Molecular binding of different classes of organophosphates to methyl parathion hydrolase from Ochrobactrum species.

Methyl parathion hydrolase (MPH) is an enzyme of the metallo-ß-lactamase superfamily, which hydrolyses a wide range of organophosphates (OPs). Recently, MPH has attracted attention as a promising enzymatic bioremediator. The crystal structure of MPH enzyme shows a dimeric form, with each subunit containing a binuclear metal ion center. MPH also demonstrates metal ion-dependent selectivity patterns. The origins of these patterns remain unclear but are linked to open questions about the more general role of metal ions in functional evolution and divergence within enzyme superfamilies. We aimed to investigate and compare the binding of different OP pesticides to MPH with cobalt(II) metal ions. In this study, MPH was modeled from Ochrobactrum sp. with different OP pesticides bound, including methyl paraoxon and dichlorvos and profenofos. The docked structures for each substrate optimized by DFT calculation were selected and subjected to atomistic molecular dynamics simulations for 500 ns. It was found that alpha metal ions did not coordinate with all the pesticides. Rather, the pesticides coordinated with less buried beta metal ions. It was also observed that the coordination of beta metal ions was perturbed to accommodate the pesticides. The binding free energy calculations and structure-based pharmacophore model revealed that all the three substrates could bind well at the active site. However, profenofos exhibit a stronger binding affinity to MPH in comparison to the other two substrates. Therefore, our findings provide molecular insight on the binding of different OP pesticides which could help us design the enzyme for OP pesticides degradation.

Core-shell ZnO@CoO nitrogen doped nano-composites as highly sensitive electrochemical sensor for organophosphate pesticides detection.

Core-shell ZIF-8@ZIF-67 was synthesized by growing a cobalt-based ZIF-67 on a ZIF-8 seed particle. Herein, through selective etching of the ZIF-8@ZIF-67 core and subsequent direct carbonization, core-shell hollow ZnO@CoO nitrogen-doped nanoporous carbon (HZnO@CoO-NPC) nanocomposites were prepared. HZnO@CoO-NPCs possessed a high nitrogen content, large surface area, high degree of graphitization and excellent electrical conductivity, all of which were attributed to successfully integrating the unique advantages of ZIF-8 and ZIF-67. HZnO@CoO-NPCs were used to assemble acetylcholinesterase (AChE) biosensors for organophosphorus pesticides (OPs) detection. The low detection limit of 2.74 × 10-13 M for chlorpyrifos and 7.6 × 10-15 M for parathion-methyl demonstrated the superior sensing performance. The results showed that the electrochemical biosensor constructed by HZnO@CoO-NPC provided a sensitive and efficient electrochemical strategy for OPs detection.

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.

Mitochondrial Impact of Organophosphate Pesticide-Induced Cardiotoxicity: An In Silico and In Vitro Study.

Organophosphate pesticides are widely used; however, their use is limited due to neurotoxicity and, to a lesser extent, cardiotoxicity in humans. Given the high energy demands of cardiac muscle, which is characterized by a dense population of mitochondria, any damage to these organelles can exacerbate cardiotoxicity. This study aims to elucidate whether the cardiotoxic effects of organophosphate pesticides originate from mitochondrial dysfunction. To investigate this, in silico toxicogenomic analyses were performed using various tools, such as the Comparative Toxicogenomic Database, GeneMANIA, STRING, and Cytoscape. Results revealed that 11 out of the 13 WHO-recommended Class Ia organophosphate pesticides target genes associated with cardiotoxicity. Notably, three of these genes were mitochondrial, with catalase (CAT) being the common differentially expressed gene among parathion, methyl parathion, and phorate. Furthermore, protein-protein interaction analysis indicated a strong association between CAT and superoxide dismutase 2, mitochondrial (SOD2). Subsequently, isolated heart mitochondria were utilized to assess CAT and superoxide dismutase (SOD) activities in vitro. The findings demonstrated that at a concentration of 7.5 ng/µL, both methyl parathion and phorate significantly decreased CAT activity by approximately 35%. Moreover, phorate reduced total SOD and SOD2 activities by 17% and 19%, respectively, at the same concentration. In contrast, none of the three organophosphate pesticides induced the opening of the mitochondrial permeability transition pore. These results suggest that the reduction in CAT and SOD2 activities, critical antioxidant enzymes, leads to the accumulation of reactive oxygen species within mitochondria, ultimately resulting in mitochondrial damage. This mechanism likely underlies the observed cardiotoxicity induced by these organophosphate pesticides.

