Cellular metabolism and health impacts of dichlorvos: Occurrence, detection, prevention, and remedial strategies-A review.

Dichlorvos (2,2-Dichlorovinyl dimethyl phosphate, [DDVP]) belongs to the class of organophosphates and is widely used as an insecticide in agriculture farming and post-harvest storage units. Extensive research has been conducted to assess the factors responsible for the presence of DDVP in terrestrial and aquatic ecosystems, as well as the entire food chain. Numerous studies have demonstrated the presence of DDVP metabolites in the food chain and their toxicity to mammals. These studies emphasize that both immediate and chronic exposure to DDVP can disrupt the host's homeostasis, leading to multi-organ damage. Furthermore, as a potent carcinogen, DDVP can harm aquatic systems. Therefore, understanding the contamination of DDVP and its toxicological effects on both plants and mammals is vital for minimizing potential risks and enhancing safety in the future. This review aimed to comprehensively consolidate information about the distribution, ecological effects, and health impacts of DDVP, as well as its metabolism, detection, prevention, and remediation strategies. In summary, this study observes the distribution of DDVP contaminations in vegetables and fruits, resulting in significant toxicity to humans. Although several detection and bioremediation strategies are emerging, the improper application of DDVP and the alarming level of DDVP contamination in foods lead to human toxicity that requires attention.

Dichlorvos-induced formation of isopeptide crosslinks between proteins in SH-SY5Y cells.

Chlorpyrifos oxon catalyzes the crosslinking of proteins via an isopeptide bond between lysine and glutamic acid or aspartic acid in studies with purified proteins. Our goal was to determine the crosslinking activity of the organophosphorus pesticide, dichlorvos. We developed a protocol for examining crosslinks in a complex protein mixture consisting of human SH-SY5Y cells exposed to 10 µM dichlorvos. The steps in our protocol included immunopurification of crosslinked peptides by binding to anti-isopeptide antibody 81D1C2, stringent washing of the immobilized complex, release of bound peptides from Protein G agarose with 50% acetonitrile 1% formic acid, liquid chromatography tandem mass spectrometry on an Orbitrap Fusion Lumos mass spectrometer, Protein Prospector searches of mass spectrometry data, and manual evaluation of candidate crosslinked dipeptides. We report a low quantity of dichlorvos-induced KD and KE crosslinked proteins in human SH-SY5Y cells exposed to dichlorvos. Cells not treated with dichlorvos had no detectable KD and KE crosslinked proteins. Proteins in the crosslink were low abundance proteins. In conclusion, we provide a protocol for testing complex protein mixtures for the presence of crosslinked proteins. Our protocol could be useful for testing the association between neurodegenerative disease and exposure to organophosphorus pesticides.

The pathway of 2,2-dichlorovinyl dimethyl phosphate (DDVP) degradation by Trichoderma atroviride strain T23 and characterization of a paraoxonase-like enzyme.

Dichlorvos (DDVP) is widely applied in the agricultural industry, and its residues are considered hazardous to the environment. Microbial bioremediation is an innovative technology with the potential to mitigate such pollution. Trichoderma atroviride strain T23, a filamentous fungus, is very efficient at degrading DDVP. Therefore, we used DDVP as a model organophosphate pesticide to study the mechanism by which Trichoderma degrades organophosphate pesticides, with the aim of attaining a global understanding of the molecular mechanism of enzymatic degradation of organophosphate pesticides by beneficial fungi. DDVP can be biodegraded via two routes, and the primary one involves hydrolysis of the P-O bond, which can result in the production of the novel degradation intermediate trichloroethanol. TaPon1-like showed continuously high expression during 120 h, and deletion of the gene decreased the efficiency of P-O bond hydrolysis. The enzyme produced by TaPon1-like had a low Km for DDVP (0.23 mM) and a high kcat (204.3 s-1). The enzyme was able to hydrolyze broad substrates such as organophosphate oxons and lactone and maintain stable activity in a wide range of pH and temperature values. The TaPon1-like hydrolase played an important role in the first step of DDVP degradation by strain T23 and contributed to a comprehensive understanding of the mechanism of organophosphate pesticide degradation.

