Michael C. Babin

Systemic administration of candidate antivesicants to protect against topically applied sulfur mustard in the mouse ear vesicant model (MEVM)

The mouse ear vesicant model (MEVM) provides a quantitative edema response as well as histopathological and biochemical endpoints as measurements of inflammation and tissue damage following exposure to the chemical warfare agent sulfur mustard (HD). In the MEVM, several topically applied anti‐inflammatory agents provided a significant degree of protection against HD‐induced edema and dermal–epidermal separation. This study evaluated the protective effects of three of these pharmacological compounds when administered systemically in the MEVM. Alzet osmotic pumps were used to deliver a subcutaneous dose of the appropriate anti‐inflammatory agent, starting 24 h before exposure to sulfur mustard and continuing until 24 h post‐exposure to HD. Twenty‐four hours after pump implantation, 5 μl of a 195 mM (0.16 mg) solution of sulfur mustard (density = 1.27 g ml−1; MW = 159; purity = 97.5%) in methylene chloride was applied to the inner surface of the right ear of each mouse. Sulfur mustard injury in the mouse ear was measured by both edema response (fluid accumulation) and histopathological damage (necrosis, epidermal–dermal separation). The systemic administration of hydrocortisone, indomethacin and olvanil provided a significant reduction in edema (24%, 26% and 22%, respectively) from the positive control. Compared to HD‐positive controls, hydrocortisone, indomethacin and olvanil caused a significant reduction in subepidermal blisters (71%, 52% and 57%, respectively) whereas only hydrocortisone produced a significant reduction in contralateral epidermal necrosis (41%). We show here that these anti‐inflammatory drugs are effective when administered systemically in the MEVM. Published in 2000 by John Wiley & Sons, Ltd.

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.

Immunohistochemical characterization of the basement membrane epitopes in bis(2-chloroethyl) sulfide-induced toxicity in mouse ear skin

Sulfur mustard (bis(2‐chloroethyl) sulfide (HD)), a potent cutaneous vesicant and bifunctional alkylating agent, produces significant time‐dependent histopathological changes in the skin of the mouse. The right ears of male CD1 mice were exposed topically to 5.0 μl of 195 mM (0.16 mg) HD in dichloromethane and harvested at 6, 12, 18 and 24 h. The left ear control was dosed with 5.0 μl of dichloromethane. In all controls and HD‐treated mouse ear, moderate immunofluorescence staining was seen at the epidermal–dermal junction with bullous pemphigoid (BP), epidermolysis bullosa acquisita (EBA) and laminin (Lam), and light staining was observed with bullous pemphigoid 180 (BP180), fibronectin (Fn) and type IV collagen (Coll IV). Mouse anti‐human monoclonal antibodies for GB3, L3d and 19‐DEJ‐1 (Uncein) did not cross‐react. In microvesicles, BP, BP180 and Fn showed areas of light focal epidermal staining and homogeneous dermal staining, and EBA, Lam and Coll IV showed moderate dermal staining. Both BP and Fn exhibited weak, inconsistent staining with time. Immunoelectron microscopy (IEM) revealed similar results, with an increase in cell damage from 6 to 24 h, which corresponded to a decrease in staining intensity. Cell proliferation, expressed as the growth fraction of proliferating cell nuclear antigen (PCNA), showed an increase in cell damage. The growth fraction was lower in the inner ear and showed time‐dependent differences. The immunofluorescence and IEM results indicate that HD causes an undulating inconsistent separation in the uppermost lamina lucida with focal cleavage into the lower portion of the basal keratinocytes just above the plasma membrane. Although this pattern of separation differs from other in vivo models in which the split occurs exclusively within the lamina lucida, this should not preclude its role as a screening model to study the effects and development of specific prophylactic and therapeutic strategies. Copyright

Doxycycline Hydrogels as a Potential Therapy for Ocular Vesicant Injury

PURPOSE The goals of this study were (1) to compare the injury at the basement membrane zone (BMZ) of rabbit corneal organ cultures exposed to half mustard (2 chloroethyl ethyl sulfide, CEES) and nitrogen mustard with that of in vivo rabbit eyes exposed to sulfur mustard (SM); (2) to test the efficacy of 4 tetracycline derivatives in attenuating vesicant-induced BMZ disruption in the 24-h period postexposure; and (3) to use the most effective tetracycline derivative to compare the improvement of injury when the drug is delivered as drops or hydrogels to eyes exposed in vivo to SM. METHODS Histological analysis of hematoxylin and eosin–stained sections was performed; the ultrastructure of the corneal BMZ was evaluated by transmission electron microscopy; matrix metalloproteinase-9 was assessed by immunofluorescence; doxycycline as drops or a hydrogel was applied daily for 28 days to eyes exposed in vivo to SM. Corneal edema was assessed by pachymetry and the extent of neovascularization was graded by length of longest vessel in each quadrant. RESULTS Injury to the BMZ was highly similar with all vesicants, but varied in degree of severity. The effectiveness of the 4 drugs in retaining BMZ integrity did not correlate with their ability to attenuate matrix metalloproteinase-9 expression at the epithelial–stromal border. Doxycycline was most effective on organ cultures; therefore, it was applied as drops or a hydrogel to rabbit corneas exposed in vivo to SM. Eyes were examined at 1, 3, 7, and 28 days after exposure. At 7 and 28 days after SM exposure, eyes treated with doxycycline were greatly improved over those that received no therapy. Corneal thickness decreased somewhat faster using doxycycline drops, whereas the hydrogel formulation decreased the incidence of neovascularization. CONCLUSIONS Corneal cultures exposed to 2-chloroethyl ethyl sulfide and nitrogen mustard were effective models to simulate in vivo SM exposures. Doxycycline as drops and hydrogels ameliorated vesicant injury. With in vivo exposed animals, the drops reduced edema faster than the hydrogels, but use of the hydrogels significantly reduced neovascularization. The data provide proof of principle that a hydrogel formulation of doxycycline as a daily therapy for ocular vesicant injury should be further investigated.

