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Topoisomerases are proven drug targets, but antibiotics that poison bacterial Topoisomerase 1 (Top1) have yet to be discovered. We have developed a rapid and direct assay for quantification of Top1-DNA adducts that is suitable for high throughput assays. Adducts are recovered by "RADAR fractionation", a quick, convenient approach in which cells are lysed in chaotropic salts and detergent and nucleic acids and covalently bound adducts then precipitated with alcohol. Here we show that RADAR fractionation followed by ELISA immunodetection can quantify adducts formed by wild-type and mutant Top1 derivatives encoded by two different bacterial pathogens, Y. pestis and M. tuberculosis, expressed in E. coli or M. smegmatis, respectively. For both enzymes, quantification of adducts by RADAR/ELISA produces results comparable to the more cumbersome classical approach of CsCl density gradient fractionation. The experiments reported here establish that RADAR/ELISA assay offers a simple way to characterize Top1 mutants and analyze kinetics of adduct formation and repair. They also provide a foundation for discovery and optimization of drugs that poison bacterial Top1 using standard high-throughput approaches., (Copyright © 2020 Elsevier Inc. All rights reserved.)

DNA adducts usually occur at a level of 0.1-1 adducts per 10 8 unmodified DNA bases, which can cause many adverse health effects if not normally repaired. Using microgram amounts of DNA to detect DNA adducts remains challenging due to the extremely low levels. Commercial solid phase extraction is the most widely used method for extracting DNA adducts from biological samples, but it is time consuming and costly, and the hydrophobic interactions between various alkyl-bonded phases and analytes typically lack selectivity when used to extract DNA adducts. Therefore, an effective method for DNA adducts analysis based on Fe 3 O 4 @graphene oxide (GO) nano-adsorbent and ultra-performance liquid chromatography-triple quadrupole mass spectrometry detection was developed in this study, in which the selectivity may also be increased by π-π stacking interactions between GO and adduct molecules. Several variables, including the extraction time, extraction pH, nano-sorbent amount and elution solvent were optimized, to achieve the best extraction efficiency using the proposed method. A small amount of Fe 3 O 4 @GO (2.5 mg) exhibited a good adsorption performance for the model DNA adducts, and butanol containing 4% NH 3 ·H 2 O showed good elution effects. Under the optimized conditions, satisfying recoveries (78-107%) from calf thymus DNA samples were achieved, and 2 adducts were detected in only 2 μg of blood DNA. This method can be used as an effective strategy for DNA adduct analysis, and can be extended to other biological samples., (Copyright © 2019 Elsevier B.V. All rights reserved.)

1,3-Butadiene (BD) is a known human carcinogen found in cigarette smoke, automobile exhaust, and urban air. Workers occupationally exposed to BD in the workplace have an increased incidence of leukemia and lymphoma. BD undergoes cytochrome P450-mediated metabolic activation to 3,4-epoxy-1-butene (EB), 1,2,3,4-diepoxybutane (DEB) and 1,2-dihydroxy-3,4-epoxybutane (EBD), which form covalent adducts with DNA. We have previously reported a quantitative nanoLC/ESI+-HRMS3 method for urinary N7-(1-hydroxy-3-buten-2-yl) guanine (EB-GII) adducts as a mechanism-based biomarker of BD exposure. In the present study, the method was updated to include high throughput 96-well solid phase extraction (SPE) and employed to establish urinary EB-GII biomarker stability and association with smoking. Urinary EB-GII levels were measured bimonthly for 1 year in 19 smokers to determine whether single adduct measurement provides reliable levels of EB-GII in an individual smoker. In addition, association of EB-GII with smoking was studied in 17 individuals participating in a smoking cessation program. EB-GII levels decreased 34% upon smoking cessation, indicating that it is associated with smoking status, but may also originate from sources other than exposure to cigarette smoke., (© The Author(s) 2019. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Reactive oxygen species (ROS) and chronic inflammation contribute to DNA damage of many organs, including the prostate. ROS cause oxidative damage to biomolecules, such as lipids, proteins, and nucleic acids, resulting in the formation of toxic and mutagenic intermediates. Lipid peroxidation (LPO) products covalently adduct to DNA and can lead to mutations. The levels of LPO DNA adducts reported in humans range widely. However, a large proportion of the DNA adducts may be attributed to artifact formation during the steps of isolation and nuclease digestion of DNA. We established a method that mitigates artifacts for most LPO adducts during the processing of DNA. We have applied this methodology to measure LPO DNA adducts in the genome of prostate cancer patients, employing ultrahigh-performance liquid chromatography electrospray ionization ion trap multistage mass spectrometry. Our preliminary data show that DNA adducts of acrolein, 6-hydroxy-1, N 2 -propano-2'-deoxyguanosine (6-OH-PdG) and 8-hydroxy-1, N 2 -propano-2'-deoxyguanosine (8-OH-PdG) (4-20 adducts per 10 7 nucleotides) are more prominent than etheno (ε) adducts (<0.5 adducts per 10 8 nucleotides). This analytical methodology will be used to examine the correlation between oxidative stress, inflammation, and LPO adduct levels in patients with benign prostatic hyperplasia and prostate cancer.

DNA adducts are associated with a number of diseases, including cancer. Based on a recent report by our group, the aim of this study was to test the hypothesis that DNA adducts can be removed by means of one or more of the following three intervention programmes: intermittent whole-body hyperthermia; detoxification; and cell repair. The number of DNA adducts and total DNA adduct concentrations were measured in 104 patients who underwent one or more of the three intervention programmes. DNA adduct assessments were carried out on extracted genomic DNA by gas-liquid chromatography, with any DNA adducts found being localised using DNA microarrays. The baseline median number of DNA adducts was 2. The follow-up median number of adducts was highly significantly lower at 0 (p < 0.000000000000003). The mean total DNA adduct concentration at baseline was 9.308 ng/mL, and highly significantly lower at follow-up at 1.553 ng/mL (p < 0.000000000000006). Of the three intervention programmes, only the intermittent whole-body hyperthermia was associated with a significant reduction in DNA adducts. This study offers support for the hypothesis that DNA adducts can be removed by intermittent whole-body hyperthermia. The intermittent hyperthermia used involved infrared-A (wavelength 700-1400 nm, or, equivalently, a frequency of 215-430 THz) being preferentially delivered to the whole body, apart from the head, for up to one hour per session, with gradual core body temperature elevation usually occurring during the first 20-30 min. These results may offer an explanation at the molecular level for other reported clinical benefits of intermittent whole-body hyperthermia., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Air pollution is a major environmental risk for human health. Acetaldehyde is present in tobacco smoke and vehicle exhaust. In this study, we show that [ 13 C 2 ]-acetaldehyde induces DNA modification with the formation of isotopically labeled 1, N 2 -propano-2'-deoxyguanosine adducts in the brain and lungs of rats exposed to concentrations of acetaldehyde found in the atmosphere of megacities. The adduct, with the addition of two molecules of isotopically labeled acetaldehyde [ 13 C 4 ]-1, N 2 -propano-dGuo, was detected in the lung and brain tissues of exposed rats by micro-HPLC/MS/MS. Structural confirmation of the products was unequivocally performed by nano-LC/ESI + -HRMS 3 analyses. DNA modifications induced by acetaldehyde have been regarded as a key factor in the mechanism of mutagenesis and may be involved in the cancer risks associated with air pollution.

