Activation of tyrosinase reduces the cytotoxic effects of the superoxide anion in B16 mouse melanoma cells

Tyrosinase may protect against oxidative stress by using the superoxide anion (O-2(-)) in the production of melanin. We have examined this by comparing its cytotoxic effects in B16/F10 and B16/F10-differential deficient (-DD) mouse melanoma cells that express high and low levels of tyrosinase activity respectively. Xanthine oxidase (XO) was used to generate O-2(-) and cytotoxicity assessed by measuring cell survival. XO increased O-2(-) concentrations and 3 h later dose related decreases in cell survival were seen. F10 cells were more resistant to these cytotoxic effects than the F10-DD cells. [Nle(4),DPhe(7)]MSH increased tyrosinase activity and melanin content, reduced O-2(-) concentration and increased the resistance of F10 cells to the cytotoxic effects of O-2(-). No such effects were seen in F10-DD cells. The effect of [Nle(4),DPhe(7)]MSH on the resistance of the F10 cells was time-dependent and noticeable when tyrosinase activity but not melanin was increased. This suggests that it was the activation of tyrosinase rather than the increase in the melanin that provided the protection against O-2(-). In support of this, inhibition of tyrosinase with phenylthiocarbamide reduced the increased resistance induced by [Nle(4),DPhe(7)]MSH. Moreover, although melanin was capable of scavenging Of it had little effect at concentrations comparable to those in the activated F10 cells. XO also increased the melanin content of F10 but not F10-DD cells. We conclude that tyrosinase is able to utilise O-2(-) to produce melanin and this provides pigment cells with a unique anti-oxidant mechanism.