Please note that co-author Rajesh Agarwal is a PLOS ONE Editorial Board member. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.
Conceived and designed the experiments: NTS CWW RA. Performed the experiments: NTS AKJ. Analyzed the data: NTS DO CWW RA. Wrote the paper: NTS AKJ DO CWW RA.
To identify effective therapies against sulfur mustard (SM)-induced skin injuries, various animals have been used to assess the cutaneous pathology and related histopathological changes of SM injuries. However, these efforts to establish relevant skin injury endpoints for efficacy studies have been limited mainly due to the restricted assess of SM. Therefore, we employed the SM analog nitrogen mustard (NM), a primary vesicating and bifunctional alkylating agent, to establish relevant endpoints for efficient efficacy studies. Our published studies show that NM (3.2 mg) exposure for 12–120 h in both the hairless SKH-1 and haired C57BL/6 mice caused clinical sequelae of toxicity similar to SM exposure in humans. The NM-induced cutaneous pathology-related structural changes were further analyzed in this study and quantified morphometrically (as percent length or area of epidermis or dermis) of skin sections in mice showing these lesions. H&E stained skin sections of both hairless and haired mice showed that NM (12–120 h) exposure caused epidermal histopathological effects such as increased epidermal thickness, epidermal-dermal separation, necrotic/dead epidermis, epidermal denuding, scab formation, parakeratosis (24–120 h), hyperkeratosis (12–120 h), and acanthosis with hyperplasia (72–120 h). Similar NM exposure in both mice caused dermal changes including necrosis, edema, increase in inflammatory cells, and red blood cell extravasation. These NM-induced cutaneous histopathological features are comparable to the reported lesions from SM exposure in humans and animal models. This study advocates the usefulness of these histopathological parameters observed due to NM exposure in screening and optimization of rescue therapies against NM and SM skin injuries.
Upon cutaneous exposure, only 20% of sulfur mustard (SM) is absorbed, leading to acute and chronic skin lesions
Several animal models display many cutaneous lesions and structural changes with SM that are seen in humans
In the current study, we sought to further examine the histopathological effects and structural changes responsible for the clinical sequelae of NM-induced toxicity observed in our earlier reported study. Similar to this reported study, both hairless (SKH-1) and haired (C56BL/6) mice were used in this study to analyze the NM-induced histopathological changes. The current assessment will help define any differences in the injury response due to uptake or absorption of NM in the skin of both strains. This study demonstrates that NM cutaneous exposure resulted in epidermal thickness, epidermal-dermal separation, necrolytic/dead epidermis, epidermal denudation and ulceration, scab formation, parakeratosis, hyperkeratosis, and acanthosis associated with hyperplasia in both haired and hairless mice. NM exposure in these mice also caused dermal necrosis and edema, increase in inflammatory cells, and red blood cell extravasation. Overall, NM-induced structural changes in hairless and haired mice evaluated in this study parallel the reported lesions from SM exposure in humans and animal models.
Both SKH-1 hairless and C57BL/6 (haired) mice (5–6 weeks of age) were purchased from Charles River Labs (Wilmington, MA), and housed under standard conditions and acclimatized before their use in our experimental studies. The Institutional Animal Care and Use Committee (IACUC) of the University of Colorado Denver, CO have specifically approved the studies with mice reported in this article. Mice (n = 5) were either exposed topically to 200 µL acetone alone or to 3.2 mg NM (mechorethamine hydrochloride; Sigma-Aldrich Chemical Co., St. Louis MO) in 200 µL acetone for 12 h, 24 h, 72 h and 120 h. Respective untreated control group was included in the study. C57BL/6 mice were shaved 3 days prior to NM exposure. Following the above mentioned exposures, mice were euthanized, the dorsal skin was collected and either snap frozen in liquid nitrogen or fixed in 10% phosphate buffered formalin as detailed earlier
The formalin-fixed dorsal skin sample was dehydrated in ascending concentration of ethanol, cleared in xylene, and embedded in PolyFin (Triangle Biomedical Sciences, Durham, NC) as detailed earlier
