ABSTRACT
N-Nitrosodimethylamine is a liver toxin and mutagen following activation by cytochrome P450. The role of the cytosol in N-nitrosodimethylamine metabolism is not well understood. The effect of cytosol on N-nitrosodimethylamine metabolism was investigated using microsomes and cytosol from rat liver in in vitro reactions with N-nitrosodimethylamine and an NADPH generating system. Studies in which [(14)C]-N-nitrosodimethylamine and calf thymus DNA were used indicated that the addition of cytosol to the microsomal reaction mixture resulted in >200% enhancement of the radioactivity associated with DNA after the DNA was isolated from the reaction mixture by phenol extraction followed by ethanol precipitation. This stimulatory effect was associated with a cytosolic protein and was found to be dependent on both the microsomes and the carbohydrate used in the glucose-6-phosphate dehydrogenase system for the generation of NADPH. The carbohydrate requirement was found to be specific for intermediates of the pentose phosphate pathway, and maximum stimulation occurred with ribulose 5-phosphate. Most of the counts from [(14)C]-N-nitrosodimethylamine which were isolated with DNA after the addition of cytosol to reaction mixtures were not covalently bound to the DNA. HPLC analysis identified four radiolabeled metabolites derived from [(14)C]-N-nitrosodimethylamine following the in vitro incubations. One of the four products was formed only when both cytosol and ribulose 5-phosphate were added to the enzymatic incubations. This product also formed from [(14)C]-alpha-acetoxy nitrosodimethylamine in the absence of microsomes, only when cytosol and ribulose 5-phosphate were added to the reaction mixtures. Thus, these data demonstrate that an enzyme in the cytosol catalyzes a reaction involving a metabolite of N-nitrosodimethylamine (which is formed following cytochrome P450-mediated activation) and a carbohydrate related to the pentose phosphate pathway. A similar reaction also occurs with N-diethylnitrosamine but not with N-dipropylnitrosamine or N-dibutylnitrosamine.