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 Ellmans 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.

Modulation of sulfur mustard-induced inflammation and gene expression by olvanil in the hairless mouse vesicant model

Cutaneous exposure to sulfur mustard [bis(2-chloroethyl) sulfide (SM)] produces a delayed inflammatory skin response that is followed by severe dermal injury. Assessment of anti-inflammatory therapies against SM-induced skin injury has mainly relied on qualitative histopathological evaluation. The goal of this study was to identify proinflammatory biomarkers in the hairless mouse vesicant model that could be used as additional indicators of SM-induced skin injury for evaluating anti-inflammatory treatment. SM-induced inflammation was determined at 2, 6, and 24 hr postexposure by changes in edema. Ribonuclease protection assay (RPA) was used to determine changes in gene expression of inflammatory mediators. At 2, 6, and 24 hr postexposure, a time-dependent increase in edema was observed in SM-exposed skin, which was significant at 6 and 24 hr when compared to unexposed controls. Ribonuclease protection assay analysis revealed a two-fold or greater increase in monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-2 (MIP-2), MIP-1α, tumor necrosis factor-α, and interleukin (IL)-1β following exposure to SM when compared to unexposed controls. A significant time-dependent increase was observed in MCP-1, MIP-1α, and IL-1β over the 24 hr time period. At 24 hr postexposure, skin treated with the anti-inflammatory drug olvanil showed a significant decrease in SM-induced edema. Additionally, mRNA levels of MCP-1, MIP-2, and IL-1β were decreased when compared to skin exposed to SM alone. In this study, we identified molecular biomarkers at the mRNA level, up-regulated in skin exposed to SM, which can be partially suppressed by olvanil. Further characterization of the mRNA and protein expression patterns of proinflammatory biomarkers may enable the use of other classes of anti-inflammatory drugs or therapeutic treatments against SM dermal injury.

Alterations of Gene Expression in Sulfur Mustard‐Exposed Skin Topically Treated with Vanilloids

Sulfur mustard [bis(2‐chloroethyl)sulfide, SM] is a chemical warfare agent that penetrates the skin rapidly and causes extensive blistering. Using the mouse ear vesicant model (MEVM), we evaluated the effect of topically applied anti‐inflammatory agents (octyl homovanillamide and heptyl isovanillamide) on ear edema formation and gene expression following SM exposure. Relative ear weight and real‐time reverse transcriptase polymerase chain reaction of GM‐CSF, IL‐1β, and IL‐6 were used to evaluate the effects of octyl homovanillamide and heptyl isovanillamide. Both vanilloids significantly reduced SM‐induced edema. At the single dose and number of animals/group tested, octyl homovanillamide produced a trend of reduced mRNA levels; however, the reduction was not significant for GM‐CSF, IL‐1β, or IL‐6. Heptyl isovanillamide significantly reduced (p ≤ 0.05) GM‐CSF, IL‐1β, and IL‐6 mRNA levels. These results show that octyl homovanillamide and heptyl isovanillamide reduce skin edema and heptyl isovanillamide significantly reduced cytokine mRNA expression following SM exposure. In addition to measuring edema formation, monitoring expression of biomarkers such as GM‐CSF, IL‐1β, and IL‐6 may also serve to evaluate therapeutic treatments against SM‐induced dermal injury.

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.

A Combination Drug Treatment Against Ocular Sulfur Mustard Injury

The eye is considered to be one of the most sensitive organs to sulfur mustard [bis(2‐chloroethyl) sulfide (SM)], with injuries ranging from mild conjunctivitis to advanced corneal disease. Even mild ocular involvement from sulfur mustard exposure can result in both physical and psychological incapacitation. In this study we explored the use of Food and Drug Administration (FDA) approved medications (prednisolone acetate ophthalmic suspension, triamcinolone, and cefazolin) as ocular treatments for sulfur mustard injury. Female New Zealand White rabbits were divided into a SM positive control group (n = 8) and a single treatment group (n = 7). At 10, 20, 30, 60, 90, and 120 min after SM exposure, two drops of prednisolone acetate ophthalmic suspension was administered to each treatment group rabbit while the control group received saline drops. At 120 min after SM exposure, each treatment group animal received a single 1.0 mL sub‐Tenons injection containing 20 mg triamcinolone and 50 mg cefazolin. Control group rabbits did not receive an injection. Rabbits were observed for a total of 16 weeks after SM exposure. Corneal thickness, corneal stromal injury, neovascularization (NV), eyelid notching, and chemosis were recorded weekly for 6 consecutive weeks and on week 16 after exposure. The SM treatment group at weeks 2, 3, and 4 had a significantly lower index value for corneal thickness than the SM positive control group. For corneal stromal injury, NV, eyelid notching, and chemosis, significant evidence of a protective effect due to treatment was seen at weeks 4, 5, and 6. In addition, corneal stromal injury was reduced at weeks 2 and 3 and notching at week 2. By week 3, all SM positive control animals developed NV in contrast to 1 of 7 treatment animals. By week 6 all positive control animals still exhibited NV compared to 2 of 7 treatment animals. These data suggest that prednisolone acetate suspension dosed for the first 2 h after SM exposure followed by a single sub‐Tenons injection of a triamcinolone/cefazolin combination is effective in treating the early stages of corneal injury from SM exposure.

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.