Wagner J R, Blount B C, Weinfeld M
Centre de Recherche en Gérontologie et Gériatrie, Hôpital d'Youville, Sherbrooke, Québec, Canada.
Anal Biochem. 1996 Jan 1;233(1):76-86. doi: 10.1006/abio.1996.0010.
The possible release from gamma-irradiated DNA of eight oxidatively modified cytosine bases by Escherichia coli endonuclease III was examined by trimethylsilylation and gas chromatography/electron impact/mass spectrometry. The results indicated that endonuclease III induced the release of 5-hydroxyhydantoin (1), 5-hydroxyuracil (2), cis-uracil 5,6-glycol (3), 5-hydroxycytosine (4), trans-uracil 5,6-glycol (5), and trans-1-carbamoyl-2-oxo-4,5-dihydroxyimidazolidine (8). The release of these products increased with the initial amount of damage in DNA, i.e., the dose of gamma-radiation (0-100 Gy), giving 4.6 +/- 1.0 fmol of 1, 5.8 +/- 0.3 fmol of 2, 4.9 +/- 0.5 fmol of 3, 11.2 +/- 1.2 fmol of 4, 10.7 +/- 2.1 fmol of 5, and 1.5 +/- 0.5 fmol of 8, per microgram DNA per 10 Gy. In addition, we estimated that the relative rates of excision were 5 approximately equal to 3 > (1.2-fold) 1 > (1.5-fold) 4 > (3.3-fold) 2 on the basis of their initial yields in DNA and initial rates of release as a function of incubation time. The excision of 5-hydroxyuracil (2) and 5-hydroxycytosine (4) lesions was studied in greater detail by enzymatic digestion and HPLC coupled to electrochemical (EC) detection which determines the amounts of these products in DNA. The results showed that the excision of 4 was more efficient than that of 2 (2.7-fold) with greater than 50% of the lesions remaining in DNA after treatment. Finally, we examined the excision of products 2 and 4 from irradiated DNA (50 Gy) by whole human cell extracts. The release of product 2 into the hydrosylate was 5.2 +/- 1.4 fmol per microgram of DNA as measured by fluorobenzylation coupled to gas chromatography/electron capture negative-ion chemical ionization/mass spectrometry. In identical samples, the amount of product 2 was reduced by 45.0 +/- 2.6% (225 from 500 fmol per microgram of DNA) and that of product 4 by 7.0 +/- 3.1% (42 from 600 fmol per microgram of DNA) as measured by HPLC/EC analysis.
通过三甲基硅烷化以及气相色谱/电子轰击/质谱联用技术，检测了经γ射线辐照的DNA中8种氧化修饰的胞嘧啶碱基被大肠杆菌内切酶III切割后可能的释放情况。结果表明，内切酶III可诱导释放5-羟基乙内酰脲（1）、5-羟基尿嘧啶（2）、顺式尿嘧啶5,6-二醇（3）、5-羟基胞嘧啶（4）、反式尿嘧啶5,6-二醇（5）以及反式-1-氨基甲酰基-2-氧代-4,5-二羟基咪唑烷（8）。这些产物的释放量随DNA初始损伤量增加，即γ射线辐射剂量（0 - 100 Gy）增加而增加，每微克DNA每10 Gy可产生4.6±1.0 fmol的1、5.8±0.3 fmol的2、4.9±0.5 fmol的3、11.2±1.2 fmol的4、10.7±2.1 fmol的5以及1.5±0.5 fmol的8。此外，基于它们在DNA中的初始产量以及作为孵育时间函数的初始释放速率，我们估计切除的相对速率为5≈3＞（1.2倍）1＞（1.5倍）4＞（3.3倍）2。通过酶消化以及与电化学（EC）检测联用的高效液相色谱法（HPLC）对5-羟基尿嘧啶（2）和5-羟基胞嘧啶（4）损伤的切除情况进行了更详细的研究，该方法可测定DNA中这些产物的含量。结果表明，4的切除效率比2高（2.7倍），处理后DNA中仍有超过50%的损伤残留。最后，我们检测了全人细胞提取物对辐照DNA（50 Gy）中产物2和4的切除情况。通过与气相色谱/电子捕获负离子化学电离/质谱联用的氟苄基化方法测定，产物2释放到水解产物中的量为每微克DNA 5.2±1.4 fmol。在相同样品中，通过HPLC/EC分析测定，产物2的量减少了45.0±2.6%（从每微克DNA 500 fmol降至225 fmol），产物4的量减少了7.0±3.1%（从每微克DNA 600 fmol降至42 fmol）。
