Exposure to heavy metals, antioxidant status, and the interaction of single nucleotide polymorphisms in the genes CAT rs7943316, GSTP1 rs1695, as well as GSTM1 and GSTT1 genes, among workers in occupational settings.

Individuals working in diverse fields are consistently exposed to work-related pollutants that can impact their overall health. The current study investigated the presence of pollutants in seven different occupational groups and their impact on human health. Biochemical and genetic approaches were employed. Heavy metals were determined by ICP-MS technique. Oxidative stress biochemical markers and molecular analysis of the glutathione transferases gene SNPs (GSTT1, GSTM1, GSTP1), catalase (CAT, rs7943316), and superoxide dismutase (SOD, rs17880487) was carried out. The results revealed a significantly higher quantity of Cd among five occupational groups. Catalase, malonaldehyde, and glutathione was significantly dysregulated. Molecular analysis of the gene SNPs suggests a probable relationship between the antioxidants and the phenotypic expression of the CAT, GSTP1, GSTT1, and GSTM1 SNPs. It is concluded that chronic exposure to occupational contaminants like Cd affects human health through oxidative stress in association with some of their gene SNPs.

Chronic exposure to electronic waste poses risk to liver toxicity with molecular interaction of GSTM1, GSTT1 null variants, and GSTP1.

Chronic exposure to electronic waste (e-waste) is becoming a serious concern for health among individuals exposed to it. E-waste has been reported to contain heavy metals, trace elements, and persistent organic pollutants which can trigger health issues through different biological pathways. The liver is a major metabolic and detoxifying organ in the body. Glutathione S-transferase (GST) is a liver enzyme for phase II detoxification that catalyzes glutathione (GSH) conjugation with environmental pollutants. This research aimed to investigate the liver toxicity caused by long-term exposure to e-wastes, exploring the potential association with null variants of GSTT1 and GSTMI, as well as GSTP1. The study was designed as a cross-sectional investigation, in which 256 adult males who were chronically exposed to e-waste and 200 non-exposed control participants, matched for age and gender, were recruited randomly. Standard colorimetric and enzymatic methods were used to analyze biochemical parameters such as serum alkaline phosphatase (ALP), alanine transaminase (ALT), total bilirubin (T. Bil), albumin, and reduced glutathione. Genotypic analysis of the null variant GSTM1, GSTT1, and GSTP1 genes was conducted by standard molecular methods. The study findings indicated a notable surge in ALP, ALT, and albumin levels while T. Bil and GSH levels showed a reduction, suggesting a potential risk of liver toxicity. Additionally, analysis of GSTM1, GSTT1, and GSTP1 genotypes revealed a possible association with GSH levels and the hepatotoxicity risk. The study concluded that the individuals exposed to e-waste exhibited dysregulation of liver enzymes that results in liver toxicity. Moreover, analysis of GSTM1, GSTT1, and GSTP1 at a molecular level revealed that these genes could potentially serve as risk factors for liver toxicity in e-waste chronic exposure.

Acetylcholinesterase, pro-inflammatory cytokines, and association of ACHE SNP rs 17228602 with male infertility.

Male infertility is a complex and polygenic reproductive disease. 10-15% of the males are affected by idiopathic infertility conditions. Acetylcholine (ACh), a major neurotransmitter has been reported to play a non-neuronal role as well. Acetylcholinesterase (AChE) is the primary ACh hydrolyzing enzyme whose over or lower expression influence the availability of ACh for physiological roles. The purpose of the study was to find the possible impact and association of acetylcholinesterase, ACHE gene variant rs 17228602, and pro-inflammatory cytokines in clinically diagnosed infertile males. The study includes clinically diagnosed fifty non-infertile (control) and forty-five infertile males. Whole blood AChE enzymatic activity was measured. Genotyping of rs17228602 was carried out from peripheral blood by standard molecular methods. Pro-inflammatory cytokines were determined by the ELISA method. AChE enzyme was found to be significantly elevated in infertile than non-infertile males. ACHE SNP rs17228602 had shown significant association in dominant model (odd ratio = 0.378, 95% CI = 0.157-0.911, p-value 0.046). Pro-inflammatory cytokine IL-1ß was notably increased with statistical significance (p ≤0.05) in male infertile patients. The study concludes and speculates that AChE plays role in the pathogenesis of male infertility through the modulation of inflammatory pathways. Further studies in this direction may resolve the idiopathic cases of male infertility. Other variants of ACHE and the association of miRNA for the regulation of AChE in male infertility are suggested for further insight.

