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DNA Repair Jan 2019A variety of agents cause DNA base alkylation damage, including the known hepatocarcinogen aflatoxin B (AFB) and chemotherapeutic drugs derived from nitrogen mustard...
A variety of agents cause DNA base alkylation damage, including the known hepatocarcinogen aflatoxin B (AFB) and chemotherapeutic drugs derived from nitrogen mustard (NM). The N7 site of guanine is the primary site of alkylation, with some N7-deoxyguanosine adducts undergoing imidazole ring-opening to stable mutagenic N-alkyl formamidopyrimidine (Fapy-dG) adducts. These adducts exist as a mixture of canonical β- and unnatural α-anomeric forms. The β species are predominant in double-stranded (ds) DNA. Recently, we have demonstrated that the DNA glycosylase NEIL1 can initiate repair of AFB-Fapy-dG adducts both in vitro and in vivo, with Neil1 mice showing an increased susceptibility to AFB-induced hepatocellular carcinoma. Here, we hypothesized that NEIL1 could excise NM-Fapy-dG and that NEIL3, a closely related DNA glycosylase, could excise both NM-Fapy-dG and AFB-Fapy-dG. Product formation from the reaction of human NEIL1 with ds oligodeoxynucleotides containing a unique NM-Fapy-dG followed a bi-component exponential function under single turnover conditions. Thus, two adduct conformations were differentially recognized by hNEIL1. The excision rate of the major form (∼13.0 min), presumed to be the β-anomer, was significantly higher than that previously reported for 5-hydroxycytosine, 5-hydroxyuracil, thymine glycol (Tg), and AFB-Fapy-dG. Product generation from the minor form was much slower (∼0.4 min), likely reflecting the rate of conversion of the α anomer into the β anomer. Mus musculus NEIL3 (MmuNEIL3Δ324) excised NM-Fapy-dG from single-stranded (ss) DNA (turnover rate of ∼0.4 min), but not from ds DNA. Product formation from ss substrate was incomplete, presumably because of a substantial presence of the α anomer. MmuNEIL3Δ324 could not initiate repair of AFB-Fapy-dG in either ds or ss DNA. Overall, the data suggest that both NEIL1 and NEIL3 may protect cells against cytotoxic and mutagenic effects of NM-Fapy-dG, but NEIL1 may have a unique role in initiation of base excision repair of AFB-Fapy-dG.
Topics: Animals; DNA Adducts; DNA Glycosylases; Mice; N-Glycosyl Hydrolases; Pyrimidines
PubMed: 30448017
DOI: 10.1016/j.dnarep.2018.11.001 -
Archives of Toxicology Feb 2019N-nitroso compounds are alkylating agents, which are widespread in our diet and the environment. They induce DNA alkylation adducts such as O-methylguanine (O-MeG),...
N-nitroso compounds are alkylating agents, which are widespread in our diet and the environment. They induce DNA alkylation adducts such as O-methylguanine (O-MeG), which is repaired by O-methylguanine-DNA methyltransferase (MGMT). Persistent O-MeG lesions have detrimental biological consequences like mutagenicity and cytotoxicity. Due to its pivotal role in the etiology of cancer and in cytotoxic cancer therapy, it is important to detect and quantify O-MeG in biological specimens in a sensitive and accurate manner. Here, we used immunological approaches and established an ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to monitor O-MeG adducts. First, colorectal cancer (CRC) cells were treated with the methylating anticancer drug temozolomide (TMZ). Immunofluorescence microscopy and an immuno-slot blot assay, both based on an adduct-specific antibody, allowed for the semi-quantitative, dose-dependent assessment of O-MeG in CRC cells. Using the highly sensitive and specific UPLC-MS/MS, TMZ-induced O-MeG adducts were quantified in CRC cells and even in peripheral blood mononuclear cells exposed to clinically relevant TMZ doses. Furthermore, all methodologies were used to detect O-MeG in wildtype (WT) and MGMT-deficient mice challenged with the carcinogen azoxymethane. UPLC-MS/MS measurements and dose-response modeling revealed a non-linear formation of hepatic and colonic O-MeG adducts in WT, whereas linear O-MeG formation without a threshold was observed in MGMT-deficient mice. Collectively, the UPLC-MS/MS analysis is highly sensitive and specific for O-MeG, thereby allowing for the first time for the determination of a genotoxic threshold upon exposure to O-methylating agents. We envision that this method will be instrumental to monitor the efficacy of methylating chemotherapy and to assess dietary exposures.
