Colibactin is a recently characterized pro-carcinogenic genotoxin produced by pks+ Escherichia coli. We hypothesized that cystic fibrosis (CF)-associated dysfunctional mucus structure increases the vulnerability of host mucosa to colibactin-induced DNA damage. In this pilot study, we tested healthy-appearing mucosal biopsy samples obtained during screening and surveillance colonoscopies of adult CF and non-CF patients for the presence of pks+ E. coli, and we investigated the possibility of detecting a novel colibactin-specific DNA adduct that has not been yet been demonstrated in humans. While CF patients had a lower incidence of pks+ E. coli carriage (~8% vs 29%, p = 0.0015), colibactin-induced DNA adduct formation was detected, but only in CF patients and only in those who were not taking CFTR modulator medications. Moreover, the only patient found to have colon cancer during this study had CF, harbored pks+ E. coli, and had colibactin-induced DNA adducts in the mucosal samples. Larger studies with longitudinal follow-up should be done to extend these initial results and further support the development of colibactin-derived DNA adducts to stratify patients and their risk.

Balkan endemic nephropathy (BEN) is a chronic kidney disease that predominantly affects inhabitants of rural farming communities along the Danube River tributaries in the Balkans. Long-standing research has identified dietary exposure to aristolochic acids (AAs) as the principal toxicological cause. This study investigates the pathophysiological role of anemia in BEN, noting its earlier and more severe manifestation in BEN patients compared to those with other chronic kidney diseases. Utilizing a mouse model, our research demonstrates that prolonged exposure to aristolochic acid I (AA-I) (the most prevalent AA variant) leads to significant red blood cell depletion through DNA damage, such as DNA adduct formation in bone marrow, prior to observable kidney function decline. Furthermore, in vitro experiments with kidney cells exposed to lowered oxygen and pH conditions mimicking an anemia environment show enhanced DNA adduct formation, suggesting increased AA-I mutagenicity and carcinogenicity. These findings indicate for the first time a positive feedback mechanism of AA-induced anemia, DNA damage, and kidney impairment in BEN progression. These results not only advance our understanding of the underlying mechanisms of BEN but also highlight anemia as a potential target for early BEN diagnosis and therapy.

Environmental carcinogens exert their carcinogenic effects by forming DNA adducts. This type of DNA damage can also be formed endogenously as a result of, e.g., oxidative damage. Unrepaired  DNA adducts may induce mutations in critical genes, leading to the initiation of chemical carcinogenesis. Therefore,  detection, identification, and quantification of DNA adducts is essential for cancer risk assessment. Over the last 50 years, the major DNA adducts formed by different classes of environmental carcinogens were characterized. With the development of techniques such as 32P-postlabeling, their measurement was implemented into molecular epidemiology. Advances in liquid chromatography-tandem mass spectrometry (LC-MS ) made the measurement of adducts more precise  and allowed to gain knowledge about their identity and structures. Therefore,  opened the way to  DNA adductomics, the  \"omics\" approach investigating DNA adducts comprehensively, similarly to proteomics. This review presents the historical perspective of DNA adducts research and the emerging field of adductomics.

It is generally accepted that adjacent guanine residues in DNA are the primary target for platinum antitumor drugs and that differences in the conformations of the Pt-DNA adducts can play a role in their antitumor activity. In this study, we investigated the effect of the carrier ligand cis-1,3-diaminocyclohexane (cis-1,3-DACH) upon formation, stability, and stereochemistry of the (cis-1,3-DACH)PtG2 and (cis-1,3-DACH)Pt(d(GpG)) adducts (G = 9-EthlyGuanine, guanosine, 5\'- and 3\'-guanosine monophosphate; d(GpG) = deoxyguanosil(3\'-5\')deoxyguanosine). A peculiar feature of the cis-1,3-DACH carrier ligand is the steric bulk of the diamine, which is asymmetric with respect to the Pt-coordination plane. The (cis-1,3-DACH)Pt(5\'GMP)2 and (cis-1,3-DACH)Pt(3\'GMP)2 adducts show preference for the ΛHT and ∆HT conformations, respectively (HT stands for Head-to-Tail). Moreover, the increased intensity of the circular dichroism signals in the cis-1,3-DACH derivatives with respect to the analogous cis-(NH3)2 species could be a consequence of the greater bite angle of the cis-1,3-DACH carrier ligand with respect to cis-(NH3)2. Finally, the (cis-1,3-DACH)Pt(d(GpG)) adduct is present in two isomeric forms, each one giving a pair of H8 resonances linked by a NOE cross peak. The two isomers were formed in comparable amounts and had a dominance of the HH conformer but with some contribution of the ΔHT conformer which is related to the HH conformer by having the 3\'-G base flipped with respect to the 5\'-G residue.