Influence of the chronic groundwater fluoride consumption on cholinergic enzymes, ACHE and BCHE gene SNPs and pro-inflammatory cytokines: A study with Pakistani population groups.

Fluoride is one of the abundant elements found in the Earth's crust and is a global environmental issue. The present work aimed to find the impact of chronic consumption of fluoride contained groundwater on human subjects. Five hundred and twelve volunteers from different areas of Pakistan were recruited. Cholinergic status, acetylcholinesterase and butyrylcholinesterase gene SNPs and pro-inflammatory cytokines were examined. Association analysis, regression and other standard statistical analyses were performed. Physical examination of the fluoride endemic areas' participants revealed the symptoms of dental and skeletal fluorosis. Cholinergic enzymes (AChE and BChE) were significantly increased among different exposure groups. ACHE gene 3'-UTR variant and BCHE K-variant showed a significant association with risk of fluorosis. Pro-inflammatory cytokines (TNF-α, IL-1ß and IL-6) were found to be increased and have a significant correlation in response to fluoride exposure and cholinergic enzymes. The study concludes that chronic consumption of high fluoride-contained water is a risk factor for developing low-grade systemic inflammation through the cholinergic pathway and the studied cholinergic gene SNPs were identified to be associated with the risk of flurosis.

Dysregulation of butyrylcholinesterase, BCHE gene SNP rs1803274, and pro-inflammatory cytokines in occupational workers.

BACKGROUND: People in different occupations are exposed to a variety of xenobiotics which affect the health and physiological processes of the body. Butyrylcholinesterase (BChE), has been reported to play neuronal and non-neuronal roles, though its exact function is yet to be established. This study aimed to find the status and role of BChE in seven different occupational groups; gasoline fillers, auto-mechanics, carpenters, textile shop workers, furniture shop workers, electricians, and office workers. METHODS: A total of 400 samples were screened. BChE activity was determined by Worek et al. method based on Ellman's principle. Pro-inflammatory cytokines were determined by ELISA. Genotypic analysis of the K-variant of BCHE gene SNP was carried out by standard molecular methods. Among seven groups, office workers were taken as a control to compare the results with all other occupational groups. RESULTS: The results revealed a significant decrease in BChE activity in gasoline fillers (79.52%) followed by carpenters (73.49%), auto mechanics (39.76%), textile shop workers (18.07%), electricians (10.84%), and furniture shop workers (7.23%). TNF-α, IL-6, and IL1-ß were elevated in all groups. IL-6 and IL1-ß in gasoline fillers, and electricians were not statistically significantly increased. Binomial regression to determine the odd ratio was found to be significant (p < 0.05) in all groups. However, correlation (Pearson) did not reveal significance between different biochemical parameters. Genotypic analysis of the K-variant SNP of the BCHE gene showed a significant association with occupational groups when compared with control which indicates a possible association with xenobiotics exposure and the physiological role of K-variant in understudied occupational groups. CONCLUSION: The study concluded that BChE and its gene SNP rs 1803274 and proinflammatory cytokines significantly dysregulates under the exposure to cumulative multiple xenobiotics in different occupational groups which may lead to pathophysiological conditions.

Association analysis of miRNA-146a and miRNA-499 polymorphisms with rheumatoid arthritis: a case-control and trio-family study.

