In vitro and in vivo inhibition of chicken brain neurotoxic esterase by leptophos analogs

Inhibition of chicken brain neurotoxic esterase (NTE) by a series of O-halogenated-phenyl-O-alkyl phenylphosphonates was studied in vitro. The “apparent” activity was found to consist of “true” NTE (sensitive to mipafox) plus a minor mipafox-resistant component. The pI₅₀ of O-(2,6-dichlorophenyl) O-methyl phenylphosphonate for “true” NTE was 6.65, whereas it was about 3 for mipafox-resistant hydrolysis of phenyl valerate. This compound is suitable as an alternative to mipafox in the assay of “true” NTE, whereas the use of leptophos oxon gives a less accurate measure. The ethoxy analogs are about as potent in vitro as the corresponding methoxy compounds. Leptophosoxon and ethoxyleptophosoxon are more potent in vitro inhibitors than desbromoleptophosoxon. Within a like group of chlorinated phenylphosphonates, a reasonable correlation between in vitro neurotoxic esterase inhibition of the oxon and in vivo delayed neurotoxic potential by the corresponding phosphonothionate exists. In vivo inhibition of “apparent” NTE from chicken brain, studied 24 hr after an oral dose, is dose dependent for leptophos, ethoxyleptophos, and desbromoleptophos, the latter one being a very potent in vivo inhibitor. Ethoxyleptophos and leptophos have about equal in vivo esterase inhibitory properties. For desbromoleptophos and leptophos there is good agreement between the minimum dose causing delayed neurotoxicity and the dose leading to substantial inhibition of “apparent” NTE; ethoxyleptophos, on the other hand, inhibits the esterase at a dose much lower than the one which is neurotoxic. Several possible explanations for this discrepancy are considered.     

Effect of disposition on the relative delayed neurotoxicity potency among leptophos analogs

The oxons of leptophos, its desbromo analog, and its ethoxy analog all inhibit hen brain neurotoxic esterase (NTE) to a similar degree in vitro, but have large differences in minimum effective oral doses for organophosphorus-induced delayed neurotoxicity. The potencies of leptophos and ethoxyleptophos are increased 10-fold when administered to hens intravenously (iv), but the potency of desbromoleptophos is nearly the same whether administered iv or po. Leptophos distributes nearly five times more rapidly from the central compartment than does desbromoleptophos, and its attenuated oral potency may be due to slower net absorption and/or dilution by compartments other than nervous tissue. Intravenously administered phenylphosphonothionates are excreted rapidly into the gastrointestinal tract and a large proportion of the dose is eliminated in the first 48 hr. Indirect evidence indicates that ethoxyleptophos is more rapidly degraded and ethoxyleptophos-inhibited NTE recovers more rapidly than does NTE inhibited by leptophos or desbromomleptophos. It is proposed that differential aging of ethoxyleptophos chiral isomers as well as pharmacokinetic factors may contribute to the apparently anomalous behavior of these three analogs.     

Interaction of some unsubstituted phosphoramidate analogs of methamidophos (O,S-dimethyl phosphorothioamidate) with acetylcholinesterase and neuropathy target esterase of hen brain

