Developmental Expression of Cytochrome P450 Enzymes in Human Liver

Abstract: Drug–metabolizing cytochrome P450 enzymes, the major phase I enzymes, are active in human liver already at very early stages of intrauterine development, although presumably at fairly low concentrations and in low numbers. During maturation, these enzymes go through various developmental programmes towards adulthood. The major increase both in abundance as well as in number of different enzymes takes place after birth, probably during the first year of life. Detailed information concerning these developmental changes is still limited. The major drug–metabolizing P450 enzymes appear to be primarily members of the CYP3A subfamily in all stages of development. The balance between different members of this subfamily, however, undergoes significant switches from the foetal predominant CYP3A7 to the major adult form CYP3A4. The ontogeny of the other cytochrome P450 enzymes is less well characterized, but the major switchon appears to occur mainly after birth. Developmental expression of P450 enzymes is one of the key factors determining the pharmacokinetic status of developing individuals both pre– and postnatally.     

因宁片对甲亢性肝损害模型大鼠肝脏细胞色素P450基因表达的影响

目的:研究因宁片对甲亢性肝损害模型大鼠肝脏细胞色素P450(CYP450)基因表达的影响。方法:采用L-甲状腺激素钠灌胃以复制甲亢性肝损害大鼠模型。实验分为6组,即空白对照、模型、甲亢灵和因宁片高、中、低剂量(2.4、1.2、0.6g.kg-1)组。通过实时荧光定量聚合酶联反应(PCR)方法检测因宁片对肝组织内CYP1A2、CYP2E1和CYP3A2基因表达的影响。结果:与模型组比较,因宁片高、中剂量组大鼠肝脏CYP2E1的表达显著减弱(P<0.05),CYP1A2和CYP3A2的表达无明显改变(P>0.05)。结论:因宁片能下调甲亢大鼠肝脏CYP2E1基因的表达,这可能是因宁片治疗甲亢性肝损害的分子作用机制之一。     

人细胞色素P450 1A2的异源表达及代谢活性测定

目的获得单一的具有活性的人细胞色素P450 1A2（CYP1A2）重组酶,以进一步进行该酶参与的药物代谢研究。方法以人肝组织提取的总RNA为模板,RT-PCR扩增全长的人类CYP1A2基因片段,插入转座载体pFastBac构建重组转座质粒pFastBac-CYP1A2,转化入DH10Bac大肠杆菌,获得重组杆状病毒穿梭质粒Bacmid-CYP1A2,以之转染草地夜蛾细胞（Sf9）,与细胞色素氧化还原酶（CYPOR）进行共表达。利用蛋白质印迹分析鉴定其表达,并以非那西丁为底物,利用HPLC对其进行代谢活性测定。结果蛋白质印迹分析表明人CYP1A2蛋白在Sf9细胞中成功表达,且该重组酶对非那西丁的Km值为（82.04±11.39）μmol·L^-1（n=3）,Vmax值为（1.78±0.26）nmol·min^-1·mg^-1蛋白（n=3）,表观清除率Clint值为0.02mL·min^-1·mg^-1蛋白。结论利用杆状病毒/昆虫细胞系统,可获得具代谢活性的人CYP1A2重组酶,该酶可进一步用于其他底物的I相代谢研究。     

芳香化酶细胞色素P450在子宫内膜异位症中的检测与临床意义

目的:探讨芳香化酶细胞色素P450(P450arom)在子宫内膜异位症(EM)患者子宫内膜中的表达及临床意义.方法:采用免疫组织化学方法检测63例EM患者、40例妇科良性疾病患者及30例正常对照组患者子宫内膜中P450arom蛋白的表达,并结合EM患者的临床分期等进行比较分析.结果:P450arom蛋白在正常对照组子宫内膜中无表达;在良性疾病对照组中阳性检出率为17.50%(7140),表达水平(0.22±0.16);在EM患者子宫内膜中阳性检出率为81%,表达水平(0.57±0.26),差异有统计学意义(P＜0.05).P450arom蛋白表达在增生期与分泌期差异无统计学意义(P＞0.05).P450arom蛋白表达随EM临床分期增加呈增强趋势,但差异无统计学意义(r=0.226,P＞0.05).结论:P450arom在EM患者子宫内膜中的高表达与EM的发生有关;检测P450arom蛋白在子宫内膜的表达可作为诊断EM新的参考指标.     

