Characterization of Anopheles minimus CYP6AA3 expressed in a recombinant baculovirus system

Metabolism by cytochrome P450 monooxygenases is a major mechanism implicated in resistance of insects to insecticides, including pyrethroids. We previously isolated the cytochrome P450 CYP6AA3 from deltamethrin-selected resistant strain of Anopheles minimus mosquito, a major malaria vector in Thailand. In the present study, we further investigated the role of CYP6AA3 enzyme in deltamethrin metabolism in vitro. The CYP6AA3 was expressed in Spodoptera frugiperda (Sf9) insect cells via baculovirus-mediated expression system. The enzymatic activity of CYP6AA3 in deltamethrin metabolism was characterized after being reconstituted with An. minimus NADPH-cytochrome P450 reductase and a NADPH-regenerating system. The contribution of CYP6AA3 responsible for deltamethrin metabolism was determined by measurement of deltamethrin disappearance following the incubation period and deltamethrin-derived compounds were detected using combined gas chromatography mass spectrometry analysis. 3-Phenoxybenzaldehyde was a major product of CYP6AA3-mediated deltamethrin metabolism. Deltamethrin degradation and formation of metabolites were NADPH-dependent and inhibited by piperonyl butoxide. Deltamethrin was catalyzed by CYP6AA3 with an apparent K(m) of 80.0 +/- 2.0 and V(max) of 60.2 +/- 3.6 pmol/min/pmol P450. Furthermore, deltamethrin cytotoxicity assays by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and trypan blue dye exclusion were examined in Sf9 insect cells, with and without expression of CYP6AA3. Results revealed that CYP6AA3 could play a role in detoxifying deltamethrin in the cells. Thus, the results of this study support the role of CYP6AA3 in deltamethrin metabolism.     

棉铃虫P450基因CYP9A12酵母表达产物对拟除虫菊酯的代谢作用

细胞色素P450氧化酶解毒代谢作用增强是棉铃虫Helicoverpa armigera对拟除虫菊酯类杀虫剂产生抗性的主要原因,棉铃虫细胞色素P450氧化酶基因CYP9A12的组成型过量表达与拟除虫菊酯抗性相关。为了进一步明确棉铃虫细胞色素P450氧化酶基因CYP9A12与拟除虫菊酯类杀虫剂抗性的关系,采用酿酒酵母Saccharomyces cerevisiae表达系统异源表达了CYP9A12基因,检测了该基因的酵母表达产物对溴氰菊酯、氟氯氰菊酯、甲氰菊酯和联苯菊酯4种药剂的离体代谢作用。结果表明：含有CYP9A12外源基因的重组酵母细胞裂解液对溴氰菊酯、氟氯氰菊酯和联苯菊酯的代谢率分别为8.58,5.85和3.94 pmol/min·mg protein,而没有检测到对甲氰菊酯的代谢。本研究表明了CYP9A12具有代谢多种拟除虫菊酯的能力,也为CYP9A12参与拟除虫菊酯的解毒代谢提供了直接证据。     

Influence of Cobalt and Zinc Exposure on mRNA Expression Profiles of Metallothionein and Cytocrome P450 in Rainbow Trout

The present research aims to evaluate the effects of cobalt and zinc exposure of rainbow trout (Oncorhynchus mykiss) on metallothioneins and cytocrome P450. Mature rainbow trouts were exposed to 10?mg/L CoCl(2).6H(2)O and 1?mg/L ZnSO(4).7H(2)O. After 6, 12, 24, and 48?h of treatment, expressions of muscle MT-A, MT-B, and CYP P4501A1 mRNAs were measured by means of quantitative real-time polymerase chain reaction. During the exposure experiments, no mortalities occurred. We observed that expression levels of all genes increased with exposure time. Since the organism has not learned how to completely dispose of heavy metals and tends to bioaccumulate them, our results indicate that cobalt and zinc exposure may result in accumulation of the non-eliminated metals which may lead to fish death.     

