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A comparison of one mono- and seven bisquaternary acetylcholinesterase (AChE) reactivators of acetylcholinesterase inhibited by VX agent was performed. As a source of the acetylcholinesterase, a rat brain homogenate was taken. There were significant differences in reactivation potency of all tested oximes. The oxime TO205 seems to be the most efficacious followed by TO046, HI-6, HS-6, K027, obidoxime, MMC and 2-PAM. In addition, the results of this study showed that the reactivation potency of the tested reactivators depends on many factors-such as the number of pyridinium rings, the number of oxime groups and their position, as well as the length and the shape of linkage bridge between two pyridinium rings.

Summary Depleted uranium is a byproduct of the uranium enrichment process and has been used for decades in different applications. Recently depleted uranium is used as an anti-armour munition because of its profitable physical properties. One of these, the ability to ignite and burn at very high temperatures, may result in the formation of an aerosol of very small uranium oxide clusters, which may be inhaled. It is alleged that these particles represent a new battlefield hazard because of the radioactivity and chemical toxicity of uranium.

To study the influence of single or repeated low-level sarin inhalation exposure on immune functions, inbred BALB/c mice were exposed to low clinically asymptomatic concentrations of sarin for 60 min. in the inhalation chamber. The evaluation of immune functions was carried out using phenotyping of CD3 (T-lymphocytes), CD4 (helper T-lymphocytes), CD8 (cytotoxic T-lymphocytes) and CD19 (B-lymphocytes) in the lungs, blood and spleen, lymphoproliferation of spleen cells stimulated in vitro by various mitogens (concanavalin A, lipopolysaccharides), phagocyte activity of peritoneal and alveolar macrophages, production of N-oxides by peritoneal macrophages and the measurement of the natural killer cell activity at one week after sarin exposure. The results were compared to the values obtained from control mice exposed to pure air instead of sarin. The results indicate that an asymptomatic dose of sarin is able to alter the reaction of the immune system at one week after exposure to sarin. While the number of CD3 cells in lung was significantly decreased, a slight increase in CD19 cells was observed especially in the lungs after a single sarin inhalation exposure. Lymphoproliferation was significantly decreased regardless of the mitogen and sarin concentration used and the number of low-level sarin exposures. The ability of peritoneal and alveolar macrophages to phagocyte the microbes was also decreased regardless of the number of low-level sarin exposures. The production of N-oxides by peritoneal macrophages was decreased following a single low-level sarin exposure but increased following repeated low-level sarin inhalation exposure. Nevertheless, the changes in the production of N-oxides that reflects a bactericidal activity of peritoneal macrophages was not significant. The natural killer cell activity was significantly higher in the case of inhalation exposure of mice to low concentration of sarin regardless of the number of exposures. Thus, not only organophosphorous insecticides but also nerve agents such as sarin are able to alter immune functions following a single inhalation exposure even at a dose that does not cause clinically manifested intoxication. Generally, the repeated exposure to low concentrations of sarin does not increase the alteration of immune functions compared to the single low-level sarin exposure with the exception of phagocyte activity of alveolar macrophages and natural killer cell activity.

Long-term effects of low doses of highly toxic organophosphorus agent sarin on various hematological and biochemical markers and physiological functions were studied in rats exposed to sarin by inhalation. The results indicate that low-level sarin-exposed rats show long-term increase in studied markers of stress and decrease in synthesis of DNA de novo without the disturbance of the functions of cholinergic nervous system. Moreover, sarin at low doses is able to induce some neurotoxic effects including an increase in the excitability of central nervous system in rats at 3 mo following inhalation exposure. Relatively long-term spatial discrimination impairments in rats exposed to low-level sarin was demonstrated too. Therefore, nerve agents such as sarin seem to be harmful not only at high, clinically symptomatic doses but also at low doses without acute clinical manifestation of overstimulation of cholinergic nervous system because of long-term manifestation of alteration of neurophysiological and neurobehavioral functions in sarin-exposed rats.

The efficacy of a new bispyridinium oxime 1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium)butane dibromide, called K048, and currently used oximes (pralidoxime, obidoxime, the oxime HI-6) to reactivate acetylcholinesterase inhibited by various nerve agents (sarin, tabun, cyclosarin, VX) was tested by in vitro methods. The new oxime K048 was found to be a more efficacious reactivator of nerve agent-inhibited acetylcholinesterase than pralidoxime (in the case of VX, tabun and cyclosarin), obidoxime (cyclosarin and tabun) and HI-6 (tabun) but it did not reach the efficacy of currently used oximes for the reactivation of acetylcholinesterase inhibited by sarin. Thus, the oxime K048 seems to be a relatively efficacious broad spectrum acetylcholinesterase reactivator and, therefore, it could be useful for the treatment of a nerve agent-exposed population if information about detection of the type of nerve agent is not available.