Topics: Animals; Antineoplastic Agents, Alkylating; Azoxymethane; Chromatography, Liquid; DNA Adducts; DNA Modification Methylases; DNA Repair Enzymes; Dose-Response Relationship, Drug; Guanine; HCT116 Cells; Humans; Immunoblotting; Leukocytes, Mononuclear; Mice, Inbred C57BL; Mice, Mutant Strains; Microscopy, Fluorescence; Sensitivity and Specificity; Tandem Mass Spectrometry; Temozolomide; Tumor Suppressor Proteins
PubMed: 30446773
DOI: 10.1007/s00204-018-2355-0 -
Chemistry (Weinheim An Der Bergstrasse,... Oct 2021To study the DNA damage caused by a potent platinum-acridine anticancer agent (PA) in cancer cells, an assay based on biorthogonal post-labeling using a click...
DNA Adduct Detection after Post-Labeling Technique with PCR Amplification (DNA-ADAPT-qPCR) Identifies the Pre-ribosomal RNA Gene as a Direct Target of Platinum-Acridine Anticancer Agents.
To study the DNA damage caused by a potent platinum-acridine anticancer agent (PA) in cancer cells, an assay based on biorthogonal post-labeling using a click chemistry-enabled, azide-modified derivative (APA) was developed. The method involves biotinylation, affinity capture, and bead-based enrichment of APA-modified genomic DNA. The key steps of the assay were validated and optimized in model duplexes, including full-length plasmids, restriction fragments, and a DNA ladder. Native DNA treated with APA and subsequently subjected to post-labeling with a biotin affinity tag was enzymatically digested and fragments were analyzed by in-line LC-MS and MS/MS. The monofunctional-intercalative adducts formed by APA in 5'-pyrimidine/guanine sequences in double-stranded DNA were quantitatively biotinylated by strain-promoted 1,3-dipolar cycloaddition chemistry. When applied to DNA extracted from A549 lung cancer cells, the assay in combination with qPCR amplification demonstrates that platinum-acridines form adducts in the gene sequences encoding pre-ribosomal RNA, a potential pharmacological target of these agents.
Topics: Acridines; Antineoplastic Agents; DNA; DNA Adducts; Genes, rRNA; Platinum; Polymerase Chain Reaction; Tandem Mass Spectrometry
PubMed: 34375484
DOI: 10.1002/chem.202102263 -
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 -
Food and Chemical Toxicology : An... Jul 2019Formation of DNA adducts by five alkenylbenzenes, safrole, methyl eugenol, eugenol, and asarone with either α- or β-conformation, was analyzed in fetal avian livers in...
Formation of DNA adducts by five alkenylbenzenes, safrole, methyl eugenol, eugenol, and asarone with either α- or β-conformation, was analyzed in fetal avian livers in two in ovo models. DNA reactivity of the carcinogens safrole and methyl eugenol was previously demonstrated in the turkey egg model, whereas non-genotoxic eugenol was negative. In the current study, alkenylbenzenes were also tested in the chicken egg model. Injections with alkenylbenzenes were administered to fertilized turkey or chicken eggs for three consecutive days. Three hours after the last injection, liver samples were evaluated for DNA adduct formation using the P-nucleotide postlabeling assay. DNA samples from turkey livers were also analyzed for adducts using mass spectrometry. In both species, genotoxic alkenylbenzenes safrole, methyl eugenol, α- and β-asarone produced DNA adducts, the presence and nature of which, with exception of safrole, were confirmed by mass spectrometry, validating the sensitivity of the P-postlabeling assay. Overall, the results of testing were congruent between fetal turkey and chicken livers, confirming that these organisms can be used interchangeably. Moreover, data obtained in both models is comparable to genotoxicity findings in other species, supporting the usefulness of avian models for the assessment of genotoxicity as a potential alternative to animal models.
