One of the most prevalent exocyclic DNA adducts is 3-(2-deoxy-β-D-erythro-pentofuranosyl) pyrimido[1,2-α]purin-10(3H)-one (M1dG), an oxidative DNA lesion that forms by the reaction of guanines in nucleic acids and pool nucleotides with oxidation-induced base propenals or with the lipid peroxidation product, malondialdehyde. Further oxidation converts M1dG to 6-oxo-M1dG, an even more deleterious lesion whose genotoxic and mutagenic properties have been characterized in vitro. The present work uses a site-specifically modified viral genome to evaluate the biochemical consequences of 6-oxo-M1dG in Escherichia coli cells and contrast them with the properties of the M1dG parent lesion. We found that 6-oxo-M1dG strongly inhibited replication, with a bypass efficiency of 1-2%, relative to an unmodified guanine. By contrast, under the same experimental conditions, the bypass efficiency of M1dG was 30-40%. Beyond its low bypass rate, 6-oxo-M1dG was 20 times more mutagenic than M1dG, with the majority of mutations being single base deletions. However, when the levels of bypass polymerases were increased by inducing the SOS response, the proportion of deletions decreased, at the expense of additional single base substitutions, primarily G → T and G → C mutations. Finally, two DNA repair pathways─the direct reversal dioxygenase AlkB and glycosylase MutY─were investigated for their putative activity on 6-oxo-M1dG. The results indicated that neither system was capable of repairing this highly mutagenic lesion.

N-Nitrosodimethylamine (NDMA) is classified as an animal and probable human carcinogen. Murine liver DNA adducts, mutations, O 6-methylguanine DNA methyltransferase (MGMT), and CYP2E1 were evaluated following chronic administration of NDMA in drinking water. In a dose-escalation study, 7-methylguanine (m7G) increased linearly with NDMA dose. O 6-Methylguanine (m6G) remained near background for NDMA doses up to ∼1 ppm, beyond which its level, and corresponding mutations, rose steeply. An extended study was done with 5 ppm NDMA, in which adducts were measured at 3 and 10 weeks and mutations at 10 weeks. We found that both sexes experienced elevated levels of point mutations closely tracking with the levels of m6G, which emerged as the dominant mutagenic adduct under chronic dosing with NDMA. Homologous recombination-mediated chromosomal rearrangements, however, did not increase over background. Mutational analysis over 96 trinucleotide contexts revealed predominantly GC→AT mutations in 5'-purine-G-3' contexts in a pattern matching human COSMIC cancer mutational signature SBS11, with secondary features resembling SBS119 (AT→GC). Moreover, we identified the m6G level (∼2000 adducts/diploid genome) above which its dedicated repair protein, MGMT, became saturated. The coordinated application of DNA adduct, mutational, and biochemical analyses provides a new approach for studying mechanisms of carcinogenesis, with relevance to early cancer detection and cancer management.

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.

Glyphosate-based herbicides (GBHs) are widely used and raise concerns about human health risks due to the detection of glyphosate (GLY) in body fluids. Their toxicity remains the subject of ongoing debate, especially regarding their carcinogenic potential, while epidemiological evidence associates GBHs exposure with increased non-Hodgkin lymphoma incidence. This study addresses key gaps by characterizing the kinetic profiles of GLY, its metabolites, and oxidative stress biomarkers in urine from 11 agricultural workers over 14 days, alongside evaluating mutagenicity using the AS52 cell model. 1H NMR metabolomic analysis identified GLY and several downstream metabolites, with formaldehyde showing the strongest associations with oxidative damage biomarkers. AS52 cells exhibited concurrent oxidative and mutagenic responses, indicating involvement of GBHs in reactive oxygen species generation, DNA damage, and apoptosis. These integrated findings demonstrate biologically relevant GLY transformation and implicate both GLY and its reactive metabolites in oxidative and genotoxic stress, advancing understanding of GBHs' toxicity in humans.

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.