Bisphenol A (BPA) is a widely used additive in the plastic industry and has been reported to have genotoxicity. A hypothesis that BPA may enhance breast cancer risk through the formation of its metabolic intermediate or DNA adduct has been proposed. In this study, breast cancer cell MCF-7 was cultured and the cellular DNA was extracted from the cells. The adducts of bisphenol A 3,4-quinone (BPAQ) with 2'-deoxyguanosine (dG), calf thymus DNA and MCF-7 cell DNA were investigated. DNA adducts were characterized by using electrospray ionization Orbitrap high-resolution mass spectrometry and tandem mass spectrometry. The BPA-DNA adducts of BPAQ with dG, calf thymus and MCF-7 cell DNA were identified as 3-hydroxy-bisphenol A-N7-guanine (3-OH-BPA-N7Gua). The MS/MS fragmentation pathway of 3-OH-BPA-N7Gua was proposed based on obtained accurate mass data. BPA quinone metabolites can react with MCF-7 cell DNA in vitro. The findings provide evidence that BPA might covalently bind to DNA in MCF-7 cells mediated by quinone metabolites, which may increase our understanding of health risk associated with BPA exposure., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Three alkylated DNA adducts, N 3 ‑methyladenine, N 3 ‑ethyladenine and N 7 ‑ethylguanine, have been proved to be potential biomarkers for DNA injury caused by exposure to cigarette smoke. In this study, a highly specific and sensitive method using a new mixed-mode sulfonate-functionalized poly(glycidyl methacrylate-divinylbenzene) as a solid-phase extraction sorbent was developed for the analysis of these three alkylated-purine adducts in human urine. Under optimized conditions, the prepared sorbent interacts strongly with these urinary adducts, demonstrating high clean-up efficiency and extraction recovery. The method detection limits (S/N ≥ 3) of N 3 -MeA, N 3 -EtA and N 7 -EtG were 1.75, 0.20, and 0.15 pg mL -1 , respectively, while the method quantitation limits were found to be 5.78, 0.66, and 0.49 pg mL -1 for N 3 -MeA, N 3 -EtA and N 7 -EtG, respectively. The intra-day and inter-day precisions were investigated, of which were in the range of 1.6-3.8% and 3.2-5.6%, respectively. The recovery values of the alkylated DNA adducts in spiked urine sample were ranged 89.7-104.5%. Their concentrations were statistically significantly higher in smokers than in nonsmokers. These results show that the proposed method is suitable for the analysis of alkylated DNA adducts., (Copyright © 2018 Elsevier B.V. All rights reserved.)

This unit contains a complete procedure for the detection and structural characterization of DNA protein crosslinks (DPCs). The procedure also describes an approach for the quantitation of the various structurally distinct DPCs. Although various methods have been described in the literature for labile DPCs, characterization of nonlabile adducts remain a challenge. Here we present a novel approach for characterization of both labile and non-labile adducts by the use of a combination of chemical, enzymatic, and mass spectrometric approaches. A Raney Ni-catalyzed reductive desulfurization method was used for removal of the bulky peptide adducts, enzymatic digestion was used to digest the protein to smaller peptides and DNA to nucleosides, and finally LC-ESI-tandem mass spectrometry (MS) was utilized for detection and characterization of nucleoside adducts., (Copyright © 2015 John Wiley & Sons, Inc.)

Malondialdehyde (MDA), an endogenous genotoxic product formed upon lipid peroxidation and prostaglandin biosynthesis, can react with DNA to form stable adducts. These adducts may contribute to the development of such inflammation-mediated diseases as cancer and cardiovascular and neurodegenerative diseases. The predominant MDA-derived DNA adduct formed under physiological conditions is 3-(2-deoxy-β-d-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine (M1dG). In this study, we developed a novel liquid chromatography (LC)-nanoelectrospray ionization (NSI)-high-resolution tandem mass spectrometry (HRMS/MS) method for the analysis of M1dG in human leukocyte DNA. After enzymatic hydrolysis of DNA, M1dG and the added internal standard [(13)C3]M1dG were reduced to their 5,6-dihydro derivatives by addition of sodium borohydride to the hydrolysate and purified by solid-phase extraction and column chromatography. The 5,6-dihydro derivatives in the purified samples were analyzed by LC-NSI-HRMS/MS using higher-energy collisional dissociation (HCD) fragmentation, isolation widths of 1 Da for both the analyte and internal standard, and a resolution of 50 000. The detection limit of the developed method is 5 amol on-column, and the limit of quantitation is 0.125 fmol/mg DNA starting with 200 μg of DNA. Method accuracy and precision were characterized. The developed method was further applied to the analysis of leukocyte DNA from 50 human subjects. M1dG was detected in all samples and ranged from 0.132 to 275 fmol/mg DNA, or 0.004 to 9.15 adducts per 10(8) bases. This unique and highly sensitive HRMS/MS-based method can be used in future studies investigating the pathophysiological role of M1dG in human diseases.

DNA adducts are a measure of internal exposure to genotoxicants. However, the measurement of DNA adducts in molecular epidemiology studies often is precluded by the lack of fresh tissue. In contrast, formalin-fixed paraffin-embedded (FFPE) tissues frequently are accessible, although technical challenges remain in retrieval of high quality DNA suitable for biomonitoring of adducts. Aristolochic acids (AA) are human carcinogens found in Aristolochia plants, some of which have been used in the preparation of traditional Chinese herbal medicines. We previously established a method to measure DNA adducts of AA in FFPE tissue. In this study, we examine additional features of formalin fixation that could impact the quantity and quality of DNA and report on the recovery of AA-DNA adducts in mice exposed to AA. The yield of DNA isolated from tissues fixed with formalin decreased over 1 week; however, the levels of AA-DNA adducts were similar to those in fresh frozen tissue. Moreover, DNA from FFPE tissue served as a template for PCR amplification, yielding sequence data of comparable quality to DNA obtained from fresh frozen tissue. The estimates of AA-DNA adducts measured in freshly frozen tissue and matching FFPE tissue blocks of human kidney stored for 9 years showed good concordance. Thus, DNA isolated from FFPE tissues may be used to biomonitor DNA adducts and to amplify genes used for mutational analysis, providing clues regarding the origin of human cancers for which an environmental cause is suspected., (© The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Enzymes that form transient DNA-protein covalent complexes are targets for several potent classes of drugs used to treat infectious disease and cancer, making it important to establish robust and rapid procedures for analysis of these complexes. We report a method for isolation of DNA-protein adducts and their identification and quantification, using techniques compatible with high-throughput screening. This method is based on the RADAR assay for DNA adducts that we previously developed (Kiianitsa and Maizels (2013) A rapid and sensitive assay for DNA-protein covalent complexes in living cells. Nucleic Acids Res., 41:e104), but incorporates three key new steps of broad applicability. (i) Silica-assisted ethanol/isopropanol precipitation ensures reproducible and efficient recovery of DNA and DNA-protein adducts at low centrifugal forces, enabling cell culture and DNA precipitation to be carried out in a single microtiter plate. (ii) Rigorous purification of DNA-protein adducts by a procedure that eliminates free proteins and free nucleic acids, generating samples suitable for detection of novel protein adducts (e.g. by mass spectroscopy). (iii) Identification and quantification of DNA-protein adducts by direct ELISA assay. The ELISA-based RADAR assay can detect Top1-DNA and Top2a-DNA adducts in human cells, and gyrase-DNA adducts in Escherichia coli. This approach will be useful for discovery and characterization of new drugs to treat infectious disease and cancer, and for development of companion diagnostics assays for individualized medicine., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Induction of pyrimidine dimers in DNA by solar UV radiation has drastic effects on microorganisms. To better define the nature of these DNA photoproducts in marine bacterioplankton and eukaryotes, a study was performed during a cruise along a latitudinal transect in the Pacific Ocean. The frequency of all possible cyclobutane pyrimidine dimers, pyrimidine (6-4) pyrimidone photoproducts (64PPs) and their related Dewar valence isomers (DEWs) was determined by high-performance liquid chromatography-mass spectrometry. Studied samples were bacterioplankton and eukaryotic fractions isolated from sea water either collected before sunrise or exposed to ambient sunlight from sunrise to sunset. Isolated DNA dosimeters were also exposed to daily sunlight for comparison purposes. A first major result was the observation in all samples of large amounts of DEWs, a class of photoproducts rarely considered outside photochemical studies. Evidence was obtained for a major role of UVA in the formation of these photoisomerization products of 64PPs. Considerations on the ratio between the different classes of photoproducts in basal and induced DNA damage suggests that photoenzymatic repair (PER) is an important DNA repair mechanism used by marine microorganisms occupying surface seawater in the open ocean. This result emphasizes the biological role of DEWs which are very poor substrate for PER., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Accelerator mass spectrometry (AMS) is a highly sensitive technique used for the quantification of adducts following exposure to carbon-14- or tritium-labeled chemicals, with detection limits in the range of one adduct per 10(11)-10(12) nucleotides. The protocol described in this chapter provides an optimal method for isolating and preparing DNA samples to measure isotope-labeled DNA adducts by AMS. When preparing samples, special precautions must be taken to avoid cross-contamination of isotope among samples and produce a sample that is compatible with AMS. The DNA isolation method described is based upon digestion of tissue with proteinase K, followed by extraction of DNA using Qiagen isolation columns. The extracted DNA is precipitated with isopropanol, washed repeatedly with 70 % ethanol to remove salt, and then dissolved in water. DNA samples are then converted to graphite or titanium hydride and the isotope content measured by AMS to quantify adduct levels. This method has been used to reliably generate good yields of uncontaminated, pure DNA from animal and human tissues for analysis of adduct levels.

