Sarin-Induced Neuroinflammation in Mouse Brain Is Attenuated by the Caspase Inhibitor Q-VD-OPh.

Organophosphates cause hyperstimulation of the central nervous system, leading to extended seizures, convulsions, and brain damage. Sarin is a highly toxic organophosphate nerve agent that has been employed in several terrorist attacks. The prolonged toxicity of sarin may be enhanced by the neuroinflammatory response initiated by the inflammasome, caspase involvement, and generation/release of proinflammatory cytokines. Since neurodegeneration and neuroinflammation are prevalent in sarin-exposed animals, we were interested in evaluating the capacity of quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methyl ketone (Q-VD-OPh), a pan caspase inhibitor to attenuate neuroinflammation following sarin exposure. To test this hypothesis, sarin-exposed C57BL/6 mice were treated with Q-VD-OPh or negative control quinolyl-valyl-O-methylglutamyl-[-2,6-difluorophenoxy]-methyl ketone, sacrificed at 2- and 14-day time points, followed by removal of the amygdala and hippocampus. A Bio-Rad 23-Plex cytokine analysis was completed on each tissue. The results suggest that exposure to sarin induced a dramatic increase in interleukin-1ß and 6 other cytokines and a decrease in 2 of the 23 cytokines at 2 days in the amygdala compared with controls. Q-VD-OPh attenuated these changes at the 2-day time point. At 14 days, six of these cytokines were still significantly different from controls. Hippocampus was less affected at both time points. Diazepam, a neuroprotective drug against nerve agents, caused an increase in several cytokines but did not have a synergistic effect with Q-VD-OPh. Treatment of sarin exposure with apoptosis inhibitors appears to be a worthwhile approach for further testing as a comprehensive counteragent against organophosphate exposure. SIGNIFICANCE STATEMENT: A pan inhibitor of caspases (Q-VD-OPh) was proposed as a potential antidote for sarin-induced neuroinflammation by reducing the level of inflammation via inflammasome caspase inhibition. Q-VD-OPh added at 30 minutes post-sarin exposure attenuated the inflammatory response of a number of cytokines and chemokines in the amygdala and hippocampus, two brain regions sensitive to organophosphate exposure. Apoptotic marker reduction at 2 and 14 days further supports further testing of inhibitors of apoptosis as a means to lessen extended organophosphate toxicity in the brain.

Efficacy of a combined anti-seizure treatment against cholinergic established status epilepticus following a sarin nerve agent insult in rats.

The development of refractory status epilepticus (SE) following sarin intoxication presents a therapeutic challenge. Here, we evaluated the efficacy of delayed combined double or triple treatment in reducing abnormal epileptiform seizure activity (ESA) and the ensuing long-term neuronal insult. SE was induced in rats by exposure to 1.2 LD50 sarin followed by treatment with atropine and TMB4 (TA) 1 min later. Double treatment with ketamine and midazolam or triple treatment with ketamine, midazolam and levetiracetam was administered 30 min post-exposure, and the results were compared to those of single treatment with midazolam alone or triple treatment with ketamine, midazolam, and valproate, which was previously shown to ameliorate this neurological insult. Toxicity and electrocorticogram activity were monitored during the first week, and behavioral evaluations were performed 2 weeks post-exposure, followed by biochemical and immunohistopathological analyses. Both double and triple treatment reduced mortality and enhanced weight recovery compared to TA-only treatment. Triple treatment and, to a lesser extent, double treatment significantly ameliorated the ESA duration. Compared to the TA-only or the TA+ midazolam treatment, both double and triple treatment reduced the sarin-induced increase in the neuroinflammatory marker PGE2 and the brain damage marker TSPO and decreased gliosis, astrocytosis and neuronal damage. Finally, both double and triple treatment prevented a change in behavior, as measured in the open field test. No significant difference was observed between the efficacies of the two triple treatments, and both triple combinations completely prevented brain injury (no differences from the naïve rats). Delayed double and, to a greater extent, triple treatment may serve as an efficacious delayed therapy, preventing brain insult propagation following sarin-induced refractory SE.

The estimation of acute oral toxicity (LD50) of G-series organophosphorus-based chemical warfare agents using quantitative and qualitative toxicology in silico methods.

