title: Replacement of media in cell culture alters oxygen toxicity: possible role of lipid aldehydes and glutathione transferase in oxygen toxicity.
abstract: Replacement of media in cell cultures during exposure to hyperoxia was found to alter oxygen toxicity. Following 100 hr of exposure to 95% or 80% O2, the surviving fraction (SF) of Chinese hamster fibroblasts, as assayed by clonogenicity, was less than 1 x 10(-3) when the culture media was replaced only at the onset of the O2 exposure. Media replacement every 24 hr throughout the hyperoxic exposure resulted in SFs of 1.7 x 10(-1) (95% O2) and 1.9 x 10(-1) (80% O2) at 95 hr. Cellular resistance to and metabolism of 4-hydroxy-2-nonenal (4HNE), a cytotoxic byproduct of lipid peroxidation, was examined in cells 24 hr following exposure to 80% O2 for 144 hr with media replacement. These O2-exposed cells were resistant to 4HNE, requiring 2.6 times as long in 80 microM 4HNE to reach 30% survival as compared to density-matched normoxia control. Furthermore, during 40 and 60 min of exposure to 4HNE, the O2-preexposed cells metabolized greater quantities of 4HNE (fmole/cell) relative to control. The activity of glutathione S-transferase (GST), an enzyme believed to be involved with the detoxification of 4HNE, was significantly increased in the O2-preexposed cells compared with controls. Catalase activity was significantly increased, but no change was found in total glutathione content, glutathione peroxidase, manganese superoxide dismutase, and copper-zinc superoxide dismutase activities at the time of 4HNE treatment in the O2-preexposed cells relative to density-matched control. The results demonstrate that in vitro tolerance to the cytotoxic effects of hyperoxia can be achieved through media replacement during O2 exposure. Tolerance to oxygen toxicity conferred resistance to the cytotoxic effects of 4HNE, possibly through GST-catalyzed detoxification. These results provide further support for the hypothesis that toxic aldehydic byproducts of lipid peroxidation contribute to hyperoxic injury.
authors.affiliation: Department of Pediatrics, University of Virginia Hospital, Charlottesville 22908.
authors.forename: S J
authors.initials: SJ
authors.lastname: Sullivan
authors.forename: R J
authors.initials: RJ
authors.lastname: Roberts
authors.forename: D R
authors.initials: DR
authors.lastname: Spitz
mesh_terms: D000447:Aldehydes / Q000378:metabolism / Q000494:pharmacology* / Q000633:toxicity; D000818:Animals; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D003470:Culture Media / Q000032:analysis / Q000494:pharmacology; D005347:Fibroblasts / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D005978:Glutathione / Q000032:analysis / Q000378:metabolism; D005979:Glutathione Peroxidase / Q000378:metabolism / Q000502:physiology; D005982:Glutathione Transferase / Q000378:metabolism / Q000502:physiology*; D015227:Lipid Peroxidation; D010100:Oxygen / Q000378:metabolism / Q000633:toxicity*; D013482:Superoxide Dismutase / Q000378:metabolism
chemical_list: D000447:Aldehydes; D003470:Culture Media; D005979:Glutathione Peroxidase; D013482:Superoxide Dismutase; D005982:Glutathione Transferase; D005978:Glutathione; C027576:4-hydroxy-2-nonenal; D010100:Oxygen