Abstracts, American Chemical Society Division of Chemical

Chem. Res. Toxicol. 2005, 18, 1967-1991

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Abstracts, American Chemical Society Division of Chemical Toxicology, 230th ACS National Meeting, Washington, DC, August 28-September 1, 2005 Trevor M. Penning*,† Department of Pharmacology, University of Pennsylvania School of Medicine, 3620 Hamilton Walk, 130C John Morgan Building, Philadelphia, Pennsylvania 19104-6084 Received October 19, 2005

1. Biological Responses to DNA Damage: A Burgeoning Field. Errol C. Friedberg. Department of Pathology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, Texas 75390-9072. Fax: 214-648-4067. E-mail: friedberg.errol@ pathology.swmed.edu. What was once a defined field of scientific investigation called DNA repair is now a subset of a large field more appropriately called biological responses to DNA damage. This burgeoning field now embraces multiple mechanisms for the repair and tolerance of DNA damage. Additionally, new inroads are being made into transcriptional regulation in response to DNA damage, posttranslational protein modification, notably monoubiquitination and SUMOylation, cell cycle checkpoint activation, and apoptosis in response to DNA damage. My talk will summarize these areas of active investigation. 2. Processing of Oxidative DNA Base Damage. Sylvie Doublie´, Viswanath Bandaru, and Susan S. Wallace. Department of Microbiology and Molecular Genetics, University of Vermont, The Markey Center for Molecular Genetics, Stafford Hall, Burlington, Vermont 05405. Fax: 802-656-8749. E-mail: [email protected]. Lesions produced by highly reactive free radicals are removed via base excision repair (BER). The first step of the BER pathway is catalyzed by DNA glycosylases, which hydrolyze the N-glycosylic bond between a damaged base and its deoxyribose, leaving an apurinic/ apyrimidinic site in DNA. In prokaryotes, two DNA glycosylases recognize and excise oxidized pyrimidines: endonuclease VIII (Nei) and endonuclease III (Nth). NEIL1 is a human DNA repair enzyme, which is orthologous to Escherichia coli Nei. The structure of human NEIL1 revealed that it harbors a novel motif that mimics the antiparallel β-hairpin zinc finger found in the other members of the Nei family, despite the absence of zincbinding residues. Our aim is to delineate the structural features of the DNA glycosylases involved in recognition of DNA base damages produced by ionizing radiation. Results of these structural studies involving NEIL1 and other BER DNA glycosylases will be presented. 3. Mismatch Repair Proteins in Repair and DNA Damage Response. Karin Drotschmann. Cancer Biology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, North Carolina 27157. Fax: 336-716-0255. E-mail: [email protected]. The mismatch repair protein MutS and its eukaryotic homologues interact with DNA adducts and initiate cell * To whom correspondence should be addressed. Tel: 215-898-9445. Fax: 215-898-7180. E-mail: [email protected]. † Program Chair.

death. This function has significant implications for the success of chemotherapy. Altered MutS-DNA interactions may distinguish cell death from repair. Molecular dynamics simulations are useful in approximating the structure of MutS with damaged DNA and determining alterations in protein-DNA contacts. DNA flexibility, as one of the key factors in repair, can be significantly altered in the MutS-damaged DNA complex. Variations in this flexibility, together with altered protein-DNA interactions, provoke distinct conformational changes throughout the protein. These affect residues in functionally and structurally important parts of the MutS protein and may contribute to changes in protein-protein interactions. Overall, conformational changes induced by the recognition of DNA damage by MutS, as opposed to mismatched DNA, provide a compelling model for the contribution of mismatch repair proteins to cell death rather than repair. 4. The Structure/Function of TFIIH Explains the XP Disorders. Jean-Marc Egly. Institut de Geńe´tique et de Biologie Molećulaire et Cellulaire, 1 rue Laurent Fries, BP 10142, 67404 Illkirch Cedex, France. Fax: 33(0)3 88 65 32 01. E-mail: [email protected]. To understand the functional involvement of TFIIH in RNA pol II transcription as well as in DNA repair, we have carried out investigations on its structure using electronmicroscopy, X-ray, or NMR. A better structure/ function analysis would help us to explain some of the phenotypes of xeroderma pigmentosum and trichothiodystrophy patients, the disorder of which is originated by mutations in the XPB, XPD, and p8 subunits of TFIIH. After we characterized the molecular structure of the human TFIIH, we focused on several TFIIH subunits: We are now in the position to explain why the XPD mutations not only prevent some steps of NER but also prevent transcription activation. Similarly, we show why the p8 subunit allows TFIIH to operate in NER. 5. How Does it Cut? Insights into the Incision Reactions During Nucleotide Excision Repair. Caroline Kisker,1 James J. Truglio,1 Benjamin Rhau,1 Erkan Karakas,1 Deborah L. Croteau,2 and Bennett Van Houten.2 (1) Department of Pharmacology, SUNY Stony Brook, Stony Brook, New York 11795-5115. Fax: 631 632 1555. E-mail: [email protected]. (2) Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, NIH. Nucleotide excision repair is a highly conserved DNA repair mechanism present in all kingdoms of life. After the damage has been recognized, damage removal is achieved by two incision reactions 3′ and 5′ to the lesion. The incision reaction is a critical step for damage removal

10.1021/tx0502894 CCC: $30.25 © 2005 American Chemical Society Published on Web 11/12/2005

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and is accomplished by the UvrC protein in eubacteria. No structural information is so far available for the 3′ or 5′ incision reaction. The structure of the N-terminal catalytic domain of UvrC catalyzes the 3′ incision reaction and shares homology with the catalytic domain of the GIY-YIG family of intron-encoded homing endonucleases. The structure reveals a patch of highly conserved residues surrounding a catalytic magnesium-water cluster, suggesting that the metal binding site is an essential feature of UvrC. Structural and biochemical data strongly suggest that the N-terminal endonuclease domain of UvrC utilizes a novel one-metal mechanism to cleave the phosphodiester bond. 6. Damage Specificity and Conformational Controls in the DNA Repair, Replication, and Recombination Interface. John A. Tainer. Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, MB4, San Diego, California 92037. Fax: 858-784-2289. E-mail: [email protected]. We aim to understand how machines for genome integrity are coordinated by reversible, multiprotein complexes. We find that these reversible complexes often form composite interfaces from preformed and unstructured regions. Such reversible interfaces provide strong, specific contacts from the combination of relatively weak, modular interactions. These modular interaction sites, which allow protein exchanges and pathway progression, also provide targets for new therapeutic strategies, as small molecular ligands may rationally block such individually weak interfaces. To achieve accurate structural information on these biologically relevant complexes, we have designed and developed the Structurally Integrated Biology for Life Sciences (SIBYLS) beamline at the Advanced Light Source (ALS). This provides a unique resource for X-ray diffraction characterizations of both static and dynamic solution states of macromolecular machines. Results will be presented from this SIBYLS beamline and that focus upon the molecular machines acting at the interfaces for the replication-repair and recombination-repair pathways. 7. Translesion DNA Synthesis. Wei Yang. Laboratory of Molecular Biology, National Institutes of Health, 9000 Rockville Pike, Building 5, Room B1-03, Bethesda, Maryland 20892. Fax: 301-496-0201. E-mail: wei.yang@ nih.gov. Y-family DNA polymerases are specialized to synthesize DNA bypassing a variety of lesions. Although sharing no detectable amino acid sequence homology with other replicative and repair polymerases, crystal structures of archaeal and eukaryotic Y-family polymerases reveal a catalytic core consisting of conserved “palm”, “finger”, and “thumb” domains found in all DNA and RNA polymerases. To elucidate the mechanism for translesion synthesis by a Y-family polymerase, we have determined the crystal structure of Dpo4, an archaeal Y-family polymerase, complexed with DNA substrate that contains a polyaromatic hydrocarbon modified base, UVcross-linked pyrimidine, or abasic lesion in the template strand. The preformed and open active site of Dpo4, as revealed by earlier studies, appears to be able to accommodate bulky lesions and also to induce unconventional conformations of DNA substrate to achieve hydrogen bonding between damaged template base and incoming nucleotide. We have further investigated the structural and kinetic properties of misincorporation and extension of a mismatched base pair by Dpo4. New insights in

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lesion selection and fidelity of this Y-family polymerase will be presented. 8. Introduction: Replication of Undamaged DNA by Replicative Polymerases. Thomas E. Spratt. Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Room C5711B Mail Code H171, 500 University Drive, Hershey, Pennsylvania 17033. Fax: 717-531-7072. E-mail: [email protected]. Replication of carcinogen-damaged DNA is the decisive step in mutagenesis. Replicative polymerases are designed to recognize base pairs with the Watson-Crick topography. However, carcinogen-damaged DNA cannot take on Watson-Crick topography, and consequently, the polymerases must use other properties of the carcinogendamaged base pairs to control fidelity of replication. The topic of this symposium is the mechanisms by which the replicative and bypass polymerases control the fidelity of dNTP insertion opposite and extension past damaged DNA. This introductory seminar will review the mechanisms by which replicative polymerases control fidelity of replication. Specific examples with Escherichia coli DNA polymerase I will be used to illustrate how interactions between the polymerase and the minor groove of the DNA can select for Watson-Crick geometry of the DNA. 9. Observation of DNA Replication Past Damaged DNA by X-ray Crystallography. Lorena S. Beese. Department of Biochemistry, Duke University, Nanaline Duke Building, Room 212, Research Drive, Durham, North Carolina 27705. Fax: 919-684-8885. E-mail: lsb@ biochem.duke.edu. Crystals of a high fidelity polymerase from a thermostable strain of Bacillus stearothermophilus are capable of several rounds of DNA replication. We have examined the replication of mispairs and the accurate and mutagenic synthesis past 8-oxoguanine (8oxoG), N2-(2′deoxyguanosin-8-yl)aminofluorene (G-AF), N2-(2′-deoxyguanosin-8-yl)acetylaminofluorene (G-AAF), and the benzo[a]pyrene diol epoxide-derived N2-guanine adduct (BP-dG). These studies reveal critical polymerase-nucleotide interactions that affect correct vs error-prone replication as well as interactions that inhibit DNA replication 10. Mechanism of DNA Lesion Bypass Catalyzed by a Y-Family DNA Polymerase. Zucai Suo. Department of Biochemistry, The Ohio State University, 484 West 12th Avenue, 749A Biological Sciences, Columbus, Ohio 43210. Fax: 614-292-6773. E-mail: [email protected]. Both endogenous and exogenous agents continually damage cellular DNA and generate a variety of lesions. Unrepaired lesions block replicative polymerases but can be bypassed by members of the novel Y-family polymerases. These Y-family polymerases lack intrinsic proofreading exonuclease activities and catalyze DNA synthesis with both a high error rate and a low processivity. To kinetically understand lesion bypass processes, we have employed presteady state kinetic methods to decipher the detailed mechanisms of incorporations of correct and incorrect nucleotides opposite both undamaged and damaged DNA templates catalyzed by Dpo4, a model Y-family polymerase from Sulfolobus solfataricus strain P2. We have determined the kinetic effects of both an abasic site and a cisplatin-GpG adduct on nucleotide incorporation. Dpo4 is found to pause strongly at these lesion sites where its fidelity decreases significantly. Our results provide a kinetic picture of lesion bypass by a Y-family DNA polymerase.

Abstracts, ACS Division of Chemical Toxicology

11. Are Abasic Lesions Noninstructive? Marc M. Greenberg. Department of Chemistry, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218. Fax: 410-516-7044. E-mail: mgreenberg@ jhu.edu. Abasic lesions (AP) are produced in DNA as a result of endogenous and exogenous agents. Oxidized abasic lesions (e.g., L, C4-AP, C2-AP) are also produced as a consequence of oxidative stress. AP sites are often referred to as noninstructive lesions because they are incapable of forming Watson-Crick hydrogen bonds. However, recent in vitro and in vivo studies on oxidized abasic lesions have revealed that each type of DNA damage has distinctive effects on DNA replication and/ or repair. Our group’s research in this area will be presented.

12. Influence of Base Pair Geometry, H-Bonding, and Base Stacking on Nucleotide Insertion Opposite Pyrimidine Dimers by Yeast Pol η. J. S. Taylor, Hanshin Hwang, and Hussam M. Bdour. Department of Chemistry, Washington University, St. Louis, Missouri 63130-4899. E-mail: [email protected]. How replicative and DNA damage polymerases select nucleotides for insertion opposite normal and damaged bases is important to the understanding of mutagenesis. We recently discovered that the hydrophobic non-Hbonding pyrene nucleotide is preferentially inserted opposite an abasic site, the 3′-T of a thymine dimer, and most undamaged bases by yeast DNA polymerase η (pol η). This preference can be ascribed to the superior base stacking ability of pyrene and the loose grip of pol η on the templating base. We also find that the insertion efficiencies of base-modified nucleotides opposite normal T and a thymine dimer by pol η depend on their ability to form H-bonded Watson-Crick base pairs. Interestingly, the relative insertion efficiencies of the basemodified nucleotides capable of forming Watson-Crick base pairs are fairly similar opposite normal nucleotides for both pol η and the Klenow fragment and parallel their H-bonding ability. Mismatches are much less strongly discriminated against by pol η, however, in accord with a more open and less shape selective active site. 13. Bypass of Bulky DNA Lesions by Thermophilic Y-Family and Other Polymerases. N. E. Geacintov. Chemistry Department, New York University, 31 Washington Place, New York, New York 10003-5180. Fax: 212-998-8421. E-mail: [email protected]. The ability of Y-family polymerases to bypass bulky DNA lesions is well-established, but the efficiency of the bypass and the nucleotide inserted opposite the lesion often depend on the structure of the DNA adduct and the local base sequence context. Mutation hot spots observed in vivo can arise because of the impact of base sequence effects and adduct structure on translesion bypass, but it is not yet clear if there is a correlation between mutation hot spots and cold spots of nucleotide excision repair. Primer extension experiments in vitro using purified polymerases and site specifically modified oligonucleotides with defined base sequence contexts are model systems that can provide insights into the mechanisms of error-free or error-prone bypass of different

