Suitability of human butyrylcholinesterase as therapeutic marker and pseudo catalytic scavenger in organophosphate poisoning: A kinetic analysis

The widespread use of organophosphorus compounds (OPs) as pesticides and the frequent misuse of OP nerve agents in military conflicts or terrorist attacks emphasize the high clinical relevance of OP poisoning. The toxic symptomatology is caused by inhibition of acetylcholinesterase (AChE). A mainstay of standard antidotal treatment is atropine for antagonizing effects mediated by over stimulation of muscarinic ACh-receptors and oxime to reactivate OP-inhibited AChE. For therapeutic monitoring of oxime treatment in OP poisoning, measurement of erythrocyte AChE is suitable because erythrocyte AChE is an easily accessible surrogate for synaptic AChE. However, measurement of erythrocyte AChE is not standard practice. In contrast, determination of plasma butyrylcholinesterase (BChE) activity is in routine use for monitoring the benefit of oxime therapy. As oxime efficacy is limited with certain OPs (e.g. dimethoate, tabun, soman) alternative therapeutic approaches, e.g. the application of scavengers (BChE) which may sequester OPs before they reach their physiological target, are under investigation.     

Two possible orientations of the HI-6 molecule in the reactivation of organophosphate-inhibited acetylcholinesterase

The inhibition of acetylcholinesterase (AChE) by organophosphorus compounds (OPs) causes acute toxicity or death of the intoxicated individual. One group of these compounds, the OP nerve agents, pose an increasing threat in the world due to their possible use in the battlefield or terrorist acts. Antidotes containing oxime compounds to reactivate the inhibited enzyme are highly valued for treatment against OP poisoning. One of these reactivators, HI-6, was shown to be significantly more effective in treating soman toxicity than other oximes, such as 2-PAM, TMB4, and obidoxime. However, HI-6 was less effective in reactivating AChE inhibited by the OP pesticide, paraoxon. In this study, the mechanism for HI-6-induced reactivation of OP-AChE conjugates was investigated using mouse mutant AChEs inhibited with different OPs including organophosphate paraoxon, and several methylphosphonates. Results indicate that the HI-6 molecule may assume two different orientations in the reactivation of AChE inhibited by organophosphate and Sp methylphosphonates. These conclusions were further corroborated by reactivation studies using an analog of HI-6 in which the bispyridinium moieties are linked by a methylene bridge rather than an ether oxygen.     

Kinetic analysis of interactions between human acetylcholinesterase, structurally different organophosphorus compounds and oximes

The wide-spread use of organophosphorus compounds (OP) as pesticides and the availability of highly toxic OP-type chemical warfare agents (nerve agents) underlines the necessity for an effective medical treatment. Acute OP toxicity is primarily caused by inhibition of acetylcholinesterase (AChE, EC 3.1.1.7). Reactivators (oximes) of inhibited AChE are a mainstay of treatment, however, the commercially available compounds, obidoxime and pralidoxime, are considered to be rather ineffective against various nerve agents. The antidotal efficacy of new oximes is primarily tested in animals for ethical reasons. However, the various interactions between AChE, OP and oximes can be investigated with human AChE which enables the direct assessment of oxime potency, thus excluding species differences. The kinetics of inhibition, reactivation and aging were investigated with human erythrocyte AChE, various structurally different OP (organophosphates, -phosphonates and phosphoramidates) and oximes (obidoxime, pralidoxime, HI 6, HL? 7). The inhibitory potency of OPs, reactivating potency of oximes and spontaneous reactivation and aging were strongly affected by the structural characteristics of the OPs and of the phosphyl-AChE-complex. The kinetic data emphasize the superior inhibitory potency of organophosphonates. AChE inhibited by various phosphoramidates was mostly resistant towards reactivation by oximes while phosphonylated AChE was easily reactivated. HL? 7 was most potent with phosphonylated AChE and obidoxime with AChE inhibited by organophosphates and phosphoramidates. With the exception of soman, OP-inhibited AChE aged rather slowly (t(1/2) 3-231 h) and reactivated spontaneously with some compounds. These results indicate that there is obviously no direct structure-activity relationship for the various interactions of human AChE, OPs and oximes.     

