A retrospective screening method for carbamate toxicant exposure based on butyrylcholinesterase adducts in human plasma with ultra-high performance liquid chromatography-tandem mass spectrometry.

Carbamate pesticides are extensively used in agriculture for their inhibition to acetylcholinesterase and damages to the insects' neural systems. Because of their toxicity, human poisoning incidents caused by carbamate pesticide exposure have occurred from time to time. What's more, some lethally toxic carbamate toxicants known as carbamate nerve agents (CMNAs) have been supplemented in Schedule 1 of the Annex on Chemicals in the Chemical Weapons Convention (CWC) by Organisation of the Prohibition of Chemical Weapons (OPCW) from 2020. And some other carbamates, like physostigmine, have been used in clinical treatment as anticholinergic drugs and their misuse may also cause damages to the body. Similar to organophosphorus toxicants, carbamate toxicants would react with butyrylcholinesterase (BChE) in plasma when entering the human body, resulting in the BChE adducts, based on which the exposure of carbamate toxicants could be detected retrospectively. In this study, methylcarbamyl nonapeptide and dimethylcarbamyl nonapeptide from pepsin digestion of BChE adducts were identified with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) in product ion scan mode. Carbofuran was chosen as the target to establish the detection method of carbamate toxicant exposure based on methylcarbamyl nonapeptide digested from methylcarbamyl BChE. Procainamide-gel affinity purification, pepsin digestion and UHPLC-MS/MS analysis in multiple reaction monitoring (MRM) mode were applied. Under the optimized conditions of sample preparation and UHPLC-MS/MS MRM analysis, the limits of detection (LODs) reached 10.0 ng/mL of plasma exposed to carbofuran with satisfactory specificity. The quantitation approach was established with d3-carbofuran-exposed plasma as the internal standard (IS) and the linearity range was 30.0-1.00 × 103 nmol/L (R2 >0.998) with the accuracy of 95.6%-107% and precision of ≤9% relative standard deviation (RSD). The applicability was also evaluated by N,N-dimethyl-carbamates with the LODs of 30.0 nmol/L for pirimicarb-exposed plasma based on dimethylcarbamyl nonapeptide. Because most of carbamate toxicants has methylcarbamyl or dimethylcarbamyl groups, this approach could be applied on the retrospective screening of carbamate toxicant exposure including CMNAs, carbamate pesticides or carbamate drugs. This study could provide an effective means in the fields of CWC verification, toxicological mechanism investigation and down-selection of potential treatment options.

Evaluation of 6-OxP-CD, an Oxime-based cyclodextrin as a viable medical countermeasure against nerve agent poisoning: Experimental and molecular dynamic simulation studies on its inclusion complexes with cyclosarin, soman and VX.

The ability of the cyclodextrin-oxime construct 6-OxP-CD to bind and degrade the nerve agents Cyclosarin (GF), Soman (GD) and S-[2-[Di(propan-2-yl)amino]ethyl] O-ethyl methylphosphonothioate (VX) has been studied using 31P-nuclear magnetic resonance (NMR) under physiological conditions. While 6-OxP-CD was found to degrade GF instantaneously under these conditions, it was found to form an inclusion complex with GD and significantly improve its degradation (t1/2 ~ 2 hrs) relative over background (t1/2 ~ 22 hrs). Consequently, effective formation of the 6-OxP-CD:GD inclusion complex results in the immediate neutralization of GD and thus preventing it from inhibiting its biological target. In contrast, NMR experiments did not find evidence for an inclusion complex between 6-OxP-CD and VX, and the agent's degradation profile was identical to that of background degradation (t1/2 ~ 24 hrs). As a complement to this experimental work, molecular dynamics (MD) simulations coupled with Molecular Mechanics-Generalized Born Surface Area (MM-GBSA) calculations have been applied to the study of inclusion complexes between 6-OxP-CD and the three nerve agents. These studies provide data that informs the understanding of the different degradative interactions exhibited by 6-OxP-CD with each nerve agent as it is introduced in the CD cavity in two different orientations (up and down). For its complex with GF, it was found that the oxime in 6-OxP-CD lies in very close proximity (PGF⋯OOxime ~ 4-5 Å) to the phosphorus center of GF in the 'downGF' orientation for most of the simulation accurately describing the ability of 6-OxP-CD to degrade this nerve agent rapidly and efficiently. Further computational studies involving the center of masses (COMs) for both components (GF and 6-OxP-CD) also provided some insight on the nature of this inclusion complex. Distances between the COMs (ΔCOM) lie closer in space in the 'downGF' orientation than in the 'upGF' orientation; a correlation that seems to hold true not only for GF but also for its congener, GD. In the case of GD, calculations for the 'downGD' orientation showed that the oxime functional group in 6-OxP-CD although lying in close proximity (PGD⋯OOxime ~ 4-5 Å) to the phosphorus center of the nerve agent for most of the simulation, adopts another stable conformation that increase this distance to ~ 12-14 Å, thus explaining the ability of 6-OxP-CD to bind and degrade GD but with less efficiency as observed experimentally (t1/2 ~ 4 hr. vs. immediate). Lastly, studies on the VX:6-OxP-CD system demonstrated that VX does not form a stable inclusion complex with the oxime-bearing cyclodextrin and as such does not interact in a way that is conducive to an accelerated degradation scenario. Collectively, these studies serve as a basic platform from which the development of new cyclodextrin scaffolds based on 6-OxP-CD can be designed in the development of medical countermeasures against these highly toxic chemical warfare agents.

