Erythrocyte Acetylcholinesterase Research Articles

Improvement by Satureja khuzestanica essential oil of malathion-induced red blood cells acetylcholinesterase inhibition and altered hepatic mitochondrial glycogen phosphorylase and phosphoenolpyruvate carboxykinase activities

The aim of this study was to examine whether Satureja khuzestanica (Lamiaceae) essential oil (SKEO) might have protective effects on toxicity of malathion, a commonly used organophosphorus (OP), by measuring the activities of hepatic cells mitochondrial glycogen phosphorylase (GP) and phosphoenolpyruvate carboxykinase (PEPCK) activities and blood levels of glucose and acetylcholinesterase (AChE) in rats. Malathion (20 mg/kg/day) and SKEO (225 mg/kg/day) were administered alone or in combination by intragastric intubation for 28 days. Treatment by malathion increased blood glucose as measured at days 18 and 28 of treatment. Malathion inhibited erythrocyte AChE and increased hepatic cells GP and PEPCK activities. Coadministration SKEO resulted in restoration of malathion-induced changes in hepatic cells GP and PEPCK activities and levels of blood AChE and glucose. It is concluded that SKEO interferes with malathion-induced stimulation of hepatic cells glycogenolysis and gluconeogenesis through its antioxidant potential and increasing AChE activity.

Effect of coumaphos on cholinesterase activity, hematology, and biochemical blood parameters of bovines in tropical regions of Mexico

To assess the effect of coumaphos [O-(3-chloro-4-methyl-2-oxo-2H-1-benzopyran-7-yl) O,O-diethyl phosphorothioate] exposure on physiological responses during bovine production, acetylcolinesterase (AChE) and butyrylcholinesterase (BuChE) activities were measured in whole blood, erythrocytes, and plasma of healthy male steers (Bos Taurus x Bos indicus) sprayed with coumaphos at a non-lethal dose of 1 mg kg− 1 body weight per day once every 14 (in vivo group) or 21 days (southern and central groups). Coumaphos topically administered at 1 mg/kg body weight per day to cattle under normal management practices in tropical areas produced a significant inhibition in erythrocyte (RBC) AChE and BuAChE activities when compared to baseline levels. RBC-AChE activity for the in vivo group decreased 71.3% (P < 0.05) and BuChE activity 59.1% (P < 0.05); RBC-AChE activity decreased 55.1% (P < 0.05) (southern group) and 43.4% (P < 0.05) (central group). Compared to the control specimens, steers from in vivo, southern, and central groups after 150 days of exposure had lower (P < 0.05) leukocyte count, absolute lymphocyte, erythrocyte, and platelet counts. Decreases in RBC-AChE activities correlated with decreased lymphocyte (r = 1.000, p = 0.01), erythrocyte (r = 1.000, p = 0.003), and platelet counts (r = 0.841, p = 0.036). Significantly increased BUN levels (P < 0.05) correlated with the decrease in RBC-AChE activities (r = − 0.997, p = 0.047) and with the decrease in absolute red blood cell (r = − 0.883, p = 0.020) and lymphocyte (r = − 0.825, p = 0.043) counts; increased (P < 0.05) total plasma protein levels correlated with the decrease in RBC-AChE activities (r = −0.998, p = 0.043), absolute red blood cell (r = − 0.998, p = 0.040), lymphocyte (r = − 0.893, p = 0.017), and platelet (r = -0.855, p = 0.030) counts. The physiological responses correlated with the erythrocyte acetylcholinesterase inhibition could be considered as early indicators or warning responses of bovine exposures to organophosphorus pesticides (OPs).

How thionphosphonates inhibit activity of different cholinesterases

Analysis of mechanism of reversible inhibition of human erythrocyte acetylcholinesterase (AChE), of horse serum cholinesterase (ChE), and ChE of optical ganglia tissue of individuals of the Commander squid Berryleuthis magister from various habitat zones was studied under effect of thionphosphonates (P=S), derivatives of piperidine, morpholine, perhydroazepine as well as several heterocyclic model compounds. Data of comparative inhibitory specificity have allowed us to suggest that thionphosphonates are sorbed in the area of cholinesterase esterase center through the phosphoryl part of the inhibitor molecule, rather than through its heterocyclic grouping. An advantage in the antienzyme efficiency of thionphosphonates (P=S) over phosphonates (P=O) is revealed. Effect of the ion strength is used for analysis of contribution of the hydrophobic—hydrophilic interaction in the enzyme—inhibitor system.

