Transcriptional activation of cytochrome P450 CYP2C45 by drugs is mediated by the chicken xenobiotic receptor (CXR) interacting with a phenobarbital response enhancer unit

Abstract

Cytochromes P450 (CYP)-2C enzymes fulfill an important role in xenobiotic metabolism and therefore have extensively been studied in rodents and humans. However, no CYP2C genes have been described in avian species to date. In this paper, we report the cloning, functional analysis, and regulation of chicken CYP2C45. The sequence shares up to 58% amino acid identity with CYP2Cs in other species. The overexpression of CYP2C45 in chicken hepatoma cells leghorn male hepatoma (LMH) led to increased scoparone metabolism. CYP2C45 regulation was studied in LMH cells at the mRNA level and in reporter gene assays using a construct containing 2.6 kb of its 5'-flanking region. Exposure of LMH cells to phenobarbital or metyrapone led to a 95- or 210-fold increase in CYP2C45 mRNA and a 140- or 290-fold increase in reporter gene expression, respectively. A phenobarbital response enhancer unit (PBRU) of 239 bp containing a DR-4 nuclear receptor binding site was identified within the 2.6-kb fragment. Site-specific mutation of the DR-4 revealed the requirement of this motif for CYP2C45 induction by drugs. The chicken xenobiotic receptor CXR interacted with the PBRU in electromobility shift and transactivation assays. Furthermore, the related nuclear receptors, mouse PXR and mouse CAR, transactivated this enhancer element, suggesting evolutionary conservation of nuclear receptor-DNA interactions in CYP2C induction.