Transient Adaptation to Oxidative Stress in Mammalian Cells

Abstract

We report a transient adaptation to the oxidative stress of hydrogen peroxide (H2O2) exposure in several mammalian cell lines: Chinese hamster ovary fibroblast (CHO) cells, HA-1 cells (a defined CHO subclone), C3H 10T12 cells (embryonic mouse fibroblasts), V79 cells (Chinese hamster lung fibroblasts), and Clone 9 liver cells (rat liver epithelial cells). Up to 40-fold adaptive increases in resistance to H2O2 challenge occurred following pretreatment with relatively low H2O2 "priming" doses, from as little as 1.9% cell viability for untreated cells to as much as 76.5% viability for H2O2 pretreated cells. Detailed studies with HA-1 cells revealed the following pattern of responses to H2O2: very low H2O2 concentrations of 0.1 to 0.5 μmo1/107 cells (3 to 15 μM) stimulated cell growth by 25 to 45%; low H2O2 concentrations of 2-5 μmo1/107 cells (120 to 150 μM) induced a temporary growth-arrest, a lengthening of cell cycle from 18 h to approximately 26 h, and marked adaptive increases in H2O2 resistance; intermediate H2O2 concentrations of 9 to 14 μmo1/107 cells (250 to 400 μM) caused permanent growth-arrest (i.e., permanent loss of replicative or divisional competence) with no evidence of necrosis; high H2O2 concentrations of 30 μmol/107 cells or greater (≥ 1 mM) caused an apoptotic-like necrotic cell death and destruction. The adaptive response to low H2O2 concentrations of 2-5 μmol/107 (120 to 150 μM) was maximal 18 h after pretreatment of HA-1 cells, declined thereafter toward baseline sensitivity, and was observed with both 7-day fix and stain procedures and clonogenic viability assays. Transient adaptation following H2O2 pretreatment of 4.15 μmol/107 (150 μM) involved the de novo synthesis of at least 20 proteins and was blocked by the translation inhibitor, cycloheximide. During the 18-h adaptation in HA-1 cells proteins were synthesized in three phases; early (0-4 h), middle (4-8 h), and late (8-15 h). No H2O2 response proteins were synthesized beyond 18 h after pretreatment, by which time adaptation had already maximized. Selective translational inhibition of the early, middle, or late proteins revealed that all three sets were necessary for a maximal adaptive increase in H2O2 resistance. Northern blot and enzyme activity analyses revealed no significant increases in transcription or translation of the classical antioxidant enzymes catalase, glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase, Cu, Zn superoxide dismutase, or Mn superoxide dismutase in H2O2-adapted HA-1 cells. Furthermore, the total H2O2-consuming capacity of HA-1 cells was unchanged by H2O2 adaptation. Since resistance to oxidative stress is determined by both antioxidant capacity and the activities of various damage removal and repair enzymes, transiently adapted HA-1 cells should provide a good model in which to study the induction of damage removal and repair enzymes.