Induction of insulin resistance by malathion: Evidence for disrupted islets cells metabolism and mitochondrial dysfunction

Hyperglycemia is observed with exposure to organophosphorus (OP) pesticides. The aim of this study was to investigate the effects of malathion on secretion of insulin from rat pancreatic islets in vitro and in vivo. Malathion was administered through food for 4 weeks at concentrations of 100, 200, and 400 ppm. For in vivo experiment, at the end of treatment, blood sample was obtained and plasma was separated. For in vitro experiment, the treated rats were anesthetized and underwent a laparatomy. The common bile duct was cannulated and the pancreas distended by injecting of cold collagenase V using peristaltic infusion pump. Islets were then hand picked under a stereomicroscope and cultured in the presence of various doses of glucose and KCl. Malathion at doses of 200 and 400 ppm increased plasma glucose and insulin concentrations and lowered activity of erythrocyte acetylcholinesterase. The isolated islets from pretreated animals with malathion 200 and 400 ppm showed lower glucose-stimulated insulin secretion while no change was observed in the presence of KCl. Light microscopic examination revealed that malathion causes patchy degenerative changes in pancreatic islets. Combination of in vivo and in vitro findings suggests that malathion induces a kind of insulin resistance that cannot overwhelm hyperglycemia. This action of malathion is mediated through disruption of islets mitochondrial function.

Structure-toxicity relationships of thiono and seleno phosphoramidate compounds: new type of acetylcholinesterase inhibitors

Novel thiono and seleno phosphoramidate compounds with the general formula (X)(Y)P(C(6)H(5))(2); (X = NMe(2) & Y = S, 1a; X = NEt(2) & Y = S, 2a; X = NMe(CH(2)Ph) & Y = S, 3a; X = NH(CH(2)Ph) & Y = S, 4a; X = NEt(CH(2)Ph) & Y = S, 5a; X = N(C(Me)(3)) (CH(2)Ph) & Y = S, 6a; X = N(CH(2)Ph)(2) & Y = S, 7a; X = NMe(2) & Y = Se, 1b; X = NEt(2) & Y = Se, 2b; X = NMe(CH(2)Ph) & Y = Se, 3b; X = NH (CH(2)Ph) & Y = Se, 4b; X = NEt(CH(2)Ph) & Y = Se, 5b; X = N(C(Me)(3))(CH(2)Ph) & Y = Se, 6b and X = N(CH(2)Ph)(2) & Y = Se, 7b) were prepared and characterized by (1)H, (31)P and( 13)C NMR and IR spectroscopy and elemental analysis. (31)P chemical shift of thiono and seleno derivatives didn't show significant different because of their little difference in electronegativity sulfur and selenium. Hydrophobic parameter of compounds was determined by measurement of octanol-water partition coefficient by shake-flask technique. Determination of human erythrocyte acetylcholinesterase (hAChE) activity was carried out according to the Ellman's modified kinetic method. IC(50 )values of the selected thiono and seleno compounds varied from 3.4 to 0.11 and 9.9 to 5.1 mM, respectively. The seleno compounds show lower affinities for hAChE relative to the thino compounds. These results demonstrate that hydrophobic and electronic factors of the organophosphorus compounds play a key role on the inhibitory potency.

Changes of Cholinesterase Activities in the Rat Blood and Brain After Sarin Intoxication Pretreated with Butyrylcholinesterase

After sarin inhalation exposure of rats pretreated with equine serum butyrylcholinesterase (EqBuChE), cholinesterase activities of the whole blood, acetylcholinesterase (AChE) in erythrocytes, pontomedullar area, frontal cortex, and striatum of the brain, and plasma butyrylcholinesterase (BuChE) were determined. Using different doses of EqBuChE as a pretreatment (intraperitoneal injection), dose-dependent increases in plasma BuChE activity and no changes in the erythrocyte and brain AChE activities were demonstrated. Decreases in plasma BuChE activity and red blood cells (RBC) and brain AChE activities were observed in control rats after sarin inhalation exposure without EqBuChE pretreatment. In rats pretreated with EqBuChE, this inhibition was lower compared with control animals not only in the blood but also in the brain structures studied. These results demonstrate protective effects of EqBuChE pretreatment in rats intoxicated with sublethal concentrations of sarin by inhalation.

