Erythrocyte Acetylcholinesterase Activity Research Articles

Acute poisoning by chlorpyrifos differentially impacts survival and cardiorespiratory function in normotensive and hypertensive rats

Hypertension is the most important and well-known risk factor for cardiovascular disease (CVD). Recently, acute organophosphate (OP) poisoning has also been pointed as a CVD risk factor. Despite this evidence, no studies have contrasted the acute toxicosis and cardiovascular (CV) effects of OP poisoning under conditions of normotension and hypertension. In this work, adult male normotensive Wistar and Spontaneously Hypertensive rats (SHR) were intraperitoneally injected with saline or chlorpyrifos (CPF), an OP compound, monitored for acute toxicosis signs and 24-h survival. After poisoning, blood pressure, heart rate and ventilation were recorded, the Bezold-Jarisch Reflex (BJR), the Chemoreflex (CR) were chemically activated, as well as the cardiac autonomic tone (AUT) was assessed. Erythrocyte and brainstem acetylcholinesterase and plasmatic butyrylcholinesterase (BuChE) activities were measured as well as lipid peroxidation, advanced oxidation protein products (AOPP), nitrite/nitrate levels, expression of catalase, TNFα and angiotensin-I converting enzyme (ACE-1) within the brainstem. CPF induced a much more pronounced acute toxicosis and 33 % lethality in SHR. CPF poisoning impaired ventilation in SHR, the BJR reflex responses in Wistar rats, and the chemoreflex tachypneic response in both strains. CPF inhibited activity of cholinesterases in both strains, increased AOPP and nitrite/nitrate levels and expression of TNFα and ACE-1 in the brainstem of Wistar rats. Interestingly, SHR presented a reduced intrinsic BuChE activity, an important bioscavenger. Our findings show that, CPF at sublethal doses in normotensive rats lead to lethality and much more pronounced acute toxicity signs in the SHR. We also showed that cardiorespiratory reflexes were differentially impacted after CPF poisoning in both strains and that the cardiorespiratory disfunction seems to be associated with interference in cholinergic transmission, oxidative stress and inflammation. These results points to an increased susceptibility to acute toxicosis in hypertension, which may impose a significant risk to vulnerable populations.

Pesticide exposure and related health problems among farmworkers' children: a case-control study in southeast Iran.

Pesticides are potentially hazardous chemicals that can cause injury to human health and the environment. The purpose of this study was to evaluate organophosphate pesticides (OPPs) and organochlorine pesticides (OCPs) exposure in farmworkers' children aged 6 to 11years in Jiroft city in southeastern Iran. One hundred twenty farmworkers' children as case and 53 non-farmworkers' children aged 6 to 11years as control were selected and the serum levels of OCPs were measured by using gas chromatography in all participants. In addition, erythrocyte acetylcholinesterase (AChE) and arylesterase activity of paraoxonase-1 (PON-1) were measured to evaluate OPPs effects. Catalase (CAT), superoxide dismutase3 (SOD3), glutathione peroxidase (GPx3) activities, and the levels of serum malondialdehyde (MDA), total antioxidant capacity (TAC), nitric oxide (NO), and protein carbonyl (PC) were measured to investigate OCPs and OPPs effects on oxidative stress (OS). The serum levels of beta-HCH, 4,4 DDE, and 4,4 DDT in the case group were significantly higher than the control group. In addition, in the case group, AChE, PON-1, CAT, SOD3, and GPx3 activities and the levels TAC were significantly lower, while MDA, PC, and NO levels were significantly higher than the control group. OCPs as illegal pesticides are present in southeast Iran and children are exposed to OCPs and OPPs in the studied area. In addition, higher serum levels of pesticides may be a major contributor in OS development, as a cause of many diseases.

Investigation of Serum and Leukocyte Ascorbic Acid, Plasma and Intra-Erythrocyte Cholinesterase Levels in Obese Persons