The idea of this study was the estimation of the theoretical acute toxicity (t-LD50, rat, oral dose) of organophosphorus-based chemical warfare agents from the G-series (n = 12) using different in silico methods. Initially identified in Germany, the G-type nerve agents include potent compounds such as tabun, sarin, and soman. Despite their historical significance, there is a noticeable gap in acute toxicity data for these agents. This study employs qualitative (STopTox and AdmetSAR) and quantitative (TEST; CATMoS; ProTox-II and QSAR Toolbox) in silico methods to predict LD50 values, offering an ethical alternative to animal testing. Additionally, we conducted quantitative extrapolation from animals, and the results of qualitative tests confirmed the acute toxicity potential of these substances and enabled the identification of toxicophoric groups. According to our estimations, the most lethal agents within this category were GV, soman (GD), sarin (GB), thiosarin (GBS), and chlorosarin (GC), with t-LD50 values (oral administration, extrapolated from rat to human) of 0.05 mg/kg bw, 0.08 mg/kg bw, 0.12 mg/kg bw, 0.15 mg/kg bw, and 0.17 mg/kg bw, respectively. On the contrary, compounds with a cycloalkane attached to the phospho-oxygen linkage, specifically methyl cyclosarin and cyclosarin, were found to be the least toxic, with values of 2.28 mg/kg bw and 3.03 mg/kg bw. The findings aim to fill the knowledge gap regarding the acute toxicity of these agents, highlighting the need for modern toxicological methods that align with ethical considerations, next-generation risk assessment (NGRA) and the 3Rs (replacement, reduction and refinement) principles.

Brominated oxime nucleophiles are efficiently reactivating cholinesterases inhibited by nerve agents.

Six novel brominated bis-pyridinium oximes were designed and synthesized to increase their nucleophilicity and reactivation ability of phosphorylated acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Their pKa was valuably found lower to parent non-halogenated oximes. Stability tests showed that novel brominated oximes were stable in water, but the stability of di-brominated oximes was decreased in buffer solution and their degradation products were prepared and characterized. The reactivation screening of brominated oximes was tested on AChE and BChE inhibited by organophosphorus surrogates. Two mono-brominated oximes reactivated AChE comparably to non-halogenated analogues, which was further confirmed by reactivation kinetics. The acute toxicity of two selected brominated oximes was similar to commercially available oxime reactivators and the most promising brominated oxime was tested in vivo on sarin- and VX-poisoned rats. This brominated oxime showed interesting CNS distribution and significant reactivation effectiveness in blood. The same oxime resulted with the best protective index for VX-poisoned rats.

[Discussion on the relationship between pathological changes of sciatic nerve and Sarm1 protein expression in rats with n-hexane poisoning].

Objective: To explore the potential evidence of active peripheral nerve necrosis when n-hexane produces toxic effects on peripheral nerves. Methods: In May 2023, 36 SPF grade SD male rats with a body weight of 200-220 g were divided into 4 groups with 9 rats in each group and given normal saline and different doses of n-hexane (168, 675, 2 700 mg/kg) by gavage for 6 consecutive weeks (5 days/week). Three rats in each group were killed at the 2nd, 4th and 6th week, respectively. The spinal cord to sciatic nerve tissue was broken and the supernatant was extracted for SDS-PAGE protein isolation. The expression level of Sarm1 protein was analyzed with the ß-Actin color strip of internal reference protein by Western blot. The expression of Sarm1 protein was analyzed by the gray ratio of the two. At the 6th week, the sciatic nerve sections of the each group were observed by light microscope and electron microscope. Results: The number of axons was obviously reduced by light microscopy. According to electron microscope, myelin lesions were mainly local disintegration, deformation, and different thickness. The deformation of axonal surface became smaller. The axons in the nerve bundle membrane showed degeneration and reduction. The gray ratio of Sarm1 protein and internal reference protein bands in each group had no significant change at the second week of exposure, and the ratio of SARM1 protein to internal reference protein bands was 1.47 in the high dose group at the fourth week, and 1.51 and 1.89 in the middle and high dose group at the sixth week, respectively. Conclusion: Waller's degeneration was observed in sciatic neuropathologic manifestations of n-hexane-poisoned rats, and the expression level of Sarm1 protein increased.

Loading and Sustained Release of Pralidoxime Chloride from Swellable MIL-88B(Fe) and Its Therapeutic Performance on Mice Poisoned by Neurotoxic Agents.

