A single post-exposure oxime RS194B treatment rapidly reactivates acetylcholinesterase and reverses acute symptoms in macaques exposed to diethylphosphorothioate parathion and chlorpyrifos insecticides.

Millions of individuals globally suffer from inadvertent, occupational or self-harm exposures from organophosphate (OP) insecticides, significantly impacting human health. Similar to nerve agents, insecticides are neurotoxins that target and inhibit acetylcholinesterase (AChE) in central and peripheral synapses in the cholinergic nervous system. Post-exposure therapeutic countermeasures generally include administration of atropine with an oxime to reactivate the OP-inhibited AChE. However, animal model studies and recent clinical trials using insecticide-poisoned individuals have shown minimal clinical benefits of the currently approved oximes and their efficacy as antidotes has been debated. Currently used oximes either reactivate poorly, do not readily cross the blood-brain barrier (BBB), or are rapidly cleared from the circulation and must be repeatedly administered. Zwitterionic oximes of unbranched and simplified structure, for example RS194B, have been developed that efficiently cross the BBB resulting in reactivation of OP-inhibited AChE and dramatic reversal of severe clinical symptoms in mice and macaques exposed to OP insecticides or nerve agents. Thus, a single IM injection of RS194B has been shown to rapidly restore blood AChE and butyrylcholinesterase (BChE) activity, reverse cholinergic symptoms, and prevent death in macaques following lethal inhaled sarin and paraoxon exposure. The present macaque studies extend these findings and assess the ability of post-exposure RS194B treatment to counteract oral poisoning by highly toxic diethylphosphorothioate insecticides such as parathion and chlorpyrifos. These OPs require conversion by P450 in the liver of the inactive thions to the active toxic oxon forms, and once again demonstrated RS194B efficacy to reactivate and alleviate clinical symptoms within 60 mins of a single IM administration. Furthermore, when delivered orally, the Tmax of RS194B at 1-2 h was in the same range as those administered IM but were maintained in the circulation for longer periods greatly facilitating the use of RS194B as a non-invasive treatment, especially in isolated rural settings.

Recombinant Organophosphorus acid anhydrolase (OPAA) enzyme-carbon quantum dot (CQDs)-immobilized thin film biosensors for the specific detection of Ethyl Paraoxon and Methyl Parathion in water resources.

Organophosphates pesticide (OP) toxicity through water resources is a large concern globally among all the emerging pollutants. Detection of OPs is a challenge which needs to be addressed considering the hazardous effects on the health of human beings. In the current research thin film biosensors of recombinant, Organophosphorus acid anhydrolase (OPAA) enzyme along with carbon quantum dots (CQDs) immobilized in thin films were developed. OPAA-CQDs thin film biosensors were used for the specific detection of two OPs Ethyl Paraoxon (EP) and Methyl Parathion (MP) in river water and household water supply. Recombinant OPAA enzyme was expressed in E. Coli, purified and immobilized on the CQD containing chitosan thin films. The CQDs used for this purpose were developed by a one-pot hydrothermal method from phthalic acid and Tri ethylene diamine. The properties of CQDs, OPAA and thin films were characterized using techniques like XPS, TEM, XRD, enzyme activity and CLSM measurements. Biosensing studies of EP and MP were performed by taking fluorescence measurements using a fiber optic spectrometer. The analytical parameters of biosensing were compared against an estimation carried out using the HPLC method. The biosensing performance indicates that the OPAA-CQDs thin film-based biosensors were able to detect both EP and MP in a range of 0-100 µM having a detection limit of 0.18 ppm/0.69 ppm for EP/MP, respectively with a response time of 5 min. The accuracy of estimation of EP/MP when spiked in water resources lie in the range of ∼100-102% which clearly indicates the OPAA-CQD based thin film biosensors can function as a point-of-use method for the detection of OP pesticides in complex water resources.

Deep eutectic solvent-based pressurized liquid extraction combined with dispersive liquid-liquid microextraction of organophosphorus pesticide residues in egg powder prior to high-performance liquid chromatography analysis.

