Immobilization of Russian VX skin depots by localized cooling: implications for decontamination and medical countermeasures.

The chemical weapon nerve agent known as Russian VX (VR) is a potent organophosphorus (OP) compound that is much less studied than its VX analogue with respect to toxicity, as well as to the effectiveness of several known countermeasures against it. An anaesthetized domestic swine model was utilized to assess several approaches in mitigating its toxicity, including the utility of cooling VR treated skin to increase the therapeutic window for treatment. The 6h LD50 for VR topically applied on the ear was 100 µg/kg. Treatment of VR exposed animals (5 × LD50) with pralidoxime (2PAM) very poorly regenerated inhibited blood cholinesterase activity, but was partially effective in preventing signs of OP poisoning and increasing survival. In contrast, treatment with the Hagedorn oxime HI-6 reactivated cholinesterase, eliminated all signs of poisoning and prevented death. Decontamination with the Reactive Skin Decontaminant Lotion (RSDL) 15 min after VR exposure was completely effective in preventing death. Cooling of the VR exposure sites for 2 or 6h prevented signs of OP poisoning and death during the cooling period. However, these animals died very quickly after the cessation of cooling, unless they were treated with oxime or decontaminated with RSDL. Blood analyses showed that cooling of agent exposure sites delayed the entry of VR into the bloodstream. Medical treatment with HI-6 and to a lesser extent 2PAM, or decontamination with RSDL are effective in protecting against the toxic effects of cutaneous exposure to VR. Immobilizing this agent (and related compounds) within the dermal reservoir by cooling the exposure sites, dramatically increases the therapeutic window in which these medical countermeasures are effective.

The therapeutic use of localized cooling in the treatment of VX poisoning.

The organophosphate (OP) nerve agent VX is a weaponized chemical warfare agent that has also been used by terrorists against civilians. This contact poison produces characteristic signs of OP poisoning, including miosis, salivation, mastication, dysrhythmias and respiratory distress prior to death. Although successful treatment of OP poisoning can be obtained through decontamination and/or oxime reactivation of agent-inhibited cholinesterase, medical countermeasures that increase the therapeutic window for these measures would be of benefit. An anaesthetized swine model was utilized to examine the effects of lethal VX exposure to the skin, followed by cooling the exposure site prior to decontamination or treatment. The cooling was simply accomplished by using crushed ice in grip-seal plastic bags applied to the exposure sites. Cooling of skin exposed to lethal doses of VX significantly increased the window of opportunity for successful decontamination using the Reactive Skin Decontaminant Lotion(®) (RSDL(®)) or treatment with the oxime antidotes HI-6 and 2PAM. Analyses of blood VX levels showed that cooling acted to slow or prevent the entry of VX into the bloodstream from the skin. If the exposure site is known, the simple and non-invasive application of cooling provides a safe means with which to dramatically increase the therapeutic window in which decontamination and/or antidote treatment against VX are life-saving.

Comparison of selected skin decontaminant products and regimens against VX in domestic swine.

An anesthetized domestic swine model was used to compare the efficacy and cross-contamination potential of selected skin decontaminant products and regimens against the chemical warfare agent, VX. Animals topically exposed to 2x, 3x or 5x LD(50) VX showed typical signs of organophosphate nerve agent poisoning, including miosis, salivation, mastication, dysrhythmias, and respiratory distress prior to death. Animals were exposed to 5x LD(50) VX and then decontaminated 45 min later with the reactive skin decontamination lotion (RSDL), Fuller's earth (FE), 0.5% hypochlorite, or soapy water. Survival was 100% when the reactive skin decontamination lotion or FE was utilized, although 50% of Fuller's earth-decontaminated animals exhibited serious signs of VX poisoning. Decontamination of VX-treated animals with 0.5% hypochlorite was less effective but also increased survival. Soapy water was ineffective in preventing lethality. Blood cholinesterase levels were not predictive of clinical outcome in decontaminated animals. The potential of "decontaminated" VX in open wounds to cause poisoning was assessed by vigorously mixing 5x LD(50) VX with the test decontaminants for 5 min and then placing the mixture onto a full-thickness skin wound. Soapy water was ineffective in preventing lethality. Although treatment with dry Fuller's earth prevented death and all signs of organophosphate poisoning, a significant proportion of treated animals decontaminated with Fuller's earth in aqueous suspension exhibited serious signs of organophosphate poisoning, suggesting that live agent may be desorbed from Fuller's earth when it is exposed to a liquid environment. Animals treated with reactive skin decontamination lotion or 0.5% hypochlorite-VX mixtures showed no signs of organophosphate poisoning during the 6- h test period.

