Abstracts, American Chemical Society Division of Chemical

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Abstracts, American Chemical Society Division of Chemical Toxicology, 228th ACS National Meeting, Philadelphia, Pennsylvania, August 22-26, 2004 Peter C. Dedon*,†,‡ and Trevor M. Penning‡,§ Biological Engineering Division, Massachusetts Institute of Technology, NE47-277 at 500 Technology Square, Cambridge, Massachusetts 02139, and Department of Pharmacology, University of Pennsylvania School of Medicine, 3620 Hamilton Walk, 130C J. Morgan Building, Philadelphia, Pennsylvania 19104-6084 Received September 22, 2004

1. Quantitation of Macromolecular Targets of Heterocyclic Amine Carcinogens at Low Dose. Karen H. Dingley,1 Esther Ubick,1 Nicholas P. Lang,2 David O. Nelson,1 and Kenneth W. Turteltaub.1 (1) Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, California 94550. Fax: 925-422-2282. E-mail: [email protected]. (2) University of Arkansas for Medical Sciences. Our aim is to understand the health risks posed by exposure to low levels of chemicals in the environment and to develop strategies for reducing cancer risk, with particular emphasis on the heterocyclic amine carcinogen PhIP. We used the highly sensitive technique of accelerator mass spectrometry to study the dosimetry of DNA adduct formation by PhIP in rats and humans following low exposure doses of 14C-PhIP. To establish the factors that influence adduct levels and potential susceptibility factors for PhIP-induced colon cancer, we established the variability in adduct levels in humans and correlated them to several metabolic polymorphisms, as well as the age of subjects. In preparation for future chemoprevention studies, rodents were treated with several putative chemopreventive agents to determine if they reduce PhIP adduct levels. This work was performed under the auspices of the U.S. DOE (W-7405-ENG-48) and partially supported by the NIH and DOD Prostate Cancer Program. 2. AMS Analysis of Biological Samples Using Online Combustion Coupled to a Gas-Accepting Ion Source. Paul L. Skipper, Rosa G. Liberman, and Steven R. Tannenbaum. Biological Engineering Division, Massachusetts Institute of Technology, Building 56, Room 753, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139. Fax: 617-252-1787. E-mail: [email protected]. An apparatus and methods were developed for direct analysis of 14C in biological specimens by accelerator mass spectrometry (AMS). Plasma and urine specimens, as well as other liquid samples such as HPLC fractions, are deposited into a bed of CuO powder supported by alumina fiberboard. H2O, and any other volatiles, are removed under reduced pressure. The CuO matrix is locally heated with an IR laser while it is contained within a sealed chamber that is swept with He carrier * To whom correspondence should be addressed. Tel: 617-253-8017. Fax: 617-324-7554. E-mail: [email protected]. † Massachusetts Institute of Technology. ‡ Program Chairs. § University of Pennsylvania School of Medicine.

gas. Heating induces combustion of the samples; carrier gas transports the resulting CO2 to the AMS ion source, which is appropriately modified to accept gas input. Application of this system for analysis of samples from several pharmacokinetics, toxicology, and metabolism studies will be described. Results confirm the potential utility of the combination of on-line combustion with a gas-accepting ion source as an alternative to graphite production for AMS analysis of radiocarbon. Anticipated benefits include a reduced risk of sample cross-contamination, shorter analysis time, and greater compatibility with robotics. 3. 14C-Labeled BCNU Release Kinetics from a Drug Delivery MEMS Device. Yawen Li,1 Rebecca S. Shawgo,1 Betty Tyler,2 Paul T. Henderson,3 John S. Vogel,3 Robert S. Langer,4 Henry Brem,2 and Michael J. Cima.1 (1) Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139. E-mail: [email protected]. (2) Department of Neurological Surgery, Johns Hopkins School of Medicine. (3) Biology and Biotechnology Research Program and Center for Accelerator Mass Spectrometry, Lawrence Livermore National Laboratory. (4) Department of Chemical Engineering, Massachusetts Institute of Technology. We describe the first in vivo use of a MEMS drug delivery device consisting of microreservoirs etched into a silicon substrate containing individual doses of drug. The drug is released by electrochemical dissolution of gold membranes that cover the reservoirs. Drug delivery by MEMS devices can enable precise control of the temporal and spatial release profiles of multiple substances and has the potential to improve survival. Subcutaneous release of the chemotherapeutic drug 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) from the MEMS device was evaluated in a rat model. Accelerator mass spectrometry was used to measure the plasma concentration and temporal release kinetics of 14C-labeled BCNU from the device. The in vivo release profile from the activated device was similar to that of the in vitro and subcutaneously injected controls. The time to reach a steady state plasma 14C concentration was on the order of 1 h. The work was performed in part at LLNL under the auspices of the U.S. DOE (Contract W-7405-ENG-48). 4. Evaluation of Microdosing Strategies Utilizing Accelerator Mass Spectrometry for Pharmacokinetic Studies in Preclinical Drug Development. Punam Sandhu,1 J. S. Vogel,2 Mark J. Rose,1 Esther A. Ubick,3 Janice E. Brunner,1 Michael A. Wallace,1 Jennifer

10.1021/tx0497346 CCC: $27.50 © 2004 American Chemical Society Published on Web 11/05/2004

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K. Adelsberger,1 Maribeth P. Baker,1 Paul T. Henderson,3 Paul G. Pearson,1 and Thomas A. Baillie.1 (1) Department of Drug Metabolism, Merck Research Laboratories, West Point, Pennsylvania 19486. E-mail: punam_sandhu@ merck.com. (2) Center for Accelerator Mass Spectrometry, Lawrence Livermore National Laboratory. (3) Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory. The goals of this study were to validate the technique of accelerator mass spectrometry (AMS) for examining the disposition properties of compound A and to further utilize AMS to assess whether compound A displayed linear kinetics across subpharmacological (microdose) and pharmacological dose ranges in an animal model, prior to initiation of a human microdose study. The AMSderived disposition properties of compound A were comparable to data obtained via conventional techniques (LCMS/MS and LSC). The study highlights for the first time a new benefit of AMS for pharmaceutical applications, namely, the definition of the full pharmacokinetic profile of a test compound following a microdose. The use of AMS revealed aspects of the disposition of this agent that would not have been possible by conventional techniques due to limitations of sensitivity. This work was supported in part by work done at LLNL under the auspices of the U.S. DOE Contract W-7405-ENG-48 and the NIH/NCRR Resource for Biomedical AMS Grant P41 RR13461. 5. Use of Accelerator Mass Spectrometry to Detect the Oxidation/Nitration of 7,8-Dihydro-8-oxo2′-deoxyguanosine. Paul T. Henderson,1 Steve Kim,1 and Sang Soo Hah.2 (1) Lawrence Livermore National Laboratory, Mail Code L-441, 7000 East Avenue, Livermore, California 94550-9698. E-mail: henderson48@ llnl.gov. (2) Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory. The mutagenic DNA lesion 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxoG) is repaired efficiently in vivo but has recently been shown to be capable of forming highly mutagenic secondary oxidation/nitration products in vitro. We are developing novel assays for several of these products using accelerator mass spectrometry (AMS), with the goal of correlating 8-oxoG repair or degradation to breast cancer progression in a mouse model of human ductal carcinoma in situ. Currently, we are developing the assays by growing MCF-7 human breast cancer cells in the presence of 14C-labeled 8-oxoG in order to incorporate the nucleoside into cellular DNA. The 14C label facilitated the monitoring of 8-oxodG repair or conversion to secondary oxidation/nitration products with AMS. This work was performed at UC LLNL under the auspices of the U.S. DOE Contract W-7405-ENG-48. 6. DNA-DNA and DNA-Protein Cross-Linking by Diepoxybutane. Natalia Tretyakova. Department of Medicinal Chemistry and Cancer Center, University of Minnesota, 760E CCRB, 806 Mayo, 420 Delaware Street SE, Minneapolis, Minneapolis 55455. Fax: 612-626-5135. E-mail: [email protected]. 1,2,3,4-Diepoxybutane (DEB) is a carcinogenic metabolite of 1,3-butadiene (1,3-BD), a major industrial chemical and an environmental pollutant. The cytotoxicity and genotoxicity of DEB are likely to result from its ability to form DNA-DNA and DNA-protein cross-links. In the present work, the structures of the major DNA-DNA cross-links of DEB were identified as bis-1,4-(guan-7-yl)2,3-butanediol, 1-(guan-7-yl)-4-(aden-1-yl)-2,3-butanediol (N7G-N1A-BD), 1-(guan-7-yl)-4-(aden-3-yl)-2,3-butanediol

Dedon and Penning

(N7G-N3A-BD), 1-(guan-7-yl)-4-(aden-7-yl)-2,3-butanediol (N7G-N7A-BD), and 1-(guan-7-yl)-4-(aden-N6-yl)-2,3-butanediol (N7G-N6A-BD). HPLC-ESI-MS/MS analyses of guanine-adenine DEB cross-links induced in synthetic oligodeoxynucleotides demonstrate that while all four G-A regioisomers are produced intrastrand, no interstrand N7G-N3A-BD cross-linking is possible. Interestingly, the ability of DEB to cross-link DNA is strongly dependent on its stereochemistry. A stable isotope labeling HPLC-MS/MS approach was used to demonstrate that the S,S stereoisomer induces the highest number of interstrand DNA-DNA lesions, while meso DEB forms primarily intrastrand cross-links. Finally, our preliminary results for DNA-protein cross-linking by DEB will be presented. 7. Identification of Novel Heterocyclic Aromatic Amines in Grilled Meats and Human Urine. R. J. Turesky, J. Taylor, T. Gehring, and R. D. Holland. Division of Chemistry, NCTR, 3900 NCTR Road, Jefferson, Arkansas 72079. Fax: 870-543-7686. E-mail: Rturesky@ nctr.fda.gov. A tandem solvent solid phase extraction method was used to isolate genotoxic heterocyclic aromatic amines (HAAs) from grilled meats and urine. Analysis by HPLC/ ESI-MS/MS revealed the presence of 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (8-MeIQx), 2-amino3,4,8-trimethylimidazo[4,5-f]quinoxaline, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine, 2-amino-R-carboline, 2-amino-3-methylimidazo[4,5-f]quinoxaline, and two previously unreported HAAs; one is an isomer of 8-MeIQx whose structure remains to be elucidated and 2-amino1-methylimidazo[4,5-b]quinoline (IQ[4,5-b]) in grilled meats at concentrations from 0.02 to 15 ppb. These HAAs were detected in the urine of subjects following consumption of grilled meats, and the amounts increased following base hydrolysis of putative phase II conjugates. The levels of IQ[4,5-b] in urine exceeded the amounts ingested. Moreover, IQ[4,5-b] was also present in base-treated urine of subjects who refrained from grilled meat consumption, while other HAAs were not detected. IQ[4,5-b] formation occurs through the reaction of creatinine with 2-aminobenzaldehdye. The formation of IQ[4,5-b] under physiological conditions is under investigation to determine whether this HAA may form endogenously in the urinary bladder. 8. Identification, Structure Elucidation, and in Vitro Replication Studies of Novel Oxidative CrossLink Lesions of DNA. Yinsheng Wang, Qibin Zhang, Chunang Gu, and Yu Zeng. Department of Chemistry, University of California at Riverside, Mail Drop 027, Riverside, California 92521-0403. Fax: 909-787-4713. E-mail: [email protected]. Reactive oxygen species (ROS) are produced by both endogenous and exogenous processes including ionizing radiation. DNA damage induced by ROS has been implicated in the pathogenesis of a number of human diseases including cancer and aging. Although a multitude of single base lesions induced by ROS have been identified and characterized, only a small number of intrastrand cross-link lesions, in which two neighboring DNA bases in the same strand are covalently bonded, had been previously characterized. The latter lesions may have implications in the CCfTT and mCGfTT tandem double mutations observed previously. In our laboratory, we have isolated several novel oxidative cross-link lesions formed between adjacent nucleobases in the same DNA

Abstracts, ACS Division of Chemical Toxicology

strand and determined their structures in dinucleoside monophosphates by extensive mass spectrometric and NMR spectroscopic methods. In addition, we have demonstrated by LC-MS/MS that some of these cross-link lesions can be induced in duplex DNA by γ-irradiation. Moreover, we have carried out in vitro replication studies with these cross-link lesion-bearing substrates by using purified DNA polymerases. Our results show that yeast polymerase h can bypass some of the cross-link lesions, and sometimes, the bypass is error-prone. 9. Idiosyncratic Drug Reactions: Approaches for Reducing Reactive Intermediates in Drug Discovery and Development. Ala Nassar. Drug Safety and Metabolism, Wyeth Research, 500 Arcola Road, Collegeville, Pennsylvania 19426. Idiosyncratic drug reactions (IDRs) are a major complication of drug therapy that needs to be addressed during drug discovery and development. The lack of an accepted approach to predict them hampers efforts to improve drug safety. The major reasons for this are the low incidence of their occurrence and the various potential mechanisms involved in the reactions. The concept of IDRs, a specific type of drug toxicity characterized by their delayed onset, relative rarity, and reactive metabolite formation, is briefly described. Hypothetical chemical mechanisms for the formation of reactive metabolites are summarized, including a classification of adverse drug reactions and types of reactive metabolites. This review examines the relative merits of current and potential strategies for dealing with reactive intermediates in drug discovery and development. The significance of covalent binding in drug discovery/development in vitro and in vivo systems is discussed. We will discuss the merits of tools (screening methods to trap reactive intermediates, enzyme inhibition, and covalent binding) and strategies to try to predict which new drugs have the potential to produce reactive intermediates and IDRs as they relate to major stages of drug discovery and development. These approaches may be considered to potentially improve the overall safety profile of drug candidates at various stages of the drug discovery and development process. 10. Lipoxygenase-Mediated Endogenous DNA Damage. Wenying Jian, Seon Hwa Lee, and Ian A. Blair. Center for Cancer Pharmacology, University of Pennsylvania, 857 Biomedical Research Building II, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104. Fax: 215573-9889. E-mail: [email protected]. Polyunsaturated fatty acids can be converted into lipid hydroperoxides by the action of lipoxygenases (LOXs) or cyclooxygenases (COXs). Homolytic decomposition of lipid hydroperoxides gives rise to endogenous genotoxins, which cause DNA adducts. The hypothesis of this research is that endogenous genotoxins derived from LOX activity are mediators of DNA damage in leukocytes, components of atherosclerotic plaques. Mouse macrophage cell line RAW 264.7 stably transfected with human 15-LOX gene and a human lymphocyte cell line CESS expressing 5-LOX were the two models tested in this study. Lipid profile analysis and DNA adducts analysis were conducted with LC/electron capture APCI/multiple reaction monitoring MS. It was revealed that heptanone2′-deoxyguanosine remained at the basal level (2.0 adducts/107 normal bases) in the first model while it increased 3.5 times in CESS cells stimulated with calcium ionephore. The result implied that cellular