Topics: Animals; Benzene Derivatives; Chickens; DNA Adducts; Fetus; Liver; Structure-Activity Relationship; Turkeys
PubMed: 31077736
DOI: 10.1016/j.fct.2019.05.010 -
The Journal of Organic Chemistry Jan 2016N(2)-Furfuryl-deoxyguanosine (fdG) is carcinogenic DNA adduct that originates from furfuryl alcohol. It is also a stable structural mimic of the damage induced by the...
N(2)-Furfuryl-deoxyguanosine (fdG) is carcinogenic DNA adduct that originates from furfuryl alcohol. It is also a stable structural mimic of the damage induced by the nitrofurazone family of antibiotics. For the structural and functional studies of this model N(2)-dG adduct, reliable and rapid access to fdG-modified DNAs are warranted. Toward this end, here we report the synthesis of fdG-modified DNAs using phosphoramidite chemistry involving only three steps. The functional integrity of the modified DNA has been verified by primer extension studies with DNA polymerases I and IV from E. coli. Introduction of fdG into a DNA duplex decreases the Tm by ∼1.6 °C/modification. Molecular dynamics simulations of a DNA duplex bearing the fdG adduct revealed that though the overall B-DNA structure is maintained, this lesion can disrupt W-C H-bonding, stacking interactions, and minor groove hydrations to some extent at the modified site, and these effects lead to slight variations in the local base pair parameters. Overall, our studies show that fdG is tolerated at the minor groove of the DNA to a better extent compared with other bulky DNA damages, and this property will make it difficult for the DNA repair pathways to detect this adduct.
Topics: Base Pairing; DNA; DNA Adducts; DNA, B-Form; Deoxyguanosine; Escherichia coli; Hydrogen Bonding; Magnetic Resonance Spectroscopy; Molecular Dynamics Simulation
PubMed: 26650891
DOI: 10.1021/acs.joc.5b02341 -
Toxins May 2022Pyrrolizidine alkaloids (PAs) have been found in over 6000 plants worldwide and represent the most common hepatotoxic phytotoxins. Catalyzed by hepatic cytochrome P450...
Pyrrolizidine alkaloids (PAs) have been found in over 6000 plants worldwide and represent the most common hepatotoxic phytotoxins. Catalyzed by hepatic cytochrome P450 enzymes, PAs are metabolized into reactive pyrrolic metabolites, which can alkylate cellular proteins and DNA to form pyrrole-protein adducts and pyrrole-DNA adducts, leading to cytotoxicity, genotoxicity, and tumorigenicity. To date, the correlation between these PA-derived pyrrole-protein and pyrrole-DNA adducts has not been well investigated. Retrorsine is a representative hepatotoxic and carcinogenic PA. In the present study, the correlations among the PA-derived liver DNA adducts, liver protein adducts, and serum protein adducts in retrorsine-treated mice under different dosage regimens were studied. The results showed positive correlations among these adducts, in which serum pyrrole-protein adducts were more accessible and present in higher abundance, and thus could be used as a suitable surrogate biomarker for pyrrole-DNA adducts to indicate the genetic or carcinogenic risk posed by retrorsine.
Topics: Animals; Carcinogens; DNA; DNA Adducts; Liver; Male; Mice; Mice, Inbred ICR; Proteins; Pyrroles; Pyrrolizidine Alkaloids
PubMed: 35737038
DOI: 10.3390/toxins14060377 -
Lung Cancer (Amsterdam, Netherlands) Jul 2015In this exploratory study, we aimed to investigate whether polymorphisms in excision repair cross-complementing group 1 (ERCC1) and excision repair cross-complementing...
