Rat liver cytosol catalyzes a reaction involving activated N-nitrosodimethylamine and a carbohydrate from the pentose phosphate pathway

N-Nitrosodimethylamine is a liver toxin and mutagen following activation by cytochrome P450. The role of the cytosol in N-nitrosodimethylamine metabol ism 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 sys tem. Studies in which [C-14]-N-nitrosodimethylamine and calf thymus DNA wer e used indicated that the addition of cytosol to the microsomal reaction mi xture resulted in > 200% enhancement of the radioactivity associated with D NA after the DNA was isolated from the reaction mixture by phenol extractio n followed by ethanol precipitation. This stimulatory effect was associated with a cytosolic protein and was found to be dependent on both the microso mes and the carbohydrate used in the glucose-6-phosphate dehydrogenase syst em for the generation of NADPH. The carbohydrate requirement was found to b e specific for intermediates of the pentose phosphate pathway, and maximum stimulation occurred with ribulose 5-phosphate. Most of the counts from [C- 14]-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 [C-14]-N-nit rosodimethylamine following the in vitro incubations. One of the four produ cts was formed only when both cytosol and ribulose 5-phosphate were added t o the enzymatic incubations. This product also formed from [C-14]-alpha-ace toxy nitrosodimethylamine in the absence of microsomes, only when cytosol a nd ribulose 5-phosphate were added to the reaction mixtures. Thus, these da ta demonstrate that an enzyme in the cytosol catalyzes a reaction involving a metabolite of N-nitrosodimethylamine (which is formed following cytochro me P450-mediated activation) and a carbohydrate related to the pentose phos phate pathway. A similar reaction also occurs with N-diethylnitrosamine but not with; N-dipropylnitrosamine or N-dibutylnitrosamine.