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localization of LOXs might determine their ability to mediate DNA damage. 11. Nitrosative DNA Base Deamination: What Can We Learn from Gas Phase Ion Chemistry? Rainer Glaser, Ming Qian, Papiya Majumdar, Hong Wu, and Nathan D. Leigh. Department of Chemistry, University of MissourisColumbia, Columbia, Missouri 65211. Fax: 573-882-2754. E-mail: [email protected]. The results are reported of mass spectrometric studies of the nucleobases adenine 1h (1, R ) H at N9), guanine 2h (2, R ) H at N9), and cytosine 3h (3, R ) H at N1). The protonated nucleobases are generated by electrospray ionization of adenosine 1r (1, R ) ribose), guanosine 2r, and deoxycytidine 3d (3, R ) deoxyribose), and their fragmentations were studied with tandem mass spectrometry. The elimination of ammonia presents a major fragmentation path for all of the ions [1h + H]+, [2h + H]+, and [3h + H]+. The ion [1h + H - NH3]+ subsequently loses HCN or HNC whereas decarbonylation occurs for [2h + H - NH3]+ and [3h + H - NH3]+. These fragmentation paths provide compelling evidence for pyrimidine ring-opened structures of the deaminated nucleobases. Because the ions [1h + H - NH3]+, [2h + H - NH3]+, and [3h + H - NH3]+ also are the products of dediazoniation of the respective nucleobase diazonium ions, the results provide the first direct experimental evidence for the existence of the pyrimidine ring-opened cations that we have proposed on the basis of theoretical studies as intermediates in nitrosative nucleobase deamination. 12. Possible Model for Cross-Linking of DNA and Peptides by Ionizing Radiation: A Density Functional Theory (DFT) Study. Petar M. Mitrasinovic. Department of Chemistry, Dalhousie University, Halifax NS B3H 4J3, Canada. E-mail: [email protected]. As an alternative to a fast electron (or hydrogen atom) transfer from the DNA to the oxidized tryptophan, an addition of tryptophan radical to DNA followed by a consecutive rearrangement may lead to DNA-peptide cross-linking. We propose the reaction mechanisms for thymine-tryptophan radical cross-linking as a model for DNA-peptide cross-linking in γ-irradiated aqueous solutions of tryptophan peptides in the presence of DNA. DFT methods are employed to investigate the feasibility of the cross-linking mechanisms. A possible complete reaction mechanism consists of a radical combination forming the initial cross-linked product, a hydrogen transfer within the initial cross-linked product by way of a bridging water molecule, and a dehydration step. Hydrogen-bonding interactions are suggested to play a key role in the hydrogen transfer reaction step. The temperature is suggested to be a key factor influencing the overall mechanism. The cross-link between the tryptophan radical (donor) and the thymine (acceptor) moieties in the final product is covalent. 13. Quantitation of Endogenous Retinoids: Toward Elucidation of Retinoid Function in the Central Nervous System. Maureen A. Kane, Na Chen, and Joseph L. Napoli. Department of Nutritional Sciences and Toxicology, University of California, Berkeley, 150 Morgan Hall, MC #3104, Berkeley, California 94720-3104. Fax: 510-642-0535. E-mail: makane@ uclink.berkeley.edu. Retinoic acid (RA) has been implicated to play a role in the maintenance and regulation of mature central nervous system function. Because the actual role of

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retinoids is unclear, there is a need to directly quantify local levels of RA as well as RA synthesis and metabolism in the brain. The development of a sensitive, specific LC/ MS/MS assay to distinguish, identify, and quantitate endogenous retinoids in biological tissues provides an essential tool toward the measurement of endogenous levels of RA and addresses the hypothesis that RA will colocalize with candidate enzymes in the pathway of RA biosynthesis. Quantitative data are considered in conjunction with immunohistochemistry and in situ hybridization approaches to identify the main RA synthesizing loci in the brain and to help clarify the role of RA and RA signaling in adult central nervous system function. Abnormal RA signaling has been linked to age-related memory dysfunction, Parkinson’s Disease, and schizophrenia. 14. Redox Chemistry of C8-Arylamine Adducts of 2′-Deoxyguanosine. James Stover,1 Carmelo J. Rizzo,1 David E. Cliffel,2 and Ma˜da˜lina Ciobanu.2 (1) Department of Chemistry and Center in Molecular Toxicology, Vanderbilt University, VU Station B 351822, Vanderbilt University, Nashville, Tennessee 37235-1822. E-mail: [email protected]. (2) Department of Chemistry, Vanderbilt University. It has been shown in the literature that 8-oxo-deoxyguanosine can undergo oxidative rearrangement to form ring-opened and dramatically modified products that are much more mutagenic than the parent modified nucleoside. Because of the ease of this oxidation from 8-oxodeoxyguanosine’s low redox potential and the ability of DNA to transfer electrons over long molecular distances, the subsequent chemistry and repair of these compounds are of particular interest. We have investigated the redox chemistry of C8-arylamine adducts of deoxyguanosine using cyclic voltammetry and square-wave voltammetry in order to demonstrate their potential for rearrangement. We have also characterized the oxidative rearrangement products of the C8-deoxyguanosine adduct of the food mutagen IQ via chemical oxidation. We find that this oxidation is facile and gives similar rearrangement products as 8-oxo-dG. 15. Spontaneous Cleavage of a 3′-DNA Radical under Aerobic Conditions. Georges Lahoud and Amanda C. Bryant-Friedrich. Department of Chemistry, Oakland University, 2200 Squirrel Road, Rochester, Michigan 48309. Fax: 248-370-2321. E-mail: galahoud@ oakland.edu and [email protected]. Over the past decade, a great deal of research effort has been devoted to showing that free radical oxidants, derived from endogenous or exogenous sources, are the primary instigators of DNA damage. The resulting lesions have been implicated in the physiopathology of cancer and the diseases of aging. This presentation will cover recent progress in understanding the reaction pathways of DNA strand breakage under physiological aerobic conditions. Oligomers containing a single C-3′pivaloyl substituent were synthesized using the Hphosphonate method. Through photolytic activation, the C-3′-radical was generated in the presence of oxygen, and the resulting degradation products were analyzed by MALDI-ToF MS. Through these investigations, we have found that at low H-donor concentrations spontaneous strand scission occurs. On the basis of the nature of the products formed, two competing pathways are postulated. One involves the formation of the potentially genotoxic 3′-phosphoglycaldehyde and the second involves the

Dedon and Penning

production of a 3′-ketosugar that undergoes subsequent base release. 16. Structural Basis of Genotoxicity of the Aflatoxin B1 FAPY Lesion. Thomas M. Harris, Constance M. Harris, Michael P. Stone, Rajkumar S. Iyer, Lalitha G. Iyer, James Z. Deng, Markus W. Voehler, and Kyle L. Brown. Department of Chemistry and Center in Molecular Toxicology, Vanderbilt University, Nashville, Tennessee 37235. Fax: 615-322-7591. E-mail: [email protected]. The deoxyguanosine N7 adduct of the 8,9-epoxide of the potently carcinogenic fungal toxin aflatoxin B1 (AFB) is formed in DNA with high efficiency but is neither stable nor highly mutagenic. Spontaneous hydrolysis gives the persistent and more mutagenic formamidopyrimidine (FAPY) derivative, which exists in multiple forms, only one of known structure. The recent report by Essigmann and co-workers that mutagenicity is due to a minor form while the major one is a complete block to replication has highlighted the importance of structural isomerism of the AFB-FAPY lesion. Using adducted bases, nucleosides, and oligonucleotides, we have conducted detailed studies of four equilibria of AFB-FAPY species: structural and geometrical isomerization of the formamide linkage, atropisomerization at the C5-N5 bond, and anomerization of deoxyribose. Key equilibria are dependent on the ss/ds status of the DNA. The results permit us to propose the structural basis for differential replicative response to the two major forms of AFBFAPY. This work was supported by USPHS ES03755 and CA55678. 17. Synthesis and in Vivo Mutational Properties of the Guanine Oxidation-Nitration Product 5-Guanidino-4-nitroimidazole. William L. Neeley,1 Paul T. Henderson,2 James C. Delaney,1 and John M. Essigmann.1 (1) Department of Chemistry and Biological Engineering Division, Massachusetts Institute of Technology, Room 56-669, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139. (2) Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory. Primary oxidation of guanine residues in DNA by peroxynitrite yields several products including 5-guanidino-4-nitroimidazole (NI). In contrast to other oxidized guanine derivatives, NI is both chemically stable and resistant to tested forms of enzymatic repair in vitro, suggesting that the lesion may accumulate in cells. Additionally, previous in vitro primer extension assays indicated that this lesion might be well bypassed by polymerases and mutagenic. NI therefore may contribute to the mutagenic spectrum of DNA damage induced by peroxynitrite. To facilitate the study of this DNA lesion, we have designed an efficient and versatile synthesis of DNA containing NI using the convertible nucleoside approach. Using a bacteriophage genome site specifically containing NI, the DNA polymerase bypass efficiency and mutation frequency of the DNA lesion were determined in normal and SOS-induced wild-type Escherichia coli cells. 18. Synthesis and Sequence Preference of Acetaldehyde-Derived Oligonucleotide Interstrand CrossLink. Yanbin Lao. Department of Medicinal Chemistry and the Cancer Center, University of Minnesota, MMC 806, 420 Delaware Street SE, Minneapolis, Minnesota 55455. Fax: 612-626-5135. E-mail: [email protected]. Masaaki Moriya. Department of Pharmacological Sci-


ences, State University of New York at Stony Brook. Stephen S. Hecht. University of Minnesota Cancer Center. Acetaldehyde (AA) is mutagenic and carcinogenic. Human exposure to AA is widespread and substantial. AA can react with DNA to form AA-DNA adducts. DNA adducts are central to chemical carcinogenesis. Our laboratory has identified several previously unknown AA-DNA adducts, including an interstrand cross-link, 3-(2-deoxyribos-1-yl)-5,6,7,8-tetrahydro-8-(N2-deoxyguanosyl)-6-methylpyrimido[1,2-a]purine-10(3H)one. We have now synthesized this cross-link in a double-stranded oligonucleotide appropriate for studies of mutagenicity and repair in human cells. The cross-linked oligonucleotide was characterized by mass spectrometry and enzymatic hydrolysis experiments. Another oligonucleotide containing the irreversible analogue of the cross-link, 1,3bis(2x-deoxyguanos-N2-yl)butane, has also been synthesized and characterized. It is being used as a control in the mutagenicity studies. The sequence preference for AA-derived cross-link formation has also been determined. 19. Integrated Discrete Multiorgan Culture: A Novel in Vitro Model for Toxicity Testing. Albert P. Li and Yumiko Sakai. Advanced Pharmaceutical Sciences, Inc., 6400 Baltimore National Pike, PMB#146, Baltimore, Maryland 21228. Fax: 410-869-9560. Email: [email protected]. In vitro assays involving primary cells are used routinely to evaluate organ specific toxic effects, for instance, the use of primary hepatocytes to evaluate hepatotoxicity. A major drawback of an in vitro system is the lack of multiple organ interactions as observed in a whole organism. A novel cell culture system, the Integrated Discrete Multiorgan Culture (idMOC; patent pending), is described here, which allows the culturing of primary cells from multiple organs in a single plate. The idMOC is specially manufactured to have wells inside a well, thereby allowing each cell type to be placed in an inner well in a medium developed for optimal growth and differentiation. As each cell type may have different growth requirements, the cells are cultured so that the multiple cell types are ready for toxicity testing on the same day. On the day of testing, the wells are connected via an overlying medium with the test chemical. After testing, the overlying medium is removed and each cell type is evaluated for toxicity using appropriate end points. Results with an idMOC with primary cells from multiple organs including liver (hepatocytes), kidney (kidney cortical cells), CNS (neuronal cells), and muscle (skeletal muscle cells) and the results with a battery of chemical toxicants with known organ specific toxicity (e.g., tamoxifen, cyclophosphamide) demonstrate that the idMOC represents a useful in vitro model for the evaluation of chemical toxicity. The use of idMOC with primary human cells (MOC-man), for instance, allows the development of data useful in the prediction of human drug toxicity. 20. Oxidative DNA Damage: New Approaches to a Very Old Problem. James Swenberg, Yo Chan Jeong, Shengkai Liao, Irene Baskerville, Jun Nakamura, Amy Ham, Eric Morinello, and Ivan Rusyn. Laboratory of Molecular Carcinogenesis and Mutagenesis, University of North Carolina, CB# 7432, Room 253c, Rosenau Hall, Chapel Hill, North Carolina 27599. Fax: 919-966-6123. E-mail: [email protected].


Oxidative stress is thought to play important roles in many diseases including cancer, aging, and neurodegeneration. Oxidative DNA damage represents an important biomarker for studying the role of oxidative stress. Unfortunately, much of the published literature is plagued by artifactual data. Our laboratory has been developing and applying several new assays that minimize artifacts and the amount of DNA required. These include the use of TEMPO during DNA isolation, novel mass spectrometric approaches for oxidized nucleosides and bases, slot blot approaches for abasic sites and oxidized bases, and gene expression studies of DNA repair pathways. These studies demonstrate that the number of oxidized DNA lesions is lower than previously reported, dose-response relationships can be complex, and both direct oxidation and secondary products such as lipid peroxidation and base propenal are important. The use of radical trapping agents and iron chelators is particularly important for accurate measures of oxidized bases. 21. DNA Damage by Tobacco Smoke Carcinogens. Stephen S. Hecht, Mingyao Wang, Guang Cheng, Yongli Shi, Peter W. Villalta, Nanxiong Yu, Shana J. Sturla, and Pramod Upadhyaya. University of Minnesota Cancer Center, MMC 806, 420 Delaware Street SE, Minneapolis, Minnesota 55455. Fax: 612-626-5135. E-mail: hecht002@ umn.edu. Tobacco products cause 30% of human cancer mortality. The carcinogens in tobacco products are responsible for this deadly toll. Among these carcinogens, we have focused on tobacco specific nitrosamines, cyclic nitrosamines, and aldehydes. Nitrosamines are enzymatically activated to diazohydroxides and related intermediates, which bind to DNA, forming adducts that cause mutations in critical growth control genes. The chemistry of cyclic and tobacco specific nitrosamine DNA adduct formation is unique because a diazohydroxide and aldehyde or ketone separated by four carbons are present in the same molecule. These reactions also generate a series of reactive aldehydes including formaldehyde, acetaldehyde, and crotonaldehyde, which produce a cascade of DNA modifications. Among the adduct structures that have been elucidated are exocyclic deoxyguanosine adducts formed at the 1,N2- and 7,8-positions, cross-links from acetaldehyde and formaldehyde, and pyridyloxobutyl adducts with a variety of DNA bases. This chemistry will be reviewed, and recent results will be presented. 22. Oxidation Reactions Induced by UVA and Ionizing Radiations within Cellular DNA. Jean Cadet, Thierry Douki, and Jean Luc Ravanat. Laboratoire “Le´sions des Acides Nucle´iques”, SCIB/DRFMC, CEA/Grenoble, F-38054, Grenoble Cedex 9, France. Fax: +33-4-38785090. E-mail: [email protected]. There is still a paucity of information on the formation of oxidative damage in cellular DNA. This may be explained by difficulties in the measurement of oxidized purine and pyrimidine bases together with the low levels of such lesions (in the range of a few lesions per 107 normal bases). So far, only 11 modified nucleobases of the 70 lesions identified in model studies have been accurately measured in the nuclear DNA of cells. This was achieved using the recently available HPLC-electrospray ionization-tandem mass spectrometry technique. The association of base excision DNA repair enzymes, including bacterial formamidopyrimidine glycosylase and endonuclease III, with the comet assay represents a better alternative for assessing low levels of base damage.

