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Chemical Research in Toxicology Jan 2017DNA nucleobases are the prime targets for chemical modifications by endogenous and exogenous electrophiles. Alkylation of the N7 position of guanine and adenine in DNA...
DNA nucleobases are the prime targets for chemical modifications by endogenous and exogenous electrophiles. Alkylation of the N7 position of guanine and adenine in DNA triggers base-catalyzed imidazole ring opening and the formation of N-substituted formamidopyrimidine (N-R-FAPy) lesions. Me-FAPy-dG adducts induced by exposure to methylating agents and AFB-FAPy-dG lesions formed by aflatoxin B have been shown to persist in cells and to contribute to toxicity and mutagenicity. In contrast, the biological outcomes of other N-substituted FAPy lesions have not been fully elucidated. To enable their structural and biological evaluation, N-R-FAPy adducts must be site-specifically incorporated into synthetic DNA strands using phosphoramidite building blocks, which can be complicated by their unusual structural complexity. N-R-FAPy exist as a mixture of rotamers and can undergo isomerization between α, β anomers and furanose-pyranose forms. In this Perspective, we will discuss the main types of N-R-FAPy adducts and summarize the strategies for their synthesis and structural elucidation. We will also summarize the chemical biology studies conducted with N-R-FAPy-containing DNA to elucidate their effects on DNA replication and to identify the mechanisms of N-R-FAPy repair.
Topics: Animals; DNA Adducts; DNA Repair; Formamides; Humans; Oligodeoxyribonucleotides; Pyrimidines
PubMed: 27959490
DOI: 10.1021/acs.chemrestox.6b00392 -
The Journal of Biological Chemistry May 2020Platinum-based chemotherapies, including oxaliplatin, are a mainstay in the management of solid tumors and induce cell death by forming intrastrand dinucleotide DNA...
Platinum-based chemotherapies, including oxaliplatin, are a mainstay in the management of solid tumors and induce cell death by forming intrastrand dinucleotide DNA adducts. Despite their common use, they are highly toxic, and approximately half of cancer patients have tumors that are either intrinsically resistant or develop resistance. Previous studies suggest that this resistance is mediated by variations in DNA repair levels or net drug influx. Here, we aimed to better define the roles of nucleotide excision repair and DNA damage in platinum chemotherapy resistance by profiling DNA damage and repair efficiency in seven oxaliplatin-sensitive and three oxaliplatin-resistant colorectal cancer cell lines. We assayed DNA repair indirectly as toxicity and directly measured bulky adduct formation and removal from the genome by slot blot and repair capacity in an excision assay, and used excision repair sequencing (XR-seq) to map repair events genome-wide at single-nucleotide resolution. Using this combinatorial approach and proxies for oxaliplatin-DNA damage, we observed no significant differences in repair efficiency that could explain the relative sensitivities and chemotherapy resistances of these cell lines. In contrast, the levels of oxaliplatin-induced DNA damage were significantly lower in the resistant cells, indicating that decreased damage formation, rather than increased damage repair, is a major determinant of oxaliplatin resistance in these cell lines. XR-seq-based analysis of gene expression revealed up-regulation of membrane transport pathways in the resistant cells, and these pathways may contribute to resistance. In conclusion, additional research is needed to characterize the factors mitigating cellular DNA damage formation by platinum compounds.
