Lantz R C, Lemus R, Lange R W, Karol M H
Department of Cell Biology and Anatomy, The University of Arizona, Tucson, Arizona 85724, USA.
Toxicol Sci. 2001 Apr;60(2):348-55. doi: 10.1093/toxsci/60.2.348.
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.
甲苯二异氰酸酯（TDI）是一种公认的化学致喘物，但其毒性机制及相关分子反应尚未阐明。我们之前已表明，TDI蒸汽与呼吸道上皮的顶端表面形成加合物，且与纤毛微管蛋白共定位。在体外，我们已表明TDI与谷胱甘肽（GSH）快速反应，并将双GS-TDI加合物转移至含巯基的主要组织相容性复合体肽。本研究旨在确定暴露于TDI后细胞内GSH是否发生改变。我们使用染料CellTracker Green（氯甲基荧光素，CMFDA）检测谷胱甘肽。将人支气管上皮细胞（HBE）的1天和6天气液培养物暴露于20 - 100 ppb的TDI蒸汽中5、15或30分钟。随后使用共聚焦显微镜对细胞进行成像。1天和6天的培养物均显示，随着TDI暴露剂量增加，硫醇染色强度降低。低至20 ppb（当前TDI的允许暴露限值）的剂量会导致荧光快速（5分钟内）降低。荧光降低并非由于细胞毒性或酯酶或谷胱甘肽-S-转移酶活性降低，而这两种酶是激活CMFDA荧光所必需的。使用另一种荧光标记物ThioGlo(TM) 1和细胞提取物验证了谷胱甘肽水平的降低。此外，6天气液界面HBE细胞在暴露于TDI后产生的黏液似乎具有保护作用，因为黏液下方的HBE细胞比未被黏液覆盖的同一培养物中的细胞保留了更多荧光。这些结果与先前的数据强烈表明，TDI进入肺细胞并与细胞内GSH快速反应，且这一过程可在当前20 ppb的允许暴露限值下发生。这种快速反应表明细胞硫醇在TDI诱导的肺部疾病中具有重要作用。
