Elucidating the detoxification efficacy of Periplaneta americana delta glutathione S-transferase 1 (PaGSTd1) against organophosphates.

As an important class of detoxifying enzymes, glutathione S-transferases (GSTs) are pivotal in decreasing insecticide toxicity to insects. Periplaneta americana GSTd1 (PaGSTd1) has been verified as a key enzyme in detoxifying pyrethroid insecticides, but its detoxification capability against a broader spectrum of insecticides has never been investigated. It is revealed that PaGSTd1 expression showed a rapid and significant increase upon exposure to various insecticides (organophosphates, neonicotinoids, and fipronil). Subsequent in vitro metabolic assays indicated that organophosphates, particularly chlorpyrifos-methyl, can be effectively metabolized by PaGSTd1. Further knockdown of PaGSTd1 via RNA interference significantly heightened the susceptibility of P. americana to chlorpyrifos-methyl, underscoring the enzyme's key role in detoxifying chlorpyrifos-methyl. Additionally, this study confirmed that PaGSTd1 cannot mitigate insecticide toxicity through countering oxidative stress. Collectively, these findings elucidate the involvement of PaGSTd1 in the detoxification processes for organophosphates, offering a comprehensive insight into the metabolic mechanisms mediated by GSTs in P. americana. This research provides a foundational understanding for managing GSTs-mediated metabolic resistance in this species, which is crucial for effective pest control strategies.

Patterns of cardio-respiratory motor outputs during acute and subacute exposure to chlorpyrifos in an ex-vivo in situ preparation in rats.

While a considerable body of literature has characterized the clinical features induced by organophosphate pesticides, the field lacks scrutiny into cardio-respiratory changes in different phases of poisoning. Herein, we evaluated the impact of chlorpyrifos (CPF) and its active metabolite chlorpyrifos-oxon (CPO) on the cardiorespiratory system during acute and subacute phases of poisoning using an in situ experimental rodent model. CPF (30 mg/kg) was injected intraperitoneally to rats beforehand (24 h) whereas CPO (15 mg/kg) was added into the perfusate reservoir to evaluate the effects on the motor outputs throughout the three phases of the respiratory cycle: inspiration, post-inspiration and late expiration. Phrenic, recurrent laryngeal (RLN) and thoracic sympathetic nerve activity (tSNA) were recorded. Heart rate was derived from the electrocardiogram (ECG) and the baro- and chemo-reflexes tested. CPF and CPO led to a time-dependent change in cardiorespiratory motor outputs. In the acute phase, the CPO induced bradypnea, transiently reduced the inspiratory time (TI), and increased the amplitude of phrenic. Post-inspiratory (PI) discharge recorded from the RLN was progressively reduced while tSNA was increased. CPO significantly depressed the chemoreflex but had no effect on baroreflex. During subacute phase, CPF prolongated TI with no effect on respiratory rate. Both the RLN PI discharge, the chemoreflex and the baroreflex sympathetic gain were reduced. In addition, both CPF and CPO shifted the cardiac sympatho-vagal balance towards sympathetic dominance. Our data show that different phases of poisoning are associated with specific changes in the cardio-respiratory system and might therefore demand distinct approaches by health care providers.

Organophosphorus Pesticides Promote Protein Cross-Linking.

Exposure to organophosphorus pesticides (OP) can have chronic adverse effects that are independent of inhibition of acetylcholinesterase, the classic target for acute OP toxicity. In pure proteins, the organophosphorus pesticide chlorpyrifos oxon induces a cross-link between lysine and glutamate (or aspartate) with loss of water. Tubulin is particularly sensitive to OP-induced cross-linking. Our goal was to explore OP-induced cross-linking in a complex protein sample, MAP-rich tubulin from Sus scrofa and to test 8 OP for their capacity to promote isopeptide cross-linking. We treated 100 µg of MAP-rich tubulin with 100 µM chlorpyrifos, chlorpyrifos oxon, methamidophos, paraoxon, diazinon, diazoxon, monocrotophos, or dichlorvos. Each sample was separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and stained with Coomassie blue. Five gel slices (at about 30, 50, 150, and 300 kDa, and the top of the separating gel) were removed from the lanes for each of the eight OP samples and from untreated control lanes. These gel slices were subjected to in-gel trypsin digestion. MSMS fragmentation spectra of the tryptic peptides were examined for isopeptide cross-links. Sixteen spectra yielded convincing evidence for isopeptide cross-linked peptides. Ten were from the chlorpyrifos oxon reaction, 1 from dichlorvos, 1 from paraoxon, 1 from diazinon, and 3 from diazoxon. It was concluded that catalysis of protein cross-linking is a general property of organophosphorus pesticides and pesticide metabolites. Data are available via ProteomeXchange with identifier PXD034529.

