Inhibitor Report for: VX

Organophosphorus nerve agents (OPNAs) are highly toxic compounds inhibiting cholinergic enzymes in the central and autonomic nervous systems and neuromuscular junctions, causing severe intoxications in humans. Medical countermeasures and efficient decontamination solutions are needed to counteract the toxicity of a wide spectrum of harmful OPNAs including G, V and Novichok agents. Here, we describe the use of engineered OPNA-degrading enzymes for the degradation of various toxic agents including insecticides, a series of OPNA surrogates, as well as real chemical warfare agents (cyclosarin, sarin, soman, tabun, VX, A230, A232, A234). We demonstrate that only two enzymes can degrade most of these molecules at high concentrations (25 mM) in less than 5 min. Using surface assays adapted from NATO AEP-65 guidelines, we further show that enzyme-based solutions can decontaminate 97.6% and 99.4% of 10 gm(-)(2) of soman- and VX-contaminated surfaces, respectively. Finally, we demonstrate that these enzymes can degrade ethyl-paraoxon down to sub-inhibitory concentrations of acetylcholinesterase, confirming their efficacy from high to micromolar doses.

Sheep were studied for the possibility of treatment after parenteral (intramuscular) intoxication with EDMM (methylthiophosphorous acid O-ethyl-S-2-dimethylamino-ethylester) and with EDIM (methylthiophosphorous acid O-ethyl-S-2-diisopropyl-aminoethylester). In both cases of intoxication, the therapy was based on a system of an anticholinergic and cholinesterase reactivator administered singly at a time of the maximum development of the clinical signs of poisoning and maximum inhibition of both erythrocytic (AChE, E.C.3.1.1.7.) and plasma (BChE, E.C.3.1.1.8.) cholinesterase. The optimum therapeutic system requires the administration of 20.0 mg atropine s. c. pro toto and 10.0 mg trimedoxim per kg 1. w. i. v. In both cases of poisoning with doses = LD50 in i. m. administration, the mentioned system was actually positive. In a single administration irrespective of the doses of the used drugs, the system does not guarantee survival after ingestion of anticholinesterasic doses above LD50.

The active sites of acetylcholinesterase multiple forms from four widely different zoological species (Electrophorus, Torpedo, rat and chicken) were titrated using a stable, irreversible phosphorylating inhibitor (O-ethyl-S2-diisopropylaminoethyl methyl-phosphonothionate). In all cases, we found that within a given species, the molecular forms we examined were equivalent in their catalytic activity per active site. As pure preparations of the molecular forms of Electrophorus acetylcholinesterase were available, we were able to establish that one inhibitor molecule binds per monomer unit for each of them. This had already been shown by several authors for the tetrameric globular form, but not for the tailed molecules. Analysis of the phosphorylation reaction showed that they are equally reactive. Under our experimental conditions, their turnover number per site was 4.4 x 10(7) mol of acetylthiocholine hydrolysed . h-1 at 28 degrees C, pH 7.0. The corresponding value was less than half for Torpedo (1.64 x 10(7) mol . h-1), and again lower for rat (1.32 x 10(7) mol . h-1) and chicken (1.05 x 10(7) mol . h-1). In the case of rat acetylcholinesterase, the activity per active site of solubilized (with or without Triton X-100) and membrane-bound enzyme were identical. We discuss the implications of these findings with respect to the quaternary structure of acetylcholinesterase, and to the physico-chemical state and physiological properties of its molecular forms.

Organophosphorus nerve agents (OPNAs) are highly toxic compounds inhibiting cholinergic enzymes in the central and autonomic nervous systems and neuromuscular junctions, causing severe intoxications in humans. Medical countermeasures and efficient decontamination solutions are needed to counteract the toxicity of a wide spectrum of harmful OPNAs including G, V and Novichok agents. Here, we describe the use of engineered OPNA-degrading enzymes for the degradation of various toxic agents including insecticides, a series of OPNA surrogates, as well as real chemical warfare agents (cyclosarin, sarin, soman, tabun, VX, A230, A232, A234). We demonstrate that only two enzymes can degrade most of these molecules at high concentrations (25 mM) in less than 5 min. Using surface assays adapted from NATO AEP-65 guidelines, we further show that enzyme-based solutions can decontaminate 97.6% and 99.4% of 10 gm(-)(2) of soman- and VX-contaminated surfaces, respectively. Finally, we demonstrate that these enzymes can degrade ethyl-paraoxon down to sub-inhibitory concentrations of acetylcholinesterase, confirming their efficacy from high to micromolar doses.

Corrected : Organophosphate (OP) intoxications from nerve agent and OP pesticide exposures are managed with pyridinium aldoxime-based therapies whose success rates are currently limited. The pyridinium cation hampers uptake into the central nervous system (CNS). Furthermore, it frequently binds to aromatic residues of OP-inhibited acetylcholinesterase (AChE) in orientations that are non-productive for AChE reactivation, and the structural diversity of OPs impedes efficient reactivation. Improvements of OP antidotes need to include much better access of AChE reactivators to the CNS and optimized orientation of the antidotes' nucleophile within the AChE active-center gorge. On the basis of X-ray structures of a CNS-penetrating reactivator, monoxime RS194B, reversibly bound to native and venomous agent X (VX)-inhibited human AChE (hAChE), here we created seven uncharged acetamido bis-oximes as candidate antidotes. Both oxime groups in these bis-oximes were attached to the same central, saturated heterocyclic core. Diverse protonation of the heterocyclic amines and oxime groups of the bis-oximes resulted in equilibration among up to 16 distinct ionization forms, including uncharged forms capable of diffusing into the CNS and multiple zwitterionic forms optimal for reactivation reactions. Conformationally diverse zwitterions that could act as structural antidote variants significantly improved in vitro reactivation of diverse OP-hAChE conjugates. Oxime group re-orientation of one of the bis-oximes, forcing it to point into the active center for reactivation, was confirmed by X-ray structural analysis. Our findings provide detailed structure-activity properties of several CNS-directed, uncharged aliphatic bis-oximes holding promise for use as protonation-dependent, conformationally adaptive, "smart" accelerated antidotes against OP toxicity.

7-methoxytacrine-4-pyridinealdoxime (7-MEOTA-4-PA, named hybrid 5C) is a compound formerly synthesized and evaluated in vitro, together with 4-pyridine aldoxime (4-PA) and commercial reactivators of acetylcholinesterase (AChE). This compound was designed with the purpose of being a prophylactic reactivator, capable of interacting with different subdomains of the active site of AChE. To investigate these interactions, theoretical results from docking were first compared with experimental data of hybrid 5C, 4-PA, and two commercial oximes, on the reactivation of human AChE (HssAChE) inhibited by VX. Then, further docking studies, molecular dynamics simulations, and molecular mechanics Poisson-Boltzmann surface area calculations, were carried out to investigate reactivation performances, considering the near attack conformation (NAC) approach, prior to the nucleophilic substitution mechanism. Our results helped to elucidate the interactions of such molecules with the different subdomains of the active site of HssAChE. Additionally, NAC poses of each oxime were suggested for further theoretical studies on the reactivation reaction.

The high toxicity of organophosphorus compounds originates from covalent inhibition of acetylcholinesterase (AChE), an essential enzyme in cholinergic neurotransmission. Poisonings that lead to life-threatening toxic manifestations require immediate treatment that combines administration of anticholinergic drugs and an aldoxime as a reactivator of AChE. An alternative approach to reduce the in vivo toxicity of OPs focuses on the use of bioscavengers against the parent organophosphate. Our previous research showed that AChE mutagenesis can enable aldoximes to substantially accelerate the reactivation of OP-enzyme conjugates, while dramatically slowing down rates of OP-conjugate dealkylation (aging). Herein, we demonstrate an efficient HI-6-assisted VX detoxification, both ex vivo in human blood and in vivo in mice by hAChE mutants modified at the choline binding site (Y337A and Y337A/F338A). The catalytic scavenging of VX in mice improved therapeutic outcomes preventing lethality and resulted in a delayed onset of toxicity symptoms.

It is believed that information about the molecular structure of highly toxic O-alkyl-S-2(N,N-dialkylamino) ethyl alkylthiophosphonates (V-gases) obtained from their EI mass spectra is too insufficient. In particular, the determination of molecular weights and structures of radicals at phosphorus and oxygen atoms causes great difficulties. In this paper, solutions of these problems are proposed.

To support the effort to eliminate the Syrian Arab Republic chemical weapons stockpile safely, there was a requirement to provide scientific advice based on experimentally derived information on both toxicity and medical countermeasures (MedCM) in the event of exposure to VM, VX or VM-VX mixtures. Complementary in vitro and in vivo studies were undertaken to inform that advice. The penetration rate of neat VM was not significantly different from that of neat VX, through either guinea pig or pig skin in vitro. The presence of VX did not affect the penetration rate of VM in mixtures of various proportions. A lethal dose of VM was approximately twice that of VX in guinea pigs poisoned via the percutaneous route. There was no interaction in mixed agent solutions which altered the in vivo toxicity of the agents. Percutaneous poisoning by VM responded to treatment with standard MedCM, although complete protection was not achieved.

