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Toluene diisocyanate (TDI) is a recognized chemical asthmogen, yet the mechanism of this toxicity and the molecular reactions involved have not been elucidated. We have previously shown that TDI vapor forms adducts with the apical surface of the respiratory epithelium, and that it colocalizes with ciliary tubulin. In vitro, we have shown rapid reaction of TDI with glutathione (GSH) and transfer of the bisGS-TDI adduct to a sulfhydryl-containing major histocompatibility complex peptide. This study sought to determine if intracellular GSH is altered following exposure to TDI. We used the dye CellTracker Green (chloromethylfluorescein, CMFDA) for detection of glutathione. One-day and 6-day air-liquid cultures of human bronchoepithelial cells (HBE) were exposed to 20-100 ppb TDI vapor for 5, 15, or 30 min. Cells were subsequently imaged using a confocal microscope. Both 1- and 6-day cultures showed a decrease in intensity of the thiol staining as a function of the TDI exposure dose. Doses as low as 20 ppb, the current permissible exposure limit (PEL) to TDI, resulted in rapid (within 5 min) decreases in fluorescence. The decreased fluorescence was not due to cytotoxicity or decrease in either esterase or glutathione-S-transferase activity, enzymes necessary for activation of the fluorescence of CMFDA. The decrease in glutathione levels was verified using another fluorescent label, ThioGlo(TM) 1, and cell extracts. In addition, the mucus produced by 6-day air-liquid interface HBE cells in response to TDI exposure appeared to be protective, as HBE cells underlying mucus retained more fluorescence than did cells in the same cultures that were not covered with mucus. These results, along with previous data, strongly suggest that TDI enters pulmonary cells and reacts rapidly with intracellular GSH, and that this can occur at the current PEL of 20 ppb. This rapid reaction suggests the importance of cellular thiols in TDI-induced pulmonary disease.

Acute inhalation of diesel fuel-polycarbonate plastic (DFPP) smoke causes severe lung injury, leading to acute respiratory distress syndrome (ARDS) and death. It has been reported that the initiation of acute lung injury is associated with the activation of pulmonary alveolar macrophages (PAM). To further explore the pathogenesis, alveolar macrophages (AM) of New Zealand rabbits ventilated and exposed to a 60 tidal volume of DFPP smoke in vivo were recovered at 1 h post-smoke. Smoke exposure induced significant increases in both mRNA and protein levels for PAM tumor necrosis factor-alpha (TNF-alpha), when compared to smoke control. Smoke also induced a biphasic response (inhibited at 2 h, enhanced at 24 h after cell isolation) in the production of superoxide (O2-) by PAM. However, aerosolized lazaroid, U75412E (1.6 mg/kg body weight), significantly attenuated smoke-induced expression in AM TNF-alpha at the protein level but not at the mRNA level, and smoke-induced changes in AM production of O2-. This study suggests that highly expressing AM TNF-alpha following smoke may be a key contributor to the cascade that establishes an acute injury process and exacerbates oxidant-derived cell injury. Whereas, the lazaroid may ameliorate smoke-induced lung injury by attenuating AM TNF-alpha release, in addition to its primary antioxidative mechanism.

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