Characterization of monochloramine toxicity on PC12 cells and protective effect of tocopherol via antioxidative function

Monochloramine (NH(2)Cl) is a physiological oxidant produced by activated neutrophils. In the present work, we studied the underlying mechanism of cytotoxic effects of NH(2)Cl on an undifferentiated rat pheochromocytoma PC12 cell line and the protective effects of antioxidants. The cells treated with 100 microM NH(2)Cl exhibited signs of apoptotic cell death such as phosphatidylserine exposure and caspase activation. To understand the mechanism of NH(2)Cl cytotoxicity, we examined the effect of various kinds of antioxidants including alpha-tocopherol (alpha-Toc) and beta-tocopherol (beta-Toc). These antioxidants exerted a protective effect against NH(2)Cl-induced cell death, and alpha-Toc exhibited the most potent inhibitory effect among the antioxidants used. A loss of cellular glutathione was observed in the cells treated with 100 microM NH(2)Cl. The formation of reactive oxygen species (ROS) was also measured using the fluorescent probe dichlorofluorescin diacetate. The fluorescence intensity increased prior to cell death and an antioxidant, such as alpha-Toc, suppressed the increase in ROS. Interestingly, beta-Toc also exerted similar inhibitory effects on cytotoxicity and caspase activation. These results suggest that free radical mediated process is involved in NH(2)Cl-induced PC12 cell death and that tocopherols inhibit this cell death via antioxidative function.