DIHYDRO-1,4-BENZOTHIAZINE-6,7-DIONE, THE ULTIMATE TOXIC METABOLITE OF4-S-CYSTEAMINYLPHENOL AND 4-S-CYSTEAMINYLCATECHOL

4-S-Cysteaminylphenol (4-S-CAP) and the corresponding catechol 4-S-cys teaminylcatechol (4-S-CAC) have been evaluated for melanocytotoxicity. It was shown recently that tyrosinase oxidation of these substrates p roduces a violet pigment, dihydro-1,4-benzothiazine-6,1-dione (BQ). In this study we examined whether BQ is the ultimate toxic metabolite pr oduced in melanoma cells from 4-S-CAP/4-S-CAC. Biochemical experiments showed that (1) BQ was formed by autoxidation of 4-S-CAC as well as b y tyrosinase oxidation of 4-S-CAP/4-S-CAC, (2) BQ reacted rapidly with thiols such as reduced glutathione (GSH), and (3) BQ inhibited the ac tivity of alcohol dehydrogenase, an SH enzyme. In vitro experiments sh owed that (1) the cytotoxicity of 4-S-CAC was mostly prevented by cata lase and superoxide dismutase, (2) Ba was highly cytotoxic to B16 mela noma cells (IC50 being 3.9 mu M as compared with 507 mu M for 4-S-CAP) , (3) BQ was metabolized rapidly to a GSH adduct in melanoma cells, an d (4) the same GSH adduct was also formed upon incubation of melanoma cells with 4-S-CAP, the reaction being tyrosinase dependent. In vivo e xperiments showed that intratumoral administration of BQ (0.5 mu mol) inhibited the subcutaneous growth of B16 melanoma nearly as effectivel y as 4-S-CAP/4-S-CAC (20 mu mol). These results indicate that BQ is th e ultimate toxic metabolite produced by tyrosinase oxidation of 4-S-CA P/4-S-CAC. BQ deprives melanoma cells of GSH and may inactivate SH enz ymes essential for DNA synthesis and cell proliferation by covalent bi nding through their cysteine residues, thereby exerting melanocytotoxi city. Cytotoxicity of 4-S-CAC depends mostly on autoxidation producing BQ and active oxygens. (C) 1997 Elsevier Science Inc.