Pulmonary injury and oxidative stress in rats induced by inhaled sulfur mustard is ameliorated by anti-tumor necrosis factor-α antibody.

Sulfur mustard (SM) is a bifunctional alkylating agent that causes severe injury to the respiratory tract. This is accompanied by an accumulation of macrophages in the lung and the release of the proinflammatory cytokine, tumor necrosis factor (TNF)α. In these studies, we analyzed the effects of blocking TNFα on lung injury, inflammation and oxidative stress induced by inhaled SM. Rats were treated with SM vapor (0.4 mg/kg) or air control by intratracheal inhalation. This was followed 15-30 min later by anti-TNFα antibody (15mg/kg, i.v.) or PBS control. Animals were euthanized 3 days later. Anti-TNFα antibody was found to blunt SM-induced peribronchial edema, perivascular inflammation and alveolar plasma protein and inflammatory cell accumulation in the lung; this was associated with reduced expression of PCNA in histologic sections and decreases in BAL levels of fibrinogen. SM-induced increases in inflammatory proteins including soluble receptor for glycation end products, its ligand, high mobility group box-1, and matrix metalloproteinase-9 were also reduced by anti-TNFα antibody administration, along with increases in numbers of lung macrophages expressing TNFα, cyclooxygenase-2 and inducible nitric oxide synthase. This was correlated with reduced oxidative stress as measured by expression of heme oxygenase-1 and Ym-1. Together, these data suggest that inhibiting TNFα may represent an efficacious approach to mitigating acute lung injury, inflammatory macrophage activation, and oxidative stress induced by inhaled sulfur mustard.

Progressive Lung Injury, Inflammation, and Fibrosis in Rats Following Inhalation of Sulfur Mustard.

Sulfur mustard (SM) inhalation causes debilitating pulmonary injury in humans which progresses to fibrosis. Herein, we developed a rat model of SM toxicity which parallels pathological changes in the respiratory tract observed in humans. SM vapor inhalation caused dose (0.2-0.6 mg/kg)-related damage to the respiratory tract within 3 days of exposure. At 0.4-0.6 mg/kg, ulceration of the proximal bronchioles, edema and inflammation were observed, along with a proteinaceous exudate containing inflammatory cells in alveolar regions. Time course studies revealed that the pathologic response was biphasic. Thus, changes observed at 3 days post-SM were reduced at 7-16 days; this was followed by more robust aberrations at 28 days, including epithelial necrosis and hyperplasia in the distal bronchioles, thickened alveolar walls, enlarged vacuolated macrophages, and interstitial fibrosis. Histopathologic changes were correlated with biphasic increases in bronchoalveolar lavage (BAL) cell and protein content and proliferating cell nuclear antigen expression. Proinflammatory proteins receptor for advanced glycation end product (RAGE), high-mobility group box protein (HMGB)-1, and matrix metalloproteinase (MMP)-9 also increased in a biphasic manner following SM inhalation, along with surfactant protein-D (SP-D). Tumor necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS), inflammatory proteins implicated in mustard lung toxicity, and the proinflammatory/profibrotic protein, galectin (Gal)-3, were upregulated in alveolar macrophages and in bronchiolar regions at 3 and 28 days post-SM. Inflammatory changes in the lung were associated with oxidative stress, as reflected by increased expression of heme oxygenase (HO)-1. These data demonstrate a similar pathologic response to inhaled SM in rats and humans suggesting that this rodent model can be used for mechanistic studies and for the identification of efficacious therapeutics for mitigating toxicity.

Debridement of Sulfur Mustard Skin Burns: A Comparison of Three Methods.

Sulfur mustard burns are characterized by delayed symptoms, slow healing, and recurrence after closure. Incomplete debridement at the level of the basement membrane is the postulated cause. Graham pioneered laser debridement of mustard burns. For field or mass-casualty use, saline wet-to-wet or antibiotic-soak debridement is more practical. In this study, we compared laser, saline, and antibiotic debridement in a porcine model of deep partial-thickness injury. Deep-dermal sulfur mustard burns were produced in 18 anesthetized Gottingen minipigs using 10-µl saturated vapor cap exposure time of 90 minutes. Debridement was started 48 hours postinjury and consisted of a single laser treatment; 5 days of 5% aqueous mafenide acetate wet-to-wet dressings; or 7 to 12 days of saline wet-to-wet dressings. Wounds were treated with daily silver sulfadiazine for 30 days and, then, assessed by histopathology, silver-ion analysis, colorimetry, and evaporimetry. All wounds healed well with no sign of infection. Antibiotic debridement showed no advantage over saline debridement. There were no significant differences between groups for colorimetry or evaporimetry. Histopathology was graded on a mustard-specific scale of 1 to 15 where higher values indicate better healing. Mean histology scores were 13.6 for laser, 13.9 for mafenide, and 14.3 for saline. Saline debridement statistically outperformed laser (P < .05) but required the longest debridement time. Laser debridement had the benefit of requiring a single treatment rather than daily dressing changes, significantly decreasing need for wound care and personnel resources. Development of a ruggedized laser for field use is a countermeasures priority.

Blood and Tissue Silver Levels Following Application of Silver-Based Dressings to Sulfur Mustard Chemical Burns.

Silver-based dressings are commonly used in burn care. Silver sulfadiazine use is associated with elevated blood, urine, and tissue levels of silver ion. We examined wound and tissue levels of silver ion in a two-species model of sulfur mustard chemical burn injury treated with two different silver-based dressings. Superficial dermal and moderate thickness dermal chemical burns were induced in 16 hairless guinea pigs and in 16 Gottingen minipigs by exposure to sulfur mustard vapor. After debridement, silver-nylon burn dressings or silver-calcium alginate dressings were applied and changed every 7 days until wound healing or a maximum of 60 days post exposure. At autopsy, liver, spleen, and wound samples were harvested. Silver ion was measured using inductively coupled plasma-mass spectrography with a lower level of detection of 0.02 parts per billion. Negligible silver ion levels were found in the liver (mean < 0.003 µg/g of tissue) and spleen (mean < 0.05 µg/g) of all 32 animals. Wound biopsies showed silver ion levels ranging from 0.07 to 19.5 µg/g of tissue. Wound levels were higher in minipigs than in hairless guinea pigs and were higher in animals treated with silver-nylon burn wound dressings than with silver-calcium alginate dressings. Silver ion could be detected in some wounds 40 days after dressings were removed. In a chemical burn model, application of silver-nylon or silver-calcium alginate dressings is associated with elevated wound levels but negligible tissue levels of silver ion.