross1
1 Axion BioSystems , Atlanta, Georgia, United States of America
The lack of advancement in anti-epileptic drugs (AEDs) over the last 30 years, along with the continued need for improved proconvulsant screening in drug safety, motivates the need for new assays of seizurogenic neural activity. Previous work has established an in vitro approach for detecting and quantifying seizurogenic activity using multiwell microelectrode array (MEA) technology, providing a predictive and high-throughput avenue for the evaluation of the efficacy of AEDs and the proconvulsant risk of other drug candidates. Here, we present an updated assay of seizurogenic activity based upon guidelines developed in the Translational Biomarkers of Neurotoxicity (NeuTox) Committee of Health and Environmental Science Institute (HESI), a consortium of academic, commercial and pharmaceutical representatives working towards the development of in vitro assessment of proconvulsant risk. We used previously published metrics for detection of burst spiking events and the quantification of synchronisation across a neural population, in spontaneous and evoked conditions. Data are included from cryopreserved rat cortical neurons evaluated with the 10 compounds selected by NeuTox consortium, which include reference compounds with known proconvulsant risk via multiple mechanisms and negative control compounds. Our results support the combined use of spontaneous and evoked neural activity, collected using multiwell MEA technology, for the high throughput evaluation of complex neuronal networks in vitro to quantify the proconvulsant risk of candidate pharmaceuticals in a pre-clinical setting.
Keywords: Microelectrode array, MEA, Neurotoxicity, Safety assessment, Seizurogenic activity
Prediction of human cardiotoxicity of methadone by a combined in vitro – physiologically based kinetic (PBK) modelling- based reverse dosimetry approach (#437)
M. Shi1, M. Strikwold2, I. M. C. M. Rietjens1, H. Bouwmeester1
1 Wageningen University, Division of Toxicology, Wageningen, Netherlands
Cardiotoxicity is a leading cause of drug failure during development and an adequate preclinical strategy that predicts in vivo human cardiotoxicity would thus be of great value. The aim of the present study was to develop an integrated animal alternative testing strategy to predict human cardiotoxicity of chemicals. We have shown before that the combination of an in vitro assay and PBK modelling-based reverse dosimetry can be very powerful to predict in vivo dose-response curves for different toxicological endpoints. In the present study we provide data extending this principle to cardiotoxicity in humans. Methadone was used as a model compound as several human case studies report cardiotoxic side effects in clinical settings. Here we assessed the effect of methadone on cardiac electrophysiology using the multi-electrode array (MEA) combined with human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). The PBK model was developed based on metabolic parameters obtained from in vitro liver microsomal incubations and parameters derived from in silico simulation and the literature. Using PBK modelling-based reverse dosimetry, the in vitro concentration dependent prolongation of field potential duration was translated into in vivo dose dependent prolonged QT interval. The in vitro effective concentration was corrected for protein binding (unbound fraction) to extrapolate to the real-life conditions in humans. The predicted in vivo 10% effective dose was used as point of departure (PoD) to evaluate the in vitro PBK modelling-based reverse dosimetry approach. Our results show that the PoDs derived from our in vitro studies were comparable with the PoDs derived from published clinical studies with less than a 4.3-fold difference. We also found that protein binding in plasma is an influential factor in the adverse cardiac effects of methadone. Therefore the individual variation in plasma binding might provide an important factor in a personalized prediction of undesirable side effects of the clinical treatment. The results provide a proof of principle that PBK modelling-based reverse dosimetry of in vitro data obtained using the MEA and hiPSC-CM can well predict the electrophysiological cardiotoxicity in humans and provide a promising tool for detecting cardiac safety liabilities during drug development.
Keywords: Physiologically based kinetic modelling, reverse dosimetry, in vitro in vivo extrapolation (IVIVE), cardiac electrophysiology, methadone
Hyperoxia reduces benzo[a]pyrene-induced toxicity by increasing the activation of Nrf2 in HaCat cell. (#472)
Y. C. Kwon1, M. K. Kim1, J. S. Kang1, B. M. Lee1
1 SungKyunKwan University, Division of Toxicology, Suwon-si, Republic of Korea
Benzo(a)pyrene (BaP) can be exposed to skin via environment, soil, air, fire, tyres, automobile exhaust, and so on and can therefore cause skin damage including skin cancer and aging. The main factor for reducing skin damage is antioxidant and detoxifying enzymes, which are regulated by Nrf2. Hyperoxia means a situation higher than the concentration of atmospheric oxygen, and is being used in various medical fields. In this study, hyperoxia was investigated in reducing the toxicity caused by BaP in the skin. Under the condition of hyperoxia, HaCat cells treated with BaP increased Nrf2 mediated by NF-κB, GSK-3β, p38 MAPK and PPARα activities. Hyperoxia also increased the expressions of HO-1, SOD2, GPX-1/2 that reduced toxicity by BaP. Thus, hyperoxia may regulate the enzymes involved in detoxification by promoting the activity of Nrf2 in HaCat cells.
Keywords: Benzo[a]pyrene, hyperoxia, HaCat, antioxidant, Nrf2
High content in vitro assessing of cardiotoxic risk and adjuvant chemotherapy effects in breast cancer (#480)
E. Dragicevic1, K. Juhasz1, 2, O. Reinhardt3, U. Thomas1, S. Stölzle-Feix1, F. Alves3, 4, N. Fertig1
1 Nanion Technologies GmbH, Munich, Germany
New anticancer agents have led to higher life expectancy for cancer patients. However, treatment related morbidity factors such as cardiac toxicity have become important issues for long-term cancer survivors. Cardiotoxic side effects such as arrhythmia, thromboembolism and myocardial ischemia are common with anti-cancer drugs. This led to the development of cardio-oncology field, to promote cardiovascular health while providing the best cancer therapy.
Using change in impedance, we monitored breast cancer cell regrowth after chemotherapy treatment in vitro, coupled with the acute and chronic effects of this treatment on human stem cell derived cardiomyocytes (hsc-CMs). One of the standard clinical regimens for breast cancer is a combination of cyclophosphamide, adriamycin (doxorubicin) and 5-fluorouracil (CAF). Even though initially successful, tumor recurrence after this therapy remains a major cause of mortality in breast cancer patients. We investigated responses from murine H8N8 (immortal mammary carcinoma cell line with tumor stem cell properties) and H8N8 T3.2 (once-treated recurrent tumor variant) cells, to single and recurrent CAF treatment. Changes in impedance and confluency of these cells were used as a measure of toxicity, with cell viability monitored under physiological conditions for 500h. Dose- and treatment dependent effects of CAF clinical treatment on cycle- regrowth of tumor cells were observed.
We further investigated putative cardiovascular side effects of CAF mix and paclitaxel (acute and chronic) on hsc-CMs viability. We observed the cardiotoxic effects of paclitaxel (a microtuble stabilizing drug approved for the treatment of breast, ovarian and lung cancer). Paclitaxel and CAF also induced negative changes in cell contraction properties. hsc-CMs´ viability and beating patterns were monitored over 190 h. Paclitaxel showed a time and dose dependent decrease in base impedance and impedance amplitude, cyclophosphamide and 5-fluouracil shown no or small effect, while doxorubicin shown significant toxic effects in all combinations.
In summary, long-term high-resolution impedance monitoring provides amenable insights into dynamics of cell proliferation and contraction, for in vitro investigations of adjuvant chemotherapy in both cancer and cardio-oncology fields.
Keywords: cardio-oncology, label-free, impedance, toxicity, cancer
In vitro skin sensitization testing of Medical Devices using GARD® (#482)
R. - M. Jenvert1, A. Johansson1, O. Larne1, E. Aaltonen1, A. Jerre1, R. Gradin1, H. Johansson1
1 SenzaGen, Lund, Sweden
Allergic contact dermatitis can severely affect quality of life and is induced by certain substances, referred to as sensitizers. In order to prevent individuals from exposure, chemicals and materials in contact with patients are required to be safety tested. Skin sensitization is included in the Biological Evaluation of Medical Devices (ISO 10993-1:2018) as one of three biological endpoints to be evaluated for all Medical Devices. This test commonly involves animal testing (ISO 10993-10:2010), but there are growing regulatory, economic and public interests for animal-free test methods.
The GARD platform is an in vitro state-of-the-art assay developed for the identification of sensitizers. The assay is based on a human dendritic-like cell line, SenzaCells, and analysis of gene expression using pattern recognition post stimulation.
The guidance document describing preparation of sample extracts of medical devices (ISO 10993-12:2012) requires the use of both polar and non-polar extraction vehicles (typically saline and an oil); the latter a challenge for many in vitro assays. Previously, we have shown that GARD is compatible with both saline and oil.