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Application of these analytical methods provides insights into the mechanisms of oxidation reactions mediated by ionizing radiation and UVA component of solar light to cellular DNA. 23. Novel and Diverse Sources of Nucleobase Deamination in DNA. Peter C. Dedon,1 Min Dong,1 Richard P. Cunningham,2 and Nicholas E. Burgis.2 (1) Biological Engineering Division, NE47-277, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139. Fax: 617-324-7554. E-mail: [email protected]. (2) Department of Biological Sciences, State University of New York. The nucleobase analogues xanthine, hypoxanthine, and uracil represent physiologically important intermediates in purine and pyrimidine metabolism as well as genotoxic consequences of nucleobase deamination in DNA. Using recently developed chemical and biological tools, we have begun to probe the physiologically relevant pathways leading to the presence of these base analogues in DNA. In the case of nitrosative deamination associated with inflammation, we conclude that xanthine is the only major product of guanine N2-deamination under biological conditions, since oxanine has not been detected in isolated DNA and human cells exposed to nitric oxide and in Escherichia coli and mouse genomic DNA. Curiously, we have not observed substantial increases in the levels of dX, dI, or dU in tissues derived from a mouse model of inflammation and nitric oxide overproduction, which leads us to look for other DNA biomarkers of inflammation such as adducts derived from lipid peroxidation. Finally, we propose a new model for the presence of xanthine and hypoxanthine in DNA, one involving disturbances in normal purine metabolism. These and other chemical and enzymatic deamination pathways suggest that nucleobase deamination products pose a significant genotoxic burden to human cells. 24. Antibiotics: Past, Present, and Future. Christopher Walsh, Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115-5701. Fax: 617-432-0438. There is a constant need for new generations of antibiotics as bacterial pathogens inevitably develop resistance to existing antibacterial drugs. The lecture will survey past triumphs, present strategies, and future challenges to the discovery of new antibiotics. The need for new molecules to test against validated targets turned up by bacterial genomics and informatics will be discussed. 25. The Comparative Toxicogenomics Database (CTD): Comparative Molecular Approaches to Environmental Health Research. Carolyn J. Mattingly,1 Glenn Colby,1 Michael Rosenstein,1 John N. Forrest,2 and James L. Boyer.2 (1) Department of Bioinformatics, Mount Desert Island Biological Laboratory, Salisbury Cove, Maine 04672. Fax: 207-288-2130. E-mail: cmattin@ mdibl.org. (2) Department of Medicine, Yale University School of Medicine. Sequencing of the human genome has stimulated a new way of thinking in biology; however, our understanding of the sequence is limited by our ability to retrieve meaning from it. The human and other vertebrate sequencing projects have underscored the significance of comparative studies to reveal conserved sequences that may be functionally significant. The Comparative Toxicogenomics Database (CTD) will be the first publicly

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available, curated database devoted to genes and proteins of human toxicological significance. The major goals of this resource include the following: (i) curating and integrating sequence, reference, and toxicant data for toxicologically important genes and proteins; (ii) promoting comparative studies of these genes and proteins across evolutionarily diverse organisms; and (iii) integrating information from existing molecular and toxicology resources. It is the goal that comparative studies of genes and proteins of toxicological interest will provide insights into the molecular basis of toxicity. 26. Systems Toxicology Assessment of Endocrine Disruptors. Tim Zacharewski. Department of Biochemistry & Molecular Biology, Michigan State University, 223 Biochemistry Building, Wilson Road, East Lansing, Michigan 48842-1319. Fax: 517-353-9334. E-mail: [email protected]. To fully assess the potential adverse health effects of chronic and subchronic exposure to drugs, industrial chemicals, environmental pollutants, food contaminants, and dietary supplements, a more comprehensive understanding of the molecular, cellular, and physiological effects is required within the context of the whole organism, its genome, proteome, and metabonome. A systematic strategy that utilizes omic technologies and computational approaches to complement existing toxicological investigation has been adopted to further elucidate the mechanisms of action of toxicants and to facilitate the development of computational models that support drug development and risk assessment. This presentation will provide an overview of the strategies used to assess endocrine disruptors and the infrastructure necessary to support a systems toxicology approach including the development of dbZACH (http://dbzach.fst.msu.edu), a MIAME compliant relational database, the construction of orthologous human, mouse, and rat cDNA microarrays, as well as approaches to comprehensive data analysis and interpretation. More specifically, examples will be taken from computational, in vitro and in vivo microarray studies, and complementary histopathological investigations examining the tissue specific effects of estrogenic endocrine disruptors and 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD). Microarray results have uncovered the physiological significance of the dramatic increase in uterine arginase activity, a classical estrogenic biomarker, by elucidating the mechanistic relevance of arginase induction and delineating the role of arginine and ornithine utilization in estrogen induction of uterine wet weight while comparative computational studies examining DRE distributions in the human, mouse, and rat genomes suggest that responses to TCDD will be significantly different between human and rodent models. 27. A Mechanism-Based Approach to Modeling Endocrine Disruptor Ligands. Ashton Hamme. Department of Chemistry, Jackson State University, 1400 J. R. Lynch Street, P.O. Box 17910, Jackson, Mississippi 39217-0510. Fax: 601-979-3674. E-mail: ashton.t.hamme@ ccaix.jsums.edu. Naturally occurring compounds such as flavanoids and isoflavones, which are found in green tea, soybeans, and fish, have been shown to be beneficial in breast cancer treatment through binding to estrogen receptors. Other natural products such as 11-deoxyfistualrin-3 are cytotoxic against estrogen-dependent MCF-7 breast cancer tissue, but no link has been made between its efficacy


and its estrogen receptors. The purpose of the computational study is to probe the interactions of known and potential estrogen binding compounds with the active site of the estrogen receptors. This presentation will highlight the computational modeling of compounds that are cytotoxic against estrogen-dependent breast cancer tissue within estrogen receptors. 28. Love on the Rocks? Hormonal Signaling and Signal Disruption in Coral Reef Ecosystems. Ann M. Tarrant. Biology Department, Woods Hole Oceanographic Institution, 45 Water Street, Redfield Building MS-32, Woods Hole, Massachusetts 02543. Fax: 508-4572134. E-mail: [email protected]. Coral reefs are complex ecosystems that contain diverse signaling systems ranging from intracellular symbioses to multispecies spawning events. Among reefbuilding corals, regulation of gametogenesis and spawning are poorly understood, and the potential for signal disruption is unknown. While endocrine-like activity has not been systematically studied in corals, several classical vertebrate hormones have been detected in coral tissues. Specifically, estrogens are present in coral tissue, released during spawning, and can affect coral growth and reproduction. If estrogens or other steroids help to regulate coral spawning, then environmental contaminants may disrupt endogenous signals. Ongoing studies are investigating mechanisms of steroid action in corals. While several nuclear receptor genes have been cloned, none are orthologs to vertebrate steroid receptors. If steroids are active in corals, they may act through alternative mechanisms. Elucidation of regulatory pathways in corals will provide insight into the evolution of hormonal signaling and a basis for the investigation of signal disruption. 29. Phyloinformatics: A Comparative Framework to Study Hormone Active Agents in the Environment. Jennifer E. Fox. Center for Ecology and Evolutionary Biology, University of Oregon, 322 Pacific Hall, Eugene, Oregon 97403-5289. E-mail: jenfox@ uoregon.edu. John A. McLachlan. Center for Bioenvironmental Research, Tulane-Xavier Universities. Chemicals designed as insect and weed killers, plastics additives, surfactants, birth control agents, pharmaceuticals, and personal care products are produced on the order of megatons per year. Such chemicals have been detected in streams, groundwater, cattle feedlot effluent, and water used for irrigating crops at concentrations reaching µg/L. Some are termed endocrine disrupting chemicals (EDCs) based on their ability to impact sexual development and reproduction in a multitude of organisms. We have identified a novel target of EDCss symbiosis between plants and bacteria necessary for nitrogen fixation. Phytoestrogen signaling from leguminous plants (soybean, alfalfa, etc.) to symbiotic soil bacteria is inhibited by many of the same hormone active EDCs, resulting in delayed nitrogen-fixing symbiosis and reduced crop yields. Using a comparative framework to analyze impacts of EDCs on organisms ranging from humans to bacteria, we suggest a global mechanism whereby EDCs disrupt signaling communication within and between organisms and ecosystems. 30. Environmental Signaling: A Systems Biology Approach to Human and Ecological Health. John A. McLachlan. Center for Bioenvironmental Research, Tulane-Xavier Universities, 1430 Tulane Avenue, New


Orleans, Louisiana 70112. Fax: 504-585-6428. E-mail: [email protected]. Abstract text not available. 31. Approaches to Synthesize Thio Analogues of 8-Oxo-2′-deoxyguanosine (8OdG): Candidate-Modified Nucleosides for Probing DNA Damage and Repair. Mohan Halasyam and Sheila S. David. Department of Chemistry, University of Utah, 315S 1400E HEB1165, Salt Lake City, Utah 84112. Fax: 801-5818433. E-mail: [email protected]. 7,8-Dihydro-8-oxo-2′-deoxyguanosine (OG) is widely considered as one of the most deleterious oxidized DNA lesions. The high mutagenicity of OG stems from the misincorporation of 2′-deoxyadenosine (A) to form OG:A mispairs. A variety of base excision repair glycosylases are involved in preventing mutations associated with OG. In particular, the OG glycosylases Fpg and hOGG1 remove OG from OG:C base pairs, while MutY/MYH remove A from OG:A base pairs. Employment of modified nucleoside analogues is a key element that offers insights into the enzymatic processing of OG substrates by repair enzymes. Indeed, our laboratory has utilized this approach extensively in examination of MutY (J. Am. Chem. Soc. 2003, 125, 16235; Biochemistry 2004, 43, 651). 8-Thio-2′-deoxyguanosine (8SG) and 6-thio-8-oxo-2′-deoxyguanosine (6SOG) compounds were identified as potential OG nucleoside derivates for incorporation at the lesion site and to determine the effects on recognition and catalysis. This paper will report multiple approaches to the synthesis of 8SG and 6SOG nucleosides. The synthetic routes involve functionalization of 6(C) and 8(C) of appropriate OG compounds, which are further transformed to the respective phosphoramidites, suitable for making oligonucleotides. 32. Violating the Rules of Charge Migration: The Reactivity of Guanine in DNA toward Oxidation by Nitrosoperoxycarbonate Correlates Directly with Guanine Oxidation Potential. Yelena Margolin,1 Vladimir Shafirovich,2 Nicholas Geacintov,2 and Peter C. Dedon.1 (1) Biological Engineering Division, NE47-277, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139. Fax: 617-3247554. E-mail: [email protected]. (2) Department of Chemistry, New York University. As a result of the emergence of DNA charge migration models, the oxidation of guanines in DNA has received considerable attention in recent years. The general conclusion is that the 5′-G in GG steps is the most easily oxidized and thus acts as a thermodynamic sink for charge transfer from other sites in the helix. We now report an atypical DNA sequence selectivity for guanine oxidation produced by an endogenous oxidizing agent. In a manner opposite that of “classical” one-electron DNA oxidants (e.g., riboflavin-mediated photooxidation), we have observed that the reactivity of a guanine base in DNA toward the nitrosoperoxycarbonate (ONOOCO2-), a chemical mediator of inflammation in humans, increases as a function of the calculated sequence-dependent guanine oxidation potential. In contrast, guanine reactivity toward riboflavin-mediated photooxidation decreases with increasing ionization potential. The mechanistic basis for and implications of this unusual guanine oxidation will be discussed. 33. Detection of Interchain Cross-Links in Acrolein-Treated Calf Thymus DNA. Ivan D. Kozekov,1 Robert J. Turesky,2 Carmelo J. Rizzo,1 and Thomas M.

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Harris.1 (1) Department of Chemistry and Center in Molecular Toxicology, Vanderbilt University, Nashville, Tennessee 37235-1822. Fax: 615-322-2649. E-mail: [email protected]. (2) Division of Chemistry, National Center for Toxicological Research. Acrolein is a bis-electrophile, which reacts with deoxyguanosine to form primarily the 8-hydroxypropano 1,N2 cyclic adduct 1. Depending on sequence context and experimental conditions, 1 can react with the exocyclic amino group of a deoxyguanosine in the complementary strand of duplex DNA to form carbinolamine type interchain cross-links, which exist in equilibrium with imines. Enzymatic hydrolysis yields 2 or 3 if preceded by reduction with NaCNBH3. Bis-nucleosides 2 and 3 and the analogous bis-bases formed by acid hydrolysis have been evaluated for detecting acrolein-mediated interchain cross-links in DNA with 3 being found to be the best. After incubation of a range of concentrations of acrolein with calf thymus DNA for a week at 37 °C followed by reduction with NaCNBH3, enzymatic hydrolysis gave 3, which was detected by MS using hexadeutero-3 as an internal standard for quantitation. These results have important biological implications because interchain cross-links are a serious form of DNA damage.

34. Development of a Quantitative LC-MS Method for the Analysis of Aflatoxin B1 Serum Albumin Adducts. Peter F. Scholl. Environmental Health Sciences, Johns Hopkins University Bloomberg School of Public Health, 615 North Wolfe Street, Baltimore, Maryland 21205-2103. Fax: 410-955-0617. E-mail: pscholl@ jhsph.edu. Les MCoy. U.S. Centers for Disease Control and Prevention, Thomas W. Kensler, Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health. John D. Groopman. School of Public Health, Johns Hopkins University. Aflatoxin B1 (AFB1) is a potent hepatocarcinogenic mycotoxin implicated in the etiology of liver cancer. AFB1 exposure is most often estimated in molecular epidemiologic studies by measuring the AFB1-albumin adduct concentration in blood using RIA and ELISA protocols. However, these methods can yield AFB1 exposure estimates 3-10-fold greater than when HPLC with fluorescent detection is used to more specifically detect lysineAFB1. Mass spectrometry can be used to detect aflatoxinalbumin adducts with greater specificity than any of these methods. Here, we report the development of an LC-MS method utilizing 4,4,5,5-D4-lysine-AFB1 as an internal standard to measure the concentration of lysineAFB1 in serum. Pronase-digested serum samples are processed, after spiking with the internal standard, using aflatoxin immunoaffinity resin and reverse phase chromatography. Lysine-AFB1 and the D4 internal standard are detected via positive ion ESI with select reaction monitoring. This assay shows potential for use in future molecular epidemiologic studies of liver cancer to accurately and precisely measure AFB1-albumin adduct concentrations. 35. Development of Metabolically Stable Inositol Thiophosphates as Tools for Investigating the Role

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of Inositol Phosphates in Cancer Chemoprevention. Shana J. Sturla, Emily C. Moody, Peter W. Villalta, and Stephen S. Hecht. University of Minnesota Cancer Center, 420 Delaware Street SE, MMC 806, Minneapolis, Minnesota 55455. Fax: 612-626-5135. E-mail: sturl002@ umn.edu. The cyclitol myo-inositol and its corresponding hexaphosphate, phytic acid (IP6), have been identified as promising agents for cancer prevention. The ultimate chemopreventive agent and the mechanism by which it inhibits carcinogenesis, however, remain unclear. By the action of inositol kinases and phosphatases, IP6 and myoinositol are metabolized to a number of inositol phosphates. Among these, inositol 1,4,5-triphosphate (IP3) is an important second messenger that regulates intracellular calcium and may be involved in the chemopreventive activity attributed to myo-inositol and IP6. To probe the potential chemopreventive role of IP3, we have synthesized a metabolically stable analogue. The design of the synthetic model incorporates strategies of hydroxyl deletion and the use of thiophosphates as hydrolytically stable phosphate surrogates. The synthesis of the analogue, 2,3-dideoxy-myo-inositol 1,4,5-trithiophosphate, has been achieved in 14 steps from (-)-quebrachitol. Preliminary results indicate binding of this synthetic IP3 analogue to isolated bovine IP3-binding receptor. In conjunction with the IP3 analogue synthesis, an IP2 model has also been prepared and receptor-binding characterized. The thiophosphate-containing analogues prepared in this study will be valuable tools for investigating biochemical functions of IP3 and mechanisms of chemoprevention by inositol phosphates. 36. Identification of the Urinary Metabolites of Furan. Lisa A. Peterson,1 Meredith Cummings,2 Choua C. Vu,2 and Brock Matter.3 (1) Environmental Health Sciences and Cancer Center, University of Minnesota, Mayo Mail Code 806, 420 Delaware Street SE, Minneapolis, Minnesota 55455. Fax: 612-626-5135. E-mail: [email protected]. (2) Cancer Center, University of Minnesota. (3) Department of Medicinal Chemistry and Cancer Center, University of Minnesota. Furan is a liver toxicant and carcinogen in laboratory animals. The toxic effects of furan require metabolism. The proposed reactive metabolite, cis-2-butene-1,4-dial, is a microsomal metabolite of furan. To determine if this compound was an important in vivo intermediate in the metabolism of furan, rats were treated with either furan12 C4 or furan-13C4 and urine was collected for 24 h. Capillary LC/MS/MS analysis of the urine indicated that one of the metabolites was a mono-glutathione cis-2-butene1,4-dial conjugate. Chemical characterization of the molecule indicated that the R-amino group of glutathione had reacted with cis-2-butene-1,4-dial to form a thiolsubstituted pyrrole adduct. These results indicate that glutathione conjugation of the reactive metabolite of furan occurs in vivo. This work was supported by ES-10577. 37. LC/MS/MS Study of Calf Thymus DNA Adducts Modified by Benzo[a]pyrene Diol Epoxide and Benzo[a]pyrene dione. Qian Ruan,1 Hye Young H. Kim,1 Ronald G. Harvey,2 Trevor M. Penning,3 and Ian A. Blair.4 (1) Center for Cancer Pharmacology, University of Pennsylvania, 848 Biomedical Research Building, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104. Fax: 215-573-9889. E-mail: qianruan@ spirit.gcrc.upenn.edu. (2) The University of Chicago. (3) Department of Pharmacology, University of Pennsylvania


