Real-time polymerase chain reaction analysis of CYP1B1 gene expression in human liver.

Procarcinogen-activating cytochrome P450 (CYP) enzymes such as CYP1B1, CYP1A1, and CYP1A2 are considered to play an important role in chemical carcinogenesis. However, conflicting data exist with respect to CYP1B1 expression in human liver. In the present study, we measured CYP1B1 mRNA and protein expression in liver samples from 12 individuals (7 nonsmokers, 4 smokers, and 1 ex-smoker) and compared the levels to those of CYP1A1 and CYP1A2. As analyzed by real-time polymerase chain reaction, CYP1B1 mRNA was present in all samples and the inter-individual variability was 16-fold. The group mean level was 5-fold greater in smokers than nonsmokers (121 +/- 46 vs. 26 +/- 5 molecules/ng double-stranded DNA, p < 0.05). By comparison, CYP1A1 mRNA was detectable in samples from 4 of 7 nonsmokers, 3 of 4 smokers, and one ex-smoker, whereas CYP1A2 mRNA was detectable in samples from 5 nonsmokers, 4 smokers, and the ex-smoker. The mean levels of CYP1A1 and CYP1A2 mRNA were 4-fold and 9-fold greater, respectively, in smokers than nonsmokers, but the differences were not statistically significant. The inter-individual variability in CYP1A1 and CYP1A2 mRNA expression was 26-fold and 500-fold, respectively. Immunoblot analysis using several antibodies and with a larger panel (n = 27) of liver microsomes showed that CYP1A1 and CYP1B1 proteins were undetectable, whereas CYP1A2 was detectable in all samples and quantifiable in 24 of 27 samples. In summary, our novel finding indicates that CYP1B1 mRNA is expressed in human liver and the levels are increased in smokers, but the protein is undetectable.     

Preferential induction of CYP1A1 and CYP1B1 in CCSP-positive cells.

Both benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are potent ligands of aryl hydrocarbon receptors (AhR). Although animal studies indicate that both compounds induce pathological changes in the peripheral lung, the specific cell type involved remains unclear. Clara cells, expressing Clara cell specific protein (CCSP) and abundant in cytochrome P450, are nonciliated bronchiolar epithelial cells in the peripheral lung. Here we explore the hypothesis that CCSP-positive Clara cells are highly responsive to AhR ligands and are the primary cell type involved in BaP- and TCDD-induced toxicities. The responsiveness to AhR ligands was evaluated by measuring the respective mRNA and protein levels of cytochrome P450 1A1 (CYP1A1) and 1B1 (CYP1B1) using real-time RT-PCR and immunocytochemistry assays. Two in vitro models were used: primary cultures of human small airway epithelial (SAE) cells and rat lung slice cultures. In the presence of calcium, human SAE cells differentiated into CCSP-positive cells. BaP- and TCDD-induced mRNA and protein levels of CYP1A1 and CYP1B1 levels were significantly elevated in CCSP-positive cell cultures. Similarly, AhR mRNA and protein levels were increased in CCSP-positive cell cultures, as determined by real-time RT-PCR and Western blot analysis. When rat lung slice cultures were treated with BaP or TCDD for 24 h, CYP1A1 and CYP1B1 proteins were strongly induced in Clara cells. These results indicate that, in the peripheral lung of both rats and humans, CCSP-positive cells (Clara cells) may be more sensitive to AhR ligands than other cell types.     

抗癌药细胞色素P450 1B1抑制剂的研究进展

介绍了有关人细胞色素P450 1B1(CYP1B1)抑制剂的研究概况。CYP1B1是一类在导致癌变的雌激素代谢和激活芳香烃化合物致癌性中起重要作用的酶，在正常组织中通常不表达而在许多肿瘤组织中表达活跃，同时对许多抗癌药物的代谢具有重要影响。这表明CYP1B1抑制剂有望成为又一类肿瘤治疗药物。     

Bromuconazole-induced hepatotoxicity is accompanied by upregulation of PXR/CYP3A1 and downregulation of CAR/CYP2B1 gene expression

