Dose‐dependent hepatotoxicity of hydrogen peroxide in <scp>HepG2</scp> cells and its modulation by <scp>CYP450</scp> induction

In vitro liver models combined with metabolomics approaches offer promising alternatives to animal testing in toxicology. In this study, we investigated concentration‐dependent effects of hydrogen peroxide (H 2 O 2 ) on the intra‐ and extracellular metabolome of HepG2 cells using 1 H Nuclear Magnetic Resonance (NMR) spectroscopy. After cells were exposed to low, medium or high concentrations of H 2 O 2 , metabolomic analysis revealed a progressive increase in metabolic perturbation with rising toxin concentration. Significant alterations were detected in a limited subset of metabolites after low H 2 O 2 exposure, and substantially broader disruptions occurred after medium or high H 2 O 2 exposure, with most measured metabolites affected at the highest exposure level. To enhance metabolic competence, cells were pretreated with rifampicin to induce cytochrome P450 (CYP450) activity, which is typically low in HepG2 cells. Comparative analysis of rifampicin‐pretreated and untreated cells exposed to high H 2 O 2 concentrations demonstrated disruption of multiple biochemical pathways, including energy metabolism, lipid metabolism and amino acid metabolism. Notably, rifampicin pretreatment attenuated the magnitude of metabolic perturbations, as reflected by reduced intracellular alterations and minimal changes in extracellular metabolite profiles. Furthermore, rifampicin‐treated cells exhibited metabolite signatures more consistent with human liver physiology in vivo , including increased glutathione and 2‐hydroxybutyrate levels. Collectively, these findings demonstrate that pretreatment with rifampicin prior to toxin exposure enhances the physiological relevance of HepG2‐based hepatotoxicity models and improves their potential to predict human liver responses. Moreover, the results highlight the sensitivity of NMR‐based metabolomics to detect toxin induced metabolic changes across a range of exposure concentrations.