Single nucleotide polymorphism is known to alter the expression and processing of miRNAs leading to a variety of diseases including rheumatoid arthritis (RA). However, disagreement is present up to date regarding the association of miRNA-146a and miRNA-499 polymorphisms with RA. The goal of this study was to assess the association of polymorphisms at miRNA-146a and miRNA-499 with the pathogenesis of RA in patients originating from Pakistan. Initially, eleven hundred subjects (1100) comprises of 550 RA patients and 550 healthy controls were investigated in the case-control analysis. Spectrophotometric measurement of lipids and C-reactive protein was used, whereas interleukin-1 receptor associated kinase-1 and TNF-receptor associated factor-6 values were quantified by an enzyme-linked immunosorbent assay. Secondly, heritability of susceptible alleles was tested from 70 trio-families. The miRNA-146a rs2910164 and miRNA-499 rs3746444 polymorphisms were genotyped using the polymerase chain reaction followed by restriction digestion. A Significant association of miRNA-146a and miRNA-499 genotypes was observed with RA patients (P < 0.05, respectively). The miRNA-146a rs2910164 G (OR = 1.4, P < 0.05) and miRNA-499 rs3746444 C (OR = 1.6, P < 0.0001) allele was significantly associated with RA in comparison with controls, respectively. Besides, the transmission analysis revealed a significant (P < 0.05) inheritance of rs2910164 G and rs3746444 C allele from parents to affected offspring. The current research concludes that miRNA-146a (rs2910164; C > G) and miRNA-499 (rs3746444; T > C) polymorphisms are linked to RA in the population studied. Furthermore, it was demonstrated for the first time in our high-risk cohort that the rs2910164 G and rs3746444 C allele was strongly related to familial RA.

Association analysis of single nucleotide polymorphisms in autophagy related 7 (ATG7) gene in patients with coronary artery disease.

Recent experimental studies sparked the involvement of autophagy-related 7 (ATG7) in the development of atherosclerosis. However, the genetic variants and their association with coronary artery disease (CAD) are still to be unveiled. Therefore, we aimed to design a retrospective case-control study for the analysis of ATG7 gene polymorphisms and their association with CAD among the subjects originating from Pakistan. The ATG7 noncoding polymorphisms (rs1375206; Chr3:11297643 C/G and rs550744886; Chr3:11272004 C/G) were examined in 600 subjects, including 300 individuals diagnosed with CAD. Arginase-1 (ARG1) and nitric oxide metabolites were measured by the colorimetric enzymatic assay. Genotyping of noncoding ATG7 polymorphisms was accomplished by the polymerase chain reaction-restriction fragment length polymorphism method. A significant association of ATG7 (rs1375206 and rs550744886) was observed in individuals exhibiting CAD (P < .0001, for each single-nucleotide polymorphism). Moreover, variant allele G at both loci showed high occurrence and significant association with the disease phenotype as compared to the wild-type allele (odds ratio [OR] = 2.03, P < .0001 and OR = 2.08, P < .001, respectively). Variant genotypes at ATG7 rs1375206 and rs550744886 showed significant association with high concentrations of ARG1 and low nitric oxide metabolites among the patients (P < .0001 for each). A significant difference was noted in the distribution of the haplotype G-G, mapped at Chr3:11297643-11272004 between cases and controls (P < .0001). The study concludes that ATG7 polymorphisms are among the risk factors for CAD in the subjects from Pakistan. The study thus highlights the novel risk factors for high incidents of the disease and reported for the first time to the best of our knowledge.

Genetic Association of Butyrylcholinesterase with Major Depressive Disorder.

