CYP3A4基础与临床研究进展

细胞色素P4503A4(CYP3A4)是存在人类肝脏及肠道中的一种主要的细胞色素CYP450酶,约占成人肝脏CYP450酶总量的25%左右。临床中约有50%的药物是通过其代谢,并且其基因位点突变也与其多种疾病相关,知晓CYP3A4的表达水平和不同功能的遗传学基础,无论是对疾病的发病基础、临床药物的应用,会带来前所未有的启发,在药物应用过程中,通过对基因组学的认识,从而可以在基因层面了解个体代谢差异产生的原因,调整药物用量,提高疗效,最终使药物副作用降到最低限。目前对CYP3A4的研究渐趋于成熟,已逐渐阐明了其药物间相互作用的机制,它能够被多种药物竞争性抑制或者诱导,并受到某些蛋白受体的调控影响,可改变药物的药代动力学,增强或降低药效,造成个体用药差异,这也是造成药物间相互作用的重要原因。然而CYP3A4基因多态性与基因导向治疗关系,还有待进一步深入研究。该文对CYP3A4基因多态性、分布以及与临床疾病及用药的研究现状作一综述。     

Use of real-time gene-specific polymerase chain reaction to measure RNA expression of three family members of rat cytochrome P450 4A.

Exposure of rats to peroxisome proliferators induces members of the cytochrome P450 4A (CYP4A) family. In rats, the CYP4A family consists of four related genes, CYP4A1, CYP4A2, CYP4A3, and CYP4A8. We are specifically interested in examining CYP4A1, CYP4A2, and CYP4A3, each of which is expressed in a tissue-dependent and sex-dependent manner. While CYP4A1 is sufficiently different from the other two members to enable relatively easy specific quantitation, the close similarity between CYP4A2 and CYP4A3 makes quantitative discrimination difficult. We have combined a fluorescent real-time PCR assay (TaqMan) with the sequence-specific mismatch amplification mutation assay (MAMA) to allow us to carry out specific quantitation of all three members of this family. The assay is designed such that a single fluorescent TaqMan(R) probe binds to all three gene products, while specificity is conferred by sequence-specific primers. This specific MAMA technique takes advantage of the ability of Taq polymerase to distinguish between the two cDNAs based on mismatches at the 3' end of a PCR primer. In the 84-base PCR product used for this assay, there is only a single-base difference between CYP4A2 and CYP4A3. Despite this similarity, there is at least a 1000-fold discrimination between the two sequences, using CYP4A2 or CYP4A3 specific standards. Analysis of rat liver RNA from both sexes demonstrates that this discrimination is also achieved in complex RNA mixtures. This technique should be broadly applicable to other areas of research such as allelic discrimination, detecting mutational hotspots in tumors, and discrimination among closely related members of other gene families.     

Regulation and induction of CYP3A11, CYP3A13 and CYP3A25 in C57BL/6J mouse liver

This study reports that dexamethasone (DEX) significantly induces CYP3A11, CYP3A13 and CYP3A25 mRNA expression in male and female 4 days, 3 weeks and 18 weeks old C57BL/6J mice. Furthermore, CYP3A activity, as measured by erythromycin-N-demethylation, is also significantly increased. PXR, RXRalpha and CAR are known to be involved in the induction of CYP3As. Here we report nuclear receptors PXR and RXRalpha but not CAR demonstrate gender- and age-dependent expression. Also, treatment of C57BL/6J mice with DEX induces PXR but not RXRalpha or CAR. In summary, we demonstrate DEX is not only able to up-regulate CYP3A expression and activity, but also the nuclear receptor PXR through which it may exert this effect. Furthermore, the gender- and age-dependent pattern of basal PXR and RXRalpha expression is similar to the 3 CYP3As analysed.     

Cloning and expression of the Arthrobacter globiformis KZT1 fcbA gene encoding dehalogenase (4-chlorobenzoate-4-hydroxylase) in Escherichia coli

The fsbA gene controlling the first step of 4-chlorobenzoic acid (4CBA) metabolism in the Gram-positive soil bacterium Arthrobacter globiformis KZT1 has been cloned and analysed in Escherichia coli. The E. coli minicells analysis showed that a polypeptide(s) with Mr = 58 kDa (and/or Mr = 32 kDa) can be the fcbA product(s). Despite the gene dose amplification and control of the E. coli inducible Plac promoter, the level of functional expression of the fcbA gene in E. coli cells seems comparable only with that in the parental KZT1 strain. Effective 4CBA dechlorination by recombinant cells during growth in the presence of substrate within a range of concentrations 0.1 g/l to 0.7 g/l as well as a sudden reduction in the reaction efficiency at higher substrate concentrations were observed.     