Inhibition effects of pesticides on glutathione-S-transferase enzyme activity of Van Lake fish liver.

Glutathione-S-transferases (GSTs) have a function in xenobiotic metabolism. They are a significant multifunctional family with a wide variety of catalytic activities. In the current study, we determined in vitro inhibition effects of 2,4-dichlorophenoxyacetic acid dimethylamine salt (2,4-D DMA), haloxyfop-P-methyl, glyphosate isopropylamine, dichlorvos, and λ-cyhalothrin on purified GST. For this purpose, GST were purified from Van Lake fish (Chalcalburnus tarichii Pallas) liver with 29.25 EU mg-1 specific activity and 10.76% yield using GSH-agarose affinity chromatographic method. The pesticides were tested at various concentrations on in vitro GST activity. Ki constants were calculated as 0.17 ± 0.01, 0.25 ± 0.05, 3.72 ± 0.32, 0.42 ± 0.06, and 0.025 ± 0.004 mM, for 2,4-D DMA, haloxyfop-P-methyl, glyphosate isopropylamine, dichlorvos, and λ-cyhalothrin, respectively. λ-Cyhalothrin showed a better inhibitory effect compared to the other pesticides. The inhibition mechanisms of λ-cyhalothrin were competitive, while the other pesticides were noncompetitive.

Micropollutants removal and health risk reduction in a water reclamation and ecological reuse system.

As reclaimed water use is increasing, its safety attracts growing attention, particularly with respect to the health risks associated with the wide range of micropollutants found in the reclaimed water. In this study, sophisticated analysis was conducted for water samples from a water reclamation and ecological reuse system where domestic wastewater was treated using an anaerobic-anoxic-oxic unit followed by a membrane bioreactor (A2O-MBR), and the reclaimed water was used for replenishing a landscape lake. A total of 58 organic micropollutants were detected in the system, consisting of 13 polycyclic aromatic hydrocarbons (PAHs), 16 phenols, 3 pesticides, and 26 pharmaceuticals and personal care products (PPCPs). After treatment by the A2O-MBR process, effective removal of pesticides and phenols was achieved, while when the reclaimed water entered the landscape lake, PPCPs were further removed. From the physicochemical properties of micropollutants, it could be inferred that phenols and dichlorphos (the only pesticide with considerable concentration in the influent) would have been mainly removed by biodegradation and/or volatilization in the biological treatment process. Additionally, it is probable that sludge adsorption also contributed to the removal of dichlorphos. For the predominant PPCP removal in the landscape lake, various actions, such as adsorption, biodegradation, photolysis, and ecologically mediated processes (via aquatic plants and animals), would have played significant roles. However, according to their logKoc, logKow and logD (pH = 8) values, it could be concluded that adsorption by suspended solids might be an important action. Although carcinogenic and non-carcinogenic risks associated with all the detected micropollutants were at negligible levels, the hazard quotients (HQs) of PPCPs accounted for 92.03%-97.23% of the HQTotal. With the significant removal of PPCPs through the ecological processes in the landscape lake, the safety of reclaimed water use could be improved. Therefore, the introduction of ecological unit into the water reclamation and reuse system could be an effective measure for health risk reduction posed by micropollutants.

Feeding inhibition in Corbicula fluminea (O.F. Muller, 1774) as an effect criterion to pollutant exposure: Perspectives for ecotoxicity screening and refinement of chemical control.