Major depressive disorder (MDD) is characterized as clinical depression, which primarily affects the mood and behaviour of an individual. In the present study butyrylcholinesterase (BChE), a co-regulatory cholinergic neurotransmitter enzyme implicated in several putative neuronal and non-neuronal physiological roles was investigated for its role in MDD. Eighty MDD patients and sixty-one healthy controls were recruited for the study. BChE activity was measured by Ellman's method using serum while DNA samples of the patients were genotyped for BCHE polymorphisms rs3495 (c.*189G > A) and rs1803274 (c.1699G > A) by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and tetra-primer Amplification Refractory Mutation System- polymerase chain reaction (ARMS-PCR). The genotyping was further validated by Sanger Sequencing. Biochemical estimation of serum BChE levels revealed a statistically significant decrease of enzyme activity in MDD patients (69.96) as compared to healthy controls (90.97), which was independent of age and gender. BCHE single nucleotide polymorphism rs1803274 genotype GA was found to be associated with the disease under a dominant model (OR 2.32; 95% CI 1.09-4.96; p value = 0.025). Furthermore, risk allele-A frequency was higher in cases (p value = 0.013) than control. Carriers of rs1803274 GA genotype showed reduced mean BChE activity than wild-type allele GG homozygotes (p value = 0.040). Gender-based analysis revealed a protective role of rs3495 in females (χ2 = 6.87, p value = 0.032, RM: OR 0.173, CI = 0.043-0.699 (p value = 0.017). In addition, rs1803274 risk allele-A was observed to be significantly higher in males (χ2 = 4.258, p value = 0.039). In conclusion, the present study is indicative of a role of BChE in the pathophysiology of MDD where genetic polymorphisms were observed to effect BChE activity. Further replication studies in different ethnicities are recommended to validate the current observations.

Antioxidant enzymes and association of CAT SNP-21 A/T (rs7943316) with male infertility.

Infertility is a multifactorial and polygenic disease. A vast majority of infertility is still unexplained despite modern diagnostic techniques. Oxidative stress is considered a factor for male infertility but etiology in terms of functional gene polymorphism and experimental studies on human subjects is scarcely reported. The aim of the study was to investigate the status of three antioxidant enzymes; catalase, superoxide dismutase (SOD), and glutathione reduced (GSH) in clinically diagnosed infertile males and find the potential association of CAT gene variant in the promoter region -21 A/T (rs7943316). The study consisted of 55 clinically diagnosed infertile males and 50 non-infertile volunteers. The activity of antioxidant enzymes was measured through a spectrophotometer. Polymerase chain reaction-restriction fragment length polymorphism was performed for genotyping of single-nucleotide polymorphism. Catalase enzyme activity was significantly decreased while SOD and GSH were substantially increased (p ≤ 0.01) in infertile men in comparison to non-infertile. CAT gene variant rs7943316 had shown significant association in dominant, recessive model and allelic frequencies. The study concludes that rs7943316 has a substantial role in male infertility. The outcome of the study may help in resolving idiopathic infertility cases and may help in evolving novel diagnostic and therapeutic approaches. Other variants of CAT and antioxidant genes are suggested to ascertain further insight.

Experimental and Established Oximes as Pretreatment before Acute Exposure to Azinphos-Methyl.

Poisoning with organophosphorus compounds (OPCs) represents an ongoing threat to civilians and rescue personal. We have previously shown that oximes, when administered prophylactically before exposure to the OPC paraoxon, are able to protect from its toxic effects. In the present study, we have assessed to what degree experimental (K-27; K-48; K-53; K-74; K-75) or established oximes (pralidoxime, obidoxime), when given as pretreatment at an equitoxic dosage of 25% of LD01, are able to reduce mortality induced by the OPC azinphos-methyl. Their efficacy was compared with that of pyridostigmine, the only FDA-approved substance for such prophylaxis. Efficacy was quantified in rats by Cox analysis, calculating the relative risk of death (RR), with RR=1 for the reference group given only azinphos-methyl, but no prophylaxis. All tested compounds significantly (p ≤ 0.05) reduced azinphos-methyl-induced mortality. In addition, the efficacy of all tested experimental and established oximes except K-53 was significantly superior to the FDA-approved compound pyridostigmine. Best protection was observed for the oximes K-48 (RR = 0.20), K-27 (RR = 0.23), and obidoxime (RR = 0.21), which were significantly more efficacious than pralidoxime and pyridostigmine. The second-best group of prophylactic compounds consisted of K-74 (RR = 0.26), K-75 (RR = 0.35) and pralidoxime (RR = 0.37), which were significantly more efficacious than pyridostigmine. Pretreatment with K-53 (RR = 0.37) and pyridostigmine (RR = 0.52) was the least efficacious. Our present data, together with previous results on other OPCs, indicate that the experimental oximes K-27 and K-48 are very promising pretreatment compounds. When penetration into the brain is undesirable, obidoxime is the most efficacious prophylactic agent already approved for clinical use.