In this study, polymers spiked with strong, moderate or weak sensitizers and non-spiked material were used as model materials (produced by Research Institute of Sweden) to develop the GARD assay for testing of Medical Devices. Additionally, commercially available medical grade materials (supplied by Medizintechnik Promedt) were used as control materials.
All the polymers used as model materials were correctly classified; spiked material as sensitizers, and non-spiked materials as non-sensitizers. The commercial medical grade materials were all classified as non-sensitizers.
Here, we show the results from the in-house validation, confirming the extended applicability domain for GARD to facilitate testing of polar and non-polar extraction vehicles and detection of leachables in extracts from solid materials. Thus, GARD can be used as an in vitro alternative for assessment of skin sensitization for Medical Devices and medical grade materials.
Johansson H. et al. The GARD assay for assessment of chemical skin sensitizers. Tox in vitro. 2013
Keywords: GARD, Skin senstization, in vitro, Medical Devices, ISO 10993
Efficient transfection and sustained long term functionality of primary human hepatocytes (#495)
N. Kukli1, S. Schuell1, M. K. Bunger2, A. Toell1, J. Schroeder1, M. Stosik1
1 Lonza Cologne GmbH, Cologne, North Rhine-Westphalia, Germany
Purpose: Primary Human Hepatocytes (PHH) are the state-of-the art in vitro human liver model system in the field of toxicology. Just as virtually all non-dividing primary cells, PHH are difficult to transfect. Furthermore, PHH tend to lose their typical liver functions rapidly in culture. In this study, we optimized the thawing, transfection and culture procedure for cryopreserved PHH. Transfection efficiency and hepatocyte functionality were analyzed over 7 days.
Keywords: Hepatocytes, Transfection, Functionality, CRISPR, human hepatocytes
Evaluation of an in vitro assay for skin sensitization of medical devices (#499)
C. Pellevoisin4, F. Cottrez2, J. Johansson1, E. Pedersen2, K. Coleman3, H. Groux1
1 ImmunoSearch, Grasse, France
Purpose: Skin sensitization, one of three biocompatibility tests recommended for all medical devices is still based on in vivo approaches (ISO 10993-10). Yet, the recent validation of in vitro skin irritation test of medical device extracts demonstrated the added value of reconstructed human models such as SkinEthic RHE, in the context of medical devices (ISO DIS 10993-23). The goal of this study was to evaluate the capacity of SENS-IS assay, a quantitative analysis of specific genes expressed in Episkin or SkinEthic RHE models, to predict in vitro skin sensitization potential of medical device extracts.
Keywords: skin sensitization, medical devices, Skinethic RHE, SENS-IS, in vitro
Study of the effect of quaternary ammoniums on dendritic cells in vitro (#505)
M. Peyneau1, 2, M. Zeller1, M. Pallardy1, S. Chollet-Martin1, 2, L. de Chaisemartin1, 2, S. Kerdine-Römer1
1 Inserm UMR-S 996 Team 2 - Drug and Chemical Allergy, Immunotoxicology And Immunopathology, Faculté de Pharmacie, Université Paris-Sud, Châtenay-Malabry, France
*These authors participated equally to this work
Neuromuscular blocking agents (NMBA) are the first incriminated molecules in intraoperative anaphylaxis, leading to high mortality risk. These reactions often occur during the first contact with the drug, suggesting that patients have previously been sensitized by exposure to molecules with structures common to NMBA. Pholcodine, a morphine-derived molecule used for its antitussive properties, has already been suggested as a potential sensitizer for NMBA allergic patients. Indeed, after withdrowal of pholcodine in Norway, patients were clinically more tolerant to NMBA. However, despite pholcodine withdrawal, IgE sensitization to NMBA remains high, suggesting other compounds might be involved. Structure-activity studies and epidemiological analysis have suggested that quaternary ammonium compounds (QA) may play this role, but the immunological mechanism remains unknown. Moreover, QA are presents in many daily use products (cosmetics, detergents, disinfectants ...).
This work aims to document the involvement of eight commonly used molecules containing a QA and of pholcodine (tertiary amine) in the immunization towards NMBA. Since dendritic cells (DC) are essential in the initiation of the immune response, we studied the DC activating ability of these molecules using two in vitro models: DC derived from fresh human monocytes (MoDC) and THP-1 cell line considered as DC-like.
The results showed that hexadecyltrimethylammonium bromide (CTAB), ethylhexadecyldimethylammonium bromide (EHD), polyquaternium-7, polyquaternium-10 and pholcodine, induce an increased expression of activation markers CD54 and CD86 on THP-1. Moreover, CTAB and EHD also increase the expression of CXCR4 on MoDCs. We also found an induction of pro-inflammatory cytokine production (IL-8, TNFα) and an activation of MAP kinases and NFκB intracellular pathways by these MoDCs exposed to QA.
These results suggest that some molecules with a QA can induce DC maturation and thus potentially initiate a specific immune response. The following part of this study will investigate the effect of QA-activated DCs on the activation and polarization of T lymphocytes. In addition, in vivo exposure models will allow us to confirm these data and understand the mechanisms involved.
Keywords: professionnal allergies, THP1, immunotoxicity
Human-based primary neural progenitor cells as a 3D in vitro model to investigate neurodevelopmental toxicity of Chinese Herbal Medicines (#507)
J. Klose1, U. Hübenthal1, J. Tigges1, L. Li2, 3, C. C. Wang2, 3, E. Fritsche1, 4
1 IUF-Leibniz Research Institute for Environmental Medicine, Düsseldorf, Germany
Traditional Chinese Medicine (TCM) has been applied for thousands of years to treat or prevent all kinds of health problems. Specifically Chinese Herbal Medicines (CHMs) have been widely used during pregnancy to promote the health of mothers and fetuses. However, information on toxicities of most CHMs that are being used during pregnancy is sparse. Considering the fact that the nervous system, especially during development, is a sensitive target, it is essential to assess if CHMs taken during pregnancy might exert adverse effects on brain development. Currently, developmental neurotoxicity (DNT) testing is performed according to in vivo guideline studies, which is resource-intensive with regards to number of animals, time and costs and bears the issue of species extrapolation.
We have developed a 3D neurosphere in vitro model based on human primary neural progenitor cells (NPC), which mimics a variety of neurodevelopmental processes, i.e. key events (KEs), like NPC proliferation, migration and differentiation into neural effector cells (astrocytes, neurons and oligodendrocytes). Using this model we analysed the effects of selected CHMs (Tian Ma (TM) and Lei Gong Teng (LGT)) on these endpoints. According to in vivo toxicity studies, TM is classified as non-toxic, while LGT exerts toxicity to the central nervous and cardiovascular systems and is thus classified as a strong toxic CHM. Based on the results of the “Neurosphere Assay” we observe that TM does not affect any of the analysed endpoint, while LGT reduces NPC migration and differentiation.
This pilot study indicates that testing CHM with the in vitro “Neurosphere Assay” might be helpful for the assessment of CHM safety. More data are needed to substantiate these findings and in the end more tests covering a broader variety of neurodevelopmental endpoints should be performed.