Organophosphorus hydrolase (OPH) is capable of hydrolyzing a wide variety of organophosphorus pesticides and chemical warfare agents. However, the hydrolytic activity of OPH against the warfare agent VX is less than 0.1% relative to its activity against parathion and paraoxon. Based on the crystal structure of OPH and the similarities it shares with acetylcholinesterase, eight OPH mutants were constructed with the goal of increasing OPH activity toward VX. The activities of crude extracts from these mutants were measured using VX, demeton-S methyl, diisopropylfluoro-phosphate, ethyl parathion, paraoxon, and EPN as substrates. One mutant (L136Y) displayed a 33% increase in the relative VX hydrolysis rate compared to wild type enzyme. The other seven mutations resulted in 55-76% decreases in the relative rates of VX hydrolysis. There was no apparent relationship between the hydrolysis rates of VX and the rates of the other organophosphorus compounds tested.

Organophosphate (OP) anticholinesterases were found to modulate metabolic activities of human neuroblastoma cells and hepatocytes, which was detectable by the Cytosensor microphysiometer. The nerve gas ethyl-S-2-diisopropylaminoethyl methylphosphorothiolate (VX), at 10 microM, produced significant reduction in cell metabolism within 2 min, as measured by changes in the acidification rate of the medium. The reduction was dose- and time-dependent and irreversible after 4 h of exposure. Two alkaline degradation products of VX produced no cytotoxicity. Exposure for 24 h to 3 microM VX caused 36% and 94% irreversible loss of metabolism in hepatocytes and neuroblastoma cells, respectively. The insecticides parathion and chlorpyrifos stimulated hepatocyte metabolism but inhibited neuroblastoma cells. Their oxons were more active. Exposure of neuroblastoma cells for 4 h to VX, parathion, paraoxon, diisopropylfluorophosphate or chlorpyrifos gave an LC50 of 65, 775, 640, 340, or 672 microM, respectively, whereas 24 h gave an LC50 of 0.7, 3.7, 2.5, 29, and 31 microM, respectively. Preincubation of hepatocytes with phenobarbital enhanced their response to parathion and VX due to metabolic bioactivation. Atropine partially blocked the effects of VX and paraoxon on both cell types, which suggests the involvement of a muscarinic receptor as the target for cytotoxicity. There was no correlation between OP in vivo neurotoxicity and in vitro cytotoxicity. It is suggested that the former results from their cholinesterase inhibition, while the latter results from action on different targets and requires much higher concentrations.

Organophosphorus (OP) insecticides and nerve agents that contain P-S bond are relatively more resistant to enzymatic hydrolysis. Purified phenol oxidase (laccase) from the white rot fungus Pleurotus ostreatus (Po) together with the mediator 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) displayed complete and rapid oxidative degradation of the nerve agents VX and Russian VX (RVX) and the insecticide analog diisopropyl-Amiton with specific activity: k(sp) = 2200, 667 and 1833 nmol min(-1) mg(-1), respectively (pH 7.4, 37 degrees C). A molar ratio of 1:20 for OP/ABTS and 0.05 M phosphate at pH 7.4 provided the highest degradation rate of VX and RVX. The thermostable laccase purified from the fungus Chaetomium thermophilium (Ct) in the presence of ABTS caused a 52-fold slower degradation of VX with k(sp) = 42 nmol min(-1) mg(-1). The enzymatic biodegradation products were identified by 31P-NMR and GC/MS analysis.

Organophosphorus acid anhydride (OP) "nerve agents" are rapid, stoichiometric, and essentially irreversible inhibitors of serine hydrolases. By placing a His near the oxyanion hole of human butyrylcholinesterase (BChE), we made an esterase (G117H) that catalyzed the hydrolysis of several OP, including sarin and VX [Millard et al. (1995) Biochemistry 34, 15925-15930]. G117H was limited, however, because it was irreversibly inhibited by pinacolyl methylphosphonofluoridate (soman); soman is among the most toxic synthetic poisons known. This limitation of G117H has been overcome by a new BChE (G117H/E197Q) that combines two engineered features: spontaneous dephosphonylation and slow aging (dealkylation). G117H/E197Q was compared with the single mutants BChE G117H and E197Q. Each retained cholinesterase activity with butyrylthiocholine as substrate, although kcat/Km decreased 11-, 11- or 110-fold for purified G117H, E197Q, or G117H/E197Q, respectively, as compared with wild-type BChE. Only G117H/E197Q catalyzed soman hydrolysis; all four soman stereoisomers as well as sarin and VX were substrates. Phosphonylation and dephosphonylation reactions were stereospecific. Double mutant thermodynamic cycles suggested that the effects of the His and Gln substitutions on phosphonylation were additive for PSCR or PRCR soman, but were cooperative for the PSCS stereoisomer. Dephosphonylation limited overall OP hydrolysis with apparent rate constants of 0.006, 0.077, and 0.128 min-1 for the PR/SCR, PSCS, and PRCS soman stereoisomers, respectively, at pH 7.5, 25 degrees C. We conclude that synergistic protein design converted an archetypal "irreversible inhibitor" into a slow substrate for the target enzyme.

We have designed a microdialysis technique to measure acetylcholinesterase (AChE) activity in the cortex of freely moving rats while simultaneously measuring the release of acetylcholine (ACh). Our approach was validated using ethyl S-2-di-isopropylaminoethyl-phosphonothiolate (VX), an irreversible inhibitor of AChE and comparing inhibition measured by this 'in vivo' method with traditional post-mortem assays of AChE activity 120 min after an intraventricular injection of VX. Maximum inhibition of AChE occurred 30 min after injection and was followed by a slow recovery. ACh release reached its maximum 60 min after treatment and then decreased towards normal levels. This method offers a new way to develop medications against poisoning with anticholinesterasic neurotoxic and allows the evaluation of the effects of cholinergic drugs for the treatment of Alzheimer's disease.

The treatment of poisoning by highly toxic organophosphorus compounds (nerve agents) is unsatisfactory. Until now, the efficacy of new potential antidotes has primarily been evaluated in animals. However, the extrapolation of these results to humans is hampered by species differences. Since oximes are believed to act primarily through reactivation of inhibited acetylcholinesterase (AChE) and erythrocyte AChE is regarded to be a good marker for the synaptic enzyme, the reactivating potency can be investigated with human erythrocyte AChE in vitro. The present study was undertaken to evaluate the ability of various oximes at concentrations therapeutically relevant in humans to reactivate human erythrocyte AChE inhibited by different nerve agents. Isolated human erythrocyte AChE was inhibited with soman, sarin, cyclosarin, tabun or VX for 30 min and reactivated in the absence of inhibitory activity over 5-60 min by obidoxime, pralidoxime, HI 6 or HL 7 (10 and 30 microM). The AChE activity was determined photometrically. The reactivation of human AChE by oximes was dependent on the organophosphate used. After soman, sarin, cyclosarin, or VX the reactivating potency decreased in the order HL 7 > HI 6 > obidoxime > pralidoxime. Obidoxime and pralidoxime were weak reactivators of cyclosarin-inhibited AChE. Only obidoxime and HL 7 reactivated tabun-inhibited AChE partially (20%), while pralidoxime and HI 6 were almost ineffective (5%). Therefore, HL 7 may serve as a broad-spectrum reactivator in nerve agent poisoning at doses therapeutically relevant in humans.

Herein a molecular mechanic study of the interaction of a lethal chemical warfare agent, O-ethyl S-[2-(diisopropylamino)ethyl] methylphosphonothioate (also called VX), with Torpedo californica acetylcholinesterase (TcAChE) is discussed. This compound inhibits the enzyme by phosphonylating the active site serine. The chirality of the phosphorus atom induces an enantiomeric inhibitory effect resulting in an enhanced anticholinesterasic activity of the SP isomer (VXS) versus its RP counterpart (VXR). As formation of the enzyme-inhibitor Michaelis complex is known to be a crucial step in the inhibitory pathway, this complex was addressed by stochastic boundary molecular dynamics and quantum mechanical calculations. For this purpose two models of interaction were analyzed: in the first, the leaving group of VX was oriented toward the anionic subsite of TcAChE, in a similar way as it has been suggested for the natural substrate acetylcholine; in the second, it was oriented toward the gorge entrance, placing the active site serine in a suitable position for a backside attack on the phosphorus atom. This last model was consistent with experimental data related to the high inhibitory effect of this compound and the difference in activity observed for the two enantiomers.

