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Chemical Research in Toxicology Mar 2016N'-Nitrosonornicotine (NNN) is carcinogenic in multiple animal models and has been evaluated as a human carcinogen. NNN can be metabolized by cytochrome P450s through...
N'-Nitrosonornicotine (NNN) is carcinogenic in multiple animal models and has been evaluated as a human carcinogen. NNN can be metabolized by cytochrome P450s through two activation pathways: 2'-hydroxylation and 5'-hydroxylation. While most previous studies have focused on 2'-hydroxylation in target tissues of rats, available evidence suggests that 5'-hydroxylation is a major activation pathway in human enzyme systems, in nonhuman primates, and in target tissues of some other rodent carcinogenicity models. In the study reported here, we investigated DNA damage resulting from NNN 5'-hydroxylation by quantifying the adduct 2-(2-(3-pyridyl)-N-pyrrolidinyl)-2'-deoxyinosine (py-py-dI). In rats treated with NNN in the drinking water (7-500 ppm), py-py-dI was the major DNA adduct resulting from 5'-hydroxylation of NNN in vivo. Levels of py-py-dI in the lung and nasal cavity were the highest, consistent with the tissue distribution of CYP2A3. In rats treated with (S)-NNN or (R)-NNN, the ratios of formation of (R)-py-py-dI to (S)-py-py-dI were not the expected mirror image, suggesting that there may be a carrier for one of the unstable intermediates formed upon 5'-hydroxylation of NNN. Rat hepatocytes treated with (S)- or (R)-NNN or (2'S)- or (2'R)-5'-acetoxyNNN exhibited a pattern of adduct formation similar to that of live rats. In vitro studies with human liver S9 fraction or human hepatocytes incubated with NNN (2-500 μM) demonstrated that py-py-dI formation was greater than the formation of pyridyloxobutyl-DNA adducts resulting from 2'-hydroxylation of NNN. (S)-NNN formed more total py-py-dI adducts than (R)-NNN in human liver enzyme systems, which is consistent with the critical role of CYP2A6 in the 5'-hydroxylation of NNN in human liver. The results of this study demonstrate that the major DNA adduct resulting from NNN metabolism by human enzymes is py-py-dI and provide potentially important new insights into the metabolic activation of NNN in rodents and humans.
Topics: Adolescent; Adult; Aged; Aged, 80 and over; Animals; Carcinogens; Child; Cytochrome P-450 Enzyme System; DNA Adducts; Female; Humans; Hydroxylation; Male; Middle Aged; Nitrosamines; Rats; Nicotiana; Young Adult
PubMed: 26808005
DOI: 10.1021/acs.chemrestox.5b00520 -
Chemical Research in Toxicology Aug 2020The human intestine is host to a vast microbial community: the gut microbiome (GM). The GM has been considered as a key modulator of human health in the past decade. In...
The human intestine is host to a vast microbial community: the gut microbiome (GM). The GM has been considered as a key modulator of human health in the past decade. In particular, several studies have supported that altered GM is associated with cancer, such as colorectal cancer, adenocarcinoma, and pancreatic cancer. The formation of a DNA adduct is one of the key events in carcinogenesis, and whether GM can influence DNA adducts has yet to be examined. This study analyzed 10 DNA adducts (N-Me-dG, N-Me-dA, N-Et-dG, OH-Me-dG, OH-Me-dA, N-EtD-dG, O-Me-dG, 1,N-ε-dG, 8-oxo-dG, and 5-Cl-dC), attributed to various endogenous processes and physiological stressors, using highly sensitive LC-MS/MS in germ-free (GF) and conventionally raised (CONV-R) mice. Our results showed that significant differences in specific DNA adducts appeared in liver, colon, and small intestine samples between GF and CONV-R mice. The differences in adduct levels may indicate that GM can locally or systemically regulate endogenous processes including neutrophil bactericidal activity (represented by 5-Cl-dC), lipid peroxidation (1,N-ε-dG), oxidative stress generation (8-oxo-dG), and endogenous aldehyde metabolism (OH-Me-dA). Further studies are warranted to elucidate how the GM influences endogenous process, DNA damage, and the risks of developing cancer.
