Tyrosinase may protect human melanocytes from the cytotoxic effects of the superoxide an ion

Reactive oxygen species (ROS) are potentially cytotoxic and several mechanisms have evolved to protect against their damaging effects. In melanocytes, tyrosinase may have such a rôle by utilising the superoxide anion (O2-) in the production of melanin. In the present study, we have examined the cytotoxic effects of O2- and hydrogen peroxide (H2O2) in human melanocytes both before and following the activation of tyrosinase. Xanthine oxidase (XO, 5-150 mU.ml-1) and glucose oxidase (GO, 0.1-20 mU.ml-1) were used to generate the O2- and H2O2 respectively, and the cytotoxic effects assessed by measuring cell survival using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium (MTT) assay. 3 h later, dose-related decreases in melanocyte survival were seen. Similar experiments with keratinocytes and fibroblasts showed that these cells were more resistant to the cytotoxic effects of O2- than were the melanocytes. The effect of increasing tyrosinase activity was examined by growing the melanocytes in the presence of an analogue of melanocyte-stimulating hormone (MSH) Nle4DPhe7 alpha-MSH (10(-8) M), for 48 h. This increased tyrosinase activity, melanin content, the ability to trap O2- and the resistance of the melanocytes to the cytotoxic effects of this ROS, but failed to alter their susceptibility to the damaging effects of H2O2.Nle4DPhe7 alpha-MSH had no effect on the resistance of keratinocytes and fibroblasts to either O2- or H2O2. After 3 h, XO, as opposed to GO, also increased the melanin content of human melanocytes; this effect was not accompanied by an increase in tyrosinase activity. The present results suggest that tyrosinase may utilise O2- to produce melanin and that this process may protect melanocytes from the potentially damaging effects of this ROS.