Regeneration of cholinesterase activities in humans and rats after inhibition by O,O-dimethyl-2,2-dichlorovinyl phosphate.

By applying rate constants determined in vitro for spontaneous reactivation and aging of O,O-dimethyl-2,2-dichlorovinyl phosphate (DDVP)-inhibited cholinesterases to enzyme activities measured in vivo after inhibition by DDVP, we evaluated whether regeneration in vivo is due to spontaneous reactivation of the inhibited enzyme or whether it must be attributed to enzyme synthesis. Regeneration in vivo of rat brain and plasma cholinesterase activities can be entirely attributed to spontaneous reactivation of the inhibited enzymes, with half-times of 2 and 2.5 hr for the brain and plasma enzyme, respectively. Regeneration in vivo of human erythrocyte and plasma cholinesterase activities is much slower than that predicted from spontaneous reactivation. It was therefore attributed to enzyme synthesis; the calculated half-times for the synthesis of erythrocyte and plasma cholinesterase are 15 and 6.7 days, respectively.     

Effect of Different Oximes on Rat and Human Cholinesterases Inhibited by Methamidophos: A Comparative In Vitro and In Silico Study

Methamidophos is one of the most toxic organophosphorus (OP) compounds. It acts via phosphorylation of a serine residue in the active site of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), leading to enzyme inactivation. Different oximes have been developed to reverse this inhibition. Thus, our work aimed to test the protective or reactivation capability of pralidoxime and obidoxime, as well as two new oximes synthesised in our laboratory, on human and rat cholinesterases inhibited by methamidophos. In addition, we performed molecular docking studies in non‐aged methamidophos‐inhibited AChE to understand the mechanisms involved. Our results suggested that pralidoxime protected and reactivated methamidophos‐inhibited rat brain AChE. Regarding human erythrocyte AChE, all oximes tested protected and reactivated the enzyme, with the best reactivation index observed at the concentration of 50 μM. Concerning BChE, butane‐2,3‐dionethiosemicarbazone oxime (oxime 1) was able to protect and reactivate the methamidophos‐inhibited BChE by 45% at 50 μM, whereas 2(3‐(phenylhydrazono)butan‐2‐one oxime (oxime 2) reactivated 28% of BChE activity at 100 μM. The two classical oximes failed to reactivate BChE. The molecular docking study demonstrated that pralidoxime appears to be better positioned in the active site to attack the O‐P moiety of the inhibited enzyme, being near the oxyanion hole, whereas our new oximes were stably positioned in the active site in a manner similar to that of obidoxime. In conclusion, our work demonstrated that the newly synthesised oximes were able to reactivate not only human erythrocyte AChE but also human plasma BChE, which could represent an advantage in the treatment of OP compounds poisoning.     

Comparison of oxime-initiated reactivation of organophosphorous-inhibited acetylcholinesterase in brains of avian embryos

Organophosphorous (OP) insecticide-induced inhibition and oxime reactivation of acetylcholinesterase (AChE) was determined in whole-brain homogenates prepared from 15-d-old chick embryos. Doses of chlorpyrifos, parathion, acephate, and trichlorfon that inhibited AChE >70% were administered to the embryos. Following insecticide exposure, an in vitro system compared the capability of the oximes pralidoxime (2-PAM), obidoxime, TMB-4, and HI-6 to reactivate the OP-inhibited AChE. Concentration-related increases in AChE activities were noted in embryo brains reactivated with 2-PAM, TMB, and HI-6. 2-PAM was the most effective reactivator of trichlorfon-inhibited AChE; 2-PAM and obidoxime were relatively similar in effectiveness for reactivation of AChE inhibited with the other OP insecticides used as test agents. All oximes were similarly effective against acephate, but HI-6 was the least effective reactivator of AChE in chick embryo brain homogenates inhibited by the other OP insecticides. These results suggest that both the OP insecticide inhibiting AChE and the oxime reactivating this enzyme can contribute to the effectiveness of the avian brain AChE reactivation.     

