7-hydroxy-4-trifluoromethylcoumarin and 7-ethoxy-4-trifluoromethylcoumarin

7-Ethoxy-4-trifluoromethylcoumarin (7-EFC) was examined as a substrate for cytochrome P450 (P450) in microsomes from human livers and expressed in B-lymphoblastoid cells. The O-deethylation of 7-EFC to 7-hydroxy-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 with 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 (r2 = 0.86, p < 0.0001). alpha-Napthoflavone (ANF), diethyldithiocarbamate (DDC) 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, CYP2B6 and CYP1A2 were found to produce substantial levels of 7-HFC whereas 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 (average K(m) = 3.3 microM) demonstrating allosteric activation. Five livers with low relative rates of 7-HFC formation also exhibited biphasic kinetics but lacked evidence of an allosteric mechanism being involved in the low K(m) component (average K(m) = 2.4 microM). Furthermore, expressed CYP2B6 and CYP2E1 converted 7-EFC to 7-HFC with allosteric activation indicated, while CYP1A2 mediated metabolism of 7-EFC to 7-HFC best fit the classic Michaelis-Menten model. A commercially available antibody to rat CYP2B, suggested to be specific for CYP2B6, was found to cross react with all members to the CYP2 family examined including CYP2C19, which possessed a nearly identical electrophoretic mobility to that of CYP2B6 in the system examined. In total, the evidence presented indicates that multiple P450s are involved in the formation of 7-HFC from 7-EFC, therefore this does not appear to be a useful or a selective probe of CYP2B6 catalytic activity. Furthermore, the specificity of both antibody and chemical inhibitor (ORP) prob

Topics: Aryl Hydrocarbon Hydroxylases; B-Lymphocytes; Benzoflavones; Blotting, Western; Cell Line; Chloramphenicol; Chromatography, High Pressure Liquid; Coumarins; Cytochrome P-450 CYP2B6; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Ditiocarb; Humans; In Vitro Techniques; Kinetics; Microsomes; Microsomes, Liver; Molecular Probes; Orphenadrine; Oxidoreductases, N-Demethylating; Substrate Specificity

The O-deethylation of 7-ethoxy-4-trifluoromethylcoumarin (EFC) by liver microsomes has been assessed as a method for monitoring the activity of cytochrome P450. The principle advantage of this substrate is the formation of a fluorescent product 7-hydroxy-4-trifluoromethylcoumarin (HFC) which can be assayed directly in the reaction medium. For rat microsomes the deethylated product was confirmed as the main metabolite, the reaction rate was linear with respect to both time and microsomal protein concentration and was independent of small changes in the added co-factors. A linear formation rate for the deethylated metabolite was also confirmed with dog and human microsomes. The intra-assay precision for rat, dog and human microsomes was 3, 5 and 4%, respectively. Hanes transformations of the dog and human data showed two phases, in contrast to a linear decline seen for the rat. Hybrid parameters for Vmax and Km, calculated from the apparently linear portions of these curves, gave interday SD for the Vmax of rat, dog and man of 2, 14 and 4%, respectively, and approximately 15% for the Km in all species. The Vmax in rat, dog and human microsomes was 1.4 +/- 0.2, 4.3 +/- 1.5 and 0.9 +/- 0.5 nmol HFC/min/nmol P450, respectively. The Km was 11.0 +/- 3.1, 67 +/- 19 and 6.8 +/- 2.5 microM, respectively. Direct evidence that at least two isoenzymes (cytochrome P450 1A2 and 2E1) metabolize EFC was obtained by experiments with competitive, suicide and immuno-inhibitors. Compared with ethoxycoumarin, the involvement of P450 2E1 in O-deethylation seemed similar in the rat. In conclusion, EFC provides a straightforward and reproducible assay for microsomal enzyme activity, requiring at most 25 pmol/mL of cytochrome P450.

Topics: Acylation; Animals; Coumarins; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Dogs; Humans; Isoenzymes; Kinetics; Male; Microsomes, Liver; Rats; Species Specificity; Spectrometry, Fluorescence