Dietary chlorpyrifos-methyl exposure impair transcription of immune-, detoxification- and redox signaling genes in leukocytes isolated from cod (Gadus morhua).

Inclusion of new environmental toxicants increase with the amount of plant ingredients substituting marine proteins and oils in feed for farmed Atlantic salmon (Salma salar). Agricultural pesticides like chlorpyrifos-methyl, present in commercial salmon feeds, may affect salmon immune and detoxification responses. Atlantic cod (Gadus morhua), surrounding the net pens, grazing on feces and uneaten pellets may be affected accordingly. The aim of this study was to analyze transcription responses in Atlantic cod head kidney tissue and isolated leukocytes following dietary chlorpyrifos-methyl inclusions and possible interactions with proinflammatory signals. Head kidney tissues and leukocytes were isolated from cod fed diets contaminated with chlorpyrifos-methyl (0.5 mg/kg, 2.4 mg/kg, 23.2 mg/kg) for 30 days. The isolated leukocytes were further challenged with bacteria (lipopolysaccharide (LPS), virus (polyinosinic acid:polycytidylic acid (PIC) mimic and l-arginine, an immuno-modulating amino acid, in vitro. The LPS-induced transcription of the interleukin genes il-1ß, il-6, il-8 increased in leukocytes isolated from cod fed chlorpyrifos-methyl 23.2 mg/kg, compared to cod fed the control diet, indicating increased inflammation. Transcriptional levels of carnitine palmitoyl transferase (cpt1a), aryl hydrogen receptor (ahr) and catalase (cat) were all reduced by dietary inclusions of chlorpyrifos-methyl in the leukocytes. The findings suggests that dietary chlorpyrifos-methyl exposure impair inflammation, detoxification and redox signaling in cod leukocytes.

Determination of chlorpyrifos-methyl, lambda-cyhalothrin and tebuconazole residues in Sultana seedless grapes sprayed with pesticides under farmer's conditions.

The aim of this study was to investigate pesticide residues on Sultana Seedless Grapes harvested at different times and consequently evaluate the risk. Analyses were performed with the Quick-Easy-Cheap-Efficient-Rugged-Safe (QuEChERS)-liquid chromatography/tandem mass spectrometry (LC-MS/MS) procedure. Pesticide-free grapes were spiked at 3 levels. Chlorpyrifos-methyl, lambda-cyhalothrin and tebuconazole detection limits were 20, 10 and 1 µg kg-1, respectively. These values were below maximum residue levels (MRL) of 1000, 80 and 500 µg kg-1, respectively. The overall recovery of the method was 108.60%. Present values were within acceptable recovery (60-140%) and repeatability (≤20%) ranges set by the Directorate-General for Health and Food Safety (SANTE). The vineyards were sprayed 4 times. Grapes were harvested in the 1st, 3rd, 5th, 7th and 14th days from the last spray. The residues in the samples taken from 5 vine stock groups were determined. MRL-exceeding chlorpyrifos-methyl residue of 1140.09 µg kg-1 was detected in the 1st day. The MRL-exceeding lambda-cyhalothrin (381.15 and 307.39 µg kg-1) and tebuconazole residues (650.58 and 570.85 µg kg-1) were detected in the 1st and 3rd days, respectively. The residues of 7th and 14th day samples were significantly different from the others. Excessive pesticide concentrations did not pose any health risks on consumers.

Rabbit Antidiethoxyphosphotyrosine Antibody, Made by Single B Cell Cloning, Detects Chlorpyrifos Oxon-Modified Proteins in Cultured Cells and Immunopurifies Modified Peptides for Mass Spectrometry.

