环氧虫啶对苜蓿蚜的毒力及对其体内解毒酶活性的影响

为研究桥环新烟碱类化合物对苜蓿蚜的影响,以吡虫啉为对照药剂,采用带虫浸叶法测定了环氧虫啶等桥环新烟碱类化合物对苜蓿蚜的毒力及对其体内解毒酶活性的影响。结果表明:以环氧虫啶为代表的七元桥环新烟碱类化合物对苜蓿蚜具有较好的杀虫活性,其中,环氧虫啶的LC50值为3.454 mg/L,高于八元桥环新烟碱化合物。酶抑制剂顺丁烯二乙酯（DEM）和胡椒基丁醚（PBO）对环氧虫啶均具有显著的增效作用,增效比分别为4.02和3.22;而对照药剂吡虫啉仅PBO对其具有明显增效作用。与空白对照组相比,经LC50浓度环氧虫啶和吡虫啉处理后,存活苜蓿蚜体内谷胱甘肽S-转移酶（GSTs）和细胞色素P450s活力均显著升高（P<0.05）,其中,环氧虫啶处理组GSTs和P450s活力分别达到（2.730±0.012）和（0.239±0.009）μmol/（mg pro.·min）,诱导能力弱于吡虫啉;而苜蓿蚜体内羧酸酯酶（CarE）活性则无明显变化。研究显示,在苜蓿蚜对环氧虫啶的解毒代谢过程中,GSTs和细胞色素P450s可能发挥着主要作用。     

稻稗HJHL-715种群对五氟磺草胺的抗药性水平及抗性机理分析

为明确东北稻区稻稗Echinochloa oryzoides HJHL-715种群对五氟磺草胺的抗性水平及抗性机制,采用整株生物测定法测定稻稗种群对五氟磺草胺的敏感性,明确抗性种群的交互抗性和多抗性情况,研究3种细胞色素P450抑制剂对其敏感性的影响;并应用分子生物学方法进行稻稗的乙酰乳酸合酶(acetolactate synthase,ALS)离体活性测定、ALS基因序列分析及其表达量测定。结果表明:在东北稻区,五氟磺草胺对稻稗HJHL-715种群鲜重的抑制中剂量GR50为62.53 g/hm^2;稻稗HJHL-715的ALS基因序列中未发现氨基酸突变,其ALS离体活性与敏感种群的ALS离体活性无显著性差异,ALS基因表达量显著低于敏感种群。1-氨基苯并三唑(1-aminobenzotriazole,ABT)、胡椒基丁醚(piperomyl butoxide,PBO)、马拉硫磷3种P450抑制剂显著提高了稻稗HJHL-715种群对五氟磺草胺的敏感性,使其对五氟磺草胺的GR50由原来的62.53 g/hm^2分别下降到5.78、5.02、3.53 g/hm^2。表明东北稻区已经出现了对五氟磺草胺具有高水平抗性的稻稗种群,稻稗HJHL-715种群对五氟磺草胺的抗性很可能是由细胞色素P450介导的代谢增强所致。     

小麦田大穗看麦娘对精噁唑禾草灵的抗性

为明确小麦田大穗看麦娘对精噁唑禾草灵的抗性水平及产生抗性的机理,采用整株法测定了河南省小麦田大穗看麦娘种群对精噁唑禾草灵的抗性水平,以及细胞色素P450s抑制剂胡椒基丁醚(PBO)对精噁唑禾草灵的增效作用,并通过基因测序技术研究了其靶标ACCase基因的突变位点。结果显示:与敏感种群HN-06相比,抗性种群HN-05对精噁唑禾草灵的抗性倍数为52.2,其ACCase基因存在Ile-2041-Asn和Gly-2096-Ala位点突变;喷施PBO后,精噁唑禾草灵对大穗看麦娘的GR50值(有效成分)为5.4 g/hm^2,表现出明显的增效作用,与未喷施PBO处理的差异倍数为161.3。研究表明,抗性种群HN-05对精噁唑禾草灵已产生高水平抗性,该抗性的产生可能是由于其靶标基因突变和P450s介导的代谢增强同时导致的,即表现出了靶标抗性和非靶标抗性共存的现象。     