Obesity is a complicated disease, including excessive amounts of body fat. Obesity is not just an aesthetic problem. It is also a medical issue that increases your risk of other diseases and health conditions, like heart disease, diabetes, and certain cancers. Ascorbic acid (A.A.) produced analog enhancing effects in superoxide generation, DNA strand cutting, and cytotoxicity. In leukocytes, toxicity due to mitochondrial permeability was seen in cells exposed to A.A./peroxynitrite, and these responses were determined to occur independently of Ca2 +. Plasma cholinesterase and erythrocyte acetylcholinesterase activities have a long history of use, both in monitoring workers at risk for organophosphorus pesticide (O.P.) exposure and investigating accidental exposures to O.P.s. Due to the wide variation between individuals, it is necessary to establish unexposed, essential enzyme activities for correct interpretation. Forty-four females and 32 males with a mean age of 40 ± 11.4 years were included in our study. In our study, those with a BMI of 18-24 were considered as control or non-obese (group 1), those between 25 and 30 as obese (group 2), and those between 31 and 42 as extremely obese (group 3). With a moderate standard deviation of 70 and 20 % erosion rate, 76 subjects were determined for 93 % power at the 5 % level of significance. Baseline comparisons among treatment sequences were analyzed using a chi-square test for categorical variables and an unpaired t-test for continuous variables. Mean and standard deviation values were assessed using the SPSS package program, and a statistically significant difference was determined at p-level below 0.05 value. As a result; Although the increase in PCE levels and the decrease in ECE, PTA, and P.A. levels were significantly different. The difference between group 2 and group 3 revealed the importance of these differences between obese and non-obese patients. It has been determined that as obesity increases, the risk of vitamin deficiency also increases.

Erythrocyte Acetylcholinesterase Enzyme Activity, Serum Interleukin-6 Level and Respiratory Function of Myanmar Agricultural Workers Exposed to Organophosphate Pesticides

Background and Aim: Myanmar is one of the countries in production of agricultural plant commodities. Nowadays, pesticides are commonly used to increase agricultural production and productivity. Organophosphate pesticides exposure can cause many physiological changes within the body including impaired respiratory function. Therefore, the present study aimed to find out the relationship between the erythrocyte acetylcholinesterase enzyme activity, serum interleukin-6 level and respiratory functions such as forced expiratory volume in first second (FEV1), forced vital capacity (FVC), ratio of forced expiratory volume in first second and forced vital capacity (FEV1/FVC), peak expiratory flow (PEF) and average forced expiratory flow between 25% and 75% of FVC (FEF25-75%) among agricultural workers who exposed to organophosphate pesticides and non-exposed control subjects. Methods: A community-based cross-sectional, comparative study was done in agricultural workers (n = 40) who live in A-lal-chaung village, Magway Township and age/BMI matched non-exposed subjects (n = 40) who live in Yan-Way Quarter, Magway Township. All the participants were selected according to inclusion and exclusion criteria by simple random sampling method. Erythrocyte acetylcholinesterase (AchE) enzyme activity was measured by spectrophotometric method and serum interleukin-6 levels were measured by enzymelinked immunosorbent assay (ELISA) method. Respiratory function parameters were measured by Spirobank II spirometer. Results: The mean erythrocyte acetylcholinesterase enzyme activity was significantly lower in exposed group than that of non-exposed group (3354.43±589.81 U/L vs 4515.83±759.33 U/L; p<0.001). The percentage of predicted value of respiratory function parameters of exposed group were significantly lower than that of non-exposed group (FEV1: 72.13±4.40 vs 75.95±4.05; FVC: 66.90±4.40 vs 70.60±4.20; FEV1/FVC: 110.35±2.69 vs 111.32±1.33; PEF: 5.52±8.66 vs 79.40±5.09) (p<0.05). There were significant positive correlation between erythrocyte acetylcholinesterase activity and respiratory function parameters in all groups (n=80) (FEV1: r=0.425, p<0.001; FVC: r=0.301, p<0.05 and PEF: r=0.316, p<0.05). There were significant negative correlation between erythrocyte acetylcholinesterase activity and serum IL-6 level (r= -0.374, n= 80, p<0.05). Serum IL-6 level was significantly higher in exposed group than non-exposed group (34.77±3.3 pg/mL vs 28.75±2.0 pg/mL; p <0.001). There was a significant negative correlation between serum IL-6 level and respiratory function parameters in all group (n=80) such as FEV1 (r= 0.300, p<0.05) and FVC (r= 0.331, p<0.05). Conclusion: It was concluded that agricultural workers who exposed to organophosphate pesticides have higher serum IL-6 level and lower respiratory functions than that of non-exposed subjects. The present findings highlighted that there has an impact of chronic low dose OP exposure on respiratory health and there has a role of IL-6 in respiratory function decline.

