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Methods in Molecular Biology (Clifton,... 2021Aldehydes are abundantly present in tobacco smoke and in urban air pollution and are endogenously generated as products of the lipid peroxidation process. These...
Aldehydes are abundantly present in tobacco smoke and in urban air pollution and are endogenously generated as products of the lipid peroxidation process. These molecules can react with DNA bases forming mutagenic exocyclic adducts, which have been used as biomarkers of aldehyde exposure and as potential tools for the study of inflammation, metal storage diseases, neurodegenerative disorders, and cancer. High-performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS) provides a highly precise, specific and ultrasensitive method for the detection of exocyclic DNA adducts. Here we present and describe a validated micro-HPLC-Electro Spray Ionization (ESI)-MS/MS method for the quantification of 1,N-propanodGuo, an adduct produced following the reaction between 2'-deoxyguanosine and acetaldehyde or crotonaldehyde.
Topics: Animals; Chromatography, High Pressure Liquid; DNA Adducts; DNA Damage; Deoxyguanosine; Lung; Rats; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry
PubMed: 33683698
DOI: 10.1007/978-1-0716-1278-1_18 -
International Journal of Molecular... Sep 2021The plant extract aristolochic acid (AA), containing aristolochic acids I (AAI) and II (AAII) as major components, causes aristolochic acid nephropathy (AAN) and Balkan...
The plant extract aristolochic acid (AA), containing aristolochic acids I (AAI) and II (AAII) as major components, causes aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN), unique renal diseases associated with upper urothelial cancer. Recently (Chemical Research in Toxicology 33(11), 2804-2818, 2020), we showed that the metabolism of AAI and AAII in Wistar rats is influenced by their co-exposure (i.e., AAI/AAII mixture). Using the same rat model, we investigated how exposure to the AAI/AAII mixture can influence AAI and AAII DNA adduct formation (i.e., AA-mediated genotoxicity). Using P-postlabelling, we found that AA-DNA adduct formation was increased in the livers and kidneys of rats treated with AAI/AAII mixture compared to rats treated with AAI or AAII alone. Measuring the activity of enzymes involved in AA metabolism, we showed that enhanced AA-DNA adduct formation might be caused partially by both decreased AAI detoxification as a result of hepatic CYP2C11 inhibition during treatment with AAI/AAII mixture and by hepatic or renal NQO1 induction, the key enzyme predominantly activating AA to DNA adducts. Moreover, our results indicate that AAII might act as an inhibitor of AAI detoxification . Consequently, higher amounts of AAI might remain in liver and kidney tissues, which can be reductively activated, resulting in enhanced AAI DNA adduct formation. Collectively, these results indicate that AAII present in the plant extract AA enhances the genotoxic properties of AAI (i.e., AAI DNA adduct formation). As patients suffering from AAN and BEN are always exposed to the plant extract (i.e., AAI/AAII mixture), our findings are crucial to better understanding host factors critical for AAN- and BEN-associated urothelial malignancy.
Topics: Animals; Aristolochic Acids; Carcinogenesis; Carcinogens; DNA Adducts; DNA, Neoplasm; Male; Rats; Rats, Wistar
PubMed: 34638820
DOI: 10.3390/ijms221910479 -
Journal of Chemical Information and... May 2021Human exposure to aromatic amines (AAs) can result in carcinogenic DNA adducts. To complement previous work geared toward understanding the mutagenicity of AA-derived...