Chronic Exposure to Organophosphates Pesticides and Risk of Metabolic Disorder in Cohort from Pakistan and Cameroon.

(1) Background: Organophosphorus pesticides (OPPs) are major chemicals used in agriculture for eradication of insecticides/pesticides. Unfortunately, the longtime exposure of human beings to OPPs could lead to metabolic disorder such as high blood pressure, hyperglycemia, overweight or dyslipidemia. The aim of this research is to evaluate the possible metabolic dysregulations as a consequence of chronic OPPs exposure to individuals in Cameroon and Pakistan. (2) Methods: Blood samples were collected from 300 participants in each country, into ethylenediaminetetraacetic acid (EDTA) tubes. The samples were extracted with solid phase extraction (methanol/water) for analysis of OPPs with gas chromatography mass spectrometry. The spectrophotometry and Enzyme Linked ImmunoSorbent Assay (ELISA) were used to measure the hepatic, renal, pancreatic and cardiovascular functions. The atherogenic index (AI) was also determined in OPPs exposed and nonexposed cohorts. (3) Results: The results showed the presence of malathion, parathion and chlorpyrifos OPPs residues in Cameroonians, and malathion and chlorpyrifos in Pakistani samples, respectively. Elevated Body Mass Index (BMI), insulin, blood glucose, dyslipidemia and hypertension were noted in OPPs chronic exposed groups. In addition, dysregulated liver and kidney function profiles were observed in all participants regardless of gender and age groups. (4) Conclusions: The study concludes that both the study cohorts showed several metabolic dysregulations attributable to chronic exposure to a mixture of OPPs which may provide precursors for establishment of metabolic syndrome and other chronic diseases. Further different extended population-based studies are suggested to understand the differential metabolic dysfunctions caused by structurally different OPPs mixtures exposure.

Mixture of Organophosphates Chronic Exposure and Pancreatic Dysregulations in Two Different Population Samples.

Organophosphates (OP) are a major agrochemical. The application of OP pesticides is expected to increase multifold in the coming decades. The etiology of diabetic diseases is attributed to multiple factors including OP pesticide exposure. The present study investigates pancreatic dysregulation with respect to exocrine enzymes and diabesity in groups of Pakistani and Cameroonian people exposed to a mixture of OP pesticides. Nine hundred and four OP exposed individuals were enrolled for this cross-sectional study after due consent and approval from an ethical review committee. Pesticides' residues were measured by GC-MS spectrometry. Cholinergic enzymes were measured by Elman's method. Serum glucose, insulin, serum amylase, lipase, and triglyceride were measured by spectrophotometry and ELISA; HOMA-IR was determined in OP exposed and non-exposed participants. Stata 15 and R 3.2.0 software were used for statistical analysis of the data. Malathion, chlorpyrifos, and parathion residues were evident in plasma samples. RBC-acetylcholinesterase was significantly depressed in OP exposed groups. In both population samples, investigated pancreatic functions were found to be statistically significantly more dysregulated than non-exposed. OP exposure indicated risk of diabetes and insulin, glycaemia, adiponectin, triglycerides, and TNF-α dysregulations. The study concludes that both OP exposed population groups exhibited a mixture of OP residues and pancreatic dysregulation, although the effect was more pronounced in the Cameroonian population. In addition, serum lipase has a positive correlation with OP exposure and diabetes and may be suggested as an alternate/additional diagnostic marker for diabesity under OP exposure. However, screening of other environmental co-factors with OP for pancreatic dysregulation is suggested.

