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.

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.

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.