Pesticide exposure and related health problems among family members of farmworkers in southeast Iran. A case-control study

BackgroundPesticides used in agriculture are some of the most common pollutants in the world. This study aimed to investigate the effects of Organophosphorus Pesticides (OPPs) and Organochlorine Pesticides (OCPs) on the families of farmworkers in the southeast of Iran. MethodsIn the present case-control study, 141 family members of farmworkers (as the case group) and 59 family members of non-farmworkers (as the controls) were recruited. Serum levels of OCPs such as α-HCH, β-HCH, γ-HCH, 2,4-DDE, 4,4-DDE, 2,4-DDT, and 4,4-DDT were determined. In addition, erythrocyte acetylcholinesterase (AChE) activity, malondialdehyde (MDA), total antioxidant capacity (TAC), protein carbonyl (PC), nitric oxide (NO) serum levels, arylesterase activity of paraoxonase 1 (PON-1), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activity were determined in all participants. Furthermore, distance to farmlands, education, crops, type, and the number of consumed fruits were evaluated for each individual separately. ResultsThe erythrocyte AChE activity and serum activities of GPx, SOD, and PON-1 and TAC levels were significantly decreased, whereas the concentration of MDA, PC, NO, and seven OCPs were significantly increased in the farmworkers’ families as compared to the controls. Spearman correlation and linear regression suggest that OCPs increase the oxidative stress in farmworkers’ family members. Moreover, distance, education, farming precedence, products, and ventilation had significant effects on the OCP levels and increased the odds ratio of OCP levels in farmworkers’ families. ConclusionWith regards to the data obtained in this study, it was revealed that OCPs as illegal pesticides and OPPs were higher than expected in the farmworkers’ family members. Furthermore, exposure to OCPs and OPPs, apart from the other effects on the body, leads to oxidative stress (OS) that may cause serious diseases in the exposed populations.

Correlation Study Between Erythrocyte Acetylcholinesterase Activity, Serum Malondialdehyde and Insulin Sensitivity in Agricultural Workers and Non-agricultural Workers in Nat-Kan Village, Magway Township.

ObjectiveThis study determined the correlation between erythrocyte acetylcholinesterase (AChE) activity, serum malondialdehyde (MDA) and insulin sensitivity in agricultural workers and non-agricultural workers.MethodologyThe cross-sectional comparative study was undertaken in 45 agricultural and 45 non-agricultural workers from Nat-Kan Village, Magway Township. Erythrocyte acetylcholinesterase activity and serum malondialdehyde were measured by spectrophotometric method. Insulin sensitivity was calculated by homeostasis model assessment (HOMA-IR).ResultsMean erythrocyte AChE activity was significantly lower in agricultural (3553.99 IU/L) compared with nonagricultural workers (4432.68 IU/L) (p<0.001). A significant high level of mean serum MDA was observed in agricultural workers (0.74 versus 0.28 μmol/L, p<0.001). Median HOMA-IR value was significantly higher in agricultural (2.74) than that of non-agricultural workers (2.28) (p<0.05). The risk of insulin resistance was 2.8 times greater in agricultural workers than non-agricultural workers (OR 2.8, 95% CI, 1.18 to 6.72). Erythrocyte AChE activity had weak negative correlation with serum MDA level (r=-0.357, p<0.001) and HOMA-IR (ρ= -0.305, p<0.05). There was a significant but weak positive correlation between serum MDA level and HOMA-IR (ρ=0.355, p<0.001).ConclusionOrganophosphate pesticide exposure lowered erythrocyte AChE activity and increased oxidative stress. Oxidative stress is partly attributed to the development of insulin resistance.

Positive and inverse correlation of blood lead level with erythrocyte acetylcholinesterase and intelligence quotient in children: implications for neurotoxicity.

Blood lead level (BLL) is insufficiently sensitive for early detection of Lead-induced neurotoxicity (LIN). This study determined the possible role of the combination of BLL, intelligent quotient (IQ) and erythrocyte acetylcholinesterase (AChE) activity in the early detection of LIN in Children. Apparently healthy children (n=309) from eight public primary schools in Ibadan, Nigeria were recruited and classified into: children with Elevated BLL (EBLL) and children with Acceptable BLL (control) based on CDC cut-off for childhood lead exposure. Neurological indices (speech, memory, cranial nerves and cerebellar functions), IQ, BLL and erythrocyte AChE activity were assessed using standard methods, Standard Progressive Matrices, AAS and HPLC respectively. Statistical analysis involved Student’s t-test, Pearson’s correlation and multivariate regression. p<0.05 was considered significant. There were 169 (54.7%) children with EBLL while there were 140 (45.3%) control children. Both groups exhibited normal speech, memory, cranial nerves and cerebellar functions. However, IQ was lower in EBLL children (85.9±11.6) compared with control (91.5±14.0) while BLL and AChE activity were higher in EBLL children (0.4±0.1 μmol/l; 117.5±25.5 μkat/l) compared with control (0.2±0.0 μmol/l; 59.4±10.2 μkat/l). BLL showed inverse correlation with IQ (r=–0.134, p=0.019) but positive correlation with AChE (r=0.978, p≤0.001). 16.2% of the observed variation in BLL could be accounted for by AChE using the equation; [BLL=–0.007+0.003 AChE] p<0.05. Elevated blood lead level is prevalent among the school children and appears to have adverse effect on their IQ. Erythrocyte AChE could be a promising marker for early recognition of significant environmental lead exposure and lead-induced neurotoxicity in children.