Keywords: in vitro, 3D in vitro model, developmental neurotoxicity, neural progenitor cells, chinese herbal medicine
Comparison of the transport of sulfated and non-sulfated bile salts by rat and human Mrp2/MRP2 and Bsep/BSEP transporters (#526)
N. Szilvasy1, B. Mártonné Tóth1, P. Pádár2, Z. Tímár2, Z. Gáborik1, E. Kis2, P. Krajcsi1
1 SOLVO Biotechnology, Budapest, Hungary
The aim of the study was to investigate the transport of chenodeoxycholate (CDC), its glycine-conjugated form (GCDC), and the sulfated forms of both (3S-CDC and 3S-GCDC) by rat and human Mrp2/MRP2 and Bsep/BSEP to map similarities between rat and human transporter affinities. In addition, CDC, GCDC, 3S-CDC, and 3S-GCDC transport was compared to taurocholic acid (TCA) transport. Vesicular transport assay allows the investigation of efflux transporters in vitro. Plasma membrane prepared from rat and human Mrp2/MRP2 and Bsep/BSEP overexpressing human embryonic kidney 293 cells form inside-out vesicles, that enables efflux transporters to pump their substrates into the vesicle. Bile salt export pump (BSEP) is the most important transporter of bile acids across the canalicular membrane of hepatocytes and, because of this, the functional deficiency of BSEP transporter resulting from BSEP mutations leads to progressive familiar intrahepatic cholestasis type 2 or type 2 benign intrahepatic cholestasis. Similar to BSEP, multidrug resistance-associated protein 2 (MRP2) is also localized in the canalicular membrane of hepatocytes but it is also expressed in renal proximal tubule cells, enterocytes (luminal side) and solid tumors as well. MRP2 is responsible for the transport of conjugated bilirubin and divalent bile salts from the hepatocytes. MRP2 mutation in human causes Dubin-Johnson syndrome, which involves chronic conjugated hyperbilirubinemia. Rat Mrp2 and Bsep transporter genes correspond to 88% and 91% to human MRP2 and BSEP genes, respectively, although there might be dissimilarities in their substrate affinity/specificity. To obtain the best prediction of the function of bile acid transporters of human based on animal experiments, differences in substrate affinity need to be mapped. Despite the thorough preclinical testing, drug-induced cholestasis is still frequent in humans. Currently, the most commonly used substrate for examining bile acid transporters is TCA, however, TCA is not the most relevant bile acid in human. Focusing on a bile acid that is more specific to human and transported with similar affinity on rat and human Bsep/BSEP or Mrp2/MRP2 could be more predictive than examining TCA. The results (KM, µM) showed no significant CDC transport on any of the transporters. KM of GCDC transport on rat and human Bsep/BSEP is similar (Bsep: 2.506; BSEP: 2.652), while human BSEP shows more than 25 times higher affinity for TCA than rat Bsep (Bsep: 40.71; BSEP: 1.460). Human BSEP and MRP2 also have high affinity for 3S-CDC (BSEP: 10.38; MRP2: 14.67). In rat, the transport of 3S-CDC and 3S-GCDC was only significant on Mrp2 with KM=47.67 and 14.48, respectively. Human BSEP and MRP2 also transported 3S-GCDC with KM=8.716 and 13.61. In summary, both rat and human Mrp2/MRP2 transported only the sulfated forms with similar affinity, while rat and human Bsep/BSEP showed significant difference in substrate specificity.
Emese Kis, E.I., Zsuzsa Rajnai, Márton Jani, Dóra Méhn, Krisztina Herédi-Szabó, Peter Krajcsi, , BSEP inhibition – In vitro screens to assess cholestatic potential of drugs. Toxicology in Vitro, 2012. 26(8): p. 1294–1299.
ASHY A.A., A.M.A.M., MECCAWY A.A., BANJAR Z.M., ABDULRAFEE A.A. and NASR H.A. , Composition of human hepatic bile. ArabJjournal of Laboratory Medicine, 1993. 19(1): p. 189-193.
GERD A. KULLAK– UBLICK, B.S., PETER J. MEIER, Enterohepatic Bile Salt Transporters in Normal Physiology and Liver Disease. Gastroenterology, 2004. 126: p. 322–342.
Manmeet S. Padda, M.D., Mayra Sanchez, M.D., Abbasi J. Akhtar, M.D., James L. Boyer, M.D., Drug Induced Cholestasis. Hepatology, 2011. 53(4): p. 1377–1387.
Szakács, V.A., Ozvegy-Laczka C, Sarkadi B., The role of ABC transporters in drug absorption, distribution, metabolism, excretion and toxicity (ADME-Tox). Drug discovery today, 2008. 13(9-10): p. 379-93.
Waddah A. Alrefai, R.K.G., Bile Acid Transporters: Structure, Function, Regulation and Pathophysiological Implications. 2007. 24(10).
Bruno Stieger, Y.M., Peter J. Meier, The bile salt export pump. Pflügers Archiv - European Journal of Physiology, 2007. 453(5): p. 611–620.
Hisamitsu Hayashia, T.T., Hiroshi Suzukib, Reiko Onukia, Alan F. Hofmannc, Yuichi Sugiyama, Transport by vesicles of glycine- and taurine-conjugated bile salts and taurolithocholate 3-sulfate: A comparison of human BSEP with rat Bsep. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2005. 1738(1-3): p. 54–62.
Ronald Oude Elferink, A.K.G., Genetic defects in hepatobiliary transport. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 2002. 1586(2): p. 129–145.
Cheng, Y., et al., In vitro model systems to investigate bile salt export pump (BSEP) activity and drug interactions: A review. Chem Biol Interact, 2016. 255: p. 23-30.
Akita H1, S.H., Ito K, Kinoshita S, Sato N, Takikawa H, Sugiyama Y., Characterization of bile acid transport mediated by multidrug resistance associated protein 2 and bile salt export pump. Biochimica et biophysica acta, 2001. 1511(1): p. 7-16.
Emmanuelle Martinota, L.S., Marine Baptissarta, Jean-Marc Lobaccaroa, Françoise Cairaa, Claude Beaudoina, David H. Volle, Bile acids and their receptors. Molecular Aspects of Medicine, 2017: p. 2.
Bhogal HK1, S.A., The molecular pathogenesis of cholestasis in sepsis. Frontiers in bioscience, 2013. 1(5): p. 87-96.
Floriane Montanari, G.F.E., Prediction of drug–ABC-transporter interaction — Recent advances and future challenges. Advanced Drug Delivery Reviews, 2015. 86(23): p. 17-26.
Keywords: bile salt, BSEP, MRP2, KM
ALT4EI: Evaluation of eye irritating potential of 59 chemicals using EpiOcular™ time-to-toxicity (EpiOcular ET-50) neat and dilution protocols (#530)
S. Letasiova1, H. Kandarova1, E. Adriaens2, S. Verstraelen3, A. R. Van Rompay3
1 MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia
Evaluation of the acute eye irritation potential is part of the international regulatory requirements for testing of chemicals. The objective of the ALT4EI (ALTernatives for Eye Irritation) project was to confirm the testing strategy developed in the CON4EI (CONsortium for in vitro Eye Irritation testing strategy) project. These projects focussed on the development of tiered testing strategies for eye irritation assessment for all drivers of classification and evaluation of whether the test methods can discriminate chemicals not requiring classification for serious eye damage/eye irritancy (No Category) from chemicals requiring classification and labelling for Category 1 (Cat 1) and Category 2 (Cat 2).
A new set of 59 chemicals (41 liquids: (un)diluted, and 18 solids) was tested using the reconstructed human cornea-like epithelium (RhCE), EpiOcular, in two EpiOcular time-to-toxicity Tests (Neat and Dilution ET-50 protocols). The set of chemicals contained 32 chemicals not requiring classification (No Cat) and 27 chemicals requiring classification (16 Cat 2 and 11 Cat 1). The chemicals were tested blinded in two independent runs by MatTek In Vitro Life Science Laboratories. In this study, a testing strategy to achieve optimal prediction for all three classes that was developed in CON4EI project (which combines the most predictive time-points of both protocols and which tests liquids and solids separately) was used.
Using the CON4EI testing strategy, we were able to identify correctly 63,6 % of the Cat 1 chemicals, 56,6 % of the Cat 2, and 76,6 % of No Cat chemicals. Reproducibility between both runs was 88,7 %. The combination of the EpiOcular ET-50 neat and dilution protocols seem to be promising in an integrated testing strategy (ITS) for eye irritation assessment.
Keywords: ALT4EI, EpiOcular ET-50, ocular irritation assay, in vitro, testing strategy
Mitochondrial impairment and oxidative stress play an important role in the toxicity of synthetic cathinones to dopaminergic SH-SY5Y cells (#537)
J. Soares1, V. M. Costa1, H. Gaspar2, S. Santos3, M. D. L. Bastos1, F. D. Carvalho1, J. P. Capela1, 4
1 Faculdade de Farmácia da Universidade do Porto, UCIBIO, REQUIMTE (Rede de Química e Tecnologia), Laboratório de Toxicologia, Departamento de Ciências Biológicas, Porto, Portugal
β-keto amphetamines, widely used as alternatives to amphetamines, with which they share the phenethylamine backbone, have been shown to display neurotoxic properties. In this study, the mechanisms by which two synthetic cathinones, 3,4-dimethylmethcatinone (3,4-DMMC) and 4-methylmethcathinone (4-MMC), exert their toxicity in vitro were evaluated, using methamphetamine (METH) as comparative agent, in differentiated SH-SY5Y cells.
The dopaminergic phenotype was achieved by treatment of SH-SY5Y cells with retinoic acid and 12-O-tetradecanoyl-phorbol-13-acetate. These differentiated cells were exposed to 0-5 mM 3,4-DMMC, 4-MMC or METH, for 6, 12 or 24 h. In addition, cells were pre-treated with 100 nM clorgyline, rasagiline or selegiline, 1mM NAC, or 1 µM trolox, 30 min prior to their exposure to the tested drugs, in neuroprotection experiments. The production of reactive oxygen and nitrogen species (ROS/RNS) was measured, as well as total gluthatione (tGSH) intracellular levels. Mitochondrial membrane potential and ATP intracellular levels, as well as caspase 3 activity, were also assessed.
