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High-throughput Assessment of Increased Chemical Toxicity Due to Hepatic Steatosis
Citation:
Tucker, N., G. Nelson, J. Harrill, AND B. Chorley. High-throughput Assessment of Increased Chemical Toxicity Due to Hepatic Steatosis. Environmental Mutagenesis and Genomics Society meeting September 2021 (EMGS) Virtual Annual meeting, Research Triangle Park, NC, September 22 - 25, 2021. https://doi.org/10.23645/epacomptox.17318912
Impact/Purpose:
Poster presented to the Environmental Mutagenesis and Genomics Society meeting September 2021. The Agency is interested in developing higher-throughput, high content assays that can quantitatively measure the impact of pre-existing susceptibilities to environmental chemical exposure. This research focuses on developing a hepatocyte-based assay that measures the impact of steatosis (abnormal fat retention in the liver that impacts ~30% of the US population) on toxicity of chemical exposure. This assay is high-content and measure multiple measures of cell loss, nuclear morphology, oxidative stress, mitochondrial stress, and cellular membrane integrity. Long-term, this assay should be integrated into a Tier 1 screening paradigm to assess a common vulnerability in the US population to environmental chemical exposure.
Description:
Hepatic steatosis alters native liver xenobiotic metabolism, which may alter sensitivity to chemical toxicity through impacting the bioactivation or detoxification of chemicals. Here, we modeled fatty human hepatic cell culture model to assess the impact of steatosis on chemical toxicity in a quantitative and high-throughput manner. We induced steatosis in human hepatoma-derived cells, HepaRGs, by dosing maintenance media with 1 mM of 1:2 oleic:palmitic free fatty acid for 1 week. Cytochrome P450 (CYP) gene expression and metabolic activity (CYPs 1A1, 1A2, 2B6, 2C9, 2E1, 3A4) were significantly altered in the steatotic culture condition. Relative culture viability was determined by Cell Titer Glo (CTG), lactate dehydrogenase (LDH) release, and multiplexed fluorometric measurements of nuclear morphology using the Opera Phenix high-content screening (HCS) system. Naive and steatotic HepaRG cells were exposed to known hepatotoxicant (rotenone) over a 5-point dose range for 24 or 48 hrs. Rotenone toxicity (IC50) shifted from a baseline 0.64 μM to 0.48 μM in steatotic cells as measured by CTG, from 0.83 μM to 0.57 μM measuring LDH, and 0.80 μM to 0.62 μM using cell counts derived from HCS. Additional high-throughput toxicity measures – including morphology, reactive oxygen species generation, and mitochondrial membrane potential – as well as additional chemicals known to be impacted by CYP-mediated metabolism are currently being assessed. The results of this study will help outline methods to quantitate the impact of pre-existing conditions on environmental chemical toxicity in a high-throughput manner. These efforts will contribute to ongoing Agency efforts to assess environmental exposure risks in susceptible subpopulations. This abstract does not necessarily reflect EPA policy. Mention of trade names is not an endorsement or recommendation for use.
URLs/Downloads:
DOI: High-throughput Assessment of Increased Chemical Toxicity Due to Hepatic Steatosis
2021_NTUCKER_EMGS_TOSTICS.PDF (PDF, NA pp, 2456.54 KB, about PDF)