The neuroprotective effects of antidotes (atropine, pralidoxime/atropine, obidoxime/atropine and HI-6/atropine mixtures) on rats poisoned with tabun at a lethal dose (220 μg/kg intramuscularly; 100% of LD50 value) were studied. The tabun-induced neurotoxicity was monitored using a functional observational battery and an automatic measurement of motor activity. The neurotoxicity of tabun was monitored at 24 hr and 7 days after tabun challenge. The results indicate that atropine alone is not able to protect the rats from the lethal effects of tabun. Three non-treated tabun-poisoned rats and one tabun-poisoned rat treated with atropine alone died within 24 hr. On the other hand, atropine combined with all tested oximes allows all tabun-poisoned rats to survive at least 7 days following tabun challenge. Obidoxime combined with atropine seems to be the most effective antidotal treatment for the elimination of tabun-induced neurotoxicity in the case of lethal poisoning among tested antidotal mixtures. The antidotal mixture consisting of atropine and HI-6 is significantly less effective than the combination of atropine with obidoxime in the elimination of tabun-induced neurotoxicity in rats at 24 hr following tabun challenge. Pralidoxime in combination with atropine appears to be practically ineffective to decrease tabun-induced neurotoxicity at 24 hours as well as 7 days following tabun poisoning. Due to its neuroprotective effects, obidoxime seems to be the most effective and most suitable oxime for the antidotal treatment of acute tabun exposure among currently used oximes. Thus, the replacement of obidoxime by a more effective acetylcholinesterase reactivator for soman poisoning, the oxime HI-6, can to a small extent diminish the neuroprotective efficacy of antidotal treatment in the case of acute tabun poisonings.

1. To study the influence of pharmacological pretreatment (PANPAL or pyridostigmine combined with biperiden) and antidotal treatment (the oxime HI-6 plus atropine) on soman-induced neurotoxicity, male albino rats were poisoned with a lethal dose of soman (54 (g/kg i.m.; 100% of LD50 value) and observed at 24 hours and 7 days following soman challenge. The neurotoxicity of soman was evaluated using a Functional observational battery and an automatic measurement of motor activity. 2. Pharmacological pretreatment as well as antidotal treatment were able to eliminate some of soman-induced neurotoxic effects observed at 24 hours following soman poisoning. The combination of pharmacological pretreatment (PANPAL or pyridostigmine combined with biperiden) and antidotal treatment was found to be more effective in the elimination of soman-induced neurotoxicity in rats at 24 hours following soman challenge in comparison with the administration of pharmacological pretreatment or antidotal treatment alone. To compare both pharmacological pretreatments, the combination of pyridostigmine with biperiden seems to be more efficacious to eliminate soman-induced signs of neurotoxicity than PANPAL. 3. At 7 days following soman poisoning, the combination of pharmacological pretreatment involving pyridostigmine and biperiden with antidotal treatment was only able to completely eliminate somaninduced neurotoxic signs. 4. Thus, our findings confirm that the combination of pharmacological pretreatment and antidotal treatment is able not only to protect the experimental animals from the lethal effects of soman but also to eliminate most soman-induced signs of neurotoxicity in poisoned rats. The pharmacological pretreatment containing pyridostigmine and biperiden appears to be more efficacious to eliminate soman-induced neurotoxic sings than PANPAL.

To study the influence of low-level sarin exposure on cognitive functions, the rats were exposed to three various low concentrations of sarin (Levels 1-3) for 60 min in the inhalation chamber. In addition, one group of rats was exposed to Level 2 of sarin repeatedly. Testing of cognitive functions was carried out using the Y-maze evaluating learning and spatial memory. The correct averse behavior of sarin-exposed rats in the Y-maze was tested several times within 6 weeks following sarin inhalation exposure to look for any cognitive impairments. The results were compared to the Y-maze performance of control rats exposed to pure air instead of sarin. While a subtle and short-term deficiency in the Y-maze performance was observed in rats exposed to the Levels 1 and 2 of sarin, the exposure to the Level 3 of sarin caused a significant decrease in the Y-maze performance for a relatively long time. Similar sarin-induced spatial memory impairments were demonstrated in rats exposed repeatedly to the Level 2. A decrease in the Y-maze performance was observed until the end of the third week following the last exposure to sarin. Thus, our findings confirm that both nonconvulsive symptomatic and clinically asymptomatic concentrations of sarin can cause relatively long-term memory impairments in sarin-poisoned rats when the rats are exposed to clinically asymptomatic sarin concentration repeatedly.

