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Abstracts, American Chemical Society Division of Chemical Toxicology, 224th ACS National Meeting, Boston, Massachusetts, August 18-22, 2002 Nicholas E. Geacintov and Peter C. Dedon, Program Chairs Department of Chemistry, New York University, 31 Washington Place, New York, New York 10003, and Biological Engineering Division, Massachusetts Institute of Technology, 56-787, Cambridge, Massachusetts 02139

Symposium 1: Genomic Approaches to Toxicology Leona Samson, Organizer 1. Genomic approaches to understanding how cells respond to damage. Leona Samson,1 Mark Ambrose,2 Kiyoshi Kobayash,3 Lisiane Meira,2 Annop Rao,2 and Ivan Rusyn.2 1Biological Engineering Division and Center for Environmental Health Sciences, MIT, 77 Massachusetts Avenue, 56-235, Cambridge, MA 02139, 617-253-8099, [email protected]. 2Biological Engineering Division, MIT, 3Mitsubishi Pharmaceuticals. We have explored the transcriptional responses elicited upon exposure of Escherichia coli, Saccharomyces cerevisiae, murine, and human cells to alkylating agents, and selected other agents. Computational analysis of these transcriptional responses has so far revealed a number of unexpected pathways that appear to play important roles in helping cells recover from such exposure. These studies will contribute to an integrated view of the way that prokaryotic and eukaryotic cells do or do not avoid the killing and mutagenic and carcinogenic effects of environmental toxicants. 2. Pathway Processor: a method for integrating whole-genome expression results into biological networks. Duccio Cavalieri,1 Paul Grosu,1 Carlotta De Filippo,2 Jeffrey P. Townsend,1 and Daniel L. Hartl.3 1Bauer Center for Genomics Research, Harvard University, 7 Divinity Avenue, Cambridge, MA, (fax) 617-4952196, [email protected], 2Department of Pharmacology, University of Florence, 3Department of Organismic and Evolutionary Biology, Harvard University We have developed a new tool to visualize expression data on metabolic pathways and to evaluate which metabolic pathways are most affected by transcriptional changes in whole-genome expression experiments. Using the Fisher Exact Test, the method scores biochemical pathways according to the probability that as many or more genes in a pathway would be significantly altered in a given experiment by chance alone. Results from multiple experiments can be compared, reducing the analysis from the full set of individual genes to a limited number of pathways of interest. The relationship between genes in the pathways can be examined in detail displaying differences in expression on charts of the cellular pathways to which the ORFs are assigned. This method has been used to revisit the diauxic shift experiments, the segregation of expression profiles in a tetrad of a wine S. cerevisiae strain, and the effect of neocarzinostatin and MMS on yeast cells.

3. Novel resistance pathways identified using high throughput genomic phenotyping of Saccharomyces cerevisiae. Thomas J. Begley,1 Ari S. Rosenbach,1 Trey Ideker,2 and Leona D. Samson.1 1Biological Engineering Division, MIT, 77 Massachusetts Avenue, 56-230, Cambridge, (fax) 617-258-5424, [email protected]. 2Whitehead Institute for Biomedical Research Established cellular mechanisms have long been known to prevent or repair damage produced after exposure to toxic agents. Using high-throughput screens we have identified a plethora of additional eukaryotic pathways that contribute to recovery after exposure to the alkylating agent methyl methanesulfonate, the oxidizing agent tert-butyl hydroperoxide, the bulky damaging agent 4-nitroquinoline-N-oxide, and ultraviolet radiation. Triplicate screening of an ordered library of yeast gene deletion strains against these agents, together with computational techniques, have allowed us to generate an Internet accessible database for analysis of the over 90 000 data points compiled in this study. This “genomic phenotyping” approach was used to identify over 500 genes that affect recovery after agent exposure and to assign phenotypes to large number of genes of hitherto unknown function. Systematic integration of our data with the yeast protein interactome has allowed us to pinpoint multi-protein nodes important for recovery and identify novel systems that mediate agent resistance. 4. So many poisons, so little time. Kevin R. Hayes,1 Russell S. Thomas,1 David R. Rank,2 Sharron G. Penn,2 Gina M. Zastrow,1 Kalyan Pande,2 Edward Glover,2 Tomi Silander,3 Mark W. Craven,2 Stevan B. Jovanovich,2 and Christopher A. Bradfield.1 1McArdle Laboratory for Cancer Research, University of Wisconsin, 1400 University Avenue, Madison, WI 53706, (fax) 608-262-2824, [email protected], 2Amersham Biosciences, (3) University of Helsinki One of the major objectives of toxicology is to understand the adverse health effects of chemicals in humans. This understanding could be aided by the ability to group chemicals that generally act the same or are believed to produce their toxic endpoints through similar mechanisms. In an attempt to identify these toxicologically relevant gene expression patterns, we initiated a survey of chemically induced changes in liver gene expression through the use of custom cDNA microarrays. Five broad chemical classes were selected for study. Microarray analysis was performed on various chemicals within these groups and the results analyzed using a Bayesian analysis. Using a forward parameter selection scheme, a ‘minimal′ set of 5 genes was identified that

10.1021/tx020089h CCC: $22.00 © 2002 American Chemical Society Published on Web 11/09/2002

Abstracts, ACS Division of Chemical Toxicology

allowed 100% accuracy in classifying the treatments and an ‘optimal′ set of 12 genes which still provided 100% accuracy, but also gave more robust predictions. These results provide significant evidence that the classification of chemicals according to their gene expression profiles is possible and opens the door to a potentially new era of toxicological testing. 5. Identification of critical determinants of agonism and antagonism in the breast by transcriptional profiling. Cheryl Lyn Walker,1 Leslie Hodges,1 Jennifer Cook,1 Ed Lobenhofer,2 Leping Li,2 Lee Bennett,2 Pierre Bushel,2 and Cindy Afshari.2 1Department of Carcinogenesis, Science Park Research Division, UT MD Anderson Cancer Center, Park Road 1C, Smithville, TX, (fax) 512-237 2475, [email protected], 2 National Institute of Environmental Health Sciences Tamoxifen is an antagonist in the breast and a widely used breast cancer therapeutic and preventative agent. Transcriptional profiling was used to identify the molecular determinants of agonist and antagonist activity of estrogen and tamoxifen in breast cancer cells. Surprisingly, tamoxifen induced a transcriptional profile similar to estrogen in breast cancer cells, including induction of many cell cycle-associated genes. Tamoxifen recapitulation of estrogen-induced gene expression patterns demonstrates that even in cells where tamoxifen functions as an antagonist, at the molecular level it acts as an agonist, an attribute that may contribute to the development of tamoxifen-resistant breast cancer. The ability of tamoxifen to function like estrogen at the molecular level may underlie agonism of this compound observed in vivo in other estrogen-responsive tissues such as the endometrium. Importantly, although the vast majority of estrogen-responsive genes were also induced by tamoxifen, we identified several key genes, including cyclinD1, that were induced solely by estrogen and appear to function as “cellular gatekeepers” to determine agonist versus antagonist activity in breast cancer cells. 6. Combined functional genomic maps of the C. elegans DNA damage response. Marc Vidal,1 Simon J. Boulton,1 Anton Bartner,2 Jerome Reboul,1 Philippe Vaglio,1 Nick Dyson,3 and David E. Hill.1 1Department of Genetics, Dana-Darber Cancer Institute, 44 Binney Street, Boston, MA 02115, (fax) 617-632-5739, [email protected], 2Max Planck Institute for Biochemistry, 3Massachusetts General Hospital Cancer Center Many human cancers originate from defects in the DNA damage response (DDR). Although much is known about this process, it is likely that additional DDR genes remain to be discovered. To identify such genes, we used a strategy that combines protein-protein interaction mapping and large-scale phenotypic analysis in Caenorhabditis elegans. Together, these approaches identified 12 worm DDR orthologs and 11 novel DDR genes. One of these is the putative ortholog of hBCL3, a gene frequently altered in chronic lymphocytic leukemia. Thus, the combination of functional genomic mapping approaches in model organisms may facilitate the identification and characterization of genes involved in cancer and, perhaps, other human diseases.

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Symposium 2: Bioinformatics, Genomics, and Proteomics R. Sasisekharan, Organizer 7. Structure and sequence of glycosaminoglycans. Ganesh Venkataraman, Director, Bioinformatics, Consortium for Functional Glycomics, Massachusetts Institute of Technology, 16-561, 77 Massachusetts Avenue, Cambridge, MA 02139 Heparin/heparan sulfate-like glycosaminoglycans are acidic complex polysaccharides found on the cell surface and the extracellular matrix. Recent progress in biology has resulted in a virtual explosion of important roles of these biopolymers in fundamental biological processes. Advances in the understanding of biosynthesis and structure, development of novel analytical methods for composition and sequence analysis of these biopolymers have provided remarkable insights into structure-function relationships of these complex and once elusive polysaccharides. This talk will focus on important advances in our understanding of structure and sequences of glycosaminoglycans. 8. Chemical glycomics. Peter Seeberger, Department of Chemistry, Massachusetts Institute of Technology, Building T-18-211, 77 Massachusetts Avenue, Cambridge, MA 02139, [email protected] The importance of cell surface oligosaccharides and glycosaminoglycans in signal transduction processes of biomedical significance is now well established. A major impediment to the rapidly growing field of molecular glycobiology is the lack of pure, structurally defined carbohydrates and glycoconjugates. Detailed biophysical and biochemical studies of complex carbohydrates require sufficient quantities of defined oligosaccharides. Described is the application of an automated solid-phase oligosaccharide synthesizer we developed recently to the preparation of even complex carbohydrates from monosaccharide building blocks. Application of this new synthetic strategy, that should be attractive to the non-specialist, to the assembly of carbohydrate chips containing oligosaccharides involved in cancer, infectious diseases, tropical diseases and HIV will be discussed. Particular emphasis will be placed on the development of an antitoxin malaria vaccine using the automated synthesizer to prepare complex oligosaccharides. 9. Heparan sulfate: Biosynthetic control of biologic diversity. Nicholas W. Shworak, Angiogenesis Research Center, Department of Medicine, Dartmouth Medical School, One Medical Center Drive, Lebanon, NH 03756, (fax) 603-653-0510, [email protected] Heparan sulfate (HS) is an extremely complex linear biopolymer. Functional properties are defined by short sequence motifs that selectively bind and activate a diverse range of biologic effector proteins. Specificity of HS protein interactions is largely defined by the order and ring position of sulfate substituents. HS functional properties are thus defined by the biosynthetic machinery. A single enzyme 2-O-sulfates uronyl residues; where as, three distinct families of sulfotransferases respectively modify glucosamine residues at amino-, 6-O-, or 3-O-positions. A multiplicity of biosynthetic isozymes

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both expands the structural diversity of HS and allows for controlled production of distinct structures in distinct cell and tissue-types. Gene knockouts of such enzymes are providing unanticipated insights into the biologic roles of HS structures. These principles shall be explored by examining the roles of HS biosynthesis in hypoxia stimulated FGF signaling, herpes virus infection and novel activities of antithrombin. 10. Do quantitative and qualitative differences in organ tissue distribution of heparan sulfate in mice solve some mysteries of protein expression? Robert Linhardt,1 Mohamad Warda,1 Wenjun Mao,1 and Toshihiko Toida.2 1Division of Medicinal and Natural Products Chemistry, University of Iowa, College of Pharmacy, 115 S. Grand Ave, Iowa City, IA 52242, (fax) 319-335-6634, [email protected], 2Faculty of Pharmaceutical Science, Chiba University Measuring gene expression at the protein level (Proteomics) is more informative than the corresponding measurement at the gene level (Genomics). Proteomics, however, cannot solve questions that arise in posttranslational modifications. Glycomics, deals with the structures of glycosylated proteins, and may explain many mysteries that are unsolved because of the posttranslation modification process. The current studies should provide a better understanding of the role of expressed glycoproteins and proteoglycans in cell-cell interactions, as well as providing a fundamental understanding of certain aspects of protein-carbohydrate interactions. Our study aims at determining quantitative and qualitative tissue distributions of heparan sulfate and heparin (HS/HP) in the ICR mouse strain. The entire mouse genome has already been sequenced. Data obtained from the current study on HS/HP concentrations in different organs should provide information on the many roles of glycosaminoglycans (GAGs) in different cell-cell interaction processes, cell signaling and subcellular protein trafficking. 11. Glycosaminoglycans and arthritis. Alan Grodzinsky,1 Moonsoo Jin,2 Parth Patwari,3 and Eliot Frank.3 1 Departments of Electrical, Mechanical, and Biological Engineering, Massachusetts Institute of Technology, 50 Vassar Street, Room 38-377, Cambridge, MA 02139, [email protected], 2Mechanical Engineering, MIT, 3Electrical Engineering, MIT Negatively charged chondroitin sulfate glycosaminoglycans (CS-GAGs) contribute more than 50% of the compressive and shear stiffness of cartilage. The loss of CS-GAGs from cartilage is one of the earliest events in the progression of the joint disease, osteoarthritis (OA). Measurements have quantified the contribution of CSGAG electrostatic interactions to cartilage’s compressive and shear properties. Recent studies also suggest that there are multiple regulatory pathways by which chondrocytes in articular cartilage sense and respond to mechanical stimuli, including upstream signaling pathways and mechanisms that may lead to direct changes at the level of transcription, translation, post-translational modifications, and cell-mediated extracellular assembly and degradation of the tissue matrix, specifically the glycosaminoglycans. This talk focuses on the effects


of mechanical loading on cartilage and the resulting chondrocyte-mediated biosynthesis remodeling, degradation, and repair of the GAG-rich extracellular matrix of this tissue. 12. Glycosaminoglycans and cancer: Tumor cell surface heparan sulfate as cryptic promoters or inhibitors of tumor growth and metastasis. Zachary Shriver, Division of Biological Engineering, Massachusetts Institute of Technology, Building 16-560, 77 Massachusetts Avenue, Cambridge, MA 02139, (fax) 617-2589409, [email protected]. Heparan sulfate glycosaminoglycans, present at the cell surface and in the extracellular matrix that surrounds cells, are important mediators of complex biological processes. Furthermore, it is now apparent that cells dynamically regulate the structure of their heparan sulfate “coat” to differentially regulate extracellular signals. In the present study, the importance of sequence information contained within tumor cell surface heparan sulfate was investigated. Herein, we demonstrate that the complex polysaccharide heparan sulfate glycosaminoglycan coat present on tumor cells contains bioactive sequences that impinge on tumor cell growth and metastasis. Importantly, we find that growth promoting, as well as growth inhibiting, sequences are contained within the polysaccharide coat. Furthermore, we find that the dynamic balance between these distinct polysaccharide populations regulates specific intracellular signal transduction pathways. The present study not only provides a framework for the development of polysaccharide-based novel anti-cancer molecules, but also underscores the importance of understanding a cell’s polysaccharide array, in addition to its protein complement, to understand how genotype translates to phenotype in this postgenomic age. 13. Functions of heparan sulfate proteoglycans in airway disease. Pyong Woo Park, Baylor College of Medicine, One Baylor Plaza, BCM 286-N1319, Houston, TX 77030, (fax) 713-798-8948, [email protected] Heparan sulfate proteoglycans (HSPGs) have been implicated in the pathogenesis of various diseases, such as infections, non-infectious inflammatory disorders and cancer. However, the molecular mechanisms as to how HSPGs influence the outcome of these diseases are not understood. We have found that shedding of syndecan1, the predominant HSPG of epithelia, is activated during murine experimental lung infections, lung fibrosis and asthma. Our results also show that shed syndecan-1 ectodomains inhibit host defense and inflammatory mediators (e.g., antimicrobials, cytokines and collectins) via their heparan sulfate (HS) chains. Furthermore, the syndecan-1 null mice resist lung infections but show an exacerbated asthma phenotype, implicating a central role for syndecan-1 HS chains in these diseases. Studies with chemically or enzymatically-modified heparinoids indicate that these activities of syndecan-1 ectodomains are mediated by specific HS domains. These findings suggest that specific HS structures on HSPGs are potential targets for novel approaches to anti-bacterial and antiinflammatory therapies.


