Development of in vitro drug-induced hepatotoxicity evaluation method based on multi-omics analysis using human primary hepatocytes

Abstract

Drug-induced liver injury (DILI) is a major cause of drug development discontinuation. The application of metabolomics and proteomics-based multi-omics analysis for DILI evaluation is expected to further elucidate the toxicity mechanisms. However, primary human hepatocyte (PHH), suitable for DILI evaluation, have not been applied to multi-omics studies because PHHs is expensive and limited in availability. In this study, we established a 96-well plate sample preparation method that allows for multi-omics analysis using 5 × 10⁴ cells. The sample preparation protocol was optimized so that drug metabolites, metabolomes, and proteomes could be measured using the same PHH sample. All steps in this method, from pretreatment on a 96-well culture plate to introduction of the sample into the LC/MS, were completed on a 96-well plate. The method was applied to PHHs exposed to acetaminophen (APAP) at a 10% inhibitory concentration. The results showed that APAP and its metabolites were detected only in the APAP-exposed group, and the expression levels of some enzymes involved in CYP and conjugation reactions also significantly increased compared to those in the control group. Furthermore, pathway analysis using the in vitro multi-omics information reproduced endogenous metabolic changes observed in vivo (e.g. GSH depletion). Future evaluation of various drugs using this method is expected to lead to a more detailed elucidation of toxicity mechanisms.