Urinary para-nitrophenol levels of pregnant women and cognitive and motor function of their children aged 2 years: Evidence from the SMBCS (China).

BACKGROUND: Urinary para-nitrophenol (PNP), an exposure biomarker of ethyl parathion (EP) and methyl parathion (MP) pesticides, was still pervasively detected in the general population even after global restriction for years. And the concern whether there is an association of PNP level with child development of the nervous system is increasing. The current study aimed to evaluate the maternal urinary PNP concentrations during late pregnancy and the associations of PNP levels with cognitive and motor function of their children at the age of 2 years. METHODS: 323 mother-child pairs from the Sheyang Mini Birth Cohort Study were included in the current study. Gas chromatography-tandem mass spectrometry was used to measure concentrations of PNP, the specific metabolite of EP and MP, in maternal urine samples during pregnancy. Developmental quotients (DQs) scores measured with Gesell Developmental Scales were employed to evaluate cognitive and motor function of children aged 2 years. Generalized linear models were performed to analyze the associations of PNP concentrations in pregnant women's urine samples with cognitive and motor function of their children. RESULTS: Maternal PNP was detected in all urine samples with a median of 4.11 µg/L and a range from 0.57 µg/L to 109.13 µg/L, respectively. Maternal urinary PNP concentrations showed a negative trend with DQ of motor area [regression coefficient (ß) = - 1.35; 95 % confidence interval (95 %CI): - 2.37, - 0.33; P < 0.01], and the children whose mothers were in the fourth quartile exposure group performed significantly worse compared to the reference group (ß = - 1.11; 95 %CI: - 1.80, - 0.42; P < 0.01). As for average DQ score, children with their mothers' urinary PNP concentrations in the third quartile group had higher scores than those in the first quartile group (ß = 0.39; 95 %CI: 0.03, 0.75; P = 0.04). In sex-stratified analyses, a negative trend between maternal urinary PNP concentrations and DQ scores in motor area of children was only observed in boys (ß = - 1.62; 95 %CI: - 2.80, - 0.43; P < 0.01). Boys in the third quartile group had higher DQ average scores than those in the lowest quartile as reference (ß = 0.53; 95 %CI: 0.02, 1.04; P = 0.04). CONCLUSIONS: The mothers from SMBCS may be widely exposed to EP and/or MP, which were associated with the cognitive and motor function of their children aged 2 years in a sex-specific manner. Our results might provide epidemiology evidence on the potential effects of prenatal exposure to EP and/or MP on children's cognitive and motor function.

Proteomic analysis of Burkholderia zhejiangensis CEIB S4-3 during the methyl parathion degradation process.