Symposium 3: The Emerging Role of Aldo-Keto Reductases in the Metabolism of Toxic Substances T. M. Penning and J. M. Petrash, Organizers 14. Introduction and overview of the Aldo-Keto reductase superfamily. Trevor M. Penning, Department of Pharmacology, University of Pennsylvania, Room 130C John Morgan Building, 3620 Hamilton Walk, Philadelphia, PA 19104-6084, (fax) 215-573-2236, [email protected]. Aldo-Keto Reductases (AKRs) are a rapidly growing gene superfamily involved in the formation of hyperosmotic sugars, which can contribute to diabetic complications. They also metabolize chemical carcinogens, reactive aldehydes, prostaglandins, steroid hormones and drugs. These enzymes (>150) are monomeric, cytosolic, NAD(P)H-dependent- oxidoreductases, that share high sequence identity (>46%) and similar 3D-structures e.g. (R/ β)8-barrels. For a complete listing of the 12 families visit: www.med.upenn.edu/akr. AKRs play a role in tobacco-carcinogenesis since they oxidize polycyclic aromatic trans-dihydrodiols to reactive and redox active ortho-quinones and catalyze the detoxication of nicotine derived nitrosamino-ketones (NNK). Discrete isozymes also detoxify aflatoxin dialdehyde and protect against hepatocellular carcinoma. The aflatoxin reductases are induced by cancer chemopreventive agents e.g., ethoxyquin. AKRs also detoxify mutagenic bifunctional electrophiles derived from the decomposition of lipid hydroperoxides. Evidence is thus mounting that AKRs are stress regulated genes and play a central role in the cellular response to drug and toxicant exposure. 15. Aldo-keto reductase-catalyzed detoxication of endogenous aldehydes associated with diabetic complications. David L Vander Jagt and Lucy A. Hunsaker. Department of Biochemistry and Molecular Biology, University of New Mexico School of Medicine, Albuquerque, NM 87131, (fax) 505-272-3518, [email protected]. Numerous reactive aldehydes elevated in diabetic patients are key intermediates in the formation of Advanced Glycation Endproducts and likely contribute to development of long-term diabetic complications. These aldehydes include formaldehyde, glyoxal, methylglyoxal, glucosone, 3-deoxyglucosone, xylosone, 3-deoxyxylosone, and 4-hydroxynonenal. All of these aldehydes are substrates of aldose and aldehyde reductases, two members of the aldo-keto reductase superfamily. The broad specificity of aldose and aldehyde reductases for these endogenous aldehydes suggests that detoxication of reactive aldehydes is one of their main functions. The structural features that contribute to substrate recognition include the presence of an oxidized carbon at the 2-position. Remaining structural features can vary widely. Thus, aldose and aldehyde reductases mainly appear to recognize a reactive aldehyde functional group. This imparts to these reductases an exceptionally broad protective function against the toxicity of reactive aldehydes. 16. Aldose reductase detoxifies lipid aldehydes and their conjugates with glutathione. Satish K Srivastava, Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, 6.644, Basic Science Building, Galveston, TX 77555, (fax) 409772-9679, [email protected].


Reduction of glucose by aldose reductase (AR) has been implicated in the development of diabetic complications. However, the structural and kinetic studies of the purified AR demonstrate that this enzyme does not favor hydrophilic glucose as an efficient substrate. We have shown that AR reduces short to medium chain aldehydes and R, β-unsaturated alkenals such as 4-hydroxy-trans2-nonenal (HNE), which is one of the most abundant and cytotoxic products of lipid peroxidation. Further, our studies indicate that AR reduces several glutathiolated aldehydes with 4-1000 fold higher efficiency than their parent free aldehydes. Our kinetic, site-directed mutagenesis, and computer modeling studies indicate that the active site of AR is compatible with glutathione and that residues other than Cys-298 interact with and recognize glutathione at the AR active site. Our results suggest that under euglycemia, AR may be involved in the metabolism and the detoxification of aldehydes and glutathionyl conjugates of xenobiotic aldehydes. 17. Aldose reductase and the stress response. Aruni Bhatnagar, Department of Medicine, University of Louisville, Room 107, Jewish Cardiovascular Research Center, 500 South Floyd, Louisville, KY 40202, (fax) 502852-1795, [email protected] Recent evidence demonstrates that aldose reductase (AR) is an efficient catalyst for the reduction of a variety of electrophilic aldehydes and their glutathione conjugates, suggesting that it may be involved in antioxidant defense. However, the participation of AR in protection against oxidative injury in vivo has not been examined. We find that in rabbit hearts brief episodes of ischemia (ischemic preconditioning) increase the expression of AR and its association with the particulate fraction. AR was also upregulated in the hearts of rats chronically fed 6% ethanol and increased association of AR with the particulate fraction was also observed in isolated perfused rat hearts subjected to global ischemia. Inhibition of AR did not affect ischemic injury, but abolished the cardioprotective effects of ischemic preconditioning and increased the ischemia-induced accumulation of the lipid peroxidation product 4-hydroxy-trans-2-nonenal. Together, these observations suggest that AR is a critical component of defense against ischemic or electrophilic injury. 18. Competing roles of reductases in the detoxication of tobacco-specific nitrosamine ketones. Edmund Maser, Institute for Pharmacology and Toxicology, Philipps-University of Marburg, Karl-von-Frisch-Strasse 1, Marburg 35033, Germany, (fax) +49-6421-28-65600, [email protected], and Ursula Breyer-Pfaff, Institute for Toxicology, University of Tuebingen The balance between metabolic activation and detoxication is critical in determining the susceptibility to lung cancer upon exposure to the tobacco-specific nitrosamine 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Activation occurs by CYP-mediated oxidation, whereas detoxication in man is initiated by carbonyl reduction of NNK to its corresponding alcohol NNAL. While the relationship between lung cancer risk, tobacco smoke and CYP activities is controversial, we focussed on the identification, expression and activity of NNK carbonyl reducing enzymes. Five different enzymes mediating NNK carbonyl reduction in man have been

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identified: microsomal 11b-hydroxysteroid dehydrogenase type 1, cytosolic carbonyl reductase (both belonging to the short-chain dehydrogenases/reductases), and three members of the aldo-keto reductases (AKR), namely AKR1C1, AKR1C2 and AKR1C4, previously designated as dihydrodiol dehydrogenases DD1, DD2 and DD4, respectively. All enzymes have been purified to homogeneity from human liver. We propose that the extent of expression and activity of these enzymes strongly influences the tissue selectivity and interindividual susceptibility to NNK-mediated cancer. This presentation focusses on the tissue distribution, activities, stereoselectivity and interindividual differences of these SDR and AKR enzymes regarding NNK metabolism. Their potential role as a determinant in the protection against tobacco-smoke related lung cancer will be discussed. 19. Aldo-keto reductases and the metabolic activation of polycyclic aromatic hydrocarbons. Trevor M. Penning,1 Nisha T. Palackal,2 Seon-Hwa Lee,1 Ian A. Blair,1 Deshan Yu,1 Jesse A. Berlin,3 Jeffrey M. Field,1 and Ronald G. Harvey4. 1Department of Pharmacology, University of Pennsylvania, Room 130C John Morgan Building, 3620 Hamilton Walk, Philadelphia, PA 191046084, (fax) 215-573-2236, [email protected], 2 Department of Biochemistry and Biophysics, University of Pennsylvania, 3Center for Clinical Epidemiology and Biostatistics, University of Pennsylvania, 4The Ben-May Institute for Cancer Research, University of Chicago Polycyclic aromatic hydrocarbons (PAH) are suspect human lung carcinogens that are metabolically activated. Five human aldo-keto reductases [AKR1A1 (aldehyde reductase) and AKR1C1-AKR1C4] are implicated in this process since they oxidize PAH-trans-dihydrodiol proximate carcinogens to yield reactive and redox-active PAHortho-quinones. These ortho-quinones enter into futile redox-cycles to amplify reactive oxygen species (ROS) and cause DNA-lesions. AKR1C1 is highly overexpressed in non-small cell lung carcinoma patients (Cancer Res. (2001) 61: 2727) and in the human lung adenocarcinoma cell line A-549. AKR1C activity in these cells is sufficient to convert dimethylbenz[a]anthracene (DMBA)-3,4-diol to DMBA-3,4-dione. We show that AKR derived PAH ortho-quinones produce sufficient ROS to mutate the major lung tumor suppressor gene (p53) in vitro. These mutations eliminate the transcriptional competency of p53 in a yeast-reporter gene system and this change-infunction results from G to T transversions (p dATP (0.01). dTTP (0.0001). The relative extension past the 3DG/N base pair by the incorporation of dCTP opposite guanine was C (finc)1) . T (0.001), G (0.0007), A (0.0005). In conclusion, the hydrogen bond between Arg668 and the minor groove of the primer terminus was involved in the fidelity of the insertion step but was not in the decrease in rate past a mispair. (Supported by NIH grant CA 75074). 37. Nuclear Magnetic Resonance studies of the structure of the (2R,3R)-2′-deoxy-N6-(2,3,4-trihydroxybutyl)-major groove adduct onto the n-ras 61 codon reveals localized structural perturbation. W. Keither Merritt, Lubomir N. Nechev, Constance M. Harris, Thomas M. Harris, and Michael P. Stone, Department of Chemistry, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center, Vanderbilt University, Nashville, TN 37235 1,3-butadiene, a suspected human carcinogen, is oxidized by cytochrome P450 2E1 to epoxides that can adduct DNA. The present work utilizes 1H NMR to study the structural perturbation in DNA caused by the (2R,3R)-2′-deoxy-N6-(2,3,4-trihydroxybutyl)adenosine adduct at position 6 of the n-ras 61 sequence d(CGGACAAGAAG).d(CTTCTTGTCCG). NMR data indicates that the perturbation is localized to the general site of adduction and that there is not global perturbation of the oligonucleotide, that the adduct is oriented in the major groove, and that Watson-Crick base pairing is not distorted. Through the use of restrained molecular dynamics calculations, a refined structure of the n-ras


61 sequence with this particular butadiene adduct has been determined. These results appear to corroborate site-specific mutagenesis studies showing that this lesion is weakly mutagenic, but does give low levels of A f G mutations. This data is of particular interest, as the structural model indicates clear differences between this and the structure of the (2S,3S)-2′-deoxy-N6-(2,3,4-trihydroxybutyl)adenosine adduct which gives low level A f C mutations and has been studied by this lab. Supported by N. I. H. grant ES-05509. 38. Photochemical generation of site-specific nitroimidazole and nitroguanine adducts via addition of nitrogen dioxide to neutral guanine radicals in DNA. Vladimir Y. Shafirovich, Steven Mock, Alexander Kolbanovskiy, and Nicholas E. Geacintov, Chemistry Department, New York University, 31 Washington Place, New York, NY 10003, (fax) 212 998 8421, [email protected] The long-lived neutral guanine radical derived from the rapid deprotonation of the guanine radical cation is believed to be an important intermediate in the formation of diverse lesions in DNA. The photochemical synthesis of nitroguanine lesions is triggered by the selective photodissociation of persulfate anions to sulfate radicals induced by either UV laser pulses, or by continuous UV irradiation. The carbonate and nitrogen dioxide radicals arising from the quantitative scavenging of sulfate radicals by bicarbonate and nitrite anions are employed for the generation of guanine lesions. The neutral guanine radicals are produced by site-selective oxidation of guanine residues in oligonucleotides by the carbonate radicals. The nitrogen dioxide radicals, with do not interact with intact DNA bases, combine rapidly with the guanine radicals either at the C8 or the C5 positions. The C8 addition generates the well known 8-nitroguanine lesion, whereas the C5 attack produces unstable adducts which rapidly decomposes to a stable 4-nitroimidazole lesion. The 4-nitroimidazole and 8-nitroguanine adducts derived from the site-selective photochemical nitration of single-stranded oligonucleotides containing a single guanine were isolated by reverse phase HPLC and characterized by MALDI-TOF and ESI mass spectroscopic techniques. In neutral solutions, the 8-nitroguanine adducts slowly depurinate with release of free 8-nitroguanine and the formation of an abasic abasic site (∼20 h half-live at 20 °C). In contrast, 4-nitro-imidazole lesions are much more stable and slowly decompose even at 90 °C (10-15%, after 6 h). The biological implications of these oxidative DNA modifications are discussed. 39. Properties of the leinamycin-DNA adduct. Tony Nooner and Kent Gates, Department of Chemistry, University of Missouri, 125 Chemistry, Columbia, MO 65211, (fax) 573-882-2754, [email protected] The natural product leinamycin has been shown to possess potent antitumor activity. The DNA damaging properties of leinamycin are unlocked by the attack of thiols on its 1,2-dithiolan-3-one 1-oxide heterocycle, leading to an activated episulfonium ion form of the antibiotic. This activated form of leinamycin alkylates DNA specifically in the major groove at the N7-position of guanine. Here we report on the stability and repair of the leinamycin-DNA adduct under various conditions,


which has led to further insight into the Leinamycin adducted DNA’s degradation mechanism.

