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High-throughput Assessment of Increased Chemical Toxicity Due to Hepatic Steatosis (SOT 2021)
Citation:
Tucker, N., G. Nelson, J. Harrill, AND B. Chorley. High-throughput Assessment of Increased Chemical Toxicity Due to Hepatic Steatosis (SOT 2021). Society of Toxicology 2021 Annual meeting, NA, Virtual, March 12 - 26, 2021. https://doi.org/10.23645/epacomptox.18127739
Impact/Purpose:
Poster presented to the Society of Toxicology virtual annual seminar March 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, impacts the bioactivation or detoxification of exposed chemicals, and changes baseline chemical toxicity. Here, we modeled this state in a human hepatocyte cell culture model to assess the impact of chemical toxicity in a quantitative and high-throughput manner. We induced steatosis in human hepatoma-derived cells, HepaRGs, by dosing maintenance media with1 mM 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 steatotic culture. 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 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 indicated that we can measure the impact of pre-existing conditions on environmental chemical toxicity, which 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 (SOT 2021)
2021_02_24_SOT_TUCKER.PDF (PDF, NA pp, 2854.198 KB, about PDF)