School of Medicine. (4) Department of Pharmacology, University of Pennsylvania. Benzo[a]pyrene, B[a]P, can be successively metabolized to 7,8-dihydroxy-9,10-epoxybenzo[a]pyrene (B[a]PDE) mediated by P-450 or to the benzo[a]pyrene-7,8-dione (B[a]P-7,8-dione) mediated by P-450 and aldo-ketose (AKR). To investigate the diol-epoxide pathway, [15N] isotope-labeled anti-B[a]PDE-N6-dAdo and N2-dGuo adduct stereoisomers were synthesized and characterized by HPLC and spectroscopy. For the first time, the LC/ MS/MS method was developed for fast separation and quantification of all of the eight B[a]PDE-N6-dAdo and B[a]PDE-N2-dGuo adduct stereoisomers. Reactions of B[a]PDE with calf thymus DNA (ctDNA) were conducted in vitro. The fully hydrolyzed B[a]PDE-ctDNA adducts were analyzed by LC/MS/MS using the isotope mononucleoside adduct standards. (+)-trans-B[a]PDE-dGuo was identified as the major B[a]PDE-ctDNA adduct, and (-)-trans-B[a]PDE-dGuo and four B[a]PDE-dAdo adducts were the minor adducts. To study the AKR pathway, direct synthesis of B[a]P-7,8-dione-dGuo and dAdo adducts in various reaction conditions was employed. Reactions of B[a]P-7,8-dione with mononucleosides and ctDNA were also investigated by LC/MS/MS. This work was supported by NIH Grant PO1 CA92537. 38. Molecular Design for Hazard Reduction Using Green Chemistry. Nicholas Anastas and John Warner. Center for Green Chemistry, University of Massachusetts Boston, 100 Morrissey Boulevard, Boston, Massachusetts 02125. E-mail: [email protected]. In business settings, synthetic chemists make molecules and therefore have a responsibility to design chemicals that are inherently less hazardous to human health and the environment. Traditionally, minimizing these hazards has not generally been an “up front” design criterion for target molecules nor for the chemicals that are used as part of the synthetic scheme. With the recent increase in the body of knowledge about mechanisms of toxicity and other types of hazards, the potential for establishing design rules has increased. Expanding on the 12 Principles of Green Chemistry, a framework for systematically evaluating hazards with the intended purpose of developing heuristic design rules for making intrinsically safer chemicals can be developed and is presented as part of this work. This framework is based on a “hierarchy of knowledge” that is dependent on the quality and quantity of the available hazard information (i.e., toxicity, both global and physical). 39. N-Nitrosoproline, a Food Borne Contaminant, Is Converted to Other Nitrosamines and Hydrazides on Mild Heating. Richard N. Loeppky and Sridharan Rajagopal. Department of Chemistry, University of Missouri, 125 Chemistry Building, Columbia, Missouri 65211. Fax: 573-882-2754. E-mail: [email protected]. N-Nitrosoproline 1 is a noncarcinogenic constituent of food, is produced by endogenous nitrosation in humans, and, along with other N-nitrosamino acids, is excreted in the urine of most humans. It has been used as an epidemiological marker of endogenous nitrosation. We report here that 1 is converted to other more complex nitrosamines and hydrazine derivatives when heated in the range of 80-100 °C in a variety of solvents. When 1 is heated at reflux in acetonitrile, it is converted into a mixture of products from which we have isolated and characterized three. Spectral data led to the assignment of structure 2 as the major product. Structural confirma-


tion was achieved by the independent synthesis of 2 from N-nitrosoprolylhydrazide and succinaldehyde. The structure of 3, the next most abundant product, was obtained by X-ray crystallography. Another product has tentatively been assigned structure 4. The facile formation of these substances suggests that they could be formed during the cooking of food. We propose that they arise by highly novel chemistry, which involves diazenium ion intermediates.

40. Oxidative DNA Damage Derived from a Combination of Superoxide with Guanine and 8-Oxo7,8-dihydroguanine Radicals. Richard Misiaszek, Conor Crean, Avrum Joffe, Nicholas E. Geacintov, and Vladimir Shafirovich. Department of Chemistry, New York University, 29 Washington Place, New York, New York 10003. Fax: 212-998-8421. E-mail: [email protected]. Overproduction of superoxide radicals associated with oxidative stress developed in response to inflammatory conditions is known to cause damage of cellular DNA. However, the mechanisms of action are poorly understood. It is shown here that the bimolecular combination of superoxide radicals with guanine neutral radicals in single- or double-stranded oligodeoxyribonucleotides culminates in the formation of oxidatively modified guanine bases (major product, imidazolone; minor product, 8-oxo7,8-dihydroguanine, 8-oxoGua). The combination of superoxide radicals with 8-oxoGua neutral radicals generates another major product, dehydroguanidinohydantoin lesions, that slowly hydrolyze to the oxaluric acid lesions. Free radical species were generated by intense 308 nm excimer laser pulses resulting in the one-electron oxidation and deprotonation of either guanine or 8-oxodGua in the oligonucleotide and the trapping of the ejected electrons by molecular oxygen. The mechanistic aspects of the radical reactions that culminate in the formation of oxidatively modified guanine bases are discussed. 41. Spirodihydantoin Is a Minor Product from 5-Hydroxyisourate in Urate Oxidation. Hongbin Yu,1 Jacquin C. Niles,1 John S. Wishnok,1 and Steven R. Tannenbaum.2 (1) Biological Engineering Division, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Room 56-731, Cambridge, Massachusetts 02139. Fax: 617-252-1787. E-mail: [email protected]. (2) Biological Engineering Division and Department of Chemistry, Massachusetts Institute of Technology. Spirodihydantoin is a minor product from the oxidation of uric acid (∼0.15% yield, pH 10.2) while spiroiminodihydantoin is a major product from the oxidation of 8-oxoguanine (37% yield, pH 10.2). High pH and high temperature favor the formation of the spiro compounds. 18O labeling experiments and in situ generation and decomposition of 5-hydroxy-N7-methylisourate indicate that spirodihydantoin and allantoin, guanidinohydantoin, and spiroiminodihydantoin are derived from 5-hydroxyisourate and 5-hydroxy-8-oxo-guanine, respectively. The difference between the yield of the spiro compound from oxidation of uric acid and 8-oxo-guanine is likely related to the ionization states of the 5-hydroxyisourate and 5-hydroxy-8-oxo-guanine intermediates. Ionization of N9-H of 5-hydroxy-8-oxo-guanine facilitates deprotonation

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of the 5-hydroxy group and subsequent rearrangement to spiroiminodihydantoin. In 5-hydroxy-isourate, hydration and decarboxylation of C6 carbonyl are facilitated by the C2 carbonyl and lead to allantoin as the major product.

42. Structures of the 1S and 1R trans-Opened Benzo[c]phenanthrene Diol Epoxide dG Adducts in a Frameshift-Prone (CpG)3 Repeat Sequence from the Salmonella typhimurium hisD3052 Gene. Yazhen Wang,1 Nathalie C. Schnetz-Boutaud,1 Heiko Kroth,2 Haruhiko Yagi,2 Jane M. Sayer,2 Subodh Kumar,3 Jerina M. Donald,2 and Michael P. Stone.1 (1) Chemistry Department, Center in Molecular Toxicology, and the Vanderbilt-Ingram Cancer Center, Vanderbilt University, West End Avenue, Nashville, Tennessee 37235. (2) Laboratory of Bioorganic Chemistry, National Institutes of Health, NIDDK. (3) Great Lakes Laboratory, State University College at Buffalo. The oligodeoxynucleotide 5′-d(ATCGCGCGGCATG)-3′‚ 5′-d(CATGCCGCGCGAT)-3′, derived from the Salmonella typhimurium hisD3052 gene, is a hot spot for frameshift mutagenesis. Frameshift mutations at this locus probably result from transient strand slippage during error-prone lesion bypass. The 1S and 1R trans-anti-benzo[c]phenanthrene [BcPh] adducts were introduced into 5′-d(ATCGCXCGGCATG)-3′‚5′-d(CATGCCGCGCGAT)-3′, X ) trans-anti-[BcPh]-N2-deoxyguanosine. The solution structures for the two adducts were refined using molecular dynamics calculations restrained by 1H NOEs and 3J coupling constants. The 1R adduct intercalated from the minor groove. Intercalation occurred to the 3′-side of the modified strand. We previously reported [226th National Meeting of the American Chemical Society, New York, 2003] that the 1S adduct intercalated with the BcPh moiety oriented 5′ to the adducted dG. Subsequent analyses of the NMR data revealed that our earlier conclusion was incorrect. In this iterated repeat sequence, the 1S adduct is not intercalated but orients instead in the minor groove, facing in the 5′-direction from the site of adduction. In contrast, when these two adducts were introduced into 5′-d(CCATCXCTACC)-3′‚5′-d(GGTAGCGATGG)-3′, a noniterated sequence that contained the same 5′-CXC-3′ flanking bases, both adducts intercalated [Lin, C. H., Huang, X., Kolbanovskii, A., Hingerty, B. E., Amin, S., Broyde, S., Geacintov, N. E., and Patel, D. J. (2001) J. Mol. Biol. 306, 1059-1080]. These results illustrate the complex interplay of PAH adduct stereochemistry, the topology of the PAH ring system, and the DNA sequence context, in determining PAH adduct structure in DNA. This work was supported by NIH Grant CA-55678 (M.P.S.). 43. Detection of DNA Damage Using Films Containing [Os(bpy)2(PVP)10Cl]+ and [Ru(bpy)2(PVP)10Cl]+ Metallopolymers. Amos Mugweru,1 Wang Bingquan,2 and J. Rusling.1 (1) Department of Chemistry,

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University of Connecticut, 55 North Eagleville Road, Box U-60, Storrs, Connecticut 06269-3060. E-mail: [email protected]. (2) Chemistry, University of Connecticut. Layer by layer films containing [Os(bpy)2(PVP)10Cl]+ and [Ru(bpy)2(PVP)10Cl]+ metallopolymer catalysts were assembled on a pyrolytic graphite electrode and used to catalyze the oxidation of guanine and 8-oxoguanine, an important biomarker of DNA oxidation. These films exhibited reversible electrochemistry for both Os(III)/ Os(II) and Ru(III)/Ru(II) centers, typical of individual compounds although in the same film. The Os(III)/Os(II) center had a formal potential of 0.336 V vs SCE and was found to catalyze the oxidation of 8-oxoguanine formed from reaction of DNA with hydroxyl radicals generated after incubation of DNA with the Fenton reagent both in films and in solution. Films analyzed in oxidatively damaged DNA showed an increase of square wave voltammetric catalytic current at the Os(III)/Os(II) center and at the Ru(III)/Ru(II) centers. DNA not oxidized with Fentons reagent gave a large catalytic peak current at the Ru(III)/Ru(II) center while no change in peak current was observed at the Os(III)/Os(II) center. Only guanine was available for oxidation at the Ru(III)/Ru(II)center before the formation of 8-oxoguanine. After incubation with Fentons reagent, to form 8-oxoguanine, a catalytic current was observed at the Os(III)/Os(II) center. The Ru(III)/Ru(II) center with a formal potential of approximately 0.75 vs SCE oxidized DNA bases and oxidized 8-oxoguanine at a faster rate than guanine because 8-oxoguanine has a lower oxidation potential than guanine and the other bases. Incubation of DNA with the Fenton reagent at different times yielded oscillating ratios of final to initial peak currents. This phenomena was also observed using polyguanilic acid and salmon testes DNA. Control experiments using DNA and FeSO4 without H2O2 and using DNA with H2O2 but without FeSO4 did not yield any significant increase in catalytic current. 44. Structural Perturbations Arising from Butadiene-Induced Adducts in Duplex DNA. W. Keither Merritt, Tandace A. Scholdberg, Lubomir N. Nechev, Stephen M. Dean, Thomas M. Harris, Constance M. Harris, R. Stephen Lloyd, and Michael P. Stone. Department of Chemistry, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, Tennessee 37235. Fax: 615-322-7591. E-mail: [email protected]. Butadiene is oxidized to mono- and diepoxides, which react with the N1 position of dA. These N1-dA adducts rearrange to N6-dA adducts or deaminate to N1-inosine adducts. The N6-dA R,R- and S,S-trihydroxybutyl adducts arising from butadiene diol epoxide were accommodated in the major groove with minimal perturbation of the DNA. The glycosyl bonds of N1-inosine R- and S-βadducts arising from butadiene epoxide were in the syn conformation placing the butadiene moiety into the major groove. An N6-dA-N6-dA intrastrand cross-link that might arise from reaction with butadiene diepoxide induced a kink in the duplex. These data were evaluated with respect to site specific mutagenesis data that revealed weak mutagenicity for the N6-dA adducts and a preference for insertion of dC opposite the N1-inosine adducts. The structural data also suggest that formation of N6-dA-N6-dA cross-links in DNA may not be facile. This work was supported by NIH Grant ES-05509 (M.P.S).


45. DIGE and SILAM Analysis of Oxidative Stress Response between Nonmalignant and Malignant Human Mammary Cells. Yuan Yan, Valerie M. Weaver, and Ian A. Blair. Center for Cancer Pharmacology, University of Pennsylvania, 1246 BRB II/III, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104-6160. Fax: 215-573-9889. E-mail: [email protected]. Two-dimensional fluorescence difference gel electrophoresis (2D DIGE), which resolves multiple samples labeled with distinct fluorescent dyes on the same 2D gel, provides a rapid and reproducible way to compare the proteome. Stable isotope labeling by metabolism (SILAM), on the other hand, allows for more accurate protein quantification using mass spectrometry. To explore the effect of oxidative stress on breast cancer cell lines, we performed both 2D DIGE and SILAM experiments on the whole cell proteome. Using LC/ESI/MS/MS, we were able to identify and accurately quantify differentially expressed proteins, both novel and previously associated with the oxidative stress response. Additional experiments are underway to explore the relevance of these observations to breast cancer prevention and treatment. This work is supported by NIH RO-1 CA95586. 46. cis-2-Butene-1,4-dial, a Reactive Metabolite of Furan, Forms Substituted N1,N6-Etheno-2’-deoxyadenosine and N1,N2-Etheno-2’-deoxyguanosine Adducts. Michael C. Byrns, Choua C. Vu, and Lisa A. Peterson. Division of Environmental Health Sciences and Cancer Center, University of Minnesota, Minneapolis, Minnesota 55455. Fax: 612-626-5135. E-mail: byrns001@ umn.edu. Furan induces liver toxicity and tumor formation in rodents and is classified as a probable human carcinogen. Its metabolite, cis-2-butene-1,4-dial, is mutagenic in the Ames assay, and previous studies show that it reacts with dCyd, dAdo, and dGuo. The initial dCyd adducts are stable, while the dAdo and dGuo adducts rearrange through a pH-dependent mechanism to secondary products. On the basis of UV absorbance, fluorescence, NMR, and mass spectral data, the rearrangement product of the dAdo adduct was identified as a substituted ethenodAdo adduct. The NMR characterization of the O-methyl oxime derivative of the secondary dGuo adduct was the key step in determining its structure as a substituted etheno-dGuo adduct. Both of these adducts retain reactive aldehydes with the potential to form cross-links, likely contributing to furan’s toxicological effects. The characterization of these secondary products is an important step toward understanding the mechanism of furan-induced carcinogenesis. 47. Toxicity and Mechanical Properties of Peroxide-Cured Silicone Rubber Foams. Hea Sun Yang. Institute of Allergy, Department of Internal Pediatrics, Yonsei University College of Medicine, Seoul 120-749, South Korea. E-mail: [email protected]. Hyo Joo Bang. Department of Biology, Sangmyoung University. Eun Soo Park. Department of Product Development, Youngchang Silicone Co., Ltd. Silicones comprise a wide variety of materials such as fluids, elastomers, resins, and foams. Traditional curing agents for silicone rubber compounds are organic peroxides, which, when heated, decompose to form free radicals that react with the pendant organic groups on the silicone polymer. However, the peroxide reaction leaves a toxic acid residue in the rubber that can deposit a powder or bloom on the part surface. The acidic byproducts play an


