Nonsteroidal anti-inflammatory drug (NSAID) use is associated with adverse consequences, including hepatic injury. The detrimental hepatotoxicity of diclofenac, a widely used NSAID, is primarily connected to oxidative damage in mitochondria, which are the primary source of reactive oxygen species (ROS). The primary ROS responsible for inducing diclofenac-related hepatocellular toxicity and the principal antioxidant that mitigates these ROS remain unknown. Peroxiredoxin III (PrxIII) is the most abundant and potent H₂O₂-eliminating enzyme in the mitochondria of mammalian cells. Here, we investigated the role of mitochondrial H₂O₂ and the protective function of PrxIII in diclofenac-induced mitochondrial dysfunction and apoptosis in hepatocytes. Mitochondrial H₂O₂ levels were differentiated from other types of ROS using a fluorescent H₂O₂ indicator. Upon diclofenac treatment, PrxIII-knockdown HepG2 human hepatoma cells showed higher levels of mitochondrial H₂O₂ than PrxIII-expressing controls. PrxIII-depleted cells exhibited higher mitochondrial dysfunction as measured by a lower oxygen consumption rate, loss of mitochondrial membrane potential, cardiolipin oxidation, and caspase activation, and were more sensitive to apoptosis. Ectopic expression of mitochondrially targeted catalase in PrxIII-knockdown HepG2 cells or in primary hepatocytes derived from PrxIII-knockout mice suppressed the diclofenac-induced accumulation of mitochondrial H₂O₂ and decreased apoptosis. Thus, we demonstrated that mitochondrial H₂O₂ is a key mediator of diclofenac-induced hepatocellular damage driven by mitochondrial dysfunction and apoptosis. We showed that PrxIII loss results in the critical accumulation of mitochondrial H₂O₂ and increases the harmful effects of diclofenac. PrxIII or other antioxidants targeting mitochondrial H₂O₂ could be explored as potential therapeutic agents to protect against the hepatotoxicity associated with NSAID use.

Ultraviolet B (UVB) irradiation generates reactive oxygen species (ROS), which can damage exposed skin cells. Mitochondria and NADPH oxidase are the two principal producers of ROS in UVB-irradiated keratinocytes. Peroxiredoxin V (PrxV) is a mitochondrial and cytosolic cysteine-dependent peroxidase enzyme that robustly removes H₂O₂. We investigated PrxV’s role in protecting epidermal keratinocytes against UVB-induced ROS damage. We separated mitochondrial and cytosolic H₂O₂ levels from other types of ROS using fluorescent H₂O₂ indicators. Upon UVB irradiation, PrxV-knockdown HaCaT human keratinocytes showed higher levels of mitochondrial and cytosolic H₂O₂ than PrxV-expressing controls. PrxV depletion enhanced hyperoxidation-mediated inactivation of mitochondrial PrxIII and cytosolic PrxI and PrxII in UVB-irradiated keratinocytes. PrxV-depleted keratinocytes exhibited mitochondrial dysfunction and were more susceptible to apoptosis through decreased oxygen consumption rate, loss of mitochondrial membrane potential, cardiolipin oxidation, cytochrome C release, and caspase activation. Our findings show that PrxV serves to protect epidermal keratinocytes from UVB-induced damage such as mitochondrial dysfunction and apoptosis, not only by directly removing mitochondrial and cytosolic H₂O₂ but also by indirectly improving the catalytic activity of mitochondrial PrxIII and cytosolic PrxI and PrxII. It is possible that strengthening PrxV defenses could aid in preventing UVB-induced skin damage.