At 37°C and pH 7.4–8.0, five higher O-alkyl analogs of methamidophos and four O-alkyl O-2,5-dichlorophenyl phosphoramidates all were more potent progressive inhibitors of hen brain AChE and neuropathy target esterase (NTE) than was methamidophos itself. For AChE, k_a increased from 7.2 × 10² to 1.0 × 10⁵ M⁻¹ min⁻¹ between methyl and n-hexyl S-methyl esters and from 9.3 × 10³ to 8.9 × 10⁵ M⁻¹ min⁻¹ between ethyl and n-hexyl dichlorophenyl analogs. For NTE, the ranges were from 16 to 7.9 × 10⁴ for S-methyl esters, and were 9.7 × 10⁴ to 7.8 × 10⁶ M⁻¹ min⁻¹ for dichlorophenyl. S-methyl esters were more active against AChE than against NTE and all the dichlorophenyl esters were more active against NTE than against AChE. Spontaneous reactivation of 75–100% activity without aging of AChE was found after 19 hr incubation at 37°C after inhibition by all nine straight-chain alkyl analogs. After inhibition by O-isopropyl S-methyl phosphorothioamidate, some spontaneous reactivation with complete aging of all remaining inhibited AChE occurred during 19 hr. No spontaneous reactivation or aging of inhibited NTE was detected. It was concluded that the molecular structures of the inhibited enzymes obtained from equivalent compounds in the two series of inhibitors were identical and that the leaving groups were, therefore, S-methyl and O-2,5-dichlorophenyl, respectively. Although hen brain NTE inhibited by methamidophos in vitro did not age, cases of delayed neuropathy in man have been reported and, presumably, require aging as well as inhibition of NTE. Possible explanations of this apparent discrepancy include (i) the fact that methamidophos consists of two chiral forms and that the form seen to be active in vitro may be disposed of preferentially in vivo, (ii) the possibility of activation in vivo to a different inhibitor, (iii) differences between conformation and ease of aging of inhibited NTE in vitro and in vivo, and (iv) species differences.     

Status of insecticide resistance in Culex pipiens field populations from north-eastern areas of Italy before the withdrawal of OP compounds

BACKGROUND: Heavy and constant use of organophosphorus (OP) larvicides selected Culex pipiens L. resistant populations through two main mechanisms of genetic resistance, the increased activity of detoxifying esterase and the production of alterate acetylcholinesterase‐1 (AChE1) by G119S mutation. The aim of this study was the assessment of the distribution of Cx. pipiens populations resistant to temephos and chlorpyrifos in the north‐eastern regions of Italy and the occurrence of the insensitive AChE in these populations. Data describe the situation in the last years before European legislation prohibited the use of OP larvicides in mosquito control, up until 2007. RESULTS: For the first time a high level of OP resistance in the samples from Ravenna (182‐fold, 80% A4/B4 or A5/B5 esterases and 38.3% Ester⁵), Emilia Romagna region, was detected; therefore, new data from the Veneto and Friuli Venezia Giulia regions were obtained and reinforced existing knowledge about resistance previously studied along the Adriatic coast. Nearby, in the Villa Verucchio locality, the highest (87.5%) AChE1R was found. CONCLUSION: Cx. pipiens resistance esterases A5/B5 and A4/B4 spread southward along the Adriatic coastal plain while OPs were being used in mosquito control, as confirmed by the first molecular screening of the AChE1 gene in these populations. Copyright © 2010 Society of Chemical Industry     

Oral administrations of methamidophos and TOCP have different effects on in vitro protein phosphorylation levels from subcellular fractions of hen brain

The effects of methamidophos and tri-o-cresyl phosphate (TOCP) on the endogenous phosphorylation of specific brain proteins were studied in Beijing white laying hens during the early stage of delayed neurotoxicity. Phosphorylation of mitochondrial and synaptosomal proteins was assayed in vitro by using [γ-³²P]ATP as phosphate donor. Tri-o-cresyl phosphate (TOCP) administration enhanced the phosphorylation of synaptosomal proteins with molecular weight of 40 and 55 kDa by as much as 36% and 65%, respectively, and decreased the phosphorylation of mitochondrial protein (35 kDa) by 33%. A single dose of methamidophos enhanced the phosphorylation of 32-kDa synaptosomal protein by 44%; however, it had no effect on brain mitochondrial proteins. The activity of neuropathy target esterase (NTE) in dosed hens’ brain and spinal cord was assayed for the effects of methamidophos and TOCP. These results showed that methamidophos inhibited brain NTE by 41% compared with that of control after 7-day exposure, while TOCP inhibited brain NTE by 66%. Moreover, NTE activity from spinal cord in treated hens also exhibited a similar trend of activity inhibition. Together, these results suggested that methamidophos and TOCP induced changes of protein phosphorylation level from hen brain and resulted in different kinds of neurotoxicity.     

