-
Chemical Research in Toxicology Oct 2022Tobacco smoke is a complex mixture of more than 7000 chemicals, of which many are toxic and/or carcinogenic. Many hazard assessments of tobacco have focused on...
Tobacco smoke is a complex mixture of more than 7000 chemicals, of which many are toxic and/or carcinogenic. Many hazard assessments of tobacco have focused on individual chemical exposures without consideration of how the chemicals may interact with one another. Two chemicals, the human carcinogen 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) and a possible human carcinogen, acrolein, were hypothesized to interact with one another, possibly owing to the additive effects of DNA adduct formation or influence on the repair of mutagenic DNA adducts. To test our hypothesis that coexposure to NNK and acrolein is more carcinogenic than either chemical alone, A/J mice were exposed to NNK (i.p., 0, 2.5, or 7.5 μmol in saline) in the presence or absence of inhaled acrolein (15 ppmV). While the single 3 h exposure to acrolein alone did not induce lung adenomas, it significantly enhanced NNK's lung carcinogenicity. In addition, mice receiving both NNK and acrolein had more adenomas with dysplasia or progression than those receiving only NNK, suggesting that acrolein may also increase the severity of NNK-induced lung adenomas. To test the hypothesis that the interaction was due to effects on DNA adduct formation and repair, NNK- and acrolein pulmonary DNA adduct levels were assessed. There was no consistent effect of the coexposure on NNK-derived DNA adducts, and acrolein DNA adducts were not elevated above endogenous levels. This study supports the hypothesis that tobacco smoke chemicals combine to contribute to the carcinogenic potency of tobacco smoke, and the mechanism of interaction cannot be explained by alterations of DNA adduct levels.
Topics: Acrolein; Adenoma; Animals; Butanones; Carcinogenesis; Carcinogens; DNA Adducts; Humans; Lung; Lung Neoplasms; Mice; Nitrosamines; Smoke; Nicotiana; Tobacco Smoke Pollution
PubMed: 36149460
DOI: 10.1021/acs.chemrestox.2c00135 -
Analytical Chemistry Nov 2017Damage to DNA from the metabolites of drugs and pollutants constitutes a major human toxicity pathway known as genotoxicity. Metabolites can react with metal ions and...
Damage to DNA from the metabolites of drugs and pollutants constitutes a major human toxicity pathway known as genotoxicity. Metabolites can react with metal ions and NADPH to oxidize DNA or participate in S2 reactions to form covalently linked adducts with DNA bases. Guanines are the main DNA oxidation sites, and 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) is the initial product. Here we describe a novel electrochemiluminescent (ECL) microwell array that produces metabolites from test compounds and measures relative rates of DNA oxidation and DNA adduct damage. In this new array, films of DNA, metabolic enzymes, and an ECL metallopolymer or complex assembled in microwells on a pyrolytic graphite wafer are housed in dual microfluidic chambers. As reactant solution passes over the wells, metabolites form and can react with DNA in the films to form DNA adducts. These adducts are detected by ECL from a RuPVP polymer that uses DNA as a coreactant. Aryl amines also combine with Cu and NADPH to form reactive oxygen species (ROS) that oxidize DNA. The resulting 8-oxodG was detected selectively by ECL-generating bis(2,2'-bipyridine)-(4-(1,10-phenanthrolin-6-yl)-benzoic acid)Os(II). DNA/enzyme films on magnetic beads were oxidized similarly, and 8-oxodG determined by LC/MS/MS enabled array standardization. The array limit of detection for oxidation was 720 8-oxodG per 10 nucleobases. For a series of aryl amines, metabolite-generated DNA oxidation and adduct formation turnover rates from the array correlated very well with rodent 1/TD and Comet assay results.
Topics: Amines; Copper; Cytochrome P-450 Enzyme System; DNA; DNA Adducts; DNA Damage; Electrochemical Techniques; Humans; Luminescent Measurements; Microfluidic Analytical Techniques; NADP; Oxidation-Reduction
PubMed: 29083162
DOI: 10.1021/acs.analchem.7b03528 -
International Journal of Environmental... Nov 2021Exposure to benzo[a]pyrene (B[a]P) may be a risk factor for pulmonary diseases. To investigate the correlations among B[a]P exposure level, DNA strand breaks and...