Topics: Animals; Carcinogenesis; Carcinogens; DNA Adducts; DNA Damage; Female; Gastrointestinal Microbiome; Mice; Mice, Inbred C57BL; Molecular Structure
PubMed: 32677427
DOI: 10.1021/acs.chemrestox.0c00142 -
Chemical Society Reviews Nov 2015Exogenous and endogenous sources of chemical species can react, directly or after metabolic activation, with DNA to yield DNA adducts. If not repaired, DNA adducts may... (Review)
Review
Exogenous and endogenous sources of chemical species can react, directly or after metabolic activation, with DNA to yield DNA adducts. If not repaired, DNA adducts may compromise cellular functions by blocking DNA replication and/or inducing mutations. Unambiguous identification of the structures and accurate measurements of the levels of DNA adducts in cellular and tissue DNA constitute the first and important step towards understanding the biological consequences of these adducts. The advances in mass spectrometry (MS) instrumentation in the past 2-3 decades have rendered MS an important tool for structure elucidation, quantification, and revelation of the biological consequences of DNA adducts. In this review, we summarized the development of MS techniques on these fronts for DNA adduct analysis. We placed our emphasis of discussion on sample preparation, the combination of MS with gas chromatography- or liquid chromatography (LC)-based separation techniques for the quantitative measurement of DNA adducts, and the use of LC-MS along with molecular biology tools for understanding the human health consequences of DNA adducts. The applications of mass spectrometry-based DNA adduct analysis for predicting the therapeutic outcome of anti-cancer agents, for monitoring the human exposure to endogenous and environmental genotoxic agents, and for DNA repair studies were also discussed.
Topics: Chromatography, Liquid; DNA Adducts; Spectrometry, Mass, Electrospray Ionization
PubMed: 26204249
DOI: 10.1039/c5cs00316d -
Environmental and Molecular Mutagenesis Mar 2019The interpretation and significance of DNA adduct data, their causal relationship to mutations, and their role in risk assessment have been debated for many years. An... (Review)
Review
Understanding the importance of low-molecular weight (ethylene oxide- and propylene oxide-induced) DNA adducts and mutations in risk assessment: Insights from 15 years of research and collaborative discussions.
The interpretation and significance of DNA adduct data, their causal relationship to mutations, and their role in risk assessment have been debated for many years. An extended effort to identify key questions and collect relevant data to address them was focused on the ubiquitous low MW N7-alkyl/hydroxyalkylguanine adducts. Several academic, governmental, and industrial laboratories collaborated to gather new data aimed at better understanding the role and potential impact of these adducts in quantifiable genotoxic events (gene mutations/micronucleus). This review summarizes and evaluates the status of dose-response data for DNA adducts and mutations from recent experimental work with standard mutagenic agents and ethylene oxide and propylene oxide, and the importance for risk assessment. This body of evidence demonstrates that small N7-alkyl/hydroxyalkylguanine adducts are not pro-mutagenic and, therefore, adduct formation alone is not adequate evidence to support a mutagenic mode of action. Quantitative methods for dose-response analysis and derivation of thresholds, benchmark dose (BMD), or other points-of-departure (POD) for genotoxic events are now available. Integration of such analyses of genetox data is necessary to properly assess any role for DNA adducts in risk assessment. Regulatory acceptance and application of these insights remain key challenges that only the regulatory community can address by applying the many learnings from recent research. The necessary tools, such as BMDs and PODs, and the example datasets, are now available and sufficiently mature for use by the regulatory community. Environ. Mol. Mutagen. 60: 100-121, 2019. © 2018 The Authors. Environmental and Molecular Mutagenesis published by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.
Topics: DNA Adducts; DNA Damage; Epoxy Compounds; Ethylene Oxide; Humans; Molecular Weight; Mutagenesis; Mutagens; Mutation; Risk Assessment
PubMed: 30536466
DOI: 10.1002/em.22248 -
Chemical Research in Toxicology Jun 2011This perspective reviews our work on the identification of DNA adducts of N-nitrosopyrrolidine and some related aldehydes. The research began as a focused project to... (Review)
Review
This perspective reviews our work on the identification of DNA adducts of N-nitrosopyrrolidine and some related aldehydes. The research began as a focused project to investigate mechanisms of cyclic nitrosamine carcinogenesis but expanded into other areas, as aldehyde metabolites of NPYR were shown to have their own diverse DNA adduct chemistry. A total of 69 structurally distinct DNA adducts were identified, and some of these, found in human tissues, have provided intriguing leads for investigating carcinogenesis mechanisms in humans due to exposure to both endogenous and exogenous agents.
Topics: Aldehydes; Animals; DNA; DNA Adducts; Humans; N-Nitrosopyrrolidine
PubMed: 21480629
DOI: 10.1021/tx200064a -
International Journal of Molecular... Mar 2020Benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon, is a group 1 carcinogen that introduces mutagenic DNA adducts into the genome. In this study, we investigated...
Benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon, is a group 1 carcinogen that introduces mutagenic DNA adducts into the genome. In this study, we investigated the molecular mechanisms underlying the involvement of silymarin in the reduction of DNA adduct formation by B[a]P-7,8-dihydrodiol-9,10-epoxide (BPDE), induced by B[a]P. B[a]P exhibited toxicity in HepG2 cells, whereas co-treatment of the cells with B[a]P and silymarin reduced the formation of BPDE-DNA adducts, thereby increasing cell viability. Determination of the level of major B[a]P metabolites in the treated cells showed that BPDE levels were reduced by silymarin. Nuclear factor erythroid 2-related factor 2 (Nrf2) and pregnane X receptor (PXR) were found to be involved in the activation of detoxifying genes against B[a]P-mediated toxicity. Silymarin did not increase the expression of these major transcription factors, but greatly facilitated their nuclear translocation. In this manner, treatment of HepG2 cells with silymarin modulated detoxification enzymes through NRF2 and PXR to eliminate B[a]P metabolites. Knockdown of Nrf2 abolished the preventive effect of silymarin on BPDE-DNA adduct formation, indicating that activation of the Nrf2 pathway plays a key role in preventing B[a]P-induced genotoxicity. Our results suggest that silymarin has anti-genotoxic effects, as it prevents BPDE-DNA adduct formation by modulating the Nrf2 and PXR signaling pathways.
Topics: 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; Benzo(a)pyrene; Chemical and Drug Induced Liver Injury; DNA Adducts; DNA Damage; Hep G2 Cells; Humans; Mutagens; Protective Agents; Signal Transduction; Silymarin
PubMed: 32235460
DOI: 10.3390/ijms21072369 -
Environmental Health Perspectives Jun 1997Quantitation of DNA adducts in human tissues has been achieved with highly sensitive techniques based on adduct radiolabeling, antisera specific for DNA adducts or... (Review)
Review
Quantitation of DNA adducts in human tissues has been achieved with highly sensitive techniques based on adduct radiolabeling, antisera specific for DNA adducts or modified DNA, and/or adduct structural characterization using chemical instrumentation. Combinations of these approaches now promise to elucidate specific adduct structures and provide detection limits in the range of 1 adduct/10(9) nucleotides. Documentation of human exposure and biologically effective dose (i.e., chemical bound to DNA) has been achieved for a wide variety of chemical carcinogens, including polycyclic aromatic hydrocarbons (PAHs), aromatic amines, heterocyclic amines, aflatoxins, nitrosamines, cancer chemotherapeutic agents, styrene, and malondialdehyde. Due to difficulties in exposure documentation, dosimetry has not been precise with most environmental and occupational exposures, even though increases in human blood cell DNA adduct levels may correlate approximately with dose. Perhaps more significant are observations that lowering exposure results in decreasing DNA adduct levels. DNA adduct dosimetry for environmental agents has been achieved with dietary contaminants. For example, blood cell polycyclic aromatic hydrocarbon-DNA adduct levels were shown to correlate with frequency of charbroiled meat consumption in California firefighters. In addition, in China urinary excretion of the aflatoxin B1-N7-guanine (AFB1-N7-G) adduct was shown to increase linearly with the aflatoxin content of ingested food. Assessment of DNA adduct formation as an indicator of human cancer risk requires a prospective nested case-control study design. This has been achieved in one investigation of hepatocellular carcinoma and urinary aflatoxin adducts using subjects followed by a Shanghai liver cancer registry. Individuals who excreted the AFB1-N7-G adduct had a 9.1-fold adjusted increased relative risk of hepatocellular carcinoma compared to individuals with no adducts. Future advances in this field will be dependent on chemical characterization of specific DNA adducts formed in human tissues, more-precise molecular dosimetry, efforts to correlate DNA adducts with cancer risk, and elucidation of opportunities to reduce human DNA adduct levels.
Topics: Biomarkers; DNA Adducts; Humans; Neoplasms; Risk Assessment
PubMed: 9255579
DOI: 10.1289/ehp.97105s4907 -
The Journal of Biological Chemistry Aug 2023The DNA adduct 6-oxo-MdG, (3-(2'-deoxy-β-D-erythro-pentofuranosyl)-6-oxo-pyrimido(1,2alpha)purin-10(3H)-one) is formed in the genome via oxidation of the...