Despite widespread use of bromuconazole as a pesticide for food crops and fruits, limited studies have been done to evaluate its toxic effects. Here, we evaluated the hepatotoxic effect of bromuconazole using classical toxicological (biochemical analysis and histopathological examination) and gene-based molecular methods. Male rats were treated either orally or topically with bromuconazole at doses equal to no observed adverse effect level (NOAEL) and 1/10 LD50 for 90?d. Bromuconazole increased activities of liver enzymes (ALT, AST, ALP, and ACP), and levels of bilirubin. It also induced hepatic oxidative stress as evidenced by significant decrease in the activities of superoxide dismutase (SOD), and significant increase in levels of malondialdehyde (MDA) in liver. In addition, bromuconazole caused an increase in liver weights and necrobiotic changes (vacuolation and hepatocellular hypertrophy). It also strongly induced the expression of PXR and its downstream target CYP3A1 gene as well as the activity of CYP3A1. However, it inhibited the expression of CAR and its downstream target CYP2B1 gene without significant changing in CYP2B1 activity. Overall, the oral route showed higher hepatotoxic effect and molecular changes than the dermal route and all changes were dose dependent. This is the first investigation to report that bromuconazole-induced liver oxidative damage is accompanied by upregulation of PXR/CYP3A1 and downregulation of CAR/CYP2B1.     

苯乙烯基萘类化合物的合成及其CYP1B1酶抑制活性研究

目的寻找CYP1B1酶的高活性高选择性抑制剂。方法以α-萘黄酮为先导物,结合二苯乙烯类CYP1B1酶抑制剂的结构特征,设计了先导物的结构类似物α-(E)-苯乙烯基萘类(Ⅰ)及β-(E)-苯乙烯基萘类(Ⅱ、Ⅲ)化合物。目标化合物通过萘甲醛(6、7或8)及苄基膦酸酯(Ⅴ1～Ⅴ10)的Horner-Wadsworth-Em-mons反应制备。利用7-乙氧基-3H-吩口恶嗪-3-酮-脱乙基(EROD)试剂评价目标化合物对人重组CYP1A1和CYP1B1酶的抑制活性。结果合成了26个新型苯乙烯基萘类化合物,其结构通过1H-NMR确证。酶抑制活性试验表明,与阳性对照物白藜芦醇相比,大多数目标化合物对CYP1B1酶表现出较强的抑制活性及选择性。化合物Ⅰ2、Ⅰ8、Ⅲ2、Ⅲ4较α-萘黄酮显示更强的酶抑制活性及选择性。其中,化合物Ⅰ2对CYP1B1酶的抑制活性最强(IC50为0.31 nmol.L-1)。结论合成了新型苯乙烯基萘类CYP1B1酶抑制剂,并初步分析了其构效关系,为进一步的结构优化及新抑制剂的研究提供依据。     

Linked expression of Ah receptor, ARNT, CYP1A1, and CYP1B1 in rat mammary epithelia, in vitro, is each substantially elevated by specific extracellular matrix interactions that precede branching morphogenesis.

Cytochrome P4501B1 (CYP1B1), the major constitutively expressed CYP in the rat mammary gland, is induced by Ah-receptor (AhR) ligands, while CYP1A1 is predominantly expressed only after induction. These CYPs contribute to carcinogenic activation of polycyclic aromatic hydrocarbons (PAHs). AhR, ARNT, and CYP1B1 were only weakly expressed, even after 2,3,7,8-tetrachlorodibenzo-p-dioxin induction, when rat mammary epithelial cells (RMEC) were cultured on plastic. RMEC cultured on the extracellular matrix (ECM), Matrigel, or on a floating gel of collagen I demonstrated branching morphogenesis and substantially increased basal CYP1B1 and induced CYP1A1 expression, in parallel with large increases in AhR and ARNT expression. Branching was more pronounced in the Wistar Kyoto than in the Wistar Furth rat strain. Although EGF enhanced branching, neither strain nor growth factor treatment substantially impacted CYP expression. Increased AhR and ARNT expression is observed within 24 h of dispersal on Matrigel, substantially prior to branch formation. Culture on thin layers of collagen I, collagen IV, and laminin, respectively, failed to reproduce the branching morphogenesis or increases in AhR, ARNT, or CYP expression. However, adherent, gelled collagen I recapitulated the increased protein expression, without supporting branching. This increased protein expression was closely paralleled by enhanced expression of beta-catenin and E-cadherin, components of cell-cell adhesion complexes. A synthetic peptide that selectively antagonizes integrin-ECM interactions reduced branch formation, without diminishing AhR, ARNT, and CYP expression. These data demonstrate that early ECM surface adhesion interactions mediate AhR and ARNT expression, which enhances CYP expression, independent of branching morphogenesis.     