Topics: Colorectal Neoplasms; DNA Adducts; DNA Damage; DNA Repair; DNA, Neoplasm; Drug Resistance, Neoplasm; HCT116 Cells; Humans; Oxaliplatin
PubMed: 32299912
DOI: 10.1074/jbc.RA120.013347 -
The Journal of Biological Chemistry Feb 2018Acrolein, an α,β-unsaturated aldehyde, is generated as the end product of lipid peroxidation and from metabolic oxidation of polyamines, and it is a ubiquitous... (Comparative Study)
Comparative Study
Acrolein, an α,β-unsaturated aldehyde, is generated as the end product of lipid peroxidation and from metabolic oxidation of polyamines, and it is a ubiquitous environmental pollutant. The reaction of acrolein with the N2 of guanine in DNA leads to the formation of γ-hydroxy-1--propano-2' deoxyguanosine (γ-HOPdG), which can exist in DNA in a ring-closed or a ring-opened form. Here, we identified the translesion synthesis (TLS) DNA polymerases (Pols) that conduct replication through the permanently ring-opened reduced form of γ-HOPdG ((r) γ-HOPdG) and show that replication through this adduct is mediated via Rev1/Polη-, Polι/Polκ-, and Polθ-dependent pathways, respectively. Based on biochemical and structural studies, we propose a role for Rev1 and Polι in inserting a nucleotide (nt) opposite the adduct and for Pols η and κ in extending synthesis from the inserted nt in the respective TLS pathway. Based on genetic analyses and biochemical studies with Polθ, we infer a role for Polθ at both the nt insertion and extension steps of TLS. Whereas purified Rev1 and Polθ primarily incorporate a C opposite (r) γ-HOPdG, Polι incorporates a C or a T opposite the adduct; nevertheless, TLS mediated by the Polι-dependent pathway as well as by other pathways occurs in a predominantly error-free manner in human cells. We discuss the implications of these observations for the mechanisms that could affect the efficiency and fidelity of TLS Pols.
Topics: Acrolein; Amino Acid Substitution; Cell Line; DNA Adducts; DNA Damage; DNA Replication; DNA-Directed DNA Polymerase; Deoxyguanosine; Environmental Pollutants; Humans; Mutagens; Mutation; Nuclear Proteins; Nucleotidyltransferases; Organophosphorus Compounds; Protein Multimerization; RNA Interference; Recombinant Fusion Proteins; Recombinant Proteins; DNA Polymerase iota
PubMed: 29330301
DOI: 10.1074/jbc.RA117.000962 -
Mutagenesis Apr 2021In vitro genotoxicity assays utilising human skin models are becoming important tools for the safety assessment of chemicals whose primary exposure is via the dermal...
In vitro genotoxicity assays utilising human skin models are becoming important tools for the safety assessment of chemicals whose primary exposure is via the dermal route. In order to explore metabolic competency and inducibility of CYP450 activating enzymes, 3D reconstructed human skin tissues were topically treated with 2-acetylaminofluorene (2-AAF) and its genotoxic metabolites, N-hydroxy-2-acetylaminofluorene (N-OH-2-AAF) and N-hydroxy-2-aminofluorene (N-OH-2-AF), which primarily cause DNA damage by forming DNA adducts. 2-AAF did not increase DNA damage measured in the reconstructed skin micronucleus (RSMN) assay when administered in multiple applications at 24 h intervals but was detected in the skin comet assay in the presence of the DNA polymerase inhibitor aphidicolin (APC). Similarly, no increase was found with N-OH-2-AAF in the RSMN assay after multiple treatments whereas a single 3 h exposure to N-OH-2-AAF caused a large dose-related increase in the skin comet assay. A significant increase in the RSMN assay was only obtained with the highly reactive N-OH-2-AF metabolite after multiple treatments over 72 h, whereas N-OH-2-AF caused a strong increase after a single 3 h exposure in the skin comet assay. In support of these results, DNA adduct formation, measured by the 32P-postlabelling assay, was examined. Adduct levels after 2-AAF treatment for 3 h were minimal but increased >10-fold after multiple exposures over 48 h, suggesting that enzyme(s) that metabolise 2-AAF are induced in the skin models. As expected, a single 3 h exposure to N-OH-2-AAF and N-OH-2-AF resulted in adduct levels that were at least 10-fold greater than those after multiple exposures to 2-AAF despite ~100-fold lower tested concentrations. Our results demonstrate that DNA damage caused by 2-AAF metabolites is more efficiently detected in the skin comet assay than the RSMN assay and after multiple exposures and enzyme induction, 2-AAF-induced DNA damage can be detected in the APC-modified comet assay.
