TY - JOUR T1 - Permeabilized cryopreserved human hepatocytes as an exogenous metabolic system in a novelmetabolism-dependent cytotoxicity assay (MDCA) for the evaluation of metabolic activation anddetoxification of drugs associated with drug induced liver injuries: Results with acetaminophen,amiodarone, cyclophosphamide, ketoconazole, nefazodone, and troglitazone JF - Drug Metabolism and Disposition JO - Drug Metab Dispos DO - 10.1124/dmd.121.000645 SP - DMD-AR-2021-000645 AU - Hong Wei AU - Albert P. Li Y1 - 2021/01/01 UR - http://dmd.aspetjournals.org/content/early/2021/11/08/dmd.121.000645.abstract N2 - We report here a novel in vitro experimental system, the metabolism-dependent cytotoxicity assay (MDCA), for the definition of the roles of hepatic drug metabolism in toxicity. MDCA employs permeabilized cofactor-supplemented cryopreserved human hepatocytes (MetMax{trade mark, serif} human hepatocytes, MMHH), as an exogenous metabolic activating system, and HEK-293 cells, a cell line devoid of drug metabolizing enzyme activity, as target cells for the quantification of drug toxicity. The assay was performed in the presence and absence of cofactors for key drug metabolism pathways known to play key roles in drug toxicity: NADPH/NAD+ for phase 1 oxidation, UDPGA for UGT mediated glucuronidation, PAPS for SULT mediated sulfation, and GSH for GST mediated GSH conjugation. Six drugs with clinically significant hepatoxicity, resulting in liver failure or a need for liver transplantation: acetaminophen, amiodarone, cyclophosphamide, ketoconazole, nefazodone and troglitazone were evaluated. All six drugs exhibited cytotoxicity enhancement by NADPH, suggesting metabolic activation via phase 1 oxidation. Attenuation of cytotoxicity by UDPGA was observed for acetaminophen, ketoconazole and troglitazone, by PAPS for acetaminophen, ketoconazole and troglitazone, and by GSH for all six drugs. Our results suggest that MDCA can be applied towards the elucidation of metabolic activation and detoxification pathways, providing information that can be applied in drug development to guide structure optimization to reduce toxicity and to aid the assessment of metabolism-based risk factors for drug toxicity. GSH detoxification represents an endpoint for the identification of drugs forming cytotoxic reactive metabolites, a key property of drugs with idiosyncratic hepatotoxicity. Significance Statement Application of the metabolism-dependent cytotoxicity assay (MDCA) for the elucidation of the roles of metabolic activation and detoxification pathways in drug toxicity may provide information to guide structure optimization in drug development to reduce hepatotoxic potential, and to aid the assessment of metabolism-based risk factors. GSH detoxification represents an endpoint for the identification of drugs forming cytotoxic reactive metabolites may be applied towards the evaluation of idiosyncratic hepatotoxicity. ER -