OBJECTIVE: In East Asia, the incidence of breast cancer has been increasing rapidly, particularly among premenopausal women. An elevated ratio of estrogen-DNA adducts was linked to a higher risk of breast cancer. The present study explored the influence of the interaction between base excision repair (BER) gene polymorphisms and estrogen-DNA adducts on breast cancer risk.
METHODS: We conducted a case-control study comprising healthy volunteers and individuals with benign breast disease (control arm, n = 176) and patients with invasive carcinoma or carcinoma in situ (case arm, n = 177). Genotyping for BER-related genes, including SMUG1, OGG1, ERCC5, and APEX1, was performed. A logistic regression model, incorporating interactions between gene polymorphisms, estrogen-DNA adduct ratio, and clinical variables, was used to identify the risk factors for breast cancer.
RESULTS: Univariate analysis indicated marginal associations between breast cancer risk and APEX1 rs1130409 T > G (P = 0.057) and APEX1 rs1760944 T > G (P = 0.065). Multivariate regression analysis revealed significant associations with increased breast cancer risk for APEX1_rs1130409 (GT/GG versus TT) combined with a natural logarithmic value of the estrogen-DNA adduct ratio (estimated OR 1.164, P = 0.023) and premenopausal status with an estrogen-DNA adduct ratio > 2.93 (estimated OR 2.433, P = 0.001).
CONCLUSIONS: APEX1_rs1130409 (GT/GG versus TT) polymorphisms, which are related to decreased BER activity, combined with an increased ratio of estrogen-DNA adducts, increase the risk of breast cancer in East Asian women.

DNA adducts are widely recognized as biomarkers of exposure to environmental carcinogens and associated health effects in toxicological and epidemiological studies. This study presents a targeted and sensitive method for comprehensive DNA adductome analysis using ultra-high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-QqQ-MS/MS). The method was developed using calf thymus DNA, with careful optimization of mass spectrometric parameters, chromatographic separation conditions, and pretreatment methods. Ultimately, a targeted method was established for 41 DNA adducts, which showed good linearity (R2 ≥0.992), recovery (80.1-119.4 %), accuracy (81.3-117.8 %), and precision (relative standard deviation <14.2 %). The established method was employed to analyze DNA adducts in peripheral blood cells from pregnant women in Shanxi and Beijing. Up to 23 DNA adducts were successfully detected in samples of varying sizes. From 2 μg of maternal DNA samples, seven specific adducts were identified: 5-methyl-2\'-deoxycytidine (5-MedC), 5-hydroxymethyl-2\'-deoxycytidine (5-HmdC), N6-methyl-2\'-deoxyadenosine (N6-MedA), 8-hydroxy-2\'-deoxyguanosine (8-OHdG), 5-hydroxy-2\'-deoxycytidine (5-OHdC), 1,N6-etheno-2\'-deoxyadenosine (1,N6-εdA), and N2-methyl-2\'-deoxyguanosine (N2-MedG). This study reveals that exposure to higher concentrations of ambient air pollutants may elevate the levels of DNA methylation and oxidative damage at different base sites, highlighting the application potential of DNA adducts as sensitive biomarkers of air pollution exposure.

Cigarette smoking is the acknowledged major cause of cancers of the lung and oral cavity and is an established important risk factor for multiple other cancers. DNA addition products (DNA adducts) caused by cigarette smoking are critical factors in its mechanism of carcinogenesis. However, most DNA adducts detected to date in humans cannot be specifically ascribed to smoking but rather have multiple exogenous and endogenous sources. In the study reported here, we prepared [13C]-labeled tobacco to address this problem. We report for the first time the successful growth from seeds to flowering under hydroponic conditions of highly [13C]-labeled tobacco in a controlled 13CO2 environment. The standard growth procedure with optimized conditions is described in detail. The [13C]-enrichment rate was assessed by quantifying nicotine and sugars and their [13C]-isotopologues in this tobacco using high-resolution mass spectrometry, reaching >94% in the tobacco leaves. The [13C]-labeled leaves after curing will be used to make cigarettes, allowing investigation of the specific contributions of tobacco smoke carcinogens to identified DNA adducts in smokers.

