Factors influencing survival of mammalian cells exposed to hypothermia: V. Effects of hepes, free radicals, and H 2O 2 under light and dark conditions

Abstract

Cytotoxicity resulting from the interaction of fluorescent light from a flow hood with Hepes-buffered cell culture medium at room temperature was demonstrated. Toxicity was prevented by keeping both cells (V79 Chinese hamster) and medium shielded from direct fluorescent light (“dark conditions”) or by supplementing the medium with 10 μg/ml catalase; this suggests that extracellular hydrogen peroxide is a major cause of the lethal effect under “lighted conditions.” No sensitization resulted from the exposure of cells in a sodium bicarbonate (SBC)-buffered medium to fluorescent light, nor in a catalase supplemented SBC-buffered medium. The Hepes/light reaction during routine cell manipulations presensitized cells to hypothermia damage in the dark with the presensitization being more severe for 5 than for 10 °C hypothermic exposure. Presensitization was prevented by performing the complete experiment under dark conditions or by supplementing the medium with 10 μg/ml catalase. However, catalase did not improve the hypothermic survival when experiments were performed under dark conditions. Hence, 10 μg/ml catalase does not protect cells from hypothermic (5 and 10 °C) damage per se, but rather from Hepes/light sublethal damage which interacts with hypothermic sublethal damage to result in lethal lesions. Additionally, under dark conditions, superoxide dismutase (SOD), allopurinol, catalase plus SOD, DMSO, or mannitol did not improve survival when present during hypothermic storage, suggesting that extracellular superoxide anion, hydrogen peroxide, or hydroxyl radicals are not the cause of cell killing under conditions of pure hypothermia uncomplicated by prehypothermic ischemia or hypoxia.