Acute and long-term effects of VX in rat brain cell aggregate culture.

The contact poison VX (O-ethyl S-(2-diisopropylaminoethyl) methylphosphonothioate) is a chemical warfare agent that is one of the most toxic organophosphorus compounds known. Its primary mechanism of toxic action is through the inhibition of acetylcholinesterase and resultant respiratory paralysis. The majority of work on VX has thus concentrated on its potent anticholinesterase activity and acute toxicity, with few studies investigating potential long-term effects. In this report we describe the effects of VX in aggregating rat brain cell cultures out to 28 days post-exposure. Cholinesterase activity was rapidly inhibited (60 min IC50 = 0.73 +/- 0.27 nM), but recovered towards baseline values over the next four weeks. Apoptotic cell death, as measured using caspase-3 activity was evident only at 100 µM concentrations. Cell type specific enzymatic markers (glutamine synthase, choline acetyltransferase and 2',3'-cyclic nucleotide 3'-phosphodiesterase) showed no significant changes. Total Akt levels were unchanged, while an increased phosphorylation of this protein was noted only at the highest VX concentration on the first day post-exposure. In contrast, significant and delayed (28 days post-exposure) decreases were noted in vascular endothelial growth factor (VEGF) levels, a protein whose reduced levels are known to contribute to neurodegenerative disorders. These observations may indicate that the long-term effects noted in some survivors of nerve agent intoxication may be due to VX-induced declines in brain VEGF levels.

Sulphur mustard induces progressive toxicity and demyelination in brain cell aggregate culture.

Sulphur mustard (H; bis(2-chloroethyl) sulphide) is a vesicant chemical warfare (CW) agent that has been well documented as causing acute injury to the skin, eyes and respiratory system. Although a great deal of research effort has been expended to understand how H exerts these effects, its mechanism of action is still poorly understood. At high exposures, H also causes systemic toxicity with chronic and long-term effects to the immune, cardiovascular and central nervous systems, and these aspects of H poisoning are much less studied and comprehended. Rat aggregate cultures comprised of multiple brain cell types were exposed to H and followed for four weeks post-exposure to assess neurotoxicity. Toxicity (LDH, caspase-3 and aggregate diameter) was progressive with time post-exposure. In addition, statistically significant changes in neurofilament heavy chain (NFH), glial fibrillary acidic protein (GFAP), Akt phosphorylation, IL-6, GRO-KC and TNF-α were noted that were time- and concentration-dependent. Myelin basic protein, CNPase and vascular endothelial growth factor (VEGF) were found to be especially sensitive to H exposure in a time- and concentration-dependent fashion, with levels falling to ∼50 % of control values at ∼10 µM H by 8 days post-exposure. Demyelination and VEGF inhibition may be causal in the long-term neuropsychological illnesses that have been documented in casualties exposed to high concentrations of H, and may also play a role in the peripheral neuropathy that has been observed in some of these individuals.

N-Acetylcysteine as a treatment for sulphur mustard poisoning.

In the long and intensive search for effective treatments to counteract the toxicity of the chemical warfare (CW) agent sulphur mustard (H; bis(2-chloroethyl) sulphide), the most auspicious and consistent results have been obtained with the drug N-acetylcysteine (NAC), particularly with respect to its therapeutic use against the effects of inhaled H. It is a synthetic cysteine derivative that has been used in a wide variety of clinical applications for decades and a wealth of information exists on its safety and protective properties against a broad range of toxicants and disease states. Its primary mechanism of action is as a pro-drug for the synthesis of the antioxidant glutathione (GSH), particularly in those circumstances where oxidative stress has exhausted intracellular GSH stores. It impacts a number of pathways either directly or through its GSH-related antioxidant and anti-inflammatory properties, which make it a prime candidate as a potential treatment for the wide range of deleterious cellular effects that H is acknowledged to cause in exposed individuals. This report reviews the available literature on the protection afforded by NAC against the toxicity of H in a variety of model systems, including its efficacy in treating the long-term chronic lung effects of H that have been demonstrated in Iranian veterans exposed during the Iran-Iraq War (1980-1988). Although there is overwhelming evidence supporting this drug as a potential medical countermeasure against this CW agent, there is a requirement for carefully controlled clinical trials to determine the safety, efficacy and optimal NAC dosage regimens for the treatment of inhaled H.

Hair follicle miRNAs: a novel biomarker for primary blast Induced-Mild traumatic brain injury.