Herein, a deep eutectic solvent (DES)-based miniaturized pressurized liquid extraction in combination with DES-based dispersive liquid-liquid microextraction (DLLME) was developed for the extraction of organophosphorus pesticides (parathion-methyl, triazophos, parathion, diazinon, and phoxim) from egg powder samples prior to their analysis by a high-performance liquid chromatography-diode array detector. In this work, first, the analytes' extraction was done by a pressurized liquid phase extraction for effective extraction of the analytes from the solid matrix, and then they were concentrated on a DLLME for more concentration of the analytes to reach low limits of detections. The use of DESs was done in both steps to omit the use of toxic organic solvents. Satisfactory results including high extraction recoveries (74-90%), great repeatability (relative standard deviations equal or less than 4.3% and 5.3% for intra- and inter-day precisions), and low limits of detection (0.11-0.29 ng/g) and quantification (0.38-0.98 ng/g) were attained under the optimum conditions. Lastly, the suggested approach was utilized for the determination of the studied pesticides in various egg powder samples marketed in Tabriz, Iran.

Activation of persulfate for the degradation of ethyl-parathion in soil: Combined effects of microwave with biochar.

Sulfate radical (SO4•-), formed by persulfate (PS) activation during advanced oxidation process (AOPs), can be used for the remediation of organic contaminated soil. However, the role of biochar and microwave (MW) in the activation of PS is not fully understood, especially the corresponding mechanism. Herein, biochar combined with MW was used to activate PS for the remediation of ethyl-parathion (PTH)-polluted soil. The dynamic evolutions of PTH under different conditions, such as biochar content, particle size, reaction temperature, and the degradation mechanisms of PTH were also systematically investigated. Significant enhancement performance on PTH removal was observed after adding biochar, which was 88.78% within 80 min. Meanwhile, activating temperature exhibited remarkable abilities to activate PS for PTH removal. The higher content of adsorption sites in nano-biochar facilitated the removal of PTH. Furthermore, chemical probe tests coupled with quenching experiments confirmed that the decomposition of PS into active species, such as SO4•-, •OH, O2•- and 1O2, contributed to the removal of PTH in biochar combined with MW system, which could oxidize PTH into oxidative products, including paraoxon, 4-ethylphenol, and hydroquinone. The results of this study provide valuable insights into the synergistic effects of biochar and MW in the PS activation, which is helpful for the potential application of biochar materials combined with MW-activated PS in the remediation of pesticide-polluted soils.

Heterogeneous kinetics of the OH-initiated degradation of fenthion and parathion.

Fenthion and parathion are two representative kinds of organophosphorus pesticides and widely used in agriculture. They are directly or indirectly released into the atmosphere by spraying or volatilization processes. However, their heterogeneous reactivity toward OH radicals has not yet been well understood. Therefore, this work investigated the heterogeneous kinetics of the OH-initiated degradation of surface-bound fenthion and parathion using a flow reactor. The results showed that OH radicals played an important role in the atmospheric degradation of fenthion and parathion. Their average rate constants were (7.20 ± 0.77) × 10-12 and (10.40 ± 0.60) × 10-12 cm3/(mol· sec) at a relative humidity (RH) and temperature of 35% and 20 °C, respectively, suggesting that they have relatively short lifetimes in the atmosphere. In addition, a negative RH dependence and a positive temperature dependence of the rate constants were observed. The Arrhenius expressions of fenthion and parathion were k2 = (1.34 ± 0.48) × 10-9exp[-(1432.59 ± 105.29)/T] and k2 = (1.96 ± 1.38) × 10-9exp[-(1619.98 ± 222.02)/T], respectively, and their overall activation energy was estimated to be (11.88 ± 0.87) and (13.48 ± 1.83) kJ/mol. The experimental results will update the kinetic data of fenthion and parathion in the atmosphere and be helpful to further understand their atmospheric transportation processes.

Localized Surface Plasmon Resonance-Based Colorimetric Assay Featuring Thiol-Capped Au Nanoparticles Combined with a Mobile Application for On-Site Parathion Organophosphate Pesticide Detection.

