Species Differences in Blood-mediated Nitrosocimetidine Denitrosation 1

Nitrosocimetidine (NC) is the nitrosated derivative of cimetidine ( 1agamet), a p.o. administered drug used widely in the treatment of stomach ulcers. NC is capable of methylating DNÃ• in vitro and in cultured cells in a manner similar to that of the laboratory carcinogens l-methyl-2nitro-1-nitrosoguanidine and methylnitrosourea (MNU) and gives posi tive indications in short-term in vitro tests for genotoxicity, generally held to be prognostic of compound carcinogenic potential. Nevertheless NC has been found to be a weak or non-carcinogen in the rat and mouse model systems and to produce minimal levels of tissue DNA alkylation when dosed p.o. or i.v. to rats. The results from our earlier experiments (D. E. Jensen, Cancer Resâ€ž43: 5258-5267, 1983) indicated that com pound denitrosation is the primary fate of NC in the rat and suggested that denitrosation in the blood, mediated by hemoglobin sulfhydryl resi dues, is perhaps the major detoxification mechanism. We now report that whole blood and hemoglobin isolated from various mammalian species differ in their capacity for NC degradation rate enhancement and for compound denitrosation. The observed whole blood activity in the degradation reaction (rat > mouse/guinea pig > human/hamster) paral leled the hemoglobin activity. The NC half-life in isolated rat blood, 37Â°C,was found to be about 2 min and in hamster or human blood 27 min. For reference, the MNU half-life in isolated blood is 8 min. Compound denitrosation accounted for at least 75% of the degradation in rat blood and 40 to 55% in human and hamster blood. Parallel NC denitrosation activity was found in the various hemoglobin preparations. The NC degradation rates in the presence of the several hemoglobin species were roughly proportional to the number of sulfhydryls on the hemoglobin tetramere available for reaction with p-chloromercuribenzoate and approximated the rates observed in solutions containing equiv alent concentrations of L-cysteine. The percentage of total decomposition due to compound denitrosation in the presence of rat hemoglobin, 95%, was found to be unique relative to the L-cysteine-mediated reactions (about 35%) and the reactions mediated by the other hemoglobins con sidered. In NC-cysteine reactions studied over the pH range 6 through 10, the denitrosation process never accounted for more than 50% of the total degradation. Chemically blocking the sulfhydryls on human hemo globin using iodoacetamide deleted the NC degradation rate enhancement. We found no evidence for nitrosylhemoglobin formation. The results from tissue DNA methylation studies in which the yields generated by MNU and by NC in the rat and in the hamster were compared demonstrated that in spite of the relatively long NC half-life in in vitro hamster blood, the compound produces low DNA alkylation yields relative to MNU in the hamster, suggesting that other denitrosating activities besides hemo globin are active in the intact animal. Determinations of the concentra tions of NC and cimetidine in the circulating blood of hamsters at times after i.v. dosing indicated that the in vivo half-life of the compound is somewhat less than 5 min and that the degradation approaches 100%