Abstract
The liver is constantly exposed to mixtures of hepatotoxic compounds, such as food contaminants and pesticides. Dose addition is regularly assumed for mixtures in risk assessment, which however might not be sufficiently protective in case of synergistic effects. Especially the prediction of combination effects of substances which do not share a common adverse outcome (AO) might be problematic. In this study, the focus was on the endpoint liver triglyceride accumulation in vitro, an indicator of hepatic fatty acid changes. The hepatotoxic compounds difenoconazole, propiconazole and tebuconazole were chosen which cause hepatic fatty acid changes in vivo, whereas fludioxonil was chosen as a hepatotoxic substance not causing fatty acid changes. Triglyceride accumulation was analyzed for combinations of steatotic and non-steatotic pesticides in human HepaRG hepatocarcinoma cells. Investigations revealed a potentiation of triglyceride accumulation by mixtures of the steatotic compounds with the non-steatotic fludioxonil, as compared to the single compounds. Mathematical modeling of combination effects indicated more than additive effects for the tested combinations if the method by Chou was applied, and a decrease in EC50 values of the steatotic compounds when applied in mixtures. Use of an adverse outcome pathway (AOP)-driven testing strategy for liver steatosis showed interactions of the test compounds with the nuclear receptors AHR, CAR and PXR, as well as a downregulation of ACOX2. An ACOX2-dependent mechanism underlying the observed mixture effect could not be verified using a siRNA approach. By contrast, a toxicokinetic interaction was identified including an inhibition of the metabolic enzyme CYP3A4 by fludioxonil and a decreased metabolic conversion of the CYP3A4 substrate difenoconazole when used in mixture experiments. In conclusion, an interaction by a steatotic and a non-steatotic compound at the toxicokinetic level on the endpoint triglyceride accumulation in vitro was described.
Highlights
The constant exposure of consumers to mixtures of chemicals is of concern for risk assessment as they could, in principle, cause toxic effects in humans different from the effects of individual compounds
The aim of this study was to investigate binary mixtures of steatotic and non-steatotic compounds and the influence of a non-steatotic compound on steatosis caused by the steatotic substance
Initial analysis of cytotoxicity was performed for all test compounds and their mixtures to ensure the use of nontoxic concentrations in all experiments
Summary
The constant exposure of consumers to mixtures of chemicals is of concern for risk assessment as they could, in principle, cause toxic effects in humans different from the effects of individual compounds. Liver steatosis is a widespread disease and has gained more and more attention over the last years (Perumpail et al 2017) It is characterized by an increased intracellular triglyceride content in hepatocytes. The interaction of chemical agonists with those receptors leads to several intermediate effects like altered gene expression (MLXIPL, SREBF1, SCD, FASN, CD36), increase of fatty acid influx, de novo synthesis of fatty acids, inhibition of respiration and mitochondrial/ microsomal β-oxidation and to liver triglyceride accumulation (Mellor et al 2016)
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