The stimulatory role of human cytochrome b5 in the bioactivation activities of human CYP1A2, 2A6 and 2E1: a new cell expression system to study cytochrome P450 mediated biotransformation

Cytochrome b₅ (b₅) is increasingly recognized to be of importancefor specific cytochrome P450 (CYP) activities. We developedhuman b₅/CYP-competent mutagenicity tester bacteria to studythe role of b₅ in the bioactivation activity of human CYP. Thesenew tester bacteria were derived from the previously engineeredhuman CYP-competent Escherichia coli K12 tester strain MTC,containing a bi-plasmid system for the co-expression of a specificCYP form (CYP1A2, 2A6 or 2E1) with human b₅, and human NADPHcytochrome P450 reductase (RED), resulting in the strain BTC-b₅-1A2,BTC-b₅-2A6 and BTC-b₅-2E1, respectively. The relative contentof b₅ with CYP and RED in these three BTC-b₅-CYP strains demonstratedphysiologically relevant co-expression levels and typical CYP-specificactivities could be determined with their specific chemicalprobes. These strains were applied in mutagenicity assays alongwith their corresponding b₅-void strains to determine the effectof b₅ on the CYP1A2-, CYP2A6- and CYP2E1-mediated bioactivationof several promutagens. For CYP1A2, of the 5 compounds tested[2-aminoanthracene (2AA), 1-aminopyrene, 6-aminochrysene, 2-amino-3-methylimidazo(4,5-f)quinolineand 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)], onlythe mutagenicity of 2AA was slightly increased (     

Molecular interactions between human cytochrome P450 1A2 and flavone derivatives

Activation by human cytochrome P450 1A2 (hCYP1A2) of heterocyclic amines is assumed to trigger of a number of carcinogenic processes. In this work, a group of natural inhibitors of human cytochrome P450 1A2 reported in literature has been theoretically analysed. These consist of flavone hydroxylated derivatives, natural compounds that exist in plants and associated products. Different theoretical/computational tools were used to describe the specific molecular interactions between these compounds and hCYP1A2. Based on this analysis, a method is proposed for helping the selection of specific molecular features that enhance protein-inhibitor interaction.     

广州地区汉族人群细胞色素P450 1A1和细胞色素P450 2E1基因多态性

目的 探讨广州地区汉族人群细胞色素Ｐ４５０ １Ａ１（ＣＹＰ１Ａ１）和细胞色素Ｐ４５０ ２Ｅ１（ＣＹＰ２Ｅ１）基因的多态性分布规律。方法 用ＰＣＲ－ＲＦＬＰ和等位基因特异性扩增技术,对１５０名广州地区汉族正常人的ＣＹＰ１Ａ１和ＣＹＰ２Ｅ１基因多态性进行了检测,并与其他人群进行了比较。结果 ＣＹＰ１Ａ１基因３’端非翻译区的Ｍｓｐ１多态位点ｍ１（ＭＳＰⅠ－）、ｍ２（ＭｓｐⅠ＋）等位基因频率分别为６２．３     

New specific marker of cytochrome P450 1A2 activity

Various methods were proposed for phenotyping of patients by activity of cytochrome P450 1A2, each has some advantages and disadvantages. However, no reference parameters were developed for measuring CYP1A2 activity that could be used as a unified standard for phenotyping of patients. We propose a mathematic model of caffeine metabolism allowing calculation of rate constants for the formation of its primary metabolites. First-order rate constant of paraxanthine formation was tested as a new specific marker of isoenzyme 1A2 in healthy volunteers.     

Recombinant human P450 forms 1A1, 1A2, and 1B1 catalyze the bioactivation of heterocyclic amine mutagens in Escherichia coli lacZ strains

Three recombinant human P450 enzymes, forms 1A1, 1A2, and 1B1, were coexpressed with NADPH-cytochrome P450 reductase in an E. coli lacZ strain suitable for detection of the mutagenicity of heterocyclic and aromatic amines. The resulting strains expressed the recombinant P450 holoenzymes at high levels. MeIQ (2-amino-3,4-dimethylimidazo[4,5-f]quinoline) was activated effectively by P450 1A2, weakly by P450 1A1, and not detectably by P450 1B1. MeIQx (2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline) and Trp-P-2 (3-amino-1-methyl-5H-pyrido[4,3-b]indole) were activated by all three enzymes, with form 1A2 the most effective. These strains facilitate analysis of the substrate specificity of human P450 forms that participate in the metabolic activation of carcinogens.     

