An inhalational swine model for the characterization of physiological effects and toxicological profile associated with cyanide poisoning.

Cyanides are highly toxic compounds that have been used as weapons of terrorism throughout history. Cyanide (CN) is acutely toxic by all routes of administration; however, inhalation is the main exposure route. To adequately test effective countermeasures against inhalational CN threats, robust and well-characterized animal models are needed. This paper describes the initial development of a hydrogen cyanide (HCN) exposure swine model for documenting the physiological effects and toxicological profile during and after HCN inhalation exposure. Animals were implanted with telemetry transmitters for heart rate (HR), blood pressure, and electrocardiogram monitoring, and vascular access ports for serial blood collections. Nine female swine were exposed to HCN concentrations of 500 ± 6 ppm while breathing parameters were monitored real-time. Inhaled HCN doses ranged from 2.02 to 2.83 mg/kg. Clinical signs included vocalization, agitation, salivation, respiratory distress and apnea. After HCN exposure initiation, systemic arterial pressure fell dramatically with a concomitant increase in HR. Blood samples were collected to determine CN blood levels using LC-MS/MS and blood gas analysis. In summary, the developed HCN inhalation swine model permitted documentation of the physiological effects associated with CN poisoning. This model could be used to evaluate potential CN medical countermeasures in the event of a public health emergency stemming from inhalational CN threats.

Development of a hydrogen cyanide inhalation exposure system and determination of the inhaled median lethal dose in the swine model.

OBJECTIVE: Cyanide is a highly toxic chemical, and acute exposure depletes cells and tissue of oxygen, depressing the respiratory, cardiovascular and neurological systems and potentially leading to death. Cyanide has been used as a weapon since ancient Rome and continues to pose a potential threat today. A well-characterized animal model is necessary for the development of novel methods of rapid detection and treatment. This manuscript describes the development of an inhalation exposure system designed to evaluate the lethality of acute cyanide inhalation in the porcine model. MATERIALS AND METHODS: A custom designed hydrogen cyanide (HCN) inhalation exposure system provided stable cyanide concentrations to un-anesthetized swine while monitoring respiratory parameters. Real-time respiratory monitoring, cyanide concentration and body weight were used to calculate inhaled doses. RESULTS: The inhalation exposure system generated controlled HCN ranging from 260 to 986 ppm to achieve inhaled doses between 1.78 and 3.97 mg/kg. Based on survival outcomes, the median lethal dose was determined to be 2.21 mg/kg, and the median lethal exposure level was 5893 mg min/m3. DISCUSSION: The ability of the HCN inhalation exposure system to deliver target inhaled doses and the determination of the inhaled median lethal dose in swine support the use of the exposure system and animal model for the evaluation of medical countermeasures of acute inhaled HCN toxicity.

Kinetic analysis of oxime-assisted reactivation of human, Guinea pig, and rat acetylcholinesterase inhibited by the organophosphorus pesticide metabolite phorate oxon (PHO).

Phorate is a highly toxic agricultural pesticide currently in use throughout the world. Like many other organophosphorus (OP) pesticides, the primary mechanism of the acute toxicity of phorate is acetylcholinesterase (AChE) inhibition mediated by its bioactivated oxon metabolite. AChE reactivation is a critical aspect in the treatment of acute OP intoxication. Unfortunately, very little is currently known about the capacity of various oximes to rescue phorate oxon (PHO)-inhibited AChE. To help fill this knowledge gap, we evaluated the kinetics of inhibition, reactivation, and aging of PHO using recombinant AChE derived from three species (rat, guinea pig and human) commonly utilized to study the toxicity of OP compounds and five oximes that are currently fielded (or have been deemed extremely promising) as anti-OP therapies by various nations around the globe: 2-PAM Cl, HI-6 DMS, obidoxime Cl2, MMB4-DMS, and HLö7 DMS. The inhibition rate constants (ki) for PHO were calculated for AChE derived from each species and found to be low (i.e., 4.8×103 to 1.4×104M-1min-1) compared to many other OPs. Obidoxime Cl2 was the most effective reactivator tested. The aging rate of PHO-inhibited AChE was very slow (limited aging was observed out to 48h) for all three species. CONCLUSIONS: (1) Obidoxime Cl2 was the most effective reactivator tested. (2) 2-PAM Cl, showed limited effectiveness in reactivating PHO-inhibited AChE, suggesting that it may have limited usefulness in the clinical management of acute PHO intoxication. (3) The therapeutic window for oxime administration following exposure to phorate (or PHO) is not limited by aging.

