Contribution of reactive oxygen species to para-aminophenol toxicity in LLC-PK1 cells

para-aminophenol (PAP) causes nephrotoxicity by biochemical mechanisms that have not been fully elucidated. PAP can undergo enzymatic or non-enzymatic oxidation to form reactive intermediates. Using modulators of reactive oxygen species (ROS), the role of ROS in PAP toxicity in LLC-PK1 cells was investigated. ROS formation was determined using a fluorescein derivative and viability using alamarBlue. Following treatment of cells with PAP, ROS formation occurred prior to loss of cell viability. Several modulators of ROS were used to identify the pathways involved in PAP toxicity. Viability was improved with catalase treatment, while viability was decreased when cells were treated with superoxide dismutase (SOD). Both catalase and SOD exert their effects outside of cells in the incubation medium, since there was no evidence of uptake of these enzymes in LLC-PK1 cells. In cell-free incubations, hydrogen peroxide (H2O2) was produced when 0.5 mM PAP was included in the incubation medium. Further, SOD greatly increased and catalase greatly decreased H2O2 production in these cell-free incubations. These data suggest that H2O2 formed in the incubation medium contributes to loss of viability following PAP treatment. When cells were coincubated with 0.5 mM PAP and tiron, pyruvate, bathocuproine, 1, 10-phenanthroline, or dimethylthiourea (DMTU), ROS formation was decreased. However, there was minimal improvement in cell viability. Paradoxically, DMTU exacerbated PAP-induced loss of viability. These data suggest that ROS are generated in cells exposed to PAP but these species are not the predominant cause of cellular injury.