鸡细胞色素P450 1A5的原核表达与活性重组

旨在对鸡细胞色素P450 1A5(CYP1A5)蛋白进行体外功能研究,采用大肠杆菌系统进行CYP1A5的异源表达。以鸡的cDNA为模板,扩增出CYP1A5基因,将该基因的N端编码区进行修饰,并连接到pCW载体中构建His-CYP1A5,经IPTG诱导在大肠杆菌中表达。经CO-差示光谱检测,所获得的His-CYP1A5具有典型的P450吸收峰。该蛋白与细胞色素P450还原酶(CPR)进行体外重组,构成的重组酶系表现出乙氧基试卤灵-O-脱乙基酶活性。结果表明,所采用的表达策略可以成功产生出具有催化活性的鸡细胞色素P450 1A5(CYP1A5)蛋白。     

Adrenodoxin (Adx) and CYP11A1 (P450scc) induce apoptosis by the generation of reactive oxygen species in mitochondria

Mitochondrial cytochrome P450 systems are an indispensable component of mammalian steroid biosynthesis; they catalyze regio- and stereo-specific steroid hydroxylations and consist of three protein entities: adrenodoxin reductase (AdR), adrenodoxin (Adx), and a mitochondrial cytochrome P450 enzyme, e.g., CYP11A1 (P450 side chain cleavage, P450scc). It is known that the latter two are able to generate reactive oxygen species (ROS) in vitro . In this study, we investigated whether this ROS generation also occurs in vivo and, if so, whether it leads to the induction of apoptosis. We found that overexpression of either human or bovine Adx causes a significant loss of viability in 11 different cell lines. This loss of viability does not depend on the presence of the tumor suppressor protein p53. Transient overexpression of human Adx in HCT116 cells leads to ROS production, to a disruption of the mitochondrial transmembrane potential (DeltaPsi), to cytochrome c release from the mitochondria, and to caspase activation. In contrast, the effect of transient overexpression of human CYP11A1 on cell viability varies in different cell lines, with some being sensitive and others not. We conclude that mitochondrial cytochrome P450 systems are a source of mitochondrial ROS production and can play a role in the induction of mitochondrial apoptosis.     

A retinoic acid binding cytochrome P450: CYP120A1 from Synechocystis sp. PCC 6803

At least 35 cytochrome P450 (P450, CYP) or cytochrome P450-like genes have been identified in 10 cyanobacterial genomes yet none have been functionally characterized. CYP110 and CYP120 represent the two largest cyanobacterial P450 families with 16 and four members, respectively, identified to date. The Synechocystis sp. PCC 6803 CYP120A1 protein sequence shares high degrees of conservation with CYP120A2 from Trichodesmium erythraeum IMS101 and CYP120B1 and CYP120C1 from Nostoc punctiforme PCC 73102. In this communication, we report the cloning, expression, purification, and characterization of CYP120A1 from Synechocystis. Homology modeling predictions of the three-dimensional structure of CYP120A1 coupled with in silico screening for potential substrates and experimental spectroscopic analyses have identified retinoic acid as a compound binding with high affinity to this P450's catalytic site. These characterizations of Synechocystis CYP120A1 lay the initial foundations for understanding the basic role of cytochrome P450s in cyanobacteria and related organisms.     

An overexpressed cytochrome P450 CYP439A1v3 confers deltamethrin resistance in Laodelphax striatellus Fallén (Hemiptera: Delphacidae)