important role in the toxicity of the final product. In this study, various silicone rubber foams were prepared by peroxide curing of a silicone compound in the presence of 2,2′-azobisisobutyronitrile (AIBN) blowing agent and various peroxide curing agents such as dicumyl peroxide, di-tert-butyl peroxide, 2,4-dichloro benzoyl peroxide, or p-methyl benzoyl peroxide. The toxicity and mechanical properties of the silicone rubber foams have been evaluated. 48. Accelerator Mass Spectrometry for Ultrasensitive Quantification of Modified Tyrosine Residues following Selective Introduction of 14C-Labeled Compounds into Peptides. Sang Soo Hah,1 Steve Kim,1 Constanze Bergt,2 John Vogel,3 Jay Heinecke,2 and Paul T. Henderson.1 (1) Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Mail Code L-441, 7000 East Avenue, Livermore, California 94550-9698. Fax: 925-424-2151. E-mail: [email protected]. (2) Department of Metabolism, University of Washington. (3) Center for Accelerator Mass Spectrometry, Lawrence Livermore National Laboratory. Nitration and chlorination of protein-bound tyrosine residues are implicated in oxidative stress and the pathogenesis of human disease. While characterization of tyrosine chlorination and nitration in amino acids is well-established, it has been difficult to establish robust methods to identify oxidative modifications of proteins isolated from human tissue. We have developed ultrasensitive methods to quantify specific sites of amino acid modification in peptides. The overall approach has been to selectively introduce a 14C-labeled acetyl group onto tyrosine residues followed by accelerator mass spectrometry, which readily detects subattomole quantities of radiolabeled material. We are currently investigating the application of these methods to the quantification of oxidized tyrosine residues in modified lipoproteins that may play a critical role in atherosclerosis. This work was performed at UC LLNL under the auspices of the U.S. DOE Contract W-7405-ENG-48. 49. Alkylation Profile of dA, dG, and DNA by n-Propyl Diazonium Ion. Jacqueline M. Heilman and James C. Fishbein. Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250. Fax: 410-455-2608. E-mail: [email protected]. To elucidate factors contributing to atom site selectivity, alkylation by n-propyl diazonium ion is currently being investigated. Reactions of primary diazonium ions with nucleosides and DNA have been thought to be dependent only on the nucleophilic character of the atom site. Recently, in the case of secondary diazonium ions, nucleophilicity has been shown to be unimportant. The importance of nucleophilicity and other factors may begin to be evaluated from the product distributions arising from use of the n-propyl diazonium ion, as this alkylating agent can partition between direct reaction, forming n-propyl adducts, or undergo rearrangement with subsequent reaction, forming isopropyl adducts (below). A quantitative profile of product distribution from this system will be presented for dA, dG, and DNA.

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50. Alkylation Stress Triggers Decrease in Enzymatic Activity of Protein Phosphatase 2A (PP2A). Simona G. Codreanu,1 Deanna G. Adams,2 Brian E. Wadzinski,2 and Daniel C. Liebler.1 (1) Mass Spectrometry Research Center, Biochemistry Department, Vanderbilt University, 9264 Medical Research Building III, 465 21st Avenue South, Nashville, Tennessee 372322653. Fax: 615-343-8372. E-mail: simona.codreanu@ vanderbilt.edu. (2) Pharmacology Department, Vanderbilt University. Protein serine/threonine phosphatase 2A (PP2A) is a potential target for reactive electrophiles that dysregulate phosphorylation specific signal transduction cascades. PP2A holoenzymes are composed of a catalytic (C) subunit, a structural (A) subunit, and a variable (B) regulatory subunit. We studied the modification of two purified PP2A holoenzymes (ABRC and ABδC) with the thiol reactive electrophiles PEO-iodoacetylbiotin and the N-alkylmaleimide biotin-BMCC. Electrophile treatment produced a dose-dependent increase in adduct formation, as observed by Western blot analysis. Although both electrophiles labeled all PP2A subunits, only biotinBMCC caused a decreased catalytic activity for both holoenzymes. Adduction of Cys residues in all three subunits was characterized by LC-MS-MS. Studies with stable isotope tag methods were used to measure the kinetic reactivity of target Cys residues. Comparisons of adduction patterns and kinetics of target modifications are directed at identifying specific residues responsible for PP2A enzymatic function. This work was supported by NIH Grants ES10056, GM51366, and ES000267. 51. Analysis of Conjugated Estrogens in Plasma by LC/Electron Capture Atmospheric Pressure Chemical Ionization/MS. Xingpin Cui, Seon Hwa Lee, Peter O’Dwyer, Peter Moate, Ray Boston, and Ian A. Blair. Department of Pharmacology, Center for Cancer Pharmacology, University of Pennsylvania, 846 BRB II/III, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104. Fax: 215-573-9889. E-mail: xingpin@ spirit.gcrc.upenn.edu. We have recently developed a stable isotope dilution LC/electron capture atmospheric pressure chemical ionization/tandem mass spectrometry (LC/ECAPCI/MS/MS) method for analyzing free estradiol and its metabolites in human plasma. This method was used to quantify estrogens in plasma after sublingual dosing with estradiol. The major circulating metabolites were estrone and 2-methoxy-estrone. Estradiol concentrations were very low, which suggested that substantial metabolism had occurred in the buccal epithelial system. Methodology was developed to analyze the plasma conjugates by coupling LC/ECAPCI/MS/MS analysis with enzymatic hydrolysis using arylsulfatase/×c4′′-glucuronidase. The limit of detection for estrogen metabolites was 4 pg on column. This assay revealed that substantial presystemic conversion of estradiol to estrone occurred and that both estradiol and estrone were conjugated. Therefore, the buccal epithelial cells must contain a substantial capacity to oxidize estradiol (through 17×c4′′-hydroxysteroid dehydrogenase) and to conjugate both estrogens (through sulfotransferase and/or UDP glucuoronosyl transferase). This work was supported by NIH Grant PO1CA082707. 52. Anisotropic Effects of the DNA Environment on the Rotational Barrier of 5-Cyanoamino-1Himidazole-4-carboxylic Acid (CAICA). Rainer Glaser and Hong Wu, Department of Chemistry, University of

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MissourisColumbia, Columbia, Missouri 65211. Fax: 573-882-2754. E-mail: [email protected] and hwc8a@ mizzou.edu. It is our hypothesis that 5-cyanoimino-1H-imidazole4-oxomethylene is formed by nitrosative deamination of guanine and that subsequent 1,4-addition of water leads to 5-cyanoamino-1H-imidazole-4-carboxylic acid. CAICA is the precursor for formation of oxanosine, and 18Olabeling studies of guanosine deamination fully support this hypothesis (J. Am. Chem. Soc., in press). The interpretation of the isotopomer ratios requires the knowledge of the rotational barrier about the C-COOH bond in CAICA. A low rotational barrier ensures an equal distribution of 18O between both acid O-atoms and an isotopomer ratio of unity. On the other hand, a high rotational barrier would give rise to an isotopomer ratio that differs from unity. Here, we present the results of ab initio studies that show large increases of the rotational barrier in the DNA environment as the result of the interaction of CAICA with neutral or protonated cytosine (the aggregate between CAICA and protonated cytosine is shown).

53. Characterization of Carrageenan-Derived Oligosaccharides and Its Effect on T Cell Function and Subset in Irradiated Mice. Yi Li. Navy 401 Hospital, 6 Minjiang Road, Qingdao 266071, China. E-mail: [email protected]. Wenjun Mao. Marine Drugs and Foods Institute, Ocean University of China, 5 Yushan Road, Qingdao 266003, China. E-mail: wenjunmqd@ hotmail.com. Carrageenan is a sulfated polysaccharide, which contains alternate β-D-galactose moieties connected by R-1,3 and β-1,4 linkages. Wide attention has been paid to carrageenan and the characterization of its special structure. In this paper, carrageenan was modified into carrageenan-derived oligosaccharides by degradation, and the characterization of carrageenan-derived oligosaccharides and their effects on T cell function and subset in irradiated mice were investigated. The molecular weight of carrageenan-derived oligosaccharides was determined by HPGPC. The structures of the carrageenan-


derived oligosaccharides were elucidated by NMR, IR, and MS. The animal experiment revealed that the carrageenan-derived oligosaccharides increased ConAand LPS-stimulated transformation of splenic T lymphocyte of mice, which was characterized by marked upregulation of CD69 expression in the thymus lymphocytes. It also significantly enhanced the ability of PBMC to produce IL-2 and IFN-R, decreased the expression of TNF-R by T-cells in PBMC, and overexpressed CD4+ in T lymphocytes, which was followed by an increment change in the ratio of CD4+/CD8+ T lymphocytes. Thus, carrageenan-derived oligosaccharides can markedly adjust T cell function and subset in irradiated mice. 54. Chemistry of the Diazeniumdiolates. RHN[N(O)NO]- Ions as Progenitors of Both Nitroxyl and Nitric Oxide. Aloka Srinivasan,1 Michael L. Citro,2 Keith M. Davies,3 Michael G. Espey,4 Larry K. Keefer,1 Katrina M. Miranda,5 Lisa A. Ridnour,4 Joseph E. Saavedra,2 Lynta Thomas,5 Douglas D. Thomas,4 David J. Waterhouse,1 and David A. Wink.4 (1) Chemistry Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute at Frederick, Building 538, Room 205E, Frederick, Maryland 21702. Fax: 301-846-5946. Email: [email protected]. (2) BRP, SAIC-Frederick, Inc., National Cancer Institute at Frederick. (3) Department of Chemistry, George Mason University. (4) Radiation Biology Branch, National Cancer Institute. (5) Department of Chemistry, University of Arizona. There is growing evidence that nitroxyl (HNO and/or its conjugate base NO-) has a rich pharmacological potential, which differs from that of its one-electron oxidation product, nitric oxide (NO). Here, we show that the isopropylamine/nitric oxide adduct ion iPrHN[N(O)NO]- (IPA/NO) dissociates to both NO and nitroxyl in a pH- and concentration-dependent partition favoring increasing nitroxyl as [IPA/NO] increased or as the pH was raised. Byproducts of the nitroxyl-forming path included its dimerization/dehydration (N2O) and autoxidation (ONOO-) products as well as 2-propanol and (when nitrosobenzene was present) Ph[N(O)NO]-. Isopropylamine was isolated as the byproduct of the NOforming pathway. The results suggest that prodrugs of structure RHN[N(O)NO]- that cogenerate nitroxyl, NO, diazonium ions (RN2+), and potentially bioactive primary amines simultaneously may have uniquely beneficial pharmacological effects. 55. Comparative Proteomic Analysis of Control and RcsX Tumor-Bearing SJL Mouse Plasma by Gel-LC-MS/MS. Vadiraja B. Bhat, Man Ho Choi, John S. Wishnok, and Steven R. Tannenbaum. Biological Engineering Division, Massachusetts Institute of Technology, 77 Massachusetts Avenue, 56-738A, Cambridge, Massachusetts 02139. Fax: 617-252-1787. E-mail: [email protected]. In SJL mice, the growth of RcsX lymphoma cells induces an inflammatory response by stimulating Vβ16+T cells. During inflammation, given serum protein levels can increase (e.g., acute phase reactants) or decrease (e.g., albumin), and most of these altered proteins are thus potential biomarkers. Although blood plasma is a valuable sample for biomarker discovery for diseases or for novel drug targets, it is a highly complex proteome. We have focused on a comprehensive comparison of the plasma proteomes from control and RcsX tumor-bearing SJL mice and have developed a simple immunoaffinity spin filter method to remove albumin and IgG from 2 µL


of plasma. Depleted samples were resolved by gradient 1D SDS-PAGE, and the entire gel lane was cut into 30 slices and in-gel digested with trypsin. Extracted peptides were analyzed by using an Applied Biosystems nano-LC/ MS/MS system, and the MS/MS spectra were analyzed on the Agilents Spectrum Mill MS Proteomics Workbench. This analysis resulted in the identification of a total of 1113 nonredundant mouse plasma proteins, more than 498 in control and 816 in RcsX tumor-bearing SJL mouse plasma. Of these, only 201 proteins were found in common. The molecular masses ranged from 2 to 876 kDa, covering the PI values between 4.22 and 12.09, and included proteins with predicted transmembrane domains. Comparison of plasma proteomic profiles of control and RcsX tumor-bearing SJL mice revealed significant changes in the levels of many proteins in RcsX tumor-bearing mouse plasma. Most of the up-regulated proteins were identified as acute phase reactants. Also, several unique proteins, i.e., Proteasome subunits, Lselectin, Fetuin-B, 14-3-3 ζ, melanoma-associated antigen B4, etc., were found only in RcsX tumor-bearing SJL mouse plasmaseither secreted, shed by membrane vesicles, or externalized due to cell death. These results affirm the effectiveness of this approach for protein identification from small samples and for comparative proteomics in animal models of human disorders. 56. Comparisons of DNA Replication of Templates with Single (+)-trans-BPDE-dG Lesions Catalyzed by a Pair of Thermophilic A Family and Y Family Polymerases. Lida Oum,1 Xuanwei Huang,1 Alexander Kolbanovskii,1 Jacek Krzeminiski,2 Shantu Amin,2 and Nicholas Geacintov.1 (1) Department of Chemistry, New York University, 29 Washington Place, New York, New York 10003. E-mail: [email protected]. (2) Institute for Cancer Prevention. The influence of (+)-trans-anti-B[a]P-N2-Gua lesions (G*) in site specifically modified oligonucleotides (43mers) on translesion bypass of two thermophilic polymerases from the A and Y families was compared. The A family polymerases are normal replicative polymerases, while Y family polymerases are involved in translesion bypass. We have used the polymerases Bst and DpO4 as representative polymerases from the A and Y families, respectively, to compare their activities using two template-primer complexes (5′-...TG*G... and 5′-...CG*G...) that were identical in composition except for the base (and its complement) flanking the lesion G* on the 5′side. There is a significant base sequence effect on primer extension in the case of DpO4, but not Bst, and the effect of temperature, and nucleotide insertion selectivities can also vary as a function of temperature (37 and 55 °C). While Bst cannot extend the primer beyond the lesion site, DpO4 readily extends the primer in a sequence- and temperature-dependent manner. 57. Competing Roles of Cytochrome P450 1A1/1B1 and Aldo-Keto Reductase 1A1 in the Metabolic Activation of (()-7,8-Dihydroxy-7,8-dihydro-benzo[a]pyrene in Human Lung Cells. Hao Jiang, Amy M. Quinn, and Trevor M. Penning. Department of Pharmacology, University of Pennsylvania School of Medicine, 130C John Morgan Building, 3620 Hamilton Walk, Philadelphia, Pennsylvania 19104-6084. E-mail: penning@ pharm.med.upenn.edu. CYP1A1 and CYP1B1 are NADPH-dependent monoxygenases involved in the metabolic activation of (()7,8-dihydroxy-7,8-dihydro-benzo[a]pyrene (BP-7,8-diol),

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generating (()-anti-benzo[a]pyrene-diol-epoxide (antiBPDE). In contrast, aldo-keto-reductases (AKR1A1, AKR1C1-AKR1C4) are NAD(P)+-dependent enzymes that play competing roles in BP-7,8-diol activation by forming the reactive and redox active benzo[a]pyrene-7,8-dione (BPQ) and formation of reactive oxygen species (ROS). The relative contributions of CYPs and AKRs in BP-7,8diol activation were compared in bronchoalveolar H358 cell lysates containing inducible CYP1A1/CYP1B1 (via TCDD induction) and recombinant AKR1A1 (via stable transfection with AKR1A1) as the NAD+/NADPH ratios were varied. Systems containing a higher ratio of NAD+/ NADPH favored formation of BPQ while the systems containing the lower ratio of NAD+/NADPH favored formation of anti-BPDE. These data suggest that the dominant pathway of PAH activation may be dependent upon redox state. These systems will permit a comparison of the DNA lesions caused by the ultimate carcinogenic products generated by CYP1A1 (anti-BPDE) and AKR1A1 (BPQ and ROS).