New Safe Method for Preparation of Sarin-Exposed Human Erythrocytes Acetylcholinesterase Using Non-Toxic and Stable Sarin Analogue Isopropyl p-Nitrophenyl Methylphosphonate and its Application to Evaluation of Nerve Agent Antidotes

A non-toxic and stable sarin analogue, isopropyl p-nitrophenyl methylphosphonate (INMP), was synthesized for safe preparation of sarin-exposed acetylcholinesterase (AChE). This agent was stable for years, able to be handled in an ordinary laboratory without special care, and its 50% inhibitory concentration (IC50) on 0.04 U/ml human erythrocytes AChE was 15 nM. This reagent was thought to be especially useful since it enables experiments that require sarin-inhibited AChE, such as the development of antidotes for sarin, in a usual laboratory. To demonstrate the usefulness of this method, 40 known and novel pyridinealdoxime methiodide (PAM)-type oxime antidotes were synthesized, and their reactivation activities to INMP-exposed AChE and structure-activities correlation were studied. Among the antidotes tested in this experiment except for 2-PAM, the compound found to have the highest reactivation activity, was the novel hydrophobic 2-PAM-type compound, 2-[(hydroxyimino)methyl]-1-[4-(tert-butyl)benzyl] pyridinium bromide.

Prognostic value of human erythrocyte acetyl cholinesterase in acute organophosphate poisoning

Acute organophosphate poisoning (OPP) such as dichlorvos may be monitored by the measurement of the erythrocyte acetyl cholinesterase (EAChE) and the serum cholinesterase (SChE) activities. The aim of this study was to look at correlation between the severity of the OPP judged by certain parameters such as coma, hemodynamic disturbances, respiratory failure, and the decrease of cholinesterases enzymes including EAChE and SChE at admission. Cholinesterase activity was determined upon admission and then on days 3 and 15 in the morning. Clinical effects, EAChE, and SChE activities data were investigated in 42 patients with OPP aged of 29.6 ± 11.8 years with acute cholinergic crisis in all cases. They were comatose in 29% of cases, presenting both hypotension or shock and hypoxemia in 17% of cases. Fifteen of them (36%) required mechanical ventilation. The mean EAChE activity at admission was 24.3 ± 11.6 μmol/mL per hour at 37 °C; it was 1260 ± 2204 IU/L for SChE. There were no correlations between the EAChE and the SChE activities. The EAChE was decreased only in comatose patients and those presenting hypotension, hypoxemia, and bradycardia with a cutoff of 23.5 μmol/mL per hour at 37 °C. Death was observed in 2 patients with a deep decrease of the EAChE at 5 μmol/mL per hour at 37 °C in 1 case and 9 μmol/mL per hour at 37 °C in another. The kinetics of improvement of the EAChE activity below the cutoff showed the absence of statistical improvement of the EAChE activity on day 3 (16.6 ± 9 vs 19.5 ± 5.7 μmol/mL per hour at 37 °C); this improvement was remarkable on day 15 (16.6 ± 9 vs 27.5 ± 6.5 μmol/mL per hour at 37 °C, P = .0004). In summary, the marked decrease of EAChE activity appears in this study as prognostic factor in acute OPP, and coma, respiratory failure, hemodynamic disturbances, and death are associated with a decrease of the EAChE of less than 23.5 μmol/mL per hour at 37 °C.

Organophosphate Pesticides Overexposure on Children Working and Living in an Agricultural Area in Brazil