OBJECTIVES
In this exploratory study, we aimed to investigate whether polymorphisms in excision repair cross-complementing group 1 (ERCC1) and excision repair cross-complementing group 2/xeroderma pigmentosum group D (ERCC2/XPD) in the nucleotide excision repair (NER) pathways associated with DNA adducts in human lung tissue. We also analyzed the association stratified by the major histologic subtypes of non-small cell lung cancer (NSCLC): adenocarcinoma (ADC) and squamous cell carcinoma (SQCC).
METHODS
The study population consisted of 107 early stage NSCLC patients from the Massachusetts General Hospital (MGH) in Boston who underwent curative surgical resection. Genotyping was completed for SNPs in ERCC1 [C8092A (rs3212986) and C118T (rs11615)] and ERCC2/XPD [Asp312Asn (rs1799793) and Lys751Gln (rs1052559)] using a PCR-RFLP method and the PCR with fluorescent allele-specific oligonucleotide probes (Taqman). DNA adduct levels were measured as relative adduct levels per 10(10) nucleotides by (32)P-postlabeling in non-tumor lung tissue.
RESULTS
After adjusting for potential confounders, lung DNA adduct levels increased by 103.2% [95% confidence interval (CI), -11.5 to 366.6] for ERCC2/XPD rs1799793AA genotype compared with their corresponding wild type homozygous genotypes in overall NSCLC, but the difference did not reach statistical significance. When we stratified by the subtypes of NSCLC, we found that DNA adducts levels in lung increased by 204.9% (95% CI, 0.8 to 822.2, P=0.059) for ERCC2/XPD rs1799793AA genotype in subjects with SQCC and the trend was statistically significant (P for trend=0.0489).
CONCLUSIONS
Polymorphisms in ERCC2/XPD Asp312Asn may be associated with increased DNA adduct levels in the lung, especially among subjects with SQCC. Further large scale studies are needed to confirm our findings.
Topics: Adenocarcinoma; Aged; Carcinoma, Non-Small-Cell Lung; Carcinoma, Squamous Cell; DNA Adducts; DNA Repair; DNA-Binding Proteins; Endonucleases; Female; Genotype; Humans; Lung Neoplasms; Male; Middle Aged; Polymorphism, Single Nucleotide; Xeroderma Pigmentosum Group D Protein
PubMed: 26001533
DOI: 10.1016/j.lungcan.2015.05.001 -
Molecular Nutrition & Food Research Sep 2014Epidemiological and clinical studies have demonstrated that the consumption of red haem-rich meat may contribute to the risk of colorectal cancer. Two hypotheses have...
SCOPE
Epidemiological and clinical studies have demonstrated that the consumption of red haem-rich meat may contribute to the risk of colorectal cancer. Two hypotheses have been put forward to explain this causal relationship, i.e. N-nitroso compound (NOC) formation and lipid peroxidation (LPO).
METHODS AND RESULTS
In this study, the NOC-derived DNA adduct O(6)-carboxymethylguanine (O(6)-CMG) and the LPO product malondialdehyde (MDA) were measured in individual in vitro gastrointestinal digestions of meat types varying in haem content (beef, pork, chicken). While MDA formation peaked during the in vitro small intestinal digestion, alkylation and concomitant DNA adduct formation was observed in seven (out of 15) individual colonic digestions using separate faecal inocula. From those, two haem-rich meat digestions demonstrated a significantly higher O(6)-CMG formation (p < 0.05). MDA concentrations proved to be positively correlated (p < 0.0004) with haem content of digested meat. The addition of myoglobin, a haem-containing protein, to the digestive simulation showed a dose-response association with O(6)-CMG (p = 0.004) and MDA (p = 0.008) formation.