COMPARISON OF OXIME-INITIATED REACTIVATION OF ORGANOPHOSPHOROUS-INHIBITED ACETYLCHOLINESTERASE IN BRAINS OF AVIAN EMBRYOS

Organophosphorous (OP) insecticide-induced inhibition and oxime reactivation of acetylcholinesterase (AChE) was determined in whole-brain homogenates prepared from 15-d-old chick embryos. Doses of chlorpyrifos, parathion, acephate, and trichlorfon that inhibited AChE>70% were administered to the embryos. Following insecticide exposure, an in vitro system compared the capability of the oximes pralidoxime (2-PAM), obidoxime, TMB-4, and HI-6 to reactivate the OP-inhibited AChE. Concentration-related increases in AChE activities were noted in embryo brains reactivated with 2-PAM, TMB, and HI-6.2-PAM was the most effective reactivator of trichlorfon-inhibited AChE; 2-PAM and obidoxime were relatively similar in effectiveness for reactivation of AChE inhibited with the other OP insecticides used as test agents. All oximes were similarly effective against acephate, but HI-6 was the least effective reactivator of AChE in chick embryo brain homogenates inhibited by the other OP insecticides. These results suggest that both the OP insecticide inhibiting AChE and the oxime reactivating this enzyme can contribute to the effectiveness of the avian brain AChE reactivation.     

Effects of oximes on rate of decarbamylation of human red blood cell AChE measured with two different methods

Treatment regimen of poisonings by organophosphorus (OP) compounds usually includes oxime therapy. The treatment options in soman poisoning are very limited due to rapid aging of the inhibited acetylcholinesterase (AChE), when the enzyme species is considered as irreversibly inhibited and resistant towards reactivation by oximes. 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. One means of protecting against soman poisoning is the prophylactic use of certain reversible inhibitors (carbamates) of AChE. The question whether there is a possibility of an interaction between pre-treating carbamates and oximes at AChE arises. Therefore we studied the effects of the oximes obidoxime, HI 6 and MMB-4 on the rate of decarbamylation for physostigmine- and pyridostigmine-inhibited human erythrocyte AChE both in a dynamically working in vitro model and a static cuvette system. Our results show that HI 6 increased the rate of decarbamylation for both physostigmine- and pyridostigmine-inhibited enzyme in both systems, the observed effect by HI 6 increasing with higher doses. Obidoxime had a slightly accelerating effect on the pyridostigmine-inhibited enzyme. MMB-4 applied to pyridostigmine-inhibited AChE in the static system only showed no difference to the experiments made in absence of oxime. No oxime showed a tendency to retard the rate of decarbamylation.     

Potential of two new oximes in reactivate human acetylcholinesterase and butyrylcholinesterase inhibited by organophosphate compounds: An in vitro study

Organophosphate (OP) compounds exert inhibition on cholinesterase (ChE) activity by irreversibly binding to the catalytic site of the enzyme. Oximes are compounds generally used to reverse the ChE inhibition caused by OP agents. In this study, we compared the in vitro reactivation potency of two new oximes (oxime 1: butane-2,3-dionethiosemicarbazone; oxime 2: 3-(phenylhydrazono) butan-2-one) against the inhibition on acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities induced by chlorpyrifos, diazinon and malathion. Oximes used clinically (obidoxime and pralidoxime) were used as positive control. For this study, human blood (erythrocytes for AChE determination and plasma for BChE determination) was used and different concentrations of oximes (1–100 μM) were tested. The concentrations of OP used were based on the IC₅₀ for AChE and BChE. Results demonstrated that obidoxime was more effective in reactivate the AChE inhibition induced by OP compounds. However, both newly developed oximes achieved similar reactivations rates that pralidoxime for chlorpyrifos and diazinon-inhibited AChE. For BChE reactivation, none of evaluated oximes achieved positives rates of reactivation, been obidoxime able to reactivate malathion-inhibited BChE only in 24% at the highest concentration. We conclude that both newly developed oximes seem to be promising reactivators of OP-inhibited AChE.     