Bivalves are commonly used in biomonitoring programs to track pollutants. Several features, including its filter-feeding abilities, cumulatively argue in favour of the use of the Asian clam (Corbicula fluminea) as a biosentinel and an ecotoxicological model. Filtration in bivalves is very sensitive to external stimuli and its control is dictated by regulation of the opening/closure of the valves, which may be used as an avoidance defence against contaminants. Here, we investigate the filter-feeding behaviour of the Asian clam as an endpoint for assessing exposure to pollutants, driven by two complementary goals: (i) to generate relevant and sensitive toxicological information based on the ability of C. fluminea to clear an algal suspension, using the invasive species as a surrogate for native bivalves; (ii) to gain insight on the potential of exploring this integrative response in the refinement of chemical control methods for this pest. Clearance rates and proportion of algae removed were measured using a simple and reproducible protocol. Despite some variation across individuals and size classes, 50-90% of food particles were generally removed within 60-120 min by clams larger than 20 mm. Removal of algae was sensitive to an array of model contaminants with biocide potential, including fertilizers, pesticides, metals and salts: eight out of nine tested substances were detected at the µg l-1 or mg l-1 range and triggered valve closure, decreasing filter-feeding in a concentration-dependent manner. For most toxicants, a good agreement between mortality (96 h - LC50 within the range 0.4-5500 mg l-1) and feeding (2 h - IC50 within the range 0.005-2317 mg l-1) was observed, demonstrating that a 120-min assay can be used as a protective surrogate of acute toxicity. However, copper sulphate was very strongly avoided by the clams (IC50 = 5.3 µg l-1); on the contrary, dichlorvos (an organophosphate insecticide) did not cause feeding depression, either by being undetected by the clams' chemosensors and/or by interfering with the valve closure mechanism. Such an assay has a large potential as a simple screening tool for industry, environmental agencies and managers. The ability of dichlorvos to bypass the Asian clam's avoidance strategy puts it in the spotlight as a potential agent to be used alone or combined with others in eradication programs of this biofouler in closed or semi-closed industrial settings.

Hepatic and branchial xenobiotic biomarker responses in Solea spp. from several NW Mediterranean fishing grounds.

The common sole, Solea solea and the Senegalese sole, Solea senegalensis are two important commercial benthic species that coexist in the NW Mediterranean Sea. Several common biomarkers of chemical exposure were measured in two organs (liver and gills) involved in a different degree in biotransformation and detoxification processes. These parameters were: phase I cytochrome P450 CYP1A-dependent ethoxyresorufin O-deethylase and carboxylesterase activities, phase II glutathione S-transferase activity and the enzymatic antioxidants: catalase, glutathione reductase and glutathione peroxidase. Principal Component Analysis (PCA) considering biometric variables (size and weight) and all liver and gill biomarkers discriminated at a certain extent individuals of both species collected at the different fishing grounds. Esterase inhibition by the organophosphorus pesticides dichlorvos and diazinon was also compared in vitro in muscle, liver and gill of the two species revealing a differential sensitivity. The use of benthic sole in pollution monitoring of Southern Europe is discussed as local sentinel in respect to other benthic fish from more Northern latitudes.

Molecular dynamics simulations of acylpeptide hydrolase bound to chlorpyrifosmethyl oxon and dichlorvos.

Acylpeptide hydrolases (APHs) catalyze the removal of N-acylated amino acids from blocked peptides. Like other prolyloligopeptidase (POP) family members, APHs are believed to be important targets for drug design. To date, the binding pose of organophosphorus (OP) compounds of APH, as well as the different OP compounds binding and inducing conformational changes in two domains, namely, α/ß hydrolase and ß-propeller, remain poorly understood. We report a computational study of APH bound to chlorpyrifosmethyl oxon and dichlorvos. In our docking study, Val471 and Gly368 are important residues for chlorpyrifosmethyl oxon and dichlorvos binding. Molecular dynamics simulations were also performed to explore the conformational changes between the chlorpyrifosmethyl oxon and dichlorvos bound to APH, which indicated that the structural feature of chlorpyrifosmethyl oxon binding in APH permitted partial opening of the ß-propeller fold and allowed the chlorpyrifosmethyl oxon to easily enter the catalytic site. These results may facilitate the design of APH-targeting drugs with improved efficacy.