Treatment of cholinergic-induced status epilepticus with polytherapy targeting GABA and glutamate receptors.

Despite new antiseizure medications, the development of cholinergic-induced refractory status epilepticus (RSE) continues to be a therapeutic challenge as pharmacoresistance to benzodiazepines and other antiseizure medications quickly develops. Studies conducted by Epilepsia. 2005;46:142 demonstrated that the initiation and maintenance of cholinergic-induced RSE are associated with trafficking and inactivation of gamma-aminobutyric acid A receptors (GABAA R) thought to contribute to the development of benzodiazepine pharmacoresistance. In addition, Dr. Wasterlain's laboratory reported that increased N-methyl-d-aspartate receptors (NMDAR) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR) contribute to enhanced glutamatergic excitation (Neurobiol Dis. 2013;54:225; Epilepsia. 2013;54:78). Thus, Dr. Wasterlain postulated that targeting both maladaptive responses of reduced inhibition and increased excitation that is associated with cholinergic-induced RSE should improve therapeutic outcome. We currently review studies in several animal models of cholinergic-induced RSE that demonstrate that benzodiazepine monotherapy has reduced efficacy when treatment is delayed and that polytherapy with drugs that include a benzodiazepine (eg midazolam and diazepam) to counter loss of inhibition, concurrent with an NMDA antagonist (eg ketamine) to reduce excitation provide improved efficacy. Improved efficacy with polytherapy against cholinergic-induced seizure is demonstrated by reduction in (1) seizure severity, (2) epileptogenesis, and (3) neurodegeneration compared with monotherapy. Animal models reviewed include pilocarpine-induced seizure in rats, organophosphorus nerve agent (OPNA)-induced seizure in rats, and OPNA-induced seizure in two mouse models: (1) carboxylesterase knockout (Es1-/- ) mice which, similarly to humans, lack plasma carboxylesterase and (2) human acetylcholinesterase knock-in carboxylesterase knockout (KIKO) mice. We also review studies showing that supplementing midazolam and ketamine with a third antiseizure medication (valproate or phenobarbital) that targets a nonbenzodiazepine site rapidly terminates RSE and provides further protection against cholinergic-induced SE. Finally, we review studies on the benefits of simultaneous compared with sequential drug treatments and the clinical implications that lead us to predict improved efficacy of early combination drug therapies. The data generated from seminal rodent studies of efficacious treatment of cholinergic-induced RSE conducted under Dr. Wasterlain's guidance suggest that future clinical trials should treat the inadequate inhibition and temper the excess excitation that characterize RSE and that early combination therapies may provide improved outcome over benzodiazepine monotherapy.

Supplemental treatment to atropine improves the efficacy to reverse nerve agent induced bronchoconstriction.

