-
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 -
PloS One 2016Acetylcholinesterase (AChE) is an important enzyme in the control of the neuronal action potential and sensitive to organophosphate inhibition. Brain fish AChE is less...
Acetylcholinesterase (AChE) is an important enzyme in the control of the neuronal action potential and sensitive to organophosphate inhibition. Brain fish AChE is less sensitive to organophosphate inhibition than AChE from terrestrial animals, although this sensitivity is variable among species and has not yet been fully evaluated in fish species. In this setting, inhibition kinetic constants for progressive irreversible inhibition of brain acetylcholinesterase due to methyl-paraoxon exposure were determined in three fish species (Mugil liza, Genidens genidens and Lagocephalus laevigatus) and hen (Gallus domesticus). Enzyme extraction using a detergent was shown to be adequate, and samples presented activity inhibition in high substrate concentrations and suppression of inhibition by methyl-paraoxon in the presence of the substrate, similar to kinetic patterns from purified enzyme preparations. Catfish (G. genidens) AChE presented the highest sensitivity among the evaluated fish species (IC50 = 1031.20 nM ± 63.17) in comparison to M. liza and L. laevigatus (IC50: 2878.83 ± 421.94 and 2842.5 ± 144.63 nM respectively). The lower dissociation constant (Kd = 20.3 ± 2.95 μM) of catfish AChE showed greater enzyme affinity for methyl-paraoxon, explaining this species higher sensitivity to organophosphates. Hen AChE presented higher ki (900.57 ± 65.3 mM-1min-1) and, consequently, greater sensitivity to methyl-paraoxon, explained by a lower Kd (0.6 ± 0.13 μM). Furthermore, hen AChE did not differentiate between the propionylthiocholine and acetylthiocholine substrates, indicating easier access of methyl-paraoxon to the hen enzyme activity site. The results obtained herein indicate a suitable extraction of AChE and, despite different inhibition kinetic constants, demonstrate that fish AChE is less sensitive to methyl-paraoxon, probably due to reduced access to the catalytic center which provides greater enzyme substrate selectivity.
PubMed: 27655611
DOI: 10.1371/journal.pone.0163317 -
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 -
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 -
Applied and Environmental Microbiology Sep 2014Phosphotriesterases catalyze the first step of organophosphorus triester degradation. The bacterial phosphotriesterases purified and characterized to date hydrolyze...
Phosphotriesterases catalyze the first step of organophosphorus triester degradation. The bacterial phosphotriesterases purified and characterized to date hydrolyze mainly aryl dialkyl phosphates, such as parathion, paraoxon, and chlorpyrifos. In this study, we purified and cloned two novel phosphotriesterases from Sphingomonas sp. strain TDK1 and Sphingobium sp. strain TCM1 that hydrolyze tri(haloalkyl)phosphates, and we named these enzymes haloalkylphosphorus hydrolases (TDK-HAD and TCM-HAD, respectively). Both HADs are monomeric proteins with molecular masses of 59.6 (TDK-HAD) and 58.4 kDa (TCM-HAD). The enzyme activities were affected by the addition of divalent cations, and inductively coupled plasma mass spectrometry analysis suggested that zinc is a native cofactor for HADs. These enzymes hydrolyzed not only chlorinated organophosphates but also a brominated organophosphate [tris(2,3-dibromopropyl) phosphate], as well as triaryl phosphates (tricresyl and triphenyl phosphates). Paraoxon-methyl and paraoxon were efficiently degraded by TCM-HAD, whereas TDK-HAD showed weak activity toward these substrates. Dichlorvos was degraded only by TCM-HAD. The enzymes displayed weak or no activity against trialkyl phosphates and organophosphorothioates. The TCM-HAD and TDK-HAD genes were cloned and found to encode proteins of 583 and 574 amino acid residues, respectively. The primary structures of TCM-HAD and TDK-HAD were very similar, and the enzymes also shared sequence similarity with fenitrothion hydrolase (FedA) of Burkholderia sp. strain NF100 and organophosphorus hydrolase (OphB) of Burkholderia sp. strain JBA3. However, the substrate specificities and quaternary structures of the HADs were largely different from those of FedA and OphB. These results show that HADs from sphingomonads are novel members of the bacterial phosphotriesterase family.
