Sulfur mustard vapor effects on differentiated human lung cells

Abstract

Context: Sulfur mustard (SM) causes skin blistering and long-term pulmonary dysfunction. Its adverse effects have been studied in battlefield-exposed humans, but lack of knowledge regarding confounding factors makes interpretation challenging. Animal studies are critical to understanding mechanisms, but differences between animals and humans must be addressed. Studies of cultured human cells can bridge animal studies and humans.

Objective: Evaluate effects of SM vapor on airway cells.

Materials and methods: We examined responses of differentiated human tracheal/bronchial epithelial cells, cultured at an air-liquid interface, to SM vapors. SM effects on metabolic activity (Water Soluble Tetrazolium (WST) assay), cytokine and metalloproteinase secretion, and cellular heme oxygenase 1 (HO-1), an oxidative stress indicator, were measured after 24 h.

Results: At noncytotoxic levels of exposure, interleukin 8 and matrix metalloproteinase-13 were significantly increased in these cultures, but HO-1 was not significantly affected.

Discussion and conclusion: Exposure of differentiated airway epithelial cells to sub-cytotoxic levels of SM vapor induced inflammatory and degradative responses that could contribute to the adverse health effects of inhaled SM.

Figure 1

Viability of primary human tracheo-bronchial epithelial cells assessed as tetrazolium conversion to formazan (WST assay) 24 h after treatment with sulfur mustard (SM) vapor. Incubator control (inc ctl): cells maintained in the CO₂ incubator during exposure of the remaining cultures in a glove box. 0: Cells exposed to the vapor from 10 μl of ethanol. SM concentrations are the estimated maximal concentration possible in the vapor phase as the result of the amount present in 10 μl of the dilution applied to the filter. OD, optical density.

Figure 2

Cytokine production by primary human tracheo-bronchial epithelial cells 24 h after treatment with sulfur mustard (SM) vapor. Incubator control (inc ctl): cells maintained in the CO₂ incubator during exposure of the remaining cultures in a glove box. 0: Cells exposed to the vapor from 10 μl of ethanol. SM concentrations are the estimated maximal concentration possible in the vapor phase as the result of the amount present in 10 μl of the dilution applied to the filter. *Indicates a value significantly different from both the inc ctl and the control cultures (0). G-CSF, granulocyte colony-stimulating factor; IL, interleukin; IP, interferon-inducible protein; MCP-1, monocyte chemotactic protein 1; VEGF, vascular endothelial growth factor.

Figure 3

Matrix metalloproteinase (MMP) release from primary human tracheo-bronchial epithelial cells 24 h after treatment with sulfur mustard (SM) vapor. MMP levels in the medium were measured by multiplex technology. Incubator control (inc ctl): cells maintained in the CO₂ incubator during exposure of the remaining cultures in a glove box. 0: Cells exposed to the vapor from 10 μl of ethanol. SM concentrations are the estimated maximal concentration possible in the vapor phase as the result of the amount present in 10 μl of the dilution applied to the filter.

Figure 4

Intracellular heme oxygenase 1 (HO-1) in primary human tracheo-bronchial epithelial cells 24 h after treatment with sulfur mustard (SM) vapor. HO-1 levels in the medium were measured by western blotting, and normalized to β-actin levels in the same blots following stripping and re-probing. Incubator control (inc ctl): cells maintained in the CO₂ incubator during exposure of the remaining cultures in a glove box. 0: Cells exposed to the vapor from 10 μl of ethanol. SM concentrations are the estimated maximal concentration possible in the vapor phase as the result of the amount present in 10 μl of the dilution applied to the filter.