40. The major malondialdehyde-guanine adduct pyrimido[1,2-r]purin-10(3H)-one (M1G) is formed from N2-(3-oxo-1-propenyl)-guanine (N2OPG) by a general acid-catalyzed mechanism. James N. Riggins,1 J. Scott Daniels,2 Derek A Pratt,1 and Lawrence J Marnett.3 1Department of Chemsitry, Vanderbilt University, Nashville, TN 37232, [email protected], 2Department of Biochemistry, Vanderbilt University, 3Departments of Chemistry and Biochemistry, Vanderbilt University Medical Center M1GdR is the major reaction product of deoxyguanosine with malondialdehyde (MDA), an endogenous product of lipid peroxidation, and with base propenals, direct products of DNA oxidation. M1GdR has been determined to be a block to replication by various DNA polymerases in vitro and is mutagenic in bacterial and mammalian cells. The M1GdR adduct is a reactive electrophile that has been shown to undergo a secondorder ring-opening reaction with hydroxide to form the acyclic adduct N2-(3-oxo-1-propenyl)-deoxyguanosine (N2OPGdR-). N2OPGdR- then ring-closes to form the M1GdR adduct under acidic and neutral conditions. Here we describe work detailing the ring-opening and ringclosing reactions of these two adducts in both nucleosides and in oligonucleotides. UV and fluorescence tracing experiments show ring-closure is a biphasic, general acidcatalyzed reaction. An initial rapid phase, which is shown to be dependent upon protonation and oxopropenyl tautomerization, is followed by a slow phase of ring-closure and loss of water to form M1GdR. 1H-NMR experiments demonstrate a number of intermediates are formed in solution. These intermediates are thought to be N2OPGdR tautomers in the mechanism for ring-closure to M1GdR. Density functional theory (DFT) calculations of the proposed intermediates were performed to provide insights into the relative energy surface for M1GdR formation. These experiments reveal the complexity of MDA-dG adduct chemical transformations and the dependence of the reaction coordinate on small changes in pH. 41. Tobacco carcinogen-DNA adduct formation at specific sites within K-ras and p53 gene sequences. Natalia Tretyakova, Brock Matter, and Rebecca Ziegel. Department of Medicinal Chemistry and Cancer Center, University of Minnesota, 760E CCRB, 806 Mayo, 420 Delaware St. SE, Minneapolis, MN 55455, (fax) 612-6265135, [email protected] Direct quantitative analysis of tobacco carcinogen-DNA adducts originating from specific sites within p53 and K-ras derived DNA sequences by using a stable isotope labeling-mass spectrometry approach is reported. Gua-


nine adducts induced by 7r,8t-dihydroxy-t9,10-epoxy7,8,9,10-tetrahydro benzo[a]pyrene (BPDE) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were produced non-randomly along K-ras and p53 gene-derived DNA sequences. N2-BPDE-dG adduct yield was increased by the neighboring 5-Me-cytosines, while the formation of O6-methyl-dG and N7-methyl-dG was inhibited by cytosine methylation. In K-ras derived DNA sequence, the majority of N2-BPDE-dG adducts originated from the first position of the codon 12 (GGT), consistent with the large number of G to T transversions observed at this nucleotide. In contrast, both O6-methyl-dG and O6pyridyloxobutyl-dG were overproduced at the second position of K-ras codon 12, a predominant hot spot for G to A transitions. The experimental approach described in this work is applicable to studies of DNA modification by other carcinogens and alkylating drugs. 42. Toxicity and SAR's of arylphosphonium salts as antibacterials, acetylcholinesterase inhibitors, and DNA binders. John C. Williams,1 Christopher Zabbo,1 Anzel Bio,1 Rhett Shumway,1 Dave Dawson,2 Sarah Stevenson,2 Karen Robinson,1 Carla Moiera,1 Marisa DiDonato,1 and Charles Owens.2 1Physical Sciences Dept, Rhode Island College, 600 Mt Pleasant Ave, Providence, RI 02908, (fax) 401-456-8396, [email protected], 2Biology Dept, Rhode Island College Arylphosphonium salts have been studied for antibacterial effects, inhibition of acetylcholinesterase in vitro, and shifting of DNA melting curves. The SAR’s are consistent in each case; p-CH3O > p-CH3 > H > p-F >pCl, and effects were observed at 10 microgram/mL for some compounds. There is a strong positive correlation between antibacterial activity, acetylcholinesterase inhibition, and DNA melting curve shifts, with the exception of bis(triaryl)phosphoniaxylene salts. The ortho isomer has no effect on DNA melting. The goal is to prepare a compound with potent antibacterial activity that will not inhibit acetylcholinesterase or bind to DNA. 43. 5-Cyanoimino-4-imidazolecarboxamide and nitrosative guanine deamination: Experimental evidence for pyrimidine ring-opening during deamination. Rainer Glaser and Ming Qian. Department of Chemistry, University of Missouri-Columbia, Columbia, MO 65211, (fax) 573 882 2754, [email protected], [email protected] Our theoretical studies suggested that the dediazoniation of guaninediazonium ion occurs with comcommitant pyrimidine ring-opening and leads to cyanoimines 1 after deprotonation. We are now testing our hypothesis experimentally. Since compounds 1 are extremely reactive, we are currently exploring the chemistry of their addition products, the cyanoamines 2. Specifically, we have synthesized and fully characterized (NMR, IR, MS/MS) the cyanoimine 2 with R ) CH2-O-CH2-CH2-OH and R′ ) H. The MS/MS spectrum of 2 shows a peak for protonated 1 and this finding provides the first direct experimental observation of the key intermediate we postulated based on our prior theoretical studies. We have found that 2 easily cyclizes to form guanines 3. If the reaction of 1 to 2 involved a DNA base (G, C or A) as the primary amine, then this guanine formation would result in a crosslink. The model reaction of 2 to 3 with R′)H thus provides a

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first indication of the possible existence of a new class of crosslinks.

44. Possible model for thiourea-based toxicities via metabolic activation. Reuben H. Simoyi, Department of Chemistry, West Virginia University, Box 6045, Morgantown, WV 26506, (fax) 304-293-4904, [email protected] Substituted thioureas and thiourea itself are known to exhibit a number of pharmacological effects in toxicity and metabolic effects. The main in vivo transformations of thioureas involve the oxygenation of the sulfur atom followed by nucleophilic substitution or elimination reactions to give sulfinic and sulfonic acids. It had been conjectured that S-oxygenation of thioureas results in the formation of genotoxic products. If S-oxygenation is a prerequisite for the expression of toxicities associated with thioureas, then the study of decompositions and metabolic activations of sulfinic and sulfonic acids should lead to reactions that generate these toxicities. Aminoiminomethanesulfinic acids decompose to give dithionite, S2O42-. The precursors to dithionite, however, appear to be the very damaging and reactive oxygen species: superoxide, O2-, peroxide, O22- and hydroxyl radical, HO. It appears the heterolytic cleavage of the C-S bond in S-oxygenated metabolites of thioureas might be responsible for the toxicities observed with thioureas. 45. Deamination of cytosine revisited: Experimental evidence for uracil formation by way of ringopening and recyclization. Rainer Glaser and Sundeep Rayat. Department of Chemistry, University of Missouri-Columbia, Columbia, MO 65211, (fax) 573 882 2754, [email protected], [email protected] Our recent theoretical studies have shown that the dediazoniation of the cytosinediazonium ion is an extremely facile process. Our studies further revealed that the resulting cation undergoes exothermic pyrimidine ring-opening with hardly any activation energy to form the acyclic cation 1 (R1dR2dH). This new insight suggests an entirely new pathway for the deamination of cytosine leading to uracil. It is our hypothesis that 1 can cyclize to uracil. We have now synthesized and fully characterized 2 (R1dH, R2)tert-butyl, R3)ethyl), a derivative of the ring-opened intermediate. The hydrolysis of the cyano group of 2 is followed by cyclization to the uracil 3. The formation of 3 has been confirmed by LCMS. The synthesis of 2 and its cyclization to uracil 3 will be discussed. Clearly, the direct nucleophilic aromatic substitution of cytosinediazonium ion by water is not the only possible pathway to uracil.


46. Developing a predictive simulation model for antiandrogen impacts on rodent prostate. Hugh A. Barton,1 R. Woodrow Setzer,1 and Laura K. Potter.2 1ORD/NHEERL/ETD/Pharmacokinetics Branch, U.S. EPA, 86 TW Alexander Drive, Research Triangle Park, NC 27711, (fax) 919-541-4284, [email protected], 2Curriculum in Toxicology, University of North Carolina Alterations in rodent prostate weight and function are very sensitive endpoints associated with adult and pubertal exposures to several classes of antiandrogens. We are extending a published model for the adult rat central axis (testosterone and luteinizing hormone pharmacokinetics and feedback regulation) to include the prostate and 5alpha-reductase conversion of testosterone to dihydrotestosterone. The goal is to predict how perturbations of the biological system create dose-response behaviors and evaluate assumptions of thresholds for the antiandrogenic impacts on prostate. The targets of antiandrogens may be androgen receptor binding (e.g. vinclozolin or flutamide), testosterone synthesis (e.g. phthalates), and dihydrotestosterone synthesis (e.g. finasteride). Impacts on prostate being modeled include alterations in the expression of androgen receptor, 5alphareductase, and other genes that result in decreased fluid production, decreased cell proliferation, and increased apoptosis. The model will be a system of delayed differential algebraic equations and may be extended to describe pubertal animals. 47. Effect of halogenated substituents on the metabolism and estrogenic effects of the equine estrogen, equilenin. Xuemei Liu, Fagen Zhang, Emily Pisha, Yan Li, Jiaqin Yao, Richard B. van Breemen, and Judy L. Bolton. Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 833 S Wood St, Chicago, IL 60612, (fax) 312-996-7107 ERT has been correlated with an increased cancer risk. Previously we showed that equilin and equilenin are metabolized to the catechol, 4-hydroxyequilenin which autoxidizes to an o-quinone causing oxidation and alkylation of DNA in vitro and in vivo. In order to block the catechol metabolism of equilenin, we synthesized 4-halogenated equilenin derivatives. These derivatives were tested for their estrogenic effects, estrogen receptor binding affinities, and their potential to form catechol metabolites. We found that 4-FEN was more estrogenic than 4-ClEN and 4-BrEN as demonstrated by a higher binding affinity for ER and an enhanced induction in activity of alkaline phosphatase in Ishikawa cells. Incubation of these compounds with tyrosinase in the presence of GSH showed that the halogenated equilenin compounds formed less catechol GSH conjugates than equilenin. In addition, these halogenated compounds showed less cytotoxicity than equilenin in S30 cells. These data suggest that the 4-halogenated equilenin derivatives have promise as alternatives to traditional estrogen replacement therapy. (Supported by NIH CA 73638). 48. Formation of DNA interchain crosslinks by the deoxyguanosine adducts of 2,3-unsaturated aldehydes. Ivan D. Kozekov,1 Lubomir Nechev,1 Hao Wang,1 Ana Sanchez,2 R. Stephen Lloyd,2 Constance M. Harris,1 Thomas M. Harris,1 and Carmelo J. Rizzo.1 1Department


of Chemistry and Center in Molecular Toxicology, Vanderbilt University, Nashville, TN 37235-1822, (fax) 615-3222649, [email protected], 2Sealy Center for Moelecular Sciences, UTMBsGalveston Alpha, beta-unsaturated aldehydes are pervasive environmental pollutants and also arise endogenously as products of oxidation of unsaturated fatty acids. Their reactivity with DNA raises concern with respect to their potential to be mutagens and carcinogens. We have examined the ability of the deoxyguanosine adducts of acrolein (1), crotonaldehyde (2) and 4-hydroxy-2-nonenal (3, 4-HNE) to form DNA crosslinks. Key observations are as follows: (1) The acrolein adduct forms interchain crosslinks in a CpG context but not GpC. (2) Crotonaldehyde adduction yields two diastereomers, both of which form crosslinks. However, crosslinking efficiency of the R-diastereomer is much higher than the S. (3) Four diastereomers of the HNE adduct arise from reaction of racemic 4-HNE with deoxyguanosine. Only the 6S, 8R, 11S diastereomer forms crosslinks but it reacts with high efficiency.


geometry is required for MAO-B inhibiting activity and that benzimidazolyl analogs, while active, are less potent than the corresponding xanthinyl analogs. The results of these studies suggest that MAO-B inhibition may contribute to the neuroprotective potential of A2A receptor antagonists such as KW-6002. 50. Lipid peroxidation induced DNA damage and mutagenesis in human cells. John Termini, Gerald E. Wuenschell, Vanessa R. Holland, and Punnajit Lim. Molecular Biology, Beckman Research Institute of the City of Hope Med Center, 1450 East Duarte Road, Duarte, CA 91001, (fax) 626-301-8271, [email protected] Diets high in polyunsaturated fats have been associated with an increased risk of certain cancers. This promotional effect results either directly or indirectly from the oxidation of polyunsaturated fat in phospholipid containing membranes, the composition of which is profoundly influenced by diet. Lipid peroxidation generates reactive free radical intermediates (peroxyl and alkoxyl radicals) which can cause oxidative base damage and strand breaks in DNA. Free radical termination reactions of lipid peroxidation create electrophilic aldehydes (malondialdehyde, hydroxynonenal) which can condense with DNA to form genotoxic, mutagenic and carcinogenic adducts. The results of studies of peroxyl radical oxidation of DNA and the structures of some newly characterized adducts will be described. We present results from mutagenesis studies in human cells which suggest that the relative contributions of the oxidative vs the aldehyde damage pathways varies with the degree of lipid unsaturation.

49. Inhibition of monoamine oxidase B by selective A2A adenosine receptor antagonists. Neal Castagnoli,1 Jacobus P. Petzer,1 Kay Castagnoli,1 Michael A. Schwarzschild,2 and Jiang-Fan Chen.3 1Department of Chemistry, Virginia Tech, 107 Davidson Hall, Blacksburg, VA 24061, (fax) 540-231-8890, [email protected], (2) Department of Neurology, Massachusetts General Hospital, 3Department of Neurology, University of Medicine and Dentistry of New Jersey

51. Nitrosamine carcinogenesis: Synthesis, hydrolysis, and dG adducts of two r-acetoxynitrosamine models of NDELA and NMOR reactive metabolites. Richard N. Loeppky, and Sunil Sukhtankar, Department of Chemistry, University of Missouri, 125 Chemistry Building, Columbia, MO 65211, (fax) 573882-2754, [email protected]

Adenosine receptor antagonists that are selective for the A2A receptor subtype (A2A antagonists) are under investigation as possible therapeutic agents for the symptomatic treatment of the motor deficits associated with Parkinson’s Disease (PD). Results of recent studies in the MPTP mouse model of PD suggest that A2A antagonists may possess neuroprotective properties. Since monoamine oxidase B (MAO-B) inhibitors also protect against the nigrostriatal toxicity of MPTP, we have examined the MAO-B inhibiting properties of several A2A antagonists and related compounds to determine to what extent inhibition of MAO-B may contribute to the observed neuroprotection. The results of these studies have established that all of the 8-styrylxanthinyl-based A2A antagonists examined display significant MAO-B inhibitory properties in vitro with Ki values in the low-µM to nM range. Included in this series is (E)-1,3-diethyl-8-(3,4-dimethoxystyryl)-7-methylxanthine (KW-6002), a potent A2A antagonist that is undergoing clinical trials as a potential antiparkinsonian agent. SAR studies have established that the trans

NHMOR is a metabolite of both N-nitrosodiethanolamine (NDELA) and N-nitrosomorpholine (NMOR). We have evidence that NHMOR is microsomally further oxidized at the carbons adjacent to the ring nitrogen to give reactive R-hydroxynitrosamines which decompose to diazonium ions. In order to further test these hypotheses we have synthesized 1 and 2 from N-nitrosodehydromorpholine and 2-hydroxyethylvinylnitrosamine, respectively, and determined their decomposition products and rates as a function of pH. The aqueous decomposition of 1 occurs much more rapidly than 2 suggesting neighboring group participation in the hydrolysis of the former. Decomposition products of 1 are glyoxal (95%), ethylene glycol (55%) and acetaldehyde (7%). Its regioisomer 2 gives glycoladehyde (15%), glyoxal (10%) and 2-acetoxyethanal (62%) as the major product. The latter is formed by rearrangement of the incipient carbocation resulting from the diazonium ion. The decomposition of 2 in the presence of an excess of deoxyguanosine (dG) gives the N1,N2-etheno as the main adduct. The glyoxal-dG adduct is produced in minor amounts, but is the major adduct

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from 1 along with smaller amounts of the O6-hydroxyethyl-, and 7-hydroxyethyl- dG adducts.

52. Nitrosative guanine deamination: An ab initio study of the carbodiimide-cyanoamine tautomerization in the pyrimidine ring-opened intermediate. Rainer Glaser and Sundeep Rayat. Department of Chemistry, University of Missouri-Columbia, Columbia, MO 65211, (fax) 573 882 2754, [email protected] Our theoretical studies have revealed that the DNA base diazonium ions are very unstable and the dediazoniation of guaninediazonium ion occurs in concert with pyrimidine ring-opening to form intermediate 1. This key intermediate is highly reactive and has a variety of options to react. One of these possibilities is the tautomerization to the cyano-amine 2. We have studied this tautomerization at the RHF/6-31G* and B3LYP/6-31G* levels of theory for models with several R-groups (H, CH3, CH2OCH3). The thermodynamics of this tautomerization has been determined for all the rotational isomers. Unexpectedly, the carbodiimide tautomer was found to be more stable than the cyano-amine in the model cations studied, unless the cyanoamine benefits from hydrogen bonding. Electronic structure analysis reveals that 1 and 2 both are best considered as protonated cyano-imines. Hence, there is no dilemma between our results and the known preference of the parent cyanoamine over the parent carbodiimide.

