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.     

Brain neuronal RNA metabolism during sustained low-level soman toxication

Quantitative azure B-RNA cytophotometry was used to monitor metabolic responses of cholinergic elements of the rat brain during sustained low-level administration of soman (0.25-0.50 LD50, sc). RNA contents of caudate and cerebrocortical (Layers III and V) neurons were measured 60 min following 1-5 soman dosages given at 24 h intervals. Marked and progressive RNA depletion was evidenced after 1-4 soman injections, whereas partial or complete restitution of RNA levels was observed following the fifth injection. These data indicate that repetitive soman toxication is associated with metabolic correlates of impaired rather than accentuated activation of CNS cholinergic systems, and that tolerance is developed to CNS actions of the agent. It is postulated that impaired neuronal activation is related to soman or ACh-induced transmission block, and that the same adaptive processes responsible for recovery during acute poisoning may underlie the development of tolerance during repetitive administration of organophosphates.     

Soman intoxication in hypothyroid rats: alterations in brain neuronal RNA,acetylcholinesterase and survival

Studies were conducted to investigate relationships among soman (pinacolyl methylphosphonofluoridate) induced seizure activity, central metabolic impairments and lethality in normal vs thyroid-deficient rats. Quantitative cytophotometric measurements of individual cerebrocortical (layer V) and striatal neuron RNA contents were made following dosages of 0.5, 0.9 and 1.5 LD₅₀ soman (LD₅₀ = 135 μg/kg, sc). Hypothyroidism was associated with a marked diminution of overt convulsive activity and reduced susceptibility to lethal actions of soman as indicated by enhanced 24- and 48-h survival rates at 0.9, 1.2 and 1.5 LD₅₀. Hypothyroidism per se produced RNA depletion in both cortical and striatal neurons. Soman treatment diminished cortical RNA to essentially the same extent in thyroid-deficient rats as in euthyroids, whereas there was no further reduction of striatal neuron RNA. It was found that amelioration of convulsive activity and lethal- ity in hypothyroid rats was accompanied by reduced cerebral acetylcholinesterase (AChE, EC 3.1.1.7) inactivation, and that plasma cholinesterase (EC 3.1.1.8) and aliesterase (EC 3.1.1.1) levels were significantly higher in hypothyroid than in euthyroid saline-control rats. The overall data indicate that soman- induced central metabolic impairments can occur independent of paroxysmal neural activity and lethal actions of the agent. Resistance to soman observed with thyroid deficiency may be due in large part to increased binding to plasma enzymes and diminished delivery of soman to AChE in vital cholinergic sites.     

Effects of subchronic pyridostigmine pretreatment on the toxicity of soman

The effect of subchronic pyridostigmine pretreatment on the toxicity of soman, in the absence of supporting therapy (atropine, oxime, and (or) anticonvulsant), as well as its effect on muscarinic cholinoceptor binding characteristics was assessed in the rat. Pretreatment with pyridostigmine by means of an implanted Alzet osmotic minipump for a 5-day total exposure dose of 12 mg/kg inhibited whole blood acetylcholinesterase activity by 73%. This pyridostigmine pretreatment lowered the soman LD50 from 104 micrograms/kg in control animals to 82 micrograms/kg. In addition, the time to onset of soman-induced convulsions in pyridostigmine pretreated animals was significantly (p less than 0.001) reduced. Pyridostigmine pretreatment produced no significant effect on muscarinic cholinoceptor binding in brain or ileum. Lower doses of pyridostigmine pretreatment inhibited acetylcholinesterase activity (65 and 25%); however, LD50 and time to onset of convulsions following soman (140 micrograms/kg) were not significantly different from controls.     

