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Food Chemistry Aug 2023Herein, we developed a method coupling TLC and enzyme inhibition principles to rapidly detect OPs (dichlorvos, paraoxon and parathion). After the removal of the organic...
Herein, we developed a method coupling TLC and enzyme inhibition principles to rapidly detect OPs (dichlorvos, paraoxon and parathion). After the removal of the organic solvent from the samples using TLC and paper-based chips, the enzyme was added to the detection system. The results showed that the current method effectively reduced the effects of solvents on enzyme behavior. Moreover, the pigments could be successfully retained on TLC with 40% ddHO/ACN solution (v/v) as a developing solvent. Additionally, the detection limits (LODs) were 0.002 µg/mL for dichlorvos, 0.006 µg/mL for paraoxon, and 0.003 µg/mL for parathion. Finally, the method was applied to spiked cabbage, cucumber, and spinach and showed good average recoveries ranging between 70.22% and 119.79%. These results showed that this paper-based chip had high sensitivity, precleaning, and elimination of organic solvent properties. Furthermore, it provides a valuable idea for sample pretreatment and rapid determination of pesticide residues in food.
Topics: Pesticides; Dichlorvos; Chromatography, Thin Layer; Paraoxon; Pesticide Residues; Parathion; Solvents
PubMed: 36893638
DOI: 10.1016/j.foodchem.2023.135822 -
Analytical Sciences : the International... Sep 2023Fusion proteins composed of an organophosphorus hydrolase (OPH) and pHluorin, a pH-sensitive green fluorescent protein variant, were constructed as whole-cell biosensors...
Fusion proteins composed of an organophosphorus hydrolase (OPH) and pHluorin, a pH-sensitive green fluorescent protein variant, were constructed as whole-cell biosensors to measure organophosphorus pesticides. pHluorin was used to detect the pH changes because of the hydrolase of paraoxon by OPH. To examine the order of fusion of OPH and pHluorin, pHluorin-OPH and OPH-pHluorin fusion proteins were constructed. In addition, a peptide linker consisting of 15 amino acid was inserted between pHluorin and OPH to reduce steric hindrance. OPH and pHluorin activities were evaluated in cells expressing the four fusion proteins. The both activities of pHluorin-OPH and pHluorin-linker-OPH were higher than that of OPH-pHluorin and OPH-linker-pHluorin. Effects of the peptide linker on the activities were slight. Therefore, pHluorin-OPH and pHluorin-linker-OPH were found to be suitable for organophosphorus pesticide measurements. Using cells expressing pHluorin-linker-OPH, 0.5 μg/mL of paraoxon could be measured.
Topics: Pesticides; Biosensing Techniques; Paraoxon; Single-Cell Analysis
PubMed: 37264267
DOI: 10.1007/s44211-023-00369-7 -
RSC Advances Oct 2023The use of organophosphate (OPs) pesticides is widespread in agriculture and horticulture, but these chemicals can be lethal to humans, causing fatalities and deaths...
The use of organophosphate (OPs) pesticides is widespread in agriculture and horticulture, but these chemicals can be lethal to humans, causing fatalities and deaths each year. The inhibition of acetylcholinesterase (AChE) by OPs leads to the overstimulation of cholinergic receptors, ultimately resulting in respiratory arrest, seizures, and death. Although 2-pralidoxime (2-PAM) is the FDA-approved drug for treating OP poisoning, there is difficulty in blood-brain barrier permeation. To address this issue, we designed and evaluated a series of 2-PAM analogs by substituting electron-donating groups on the and/or positions of the pyridinium core using techniques. Our PCM-ONIOM2 (MP2/6-31G*:PM7//B3LYP/6-31G*:UFF) binding energy results demonstrated that 13 compounds exhibited higher binding energy than 2-PAM. The analog with phenyl and methyl groups substituted on the and positions, respectively, showed the most favorable binding characteristics, with aromatic residues in the active site (Y124, W286, F297, W338, and Y341) and the catalytic residue S203 covalently bonding with paraoxon. The results of DS-MD simulation revealed a highly favorable apical conformation of the potent analog, which has the potential to enhance reactivation of AChE. Importantly, newly designed compound demonstrated appropriate drug-likeness properties and blood-brain barrier penetration. These results provide a rational guide for developing new antidotes to treat organophosphate insecticide toxicity.
PubMed: 37928857
DOI: 10.1039/d3ra03087c -
The Journal of Pharmacology and... Jan 2024Organophosphate (OP) compounds are highly toxic and include pesticides and chemical warfare nerve agents. OP exposure inhibits the acetylcholinesterase enzyme, causing...
