Evidence for the accumulation of oxidative stress during cellular ageing of human diploid fibroblasts.

We have examined the level of 8-hydroxydeoxyguanosine (8-OH-dG) in the DNA of human diploid fibroblast TIG-1 cells at various stages of cellular ageing. Cellular DNA was obtained from cell lysates by simple precipitation with ethanol and analyzed 8-OH-dG levels by an electrochemical detection system. The data reveals that this method is valid for the detection of 8-OH-dG at levels of 1 x 10(-6) 8-OH-dG/dG without altering the recovery of other deoxynucleosides. Using this method, we demonstrate a significant increase in 8-OH-dG content during cellular ageing up to a 50 population doubling level (PDL) of TIG-1 cells, with levels of 0.5 x 10(-6) and 1.6 x 10(-6) 8-OH-dG/dG in young (22 PDL) and old (50 PDL) cells, respectively. The level decreases with further ageing and is 1.3 x 10(-6) 8-OH-dG/dG in senescent cells. We also show that the repair activity for the 8-OH-dG lesion declines significantly during cellular ageing, a phenomenon that might be associated with the increase in 8-OH-dG levels.