Analysis of PON1 gene polymorphisms (rs662 and rs854560) and inflammatory markers in organophosphate pesticides exposed cohorts from two distinct populations.

BACKGROUND AND OBJECTIVES: Organophosphate (OPs) anticholinesterases are one of the main groups of pesticides used in agriculture. Harmful effects of OPs on health have been attributed primarily for irreversible inhibition of acetylcholinesterase (AChE) at nerve synapse. However, studies have shown that inhibition of AChE alone cannot explain all the maladies encountered in prolonged exposure to OPs. Predisposition to population heterogeneity and irregularities in various biochemicals like paraoxonases and inflammatory biochemicals are the possible affects of OPs long term exposure that may lead to sequels of diseases and are less addressed in literature. The study was aimed to assess the cholinergic enzymes (AChE and BChE), PON1, and inflammatory markers (IL1ß, IL6, TNFα, CRP, Apo AI, Apo B) and determine the toxicogenetics association of PON1 gene (rs 662 and rs 85456) to chronically OPs exposed groups from Pakistan and Cameroon. MATERIALS AND METHODS: AChE, BChE and PON1 were measured by colorimetric method using spectrophotometry. Inflammatory markers were determined by Elisa assay. PCR-restriction fragment length polymorphism (PCR-RFLP) using salting out method was employed for SNP genotyping. RESULTS: The results revealed the significant (p ≤ 0.05) inhibition of cholinergic enzymes PON 1 was found to be 6.91 ng/mL±1.03 and 2.84 ng/mL±1.40 (mean ± SD) in Pakistan and Cameroon groups respectively. IL6, TNFα, CRP were increased and Apo AI was less while Apo B was increased in OP exposed groups in both population groups. SNPs analysis of PON1 showed significant differences in allelic and genotype frequencies of OPs exposed and non-exposed groups. CONCLUSIONS: PON1 was noticeably less in Cameroonian than Pakistani, albeit both groups have significant decrease in PON1 actity. In addition, the study concludes that OPs induce low grade inflammation, an aetiology of many diseases. Selected PON1 SNPs analysis showed a significant toxicogenetics association with OPs exposure marker enzymes. The results of this study may help in regulation of usage of OPs anticholinesterases in different populations. The study will further open new avenues in toxicogenetic and exploration of SNPs based strategies on organophosphate intoxication.

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.

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.

Molecular docking analyses of CYP450 monooxygenases of Tribolium castaneum (Herbst) reveal synergism of quercetin with paraoxon and tetraethyl pyrophosphate: in vivo and in silico studies.

Pest management in stored grain industry is a global issue due to the development of insecticide resistance in stored grain insect pests. Excessive use of insecticides at higher doses poses a serious threat of food contamination and residual toxicity for grain consumers. Since the development of new pesticide incurs heavy costs, identifying an effective synergist can provide a ready and economical tool for controlling resistant pest populations. Therefore, the synergistic property of quercetin with paraoxon and tetraethyl pyrophosphate has been evaluated against the larvae and adults of Tribolium castaneum (Herbst). Comparative molecular docking analyses were carried out to further identify the possible mechanism of synergism. It was observed that quercetin has no insecticidal when applied at the rate of 1.5 and 3.0 mg/g; however, a considerable synergism was observed when applied in combination with paraoxon. The comparative molecular docking analyses of CYP450 monooxygenase (CYP15A1, CYP6BR1, CYP6BK2, CYP6BK3) family were performed with quercetin, paraoxon and tetraethyl pyrophosphate which revealed considerable molecular interactions, predicting the inhibition of CYP450 isoenzyme by all three ligands. The study concludes that quercetin may be an effective synergist for organophosphate pesticides depending upon the dose and type of the compound. In addition, in silico analyses of the structurally diversified organophosphates can effectively differentiate the organophosphates which are synergistic with quercetin.