Transplacental exposure to indole-3-carbinol induces sex-specific expression of CYP1A1 and CYP1B1 in the liver of Fischer 344 neonatal rats.

Indole-3-carbinol (I3C), a naturally occurring component of broccoli, cabbage, and other members of the family Cruciferae, is a tumor modulator in several animal models that demonstrates significant chemoprevention against development of both spontaneous and chemically induced cancers while conversely eliciting tumor promoter effects in others. This study examines the disposition of I3C in the pregnant rat model, specifically to determine whether I3C can traverse the maternal placenta, and what effects, if any, are elicited in the neonate. We now report that dietary I3C treatment of pregnant female rats results in appearance of I3C acid condensation products in both maternal and neonatal livers. Livers from I3C-fed maternal rats showed CYP1A1 protein induction; however, no CYP1B1 protein was detected. No CYP1A1 or CYP1B1 protein was detected in the livers of pregnant controls or their offspring. We also report a sex-specific induction of CYP1A1 and CYP1B1 protein in livers from newborns born to I3C-fed dams. CYP1A1 protein was significantly induced in male neonatal liver, but not in females. Conversely, hepatic CYP1B1 protein was induced to high levels in female neonates, with no CYP1B1 protein detected in male littermates. Our results demonstrate that dietary I3C acid condensation products can cross the maternal placenta and differentially induce neonatal hepatic CYP1A1 and CYP1B1 in a sex-specific manner. The results highlight the potential of I3C to effect changes in the overall metabolic profile of xenobiotics to which the fetus is exposed transplacentally and indicate the possible involvement of sex-specific modulators in Ah receptor-mediated responses in this model.     

草甘膦对大鼠细胞色素P450 2B1和P450 2C11基因表达的影响

采用酶学和分子生物学方法，研究了两种草甘膦除草剂对大鼠细胞色素Ｐ４５０２Ｂ１和２Ｃ１１（ＣＹＰ４５０２Ｂ１和２Ｃ１１）ｍＲＮＡ表达的影响以探讨其作用机理。结果表明，草甘膦Ⅱ可增加肝细胞中ＣＹＰ４５０２Ｂ１ｍＲＮＡ转录水平，与对照组比较有显著性差异（Ｐ＜０．０５）；而草甘膦Ⅰ对肝细胞ＣＹＰ４５０２Ｂ１ｍＲＮＡ没有明显影响（Ｐ＞０．０５）。两种草甘膦均可显著降低肾脏ＣＹＰ４５０２Ｂ１ｍＲＮＡ表达水平，与对照组比较均有极显著差异（Ｐ＜０．０１）。草甘膦Ⅰ可使大鼠肝细胞中ＣＹＰ４５０２Ｃ１１ｍＲＮＡ转录水平显著降低，与对照组比较有显著性差异（Ｐ＜０．０５）；而草甘膦Ⅱ对大鼠肝细胞中ＣＹＰ４５０２Ｃ１１ｍＲＮＡ没有明显影响。     

膀胱癌患者CYP1A1基因MSPI多态性的相关分析

目的探讨细胞色素CYP1A1基因MSPI多态性与膀胱癌遗传易感性的关系。方法应用聚合酶链式反应-限制性片段长度多态性(PCR-RFLP)分析技术,检测44例膀胱癌患者(病例组)和85例同期住院非膀胱癌患者(对照组),检测CYP1A1基因MSPI多态性位点的3种基因型及等位基因的分布频率。结果在病例组CYP1A1基因MSPI位点基因型分布频率为:TT(54·5%)、TC(36·4%)、CC(9·1%),等位基因分布频率为T(72·7%)、C(27·3%);在对照组CYP1A1基因MSPI位点基因型的分布频率为TT(61·2%)、TC(31·2%)、CC(7·1%),等位基因分布频率为T(77·1%)、C(22·9%)。各个基因型在两组中所占的比例差异无统计学意义(P>0·05);T、C等位基因频率两组比较差异亦无统计学意义(P>0·05)。结论CYP1A1基因MSPI位点多态性的单独存在可能与本地区膀胱癌易感性无关。     

