EXAMINATION OF PURPORTED PROBES OF HUMAN CYP2B6

7-Ethoxy-4-trifluoromethylcoumarin (7-EFC) was examined as a substrate for cytochrome P450 (P450) in microsomes from human livers and expres sed in B-lymphoblastoid cells, The O-deethylation of 7-EFC to 7-hydrox y-4-trifluoromethylcoumarin (7-HFC) varied over a liver bank (n = 19) by a factor of 13 (40-507 pmol min(-1)mg(-1) protein), When compared w ith the ability of the bank of human liver samples to metabolize form- selective substrates of the P450, 7-HFC formation correlated strongly with the formation of the S-mephenytoin metabolite, nirvanol (r(2) = 0 .86, p < 0.0001), alpha-Napthoflavone (ANF), diethyldithiocarbamate (D DC) and chloramphenicol (CAP) inhibited the O-deethylation of 7-EFC by microsomes from human livers by greater than 60%, Orphenadrine (ORP), a reported specific CYP2B6 inhibitor, was a Less potent inhibitor of 7-HFC formation by microsomes from human liver than DDC or ANF. Using microsomes from B-lymphoblastoid cells expressing specific P450s, CYP2 B6 and CYP1A2, were found to produce substantial levels of 7-HFC where as CYP2E1 and CYP2C19 produced detectable amounts of this metabolite, ORP inhibited expressed CYP2E1 and CYP2B6 mediated 7-HFC formation to a greater extent than the inhibition observed for CYP1A2. Methoxychlor and S-mephenytoin inhibited expressed CYP2B6 but not CYP1A2 mediated 7-EFC O-deethylation. Livers (n = 5) with high relative rates of 7-HFC formation displayed biphasic enzyme kinetics with the low K-m site (a verage K-m = 3.3 mu M) demonstrating allosteric activation, Five Liver s with low relative rates of 7-HFC formation also exhibited biphasic k inetics but lacked evidence of an allosteric mechanism being involved in the low K-m component (average K-m = 2.4 mu M). Furthermore, expres sed CYP2B6 and CYP2E1 converted 7-EFC to 7-HFC with allosteric activat ion indicated, while CYP1A2 mediated metabolism of 7-EFC to 7-HFC best fit the classic Michaelis-Menten model. A commercially available anti body to rat CYP2B, suggested to be specific for CYP2B6, was found to c ross react with all members of the CYP2 family examined including CYP2 C19, which possessed a nearly identical electrophoretic mobility to th at of CYP2B6 in the system examined, In total, the evidence presented indicates that multiple P450s are involved in the formation of 7-HFC f rom 7-EFC, therefore this does not appear to be a useful or a selectiv e probe of CYP2B6 catalytic activity Furthermore, the specificity of b oth antibody and chemical inhibitor (ORP) probes previously suggested to be specific for CYP2B6 is also questioned.