In this study, we employed a dual strategy for parathion organophosphate pesticide (parathion) detection; first, we used a localized surface plasmon resonance (LSPR)-based colorimetric sensor featuring thiol-capped Au NPs, namely cysteine (Cys)@Au NPs, 11-mercaptoundecanoic acid (11-MUA)@Au NPs, and glutathione (GSH)@Au NPs, via acetylcholinesterase (ACHE) and acetylthiocholine (ATCH) enzyme-mediated hydrolysis reactions; second, we developed a color analysis toxicity-sensing app (Toxin APP). Positively charged thiocholine (TCH) molecules, which were continuously generated via hydrolysis, subsequently conjugated with thiol-capped Au NPs, causing Au NP aggregation through electrostatic attractions. The degree of aggregation of the thiol-capped Au NPs was influenced by parathion concentrations in the range 0 to 108 ppt, because parathion acted as an ACHE inhibitor by controlling the amount of TCH generated. Based on the values of LSPR absorbance ratio, the limits of detection (LODs) of three types thiol-capped Au NPs were determined to be 100 ppt using ultraviolet-visible spectroscopy measurements. However, the aggregation efficiency of GSH@Au NPs was lower than that of the others regarding gradual changes in their color and LSPR absorbance band. Furthermore, we designed Toxin APP for color analysis which consists of three modules: processing, database collection, and communication. Toxin APP could on-site and precisely detect the color changes of GSH@Au NPs at parathion concentrations in the ranges of 100 ppt to 1, 10, and 100 ppm and could distinguish between OP and non-OP pesticides (e.g., fipronil) in tap water samples with high sensitivity and selectivity. Moreover, the concentration of residual parathion in real samples (tomato and strawberry) was quantified based on the color changes of GSH@Au NPs detected using Toxin APP. Therefore, the combination of an LSPR-based colorimetric assay and Toxin APP can be a reliable method for the facile and rapid detection of parathion in food and water samples.

High-performance thin-layer chromatography in combination with an acetylcholinesterase-inhibition bioassay with pre-oxidation of organothiophosphates to determine neurotoxic effects in storm, waste, and surface water.

Pesticides such as organothiophosphates (OTPs) are neurotoxically active and enter the aquatic environment. Bioassays, using acetylcholinesterase (AChE), a suitable substrate and reactant, can be applied for the photometric detection of AChE-inhibiton (AChE-I) effects. The oxidized forms of OTPs, so-called oxons, have higher inhibition potentials for AChE. Therefore, a higher sensitivity is achieved for application of oxidized samples to the AChE assay. In this study, the oxidation of malathion, parathion, and chlorpyrifos by n-bromosuccinimide (NBS) was investigated in an approach combining high-performance thin-layer chromatography (HPTLC) with an AChE-I assay. Two AChE application approaches, immersion and spraying, were compared regarding sensitivity, precision, and general feasibility of the OTP effect detection. The oxidation by NBS led to an activation of the OTPs and a strong increase in sensitivity similar to the oxons tested. The sensitivity and precision of the two application techniques were similar, although the spray method was slightly more sensitive to the oxidized OTPs. The 10% inhibition concentrations (IC10) for the spray approach were 0.26, 0.75, and 0.35 ng/spot for activated malathion, parathion, and chlorpyrifos, respectively. AChE-I effect recoveries in samples from a stormwater retention basin and receiving stream were between 69 and 92% for malathion, parathion, and chlorpyrifos. The overall workflow, including sample enrichment by solid-phase extraction, HPTLC, oxidation of OTPs, and AChE-I assay, was demonstrated to be suitable for the detection of AChE-I effects in native water samples. An effect of unknown origin was found in a sample from a stormwater retention basin.

Carvacrol loaded beta cyclodextrin-alginate-chitosan based nanoflowers attenuates renal toxicity induced by malathion and parathion: A comparative toxicity.

Most of approximately 1.8 billion people involved in agriculture protect their food products using pesticides especially insecticides which may remain in foods as pesticide residues. Among insecticides organophosphates such as malathion have been widely used around the world and others such as parathion has been restricted because of their toxicity. Carvacrol (CAR) is the main component of Satureja khuzestanica. Since chemical composition of foods can alter toxicity of pesticides, in this work, the effect of coadministration of CAR and organophosphates on renal function has been studied and compared with the effect of coadministration of carvacrol loaded beta cyclodextrin-alginate-chitosan (BAC) based nanoflowers. Serum levels of urea and creatinine and histological examination were analyzed after 10 days of administration of chemicals. Malathion and parathion significantly increased urea and creatinine and induced renal inflammation. However, coadministration of CAR or BAC-CAR modified urea and creatinine and improved renal inflammation. BAC-CAR modified serum levels of urea more efficient than CAR (P < 0.05). It is concluded that BAC could be considered as a carrier for drugs used to treat renal disorders. Carvacrol can be used in the formulation of organophosphate pesticides, which may control pests more efficiently than conventional organophosphate pesticides.