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lesions in the absence of DNA repair. Our recent efforts have been directed toward gaining a better understanding of these factors using selected purified Y-family and A-family polymerases and DNA adducts of known characteristics incorporated into oligonucleotides with defined and variable sequence contexts. Some of these polymerases are thermophilic that allow for studies of temperature dependence effects that may be traceable to local conformational motions at the polymerase active sites. The results are, whenever feasible, compared with the insights derived from molecular modeling with molecular dynamics simulations to obtain a deeper understanding of adduct structure-function relationships and local bases sequence effects. 14. Elucidating Structure-Function Relationships Through Modeling of DNA Bulky Adducts in Replicative and Bypass Polymerases. Suse Broyde. Department of Biology, New York University, 100 Washington Square East, New York, New York 10003. Fax: 212-995-4015. E-mail: [email protected]. We employ molecular modeling with molecular dynamics simulations to gain understanding of the treatment of bulky lesions by DNA polymerases. These types of covalent DNA adducts primarily block replicative polymerases, but a low level of mutagenic bypass is observed. Blockage is now understood to lead to polymerase switch to error-prone bypass polymerases for translesion synthesis. Our goal is to delineate on a dynamic, molecular level the structural features of the adducts and polymerase active site regions that interpret experimental data and make predictions. The dynamics trajectories produce ensembles of thousands of structures through time, which are analyzed in detail and compared with unmodified control simulations. These studies for a number of systems have begun to define the interplay in dynamic structural features that can produce mutagenic lesion bypass. 15. Withdrawn. 16. Role of Contemporary Drug Metabolism in Drug Discovery. Thomas A. Baillie. Department of Drug Metabolism, Merck Research Laboratories, WP75A303, West Point, Pennsylvania 19486. Fax: 215-6529427. E-mail: [email protected]. The past decade has witnessed a transformation in the use of drug metabolism and pharmacokinetic (DMPK) data to support the discovery and development of novel therapeutic agents. In contrast to the traditional role of such information in qualifying candidates already selected for development, the DMPK characteristics of new chemical entities (NCEs) play a key role, together with evaluations of potency and selectivity against the biological target, in the selection process itself. Criteria such as appropriate PK of the NCE in animal species, interactions with drug metabolizing enzymes and transporters, plasma protein binding, identities and pharmacological activities of mammalian metabolites, and formation of chemically reactive, potentially toxic metabolites all play a role in the choice of candidates for development. This presentation will highlight Merck’s integrated approach to drug discovery, in which chemical, pharmacological, and DMPK data all play a key role in the early development process. 17. Assessing Bioactivation in Lead Optimization Stages of Drug Discovery. Cyrus Khojasteh. Drug Metabolism and Pharmacokinetics, Genentech, Inc.,

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South San Francisco, California 94080. E-mail: [email protected]. The body is armed with drug metabolism enzymes, many of which are strategically located in the liver. These enzymes are capable of functionalizing, unmasking, or conjugating the drugs and thereby enhance their ability to be removed. This gating mechanism works in most situations, but in some cases, the body may convert an inert compound to a toxic one. This has been studied in metabolism by cytochrome P450, where oxidation of certain moieties has led to the formation of reactive electrophiles. These electrophiles can subsequently form protein adducts that eventually may lead to toxicity. There are major ongoing efforts in the pharmaceutical industries to evaluate the formation of protein adducts at an early stage. Drug-protein adduction is assessed by using in vivo and in vitro radiolabel studies and also glutathione trapping experiments using liver microsomes or hepatocytes. If these assays are used judiciously, it is thought to lead to safer drugs. In this presentation, new methods to assess the formation of electrophiles under enzymatic and nonenzymatic conditions will be discussed. 18. Utility of Metabolite Identification Studies in Minimizing ADME Liabilities and Safety Concerns. Amit S. Kalgutkar. Pharmacokinetics, Dynamics, and Metabolism, Pfizer Global Research and Development, Eastern Point Road, Groton, Connecticut 06340. Fax: 860-686-1059. E-mail: [email protected]. Characterization of the biotransformation pathways of NCEs is an integral part of drug discovery not only in optimizing ADME properties but also in eliminating potential safety concerns. In several instances, biotransformation studies in early discovery have been used to identify metabolic soft spots that render high metabolic instability. The availability of such information has aided in the rational design of compounds with increased resistance to metabolism and overall improvements in oral bioavailability and -activity. Furthermore, there are circumstances wherein the presence of structural alerts in otherwise attractive NCEs creates uncertainty around the potential of the drug candidate to cause ADRs. Metabolism studies have proved particularly invaluable in this arena. For instance, characterization of stable conjugates derived from bioactivation of NCEs provides indirect information on the structure of the electrophilic species, thereby providing insight into the bioactivation mechanism and hence a rationale on which to base subsequent chemical intervention strategies. Literature and in-house examples will be used to illustrate these principles. 19. Drug Metabolism and Drug Toxicities. Gerald Miwa. Department of Drug Metabolism and Pharmacokinetics, Millennium Pharmaceuticals, Cambridge, Massachusetts 02139. E-mail: [email protected]. The metabolites of drug molecules may possess both pharmacological and toxicological potential. Consequently, the DMPK functions in pharmaceutical companies often work to meet regulatory agency expectations on characterization of metabolites. To minimize drug safety risks to humans, preclinical toxicology studies in animals precede comparable dosing regimens in human subjects. Frequently, species specific toxicities are observed and their relevance to human subjects becomes a critical issue for the initiation or continuation of studies in the clinic. Drug metabolism can play a causal role in species specific drug-induced toxicities. When this can be

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deduced in a timely manner, it can have profound influence on the successful development of a new drug and the regulatory agency perspective of the relevance of nonclinical studies to human safety. Two examples of the role of drug metabolism in drug toxicities will be reviewed, and the implications of the research on drug metabolism to the manifestation of toxicities, the successful definition of human safety, and in redefining a guidance from a regulatory agency will be discussed. 20. Activation of (()-Benzo[a]pyrene-7,8-diol by AKR1A1 Elevates anti-BPDE Generation in Human Bronchoalveolar H358 Cells: Functional Induction of CYP1B1 by Benzo[a]pyrene-7,8-dione. Hao Jiang,1 Daljit Vudathala,2 Ian A. Blair,2 and Trevor M. Penning.1 (1) Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104-6084. E-mail: haojiang@ mail.med.upenn.edu. (2) Center for Cancer Pharmacology, University of Pennsylvania. Benzo[a]pyrene-7,8-diol (BP-7,8-diol), a proximate carcinogen, requires activation to exert its deleterious effects. Formation of electrophilic BP-7,8-dione (via AKR1A1) and anti-BPDE (via CYP1B1) was confirmed by HPLC/ MSn and quantitatively measured by HPLC/UV/β-RAM in human bronchoalveolar cell models manipulated to express one or both pathways. Unexpectedly, the generation of BP-tetrols (hydrated anti-BPDE) was observed in AKR1A1+/CYP1- cells. BP-7,8-diol was found to be a robust inducer of CYP1B1 in these cells but not in AKR1A1-/CYP1- cells as measured by Northern and Western blot analysis. CYP1B1 induction was blocked by the AKR1A1 inhibitor (sulfonylnitromethane), the o-quinone scavenger (N-acetyl-L-cysteine), or the AhR antagonist (diflubenzuron). Attenuation of BP-tetrol formation in cells by these antagonists was verified by metabolic profiling. Therefore, the role of AKR1A1 in the activation of BP-7,8-diol is bifunctional; that is, it directly activates BP-7,8-diol to the redox active PAH o-quinone (BP-7,8-dione), and it indirectly trans-activates the CYP1B1 gene by generating the AhR ligand BP-7,8dione. 21. Kinetic and Trapping Studies of 2′-Deoxynucleoside Alkylation by a Quinone Methide. Emily E. Weinert and Steven E. Rokita. Department of Chemistry and Biochemistry, University of Maryland, College Park, College Park, Maryland 20742. E-mail: eweinert@ wam.umd.edu. Quinone methides (QM) are reactive intermediates generated in vivo by a variety of natural products and synthetic chemicals, such as mitomycin C and butylated hydroxytoluene. The apparent selectivity of QM alkylation of 2′-deoxynucleosides depends on the stability of the resulting adducts as well as the time frame of analysis. The reversibility of QM adduct formation can be altered in a predictable manner by choice of aromatic substituents. Addition of an electron-donating group stabilizes QM formation, while addition of an electron-withdrawing group destabilizes QM formation. Kinetic analysis of DNA alkylation, in contrast to 2′-deoxynucleoside alkylation, has been hampered by the lability of the QM adducts since these adducts decompose under the digestion conditions necessary for analysis. Oxidation of QM adducts formed in DNA can effectively trap the labile adducts, thus enabling analysis of alkylation despite its reversibility.


22. Cyclization of the Malondialdehdye OPdG Adduct to M1dG When Placed Opposite a Two-Base Deletion in the (CpG)3 Frameshift Hotspot of the Salmonella typhimurium hisD3052 Gene. Yazhen Wang, Sam Selch, Lawrence J. Marnett, and Michael P. Stone. Chemistry Department, Vanderbilt University, Nashville, Tennessee 37235. The 3-(β-D-ribofuranosyl)pyrimido[1,2a]purin-10(3H)one (M1dG adduct) was formed in DNA exposed to malondialdehyde. The M1dG adduct exists in pH-dependent equilibrium with the ring-opened N2-(3-oxo-1-propenyl) deoxyguanosine adduct (OPdG). The OPdG adduct was annealed into 5′-d(ATCGCOCGGCATG)-3′. 5′-d(CATGCCGCGAT)-3′ at pH 7; OdOPdG. This duplex was derived from a frameshift hotspot of the Salmonella typhimuium hisD3052 gene and contained a two-base deletion (2BD) in the complementary strand. The conversion of OPdG to M1dG was monitored as a function of time, using NMR spectroscopy. This occurred slowly, with equilibrium reached over a period of approximately 4 months at 25 °C. At equilibrium, both M1dG and OPdG were observed at a ratio of ∼2:1 M1dG:OpdG. The results suggested that in this two-base deletion sequence the two-base bulge in the OPdG-adducted strand migrates at a rate that is slow as compared to the acid-catalyzed formation of the pyrimidopurinone of M1 dG. We propose that bulge migration transiently positions OPdG or, alternatively, reverts rapidly formed intermediates in the OpdG to M1dG reaction pathway, into a fully duplex conformation opposite deoxycytosine, thus maintaining the N2-(3-oxo-1-propenyl) moiety within the minor groove, effectively slowing the conversion of OPdG to M1dG. Supported by NIH Grants CA-55678 (M.P.S.) and CA87819 (L.J.M.), the Center in Molecular Toxicology ES00267, and the Vanderbilt-Ingram Cancer Center CA68485. 23. Nitrosative Guanosine Deamination: 17OLabeling Studies as an Integral Accompaniment to 18 O-Studies. Rainer Glaser and Papiya Majumdar. Department of Chemistry, University of Missouris Columbia, Columbia, Missouri 65211. Fax: 573-8822754. E-mail: [email protected] and pm244@ mizzou.edu. Nitrosative deamination represents an important mechanism of endogenous DNA oxidation. We recently reported 18O-labeling studies of the nitrosative deamination of guanosine (1) to explore the mechanisms of formation of xanthosine 2 and oxanosine 3. The results showed that most of 2 is formed by direct nucleophilic substitution of the diazonium ion and that 3 is formed via 5-cyanoimino4-oxomethylene-4,5-dihydroimidazole (4) and 5-cyanoamino-4-imidazolecarboxylic acid (5), i.e., the water addition to the ketene moiety of 4 (path A). The alternative path B involves water addition to the cyanoimino group of 4 to form the urea derivative (6), which closes to 2 and/or 3. Path B affects the ratio of the isotopomers of 3. While our previous assessment of this ratio relied on the integration of 18O-isotopically shifted 13C NMR signals, we have now determined this ratio by integration of the 17 O NMR signals. The O(dC7) and O6 signals of 3 are well-separated, and their integrals are identical. This finding provides compelling evidence that under “free nucleobase” conditions (a) oxanosine is formed exclusively via 4 and that (b) all xanthosine is formed by direct nucleophilic substitution of the diazonium ion.


24. Stereospecific Interstrand Carbinolamine Cross-Linking by Crotonaldehyde-Derived 1,N2Propano-2′-Deoxyguanosine DNA Adducts in the 5′CpG-3′ Sequence. Young-Jin Cho,1 Jaison Jacob,2 Markus Voehler,1 Hai Huang,1 Hye-Young Kim,1 Ivan D. Kozekov,1 Hao Wang,1 Andrew J. Kurtz,3 Thomas M. Harris,1 Carmelo J. Rizzo,1 R. Stephen Lloyd,4 and Michael P. Stone.1 (1) Department of Chemistry, Center in Molecular Toxicology, and Vanderbilt-Ingram Cancer Center, Vanderbilt University, VU Station B Box 1822, Nashville, Tennessee 37235. E-mail: young.j.cho@ vanderbilt.edu. (2) Department of Biochemistry, Center in Molecular Toxicology, and Vanderbilt-Ingram Cancer Center, Vanderbilt University, VU Station B Box 1822, Nashville, Tennessee 37235. (3) Sealy Center for Molecular Science and the Department of Human Biological Chemistry and Genetics, University of Texas Medical Branch, Galveston, Texas 77555. (4) Center for Research on Environmental and Occupational Toxicology, Oregon Health and Science University, Portland, Oregon 97239. Equilibria between 1,N2-dG cyclic, N2-(R-CH3-γ-oxopropyl)dG aldehydic, and interstrand N2,N2-dG 5′-CpG3′ cross-links formed by crotonaldehyde in 5′-d(GCTAGCXAGTCC)-3′‚5′-d(GGACTGYCTAGC)-3′ (X ) R- or S-RCH3-γ-13C-OH-PdG; Y ) 15N2-dG) were monitored by NMR. In 5′-d(GCTAGCXAGTCC)-3′ epimeric mixtures of the 1,N2 -dG cyclic lesions were in slow exchange on the NMR time scale. When either was annealed into the duplex oligodeoxynucleotide complementary to dC, pHdependent ring-opening to diastereomeric N2-(R-CH3-γoxopropyl)dG aldehydes was observed at temperatures below the Tm of the duplexes, but when annealed opposite T, 1,N2 -dG cyclic lesions were stable. The aldehydes equilibrated with their geminal diol hydrates. After 3 weeks at 37 °C, equilibrium between the N2-(RR-CH3-γ-oxopropyl)dG aldehyde and two diastereomeric 5′-CpG-3′ cross-links was achieved; ∼26% of the oligodeoxynucleotide was cross-linked. The cross-links were assigned as diastereomeric carbinolamines, with the corresponding imine remaining below the level of detection. Molecular modeling suggested the carbinolamines maintained Watson-Crick hydrogen bonding at both of the tandem cross-linked C‚G base pairs, with minimal distortion of the duplexes. Dehydration of the carbinolamines to the imine, or subsequent cyclization of the imine to pyrimidopurinones, required disruption of Watson-Crick hydrogen bonding at the tandem cross-linked C‚G base pairs. Structural analysis of fully reduced Rand S-R-CH3-crotonaldehyde cross-links revealed stable duplexes without disruption of Watson-Crick hydrogen bonding. The N2-(S-R-CH3-γ-oxopropyl)dG aldehyde oriented in the minor groove, with the methyl group proximate to N2 -dG in the complimentary strand. Supported by NIH Grants ES-05355 (M.P.S., C.M.J., T.M.H., and R.S.L.), the Center in Molecular Toxicology ES-00267, and the Vanderbilt-Ingram Cancer Center CA68485. 25. DNA Damage by Fasicularin. Sanjay Dutta,1 Hideki Abe,2 Sakae Aoyagi,2 Chihiro Kibayashi,2 and

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Kent S. Gates.3 (1) Department of Chemistry, University of MissourisColumbia, 601 South College Avenue, Columbia, Missouri 65211. E-mail: [email protected]. (2) School of Pharmacy, Tokyo University of Pharmacy & Life Sciences. (3) Departments of Chemistry and Biochemistry, University of MissourisColumbia. Fasicularin (1) is a structurally novel alkaloid with potent cytotoxic properties isolated from the ascidian Nephteis fasicularis. Early biological assays suggested that this compound’s cytotoxicity may be derived from its ability to damage cellular DNA. Here, we report the first evidence that fasicularin can cause strand cleavage at guanine residues in duplex DNA. Further experiments reveal that generation of base labile DNA lesions involves alkylation of guanine residues by a fasicularin-derived aziridinium ion. Thus, our work indicates that fasicularin may be the first natural product that forms a DNAalkylating aziridinium ion in a manner analogous to the clinically used drugs mechlorethamine and chlorambucil.

