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DNA Repair Feb 2021Genome integrity is essential for life and, as a result, DNA repair systems evolved to remove unavoidable DNA lesions from cellular DNA. Many forms of life possess the... (Review)
Review
Genome integrity is essential for life and, as a result, DNA repair systems evolved to remove unavoidable DNA lesions from cellular DNA. Many forms of life possess the capacity to remove interstrand DNA cross-links (ICLs) from their genome but the identity of the naturally-occurring, endogenous substrates that drove the evolution and retention of these DNA repair systems across a wide range of life forms remains uncertain. In this review, we describe more than a dozen chemical processes by which endogenous ICLs plausibly can be introduced into cellular DNA. The majority involve DNA degradation processes that introduce aldehyde residues into the double helix or reactions of DNA with endogenous low molecular weight aldehyde metabolites. A smaller number of the cross-linking processes involve reactions of DNA radicals generated by oxidation.
Topics: Animals; DNA Adducts; DNA Repair; Humans
PubMed: 33385969
DOI: 10.1016/j.dnarep.2020.103029 -
Basic & Clinical Pharmacology &... Sep 2017DNA-protein cross-links (DPCs) are unusually bulky DNA adducts that form in cells as a result of exposure to endogenous and exogenous agents including reactive oxygen... (Review)
Review
DNA-protein cross-links (DPCs) are unusually bulky DNA adducts that form in cells as a result of exposure to endogenous and exogenous agents including reactive oxygen species, ultraviolet light, ionizing radiation, environmental agents (e.g. transition metals, formaldehyde, 1,2-dibromoethane, 1,3-butadiene) and common chemotherapeutic agents. Covalent DPCs are cytotoxic and mutagenic due to their ability to interfere with faithful DNA replication and to prevent accurate gene expression. Key to our understanding of the biological significance of DPC formation is identifying the proteins most susceptible to forming these unusually bulky and complex lesions and quantifying the extent of DNA-protein cross-linking in cells and tissues. Recent advances in bottom-up mass spectrometry-based proteomics have allowed for an unbiased assessment of the whole protein DPC adductome after in vitro and in vivo exposures to cross-linking agents. This MiniReview summarizes current and emerging methods for DPC isolation and analysis by mass spectrometry-based proteomics. We also highlight several examples of successful applications of these novel methodologies to studies of DPC lesions induced by bis-electrophiles such as formaldehyde, 1,2,3,4-diepoxybutane, nitrogen mustards and cisplatin.
Topics: Cisplatin; Cross-Linking Reagents; DNA; DNA Adducts; DNA Damage; Environmental Exposure; Epoxy Compounds; Formaldehyde; Mass Spectrometry; Mutagenicity Tests; Mutagens; Nitrogen Mustard Compounds; Proteins; Proteomics
PubMed: 28032943
DOI: 10.1111/bcpt.12751 -
Analytical Chemistry Jan 2018
Review
Topics: Alkylating Agents; Amines; Animals; Chemistry Techniques, Analytical; DNA; DNA Adducts; DNA Damage; Heterocyclic Compounds; Humans; Reactive Oxygen Species
PubMed: 29084424
DOI: 10.1021/acs.analchem.7b04247 -
Nucleic Acids Research Nov 2023DNA damage causes genomic instability underlying many diseases, with traditional analytical approaches providing minimal insight into the spectrum of DNA lesions in...
DNA damage causes genomic instability underlying many diseases, with traditional analytical approaches providing minimal insight into the spectrum of DNA lesions in vivo. Here we used untargeted chromatography-coupled tandem mass spectrometry-based adductomics (LC-MS/MS) to begin to define the landscape of DNA modifications in rat and human tissues. A basis set of 114 putative DNA adducts was identified in heart, liver, brain, and kidney in 1-26-month-old rats and 111 in human heart and brain by 'stepped MRM' LC-MS/MS. Subsequent targeted analysis of these species revealed species-, tissue-, age- and sex-biases. Structural characterization of 10 selected adductomic signals as known DNA modifications validated the method and established confidence in the DNA origins of the signals. Along with strong tissue biases, we observed significant age-dependence for 36 adducts, including N2-CMdG, 5-HMdC and 8-Oxo-dG in rats and 1,N6-ϵdA in human heart, as well as sex biases for 67 adducts in rat tissues. These results demonstrate the potential of adductomics for discovering the true spectrum of disease-driving DNA adducts. Our dataset of 114 putative adducts serves as a resource for characterizing dozens of new forms of DNA damage, defining mechanisms of their formation and repair, and developing them as biomarkers of aging and disease.
