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Long-term effects of the anticholinesterases sarin and soman on latencies of muscle action potentials in mouse diaphragm muscle.

In-vivo administration of the irreversible anticholinesterases sarin and soman has been shown to produce long-term effects on latency and variability of latency of muscle action potentials in in-vitro mouse diaphragm muscle preparations. The maximum observed effects occurred three days post-soman administration and seven days post-sarin administration, and were no longer detectable 28 days later. With both anticholinesterases the increase in latency, and variability of latency, was reduced by pyridostigmine pretreatment. Therapeutic administration of pralidoxime mesylate effectively prevented the sarin-induced effects when given after a delay of 24 h. In contrast, the effectiveness of pralidoxime mesylate declined rapidly when its administration was delayed following soman. These findings are consistent with this action of soman and sarin being a product of acetylcholinesterase inhibition. The results obtained with sarin suggest that a period of acetylcholinesterase inhibition in excess of 24 h is required to trigger the events leading to the production of this long-term effect.

Action Potentials↗

Effects of sarin on temperature and activity of rats as a model for gulf war syndrome neuroregulatory functions.

Coexposure to subclinical levels of nerve gas and to heat stress may have induced some of the clinical symptoms of the Gulf War Syndrome. We tested the hypothesis that single or repeated subclinical exposure to sarin, particularly under conditions of heat stress, would impair regulation of body temperature and locomotor activity. Male F344 rats were housed at 25 degrees C or under mild heat stress at 32 degrees C and were exposed 1 h/day for 1, 5, or 10 days to 0, 0.2, or 0.4 mg/m(3) of sarin in a nose-only exposure system. Body temperature and activity were monitored continuously by telemetry during exposure and 1 month postexposure. Exposed rats showed no clinical symptoms of toxicity such as tremors, despite evidence of reduced red blood cell cholinesterase activity. Heat stress consistently elevated body temperature in unexposed animals, particularly during the dark period when animals are most active. Inhalation of sarin gas at the two subclinical levels did not affect body temperature acutely in a biologically meaningful manner after the first exposure nor after 5 or 10 repeated exposures, either at thermoneutral ambient temperature or during chronic heat stress. There were no consistent effects of sarin or housing temperature on activity. The data suggest that subclinical levels of sarin have minimal effects on temperature regulation and locomotor activity under these observation conditions.

Animals↗

Biodegradation of neutralized sarin.

This research investigated the biotransformation of IMPA, the neutralization product of the nerve agent Sarin, by a microbial consortia. As mandated by the Chemical Weapons Convention signed by 132 countries in 1993, all chemical warfare agents are to be destroyed within ten years of ratification. Technologies must be developed to satisfy this commitment. This paper presents data from a biodegradation kinetics study and background information on the biological transformation of IMPA. Microbial transformation of organophosphate nerve agents and organophosphate pesticide intermediates can be incorporated into a treatment process for the fast and efficient destruction of these similar compounds. Sarin (isopropyl methylphosphonofluoridate), also known as GB, is one of several highly neurotoxic chemical warfare agents that have been developed over the past 50 to 60 years. Four mixed cultures were acclimated to the Sarin hydrolysis product, isopropyl methylphosphonic acid (IMPA). Two of these cultures, APG microorganisms and SX microorganisms, used IMPA as the sole phosphorus source. Extended exposure to IMPA improved the cultures' abilities to degrade IMPA to form methylphosphonic acid (MPA) and inorganic phosphate. The presence of free phosphate in the reactor suppressed the degradation of IMPA. IMPA did not inhibit either cultural consortia within the tested concentration range (0 to 1250 mg/L). The numax was 120.9 mg/L/day for the SX microorganisms and 118.3 mg/L/day for the APG microorganisms. Initial IMPA concentrations of 85 to 90 mg/L were degraded to nondetectable levels within 75 h. These results demonstrate the potential for biodegradation to serve as a complementary treatment process for the destruction of stockpiled Sarin.

Biodegradation, Environmental↗

Protection and inflammatory markers following exposure of guinea pigs to sarin vapour: comparative efficacy of three oximes.