(32)P-Postlabeling analysis is an ultra-sensitive method for the detection of DNA adducts, such as those formed directly by the covalent binding of carcinogens and mutagens to bases in DNA and other DNA lesions resulting from modification of bases by endogenous or exogenous agents (e.g., oxidative damage). The procedure involves four main steps: enzymatic digestion of the DNA sample; enrichment of the adducts; radiolabeling of the adducts by T4 kinase-catalyzed transference of (32)P-orthophosphate from [γ-(32)P]ATP; chromatographic separation of labeled adducts; and detection and quantification by means of their radioactive decay. Using 10 μg of DNA or less, it is capable of detecting adduct levels as low as 1 adduct in 10(9)-10(10) normal nucleotides. It is applicable to a wide range of investigations, including monitoring human exposure to environmental or occupational carcinogens, determining whether a chemical has genotoxic properties, analysis of the genotoxicity of complex mixtures, elucidation of the pathways of activation of carcinogens, and monitoring DNA repair.

Repair of DNA lesions must occur within the chromatin landscape and is associated with alterations in histone modifications and nucleosome rearrangement. To directly associate these chromatin features with DNA damage and repair, it is necessary to be able to map DNA adducts. We have developed a cyclobutane pyrimidine dimer (CPD)-specific immunoprecipitation method and mapped ultraviolet damage hotspots across human chromosomes 1 and 6. CPD hotspots occur almost equally in genic and intergenic regions. However, these hotspots are significantly more prevalent adjacent to repeat elements, especially Alu repeats. Nucleosome mapping studies indicate that nucleosomes are consistently positioned at Alu elements where CPD hotspots form, but by 2 h post-irradiation, these same regions are significantly depleted of nucleosomes. These results indicate that nucleosomes associated with hotspots of CPD formation are readily rearranged, potentially making them accessible to DNA repair machinery. Our results represent the first chromosome scale map of ultraviolet-induced DNA lesions in the human genome, and reveal the sequence features and dynamic chromatin changes associated with CPD hotspots.

Acetaldehyde and crotonaldehyde are genotoxic aldehydes present in tobacco smoke and vehicle exhaust. The reaction of these aldehydes with 2'-deoxyguanosine in DNA produces α-methyl-γ-hydroxy-1,N(2)-propano-2'-deoxyguanosine (1,N(2)-propanodGuo). Online HPLC-tandem mass spectrometry was utilized to accurately quantify 1,N(2)-propanodGuo in human urinary samples from 47 residents of São Paulo City (SP) and 35 residents of the rural municipality of São João da Boa Vista (SJBV) in the state of São Paulo. Significantly higher 1,N(2)-propanodGuo levels were found in the samples from SP donors than in samples from SJBV donors. Our results provide the first evidence that elevated levels of 1,N(2)-propanodGuo in urinary samples may be correlated with urban air pollution.

Currently there are three major hypotheses that have been proposed for estrogen induced carcinogenicity, however exact etiology remains unknown. Based on the chemical logic, studies were undertaken to investigate if estrogens could generate quinone methides in an oxidative environment which then could cause DNA damage in humans. In presence of MnO2 estrogens were oxidized to quinone methides. Surprisingly quinone methides were found to be stable with t1/2 of 20.8 and 4.5 min respectively. Incubation of estrogens with lactoperoxidase (LPO) and H2O2 resulted in formation of respective quinone methides (E1(E2)-QM). Subsequent addition of adenine to the assay mixture lead to trapping of E1(E2)-QM, resulting in formation of adenine adducts of estrogens, E1(E2)-9-N-Ade. Targeted ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) based metabolomic analysis of the breast tissue extracts showed the presence of adenine adducts of estrogens, E1(E2)-9-N-Ade, along with other estrogen related metabolites. Identity of E1(E2)-N-Ade in LPO assay extracts and breast tissue extracts were confirmed by comparing them to pure synthesized E1(E2)-9-N-Ade standards. From these results, it is evident that peroxidase enzymes or peroxidase-like activity in human breast tissue could oxidize estrogens to electrophilic and stable quinone methides in a single step that covalently bind to DNA to form adducts. The error prone repair of the damaged DNA can result in mutation of critical genes and subsequently cancer. This article reports evidence for hitherto unknown estrogen metabolic pathway in human breast, catalyzed by peroxidase, which could initiate cancer.

Quantitation of DNA adducts could provide critical information on the relationship between exposure to tobacco smoke and cancer risk in smokers. In this study, we developed a robust and sensitive liquid chromatography-tandem mass spectrometry method for the analysis of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing DNA adducts in human oral cells, a noninvasive source of DNA for biomarker studies. Isolated DNA undergoes acid hydrolysis, after which samples are purified by solid-phase extraction and analyzed by LC-ESI-MS/MS. The developed method was applied to the analysis of samples obtained via collection with a commercial mouthwash from 30 smokers and 15 nonsmokers. In smokers, the levels of HPB-releasing DNA adducts averaged 12.0 pmol HPB/mg DNA (detected in 20 out of 28 samples with quantifiable DNA yield), and in nonsmokers, the levels of adducts averaged 0.23 pmol/mg DNA (detected in 3 out of 15 samples). For the 30 smoking subjects, matching buccal brushings were also analyzed, and HPB-releasing DNA adducts were detected in 24 out of 27 samples with quantifiable DNA yield, averaging 44.7 pmol HPB/mg DNA. The levels of adducts in buccal brushings correlated with those in mouthwash samples of smokers (R = 0.73, p < 0.0001). Potentially, the method can be applied in studies of individual susceptibility to tobacco-induced cancers in humans.

³²P-Postlabeling analysis is an ultrasensitive method for the detection and quantitation of DNA adducts and covalent modifications of the DNA. It consists of four main steps: (1) enzymatic digestion of DNA to 3'-monophosphate nucleosides; (2) enrichment of the adducts; (3) 5'OH-labeling of the adducts by T4 polynucleotide kinase-catalyzed transfer of ³²P-orthophosphate from [γ-³²P]ATP; and (4) chromatographic or electrophoretic separation of labeled adducts and detection and quantification by means of their radioactive decay. The assay requires only micrograms of DNA and is capable of detecting adduct levels as low as one adduct in 10⁹-10¹⁰ normal nucleotides. It is applicable to a wide range of investigations in human, animal, and in vitro studies including monitoring exposure to environmental or occupational carcinogens, determining whether a chemical or a complex mixture has genotoxic properties, elucidation of the toxicological pathways of carcinogens, and monitoring DNA repair.

