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.

Pyrrolizidine Alkaloids Disturb Bile Acid Homeostasis in the Human Hepatoma Cell Line HepaRG.

1,2-unsaturated pyrrolizidine alkaloids (PAs) belong to a group of secondary plant metabolites. Exposure to PA-contaminated feed and food may cause severe hepatotoxicity. A pathway possibly involved in PA toxicity is the disturbance of bile acid homeostasis. Therefore, in this study, the influence of four structurally different PAs on bile acid homeostasis was investigated after single (24 h) and repeated (14 days) exposure using the human hepatoma cell line HepaRG. PAs induce a downregulation of gene expression of various hepatobiliary transporters, enzymes involved in bile acid synthesis, and conjugation, as well as several transcription regulators in HepaRG cells. This repression may lead to a progressive impairment of bile acid homeostasis, having the potential to accumulate toxic bile acids. However, a significant intracellular and extracellular decrease in bile acids was determined, pointing to an overall inhibition of bile acid synthesis and transport. In summary, our data clearly show that PAs structure-dependently impair bile acid homeostasis and secretion by inhibiting the expression of relevant genes involved in bile acid homeostasis. Furthermore, important biliary efflux mechanisms seem to be disturbed due to PA exposure. These mole-cular mechanisms may play an important role in the development of severe liver damage in PA-intoxicated humans.

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

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.

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.

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.

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.

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.

In vitro characterization of hepatic toxicity of Alternaria toxins.

Alternaria mycotoxins are secondary fungal metabolites which can contaminate food and feed. They are produced by Alternaria species with alternariol (AOH), alternariol monomethyl ether (AME), tenuazonic acid (TeA), and tentoxin (TEN) as the main representatives for Alternaria mycotoxins in food. Once passing the intestinal barrier, Alternaria toxins can reach the liver to exert yet uncharacterized molecular effects. Therefore, hepatic in vitro systems were used to examine selected Alternaria mycotoxins for their induction of metabolism-dependent cytotoxicity, phosphorylation of the histone H2AX as a surrogate marker for DNA double-strand breaks, and relevant marker genes for hepatotoxicity. Analysis of cell viability as well as the induction of H2AX phosphorylation in the hepatocarcinoma cell line HepG2 revealed a detoxification of 100 µmol/l AME and AOH by pre-treatment with S9 liver homogenate as shown by a decrease in cytotoxicity and H2AX histone phosphorylation to levels observed in control cells. Concentrations up to 100 µmol/l TeA and TEN did not induce H2AX phosphorylation whether metabolized or not. In the metabolically competent human hepatoma cell line HepaRG, no cytotoxicity of Alternaria toxins occurred even at high concentrations up to 100 µmol/l, which indicates a low cytotoxic potential. Induction of gene expression associated with liver toxicity was analyzed by quantitative real-time PCR using a specific hepatotoxicity PCR array in HepaRG cells: here, an evidence was found that 50 µmol/l of AOH, AME, TeA, and TEN might be associated with hepatotoxic effects, necrosis, and the development of diseases like cholestasis and phospholipidosis.

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 RARα 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.

PXR: Structure-specific activation by hepatotoxic pyrrolizidine alkaloids.

Pyrrolizidine alkaloids (PAs) comprise a large group of more than 660 secondary metabolites found in more than 6000 plant species worldwide. Acute PA intoxication induces severe liver damage. Chronic exposure to sub-lethal doses may cause cumulative damage or cancer. Nuclear receptor activation often constitutes a molecular event for xenobiotic-induced toxicity. However, so far nothing is known about potential interactions of PAs with nuclear receptors as a toxicological mode of action. Thus, in the present study PA-dependent activation of a comprehensive panel of nuclear receptors (PPARs, LXRα, RARα, RXRα, FXR, CAR, PXR, ERα/ß) was investigated using GAL4/UAS-based transactivation reporter gene assays. To cover the most frequently occurring PA structure types (retronecine, heliotridine and otonecine type; as well as monoester, open-chain diester and cyclic diester) different PAs were analyzed for interaction with nuclear receptors. Most of the nuclear receptors investigated were not affected by the tested PAs. However, significant activation was found for PXR, which was exclusively activated by the open-chain diesters, echimidine and lasiocarpine. Induction of the model PXR target gene CYP3A4 by PAs was verified at the mRNA, protein and enzyme activity level. In conclusion, PXR activation and PXR-mediated induction of CYP3A4 expression by PAs seem to be structure-dependent. Data suggest that only open-chain diesters act as PXR agonists. This might imply that a PXR-mediated mode of action may contribute to the hepatotoxicity of PAs that is dependent on PA structure.