Exposure to highly toxic organophosphorus compounds causes inhibition of the enzyme acetylcholinesterase resulting in a cholinergic toxidrome and innervation of receptors in the neuromuscular junction may cause life-threatening respiratory effects. The involvement of several receptor systems was therefore examined for their impact on bronchoconstriction using an ex vivo rat precision-cut lung slice (PCLS) model. The ability to recover airways with therapeutics following nerve agent exposure was determined by quantitative analyses of muscle contraction. PCLS exposed to nicotine resulted in a dose-dependent bronchoconstriction. The neuromuscular nicotinic antagonist tubocurarine counteracted the nicotine-induced bronchoconstriction but not the ganglion blocker mecamylamine or the common muscarinic antagonist atropine. Correspondingly, atropine demonstrated a significant airway relaxation following ACh-exposure while tubocurarine did not. Atropine, the M3 muscarinic receptor antagonist 4-DAMP, tubocurarine, the ß2-adrenergic receptor agonist formoterol, the Na+-channel blocker tetrodotoxin and the K+ATP-channel opener cromakalim all significantly decreased airway contractions induced by electric field stimulation. Following VX-exposure, treatment with atropine and the Ca2+-channel blocker magnesium sulfate resulted in significant airway relaxation. Formoterol, cromakalim and magnesium sulfate administered in combinations with atropine demonstrated an additive effect. In conclusion, the present study demonstrated improved airway function following nerve agent exposure by adjunct treatment to the standard therapy of atropine.

A delayed treatment model for the evaluation of scopolamine for VX nerve agent intoxication.

Most research on medical countermeasures for nerve agent exposure assumes a military scenario, in which (autoinjector) treatment is envisaged to be available immediately. In a civilian setting however, treatment is delayed until arrival of first-aid responders. This may significantly affect treatment efficacy and the requirements for secondary intensive care. The aim of the current study was to develop a guinea pig model to evaluate the efficacy of delayed treatment following nerve agent exposure. We identified a trigger-to-treat based on a progressive stage of the toxidrome following VX exposure, which was associated with the subsiding of clonic movements. This paradigm resulted in treatment consistently being administered between 15 and 25 min post-exposure. Using the model, we investigated the potential for the anticholinergic scopolamine to act as a delayed treatment either as a standalone treatment, or as an adjunct to delayed treatment with Standard of Care (SOC), containing atropine, 2-PAM, and midazolam. The study provides a framework for a small animal model for evaluating the efficacy of treatment administered at a specific stage of the toxidrome, when immediate treatment is absent. As an adjunct, scopolamine treatment did not result in improved survival, but did show a beneficial effect on recovery, in terms of general posture. As a standalone treatment, scopolamine showed a significant, dose-responsive, beneficial effect on survival and recovery. These promising results warrant additional studies to investigate which observed physiological improvements are relevant for the recovery process and residual injury.

Widow spiders in the New World: a review on Latrodectus Walckenaer, 1805 (Theridiidae) and latrodectism in the Americas

Humankind has always been fascinated by venomous animals, as their toxic substances have transformed them into symbols of power and mystery. Over the centuries, researchers have been trying to understand animal venoms, unveiling intricate mixtures of molecules and their biological effects. Among venomous animals, Latrodectus Walckenaer, 1805 (widow spiders) have become feared in many cultures worldwide due to their extremely neurotoxic venom. The Latrodectus genus encompasses 32 species broadly spread around the globe, 14 of which occur in the Americas. Despite the high number of species found in the New World, the knowledge on these spiders is still scarce. This review covers the general knowledge on Latrodectus spp. from the Americas. We address widow spiders' taxonomy; geographical distribution and epidemiology; symptoms and treatments of envenomation (latrodectism); venom collection, experimental studies, proteome and transcriptome; and biotechnological studies on these Latrodectus spp. Moreover, we discuss the main challenges and limitations faced by researchers when trying to comprehend this neglected group of medically important spiders. We expect this review to help overcome the lack of information regarding widow spiders in the New World.(AU)

Discovery of treatment for nerve agents targeting a new metabolic pathway.

The inhibition of acetylcholinesterase is regarded as the primary toxic mechanism of action for chemical warfare agents. Recently, there have been numerous reports suggesting that metabolic processes could significantly contribute to toxicity. As such, we applied a multi-omics pipeline to generate a detailed cascade of molecular events temporally occurring in guinea pigs exposed to VX. Proteomic and metabolomic profiling resulted in the identification of several enzymes and metabolic precursors involved in glycolysis and the TCA cycle. All lines of experimental evidence indicated that there was a blockade of the TCA cycle at isocitrate dehydrogenase 2, which converts isocitrate to α-ketoglutarate. Using a primary beating cardiomyocyte cell model, we were able to determine that the supplementation of α-ketoglutarate subsequently rescued cells from the acute effects of VX poisoning. This study highlights the broad impacts that VX has and how understanding these mechanisms could result in new therapeutics such as α-ketoglutarate.