Perikaryal RNA changes within neurons of the caudate-putamen and substantia nigra in soman intoxicated rats

Quantitative azure B cytophotometry was employed to monitor neuronal ribonucleic acid (RNA) metabolism within cholinergic and noncholinergic brain compartments following single sc injections of either 0.5, 0.9 or 1.5 LD₅₀ soman (pinacolyl methylphosphonofluoridate). Dose-dependent losses in neuronal RNA were observed within the cholinergic caudate-putamen (CP) and dopaminergic substantia nigra pars compacta (SNPC), whereas levels of RNA were generally maintained or elevated within the gabaergic substantia nigra pars reticulata (SNPR). CP acetylcholinesterase was inhibited in a dose-dependent fashion. These neuronal RNA changes are perhaps related to seizure activity. The overall data lend support to the hypothesis that alterations in noncholinergic activity contribute to certain manifestations of soman neurotoxicity, such as seizures, which probably stem from an impairment in the functional integrity of both excitatory and inhibitory neuronal elements.     

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.     

Cytophotometric analyses of thalamic neuronal RNA in soman intoxicated rats

Quantitative azure B-RNA cytophotometry was used to monitor metabolic responses of individual neurons within the ventrobasal nuclear complex (VBC) and nucleus reticularis (NR) of the rat thalamus following administration of soman (0.5, 0.9 or 1.5 LD₅₀, sc). A dose-dependent depression in brain acetylcholinesterase (AChE) was evidenced. With respect to thalamic RNA responses, a complex pattern of RNA alterations was evidenced, with these two regions generally exhibiting opposite patterns of dose-related RNA changes. With sub-lethal dosages of soman, RNA accumulation was evidenced in the acetylcholine (ACh) mediated excitatory VBC region and RNA depletion in the ACh mediated inhibitory NR neurons. With a lethal dose, an opposite RNA response pattern observed in both thalamic regions. It is postulated that the observed RNA response pattern with sub-lethal dosages of soman is what one would anticipate with cholinergic brainstem reticular formation activation. The absence of such a response with lethal doses strongly suggests some disruption of functional excitatory cholinergic activity and perhaps also an impairment of inhibitory cholinergic synaptic activity.     

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.     

Ketamine pretreatment exacerbated 3,4-methylenedioxymethamphetamine-induced central dopamine toxicity

Currently, joint use of ketamine and 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) represents a specific combination of polydrug abuse. Long-lasting and even aggravated central neuronal toxicity associated with mixing ketamine and MDMA use is of special concern. This study was undertaken to examine the modulating effects of ketamine treatment on later MDMA-induced dopamine and serotonin neurotoxicity. We found that repeated administration of ketamine (50 mg/kg x 7) at 1.5-h intervals did not render observable dopamine or serotonin depletion in catecholaminergic target regions examined. In contrast, three consecutive doses of MDMA (20 mg/kg each) at 2-h intervals produced long-lasting dopamine and serotonin depletions in striatum, nucleus accumbens and prefrontal cortex. More importantly, pretreatment with binge doses of ketamine (50 mg/kg x 7 at 1.5-h intervals) 12 h prior to the MDMA dosing regimen (20 mg/kg x 3 at 2-h intervals) aggravated the MDMA-induced dopaminergic toxicity. Nonetheless, such binge doses of ketamine treatment did not affect MDMA-induced serotonergic toxicity. These results, taken together, indicate that binge use of ketamine specifically enhances the MDMA-induced central dopaminergic neurotoxicity in adult mouse brain.     

Viral neuroinvasion as a marker for BBB integrity following exposure to cholinesterase inhibitors

