乙酰胆碱酯酶酶抑制法快速检测农药残留的研究进展

概述了乙酰胆碱酯酶酶抑制法快速检测农药残留技术的研究现状．指出了酶抑制法在使用中存在的问题和发展趋势。     

Susceptibilities to methamidophos and enzymatic characteristics in 18 species of pest insects and their natural enemies in crucifer vegetable crops

The susceptibilities to methamidophos as well as the kinetic and inhibitory parameter of acetylcholinesterases (AChE) and the activities of carboxyestsrases (CarE) and glutathione-S-transferases (GST) were studied in 18 species field populations of insects collected in Fuzhou, China during April and May 2000 and 2001. The insect species included five hymenopteran endoparasitoids, one hymenopteran exoparasitoid, one hymenopteran hyperparasitoid, one dipteran predator, four coleopteran predator ladybirds, six herbivorous pest insects of lepidoptera, diptera, homoptera, and coleoptera, respectively. There existed significant correlations between the susceptibility to methamidophos and the k_i values of AChE to methamidophos, dichlorvos, and carbofuran and between the k_i and V_max values of AChE among 18 species of insects. The six herbivorous pests and four ladybirds showed significantly low k_i and V_max values of AChE compared to the seven parasitoids and predator Epistrophe balteate. It was difficult to correlate the susceptibility to methamidophos or the k_i values with the K_m values of AChE, or with the activity of CarE and GST. The activities of CarE and GST varied depending on the different insect species. Significant synergisms of piperonyl butoxide (PB), triphenyl phosphate (TPP), and diethyl maleate (DEM) with methamidophos were observed in 14 pest insects and their natural enemies. Synergisms of PB were found to be the greatest. Reduced k_i values suggested that insensitive AChE might play a critical role in the tolerance to methamidophos in the 18 insect species. The detoxification enzymes, mixed-function oxidase (MFO), CarE, and GST, were believed to be involved in the tolerance to methamidophos. MFO might play the most important role, and CarE or GST might be important in the tolerance in some insect species. Different models of tolerance to methamidophos and enzymatic potential were existed in parasitoids, predators, and herbivores based on the different selection of insecticide pressure (either directly by exposing to the spray in the field, or indirectly by the insecticides penetrated into the body of host insects) as well as different ecological and biological habitats.     

Enzyme inhibition by molluscicidal component of Areca catechu and Carica papaya in the nervous tissue of vector snail Lymnaea acuminata

In the present study toxic effects of active molluscicidal component of Areca catechu and Carica papaya was studied on certain enzymes in the nervous tissue of freshwater snail Lymnaea acuminata. In in vivo and in vitro exposure of arecoline (active component of Areca catechu seed) and papain (C. papaya latex and seed) significantly inhibited the acetylcholinesterase (AChE), acid and alkaline phosphatase (ACP/ALP) activity in the nervous tissue of L. acuminata. The inhibition kinetics of these enzymes indicate that arecoline and papain caused competitive and uncompetitive inhibition of AChE, respectively, whereas arecoline caused competitive-non-competitive inhibition of ACP/ALP and papain caused non-competitive inhibition of ACP/ALP. Thus the inhibition of AChE, ACP and ALP by arecoline and papain in the nervous tissue of L. acuminata may be the cause of molluscicidal activity of A. catechu and C. papaya, respectively.     

Synergetic effect of organic solvents and paraoxon on the immobilized acetylcholinesterase

The toxicity of organophosphorus pesticides is attributed to their ability to inhibit acetylcholinesterase (AChE), which catalyses the hydrolysis of the neurotransmitting agent acetylcholine (ACh). A simple, reproducible and stable AChE-based amperometric bioelectrode was used, for measuring the enzyme activity before and after the incubation with paraoxon dissolved in organic solvents.The inhibition percentage induced by a paraoxon in organic solvent solutions increases in the following sequence: acetonitrile < water < hexane, suggesting that the paraoxon repartition between the organic solvent and the essential water for enzyme activity plays an important role in establishing the analytical and kinetic parameters of the bioelectrode. Considering the AChE denaturation, induced only by the organic solvent, as a pseudo-inhibition process, the kinetic treatment of the bioelectrode response revealed that it follows a mixed competitive/non-competitive mechanism, while the inhibition due to a non-aqueous solution of paraoxon corresponds to a non-competitive/uncompetitive mechanism. In the last case, the synergetic effect of denaturation and inhibition of enzyme was dominated by the inhibitor presence.     

Boric acid toxicity to the German cockroach, Blattella germanica: Alterations in midgut structure, and acetylcholinesterase and glutathione S-transferase activity

Oral toxicity of boric acid, an inorganic insecticide, was evaluated on German cockroach, Blattella germanica L. (Dictyoptera, Blattellidae). Newly emerged adults were exposed to various concentrations of boric acid incorporated into the diet. Results showed that treated insects exhibited toxic symptoms with a dose-dependent mortality. Histological study of midgut revealed alterations in the epithelial cells and a significant increase in the epithelium thickness. In a second series of experiments, the compound was investigated on the activities of acetylcholinesterase (AChE) and glutathione S-transferases (GSTs). Data showed that the compound induced GSTs and reduced the activity of AChE. From this experiment, it may be concluded that ingested boric acid caused death of insects perhaps ultimately by starvation via alterations of the midgut. Additionally, the compound seemed to present a neurotoxic action as evidenced by the symptoms of poisoning and the reduction in AchE activity.     

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.     