In this manuscript, an efficient high resolution reversed phase-ion pairing-liquid chromatography electrospray ionization tandem mass spectrometry (RP-IP-LC-MS/MS) method for separation of isomeric modified oligonucleotides using a polymeric (styrene-divinylbenzene) monolithic capillary column is presented. The effects of different ion pairing reagents (IPR), their concentration, mobile phase additives and conditions were evaluated towards achieving the highest possible resolution and chromatographic separation of isomeric oligonucleotides. Ion pairing reagents and mobile phase conditions were evaluated using as model N-acetylaminofluorene [AAF] adducted ss-oligonucleotides (CCC CGA GCA ATC TCA AT). The optimized mobile phase conditions were then applied for the mapping of sites of base modification of AAF adducted 15-base pair oligonucleotide fragments containing codon 135 of the p53 gene and for profiling a complex synthetic oligonucleotide mixture. The optimized method utilizes a monolithic poly(styrene-divinylbenzene) capillary column, triethylammonium bicarbonate as ion pairing reagent and methanol as organic modifier to perform IP-RPLC-ESI-MS/MS separation. The results show that the method is simultaneously applicable not only to oligonucleotide fragments adducted separately by different carcinogens but also to the analysis of multiple adducts in the same oligonucleotide fragment in a single experiment. The method presents itself as a tool for the identification, characterization and mapping of oligonucleotide adducts as biomarkers for DNA damage from carcinogens., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Methanol is a large volume industrial chemical and widely used solvent and fuel additive. Methanol's well known toxicity and use in a wide spectrum of applications has raised long-standing environmental issues over its safety, including its carcinogenicity. Methanol has not been listed as a carcinogen by any regulatory agency; however, there are debates about its carcinogenic potential. Formaldehyde, a metabolite of methanol, has been proposed to be responsible for the carcinogenesis of methanol. Formaldehyde is a known carcinogen and actively targets DNA and protein, causing diverse DNA and protein damage. However, formaldehyde-induced DNA adducts arising from the metabolism of methanol have not been reported previously, largely due to the absence of suitable DNA biomarkers and the inability to differentiate what was due to methanol compared with the substantial background of endogenous formaldehyde. Recently, we developed a unique approach combining highly sensitive liquid chromatography-mass spectrometry methods and exposure to stable isotope labeled chemicals to simultaneously quantify formaldehyde-specific endogenous and exogenous DNA adducts. In this study, rats were exposed daily to 500 or 2000 mg/kg [¹³CD₄]-methanol by gavage for 5 days. Our data demonstrate that labeled formaldehyde arising from [¹³CD₄]-methanol induced hydroxymethyl DNA adducts in multiple tissues in a dose-dependent manner. The results also demonstrated that the number of exogenous DNA adducts was lower than the number of endogenous hydroxymethyl DNA adducts in all tissues of rats administered 500 mg/kg per day for 5 days, a lethal dose to humans, even after incorporating an average factor of 4 for reduced metabolism due to isotope effects of deuterium-labeled methanol into account.

Tobacco smoking is one of the leading causes for oral cancer. Dibenzo[a,l]pyrene (DB[a,l]P), an environmental pollutant and a tobacco smoke constituent, is the most carcinogenic polycyclic aromatic hydrocarbon (PAH) tested to date in several animal models (target organs: skin, lung, ovary, and mammary tissues). We have recently demonstrated that DB[a,l]P is also capable of inducing oral cancer in mice; however, its metabolic activation to the ultimate genotoxic metabolite dibenzo[a,l]pyrene-11,12-dihydrodiol-13,14-epoxide (DB[a,l]PDE) in mouse oral cavity has not been examined. Here we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to detect and quantify (±)-anti-DB[a,l]PDE-dA adducts in oral tissues of mice treated with DB[a,l]P. [(15)N(5)]-(±)-anti-DB[a,l]PDE-N(6)-dA adducts were synthesized as internal standards. The stereoisomeric adducts were characterized by MS, NMR, and CD analysis. The detection limit of the method is 8 fmol with 100 μg of digested DNA as the matrix. Two adducts were detected and identified as (-)-anti-cis and (-)-anti-trans-DB[a,l]PDE-dA in the oral tissues of mice following the direct application of DB[a,l]P (240 nmol per day, for 2 days) into the oral cavity, indicating that DB[a,l]P is predominantly metabolized into (-)-anti-DB[a,l]PDE in this target organ. We also compared the formation and removal of adducts as a function of time, following the direct application of DB[a,l]P (24 nmol, 3 times per week for 5 weeks) into the oral cavity of mice. Adducts were quantified at 48 h, 1, 2, and 4 weeks after the last dose. Maximal levels of adducts occurred at 48 h, followed by a gradual decrease. The levels (fmol/μg DNA) of (-)-anti-trans adducts (4.03 ± 0.27 to 1.77 ± 0.25) are significantly higher than (-)-anti-cis-DB[a,l]PDE-dA adduct (1.63 ± 0.42 to 0.72 ± 0.04) at each time point (p < 0.005). The results presented here indicate that the formation and persistence of (-)-anti-DB[a,l]PDE-dA adducts may, in part, contribute to the initiation of DB[a,l]P-induced oral carcinogenesis.

Here, we demonstrate the potential for high-resolution electrophoretic separations of ssDNA-protein conjugates in borosilicate glass microfluidic chips, with no sieving media and excellent repeatability. Using polynucleotides of two different lengths conjugated to moderately cationic protein polymer drag-tags, we measured separation efficiency as a function of applied electric field. In excellent agreement with prior theoretical predictions of Slater et al., resolution is found to remain constant as applied field is increased up to 700 V/cm, the highest field we were able to apply. This remarkable result illustrates the fundamentally different physical limitations of free-solution conjugate electrophoresis (FSCE)-based DNA separations relative to matrix-based DNA electrophoresis. ssDNA separations in "gels" have always shown rapidly declining resolution as the field strength is increased; this is especially true for ssDNA > 400 bases in length. FSCE's ability to decouple DNA peak resolution from applied electric field suggests the future possibility of ultra-rapid FSCE sequencing on chips. We investigated sources of peak broadening for FSCE separations on borosilicate glass microchips, using six different protein polymer drag-tags. For drag-tags with four or more positive charges, electrostatic and adsorptive interactions with poly(N-hydroxyethylacrylamide)-coated microchannel walls led to appreciable band-broadening, while much sharper peaks were seen for bioconjugates with nearly charge-neutral protein drag-tags., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Catechol estrogen-derived DNA adducts are formed as a result of the reaction of catechol estrogen metabolites (e.g., catechol estrogen quinones) with DNA to form depurinating adducts. Developing a new methodology for the detection of various DNA adducts is essential for medical diagnostics, and to this end, we demonstrate the applicability of on-chip capillary electrophoresis with an integrated electrochemical system for the separation and amperometric detection of various catechol estrogen-derived DNA adducts. A hybrid PDMS/glass microchip with in-channel amperometric detection interfaced with in situ palladium decoupler is utilized and presented. The influence of buffer additives along with the effect of the separation voltage on the resolving power of the microchip is discussed. Calibration plots were constructed in the range 0.4-10 μM with r(2) ≥ 0.999, and detection limits in the attomole range are reported. These results suggest that on-chip analysis is applicable for analyzing various DNA adducts as potential biomarkers for future medical diagnostics.

Modifications of genomic DNA may change gene expression and cause adverse health effects. Here we for the first time demonstrate a particle counting immunoassay for rapid and sensitive detection of DNA modifications using benzo[a]pyrenediol epoxide (BPDE)-DNA adducts as an example. The BPDE-adducted DNA is specifically captured by immunomagnetic particles and then isolated from unmodified DNA by applying an external magnetic field. By taking advantage of the fluorescence signal amplification through multiple labeling of captured DNA by OliGreen dye, the captured BPDE-DNA adducts can be quantified by particle counting from fluorescence imaging. This clearly demonstrates that the number of fluorescently countable particles is proportional to the modification content in genomic DNA. It is interesting to note that the background fluorescence signal caused by nonspecific adsorption of OliGreen dye can be more effectively quenched than that induced by the binding of OliGreen dye to ssDNA, allowing for significant reduction in the background fluorescence and further enhancing the detection sensitivity. The developed method can detect trace BPDE-DNA adducts as low as 180 fM in the presence of 1 billion times more normal nucleotides in genomic DNA and has a dynamic range over 4 orders of magnitude. By using anti-5-methylcytosine antibody, the method is extended to the detection of global DNA methylation. With high sensitivity and specificity, this rapid and easy-to-perform analytical method for DNA modifications shows a broad spectrum of potential applications in genotoxical and epigenetic analysis.