Effect of TCDD exposure on CYP1A1 and CYP1B1 expression in explant cultures of human endometrium.

Endometriosis is a debilitating disease estimated to affect 10% of reproductive-age women and characterized by the growth of endometrial tissue outside of the uterus. The present study characterizes a human endometrial explant culture model for studying the direct effects of TCDD exposure by assessing the expression of CYP1A1 and CYP1B1 mRNA (Northern blotting), protein (Western blotting), and activity (7-ethoxyresorufin-O-deethylase; EROD) in explants cultured with and without TCDD. Explants were obtained at laparoscopy or laparotomy from women undergoing surgery for tubal ligation, endometriosis, or pelvic pain unrelated to endometriosis. The explants were cultured with 10 nM estradiol (E(2)) or 1 nM E(2) plus 500 nM progesterone (P(4)) with or without TCDD (first 24 h). The expression of CYP1A1 and CYP1B1 mRNA was greatest with 10 nM TCDD and increased up to 72 h after initial exposure. EROD activity increased up to 120 h. Explants from a secretory phase biopsy became reorganized in culture and formed a new epithelial membrane, while maintaining basic endometrial morphology and viability for up to 120 h. At 24 h, TCDD significantly increased CYP1A1 and CYP1B1 mRNA, and at 72 h, TCDD significantly increased EROD activity and CYP1B1 protein compared to explants cultured without TCDD for similar times. CYP1B1 protein also exhibited substantial constitutive expression that was similar in uncultured biopsies, where CYP1B1 protein was immunolocalized in the cytoplasm of epithelial glands, with only occasional patches of protein in the surface epithelial membrane. In explants cultured with and without TCDD exposure, CYP1B1 protein was localized in the cytoplasm of the new surface epithelial membrane and glands closest to the surface. CYP1A1 protein was not detected in uncultured biopsies or explants. Both younger age (age 30 and under) and proliferative phase were associated with higher TCDD-induced EROD activity in specimens treated with E(2):P(4). No significant endometriosis-related differences were observed for any of the biomarkers, but the detection of disease-specific change was limited by small sample size and variability in tissue-cycle phase. The human endometrial explant culture model will be useful for future studies of the effects of dioxin-like compounds on human endometrium in relationship to cycle phase and hormonal exposure.     

The potential of targeting CYP11B

Introduction: Members of the CYP11B subfamily participate in the biosynthesis of important steroid hormones. CYP11B1 catalyzes the formation of cortisol, while CYP11B2 realizes the biosynthesis of aldosterone. Overproduction of cortisol is related to Cushing’s disease, whereas overproduction of aldosterone leads to hypertension and end-organ damage such as cardiac and renal hypertrophy. Therefore, CYP11B1 and CYP11B2 have been defined as interesting targets for the development of novel drugs.

Areas covered: The paper describes the CYP11B1 and CYP11B2 genes and proteins, giving special attention to their functional and structural properties, the development of efficient test systems for potential inhibitors of both CYPs and the development and testing of novel potential drugs on the basis of selective inhibition of CYP11B1 and CYP11B2.

Expert opinion: The availability of relevant and efficient screening systems for testing the effects of inhibitors of human CYP11B1 and CYP11B2, combined with experiences and success in synthesizing selective and efficient inhibitors of these isoenzymes, provides a realistic basis for a successful development of drugs using CYP11B1 and CYP11B2 as targets. The first clinical trials with the CYP11B2 inhibitor LCI699 demonstrated some side effects but showed inhibition of end-organ damage in animals, indicating that it might be a useful lead compound for future developments.     

