Nature of N-nitrosodimethylamine demethylase and its inhibitors

The present study was undertaken to examine the nature of the low Km (KmI) form of rat liver microsomal N-nitrosodimethylamine demethylase (NDMAd) and its inhibition by organic compounds which are commonly present in the assay mixture. Using radiometric and colorimetric assay methods with an NADPH-generating system consisting of 0.4 mM NADP, 10 mM glucose-6-phosphate, and glucose-6-phosphate dehydrogenase (0.4 units/ml), Km values of 40-50 microM were obtained. These Km values were lower than the values of 60-80 microM reported previously. This decrease was due to the elimination of inhibitors such as glycerol in the assay mixture. Glycerol was a competitive inhibitor, and this observation explained in part why purified P-450ac (acetone-inducible form of P-450), displayed a higher Km value in a reconstituted NDMAd system, which contained glycerol, than in microsomes. Semi-carbazide which had been used in many previous assays of NDMAd was also found to be a competitive inhibitor of this enzyme. Other inhibitors studied include the commonly used solvents dimethylsulfoxide, acetone, ethylene glycol, dimethylformamide, ethyl acetate, benzene, and hexane as well as thiol compounds dithiothreitol and mercaptoethanol. Although very low Km values (10-20 microM) for N-nitrosodimethylamine metabolism were reported in studies with perfused liver, liver slices, and isolated liver cells, we believe that the KmI form of liver NDMAd is responsible for the metabolism and activation of N-nitrosodimethylamine in the rat liver.