Exposure to benzo[a]pyrene (B[a]P) may be a risk factor for pulmonary diseases. To investigate the correlations among B[a]P exposure level, DNA strand breaks and pulmonary inflammation, we recruited 83 children diagnosed with pulmonary diseases and 63 healthy children from Guangzhou, China. Results showed that the levels of Benzo[a]pyrene diol epoxide (BPDE) DNA adduct in blood and IL-8 in serum in case group were significantly higher than those in control group ( < 0.01). Moreover, levels of atmospheric B[a]P in case group was about twice of those in control group, which was consistent with the levels of BPDE-DNA adduct in blood. Significant positive correlations were observed among the levels of BPDE-DNA adduct, IL-8 and DNA strand breaks ( < 0.05). Our findings indicate that environmental air is an important exposure source of B[a]P and higher B[a]P exposure may contribute to the occurrence of pulmonary inflammation and lead to high health risks.
Topics: 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; Adolescent; Air Pollutants; Biological Monitoring; Child; Child, Preschool; China; Comet Assay; DNA Adducts; DNA Breaks; Female; Humans; Interleukin-8; Lung Diseases; Lymphocytes; Male; Polycyclic Aromatic Hydrocarbons; Risk Assessment
PubMed: 31722538
DOI: 10.1080/09603123.2019.1690638 -
Journal of the American Chemical Society Nov 20223-Methylthymine (m3T) is a long-known chemically stable but strongly mutagenic DNA base adduct; however, the sequencing method to determine m3T is lacking so far. Two of...
3-Methylthymine (m3T) is a long-known chemically stable but strongly mutagenic DNA base adduct; however, the sequencing method to determine m3T is lacking so far. Two of the main obstacles include the capacity of m3T to stall DNA elongation and its low abundance. To address the challenges, we report an unnatural base pairing strategy in this paper. A new m3T-TPT3 base pair was developed with a Vmax/Km value 82-fold higher than that of the m3T-A pair. The TPT3 nucleobase can be specifically incorporated opposite the m3T base, and the resulting m3T-TPT3 base pair can be effectively extended to give full-length products in the presence of commercial DNA polymerases. Importantly, the feature of the m3T-TPT3 pair enables a replacement PCR amplification to transfer m3T lesions at the exact positions into unnatural base pairs, which permits Sanger sequencings as well as biotin-streptavidin-based enrichments of m3T lesions. Taken together, this work offers a simple, convenient, and practical method for amplification, enrichment, and sequencing of m3T for the first time.
Topics: DNA Adducts; Mutagens; Base Pairing; DNA-Directed DNA Polymerase; DNA
PubMed: 36287063
DOI: 10.1021/jacs.2c06110 -
Bioorganic & Medicinal Chemistry Letters Mar 2021Current techniques for the identification of DNA adduct-inducing and DNA interstrand crosslinking agents include electrophoretic crosslinking assays, electrophoretic gel...
Current techniques for the identification of DNA adduct-inducing and DNA interstrand crosslinking agents include electrophoretic crosslinking assays, electrophoretic gel shift assays, DNA and RNA stop assays, mass spectrometry-based methods and P-post-labelling. While these assays provide considerable insight into the site and stability of the interaction, they are relatively expensive, time-consuming and sometimes rely on the use of radioactively-labelled components, and thus are ill-suited to screening large numbers of compounds. A novel medium throughput assay was developed to overcome these limitations and was based on the attachment of a biotin-tagged double stranded (ds) oligonucleotide to Corning DNA-Bind plates. We aimed to detect anthracycline and anthracenedione DNA adducts which form by initial non-covalent intercalation with duplex DNA, and subsequent covalent adduct formation which is mediated by formaldehyde. Following drug treatment, DNA samples were subjected to a denaturation step, washing and then measurement by fluorescence to detect remaining drug-DNA species using streptavidin-europium. This dissociation-enhanced lanthanide fluorescent immunoassay (DELFIA) is a time-resolved fluorescence intensity assay where the fluorescence signal arises only from stabilised drug-DNA complexes. We applied this new methodology to the identification of anthracycline-like compounds with the ability to functionally crosslink double-strand oligonucleotides. The entire procedure can be performed by robotics, requiring low volumes of compounds and reagents, thereby reducing costs and enabling multiple compounds to be assessed on a single microtitre plate.
Topics: Automation; Cross-Linking Reagents; DNA Adducts; Dose-Response Relationship, Drug; Drug Development; Molecular Structure; Structure-Activity Relationship
PubMed: 33486050
DOI: 10.1016/j.bmcl.2021.127813 -
Carcinogenesis Dec 20144-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is metabolized to enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), found in the urine of...
Carcinogenicity and DNA adduct formation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and enantiomers of its metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol in F-344 rats.
