Cell Metabolomics Reveals the Hepatotoxic Mechanism of Copper in Normal Rat Liver Cells Using Reversed-Phase and Hydrophilic Interaction Liquid Chromatography-Quadrupole-Time-of-Flight Mass Spectrometry

Copper (Cu) is an essential trace element for maintaining normal cellular functions; however, excessive Cu accumulation has been confirmed to induce hepatotoxicity, while the metabolic mechanisms underlying Cu-induced hepatotoxicity remain unclear. In this study, an innovative integrated separation strategy was established, combining hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography (RPLC), coupled with quadrupole-time-of-flight mass spectrometry (Q-TOF/MS), to systematically resolve metabolomic perturbations in CuCl₂-exposed rat BRL-3A hepatocytes. Based on their complementary separation mechanisms-HILIC enables efficient retention and separation of polar metabolites via hydrophilic interactions, while RPLC separates nonpolar/weakly polar lipid molecules based on hydrophobic interactions-this analytical strategy significantly expanded the coverage of detectable metabolites and improved the reliability of metabolite identification through cross-validation between the two chromatographic platforms. The results showed that a total of 25 metabolites with significant changes were identified when BRL-3A cells were exposed to 50 µM CuCl₂ (with a cell viability of 85%). These changes were mainly enriched in metabolic pathways such as glutathione metabolism (characterized by a significant decrease in the GSH/GSSG ratio, p < 0.01), arachidonic acid (AA) metabolism (a 42% reduction in AA, p < 0.05), and glycerophospholipid metabolism (a 1.8-fold increase in the levels of lysophospholipids [LysoPCs/LysoPEs], p < 0.05). These findings reveal that oxidative stress, membrane structure damage, and energy metabolism imbalance are the core mechanisms of Cu-induced hepatotoxicity. The integrated liquid chromatography-mass spectrometry (LC-MS) analytical framework established in this study not only provides a novel molecular perspective for elucidating the mechanisms of Cu-induced hepatotoxicity but also demonstrates the application potential of advanced complementary separation technologies in the risk assessment of environmental pollutants.

HILIC‐Q‐TOF/MS; RPLC‐Q‐TOF/MS; copper exposure; hepatotoxicity; metabolomics.