CONTEXT: Due to the wide use of improvised explosive devices during modern warfare, primary blast-derived mild traumatic brain injury (mTBI) has become a major medical condition in the military. With minimal visually identifiable symptoms, an effective molecular biomarker system is desirable. OBJECTIVE: We assessed the potential of mammalian hair follicle miRNAs as an mTBI biomarker. MATERIALS AND METHODS: Due to their well-established roles in mTBI molecular pathology, the expression level of miR-183, miR-26a, miR-181c, miR-29a, miR-34a and miR-27b was determined using qRT-PCR in whisker hair follicles from rats subject to head-only exposure to a single-pulse shock wave. Based on established transcriptomics profiles, sub-network enrichment analysis (SNEA) was also conducted. RESULTS: The results revealed that molecular networks involving miR-183, miR-26a and miR-181c were enriched in multiple treatments, whereas sub-networks of miR-29a, miR-34a and miR-27b were unique to individual exposure groups. DISCUSSION: Our study showed that all six miRNAs were reflective of the mTBI signature involved in cellular responses. Noteworthy was that the decrease in the transcript levels of miR-181c was correlated with shockwave exposure severity. CONCLUSION: This study demonstrates for the first time that mammalian hair follicles are capable of housing miRNA biomarkers for TBI.

Comprehensive assessment of shockwave intensity: Transcriptomic biomarker discovery for primary blast-induced mild traumatic brain injury using the mammalian hair follicle.

OBJECTIVE: Primary blast-induced mild traumatic brain injury (mTBI) is an injury experienced during modern warfare due to exposure to the pressure waves produced by the detonation of explosives. With virtually no apparent physical damage or symptoms presented, there is a need for more objective and accessible mTBI biomarkers posing minimal invasiveness risk. METHODS: We measured the transcriptomic sensitivity of the hair follicles in relation to the severity of primary blast-derived TBI. An Advanced Blast Simulator system was used to expose male rats to single pulse shock waves (intensities ranging from 15 to 30 psi) in a head-only fashion. Gene differential expression (DE) and gene set (GS) analyses were conducted in the rat whisker hair follicles and the whole blood samples. RESULTS: While shared cellular function, themes were found across the exposure groups, some gene sets under such themes were unique to the exposure conditions. Intensity-specific pathway enrichment patterns within shared GS themes were also identified. Furthermore, while exhibited shared pathways, the blood transcriptome showed substantially fewer enriched gene sets compared with the hair follicles across all exposure conditions. CONCLUSIONS: Accordingly, we demonstrate the potential of mammalian hair follicles serving as an additional source for biomarker discovery and for diagnosing mTBI with high accessibility.

Primary Blast Causes Delayed Effects without Cell Death in Shell-Encased Brain Cell Aggregates.

Previous work in this laboratory used underwater explosive exposures to isolate the effects of shock-induced principle stress without shear on rat brain aggregate cultures. The current study has utilized simulated air blast to expose aggregates in suspension and enclosed within a spherical shell, enabling the examination of a much more complex biomechanical insult. Culture medium-filled spheres were exposed to single pulse overpressures of 15-30 psi (∼6-7 msec duration) and measurements within the sphere at defined sites showed complex and spatially dependent pressure changes. When brain aggregates were exposed to similar conditions, no cell death was observed and no changes in several commonly used biomarkers of traumatic brain injury (TBI) were noted. However, similarly to underwater blast, immediate and transient increases in the protein kinase B signaling pathway were observed at early time-points (3 days). In contrast, the oligodendrocyte marker 2',3'-cyclic nucleotide 3'-phosphodiesterase, as well as vascular endothelial growth factor, both displayed markedly delayed (14-28 days) and pressure-dependent responses. The imposition of a spherical shell between the single pulse shock wave and the target brain tissue introduces greatly increased complexity to the insult. This work shows that brain tissue can not only discriminate the nature of the pressure changes it experiences, but that a portion of its response is significantly delayed. These results have mechanistic implications for the study of primary blast-induced TBI and also highlight the importance of rigorously characterizing the actual pressure variations experienced by target tissue in primary blast studies.

Primary Blast-Induced Changes in Akt and GSK3ß Phosphorylation in Rat Hippocampus.