The purpose of the present study was to compare the antidotal efficacy and the combined effects on inflammatory markers of three oximes--toxogonine, TMB4 and 2-PAM--in combination with anticholinergic drugs following exposure to sarin vapour by inhalation. Guinea pigs restrained in plethysmographs were exposed to various doses of sarin vapour (in the range of 1.4-4.4LD50). The antidotal mixture was injected immediately (5-20 s) following exposure (3 mg kg(-1) atropine and 1 mg kg(-1) benactyzine in combination with 6 mg kg(-1) toxogonine, 2 mg kg(-1) TMB4 or 12 mg kg(-1) 2-PAM). Bronchoalveolar lavage (BAL) samples were taken from surviving animals 24 h after exposure to determine the levels of inflammatory markers. A differential cell count was performed in BAL samples on Giemsa-stained slides. The inflammatory markers--histamine and prostaglandins (PGE)--were measured in BAL using radioimmunoassay (RIA) techniques. The survival rate in the various treatment groups and analysis of BAL samples showed that: (i) Toxogonine, TMB4 and 2-PAM, without pyridostigmine pretreatment, at doses that were proportional to their doses in the respective auto-injectors, exhibited similar antidotal efficacy against sarin exposure. (ii) The results demonstrated that a centrally acting anticholinergic drug is essential in the antidotal mixture to ensure survival. (iii) Histamine release and eosinophilia following sarin inhalation might require additional intervention, aimed at reducing the symptoms of allergic reaction and possibly expediting recovery.

Animals↗

Comparison of in vitro potency of oximes (pralidoxime, obidoxime, HI-6) to reactivate sarin-inhibited acetylcholinesterase in various parts of pig brain.

The potency of currently used oximes to reactivate sarin-inhibited acetylcholinesterase (AChE) in various parts of pig brain and whole pig brain was evaluated using in vitro methods. Significant differences in reactivation potency among all tested oximes were observed. At concentrations (10(-4) M) corresponding to recommended doses in vivo, the oxime HI-6 seems to be a more efficacious reactivator of sarin-inhibited AChE in whole pig brain as well as in cerebral hemispheres and cerebellum compared with the other oximes studied. Nevertheless, there are not any differences in the potency of oximes tested to reactivate sarin-inhibited AChE in medulla oblongata. Thus, the oxime HI-6 appears to be the most promising oxime among currently available oximes for the antidotal treatment of acute sarin poisoning, although it is not more efficacious than other currently used oximes in medulla oblongata, whose function is necessary for the vital functions of respiration and circulation.

Acetylcholinesterase↗

Interaction of obidoxime with sarin in aqueous solution.

The interaction of obidoxime (Toxogonin) with sarin was shown by different analytical methods. The UV spectrum of obidoxime at pH 7.4 yields two absorption maxima, lambda 1 = 284 nm and lambda 2 = 353 nm. The peak at lambda 2 = 353 nm is representative for the amount of zwitter-ionic obidoxime, i.e. the active form of obidoxime. By addition of sarin, lambda 1 shifts immediately to 278 nm and the intensity at lambda 2 decreases, thus indicating an interaction. TLC and 31P-NMR evidence shows that both mono-phosphonylated and diphosphonylated obidoximes are present. Decomposition of phosphonylated obidoxime in MOPS (3-[N-morpholino] propanesulfonic acid) buffered D2O at pH 7.4 occurs with t1/2 = 13.3 min at 24 degrees C. Decomposition of di-phosphonylated obidoxime is faster. It is suggested that decomposition of di-phosphonylated obidoxime occurs through the mono-phosphonylated form. Formation and decomposition of mono- and di-phosphonylated obidoxime is pH dependent. We conclude that obidoxime exerts a detoxifying effect by capturing free sarin molecules and thus increasing its polarity. Thereby the transition of sarin through the blood-brain barrier is restricted and its renal elimination facilitated.

Chromatography, Thin Layer↗

Effect of human plasma on the reactivation of sarin-inhibited human erythrocyte acetylcholinesterase.

The reactivation of organophosphate-inhibited acetylcholinesterase (AChE) by oximes inevitably results in the formation of highly reactive phosphoryloximes (POX), which are able to re-inhibit the enzyme. In this study, the dependence of POX formation on AChE concentration was investigated with sarin-inhibited human erythrocyte AChE (EryAChE). A marked dependence was found with obidoxime but not with the experimental oxime HI 6, suggesting great differences in the decomposition rates of the respective POXs. At a physiological erythrocyte content the reactivation of EryAChE was markedly affected by POX with obidoxime and pralidoxime (2-PAM) but not with the newer oximes HI 6 and HLö 7. Addition of extensively dialysed, sarin-treated human plasma reduced the reactivation by obidoxime and 2-PAM even more. Obidoxime and 2-PAM were superior to HI 6 and HLö 7 in reactivating butyrylcholinesterase (BChE). This effect was pronounced in diluted plasma, but was obscured in concentrated plasma, probably because of re-inhibition by the generated POX. Addition of native erythrocytes to sarin-treated plasma resulted in marked inhibition of EryAChE in the presence of obidoxime, suggesting a higher affinity of the POX for EryAChE. The results indicate that obidoxime and 2-PAM may reactivate sarin-inhibited AChE insufficiently due to re-inhibition by the POX formed. In addition, the re-inhibition of Ery-AChE may be aggravated by the POX that is produced during BChE reactivation. These reactions must be regarded as therapeutically detrimental and disqualify those oximes which are capable of forming stable POX by reactivation of BChE.

