Effect of phenytoin on cytochrome P450 2B mRNA expression in primary rat astrocyte cultures.

Abstract

Studies on cytochrome P450 2B (CYP2B) in the brain have essentially been focused on protein characterization and regional distribution. Due to the high sequence homology between the closely related CYP2B1 and 2B2 isoforms and the low amounts of the corresponding mRNAs few efforts have been made to analyze the expression, regulation, and inducibility of these P450 genes in a specific cell type. In the present study, we investigated CYP2B mRNA expression in primary rat astrocyte cultures under the influence of the anti-epileptic drug phenytoin, which is known to be a CYP2B inducing agent in liver. In situ hybridization with a digoxigenin (DIG)-labeled cRNA probe demonstrated that 30-40% of the astrocytes strongly expressed a CYP2B mRNA-specific signal within the first week of cultivation. With increasing age (> 14 days) a greater percentage of cells (>90%) expressed mRNA for P450 2B. However, the level of transcriptional activity was substantially lower than in younger cultures. To discriminate between the 2B1 and 2B2 isoforms the reverse transcription/polymerase chain reaction (RT/PCR) procedures were proved for rat hepatic mRNA as a control assay. Subsequently, the application of this method on cultured astrocytes confirmed that these brain cells may express CYP2B1 mRNA. CYP2B2 mRNA could not be detected in astrocyte cultures at any age examined. Phenytoin led to the down regulation of CYP2B1 mRNA, which contrasts with the drug inducing effect on hepatic CYP2B1 and 2B2 levels. After 4 hr of exposure of phenytoin to the astrocytes no amplification product could be detected at all. Phenytoin did not induce either CYP2B1 or 2B2 expression.