53. Inhibition of monoamine oxidase by tobacco smoke. Kay Castagnoli, Izel Fourie, Cornelis J. Van der Schyf, Thangaraju Murugesan, and Neal Castagnoli. Department of Chemistry, Virginia Tech, 107 Davidson Hall, Blacksburg, VA 24061, (fax) 540-231-8890, [email protected] Recent studies using PET imaging techniques have established that brain MAO-A and MAO-B activities are significantly reduced in smokers compared to non-smokers, a finding consistent with the lower levels of MAO-B activity in blood platelets of smokers. In view of the important roles that these enzymes play in the regulation of brain biogenic amines, these effects on MAO activity may be related to the well-documented inverse relationship between smoking and the incidence of Parkinson’s disease (PD). Our interest in this area is based in part on the neuroprotective effects of MAO-B inhibitors such as (R)-deprenyl, a potent and selective mechanism based inactivator of MAO-B that is of therapeutic value in the treatment of PD. Therefore, we have initiated studies to characterize inhibitors of MAO-A and MAO-B in ciga-


rette tobacco smoke. Crude isolates of tobacco smoke were found to be effective, irreversible inhibitors of baboon and human liver MAO-B and, less potently, human placental MAO-A. Extensive fractionation/isolation studies are underway to characterize the specific substances that mediate the inhibition. We also are investigating their in vivo MAO inhibiting properties by measuring enzyme activities in rat liver and brain mitochondrial homogenates obtained following 28-day exposure via osmotic minipumps. The results of these studies and their potential significance with regard to neuroprotective mechanisms will be discussed. 54. Reaction of aflatoxin B1 oxidation products with lysine. F. P. Guengerich,1 M. Voehler,2 K. M. Williams,3 K. O. Arneson,1 Z. Deng,4 and T. M. Harris.5 1 Dept. of Biochemistry & Center in Molecular Toxicology, Vanderbilt University, 638 Robinson Research Bldg, 23rd & Pierce Avenues, Nashville, TN 37232, (fax) 615-3223141, [email protected], 2Dept. of Chemistry & Center in Molecular Toxicology, Vanderbilt University, 3Dept. of Biochemistry & Center in Molecular Toxicology, Vanderbilt University, 4Chemistry, Vanderbilt University, 5Department of Chemistry & Center in Molecular Toxicology, Vanderbilt University Aflatoxin (AF) B1 exo-8,9-epoxide hydrolysis yields AFB1 dihydrodiol, which undergoes base-catalyzed rearrangement to AFB1 dialdehyde. Pronase digestion of bovine albumin serum treated with AFB1 dialdehyde and HPLC yielded the adduct, identified by its characteristic UV and mass as (S)-alpha-amino-2,3-dihydro-2-oxo-4(1,2,3,4-tetrahydro-7-hydroxy-9-methoxy-3,4-dioxocyclopenta[c][1]benzopyran-6-yl)-1H-pyrrole-1-hexanoic acid, based on its spectral properties and comparison with the methylamine adduct, the structure of which was established by NOESY NMR spectroscopy. Kinetic analysis of the reaction of AFB1 dialdehyde with N2-acetylLys at pH 7.2 was best described by the reaction of AFB1 dialdehyde with the N6 atom of N2-acetylLys (k)2600 M-1 min-1) followed by carbinolamine formation (k)7.6 min-1). Competition experiments with the hydrolysis of AFB1 exo-8,9-epoxide indicate that N2-acetylLys also reacts with the epoxide at pH 7. Mass spectral analysis of trypsin digests of bovine serum albumin modified with AFB1 dialdehyde indicated selective modification of Lys455 and Lys548. (Supported in part by USPHS R01 CA90426, P30 ES00267). 55. Toxicity of methyl-tert butyl ether to higher plants. Youn-Joo An, Department of Environmental Science and Engineering, Ewha Womans University, 11-1 Daehyun-dong, Seodaemun-gu, Seoul 120-750, South Korea, (fax) 82-2-3277-3275, [email protected], Donald H. Kampbell, National Risk Management Research Laboratory, U.S. Environmental Protection Agency, and Mary E. McGill, Robert S. Kerr Environmental Research Center Toxicity of methyl tert-butyl ether (MTBE) to higher plants were studied in laboratory experiments. Test plants were wild oats (Avena sativa), sweet corn (Zea mays), wheat (Triticum aestivum), and lettuce (lactuca sativa). The endpoints measured were percent germination, shoot growth, and root elongation. The LC50 values for seed germination tests and the EC50 values for shoot or root growth were calculated. The values for lettuce,


wild oats, and wheat, and sweet corn were in the range of 18-91, 362-459, 432-751, and 672-964 mg MTBE/ kg soil as dry weight, respectively. MTBE concentration in soil at 60 mg/kg would affect the seedling growth of a plant as sensitive as lettuce. Smaller seeds are generally more sensitive because they are likely to absorb more MTBE based on their mass Our plant growth test should be a good protocol of how an autotroph might respond to a chemical stressor such as a MTBE. T. Penning Symposium Poster Session: Aldo-Keto Reductases 56. Chemistry of PAH o-quinones generated by the AKR pathway of PAH activation. Sridhar R. Gopishetty,1 Ronald. G Harvey,2 Seon-Hwa Lee,3 Ian. A Blair,3 and Trevor. M Penning.1 1Deparment of Pharmacology, Univeristy of Pennsylvania, 135 John Morgan Bldg, 3620 Hamilton Walk, Philadelphia, PA 19104, (fax) 215-5732236, [email protected], 2The Ben May Institute for Cancer Research, University of Chicago, 3Center for Cancer Pharmacology, University of Pennsylvania Carcinogenic polycyclic aromatic hydrocarbons (PAHs) can be metabolically activated by human aldo - keto reductases (AKR) to three classes of o-quinones of different reactivity and redox-activity. Class I o-quinones [e.g. naphthalene-1,2-dione and 7,12-dimethylbenz[a]anthracene-3,4-dione (DMBA-3,4-dione)] are among the most reactive AKR products and have the potential to form conjugates with cellular nucleophiles and stable and depurinating adducts with DNA. We now show that DMBA-3,4-dione will react with 2-mercaptoethanol to yield, mono- and bis-thioether conjugates by undergoing sequential 1,6- and 1,4-Michael addition reactions. Similar reactions should occur with cellular GSH. We also describe methods for the synthesis of stable naphthalene1,2-dione-N2-dGuo and N6-dAdo adducts. These methods involve coupling bromo-o-quinones to protected deoxyribonucleosides or coupling amino-o-quinones to halogenated deoxyribonucleosides. These methods can be applied to other AKR derived o-quinones, and should provide synthetic standards for the detection of these adducts in AKR expressing cells. 57. LC/APCI/MS analysis of etheno 2′-deoxyguanosine adducts as a dosimeter for AKR mediatedoxidative stress. Seon Hwa Lee, Tomoyuki Oe, and Ian A. Blair. Center for Cancer Pharmacology, University of Pennsylvania, 1246 BRB II/III, 421 Curie Blvd., Philadelphia, PA 19104-6160, (fax) 215-573-9889, [email protected] Aldo-keto reductases (AKRs) oxidize structurally diverse PAH trans-dihydrodiols to the corresponding oquinones. NADP+-dependent oxidation of trans-dihydrodiol initially results in a ketol, which spontaneously rearranges to form a catechol. Reactive oxygen species (ROS) are produced during the subsequent conversion of catechols to quinones. The first one-electron oxidation of the catechol to an o-semiquinone radical anion results in the formation of hydrogen peroxide. The second oneelectron oxidation of the o-semiquinone radical anion to the o-quinone results in the formation of superoxide radical anion. The resulting quinones can undergo a two-


electron reduction to regenerate the original catechol. This sets up a futile redox cycle and amplifies the formation of ROS, which leads to direct DNA damage or initiates the formation of lipid hydroperoxides. Lipid hydroperoxides undergo transition metal ion or vitamin C-induced decomposition to the R,β-unsaturated aldehydes, which react with DNA bases to form DNAadducts. Lipid hydroperoxide-derived DNA-adducts are thought to be important mediators of the degenerative diseases of aging including cancer and cardiovascular disease. The liquid chromatography/mass spectrometry (LC/MS) analysis of the products from the reaction between 13-hydroperoxyoctadecadienoic acid (13-HPODE; a prototypic ω-6 lipid hydroperoxide) and 2′deoxyguanosine (dGuo) in the presence of vitamin C revealed the formation of three major products with protonated molecular (MH+) ions at m/z 292 (ethenodGuo), m/z 476 (carboxynonanone etheno-dGuo), and m/z 404 (heptanone etheno-dGuo). After the pentafluorobenzyl (PFB) derivatization, we have developed a highly sensitive analytical method to quantify the three adducts from biological matrices using LC/atmospheric pressure chemical ionization (APCI)/MS. This assay will provide a comprehensive etheno-dGuo adduct profile and make it possible to establish the relative potency of each PAH o-quinone to stimulate lipid hydroperoxide formation and its breakdown to bifunctional electrophiles. Supported by NIH grant RO1-CA91016. 58. Protein adduct formed from the reaction of a dialdehyde derivative of aflatoxin B1 with BSA. F. P. Guengerich,1 M. Voehler,2 K. M. Williams,3 K. O. Arneson,1 Z. Deng,2 and T. M. Harris.1 1Dept. of Biochemistry & Center in Molecular Toxicology, Vanderbilt University, 638 Robinson Research Bldg, 23rd & Pierce Avenues, Nashville, TN 37232, (fax) 615-322-3141, [email protected], 2Chemistry, Vanderbilt University, 3Dept. of Biochemistry & Center in Molecular Toxicology, Vanderbilt University The structure of the products derived from the reactions of AFB1 dialdehyde with bovine serum albumin was identified by a combination of HPLC, UV, mass spectrometry, and NOESY NMR techniques. 59. Lipid hydroperoxide-mediated covalent modifications to sulfhydryl groups. Amy M. Quinn, Tomoyuki Oe, Seon Hwa Lee, and Ian A. Blair. Center for Cancer Pharmacology, University of Pennsylvania, Room 1246, BRB II/III, 421 Curie Boulevard, Philadelphia, PA 19104-6160, (fax) 215-573-9889, [email protected] Protein sulfhydryl groups are important structural features necessary for a diversity of biological effects including catalysis, regulation, electron transfer, and metal binding. Therefore, irreversible modification to sulfhydryl groups causes a deterioration in the function of many proteins. In previous studies, we have shown that lipid hydroperoxide-derived 4-oxo-2-nonenal can react with DNA-bases and the amino acid arginine to form stable adducts. Adduct formation is initiated by nucleophilic attack of the DNA-base or arginine on the 4-oxo-2-nonenal to form a Schiff base and/or a Michael addition product. This led us to speculate that sulfhydryl groups would also be modified by lipid hydroperoxidederived 4-oxo-2-nonenal. We have now shown that 4-oxo-

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2-nonenal will cause covalent modifications to free sulfhydryl groups, which results in the formations of stable adducts. 4-Oxo-2-nonenal was reacted with N-acetylcysteine in transition metal ion-free buffer (pH 7) at 37 °C. The reaction products were analyzed by reversed phase liquid chromatography/electrospray ionization/ mass spectrometry (LC/ESI/MS) using an ion trap instrument. The initial product of N-acetyl-cysteine with 4-oxo-2-nonenal appeared to be a simple Michael addition adduct based on the mass spectrum. This subsequently rearranged to a more stable adduct. Based on MSn and 1 H-NMR studies, its structure was consistent with a novel bicyclic thiomorpholine derivative. Glutathione and cysteine reacted with 4-oxo-2-nonenal to give more complex LC/MS profiles, which contained additional Schiff base products from reactions with the a-amino groups. These modifications have potential utility as molecular dosimeters for oxidative damage to sulfhydryl groups in plasma proteins. The functional consequences of such modifications to intracellular proteins may have relevance to pathophysiology of the degenerative diseases of aging such as cancer and cardiovascular disease. Supported by NIH RO1 CA95586. 60. Modification of arginine-containing peptides derived from lipid hydroperoxides. Tomoyuki Oe, Seon Hwa Lee, Amy M. Quinn, and Ian A. Blair. Center for Cancer Pharmacology, University of Pennsylvania, Room 1246, BRB II/III, 421 Curie Boulevard, Philadelphia, PA 19104-6160, (fax) 215-573-9889, [email protected] Metabolic activation of polycyclic aromatic hydrocarbons is required before they become carcinogenic. One pathway of activation involves aldo-keto reductases (AKRs) that convert CYP1A1-derived trans-dihydrodiols to o-quinones. o-Quinones are known to not only give rise to a series of stable DNA adducts but also redox cycle to generate reactive oxygen species that causes lipid hydroperoxide formation. We have previously shown the bifunctional electrophiles derived from lipid hydroperoxides also can react with DNA bases to form stable adducts. This suggests that they can also cause covalent modifications to functionally important proteins. We have now demonstrated that products of transition metal ion or vitamin C mediated decomposition of lipid hydroperoxides can react with arginine. 4-Oxo-2-nonenal, one of the major degradation products of lipid hydroperoxide was reacted with model compounds [t-Boc-arginine, MetArg-Phe-Ala, and human serum albumin (HSA)] in transition metal ion-free MOPS buffer (pH 7.4) at 37°C. The protein samples were digested by trypsin or V8 protease before analysis by liquid chromatography/electrospray/mass spectrometry (LC/ESI/MS) using an ion trap instrument. Separations were performed using an ODS-column with a gradient elution using trifluoroacetic acid containing water-acetonitrile system. The initial product of t-Boc-arginine appeared to be a hydroxyethano adduct based on the mass spectrum. The intially formed adduct was then converted to a stable dehydrated form. The structure was identified by MSn as an imidazole arginine derivative. The same type of adduct was identified from reaction with a tetrapeptide (Met-Arg-Phe-Ala). Covalent modifications to HSA have also been identified from in situ generation of 4-oxo-2-nonenal using a 13HPODE (13-hydroperoxyoctadienoic acid)/vitamin C system. These modifications will be utilized as a molecular


dosimeter of oxidative damage in vivo. Supported by NIH RO1 CA95586. 61. Role of AKR7A aldo-keto reductases in the detoxication of aldehydes and ketones. Elizabeth M. Ellis, Rachel A. Gardner, Dan Li, and Anne W. Grant. Department of Bioscience, University of Strathclyde, Royal College, 204 George Street, Glasgow, United Kingdom, (fax) 44-141-553-4124, [email protected] Aldehydes and ketones are present in a diverse range of compounds including drugs, dietary components, products of lipid peroxidation, steroids. Many aldehydes, particularly R,β-unsaturated compounds are extremely toxic, causing damage to protein and DNA. To counter the effects of these compounds, enzyme systems have evolved which can either oxidize, reduce or conjugate them to less reactive metabolites. The AKR7A sub-family of enzymes was originally identified through their ability to reduce a cytotoxic metabolite of aflatoxin B1 but these enzymes have subsequently been shown to reduce a wide range of aldehydes and diketones. We have constructed cell lines that express individual members of the AKR7A family, and have tested the ability of these enzymes to protect cells against the toxic effects of a range of aldehyde-containing compounds. This data suggests that these enzymes can detoxify lipid peroxidation products, and may have a role in protecting against oxidative stress. 62. Aldose reductase interrupts reactive oxygen species-mediated inflammatory signals. Kota V. Ramana,1 Deepak Chandra,1 Sanjay Srivastava,2 Aruni Bhatnagar,2 Bharat B. Aggarwal,3 and Satish K. Srivastava.1 1Human Biological Chemistry and Genetics, University of Texas Medical Branch, 6.644, Basic Science Building, Galveston, TX 77555, (fax) 409-772-9679, [email protected], 2Department of Medicine, University of Louisville, 3Department of Bioimmunotherapy, University of Texas M. D. Anderson Cancer Center Growth factor or cytokine stimulation results in the activation of the transcription factor - NF-κB, which is a key determinant of proliferation and apoptosis. We have demonstrated that inhibition of aldose reductase (AR) causes a 50% decrease in the extent of restenosis of balloon-injured rat carotid arteries. Moreover, inhibition of AR prevents TNF-alpha-induced proliferation of vascular smooth muscle cells (VSMC) and apoptosis of vascular endothelial cells (VEC) in culture. We have shown that attenuation of VSMC proliferation by AR inhibitors is not due to activation of caspase-3 or apoptosis. On the other hand, cytokine- induced caspase-3 activation was attenuated by AR inhibitors in VEC. These results suggests that the mitogenic or cytotoxic signals are due to the modulation of the activity of the redox-sensitive transcription factors such as NF-κB and AP1 and the interruption of these signals by AR inhibition suggests an antioxidant role of AR in the detoxification of proinflammatory ROS products. 63. Role of aldose reductase in the detoxification of oxidized phospholipids. Sanjay Srivastava,1 Kota V Ramana,2 Satish K Srivastava,3 and Aruni Bhatnagar.1 1 Department of Medicine, University of Louisville, 500 South Floyd, Louisville, KY 40202, (fax) 502-852-1795,