Cytotoxicity was observed for cathinones and METH in a concentration- and time-dependent manner, both in MTT reduction and NR uptake assays. At 24 h of exposure, and according to MTT reduction assay, the following order of toxic potencies was 3,4-DMMC > 4-MMC > METH. The decrease in intracellular tGSH levels elicited by 3,4-DMMC and 4-MMC, in addition to an increase in ROS/RNS production induced by of these two cathinones confirmed the oxidative stress elicited by these drugs. Clorgyline, rasagiline, selegiline and trolox provided partial protection for all tested drugs, while NAC only prevented the toxicity induced by cathinones, in the MTT reduction assay. The significant increase in ROS/RNS production elicited by cathinones was lessened by the putative protectors, with NAC totally preventing it. Both cathinones and METH caused mitochondrial dysfunction due to mitochondrial membrane depolarization and depletion of ATP intracellular levels. Moreover, caspase-3 activation was triggered by cathinones and METH.
In conclusion, under the present experimental conditions, mitochondrial activity appeared to be a main target for the toxicity of the studied cathinones, leading to cell death.
Acknowledgments: Jorge Soares acknowledges University of Porto/FMUP through FSE, NORTE2020 for his grant (NORTE-08-5369-FSE-000011). This work received financial support from the European Union (FEDER funds POCI/01/0145/FEDER/007728) and National Funds (FCT/MEC) under the Partnership Agreement PT2020 UID/MULTI/04378/2013, UID/MULTI/04046/2013 and UID/MULTI/00612/2013. Vera Marisa Costa acknowledges FCT for her grant (SFRH/BPD/110001/2015). This work is included in and supported by TOX-OER Project (https://toxoer.com/) that was funded by the European Commission and co-funded by the Erasmus+ Programme of the European Union.
Keywords: SH-SY5Y cells
Enantioselective absorption of cathinones by intestinal ephitelial: studies in Caco-2 cells (#544)
B. Silva1, C. Fernandes2, P. G. Pinho1, F. Remião1
1 Pharmacy Faculty, University of Porto, UCIBIO-REQUIMTE, Toxicology Lab., Departament of Biological Sciences,, porto, Portugal
The in vitro model of the intestinal barrier, Caco-2, has been frequently used to evaluate drug permeability . This model represents a relatively reproducible and inexpensive tool and shows a good relation to in vivo data . Synthetic cathinones are psychoactive substances derivatives of cathinone, a naturally occurring β-ketone amphetamine found in Catha edulis (khat) [3-5]. Being chiral molecules, each enantiomer of cathinone derivatives may have different binding to proteins, or other chiral biomolecules, leading to many kinetic or dynamic variations [6, 7]. The absorption of these compounds occurs mainly through the oral mucosa and the second route takes place in the stomach and small intestine . The most common gastrointestinal effects reported by the consumers of ‘‘bath salts’’ are abdominal pain, nausea and liver failure [8-11]. However, the level to which these compounds cross the intestinal barrier have not yet been determined, and as chiral molecules, it could be expected a differentiated permeability between enantiomers. The present study aimed to develop and validate an HPLC-UV method for the determination and quantification of racemic form and enantiomers of pentedrone and methylone to study the intestinal permeability of these drugs. Both cathinones were efficiently separated and determined with a single 7 minutes chromatographic run-time. The method was validated concerning selectivity, linearity (coefficients always > 0.999), accuracy (88.62-106.48 %), inter-day and intra-day precisions (always below 10 %), limits of detection and quantification and stability. In Caco-2 cell line, the kinetic studies were performed to evaluate the ability of pentedrone and methylone (racemate and enantiomers) to pass across the intestinal barrier model. Pentedrone and methylone enantiomers were obtained by our group with a chiral semi-preparative liquid chromatography method . During the experience, the cells were incubated with 500 μM of pentedrone and methylone and 200 μL were collected at 7 time points. It was possible to observe a differentiated passage of the cathinones enantiomers through intestinal membrane. For pentedrone, this difference is observed after the first hour, being R-(-)-pentedrone the most permeable compound. Concerning methylone, the difference is noted after the fourth hour, with R-(+)-methylone being the most absorbed. In conclusion, we developed and fully validated a method that allowed the identification and quantification of pentedrone and methylone. The method was successfully applied for the analysis of Caco-2 cell samples, which allowed to discover the enantioselectivity of these cathinones in intestinal permeability.
Financial supported from Universidade do Porto/FMUP through FSE, NORTE 2020 (NORTE-08-5369-FSE-000011). Included in and supported by TOX-OER Project (https://toxoer.com/) that was funded by the European Commission and co-funded by the Erasmus+ Programme of the European Union.
Keywords: Caco-2, pentedrone, methylone, enantiomers, permeability assay
In vitro toxicity assessment of toxic cyanobacteria as an emerging environmental risk in Europe (#545)
V. B. Ilieva1, 2, T. P. Georgieva2, M. S. Kondeva-Burdina1, D. Aulani1, V. Tzankova1
1 Medical University Sofia, Pharmacology, Pharmacotherapy and Toxicology, Sofia, Bulgaria
Toxigenic Cyanobacteria is one of the main health risks associated with European water resources. According to the European Safety Authority and Food (EFSA) and Agriculture Organization of the United Nation (FAO), cyanobacterial blooms are classified as an emerging risk.
Keywords: cyanotoxin, emerging risk, in vitro toxicity
Co-culture model Caco-2/HT29-MTX: a promising tool for toxicity investigation of phycotoxins on the intestinal barrier (#558)
O. Reale1, A. Huguet1, V. Fessard1
1 French Agency for Food, Environmental and Occupational Health & Safety, 1Toxicology of contaminants, Javené, France
Lipophilic phycotoxins produced by marine microalgae can accumulate in edible shellfish. Some of them are documented to affect the gastrointestinal tract provoking acute intoxications in humans. However, for some toxins, the absence of proven human intoxications makes it difficult by public health authorities to estimate the risk for humans following acute exposure. Investigation of toxins toxicity through both in vitro and in vivo studies can provide key information. In fact, several phycotoxins have been shown in vivo to induce toxic effects on the intestinal epithelium such as cell detachment, fluid accumulation and villous erosion. Nevertheless, most of the toxicity data have been obtained in vitro on intestinal epithelial cell monolayers with a single cell type. Recently, co-culture models have been developed to mimic more closely the human intestinal barrier and are expected to improve evaluation of the toxicity of ingested compounds. Using such relevant co-culture model with enterocytic Caco-2 cells and HT29-MTX goblet cells, we investigated the effects of four phycotoxins (okadaic acid (OA), yessotoxin (YTX), pectenotoxin-2 (PTX2) and azaspiracid-1 (AZA1)). Cell viability, permeability, production of mucus and inflammation were evaluated using various approaches such as TEER, ELISA, histology and High Content Analysis. Our results showed that OA and PTX2 affected the monolayer permeability and that YTX and AZA1 increased the mucus layer through histological analysis. Only OA seems to induce inflammation through IL8 cytokine release. Additional results using RT-PCRq will highlight the pathways and genes affected by these toxins on the investigated processes. This co-culture model appears to be a promising tool to evaluate and compare the effects of phycotoxins on the human intestinal barrier.
Keywords: intestinal epithelium, co-culture, phycotoxin, toxicity, mucus
Aerosol bubbled extracts of next generation products show significantly reduced toxicity compared to cigarettes in a series of in vitro assays. (#570)
L. D. Simms1, R. Wieczorek2, E. Trelles Sticken2, J. Pani2, L. M. Bode2, G. Cava1, M. Stevenson1
1 Imperial Brands, Product Science , Bristol, United Kingdom
To assess the potential harm reduction of tobacco-based and tobacco-free Next Generation Products (NGPs), three different products were compared to conventional cigarettes (3R4F) in a series of in vitro assays. The trapping of smoke/aerosols in phosphate buffered saline (PBS) was used, to enable the use of in vitro systems where direct exposure to smoke/aerosol is not possible. The objective of this study was to assess the smoke chemistry and in vitro biological activity of PBS which had either cigarette smoke or a selection of NGP aerosols bubbled through it. The products investigated were the Kentucky reference cigarette (3R4F, 1.8 puffs/ml), a tobacco heating product (THP), a hybrid product (HYB) and a myblu™ e-cigarette (Tobacco Flavour 1.6% Nicotine) all at 4 puffs/ml of PBS. The 3R4F and THP were smoked using the HCI Intense smoking regime, with HYB and mybluTM vaped according to CORESTA Recommended Method N°81. The cigarette smoke and NGP aerosols were bubbled through a series of three impingers (10mls each) containing PBS and combined to form a mixed sample.
