Skin Models Used to Define Mechanisms of Action of Sulfur Mustard

Abstract

Sulfur mustard (SM) is a threat to both civilian and military populations. Human skin is highly sensitive to SM, causing delayed erythema, edema, and inflammatory cell infiltration, followed by the appearance of large fluid-filled blisters. Skin wound repair is prolonged following blistering, which can result in impaired barrier function. Key to understanding the action of SM in the skin is the development of animal models that have a pathophysiology comparable to humans such that quantitative assessments of therapeutic drugs efficacy can be assessed. Two animal models, hairless guinea pigs and swine, are preferred to evaluate dermal products because their skin is morphologically similar to human skin. In these animal models, SM induces degradation of epidermal and dermal tissues but does not induce overt blistering, only microblistering. Mechanisms of wound healing are distinct in these animal models. Whereas a guinea pig heals by contraction, swine skin, like humans, heals by re-epithelialization. Mice, rats, and rabbits are also used for SM mechanistic studies. However, healing is also mediated by contraction; moreover, only microblistering is observed. Improvements in animal models are essential for the development of therapeutics to mitigate toxicity resulting from dermal exposure to SM.

Sulfur mustard (SM, bis 2-chloroethyl sulfide) is a potent skin vesicant synthesized for chemical warfare. As a bifunctional alkylating agent, SM initiates its action by modifying and disrupting cellular macromolecules, including DNA and proteins. ^1–5 Acute responses of skin to SM are typically characterized by delayed onset erythema and intense itching, followed by the formation of small fluid-filled vesicles; with time, these vesicles coalesce to form pendulous blisters. ^1,6,7 A necrotic layer and ulceration can form on the affected skin surface following rupture of the blisters. Responses of human skin to SM are multifactorial and depend on the dose and time following exposure, as well as environmental conditions such as temperature and humidity. ^7,8 Location of exposure sites on the body, variations in skin properties, and underlying disease states, along with age and sex, are all determinants of skin responses to SM. ⁸

To understand the mechanism of action of SM and develop medical countermeasure, various animal models have been utilized, including mice, rats, guinea pigs, rabbits, and pigs. ^9–12 Unfortunately, there are no simple or common animal models for SM injury that produce true blisters like humans. In this context, in describing early reporting on the use of human subjects for mustard research in 1919, Sollman explained that “experiments on animals was [sic] abandoned after a few trials, since their skin does not react in the same manner as human skin, and the effects that do occur are not easily graded.” ⁸ Blistering is not commonly observed in animals. ^13,14 To produce true blistering, either unconventional species must be used, or multistep procedures must be undertaken in common animal models. ¹⁴ For example, it has been reported that blisters can be produced on the skin of frogs, birds, and the inner ears of rabbits, ¹⁵ on the skin of isolated perfused pig flaps, ^15,16 and on guinea pig skin that has been thermally burned and allowed to re-epithelialize. ^16,17 Studies performed with SM on birds and frogs are limiting as their skin is not similar to human skin. For this reason, SM research has relied on the surrogate marker of microblistering or subepidermal blister formation at the dermal-epidermal junction, which occurs in rodents, rabbits, and pigs. ^18–20 SM is known to damage not only epidermal structures, including the basement membrane, but also stromal and vascular components of the skin tissue. ^21–23

Translating SM data from animals to humans has been challenging not only because there is little or no blistering, but also to additional factors such as distinct structural differences in the tissue, unique aspects of the immune system, and mechanisms of wound healing. For example, in mice, the skin and epidermis are thinner when compared to humans, there are fewer epidermal cell layers, a lack of epidermal ridges and eccrine sweat glands, and limited adherence to underlying tissues. ²⁴ In humans and pigs, wounds close by formation of granulation tissue followed by re-epithelialization ²⁴ ; in contrast, wound closure in rodents and rabbits is primarily by contraction, in part due to the presence of the panniculus carnosus. ²⁵ At later stages, tissue remodeling during wound healing occurs via fibroblast migration and myofibroblast activity. ²⁶ It should be noted that contraction is usually defined for incisional wounds ²⁷ ; the role of contraction in thermal and SM injury is not clear since the extent of tissue damage may not allow wound closure by the panniculus carnosus.