Sodium Nitrite and Sodium Thiosulfate Are Effective Against Acute Cyanide Poisoning When Administered by Intramuscular Injection.

STUDY OBJECTIVE: The 2 antidotes for acute cyanide poisoning in the United States must be administered by intravenous injection. In the out-of-hospital setting, intravenous injection is not practical, particularly for mass casualties, and intramuscular injection would be preferred. The purpose of this study is to determine whether sodium nitrite and sodium thiosulfate are effective cyanide antidotes when administered by intramuscular injection. METHODS: We used a randomized, nonblinded, parallel-group study design in 3 mammalian models: cyanide gas inhalation in mice, with treatment postexposure; intravenous sodium cyanide infusion in rabbits, with severe hypotension as the trigger for treatment; and intravenous potassium cyanide infusion in pigs, with apnea as the trigger for treatment. The drugs were administered by intramuscular injection, and all 3 models were lethal in the absence of therapy. RESULTS: We found that sodium nitrite and sodium thiosulfate individually rescued 100% of the mice, and that the combination of the 2 drugs rescued 73% of the rabbits and 80% of the pigs. In all 3 species, survival in treated animals was significantly better than in control animals (log rank test, P<.05). In the pigs, the drugs attenuated an increase in the plasma lactate concentration within 5 minutes postantidote injection (difference: plasma lactate, saline solution-treated versus nitrite- or thiosulfate-treated 1.76 [95% confidence interval 1.25 to 2.27]). CONCLUSION: We conclude that sodium nitrite and sodium thiosulfate administered by intramuscular injection are effective against severe cyanide poisoning in 3 clinically relevant animal models of out-of-hospital emergency care.

Efficacy of Recommended Prehospital Human Equivalent Doses of Atropine and Pralidoxime Against the Toxic Effects of Carbamate Poisoning in the Hartley Guinea Pig.

PURPOSE: Aldicarb and methomyl are carbamate pesticides commonly implicated in human poisonings. The primary toxic mechanism of action for carbamate poisoning is cholinesterase (ChE) inhibition. As such, it is logical to assume that the currently accepted therapies for organophosphate poisoning (muscarinic antagonist atropine and the oxime acetylcholinesterase reactivator pralidoxime chloride [2-PAM Cl]) could afford therapeutic protection. However, oximes have been shown to be contraindicated for poisoning by some carbamates. METHODS: A protective ratio study was conducted in guinea pigs to evaluate the efficacy of atropine and 2-PAM Cl. The ChE activity was determined in both the blood and the cerebral cortex. RESULTS: Coadministration of atropine free base (0.4 mg/kg) and 2-PAM Cl (25.7 mg/kg) demonstrated protective ratios of 2 and 3 against aldicarb and methomyl, respectively, relative to saline. The data reported here show that this protection was primarily mediated by the action of atropine. The reactivator 2-PAM Cl had neither positive nor negative effects on survival. Both blood acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) activities were significantly reduced at 15 minutes postchallenge but gradually returned to normal within 24 hours. Analysis of cerebral cortex showed that BChE, but not AChE, activity was reduced in animals that succumbed prior to 24 hours after challenge. CONCLUSION: The results suggest that coadministration of atropine and 2-PAM Cl at the currently recommended human equivalent doses for use in the prehospital setting to treat organophosphorus nerve agent and pesticide poisoning would likely also be effective against aldicarb or methomyl poisoning.

Characterization of a nose-only inhaled phosgene acute lung injury mouse model.