Cloning,characterization, and RNAi effect of the chitin synthase B gene in Cnaphalocrocis medinalis

The rice leaf folder (RLF), Cnaphalocrocis medinalis, is one of the major pests of rice, and chitin synthase is a key enzyme for the chitin synthesis pathway in insects. In this study, the chitin synthase B gene from C. medinalis (CmCHSB) was cloned and characterized. The cDNA of CmCHSB is 4824 bp in length, containing an open reading frame of 4578 nucleotides that encodes 1525 amino acids. The CmCHSB zymoprotein consists of 10 transmembrane domains (TMDs) in the N-terminus, a middle conserved catalytic domain, and 7 TMDs in the C-terminus. Homology and phylogenetic analyses revealed that CmCHSB possesses the closest relationship with its homolog in Ostrinia furnacalis. CmCHSB was expressed throughout development and in all of the adult tissues tested, with the highest expression level in the adult and in the midgut. Silencing of CmCHSB through RNA interference (RNAi) severely affected RLF larval growth and caused larval lethality. Our results revealed that CmCHSB is essential for RLF growth and development, which sheds new light on the characteristics and functions of this gene. These findings will be helpful for green control of RLF, by targeting the CmCHSB gene using RNAi technology.     

氯吡格雷对大鼠肝药物酶CYP3A4 和CYP1A2 表达的影响

目的：氯吡格雷主要由CYP3A4 催化使其激活,CYP1A2 也参与氯吡格雷活化。关于氯吡格雷对肝微粒体酶的影响国内外 文献报道不多,因此本实验通过检测肝细胞色素氧化酶CYP3A4 和CYP1A2 的表达,探讨氯吡格雷对大鼠肝药物酶的影 响。方法：生理盐水为对照组,氯吡格雷设高、中、低三个剂量组（27,13.5,6.75mg/kg/d）,雄性健康大鼠连续灌胃给药7天,脱臼处 死,取肝组织,通过western blot法检测大鼠肝脏CYP3A4 和CYP1A2 蛋白表达情况。结果：1）、氯吡格雷抑制大鼠CYP3A4 蛋白 表达,氯吡格雷高中低剂量组分别比生理盐水组大鼠CYP3A4 蛋白表达量降低（P<0.05）;氯吡格雷低中高剂量组间进行比较,大 鼠CYP3A4 蛋白表达量呈梯度减少（P<0.05）;2）、氯吡格雷抑制大鼠CYP1A2 蛋白表达,氯吡格雷高中低剂量组分别比生理盐水 组大鼠CYP1A2 蛋白表达量降低（P<0.05）,氯吡格雷低中高剂量组间进行比较,大鼠CYP1A2 蛋白表达量呈梯度减少（P<0.05）。 结论：氯吡格雷使肝细胞色素氧化酶CYP3A4 和CYP1A2 的表达量减少,因此氯吡格雷高、中、低3 个剂量组均不同程度的抑制 大鼠肝脏CYP3A4 和CYP1A2 的表达,提示当氯吡格雷与某些主要经CYP3A4 和CYP1A2 代谢的药物合用时,发生代谢性相关 作用的可能性大。     

