Induction of CYP3A gene and enzyme activity expression in kidney cell of grass carp (Ctenopharyngodon idellus)

Abstract

Cytochrome P450 3A(CYP3A) is the major cytochrome P450 responsible for the metabolism of en-dogenous and xenobiotic substrates.Unexpected drug–drug interactions in fish are generally associated with the induction of CYP3A activity,and can lead to the formation of drug residues,which threaten the safety of fishery products.We determined the in vitro effects of rifampicin(RIF) on CYP3A mRNA expression and enzyme activity in a freshwater teleost,the grass carp(Ctenopharyngodon idellus).Recently,a number of CYP3A genes have been isolated from several teleost species.Although CYP3A genes in fish contain multiple paralogs differing in gene expression pattern and tissue distribution,CYP3A enzymes exhibit similar catalytic properties because of their structural similarities.The CYP3A gene and its full-length cDNA of 1 849 bp were cloned from grass carp by RT-PCR.The CYP3A gene has an open reading frame(ORF) of 1 542 bp,a stop codon,a 3'-untranslated regions(3' UTR),and a polyA signal is present in the 3'UTR region.The ORF encodes a 513 amino acid(aa) protein,which has a signal peptide(29aa),two transmembrane helixes(23aa and 23aa),a heme binding domain(HBD,21aa),and six substrate recognition sites(SRS1-6).Multiple alignments showed that the CYP3A amino acid se-quence from grass carp is 60% to 92% similar to other vertebrate CYP3As.CYP3A gene expression after induc-tion by RIF in grass carp kidney cells(CIK) was assayed by quantitative real-time PCR.CYP3A-dependent erythromycin N-demethylase(ERND) activity was determined by spectrophotometry,and CYP3A activity was assessed by measuring the formation of formaldehyde.In the induced group,CYP3A mRNA expression reached a plateau at 8h,while the highest level of CYP3A activity occurred at 10h.The results indicated that CYP3A mRNA expression and enzyme activity were significantly higher than in the control group(P0.05).The highest level of CYP3A mRNA expression appeared 2 hours before the maximum of enzyme activity.In addition,the transcription of level CYP3A was apparently higher than the enzyme activity,implying that induction by RIF affected enzyme activity by transcriptional regulation of the CYP3A gene.