Confluence and antioxidant vitamin protection determine the effects of hyperoxia on mdck epithelial cells

Effects of hyperoxia and antioxidant vitamin protection on epithelial cell proliferation and death were examined, using a MDCK canine epithelial cell line. Subconfluent and confluent MDCK cells were cultured under normoxia or hyperoxia (95% O: and 5% CO3) for 0, 1, 2 days and changes in viable cell number, mitochondrial enzymatic activity (MTT assay), DNA synthesis (thymidine incorporation), LDH release, and DNA fragmentation and morphological changes by acridine orange (AO) staining were studied. Lipidsoluble vitamin E, carotenoids (-carotene and astaxanthin) and water-soluble vitamin C were tested as antioxidant vitamins. In subconfluent cells, cell number declined markedly following 1-2 days of hyperoxia in parallel to declines of MTT activity and thymidine incorporation. LDH release and apoptotic changes were minimal. In confluent cells, hyperoxic injury increased LDH levels and apoptosis detected by AO staining, but the decline in cell numbers, MTT activity and thymidine incorporation was less than in subcpnfluent cells. Vitamin E was most effective in protecting MTT' activity and also prevented increase in LDH release in confluent cells. Vitamin C protected changes in these parameters in subconfluent cells but not in confluent cells. Carotenoids showed no protection. These results indicate that confluence and antioxidant vitamin protection determine the effects of hyperoxic injury on MDCK epithelial cells. Supported by grants from the Minnesota Medical Foundation and Viking Children's Foundation.