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.     

HI-6 in man: efficacy of the oxime in poisoning by organophosphorus insecticides.

The efficacy of the oxime HI-6 was studied as a treatment for organophosphorus poisoning. HI-6 was given four times daily as a single intramuscular injection of 500 mg accompanied by atropine and diazepam therapy. Oxime treatment was started on admission and continued for a minimum of 48 h and a maximum of 7 d. HI-6 rapidly reactivated human blood acetylcholinesterase inhibited by dimethoxy organophosphorus compounds, while the dimethoxy-inhibited enzyme was mainly resistant to the treatment by HI-6. Although both HI-6 and pralidoxime chloride reactivated the red blood cell cholinesterase in quinalphos-poisoned subjects, the return of enzyme activities was more rapid following the use of HI-6. The general improvement of poisoned patients, which was sometimes more rapid than the rise of acetylcholinesterase activity, pointed to direct pharmacological effects of HI-6. No undesirable side-effects were noted in patients when HI-6 plasma concentrations were maintained at levels far above the 'therapeutic' concentration for up to 7 d.     

Evaluation of oxime efficacy in nerve agent poisoning: development of a kinetic-based dynamic model

The widespread use of organophosphorus compounds (OP) as pesticides and the repeated misuse of highly toxic OP as chemical warfare agents (nerve agents) emphasize the necessity for the development of 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. By combining enzyme kinetics (inhibition, reactivation, aging) with OP toxicokinetics and oxime pharmacokinetics a dynamic in vitro model was developed which allows the calculation of AChE activities at different scenarios. This model was validated with data from pesticide-poisoned patients and simulations were performed for intravenous and percutaneous nerve agent exposure and intramuscular oxime treatment using published data. The model presented may serve as a tool for defining effective oxime concentrations and for optimizing oxime treatment. In addition, this model can be useful for the development of meaningful therapeutic animal models.     

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, HL? 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. HL? 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.     

Dose-related inhibition of brain and plasma cholinesterase in neonatal and adult rats following sublethal organophosphate exposures.

Developing mammals are markedly more sensitive to acute toxicity from exposure to a variety of organophosphorus (OP) pesticides. The present study examined dose-related inhibition of both brain and plasma cholinesterase activity in neonatal and adult rats exposed to sublethal doses of one of three common OP pesticides, methyl parathion, parathion and chlorpyrifos. Effective dose 50 (i.e., ED50 or dose which would inhibit 50% of the cholinesterase activity) values were determined and then correlated with an indicator of acute toxicity, the maximal tolerated dose (MTD). It was found that ED50 estimates for both brain and plasma cholinesterase correlated highly (r = 0.932-0.992) with previously derived MTD values. In no case was there a significant difference between in vivo brain and plasma cholinesterase inhibition across doses in neonatal rats was high (r = 0.962-0.975) but lower in adults (r = 0.700-0.943). The results suggest that in vivo inhibitory potency of the three OPs towards either brain or plasma ChE activity is highly correlated with sensitivity to acute toxicity in both neonatal and adult rats. Additionally, under defined experimental conditions, plasma ChE inhibition may be a useful quantitative index for the degree of brain cholinesterase inhibition following OP exposures.     

Effects of 1,1'-oxydimethylene bis-(4-tert-butylpyridinium chloride) (SAD-128) and decamethonium on reactivation of soman- and sarin-inhibited cholinesterase by oximes