Abstracts, ACS Division of Chemical Toxicology 2

Human Biological Chemistry and Genetics, University of Texas Medical Branch, 3Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch Oxidation of phospholipids generates products in which unsaturated fatty acids at the sn-2 position are oxidized to short chain aldehydes or epoxides. Such phospholipids containing phosphatidylcholine display biological activity similar to platelet activating factor (PAF) and stimulate monocytes adhesion to endothelial cells. These lipids can serve also as recognition sites for scavenger receptors. However, the mechanisms by which cells metabolize and detoxify oxidized phospholipids are not known. We therefore examined the possibility that oxidized phospholipids are removed by metabolism via aldose reductase (AR). Recombinant AR was an excellent catalyst for the reduction of 1-palmitoyl-2-(5-oxovaleroyl) phosphatidylcholine (POVPC). The enzyme also reduced short to medium chain aldehydes that are likely to be derived from POVPC by phospholipase A2-mediated metabolism. These observations suggest that AR could be a potential mechanism for the detoxification of oxidized phospholipids that appear during oxidative stress caused a variety of disease states. 64. Molecular cloning and characterization of dihydrodiol dehydrogenase from mouse liver. Yoshihiro Deyashiki, Takahiro Takatsuji, and Akira Hara. Laboratory of Biochemistry, Gifu Pharmaceutical University, 5-6-1, Mitahora-higashi, Gifu 502-8585, Japan, (fax) 81-58-237-5979, [email protected] Two novel cDNA species have been cloned by the screening of mouse liver cDNA library with human dihydrodiol dehydrogenase isoform (AKR1C1) cDNA. One contained an open reading frame of mouse dihydrodiol dehydrogenase (mDD) consisting of 323 amino acids. The other was supposed to encode the COOH-terminal part of an unknown protein that is similar to AKR1C6. mDD exhibited more than 85% amino acid sequence identity with the aldo-keto reductase superfamily proteins from mouse (AKR1C12, AKR1C13, mAKRa) and rat (RAKb, RAKd, RAKf). Recombinant mDD oxidized benzene dihydrodiol and reduced various carbonyl compounds including isatin and diacetyl, and preferred NAD(H) to NADP(H) as the coenzymes. mRNA for mDD was predominantly expressed in liver and small intestine. The results indicate that mDD is a new member of the aldoketo reductase superfamily and may be involved in the detoxification of xenobiotics and in the metabolism of some endogenous carbonyl compounds. Poster Session 65. Cimicifuga racemosa L. (Nutt.) (black cohosh) scavenges free radicals and protects DNA against menadione induced oxidative damage. Joanna E. Burdette,1 Shao-Nong Chen,1 Daniel Fabricant,2 Haiyan Xu,3 Bethany White,3 Andreas I. Constantinou,3 Norman Farnsworth,2 John Pezzuto,2 and Judy Bolton.1 1Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 833 S. Wood St., Chicago, IL 60612, (fax) 312-996-7107, 2Program for Collaborative Research in the Pharmaceutical Sciences and Department of Medicinal Chemistry and Pharmacognosy, Uni-


versity of Illinois at Chicago, 3Department of Surgical Oncology, University of Illinois at Chicago Compounds were isolated from the rhizome of Cimicifuga racemosa L. (Nutt.) (black cohosh) using bioassay guided fractionation and investigated for their free radical scavenging properties and their ability to protect against menadione induced DNA damage. In this study the DPPH (1,1-diphenyl-2-picryl-hydrazyl) radical scavenging activity was utilized to identify active plant compounds. These compounds were then investigated to determine if they could be used to prevent menadioneinduced single strand DNA breaks using the alkaline single-cell gel electrophoresis assay (comet). The FLARE (fragment length analysis using repair enzymes) assay was employed to detect compounds that protected against oxidative damage to the DNA bases induced by menadione. Caffeic acid, methyl caffeate, ferulic acid, and cimicifugate A and B were identified as the pure compounds responsible for the antioxidant effects. These findings demonstrate that black cohosh functioned as an antioxidant and protected against menadione induced DNA single-strand breaks and oxidative damage to the DNA bases. 66. Antiestrogens, oxidative metabolites, NO, and peroxynitrite. Gregory R J Thatcher,1 Violeta Toader,1 Adrian Nicolescu,1 and Judy L. Bolton.2 1Department of Chemistry, Queen’s University, Queen’s University, Kingston, ON K7L 3N6, Canada, (fax) 613 533 6669, [email protected], 2Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago Antiestrogens or Selective Estrogen Receptor Modulators (SERMs) such as tamoxifen and raloxifene are chemopreventive agents that show varying degrees of endometrial carcinogenesis. Many biological actions of SERMs, including beneficial cardiovascular effects are mediated via elevation of tissue NO/cGMP levels. SERMs are particularly sensitive to oxidative metabolism, in some cases generating superoxide. Under conditions of elevated NO production, the rapid reaction of NO with superoxide generates the potent cytotoxic oxidant peroxynitrite. Thus there appears an intrinsic link between SERM metabolism, oxidative stress and reactve nitrogen/ oxygen species. SERMs undergo oxidation and other reactions in the presence of peroxynitrite and N,Ospecies, the products of which have been characterized and compared to SERMs in their ability to inhibit lipid peroxidation. The influence of peroxynitrite and other N,O-species on tissue relaxation, elevation of cGMP, and formation of nitrotyrosine has been examined in tissue treated with SERMs. 67. Conformational analysis of model estrogenDNA adducts, N2-(2-hydroxyestron-6(r,β)-yl)-2′-deoxyguanosine and N6-(2-hydroxyestron-6(r,β)-yl)-2′deoxyadenosine in duplex B-DNA. Lihua Wang, Biology Department, New York University, 1009 Main Bldg, 100 Washington Sqare East, New York, NY 10003, (fax) 212-995-4015, [email protected], Brian E. Hingerty, Life Sciences Division, Oak Ridge National Laboratory, and Suse Broyde, Department of Biology, New York University An extensive conformational analysis has been carried out for two stereoisomeric pairs of model estrogen-DNA

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adducts, N2-(2-hydroxyestron-6(R,β)-yl)-2′-deoxyguanosine and N6-(2-hydroxyestron-6(R,β)-yl)-2′-deoxyadenosine, employing our nucleic acids torsion angle space molecular mechanics program DUPLEX with an improved treatment of solvent and counterions. In vitro studies have shown that these adducts can cause mutations. We found that the four-ring non-planar estrogen moiety can reside only in the minor groove of the guanine adducts or the major groove of the adenine adducts, due to its nonaromaticity. A kink is created in the guanine adducts to accommodate the estrogen ring system in the minor groove, and a pocket is formed around the adenine-bound estrogen moiety for maximum shielding from solvent exposure. No low-energy classically or base-displaced intercalated conformations were located in the conformational search. The apparent proclivity for such bulky, non-planar adducts to reside at the DNA helix exterior is a plausible conformational feature of other structurally similar estrogen-DNA adducts. 68. Formation and reactivity of a raloxifene quinone methide. Linning Yu,1 Fagen Zhang,1 Dejan Nikolic,2 Wenkui Li,3 Richard B. van Breemen,1 and Judy L. Bolton.1 1Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 833 S Wood St, M/C 781, Chicago, IL 60612, (fax) 312-996-7107, [email protected], 2Department of Medicinal Chemistry & Pharmacognosy, University of Illinois, 3Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago Long-term usage of the antiestrogen tamoxifen has been linked to increased risk of developing endometrial cancer in women. One of the suggested pathways leading to toxicity of tamoxifen involves its metabolism to an electrophilic quinone methide which could attack cellular macromolecules leading to initiation of carcinogenesis. Another antiestrogen raloxifene was recently approved by the FDA for treatment of osteoporosis in postmenopausal women and it is currently in clinical trials for chemoprevention of breast cancer. Before it is widely used for healthy individuals, it is crucial to fully understand its potential cytotoxic mechanisms since raloxifene has a similar structure to tamoxifen. In this study, incubations of raloxifene with GSH in the presence of rat liver microsomes or tyrosinase were carried out and analyzed by LC/MS-MS. The results showed that incubations with raloxifene formed GSH conjugates which were identified as the raloxifene quinone methide GSH conjugates. These preliminary results suggest that raloxifene could be metabolized to an electrophilic quinoid which has the potential to cause toxicity in vivo. (Supported by NIH grant CA79870). 69. Conformational analysis of the 4-hydroxyequilenin base adducts using density functional theory. Shuang Ding,1 Shixiang Yan,1 Min Wu,1 Nicholas E. Geacintov,1 and Suse Broyde.2 1Department of Chemistry, Department of Chemistry, New York University, 31 Washington Place, Main 1001, New York, NY 10003, [email protected], 2Department of Biology, New York University Equilin and equilenin, the major components of the drug Premarin (Wyeth-Ayerst), can be metabolized to the catechol, 4-hydroxyequilenin (4-OHEN). 4-OHEN can react in vitro with 2′-deoxynucleosides to form unusual


cyclic adducts with dG, dA and dC. Four stereoisomers are formed in each case. However, the conformations of the adducts are not available. Our objective in this work was to compute the conformations for each of the four stereoisomers of the 4-hydroxyequilenin base adducts, 4-OHEN-G, 4-OHEN-A, and 4-OHEN-C, since investigation of the base adduct structures can aid in understanding the structures on the DNA level. Initial models were built, based on the geometric constraints of the connection ring and adjacent cyclohexene A ring. These were representative of all the possible conformational families. A computational investigation was then carried out, employing high level quantum mechanical geometry optimization using density functional theory (DFT). The structures suggest that these adducts would not be able to intercalate into the DNA double helix. 70. Dioxin’s role in altering human low-density and very low-density lipoprotein structure. Eric Arehart, Department of Biochemistry, University of Maine, Orono, ME 04469, [email protected], and Howard H. Patterson, Department of Chemistry, University of Maine Dioxins are a class of organic pollutants that are ubiquitous in the environment, which may be taken up either through the food supply or in contaminated water. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is by far the most toxic member of this group. Although TCDD exposure is linked to many forms of cancer, recent investigations have correlated TCDD exposure in humans to heart disease. To date there has been no adequate explanation for this trend. Fluorescence spectroscopy was employed to measure the binding affinity between lipoproteins and TCDD as well as various PCB's. Here we show the ability of TCDD to tightly bind human very low-density and lowdensity lipoproteins. We found significant binding and conformation change within the lipoprotein molecule upon exposure to TCDD. We performed molecular mechanics calculations to describe the energetics of these toxins in solution. It is our belief that destruction of the lipoprotein molecule inactivates the cellular recognition pathway leading to a buildup of serum lipoproteins. 71. GC/MS analysis of deoxyribose oxidation products induced by radiation and Fe-EDTA: Quantitative comparison of phosphoglycolaldehyde and phosphoglycolate. Christiane Struve, Mohamad Awada, and Peter C. Dedon. Biological Engineering Division, MIT, 56-786, 400 Main St, Cambridge, MA 02139, [email protected] Oxidation of 2′-deoxyribose (dR) represents an important component of oxidative DNA damage, both for the toxicity of strand breaks and oxidized abasic sites and for the reaction of the electrophilic products of deoxyribose oxidation with bases to form mutagenic adducts. To better understand the chemistry and biology of dR oxidation, we have developed a sensitive GC-MS assay to identify and quantify the spectrum of carbonylcontaining deoxyribose oxidation products. We now present the results of studies comparing the quantities of 3′phosphoglycolaldehyde (3′-oxidation of dR) and 3′-phosphoglycolate (4′-oxidation of dR) produced by ionizing radiation and Fe-EDTA. In this method, residues containing aldehydes and ketones are enzymatically released from oxidized DNA and converted to oximes by reaction with pentafluorobenzylhydroxylamine. Following addi-


tion of [2H] or [13C]-labeled internal standards, the oximes are silylated with BSTFA and quantified by GC/MS/SIM. The method is both sensitive and generally applicable to the variety of deoxyribose oxidation products. 72. Oxidation of alkylanilines by recombinant human cytochrome P450s and human liver microsomes. H. Patty Sun,1 Jinping Gan,2 Paul L Skipper,1 John S Wishnok,1 and Steven R. Tannenbaum.1 1Biological Engineering Division, MIT, 77 Massachusetts Ave, 56-738, Cambridge, MA 02139, (fax) 617-252-1787, [email protected], 2Vertex Pharmaceuticals, Inc. From previous studies, 2,6-dimethylaniline, 3,5-dimethylaniline, and 3-ethylaniline have been associated with an increased risk for human bladder cancers, independent of cigarette smoking. To provide further information of the mechanisms and pathway of the carcinogenicity of these three compounds, we have chosen to focus on studying their oxidation by recombinant human P450 enzymes and human liver microsomes based on the hypothesis that the oxidized metabolites are the proximal intermediates in the formation of DNA and protein adducts. In addition, we have also studied the metabolism of their isomers, including 2,3-dimethylaniline, 2,4-dimethylaniline, 2,5-dimethylaniline, 3,4-dimethylaniline, 3,5-dimethylaniline, and also 2-ethylaniline and 4-ethylaniline to gain some insight into the comparative biochemistry of this group of aromatic amines. The results of these studies will be presented with data on the relative rates of oxidation of the amino group, the aromatic ring, and the alkyl side-chains. 73. Transcriptional response to radical-mediated DNA damaging agents in S. cerevisiae. Joseph P. Cosgrove, Thomas J. Begley, and Peter C. Dedon. Biological Engineering Division, MIT, 77 Massachusetts Avenue, 56-786, Cambridge, MA 02139, (fax) 617-2580225, [email protected] Among free radical agents selective for DNA, the chemistry and location of damage varies considerably. To determine if the cellular response to these agents depends on the specific damage produced, we have studied the transcriptional response of S. cerevisiae to a series of enediyne antibiotics (calicheamicin, esperamicinA1, and neocarzinostatin) that specifically attack the deoxyribose of DNA but produce different degradation products and different proportions of single- and doublestranded lesions. These results are compared to the nonspecific damage caused by gamma-radiation. Preliminary results with an exposure of 15 min to 0.1 and 0.5 nM calicheamicin show that both doses cause >2-fold up regulation of repair genes like RAD 51 and HDF1, a DNA end-joining protein. After 1 h, most of these genes are down regulated, including many stress response genes. Studies with enediynes and agents such as bleomycin will reveal the details of cellular responses to different kinds of DNA damage. 74. trans-2-Butene-1,4-dial as a product of deoxyribose oxidation in DNA. Tonika Chatterji, BEH, MIT, 56-786 MIT, 77 Massachusetts Avenue, Cambridge, MA 02139, (fax) 617-258-0225, [email protected], and Peter C. Dedon, Biological Engineering Division, MIT Oxidation of the 5′ position of deoxyribose in DNA yields either a nucleoside 5′-aldehyde residue or a 3′-