Chronic low-dose exposure to organophosphorus pesticides is associated with the risk of neurodegenerative disease. The mechanism of neurotoxicity is independent of acetylcholinesterase inhibition. Adducts on tyrosine, lysine, threonine, and serine can occur after exposure to organophosphorus pesticides, the most stable being adducts on tyrosine. Rabbit monoclonal 1C6 to diethoxyphosphate-modified tyrosine (depY) was created by single B cell cloning. The amino acid sequence and binding constant (Kd 3.2 × 10-8 M) were determined. Cultured human neuroblastoma SH-SY5Y and mouse neuroblastoma N2a cells incubated with a subcytotoxic dose of 10 µM chlorpyrifos oxon contained depY-modified proteins detected by monoclonal 1C6 on Western blots. depY-labeled peptides from tryptic digests of cell lysates were immunopurified by binding to immobilized 1C6. Peptides released with 50% acetonitrile and 1% formic acid were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) on an Orbitrap Fusion Lumos mass spectrometer. Protein Prospector database searches identified 51 peptides modified on tyrosine by diethoxyphosphate in SH-SY5Y cell lysate and 73 diethoxyphosphate-modified peptides in N2a cell lysate. Adducts appeared most frequently on the cytoskeleton proteins tubulin, actin, and vimentin. It was concluded that rabbit monoclonal 1C6 can be useful for studies that aim to understand the mechanism of neurotoxicity resulting from low-dose exposure to organophosphorus pesticides.

Susceptibility of different developmental stages of Trichogramma parasitoids to insecticides commonly used in the Mediterranean olive agroecosystem.

Insecticide application and augmentative parasitoid releases are often considered incompatible. However, pesticide applications and parasitoid releases can be integrated into a pest management scheme if there is careful time scheduling of these interventions. In this study, we assessed the influence of commonly used insecticides (chlorpyrifos-methyl, deltamethrin, pyriproxyfen, thiamethoxam) in olive agroecosystems to two currently present Trichogramma parasitoids in the Mediterranean basin. Exposure to insecticides in relation to parasitoid's development was also tested. Both, insecticide type and application time influenced parasitism and the emergence rates of the two parasitoid species. Chlorpyrifos-methyl had the strongest impact on parasitoids resulting in low numbers of emerged adults followed by deltamethrin. The two parasitoids also exhibited different levels of susceptibility to the insecticides used. Potential integration of insecticides to integrated pest management using Trichogramma parasitoids is discussed.

Transgenerational effects of developmental exposure to chlorpyrifos-oxon in zebrafish (DANIO RERIO).

The organophosphate chlorpyrifos, and its active metabolite chlorpyrifos-oxon (CPO), have been attributed to a number of neurodevelopmental disorders. It is unclear if the adverse effects associated with developmental exposure to the active CPO persist into adulthood and future generations. The goal of this study was to investigate whether CPO-associated changes in embryo-larval zebrafish (ZF) behavior at the F0 5 dpf were manifest throughout the life of the exposed F0, and are inherited by subsequent generations. For this study, embryos were exposed to chlorpyrifos-oxon at the environmentally relevant concentration of 0.01 µg/L and a high concentration of 50 µg/L starting at 4 hpf to 5 dpf, and then raised to F2. There was a significant decrease in distance traveled with 5 dpf F0 ZF exposed to the 50 µg/L CPO, with alterations in noncholinergic genes CFOS and LINGO, and alterations in global DNA methylation. CPO-related behavioral effects were ameliorated by day 21 through the F1 generation. This trend changed with hyperactive behavior, increase acetylcholine concentration in F2 zebrafish that were exposed to 50 µg/L CPO during the F0 development. There was also an increase in AChE activity and hypermethylation in F2 0.01 µg/L exposure larvae, indicating that even low dose exposures can have transgenerational effects. Results from this study demonstrate that early life stage exposures to CPO can lead to epigenetic changes in neurological activity, which may lead to alterations in response to CPO in future generations. ABSTRACT SUMMARY: This study identified a correlation between CPO exposure during F0 development and significant differences in F2 behavioral, AChE activity and neurotransmitter concentration.

Neurite outgrowth inhibitory levels of organophosphates induce tissue transglutaminase activity in differentiating N2a cells: evidence for covalent adduct formation.

