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

摘要

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 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 release of either malondialdehyde and free base or a base propenal. Highly sensitive gas chromatography-mass spectrometry (GC/MS) 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 pre-purification, both PG and HMP were silylated and quantified by GC-MS with limits of detection of 100 and 200 fmol, and sensitivities of 2 and 4 lesions per 10⁶ nucleotides (nt) in 250 μg 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 γ-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 lesion frequency against total 2-deoxyribose oxidation events, the latter determined by a plasmid nicking assay. 3PG residues and oxAB were produced at the rate of 32 and 12 lesions per 10⁶ nt per μM, respectively. For γ-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).