High Throughput Metabolomics In vitro Platform for The Identification of Hepatotoxicity Modes of Action

Abstract Cell-based metabolomics provides multiparametric physiologically relevant readouts that can be highly advantageous for improved, biologically based decision making in early stages of compound development. Here we present the development of a 96-well plate LC-MS-based targeted metabolomics screening platform for the classification of liver toxicity MoAs in HepG2 cells. Different parameters of the workflow (cell seeding density, passage number, cytotoxicity testing, sample preparation, metabolite extraction, analytical method, and data processing) were optimized and standardized to increase the efficiency of the testing platform. The applicability of the system was tested with seven substances known to be representative of three different liver toxicity MoAs (peroxisome proliferation, liver enzyme induction and liver enzyme inhibition). Multivariate and univariate analyses showed a dose response of the metabolic effects, a clear differentiation between liver toxicity MoAs and resulted in the identification of metabolite patterns specific for each MoA. Key metabolites indicative of both, general and mechanistic specific hepatotoxicity were identified. The method presented here offers a multiparametric, mechanistic-based and cost-effective hepatotoxicity screening that provides MoA classification and sheds light into the pathways involved in the toxicological mechanism. This assay can be implemented as a reliable compound screening platform for improved safety assessment in early compound development pipelines.