1-Naphthyl acetate: A chromogenic substrate for the detection of erythrocyte acetylcholinesterase activity

Erythrocyte acetylcholinesterase (AChE) is a preferred biomarker for the detection of organophosphorus poisoning. Acetylthiocholine (ATCh) is the most popular substrate for the detection of AChE activity. However, oximolysis is a prominent feature with ATCh. In this context, we have searched alternative substrates for AChE using in silico tools for screening of a better substrate. The in silico approach was performed to understand the fitness and the Total Interaction Energy (TIE) of substrates for AChE. The alternative substrates for AChE were screened in terms of high Goldscore and favorable TIE in comparison to acetylcholine (ACh)-AChE complex and other relevant esterases. Among the screened substrates, 1-Naphthyl acetate (1-NA) exhibited the most favorable interaction with AChE in terms of highest TIE and corresponding high Goldscore. The Molecular Dynamic (MD) simulation of the 1-NA-AChE complex showed a stable complex formation over a period of 5 ns. The results obtained in the in silico studies were validated in vitro using pure erythrocyte AChE and hemolysate. We observed 1-NA to be a better alternative substrate for AChE than ATCh in terms of lower Km value. Its specificity appeared at least similar to ATCh. Therefore, we propose that 1-NA can be an attractive chromogenic substrate for the measurement of AChE activity, and it possess the potential to detect organophosphorus pesticide (OP) poisoning.

Assessment of mouse strain differences in baseline esterase activities and toxic response to sarin

Genetics likely play a role in various responses to nerve agent (NA) exposure, as genetic background plays an important role in behavioral, neurological, and physiological responses. This study uses different mouse strains to identify if mouse strain differences in sarin exposure exist. In Experiment 1, basal levels of acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and carboxylesterase (CE) were measured in different strains of naïve mice to account for potential pharmacokinetic determinants of individual differences. In Experiment 2, median lethal dose (MLD) levels were estimated in 8 inbred mouse strains following subcutaneous (s.c.) administration of sarin. Few strain or sex differences in esterase activity levels were observed, with the exception of erythrocyte AChE activity in the C57BL/6J strain. Both sex and strain differences in toxicity were observed, with the most resistant strains being the BALB/cByJ and FVB/NJ strains and the most sensitive strain being the DBA/2J strain. These findings can be expanded to explore pathways involved in NA response, which may provide an avenue to develop therapeutics for preventing and treating the damaging effects of NA exposure.

Erythrocyte acetylcholinesterase as biomarker of pesticide exposure: new and forgotten insights

Acetylcholinesterase (AChE) acts on the hydrolysis of acetylcholine, rapidly removing this neurotransmitter at cholinergic synapses and neuromuscular junctions as well as in neuronal growth and differentiation, modulation of cell adhesion ("electrotactins") and aryl-acylamidase activity (AAA). This enzyme is also found in erythrocyte, as 160kDa dimer that anchors to the plasma membrane via glycophosphatidylinositol. The function of this enzyme in erythrocytes has not yet been elucidated; however, it is suspected to participate in cell-to-cell interactions. Here, a review on erythrocyte AChE characteristics and use as biomarker for organophosphorus and carbamate insecticides is presented since it is the first specific target/barrier of the action of these pesticides, besides plasma butyrylcholinesterase (BChE). However, some past and current methods have disadvantages: (a) not discriminating the activities of AChE and BChE; (b) low accuracy due to interference of hemoglobin in whole blood samples. On the other hand, extraction methods of hemoglobin-free erythrocyte AChE allows: (a) the freezing and transporting of samples; (b) samples free of colorimetric interference; (c) data from only erythrocyte AChE activity; (d) erythrocyte AChE specific activity presents higher correlation with the central nervous system AChE than other peripheral ChEs; (e) slow spontaneous regeneration against anti-ChEs agents of AChE in comparison to BChE, thus increasing the chances of detecting such compounds following longer interval after exposure. As monitoring perspectives, hemoglobin-free methodologies may be promising alternatives to assess the degree of exposure since they are not influenced by this interfering agent.

