Phenytoin induction of cytochrome P4502B in mice: effects on hexobarbital hydroxylase activity

Abstract

1. Treatment of ICR and C57BL/6 mice with phenytoin (50 mg/kg, i.p., 3 days) resulted in β33 and 43% increases in hepatic cytochrome P450 levels relative to uninduced microsomes, respectively. Phenytoin treatment caused a 63% decrease in hexobarbital sleeping time in ICR mice (19 versus 52min).

2. Both Western immunoblot analysis and solid phase radioimmunoassay using monoclonal anti-rat P4502B antibody showed that P4502B was increased significantly in phenytoin-induced mouse microsomes compared with uninduced mice. P4502B9 was the predominantly induced form whereas 2B10 was elevated marginally. Phenytoin was as efficacious as phenobarbital in increasing P4502B.

3. Phenytoin treatment resulted in an β8-fold increase in hexobarbital hydroxylase activity whereas phenobarbital treatment caused an β13-fold increase. Addition of anti-P4502B antibody produced complete inhibition of hexobarbital oxidation in phenytoin-induced microsomes, indicating that raised P4502B in phenytoin-induced microsomes is associated with the increased hexobarbital hydroxylase activity.

4. Phenytoin failed to increase P4501A in either C57BL/6 or ICR mice, as assessed by both immunoblot analysis and metabolic activities. Although both aryl hydrocarbon hydroxylase and 7-ethoxycoumarin deethylase activities were raised βtwo-fold following phenytoin treatment, the metabolic activities were not inhibited by anti-P4501A antibody.

5. These results provide evidence that phenytoin induces P4502B in mice with pronounced increase in hexobarbital hydroxylase activity, and fails to induce P4501A in either C57BL/6 or ICR mice.