Bingzi Chen

Postdoctoral Fellow

Genomic DNA is continuously exposed to endogenous and exogenous oxidative agents (Fe2+, peroxynitrite, etc.), ionizing radiation, and secondary oxidative reactive species generated by them. There is growing evidence that oxidation of DNA deoxyribose plays a critical role in the genetic toxicology of oxidative stress, including involvement in complex DNA lesions, cross-linking with DNA repair proteins and the formation of endogenous DNA adducts.

Oxidation of deoxyribose in DNA produces a wide range of lesions, with a different spectrum of products for each position in the sugar.  For example, due to both solvent accessibility and issues of site-specificity, oxidation of the C4´ and C5´ positions of deoxyribose are common to many DNA damaging agents. At least two lesions are initiated by hydrogen abstraction from the deoxyribose C4' carbon, depending on reaction with oxygen: the abasic 2-deoxypentos-4-ulose (C4-keto-aldehyde) without chain breakage, or a 3'-phosphoglcolate on one side of a strand break. 3´-Phosphoglycolate (PGL) is a major 3'- terminal blocking group, which must be removed to create a single nucleotide gap before base excision repair (BER).  With two free aldehyde/ketone groups, 4´-ketoaldehyde abasic site is a strong electrophile, which can react with a variety of nucleophiles (protein, DNA bases) to form crosslink products.

Chemical reactions following abstraction of the C5´-hydrogen atom partition to form either a nucleoside 5´-aldehyde residue attached to the 5´-end of the DNA strand or a 5´-formyl phosphate residue attached to the 3´-end of the DNA strand that is accompanied by a four-carbon fragment on the 5´-end.

As part of a larger effort to systematically quantify all deoxyribose lesions and investigate the secondary lesions arising from them, we have developed sensitive analytical techniques to quantify 4´ and 5´-deoxyribose (dR) oxidation products.

By applying our newly developed methods for analysis of 4´-oxidation products, we found that phosphoglycolate accounts for about 11% of total DNA deoxyribose oxidation events caused by g-radiation. Phosphoglycolate arises at levels 10-times higher than the 3´-oxidaiton product of deoxyribose, the 3´-phosphoglycoaldehyde residue. Bleomycin specifically targets the 4´ position of deoxyribose in DNA and induces the formation of both 3´-phosphoglycolate and 4´-ketoaldehyde abasic sites.  Our data show that 3´-phosphoglycolate (75%) is the major product under aerobic conditions as expected.  The 4´-ketoaldehyde abasic site accounts for only 25% of the detected C4´ damage.

To identify and quantify an A-/B-unsaturated dicarbonyl product of 5´-oxidation of dR in DNA: 2-phosphoryl-1,4-dioxobutane. We approached this problem by subjecting oxidized DNA to reaction with hydrazine to form the B-elimination derivative pyridazine which was then quantified by isotope dilution GC/MS. With a limit of detection of 50 fmol, the technique is illustrated with DNA oxidized by neocarzinostatin (NCS; causes 5´-oxidation of dR); Fe(II)-bleomycin (bleo; causes only 4´-oxidation of dR); and Fe(II)-EDTA and g-radiation (both oxidize all positions in dR). The results revealed a linear dose-response with g-radiation and NCS. However, bleo does not produce detectable amounts of pyridazine. The results have verified several hypotheses related to 4´- and 5´-chemistry of dR oxidation in DNA and provided new approaches to studying dR oxidation in biological systems.

We are now combining these approaches with quantification of total DNA deoxyribose oxidation events and quantification of products from other deoxyribose positions.  This will offer valuable insights into the spectrum of DNA deoxyribose oxidation products and the chemical mechanisms behind the partitioning.

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