Lack of a role for creatine phosphate kinase in sulphur mustard-induced cytotoxicity.

Several compounds involved in the creatine phosphate kinase (CPK) pathway were evaluated for their protective effects against the chemical warfare (CW) agent sulphur mustard (HD), in primary chick embryo neuron and first passage human skin keratinocyte cultures. High concentrations of both creatine and creatine phosphate were found to be protective under all culture conditions and increased the LC(50) of HD in both culture systems up to approximately 250%. Little difference was observed in the protective activity of these compounds in undifferentiated versus differentiated neuronal culture, or in proliferating versus differentiating cultures of keratinocytes. The protective effect of these compounds was found to be strictly prophylactic in nature. Although a modest decline in HD half-life was measured in buffer containing creatine phosphate, this did not account for the protective effects of this compound. In contrast to historical literature reporting 90-100% HD-induced CPK inhibition of purified enzyme, less than 30% of CPK activity was found to be inhibited by HD in both human keratinocytes and in swine blood plasma. Incubation of keratinocyte cultures with creatine or creatine phosphate prior to HD exposure did not alter CPK activity, compared with HD-only treated cultures. Although high mM concentrations of both creatine and creatine phosphate exert significant protective effects against HD, these results do not support a role for CPK in its toxicity or in the development of medical countermeasures against this CW agent.

Swine models in the design of more effective medical countermeasures against organophosphorus poisoning.

Although the three most commonly used large mammal species in the safety assessment of drugs remain the dog, the macaque and the marmoset, swine, especially minipigs, have also been widely used over the years in many toxicological studies. Swine present a number of interesting biological and physiological characteristics. Similarities in skin properties with humans have led to extensive in vitro and in vivo studies. There is a specific interest in cardiovascular research, as well as in anaesthesiology and critical care medicine due to common features of swine and human physiology. Although knowledge of swine brain structure and functions remains incomplete, data does exist. The multiple blood sampling that is necessary in pharmacokinetic and toxicokinetic studies are possible, as well as multiparametric monitoring and interventions with equipment used in human clinical settings. Practicality (handling), scientific (stress reduction) and ethical (invasive monitoring) reasons have led research teams to incorporate anaesthesia into their paradigms which makes the analysis of data increasingly difficult. Although not substantiated by scientific data, the swine appears to have an intermediate position in the scale of public perception between non-human primates and animals commonly referred to as pets (i.e. dogs and cats) and rodents. The benefits of the swine model justify the use of these animals in the design of more effective medical countermeasures against known chemical warfare agents (nerve agents, vesicants and lung damaging agents). Exposure to organophosphorus (OP) pesticides represents a severe health issue in developing countries, while OP intoxication with the more lethal military nerve agents is not only of military concern but also a terrorist threat. Tailoring therapeutic regimens to the reality of OP poisoning is of the utmost importance when little experimental data and sparse human clinical data are available in the decision making process. We will present some of the advantages and disadvantages of the swine model in OP countermeasures elaborating on two examples. First, we will present the issues related to the use of anaesthesia during experimental OP poisoning and second we will show how results from experiments with swine can be integrated into a kinetic-based dynamic model to evaluate oxime efficacy. A better knowledge of OP poisoning in swine (comparative toxicokinetics, pharmacokinetics and biochemistry) is definitely necessary before accepting it as a first choice non-rodent model. However, there exists a large amount of data in the model on anaesthesia and different types of shock favouring their use for evaluation of complex situations such as the anaesthesia of OP poisoned patients and combined injuries.

Volatile anesthetics give a false-positive reading in chemical agent monitors in the "H" mode.

