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Current Opinion in Biotechnology Feb 2019Detection and characterization of DNA damage is essential for evaluating genotoxicity, monitoring DNA repair, developing biomarkers for exposures, and evaluating the... (Review)
Review
Detection and characterization of DNA damage is essential for evaluating genotoxicity, monitoring DNA repair, developing biomarkers for exposures, and evaluating the efficacy of chemotherapies. These diverse applications for DNA damage measurements have spurred the continual development and refinement of methodologies for detecting, characterizing, and quantifying DNA damage from isolated DNA and in cells and tissues. Current damage detection methods cover a wide range of techniques from radiolabeling to mass spectrometry, and use of these techniques varies widely based on expense, expertise, and knowledge of adduct formation. More generalizable, easy-to-use methods for detecting and quantifying DNA damage are needed, and there has been an emergence of fluorescence-based methodologies to address this need. Developments in these fluorescence-based strategies are reviewed here.
Topics: Biomarkers; DNA Adducts; DNA Damage; DNA Repair; Enzyme Assays; Fluorescence; Humans
PubMed: 30114673
DOI: 10.1016/j.copbio.2018.08.001 -
Chemical Research in Toxicology May 20171,2-Dibromoethane (DBE, ethylene dibromide) is a potent carcinogen due at least in part to its DNA cross-linking effects. DBE cross-links glutathione (GSH) to DNA,...
1,2-Dibromoethane (DBE, ethylene dibromide) is a potent carcinogen due at least in part to its DNA cross-linking effects. DBE cross-links glutathione (GSH) to DNA, notably to sites on 2'-deoxyadenosine and 2'-deoxyguanosine ( Cmarik , J. L. , et al. ( 1991 ) J. Biol. Chem. 267 , 6672 - 6679 ). Adduction at the N6 position of 2'-deoxyadenosine (dA) had not been detected, but this is a site for the linkage of O-alkylguanine DNA alkyltransferase ( Chowdhury , G. , et al. ( 2013 ) Angew. Chem. Int. Ed. 52 , 12879 - 12882 ). We identified and quantified a new adduct, S-[2-(N-deoxyadenosinyl)ethyl]GSH, in calf thymus DNA using LC-MS/MS. Replication studies were performed in duplex oligonucleotides containing this adduct with human DNA polymerases (hPols) η, ι, and κ, as well as with Sulfolobus solfataricus Dpo4, Escherichia coli polymerase I Klenow fragment, and bacteriophage T7 polymerase. hPols η and ι, Dpo4, and Klenow fragment were able to bypass the adduct with only slight impedance; hPol η and ι showed increased misincorporation opposite the adduct compared to that of unmodified 2'-deoxyadenosine. LC-MS/MS analysis of full-length primer extension products by hPol η confirmed the incorporation of dC opposite S-[2-(N-deoxyadenosinyl)ethyl]GSH and also showed the production of a -1 frameshift. These results reveal the significance of N-dA GSH-DBE adducts in blocking replication, as well as producing mutations, by human translesion synthesis DNA polymerases.
Topics: Animals; Cattle; Chromatography, Liquid; DNA Adducts; DNA Replication; DNA-Directed DNA Polymerase; Ethylene Dibromide; Glutathione; Tandem Mass Spectrometry
PubMed: 28395138
DOI: 10.1021/acs.chemrestox.7b00022 -
Current Medicinal Chemistry 2017Maintenance of genomic stability is a critical determinant of cell survival and is necessary for growth and progression of malignant cells. Interstrand crosslinking... (Review)
Review
Maintenance of genomic stability is a critical determinant of cell survival and is necessary for growth and progression of malignant cells. Interstrand crosslinking (ICL) agents, including platinum-based agents, are first-line chemotherapy treatment for many solid human cancers. In malignant cells, ICL triggers the DNA damage response (DDR). When the damage burden is high and lesions cannot be repaired, malignant cells are unable to divide and ultimately undergo cell death either through mitotic catastrophe or apoptosis. The activities of ICL agents, in particular platinum-based therapies, establish a "molecular landscape," i.e., a pattern of DNA damage that can potentially be further exploited therapeutically with DDR-targeting agents. If the molecular landscape created by platinum-based agents could be better defined at the molecular level, a systematic, mechanistic rationale(s) could be developed for the use of DDR-targeting therapies in combination/maintenance protocols for specific, clinically advanced malignancies. New therapeutic drugs such as poly(ADP-ribose) polymerase (PARP) inhibitors are examples of DDR-targeting therapies that could potentially increase the DNA damage and replication stress imposed by platinum-based agents in tumor cells and provide therapeutic benefit for patients with advanced malignancies. Recent studies have shown that the use of PARP inhibitors together with platinum-based agents is a promising therapy strategy for ovarian cancer patients with "BRCAness", i.e., a phenotypic characteristic of tumors that not only can involve loss-of-function mutations in either BRCA1 or BRCA2, but also encompasses the molecular features of BRCA-mutant tumors. On the basis of these promising results, additional mechanism-based studies focused on the use of various DDR-targeting therapies in combination with platinum-based agents should be considered. This review discusses, in general, (1) ICL agents, primarily platinum-based agents, that establish a molecular landscape that can be further exploited therapeutically; (2) multiple points of potential intervention after ICL agent-induced crosslinking that further predispose to cell death and can be incorporated into a systematic, therapeutic rationale for combination/ maintenance therapy using DDR-targeting agents; and (3) available agents that can be considered for use in combination/maintenance clinical protocols with platinum-based agents for patients with advanced malignancies.