Organophosphate compounds (OPs) induce both acute and delayed neurotoxic effects, the latter of which is believed to involve their interaction with proteins other than acetylcholinesterase. However, few OP-binding proteins have been identified that may have a direct role in OP-induced delayed neurotoxicity. Given their ability to disrupt Ca2+ homeostasis, a key aim of the current work was to investigate the effects of sub-lethal neurite outgrowth inhibitory levels of OPs on the Ca2+-dependent enzyme tissue transglutaminase (TG2). At 1-10 µM, the OPs phenyl saligenin phosphate (PSP) and chlorpyrifos oxon (CPO) had no effect cell viability but induced concentration-dependent decreases in neurite outgrowth in differentiating N2a neuroblastoma cells. The activity of TG2 increased in cell lysates of differentiating cells exposed for 24 h to PSP and chlorpyrifos oxon CPO (10 µM), as determined by biotin-cadaverine incorporation assays. Exposure to both OPs (3 and/or 10 µM) also enhanced in situ incorporation of the membrane permeable substrate biotin-X-cadaverine, as indicated by Western blot analysis of treated cell lysates probed with ExtrAvidin peroxidase and fluorescence microscopy of cell monolayers incubated with FITC-streptavidin. Both OPs (10 µM) stimulated the activity of human and mouse recombinant TG2 and covalent labelling of TG2 with dansylamine-labelled PSP was demonstrated by fluorescence imaging following SDS-PAGE. A number of TG2 substrates were tentatively identified by mass spectrometry, including cytoskeletal proteins, chaperones and proteins involved protein synthesis and gene regulation. We propose that the elevated TG2 activity observed is due to the formation of a novel covalent adduct between TG2 and OPs.

Seasonal variations in the dose-response relationship of acetylcholinesterase activity in freshwater fish exposed to chlorpyrifos and glyphosate.

The herbicide glyphosate [N- (phosphonomethyl) glycine; PMG] and the insecticide chlorpyrifos [O, O-diethyl O- (3,5,6-trichloro-2-pyridinyl) -phosphorothioate, CPF] are widely used in agricultural practices around the world and can reach aquatic environments. Therefore, it is necessary to characterize the toxicity of these pesticides on non-target species. The use of biomarkers as a tool to assess responses of organisms exposed to pollutants requires the understanding of their natural fluctuation and the dose-response relationship. In the present work, the effect of the exposure to PMG and CPF on the acetylcholinesterase activity (AChE, biomarker of neurotoxicity) in Cnesterodon decemmaculatus, a native teleost, was evaluated in different environmental conditions. Semi-static bioassays of acute toxicity were carried out under controlled conditions during the four weather seasons of the year using animals of homogeneous size. Circannual rhythms in the basal levels of AChE activity in homogenates of the anterior section were confirmed. Statistically significant average inhibition of AChE activity (47.1 ±â€¯0.7% for 1 µg CPF × L-1; 69.7 ±â€¯2.5% for 5 µg CPF × L-1; 23.1 ±â€¯1.1% for 1 mg PMG × L-1 and 32.9 ±â€¯3.3% for 10 mg PMG × L-1) was determined during summer, winter and spring weather seasons. Interestingly, animals exhibit an increased susceptibility to exposure during the autumn season (inhibition of 55.4 ±â€¯0.6% for 1 µg CPF × L-1; 81.9 ±â€¯3.3% for 5 µg CPF × L-1; 41.4 ±â€¯1.7% for 1 mg PMG × L-1 and 61.1 ±â€¯0.3% for 10 mg PMG × L-1). A different sensitivity of the enzyme between seasons was evaluated by in vitro tests. The inhibition pattern for chlorpyrifos-oxon (CPF-oxon, the active metabolite of CPF) was not affected when test was performed using homogenates of unexposed specimens of summer or autumn. Otherwise, PMG in vitro inhibitory effect was not observed in a wide range of concentrations. The results confirm that AChE activity is a sensitive biomarker for exposure to CPF and PMG, even at environmentally relevant concentrations. Finally, this work highlights the existence of seasonal variations in the dose-response relationship, which could be due to variations in the metabolism of the pollutants.

Chlorpyrifos Oxon-Induced Isopeptide Bond Formation in Human Butyrylcholinesterase.