Cannabis Constituents and Acetylcholinesterase Interaction: Molecular Docking, In Vitro Studies and Association with CNR1 rs806368 and ACHE rs17228602.

The study documented here was aimed to find the molecular interactions of some of the cannabinoid constituents of cannabis with acetylcholinesterase (AChE). Molecular docking and LogP determination were performed to predict the AChE inhibitory effect and lipophilicity. AChE enzyme activity was measured in the blood of cannabis addicted human subjects. Further, genetic predisposition to cannabis addiction was investigated by association analysis of cannabinoid receptor 1 (CNR1) single nucleotide polymorphism (SNP) rs806368 and ACHE rs17228602 using restriction fragment length polymorphism (RFLP) method. All the understudied cannabis constituents showed promising binding affinities with AChE and are lipophilic in nature. The AChE activity was observed to be indifferent in cannabis addicted and non-addicted healthy controls. There was no significant association with CNR1 SNP rs806368 and ACHE rs17228602. The study concludes that in silico prediction for individual biomolecules of cannabis is different from in vivo physiological action in human subjects when all are present together. However, for a deeper mechanistic insight into these interactions and association, multi-population studies are suggested. Further studies to explore the inhibitory potential of different cannabis constituents for intended AChE inhibitor-based drug are warranted.

Combined Pre- and Posttreatment of Paraoxon Exposure.

AIMS: Organophosphates (OPCs), useful agents as pesticides, also represent a serious health hazard. Standard therapy with atropine and established oxime-type enzyme reactivators is unsatisfactory. Experimental data indicate that superior therapeutic results can be obtained when reversible cholinesterase inhibitors are administered before OPC exposure. Comparing the protective efficacy of five such cholinesterase inhibitors (physostigmine, pyridostigmine, ranitidine, tacrine, or K-27), we observed best protection for the experimental oxime K-27. The present study was undertaken in order to determine if additional administration of K-27 immediately after OPC (paraoxon) exposure can improve the outcome. METHODS: Therapeutic efficacy was assessed in rats by determining the relative risk of death (RR) by Cox survival analysis over a period of 48 h. Animals that received only pretreatment and paraoxon were compared with those that had received pretreatment and paraoxon followed by K-27 immediately after paraoxon exposure. RESULTS: Best protection from paraoxon-induced mortality was observed after pretreatment with physostigmine (RR = 0.30) and K-27 (RR = 0.34). Both substances were significantly more efficacious than tacrine (RR = 0.67), ranitidine (RR = 0.72), and pyridostigmine (RR = 0.76), which were less efficacious but still significantly reduced the RR compared to the no-treatment group (paraoxon only). Additional administration of K-27 immediately after paraoxon exposure (posttreatment) did not further reduce mortality. Statistical analysis between pretreatment before paraoxon exposure alone and pretreatment plus K-27 posttreatment did not show any significant difference for any of the pretreatment regimens. CONCLUSIONS: Best outcome is achieved if physostigmine or K-27 are administered prophylactically before exposure to sublethal paraoxon dosages. Therapeutic outcome is not further improved by additional oxime therapy immediately thereafter.

Oximes as pretreatment before acute exposure to paraoxon.