In rodent models, both haired and hairless strains have been used; hairless animals are advantageous largely due to the ease of visualizing a cutaneous response. ²⁸ Hair removal and associated inflammation are avoided with these animals. ²⁹ However, it should be noted that the skin of haired and hairless animal strains can be morphologically different. For example, the epidermis of mice of the most commonly used hairless strain, SKH1, is thicker than haired strains. ⁹ Haired and hairless strains are also genetically and immunologically distinct, complicating efforts to compare results from different laboratories. ³⁰ Little information is available on differences in wound healing in response to chemical and thermal injury in haired and hairless mouse strains.

In most animal models, different phases of SM injury can be defined, including latency, erythema/inflammation, microblistering, ulceration/eschar formation, and wound healing. The extent of injury depends on several factors, including the model, location and area of skin exposed to SM, as well as SM dose and methods of administration and environmental conditions when applying SM. Targeting one or more phases of injury is essential in the development of effective countermeasures to mitigate SM toxicity. Both clinical signs and morphological/biochemical parameters have been used to characterize the action of SM in animal models. Clinical signs are evident by visual inspection; at early times, this includes erythema, edema and transepidermal water loss (TEWL), and, at later times, extent of injury and whether injury is superficial, intermediate in depth, or deep dermal injury. ²⁹ The integrity of the dermal-epidermal junction, measured by dermal torque, has been demonstrated in SM-treated guinea pigs. ³¹ Laser Doppler imaging has also been used to assess cutaneous blood flow and ballistometry to evaluate mechanical properties of the skin, including rigidity and elasticity in pig models. ^32,33

Techniques in histology, electron microscopy, and immunohistochemistry have been used to analyze structural alterations in skin exposed to SM. These studies have largely focused on the epidermis, basement membrane, and accessary structures, including hair follicles and sebaceous glands. Early effects of SM in basal cells of the epidermis, as reported in guinea pig skin include nuclear condensation and mitochondrial swelling, disorganization of desmosomes and hemidesmosomes, and widening of intracellular spaces in the basal cell layer. ¹⁹ At later times, nuclear pyknosis, cell fragmentation, and necrosis extending into suprabasal cells are evident. ³⁴ Markers of DNA damage and apoptosis and necrosis also appear in epidermal cells. ³⁵ Mediators of inflammation, including prostaglandins and cytokines, are also expressed after SM-induced injury. ^11,22 Microvesicles appear in the lamina lucida of the basement membrane as a consequence of degeneration of the basal layer. ^36,37 Proteolysis of basement membrane components, including laminins, collagens, and other anchoring proteins by matrix metalloproteinases, contributes to the disruption of the basal cell layer, microvesication, and ulceration. ^38,39 At later times, in minipig skin, aberrant epidermal proliferation and differentiation are associated with re-epithelialization including hyperplasia, hyperkeratosis, and parakeratosis. This is thought to contribute to prolonged wound healing. ^40,41

The dermis and hypodermis are also targets for SM. This is important as the integrity of these tissues is critical for wound healing. ^22,23,42,43 Leukocyte infiltration, a marker of inflammation, has been observed in the dermis post-SM exposure in all animals studied. ^19,44–46 In mouse and guinea pig skin, mast cell degranulation is also evident, along with alterations in collagen deposition. ^38,47,48 In pig skin, SM also disrupts the dermal vasculature and subsequent blood flow, and responses can affect tissue oxygenation, possibly leading to reperfusion injury. ^49,50 These pathologic responses can impair wound healing, lead to infection, and initiate scarring.

Guinea Pig Skin Model of SM Toxicity

Both haired and hairless guinea pigs have been used to assess SM toxicity with generally similar results (Table 1). Hairless guinea pigs have been reported to be more sensitive to SM in terms of the extent of dermal injury. ⁵¹ These animals are also more sensitive to SM-induced epidermal necrosis compared to other animal models, including the weanling pig, mouse ear, and hairless mice. ¹⁸ The hairless guinea pig skin is considered morphologically more like human skin, ^31,52 which has prompted greater use of these animals to understand the mechanism of action of SM and for the development of countermeasures. ⁵³