Topics: Animals; Female; Humans; Male; Rats; Chromatography, Liquid; DNA; DNA Adducts; Rodentia; Tandem Mass Spectrometry
PubMed: 37843128
DOI: 10.1093/nar/gkad822 -
Advances in Pharmacology (San Diego,... 2023Hexavalent chromium is a firmly established human carcinogen with documented exposures in many professional groups. Environmental exposure to Cr(VI) is also a...
Hexavalent chromium is a firmly established human carcinogen with documented exposures in many professional groups. Environmental exposure to Cr(VI) is also a significant public health concern. Cr(VI) exists in aqueous solutions as chromate anion that is unreactive with DNA and requires reductive activation inside the cells to produce genotoxic and mutagenic effects. Reduction of Cr(VI) in cells is nonenzymatic and in vivo principally driven by ascorbate with a secondary contribution from nonprotein thiols glutathione and cysteine. In addition to its much faster rate of reduction, ascorbate-driven metabolism avoids the formation of Cr(V) which is the first intermediate in Cr(VI) reduction by thiols. The end-product of Cr(VI) reduction is Cr(III) which forms several types of Cr-DNA adducts that are collectively responsible for all mutagenic and genotoxic effects in Cr(VI) reactions with ascorbate and thiols. Some Cr(V) forms can react with HO to produce DNA-oxidizing peroxo species although this genotoxic pathway is suppressed in cells with physiological levels of ascorbate. Chemical reactions of Cr(VI) with ascorbate or thiols lack directly DNA-oxidizing metabolites. The formation of oxidative DNA breaks in early studies of these reactions was caused by iron contamination. Production of Cr(III)-DNA adducts in cells showed linear dose-dependence irrespective of the predominant reduction pathway and their processing by mismatch repair generated more toxic secondary genetic lesions in euchromatin. Overall, Cr(III)-DNA adduction is the dominant pathway for the formation of genotoxic and mutagenic DNA damage by carcinogenic Cr(VI).
Topics: Humans; Carcinogens; DNA Adducts; Hydrogen Peroxide; DNA Damage; Ascorbic Acid
PubMed: 36858775
DOI: 10.1016/bs.apha.2022.07.003 -
Nucleic Acids Research Mar 2022Two families of DNA glycosylases (YtkR2/AlkD, AlkZ/YcaQ) have been found to remove bulky and crosslinking DNA adducts produced by bacterial natural products. Whether DNA...
Two families of DNA glycosylases (YtkR2/AlkD, AlkZ/YcaQ) have been found to remove bulky and crosslinking DNA adducts produced by bacterial natural products. Whether DNA glycosylases eliminate other types of damage formed by structurally diverse antibiotics is unknown. Here, we identify four DNA glycosylases-TxnU2, TxnU4, LldU1 and LldU5-important for biosynthesis of the aromatic polyketide antibiotics trioxacarcin A (TXNA) and LL-D49194 (LLD), and show that the enzymes provide self-resistance to the producing strains by excising the intercalated guanine adducts of TXNA and LLD. These enzymes are highly specific for TXNA/LLD-DNA lesions and have no activity toward other, less stable alkylguanines as previously described for YtkR2/AlkD and AlkZ/YcaQ. Similarly, TXNA-DNA adducts are not excised by other alkylpurine DNA glycosylases. TxnU4 and LldU1 possess unique active site motifs that provide an explanation for their tight substrate specificity. Moreover, we show that abasic (AP) sites generated from TxnU4 excision of intercalated TXNA-DNA adducts are incised by AP endonuclease less efficiently than those formed by 7mG excision. This work characterizes a distinct class of DNA glycosylase acting on intercalated DNA adducts and furthers our understanding of specific DNA repair self-resistance activities within antibiotic producers of structurally diverse, highly functionalized DNA damaging agents.