细胞色素P450s酶系催化除草剂代谢的研究进展

综述了细胞色素P450s酶系催化的单加氧反应机理,细胞色素P450s酶系在酰胺类、三氮苯类、磺酰脲类、脲类、苯氧羧酸类等除草剂的活性或降解代谢中的催化反应。讨论了研究细胞色素P450s酶系代谢作用在除草剂选择性、抗药性机理,抗除草剂作物的培育以及除草剂安全剂的解毒机理等方面的意义。     

广东省水稻田稗对五氟磺草胺的抗性分析

为明确广东省水稻田杂草稗Echinochloa crus-galli对五氟磺草胺的抗性现状及其可能的抗性机理,采用整株剂量反应法测定不同地区稗种群对五氟磺草胺的抗性水平,对不同稗种群的乙酰乳酸合成酶（acetolactate synthase,ALS）基因片段进行扩增测序,分析细胞色素P450酶（cytochrome P450 monooxygenase,P450）和谷胱甘肽-S-转移酶（glutathione-S-transferase,GST）抑制剂胡椒基丁醚（piperonylbutoxide,PBO）和4-氯-7-硝基-2,1,3-苯并氧杂噁二唑（4-chloro-7-nitro-1,2,3-benzoxadiazole,NBD-Cl）对不同稗种群抗性水平的影响,并对替代药剂进行筛选。结果显示,广东省水稻田多数稗种群对五氟磺草胺仍表现敏感,但采自湛江市的1个种群BC-7对五氟磺草胺产生了抗性,抗性倍数达6.5倍。与敏感种群BC-2相比,BC-7种群并未发生已报道的ALS靶标抗性相关突变。PBO和NBD-Cl均可显著提高BC-7种群对五氟磺草胺的敏感性,其干重抑制中量GR₅₀由31.1 g/hm²分别降为11.0 g/hm²和24.7 g/hm²。BC-7种群对氰氟草酯和噁唑酰草胺仍较敏感,但对二氯喹啉酸和双草醚产生了抗性。表明P450和GST介导的代谢抗性是稗BC-7种群产生抗性的重要原因,氰氟草酯和噁唑酰草胺适用于治理该抗性种群。     

新烟碱杀虫剂环氧虫啶对苜蓿蚜的毒力及其在苜蓿蚜和水稻中的降解转化

为明确顺硝烯新烟碱杀虫剂环氧虫啶对苜蓿蚜Aphis craccivora Koch的毒力及其在苜蓿蚜体内和水稻中的降解转化趋势,采用带虫浸液法测定环氧虫啶及其降解产物NTN23692对苜蓿蚜的毒力,并采用超高效液相色谱检测法研究苜蓿蚜体内不同酶对环氧虫啶降解转化的影响以及环氧虫啶在水稻中降解转化为NTN23692的情况。结果表明:环氧虫啶及其降解产物NTN32692对苜蓿蚜的LC_(50)分别为3.45 mg/L和0.79 mg/L,NTN32692的毒力明显高于环氧虫啶。与对照组相比,在添加酶抑制剂的条件下,苜蓿蚜谷胱甘肽S-转移酶孵育液中环氧虫啶和NTN32692的浓度均显著降低,羧酸酯酶和细胞色素P450酶孵育液中环氧虫啶和NTN32692的浓度变化不显著。用不同浓度环氧虫啶处理水稻24 h后,叶片中环氧虫啶和NTN32692的累积量随着浓度提高均显著增加;在水稻中的残留动态表明,培养7 d后,叶片中环氧虫啶和NTN32692的浓度皆显著降低,降幅分别达到67.33%和89.42%。     