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is metabolized to enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), found in the urine of virtually all people exposed to tobacco products. We assessed the carcinogenicity in male F-344 rats of (R)-NNAL (5 ppm in drinking water), (S)-NNAL (5 ppm), NNK (5 ppm) and racemic NNAL (10 ppm) and analyzed DNA adduct formation in lung and pancreas of these rats after 10, 30, 50 and 70 weeks of treatment. All test compounds induced a high incidence of lung tumors, both adenomas and carcinomas. NNK and racemic NNAL were most potent; (R)-NNAL and (S)-NNAL had equivalent activity. Metastasis was observed from primary pulmonary carcinomas to the pancreas, particularly in the racemic NNAL group. DNA adducts analyzed were O (2)-[4-(3-pyridyl)-4-oxobut-1-yl]thymidine (O (2)-POB-dThd), 7-[4-(3-pyridyl)-4-oxobut-1-yl]guanine(7-POB-Gua),O (6)-[4-(3-pyridyl)-4-oxobut-1-yl]deoxyguanosine(O (6)-POB-dGuo),the 4-(3-pyridyl)-4-hydroxybut-1-yl(PHB)adductsO (2)-PHB-dThd and 7-PHB-Gua, O (6)-methylguanine (O (6)-Me-Gua) and 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing adducts. Adduct levels significantly decreased with time in the lungs of rats treated with NNK. Pulmonary POB-DNA adducts and O (6)-Me-Gua were similar in rats treated with NNK and (S)-NNAL; both were significantly greater than in the (R)-NNAL rats. In contrast, pulmonary PHB-DNA adduct levels were greatest in the rats treated with (R)-NNAL. Total pulmonary DNA adduct levels were similar in (S)-NNAL and (R)-NNAL rats. Similar trends were observed for DNA adducts in the pancreas, but adduct levels were significantly lower than in the lung. The results of this study clearly demonstrate the potent pulmonary carcinogenicity of both enantiomers of NNAL in rats and provide important new information regarding DNA damage by these compounds in lung and pancreas.
Topics: Adenocarcinoma; Adenoma; Animals; Apoptosis; Carcinogens; Chromatography, High Pressure Liquid; DNA Adducts; DNA Damage; Humans; Lung Neoplasms; Male; Nitrosamines; Pancreatic Neoplasms; Prohibitins; Pyridines; Rats; Rats, Inbred F344; Spectrometry, Mass, Electrospray Ionization; Stereoisomerism
PubMed: 25269804
DOI: 10.1093/carcin/bgu204 -
The Analyst Oct 2016Exposure to chemical pollutants and pharmaceuticals may cause health issues caused by metabolite-related toxicity. This paper reports a new microfluidic electrochemical...
Exposure to chemical pollutants and pharmaceuticals may cause health issues caused by metabolite-related toxicity. This paper reports a new microfluidic electrochemical sensor array with the ability to simultaneously detect common types of DNA damage including oxidation and nucleobase adduct formation. Sensors in the 8-electrode screen-printed carbon array were coated with thin films of metallopolymers osmium or ruthenium bipyridyl-poly(vinylpyridine) chloride (OsPVP, RuPVP) along with DNA and metabolic enzymes by layer-by-layer electrostatic assembly. After a reaction step in which test chemicals and other necessary reagents flow over the array, OsPVP selectively detects oxidized guanines on the DNA strands, and RuPVP detects DNA adduction by metabolites on nucleobases. We demonstrate array performance for test chemicals including 17β-estradiol (E), its metabolites 4-hydroxyestradiol (4-OHE), 2-hydroxyestradiol (2-OHE), catechol, 2-nitrosotoluene (2-NO-T), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), and 2-acetylaminofluorene (2-AAF). Results revealed DNA-adduct and oxidation damage in a single run to provide a metabolic-genotoxic chemistry screen. The array measures damage directly in unhydrolyzed DNA, and is less expensive, faster, and simpler than conventional methods to detect DNA damage. The detection limit for oxidation is 672 8-oxodG per 10 bases. Each sensor requires only 22 ng of DNA, so the mass detection limit is 15 pg (∼10 pmol) 8-oxodG.
Topics: DNA; DNA Adducts; DNA Damage; Microfluidic Analytical Techniques; Oxidation-Reduction
PubMed: 27517117
DOI: 10.1039/c6an01237j -
Metallomics : Integrated Biometal... Mar 2019Cisplatin is an anticancer drug widely used in clinics; it induces the apoptosis of cancer cells by targeting DNA. However, its interaction with proteins has been found...