Organophosphates, useful agents as pesticides, also represent a serious danger due to their high acute toxicity. There is indication that oximes, when administered before organophosphate exposure, can protect from these toxic effects. We have tested at equitoxic dosage (25% of LD01 ) the prophylactic efficacy of five experimental (K-48, K-53, K-74, K-75, K-203) and two established oximes (pralidoxime and obidoxime) to protect from mortality induced by the organophosphate paraoxon. Mortalities were quantified by Cox analysis and compared with those observed after pretreatment with a strong acetylcholinesterase inhibitor (10-methylacridine) and after the FDA-approved pretreatment compound pyridostigmine. All nine tested substances statistically significantly reduced paraoxon-induced mortality. Best protection was conferred by the experimental oxime K-48, reducing the relative risk of death (RR) to 0.10, which was statistically significantly superior to pyridostigmine (RR = 0.31). The other oximes reduced the RR to 0.13 (obidoxime), 0.20 (K-203), 0.21 (K-74), 0.24 (K-75) and 0.26 (pralidoxime), which were significantly more efficacious than 10-methylacridine (RR = 0.65). These data support the hypothesis that protective efficacy is not primarily due to cholinesterase inhibition and indicate that the tested experimental oximes may be considered promising alternatives to the established pretreatment compound pyridostigmine.

Reactivation potency of two novel oximes (K456 and K733) against paraoxon-inhibited acetyl and butyrylcholinesterase: In silico and in vitro models.

Organophosphates (OPs) irreversibly inhibit acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes. The reactivation of these inhibited enzymes is paramount for their normal function. Present study evaluates reactivation potency of two newly developed oximes, K456 and K733, against paraoxon (POX)-inhibited human-RBC-AChE and human-plasma-BChE in comparison to reported reactivator, pralidoxime (2-PAM). In vitro studies showed higher intrinsic toxicities of both oximes than 2-PAM for AChE. No substantial reactivation of hBChE was noted by tested concentration. Contrary to 2-PAM, the in silico study predicted lower binding free energies for both oximes. However, the detailed interaction study revealed inability of oximes to interact with catalytic anionic site of AChE and hBChE in contrast to 2-PAM. Both in vitro and in silico studies conclude that K456 and K733 are unlikely to be used as reactivators of paraoxon-inhibited AChE or BChE.

Association of status of acetylcholinesterase and ACHE gene 3' UTR variants (rs17228602, rs17228616) with drug addiction vulnerability in pakistani population.

Substance addiction is a chronic, relapsing mental disorder Characterized by compulsive drug seeking, and loss of control over drug intake and relapse after prolonged abstinence. Genetics has been shown to contribute towards an individual's vulnerability to addiction. Acetylecholine (ACh), a cholinergic neurotransmitter hydrolyzed by acetylcholinesterase (AChE), is an essential neurotransmitter and neuromodulator in central and peripheral nervous system and has regulatory influence on numerous neuronal functions including addiction. The present study was carried out to investigate the role of acetylcholinesterase (AChE) in addiction through measurement of enzyme activity and to find potential association of ACHE gene 3'UTR variants rs17228602 and rs17228616 in heroin, hashish and poly drug addicts. Both SNPs are located within microRNA (miRNA) recognition sites with potential to affect miRNA/transcript interaction. A total of 122 addicts of heroin, hashish and polydrug were recruited from local rehabilitation centers to participate in this study. AChE activity was measured in blood by Ellman's method. SNP genotyping was performed by restriction fragment length polymorphism (PCR-RFLP) and Sanger sequencing. The AChE activity was found significantly higher (p ≤ 0.005) in addicted cohort (mean ±â€¯standard error of mean 0.020 ±â€¯0.001 µmol/L/min; 95% confidence interval (CI) 0.018-0.022) in comparison to non-addicted healthy subjects (0.011 ±â€¯0.001 µmol/L/min; 95% confidence interval CI 0.010-0.013). A statistically significant association of ACHE rs17228602 SNP with addiction vulnerability in dominant (DM: Odd's ratio OR = 2.095, 95% CI = 1.157-3.807 p = 0.009) and allelic genetic models (OR = 1.854 95% CI = 1.082-3.187, p = 0.016) was observed. However, no statistically significant association of rs17228616 SNP with substance abuse disorder was found. The data presented here shows that AChE could play significant role in substance addiction. Further studies with larger sample size and other variants of AChE are recommended to identify novel therapeutic approaches for cholinergic based treatment of addiction.