26. Sequence Context Affects the Repair of O6Pyridyloxobutylguanine by Human O6-Alkylguanine-DNA Alkyltransferase Variants. Reneé S. Mijal,1 Sreenivas Kanugula,2 Choua C. Vu,3 Qingming Fang,2 Anthony E. Pegg,4 and Lisa A. Peterson.1 (1) Environmental Health Sciences and Cancer Center, University of Minnesota, 420 Delaware Street Southeast, Mayo Mail Code 806, Minneapolis, Minnesota 55455. Fax: 612-6265135. E-mail: [email protected]. (2) Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine. (3) Cancer Center, University of Minnesota. (4) The Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine. The tobacco-specific nitrosamine 4-(methylnitrosamino)1-(3-pyridyl)-1-butanone (NNK) is a potent pulmonary carcinogen in rodents and a likely human carcinogen. Metabolic activation of NNK results in the production of genotoxic alkylating agents that methylate or pyridyloxobutylate DNA. Both O6-methylguanine (O6-meG) and O6pyridyloxobutylguanine (O6-pobG) are important to the mutagenic activity of the methylation and pyridyloxobutylation pathways, respectively. The DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) repairs O6-alkylguanine adducts including O6-meG and O6-pobG. AGT repair protects cells from the mutagenic effects of alkylating agents. The human AGT gene is polymorphic, encoding several variants including the L84F and I143V/K178R proteins. The variant proteins repair O6-meG adducts as efficiently as the wild-type protein. Using a competitive repair assay, we determined that human AGTs differ in their repair of O6-pobG in a sequence-dependent fashion. This variation in sequencedependent repair could offer a biochemical explanation for increased lung cancer risk associated with the I143V/ K178R genotype. 27. Phototoxicity and Photomutagenicity Studies of 4-Chloro-Phenylenediamine and 2-Phenylbenzimidazole. Hongtao Yu,1 Charity N. Mosley,2 and Lei

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Wang.1 (1) Department of Chemistry, Jackson State University, 1400 J. R. Lynch Street, Jackson, Mississippi 39217. E-mail: [email protected]. (2) Department of Chemistry, Jackson State University, 1400 Valley Street Apartment 422B, Jackson, Mississippi 39204. E-mail: [email protected]. CPD, 4-chloro-1,2-phenylenediamine, is an active ingredient in the manufacture of hair dyes. PBI, 2-phenylbenzimidazole, is an ingredient found in most sunscreen agents. Because each absorbs UVA light, it is a possibility that, upon UVA exposure, these compounds undergo adverse reactions. Therefore, we studied the chemical and photochemical toxicities of these compounds on the Salmonella typhimurium (TA102) bacteria. We show that CPD is both phototoxic and photomutagenic. Results from the photochemical toxicity suggest CPD is more phototoxic than PBI, although PBI does exhibit chemical toxicity. CPD is strongly photomutagenic in TA102 when concomitantly exposed to light as compared to the negative dark control. At 600 µM and an irradiation time of 30 min, the revertant colonies per plates were more than 2 orders of magnitude lower in CPD as compared to that of PBI, which remained relatively the same. Further investigations are in progress to determine the photomutagenicity, cytotoxicity, and genotoxicity of the aforementioned compounds. 28. Modification of Amino Acid Side Chains and Proteins by Lipoxidation Derived Aldehydes. De Lin and Lawrence M. Sayre. Department of Chemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106. E-mail: [email protected]. Electrophilic aldehydes, generated from oxidation of polyunsaturated fatty acids (PUFAs) under conditions of oxidation stress, are thought to play an important role in the pathology of degenerative disease. We report here that the fastest reaction of 4-oxo-2-nonenal (ONE) with proteins involves Schiff base formation at Lys -amino groups, whereas Schiff base formation is not spectroscopically apparent for 4-hydroxy-2-nonenal (HNE). Detailed kinetic studies of the initial reactions of HNE and ONE with amino acids and amino acid surrogates, in conjunction with NMR, reveal that ONE Schiff base formation and HNE amine conjugate addition are reversible, that conjugate addition of imidazole and thiol nucleophiles to both aldehydes (ONE > HNE) is not detectably reversible, and that conjugate addition of amines to ONE is relatively noncompetitive. Additionally, 4,5-epoxy-3-heptenal (EH), another oxidation product of PUFA, is reported to induce protein cross-links between Lys and His. 29. Systems Biology: Integrating Biology, Technology, and Computation. Leroy Hood, Institute for Systems Biology, 4225 Roosevelt Way Northeast, Suite 200, Seattle, Washington 98105. Fax: 206-732-1254. E-mail: [email protected]. Systems biology is predicated on the idea that all of the elements in a system must be analyzed with respect to one another in response to a wide range of systems perturbations. This requires the computational integration of a variety of global sets of data (e.g., DNA, mRNA, protein interactions, etc.). I will discuss this approach for physiological systems in yeast and Halobacterium and a developmental system in the sea urchin. I will also discuss some of the new global technologies being developed for the acquisition of global data sets.


30. Overview of Tobacco Carcinogenesis. Stephen S. Hecht. University of Minnesota Cancer Center, MMC 806, 420 Delaware Street Southeast, Minneapolis, Minnesota 55455. Fax: 612-626-5135. E-mail: hecht002@ umn.edu. That tobacco causes cancer is common knowledge, even to school children. Cigarette smoking causes 30% of cancer death in developed countries, and smokeless tobacco products are a major cause of cancer in some parts of the world. With these facts established beyond any reasonable doubt, why is it necessary to conduct research on tobacco carcinogenesis? There are still over 1 billion smokers in the world and hundreds of millions of smokeless tobacco users. An understanding of mechanisms by which tobacco products cause cancer can provide new insights on approaches to tobacco control, individual susceptibility to cancer, and cancer prevention in general. This presentation will give an overview of current topics in tobacco carcinogenesis including chemical mechanisms by which tobacco constituents cause cancer, the role of volatile and particulate phase constituents of cigarette smoke as causes of cancer, and biomarkers for assessing carcinogen uptake by people who use tobacco products. 31. Tobacco Addiction: Interface Between Tobacco and Cancer. Jack E. Henningfield. Pinney Associates, 3 Bethesda Metro Center Suite 1400, Bethesda, Maryland 20814. Fax: 301-718-0034. E-mail: jhenning@ pinneyassociates.com. The risk of cancer produced by tobacco is related to daily exposure level and years of exposure. The amount and persistence of tobacco use, in turn, is related to nicotine addiction. Exposure to substances in tobacco increases the risk of a variety of cancers, due to the pathogenic consequences of exposures at the cellular and subcellular level. Similarly, exposure to nicotine and other substances in tobacco produces pathogenic effects including tolerance, dependence, reinforcement, and powerful behavioral conditioning, more commonly referred to as “addiction”. Addiction helps explain why most tobacco users use the products repeatedly throughout every day, for several decades, despite their desire to quit and regret at having ever begun to use tobacco. From this perspective, tobacco-caused cancers, may be considered side effects of addiction. Understanding the pharmacological, physiological, and environmental forces driving addiction are key to its control and, in turn, to the control of tobacco-caused cancer. 32. Properties that Alter the Levels of Carcinogenic Compounds in Tobacco and Tobacco Smoke. David L. Ashley. National Center for Environmental Health, Centers for Disease Control and Prevention, Mailstop F-47, 4770 Buford Highway, Atlanta, Georgia 30341. Fax: 770-488-0181. E-mail: [email protected]. Cigarette smoke is a complex mixture of chemical compounds bound to aerosol particles and free in the gas phase. Chemical compounds can be distilled directly from tobacco into smoke or react to form other materials. Many of these chemicals in tobacco smoke are mutagenic, carcinogenic, addictive, or promote these harmful properties. Although the list of carcinogens is not exhaustive, it includes the tobacco-specific nitrosamines, polyaromatic hydrocarbons, volatiles, aminobiphenyls, and heavy metals. The levels of these chemicals in smoke can be altered by cigarette design, chemical constituents of the product, and the way people consume the cigarette. At


the Centers for Disease Control and Prevention, we have developed analytical methods to measure many of these chemicals in tobacco, tobacco smoke, and the blood and urine of smokers and those exposed to second-hand smoke. By comparing the levels found in the product, the properties of the products, and how people smoke, we can better understand how these influence the delivery of carcinogens and, thus, cancer resulting from the exposure. 33. Mechanisms of Carcinogenesis: Polycyclic Aromatic Hydrocarbons (PAH). Trevor M. Penning. Department of Pharmacology, University of Pennsylvania, 135 John Morgan Building, 3620 Hamilton Walk, Philadelphia, Pennsylvania 19104-6084. Fax: 215-8987180. E-mail: [email protected]. Polycyclic aromatic hydrocarbons (PAH) exist as a mixture in tobacco smoke and are suspect human carcinogens. Benzo[a]pyrene (BP) is a representative PAH and is present at concentrations of about 20-40 ng/ cigarette. BP requires metabolic activation to electrophiles to exert its deleterious effects by one of three pathways. Evidence for these pathways (cytochrome P450-mediated formation of diol-epoxides, aldo-keto reductase formation of reactive and redox-active o-quinones, and peroxidase-mediated formation of radical cations) in human lung will be reviewed. The ability of these reactive metabolites to form DNA adducts and mutate the two most common genes mutated in lung cancer, e.g., K-ras and the p53 tumor suppressor gene, will be examined. PAHs act as multisite and multispecies carcinogens in animal models, and this provides a basis for human risk assessment. The success/failure to correlate biomarkers of PAH exposure to human lung cancer incidence will be explored. Supported by Grants R01CA39504 and P01-CA-092537 to T.M.P. 34. Metabolic Deactivation of Nicotine and Activation of Tobacco-Specific Nitrosamines by Cytochrome P450 Enzymes. Sharon E. Murphy. Cancer Center, University of Minnesota, MMC 806, 420 Delaware Street Southeast, Minneapolis, Minnesota 55455. Fax: 612-626-5135. E-mail: [email protected]. Nicotine is the primary addictive agent in tobacco. The structurally similar tobacco-specific compounds N′-nitrosonornicotine (NNN) and 4-(methylnitrosamine)-1-(3pyridyl)-1-butanone (NNK) are likely contributors to esophageal and lung cancers in smokers. Nicotine is deactivated by P450 enzymes, whereas both NNK and NNN are metabolically activated by P450s to their carcinogenic forms. P450 2A6 is an efficient catalyst of both the 5′-oxidation of nicotine and the 5′-hydroxylation of NNN, but it is not a good catalyst of NNK R-hydroxylation. However, the predominantly extrahepatic P450, 2A13, which is 94% identical to P450 2A6, is an outstanding catalyst of NNK R-hydroxylation. We recently reported that P450 2A13 is the catalyst of NNK R-hydroxylation in human fetal nasal mucosa. Despite the structural similarities of nicotine, NNN, and NNK, the efficiency of P450-catalyzed metabolism of each is quite different. The relative importance of P450 2A6 and P450 2A13 in the metabolism of these three compounds will be discussed. 35. Biomarkers of Carcinogen Uptake and Activation. Peter G. Shields. Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, 3800 Reservoir Road Northwest, Washington, DC 200571465. Fax: 202-687-0004. E-mail: [email protected].

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The prediction of tobacco-related cancer risks in individual smokers has been challenging for several reasons. What governs exposure in a person depends on what level of nicotine they need in their bodies, which affects how they smoke. Internal exposure can be assessed through biomarkers, some of which are chemically specific and others that represent complex exposures. However, the demonstration that these biomarkers sufficiently predict risk has not been shown; a panel of biomarkers likely will be needed for use in a clinical setting or to predict differential effects of tobacco products. Data will be presented for a study of 290 African Americans and Caucasians, smoking behavior (topography, nicotine boosts, cigarette brand, and other variables), genetics of addiction, exposure biomarkers (chromosomal aberrations and urine mutagenicity), biologically effective doses (DNA adducts by 32P-postlabeling, malonaldeyhyde adducts, and 8-OHdG), and biomarkers of effect (RNA expression arrays in circulating lymphocytes). 36. Molecular Epidemiology of Tobacco and Cancer. Mimi C. Yu. Department of Preventive Medicine, University of Southern California Keck School of Medicine, 1441 Eastlake Avenue, NOR 7346, Los Angeles, California 90033. Fax: 323-865-0104. E-mail: mimiyu@ usc.edu. The two major types of human cancer most closely related to tobacco smoking are cancers of the lung and bladder. This talk will focus on recent human data indicating the roles of host and environmental factors in modifying the tobacco-lung/bladder cancer risk relationships in smokers. Specifically, data indicating a major role for glutathione S-transferase genotypes in modifying the interplay of dietary isothiocyanates, known to inhibit lung cancer in animals, and tobacco smoking in relation to lung cancer risk will be reviewed. 4-Aminobiphenyl (4-ABP), which is present in tobacco smoke, is a known human bladder carcinogen. Data indicating gender (higher risk in women) and dietary carotenoids (lower risk in high consumers) as modifiers of the association between tobacco smoking and bladder cancer risk will be reviewed. 37. Smoke Chemistry: Is There a Correlation with Lung Cancer Epidemiology? John H. Lauterbach. Lauterbach & Associates, LLC, 211 Old Club Court, Macon, Georgia 31210-4708. Fax: 478-474-0117. Email: [email protected]. Fan D. Chen. Master of Public Health Program, Mercer University. Cigarette smoking causes lung cancer and other serious diseases. Many believe there is little difference in health risks posed by different types of cigarettes although there is epidemiological evidence that the risks associated with unfiltered cigarettes are greater than those associated with filtered cigarettes with the same type of tobacco. However, what about cigarettes with similar deliveries made with different types of tobacco? Epidemiology has shown that smokers of black (dark aircured) tobacco cigarettes have a 4-fold or higher risk of lung cancer than smokers of blond tobacco cigarettes (Joly et al., 1983; Benhamou et al., 1985, 1989; Armadans-Gil et al., 1999; Lee, 2001). The chemistry of black tobacco cigarettes is different from that of blond and American-blend cigarettes, and these differences result in changes in smoke chemistry among the three cigarette types. The impact of changes in chemistry will be discussed in terms of the epidemiology. 38. Deoxyguanosine and DNA Adducts Resulting from 5′-Hydroxylation of N′-Nitrosonornicotine.

