| Effects of Hexamethylene Diisocyanate Exposure on Human Airway Epithelial Cells: In Vitro Cellular and Molecular Studies Adam V. Wisnewski, Qing Liu, Jing-Jing Miller, Nadine Magoski, and Carrie A. Redlich Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut, USA Abstract
In this study we developed an in vitro exposure model to investigate the effects of hexamethylene diisocyanate (HDI) on human airway epithelial cells at the cellular and molecular level. We used immunofluorescence analysis (IFA) to visualize the binding and uptake of HDI by airway epithelial cell lines (A549 and NCI-NCI-H292) and microarray technology to identify HDI sensitive genes. By IFA, we observed that subcytotoxic concentrations of HDI form microscopic micelles that appear to be taken up by cells over a 3-hr period postexposure. Microarray analysis (4.6K genes) of parallel cultures identified four genes (thioredoxin reductase, dihydrodiol dehydrogenase, TG interacting factor, and stanniocalcin) whose mRNA levels were up-regulated after HDI exposure. Northern analysis was used to confirm that HDI increased message levels of these four genes and to further explore the dose dependence and kinetics of the response. The finding that HDI exposure increases thioredoxin reductase expression supports previous studies suggesting that HDI alters thiol-redox homeostasis, an important sensor of cellular stress. Another of the HDI-increased genes, a dihydrodiol dehydrogenase, encodes a protein previously shown to be specifically susceptible to HDI conjugation, and known to detoxify other hydrocarbons. Together, the data describe a novel approach for investigating the effects of HDI binding and uptake by human airway epithelial cells and begin to identify genes that may be involved in the acute response to exposure. Key words: exposure, hexamethylene diisocyanate, human airway epithelial cell, redox, thiol, thioredoxin reductase. Environ Health Perspect 110:901-907 (2002) . [Online 25 July 2002] http://ehpnet1.niehs.nih.gov/docs/2002/110p901-907wisnewski/ abstract.html Address correspondence to A.V. Wisnewski, Yale University School of Medicine, 333 Cedar Street, LCI-105, New Haven, CT 06520 USA. Telephone: (203) 737-2544. Fax: (203) 785-3826. E-mail: adam.wisnewski@yale.edu We thank K. Bottomly, C. Herrick, and M. Karol for helpful criticisms and thoughtful discussions on these studies, and the Yale Microarray Center for performing the microarray analysis. The work was supported by grants from the National Institutes of Health (1P01HL56389, 1R01HL62622, and K24-ES00355) and the American Lung Association. Received 7 November 2001 ; accepted 21 February 2002.
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