Deltamethrin resistance in Laodelphax striatellus had been associated with its oxidative detoxification by overexpression of four cytochrome P450 monooxygenases like CYP353D1v2, CYP6FU1, CYP6AY3v2, and CYP439A1v3. The first three P450s have been validated for insecticide‐metabolizing capability and only CYP6FU1 was found to degrade deltamethrin. In this study, an investigation was conducted to confirm the capability of CYP439A1v3 to degrade deltamethrin. The CYP439A1v3 was first expressed in Sf9 cell line and its recombinant enzyme was tested for metabolic activity against different insecticides using substrate depletion assay combined with metabolite identification. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and carbon monoxide (CO)‐difference spectra analysis showed that the intact cytochrome P450 protein was successfully expressed. Tests with probe substrates proved its enzyme activity, as p‐nitroanisole, ethoxycoumarin, and ethoxyresorufin were preferentially metabolized (specific activity 7.767 ± 1.22, 1.325 ± 0.37, and 0.355 ± 0.37 nmol/min per mg of protein, respectively) while only luciferin‐HEGE was not. In vitro incubation of the recombinant CYP439A1v3 protein with deltamethrin revealed hydroxylation by producing hydroxydeltamethrin. On the contrary, no metabolite/metabolism was seen with nonpyrethroid insecticide, including imidacloprid, buprofezin, chlorpyrifos, and fipronil. To the best of our knowledge, this is the first study to link a CYP450 from family 439 to confer pyrethroid resistance to L. striatellus. This finding should help in the design of appropriate insecticide resistance management for control of this strain of L. striatellus.     

Induction of P450 monooxygenases in the German cockroach,Blattella germanica L

The cytochrome P450 monooxygenases are an important metabolic system whose level of activity can be influenced by several dietary constituents. We examined the effects of six known P450 inducers on the levels of total cytochromes P450, cytochrome b(5), and six monooxygenase activities in adult German cockroaches. In addition, the levels of CYP6L1 and CYP9E2 mRNA were also investigated. Phenobarbital treatment resulted in increases in total cytochromes P450 and metabolism of three resorufin analogues, but not CYP6L1 nor CYP9E2 mRNA. There was no significant effect of the other five inducers on any of the monooxygenase parameters we measured. In comparison with other insects, the German cockroach seems unusually refractory to most inducing agents.     

Induction of cytochrome P450 3A by Shexiang Baoxin Pill and its main components

The expression of cytochrome P450 is regulated by both endogenous factors and xenobiotics including chemical drugs and natural medicines. Induction on cytochrome P450 can reduce the therapeutic efficacy from drugs inactivated by this enzyme system, but may increase the efficacy or lead to intoxication for prodrugs. Shexiang Baoxin Pill (SBP) is a traditional Chinese medicine widely used for the treatment of angina pectoris and myocardial infarction in China and other oriental countries. To assess the potential of SBP to alter the activity and expression of cytochrome P450 3A (CYP3A) extensively involved in drug metabolism, we investigated the enzyme-inducing effects of SBP in HepG2 cells and in rats. The results showed that treatment with SBP increased the enzyme activity, mRNA levels and protein expression of CYP3A4 in a concentration-dependent manner in HepG2 cells. Moreover, treatment with SBP enhanced the activities and mRNA expressions of CYP3A1 and CYP3A2 ex vivo in rats. Furthermore, we utilized HepG2 cell line to identify individual components in SBP as potential inducers of CYP3A4. It was found that bufalin, cinobufagin, and resibufogenin were novel CYP3A4 inducers. Among them, bufalin and cinobufagin significantly promoted the CYP3A4 enzyme activity, mRNA and protein levels, with the maximal induction challenging or exceeding that of the induction by rifampicin, indicating that they might play a critical role in CYP3A4 enzyme-inducing effects of SBP. In addition, the metabolic studies with specific inhibitors of CYP isoforms suggested that the three CYP3A4 inducers in SBP are also the substrates for the enzyme. Overall, our results show that SBP contains constituents that can potently induce CYP3A and suggest that this traditional Chinese medicine should be examined clinically for potential drug metabolic interactions.     