Methyl parathion is an organophosphorus pesticide widely employed worldwide to control pests in agricultural and domestic environments. However, due to its intensive use, high toxicity, and environmental persistence, methyl parathion is recognized as an important ecosystem and human health threat, causing severe environmental pollution events and numerous human poisoning and deaths each year. Therefore, identifying and characterizing microorganisms capable of fully degrading methyl parathion and its degradation metabolites is a crucial environmental task for the bioremediation of pesticide-polluted sites. Burkholderia zhejiangensis CEIB S4-3 is a bacterial strain isolated from agricultural soils capable of immediately hydrolyzing methyl parathion at a concentration of 50 mg/L and degrading the 100% of the released p-nitrophenol in a 12-hour lapse when cultured in minimal salt medium. In this study, a comparative proteomic analysis was conducted in the presence and absence of methyl parathion to evaluate the biological mechanisms implicated in the methyl parathion biodegradation and resistance by the strain B. zhejiangensis CEIB S4-3. In each treatment, the changes in the protein expression patterns were evaluated at three sampling times, zero, three, and nine hours through the use of two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and the differentially expressed proteins were identified by mass spectrometry (MALDI-TOF). The proteomic analysis allowed the identification of 72 proteins with differential expression, 35 proteins in the absence of the pesticide, and 37 proteins in the experimental condition in the presence of methyl parathion. The identified proteins are involved in different metabolic processes such as the carbohydrate and amino acids metabolism, carbon metabolism and energy production, fatty acids ß-oxidation, and the aromatic compounds catabolism, including enzymes of the both p-nitrophenol degradation pathways (Hydroquinone dioxygenase and Hydroxyquinol 1,2 dioxygenase), as well as the overexpression of proteins implicated in cellular damage defense mechanisms such as the response and protection of the oxidative stress, reactive oxygen species defense, detoxification of xenobiotics, and DNA repair processes. According to these data, B. zhejiangensis CEIB S4-3 overexpress different proteins related to aromatic compounds catabolism and with the p-nitrophenol  degradation pathways, the higher expression levels observed in the two subunits of the enzyme Hydroquinone dioxygenase, suggest a preferential use of the Hydroquinone metabolic pathway in the p-nitrophenol degradation process. Moreover the overexpression of several proteins implicated in the oxidative stress response, xenobiotics detoxification, and DNA damage repair reveals the mechanisms employed by B. zhejiangensis CEIB S4-3 to counteract the adverse effects caused by the methyl parathion and p-nitrophenol exposure.

Electrochemical detection of methyl parathion using calix[6]arene/bismuth ferrite/multiwall carbon nanotube-modified fluorine-doped tin oxide electrode.

A highly sensitive electrochemical sensor using a calix[6]arene/bismuth ferrite/multiwall carbon nanotube-modified fluorine-doped tin oxide electrode (CA6/BFO/MWCNTs/FTO) was fabricated for the detection of methyl parathion. The MWCNTs, BFO, and CA6 were consecutively cast onto the FTO electrode surface to enhance the surface area, electron transfer, and selectivity of sensors. The electrochemical behavior of CA6/BFO/MWCNTs/FTO was studied via cyclic voltammetry and electrochemical impedance spectroscopy. MP was detected via cyclic voltammetry in a phosphate buffer solution at pH 7.0. The working principle of the sensor involves a linear decrease in the anodic peak current of BFO with increasing MP concentration. The linear working ranges are 0.005-0.05 nM and 0.07-1.5 nM. The CA6/BFO/MWCNTs/FTO sensor provides a low detection limit (S/N = 3) of 5 pM and a high electrochemical sensitivity of 1.23 A µM-1 cm-2. The fabricated sensor was successfully applied to assess the presence and amount of MP in vegetables and fruits (recoveries of 82.0-106.8%), with results comparable to high-performance liquid chromatography.

Electrochemical Organophosphorus Pesticide Detection Using Nanostructured Gold-Modified Electrodes.