绿磺隆、醚苯磺隆及其体外代谢物对乙酰乳酸合成酶的离体抑制作用

利用异源表达于酵母细胞中的小麦细胞色素P450cDNA(CYP71C6v1)研究了磺酰脲类除草剂绿磺隆、醚苯磺隆的代谢作用。结果表明,代谢产物5-羟基绿磺隆和5-羟基醚苯磺隆能够抑制乙酰乳酸合成酶(ALS酶)活性,且代谢产物与母体化合物绿磺隆、醚苯磺隆抑制ALS酶活性的IC50值差异小,但是代谢产物在茎叶喷雾小麦和菜豆时,均未表现出活性。绿磺隆及其代谢产物抑制小麦ALS酶活性的IC50值分别为7.1×10-9和7.9×10-9mol/L,抑制菜豆ALS酶活性的IC50分别为3.6×10-9和4.1×10-9mol/L;醚苯磺隆及其代谢产物抑制小麦ALS酶活性的IC50分别为4.6×10-9和5.3×10-9mol/L,抑制菜豆ALS酶活性的IC50分别为4.7×10-9和4.9×10-9mol/L。结果表明,在磺酰脲类分子苯环5位上进行结构改造,有可能得到高活性的化合物。     

有机磷类杀虫剂代谢机制研究进展

文章对有机磷类杀虫剂代谢机制的研究进展以及昆虫对此类杀虫剂的相关代谢抗性机制进行了总结,阐述了有机磷杀虫剂的生物代谢途径及相关代谢酶系。在生物体中,有机磷类杀虫剂主要发生氧化代谢、水解代谢和轭合代谢等反应。其氧化代谢主要在细胞色素P450酶系(P450s)的催化作用下进行,其中,最重要的氧化反应是硫代有机磷酸酯类杀虫剂氧化脱硫形成生物毒性更高的有机磷氧化物的反应,以及氧化脱芳(烷)基化的反应;有机磷杀虫剂及其氧化产物在生物体内还可发生水解代谢反应,在对氧磷酶PON1等磷酸三酯酶的催化作用下,水解生成低毒性或者无毒的代谢物;有机磷杀虫剂的轭合代谢主要是在谷胱甘肽硫转移酶(GSTs)的催化下进行的。昆虫对有机磷类杀虫剂的代谢抗性与昆虫中参与此类杀虫剂代谢的解毒酶的改变密切相关,其中,与有机磷类杀虫剂代谢相关的P450s基因的过量表达和酶活性增强、丝氨酸水解酯酶的过量表达及基因突变、GSTs基因的过量表达等,均可导致铜绿蝇Lucilia cuprina、桃蚜Myzus persicae等昆虫对二嗪磷和马拉硫磷等有机磷类杀虫剂的代谢抗性。明确有机磷类杀虫剂的结构特点、代谢途径以及昆虫对此类杀虫剂的代谢抗性机制,对掌握有机磷类杀虫剂的毒理学机制,安全有效地使用此类杀虫剂,有效治理害虫对有机磷类杀虫剂的抗药性,以及开发生物选择性好的新型有机磷类杀虫剂,均具有重要意义。     

西花蓟马抗甲氨基阿维菌素苯甲酸盐种群的交互抗性与生化抗性机制

为了解西花蓟马Frankliniella occidentalis(Pergande)对甲氨基阿维菌素苯甲酸盐的抗性风险,采用生物和生化测定方法研究了西花蓟马甲维盐抗性种群与其它杀虫剂的交互抗性和生化抗性机制。西花蓟马甲维盐抗性种群对阿维菌素有高水平交互抗性,抗性倍数为31.656,对啶虫脒有中等水平交互抗性,为12.182,对吡虫啉、溴虫腈、氯氟氰菊酯、毒死蜱和灭多威有低水平交互抗性,为5.517~8.568,而对多杀菌素无明显交互抗性。增效剂胡椒基丁醚(PBO)、马来酸二乙酯(DEM)、三丁基三硫磷酸酯(DEF)和磷酸三苯酯(TPP)对甲维盐抗性种群和田间种群均有显著增效作用。甲维盐抗性种群多功能氧化酶细胞色素P₄₅₀和b₅含量、O-脱甲基酶、谷胱甘肽S-转移酶和羧酸酯酶活性均显著提高,分别为敏感种群的3.89、3.61、5.32、4.42和1.30倍,表明多功能氧化酶、谷胱甘肽S-转移酶和羧酸酯酶等解毒代谢酶活性的提高是西花蓟马对甲维盐产生抗性的重要机制。     

