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.

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.

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.

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.

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.

Cu(I) from tetrakis(acetonitrile)copper(I) hexafluorophosphate ([Cu(MeCN)4]PF6) was complexed with five structurally related phosphines containing N-heterocycles. The interactions between the resulting complexes and some N-nitrosamines were studied using X-ray crystallography as well as emission spectroscopy. Upon complexation, three phosphine ligands bridge two Cu(I) centers to give paddlewheel type structures that displayed a range of emission wavelengths spanning the visible region. N-Nitrosodimethylamine (NDMA) was shown to coordinate to one of the two copper centers in some of the paddlewheel complexes in the solid state and this interaction also quenches their emissions in solution. The influence of the weakly coordinating anion on crystal and spectroscopic properties of one of the paddlewheel complexes was also examined using tetrakis(acetonitrile)copper(I) perchlorate ([Cu(MeCN)4]ClO4) as an alternative Cu(I) source. Similarly, copper(II) perchlorate hexahydrate (Cu(ClO4)2·6H2O) was used for complexation to observe the impact of metal oxidation state on the two aforementioned properties. Lastly, the spectroscopic properties of the complex between Ph2P(1-Isoquinoline) and Cu(I) was shown to exhibit solvent dependence when the counterion is ClO4-. These Cu(I) complexes are bench stable solids and may be useful materials for developing a fluorescence based detection method for N-nitrosamines.

Abstract DNA-methylating environmental carcinogens such as N-nitrosodimethylamine (NDMA) and certain alkylators used in chemotherapy form O6-methylguanine (m6G) as a functionally critical intermediate. NDMA is a multi-organ carcinogen found in contaminated water, polluted air, preserved foods, tobacco products, and many pharmaceuticals. Only ten weeks after exposure to NDMA, neonatally-treated mice experienced elevated mutation frequencies in liver, lung and kidney of ∼35-fold, 4-fold and 2-fold, respectively. High-resolution mutational spectra (HRMS) of liver and lung revealed distinctive patterns dominated by GC→AT mutations in 5’-Pu-G-3’ contexts, very similar to human COSMIC mutational signature SBS11. Commonly associated with alkylation damage, SBS11 appears in cancers treated with the DNA alkylator temozolomide (TMZ). When cells derived from the mice were treated with TMZ, N-methyl-N-nitrosourea, and streptozotocin (two other therapeutic methylating agents), all displayed NDMA-like HRMS, indicating mechanistically convergent mutational processes. The role of m6G in shaping the mutational spectrum of NDMA was probed by removing MGMT, the main cellular defense against m6G. MGMT-deficient mice displayed a strikingly enhanced mutant frequency, but identical HRMS, indicating that the mutational properties of these alkylators is likely owed to sequence-specific DNA binding. In sum, the HRMS of m6G-forming agents constitute an early-onset biomarker of exposure to DNA methylating carcinogens and drugs.