细胞色素P450 1B1在上皮性卵巢癌组织中的表达

目的：检测卵巢癌组织、卵巢良性上皮性肿瘤及正常卵巢组织中细胞色素P4501B1（cytochrome P4501B1，CYP1B1）的表达，为以CYP1B1为靶点进行卵巢癌治疗提供理论基础。方法：采用免疫组化SP法检测53例上皮性卵巢癌、30例卵巢良性上皮性肿瘤及19例正常卵巢组织中CYP1B1的表达。结果：53例卵巢癌中CYP1B1阳性率92．45％，明显高于卵巢良性肿瘤组织（13．33％），差异有统计学意义（P〈0．01）。正常卵巢组织CYP1B1呈阴性表达。14例同时收集的卵巢癌转移灶组织有13例CYP1B1阳性表达，阳性率为92．86％。结论：CYP1B1可作为一个抗癌药开发及逆转化疗药耐药的新靶点。     

Characterization of the Effects of Musk Ketone on Mouse Hepatic Cytochrome P450 Enzymes

Nitroaromatic musks, including musk ketone (MK; 2,6-dimethyl-3,5-dinitro-4-t-butylacetophenone),are chemicals used as perfume ingredients in household products,cosmetics, and toiletries. Musk xylene (MX; 1,3,5-trinitro-2-t-butylxylene),another nitromusk, is not genotoxic but has been reported toproduce mouse liver tumors in a chronic bioassay. In addition,MX has been shown to both induce and inhibit mouse liver cytochromeP450 2B (CYP2B) isozymes. The ability of MX to inhibit CYP2Benzyme activity is attributable to inactivation of the enzymeby a specific amine metabolite. MK is structurally similar toMX, but lacks the nitro substitution that is reduced to theinactivating amine metabolite. Therefore, we hypothesized thatMK would induce, but not inhibit, CYP2B isozymes. To test thishypothesis, and to evaluate the effects of MK on mouse livercytochrome P450 enzymes, two sets of experiments were performed.To evaluate the ability of MK to induce cytochromes P450, micewere dosed daily by oral gavage at dosages ranging from 5 to500 mg/kg MX for 7 days. This treatment resulted in a pleiotropicresponse in mouse liver, including increased liver weight, increasedtotal microsomal protein, and centrilobular hepatocellular hypertrophy.At the highest dose tested, MK caused a 28-fold increase inCYP2B enzyme activity and a small (approximately 2-fold) increasein both cytochromes P450 1A and 3A (CYP1A and CYP3A) enzymeactivities over control levels. Protein and mRNA analyses confirmedthe relative levels of induction for CYP2B, CYP1A, and CYP3A.In addition, the no-observable-effect level (NOEL) for CYP2Binduction by MK was 20 mg/kg. To evaluate the ability of MKto inhibit phenobarbitalinduced CYP2B activity, mice were given500 ppm phenobarbital (PB) in the drinking water for 5 daysto induce CYP2B isozymes, followed by a single equimolar (0.67mmol/kg) oral gavage dose of either MK (198 mg/kg) or MX (200mg/kg), and microsomes were prepared 18 h later. While MX inhibitedmore than 90% of the PB-induced CYP2B activity in the microsomes,MK caused only a small (about 20%) reduction in PB-induced CYP2Benzyme activity. These results indicate that, like MX, MK isa PB-type inducer of mouse liver CYP2B isozymes, but unlikeMX, MK does not effectively inhibit PB-induced CYP2B enzymeactivity.     

