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A Multiplex Automated Screen for Mitotoxicity in Human Hepatocytes and HepG2 Cells Poster

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Abstract

Timothy A. Moeller1, Tracy Worzella2 and Brad Larson3
1Celsis In Vitro Technologies (Baltimore, MD) 2Promega (Madison, WI) 3 BioTek (Winooski, VT)

Many drugs and environmental chemicals have been implicated in mitochondrial toxicity resulting in myopathies, hepatotoxicities, peripheral neuropathies and cardiovascular disorders. The mechanisms of mitochondrial toxicities are complex due to multiple modes of action such as modulating mitochondrial replication and disruption of electron transport chain, and by organ-specific susceptibility. For example, the mitotoxin propionate preferentially targets the liver over muscle due to metabolic activation in hepatocytes. Therefore, multiple approaches are needed to assess a chemical’s mitochondrial liabilities in specific cellular systems. Herein, we present one approach to utilize two markers of cellular viability in an automated multiplexed format for highthroughput screening to monitor hepatotoxicity that can discriminate primary mitochondrial toxicity from general cytotoxicity. Primary human hepatocytes and HepG2 cells were used in a 384-well suspension culture format in serum-free, glucose-free galacatose-containing medium for up to 6 hour exposure with cytotoxins in an 11-point response curve. ATP Detection Reagent and Cytotoxicity Reagent were used to measure ATP levels and cell membrane integrity, respectively. A mitochondrial toxic effect was defined as a reduction of ATP relative to untreated control, with no or minor changes in cell membrane integrity. CCCP and antimycin were used as model compounds to disrupt mitochondrial activity. Digitonin cytotoxicity was caused by solubilizing the cell membrane, resulting in decreased ATP and loss of cell membrane integrity. Staurosporine and tamoxifen were also used as cytotoxins to validate the system. Similar results were observed between HepG2 and human hepatocytes except for antimycin where a Crabtree effect was implicated for the difference in its potency. In summary, the multiplex system provided a robust and informative platform to screen drugs and chemicals for hepatic mitotoxicity by measuring ATP and cell membrane integrity.

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  • Part# PS151
  • Printed in USA.

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