Traumatic brain injury (TBI) due to blast from improvised explosive devices has been a leading cause of morbidity and mortality in recent conflicts in Iraq and Afghanistan. However, the mechanisms of primary blast-induced TBI are not well understood. The Akt signal transduction pathway has been implicated in various brain pathologies including TBI. In the present study, the effects of simulated primary blast waves on the phosphorylation status of Akt and its downstream effector kinase, glycogen synthase kinase 3ß (GSK3ß), in rat hippocampus, were investigated. Male Sprague-Dawley (SD) rats (350-400 g) were exposed to a single pulse shock wave (25 psi; ~7 ms duration) and sacrificed 1 day, 1 week, or 6 weeks after exposure. Total and phosphorylated Akt, as well as phosphorylation of its downstream effector kinase GSK3ß (at serine 9), were detected with western blot analysis and immunohistochemistry. Results showed that Akt phosphorylation at both serine 473 and threonine 308 was increased 1 day after blast on the ipsilateral side of the hippocampus, and this elevation persisted until at least 6 weeks postexposure. Similarly, phosphorylation of GSK3ß at serine 9, which inhibits GSK3ß activity, was also increased starting at 1 day and persisted until at least 6 weeks after primary blast on the ipsilateral side. In contrast, p-Akt was increased at 1 and 6 weeks on the contralateral side, while p-GSK3ß was increased 1 day and 1 week after primary blast exposure. No significant changes in total protein levels of Akt and GSK were observed on either side of the hippocampus at any time points. Immunohistochemical results showed that increased p-Akt was mainly of neuronal origin in the CA1 region of the hippocampus and once phosphorylated, the majority was translocated to the dendritic and plasma membranes. Finally, electrophysiological data showed that evoked synaptic N-methyl-d-aspartate (NMDA) receptor activity was significantly increased 6 weeks after primary blast, suggesting that increased Akt phosphorylation may enhance synaptic NMDA receptor activation, or that enhanced synaptic NMDA receptor activation may increase Akt phosphorylation.

Comparative toxicity of mono- and bifunctional alkylating homologues of sulphur mustard in human skin keratinocytes.

Sulphur mustard (bis(2-chloroethyl) sulphide; agent H) is a vesicant chemical warfare (CW) agent whose mechanism of action is not known with any certainty and for which there are no effective antidotes. It has a pronounced latent period before signs and symptoms of poisoning appear which it shares with the nitrogen mustards, and that differentiates it from other classes of vesicant agents. Sulphur mustard, the sulphur mustard CW agents Q (1,2-bis(2-chloroethylthio) ethane) and T (1,1 bis(2-chloroethylthioethyl) ether), the H partial hydrolysis product hemi-sulphur mustard (2-chloroethyl 2-hydroxyethyl sulphide; HSM), and the commercially available 2-chloroethyl ethyl sulphide (CEES) were characterized with respect to their toxicity in first passage cultures of proliferating human skin keratinocytes, the target cell of H-induced skin vesication. Agents H and T were equitoxic and half as toxic as agent Q. Hemi-sulphur mustard and CEES were approximately six times and seventeen times, respectively less cytotoxic than H. 2-Chloroethyl ethyl sulphide was only slightly less toxic in confluent cultures compared to actively proliferating cells. In contrast, the toxicity of H, Q, T and HSM significantly decreased as the cultures became confluent, paralleling the decreasing sensitivity of skin keratinocytes to H as they leave the basement membrane of the skin. The toxicity of CEES was maximal by 24h. In contrast, the maximal toxicity of the other four agents occurred at 48h, mirroring the latent period observed for these agents in vivo. The markedly different characteristics of toxicity between CEES and the other four test compounds indicate that it is likely that different mechanisms of action are operative between them. Caution should therefore be taken when interpreting the results of studies utilizing CEES as a simulant for the mechanistic study of H, or in the elucidation of medical countermeasures against this CW agent. It is also notable that the toxicity characteristics of the mono-alkylating HSM mirrors those of H, Q and T, suggesting that the bi-alkylating characteristics of these latter compounds may not play as large a role in their toxic effects as commonly thought.

The Effect of Underwater Blast on Aggregating Brain Cell Cultures.