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Pramod Upadhyaya. University of Minnesota Cancer Center, 420 Delaware Street Southeast, MMC 806, Minneapolis, Minnesota 55455. Fax: 612-626-5135. Email: [email protected]. N′-Nitrosonornicotine (NNN) is a tobacco-specific nitrosamine, which causes esophageal and nasal tumors in rats and is believed to play a significant role in cancer induction in people who use tobacco products. NNN requires metabolic activation to exert its carcinogenic activity. DNA adducts of NNN resulting from 2′-hydroxylation are expected to be the same as those formed upon methyl hydroxylation of the related nitrosamine NNK, and these have been previously characterized. In this study, we characterized adducts resulting from 5′-hydroxylation of NNN, through reactions of 5′-acetoxyNNN with deoxyguanosine (dG) and DNA. 5′-Hydroxylation produces either a carbocation or an oxonium ion intermediate. Adducts from the carbocation were identified as diastereomers of 2-[2-(3-pyridyl)pyrrolidin-1-yl]deoxyinosine (after NaBH3CN treatment) while those from the oxonium ion were diastereomers of N2-[5-(3-pyridyl)-2hydroxytetrahydrofuran-2-yl]dG. The absolute configuration of the adducts at the 3-pyridyl- substituted carbon was determined. These results demonstrated diverse dG and DNA adduct formation by 5′-hydroxylation of NNN. 39. Development of a Sensitive Method for Quantitation of DNA Pyridyloxobutylation Adducts Derived from the Tobacco Specific Nitrosamine, 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Yanbin Lao,1 Peter W. Villalta,2 Mingyao Wang,2 Guang Cheng,2 Shana J. Sturla,1 and Stephen S. Hecht.2 (1) Department of Medicinal Chemistry and the Cancer Center, University of Minnesota, MMC 806, 420 Delaware Street Southeast, Minneapolis, Minnesota 55455. Fax: 612-626-5135. E-mail: [email protected]. (2) University of Minnesota Cancer Center. NNK, a tobacco specific nitrosamine, is a potent pulmonary carcinogen in rodents and has been recently evaluated as a human carcinogen by IARC. NNK requires cytochrome P450-mediated metabolic activation to exert its carcinogenicity. One of the bioactivation pathways, R-methyl hydroxylation, results in the formation of a pyridyloxobutyl diazonium ion, which alkylates DNA to form pyridyloxobutyl (POB) adducts, i.e., O6-POB-dGuo, 7-POB-Gua, O2-POB-Thy, and O2-POB-Cyt. In this study, an analytical method was developed for quantitation of these POB-DNA adducts. With deuterated internal standards, DNA was sequentially subjected to neutral thermal hydrolysis, enzymatic hydrolysis, and solid phase extraction, followed by capillary HPLC-ESI-MS/MS-SRM (selected reaction monitoring) analysis. This method allows quantitation of high attomole to low femtomole levels of the POB-DNA adducts in a 1 mg DNA matrix. The method is suitably sensitive, accurate, precise, and of high recovery for evaluation of DNA samples isolated from smokers’ bronchial washings/brushings and tissues from NNK-treated rats. 40. Formation and QSAR Analysis of DNA Adducts with Pesticides. Donald W. Boerth,1 Erwin Eder,2 John R. Stanks,3 and Paul Wanek.2 (1) Department of Chemistry and Biochemistry, University of Massachusetts, North Dartmouth, Massachusetts 02747. Fax: 508-999-9167. E-mail: [email protected]. (2) Institute of Toxicology, University of Wu¨rzburg. (3) Department of Chemistry and Biochemistry, University of Massachusetts Dartmouth.


Pesticides pose a potential for risk for stress and DNA damage to agricultural plants, as well as to humans and animals. These chemical agents are frequently halogenated compounds or contain electrophilic groups, which can react at one or more of the nucleophilic sites of the DNA bases to form adducts. In this study, experimental and computational modeling methods were developed for screening pesticides and assessing the risk of pesticide damage to DNA. [32P]-Postlabeling studies in our laboratories provide evidence of DNA adduct formation in a number of crop plants. Both direct and indirect adducts were detected in DNA extracted from crop plants after treatment with pesticides. Direct adducts of DNA with pesticide molecules or their metabolites, as well as indirect adducts of DNA with products of lipid peroxidation, were observed with common pesticides, chlorothalonil, diazinon, esfenvalerate, carbaryl, etc. DNA products with 4-hydroxy-2-nonenal and 2-hexenal are prominent features of the postlabeling radiochromatograms, indicating that these plant systems have been subjected to oxidative stress. These adducts serve as biomarkers for stress in plants. Theoretical molecular modeling has been employed to identify the nature, degree, and site(s) of interactions between DNA bases and a series of pesticide molecules. Approaches to the development of QSAR methods for screening pesticides for potential risk of DNA damage are discussed. 41. Factors that Dictate Atom Site Selectivity in the Diazonium Ion-Mediated Alkylation of Nucleosides and DNA. Xuefang Lu, Jacqueline M. Heilman, Patrick Blans, and James C. Fishbein. Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250. Fax: 410-455-2608. E-mail: jfishbei@ umbc.edu. Studies of the alkylation of heteroatoms of purine nucleosides and the purine nucleobases of DNA by 1-propyl- and 2-propyl-diazonium ions have been completed. There are differences in the product yields at different atom sites for reactions of the monomers as compared to the polymer. The yields and these differences between monomers and polymers allow an analysis of factors that contribute to atom site selectivity. 42. Thermodynamic Stability and Biological Effects of a Single Urea Lesion. Alvin A. Altamirano and Ashis K. Basu. Department of Chemistry, University of Connecticut, 55 North Eagleville Road, Storrs, Connecticut 06269. Fax: 860-486-2981. E-mail: [email protected]. The mutagenic effect of urea lesion (Ur) was investigated using an M13 bacteriophage genome containing a mixture of the two Ur isomers at a specific site. Replication of the lesion-containing M13 genome in both uninduced and SOS-induced Escherichia coli showed significant reduction (∼97%) in the viability of the modified M13 genome. Analysis of the progeny phage showed that Ur was preferentially replaced by a T. However, Ur-to-C point mutations (at ∼3% frequency) and targeted onebase deletions (at ∼4% frequency) were also detected. SOS induction caused the Ur-to-C mutations to increase severalfold, although frequency of deletions did not increase. Thermal melting studies showed that the presence of Ur destabilized the duplex DNA, except when the base opposite Ur was deleted. This suggests that Ur is an instructional lesion and that the thermodynamic stability may play a role in its replicative bypass in vivo.


43. Free Radical Nitration and Oxidation of Guanine and 8-Oxo-7,8-Dihydroguanine in DNA. Richard Misiaszek,1 Conor Crean,1 Nicholas E. Geacintov,1 and Vladimir Y. Shafirovich.2 (1) Department of Chemistry, New York University, 29 Washington Place. Fax: 212-998-8421. (2) Chemistry Department, New York University, 31 Washington Place. Fax: 212-9988421. E-mail: [email protected]. In living tissues under inflammatory conditions, reactive oxygen and nitrogen species are known to cause the oxidation and nitration of cellular DNA. However, the mechanisms of action are poorly understood. The oxidation and nitration reactions in DNA associated with the combination of nitrogen dioxide radicals with 8-oxo-7,8dihydroguanine (8-oxoGua) and guanine radicals have been explored by kinetic laser spectroscopy and mass spectrometry methods. These reactions occur with similar rate constants (∼4.3 × 108 M-1) in both single- and double-stranded DNA. In the case of 8-oxoGua, the major end products of this bimolecular radical-radical addition are spiroiminodihydantoin lesions, the products of 8-oxoGua oxidation. Oxygen-18 isotope labeling experiments reveal that the O-atom in the spiroiminodihydantoin lesion originates from water molecules, not from nitrogen dioxide radicals. In contrast, the combination of nitrogen dioxide and guanine neutral radicals generated under the same conditions results in the formation of the nitro products, 5-guanidino-4-nitroimidazole and 8-nitroguanine adducts. The mechanistic aspects of the oxidation/nitration processes and their biological implications are discussed. Supported by NIH Grant 5 R01 ES11589. 44. Sequence Effects on the Conformational Heterogeneity of Aminofluorene-Derived DNA Adducts and Incision by UvrABC Nuclease Repair. Srinivasa Rao Meneni,1 Steven M. Shell,2 Yue Zou,2 and Bongsup P. Cho.1 (1) Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, 41 Lower College Road, Kingston, Rhode Island 02881. Fax: 401874-5766. E-mail: [email protected] and [email protected]. (2) Department of Biochemistry and Molecular Biology, East Tennessee State University. The well-known carcinogen N-acetyl-2-aminofluorene produces a dG-C8-AF lesion as the most persistent DNA adduct. This N-deacetylated lesion is known to exist in two prototype conformers: B type (B) and stacked (S). To probe the sequence dependence of S/B heterogeneity, we carried out 19F NMR experiments using fluorinated AF (FAF)-modified DNA duplexes with different bases flanking the adduct. Our data indicates that (a) the AFinduced S/B equilibrium is markedly sequence dependent; (b) the stacking ability of the AF ring of S-conformers is sequence dependent; (c) all of the duplexes adopt a typical two-site chemical exchange, suggesting that repair and replication enzymes are likely to be confronted with the S/B heterogeneity in vivo. The incision efficiency of the FAF adducts by UvrABC nuclease was found to be strongly sequence dependent. These results provide valuable quantitative and dynamic insights into the sequence effects on the S/B population balance and their NER efficiency (NIH R01CA98296). 45. Solution Conformations of the Major (+)trans-anti-BPDE-N2-Guanine Adducts in GG Dinucleotide Mutation Hot Spots. Fabian A. Rodriguez,1 Qian Ruan,1 Chin Lin,1 S. Amin,2 and Nicholas E. Geacintov.1 (1) Department of Chemistry, New York

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University, 29 Washington Place, Brown Building, Room 453, New York, New York 10003. E-mail: [email protected]. (2) Department of Pharmacology, Penn State College of Medicine. The 10S (+)-trans-anti-BPDE-N2-dG DNA adduct (G*) gives rise to mutation hot spots when present on the 5′guanine in 5′-G*G dinucleotide steps in prokaryotic and on the 3′-guanine in eukaryotic cells. Electrophoretic mobility experiments in nondenaturing gels show that 5′-‚‚‚GG*‚‚‚ duplexes exhibit a markedly slower mobility, characteristic of kinks at the lesion site, than the sequence isomeric adduct in the duplex 5′-‚‚‚G*G‚‚‚. We investigated the solution NMR properties of two duplexes

in an otherwise identical sequence context with the lesion either at G6*G7 or at G6G7*. In both cases, the BPDE residue is located in the minor groove and is oriented toward the 5′-end of the modified strand. Chemical shifts of sugar protons of nucleotides on the complementary strand clearly show that the orientations of the BPDE residues in the minor groove are significantly different in the two duplexes. These results suggest that the exocyclic amino group in the minor groove, N2-dG6, flanking the adduct G7* in the G6G7* duplex, displaces the bulky BPDE residue from the position observed in G6*G7 and gives rise to the unusual bend or kink. Research supported by NIH Grant CA099194. 46. Base Sequence and Conformational Effects on Time-Resolved Fluorescence Decay Profiles of Covalent BPDE-N2-dG Adducts in DNA. Yijin Tang, Alexander Durandin, Nathaniel Stanley, Sofia Fayngold, Alexander Kolbanovskiy, and Nicholas E. Geacintov. Department of Chemistry, New York University, New York University, 31 Washington Place, New York, New York 10003-5180. E-mail: [email protected]. The biologically most active metabolite of the ubiquitous environmental pollutant benzo[a]pyrene (B[a]P), the diol epoxide 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydroB[a]P (B[a]PDE), binds covalently to N2-dG in DNA to form potentially mutagenic lesions. The conformational characteristics of a number of these B[a]PDE-N2-dG adducts (G*) in DNA in aqueous solutions and the effects of local base sequence context on these conformations have been investigated by several different laboratories utilizing high resolution NMR methods. However, milligram quantities of purified site-specifically modified oligonucleotides are needed for such studies and are often not available. Fluorescence methods, on the other hand, although providing only low-resolution information, are highly sensitive to the local environment of the pyrenyl residues and require only submicrograms of material. We have therefore reinvestigated the effects of B[a]PDE-N2dG adduct conformation and flanking base sequence effects on the fluorescence of the pyrenyl residue excited by 355 nm femtosecond laser pulse pulse trains (∼2 × 107 Hz frequency) using time-correlated single-photon counting methods. In double-stranded DNA, the fluorescence of the pyrenyl residue is highly quenched, and three different decay components with subnanosecond to nanosecond lifetimes are observed. These lifetimes are modulated by the flanking bases, especially by flanking adenines that enhance the mean fluorescence decay time