Topics: Ataxia Telangiectasia Mutated Proteins; Cell Cycle Proteins; Coordination Complexes; DNA Adducts; DNA Damage; DNA Repair; Humans; Neoplasms; Nuclear Proteins; Platinum; Poly(ADP-ribose) Polymerase Inhibitors; Protein-Tyrosine Kinases
PubMed: 27978798
DOI: 10.2174/0929867323666161214114948 -
Molecular Biology Reports Feb 2017It is well known that a connection between xenobiotics inhalation, especially tobacco combustion and Lung Cancer development is strongly significant and indisputable.... (Review)
Review
It is well known that a connection between xenobiotics inhalation, especially tobacco combustion and Lung Cancer development is strongly significant and indisputable. However, recent studies provide evidence indicating that another factors such as, estrogens are also involved in lung carcinoma biology and metabolism. Although the status of estrogen receptors (ER), in both cancerous and healthy lung tissue has been well documented, there is still inconclusive data with respect of which isoform of the receptor is present in the lungs. However according to several studies, ERβ appears to be predominant form. Apart from ERs, estrogens can work through a recently discovered G-coupled estrogen receptor. Binding with both types of the receptors causes a signal, which leads to i.e. enhanced cell proliferation. There are many published reports which suggest that estrogen can be synthesized in situ in lung cancer. Some disturbances in the activity and expression levels of enzymes involved in estrogen synthesis were proved. This suggests that increased amounts of sex-steroid hormones can affect cells biology and be the reason of the accelerated development and pathogenesis of lung cancer. There also exist phenomena which associate estrogenic metabolism and tobacco combustion and its carcinogenic influence on the lungs. Compounds present in cigarette smoke induce the activity of CYP1B1, the enzyme responsible for estrogenic metabolism and synthesis of their cateholic derivatives. These structures during their redox cycle are able to release reactive oxygen species or form DNA adduct, which generally leads to destruction of genetic material. This process may explain the synergistic effect of smoking and estrogens on estrogen-dependent lung cancer development.
Topics: Animals; Cell Proliferation; Cytochrome P-450 CYP1B1; DNA Adducts; Estrogens; Female; Gene Expression Regulation, Neoplastic; Humans; Lung Neoplasms; Male; Reactive Oxygen Species; Receptors, Estrogen; Smoking
PubMed: 27783191
DOI: 10.1007/s11033-016-4086-8 -
Advances in Experimental Medicine and... 2019Prostate cancer (PC) is the most commonly diagnosed non-cutaneous cancer and the second leading cause of cancer-related to death in men. The major risk factors for PC... (Review)
Review
Prostate cancer (PC) is the most commonly diagnosed non-cutaneous cancer and the second leading cause of cancer-related to death in men. The major risk factors for PC are age, family history, and African American ethnicity. Epidemiological studies have reported large geographical variations in PC incidence and mortality, and thus lifestyle and dietary factors influence PC risk. High fat diet, dairy products, alcohol and red meats, are considered as risk factors for PC. This book chapter provides a comprehensive, literature-based review on dietary factors and their molecular mechanisms of prostate carcinogenesis. A large portion of our knowledge is based on epidemiological studies where dietary factors such as cancer promoting agents, including high-fat, dairy products, alcohol, and cancer-initiating genotoxicants formed in cooked meats have been evaluated for PC risk. However, the precise mechanisms in the etiology of PC development remain uncertain. Additional animal and human cell-based studies are required to further our understandings of risk factors involved in PC etiology. Specific biomarkers of chemical exposures and DNA damage in the prostate can provide evidence of cancer-causing agents in the prostate. Collectively, these studies can improve public health research, nutritional education and chemoprevention strategies.
