Methimazole is an inhibitor of melanin synthesis in cultured B16 melanocytes.

Recently, we have demonstrated in vivo that topical application of the antithyroid agent methimazole (1methyl-2-mercaptoimidazole: MMI) on dark brown guineapig skin produces cutaneous depigmentation (Kasraee, 2002b). The DOPA incubation of the depigmented specimens revealed that MMI induces morphology changes of melanocytes in the treated skin. But unlike most depigmenting chemicals that act as melanocytotoxins, MMI did not cause a significant decrease in the numerical count of melanocytes. This indicated that MMI-induced depigmentation in vivo might result from the inhibition of the melanogenesis pathway in viable melanocytes, rather than a melanocytotoxic effect. To verify this in vitro, we compared the depigmenting and melanocytotoxic effects of MMI with those of kojic acid, hydroquinone, and arbutin, on cultured B16 murine melanocytes. B16 melanocytes were grown in DMEM/10% fetal bovine serum (FBS) supplemented with 10 nM Cholera toxin, and the murine BDVII keratinocytes were grown in RPMI/10% FBS. MMI was dissolved in vehicle (propylene glycol:ethanol:water (5:3:2)) and was exposed to the cells at final concentrations in medium ranging from 0 to 100 mg per mL (0–880 mM) during four consecutive days according to the Dooley method (Dooley et al, 1994). On the fifth day, the melanocytes were harvested, sonicated, and centrifuged. DMSO was added to the pellet, the suspension was sonicated, and melanin determination was performed spectrophotometrically at 405 nm. The results from triplicate samples were analyzed as percent of control. Cell viability was determined on the fifth day for both keratinocytes and melanocytes, using MTT assay (Sieuwerts et al, 1995). For melanocytes, the specificity of melanogenesis inhibition was assessed by dividing the melanin content by the amount of reduced MTT. At the highest concentration examined (880 mM), MMI was still well tolerated, as shown by the MTT assay, although it inhibited the melanogenesis activity of B16 melanocytes by more than 50% when compared with control (Fig 1A). The (melanin/MTT) ratio showed a concentration-dependent decrease, indicating that the depigmenting effect of MMI on cultured melanocytes is a result of the inhibition of melanin synthesis and not a melanocytotoxic effect (Fig 1B). Hydroquinone, a wellknown depigmenting agent, was shown to be efficient as an inhibitor of melanogenesis in B16 melanocytes (Figs 2A and 2B); however, hydroquinone was highly toxic at 10 mM (Fig 2A), indicating that its observed depigmenting action in vivomay be due, at least in part, to its melanocytotoxicity. Arbutin, a phenolic compound used as a skin-lightening Figure1 Effect of MMI on melanogenesis. MMI was added at various concentrations on B16 melanocytes, and then melanin content and MTTassay were performed (A). For MTT assay, 100 ml of a 5 mg per ml MTTsolution was added to each well and the plates were incubated for 3 h at 371C. The formed MTT formazan crystals were dissolved by adding 500 mL DMSO, and spectrophotometric assay was carried out at 500 nm. (B) Ratio of melanin content (mg) per mmol of MTT formazan produced, for the same number of cells.