In this study, nanostructured gold was successfully prepared on a bare Au electrode using the electrochemical deposition method. Nanostructured gold provided more exposed active sites to facilitate the ion and electron transfer during the electrocatalytic reaction of organophosphorus pesticide (methyl parathion). The morphological and structural characterization of nanostructured gold was conducted using field-emission scanning electron microscopy (FESEM), X-ray photoelectron spectroscopy (XPS), and X-ray diffraction (XRD), which was further carried out to evaluate the electrocatalytic activity towards methyl parathion sensing. The electrochemical performance of nanostructured gold was investigated by electrochemical measurements (cyclic voltammetry (CV) and differential pulse voltammetry (DPV)). The proposed nanostructured gold-modified electrode exhibited prominent electrochemical methyl parathion sensing performance (including two linear concentration ranges from 0.01 to 0.5 ppm (R2 = 0.993) and from 0.5 to 4 ppm (R2 = 0.996), limit of detection of 5.9 ppb, excellent selectivity and stability), and excellent capability in determination of pesticide residue in real fruit and vegetable samples (bok choy and strawberry). The study demonstrated that the presented approach to fabricate a nanostructured gold-modified electrode could be practically applied to detect pesticide residue in agricultural products via integrating the electrochemical and gas chromatography coupled with mass spectrometry (GC/MS-MS) analysis.

Higher-order epistasis shapes the fitness landscape of a xenobiotic-degrading enzyme.

Characterizing the adaptive landscapes that encompass the emergence of novel enzyme functions can provide molecular insights into both enzymatic and evolutionary mechanisms. Here, we combine ancestral protein reconstruction with biochemical, structural and mutational analyses to characterize the functional evolution of methyl-parathion hydrolase (MPH), an organophosphate-degrading enzyme. We identify five mutations that are necessary and sufficient for the evolution of MPH from an ancestral dihydrocoumarin hydrolase. In-depth analyses of the adaptive landscapes encompassing this evolutionary transition revealed that the mutations form a complex interaction network, defined in part by higher-order epistasis, that constrained the adaptive pathways available. By also characterizing the adaptive landscapes in terms of their functional activities towards three additional organophosphate substrates, we reveal that subtle differences in the polarity of the substrate substituents drastically alter the network of epistatic interactions. Our work suggests that the mutations function collectively to enable substrate recognition via subtle structural repositioning.

The Effect of Organophosphate Exposure on Neuronal Cell Coenzyme Q10 Status.

Organophosphate (OP) compounds are widely used as pesticides and herbicides and exposure to these compounds has been associated with both chronic and acute forms of neurological dysfunction including cognitive impairment, neurophysiological problems and cerebral ataxia with evidence of mitochondrial impairment being associated with this toxicity. In view of the potential mitochondrial impairment, the present study aimed to investigate the effect of exposure to commonly used OPs, dichlorvos, methyl-parathion (parathion) and chloropyrifos (CPF) on the cellular level of the mitochondrial electron transport chain (ETC) electron carrier, coenzyme Q10 (CoQ10) in human neuroblastoma SH-SY5Y cells. The effect of a perturbation in CoQ10 status was also evaluated on mitochondrial function and cell viability. A significant decreased (P < 0.0001) in neuronal cell viability was observed following treatment with all three OPs (100 µM), with dichlorvos appearing to be the most toxic to cells and causing an 80% loss of viability. OP treatment also resulted in a significant diminution in cellular CoQ10 status, with levels of this isoprenoid being decreased by 72% (P < 0.0001), 62% (P < 0.0005) and 43% (P < 0.005) of control levels following treatment with dichlorvos, parathion and CPF (50 µM), respectively. OP exposure was also found to affect the activities of the mitochondrial enzymes, citrate synthase (CS) and mitochondrial electron transport chain (ETC) complex II+III. Dichlorvos and CPF (50 µM) treatment significantly decreased CS activity by 38% (P < 0.0001) and 35% (P < 0.0005), respectively compared to control levels in addition to causing a 54% and 57% (P < 0.0001) reduction in complex II+III activity, respectively. Interestingly, although CoQ10 supplementation (5 µM) was able to restore cellular CoQ10 status and CS activity to control levels following OP treatment, complex II+III activity was only restored to control levels in neuronal cells exposed to dichlorvos (50 µM). However, post supplementation with CoQ10, complex II+III activity significantly increased by 33% (P < 0.0005), 25% (P < 0.005) and 35% (P < 0.0001) in dichlorvos, parathion and CPF (100 µM) treated cells respectively compared to non-CoQ10 supplemented cells. In conclusion, the results of this study have indicated evidence of neuronal cell CoQ10 deficiency with associated mitochondrial dysfunction following OP exposure. Although CoQ10 supplementation was able to ameliorate OP induced deficiencies in CS activity, ETC complex II+III activity appeared partially refractory to this treatment. Accordingly, these results indicate the therapeutic potential of CoQ10 supplementation in the treatment of OP poisoning. However, higher doses may be required to engender therapeutic efficacy.