The role of B‐type esterases in conferring insecticide resistance in the tobacco whitefly,Bemisia tabaci (Genn)

Separation of non‐specific esterases on electrophoretic gels has played a key role in distinguishing between races or biotypes of the tobacco whitefly, Bemisia tabaci. One intensively staining esterase in particular (termed E_0.14) has assumed significance as a diagnostic of B‐type whiteflies (aka Bemisia argentifolii), despite any knowledge of its biological function. In this study, a whitefly strain (B‐Null) homozygous for a null allele at the E_0.14 locus that had been isolated from a B‐type population was used to demonstrate a significant role for E_0.14 in resistance of B‐type populations to pyrethroids but not to organophosphates (OPs). Bioassays with pyrethroids, following pre‐treatment with sub‐lethal doses of the OP profenofos (to inhibit esterase activity), coupled with metabolism studies with radiolabelled permethrin, supported the conclusion that pyrethroid resistance in a range of B‐type strains expressing E_0.14 was primarily due to increased ester hydrolysis. In the same strains, OP resistance appeared to be predominantly conferred by a modification to the target‐site enzyme acetylcholinesterase. © 2000 Society of Chemical Industry     

上海地区菜缢管蚜对有机磷的抗药性及其生化检测

呼伦贝尔盟农田杂草计49科、198属、348种,其中包括6个亚种、29个变种和1个变型。杂草发生特点:种类多、多年生杂草多、恶性杂草多、群落结构特殊、危害严重。主要优势群落:茅香和光稃茅香群落、匍匐冰草群落、野燕麦群落与裂边鼬瓣花群落。防治措施与策略:适当压缩小麦,扩大油菜种植面积,在休闲地应用草甘膦防治多年生杂草,用2甲4氯取代2,4-D丁酯防治小麦田杂草,发展应用磺酰脲类除草剂与禾草灵混用,以兼治野燕麦与宽叶杂草。     

Acetylcholinesterase point mutations in European strains of Tetranychus urticae (Acari: Tetranychidae) resistant to organophosphates

BACKROUND: In Tetranychus urticae Koch, acetylcholinesterase insensitivity is often involved in organophosphate (OP) and carbamate (CARB) resistance. By combining toxicological, biochemical and molecular data from three reference laboratory and three OP selected strains (OP strains), the AChE1 mutations associated with resistance in T. urticae were characterised. RESULTS: The resistance ratios of the OP strains varied from 9 to 43 for pirimiphos‐methyl, from 78 to 586 for chlorpyrifos, from 8 to 333 for methomyl and from 137 to 4164 for dimethoate. The insecticide concentration needed to inhibit 50% of the AChE1 activity was, in the OP strains, at least 2.7, 55, 58 and 31 times higher for the OP pirimiphos‐methyl, chlorpyrifos oxon, paraoxon and omethoate respectively, and 87 times higher for the CARB carbaryl. By comparing the AChE1 sequence, four amino acid substitutions were detected in the OP strains: (1) F331W (Torpedo numbering) in all the three OP strains; (2) T280A found in the three OP strains but not in all clones; (3) G328A, found in two OP strains; (4) A201S found in only one OP strain. CONCLUSIONS: Four AChE1 mutations were found in resistant strains of T. urticae, and three of them, F331W, G328A and A201S, are possibly involved in resistance to OP and CARB insecticides. Among them, F331W is probably the most important and the most common in T. urticae. It can be easily detected by the diagnostic PCR‐RLFP assay developed in this study. Copyright © 2009 Society of Chemical Industry     

Variation in the sensitivity of Callosobruchus (Coleoptera: Bruchidae) acetylcholinesterase to the organophosphate insecticide malaoxon: effect of species, geographical strain and food type