Synthesis and in vitro evaluation of novel non-oximes for the reactivation of nerve agent inhibited human acetylcholinesterase.

Since several decades oximes have been used as part of treatment of nerve agent intoxication with the aim to restore the biological function of the enzyme acetylcholinesterase after its covalent inhibition by organophosphorus compounds such as pesticides and nerve agents. Recent findings have illustrated that, besides oximes, certain Mannich phenols can reactivate the inhibited enzyme very effectively, and may therefore represent an attractive complementary class of reactivators. In this paper we further probe the effect of structural variation on the in vitro efficacy of Mannich phenol based reactivators. Thus, we present the synthesis of 14 compounds that are close variants of the previously reported 4-amino-2-(1-pyrrolidinylmethyl)-phenol, a very effective non-oxime reactivator, and 3 dimeric Mannich phenols. All compounds were assessed for their ability to reactivate human acetylcholinesterase inhibited by the nerve agents VX, tabun, sarin, cyclosarin and paraoxon in vitro. It was confirmed that the potency of the compounds is highly sensitive to small structural changes, leading to diminished reactivation potency in many cases. However, the presence of 4-substituted alkylamine substituents (as exemplified with the 4-benzylamine-variant) was tolerated. More surprisingly, the dimeric compounds demonstrated non-typical behavior and displayed some reactivation potency as well. Both findings may open up new avenues for designing more effective non-oxime reactivators.

Rational polytherapy in the treatment of cholinergic seizures.

The initiation and maintenance phases of cholinergic status epilepticus (SE) are associated with maladaptive trafficking of synaptic GABAA and glutamate receptors. The resulting pharmacoresistance reflects a decrease in synaptic GABAA receptors and increase in NMDA and AMPA receptors, which tilt the balance between inhibition and excitation in favor of the latter. If these changes are important to the pathophysiology of SE, both should be treated, and blocking their consequences should have therapeutic potential. We used a model of benzodiazepine-refractory SE (RSE) (Tetz et al., 2006) and a model of soman-induced SE to test this hypothesis. Treatment of RSE with combinations of the GABAAR agonists midazolam or diazepam and the NMDAR antagonists MK-801 or ketamine terminated RSE unresponsive to high-dose monotherapy with benzodiazepines, ketamine or other antiepileptic drugs (AEDs). It also reduced RSE-associated neuronal injury, spatial memory deficits and the occurrence of spontaneous recurrent seizures (SRS), tested several weeks after SE. Treatment of sc soman-induced SE similarly showed much greater reduction of EEG power by a combination of midazolam with ketamine, compared to midazolam monotherapy. When treating late (40 min after seizure onset), there may not be enough synaptic GABAAR left to be able to restore inhibition with maximal GABAAR stimulation, and further benefit is derived from the addition of an AED which increases inhibition or reduces excitation by a non-GABAergic mechanism. The midazolam-ketamine-valproate combination is effective in terminating RSE. 3-D isobolograms demonstrate positive cooperativity between midazolam, ketamine and valproate, without any interaction between the toxicity of these drugs, so that the therapeutic index is increased by combination therapy between GABAAR agonist, NMDAR antagonist and selective AEDs. We compared this drug combination based on the receptor trafficking hypothesis to treatments based on clinical practice. The midazolam-ketamine-valproate combination is far more effective in stopping RSE than the midazolam-fosphenytoin-valproate combination inspired from clinical guidelines. Furthermore, sequential administration of midazolam, ketamine and valproate is far less effective than simultaneous treatment with the same drugs at the same dose. These data suggest that we should re-evaluate our traditional treatment of RSE, and that treatment should be based on pathophysiology. The search for a better drug has to deal with the fact that most monotherapy leaves half the problem untreated. The search for a better benzodiazepine should acknowledge the main cause of pharmacoresistance, which is loss of synaptic GABAAR. Future clinical trials should consider treating both the failure of inhibition and the runaway excitation which characterize RSE, and should include an early polytherapy arm.