Exposure to the nerve agent soman, an irreversible cholinesterase (ChE) inhibitor, results in changes in blood-brain barrier permeability attributed to its seizure-induced activity. However, smaller BBB changes may be independent of convulsions. Such minor injury may escape detection. A nonneuroinvasive neurovirulent Sindbis virus strain (SVN) was used as a marker for BBB permeability. Peripheral inoculation of mice with 2 x 10(3) plaque forming units (PFU) caused up to 10(5) PFU/ml viremia after 24 hours with no signs of central nervous system (CNS) infection and with no virus detected in brain tissue. Intra-cerebral injection of as low as 1-5 PFU of the same virus caused CNS infection, exhibited 5-7 days later as hind limb paralysis and death. Soman (0.1-0.7 of the LD50) was administered at peak viremia (1 day following peripheral inoculation). Sublethal soman exposure at as low as 0.1 LD50 resulted in CNS infection 6-8 days following inoculation in 30-40% of the mice. High virus titer were recorded in brain tissue of sick mice while no virus was detected in healthy mice subjected to the same treatment. No changes in the level of viremia or changes in viral traits were observed in the infected mice. The reversible anticholinesterases physostigmine (0.2 mg/kg, s.c.) and pyridostigmine (0.4 mg/kg, i.m.) injected at a dose equal to 0.1 LD50, induced similar results. Thus, both central and peripheral anticholinesterases (anti-ChEs) induce changes in BBB permeability sufficient to allow, at least in some of the mice, the invasion of this otherwise noninvasive but highly neurovirulent virus. This BBB change is probably due to the presence of cholinesterases in the capillary wall. SVN brain invasion served here as a highly sensitive and reliable marker for BBB integrity.     

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.     

Changes of acetylcholinesterase activity in different rat brain areas following intoxication with nerve agents: biochemical and histochemical study

Acetylcholinesterase activity in defined brain regions was determined using biochemical and histochemical methods 30 min after treating rats with sarin, soman or VX (0.5 x LD(50)). Enzyme inhibition was high in the pontomedullar area and frontal cortex, but was low in the basal ganglia. Histochemical and biochemical results correlated well. Determination of the activity in defined brain structures was a more sensitive parameter than determination in whole brain homogenate where the activity was a "mean" of the activities in different structures. The pontomedullar area controls respiration, so that the special sensitivity of acetylcholinesterase to inhibition by nerve agents in this area is important for understanding the mechanism of death caused by nerve agents. Thus, acetylcholinesterase activity is the main parameter investigated in studies searching for target sites following nerve agent poisoning.     

Effect of soman (pinacolyl methylphosphonofluoridate) on the blood levels of corticosterone and adrenocorticotropin in mice

Mice were poisoned by an extremely toxic organophosphate anticholinesterase soman (pinacolyl methylphosphonofluoridate), 50 or 100 micrograms/kg at 1000, and the serum concentrations of corticosterone were determined fluorometrically at 3-h intervals for at least 24 h. The lower soman dose (50 micrograms/kg) produced a modest increase in serum corticosterone concentrations but by 24 h the levels were not significantly different from control. Following the higher soman dose (100 micrograms/kg) the serum corticosterone levels were elevated significantly (p less than 0.05), for at least 27 h. However, ACTH concentrations were not elevated. It is possible that the elevated levels of corticosterone were due to a reduced metabolism and excretion of corticosterone resulting from the intense hypothermia, following soman poisoning which may change cardiac output and organ (liver and kidney) perfusion and not due to an enhanced release from the adrenal gland.     

Cytophotometric analyses of mesencephalic reticular formation neuronal RNA in soman intoxicated rats

Quantitative azure B cytophotometry was used to monitor ribonucleic acid (RNA) responses of individual neurons within the nucleus cuneiformis (NC) and ventrotegmental nucleus (VTN) of the rat mesencephalic reticular formation following single subcutaneous soman (pinacolyl methylphosphonofluoridate) injections (0.5, 0.9 or 1.5 LD₅₀). The sub-lethal (0.5 LD₅₀) dosage of soman produced RNA accumulation in NC neurons, but VTN-RNA levels were not significantly altered. In contrast, both reticular nuclei exhibited prominent RNA depletion with higher soman dosages, the severity of which was greater with lethal (1.5 LD₅₀) than near-lethal (0.9 LD₅₀) dosages. These data indicate that metabolic correlates of enhanced activation of cholinergic reticular nuclei are present only with sub-lethal dosages, and that higher dosages produce characteristics of impaired activation of ascending cholinergic pathways. At present, mechanisms underlying soman-induced metabolic and neurologic deficits remain speculative.     