The female Wistar rats were intoxicated (i.m.) with sarin, soman and VX in doses equal to 1xLD50 and pontomedullar areas of the brain were prepared, homogenized, centrifuged and in these samples, acetylcholinesterase (AChE, EC 3.1.1.7) activities were determined. In the same samples, AChE was separated using polyacrylamide gel electrophoresis and AChE molecular forms were detected and densitometrically evaluated. In control animals, AChE was separated into four forms differing in their electrophoretic mobility and their quantitative content in the sample. The form with lowest electrophoretic mobility represent the main part of AChE activity constituting the whole enzymatic activity. Following intoxication with the nerve agents mentioned, the whole AChE activity in the pontomedullar area of the brain was decreasing in intervals of ten minutes (soman and sarin) or one hour (VX). The AChE activity at the time of death (or terminal stage) was represented 5-30% of controls. Molecular forms of AChE were inhibited in different extent: the form with lowest electrophoretic mobility was diminished to zero level while the form with the highest mobility was practically unaffected, independently on the type of nerve agent. From quantitative expression of percentage content of the forms vs their activity we can imply that value of the total AChE activity represent the "mean" activity of the forms determined.

We present here a gas chromatography technique allowing the detection and quantification of VX [O-ethyl S-(2-diisopropylaminoethyl)methylphosphonothiolate] as well as its P-S bond hydrolysis product diisopropylaminoethanethiol directly from spiked rat plasma. This technique was applied to study VX hydrolysis in rat plasma. We observed that 53 +/- 4% of 374 microM VX disappeared from spiked plasma after 2 h. VX disappearance was mainly related to enzymatic cleavage of the P-S bond (Km = 2.5 mM and Vmax = 13.3 nmol min-1 ml-1 of rat plasma). The activity was totally inhibited by 1 mM Hg2+ and was also inhibited by metal chelators.

Long-term observation of workers engaged into production of VX chemical diagnosed slow progressing manifestation of chronic occupational poisoning with the chemical. The characteristic nervous, digestive, locomotory, visual and cardiovascular symptoms were revealed. The authors presented laboratory and instrumental data on the cases. As the treatment appeared ineffective, further adjustment and improvement of the therapy is required. The article demonstrated the main underlying metabolic disorders that could be addressed by the pathogenetic therapy.

The authors analyzed over 200 cases of acute poisoning with sarin, soman and VX chemical, determined risk of the poisoning in various conditions. The clinical manifestations of acute poisoning and the long-term effects are presented.

BACKGROUND Causal antidotal therapy of acute intoxications with organophosphorus compounds involving administration of the parasympatholytic and cholineesterase reactivator (oxime) has not been resolved so far satisfactorily despite knowledge of the basic mechanism of action of these noxious substances. METHODS AND RESULTS: In experiments on mice the therapeutic effect of parasympatholytics atropine, benactyzine and biperidene (Akineton) combined with oxime HI-6 on the toxicity of highly toxic organophosphates soman and substance VX and the organophosphorus insecticide phosdrine was compared as regards their influence on the LD50 of these noxious substances during 24-hour survival of experimental animals. Two levels of antidotes were tested. These findings confirm that the LD50 value of untreated intoxication with all three organophosphorus compounds is most increased by oxime HI-6 combined with benactyzine regardless of the antidote dosage.
CONCLUSIONS: Oxime HI-6 is the most effective against highly toxic organophosphates and organophosphorus insecticides when combined with the centrally acting parasympatholytic benactyzine.

For the unequivocal proof of the use of a nerve agent O-ethyl S-2-diisopropylaminoethyl methylphosphonothiolate (VX), a rapid, accurate and sensitive method which allows us to identify its main hydrolysis product ethyl methylphosphonic acid (EMPA) in human serum was explored by GC-MS. GC-MS analysis was performed after solvent extraction with acetonitrile in acidic conditions from the serum sample, which was previously deproteinized by micro-ultrafiltration, and subsequent tert.-butyldimethylsilyl derivatization with N-methyl-N-(tert.-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) with 1% tert.-butyldimethylsilyl chloride (t-BDMSC). Linear calibration curves were obtained in the concentration range from 50 to 500 ng/ml for EMPA in the full-scan EI mode and from 5 to 50 ng/ml for EMPA in the SIM EI mode. The relative standard deviation obtained at a sample concentration of 50 ng/ml was 8.4% in the full-scan mode and 7.3% in the SIM mode. Upon applying the full-scan EI and CI mode, 40 ng/ml and 80 ng/ml were the detection limits. Using the SIM-EI mode, in which the ion at m/z 153 was chosen, the limit was 3 ng/ml.

Bioscavengers of organophophates (OP) have been examined as potential substitutes for the currently approved drug treatment against OP toxicity. The present work was designed to assess the ability of butyrylcholinesterase, purified from human serum (HuBChE), to prevent the toxicity induced by soman and VX in rhesus monkeys. The consistency of the data across species was then evaluated as the basis for the extrapolation of the data to humans. The average mean residence time of the enzyme in the circulation of monkeys following an intravenous loading was 34 hr. High bioavailability of HuBChE in blood (>80%) was demonstrated after intramuscular injection. A molar ratio of HuBChE:OP approximately 1.2 protected against an i.v. bolus injection of 2.1 x LD50 VX, while a ratio of 0.62 was sufficient to protect monkeys against an i.v. dose of 3.3 x LD50 of soman, with no additional postexposure therapy. A remarkable protection was also seen against soman-induced behavioral deficits detected in the performance of a spatial discrimination task. The consistency of the results across several species offers a reliable prediction of both the stoichiometry of the scavenging and the extent of prophylaxis with HuBChE against nerve agent toxicity in humans.

An enzyme termed organophosphorus hydrolase (OPH), derived from Pseudomonas diminuta, had been found previously to hydrolyze the powerful acetylcholinesterase (AChE) inhibitor O-ethyl S-(2-diisopropylaminoethyl) methylphosphonothiolate (VX). This enzyme has now been shown to be correlated with the loss of AChE inhibitory potency (detoxication). OPH also hydrolyzed and detoxified the VX analogue, O,O-diisopropyl S-(2-diisopropylaminoethyl) phosphorothiolate (Tetriso), also a potent AChE inhibitor, about five times faster than VX. The Km for the hydrolysis of the P-S bond of Tetriso was 6.7 x 10(-3) M. OPH also hydrolyzed diisopropylphosphorofluoridate (DFP) 50-60 times faster than Tetriso, and 1,2,2-trimethylpropyl methylphosphonofluoridate (Soman) about seven times faster than Tetriso. DFP was a non-competitive inhibitor of Tetriso hydrolysis, Ki = 8.7 x 10(-4) M. The DFP hydrolysis product, diisopropyl phosphate, was a competitive inhibitor, Ki = 2.3 x 10(-4) M. The rate of detoxication of Tetriso compared with the rate of hydrolysis suggests that OPH may not be totally specific for P-S bond cleavage. OPH was inhibited completely by 1.5 x 10(-4) M 8-hydroxyquinoline-5-sulfonate or 1,10-phenanthroline, both transition element chelators, but inhibited only partially by EDTA, a much more potent chelator.

Studies on the mechanism of detoxication against VX toxicity by purified rabbit anti-VX antibodies were carried out in mice. VX (25 micrograms/kg) was injected subcutaneously immediately following intravenous injection of purified anti-VX antibodies. Blood concentration of free VX declined linearly as the antibody dose increased. Free VX concentration in brain was far lower than that in blood. When the dose of purified anti-VX antibodies was 8 and 6 mg/kg, respectively, free VX concentration in brain and blood approached 0. At the same time, the cholinesterase (ChE) activity in brain increased along with increasing antibody dose, with 70% of normal control value detected when 8 mg/kg antibodies were administered. On the other hand, anti-VX antibodies were not able to protect blood ChE unless the VX dose was lower than 10 micrograms/kg. The studies suggest that the protective action of purified rabbit anti-VX antibodies is from VX antibodies combining with the free VX in blood and extravascular tissue to reduce the amount of VX entering into the brain.

The efficacy of clay or alcoholate as decontaminants in pigs percutaneously poisoned with 6 LD50 of O-ethyl S-2-diisopropylaminoethyl methylphosphonothioate (VX) and 3 LD50 of 1,2,2-trimethylpropyl methylphosphonofluoridate (soman) nerve gases was tested. It was assessed by the time of onset of the first signs of poisoning and death, as well as by the activity of blood cholinesterase (ChE). No toxic signs or fatalities were observed in decontaminated pigs, regardless of the decontaminant used. In VX poisoning up to 240 min. both decontaminants kept ChE values at normal level. Twenty four hours later, ChE activity in pigs decontaminated with clay was 71%, significantly higher than in pigs decontaminated with alcoholate (49%). In soman poisoning the activity in control group was maintained at almost normal level up to 60 min, followed by rapid fall to 58%. Further readings were impossible due to the death of all animals. No significant difference between decontaminants could be noticed throughout the observation of 24 hr. The values were kept between 80 and 100%, with the trend of rising after 120 min.