N-Nitrosodimethylamine (NDMA) is present in food, water, and drugs and is considered a probable human carcinogen by the International Agency for Research on Cancer. The mechanism of action of NDMA involves the generation of carcinogenic methyl lesions such as 3-methyladenine (3MeA) on DNA bases. Alkyladenine DNA Glycosylase (AAG; a.k.a. N-methylpurine DNA glycosylase, MPG) removes 3MeA to initiate Base Excision Repair, leaving an abasic site that is resolved by backbone cleavage, nucleotide insertion, and backbone ligation. The intermediate steps following base removal produce potentially toxic and mutagenic abasic sites and single-strand DNA breaks. Here, we explored differences between males and females regarding downstream DNA damage, toxicity, mutations and cancer arising from 3MeA in the livers of WT, Aag-/-, and Aag-overexpressing (AagTg) mice. We found that males were more susceptible to NDMA-induced mutations (WT and Aag-/-) and cancer (all genotypes). In contrast, AagTg females were more prone to micronucleus induction. As we showed in our prior analyses where data were pooled for males and females, Aag-/- mice were significantly more susceptible to NDMA-induced mutations and cancer, and AagTg mice displayed significantly greater toxicity. Building on these findings, our analyses of sex-related differences show that Aag deficiency and maleness are both susceptibility factors for NDMA-induced hepatic cancer, while Aag overexpression drives toxicity, potentially with a greater effect in females. This study reveals a deeper understanding of the underpinnings for a well-known increased risk of hepatic cancer in men versus women by demonstrating a higher susceptibility of male mice to both mutations and cancer.

N-Nitrosodimethylamine (NDMA) is a water-soluble carcinogen typically quantified using mass spectrometry coupled with chromatographic separation, which requires extensive sample preparation and sophisticated instrumentation. Here, we report a fluorescence-based detection strategy for NDMA in aqueous media that replaces column-based separation with affinity capture. Photochemical transformation of NDMA, followed by reaction with a biotinylated polymeric reagent, generates a biotin-labeled fluorescent product that can be selectively enriched through streptavidin-biotin interaction, enabling effective separation of the analytical signal from background. The assay is performed entirely in water without sample pretreatment, preconcentration, or solvent exchange. Under optimized conditions, quantitative detection is achieved in a plate-based format using only 100 mu L samples, affording a limit of detection of 235 ppb. The chemical transformation is completed within 22 min, followed by rapid affinity capture via streptavidin-coated agarose beads or plates. The method is rigorously validated through spectroscopic characterization, selective capture experiments, and quantitative analysis, and demonstrates consistent performance in spiked commercial water samples. These results establish a chemically defined platform for chromatography-independent optical NDMA detection and provide a basis for further development toward scalable sensing formats built on streptavidin-functionalized solid phases.

While optimal fluoride (F) levels support oral health, chronic exposure to high concentrations can lead to skeletal and dental fluorosis, especially in children. Emerging evidence suggests that excessive fluoride intake may disrupt systemic physiology, yet the underlying mechanisms remain poorly understood. To address this gap, we performed a comparative urinary proteomic analysis using LC-MS/MS in schoolchildren residing in a high-fluoride region, categorizing participants into high-fluoride (HF) and low-fluoride (LF) groups based on urinary fluoride excretion. Among 460 quantified proteins, ten were differentially expressed in the HF group, six upregulated (PHPT1, SPP1, COLEC12, CST4, DCHS1, LDHB) and four downregulated (CTSH, NECTIN1, TNC, KLK1). Gene Ontology enrichment highlighted associations with cell adhesion, ossification, and tissue development. Notably marked alterations in osteopontin (SPP1) and tenascin-C (TNC), key regulators of bone remodeling and dental matrix organization, suggesting disrupted focal adhesion signaling, impaired matrix integrity, and dysregulated biomineralization. Additional changes in proteins associated with enamel formation, oxidative stress, and immune regulation suggest that high fluoride exposure may broadly disrupt extracellular matrix organization and trigger inflammatory pathways. These findings demonstrate that excessive fluoride exposure induces systemic molecular disturbances in children, with implications for bone and soft tissue homeostasis. This research adds to the body of human evidence concerning fluoride's biological impact, advocating for vigilant exposure monitoring.

