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High Content Imaging Assessment of Chemical Hepatotoxicity and Differential Impact of Pre-Existing Hepatic Steatosis
Citation:
Tucker, N., G. Nelson, J. Harrill, AND B. Chorley. High Content Imaging Assessment of Chemical Hepatotoxicity and Differential Impact of Pre-Existing Hepatic Steatosis. Society of Toxicology 62nd Annual Meeting and ToxExpo 2023, Nashville, NC, March 19 - 23, 2023. https://doi.org/10.23645/epacomptox.24917250
Impact/Purpose:
This abstract will be submitted to the Society of Toxicology for consideration for a poster or platform presentation at the 2023 Annual meeting. The research outlined describe the development of a high-throughput, high-content imaging assay using a hepatic cell culture to assess the impact of steatosis on chemical-mediated toxicity. The implications, if successful in development, are a screening assay which can quantitatively measure the impact of a pre-existing susceptibility that currently affects 1/3 of the population. The New Approach Methodology will be a valuable tool for hazard identification and risk assessment of vulnerable subpopulations.
Description:
A latent liver condition experienced by an estimated third of adults consuming a Western (American) diet, hepatic steatosis, alters native liver metabolism impacting xenobiotic bioactivation/detoxification and may alter response sensitivity to chemical insults. Here, we modeled this disease state in a human hepatoma-derived cell (HepaRG) culture model to assess the impact of steatosis on chemical toxicity using high-content imaging (HCI) approaches. HCI may be used to enhance high-throughput screening efforts to include a pre-existing susceptibility affecting a large proportion of our population. We induced steatosis in HepaRG by adding 1 mM 1:2 oleic:palmitic free fatty acid to maintenance media for 1 week. Cytochrome P450 (CYP) gene expression measured by RT-qPCR (CYPs 1A1, 1A2, 2B6, 2C9, 2E1, 3A4) and metabolic activity measured by LC-MS (CYPs 2B6, 2C19, 3A4) were significantly altered in the steatotic culture condition, and elements of these alterations persisted for up to 48 h following a return to control maintenance media. Cell viability was determined by Cell Titer Glo (CTG), lactate dehydrogenase (LDH) release, and multiplexed fluorometry determined by high content confocal microscopy using the Opera Phenix system. Naive and steatotic HepaRG cells were exposed to known mitochondrial hepatotoxicant rotenone over a 5-point dose range for 24 or 48 h. Rotenone toxicity (IC50) shifted from a baseline of 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 HCI. Additional toxicity measures included probes for cell and nuclear morphology (Hoechst33342), evaluations of mitochondrial membrane permeability and potential (TOTO-3, TMRM), and reactive oxygen species generation (H2-DCFDA). Additionally, we screened a suite of chemicals with known CYP-mediated bioactivation, detoxification, or non-impact on hepatotoxicity. Results of this study indicate that we can evaluate the impact of a pre-existing health condition on environmental chemical toxicity, which will contribute to assessment of health risks in susceptible subpopulations. This abstract does not necessarily reflect EPA policy.
URLs/Downloads:
DOI: High Content Imaging Assessment of Chemical Hepatotoxicity and Differential Impact of Pre-Existing Hepatic Steatosis
20230315_TUCKER_SOT23_FIN.PDF (PDF, NA pp, 7815.921 KB, about PDF)