The MCRA toolbox of models and data to support chemical mixture risk assessment

A model and data toolbox is presented to assess risks from combined exposure to multiple chemicals using probabilistic methods. The Monte Carlo Risk Assessment (MCRA) toolbox, also known as the EuroMix toolbox, has more than 40 modules addressing all areas of risk assessment, and includes a data repository with data collected in the EuroMix project. This paper gives an introduction to the toolbox and illustrates its use with examples from the EuroMix project. The toolbox can be used for hazard identification, hazard characterisation, exposure assessment and risk characterisation. Examples for hazard identification are selection of substances relevant for a specific adverse outcome based on adverse outcome pathways and QSAR models. Examples for hazard characterisation are calculation of benchmark doses and relative potency factors with uncertainty from dose response data, and use of kinetic models to perform in vitro to in vivo extrapolation. Examples for exposure assessment are assessing cumulative exposure at external or internal level, where the latter option is needed when dietary and non-dietary routes have to be aggregated. Finally, risk characterisation is illustrated by calculation and display of the margin of exposure for single substances and for the cumulation, including uncertainties derived from exposure and hazard characterisation estimates.</p

Impairment of bile acid metabolism by perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) in human HepaRG hepatoma cells

Perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) are man-made chemicals that are used for the fabrication of many products with water- and dirt-repellent properties. The toxicological potential of both substances is currently under debate. In a recent Scientific Opinion, the European Food Safety Authority (EFSA) has identified increased serum total cholesterol levels in humans as one major critical effect being associated with exposure to PFOA or PFOS. In animal studies, both substances induced a decrease of serum cholesterol levels, and the underlying molecular mechanism(s) for these opposed effects are unclear so far. In the present study, we examined the impact of PFOA and PFOS on cholesterol homoeostasis in the human HepaRG cell line as a model for human hepatocytes. Cholesterol levels in HepaRG cells were not affected by PFOA or PFOS, but both substances strongly decreased synthesis of a number of bile acids. The expression of numerous genes whose products are involved in synthesis, metabolism and transport of cholesterol and bile acids was strongly affected by PFOA and PFOS at concentrations above 10 µM. Notably, both substances led to a strong decrease of CYP7A1, the key enzyme catalyzing the rate-limiting step in the synthesis of bile acids from cholesterol, both at the protein level and at the level of gene expression. Moreover, both substances led to a dilatation of bile canaliculi that are formed by differentiated HepaRG cells in vitro. Similar morphological changes are known to be induced by cholestatic agents in vivo. Thus, the strong impact of PFOA and PFOS on bile acid synthesis and bile canalicular morphology in our in vitro experiments may allow the notion that both substances have a cholestatic potential that is connected to the observed increased serum cholesterol levels in humans in epidemiological studies

Transcriptomics analysis of hepatotoxicity induced by the pesticides imazalil, thiacloprid and clothianidin alone or in binary mixtures in a 28-day study in female Wistar rats

Co-occurrence of pesticide residues in food commodities raises a potential safety issue as their mixture effects on human health are largely unknown. In a previous study, we reported the toxicological effects (pathology and histopathology) of imazalil (IMZ), thiacloprid (THI), and clothianidin (CTD) alone and in binary mixtures in a 28-day oral gavage study in female Wistar rats. Five dose levels (up to 350 mg/kg body weight/day) ranging from a typical toxicological reference value to a clear effect dose were applied. In the present study, we undertook a transcriptomics analysis of rat livers by means of total RNA sequencing (RNA-Seq). Bioinformatic data analysis involving Ingenuity Pathway Analysis (IPA) was used to gain mechanistic information on hepatotoxicity-related pathways affected after treatment with the pesticides, alone and in mixtures. Our data show that 2986 genes were differentially regulated by CTD while IMZ and THI had effects on 194 and 225 genes, respectively. All three individual compounds shared a common subset of genes whose network is associated with xenobiotic metabolism and nuclear receptor activation. Similar networks were retrieved for the mixtures. Alterations in the expression of individual genes were in line with the assumption of dose addition. Our results bring new insight into the hepatotoxicity mechanisms of IMZ, THI, and CTD and their mixtures

Transcript and protein marker patterns for the identification of steatotic compounds in human HepaRG cells

Lichtenstein D, Mentz A, Schmidt FF, et al. Transcript and protein marker patterns for the identification of steatotic compounds in human HepaRG cells. Food and Chemical Toxicology. 2020;145: 111690.Non-alcoholic fatty liver disease is a major health concern especially in Western countries. Animal studies suggest that certain chemicals may contribute to hepatocellular triglyceride accumulation, among them a number of hepatotoxic pesticidal active compounds. In order to improve the identification of potential liver steatosis inducers in vitro in a human cell culture system, HepaRG cells were treated with a selection of 30 steatotic or non-steatotic pesticides. Induction of triglyceride accumulation was monitored, and changes in the expression of hepatotoxicity marker genes were measured at the mRNA and protein levels. Based on these data, transcript and protein marker signatures predictive of triglyceride accumulation in HepaRG cells were derived. The predictive transcript set consisted of POR, ANXA10, ARG1, CCL20, FASN, INSIG1, SREBF1, CD36, CYP2D6, and SLCO1B1. The predictive protein set consisted of NCPR (POR), CYP2E1, CYP1A1, ALDH3A1, UGT2B7, UGT2B15, S100P, LMNA, and PRKDC. In conclusion, the present study presents for the first time transcript and protein marker patterns to separate steatotic from non-steatotic compounds in a human liver cell line

