Oxidative Stress and Analysis of Selected SNPs of ACHE (rs 2571598), BCHE (rs 3495), CAT (rs 7943316), SIRT1 (rs 10823108), GSTP1 (rs 1695), and Gene GSTM1, GSTT1 in Chronic Organophosphates Exposed Groups from Cameroon and Pakistan.
ABSTRACT: The detrimental effects of organophosphates (OPs) on human health are thought to be of systemic, i.e., irreversible inhibition of acetylcholinesterase (AChE) at nerve synapses. However, several studies have shown that AChE inhibition alone cannot explain all the toxicological manifestations in prolonged exposure to OPs. The present study aimed to assess the status of antioxidants malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH) (reduced), catalase, and ferric reducing antioxidant power (FRAP) in chronic OP-exposed groups from Cameroon and Pakistan. Molecular analysis of genetic polymorphisms (SNPs) of glutathione transferases (GSTM1, GSTP1, GSTT1), catalase gene (CAT, rs7943316), sirtuin 1 gene (SIRT1, rs10823108), acetylcholinesterase gene (ACHE, rs2571598), and butyrylcholinesterase gene (BCHE, rs3495) were screened in the OP-exposed individuals to find the possible causative association with oxidative stress and toxicity. Cholinesterase and antioxidant activities were measured by colorimetric methods using a spectrophotometer. Salting-out method was employed for DNA extraction from blood followed by restriction fragment length polymorphism (RFLP) for molecular analysis. Cholinergic enzymes were significantly decreased in OP-exposed groups. Catalase and SOD were decreased and MDA and FRAP were increased in OP-exposed groups compared to unexposed groups in both groups. GSH was decreased only in Pakistani OPs-exposed group. Molecular analysis of ACHE, BCHE, Catalase, GSTP1, and GSTM1 SNPs revealed a tentative association with their phenotypic expression that is level of antioxidant and cholinergic enzymes. The study concludes that chronic OPs exposure induces oxidative stress which is associated with the related SNP polymorphism. The toxicogenetics of understudied SNPs were examined for the first time to our understanding. The findings may lead to a newer area of investigation on OPs induced health issues and toxicogenetics.
Project description:Agricultural pesticide handlers are at an elevated risk for overexposure to organophosphate (OP) pesticides, but symptoms can be difficult to recognise, making biomarkers invaluable for diagnosis. Occupational monitoring programmes for cholinesterase depression generally rely on measuring activity of either of the two common blood cholinesterases which serve as proxy measurements for nervous-system acetylcholinesterase activity: red blood cell acetylcholinesterase (AChE) and plasma butyrylcholinesterase (BChE). These biomarkers, however, may be affected differentially by some OPs and the relationship between them has not been well characterised. We aim to determine the association between blood AChE and BChE activity levels and assess whether they produce comparable classifications of clinical cholinesterase depression among OP pesticide handlers.Using blood samples from 215 participants of the Washington State Cholinesterase Monitoring Program, we quantified changes in AChE and BChE activity from before and after exposure to OP pesticides and calculated Pearson correlation statistics for correlation of AChE and BChE changes in activity, as well as weighted ? statistics for agreement of classification of clinical cholinesterase depression based on AChE versus BChE measurements.AChE and BChE activity measurements are weakly negatively correlated in our study population. Reaching a clinical threshold for diagnosis of cholinesterase depression based on the AChE marker did not correlate with reaching clinical depression based on the BChE marker.Both AChE and BChE should be measured in monitoring programmes because they may both give potentially important but disparate classifications of clinical cholinesterase depression.