To study the influence of low-level sarin exposure on cognitive functions, male albino Wistar rats were exposed to three various low concentrations of sarin (LEVEL 1–3) for 60 minutes in the inhalation chamber. Testing of cognitive functions was carried out using the T-maze evaluating learning and spatial memory. The behavior of sarin-exposed rats in the T-maze was tested several times within five weeks following sarin inhalation exposure to look for any cognitive impairments. The alteration of cognition was evaluated by using a method studying memory elicitation in response to appetitive motivation in a multiple T-maze. 2. Statistically significant, short-term deficiency in the T-maze performance was observed in rats exposed to symptomatic (LEVEL 3) as well as clinically asymptomatic concentration (LEVEL 2) of sarin. The repeated exposure of rats to clinically asymptomatic dose of sarin (LEVEL 2R) did not change the effect of lowlevel sarin exposure on spatial memory compared to the single exposure to the same dose of sarin. 3. Thus, sarin is able to influence the cognitive functions (e.g. spatial memory) even at low doses that do not cause clinically manifested intoxication following the inhalation exposure. Nevetheless, the alteration of spatial memory lasts for a short time only, in contrast with the severe sarin poisoning.

1. Long term effects of low doses of highly toxic organophosphorus agent sarin on behavioral and neurophysiological functions were studied in rats exposed to sarin by inhalation. The toxic effects of sarin were monitored using a functional observational battery (FOB), an automatic measurement of motor activity and a test of excitability of central nervous system at 3, 6 and 12 months following sarin exposure. 2. The results indicate that sarin at symptomatic as well as asymptomatic doses (level 2 and 3) is able to induce some neurotoxic effects (a decrease in activity and mobility, an alteration of gait, an increase in stereotyped behavior) including an increase in the excitability of central nervous system (an increase in convulsive activity following the administration of pentamethylenetetrazole) in rats at 3 months following inhalation exposure. Some sings of increased excitability were also observed in sarin-exposed rats following 6 or 12 months (an increase in exploratory activity, body temperature and a hindlimb grip strength at 6 months following exposure to sarin at asymptomatic doses, an increase in tail-pinch response at 12 months following exposure to sarin at symptomatic doses). 3. Therefore, nerve agents such as sarin seem to be harmful not only at high, clinically symptomatic doses but also at low, clinically asymptomatic doses because of long term manifestation of alteration of neurophysiological functions in sarin-exposed rats without disruption of cholinergic nervous system.

A rapid and sensitive method for simultaneous determination of 3-hydroxytyramine (dopamine), 5-hydroxytryptamine (serotonin) and their metabolites - 3,4-dihydroxyphenylacetic acid, 3-methoxytyramine, 4-hydroxy-3-methoxyphenylacetic acid (homovanillic acid) and 5-hydroxyindole-3-acetic acid in the rat brain was developed. Brain samples with the internal standard and heparin were deproteinized by perchloric acid with ethylenediaminetetraacetic acid disodium salt and sodium sulfite. Following homogenization, centrifugation and filtration, the supernatant was directly injected into a reversed-phase HPLC system with coulometric detector. The response of the detected substances was linear in the range 12-700 ng/g of cerebellum homogenate (24-1 400 pg on column). Total recovery of the method was higher than 95%. The method was used for the determination of catecholamines and their metabolites in the chosen part of rat brain following the inhalation exposure to sarin (organophosphate).

1. The neuroprotective effects of antidotes (atropine, obidoxime/atropine mixture, HI-6/atropine mixture) on rats poisoned with soman at a sublethal dose (48 μg/kg i.m.; 60% of LD50value) were studied. The neurotoxicity was monitored using a functional observational battery (FOB) and an automatic measurement of motor activity. The neurotoxicity of soman was monitored at 24h and 7d following soman poisoning. 2. The results indicate that atropine alone and the oxime HI-6 in combination with atropine seem to be effective antidotal treatment for the elimination of soman-induced neurotoxicity in the case of sublethal poisonings. 3. On the other hand, the combination of obidoxime with atropine appears to be practically ineffective in diminishing neurotoxic soman-induced symptoms. 4. Dealing with neuroprotective effects of antidotes, the oxime HI-6 in combination with atropine seems to be more suitable antidotal mixture than obidoxime in combination with atropine even in the case of sublethal poisoning with nerve agents.

Inhibition of plasma cholinesterase by three methylfluorophosphonates (MFF), sarin, soman and cyclosin, and by the products of their hydrolysis and alcoholysis was examined. Inhibition by phosphonic acids and by methyl esters derived from MFF was purely competitive while that by MFF was irreversible. The rate of phosphorylation of cholinesterase by MFF differs, depending on the structure of the alkoxy group in the MFF and decreases in the sequence soman-sarin-cyclosin. The affinity values of MFF, phosphonic acids and methyl esters of phosphonic acid for cholinesterase are comparable. The in vitro kinetic parameters suggest that plasma cholinesterase might act as a natural detoxicating agent in cases of poisoning with the above inhibitors of acetylcholinesterase.

