Alkylation of cellular macromolecules and target specificity of carcinogenic nitrosodialkylamines: metabolic activation by cytochromes P450 2B1 and 2E1.

Abstract

The alkylation of DNA, RNA and protein by labeled metabolites of [alpha-14C]nitrosodimethylamine (NDMA), [alpha-14C]nitrosodipropylamine (NDPA) and [alpha-14C]nitrosodibutylamine (NDBA) was determined as a measure of the metabolic activation of these nitrosamine carcinogens in vitro using microsomes prepared from freshly isolated rat hepatocytes as well as in intact cells using primary cultured rat hepatocytes. The abilities of these nitrosodialkylamines to alkylate cellular macromolecules were significantly affected by pretreatment of rats with inducers of cytochrome P450 and were related to the specific activities of cytochrome P450 2B1 or 2E1 in rat hepatocytes. Pretreatment of rats with phenobarbital (PB) substantially increased the catalytic activity of pentoxyresorufin (PR) O-depentylase, an activity catalyzed by cytochrome P450 2B1, in rat hepatocytes. The increase in the PR O-depentylase activity was associated with a significant increase in the alkylation of DNA or RNA by NDPA, and in alkylation by NDBA, particularly of proteins. However, induction of cytochrome P450 2B1 resulted in a significant decrease in alkylation of cellular macromolecules by NDMA in all cases. In contrast, enhancement of the catalytic activity of the p-nitrophenol (pNP) hydroxylase (P450 2E1) due to pretreatment of rats with pyridine (PYR) resulted in a significant increase in the alkylation of cellular DNA by NDMA. The induction of cytochrome P450 2E1 also increased the alkylation of DNA and RNA by NDPA, but to a lesser extent. Inhibition studies using the chemical inhibitors orphenadrine (OP) and diethyldithiocarbamate (DDC), which are specific for cytochromes P450 2B1 and 2E1, respectively, indicated that cytochrome P450 2B1 was not involved in the metabolic activation of NDMA and that cytochrome P450 2E1 was not responsible for the bioactivation of NDBA. The results presented here demonstrate the substrate specificity and important role of cytochromes P450 2B1 and 2E1 in the bioactivation of nitrosodialkylamines, and suggest that multiple mechanisms may be involved in carcinogenesis induced by nitrosodialkylamines.