BACKGROUND: Bruchid beetles, Callosobruchus species, are serious pests of economically important grain legumes; their activity in stores is often controlled by the use of synthetic insecticides. Esterases are known to be involved in insecticide resistance in insects. However, there is a dearth of information on esterase activity in the genus Callosobruchus. In this study, the effect of species, geographical strain and food type on the variation in acetylcholinesterase (AChE) activity and its inhibition by malaoxon (malathion metabolite) was investigated using an in vitro spectrophotometric method. RESULT: AChE activity varied significantly among species and strains and also among legume type used for rearing them. Generally, irrespective of species, strain or food type, the higher the AChE activity of a population, the higher is its inhibition by malaoxon. C. chinensis had the highest AChE activity of the species studied, and in the presence of malaoxon it had the lowest remaining AChE activity, while C. rhodesianus retained the highest activity. CONCLUSION: A first‐hand knowledge of AChE activity in regional Callosobruchus in line with the prevailing food types should be of utmost importance to grain legume breeders, researchers on plant materials for bruchid control and pesticide manufacturer/applicators for a robust integrated management of these bruchids. Copyright © 2012 Society of Chemical Industry     

Carbofuran occupational dermal toxicity, exposure and risk assessment

BACKGROUND: Carbofuran is a carbamate insecticide that inhibits AChE. Although toxic by ingestion in mammals, it has low dermal toxicity, with relatively few confirmed worker illnesses. This risk assessment describes its time of onset, time to peak effect and time to recovery in rats using brain AChE inhibition in acute and 21 day dermal studies; in vitro rat/human relative dermal absorption for granular (5G) and liquid (4F) formulations; occupational exposure estimates using the Pesticide Handlers' Exposure Database and Agricultural Handlers' Exposure Database (PHED/AHED). RESULTS: The point of departure for acute risk calculation (BMDL₁₀) was 6.7 mg kg^?1 day^?1 for brain AChE inhibition after 6 h exposure. In a 21 day study, the BMDL₁₀ was 6.8 mg kg^?1 day^?1, indicating reversibility. At 75 mg kg^?1 day^?1, time of onset was ?30 min and time to peak effect was 6–12 h. Rat skin had ca tenfold greater dermal absorption of carbofuran (Furadan^® 5G or 4F) than human skin. Exposure estimates for 5G in rice and 4F in ten crops had adequate margins of exposure (>100). CONCLUSION: Rat dermal carbofuran toxicity was assessed in terms of dose and time‐related inhibition of AChE. Comparative dermal absorption in rats was greater than in humans. Worker exposure estimates indicated acceptable risk for granular and liquid formulations of carbofuran. Copyright © 2011 Society of Chemical Industry     

Variability in resistance-related enzyme activities in field populations of the tarnished plant bug, Lygus lineolaris

Widespread use of Bt crops for control of lepidopterous pests has reduced insecticide use and provided the tarnished plant bug the opportunity to become a serious pest on mid-South cotton. Organophosphate insecticides have predominantly been used against plant bugs in recent years due to the reduced efficacy of other insecticides. In this study, a biochemical approach was developed to survey enzymatic levels associated with organophosphate resistance levels in field populations of the tarnished plant bug. Forty-three populations were collected from the delta areas of Arkansas, Louisiana, and Mississippi. Three esterase substrates and one substrate each of glutathione S-transferase (GST) and acetylcholinesterase (AChE) were used to determine corresponding detoxification enzyme activities in different populations. Compared to a laboratory susceptible colony, increases up to 5.29-fold for esterase, 1.96-fold for GST, and 1.97-fold for AChE activities were detected in the field populations. In addition to the survey of enzyme activities among the populations, we also examined the susceptibility of major detoxification enzymes to several inhibitors which could be used in formulations to synergize insecticide toxicity against the target pests. As much as 52-76% of esterase, 72-98% of GST, and 93% of AChE activities were inhibited in vitro. Revealing variable esterase and GST activities among field populations may lead to a better understanding of resistance mechanisms in the tarnished plant bug. This study also reports effective suppression of detoxification enzymes which may be useful in future insecticide resistance management program for the tarnished plant bug and other Heteropteran pests on Bt crops.     