小菜蛾CYP6BG1基因体外真核表达及其对氯虫苯甲酰胺的代谢

小菜蛾Plutella xylostella是为害十字花科作物的世界性重要害虫,对氯虫苯甲酰胺产生了严重抗性。已经明确小菜蛾CYP6BG1的过量表达与其对氯虫苯甲酰胺的抗性相关。本文进一步通过昆虫杆状病毒表达系统在Sf9细胞中表达了小菜蛾CYP6BG1和NADPH-细胞色素P450还原酶(CPR)蛋白,并检测Sf9细胞表达CYP6BG1蛋白后对氯虫苯甲酰胺的敏感变化及氯虫苯甲酰胺经CYP6BG1蛋白代谢后对小菜蛾幼虫毒力的变化。结果显示:Sf9细胞中与CPR共表达的CYP6BG1具有7-乙氧基香豆素O-脱乙基酶活性,且细胞对氯虫苯甲酰胺的敏感性显著下降。将表达的蛋白与不同浓度的氯虫苯甲酰胺孵育24 h后,对3龄小菜蛾幼虫致死率显著低于对照组。本研究结果间接表明CYP6BG1能够降解氯虫苯甲酰胺,从而增强小菜蛾对氯虫苯甲酰胺的抗性。     

Enhancement of thiazopyr bioefficacy by inhibitors of monooxygenases

The preceding paper described inhibition of thiazopyr metabolism in plant seedlings by inhibitors of cytochrome P450 monooxygenases, and the lack of inhibition by esterase inhibitors. We now describe greenhouse evaluation of the effects of these metabolic inhibitors on the bioefficacy of thiazopyr. Inhibitors of cytochrome P450 monooxygenases, piperonyl butoxide (PBO), 1-aminobenzotriazole (ABT), metyrapone (MET) and tetcyclacis (TET), all enhanced the bioefficacy of thiazopyr against pigweed and other plant species. In contrast, inhibitors of esterases, tributyl phosphate (TBP) and triphenyl phosphate (TPP), produced only slight enhancement of thiazopyr activity. The effect of PBO was dose-dependent and was demonstrated against barnyardgrass, grain sorghum, redroot pigweed, seedling johnsongrass, and giant foxtail. PBO demonstrated no enhancement of thiazopyr activity in velvetleaf, tall morningglory, cotton, or soybeans. Bioefficacy was most enhanced via exposure of seedling shoots to PBO and thiazopyr. The combination of results from the present and the preceding papers suggests that PBO enhances thiazopyr bioefficacy by effectively inhibiting thiazopyr metabolism in plants.     

Involvement of cytochrome P‐450 enzyme activity in the selectivity and safening action of pyrazosulfuron‐ethyl

To investigate the selectivity and safening action of the sulfonylurea herbicide pyrazosulfuron‐ethyl (PSE), pyrazosulfuron‐ethyl O‐demethylase (PSEOD) activity involving oxidative metabolism by cytochrome P‐450 was studied in rice (Oryza sativa L cv Nipponbare) and Cyperus serotinus Rottb. Cytochrome P‐450‐dependent activity was demonstrated by the use of the inducers 1,8‐naphthalic anhydride and ethanol, the herbicides PSE, bensulfuron‐methyl, dimepiperate and dymron, or the inhibitor piperonyl butoxide (PBO). Growth inhibition in C serotinus seedlings was more severe than that in rice seedlings. O‐Dealkylation activities of PSE were induced differently in rice and in C serotinus, with distinctly higher activity in rice seedlings. The induced PSEOD activities were slightly inhibited by PBO in rice seedlings, whereas they were strongly inhibited in C serotinus seedlings. Dimepiperate and dymron were effective safeners of rice against PSE treatment. Treatments with herbicide alone resulted in less induction of PSEOD activity compared with combined treatments of the herbicide and safener. PSEOD activity in rice seedlings induced with herbicide alone was strongly inhibited by PBO, whereas it was weakly inhibited in rice seedlings induced with combinations of PSE and two safeners. These results suggest that O‐demethylation by cytochrome P‐450 enzymes may be involved in the metabolism of PSE and may contribute to its selectivity and safening action. Furthermore, these results suggest the existence of a multiple form of cytochrome P‐450 in plants. © 2001 Society of Chemical Industry     