formylphosphate residue plus a four-carbon fragment. We have now identified trans-2-butene-1,4-dial as a product of DNA oxidation by gamma-radiation. We propose that this species arises by beta-elimination of a 2-phospho1,4-dioxobutane precursor, the missing four-carbon residue. We have also developed a sensitive GC/MS/SIM method for quantifying both cis-(furan metabolite) and trans-2-butene-1,4-dial. Following DNA oxidation, aldehyde residues are converted to dioximes by reaction with benzylhydroxylamine and the trans-2-butene-1,4-dial is released by heating. Authentic samples of cis- and trans2-butene-1,4-dioxime species each produce a characteristic pattern of three GC/MS/SIM signals. Using a [2H]labeled cis-2-butene-1,4-dial internal standard, we determined that gamma-irradiation of DNA produced ∼5 trans-2-butene-1,4-dial residues per 106 nucleotides per Gray. This is a conservative estimate since trans-2butene-1,4-dial readily reacts with dC to form the oxadiazabicyclo(3.3.0)octaimine adduct observed in earlier studies. 75. Activation of benzo[a]pyrene to electrophilic species by the action of singlet oxygen on benzo[a]pyrene-7,8-dihydrodiol. Kevin H. Stansbury and Michael A. Trush Dept. of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Room 7032, Baltimore, MD 21205, (fax) 410-955-0116, [email protected] The ubiquitous carcinogen benzo[a]pyrene (BP) is activated to its ultimate carcinogenic form through a series of oxidation reactions. Since singlet oxygen (SOX) reacts with isolated double bonds by different mechanisms, an investigation into the products formed from the action of SOX on the 7,8-dihydrodiol of BP was undertaken. Using a rose bengal suspended sensitizer system as the SOX generator, both metabolites and electrophilic products were studied. The anti-/syn- BP diolepoxide (BPDE) ratio was determined to be much greater than that previously reported from various cytochrome P450 studies. Three DNA adducts were seen by 32P-postlabeling, with one adduct co-chromatographing with the major deoxyguanosine (dG) adduct formed by anti-BPDE. The data indicates that the formation of both the BP tetrol metabolites and the BPDE-dG DNA adduct by SOX most likely proceeds stereospecifically through a perepoxide-like mechanism. Supported by NIH Grant ES03760. 76. Synthesis and Introduction into DNA of Benzo[a]pyrene adducts at the N2-position of deoxyguanosine and the N6-position of deoxyadenosine. Francis Johnson, Radha R Bonala, Deepak S Tawde, and M Cecilia Torres. Department of Pharmacology, SUNY at Stony Brook, Stony Brook, NY 11794, (fax) 631-6327394, [email protected] Benzo[a]pyrene, one of the most studied carcinogenic polycyclic aromatic hydrocarbons (PAH), is metabolically activated to form bay-region diol epoxides (1). It is believed that these diol epoxides of the PAH exert their carcinogenic properties by forming stable DNA adducts, primarily at the exocyclic amino groups of deoxyguanosine (dG) and deoxyadenosine (dA). This poster described the new methods for the synthesis of Benzo[a]pyrene adducts of dG (2) and dA (3) in large quantities. These adducts were easily incorporated into oligomeric

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DNA using the standard protocol. In each case the two diastereomers could be separated without difficulty at the level of monomers (chromatography) and at the level of oligomers (HPLC). Each of these adducts have characterized by circular dicroism, NMR and mass spectra. Studies on both the mutagenic spectra and conformational structures of several of the oligomers are currently underway.

77. Efficient HPLC separation of diastereomers of modified oligodeoxynucleotides. M. Cecilia Torres and Charles R. Iden. Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, NY 11794-3400, (fax) 631-632-7394, [email protected] Both endogenous and exogenous chemical agents react with DNA and in many instances modify the structure of the nucleobase or the deoxyribose moiety. A single agent may produce products that are mixtures of stereoisomers, each of which may adopt a distinct chemical structure, have unique chemical stabilities and undergo reactions with specific DNA polymerases and repair enzymes. Modified oligodeoxynucleotides are used frequently to study the properties of these DNA adducts, and, ideally, each stereoisomer of a specific adduct should be isolated in order to elucidate differences in genotoxicity. We report several examples of the efficient separation of oligonucleotides containing diastereomers of critical DNA adducts using X-TerraTM MS C18 columns (Waters Corporation). These include adducts from the reaction of acrolein, a reactive, R,β-unsaturated aldehyde; oligodeoxynucleotides containing the N2-benzo[a]pyrene diol epoxide (BPDE) adduct of deoxyguanosine; and diastereomers of thymine glycol in oligomers (23-mers). Excellent separation was achieved for these adducts resulting in HPLC fractions with oligomers containing a single stereoisomer. 78. Solute-CSP interactions in the enantiomeric resolution of benzo[a]pyrene dihydrodiol and 6-halo analogs on cellulose-based stationary phases. Barbara Zajc,1 Rok Grahek,2 Andrej Kocijan,2 Mahesh K. Lakshman,1 Janez Kosmrlj,3 and Jure Lah.3 1Department of Chemistry, City College of CUNY, 138th Street and Convent Avenue, New York, NY 10031, (fax) 212 650 6107, [email protected], 2Research and Development, Lek Pharmaceutical Company, 3Faculty of Chemistry and Chemical Technology, University of Ljubljana Enantiomeric resolution and elution order of benzo[a]pyrene 7,8-dihydrodiol, as well as its 6-fluoro and 6-bromo derivatives were compared on three different polysaccharide-based chiral columns in light of the conformational preferences of the dihydrodiol analogues. Introduction of a C-6 halogen causes the dihydrodiol to adopt a quasi-diaxial arrangement of the hydroxyls whereas in the protio analog these are quasi-diequatorial. The


polysaccharide columns chosen for the study were Daicel Chiralcel CA-I (microcrystalline cellulose triacetate), OF (cellulose tris(4-chlorophenylcarbamate) derivative) and OG (cellulose tris(4-methylphenylcarbamate) derivative). To understand better the possible sources of interactions leading to separation of (()- -7,8-dihydrodiols of 6-halobenzo[a]pyrene, the separation of the well-studied (()1,1′-bi-2-naphthol was evaluated on the two phenylcarbamate columns. Results of these experiments as well as molecular modeling provide evidence that there may be similarities between the interactions of the (S)-1,1′bi-2-naphthol and (7S,8S) isomers of 6-halo dihydrodiols with the chiral stationary phase.

79. Stereoselective synthesis of 1,N2-deoxygunaosine adducts of enals. Mansoureh Rezaei, Kris H. Franklin, Thomas M. Harris, and Carmelo J. Rizzo. Department of Chemistry and Center in Molecular Toxicology, Vanderbilt University, VU Station B35 1822, Nashville, TN 37235-1822, (fax) 615-322-7591, [email protected] alpha,beta-Unsaturated aldehydes (enals) are products of lipid peroxidation and are pervasive in the environment. Simple enals react with deoxyguanosine to give two diastereomeric 1,N2-hydroxypropanos adducts (3). These adducts can be synthesized at the nucleosides and oligonucleotide through the reaction of O6-protected 2-fluoroinosine (1) with an appropriate amino diol (2) followed by periodate cleavage. Stereospecific synthesis of these adducts require the synthesis of enantiomerically pure amino diols. We have devised general strategies for the preparation of these amino diol for the stereocontrolled syntheses of 1,N2-hydroxylpropano adducts of deoxyguanosine.

80. Structural differentiation of diastereomeric benzo[ghi]fluoranthene adducts of deoxyadenosine by matrix-assisted laser desorption/ionization timeof-flight mass spectrometry and post-source decay. Hui-Fang Chang,1 Duane Huffer,2 Ken Olsen,2 Paul Chiarelli,2 Damen Barbucci,3 and Bongsup Cho.4 1Department of Biomedical Sciences, University of Rhode Island, Kingston, RI 02881, 2Department of Chemistry, Loyola University, Chicago, IL 60626, 3Department of


Chemistry, Washington University, 4Department of Biomedical Sciences, Univeristy of Rhode Island Product ion studies of four diastereomeric deoxyadenosine (dA) adducts of benzo[ghi]fluoranthene-3,4- dihydrodiol-5,5a-epoxide (B[ghi]FDE) were conducted to determine if their structures could be differentiated using mass spectrometry. The two adduct structures that have the dA at 5a and B[ghi]F 3-hydroxyl group cis with respect to each other produce substantial abundances of product ions formed by water loss. When these two groups are trans with respect to each other, no such product ions are observed. The data suggests that water loss proceeds through a transition state where the charge-bearing proton on the adenine N1 nitrogen is hydrogen-bonded to the O3 oxygen of B[ghi]F. The relative stabilities of the hydrogen-bonded transition states will be calculated using molecular modeling methods (Spartan) in order to test the feasibility of this mechanism (Supported in part by a grant from the American Cancer Society).

81. The Malondialdehyde M1G Adduct Alters the Structure of a 2-Base Bulge in the Salmonella typhimurium hisD3052 gene (CG)3 iterated repeat sequence. Yazhen Wang, Chemistry Department, Vanderbilt University, P.O. Box 1822 B, Vanderbilt University, Nashville, TN 37235, (fax) 615-3430384, [email protected], Nathalie Schnetz-Boutaud, Biochemistry Department, Vanderbilt University, and Micheal P. Stone, Chemistry Department, Vanderbilt University The unmodified hisD3052 (CG)3 iterated repeat sequence 5′-d(ATCGCGCGGCATG)-3′:5′-d(TAGCGCCGTAC)-3′ containing a two-base CG bulge was investigated using NMR spectroscopy. The results were compared with those from the modified duplex 5′-d(ATCGCMCGGCATG)-3′:5′-d (TAGCGCCGTAC)-3′ containing the malondialdehyde deoxyguanosine adduct opposite the two base deletion in the (CpG)3 frameshift hotspot of Salmonella typhimurium hisD3052, where M)M1G [3-(2′deoxy-b-D-erythro-pentofuranosyl) pyrimido[1,2-a]-purin-10(3H)-one]. [Schnetz-Boutaud N. C., et al. Biochemistry 2001, 40, 15638-15649]. For both the modified M1G duplex and the unmodified duplex, only one set of resonances was observed in the 1H NMR spectra, exchangeable and nonexchangeable sequential NOEs were interrupted at the location of the bulge and specific NOEs were missing at the site of the bulge. This suggested a single bulge conformation existed. However, the two-base bulge was localized at M6C7 for the modified duplex, but localized at the neighboring positions C3G4 for the unmodified duplex. Experiments are underway to further characterize the differences between the unmodified and modified duplex. These may play a role in modulating two-base strand slippage deletions by the M1G adduct. Supported by NIH grant CA-55678.


82. Enhancement of toxic and mutagenic effects of ethylene dibromide by alkylguanine-DNA alkyltransferase. K. M. Williams,1 L. Liu,2 A. E. Pegg,2 and F. P. Guengerich.3 1Department of Biochemistry & Center in Molecular Toxicology, Vanderbilt University, School of Medicine, Nashville, TN 37232, (fax) 615-322-3141, [email protected], 2Department of Physiology, Pennsylvania State University, 3Department of Biochemistry & Center in Molecular Toxicology, Vanderbilt University The mutagenicity and cytotoxicity of ethylene dibromide (EDB) in E. coli was enhanced by expression of the DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT). This increase was blocked by mutation of the active site Cys145 residue in AGT. AGT reaction with EDB resulted in loss of activity. AGT was labeled when incubated with [14C]EDB, indicating an AGT-EDB conjugate at Cys145. Mass spectrometry (MS) confirmed that a 2-hydroxyethyl adduct was present in the peptide. Incubation of AGT with EDB and oligonucleotide yielded covalent AGT-oligo complexes as shown with gel electrophoresis. MS of the tryptic digest of the reaction product of EDB and AGT gave a signal corresponding to the Gly136-Arg147 peptide cross-linked to a guanine. These results are consistent with a mechanism by which EDB reacts at the active site of AGT to generate an S-(2bromoethyl) half-mustard at Cys145. Subsequent depurination of the adduct would lead to point mutations and cytotoxicity. 83. Mitotic homologous recombination in mammalian cells: A double-edged sword. Karen H. Almeida, Carrie A. Hendricks, Laurel Vuong, Jeffery T. Loh, and Bevin P. Engelward. Division of Bioengineering and Environmental Health, Massachusetts Institute of Technology, 77 Massachusetts Ave., 56-638, Cambridge, MA 02139, (fax) 617-258-0499, [email protected] Mitotic homologous recombination is an effective strategy for repairing damaged DNA, although it is employed judiciously since recombination between misaligned sequences can lead to loss of genetic information. We have developed systems that exploit fluorescence to rapidly quantify mitotic recombination in mammalian cells. Using recombinant DNA technology, we have constructed vectors containing two non-functional, partially-truncated repeats of the Enhanced Yellow Fluorescence Protein (EYFP). Intrachromosomal recombination between these non-functional EYFP cassettes restores expression of EYFP thus producing a yellow fluorescent cell. We are currently employing these systems to investigate both endogenous and environmental DNA damaging agents for their ability, either directly through DNA interaction or indirectly through subsequent enzyme action, to induce homologous recombination. Our primary focus is the effect of DNA damaging drugs on cell lines of differing genetic backgrounds in an effort to better understand the role of homologous recombination in maintaining genetic stability. 84. Prokaryotic nucleotide excision repair of BPDEmodified DNA duplexes flanked by A:T or G:C base pairs depends on local structural disturbances caused by the lesions. Qian Ruan,1 Milan Skorvaga,2 Yue Zou,3 Bennett Van Houten,2 Shantu Amin,4 and Nicholas E. Geacintov.1 1Chemistry Department, New

