Glutathione and ascorbate are negatively correlated with oxidative DNA damage in human lymphocytes

Intracellular antioxidants, glutathione and ascorbate, and two molecular ma rkers of oxidative DNA damage, 5-hydroxy-2 '-deoxycytidine (5-OH-dCyd) and 8-oxo-7,8dihydro-2 ' -deoxyguanosine (8-oxo-dGuo), were measured in lymphoc ytes from 105 healthy volunteers. The analysis of 5-OH-dCyd and 8-oxo-dGuo was carried out by HPLC with electrochemical detection such that both compo unds were detected on the same chromatography run. There was no significant difference in oxidative DNA damage when the extraction of DNA from cells u sing phenol was carried out under anaerobic conditions or in the presence o f metal ion chelators, This indicates that auto-oxidation of DNA during sam ple preparation was minimal. Using the above methods, the average level of oxidative DNA damage in lymphocytes was 2.9 +/- 1.4 for 5-OH-dCyd and 4.5 /- 1.8 for 8-oxo-dGuo lesions per 10(6) dGuo (n = 105), It is unlikely that artifactual oxidation contributed to the observed damage because the level of 5-OH-dCyd was comparable with that of 8-oxo-dGuo in lymphocyte DNA, whe reas 8-oxo-dGuo outnumbers 5-OH-dCyd by a ratio of >5:1 when DNA is exposed to various oxidants, including ionizing radiation or Fenton reagents, Rath er, the nearly equal levels of 5-OH-dCyd and 8-oxo-dGuo in cellular DNA imp lies that 8-oxo-dGuo may be more efficiently removed by DNA repair. Finally , and most importantly, the correlation of our endpoints revealed that the naturally occurring level of intracellular antioxidants was negatively corr elated to the level of oxidative DNA damage with the strongest correlation observed for glutathione and 8-oxo-dGuo (r = -0.36; P < 0.001), These resul ts strongly suggest that intracellular glutathione and ascorbate protect hu man lymphocytes against oxidative DNA damage.