A novel polymorphism in ABCB1 gene, CYP2B6*6 and sex predict single-dose efavirenz population pharmacokinetics in Ugandans

AIMS

Efavirenz exhibits pharmacokinetic variability causing varied clinical response. The aim was to develop an integrated population pharmacokinetic/pharmacogenetic model and investigate the impact of genetic variations, sex, demographic and biochemical variables on single-dose efavirenz pharmacokinetics among Ugandan subjects, using nonmem.

METHODS

Efavirenz plasma concentrations (n= 402) from 121 healthy subjects were quantified by high-performance liquid chromatography. Subjects were genotyped for 30 single nucleotide polymorphisms (SNPs), of which six were novel SNPs in CYP2B6, CYP3A5 and ABCB1. The efavirenz pharmacokinetics was described by a two-compartment model with zero- followed by first-order absorption.

RESULTS

Apparent oral clearance (95% confidence interval) was 4 l h l⁻¹ (3.5, 4.5) in extensive metabolizers. In the final model, incorporating multiple covariates, statistical significance was found only for CYP2B6*6 and CYP2B6*11 on apparent oral clearance as well as ABCB1 (rs3842) on the relative bioavailability. Subjects homozygous for CYP2B6*6 (G516T, A785G) and *11 displayed 21 and 20% lower apparent oral clearance, respectively. Efavirenz relative bioavailability was 26% higher in subjects homozygous for ABCB1 (rs3842). The apparent peripheral volume of distribution was twofold higher in women compared with men.

CONCLUSIONS

The model identified the four factors CYP2B6*6, CYP2B6*11, a novel variant allele in ABCB1 (rs3842) and sex as major predictors of efavirenz plasma exposure in a healthy Ugandan population after single-dose administration. Use of mixed-effects modelling allowed the analysis and integration of multiple pharmacogenetic and demographic covariates in a pharmacokinetic population model.     

三氯乙烯对3种细胞色素P450酶基因表达的影响

目的 探讨三氯乙烯（Trichloroethylene,TCE)对人体淋巴细胞瘤细胞株（MCL-5）中3种细胞色素P450酶基因（CYP1A1、CYP2E1、CYP3A4）表达的影响,并研究剂量反应关系和时间反应关系,方法 用常规的细胞培养方法,0.5、1.0、1.5、2.0mmol/L TCE处理细胞12、24、48、72h。利用提纯RNA和cDNA的药盒,合成cDNA,然后逆转录聚合酶反应（RT-PCR）表达3种CYP450基因,以β-Actin作为内对照,分析不同处理剂量和时间时基因表达的强度。结果 在MCL-5细胞株中都有基本的表达,CYP1A1表达在用1.0、1.5、2.0mmol/LTCE处理48h后有被上调的趋势,而且上调趋势随处理时间延长耐加强;CYP2E1、CYP3A4表达不受TCE处理时间长短的影响。3种CYP450酶基因的表达受TCE剂量的影响。3随0.5mol/L,1.0、1.5、2.0mmol/L剂量的增加有上调的趋势,结论 TCE对CYP450酶系统中的CYP2E1、CYP1A1、CYP3A4基因有明显的诱导作用。这些基因被诱导后的结果。可能会导致相对应酶活性的增加,同时加强对TCE的代谢,使TCE的代谢产物增加。     

Importance of CYP1A1 and CYP1B1 in bioactivation of benzo[a]pyrene in human lung cell lines

