Improved preparation and identification of aristolochic acid-DNA adducts by solid-phase extraction with liquid chromatography-tandem mass spectrometry

Aristolochic acid （AA） is a known nephrotoxin and potential carcinogen, which can form covalent DNA adducts after metabolic activation in vivo and in vitro. A simple method for preparation and characterization of aristolochic acid-DNA adducts was developed. Four AA-adducts were synthesized by a direct reaction of AAI/AAII with 2′-deoxynucleosides. The reaction mixture was first cleaned-up and pre-concentrated using solid phase extraction （SPE）, and further purified by a reversed-phase high performance liquid chromatography （HPLC）. By the application of developed SPE procedure, matrices and byproducts in reaction mixture could be greatly reduced and adducts of high purity （more than 94% as indicated by HPLC） were obtained. The purified AA-DNA adducts were identified and characterized with liquid-electrospray ionization-quadrupole-time of flight-mass spectrometry （LC-ESI-Q-TOF-MS/MS） and LC-Diode array detector-fluorescence （LC-DAD-FL） analysis. This work provides a robust tool for possible large-scale preparation of AA-DNA adduct standards, which can promote the further studies on carcinogenic and mutagenic mechanism of aristolochic acids.

STUDY ON GMA-DNA ADDUCTS

Objective. DNA modification fixed as mutations in the cells may be an essential factor in the initiation step of chemical carcinogenesis. In order to explore the mechanism of gene mutation and cell transformation induced by glycidyl methacrylate (GMA), the current test studied the characteristics of GMA DNA adducts formation in vitro. Methods. In vitro test, dAMP, dCMP, dGMP, dTMP and calf thymus DNA were allowed to react with GMA (Glycidyl Methacrylate). After the reaction, the mixtures were detected by UV and subjected to reversed phase HPLC on ultrasphere ODS reversed phase column, the reaction products were eluted with a linear gradients of methanol (solvent A) and 10mmol/L ammonium formate, pH5 0 (solvent B). The synthesized adducts were then characterized by UV spectroscopy in acid (pH1 0), neutral (pH7 2), alkaline (pH11 0) and by mass spectroscopy. Results. The results showed that GMA could bind with dAMP, dCMP, dGMP and calf thymus DNA by covalent bond, and the binding sites were specific (N 6 of adenine, N 3 of cytosine). Meanwhile, a main GMA DNA adduct in the reaction of GMA with calf thymus DNA was confirmed as N 3 methacrylate 2 hydroxypropy1 dCMP. Conclusions. GMA can react with DNA and /or deoxynucleotide monophosphate and generate some adducts such as N 6 methacrylate 2 hydroxypropyl dAMP and N 3 methacrylate 2 hydroxypropyl dCMP, ets. Formation of GMA DNA adducts is an important molecular event in gene mutation and cell transformation induced by GMA.

Leptin influences estrogen metabolism and increases DNA adduct formation in breast cancer cells

Objective: The elevated incidence of obesity has been paralleled with higher risks of breast cancer. High adiposity increases leptin secretion from adipose tissue, which in turn increases cancer cell proliferation. The interplay between leptin and estrogen is one of the mechanisms through which leptin influences breast carcinogenesis. An unbalanced estrogen metabolism increases the formations of catechol estrogen quinones, DNA adducts, and cancer mutations. This study aims to investigate the effect of leptin on some estrogen metabolic enzymes and DNA adduction in breast cancer cells.Methods: High performance liquid chromatography(HPLC) was performed to analyze the DNA adducts 4-OHE1[E2]-1-N3 adenine and 4-OHE1[E2]-1-N7 guanine. Reporter gene assay, real time reverse transcription polymerase chain reaction(real time RT-PCR), and Western blot were used to assess the expression of estrogen metabolizing genes and enzymes: Cytochrome P-4501B1(CYP1B1), Nicotinamide adenine dinucleotide phosphate-quinone oxidoreductase1(NQO1), and Catechol-O-methyl transferase(COMT).Results: Leptin significantly increased the DNA adducts 4-OHE1[E2]-1-N3 adenine and 4-OHE1[E2]-1-N7 guanine.Furthermore, leptin significantly upregulated CYP1B1 promoter activity and protein expression. The luciferase promoter activities of NQO1 and m RNA levels were significantly reduced. Moreover, leptin greatly reduced the reporter activities of the COMT-P1 and COMT-P2 promoters and diminished the protein expression of COMT.Conclusions: Leptin increases DNA adduct levels in breast cancer cells partly by affecting key genes and enzymes involved in estrogen metabolism. Thus, increased focus should be directed toward leptin and its effects on the estrogen metabolic pathway as an effective approach against breast cancer.