Selective Downregulation of Hepatic Cytochrome P450 Expression and Activity in a Rat Model of Inflammatory Pain

No HeadingPurpose. This study was designed to examine the effect of Freunds complete adjuvant (FCA)-induced inflammation on liver P450 expression and activities in the first 7 days that followed a single FCA injection in the rat hindpaw.Methods. Rats were humanely sacrificed at regular time points, plasma and liver samples were collected, liver mRNA extracted, and liver microsomes prepared.Results. FCA injection led to the development of an acute inflammatory response evidenced by paw edema and increased alpha-1-acid glycoprotein (AGP) and total-nitrite (NOx) plasma concentrations. Plasma IL-6 levels were significantly higher in FCA-treated rats than in controls at 8 h post-FCA. Within 24 h, these changes were accompanied by a rapid decrease in total P450 contents in FCA-treated rat liver and the selective downregulation of specific CYP isoforms, as illustrated by decreased mRNA levels (CYP2B, CYP2C11, CYP3A1, and CYP2E1), protein contents (CYP2B, CYP2C11, and CYP2E1) or catalytic activities (CYP2C6, CYP2C11, and CYP2E1). CYP3A1 mRNA levels were severely decreased by FCA administration, whereas CYP3A2 mRNA and protein levels remained unchanged.Conclusions. These early biochemical and metabolic modifications may have pharmacokinetic and pharmacodynamic consequences when hepatically cleared drugs are administered to FCA-treated rats, especially within the first 24–72 h post-FCA.     

Comparative 1-Substituted Imidazole Inhibition of Cytochrome P450 Isozyme-Selective Activities in Human and Mouse Hepatic Microsomes

Inhibition of cytochrome P450(CYP)-selective reactions in a single human and a single mouse hepatic microsome preparation by fourteen 1-substituted imidazoles provides a simultaneous ranking of reaction susceptibility to a specific imidazole and the relative inhibitory potency of the imidazoles for a given reaction. CYP3A4/5 activity was inhibited (IC₅₀ <5 μM) by the greatest number of imidazoles, followed closely by CYP2C9. Seven imidazoles exhibited IC₅₀ values for CYP3A4/5 <0.3 μM (none for CYP2C9) and were exclusively above 300 MW. Nafimidone (MW, 236) exhibited an IC₅₀ value <0.3 μM towards CYP2D6 and CYP1A2 reactions. CYP2E1 and CYP2A6 were exclusively inhibited (IC₅₀ <5 μM) by imidazoles with MWs below ～200. In general, mouse activities exhibited lower IC₅₀ values than in human microsomes.     

Expression and Regulation of Xenobiotic-Metabolizing Cytochrome P450 (CYP) Enzymes in Human Lung

Pathogenesis of lung diseases, such as lung cancer and chronic obstructive pulmonary disease, is tightly linked to exposure to environmental chemicals, most notably tobacco smoke. Many of the compounds associated with these diseases require an enzymatic activation to exert their deleterious effects on pulmonary cells. These activation reactions are mostly catalyzed by cytochrome P450 (CYP) enzymes. Interindividual differences in the in situ activation and inactivation of chemical toxicants may contribute to the risk of developing lung diseases associated with these compounds. This review summarizes in detail the expression of individual CYP forms in human pulmonary tissue and gives a view on the significance of the pulmonary expression of CYP enzymes. The localization of individual CYP enzymes in various cell types of human lung and the emerging field of regulation of human pulmonary CYP enzymes are discussed. At least CYP1A1 (in smokers), CYP1B1, CYP2B6, CYP2E1, CYP2J2, and CYP3A5 proteins are expressed in human lung, and also other CYP forms are likely to be expressed. Xenobiotic-metabolizing CYP enzymes are mostly expressed in bronchial and bronchiolar epithelium, Clara cells, type II pneumocytes, and alveolar macrophages in human lung, although individual CYP forms have different patterns of localization in pulmonary tissues. Problems in animal to human lung toxicity extrapolation and several specific aspects requiring more detailed assessment are identified.     