Chemical analysis of the 3R4F stock solution, quantified nicotine at 64 µg/ml and a selection of carbonyls ranging between 5.9 - 157 µg/ml. The three NGP stock solutions contained nicotine levels ranging from 46 - 169 µg/ml and had marked reductions in carbonyls when compared to 3R4F (mybluTM had no detectable carbonyls present).
3R4F extract was cytotoxic in the Neutral Red Uptake assay and mutagenic in the Ames assay with both strains TA100 and TA98 with S9 activation. The THP extract was less cytotoxic than 3R4F extract, with only a weak positive response observed in the Ames test with TA100+S9. The HYB and mybluTM extracts were both non-cytotoxic and mybluTM non-mutagenic at the maximum tested concentration of 10% in PBS, under the conditions of test. None of the PBS samples were active in the in vitro micronucleus assay. Only the 3R4F extract was classified as having tumour promoting activity in the Cellular Transformation Assay (Bhas 42 strain).
Both the HYB and mybluTM extracts were non-cytotoxic and mybluTM non-mutagenic under the conditions of this study. Using a core battery of in vitro tests, mybluTM demonstrated the lowest biological response compared to 3R4F and the other NGPs tested.
Keywords: In vitro assays, smoke chemistry, mutagenicity, next generation products.
Development of an in vitro photosensitization assay using reconstituted 3D human epidermis and a genomic signature: the PhotoSENS-IS assay. (#583)
H. Groux1, F. Cottrez1, E. Boitel1, B. van de Waart2, W. Westerink2
1 ImmunoSearch, Grasse, France
Chemical photosensitivity can be elicited by exposure of the skin to various pharmaceutical substances, foods, cosmetics and other environmental chemicals, followed by exposure to sunlight. In order to develop an in vitro test for photosensitization, we decided to use the advantages of the SENS-IS assay in terms of chemical sensitization potency measurement and skin metabolism capabilities. After optimizing the time of contact between the chemical product and the 3D human epidermis (Large Episkin model) and the intensity of UV irradiation using 6-methyl coumarin as a prototypical photosensitizer, we have tested 8 phototoxic or photosensitizer chemicals. All chemicals were tested at 50% or 10% in DMSO except for Chlopromazine which was tested diluted in PBS. 2-ethylhexyl-p-methoxycinnamate, 2-tert-butyl-4,6-dinitro-5-methylanisole, para-amino-benzoic acid, bithionol and 6-methyl coumarin used as a positive control, were all detected as moderate photosensitizer, i.e. positive at up to 10%. Bithionol was also detected as an irritant and para-amino-benzoic acid as a weak sensitizer without UV irradiation. Sulphanilamide was measured as a weak photosensitizer, i.e. positive only at 50%. Carprofin and Chlopromazine were too irritant and could not be analyzed at the concentrations tested. These very encouraging preliminary results suggest that the PhotoSENS-IS assay may be a promising in vitro tool for the assessment of potential photosensitizers.
Keywords: Photosensitization, SENS-IS, in vitro assay
Dosing considerations for in vitro inhalation testing of VOCs in air-lifted Interphase (ALI) cultures (#586)
T. Hansen1, D. Ritter1, J. Knebel1
1 Fraunhofer ITEM, Preclinical Pharmacology and In VitroToxicology, Hannover, Germany
VOCs are difficult to dose in submerged in vitro systems, since evaporation may lead to rapid changes in the available concentration. Moreover, due to high vapor pressures, inhalation is an important route of exposure and, thus, ALI exposure of cells from the respiratory tract seems to be a highly relevant setup. In an ALI setup, it is reasonable to assume that the applied dose (D) is defined by the general equation D = c x t x Q, with the concentration c, the exposure time t and the exposure volume flow Q, which quantifies the flow that is conducted over the surface of the exposed cells or tissues. This study aimed at the identification of an experimental window within which this correlation is valid. Formaldehyde was chosen as a hydrophilic, relatively high toxic model compound with a high vapor pressure and low boiling point. Human lung epithelial A549 cells were exposed to formaldehyde vapor at ALI conditions using the P.R.I.T.®ExpoCube® device. This unique exposure device provides a highly efficient exposure situation by preventing contact between the test compound and the culture medium. Test atmospheres were generated by evaporation of the volatile test compounds and diluted in clean air. FT-IR spectroscopy enabled online analysis of the exposure concentration. Dose-response curves were obtained using WST as readout for cytotoxicity and compared to a comprehensive historical data set available at ITEM. Clear and conclusive results were evaluated leading to a simple definition of an experimental window within which the above named general formula for calculation of the dosage was valid. It is defined by
8 min ≤ t ≤ 60 min and 3 ml/min/cm² ≤ Q ≤ 10 ml/min/cm²
The efficiencies of low exposure flows (1.5 to 2.5 ml/min) were clearly lower as during historical experiments. The reason for this may be a lack of gas phase concentration equilibration or the stability of fluid dynamical boundary layers such as the Prandtl’s boundary layer over the cellular surface.
In summary, in conclusive explorative experiments it could be shown that the general formula for the dosage D as defined above was valid in the experimental setup used and in an experimental window defined in this study.
This project received funding from NC3Rs.
Keywords: in vitro testing, inhalation toxicity, air-liquid interface
Differentiation and freeze-thawing of human iPS cell-derived brain microvascular endothelial cells (#587)
M. Yamashita1, H. Aoki1, T. Hashita1, T. Iwao1, T. Matsunaga1
1 Nagoya City University, Graduate School of Pharmaceutical Sciences, Clinical Pharmacy, Aichi, Japan
[Purpose] The blood-brain barrier (BBB) is composed of brain microvascular endothelial cells (BMECs) that are surrounded by pericytes, astrocytes and neurons. The BMECs, which are characterized by robust tight junctions and the enrichment of efflux transporters, have an essential biological barrier function to protect the brain from toxic factors and pathogens. In drug development, accurate evaluation of BBB permeability is required to predict not only efficacy but safety of drugs. Although BBB permeability has been evaluated by experimental animals, accurate prediction in human is difficult because of species differences. Therefore, a human induced pluripotent stem (iPS) cell-derived BBB model has been developed for preclinical drug screening. However, in previous study, human iPS cell-derived BMECs (iBMECs) express the low levels of endothelial cell markers and are difficult to maintain the barrier function after freeze-thawing. In this study, we attempted to promote differentiation of iBMECs and investigated the effect on freeze-thawing by compounds X.
[Methods] Differentiation to iBMECs was performed with reference to previous report (Lippmann et al., 2012) and compounds X were added to the differentiated media for appropriate period.
[Results] As the results of immunofluorescence staining and tube formation assay, compounds X remarkably increased the protein expression level of vascular endothelial cell marker and enhanced the ability of blood vessel-like structure formation. Moreover, transendothelial electrical resistance (TEER) values of iBMECs were significantly increased by compounds X. Although TEER values were significantly decreased in frozen cells without compounds X, we succeeded in maintaining TEER values in frozen cells by compounds X.
[Conclusion] We have succeeded in discovering compounds X, which enhanced the barrier functions of iBMECs and suppressed the cell damage by freeze-thawing. We concluded that compounds X would be useful for developing in vitro BBB models from iPS cells.
Keywords: human induced pluripotent stem cells, in vitro BBB models, freeze-thawing, tight junctions, drug development
Development of in vitro cholestatic drug-induced liver injury evaluation system using HepG2-hNTCP-C4 cells with sandwich culture (#588)
Y. Sakai1, H. Okumura1, T. Iwao1, K. Watashi2, K. Ito3, T. Matsunaga1
1 Nagoya City University, Pharmaceutical Sciences, Nagoya, Japan
Introduction and Purpose: Toxicological approaches for screening of drug candidates causing drug-induced liver injury (DILI) during early stage of drug development studies are needed to reduce risk and cost. It is thought that some kinds of cholestatic DILI cases are caused by the accumulation of bile acids (BAs) in hepatocytes due to inhibition of transporters. Then, the expression of Na⁺-taurocholate cotransporting polypeptide (NTCP), which incorporates BAs into hepatocytes, is essential for properly constructing cholestatic DILI evaluation systems.
Methods: We evaluated the expression of mRNA and protein and functions in SCHepG2-hNTCP-C4 cells. We also exposed 22 compounds, whose clinical DILI risks are known, under the optimal conditions.