In the present paper the effect of zeolite tuff (61% clinoptilolite) was investigated on cholinesterase activity in brain, liver, spleen, femoral muscle, heart, stomach, duodenum, colon and erythrocytes in sewer-rats after peroral intoxication with VX substance (65.5 micrograms/kg). Fig. 1 shows the ChE activity in the tissues and erythrocytes in the animals of control group and in the group of animals after intoxication with VX substance. The highest activity in the control group was found in brain and duodenum. The enzyme activity in the femoral muscle had the lowest values. A significant decrease in the ChE activity (P < 0.001 or P < 0.01) occurred in all the investigated samples in the group of animals intoxicated with the VX substance. highest enzyme inhibition was observed in erythrocytes (97.9%), stomach (97.9%), brain (95.4%) and liver (94.7%) if compared with the control group. The relatively lowest inhibition was found out in duodenum and colon. In the group administered zeolite before intoxication (1.0 g/kg five minutes before intoxication) the ChE activity was significantly higher in almost all investigated samples than in the group without zeolite (P < 0.001 or P < 0.01)-Fig. 2. The duodenum is an exception, in which the ChE activity in the zeolite group was lower than in the zeolite-free group (P < 0.001), as well as the colon, in which there were no significant differences in the activity between the groups.

The inhibitory effect of VX, an organophosphate nerve agent, on tissue and erythrocyte cholinesterase (ChE) and the protective effect of clinoptilolite (Cli) in male Wistar rats were studied. The tissue and erythrocyte ChE activity were significantly inhibited 30 min after 65.5 micrograms VX/kg body weight administration. The greatest enzyme inhibition was observed in erythrocytes, stomach, brain and liver. The ChE activity after pretreatment with 1.0 g Cli/kg was significantly higher than that in the VX without Cli group in all samples except duodenum and colon.

The aim of this study was to evaluate the efficiency of hemoperfusion (HP) through coated resin adsorbent Synachrom E-5 in animal intoxications with organophosphate inhibitors of cholinesterases type of nerve agents. Five anesthetized dogs were intoxicated with 2 to 6 LD50 of VX substance and another 4 with 2 to 3 LD50 sarine. Both nerve agents were given i.m. after starting 5 h HP. The clinical and laboratory tests were monitored during each HP. HP therapy prevented the development of serious signs of intoxication provided that the administered quantity of both sarine and the VX substance was only 2 doses of LD50. Specific antidote therapy was necessary to prevent cardiorespiratory failure in animals intoxicated with a higher dose of poison. The results obtained show that HP through Synachrom E-5 in intoxication with nerve agents sarine and the VX type is only partially successful.

In a guinea-pig model with on-line respiratory and circulatory monitoring the therapeutic efficacy of atropine, HL 7 and HI 6 in VX poisoning was compared. In female urethane-anaesthetized Pirbright-white guinea-pigs the a. carotis, v. jugularis and trachea were cannulated. After base line measurements the animals received VX (22.5, 45 or 90 micrograms/kg = 5, 10 or 20 x LD50) intravenously and 2 min. later the antidotes: HL 7 or HI 6 (30 mumol/kg, each) or atropine 10 mg/kg or a combination of atropine and one of the oximes (all intravenously). Respiratory and circulatory parameters were recorded for 60 min. or until death of the animal. Erythrocyte, brain and diaphragm acetylcholinesterase (AChE) activity was determined after the experiment. VX poisoning caused a rapid respiratory arrest within 4-5 min. Atropine treatment was effective in improving the respiratory function after VX, 22.5 micrograms/kg, but had only a small effect after the higher VX doses. The treatment of VX (10 or 20 x LD50) poisoned animals with oxime plus atropine improved respiration to various extents, restored circulation and prolonged the survival time, HL 7 being more effective than HI 6 after VX 90 micrograms/kg. Oximes alone were completely ineffective. Erythrocyte and diaphragm AChE was reactivated by HL 7 and, less effectively, by HI 6, while brain AChE remained almost completely inhibited in all groups. The results of this investigation demonstrate a reasonable efficacy of atropine after lower VX doses and of HL 7 and HI 6 (plus atropine) after high-dose VX poisoning, HL 7 being slightly more effective than HI 6.

Changes of acetylcholinesterase activity in the blood and different organs of the rat following intoxication with sarin, soman, VX and 2-dimethylamino-ethyl-(dimethylamido)phosphonofluoridate (GV) in doses of approximately 2 x LD50 (i.m.) were obtained from literature data and by experiment. The time course of acetylcholinesterase inhibition in the blood, regions of brain and diaphragm and the occurrence of signs and symptoms of poisoning (none, salivation, disturbed ventilation and fasciculations, convulsions or death) were summarized and compared. When blood enzyme activities were 70-100% normal, no signs were seen; at 60-70%, salivation occurred; at less than 30-55%, disturbed ventilation and fasciculations were seen while at 15-30%, convulsions occurred. Less than 10% was fatal. In experiments with narcotized dogs, the blood acetylcholinesterase activity and its reactivatability with trimedoxime were determined following intoxication (i.m.) with the above mentioned four compounds. It can be concluded that acetylcholinesterase activity in the blood corresponds to that in the target organs and can be considered as an appropriate parameter for biological monitoring of nerve gas exposure. Moreover, determination of reactivatability of blood acetylcholinesterase indicates more information than simple enzyme activity determination. determination

A strategy is described to raise high-affinity antibodies directed against the organophosphorus nerve agent VX [O-ethyl S-(2-diisopropylamino)ethyl)methyl phosponothionate]. Ten chemical derivatives of VX (haptens) have been synthesized. Their structures differ principally from VX structure by substitution of S-atom by an O-atom or CH2-group and by introduction of a reactive group (carboxylic acid, arylamine or primary amine) on the O-ethyl side chain. None of these haptens, except one, exhibit potential toxicity as tested by their inhability to inhibit acetylcholinesterase (E.C. 3.1.1.7.). After coupling with a protein carrier, they were injected intradermally to rabbits. Nine of these immunogenic conjugates led to the appearance of antibodies able to bind VX in a competitive solid phase immunoassay. The apparent titer and affinity of the antisera differed greatly depending on the hapten used. The highest affinity (9 nM) was observed with the VX derivative bearing O-S substitution and O-ethyl-carboxylic side chains. The antibodies appear specific for VX, since cross-reactivity with other nerve agents (Soman, Sarin or Tabun) was low. However, two haptens elicited antibodies with affinity to Soman or Sarin in the micromolar range. Antibodies were able to neutralize VX inhibition of acetylcholinesterase in vitro but not in vivo.

Iodination of fasciculin 3 (FAS3) from Dendroaspis viridis venom provided us with a fully active specific probe of fasciculin binding sites on rat brain acetylcholinesterase (AChE). Binding and inhibition are concomitant, as association and inhibition rate constants k1 and ki are identical. The 125I-FAS3.AChE complex dissociates very slowly (t 1/2 = 48 h) and is characterized by a dissociation constant, Kd, of 0.4 pM. All the specific binding of 125I-FAS3 to AChE is prevented by FAS3 as from D. angusticeps venom (Kd = 0.4, 14, and 25 pM, respectively). It is also prevented by propidium iodide, BW284C51, and d-tubocurarine, which bind to peripheral anionic sites of AChE, by Ca2+ and Mg2+, known to enhance AChE activity through an allosteric phenomenon and by acetylthiocholine concentrations which lead to excess substrate inhibition of the enzyme. Diisopropyl fluorphosphate and paroxon, which inhibit AChE by phosphorylating the catalytic serine, have no effect on either the binding rate or the number of binding sites of 125I-FAS3. O-Ethyl-S2-diisopropylaminoethyl methylphosphonothionate, however, which binds irreversibly to the AChE catalytic site but reversibly to a peripheral site, induces a 130% increase in the binding rate of 125I-FAS3, without changing the total number of 125I-FAS3 binding sites. Our results demonstrate that fasciculins bind on a peripheral site of AChE, distinct from the catalytic site and, at least partly, common with the sites on which some cationic inhibitors and the substrate in excess bind. Since phosphorylation of the catalytic serine (esteratic subsite) by [1,3-3H]diisopropyl fluorophosphate can still occur on the FAS3.AChE complex, the structural modification induced by fasciculins may affect the anionic subsite of AChE catalytic site.