Regulation of Cytochrome b 5 Expression by miR-223 in Human Liver: Effects on Cytochrome P450 Activities

Purpose

Cytochrome b ₅ (b ₅) is a hemoprotein that transfers electrons to several enzymes to fulfill functions in fatty acid desaturation, methemoglobin reduction, steroidogenesis, and drug metabolism. Despite the importance of b ₅, the regulation of b ₅ expression in human liver remains largely unknown. We investigated whether microRNA (miRNA) might be involved in the regulation of human b ₅.

Methods

Twenty-four human liver specimens were used for correlation analysis. In silico analysis and luciferase assay were performed to determine whether the predicted miRNAs functionally target to b ₅. The miR-223 was overexpressed into HepG2 cells infected with adenovirus expressing human cytochrome P450.

Results

In human livers, the b ₅ protein levels were not positively correlated with the b ₅ mRNA levels, and miR-223 levels were inversely correlated with the b ₅ mRNA levels or the translational efficiencies. The luciferase assay showed that miR-223 functionally binds to the element in the 3′-untranslated region of b ₅ mRNA. The overexpression of miR-223 significantly reduced the endogenous b ₅ protein level and the mRNA stability in HepG2 cells. Moreover, the overexpression of miR-223 significantly reduced CYP3A4-catalyzed testosterone 6β-hydroxylation activity and CYP2E1-catalyzed chlorzoxazone 6-hydroxylase activity but not CYP1A2-catalyzed 7-ethoxyresorufin O-deethylase activity.

Conclusions

miR-223 down-regulates b ₅ expression in the human liver, modulating P450 activities.     

细胞色素P450酶系介导的甾体羟化反应及其在分子水平上的研究进展

细胞色素P450单加氧酶系(cytochrome P450 monooxygenases,P450s)是主要位于某些细胞的内质网上的一个氧化酶系.P450酶系介导羟化反应是微生物甾体化合物转化反应中最重要的反应.本文概述了微生物细胞色素P450酶系,并注重综述了细胞色素P450酶系基因在转录水平调控、克隆表达以及在甾体转化应用、药物代谢等分子水平方面的研究进展.     

Pharmacokinetic and Tissue Distribution Mechanism of Mouse Recombinant Heat Shock Protein 70 in Mice

No HeadingPurpose. To investigate the in vivo pharmacokinetics and uptake mechanisms of recombinant mouse heat shock protein 70 (Hsp70) by hepatocytes in mice.Methods. The tissue distribution and intrahepatic localization of Hsp70 were determined after an intravenous injection of ¹¹¹In-Hsp70 (¹¹¹In-Hsp70) into mice. Ligands of CD91 or scavenger receptors were injected prior to Hsp70 to examine the involvement of these molecules on the distribution of ¹¹¹In-Hsp70. The uptake of ¹¹¹In-Hsp70 by primary mouse hepatocytes was also examined.Results. After intravenous injection, ¹¹¹In-Hsp70 was rapidly eliminated from the circulation and taken up mainly by the liver. The hepatic uptake was significantly inhibited by preinjection of ligands for CD91 or scavenger receptors. The separation of liver-constituting cells revealed a major contribution of hepatocytes to the overall hepatic uptake of ¹¹¹In-Hsp70. The uptake of ¹¹¹In-Hsp70 by cultured hepatocytes was inhibited by a CD91 ligand or anti-CD91 anibody. In addition, after subcutaneous injection, ¹¹¹In-Hsp70 gradually disappeared from the injection site and accumulated in primary lymph nodes.Conclusions. These results indicate for the first time that intravenous Hsp70 is, at least partially, recognized by CD91 and eliminated by hepatocytes, whereas subcutaneous Hsp70 is efficiently delivered to regional lymph nodes.     