N-Nitrosodimethylamine (NDMA) is a water-soluble carcinogen typically quantified using mass spectrometry coupled with chromatographic separation, which requires extensive sample preparation and sophisticated instrumentation. Here, we report a fluorescence-based detection strategy for NDMA in aqueous media that replaces column-based separation with affinity capture. Photochemical transformation of NDMA, followed by reaction with a biotinylated polymeric reagent, generates a biotin-labeled fluorescent product that can be selectively enriched through streptavidin-biotin interaction, enabling effective separation of the analytical signal from background. The assay is performed entirely in water without sample pretreatment, preconcentration, or solvent exchange. Under optimized conditions, quantitative detection is achieved in a plate-based format using only 100 μL samples, affording a limit of detection of 235 ppb. The chemical transformation is completed within 22 min, followed by rapid affinity capture via streptavidin-coated agarose beads or plates. The method is rigorously validated through spectroscopic characterization, selective capture experiments, and quantitative analysis, and demonstrates consistent performance in spiked commercial water samples. These results establish a chemically defined platform for chromatography-independent optical NDMA detection and provide a basis for further development toward scalable sensing formats built on streptavidin-functionalized solid phases.

Aflatoxin B1 (AFB1) and sterigmatocystin (ST) are mycotoxins that pose significant threats to human and animal health owing to their mutagenic, carcinogenic, and toxic properties. They are structurally similar and widely believed to exert their biological effects via the generation of DNA-damaging epoxides at their respective terminal furan rings. Despite structural identity in the warhead portion of each toxin, this work shows that distal parts of each molecule are responsible for the distinctive mutational fingerprints seen in gptΔ C57BL/6J mouse embryo fibroblasts (MEFs). The two toxins differ structurally in the puckered cyclopentenone ring of AFB1 and in the planar xanthone functionality of ST. While both toxins mainly induce GC→TA mutations, the aforementioned differences in structure apparently trigger unique patterns of mutations, as revealed by high-resolution duplex sequencing of MEF genomes. AFB1 is more mutagenic than ST and displays its transversion mutations in a pattern with primary and secondary hotspots (underscored) in 5′-CGC-3′ and 5′-CGG-3′ contexts, respectively. ST displays a modest 5′-CGG-3′ hotspot while its other GC→TA transversions are more uniformly distributed in a pattern resembling established oxidative stress mutational spectra. This research delineates the mutational spectra of AFB1 and ST, establishing these patterns as possible early-onset biomarkers of exposure.

N-Nitrosodimethylamine (NDMA) is present in food, water, and drugs and is considered a probable human carcinogen by the International Agency for Research on Cancer. The mechanism of action of NDMA involves the generation of carcinogenic methyl lesions such as 3-methyladenine (3MeA) on DNA bases. Alkyladenine DNA Glycosylase (AAG) removes 3MeA to initiate Base Excision Repair, leaving an intermediary lesion that is subsequently resolved by backbone cleavage, nucleotide insertion, and backbone ligation. The intermediate steps following lesion removal produce potentially toxic and mutagenic single-strand DNA breaks. Here, we explored differences between males and females regarding downstream DNA damage, toxicity, mutations and cancer arising from 3MeA in the livers of WT, Aag -/-, and Aag-overexpressing (AagTg) mice. We found that males were more susceptible to NDMA-induced mutations (WT and Aag -/-) and cancer (all genotypes). In contrast, AagTg females were more prone than males to micronucleus induction. As we showed in our prior analyses where data were pooled for males and females, Aag -/- mice were significantly more susceptible to NDMA-induced mutations and cancer, and AagTg mice displayed significantly greater toxicity. Building on these findings, our analyses of sex-related differences show that Aag deficiency and maleness are both susceptibility factors for NDMA-induced liver cancer, while Aag overexpression drives toxicity, potentially with a greater effect on females. By assessing differences between males and females, this study reveals a deeper mechanistic understanding of the underpinnings for a well-known increased risk of liver cancer in men versus women by demonstrating a higher susceptibility of male mice to both mutations and cancer.