Cisplatin is an anticancer drug widely used in clinics; it induces the apoptosis of cancer cells by targeting DNA. However, its interaction with proteins has been found to be crucial in modulating the pre and post-target activity. Nuclear DNA is tightly assembled with histone proteins to form nucleosomes in chromatin; this can impede the drug to access DNA. On the other hand, the linker histone H1 is considered 'the gate to nucleosomal DNA' due to its exposed location and dynamic conformation; therefore, this protein can influence the platination of DNA. In this study, we performed a reaction of cisplatin with histone H1 and investigated the interaction of the H1/cisplatin adduct with DNA. The reactions were conducted on the N-terminal domains of H1.4 (sequence 1-90, H1N90) and H1.0 (sequence 1-7, H1N7). The results show that H1 readily reacts with cisplatin and generates bidentate and tridentate adducts, with methionine and glutamate residues as the preferential binding sites. Chromatographic and NMR analyses show that the platination rate of H1 is slightly higher than that of DNA and the platinated H1 can form H1-cisplatin-DNA ternary complexes. Interestingly, cisplatin is more prone to form H1-Pt-DNA ternary complexes than trans-oriented platinum agents. The formation of H1-cisplatin-DNA ternary complexes and their preference for cis- over trans-oriented platinum agents suggest an important role of histone H1 in the mechanism of action of cisplatin.
Topics: Binding Sites; Cisplatin; DNA Adducts; Histones; Humans; Protein Binding
PubMed: 30672544
DOI: 10.1039/c8mt00358k -
Marine Environmental Research Apr 2017DNA adducts in fish represent a very important genotoxicity endpoint in environmental monitoring, being a pre-mutagenic lesion that plays an essential role in the... (Review)
Review
DNA adducts in fish represent a very important genotoxicity endpoint in environmental monitoring, being a pre-mutagenic lesion that plays an essential role in the initiation of carcinogenesis. The analysis of DNA adducts is a challenging task due to the low concentration of the analyte. Methods are available to determine the presence of DNA adducts, although further knowledge is required to fully understand the nature of the adducts and responsible xenobiotics (i.e. position of adduct in DNA, most active xenobiotic and metabolite forms, structural information). At present, P-postlabeling is the most used method that has the required sensitivity for DNA adduct analyses in both human health and environmental monitoring. Development of new mass spectrometry based methods for identifying DNA adducts in complex matrixes is now considered as a necessary mission in toxicology in order to gain the necessary information regarding adduct formation and facilitate tracking sources of contamination. Mass spectrometry therefore represents the future of DNA adduct detection, bringing along a series of challenges that the scientific community is facing at present.
Topics: Animals; Biomarkers; DNA Adducts; Environmental Monitoring; Fishes; Mutagens; Water Pollutants, Chemical
PubMed: 28167386
DOI: 10.1016/j.marenvres.2017.01.002 -
Chemical Research in Toxicology Feb 2020Characterization of the chemistry, structure, formation, and metabolism of DNA adducts has been one of the most significant contributions to the field of chemical... (Review)
Review
Characterization of the chemistry, structure, formation, and metabolism of DNA adducts has been one of the most significant contributions to the field of chemical toxicology. This work provides the foundation to develop analytical methods to measure DNA adducts, define their relationship to disease, and establish clinical tests. Monitoring exposure to environmental and endogenous toxicants can predict, diagnose, and track disease as well as guide therapeutic treatment. DNA adducts are one of the most promising biomarkers of toxicant exposure owing to their stability, appearance in numerous biological matrices, and characteristic analytical properties. In addition, DNA adducts can induce mutations to drive disease onset and progression and can serve as surrogate markers of chemical exposure. In this perspective, we highlight significant advances made within the past decade regarding DNA adduct quantitation using mass spectrometry. We hope to expose a broader audience to this field and encourage analytical chemistry laboratories to explore how specific adducts may be related to various pathologies. One of the limiting factors in developing clinical tests to measure DNA adducts is cohort size; ideally, the cohort would allow for model development and then testing of the model to the remaining cohort. The goals of this perspective article are to (1) provide a summary of analyte levels measured using state-of-the-art analytical methods, (2) foster collaboration, and (3) highlight areas in need of further investigation.
Topics: Biomarkers; DNA Adducts; Diabetes Mellitus, Type 2; Environmental Monitoring; Humans; Mass Spectrometry; Molecular Structure; Neoplasms
PubMed: 31638384
DOI: 10.1021/acs.chemrestox.9b00295