Characterization of covalent binding of N'-nitrosonornicotine in rat liver microsomes.
The metabolism of the carcinogenic nitrosamine, N'-nitrosonornicotine (NNN), to reactive intermediates which bind covalently was assessed using male Sprague-Dawley rat liver microsomes. The NADPH-dependent covalent binding of [14C]NNN was linear with time up to 90 min and protein concentration up to 3.0 mg/ml. The apparent Km and Vmax of the binding were determined from the initial velocities and found to be 0.91 mM and 4.7 pmol/min/mg protein, respectively. Although NNN is not a hepatocarcinogen, this amount of NADPH-dependent covalent binding is 7-fold greater than that reported for dimethylnitrosamine, a potent hepatocarcinogen. Extensive covalent binding of [14C]NNN to liver and muscle microsomal protein was also present in the absence of an NADPH-generating system and in the presence of 50% methanol, indicating a non-enzymatically mediated reaction. Addition of the nucleophiles glutathione, cysteine and N-acetylcysteine significantly decreased (p less than 0.01) the non-NADPH-dependent binding, but did not affect NADPH-dependent binding. In vitro addition of the cytochrome P-450 inhibitors metyrapone, piperonyl butoxide and SKF-525A significantly decreased (p less than 0.05) NADPH-dependent binding of [14C]NNN by 27-40%. NADH did not replace NADPH in supporting covalent binding. Replacement of an air atmosphere with nitrogen or CO:O2 (8:2) significantly decreased (p less than 0.05) NADPH-dependent binding of [14C]NNN by 40 and 27%, respectively. Aroclor 1254 pre-treatment of the rats did not enhance the NADPH-dependent binding of [14C]NNN. These data indicate that cytochrome P-450 is at least in part responsible for the metabolic activation of the carcinogen NNN but also suggest additional mechanisms of activation.
D000596:Amino Acids / Q000494:pharmacology; D000818:Animals; D002250:Carbon Radioisotopes; D002273:Carcinogens / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism*; D009249:NADP / Q000378:metabolism; D009602:Nitrosamines / Q000378:metabolism*; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism*; D011865:Radioisotope Dilution Technique; D051381:Rats; D011919:Rats, Inbred Strains
D000596:Amino Acids; D002250:Carbon Radioisotopes; D002273:Carcinogens; D009602:Nitrosamines; D011506:Proteins; D009249:NADP; C008655:N'-nitrosonornicotine