Acrolein (AC) and 4-hydroxy-2-nonenal (HNE) are endogenous bis-electrophiles that arise from the oxidation of polyunsaturated fatty acids. AC is also found in high concentrations in cigarette smoke and automobile exhaust. These reactive α,β-unsaturated aldehyde (enal) covalently modify nucleic acids, to form exocyclic adducts, where the three-carbon hydroxypropano unit bridges the N1 and N(2) positions of deoxyguanosine (dG). The bifunctional nature of these enals allows them to undergo reaction with a second nucleophilic group and form DNA cross-links. These cross-linked enal adducts are likely to contribute to the genotoxic effects of both AC and HNE. We have developed a sensitive mass spectrometric method to detect cross-linked adducts of these enals in calf thymus DNA (CT DNA) treated with AC or HNE. The AC and HNE cross-linked adducts were measured by the stable isotope dilution method, employing a linear quadrupole ion trap mass spectrometer and consecutive reaction monitoring at the MS(3) or MS(4) scan stage. The lower limit of quantification of the cross-linked adducts is ∼1 adduct per 10(8) DNA bases, when 50 μg of DNA is assayed. The cross-linked adducts occur at levels that are ∼1-2% of the levels of the monomeric 1,N(2)-dG adducts in CT DNA treated with either enal.

Leukotriene A(4) (LTA(4)), a reactive electrophilic intermediate formed during the biosynthesis of inflammation-related lipid mediators, has been found to bind covalently to DNA. The major DNA adducts formed by LTA(4) in vitro and human cells have been identified by mass spectrometry on the nucleoside level. Here we investigated whether the thin-layer chromatography (TLC) (32)P-postlabeling method is suitable for the detection of LTA(4)-DNA adducts. The reaction of individual deoxynucleoside 3'-monophosphates with LTA(4) in aqueous basic solution yielded numerous adduct spots when analyzed by the two enrichment procedures of the (32)P-postlabeling method-nuclease P1 digestion and butanol extraction. Highest LTA(4)-adduct levels were found with deoxyguanosine 3'-phosphate (around one adduct per 10(4) normal nucleotides). Under similar reaction conditions LTA(4) (25-320 microM) was incubated with calf thymus DNA, then DNA adduct patterns and levels were determined with the TLC (32)P-postlabeling method using both enrichment versions. The same DNA adduct pattern consisting of up to seven spots was observed with both enrichment versions. DNA adduct formation by LTA(4) was concentration-dependent with major adducts being derived from deoxyguanosine. When a human monocytic cell line (Mono Mac 6) was stimulated with arachidonic acid and calcium ionophore LTA(4)-DNA adducts were detected by (32)P-postlabeling. However, the level of these endogenously formed DNA adducts was close to the detection limit (3 +/- 2 adducts per 10(8) normal nucleotides). In summary, the TLC (32)P-postlabeling method is suitable for studying DNA adduct formation by LTA(4) and can be used for further investigations on the link between inflammation and cancer.

The reactions of adenosine with malonaldehyde and glyoxal, and with malonaldehyde and methylglyoxal resulted in the formation of one malonaldehyde-glyoxal and one malonaldehyde-methylglyoxal conjugate adduct, respectively. These adducts were isolated and purified by reversed-phase liquid chromatography, and structurally characterized by UV, (1)H- and (13)C-NMR spectroscopy, and mass spectrometry. The malonaldehyde-glyoxal adduct was identified as 8-(diformylmethyl)-3-(beta-D-ribofuranosyl)imidazo[2,1-i]purine (M(1)Gx-A), while the malonaldehyde-methylglyoxal one as 8-(diformylmethyl)-7-methyl-3-(beta-D-ribofuranosyl)imidazo[2,1-i]purine (M(1)MGx-A). Both adducts were also observed in calf thymus DNA when incubated in the respective aldehydes under physiological pH and temperature. Moreover, in the reaction of methylglyoxal and malonaldehyde with adenosine, an additional adduct was formed. This adduct was found to consist of one unit derived from methylglyoxal and one unit from formaldehyde. The adduct was identified as N(6)-(2,3-dihydroxy-2-methylpropanoyl)-9-(beta-D-ribofuranosyl)purine (MGxFA-A). Formaldehyde was found to originate from the commercial methylglyoxal in which it was present as an impurity.

The potent renal carcinogenicity of ochratoxin A (OTA) in rats, principally in the male, raises questions about mechanism. Chromatographic evidence of DNA adducts after (32)P-postlabeling analysis contrasts with experimental attempts to demonstrate the absence of OTA in such adducts. Proffered schemes for alternative epigenetic mechanisms in OTA carcinogenicity remain unsatisfying, while structural data substantiating DNA-OTA adducts has also been lacking. We report refined (32)P-postlabeling methodology revealing one principal adduct isolated in small amounts from the kidneys of all five Fischer and five Dark Agouti rats to which OTA had been given on four consecutive days. We also describe structural data for the principal adduct from OTA/DNA interaction in vitro and its subsequent preparative isolation by the postlabeling methodology (as C-C8 OTA 3'dGMP), essentially creating an ochratoxin B-guanine adduct. Reasoning for the unsuitability of experimental protocols in published evidence claiming nongenotoxicity of OTA is given. In vivo exposure of renal DNA to cycles of adduction with OTA, necessarily protracted for carcinogenesis to occur, can reasonably explain an occasional focal neoplasm from which metastasizing carcinoma could develop.

There have been many attempts in the past to determine whether significant levels of Adriamycin-DNA adducts form in cells and contribute to the anticancer activity of this agent. Supraclincal drug levels have been required to study drug-DNA adducts because of the lack of sensitivity associated with many of the techniques employed, including liquid scintillation counting of radiolabeled drug. The use of accelerator mass spectrometry (AMS) has provided the first direct evidence of Adriamycin-DNA adduct formation in cells at clinically relevant Adriamycin concentrations. The exceedingly sensitive nature of AMS has enabled over three orders of magnitude increased sensitivity of Adriamycin-DNA adduct detection (compared to liquid scintillation counting) and has revealed adduct formation within an hour of drug treatment. The rigorous protocol required for this approach, together with many notes on the precautions and procedures required in order to ensure that absolute levels of Adriamycin-DNA adducts can be determined with good reproducibility, is outlined in this chapter.

Dibenzo[a,l]pyrene (DBP) is the most potent tumor initiating polycyclic aromatic hydrocarbon tested to date in rodent tumor models. To investigate how DBP adduct formation and removal might influence carcinogenesis, we have examined the effects of treatment of several nucleotide excision repair (NER)-proficient (NER(+)) and -deficient (NER(-)) cell lines with the carcinogenic metabolite (+/-)-anti-DBP-11,12-diol-13,14-epoxide (DBPDE). The treatment of NER(-) cells with (+/-)-anti-DBPDE for 0.5, 1, or 2 h yielded similar total adduct levels, indicating that adduct formation was essentially complete during a 2 h treatment period with no additional adducts produced after replacement of media. In all cell lines, treatment with (+/-)-anti-DBPDE generated five major and at least two minor adducts that were chromatographically identical to those formed by direct treatment of 3'-GMP and 3'-AMP with (+/-)-anti-DBPDE. When adduct levels were assessed in NER(-) cells, the number of adducts/10(9) nucleotides decreased over time, suggesting that DNA replication was ongoing, so we incorporated a normalization strategy based on DNA synthesis. This strategy indicated that DBPDE-DNA adduct levels in NER(-) cells are stable over time. After normalization for DNA synthesis in the NER(+) cells, our data indicated that three adducts showed biphasic repair kinetics. A faster rate of removal was observed during the first 6 h following DBPDE removal followed by a slower rate for up to 34 h. Importantly, two of the major guanine adducts were particularly refractory to removal in the NER(+) cells. Our results suggest that the extreme carcinogenicity of DBPDE may result from the ability of a substantial percentage of two structurally distinct DBPDE-DNA adducts to escape repair.

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.