Fenitrothion toxicity in triatoma infestans synergized by quercetin or thymol blue

Quercetin and thymol blue were shown to synergize the toxicity of fenitrothion to Triatoma infestans with synergistic ratios of 1.89 and 2.65 respectively. These synergistic ratios were statistically significant at P<0.05. Both compounds inhibited glutathione S-transferases (GST) in vitro, with pI₅₀ values of 6.1 and 5.1 respectively. Quercetin or thymol blue caused in-vivo GST inhibition without affecting non-specific esterase (NSE) or acetylcholinesterase (AChE) activity. Incubation of [¹⁴C]fenitrothion with T. infestans or rabbit liver GST produced desmethylfenitrothion as the major metabolite, which was specifically diminished in the presence of 0.3 mM quercetin. [¹⁴C]Fenitrothion toxicokinetics study showed a significant decrease (P<0.05) in radioactivity due to polar metabolites when insects were pre-treated with quercetin. These facts suggest that both assayed chemicals may be active in synergizing fenitrothion toxicity due to their ability to prevent the detoxification of organophosphorus insecticides by GSH conjugation. © 1999 Society of Chemical Industry     

Resistance management strategies in malaria vector mosquito control. A large-scale field trial in Southern Mexico

A resistance management programme comparing rotations, mosaics and single use of insecticides for residual house-spraying against the insect vectors of malaria is being carried out in Southern Mexico. The area was chosen because of its prior history of insecticide use, relatively sedentary vector, and physical features of the area which limit inward migration of insects to the study area. A high level of resistance to DDT and low levels of organophosphorus (OP), carbamate and pyrethroid resistance were detected by WHO discriminating-dose assays in field populations of Anopheles albimanus in the pre-spray period in the region where this resistance management project is being undertaken. After the first year of spraying, resistance, as measured by a discriminating-dose assay, was still at a high level for DDT and had risen for all the other insecticides. Biochemical assays showed that DDT resistance was primarily caused by elevated levels of glutathione S-transferase (GST) activity leading to increased rates of metabolism of DDT to DDE. The numbers of individuals with elevated GST and DDT resistance were well correlated, suggesting that this is the only major DDT resistance mechanism in this population. The carbamate resistance in this population was conferred by an altered acetylcholinesterase (AChE) mechanism. The level of resistance in bioassays correlated well with the frequency of individuals homozygous for the altered AChE allele. This suggests that the level of resistance conferred by this mechanism in its heterozygous state is below the level of detection of the bioassay. The low levels of OP and pyrethroid resistance could be conferred by either the elevated esterase or monooxygenase enzymes. The esterases, however, are elevated only with p-nitrophenyl acetate (PNPA), and are unlikely to be causing broad-spectrum OP resistance. The altered AChE mechanism may also be contributing to the OP but not the pyrethroid resistance. There were significant differences in some resistance gene frequencies for insects obtained by different indoor and outdoor trapping methods. To determine whether the different sampling methods were effectively sampling the same interbreeding population, RAPD analysis of insects obtained by different collection methods in different villages was undertaken. There was no observed variability in the RAPD patterns for the different mosquito samples with a number of primers. ©1997 SCI     

Biochemical studies on sheep body louse Bovicola ovis (Schrank) (Phthiraptera: Trichodectidae): comparison of pyrethroid and organophosphate resistant and susceptible strains

Strains of sheep louse Bovicola ovis (Schrank) with various levels of resistance to pyrethroid and one strain with high degree of resistance to organophosphate (OP) insecticides were used to investigate the biochemical mechanisms of insecticide resistance, i.e., enhanced levels of general esterases, specific acetylcholinesterases (AChE), glutathione S-transferase (GST), and mixed function oxidases. Native gel electrophoresis combined with quantitative enzyme assays showed analogous expression profiles of several esterase isozymes in all the strains tested. The determination of the sensitivity of each esterase isozyme to five inhibitors (acetylthiocholine iodide, butyrylthiocholine iodide, paraoxon eserine sulfate, and pCMB) led to the identification of nine esterases in the B. ovis strain. Gel electrophoresis results are supported by enzyme assay studies where, except for the OP resistant strain, no differences in esterase activities were detected in all the pyrethroid resistant and susceptible strains assayed. Statistical analyses demonstrated that some strains have elevated GST activities compared to the susceptible reference strain.     