Topics: 2-Acetylaminofluorene; Carcinogens; DNA Adducts; DNA Damage; Fluorenes; Humans; Hydroxyacetylaminofluorene; Micronucleus Tests; Mutagens; Skin
PubMed: 31816077
DOI: 10.1093/mutage/gez044 -
Chemical Research in Toxicology Jan 2017The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent pulmonary carcinogen in laboratory animals. It is classified as a Group... (Review)
Review
The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent pulmonary carcinogen in laboratory animals. It is classified as a Group 1 human carcinogen by the International Agency for Cancer Research. NNK is bioactivated upon cytochrome P450 catalyzed hydroxylation of the carbon atoms adjacent to the nitrosamino group to both methylating and pyridyloxobutylating agents. Both pathways generate a spectrum of DNA damage that contributes to the overall mutagenic and toxic properties of this compound. NNK is also reduced to form 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), which is also carcinogenic. Like NNK, NNAL requires metabolic activation to DNA alkylating agents. Methyl hydroxylation of NNAL generates pyridylhydroxybutyl DNA adducts, and methylene hydroxylation leads to DNA methyl adducts. The consequence of this complex metabolism is that NNK generates a vast spectrum of DNA damage, any form of which can contribute to the overall carcinogenic properties of this potent pulmonary carcinogen. This Perspective reviews the chemistry and genotoxic properties of the collection of DNA adducts formed from NNK. In addition, it provides evidence that multiple adducts contribute to the overall carcinogenic properties of this chemical. The adduct that contributes to the genotoxic effects of NNK depends on the context, such as the relative amounts of each DNA alkylating pathway occurring in the model system, the levels and genetic variants of key repair enzymes, and the gene targeted for mutation.
Topics: Animals; DNA; DNA Adducts; Humans; Mutagens; Nitrosamines; Smoke; Nicotiana
PubMed: 28092943
DOI: 10.1021/acs.chemrestox.6b00386 -
Free Radical Biology & Medicine Feb 2020Fuchs Endothelial Corneal Dystrophy (FECD) is an age-related genetically complex disease characterized by increased oxidative DNA damage and progressive degeneration of...
Fuchs Endothelial Corneal Dystrophy (FECD) is an age-related genetically complex disease characterized by increased oxidative DNA damage and progressive degeneration of corneal endothelial cells (HCEnCs). FECD has a greater incidence and advanced phenotype in women, suggesting a possible role of hormones in the sex-driven differences seen in the disease pathogenesis. In this study, catechol estrogen (4-OHE), the byproduct of estrogen metabolism, induced genotoxic estrogen-DNA adducts formation, macromolecular DNA damage, and apoptotic cell death in HCEnCs; these findings were potentiated by menadione (MN)-mediated reactive oxygen species (ROS). Expression of NQO1, a key enzyme that neutralizes reactive estrogen metabolites, was downregulated in FECD, indicating HCEnC susceptibility to reactive estrogen metabolism in FECD. NQO1 deficiency in vitro exacerbated the estrogen-DNA adduct formation and loss of cell viability, which was rescued by the supplementation of N-acetylcysteine, a ROS scavenger. Notably, overexpression of NQO1 in HCEnCs treated with MN and 4-OHE quenched the ROS formation, thereby reducing the DNA damage and endothelial cell loss. This study signifies a pivotal role for NQO1 in mitigating the macromolecular oxidative DNA damage arising from the interplay between intracellular ROS and impaired endogenous estrogen metabolism in post-mitotic ocular tissue cells. A dysfunctional Nrf2-NQO1 axis in FECD renders HCEnCs susceptible to catechol estrogens and estrogen-DNA adducts formation. This novel study highlights the potential role of NQO1-mediated estrogen metabolite genotoxicity in explaining the higher incidence of FECD in females.
Topics: DNA Adducts; DNA Damage; Endothelial Cells; Endothelium, Corneal; Estrogens; Female; Fuchs' Endothelial Dystrophy; Humans; NAD(P)H Dehydrogenase (Quinone)
PubMed: 31857234
DOI: 10.1016/j.freeradbiomed.2019.12.014 -
Chemical Research in Toxicology Jan 2019Dietary exposure to aflatoxin B (AFB) is a significant contributor to the incidence of hepatocellular carcinomas globally. AFB exposure leads to the formation of...
Aflatoxin-Guanine DNA Adducts and Oxidatively Induced DNA Damage in Aflatoxin-Treated Mice in Vivo as Measured by Liquid Chromatography-Tandem Mass Spectrometry with Isotope Dilution.