58. Computational Modeling of Covalent Deoxyguanosine Adducts. Robert Q. Topper. Department of Chemistry, Medical Technology and Physics, Monmouth University, Thomas A. Edison Hall of Science, West Long Branch, New Jersey 07764-1898. Fax: 732-263-5213. E-mail: [email protected]. Timothy A. Isgro. Department of Physics, University of Illinois at UrbanaChampaign. Nitin Mathew. Department of Chemistry, Cooper Union for the Advancement of Science and Art. After activation, the widely studied hepatic carcinogen N-acetyl-2-aminofluorene (AAF) may form a major or a minor adduct with DNA either in vivo or in vitro. The structure of the minor adduct has not been experimentally determined and is believed to be of importance due to its persistence in vivo. In the present study, four varieties of modified AAF have been studied computationally: AAF bonded to C8 of guanine (G) or deoxyguanosine (dG) (in analogy with the major adduct) and AAF bonded to N2 of guanine or deoxyguanosine (minor adduct). A computational cascade was employed to search for low-energy conformers of each molecule. Each adduct was subjected to a series of systematic searches and molecular mechanics geometry optimizations of thousands of structures. After convergence with respect to the search parameters was obtained, the resulting conformers were then geometry optimized via benchmarked quantum mechanical methods, including density functional theory and semiempirical molecular orbital theory.

Dedon and Penning

These results provide substantial motivation for experimental work on model AAF adduct complexes, highlight important local stresses in the complexes, and yield insight into which computational models may be reliable for simulations of modified DNA. Other modified guanosine and deoxyguanosine systems will also be discussed. 59. Computational Studies of Spiroiminodihydantoin Lesions. Lei Jia,1 Vladimir Shafirovich,1 Brian E. Hingerty,2 Robert Shapiro,1 N. E. Geacintov,1 and Suse Broyde.3 (1) Department of Chemistry, New York University, New York, New York 10003. (2) Life Sciences Division, Oak Ridge National Laboratory. (3) Department of Biology, New York University. Oxidative stress in cellular environments generates DNA damage that involves, predominantly, the formation of the oxidation product 8-oxo-7,8-dihydroguanine (8-oxoG), which can be further oxidized to spiroiminodihydantoin (Sp). The Sp R- and S-stereoisomers are highly mutagenic, both in primer extension experiments in vitro and in vivo, causing GfT and GfC transversions. Hence, their structural properties are of great interest. To elucidate the structures of these novel propeller-like lesions, we have carried out computational investigations on the level of the modified base, the nucleoside, and the Sp lesion positioned within duplex DNA. On the base level, quantum mechanical geometry optimization studies revealed exact mirror image symmetry in the R- and S-stereoisomers, with near-perpendicular geometry of the two rings. On the nucleoside level, an extensive survey of the potential energy surface with molecular mechanics calculations using AMBER provided three-dimensional energy maps. These maps revealed that the range of glycosidic torsion angles is more restricted in both stereoisomers than in unmodified dG and that both syn and anti domains are feasible. Moreover, there are subtle differences between the R- and the S-forms. Molecular dynamics simulations for DNA duplexes incorporating the R- and S-stereoisomers, using AMBER, further elucidated the conformational features of these lesions and suggested possible structural elements that may stabilize mismatches opposite these damaged guanines. This work was supported by the NIH. 60. Covalent Modification of Cytochrome c by Lipid Hydroperoxide-Derived Bifunctional Electrophile. Roman Shimanovich. Department of Pharmacology, University of Pennsylvania, 421 Curie Boulvard, BRB II/III Room 843, Philadelphia, Pennsylvania 19104. E-mail: [email protected]. Ian. A Blair. Center for Cancer Pharmacology, University of Pennsylvania. Lipid hydroperoxide-derived bifunctional electrophiles arise in cells under conditions of oxidative stress, can covalently modify proteins, and have been associated with a number of important pathological conditions. The discovery of modified proteins, involved in apoptosis, can demonstrate the involvement of bifunctional electrophiles in cell death and offer new insights into the role of lipid peroxidation in cell pathophysiology. Because of the centrality of mitochondria in cellular metabolism, changes to mitochondrial components have profound effects on cellular biology. In particular, modifications of proteins in the mitochondrial matrix, as well as the changes in membrane properties, such as mitochondrial membrane permeability transition (MMPT), can lead to initiation of apoptosis. We have observed the covalent modification of cytochrome c by 4-oxy-2-nonenal (4-ONE), a lipid


hydroperoxide-derived bifunctional electrophile, in a concentration-dependent manner. LC/MS/MS analysis of modified protein digests points to the adduct formation at lysine residues through Michael addition. HPLC data show that the modification of cytochrome c by 4-ONE significantly modifies its surface hydrophobicity. The discovery of covalent modification of cytochrome c by 4-ONE provides a mechanism for its release from the mitochondrial membrane, which initiates MMPT, a key step in apoptosis. 61. Cyanoimine or Pyrimidine? Synthesis, Reactions, and Toxicological Relevance of 5-Cyanoamino-4-imidazolecarboxamide. Rainer Glaser and Ming Qian. Department of Chemistry, University of MissourisColumbia, Columbia, Missouri 65211. Fax: 573-882-2754. E-mail: [email protected] and qianm@ missouri.edu. Theoretical and experimental studies have shown that nitrosative guanine deamination can lead to 4-oxomethylene-5-cyanoimino-4,5-dihydroimidazole (1). The hydrolysis of 1 is fast and leads to the acid 2, the precursor to oxanine formation (J. Am. Chem. Soc., in press). In this context, we have been studying the chemistry of the amide 3. We recently communicated (J. Am. Chem. Soc. 2004, 126, 2274) the synthesis of pure 5-cyanoamino-4imidazolecarboxamide (3), its pH-dependent cyclization to guanine 7 and isoguanine 8, and the cross-link formation by reaction of 3 with guanosine. Here, we report details of the base-catalyzed reaction of 4. It was found that 3 is formed together with the pyrimidines 9-(2hydroxyethoxymethyl)-2-methylthioadenine (5) and 9-(2hydroxyethoxymethyl)-2-methylthiohypoxanthine (6). The relative yields of the three products can be influenced by variation of the pH and the reaction temperature. Pyrimidine 6 might be formed directly from 4 or indirectly via 5, and these options are discussed. In particular, the insights gained in these studies suggest plausible strategies for the synthesis of 2.

62. Defining the Source of Endogenous DNA Adducts: Base Propenals as the Source of M1G in Cells. Xinfeng Zhou and Peter C. Dedon. Biological Engineering Division, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139. E-mail: [email protected]. The oxidation of cellular macromolecules yields a variety of electrophiles that react to form DNA adducts.


For example, the pyrimidopurinone adduct of guanine, M1G, arises in reactions of DNA in vitro with malondialdeyde (MDA) from the peroxidation of polyunsaturated fatty acids (PUFA). We have shown that M1G also arises from reactions of DNA with base propenals, products of 4′-oxidation of deoxyribose by bleomycin, peroxynitrite, and other oxidants. We now report the results of a series of in vitro and cellular studies in Escherichia coli and human TK6 cells aimed at defining the source of M1G. Using HPLC with postcolumn TBA derivatization for base propenals and MDA, LC/MS/MS for etheno-dA, and an immunoblot assay for M1G, in vitro studies revealed that the generation of base propenals correlates directly with M1G formation for peroxynitrite and bleomycin, while γ-radiation and Fe(II)-EDTA produced malondialdehyde instead of base propenals and that there was no M1G formed in DNA treated with either of these oxidants. These in vitro studies set the stage for work with E. coli and human TK6 cells in which PUFA content was varied by growth in defined medium. In E. coli treated with peroxynitrite, there was an inverse correlation between M1G and PUFA content and thus MDA formation. Similar results were obtained with TK6 cells exposed to SIN-1, a peroxynitrite generator, although the level of PUFA variation was limited by a cellular requirement for PUFA. The results with M1G are compared to the formation of etheno-dA as a biomarker for lipid peroxidation-derived DNA lesions. These results suggest that base propenals derived from DNA oxidation represent an important source of M1G adducts. 63. Deoxyribose Oxidation in DNA as a Source Adducts in Histone Proteins. Tao Jiang, Xinfeng Zhou, Min Dong, and Peter C. Dedon. Biological Engineering Division, NE47-277, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139. Fax: 617-324-7554. E-mail: taoj@ mit.edu. DNA oxidation has been implicated in the pathophysiology of cancer and aging, with recent evidence indicating a critical role for deoxyribose oxidation in these processes. Oxidation of each position in deoxyribose produces a set of potentially genotoxic electrophiles that can react with DNA and proteins. For example, 5′-oxidation leads to strand breaks containing 5′-(2-phosphoryl-1,4-dioxobutane) and 3′-formyl phosphate residues, the latter being a highly reactive acylating species. The proximity of lysine-rich histone proteins to DNA in nuclei suggested that oxidative DNA damage could result in the transfer of these electrophiles to the histones. This hypothesis was tested by labeling DNA in human TK6 cells with [5′-3H]or [methyl-3H]thymidine (-thy) and treating the nuclei with γ-radiation and neocarzinostatin (NCS), an enediyne that causes 5′-chemistry. Following quantification of radioactivity in each class of histone, it was observed that NCS caused selective transfer of 3H-labels from [5′-3H]thy to the nucleosome linker histone H1, with lesser amounts associated with core histones H2B and H3. This is consistent with the linker selective DNA cleavage produced by NCS. Both the degree and the selectivity of labeling decreased when [methyl-3H]thy was used, which is consistent with a role for the 5′-carbon of deoxyribose in labeling of histone proteins. These results suggest that the products of oxidative DNA damage can react with nuclear proteins with the possible disruption of the normal function of the proteins. In the case of the 3′-formyl phosphate residues, acylation of the side chain

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amines of lysines would result in N-formylation analogous to the physiologically important N-acetylation that controls gene expression. 64. Detection of Protein-Metabolite Adducts Using Natural Isotopic Signatures. Weimin Yue, Yakov M. Koen, and Robert P. Hanzlik. Department of Medicinal Chemistry, The University of Kansas, Malott Hall 4048, 1251 Wescoe Hall Drive, Lawrence, Kansas 66045. Fax: 785-864-5326. E-mail: [email protected]. The hepatotoxicity of bromobenzene (BB) is associated with, and believed to be triggered by, the covalent binding of its chemically reactive metabolites to hepatocellular proteins. Our previous chemical analysis of unfractionated liver protein revealed Cys, His, and Lys adducts of BB-derived epoxide and quinone metabolites, and proteomic analysis by peptide mass mapping identified a number of individual proteins as targets for reactive [14C]BB metabolites [(2002) Chem. Res. Toxicol. 15, 699-706]. To facilitate searching for specific adducts on individual target proteins, we constructed model adducted proteins by EDC coupling of 1 and 2 to lysine side chains on RNase and analyzed the conjugates [RNase-(1)n and RNase-(2)n, respectively] and their tryptic digests by MALDI-TOF/MS and ESI MS/MS. RNase-(1)n showed primarily n ) 0-2 species with n ) 1 predominating, while RNAse-(2)n showed n ) 0-6 species with n ) 4 predominating. After tryptic digestion, analysis of unmodified RNase showed 93% coverage including nine of 10 lysines. Similar analyses of RNase-(1)n and RNAse-(2)n showed adduction at K1, K7, K37, K91, and K98: the isotopic signature of bromine was very conspicuous in the adducted peptides. Failure to observe modification at K31, K41, K61, K66, and K104 might be due more to the large size of their tryptic peptides than to a lack of intrinsic reactivity. This work was supported by NIH-GM21784.

65. Determination of 14C-Labeled 7,8-Dihydro-8oxo-2′-deoxyguanosine Incorporation into MCF-7 Human Breast Cancer Cells by Accelerator Mass Spectrometry. Sang Soo Hah, Steve Kim, and Paul T. Henderson. Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory, Mail Code L-441, 7000 East Avenue, Livermore, California 94550-9698. Fax: 925-424-2151. E-mail: hah2@ llnl.gov. Accelerator mass spectrometry (AMS) is a wellestablished analytical technique for detection of extremely low abundance stable and long-lived radioactive isotopes in various research fields. We report the use of AMS to characterize the oxidation of DNA, particularly 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-OxodG), a wellknown biomarker of oxidative stress in DNA, and the products of 8-oxodG oxidation in vivo. We report the incorporation of 14C-labeled 8-oxodG into the DNA of MCF-7 cells, which occurs to a low extent as compared to 14C-labeled 2′-deoxyguanosine. The incorporation of both nucleosides into cellular DNA was readily measurable by AMS, and the conversion of 8-oxodG into secondary oxidation products in vivo is currently under investigation. This work was performed at UC LLNL under the auspices of the U.S. DOE Contract W-7405-ENG-48.

Dedon and Penning

66. Determining Reaction Rates of Protein Adduction at Different Sites on the Same Protein by Quantitative Mass Spectrometry. Christopher R. Orton,1 Amy Joan L. Ham,1 and Daniel C. Liebler.2 (1) Department of Biochemistry, Vanderbilt University, 9264 Medical Research Building III, 465 21st Avenue South, Nashville, Tennessee 37232-8575. Fax: 615-343-8372. E-mail: [email protected]. (2) Department of Biochemistry, Vanderbilt University Medical Center. Reaction of glutathione S-transferase P1-1 (GSTP1-1) with the N-alkylmaleimide biotin-BMCC forms adducts at Cys-14 and Cys-47 and decreases GSTP1-1 activity. Kinetic reactivities of these two targets were measured by stable isotope tagging and LC-MS-MS. Following alkylation and tryptic digestion, GSTP1-1 peptides were tagged at their N-termini with unlabeled phenyl isocyanate (PIC). Samples of maximally adducted GSTP1-1 peptides (e.g., the longest incubation time) were treated with 13C6-PIC where aliquots of this sample were paired with samples from earlier time points and analyzed by LC-MS-MS. Plots of ratios of light/heavy forms vs time were used to estimate rate constants for alkylation reactions. These analyses generated kobs values of 0.0154 and 0.0547 µΜ-1 min-1 for adduction at Cys-14 and Cys47, respectively. This approach provides a quantitative method to simultaneously determine multiple site specific rates of protein adduction by reactive electrophiles. This work was supported by NIH Grants ES10056 and ES000267. 67. Development of an LC/MS Method to Quantify Homocysteine Concentrations in Human Plasma. Yuehua Huang. Pharmacology, University of Pennsylvania, 421 Curie Boulevard, 845 BRB II/III, Philadelphia, Pennsylvania 19104-6160. E-mail: [email protected]. A. Steven Whitehead. Pharmacogenetics, University of Pennsylvania, Philadelphia, Pennsylvania 190104. Ian Blair. Department of Pharmacology, University of Pennsylvania. Elevated homocysteine is considered to be an important risk factor for cardiovascular diseases and cancer. Therefore, it is important to be able to determine its concentration in plasma with high specificity and accuracy. For reducing homocystine to homocysteine, TECP [tris-(2-carboxyethyl)phosphine] was used to chemically reduce 1 mol of homocystine to 2 mol of homocysteine. After it was reduced, a triple quadrupole tandem mass spectrometer (Theromo Finnigan TSQ7000) was used to monitor the homocysteine using the transition m/z 136 to m/z 90 and D4homocysteine using the transition m/z 140 to m/z 94 in the multiple reaction monitoring mode. The recovery of homocysteine from plasma was 90% or higher. The limit of detection was 15 pg (110 fmol) on column. From 3 days of validation, the intraday precision for LLQC, LQC, MQC, and HQC was 1.8, 3.8, 0.8, and 1.8%, respectively. The interday precision for LLQC, LQC, MQC, and HQC was 7.3, 4.3, 1.7, and 3.1%, respectively. A simple, sensitive, and accurate method has been developed for homocysteine quantitation. This work was supported by NIH Grant RO1 AR47663. 68. Development of Methods to Quantitatively Compare the Respective Contributions of CYPs and AKRs to the Metabolic Activation of PAH trans-Dihydrodiols in Vitro. Amy M. Quinn, Hao Jiang, and Trevor M. Penning. Department of Pharmacology, University of Pennsylvania School of Medicine, 135 John Morgan Building, 3620 Hamilton Walk, Phila-


