The comet assay is a versatile assay for detecting DNA damage in eukaryotic cells. The assay can measure the levels of various types of damage, including DNA strand breaks, abasic sites and alkali-sensitive sites. Furthermore, the assay can also be modified to include purified DNA glycosylases so that alkylated and oxidized bases can be detected. The CometChip is a higher throughput version of the traditional comet assay and has been used to study cultured cells. Here, we have tested its utility for studies of DNA damage present in vivo. We show that the CometChip is effective in detecting DNA damage in multiple tissues of mice exposed to the direct-acting methylating agent methylmethane sulfonate (MMS) and to the metabolically activated methylating agent N-nitrosodimethylamine (NDMA), which has been found to contaminate food, water, and drugs. Specifically, results from MMS-exposed mice demonstrate that DNA damage can be detected in cells from liver, lung, kidney, pancreas, brain and spleen. Results with NDMA show that DNA damage is detectable in metabolically competent tissues (liver, lung, and kidney), and that DNA repair in vivo can be monitored over time. Additionally, it was found that DNA damage persists for many days after exposure. Furthermore, glycosylases were successfully incorporated into the assay to reveal the presence of damaged bases. Overall, this work demonstrates the efficacy of the in vivo CometChip and reveals new insights into the formation and repair of DNA damage caused by MMS and NDMA.

N-Nitrosodimethylamine (NDMA) is a probable human carcinogen found in contaminated pharmaceuticals and drinking water, yet the impact of age on NDMA susceptibility remains poorly understood. Using DNA repair-deficient (Aag^-/-;Mgmt^-/-) and wild-type mice, we systematically compare the effects of NDMA exposure in juveniles and adults. Juvenile Aag^-/-;Mgmt^-/- mice are profoundly more vulnerable, exhibiting persistent DNA damage, inflammation, and mutations that lead to liver pathology and tumorigenesis, particularly in males. Adults, by comparison, are resistant to NDMA. Wild-type mice show similar, attenuated trends. NDMA-induced DNA adduct levels are comparable across age groups, implicating proliferation-dependent responses to adducts, rather than adduct formation, as the primary driver of age-related risk. Supporting this mechanism, triiodothyronine-stimulated cell proliferation in adults partially recapitulates juvenile sensitivity, linking cell division to NDMA genotoxicity. Our findings identify developmental stage, sex, and DNA repair capacity as key modifiers of NDMA-induced carcinogenesis, with potential implications for environmental risk assessment and regulatory policy.

N-Nitrosodimethylamine (NDMA), a probable human carcinogen, induces toxic and mutagenic O ⁶ -methylguanine lesions that are repaired by the O ⁶ -methylguanine methyltransferase (MGMT). To elucidate mechanisms of NDMA-induced liver cancer progression, we performed longitudinal analyses of phenomic, transcriptomic, and phosphoproteomic changes in wild-type and MGMT-deficient mice, observing amplified responses in the deficient genotype. Early molecular rewiring indicative of a DNA damage response was detected by phosphoproteomic and transcriptomic profiling within days post-exposure. Transcriptomic analyses identified a persistent and robust interferon response as the dominant activated pathway. This chronic interferon signaling, which remained unresolved, correlated with extensive clonal expansion, an early hallmark of oncogenesis. Spatial transcriptomics further revealed pathway alterations favoring tumorigenesis within clonally expanded cells. These findings delineate the cascade of molecular events triggered by acute early-life NDMA exposure, culminating in cancer development months later. Our study unveils potential predictive biomarkers and strategies for disease mitigation.

Maintaining genome stability is crucial for human health and it is of particular importance in neural cells during early brain development. Genome maintenance occurs at two broad stages; surveillance during DNA replication and DNA damage repair in differentiating and mature cells. Neural cells are particularly sensitive to DNA strand breaks and defective DNA damage responses can result in detrimental effects on the nervous system, including cancer. Multiple DNA repair pathways play critical roles in preventing DNA damage accumulation in stem and neural progenitor cells. The mechanisms that protect progenitor genomes also suppress DNA mutations that can result in cancer. A primary objective of this dissertation is to understand the relative contributions of key DNA repair factors that prevent tumorigenesis during cortical development. We have compared the differential effects of inhibition of homologous recombination (HR), via BRCA2-inactivation and non-homologous end-joining (NHEJ), via LIG4-inactivation towards tumorigenesis by directing their deletion specifically to early cortical progenitors using an Emx1-cre recombinase driver. We find that coincident loss of either of these repair pathways with p53 inhibition result in distinct high-grade glioma (HGG) formation resulting from elevated genome instability by DNA damage accumulation during embryogenesis. Furthermore, the presence of the oncohistone H3K27M mutation, commonly found in pediatric HGGs, enhances genome instability and accelerates cortical gliomagenesis with p53 inactivation and defective HR or NHEJ. Additionally, the H3K27M resultant gliomas showed distinctive differences in increased brain tumor penetrance and diffusion. Through RNA-sequencing and whole exome sequencing we identify upregulation of genes normally controlled by bivalent gene promoter post-translational modifications, which result in transcriptional alterations in genes important for both neural development and tumorigenesis. Mechanistically, this is done by targeting specific populations of cortical cells that are more susceptible to DNA damage and transformations that may cause additional critical mutations during a limited timeframe of early cortical development which eventually result in HGGs. We provide evidence supporting that BRCA2 functions to provide DSBR and genome stability to the early-born proliferating cortical progenitor cell population, while LIG4 provides the same function but to a lesser extent to progenitor cells and more so to post-mitotic neurons. Since, epigenetic regulation is tightly connected with neural development and differentiation, we propose the specific genes that H3K27M effects may differ depending on the time period and particular cell state from which the HGG initiates. We believe this contributes to reduced heterogeneity in glioma expression signatures with H3K27M in addition to either HR- or NHEJ-deficiency. Ultimately this work highlights the power of inducible genetically engineered mouse models as an approach to better understand the complexities of providing a connection between genome instability and gliomagenesis.

N-Nitrosodimethylamine (NDMA), a probable human carcinogen, induces toxic and mutagenic DNA O6-methylguanine (O6MeG) adducts that are repaired by O6-methylguanine-DNA methyltransferase (MGMT). To elucidate how early-life environmental mutagenic exposures promote latent liver tumorigenesis, we performed longitudinal studies in wild-type and MGMT-deficient (Mgmt−/−) mice. Neonatal mice received NDMA intraperitoneally on postnatal days 8 and 15 and were followed for up to 10 months post-exposure. Phenomics, transcriptomics, phosphoproteomics, spatial transcriptomics, and histopathology revealed that NDMA exposure in Mgmt−/− mice produced a nine-fold increase in liver tumors, including hepatocellular carcinomas and adenomas. Early molecular profiling revealed elevated O6MeG adducts, sustained γH2AX activation, and increased micronucleus formation in Mgmt−/− livers. Transcriptomics showed persistent interferon response and immune cell infiltration, particularly in Mgmt−/− mice, up to 10 months post-exposure. Clonal expansion, quantified by RaDR-GFP expression, was significantly higher in NDMA-exposed Mgmt−/− mice. Spatial transcriptomics of clonally expanded cells demonstrated activation of oncogenic and inflammatory pathways. These findings demonstrate that acute early-life NDMA exposure drives persistent DNA damage signaling, chronic interferon activation, and clonal expansion that together promote MGMT-dependent hepatocarcinogenesis. Collectively, these processes link early environmental mutagen exposure to long-term premalignant evolution and tumor emergence, and define mechanistic biomarkers and potential interception targets for NDMA-induced liver cancer.