Maintaining a long-term continuous and stable reactivator blood concentration to treat organophosphorus nerve agent poisoning using acetylcholinesterase (AChE) reactivator pralidoxime chloride (2-PAM) is very important yet difficult. Because the flexible framework of MIL-88B(Fe) nanoparticles (NPs) can swell in polar solvents, pralidoxime chloride (2-PAM) was loaded in MIL-88B(Fe) NPs (size: ca. 500 nm) by stirring and incubation in deionized water to obtain 2-PAM@MIL-88B(Fe), which had a maximum drug loading capacity of 12.6 wt %. The as-prepared composite was characterized by IR, powder X-ray diffraction (P-XRD), scanning electron microscopy (SEM), ζ-potential, Brunauer-Emmett-Teller (BET), and thermogravimetry/differential thermal analysis (TG/DTA). The results showed that under constant conditions, the maximum drug release rates of 2-PAM@MIL-88B(Fe) in absolute ethanol, phosphate-buffered saline (PBS) solution (pH = 7.4), and PBS solution (pH = 4) at 150 h were 51.7, 80.6, and 67.1%, respectively. This was because the composite showed different swelling behaviors in different solvents. In PBS solution with pH = 2, the 2-PAM@MIL-88B(Fe) framework collapsed after 53 h and released 100% of 2-PAM. For mice after intragastric poisoning with sarin (a neurotoxic agent), an atropine-assisted 2-PAM@MIL-88B(Fe) treatment experiment revealed that 2-PAM@MIL-88B(Fe) continuously released 2-PAM for more than 72 h so that poisoned AChE was continuously and steadily reactivated. The reactivation rate of AChE was 56.7% after 72 h. This composite is expected to provide a prolonged, stable therapeutic drug for the mid- and late-stage treatment of neurotoxic agent poisoning.

Discovery of Novel Non-Oxime Reactivators Showing In Vivo Antidotal Efficiency for Sarin Poisoned Mice.

A family of novel efficient non-oxime compounds exhibited promising reactivation efficacy for VX and sarin inhibited human acetylcholinesterase was discovered. It was found that aromatic groups coupled to Mannich phenols and the introduction of imidazole to the ortho position of phenols would dramatically enhance reactivation efficiency. Moreover, the in vivo experiment was conducted, and the results demonstrated that Mannich phenol L10R1 (30 mg/kg, ip) could afford 100% 48 h survival for mice of 2*LD50 sarin exposure, which is promising for the development of non-oxime reactivators with central efficiency.

Substrate Analogues for the Enzyme-Catalyzed Detoxification of the Organophosphate Nerve Agents-Sarin, Soman, and Cyclosarin.

The G-type nerve agents, sarin (GB), soman (GD), and cyclosarin (GF), are among the most toxic compounds known. Much progress has been made in evolving the enzyme phosphotriesterase (PTE) from Pseudomonas diminuta for the decontamination of the G-agents; however, the extreme toxicity of the G-agents makes the use of substrate analogues necessary. Typical analogues utilize a chromogenic leaving group to facilitate high-throughput screening, and substitution of an O-methyl for the P-methyl group found in the G-agents, in an effort to reduce toxicity. Till date, there has been no systematic evaluation of the effects of these substitutions on catalytic activity, and the presumed reduction in toxicity has not been tested. A series of 21 G-agent analogues, including all combinations of O-methyl, p-nitrophenyl, and thiophosphate substitutions, have been synthesized and evaluated for their ability to unveil the stereoselectivity and catalytic activity of PTE variants against the authentic G-type nerve agents. The potential toxicity of these analogues was evaluated by measuring the rate of inactivation of acetylcholinesterase (AChE). All of the substitutions reduced inactivation of AChE by more than 100-fold, with the most effective being the thiophosphate analogues, which reduced the rate of inactivation by about 4-5 orders of magnitude. The analogues were found to reliably predict changes in catalytic activity and stereoselectivity of the PTE variants and led to the identification of the BHR-30 variant, which has no apparent stereoselectivity against GD and a kcat/Km of 1.4 × 106, making it the most efficient enzyme for GD decontamination reported till date.