Time-Dependent Induction of Hepatic Cytochrome P450 Enzyme Activity and mRNA Expression by Bilobalide in Rats

A single dose by gavage of bilobalide (30 mg/kg) was found to produce a time-dependent induction of hepatic cytochrome P450 (CYP) enzyme activity and protein expression in rats. An RT-PCR study further showed that mRNA expression of CYP2B was maximal at 6 h. Plasma and liver bilobalide concentration in rats following administration of Ginkgo biloba extract equivalent to bilobalide of approximately 40 mg/kg showed a similar response to that exhibited by mRNA expression. These findings suggest that bilobalide markedly induced hepatic CYPs, but the induction could be mitigated due to rapid elimination from the liver.     

Cytochrome P450 and therapeutic drug monitoring with respect to clozapine

Clozapine is an atypical antipsychotic drug that is mainly used for the treatment of refractory schizophrenia. Clozapine is eliminated by oxidation in the liver, predominantly by cytochrome P4501A2 (CYP1A2). Due to the influence of inhibitors, inducers and genetic factors on CYP1A2-activity, several studies have reported a very large interindividual variability in clozapine plasma concentrations at a fixed dose. A number of methods have been published for the measurement of clozapine and metabolites in plasma. Plasma concentrations are most frequently measured by high-performance liquid chromatography. Most methods measure clozapine and the main metabolite, norclozapine, whereas two methods measure clozapine and two metabolites. Several studies suggest that a minimum effective clozapine plasma concentration of >350 μg/l must be achieved in order to ensure acceptable clinical response, whereas the upper limit of the therapeutic interval not yet has been clearly defined. The occurrence of agranulocytosis, the most serious side-effect of clozapine treatment does not seem to be dose-related and it is not possible to predict which patients are at risk of developing agranulocytosis. The risk of central nervous system side-effects seems to increase with concentrations above 1300 μg/l. Monitoring of clozapine plasma concentrations is recommended during concomitant use of other drugs that are known to interact with the oxidation of clozapine, such as carbamazepine (inducer) or fluvoxamine (inhibitor). Overall, it is concluded that therapeutic drug monitoring may be of value in the clinical management of clozapine.     

Gender-specific induction of cytochrome P450s in nonylphenol-treated FVB/NJ mice

Nonylphenol (NP) is a breakdown product of nonylphenol ethoxylates, which are used in a variety of industrial, agricultural, household cleaning, and beauty products. NP is one of the most commonly found toxicants in the United States and Europe and is considered a toxicant of concern because of its long half-life. NP is an environmental estrogen that also activates the pregnane X-receptor (PXR) and in turn induces P450s. No study to date has examined the gender-specific effects of NP on hepatic P450 expression. We provided NP at 0, 50 or 75 mg/kg/day for 7 days to male and female FVB/NJ mice and compared their P450 expression profiles. Q-PCR was performed on hepatic cDNA using primers to several CYP isoforms regulated by PXR or its relative, the constitutive androstane receptor (CAR). In female mice, NP induced Cyp2b10 and Cyp2b13, and downregulated the female-specific P450s, Cyp3a41 and Cyp3a44. In contrast, male mice treated with NP showed increased expression of Cyp2a4, Cyp2b9, and Cyp2b10. Western blots confirmed induction of Cyp2b subfamily members in both males and females. Consistent with the Q-PCR data, Western blots showed dose-dependent downregulation of Cyp3a only in females and induction of Cyp2a only in males. The overall increase in female-predominant P450s in males (Cyp2a4, 2b9) and the decrease in female-predominant P450s in females (Cyp3a41, 3a44) suggest that NP is in part feminizing the P450 profile in males and masculinizing the P450 profile in females. Testosterone hydroxylation was also altered in a gender-specific manner, as testosterone 16alpha-hydroxylase activity was only induced in NP-treated males. In contrast, NP-treated females demonstrated a greater propensity for metabolizing zoxazolamine probably due to greater Cyp2b induction in females. In conclusion, NP causes gender-specific P450 induction and therefore exposure to NP may cause distinct pharmacological and toxicological effects in males compared to females.