Regulation of Drug Metabolism by the Interplay of Inflammatory Signaling, Steatosis, and Xeno-Sensing Receptors in HepaRG Cells.

Nonalcoholic fatty liver disease (NAFLD), which is characterized by triglyceride deposition in hepatocytes resulting from imbalanced lipid homeostasis, is of increasing concern in Western countries, along with progression to nonalcoholic steatohepatitis (NASH), liver fibrosis, and cirrhosis. Previous studies suggest a complex, mutual influence of hepatic fat accumulation, NASH-related inflammatory mediators, and drug-sensing receptors regulating xenobiotic metabolism. Here, we investigated the suitability of human HepaRG hepatocarcinoma cells as a model for NAFLD and NASH. Cells were incubated for up to 14 days with an oleate/palmitate mixture (125 µM each) and/or with 10 ng/ml of the inflammatory mediator interleukin-6 (IL-6). Effects of these conditions on the regulation of drug metabolism were studied using xenobiotic agonists of the aryl hydrocarbon receptor (AHR), pregnane X receptor (PXR), constitutive androstane receptor (CAR), nuclear factor (erythroid-derived 2)-like 2, and peroxisome proliferator-activated receptor α (PPARα). Results underpin the suitability of HepaRG cells for NAFLD- and NASH-related research and constitute a broad-based analysis of the impact of hepatic fatty acid accumulation and inflammation on drug metabolism and its inducibility by xenobiotics. IL-6 exerted pronounced negative regulatory effects on basal as well as on PXR-, CAR-, and PPARα-, but not AHR-dependent induction of drug-metabolizing enzymes. This inhibition was related to diminished transactivation potential of the respective receptors rather than to reduced transcription of nuclear receptor-encoding mRNAs. The most striking effects of IL-6 and/or fatty acid treatment were observed in HepaRG cells after 14 days of treatment, making these cultures appear a suitable model for studying the relationship of fatty acid accumulation, inflammation, and xenobiotic-induced drug metabolism.

Comparative analysis of 3D culture methods on human HepG2 cells.

Human primary hepatocytes represent a gold standard in in vitro liver research. Due to their low availability and high costs alternative liver cell models with comparable morphological and biochemical characteristics have come into focus. The human hepatocarcinoma cell line HepG2 is often used as a liver model for toxicity studies. However, under two-dimensional (2D) cultivation conditions the expression of xenobiotic-metabolizing enzymes and typical liver markers such as albumin is very low. Cultivation for 21 days in a three-dimensional (3D) Matrigel culture system has been reported to strongly increase the metabolic competence of HepG2 cells. In our present study we further compared HepG2 cell cultivation in three different 3D systems: collagen, Matrigel and Alvetex culture. Cell morphology, albumin secretion, cytochrome P450 monooxygenase enzyme activities, as well as gene expression of xenobiotic-metabolizing and liver-specific enzymes were analyzed after 3, 7, 14, and 21 days of cultivation. Our results show that the previously reported increase of metabolic competence of HepG2 cells is not primarily the result of 3D culture but a consequence of the duration of cultivation. HepG2 cells grown for 21 days in 2D monolayer exhibit comparable biochemical characteristics, CYP activities and gene expression patterns as all 3D culture systems used in our study. However, CYP activities did not reach the level of HepaRG cells. In conclusion, the increase of metabolic competence of the hepatocarcinoma cell line HepG2 is not due to 3D cultivation but rather a result of prolonged cultivation time.

Utility of an appropriate reporter assay: Heliotrine interferes with GAL4/upstream activation sequence-driven reporter gene systems.

Reporter gene assays are widely used for the assessment of transcription factor activation following xenobiotic exposure of cells. A critical issue with such assays is the possibility of interference of test compounds with the test system, for example, by direct inhibition of the reporter enzyme. Here we show that the pyrrolizidine alkaloid heliotrine interferes with reporter signals derived from GAL4-based nuclear receptor transactivation assays by a mechanism independent of luciferase enzyme inhibition. These data highlight the necessity to conduct proper control experiments in order to avoid perturbation of reporter assays by test chemicals.