The DNA adduct 6-oxo-MdG, (3-(2'-deoxy-β-D-erythro-pentofuranosyl)-6-oxo-pyrimido(1,2alpha)purin-10(3H)-one) is formed in the genome via oxidation of the peroxidation-derived adduct MdG. However, the effect of 6-oxo-MdG adducts on subsequent DNA replication is unclear. Here we investigated the ability of the human Y-family polymerase hPol η to bypass 6-oxo-MdG. Using steady-state kinetics and analysis of DNA extension products by liquid chromatography-tandem mass spectrometry, we found hPol η preferentially inserts a dAMP or dGMP nucleotide into primer-templates across from the 6-oxo-MdG adduct, with dGMP being slightly preferred. We also show primer-templates with a 3'-terminal dGMP or dAMP across from 6-oxo-MdG were extended to a greater degree than primers with a dCMP or dTMP across from the adduct. In addition, we explored the structural basis for bypass of 6-oxo-MdG by hPol η using X-ray crystallography of both an insertion-stage and an extension-stage complex. In the insertion-stage complex, we observed that the incoming dCTP opposite 6-oxo-MdG, although present during crystallization, was not present in the active site. We found the adduct does not interact with residues in the hPol η active site but rather forms stacking interactions with the base pair immediately 3' to the adduct. In the extension-stage complex, we observed the 3' hydroxyl group of the primer strand dGMP across from 6-oxo-MdG is not positioned correctly to form a phosphodiester bond with the incoming dCTP. Taken together, these results indicate 6-oxo-MdG forms a strong block to DNA replication by hPol η and provide a structural basis for its blocking ability.
Topics: Humans; DNA Adducts; DNA-Directed DNA Polymerase; DNA; DNA Replication
PubMed: 37468099
DOI: 10.1016/j.jbc.2023.105067 -
Cancer Letters Aug 2006The evidence for polycyclic aromatic hydrocarbons (PAH) playing a role in prostate carcinogenesis comes mainly from associations between reported PAH exposures and... (Review)
Review
The evidence for polycyclic aromatic hydrocarbons (PAH) playing a role in prostate carcinogenesis comes mainly from associations between reported PAH exposures and prostate cancer in epidemiologic studies. Associations between prostate cancer and DNA repair genotypes and phenotypes have also been reported, lending further credence to a PAH-induced carcinogenesis pathway in prostate cancer. Recent work that demonstrates the human prostate has metabolic enzyme activity necessary for PAH activation and will form DNA adducts upon exposure to PAH further supports PAH carcinogenesis. We have demonstrated the presence of PAH-DNA adducts in prostate cancer cases, but further validation of this biomarker as a carcinogenic agent in human prostate is needed.
Topics: DNA Adducts; DNA Damage; DNA Repair; Environmental Exposure; Humans; Male; Polycyclic Compounds; Prostatic Neoplasms
PubMed: 16154258
DOI: 10.1016/j.canlet.2005.07.029 -
Mutagenesis Mar 2017The frequency of stable DNA adducts in a target tissue can be used to assess biologically effective dose; however, the utility of the metric in a risk assessment context...
The frequency of stable DNA adducts in a target tissue can be used to assess biologically effective dose; however, the utility of the metric in a risk assessment context depends on the likelihood that the DNA damage will be manifested as mutation. Previously, we employed the Muta™Mouse system to examine the induction of lacZ mutants and DNA adducts following exposure to the well-studied mutagenic carcinogen 3-nitrobenzanthrone (3-NBA). In this follow-up work, we examined the empirical relationships between total adduct frequency and mutant frequency (MF) in tissues and cultured cells following acute 3-NBA exposure. The results show a significant induction of DNA damage and lacZ mutants in liver, colon and bone marrow, as well as FE1 pulmonary epithelial cells. In contrast, lung and small intestine samples had low, but significantly elevated adduct levels, with no significant increases in lacZ MF. Additional analyses showed a significant relationship between the mutagenic efficiency of total adducts, measured as the slope of the relationships between MF and total adduct frequency, and tissue-specific mitotic index (MI). The lack of mutation response in lung, in contrast to the high in vitro MF in FE-1 lung cells, is likely related to the 100-fold difference in MI. The lack of small intestine mutagenic response may be related to limited metabolic capacity, differences in DNA repair, and /or chemically induced apoptosis that has been observed for other potent mutagens. The results indicate that interpretation of adduct frequency values in a risk assessment context can be improved by considering the MI of the target tissue; however, more generalised interpretation is hampered by tissue-specific variations in metabolic capacity and damage processing. The work provides a proof of principle regarding the use of the Muta™Mouse system to critically examine the health risks associated with tissue-specific adduct loads.
Topics: Animals; Benz(a)Anthracenes; DNA Adducts; DNA Damage; DNA Repair; Lac Operon; Male; Mice; Mutagenicity Tests; Mutation; Organ Specificity; Transgenes
PubMed: 28096451
DOI: 10.1093/mutage/gew067