Biological Fates of DNA and RNA Damage Products

A major hurdle to the development of damaged biomolecules as biomarkers is our lack of understanding of the metabolic fate of the damage products that are formed in tissues. This problem is framed with four questions:

(1) After formation in a tissue, is the product altered by metabolism during its course to the clinically relevant blood and urine sampling compartments? (2) What is the fate of damage products that are reactive electrophiles, in terms of local reactions and stable metabolites appearing the blood and urine sampling compartments? (3) Are there nuclear or cellular metabolic reactions that constitute a form of DNA repair in capturing locally generated reactive electrophiles before they can form DNA adducts?

With much of the DNA and RNA damage chemistry now characterized in terms of product structure, we have now turned our attention to the biological fates of these damage products, with the goal of developing biomarkers of mechanism, source and risk. This effort is illustrated in our studies of the spectrum of products arising from oxidation of 2-deoxyribose in DNA, both in vitro and in vivo. An important facet of this research has involved the development of sensitive and quantitative analytical methods for the various products, including gel-based approaches, GC-MS and LC-MS. Oxidation of each of five carbons of 2-deoxyribose leads to the formation of a unique set of products, many of which are highly reactive electrophiles. We have now identified a host of biologically important adducts formed by reaction of these electrophiles with nucleophilic sites in DNA, RNA and protein.

DNA adducts -- One consequence of the generation of reactive electrophiles during oxidation of 2-deoxyribose in DNA is the formation of DNA adducts. This phenomenon is illustrated with 3', 4'- and 5'-oxidation products. For example, the base propenals derived from 4'-oxidation are structural analogs of the DNA-reactive malondialdehyde and react with guanine to form the pyimidopurinone adduct, M1G. Oxidation of the 3'-position yields a 3'-phosphoglycolaldehyde residue that undergoes a radical-independent phosphate-phosphonate rearrangement to form glyoxal and subsequently the variety of glyoxal adducts of DNA bases. The electrophilic 5'-(2-phosphoryl-1,4-dioxobutane) species generated by 5'-oxidation has been found to form both DNA and protein adducts, with bicyclic oxadiazabicyclo-(3.3.0)octaimine adducts formed from dC, dA and dG.

Protein adducts -- A second biological consequence of 2-deoxyribose oxidation products involves their reaction with nucleophilic amino acids to form protein adducts and protein -- DNA cross-links. For example, the reaction of the 3'-formylphosphate residue with e-amino group of lysine in histone proteins to form an N6-formyllysine residue. Its chemical analogy to the physiologically important lysine N-acetylation suggests that lysine N-formylation may interfere with signaling mediated by histone modifications.

Metabolic fate of 2-deoxyribose oxidation products -- With the goal of identifying candidate biomarkers of oxidative stress and inflammation, we are shifting our focus from the formation of DNA damage products to their fate in biological systems. For example, the electrophilic species arising from 2-deoxyribose oxidation in DNA are ideal candidates for glutathione conjugation by glutathione transferases, which points to mercapturic acid metabolites as potential biomarkers of oxidatively damaged DNA arising from oxidative stress and inflammation. Given the presence of glutathione transferase activities in mammalian nuclei, such activity may represent a form of DNA protection or repair by reacting with base propenals and other DNA-bound or freely diffusible deoxyribose oxidation products, as well as the more commonly recognized lipid peroxidation targets.