MX100, a new Escherichia coli tester strain for use in genotoxicity studies

The development of a new Escherichia coli tester strain foruse in metabolic and mechanistic studies of genotoxins, strainMR2101/pKR11, has recently been reported. This strain, a derivativeof the E.coli K12 laboratory strain AB1157, has sensitivitytowards the detection of basesubstitution mutagenesis, monitoredby the reversion of arginine auxotrophy [argE3, (ochre)]. Besidesarginine, MR2101/pKR11 is auxotrophic for histidine (hisG4),leucine (leuB6), proline (     

乙醇对肝细胞色素P-450氧化酶2E1的诱导

目的: 研究摄入乙醇对肝脏CYP450 2E1的诱导。方法: 18例不同程度酒精性脂肪性肝炎患者, 其中12例酗酒持续至肝活检术前1-3日内, 6例则已戒酒3周至7个月; 4例病因不明脂肪性肝炎患者, 但无酗酒史。采用免疫组化方法对肝组织切片进行CYP450 2E1染色及程度评估。结果: 近日仍酗酒的患者中肝组织切片CYP450 2E1的免疫组化染色显著强于已戒酒、无酗酒者。结论: 持续摄入乙醇能显著诱导CYP450 2E1, 但戒酒后乙醇的诱导作用可以随之减弱、消失。     

The stimulatory role of human cytochrome b5 in the bioactivation activities of human CYP1A2, 2A6 and 2E1: a new cell expression system to study cytochrome P450-mediated biotransformation (a corrigendum report on Duarte et al. (2005) Mutagenesis 20, 93-100)

This corrigendum report describes the study of the comparison of human cytochrome b(5) (b(5)) with rat b(5) when coupled with human cytochrome P450 CYP1A2, 2A6 or 2E1. Results indicate a role of the N-terminal part of b(5) in the coupling with CYP. Indeed, the plasmid pLCM-b(5)-RED used in our former study on b(5) [Duarte et al. (2005) Mutagenesis, 20(2), 193-100] erroneously contained rat b(5). Plasmid pLCM-b(5)-RED was corrected with human b(5) and subsequently all experimental work was repeated as was described for the rat b(5) plasmid. Although absolute values of contents and activities were lower, all key-findings as found for rat b(5) could be confirmed using human b(5). The physiological relevant co-expression of the members of the cytochrome P450 complex, CYP, NADPH-cytochrome P450 oxidoreductase (RED) and human b(5) could be demonstrated in the different BTC strains, as was found before. The stimulatory effect of human b(5) on the activity of CYP1A2, CYP2A6 and CYP2E1 was in general similar, when compared with rat b(5), though less quantitatively pronounced. This was both the case when using membrane preparations as well as by the bioactivation of procarcinogens using the bacterial mutagenicity assay. Human b(5) stimulated the bioactivation of all compounds as described for rat b(5), except for CYP1A2 mediated bioactivation of 2-aminoanthracene (2AA), which was not stimulated by human b(5). All other main findings of the effect of rat b(5) were confirmed with human b(5), i.e. for CYP2A6: N-nitrosodiethylamine (NNdEA): approximately 14-fold increase ( approximately 23-fold with rat b(5)) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK): approximately 3-fold ( approximately 9-fold with rat b(5)); for CYP2E1: NNdEA: approximately 1.5-fold increase ( approximately 3-fold with rat b(5)); NNK: no mutagenicity with or without human b(5). Both CYP2A6 and CYP2E1 demonstrated total dependence on the presence of human b(5) for N-nitrosodi-n-propylamine (NNdPA) mutagenicity, as was shown before with rat b(5).     

