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 (O 2 ?- ) production, we investigated the consequences of benzbromarone-induced mitochondrial oxidative stress on the hepatic anti - oxidative defense system. We exposed HepG2 cells (a human hepatocellular carcinoma cell line) to increasing concentrations of benzbromarone (1-100 ?M) for di fferent durations (2-24 h), and investigated markers of an- tioxidative defense and oxidative damage. At high concentrations ( ?50 ?M), benzbromarone caused accumu- lation of mitochondrial superoxide (O 2 ?- ) and cellular reactive oxygen species (ROS). At concentrations 50 ?M, benzbromarone increased the mitochondrial and cellular GSSG/GSH ratio and in- creased 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 da- mage and ATP depletion. In conclusion, benzbromarone increased mitochondrial O 2 ?- accumulation and acti- vates the NRF2 signaling pathway in HepG2 cells, thereby strengthening the cytosolic and mitochondrial an- tioxidative defense. Impaired antioxidative defense may represent a risk factor for benzbromarone-induced hepatotoxicity.