Human exposure to aromatic amines (AAs) can result in carcinogenic DNA adducts. To complement previous work geared toward understanding the mutagenicity of AA-derived adducts, which has almost exclusively studied (monoadducted) DNA containing a single lesion, the present work provides the first in-depth comparison of the structure of monoadducted and diadducted DNA duplexes. Specifically, molecular dynamics (MD) simulations were initially performed on DNA containing the nonmutagenic single-ringed -(deoxyguanosin-8-yl)-aniline (dG) or the mutagenic four-ringed -(deoxyguanosin-8-yl)-1-aminopyrene (dG) lesion at G, G, or G in the AA deletion hotspot (5'-GGCGCC) in the or glycosidic orientation (B/S duplex conformation). Subsequently, diadducted strands were assessed that span each combination of damaged sites (GG (nearest neighbors), GG (next-nearest neighbors), and GG (two intervening nucleotides)) and / lesion glycosidic orientations. Despite other N-linked C8-dG adducts exhibiting sequence dependence conformational heterogeneity, a single dG or dG lesion induces helical conformational homogeneity that is exclusively controlled by aryl moiety size. However, the preferred damaged DNA conformation can change upon the addition of a second adduct depending on lesion separation, with neighboring lesions stabilizing a nonmutagenic conformation and next-nearest damaged sites stabilizing a promutagenic conformation regardless of adduct size. As a result, diadducted DNA is found to adopt conformations that are unfavored for the corresponding monoadducted system, pointing to differential replication and repair outcomes for diadducted DNA compared to those for monoadducted DNA. Thus, although the toxicity of monoadducted DNA is most significantly dictated by lesion size, the toxicity can increase or decrease upon a second damaging event depending on lesion size and relative position. Overall, our work adds the number of lesions and their spatial separation to the growing list of factors that determine the structure and biological outcomes of adducted DNA.
Topics: Amines; DNA; DNA Adducts; Humans; Molecular Dynamics Simulation; Nucleic Acid Conformation
PubMed: 33977716
DOI: 10.1021/acs.jcim.1c00202 -
International Journal of Molecular... Oct 2021DNA-dependent DNA and RNA polymerases are important modulators of biological functions such as replication, transcription, recombination, or repair. In this work...
Processing and Bypass of a Site-Specific DNA Adduct of the Cytotoxic Platinum-Acridinylthiourea Conjugate by Polymerases Involved in DNA Repair: Biochemical and Thermodynamic Aspects.
DNA-dependent DNA and RNA polymerases are important modulators of biological functions such as replication, transcription, recombination, or repair. In this work performed in cell-free media, we studied the ability of selected DNA polymerases to overcome a monofunctional adduct of the cytotoxic/antitumor platinum-acridinylthiourea conjugate [PtCl(en)(L)](NO) (en = ethane-1,2-diamine, L = 1-[2-(acridin-9-ylamino)ethyl]-1,3-dimethylthiourea) (ACR) in its favored 5'-CG sequence. We focused on how a single site-specific ACR adduct with intercalation potency affects the processivity and fidelity of DNA-dependent DNA polymerases involved in translesion synthesis (TLS) and repair. The ability of the G(N7) hybrid ACR adduct formed in the 5'-TCGT sequence of a 24-mer DNA template to inhibit the synthesis of a complementary DNA strand by the exonuclease-deficient Klenow fragment of DNA polymerase I (KF) and human polymerases eta, kappa, and iota was supplemented by thermodynamic analysis of the polymerization process. Thermodynamic parameters of a simulated translesion synthesis across the ACR adduct were obtained by using microscale thermophoresis (MST). Our results show a strong inhibitory effect of an ACR adduct on enzymatic TLS: there was only small synthesis of a full-length product (less than 10%) except polymerase eta (~20%). Polymerase eta was able to most efficiently bypass the ACR hybrid adduct. Incorporation of a correct dCMP opposite the modified G residue is preferred by all the four polymerases tested. On the other hand, the frequency of misinsertions increased. The relative efficiency of misinsertions is higher than that of matched cytidine monophosphate but still lower than for the nonmodified control duplex. Thermodynamic inspection of the simulated TLS revealed a significant stabilization of successively extended primer/template duplexes containing an ACR adduct. Moreover, no significant decrease of dissociation enthalpy change behind the position of the modification can contribute to the enzymatic TLS observed with the DNA-dependent, repair-involved polymerases. This TLS could lead to a higher tolerance of cancer cells to the ACR conjugate compared to its enhanced analog, where thiourea is replaced by an amidine group: [PtCl(en)(L)](NO) (complex AMD, en = ethane-1,2-diamine, L = N-[2-(acridin-9-ylamino)ethyl]-N-methylpropionamidine).
