Metabolism of the herbicides chlorotoluron, diuron, linuron, simazine, and atrazine by CYP1A9 and CYP1C1 from Japanese eel (Anguilla japonica)

Through the use of a number of bioconversion experiments we demonstrated that P450 proteins (CYP1A9 and CYP1C1) from Japanese eel (Anguilla japonica) metabolized a number of herbicides and the drug phenacetin. We performed bioconversion experiments in which substrates were added directly to incubation medium. The resulting metabolites were extracted and analyzed by high-performance liquid chromatography. Proteins CYP1A9 and CYP1C1 metabolized 50 nmol of the drug phenacetin to yield 12.1 and 1.1 nmol of product (acetaminophen), respectively. Further incubation of CYP1A9 with 50 nmol of the herbicides chlorotoluron, diuron, linuron, simazine, or atrazine yielded 16.5, 18.5, 7.3, 1.6, or 0.8 nmol of product, respectively. CYP1C1 also metabolized linuron, diuron, and simazine yield 5.4, 4.6, or 0.7 nmol of product, respectively. Next, polyclonal antibody was isolated by immunizing with two conjugated-peptides (amino acid residues 272–290 and 294–310) of CYP1A9. This antibody did not recognize human CYP1A2 or CYP1C1. Western blotting using the antibody revealed one band in the livers of Japanese eel and tilapia (Oreochromis niloticus). Theses results suggest that CYP1A9 and CYP1C1 metabolize herbicides, and that CYP1A9 is an useful biomarker of contamination when detected with this antibody.     

Functional expression of Helicoverpa armigera CYP9A12 and CYP9A14 in Saccharomyces cerevisiae

Elevated oxidative detoxification is a major mechanism responsible for pyrethroid resistance in Helicoverpa armigera from Asia. Constitutive overexpression of CYP9A12 and CYP9A14 was associated with pyrethroid resistance in the YGF strain of H. armigera. CYP9A12 and CYP9A14 were functionally expressed in the W(R) strain of yeast (Saccharomyces cerevisiae) transformed with a plasmid shuttle vector pYES2. The cell lysates prepared from yeast transformed with CYP9A12 and CYP9A14, respectively, exhibited considerable O-demethylation activities against two model substrates p-nitroanisole (0.59 and 0.42 nmol p-nitrophenol min⁻¹ mg protein⁻¹) and methoxyresorufin (2.98 and 5.41 pmol resorufin min⁻¹ mg protein⁻¹), and clearance activity against the pyrethroid esfenvalerate (8.18 and 4.29 pmol esfenvalerate min⁻¹ mg protein⁻¹). These results provide important evidence on the role of CYP9A12 and CYP9A14 in conferring pyrethroid resistance in H. armigera, and also demonstrate that the yeast expression system can provide necessary redox environment for insect P450s to metabolize xenobiotics.     

Modulation of Mouse P450 Isoforms CYP1A2, CYP2B10, CYP2E1, and CYP3A by the Environmental Chemicals Mirex, 2,2-Bis(p-chlorophenyl)-1,1-dichloroethylene, Vinclozolin, and Flutamide

Several environmental chemicals are disruptive to the reproductive and endocrine systems of many species, including humans. Mechanisms for endocrine disruption are presently under scrutiny. Xenobiotic inducible mammalian cytochrome P450 (CYP) enzymes metabolize a variety of substrates including environmental chemicals, pesticides, and drugs. The metabolism, and thus the effect, of endogenous chemicals including steroid hormones, vitamins, etc. that are transformed by CYP enzymes can be influenced by environmental exposure to CYP-inducing chemicals. This study demonstrated that structurally diverse environmental chemicals including mirex, 2,2-Bis(p-chlorophenyl)-1,1-dichloroethylene (DDE), vinclozolin, and flutamide are capable of inducing several mouse liver CYP isozymes. As demonstrated by Western blotting, mirex induced CYP1A2, 2B10, 2E1, and 3A and vinclozolin induced 1A2 and 2B10. The only isoforms significantly induced by DDE and flutamide were 3A and 1A2, respectively. Since some of these isoforms are known to be involved in metabolism of endogenous hormones, we also studied the effects of these CYP inducers on testosterone metabolism and seminal vesicle weights. Mirex and DDE treatments had profound effects on the metabolism of testosterone, resulting in 2.5- to 3-fold more hydroxylated products than controls. Lesser, but significant, increases in specific metabolites of testosterone were also observed following treatment with vinclozolin and flutamide. Seminal vesicle weights were lower for all treatment groups except DDE. Results of this study demonstrate that, due to their CYP-inducing potential, these chemicals may significantly impact testosterone metabolism and this may be a contributing factor in their antiandrogenic effects.     

