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EFSA Journal. European Food Safety... Dec 2022The utility of pesticides in the agricultural field is unquestionable, but at the same time pesticide use presents serious hazards to the environment and the human...
The utility of pesticides in the agricultural field is unquestionable, but at the same time pesticide use presents serious hazards to the environment and the human health. For that reason, detection of pesticides and their biotransformation products in food is of utmost importance. According to previous studies, esterase-based biosensors have been proposed as a viable and efficient solution for the detection of organophosphate pesticides. In this project, a double mutant of the thermostable esterase-2 (EST2) from was studied as a potential biosensor, for its ability to detect residual amounts of pesticides. Initial characterisation of the enzyme was performed, that included determination of optimal pH, thermophilicity, as well as kinetic analysis. Subsequently, the enzyme was studied by enzymatic activity assays with and without the presence of various organophosphate compounds. The effect of the organophosphates on the enzymatic activity was measured and complete inhibition of the enzyme was observed after incubation with paraoxon. These experiments were followed by an additional method involving labelling of the enzyme with a fluorescent probe. In this case, the effect of different pesticides on the EST2 enzyme was monitored by measuring the fluorescence quenching upon addition to the enzyme. Fourteen compounds were screened with this method and significant fluorescence quenching was observed in the presence of paraoxon and methyl-paraoxon when used in equimolar amounts with the enzyme in the range of nanomolar. This biosensor has been also used to test the presence of pesticides in real food samples, like fruits and juices. This research represents a starting point to develop effective fluorescence-based biosensors aiming at the screening of mutants with different pesticide selectivity profiles. The use of this enzyme-based biosensor can have applications in the field of food traceability as well as environmental monitoring, to control the presence of toxic chemicals, in particular organophosphate pesticides.
PubMed: 36531285
DOI: 10.2903/j.efsa.2022.e200922 -
PloS One 2023Organophosphate intoxication via acetylcholinesterase inhibition executes neurotoxicity via hyper stimulation of acetylcholine receptors. Here, we use the...
Organophosphate intoxication via acetylcholinesterase inhibition executes neurotoxicity via hyper stimulation of acetylcholine receptors. Here, we use the organophosphate paraoxon-ethyl to treat C. elegans and use its impact on pharyngeal pumping as a bio-assay to model poisoning through these neurotoxins. This assay provides a tractable measure of acetylcholine receptor mediated contraction of body wall muscle. Investigation of the time dependence of organophosphate treatment and the genetic determinants of the drug-induced inhibition of pumping highlight mitigating modulation of the effects of paraoxon-ethyl. We identified mutants that reduce acetylcholine receptor function protect against the consequence of intoxication by organophosphates. Data suggests that reorganization of cholinergic signalling is associated with organophosphate poisoning. This reinforces the under investigated potential of using therapeutic approaches which target a modulation of nicotinic acetylcholine receptor function to treat the poisoning effects of this important class of neurotoxins.
Topics: Animals; Organophosphate Poisoning; Paraoxon; Cholinesterase Inhibitors; Caenorhabditis elegans; Acetylcholinesterase; Receptors, Nicotinic; Neurotoxins; Organophosphates
PubMed: 37083685
DOI: 10.1371/journal.pone.0284786 -
Archives of Toxicology Jan 2022Organophosphorus compound pesticides (OP) are widely used in pest control and might be misused for terrorist attacks. Although acetylcholinesterase (AChE) inhibition is...
Organophosphorus pesticides exhibit compound specific effects in rat precision-cut lung slices (PCLS): mechanisms involved in airway response, cytotoxicity, inflammatory activation and antioxidative defense.
Organophosphorus compound pesticides (OP) are widely used in pest control and might be misused for terrorist attacks. Although acetylcholinesterase (AChE) inhibition is the predominant toxic mechanism, OP may induce pneumonia and formation of lung edema after poisoning and during clinical treatment as life-threatening complication. To investigate the underlying mechanisms, rat precision-cut lung slices (PCLS) were exposed to the OP parathion, malathion and their biotransformation products paraoxon and malaoxon (100-2000 µmol/L). Airway response, metabolic activity, release of LDH, cytokine expression and oxidative stress response were analyzed. A concentration-dependent inhibition of airway relaxation was observed after exposure with the oxon but not with the thion-OP. In contrast, cytotoxic effects were observed for both forms in higher concentrations. Increased cytokine expression was observed after exposure to parathion and paraoxon (IL-6, GM-CSF, MIP-1α) and IL-6 expression was dependent on NFκB activation. Intracellular GSH levels were significantly reduced by all four tested OP but an increase in GSSG and HO-1 expression was predominantly observed after malaoxon exposure. Pretreatment with the antioxidant N-acetylcysteine reduced malaoxon but not paraoxon-induced cytotoxicity. PCLS as a 3D lung model system revealed OP-induced effects depending on the particular OP. The experimental data of this study contribute to a better understanding of OP toxicity on cellular targets and may be a possible explanation for the variety of clinical outcomes induced by different OP.