CONTEXT: Phosgene's primary mode of action is as a pulmonary irritant characterized by its early latent phase where life-threatening, non-cardiogenic pulmonary edema is typically observed 6-24 h post-exposure. OBJECTIVE: To develop an inhaled phosgene acute lung injury (ALI) model in C57BL/6 mice that can be used to screen potential medical countermeasures. METHODS: A Cannon style nose-only inhalation exposure tower was used to expose mice to phosgene (8 ppm) or air (sham). An inhalation lethality study was conducted to determine the 8 ppm median lethal exposure (LCt50) at 24 and 48 h post-exposure. The model was then developed at 1.2 times the 24 h LCt50. At predetermined serial sacrifice time points, survivors were euthanized, body and lung weights collected, and lung tissues processed for histopathology. Additionally, post-exposure clinical observations were used to assess quality of life. RESULTS AND DISCUSSION: The 24-hour LCt50 was 226 ppm*min (8 ppm for 28.2 min) and the 48-hour LCt50 was 215 ppm*min (8 ppm for 26.9 min). The phosgene exposed animals had a distinct progression of clinical signs, histopathological changes and increased lung/body weight ratios. Early indicators of a 1.2 times the 24-hour LCt50 phosgene exposure were significant changes in the lung-to-body weight ratios by 4 h post-exposure. The progression of clinical signs and histopathological changes were important endpoints for characterizing phosgene-induced ALI for future countermeasure studies. CONCLUSION: An 8 ppm phosgene exposure for 34 min (1.2 × LCt50) is the minimum challenge recommended for evaluating therapeutic interventions. The predicted higher mortality in the phosgene-only controls will help demonstrate efficacy of candidate treatments and increase the probability that a change in survival rate is statistically significant.

A comprehensive evaluation of the efficacy of leading oxime therapies in guinea pigs exposed to organophosphorus chemical warfare agents or pesticides.

The currently fielded pre-hospital therapeutic regimen for the treatment of organophosphorus (OP) poisoning in the United States (U.S.) is the administration of atropine in combination with an oxime antidote (2-PAM Cl) to reactivate inhibited acetylcholinesterase (AChE). Depending on clinical symptoms, an anticonvulsant, e.g., diazepam, may also be administered. Unfortunately, 2-PAM Cl does not offer sufficient protection across the range of OP threat agents, and there is some question as to whether it is the most effective oxime compound available. The objective of the present study is to identify an oxime antidote, under standardized and comparable conditions, that offers protection at the FDA approved human equivalent dose (HED) of 2-PAM Cl against tabun (GA), sarin (GB), soman (GD), cyclosarin (GF), and VX, and the pesticides paraoxon, chlorpyrifos oxon, and phorate oxon. Male Hartley guinea pigs were subcutaneously challenged with a lethal level of OP and treated at approximately 1 min post challenge with atropine followed by equimolar oxime therapy (2-PAM Cl, HI-6 DMS, obidoxime Cl2, TMB-4, MMB4-DMS, HLö-7 DMS, MINA, and RS194B) or therapeutic-index (TI) level therapy (HI-6 DMS, MMB4-DMS, MINA, and RS194B). Clinical signs of toxicity were observed for 24 h post challenge and blood cholinesterase [AChE and butyrylcholinesterase (BChE)] activity was analyzed utilizing a modified Ellman's method. When the oxime is standardized against the HED of 2-PAM Cl for guinea pigs, the evidence from clinical observations, lethality, quality of life (QOL) scores, and cholinesterase reactivation rates across all OPs indicated that MMB4 DMS and HLö-7 DMS were the two most consistently efficacious oximes.

Structural changes in hair follicles and sebaceous glands of hairless mice following exposure to sulfur mustard.