Involvement of rat cytochrome 1A1 in the biotransformation of kaempferol to quercetin: relevance to the genotoxicity of kaempferol

Kaempferol is a flavonoid widely distributed in edible plantsand has been shown to be genotoxic to V79 cells in the absenceof external metabolizing systems. The presence of an externalmetabolizing system, such as rat liver homogenates (S9 mix),leads to an increase in its genotoxicity, which is attributedto its biotransformation to the more genotoxic flavonoid quercetin,via the cytochrome P450 (CYP) mono-oxygenase system. In thepresent work we investigated the mechanisms of the genotoxicityof kaempferol further. Special attention has been given to therole of CYP in the genotoxicity of this flavonoid. We studiedthe induction of mutations in Salmonella typhimurium TA98 inthe presence and in the absence of S9 mix and the inductionof chromosomal aberrations (CAs) and micronuclei (MN) by kaempferolin V79 cells in the presence and in the absence of S9 mix. Toevaluate the role of different CYP in the biotransformationof kaempferol we studied the induction of CAs and MN in V79cells genetically engineered for the expression of rat CYP 1A1,1A2 and 2B1. In addition we performed CYP inhibition studiesusing the above-mentioned indicators and high performance liquidchromatography (HPLC) analysis. The results obtained in thiswork suggest that rat CYP 1A1 is, among the cytochromes studied,the one that plays the major role in the transformation of kaempferolinto quercetin. The relevance of these findings to the humansituation is discussed. ⁴To whom correspondence should be addressed     

Expression of Cytochrome P450nor in Escherichia coli , Purification and Preparation of Polyclonal Antibodies

The term “cytochrome P450” was characterized of aunique 450 nm optical absorption peak of its carbon mon oxide bound form. Cytochrome P450s constitute a super gene family of heme containing proteins that are involvedin the metabolism of a variety o…     

Immunohistochemical localization of cytochrome P450 2E1 in human pulmonary carcinoma and normal bronchial tissue

Cytochrome P450 2E1 (CYP2E1) is a major xenobiotic-metabolizing enzyme but data concerning its extrahepatic expression are few. CYP2E1 can metabolically activate many procarcinogens and therefore its presence in the lung might play a role in bioactivation of procarcinogens, so we studied the expression and localization of CYP2E1 in primary pulmonary carcinomas and surrounding normal bronchial tissue from 28 patients. Seromucous glands showed expression of CYP2E1 in 19 and bronchial epithelium in 18 of the 28 samples of normal bronchial tissue. Thirteen of the corresponding cases of primary pulmonary carcinoma showed staining for CYP2E1. In 11 of these 13 cases, CYP2E1 was also present in normal bronchial tissue. There was no statistically significant difference in the expression of CYP2E1 between adenocarcinomas and squamous cell carcinomas. No association was observed between the expression of CYP2E1 in tumour tissue and normal bronchial tissue. However, there was a significant correlation between the expression of CYP2E1 in seromucous glands and bronchial epithelium (r=0.61, P<0.01) of normal tissue. We conclude that CYP2E1 can be present in both normal and neoplastic bronchial tissue.     

Towards a physiological role for cytochrome P450 aromatase in ejaculated human sperm