Although the deleterious effects of primary blast on gas-filled organs are well accepted, the effect of blast-induced shock waves on the brain is less clear because of factors that complicate the interpretation of clinical and experimental data. Brain cell aggregate cultures are comprised of multiple differentiated brain cell types and were used to examine the effects of underwater blast. Suspensions of these cultures encased in dialysis tubing were exposed to explosive-generated underwater blasts of low (∼300 kPa), medium (∼2,700 kPa), or high (∼14,000 kPa) intensities and harvested at 1-28 days post-exposure. No changes in gross morphology were noted immediately or weeks after blast wave exposure, and no increases in either apoptotic (caspase-3) or necrotic (lactate dehydrogenase) cell death were observed. Changes in neuronal (neurofilament H, acetylcholinesterase, and choline acetyltransferase) and glial (glial fibrillary acidic protein, glutamine synthetase) endpoints did not occur. However, significant time- and pressure-related increases in Akt (protein kinase B) phosphorylation were noted, as well as declines in vascular endothelial growth factor levels, implicating pathways involved in cellular survival mechanisms. The free-floating nature of the aggregates during blast wave exposure, coupled with their highly hydrolyzed dialysis tubing containment, results in minimized boundary effects, thus enabling accurate assessment of brain cell response to a simplified shock-induced stress wave. This work shows that, at its simplest, blast-induced shock waves produce subtle changes in brain tissue. This study has mechanistic implications for the study of primary blast-induced traumatic brain injury and supports the thesis that underwater blast may cause subtle changes in the brains of submerged individuals.

High-Fidelity Simulation of Primary Blast: Direct Effects on the Head.

The role of primary blast in blast-induced traumatic brain injury (bTBI) is controversial in part due to the technical difficulties of generating free-field blast conditions in the laboratory. The use of traditional shock tubes often results in artifacts, particularly of dynamic pressure, whereas the forces affecting the head are dependent on where the animal is placed relative to the tube, whether the exposure is whole-body or head-only, and on how the head is actually exposed to the insult (restrained or not). An advanced blast simulator (ABS) has been developed that enables high-fidelity simulation of free-field blastwaves, including sharply defined static and dynamic overpressure rise times, underpressures, and secondary shockwaves. Rats were exposed in head-only fashion to single-pulse blastwaves of 15 to 30 psi static overpressure. Head restraints were configured so as to eliminate concussive and minimize whiplash forces exerted on the head, as shown by kinematic analysis. No overt signs of trauma were present in the animals post-exposure. However, significant changes in brain 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNPase) and neurofilament heavy chain levels were evident by 7 days. In contrast to most studies of primary blast-induced TBI (PbTBI), no elevation of glial fibrillary acidic protein (GFAP) levels was noted when head movement was minimized. The ABS described in this article enables the generation of shockwaves highly representative of free-field blast. The use of this technology, in concert with head-only exposure, minimized head movement, and the kinematic analysis of the forces exerted on the head provide convincing evidence that primary blast directly causes changes in brain function and that GFAP may not be an appropriate biomarker of PbTBI.

Decontamination Efficacy and Skin Toxicity of Two Decontaminants against Bacillus anthracis.

Decontamination of bacterial endospores such as Bacillus anthracis has traditionally required the use of harsh or caustic chemicals. The aim of this study was to evaluate the efficacy of a chlorine dioxide decontaminant in killing Bacillus anthracis spores in solution and on a human skin simulant (porcine cadaver skin), compared to that of commonly used sodium hypochlorite or soapy water decontamination procedures. In addition, the relative toxicities of these decontaminants were compared in human skin keratinocyte primary cultures. The chlorine dioxide decontaminant was similarly effective to sodium hypochlorite in reducing spore numbers of Bacillus anthracis Ames in liquid suspension after a 10 minute exposure. After five minutes, the chlorine dioxide product was significantly more efficacious. Decontamination of isolated swine skin contaminated with Bacillus anthracis Sterne with the chlorine dioxide product resulted in no viable spores sampled. The toxicity of the chlorine dioxide decontaminant was up to two orders of magnitude less than that of sodium hypochlorite in human skin keratinocyte cultures. In summary, the chlorine dioxide based decontaminant efficiently killed Bacillus anthracis spores in liquid suspension, as well as on isolated swine skin, and was less toxic than sodium hypochlorite in cultures of human skin keratinocytes.

Primary blast-induced traumatic brain injury in rats leads to increased prion protein in plasma: a potential biomarker for blast-induced traumatic brain injury.