Project description:The freshwater planarian Dugesia japonica has recently emerged as an animal model for developmental neurotoxicology and found to be sensitive to organophosphorus (OP) pesticides. While previous activity staining of D. japonica, which possess a discrete cholinergic nervous system, has shown acylthiocholine catalysis, it is unknown whether this is accomplished through an acetylcholinesterase (AChE), butyrylcholinesterase (BChE), or a hybrid esterase and how OP exposure affects esterase activity. Here, we show that the majority of D. japonica cholinesterase (DjChE) activity departs from conventional AChE and BChE classifications. Inhibition by classic protonable amine and quaternary reversible inhibitors (ethopropazine, donepezil, tacrine, edrophonium, BW284c51, propidium) shows that DjChE is far less sensitive to these inhibitors than human AChE, suggesting discrete differences in active center and peripheral site recognition and structures. Additionally, we find that different OPs (chlorpyrifos oxon, paraoxon, dichlorvos, diazinon oxon, malaoxon) and carbamylating agents (carbaryl, neostigmine, physostigmine, pyridostigmine) differentially inhibit DjChE activity in vitro. DjChE was most sensitive to diazinon oxon and neostigmine and least sensitive to malaoxon and carbaryl. Diazinon oxon-inhibited DjChE could be reactivated by the quaternary oxime, pralidoxime (2-PAM), and the zwitterionic oxime, RS194B, with RS194B being significantly more potent. Sodium fluoride (NaF) reactivates OP-DjChE faster than 2-PAM. As one of the most ancient true cholinesterases, DjChE provides insight into the evolution of a hybrid enzyme before the separation into distinct AChE and BChE enzymes found in higher vertebrates. The sensitivity of DjChE to OPs and capacity for reactivation validate the use of planarians for OP toxicology studies.
Project description:In Chile, agriculture is a relevant economic activity and is concomitant with the use of pesticides to improve the yields. Acute intoxications of agricultural workers occur with some frequency and they must be reported to the surveillance system of the Ministry of Health. However the impacts of chronic and environmental pesticide exposure have been less studied. Among pesticides frequently used in Chile for insects control are organophosphates (OP) and carbamates (CB). They are inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). In this study we determined the pattern of both biomarkers activity in three populations with different type of chronic exposure to OP/CB: environmentally exposed (EE), occupationally exposed (OE) and a reference group (RG) without exposure. Besides this, we also measured the activity of acylpeptide hydrolase (APEH), an enzyme involved in relevant functions in the central synapses that is also expressed in erythrocytes and previously reported to be highly inhibited by some OP. A baseline measurement was done in both exposure groups and then a second measurement was done during the spraying season. The RG was measured only once at any time of the year. Our results indicate that people under chronic OP/CB exposure showed an adaptive response through an increase of basal BChE activity. During the spray season only BChE activity was decreased in the EE and OE groups (p<0.05 and p<0.01, respectively) and the higher magnitude of BChE inhibition was observed in the EE group. The analysis of the frequencies of inhibition above 30% (biological tolerance limit declared by Chilean legislation) indicated that BChE was most frequently inhibited in the EE group (53% of the individuals displayed inhibition) and AChE in the OE group (55% of the individuals displayed AChE inhibition). APEH activity showed the highest frequency of inhibition in the EE group independent of its magnitude (64%). Our results demonstrate that the rural population living nearby agricultural settings displays high levels of environmental exposure. APEH activity seems to be a sensitive biomarker for acute low-level exposure and its usefulness as a routine biomarker must to be explored in future studies. Systematic biomonitoring and health outcomes studies are necessary as well as obtaining the baseline for BChE and AChE activity levels with the aim to improve environmental and occupational health policies in Chile.
Project description:Acetylcholinesterase (AChE) is the physiologically important target for organophosphorus toxicants (OP) including nerve agents and pesticides. Butyrylcholinesterase (BChE) in blood serves as a bioscavenger that protects AChE in nerve synapses from inhibition by OP. Mass spectrometry methods can detect exposure to OP by measuring adducts on the active site serine of plasma BChE. Genetic variants of human AChE and BChE do exist, but loss of function mutations have been identified only in the BCHE gene. The most common AChE variant, His353Asn (H322N), also known as the Yt blood group antigen, has normal AChE activity. The most common BChE variant, Ala567Thr (A539T) or the K-variant in honor of Werner Kalow, has 33% reduced plasma BChE activity. The genetic variant most frequently associated with prolonged response to muscle relaxants, Asp98Gly (D70G) or atypical BChE, has reduced activity and reduced enzyme concentration. Early studies in young, healthy males, performed at a time when it was legal to test nerve agents in humans, showed that individuals responded differently to the same low dose of sarin with toxic symptoms ranging in severity from minimal to moderate. Additionally, animal studies indicated that BChE protects from toxicants that have a higher reactivity with AChE than with BChE (e.g., nerve agents) but not from toxicants that have a higher reactivity with BChE than with AChE (e.g., OP pesticides). As a corollary, we hypothesize that individuals with genetic variants of BChE may be at increased risk of toxicity from nerve agents but not from OP pesticides.