Topics: Aminoglycosides; Anti-Bacterial Agents; DNA Adducts; DNA Damage; DNA Glycosylases; DNA Repair
PubMed: 35191495
DOI: 10.1093/nar/gkac085 -
Basic & Clinical Pharmacology &... Sep 2017Human beings are exposed to many reactive electrophiles, both formed endogenously and from exogenous exposures. Such compounds could react with cellular biomolecules and... (Review)
Review
Human beings are exposed to many reactive electrophiles, both formed endogenously and from exogenous exposures. Such compounds could react with cellular biomolecules and form stable reaction products, adducts, at nucleophilic sites in proteins and DNA, constituting a risk for toxic effects. Adductomic approaches aim to study the totality of adducts, to specific biomolecules, by mass spectrometric screening. This Mini-Review focuses on the development and application of an adductomic approach for the screening of unknown adducts to N-terminal valine (Val) in haemoglobin (Hb) by liquid chromatography tandem mass spectrometry (LC-MS/MS). The approach is based on the FIRE procedure, a modified Edman procedure for the analysis of adducts to N-terminal Val in Hb by LC-MS/MS. In the first application of the approach, samples from 12 smokers/non-smokers were screened for Hb adducts, and six previously identified adducts and 20 unknown adducts were detected. To confirm the observation of the detected unknown adducts, targeted screenings were performed in larger sets of blood samples (n = 50-120) from human cohorts. The majority of the previously detected unknown adducts was found in all analysed samples, with large interindividual variations in adduct levels. For structural identification of unknown adducts, a strategy using adductome LC-MS/MS data was formulated and applied. Six identified adducts correspond to ethylation and the precursor electrophiles ethyl vinyl ketone, glyoxal, methylglyoxal, acrylic acid and 1-octen-3-one. The observation of these adducts in human blood motivate further studies to evaluate possible contributions to health risks, as well as their potential as biomarkers of exposure.
Topics: Aldehydes; Biomarkers; Chromatography, Liquid; DNA Adducts; Environmental Exposure; Epoxy Compounds; Hemoglobins; Humans; Nitrosamines; Tandem Mass Spectrometry; Valine
PubMed: 27889941
DOI: 10.1111/bcpt.12715 -
Chemical Research in Toxicology Apr 2021The tobacco-specific nitrosamines '-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are considered to be two of the most important...
The tobacco-specific nitrosamines '-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are considered to be two of the most important carcinogens in unburned tobacco and its smoke. They readily cause tumors in laboratory animals and are classified as "carcinogenic to humans" by the International Agency for Research on Cancer. DNA adduct formation by these two carcinogens is believed to play a critical role in tobacco carcinogenesis. Among all the DNA adducts formed by NNN and NNK, 2'-deoxyadenosine (dAdo)-derived adducts have not been fully characterized. In the study reported here, we characterized the formation of -[4-(3-pyridyl)-4-oxo-1-butyl]-2'-deoxyadenosine (-POB-dAdo) and its reduced form -PHB-dAdo formed by NNN 2'-hydroxylation in rat liver and lung DNA. More importantly, we characterized a new dAdo adduct -[4-hydroxy-1-(pyridine-3-yl)butyl]-2'-deoxyadenosine (-HPB-dAdo) formed after NaBHCN or NaBH reduction both in calf thymus DNA reacted with 5'-acetoxy-'-nitrosonornicotine and in rat liver and lung upon treatment with NNN. This adduct was specifically formed by NNN 5'-hydroxylation. Chemical standards of -HPB-dAdo and the corresponding isotopically labeled internal standard [pyridine-]-HPB-dAdo were synthesized using a four-step method. Both NMR and high-resolution mass spectrometry data agreed well with the proposed structure of -HPB-dAdo. The new adduct coeluted with the synthesized internal standard under various LC conditions. Its product ion patterns of MS and MS transitions were also consistent with the proposed fragmentation patterns. Chromatographic resolution of the two diastereomers of -HPB-dAdo was successfully achieved. Quantitation suggested a dose-dependent response of the levels of this new adduct in the liver and lung of rats treated with NNN. However, its level was lower than that of 2-[2-(3-pyridyl)--pyrrolidinyl]-2'-deoxyinosine, a previously reported dGuo adduct that is also formed from NNN 5'-hydroxylation. The identification of -HPB-dAdo in this study leads to new insights pertinent to the mechanism of carcinogenesis by NNN and to the development of biomarkers of NNN metabolic activation.