Temozolomide (TMZ) is the standard treatment for nearly all glioblastoma (GBM) patients, as it is the only chemotherapy shown to extend overall survival. However, this benefit is limited to a few months, underscoring the need for combination strategies to improve its efficacy. While TMZ-induced DNA damage can both mediate cytotoxicity and promote resistance, DNA damage more broadly can also stimulate immune activation. To evaluate its immunomodulatory potential, we characterized the previously unexplored early, cell-intrinsic consequences of TMZ in GBM cells, spanning DNA damage, stress responses, and antigen presentation. A multi-omics approach combining RNA sequencing and quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) profiled changes in gene expression, nascent protein translation, steady-state protein levels, kinase-substrate phosphorylation patterns, and MHC-I peptide presentation in GBM cells within 72 hours of TMZ exposure. This analysis revealed rapid activation of DNA damage signaling and p53-associated stress pathways, alongside dynamic changes in protein synthesis and antigen presentation. A set of TMZ treatment-associated peptide antigens (TAPAs) was identified, including peptides derived from stress response proteins, phosphorylated MHC-I peptides, and those induced by other genotoxic treatments such as radiation. Several of these peptides were also detected in recurrent GBM patient tumors. Our findings suggest that TMZ not only triggers early adaptive and potentially resistance-associated stress programs but may also enhance the immune visibility of GBM cells. These data highlight potential windows for combination therapies with TMZ that bolster immune recognition of GBM, while the systems approach provides a framework to examine how genotoxic therapies across cancers alter tumor immunogenicity.

N-Nitrosamines are contaminants found throughout the environment, including in drinking water, and many nitrosamines are likely potent carcinogens. Correspondingly, there is a need for rapid and cost-effective in-field detection methods that can provide timely information about their contamination levels in water. This study details a colorimetric assay for detecting aqueous N-nitrosodimethylamine (NDMA) by photochemical nitrosation of a commercial naphtholsulfonate, to offer an attractive alternative to traditional laboratory-based analysis. The resulting naphthoquinone-oxime coordinates to aqueous iron(II) ions to form a green complex, allowing for direct visual detection. Characterization via Mossbauer and electron paramagnetic resonance (EPR) spectroscopy, alongside single-crystal structure determination, provides comprehensive structure information on the iron indicator complex. Optimization of detection conditions, including UV irradiation and response times, led to an improved colorimetric detection method with a limit of detection of 0.66 ppm for NDMA. The practical applicability and selectivity of this colorimetric detection scheme make it a promising candidate for the development of field-deployable sensors for NDMA in environmental water samples.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common monogenic cause of chronic kidney disease and the fourth leading cause of end-stage kidney disease, accounting for over 50% of prevalent cases requiring renal replacement therapy. There is a pressing need for improved therapy for ADPKD. Recent insights into the pathophysiology of ADPKD revealed that cyst cells undergo metabolic changes that up-regulate aerobic glycolysis in lieu of mitochondrial respiration for energy production, a process that ostensibly fuels their increased proliferation. The present work leverages this metabolic disruption as a way to selectively target cyst cells for apoptosis. This small-molecule therapeutic strategy utilizes 11beta-dichloro, a repurposed DNA-damaging anti-tumor agent that induces apoptosis by exacerbating mitochondrial oxidative stress. Here, we demonstrate that 11beta-dichloro is effective in delaying cyst growth and its associated inflammatory and fibrotic events, thus preserving kidney function in perinatal and adult mouse models of ADPKD. In both models, the cyst cells with homozygous inactivation of Pkd1 show enhanced oxidative stress following treatment with 11beta-dichloro and undergo apoptosis. Co-administration of the antioxidant vitamin E negated the therapeutic benefit of 11beta-dichloro in vivo, supporting the conclusion that oxidative stress is a key component of the mechanism of action. As a preclinical development primer, we also synthesized and tested an 11beta-dichloro derivative that cannot directly alkylate DNA, while retaining pro-oxidant features. This derivative nonetheless maintains excellent anti-cystic properties in vivo and emerges as the lead candidate for development.