P-350 Introduction: Considerable efforts have to be made to evaluate impact of pesticides exposure on health of children working on or living near agricultural areas. This work aimed the assessment of epidemiological and biological monitoring data to evaluate pesticides exposure in a group of children directly involved with working activities in the field at an agricultural community in Brazil. Methods: A group of 73 children, age 13,65 ± 2,36 (mean ± s.d), under multiple exposure to pesticides, including organophosphates methamidophos, methyl parathion and fenitrothion, were selected at this study. Plasma and erythrocyte acetylcholinesterase activities were measured using EQM testmate kit. Blood cholinesterase levels were dichotomized into low and normal categories for statistical analyses, which was performed using bivariate and multivariate models (student’s t-test, linear and logistic regression). Urinary p-nitrophenol and p-aminophenol levels were analysed by an HPLC method developed and validated in our laboratory. Urine and blood samples were obtained by the same time, in the morning after one usual spraying shift. Interviews were also performed to assess important occupational exposure variables and to investigate possible associations between biological monitoring and epidemiological data. Results: In relation to erythrocyte acetylcholinesterase, our results showed significant associations in the low category group as compared to the normal category group in the following situations: 1- Activity of applying or mixing pesticides (OR=2.33 - 95% CI =1.13–4.44) and 2- More than 6 hours work shift spraying pesticides OR=3.67 (1.79–6.32). Results from plasma cholinesterase activity showed significant associations with: 1- No protective measures to mix pesticides formulations OR=4.24 (1.62–7.87); 2- More than 6 hours work shift spraying pesticides OR=2.88 (1.08–5.49) and 3- Skin contact with pesticides while applying formulations on crop OR=2.48 (1.39–8.43). Urinary analyses of p-nitrophenol and p-aminophenol (respective ending products from methyl parathion and fenitrothion metabolism) from 12 urine samples showed significant detection levels of p-nitrophenol, which was significantly correlated with lower levels of cholinesterases. Conclusion: Evaluation of organophosphate pesticides overexposure has to be determined with as many sensitive indicators as possible to assess an actual profile of health status of exposed individuals. Children overexposure to pesticides becomes a major threat in rural communities as they are disproportionally exposed in comparison to adults due to their great intake of food, water and air per unit of body weight, and different metabolism rates. More studies and public health policies have to address the minimization of impact of pesticides exposure on health of children working in agriculture.

Reversible inhibition of acetylcholinesterase by carbamates or huperzine A increases residual activity of the enzyme upon soman challenge

The treatment options in soman poisoning are very limited due to rapid aging of the inhibited acetylcholinesterase, which makes the enzyme essentially intractable. Hence, oxime treatment probably comes too late in realistic scenarios. As an alternative, protecting part of the enzyme by reversible inhibition prior to soman exposure has been proposed. This strategy was successfully tested in animal experiments, but its efficacy still awaits complete understanding. In particular, it is unclear whether survival is improved by a higher residual activity of acetylcholinesterase during the acute phase, when the reversible and irreversible inhibitors are present together. In previous experiments with carbamate pre-treatment and paraoxon challenge we noticed an increased residual activity of erythrocyte acetylcholinesterase compared to non-pre-treatment. This result was encouraging to also test for comparable effects when using soman. Immobilized human erythrocytes were continuously perfused for real-time measurement of acetylcholinesterase activity by a modified Ellman method using 0.45 mM acetylthiocholine. After having established the inhibition rate constant of soman, we tested the prophylactic potential of physostigmine, pyridostigmine and huperzine A. Pre-treatment with the reversible inhibitors inhibited the enzyme by 20–95%. Additional perfusion with 10 nM soman for 30 min resulted in a residual activity of 1–5%, at low and high pre-inhibition, respectively. The residual activity was markedly higher than in the absence of reversibly blocking agents (0.1%). After discontinuation of soman and the reversible inhibitors, enzyme activity recovered up to 30% following pre-inhibition by 50%. The experimental data agreed with computer simulations when feeding the kinetic-based model with the established rate constants. The results with soman essentially agreed with those obtained previously with paraoxon.

Development of methods to measure humoral immune responses against selected antigens in the common marmoset ( Callithrix jacchus) and the effect of pyridostigmine bromide administration

This methodological study was carried out in preparation for a major long term study, also reported in this volume, which was designed to investigate whether the combination of vaccines and pyridostigmine bromide (PB) could have been responsible for adverse signs and symptoms reported by a number of veterans of the 1990/1991 Gulf conflict. In this context, the marmoset has been used to model aspects of the human immune system. The purposes of this methodological study were to select appropriate immunochemical reagents to measure humoral responses induced in marmosets in response to selected health and hygiene and biological warfare vaccines and to initially assess the effects of PB on the responses recorded. Vaccines were administered at 1/5th of a human dose, and also investigated in combination with the nerve agent pretreatment compound PB. PB dosing was selected to induce an inhibition of erythrocyte acetylcholinesterase by 30%. In order to assess the functionality of the immune system, antibody responses to a neo-antigen (keyhole limpet haemocyanin—KLH), administered some 2 months following the completion of the vaccination schedule, were measured. The present study identified appropriate isotyping reporter reagents which cross-reacted with equivalent marmoset immunoglobulins. Robust antibody responses were identified against anthrax protective antigen (PA), whole cell pertussis vaccine and KLH, while weaker responses were measured against cholera and typhoid vaccines. The killed whole cell plague vaccine induced a response which was at the limit of detection of the assay. Coadministered PB had no discernable effect on immunological responses in this study.