Reactive oxygen species alter autocrine and paracrine signaling

Cytochrome P450 (P450) 3A4 (CYP3A4) is the most abundant P450 protein in human liver and intestine and is highly inducible by a variety of drugs and other compounds. The P450 catalytic cycle is known to uncouple and release reactive oxygen species (ROS), but the effects of ROS from P450 and other enzymes in the endoplasmic reticulum have been poorly studied from the perspective of effects on cell biology. In this study, we expressed low levels of CYP3A4 in HepG2 cells, a human hepatocarcinoma cell line, and examined effects on intracellular levels of ROS and on the secretion of a variety of growth factors that are important in extracellular communication. Using the redox-sensitive dye RedoxSensor red, we demonstrate that CYP3A4 expression increases levels of ROS in viable cells. A custom ELISA microarray platform was employed to demonstrate that expression of CYP3A4 increased secretion of amphiregulin, intracellular adhesion molecule 1, matrix metalloprotease 2, platelet-derived growth factor (PDGF), and vascular endothelial growth factor, but suppressed secretion of CD14. The antioxidant N-acetylcysteine suppressed all P450-dependent changes in protein secretion except for CD14. Quantitative RT-PCR demonstrated that changes in protein secretion were consistently associated with corresponding changes in gene expression. Inhibition of the NF-κB pathway blocked P450 effects on PDGF secretion. CYP3A4 expression also altered protein secretion in human mammary epithelial cells and C10 mouse lung cells. Overall, these results suggest that increased ROS production in the endoplasmic reticulum alters the secretion of proteins that have key roles in paracrine and autocrine signaling.     

Homotropic cooperativity of monomeric cytochrome P450 3A4 in a nanoscale native bilayer environment

Mechanistic studies of mammalian cytochrome P450s are often obscured by the phase heterogeneity of solubilized preparations of membrane enzymes. The various protein-protein aggregation states of microsomes, detergent solubilized cytochrome or a family of aqueous multimeric complexes can effect measured substrate binding events as well as subsequent steps in the reaction cycle. In addition, these P450 monooxygenases are normally found in a membrane environment and the bilayer composition and dynamics can also effect these catalytic steps. Here, we describe the structural and functional characterization of a homogeneous monomeric population of cytochrome P450 3A4 (CYP 3A4) in a soluble nanoscale membrane bilayer, or Nanodisc [Nano Lett. 2 (2002) 853]. Cytochrome P450 3A4:Nanodisc assemblies were formed and purified to yield a 1:1 ratio of CYP 3A4 to Nanodisc. Solution small angle X-ray scattering was used to structurally characterize this monomeric CYP 3A4 in the membrane bilayer. The purified CYP 3A4:Nanodiscs showed a heretofore undescribed high level of homotropic cooperativity in the binding of testosterone. Soluble CYP 3A4:Nanodisc retains its known function and shows prototypic hydroxylation of testosterone when driven by hydrogen peroxide. This represents the first functional characterization of a true monomeric preparation of cytochrome P450 monooxygenase in a phospholipid bilayer and elucidates new properties of the monomeric form.     

温肾咳喘片主要成分对HepG2细胞中CYP相关基因表达的影响

观察温肾咳喘片组方中5种主要单体成分甘草酸、厚朴酚、和厚朴酚、蛇床子素和 欧前胡素对细胞色素P450(cytochrome P450, CYP) 1A2,2D6,2E1和3A4基因表达的影 响. 采用实时荧光定量PCR技术检测HepG2细胞中药物处理后各CYP mRNA的表达.厚朴酚 、和厚朴酚、蛇床子素和欧前胡素在不同浓度均能明显的诱导CYP2E1和CYP3A4,同时欧 前胡素也能诱导CYP1A2的表达,而甘草酸、厚朴酚、和厚朴酚、蛇床子素和欧前胡素在 不同浓度对CYP2D6的表达均具有较弱的抑制作用.甘草酸、厚朴酚、和厚朴酚、蛇床子 素和欧前胡素能明显影响CYP1A2、2D6、2E1或3A4的表达.此研究为中西药物代谢性相互 作用及毒理学的研究提供实验依据.     

