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 (/sup 14/C)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 (/sup 14/C)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 (/sup 14/C)NNN by 40 and 27%, respectively. Aroclor 1254 pre-treatment of the rats did not enhance the NADPH-dependent binding of (/sup 14/C)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.
- Research Organization:
- Univ. of Kentucky, Lexington
- OSTI ID:
- 5631974
- Journal Information:
- Carcinogenesis; (United States), Vol. 1
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
NITROSAMINES
METABOLISM
AMINO ACIDS
BIOCHEMICAL REACTION KINETICS
CARCINOGENS
CHEMICAL BONDS
LIVER
METABOLITES
MICROSOMES
NADP
PROTEINS
RATS
REACTION INTERMEDIATES
AMINES
ANIMALS
BODY
CARBOXYLIC ACIDS
CELL CONSTITUENTS
COENZYMES
DIGESTIVE SYSTEM
GLANDS
KINETICS
MAMMALS
NITROSO COMPOUNDS
NUCLEOTIDES
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANOIDS
ORGANS
REACTION KINETICS
RODENTS
VERTEBRATES
560305* - Chemicals Metabolism & Toxicology- Vertebrates- (-1987)