delphia, Pennsylvania 19104-6084. Fax: 215-573-2236. E-mail: [email protected]. Polycyclic aromatic hydrocarbons (PAH) are environmental pollutants that require metabolic activation to exert their carcinogenic effects. The metabolism of benzo[a]pyrene (B[a]P) yields the proximate carcinogen (-)trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene [(-)-B[a]P7,8-diol]. Monooxygenation of (-)-B[a]P-7,8-diol by the human cytochrome P450 (CYP) enzymes CYP1A1 and CYP1B1 produces (+)-anti-benzo[a]pyrene diol epoxide which is readily hydrolyzed in vitro to tetraol products. Human members of the aldo-keto reductase (AKR) superfamily also oxidize PAH trans-dihydrodiols to generate cytotoxic and mutagenic o-quinones. We have established in vitro reconstituted systems to measure (()-B[a]P-7,8diol metabolism using RP-HPLC methods to quantitate product formation. Rates of tetraol production were measured in systems containing CYP1A1 or CYP1B1 Supersomes. Purified recombinant AKR1A1 and AKR1C1AKR1C4 enzymes were also used to activate B[a]P-7,8diol to the o-quinone product, which was quantitatively trapped as a thioether conjugate. Assignment of kcat and kcat/Km values by these methods will facilitate determination of the relative kinetic contributions of the AKR and CYP pathways to PAH trans-dihydrodiol activation in vitro. 69. Development of Sensitive Analytical Methods to Quantify the Products of 4′-Oxidation of Deoxyribose in Isolated and Cellular DNA. Bingzi Chen, Xinfeng Zhou, and Peter C. Dedon. Biological Engineering Division, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139. Fax: 617-324-7554. E-mail: [email protected]. DNA oxidation plays a role in the pathophysiology of cancer and other diseases. While base lesions have received the greatest attention, recent evidence indicates that deoxyribose oxidation plays a critical role in the cellular response to oxidative stress. Oxidation of each position in deoxyribose produces a unique set of potentially genotoxic electrophiles that can react with DNA and proteins. For example, oxidation of the 4′-position of deoxyribose in DNA results in the formation of either a strand break containing a 3′-phosphoglycolate residue and a base propenal (or malondialdehyde and free base) or a 4′-keto-1′-aldehyde abasic site. To explore the role of the cellular environment and the chemistry of the oxidizing agent in the extent of and partitioning of this 4′-chemistry, we have undertaken the development of sensitive GC/MS methods to identify and quantify the 3′-phosphoglycolate residue and the 4′-keto-1′-aldehyde abasic site. Both methods, with detection limits of ∼100 fmol using 170 µg of DNA, were applied to study isolated and cellular DNA damaged by ionizing radiations, bleomycin, enediyne antibiotics, and peroxynirite. Considered in the context of total deoxyribose oxidation under the same conditions, the results provide rigorous insights into the chemistry of 4′-oxidation of deoxyribose and its relationship to oxidation of other sites in the sugar, as well as a new approach to studying DNA oxidation in biological systems. 70. Different Translesion Bypass of Guanine-N2 Monoadducts of Mitomycin C and Guanine-N7 Monoadducts of 2,7-Diaminomitosene by η, Klenow exo-, Klenow exo+, and T7 exo-DNA Polymerases. Cristina C. Clement and Maria Tomasz. Chemistry Department, Hunter College, City University of New


York, 695 Park Avenue, New York City, New York 10021. E-mail: [email protected]. The guanine (G)-N2 DNA monoadduct of mitomycin C (MC), a cytotoxic anticancer drug, inhibits translesion bypass by DNA polymerases as shown previously. The noncytotoxic MC metabolite 2,7-diaminomitosene (2,7DAM) forms a G-N7 DNA monoadduct in vitro and in vivo. We tested a potential correlation between the relative ease of bypass of this adduct (as compared with the MC adduct) and the lack of cytotoxicity of 2,7-DAM. 24-mer and 27-mer templates, adducted at a single guanine either with MC or 2,7-DAM, were synthesized and submitted to extension of primers by T7 exo-, Klenow exo-, Klenow exo+, and η DNA polymerases. In the 27mer template/15-mer and 24-mer template/15-mer primer systems, the G-N7-2, 7-DAM adduct was bypassed by all four polymerases, resulting in the production of a fully extended primer. However, the extension was at a slower rate as compared with the control, nonalkylated template and each of the four polymerase had a different extension efficiency. In sharp contrast, the G-N2-MC monoadduct was not bypassed beyond the adduct position under the same condition by none of the four polymerases. These results suggest a positive correlation between cytotoxicity and inhibition of DNA synthesis in the case of the two mitomycin monoadducts. 71. Effect of Temperature and pH on the Secondary Structure of Organophosphorus Hydrolase (OPH) by Circular Dichroism. Jiayin Zheng,1 Xihui Cao,1 Roger M. Leblanc,1 Vipin K. Rastogi,2 Tu chen Cheng,2 and Joseph J. DeFrank.3 (1) Department of Chemistry, University of Miami, 1301 Memorial Drive Room 315, Department of Chemistry, Coral Gables, Florida 33124. E-mail: [email protected]. (2) U.S. Army Edgewood Chemical Biological Center. (3) U.S. Army. Organophosphorus hydrolase (OPH) is a bacterial enzyme that has been shown to degrade a wide range of neurotoxic organophosphate nerve agents. OPH-based biosensors have a promising future in the detection of the organophosphorus compounds (OPs), which are widely used in agriculture as pesticides and insecticides. In this study, the secondary structure of OPH was studied using circular dichroism (CD) in the far-ultraviolet region based on the optimum conditions for developing a stable monolayer of OPH at the air-water interface. A comparative study of the thermal treatment on the secondary structure of OPH in solution, as a Langmuir-Blodgett film and as a dry film, showed that the molecular packing plays a dominant role in the thermal stability of OPH. The effect of pH on the secondary structure of OPH solution was examined over the pH range from 2.53 to 9.50. As shown on the CD spectra, the secondary structure of the enzyme is almost intact at pH values near the isoelectric point (7.6) of OPH and is distorted more as the pH values increase or decrease further. This is explained by a loss of helical regions in OPH. 72. Effects of Brief Exposure to Hydrogen Chloride on Breathing Pattern in Conscious Rats. Zengfa Gu and Adolph J. Januszkiewicz. Department of Respiratory Research, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, Maryland 20910. E-mail: [email protected]. Gaseous hydrogen chloride (HCl) has been suspected to impair cardiopulmonary function due to its irritant and other toxicities. However, our understanding of the effects

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on breathing pattern is limited. This study addressed the changes of respiratory durations following a brief exposure (5 min) to 1000 and 16000 ppm HCl. Male SpragueDawley rats (250-380 g) were placed into the tube restrainers attached to a static exposure chamber. Respiratory measurements were made using partial body flow plethysmographs. Before exposure, animals breathed with UPS air for 5 min. The breathing data served as the control. Breathings were also measured immediately after the exposure and at the 24 h after the exposure for 5 min, respectively. All data were analyzed by a DATAQ/ ViewCodas system. In the 1000 ppm group, inspiratory durations were 0.14 ( 0.01, 0.18 ( 0.04, and 0.17 ( 0.01 s in the periods of the control, after exposure, and 24 h later, respectively; expiratory durations were found to be 0.13 ( 0.01, 0.24 ( 0.07, 0.15 ( 0.01 s, respectively. In the 16000 ppm group, inspiratory durations were 0.17 ( 0.02, 0.28 ( 0.05, and 0.27 ( 0.04 s for the periods of the control, after exposure, and at 24 h later, respectively, while expiratory durations were 0.16 ( 0.02, 0.20 ( 0.04, and 0.27 ( 0.06 s, respectively. These results suggested that a brief exposure to a high concentration of HCl could cause both acute and delayed respiratory injury, prolong inspiratory or expiratory durations, and result in a change of the breathing pattern. 73. Examination of 1-(2-Ethoxyacetaldehyde)benzimidazole. Charles N. Zink and James C. Fishbein. Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250. Fax: 410-455-2608. Email: [email protected]. N-Nitroso-3-hydroxymorpholine, a metabolite of the carcinogen N-nitrosomorpholine, has been proposed to decompose in aqueous media to form a 2-ethoxyacetaldehyde diazonium ion, as indicated by the scheme below. We are interested in the chemical stability of the potential adducts of this diazonium ion, and we wish to determine if such adducts can decompose to form a 2-hydroxyethyl adduct. This is of interest because there is one reported isolation of a 2-hydroxyethyl adduct at the N7 position of guanine in rats treated with Nnitrosomorpholine. Using benzimidazole as a model for the DNA bases adenine and guanine, we have synthesized 1-(2-ethoxyacetaldehyde)benzimidazole and are examining its stability in aqueous media. The stability and products of decomposition of this compound in aqueous media will be reported.

74. Facile Synthesis of Bifunctional Electrophiles to Study the Oxidative Damage to Proteins and DNA. Jasbir S. Arora, Tomoyuki Oe, Seon Hwa Lee, and Ian A. Blair. Center for Cancer Pharmacology, University of Pennsylvania, 841 Biomedical Research Building, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104-6160. Fax: 215-573-9889. E-mail: jasbir@ spirit.gcrc.upenn.edu. Oxidative stress causes damage to cellular macromolecules such as proteins and DNA by reactive oxygen species (ROS) or ROS-derived lipid hydroperoxide breakdown products. Lipid hydroperoxides are also formed

Dedon and Penning

enzymatically during oxidative stress. Both 15-lipoxygenase and cyclooxygenase-2 convert linoleic acid into 13(S)-hydroperoxyoctadecadienoic acid (13-HPODE), the prototypic w-6 polyunsaturated fatty acid hydroperoxide. We have shown that 4-hydroperoxy-2-nonenal, 4-oxo-2nonenal, 4-hydroxy-2-nonenal, and 4,5-epoxy-2-decenal are formed from 13-HPODE with a variety of initiators. Also, the reaction of these genotoxic bifunctional electrophiles with these species can generate several regioisomers that require extensive NMR studies to determine the correct structures. We report the preparation and utilization of unlabeled and labeled bifunctional electrophiles and their use for mechanistic studies of lipid hydroperoxide-derived modifications to proteins. This work was supported by NIH Grants CA91016 and CA95586. 75. Formation of 8-Oxo-dGuo by PAH o-Quinones and Its Mutagenic Effect on the Tumor Suppressor Gene p53: Involvement of Reactive Oxygen Species and Copper. Jong Heum Park,1 Yumin Shen,1 Jeffrey M. Field,2 and Trevor M. Penning.3 (1) Department of Pharmacology, University of Pennsylvania School of Medicine, 135 John Morgan building, 3620 Hamilton Walk, Philadelphia, Pennsylvania 19104-6084. Fax: 215573-2236. E-mail: [email protected]. (2) Department of Pharmacology, University of Pennsylvania. (3) Department of Pharmacology, University of Pennsylvania. PAHs (polycyclic aromatic hydrocarbons) require metabolic activation to genotoxins that can mutate the p53 gene, which is most mutated in lung cancer. Analysis of the p53 gene database shows that G to T transversions are characteristic of the disease. One route to the transversions is via oxidative DNA damage. Aldo-keto reductases activate PAH trans-dihydrodiols to the corresponding o-quinones, which may cause oxidative DNA damage. PAH o-quinones (BP-7,8-dione, BA-3,4-dione, and DMBA-3,4-dione) in the presence of NADPH and CuCl2 produced 8-oxo-dGuo in salmon testis DNA and mutated p53 cDNA. Submicromolar concentrations of PAH o-quinones increased 8-oxo-dGuo adduct/105 dGuo as measured by EC-HPLC, and this was blocked by catalase, tiron, or bathocuproine. Mutations in p53 cDNA were detected as red colonies in a yeast reporter gene system. Sequencing of the recovered plasmids showed a preference for G to T transversions with BP-7,8-dione, showing that 8-oxo-dGuo is the lesion that may account for the mutation. 76. HPLC/Tandem MS for the Analysis of 8-Oxo2′-deoxyguanosine. Daljit Vudathala and Ian Blair. Department of Pharmacology, University of Pennsylvania, 846 BRB II/III, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104. E-mail: [email protected]. Oxidation reactions that involve both oxygen (•OH radical, H2O2, 1O2, and O3) and nitrogen (NO•, -OONO) reactive species together with one-electron abstraction processes generate several different types of damage to DNA. Oxidative DNA damage results in the formation of 8-oxo-2′-deoxyguanosine (8-oxo-dGuo), which has been used extensively as a biomarker of DNA base damage. However, artifacts produced during sample analysis have been a major obstacle to the accurate analysis of 8-oxodGuo. This results from the huge excess of dGuo that is present during DNA hydrolysis and its subsequent oxidation by Fenton type chemistry. Also, several studies have shown that 8-oxo-dGuo is highly susceptible to


further oxidation. We have developed a sensitive and highly specific assay aimed at measuring 8-oxo-dGuo by combining a reversed phase liquid chromatographic separation with mass spectrometric detection. 77. Identification of Lipid Oxidation-Induced Adducts to Human Serum Albumin: Potential Markers of Oxidative Stress. James N. Riggins and Daniel C. Liebler. Department of Biochemistry, Vanderbilt University Medical Center, 9264 Medical Research Building III, 465 21st Avenue South, Nashville, Tennessee 37232. E-mail: [email protected]. The electrophilic lipid oxidation product 4-hydroxynonenal (HNE) modifies proteins on the nucleophilic residues H, K, and C. To identify potential serum biomarkers of oxidative stress, human serum albumin (HSA) was incubated with either HNE or liposomes containing the unsaturated fatty acids linoleic acid (LA) or arachidonic acid (AA). Liposome oxidation was initiated with 4,4′-azobis-2,4-dimethylvaleric acid. Modified protein was digested and analyzed by LC-MS-MS and MS-MS spectra corresponding to modified HSA sequences, which were identified with the P-Mod program. Both Western blot analysis and LC-MS-MS reveal HNE Michael adducts formed in a dose-dependent manner on H67, K310, K323, and K403. HSA modification with initiated liposomes involved many of the same targets, but several different adduct masses were formed during liposome oxidation. These findings suggest that lipid oxidation-derived adducts can be readily observed on HSA and that they may provide biomarkers of oxidative stress. This work was supported by NIH Grants ES10056 and ES000267. 78. Identification of Nuclear and Cytosolic Protein Targets of Thiol Reactive Electrophiles. Michelle K. Dennehy, Karolyn A. Richards, and Daniel C. Liebler. Department of Biochemistry, Vanderbilt University Medical Center, 9264 Medical Research Building III, 465 21st Avenue South, Nashville, Tennessee 37212. Fax: 615343-0704. E-mail: [email protected] and [email protected]. Alkylation of cellular proteins may provide a mechanism to sense stress stimuli and trigger adaptive changes in gene expression. Nuclear and cytoplasmic fractions from HEK293 cells were incubated with thiol reactive biotin-linked electrophiles and digested with trypsin. Detection of low abundance biotinylated peptides was enhanced by affinity enrichment using neutravidin beads. Adducted peptide mixtures were analyzed by multidimensional LC-MS-MS. Data mining with Sequest identified target protein sequences. Specific Cys residue targets were reproducibly detected in replicate analyses. Major susceptible targets in the nuclei included DNA maintenance proteins (PARP-1), RNA binding proteins (hnRNPs), and structural proteins (tubulin, actin). Cytoplasmic protein targets included structural proteins (tubulin), ribosomal proteins, glutathione transferases (GSTP1-1), components of the ubiquitin/proteasome system (UBCH-L and proteasome subunits), chaperones, and metabolic enzymes. Sequence specific adduct mapping is directed at identifying protein domain structures and motifs with high susceptibility to modification. This work was supported by NIH Grants ES10056, ES11811, and ES000267. 79. Mechanistic Interpretation of the Hydroxyl Radical Detoxifying Effect of Estrogen and Estrogen Derivatives. Laszlo Prokai,1 Katalin Prokai-Tatrai,2