Table 1. Effects of sulfur mustard on guinea pig skin

As indicated above, a characteristic early response of guinea pig skin to SM is a marked inflammatory response, notably, infiltration of neutrophils and macrophages into the tissue. ⁵⁴ Mustards cause the release of inflammatory mediators, including reactive oxygen and reactive nitrogen species, and cytokines such as TNFα and IL-1α, which activate macrophages contributing to tissue injury. ^2,55 This is followed by the appearance of anti-inflammatory/wound repair macrophages. ⁵⁶ That macrophages can contribute to wound repair is evidenced by findings that intradermal injection of activated human macrophages into SM-treated guinea pig skin can significantly improve clinical signs of tissue damage. ⁵⁷

Of interest are studies by Graham et al. ⁴⁷ showing that SM reduces mast cell numbers in hairless guinea pig skin, suggesting that degranulation may be an early marker of toxicity. These investigators hypothesized that histamine and other mediators released by mast cells may play a role in SM-induced injury. These data are in accord with studies by ours and other laboratories demonstrating mast cell degranulation and reduced number of mast cells in SM-exposed hairless mouse skin. ^38,48 The use of antihistamine promethazine, in combination with the PARP inhibitor niacinamide, and the non-steroidal anti-inflammatory agent indomethacin in guinea pig skin, decreases mast cell degranulation. ^58–60

Rat, Mouse, and Rabbit Models of SM Toxicity

In these models, exposure to SM is either by direct application of liquid to the skin or as a vapor (Tables 2–5). Vapor exposures are typically preferred since vapor is the more likely route of exposure during a mass causality scenario. Depending on the dose and environmental conditions, generally similar characteristic responses are observed following treatment of the dorsal skin of rats, mice, and rabbits with SM. Initially, there is a latency period, which is followed by a cutaneous inflammatory response characterized by erythema, edema, and leukocyte infiltration. Subsequently, there is microblister formation, tissue granulation, epidermal necrosis, and, finally, wound repair and tissue remodeling. ^15,61,62 More detailed information has been reported on the effects of SM on hair follicles and sebaceous glands in the mouse model. ^23,63 In hair follicles, SM induces epithelial cell karyolysis within the hair root sheath, infundibulum, and isthmus and reduces the numbers of sebocytes in sebaceous glands. ⁶⁴ Significant DNA damage and apoptosis are evident around pilosebaceous units with increased numbers of inflammatory cells surrounding utriculi. These findings may explain, at least in part, depletion of hair follicles in human skin following exposure to SM.

Table 2. Effects of sulfur mustard on rat skin

Table 3. Effects of sulfur mustard on mouse skin

Table 4. Effects of sulfur mustard in the mouse ear vesicant model

Table 5. Effects of sulfur mustard on rabbit skin

An important method that can partially overcome wound contraction and the need for fur removal is the use of the mouse ear vesicant model (see Table 4). This method is largely based on early studies showing that biological and biochemical processes associated with inflammation can easily be measured following exposure to cutaneous irritants or allergens. ^36,65–67 In this model, SM is applied to the inner surface of the mouse ear, which is largely free of hair. Ear cartilage appears to prevent wound contraction. ⁶⁸ After a latency period, edema, measured by changes in ear weight, epidermal necrosis, and epidermal-dermal separation are assessed. ^65,69 Transmission electron microscopy and immunohistochemistry have been used to identify biomarkers of injury, as well as mechanisms of subepidermal blister formation. ^36,70

In rabbits, dorsal and ventral skin and ear skin have been used to investigate SM injury and the formation of microblisters. ^71–75 In each exposure scenario, SM damage has been assessed visually by monitoring erythema, wound healing, and histopathology. ^19,73,75,76 In the rabbit models, depending on the dose, SM damages the superficial microvasculature as measured by Evans blue dye extravasation and leakage of erythrocytes. ^19,77 SM also damages fibroblasts, possibly disrupting the extracellular matrix. In contrast to the dorsal and ventral skin, rabbit ears have no panniculus carnosus; thus, wound contraction does not contribute to the healing process. ⁷⁸ This model is thought to better reflect wound healing in humans. However, in a continuous flow vapor exposure model, rabbit ears have been reported to be significantly less sensitive than human skin to SM injury. ⁷⁶