Impact of Pesticide Type and Emulsion Fat Content on the Bioaccessibility of Pesticides in Natural Products.

There is interest in incorporating nanoemulsions into certain foods and beverages, including dips, dressings, drinks, spreads, and sauces, due to their potentially beneficial attributes. In particular, excipient nanoemulsions can enhance the bioavailability of nutraceuticals in fruit- and vegetable-containing products consumed with them. There is, however, potential for them to also raise the bioavailability of undesirable substances found in these products, such as pesticides. In this research, we studied the impact of excipient nanoemulsions on the bioaccessibility of pesticide-treated tomatoes. We hypothesized that the propensity for nanoemulsions to raise pesticide bioaccessibility would depend on the polarity of the pesticide molecules. Bendiocarb, parathion, and chlorpyrifos were therefore selected because they have Log P values of 1.7, 3.8, and 5.3, respectively. Nanoemulsions with different oil contents (0%, 4%, and 8%) were fabricated to study their impact on pesticide uptake. In the absence of oil, the bioaccessibility increased with increasing pesticide polarity (decreasing Log P): bendiocarb (92.9%) > parathion (16.4%) > chlorpyrifos (2.8%). Bendiocarb bioaccessibility did not depend on the oil content of the nanoemulsions, which was attributed to its relatively high water-solubility. Conversely, the bioaccessibility of the more hydrophobic pesticides (parathion and chlorpyrifos) increased with increasing oil content. For instance, for chlorpyrifos, the bioaccessibility was 2.8%, 47.0%, and 70.7% at 0%, 4%, and 8% oil content, respectively. Our findings have repercussions for the utilization of nanoemulsions as excipient foods in products that may have high levels of undesirable non-polar substances, such as pesticides.

Organophosphorus Pesticides Induce Cytokine Release from Differentiated Human THP1 Cells.

Epidemiologic studies link organophosphorus pesticides (OPs) to increased incidence of asthma. In guinea pigs, OP-induced airway hyperreactivity requires macrophages and TNF-α. Here, we determined whether OPs interact directly with macrophages to alter cytokine expression or release. Human THP1 cells were differentiated into macrophages and then exposed to parathion, chlorpyrifos, or diazinon, or their oxon, phosphate, or phosphorothioate metabolites for 24 hours in the absence or presence of reagents that block cholinergic receptors. TNF-α, IL-1ß, platelet-derived growth factor, and transforming growth factor-ß mRNA and protein were quantified by qPCR and ELISA, respectively. The effects of OPs on NF-κB, acetylcholinesterase, and intracellular calcium were also measured. Parent OPs and their oxon metabolites upregulated cytokine mRNA and stimulated cytokine release. TNF-α release, which was the most robust response, was triggered by parent, but not oxon, compounds. Cytokine expression was also increased by diethyl dithiophosphate but not diethyl thiophosphate or diethyl phosphate metabolites. Parent OPs, but not oxon metabolites, activated NF-κB. Parent and oxon metabolites decreased acetylcholinesterase activity, but comparable acetylcholinesterase inhibition by eserine did not mimic OP effects on cytokines. Consistent with the noncholinergic mechanisms of OP effects on macrophages, pharmacologic antagonism of muscarinic or nicotinic receptors did not prevent OP-induced cytokine expression or release. These data indicate that phosphorothioate OP compounds directly stimulate macrophages to release TNF-α, potentially via activation of NF-κB, and suggest that therapies that target NF-κB may prevent OP-induced airway hyperreactivity.

Colorimetric bio-barcode immunoassay for parathion based on amplification by using platinum nanoparticles acting as a nanozyme.