Topics: Animals; DNA; DNA Adducts; Deoxyadenosines; Liver; Lung; Molecular Structure; Nitrosamines; Prohibitins; Rats
PubMed: 33705110
DOI: 10.1021/acs.chemrestox.1c00013 -
Redox Biology Aug 2016The accurate and sensitive detection of biological free radicals in a reliable manner is required to define the mechanistic roles of such species in biochemistry,... (Review)
Review
The accurate and sensitive detection of biological free radicals in a reliable manner is required to define the mechanistic roles of such species in biochemistry, medicine and toxicology. Most of the techniques currently available are either not appropriate to detect free radicals in cells and tissues due to sensitivity limitations (electron spin resonance, ESR) or subject to artifacts that make the validity of the results questionable (fluorescent probe-based analysis). The development of the immuno-spin trapping technique overcomes all these difficulties. This technique is based on the reaction of amino acid- and DNA base-derived radicals with the spin trap 5, 5-dimethyl-1-pyrroline N-oxide (DMPO) to form protein- and DNA-DMPO nitroxide radical adducts, respectively. These adducts have limited stability and decay to produce the very stable macromolecule-DMPO-nitrone product. This stable product can be detected by mass spectrometry, NMR or immunochemistry by the use of anti-DMPO nitrone antibodies. The formation of macromolecule-DMPO-nitrone adducts is based on the selective reaction of free radical addition to the spin trap and is thus not subject to artifacts frequently encountered with other methods for free radical detection. The selectivity of spin trapping for free radicals in biological systems has been proven by ESR. Immuno-spin trapping is proving to be a potent, sensitive (a million times higher sensitivity than ESR), and easy (not quantum mechanical) method to detect low levels of macromolecule-derived radicals produced in vitro and in vivo. Anti-DMPO antibodies have been used to determine the distribution of free radicals in cells and tissues and even in living animals. In summary, the invention of the immuno-spin trapping technique has had a major impact on the ability to accurately and sensitively detect biological free radicals and, subsequently, on our understanding of the role of free radicals in biochemistry, medicine and toxicology.
Topics: DNA Adducts; Electron Spin Resonance Spectroscopy; Free Radicals; Nitrogen Oxides; Organelles; Proteins; Spin Trapping
PubMed: 27203617
DOI: 10.1016/j.redox.2016.04.003 -
International Journal of Molecular... Dec 2022Organoids are 3D cultures that to some extent reproduce the structure, composition and function of the mammalian tissues from which they derive, thereby creating in...
Organoids are 3D cultures that to some extent reproduce the structure, composition and function of the mammalian tissues from which they derive, thereby creating in vitro systems with more in vivo-like characteristics than 2D monocultures. Here, the ability of human organoids derived from normal gastric, pancreas, liver, colon and kidney tissues to metabolise the environmental carcinogen benzo[]pyrene (BaP) was investigated. While organoids from the different tissues showed varied cytotoxic responses to BaP, with gastric and colon organoids being the most susceptible, the xenobiotic-metabolising enzyme (XME) genes, and , were highly upregulated in all organoid types, with kidney organoids having the highest levels. Furthermore, the presence of two key metabolites, BaP--7,8-dihydrodiol and BaP-tetrol-l-1, was detected in all organoid types, confirming their ability to metabolise BaP. BaP bioactivation was confirmed both by the activation of the DNA damage response pathway (induction of p-p53, pCHK2, p21 and γ-H2AX) and by DNA adduct formation. Overall, pancreatic and undifferentiated liver organoids formed the highest levels of DNA adducts. Colon organoids had the lowest responses in DNA adduct and metabolite formation, as well as XME expression. Additionally, high-throughput RT-qPCR explored differences in gene expression between organoid types after BaP treatment. The results demonstrate the potential usefulness of organoids for studying environmental carcinogenesis and genetic toxicology.
Topics: Humans; Activation, Metabolic; Benzo(a)pyrene; Cytochrome P-450 CYP1A1; DNA Adducts; Liver; Organoids
PubMed: 36614051
DOI: 10.3390/ijms24010606