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Adverse outcome pathway-based analysis of liver steatosis in vitro using human liver cell lines.

ABSTRACT: Here, we present an in vitro test battery to analyze chemicals for their potential to induce liver triglyceride accumulation, a hallmark of liver steatosis. We describe steps for using HepG2 and HepaRG human hepatoma cells in conjunction with a combination of several in vitro assays covering the different molecular initiating events and key events of the respective adverse outcome pathway. This protocol is suitable for assessing single substance effects as well as mixtures allowing their classification as steatotic or non-steatotic. For complete details on the use and execution of this protocol, please refer to Luckert et al. (2018),¹ Lichtenstein et al. (2020),² and Knebel et al. (2019).³.

SUBMITTER: Karaca M

PROVIDER: S-EPMC10463250 | biostudies-literature | 2023 Aug

REPOSITORIES: biostudies-literature

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Adverse outcome pathway-based analysis of liver steatosis in vitro using human liver cell lines.

Karaca Mawien M Fritsche Kristin K Lichtenstein Dajana D Vural Özlem Ö Kreuzer Katrin K Alarcan Jimmy J Braeuning Albert A Marx-Stoelting Philip P Tralau Tewes T

STAR protocols 20230823 3

Here, we present an in vitro test battery to analyze chemicals for their potential to induce liver triglyceride accumulation, a hallmark of liver steatosis. We describe steps for using HepG2 and HepaRG human hepatoma cells in conjunction with a combination of several in vitro assays covering the different molecular initiating events and key events of the respective adverse outcome pathway. This protocol is suitable for assessing single substance effects as well as mixtures allowing their classif ...[more]

PMID: 37616165

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Project description:Exposure to chemicals contributes to the development and progression of fatty liver, or steatosis, a process characterized by abnormal accumulation of lipids within liver cells. However, lack of knowledge on how chemicals cause steatosis has prevented any large-scale assessment of the 80,000+ chemicals in current use. To address this gap, we mined a large, publicly available toxicogenomic dataset associated with 18 known steatogenic chemicals to assess responses across assays (in vitro and in vivo) and species (i.e., rats and humans). We identified genes that were differentially expressed (DEGs) in rat in vivo, rat in vitro, and human in vitro studies in which rats or in vitro primary cell lines were exposed to the chemicals at different doses and durations. Using these DEGs, we performed pathway enrichment analysis, analyzed the molecular initiating events (MIEs) of the steatosis adverse outcome pathway (AOP), and predicted metabolite changes using metabolic network analysis. Genes indicative of oxidative stress were among the DEGs most frequently observed in the rat in vivo studies. Nox4, a pro-fibrotic gene, was down-regulated across these chemical exposure conditions. We identified eight genes (Cyp1a1, Egr1, Ccnb1, Gdf15, Cdk1, Pdk4, Ccna2, and Ns5atp9) and one pathway (retinol metabolism), associated with steatogenic chemicals and whose response was conserved across the three in vitro and in vivo systems. Similarly, we found the predicted metabolite changes, such as increases of saturated and unsaturated fatty acids, conserved across the three systems. Analysis of the target genes associated with the MIEs of the current steatosis AOP did not provide a clear association between these 18 chemicals and the MIEs, underlining the multi-factorial nature of this disease. Notably, our overall analysis implicated mitochondrial toxicity as an important and overlooked MIE for chemical-induced steatosis. The integrated toxicogenomics approach to identify genes, pathways, and metabolites based on known steatogenic chemicals, provide an important mean to assess development of AOPs and gauging the relevance of new testing strategies.

Project description:BackgroundPer- and polyfluoroalkyl substances (PFAS) are persistent organic pollutants with myriad adverse effects. While perfluorooctanoic acid (PFOA) and perfluorooctane sulfonic acid (PFOS) are the most common contaminants, levels of replacement PFAS, such as perfluoro-2-methyl-3-oxahexanoic acid (GenX), are increasing. In rodents, PFOA, PFOS, and GenX have several adverse effects on the liver, including nonalcoholic fatty liver disease.ObjectiveWe aimed to determine human-relevant mechanisms of PFAS induced adverse hepatic effects using FRG liver-chimeric humanized mice with livers repopulated with functional human hepatocytes.MethodsMale humanized mice were treated with 0.067 mg/L of PFOA, 0.145 mg/L of PFOS, or 1 mg/L of GenX in drinking water for 28 days. Liver and serum were collected for pathology and clinical chemistry, respectively. RNA-sequencing coupled with pathway analysis was used to determine molecular mechanisms.ResultsPFOS caused a significant decrease in total serum cholesterol and LDL/VLDL, whereas GenX caused a significant elevation in LDL/VLDL with no change in total cholesterol and HDL. PFOA had no significant changes in serum LDL/VLDL and total cholesterol. All three PFAS induced significant hepatocyte proliferation. RNA-sequencing with alignment to the human genome showed a total of 240, 162, and 619 differentially expressed genes after PFOA, PFOS, and GenX exposure, respectively. Upstream regulator analysis revealed inhibition of NR1D1, a transcriptional repressor important in circadian rhythm, as the major common molecular change in all PFAS treatments. PFAS treated mice had significant nuclear localization of NR1D1. In silico modeling showed PFOA, PFOS, and GenX potentially interact with the DNA-binding domain of NR1D1.DiscussionThese data implicate PFAS in circadian rhythm disruption via inhibition of NR1D1. These studies show that FRG humanized mice are a useful tool for studying the adverse outcome pathways of environmental pollutants on human hepatocytes in situ.

Dataset Information

Adverse outcome pathway-based analysis of liver steatosis in vitro using human liver cell lines.

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Adverse outcome pathway-based analysis of liver steatosis in vitro using human liver cell lines.

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