Inhibition of melanogenesis in response to oxidative stress: transient downregulation of melanocyte differentiation markers and possible involvement of microphthalmia transcription factor

H2O2 and other reactive oxygen species are key regulators of many intracell ular pathways. Within mammalian skin, H2O2 is formed as a byproduct of mela nin synthesis, and following u.v. irradiation. We therefore analyzed its ef fects on melanin synthesis. The activity of the rate-limiting melanogenic e nzyme, tyrosinase, decreased in H2O2-treated mouse and human melanoma cells , This inhibition was concentration- and time-dependent in the B16 melanoma model. Maximal inhibition (50-75%) occurred 8-16 hours after a 20 minute e xposure to 0.5 mM H2O2, B16 cells withstand this treatment adequately, as s hown by a small effect on glutathione levels and a rapid recovery of basal lipid peroxidation levels. Enzyme activities also recovered, beginning to i ncrease 16-20 hours after the treatment. Inhibition of enzyme activities re flected decreased protein levels. mRNAs for tyrosinase, tyrosinase-related protein 1, dopachrome tautomerase, silver protein and melanocortin 1 recept or also decreased after H2O2 treatment, and recovered at different rates. D ownregulation of melanocyte differentiation markers mRNAs was preceded by a decrease in microphthalmia transcription factor (Mitf) gene expression, wh ich was quantitatively similar to the decrease achieved using 12-O-tetradec anoylphorbol-13-acetate. Recovery of basal Mitf mRNA levels was also observ ed clearly before that of tyrosinase, Therefore, oxidative stress may lead to hypopigmentation by mechanisms that include a downregulation of a the mi crophthalmia-dependent melanogenic enzymes.