Oxidative damage to DNA and replicative lifespan in cultured adrenocortical cells

Abstract

Oxidative damage to DNA in cultured bovine adrenocortical cells was investigated by exposing cells to a sublethal concentration (10 μM) of cumene hydroperoxide under conditions previously shown to be deficient in the biological antioxidants selenium and α-tocopherol (vitamin E). DNA prepared from cells incubated for 4 h with 10 μM cumene hydroperoxide had a greater fraction showing resistance to S1 nuclease after denaturation and reassociation to a log C 0 t of −3. Cross-linking by cumene hydroperoxide was abolished in cells that had been grown in the presence of 20 nM selenite or 1 μM α-tocopherol for 96 h prior to peroxide addition, whereas such cells remained susceptible to crosslinking by nitrogen mustard. Extensive strand breaks in DNA from peroxide-treated cells as assessed by alkaline sucrose gradient centrifugation were greatly reduced in cells grown in selenite or α-tocopherol. Despite the evidence of damage to DNA, cumene hydroperoxide was not detectably mutagenic, in contrast to 5 μM methylnitronitrosoguanidine (MNNG), when assessed as the incidence of resistance to 25 μM ouabain. We confirmed that cumene hydroperoxide at >10 μM lowers cloning efficiency and that this is largely prevented by selenite or α-tocopherol. Additionally, selenite or α-tocopherol produced increased clonogenicity in cells not incubated with peroxide. To examine effects of the biological antioxidants on replicative lifespan, cells were grown continuously in fetal bovine serum (FBS), fibroblast growth factor (FGF), and selenite or α-tocopherol. Selenium increased replicative lifespan by 10–20% and α-tocopherol by 22–30%. Levels of DNA cross-links and strand breaks did not differ under any circumstances between early (second) passage and late (30th) passage cells. The experiments on replicative potential were all performed in the presence of FGF. When FGF was omitted from the culture medium, replicative lifespan was reduced by 85%. We conclude that types of damage to DNA resulting from peroxide exposure are not present in cells under standard culture conditions at early or late stages of the lifespan. Other work has noted a relationship between clonogenicity and replicative lifespan; thus, the increase in cloning efficiency seen with selenium and α-tocopherol may cause the observed slight increase in replicative lifespan. Oxidative damage does not appear to be a major determinant of cellular senescence in adrenocortical cells.