GCMS and FTIR spectral analysis of aqueous methylparathion biotransformation by the microbial mpd strains of Pseudomonas aeruginosa and Fusarium spp.

This study aimed to find out the effective biotreatment processes of aqueous methyl parathion in stipulations of its biotransformation by potential indigenous mpd strains of Pseudomonas aeruginosa and Fusarium spp. Microbial biotransformation study of methyl parathion in contaminated synthetic wastewater at optimum conditions by P. aeruginosa mpd-5 (at temperature 33 °C, pH 7, under the aerobic condition with inoculum density of 108cells/mL) and by Fusarium spp mpd-1 (at temperature 30 °C, pH 7, under the aerobic condition with inoculum density of 25 mgL-1 dry biomass) was carried out. The major biodegradation compounds formed during the biotreatment process were analyzed by spectral studies using GCMS and FTIR. GCMS analysis of bacterial transformed compounds was p-nitrophenol, dimethylaminophenol, and glycyl-L-proline of 2-(3-methylpiperidino-4-5, 6-benzothiazin-4-one) and 2, 5-piperazinodione 3, 6-bis (2-methylpropyl). And the fungal transformed compounds were observed to be p-nitrophenol, phenol,2,4-Bis(1,1-Dimethylethyl), beta-L-arabinopyranoside-methyl, Cyclobutanethiol, 3-2(4)-thiophenone, dihydro-5-(1-methylethyl), Benzene,1,3-bis(1,1-dimethyl ethyl), Butanoic acid, 2-methyl methyl ester, and L-(+)-Ascorbic acid. The FTIR spectra of the molecule and the products of methyl parathion are observed to be changed in structures. It can be concluded from the aforementioned results and discussions that P. aeruginosa mpd-5 and Fusarium spp mpd-1 can be used in the biotreatment of pesticide wastewater having the high methyl parathion concentration and is possible by the indigenous microbial strains; they utilize as phosphate and carbon source of energy. Hence the strains can be used as a whole microbial cell, or its bioactive metabolites can be applied for the biotreatment of pesticide wastewater and potentially degrade methylparathion.

Recombinant organophosphorus hydrolase (OPH) expression in E. coli for the effective detection of organophosphate pesticides.

Accumulation and exposure of organophosphate pesticides are of great concern today owing to their abundant usage and potential health hazards. Harmful effects of organophosphate pesticide exposure and limitations of the available treatment methods necessitate the development of reliable, selective, cost-effective, and sensitive methods of detection. We developed a novel biosensor based on the enzymatic action of recombinant organophosphorus hydrolase (OPH) expressed in E. coli. We report the development of colorimetric biosensors made of His-Nus-OPH as well as His-Nus-OPH loaded alginate microspheres. The colorimetric detection method developed using solution-phase and alginate-encapsulated His-Nus-OPH exhibited detection limits of 0.045 and 0.039 mM, respectively, for ethyl paraoxon, and 0.101 and 0.049 mM, respectively, for methyl parathion. Additionally, fluorescence measurement using pH-sensitive fluorescein isothiocyanate (FITC) was used to sense the quantity of organophosphorus pesticides. The fluorometric detection method using solution-phase His-Nus-OPH, with ethyl paraoxon and methyl parathion as the substrate, reveals the lower limit of detection as 0.014 mM and 0.044 mM, respectively. Our results demonstrate the viability of His-Nus-OPH for OP detection with good sensitivity, LOD, and linear range. We report the first use of N-terminal His-NusA-tagged OPH, which enhances solubility significantly and presents a significant advance for the scientific community.