This study was done to assess the effects of pyridostigmine (PYR) on a) the accumulation of labelled VX and soman within the brain, b) the therapeutic efficacy of atropine and oxime (2-PAM or HI-6) against intoxication by VX and soman and c) oxime-induced reactivation of inhibited acetylcholinesterase (AChE). In all experiments, rats were given PYR (131 micrograms/kg, im; I70 dose for whole blood AChE) or vehicle 30 min prior to nerve agent. In estimating 3H-agent the accumulation in the brain or estimating blood AChE activity, sufficient soman (47 micrograms/kg, iv) or VX (21.3 micrograms/kg, iv) was given to inhibit 50% of brain AChE activity. In assessing therapeutic efficacy and oxime-induced reactivation of blood AChE, rats were pretreated with PYR, challenged with agent and treated with atropine (16 mg/kg, im) and HI-6 or 2-PAM (100 umoles/kg, im) 30 sec post agent. Whole blood was collected by tail bleeding to monitor peripheral AChE activity at various time points before and after PYR and challenge. Pyridostigmine failed to alter covalent binding of labelled VX or soman in the brain. The 24-hr survival data showed that PYR reduced the therapeutic benefit of atropine and oxime against VX intoxication (but not soman). Protective ratios in VX-challenged rats given vehicle or PYR and treated with atropine + 2-PAM decreased slightly from 2.5 to 2.1 (p > .05), whereas with atropine + HI-6 they decreased significantly from 3.8 to 2.4. Also, AChE reactivation by HI-6 in VX-challenged rats was greater (p < .05) in vehicle- than in PYR-pretreated rats. HI-6 significantly reactivated AChE activity in both pretreatment groups (PYR or vehicle) given soman. The data suggest that PYR decreases the overall recovery of inhibited AChE in VX-challenged rats given HI-6; under the conditions used, this adverse effect decreases atropine+oxime efficacy against VX-induced lethality.

Male Sprague-Dawley rats when administered sc a sublethal dose of organophosphorus cholinesterase inhibitors such as the nerve agents, soman (100 micrograms/kg, sc), sarin (110 micrograms/kg, sc), tabun (200 micrograms/kg, sc), or VX (12 micrograms/kg, sc), developed seizures and severe muscle fasciculations within 15-20 min, lasting for 4-6 hr. Marked inhibition of acetylcholinesterase (AChE) and necrotic lesions in skeletal muscles such as soleus, extensor digitorum longus, and diaphragm were evident between 1-24 hr following injection. Pretreatment with memantine HCl (MEM, 18 mg/kg, sc) together with atropine sulfate (ATS, 16 mg/kg, sc), 60 min and 15 min, respectively, prior to nerve agents attenuated AChE inhibition, prevented myonecrosis, and muscle fasciculations as well as other signs of cholinergic toxicity. Pretreatment combining d-tubocurarine (d-TC, 0.075 mg/kg, sc) and ATS (16 mg/kg, sc) prevented the myonecrosis and fasciculation without protecting AChE against inhibition by these nerve agents. Neither MEM, d-TC, nor ATS in the concentration given interfered with the normal behavior of the rats. The role of d-TC and ATS interaction with presynaptic receptors regulating ACh release and MEM's role in modulating neural hyperactivity as protective mechanisms are discussed.

1. The effects of VX (10 microM) were examined on sympathetic ganglion neurons from the bullfrog using intracellular recording techniques. 2. VX significantly increased the amplitude of the residual EPSP from 4.8 +/- 0.86 mV (n = 4) to 13.7 +/- 1.23 mV (n = 4). 3. VX significantly decreased the membrane potential 5.2 +/- 0.75 mV (n = 6). The input resistance and the duration of the spike afterhyperpolarization (AHP) were also reduced 69.8% and 69.6% of control, respectively. 4. VX increased neuronal excitability greater than 200% (n = 5) of control. 5. The VX-induced neuronal excitability may result from a reduction in the duration of the AHP and contribute to the CNS toxicity.

This study concerned the effect of pyridostigmine pretreatment on (a) the antidotal efficacy of atropine and 2-PAM in sarin, tabun, and VX poisoning in mice and guinea pigs and on (b) the oxime-induced reactivation of VX-inhibited whole blood acetylcholinesterase (AChE) of guinea pigs. One hour prior to organophosphate (OP) challenge with sarin, tabun, or VX, animals were given oral doses of pyridostigmine to induce approximately 30 and 60% inhibition of whole blood AChE; controls received vehicle. Mice were challenged im and guinea pigs sc with the OP compounds. Treatment with atropine (11.2 mg/kg to mice; 32 mg/kg to guinea pigs) plus 2-PAM (25 mg/kg) was given im at 10 sec postchallenge in mice and 1 min postchallenge in guinea pigs. In the reactivation experiments, pyridostigmine or saline was given im to guinea pigs 30 min prior to VX (8.24 micrograms/kg, sc), atropine (16 mg/kg) was given im at 1 min, and 2-PAM (25 mg/kg) at 16 min postchallenge. Pyridostigmine significantly enhanced the efficacy of atropine and 2-PAM against tabun in both species. In contrast, pyridostigmine reduced or did not increase the efficacy of atropine and 2-PAM against sarin or VX in both species. Recovery of VX-inhibited AChE by 2-PAM was decreased significantly in pyridostigmine pretreated animals. The results suggest that pyridostigmine pretreatment may adversely effect the efficacy of atropine and 2-PAM as antidotes for VX and sarin intoxication.

Chemical pretreatment is effective against a 2 LD50 challenge of soman, sarin or VX or a 5 LD50 challenge of tabun. Chemical pretreatment followed by post challenge therapy should be effective against greater levels of agent. Such tests in guinea pigs are reported here; pretreatment regimens (PRGs) consisted of physostigmine (0.15 mg/kg, im) and an adjunct. The adjuncts [mg/kg, im] used were aprophen [8], atropine (AT)[16], azaprophen (AZA)[5], benactyzine [1.25], benztropine (BT) [4], scopolamine [0.08] and trihexyphenidyl [2]. Pretreatment was given 30 min before, and atropine (16 mg/kg, im) and 2-PAM (25 mg/kg, im) therapy (T) at one min after, 5 LD50s of agent. Results indicate that, all of the PRG+T regimens, except BT-not tested with T, prevent lethality by soman; trihexyphenidyl and scopolamine (the only adjuncts used therein) regimens each prevent lethality by sarin and VX. Against soman, all PRG+T regimens (vs PRG only) may shorten the median recovery time to 2 hrs or less. Even without therapy, the PRGs containing AT, AZA or BT prevent lethality by 5 LD50s of soman; however, used alone, only the PRG containing AZA reduces the incidence of convulsions at this level of soman.

Visual observations were made to compare the pretreatment benefits of subacute (75 micrograms/hr, sc) and acute (146 micrograms/kg, im, at 30 min) deliveries of physostigmine salicylate (Phy) against 2 or 5 LD50s (60 or 150 micrograms/kg, sc) of soman in guinea pigs; scopolamine, 80 micrograms/kg, im, was given routinely at 30 min. In a second set of studies, pretreatment with subacute carbamate [sc, Phy 36 micrograms/hr or pyridostigmine (Pyr), 50 micrograms/hr] and acute adjunct (im, scopolamine, 0.48 mg/kg, or trihexyphenidyl, 2 mg/kg) at 30 min, was used against soman (5 LD50s, sc) and VX (18.4 micrograms/kg, sc; 2 LD50s); atropine (16 mg/kg, im) and 2-PAM (25 mg/kg, im) were given at 1 min post soman. In all studies, lethality, % convulsing, convulsive/subconvulsive score, and recovery time were noted. Subacute dosing for 7 days was done via 14-day osmotic minipumps (OMPs). Results of the first set of studies indicate that subacute and acute deliveries of Phy give essentially comparable protection against 2 or 5 LD50s of soman. The second set of studies show that against soman, the adjuncts scopolamine and trihexyphenidyl when compared, and the carbamates, Phy and Pyr when compared, gave similar protective benefits as indicated by all four monitored measures of toxicity. Phy with either adjunct provided excellent protection against VX induced mortality and convulsions. With both carbamates, trihexyphenidyl gave similar protective benefits against VX. Scopolamine, however, under the conditions used herein, failed to act beneficially with Pyr against VX.