Acetylcholinesterase↗

The Arg192 isoform of paraoxonase with low sarin-hydrolyzing activity is dominant in the Japanese.

The high-density-lipoprotein-associated enzyme paraoxonase, which has a role in the detoxification of organophosphorus compounds, is known to be polymorphic in humans. The Arg192 isoform of paraoxonase hydrolyzes paraoxon more rapidly than the Gln192 isoform. However, with respect to the hydrolysis of toxic nerve agents, such as diazoxon, soman, and sarin, the Arg192 isoform displays a lower activity than the other isoform. To evaluate the possibility that the genetic polymorphism was involved in the aggravated extent of human injury in the sarin gas poisoning incident in the Tokyo subway in March 1995, we investigated the prevalance of this polymorphism in the Japanese population. We found that the Arg192 allele is more common in the Japanese (allele frequency: 0.66) than in people of other races (ranging 0.24-0.31). In the Japanese, 135 out of the 326 subjects (41.4%) investigated were homozygous for the Arg192 allele, which shows a very low hydrolysis activity for sarin. Thus, there seems to be a racial difference in vulnerability to toxic nerve agents, such as sarin. The dominance of the Arg192 allele in the Japanese population probably worsened the tragedy of March 1995 in the Tokyo subway.

Alleles↗

Soman and sarin induce a long-lasting naloxone-reversible analgesia in mice.

Soman (50 micrograms/kg) and sarin (120 micrograms/kg), potent organophosphate anticholinesterase agents, produced an analgesic response in the mouse hotplate latency test. Naloxone antagonized but did not completely reverse the soman- and sarin-induced analgesia, whereas atropine antagonized completely the soman-and sarin-induced analgesia. Soman poisoning did not potentiate morphine-induced analgesia. It was simply an additive response. In survivors of soman (287 micrograms/kg) poisoning, the analgesia was more pronounced and was still apparent 96 hr after administration. This analgesia was completely antagonized by naloxone. Similar results were found in survivors of sarin (510 micrograms/kg) poisoning. The organophosphate-induced analgesia was not due to physical incapacitation as evidenced by performance on the accelerating rotorod. It is suggested that the organophosphate-induced analgesia is due to a combination of an increased concentration of acetylcholine due to inhibition of acetylcholinesterase combined with a reduced destruction of endogenous opioid-like substances due to organophosphate inhibition of proteases.

Analgesics↗

Segmental synaptic depression caused by diisopropylphosphorofluoridate and sarin is reversed by thyrotropin-releasing hormone in the neonatal rat spinal cord.

The organophosphorus compounds diisopropylphosphorofluoridate (DFP) and isopropylmethylphosphonofluoridate (sarin) depressed the monosynaptic reflex (MSR) in spinal cords from 7- to 9-day-old male rats. The concentrations of DFP and sarin which depressed the MSR by nearly 50% were 100 microM and 100 nM, respectively. Simultaneous superfusion of the cords with thyrotropin-releasing hormone (TRH) with either DFP or sarin resulted in a reversal of the depression. The depression caused by DFP was reversed to 95% of control by 100 nM TRH whereas similar reversal of sarin-induced depression required a 10-fold greater concentration of TRH. The potentiating effect of TRH was not affected by atropine even at a high concentration (1 microM) although atropine easily reversed organophosphorus-induced depression of the MSR. It appears that reversal of organophosphorus-induced depression by TRH might occur through a noncholinergic, TRH-sensitive receptor mechanism and may be unrelated to acetylcholinesterase activity. This action represents a possible utility of TRH as an adjunct in organophosphorus toxicity.

Animals↗

Acute inhalation toxicity of soman and sarin in baboons.