Using DNA damage to monitor water environment

DNA damage of aquatic organisms living in polluted environments can be used as a biomarker of the genotoxicity of toxic agents to organisms. This technique has been playing an important role in ecotoxi- cological study and environmental risk assessment. In this article, main types of DNA damage caused by pollut- ants in water environments were reviewed; methods of detecting DNA damage were also documented for water environmental monitoring.

Targeting Tyrosyl-DNA phosphodiesterase I to enhance toxicity of phosphodiester linked DNA-adducts

Our genomic DNA is under constant assault from endogenous and exogenous sources,which needs to be resolved to maintain cellular homeostasis.The eukaryotic DNA repair enzyme Tyrosyl-DNA phosphodiesterase I(Tdp1)catalyzes the hydrolysis of phosphodiester bonds that covalently link adducts to DNA-ends.Tdp1 utilizes two catalytic histidines to resolve a growing list of DNA-adducts.These DNA-adducts can be divided into two groups:small adducts,including oxidized nucleotides,RNA,and non-canonical nucleoside analogs,and large adducts,such as(drug-stabilized)topoisomerase-DNA covalent complexes or failed Schiff base reactions as occur between PARP1 and DNA.Many Tdp1 substrates are generated by chemotherapeutics linking Tdp1 to cancer drug resistance,making a compelling argument to develop small molecules that target Tdp1 as potential novel therapeutic agents.Tdp1’s unique catalytic cycle,which is centered on the formation of Tdp1-DNA covalent reaction intermediate,allows for two principally different targeting strategies:(1)catalytic inhibition of Tdp1 catalysis to prevent Tdp1-mediated repair of DNA-adducts that enhances the effectivity of chemotherapeutics;and(2)poisoning of Tdp1 by stabilization of the Tdp1-DNA covalent reaction intermediate,which would increase the half-life of a potentially toxic DNA-adduct by preventing its resolution,analogous to topoisomerase targeted poisons such as topotecan or etoposide.The catalytic Tdp1 mutant that forms the molecular basis of the autosomal recessive neurodegenerative disease spinocerebellar ataxia with axonal neuropathy best illustrates this concept;however,no small molecules have been reported for this strategy.Herein,we concisely discuss the development of Tdp1 catalytic inhibitors and their results.

Evaluation of DNA adduct damage using G-quadruplex-based DNAzyme

Toxicity assessment is a major problem in pharmaceutical candidates and industry chemicals development.However,due to the lack of practical analytical methods for DNA adduct analysis,the safety evaluation of drug and industry chemicals was severely limited.Here,we develop a DNAzyme-based method to detect DNA adduct damage for toxicity assessment of drugs and chemicals.Among 18 structural variants of G4 DNAzyme,EA2 DNAzyme exhibits an obvious DNA damaging effect of styrene oxide(SO)due to its unstable structure.The covalent binding of SO to DNAzyme disrupts the Hoogsteen hydrogen bonding sites of G-plane guanines and affects the formation of the G4 quadruplex.DNA damage chemicals reduce the peroxidase activity of the G4 DNAzyme to monitor the DNA adduct damage by disrupting the structural integrity of the G4 DNAzyme.Our method for genotoxic assessment of pharmaceutical candidates and industrial chemicals can elucidate the complex chemical pathways leading to toxicity,predict toxic effects of chemicals,and evaluate possible risks to human health.

Nuclear DNA damages generated by reactive oxygen molecules (ROS) under oxidative stress and their relevance to human cancers, including ionizing radiation-induced neoplasia part II: Relation between ROS-induced DNA damages and human cancer