Literature survey dealing with cholinesterases and effects of highly toxic organophosphorus compounds suitable for use as chemical weapons is given in introductory part of this work. There are nerve paralytical agents (NPA)--sarin, soman, VX and a model compound O-ethyl-S-(2-dimethylaminoethyl)-methyl-phosphonothioate (EDMM). On the base of described scheme of intoxication with NPA, inhibition effect on cholinesterases, preferably on AChE as the most important factor involved in the mechanism of acute intoxication with NPA was studied. Intoxication of mice or rats with sarin and soman (2 x LD50) showed that time course of poisoning is faster than that for VX or EDMM. Inhibition of AChE in the blood was in good correlation with symptoms of intoxication and also with inhibition of AChE in the brain. The differences between inhibition effect of soman preferably uniform character of inhibition in the brain parts) and sarin (selective inhibition in the brain parts, with maximum in the frontal cortex and pontomedullar area) were observed. This selectivity was most marked for VX and EDMM intoxication (maximal inhibition in the part of the pontomedullar area containing reticular formation). The dose causing inhibition effect in the brain was assessed to be about 1% of the dose administered. The study of the effect of antidotal therapy (combination of atropine and reactivator) in vivo showed in mice and rats intoxicated with sarin non-uniform increase of AChE activity in the pontomedullar part depending on the dose and type of reactivator. The most marked effect was observed for methoxime. It was demonstrated that there exists good correlation between survival of experimental animals and the rest AChE activity in the pontomedullar part of the brain. AChE activity level critical for survival or death of the organism poisoned with NPA was assessed from these experiments; it was about 1-5% of normal values. By means of original method allowing continual monitoring of AChE activity in the blood, similar AChE reactivation was demonstrated, with highest effect for trimedoxime and methoxime. Using continual determination of the blood AChE activity following sarin, soman, VX and EDMM intoxication demonstrated that only a part of the dose administered caused inhibition effect in the blood; this part was determined to be practically 100% (i. v. administration); for other routes of administration this ratio was as follows: 50-80% (i. m.), 20-40% (i. p.), 6-16% (p. o.) and 1-5% (p. c.), respectively. Using this continual monitoring, the detoxication of sarin and soman was demonstrated. Detoxication of VX and EDMM was not observed.

The aim of this study was to compare the anticonvulsive and protective effects of diazepam and midazolam in rats poisoned by chemical warfare agents. In rats treated with soman, sarin or VX, the anticonvulsive effects of midazolam and diazepam were of similar magnitude. Atropine and oxime HI-6 decreased the toxicity of soman, sarin and VX 1.65, 2.06 and 18.3 times, respectively. The introduction of diazepam and midazolam in the therapy of rats poisoned by VX and sarin led to further improvement of protective indices. Midazolam was even more effective than diazepam. A reliable protective effect was obtained with the lowest dose of both benzodiazepines used (0.5 mg/kg). The specific benzodiazepine antagonist flumazenil abolished, almost completely, the protective effect of both benzodiazepines. These data confirmed a significant role of the gabaergic system in poisoning with organophosphorus compounds, especially during the initial stage of intoxication.

We have successfully demonstrated that exogenously administered acetyl- or butyrylcholinesterase (AChE, BChE respectively) will sequester organophosphates (OPs) before they reach their physiological targets. In addition, a third enzyme, endogenous carboxylesterase is known to be capable of scavenging OPs. In these studies, we have administered AChE and BChE to three different species of animals (mice, marmosets and monkeys) which were challenged with three different OPs (VX, MEPQ and soman). Results obtained from these systematic studies demonstrate that: (a) a quantitative linear correlation exists between blood AChE levels and the protection afforded by exogenously administered ChEs in animals challenged with OP, (b) approximately one mole of either AChE or BChE sequesters one mole of OP, (c) such prophylactic measures are sufficient to protect animals against OPs without the administration of any supportive drugs. Thus the OP dose, the blood-level of esterase, the ratio of the circulating enzyme to OP challenge, and the rate of reaction between them determine the overall efficacy of an enzyme as a pretreatment drug. The biochemical mechanism underlying the sequestration of various OPs by the use of exogenously administered scavenging esterases is the same in all species of animals studied. Therefore, the extrapolation of the results obtained by the use of ChE prophylaxis in animals to humans should be more reliable and effective than extrapolating the results from currently used multidrug antidotal modalities.

Male Sprague-Dawley rats injected with a sublethal sc dosage of 110 micrograms/kg of sarin (isopropyl methylphosphonofluoridate), or 12 micrograms/kg of VX (S-(2-diisopropylaminoethyl) O-ethyl methylphosphonothioate), developed severe toxic signs within 5-15 min after sarin and 20-50 min after VX lasting for 5 to 7 hr. Myonecrotic lesions were seen in soleus and diaphragm muscles within 1 hr. A maximum number of lesions had developed after 24 hr, and lesions were also present in extensor digitorum longus (EDL) at this time. Regeneration of muscle fibers was slow since lesions were still evident past 7 days of treatment. Within 1 hr following VX, AChE activity was reduced to 8, 12, and 17% of control activity in soleus, diaphragm, and EDL, respectively, whereas with sarin the enzyme activity was reduced to 23, 48, and 82% of control. A still greater inhibition was seen 24 hr after sarin when AChE activity was reduced to 19, 13, and 43% in these muscles. In skeletal muscles the different molecular forms of AChE, such as 16 S, 12 S, 10 S, and 4 S vary in location and functional importance with the 16 S form highly concentrated at the neuromuscular junction. All forms in a given muscle were equally sensitive to the inhibitors. In EDL, sarin was the least effective in reducing AChE or its molecular forms. In the brain structures (cortex, brain stem, striatum, and hippocampus), AChE activity was reduced to 1-6% of control by sarin and VX with the exception that following VX striatal AChE was reduced to only 41% of control activity. AChE activity in the brain cortex following either of the agents was maximally affected (1%). A slow but significant recovery of brain AChE was evident after 24 hr and more so after Day 7. Butyrylcholinesterase (BCHE) activity was less sensitive to inhibition by both inhibitors compared to AChE activity and showed a rapid recovery. Based on the equitoxic doses (toxic signs of similar magnitude), VX was found to be 10 times more toxic than sarin. The mechanisms of this disparity may be due to differences in rate of uptake, circulation, susceptibility to hydrolysis, and reactivity with nonspecific binding sites.

A pretreatment for organophosphorus (OP) anticholinesterase (e.g., soman) intoxication should prevent lethality and convulsions (CNV) at 2 LD50s and be behavioral-decrement-free when given alone. Behavioral-deficit-free pretreatment regimens (PRGs) for guinea pigs consisted of Physostigmine (0.15 mg/kg, im) and adjunct. Adjuncts [mg/kg, im] tested were akineton [0.25], aprophen [8], trihexyphenidyl [2], atropine [16], azaprophen [5], benactyzine [1.25], cogentin [4], dextromethorphan [7.5], ethopropazine [12], kemadrin [1], memantine [5], promethazine [5], scopolamine [0.08] and vontrol [2]. PRGs were given 30 min before soman (60 micrograms/kg, sc; 2 LD50s) or other OP agents. Animals were then observed and graded for signs of intoxication, including CNV at 7 time points and at 24 hr. Physostigmine alone reduced the incidence of CNV and lethality induced by 2 LD50s of soman by 42 and 60%, respectively. All of the PRGs tested abolished lethality and 12 shortened recovery time to 2 hr or less. Also, PRGs including azaprophen or atropine prevented CNV. When selected PRGs were tested against intoxication by sarin, tabun or VX, the efficacy was generally superior to that for soman. The data show that several PRGs are effective against soman intoxication in guinea pigs.

The effects of soman, sarin and VX were examined on ganglionic transmission through paravertebral chain ganglia of the bullfrog, Rana catesbeiana. Low frequency (0.1 Hz), short (2 sec) and long (10 sec) trains of preganglionic stimulation, after exposure to the agents, induced repetitive activity in the extracellularly recorded compound action potential. An irreversible transient depression was observed after exposure to the agents during the first second of short and long stimulus trains. Long stimulus trains of high frequency were required to produce a rundown in the amplitude of the compound action potential, whether recorded in the presence of each agent (10 microM) or following a wash with agent-free solution. The rundown of the compound action potential was use-dependent and not blocked or reversed by atropine (10 microM). Intracellular recordings, in the presence of either soman or VX, demonstrated (1) an increase in the amplitude of the residual excitatory postsynaptic potential or current evoked by synaptic stimulation, (2) an increase in the amplitude and duration of the acetylcholine-induced potential, (3) no increase in either the amplitude or duration of the carbachol-induced potential, (4) repetitive firing with orthodromic but not antidromic stimulation and (5) a concentration- and frequency-dependent depolarization of individual ganglion neurons with orthodromic stimulation which resulted in a decrease in the generation of action potentials. These results suggest that the agent-induced decrease in the compound action potential occurred as a consequence of activity-dependent depolarization of ganglion neurons, which occurs after inhibition of cholinesterase.