稻瘟菌CYP51蛋白表达、纯化及其与抑制剂的复合体的结晶条件筛选

为阐明稻瘟菌Magnaporthe oryzae甾醇14α-脱甲基酶（sterol 14α-demethylase，CYP51）与抑制剂的互作机制，首先通过分子生物学软件预测并分析稻瘟菌CYP51蛋白的跨膜域、二级结构以及氨基酸序列保守性，对CYP51蛋白的氮端跨膜域序列进行截除处理并以此构建蛋白表达质粒；其次对表达质粒进行原核表达，并使用亲和层析、酶切、透析和凝胶过滤层析等多种蛋白纯化手段得到目的蛋白；最后利用坐滴法对CYP51蛋白与抑制剂的复合体的结晶进行筛选。结果表明，稻瘟菌CYP51A和CYP51B蛋白均有两段跨膜域，在不破坏CYP51蛋白底物和抑制剂结合区域的前提下，将稻瘟菌CYP51A蛋白氮端1~100位氨基酸以及稻瘟菌CYP51B蛋白氮端1~110位氨基酸截除，构建蛋白表达质粒pET28a-His6-MBP-TEV-CYP51A和pET28a-MBP-TEV-CYP51B-His6。经原核表达与多种纯化方法成功获得质量佳且纯度高的稻瘟菌CYP51A和CYP51B-His6单体蛋白，并分别与氯氟醚菌唑、烯唑醇及戊唑醇等抑制剂孵育获得复合体，稻瘟菌CYP51B蛋白与烯唑醇复合体在0.1 mol/L酒石酸钾钠、0.1 mol/LTris-HCl （pH 8.5）、0.4 mol/L水合硫酸镁结晶条件下长出晶体。     

Resistance evolution in Drosophila: the case of CYP6G1

The massive use of DDT as an insecticide between 1940 and 1970 has resulted in the emergence of a resistant population of insects. One of the main metabolic mechanisms developed by resistant insects involves detoxification enzymes such as cytochrome P450s. These enzymes can metabolise the insecticide to render it less toxic and facilitate its elimination from the organism. The P450 Cyp6g1 was identified as the major factor responsible for DDT resistance in Drosophila melanogaster field populations. In this article, we review the data available for this gene since it was associated with resistance in 2002. The knowledge gained on Cyp6g1 allows a better understanding of the evolution of insecticide resistance mechanisms and highlights the major role of transposable elements in evolutionary processes. © 2016 Society of Chemical Industry     

北京、山东和湖南地区烟粉虱抗药性及CYP4v2和CYP6CX1 mRNA水平表达量分析

于2011年采集北京、山东和湖南三地的烟粉虱,进行B、Q隐种鉴定,并测定4种杀虫剂的抗药性,同时通过荧光定量PCR分析CYP4v2和CYP6CX1两个基因的mRNA水平的表达量。结果表明,北京、湖南和长沙烟粉虱均为Q隐种。抗药性监测表明,北京和湖南种群对阿维菌素敏感,山东种群抗性水平较低,而对烟碱类药剂噻虫嗪出现不同程度的抗药性,其中湖南地区烟粉虱对噻虫嗪的抗药性达到49.08倍的高抗水平,北京和山东地区也达到中抗水平。另外,这3个地区的种群对毒死蜱和联苯菊酯抗性水平都较低。通过qRT-PCR分析三地的CYP4v2和CYP6CX1基因表达量,发现相对于敏感种群CYP4v2基因在北京、山东和湖南3个地理种群中分别过量表达3.85倍、19.57倍和10.78倍,而CYP6CX1基因在北京种群中过量表达20.55倍。结果提示田间烟粉虱的细胞色素P450基因CYP4v2和CYP6CX1过量表达可能会是烟粉虱抗药性的形成机制之一。     

黏虫CYP9A134基因的克隆及其解毒功能

昆虫体内的细胞色素P450酶系统在昆虫解毒过程中发挥着重要作用。本次试验通过转录组测序获得一条新的黏虫P450基因，经国际委员会命名为CYP9A134（登录号为MT990973）。该序列全长为1801 bp，开放序列长度为1596 bp，编码531个氨基酸，分子质量为61.42 kDa，等电点为4.90。在黏虫幼虫阶段用2.5%高效氯氟氰菊酯和20%氯虫苯甲酰胺诱导时该基因表达量会有不同程度上升，最高的可分别达对照组的2.6倍和6.5倍。RNA干扰后，该基因表达量最低下降了70%，以上2种杀虫剂LD₃₀杀虫效果分别提高了13%和25%；在黏虫成虫阶段用20%氯虫苯甲酰胺LD₃₀诱导时，该基因表达量最高可达7.8倍。RNA干扰后，该基因表达量最低下降了61%，LD₃₀剂量的氯虫苯甲酰胺杀虫效果提高26%。结果表明，该基因可能在杀虫剂诱导的解毒代谢过程中发挥重要作用。     

