Detection of endonuclease III- and 8-oxoguanine glycosylase-sensitive base modifications in γ-irradiated DNA and cells by the aldehyde reactive probe (ARP) assay
Detection of endonuclease III- and 8-oxoguanine glycosylase-sensitive base modifications in γ-irradiated DNA and cells by the aldehyde reactive probe (ARP) assay
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DOI：
10.1269/jrr.45.229
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发表时间：
2004-06-01
影响因子：
2
通讯作者：
Ide, H
中科院分区：
文献类型：
--
作者：
Ali, MM;Kurisu, S;Ide, H
电离辐射会产生不同的 DNA 损伤，从而不同程度地诱导细胞死亡和突变。在本研究中，小牛胸腺 DNA (400 mug/ml) 和 HeLa 细胞经 (60)Co 伽马射线照射，通过醛反应探针 (ARP) 测定对 DNA 中的脱碱基 (AP) 位点以及核酸内切酶 (Endo)III-和 8-氧代鸟嘌呤糖基化酶 (hOGG1) 敏感的碱基修饰进行定量。在磷酸盐缓冲液中照射小牛胸腺 DNA 会产生 91 个 Endo III 和 100 个 hOGG1 敏感碱基修饰，以及每 Gy 每 10(6) 个碱基对 (bp) 110 个 AP 位点。 Tris 缓冲液中的损伤发生率比磷酸盐中的损伤发生率低 41 至 91 倍，这证明了 Tris 的辐射防护作用。 HeLa 细胞染色体 DNA 含有 12 个 Endo III 敏感碱基修饰和 3.8 个 hOGG1 敏感碱基修饰，以及每 10(6) bp 少于 1 个 AP 位点作为内源性损伤，并且其水平通过辐射而增加。 1 Gy（大致相当于 HeLa 细胞的致死剂量 [1.6-1.8 Gy]）时的损伤产量为每 10(6) bp 0.13 Endo III、0.091 hOGG1 和 0.065 个 AP 位点，表明与内源性损伤相比，致死剂量的照射仅导致基础损伤略有增加。将现有数据与报道的 DNA 链断裂数据进行比较，支持双链断裂和聚集性损伤作为电离辐射形成的致命损伤的首要重要性。
Ionizing radiation generates diverse DNA lesions that differentially induce cell death and mutations. In the present study, calf thymus DNA (400 mug/ml) and HeLa cells were irradiated by (60)Co gamma-rays, and abasic (AP) sites and endonuclease (Endo)III- and 8-oxoguanine glycosylase (hOGG1)-sensitive base modifications in DNA were quantitated by the aldehyde reactive probe (ARP) assay. The irradiation of calf thymus DNA in phosphate buffer generated 91 Endo III- and 100 hOGG1-sensitive base modifications and 110 AP sites per 10(6) base pairs (bp) per Gy. The yield of the lesions in Tris buffer was 41- to 91-fold lower than that in phosphate, demonstrating a radioprotective effect of Tris. The HeLa cell chromosomal DNA contained 12 Endo III- and 3.8 hOGG1-sensitive base modifications and less than 1 AP sites per 10(6) bp as endogenous damage, and their level was increased by irradiation. The yields of the damage at 1 Gy (roughly equivalent to the lethal dose of HeLa cells [1.6-1.8 Gy]) were 0.13 Endo III, 0.091 hOGG1, and 0.065 AP sites per 10(6) bp, showing that irradiation with a lethal dose brought about only a marginal increase in base damage relative to an endogenous one. A comparison of the present data with those reported for DNA strand breaks supports the primary importance of double-strand breaks and clustered lesions as lethal damages formed by ionizing radiation.