Organophosphate (OP) compounds are highly toxic and include pesticides and chemical warfare nerve agents. OP exposure inhibits the acetylcholinesterase enzyme, causing cholinergic overstimulation that can evolve into status epilepticus (SE) and produce lethality. Furthermore, OP-induced SE survival is associated with mood and memory dysfunction and spontaneous recurrent seizures (SRS). In male Sprague-Dawley rats, we assessed hippocampal pathology and chronic SRS following SE induced by administration of OP agents paraoxon (2 mg/kg, s.c.), diisopropyl fluorophosphate (4 mg/kg, s.c.), or O-isopropyl methylphosphonofluoridate (GB; sarin) (2 mg/kg, s.c.), immediately followed by atropine and 2-PAM. At 1-hour post-OP-induced SE onset, midazolam was administered to control SE. Approximately 6 months after OP-induced SE, SRS were evaluated using video and electroencephalography monitoring. Histopathology was conducted using hematoxylin and eosin (H&E), while silver sulfide (Timm) staining was used to assess mossy fiber sprouting (MFS). Across all the OP agents, over 60% of rats that survived OP-induced SE developed chronic SRS. H&E staining revealed a significant hippocampal neuronal loss, while Timm staining revealed extensive MFS within the inner molecular region of the dentate gyrus. This study demonstrates that OP-induced SE is associated with hippocampal neuronal loss, extensive MFS, and the development of SRS, all hallmarks of chronic epilepsy. SIGNIFICANCE STATEMENT: Models of organophosphate (OP)-induced SE offer a unique resource to identify molecular mechanisms contributing to neuropathology and the development of chronic OP morbidities. These models could allow the screening of targeted therapeutics for efficacious treatment strategies for OP toxicities.
Topics: Rats; Male; Animals; Rats, Sprague-Dawley; Mossy Fibers, Hippocampal; Organophosphates; Acetylcholinesterase; Status Epilepticus; Epilepsy; Seizures; Disease Models, Animal
PubMed: 37643794
DOI: 10.1124/jpet.123.001739 -
Biomolecules May 2024Olesoxime, a cholesterol derivative with an oxime group, possesses the ability to cross the blood-brain barrier, and has demonstrated excellent safety and tolerability...
Olesoxime, a cholesterol derivative with an oxime group, possesses the ability to cross the blood-brain barrier, and has demonstrated excellent safety and tolerability properties in clinical research. These characteristics indicate it may serve as a centrally active ligand of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), whose disruption of activity with organophosphate compounds (OP) leads to uncontrolled excitation and potentially life-threatening symptoms. To evaluate olesoxime as a binding ligand and reactivator of human AChE and BChE, we conducted kinetic studies with the active metabolite of insecticide parathion, paraoxon, and the warfare nerve agents sarin, cyclosarin, tabun, and VX. Our results showed that both enzymes possessed a binding affinity for olesoxime in the mid-micromolar range, higher than the antidotes in use (i.e., 2-PAM, HI-6, etc.). While olesoxime showed a weak ability to reactivate AChE, cyclosarin-inhibited BChE was reactivated with an overall reactivation rate constant comparable to that of standard oxime HI-6. Moreover, in combination with the oxime 2-PAM, the reactivation maximum increased by 10-30% for cyclosarin- and sarin-inhibited BChE. Molecular modeling revealed productive interactions between olesoxime and BChE, highlighting olesoxime as a potentially BChE-targeted therapy. Moreover, it might be added to OP poisoning treatment to increase the efficacy of BChE reactivation, and its cholesterol scaffold could provide a basis for the development of novel oxime antidotes.
Topics: Humans; Butyrylcholinesterase; Acetylcholinesterase; Ligands; Oximes; Cholinesterase Reactivators; Cholinesterase Inhibitors; Cholestenones; Kinetics; Sarin; GPI-Linked Proteins; Antidotes; Cholesterol; Organophosphorus Compounds
PubMed: 38785995
DOI: 10.3390/biom14050588 -
Bioorganic & Medicinal Chemistry Letters Nov 2023This study aimed to explore non-pyridinium oxime acetylcholinesterase (AChE) reactivators that could hold the potential to overcome the limitations of the currently...