Chemical agent monitors (CAMs) are routinely used by the armed forces and emergency response teams of many countries for the detection of the vesicant sulfur mustard (HD) and the G series of organophosphate nerve agents. Ambient operating room isoflurane levels were found to produce strong positive signals in the "H" mode when the CAM was used to monitor the efficacy of decontamination procedures during routine surgical procedures on HD-poisoned animals requiring up to 8 hours of general anesthesia. Subsequent testing showed that isoflurane, as well as desflurane, sevoflurane, halothane and methoxyflurane, produce two ionization peaks in the CAM response. One of these peaks is interpreted by the CAM processing software as HD, resulting in a CAM "H" mode bar response. No interference was encountered with isoflurane, desflurane, and sevoflurane when the CAM was set to the "G" mode, although extremely high (nonclinical) concentrations of halothane and methoxyflurane yielded a weakly positive bar response. These findings have potentially serious ramifications for the medical management of patients resulting from terrorist, military, or chemical agent decommissioning activity when concomitant chemical injuries are also possible.

A comparison of the effects of sarin and succinylcholine on respiratory parameters in anesthetized domestic swine.

Differences in the "respiratory paralysis" caused by sarin (GB) and succinylcholine (SDC) were observed in a domestic swine model using a bedside pulmonary dynamics monitor. GB was administered intravenously (9 micrograms/kg/30 min) and compared with SDC administered intravenously (20 mg/30 min). All animals developed respiratory insufficiency indicated by decreased respiratory frequency. Minute ventilation was relatively maintained in animals that received GB by increasing tidal volume, whereas both of these parameters decreased in animals that received SDC. GB animals showed an increase in airway resistance and work of breathing. The former was unchanged and the latter was decreased in animals that received SDC. Mouth occlusion pressure at 100 milliseconds and tidal volume were relatively maintained in GB animals but decreased in SDC animals, suggesting a central mechanism for respiratory paralysis with GB and a peripheral mechanism for respiratory paralysis with SDC.

Domestic swine model for the assessment of chemical warfare agent-anesthetic interactions: some effects of sulfur mustard.

A domestic swine model was developed to examine the interaction of chemical warfare agents with anesthetics and other drugs used during general anesthesia. Animals were fully instrumented, and clinically relevant physiological parameters were monitored throughout the experimental procedures. Exposure of animals under halothane anesthesia to the chemical warfare agent sulfur mustard (HD; 1 mg/kg intravenous) produced mild signs of systemic intoxication during the subsequent 5 hours. Induction doses of ketamine 1 hour after HD exposure resulted in periods of profound apnea, with continued respiratory distress for the next 2 hours. When animals were treated with HD 1 hour after the initiation of ketamine anesthesia, severe and persistent convulsion-like muscular activity was observed within 45 minutes of HD administration. This nonpurposeful activity was not ameliorated by diazepam but was dramatically reduced or eliminated by resumption of halothane anesthesia. Treatment of HD-intoxicated pigs with succinylcholine produced a prolonged apnea resulting in death. In these apparently mildly HD-intoxicated animals, the introduction of ketamine or succinylcholine can rapidly induce potentially life-threatening situations.

Effect of intracellular calcium modulation on sulfur mustard cytotoxicity in cultured human neonatal keratinocytes.

Previous studies in human skin keratinocyte cultures have shown that sulfur mustard (HD) induces an immediate and irreversible increase in internal free calcium levels that was independent of external calcium concentrations. These findings suggested a role for calcium in the aetiology of HD-induced cell death and that modulation of intracellular calcium concentrations may assist in providing protection against this agent. In the current work, actively proliferating and confluent cultures of first passage neonatal human skin keratinocytes were used to assess the effect of altered intra- and extracellular calcium levels on HD toxicity. Treatment of cultures with the endoplasmic reticulum calcium ATPase inhibitor thapsigargin, or the calcium chelator BAPTA-AM, which reduce HD-induced elevation of intracellular free calcium, did not modulate the toxicity of HD. Furthermore, alteration of external calcium concentrations during these same experiments failed to elicit any change in the viability of HD-exposed cells. Treatment of confluent cultures with ionomycin at either low (100 microM) or high (1.2 mM) external calcium concentrations also failed to modulate the toxicity of HD in any way. It appears that in neonatal human skin keratinocytes in culture, HD-induced intracellular calcium perturbation does not play a major role in HD-induced cytotoxicity.

Effects of selected arginine analogues on sulphur mustard toxicity in human and hairless guinea pig skin keratinocytes.

