Identification of the cytochrome P450 isozymes involved in the metabolism of N-nitrosodipropyl-,N-nitrosodibutyl- and N-nitroso-n-butyl-n-propylamine.

Abstract

The metabolism of N-nitrosodipropylamine (NDPA), N-nitrosodibutylamine (NDBA) and N-nitroso-n-butyl-n-propylamine (NBPA) was investigated in vitro using liver microsomes and purified isoforms of cytochrome P450 in a reconstituted system. Liver microsomes were prepared from rats pretreated with phenobarbital (PB), pyridine (PYR), beta-naphthoflavone (BNF), butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), clofibrate (CLO) or from untreated rats. The purified cytochrome P450s used in the reconstituted system were rat 1A1 and 2B1 and rabbit 2E1. The rates of metabolism and the product profiles for NDPA, NDBA and NBPA changed significantly depending on the pretreatment of the rats or the identity of the purified cytochrome P450 isoforms. Induction by PB dramatically increased cleavage of NDPA, NDBA and NBPA at C-N bonds, leading to substantial increases in formation of the respective aldehydes and the overall metabolic rates. Microsomes from PYR-pretreated rats exhibited increased activities for formation of formaldehyde and propionaldehyde from NDPA and NBPA. Microsomes from BHT-pretreated rats showed a slight increase in activity for N-dealkylation of NDBA and BNPA. Treatment with BHA decreased the overall metabolism of NDBA, but slightly increased N-dealkylation of NBPA. Microsomal metabolism of NDPA, NDBA and NBPA was decreased by pretreatment with BNF and CLO. Results from studies using the reconstituted system with purified cytochrome P450 isoforms demonstrated that cytochrome P450 2B1 specifically catalyzed alpha-hydroxylation of these three long chain nitrosamines with high activity. Cytochrome P450 2E1 catalyzed formation of formaldehyde and propionaldehyde from NDPA and NBPA, but did not catalyze formation of acetaldehyde or butyraldehyde. Cytochrome P450 1A1 exhibited no activity for metabolism of NDPA, NDBA and NBPA. The contributions of cytochrome P450 2B1 and 2E1 to N-dealkylation reactions were determined using inhibitory monoclonal antibodies (mAb). With microsomes from PB-pretreated rats, inhibition by mAb-2B1 indicated a 62% contribution by cytochrome P450 2B1 to debutylation of NDBA and 65% to depropylation of NDPA. In microsomes from PYR-pretreated rats inhibition by mAbs also showed a role for cytochrome P450 2E1 in depropylation of NDPA. These studies provide a better understanding of the role of various forms of cytochrome P450 in metabolic activation of these long chain N-nitrosodialkylamines to potentially toxic, mutagenic and carcinogenic intermediates.