Topics: Animals; Carcinogens; DNA Adducts; Diet; Humans; Male; Meat; Prostatic Neoplasms; Risk Factors
PubMed: 31900903
DOI: 10.1007/978-3-030-32656-2_2 -
Pharmacology & Therapeutics Apr 2016The repair of DNA damage is a complex process that relies on particular pathways to remedy specific types of damage to DNA. The range of insults to DNA includes small,... (Review)
Review
The repair of DNA damage is a complex process that relies on particular pathways to remedy specific types of damage to DNA. The range of insults to DNA includes small, modest changes in structure including mismatched bases and simple methylation events to oxidized bases, intra- and interstrand DNA crosslinks, DNA double strand breaks and protein-DNA adducts. Pathways required for the repair of these lesions include mismatch repair, base excision repair, nucleotide excision repair, and the homology directed repair/Fanconi anemia pathway. Each of these pathways contributes to genetic stability, and mutations in genes encoding proteins involved in these pathways have been demonstrated to promote genetic instability and cancer. In fact, it has been suggested that all cancers display defects in DNA repair. It has also been demonstrated that the ability of cancer cells to repair therapeutically induced DNA damage impacts therapeutic efficacy. This has led to targeting DNA repair pathways and proteins to develop anti-cancer agents that will increase sensitivity to traditional chemotherapeutics. While initial studies languished and were plagued by a lack of specificity and a defined mechanism of action, more recent approaches to exploit synthetic lethal interaction and develop high affinity chemical inhibitors have proven considerably more effective. In this review we will highlight recent advances and discuss previous failures in targeting DNA repair to pave the way for future DNA repair targeted agents and their use in cancer therapy.
Topics: Animals; Antineoplastic Agents; DNA; DNA Adducts; DNA Breaks, Double-Stranded; DNA Repair; Neoplasms
PubMed: 26896565
DOI: 10.1016/j.pharmthera.2016.02.003 -
Carcinogenesis Jun 2022DNA adducts are central in the mechanism of carcinogenesis by genotoxic agents. We compared levels of a DNA adduct of acrolein, a genotoxic carcinogen found in...
DNA adducts are central in the mechanism of carcinogenesis by genotoxic agents. We compared levels of a DNA adduct of acrolein, a genotoxic carcinogen found in e-cigarette vapor, in oral cell DNA of e-cigarette users and non-users of any tobacco or nicotine product. e-Cigarette users and non-users visited our clinic once monthly for 6 months, and oral brushings and urine samples were collected. For this study, we analyzed oral cell DNA adducts from three monthly visits in e-cigarette users and non-users as confirmed by urinary cyanoethyl mercapturic acid and total nicotine equivalents. DNA was isolated from the oral brushings and analyzed by a validated liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry method for the acrolein DNA adduct 8R/S-3-(2'-deoxyribos-1'-yl)-5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2-a]purine-10-(3H)-one (γ-OH-Acr-dGuo). The median value of this DNA adduct in the e-cigarette users was 179 fmol/µmol dGuo (range 5.0 - 793 fmol/µmol dGuo) while that for non-users was 21.0 fmol/µmol dGuo (range 5.0 - 539 fmol/µmol dGuo), P = 0.001. These results demonstrate for the first time that e-cigarette users have elevated levels of a carcinogen-DNA adduct in their oral cells.