氯吡格雷对大鼠肝药物酶CYP3A4和CYP1A2表达的影响

目的：氯吡格雷主要由CYP3A4催化使其激活,CYPlA2也参与氯吡格雷活化。关于氯吡格雷对肝微粒体酶的影响国内外文献报道不多,因此本实验通过检测肝细胞色素氧化酶CYP3A4和CYPlA2的表达,探讨氯吡格雷对大鼠肝药物酶的影响。方法：生理盐水为对照组,氯吡格雷设高、中、低三个剂量组（27,13．5,6．75mg／kg／d）,雄性健康大鼠连续灌胃给药7天,脱臼处死,取肝组织,通过westernblot法检测大鼠肝脏CYP3A4和CYPlA2蛋白表达情况。结果：1）、氯吡格雷抑制大鼠CYP3A4蛋白表达,氯吡格雷高中低剂量组分别比生理盐水组大鼠CYP3A4蛋白表达量降低（P〈0．05）;氯吡格雷低中高剂量组间进行比较,大鼠CYP3A4蛋白表达量呈梯度减少（P〈0．05）;2）、氯吡格雷抑制大鼠CYPlA2蛋白表达,氯吡格雷高中低剂量组分别比生理盐水组大鼠CYPlA2蛋白表达量降低（P〈0．05）,氯吡格雷低中高剂量组间进行比较,大鼠CYPlA2蛋白表达量呈梯度减少（P〈0．05）。结论：氯吡格雷使肝细胞色素氧化酶CYP3A4和CYPlA2的表达量减少,因此氯吡格雷高、中、低3个剂量组均不同程度的抑制大鼠肝脏CYP3A4和CYPlA2的表达,提示当氯吡格雷与某些主要经CYP3A4和CYPlA2代谢的药物合用时,发生代谢性相关作用的可能性大。     

Renal localization, expression, and developmental regulation of P450 4F cytochromes in three substrains of spontaneously hypertensive rats

Cytochrome P450 4F isoforms have been shown to metabolize arachidonic acid to generate 20-hydroxyeicosatetraenoic acid (20-HETE), a potent eicosanoid that modulates vascular tone and renal tubular function. 20-HETE production in the kidney is implicated in the development of essential hypertension in the spontaneously hypertensive rat (SHR). In this study, we determined CYP4F mRNA localization and distribution in rat liver and kidney by in situ hybridization and real time quantitative PCR. CYP4Fs are regionally distributed in the kidney with CYP4F1, 4F4, and 4F5 being expressed more in the renal cortex than medulla while CYP4F6 shows higher medullary expression. We investigated developmental CYP4F gene expression in three different substrains of SHR. Distinct age-dependent patterns of expression were seen for individual CYP4F isoforms in Wistar-Kyoto (WKY) and three SHR substrains (B2, C, and A3). A steady increase in CYP4F1 expression with age was seen in each of the three substrains which correlate well with increased 20-HETE levels and elevated blood pressure seen in these animals. CYP4F4 expression increased significantly at 8 weeks followed by a precipitous fall in WKY and A3 strains at 12 weeks of age. In strains B2 and C, CYP4F4 levels started declining as early as 8 weeks of age. CYP4F5 and 4F6 levels fluctuated with age in a biphasic manner with a different profile for each sub-strain. Based on the expression profile and catalytic activity, CYP4F1 seems to be the most critical 4F isoform involved in the production of 20-HETE in the SHR kidney.     

Modulation of CYP3A4 expression by ceramide in human colon carcinoma HT-29 cells

Cytochrome P450 3A4 (CYP3A4) enzyme is responsible for the metabolic activation and inactivation of the majority of clinically used drugs in human liver and intestines. Recent studies have increasingly implicated various inflammatory stimuli to cause changes in the activities and expression levels of CYPs. However, the underlying mechanisms are largely unknown. In the present study, our studies investigated the effects of ceramide on CYP3A4 expression in human colon carcinoma HT-29 cells. Treatment with the cell-permeable ceramide analog C(6)-ceramide to the cells significantly decreased the expression of CYP3A4. By contrast, C(6)-dihydroceramide, a biologically inactive analog of C(6)-ceramide, did not affect CYP3A4 expression. We found that bacterial sphingomyelinase (SMase) and tumor necrosis factor-alpha (TNF), which are known to increase intracellular ceramide levels, also markedly suppressed the synthesis of CYP3A4. To elucidate whether nitric oxide (NO) participates in suppression of CYP3A4 expression by ceramide, the effects of NO modulators were determined. Treatment with N(G)-monomethyl-L-arginine, a competitive inhibitor of inducible nitric oxide synthase (iNOS), was able to protect ceramide-dependent CYP3A4 suppression. In contrast, the addition of S-nitroso-N-acetylpenicillamine, a NO donor, to HT-29 cells reduced CYP3A4 expression. The addition of iNOS antisense oligonucleotide prevented ceramide-mediated induction of iNOS expression and restored CYP3A4 expression. Wortmannin which is known to inhibit phosphatidylinositol 3-kinase (PI3-K) blocked CYP3A4 suppression by ceramide. Taken together, our results demonstrate that ceramide-mediated suppression of CYP3A4 is due to production of NO, which might result from activation of PI3-K.     