A newly recognized action of organophosphates (OP) is the ability to crosslink proteins through an isopeptide bond. The first step in the mechanism is covalent addition of the OP to the side chain of lysine. This activates OP-lysine for reaction with a nearby glutamic or aspartic acid to make a gamma glutamyl epsilon lysine bond. Crosslinked proteins are high molecular weight aggregates. Our goal was to identify the residues in the human butyrylcholinesterase (HuBChE) tetramer that were crosslinked following treatment with 1.5 mM chlorpyrifos oxon. High molecular weight bands were visualized on an SDS gel. Proteins in the gel bands were digested with trypsin, separated by liquid chromatography and analyzed in an Orbitrap mass spectrometer. MSMS files were searched for crosslinked peptides using the Batch-Tag program in Protein Prospector. MSMS spectra were manually evaluated for the presence of ions that supported the crosslinks. The crosslink between Lys544 in VLEMTGNIDEAEWEWK544AGFHR and Glu542 in VLEMTGNIDEAEWE542WK satisfied our criteria including that of spatial proximity. Distances between Lys544 and Glu542 were 7.4 and 9.5 Å, calculated from the cryo-EM (electron microscopy) structure of the HuBChE tetramer. Paraoxon ethyl, diazoxon, and dichlorvos had less pronounced effects as visualized on SDS gels. Our proof-of-principle study provides evidence that OP have the ability to crosslink proteins. If OP-induced protein crosslinking occurs in the brain, OP exposure could be responsible for some cases of neurodegenerative disease.

Optimization of a Simplified and Effective Analytical Method of Pesticide Residues in Mealworms (Tenebrio molitor Larvae) Combined with GC-MS/MS and LC-MS/MS.

An effective analytical method was optimized for residues including chlorpyrifos-methyl, deltamethrin, fenoxanil, thiobencarb and fludioxonil in mealworms, the larval form of Tenebrio molitor. They are listed for pest control during wheat cultivation and can be found in wheat-bran feed for growing mealworms in South Korea. Analytes were extracted using acetonitrile and salt packet. Four clean-up methods ((1) MgSO4 + 25 mg PSA + 25 mg C18; (2) MgSO4 + 50 mg PSA + 50 mg C18; (3) EMR-lipidTM tube; and (4) 10 mL n-hexane) were investigated and the method (1) was selected due to its robustness. Low-temperature precipitation of fat and proteins improved the recoveries. Recoveries from the Method (1) were satisfying with 70-120% with <20% relative SD at a spiking level of 0.01 mg/kg. With the simultaneous sample preparation, fenoxanil, thiobencarb and fludioxonil were analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) and chlorpyrifos-methyl and deltamethrin by gas chromatography tandem mass spectrometry (GC-MS/MS). Quantification limits for LC-MS/MS and GC-MS/MS were 0.5 and 2.5 µg/L, respectively. No pesticides of interest were detected in 30 real samples collected across the nation. However, the data can be provided for establishing maximum residue limits for the pesticides in mealworms in response to the positive list system.

Qualitative enzymatic detection of organophosphate and carbamate insecticides.

In this study, new polymers containing amides (TrisPS-Ntaa, and TrisPS-Ntaa-Fc) were synthesized by condensation reaction for qualitative identification of insecticides. The synthesized polymers, including amides were investigated by infrared spectroscopy (IR), scanning electron microscopy-energy dispersion X- ray spectrometry (SEM-EDX), and gel permeation chromatography (GPC). Then, acetylcholinesterase enzyme (AChE) was covalently immobilized on these polymers to improve properties (including activity, reusability, and storage stability). Accordingly, organophosphate (malathion, acephate, chlorpyrifos methyl) and carbamate (carbofuran, methiocarb, methomyl), which are used to prevent harmful organisms in some agricultural products were enzymatically determined based on their inhibitory activity on AChE.

Chlorpyrifos oxon promotes tubulin aggregation via isopeptide cross-linking between diethoxyphospho-Lys and Glu or Asp: Implications for neurotoxicity.

Exposure to organophosphorus toxicants (OP) can have chronic adverse effects that are not explained by inhibition of acetylcholinesterase, the cause of acute OP toxicity. We therefore hypothesized that OP-induced chronic illness is initiated by the formation of organophosphorus adducts on lysine residues in proteins, followed by protein cross-linking and aggregation. Here, Western blots revealed that exposure to the OP chlorpyrifos oxon converted porcine tubulin from its original 55-kDa mass to high-molecular-weight aggregates. Liquid chromatography-tandem MS analysis of trypsin-digested samples identified several diethoxyphospho-lysine residues in the OP-treated tubulin. Using a search approach based on the Batch Tag program, we identified cross-linked peptides and found that these chemically activated lysines reacted with acidic amino acid residues creating γ-glutamyl-ϵ-lysine or aspartyl-ϵ-lysine isopeptide bonds between ß- and α-tubulin. Of note, these cross-linked tubulin molecules accounted for the high-molecular-weight aggregates. To the best of our knowledge, this is the first report indicating that chlorpyrifos oxon-exposed tubulin protein forms intermolecular cross-links with other tubulin molecules, resulting in high-molecular-weight protein aggregates. It is tempting to speculate that chronic illness from OP exposure may be explained by a mechanism that starts with OP adduct formation on protein lysines followed by protein cross-linking. We further speculate that OP-modified or cross-linked tubulin can impair axonal transport, reduce neuron connections, and result in neurotoxicity.

