Inhibition of BROD and BUP hydroxylase activities in BNF- and PB-pretreated RLM and cDNA-expressed CYP2B1 by ANF, proadifen, and metyrapone
BUP hydroxylase activity was determined by incubating 500 μM BUP, 0.05 mg of microsomal protein, and 1 mM NADPH for 30 min. HBUP formation was quantitated by HPLC. BROD activity was determined by incubating with 20.5 μM benzyloxyresorufin and 1 mM NADPH for 2 min. Resorufin formation was quantitated by spectrofluorometry. IC50 values are the mean ± S.E.
| Reactions | Liver Microsomes | Inhibitory Effects, IC50 | ||
|---|---|---|---|---|
| ANF | Proadifen | Metyrapone | ||
| μM | ||||
| BROD | BNF pretreated | 0.00248 ± 0.00084 | 47.1 ± 3.7 | 40.8 ± 4.6 |
| PB pretreated | >100 | 0.688 ± 0.161 | 0.806 ± 0.663 | |
| cDNA-expressed CYP2B1 | N.D. | 38.1 ± 1.3 | 1.85 ± 1.11 | |
| BUP hydroxylation | BNF pretreated | >100 | >100 | 41.8 ± 3.4 |
| PB pretreated | >100 | 1.21 ± 0.49 | 3.89 ± 1.54 | |
| cDNA-expressed CYP2B1 | N.D. | 14.9 ± 3.7 | 7.52 ± 0.54 | |
N.D., not determined.