A competitive bio-barcode immunoassay is described for the trace detection of parathion in water, pear, cabbage, and rice samples. It is based on amplification by platinum nanoparticle acting as a nanozyme. Gold nanoparticles (AuNPs) were modified with (a) monoclonal antibodies (mAbs) against parathion, and (b) thiolated single-stranded DNA (ssDNA) oligonucleotides. Magnetic nanoparticles (MNPs) were functionalized with ovalbumin coupled with parathion hapten. Parathion and its hapten compete with mAbs on the surface of the AuNPs. Subsequently, the platinum nanoparticles (PtNPs) probe, which was functionalized with the complementary thiolated ssDNA (C-ssDNA), was added to the reaction mixture for the detection of parathion. The signal was catalytically amplified by coupling with platinum nanozyme using teramethylbenzidine and H2O2 as the chromogenic system. The immunoassay has a linear range that extends from 0.01-50 µg·L-1, and the limit of detection is 2.0 × 10-3 µg·L-1. The recoveries and relative standard deviations (RSDs) ranged from 91.1-114.4% and 3.6-15.8%, respectively. The method correlates well with data obtained by gas chromatography-tandem mass spectrometry (GC-MS/MS). Graphical abstract The parathion and the magnetic nanoparticles (MNPs) labelled with hapten-OVA competitively reacted to AuNPs modified with mAbs and thiolated DNA for the detection of parathion. The signal was catalyzed by platinum nanozyme. The limit of detection for parathion is 2.0 ng·L-1.

Safety Assessment of Compounds after In Vitro Metabolic Conversion Using Zebrafish Eleuthero Embryos.

Zebrafish-based platforms have recently emerged as a useful tool for toxicity testing as they combine the advantages of in vitro and in vivo methodologies. Nevertheless, the capacity to metabolically convert xenobiotics by zebrafish eleuthero embryos is supposedly low. To circumvent this concern, a comprehensive methodology was developed wherein test compounds (i.e., parathion, malathion and chloramphenicol) were first exposed in vitro to rat liver microsomes (RLM) for 1 h at 37 °C. After adding methanol, the mixture was ultrasonicated, placed for 2 h at -20 °C, centrifuged and the supernatant evaporated. The pellet was resuspended in water for the quantification of the metabolic conversion and the detection of the presence of metabolites using ultra high performance liquid chromatography-Ultraviolet-Mass (UHPLC-UV-MS). Next, three days post fertilization (dpf) zebrafish eleuthero embryos were exposed to the metabolic mix diluted in Danieau's medium for 48 h at 28 °C, followed by a stereomicroscopic examination of the adverse effects induced, if any. The novelty of our method relies in the possibility to quantify the rate of the in vitro metabolism of the parent compound and to co-incubate three dpf larvae and the diluted metabolic mix for 48 h without inducing major toxic effects. The results for parathion show an improved predictivity of the toxic potential of the compound.

Isolation of Bactrian Camel Single Domain Antibody for Parathion and Development of One-Step dc-FEIA Method Using VHH-Alkaline Phosphatase Fusion Protein.

A heavy chain variable fragment of heavy chain only antibodies derived from camelids termed VHH shows beneficial characteristics for immunoassay in terms of high sensitivity, outstanding stability and ease in expression. In the present study, we isolated six VHHs from phage display library against parathion, which is a widely used organophosphorus pesticide with high toxicity and persistence. One of six selected VHHs named VHH9, showed highest specificity and superior thermo-stability. A VHH9-alkaline phosphatase (AP) fusion was constructed and used to establish a one-step direct competitive fluorescence enzyme immunoassay (dc-FEIA) with a half maximal inhibitory concentration (IC50) of 1.6 ng/mL and a limit of detection of 0.2 ng/mL which was 4-fold or 3-fold higher sensitivity than direct competitive enzyme-linked immunoassay (dc-ELISA) and indirect competitive enzyme-linked immunoassay (ic-ELISA) for parathion. Furthermore, our assay indicated a 50% reduction on operation time compared with the ic-ELISA method. The presented immunoassay was validated with spiked Chinese cabbage, cucumber, and lettuce samples, and confirmed by UPLC-MS/MS. The results indicated that the VHH-AP-based dc-FEIA is a reproducible detection assay for parathion residues in vegetable samples.