Oxidative stress(OS)occurs when the production of reactive oxygen species(ROS)overrides the body’s natural defence.When the cell nucleus represents the target,macromolecular damage may result in mutations.Cancer is a disease of mutations,and DNA damages that are not repaired or mis-repaired during cell proliferation are necessary but not sufficient for cancer development.A role of ROS for cancer initiation depends on the likelihood of interaction between reactive electrophilic molecules and nuclear DNA.As described in part one of this presentation,the physico-chemical properties of the ROS involved in OS and of the ensuing DNA lesions are of major importance.Current knowledge dictates that emphasis should be shifted from oxidative DNA damages of low genotoxicity towards pro-mutagenic lesions induced by reaction products of nitrogen monoxide and complex highly reactive carbonyls,e.g.from the peroxidation of lipids.Based on the determination of pro-mutagenic DNA adducts in human tissues there is compelling evidence for a causal relation between OS and cancers of the liver,colon/rectum,cervix,pancreas and stomach.However,modulation by the simultaneous presence of an ubiquitous high background of potent pro-carcinogenic DNA adducts,which are not generated by ROS should be taken into account.Ionizing radiation is established human carcinogenic agent,and generate some of the same oxidative ROS as those involved in OS.However,the cancer spectrum from whole body radiation exposure differs in some important respects from that associated with OS.The scientific support for a causal link between exposure to non-ionizing electromagnetic radiation and human cancer is judged to be insufficient.As exemplified by diabetes,a common shortcoming when assessing the role of OS in disease is the failure to distinguish between cause and effect-i.e.could the indicators of harmful oxidative stress be the result of the pathological condition in question,rather than its cause.

Nuclear DNA damages generated by reactive oxygen molecules (ROS) under oxidative stress and their relevance to human cancers, including ionizing radiation-induced neoplasia part I: Physical, chemical and molecular biology aspects

Oxidative stress(OS)occurs when the production of reactive oxygen species(ROS)overpowers the body’s natural defence,causing macromolecular damage.The role of OS in cancer initiation will depend on the likelihood of interaction between short lived ROS and nuclear DNA.For this reason,a description of the physico-chemical properties of the various ROS that have been suggested to be involved is included.DNA damages that are not repaired or mis-repaired during cell proliferation are necessary but not sufficient for cancer initiation.The characteristics of DNA pro-mutagenic lesions and their potential role in cancer induction will be assessed,while stressing quantitative aspects as well as the importance of DNA repair.A low level of a specific DNA adduct can be compensated for by its persistence and high pro-mutagenic potency.Because ionizing radiations generate some of the same oxidative ROS as those involved in OS,the cancer spectrum from whole body radiation exposure should be compared with that associated with OS.A causal link between electromagnetic radiations and human cancer lacks adequate scientific support.Current knowledge dictates that emphasis should be shifted from oxidative damages of low genotoxicity towards pro-mutagenic lesions induced by reaction products of nitrogen monoxide and complex highly reactive carbonyls,e.g.from the peroxidation of lipids.A common shortcoming when assessing the role of OS in disease is the failure to distinguish between cause and effect-i.e.could the indicators of harmful OS be the result of the pathological condition in question,rather than its cause?Further,little attention has been paid to exposure in food to some of the same ROS(e.g.reactive carbonyl compounds),as are generated endogenously by OS.Nor have the simultaneous presence of an ubiquitous high background of potent pro-carcinogenic DNA adducts which are not generated by ROS been taken into account.

Ecogenotoxicology in earthworms： A review

Pollutant dynamics and bioavailability greatly differ in soil and aquatic systems. Therefore, specific approaches and models are needed to assess the impact of soil contamination to terrestrial ecosystems. Earthworms among other soil invertebrates have received more attention because of their ecological importance. They represent a dominant part of the soil biomass and are soil engineers regulating important soil processes, notably fertilization. The release in soils of pollutants known for their persistence and/or their toxicity is a concern. Exposure of terrestrial species to pollutants that may alter genomic function has become an increasing topic of research in the last decade. Indeed, genome disturbances due to genetic and epigenetic mechanisms may impair growth, as well as reproduction and population dynamics in the long term. Despite their importance in gene expres- sion, epigenetic mechanisms are not yet understood in soil invertebrates. Until now, pollutant-induced changes in genome expression in natural biota are still being studied through structural alteration of DNA. The first biomarker relating to genotoxicant exposure in earthworms from multi-contaminated soils reported is DNA adducts measurements. It has been replaced by DNA breakage measured by the Comet assay, now more commonly used. Functional genomic changes are now being explored owing to molecular ＂omic＂ technologies. Approaches, objectives and results are overviewed herein. The focus is on studies dealing with genotoxicity and populational effects established from environmentally-relevant experiments and in situ studies [Current Zoology 60 （2）： 255-272, 2014].