An attempt to assess functionally minimal acetylcholinesterase activity necessary for survival of rats intoxicated with nerve agents

Acetylcholinesterase (AChE, EC 3.1.1.7) is an important enzyme for cholinergic nerve transmission. The action of toxic organophosphates such as nerve agents is based on AChE inhibition. The death following acute nerve agent poisoning is due to central or peripheral respiratory/cardiac failure. Therefore, the changes in AChE activity following nerve agents acting predominantly on the central (sarin, soman) or peripheral (VX) level were studied. It is known that AChE activity in different structures exists in relative excess. Female Wistar rats intoxicated with sarin, soman, and VX in different doses (0.5-2.0xLD(50)) were divided into groups of survived and died animals. AChE activities in diaphragm, brain parts (pontomedullar area, frontal cortex, basal ganglia, in some cases other parts of the brain) were determined and the rest of activity (in %) was correlated with survival/death of animals. More precise elucidation of action of nerve agents and the assessment of minimal AChE activity in different organs compatible with the survival of organism poisoned with nerve agents were the aims of this study.     

Involvement of caspase-3-like protease in the mechanism of cell death following focally evoked limbic seizures

The cysteine protease caspase-3 may be involved in the mechanism of cell death following seizures. Using a rat model of focally evoked limbic epilepsy with continuous electroencephalography monitoring, we investigated seizure-induced changes in caspase-3 protein expression and processing, enzyme activity, and the in vivo effect of caspase-3 inhibition. Seizures were induced by intraamygdaloid injection of kainic acid (0.1 microg) and were terminated after 45 min by diazepam (30 mg/kg) administration. Animals were killed 0-72 h following diazepam administration. Levels of the 32-kDa proenzyme form of caspase-3 were unaffected by seizures. Levels of the 17-kDa cleaved (active) fragment of caspase-3 were almost undetectable in control brain, but were increased significantly at 4 and 24 h within ipsilateral hippocampus and cortex in seizure animals. Caspase-3-like protease activity was increased within the ipsilateral hippocampus at 8 and 24 h following seizures. Caspase-3 immunoreactivity was increased within the vulnerable ipsilateral CA3/CA4 subfield at 24 and 72 h following seizures and was associated predominantly, but not exclusively, with neurons exhibiting DNA fragmentation. The putatively selective caspase-3 inhibitor N-benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-fluoromethyl ketone significantly improved neuronal survival bilaterally within the hippocampal CA3/CA4 subfields following seizures. Collectively, these data suggest that caspase-3 may play a significant role in the mechanism by which neurons die following seizures.     

+]-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.     

Immuno-detection of aluminium and aluminium induced conformational changes in calmodulin - implications in Alzheimer's disease

Studies were conducted to ascertain any involvement of free radical mediated prooxidative processes in different brain regions following diazopam administration. A significant decrease in TBA reactive substance formation was observed in cerebral cortex, cerebellum and brain stem regions after single doses of 1.5, 3 and 6 mg/kg b.wt. For further studies rats were given diazepam (i.p.) at 3 mg/kg body weight dose and sacrificed after 1 h to follow changes in the pro/antioxidant status. An enhancement in the TBARS formation was found in the mitochondrial fractions from cerebral cortex and brain stem. This effect was highest in brain stem being 107% as compared to controls. In the post mitochondrial fraction, cerebellum showed 49% enhancement whereas decreased formation of thiobarbituric acid reactive substances was observed in cerebral cortex and brain stem. Isozymes of superoxide dismutase showed a decrease in activity which was region dependent. Even though, total thiols were not significantly altered, free thiols showed depletion in cerebellum (39.8%) and brain stem (50%). Glutathione reductase activity was also decreased in cerebellum and brain stem. The results indicate that a single dose of diazepam causes free radical mediated changes and the modulatory response of antioxidant defences appears to be region specific.