DNA base damage is a major source of oncogenic mutations1. Such damage can produce strand-phased mutation patterns and multiallelic variation through the process of lesion segregation2. Here we exploited these properties to reveal how strand-asymmetric processes, such as replication and transcription, shape DNA damage and repair. Despite distinct mechanisms of leading and lagging strand replication3,4, we observe identical fidelity and damage tolerance for both strands. For small alkylation adducts of DNA, our results support a model in which the same translesion polymerase is recruited on-the-fly to both replication strands, starkly contrasting the strand asymmetric tolerance of bulky UV-induced adducts5. The accumulation of multiple distinct mutations at the site of persistent lesions provides the means to quantify the relative efficiency of repair processes genome wide and at single-base resolution. At multiple scales, we show DNA damage-induced mutations are largely shaped by the influence of DNA accessibility on repair efficiency, rather than gradients of DNA damage. Finally, we reveal specific genomic conditions that can actively drive oncogenic mutagenesis by corrupting the fidelity of nucleotide excision repair. These results provide insight into how strand-asymmetric mechanisms underlie the formation, tolerance and repair of DNA damage, thereby shaping cancer genome evolution.

Tobacco smoke exposure significantly increases the level of global nucleoside damage. To evaluate all aspects of nucleic acid (NA) modifications, NA adductomics analyzes DNA, RNA and nucleobase adducts and provides comprehensive data. Liquid chromatography-tandem triple quadrupole mass spectrometry (LC-QQQ-MS/MS) and LC-Zeno-TOF-MS/MS were employed to screen for DNA, RNA and nucleobase adducts, as part of the analytical platform that was designed to combine high sensitivity and high resolution detection. We identified and distinguished urine nucleoside adducts via precursor ion and neutral loss scanning. A total of 245 potential adducts were detected, of which 28 were known adducts. The smoking group had significantly higher concentrations of nucleoside adducts in rat urine than the control group, based on MRM scanning, which was then used to perform relative quantitative analysis of these adducts. Urine nucleoside adducts were further confirmed using LC-Zeno-TOF-MS/MS. This highlights the potential of untargeted detection methods to provide comprehensive data on both known and unknown adducts. These approaches can be used to investigate the interactions among oxidative and alkylation stresses, and epigenetic modifications caused by exposure to tobacco smoke.

Aristolochic acids I and II (AA-I/II) are carcinogenic principles of Aristolochia plants, which have been employed in traditional medicinal practices and discovered as food contaminants. While the deleterious effects of AAs are broadly acknowledged, there is a dearth of information to define the mechanisms underlying their carcinogenicity. Following bioactivation in the liver, N-hydroxyaristolactam and N-sulfonyloxyaristolactam metabolites are transported via circulation and elicit carcinogenic effects by reacting with cellular DNA. In this study, we apply DNA adduct analysis, X-ray crystallography, isothermal titration calorimetry, and fluorescence quenching to investigate the role of human serum albumin (HSA) in modulating AA carcinogenicity. We find that HSA extends the half-life and reactivity of N-sulfonyloxyaristolactam-I with DNA, thereby protecting activated AAs from heterolysis. Applying novel pooled plasma HSA crystallization methods, we report high-resolution structures of myristic acid-enriched HSA (HSAMYR) and its AA complexes (HSAMYR/AA-I and HSAMYR/AA-II) at 1.9 Å resolution. While AA-I is located within HSA subdomain IB, AA-II occupies subdomains IIA and IB. ITC binding profiles reveal two distinct AA sites in both complexes with association constants of 1.5 and 0.5 · 106 M-1 for HSA/AA-I versus 8.4 and 9.0 · 105 M-1 for HSA/AA-II. Fluorescence quenching of the HSA Trp214 suggests variable impacts of fatty acids on ligand binding affinities. Collectively, our structural and thermodynamic characterizations yield significant insights into AA binding, transport, toxicity, and potential allostery, critical determinants for elucidating the mechanistic roles of HSA in modulating AA carcinogenicity.