Ratiometric Detection of Nerve Agents by Coupling Complementary Properties of Silicon-Based Quantum Dots and Green Fluorescent Protein.

Ratiometric photoluminescent detection of the toxicologically potent organophosphate ester nerve agents paraoxon (PX) and parathion (PT) using the complementary optical and chemical properties of the long Stokes shift green fluorescent protein variant, mAmetrine1.2 (mAm), and red-emitting silicon-based quantum dots (SiQDs) is reported. PX and PT selectively quench SiQD photoluminescence (PL) through a dynamic quenching mechanism, thereby, facilitating the development of a ratiometric sensor platform that shows micromolar limits of detection for PX and PT and that is unaffected by the presence of common inorganic and organic interferents. As a part of the present study, we also demonstrate that the paper-based sensors derived from mAm and SiQDs detect PX and PT at concentrations as low as 5 µM using a readily available commercial color analysis smartphone "app". The ratiometric sensor reported herein can potentially be used for the convenient and rapid on-site detection and quantification of PX and PT in real-world samples.

Nerve agent markers screening after accumulation in garden cress (Lepidium sativum) used as a model plant object.

In this research an accumulation of nerve agent markers in garden cress (Lepidium sativum) as a model plant object was studied using LC-QTOF hybrid system. For the determination of methylphosphonic acid and alkyl methylphosphonates, which are specific markers of sarin, soman, VR and VX, simple and sensitive approach was developed. Direct analysis of aqueous extracts on the reversed phase column with polar endcapping allowed to achieve satisfactory retention factor for methylphosphonic acid, which has high polarity and is usually very weakly retained on the ordinary reversed phase columns. Application of the QTOF mass spectrometer with high mass resolution led to the increase in the accuracy of the conducted measurements. The HPLC-HRMS technique developed exclusively for this study has been validated for linearity, limit of detection, limit of quantification, precision, accuracy and matrix effect prior to the analysis of plant extract samples. Hydroponic growth model was employed to examine accumulation of nerve agent markers in garden cress. It was found that after elimination of nerve agent markers from the plant growth medium, garden cress was able to store these substances for at least 5 weeks providing high retrospectivity of the analysis. Moreover, during the cress growth, no metabolization of alkyl methylphosphonates was observed. This allows not only to reveal the fact of nerve agents release into environment, but also to define its type after a long period of time.

Structure-Based Optimization of Nonquaternary Reactivators of Acetylcholinesterase Inhibited by Organophosphorus Nerve Agents.

Acetylcholinesterase (AChE), a key enzyme in the central and peripheral nervous systems, is the principal target of organophosphorus nerve agents. Quaternary oximes can regenerate AChE activity by displacing the phosphyl group of the nerve agent from the active site, but they are poorly distributed in the central nervous system. A promising reactivator based on tetrahydroacridine linked to a nonquaternary oxime is also an undesired submicromolar reversible inhibitor of AChE. X-ray structures and molecular docking indicate that structural modification of the tetrahydroacridine might decrease inhibition without affecting reactivation. The chlorinated derivative was synthesized and, in line with the prediction, displayed a 10-fold decrease in inhibition but no significant decrease in reactivation efficiency. X-ray structures with the derivative rationalize this outcome. We thus show that rational design based on structural studies permits the refinement of new-generation pyridine aldoxime reactivators that may be more effective in the treatment of nerve agent intoxication.

Evaluation of the Influence of Three Newly Developed Bispyridinium Anti-nicotinic Compounds (MB408, MB442, MB444) on the Efficacy of Antidotal Treatment of Nerve Agent Poisoning in Mice.

The influence of three newly developed bispyridinium antinicotinic compounds (the non-oximes MB408, MB442 and MB444) on the therapeutic efficacy of a standard antidotal treatment (atropine in combination with an oxime) of acute poisoning by the organophosphorus nerve agents tabun and soman was studied in mice. The therapeutic efficacy of atropine in combination with an oxime with or without one of the bispyridinium non-oximes was evaluated by determination of the LD50 values of the nerve agents and measurement of the survival time after supralethal poisoning. Addition of all the tested non-oximes increased significantly the therapeutic efficacy of atropine in combination with an oxime against tabun poisoning. They also positively influenced the number of surviving mice 6 hr after supralethal poisoning with tabun. However, they were only slightly effective for the treatment of soman poisoning. The benefit of the tested bispyridinium non-oximes was dose-dependent. To conclude, the addition of bispyridinium non-oximes to the standard antidotal treatment of acute poisoning with tabun was beneficial regardless of the chosen non-oxime, but only slightly beneficial in the case of soman poisoning.