Oxidation of diazinon and malathion by myeloperoxidase

The aim of the work was to investigate the in vitro oxidation of diazinon and malathion, organophosphorous pesticides (OPs) containing phosphorthioate group, catalyzed by enzyme myeloperoxidase (MPO). The oxidation was performed in the presence of hydrogen peroxide. The products were identified as oxon derivatives (phosphates), where the sulfur atom from thioate group was substituted by an oxygen atom. No hydrolysis products were detected after enzyme - induced oxidation. The oxidation efficiency was controlled using acethylcholinesterase (AChE) bioassay for determination of oxon derivatives concentration. The influence of OPs concentration, incubation time of OPs with MPO, as well as MPO concentration on the yield of oxo forms was investigated. Kinetic constants of MPO in oxidation of malathion and diazinon were estimated. The maximum concentration of oxo forms was achieved after 10 min incubation of OPs in 50 mM phosphate buffer (pH 6.0) with 100 nM MPO.     

Synergism of resistance to phosalone and comparison of kinetic properties of acetylcholinesterase from four field populations and a susceptible strain of Colorado potato beetle

The susceptibility to phosalone and biochemical characteristics of acetylcholinesterase (AChE) were compared between susceptible (SS) strain and four field populations of Colorado potato beetle (CPB) collected from commercial potato fields of Hamedan Province in west of Iran. Bioassays involving topical application of phosalone to fourth instars revealed up to 252 fold resistance in field populations compared with the SS strain. Synergism studies showed that although esterase and/or glutathione S-transferase metabolic pathways were present and active against phosalone, they were not selected for and did not have a major role in resistance. It is likely that piperonyl butoxide (PBO) reduced phosalone toxicity by inhibiting bio-activation of phosalone. The affinity (K_m) and hydrolyzing efficiency (V_max) of AChE to selected substrates, namely, acetylthiocholine iodide (ATC), propionylthiocholine iodide (PTC), and butyrylthiocholine iodide (BTC) were examined. AChE inhibition by higher substrate concentration was evident only in the SS strain. In resistant field populations, Aliabad (Aa), Bahar (B) and Dehpiaz (Dp), substrate inhibition at higher concentrations was not seen. There was no definite optimal concentration found for any of the substrates examined. When ATC, PTC, and BTC were used as substrate, the reaction rates of AChE from Yengijeh (Yg) population increased as the concentration of all three substrates were increased, but were almost constant at concentration of ATC ? 3.98, PTC ? 2.8, and BTC ? 5 mM. The susceptible form of AChE had the most efficient ATC hydrolysis but very low BTC hydrolysis activity. In contrast, AChEs from field populations elicited relatively reduced ATC hydrolysis, but relatively increased BTC hydrolysis. The in vitro inhibition potency of some organophosphates (OPs), on AChEs of the field populations and SS strain was determined. The rank order from the most potent inhibitor to the least as determined by their bimolecular reaction constants (K_i) was ethyl paraoxon > diazoxon > methyl paraoxon for AChE from Aa, B, Dp, and Yg populations, respectively, whereas the rank order for the susceptible strain was methyl paraoxon > ethyl paraoxon > diazoxon.     