SensiPath: computer-aided design of sensing-enabling metabolic pathways.

Genetically-encoded biosensors offer a wide range of opportunities to develop advanced synthetic biology applications. Circuits with the ability of detecting and quantifying intracellular amounts of a compound of interest are central to whole-cell biosensors design for medical and environmental applications, and they also constitute essential parts for the selection and regulation of high-producer strains in metabolic engineering. However, the number of compounds that can be detected through natural mechanisms, like allosteric transcription factors, is limited; expanding the set of detectable compounds is therefore highly desirable. Here, we present the SensiPath web server, accessible at http://sensipath.micalis.fr SensiPath implements a strategy to enlarge the set of detectable compounds by screening for multi-step enzymatic transformations converting non-detectable compounds into detectable ones. The SensiPath approach is based on the encoding of reactions through signature descriptors to explore sensing-enabling metabolic pathways, which are putative biochemical transformations of the target compound leading to known effectors of transcription factors. In that way, SensiPath enlarges the design space by broadening the potential use of biosensors in synthetic biology applications.

A sensitive chemiluminescence enzyme immunoassay based on molecularly imprinted polymers solid-phase extraction of parathion.

The chemiluminescence enzyme immunoassay (CLEIA) method responds differently to various sample matrices because of the matrix effect. In this work, the CLEIA method was coupled with molecularly imprinted polymers (MIPs) synthesized by precipitation polymerization to study the matrix effect. The sample recoveries ranged from 72.62% to 121.89%, with a relative standard deviation (RSD) of 3.74-18.14%.The ratio of the sample matrix-matched standard curve slope rate to the solvent standard curve slope was 1.21, 1.12, 1.17, and 0.85 for apple, rice, orange and cabbage in samples pretreated with the mixture of PSA and C18. However, the ratio of sample (apple, rice, orange, and cabbage) matrix-matched standard-MIPs curve slope rate to the solvent standard curve was 1.05, 0.92, 1.09, and 1.05 in samples pretreated with MIPs, respectively. The results demonstrated that the matrices of the samples greatly interfered with the detection of parathion residues by CLEIA. The MIPs bound specifically to the parathion in the samples and eliminated the matrix interference effect. Therefore, the CLEIA method have successfully applied MIPs in sample pretreatment to eliminate matrix interference effects and provided a new sensitive assay for agro-products.

Graphene quantum dot modified screen printed immunosensor for the determination of parathion.

The widespread use of pesticides has immense effect on increased crop productions. However, they are also responsible for posing detrimental health hazards and/or for contaminating the environment with chemical residues. A routine and an on-field detection of pesticide residues in different food, water, and soil samples has become a need of the hour for which biosensors can offer a viable alternative. The present work reports a functionalized graphene quantum dot (GQD) based screen printed electrochemical immunosensor for the detection of parathion. The application of GQDs has permitted the realization of a sensitive, robust, and reproducible sensor unlike those carried out earlier for the similar purposes. This immunosensor exhibited a dynamic linear response for parathion within the range of 0.01-106 ng/L with a very low detection limit of 46 pg/L. According to the analysis of potential interferences, the proposed sensor was specifically detecting parathion even in the presence of its metabolite, paraoxon. The investigations of the proposed sensing approach with respect to stability, response reproducibility, and regeneration have fully supported its potential practical applicability.

A method for quantitative risk appraisal for pesticide risk assessments.

Pesticide risk assessments are fraught with uncertainties that arise from the process of estimating exposure to and toxicity of chemicals. Regulatory agencies resolve those uncertainties in a health-protective (conservative) manner, typically acknowledging only inter- and intraspecies uncertainties quantitatively. Other uncertainties may be acknowledged qualitatively, but those safety factors (SF) are not enumerated. Quantitative risk appraisal may be used to enumerate the multiplicative SF generated by conservative assumptions regarding uncertainties. The magnitude of SF derived from decision points dealing with historically unquantified uncertainty may far exceed explicit SF used to gauge acceptable margins of exposure (MoE). Examination of the basis for some previously unenumerated SF may justify potential changes in regulatory practices and policies. Using past risk assessments of 3 pesticides (mevinphos, parathion, and methyl iodide) for which the California Department of Pesticide Regulation found unacceptable risk as examples, the previously unquantified SF ranged from 47 to 1 × 106 for scenarios involving handlers, reentry workers, and bystanders.