Pretreatment with pyridinium oximes improves antidotal therapy against tabun poisoning

Oximes K033 [1,4-bis(2-hydroxyiminomethylpyridinium) butane dibromide] and K048 [1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium) butane dibromide] were tested as pretreatment drugs in tabun-poisoned mice followed by treatment with atropine plus K033, K048, K027 [1-(4-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium) propane dibromide], TMB-4 [1,3-bis(4-hydroxyiminomethylpyridinium) propane dibromide] and HI-6 [(1-(2-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium)-2-oxapropane dichloride)]. Oxime doses of 25% or 5% of its LD 50 were used for pretreatment 15 min before tabun-poisoning and for treatment 1 min after tabun administration to mice. The best therapeutic effect was obtained when oxime K048 (25% of its LD 50) was used in both pretreatment and treatment with atropine. This regiment insured survival of all tested animals after the application of 10 LD 50 of tabun. In addition, since butyrylcholinesterase (BChE; EC 3.1.1.8) is considered an endogenous bioscavenger of anticholinesterase compounds and its interactions with oximes could be masked by AChE interactions, we evaluated kinetic parameters for interactions of tested oximes with native and tabun-inhibited human plasma BChE and compared them with results obtained previously for human erythrocyte acetylcholinesterase (AChE; EC 3.1.1.7). Progressive inhibition of BChE by tabun was slightly faster than that of AChE. The reactivation of tabun-inhibited BChE by oximes was very slow, and BChE binding affinity for oximes was lower than AChE's. Therefore, BChE could scavenge tabun prior to AChE inhibition, but fast oxime-assisted reactivation of tabun-inhibited AChE or protection of AChE by oxime against inhibition with tabun would not be obstructed by interaction between BChE and oximes.

Evaluation of reactivation test in anaesthetized dogs with experimental intoxication with nerve agents

Following repeated antidotal treatment of anaesthetized dogs (1 min with atropine, 10 min with atropine and obidoxime, 60 min with atropine and obidoxime) after the intoxication with soman, sarin and VX (1 x LD50, i.m.), the blood cholinesterases (erythrocyte, whole blood, plasma) were monitored and their reactivatability (whole blood) was determined. During this treatment, the activities of erythrocyte acetylcholinesterase (AChE), plasma butyrylcholinesterase (BuChE) and whole blood cholinesterases were monitored. Atropine and obidoxime did not affect cholinesterase activities in control animals, whereas administration of obidoxime to dogs intoxicated with nerve agent caused an increase in the cholinesterase activities. The sensitivity of cholinesterases decreased in the order erythrocyte AChE > whole blood cholinesterases > plasma BuChE, respectively. Following sarin intoxication, blood cholinesterases were increased after the obidoxime administration. Intoxication with VX showed a similar picture but reactivation after the obidoxime administration was greater. In soman intoxication, the picture of cholinesterase changes was similar during the first 30 min of treatment. Then the increase in AChE activity following obidoxime administration was not as high as in the case of sarin and VX intoxication. Thus, the reactivation efficacy of obidoxime during nerve agent intoxication indicates that its repeated administration could be easily monitored using the reactivation test.

Evaluation of monoquaternary pyridinium oximes potency to reactivate tabun-inhibited human acetylcholinesterase

Monoquaternary N-benzyl-4-hydroxyiminomethylpyridinium bromide (Py-4-H) and its analogous with diverse substituents introduced into the phenyl ring (Py-4-CH 3, Py-4-Br, Py-4-Cl and Py-4-NO 2) were synthesized in order to examine their potency as reactivators of tabun-inhibited human erythrocyte acetylcholinesterase (AChE; EC 3.1.1.7). Within 24 h, the reactivation of tabun-inhibited AChE reached 80% with Py-4-CH 3, Py-4-Br and Py-4-Cl, 40% with Py-4-NO 2, and 30% with Py-4-H. The overall reactivation rate constants were up to 5.0 min −1 M −1. All oximes inhibited human AChE reversibly, and the inhibition potency increased in the following order Py-4-Br < Py-4-Cl < Py-4-CH 3 < Py-4-H < Py-4-NO 2. Although oximes Py-4-H and Py-4-NO 2 did not show significant reactivation ability, these oximes might be of interest as pre-treatment drugs due to their high affinity for the native AChE. Docking studies were carried out to elucidate the differences in oximes potency. The orientations of all studied oximes in the active site of human AChE have been proposed by flexible ligand docking with AutoDock 3.0. Analyses of the obtained complexes revealed the presence of numerous hydrogen bonds and close contacts between the oximes and the residues in the active site. Final docked energies predicted correctly the relative order of the inhibition potency of compounds (except in the case of Py-4-CH 3) as well as the most probable orientation of the best reactivator, Py-4-Br, which can result in an attack on the phosphorus atom of the tabun-phosphorylated human AChE.