Disturbance of gene expression in primary human hepatocytes by hepatotoxic pyrrolizidine alkaloids: A whole genome transcriptome analysis.

1,2-unsaturated pyrrolizidine alkaloids (PA) are plant metabolites predominantly occurring in the plant families Asteraceae and Boraginaceae. Acute and chronic PA poisoning causes severe hepatotoxicity. So far, the molecular mechanisms of PA toxicity are not well understood. To analyze its mode of action, primary human hepatocytes were exposed to a non-cytotoxic dose of 100 µM of four structurally different PA: echimidine, heliotrine, senecionine, senkirkine. Changes in mRNA expression were analyzed by a whole genome microarray. Employing cut-off values with a |fold change| of 2 and a q-value of 0.01, data analysis revealed numerous changes in gene expression. In total, 4556, 1806, 3406 and 8623 genes were regulated by echimidine, heliotrine, senecione and senkirkine, respectively. 1304 genes were identified as commonly regulated. PA affected pathways related to cell cycle regulation, cell death and cancer development. The transcription factors TP53, MYC, NFκB and NUPR1 were predicted to be activated upon PA treatment. Furthermore, gene expression data showed a considerable interference with lipid metabolism and bile acid flow. The associated transcription factors FXR, LXR, SREBF1/2, and PPARα/γ/δ were predicted to be inhibited. In conclusion, though structurally different, all four PA significantly regulated a great number of genes in common. This proposes similar molecular mechanisms, although the extent seems to differ between the analyzed PA as reflected by the potential hepatotoxicity and individual PA structure.

Dose-dependent induction of signaling pathways by the flavonoid quercetin in human primary hepatocytes: A transcriptomic study.

SCOPE: Quercetin is widespread in plant kingdom and consumed regularly with human diet (16 mg/day). Due to reported positive effects on health, quercetin supplements with recommended doses up to 2 g/day are offered. However, molecular effects of such high doses on human liver have not been assessed yet. Therefore, molecular effects on human hepatocytes were analyzed to help assessing the risk of quercetin supplementation. METHODS AND RESULTS: Molecular effects of three different quercetin concentrations on gene expression in human hepatocytes were investigated by microarray analysis. Possible new signaling pathways were investigated using reporter gene assays. Quercetin concentrations representing the normal intake showed weak effects on mRNA expression in liver cells. In contrast, supplemental doses affect immune response and p53 signaling and might be associated with cancer. Additionally, quercetin showed inhibition of transcriptional activation and mRNA-expression of HNF4α and its target genes. Inhibitory effects were also found for FXR, LXRα, and PXR. CONCLUSION: Normal intake of quercetin seems to play a minor regulatory role, while supplement doses may have great effects on gene expression in hepatocytes. However, since it is not clarified whether such high doses of quercetin exert positive or negative effects, a careful handling of quercetin supplements is advised.

Polycyclic aromatic hydrocarbons stimulate human CYP3A4 promoter activity via PXR.

Metabolic activation of polycyclic aromatic hydrocarbons (PAH) is mediated mainly by cytochrome P450 monooxygenases (CYP) CYP1A1, 1A2 and 1B1. Several PAH are known to induce these CYP via aryl hydrocarbon receptor (AhR) signaling. Recently, it was shown that the PAH benzo[a]pyrene (BaP) can induce CYP3A4 as well. The induction was suggested to be mediated by the pregnane X receptor (PXR) rather than AhR. Metabolism by CYP3A4 is only known for dihydrodiol metabolites of PAH but not for their parent compounds. In the present study, a CYP3A4 reporter gene assay, requiring the overexpression of PXR, was used to investigate whether the PAH parent compounds BaP, benzo[c]phenanthrene (BcP) and dibenzo[a,l]pyrene (DBalP) as well as their corresponding phase I metabolites, the respective dihydrodiols and diol epoxides, can induce CYP3A4 promoter activity. BaP, BcP and their dihydrodiols were found to significantly activate the CYP3A4 promoter. Moreover, activation of PXR by all four compounds was detected by using a PXR transactivation assay, supporting that PXR mediates CYP3A4 induction by PAH. Taken together, these results show that both PAH parent compounds as well as their phase I metabolites induce CYP3A4 promoter via the transcription factor PXR.