Intracellular S-glutathionyl adducts in murine lung and human bronchoepithelial cells after exposure to diisocyanatotoluene.
Diisocyanatotoluene (toluene diisocyanate, TDI), a 4:1 mixture of 2, 4- and 2,6-isomers used in the preparation of polyurethanes, causes occupational asthma by an as yet unknown mechanism. We previously showed that it forms adducts with the apical surface of the bronchoepithelium in vivo, and with ciliary microtubules in cultured human bronchoepithelial (HBE) cells. These results suggested that TDI may not enter HBE cells. In vitro studies, however, showed that TDI avidly forms bis adducts with glutathione (GSH) and that these adducts transfer monoisocyanato-monoglutathionyl-TDI to a sulfhydryl-containing peptide. This study sought to elucidate intracellular reactions of TDI. Using an electron paramagnetic resonance spectrometric (EPR) method, we established that the level of thiol-dependent quenching of phenoxyl radicals of etoposide was decreased >40% in pulmonary tissue of mice that received TDI intrabronchially. Similarly, HBE cells exposed to 100 ppb TDI vapor experienced a >30% reduction in thiol levels as determined with a thiol-specific fluorescent probe (ThioGlo 1). HPLC/UV analysis of lysates from HBE cells exposed to 200 and 500 ppb TDI vapor suggested a dose-related formation of S-glutathionyl adducts. Data from the 500 ppb TDI-treated HBE cells verified the identity of the 2-monoglutathionyl-4-monoisocyanato adduct. The results provide firm evidence that TDI enters pulmonary cells and reacts with GSH. This rapid reaction leading to formation of S-glutathionyl adducts of TDI suggests the importance of cellular thiols in TDI-induced pulmonary disease.
Lange
R W
RW
Department of Environmental and Occupational Health, Department of Pharmaceutical Sciences, and Department of Pharmacology, University of Pittsburgh, 260 Kappa Drive, Pittsburgh, Pennsylvania 15238, USA.
Day
B W
BW
Lemus
R
R
Tyurin
V A
VA
Kagan
V E
VE
Karol
M H
MH
D000818:Animals; D000972:Antineoplastic Agents, Phytogenic / Q000633:toxicity; D001980:Bronchi / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004578:Electron Spin Resonance Spectroscopy; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D005047:Etoposide / Q000633:toxicity; D005260:Female; D016166:Free Radical Scavengers / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism*; D006801:Humans; D007509:Irritants / Q000633:toxicity*; D008168:Lung / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D011506:Proteins / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013057:Spectrum Analysis; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D014051:Toluene 2,4-Diisocyanate / Q000633:toxicity*
D000972:Antineoplastic Agents, Phytogenic; D016166:Free Radical Scavengers; D007509:Irritants; D011506:Proteins; D013438:Sulfhydryl Compounds; D014051:Toluene 2,4-Diisocyanate; D005047:Etoposide; D005978:Glutathione