CYP isoform induction screening in 96-well plates: use of 7-benzyloxy-4-trifluoromethylcoumarin as a substrate for studies with rat hepatocytes

1. In this study, 7-benzyloxy-4-trifluoromethylcoumarin (BFC) was evaluated as a substrate to assess the induction of cytochrome P450 (CYP) isoform en zyme activities in rat hepatocytes using a 96-well plate format. 2. BFC was metabolized by both untreated and sodium phenobarbitone (NaPB)tr eated rat hepatocytes in a time- and concentration-dependent manner to the highly fluorescent product 7-hydroxy-4-trifluoromethylcoumarin (HFC). 3. HFC was extensively conjugated with D-glucuronic acid and/or sulphate in both untreated and NaPB-treated rat hepatocytes, thus necessitating the in clusion of an enzymatic deconjugation step in the assay procedure. 4. The time-course of induction of 7-ethoxyresorufin metabolism by the CYP1 A inducer beta-naphthoflavone (BNF), 7-benzyloxyresorufin metabolism by the CYP2B inducer NaPB and BFC metabolism by both BNF and NaPB was studied in rat hepatocytes treated for 24-96 h. The optimal time for induction of meta bolism of all three substrates was 72 h, with no medium changes being neces sary during this period. 5. The effect of treatment with 0.5-20 mu M BNF, 50-2000 mu M NaPB, 2-20 mu M dexamethasone (DEX), 20-100 mu M methylclofenapate (MCP), and 50 and 200 mu M isoniazid (ISN) for 72 h on BFC metabolism in cultured rat hepatocyte s was studied. BFC metabolism was induced by treatment with BNF, NaPB and M CP, but not with either DEX or ISN. 6. The metabolism of BFC in liver microsomes from the control rat and rat t reated with CYP isoform inducers was also studied. BFC metabolism was induc ed by treatment with NaPB, BNF and DEX. 7. The metabolism of BFC was also studied using microsomes from baculovirus -infected insect cells containing rat cDNA-expressed CYP1A, CYP2B, CYP2C an d CYP3A isoforms. Whereas BFC was metabolized to some extent by all the rat cDNA-expressed CYP isoforms examined, at a substrate concentration of 2.5 mu M the greatest rates of BFC metabolism were observed with the CYP1A1, CY P1A2 and CYP2B1 preparations. 8. In summary, the results demonstrate that BFC is a good substrate for ass essing the induction of CYP1A and CYP2B isoforms in rat hepatocytes in a 96 -well plate format.