BACKGROUND: Advances in the definition of the function and the mechanism of estrogen action in different tissues have come from human and animal models of estrogen insufficiency. Recently we have demonstrated that aromatase is present and biologically active in human ejaculated sperm, suggesting that autonomous estradiol sperm production may influence sperm functions. In the present study we investigate a possible physiological role for enzymatically active P450 aromatase in human ejaculated sperm. METHODS AND RESULTS: To confirm the presence of mRNA coding for P450 aromatase, total RNA isolated from human sperm underwent RT-PCR and then Southern blot analysis. In non-capacitating medium, we observed that only estradiol and aromatizable steroids were able to increase sperm motility/migration; concomitantly they enhanced protein tyrosine phosphorylation and increased p-44/42 extracellular signal-regulated kinase activity. When we tested acrosin activity, it emerged that estradiol and aromatizable androgens were also able to induce the acrosome reaction evaluated by two different cytological staining techniques (triple-stain and fluorescein isothiocyanate-Pisum sativum agglutinin). All these events were enhanced by the 2'-O-dibutyryladenosine-3',5'-cyclic monophosphate and inhibited in the presence of the specific aromatase inhibitor, letrozole. CONCLUSIONS: From this study, it appears that a link exists between the locally produced estradiol (from ejaculated sperm), sperm capacitation and the acrosome reaction. The induction of both events by aromatizable androgens in the absence of exogenous mediators suggests that estrogen biosynthesis in ejaculated sperm is a process that may influence the intrinsic sperm fertilizing capability.     

Use of genetically engineered Salmonella typhimurium OY1002/1A2 strain coexpressing human cytochrome P450 1A2 and NADPH-cytochrome P450 reductase and bacterial O-acetyltransferase in SOS/umu assay

The major pathway of bioactivation of procarcinogenic heterocyclic aromatic amines (HCAs) is cytochrome P450 1A2 (CYP1A2)-catalyzed N-hydroxylation and subsequent esterification by O-acetyltransferase (O-AT). We have previously reported that an umu tester strain, Salmonella typhimurium OY1001/1A2, endogenously coexpressing human CYP1A2 and NADPH-P450 reductase (reductase), is able to detect the genotoxicity of some aromatic amines [Aryal et al., 1999, Mutat Res 442:113-120]. To further enhance the sensitivity of the strain toward HCAs, we developed S. typhimurium OY1002/1A2 by introducing pCW"/1A2:hNPR (a bicistronic construct coexpressing human P450 1A2 and the reductase) and pOA102 (constructed by subcloning the Salmonella O-AT gene in the pOA101-expressing umuC"lacZ gene) in S. typhimurium TA1535. In addition, as an O-AT-deficient strain, we developed the OY1003/1A2 strain by introducing pCW"/1A2:hNPR and pOA101 into O-AT-deficient S. typhimurium TA1535/1,8-DNP. Strains OY1001/1A2, OY1002/1A2, and OY1003/1A2 expressed, respectively, about 150, 120, and 140 nmol CYP1A2/l culture (in whole cells), and respective cytosolic preparations acetylated 15, 125, and > or = 0 nmol isoniazid/min/mg protein as the O-AT activities of cytosolic preparations, respectively. We compared the induction of umuC gene expression as a measure of genotoxicity and observed that the OY1002/1A2 strain was more sensitive than OY1001/1A2 strain toward the genotoxicity of 2-amino-1,4-dimethylimidazo[4,5-f]quinol ine(MeIQ), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ),2-amino-3, 8-dimethylimidazo[4,5-f]quinoxaline (MeIQx),2-aminoanthracene, 2-amino-6-methyldipyrido[1,2-a::3,2'-d]i midazole,3-amino-1, 4-dimethyl-5H-pyrido[4,3-b]indole, and 3-amino-1-methyl-5H-pyrido[4, 3-a]indole. However, the genotoxicity of MeIQ, IQ, and MeIQx was not detected with the OY1003/1A2 strain. These results indicate that the newly developed strain OY1002/1A2 can be employed in detecting potential genotoxic aromatic amines requiring bioactivation by CYP1A2 and O-acetyltransferase.     

Immunohistochemical study of cytochrome P450 2C and 3A in human non-neoplastic and neoplastic tissues

