GC/MS methods to quantify the 2-deoxypentos-4-ulose and 3′-phosphoglycolate pathways of 4′ oxidation of 2-deoxyribose in DNA:: application to DNA damage produced by γ radiation and bleomycin
GC/MS methods to quantify the 2-deoxypentos-4-ulose and 3′-phosphoglycolate pathways of 4′ oxidation of 2-deoxyribose in DNA:: application to DNA damage produced by γ radiation and bleomycin
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DOI：
10.1021/tx700164y
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发表时间：
2007-11-01
影响因子：
4.1
通讯作者：
Dedon, Peter C.
中科院分区：
文献类型：
--
作者：
Chen, Bingzi;Zhou, Xinfeng;Dedon, Peter C.
DNA氧化在癌症等人类疾病的病理生理学中起着重要作用。虽然碱基损伤的化学一直主导着DNA损伤的研究，但越来越多的证据表明，DNA中2-脱氧核糖的氧化在氧化应激的遗传毒理学中发挥着关键作用。作为确定体内和体外2-脱氧核糖氧化产物谱的一部分，我们现在描述DNA中2-脱氧核糖4‘氧化产物的定量方法。4‘氧化分配在两条途径中的任一条之间的化学作用，形成2-脱氧五磷酸基(OxAB)或由3’-磷酸乙醇酸(3PG)残基和5‘-磷酸组成的链断裂，释放丙二醛和游离碱或丙二醛。开发了高灵敏的气相色谱/质谱仪(GUMS)方法来量化这两种损伤。用肼处理DNA损伤后，碱基位置转变为3‘-磷-3-哒嗪甲基衍生物，DNA经酶解后释放为3-羟甲基哒嗪(HMP)。同样，3PG通过酶解释放为2-磷酸乙醇酸(PG)。经高效液相色谱预纯化后，PG和HMP被硅烷化，并用胶体定量，检测限分别为100和200fmol，灵敏度分别为2个和4个斑点/106个核苷酸(M)/250mDNA。在验证了含有这两个损伤的寡核苷酸的方法后，这些方法被应用于博莱霉素和伽玛射线造成的DNA损伤。如预期的那样，对于一种仅产生DNA 4‘氧化的试剂，3PG和oxAB的数量占所有2-脱氧核糖氧化事件的数量，如斜率分别为0.8和0.3所示，在损伤频率与总的2-脱氧核糖氧化事件的曲线图上，后者确定为质粒划痕试验。3pg残留物和oxAB的产量分别为32个/10nT/亩M和12个/亩M。另一方面，对于伽马辐射，4‘氧化仅占2-脱氧核糖氧化化学的13%，3%的oxAB(每106nT/Gy4个)和10%3Pg(每106nT/Gy13个)。
DNA oxidation plays a substantive role in the pathophysiology of human diseases, such as cancer. While the chemistry of nucleobase lesions has dominated studies of DNA damage, there is growing evidence that the oxidation of 2-deoxyribose in DNA plays a critical role in the genetic toxicology of oxidative stress. As part of an effort to define the spectrum of 2-deoxyribose oxidation products arising in vitro and in vivo, we now describe methods for quantifying products arising from 4' oxidation of 2-deoxyribose in DNA. The chemistry of 4' oxidation partitions between either of two pathways to form either a 2-deoxypentos-4-ulose abasic site (oxAB) or a strand break comprised of a 3'-phosphoglycolate (3PG) residue and a 5'-phosphate, with the release of either malondialdehyde and free base or a base propenal. Highly sensitive gas chromatography/mass spectrometry (GUMS) methods were developed to quantify both lesions. The abasic site was converted to a 3'-phosphoro-3-pyridazinylmethylate derivative by treatment of the damaged DNA with hydrazine, which was released from DNA as 3-hydroxymethylpyridazine (HMP) by enzymatic hydrolysis. Similarly, 3PG was released as 2-phosphoglycolic acid (PG) by enzymatic hydrolysis. Following HPLC prepurification, both PG and HMP were silylated and quantified by GUMS, with limits of detection of 100 and 200 fmol and sensitivities of 2 and 4 lesions per 106 nucleotides (m) in 250 mu g of DNA, respectively. Following validation of the methods with oligodeoxynucleotides containing the two lesions, the methods were applied to DNA damage produced by bleomycin and gamma radiation. As expected for an agent known to produce only 4' oxidation of DNA, the quantities of 3PG and oxAB accounted for all 2-deoxyribose oxidation events, as indicated by slopes of 0.8 and 0.3, respectively, in plots of the lesion frequency against total 2-deoxyribose oxidation events, with the latter determined a plasmid-nicking assay. 3PG residues and oxAB were produced at the rate of 32 and 12 lesions per 10 nt per mu M, respectively. For gamma radiation, on the other hand, 4' oxidation was found to comprise only 13% of 2-deoxyribose oxidation chemistry, with 3% oxAB (4 per 106 nt per Gy) and 10% 3PG (13 per 106 nt per Gy).