The toxicity of sulphur mustard (HD) was characterized in first passage cultures of human neonatal foreskin keratinocytes and then several arginine analogues were investigated to ascertain their efficacies in protecting against HD toxicity in this system. d- and l-nitroarginine methyl ester (d/l-NAME), l-phosphoarginine, and l-nitroarginine were all found to confer concentration-related protective effects against HD in confluent cultures. l-NAME was used to further characterize this protection and was only effective in cultures that were not actively proliferating. This compound was also found to be efficacious when added to the cultures up to several hours after HD exposure, although its continued presence was required in order for protection to be effective. The protective effects of l-thiocitrulline (l-TC) against HD toxicity were also assessed. This arginine analogue was extremely potent in preventing HD toxicity in actively proliferating, just-confluent, and postconfluent cultures in a concentration-dependent fashion (0.1-15 mM), with little HD toxicity apparent at high l-TC concentrations. Protection was prophylactic in nature, with l-TC having almost maximal effect when added to the cultures only 1 min prior to HD culture exposure. Efficacy then declined rapidly so that no protection was evident when l-TC was added 30 min post-HD. The effects of this drug were persistent, with no decrease in protective efficacy up to 4 days after HD exposure, even when l-TC was removed from the cultures. l-TC did not protect against the antimitotic effects of HD; while l-TC-protected cells were subcultured successfully, they displayed no clonogenic activity. Although l-TC is closely related structurally to protective nitroarginine derivatives, the characteristics of l-TC protection against HD were markedly different and suggest that they exert their protective activities at different sites. Administration of l-NAME and l-TC by a variety of routes did not result in consistent protection against topical vapor challenges of HD in hairless guinea pigs. However, both compounds were effective in preventing the toxicity of HD in primary cultures of hairless guinea pig skin keratinocytes, indicating that species differences were not likely to be responsible for the poor efficacy of these compounds in vivo.

Effect of lowered temperature on the toxicity of sulphur mustard in vitro and in vivo.

Primary cultures of chick embryo neurons were exposed to sulphur mustard (HD) and L-nitroarginine methyl ester (L-NAME) and then incubated at either 25 or 37 degrees C. Lowering the temperature of the cultures decreased the 24-h toxicity of HD, but did not increase the efficacy of L-NAME protection. However, the length of time post-HD treatment in which L-NAME was maximally effective in protecting against HD toxicity was dramatically enhanced, out to 12 h after HD exposure. In addition, the persistence of L-NAME protection of the cells against HD was significantly lengthened. Tests conducted in human skin keratinocytes also showed that lowering the incubation temperature of actively proliferating, just-confluent or post-confluent cultures significantly and persistently decreased the cytotoxicity of HD. The persistence of L-NAME protection was increased in non-proliferating cells. Finally, cooling of HD-vapour exposed sites on hairless guinea pigs for 4.5 h decreased the severity of the resultant lesions out to 72 h post-exposure.

Synergistic protective effects of selected arginine analogues against sulphur mustard toxicity in neuron culture.

Previous studies in this laboratory have shown that the arginine analogues L-thiocitrulline (L-TC) and L-nitroarginine methyl ester (L-NAME) have potent protective activity against sulphur mustard (HD) toxicity that was not related to their nitric oxide synthase inhibiting activities. Furthermore, their characteristics of action suggested that they act at different sites to exert their protection. L-TC acted rapidly (minutes of preincubation) and was equipotent in protecting either immature or mature cultures of chick embryo neurons against the toxicity of HD while L-NAME was only effective in mature cultures. Maximal protection occurred at mM drug concentrations and increased the LC50 of HD by approximately 200% (L-NAME) to approximately 800% (L-TC). L-NAME did not alter the efficacy of L-TC in immature cultures but increased the LC50 up to 1500% in mature cultures. Removal of L-NAME eliminated this synergism, leaving only the persistent protection of L-TC. L-Nitroarginine and d-NAME also increased the protective efficacy of L-TC in a concentration-related manner in mature cultures. The timing of drug administration before or after HD culture exposure was critical. Drug coadministration resulted in synergistic protection only when L-TC was added to the cultures prior to HD treatment. Thus, synergistic protective effects were also achieved when L-NAME was added up to 8 h after HD exposure, if they were pretreated with L-TC. Based on these findings, it is proposed that HD initiates its toxicity extremely rapidly through a cell surface-mediated event that can be blocked by L-TC. A signal is transduced into the cell that results in an additional event or lesion that manifests itself several hours downstream. This event/lesion progresses to cell death unless blocked reversibly by L-NAME.