Project description:Although non-innervated, the placenta contains both cholinesterases (ChEs), butyrylcholinesterase (BChE) and acetylcholinesterase (AChE). These enzymes are well-known for their multiple molecular forms. In a first approach, we used recognized specific inhibitors, substrate preferences and non-denaturating gel electrophoresis in order to characterize the ChE profile of term placenta from uncomplicated pregnancy. Results strongly suggest that the predominant cholinesterasic form present was tetrameric BChE. It is well established that both ChEs are targets of cholinesterase-inhibiting organophosphates (OP), one of the most important classes of chemicals actively applied to the environment. However, we have previously reported increased ChEs activity in placenta of rural residents exposed to OP. In the present work, we have studied: 1) whether this finding was reproducible and, 2) whether AChE or BChE up regulation is behind the increase of placental ChE activity. The population studied included forty healthy women who live in an agricultural area. Samples were collected during both the OP pulverization period (PP) and the recess period (RP). The placental ChEs activity increased in PP, evidencing reproducibility of previous results. The analysis of non-denaturating gels revealed that increased activity of total ChE activity in placenta from women exposed to OP may be attributable to tetrameric BChE up-regulation.
Project description:Organophosphate (OP) nerve agents and pesticides trigger a common mechanism of neurotoxicity resulting from critical targeting and inhibition of acetylcholinesterases (AChE) in central and peripheral synapses in the cholinergic nervous system. Therapeutic countermeasures have thus focused on either administering an oxime post-exposure, that can rapidly reactivate OP-inhibited AChE, or by preventing OP poisoning through administering pre-exposure treatments that scavenge OPs before they inhibit their physiological AChE targets. While several pyridinium aldoxime antidotes are currently approved, their utility is impaired due to their inability to cross the blood-brain barrier (BBB) efficiently. The present study utilized a macaque (Ma) model to demonstrate the efficacy of a novel zwitterionic and centrally acting oxime RS194B to reactivate sarin- and paraoxon-inhibited macaque AChE and butyrylcholinesterase (BChE) in vitro and to further assess the capacity of RS194B to effect a reversal of clinical symptoms following sarin inhalation in vivo. In vitro, oxime reactivation of MaAChE and MaBChE was shown to be comparable to their human orthologs, while the macaque studies indicated that IM administration of 62.5 mg/kg of RS194B and 0.28 mg/kg atropine after continuous exposure to 49.6 ?g/kg sarin vapor, rapidly reactivated the inhibited AChE and BChE in blood and reversed both early and advanced clinical symptoms of sarin-induced toxicity following pulmonary exposure within 1 h. The rapid cessation of autonomic and central symptoms, including convulsions, observed in macaques bodes well for the use of RS194B as an intra- or post-exposure human treatment and validates the macaque model in generating efficacy and toxicology data required for approval under the FDA Animal rule.
Project description:Acetylcholinesterase (AChE; EC 22.214.171.124), an essential enzyme of cholinergic neurotransmission in vertebrates, is a primary target in acute nerve agent and organophosphate (OP) pesticide intoxication. Catalytically inactive OP-AChE conjugates formed between the active-center serine and phosphorus of OPs can, in principle, be reactivated by nucleophilic oxime antidotes. Antidote efficacy is limited by the structural diversity of OP-AChE conjugates resulting from differences in the structure of the conjugated OP, the different active-center volumes they occupy when conjugated to the active-center serine of AChE, and the distinct chemical characteristics of both OPs and oximes documented in numerous X-ray structures of OP-conjugated AChEs. Efforts to improve oxime reactivation efficacy by AChE structure-based enhancement of oxime structure have yielded only limited success. We outline here the potential limitations of available AChE X-ray structures that preclude an accurate prediction of oxime structures, which are necessary for association in the OP-AChE gorge and nucleophilic attack of the OP-conjugated phosphorus.