Combined in silico and in vivo studies shed insights into the acute acetylcholinesterase response in rat and human brain.

Combined in vivo and in silico studies were undertaken to gain insights into the change in mammalian brain acetylcholinesterase (AChE) activity under acute toxicity conditions in response to two representatives of organophosphates (OPs)--dichlorvos (DCV) and dimethoate (DM). In vivo experiments elucidated that DCV, at multiple sublethal doses for acute time periods, markedly reduced (10-25%) AChE activity, whereas with DM intoxication, a decrease in enzyme activity appeared to be lower, that is, (2-15%), in contrast to respective normal control (100%). Furthermore, a significant inhibition (P < 0.01) in the brain esterase activity was recorded for positive control animals treated with an alkylating agent-cyclophosphamide, with spontaneous reactivation at later time periods. In vivo results were further substantiated with in silico molecular docking analysis using "Autodock 4.2." The lowest binding energy obtained through the computational study strongly augment that DCV binds to brain AChE with greater affinity compared with DM with reference to ∆G and Ki values. Thus, the animal biochemical assay and computational assessment suggest that DM is better to be used over DCV. The precautionary antidote for exposed humans can be developed prior to dealing with OPs. The study will aid in efficacious and safe clinical use of the above-mentioned compounds.

Lab-on-a-drop: biocompatible fluorescent nanoprobes of gold nanoclusters for label-free evaluation of phosphorylation-induced inhibition of acetylcholinesterase activity towards the ultrasensitive detection of pesticide residues.

A simple, sensitive, selective, and "lab-on-a-drop"-based fluorimetric protocol has been proposed using biocompatible fluorescent nanoprobes of gold nanoclusters (AuNCs) for the label-free evaluation of the catalytic activity and phosphorylation of acetylcholinesterase (AChE) under physiologically simulated environments. Protein-stabilized AuNCs were prepared and mixed with acetylthiocholine (ATC) serving as "a drop" of fluorimetric reaction substrate. The AChE-catalyzed hydrolysis of ATC releases thiocholine to cause the aggregation of the AuNCs towards a dramatic decrease in fluorescence intensities, which could be curbed by the phosphorylation-induced inhibition of AChE activity when exposed to organophosphorus compounds (OPs). The reaction procedures and conditions of AChE catalysis and phosphorylation were monitored by fluorimetric measurements and electron microscopy imaging. Moreover, a selective and ultrasensitive fluorimetric assay has been tailored for the detection of pesticide residues using dimethyl-dichloro-vinyl phosphate (DDVP) as an example. Investigation results indicate that the specific catalysis and irreversible OP-induced phosphorylation of AChE, in combination with sensitive fluorimetric outputs could facilitate the detection of total free OPs with high selectivity and sensitivity. A linear concentration of DDVP ranging from 0.032 nM to 20 nM could be obtained with a detection limit of 13.67 pM. Particularly, pesticide residues of DDVP in vegetable samples were quantified down to ~36 pM. Such a label-free "lab-on-a drop"-based fluorimetry may promise wide applications for the evaluation of the physiological catalytic activity of various enzymes (i.e., cholinesterase), and especially for monitoring the direct phosphorylation biomarkers of free OPs towards rapid and early warning, and accurate diagnosis of OP exposure.

Detection of dichlorvos adducts in a hepatocyte cell line.