Timolol effects on erythrocyte deformability and nitric oxide metabolism.

Timolol maleate is a compound used in treatment for reducing increased intra-ocular pressure by limiting aqueous humor production. Decreased erythrocyte deformability (ED), increased activity of erythrocyte acetylcholinesterase (AChE), increased values of nitrosoglutathione (GSNO) and nitic oxide (NO) and decreased plasma levels of NO metabolites, were described in primary open angle glaucoma patients. In healthy human red blood cells (RBCs), timolol is an inhibitor of AChE and induces NO efflux and GSNO efflux from that blood component in lower concentration than those obtained in presence of the natural AChE substrate, acetylcholine (ACh). The signal transduction pathway in RBCs described for NO in dependence of AChE-ACh active complex involves Gi protein, protein tyrosine kinase (PTK like Syk and p53/56Lyn), protein tyrosine phosphatase (PTP) and adenylyl cyclase (AC).The aim of this in vitro study was to verify the effect of timolol maleate in ED, NO efflux and NO derivatives molecules (NOx) like nitrite (NO2-), nitrate (NO3-, peroxynitrite (-ONOO) and GSNO under the presence of PTK, PTP, AC and guanylyl cyclase (GC) enzyme proteins inhibitors.Blood samples from healthy donors were each one divided and were performed aliquots in absence (control aliquots) and presence of timolol or timolol plus each inhibitor and Gi protein uncoupling. No significant differences in erythrocyte NO efflux, GSNO, peroxynitrite, nitrite and nitrate concentrations in response to timolol when compared with the untreated blood samples aliquots were obtained.It was observed an increase in erythrocyte deformability at high shear stresses induced by the simultaneous presence of timolol and band 3 protein dephosphorylation by PTK syk inhibitor. No significant differences where verified in peroxynitrite levels in the blood aliquots in presence of timolol plus each enzyme inhibitor and Gi protein uncoupling in relation to the control aliquots. No variation of GSNO concentration occurs under the presence of timolol and AMGT (PTK lyn inhibitor) besides the significant higher values observed with each one of the other inhibitors. Nitrate concentration increases significantly in all aliquots with timolol plus each one of the inhibitors. The same was observe with nitrite levels with exception of the aliquots with timolol plus AMGT or timolol plus Gi protein uncoupling showing no significant values in relation to the control aliquots.Besides the changes in NO derivative molecules and NO efflux from RBCs obtained in this study with blood samples of healthy donors under the effect of timolol plus each inhibitor of the proteins participants in NO signal transduction mechanism, further analogue studies must be promoted with blood samples of patients with glaucoma or any other inflammatory vascular disease.

The efficacy of HI-6 DMS in a sustained infusion against percutaneous VX poisoning in the guinea-pig

Post-exposure nerve agent treatment usually includes administration of an oxime, which acts to restore function of the enzyme acetylcholinesterase (AChE). For immediate treatment of military personnel, this is usually administered with an autoinjector device, or devices containing the oxime such as pralidoxime, atropine and diazepam. In addition to the autoinjector, it is likely that personnel exposed to nerve agents, particularly by the percutaneous route, will require further treatment at medical facilities. As such, there is a need to understand the relationship between dose rate, plasma concentration, reactivation of AChE activity and efficacy, to provide supporting evidence for oxime infusions in nerve agent poisoning.Here, it has been demonstrated that intravenous infusion of HI-6, in combination with atropine, is efficacious against a percutaneous VX challenge in the conscious male Dunkin-Hartley guinea-pig. Inclusion of HI-6, in addition to atropine in the treatment, improved survival when compared to atropine alone. Additionally, erythrocyte AChE activity following poisoning was found to be dose dependent, with an increased dose rate of HI-6 (0.48mg/kg/min) resulting in increased AChE activity. As far as we are aware, this is the first study to correlate the pharmacokinetic profile of HI-6 with both its pharmacodynamic action of reactivating nerve agent inhibited AChE and with its efficacy against a persistent nerve agent exposure challenge in the same conscious animal.

