Superoxide-hydrogen peroxide imbalance differentially modulates the keratinocytes cell line (HaCaT) oxidative metabolism via Keap1-Nrf2 redox signaling pathway

The purpose of this study was to investigate the effect of a superoxide-hydrogen peroxide (S-HP) imbalance of the superoxide dismutase manganese dependent (SOD2) gene, generated by paraquat and porphyrin exposure, on the keratinocytes cell line (HaCaT) oxidative metabolism. Paraquat acts increasing superoxide (O 2 ·− ) levels, while porphyrin increases hydrogen peroxide (H 2 O 2 ) levels, acting as VV-SOD2-like and AA-SOD2-like molecules, respectively. First of all, HaCAT cells were treated with different concentrations of paraquat and porphyrin (1; 10; 30, and 70 μM) to determine the concentration of both that causes imbalance. After defining the concentration of paraquat and porphyrin (70 μM), a time curve was performed (1, 3, 6, and 24 h) to evaluate ROS production levels. Other oxidative parameters, such as nitric oxide (NO), lipoperoxidation (TBARS) and protein carbonyl, were evaluated after 24 h of incubation, as well as genotoxic analyses, apoptosis detection, and gene expression. Our findings revealed that paraquat exposure decreased cell viability, increasing lipoperoxidation, DNA damage, and apoptosis. On the other hand, porphyrin treatment increased cell viability and proliferation, ROS and NO production, triggering protein and DNA damage. In addition, porphyrin up-regulated Keap1 and Nrf2 gene expression, while paraquat decreased Nrf2 gene expression. In this sense, we suggested that the superoxide-hydrogen peroxide imbalance differentially modulates oxidative stress on keratinocytes cell line via Keap1-Nrf2 gene expression pathway.