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Current Protocols Feb 2023Adductomics is epidemiology at the molecular level. Untargeted adductomics compares levels of chemical adducts on albumin, hemoglobin, and DNA between healthy and...
Adductomics is epidemiology at the molecular level. Untargeted adductomics compares levels of chemical adducts on albumin, hemoglobin, and DNA between healthy and exposed individuals. The goal is to determine a cause-and-effect relationship between chemical exposure and illness. Chemical exposures are not necessarily due to synthetic chemicals but are often due to oxidation products of naturally occurring lipids, for example, 4-hydroxynonenal and acrolein produced by lipid peroxidation of arachidonic and linoleic acids. The preferred method used in adductomics is ultra-high pressure liquid chromatography coupled to with nanoelectrospray tandem mass spectrometry. The mass of the adduct indicates its structure and identifies the chemical. The advantages of molecular epidemiology include information about the many toxicants to which a person is exposed over a period of weeks or months and the relative exposure levels. The disadvantage is the absence of information about the mechanism of toxicity. Untargeted adductomics examines albumin and hemoglobin adducts, which serve as biomarkers of exposure but do not identify the proteins and genes responsible for the toxicity. Targeted adductomics is used when the origin of the toxicity is known. This can be either an adducted protein, such as the butyrylcholinesterase protein modified by nerve agents, or a toxicant, such as acetaminophen. Untargeted adductomics methods have identified potential protein adduct biomarkers of breast cancer, colorectal cancer, childhood leukemia, and lung cancer. Adductomics is a new research area that offers structural insights into chemical exposures and a platform for the discovery of disease biomarkers. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.
Topics: Humans; Albumins; Biomarkers; Butyrylcholinesterase; DNA; DNA Adducts
PubMed: 36799690
DOI: 10.1002/cpz1.672 -
DNA Repair May 2020Topoisomerases are essential enzymes solving DNA topological problems such as supercoils, knots and catenanes that arise from replication, transcription, chromatin... (Review)
Review
Topoisomerases are essential enzymes solving DNA topological problems such as supercoils, knots and catenanes that arise from replication, transcription, chromatin remodeling and other nucleic acid metabolic processes. They are also the targets of widely used anticancer drugs (e.g. topotecan, irinotecan, enhertu, etoposide, doxorubicin, mitoxantrone) and fluoroquinolone antibiotics (e.g. ciprofloxacin and levofloxacin). Topoisomerases manipulate DNA topology by cleaving one DNA strand (TOP1 and TOP3 enzymes) or both in concert (TOP2 enzymes) through the formation of transient enzyme-DNA cleavage complexes (TOPcc) with phosphotyrosyl linkages between DNA ends and the catalytic tyrosyl residue of the enzymes. Failure in the self-resealing of TOPcc results in persistent TOPcc (which we refer it to as topoisomerase DNA-protein crosslinks (TOP-DPC)) that threaten genome integrity and lead to cancers and neurodegenerative diseases. The cell prevents the accumulation of topoisomerase-mediated DNA damage by excising TOP-DPC and ligating the associated breaks using multiple pathways conserved in eukaryotes. Tyrosyl-DNA phosphodiesterases (TDP1 and TDP2) cleave the tyrosyl-DNA bonds whereas structure-specific endonucleases such as Mre11 and XPF (Rad1) incise the DNA phosphodiester backbone to remove the TOP-DPC along with the adjacent DNA segment. The proteasome and metalloproteases of the WSS1/Spartan family typify proteolytic repair pathways that debulk TOP-DPC to make the peptide-DNA bonds accessible to the TDPs and endonucleases. The purpose of this review is to summarize our current understanding of how the cell excises TOP-DPC and why, when and where the cell recruits one specific mechanism for repairing topoisomerase-mediated DNA damage, acquiring resistance to therapeutic topoisomerase inhibitors and avoiding genomic instability, cancers and neurodegenerative diseases.
Topics: DNA; DNA Adducts; DNA Repair; DNA Topoisomerases, Type I; Humans; Phosphoric Diester Hydrolases; Topoisomerase Inhibitors
PubMed: 32200233
DOI: 10.1016/j.dnarep.2020.102837 -
Science Advances Dec 2023The catalytic cycle of topoisomerase 2 (TOP2) enzymes proceeds via a transient DNA double-strand break (DSB) intermediate termed the TOP2 cleavage complex (TOP2cc), in...