This study aimed to explore non-pyridinium oxime acetylcholinesterase (AChE) reactivators that could hold the potential to overcome the limitations of the currently available compounds used in the clinic to treat the neurologic manifestations induced by intoxication with organophosphorus agents. Fifteen compounds with various non-pyridinium oxime moieties were evaluated for AChE activity at different concentrations, including aldoximes, ketoximes, and α-ketoaldoximes. The therapeutic potential of the oxime compounds was evaluated by assessing their ability to reactivate AChE inhibited by paraoxon. Among the tested compounds, α-Ketoaldoxime derivative 13 showed the highest reactivation (%) reaching 67 % and 60 % AChE reactivation when evaluated against OP-inhibited electric eel AChE at concentrations of 1,000 and 100 μM, respectively. Compound 13 showed a comparable reactivation ability of AChE (60 %) compared to that of pralidoxime (56 %) at concentrations of 100 μM. Molecular docking simulation of the most active compounds 12 and 13 was conducted to predict the binding mode of the reactivation of electric eel AChE. As a result, a non-pyridinium oxime moiety 13, is a potential reactivator of OP-inhibited AChE and is taken as a lead compound for the development of novel AChE reactivators with enhanced capacity to freely cross the blood-brain barrier.
Topics: Oximes; Paraoxon; Acetylcholinesterase; Cholinesterase Reactivators; Cholinesterase Inhibitors; Molecular Docking Simulation; Pyridinium Compounds; Acetamides; Organophosphorus Compounds
PubMed: 37838342
DOI: 10.1016/j.bmcl.2023.129504 -
Drug and Chemical Toxicology Jan 2024A series of uncharged salicylaldehyde oximes were synthesized and evaluated for the reactivation of organophosphorus (OP) nerve agents simulants Diethylchlorophosphonate...
A series of uncharged salicylaldehyde oximes were synthesized and evaluated for the reactivation of organophosphorus (OP) nerve agents simulants Diethylchlorophosphonate (DCP) & Diethylcyanophosphonate (DCNP) and pesticides (paraoxon & malaoxon) inhibited Acetylcholinesterase AChE). The computational software Swiss ADME and molinspiration were used to unfold the probability of drug-likeness properties of the oximes derivatives. Substituted aromatic oximes with diethylamino or bromo group with free hydroxyl group ortho to oxime moiety were found efficient to regenerate the enzymatic activity in AChE assay. The alkylation of the ortho hydroxyl group of derivatives led to the loss of reactivation potential. The derivatives with a hydroxyl group and without oxime group and vice versa did not show significant reactivation potency against tested OP toxicants. Further, we also evaluated the reactivation potential of these selected molecules on the rat brain homogenate against different OPs inhibited ChE and found maximum reactivation potency of oxime . The results were further validated by molecular docking and dynamic studies which showed that the hydroxyl group interacted with serine amino acids in the catalytic anionic site of AChE enzyme and was stable up to 200 ns consequently providing proper orientation to oxime moiety for reactivating the OP inhibited enzyme. It has thus been proved by the structure-activity relationship of oximes derivatives that hydroxyl group ortho to oxime is essential for reactivating OP inhibited AChE. Amongst the twenty-one oximes derivatives, was found to be most active in regenerating the paraoxon, malaoxon, DCP and DCNP inhibited AChE enzyme.
Topics: Cholinesterase Inhibitors; Paraoxon; Cholinesterase Reactivators; Acetylcholinesterase; Molecular Docking Simulation; Oximes; Organophosphorus Compounds; Malathion; Nitrophenols
PubMed: 36514993
DOI: 10.1080/01480545.2022.2150210 -
International Journal of Biological... Jun 2024In this present study, characteristics and structure-function relationship of an organophosphate-degrading enzyme from Bacillus sp. S3wahi were described. S3wahi...
Identification and characterization of a promiscuous metallohydrolase in metallo-β-lactamase superfamily from a locally isolated organophosphate-degrading Bacillus sp. strain S3wahi.
In this present study, characteristics and structure-function relationship of an organophosphate-degrading enzyme from Bacillus sp. S3wahi were described. S3wahi metallohydrolase, designated as S3wahi-MH (probable metallohydrolase YqjP), featured the conserved αβ/βα metallo-β-lactamase-fold (MBL-fold) domain and a zinc bimetal at its catalytic site. The metal binding site of S3wahi-MH also preserves the H-X-H-X-D-H motif, consisting of specific amino acids at Zn1 (Asp69, His70, Asp182, and His230) and Zn2 (His65, His67, and His137). The multifunctionality of S3wahi-MH was demonstrated through a steady-state kinetic study, revealing its highest binding affinity (K) and catalytic efficiency (k/K) for OP compound, paraoxon, with values of 8.09 × 10 M and 4.94 × 10 M s, respectively. Using OP compound, paraoxon, as S3wahi-MH native substrate, S3wahi-MH exhibited remarkable stability over a broad temperature range, 20 °C - 60 °C and a broad pH tolerance, pH 6-10. Corresponded to S3wahi-MH thermal stability characterization, the estimated melting temperature (T) was found to be 72.12 °C. S3wahi-MH was also characterized with optimum catalytic activity at 30 °C and pH 8. Additionally, the activity of purified S3wahi-MH was greatly enhanced in the presence of 1 mM and 5 mM of manganese (Mn), showing relative activities of 1323.68 % and 2073.68 %, respectively. The activity of S3wahi-MH was also enhanced in the presence of DMSO and DMF, showing relative activities of 270.37 % and 307.41 %, respectively. The purified S3wahi-MH retained >60 % residual activity after exposure to non-ionic Tween series surfactants. Nevertheless, the catalytic activity of S3wahi-MH was severely impacted by the treatment of SDS, even at low concentrations. Considering its enzymatic properties and promiscuity, S3wahi-MH emerges as a promising candidate as a bioremediation tool in wide industrial applications, including agriculture industry.