Nerve agents belong to highly toxic organophosphorus compounds misused as chemical warfare agents for military as well as terroristic purposes. Basic mechanism of action of nerve agents is based on acetylcholinesterase (AChE, EC 3.1.1.7) inhibition and subsequent accumulation of neuromediator acetylcholine at the cholinergic synapses, either peripheral or central leading to cholinergic hyperstimulation and development of symptoms of poisoning, followed by metabolic dysbalance and death without effective prophylaxis/treatment. The antidotal treatment of acute poisonings with nerve agents still represents a serious problem and, therefore, we are searching how to satisfactorily protect people against acute toxicity of nerve agents. There are two approaches how to improve the medical protection against nerve agents: to increase the resistance of nerve agent-exposed organism and the efficacy of post-exposure antidotal treatment by pharmacological prophylaxis the development of new, more effective antidotes, expecially AChE reactivators, to achieve the satisfactorily effective antidotal treatment of acute poisonings with nerve agents.

Tabun (O-ethyl-N,N-dimethyl phosphoramidocyanidate) belongs to highly toxic organophosphorus compounds misused as chemical warfare agents for military as well as terroristic purposes. It differs from other highly toxic organophosphates by its chemical structure and by the fact that tabun-inhibited acetylcholinesterase is extraordinarily difficult to reactivate. The potency of trimedoxime and other commonly used oximes (pralidoxime, obidoxime, the oxime HI-6) to reactivate tabun-inhibited acetylcholinesterase and to eliminate tabun-induced acute effects was evaluated using in vitro and in vivo methods. In vitro calculated kinetic parameters of reactivation of tabun-inhibited acetylcholinesterase from rat brain homogenate and in vivo determined percentage of reactivation of tabun-inhibited blood and tissue acetylcholinesterase in poisoned rats show that trimedoxime seems to be the most efficacious reactivator in the case of tabun poisonings. Trimedoxime was also found to be the most efficacious oxime in the elimination of acute lethal toxic effects in tabun-poisoned rats and mice. The oxime HI-6, so efficacious against soman, does not seem to be sufficiently effective oxime to reactivate tabun-inhibited acetylcholinesterase and to counteract acute lethal effects of tabun.

The neuroprotective effects of newly developed oximes (K027, K048) and currently available oximes (obidoxime, HI-6) in combination with atropine in rats poisoned with tabun at a sublethal dose (170 microg kg(-1) i.m.; 80% of LD(50) value) were studied. The tabun-induced neurotoxicity was monitored using a functional observational battery and an automatic measurement of motor activity. The neurotoxicity of tabun was monitored at 24 h and 7 days following tabun challenge. The results indicate that the oxime HI-6 in combination with atropine was not able to protect the rats from the lethal effects of tabun. Two non-treated tabun-poisoned rats and one tabun-poisoned rat treated with atropine combined with HI-6 died within 2 h. On the other hand, all other tested oximes combined with atropine allowed all the tabun-poisoned rats to survive 7 days following tabun challenge. Both newly developed oximes combined with atropine seem to be sufficiently effective antidotes for a decrease in tabun-induced neurotoxicity in the case of sublethal poisoning although they are not able to eliminate tabun-induced neurotoxicity completely. The neuroprotective efficacy of obidoxime in combination with atropine approached the potency of newly developed oximes but the ability of the oxime HI-6 to counteract tabun-induced acute neurotoxicity was significantly lower, especially at 24 h after tabun poisoning. Due to their neuroprotective effects, both newly developed oximes appear to be suitable oximes for the antidotal treatment of acute tabun poisoning.

In this work, the ability of four newly synthesized oximes - K005 (1,3-bis(2-hydroxyiminomethylpyridinium) propane dibromide), K027 (1-(4-hydroxyiminomethylpyridinium)-3-(4-carbamoylpyridinium) propane dibromide), K033 (1,4-bis(2-hydroxyiminomethylpyridinium) butane dibromide) and K048 (1-(4-hydroxyiminomethylpyridinium)-4-(4- carbamoylpyridinium) butane dibromide) to reactivate acetylcholinesterase (AChE, EC 3.1.1.7) inhibited by nerve agents is summarized. Reactivation potency of these compouds was tested using standard in vitro reactivation test. Tabun, sarin, cyclosarin and VX agent were used as appropriate testing nerve agents. Rat brain AChE was used as a source of the enzyme. Efficacies of new reactivators to reactivate tabun-, sarin-, cyclosarin- and VX-inhibited AChE were compared with the currently used AChE reactivators (pralidoxime, obidoxime and HI-6). Oxime K048 seems to be promising reactivator of tabun-inhibited AChE. Its reactivation potency is significantly higher than that of HI-6 and pralidoxime and comparable with the potency of obidoxime. The best reactivator of sarin-inhibited AChE seems to be oxime HI-6. None of the new AChE reactivators reached comparable reactivation potency. The same results were obtained for cyclosarin-inhibited AChE. However, oxime K033 is also potent reactivator of AChE inhibited by this nerve agent. In the case of VX inhibition, obidoxime and new oximes K027 and K048 seem to be the best AChE reactivators. None from the currently tested AChE reactivators is able to reactivate AChE inhibited by all nerve agents used and, therefore, the search for new potential broad spectrum AChE reactivators is needed.

