Enzymic denitrosation of 1,3-dimethyl-2-cyano-1-nitrosoguanidine in rat liver cytosol and the fate of the immediate product S-nitrosoglutathione

Abstract

The tumorigenicity of certain N-nitrosoguanidinium compounds is limited, in rodents, by the propensity of these agents to be detoxified by denitrosation. Previous studies have revealed that rodent glutathione transferase isoenzymes are capable of catalyzing this process, generating exclusively the denitrosated guanidinium compound and S-nitrosoglutathione (GSNO). Experiments considering the denitrosation of 1,3-dimethyl-2-cyano-1-nitrosoguanidine (CyanoDMNG) in rat liver cytosol incubates are reported, with emphasis on the fate of GSNO. Incubates composed with equimolar CyanoDMNG and reduced glutathione (GSH) effected 100% denitrosation; the GSNO yield was less than expected as was the quantity of GSH consumed. When the anticipated 100% yield concentration of GSNO was applied to cytosol incubates, 20–40% of it rapidly disappeared. Nitrosated protein thiols accounted for 35% of the NO moiety released, nitrite ion 30%, and nitric oxide production was detectable. Concomitant with GSNO loss, GSH and oxidized glutathione (GSSG) were generated in yields similar to those detected in the CyanoDMNG/GSH incubates. Thus, the fate of GSNO in cytosol determines the yields of glutathione-based products, and the stoichiometry of the glutathione transferase reaction is demonstrated. In incubates composed with equimolar CyanoDMNG, GSH, and NADPH, denitrosation was again 100%, but GSNO yields were very low and residual GSH increased. Inclusion of NADPH in incubates containing the anticipated 100% yield concentration of GSNO resulted in rapid GSNO degradation, producing GSH and a detected but unidentified product; S-nitrosated protein, nitrite, and nitrate yields were minimal, nitric oxide production was abolished, and incubate response to a mercuric chloride/azo dye assay approached zero. The fate of the NO moiety consequent to this GSNO catabolism is presently unknown.