Effect of Oxime Encapsulation on Acetylcholinesterase Reactivation: Pharmacokinetic Study of the Asoxime-Cucurbit[7]uril Complex in Mice Using Hydrophilic Interaction Liquid Chromatography-Mass Spectrometry.

Oxime-based molecules are used for the treatment of patients to reactivate acetylcholinesterase (AChE) function after organophosphate intoxication. However, their efficacy is limited by low penetration through the blood-brain barrier and fast elimination. In this work, the cucurbit[7]uril (CB[7]) carrier was used for the encapsulation of the clinical agent asoxime to enhance brain bioavailability and the treatment window. We present a pharmacokinetic study of asoxime and the asoxime-CB[7] complex in an in vivo mouse model. Ultrahigh-performance liquid chromatography with electrospray ionization-mass spectrometry detection was developed to determine asoxime and CB[7] in biological fluids and tissues after thorough optimization of chromatographic conditions. The dihydroxypropane-silica stationary phase using hydrophilic interaction liquid chromatography conditions provided the best chromatographic performance. The final method was validated and applied for the pharmacokinetic study of mouse plasma, urine, bile, liver, kidney, and brain samples at different times after administration of asoxime and the asoxime-CB[7] complex. The results showed a greater than 3-fold increase in the area under the curve (AUC) in the brain for asoxime administered as a complex with CB[7] relative to that for the administration of asoxime alone. The effectiveness of the treatment strategy was evaluated using a reactivation study and a functional observatory battery. Protection of brain AChE activity is crucial for saving human lives or reducing the consequences of poisoning. The asoxime administered as a complex increased the brain activity by approximately 30% compared to that with atropine alone. CB[7] coadministration improved the AChE activity by 11%, which agrees with the higher asoxime AUC assessed in the pharmacokinetic study.

Battling Chemical Weapons with Zirconium Hydroxide Nanoparticle Sorbent: Impact of Environmental Contaminants on Sarin Sequestration and Decomposition.

The promising reactive sorbent zirconium hydroxide (ZH) was challenged with common environmental contaminants (CO2, SO2, and NO2) to determine the impact on chemical warfare agent decomposition. Several environmental adsorbates rapidly formed on the ZH surface through available hydroxyl species and coordinatively unsaturated zirconium sites. ZH decontamination effectiveness was determined using a suite of instrumentation including in situ diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS) to monitor sarin (GB) decomposition in real time and at ambient pressure. Surface products were characterized by ex situ X-ray photoelectron spectroscopy (XPS). The adsorption enthalpies, entropies, and bond lengths for environmental contaminants and GB decomposition products were estimated using density functional theory (DFT). Consistent with the XPS and DRIFTS results, DFT simulations predicted the relative stabilities of molecular adsorbates and reaction products in the following order: CO2 < NO2 < GB ≈ SO2. Microbreakthrough capacity measurements on ZH showed a 7-fold increase in the sorption of NO2 vs SO2, which indicates differences in the surface reactivity of these species. GB decomposition was rapid on clean and CO2-dosed ZH and showed reduced decomposition on SO2- and NO2-predosed samples. Despite these findings, the total GB sorption capacity of clean and predosed ZH was consistent across all samples. These data provide insight into the real-world use of ZH as a reactive sorbent for chemical decontamination applications.

Effects of novel brain-penetrating oxime acetylcholinesterase reactivators on sarin surrogate-induced changes in rat brain gene expression.

Past assassinations and terrorist attacks demonstrate the need for a more effective antidote against nerve agents and other organophosphates (OP) that cause brain damage through inhibition of acetylcholinesterase (AChE). Our lab has invented a platform of phenoxyalkyl pyridinium oximes (US patent 9,277,937) that demonstrate the ability to cross the blood-brain barrier in in vivo rat tests with a sarin surrogate nitrophenyl isopropyl methylphosphonate (NIMP) and provide evidence of brain penetration by reducing cessation time of seizure-like behaviors, accumulation of glial fibrillary acidic protein (GFAP), and hippocampal neuropathology, as opposed to the currently approved oxime, 2-pyridine aldoxime methyl chloride (2-PAM). Using two of the novel oximes (Oximes 1 and 20), this project examined whether gene expression changes might help explain this protection. Expression changes in the piriform cortex were examined using polymerase chain reaction arrays for inflammatory cytokines and receptors. The hippocampus was examined via quantitative polymerase chain reaction for the expression of immediate-early genes involved in brain repair (Bdnf), increasing neurotoxicity (Fos), and apoptosis control (Jdp2, Bcl2l1, Bcl2l11). In the piriform cortex, NIMP significantly stimulated expression for the macrophage inflammatory proteins CCL4, IL-1A, and IL-1B. Oxime 20 by itself elicited the most changes. When it was given therapeutically post-NIMP, the largest change occurred: a 310-fold repression of the inflammatory cytokine, CCL12. In the hippocampus, NIMP increased the expression of the neurotoxicity marker Fos and decreased the expression of neuroprotective Bdnf and antiapoptotic Bcl2l1. Compared with 2-PAM, Oxime 20 stimulated Bcl2l1 expression more and returned expression closer to the vehicle control values.