Keywords: bile acids, sandwich culture, HepG2-hNTCP-C4 cells, drug-induced liver injury, in vitro model
Differentiation of human iPS cell-derived endothelial progenitor cells into brain microvascular endothelial cells. (#590)
H. Aoki1, M. Yamashita1, T. Hashita1, T. Iwao1, T. Matsunaga1
1 Nagoya City University, Clinical Pharmacy, Aichi, Japan
[Purpose] Brain microvascular endothelial cells (BMECs), which are one of the constituents of the blood brain barrier (BBB), inhibit the non-specific entry of substances into the brain parenchyma through strong intercellular adhesions and expression of multidrug efflux transporters. Recently, human induced pluripotent stem (hiPS) cell-derived BMECs (iBMECs) were developed as new resources for the human BBB models. However, in iBMECs previously reported, the expression levels of endothelial markers are low. Previously, we succeeded in differentiation, expansion and cryopreservation of human iPS cell-derived endothelial progenitor cells (iEPCs). Therefore, the aim of this study was establishment of original method for differentiation of iBMECs with endothelial phenotypes from cryopreserved iEPCs.
[Methods] The iEPCs were differentiated and cryopreserved. Further, the cryopreserved iEPCs were thawed and differentiated into iBMECs. Expression of genes and proteins were determined by RT-qPCR and immunofluorescence analysis, respectively. Furthermore, transendothelial electrical resistance (TEER) values, which represent the intensity of tight junction, were measured in the iEPCs and iBMECs.
[Results and Discussion] The iEPC-derived cells strongly expressed endothelial markers. Further, they also expressed multidrug efflux transporters and tight junction makers. Besides, iEPC-derived cells showed higher TEER values and lower permeability of FITC-Dextran 4,000, which is index of paracellular pathway, than iEPCs. These results suggest that iEPC-derived cells have a stronger barrier function than the iEPCs. Thus, we conclude that iEPC-derived cells have features similar to those of BMECs in vivo and the iBMECs can be differentiated from cryopreserved iEPCs.
[Conclusion] we succeeded in differentiation of cryopreserved iEPCs into iBMECs with endothelial phenotypes.
Keywords: Human induced pluripotent stem cells, Endothelial progenitor cells, Blood-brain barrier, Brain microvascular endothelial cells, Differentiation
Effect of glyphosate at low concentrations on chromosome missegregation and aneuploidy induction in human peripheral blood lymphocytes in vitro (#610)
V. Mužinić1, D. Želježić1
1 Institute for Medical Research and Occupational Health, Zagreb, Croatia
Aneuploidy, a state of imbalance in chromosome numbers implicated in cancer development and progression, arises after erroneous chromosome missegregation during anaphase. Glyphosate is the world's most commonly used herbicide, frequently examined for its potential toxicity in non-target organisms. To assess whether exposure to glyphosate might affect chromosome segregation fidelity in human cells, we have treated human whole peripheral blood with solutions of glyphosate in vitro for 24 h at final concentrations equivalent to acceptable daily intake (ADI; 0,5 µg/mL) and acceptable operator exposure level (AOEL; 3,5 µg/mL). After processing whole blood cultures according to cytokinesis-block micronucleus assay, we have performed fluorescence in situ hybridization with directly labeled pancentromeric probes for chromosomes 18, 9, X and Y. We have noticed a significant increase in chromosome loss in binucleate lymphocytes at both concentrations for chromosomes 18, 9, and X as well as for chromosome Y at AOEL. We conclude that glyphosate exposure affects chromosome segregation, potentially increasing the risk of malign transformation. These results will be useful for future risk assessments of this herbicide.
Keywords: glyphosate, chromosome missegregation, fluorescence in situ hybridization, micronucleus assay
Identification of key transcription factor networks mediating valproic acid-induced mitochondrial dysfunctioning in primary human hepatocytes (#611)
F. Caiment1, J. Wolters1, E. Smit1, Y. Schrooders1, J. C. S. Kleinjans1, T. van den Beucken1
1 Maastricht University, Toxicogenomics, Maastricht, Netherlands
Valproic acid (VPA) is one of the most frequently prescribed antiepileptic drugs worldwide. Besides its pharmacological activity, it may cause liver toxicity and steatosis through mitochondrial dysfunctioning. Nevertheless the mechanisms underlying these adverse effects are incompletely understood. We and others have previously studied the effect of VPA on gene expression and DNA methylation profiles in primary human hepatocytes (PHH). In the current study we aimed to link changes in gene expression to mitochondrial dysfunctioning by performing an RNA interference (RNAi) screening strategy. For this we predicted which transcription factors needed to be activated in order to explain the VPA-induced expression profiles. This resulted in the identification of a panel of 18 TFs that were constitutively activated during 3 days of repetitive VPA exposure. Next, we applied RNAi, using lentiviral-based shRNAs, to knockdown the expression of 16/18 TFs identified and determine their effect on mitochondrial dysfunctioning. For each of the knockdown models we determine oxygen consumption rates (OCR) using Seahorse technology. Importantly we found a dose-dependent decrease in OCR in PHH after 24, 48 and 72 hours of repetitive VPA administration. There was no acute effect of VPA administration on OCR, indicating the need for prolonged exposure. Knockdown of several TFs modified the response of PHH to VPA treatment after 72 hours. CEBPA was one of the most prominent TFs of which the expression affected the cellular response to VPA. Knockdown of CEBPA increased basal and maximal respiration rates in PHH upon VPA exposure compared to control shRNAs. In order to identify CEBPA-dependent gene expression, RNA-seq was performed on CEBPA knockdown and control cells under both control conditions and after VPA exposure. Out of the 27 transcriptional targets initially used to identify CEBPA activation we could confirm 23. Four of the previously identified transcriptional targets of CEBPA did not respond to CEBPA depletion, suggesting that these targets are not regulated by CEBPA in the cellular context of the PHH we used. Altogether, our study demonstrates that VPA-induced changes in gene expression can be causally linked to mitochondrial dysfunctioning in PHH.
Keywords: Valproic acid, Mitochondrial dysfunction, RNA-seq, RNA interference
Investigation into the cross-species sensitivity of erythrocytes in vitro to hydroxylamine-mediated stress and cytotoxicity. (#617)
G. Sohanpal1, P. K. Allen1, J. D. Parry1
1 GlaxoSmithKline, Investigative Safety, In vitro / In vivo Translation, HERTFORDSHIRE, United Kingdom
Rheumatoid arthritis (RA) is a chronic autoimmune condition which causes inflammation and destruction of the diarthrodial joints of the hands and feet. The pathogenesis of RA involves complex interactions between environmental and genetic factors, leading to the permeation and aggregation of immune cells and macrophages in the synovium. Infiltrating macrophages produce a variety of pro-inflammatory cytokines that contribute to the destruction of cartilage and bone. RA is the most common inflammatory arthritis, affecting around 400 000 adults in the UK.
GSK Compound X has the potential to be an effective agent in the treatment of RA. However, the production of the carboxylic acid of the hydrolysed hydroxamic acid of Compound X was identified in rat, monkey and human hepatocytes, indicating the potential formation of hydroxylamine (HA). HA is associated with haematotoxicity in vivo, methaemoglobinaemia, haemolytic anaemia and haemosiderosis. It also a potent non-genotoxic rodent carcinogen with the potential to cause splenic haemangiosarcomas (HS) in male rats, as described in the adverse outcome pathway.
To determine whether there are species differences in the sensitivity of erythrocytes in vitro to HA-mediated cytotoxicity, a series of erythrotoxicity and viability studies were conducted. These included measurement of glutathione/oxidised glutathione (GSH/GSSG), observation of Heinz bodies and intracellular reactive oxygen species (ROS) detection via flow cytometry.
Results suggest rat red blood cells are most sensitive to HA-mediated toxicity followed by human, then dog. A favourable ranking of human versus other species (particularly the rat and dog) could be of value in defining the relevance of any eventual findings in the 2-year rodent carcinogenicity studies. The data generated was integrated into the assessment of the translational risk of HA-mediated haematotoxicity and haemangiosarcoma after chronic exposure to Compound X in humans. Overall, the data added to the weight of evidence that the risk in humans is likely to be lower than what has been observed in the rat following HA exposure.
LUDBROOK, V., LEWIS, H., PATEL, A., FERNANDO, D., PARRY, J., FURZE, R., SIMEONI, M., CHALKER, M., POTHET, C., SOAMES, E., HAMER, M. 2016. Compound X Investigator's Brochure, GlaxoSmithKline.
MCINNES, I. B. & SCHETT, G. 2011. The pathogenesis of rheumatoid arthritis. N Engl J Med, 365, 2205-19.
SPOOREN, A. A. M. G. 2000. Erythrotoxicity of Aliphatic Hydroxylamines: Mechanistic Aspects and Parameters for Biological Effect Monitoring, Universiteit Maastricht.
SYMMONS, D. 2002. The prevalence of rheumatoid arthritis in the United Kingdom: new estimates for a new century. Rheumatology, 41, 793-800.
