The effect of cytosol on liver microsomal metabolic activation and demethylation of N-nitrosodimethylamine.

Abstract

The role of rat liver cytosol in the demethylation and metabolic activation of N-nitrosodimethylamine (NDMA) was examined. Addition of cytosol to liver microsomes from pyridine-pretreated rats enhanced DNA alkylation by NDMA 10- to 14-fold over microsomes alone, while cytosol alone had little DNA alkylating activity. The cytosolic activity responsible for the enhancement of DNA alkylation was heat labile, required NADPH, and was not a general protein effect. Addition of cytosol to purified rabbit liver cytochrome P450 2E1 in a reconstituted system consisting of NADPH-cytochrome P450 reductase, 2E1, and phospholipid produced an 18-fold increase in DNA alkylation over that observed with the reconstituted system alone. The cytosolic activity responsible for the enhancement of DNA alkylation did not work by inhibition of lipid peroxidation, nor did the addition of cytosol affect the level of NADPH present in the reaction mixtures. Attempts to identify the cytosolic component(s) responsible for the DNA alkylation enhancing activity demonstrated no evidence for the involvement of sulfhydryl-dependent enzymes, a flavoprotein, or conjugating enzymes. Studies with semicarbazide and phenylhydrazine suggest that carbonyl groups may be involved in the cytosolic activity. Measurements of NDMA demethylation demonstrated that cytosol addition led to a significant decrease in formaldehyde production, indicating that cytosol was not enhancing the activation of NDMA to a DNA alkylating species by facilitating the cytochrome P450-catalyzed demethylation reaction, and suggested that a cytosolic reaction might be occurring at the expense of formaldehyde formation.