The toxicity of dichlorvos (DDVP), an organophosphate (OP) pesticide, classically results from modification of the serine in the active sites of cholinesterases. However, DDVP also forms adducts on unrelated targets such as transferrin and albumin, suggesting that DDVP could cause perturbations in cellular processes by modifying noncholinesterase targets. Here we identify novel DDVP-modified targets in lysed human hepatocyte-like cells (HepaRG) using a direct liquid chromatography-mass spectrometry (LC-MS) assay of cell lysates incubated with DDVP or using a competitive pull-down experiments with a biotin-linked organophosphorus compound (10-fluoroethoxyphosphinyl-N-biotinamidopentyldecanamide; FP-biotin), which competes with DDVP for similar binding sites. We show that DDVP forms adducts to several proteins important for the cellular metabolic pathways and differentiation, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin. We validated the results using purified proteins and enzymatic assays. The study not only identified novel DDVP-modified targets but also suggested that the modification directly inhibits the enzymes. The current approach provides information for future hypothesis-based studies to understand the underlying mechanism of toxicity of DDVP in non-neuronal tissues. The MS data have been deposited to the ProteomeXchange with identifier PXD001107.

Colorimetric detection of dichlorvos using polydiacetylene vesicles with acetylcholinesterase and cationic surfactants.

Widespread use of dichlorvos in agriculture has posed serious concern for food and water contamination. A new colorimetric method for the detection of dichlorvos based on polydiacethylene and acetylcholinesterase inhibition is developed. The blue-to-red color transition of poly(10,12-pentacosadynoic acid) vesicles can be induced by myristoylcholine which is enzymatically hydrolyzed by acetylcholinesterase to myristic acid and choline to prevent the color transition. In the presence of dichlorvos, the hydrolytic activity of the enzyme is inhibited that the blue-to-red color transition is restored with a linear correlation to the dichlorvos concentration. Using UV-vis absorption spectrometer, the limit of dichlorvos detection is 6.7 ppb. A naked eye detection of 50 ppb dichlorvos is achievable by using dimiristoylphosphatidylcholine to the diacetylene mixed lipid vesicles.

Use of OpdA, an organophosphorus (OP) hydrolase, prevents lethality in an African green monkey model of acute OP poisoning.

Organophosphorus (OP) pesticides are a diverse class of acetylcholinesterase (AChE) inhibitors that are responsible for tremendous morbidity and mortality worldwide, killing approximately 300,000 people annually. Enzymatic hydrolysis of OPs is a potential therapy for acute poisoning. OpdA, an OP hydrolase isolated from Agrobacterium radiobacter, has been shown to decrease lethality in rodent models of OP poisoning. This study investigated the effects of OpdA on AChE activity, plasma concentrations of OP, and signs of toxicity after administration of dichlorvos to nonhuman primates. A dose of 75 mg/kg dichlorvos given orally caused apnea within 10 min with a progressive decrease in heart rate. Blood AChE activity decreased to zero within 10 min. Respirations and AChE activity did not recover. The mean dichlorvos concentration rose to a peak of 0.66 µg/ml. Treated monkeys received 1.2mg/kg OpdA iv immediately after poisoning with dichlorvos. In Opda-treated animals, heart and respiratory rates were unchanged from baseline over a 240-minute observation period. AChE activity slowly declined, but remained above 25% of baseline for the entire duration. Dichlorvos concentrations reached a mean peak of 0.19 µg/ml at 40 min after poisoning and decreased to a mean of 0.05 µg/ml at 240 min. These results show that OpdA hydrolyzes dichlorvos in an African green monkey model of lethal poisoning, delays AChE inhibition, and prevents lethality.

Design, synthesis and characterization of a hexapeptide bio-inspired by acetylcholinesterase and its interaction with pesticide dichlorvos.

This paper describes the molecular modeling design, synthesis and characterization of a new bio-inspired hexapeptide of acetylcholinesterase enzyme and its interaction with the organophosphate pesticide dichlorvos monitored by UV-Vis spectroscopy and mass spectrometry. This strategy can contribute to the development of synthetic receptors to be coupled to biosensor transducers, avoiding the issues associated with proteins such as low stability under different pH and temperature conditions and high production cost. The resulting data of this work indicate a strong interaction between the pesticide dichlorvos and the hexapeptide (NH3(+)-Glu-His-Gly-Gly-Pro-Ser-COO(-)) with a binding constant of 4.10 × 10(5) M(-1) and the formation of an adduct by covalent binding on the serine residue from the hexapeptide.