Activity Based Protein Profiling Leads to Identification of Novel Protein Targets for Nerve Agent VX.

Organophosphorus (OP) nerve agents continue to be a threat at home and abroad during the war against terrorism. Human exposure to nerve agents such as VX results in a cascade of toxic effects relative to the exposure level including ocular miosis, excessive secretions, convulsions, seizures, and death. The primary mechanism behind these overt symptoms is the disruption of cholinergic pathways. While much is known about the primary toxicity mechanisms of nerve agents, there remains a paucity of information regarding impacts on other pathways and systemic effects. These are important for establishing a comprehensive understanding of the toxic mechanisms of OP nerve agents. To identify novel proteins that interact with VX, and that may give insight into these other mechanisms, we used activity-based protein profiling (ABPP) employing a novel VX-probe on lysates from rat heart, liver, kidney, diaphragm, and brain tissue. By making use of a biotin linked VX-probe, proteins covalently bound by the probe were isolated and enriched using streptavidin beads. The proteins were then digested, labeled with isobarically distinct tandem mass tag (TMT) labels, and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Quantitative analysis identified 132 bound proteins, with many proteins found in multiple tissues. As with previously published ABPP OP work, monoacylglycerol lipase associated proteins and fatty acid amide hydrolase (FAAH) were shown to be targets of VX. In addition to these two and other predicted neurotransmitter-related proteins, a number of proteins involved with energy metabolism were identified. Four of these enzymes, mitochondrial isocitrate dehydrogenase 2 (IDH2), isocitrate dehydrogenase 3 (IDH3), malate dehydrogenase (MDH), and succinyl CoA (SCS) ligase, were assayed for VX inhibition. Only IDH2 NADP+ activity was shown to be inhibited directly. This result is consistent with other work reporting animals exposed to OP compounds exhibit reduced IDH activity. Though clearly a secondary mechanism for toxicity, this is the first time VX has been shown to directly interfere with energy metabolism. Taken together, the ABPP work described here suggests the discovery of novel protein-agent interactions, which could be useful for the development of novel diagnostics or potential adjuvant therapeutics.

α-Linolenic Acid, A Nutraceutical with Pleiotropic Properties That Targets Endogenous Neuroprotective Pathways to Protect against Organophosphate Nerve Agent-Induced Neuropathology.

α-Linolenic acid (ALA) is a nutraceutical found in vegetable products such as flax and walnuts. The pleiotropic properties of ALA target endogenous neuroprotective and neurorestorative pathways in brain and involve the transcription factor nuclear factor kappa B (NF-κB), brain-derived neurotrophic factor (BDNF), a major neuroprotective protein in brain, and downstream signaling pathways likely mediated via activation of TrkB, the cognate receptor of BDNF. In this review, we discuss possible mechanisms of ALA efficacy against the highly toxic OP nerve agent soman. Organophosphate (OP) nerve agents are highly toxic chemical warfare agents and a threat to military and civilian populations. Once considered only for battlefield use, these agents are now used by terrorists to inflict mass casualties. OP nerve agents inhibit the critical enzyme acetylcholinesterase (AChE) that rapidly leads to a cholinergic crisis involving multiple organs. Status epilepticus results from the excessive accumulation of synaptic acetylcholine which in turn leads to the overactivation of muscarinic receptors; prolonged seizures cause the neuropathology and long-term consequences in survivors. Current countermeasures mitigate symptoms and signs as well as reduce brain damage, but must be given within minutes after exposure to OP nerve agents supporting interest in newer and more effective therapies. The pleiotropic properties of ALA result in a coordinated molecular and cellular program to restore neuronal networks and improve cognitive function in soman-exposed animals. Collectively, ALA should be brought to the clinic to treat the long-term consequences of nerve agents in survivors. ALA may be an effective therapy for other acute and chronic neurodegenerative disorders.