紫茎泽兰CYP75基因cDNA片段的克隆与鉴定

根据菊科植物P450基因CYP75 B5(GenEMBL AF313489)和C YP75 B6(GenEMBL AF313488)核酸序列同源区设计引物,用RT-PCR方法从紫茎泽兰植株中获得一大小为378bp的细胞色素P450基因片段。经克隆、测序及氨基酸序列同源性比较,发现由该片段推导出的氨基酸序列与C YP75 B5和C YP75 B6的氨基酸序列分别具有79.5%和85.6%的同源性,与C YP76 B1、C YP81 B1 v1、C YP81 E1 v2同源性分别为40.8%、35.2%、35.0%,与C YP73 A家族的同源性在28.9%～31.4%;所绘制的系谱树与同源性分析结果一致。因此,初步确定该序列为C YP75家族中某一成员的结构基因片段。     

桃蛀螟细胞色素P450基因CYP4G113时空表达及功能研究

CYP家族基因是昆虫体内重要的解毒酶基因,本研究拟明确桃蛀螟体内解毒酶基因CYP4G113的基本序列特征、表达模式及其在桃蛀螟适应性进化中的功能.基于桃蛀螟转录组数据信息,克隆获得桃蛀螟CYP4G113的开放阅读框;利用实时荧光定量PCR(Quantitative real-time PCR,qRT-PCR)技术,测定...     

甘蓝夜蛾CYP9A90基因的克隆及溴氰菊酯对其诱导表达

P450 CYP9A家族基因与昆虫对拟除虫菊酯类杀虫剂的抗性相关。为证实溴氰菊酯对甘蓝夜蛾CYP9A基因的诱导效果,本研究采用RT-PCR和RACE技术克隆获得甘蓝夜蛾CYP9A基因,real-time PCR检测该基因在甘蓝夜蛾不同组织中的表达差异及溴氰菊酯处理甘蓝夜蛾5龄幼虫不同时间后该基因相对表达量变化,研究结果可为甘蓝夜蛾对溴氰菊酯的抗性治理提供依据。结果表明:克隆得到甘蓝夜蛾CYP9A基因cDNA全长序列,该序列包含1 828bp,包括1个125bp的5′非编码区,1个104bp的3′非编码区和1个1 599bp的开放阅读框,编码532个氨基酸,分子量约为61.1kDa,等电点为8.84,GenBank登录号为KR676343,被国际P450命名委员会命名为CYP9A90。Real-time PCR分析结果表明,该基因在甘蓝夜蛾5龄幼虫6个组织中表达情况不同,其中在脂肪体中表达量最高。低剂量溴氰菊酯作用后不同时间点CYP9A90mRNA总体呈现诱导表达趋势。     

Differential metabolism of sulfoximine and neonicotinoid insecticides by Drosophila melanogaster monooxygenase CYP6G1

Sulfoxaflor [N-[methyloxido[1-[6-(trifluoromethyl)-3-pyridinyl]ethyl]-λ⁴-sulfanylidene] cyanamide] is in development as the first product from the new sulfoximine class of insect control agents. Highly effective against a variety of sap-feeding pest insects, available data indicate no cross-resistance to sulfoxaflor in pest insect strains that exhibit high levels of resistance to neonicotinoids and other insecticides. In vitro studies of the cytochrome P450 monooxygenase CYP6G1 from Drosophila melanogaster, expressed in a Drosophila cell line, show very high levels of metabolism for a variety of neonicotinoids, but not for sulfoxaflor and its chloropyridine-analog. A sulfoxaflor analog with nitrogen in place of the carbon in the bridge between the pyridine and sulfoximine moiety shows a modest degree of metabolism. In silico homology modeling of the CYP6G1 with the sulfoximines and neonicotinoids suggests that steric effects may limit interactions of the sulfoximines with the reactive heme-oxo complex. A distinct relationship was identified for the summed Hückel charges and the degree of metabolism observed. These observations help explain the lack of sulfoxaflor metabolism by CYP6G1, and in turn provide a basis for the lack of cross-resistance to sulfoxaflor in insecticide resistant strains of pest insects.