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in a manner that depends on adduct stereochemistry. This approach should be very convenient for monitoring the dynamics of conformational changes because the technique is highly sensitive, and complete decay profiles can be accumulated in a matter of minutes. Research supported by NIH Grant CA 099194. 47. Molecular Dynamics Studies of the Major DNA Adduct From a Tumorigenic Metabolite of Benzo[a]pyrene in Replicative and Bypass Polymerases. Pingna Xu,1 Olga Rechkoblit,2 Dinshaw J. Patel,2 Nicholas E. Geacintov,3 and Suse Broyde.1 (1) Department of Biology, New York University, 100 Washington Square East, New York, New York 10003. Email: [email protected]. (2) Cellular Biochemistry & Biophysics Program, Memorial Sloan-Kattering Cancer Center. (3) Department of Chemistry, New York University. Current evidence suggests that bulky carcinogen-DNA adducts primarily block replicative DNA polymerases, but translesion synthesis catalyzed by error-prone Yfamily bypass polymerases has been observed. Benzo[a]pyrene (BP), a combustion-derived polycyclic aromatic hydrocarbon (PAH) found widely in the environment, can be metabolically activated to reactive derivatives that can attack DNA. The tumorigenic (+)-anti-BPDE metabolite forms a major mutagenic adduct with guanine in DNA, 10S(+)-trans-anti-[BP]-N2 -dG ([BP]G). Our goal is to elucidate, on a structural level, the observed predominant blockage and rare bypass of this lesion in a replicative polymerase and the comparatively efficient bypass of the same DNA adduct by a Y-family polymerase. We have carried out computer modeling and molecular dynamics studies of this adduct in a thermophilic bacterial replicative polymerase, the bacillus fragment (BF), with this adduct positioned in the active site of BF; we considered both open binary and closed ternary complexes, as well as preinsertion, insertion, and postinsertion site models, with C or A opposite the lesion in the insertion and postinsertion cases. In addition, we investigated this lesion in a thermophilic archaeal Y-family polymerase, Dpo4, and considered both mismatched A and -1 deletions. Crystal structures of the polymerases served as initial models for the simulations [Johnson et al. (2003) PNAS 100, 3895-3900. Ling et al. (2001) Cell 107, 91102. Rechkoblit et al. Manuscript in preparation]. Our studies show how active site structural differences in the two enzyme families explain their differential treatment of this bulky adduct. Supported by NIH Grant 2RO1 CA 28038 (S.B.), CA 46533 (D.J.P), and CA 099194 (N.E.G.). 48. 4-OHEN-C Adduct Structures in DNA Duplexes: Molecular Dynamics Studies. Shuang Ding,1 Robert Shapiro,1 Nicholas E. Geacintov,1 and Suse Broyde.2 (1) Department of Chemistry, New York University, 100 Washington Square East, Room 1001, New York, New York 10003. E-mail: [email protected]. (2) Department of Biology, New York University. The Premarin family of drugs are the most widely used formulas for hormone replacement therapy. However, long term exposure to estrogens from the Premarin drugs has been determined to increase the risk of breast cancer. Equilin and equilenin, major components of Premarin, are predominantly metabolized to 4-hydroxyequilenin (4OHEN). The quinoids produced by 4-OHEN oxidation react with dG, dA, and dC to form unusual stable cyclic bulky adducts (Shen et al. 1998), with four stereoisomers identified for each base adduct. In vitro primer extension


studies conducted with several Y-family bypass polymerases suggest that the 4-OHEN-dC lesions can be bypassed but with a sequence-dependent insertion of the incorrect dNTP opposite the lesion in some cases (Chen, D., Oum, L., and Geacintov, N. E. To be published). Furthermore, 4-OHEN-dC adducts have been found in human tumor tissue. We have carried out molecular modeling and molecular dynamics simulations to investigate structures and thermodynamics of the four 4-OHEN-dC stereoisomeric adducts in DNA duplexes. Our results show that the structure of each stereoisomer adduct in duplex DNA is specifically governed by its unique stereochemistry. The adducts are located in the B-DNA major groove, but stereochemistry determines their orientation with respect to the 5′ to 3′ direction of the modified strand, as well as the positioning of the equilenin moiety’s methyl and hydroxyl groups. These stereochemical effects are likely to play a role in the biological processing of these lesions. Supported in part by NIH Grant 2RO1 CA 73638 (subcontract from the University of Illinois at Chicago, J. Bolton, P.I.) and NIH 2RO1 CA 75449 (S.B.). 49. Replication of DNA Sequences with Equine Estrogen Metabolite 4-OHEN-dC Adducts Catalyzed by A- and Y-Family Polymerases In Vitro. Dandan Chen,1 Alexander Kolbanovskiy,2 Anant Shastry,1 Minsun Chang,3 Judy L. Bolton,4 and N. E. Geacintov.5 (1) Chemistry Department, New York University, 29 Washington Place, Room 453, New York, New York 10003. Fax: 212-998-8421. E-mail: [email protected]. (2) Department of Chemistry, New York University. (3) College of Pharmacy, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago. (4) Department of Medicinal Chemistry & Pharmacognosy, University of Illinois College of Pharmacy. (5) Chemistry Department, New York University. The equine estrogen equilenin, a component of a widely prescribed formulation that is used as a hormone replacement treatment for postmenopausal women, is metabolized by mammalian P450 enzymes to the catechol 4-hydroxyequilenin (4-OHEN). The latter is autooxidized to o-quinones that form unusual cyclic adducts with cytidine and adenine in DNA. These lesions have been incorporated site-specifically into 43-mer oligonucleotides that were used as templates in primer extension reactions catalyzed by purified polymerases in vitro. Although all of the stereoisomeric covalent 4-OHEN-dC lesions significantly distort the normal B-DNA duplex structure and thus, presumably, the normal characteristics of the DNA at the active sites of polymerases, small extents of translesion bypass are observed even in the case of the A-family polymerase Bst. However, significant errorprone translesion bypass catalyzed by the Y-family polymerases Dpo4, pol η and pol κ is observed. In the case of Dpo4, the mutagenic insertion of the incorrect nucleotides depends on the local base sequence context. Research supported by NIH Grants CA 73638 (J. L. Bolton, P.I.) and CA112412. 50. Mass Spectral Analysis of DNA Polymerase Dpo4 Bypass Reaction Products Formed with 1,N2Ethenoguanine. Hong Zang,1 Angela K. Goodenough,2 Joeng-Yun Choi,3 Martin Egli,3 and F. P. Guengerich.1 (1) Department of Biochemistry & Center in Molecular Toxicology, Vanderbilt University, 638 Robinson Research Building, 23rd and Pierce Avenues, Nashville, Tennessee 37232-0146. Fax: 615-322-3141. E-mail:


[email protected]. (2) Chemistry, Vanderbilt University. (3) Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University. 1,N2-Etheno()guanine is a mutagenic DNA adduct. Gel electrophoretic analysis of the products of primer extension by Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) indicated preferential incorporation of A opposite 3′-(1,N2--G)TACT-5′, among the four dNTPs tested individually. When primer extension was done in the presence of a mixture of all four dNTPs, LC-mass spectrometry (MS) analysis of the products indicated that, opposite 3′-(1,N2--G)CACT-5′, the major product was 5′GTCA-3′ and the minor one was 5′-AGTCA-3′. With the template 3′-(1,N2--G)TACT- 5′, four products were identified by LC-MS: 5′-AATGA-3′, 5′-ATTGA-3′, 5′ATGA-3′, and 5′-TGA-3′. We conclude that Dpo4 uses several mechanisms, including A incorporation opposite 1,N2--G and also a variation of dNTP-stabilized misalignment, to generate both base-pair and frameshift mutations. X-ray structures of three intermediates were solved. 51. Elucidating the Incorporation of dCTP Opposite an N-(Deoxyguanosin-8-yl)-2-Acetyl-Aminofluorene Adduct (AAF-dG) within the Y-Family DNA Polymerase Dpo4 by Molecular Dynamics. Lihua Wang. Biology Department, New York University, 100 Washington Square East, 1009 Silver Center, New York, New York 10003. Fax: 212-995-4015. Suse Broyde. Department of Biology, New York University. The Y-family DNA polymerase Dpo4 can bypass the AAF-dG adduct with preferential incorporation of the normal partner C in primer extension studies [Boudsocq et al. (2001) Nucleic Acids Res. 29, 4607]. However, many decades of studies have indicated that AAF-dG favors the syn glycosidic torsion conformation, although the possibility of an unusual anti conformation had been predicted [Hingerty and Broyde (1982) Biochemistry 21, 3243]. The syn conformation would preclude WatsonCrick base-pairing in B-DNA, where the glycosidic bond is normally anti. Our goal in the present study was to elucidate on a structural level how the normal partner C may be incorporated opposite AAF-dG, in the Dpo4 DNA polymerase active site. We carried out molecular modeling and molecular dynamics simulations to study feasible conformations of the AAF-dG adduct opposite an incoming dCTP in the Dpo4 active site. Both anti and syn conformations of the templating AAF-dG in Dpo4 were investigated, employing a Dpo4 ternary complex crystal structure [Ling et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 2265] as the initial model. We found that a structure with unusual anti glycosidic and sugar conformations allows AAF-dG to be Watson-Crick hydrogenbonded with dCTP; in this structure, the AAF-dG is comfortably accommodated in the Dpo4 active site, with the AAF ring system in the major groove open pocket. With AAF-dG syn, however, the active site region is very distorted. Thus, flexibilities inherent in DNA structure can be utilized within the spacious Dpo4 DNA polymerase enzyme to facilitate normal partner bypass of a bulky adduct. This work is supported by NIH 2R01 CA75449. 52. 19F NMR and CD Studies on the Sequence Effect of Aminofluorene and FluoroaminofluoreneModified NarI Sequences. Yuyuan Li,1 Li Zhang,1 Lan Gao,2 M. Paul Chiarelli,2 and Bongsup P. Cho.1 (1) Department of Biomedical and Pharmaceutical Sciences,

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University of Rhode Island, Kingston, Rhode Island 02881. Fax: 401-764-5766. E-mail: [email protected] and [email protected]. (2) Department of Chemistry, Loyola University. The NarI sequence (5′-G1G2CG3CX-3′) is a strong mutational hotspot for -2 frameshift mutagenesis in Escherichia coli. Modification at G3 by the aromatic amines AAF and AF dramatically increases the rate of -2 deletion mutations even though each G shows similar reactivity. The nature of nucleotide X is known to strongly modulate the frequency of -2 deletions. To understand the origin of this sequence effect, we carried out CD and UV thermomelting experiments on a series of complementary and -2 deletion DNA duplexes that are site-specifically modified by AF and the fluoro analogue FAF. 19F NMR experiments were conducted to probe the adduct-induced conformational heterogeneity. The results are discussed in terms of how the nature of nucleotide X influences the propensity to form a slipped mutagenic intermediate, which could explain the large difference in -2 frameshift mutagenesis in the NarI sequence (NIH R01-CA098296 and RI-INBRE Core Facility #P20 RR016457). 53. Sequence Effect on the Conformational Heterogeneity of Aminofluorene and Fluoroaminofluorene-Modified DNA: UV Thermomelting and CD Studies. Rhijuta D’Mello,1 Srinivasa Rao Meneni,1 Lan Gao,2 Paul Chiarelli,2 and Bongsup P. Cho.1 (1) Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, Kingston, Rhode Island 02881. Fax: 401-874-5766. E-mail: [email protected]. (2) Department of Chemistry, Loyola University. N-Acetyl-2-aminofluorene is a prototype arylamine carcinogen that, when activated, forms a persistent adduct by attachment to the C8 position of a guanine base. NMR studies indicate that the N-deacetylated lesion in DNA exists in two distinct conformers: basedisplaced stacked (S) and external B type (B). In this study, we prepared a series of 12-mer DNA duplexes where sequences flanking the AF- or FAF-lesion (G*) were varied systematically (5′-CTTCTXG*NCCTC-3′; X ) dG, dA; N ) dG, dA, dC, dT). CD and UV melting experiments were conducted to investigate the sequence context effects of these lesions on DNA duplex structures. The position of the lesion in the DNA strands was determined by time-dependent exonuclease digestion-LC/ MS-TOF. The presence of the AF-lesion induces significant thermal and enthalpic destabilization. The results will be discussed in terms of the sequence context and the S/B population data obtained from 19F NMR studies (NIH R01-CA098296 and RI-INBRE Core Facility #P20 RR016457). 54. Probing the Sequence Effect on the Conformational Heterogeneity of FluoroaminofluoreneModified DNA Duplexes by 19F NMR. Srinivasa Rao Meneni and Bongsup P. Cho. Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, 41 Lower College Road, Kingston, Rhode Island 02881. Fax: 401-874-5766. E-mail: [email protected]. The sequence effects of aminofluorene (AF) modification on the base-displaced stacked (S)/external B-type (B) conformational heterogeneity have been investigated for a number of DNA duplexes using 19F NMR. We have prepared 16 fluorinated AF (FAF)-modified DNA duplexes as 19F model probes in which the nucleotides flanking the lesion (CTTCTNG*NCCTC: G*)dG-C8-

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FAF; N ) dG, dC, dA, dT) are varied systematically. Twodimensional chemical exchange/NOESY, H/D isotope, and dynamic exchange 19F NMR experiments, along with complete line-shape analysis, were carried out for each of the fully complementary DNA duplexes. The results indicate that the extent of S-conformer population is markedly sequence dependent and that the identity of bases flanking the adduct influences greatly the stacking ability of the base-inserted carcinogenic ring of S-conformers. Molecular mechanics simulation provides valuable structural insights into the sequence-dependent S/B conformational heterogeneity and the base stacking preference (NIH R01CA98296 and RI-INBRE Core Facility #P20 RR016457). 55. Electrophoretic Mobility Shift Assay on the Formation of Klenow Fragment Exo-/Fluoroaminofluorene-Modified Template-Primer Complexes Using IRDye-700-Labeled Oligonucleotides. Fengting Liang, Aftab Ahmed, and Bongsup P. Cho. Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, Kingston, Rhode Island 02881. Fax: 401-874-5766. E-mail: [email protected]. Studies of adduct-induced conformational heterogeneity are motivated by the need to understand different mutagenic outcomes. An ultimate challenge is to conduct conformation-specific adduct mutagenesis in a biologically relevant environment. As part of a program to achieve that goal, we developed a new fluorescence-based gel-shift assay procedure to measure the complex formation of Klenow fragment exo- (KFexo-) with a carcinogenmodified template-primer DNA. The 68kDa KFexo- of Escherichia coli DNA polymerase I is one of the most extensively studied polymerases that lacks the 3′-5 editing activity but exhibits normal polymerase activity. The substrate was a 16-mer DNA modified by 7-fluoro2-aminofluorene annealed with a primer whose 5′-end is linked with an IRDye. The presence of the lesion causes a decrease in Kd and the formation of dimeric complexes is increased with increasing protein concentration. Preliminary 19F NMR and synthetic work will also be presented as well (NIH R01-CA098296 and RI-INBRE Core Facility #P20 RR016457). 56. Kinetic Analysis of the Sequence Context Effects on Polymerization Catalyzed by HIV-1 Reverse Transcriptase. Rio Yamanaka and John Termini. Division of Molecular Biology, Beckman Research Institute of the City of Hope, 1450 East Duarte Road, Duarte, California 91010. E-mail: [email protected]. The crystal structure of the HIV-1 RT complex with template/primer reveals extremely significant interactions with positions 2-6 base pairs upstream from the polymerase active site in the minor groove binding track, regarded as important in DNA/RNA binding and processivity. We examined the effects of RNA template sequence context on HIV-1 RT-catalyzed polymerization. Specific base substitutions were made to the template at either the -2 or -6 positions of the nucleotide insertion site. Substitution of adenine for cytosine at the -6 position results in a 14-fold increase in the steady state kinetic parameter (kcat/Km) for incorporation of dATP opposite U. Presteady state experiments were employed to determine KDdATP, KDTemplatePrimer, and kpol in the reactions for both templates. The base pair at the -6 position has a significant effect on the efficiency of nucleotide insertion by HIV-1 RT, presumably by modulating enzyme binding to the primer/template complex.


