Cytochrome P450 3a And Pregnane X Receptor Gene Expression And Function Analysis In Grass Carp (cetnopharyngodon Idellus)

Cytochrome P450 (CYP) is an important animal drug metabolism enzyme. Cytochrome P450 3A (CYP3A), responsible for the metabolism of the majority of drugs, comprises the largest portion of the liver, small intestinal and kidney P450s in animals. CYP1A are responsible for the biotransformation of drug, CYP1A and CYP3A are induced or inhibited by drug. Pregnane X receptor (PXR) belongs to the nuclear receptor superfamily (NR) of the NR1Ⅰfamily, and PXR are responsible in the regulatory regions of CYP3A genes. However, little information is available about the stable stage on CYP3A expression and available on the correlation between PXR and CYP3A involved in the metabolic processes of medicines in fish.In the paper, We used biochemistry and molecular biology methods. Firstly, We cloned CYP3A gene, analysed the gene structure and function domains, and detected the gene expression level in grass carp tissues. We cloned the PXR gene and detected the expression level in tissues. Secondly, We analysed the correlation of CYP3A mRNA and enzyme activity depending on temporal dynamics for determination of the stable expression in optional induction of grass carp in vitro model. In drug treatment conditions, we studied the different expression and analyzed the correlation between the levels of CYP3A enzyme, CYP3A and PXR mRNA. In initial induced platform, ofloxacin (OFLX) is both a substrate and inducer for CYP3A, we verified the CYP3A stable phase and the correlation of CYP3A activity -CYP3A mRNA-PXR mRNA in the OFLX metabolism process. The major results are as follows:1. Cloning, sequencing and different mRNA expression of CYP3A gene in Grass carpAccording to the sequences of CYP3A gene in zebra fish and rainbow trout from the GenBank, we designed the degenerate primers using premier primer 5.0, then we used primer for polymerase chain reaction (PCR) amplification and gained the partial conserved sequences of CYP3A. According to the conserved sequences, we re-designed the primer to amplify the ORF, then we designed primers for 3' Rapid Amplification of cDNA Ends followed by the conserved sequences. We designed qRT-PCR primers to detect CYP3A mRNA expression in different tissues by fluorescent dye SYBR. On the basis of crucian carp CYP1A, we analyzed the results of different genetic characteristics of different subtypes in fish. The results showed that: (1) The partial CYP3A gene cDNA nucleotide sequences of grass carp is 1847 bp, including conserved coding region and 3'–untranslated region(3' UTR). The CYP3A gene has an open reading frame (ORF) of 1542bp. The ORF encodes a 513 amino acid (aa) protein, which has a signal peptide (29aa) and a stop codon. (2) The main domain of grass carp CYP3A is composed of 3 transmembrane helix, 9 phosphorylation sites, 6 substrate binding site, a heme protein binding and containing a cysteine (Cys, C-448aa) of iron-heme ligand binding site. (3) The quantitative real time polymerase chain reaction (qRT-PCR) assay was developed to estimate the mRNA expression levels in 9 tissues, its expression abundance in turn are as follows: anterior intestine> liver> kidney> gills> muscle> hindgut> gonad> heart> spleen. The Foregut, liver and kidney are the main expression sites,however weak expression was detected in the other tissues. CYP1A expression in curcian carp was assayed with the same method, the results were as follows: liver> foregut> gills> kidney> hindgut > gonads> heart> muscle> spleen, where liver, intestine and gills are the main expression sites. However weak expression were detected in other organs. The distribution of Cytochome P450 isoforms in tissues consist with the main site of drug metabolism.2. Cloning, sequencing and different mRNA expression of PXR gene in Grass carpAccording to the sequences of PXR gene in zebra fish and rainbow trout from the GenBank, we designed the degenerate primers using premier primer 5.0, then we used primer for polymerase chain reaction (PCR) amplification and gained the partial conserved sequences of PXR. We designed qRT-PCR primers to detect PXR mRNA expression in different tissues using fluorescent dye SYBR. The results showed that: (1) The partial PXR gene cDNA nucleotide sequences was 509 bp with the deduced 169 aa for grass carp. (2) the results of qRT-PCR assay showed high similarity with CYP3A gene,that is, anterior intestine> liver> kidney> gills> muscle> hindgut> gonad> heart> spleen.3. Toxicity test by RIF and OFLX in CIKConventional methods were employed for cell continuous culture, resuscitation and cryopreservation. Protein contents was determined by BCA method. MTT reduction was used to optimize the sublethal concentrations at 10-,20-,40-,80 uM, and time for 1-, 2-, 4-, 6-, 12-, 24-, 48-,72 h for RIF test. MTT reduction was used to optimize concentrations at 25 -, 50 -, 100 -, 200 -, 400 and 800μM, and time for 1 -, 2 -, 4 -, 6 -, 8 -, 10 -, 12 -, 24 and 48 h for ofloxacin test. The relative viability of CIK Sharply decreased when the concentrations were set at 200 uM and with incubation time for 48h above incubation with OFLX. The results showed that RIF incubation at 40 uM and 24 h was the best induction model for experiment. The OFLX incubation at 50 uM and 24 h for the further study. It can offer the information to further study RIF and OFLX in CIK.4. Assay of CYP3A gene transcription and CYP3A enzyme activity with optimal induction model in CIKIn order to evaluate the CYP3A expression in kidney of grass carp, the study determined the CYP3A gene transcription and enzyme activity. CYP3A gene expression after induction 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 those in the control group (p<0.05). The highest level of CYP3A mRNA expression appeared 2 hours before the maximum of enzyme activity. In addition, the transcription level of CYP3A was apparently higher than the enzyme activity, implying that induction by RIF affect enzyme activity by transcriptional regulation of the CYP3A gene.5. Establishment of the model for CYP3A activity, CYP3A and PXR mRNA correlation in fishCYP3A mRNA transcription was up-regulated after 2 h, while PXR was up-regulated after 10 h. The results indicated that the expression of CYP3A was prior to PXR. In addition, the transcription level of CYP3A was apparently higher than the enzyme activity. The PXR transcription level was involved in the activation and induction of CYP3A mRNA expression, but not directly on the enzyme activity. Therefore, we hypothesized that the induction of CYP3A activity in grass carp was dependent on PXR. That induction by RIF affected enzyme activity by transcriptional regulation of the CYP3A gene.6. The correlation between OFLX ,CYP3A and PXR We study the correlation between CYP3A and PXR mRNA after incubation by OFLX in CIK against time. CYP3A gene expression was assayed by quantitative real-time PCR, and CYP3A activity was assessed by measuring the formation of formaldehyde. The results showed that: (1) The CYP3A transcription level was up-regulated after 4 h but acted as a weak induction of CYP3A enzyme activities. (2) CYP3A mRNA transcript was up-regulated after 12 h. PXR mRNA have been down-regulated from 1-10 h, and up-regulated after 12 h treated by OFLX. (3) Thus, our work confirmed that OFLX is both the substrate and inducer of CYP3A.