DNA cross-links severely challenge replication and transcription in cells, promoting senescence and cell death. In this paper, we report a novel type of DNA interstrand cross-link (ICL) produced as a side product during the attempted repair of 1,N-6-ethenoadenine (epsilon A) by human alpha-ketoglutarate/Fe(II)-dependent enzyme ALKBH2. This stable/nonreversible ICL was characterized by denaturing polyacrylamide gel electrophoresis analysis and quantified by high-resolution LC-MS in well-matched and mismatched DNA duplexes, yielding 5.7% as the highest level for cross-link formation. The binary lesion is proposed to be generated through covalent bond formation between the epoxide intermediate of epsilon A repair and the exocyclic N-6-amino group of adenine or the N-4-amino group of cytosine residues in the complementary strand under physiological conditions. The cross-links occur in diverse sequence contexts, and molecular dynamics simulations rationalize the context specificity of cross-link formation. In addition, the cross-link generated from attempted epsilon A repair was detected in cells by highly sensitive LC-MS techniques, giving biological relevance to the cross-link adducts. Overall, a combination of biochemical, computational, and mass spectrometric methods was used to discover and characterize this new type of stable cross-link both in vitro and in human cells, thereby uniquely demonstrating the existence of a potentially harmful ICL during DNA repair by human ALKBH2.

The biomarker 5-chlorocytosine (5ClC) appears in the DNA of inflamed tissues. Replication of a site-specific 5ClC in a viral DNA genome results in C -> T mutations, which is consistent with 5ClC acting as a thymine mimic in vivo. Direct damage of nucleic acids by immune-cell-derived hypochlorous acid is one mechanism by which 5ClC could appear in the genome. A second, nonmutually exclusive mechanism involves damage of cytosine nucleosides or nucleotides in the DNA precursor pool, with subsequent utilization of the 5ClC deoxynucleotide triphosphate as a precursor for DNA synthesis. The present work characterized the mutagenic properties of 5ClC in the nucleotide pool by exposing cells to the nucleoside 5-chloro-2 '-deoxycytidine (5CldC). In both Escherichia coli and mouse embryonic fibroblasts (MEFs), 5CldC in the growth media was potently mutagenic, indicating that 5CldC enters cells and likely is erroneously incorporated into the genome from the nucleotide pool. High-resolution sequencing of DNA from MEFs derived from the gpt Delta C57BL/6J mouse allowed qualitative and quantitative characterization of 5CldC-induced mutations; CG -> TA transitions in 5 '-GC(Y)-3 ' contexts (Y = a pyrimidine) were dominant, while TA -> CG transitions appeared at a much lower frequency. The high-resolution mutational spectrum of 5CldC revealed a notable similarity to the Catalogue of Somatic Mutations in Cancer mutational signatures SBS84 and SBS42, which appear in human lymphoid tumors and in occupationally induced cholangiocarcinomas, respectively. SBS84 is associated with the expression of activation-induced cytidine deaminase (AID), a cytosine deaminase associated with inflammation, as well as immunoglobulin gene diversification during antibody maturation. The similarity between the spectra of AID activation and 5CldC could be coincidental; however, the administration of 5CldC did induce some AID expression in MEFs, which have no inherent expression of its gene. In summary, this work shows that 5CldC induces a distinct pattern of mutations in cells. Moreover, that pattern resembles human mutational signatures induced by inflammatory processes, such as those triggered in certain malignancies.

An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.