The efficacy of various oximes to reactivate acetylcholinesterase phosphorylated by tabun (O-ethyl-N,N-dimethyl phosphoramidocyanidate) was tested by in vitro and in vivo methods. The oximes commonly used for the treatment of acute poisonings with highly toxic organophosphates appeared to be almost ineffective (HI-6, pralidoxime, methoxime) or just slightly effective (obidoxime) against tabun. On the other hand, trimedoxime seemed to be a significantly more efficacious reactivator than the others in the case of tabun poisonings. In vitro, the concentration of trimedoxime corresponding to 1.0 mmol/l was able to reach 50% reactivation of tabun-inhibited brain acetylcholinesterase. Higher reactivating potency of trimedoxime in comparison with the other commonly used oximes was demonstrated by in vivo method, too. In addition, other structural analogues of trimedoxime were found to be efficacious in counteracting tabun-induced acetylcholinesterase inhibition although not as efficacious as trimedoxime itself. Some effective acetylcholinesterase reactivators were characterised by dissociation constant of enzyme-reactivator complex as well as enzyme-inhibitor-reactivator complex and by rate constant of reactivation.

To study the influence of low-level sarin inhalation exposure on immune functions, inbred BALB/c mice were exposed to low concentrations of sarin for 60 min in the inhalation chamber. The evaluation of immune functions was carried out using phenotyping of CD3 (T lymphocytes), CD4 (helper T lymphocytes), CD8 (cytotoxic T lymphocytes), and CD19 cells (B lymphocytes) in the lungs, blood, and spleen, lymphoproliferation of spleen cells stimulated in vitro by various mitogens (concanavalin A, lipopolysaccharides), phagocyte activity of peritoneal and alveolar macrophages, production of N-oxides by peritoneal macrophages, and the measurement of the natural killer cell activity at 1 wk following sarin exposure. The results were compared to the values obtained from control mice exposed to pure air instead of sarin. The results indicate that low doses of sarin are able to alter the reaction of immune system at one week following exposure to sarin. While the numbers of CD3 cells in the lungs, blood, and spleen were slightly decreased, an increase in CD19 cells was observed, especially in the lungs and blood. The reduced proportion of T lymphocytes is caused by decay of CD4-positive T cells. Lymphoproliferation was significantly decreased regardless of the mitogen and sarin concentration used. The production of N-oxides by peritoneal macrophages was stimulated after exposure to the highest dose of sarin, whereas their ability to phagocytize the microbes was increased after exposure to the lowest dose of sarin. The natural killer cell activity was significantly higher in the case of inhalation exposure of mice to the highest level of sarin. Thus, not only organophosphorus insecticides but also nerve agents such as sarin are able to alter immune functions even at a dose that does not cause clinically manifested disruption of cholinergic nervous system in the case of inhalation exposure. Nevertheless, the alteration of immune functions following the inhalation exposure to a symptomatic concentration of sarin seems to be more pronounced.

Synthesis of a new asymmetric bisquaternary reactivator of tabun-inhibited acetylcholinesterase-1-(4-hydroxyiminomethylpyridinium)-4-(4-carbamoylpyridinium) butane dibromide is described. Reactivation potency of this oxime is compared to the currently used reactivators-pralidoxime, obidoxime and H-oxime HI-6.