The catalytic cycle of topoisomerase 2 (TOP2) enzymes proceeds via a transient DNA double-strand break (DSB) intermediate termed the TOP2 cleavage complex (TOP2cc), in which the TOP2 protein is covalently bound to DNA. Anticancer agents such as etoposide operate by stabilizing TOP2ccs, ultimately generating genotoxic TOP2-DNA protein cross-links that require processing and repair. Here, we identify RAD54 like 2 (RAD54L2) as a factor promoting TOP2cc resolution. We demonstrate that RAD54L2 acts through a novel mechanism together with zinc finger protein associated with tyrosyl-DNA phosphodiesterase 2 (TDP2) and TOP2 (ZATT/ZNF451) and independent of TDP2. Our work suggests a model wherein RAD54L2 recognizes sumoylated TOP2 and, using its ATPase activity, promotes TOP2cc resolution and prevents DSB exposure. These findings suggest RAD54L2-mediated TOP2cc resolution as a potential mechanism for cancer therapy resistance and highlight RAD54L2 as an attractive candidate for drug discovery.
Topics: Humans; DNA Adducts; DNA-Binding Proteins; Phosphoric Diester Hydrolases; DNA Topoisomerases, Type II; DNA; Genomic Instability; DNA Helicases
PubMed: 38055822
DOI: 10.1126/sciadv.adl2108 -
International Journal of Molecular... Feb 2023Nitrosamines occur widespread in food, drinking water, cosmetics, as well as tobacco smoke and can arise endogenously. More recently, nitrosamines have been detected as... (Review)
Review
Nitrosamines occur widespread in food, drinking water, cosmetics, as well as tobacco smoke and can arise endogenously. More recently, nitrosamines have been detected as impurities in various drugs. This is of particular concern as nitrosamines are alkylating agents that are genotoxic and carcinogenic. We first summarize the current knowledge on the different sources and chemical nature of alkylating agents with a focus on relevant nitrosamines. Subsequently, we present the major DNA alkylation adducts induced by nitrosamines upon their metabolic activation by CYP450 monooxygenases. We then describe the DNA repair pathways engaged by the various DNA alkylation adducts, which include base excision repair, direct damage reversal by MGMT and ALKBH, as well as nucleotide excision repair. Their roles in the protection against the genotoxic and carcinogenic effects of nitrosamines are highlighted. Finally, we address DNA translesion synthesis as a DNA damage tolerance mechanism relevant to DNA alkylation adducts.
Topics: Nitrosamines; DNA Damage; Alkylation; DNA Repair; Alkylating Agents; DNA Adducts
PubMed: 36902118
DOI: 10.3390/ijms24054684 -
Journal of Applied Genetics Aug 2017Investigations on the impact of chemicals on the environment and human health have led to the development of an exposome concept. The exposome refers to the totality of... (Review)
Review
Investigations on the impact of chemicals on the environment and human health have led to the development of an exposome concept. The exposome refers to the totality of exposures received by a person during life, including exposures to life-style factors, from the prenatal period to death. The exposure to genotoxic chemicals and their reactive metabolites can induce chemical modifications of DNA, such as, for example, DNA adducts, which have been extensively studied and which play a key role in chemically induced carcinogenesis. Development of different methods for the identification of DNA adducts has led to adopting DNA adductomic approaches. The ability to simultaneously detect multiple PAH-derived DNA adducts may allow for the improved assessment of exposure, and offer a mechanistic insight into the carcinogenic process following exposure to PAH mixtures. The major advantage of measuring chemical-specific DNA adducts is the assessment of a biologically effective dose. This review provides information about the occurrence of the polycyclic aromatic hydrocarbons (PAHs) and their influence on human exposure and biological effects, including PAH-derived DNA adduct formation and repair processes. Selected methods used for determination of DNA adducts have been presented.
Topics: Biotransformation; DNA Adducts; DNA Damage; DNA Repair; Environmental Exposure; Humans; Polycyclic Aromatic Hydrocarbons
PubMed: 27943120
DOI: 10.1007/s13353-016-0380-3 -
DNA Repair Oct 2020Proteins that act on DNA, or are in close proximity to it, can become inadvertently crosslinked to DNA and form highly toxic lesions, known as DNA-protein crosslinks...
