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

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 (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 prepurification, 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 the lesion frequency against total 2-deoxyribose oxidation events, with 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).