Traumatic brain injury (TBI) is deemed the "signature injury" of recent military conflicts in Afghanistan and Iraq, largely because of increased blast exposure. Injuries to the brain can often be misdiagnosed, leading to further complications in the future. Therefore, the use of protein biomarkers for the screening and diagnosis of TBI is urgently needed. In the present study, we have investigated the plasma levels of soluble cellular prion protein (PrPC) as a novel biomarker for the diagnosis of primary blast-induced TBI (bTBI). We hypothesize that the primary blast wave can disrupt the brain and dislodge extracellular localized PrPC, leading to a rise in concentration within the systemic circulation. Adult male Sprague-Dawley rats were exposed to single pulse shockwave overpressures of varying intensities (15-30 psi or 103.4-206.8 kPa] using an advanced blast simulator. Blood plasma was collected 24 h after insult, and PrPC concentration was determined with a modified commercial enzyme-linked immunosorbent assay (ELISA) specific for PrPC. We provide the first report that mean PrPC concentration in primary blast exposed rats (3.97 ng/mL ± 0.13 SE) is significantly increased compared with controls (2.46 ng/mL ± 0.14 SE; two tailed test p < 0.0001). Furthermore, we report a mild positive rank correlation between PrPC concentration and increasing blast intensity (psi) reflecting a plateaued response at higher pressure magnitudes, which may have implications for all military service members exposed to blast events. In conclusion, it appears that plasma levels of PrPC may be a novel biomarker for the detection of primary bTBI.

Transcriptional profiling in rat hair follicles following simulated Blast insult: a new diagnostic tool for traumatic brain injury.

With wide adoption of explosive-dependent weaponry during military activities, Blast-induced neurotrauma (BINT)-induced traumatic brain injury (TBI) has become a significant medical issue. Therefore, a robust and accessible biomarker system is in demand for effective and efficient TBI diagnosis. Such systems will also be beneficial to studies of TBI pathology. Here we propose the mammalian hair follicles as a potential candidate. An Advanced Blast Simulator (ABS) was developed to generate shock waves simulating traumatic conditions on brains of rat model. Microarray analysis was performed in hair follicles to identify the gene expression profiles that are associated with shock waves. Gene set enrichment analysis (GSEA) and sub-network enrichment analysis (SNEA) were used to identify cell processes and molecular signaling cascades affected by simulated bomb blasts. Enrichment analyses indicated that genes with altered expression levels were involved in central nervous system (CNS)/peripheral nervous system (PNS) responses as well as signal transduction including Ca2+, K+-transportation-dependent signaling, Toll-Like Receptor (TLR) signaling and Mitogen Activated Protein Kinase (MAPK) signaling cascades. Many of the pathways identified as affected by shock waves in the hair follicles have been previously reported to be TBI responsive in other organs such as brain and blood. The results suggest that the hair follicle has some common TBI responsive molecular signatures to other tissues. Moreover, various TBI-associated diseases were identified as preferentially affected using a gene network approach, indicating that the hair follicle may be capable of reflecting comprehensive responses to TBI conditions. Accordingly, the present study demonstrates that the hair follicle is a potentially viable system for rapid and non-invasive TBI diagnosis.

Non-cholinergic intervention of sarin nerve agent poisoning.

The protective effects of selected anesthetic regimens on sarin (GB) were investigated in domestic swine. At 30% oxygen, the toxicity of this agent in isoflurane anesthetized animals (LD(50)=10.1µg/kg) was similar to literature sited values in awake swine (LD(50)=11.8µg/kg) and slightly higher than that of both ketamine (LD(50)=15.6µg/kg) and propofol (LD(50)=15.3µg/kg) anesthetized swine. Use of 100% oxygen in ketamine anesthetized animals resulted in three-fold protective effects compared to 30% oxygen. Use of 100% oxygen in both isoflurane and propofol anesthetized animals, compared to 30% resulted in profound protection against GB poisoning (>33×). There were no differences in the severity of the poisoning or recovery time in animals treated over dose ranges of 10-350µg/kg (isoflurane) or 15-500µg/kg GB (propofol). Survivors of high GB challenges that were revived from propofol anesthetic exhibited no signs of cognitive impairment seven days later. Protective treatments did not attenuate cholinesterase (ChE) inhibition; survivors of otherwise supralethal GB concentrations exhibited very low blood ChE activities. This work indicates that propofol has protective effects against GB, and that oxygen tension may have an important role in treating nerve agent casualties. More importantly, it demonstrates that non-cholinergic protective mechanisms exist that may be exploited in the future development of medical countermeasures against organophosphorous nerve agents.

Comparative protective effects of HI-6 and MMB-4 against organophosphorous nerve agent poisoning.