Piperonyl butoxide-mediated inhibition of cytochrome P450-catalysed insecticide metabolism: a rational approach

The inhibitory effect of piperonyl butoxide (PBO) on cytochrome P450 was studied by theoretical methods. Binding conformations of PBO were obtained by the recently developed low-mode conformational search within the active site of cytochrome P450_cam. Increased activity of PBO relative to other methylendioxyphenyl inhibitors was rationalized by its decreased conformational mobility and the steric block created by its long side-chain in the substrate access channel of the enzyme. © 1999 Society of Chemical Industry     

Effect of monooxygenase inhibitors on bentazon uptake and metabolism in maize cell suspension cultures

Maize “black Mexican sweet” (BMS) cell suspension cultures were used to study the effects of various cytochrome P450 monooxygenase inhibitors on the uptake and metabolism of the herbicide bentazon. Maize cells rapidly absorbed bentazon and metabolized it via aryl hydroxylation and glycosylation to a glycosyl conjugate of 6-hydroxybentazon. BMS cells accumulated bentazon to levels approximately 20-fold greater than those in the external medium. When BMS cells were incubated in an external medium containing 25 μM bentazon, the formation of the glycosyl conjugate (ca. 2 nmol/min/g fresh wt) was rate-limited by aryl hydroxylation. Tetcyclacis, a plant growth retardant, phenylhydrazine, a mechanism-based cytochrome P450 inhibitor, and piperonyl butoxide, an insecticide synergist, inhibited bentazon metabolism with I₅₀ values of approximately 0.1, 1.0, and 1.0 μM, respectively. Other mechanism-based cytochrome P450 inhibitors, 3(2,4-dichlorophenoxy)-1-propyne and aminobenzotriazole, also inhibited bentazon metabolism but were less effective. The results obtained with selected inhibitors are consistent with the hypothesis that aryl hydroxylation of bentazon is catalyzed by a cytochrome P450 monooxygenase.     

Inhibition of thiazopyr metabolism in plant seedlings by inhibitors of monooxygenases

Metabolism of the herbicide thiazopyr [methyl 2-(difluoromethyl)-5-(4, 5-dihydroO-lhiazo!vt)-4-(2-methylpropy!)-6-(trinuorornethyl)-3-pyridinecarboxy-late] was examined in young seedlings of redroot pigweed, grain sorghum, sunflower, corn and soybean. As previously observed with rat liver microsomes plants predominantly metabolized thiazopyr via oxidation reactions. Sulfur and carbon atoms in the thiazoline ring were the primary sites of plant oxygenases. De-esterification was also identified as an important pathway of transformations in plants. Although similar pathways of thiazopyr metabolism were observed among plants, our data indicated species differences in rates of thiazopyr degradation. Among species examined, pigweed (Amaranthus retroflexus L.) showed the fastest metabolism. Thiazopyr metabolism in pigweed was significantly inhibited by several cytochrome P450 monooxygenase inhibitors, among which tetcyclacis (TET) and piperonyl butoxide (PBO) were the most inhibitory. Thiazopyr metabolism in pigweed was not inhibited by organophosphates, known inhibitors of esterases. The results suggest that thiazopyr metabolism in plants is predominantly mediated via plant mono-oxygenases.     