Adult baboons (Papio sp.; 8-12 kg) were anesthesized with sodium pentobarbital (20 mg/kg iv). The animals were instrumented for measurement of mean blood pressure (MBP), pulmonary artery pressure (PAP), ECG, arterial and mixed venous blood gases, lung volumes, lung pressures, and efferent phrenic nerve activity. Bronchoalveolar lavage (BAL) was performed. Studies were done prior to exposure, at intervals during the first 4 hr postexposure, and at 4 and 28 days after exposure. Control animals received a sham exposure to 2-propanol (N = 5). Soman (pinacolyl methylphosphonofluoridate) at 13.14 micrograms/kg (2 X LD50) was vaporized into the upper airway in a second group of animals (N = 5), and sarin (isopropyl methylphosphonofluoride) 30 micrograms/kg (2 X LD50) was vaporized into a third group of animals (N = 4). Controls showed no change in any parameter either immediately after diluent exposure or during the monitoring period. Soman and sarin produced a decline in MBP and bradyarrhythmias that were reversed with atropine. Apnea occurred in all soman- and sarin-exposed animals within 5 min postexposure, and was associated with absence of phrenic nerve signal. Ventilation was mechanically supported until the animal could maintain normal arterial blood gases during spontaneous breathing. BAL studies revealed an increase in total white cell population and neutrophils at 4 hr in all three groups. There were signs of impaired hemodynamics and persistent lung injury for 4 days that resolved by 28 days after exposure. In conclusion, inhalation of soman and sarin in the baboon is associated with cardiac arrhythmias, development of apnea, and a significant decrease in MBP. Inhalation exposure also resulted in a persistent influx of neutrophils and hypoxemia.

Administration, Inhalation↗

Reduction by pyridostigmine pretreatment of the efficacy of atropine and 2-PAM treatment of sarin and VX poisoning in rodents.

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.

Acetylcholinesterase↗

Changes in spinal cord reflexes following subchronic exposure to soman and sarin.

The organophosphorus agents soman and sarin were administered subchronically and the spinal monosynaptic (MSR) and dorsal root (DRR) reflexes were assessed in spinal cord-transected cats. Neither soman nor sarin produced behavioral signs of delayed neurotoxicity. However, both soman and sarin significantly reduced the area under the MSR and DRR with only minimal changes in the excitability of the potentials as determined using quipazine, a serotonin agonist. The changes observed in these reflexes resulting from soman or sarin exposure are discussed in relation to alterations in the central terminals of primary afferent neurons.

Action Potentials↗

Anticonvulsant treatment of sarin-induced seizures with nasal midazolam: an electrographic, behavioral, and histological study in freely moving rats.

Centrally mediated seizures and convulsions are common consequences of exposure to organophosphates (OPs). These seizures rapidly progress to status epilepticus (SE) and contribute to profound brain injury. Effective management of these seizures is critical for minimization of brain damage. Nasal application of midazolam (1.5 mg/kg) after 5 min of sarin-induced electrographic seizure activity (EGSA) ameliorated EGSA and convulsive behavior (238 +/- 90 s). Identical treatment after 30 min was not sufficient to ameliorate ECoG paradoxical activity and convulsive behavior. Nasal midazolam (1.5 mg/kg), together with scopolamine (1 mg/kg, im) after 5 min of EGSA, exerted a powerful and rapid anticonvulsant effect (53 +/- 10 s). Delaying the same treatment to 30 min of EGSA leads to attenuation of paroxysmal ECoG activity in all cases but total cessation of paroxysmal activity was not observed in most animals tested. Cognitive tests utilizing the Morris Water Maze demonstrated that nasal midazolam alone or together with scopolamine (im), administered after 5 min of convulsions, abolished the effect of sarin on learning. Both these treatments, when given after 30 min of convulsions, only decreased the sarin-induced learning impairments. Whereas rats which were not subject to the anticonvulsant agents did not show any memory for the platform location, both treatments (at 5 min as well as at 30 min) completely abolished the memory deficits. Both treatments equally blocked the impairment of reversal learning when given at 5 min. However, when administered after 30 min, midazolam alone reversed the impairments in reversal learning, while midazolam with scopolamine did not. Rats exposed to sarin and treated with the therapeutic regimen with the exclusion of midazolam exhibited severe brain lesions that encountered the hippocampus, pyriform cortex, and thalamus. Nasal midazolam at 5 min prevented brain damage, while delaying the midazolam treatment to 30 min of EGSA resulted in brain damage. The addition of scopolamine to midazolam did not alter the above observation. In summary, nasal midazolam treatment briefly after initiation of OP-induced seizure leads to cessation of EGSA and prevented brain lesions and behavioral deficiencies in the rat model.