The oximes pralidoxime (2-PAM), its dimethanesulphonate salt derivative P2S, and obidoxime (toxogonin) are currently licensed and fielded for the treatment of chemical warfare (CW) organophosphorous (OP) nerve agent poisoning. While they are effective against several of the identified threat CW OP agents, they have little efficacy against others such as soman (GD) and cyclosarin (CF). In addition, they are also significantly less effective than other investigational oximes against the nerve agent known as Russian VX (RVX). Among the oximes currently being investigated, two in particular, HI-6 (asoxime) and MMB-4 (ICD-039, methoxime) have been proposed as replacement therapies for the currently licensed oximes. HI-6 has been safely used in individuals to treat OP insecticide poisoning, as well as in human volunteers, although its efficacy against OP nerve agent poisoning in humans cannot be demonstrated due to ethical considerations. It is currently available for use in defined military settings in Canada, Sweden and the Czech Republic, and is also under development in a number of other countries. The oxime MMB-4 has not yet been studied clinically, but is fielded by the Czech Republic, and is being developed by the United States armed services as a replacement for the currently fielded 2-PAM. This review compares the effectiveness of HI-6 and MMB-4 against nerve agent threats where comparisons can be made. HI-6 has been demonstrated to be generally a superior reactivator of nerve agent inhibited enzyme, particularly with human and non-human primate derived enzyme, and has also shown better protective effects against the lethality of most OP agents in a variety of species. Both compounds appear to be clearly superior to the available oximes, obidoxime and 2-PAM.

Ionic dependence of sulphur mustard cytotoxicity.

The effect of ionic environment on sulphur mustard (bis 2-chloroethyl sulphide; HD) toxicity was examined in CHO-K1 cells. Cultures were treated with HD in different ionic environments at constant osmolar conditions (320 mOsM, pH 7.4). The cultures were refed with fresh culture medium 1h after HD exposure, and viability was assessed. Little toxicity was apparent when HD exposures were carried out in ion-free sucrose buffer compared to LC(50) values of approximately 100-150 microM when the cultures were treated with HD in culture medium. Addition of NaCl to the buffer increased HD toxicity in a salt concentration-dependent manner to values similar to those obtained in culture medium. HD toxicity was dependent on both cationic and anionic species with anionic environment playing a much larger role in determining toxicity. Substitution of NaI for NaCl in the treatment buffers increased HD toxicity by over 1000%. The activity of the sodium hydrogen exchanger (NHE) in recovering from cytosolic acidification in salt-free and in different chloride salts did not correlate with the HD-induced toxicity in these buffers. However, the inhibition by HD of intracellular pH regulation correlated with its toxicity in NaCl, NaI and sucrose buffers. Analytical chemical studies and the toxicity of the iodine mustard derivative ruled out the role of chemical reactions yielding differentially toxic species as being responsible for the differences in HD toxicity observed. This work demonstrates that the early events that HD sets into motion to cause toxicity are dependent on ionic environment, possibly due to intracellular pH deregulation.

Role of the sodium hydrogen exchanger in maitotoxin-induced cell death in cultured rat cortical neurons.

Maitotoxin (MTX) is one of the most potent toxins known to date. It causes massive calcium (Ca(2+)) influx and necrotic cell death in various tissues. However, the exact mechanism(s) underlying its cellular toxicity is not fully understood. In the present study, the role of the sodium hydrogen exchanger (NHE) in MTX-induced increases in intracellular Ca(2+) and subsequent cell death were investigated in cultured rat cortical neurons. Intracellular Ca(2+) concentrations ([Ca(2+)](i)) were measured fluorimetrically using FURA-2 as the fluorescence indicator. Cell death was measured with the alamarBlue cell viability assay and the vital dye ethidium bromide (EB) uptake assay. Results showed that MTX increased, in a concentration dependent manner, both [Ca(2+)](i) and cell death in cortical neurons. Decreasing the pH of the treatment medium from 7.5 to 6.0 diminished MTX-induced cell death. The protection offered by lowering extracellular pH was not due to MTX degradation, because it was still effective even if the cells were treated with MTX in normal pH and then switched to a lower pH. Pretreatment of cells with the specific NHE inhibitor, 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), prevented MTX-induced increases in [Ca(2+)](i), as well as cell death in a concentration dependent manner. Furthermore, knockdown of NHE1 by SiRNA transfection suppressed MTX-induced cell death in human embryonic kidney (HEK) cells. Together, these results suggest that NHE1 plays a major role in MTX-induced neurotoxicity.