Mass Spectrometry Identifies Isopeptide Cross-Links Promoted by Diethylphosphorylated Lysine in Proteins Treated with Chlorpyrifos Oxon.

Exposure to chlorpyrifos at doses that do not inhibit acetylcholinesterase can be followed by chronic illness in adults and developmental deficits in children. A mechanism to explain these effects is not available. Using mass spectrometry, we have found that chlorpyrifos oxon is a cross-linking agent. Pure proteins incubated with 1.5 mM chlorpyrifos oxon were diethylphosphorylated on lysine and tyrosine. The diethylphospho-lysine reacted with the carboxyl side-chain of aspartic and glutamic acid to form an isopeptide cross-link, releasing diethylphosphate in the process. Of the 14 proteins tested, 9 had cross-links between distinct proteins or between monomers of the same protein, whereas 8 had a cyclic structure created by joining side-chains of nearby residues through an isopeptide bond. The precursor lysine in the isopeptide bond was diethylphosphorylated on the ε-amino group. Tubulin was more susceptible to chlorpyrifos-oxon-induced cross-linking than the other proteins (10 cross-links in tubulin, 2 in human albumin). The role of diethylphospho-tyrosine was not examined. We hypothesize that the protein misfolding and protein cross-linking induced by exposure to chlorpyrifos oxon, via metabolism of chlorpyrifos, could disrupt function, particularly of tubulin, thus leading to chronic illness. Our proposed mechanism is hypothetical until the many questions it raises have been addressed.

LC-MS/MS method for the determination of organophosphorus pesticides and their metabolites in salmon and zebrafish fed with plant-based feed ingredients.

The composition of Atlantic salmon feed has changed considerably over the last two decades from being marine-based (fishmeal and fish oil) to mainly containing plant ingredients. Consequently, concern related to traditional persistent contaminants typically associated with fish-based feed has been replaced by other potential contaminants not previously associated with salmon farming. This is the case for many pesticides, which are used worldwide to increase food production, and may be present in plant ingredients. Earlier studies have identified two organophosphorus pesticides, chlorpyrifos-methyl and pirimiphos-methyl, in plant ingredients used for aquafeed production. In the present study, we developed a reliable and sensitive analytical method, based on liquid chromatography coupled to tandem mass spectrometry, for the determination of these pesticides and their main metabolites in warm water (zebrafish) and cold water (Atlantic salmon) species, where possible differences in metabolites could be expected. The method was tested in whole zebrafish and in different salmon tissues, such as muscle, bile, kidney, fat, and liver. The final objective of this work was to assess kinetics of chlorpyrifos-methyl and pirimiphos-methyl and their main metabolites in fish tissue, in order to fill the knowledge gaps on these metabolites in fish tissues when fed over prolonged time.

Exploring the potential enzymatic bioremediation of vermicompost through pesticide-detoxifying carboxylesterases.

Vermicompost is a known biofertilizer of potential use in soil bioremediation. This study was undertaken to explore the capacity of grape marc-derived vermicompost to inactivate methyl carbamate (MC) and organophosphorus (OP) pesticides via exploring the carboxylesterase (CE) activity level and its response to pesticide exposure. We first optimized the method for enzyme activity assay comparing the CE activity in two contrasting homogenization procedures (30-min mixing and mortar grinding). Thereafter, we assessed the sensitivity of the enzyme by both in vitro and vermicompost incubation trials with selected pesticides. The main findings can be summarized as follows: i) grinding the vermicompost in water (2% w/v) yielded maximum enzyme activity; ii) at concentrations around 10-4 M, highly toxic oxygen-analog metabolites of OPs strongly inhibited the CE activity (76-93% inhibition), but MC did not inhibit the enzyme activity; iii) liquid vermicompost was able to degrade chlorpyrifos and inactivate its highly toxic metabolite chlorpyrifos-oxon. Our results suggest that liquid vermicompost is the most appropriate preparation to increase the enzymatic potential of vermicompost in pesticide-contaminated soils.