MPH-GST sensing microplate for easy detection of organophosphate insecticides.

OBJECTIVE: To develop a convenient and efficient means for organophosphate (OP) insecticide detection, a simple, cost-effective, and easy-to-use absorbance-based sensing device was generated using methyl parathion hydrolase fused with glutathione-S-transferase (MPH-GST) covalently immobilized onto a chitosan film-coated microplate. RESULTS: With methyl parathion (MP) as a representative substrate, this MPH-GST sensing microplate had the detection limit of 0.1 µM and the linear range of 0.1-50 µM. Despite its highest stability at 4 °C, it was considerably stable at 25 °C with high activity for 30 days. It was also most stable at pH 8.0 and could be efficiently reused up to 100 rounds. The device revealed a high percentage of recovery for tap water spiked with a known concentration of MP, which was also comparable to the result obtained from gas chromatography-mass spectrometry. It also showed a high recovery of 82-100% with MP spiked agricultural products and satisfactory results with non-spiked samples. This immobilized enzyme sensing system was more sensitive and efficient than the whole cell system from our previous work. CONCLUSIONS: All of the advantages of the MPH-GST sensing microplate developed have rendered it suitable for rapid and convenient OP screening, and for being a bio-element for fabricating a potential optical biosensor in the future.

Graphene oxide@Ce-doped TiO2 nanoparticles as electrocatalyst materials for voltammetric detection of hazardous methyl parathion.

A sensitive voltammetric sensor has been developed for hazardous methyl parathion detection (MP) using graphene oxide@Ce-doped TiO2 nanoparticle (GO@Ce-doped TiO2 NP) electrocatalyst. The GO@Ce-doped TiO2 NPs were prepared through the sol-gel method and characterized by various physicochemical and electrochemical techniques. The GO@Ce-doped TiO2 NP-modified glassy carbon electrode (GCE) addresses excellent electrocatalytic activity towards MP detection for environmental safety and protection. The developed strategy of GO@Ce-doped TiO2 NPs at GCE surfaces for MP detection achieved excellent sensitivity (2.359 µA µM-1 cm-2) and a low detection limit (LOD) 0.0016 µM with a wide linear range (0.002 to 48.327 µM). Moreover, the fabricated sensor shows high selectivity and long-term stability towards MP detection; this significant electrode further paves the way for real-time monitoring of environmental quantitative samples with satisfying recoveries.

A dual-mode nanoprobe for the determination of parathion methyl based on graphene quantum dots modified silver nanoparticles.

We developed a highly sensitive and selective method for double-signal analysis (fluorescence and ultraviolet-visible spectrophotometry) of organophosphorus pesticides (OPs), based on reversible quenching of graphene quantum dots (GQDs; fluorophores) with silver nanoparticles (AgNPs; absorbers). We used acetylcholinesterase to catalytically convert acetylthiocholine into thiocholine. In turn, by competitive binding to the AgNPs, the produced thiocholine displaces AgNPs from the GQDs and thus induces fluorescence recovery. However, OP analytes inhibit the activity of acetylcholinesterase and, in so doing, retain the silver-graphene nanoparticle complex and fluorescence quenching. The degree of quenching is proportional to the concentration of OPs; the detection limit is as low as 0.017 µg/L. The ultraviolet-visible absorption of GQDs/AgNPs at 390 nm decreases-because of AgNP aggregation that occurs after desorption from the GQDs-and the absorbance is linearly proportional to the OP concentration. Our system has good selectivity to substances that are commonly present in water and vegetables. We successfully applied our method to OP analysis in water, apple, and carrot samples.