Organophosphorus poisoning: A comparative study of the toxicity of chlorfenvinphos [2-chloro-1- (2′, 4′, -dichlorophenyl) vinyl diethyl phosphate] to the pigeon,the pheasant and the Japanese Quail

The acute oral toxicity of chlorfenvinphos [2-chloro-1-(2′, 4′-dichlorophenyl)vinyl diethyl phosphate] was measured in pigeon (Columba livia), pheasant (Phasianus colchicus), and quail (Coturnix coturnix japonica) and the compound was shown to be particularly toxic to pigeons. Additionally, all three species were fed chlorfenvinphos at 100 ppm in their diet for two or four weeks and esterase measurements were made by conventional and electrophoretic methods on extracts of liver, kidney and brain. The conventionally measured esterase inhibition correlated well with the acute oral toxicity figures. A more detailed study of the histochemically stained electrophoregams showed some discrepancies compared with conventional methods but offered a possible explanation of the inter-species toxicity difference in that it revealed differential inhibition of some brain iso-esterases in pigeon but not in pheasant or quail.     

Oxidation of chlorpyrifos, azinphos-methyl and phorate by myeloperoxidase

The present paper deals with the investigations of optimal conditions for the myeloperoxidase (MPO) mediated oxidation of chlorpyrifos, azinphos-methyl and phorate, organophosphorous pesticides (OPs) containing phosphorothionate group, from thio- to oxo-forms, in the presence of hydrogen peroxide. The aim of the work was to apply this oxidation method in the AChE based bioanalytical tests for OPs determination. The maximum concentration of oxo-forms for all tested pesticides was achieved after 10 min incubation of OPs in 50 mM phosphate buffer (pH 6.0) with 100 nM MPO in the presence of 50 μM H₂O₂. Optimal temperature for obtaining maximal concentration of oxo-forms was 37 °C. Only the parent compounds and their oxo-forms were identified chromatographically in the OPs samples after their exposure to MPO. Moreover, no hydrolysis products were detected in the time interval of 1 h after the MPO catalyzed reaction was stopped by catalase. The efficiency of OPs transformation from thio- to oxo-forms was measured using acethylcholinesterase (AChE) test, by comparison of percent of AChE inhibition before and after exposure to the oxidized sample.     

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。     

Fipronil metabolism,oxidative sulfone formation and toxicity among organophosphate‐ and carbamate‐resistant and susceptible western corn rootworm populations

Fipronil toxicity and metabolism were studied in two insecticide‐resistant, and one susceptible western corn rootworm (Diabrotica virgifera virgifera, LeConte) populations. Toxicity was evaluated by exposure to surface residues and by topical application. Surface residue bioassays indicated no differences in fipronil susceptibility among the three populations. Topical bioassays were used to study the relative toxicity of fipronil, fipronil + the mono‐oxygenase inhibitor piperonyl butoxide, and fipronil's oxidative sulfone metabolite in two populations (one resistant with elevated mono‐oxygenase activity). Fipronil and fipronil‐sulfone exhibited similar toxicity and application of piperonyl butoxide prior to fipronil resulted in marginal effects on toxicity. Metabolism of [¹⁴C]fipronil was evaluated in vivo and in vitro in the three rootworm populations. In vivo studies indicated the dominant pathway in all populations to be formation of the oxidative sulfone metabolite. Much lower quantities of polar metabolites were also identified. In vitro studies were performed using sub‐cellular protein fractions (microsomal and cytosolic), and glutathione‐agarose purified glutathione‐S‐transferase. Oxidative sulfone formation occurred almost exclusively in in vitro microsomal reactions and was increased in the resistant populations. Highly polar metabolites were formed exclusively in in vitro cytosolic reactions. In vitro reactions performed with purified, cytosolic glutathione‐S‐transferase (MW = 27 kDa) did not result in sulfone formation, although three additional polar metabolites not initially detectable in crude cytosolic reactions were detected. Metabolism results indicate both cytochromes P450 and glutathione‐S‐transferases are important to fipronil metabolism in the western corn rootworm and that toxic sulfone formation by P450 does not affect net toxicity. © 2000 Society of Chemical Industry