Establishing the background level of base oxidation in human lymphocyte DNA: results of an interlaboratory validation study

Abstract

Accurate measurement of low levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) in DNA is hampered by the ease with which guanine is oxidized during preparation of DNA for analysis. ESCODD, a consortium of mainly European laboratories, has attempted to minimize this artifact and to provide standard, reliable protocols for sample preparation and analysis. ESCODD has now analyzed 8-oxodGuo in the DNA of lymphocytes isolated from venous blood from healthy young male volunteers in several European countries. Two approaches were used. Analysis of 8-oxodGuo by HPLC with electrochemical detection was performed on lymphocytes from 10 groups of volunteers, in eight countries. The alternative enzymic approach was based on digestion of DNA with formamidopyrimidine DNA glycosylase (FPG) to convert 8-oxo-7,8-dihydroguanine (8-oxoGua) to apurinic sites, subsequently measured as DNA breaks using the comet assay (7 groups of volunteers, in six countries). The median concentration of 8-oxodGuo in lymphocyte DNA, calculated from the mean values of each group of subjects as determined by HPLC, was 4.24 per 10(6) guanines. The median concentration of FPG-sensitive sites, measured with the comet assay, was 0.34 per 10(6) guanines. Identical samples of HeLa cells were supplied to all participants as a reference standard. The median values for 8-oxodGuo in HeLa cells were 2.78 per 10(6) guanines (by HPLC) and 0.50 per 10(6) guanines (by enzymic methods). The discrepancy between chromatographic and FPG-based approaches may reflect overestimation by HPLC (if spurious oxidation is still not completely controlled) or underestimation by the enzymic method. Meanwhile, it is clear that the true background level of base oxidation in DNA is orders of magnitude lower than has often been claimed in the past.