Data were analyzed using one-way analysis of variance (one-way ANOVA) to get the statistically significant difference in control versus treated groups, with Tukey or Bonferroni t-test for multiple comparisons (SigmaStat 2.03). Differences were considered significant if the
Our published study showed that NM exposure (3.2 mg) for 12–120 h caused edema, erythema, microblister formation, pigmentation changes, dry skin and wounded areas on the skin of SKH-1 hairless and C57BL/6 mice
In the epidermis, death of keratinocytes and epidermal dermal separation, as well as inflammation-related changes with SM exposure were observed
Our clinical observations in NM (3.2 mg) exposed haired and hairless mice skin revealed increased bi-fold thickness and microblister like appearance
Dorsal skin of mice was exposed topically to either 200 µL of acetone or NM (3.2 mg) in 200 µL acetone. After 12, 24, 72 and 120 h of NM exposure, mice were sacrificed and dorsal skin tissue sections (5 µm) were processed, H&E stained and analyzed as detailed under Materials and Methods. Panels A and C are representative H&E stained skin sections (400× magnification) showing epidermal thickness from vehicle control and 24 h exposed NM exposed skin tissue in SKH-1 and C57BL/6 mice, respectively. Panel E (i–iii) and G (i–iii) are representative H&E stained skin sections (100× magnification), and E (iv–vi) and G (iv–vi) are representative H&E stained skin sections (400× magnification) showing microvesication from vehicle control and 12–120 h NM exposed skin tissue in mice. These NM-related histopathological changes were assessed as detailed under materials and methods, and calculated as percent length of mice skin epidermis showing epidermal thickness (B and D) and microvesication (F and H). Data presented are mean ± SEM of 3–5 animals in each group. *, p<0.05 compared to respective vehicle control; VC, vehicle control; NM, nitrogen mustard; e, epidermis; d, dermis; red arrows, microvesication (epidermal and dermal separation); pink arrows, microvesication within the epidermal layer.
Skin pathology effects after 12–120 h NM (3.2 mg) exposure included erythema, broken skin (ulceration) and dead peeled skin areas
Dorsal skin of mice was exposed topically to either 200 µL of acetone or NM (3.2 mg) in 200 µL acetone. After 12, 24, 72 and 120 h of NM exposure, mice were sacrificed and dorsal skin tissue sections (5 µM) were processed, H&E stained and analyzed as detailed under Materials and Methods. Panels A and C, E and G and I and K are representative H&E stained skin sections (100 or 400× magnification) showing epidermal cell death, epidermal denuding and scab formation, respectively from vehicle control and 72 or 120 h NM exposed skin tissue in SKH-1 and C57BL/6 mice. These NM-related histopathological changes were assessed as detailed under materials and methods, and calculated as percent length of mice skin epidermis showing epidermal cell death (B and D) epidermal denuding and ulceration (F and H), or as incidences of scab formation/outgrowths (J and L). Data presented are mean ± SEM of 3–5 animals in each group. *, p<0.05 compared to respective vehicle control; VC, vehicle control; NM, nitrogen mustard; e, epidermis; d, dermis; green arrows, necrosis/dead epidermal layer or epidermal denuding or scab formation.
Gross examination of NM treated hairless and haired mice showed thick leathery, scaly and desquamating skin as well as pigmentation changes at 72 and 120 h post-NM exposure
Dorsal skin of mice was exposed topically to either 200 µL of acetone or NM (3.2 mg) in 200 µL acetone. After 12, 24, 72 and 120 h of NM exposure, mice were sacrificed and dorsal skin tissue sections (5 µM) were processed, H&E stained and analyzed as detailed under Materials and Methods. Panels A and C (i–iii) and E and G (i–iii) are representative H&E stained skin sections (100 or 400× magnification) showing parakeratosis and hyperkeratosis, respectively, from vehicle control as well as NM exposed (72 and 120 h) skin tissue in mice. These NM-related histopathological changes were assessed as detailed under materials and methods, and calculated as percent length of mice skin epidermis showing parakeratosis (B and D) and hyperkeratosis (F and H). Data presented are mean ± SEM of 3–5 animals in each group. *, p<0.05 compared to respective vehicle control; VC, vehicle control; NM, nitrogen mustard; e, epidermis; d, dermis; green arrows, parakeratosis, hypercornification or acanthosis.