Project description:Organophosphorus agents (OPs) are irreversible inhibitors of acetylcholinesterase (AChE). OP poisoning causes major cholinergic syndrome. Current medical counter-measures mitigate the acute effects but have limited action against OP-induced brain damage. Bioscavengers are appealing alternative therapeutic approach because they neutralize OPs in bloodstream before they reach physiological targets. First generation bioscavengers are stoichiometric bioscavengers. However, stoichiometric neutralization requires administration of huge doses of enzyme. Second generation bioscavengers are catalytic bioscavengers capable of detoxifying OPs with a turnover. High bimolecular rate constants (kcat/Km > 106 M-1min-1) are required, so that low enzyme doses can be administered. Cholinesterases (ChE) are attractive candidates because OPs are hemi-substrates. Moderate OP hydrolase (OPase) activity has been observed for certain natural ChEs and for G117H-based human BChE mutants made by site-directed mutagenesis. However, before mutated ChEs can become operational catalytic bioscavengers their dephosphylation rate constant must be increased by several orders of magnitude. New strategies for converting ChEs into fast OPase are based either on combinational approaches or on computer redesign of enzyme. The keystone for rational conversion of ChEs into OPases is to understand the reaction mechanisms with OPs. In the present work we propose that efficient OP hydrolysis can be achieved by re-designing the configuration of enzyme active center residues and by creating specific routes for attack of water molecules and proton transfer. Four directions for nucleophilic attack of water on phosphorus atom were defined. Changes must lead to a novel enzyme, wherein OP hydrolysis wins over competing aging reactions. Kinetic, crystallographic, and computational data have been accumulated that describe mechanisms of reactions involving ChEs. From these studies, it appears that introducing new groups that create a stable H-bonded network susceptible to activate and orient water molecule, stabilize transition states (TS), and intermediates may determine whether dephosphylation is favored over aging. Mutations on key residues (L286, F329, F398) were considered. QM/MM calculations suggest that mutation L286H combined to other mutations favors water attack from apical position. However, the aging reaction is competing. Axial direction of water attack is not favorable to aging. QM/MM calculation shows that F329H+F398H-based multiple mutants display favorable energy barrier for fast reactivation without aging.
Project description:We developed a new magnetic nanoparticle sandwich-like immunoassay using protein cage nanoparticles (PCN) for signal amplification together with graphite furnace atomic absorption spectrometry (GFAAS) for the quantification of an organophosphorylated acetylcholinesterase adduct (OP-AChE), the biomarker of exposure to organophosphate pesticides (OPs) and nerve agents. OP-AChE adducts were firstly captured by titanium dioxide coated magnetic nanoparticles (TiO2-MNPs) from the sample matrixes through metal chelation with phospho-moieties, and then selectively recognized by anti-AChE antibody labeled on PCN which was packed with lead phosphate in its cavity (PCN-anti-AChE). The sandwich-like immunoreaction was performed among TiO2-MNPs, OP-AChE and PCN-anti-AChE to form a TiO2-MNP/OP-AChE/PCN-anti-AChE immunocomplex. The complex could be easily isolated from the sample solution with the help of magnet, and the released lead ions from PCN were detected by GFAAS for the quantification of OP-AChE. Greatly enhanced sensitivity was achieved because PCN increased the amount of metal ions in the cavity of each apoferritin. The proposed immunoassay yielded a linear response over a broad range of OP-AChE concentrations from 0.01 nM to 2 nM, with a detection limit of 2 pM, which has enough sensitivity for monitoring of low-dose exposure to OPs. This new method showed an acceptable stability and reproducibility and was validated with OP-AChE spiked human plasma.
Project description:Nerve agents are chiral organophosphate compounds (OPs) that exert their acute toxicity by phosphorylating the catalytic serine of acetylcholinesterase (AChE). The inhibited cholinesterases can be reactivated using oximes, but a spontaneous time-dependent process called aging alters the adduct, leading to resistance toward oxime reactivation. Human butyrylcholinesterase (BChE) functions as a bioscavenger, protecting the cholinergic system against OPs. The stereoselectivity of BChE is an important parameter for its efficiency at scavenging the most toxic OPs enantiomer for AChE. Crystals of BChE inhibited in solution or in cristallo with racemic V-agents (VX, Russian VX, and Chinese VX) systematically show the formation of the P(S) adduct. In this configuration, no catalysis of aging seems possible as confirmed by the three-dimensional structures of the three conjugates incubated over a period exceeding a week. Crystals of BChE soaked in optically pure VX(R)-(+) and VX(S)-(-) solutions lead to the formation of the P(S) and P(R) adduct, respectively. These structural data support an in-line phosphonylation mechanism. Additionally, they show that BChE reacts with VX(R)-(+) in the presence of racemic mixture of V-agents, at odds with earlier kinetic results showing a moderate higher inhibition rate for VX(S)-(-). These combined results suggest that the simultaneous presence of both enantiomers alters the enzyme stereoselectivity. In summary, the three-dimensional data show that BChE reacts preferentially with P(R) enantiomer of V-agents and does not age, in complete contrast to AChE, which is selectively inhibited by the P(S) enantiomer and ages.