Polycyclic aromatic hydrocarbons are ubiquitous environmental pollutants classified as carcinogens in humans and rodents. The cytochromes P4501A1 and 1B1 have both shown capacity to carry out bioactivation of the prototype PAH, benzo[a]pyrene (B[a]P) to its ultimate carcinogenic B[a]P-diol-epoxide-I-1 form. The part played by each enzyme in human lung cells, however, has not been clarified. To get further insight into their individual role in the metabolic activation of B[a]P, RNA-interference was used to down-regulate CYP1A1 and/or CYP1B1 gene expression in the human lung cell lines BEP2D and NCIH2009. Fluorescence-HPLC analysis revealed that formation of B[a]P-tetrol-I-1 (hydrolyzed form of the corresponding diol-epoxide) was dependent primarily on CYP1A1. In cells without down-regulation of CYP1A1, the B[a]P-tetrol-I-1 was the major tested isomer formed. In contrast, the B[a]P-cis- and trans-7,8-dihydrodiol isomers were readily formed in cells expressing high levels of either CYP-gene. Simultaneous down-regulation of CYP1A1 and CYP1B1 mRNA resulted in low levels of metabolites overall. Residual unmetabolized B[a]P levels followed the expression of CYP1A1 in an inverse manner. In conclusion, these results indicate a major role of CYP1A1 in the bioactivation of B[a]P to carcinogenic B[a]P-diol-epoxides and in overall metabolism of B[a]P in human lung cell lines. In contrast, both CYP1A1 and CYP1B1 contribute significantly to the formation of the B[a]P-cis- and trans-7,8-dihydrodiol isomers.     

Human hepatic CYP2B6 developmental expression: The impact of age and genotype

Although CYP2B6 is known to metabolize numerous pharmaceuticals and toxicants in adults, little is known regarding CYP2B6 ontogeny or its possible role in pediatric drug/toxicant metabolism. To address this knowledge gap, hepatic CYP2B6 protein levels were characterized in microsomal protein preparations isolated from a pediatric liver bank (N = 217). Donor ages ranged from 10 weeks gestation to 17 years of age with a median age of 1.9 months. CYP2B6 levels were measured by semi-quantitative western blotting. Overall, CYP2B6 expression was detected in 75% of samples. However, the percentage of samples with detectable CYP2B6 protein increased with age from 64% in fetal samples to 95% in samples from donors >10 years of age. There was a significant, but only 2-fold increase in median CYP2B6 expression after the neonatal period (birth to 30 days postnatal) although protein levels varied over 25-fold in both age groups. The median CYP2B6 level in samples over 30 postnatal days to 17 years of age (1.3 pmol/mg microsomal protein) was lower than previously reported adult levels (2.2-22 pmol/mg microsomal protein), however, this likely relates to the median age of these samples, i.e., 10.3 months. CYP2B6 expression did not vary significantly by gender. Furthermore, CYP2B6 levels did not correlate with CYP3A4, CYP3A5.1 or CYP3A7 activity, consistent with different mechanisms controlling the ontogeny and constitutive expression of these enzymes and the lack of significant induction in the pediatric samples.     

CYP1A2、CYP2D6和CYP2C19基因多态性与氯氮平及其代谢物血药浓度的相关性研究

目的：研究CYP1A2、CYP2D6和CYP2C19基因多态性与精神分裂症患者氯氮平（clozapine,CLZ）及其活性代谢物去甲氯氮平（N-desmethy clozapine,N-CLZ）血药浓度的相关性。方法：纳入156例经CLZ单药治疗1个月以上的精神分裂症患者,采集清晨服药前空腹血,采用液相色谱-串联质谱（LC-MS/MS）法测定CLZ及N-CLZ稳态谷浓度。通过Axiom基因芯片分析技术检测CYP1A2（*1C、*1F）、CYP2D6（*2、*10）、CYP2C19（*2、*3）等6个SNP位点的基因型,比较不同基因型患者CLZ及其代谢物的浓度剂量比（C/D）及代谢物与CLZ血药浓度比值（C_N-CLZ/C_CLZ）的差异。结果：CYP2D6*10基因多态性与N-CLZ C/D具有相关性（P<0.01）。CYP1A2*1C、CYP2D6（*2、*10）基因多态性与C_N-CLZ/C_CLZ具有相关性（P<0.05,P<0.01,P<0.01）。CYP1A2*1F、CYP2C19*2、CYP2C19*3基因多态性与C/D及C_N-CLZ/C_CLZ无相关性（P>0.05）。结论：CYP1A2*1C和CYP2D6（*2、*10）基因多态性对CLZ代谢存在影响,建议临床在使用CLZ治疗前检测患者CYP1A2*1C和CYP2D6（*2、*10）基因型,为CLZ个体化治疗提供参考。     

Contribution of CYP2C9, CYP2A6, and CYP2B6 to valproic acid metabolism in hepatic microsomes from individuals with the CYP2C9*1/*1 genotype.