Aristolochic acid (AA), derived from the herbal genus Aristolochia and Asarum, has recently been shown to be associated with the development of nephropathy. Upon enzyme activation, AA is metabolized to the aristolactam-nitrenium ion intermediate, which reacts with the exocyclic amino group of the DNA bases via an electrophilic attack at its C7 position, leading to the formation of the corresponding DNA adducts. The AA-DNA adducts are believed to be associated with the nephrotoxic and carcinogenic effects of AA. In this study, liquid chromatography coupled with electrospray ionization mass spectrometry (LC-MS) was used to identify and quantify the AA-DNA adducts isolated from the kidney and liver tissues of the AA-dosed rats. The deoxycytidine adduct of AA (dC-AA) and the deoxyadenosine-AA adduct (dA-AA) were detected and quantified in the tissues of rats with one single oral dose (5mg or 30mg AA/kg body weight). The deoxyguanosine adduct (dG-AA), however, was detected only in the kidney of rats that were dosed at 30mg AA/kg body weight for three consecutive days. The amount of AA-DNA adducts found in the rats correlated well with the dosage.

Oxaliplatin is a third-generation platinum complex, and has a broad spectrum of antitumor activity. Such platinum complexes with the DACH carrier ligand have recently received increasing attention since they show efficacy against cisplatin-resistant cell lines. As the foremost indication of antitumor activity of platinum drugs is the formation of adducts with genomic DNA, calf thymus DNA-oxaliplatin adducts were the major target in this study. Calf thymus DNA was incubated with oxaliplatin, resulting in the formation of a large number of platinum-DNA adducts. Treated DNA was digested into the dinucleotides with a combination of enzymes, namely, benzonase, alkaline phosphatase, and nuclease S1. Using a high-performance liquid chromatography, we carried out the separation of individual platinum-DNA adducts which were concurrently identified using electrospray ionization ion trap mass spectrometry (MS). Both 1,2-intrastrand and 1,2-interstrand cross-linked adducts were found; however, those of the intrastrand nature have a considerably higher abundance than those of the interstrand cross-links. Among them, d(GpG)-oxaliplatin was the most abundant bifuctional adduct. To a lesser extent, a few monofunctional adducts were detected as well. MS(n) experiments served to ascertain the detailed structures of oxaliplatin adducts of dinucleoside monophosphates and of dinucleotides.

Melphalan is a bifunctional alkylating agent that covalently binds to the nucleophilic sites present in DNA. In this study we investigated oligonucleotides prepared enzymatically from DNA modified with melphalan. Calf thymus DNA was incubated in-vitro with melphalan and the resulting modifications were enzymatically cleaved by means of benzonase and nuclease S1. Efficient sample preconcentration was achieved by solid-phase extraction, in which phenyl phase cartridges resulted in better recovery of the modified species than C(18). The applied enzymatic digestion time resulted in production of trinucleotide adducts which were efficiently separated and detected by use of reversed-phase HPLC coupled to an ion-trap mass spectrometer with electrospray ionization. It was assumed that melphalan could act as both a monofunctional and bifunctional alkylating agent. Mono-alkylated adducts were much more abundant, however, and the alkylation site was located on the nucleobases. On the other hand, we unequivocally identified cross-link formation in DNA, even though at low abundance and only a few adduct types were detected.

Objectives: Ellipticine is a potent antineoplastic agent exhibiting multiple mechanisms of its action. Recently, we have found that 13-hydroxyellipticine, formed from ellipticine as the predominant metabolite in human livers, is bound to deoxyguanosine in DNA, generating the major DNA adduct in vivo and in vitro. The development of the methods suitable for the preparation of this adduct in the amounts sufficient for identification of its structure and those for its isolation and partial characterization is the aim of this study., Methods: High performance liquid chromatography (HPLC) was employed for separation of 13-hydroxyellipticine-mediated deoxyguanosine adduct. The 32P-postlabeling technique was utilized to detect this adduct in DNA., Results: The formation of the 13-hydroxyellipticine-derived deoxyguanosine adduct in DNA in vitro was increased under the alkaline pH of the incubations and by the formation of the sulfate and acetate conjugates of 13-hydroxyellipticine generated by reactions with 3'-phosphoadenosine-5'-phosphosulfate (PAPS) or acetyl-coenzyme A (acetyl-CoA) catalyzed by human sulfotransferases (SULTs) 1A1 and 1A2 and N,O-acetyltransferases (NATs) 1 and 2. The HPLC method suitable for separation the 13-hydroxyellipticine-derived deoxyguanosine adduct from other reactants, deoxyguanosine and 13-hydroxyellipticine, was developed. The structure of this adduct is proposed to correspond to the product formed from ellipticine-13-ylium with the exocyclic 2-NH2 group of guanine in DNA., Conclusions: The data are the first report on HPLC isolation of the deoxyguanosine adduct formed by 13-hydroxyellipticine in DNA and its partial characterization.

The reactions of glyoxal with 2'-deoxyguanosine and calf thymus single- and double-stranded DNA in aqueous buffered solutions at physiological conditions resulted in the formation of two previously undetected adducts in addition to the known reaction product 3-(2'-deoxy-beta-D-erythro-pentofuranosyl)-5,6,7-trihydro-6,7-dihydroxyimidazo[1,2-a]purine-9-one (Gx-dG). The adducts were isolated and purified by reversed-phase liquid chromatography and structurally characterised by UV absorbance, mass spectrometry, (1)H and (13)C NMR spectroscopy. The hitherto unknown adducts were identified as: 5-carboxymethyl-3-(2'-deoxy-beta-D-erythro-pentofuranosyl)-5,6,7-trihydro-6,7-dihydroxyimidazo[1,2-a]purine-9-one (Gx(2)-dG) and N(2)-(carboxymethyl)-9-(2'-deoxy-beta-D-erythro-pentofuranosyl)-purin-6(9H)-one (Gx(1)-dG). Both adducts were shown to arise from Gx-dG. Gx-dG and Gx(2)-dG were found to be unstable and partly transformed to Gx(1)-dG, which is a stable adduct and seems to be the end-product of the glyoxal reaction with 2'-deoxyguanosine. All adducts formed in the reaction of glyoxal with 2'-deoxyguanosine were observed in calf thymus DNA. Also in DNA, Gx(1)-dG was the only stable adduct. The transformation of Gx-dG to Gx(1)-dG seemed to take place in single-stranded DNA and therefore, Gx(1)-dG may be a potentially reliable biomarker for glyoxal exposure and may be involved in the genotoxic properties of the compound.

Benzo[a]pyrene, a common environmental pollutant, can be metabolized into reactive anti-benzo[a]pyrene diol epoxide (anti-BPDE), which predominantly binds to deoxyguanine in DNA and forms four stereoisomeric adducts. To characterize the stereochemistry of these adduct isomers, preparation of single adducted deoxyguanosine (dG) is required for efficient enantiomeric analysis. Here, we demonstrate an improved method for preparation, identification, and analysis of four BPDE-adducted dGs, including (+)-trans-, (-)-trans-, (+)-cis-, and (-)-cis-anti-BPDE-N(2)-dG. These stereoisomerically adducted nucleosides were first synthesized by a direct reaction of (+/-)-anti-BPDE with dG, followed by optimized solid-phase extraction (SPE) and HPLC purification. The reaction of (+/-)-anti-BPDE and dG displayed a yield as high as 45%. The developed preparation method does not require any enzymatic digestion. Based on highly efficient separation achieved by optimization of stationary phase and mobile phase, LC-UV-MS/MS and LC-diode array detection (DAD)-fluorescence detection (FL) methods were established for characterization and analysis of the four stereoisomeric anti-BPDE-dGs. The established LC-DAD-FL method may provide characterization and analysis of four stereoisomeric anti-BPDE-dGs and two interfering anti-BPDE tetrols by taking advantage of their distinct fluorescence quenching.