57. Structures of a Food Carcinogen-DNA Adduct in Replicative and Bypass Polymerases Using Molecular Dynamics Simulations. Ling Zhang,1 Robert Shapiro,1 and Suse Broyde.2 (1) Department of Chemistry, New York University, 100 Washington Square East, New York, New York 10003. E-mail: [email protected]. (2) Department of Biology, New York University. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant of the carcinogenic heterocyclic aromatic amines in the human diet, and the major PhIPderived DNA adduct is to C8 of guanine. We have investigated this adduct in a mutational hotspot sequence, 5′-GGGA-3′, of the Apc tumor suppressor gene. This sequence is a hotspot for PhIP-induced -1 deletions, and G to T transversions caused by PhIP are also found here. It is currently believed that bulky adducts induce mutations predominantly through a mechanism involving polymerase stalling in a replicative polymerase, followed by error-prone adduct bypass in a Y family polymerase. We have carried out a molecular dynamics study to elucidate on a structural level the effect of the dG-C8-PhIP adduct on the active site of DNA polymerases. Specifically, we investigated a pol R family replicative type polymerase, RB69 (Franklin et al. 2001), and the Dpo4 Y-family bypass polymerase (Ling et al. 2001). We considered nucleotide incorporation with normal partner C or mismatched A opposite the adduct. Our results show that the adduct can reside on the major groove side of the modified DNA template in the replicative DNA polymerase. An occasionally sampled nearreaction ready distance between the 3′-terminal oxygen of primer and PR of incoming nucleotide indicates a rare possibility of error-free nucleotide incorporation and induction of a G to T transversion mutation. However, disturbance of the active site suggests that the predominant effect of the adduct in the replicative polymerase is to cause the polymerase to stall, which could lead to a switch to a bypass polymerase. Our study of the adduct in Dpo4 shows that both a G‚A mismatch and a -1 deletion can be readily accommodated in the polymerase active site. Supported by NIH, 2RO1 CA75449. 58. 2-Amino-1,7-dimethylimidazo[4,5-g]quinoxaline (7-MeIgQx): A Novel and Abundant Mutagenic Heterocyclic Aromatic Amine Formed in Grilled Beef. Robert J. Turesky,1 David LeMaster,2 Lynn Mcnaughton,2 Ricky D. Holland,3 Rebecca W. Wu,4 and James S. Felton.4 (1) Division of Environmental Disease Prevention, Wadsworth Center, NYS Department of Health, Empire Plaza, P.O. Box 509, Albany, New York 12201. Fax: 518-486-1505. (2) Division of Biomedical Sciences, Wadsworth Center, NYS Department of Health. (3) Division of Systems Biology, NCTR, U.S. Food and Drug Administration. (4) Biology and Biotechnology Research Program, Lawrence Livermore National Laboratory. Heterocyclic aromatic amines (HAAs) are potent mutagens and animal carcinogens that form in meats prepared under household cooking practices. Some epidemiological studies have reported elevated cancer risk for individuals who frequently consume meats cooked well-done that contain HAAs. 2-Amino-3,8-dimethyl-


imidazo-[4,5-f]quinoxaline (8-MeIQx) and 2-amino-1methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are two of the most prevalent HAAs formed in cooked meats. Recently, we detected six novel HAAs in grilled meats and human urine. One compound is formed in grilled beef at higher concentrations than either 8-MeIQx or PhIP and is the most abundant of all of the HAAs measured in human urine. This HAA has been identified as 7-MeIgQx, based upon chromatographic, UV, and mass spectral data that are indistinguishable to those of the synthetic compound. 7-MeIgQx is a potent bacterial mutagen in Salmonella typhimurium YG1024 strain, suggesting bioactivation by cytochrome P450 N-oxidation, followed by O-acetylation. Further investigations on the genotoxic properties of this novel HAA are underway. 59. Base-Displaced Intercalated Structure of the Food Mutagen 2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) in the Recognition Sequence of the narI Restriction Enzyme, a Hotspot for -2 bp Deletions. Feng Wang,1 C. Eric Elmquist,2 Carmelo J. Rizzo,3 and Michael P. Stone.3 (1) Department of Chemistry, Vanderbilt University, Box 1822, Vanderbilt University, Nashville, Tennessee 37235. E-mail: feng.wang.1@ vanderbilt.edu. (2) Chemistry, Vanderbilt University. (3) Department of Chemistry, Center in Molecular Toxicology, and Vanderbilt-Ingram Cancer Center, Vanderbilt University. 2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) is a prominent member of heterocyclic mutagens and carcinogens found during the cooking of meats. The C-G1-G2-C-G3C-C recognition sequence of the NarI restriction enzyme represents a hot spot for -2 frameshift mutations at G3. We prepared and purified the oligodeoxynucleotide duplex containing the [IQ]dG adduct positioned in the C-[IQ]G3-C NarI context, which was named NarIIQ3. NMR data revealed a base-displaced intercalated conformation. Watson-Crick base pairing was perturbed at the adduct site. The largest chemical shift perturbations were observed for the base aromatic protons in the complementary strand, opposite to the adduct. Chemical shift perturbations were also observed for the 31P resonances corresponding to the linkages between C6 and [IQ]G7, [IQ]G7 and C8, G17 and C18, and C18 and G19, the phosphodiesters around the adduct. There were 21 NOEs observed between the IQ moiety and the DNA protons. The [IQ]G3 aromatic protons had strong NOEs with the sugar protons of the opposite C18, G17, and G19 in the complementary strand, and the methyl protons of [IQ]G3 showed NOEs to C8 H6, G19 H8, G17 NH1, and G19 NH1. Deoxyribose pseudorotational angles (P) were estimated by examining the 3JHH of sugar protons. J1′-2′ and J1′-2′′ were measured from ECOSY spectra, while the intensities of H2′′-H3′ and H3′-H4′ cross-peaks were determined from DQF-COSY spectra. Molecular dynamics calculations on NarIIQ3, restrained by 1H NOEs and J couplings, yielded ensembles of intercalated structures in which the modified guanine was in a syn alignment and displaced. NOE intensities calculated using complete relaxation matrix theory were in agreement with experimental NOE intensities. Structural differences between NarIIQ3 and NarIAF3 could provide insight into the greater biological activity of IQ than that of AF. Funded by Grant CA-55678. 60. In vitro Replication and Repair Studies of a Novel Intrastrand Cross-Link Lesion G[8-5]C. Chunang Gu. Environmental Toxicology Graduate Pro-

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gram, University of California at Riverside, Mail Drop 027, Riverside, California 92521-0403. E-mail: cgu001@ student.ucr.edu. Yinsheng Wang. Department of Chemistry, University of California at Riverside. We recently identified a new γ-irradiation-induced intrastrand cross-link lesion G[8-5]C, in which the C8 carbon atom of guanine and the C5 carbon atom of its 3′ neighboring cytosine are covalently bonded. Primer extension assays showed that both replicative polymerases, i.e., exonuclease-deficient bacteriophage T7 DNA polymerase and HIV-RT, stopped synthesis after incorporating the correct nucleotide opposite the 3′cytosine in the G[8-5]C lesion, whereas polymerase η was able to replicate past the cross-link lesion. Moreover, we are in the process of characterizing the binding and incision of double-stranded oligodeoxynucleotides bearing a G[8-5]C or a dimeric TT photoproduct, i.e., T[c,s]T or T[6-4]T, by Escherichia coli Uvr(A)BC exonuclease. The goal for the latter experiments is to determine whether the G[8-5]C lesion can be recognized and cleaved similarly as TT photoproducts by NER enzymes. Through these replication and repair studies, we can gain further insights into the biological implications of the intrastrand cross-link lesion G[8-5]C. 61. Disulfide Trapping and Structural Characterization of Complexes of DNA Glycosylases with Undamaged DNA. Anirban Banerjee and Gregory L. Verdine. Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138. Fax: 617-495-8755. DNA glycosylases form the cornerstone of the base excision repair pathway whereby they are burdened with the dual responsibility of locating the damage and excising it from DNA by a nucleophilic reaction. There exists a number of representative cocrystal structures of DNA glycosylases bound to substrate DNA, which reveals the general mechanisms of extrusion and recognition of damaged bases. However, there is no structural characterization of any complex between DNA glycosylases and nonsubstrate undamaged DNA. By using a disulfide cross-linking strategy, we have been able to isolate and crystallize complexes of DNA glycosylases at different stages of interrogating normal DNA [(2005) Nature 434, 612-618]. Together, these structures represent intermediates in the base excision repair pathway hitherto not characterized in atomic detail. 62. Repair of Oxidized Guanines by OG Glycosylases. Nirmala Krishnamurthy,1 Kazuhiro Haraguchi,2 Carissa J. Wiederholt,2 James G. Muller,1 Cynthia J. Burrows,1 Marc M. Greenberg,3 and Sheila S. David.1 (1) Department of Chemistry, University of Utah, 315 South, 1400 East, Salt Lake City, Utah 84112. E-mail: [email protected]. (2) Johns Hopkins University. (3) Department of Chemistry, Johns Hopkins University. Oxidation of guanine in DNA generates a plethora of base lesions including 7,8-dihydro-8-oxoguanine (8-oxoG), imidazole ring-opened purines (FapydG and FapydA), and the hydantoin lesions, guanidinohydantoin(Gh) and spiroiminodihydantoin(Sp). To avoid the deleterious effects of oxidative DNA damage, organisms have evolved the base excision repair (BER) pathway initiated by the DNA glycosylases. This work is a detailed kinetic comparison of the recognition and repair of these base lesions by the bacterial glycosylase, Fpg, and the functional homologues, yOGG2 and hOGG1. The study reveals that FapydG and FapydA are removed by all of the three

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enzymes much faster than 8-oxoG. However, Gh and Sp are removed less efficiently than 8-oxoG by Fpg, while they are better substrates with yOGG2 and are not substrates for hOGG1. The removal of the oxidized base is also influenced by the base opposite (C >A) with all three enzymes. This work provides important insights into the damage recognition features of these enzymes, as well as revealing the potentially important substrates. 63. Structural and Thermodynamic Features of Spiroiminodihydantoin Oxidative Lesions in Duplex DNA by Molecular Dynamics Simulations. Lei Jia,1 Vladimir Shafirovich,1 Robert Shapiro,1 Nicholas E. Geacintov,1 and Suse Broyde.2 (1) Department of Chemistry, New York University, 100 Washington Square East, New York, New York 10003. (2) Department of Biology, New York University. Oxidation of guanine or 8-oxo-7,8-dihydroguanine can produce spiroiminodihydantoin (Sp) R and S stereoisomers. Both in vitro and in vivo experiments have shown that the Sp stereoisomers are highly mutagenic, causing G to C and G to T transversion mutations. Therefore, these oxidative DNA lesions are of interest as potential endogenous cancer-causing forms of DNA damage. However, their structural properties in DNA duplexes remain to be elucidated. We have employed computational methods to study the Sp lesions in 11mer DNA duplexes paired with A, C, and G partner bases in the complementary strands. Molecular dynamics simulations have been carried out to obtain ensembles of structures, and the trajectories were employed to analyze the structures and compute free energies. The structural and thermodynamic analyses reveal that the Sp stereoisomers favor conformations in the B-DNA major groove, irrespective of stereochemistry or partner base, and that the R and S stereoisomers adopt opposite orientations with respect to the 5′ to 3′ direction of the modified strand. The syn glycosidic bond conformation is equally or more stable than anti. The lesions adversely impact base stacking and hydrogen bonding interactions, as well as cause widening of the B-DNA major groove. These structural properties may relate to observed mutagenic properties of the Sp stereoisomers, including possible stereoisomer-dependent differences. Supported by NIH Grant 2RO1 CA 75449 (S.B.) and NIH Grant ES 11589 (V.S. and N.E.G.). 64. Biochemical Characterization of Formamidopyrimidine Lesions. Jennifer N. Patro,1 Carissa J. Wiederholt,1 Yu Lin Jiang,2 Kazuhiro Haraguchi,1 and Marc M. Greenberg.1 (1) Department of Chemistry, Johns Hopkins University, Baltimore, Maryland 21218. Email: [email protected]. (2) Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine. The formamidopyrimidine lesions (Fapy‚dG, Fapy‚dA) are formed in DNA as a result of oxidative stress. These structurally novel lesions cause misreplication by polymerases that can lead to mutations in DNA. Klenow exomisincorporates dA opposite Fapy‚dG more frequently than it does opposite native nucleotides. We investigated the replication of templates containing Fapy‚dG using Klenow exo+ in order to take into account the enzyme’s proofreading activity. We also investigated the ability of various repair enzymes to excise these lesions from DNA. Fapy‚dG and Fapy‚dA were substrates for endonuclease III and endonuclease VIII. However, the dependency on opposing nucleotide indicates that these enzymes do not play an important role in the repair of formamidopyri-


midines. Finally, the formamidopyrimidines exist as a mixture of R- and β-anomers in solution. This led us to use endonuclease IV as an enzymatic probe in order to determine the configurational composition of these lesions in duplex DNA. These experiments indicated that the formamidopyrimidines exist almost exclusively in the β-configuration in DNA.