12-O-tetradecanoylphorbol-13-acetate upregulates the Ah receptor and differentially alters CYP1B1 and CYP1A1 expression in MCF-7 breast cancer cells

Elevated expression of cytochrome P450 1B1 (CYP1B1) and estradiol 4-hydroxylation have been reported to be biomarkers of tumorigenesis in humans. The aromatic hydrocarbon receptor (AhR) regulates expression of human cytochrome P450 1A1 (CYP1A1) and CYP1B1, 17β-estradiol (E₂) 2- and 4-hydroxylases, respectively. There is also evidence that expression of estrogen receptor α (ERα) potentiates CYP1A1 inducibility in breast cancer cells. To characterize these relationships further, we examined the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), which downregulates ERα, and the high-affinity AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), on the expression of AhR, ERα, CYP1A1, and CYP1B1 in MCF-7 human breast cancer cells. Treatment with TPA, which suppressed ERα mRNA levels, caused a greater than fourfold elevation of AhR mRNA and protein levels, whereas treatment with TCDD caused a decrease in AhR protein but no change in ERα or AhR mRNA levels. In MCF-7 cells treated with TPA prior to treatment with TCDD, the AhR mRNA level was elevated, the ERα mRNA level remained suppressed, and the ratio of CYP1B1 to CYP1A1 mRNA was increased compared with treatment with TCDD alone. A corresponding increase in the ratio of the rates of 4- to 2-hydroxylation pathways of E₂ metabolism was also observed in response to pretreatment with TPA prior to the addition of TCDD. These results demonstrate differential regulation of the human CYP1A1 and CYP1B1 genes and provide a cellular model to investigate further the mechanisms that may be involved in the elevated expression of CYP1B1 in tumorigenesis. J. Cell. Biochem. 70:289–296, 1998. © 1998 Wiley-Liss, Inc.     

Development of a new genotoxicity test system with Salmonella typhimurium OY1001/1A2 expressing human CYP1A2 and NADPH-P450 reductase.

In order to develop a new tester strain detecting environmental promutagens and procarcinogens, we introduced two plasmids into Salmonella typhimurium TA1535; one contains the cDNAs of human cytochrome P450 (P450 or CYP) 1A2 and NADPH-P450 reductase and the other (pOA101) a umuC"lacZ fusion gene. The newly developed tester strain, S. typhimurium OY1001/1A2, was found to express P450 at a level of 0.15 nmol/ml in whole cell culture. Membrane fractions, when isolated from this tester strain, contained 0.04 P450 nmol/mg protein and a reductase activity of 170 nmol cytochrome c reduced/min/mg protein and were active in catalyzing CYP1A2-dependent 7-ethoxyresorufin O-deethylation and metabolic activation of heterocyclic aromatic amines to DNA-damaging products in a conventional tester S. typhimurium NM2009 strain, only when NADPH was added as a reducing equivalent. In the OA1002/1A2 strain, heterocyclic aromatic amines (e.g., IQ, MeIQ, and MeIQx) were found to be activated to reactive metabolites that cause induction of umuC gene expression in a dose-dependent manner, without addition of external NADPH. These results indicate that the newly established strain can be of use to detect mutagenic and carcinogenic potencies of environmental chemicals without addition of metabolic activation system.     

Functional characterization of medaka CYP3A38 and CYP3A40: kinetics and catalysis by expression in a recombinant baculovirus system