Avaliação da atividade de aldeídos aromáticos e suas oximas frente à enzima acetilcolinesterase eritrocitária humana

Considerando a importância terapêutica das drogas inibidoras da acetilcolinesterase, bem como dos regeneradores da enzima, em casos de intoxicações por organofosforados. A atividade de aldeídos aromáticos e suas respectivas oximas foi avaliada frente à acetilcolinesterase eritrocitária humana. As oximas foram preparadas a partir da oximação do benzaldeído, do 4-hidroxibenzaldeído, do 4-hidroxi-3-metoxibenzaldeído e do 4-dimetialminobenzaldeído, e em seguida, o ensaio de Ellman foi empregado para avaliar o comportamento destes compostos frente à acetilcolinesterase ativa e inibida por diclorvós. Os aldeídos avaliados apresentaram atividade inibidora da acetilcolinesterase nas porcentagens de 21,33%, 53,00%, 44,00% e 62,67% respectivamente para benzaldeído, 4-hidroxibenzaldeído, 4-hidroxi-3-metoxibenzaldeído e 4- dimetilminobenzaldeído. Dentre as oximas avaliadas, apenas a dimetilaminobenzaldoxima apresentou atividade inibitória (36,67%). A benzaldoxima, a 4-hidroxibenzaldoxima e a 4-hidroxi-3-metoxibenzaldoxima mostraram-se capazes de regenerar modestamente a atividade enzimática em 9,21%, 32,83% e 8,90%, respectivamente, quando a enzima se encontrava inibida por diclorvós. Os aldeídos aromáticos testados apresentaram potencial atividade inibitória da acetilcolinesterase eritrocitária, na concentração de 100mM, sendo o maior potencial demonstrado pelo 4-dimetilamibenzaldeído, com 62,67% de inibição. As oximas, com exceção da dimetilaminobenzaldoxima, se comportaram como regeneradores fracos da enzima inibida por diclorvós, mostrando a relevância do grupo oxima para a regeneração da enzima inibida por organofosforados.

Effect of deltamethrin and fluoride co-exposure on the brain antioxidant status and cholinesterase activity in Wistar rats

The study evaluated the effect of commercial preparation of deltamethrin, Butox®, and fluoride (F−) co-exposure on the brain antioxidant status and cholinesterase activity in rats. Group A was untreated. Group B was gavaged Butox®, providing deltamethrin at the dose rate of 1.28 mg per kg body weight per day. Group C was administered F−, as NaF, in drinking water providing 20 ppm F−. Group D received both deltamethrin and F− at the same dosages as groups B and C, respectively. Although, glutathione S-transferase activity was induced only in Butox® alone treated group, the activities of superoxide dismutase and catalase were inhibited in all treatment groups when compared to the control group. Elevated lipid peroxidation was observed in the groups exposed to F−. The activity of erythrocyte acetylcholinesterase (AChE) was inhibited in Butox® treated groups, whereas brain AChE activity was inhibited in all treatment groups. In conclusion, both deltamethrin (given as Butox®) and F− inhibit AChE activity and produce oxidative stress in brain with F− producing more oxidative damage. However, compared to the individual exposures, the co-exposure of these chemicals does not produce any exacerbated alteration in these biochemical parameters.

Activity of cholinesterases in a young and healthy middle-European population: Relevance for toxicology, pharmacology and clinical praxis

The activity of human cholinesterases, erythrocyte acetylcholinesterase (AChE; EC 3.1.1.7) and plasma butyrylcholinesterase (BChE; EC 3.1.1.8) represents an important marker when monitoring exposure to pesticides/nerve agents, and may also be used in occupational medicine in diagnosis and prognosis of some diseases. In this study “normal/baseline” AChE and BChE activity has been investigated in a young and healthy population, with subsequent evaluation of several intra-population factors including sex, age (categories 18–25, 26–35 and 36–45 years old) and smoker status.The modified Ellman's method was used for enzyme activity assessment in 387 young and healthy individuals (201 males and 186 females aged 18–45). A significant inter-sexual difference in AChE and BChE activity was found (AChE: 351±67 for males and 377±65 for females, (μmol/min)/(μmol of hemoglobin), p<0.001; BChE: 140±33 for males and 109±29 for females, μkat/l, p<0.001; mean±SD). Despite the finding that mean AChE activity somewhat decreased whereas BChE activity grew within the age categories of the tested subjects, no significant effect of age on cholinesterase activity was found (p>0.05). Smoking influenced cholinesterase activity – AChE activity in smokers was elevated (approx. 3% in males; 8% in females) relative to that in non-smokers (p<0.05). Smoking was found not to have any effect on BChE activity.Reference values based on confidence intervals for AChE and BChE activity were established. The presented results might be useful in routine clinical practice where the monitoring of blood AChE and plasma BChE activity is crucial for prognosis and diagnosis of organophosphate poisoning, in occupational medicine and in relevant mass casualty scenarios.

