Hydrogen peroxide or heat shock induces resistance to hydrogen peroxide in Chinese hamster fibroblasts

Abstract

Survival after H₂O₂ exposure or heat shock of asynchronous Chinese hamster ovary cells (HA-1) was assayed following pretreatment with mildly toxic doses of either H₂O₂ or hyperthermia. H₂O₂ cytotoxicity at 37°C, expressed as a function of mM H₂O₂ was found to be dependent on cell density at the time of treatment. The density dependence reflected the ability of cells to reduce the effectiveness of H₂O₂ as a cytotoxic agent. When the survival data were plotted as a function of μmoles H₂O₂/cell at the beginning of the treatment, survival was independent of cell density. Cells pretreated with 0.1 mM (3–5 μmoles/cell × 10⁻⁷) H₂O₂ for 1 hr at 37°C (30–50% survival) became resistant to a subsequent H₂O₂ treatment 16–36 hr after pretreatment [dose modifying factor (DMF) at 1% isosurvival = 4–6]. Their resistance to 43°C heating, however, was only slightly increased over controls 16–36 hr following pretreatment (DMF at 1% isosurvival = 1.2). During this same interval, the synthesis of protein migrating in the 70 kD region of a one-dimensional SDS-polyacrylamide gel was enhanced twofold in the H₂O₂-pretreated cells. When the cells were heated for 15 min at 45°C (40–60% survival), the survivors became extremely resistant to 43°C heating and somewhat resistant to H₂O₂ (DMF at 1% isosurvival = 2). The heat-induced resistance to heat developed much more rapidly (reached a maximum between 6 and 13 hr) following pretreatment than the heat-induced rsistance to H₂O₂ (16–36 hr). The enhanced synthesis of 70 kD protein after heat shock was greater in magnitude and occurred more rapidly following preheating than following H₂O₂ pretreatment. The cells that became resistant to H₂O₂ by either pretreatment (H₂O₂ or heat shock) also increased their ability to reduce the H₂O₂ cytotoxicity from the treatment medium beyond that of the untreated HA-1 cells. This may be one of the mechanisms involved in the increased resistance and a common adaptive mechanism induced by both stresses. These data indicate that mammalian cells develop resistance to H₂O₂ following mild pretreatment with H₂O₂ or heat shock. The cross-resistance induced by H₂O₂ and heat shock reinforce the hypothesis that some overlap in mechanisms exist between the cellular responses to these two stresses. However, the failure of H₂O₂ pretreatment to induce much resistance to heat indicates that there are also differences in the actions of the two agents.