Single and simultaneous exposure of acetylcholinesterase to diazinon, chlorpyrifos and their photodegradation products

In vitro inhibition of electric eel acetylcholinesterase (AChE) by single and simultaneous exposure to organophosphorus insecticides diazinon and chlorpyrifos, and their transformation products, formed due to photoinduced degradation, was investigated. Increasing concentrations of diazinon, chlorpyrifos and their oxidation products, diazoxon and chlorpyrifos-oxon, inhibited AChE in a concentration-dependent manner. IC₅₀ (20 min) values, obtained from the inhibition curves, were (in mol/l): (5.1 ± 0.3) × 10⁻⁸, (4.3 ± 0.2) × 10⁻⁶ and (3.0 ± 0.1) × 10⁻⁸ for diazoxon, chlorpyrifos and chlorpyrifos-oxon, respectively, while maximal diazinon concentration was lower than its IC₅₀ (20 min). Calculated K_I values, in mol/l, of 7.9 × 10⁻⁷, 9.6 × 10⁻⁶ and 4.3 × 10⁻⁷ were obtained for diazoxon, chlorpyrifos and chlorpyrifos-oxon, respectively. However, 2-isopropyl-4-methyl-6-pyrimidinol (IMP) and 3,5,6-trichloro-2-pyridinol, diazinon and chlorpyrifos hydrolysis products, did not noticeably affect the enzyme activity at all investigated concentrations. Additive inhibition effect was achieved for lower concentrations of the inhibitors (diazinon/diazoxon ?1 × 10⁻⁴/1 × 10⁻⁸ mol/l i.e., chlorpyrifos/chlorpyrifos-oxon ?2 × 10⁻⁶/3 × 10⁻⁸ mol/l), while an antagonistic effect was obtained for all higher concentrations of the organophosphates. Inhibitory power of 1 × 10⁻⁴ mol/l diazinon irradiated samples can be attributed mostly to the formation of diazoxon, while the presence of non-inhibiting photodegradation product IMP did not affect diazinon and diazoxon inhibitory efficiencies.     

Oxidative stress and locomotor behaviour response as biomarkers for assessing recovery status of mosquito fish, Gambusia affinis after lethal effect of an organophosphate pesticide, monocrotophos

Recovery study was performed at regular intervals to establish the time course of 50% and 100% recovery in neurotransmitter enzyme (acetylcholinesterase, AChE, EC 3.1.1.7) and locomotor behaviour response of mosquito fish, Gambusia affinis exposed to lethal concentration (20.49 mg L⁻¹) of an organophosphorous pesticide, monocrotophos (MCP) for 96 h. In vitro AChE activity studies indicated that MCP could cause 50% inhibition (I₅₀) at 10.2 × 10⁻⁵ M. A positive correlation was observed between brain AChE activity and swimming speed during the recovery study. Also, the recovery response of the antioxidant enzymes superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6) and glutathione reductase (GR, EC 1.6.4.2) as well as lipid peroxidation (LPO) as biomarkers of oxidative stress were assessed in viscera of G. affinis. The results showed that the MCP besides its inhibitory effect on target enzyme AChE activity and induction in antioxidant enzyme activities as a characteristic of oxidative stress, which can be used as biomarkers in the pesticide contaminated aquatic streams.     

Characterization of acephate resistance in the diamondback moth Plutella xylostella

Decreased acetylcholinesterase (AChE) sensitivity and metabolic detoxification mediated by glutathione S-transferases (GSTs) were examined for their involvement in resistance to acephate in the diamondback moth, Plutella xylostella. The resistant strain showed 47.5-fold higher acephate resistance than the susceptible strain had. However, the resistant strain was only 2.3-fold more resistant to prothiofos than the susceptible strain. The resistant strain included insects having the A298S and G324A mutations in AChE1, which are reportedly involved in prothiofos resistance in P. xylostella, showing reduced AChE sensitivity to inhibition by methamidophos, suggesting that decreased AChE1 sensitivity is one factor conferring acephate resistance. However, allele frequencies at both mutation sites in the resistant strain were low (only 26%). These results suggest that other factors such as GSTs are involved in acephate resistance. Expression of GST genes available in P. xylostella to date was examined using the resistant and susceptible strains, revealing no significant correlation between the expression and resistance levels.     

烟粉虱与温室白粉虱羧酸酯酶、谷胱甘肽转移酶和乙酰胆碱酯酶性质的比较研究

对温室白粉虱 Trialeurodes vaporariorum和烟粉虱 Bemisia tabaci羧酸酯酶(Car E)、乙酰胆碱酯酶 (ACh E)和谷胱甘肽转移酶 (GSTs)的生物化学性质进行了初步研究。比较 Car E活性 ,烟粉虱显著高于温室白粉虱 ,烟粉虱和温室白粉虱 Car E对底物乙酸 -α-萘酯的 Km 分别为 0 .073mmol/ L 和 3.458mmol/ L。烟粉虱 ACh E活性分布高峰在 2～3m OD/ (min·头 )之间 ,而温室白粉虱 ACh E活性分布高峰在 10～ 15 m OD/ (min·头 )之间 ,烟粉虱 ACh E的 Km 值是温室白粉虱 Km 值的 12 .6倍。烟粉虱的 GSTs比活力为2 .218OD/ (mg pro·min) ,显著高于温室白粉虱的 GSTs比活力 0 .663OD/ (mg pro· min) ,差异达极显著水平 ,烟粉虱 GSTs对底物的亲和力比温室白粉虱高。