Comparative study on the biodegradation of chlorpyrifos-methyl by Bacillus megaterium CM-Z19 and Pseudomonas syringae CM-Z6.

The strains CM-Z19 and CM-Z6, which are capable of highly degrading chlorpyrifos-methyl, were isolated from soil. They were identified as Bacillus megaterium CM-Z19 and Pseudomonas syringae CM-Z6, respectively, based on the 16S rRNA and an analysis of their morphological, physiological and biochemical characteristics. The strain CM-Z19 showed 92.6% degradation of chlorpyrifos-methyl (100 mg/L) within 5 days of incubation, and the strain CM-Z6 was 99.1% under the same conditions. In addition, the degradation characteristics of the two strains were compared and studied, and the results showed that the strain CM-Z19 had higher phosphoesterase activity and ability to degrade the organophosphorus pesticide than did the strain CM-Z6. However, the strain CM-Z19 could not degrade its first hydrolysis metabolite 3,5,6-trichloro-2-pyridinol (TCP) and could not completely degrade chlorpyrifos-methyl. The strain CM-Z6 could effectively degrade TCP and could degrade chlorpyrifos-methyl more quickly than strain CM-Z19.

Mass Spectral Detection of Diethoxyphospho-Tyrosine Adducts on Proteins from HEK293 Cells Using Monoclonal Antibody depY for Enrichment.

Chronic illness from exposure to organophosphorus toxicants is hypothesized to involve modification of unknown proteins. Tyrosine in proteins that have no active site serine readily reacts with organophosphorus toxicants. We developed a monoclonal antibody, depY, that specifically recognizes diethoxyphospho-tyrosine in proteins and peptides, independent of the surrounding amino acid sequence. Our goal in the current study was to identify diethoxyphosphorylated proteins in human HEK293 cell lysate treated with chlorpyrifos oxon. Cell lysates treated with chlorpyrifos oxon were recognized by depY antibody in ELISA and capillary electrophoresis based Western blot. Tryptic peptides were analyzed by liquid chromatography tandem mass spectrometry. Liquid chromatography tandem mass spectrometry identified 116 diethoxyphospho-tyrosine peptides from 73 proteins in immunopurified samples, but found only 15 diethoxyphospho-tyrosine peptides from 12 proteins when the same sample was not immunopurified on depY. The most abundant proteins in the cell lysate, histone H4, heat shock 70 kDa protein 1A/1B, heat shock protein HSP 90 ß, and α-enolase, were represented by several diethoxyphospho-tyrosine peptides. It was concluded that use of immobilized depY improved the number of diethoxyphospho-tyrosine peptides identified in a complex mixture. The mass spectrometry results confirmed the specificity of depY for diethoxyphospho-tyrosine peptides independent of the context of the modified tyrosine, which means depY could be used to analyze modified proteins in any species. Use of the depY antibody could lead to an understanding of chronic illness from organophosphorus pesticide exposure.

Safety of Safety Evaluation of Pesticides: developmental neurotoxicity of chlorpyrifos and chlorpyrifos-methyl.

Authorization of pesticides for market release requires toxicity testing on animals, typically performed by test laboratories on contract with the pesticide producer. The latter provides the results and summary to the regulatory authorities. For the commonly used pesticide chlorpyrifos, an industry-funded toxicity study concludes that no selective effects on neurodevelopment occur even at high exposures. In contrast, the evidence from independent studies points to adverse effects of current exposures on cognitive development in children. We reviewed the industry-funded developmental neurotoxicity test data on chlorpyrifos and the related substance chlorpyrifos-methyl. We noted treatment-related changes in a brain dimension measure for chlorpyrifos at all dose levels tested, although not been reported in the original test summary. We further found issues which inappropriately decrease the ability of the studies to reveal true effects, including a dosage regimen that resulted in too low exposure of the nursing pups for chlorpyrifos and possibly for chlorpyrifos-methyl, and a failure to detect any neurobehavioral effects of lead nitrate used as positive control in the chlorpyrifos study. Our observations thus suggest that conclusions in test reports submitted by the producer may be misleading. This discrepancy affects the ability of regulatory authorities to perform a valid and safe evaluation of these pesticides. The difference between raw data and conclusions in the test reports indicates a potential existence of bias that would require regulatory attention and possible resolution.