Topics: Acetylcholinesterase; Animals; Antioxidants; Lung; Organophosphorus Compounds; Pesticides; Rats
PubMed: 34778934
DOI: 10.1007/s00204-021-03186-x -
Journal of Enzyme Inhibition and... Dec 2021Organophosphorus poisoning caused by some pesticides and nerve agents is a life-threating condition that must be swiftly addressed to avoid casualties. Despite the...
Organophosphorus poisoning caused by some pesticides and nerve agents is a life-threating condition that must be swiftly addressed to avoid casualties. Despite the availability of medical countermeasures, the clinically available compounds lack a broad spectrum, are not effective towards all organophosphorus toxins, and have poor pharmacokinetics properties to allow them crossing the blood-brain barrier, hampering cholinesterase reactivation at the central nervous system. In this work, we designed and synthesised novel isatin derivatives, linked to a pyridinium 4-oxime moiety by an alkyl chain with improved calculated properties, and tested their reactivation potency against paraoxon- and NEMP-inhibited acetylcholinesterase in comparison to the standard antidote pralidoxime. Our results showed that these compounds displayed comparable reactivation also pointed by the studies, suggesting that they are promising compounds to tackle organophosphorus poisoning.
Topics: Acetylcholinesterase; Cholinesterase Reactivators; Computer Simulation; In Vitro Techniques; Isatin; Pyridines
PubMed: 34148470
DOI: 10.1080/14756366.2021.1916009 -
RSC Advances Dec 2022In recent years, graphene quantum dots (GQDs) received huge attention due to their unique properties and potential applicability in different area. Here, we report...
In recent years, graphene quantum dots (GQDs) received huge attention due to their unique properties and potential applicability in different area. Here, we report simple and facile method for the synthesis of GQDs and their functionalization by doping and co-doping using different heteroatom under the optimized conditions. The doping and co-doping of GQDs using boron and nitrogen have been confirmed by FTIR and TEM. The UV-visible and fluorescence techniques have been used to study the optical properties and stability of functionalized GQDs. Further, the screening for enhancement of quantum yields of all GQDs were performed with fluorescence and UV-visible spectra under the optimized conditions. The average QY was obtained as 16.0%, 83.6%, 18.2% and 29.6% for GQDs, B-GQDs, N-GQDs and B,N-GQDs, respectively. The sensor was used to determine paraoxon in water samples. The LOD was observed to be 1.0 × 10 M with linearity range of 0.001 to 0.1 M. The RSD was calculated for the developed B,N-GQDs based sensor and observed to be 2.99% with the regression coefficient as 0.997. All the doped, co-doped and un-doped GQDs possess remarkable properties as a fluorescent probe.
PubMed: 36605643
DOI: 10.1039/d2ra05275j -
Chemical Research in Toxicology Sep 2019The single residue mutation of butyrylcholinesterase (BChE) hydrolyzes a number of organophosphosphorus (OP) anticholinesterases. Whereas other BChE active site/proximal...
The single residue mutation of butyrylcholinesterase (BChE) hydrolyzes a number of organophosphosphorus (OP) anticholinesterases. Whereas other BChE active site/proximal mutations have been investigated, none are sufficiently active to be prophylactically useful. In a fundamentally different computer simulations driven strategy, we identified a surface peptide loop (residues 278-285) exhibiting dynamic motions during catalysis and modified it via residue insertions. We evaluated these loop mutants using computer simulations, substrate kinetics, resistance to inhibition, and enzyme reactivation assays using both the choline ester and OP substrates. A slight but significant increase in reactivation was noted with paraoxon with one of the mutants, and changes in and catalytic efficiency were noted in others. Simulations suggested weaker interactions between OP versus choline substrates and the active site of all engineered versions of the enzyme. The results indicate that an improvement of OP anticholinesterase hydrolysis through surface loop engineering may be a more effective strategy in an enzyme with higher intrinsic OP compound hydrolase activity.
