Unveiling the Cellular Mystery of Isoniazid–Rifampicin (IR) in CYP2E1 Overexpressing HepG2 Cells

Astratt

Background and Aims The molecular basis of liver injury observed following cotreatment with the first‐line antitubercular drugs (ATDs) isoniazid (INH) and rifampicin (RIF) is not fully understood. Cytochrome P450 2E1 (CYP2E1), a key enzyme in drug metabolism, is thought to contribute to INH‐RIF (IR)–associated hepatotoxicity. In this study, we investigated the intracellular events, including lysosomal and mitochondrial alterations, that occur in CYP2E1 overexpressing HepG2 (HepG2‐E47) cells during IR exposure. Methods HepG2‐E47 cells overexpressing CYP2E1 were cotreated with 5 μ M INH and 2.5 μ M RIF for various time points. Reactive oxygen species (ROS) generation, antioxidant defense status, and CYP2E1 activity were assessed. Lysosomal destabilization, mitochondrial depolarization, and cell death were analyzed using fluorescence spectrophotometer and flow cytometry. To explore CYP2E1 involvement, cells were pretreated with the selective CYP2E1 inhibitor chlormethiazole (CMZ). Pharmacological modulators were used to explore the role of lysosomal and mitochondrial involvement following IR treatment. Results IR treatment significantly enhanced CYP2E1 activity, ROS production, and impairment of the cellular antioxidant system. These effects were prevented by CMZ, indicating CYP2E1‐dependent oxidative stress. Increased ROS in hepatocytes was accompanied by an early lysosomal dysfunction and cathepsin D release, which were followed by mitochondrial depolarization and apoptosis of the cells. Treatment with bafilomycin, a pharmacological modulator of lysosomal function, alleviated mitochondrial damage, consistent with lysosomal destabilization preceding mitochondrial dysfunction. Conclusion CYP2E1 plays a pivotal role in IR‐associated cellular injury in CYP2E1 overexpressing HepG2 cells by driving oxidative stress that triggers lysosomal and mitochondrial dysfunction leading to apoptosis. Targeting CYP2E1 or its downstream signalling pathways may provide therapeutic potential against ATD‐associated liver injury.