Epigallocatechin-3-gallate (EGCG) protects the oocytes from methyl parathion-induced cytoplasmic deformities by suppressing oxidative and endoplasmic reticulum stress.

Methyl parathion (MP) is a commonly used organophosphorus insecticide in commercial farming. It is well known that MP exposure can affect the function of nervous, respiratory, cardiovascular and reproductive systems. In our previous report we have demonstrated that MP exposure results in poor oocyte maturation and defective embryo development which is mainly mediated through oxidative stress. The present investigation was designed to explore whether using a potent free radical scavenger like Epigallocatechin-3-gallate (EGCG) can help in reducing the detrimental effects of MP on the oocytes. For the study, germinal vesicle (GV) stage oocytes collected from the ovaries of adult Swiss albino mice were subjected to in vitro maturation (IVM) in the presence or absence of MP (100 µg/mL) and/or EGCG (0.25 µM). MP significantly reduced the nuclear maturation rate, and resulted in poor cytoplasmic organization which was evident from the altered distribution pattern of mitochondria, endoplasmic reticulum and abnormal spindle organization. These changes were associated with significant elevation in oxidative stress and expression of ER stress markers such as 78 kDa Glucose regulated protein (GRP78) as well as X-box binding protein-1 (XBP1) in the oocytes. Further, the oocytes exposed to MP had lower activation rate and developmental potential. Supplementation of EGCG during IVM not only improved the nuclear maturation rate but also reduced the cytoplasmic abnormalities. These beneficial effects appear to be due to mitigation of oxidative and ER stress in oocytes. In conclusion, results of our study indicate that EGCG can help in alleviating MP-induced oocyte abnormalities.

Magnetic solid phase extraction sorbents using methyl-parathion and quinalphos dual-template imprinted polymers coupled with GC-MS for class-selective extraction of twelve organophosphorus pesticides.

A novel magnetic dual-template molecularly imprinted polymer (DMIP) was prepared with methyl-parathion and quinalphos as templates. For comparison, a series of single-template polymers with only methyl-parathion (MPMIP) or quinalphos (QPMIP) as template as well as a non-imprinted polymer (NIP) in the absence of the template, were synthesized using the same procedure of DMIP. The obtained MIPs were characterized by scanning electron microscopy(SEM), Fourier transform infrared (FT-IR) spectroscopy, vibrating sample magnetometer (VSM), and X-ray diffraction (XRD). The properties including kinetic effect, thermodynamic effect, selectivity, and reusability of MIPs were investigated . Only DMIP possessed high affinity and good recognition for all twelve OPPs including quinalphos, isazophos, chlorpyrifos-methyl, chlorpyrifos, methidathion, triazophos, profenofos, fenthion, fenitrothion, methyl-parathion, parathion, and paraoxon in comparison to MPMIP, QPMIP, or NIP. Moreover, DMIP was used as magnetic solid phase extraction (MSPE) sorbent for the pre-concentration of twelve OPPs in cabbage samples. The developed DMIP-MSPE-GC-MS method showed high sensitivity, low LODs (1.62-13.9 ng/g), fast adsorption equilibrium (10 min), and acceptable spiked recoveries (81.5-113.4%) with relative standard deviations (RSD) in the range 0.05-7.0% (n = 3). The calibration plots were linear in the range 10-800 ng/mL with coefficients of determination (R2) better 0.99 for all twelve compounds. These results suggest that the DMIP is applicable for rapid determination and high throughput analysis of multi-pesticide residues. Graphical abstract.

Highly Efficient Abiotic Assay Formats for Methyl Parathion: Molecularly Imprinted Polymer Nanoparticle Assay as an Alternative to Enzyme-Linked Immunosorbent Assay.

