Oxidative DNA damage as a marker of aging in WI-38 human fibroblasts

Abstract

Cause–effect relationships between oxidative stress, DNA damage and aging were investigated in WI-38 human diploid fibroblasts at 21, 41 or 58 population doublings (PDs), corresponding to young, middle age or old fibroblasts, respectively. Oxidative DNA damage was evaluated by immunohistochemical detection of 8-hydroxy-2′deoxyguanosine (8-OHdG) adducts or by single cell microgel electrophoresis (COMET assay). Aging was evaluated by growth rate, senescence-associated-β-galactosidase (SA-β galactosidase) activity, cell cycle distribution, and expression of p21. Our results demonstrate that (i) oxidative DNA damage is proportional to the age of cells (ii) DNA damage in old/58 PDs cells reflects both an increased susceptibility to oxidative stress, induced by acute exposure to sub-lethal concentrations of hydrogen peroxide (H 2O 2), and a reduced efficiency of repair mechanisms. We also show that mild chronic oxidative stress, induced by prolonged exposure to 5 μM H 2O 2, accelerates aging in fibroblasts. In fact, this treatment increased 8-OHdG levels, SA-β-galactosidase activity, and G0/G1 cell cycle arrest in middle age/41 PDs, making them similar to H 2O 2-untreated old/58 PDs cells. Although other mechanisms may concur in mediating the effects of H 2O 2, these results lend support to the concept that oxidative stress may be a key determinant of aging. Measurements of oxidative DNA damage might therefore be exploited as reliable marker of cellular aging.