EVALUATION OF 8-HYDROXYDEOXYGUANOSINE, A TYPICAL OXIDATIVE DNA-DAMAGE, IN HUMAN-LEUKOCYTES

8-Hydroxydeoxyguanosine (8-OHdG) is a typical form of oxidative DNA da mage which causes mutation in vitro and in vivo. We investigated poten tial factors confounding 8-OHdG determination and, based on the result s, then determined the 8-OHdG levels in human peripheral blood leukocy tes. 8-OHdG was detected electrochemically after extraction of DNA fro m the cells without the use of phenol by a DNA extractor under helium. In the preliminary experiments, the mononuclear leukocytes (MN) in bl ood samples obtained from 19 laboratory workers and students were sepa rated from the polymorphonuclear leukocytes (PMN) with Mono-Poly resol ving medium. The 8-OHdG in the MN (1.157 +/- 0.414 molecules per 10(5) deoxyguanosine) did not differ significantly from that in PMN (1.131 +/- 0.418). The effect of red blood cells (RBC) on 8-OHdG formation du ring DNA extraction was then examined by adding RBC to the human lymph oblastoid cell line FA72. Addition of RBC at ratios of up to 4 RBC per FA72 cell did not increase 8-OHdG levels, while addition at a RBC/FA7 2 cell ratio of 20 increased the 8-OHdG level 1.43-fold over that with out RBC. The potential effect of histidine, a scavenger of both hydrox yl radicals and singlet oxygen, on reduction of artificial 8-OHdG form ation during DNA extraction was examined during DNA extraction in the human promyelocytic leukemia cell line HL60. Addition of His decreased the 8-OHdG level dose-dependently (30% reduction at 30 mM His concent ration). Based on these results, we determined the 8-OHdG levels in hu man leukocyte samples obtained from 79 healthy male factory workers ag ed 24-59 years. The leukocyte fraction containing both MN and PMN was separated from RBC with Mono-Poly resolving medium and DNA was extract ed from the leukocytes in the presence of 30 mM His. The mean 8-OHdG l evel in these samples was 1.072 +/- 0.230. To evaluate the reliability of the assay, FA72 was used as a standard sample in all assay determi nations and the 8-OHdG levels of both the leukocyte samples and the FA 72 sample(s) were measured in each determination, The inter- and intra -assay coefficients of variation (CV) were calculated to be 14.4% (n = 14) and 3.9-13.5% (n = 3-5 per assay) respectively. The 8-OHdG level was measured twice in 19 leukocyte samples; the value at the first det ermination was not correlated with that at the second determination. T he range of 8-OHdG levels in the samples was relatively smallcompared with the CV of the assay. The 8-OHdG level was correlated with neither the age nor the smoking status of the donors.