人神经生长因子基因在大肠杆菌中的克隆、表达及产物纯化

从人胎盘组织中提取总DNA, 经PCR扩增编码人β神经生长因子（β-NGF）成熟肽的基因,并克隆到大肠杆菌表达载体pET15b中。重组质粒pET15b-NGF经测序与报道的完全一致。重组质粒转化大肠杆菌BL21(DE3)pLysS,经IPTG诱导表达得到16kD的目的蛋白带,与预期的大小一致,NGF表达量约占全菌总蛋白的25%.经过亲和层析柱（Ni2+-charged IDA his-bind column）纯化后得到了单一的NGF蛋白条带,蛋白纯度可达90%以上,从每升表达菌液中可以得到4.56mgNGF。表达产物的Western 印迹鉴定结果显示：重组人神经生长因子能与兔抗人β-NGF的多克隆抗体发生特异性结合反应,在16kD处出现单一的条带,表明诱导表达的重组NGF具有免疫学活性。鸡胚背根神经节感觉神经元鉴定试验表明,本实验表达的重组NGF具有良好的生物学活性。     

Haplotype study of the CYP4A11 gene and coronary artery disease in Han and Uygur populations in China

Background

CYP4A11 converts arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE), which has a crucial role in the modulation of cardiovascular homeostasis. We assessed the association between the human CYP4A11 gene and coronary artery disease (CAD) in Han and Uygur populations in China.

Methods and Results

In the Han population, 361 CAD patients and 315 controls were genotyped for four single-nucleotide polymorphisms (SNPs) of the human CYP4A11 gene (rs9332978, rs4660980, rs3890011, rs1126742). In the Uygur population, 331 CAD patients and 182 controls were genotyped for the same four SNPs. Data were assessed via haplotype-based case–control studies. For the Han population, the significance of the recessive model of SNP3 (GG vs. CC+GC) between CAD patients and control subjects was retained after adjustment for EH, DM and smoking (for men, 95% CI: 1.173–3.013, P = 0.009). The G-G-T haplotype in CAD was significantly higher than that in the control group (P = 0.037). In the Uygur population, neither the distribution of genotypes and alleles for the four SNPs nor the distribution of haplotypes constructed with the same three SNPs showed a significant difference between CAD and control subjects.

Conclusions

The GG genotype of rs3890011 and the G-G-T haplotype in the CYP4A11 gene could be a useful genetic marker of CAD in Han populations in China.     

青杨脊虎天牛CYP4G2基因片段的克隆、序列分析与表达

根据报道的十几种昆虫CYP4家族基因的氨基酸序列保守区域设计一对引物,利用RT-PCR技术扩增编码青杨脊虎天牛Xylotechus rusticus中肠细胞色素氧化酶CYP4G2蛋白的cDNA片段,构建原核表达载体pET-CYP4G2,将其转化入大肠杆菌Escherichia coli JM109中表达。序列分析结果表明,该基因(CYP4G2,GenBank登录号为EF429250)保守区域阅读框全长387 bp,编码129个氨基酸残基,预测分子量和等电点分别为16.9 kD和5.75;推导的氨基酸序列与已报道的昆虫CYP4家族氨基酸序列一致性较高（63%～86%）,且具有细胞色素氧化酶的典型特征。IPTG诱导后,SDS-PAGE电泳检测到一条22 kD大小的外源蛋白,与预测融合蛋白的分子量大小相应。CO差光谱分析证明重组菌表达了有活性的pET-CYP4G2。     

CYP330A1 and CYP4C39 enzymes in the shore crab Carcinus maenas: sequence and expression regulation by ecdysteroids and xenobiotics