To determine the mechanism governing pulmonary edema induced by an organophosphorus compound, S-(2-diisopropylaminoethyl)-O-ethylmethyl phosphonothiolate (VX), lung lymph flow and lymph-to-plasma protein concentration ratio were measured in six anesthetized, open-chest, mechanically ventilated beagle dogs before and after intravenous injection of 6 micrograms/kg of VX. Systemic and pulmonary hemodynamic data (heart rate, aortic blood flow, and left atrial, systemic arterial, pulmonary arterial, and pulmonary capillary pressures) were continuously recorded. Arterial blood gases and pH were measured every 30 min. Histological examinations and lung water content measurements were also carried out. Following VX injection, lung lymph flow increased (from 109 +/- 38 to 179 +/- 66 microliters/min, p less than 0.05) while lymph-to-plasma protein concentration ratio remained unchanged (from 0.64 +/- 0.14 to 0.62 +/- 0.12, N.S.). Neither systemic nor pulmonary hemodynamics were changed. Lung water content expressed as blood-free wet-to-dry weight ratio increased from 4.31 +/- 0.23 to 5.35 +/- 0.26 (p less than 0.05). Histological examinations revealed in many cases diffuse congestion of lungs and interstitial edema. These results suggest that VX injection induces an increase in pulmonary capillary permeability which may lead to a high-permeability edema.

Serine-specific reagents, anticholinesterase organophosphorus compounds like Vx provoke, in the micromolar range, digitalis-like ventricular arrhythmias of non-cholinergic origin in rodent hearts. The sensitivities of the two rat cardiac Na+,K(+)-ATPase isoforms (alpha 1 and alpha 2) to Vx (0.1-100 microM) were measured in sarcolemma vesicles. At 1 microM Vx, the inhibition of the total activity averaged 18% but never exceeded 75% with 100 microM. When the alpha 2 isoform activity was inhibited by 0.1 microM ouabain, alpha 1 was 35% inhibited by 1 microM Vx, i.e. a 16 +/- 4% inhibition of the total activity. The cardiac alpha 1 being related to the digitalis-induced toxicity, its selective inhibition by a micromolar dose of Vx fully accounts for the cardiotoxicity of Vx. Inasmuch as Vx had no effect on the rat kidney alpha 1, differentially inactivated the cardiac isozymes and specifically reacted with serine residues, the putative binding-site(s) of the organophosphorus compound on the Na+-K(+)-ATPase molecules has been considered.

In our preliminary study, it has been found that VX oxidase exists in the microsome fraction of rat liver and the catalytic reaction needs the participation of molecular oxygen and coenzyme I or II. In this paper, the data showed that deoxycholate inactivated both the mixed function oxidase and VX oxidase. The specific inhibitor proadifen of the mixed function oxidase also profoundly inhibited VX oxidase activity. The complex of VX and cytochrome P-450 exhibited typical difference spectrum of type I. Aniline competitively inhibited the inactivation of VX catalyzed by microsomes. These results indicate that VX is one of the substrates of mixed function oxidase. VX oxidase in the rat liver cells is exactly the mixed function oxidase.

Effect of oxime HI-6 to rabbit miosis induced by the topical sarin and VX administration is presented. It has been found that effects of both toxins are better antagonized by parenteral than by topical HI-6 administration. The sarin antagonizing effect was found more effective. It has been also confirmed that there was no significant difference concerning the oxime HI-6 effect when topically administered either as 2.5% hypertonic or isotonic solution. Opposite to PAM-2, HI-6 effect was better in parenteral and topical administration of isotonic solutions, and less effective in local administration of hypertonic solutions.

Rabbits immunized with an artificial VX-antigen could survive 1 x LD95 of VX challenge on the 7th day and the 31st day after the last immunization. One hundred microliters of rabbit anti-VX antiserum given intravenously immediately before 1 x LD95 of VX or 200 microliters of antiserum intraperitoneally 1-10 days before 1 x LD95 VX protected all the tested mice from death. The antiserum could prevent the in vitro inhibition of Torpedo AChE activity by VX and reduced its effect on brain AChE activity in vivo. No protective effect of the antiserum was observed on the Torpedo AChE activity inhibited by sarin and soman.

The M2 subtype of muscarinic receptor is predominant in heart, and such receptors were reported to be located in muscles as well as in presynaptic cholinergic and adrenergic nerve terminals. Muscarinic receptors of rat heart were identified by the high affinity binding of the agonist (+)-[3H]cis-methyldioxolane ([3H]CD), which has been used to label a high affinity population of M2 receptors. A single population of sites (KD 2.74 nM; Bmax of 82 fmol/mg protein) was detected and [3H]CD binding was sensitive to the M2 antagonist himbacine but much less so to pirenzepine, the M1 antagonist. These cardiac receptors had different sensitivities to NiCl2 and N-ethylmaleimide from brain muscarinic receptors, that were also labeled with [3H]CD and considered to be of the M2 subtype. Up to 70% of the [3H]CD-labeled cardiac receptors had high affinities for several organophosphate (OP) anticholinesterases. [3H]CD binding was inhibited by the nerve agents soman, VX, sarin, and tabun, with K0.5 values of 0.8, 2, 20, and 50 nM, respectively. It was also inhibited by echothiophate and paraoxon with K0.5 values of 100 and 300 nM, respectively. The apparent competitive nature of inhibition of [3H]CD binding by both sarin and paraoxon suggests that the OPs bind to the acetylcholine binding site of the muscarinic receptor. Other OP insecticides had lower potencies, inhibiting less than 50% of 5 nM [3H]CD binding by 1 microM of EPN, coumaphos, dioxathion, dichlorvos, or chlorpyriphos. There was poor correlation between the potencies of the OPs in reversibly inhibiting [3H]CD binding, and their anticholinesterase activities and toxicities. Acetylcholinesterases are the primary targets for these OP compounds because of the irreversible nature of their inhibition, which results in building of acetylcholine concentrations that activate muscarinic and nicotinic receptors and desensitize them, thereby inhibiting respiration. Nevertheless, the high affinities that cardiac muscarinic receptors have for these toxicants point to their extra vulnerability. It is suggested that the success of iv administration of the muscarinic receptor inhibitor atropine in initial therapy of poisoning by OP anticholinesterases may be related in part to the extra sensitivity of M2 receptors to certain OPs.

The determination of the nerve agent O-ethyl S-2-diisopropylaminoethyl methylphosphonothioate (VX) by thermospray liquid chromatography-mass spectrometry was studied. The solvent system acetonitrile-methanol-0.25 M ammonium acetate was used on a reversed-phase C18 column. By selected ion monitoring at the protonated molecular ion of VX (m/z 268), the predominant peak in its thermospray mass spectrum, an amount of 200 pg could be detected. For the determination of VX in water at levels below 1 ng/ml, preconcentration by C18 cartridges was investigated. The applicability of the method was demonstrated by the determination of VX in spiked river waters. A concentration of 0.1 ng/ml could be detected starting from a water sample of 50 ml. A second application concerned the analysis of water extracts of spiked soil samples.

This paper describes an indirect method for the quantification of the toxic military agent O-ethyl S-(2-diisopropylaminoethyl) methylphosphonothioate (VX) in the vapor state in air or other similar gases at ng/m3 levels. The method begins with the passage of a gaseous sample through a filter impregnated with silver fluoride to convert the VX vapor to ethyl methylphosphonofluoridate. The latter compound is then trapped on a bed of Chromosorb 106, transferred to a smaller bed of the same sorbent, and desorbed thermally into a gas chromatograph equipped with a flame-photometric detector. The method is comparable in sensitivity to the principal alternative method, which is based on cholinesterase inhibition, and it is less subject to interference from common organic solvents and other cholinesterase inhibitors. The detection limit was found to be limited by, and therefore dependent on, the nature and extent of any background substances that produced a significant chromatographic signal or response at the retention time of the analyte. In the absence of such substances, the instrument provided a response to 0.19 ng of VX that was thirty times larger than the peak-to-peak noise amplitude on the chromatographic base line. Moreover, the method bias (i.e., 100% minus the percent VX recovery) was found to depend on VX concentration, with estimates of agent recovery ranging from 83% at a VX concentration of 0.67 ng/m3 to 104% at a concentration of 0.084 ng/m3. The relative standard deviation varied with VX concentration and with the nature of the test that was performed to estimate it. It ranged from 2.1% in one VX vapor-challenge test to 17% in an experiment involving spiked sampling tubes, and it was generally lower at the higher VX test concentrations.

Using a microtiter plate spectrophotometric system, an assay procedure was developed for the following toxic organophosphorus compounds: 1,2,2-trimethylpropyl ester of methylphosphonofluoridic acid (1, soman); ethyl N,N-dimethylphosphoramidocyanidate (3, tabun); O-ethyl S-[2-[bis(1-methylethyl)amino]ethyl]- methylphosphonothiolate (4, VX); the diethyl 4-nitrophenyl ester of phosphoric acid (5, paraoxon); and bis(1-methylethyl) phosphorofluoridate (6, DFP). The procedure, based on the Ellman assay method, uses inhibition of eel acetylcholinesterase (0.01 unit per well) to carry out the determination of inhibitor concentrations for both a standard curve and the unknown samples on a single 96-well microtiter plate. On a typical plate, samples of both unknowns and standards (a minimum of six concentrations were used per standard curve) were assayed five times per sample, with three control (uninhibited) enzyme activity points included for each sample. The time required for carrying out a single plate was approx 30 min. Sensitivity for the most potent acetylcholinesterase inhibitor tested was 0.4 nM under the conditions used for a typical assay. It should be noted, however, that no attempt was made to optimize the assay procedure for sensitivity.

