The uricosuric benzbromarone disturbs the mitochondrial redox homeostasis and activates the NRF2 signaling pathway in HepG2 cells

The uricosuric benzbromarone is a mitochondrial toxicant associated with severe liver injury in patients treated with this drug. Since dysfunctional mitochondria can increase mitochondrial superoxide (O₂^•-) production, we investigated the consequences of benzbromarone-induced mitochondrial oxidative stress on the hepatic antioxidative defense system. We exposed HepG2 cells (a human hepatocellular carcinoma cell line) to increasing concentrations of benzbromarone (1-100 μM) for different durations (2-24 h), and investigated markers of antioxidative defense and oxidative damage. At high concentrations (≥50 μM), benzbromarone caused accumulation of mitochondrial superoxide (O₂^•-) and cellular Reactive Oxygen Species (ROS). At concentrations >50 μM, benzbromarone increased the mitochondrial and cellular GSSG/GSH ratio and increased the oxidized portion of the mitochondrial thioredoxin 2. Benzbromarone stabilized the transcription factor NRF2 and caused its translocation into the nucleus. Consequently, the expression of the NRF2-regulated antioxidative proteins superoxide dismutase 1 (SOD1) and 2 (SOD2), Glutathione Peroxidase 1 (GPX1) and 4 (GPX4), as well as thioredoxin 1 (TRX1) and 2 (TRX2) increased. Finally, upregulation of NRF2 by siRNA-mediated knock-down of KEAP1 partially protected HepG2 cells from benzbromarone-induced membrane damage and ATP depletion. In conclusion, benzbromarone increased mitochondrial O₂^•- accumulation and activates the NRF2 signaling pathway in HepG2 cells, thereby strengthening the cytosolic and mitochondrial antioxidative defense. Impaired antioxidative defense may represent a risk factor for benzbromarone-induced hepatotoxicity.

Benzbromarone; Glutathione; HepG2 cells; Mitochondria; NRF2; Reactive oxygen species; Thioredoxin.