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Hepatic Steatosis Shifts Phase I Metabolism & Alters Susceptibility to Toxicants In Vitro
Citation:
Tucker, N., G. Nelson, AND B. CHORLEY. Hepatic Steatosis Shifts Phase I Metabolism & Alters Susceptibility to Toxicants In Vitro. NC Society of Toxicology (NC SOT), Research Triangle Park, NC, October 07, 2019. https://doi.org/10.23645/epacomptox.25676808
Impact/Purpose:
Nearly 30% of the US population have some form of fatty liver disease. The majority of these individuals have asymptomatic hepatic steatosis due to primarily diet and sedentary lifestyle, but also genetic predisposition, environmental exposure, and other factors. Given the principal function of the liver is to metabolize, detoxify, inactivate, and convert both endogenous and exogenous substances that are either excreted into bile or returned to the systemic circulation, excessive hepatic fat accumulation has the potential to alter the liver’s absorption, deposition, metabolism, and excretion (ADME) of those chemicals. This can directly affect the hepatotoxicity of these exogenous chemical exposures. In vitro assays provide a broad survey of the potential proximal biochemical and cellular targets for a chemical. The goal of this research is to measure the impact of steatosis on ADME of chemicals that are linked to liver toxicity using an in vitro model.
Description:
Hepatic steatosis (fatty liver disease) is a pathological condition that may alter xenobiotic metabolism, thereby increasing susceptibility to environmental toxicants. Here we used a metabolically competent human liver-derived cell line – HepaRG – to model steatosis for in-vitro toxicity assessment. Cells were exposed to media containing free fatty acid, complexed oleate and palmitate, to induce a steatotic state. An optimum free fatty acid ratio, concentration, and exposure time were identified and chosen to achieve measurable lipid accumulation with minimal toxicity. The hepatotoxic piscicide rotenone was selected to assess chemical toxicity in our steatotic model. After 48h chemical exposure, measurement of cell viability by an intracellular ATP assay demonstrated increased sensitivity to rotenone corresponding to varied free fatty acid ratios in a 1 mM dose- experimentally derived IC50 values of 0.67<0.53<0.33<0.40 resulted from varying dosing ratios of oleate to palmitate starting with no free fatty acid, a 1:1, 1:2, and 2:1 dose conditions. Additionally, qPCR measurement of cytochrome P450 (CYP) gene expression showed marked reduction responding to free-fatty acid addition and ratios in the expression of several CYP genes including CYP3A4, the most active P450 enzyme in rotenone metabolism. P450 activity is a major factor in limiting rotenone toxicity as rotenone metabolites are less active than the parent compound. These results suggest that our HepaRG steatosis model can be a useful tool for evaluating steatosis as a risk factor in chemical toxicity studies. Future addition of high content analysis (HCA) oxidative stress and mitochondrial dysfunction parameters to the model may enhance its predictive capability for human hepatotoxicity susceptibility screening. This poster does not necessarily reflect the policy of the US EPA.
URLs/Downloads:
DOI: Hepatic Steatosis Shifts Phase I Metabolism & Alters Susceptibility to Toxicants In Vitro
NC SOT 2019 FALL HEPATIC STEATOSIS FINAL.V.3.PDF (PDF, NA pp, 5186.254 KB, about PDF)