Topics: Acrolein; Carcinogens; Chromatography, High Pressure Liquid; DNA; DNA Adducts; Electronic Nicotine Delivery Systems; Nicotine; Spectrometry, Mass, Electrospray Ionization
PubMed: 35239969
DOI: 10.1093/carcin/bgac026 -
DNA Repair Nov 2018Repair pathways of covalent DNA damage are understood in considerable detail due to decades of brilliant biochemical studies by many investigators. An important feature... (Review)
Review
Repair pathways of covalent DNA damage are understood in considerable detail due to decades of brilliant biochemical studies by many investigators. An important feature of these experiments is the defined adduct location on oligonucleotide or plasmid substrates that are incubated with purified proteins or cell free extracts. With some exceptions, this certainty is lost when the inquiry shifts to the response of living mammalian cells to the same adducts in genomic DNA. This reflects the limitation of assays, such as those based on immunofluorescence, that are widely used to follow responding proteins in cells exposed to a DNA reactive compound. The lack of effective reagents for adduct detection means that the proximity between responding proteins and an adduct must be assumed. Since these assumptions can be incorrect, models based on in vitro systems may fail to account for observations made in vivo. Here we discuss the use of a detection tag to address the problem of lesion location, as illustrated by our recent work on replication dependent and independent responses to interstrand crosslinks.
Topics: Cross-Linking Reagents; DNA; DNA Adducts; DNA Repair; DNA Replication; Humans; Immunohistochemistry; Mutagenicity Tests
PubMed: 30166246
DOI: 10.1016/j.dnarep.2018.08.023 -
Nucleic Acids Research Jun 2023Apurinic/apyrimidinic (AP) sites are DNA lesions created under normal growth conditions that result in cytotoxicity, replication-blocks, and mutations. AP sites are...
Apurinic/apyrimidinic (AP) sites are DNA lesions created under normal growth conditions that result in cytotoxicity, replication-blocks, and mutations. AP sites are susceptible to β-elimination and are liable to be converted to DNA strand breaks. HMCES (5-hydroxymethylcytosine binding, ES cell specific) protein interacts with AP sites in single stranded (ss) DNA exposed at DNA replication forks to generate a stable thiazolidine protein-DNA crosslink and protect cells against AP site toxicity. The crosslinked HMCES is resolved by proteasome-mediated degradation; however, it is unclear how HMCES-crosslinked ssDNA and the resulting proteasome-degraded HMCES adducts are processed and repaired. Here, we describe methods for the preparation of thiazolidine adduct-containing oligonucleotides and determination of their structure. We demonstrate that the HMCES-crosslink is a strong replication blocking adduct and that protease-digested HMCES adducts block DNA replication to a similar extent as AP sites. Moreover, we show that the human AP endonuclease APE1 incises DNA 5' to the protease-digested HMCES adduct. Interestingly, while HMCES-ssDNA crosslinks are stable, the crosslink is reversed upon the formation of dsDNA, possibly due to a catalytic reverse reaction. Our results shed new light on damage tolerance and repair pathways for HMCES-DNA crosslinks in human cells.
Topics: Humans; DNA Adducts; Thiazolidines; DNA Repair; Proteasome Endopeptidase Complex; DNA; DNA Damage; DNA, Single-Stranded; DNA-(Apurinic or Apyrimidinic Site) Lyase
PubMed: 37021581
DOI: 10.1093/nar/gkad246 -
Mass Spectrometry Reviews Mar 2020Hazardous chemicals in the environment and diet or their electrophilic metabolites can form adducts with genomic DNA, which can lead to mutations and the initiation of... (Review)
Review
Hazardous chemicals in the environment and diet or their electrophilic metabolites can form adducts with genomic DNA, which can lead to mutations and the initiation of cancer. In addition, reactive intermediates can be generated in the body through oxidative stress and damage the genome. The identification and measurement of DNA adducts are required for understanding exposure and the causal role of a genotoxic chemical in cancer risk. Over the past three decades, P-postlabeling, immunoassays, gas chromatography/mass spectrometry, and liquid chromatography/mass spectrometry (LC/MS) methods have been established to assess exposures to chemicals through measurements of DNA adducts. It is now possible to measure some DNA adducts in human biopsy samples, by LC/MS, with as little as several milligrams of tissue. In this review article, we highlight the formation and biological effects of DNA adducts, and highlight our advances in human biomonitoring by mass spectrometric analysis of formalin-fixed paraffin-embedded tissues, untapped biospecimens for carcinogen DNA adduct biomarker research.
Topics: Animals; Biopsy; Chromatography, Liquid; DNA Adducts; Humans; Mass Spectrometry; Mutation; Neoplasms
PubMed: 29889312
DOI: 10.1002/mas.21570