The effects of 1,1′-oxydimethylene bis-(4-tert-butylpyridinium chloride) (SAD-128) and decamethonium on reactivation by oxime of Soman- and Sarin-inhibited erythrocyte acetylcholinesterase (AChE; EC 3.1.1.7) are reported. The inclusion of SAD-128 or decamethonium (10^?3 M) together with either TMB-4 or Toxogonin markedly augmented the reactivation of Soman- or Sarin-inhibited AChE; the recovery of enzyme activity was more than doubled in each instance when compared to the recovery in the presence of oxime alone. No reactivation was observed with SAD-128 or decamethonium in the absence of oxime. The i.v. ld₅₀ of SAD-128 in rabbits is 11.9 mg/kg. When 5 mg/kg of SAD-128 was given i.v. to rabbits 2 min before poisoning with Soman. 15–20 per cent of the blood cholinesterase was protected from inhibition by Soman. The aging rate in vitro of Soman-inhibited erythrocyte AChE was decreased by a factor of 1.9 with 10^?3 M SAD-128. A concentration of 1.4 × 10^?4M SAD-128 reduced the rate of acetylcholine hydrolysis by erythrocyte AChE in vitro to 50 per cent of control. We suggest that SAD-128 protects against inhibition by Soman due to its ability to function as a reversible inhibitor and that SAD-128 and decamethonium might be allosteric modifiers of the inhibited enzyme, rendering it more susceptible to reactivation by TMB-4 or Toxogonin.     

Protective effect of equine butyrylcholinesterase in inhalation intoxication of rats with sarin: determination of blood and brain cholinesterase activities

The effect of pretreatment with equine butyrylcholinesterase (EqBuChE) on cholinesterase inhibition in the blood and brain of rats following inhalation intoxication with low concentrations (1.25 microg/L for 60 min) of sarin were studied. Animals pretreated with different doses of equine butyrylcholinesterase showed significant increases in plasma butyrylcholinesterase activity. However, erythrocyte acetylcholinesterase activity was unchanged. The decrease in acetylcholinesterase and butyrylcholinesterase activity after inhalation intoxication was dependent on the dose of equine butyrylcholinesterase used for pretreatment and was always greater for erythrocyte acetylcholinesterase. Acetylcholinesterase activity in different brain regions was unchanged following pretreatment with equine butyrylcholinesterase. After inhalation exposure to sarin, acetylcholinesterase activity was diminished markedly in the pontomedullar area (51.5% of normal activity) and frontal cortex (72.0% of normal activity), and slightly in basal ganglia (91.4% of normal activity). Plasma levels of sarin were determined using fluoride-induced reactivation of inhibited enzyme. As expected, the amounts of sarin in plasma were almost identical in rats pretreated with EqBuChE as well as in untreated rats. In pretreated animals, the plasma amount of sarin did not depend on the dose of equine butyrylcholinesterase used for pretreatment. Our results demonstrate that equine butyrylcholinesterase pretreatment can be considered as an effective prophylaxis against nerve agents (at least with sarin) and seems to be an alternative or superior to prophylaxis provided by reversible cholinesterase inhibitors.     

Interindividual variations in enzymes controlling organophosphate toxicity in man.

1 Interindividual variations in an unexposed population have been defined for five enzymes involved in organophosphate (OP) toxicity. The enzymes measured were: red blood cell acetylcholinesterase (AChE), lymphocyte neuropathy target esterase (NTE), serum cholinesterase (ChE), serum paraoxonase and serum arylesterase. 2 AChE and arylesterase were normally distributed in the population whilst the distribution of NTE, ChE and paraoxonase deviated significantly from normal. 3 Assay precision and intra-individual variability were measured for each of the enzymes; the effect on interindividual variation was assessed. 4 Variations in enzyme activities between individuals could have profound effects on susceptibility to OP toxicity. Prior determination of these enzymes may be predictive of susceptibility. 5 Lymphocyte NTE has some limitations as an indicator of exposure to neurotoxic OPs.     