Toxicity of sulfur mustard in primary neuron culture.

The toxicity of sulfur mustard (HD) was assessed in primary cultures of chick embryo forebrain neurons using several different endpoints. Mature neurons were found to be very sensitive to the toxic effects of this agent and tritiated arachidonic acid release, as well as the MTT, neutral red and alamarBlue cytotoxicity assays all gave LC(50) values in the low mum range. Maximal toxicity was initiated within minutes of culture exposure to HD and was not found to be associated with toxic mediator release into the medium. The characteristics of toxicity were quite different when comparing immature cultures to mature ones. Mature cultures were more sensitive to the toxicity of HD than were immature cultures, and maximal toxicity in mature cultures took longer to be expressed. In addition, the toxicity was found to be dependent on the initial seeding density, as well as on the age of the cultures at the time of chemical treatment. Although the reasons for these observations are unclear, the apparent dependence of HD toxicity on the differentiative maturity of the cultures may eventually provide some clues as to the mechanism of action of this chemical agent. Furthermore, the extreme sensitivity of these cells to the toxic effects of HD makes them a useful model system with which to screen for potential protective drug regimens against this chemical warfare agent.

l-thiocitrulline: A potent protective agent against the toxicity of sulphur mustard in vitro.

Previous studies in this laboratory have shown that the well-characterized arginine analogue nitric oxide synthase (NOS) inhibitor, l-nitroarginine methyl ester (l-NAME) is protective against the cytotoxicity of the vesicating agent bis (2-chloroethyl) sulphide (HD). Furthermore, these protective effects were not mediated through the inhibition of NOS. In studies designed to investigate the efficacy of additional arginine analogue NOS inhibitors as protective agents against this compound, l-thiocitrullline (l-TC) was identified as being extremely potent. In contrast to the protection conferred by l-NAME, however, l-TC was found to protect immature cultures of neurons against HD, as well as mature cultures. In addition, l-TC was found to exert its effects prophylactically, as opposed to the therapeutic characteristics of l-NAME. l-TC gave approximately 800% protection against HD with a one-h pretreatment compared to approximately 1500% protection with a 24-h pretreatment. The protection conferred by l-TC against HD was persistent, and did not require the continued presence of l-TC after the initial HD lesion was expressed. The protective characteristics of l-TC against HD are very different than those of l-NAME and suggest that these closely related arginine analogues act at as yet unidentified and different sites to exert their effects.

Effects of bis(2-chloroethyl)sulfide on ATP receptor-mediated responses of the rat vas deferens: possible relationship to cytotoxicity.

Extracellular ATP is a broad-spectrum cytotoxic agent that produces effects via cell surface P2 purinoceptors. The ligand-gated P2X purinoceptor subtype has very high sequence homology with the RP-2 gene, which encodes for apoptosis. The P2X RNA found in rat vas deferens is expressed preferentially by apoptotic thymocytes. P2X purinoceptor-mediated phasic (twitch) motor responses of the isolated rat vas deferens to neurogenic or exogenous ATP were rapidly, specifically and irreversibly potentiated by bis(2-chloroethyl)sulfide (HD 10-100 microM). Both untreated and HD-potentiated neurogenic responses were Ca++ dependent, blocked in the absence of Ca++ plus 0.1 mM EGTA, by the neuronal Ca++ channel blocker omega-conotoxin-MVIIC (3 microM), by the P2 purinoceptor antagonist suramin (100 microM) and by tetrodotoxin (100 nM). HD also potentiated the effects of ATP on isolated guinea pig taenia caecum, where the nucleotide acts at G protein-coupled P2Y purinoceptor subtypes to cause relaxation. HD failed to inhibit the metabolism of ATP by ecto-ATPase in vas deferens or to cause the release of endogenous ATP. Potentiation of the twitch response to electric field stimulation by HD was attenuated or eliminated in tissues excised from rats previously challenged with topically applied HD, suggesting that HD absorbed into the systemic circulation had already effected maximal potentiation of ATP responses before in vitro testing. The physiological consequences of HD-induced potentiation of the extracellular actions of ATP are discussed in relation to apoptosis and necrosis.