Dietary exposure to aflatoxin B (AFB) is a significant contributor to the incidence of hepatocellular carcinomas globally. AFB exposure leads to the formation of AFB-N-guanine (AFB-N-Gua) and two diastereomers of the imidazole ring-opened 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxyaflatoxin B (AFB-FapyGua) in DNA. These adducts lead to G → T transversion mutations with the ring-opened adduct being more mutagenic than the cationic species. Accurate measurement of these three adducts as biomarkers in DNA and urine will help identify dietary exposure to AFB as a risk factor in the development of hepatocellular carcinoma worldwide. Herein, we report an improved methodology for the measurement of AFB-N-Gua and the two diastereomers of AFB-FapyGua using liquid chromatography-tandem mass spectrometry with isotope dilution. We measured the levels of these compounds in liver DNA of six control mice and six AFB-treated mice. Levels varying from 1.5 to 45 lesions/10 DNA bases in AFB-treated mice were detected depending on the compound and animal. No background levels of these adducts were detected in control mice. We also tested whether the AFB treatment caused oxidatively induced DNA base damage using gas chromatography-tandem mass spectrometry with isotope dilution. Although background levels of several pyrimidine- and purine-derived lesions were detected, no increases in these levels were found upon AFB treatment of mice. On the other hand, significantly increased levels of (5' R)- and (5' S)-8,5'-cyclo-2'-deoxyadenosines were observed in liver DNA of AFB-treated mice. The impact of this work is expected to achieve the accurate measurement of three AFB-DNA adducts and oxidatively induced DNA lesions as biomarkers of AFB exposure as germane to investigations designed for the prevention of aflatoxin-related hepatocellular carcinomas and for determining the effects of genetic deficiencies in human populations.
Topics: Aflatoxins; Animals; Chromatography, Liquid; DNA Adducts; DNA Damage; Guanine; Liver; Mice; Mice, Inbred C57BL; Mice, Knockout; Molecular Conformation; Oxidation-Reduction; Radioisotope Dilution Technique
PubMed: 30525498
DOI: 10.1021/acs.chemrestox.8b00202 -
Molecular Pharmacology Oct 2019Topoisomerase II (TOP2) poisons are effective cytotoxic anticancer agents that stabilize the normally transient TOP2-DNA covalent complexes formed during the enzyme...
Topoisomerase II (TOP2) poisons are effective cytotoxic anticancer agents that stabilize the normally transient TOP2-DNA covalent complexes formed during the enzyme reaction cycle. These drugs include etoposide, mitoxantrone, and the anthracyclines doxorubicin and epirubicin. Anthracyclines also exert cell-killing activity via TOP2-independent mechanisms, including DNA adduct formation, redox activity, and lipid peroxidation. Here, we show that anthracyclines and another intercalating TOP2 poison, mitoxantrone, stabilize TOP2-DNA covalent complexes less efficiently than etoposide, and at higher concentrations they suppress the formation of TOP2-DNA covalent complexes, thus behaving as TOP2 poisons at low concentration and inhibitors at high concentration. We used induced pluripotent stem cell (iPSC)-derived human cardiomyocytes as a model to study anthracycline-induced damage in cardiac cells. Using immunofluorescence, our study is the first to demonstrate the presence of topoisomerase II (TOP2B) as the only TOP2 isoform in iPSC-derived cardiomyocytes. In these cells, etoposide robustly induced TOP2B covalent complexes, but we could not detect doxorubicin-induced TOP2-DNA complexes, and doxorubicin suppressed etoposide-induced TOP2-DNA complexes. In vitro, etoposide-stabilized DNA cleavage was attenuated by doxorubicin, epirubicin, or mitoxantrone. Clinical use of anthracyclines is associated with cardiotoxicity. The observations in this study have potentially important clinical consequences regarding the effectiveness of anticancer treatment regimens when TOP2-targeting drugs are used in combination. These observations suggest that inhibition of TOP2B activity, rather than DNA damage resulting from TOP2 poisoning, may play a role in doxorubicin cardiotoxicity. SIGNIFICANCE STATEMENT: We show that anthracyclines and mitoxantrone act as topoisomerase II (TOP2) poisons at low concentration but attenuate TOP2 activity at higher concentration, both in cells and in in vitro cleavage experiments. Inhibition of type II topoisomerases suppresses the action of other drugs that poison TOP2. Thus, combinations containing anthracyclines or mitoxantrone and etoposide may reduce the activity of etoposide as a TOP2 poison and thus reduce the efficacy of drug combinations.