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York University, 31 Washinton Place, New York, NY 10003, [email protected], 2NIEHS-NIH, 3East Tennessee State University, 4American Health Foundation Nucleotide excision repair (NER) is a major DNA repair mechanism responsible for recognition and removal of a variety of bulky DNA lesions such as the adduct of benzo[a]pyrene diol epoxides (BPDE) to N2 of guanine (G*). The objective of this work is to determine if a structural disturbance of the DNA duplex around the lesion site is related to the efficiency of excision of the lesions by NER enzymes. We synthesized and constructed site specifically modified 43-mer oligonucleotides with the sequence context 5′-...TG*T... and 5′-...CG*C... with G* ) (+)-trans-anti-BPDE-N2-dG, hybridized with a fully complementary strand in each case. The melting points of the duplexs, Tm. reflect the extent of disorder of each of the two duplexes studied that differ from one another only by the two flanking base pairs. Based on Tm values, the TG*T sequence is locally more destabilized than the CG*C sequence. Studies of NER of 5′ and 3′ labeled adduct duplexes indicated that the TG*T lesion is more easily excised by UvrABC repair enzymes of Escherichia coli than CG*C lesion at 37 °C. The excision activities were also studied with UvrABC repair enzymes of the thermophilic organism B. caldotenax, which allows for the determination of the temperature-dependent efficiencies of adduct excision. At 55 °C, NER is similar for the two duplexes, but at 37 °C the CG*C adduct is excised with an efficiency ∼3 times lower than the TG*T adduct. These findings are consistent with a greater local disturbance at the adduct site in the case of the TG*T adduct leading to a greater efficiency of DNA repair at 37 °C. At 55 °C, both lesions are excised with nearly equal efficiencies because, at this higher temperature, heatinduced local structural disorder exceeds the different extents of structural disturbance induced by the lesions in TG*T and CG*C sequences. Thus, the hypothesis that a local structural destabilization induced by these lesions serves as a recognition motif in NER in this model system, is supported by the data [Research supported by NIH Grant CA 76660]. 85. Reproducing structural and thermodynamic properties of carcinogen-DNA adducts through molecular dynamics simulation. Shixiang Yan,1 Min Wu,1 Nicholas E. Geacintov,1 and Suse Broyde.2 1Department of Chemistry, New York University, New York, NY 10003, [email protected], [email protected], 2Department of Biology, New York University The recently developed MM-PBSA (molecular mechanics Poisson-Boltzmann surface area) methodology utilizes molecular dynamics (MD) simulations to generate ensembles of structures from which free energies can be calculated. Validation of this computational technique requires a system that has been experimental fully characterized both structurally and thermodynamically. A pair of covalent adducts to guanine residues in DNA derived from metabolic activation of benzo[a]pyrene (BP) are among the few such well studied macromolecules. Both high-resolution NMR solution structures and microcalorimetry data are available for these 10S (+)- and 10R (-)-trans-anti-[BP]-N2-dG adducts, and they therefore provide an ideal system for rigorously testing the MM-PBSA method. We have carried out 3-ns molecular dynamics simulations, using NMR solution structures as


the starting models for the 10S (+)- and 10R (-)-trans adducts in a DNA duplex 11-mer. We employed the MMPBSA method to compute the free energies, enthalpies and entropies of the two adducts, and our complete thermodynamic analysis agrees well with the full experimental thermodynamic characterization of these adducts. We also calculated the NOE distances from the MD trajectories, and compared them against the experimental NMR derived NOE distances. Our results showed that the simulated structures are in good agreement with the NMR experimental NOE data. Furthermore, new insights linking structure and thermodynamics have been gained. Specifically, the enhanced exposure to solvent of the BP aromatic ring system in the 10S (+)-trans adduct, deduced through the experimental thermodynamic investigations in the same DNA sequence, is structurally rationalized. Thus, the MM-PBSA technique is remarkably robust. 86. Base sequence-dependence of translesion bypass of BPDE-N2-dG adducts by the Y-family bypass human polymerase pol kappa. Xuanwei Huang,1 Yanbin Zhang,2 Zhigang Wang,2 Shantu Amin,3 and Nicholas E. Geacintov.1 1Chemistry Department, New York University, 31 Washington Place, New York, NY 10003, (fax) 212 998 8421, [email protected], 2Graduate Center for Toxicology, University of Kentucky, 3American Health Foundation, Valhalla, NY 10595 Human DNA polymerase κ (pol κ), encoded by the human DinB1 gene, belongs to the Y-family polymerases. In contrast to the classical, replicative polymerases, pol κ readily bypasses (+)-trans- or (-)-trans-anti-B[a]P-N2dG (G*) adducts in DNA. We have investigated the base sequence-dependence of the relative efficiencies and fidelities of bypass in 43-mer oligonucleotide sequences containing the sequence ...XG*Y... (X, Y)G, T, C, A). Our in vitro primer extension studies show that human pol κ catalyzes the translesional bypass with efficiencies and fidelities that are sequence- and adduct stereochemistrydependent. In the case of the (+)-trans-adduct, the primary base incorporated opposite the lesion is C, while in the case of the (-)-trans-adduct, the fidelity of insertion is lower. Overall, when a G flanks the adducts on the 5′-side, the bypass efficiencies are enhanced, while a G on the 3′-side greatly diminishes the bypass efficiencies. Intermediate efficiencies of translesion bypass are observed with X,YdC or T. Base sequence-dependent differences in the Vmax/Km insertion and extension parameters as high as ∼ 10-200 are observed. Comparably minor base sequence effects are observed in unmodified sequences. Supported by NIH grants CA 20851 and CA 76660. 87. Striking conformational changes induced by a 5-MeC groups 5¢ to a benzo[a]pyrene diol epoxideN2-dG adduct in a 5-MeCG* oligonucleotide sequence context. Xuanwei Huang,1 Katharine Colgate,1 Alexander Kolbanovskiy,1 Shantu Amin,2 and Nicholas E. Geacintov.1 1Chemistry Department, New York University, 31 Washington Place, New York, NY 10003, (fax) 212 998 8421, [email protected], [email protected], (2) American Health Foundation It is well known that the reactivities of the mutagenic metabolite of benzo[a]pyrene, the bay region diol epoxide r7,t8-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]-


pyrene (BPDE), with guanine, is enhanced when the cytosine in CpG dinucleotide sequences in DNA is methylated. We have investigated if there is also a change in the conformations of (+)- and (-)-trans-anti-BPDEN2-dG adducts (G*) in double stranded DNA when a methyl group is present at the 5′-flanking cytosine (5-MeC). The conformational changes were studied by optical spectroscopy methods (absorption and fluorescence). While both the (+)-trans- and (-)-trans-adducts are positioned in the minor groove of unmethylated DNA duplexes (CpG* sequence context), the conformation of the (-)-trans-adduct is strikingly different in the analogous methylated duplex (5-MeCpG*). In the latter duplex, the conformation of the (-)-trans-adduct is intercalative, while that of the (+)-trans-adduct is not affected significantly. Such remarkable conformational differences associated with the presence of a single methyl group could affect the clearance of these lesions by nucleotide excision repair enzymes (NER). Such studies have been performed with prokaryotic UvrABC NER proteins, and the efficiencies of excision have been compared with the same BPDE-N2-dG adducts in CpG* and 5-MeCpG* sequence contexts. (Supported by NIH/NCI grants CA20851 and CA76660). 88. Structural studies of DNA adducts derived from benzo[c]phenanthrene diol epoxides within a Y family bypass polymerase. Min Wu,1 Shixiang Yan,1 Nicholas E. Geacintov,1 and Suse Broyde.2 1Department of Chemistry, New York University, New York, NY 10003, [email protected], [email protected], 2Department of Biology, New York University Duplexes containing the fjord region 1S (-)-trans-antibenzo[c]phenanthrene (B[c]Ph)-N6-dA adduct within the human N-ras codon 61 sequence (CA*A) are resistant to repair by the nucleotide excision repair system in human cell free extracts, and the unrepaired adducts may therefore encounter the DNA replication machinery. Experimental observations in vitro suggest that primer extension by replicative DNA polymerases is mostly blocked by bulky polycyclic aromatic hydrocarbon (PAH) DNA adducts, but they may be bypassed by low fidelity Y family DNA polymerases. Unfaithful translesion bypass can then give rise to mutations and the initiation of cancer, which may help to account for the high tumorigenicities of the B[c]Ph diol epoxides. A computational study has been undertaken in order to rationalize the biological effects of the 1S (-)-trans-anti-B[c]Ph-N6dA adduct at the polymerase active site in the N-ras codon 61 sequence context. In the first stage, we investigated the adduct at a primer-template junction site to obtain possible conformations of the adduct. Then, we investigated the results from the first stage within a low fidelity Y family translesion DNA polymerase (Dpo4). We find that the adduct can fit in the Dpo4 DNA polymerase active site opposite an incoming nucleotide, which suggests that this polymerase may bypass the lesion despite the presence of the B[c]Ph bulky ring system. In these studies, we used molecular dynamics simulations with explicit solvent and counterions using AMBER 6.0. 89. Structure of a mismatched C: A base pair flanking in the 5¢ direction of an Aflatoxin B1 N7 cationic Guanine adduct. Indrajit Giri, Department of Chemistry & Center in Molecular Toxicology, Vanderbilt University, Nashville, TN 37235, (fax) 615-343-1234,


[email protected], and Michael P. Stone, Department of Chemistry, Center in Molecular Toxicology, VanderbiltIngram Cancer Center, Vanderbilt University We have examined the structure of d(ACATCAFBGATCT): d(AGATGAATGT) with a mismatched C:A base pair flanking in the 5′ direction of the cationic guanine N7 adduct of aflatoxin B1. The adduct stabilized the duplex, evident from 8 °C rise in melting temperature. The structure of d(ACATCAFBGATCT): d(AGATGAATGT), was refined, restrained by NOE data and dihedral restraints. The adduct stabilized a single conformation of the C: A mismatch, at neutral pH, where both the bases were in the anti conformation. The mismatched cytosine shifted towards the major groove, while the adenine amino group shifted towards the center of the helix. Based on NMR studies, and refined structure, some perturbations were evident at the mismatch site. The structure models the putative intermediate leading to a C to T transition at the 5′ neighbor base pair to a guanine N7 adduct of aflatoxin B1. Supported by NIH Grant CA55678. 90. Bioactivation of ochratoxin A and deoxyguanosine adduct formation. Richard Manderville, Jian Dai, and Gyungse Park. Department of Chemistry, Wake Forest University, Salem Hall, Winston-Salem, NC 27109, (fax) (336)-758-4656, [email protected], [email protected] Ochratoxin A (OTA, 1) is a fungal toxin that has been implicated in human kidney carcinogenesis. The toxin facilitates guanine-specific DNA adduction when metabolically activated. To provide insight into bioactivation of OTA and its subsequent reactivity toward deoxyguanosine (dG), we have examined the ability of the toxin to react with dG, cysteine (CySH) and GSH upon oxidative activation by horseradish peroxidase and rat liver microsomes. Using high-pressure liquid chromatography coupled to electrospray mass spectrometry (LC/MS), CySH- and GSH-conjugates were detected from the reaction of OTA with CySH and GSH under oxidative activation conditions. The same conjugates were obtained from the hydroquinone form (OTHQ), a metabolite intermediate of OTA. Evidence for the formation of dGadducts from OTA and OTHQ were documented by LC/ MS. The role of the reactive CySH- and GSH-conjugates in dG-adduct formation was investigated. The implications of these findings with regard to the carcinogenic properties of OTA will be presented. 91. Differential repair of S-(2-chloroethyl)-glutathione-induced adducts as a basis of the mutation spectrum of the human p53 tumor suppressor gene. J. Gerardo Valadez and F. Peter Guengerich. Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, 646 Robinson Research Building, 23rd and Pierce Avenues, Nashville, TN 37232, (fax) 615-322-31-41, [email protected] More than 200 mutated codons of the p53 gene have been identified in various human cancers and the type of mutations often bear the signature of the etiological agent. We used a double-selection yeast functional assay for identification of mutations in the DNA binding domain-encoded region of the p53 gene after in vivo exposure to S-(2-chloroethyl)-glutathione (GSCH2CH2-

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Cl). The sequence analysis revealed a high-frequency pattern of mutations at codons 152, 177, 244, and 279. An in vivo fluorescence-labeling ligation mediated-PCR assay was developed to establish if those hotspots corresponded to the sites where adducts are produced by GSCH2CH2Cl. We observed a complex pattern of adduced nucleotides, preferentially on G-rich sequences, along p53. Individual GSCH2CH2Cl-adducts disappeared at different rates. In general, the most persistent adducts were located at nucleotides where the mutations occurred most frequently, suggesting that biased DNA repair is an important factor for the mutation spectra. 92. Identification and detection of adducts formed in the reaction of cis-2-butene-1,4-dial with DNA. Michael C. Byrns, Division of Environmental and Occupational Health, University of Minnesota, Minneapolis, MN 55455, and Lisa A. Peterson, Division of Environmental and Occupational Health and Cancer Center, University of Minnesota Furan is a hepatic toxicant and carcinogen in rodents. Its microsomal metabolite, cis-2-butene-1,4-dial, is mutagenic in the Ames assay. Consistent with this observation, cis-2-butene-1,4-dial reacts with 2′-deoxycytidine (dC), 2′-deoxyguanosine (dG) and 2′-deoxyadenosine (dA) to form diastereomeric products. These adducts result from the addition of cis-2-butene-1,4-dial to the exo- and endocyclic nitrogens of each base. The dC adducts are stable, but the dA adducts undergo dehydration to form etheno adducts, and the dG adducts decompose to multiple decomposition products. All three initial nucleoside adducts were detected in cis-2-butene-1,4-dialtreated DNA. These data support the hypothesis that cis2-butene-1,4-dial is an important genotoxic intermediate in furan-induced carcinogenesis [Supported by ES10577]. 93. Identification of 7-[1-hydroxy-1-(3-pyridyl)-but4-yl]deoxyguanosine in reactions of 4-[(acetoxymethyl)nitrosamino]-1-(3-pyridyl)-1-butanol with deoxyguanosine and DNA. Pramod Upadhyaya, Peter W. Villalta, Shana Sturla, Mingyao Wang, and Stephen S. Hecht, University of Minnesota Cancer Center, 425 E. River Rd., Minneapolis, MN 55455, (fax) 612-626-5135, [email protected] 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its major metabolite 4-(methylnitrosamino)-1-(3pyridyl)-1-butanol (NNAL) are potent pulmonary carcinogens in rodents. NNAL is metabolically activated by hydroxylation at its methylene and methyl carbons, yielding methyl DNA adducts and pyridylhydroxybutyl DNA adducts, respectively. In this study we investigated the reaction of 4-[(acetoxymethyl)nitrosamino]-1-(3-pyridyl)-1-butanol (NNAL-CH2-OAc) with deoxyguanosine and DNA in order to obtain more information about the structures of the pyridylhydroxybutyl adducts. In the dG and DNA reactions we identified N2-[1-hydroxy-1-(3pyridyl) but-4-yl]deoxyguanosine and O6-[1-hydroxy-1-(3pyridyl)but-4-yl]deoxyguanosine. HPLC-MS analysis of the dG reaction resulted in detection of an adduct with m/z 417 (M+1 of pyridylhydroxylbutyl-dG). Neutral thermal or acid hydrolysis of the dG reaction mixture caused disappearance of m/z 417 and an increase in 4-hydroxy-1-(3-pyridyl)-1-butanol as well as appearance of a peak with m/z 301. This peak was identified as 7-[1-


hydroxy-1-(3-pyridyl)but-4-yl]guanine by comparison to a synthetic standard. Similar results were obtained on neutral thermal or acid hydrolysis of DNA. 94. Influence of the (S,S)(61,2) N6-adenyl butadiene adduct in an oligodeoxynucleotide containing the N-ras 61 codon. Tandace A. Scholdberg, Lubomir N. Nechev, Constance M. Harris, Thomas M. Harris, and Michael P. Stone. Department of Chemistry, Vanderbilt University, Nashville, TN 37235, [email protected] Butadiene is used in the manufacture of synthetic rubber and plastics. Chronic occupational exposure during the manufacture of these products is a potential health concern since butadiene is mutagenic in bacterial and mammalian cells, and implicated as a potential carcinogen in humans. Of interest in particular are adducts formed at the exocyclic amino goup N6 of adenine. NMR combined with molecular dynamics studies of the solution structure of the (S,S)-butadiene adduct positioned opposite dT in the sequence context: d(C1G2-G3-A4-C5-A6-A7-G8-A9-A10-G11)- d(C12-T13-T14C15-T16-T17-G18-T19-C20-C21-G22) are presented here. The exchangeable and nonexchangeable protons of the butadiene moiety and nucleic acids were assigned following analysis of two-dimensional NMR data sets. The experimentally determined structure was compared to NOE intensity data using complete relaxation matrix back-calculations, yielding an Rx value of 1.15 × 10-2. The butadiene moiety was oriented in the major groove and caused localized structural perturbation. This work was supported by NIH grant ES05509. 95. Positive DNA supercoiling, DNA structure, and base-flipping enzymes. Marita C. Barth,1 Yelena Margolin,1 Viengsai Vongchampa,1 Andrzej Stasiak,2 and Peter C. Dedon.1 1Biological Engineering Division, MIT, 77 Massachusetts Ave, 56-786, Cambridge, MA 02139, (fax) 617-258-0225, [email protected], (2) Laboratoire d’Analyse Ultrastructurale, Universite de Lausanne DNA in human cells is subjected to numerous mechanical forces, such as the torque applied by polymerases. The twisting force generates negative and positive DNA supercoiling ahead of and behind the enzyme, respectively. We now present biochemical studies of the effects of positive supercoiling on DNA structure and proteinDNA interactions. Electron microscopy of a positively supercoiled plasmid revealed a plectonemic conformation with tightly interwound helices similar to negative supercoiling. However, positively supercoiled DNA was more reactive with chemical probes of DNA structure than relaxed DNA. We also present the results of hydrogen-tritium exchange experiments to investigate base-pairing effects of positive supercoiling. Our results are consistent with an increase in solvent exposure of bases as a result of helical over-winding. Interestingly, we observed that positive supercoiling affected the activity of the base-flipping EcoRI methyltransferase, which suggests that helical over-winding may affect the activity of other base-flipping enzymes such as DNA glycosylases. 96. Reaction of O6-alkylguanine-DNA alkyltransferase variants with O6-[4-oxo-4-(3-pyridyl)butyl]guanine in oligodeoxyribonucleotides. Nicole M. Thomson,1 Anthony E. Pegg,2 and Lisa A. Peterson.1 1-