The present study investigated the role of specific human cytochrome P450 (CYP) enzymes in the in vitro metabolism of valproic acid (VPA) by a complementary approach that used individual cDNA-expressed CYP enzymes, chemical inhibitors of specific CYP enzymes, CYP-specific inhibitory monoclonal antibodies (MAbs), individual human hepatic microsomes, and correlational analysis. cDNA-expressed CYP2C9*1, CYP2A6, and CYP2B6 were the most active catalysts of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA formation. The extent of 4-OH-VPA and 5-OH-VPA formation by CYP1A1, CYP1A2, CYP1B1, CYP2C8, CYP2C19, CYP2D6, CYP2E1, CYP4A11, CYP4F2, CYP4F3A, and CYP4F3B was only 1-8% of the levels by CYP2C9*1. CYP2A6 was the most active in catalyzing VPA 3-hydroxylation, whereas CYP1A1, CYP2B6, CYP4F2, and CYP4F3B were less active. Correlational analyses of VPA metabolism with CYP enzyme-selective activities suggested a potential role for hepatic microsomal CYP2A6 and CYP2C9. Chemical inhibition experiments with coumarin (CYP2A6 inhibitor), triethylenethiophosphoramide (CYP2B6 inhibitor), and sulfaphenazole (CYP2C9 inhibitor) and immunoinhibition experiments (including combinatorial analysis) with MAb-2A6, MAb-2B6, and MAb-2C9 indicated that the CYP2C9 inhibitors reduced the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA by 75-80% in a panel of hepatic microsomes from donors with the CYP2C9*1/*1 genotype, whereas the CYP2A6 and CYP2B6 inhibitors had a small effect. Only the CYP2A6 inhibitors reduced VPA 3-hydroxylation (by approximately 50%). The extent of inhibition correlated with the catalytic capacity of these enzymes in each microsome sample. Overall, our novel findings indicate that in human hepatic microsomes, CYP2C9*1 is the predominant catalyst in the formation of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA, whereas CYP2A6 contributes partially to 3-OH-VPA formation.     

Effect of Ginkgo biloba extract on procarcinogen-bioactivating human CYP1 enzymes: identification of isorhamnetin, kaempferol, and quercetin as potent inhibitors of CYP1B1

In the present study, we investigated the effect of Ginkgo biloba extracts and some of its individual constituents on the catalytic activity of human cytochrome P450 enzymes CYP1B1, CYP1A1, and CYP1A2. G. biloba extract of known abundance of terpene trilactones and flavonol glycosides inhibited 7-ethoxyresorufin O-dealkylation catalyzed by human recombinant CYP1B1, CYP1A1, and CYP1A2, and human liver microsomes, with apparent Ki values of 2 +/- 0.3, 5 +/- 0.5, 16 +/- 1.4, and 39 +/- 1.2 microg/ml (mean +/- SE), respectively. In each case, the mode of inhibition was of the mixed type. Bilobalide, ginkgolides A, B, C, and J, quercetin 3-O-rutinoside, kaempferol 3-O-rutinoside, and isorhamentin 3-O-rutinoside were not responsible for the inhibition of CYP1 enzymes by G. biloba extract, as determined by experiments with these individual chemicals at the levels present in the extract. In contrast, the aglycones of quercetin, kaempferol, and isorhamentin inhibited CYP1B1, CYP1A1, and CYP1A2. Among the three flavonol aglycones, isorhamentin was the most potent in inhibiting CYP1B1 (apparent Ki = 3 +/- 0.1 nM), whereas quercetin was the least potent in inhibiting CYP1A2 (apparent Ki = 418 +/- 50 nM). The mode of inhibition was competitive, noncompetitive, or mixed, depending on the enzyme and the flavonol. G. biloba extract also reduced benzo[a]pyrene hydroxylation, and the effect was greater with CYP1B1 than with CYP1A1 as the catalyst. Overall, our novel findings indicate that G. biloba extract and the flavonol aglycones isorhamnetin, kaempferol, and quercetin preferentially inhibit the in vitro catalytic activity of human CYP1B1.