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Deuterium Isotope Effect on Denitrosation and Demethylation of N-Nitrosodimethylamine by Rat Liver Microsomes¹

Department of Biochemistry, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103 [D. W., C. S. Y.]; Program Resources, Inc. [C. J. M., J. M. R., J. A. H.], Information Management Services, Inc. [C. W. R.], and Chemistry Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute, Frederick Cancer Research Facility, Frederick, Maryland 21701 [T. A., L. K. K.]; and Department of Drug Metabolism, Smith, Kline & French Laboratories, Philadelphia, Pennsylvania 19101 [B. A. M.]

In an attempt to elucidate the molecular basis for the decrease in rat liver carcinogenicity and DNA-alkylating ability that accompanies deuteration of N-nitrosodimethylamine (NDMA), NDMA and its fully deuterated analogue ([²H₆]NDMA) were incubated with acetone-induced rat liver microsomes. Rates for the competing metabolic routes, denitrosation and demethylation, were determined from colorimetric data on nitrite and formaldehyde generation, respectively. The V_max calculated for demethylation of NDMA was 7.9 nmol/min/mg, while that for denitrosation was 0.83 nmol/min/mg. Deuteration of NDMA did not significantly change the V_max for either pathway, but it did increase the K_m for demethylation from 0.06 to 0.3 mM. The K_m for denitrosation was also increased from 0.06 to 0.3 mM on deuteration, as determined by incubating an equimolar mixture of amino-¹⁵N-labeled NDMA with [²H₆]NDMA and measuring the methyl[¹⁵N]amine:[²H₃]methylamine ratio by derivatization-gas chromatography-mass spectrometry. The fact that the K_m values for denitrosation were so similar to those for demethylation suggested that the two pathways were catalyzed by the same enzyme. The isotope effects calculated from these data [V_max^H/V_max^D 1 and (V_max/K_m)^H/(V_max/K_m)^D 5] show that microsomal metabolism of NDMA is not significantly shifted from demethylation to denitrosation on deuteration of substrate and may indicate a low commitment to catalysis for the enzyme. The results are consistent with the view that the metabolism of NDMA is initiated by formation of an -nitrosamino radical which either combines with a hydroxyl radical to form the -hydroxynitrosamine as the initial product of the demethylation pathway or fragments to nitric oxide and N-methylformaldimine as the first products of denitrosation.

¹ Research was supported in part by National Cancer Institute Grant CA-37037 and Contract N01-CO-23910. A portion of this work was presented at the International Agency for Research on Cancer's Ninth International Meeting on N-Nitroso Compounds: Relevance to Human Cancer, September 1–5, 1986, Baden, Austria (33).

² To whom requests for reprints should be addressed.

Received 12/15/86. Revised 3/10/87. Accepted 3/20/87.

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