Proteins that act on DNA, or are in close proximity to it, can become inadvertently crosslinked to DNA and form highly toxic lesions, known as DNA-protein crosslinks (DPCs). DPCs are generated by different chemotherapeutics, environmental or endogenous sources of crosslinking agents, or by lesions on DNA that stall the catalytic cycle of certain DNA processing enzymes. These bulky adducts impair processes on DNA such as DNA replication or transcription, and therefore pose a serious threat to genome integrity. The large diversity of DPCs suggests that there is more than one canonical mechanism to repair them. Indeed, many different enzymes have been shown to act on DPCs by either processing the protein, the DNA or the crosslink itself. In addition, the cell cycle stage or cell type are likely to dictate pathway choice. In recent years, a detailed understanding of DPC repair during S phase has started to emerge. Here, we review the current knowledge on the mechanisms of replication-coupled DPC repair, and describe and also speculate on possible pathways that remove DPCs outside of S phase. Moreover, we highlight a recent paradigm shifting finding that indicates that DPCs are not always detrimental, but can also play a protective role, preserving the genome from more deleterious forms of DNA damage.
Topics: Animals; DNA; DNA Adducts; DNA Repair; DNA Replication; Eukaryota; Humans; Proteins
PubMed: 32683310
DOI: 10.1016/j.dnarep.2020.102924 -
Rapid Communications in Mass... Jul 2021As a new approach to DNA adductomics, we directly reacted intact, double-stranded (ds)-DNA under warm conditions with an alkylating mass tag followed by analysis by...
RATIONALE
As a new approach to DNA adductomics, we directly reacted intact, double-stranded (ds)-DNA under warm conditions with an alkylating mass tag followed by analysis by liquid chromatography/mass spectrometry. This method is based on the tendency of adducted nucleobases to locally disrupt the DNA structure (forming a "DNA bubble") potentially increasing exposure of their nucleophilic (including active hydrogen) sites for preferential alkylation. Also encouraging this strategy is that the scope of nucleotide excision repair is very broad, and this system primarily recognizes DNA bubbles.
METHODS
A cationic xylyl (CAX) mass tag with limited nonpolarity was selected to increase the retention of polar adducts in reversed-phase high-performance liquid chromatography (HPLC) for more detectability while maintaining resolution. We thereby detected a diversity of DNA adducts (mostly polar) by the following sequence of steps: (1) react DNA at 45°C for 2 h under aqueous conditions with CAX-B (has a benzyl bromide functional group to label active hydrogen sites) in the presence of triethylamine; (2) remove residual reagents by precipitating and washing the DNA (a convenient step); (3) digest the DNA enzymatically to nucleotides and remove unlabeled nucleotides by nonpolar solid-phase extraction (also a convenient step); and (4) detect CAX-labeled, adducted nucleotides by LC/MS or a matrix-assisted laser desorption/ionization (MALDI)-MS technique.
RESULTS
Examples of the 42 DNA or RNA adducts detected, or tentatively so based on accurate mass and fragmentation data, are as follows: 8-oxo-dGMP, ethyl-dGMP, hydroxyethyl-dGMP (four isomers, all HPLC-resolved), uracil-glycol, apurinic/apyrimidinic sites, benzo[a]pyrene-dGMP, and, for the first time, benzoquinone-hydroxymethyl-dCMP. Importantly, these adducts are detected in a single procedure under a single set of conditions. Sensitivity, however, is only defined in a preliminary way, namely the latter adduct seems to be detected at a level of about 4 adducts in 10 nucleotides (S/N ~30).
CONCLUSIONS
CAX-Prelabeling is an emerging new technique for DNA adductomics, providing polar DNA adductomics in a practical way for the first time. Further study of the method is encouraged to better characterize and extend its performance, especially in scope and sensitivity.