Topics: Amino Acid Sequence; Cloning, Molecular; Coenzymes; Hydrocarbons, Brominated; Hydrocarbons, Chlorinated; Hydrolysis; Mass Spectrometry; Molecular Sequence Data; Molecular Weight; Organophosphates; Phosphoric Triester Hydrolases; Protein Conformation; Sequence Analysis, DNA; Sequence Homology; Sphingomonadaceae; Substrate Specificity; Zinc
PubMed: 25038092
DOI: 10.1128/AEM.01845-14 -
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 -
Proteins Jun 2012TM0077 from Thermotoga maritima is a member of the carbohydrate esterase family 7 and is active on a variety of acetylated compounds, including cephalosporin C. TM0077...
TM0077 from Thermotoga maritima is a member of the carbohydrate esterase family 7 and is active on a variety of acetylated compounds, including cephalosporin C. TM0077 esterase activity is confined to short-chain acyl esters (C2-C3), and is optimal around 100°C and pH 7.5. The positional specificity of TM0077 was investigated using 4-nitrophenyl-β-D-xylopyranoside monoacetates as substrates in a β-xylosidase-coupled assay. TM0077 hydrolyzes acetate at positions 2, 3, and 4 with equal efficiency. No activity was detected on xylan or acetylated xylan, which implies that TM0077 is an acetyl esterase and not an acetyl xylan esterase as currently annotated. Selenomethionine-substituted and native structures of TM0077 were determined at 2.1 and 2.5 Å resolution, respectively, revealing a classic α/β-hydrolase fold. TM0077 assembles into a doughnut-shaped hexamer with small tunnels on either side leading to an inner cavity, which contains the six catalytic centers. Structures of TM0077 with covalently bound phenylmethylsulfonyl fluoride and paraoxon were determined to 2.4 and 2.1 Å, respectively, and confirmed that both inhibitors bind covalently to the catalytic serine (Ser188). Upon binding of inhibitor, the catalytic serine adopts an altered conformation, as observed in other esterase and lipases, and supports a previously proposed catalytic mechanism in which Ser hydroxyl rotation prevents reversal of the reaction and allows access of a water molecule for completion of the reaction.
Topics: Acetylesterase; Catalytic Domain; Computer Simulation; Crystallography, X-Ray; Enzyme Stability; Hot Temperature; Hydrogen-Ion Concentration; Models, Molecular; Protein Conformation; Reproducibility of Results; Serine; Thermotoga maritima
PubMed: 22411095
DOI: 10.1002/prot.24041 -
Journal of Clinical and Diagnostic... Nov 2015Paraoxonase 1 (PON1) is an enzyme associated with High density lipoprotein (HDL) in blood and it is considered to have antioxidant and antiatherogenic properties. PON1...
BACKGROUND
Paraoxonase 1 (PON1) is an enzyme associated with High density lipoprotein (HDL) in blood and it is considered to have antioxidant and antiatherogenic properties. PON1 plays an important role in protecting HDL and especially low density lipoprotein (LDL) from oxidative modification by hydrolyzing lipid peroxides which are known to be associated with many vascular diseases including atherosclerosis and ischemic stroke.
AIM
The aim of the study was to evaluate and correlate serum paraoxonase (PON1) and arylesterase (ARE) activities as well as lipid profile levels in patients with ischemic stroke.
MATERIALS AND METHODS
The study population was comprised of 50 ischemic stroke patients and 50 healthy controls. The serum PON1 and ARE activities were measured spectrophotometrically by using paraoxon and phenylacetate as substrate respectively by Eckerson method. Serum lipid was measured using routine biochemical method.
RESULTS
The normality of the distribution of the parameters are assessed by Shapiro-Wilk test. Two sample t-test is applied for hypothesis testing. The serum PONI and arylesterase ARE decreased significantly in ischemic stroke patients (p<0.001). The PON1 was positively correlated with HDL.
CONCLUSION
This study strongly suggests that the estimation of HDL-C associated PON1 enzyme gives valuable information for prediction of risk of ischemic stroke due to cerebrovascular thromboembolism. The result shows that PON1 and ARE could be considered as a risk factors for ischemic stroke.
PubMed: 26673997
DOI: 10.7860/JCDR/2015/15345.6707