Erythrocyte nitric oxide in glaucoma patients - ex vivo study.

Glaucoma is an optic neuropathy associated with vascular dysregulation and increased intra-ocular pressure (IOP). Timolol is used as treatment for reducing IOP, by limiting aqueous humour production. Increased NOS expression as well as decreased levels of nitric oxide (NO) metabolites, and high activity of erythrocyte acetylcholinesterase (AChE) were observed in primary open angle glaucoma patients. This ex vivo study aims to evaluate timolol effect in NO efflux and its derivatives in glaucoma patient's erythrocytes. Venous blood from 15 glaucoma patients was collected. Erythrocyte suspensions were incubated with the AChE modulators acetylcholine (ACh) and timolol at 10 μM. Erythrocyte NO efflux and S-nitrosoglutathione (GSNO) concentration were measured. No significant differences were obtained in erythrocyte NO efflux and GSNO concentration in response to ACh or timolol when compared with the untreated erythrocytes of glaucoma patients. When comparing the same incubation conditions for erythrocyte suspensions between glaucoma patients and healthy subjects, those from glaucoma patients showed higher NO efflux in presence and absence of timolol, and higher values of GSNO in the presence of timolol. We demonstrated that erythrocytes from glaucoma patients have similar availability to release NO both in absence and presence of timolol, and have higher GSNO values in presence of timolol.

Determination of reference values of acetyl and butyryl cholinesterase activities in Serbian healthy population

Background/Aim. Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are important biomarkers of exposure to organophosphorus and carbamate insecticides. Since the estimation of the level of cholinesterase inhibition depends on the normal values which may vary in different populations, it is important to determine them in our population, which so far has not been done. Therefore, the aim of this study was to determine the reference values for AChE and BuChE in a healthy population of adults in the Republic of Serbia. Methods. The AChE activity was measured by spectrophotometry (? = 412 nm), using a modified Ellman?s method. BuChE activity was determined by the integrated chemical system (Dimension RxLMax) with ready-made reagent cartridge for analysis. The examinees were healthy voluntary blood donors from the Institute of Transfusiology and Hemobiology, Military Medical Academy in Belgrade, Serbia. Statistical Package for Social Sciences (SPSS) software program was used for data processing. Results. In the group of 851 persons, there were 728 males and 123 females. The mean age was 39.1 ? 11.6 years. For all of them, erythrocyte AChE activity was done while BuChE was determined in 205 persons (169 males and 36 females). Their mean value of acetylcholinesterase activity was 8,090.6 ? 1,976.7 IU/L, and of butyrylcholinesterase activity was 14,556.6 ? 4,078.1 U/L. Due to lack of normal data distribution in male group (both enzymes), reference ranges were estimated as 2.5 and 97.5 percentiles. Conclusion. The results of this pilot study on cholinesterase in healthy population in the Republic of Serbia which has now been done for the first time, indicate the need for considering their wider ranges of when estimating the severity of poisoning. However, further study for BuChE with the inclusion of a larger number of females and data for body weight of the examinees, in order to get more precise reference limits, is suggested.

Evaluation of chronic intoxication by organophosphate insecticides among hygiene workers in the city of Fez, Morocco

Summary Context of the study The use of pesticides in the field of public health involves a risk for both humans and the environment. Objective The purpose of this study, carried out for the first time in the center of Morocco, was to evaluate chronic intoxication by organophosphate insecticides. This was done through the evaluation of cholinesterase activity as a biomarker of pesticide intoxication among a population of hygiene workers who daily handled these products, in six districts of the city of Fez. Method This study is a descriptive cross-sectional study including 30 consenting hygiene workers carried out during a period of 4 months (February-May 2015). The study included a physical examination and biotoxicological measurements: plasma and erythrocyte acetylcholinesterase activity. Erythrocyte acetylcholinesterase activity was determined by spectrophotometric method at ambient laboratory temperature (25 °C) using the kit “Test Mat - model 400”. Results Twenty-nine subjects of the studied population were of male gender with an average age of 48 ± 8 years and a body mass index of 25.67 ± 2.09 kg/m 2 ; and only one subject was of female gender aged 56, and a body mass index of 26.25 kg/m 2 . The somatic clinical examination of the participants in this study revealed neither serious clinical history nor cases of pesticide poisoning. The mode value of erythrocyte cholinesterase activity was 54, and that of plasmatic cholinesterase activity was 65. The measured cholinesterase activity is not correlated with age but it is correlated with the body mass index. Three cases showed a decrease in cholinesterase activity of the order of (35%, 36%, and 25%) compared to 30% as recommended by the World Health Organization. Conclusion These results relating to the determination of cholinesterase activity, although preliminary, should reassure the exposed persons and the authorities concerned without neglecting the systematic control of the three cases detected. The use of organophosphates in the field of public health requires regular checking, precautions, and training of the hygiene workers to avoid any serious health risk.