Division of Environmental and Occupational Health and Cancer Center, University of Minnesota, Box 806 MAYO, 420 Delaware St SE, Minneapolis, MN 55455, (fax) 612626-5135, [email protected], 2Pennsylvania State University College of Medicine 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a potent pulmonary carcinogen and tobacco-specific nitrosamine, both methylates and pyridyloxobutylates DNA. Both reaction pathways generate promutagenic O6alkylguanine adducts. These adducts, O6-methylguanine (O6-mG) and O6-[4-oxo-4-(3-pyridyl)butyl]guanine (O6pobG), are repaired by O6-alkylguanine-DNA alkyltransferase (AGT). Modifications within the sequence of AGT can alter the ability of this protein to repair bulky O6alkylguanine adducts relative to O6-mG. Several AGT variants were studied for repair preference when presented with equal molar amounts of oligonucleotides containing site-specifically incorporated O6-mG or O6pobG adducts. Mouse and rat AGT showed no preference between these two adducts. Wild-type human AGT and two variants, I143V and N157H, preferentially repaired O6-mG but also repaired O6-pobG to a significant extent. Human variants E166G and G160R weakly repaired O6pobG whereas variants Y158H and P140K did not repair this adduct. The importance of these preferences for repair of O6-mG versus O6-pobG may translate into differences in sensitivity to the mutagenic effects of NNK or other pyridyloxobutylating nitrosamines [Supported by CA-59887 and CA-18137]. 97. Reactions of formaldehyde plus acetaldehyde with deoxyguanosine and DNA. Mingyao Wang, Yongli Shi, Guang Cheng, Peter W. Villalta, Shana J. Sturla, John R. Jalas, Edward J. McIntee, and Stephen S. Hecht. University of Minnesota Cancer Center, 425 E. River Rd., Minneapolis, MN 55455, (fax) 612-626-5135, [email protected] Formaldehyde and acetaldehyde commonly occur at relatively high concentrations in the human environment. We hypothesized that they could react with deoxyguanosine (dG) or DNA to produce the exocyclic 1,N2propanodeoxyguanosine adduct of acrolein (1), which has been observed in human DNA and certain in vitro systems. We therefore examined reactions of formaldehyde and acetaldehyde (0.01-50 mM of each) with dG and DNA.

Adduct 1 was detected in the dG reactions, however, a more prevalent product that results from the reaction of both aldehydes with dG and DNA was observed. This material was identified by UV, MS, and 1H-NMR as stereoisomers of 2, R1dH; R2dCH3. A similar product, R1dR2dCH3, was identified in the reaction of acetaldehyde with dG. Products of formaldehyde or acetaldehyde reactions with dG and DNA were also detected. These results indicate that exocyclic adducts can be formed by tandem reactions of formaldehyde and acetaldehyde with dG and DNA.


98. Endogenous nitrosation chemistry in carcinogenesis: An ab initio theoretical study of decomposition reactions of cyclic r-hydroxynitrosamines. Rainer Glaser, Hong Wu, and Richard Loeppky, Department of Chemistry, University of Missouri-Columbia, Columbia, MO 65211, (fax) 573 882 2754, [email protected], [email protected] We are discussing the nitrosation chemistry of three cyclic imines: 1 (X)CH2), 2 (X)NH), and 3 (X)O). These imines occur in a number of drugs and it is our postulate that endogenous nitrosation might convert these imines into potent carcinogens. The imines 1 - 3 are converted to the R-hydroxynitrosamines 7 - 9 via the N-nitrosoiminium ions 4 - 6. The R-hydroxy-N-nitrosamines 7 - 9 may undergo retro-ene reaction to the δ-oxoalkyl diazotic acids 10 - 12. For 8 and 9 we also explored the water-assisted E2 eliminations via 6-membered ring transition state structures to 15 and 16. These reaction channels were explored at the B3LYP/6-31G** level of theory and the results will be discussed with an emphasis on the comparison of the kinetics and thermodynamics of C-N versus C-X cleavage.

99. Endogenous nitrosation: Potentially toxic nitrosamino lactones from dietary amino acids and aldehydes. Richard N. Loeppky, Hongbin Yu, and Erqing Tang, Department of Chemistry, University of Missouri, 125 Chemistry Building, Columbia, MO 65211, (fax) 573-882-2754, [email protected], [email protected] Endogenous nitrosation reactions are known to occur in humans. Unknown nitroso compounds have also been found in human gastric juice. To test our hypothesis that gastric nitrosation of amino acids in the presence of common dietary aldehydes can lead to potentially carcinogenic N-nitroso-1,3-oazolidin-5-ones 3, we have synthesized a number of these nitrosamino lactones and examined their properties, as well as their formation under acidic nitrosation conditions from 1 and 2. For example, we have recently synthesized 3 (RdH or PhCH2, R2dCHOHCH2OH from the acetonide of glyceraldehydes and have chromatographic evidence for its formation from acidic nitrosation under simulated gastric conditions. Detailed kinetic studies demonstrate that the nitrosamino lactones are relative stable, hydrolyzing more rapidly as the pH increases above 7 to products derived from diazonium ions. We have also shown that chymotrypsin and porcine pancreatic lipase can catalyze the hydrolysis of several substrates indicating their possible transport and “activation” at other tissue sites.

100. Probing the mechanism of peroxynitriteinduced oxidation of guanine and 8-oxoguanine.

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Jacquin C. Niles, Lata M. Venkatarangan, John S. Wishnok, and Steven R. Tannenbaum. Biological Engineering Division Massachusetts Institute of Technology, 77 Massachusetts Avenue, 56-731, Cambridge, MA 02139, (fax) 617-252-1787, [email protected] Peroxynitrite, a potent oxidizing and nitrating agent, is formed via the diffusion-limited reaction of nitric oxide with superoxide, and is proposed to react with various biological compounds including proteins, lipids and DNA. We have been studying the latter reaction in order to better understand how peroxynitrite causes mutations and cellular toxicity. Oxidation of guanine (Eo)1.29 V) is thermodynamically favored, since this base has the lowest redox potential in DNA. Furthermore, 8-oxoguanine (Eo)0.58 V), which results from guanine oxidation and is present in genomic DNA at detectable levels is much more readily oxidized than its parent. In pursuing our objective, we have characterized the peroxynitriteinduced guanine and 8 oxoguanine products, and have probed the mechanism of their formation using various isotopically labeled substrates and intermediate trapping agents. Our studies indicate involvement of the radical and/or an electrophilic intermediate in the oxidation of both compounds. 101. Quantification of peroxynitrite induced guanine nucleobase damage using high-performance liquid chromatography-tandem mass spectrometry. L. M. Venkatarangan, J. C. Niles, J. S. Wishnok, and S. R. Tannenbaum. Division of Biological Engineering, MIT, 56-731, 77 Massachusetts avenue, Cambridge, MA 02139, [email protected] Peroxynitrite (ONOO-) reacts with the guanine nucleobase resulting in the formation of a wide array of primary and secondary oxidation and nitration products, many of which have been shown to act as mutagenic lesions. In an effort to directly detect some of these lesions in isolated and cellular DNA, we have developed a highly sensitive HPLC-tandem mass spectrometry method. In the present work, we studied the in vitro formation of 8-oxdG, oxazolone and 5-guanidino-4-nitroimidazole arising from the treatment of calf thymus DNA with ONOO-. First, stable, isotope-labeled standards were synthesized for the lesions of interest. Calf thymus DNA was treated with varying amounts of ONOO-, spiked with a known amount of the labeled standard of the lesion under study and finally digested down to the nucleoside level with a cocktail of enzymes. The amount of each lesion formed was then determined by selective reaction monitoring using LC-MS/MS. 102. Identification of nitrated proteins by a proteomics-based approach. George N. Nikov, Vadiraja Bhat, John S Wishnok, and Steven R. Tannenbaum. Biological Engineering Division, MIT, 77 Massachusetts Ave, 56-738, Cambridge, MA 02139, [email protected] Tyrosine nitration is a well-established protein modification occurring in vivo in various diseases states associated with oxidative stress and increased activity of NO synthases. Nitration of specific tyrosine residues has been reported to affect protein structure and function, suggesting that 3-nitrotyrosine formation may not only be a disease marker but could be involved in the pathogenesis of certain diseases. We describe a new


proteomics based approach for the characterization of nitrated proteins. Our method combines specific isolation of nitrated proteins from complex protein mixtures and mass spectrometric determination of the amino acid sequence, and the site of nitration of individual proteins. The isolation of nitrotyrosine containing peptides/proteins is achieved via specific chemical reactions that result in covalent attachment of purification tags and affinity chromatography. Using this strategy we compared the nitration of proteins in SJL mice and SJL mice bearing RCSX carcinoma. 103. Reduction of the nitro functionality in the benzazolo[3,2-A]quinolinium salts (NBQ's) is an important step for DNA binding. Iris Gisela ColonFerran,1 Marie Santiago-Quiles,2 Osvaldo Cox,3 Diogenes Herreno-Saenz,2 Fernando A. Gonzalez,1 and Antonio Alegria.4 1Department of Chemistry, University of Puerto Rico, Rio Piedras Campus, San Juan, Puerto Rico, PR 00931, PR, 2University of Puerto Rico-Medical Science Campus, 3Metropolitan University, 4University of Puerto Rico-Humacao Campus Interest in DNA binding drugs has increased in recent years due to their importance in the treatment of genomerelated diseases, like cancer. We are currently studying a new family of DNA-binding compounds, the benzazolo [3,2-R] quinolinium salts (NBQ’s). Our research efforts are aimed at NBQ compounds incorporating the nitro (-NO2) functionality because they may be able to interact with the DNA molecule upon reduction of the nitro group. This property makes these compounds potential antitumor agents under hypoxic (low oxygen) conditions, which characterize solid tumors but not normal cells. Here we present experimental data for various NBQ’s, which confirms the importance of the nitro functionality for the covalent binding of these drugs to DNA. The amount of NBQ compound bound to the DNA was measured with dialysis experiments performed under hypoxic conditions in the presence and absence of reducing agents. Under these conditions, the 3-nitro-10-methylbenzazolo [3,2-R] quinolinium salt (NBQ-91) showed an increase in covalent binding to the DNA, which increased with reducing agent (xanthine/xanthine oxidase) when compared to the another NBQ derivative without nitro substitution. 32P-post labeling analysis provided further evidence for the formation of the DNA3-nitro-10-methylbenzazolo [3,2-R] quinolinium salt (NBQ91) adduct. Our results demonstrate that these new nitrocontaining compounds could be promising candidates for the chemical treatment of solid tumors. Future experiments will include cytotoxicity determinations in cancer cells to determine if these compounds are possible effective anti-tumor agents under hypoxic conditions. 104. Identification of the cysteine residues of tubulin oxidized by peroxynitrite. Lisa M. Landino, Alex Chen, Erin Carson, and Elizabeth Doyal, Department of Chemistry, The College of William and Mary, P.O. Box 8795, Williamsburg, VA 23187-8795, (fax) 757221-2715, [email protected] Recently we reported that treatment of bovine brain microtubule protein, composed of tubulin and microtubule-associated proteins, with peroxynitrite led to a dosedependent inhibition of microtubule polymerization. The extent of cysteine oxidation, rather than tyrosine nitra-


tion or other types of peroxynitrite-induced damage, correlated well with the observed inhibition of polymerization. Thus, our current efforts have been directed at identifying the specific cysteines of tubulin and the major microtubule-associated proteins, MAP2 and tau, that are oxidized by peroxynitrite. We have developed a doublelabeling protocol in which thiol-specific fluorescent tags are incorporated into control and peroxynitrite-treated protein samples. Because peroxynitrite-induced disulfides in tubulin, rather than MAP2 or tau, correlate with inhibition of polymerization, we will present the results of our thiol labeling and peptide mapping work performed with purified tubulin. 105. Interfaces for direct analysis of isotopes by accelerator mass spectrometry. Paul L Skipper,1 Barbara J. Hughey,2 Rosa G. Liberman,1 Man Ho Choi,1 John S. Wishnok,1 Robert E. Klinkowstein,2 Ruth E. Shefer,2 Steven R. Tannenbaum,1 Shawn P. Harriman,3 and Chandra Prakash.3 1Biological Engineering Division, Massachusetts Institute of Technology, Building 56, Room 753, 77 Massachusetts Ave., Cambridge, MA 02139, (fax) 617 252 1787, [email protected], 2Newton Scientific, Inc, 3Pfizer, Inc. Accelerator Mass Spectrometry (AMS) is a proven technique for detection of 14C and 3H in a wide variety of tracer applications, but its widespread use has been restricted by instrument cost and limited methods for sample introduction. Instrument cost is being addressed with specialized designs that reduce size by limiting the detection range to low-mass isotopes. The compact instrument used in this work has already been described in detail; in this presentation, we will describe development efforts related to facilitating efficient transfer of samples from complex matrices into the AMS instrument for isotope analysis. A laser-induced sample combustion interface is used for production of CO2 for detection of 14 C. This interface is designed for continuous production and analysis of CO2, so that a sample such as the effluent stream from a chromatographic or electrophoretic separation can be analyzed without fractionation, as current practice requires. A direct injection pyrolytic interface is used for production of H2 for analysis of 3H. This interface is designed for nanoliter/min volumetric flows and operates without desolvation. Studies with both 14C and 3Hacetaminophen under conditions relevant to biomedical applications will be reported. 106. Quantifying DNA base deamination products caused by exposure to nitric oxide. Min Dong, Biological Engineering Division, Massachusetts Institute of Technology, 77 Massachusetts Ave, bd:56-rm786, Cambridge, MA 02139, (fax) 617-258-0225, [email protected], and Peter C. Dedon, Biological Engineering Division, MIT Nitric oxide, via derivative ONOO- and N2O3, causes DNA damage by oxidation and deamination, respectively. However, little is known about the relative quantities of the various products. We have undertaken studies to define the spectrum of lesions associated with NO exposure. First, we identified two sets of DNA glycosylases that differentially recognize N2O3- and ONOOinduced lesions. Using a recently developed NO delivery system that mimics biological exposure, we found that AlkA and uracil glycosylase recognize only NO-induced


DNA damage while Fpg reacts selectively with ONOOinduced lesions. These enzymes were used to map the different lesions in human genes. Second, we developed a sensitive (

Abstracts, American Chemical Society Division of Chemical

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