Novel centrally active oxime reactivators of acetylcholinesterase inhibited by surrogates of sarin and VX.

A novel oxime platform, the substituted phenoxyalkyl pyridinium oximes (US patent 9,227,937), was invented at Mississippi State University with an objective of discovering a brain-penetrating antidote to highly potent organophosphate anticholinesterases, such as the nerve agents. The goal was reactivation of inhibited brain acetylcholinesterase to attenuate the organophosphate-induced hypercholinergic activity that results in glutamate-mediated excitotoxicity and neuropathology. The currently approved oxime antidote in the US, 2-PAM, cannot do this. Using highly relevant surrogates of sarin and VX that leave acetylcholinesterase phosphylated with the same chemical moiety as their respective nerve agents, in vitro screens and in vivo tests in rats were conducted to identify the most efficacious members of this platform. The most promising novel oximes provided 24-h survival of lethal level surrogate exposure better than 2-PAM in almost all cases, and two of the oximes shortened the time to cessation of seizure-like behavior while 2-PAM did not. The most promising novel oximes attenuated neuropathology as indicated by immunohistochemical stains for both glia and neurons, while 2-PAM did not protect either glia or neurons. These results strongly suggest that these novel oximes can function within the brain to protect it, and therefore show great promise as potential future nerve agent antidotes.

Efficacy of retigabine in ameliorating the brain insult following sarin exposure in the rat.

BACKGROUND: Sarin is an irreversible organophosphate cholinesterase inhibitor. Following toxic signs, an extensive long-term brain damage is often reported. Thus, we evaluated the efficacy of a novel anticonvulsant drug retigabine, a modulator of neuronal voltage gated K+ channels, as a neuroprotective agent following sarin exposure. METHODS: Rats were exposed to 1 LD50 or 1.2 LD50 sarin and treated at onset of convulsions with retigabine (5 mg/kg, i.p.) alone or in combination with 5 mg/kg atropine and 7.5 mg/kg TMB-4 (TA) respectively. Brain biochemical and immunohistopathological analyses were processed 24 h and 1 week following 1 LD50 sarin exposure and at 4 weeks following exposure to 1.2 LD50 sarin. EEG activity in freely moving rats was also monitored by telemetry during the first week following exposure to 1.2 LD50 and behavior in the Open Field was evaluated 3 weeks post exposure. RESULTS: Treatment with retigabine following 1 LD50 sarin exposure or in combination with TA following 1.2 LD50 exposure significantly reduced mortality rate compared to the non-treated groups. In both experiments, the retigabine treatment significantly reduced gliosis, astrocytosis and brain damage as measured by translocator protein (TSPO). Following sarin exposure the combined treatment (retigabine+ TA) significantly minimized epileptiform seizure activity. Finally, in the Open Field behavioral test the non-treated sarin group showed an increased mobility which was reversed by the combined treatment. CONCLUSIONS: The M current modulator retigabine has been shown to be an effective adjunct therapy following OP induced convulsion, minimizing epileptiform seizure activity and attenuating the ensuing brain damage.

Influence of experimental end point on the therapeutic efficacy of the antinicotinic compounds MB408, MB442 and MB444 in treating nerve agent poisoned mice - a comparison with oxime-based treatment.

