Rapid reduction of intracellular glutathione in human bronchial epithelialcells exposed to occupational levels of toluene diisocyanate

Toluene diisocyanate (TDI) is a recognized chemical asthmogen, yet the mech anism of this toxicity and the molecular reactions involved have not been e lucidated. We have previously shown that TDI vapor forms adducts with the a pical surface of the respiratory epithelium, and that it colocalizes with c iliary tubulin, In vitro, we have shown rapid reaction of TDI with glutathi one (GSH) and transfer of the bisGS-TDI adduct to a sulfhydryl-containing m ajor histocompatibility complex peptide. This study sought to determine if intracellular GSH is altered following exposure to TDI, We used the dye Cel lTracker Green (chloromethylfluorescein, CMFDA) for detection of glutathion e. 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 fluoresc ence. The decreased fluorescence was not due to cytotoxicity or decrease in either esterase or glutathione-S-transferase activity, enzymes necessary f or activation of the fluorescence of CMFDA, The decrease in glutathione lev els 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 cult ures that were not covered with mucus. These results, along with previous d ata, strongly suggest that TDI enters pulmonary cells and reacts rapidly wi th 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-indu ced pulmonary disease.