Absorption, translocation and metabolism of metamitron in Chenopodium album

BACKGROUND: In recent years, common lambsquarters (Chenopodium album L.) populations from sugar beet fields in different European countries have responded as resistant to the as‐triazinone metamitron. The populations have been found to have the same D1 point mutation as known for atrazine‐resistant biotypes (Ser₂₆₄ to Gly). However, pot experiments revealed that metamitron resistance is not as clear‐cut as observed with triazine resistance in the past. The objectives of this study were to clarify the absorption, translocation and metabolic fate of metamitron in C. album. RESULTS: Root absorption and foliar absorption experiments showed minor differences in absorption, translocation and metabolism of metamitron between the susceptible and resistant C. album populations. A rapid metabolism in the C. album populations was observed when metamitron was absorbed by the roots. The primary products of metamitron metabolism were identified as deamino‐metamitron and metamitron‐N‐glucoside. PABA, known to inhibit the deamination of metribuzin, did not alter the metabolism of metamitron, and nor did the cytochrome P450 inhibitor PBO. However, inhibition of metamitron metabolism in the presence of the cytochrome P450 inhibitor ABT was demonstrated. CONCLUSION: Metamitron metabolism in C. album may act as a basic tolerance mechanism, which can be important in circumstances favouring this degradation pathway. Copyright © 2011 Society of Chemical Industry     

Mode of action of naphthalic anhydride as a safener for the herbicide AC 263222 in maize

In hydroponic experiments, seed-dressing with the herbicide safener 1,8-naphthalic anhydride (NA), significantly enhanced the tolerance of maize, (Zea mays L., cv. Monarque) to the imidazolinone herbicide, AC 263222, (2-[4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl]-5-methylnicotinic acid). Uptake, distribution and metabolism studies where [¹⁴C]AC 263222 was applied through the roots of hydroponically grown maize plants showed that NA treatment reduced the translocation of radiolabel from root to shoot tissue and accelerated the degradation of this herbicide to a hydroxylated metabolite. Reductions in the lipophilicity and, therefore, mobility of this compound following hydroxylation may account for NA-induced retention of radiolabel in the root system. Hydroxylation of AC 263222 suggested that NA may stimulate the activity of enzymes involved in oxidative herbicide metabolism, such as the cytochrome P₄₅₀ mono-oxygenases. In agreement with this theory, the cytochrome P₄₅₀ inhibitor, 1-aminobenzotriazole (ABT), synergized AC 263222 activity and inhibited its hyroxylation in vivo. NA seed-dressing enhanced the total cytochrome P₄₅₀ and b₅ content of microsomes prepared from etiolated maize shoots. Isolated microsomes catalyzed AC 263222 hydroxylation in vitro. This activity possessed the characteristics of a cytochrome P₄₅₀ mono-oxygenase, being NADPH-dependent and susceptible to inhibition by ABT. Activity was stimulated four-fold following NA seed treatment. Differential NA enhancement of AC 263222 hydroxylase and the cytochrome P₄₅₀-dependent cinnamic acid-4-hydroxylase (CA4H) activity, suggested that separate P₄₅₀ isozymes were responsible for each activity. These results indicate that the protective effects of NA result from enhancement of AC 263222 hydroxylation and concomitant reduction in herbicide translocation. This may be attributed to the stimulation of a microsomal cytochrome P₄₅₀ system. © 1998 SCI.     

Resistance selection and biochemical characterization of spinosad resistance in Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae)

A Helicoverpa armigera population was collected from Shandong province, China. After 15 generations of selection in the laboratory, the H. armigera strain developed more than 20-fold resistance to spinosad. At LD₅₀ level, no significant cross-resistance was found between spinosad and chlorpyrifos, methomyl, avermectin and chlorfenapyr except for fenvalerate with a low cross-resistance of 2.4-fold. However, LD₉₉ values of fenvalerate against the parental and resistant strains were not different significantly. After inhibitors were used, spinosad resistance could be partially suppressed by piperonylbutoxide (PBO) and triphenylphosphate (TPP), but not by diethylmaleate (DEM). Activities of p-nitroanisole O-demethylase (ODM) developed to 8.26-fold compared with the parental strain, but no obvious changes were found in activities of carboxyl esterase (CarE) and glutathione-S-transferase (GST). The results indicated that resistance to spinosad in the cotton bollworm might be associated with an increase in cytochrome P450 monooxygenase.