Therapeutic efficacy of antidotal treatment of acute poisoning by nerve agents is generally assessed by the evaluation of LD50 values of nerve agents over 24 h following poisoning without or with a single administration of antidotal treatment. In this study, LD50 values of four nerve agents (sarin, soman, tabun and cyclosarin) for non-treated and treated poisoning were evaluated in mice for two experimental end points - 6 h and 24 h. While the efficacy of atropine or oxime-based antidotal treatment was the same regardless of the experimental end point, the therapeutic efficacy of all three newly developed bispyridinium non-oxime compounds (MB408, MB442, and MB444) was mostly slightly higher at the 6 h end point compared to the 24 h end point, although the therapeutic efficacy of MB compounds was not superior to oxime-based antidotal treatment. These results contrast with a study in guinea-pigs using a structurally-related compound, MB327, which showed a striking increase in protection at 6 h compared to 24 h. It is suggested that the disparity may be due to pharmacokinetic differences between the two animal species.

Lung damage following whole body, but not intramuscular, exposure to median lethality dose of sarin: findings in rats and guinea pigs.

Objective: Sarin is an irreversible organophosphate cholinesterase inhibitor and a highly toxic, volatile warfare agent. Rats and guinea pigs exposed to sarin display cholinergic excitotoxicity which includes hyper-salivation, respiratory distress, tremors, seizures, and death. Here we focused on the characterization of the airways injury induced by direct exposure of the lungs to sarin vapor and compared it to that induced by the intramuscularly route. Materials and methods: Rats were exposed to sarin either in vapor (∼1LCT50, 34.2 ± 0.8 µg/l/min, 10 min) or by i.m. (∼1LD50, 80 µg/kg), and lung injury was evaluated by broncho-alveolar lavage (BAL). Results and discussion: BAL analysis revealed route-dependent effects in rats: vapor exposed animals showed elevation of inflammatory cytokines, protein, and neutrophil cells. These elevations were seen at 24 h and were still significantly higher compared to control values at 1 week following vapor exposure. These elevations were not detected in rats exposed to sarin i.m. Histological evaluation of the brains revealed typical changes following sarin poisoning independent of the route of administration. The airways damage following vapor exposure in rats was also compared to that induced in guinea pigs. The latter showed increased eosinophilia and histamine levels that constitutes an anaphylactic response not seen in rats. Conclusions: These data clearly point out the importance of using the appropriate route of administration in studying the deleterious effects of volatile nerve agents, as well as the selection of the appropriate animal species. Since airways form major target organs for the development of injury following inhalation toxicity, they should be included in any comprehensive evaluation of countermeasures efficacy.

Environmental challenges for the nervous system and the brain in Japan.

Japan provides many lessons for the Environmental Neurology's issues. Drama and disasters have paved the recent history of Japan. The Japanese people have been intoxicated by chemical compounds (methylmercury, sulfur dioxide, cadmium, PCBs and other dioxin-related compounds) and were the victims of several dramatic disasters (atomic bombing, nuclear disaster, sarin gas attack). They are still exposed to air pollution. Prion diseases including dura-graft-associated CJD are still an issue. In addition, continuously spreading chronic wasting disease is a worldwide challenge .

Design, synthesis and evaluation of new classes of nonquaternary reactivators for acetylcholinesterase inhibited by organophosphates.

A new series of nonquaternary conjugates for reactivation of both nerve agents and pesticides inhibited hAChE were described in this paper. It was found that substituted salicylaldehydes conjugated to aminobenzamide through piperidine would produce efficient reactivators for sarin, VX and tabun inhibited hAChE, such as L6M1R3, L6M1R5 to L6M1R7, L4M1R3 and L4M1R5 to L4M1R7. The in vitro reactivation experiment for pesticides inhibited hAChE of these new synthesized oximes were conducted for the first time. Despite they were less efficient than obidoxime, some of them were highlighted as equal or more efficient reactivators in comparison to 2-PAM. It was found that introduction of peripheral site ligands could increase oximes' binding affinity for inhibited hAChE in most cases, which resulted in greater reactivation ability.

Comparative evaluation of antidotal efficacy of 2-PAM and HNK-102 oximes during inhalation of sarin vapor in Swiss albino mice.

