-
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 -
Cancer Biology & Therapy Dec 2017Glioblastoma is a lethal form of brain tumour usually treated by surgical resection followed by radiotherapy and an alkylating chemotherapeutic agent. Key to the success... (Review)
Review
Glioblastoma is a lethal form of brain tumour usually treated by surgical resection followed by radiotherapy and an alkylating chemotherapeutic agent. Key to the success of this multimodal approach is maintaining apoptotic sensitivity of tumour cells to the alkylating agent. This initial treatment likely establishes conditions contributing to development of drug resistance as alkylating agents form the O-methylguanine adduct. This activates the mismatch repair (MMR) process inducing apoptosis and mutagenesis. This review describes key juxtaposed drivers in the balance between alkylation induced mutagenesis and apoptosis. Mutations in MMR genes are the probable drivers for alkylation based drug resistance. Critical to this interaction are the dose-response and temporal interactions between adduct formation and MMR mutations. The precision in dose interval, dose-responses and temporal relationships dictate a role for alkylating agents in either promoting experimental tumour formation or inducing tumour cell death with chemotherapy. Importantly, this resultant loss of chemotherapeutic selective pressure provides opportunity to explore novel therapeutics and appropriate combinations to minimise alkylation based drug resistance and tumour relapse.
Topics: Antineoplastic Agents, Alkylating; Apoptosis; DNA Adducts; DNA Mismatch Repair; DNA Repair; Drug Resistance, Neoplasm; Glioblastoma; Guanine; Humans; Mutation; Neoplasm Recurrence, Local
PubMed: 29020502
DOI: 10.1080/15384047.2017.1385680 -
Free Radical Biology & Medicine Jun 2017Oxygen is both necessary and dangerous for aerobic cell function. ATP is most efficiently made by the electron transport chain, which requires oxygen as an electron... (Review)
Review
Oxygen is both necessary and dangerous for aerobic cell function. ATP is most efficiently made by the electron transport chain, which requires oxygen as an electron acceptor. However, the presence of oxygen, and to some extent the respiratory chain itself, poses a danger to cellular components. Mitochondria, the sites of oxidative phosphorylation, have defense and repair pathways to cope with oxidative damage. For mitochondrial DNA, an essential pathway is base excision repair, which acts on a variety of small lesions. There are instances, however, in which attempted DNA repair results in more damage, such as the formation of a DNA-protein crosslink trapping the repair enzyme on the DNA. That is the case for mitochondrial DNA polymerase γ acting on abasic sites oxidized at the 1-carbon of 2-deoxyribose. Such DNA-protein crosslinks presumably must be removed in order to restore function. In nuclear DNA, ubiquitylation of the crosslinked protein and digestion by the proteasome are essential first processing steps. How and whether such mechanisms operate on DNA-protein crosslinks in mitochondria remains to be seen.
Topics: DNA Adducts; DNA Damage; DNA Polymerase gamma; DNA Repair; Deoxyribose; Free Radicals; Humans; Mitochondria; Oxidation-Reduction; Oxidative Stress; Proteasome Endopeptidase Complex; Ubiquitination
PubMed: 27867099
DOI: 10.1016/j.freeradbiomed.2016.11.025 -
Chemical Research in Toxicology Nov 2019Frequent exposure to chemicals in the environment, diet, and endogenous electrophiles leads to chemical modification of DNA and the formation of DNA adducts. Some DNA...
Frequent exposure to chemicals in the environment, diet, and endogenous electrophiles leads to chemical modification of DNA and the formation of DNA adducts. Some DNA adducts can induce mutations during cell division and, when occurring in critical regions of the genome, can lead to the onset of disease, including cancer. The targeted analysis of DNA adducts over the past 30 years has revealed that the human genome contains many types of DNA damages. However, a long-standing limitation in conducting DNA adduct measurements has been the inability to screen for the total complement of DNA adducts derived from a wide range of chemicals in a single assay. With the advancement of high-resolution mass spectrometry (MS) instrumentation and new scanning technologies, nontargeted "omics" approaches employing data-dependent acquisition and data-independent acquisition methods have been established to simultaneously screen for multiple DNA adducts, a technique known as DNA adductomics. However, notable challenges in data processing must be overcome for DNA adductomics to become a mature technology. DNA adducts occur at low abundance in humans, and current softwares do not reliably detect them when using common MS data acquisition methods. In this perspective, we discuss contemporary computational tools developed for feature finding of MS data widely utilized in the disciplines of proteomics and metabolomics and highlight their limitations for conducting nontargeted DNA-adduct biomarker discovery. Improvements to existing MS data processing software and new algorithms for adduct detection are needed to develop DNA adductomics into a powerful tool for the nontargeted identification of potential cancer-causing agents.