Topics: Animals; Benzo(a)pyrene; Benzyl Compounds; Cations; Cattle; Chromatography, High Pressure Liquid; DNA Adducts; Ethylamines; Guanine; Humans; Nucleotides; Phosphorus Radioisotopes; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Uracil
PubMed: 33821547
DOI: 10.1002/rcm.9095 -
Molecules (Basel, Switzerland) Aug 2019DNA polymerase (pol) kappa is a Y-family translesion DNA polymerase conserved throughout all domains of life. Pol kappa is special6 ized for the ability to copy DNA... (Review)
Review
DNA polymerase (pol) kappa is a Y-family translesion DNA polymerase conserved throughout all domains of life. Pol kappa is special6 ized for the ability to copy DNA containing minor groove DNA adducts, especially -dG adducts, as well as to extend primer termini containing DNA damage or mismatched base pairs. Pol kappa generally cannot copy DNA containing major groove modifications or UV-induced photoproducts. Pol kappa can also copy structured or non-B-form DNA, such as microsatellite DNA, common fragile sites, and DNA containing G quadruplexes. Thus, pol kappa has roles both in maintaining and compromising genomic integrity. The expression of pol kappa is altered in several different cancer types, which can lead to genome instability. In addition, many cancer-associated single-nucleotide polymorphisms have been reported in the gene, some of which are associated with poor survival and altered chemotherapy response. Because of this, identifying inhibitors of pol kappa is an active area of research. This review will address these activities of pol kappa, with a focus on lesion bypass and cellular mutagenesis.
Topics: DNA Adducts; DNA Damage; DNA Replication; DNA-Directed DNA Polymerase; G-Quadruplexes; Humans; Mutagenesis
PubMed: 31374881
DOI: 10.3390/molecules24152805 -
Photochemistry and Photobiology Sep 2020Psoralen is a furocoumarin natural product that intercalates within DNA and forms covalent adducts when activated by ultraviolet radiation. It is well known that this...
Psoralen is a furocoumarin natural product that intercalates within DNA and forms covalent adducts when activated by ultraviolet radiation. It is well known that this property contributes to psoralen's clinical efficacy in several disease contexts, which include vitiligo, psoriasis, graft-versus-host disease and cutaneous T-cell lymphoma. Given the therapeutic relevance of psoralen and its derivatives, we attempted to synthesize psoralens with even greater potency. In this study, we report a library of 73 novel psoralens, the largest collection of its kind. When screened for the ability to reduce cell proliferation, we identified two derivatives even more cytotoxic than 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT), one of the most potent psoralens identified to date. Using MALDI-TOF MS, we studied the DNA adduct formation for a subset of novel psoralens and found that in most cases enhanced DNA binding correlated well with cytotoxicity. Generally, our most potent derivatives contain positively charged substituents, which we believe increase DNA affinity and enhance psoralen intercalation. Thus, we provide a rational approach to guide efforts toward further optimizing psoralens to fully capitalize on this drug class' therapeutic potential. Finally, the structure-activity insights we have gained shed light on several opportunities to study currently underappreciated aspects of psoralen's mechanism.
Topics: Animals; Cell Line, Tumor; DNA; DNA Adducts; Furocoumarins; Mice; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Structure-Activity Relationship; Ultraviolet Rays
PubMed: 32221980
DOI: 10.1111/php.13263 -
Cancer Science Jan 2021Chemical carcinogenesis is focused on the formation of DNA adducts, a form of DNA damage caused by covalent binding of a chemical moiety to DNA. The detection of... (Review)
Review
Chemical carcinogenesis is focused on the formation of DNA adducts, a form of DNA damage caused by covalent binding of a chemical moiety to DNA. The detection of carcinogen-DNA adducts in human tissues, along with demonstration of mutagenicity/carcinogenicity in experimental systems, and validation of adducts as biomarkers of environmental exposure and indicators of cancer risk in molecular epidemiological studies suggests a pivotal role of DNA adducts in cancer development. However, accurate measurement of DNA adducts in varied biological samples is challenging. Advances in mass spectrometry have prompted the development of DNA adductome analysis, an emerging method that simultaneously screens for multiple DNA adducts and provides relevant structural information. In this review, we summarize the basic principle and applications of DNA adductome analysis that would contribute to the elucidation of the environmental causes of cancer. Based on parallel developments in several fields, including next-generation sequencing, we describe a new approach used to explore cancer etiology, which integrates analyses of DNA adductome data and mutational signatures derived from whole-genome/exome sequencing.
Topics: Animals; DNA; DNA Adducts; DNA Damage; Environmental Exposure; Humans; Mutation; Neoplasms
PubMed: 32978845
DOI: 10.1111/cas.14666