Keywords: Erythrotoxicity, Haematotoxicity, Hydroxylamine, AOP, ROS
Induced pluripotent stem cell-derived human retinal model containing microglial cells as a platform for toxicology studies (#619)
V. Chichagova1, M. Georgiou2, M. Carter2, J. Ajeian1, B. Dorgau1, E. Sernagor3, M. Nicholds1, L. Armstrong1, 2, M. Lako2
1 Newcells Biotech Ltd, Newcastle upon Tyne, United Kingdom
Microglia are the primary tissue resident immune cells in the retina. They co-exist in close interaction with Müller glial cells and are essential for normal development by regulating neuronal survival and synaptic pruning. In the adult retina, they regulate homeostasis by maintaining synaptic structure and function. Under pathological conditions, microglia-Müller cell signalling can mediate adaptive responses within the retina following injury. In addition, microglia can trigger neurodegeneration within the retina exacerbating the effect of the disease making it a potential therapeutic target. Retinal organoids derived from human induced pluripotent stem cells (hiPSC) provide a human physiologically relevant platform to study retinal development, disease modelling and compound screening. However, due to the differences in their developmental origins, microglia and retina do not arise under the same differentiation conditions. Therefore, to enhance the current retinal model, a co-culture approach is needed. We developed a differentiation protocol for deriving microglia from hiPSCs. The cells expressed key developmental markers, including CD14, CX3CR1 and IBA1. In addition, they were functional as was shown by their ability to phagocytose fluorescent beads. In parallel, we differentiated hiPSCs to retinal organoids using our established protocols. We assessed their development by confirming the expression of key markers, including RECOVERIN, HUC/D, AP2α, and PROX1. To enhance our retinal model, we incorporated hiPSC-derived microglia and tested their retinal invasion capacity and function in response to agents causing retinal degeneration. Our in vitro retinal model which incorporates immune cells represents a tissue structure with greater physiological relevance to the in vivo human retina and provides a platform for compound screening and disease modelling.
Keywords: Induced pluripotent stem cells, retina, organoids, differentiation
Simultaneous real-time monitoring of cytotoxicity and stress response pathway by means of dual color luciferase monitoring system (#629)
Y. Nakajima1, Y. Fujita1, N. Oonishi1, K. Tazumi1, T. Iwaki1, H. Abe1
1 National Institute of Advanced Industrial Science and Technology (AIST), Health Research Institute, Takamatsu, Japan
Recently, luciferase reporter assay has become one of the conventional methods for cytotoxicity evaluation. Typically, cells are destroyed at a particular time point, called the endpoint assay, enabling conventional and high-throughput assay. On the other hand, luciferases are also used in the longitudinal monitoring of such cellular events in cellulo. In addition, recent advances in luciferase technology allow us to monitor the expression of multiple genes simultaneously when luciferases are used that induce differently colored emission spectra, namely, green-emitting and red-emitting beetle luciferases that act on a single bioluminescent substrate . To improve cytotoxicity evaluation using luciferase, in this study, we developed real-time multicolor luciferase measurement method to simultaneously monitor dynamics of expression of cellular toxicity and activation of stress response pathway, including Keap1-Nrf2 pathway.
First, we generated HepG2 cells harboring mouse artificial chromosome (MAC) vector . Next, an internal control reporter plasmid, in which green-emitting luciferase is expressed under the control of TK promoter, was inserted into R4 site of the MAC vector. Finally, test reporter plasmid, in which red-emitting luciferase is expressed under the control of antioxidant response element (ARE), was inserted into φC31 site of the MAC vector.
To verify the monitoring system, luciferase-expressing HepG2 cells seeded into 96-well plates were treated with representative Nrf2 activators, including tertiary butylhydroquinone, dimethylfumarate and sulforaphane. Bioluminescence was measured for 72 h at 37˚C in 5% CO2 atmosphere under saturated humidity. As expected, luminescence intensity of green-emitting luciferase (internal control reporter) was dose and time dependently decreased. In contrast, luminescence intensity of red-emitting luciferase (ARE-dependent expression) was significantly increased. These results clearly demonstrated that the system successfully monitor dynamics of activation of Keap1-Nrf2 pathway accompanying increase of cytotoxicity. Thus, real-time monitoring system developed in this study would be useful for mechanism-based cytotoxicity assays by directly monitoring both events.
 Nakajima Y, Ohmiya Y, Expert Opin. Drug Discov., 5, 835-849 (2010)
 Wakuri S, et al., Anal. Biochem., 522, 18-29 (2017)
Keywords: artificial chromosome vector, cell-based assay, luciferase, real-time monitoring, stress response pathway
Retinal organoids derived from human induced pluripotent stem cells as an in vitro model for toxicological studies for new retinal disease treatments (#634)
B. Dorgau1, M. Georgiou2, V. Chichagova1, G. Hilgen3, E. Sernagor3, M. Nicholds1, L. Armstrong1, 2, M. Lako2
1 Newcells Biotech Ltd, Newcastle upon Tyne, United Kingdom
Vision is one of our most important sensory senses and diseases such as age related macular degeneration (AMD) and retinitis pigmentosa (RP) cause the loss or dysfunction of retinal photoreceptors and/or the underlying retinal pigment epithelium (RPE). This results either in irreversible impairment of vision or complete vision loss as the retina is unable to regenerate. To date there are no effective treatments to halt progression of retinal degeneration. The generation of 3D laminated retina derived from human pluripotent stem cells (hPSCs) provides a suitable platform and offers the opportunity to develop new strategies for treatment of these conditions, drug screening and repurposing and diseases modelling. Recent research demonstrated that retinal organoids derived from hPSCs contain all retinal cell types which are capable of forming synapses. Moreover, these organoids are physiologically functional, meaning they are responsive to light. This study investigated the applicability of human induced pluripotent stem cell (hiPSCs) derived retinal organoids as a human in vitro model for toxicological studies. Therefore retinal organoids, which are aged around 5 months and resemble the normal retina, were used to test different drugs: Digoxin, Thioridazine and Methanol are drugs with well-known side effect on the retina and Flubiprofen and Ketorolac are drugs without any reported effects serving as a control in this study. Retinal organoids were exposed to the drugs for 24 hours and subsequently analysed by Lactate Dehydrogenase (LDH) assay to determine cytotoxicity and by immunohistochemistry to evaluate possible structural and functional changes within the retina. Our results, so far, indicated that Digoxin, Thioridazine and Methanol affected the retinal structure and the control drugs (Flubiprofen and Ketorolac) showed no effects on the retinal structure and/or physiological functionality regardless of the concentration (200ug/ml – 2mg/ml). This study is a proof of principle and demonstrates that the retinal organoids derived from hiPSCs represent a good human in vitro model, which can be used for drug screening to develop new treatments for retinal diseases.
Keywords: human induced pluripotent stem cells, retina, toxicity, organoid, drugs
Protective and reparative effects of fluconazole on neuronal differentiation altered by 5HT (#643)
M. Battistoni1, C. Parravicini2, L. Palazzolo2, F. Di Renzo3, I. Eberini2, E. Menegola3
1 Università degli Studi di Milano, Department of Biomedical and Clinical Sciences, Milano, Italy
Micromass test involves exposing undifferentiated rat embryo midbrain cells put in culture at high density to test compounds and observing the subsequent effect on cell differentiation. Within these high-density cell colonies, cells that are destined to differentiate move together and form distinct foci (tridimensional aggregation of cell bodies) interconnected by bundles (aggregation of neuronal processes, differentiated foci) which can be distinguished from foci without bundles (undifferentiated foci). Recently, a pro-differentiating effect in oligodendrocyte precursors (OPC) has been demonstrated for miconazole, beside its antifungal activity. In order to test whether other antifungal azoles show the same activity on cell maturation, micromasses were incubated during the whole culture period with a known promoter of differentiation (RA), with a known differentiation inhibitor (5HT) or with azoles in clinical use. Fluconazole, itraconazole and miconazole, whose differentiative potential was first confirmed in OPCs cultured both alone and with neurons, were selected based on in silico prioritization. Results confirmed 5HT inhibitory effects at 50-100 mM. Among RA and the selected azoles, the most promising molecule in our model was fluconazole, tested at 5-100 mM. In order to test protective effects of fluconazole on 5HT inhibition, we co-exposed micromasses to both molecules during the whole culture period. Results show that co-exposed groups displayed parameters comparable to controls, suggesting a protective effect of fluconazole. A second set of experiments were devoted to the evaluation of a fluconazole-related reparative effect. Cultures were exposed during the first day to 5HT alone and during the remaining culture days to fluconazole alone. Results show that the one-day 5HT exposure affected development while after the post-exposure to fluconazole a reparative effect was evident.