Organ and cellular distribution and expression constancy of microsomal cytochrome P450 (CYP) 2C and 3A in humans were studied with new polyclonal antibodies to CYP2C (MP-1) and 3A (NF-2) active in formalin-fixed, paraffin-embedded tissues. Antibodies were raised against purified human CYP2C9 and CYP3A4. On western blotting, MP-1 reacted with 2C8, 2C9, 2C18 and 2C19, and NF-2 with 3A4. In both frozen and paraffin sections, hepatocytes showed diffuse immunoreactivity with MP-1 and centrilobular staining with NF-2. In-paraffin sections of 40 kinds of nonneoplastic tissues, epithelium of the small and large intestine, bile duct, nasal mucosa, kidney and adrenal cortex stained positively with both MP-1 and NF-2 antibodies. Epithelium of gastric fundic glands, salivary glands, tracheobronchial glands, Brunner's glands, the prostate, uterine cervix and nasopharynx showed definite reactivity with MP-1. Epithelium of the gastric mucosa with intestinal metaplasia, duodenum, gallbladder and intercalated ducts of the pancreas and chief cells of the parathyroid and the corpus luteum of the ovary reacted with NF-2. Among the neoplastic tissues, MP-1 reacted with pleomorphic adenoma of the salivary gland and carcinomas of six different organs, and NF-2 with those of 7 different organs. These results indicate that CYP2C and CYP3A are distributed widely and organ specifically, as well as being variably expressed in neoplastic and normal states.     

Lymphocyte cytochrome P450 expression: inducibility studies in male Wistar rats

The cytochrome P450 system plays a key role in the metabolism of endogenous and exogenous compounds. The system is distributed widely in body tissues, with the highest concentration of the enzymes found in liver hepatocytes. Extrahepatic expression of the P450 system has been documented in the lung, pancreas and kidney, and the enzymes are induced by many disease states, including diabetes mellitus and cancer. Little attention has been paid to the expression and inducibility of the system in peripheral blood lymphocytes. In this study, specific P450 inducers are administered in vivo to male Wistar rats. The expression and in vivo induction of the P450 isoforms CYP2B, CYP2E, CYP3A and CYP4A in liver and lymphocyte samples is determined using Western blot analysis. Following in vivo induction, the lymphocyte P450 proteins showed an average three-fold increase in expression (0.003-0.005 microg P450/microg microsomal protein), compared to the control lymphocyte samples. Expression in the induced lymphocyte samples was up to 11-fold lower than that in the induced liver samples, as expected. These results indicate that lymphocytes may provide a relatively simple method by which to monitor the P450 profile in human subjects.     

细胞色素P450 1A1在自然流产患者胎盘绒毛组织中的表达及意义

目的　探讨细胞色素P4 5 0 1A1(CYP1A1)mRNA在自然流产患者胎盘绒毛组织中的表达及其意义。方法　采用半定量逆转录聚合酶链反应 (RT -PCR)法检测 4 5例自然流产患者 (研究组 )中及 30例正常妊娠孕妇 (对照组 )胎盘绒毛组织中CYP1A1mRNA的表达。结果　自然流产及正常妊娠胎盘绒毛组织中CYP1A1mRNA相对表达强度分别为 0 .5 6± 0 .2 0、0 .35± 0 .14、两组比较差异显著 (P <0 .0 1)。自然流产患者中孕期有被动吸烟及孕期有饮茶或咖啡习惯者的胎盘绒毛组织中CYP1A1mRNA表达最强。结论　胎盘绒毛组织中CYP1A1mRNA表达增强可能与自然流产的发生相关 ,同时可在一定程度上说明孕妇吸烟、被动吸烟及摄入过多咖啡因可通过增强胎盘绒毛组织中CYP1A1的转录水平而增加自然流产的风险。     

Transcriptional activation of cytochrome P450 1A1 with α-tocopherol

Peroral administration of α-tocopherol in a daily dose of 150 mg/kg for 1, 4, 8, and 12 days leads to induction of cytochromes P450 1A in male rats. Activity of CYP1A1 and CYP1A2 increased most significantly one day after α-tocopherol administration (by 2.6 and 2.7 times, respectively). CYP1A1 was immunohistochemically detected in rat liver microsomes during this period. The content ofCYP1A1 mRNA significantly increased in the liver. The amount ofCYP1A2 mRNA and regulatory proteins for signal activation of CYP1A1 (AhR andArnt) remained unchanged after treatment with α-tocopherol. Translated fromByulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 138, No. 9, pp. 264–267, September, 2004