Epithelial cells are prone to oxidant injury, which could change epithelial cell homeostasis and lead to degenerative diseases. We examined the effects of hyperoxia on death and proliferation of Madin-Darby canine kidney (MDCK) epithelial cells and antioxidant vitamin protection. Subconfluent and near-confluent MDCK cells were cultured under normoxia or hyperoxia for two days. We measured cell number and viability, mitochondrial enzymatic activity, thymidine incorporation, necrosis [lactate dehydrogenase (LDH) release], and apoptosis (DNA fragmentation and morphological changes). When the cells were subconfluent, hyperoxia decreased the number of adherent cells, mitochondrial enzymatic activity, and thymidine incorporation, but neither LDH release nor apoptotic changes increased compared with normoxic controls. In normoxia, near-confluent cells had lower nonadherent cell numbers, mitochondrial enzymatic activity, and thymidine incorporation than sub-confluent cells; hyperoxia further decreased the latter two parameters and increased apoptotic changes and LDH release in near-confluent cells. Vitamin E protected mitochondrial enzymatic activity, apoptotic changes, and LDH release against hyperoxic injury but did not affect changes in thymidine incorporation with hyperoxia. Vitamin C partially protected the mitochondrial enzymatic activity and thymidine incorporation in subconfluence, but not in near confluence. These results indicate that cell density is a major determinant of the effects of hyperoxic injury and the profile of antioxidant vitamin protection.
Bibliographical noteFunding Information:
The authors thank Drs. Peter Bitterman and Paul Quie for critical review of the manuscript and Dr. W. Regelmann for helping us initiate the study. This study is supported in part by grants from the Minnesota Medical Foundation and Viking Children's Foundation (H. Jyonouchi) and an American Lung Association Career Investigator Award, American Heart Association Research Grant In Aid, and National Institutes of Health Specialized Center of Research in Acute Lung Injury Grant HL-50152 (D. H. Ingbar). Address reprint requests to Dr. Harumi Jyonouchi, Dept. of Pediatrics, University of Minnesota, Box 610, UMHC, 420 Delaware St., Minneapolis, MN 55455. Phone: (612) 626-3412. Fax: (612) 624-9188. E-mail: jyono001l @maroon.tc.umn.edu.