Characterization of the protective effects of L-nitroarginine methyl ester (L-NAME) against the toxicity of sulphur mustard in vitro.

The protective effects of L-nitroarginine methyl ester (L-NAME) against the toxicity of sulphur mustard (HD) was characterized in primary cultures of chick embryo forebrain neurons. These effects were not associated with the known nitric oxide synthase-inhibiting characteristics of this compound. No protection was evident in immature (1 day old) cultures. However, L-NAME pre-treatment resulted in a concentration-dependent protection against the toxicity of HD in mature (5 days and older) cultures, with maximal protection reaching greater than 220% at 5 mM L-NAME in terms of increasing the LC50 of control HD-treated cultures. Maximal protective effects were also achieved when L-NAME treatment was delayed up to 3 h post-HD exposure. These effects were reduced by 5 h and absent when the L-NAME was added to the cultures 8 h after HD exposure. Protection against the toxicity of HD was dependent on the continued presence of L-NAME in the medium and was persistent up to 48 h after HD exposure. This compound is one of the most effective drugs yet identified in protecting against the toxicity of HD and is the only one that exerts its effects therapeutically.

Modulation of sulfur mustard toxicity by arginine analogues and related nitric oxide synthase inhibitors in vitro.

The modulating effects of a series of arginine analogues and related nitric oxide synthase inhibitors against the toxicity of sulfur mustard (HD) in primary cultures of chick embryo forebrain neurons were examined. In addition to the previously identified protective compounds, D- and L-nitroarginine methyl ester, eight additional arginine analogues were shown to have significant, concentration-dependent protective characteristics against HD toxicity. Of these, L-nitroarginine was the most potent, increasing the LC50 of vehicle-pretreated HD-treated control cultures by approximately 350%. In addition to these protective agents, five compounds related to arginine were also identified that potentiated the toxicity of HD in the neuron cultures in a concentration-dependent manner. This action occurred at concentrations where these chemicals alone exhibited no toxicity. Characterization of the active compounds in this study showed that it was likely that the protective agents, as well as those compounds that potentiated HD toxicity, were exerting their effects at the same biochemical target, but not through the inhibition of nitric oxide synthase. Although the identity of this target site is as yet unknown, these studies demonstrate that subtle alterations to the arginine structure can yield compounds that differentially modulate the toxicity of HD through their activity at a common target site.

Modification of cytosolic free calcium concentrations in human keratinocytes after sulfur mustard exposure.

Exposure of confluent cultures of human skin keratinocytes to sulfur mustard (SM) induced an immediate and irreversible rise in internal free Ca(2+) levels that was independent of external Ca(2+) concentrations. The response was rapid, beginning within 1min after addition of SM to the cells, and sensitive, with significant effects observed at 100 mum. The rise in [Ca(2+)](INT) was unaffected by zero external Ca(2+) but was blocked by prior incubation with thapsigargin. The sensitivity to and irreversibility of the effects of SM on Ca(2+) levels was paralleled by cellular toxicity as assessed using three different cell viability assays. In addition, the time course of the onset of irreversible toxicity in our cultures coincides with the time course of effects on [Ca(2+)](INT). SM was also found to displace specifically bound ATP from purinergic (P(2)) receptors. These results suggest that therapies aimed at protecting internal stores of Ca(2+) from disruption by SM, perhaps at P(2) receptors, may provide substantial benefit in protecting human skin cells from the toxic effects of this vesicant.

Protective effect of an inhibitor of nitric oxide synthase on sulphur mustard toxicity in vitro.

Although sulphur mustard is one of the oldest chemical warfare agents, its mechanism of toxic action is still not understood and as a consequence, no antidotes exist that are effective against this agent. Pretreatment of chick embryo neuron cultures with the well-known nitric oxide synthase (NOS) inhibitor L-nitroarginine methyl ester (L-NAME) was found to confer significant protection against sulphur mustard-induced cell death. However, these protective effects were not mediated through the inhibition of NOS. These findings may provide clues to the eventual understanding to sulphur mustard toxicity, and also suggest that L-NAME has significant novel pharmacological effects other than the inhibition of NOS.