Administration, Intranasal↗

Ocular signs and symptoms caused by exposure to sarin gas.

PURPOSE: Ocular signs and symptoms that occurred in people exposed to sarin gas in a subway sarin gas attack were studied. METHODS: Among victims of sarin gas exposure, 96 were treated by us. RESULTS: Ocular signs and symptoms caused by sarin gas exposure included miosis, conjunctival injection, and ocular pain. CONCLUSIONS: Ocular signs and symptoms spontaneously resolved between three and 21 days after exposure in most cases. Treatment with 0.5% tropicamide ophthalmic solution was effective in decreasing ocular pain.

Conjunctival Diseases↗

Determination of sarin, soman and their hydrolysis products in soil by packed capillary liquid chromatography-electrospray mass spectrometry.

An analytical method based on aqueous ultrasonic extraction and packed capillary liquid chromatography-electrospray mass spectrometry (LC-ESI-MS) analysis was developed and compared to an existing gas chromatography(GC)-MS based method for the determination of sarin, soman and their hydrolysis products in contaminated soil. Three soils, a red clay, a tan sandy clay and a brown sandy clay loam, were spiked with sarin and soman and their initial hydrolysis products, isopropyl methylphosphonic acid and pinacolyl methylphosphonic acid, at the 10 microg/g level to assess recovery efficiency. Recovery of sarin and soman from the aqueous soil extracts was comparable to the existing analytical method, with a significant improvement in recovery being demonstrated for the chemical warfare agent hydrolysis products. Sarin and soman were recovered in the 20-90% range from the three soil types with aqueous extraction, while the hydrolysis products of these chemical warfare agents were extracted with recoveries in excess of 80%. The developed soil extraction and analysis method appears to be an attractive alternative to the GC-MS based method, since aqueous extracts containing chemical warfare agent hydrolysis products may be analysed directly, eliminating the need for additional sample handling and derivatization steps.

Chemical Warfare Agents↗

Comparative efficacy of diazepam and avizafone against sarin-induced neuropathology and respiratory failure in guinea pigs: influence of atropine dose.

This investigation compared the efficacy of diazepam and the water-soluble prodiazepam-avizafone-in sarin poisoning therapy. Guinea pigs, pretreated with pyridostigmine 0.1 mg/kg, were intoxicated with 4LD(50) of sarin (s.c. route) and 1 min after intoxication treated by intramuscular injection of atropine (3 or 33.8 mg/kg), pralidoxime (32 mg/kg) and either diazepam (2 mg/kg) or avizafone (3.5 mg/kg). EEG and pneumo-physiological parameters were simultaneously recorded. When atropine was administered at a dose of 3 mg/kg, seizures were observed in 87.5% of the cases; if an anticonvulsant was added (diazepam (2 mg/kg) or avizafone (3.5 mg/kg)), seizure was prevented but respiratory disorders were observed. At 33.8 mg/kg, atropine markedly increased the seizure threshold and prevented early respiratory distress induced by sarin. When diazepam was administered together with atropine, seizures were not observed but 62.5% of the animals displayed respiratory difficulties. These symptoms were not observed when using avizafone. The pharmacokinetic data showed marked variation of the plasma levels of atropine and diazepam in different antidote combination groups, where groups receiving diazepam exhibited the lowest concentration of atropine in plasma. Taken together, the results indicate that avizafone is suitable in therapy against sarin when an anticonvulsant is judged necessary.

Animals↗

Probing the acetylcholinesterase inhibition of sarin: a comparative interaction study of the inhibitor and acetylcholine with a model enzyme cavity.

Interaction energies have been estimated between sarin and a model enzyme cavity of acetylcholinesterase (ACHE) using the density functional and Møller-Plesset second-order perturbation (MP2) levels of theories. The calculated interaction energies have been compared with those of acetylcholine and the same model ACHE cavity. The ACHE...sarin and ACHE...acetylcholine (Ach) structures have been optimized using DFT based two-layer ONIOM hybrid calculations. The nature of interactions has been investigated in detail using an interaction energy partitioning technique. The effects of solvation on the interaction energies have also been taken into account. An inhibition mechanism during the uptake of sarin inside the ACHE cavity has been proposed from the comparison of the energetics of the ACHE...sarin and ACHE...Ach complexes.

Acetylcholine↗