CONCLUSION
The results suggest the haem-iron involvement for both the LPO and NOC pathway during meat digestion. Moreover, results unambiguously demonstrate that DNA adduct formation is very prone to inter-individual variation, suggesting a person-dependent susceptibility to colorectal cancer development following haem-rich meat consumption.
Topics: Adult; Animals; Caco-2 Cells; Chickens; DNA Adducts; Digestion; Feces; Female; Guanine; Heme; Humans; In Vitro Techniques; Lipid Peroxidation; Male; Malondialdehyde; Meat; Middle Aged; Nitroso Compounds; Swine
PubMed: 24990219
DOI: 10.1002/mnfr.201400078 -
Accounts of Chemical Research Feb 2016The genetic integrity of living organisms is constantly threatened by environmental and endogenous sources of DNA damaging agents that can induce a plethora of... (Review)
Review
The genetic integrity of living organisms is constantly threatened by environmental and endogenous sources of DNA damaging agents that can induce a plethora of chemically modified DNA lesions. Unrepaired DNA lesions may elicit cytotoxic and mutagenic effects and contribute to the development of human diseases including cancer and neurodegeneration. Understanding the deleterious outcomes of DNA damage necessitates the investigation about the effects of DNA adducts on the efficiency and fidelity of DNA replication and transcription. Conventional methods for measuring lesion-induced replicative or transcriptional alterations often require time-consuming colony screening and DNA sequencing procedures. Recently, a series of mass spectrometry (MS)-based strategies have been developed in our laboratory as an efficient platform for qualitative and quantitative analyses of the changes in genetic information induced by DNA adducts during DNA replication and transcription. During the past few years, we have successfully used these MS-based methods for assessing the replicative or transcriptional blocking and miscoding properties of more than 30 distinct DNA adducts. When combined with genetic manipulation, these methods have also been successfully employed for revealing the roles of various DNA repair proteins or translesion synthesis DNA polymerases (Pols) in modulating the adverse effects of DNA lesions on transcription or replication in mammalian and bacterial cells. For instance, we found that Escherichia coli Pol IV and its mammalian ortholog (i.e., Pol κ) are required for error-free bypass of N(2)-(1-carboxyethyl)-2'-deoxyguanosine (N(2)-CEdG) in cells. We also found that the N(2)-CEdG lesions strongly inhibit DNA transcription and they are repaired by transcription-coupled nucleotide excision repair in mammalian cells. In this Account, we focus on the development of MS-based approaches for determining the effects of DNA adducts on DNA replication and transcription, where liquid chromatography-tandem mass spectrometry is employed for the identification, and sometimes quantification, of the progeny products arising from the replication or transcription of lesion-bearing DNA substrates in vitro and in mammalian cells. We also highlight their applications to lesion bypass, mutagenesis, and repair studies of three representative types of DNA lesions, that is, the methylglyoxal-induced N(2)-CEdG, oxidatively induced 8,5'-cyclopurine-2'-deoxynucleosides, and regioisomeric alkylated thymidine lesions. Specially, we discuss the similar and distinct effects of the minor-groove DNA lesions including N(2)-CEdG and O(2)-alkylated thymidine lesions, as well as the major-groove O(4)-alkylated thymidine lesions on DNA replication and transcription machinery. For example, we found that the addition of an alkyl group to the O(4) position of thymine may facilitate its preferential pairing with guanine and thus induce exclusively the misincorporation of guanine nucleotide opposite the lesion, whereas alkylation of thymine at the O(2) position may render the nucleobase unfavorable in pairing with any of the canonical nucleobases and thus exhibit promiscuous miscoding properties during DNA replication and transcription. The MS-based strategies described herein should be generally applicable for quantitative measurement of the biological consequences and repair of other DNA lesions in vitro and in cells.
Topics: Cell Line; DNA Adducts; DNA Repair; DNA Replication; Escherichia coli; Humans; Mass Spectrometry; Transcription, Genetic
PubMed: 26758048
DOI: 10.1021/acs.accounts.5b00437