A CONVENIENT METHOD TO DISCRIMINATE BETWEEN CYTOCHROME-P450 ENZYMES AND FLAVIN-CONTAINING MONOOXYGENASES IN HUMAN LIVER-MICROSOMES

Liver microsomes are a frequently used probe to investigate the phase I metabolism of xenobiotics in vitro. Structures containing nucleophil ic heteroatoms are possible substrates for cytochrome P450 enzymes (P4 50) and flavin-containing monooxygenases (FMO). Both enzymes ire locat ed in the endoplasmatic reticulum of hepatocytes and both need oxygen and NADPH as cofactors. The common method to distinguish between the t wo enzyme systems is to use the thermal inactivation of FMO and to inh ibit P450 completely with carbon monoxide, N-octylamine or N-benzylimi dazole. In the literature no indication could be found that the heat i nactivation of FMO does not affect any of the human P450 enzymes or th at the overall P450 inhibitors inhibit the different human P450 enzyme s sufficiently and do not affect the FMO. The effect of N-benzylimidaz ole and heat inactivation was tested on specific activities of seven P 450 enzymes id human liver microsomes, 1A2, 2A6, 2C9, 2C19, 2D6, 3A4/5 , and 2E1, using methoxyresorufin O-demethylation, coumarin 7-hydroxyl ation, (S)-warfarin 4-hydroxylation, (S)-(+)-mephenytoin di-hydroxylat ion, dextrometorphan O-demethylation, oxidation of denitronifedipine, and chlorzoxazone 6-hydroxylation respectively. The sulfoxidation of m ethimazole (MMI) was used as a specific probe for the determination of FMO activity. Methimazole sulfoxidation was compared with the well kn own assay for FMO metabolism, the formation of N,N-dimethylaniline (DM A) N-oxide, to be confirmed as an exclusively FMO mediated reaction. T he participation of P450 and FMO in the sulfoxidation of four sulfur c ontaining pesticides, ametryne; terbutryne, prometryne and methiocarb was investigated using human liver microsomes. All four reactions were demonstrated to be catalysed predominantly by cytochrome P450.