In this report we show that human DNA Topoisomerase I (Top1) forms DNA-protein adducts with nicked and gapped DNA structures lacking a conventional Top1 cleavage site. The radioactively labeled crosslinking products were identified by SDS-gel electrophoresis. The chemical structure of the groups at 5' or 3' end of the nick does not have an effect on the formation of these covalent adducts. Therefore, all kinds of nicks, either directly induced by ionizing radiation or reactive oxygen species or indirectly induced in the course of base excision repair (BER) are targets for Top1 that competes with BER proteins and other nick-sensors. Top1-DNA covalent adducts formed in cells exposed to DNA damaging agents can promote genetic instability., ((c) 2008 IUBMB.)

Antibody cross-reactivity makes separation and differentiation of enantiomeric analytes one of the most challenging problems in immunoanalytical research, particularly for the analysis of structurally related biological molecules [such as benzo( a)pyrene (BP) metabolites and BP-derived DNA adducts]. It has recently been shown that the interaction of enantiomers of BP tetrols (BPT) with a promiscuous anti-polycyclic aromatic hydrocarbon ( anti-PAH) monoclonal antibody (mAb) allowed for separation of all four enantiomeric isomers using immunoaffinity capillary electrophoresis [ Grubor, N. M. , Armstrong, D. W. , and Jankowiak, R. ( 2006) Electrophoresis 27, 1078 ] and unambiguous spectral resolution using fluorescence line narrowing spectroscopy (FLNS) [ Grubor, N. M. , Liu, Y. , Han, X. , Armstrong, D.W. , and Jankowiak, R. ( 2006) J. Am.Chem. Soc. 128, 6409 ]. Here, we expand the use of the above two methodologies to the group of biologically important molecules that are products of BP diol epoxide (BPDE)-induced DNA damage. Four diastereomeric anti-BPDE-derived deoxyguanosine (dG) adducts, that is, (+)- and (-)- anti-trans-BPDE- N (2)-dG and (+)- and (-)- anti-cis-BPDE- N (2)-dG, were electrophoretically separated and spectroscopically differentiated using 8E11 mAb raised against BP-DNA conjugates. In fluorescence line narrowing spectroscopy (FLNS) experiments, complexes of BPDE-dG adducts with mAb revealed differences in fluorescence origin band positions, bandwidths, and vibrational patterns for all four BPDE- N (2)-dG adducts. Narrow fluorescence origin bands observed for (-)- trans-BPDE-dG (70 cm (-1)) and (+)- trans-BPDE- N (2)-dG (80 cm (-1)) suggest spatial constraint within the mAb binding pocket. Broader origin bands observed for cis type adducts ( approximately 120 cm (-1)) in 8E11 mAb suggest different binding geometries and/or conformational changes, as also indicated by changes in vibrational frequencies observed for the (+)- anti-cis and (-)- anti-cis adducts complexed with mAb. FLNS revealed that binding conformations and interactions within the mAb binding pocket are different for each adduct, enabling unambiguous positive identification. The methodologies described in this manuscript could also be used for analysis of DNA adducts following enzymatic hydrolysis of BPDE-adducted DNA to free nucleosides.

An ion-pair reversed-phase nano-high-performance liquid chromatography (IP-RP-nano-HPLC) method using a monolithic poly(styrene-divinylbenzene) (PS-DVB) column coupled to nanoelectrospray ionization mass spectrometry (nano-ESI-MS) was evaluated to separate and identify isomeric oligonucleotide adducts derived from the covalent binding of (+/-)-anti-7r,8t-dihydroxy-9t,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(+/-)-anti-BPDE] to double-stranded (ds) 5'-PO4--ACCCGCGTCCGCGC-3'/5'-GCGCGGGCGCGGGT-3' oligonucleotide. The influence of three different nanospray emitters on electrospray signal was evaluated in terms of analyte ion sensitivity. The best nanoelectrospray performance for the oligonucleotides was observed with the distal metal-coated emitter. The performance of three different stationary phases was also investigated. The chromatographic separation performance of the polymeric monolithic PS-DVB stationary phase significantly surpassed that of columns packed with the microparticulate sorbents C18 or PS-DVB. Different mobile phase organic solvents and ion-pairing reagents were also evaluated. An optimized mobile phase consisting of methanol and 25 mM triethylammonium bicarbonate resulted in the best chromatographic resolution and increased MS sensitivity of the oligonucleotides. By using a monolithic PS-DVB stationary phase fabricated in a nanocolumn, four positional isomeric (+/-)-BPDE-oligonucleotide adducts were separated and identified. In addition to four of the possible five positional isomers, three positional isomers were also resolved to several diastereoisomers, although their stereostructures could not be identified in the absence of reference standards.

8-hydroxy-deoxyguanosine (8-OH-dG) DNA adduct is one of the most frequently used biomarkers reporting on the oxidative stress that leads to DNA damage. More sensitive and reliable microfluidic devices are needed for the detection of these biomarkers of interest. We have developed a capillary electrophoresis (CE)-based microfluidic device with an electroplated palladium decoupler that provides significantly improved detection limit, separation efficiency, and resolving power. The poly(dimethylsiloxane) (PDMS)/glass hybrid device has fully integrated gold microelectrodes covered in situ with palladium nanoparticles using an electroplating technique. The performance and coverage of the electrodes electroplated with palladium particles were evaluated electrochemically and via scanning electron microscope (SEM) imaging, respectively. The performance of the device was tested and evaluated with different buffer systems, pH values, and electric field strengths. The results showed that this device has significantly improved resolving power, even at separation electric field strengths as high as 600 V cm-1. The detection limit for the 8-OH-dG adduct is about 20 attomoles; the concentration limit is on the order of 100 nM (S/N=3). A linear response is reported for both 8-OH-dG and dG in the range from 100 nM to 150 microM (approximately 100 pA microM-1) with separation efficiencies of approximately 120,000-170,000 plates m-1.

There is substantial evidence to suggest that polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (B[a]P) induce lung cancer through metabolic activation. As part of a program to delineate the routes of PAH activation, we have examined DNA adducts that are formed in human lung cells. A stable isotope dilution liquid chromatography/multiple reaction monitoring mass spectrometry method was used to quantify eight anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro-B[a]P (B[a]PDE)-derived DNA adducts in four H358 human bronchoalveolar cell lines with different phenotypes. In P450 1A1/P450 1B1-induced H358 cells exposed to (+/-)-B[a]P-7,8-dihydro-7,8-diol (B[a]P-7,8-dihydrodiol), (+)-anti-trans-B[a]PDE-N2-2'-deoxyguanosine [(+)-anti-trans-B[a]PDE-N2-dGuo] was the major DNA adduct, and it formed with no lag phase. In AKR1A1-transfected H358 cells, (+)-anti-trans-B[a]PDE-N2-dGuo was also the major adduct with a 3 h lag phase before significant adduct formation was detected. In AKR1A1-transfected H358 cells with induced P450 1A1/P450 1B1, (+)-anti-trans-B[a]PDE-N2-dGuo was formed with no lag phase in amounts similar to those in the H358 cells with up-regulated P450 1A1/P450 1B1. Surprisingly, the greatest amount of (+)-anti-trans-B[a]PDE-N2-dGuo was formed in the control H358 cells. Furthermore, (+)-anti-trans-B[a]PDE-N2-dGuo formation was 2-fold higher in (-)-B[a]P-7,8-dihydrodiol-exposed H358 cells when compared with (+/-)-B[a]P-7,8-dihydrodiol-exposed cells. The P450 1A1/1B1 inhibitor 2,4,3',5'-tetramethoxystilbene did not attenuate DNA adduct formation in the control H358 cells, suggesting that another P450 was responsible. These data raise the intriguing possibility that P450 1A1/P450 1B1 and AKR1A1 may be protective against (+)-B[a]PDE-mediated DNA damage.