1. To study the influence of antidotes on low-level sarin-induced impairment of cognitive functions, the rats were exposed to three various low concentrations of sarin (LEVEL 1–3) for 60 minutes in the inhalation chamber. In addition, one group of rats was exposed to LEVEL 2 of sarin repeatedly. 2. Testing of cognitive functions was carried out using the Y-maze evaluating learning and spatial memory. The correct averse behavior of sarin-exposed rats in the Y-maze was tested several times within four weeks following sarin inhalation exposure and antidotal treatment to look for any cognitive impairments. 3. The results were compared to the Y-maze performance of sarin-exposed rats without antidotal treatment and control rats exposed to pure air instead of sarin with or without antidotal treatment. While antidotal treatment was able to eliminate a short-term deficiency in the Y-maze performance in rats exposed to the LEVEL 1 of sarin, a significant decrease in the Y-maze performance in rats exposed to sarin at the LEVEL 2 and 3 was only shortened. Sarin-induced spatial memory impairments in rats exposed repeatedly to sarin at the LEVEL 2 was also shortened when rats were treated following each sarin inhalation exposure. 4. The findings confirm that antidotes currently used for nerve agent poisonings are beneficial for the treatment of rats singly or repeatedly exposed to non-convulsive symptomatic or even clinically asymptomatic concentrations of sarin.

1. To study the influence of antidotes on low-level sarin-induced alteration of cognitive functions, male albino Wistar rats were exposed to three various low concentrations of sarin for 60 minutes in the inhalation chamber. One minute following sarin exposure, the rats were i.m. treated with the oxime HI-6 in combination with atropine. Control rats were treated with antidotes as experimental rats but exposed to the pure air instead of sarin. Cognitive functions of the rats were tested using a T-maze where spatial memory and spatial orientation were evaluated. The performance of sarin-exposed and treated rats in the T-maze was tested several times within six weeks (single exposure) or five weeks (repeated exposure) following inhalation exposure to evaluate cognitive impairments. 2. In the case of single exposure to sarin, no statistically significant differencies between the performances of the control and the experimental groups in the alteration of spatial memory and spatial orientation were observed. The repeated exposure of treated rats to clinically asymptomatic dose of sarin (LEVEL 2) did not change the effect of low-level sarin exposure on spatial memory of the experimental rats compared to the single exposure to the same dose of sarin. 3. The decrease in the T-maze performance of the control rats was caused by the impairments of rat’s mobility due to the features of a solution of antidotes.

To study the influence of pharmacological pretreatment (PANPAL) and antidotal treatment (obidoxime plus atropine) on tabun-induced neurotoxicity, male albino rats were poisoned with a lethal dose of tabun (280 μg/kg i.m.; 100% of LD 50 value) and observed at 24 h and 7 days following tabun challenge. The neurotoxicity of tabun was evaluated using a functional observational battery (FOB) and an automatic measurement of motor activity. Pharmacological pretreatment as well as antidotal treatment were able to eliminate most of tabun-induced neurotoxic effects observed at 24 h following tabun poisoning. However, there was not significant difference between the efficacy of PANPAL and antidotal treatment to eliminate tabun-induced neurotoxicity in rats. The combination of PANPAL pretreatment and antidotal treatment seems to be slightly more effective in the elimination of tabun-induced neurotoxicity in rats at 24 h following tabun challenge in comparison with the administration of PANPAL pretreatment or antidotal treatment alone. At 7 days following tabun poisoning, very few neurotoxic signs in tabun-poisoned rats were observed regardless of administration of pharmacological pretreatment or antidotal treatment. Thus, our findings confirm that the combination of pharmacological pretreatment and antidotal treatment is not only able to protect the experimental animals from the lethal effects of tabun but also to eliminate most of tabun-induced signs of neurotoxicity in tabun-poisoned rats.

The cholinesterase-inhibiting organophosphorus compounds referred to as nerve agents (soman, sarin, tabun, GF agent, and VX) are particularly toxic and are considered to be among the most dangerous chemical warfare agents. Included in antidotal medical countermeasures are oximes to reactivate the inhibited cholinesterase. Much experimental work has been done to better understand the properties of the oxime antidotal candidates including the currently available pralidoxime and obidoxime, the H oximes HI-6 and Hlö-7, and methoxime. There is no single, broad-spectrum oxime suitablefor the antidotal treatment of poisoning with all organophosphorus agents. If more than one oxime is available, the choice depends primarily on the identity of the responsible organophosphorus compound. The H oximes appear to be very promising antidotes against nerve agents because they are able to protect experimental animals from toxic effects and improve survival of animals poisoned with supralethal doses. They appear more effective against nerve agent poisoning than the currently used oximes pralidoxime and obidoxime, especially in the case of soman poisoning. On the other hand, pralidoxime and especially obidoxime seem sufficiently effective to treat poisonings with organophosphorus insecticides that have relatively less toxicity than nerve agents.