Hypothermia as an adjunct therapy to vesicant-induced skin injury.

OBJECTIVE: The notion that cooling vesicant-exposed tissue may ameliorate or prevent resultant injury is not a novel concept. During both World Wars, studies were conducted that investigated this potential mode of therapy with sulfur mustard and seemed to conclude that there might be merit in pursuing this research direction. However, it does not appear that these studies were followed up vigorously, and the literature that describes this work is not readily accessible. In this report, we compare the toxicities of lewisite and sulfur mustard in vitro and in vivo and also provide an overview of historical and recent work on the effect of temperature on the toxicity of these vesicating chemical warfare agents. METHODS: Tissue culture and animal studies were utilized to examine the effects of hypothermia on vesicant-induced toxicity. RESULTS: Cytotoxicity was either significantly delayed (lewisite) or prevented (sulfur mustard) when cultures were maintained at 25 degrees C. However, the effects of hypothermia on sulfur mustard-induced cell death were reversible when the cells were returned to 37 degrees C. Despite these in vitro results, animal studies demonstrated that the therapeutic cooling of both mustard sulfur-exposed and lewisite-exposed skin resulted in dramatic and permanent protection against injury. Cooling also increased the therapeutic window in which drugs were effective against vesicant agents in tissue culture and lewisite-induced skin injury. CONCLUSIONS: The simple and noninvasive application of cooling measures may not only provide significant therapeutic relief to vesicant-exposed skin but also increase the therapeutic window in which medical countermeasures against vesicant agents are useful.

Attenuation of maitotoxin-induced cytotoxicity in rat aortic smooth muscle cells by inhibitors of Na+/Ca2+ exchange, and calpain activation.

The highly potent marine toxin maitotoxin (MTX) evoked an increase in cytosolic Ca(2+) levels in fura-2 loaded rat aortic smooth muscle cells, which was dependent on extracellular Ca(2+). This increase was almost fully inhibited by KB-R7943, a potent selective inhibitor of the reverse mode of the Na(+)/Ca(2+) exchanger (NCX). Cell viability was assessed using ethidium bromide uptake and the alamarBlue cytotoxicity assay. In both assays MTX-induced toxicity was attenuated by KB-R7943, as well as by MDL 28170, a membrane permeable calpain inhibitor. Maitotoxin-evoked contractions of rat aortic strip preparations in vitro, which persist following washout of the toxin, were relaxed by subsequent addition of KB-R7943 or MDL 28170, either in the presence of, or following washout of MTX. These results suggest that MTX targets the Na(+)/Ca(2+) exchanger and causes it to operate in reverse mode (Na(+) efflux/Ca(2+) influx), thus leading to calpain activation, NCX cleavage, secondary Ca(2+) overload and cell death.

pH-dependent toxicity of sulphur mustard in vitro.

The dependence of sulphur mustard (HD) toxicity on intracellular (pH(i)) and extracellular pH was examined in CHO-K1 cells. HD produced an immediate and significant concentration-dependent decline in cytosolic pH, and also inhibited the mechanisms responsible for restoring pH(i) to physiological values. The concentration-response of HD-induced cytosolic acidification, closely paralleled the acidification of the extracellular buffer through HD hydrolysis. A viability study was carried out in order to assess the importance of HD-induced cytosolic acidification. Cultures were exposed to HD for 1 h in media that were adjusted through a pH range (pH 5.0-10), and the 24 h LC(50) values were assessed using the viability indicator dye alamarBlue. The toxicity of HD was found to be dependent on extracellular pH, with a greater than eight-fold increase in LD(50) obtained in cultures treated with HD at pH 9.5, compared to those treated at pH 5.0. Assays of apoptotic cell death, including morphology, soluble DNA, caspase-3 activity and TUNEL also showed that as pH was increased, much greater HD concentrations were required to cause cell death. The modest decline in HD half-life measured in buffers of increasing pH, did not account for the protective effects of basic pH. The early event(s) that HD initiates to eventually culminate in cell death are not known. However, based on the data obtained in this study, we propose that HD causes an extracellular acidification through chemical hydrolysis and that this, in both a concentration and temporally related fashion, results in cytosolic acidification. Furthermore, HD also acts to poison the antiporter systems responsible for maintaining physiological pH(i), so that the cells are unable to recover from this insult. It is this irreversible decline in pH(i) that initiates the cascade of events that results in HD-induced cell death.