Topics: Biocatalysis; Butyrylcholinesterase; Catalytic Domain; Cholinesterase Inhibitors; Echothiophate Iodide; Hydrolysis; Isoflurophate; Kinetics; Molecular Dynamics Simulation; Mutation; Paraoxon; Protein Binding; Protein Engineering; Thermodynamics
PubMed: 31411024
DOI: 10.1021/acs.chemrestox.9b00146 -
Molecules (Basel, Switzerland) Jan 2021Organophosphorous nerve agents (OPNA) pose an actual and major threat for both military and civilians alike, as an upsurge in their use has been observed in the recent...
Organophosphorous nerve agents (OPNA) pose an actual and major threat for both military and civilians alike, as an upsurge in their use has been observed in the recent years. Currently available treatments mitigate the effect of the nerve agents, and could be vastly improved by means of scavengers of the nerve agents. Consequently, efforts have been made over the years into investigating enzymes, also known as bioscavengers, which have the potential either to trap or hydrolyze these toxic compounds. We investigated the previously described esterase 2 from (TtEst2) as a potential bioscavenger of nerve agents. As such, we assessed its potential against G-agents (tabun, sarin, and cyclosarin), VX, as well as the pesticide paraoxon. We report that TtEst2 is a good bioscavenger of paraoxon and G-agents, but is rather slow at scavenging VX. X-ray crystallography studies showed that TtEst2 forms an irreversible complex with the aforementioned agents, and allowed the identification of amino-acids, whose mutagenesis could lead to better scavenging properties for VX. In conjunction with its cheap production and purification processes, as well as a robust structural backbone, further engineering of TtEst2 could lead to a stopgap bioscavenger useful for in corpo scavenging or skin decontamination.
Topics: Amino Acids; Crystallography, X-Ray; Esterases; Kinetics; Nerve Agents; Organophosphates; Organophosphorus Compounds; Paraoxon; Planctomycetales; Planctomycetes; Sarin
PubMed: 33513869
DOI: 10.3390/molecules26030657 -
Nanomaterials (Basel, Switzerland) Mar 2020Gold (Au) and copper (Cu)-based nanostructures are of great interest due to their applicability in various areas including catalysis, sensing and optoelectronics....
Gold (Au) and copper (Cu)-based nanostructures are of great interest due to their applicability in various areas including catalysis, sensing and optoelectronics. Nanostructures synthesized by the galvanic displacement method often lead to non-uniform density and poor size distribution. Here, density and size-controlled synthesis of Au and Cu-based nanostructures was made possible by galvanic displacement with limited exposure to hydrofluoric (HF) acid and the use of surfactants like L-cysteine (L-Cys) and cetyltrimethylammonium bromide (CTAB). An approach involving cyclic exposure to HF acid regulated the nanostructure density. Further, the use of surfactants generated monodisperse nanoparticles in the initial stages of the deposition with increased density. The characterization of Au and Cu-based nanostructures was performed by scanning electron microscopy, atomic force microscopy, UV-Visible spectroscopy, X-ray photoelectron spectroscopy, Raman spectroscopy and X-ray diffraction. The surface enhanced Raman spectroscopic measurements demonstrated an increase in the Raman intensity by two to three orders of magnitude for analyte molecules like Rhodamine 6G dye and paraoxon.
PubMed: 32235596
DOI: 10.3390/nano10040644 -
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 -
RSC Advances Mar 2022This paper demonstrates a method to fabricate plasmonic nanostructures over a large area that can be implemented as SERS substrates. The proposed method comprises batch...
This paper demonstrates a method to fabricate plasmonic nanostructures over a large area that can be implemented as SERS substrates. The proposed method comprises batch processes such as spin coating, reactive ion etching, and thin metal deposition. These processes can be performed on large wafers, resulting in large numbers of SERS substrates in a single run. The effects of different process parameters were studied to optimize the performance of the SERS substrates. The study of sensitivity on the optimized SERS substrates was conducted using the SERS-active molecule pMBA. The SERS substrates thus fabricated were able to detect molecule concentrations as low as 100 nM. The SERS substrates were also evaluated for uniformity across the sample and for sample-to-sample reproducibility. Finally, the SERS substrates were applied to demonstrate label-free detection of organophosphorous pesticides - paraoxon ethyl and paraoxon methyl.
PubMed: 35424947
DOI: 10.1039/d2ra00407k