The effect of mild hypothermia (34°C) and mild hyperthermia (39°C) on DNA damage, repair and aging of human diploid fibroblasts

Abstract

We used mild hypothermia (34°C) and mild hyperthermia (39°C) to examine aging at the cellular level in relation to DNA damage and repair. With the filter elution technique we monitored spontaneous single-strand breaks (SSBs) and double-strand breaks (DSBs) in DNA during in vitro aging at 34°C, 37°C and 39°C of normal human diploid fibroblasts (HDF). DNA repair was assessed after ionizing and non-ionizing (ultraviolet) radiation of HDF at different population doubling levels (PDLs): the former was assayed by filter elution and the latter by unscheduled DNA synthesis. Survival was assessed by trypan blue dye exclusion and colony formation. Cells at 37°C achieve a higher cumulative PDL (67 ± 6) than cells at 39°C (60 ± 5) or at 34°C (55 ± 6). The level of spontaneous SSBs and DSBs, and radiosensitivity of DNA to either 6 Gy or 100 Gy gamma rays, do not change with in vitro age at any of the three temperatures. Repair of SSBs (induced by 6 Gy) and DSBs (induced by 100 Gy) does not change with in vitro age: rejoining is 86–104% complete by 60 min repair and generally does not differ across temperatures. Response to non-ionizing radiation (254 nm, 75, 150, 300 ergs/mm 2) does not change with in vitro age at 37°C or 39°C, whereas excision repair increases with age at 34°C even though cell survival does not. The results do not support the rate of living theory of aging (Pearl, R., The Rate of Living, University of London Press, London, 1928) as applied to temperature effects on HDF aging in vitro (as measured by proliferative lifespan) and on their response to radiation-induced DNA damage.