65. AEI: Analysis of 2-Deoxyribonolactone in DNA Using Cysteine Conjugates. Liang Xue, Kousuke Sato, and Marc M. Greenberg. Department of Chemistry, Johns Hopkins University, New Chemistry Building 336, Baltimore, Maryland 21218. E-mail: [email protected]. 2-Deoxyribonolactone is formed in greater yields in DNA than previously believed. In addition, its interactions with DNA repair enzymes and its mutagenicity in Escherichia coli are distinctive. These properties raise the level of importance for detecting this lesion in DNA. However, detection methods for different types of abasic lesions are often limited to mass spectrometric analysis of individual lesions in conjunction with chemical or enzymatic degradation of the nucleic acids. We report a novel method for selectively detecting of 2-deoxyribonolactone (L) in DNA using cysteine conjugates (e.g., 1). Both ELISA-like and fluorescent assays have been developed. The sensitivity and selectivity of these probes will be discussed.

66. In Vitro DNA Ligation of Oligodeoxynucleotides Containing Oxidized Purine Lesions Using Bacteriophage T4 DNA Ligase. Xiaobei Zhao,1 James G. Muller,1 Mohan Halasyam,2 Sheila S. David,2 and Cynthia J. Burrows.1 (1) Department of Chemistry, University of Utah, Salt Lake City, Utah 84112. E-mail: [email protected]. (2) Department of Chemistry Utah State. 8-Oxo-7,8-dihydroguanine (OG) is considered a biomarker of oxidative DNA damage caused by reactive oxygen species (ROS). Ligation experiments were carried out with OG located at four positions surrounding a single-strand nick in a DNA duplex using bacteriophage T4 DNA ligase. It was found that T4 DNA ligase is most sensitive to lesions on the 3′end of the nick. Ligations with 8-oxo-7,8-dihydroadenine (OA) and 8-oxo-7,8-dihydrohypoxanthine (OI) were used in comparison with OG. OA can base pair with T in anti-anti conformation while OI has similar ligation efficiency with OG by favoring base pairing with A in a syn-anti conformation in which the C8 carbonyl group is positioned in the minor groove. OG is readily oxidized to secondary oxidation products, such as guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) nucleosides. Gh and Sp prefer to base pair with A and G although the overall ligation efficiencies are much lower than most OG base pairs.


67. Defining the Basis for the Unusual Sequence Selectivity of Nitrosoperoxycarbonate-Induced Guanine Oxidation. Yelena Margolin,1 Vladimir Shafirovich,2 Nicholas E. Geacintov,2 and Peter C. Dedon.1 (1) Biological Engineering Division, NE47-277, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139. Fax: 617-324-7554. E-mail: [email protected]. (2) Department of Chemistry, New York University. We have previously demonstrated that nitrosoperoxycarbonate, a chemical mediator of inflammation in humans, selectively oxidizes guanine bases in 5′-GC-3′ steps that also have the highest calculated guanine ionization potentials of all other sites in double-stranded DNA. This selectivity contrasts with other classical, one-electron oxidants, such as photoactivated riboflavin, that preferentially oxidize guanines with the lowest ionization potentials as in runs of guanine (e.g., 5′-GG-3′ and 5′GGG-3′). To define the basis for this unusual behavior of nitrosoperoxycarbonate, we have undertaken several studies that address the role of solvent exposure in the sequence selectivity of this oxidant, including mispairing of guanine with adenine, thymine, and an abasic site. These studies provide an opportunity to compare the stability of the mismatch, as expressed in the melting temperature perturbation, with guanine reactivity. Additionally, we have compared the reactivity of nitrosoperoxycarbonate to that of other oxidants with different charge characteristics to assess the role of negative charge in the guanine oxidation sequence selectivity. 68. Oxidative DNA Damage Induced by Carbonate Radical Anions: Laser Flash Photolysis and Gel Electrophoresis Studies. Young Ae Lee,1 Byeong Hwa Yun,1 Conor Crean,2 Yelena Margolin,3 Peter C. Dedon,3 Nicholas E. Geacintov,2 and Vladimir Shafirovich.2 (1) Chemistry, New York University, 29 Washington Place, New York, New York 10003. E-mail: [email protected]. (2) Department of Chemistry, New York University. (3) Biological Engineering Division, NE47-277, Massachusetts Institute of Technology. Carbonate radical anions are potentially important oxidants of nucleic acids in living systems. There is growing evidence that, in vivo, these radicals are generated in reactions of peroxynitrite with carbon dioxide. However, the mechanisms of reaction of carbonate radical anions with nucleic acids are poorly understood. We have investigated the reaction rates, using laser transient absorbance techniques, of carbonate radical anions (concentrations monitored at 600 nm) with guanines in 1122-mer oligonucleotides with either single G, GG, or GGG sequence contexts surrounded by thymidines in each case. The electron transfer rate constants of DNA oxidation by carbonate radicals are in the range of (0.5-2.5) × 107 M-1 s-1 and essentially independent of the number of contiguous guanines in the oligonucleotides. The electron transfer rate is only weakly dependent on the temperature (activation energy of 0-4 kcal/mol). DNA oxidation of guanines by carbonate radicals results predominantly in the formation of diastereomeric spiroiminodihydantoin (Sp) lesions. The Sp nucleobases obtained after the chemical hydrolysis of the Sp adducts with HF in pyridine were isolated by HPLC and identified by ESIMS/MS analysis. The distributions of alkali-labile guanine lesions induced by carbonate radical anions in different sequence contexts have been analyzed by gel electrophoresis. The impact of base sequence on the yield

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of oxidative DNA damage induced by carbonate radical anions is discussed. Supported by NIH Grant 1 R01 CA110261. 69. Sensitive Methods to Quantify C4’ Deoxyribose Lesions Produced by Bleomycin and γ-Radiation. Xinfeng Zhou, Bingzi Chen, and Peter C. Dedon. Division of Biological Engineering, MIT, 500 Technology Square, Cambridge, Massachusetts 02139. Fax: 617-3247554. Oxidation of deoxyribose in DNA produces a wide range of lesions, with a different spectrum of products for each position in the sugar. Because of both solvent accessibility and issues of site-specificity, oxidation of the C4x position of deoxyribose is common to many DNA damaging agents, including ionizing radiation and the antitumor antibiotic bleomycin. The chemistry of C4′ oxidation by a variety of agents partitions along either of two pathways to form an abasic site lesion, 2-deoxypentos-4-ulose, or a strand break comprised of a 3′phosphoglycolate residue on one end with release of either malondialdehyde and free base with ionizing radiation or base propenals with bleomycin. 3x-Phosphoglycolate is a major 3′- terminal blocking group that must be removed prior to polymerase-mediated gap-filling reactions, while the 2-deoxypentos-4-ulose abasic site is a strong electrophile that has the potential to react with a variety of nucleophiles (protein, DNA bases) to form adducts. To provide direct and definitive chemical evidence for the formation of deoxyribose oxidation products in vivo and to better understand the chemical mechanisms of deoxyribose oxidation related to genotoxin chemistry, we have developed highly sensitive gas chromatography-mass spectrometry (GC/MS) methods to quantify 3x-phosphoglycolate residues and 4x-keto-1xaldehyde abasic sites, and we have applied these methods to quantify 4′-chemistry caused by γ-radiation and Fe(II)‚bleomycin. The abasic site method entails conversion of the 2-deoxypentos-4-ulose to a 3-hydroxymethylpyridazine (HMP) residue by reaction with hydrazine, which, together with the 3′-phosphoglycolate, is released by enzymatic hydrolysis and quantified by isotope dilution GC/MS. The detection limits are ∼500 fmol for 3′-phosphoglycolate and ∼100 fmol for the 2-deoxypentos-4-ulose abasic site. These methods provide a means to define the determinants of deoxyribose oxidation in isolated DNA and in cells subjected to oxidative stress. 70. DNA Oxidative Damage by Analogues of Diterpenone Catechol. Miguel A. Zuniga, Mark Wehunt, and Qibing Zhou. Department of Chemistry, Virginia Commonwealth University, 1001 West Main Street, Richmond, Virginia 23284. Fax: 804-828-8599. E-mail: [email protected]. Natural terpene quinone methides (TPQMs) exhibit broad biological activity against bacteria, fungi, and tumor cells. The antitumor activity of triterpene quinone methides has been proposed through DNA damage by reactive QM and quinone intermediates during the enzymatic oxidation. We believe that in addition to the reactive quinone intermediates, the stereochemistry and the substituent may play a key role in the biological activity. On the basis of model studies, we found that the cis-analogues had enhanced stability over the transanalogues, so were the analogues with a m-methyl substituent. These results led us to investigate the DNA damage by a series of diterpenone analogues. The DNA

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damage by diterpenone analogues in the presence of Cu2+ included direct DNA cleavage and nucleobase oxidation by in situ generated hydroxide radicals. Our DNA assays revealed that the catechol analogues resulted in more extensive DNA damage than the QM analogues, and the trans-analogues were more reactive than other diastereoisomeric analogues. In addition, the presence of a reducing agent such as NADH significantly enhanced the extent of DNA damage by all of the diterpenone analogues, which was consistent with reported redox mechanisms under a reductive environment. 71. Identification and Quantitation of Hepatic DNA Adducts in N-Nitrosopyrrolidine (NPYR)Treated Rats. Mingyao Wang,1 Yongli Shi,1 Yanbin Lao,2 Peter W. Villalta,1 and Stephen S. Hecht.1 (1) University of Minnesota Cancer Center, 420 Delaware Street Southeast, MMC 806, Minneapolis, Minnesota 55455. Fax: 612-626-5135. E-mail: [email protected]. (2) Department of Medicinal Chemistry and the Cancer Center, University of Minnesota. NPYR is a well-established carcinogen in rodents and may be formed endogenously in humans. NPYR is metabolically activated by R-hydroxylation. The resulting metabolite, R-hydroxyNPYR, rearranges to 4-oxobutane1-diazohydroxide and related electrophiles, which react with DNA forming multiple adducts with deoxyguanosine (dG). These include N2-(tetrahydrofuran-2-yl)dG (N2(THF)dG), which was previously identified. Recently, N6(THF)dA and N4 -(THF)dC were characterized in reactions of dA, dC, and DNA with R-acetoxyNPYR, a stable precursor to R-hydroxyNPYR. We now report the identification and quantitation of N2-(THF)dG and N6-(THF)dA in hepatic DNA of rats treated with NPYR. For quantitation, DNA was treated with NaBH3CN, followed by enzyme hydrolysis. N2-(4-Hydroxybut-1-yl)dG, N6-(4hydroxybut-1-yl)dA, and N4 -(4-hydroxybut-1-yl)dC were analyzed by capillary LC-ESI-MS/MS-SRM. For further confirmation, the DNA was subjected to neutral thermal hydrolysis to release 4-hydroxybutyraldehyde, determined as its 2,4-dinitrophenylhydrazone. These adducts may be important in hepatocarcinogenesis by NPYR and could be used as biomarkers to assess NPYR exposure and metabolic activation in humans. 72. Complex Products from the Nitrosation of N-Arylpyrrolidines. Richard N. Loeppky,1 Sukhjeet S. Singh,2 and Yinan Li.1 (1) Department of Chemistry, University of Missouri, 125 Chemistry Building, Columbia, Missouri 65211. Fax: 573-882-2754. E-mail: [email protected]. (2) Bristol Meyers Squibb. Compounds containing the N-substituted pyrrolidine moiety are found in both commercial and natural products. The nitrosation chemistry of these tertiary amines is poorly understood but of importance from the viewpoint of carcinogenesis. A study of the nitrosation of N-4chlorophenylpyrrolidine 1 (Ar ) 4-ClPh) showed that it rapidly reacts with nitrous acid in HOAc at room temperature to produce a solid as the major reaction product. The structure of the major product 2 was assigned on the basis of 1D and 2D H, C-13, and N-15 NMR spectral analysis and finally X-ray crystallography of the corresponding BF4 salt. The aldehyde 3 is the expected product of ring opening, and 4 is formed from it. Both 2 and 5, and perhaps 3, are thought to arise from an intermediate radical cation. The formation of the dimeric product 2 is novel in the realm of nitrosation chemistry.


73. Toxicity Screening Using Biocatalyst/DNA Films and CapLC-MS/MS. Maricar C. Tarun. Department of Chemistry, University of Connecticut, 55 North Eagleville Road, Storrs, Connecticut 06269. Fax: 860486-2981. E-mail: [email protected]. The vast number of new chemicals and drugs synthesized annually require alternate toxicity screening procedures to complement conventional microbiology and animal toxicity testing. A major genotoxicity pathway in the mammalian liver involves cytochrome P450 enzymecatalyzed bioactivation of lipophilic molecules to produce metabolites that react with DNA. The bioactivated metabolites cause DNA damage by forming covalently bound DNA adducts, which may in turn initiate mutagenesis and carcinogenesis. We have developed a method that mimics the toxicity pathway in mammalian liver and which can be used as a screening technique for genotoxic chemicals. We assembled protein biocatalysts and DNA layer-by-layer on a high surface area solid substrate, i.e., carbon cloth. These protein/DNA films were incubated with molecules that are “metabolized” by the biocatalyst and resulting DNA damage detected by LC-MS/MS. For proof of concept, we used myoglobin (Mb) as the biocatatyst in the film, and the influence of suspected human carcinogen styrene was evaluated. H2O2 activated Mb, which converts styrene to styrene oxide (SO), mimicking the action of cytochrome P450. The in situ-generated SO reacted with DNA on the film. After incubation, the films were subjected to neutral thermal hydrolysis, a procedure that selectively releases guanine adducts alklyated at N7 and adenine adducts alkylated at N3. The hydrolysate was analyzed for the presence of N7-guanine-styrene oxide (N7-GSO) using CapLC-MS/ MS with an on-line preconcentration column. MRM for the transition m/z 272 f 152 was used for the analyses, since GSO adduct was found as the major adduct. GSO was detected in films incubated in styrene and H2O2 allowing estimation of relative kinetics within ∼10 min of reaction. This new method for genotoxicity screening is simple, highly sensitive, and allows for measurement of relative rates of DNA damage with high chemical information. 74. Fluorescence-Based Assay for DNA Damage Induced by Toxic Chemicals. Srividya Kailasam. National Exposure Research Laboratory, National Research Council Associate, U.S. Environmental Protection Agency, 944 East Harmon Avenue, Las Vegas, Nevada 89119. Fax: 702-798-2107. E-mail: Kailasam.Srividya@ epa.gov. Kim R. Rogers, National Exposure Research Laboratory, U.S. Environmental Protection Agency. Many toxic industrial chemicals are not only acutely toxic but are genotoxic as well. A simple, rapid, and innovative assay to detect DNA damage resulting from exposure of surrogate DNA to toxic industrial chemicals (acrolein, allylamine, chloroacetone, formaldehyde, acrylonitrile, bromoethane, and crotonaldehyde), nanomaterials (buckminster fullerene), and some of the well-known genotoxic compounds (mitomycin C, 4-nitroquinoline-N-


oxide, chlorophyllin, and gluteraldehyde) is reported. This assay is based on changes in the melting and annealing behavior observed for damaged DNA, which is monitored using a fluorescence indicator dye, PicoGreen. We found that a 30 min exposure to these chemicals is adequate to cause significant damage to the DNA. The extent of DNA damage induced by the different chemicals will be discussed. The effect of varying different parameters such as concentrations of the chemicals under study, incubation time, and salt concentration on the extent of DNA damage will also be presented. 75. Investigation into Mechanisms of Hypoxia Selective Anticancer Agents, 3-Methyl-1,2,4-benzotriazine-1,4-dioxide and 3-Amino-1,2,4-benzotriazine-1,4-dioxide. Venkatraman Junnotula, Ujjal Sarkar, Sarmistha Sinha, Chad Inman, Charles L. Barnes, and Kent S. Gates. Department of Chemistry, University of MissourisColumbia, 601 South College Avenue, Columbia, Missouri 65211. E-mail: [email protected]. Benzotriazine 1,4-dioxides are bioreductively activated, hypoxia-selective anticancer agents. In vivo, these drugs undergo one-electron reductive activation to generate an activated drug that leads to DNA damage selectively under the low oxygen conditions found in tumor cells. The exact nature of reactive species that cause DNA strand cleavage by these drugs remains a matter of debate. Here, we present the results of various mechanistic studies supporting the idea that one-electron reductive activation of 1,2,4-benzotriazine 1,4-dioxides leads to release of the well-known DNA damaging species hydroxyl radical. Thus, these compounds serve to deliver the active species of radiation therapy selectively to hypoxic cells.