Spontaneous reactivation and aging kinetics of acetylcholinesterase inhibited by dichlorvos and diazinon.

Organophosphorus (OP) are among the most toxic of all substances that cause poisoning in food animals and are the most frequently encountered insecticides, commonly detected in agricultural products, animal-derived foodstuffs, environmental samples, and home use and represent a significant potential health risk. The first-order rate constants obtained for spontaneous reactivation (k(s)) was found to be higher in sheep compared to cattle, pig, and ranged between 0.133 to 0.323 hr⁻¹ and between 0.021 to 0.088 hr⁻¹ for dichlorvos (DDVP) and diazinon (DZN) respectively. Aging of phosphorylated acetylcholinesterase (AChE) follows the kinetics of a first-order reaction with rate constants of aging (k(a)) higher in cattle compared to sheep and pig, and ranged between 0.013 to 0.021 hr⁻¹ and between 0.009 to 0.01 hr⁻¹ for DDVP and DZN respectively. Half-time (t½) for spontaneous reactivation and aging are higher in DZN compared to DDVP and ranged from 2.3 to 85.3 hr (sheep), 3.2 to 76.3 hr (cattle), and 2.9 to 58.3 hr (pig), respectively.

Reduction of pesticide residues on fresh vegetables with electrolyzed water treatment.

Degradation of the 3 pesticides (acephate, omethoate, and dimethyl dichloroviny phosphate [DDVP]) by electrolyzed water was investigated. These pesticides were commonly used as broad-spectrum insecticides in pest control and high-residual levels had been detected in vegetables. Our research showed that the electrolyzed oxidizing (EO) water (pH 2.3, available chlorine concentration:70 ppm, oxidation-reduction potential [ORP]: 1170 mV) and the electrolyzed reducing (ER) water (pH 11.6, ORP: -860 mV) can reduce the pesticide residues effectively. Pesticide residues on fresh spinach after 30 min of immersion in electrolyzed water reduced acephate by 74% (EO) and 86% (ER), omethoate by 62% (EO) and 75% (ER), DDVP by 59% (EO) and 46% (ER), respectively. The efficacy of using EO water or ER water was found to be better than that of using tap water or detergent (both were reduced by more than 25%). Besides spinach, the cabbage and leek polluted by DDVP were also investigated and the degradation efficacies were similar to the spinach. Moreover, we found that the residual level of pesticide residue decreased with prolonged immersion time. Using EO or ER water to wash the vegetables did not affect the contents of Vitamin C, which inferred that the applications of EO or ER water to wash the vegetables would not result in loss of nutrition.

Dichlorvos, chlorpyrifos oxon and Aldicarb adducts of butyrylcholinesterase, detected by mass spectrometry in human plasma following deliberate overdose.

The goal of this study was to develop a method to detect pesticide adducts in tryptic digests of butyrylcholinesterase in human plasma from patients poisoned by pesticides. Adducts to butyrylcholinesterase in human serum may serve as biomarkers of pesticide exposure because organophosphorus and carbamate pesticides make a covalent bond with the active site serine of butyrylcholinesterase. Serum samples from five attempted suicides (with dichlorvos, Aldicarb, Baygon and an unknown pesticide) and from one patient who accidentally inhaled dichlorvos were analyzed. Butyrylcholinesterase was purified from 2 ml serum by ion exchange chromatography at pH 4, followed by procainamide affinity chromatography at pH 7. The purified butyrylcholinesterase was denatured, digested with trypsin and the modified peptide isolated by HPLC. The purified peptide was analyzed by multiple reaction monitoring in a QTRAP 4000 mass spectrometer. This method successfully identified the pesticide-adducted butyrylcholinesterase peptide in four patients whose butyrylcholinesterase was inhibited 60-84%, but not in two patients whose inhibition levels were 8 and 22%. It is expected that low inhibition levels will require analysis of larger serum plasma volumes. In conclusion, a mass spectrometry method for identification of exposure to live toxic pesticides has been developed, based on identification of pesticide adducts on the active site serine of human butyrylcholinesterase.