Four nerve agents and one therapeutic organophosphate (OP) anticholinesterase (anti-ChE) bind to acetylcholine (ACh) receptors, inhibit or modulate binding of radioactive ligands to these receptors, and modify events regulated by them. The affinity of nicotinic (n) ACh receptors of Torpedo electric organs and most muscarinic (m) ACh receptors of rat brain and N1E-115 neuroblastoma cultures for the OP compounds was usually two to three orders of magnitude lower than concentrations required to inhibit 50% (IC-50) of ACh-esterase activity. However, a small population of m-ACh receptors had an affinity as high as that of ACh-esterase for the OP compound. This population is identified by its high-affinity [3H]-cis-methyldioxolane ([3H]-CD) binding. Although sarin, soman, and tabun had no effect, (O-ethyl S[2-(diisopropylamino)ethyl)] methyl phosphonothionate (VX) and echothiophate inhibited competitively the binding of [3H]-quinuclidinyl benzilate ([3H]-QNB) and [3H]-pirenzepine ([3H]-PZ) to m-ACh receptors. However, VX was more potent than echothiophate in inhibiting this binding and 50-fold more potent in inhibiting carbamylcholine (carb)-stimulated [3H]-cGMP synthesis in N1E-115 neuroblastoma cells--both acting as m receptor antagonist. All five OPs inhibited [3H]-CD binding, with IC-50s of 3, 10, 40, 100, and 800 nM for VX, soman, sarin, echothiophate, and tabun, respectively. The OP anticholinesterases also bound to allosteric sites on the n-ACh receptor (identified by inhibition of [3H]-phencyclidine binding), but some bound as well to the receptor's recognition site (identified by inhibition of [125I]-alpha-bungarotoxin binding). Soman and echothiophate in micromolar concentrations acted as partial agonists of the n-ACh receptor and induced receptor desensitization. On the other hand, VX acted as an open channel blocker of the activated receptor and also enhanced receptor desensitization. It is suggested that the toxicity of OP anticholinesterases may include their action on n-ACh as well as m-ACh receptors if their concentrations in circulation rise above micromolar levels. At nanomolar concentrations their toxicity is due mainly to their inhibition of ACh-esterase. However, at these low concentrations, many OP anticholinesterases (eg, VX and soman) may affect a small population of m-ACh receptors, which have a high affinity for CD. Such effects on m-ACh receptors may play an important role in the toxicity of certain OP compounds.

The neurotoxicities of single doses of a chemical warfare agent VX [phosphonothioic acid, methyl-S-(2-[bis(1-methylethyl)amino/ethyl) O-ethyl ester], a metabolite of the agricultural chemical parathion, paraoxon, PO (phosphonothioic acid, diethyl paranitrophenyl ester), and the known neuropathic agents DFP] phosphorofluoridic acid, bis(1-methylethyl) ester] and TOCP (phosphoric acid, tri-o-tolyl ester) were compared in the chicken. Single injections (subcutaneous, sc) of VX as high as 150 micrograms/kg (5 times the LD50, intramuscular, im) were tolerated by laying tens if atropine and 2-pralidoxime were used as antidotes before and immediately after injection. The 150 of VX for inhibition of chicken brain acetylcholinesterase was approximately 5 X 10(-10). Plasma acetylcholinesterase, but not butyrylcholinesterase, was depressed 2 h after injections of 2-20 micrograms VX/kg im without antidotes. Levels of plasma enzymes such as creatine kinase, indicative of tissue damage, were increased after exposure to both VX and PO. Injections of up to 150 micrograms/kg of VX with antidotes did not cause locomotor or histological signs of organophosphorus-induced delayed neuropathy, but single injections of 400 mg TOCP/kg did.

We have investigated the effect of electromechanical activity on the molecular forms of acetylcholinesterase (AChE) in cultured embryonic rat myotubes. Both globular and asymmetric forms of AChE are present on the 5th day of culture when myotubes are just beginning to fibrillate. Between days 5 and 8, the 4 S (G1), 10 S (G4), and 16 S (A12) forms increase dramatically, and appreciable 12.5 S (A8) AChE appears. When fibrillation is prevented by adding tetrodotoxin on day 4, the increases in the A12 and A8 forms are prevented, and the increases in the G4 and G1 forms are significantly impaired. At 8 days, fibrillating myotubes have 19 times more A12 AChE and over 4 times more G1 and G4 enzyme than do nonfibrillating myotubes. The effect of tetrodotoxin is reversible. When tetrodotoxin is removed at 7 days, fibrillation resumes promptly, and globular and asymmetric forms recover. Light microscopic examination of fibrillating and nonfibrillating myotubes showed that tetrodotoxin does not affect the gross morphological development of the myotubes. Titration of AChE-active sites with O-ethyl-S2-diisopropyl methyl-phosphonothionate demonstrated that the increase in AChE activity associated with fibrillation is due to an increase in the number of AChE molecules present and not to an increase in the rate at which individual AChE molecules turn over acetylcholine. To evaluate AChE metabolism in fibrillating and nonfibrillating myotubes, we examined the enzyme after inactivating it with paraoxon. Paraoxon readily penetrates cells and diethylphosphorylates a serine in the active site of AChE, thereby inactivating it. The diethylphosphorylated enzyme is stable, but it can be reactivated rapidly and quantitatively with pyridine-2-aldoxime methiodide (2-PAM). After inactivating AChE with paraoxon, we simultaneously evaluated synthesis (by following the newly synthesized active AChE) and turnover (by following the 2-PAM-reactivatable AChE). Our results show that globular and asymmetric forms of AChE are both synthesized more rapidly in fibrillating than in nonfibrillating myotubes.

Atropinized rats intoxicated with ethyl-S-2-diisopropyl aminoethyl methyl phosphonothioate (VX), 15 mg/kg iv, were divided into three groups and were treated with normal saline, iv, 30 mg/kg of 2-PAM C1, iv, and 30 mg/kg of HS-6, iv. One hr after administration of therapy they were decapitated and cholinesterase (ChE) activity was determined on blood, brain and diaphragm tissue. Both 2-PAM C1 and HS-6 markedly reactivated VX-inhibited blood and diaphragm ChE. Brain ChE activity was not significantly reactivated by either oxime. The effectiveness of these oximes in restoration of VX-inactivated ChE in vivo offers an explanation as to why conventional atropine/oxime therapy is so effective against VX intoxication.

Sheep were studied for the possibility of treatment after parenteral (intramuscular) intoxication with EDMM (methylthiophosphorous acid O-ethyl-S-2-dimethylamino-ethylester) and with EDIM (methylthiophosphorous acid O-ethyl-S-2-diisopropyl-aminoethylester). In both cases of intoxication, the therapy was based on a system of an anticholinergic and cholinesterase reactivator administered singly at a time of the maximum development of the clinical signs of poisoning and maximum inhibition of both erythrocytic (AChE, E.C.3.1.1.7.) and plasma (BChE, E.C.3.1.1.8.) cholinesterase. The optimum therapeutic system requires the administration of 20.0 mg atropine s. c. pro toto and 10.0 mg trimedoxim per kg 1. w. i. v. In both cases of poisoning with doses = LD50 in i. m. administration, the mentioned system was actually positive. In a single administration irrespective of the doses of the used drugs, the system does not guarantee survival after ingestion of anticholinesterasic doses above LD50.

The active sites of acetylcholinesterase multiple forms from four widely different zoological species (Electrophorus, Torpedo, rat and chicken) were titrated using a stable, irreversible phosphorylating inhibitor (O-ethyl-S2-diisopropylaminoethyl methyl-phosphonothionate). In all cases, we found that within a given species, the molecular forms we examined were equivalent in their catalytic activity per active site. As pure preparations of the molecular forms of Electrophorus acetylcholinesterase were available, we were able to establish that one inhibitor molecule binds per monomer unit for each of them. This had already been shown by several authors for the tetrameric globular form, but not for the tailed molecules. Analysis of the phosphorylation reaction showed that they are equally reactive. Under our experimental conditions, their turnover number per site was 4.4 x 10(7) mol of acetylthiocholine hydrolysed . h-1 at 28 degrees C, pH 7.0. The corresponding value was less than half for Torpedo (1.64 x 10(7) mol . h-1), and again lower for rat (1.32 x 10(7) mol . h-1) and chicken (1.05 x 10(7) mol . h-1). In the case of rat acetylcholinesterase, the activity per active site of solubilized (with or without Triton X-100) and membrane-bound enzyme were identical. We discuss the implications of these findings with respect to the quaternary structure of acetylcholinesterase, and to the physico-chemical state and physiological properties of its molecular forms.