Topics: DNA Adducts; DNA Damage; DNA Repair; DNA Replication; DNA-Directed DNA Polymerase; Humans; Intercalating Agents; Organoplatinum Compounds; Urea
PubMed: 34639179
DOI: 10.3390/ijms221910838 -
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 -
Toxicology and Applied Pharmacology Feb 2015Phosphoramide mustard (PM), the ovotoxic metabolite of the anti-cancer agent cyclophosphamide (CPA), destroys rapidly dividing cells by forming NOR-G-OH, NOR-G and...
Phosphoramide mustard (PM), the ovotoxic metabolite of the anti-cancer agent cyclophosphamide (CPA), destroys rapidly dividing cells by forming NOR-G-OH, NOR-G and G-NOR-G adducts with DNA, potentially leading to DNA damage. A previous study demonstrated that PM induces ovarian DNA damage in rat ovaries. To investigate whether PM induces DNA adduct formation, DNA damage and induction of the DNA repair response, rat spontaneously immortalized granulosa cells (SIGCs) were treated with vehicle control (1% DMSO) or PM (3 or 6μM) for 24 or 48h. Cell viability was reduced (P<0.05) after 48h of exposure to 3 or 6μM PM. The NOR-G-OH DNA adduct was detected after 24h of 6μM PM exposure, while the more cytotoxic G-NOR-G DNA adduct was formed after 48h by exposure to both PM concentrations. Phosphorylated H2AX (γH2AX), a marker of DNA double stranded break occurrence, was also increased by PM exposure, coincident with DNA adduct formation. Additionally, induction of genes (Atm, Parp1, Prkdc, Xrcc6, and Brca1) and proteins (ATM, γH2AX, PARP-1, PRKDC, XRCC6, and BRCA1) involved in DNA repair were observed in both a time- and dose-dependent manner. These data support that PM induces DNA adduct formation in ovarian granulosa cells, induces DNA damage and elicits the ovarian DNA repair response.
Topics: Animals; Antigens, Nuclear; Antineoplastic Agents; Ataxia Telangiectasia Mutated Proteins; BRCA1 Protein; Cell Line; Cell Survival; DNA Adducts; DNA Repair; DNA-Activated Protein Kinase; DNA-Binding Proteins; Female; Gene Expression Regulation; Granulosa Cells; Histones; Ku Autoantigen; Phosphoproteins; Phosphoramide Mustards; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerases; Rad51 Recombinase; Rats
PubMed: 25497287
DOI: 10.1016/j.taap.2014.11.017 -
Chemical Research in Toxicology Feb 2023Prolonged exposure to aristolochic acid (AA) through AA-containing herbal medicines or AA-tainted food is putting a large portion of the global population at risk of...
Prolonged exposure to aristolochic acid (AA) through AA-containing herbal medicines or AA-tainted food is putting a large portion of the global population at risk of developing renal fibrosis and tumors of the upper urinary tract. In an effort to better understand the organotropic property of AA, we studied the cytotoxicity, absorption, oxidative-stress inducing potential, and DNA adduct formation capability of aristolactam I (ALI), one of the major urinary metabolites of aristolochic acid I (AAI) in human cells. Despite ALI having a slightly lower cytotoxicity than that of AAI, the analysis revealed, for the first time, that ALI is bioaccumulated 900 times more than that of AAI inside cultured kidney cells. Furthermore, ALI induced a significantly larger glutathione depletion than that of AAI in the exposed cells. Together with the formation of ALI-DNA adduct at a reasonably high abundance, results of this study unmasked a previously disregarded causative role of ALI in the organotropic tumor-targeting property of AA.
Topics: Humans; DNA Adducts; Bioaccumulation; Aristolochic Acids; Kidney Diseases; Neoplasms; Carcinogens
PubMed: 36757010
DOI: 10.1021/acs.chemrestox.2c00415 -
Chemical Research in Toxicology Jan 2018The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a powerful lung carcinogen in animal models and is considered a causative factor...