Conidia immobilization of T-DNA inserted Trichoderma atroviride mutant AMT-28 with dichlorvos degradation ability and exploration of biodegradation mechanism.

An immobilizing conidia approach was used to study the degradation ability of dichlorvos in Trichoderma atroviride T-DNA insertional mutant AMT-28. Beads with 10(7) immobilized conidia per 100 mL of Na-alginate solution exhibited the highest degradation abilities. The immobilized conidia showed enhanced degradation abilities compared with immobilized or freely suspended mycelia. The immobilized cells kept good storage capacity and reusability. Dichlorvos was confirmed to be completely removed by mycelia of AMT-28 within 7 days using HPLC analysis. The dichlorvos degradation rates in auxotrophic Burk media varied and were significantly affected by nitrogen sources. There was no detectable biosorption and the removal of dichlorvos in AMT-28 was primarily attributed to a kind of Biomineralization process.

Functional assembly of bacterial communities with activity for the biodegradation of an organophosphorus pesticide in the rape phyllosphere.

Although most pesticides sprayed on terrestrial plants remain on their leaf surfaces, the relationship between leaf-associated microbial populations and pesticide degradation remains unclear. Here we examined changes in the bacterial community composition in the rape phyllosphere after treatment with dichlorvos, an organophosphorus pesticide. Results indicate that the bacterial community showed marked changes after treatment. We evaluated the rate of dichlorvos degradation by a natural microbial community on rape leaves and found that more dichlorvos was degraded on microbial-population-inhabited leaves than on surface-sterilized leaves. Six dichlorvos-degrading bacteria with 16S rRNA gene sequences that are most similar to those of members of the genera Pseudomonas, Xanthomonas, Sphingomonas, Acidovorax, Agrobacterium and Chryseobacterium were isolated from the natural community. We report for the first time that three of these epiphytic bacterial species, from the genera Sphingomonas, Acidovorax and Chryseobacterium, can degrade organophosphorus compounds. Collectively, these results provide direct evidence that bacteria on leaves can degrade organophosphate pesticides, and demonstrate that phyllosphere bacteria have great potential for the bioremediation of pesticides in situ, where the environment is hostile to nonepiphytic bacteria.

Methamidophos, dichlorvos, O-methoate and diazinon pesticides used in Turkey make a covalent bond with butyrylcholinesterase detected by mass spectrometry.

Organophosphorus pesticides used most commonly in Turkey include methamidophos, dichlorvos, O-methoate and diazinon. These toxic chemicals or their metabolites make a covalent bond with the active site serine of butyrylcholinesterase. Our goal was to identify the adducts that result from the reaction of human butyrylcholinesterase with these pesticides. Highly purified human butyrylcholinesterase was treated with a 20-fold molar excess of pesticide. The protein was denatured by boiling and digested with trypsin. MS and MSMS spectra of HPLC-purified peptides were acquired on a MALDI-TOF-TOF 4800 mass spectrometer. It was found that methamidophos added a mass of +93, consistent with addition of methoxy aminophosphate. A minor amount of adduct with an added mass of +109 was also found. Dichlorvos and O-methoate both made dimethoxyphosphate (+108) and monomethoxyphosphate adducts (+94). Diazinon gave a novel adduct with an added mass of +152 consistent with diethoxythiophosphate. Inhibition of enzyme activity in the presence of diazinon developed slowly (15 h), concomitant with isomerization of diazinon via a thiono-thiolo rearrangement. The isomer of diazinon yielded diethoxyphosphate and monoethoxyphosphate adducts with added masses of +136 and +108. MSMS spectra confirmed that each of the pesticides studied made a covalent bond with serine 198 of butyrylcholinesterase. These results can be used to identify the class of pesticides to which a patient was exposed.