76. Structure-Activity Relationship of G-N7 and G-N2 DNA Monoadducts of Noncytotoxic Metabolite of Mitomycin C, 2,7-Diaminomitosine (2,7DAM). Cristina C. Clement and Maria Tomasz. Chemistry Department, Hunter College, City University of New York, 695 Park Avenue, New York City, New York 10021. E-mail: [email protected]. Our previous studies showed that the guanine (G)-N2 DNA monoadduct of mitomycin C (MC), a cytotoxic anticancer drug, inhibits translesion bypass by Klenow exo-, T7 exo-, and η DNA polymerases. The noncytotoxic MC metabolite 2,7-diaminomitosene (2,7-DAM) forms a G-N7 DNA monoadduct in vitro and in vivo but is noncytotoxic as compared with MC. Our studies showed fully bypassed G-N7-2,7-DAM lesion by all three polymerases, resulting in the production of a fully extended primer. However, the lesion bypass is performed at a slower rate as compared with the control, nonalkylated template; build-up of abortive products before the lesion was observed suggesting that the polymerases are losing the processivity as they encounter the bulky adducts. In this study, we are presenting the first evidence of translesion synthesis (TLS) beyond another monoadduct of 2,7-DAM, the G-N2-DNA monoadduct performed by T7 exo- and Klenow exo-DNA polymerases. The TLS of

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G-N2-2,7-DAM is slower, less efficient than N7-dG-2,7DAM adduct with both Klenow exo- and T7 exo-polymerases. These in vitro TLS studies are presented as the basis for structural investigations of the process of molecular recognition by DNA polymerases of G-N7 and G-N2 DNA monoadducts through molecular modeling.

77. Synthesis of and DNA Damage by a Small Leinamycin Analogue. Kripa Keerthi. Department of Chemistry, University of Missouri, 601, South College Avenue, Columbia, Missouri 65211. E-mail: kdkbb6@ mizzou.edu. Kent Gates. Departments of Chemistry, University of MissourisColumbia. Leinamycin (1) is an antitumor natural product whose DNA-damaging chemistry is unmasked by the reaction of the natural product with water (slow reaction) or thiols (fast reaction). The natural product causes both DNA alkylation and oxidative stress. In this poster, we report the synthesis of a simple analogue, 2, that contains the “core” functional groups found in leinamycin and the evaluation of the DNA-damaging properties of this analogue.

78. Leinamycin: A Structurally Novel DNA Intercalator. Joseph Szekely,1 Sanjay Dutta,1 and Kent Gates.2 (1) Department of Chemistry, University of MissourisColumbia, 601 South College Avenue, Columbia, Missouri 65211. Fax: 573-882-2754. E-mail: js13a@ mizzou.edu. (2) Departments of Chemistry, University of MissourisColumbia. Leinamycin (1) is a DNA-alkylating antitumor natural product. Efficient DNA alkylation by leinamycin is dependent upon initial noncovalent interaction of the natural product with duplex DNA. We will present data suggesting that leinamycin intercalates on the 3′-side of guanine alkylation sites (2). This work presents leinamycin as a structurally novel DNA intercalator.

79. Mechanistic Studies on the Photosensitizing Activities of 2,3-Diaminophenazine, a Common Contaminant in Hair Dyes. Patty K.-L. Fu,1 Yao Liu,2 Jun-Jie Yin,1 and Claudia Turro.2 (1) Center for Food

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Safety and Applied Nutrition, U.S. Food and Drug Administration, 5100 Paint Branch Parkway, Collee Park, Maryland 20740. Fax: 301-436-2897. E-mail: [email protected]. (2) Department of Chemistry, The Ohio State University. o-Phenylenediamine (OPD) is used extensively in hair dyes and in the synthesis of agricultural pesticides. Air oxidation of OPD results in the formation of 2,3-diaminophenazine (DAP). Although DAP has been shown to be mutagenic in prokaryotes and eukaryotes, its phototoxicity remains largely unexplored. We have investigated the pH-dependent photophysical properties of DAP, its ability to induce light-dependent damage in pUC19 plasmid, and its photocytotoxicity in human skin fibroblasts. The concentration of DAP, which resulted in 50% cell death, was 172 µM in the dark and 13 µM upon irradiation with visible light. The mechanism underlying the photocytotoxicity of DAP was further investigated using ESR spectroscopy. Preliminary data show that DAP photosensitizes the formation of reactive oxygen species. Bathochromic and hypochromic shifts were observed when DAP chelates with heavy metal cations. The role of metal chelation in photosensitization was also investigated. 80. Photocytotoxicity of Retinol and the Role of Photooxidative Damage. Wayne Wamer. Center for Food Safety and Applied Nutrition, FDA, 5100 Paint Branch Parkway, College Park, Maryland 20740. Email: [email protected]. Peter P. Fu. Division of Biochemical Toxicology, National Center for Toxicological Research. Daniel E. Falvey. Department of Chemistry, University of Maryland. Retinol (vitamin A) and its esters are commercially important ingredients in cosmetics applied to sunexposed skin. Therefore, an understanding of the safety of these ingredients requires knowledge of retinol’s photochemistry and photobiology. We have used an in vitro model to examine the mechanism underlying the photocytotoxicity of retinol. We determined that the growth of human dermal fibroblasts is dramatically inhibited by pretreatment for 2 h with 40 µM retinol followed by exposure to as little as 0.5 J/cm2 UVA light (320-400 nm). This photocytotoxicity is inhibited by sodium azide, R-tocopherol, and ascorbic acid. In addition, lipid peroxidation, measured as the formation of malonyldialdehyde, occurs concomitantly with photocytotoxicity. These results suggest that photooxidative damage plays a role in the photocytotoxicity of retinol. In addition, our study indicates that biomarkers for oxidative damage could be valuable end points for measurement in toxicological and clinical studies on the safety of retinol. 81. Cyclooxygenase-Mediated Metabolism of Arachidonic Acid in Rat Intestinal Epithelial Cells. Seon Hwa Lee,1 Michelle V. Williams,1 Raymond N. DuBois,2 and Ian A. Blair.1 (1) Center for Cancer Pharmacology, University of Pennsylvania, 856 BRB II/III, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104-6160. Fax: 215-573-9889. E-mail: seonhwa@ spirit.gcrc.upenn.edu. (2) Department of Medicine, Vanderbilt-Ingram Cancer Center, Vanderbilt University Medical Center. Numerous studies have implicated cyclooxygenases (COXs) as mediators of carcinogenesis. COX-mediated formation of prostaglandins (PGs) has been assumed to play a role in tumorigenesis. In a recent study, we have suggested a potential mechanism of tumorigenesis, which


involves DNA adduct formation. COX-mediated metabolism of arachidonic acid (AA) has been studied in recombinant enzymes or in intact cells after the addition of AA. However, no data exists on AA metabolites formed by rat COXs or the chirality of rat COXs-mediated HETE formation. To address these issues, HETEs and PGE2 were quantified in wild-type rat intestinal epithelial (RIE) cells and RIE cells that express the COX-2 genes permanently (RIES cells). The relative production ratio and the chirality of the AA metabolites were determined in cell lysates treated with AA and in intact cells treated with AA or ionophore A-23187. Supported by NIH Grants RO1 CA 91016 and PO1 CA 92537. 82. Characterization of Lipid HydroperoxideDerived Protein Modifications. Tomoyuki Oe, Seon Hwa Lee, Jasbir S. Arora, and Ian A. Blair. Center for Cancer Pharmacology, University of Pennsylvania, 856 BRB II/III, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104-6160. Fax: 215-573-9889. E-mail: tomo@ spirit.gcrc.upenn.edu. Lipid peroxidation of polyunsaturated fatty acids is thought to play an important role in the degenerative diseases of aging such as cancer. The decomposition of lipid hydroperoxides, which can be initiated by transition metal ions or vitamin C, results in the formation of bifunctional electrophiless4-oxo-2(E)-nonenal, 4-hydroxy2(E)-nonenal, 4,5-epoxy-2(E)-decenal, and 4-hydroperoxy2(E)-nonenal. These bifunctional electrophiles can then covalently modify DNA and proteins. We have recently shown that 4-oxo-2(E)-nonenal reacts with biologically important proteins such as histone H4 and hemoglobin to produce unique adducts. However, protein adducts formed by bifunctional electrophiles are more varied and their identification in biological samples is complicated because large numbers of target proteins and variations in each protein are possible. Here, we demonstrate the possibility for the selective proteomics approach to detect lipid hydroperoxide-derived protein modifications using tagging reagents. Supported by NIH RO1CA95586. 83. Mass Spectroscopic Characterization of Protein-Based Carbonyls Generated by Oxidative Modification. Xiaochun Zhu, Quan Yuan, and Lawrence M. Sayre. Department of Chemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106. E-mail: [email protected]. Oxidative modification of proteins plays an important role in the etiology of age-related degenerative disease. 2,4-Dinitrophenylhydrazine (DNPH)-detectable proteinbased carbonyl is the most commonly used marker of protein oxidative damage, but it is unclear whether carbonyl appearance reflects mainly metal-catalyzed oxidations (MCO) of side chains or conjugate addition of nucleophilic side chains (e.g., His) to lipoxidation-derived R,β-unsaturated aldehydes such as 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE). We have compared the extent of carbonyl generation in a variety of reaction conditions where model proteins such as β-lactoglobulin have been exposed either to MCO or lipoxidation-derived aldehydes. At the same time, we have characterized the sites of carbonyl generation under both conditions using both MALDI-TOF MS and HPLC-ESI-MS following tryptic digestion. DNPH has been previously used as a MALDI matrix. We find that use of DNPH as matrix improves the detection of DNPH-marked peptides relative to the commonly used matrix R-cyano-4-hydroxycin-


namic acid (HCCA). The heterogeneity and selectivity of the two types of oxidative modifications will be discussed. 84. Chemical Model for the Redox Regulation of Protein Tyrosine Phosphatase 1B (PTP1B). Santhosh Sivaramakrishnan, Kripa Keerthi, and Kent S. Gates. Department of Chemistry, University of Missouris Columbia, 125 Chemistry Building, 601 South College Avenue, Columbia, Missouri 65211. E-mail: ssdc9@ mizzou.edu. Protein tyrosine phosphatase 1B (PTP 1B) is inactivated under conditions of oxidative stress. Recent reports indicate that oxidative inactivation of PTP-1B involves a unique active site rearrangement in which a catalytic cysteine thiol residue becomes masked as part of an isothiazolinone heterocycle. The mechanism for this transformation is not completely clear. In this poster, we report the use of model compounds to probe the chemistry underlying oxidative inactivation of PTP-1B. We provide the first direct evidence that conversion of the cysteine residue to its sulfenic acid derivative can lead directly to the isothiazolinone form of the enzyme.

85. Alkylation Stress Triggers Ubiquitination of Modified Proteins. Elizabeth B. Burnette. Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University Medical Center, 465 21st Avenue, 9264 MRBIII, Nashville, Tennessee 37232. E-mail: [email protected]. Daniel C. Liebler, Department of Biochemistry, Vanderbilt University Medical Center. Reactive electrophiles formed by xenobiotic metabolism and oxidative stress damage proteins, but the fate of damaged proteins is poorly understood. Ubiquitination is thought to govern turnover of damaged proteins in eukaryotic cells. Our objective was to identify protein targets of reactive electrophiles and to characterize the ubiquitination of adducted proteins. HEK293 cells expressing His6-ubiquitin were treated for 2 h with 100 µM iodoacetyl-LC-biotin, a prototypical biotin-tagged reactive electrophile. Adducted and ubiquitinated proteins were captured by sequential affinity purification using immobilized streptavidin and Ni-NTA. The captured proteins were subjected to tryptic digestion, followed by multidimensional LC-MS-MS and identification of peptide sequences with Sequest. These analyses identified approximately 400 adducted proteins, at least 75 of which were shown to also be ubiquitinated. Studies are being extended to characterize the mechanism of recognition of these adducted proteins for degradation via ubiquitinmediated proteolysis. Supported by NIH Grants ES007028, ES011811, and ES000267. 86. Identification of a Peptide Adduct Formed with a Phase II Conjugate. William H. Connors,1 Jimmy Flarakos,1 Paul L. Skipper,2 J. S. Wishnok,3 and S. Tannenbaum.4 (1) Biological Engineering Division, MIT, 77 Massachusetts Avenue, Building 56-722a, Cambridge, Massachusetts 02139. Fax: 617-252-1787. Email: [email protected]. (2) