Anticonvulsant effects of phencynonate hydrochloride and other anticholinergic drugs in soman poisoning: neurochemical mechanisms

Previous studies have paid little attention to the anticonvulsant effect of anticholinergic drugs that act on both muscarinic (M) and nicotinic (N) receptors during soman-induced seizures. Therefore, with the establishment of a soman-induced seizures model in rats, this study evaluated the efficacy in preventing soman-induced convulsions of two antagonists of both the M and N receptors, phencynonate hydrochloride (PCH) and penehyclidine hydrochloride (8018), which were synthesized by our institute, and of other anticholinergic drugs, and investigated the mechanisms of their antiseizures responses. Male rats, previously prepared with electrodes to record electroencephalographic (EEG) activity, were pretreated with the oxime HI-6 (125 mg kg-1, i.p.) 30 min before they were administered soman (180 microg kg-1, s.c.). All animals developed seizures subsequent to this treatment. Different drugs were given at different times (5, 20 and 40 min after seizures onset) and their anticonvulsant effects were monitored and compared using the two variables, i.e. the dose that could totally control the ongoing seizures, as well as the speed of seizures control. The anticonvulsant effects of atropine, scopolamine and 8018 decreased with the progression of the seizures, and they eventually lost their anticonvulsant activity when the seizures had progressed for 40 min. In contrast, PCH showed good anticonvulsant effectiveness at 5 and 20 min, and especially at 40 min after seizures onset. Of the anticholinergic drugs tested, atropine, scopolamine, and 8018 showed no obvious protection against pentylenetetrazol (PTZ)-induced convulsions or N-methyl-D-aspartate (NMDA)-induced lethality in mice. However, PCH antagonized the PTZ-induced convulsions in a dose-dependant manner with an ED50 of 10.8 mg kg-1, i.p. (range of 7.1-15.2 mg kg-1) and partly blocked the lethal effects of NMDA in mice. PCH also dose-dependently inhibited NMDA-induced injury in rat primary hippocampal neuronal cultures, suggesting a possible neuroprotective action in vivo. In conclusion, our study suggests that the mechanisms of PCH action against soman-induced seizures might differ from those of the M receptor antagonists atropine and scopolamine, and that of the antagonist of both the M and N receptors, 8018. The pharmacological profile of PCH might include anticholinergic and anti-NMDA properties. Compared with the currently recommended anticonvulsant drug diazepam, with known NMDA receptor antagonists such as MK-801 and with conventional anticholinergics such as scopolamine and atropine, the potent anticonvulsant effects of PCH during the entire initial 40 min period of soman poisoning, and its fewer adverse effects, all suggest that PCH might serve as a new type of anticonvulsant for the treatment of seizures induced by soman.     

Anticonvulsants for soman-induced seizure activity

This report describes studies of anticonvulsants for the organophosphorus (OP) nerve agent soman: a basic research effort to understand how different pharmacological classes of compounds influence the expression of seizure produced by soman in rats, and a drug screening effort to determine whether clinically useful antiepileptics can modulate soman-induced seizures in rats. Electroencephalographic (EEG) recordings were used in these studies. Basic studies were conducted in rats pretreated with HI-6 and challenged with 1.6×LD₅₀ soman. Antimuscarinic compounds were extremely effective in blocking (pretreatment) or terminating soman seizures when given 5 min after seizure onset. However, significantly higher doses were required when treatment was delayed for more than 10 min, and some antimuscarinic compounds lost anticonvulsant efficacy when treatment was delayed for more than 40 min. Diazepam blocked seizure onset, yet seizures could recur after an initial period of anticonvulsant effect at doses 2.5 mg/kg. Diazepam could terminate ongoing seizures when given 5 min after seizure onset, but doses up to 20 mg/kg were ineffective when treatment was delayed for 40 min. The GABA uptake inhibitor, tiagabine, was ineffective in blocking or terminating soman motor convulsions or seizures. The glutamate receptor antagonists, NBQX, GYKI 52466, and memantine, had weak or minimal antiseizure activity, even at doses that virtually eliminated signs of motor convulsions. The antinicotinic, mecamylamine, was ineffective in blocking or stopping seizure activity. Pretreatment with a narrow range of doses of ₂-adrenergic agonist, clonidine, produced variable protection (40–60%) against seizure onset; treatment after seizure onset with clonidine was not effective. Screening studies in rats, using HI-6 pretreatment, showed that benzodiazepines (diazepam, midazolam and lorazepam) were quite effective when given 5 min after seizure onset, but lost their efficacy when given 40 min after onset. The barbiturate, pentobarbital, was modestly effective in terminating seizures when given 5 or 40 min after seizure onset, while other clinically effective antiepileptic drugs, trimethadione and valproic acid, were only slightly effective when given 5 min after onset. In contrast, phenytoin, carbamazepine, ethosuximide, magnesium sulfate, lamotrigine, primidone, felbamate, acetazolamide, and ketamine were ineffective.The animals used in studies performed in, or sponsored by, this Institute were handled in accordance with the principles stated in the Guide for the Care and Use of Laboratory Animals, proposed by the Committee to Revise the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources, National Research Council, and published by National Academy Press, 1996, and the Animal Welfare Act of 1966, as amended. The opinions or assertions contained herein are the private views of the authors, and are not to be construed as reflecting the views of the Department of the Army or the Department of Defense.     

Anticonvulsant Efficacy of Drugs with Cholinergic and/or Glutamatergic Antagonism Microinfused into Area Tempestas of Rats Exposed to Soman

A group of antiparkinson drugs (benactyzine, biperiden, caramiphen, procyclidine, and trihexyphenidyl) has been shown to possess both anticholinergic and antiglutamatergic properties, making these agents very well suited as anticonvulsants against nerve agents. The first purpose of this study was to make a comparative assessment of the anticonvulsant potencies of the antiparkinson agents when microinfused (1 μl) into the seizure controlling area tempestas (AT) of rats 20 min before subcutaneous injection of soman (100 μg/kg). The second purpose was to determine whether cholinergic and/or glutamatergic antagonism was the effective property. The results showed that only procyclidine (6 μg) and caramiphen (10 μg) antagonized soman-induced seizures. Cholinergic, and not glutamatergic, antagonism was likely the active property, since atropine (100 μg), and scopolamine (1 μg) caused anticonvulsant effects, whereas MK-801 (1 μg), and ketamine (50 μg) did not. Soman (11 nmol) injected into AT resulted more frequently in clonic convulsions than full tonic–clonic convulsions. AT may serve as both a trigger site for soman-evoked seizures and a site for screening anticonvulsant potencies of future countermeasures. Special issue article in honor of Dr. Frode Fonnum.     

Concomitant Increases in the Levels of Choline and Free Fatty Acids in Rat Brain: Evidence Supporting the Seizure-Induced Hydrolysis of Phosphatidylcholine

The main objective of this study was to determine whether the excitotoxic cholinesterase inhibitor soman increases the catabolism of phospholipids in rat brain. Injections of soman (70 micrograms/kg, s.c.), at a dose that produced toxic effects, increased the levels of both free fatty acids (175-250% of control) and free choline (250% of control) in rat cerebrum 1 h after administration. All fatty acids contained in brain phosphatidylcholine were elevated significantly including palmitic (16:0), stearic (18:0), oleic (18:1), arachidonic (20:4), and docosahexaenoic (22:6) acids. The changes observed were consistent with those reported to occur following ischemia and the administration of other convulsants. Pretreatment of rats with the anticonvulsant diazepam (4 mg/kg, i.p.) prevented both the signs of soman toxicity and the soman-induced increase of choline and free fatty acids. Diazepam alone did not affect the levels of choline or free fatty acids, cholinesterase activity, or soman-induced cholinesterase inhibition, suggesting that soman toxicity involves a convulsant-mediated increase in phosphatidylcholine catabolism. In addition, administration of the convulsant bicuculline, at a dose that produces seizures and increases the levels of free fatty acids in brain, significantly increased the levels of choline. Results suggest that excitotoxic events enhance the hydrolysis of phosphatidylcholine in brain as evidenced by a concomitant increase in the levels of choline and free fatty acids.     

Protection and induced reactivation of cholinesterase by HS-6 in rabbits exposed to soman

Rabbits intoxicated with soman were treated with various doses of HS-6 at 3 min following administration of soman to establish whether the antidotal efficacy reported for HS-6 against soman can be attributed in part to reactivation of the inhibited cholinesterase (ChE) enzymes. Within 5 min after treating animals intoxicated with soman with 15 or 30 mg/kg of HS-6 (iv) the whole blood ChE activity increased from 6.0 to 30.5 and 44.2% of control activity, respectively. Because HS-6 apparently is able to reactivate completely the unaged inhibited enzyme, HS-6, 60 mg/kg (iv) was used to measure for the first time the $\text{in vivo}$ rate of aging of whole blood ChE in soman-intoxicated rabbits. The half time for aging was determined to be 7.6 (5.8 ? 9.4) min, P = 0.05. HS-6 in combination with atropine and pyridostigmine was tested as a pretreatment against soman. When only atropine + pyridostigmine was used in the pretreatment regimen, none of the rabbits survived a 10 LD50 dose of soman (iv). However, when HS-6 (30 mg/kg, iv) was used together with atropine + pyridostigmine in the pretreatment regimen, 87% of the animals survived this high dose of soman. Since HS-6 is a powerful reactivator of unaged, soman-inhibited ChE, the antidotal effectiveness of HS-6 against soman can be attributed in part to the restoration of vital enzyme activity.     

Soman toxication in hypoxia acclimated rats: Alterations in brain neuronal RNA and survival

Effects of prior hypoxia acclimation (14-day at 380 mm Hg) on soman (pinacolyl methylphosphonofluoridate) induced brain neuronal RNA and acetylcholinesterase (AChE) depletion and lethality were monitored in rats following their return to ambient oxygenation. Quantitative cytochemical techniques were used to measure RNA and AChE changes in individual cerebrocortical (Layer III) and striatal (caudate plus putamen) neurons. In ambient P_{O 2} controls, soman eventuated in a moderate diminution of neuronal RNA in both brain regions and severe, dosedependent suppression of AChE activity. Hypoxia acclimation per se induced RNA alterations as manifested in cortical RNA depletion and increased variability of striatal neuron RNA contents. In hypoxia acclimated rats, the extent of neuronal RNA depletion following soman injection was attenuated in both brain regions, yet there were no discernible differences in saline control AChE levels or in the extent of soman-induced AChE inhibition in ambient control versus hypoxia acclimated treatment groups. Hypoxia acclimated rats, however, were found to be even more susceptible to lethal actions of soman as assessed using 24- and 48-hour survival following a three-point treatment regimen. These data indicate that while compensatory systemic and central metabolic adjustments associated with 14d acclimation to reduced oxygen availability may retard soman-induced neuronal RNA depletion, resistance to lethal or near-lethal soman exposure is not enhanced. It is postulated that hypoxia acclimation is associated with complex adaptive and maladaptive neurophysiological alterations influencing CNS responsiveness to soman toxication, and that detrimental consequences exceed protection afforded by metabolic adaptation.     

Relationship between reversible acetylcholinesterase inhibition and efficacy against soman lethality

Carbamate pretreatment (45% inhibition, reversible), combined with therapy, protected rats from soman-induced lethality [The Pharmacologist 23, 224 (1981)]. The present study was done to see if less than 45% inhibition protects and to see if reversible acetylcholinesterase (AChE) inhibition and efficacy against soman lethality are correlated. At 30 min pre-soman, guinea pigs and rats received (im) either pyridostigmine (Py) or physostigmine (Ph) to inhibit whole blood AChE from 10 to 70%; at 1 min post-soman (sc), they received (im) atropine (16 mg/kg)/2-PAMCl (50 mg/kg) and mecamylamine (0.8 mg/kg)/atropine (16 mg/kg), respectively. Protective ratios (PRs) were computed and they ranged from 3.1 to 7.7 for guinea pigs and from 1.8 to 2.4 for rats. In guinea pigs the PRs for Py + therapy were roughly similar to those of Ph + therapy. In both species at 30 min after im injection of Py and Ph, a linear relationship was found between percentage of whole blood AChE inhibition and ln dosage of carbamate. Positive correlation (p less than 0.05) was found between the degree of reversible AChE inhibition by pretreatment, coupled with therapy, and efficacy against soman lethality. The present data indicate that inhibition levels as low as 10% may provide some protection.     

Evidence that Alterations in γ-Aminobutyric Acid and Acetylcholine in Rat Striata and Cerebella Are Not Related to Soman-Induced Convulsions

Many reports have suggested that gamma-aminobutyric acid (GABA) may play a role in organophosphate-induced convulsions. The balance between GABA and acetylcholine (ACh) in the brain also has been suggested by some investigators to be related to brain excitability. We examined these questions by studying the levels of GABA and ACh and the ratios of GABA to ACh in rat striata and cerebella (two major motor control areas in the CNS) after the administration of soman, an organophosphate acetylcholinesterase inhibitor also known as nerve gas. Male Sprague-Dawley rats weighing 250-300 g were injected subcutaneously with three different doses of soman: a subconvulsive dose of 40 micrograms/kg (approximately 30% of the ED50 for convulsions in rats), a convulsive dose of 120 micrograms/kg (approximately one ED50 for convulsions), and a higher convulsive dose of 150 micrograms/kg (approximately 120% of the ED50 for convulsions). The incidence and severity of convulsions were monitored in individual rats until they were sacrificed by focused microwave irradiation of the head at the following time points after soman administration: 4 min, a time prior to the onset of convulsions; 10 min, the time of onset of convulsions; 1 h, the time of peak convulsive activity; and 6 h, a time at which rats were recovering from convulsions. Results showed that in rat striata and cerebella, neither changes in levels of GABA and ACh nor changes in ratios of GABA to ACh were related to soman-induced convulsions, i.e., none of the changes in either levels or ratios of these two neurotransmitters were related to the initiation of, maintenance of, or recovery from soman-induced convulsions.     

Involvement of glutamatergic system of amygdala in generalized seizures induced by soman: comparison with the hippocampus]

During seizures induced by soman, an organophosphorus compound, irreversible inhibitor of acetylcholinesterase, the intra-amygdaloid microdialysis of extracellular glutamate, an excitatory amino-acid, showed a sustained increase, more rapid than in hippocampus. This result suggests an early involvement of the amygdala in the development of soman-induced seizures. Moreover, the ex vivo, study by quantitative autoradiography of the binding of tritiated TCP (thienyl-phencyclidine) does not reveal an opening of ionic channels linked to N-methyl-D-aspartate (NMDA) sensitive receptors of glutamate, during seizures, unlike in the hippocampus. This difference could indicate, according to other experimental models, that in amygdala the release of glutamate could occur massively without repeated stimuli as in the hippocampus.     

+]-Huperzine A protects against soman toxicity in guinea pigs

The chemical warfare nerve agent (CWNA) soman irreversibly inhibits acetylcholinesterase (AChE) causing seizure, neuropathology and neurobehavioral deficits. Pyridostigmine bromide (PB), the currently approved pretreatment for soman, is a reversible AChE inhibitor that does not cross the blood–brain barrier (BBB) to protect against central nervous system damage. [−]-Huperzine A, a natural reversible AChE inhibitor, rapidly passes through the BBB and has numerous neuroprotective properties that are beneficial for protection against soman. However, [−]-Huperzine A is toxic at higher doses due to potent AChE inhibition which limits the utilization of its neuroprotective properties. [+]-Huperzine A, a synthetic stereoisomer of [−]-Huperzine A and a weak inhibitor of AChE, is non-toxic. In this study, we evaluated the efficacy of [+]-Huperzine A for protection against soman toxicity in guinea pigs. Pretreatments with [+]-Huperzine A, i.m., significantly increased the survival rate in a dose-dependent manner against 1.2× LD₅₀ soman exposures. Behavioral signs of soman toxicity were significantly reduced in 20 and 40 mg/kg [+]-Huperzine A treated animals at 4 and 24 h compared to vehicle and PB controls. Electroencephalogram (EEG) power spectral analysis showed that [+]-Huperzine A significantly reduces soman-induced seizure compared to PB. [+]-Huperzine A (40 mg/kg) preserved higher blood and brain AChE activity compared to PB in soman exposed animals. These data suggest that [+]-Huperzine A protects against soman toxicity stronger than PB and warrant further development as a potent medical countermeasure against CWNA poisoning.     

Long-term effects of human butyrylcholinesterase pretreatment followed by acute soman challenge in cynomolgus monkeys

Human serum butyrylcholinesterase (Hu BChE) was demonstrated previously to be an effective prophylaxis that can protect animals from organophosphate nerve agents. However, in most of those studies, the maximum dose used to challenge animals was low (<2x LD(50)), and the health of these animals was monitored for only up to 2 weeks. In this study, six cynomolgus monkeys received 75mg of Hu BChE followed by sequential doses (1.5, 2.0, 2.0x LD(50)) of soman 10h later for a total challenge of 5.5x LD(50). Four surviving animals that did not show any signs of soman intoxication were transferred to WRAIR for the continuous evaluation of long-term health effects for 14 months. Each month, blood was drawn from these monkeys and analyzed for serum chemistry and hematology parameters, blood acetylcholinesterase (AChE) and BChE levels. Based on the serum chemistry and hematology parameters measured, no toxic effects or any organ malfunctions were observed up to 14 months following Hu BuChE protection against exposure to 5.5x LD(50) of soman. In conclusion, Hu BChE pretreatment not only effectively protects monkeys from soman-induced toxicity of the immediate acute phase but also for a long-term outcome.     

Correlations Between Acetylcholinesterase Activity in Guinea-Pig Iris and Pupillary Function: A Biochemical and Pupillographic Study

: Acetylcholinesterase (AChE) in guinea pig iris was inhibited by methylisocyclopentylfluorophosphate (soman) administered topically or parenterally, and enzyme activity was correlated to pupillary diameter by infrared pupillography. After a single topical soman instillation into the conjunctival sac there was an almost linear relationship between the reduction in AChE activity and pupillary diameter. Topical administration of soman at 24-h intervals in doses capable of almost complete inhibition of AChE in iris was accompanied by a reduced miotic effect of this drug. This was indicated by a reduced rate of the soman-induced pupillary constriction, a less pronounced reduction in pupillary diameter, and a more rapid return of the pupillary diameter to normal size. The change in pupillary diameter occurred after three daily administrations and remained constant during 31 days of treatment. These observations were seen irrespective of inhibition of blood AChE. The decrease in response to repeated administration could not be explained by a reduced inhibitory effect of soman on AChE, by a more rapid de novo synthesis of AChE, or by a change in the number of the muscarinic receptors as determined by quinuclidinyl benzilate binding. When soman or DFP was administered subcutaneously in high doses a severe AChE inhibition was obtained in iris without any concomitant miosis.     

Effects of diazepam on soman-induced brain neuronal RNA depletion and lethality in rats

Studies were conducted to determine effects of the benzodiazepine anticonvulsant diazepam on soman induced brain neuronal RNA depletion and lethality in rats. Quantitative azure B-RNA cytophotometry was used to monitor RNA responses of cerebrocortical (layer V) and striatal neurons following dosages of 0.5, 0.9 and 1.5 LD50 soman (LD50 = 135 micrograms/kg, sc), whereas mean time of death and 24-h survival following 0.8, 1.2 and 1.5 LD50 were used to assess the antidotal efficacy of diazepam (2.2 mg/kg, im) pretreatment. Soman produced dose-dependent RNA depletion in both brain regions. This RNA impairment was almost completely prevented by diazepam, although neuronal RNA contents were generally slightly lower than corresponding control values. However, diazepam pretreatment was not associated with any change in mean time of death or in 24-h survival. The overall data suggest that excessive neural activity per se may underlie the genesis of soman-induced central metabolic impairments, but also appear to effectively dissociate epileptiform activity from lethal actions of soman.     

Effects of inhibitors of acetylcholine synthesis on brain acetylcholine and survival in soman-intoxicated animals

The effects of hemicholinium-3 (HC-3) or 4-(1-naphthylvinyl)pyridine (4-NVP) alone and together with cholinolytics and/or cholinesterase inhibitors on brain acetylcholine (ACh) levels and survival were studied. Intracerebroventricular (ICVT) injection of 10 μg HC-3 280 min before euthanasia by microwave irradiation reduced rat cerebral ACh levels from 28.4 to 5.4 nmoles ACh/g wet tissue. In rats pretreated with HC-3 alone or with other pretreatment drugs prior to giving up to 2.7 LD50 of soman, iv, cerebral ACh levels increased very little, but in animals not receiving HC-3, brain ACh levels increased to 67.1 nmoles. Treatment of unpoisoned rats with 4-NVP resulted in a significant (26%) reduction in ACh. The inclusion of atropine with 4-NVP resulted in a further reduction in ACh. Pretreatment with 4-NVP caused sign-free doses of physostigmine to produce toxic signs in rabbits and did not enhance the efficacy of carbamate pretreatment against soman. Pretreatment of rabbits with pyridostigmine and atropine methyl nitrate (AMN) failed to provide any protection against soman, but when HC-3, ICVT, was included with those drugs, the protective ratio (PR) against soman was increased from 0.8 to 7.3. These data are consistent with the hypothesis that excess ACh is a primary lesion in organophosphorus anticholinesterase intoxication and that the central nervous system is quite sensitive to excesses of ACh.     

Neuroprotection in epilepsy

Brain damage and neuronal loss caused by traumatic brain injury, ischemic stroke, and symptomatic status epilepticus can lead to severe long-term consequences, such as impairment in learning and memory and cognitive functions, and development of chronic epilepsy. This can be the result of morphologic and functional changes underlying temporal lobe epilepsy. Epilepsy patients have increased risk of status epilepticus. It is a life-threatening condition when seizures last for more than 30 min and trigger processes leading to neuronal apoptosis and necrosis in various parts of brain. Administration of neuroprotective drugs preventing these pathologic processes could improve the prognosis for such patients. However despite of active research of neuroprotective drugs, the effective ways to prevent brain damage resulting from prolonged seizures are yet to be found. Studies of neuroprotective properties of classic and novel anticonvulsant drugs showed that most of them do not have the sufficient neuroprotective effect and are not able to prevent epileptogenesis. Thus the studies of other potential neuroprotective drugs seem to be promising.     

Excitatory aminoacids and epileptic seizures in immature brain

Data on convulsant and anticonvulsant action of drugs influencing excitatory amino acid receptors in developing rats are reviewed. Agonists of NMDA type of receptors NMDA and homocysteic acid, elicited an age-related seizure pattern--flexion, emprosthotonic seizures--in the first three postnatal weeks of rats. Generalized clonic-tonic seizures appeared only after a longer latency. Kainic acid administration resulted in epileptic automatisms and later in minimal, clonic seizures followed by generalized tonic-clonic seizures. A decrease of sensitivity to convulsant action with age is a general rule for all agonists tested. Different anticonvulsant action of NMDA and nonNMDA antagonists was demonstrated in a model of generalized tonic-clonic seizures induced by pentetrazol, whereas their action against epileptic afterdischarges elicited by electrical stimulation of cerebral cortex was similar. Again, higher efficacy in younger animals was a rule. As far as metabotropic glutamate receptors are concerned, agonists of groups II and III were shown to protect against convulsant action of homocysteic acid in immature rats and an antagonist of group I receptors MPEP suppressed the tonic phase of generalized tonic-clonic seizures induced by pentetrazol more efficiently in younger than in more mature rat pups. Unfortunately, a higher sensitivity to the action of antagonists of ionotropic glutamate receptors was demonstrated also for unwanted side effects (motor functions were compromized). In contrast, glutamate metabotropic receptor antagonist MPEP did not exhibit any serious side effects in rat pups.     

Soman inhibition of butyrylcholinesterase in the presence of substrate: pressure and temperature perturbations.

Irreversible inhibition of butyrylcholinesterase by soman was studied in the presence of the substrate (o-nitrophenyl butyrate). Inhibition was found of the competitive complexing type. Study at different temperatures and pressures showed that the behavior of the enzyme differs from that of the inhibitor-free enzyme. In the absence of inhibitor, enzyme kinetics displayed a non-linear temperature dependence with a break at 21 degrees C. In the presence of a non-inhibitor structural analog of soman (pinacolyl dimethylphosphinate and methyl dimethylphosphinate), the Arrhenius plot break is slightly shifted (18 degrees C). On the other hand, in the presence of soman this break is abolished. The pressure-dependence of the substrate hydrolysis revealed also differences between the native enzyme and the enzyme in the presence of soman: the sign and magnitude of the apparent activation volume (delta V not equal to) were different for the two reactions. Beyond 300 bar, in the presence of soman, a plateau (delta V not equal to approx. 0) was observed over a large pressure range depending on temperature. Such a behavior with respect to temperature and pressure can reflect a soman-induced enzyme conformational state. Thus, temperature and pressure perturbations of the kinetics allow to complete the inhibition scheme of butyrylcholinesterase by soman. Our data suggest that upon soman binding, the enzyme undergoes a long-lived soman-induced-fit conformational change preceding the phosphonylation step. However, an alternative hypothesis according to which the enzyme processes a secondary soman-binding site cannot be ruled out.     

Brain neuronal RNA metabolism during acute soman toxication: Effects of antidotal pretreatments

Effects of various antidotal treatments on neuronal RNA contents and on soman induced RNA and acetylcholinesterase (AChE) depletion were monitored using quantitative cytochemical techniques. In rats treated only with antidotes, atropine depressed whereas pralidoxime (2-PAM) elevated RNA contents of both caudate and cerebrocortical (Layer V) neurons. Soman produced a virtually complete inhibition of AChE activity and a moderate decline in neuronal RNA contents. Atropine pretreatment partially restored neuronal RNA levels. Atropine+2-PAM prophylaxis eventuated in a complete restoration of RNA levels but no reactivation of AChE. Addition of physostigmine to the atropine +2-PAM treatment regimen resulted in appreciable AChE reactivation but reduced RNA levels. The overall data indicate that: (1) soman-induced neuronal RNA depletion can be completely reversed by antidotal pretreatment; (2) no precise relationship exists between the extents of antidote-induced restoration of RNA and AChE levels; and (3) 2-PAM exerts marked effects on the brain neuronal network which are unrelated to AChE reactivation. It is postulated that effects of soman and antidotes on neuronal RNA metabolism may signify alterations in acetylcholine (ACh) sensitivity and that pharmacologic manipulation of ACh responsiveness during organophosphate cholinesterase poisoning may be a mechanism for additional therapeutic intervention.     

Tenocyclidine treatment in soman-poisoned rats--intriguing results on genotoxicity versus protection

This study aimed to evaluate the antidotal potency of tenocyclidine (TCP) that probably might protect acetylcholinesterase (AChE) in the case of organophosphate poisoning. TCP was tested alone as a pretreatment or in combination with atropine as a therapy in rats poisoned with (1/4) and (1/2) of LD(50) of soman. Possible genotoxic effects of TCP in white blood cells and brain tissue were also studied. Results were compared with previous findings on the adamantyl tenocyclidine derivative TAMORF. TCP given alone as pretreatment, 5 min before soman, seems to be superior in the protection of cholinesterase (ChE) catalytic activity in the plasma than in brain, especially after administration of the lower dose of soman. Plasma activities of the enzyme after a joint treatment with TCP and soman were significantly increased at 30 min (P<0.001) and 24 h (P=0.0043), as compared to soman alone. TCP and atropine, given as therapy, were more effective than TCP administered alone as a pretreatment. The above therapy significantly increased activities of the enzyme at 30 min (P=0.046) and 24 h (P<0.001), as compared to controls treated with (1/4) LD(50) of soman alone. Using the alkaline comet assay, acceptable genotoxicity of TCP was observed. However, the controversial role of TCP in brain protection of soman-poisoned rats should be studied further.