The dynamic interplay between DNA damage and metabolism : the metabolic fate and transport of DNA lesions and novel DNA damage derived from intermediary metabolism

• Main Author: Jumpathong, Watthanachai
• Other Authors: Peter C. Dedon.
• Format: Others
• Language: English
• Published: Massachusetts Institute of Technology 2015
• Subjects: Biological Engineering.
• Online Access: http://hdl.handle.net/1721.1/93772

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Biological Engineering, 2014. === This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. === Cataloged from student-submitted PDF version of thesis. === Includes bibliographical references. === The work presented in this thesis explores two novel and complementary facets of endogenous DNA damage: the development of biomarkers of inflammation based on metabolites of DNA damage products and the formation of DNA adducts by electrophilic products of intermediary metabolism. From the first perspective, endogenous DNA damage generated by reactive oxygen and nitrogen species from inflammation and oxidative stress has shown strong mechanistic links to the pathophysiology of cancer and other human diseases, with the damage products reflecting all types of damage chemistries including oxidation, deamination, halogenation, nitration and alkylation. However, the use of DNA damage products as biomarkers has been limited by poor understanding of the damage actually arising in tissues and a lack of appreciation of the fate of DNA damage products from the moment of formation at the site of damage to release from cells to final excretion from the body. The goal of the work presented in the first part of this thesis was to investigate the metabolic fates of the base propenal products arising from 4'-oxidation of 2'-deoxyribose in DNA, one of the most common products of DNA oxidation, and to define base propenal metabolites as potential biomarkers of oxidative stress. This project was approached with systematic metabolite profiling, starting with prediction of potential base propenal metabolites based on a priori knowledge of its chemical reactivity as an [alpha],[beta]-unsaturated aldehyde toward glutathione (GSH) in non-enzymatic reactions and in rat liver cell extracts. Of 15 potential candidates predicted and identified from these in vitro studies, analysis of urine samples from rats given intravenous doses (IV) of thymine propenal revealed three major metabolites: thymine propenoic acid and two mercapturic acid derivatives, which accounted for ~6% of the injected dose. An additional four metabolites, including conjugates with GSH, cysteinylglycine and cysteine, were observed in bile and accounted for ~22% of the dose. One of the major metabolites detected in urine and bile, a bis-mercapturic acid adduct of reduced thymine propenal was detected as a background excretory product in saline-treated rats and was significantly elevated after oxidative stress caused by treatment with bleomycin and CCl₄. Our observations suggest that metabolism and disposition of damaged biomolecules should be considered as crucial factors in the development of biomarkers relevant to inflammation and oxidative stress. The second part of this thesis addresses the complementary hypothesis that electrophilic metabolites generated endogenously from intermediary metabolism can react with DNA to form adducts. This concept is illustrated here with glyoxylate from the glyoxylate metabolic cycle, whicvh plays a key role as an alternative to the TCA cycle in plants, bacteria, protists and fungi under changing conditions of environmental nutrients. The goal of this project was to characterize DNA adducts caused by glyoxylate in the mycobacterium M. smegmatis, with the studies motivated by the higher-than-expected mutation rate of mycobacteria during dormancy induced by nutrient deprivation and a shift to utilization of the glyoxylate cycle. Initially, in vitro reactions of 2'-deoxyguanosine (dG) with glyoxylate yielded N²-carboxyhydroxymethyl dG (N²-CHMdG) as the only adduct. However, the adduct proved to be unstable, so a reduction-based analytical method was developed to yield the stable amine derivative, N2-carboxymethyl dG (N²-CMdG). This stable adduct was used to develop an isotope-dilution chromatography-coupled tandem mass spectrometry method to quantify N²-CHMdG as N²-CMdG in calf thymus DNA treated with glyoxylate in vitro. This analytical method was then applied to quantify and compare the level N2-CMdG in (1) wild-type M. smegmatis grown in rich medium (7H9) or in minimal M9 medium supplemented with acetate, the latter inducing a switch from the TCA cycle to the glyoxylate cycle; and (2) the isocitrate dehydrogenase (ICD)-deficient mutant of M. smegmatis. Mycobacteria grown in the acetate medium experienced a 2-fold increase in the adduct compared to those grown in 7H9. Similarly, the adduct increased 2-fold in the ICD mutant compared to wild-type M. smegmatis grown in 7H9. The results support the idea that shifts in intermediary metabolism can lead to DNA damage that may cause mutations associated with nutrient deprivation in mycobacteria, with implications for the genetic toxicology of other metabolism-derived electrophiles. === by Watthanachai Jumpathong. === Ph. D.