Male albino Wistar rats were once or repeatedly exposed to three various low concentrations of sarin for 60 min. in the inhalation chamber. The clinical status of control as well as sarin-poisoned rats was tested 3 months after exposure to sarin using biochemical, haematological, neurophysiological, behavioural and immunotoxicological methods. While biochemical and haematological parameters, including the activities of cholinesterases in erythrocytes, plasma and various organs (brain, diaphragm), did not differ from the control values regardless of the sarin concentration used, few signs of sarin-induced neurotoxicity and immunotoxicity in sarin-poisoned rats were demonstrated. This was especially true when the single exposure of rats to non-convulsive symptomatic concentration and repeated exposure of rats to clinically asymptomatic concentration of sarin was used. In rats repeatedly poisoned with clinically asymptomatic concentrations of sarin, the alteration of the gait characterized by ataxia, the increase in the stereotyped behaviour, the increase in the excitability of the central nervous system following the administration of the convulsive drug pentamethylenetetrazol were observed. In rats poisoned with non-convulsive symptomatic concentration of sarin, the subtle supression of spontaneous, as well as lipopolysaccharides-stimulated, proliferation of spleen lymphocytes and the bactericidal activity of peritoneal macrophages was primarily observed besides the signs of neurotoxicity. Our findings confirm that both non-convulsive symptomatic and clinically asymptomatic concentrations of sarin can only cause very few, subtle long-term signs of neurotoxicity and immunotoxicity in sarin-poisoned rats when the rats were exposed to asymptomatic sarin concentrations repeatedly.

The neuroprotective effects of antidotes (atropine, obidoxime, obidoxime/atropine mixture) on rats poisoned with soman at a sublethal dose (54 μg/kg, im, 80% of LD 50 value) were studied. The soman-induced neurotoxicity was monitored using a functional observational battery (FOB) and an automatic measurement of motor activity. The neurotoxicity of soman was monitored at 24 h and 7 days following soman challenge. The results indicate that obidoxime alone is not able to protect the rats from the lethal effects of soman. Three soman-poisoned rats treated with obidoxime alone died within 24 h. On the other hand, atropine alone or combined with obidoxime allows all soman-poisoned rats to survive within 7 days following soman challenge. Atropine alone and combined with obidoxime seems to be relatively effective antidotal treatment for the elimination of soman-induced neurotoxicity in the case of sublethal poisonings, although the antidotal mixture is significantly less effective than atropine alone because obidoxime can counteract the beneficial effects of atropine. Obidoxime appears to be practically ineffective to diminish soman-induced neurotoxicity. The neuroprotective effects of antidotal mixture consisting of atropine and obidoxime depend on the antimuscarinic effects of atropine only. Thus, the replacement of obidoxime by more effective acetylcholinesterase (AChE) reactivators is necessary to increase the neuroprotective efficacy of antidotal treatment in the case of soman poisonings.

1 The reactivating and therapeutic efficacy of a new acetylcholinesterase reactivator, designated BI-6 (1-/2-hydroxyiminomethylpyridinium/-4-/carbamoylpyridinium/-2-butene dibromide), against organophosphate sarin was compared with presently used oximes (pralidoxime, obidoxime, methoxime) and H oximes (HI-6, HLö-7) by in vitro and in vivo methods. 2 Our results confirm that the new oxime BI-6 is a significantly more efficacious acetylcholinesterase reactivator than currently available pralidoxime and obidoxime but not as effective as H oximes (HI-6, HLö-7) in vitro as well as in vivo. In addition, the oxime BI-6 is able to protect supralethal sarin poisoned rats at human-relevant doses. 3 Our data also suggest that the potency of oximes tested to reactivate sarin-inhibited acetylcholinesterase in vitro closely corresponds to their reactivating efficacy in vivo and their ability to protect rats poisoned with supralethal doses of sarin.

The reactivating and therapeutic efficacy of a new acetylcholinesterase reactivator, designated BI-6(1-/2-hydroxyiminomethylpyridinium/-4-/carbamoylpyridinium/-2-butene dibromide), against the organophosphate soman was compared with oximes at present used (pralidoxime, obidoxime, methoxime) and H oximes (HI-6, HLo-7) using in vitro and in vivo methods. H oximes HI-6 and HLo-7 seem to be the most efficacious acetylcholinesterase reactivators against soman according to the evaluation of their reactivating and therapeutic efficacy in vitro as well as in vivo. The new oxime BI-6 is not as effective as the H oximes against soman, nevertheless it is significantly more effective against soman than the currently available oximes, pralidoxime, obidoxime and methoxime, which failed to protect rats poisoned with supralethal doses of soman. Our results confirm that the reactivating efficacy of oximes evaluated by the methods in vitro closely correlates not only with the potency of oximes in vivo in reactivating soman-inhibited acetylcholinesterase but also with the ability to protect rats poisoned with supralethal doses of soman.