Comparative study of cholinergic activity of some tropolone and isoquinoline alkaloids

Comparative study was performed of action of a group of 15 isoquinoline homoproaporphine and homoaporphine alkaloids, 10 tropolone colchicine alkaloids and their lumoderivatives that were isolated from corms of a representative of the lily family, the showy autumn crocus Colchicum speciosum Stev. on activity of mammalian erythrocyte acetylcholinesterase and serum butyrylcholinesterase. The studied compounds have turned out to be moderate-potency reversible inhibitors of the studied cholinesterases and to show to a certain degree some specificity of action towards different enzymes both quantitatively, by the ratio of total inhibitor constant values, and qualitatively, by the type of mechanism of the enzymatic activity inhibition. The overwhelming majority of the studied alkaloids revealed certain specificity towards butyrylcholinesterase. An exception was colchamine.

In vitro biological efficiency of tenocyclidine – TCP and its adamantane derivative TAMORF

Tenocyclidine – TCP showing a broad spectrum of pharmacological activity including antidotal effect in organophosphorus compounds poisoning, radioprotective and anticancer effects. We investigated in vitro interactions of TCP and its adamantane derivative – TAMORF with human erythrocyte acetylcholinesterase (AChE). Moreover, their genotoxicity and radioprotective activity on human white blood cells were studied using the alkaline comet assay, viability testing and the analysis of the structural chromosome aberrations. The tested compounds were found to be weak inhibitors of AChE, for TCP IC 50 = 1 × 10 −5 M and for TAMORF IC 50 > 1 × 10 −3 M, without reactivating and protective effects on AChE inhibited by soman. Results suggest that TCP modified by the replacement of the cyclohexyl ring with an adamantly ring and piperidine with morpholine group (TAMORF) have lower toxicity. Both compounds possess low cytotoxicity and radioprotective activity, but TAMORF also shows cell growth inhibitory effects. To clarify differences in their biological efficiency observed in vitro and in vivo, additional analyses are necessary. Since TAMORF was found to significantly inhibit cell growth and proliferation in vitro, it is reasonably to consider it as a source molecule promising for further modifications and development of more potent substances with antitumor properties rather then radioprotector or antidote in organophosphorus poisoning.

Enzyme-kinetic investigation of different sarin analogues reacting with human acetylcholinesterase and butyrylcholinesterase

The pertinent threat of using organophosphorus compound (OP)-type chemical warfare agents (nerve agents) during military conflicts and by non-state actors requires the continuous search for more effective medical countermeasures. OP inhibit acetylcholinesterase (AChE) and therefore standard treatment of respective poisoning includes AChE reactivators (oximes) in combination with antimuscarinic agents. Hereby, standard oximes, 2-PAM and obidoxime, are considered to be rather insufficient against various nerve agents. Numerous experimental oximes have been investigated in the last decades by in vitro and in vivo models. Recently, we studied the reactivating potency of several oximes with human AChE inhibited by structurally different OP and observed remarkable differences depending on the OP and oxime. In order to investigate structure–activity relationships we determined the various kinetic constants (inhibition, reactivation, aging) for a series of sarin analogues bearing a methyl, ethyl, n-propyl, n-butyl, i-propyl, i-butyl, cyclohexyl or pinacolyl group with human AChE and BChE. The rate constants for the inhibition of human erythrocyte AChE and plasma BChE by these OP ( k i), for the spontaneous dealkylation ( k a) and reactivation ( k s) of OP-inhibited AChE and BChE as well as for the oxime-induced reactivation of OP-inhibited AChE and BChE by the oximes obidoxime, 2-PAM, HI 6, HLö 7 and MMB-4 were determined. With compounds bearing a n-alkyl group the inhibition rate constant increased with chain length. A relation between chain length and spontaneous reactivation velocity was also observed. In contrast, no structure–activity dependence could be observed for the oxime-induced reactivation of AChE and BChE inhibited by the compounds tested. In general, OP-inhibited AChE and BChE were susceptible towards reactivation by oximes. HLö 7 was the most potent reactivator followed by HI 6 and obidoxime while 2-PAM and MMB-4 were rather weak reactivators. These data indicate a potential structure–activity relationship concerning inhibition and spontaneous reactivation but not for oxime-induced reactivation.