Topics: Bacillus; beta-Lactamases; Kinetics; Substrate Specificity; Enzyme Stability; Hydrogen-Ion Concentration; Catalytic Domain; Amino Acid Sequence; Organophosphates; Bacterial Proteins; Temperature
PubMed: 38761915
DOI: 10.1016/j.ijbiomac.2024.132395 -
Micromachines Aug 2023The ongoing advancement in the synthesis of new nanomaterials has accelerated the rapid development of non-enzymatic pesticide sensors based on electrochemical...
Development and Characterization of Nano-Ink from Silicon Carbide/Multi-Walled Carbon Nanotubes/Synthesized Silver Nanoparticles for Non-Enzymatic Paraoxon Residuals Detection.
The ongoing advancement in the synthesis of new nanomaterials has accelerated the rapid development of non-enzymatic pesticide sensors based on electrochemical platforms. This study aims to develop and characterize Nano-ink for applying organophosphorus pesticides using paraoxon residue detection. Multi-walled carbon nanotubes, silicon carbide, and silver nanoparticles were used to create Nano-ink using a green synthesis process in 1:1:0, 1:1:0.5, and 1:1:1 ratios, respectively. These composites were combined with chitosan of varying molecular weights, which served as a stabilizing glue to keep the Nano-ink employed in a functioning electrode stable. By using X-ray powder diffraction, Raman spectroscopy, energy dispersive X-ray spectroscopy, and a field emission scanning electron microscope, researchers were able to examine the crystallinity, element composition, and surface morphology of Nano-ink. The performance of the proposed imprinted working electrode Nano-ink was investigated using cyclic voltammetry and differential pulse voltammetry techniques. The Cyclic voltammogram of Ag NPs/chitosan (medium, 50 mg) illustrated high current responses and favorable conditions of the Nano-ink modified electrode. Under the optimized conditions, the reduction currents of paraoxon using the DPV techniques demonstrated a linear reaction ranging between 0.001 and 1.0 µg/mL (R = 0.9959) with a limit of detection of 0.0038 µg/mL and a limit of quantitation of 0.011 µg/mL. It was concluded that the fabricated Nano-ink showed good electrochemical activity for non-enzymatic paraoxon sensing.
PubMed: 37630149
DOI: 10.3390/mi14081613 -
ACS Applied Materials & Interfaces Jun 2024The catalytic efficiency of enzymes can be harnessed as an environmentally friendly solution for decontaminating various xenobiotics and toxins. However, for some...
The catalytic efficiency of enzymes can be harnessed as an environmentally friendly solution for decontaminating various xenobiotics and toxins. However, for some xenobiotics, several enzymatic steps are needed to obtain nontoxic products. Another challenge is the low durability and stability of many native enzymes in their purified form. Herein, we coupled peptide-based encapsulation of bacterial phosphotriesterase with soil-originated bacteria, sp. 4Hβ as an efficient system capable of biodegradation of paraoxon, a neurotoxin pesticide. Specifically, recombinantly expressed and purified methyl parathion hydrolase (MPH), with high hydrolytic activity toward paraoxon, was encapsulated within peptide nanofibrils, resulting in increased shelf life and retaining ∼50% activity after 132 days since purification. Next, the addition of sp. 4Hβ, capable of degrading para-nitrophenol (PNP), the hydrolysis product of paraoxon, which is still toxic, resulted in nondetectable levels of PNP. These results present an efficient one-pot system that can be further developed as an environmentally friendly solution, coupling purified enzymes and native bacteria, for pesticide bioremediation. We further suggest that this system can be tailored for different xenobiotics by encapsulating the rate-limiting key enzymes followed by their combination with environmental bacteria that can use the enzymatic step products for full degradation without the need to engineer synthetic bacteria.
PubMed: 38920304
DOI: 10.1021/acsami.4c06501