Topics: Biomarkers; Computational Biology; DNA Adducts; Data Analysis; Humans; Mass Spectrometry; Workflow; Xenobiotics
PubMed: 31549505
DOI: 10.1021/acs.chemrestox.9b00196 -
Chemical Research in Toxicology Jan 2021Smoking is a leading cause of lung cancer, accounting for 81% of lung cancer cases. Tobacco smoke contains over 5000 compounds, of which more than 70 have been...
Smoking is a leading cause of lung cancer, accounting for 81% of lung cancer cases. Tobacco smoke contains over 5000 compounds, of which more than 70 have been classified as human carcinogens. Of the many tobacco smoke constituents, 1,3-butadiene (BD) has a high cancer risk index due to its tumorigenic potency and its abundance in cigarette smoke. The carcinogenicity of BD has been attributed to the formation of several epoxide metabolites, of which 1,2,3,4-diepoxybutane (DEB) is the most toxic and mutagenic. DEB is formed by two oxidation reactions carried out by cytochrome P450 monooxygenases, mainly CYP2E1. Glutathione-S-transferase 1 (GSTT1) facilitates the conjugation of DEB to glutathione as the first step of its detoxification and subsequent elimination via the mercapturic acid pathway. Human biomonitoring studies have revealed a strong association between copy number and urinary concentrations of BD-mercapturic acids, suggesting that it plays an important role in the metabolism of BD. To determine the extent that genotype affects the susceptibility of individuals to the toxic and genotoxic properties of DEB, negative and positive HapMap lymphoblastoid cell lines were treated with DEB, and the extent of apoptosis and micronuclei (MN) formation was assessed. These toxicological end points were compared to the formation of DEB-GSH conjugates and 1,4--(guan-7-yl)-2,3-butanediol (-N7G-BD) DNA-DNA cross-links. negative cell lines were more sensitive to DEB-induced apoptosis as compared to positive cell lines. Consistent with the protective effect of GSH conjugation against DEB-derived apoptosis, positive cell lines formed significantly more DEB-GSH conjugate than negative cell lines. However, genotype did not affect formation of MN or -N7G-BD cross-links. These results indicate that genotype significantly influences BD metabolism and acute toxicity.
Topics: Cell Line; DNA; DNA Adducts; Epoxy Compounds; Genotype; Glutathione; Glutathione Transferase; Humans; Molecular Structure
PubMed: 33381973
DOI: 10.1021/acs.chemrestox.0c00376 -
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 -
Analytica Chimica Acta Sep 2023Although interest in characterizing DNA damage by means of DNA adductomics has substantially grown, the field of DNA adductomics is still in its infancy, with room for...
Although interest in characterizing DNA damage by means of DNA adductomics has substantially grown, the field of DNA adductomics is still in its infancy, with room for optimization of methods for sample analysis, data processing and DNA adduct identification. In this context, the first objective of this study was to evaluate the use of hydrophilic interaction (HILIC) vs. reversed phase liquid chromatography (RPLC) coupled to high resolution mass spectrometry (HRMS) and thermal acidic vs. enzymatic hydrolysis of DNA followed by DNA adduct purification and enrichment using solid-phase extraction (SPE) or fraction collection for DNA adductome mapping. The second objective was to assess the use of total ion count (TIC) and median intensity (MedI) normalization compared to QC (quality control), iQC (internal QC) and quality control-based robust locally estimated scatterplot smoothing (LOESS) signal correction (QC-RLSC) normalization for processing of the acquired data. The results demonstrate that HILIC compared to RPLC allowed better modeling of the tentative DNA adductome, particularly in combination with thermal acidic hydrolysis and SPE (more valid models, with an average Q(Y) and R(Y) of 0.930 and 0.998, respectively). Regarding the need for data normalization and the management of (limited) system instability and signal drift, QC normalization outperformed TIC, MedI, iQC and LOESS normalization. As such, QC normalization can be put forward as the default data normalization strategy. In case of momentous signal drift and/or batch effects however, comparison to other normalization strategies (like e.g. LOESS) is recommended. In future work, further optimization of DNA adductomics may be achieved by merging of HILIC and RPLC datasets and/or application of 2D-LC, as well as the inclusion of Schiff base stabilization and/or fraction collection in the thermal acidic hydrolysis-SPE sample preparation workflow.
Topics: DNA Adducts; Mass Spectrometry; Chromatography, Reverse-Phase; Hydrolysis; Hydrophobic and Hydrophilic Interactions
PubMed: 37455087
DOI: 10.1016/j.aca.2023.341578 -
DNA Repair Jan 2021While mammalian mitochondria are known to possess a robust base excision repair system, direct evidence for the existence of additional mitochondrial DNA repair pathways...
