Innovative Technologies for the Study of DNA Adducts in Aging and DNA Repair Deficiency

Abstract

Unrepaired DNA damage is an important source of mutagenesis, genomic instability, cellular dysfunction, and disease. Among the many forms of DNA damage, DNA adducts are covalent modifications of DNA generated by endogenous reactive metabolites as well as exogenous genotoxicants. If not efficiently repaired, these lesions can disrupt DNA replication and transcription, promote mutations, cause cellular senescence, and contribute to aging and disease. Although many individual DNA adducts have been linked to carcinogenic exposures, including tobacco smoke, alcohol, and environmental pollutants, systematic characterization of the broader DNA adduct landscape in mammalian tissues and cell types remains limited. In particular, methods capable of discovery-level screening, followed by characterization of adduct patterns across different biological contexts, have been lacking. In this thesis, I present an LC-MS-based adductomics framework for characterizing the DNA adductome in tissues and cells, and apply the resulting database to investigate variation associated with age, sex, tissue type, and pathology, as well as the effects of DNA repair deficiency. Chapter 1 provides background for the thesis in three parts. I first introduce DNA adducts, their chemical origins, and their structural diversity. I then review the major analytical approaches used to detect them, weighing the strengths and limitations of each. Finally, I discuss the biological and toxicological consequences of adduct formation, including its links to carcinogenesis, the mechanistic pathways through which DNA damage drives disease, and age-associated patterns of damage accumulation. Chapter 2 describes the development of a stepped multiple-reaction monitoring (stepped MRM) LC-MS/MS platform for DNA adduct discovery and targeted follow-up analysis. I outline the analytical workflow used to build tissue- and cell-type-specific discovery datasets, assess reproducibility across independent rat cohorts, validate selected signals against authentic chemical standards, and establish quantitative performance for selected adducts by isotope-dilution LC-MS/MS. Chapter 3 presents the biological findings generated with this platform, focusing on sex-, age-, tissue-, and pathology-associated variation in DNA adduct profiles across rat tissues and human heart and brain samples. The results reveal strong tissue specificity, substantial sex bias, and significant age dependence for multiple adducts, demonstrating the utility of discovery adductomics for uncovering biologically meaningful patterns of DNA damage and for expanding the landscape of potential biomarkers relevant to aging and disease. Chapter 4 examines how deficiencies in selected DNA repair pathways alter adduct profiles in fibroblasts and stem cell-derived cardiomyocytes. Using CRISPR-generated repair-deficient lines, I assess the phenotypic and metabolic consequences of pathway disruption and characterize the resulting changes in adduct patterns, closing with a discussion of their biological implications and future mechanistic experiments.