Simulation of cholinesterase status at different scenarios of nerve agent exposure

The ongoing threat of homicidal use of organophosphorus-type chemical warfare agents (“nerve agents”) during military conflicts and by terrorists underlines the necessity for effective medical countermeasures. Standard treatment with atropine and the established acetylcholinesterase (AChE) reactivators, obidoxime and pralidoxime, is considered to be ineffective with certain nerve agents due to low oxime effectiveness. From obvious ethical reasons only animal experiments can be used to evaluate new oximes as nerve agent antidotes. However, the extrapolation of data from animal to humans is hampered by marked species differences. Since reactivation of OP-inhibited AChE is considered to be the main mechanism of action of oximes, human erythrocyte AChE can be exploited to test the efficacy of new oximes. Recently, a dynamic computer model was developed which allows the calculation of AChE activities at different scenarios by combining enzyme kinetics (inhibition, reactivation, aging) with OP toxicokinetics and oxime pharmacokinetics. Now, this computer model was further extended by including the pharmaco- and enzyme kinetics of carbamate pretreatment. Simulations were performed for intravenous and percutaneous nerve agent exposure and intramuscular oxime treatment in the presence and absence of pyridostigmine pretreatment using published data. The model presented may serve as a tool for evaluating the impact of carbamate pretreatment on oxime-induced reactivation of inhibited AChE, for defining effective oxime concentrations and for optimizing oxime treatment. In addition, this model may be useful for the development of meaningful therapeutic strategies in animal experiments.

Spectral study of the peripheral site of ligand binding in the active center of cholinesterases from different natural sources

An increased ethidium bromide fluorescence at 610 nm was observed in the presence of cholinesterases from some natural sources, and a new fluorescence band appeared in the 500-570 nm region. The data obtained suggest a resonance energy transfer from the cholinesterase-ethidium bromide complex to a free ethidium bromide molecule. The structure of the peripheral ligand binding sites in the active center of bovine erythrocyte acetylcholinesterase, horse serum butyrylcholinesterase, and squid ganglia cholinesterase proved essentially similar.

Analysis of inhibition, reactivation and aging kinetics of highly toxic organophosphorus compounds with human and pig acetylcholinesterase

Organophosphorus compounds (OP) are in wide spread use as pesticides and highly toxic OP may be used as chemical warfare agents (nerve agents). OP inhibit acetylcholinesterase (AChE), therefore, standard treatment includes AChE reactivators (oximes) in combination with antimuscarinic agents. In the last decades, the efficacy of oximes has been investigated in various animal models, mostly in rodents. However, extrapolating animal data to humans is problematical because of marked differences between rodents and humans concerning the toxicokinetics of nerve agents, the pharmacokinetics of antidotes and the AChE enzyme kinetics. In order to improve the understanding of species differences and to enable a more reliable extrapolation of animal data to humans a study was initiated to investigate the effect of highly toxic nerve agents, i.e. VX, Russian VX (VR) and Chinese VX (CVX), with human and pig erythrocyte AChE. Hereby, the rate constants for the inhibition of AChE by these OP ( k i) and for the spontaneous dealkylation ( k a) and reactivation ( k s) of OP-inhibited AChE as well as for the oxime-induced reactivation of OP-inhibited AChE by the oximes obidoxime, 2-PAM, HI 6, HLö 7 and MMB-4 were determined. Compared to human AChE pig AChE showed a lower sensitivity towards the investigated OP. Furthermore, a slower spontaneous dealkylation and reactivation of pig AChE was recorded. The potency of the investigated oximes was remarkably lower with OP-inhibited pig AChE. These data may contribute to a better understanding of species differences and may provide a kinetic basis for extrapolation of data from pig experiments to humans.