Background: Apoptosis is a highly regulated type of cell death that occurs in multicellular organisms. Apoptosis can arise following exposure of cells to various types of stress, including hyperthermia, radiation, oxidative stress and chemotherapeutic agents. The mechanisms of hyperthermia-induced cell death are not entirely understood. Hyperthermia could promote an increase in oxidative stress, thus creating a redox imbalance in favour of pro-oxidants. This could arise by increasing generation and reactivity of oxidants such as superoxide and H₂O₂ and/or by inactivating cellular antioxidant defences. As a consequence, hyperthermia is likely to induce oxidative changes in cells leading to cell damage and eventually cell death. This study evaluates the by altering the level of antioxidant defenses against superoxide. We investigated (i) the ability of the antioxidants Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (MnTBAP) and polyethylene glycol-superoxide dismutase (PEG-SOD) to protect cells against hyperthermia-induced apoptosis, and (ii) the ability of diethyldithiocarbamate (DDC), an inhibitor of the antioxidant SOD, to increase hyperthermia-induced apoptosis.

Methods: Cells, with normal or altered levels of antioxidants, were heated at 42 and 43^oC relative to controls at 37^oC. Apoptosis was evaluated at the level of the stress-activated mitochondrial pathway. The proteins Bax, cytochrome c, inhibitor of caspase-activated DNase (ICAD) and poly (ADP-ribose) polymerase-1 (PARP) were analysed by Western blotting and the activity of caspase enzymes by spectrofluorimetry. Cell death by apoptosis and necrosis was monitored by fluorescence microscopy using Hoechst (chromatin condensation) and propidium iodide dyes, respectively.

Results: The antioxidants MnTBAP and PEG-SOD protected cells against hyperthermia-induced apoptosis. They inhibited hyperthermia-induced translocation of Bax and cytochrome c between mitochondria and the cytosol. These antioxidants inhibited heat-induced activation of caspase-9 and caspase-3. MnTBAP and PEG-SOD also inhibited hyperthermia-induced cleavage of ICAD and PARP and chromatin condensation. The SOD inhibitor, DDC, also inhibited hyperthermia-induced apoptosis, but instead caused an increase in hyperthermia-induced cell death by necrosis.

Conclusion: In conclusion, these results show that oxidative stress is involved in hyperthermia-induced apoptosis via the mitochondrial pathway.