Hepatotoxicity of the pesticides imazalil, thiacloprid and clothianidin – Individual and mixture effects in a 28-day study in female Wistar rats

Humans are exposed to pesticide residues through various food products. As these residues can occur in mixtures, there is a need to investigate possible mixture effects on human health. Recent exposure studies revealed the preponderance of imazalil, thiacloprid, and clothianidin in food diets. In this study, we assessed their toxicity alone and in binary mixtures in a 28-day gavage study in female Wistar rats. Five dose levels (up to 350 mg/kg bw/day) ranging from a typical toxicological reference value to a clear effect dose were applied. Data show that the liver was a target organ of all pesticides and their mixtures. Increases in liver weight were observed and histopathological examination revealed centrilobular hepatocellular hypertrophy and cytoplasm degeneration for all treatment conditions. No accumulation of hepatic triglycerides was reported. Tissue residue analysis showed altered pesticide residues in the liver and the kidney when being in mixture as compared to the levels of pesticide residues for the single compound treatment, indicating possible toxicokinetic interactions. Overall, all mixtures appeared to follow the additivity concept, even though quantitative analysis was limited for some endpoints due to the semi-quantitative nature of the data, raising no specific concern for the risk assessment of the examined pesticides.</p

Adverse outcome pathway-driven analysis of liver steatosis in vitro : a case study with cyproconazole

Adverse outcome pathways (AOPs) describe causal relationships between molecular perturbation and adverse cellular effects and are being increasingly adopted for linking in vitro mechanistic toxicology to in vivo data from regulatory toxicity studies. In this work, a case study was performed by developing a bioassay toolbox to assess key events in the recently proposed AOP for chemically induced liver steatosis. The toolbox is comprised of in vitro assays to measure nuclear receptor activation, gene and protein expression, lipid accumulation, mitochondrial respiration, and formation of fatty liver cells. Assay evaluation was performed in human HepaRG hepatocarcinoma cells exposed to the model compound cyproconazole, a fungicide inducing steatosis in rodents. Cyproconazole dose-dependently activated RAR alpha and PXR, two molecular initiating events in the steatosis AOP. Moreover, cyproconazole provoked a disruption of mitochondrial functions and induced triglyceride accumulation and the formation of fatty liver cells as described in the AOP. Gene and protein expression analysis, however, showed expression changes different from those proposed in the AOP, thus suggesting that the current version of the AOP might not fully reflect the complex mechanisms linking nuclear receptor activation and liver steatosis. Our study shows that cyproconazole induces steatosis in human liver cells in vitro and demonstrates the utility of systems-based approaches in the mechanistic assessment of molecular and cellular key events in an AOP. AOP-driven in vitro testing as demonstrated can further improve existing AOPs, provide insight regarding molecular mechanisms of toxicity, and inform predictive risk assessment

An adverse outcome pathway-based approach to assess steatotic mixture effects of hepatotoxic pesticides in vitro

Exposure to complex chemical mixtures requires a tiered strategy for efficient mixture risk assessment. As a part of the EuroMix project we developed an adverse outcome pathway (AOP)-based assay toolbox to investigate the combined effects of the liver steatosis-inducing compounds imazalil, thiacloprid, and clothianidin in human HepaRG hepatocarcinoma cells. Compound-specific relative potency factors were determined using a benchmark dose approach. Equipotent mixtures were tested for nuclear receptor activation, gene and protein expression, and triglyceride accumulation, according to the molecular initiating events and key events proposed in the steatosis AOP. All three compounds affected the activity of nuclear receptors, but not key genes/proteins as proposed. Triglyceride accumulation was observed with three different methods. Mixture effects were in agreement with the assumption of dose additivity for all the combinations and endpoints tested. Compound-specific RPFs remained similar over the different endpoints studied downstream the AOP. Therefore, it might be possible to reduce testing to a smaller battery of key tests. The results demonstrate the suitability of our in vitro assay toolbox, integrated within an AOP framework and combined with the RPF approach, for the analysis of steatotic effects of chemical mixtures. However, mRNA results suggest that the steatosis AOP still needs improvement.</p

The MCRA toolbox of models and data to support chemical mixture risk assessment

International audienceA model and data toolbox is presented to assess risks from combined exposure to multiple chemicals using probabilistic methods. The Monte Carlo Risk Assessment (MCRA) toolbox, also known as the EuroMix toolbox, has more than 40 modules addressing all areas of risk assessment, and includes a data repository with data collected in the EuroMix project. This paper gives an introduction to the toolbox and illustrates its use with examples from the EuroMix project. The toolbox can be used for hazard identification, hazard characterisation, exposure assessment and risk characterisation. Examples for hazard identification are selection of substances relevant for a specific adverse outcome based on adverse outcome pathways and QSAR models. Examples for hazard characterisation are calculation of benchmark doses and relative potency factors with uncertainty from dose response data, and use of kinetic models to perform in vitro to in vivo extrapolation. Examples for exposure assessment are assessing cumulative exposure at external or internal level, where the latter option is needed when dietary and non-dietary routes have to be aggregated. Finally, risk characterisation is illustrated by calculation and display of the margin of exposure for single substances and for the cumulation, including uncertainties derived from exposure and hazard characterisation estimates