While mammalian mitochondria are known to possess a robust base excision repair system, direct evidence for the existence of additional mitochondrial DNA repair pathways is elusive. Herein a PCR-based assay was employed to demonstrate that plasmids containing DNA-protein crosslinks are rapidly repaired following electroporation into isolated mammalian mitochondria. Several lines of evidence argue that this repair occurs via homologous recombination. First, DNA-protein crosslinks present on plasmid DNA homologous to the mitochondrial genome were efficiently repaired (21 % repair in three hours), whereas a DNA-protein crosslink present on DNA that lacked homology to the mitochondrial genome remained unrepaired. Second, DNA-protein crosslinks present on plasmid DNA lacking homology to the mitochondrial genome were repaired when they were co-electroporated into mitochondria with an undamaged, homologous plasmid DNA molecule. Third, no repair was observed when DNA-protein crosslink-containing plasmids were electroporated into mitochondria isolated from cells pre-treated with the Rad51 inhibitor B02. These findings suggest that mitochondria utilize homologous recombination to repair endogenous and xenobiotic-induced DNA-protein crosslinks. Consistent with this interpretation, cisplatin-induced mitochondrial DNA-protein crosslinks accumulated to higher levels in cells pre-treated with B02 than in control cisplatin-treated cells. These results represent the first evidence of how spontaneous and xenobiotic-induced DNA-protein crosslinks are removed from mitochondrial DNA.
Topics: Animals; Cell Line, Tumor; Cisplatin; Cricetulus; Cross-Linking Reagents; DNA Adducts; DNA, Mitochondrial; HEK293 Cells; Humans; Mitochondria; Rad51 Recombinase; Recombinational DNA Repair
PubMed: 33316746
DOI: 10.1016/j.dnarep.2020.103026 -
Chemical Research in Toxicology Apr 2019Quantitative measurement of DNA adducts in carcinogen-exposed cells provides the information about the frequency of formation and the rate of removal of DNA lesions in...
Quantitative measurement of DNA adducts in carcinogen-exposed cells provides the information about the frequency of formation and the rate of removal of DNA lesions in vivo, which yields insights into the initial events of mutagenesis. Metabolic activation of tobacco-specific nitrosamines, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its reduction product 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), leads to pyridyloxobutylation and pyridylhydroxybutylation of DNA. In this study, we employed a highly robust nanoflow liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry (nLC-nESI-MS/MS) coupled with the isotope-dilution method for simultaneous quantification of O-[4-(3-pyridyl)-4-hydroxylbut-1-yl]-2'-deoxyguanosine ( O-PHBdG) and O- and O-[4-(3-pyridyl)-4-hydroxylbut-1-yl]-thymidine ( O-PHBdT and O-PHBdT). Cultured mammalian cells were exposed to a model pyridylhydroxybutylating agent, 4-(acetoxymethylnitrosamino)-1-(3-pyridyl)-1-butanol (NNALOAc), followed by DNA extraction, enzymatic digestion, and sample enrichment prior to nLC-nESI-MS/MS quantification. Our results demonstrate, for the first time, that O-PHBdT is quantifiable in cellular DNA and naked DNA upon NNALOAc exposure. We also show that nucleotide excision repair (NER) machinery may counteract the formation of O-PHBdT and O-PHBdT, and O-alkylguanine DNA alkyltransferase (AGT) may be responsible for the repair of O-PHBdG and O-PHBdT in mammalian cells. Together, our study provides new knowledge about the occurrence and repair of NNAL-induced DNA lesions in mammalian cells.
Topics: Animals; Cattle; Cells, Cultured; DNA; DNA Adducts; DNA Repair; Esterases; Liver; Molecular Structure; Nitrosamines; Swine
PubMed: 30714728
DOI: 10.1021/acs.chemrestox.8b00374 -
Current Opinion in Pediatrics Apr 2020Exposomics studies can measure health-relevant chemical exposures during a lifetime and estimate the 'internal' environment. However, sampling limitations make these... (Review)
Review
PURPOSE OF REVIEW
Exposomics studies can measure health-relevant chemical exposures during a lifetime and estimate the 'internal' environment. However, sampling limitations make these features difficult to capture directly during the critical neonatal time period.
RECENT FINDINGS
We review the use of newborn dried bloodspots (DBS) archived from newborn screening programs for exposomic analysis in epidemiological children's health studies. Emerging 'omics technologies such as adductomics and metabolomics have been adapted for DBS analysis, and these technologies can now provide valuable etiological information on the complex interplay between exposures, biological response, and population phenotypes.
SUMMARY
Adductomics and metabolomics of DBS can provide robust measurements for retrospective epidemiological investigations. With extensive bioarchiving programs in the United States and other countries, DBS are poised to substantially aid epidemiological studies, particularly for rare and low-frequency childhood diseases and disorders.
Topics: Child; DNA; DNA Adducts; Dried Blood Spot Testing; Environmental Exposure; Exposome; Genetic Predisposition to Disease; Humans; Infant, Newborn; Metabolomics; Proteomics
PubMed: 31913157
DOI: 10.1097/MOP.0000000000000875