Pig Models of SM Toxicity

Pig skin is the most anatomically and physiologically similar to human skin, compared to rodents and rabbits, making it a preferable model for translational research (Tables 6 and 7). From a regulatory standpoint, considerable background data are available on pig skin related to the development of dermatological products, making this model ideal for SM countermeasure research. Pig skin is tightly attached to the subcutaneous connective tissue, contains a relatively thick epidermis, distinct rete ridges and, like human skin, dense elastic fibers in the dermis. ^79–81 Pig skin hair is coarser than human hair but has a similar distribution. ^41,79,82 Although humans have eccrine glands distributed throughout their skin, swine eccrine glands are primarily found in the snout, lips, and carpal organ. ⁸⁰ In the skin of both pigs and humans, re-epithelialization during wound healing is associated with basal cell proliferation and differentiation into enucleated granular cells that migrate outward toward the surface of the skin. ⁸³ However, as with other animal models, SM is unable to form true blisters, a characteristic sign of toxicity in humans following vesicant exposure. ^6,7,84

Table 6. Effects of sulfur mustard on pig skin

Table 7. Effects of sulfur mustard on pig skin

Both dorsal and ventral skin models have been used to assess SM toxicity in pig skin (see Tables 6 and 7). In general, the ventral skin of pigs is thinner and more responsive to SM than dorsal skin. ⁸⁵ The choice of dorsal versus ventral pig skin models is dependent on the type of exposure (eg, liquid vs vapor cap) and the type of injury being investigated (eg, superficial vs intermediate or deep dermal). Both models can be used to assess pharmaceutical preparations. However, dorsal skin is preferable with the use of wound dressings that must be maintained for prolonged periods of time (see further below). Both clinical and histopathological endpoints are used to assess tissue damage. Clinical changes include blood flow, elasticity, skin color, thickness, and spectral properties. ^49,50 Histopathological changes include skin structure, epithelial and basement membrane integrity, and expression of markers of proliferation and differentiation of keratinocytes during wound healing. ^40,86–88 Following these endpoints over time will provide information on the wound healing process and the effectiveness of potential countermeasures. Decontaminants, protectants, anti-inflammatory agents, and wound dressing have been evaluated for their ability to mitigate tissue damage induced by SM, often with varying degrees of success. ⁸⁹

Based on pig skin models that have been developed to assess medical countermeasures against SM-induced skin injury, one product, Silverlon® Wound Contact, Burn Contact Dressings, has been approved by the FDA. ⁹⁰ Manufactured as a non-adherent knitted nylon fiber wound dressing coated with metallic silver, Silverlon® is approved for use with decontaminated, unroofed first and second degree burns induced by SM. Silverlon® also acts as an oxygen-permeable sterile barrier, which promotes wound healing. ⁵³ Silver ions in the product also serve as an antimicrobial, reducing infections at the wound site. ⁹¹

Support for Silverlon® in the FDA approval process was based on a pathophysiological scale in the Göttingen minipig vapor cap model (Table 8). Individual endpoints indicate the extent and type of repair and include the appearance of epithelial cells, basement membrane damage, re-epithelialization of the wound, whether abnormal hair follicles are present, extent of dermal inflammation and the presence of rete ridges, vascular proliferation, and hemorrhage. ^33,92 In the case of Silverlon®, approvals were based on re-epithelialization of the skin and improved appearance of the basement membrane, as well as a reduction in dermal inflammation. Silverlon® has also been FDA approved for radiation dermatitis and cutaneous radiation injury through dry desquamation. ⁹³

Table 8. Skin histopathology scoring for evaluating sulfur mustard countermeasures using Göttingen minipigs^a

^a Scoring system^33,92

Summary

Animal models are essential not only for understanding the mechanism of action of SM, but also to develop effective therapeutics. Importantly, therapeutics may be effective at different stages of SM injury (eg, during the latency prior to a cutaneous response, during the inflammatory response, or during wound healing/tissue remodeling) and can be used alone or in combination. For example, Silverlon® is effective for wound healing following the appearance of first- and second-degree burns after exposure to SM. It remains to be determined whether treatments with anti-inflammatory agents prior to the development of SM burns will improve Silverlon®-induced wound healing. Thus far, research in the field is limited as SM is a blistering agent, and none of the animal models form overt blisters in response to this vesicant. Further studies are required to better understand differences between human and animal responses to SM so that more effective countermeasures can be developed that not only enhance wound healing, but also mitigate the blistering response.