Phylogenic analysis of the teleost genomic lineages has demonstrated the precedent for multiple genome duplications. Among many of the genes duplicated, cytochrome P450 genes have undergone independent diversification, which can be traced to a single ancestral gene. In teleosts, cytochrome P450s, from all major families, have been identified. Among these, the CYP3A family has been cloned in several teleost species and demonstrated to contain multiple paralogs differing in gene expression patterns and tissue distribution. Herein we characterized the catalytic and kinetic activities of two medaka CYP3A paralogs (CYP3A38 and CYP3A40) with benzyloxyresorufin (BFC), a fluorescent 3A-selective substrate, and testosterone, a known metabolic substrate for CYP3A enzymes. Recombinant CYP3A was produced using the baculovirus expression vector system in Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tn5) insect cells and accounted for up to 24% of total cellular protein. Following addition of a heme-albumin conjugate to log phase cells, spectral P450 content reached a maximum of 560 and 2350 pmol/mg microsomal protein for CYP3A38 and CYP3A40, respectively. Incubations containing recombinant CYP3A, human NADPH-cytochrome P-450 oxidoreductase reductase, human cytochrome b5, and a NADPH generation system catalyzed the dealkylation of BFC and hydroxylation of testosterone with a high degree of stereoselectivity. However, efficiencies and specificities were significantly different between the two isoforms. Km and Vmax activities based on BFC-catalysis were 0.116 and 0.363 muM, and 7.95 and 7.77 nmol/min/nmol P450 for CYP3A38 and CYP3A40, respectively. CYP3A38 preferentially catalyzed testosterone hydroxylation at the 6beta-, 2beta- and 16beta-positions with minor hydroxylation at other positions within the steroid nucleus. Testosterone catalysis with CYP3A40 was limited predominantly to the 6beta- and 2beta-positions. Putative identification of CYP3A substrate recognition sites (SRS) 1-6 indicates that 12 of the 49 amino acid differences between CYP3A38 and CYP3A40 OFRs occur in SRS regions previously known to be associated with steroid hydroxylation. We suggest that differences in kinetics and catalytic activities are a result of amino acid substitutions in SRS regions 1, 3 and 5 within the CYP3A38 and CYP3A40 protein sequence.     

Characterization of mosquito CYP6P7 and CYP6AA3: differences in substrate preference and kinetic properties

Cytochrome P450 monooxygenases are involved in insecticide resistance in insects. We previously observed an increase in CYP6P7 and CYP6AA3 mRNA expression in Anopheles minimus mosquitoes during the selection for deltamethrin resistance in the laboratory. CYP6AA3 has been shown to metabolize deltamethrin, while no information is known for CYP6P7. In this study, CYP6P7 was heterologously expressed in the Spodoptera frugiperda (Sf9) insect cells via baculovirus‐mediated expression system. The expressed CYP6P7 protein was used for exploitation of its enzymatic activity against insecticides after reconstitution with the An. minimus NADPH‐cytochrome P450 reductase enzyme in vitro. The ability of CYP6P7 to metabolize pyrethroids and insecticides in the organophosphate and carbamate groups was compared with CYP6AA3. The results revealed that both CYP6P7 and CYP6AA3 proteins could metabolize permethrin, cypermethrin, and deltamethrin pyrethroid insecticides, but showed the absence of activity against bioallethrin (pyrethroid), chlorpyrifos (organophosphate), and propoxur (carbamate). CYP6P7 had limited capacity in metabolizing λ‐cyhalothrin (pyrethroid), while CYP6AA3 displayed activity toward λ‐cyhalothrin. Kinetic properties suggested that CYP6AA3 had higher efficiency in metabolizing type I than type II pyrethroids, while catalytic efficiency of CYP6P7 toward both types was not significantly different. Their kinetic parameters in insecticide metabolism and preliminary inhibition studies by test compounds in the flavonoid, furanocoumarin, and methylenedioxyphenyl groups elucidated that CYP6P7 had different enzyme properties compared with CYP6AA3. © 2011 Wiley Periodicals, Inc.     

亚致死剂量毒死蜱对飞蝗细胞色素P450酶活性及基因表达的影响

【目的】研究有机磷杀虫剂毒死蜱对飞蝗体内细胞色素P450的影响。【方法】采用酶活力测定法和实时定量PCR技术分别研究了毒死蜱3种亚致死剂量(LD_(10)、LD_(30)和LD_(50))处理飞蝗3龄幼虫24 h后,体内细胞色素P450酶活性及CYP409A1和CYP408B1基因表达量的变化。【结果】不同亚致死剂量毒死蜱处理引起细胞色素P450活性显著性降低,分别为对照组的0.68、0.50和0.62倍。同时通过mRNA水平表达的差异比较显示,飞蝗的两个P450基因CYP409A1和CYP408B1的表达受到抑制,均出现表达量减少的现象。【结论】某些细胞色素P450基因表达受不同亚致死剂量毒死蜱的抑制而使酶的量被降低,从而造成飞蝗整体细胞色素P450酶活性的下降。