Dihydro-1,4-benzothiazine-6,7-dione, the ultimate toxic metabolite of 4-S-cysteaminylphenol and 4-S-cysteaminylcatechol

Abstract 4- S -Cysteaminylphenol (4- S -CAP) and the corresponding catechol 4- S -cysteaminylcatechol (4- S -CAC) have been evaluated for melanocytotoxicity. It was shown recently that tyrosinase oxidation of these substrates produces a violet pigment, dihydro-1,4-benzothiazine-6,7-dione (BQ). In this study we examined whether BQ is the ultimate toxic metabolite produced 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 by 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 activity of alcohol dehydrogenase, an SH enzyme. In vitro experiments showed that (1) the cytotoxicity of 4- S -CAC was mostly prevented by catalase and superoxide dismutase, (2) BQ was highly cytotoxic to B16 melanoma cells ( ic 50 being 3.9 μM as compared with 507 μM for 4- S -CAP), (3) BQ was metabolized rapidly to a GSH adduct in melanoma cells, and (4) the same GSH adduct was also formed upon incubation of melanoma cells with 4- S -CAP, the reaction being tyrosinase dependent. In vivo experiments showed that intratumoral administration of BQ (0.5 μmol) inhibited the subcutaneous growth of B16 melanoma nearly as effectively as 4-S- CAP 4-S- CAC (20 μmol). These results indicate that BQ is the ultimate toxic metabolite produced by tyrosinase oxidation of 4-S- CAP 4-S- CAC . BQ deprives melanoma cells of GSH and may inactivate SH enzymes essential for DNA synthesis and cell proliferation by covalent binding through their cysteine residues, thereby exerting melanocytotoxicity. Cytotoxicity of 4- S -CAC depends mostly on autoxidation producing BQ and active oxygens.