Sulfur mustard (SM) is a bifunctional alkylating agent causing skin inflammation, edema and blistering. A hallmark of SM-induced toxicity is follicular and interfollicular epithelial damage. In the present studies we determined if SM-induced structural alterations in hair follicles and sebaceous glands were correlated with cell damage, inflammation and wound healing. The dorsal skin of hairless mice was treated with saturated SM vapor. One to seven days later, epithelial cell karyolysis within the hair root sheath, infundibulum and isthmus was apparent, along with reduced numbers of sebocytes. Increased numbers of utriculi, some with connections to the skin surface, and engorged dermal cysts were also evident. This was associated with marked changes in expression of markers of DNA damage (phospho-H2A.X), apoptosis (cleaved caspase-3), and wound healing (FGFR2 and galectin-3) throughout pilosebaceous units. Conversely, fatty acid synthase and galectin-3 were down-regulated in sebocytes after SM. Decreased numbers of hair follicles and increased numbers of inflammatory cells surrounding the utriculi and follicular cysts were noted within the wound 3-7 days post-SM exposure. Expression of phospho-H2A.X, cleaved caspase-3, FGFR2 and galectin-3 was decreased in dysplastic follicular epidermis. Fourteen days after SM, engorged follicular cysts which expressed galectin-3 were noted within hyperplastic epidermis. Galectin-3 was also expressed in basal keratinocytes and in the first few layers of suprabasal keratinocytes in neoepidermis formed during wound healing indicating that this lectin is important in the early stages of keratinocyte differentiation. These data indicate that hair follicles and sebaceous glands are targets for SM in the skin.

Structural changes in the skin of hairless mice following exposure to sulfur mustard correlate with inflammation and DNA damage.

Sulfur mustard (SM, bis(2-chloroethyl)sulfide) is a bifunctional alkylating agent that causes dermal inflammation, edema and blistering. To investigate the pathogenesis of SM-induced injury, we used a vapor cup model which provides an occlusive environment in which SM is in constant contact with the skin. The dorsal skin of SKH-1 hairless mice was exposed to saturated SM vapor or air control. Histopathological changes, inflammatory markers and DNA damage were analyzed 1-14 days later. After 1 day, SM caused epidermal thinning, stratum corneum shedding, basal cell karyolysis, hemorrhage and macrophage and neutrophil accumulation in the dermis. Cleaved caspase-3 and phosphorylated histone 2A.X (phospho-H2A.X), markers of apoptosis and DNA damage, respectively, were increased whereas proliferating cell nuclear antigen (PCNA) was down-regulated after SM exposure. By 3 days, epithelial cell hypertrophy, edema, parakeratosis and loss of epidermal structures were noted. Enzymes generating pro-inflammatory mediators including myeloperoxidase and cyclooxygenase-2 were upregulated. After 7 days, keratin-10, a differentiation marker, was evident in the stratum corneum. This was associated with an underlying eschar, as neoepidermis began to migrate at the wound edges. Trichrome staining revealed increased collagen deposition in the dermis. PCNA expression in the epidermis was correlated with hyperplasia, hyperkeratosis, and parakeratosis. By 14 days, there was epidermal regeneration with extensive hyperplasia, and reduced expression of cleaved caspase-3, cyclooxygenase-2 and phospho-H2A.X. These findings are consistent with the pathophysiology of SM-induced skin injury in humans suggesting that the hairless mouse can be used to investigate the dermatoxicity of vesicants and the potential efficacy of countermeasures.

SM1997 downregulates mustard-induced enzymes that disrupt corneal epithelial attachment.

Amino-Plex (SM1997) is a spray or liquid cosmeceutical that has been used for skin dryness, aging, or sun exposure. Its formulation includes electrolytes, trace minerals, amino acids, peptides, nucleosides and nucleotides, all substances that are <10 kDa. It is designed to increase oxygen levels in cells, improve glucose transport, stimulate ATP synthesis, and stimulate collagen formation, actions that can help facilitate repair of damaged cells. It also supports collagen synthesis and formation of healthy granulation tissue, accelerating reepithelization of damaged skin. Here, SM1997 has been tested as an agent to improve the healing of mustard injury to the cornea. The results indicate that SM1997 facilitates the retention of corneal epithelial attachment when applied to corneal organ cultures after nitrogen mustard exposure. In addition, it reduces the activation of enzymes that lead to epithelial-stromal separation, namely, ADAM17 and MMP-9. Therefore, SM1997 should be further investigated as a potential therapy sulfur mustard and nitrogen mustard exposure.