Acanthosis has been reported to be induced by SM; it is defined as diffuse hyperproliferation and thickening of the stratum spinosum with an increased size of the cells in this layer
Dorsal skin of mice was exposed topically to either 200 µL of acetone or NM (3.2 mg) in 200 µL acetone. After 12, 24, 72 and 120 h of NM exposure, mice were sacrificed and dorsal skin tissue sections (5 µM) were processed, H&E stained and analyzed as detailed under Materials and Methods. Panels A and B are representative H&E stained skin sections (100 or 400× magnification) showing acanthosis from vehicle control as well as NM exposed (72 and 120 h) skin tissue in mice. This NM-related histopathological change was assessed as detailed under materials and methods, and calculated as percent length of mice skin epidermis showing acanthosis (B and D). Data presented are mean ± SEM of 3–5 animals in each group. *, p<0.05 compared to respective vehicle control; VC, vehicle control; NM, nitrogen mustard; e, epidermis; d, dermis; green arrows, acanthosis.
SM exposure causes a profound inflammatory response including dermal edema, necrosis and influx of inflammatory cells in the dermis. Hence, we next assessed these changes following NM exposure in SKH-1 and C57BL/6 mice.
Histological analysis of NM-exposed skin tissue sections from both hairless and haired mice revealed that NM-induced dermal necrosis (dead dermal areas), which was accompanied by the presence of a large number of inflammatory cells (
Dorsal skin of mice was exposed topically to either 200 µL of acetone or NM (3.2 mg) in 200 µL acetone. After 12, 24, 72 and 120 h of NM exposure, mice were sacrificed and dorsal skin tissue sections (5 µM) were processed, H&E stained and analyzed as detailed under Materials and Methods. Panels A and C, E and F, G and H, and I and J are representative H&E stained skin sections from vehicle control as well as NM exposed skin tissue in mice for dermal necrosis (with presence of breaking down inflammatory cells, mainly neutrophils), dermal thickness, inflammatory cells, and extravasated red blood cells. These NM-related histopathological changes were assessed as detailed under materials and methods, and calculated as percent area of mice skin dermis showing necrosis (B and D), inflammatory cells (
NM exposure also resulted in an influx of inflammatory cells (
Exposure | VC | 12 h NM | 24 h NM | 72 h NM | 120 h NM |
Inflammatory cells | + | +++ | +++ | +++ | ++ |
Exposure | VC | 12 h NM | 24 h NM | 72 h NM | 120 h NM |
Inflammatory cells | + | ++ | +++ | ++ | ++ |
+, Few (less than 100); ++, moderate (100–1000); +++, high (more than 1000) inflammatory cells per skin section (at 400×); VC, vehicle control.
Exposure | VC | 12 h NM | 24 h NM | 72 h NM | 120 h NM |
Extravasated RBCs | + | ++ | +++ | +++ | +++ |
Exposure | VC | 12 h NM | 24 h NM | 72 h NM | 120 h NM |
Extravasated RBCs | ++ | +++ | +++ | +++ | +++ |
+, Few (less than 10%); ++, moderate (10–30%); +++, high (more than 30%) extravasated RBCs per skin section (at 400×); vehicle control.
NM is an analog of SM with reported chemical properties and cutaneous toxic effects similar to SM. Thus far it has not been used as a weapon of mass destruction
Inflammatory response in the skin is associated with an increase in epidermal thickness, which has also been reported following SM exposure
Following 48–72 h of cutaneous SM exposure in humans, blisters in the skin burst open causing ulceration, epidermal denudation, necrosis and finally scab or eschar like formation
The presence of parakeratosis at 72 and 120 h post-NM exposure in our study is consistent with reports of parakeratosis in human and animal models with SM cutaneous exposure
Dermal changes following SM exposure in humans and animals include edema, infiltration of inflammatory cells, extravasated erythrocytes, necrosis and endothelial swelling
According to the observation from this study, immunohistochemical analyses, and other specific assays to further investigate the NM-induced inflammatory response, epidermal cell death, and microvesication are under investigation. These studies will further help in dissecting and examining mechanisms involved in NM-induced skin injuries, which will also be useful in screening treatment agents against vesicant injuries.
In summary, this study provides a detailed assessment of NM-related structural changes in both hairless and haired mice to establish that its histopathological parameters are comparable to SM exposure in humans and other animal models. We believe this histopathological analysis will be valuable for screening and identification of therapies against skin injuries induced by both SM and NM.