Pal Perjesi,1 Alevtina D. Zharikova,1 Evelyn J. Perez,3 Ran Liu,3 and James W. Simpkins.3 (1) Department of Medicinal Chemistry, University of Florida, 1600 SW Archer Road, Gainesville, Florida 32610-0485. Fax: 352392-9455. E-mail: [email protected]. (2) Department of Pharmacology and Therapeutics, University of Florida. (3) Department of Pharmacology and Neuroscience, University of North Texas Health Science Center. Free radical scavenging contributes to the receptorindependent effects of estrogen and several estrogen derivatives, which is inherently associated with the presence of a phenolic A-ring in these molecules. We report experimental data (also supported by theoretical analysis) that the capture of hydroxyl radical (•OH) produces a nonphenolic steroidal quinol with no affinity to the estrogen receptors. We also provide evidence on a previously unrecognized antioxidant cycle that maintains a “chemical shield” raised by estrogen and phenolic A-ring estrogen derivatives in vitro and in vivo against this most harmful reactive oxygen species (ROS). This cycle is due to a rapid conversion of a steroidal quinol back to the parent molecule via an enzyme-catalyzed reduction utilizing NAD(P)H as a coenzyme (reductant) and, unlike redox cycling of catechol estrogens, without the production of ROS. Therefore, protection against •OH-induced oxidative stress was also manifested by steroidal quinols that essentially acted as prodrugs for the radical-scavenging molecules. 80. Mercapturic Acid Biosynthesis of 1-Chloro2,4-dinitrobenzene in Rat Kidney. Xiangming Guan, Chandradhar Dwivedi, So Young Kim, Todd Fine, Anthony Ratzsch, Seefeldt Teresa, Greg Peitz, and Zhiling Zhang. Department of Pharmaceutical Sciences, College of Pharmacy, South Dakota State University, Box 2202 C, Brookings, South Dakota 57007. Fax: 605-688-5993. E-mail: [email protected]. Mercapturic acid biosynthesis is an important biochemical process in detoxifying reactive electrophiles. The biosynthesis has been viewed as a result of interorgan processes of the liver and kidney with the formation of a glutathione conjugate in the liver as the initial step followed by cysteine conjugate formation in the kidney and the end product mercapturic acid being excreted into urine. Although all of the enzyme systems required for mercapturic acid biosynthesis have been found in rat kidney, no report has been documented to demonstrate the independent capability of rat kidney for mercapturic acid biosynthesis. By using rat kidney homogenate and 1-chloro-2,4-dinitrobenzene as a model compound, our data demonstrated that rat kidney was able to carry out the mercapturic acid biosynthesis independently without the involvement of liver. Our investigation suggests that this independent capability of the kidney needs to be taken into account in the study of mercapturic acid biosynthesis of xenobiotics. 81. N-Nitrosooxazolidinones Derived from the Nitrosation of Amino Acids and Aldehydes. Richard N. Loeppky, Sailaja Geddam, Sridharan Rajagopal, and Yinan Li. Department of Chemistry, University of Missouri, 125 Chemistry Building, Columbia, Missouri 65211. Fax: 573-882-2754. E-mail: [email protected] and [email protected]. We have previously shown, in proof of concept, that N-nitroso-2-phenyl-4-substitutied-1,3-oxazolidin-5-ones are produced from the nitrosation of benzaldehyde-derived imines of several amino acids. Compounds such as this

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could be produced from endogenous nitrosation in the stomach and could be transported to other organs or tissues where they could be enzymatically hydrolyzed to DNA reactive electrophiles. To explore this hypothesis further, we have synthesized a group of N-nitrosooxazolidinones 1 derived from the most prevalent dietary aldehydes, formaldehyde, and acetaldehyde and glycine, alanine, leucine, isoleucine, and phenylalanine. The sodium salt of the amino acid was reacted with the aldehyde and frozen. Sodium nitrite and HCl were added at 0 °C to the mixture, and the products were extracted into ethyl acetate, chromatographed, and characterized. These markers have been examined for their chemical stability and used to determine whether they are formed under conditions simulating those in the stomach.

82. Nitrolic Acid Chemistry within a Toxicological Perspective. Richard N. Loeppky and Nicholas P. Power. Department of Chemistry, University of Missouri, 125 Chemistry Building, Columbia, Missouri 65211. Fax: 573-882-2754. E-mail: [email protected] and [email protected]. We have previously reported that the nitrosation of either alanine or phenylalanine under simulated gastric conditions produces significant yields of the corresponding nitrolic acids 1. Ethylnitrolic acid has been reported to be mutagenic. We report here a further exploration of the production of nitrolic acids from amino acid nitrosation and an examination of their chemistry from a toxicological perspective. The yield of phenethylnitrolic acid from the nitrosation of phenylalanine at pH 3 increases from 0.5 to 11% as the nitrite equivalents are increased from 1 to 6. Initial rates of formation are linearly related to nitrite concentration. Nitrolic acid formation is most rapid at pH 2. The nitrosation of leucine gives 3-methylbutylnitrolic acid, but the nitrolic acids from valine and isoleucine are more difficult to isolate. Propylnitrolic acid (1, R ) Et) decomposes on standing in chloroform to propanoic acid and propanonitrile with a half-life of 12 h at 15 °C. It reacts with one equivalent of morpholine 2 to give N-nitrosomorpholine 3, morpholinium nitrite 4, N-(1-oximinopropyl)morpholine 5, and N-propanoylmorpholine 6. Larger morpholine equivalents reduce the nitrosamine 3 and amide 6 formation and increase the yields of the other products. Reactions of nitrolic acids with adenine and guanine derivatives will also be discussed.

83. Novel Metabolites of 2-Amino-r-carboline Formed in Rats in Vivo, Rat Hepatocytes, and HepG2 Cells. Roberta S. King and Zhixin Yuan. Department of Biomedical Sciences, University of Rhode Island,

Dedon and Penning

41 Lower College Road, Kingston, Rhode Island 02881. Fax: 401-874-2516. E-mail: [email protected]. Human exposure to the genotoxic carcinogen 2-aminoR-carboline (AaC, 2-amino-9H-pyrido[2,3-b]indole) through foods (grilled/pan-fried meats), drinking water, and smoke inhalation (cigarette/wood smoke, diesel exhaust) is low but chronic. We use model systems to develop methods for efficient detection, identification, and quantification of AaC metabolites. The models provide plausible biomarkers for human studies of dietary/environmental exposure to AaC. We now report AaC metabolites formed in rat hepatocytes and human HepG2 cells. Our analytical methods include HPLC separation, UV340 and 3H detection, and MS/MS and NMR structure identification. The major metabolites were O-sulfonates. Three AaC-Osulfonates and AaC itself were acetylated at a single nitrogen, noteworthy because AaC-N-acetylation was not detected in enzyme incubations and is uncommon for heterocyclic amine carcinogens. Rat hepatocytes formed AaC-O-glucuronides and three different AaC-N-glucuronides; however, no glucuronide metabolite was detected in HepG2 cells. Thus, only N-acetylation and O-sulfonation pathways were available for detoxification of AaC in the human cell line.

84. Nrf2-Dependent Transcription of ARE-Driven Genes Is Modulated through Covalent Modification on Keap1 Triggered by Electrophiles. Fei Hong,1 Michelle K. Dennehy,1 Sekhar R. Konjeti,2 Michael Freeman,2 and Daniel C. Liebler.1 (1) Department of Biochemistry, Vanderbilt University Medical Center, 9264 Medical Research Building III, 465 21st Avenue South, Nashville, Tennessee 37232. Fax: 615-343-0704. E-mail: [email protected]. (2) Department of Radiation Oncology, Vanderbilt University Medical Center. Release of the transcription factor Nfr2 from its negative regulator keap1 regulates the expression of phase II enzymes in cellular defense against carcinogenic electrophiles. Covalent modification or oxidation of keap1 is thought to release Nrf2. Treatment of HEK293 cells with the model electrophile iodoacetyl-LC-biotin (IAB) induces Nrf2-mediated upregulation of the Nrf2 target gene heme oxygenase 1. Western blot and confocal microscopy analyses indicate that IAB also induces translocation of Nrf2 from cytoplasm into the nucleus. Immunoprecipitation of FLAG-keap1 expressed in HEK293 cells demonstrated that IAB also triggers the posttranslational modification of keap1 to a high MW form observed by western blotting. Treatment with IAB forms 14 Cys adducts with His6-keap1 in vitro, as detected by LC-MS/MS. Identification of keap1 adducts and other posttranslational modifications in vivo will be critical to understanding the mechanism of keap1-Nrf2 dissociation. This work was supported by NIH Grants ES10056, ES000267, and CA038079. 85. Opposite Orientations of 4-HydroxyequileninDerived dC Adduct Stereoisomers in a B-DNA


Duplex. Shuang Ding,1 Robert Shapiro,1 Nicholas E. Geacintov,1 and Suse Broyde.2 (1) Department of Chemistry, New York University, 100 Washington Square East, Room 1001, New York, New York 10003. E-mail: [email protected]. (2) Department of Biology, New York University. The equine estrogens, equilin and equilenin, are major components of Premarin (Wyeth), the most frequently prescribed hormone replacement therapy formulation. The catechol 4-hydroxyequilenin (4-OHEN), a major phase I metabolite of both equilin and equilenin, autoxidizes to potent cytotoxic quinoids that can react with dG, dC, and dA to form unique cyclic adducts, which obstruct the Watson-Crick hydrogen-bonding edge of these nucleobases. Each base adduct has two pairs of stereoisomers, with each pair characterized by near-mirror image configurations. On the basis of previous quantum mechanical geometry optimization studies of the base adducts, we have constructed models of the 4-OHEN-C stereoisomers on the DNA duplex level and carried out molecular dynamics simulations. In all cases, the modified cytosine adopts the syn glycosidic bond conformation, placing the bulky and nonplanar equilenin moiety into the B-DNA major groove region, with a distortion of the DNA duplex. Importantly, the stereoisomeric pairs with near-mirror image structures align in opposite directions relative to the modified base and the 5′f3′ direction of the modified strand. Such opposite orientations may result in differences in the response of these stereoisomeric lesions to DNA replication and repair enzymes, in analogy to the effects observed in the case of stereoisomeric PAH-derived adducts. This work was supported by NIH. 86. Oxanosine: A Substrate of Adenosine Deaminase. Rainer Glaser,1 Papiya Majumdar,1 Hong Wu,1 and Peter A. Tipton.2 (1) Department of Chemistry, University of MissourisColumbia, Columbia, Missouri 65211. Fax: 573-882-2754. E-mail: [email protected] and [email protected]. (2) Department of Biochemistry, University of Missouri. Oxanosine, 5-amino-3-EÄ ×c0-(D-ribofuranosyl)-3H-imidazo[4,5-d][1,3]oxazine-7-one, was isolated as a novel nucleoside antibiotic in 1981 from Streptomyces capreolus MG265-CF3. Oxanosine became relevant in toxicology in 1996 with the discovery that it is formed in nitrosative guanosine deamination. We reacted oxanosine with adenosine deaminase enzyme (ADA) under physiological conditions in an attempt to synthesize [7-18O]oxanosine in analogy to the synthesis of [6-18O]guanosine from 2-amino-6-chloropurine. Unexpectedly, we discovered that the incubation of oxanosine with ADA in sodium phosphate buffer (pH 7.4) results in the formal replacement of the C5 amino group by a hydroxyl group. The reaction product was separated by preparative RPHPLC and characterized by LC/MS analysis and 13C NMR spectroscopy. Reaction in 18OH2 leads to 18Oincorporation at C7. This reaction is consistent with the known mode of action of ADA for other substrates. Our discovery has important implications for studies of nitrosative deamination of oligonucleotides. Alkaline phosphatase enzyme generally is used to remove the phosphate groups from the nucleotides, and this enzyme frequently contains small admixtures of adenosine deaminase. Hence, small amounts of oxanosine formed by the nitrosation of the oligonucleotide might escape detection.


87. Preparation and Characterization of Heparan Sulfate from Mouse Liver with Cancer. Wenjun Mao. Marine Drugs and Foods Institute, Ocean University of China, 5 Yushan Road, Qingdao 266003, China. E-mail: [email protected]. Yi Li. Navy 401 Hospital, 6 Minjiang Road, Qingdao 266071, China. E-mail: liyiqd@ hotmail.com. Heparan sulfate is a widespread complex linear polysaccharide that consists of alternate hexuronic acid and N-substituted glucosamine residues. In vivo, heparan sulfate is usually found covalently attached to various core proteins. Heparan sulfate regulates several aspects of cancer biology, including tumorigenesis, tumor progression, and metastasis. Gaining better insight into the structure-function relationships of heparan sulfate is key to understanding how these molecules regulate different aspects of tumor biology and to developing targeted therapeutics. In this paper, the structural characterization of heparan sulfate from mouse liver with cancer was investigated. The liver tissue was homogenized, defatted with chloroform/methanol, and digested with Pronase. The supernatant was applied to Dowex macroporous SAX resin column to separate glycosaminoglycans. Following, digestion with chondroitinase ABS and endonucleases heparan sulfate was purified. The purified heparan sulfate was investigated by PAGE, CE, NMR, and MS. This work was supported by a grant from natural science foundation of Shandong province, China (Y2003C01). 88. Proteomic-Based Strategy to Identify Pharmacological Targets in Genetic MLL/AF4 Translocated Leukemias. Anastasia K. Yocum,1 Christine M. Busch,1 Carolyn A. Felix,2 and Ian. A Blair.1 (1) Center for Cancer Pharmacology, University of Pennsylvania, 1246 Biomedical Research Building II/III, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104-6160. Email: [email protected]. (2) Division of Oncology, Abramson Research Center, The Children’s Hospital of Philadelphia. DNA translocations involving the Mixed Lineage Leukemia gene (MLL) often result in aggressive forms of pediatric acute primary leukemias, myelogenous, and lymphoblastic, as well as treatment-related leukemias. One of the more common partners with MLL translocations is the AF4 gene located at chromosome band 4q21. Interindividual variability occurs within the break and recombination point of both the MLL and the AF4 genes in patients with the MLL/AF4 translocation. It is hypothesized that even though there are many genetic variations with MLL/AF4 translocations, after alternative splicing and other posttranscriptional and translational processing events, it results in the same MLL/AF4 fusion protein. If this is the case, then the fusion protein can serve as a common target for pharmacological intervention despite subclassification or etiology of the leukemia. Furthermore, this same protein is hypothesized to affect the same down stream targets that can also be identified and characterized for possible pharmacological intervention. This research is for a proteomics-based strategy to identify pharmacological targets for MLL/AF4 translocation leukemias. 89. Purine Metabolism as a Source of Endogenous DNA Damage. Min Dong,1 Nicholas E. Burgis,2 Peter C. Dedon,1 and Richard P. Cunningham.2 (1) Biological Engineering Division, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge,

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Massachusetts 02139. Fax: 617-324-7554. E-mail: mdong @mit.edu. (2) Department of Biological Sciences, State University of New York, 1400 Washington Avenue, Albany, New York 12222. Fax: 518-442-4767. Endogenous processes and chemicals are increasingly recognized as important causes of DNA damage that leads to human diseases such as cancer. The most wellstudied examples include reactions of DNA bases with reactive oxygen and nitrogen species or with electrophiles generated from lipids, proteins, and nucleic acids. We now present data consistent with a new paradigm in which perturbations of nucleobase metabolism may lead to incorporation of the purine precursors hypoxanthine and xanthine into DNA. The studies employed Escherichia coli strains containing single or combinatorial deletions of the genes encoding deoxyribonucleotide triphosphate pyrophosphohydrolase (rdgB), adenylosuccinate synthetase (purA), and endonuclease V (nfi). Purified genomic DNA from each strain was subjected to LC/MS analysis to quantify four deamination lesions: deoxyxanthosine (dX), deoxyoxanosine (dO), deoxyinosine (dI), and deoxyuridine (dU). The first observation was that dO did not occur at detectable levels (

Abstracts, American Chemical Society Division of Chemical

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