Efficacy of two oximes treatments evaluated during inhalation of sarin vapor (LCt50, 755.9 mg/min/m3) in simulated real scenario in vivo. Majority of mice either became moribund or died within 1-2 min during exposure to multifold-lethal concentrations of sarin vapor. Protection indices were determined by exposing to sarin vapor in two sessions, 1 min exposure followed by treatments with or without HNK-102 (56.56 mg/kg, im) or 2-PAM (30 mg/kg, im) and atropine (10 mg/kg, ip), and again exposed for remaining 14 min. Protection offered by HNK-102 was found to be four folds higher compared to 2-PAM in the same toxic environment. Secondly, sub-lethal concentration of sarin vapor (0.8 × LCt50 or 605 mg/min/m3), 24 h post investigations revealed that the oximes could not reactivate brain and serum acetylcholinesterase (AChE) activity. The treatments prevented increase in protein concentration (p < .05) and macrophages infiltration compared to sarin alone group in broncho-alveolar lavage fluid. Lung histopathology showed intense peribronchial infiltration and edema with desquamating epithelial lining and mild to moderate alveolar septal infiltration in sarin and atropine groups, respectively. Noticeable peeling-off observed in epithelial lining and sporadic mild infiltration of epithelial cells at bronchiolar region in 2-PAM and HNK-102 groups, respectively. The oximes failed to reactivate AChE activity; however, the mice survived up to 6.0 × LCt50, proved involvement of non-AChE targets in sarin toxicity. Atropine alone treatment was found to be either ineffective or increased the toxicity. HNK-102, exhibited better survivability with lung protection, can be considered as a better replacement for 2-PAM to treat sarin inhalation induced poisoning.

Younger rats are more susceptible to the lethal effects of sarin than adult rats: 24 h LC50 for whole-body (10 and 60 min) exposures.

Chemical warfare nerve agents (CWNA) inhibit acetylcholinesterase and are among the most lethal chemicals known to man. Children are predicted to be vulnerable to CWNA exposure because of their smaller body masses, higher ventilation rates and immature central nervous systems. While a handful of studies on the effects of CWNA in younger animals have been published, exposure routes relevant to battlefield or terrorist situations (i.e. inhalation for sarin) were not used. Thus, we estimated the 24 h LC50 for whole-body (10 and 60 min) exposure to sarin using a stagewise, adaptive dose design. Specifically, male and female Sprague-Dawley rats were exposed to a range of sarin concentrations (6.2-44.0 or 1.6-12.5 mg/m³) for either 10 or 60 min, respectively, at six different times during their development (postnatal day [PND] 7, 14, 21, 28, 42 and 70). For male and female rats, the lowest LC50 values were observed for PND 14 and the highest LC50 values for PND 28. Sex differences were observed only for PND 42 for the 10 min exposures and PND 21 and 70 for the 60 min exposures. Thus, younger rats (PND 14) were more susceptible than older rats (PND 70) to the lethal effects of whole-body exposure to sarin, while adolescent (PND 28) rats were the least susceptible and sex differences were minimal. These results underscore the importance of controlling for the age of the animal in research on the toxic effects associated with CWNA exposure.

Propagation of damage in the rat brain following sarin exposure: Differential progression of early processes.

Sarin is an irreversible organophosphate cholinesterase inhibitor and a highly toxic warfare agent. Following the overt, dose-dependent signs (e.g. tremor, hyper secretion, seizures, respiratory depression and eventually death), brain damage is often reported. The goal of the present study was to characterize the early histopathological and biochemical events leading to this damage. Rats were exposed to 1LD50 of sarin (80µg/kg, i.m.). Brains were removed at 1, 2, 6, 24 and 48h and processed for analysis. Results showed that TSPO (translocator protein) mRNA increased at 6h post exposure while TSPO receptor density increased only at 24h. In all brain regions tested, bax mRNA decreased 1h post exposure followed by an increase 24h later, with only minor increase in bcl2 mRNA. At this time point a decrease was seen in both anti-apoptotic protein Bcl2 and pro-apoptotic Bax, followed by a time and region specific increase in Bax. An immediate elevation in ERK1/2 activity with no change in JNK may indicate an endogenous "first response" mechanism used to attenuate the forthcoming apoptosis. The time dependent increase in the severity of brain damage included an early bi-phasic activation of astrocytes, a sharp decrease in intact neuronal cells, a time dependent reduction in MAP2 and up to 15% of apoptosis. Thus, neuronal death is mostly due to necrosis and severe astrocytosis. The data suggests that timing of possible treatments should be determined by early events following exposure. For example, the biphasic changes in astrocytes activity indicate a possible beneficial effects of delayed anti-inflammatory intervention.