Rabbit liver microsomal system: study of interaction with two model N-nitrosamines and their metabolism

Rabbit liver microsomes of control (non-treated) or animals induced either by ethanol (EtOH) or phenobarbital (PB) were incubated with N-nitrosodimethylamine (NDMA) or N-nitrosomethylaniline (NMA). Difference spectroscopy showed that NMA is bound to the substrate-binding site of cytochrome P-450 (CYP) isoforms as heme ligand in control and EtOH pre-treated microsomes. On the other hand, PB-induced microsomes exhibit with NMA substrate type of spectra. NDMA does not provide any type of binding spectra with used microsomal systems. Oxidative bio-activation of N-nitrosamines by the microsomal CYP isoforms was measured as formaldehyde formation. Analysis of reaction kinetics in control microsomes revealed, for both substrates, two values of Michaelis-Menten constant (K(m)) for, K(m) values of 0.03 and 0.13 mmol/l for NDMA, and 0.30 and 0.82 mmol/l for NMA. Induction of animals with EtOH resulted in a decrease in the K(m) value for both substrates. In contrast, PB treatment caused an elevation of K(m) value for NDMA. Based on these data, we conclude that EtOH-inducible microsomal CYP isoforms (mainly CYP2E1) are responsible for binding and N-demethylation metabolism of both studied N-nitrosamines in rabbit liver microsomal system. The role of the other CYP isoforms involved in the metabolism of mentioned N-nitrosamines is discussed.