Changes of acetylcholinesterase (AChE) activity in various parts of the brain (frontal cortex, medulla oblongata., pons Varoli, cerebellum, hypothalamus, and hippocampus), following im sublethal nontreated and treated soman poisoning were studied. As a treatment, two antidotal mixtures containing atropine and either obidoxime or oxime HI-6 were used. This antidotal treatment was administered im for 30 s following soman intoxication. The AChE activities in the various brain tissues were evaluated at 1 and 3 h following soman administration. As expected, the highly toxic organophosphorus compound, soman, markedly inhibited AChE activity in all the brain sections at both time intervals. Both oximes had little influence on soman-induced AChE inhibition, but only the HI-6 mixture was able to reactivate soman-inhibited AChE significantly in some of the brain parts (frontal cortex, pons Varoli, hypothalamus). In the brain, the effect, of HI-6 against soman.-induced AChE inhibition is higher in comparison with obidoxime, but not quite satisfactory. Despite its limited effectiveness in the brain, HI-6 seems to be the most effective oxime yet found against soman poisoning because of its high reactivating effect in the peripheral, compartment and other beneficial effects.

The efficacy of two new monopyridinium oximes (2-PAAM, 2-PAEM) and two bispyridinium oximes (obidoxime. HI-6) was tested in combination with atropine sulphate against acute poisoning with the organophosphorus insecticide mevinphos in mice. When mice were treated two min. after mevinphos poisoning, no significant differences in the therapeutic effect of tested oximes were observed. The oximes increased the 24 hr LD50 values of mevinphos about two times in comparison with the 24 hr LD50 values of mevinphos in mice protected with atropine sulphate alone and more than three times in comparison with non-treated intoxicated animals. On the other hand, both monopyridinium oximes were significantly more efficacious than HI-6 and as efficacious as obidoxime when they were administered 30 sec. after mevinphos poisoning. Both monopyridinium oximes and obidoxime increased the 24 hr values of mevinphos almost three times in comparison with the 24 hr values of mevinphos in mice protected with atropine sulphate alone and about twenty-five times in comparison with non-treated intoxicated animals, while the oxime HI-6 less than two times in comparison with the 24 hr values of mevinphos in mice protected with atropine sulphate alone and about fifteen times in comparison with non-treated intoxicated animals. Use of new monopyridinium oximes seems to be the improvement in the antidotal treatment of poisoning with organophosphorous insecticide mevinphos in comparison with HI-6 but not in comparison with obidoxime when oximes are used in equimolar doses.

The influence of cholinolytic drugs (atropine, benactyzine, biperiden) on the efficacy of the oxime HI-6 (1-[[[(4-aminocarbonyl)pyridinio]methoxy ]methyl]-2-[hydroxyimino)methyl]pyridinium dichloride monohydrate) on soman-induced anticholinesterase and stressogenic effects was studied in rats. Soman-induced acetylcholinesterase inhibition in blood and diaphragm and the stressogenic effects of soman, i.e. an increase in plasma corticosterone level and liver tyrosine aminotransferase activity, were more significantly diminished by HI-6 in combination with benactyzine or biperiden in comparison with HI-6 plus atropine. These findings support a hypothesis that benactyzine as well as biperiden can increase the efficacy of the oxime HI-6 in comparison with atropine. They demonstrate the importance of cholinolytic drug selection in the treatment of soman poisoning in rats. Ltd.

1 The influence of pharmacological pretreatment (pyr idostigmine, benactyzine and trihexyphenidyle), de signated PANPAL, on soman-induced cholinergic and stressogenic effects as well as on the efficacy of antidotal treatment (HI-6 plus obidoxime) in rats was studied. 2 PANPAL prophylaxis significantly decreased soman- induced cholinesterase inhibition in blood, brain and diaphragm as well as stressogenic effects of soman (an increase in plasma corticosterone level and liver tyrosine aminotransferase activity). 3 PANPAL pretreatment did not improve the efficacy of HI-6 in combination with benactyzine on soman- induced anticholinesterase and stressogenic effects. 4 These findings confirm that PANPAL prophylaxis can improve prognosis of soman poisoning especially by protection of cholinesterases.

The effects of HI-6 (1-[[[(4-aminocarbonyl) pyridinio]methoxy]methyl]-2-(hydroxyimino)-methyl]pyridinium dichloride monohydrate) on the organophosphorus agent 2-dimethylaminoethyl-(dimethylamido)-phosphonofluoridate (GV) inhibited cholinesterases in blood, brain and diaphragm and the stressogenic effect of GV in rats were compared to the effects of obidoxime. In GV-poisoned rats, HI-6 had a significantly higher reactivating effect in blood and brain as well as diaphragm than had obidoxime. HI-6 also practically eliminated the stressogenic effect of the GV compound. On the other hand, hypercorticosteronaemia and increase of liver tyrosine aminotransferase activity during treatment of poisoning with obidoxime were observed. These findings demonstrate the importance of HI-6 for treatment of GV agent poisoning in rats.