Topics: Anthracyclines; Cardiotoxicity; Cell Line, Tumor; Cell Survival; DNA Adducts; DNA Topoisomerases, Type II; Dose-Response Relationship, Drug; Doxorubicin; Etoposide; Humans; Induced Pluripotent Stem Cells; K562 Cells; Mitoxantrone; Myocytes, Cardiac; Topoisomerase II Inhibitors
PubMed: 31399497
DOI: 10.1124/mol.119.117259 -
Chemical Research in Toxicology Jan 2017Biomarker-driven drug selection plays a central role in cancer drug discovery and development, and in diagnostic strategies to improve the use of traditional...
Biomarker-driven drug selection plays a central role in cancer drug discovery and development, and in diagnostic strategies to improve the use of traditional chemotherapeutic drugs. DNA-modifying anticancer drugs are still used as first line medication, but drawbacks such as resistance and side effects remain an issue. Monitoring the formation and level of DNA modifications induced by anticancer drugs is a potential strategy for stratifying patients and predicting drug efficacy. In this perspective, preclinical and clinical data concerning the relationship between drug-induced DNA adducts and biological response for platinum drugs and combination therapies, nitrogen mustards and half-mustards, hypoxia-activated drugs, reductase-activated drugs, and minor groove binding agents are presented and discussed. Aspects including measurement strategies, identification of adducts, and biological factors that influence the predictive relationship between DNA modification and biological response are addressed. A positive correlation between DNA adduct levels and response was observed for the majority of the studies, demonstrating the high potential of using DNA adducts from anticancer drugs as mechanism-based biomarkers of susceptibility, especially as bioanalysis approaches with higher sensitivity and throughput emerge.
Topics: Animals; Antineoplastic Agents; Biomarkers; DNA Adducts; Humans; Hypoxia; Nitrogen Mustard Compounds; Oxidoreductases; Platinum Compounds; Precision Medicine; Prodrugs
PubMed: 27936622
DOI: 10.1021/acs.chemrestox.6b00380 -
Chemical Research in Toxicology Oct 2022-Methyl-2'-deoxyguanosine (-MeG) is one of the most common DNA lesions and arises as a consequence of both xenobiotic carcinogens and endogenous methylation by...
-Methyl-2'-deoxyguanosine (-MeG) is one of the most common DNA lesions and arises as a consequence of both xenobiotic carcinogens and endogenous methylation by -adenosylmethionine. -MeG frequently causes G-to-A mutations during DNA replication due to the misincorporation of dTTP and continued DNA synthesis. Efforts to detect DNA adducts such as -MeG, and to understand their impacts on DNA structure and function, have motivated the creation of nucleoside analogs with altered base moieties to afford a more favorable interaction with the adduct as compared to the unmodified nucleotide. Such analogs directed at -MeG include benzimidazolinone and benzimidazole nucleotides, as well as their extended π surface analogs naphthimidazolinone and napthimidazole derivatives. These analogs form a more stable pair with -MeG than with G, most likely due to a combination of H-bonding and stacking. While extending the π surface of the analogs enhances their performance as adduct-directed probes, the precise origins of the increased affinity between the synthetic analogs and -MeG remain unclear. To better understand relevant conformational and pairing properties, we used X-ray crystallography and analyzed the structures of the DNA duplexes with naphthimidazolinone inserted opposite G or -MeG. The structures reveal a complex interaction of the analog found either in an anti orientation and stacked inside the duplex, either above or below G or -MeG, or in a syn orientation and paired opposite G with formation of a single H-bond. The experimental structural data are consistent with the stabilizing effect of the synthetic analog observed in UV melting experiments and calculations and moreover reveal that the origin of these observations appears to be superior stacking between -MeG and the extended π system of the synthetic probe.
Topics: Benzimidazoles; Carcinogens; DNA; DNA Adducts; Deoxyguanosine; Nucleic Acid Conformation; Nucleosides; Nucleotides; S-Adenosylmethionine; Xenobiotics
PubMed: 35973057
DOI: 10.1021/acs.chemrestox.2c00165