Methyl DNA Phosphate Adduct Formation in Rats Treated Chronically with 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone and Enantiomers of Its Metabolite 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol.
The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a powerful lung carcinogen in animal models and is considered a causative factor for lung cancer in tobacco users. NNK is stereoselectively and reversibly metabolized to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which is also a lung carcinogen. Both NNK and NNAL undergo metabolic activation by α-hydroxylation on their methyl groups to form pyridyloxobutyl and pyridylhydroxybutyl DNA base and phosphate adducts, respectively. α-Hydroxylation also occurs on the α-methylene carbons of NNK and NNAL to produce methane diazohydroxide, which reacts with DNA to form methyl DNA base adducts. DNA adducts of NNK and NNAL are important in their mechanisms of carcinogenesis. In this study, we characterized and quantified methyl DNA phosphate adducts in the lung of rats treated with 5 ppm of NNK, (S)-NNAL, or (R)-NNAL in drinking water for 10, 30, 50, and 70 weeks, by using a novel liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry method. A total of 23, 21, and 22 out of 32 possible methyl DNA phosphate adducts were detected in the lung tissues of rats treated with NNK, (S)-NNAL, and (R)-NNAL, respectively. Levels of the methyl DNA phosphate adducts were 2290-4510, 872-1120, and 763-1430 fmol/mg DNA, accounting for 15-38%, 8%, and 5-9% of the total measured DNA adducts in rats treated with NNK, (S)-NNAL, and (R)-NNAL, respectively. The methyl DNA phosphate adducts characterized in this study further enriched the diversity of DNA adducts formed by NNK and NNAL. These results provide important new data regarding NNK- and NNAL-derived DNA damage and new insights pertinent to future mechanistic and biomonitoring studies of NNK, NNAL, and other chemical methylating agents.
Topics: Animals; DNA Adducts; DNA Methylation; Hydrolysis; Lung; Male; Molecular Structure; Nitrosamines; Phosphates; Rats; Rats, Inbred F344; Stereoisomerism
PubMed: 29131934
DOI: 10.1021/acs.chemrestox.7b00281 -
Carcinogenesis Nov 2019The formation of methyl DNA adducts is a critical step in carcinogenesis initiated by the exposure to methylating carcinogens. Methyl DNA phosphate adducts, formed by...
The formation of methyl DNA adducts is a critical step in carcinogenesis initiated by the exposure to methylating carcinogens. Methyl DNA phosphate adducts, formed by methylation of the oxygen atoms of the DNA phosphate backbone, have been detected in animals treated with methylating carcinogens. However, detection of these adducts in human tissues has not been reported. We developed an ultrasensitive liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry method for detecting methyl DNA phosphate adducts. Using 50 μg of human lung DNA, a limit of quantitation of two adducts/1010 nucleobases was achieved. Twenty-two structurally unique methyl DNA phosphate adducts were detected in human lung DNA. The adduct levels were measured in both tumor and adjacent normal tissues from 30 patients with lung cancer, including 13 current smokers and 17 current non-smokers, as confirmed by measurements of urinary cotinine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol. Levels of total methyl DNA phosphate adducts in normal lung tissues were higher in smokers than non-smokers, with an average of 13 and 8 adducts/109 nucleobases, respectively. Methyl DNA phosphate adducts were also detected in lung tissues from untreated rats with steady-state levels of 5-7 adducts/109 nucleobases over a period of 70 weeks. This is the first study to report the detection of methyl DNA phosphate adducts in human lung tissues. The results provide new insights toward using these DNA adducts as potential biomarkers to study human exposure to environmental methylating carcinogens.
Topics: Animals; Chromatography, Liquid; DNA Adducts; DNA, Neoplasm; Humans; Lung; Lung Neoplasms; Rats; Sensitivity and Specificity; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry; Tobacco Smoking
PubMed: 30873516
DOI: 10.1093/carcin/bgz053 -
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