Silver nanoparticle-modified electrode for the determination of nitro compound-containing pesticides.

This paper reports the electroanalytical determination of pendimethalin and ethyl parathion by square-wave adsorptive stripping voltammetry using a material comprised of chitosan-stabilized silver nanoparticles to modify a glassy carbon electrode. Under optimized experimental conditions, the peak current was found to vary linearly with the concentration of pendimethalin in the range of 70 to 2000 nmol L(-1) and with concentration of ethyl parathion in the range of 40 to 8000 nmol L(-1). Detection limits of 36 and 40 nmol L(-1) were obtained for pendimethalin and ethyl parathion, respectively. The silver - nanoparticle-modified electrode was successfully employed for the analysis of pesticides in tap and mineral water (pendimethalin) and in lettuce and honey (ethyl parathion) samples. Pendimethalin recovery was between 94 and 100 %, and ethyl parathion recovery was between 97 and 101 %, indicating no significant matrix interference effects on the analytical results. The accuracy of the electroanalytical methodology using the proposed modified electrode was also compared to that of the UV-vis spectrophotometric method.

Vitamin K3 (menadione) redox cycling inhibits cytochrome P450-mediated metabolism and inhibits parathion intoxication.

Parathion, a widely used organophosphate insecticide, is considered a high priority chemical threat. Parathion toxicity is dependent on its metabolism by the cytochrome P450 system to paraoxon (diethyl 4-nitrophenyl phosphate), a cytotoxic metabolite. As an effective inhibitor of cholinesterases, paraoxon causes the accumulation of acetylcholine in synapses and overstimulation of nicotinic and muscarinic cholinergic receptors, leading to characteristic signs of organophosphate poisoning. Inhibition of parathion metabolism to paraoxon represents a potential approach to counter parathion toxicity. Herein, we demonstrate that menadione (methyl-1,4-naphthoquinone, vitamin K3) is a potent inhibitor of cytochrome P450-mediated metabolism of parathion. Menadione is active in redox cycling, a reaction mediated by NADPH-cytochrome P450 reductase that preferentially uses electrons from NADPH at the expense of their supply to the P450s. Using human recombinant CYP 1A2, 2B6, 3A4 and human liver microsomes, menadione was found to inhibit the formation of paraoxon from parathion. Administration of menadione bisulfite (40mg/kg, ip) to rats also reduced parathion-induced inhibition of brain cholinesterase activity, as well as parathion-induced tremors and the progression of other signs and symptoms of parathion poisoning. These data suggest that redox cycling compounds, such as menadione, have the potential to effectively mitigate the toxicity of organophosphorus pesticides including parathion which require cytochrome P450-mediated activation.

Altering the substrate specificity of methyl parathion hydrolase with directed evolution.

Many organophosphates (OPs) are used as pesticides in agriculture. They pose a severe health hazard due to their inhibitory effect on acetylcholinesterase. Therefore, detoxification of water and soil contaminated by OPs is important. Metalloenzymes such as methyl parathion hydrolase (MPH) from Pseudomonas sp. WBC-3 hold great promise as bioremediators as they are able to hydrolyze a wide range of OPs. MPH is highly efficient towards methyl parathion (1 × 10(6) s(-1) M(-1)), but its activity towards other OPs is more modest. Thus, site saturation mutagenesis (SSM) and DNA shuffling were performed to find mutants with improved activities on ethyl paraxon (6.1 × 10(3) s(-1) M(-1)). SSM was performed on nine residues lining the active site. Several mutants with modest activity enhancement towards ethyl paraoxon were isolated and used as templates for DNA shuffling. Ultimately, 14 multiple-site mutants with enhanced activity were isolated. One mutant, R2F3, exhibited a nearly 100-fold increase in the kcat/Km value for ethyl paraoxon (5.9 × 10(5) s(-1) M(-1)). These studies highlight the 'plasticity' of the MPH active site that facilitates the fine-tuning of its active site towards specific substrates with only minor changes required. MPH is thus an ideal candidate for the development of an enzyme-based bioremediation system.