Effect of isopropyl methylphosphonofluoridate (IMPF) poisoning on selected immunological parameters of angiogenesis.

Acetylcholinesterase (AChE) and cholinergic receptors play an important role in the immune system, including lymphocyte-induced angiogenesis. However, their exact role is not fully understood. The presented work tests the influence of isopropyl methylphosphonofluoridate (IMPF), an irreversible inhibitor of AChE, on selected immune parameters associated with angiogenesis in mice. The levels of VEGF, bFGF, TNF-α, and IFN-γ production were measured, together with the ability of lymphoid spleen cells to induce local GvH reaction after a single dose of IMPF. Experiments were performed in male BALB/c mice. Acetylcholinesterase activity in erythrocytes was determined by the Ellman`s procedure. Levels of cytokines were measured in serum using standard commercial ELISA kits. Influence of IMPF intoxication upon angiogenesis was examined by the LIA test, according to the Sidky and Auerbach procedure. The results showed a 6- and 8-fold increase in VEGF at days 1 and 7 of the experiment, respectively, as well as a decrease (at days 14 and 21 after administration), followed by a significant increase (day 1) in bFGF levels. A statistically significant decrease in the concentration of IFN-γ was observed throughout all experiments. The maximum decrease in the level of TNF-α was found at days 1 and 7 after administration of IMPF. Additionally, a significant decrease was found in the ability to form new blood vessels following IMPF administration. This study revealed that IMPF has a significant effect on the regulation of lymphocyte-induced angiogenesis, which is related with the modulation of angiogenic and pro-inflammatory cytokines secretion. The observed differences suggest a possible derangement of certain elements of the neuronal and/or non-neuronal cholinergic system.

Post-exposure treatment of VX poisoned guinea pigs with the engineered phosphotriesterase mutant C23: A proof-of-concept study

The highly toxic organophosphorus (OP) nerve agent VX is characterized by a remarkable biological persistence which limits the effectiveness of standard treatment with atropine and oximes. Existing OP hydrolyzing enzymes show low activity against VX and hydrolyze preferentially the less toxic P(+)-VX enantiomer. Recently, a phosphotriesterase (PTE) mutant, C23, was engineered towards the hydrolysis of the toxic P(−) isomers of VX and other V-type agents with relatively high in vitro catalytic efficiency (kcat/KM=5×106M−1min−1). To investigate the suitability of the PTE mutant C23 as a catalytic scavenger, an in vivo guinea pig model was established to determine the efficacy of post-exposure treatment with C23 alone against VX intoxication. Injection of C23 (5mgkg−1 i.v.) 5min after s.c. challenge with VX (∼2LD50) prevented systemic toxicity. A lower C23 dose (2mgkg−1) reduced systemic toxicity and prevented mortality. Delayed treatment (i.e., 15min post VX) with 5mgkg−1 C23 resulted in survival of all animals and only in moderate systemic toxicity. Although C23 did not prevent inhibition of erythrocyte acetylcholinesterase (AChE) activity, it partially preserved brain AChE activity. C23 therapy resulted in a rapid decrease of racemic VX blood concentration which was mainly due to the rate of degradation of the toxic P(−)-VX enantiomer that correlates with the C23 blood levels and its kcat/KM value. Although performed under anesthesia, this proof-of-concept study demonstrated for the first time the ability of a catalytic bioscavenger to prevent systemic VX toxicity when given alone as a single post-exposure treatment, and enables an initial assessment of a time window for this approach. In conclusion, the PTE mutant C23 may be considered as a promising starting point for the development of highly effective catalytic bioscavengers for post-exposure treatment of V-agents intoxication.