Takeuchi T, Nakajima M, Ohta Y, Mure K, Takeshita T, Morimoto K
Department of Hygiene and Preventive Medicine, Osaka University School of Medicine, Japan.
Carcinogenesis. 1994 Aug;15(8):1519-23. doi: 10.1093/carcin/15.8.1519.
8-Hydroxydeoxyguanosine (8-OHdG) is a typical form of oxidative DNA damage which causes mutation in vitro and in vivo. We investigated potential factors confounding 8-OHdG determination and, based on the results, then determined the 8-OHdG levels in human peripheral blood leukocytes. 8-OHdG was detected electrochemically after extraction of DNA from the cells without the use of phenol by a DNA extractor under helium. In the preliminary experiments, the mononuclear leukocytes (MN) in blood samples obtained from 19 laboratory workers and students were separated from the polymorphonuclear leukocytes (PMN) with Mono-Poly resolving 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 during DNA extraction was then examined by adding RBC to the human lymphoblastoid 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/FA72 cell ratio of 20 increased the 8-OHdG level 1.43-fold over that without RBC. The potential effect of histidine, a scavenger of both hydroxyl radicals and singlet oxygen, on reduction of artificial 8-OHdG formation 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 concentration). Based on these results, we determined the 8-OHdG levels in human leukocyte samples obtained from 79 healthy male factory workers aged 24-59 years. The leukocyte fraction containing both MN and PMN was separated from RBC with Mono-Poly resolving medium and DNA was extracted from the leukocytes in the presence of 30 mM His. The mean 8-OHdG level 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 determinations and the 8-OHdG levels of both the leukocyte samples and the FA72 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 determination was not correlated with that at the second determination. The range of 8-OHdG levels in the samples was relatively small compared with the CV of the assay.(ABSTRACT TRUNCATED AT 400 WORDS)
8-羟基脱氧鸟苷（8-OHdG）是一种典型的氧化性DNA损伤形式，可在体内外引起突变。我们研究了影响8-OHdG测定的潜在因素，并基于这些结果测定了人类外周血白细胞中的8-OHdG水平。在氦气环境下，使用DNA提取仪从细胞中提取DNA后，不使用苯酚，通过电化学方法检测8-OHdG。在初步实验中，使用单核-多形核细胞分离培养基从19名实验室工作人员和学生的血液样本中分离出单核白细胞（MN）和多形核白细胞（PMN）。MN中的8-OHdG（每10⁵个脱氧鸟苷中有1.157±0.414个分子）与PMN中的8-OHdG（1.131±0.418）无显著差异。然后通过向人淋巴母细胞系FA72中添加红细胞（RBC），研究了RBC在DNA提取过程中对8-OHdG形成的影响。以每FA72细胞最多4个RBC的比例添加RBC不会增加8-OHdG水平，而以20个RBC/FA72细胞的比例添加则使8-OHdG水平比不添加RBC时增加了1.43倍。在人早幼粒细胞白血病细胞系HL60的DNA提取过程中，研究了组氨酸（一种羟基自由基和单线态氧的清除剂）对减少人工8-OHdG形成的潜在影响。添加组氨酸使8-OHdG水平呈剂量依赖性降低（在30 mM组氨酸浓度下降低30%）。基于这些结果，我们测定了79名年龄在24至59岁的健康男性工厂工人的白细胞样本中的8-OHdG水平。使用单核-多形核细胞分离培养基从RBC中分离出含有MN和PMN的白细胞部分，并在30 mM组氨酸存在的情况下从白细胞中提取DNA。这些样本中的平均8-OHdG水平为1.072±0.230。为评估该测定方法的可靠性，在所有测定中均使用FA72作为标准样本，并在每次测定中测量白细胞样本和FA72样本的8-OHdG水平。计算得出批间和批内变异系数（CV）分别为14.4%（n = 14）和3.9 - 13.5%（每次测定n = 3 - 5）。对19个白细胞样本进行了两次8-OHdG水平测量；第一次测定的值与第二次测定的值不相关。与测定的CV相比，样本中8-OHdG水平的范围相对较小。（摘要截断于400字）