Keywords: micromass, azoles, neural differentiation
Co-exposure to preadipocytes and TCDD increase breast cancer cells aggressiveness and leads resistance to chemotherapy (#648)
M. Koual2, C. Tomkiewicz1, 2, X. D. Coumoul2
1 INSERM, PARIS, France
Breast cancer is an important disease with high incidence as well as mortality among women. In the past 50 years, it has become a major health problem with over 2 million new cases diagnosed in 2018. This represents about 12% of all new cancer cases, 25% of all cancers in women and more than 600.000 cases of deaths worldwide in 2018. Metastatic process, or the spread of tumor cells throughout the body, is responsible for about 90% of cancer patient deaths and represents the central clinical challenge of solid tumor oncology.
The development and progression of breast cancer are complex processes that involve hormonal factors as well as numerous genetic and epigenetic alterations. During the past 10 years, many studies have focused on the role of the tumor microenvironment and the peritumoral stromal fraction. During tumor progression, cancer cells modify their microenvironment which in return will promote the growth and dissemination of the tumor. Adipose tissue, consisting of mainly mature adipocytes and progenitors, is the most abundant component surrounding breast cancer cells. It exerts a major endocrine and secretory role, and represents then an essential actor in the remodeling responses of the extracellular matrix, which influences tumor behavior. Recent studies have shown that pre-adipocytes/adipocytes may promote migration and invasion of breast cancer cells. Adipocyte cells are also responsible for the storage of persistent organic pollutants (POPs) for long periods of time. POPs are a major public health concern due to their toxic effects and their persistence in the environment and organisms.
Here, we studied the paracrine role of pre-adipocytes during metastasis in mammary cancer epithelial cells and the aggravating role of TCDD exposition. We set up an original in vitroco-culture model using mainly human mammary tumor cells (MCF-7 and MDA-MB-231) and human pre-adipose cells (hMADS, human multipotent adipose-derived stem cells), exposed or not to TCDD. The first part of this study reports the effect of the co-culture and TCDD on the phenotypical characteristics of MCF-7 cells. Next, we performed a large-scale proteomics-based experiment and identified specifically in the co-exposure condition, a stem-cell biomarker, the Aldehyde dehydrogenase 1 family, member A3 (ALDH1A3). We then monitored assays of sphere formation and studied their cancer stem-cell (CSC) like properties of MCF-7 cells. To ensure that these aggressive features were also observed in vivo, we demonstrate the ability of co-exposed cancer cells to metastasizein vivo in a Zebrafish model. We then examined if these cells display specific cellular characteristics and found that the co-exposure leads to the generation of giant polynuclear cells (PGCCs), a cancer sub-population strongly associated with aggressiveness and chemoresistance in cancer
Keywords: AhR, TCDD, breast cancer, chemoresistance, polyploid giant cells
Response of MCF7 cells to Vincristine in presence of BPA and DEHP (#663)
R. Uyar1, B. Yurdakok-Dikmen1, Y. Turgut1, A. Filazi1
1 Ankara University, Faculty of Veterinary Medicine, Ankara, Turkey
Aim: Among phthalates DEHP (Bis(2-ethylhexyl) phthalate) and Bisphenol A (BPA) are estrogen active compounds. Eventhough its ban in toys and catheters, there are still various possible exposure scenarios. In estrogen receptor (ER)-positive breast cancers, environmental estrogen active compounds pose a risk for prominent treatment. In this study, we searched the efficacy of vincristine and tamoxifen, in presence of the endocrine active compounds DEHP and BPA, in ER+ mammary tumor cancer cell line MCF7 (human breast adenocarcinoma).
1. Kim JY1, Choi HG1, Lee HM1, Lee GA1, Hwang KA1, Choi KC1. Effects of bisphenol compounds on the growth and epithelial mesenchymal transition of MCF-7 CV human breast cancer cells. J Biomed Res. 2017 Jul 13;31(4):358-369. doi: 10.7555/JBR.31.20160162.
2. Jin Hee Kim. Analysis of the in vitro effects of di-(2-ethylhexyl) phthalate exposure on human uterine leiomyoma cells. Exp Ther Med. 2018 Jun; 15(6): 4972–4978.
3. Carlos Martínez-Campa Pedro, Casado René, Rodríguez Pedro, Zuazua Juana, M. García-Pedrero Pedro, S. Lazo Sofía Ramos, Effect of Vinca alkaloids on ERα levels and Estradiol-induced responses in MCF-7 cells, Preclinical study, 23 March 2006
4. Motala Zainab, Efficacy of Tamoxifen, Alone or in Combination with Vincristine and Temsirolimus, in in vitro and in vivo Models of Rhabdomyosarcoma, Jun-2018
Keywords: Bisphenol A, dehp diethylhexyl phthalate, Vincristine, cytotoxicity, in vitro testing
Establishment of a human embryonic stem cell test with hiPSC derived cardiomyocytes for developmental toxicity testing (#667)
S. Wuttke1, D. Bartsch2, J. Tigges1, E. Fritsche1
1 Leibniz Research Institute for Environmental Medicine, Modern risk assessment and sphere biology, Duesseldorf, North Rhine-Westphalia, Germany
Developmental toxicity testing is performed with animal testing according to several guidelines like OECD or U.S. EPA. Due to the resource-intensity, testing of all registered chemicals using OECD guidelines is logistically and financially not feasible. As an alternative method for embryotoxicity testing, the European center for validation of alternative methods (ECVAM) validated the mouse embryonic stem cell test. This test is based on a permanent mouse embryonic stem cell line, which is differentiated into cardiomyocytes. Hence, the test assesses the effects of chemicals onto the early developmental aspect of cardiomyocyte differentiation. However, this test is based on mouse cells and therefore, bears the issue of species extrapolation to humans. To overcome this issue, we established a human cell based embryonic stem cell test (hEST) using human induced pluripotent stem cells (hiPSC). These hiPSC originate from somatic cells e.g. fibroblasts, which were reprogrammed into stem cells that cover all the characteristics of embryonic stem cells like self-renewal and pluripotency yet does not bear any ethical concerns. By modulation of the Wnt and BMP pathways, cardiomyocyte differentiation is induced. We show characterization of the cell culture by assessment of beating cells, as well as marker expression by PCR, FACS and immunocytochemistry. In a next step, these markers have to be established as test methods with the final goal of testing compounds for their ability to interfere with hiPSC differentiation into cardiomyocytes.
Keywords: hIPSC, cardiomyocytes, developmental toxicity
Characterization of fresh hepatocytes isolated from TK-NOG chimeric mice with humanized livers (#676)
H. Suemizu1, Y. Higuchi1, N. Yoneda1, H. Yamazaki2, S. Uehara1
1 Central Institute for Experimental Animals, Laboratory Animal Research Department, Kawasaki, Japan
Purpose: Human hepatocytes are an important tool for drug development and in vitrotoxicity studies. We developed a TK-NOG transgenic mouse which can be used for the expansion of human hepatocytes within the host liver tissue. The purpose of the present study was to evaluate hepatocytes isolated from TK-NOG chimeric mice with humanized livers(Hu-liver), and determinewhether these hepatocytes could be used as an alternative to primary human hepatocytes for in vitrotoxicity studies.
Methods: Hepatocytes were harvested from chimeric mice with Hu-liverusing a two-step collagenase perfusion method. The purity and viability of the prepared hepatocytes was analyzed using flow cytometry. The expression levels of drug-mediated cytochrome P450 genes were determined using real-time polymerase chain reaction analysis with gene-specific primers. The ability of CYP3A to induce enzymatic activity was evaluated using testosterone as a CYP3A probe and rifampicin as CYP3A inducer.
Results and Discussion: Up to 98% of the Hu-liver cells stained positive for human leukocyte antigen, with a mean viability exceeding 87% (n= 29). Monolayer-cultured Hu-liver cells were binucleated and displayed a cobblestone cell morphology. A good correlation was observed between the mRNA expression levels of 16 P450 forms belonging to P450 subfamilies 1–4 in the Hu-liver cells and those in human donor hepatocytes. Similar to the observations made using donor human hepatocytes, omeprazole/β-naphthoflavone and rifampicin/phenobarbital treatment caused a more than two-fold induction of CYP1A2 and CYP3A4 mRNA in the Hu-liver cells. We also confirmed a significant increase in the production of 6β-hydroxytestosterone in the Hu-liver cells after treatment with rifampicin, a CYP3A inducer. In long-term cultures of Hu-liver cells, both the rate of human albumin production and the expression levels of CYP3A4 mRNA were maintained for up to 4 weeks in commercially available medium. These results suggest that Hu-liver cells have characteristics similar to those of human hepatocytes. Hu-liver cells can therefore potentially be used for in vitro