Enzyme-linked immunosorbent assay (ELISA) is a widely used standard method for sensitive detection of analytes of environmental, clinical, or biotechnological interest. However, ELISA has clear drawbacks related to the use of relatively unstable antibodies and enzyme conjugates and the need for several steps such as washing of nonbound conjugates and addition of dye reagents. Herein, we introduce a new completely abiotic assay where antibodies and enzymes are replaced with fluorescent molecularly imprinted polymer nanoparticles (nanoMIPs) and target-conjugated magnetic nanoparticles, which acted as both reporter probes and binding agents. The components of the molecularly imprinted polymer nanoparticle assay (MINA) are assembled in microtiter plates fitted with magnetic inserts. We have compared the performance of a new magnetic assay with molecularly imprinted polymer (MIP)-based ELISA for the detection of methyl parathion (MP). Both assays have shown high sensitivity toward allowing detection of MP at picomolar concentrations without any cross-reactivity against chlorpyriphos and fenthion. The fully abiotic assays were also proven to detect analyte in real samples such as tap water and milk. Unlike ELISA-based systems, the novel assay required no washing steps or addition of enzyme substrates, making it more user-friendly and suitable for high throughput screening.

Catalytic Chemosensing Assay for Selective Detection of Methyl Parathion Organophosphate Pesticide.

Herein, a catalytic chemosensing assay (CCA), based on a bimetallic complex, [RuII (bpy)2 (CN)2 ]2 (CuI I)2 (bpy=2,2'-bipyridine), is described. This complex integrates a task-specific catalyst (CuI -catalyst) and a signaling unit ([RuII (bpy)2 (CN)2 ]) to specifically hydrolyze methyl parathion, a highly toxic organophosphate (OP) pesticide. The bimetallic complex catalyzed the hydrolysis of the phosphate ester to generate o,o-dimethyl thiophosphate (DTP) anion and 4-nitrophenolate. Intrinsically, 4-nitrophenolate absorbed UV/Vis light at λmax =400 nm, creating the first level of the chemosensing signal. DTP interacted with the original complex to displace the chromophore, [RuII (bpy)2 (CN)2 ], which was monitored by spectrofluorometry; this was classified as the second level of chemosensing signal. By integrating both spectroscopic and spectrofluorometric signals with a simple AND logic gate, only methyl parathion was able to provide a positive response. Other aromatic and aliphatic OP pesticides (diazinon, fenthion, meviphos, terbufos, and phosalone) and 4-nitrophenyl acetate provided negative responses. Furthermore, owing to the metal-catalyzed hydrolysis of methyl parathion, the CCA system led to the detoxification of the pesticide. The CCA system also demonstrated its catalytic chemosensing properties in the detection of methyl parathion in real samples, including tap water, river water, and underground water.

Glutathione reductase and catalase as potential biomarkers for synergistic intoxication of pesticides in fish.

Synergy occurs when chemicals give pronounced effect on combination in contrast to their individual effect. The objective of this study was to investigate the synergistic effect of pesticides carbaryl (C) and methyl parathion (MP) on oxidative stress biomarkers viz catalase (CAT), glutathione reductase (GSSG-R) including different enzymes like lactate dehydrogenase (LDH), succinate dehydrogenase (SDH) and acetyl cholinesterase (AChE) in different tissues of carps Catla catla. Fishes were exposed to 6.25 mg/L of MP and 2.3 mg/L of C in mixture (one-third of LC50 value). CAT and GSSG-R were studied in gills, brain, liver and muscle of carp were found to be elevated significantly (p < 0.005). LDH activity increased significantly (p < 0.005) in synergistic group, there was a seven-fold (748%) increase in LDH activity in muscle compared to individual studies with same pesticides. Contrary to LDH, sudden decrease in SDH activity was accounted. Significant (p < 0.005) decrease in AChE activity after initial 24 h was remarkable addressing to the shift in neurotransmission pathway in organism. Significant increase was observed in activity of CAT and GSSG-R in all tissues compared to control fishes in individual as well as synergistic (MP + C) group suggesting that CAT and GSSG-R can be a potential biomarker of oxidative stress when studied in combination.