Novel cell imaging techniques show induction of apoptosis and proliferation in mesothelial cells by asbestos.
Goldberg-JL; Zanella-CL; Janssen-YM; Timblin-CR; Jimenez-LA; Vacek-P; Taatjes-DJ; Mossman-BT
Am J Respir Cell Mol Biol 1997 Sep; 17(3):265-271
The effect of asbestos (1332214) on apoptosis and proliferation in mesothelial cells was examined using novel cell imaging techniques. Rat pleural mesothelial (RPM) cells isolated from Fischer-344-rats were cultured with a hydrogen-peroxide (H2O2) dose of 300 micromolar, crocidolite (12001284) concentrations of 0.5 to 5.0 micrograms per square centimeter (microg/cm2), a riebeckite (61105304) dose of 5.0microg/cm2, a 12-O-tetradecanoylphorbol-13- acetate (TPA) dose of 100 nanograms per milliliter (ng/ml), a tumor- necrosis-factor-alpha (TNF-alpha) dose of 10ng/ml, or an epidermal- growth-factor (EGF) concentration of 5.0ng/ml. Control cultures were sham treated. Cells were analyzed for apoptosis 24 to 72 hours after exposure. The following techniques were used: the in-situ terminal deoxynucleotidyl-transferase mediated deoxyuridine- triphosphate nick end labeling (TUNEL) assay, the 4',6-diamidino-2- phenylindole technique, and immunofluorescent microscopic examination. RPM cell proliferation was determined using the 5'- bromodeoxyuridine (BrdU)/oxazole-yellow-homodimer (YOYO) dual fluorescence technique. Treatment with the positive control H2O2 induced apoptosis in RPM cells, as indicated by TUNEL incorporation. RPM cells treated with crocidolite for 24 to 72 hours exhibited significant, dose dependent increases in apoptosis. Exposure to TNF- alpha for 24 hours produced significant increases in apoptosis in RPM cells. Riebeckite, TPA, and EGF failed to induce apoptosis in RPM cells. BrdU incorporation was significantly increased in RPM cells treated with crocidolite, TPA, EGF, and TNF-alpha. Riebeckite caused no significant increases in RPM cell proliferation. The YOYO technique failed to detect any changes in the total number of RPM cells following exposure to any agent. The authors conclude that the above cell imaging techniques allow for the measurement of apoptosis and cell proliferation in the same culture dish. RPM cells exposed to asbestos appear to exhibit a dynamic balance between cell death and cell proliferation.
NIOSH-Publication; NIOSH-Grant; Pulmonary-system-disorders; Cell-cultures; Cell-damage; Cell-division; Mesothelial-cells; Deoxyribonucleic-acids; Exposure-levels; Dose-response; Asbestos-fibers; Microscopic-analysis; Cell-culture-techniques; Laboratory-techniques; In-vitro-study
Dr. Brooke Mossman, University of Vermont, Department of Pathology, Burlington, VT 05405
1332-21-4; 12001-28-4; 61105-30-4
American Journal of Respiratory Cell and Molecular Biology
University of Vermont & St Agric College, Burlington, Vermont