In this study, we investigated the products formed following the reaction of benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (B[a]PDE) with 2'-deoxynucleoside 3'-monophosphates. The B[a]PDE plus 2'-deoxynucleotide reaction mixtures were purified using solid phase extraction (SPE) and subjected to HPLC with fluorescence detection. Fractions corresponding to reaction product peaks were collected and desalted using SPE prior to analysis for the presence of molecular ions corresponding to m/z 648, 632, 608 and 623 [M-H]- consistent with B[a]PDE adducted (either on the base or phosphate group) 2'-deoxynucleotides of guanine, adenine, cytosine and thymine, respectively, using LC-ESI-MS/MS collision-induced dissociation (CID). Reaction products were identified having CID product ion spectra containing product ions at m/z 452, 436 and 412 [(B[a]Ptriol+base)-H]-, resulting from cleavage of the glycosidic bond between the 2'-deoxyribose and base, corresponding to B[a]PDE adducts of guanine, adenine and cytosine, respectively. Further reaction products were identified having unique CID product ion spectra characteristic of B[a]PDE adduct formation with the phosphate group of the 2'-deoxynucleotide. The presence of product ions at m/z 399 and 497 were observed for all four 2'-deoxynucleotides, corresponding to [(B[a]Ptriol+phosphate)-H]- and [(2'-deoxyribose+phosphate+B[a]Ptriol)-H]-, respectively. In conclusion, this investigation provides the first direct evidence for the formation of phosphodiester adducts by B[a]PDE following reaction with 2'-deoxynucleotides.

Immuno-spin trapping is a highly sensitive method for detecting DNA radicals in biological systems. This technique involves three main steps: (i) in situ and real-time trapping of DNA radicals with the nitrone spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), thus forming DMPO-DNA nitrone adducts (referred to here as nitrone adducts); (ii) purification of nitrone adducts; and (iii) analysis of nitrone adducts by heterogeneous immunoassays using Abs against DMPO. In experiments, DMPO is added prior to the formation of free radicals. It diffuses easily through all cell compartments and is present when DNA free radicals are formed as a result of oxidative damage. Due to its low toxicity, DMPO can be used in cells at high enough concentrations to out-compete the normal reactions of DNA radicals, thus ensuring a high yield of DNA nitrone adducts. Because both protein and DNA nitrone adducts are formed, it is important that the DNA be pure in order to avoid misinterpretations. Depending on the model under study, this protocol can be completed in as few as 6 h.

Detection and analysis of DNA adducts resulting from endogenous or exogenous exposures to carcinogens are essential not only for quantifying biologically effective doses but also for establishing relationships between exposure and cancer risk. We have developed and validated a procedure of high sensitivity and specificity based on fluorescence labeling of DNA adducts combined with high-performance liquid chromatography-laser-induced fluorescence detection. The fluorescent dye 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY FL) was used to label the deoxynucleoside adducts N-(2'-deoxyguanosine-8-yl)-4-aminobiphenyl and N-(2'-deoxyguanosine-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and the base adduct aflatoxin B(1)-formamidopyrimidine by acylation. The labeling reaction was carried out on adducts at 1pmol to 30nmol concentrations at 25 degrees C for 4h in dichloromethane with 200- to 5000-fold excess of BODIPY FL. BODIPY FL and its activating agents 1,3-dicyclohexylcarbodiimide and 4-dimethylaminopyridine were used at a molar ratio of 1:2:2. Under these conditions, all of the above adducts were quantitatively converted to bis-labeled products, as confirmed by mass spectrometry. Sites of derivatization of adduct deoxynucleosides were established primarily by nuclear magnetic resonance and by collision-induced dissociation mass spectrometric analysis, which indicated that the bis-BODIPY groups were located predominantely on the 3'- and 5'-hydroxyl groups of the deoxyribose ring.

3-Nitrobenzanthrone (3-NBA) is an environmental pollutant and suspected human carcinogen found in emissions from diesel and gasoline engines and on the surface of ambient air particulate matter; human exposure to 3-NBA is likely to occur primarily via the respiratory tract. In our study female Sprague Dawley rats were treated by intratracheal instillation with a single dose of 0.2 or 2 mg/kg body weight of 3-NBA. Using the butanol enrichment version of the (32)P-postlabeling method, DNA adduct formation by 3-NBA 48 hr after intratracheal administration in different organs (lung, pancreas, kidney, urinary bladder, heart, small intestine and liver) and in blood was investigated. The same adduct pattern consisting of up to 5 DNA adduct spots was detected by thin layer chromatography in all tissues and blood and at both doses. Highest total adduct levels were found in lung and pancreas (350 +/- 139 and 620 +/- 370 adducts per 10(8) nucleotides for the high dose and 39 +/- 18 and 55 +/- 34 adducts per 10(8) nucleotides for the low dose, respectively) followed by kidney, urinary bladder, heart, small intestine and liver. Adduct levels were dose-dependent in all organs (approximately 10-fold difference between doses). It was demonstrated by high performance liquid chromatography (HPLC) that all 5 3-NBA-derived DNA adducts formed in rats after intratracheal instillation are identical to those formed by other routes of application and are, as previously shown, formed from reductive metabolites bound to purine bases. Although total adduct levels in the blood were much lower (41 +/- 27 and 9.5 +/- 1.9 adducts per 10(8) nucleotides for the high and low dose, respectively) than those found in the lung, they were related to dose and to the levels found in lung. These results show that uptake of 3-NBA by the lung induces high levels of specific DNA adducts in several organs of the rat and an identical adduct pattern in DNA from blood. Therefore, 3-NBA-DNA adducts present in the blood are useful biomarkers for exposure to 3-NBA and may help to assess the effective biological dose in humans exposed to it.

3-Nitrobenzanthrone (3-NBA) has been isolated from diesel exhaust and airborne particles and identified as a potent direct-acting mutagen in vitro and genotoxic agent in vivo. In order to evaluate the in vivo toxicity and carcinogenicity of 3-NBA in a situation corresponding to inhalation, a combined short-term and lifetime study with intratracheal (i.t.) instillation in female F344 rats was performed. DNA adduct formation, as a marker for the primary effect and analyzed by 32P-HPLC after single instillation, showed a few major DNA adducts and a rapid increase with a peak after 2 days, followed by a decline. No DNA adducts above the background level were observed after 16 days. The highest DNA adduct formation was observed in lung [approximately 250 DNA adducts/10(8) normal nucleotides (NN)] closely followed by kidney (approximately 200 DNA adducts/10(8) NN), whereas liver contained only 12% (approximately 30 DNA adducts/10(8) NN) of the levels of DNA adducts found in lung. In the tumor study, squamous cell carcinomas were found after 7-9 months in the high-dose group (total dose of 2.5 mg 3-NBA) and after 10-12 months in the low-dose group (total dose of 1.5 mg 3-NBA). The fraction of squamous cell carcinoma out of the total amount of tumors observed at the end of experiment at 18 months, corresponded to 3/16 and 11/16 in the low- and high-dose group, respectively. A single case of adenocarcinoma was also observed in each group. In the control group, no tumors were observed during the entire study of 18 months. In addition, a few cases of squamous metaplasia were also observed in the lung in both dose groups but not in the controls. In conclusion, 3-NBA forms DNA adducts in the lung immediately after i.t. administration but almost all DNA adducts were eliminated after 16 days. Tumor formation in two dose groups was observed in a dose-dependent manner with squamous cell carcinomas as the predominant tumor type at high exposure.

Reactive oxygen molecules are formed in vivo as by-products of normal aerobic metabolism. All organisms dependent on oxygen are inevitably exposed to these species so that DNA damage can occur in both genomic and mitochondrial DNA (mtDNA). In order to determine endogenous DNA damage we have developed an analytical method that involves the isolation and hydrolysis of genomic DNA or mtDNA, the labeling of modified and unmodified nucleotides and micellar electrokinetic chromatography with laser-induced fluorescence detection. With this method we have found etheno-adenine, thymine glycol, uracil, hypoxanthine, and 5-methylcytosine. These were identified by the addition of internal standards to the genomic or mtDNA. There are a large number of other signals in the electropherograms of mtDNA that we have never found in genomic DNA analysis because they are at lower concentration in the genome. In the DNA of untreated patients with chronic lymphocytic leukemia (CLL), uracil and high levels of etheno-adenine were found, which can be explained by antioxidant enzyme alterations and oxidative stress in the CLL lymphocytes.