Cytochrome P450 enzymes (CYP enzymes) catalyse important metabolic reactions of exogenous and endogenous substrates, including steroid hormones. Here, we report the first two CYP sequences from the shore crab, Carcinus maenas. Two complete cDNAs isolated from crab hepatopancreas encode CYP enzymes named CYP330A1, the first member of a new family, and CYP4C39. CYP330A1 is closest related to members of the CYP2 family (37.3% identical to mouse CYP2J6) and CYP4C39 is most identical to crayfish CYP4C15 (59.5%). CYP330A1 gene expression was induced in hepatopancreas of male green intermoult crabs by ecdysone and ponasterone A, but also by benzo(a)pyrene and phenobarbital. CYP330A1 induction was not observed in red crabs. The present results indicate that the CYP330A1 enzyme may be involved in ecdysteroid metabolism, presumably catabolism, and in the detoxification of environmental pollutants. Ecdysteroids or xenobiotics did not affect CYP4C39 gene expression. The fact that both ecdysteroids and xenobiotics affect CYP330A1 gene expression indicates that mutual interactions between chemical exposures and endocrine functions may exist in the shore crab.     

CYP3A4 and CYP2D6 inhibitory activities of Indonesian medicinal plants

Thirty samples of Indonesian medicinal plants were analyzed for their capacity to inhibit in vitro metabolism by human cytochrome P450 3A4 (CYP3A4) and CYP2D6 with a radiometric assay. The MeOH-soluble fractions of 25 samples, prepared from water extracts, demonstrated inhibitory activity more than 50% on the metabolism mediated by CYP3A4, and 21 samples on the metabolism mediated by CYP2D6. Among the MeOH-soluble fractions, Piper nigrum leaf showed the highest inhibitory activity against CYP3A4 (91.7%), and Punica granatum against CYP2D6 (98.1%). The water extracts of which MeOH-soluble fraction showed inhibitory activity more than 70% were fractionated with EtOAc. From the EtOAc-soluble fractions, Curcuma heyneana (67.0%), Pi. cubeba (75.0%), Pi. nigrum fruit (84.0%), Pi. nigrum leaf (85.8%), and Zingiber aromaticum (75.3%) demonstrated inhibitory activity more than 50% on the metabolism mediated by CYP3A4, but only Pi. nigrum fruit (72.8%) and Pi. nigrum leaf (69.1%) showed strong inhibitory activity against CYP2D6. For samples that showed more than 70% inhibition, their IC(50) values were determined. The most potent inhibitory activity against CYP3A4 (IC(50) value of 25 microg/ml) was found for the extract of Pi. nigrum leaf, while that of Catharanthus roseus showed the most potent inhibitory effect against CYP2D6 (IC(50) value of 11 microg/ml). These results should indicate once more the possibility of potential medicinal plant-drug interactions.     

Comparison of genomic and cDNA sequences of guinea pig CYP2B18 and rat CYP2B2: absence of a phenobarbital-responsive enhancer module in the upstream region of the CYP2B18 gene

Potential mechanisms were investigated whereby CYP2B18, a cytochrome P450 gene exhibiting high constitutive expression but only low levels of phenobarbital-inducibility in the guinea pig liver, may be differentially regulated versus the highly inducible rat CYP2B2 gene. To comparatively assess potential regulatory sequences associated with CYP2B18, a guinea pig genomic library was screened enabling isolation of the CYP2B18 gene. The genomic screening process resulted in the identification of at least four closely-related CYP2B18 genes, designated here as CYP2B18A-D. Of these isolates, CYP2B18A exhibited sequence identical to that of the CYP2B18 cDNA. Further, the deduced amino acid sequence of the CYP2B18 cDNA was identical to that of N-terminal and internally-derived peptide sequences obtained in this investigation from CYP2B18 protein isolated from guinea pig liver. Genomic structural sequences were derived for CYP2B18A, together with the respective 5'-upstream and intronic regions of the gene. Comparison of the CYP2B18A and CYP2B2 gene sequences revealed the lack of repetitive LINE gene sequences in CYP2B18A, putative silencing elements that effect neighboring genes, although these sequences were present in both 5'-upstream and 3'-downstream regions of CYP2B2. We determined that the phenobarbital-responsive enhancer module was absent from the 5'-upstream region as well as the intronic regions of CYP2B18A gene. We hypothesize that the compromised phenobarbital inducibility of CYP2B18A stems from its lack of a functional phenobarbital responsive enhancer module.