Protective Effect of Equine Butyrylcholinesterase in Inhalation Intoxication of Rats with Sarin: Determination of Blood and Brain Cholinesterase Activities

The effect of pretreatment with equine butyrylcholinesterase (EqBuChE) on cholinesterase inhibition in the blood and brain of rats following inhalation intoxication with low concentrations (1.25 μg/L for 60 min) of sarin were studied. Animals pretreated with different doses of equine butyrylcholinesterase showed significant increases in plasma butyrylcholinesterase activity. However, erythrocyte acetylcholinesterase activity was unchanged. The decrease in acetylcholinesterase and butyrylcholinesterase activity after inhalation intoxication was dependent on the dose of equine butyrylcholinesterase used for pretreatment and was always greater for erythrocyte acetylcholinesterase. Acetylcholinesterase activity in different brain regions was unchanged following pretreatment with equine butyrylcholinesterase. After inhalation exposure to sarin, acetylcholinesterase activity was diminished markedly in the pontomedullar area (51.5% of normal activity) and frontal cortex (72.0% of normal activity), and slightly in basal ganglia (91.4% of normal activity). Plasma levels of sarin were determined using fluoride-induced reactivation of inhibited enzyme. As expected, the amounts of sarin in plasma were almost identical in rats pretreated with EqBuChE as well as in untreated rats. In pretreated animals, the plasma amount of sarin did not depend on the dose of equine butyrylcholinesterase used for pretreatment. Our results demonstrate that equine butyrylcholinesterase pretreatment can be considered as an effective prophylaxis against nerve agents (at least with sarin) and seems to be an alternative or superior to prophylaxis provided by reversible cholinesterase inhibitors.     

Weak inhibitors protect cholinesterases from stronger inhibitors (dichlorvos): in vitro effect of tiapride

Metoclopramide is a benzamide dopamine receptor antagonist and serotonine receptor agonist widely used as an antiemetic and gastric prokinetic drug. In addition, metoclopramide is a weak and reversible inhibitor of cholinesterases. The authors have previously shown that metoclopramide has a cholinesterase protective effect against inhibition by organophosphates (OPs). The putative mode of protective action of metoclopramide is, when administered in excess, competion for the active site of the enzyme with the more potent OP. In the present paper the authors present their results using another benzamide with weak cholinesterase inhibitory properties, tiapride (TIA). The purpose of the study was to quantify in vitro the extent of TIA-conferred protection, using dichlorvos (dichlorovinyl dimethyl phosphate; DDVP) as an inhibitor. DDVP is a moderately toxic (LD50 in rats in the milligram range), non-neuropathic OP. The substance is responsible for a large number of accidental or suicidal exposures. Red blood cell (RBC) acetylcholinesterase (AChE) activities in whole blood and butyrylcholinesterase (BChE) activities in human plasma were measured photometrically in the presence of different DDVP and TIA concentrations and IC50 was calculated. Determinations were repeated in the presence of increasing TIA concentrations. The IC50 of DDVP increases with the TIA concentration in a linear manner. The protective effect of TIA on cholinesterase could be of practical relevance in the treatment of OP poisoning. The authors conclude that in vivo testing of TIA as an OP protective agent is warranted.     

Efficacy of trimedoxime in mice poisoned with dichlorvos, heptenophos or monocrotophos

The aim of the study was to examine antidotal potency of trimedoxime in mice poisoned with three direct dimethoxy-substituted organophosphorus inhibitors. In order to assess the protective efficacy of trimedoxime against dichlorvos, heptenophos or monocrotophos, median effective doses and efficacy half-times were calculated. Trimedoxime (24 mg/kg intravenously) was injected 5 min. before 1.3 LD50 intravenously of poisons. Activities of brain, diaphragmal and erythrocyte acetylcholinesterase, as well as of plasma carboxylesterases were determined at different time intervals (10, 40 and 60 min.) after administration of the antidotes. Protective effect of trimedoxime decreased according to the following order: monocrotophos > heptenophos > dichlorvos. Administration of the oxime produced a significant reactivation of central and peripheral acetylcholinesterase inhibited with dichlorvos and heptenophos, with the exception of erythrocyte acetylcholinesterase inhibited by heptenophos. Surprisingly, trimedoxime did not induce reactivation of monocrotophos-inhibited acetylcholinesterase in any of the tissues tested. These organophosphorus compounds produced a significant inhibition of plasma carboxylesterase activity, while administration of trimedoxime led to regeneration of the enzyme activity. The same dose of trimedoxime assured survival of experimental animals poisoned by all three organophosphorus compounds, although the biochemical findings were quite different.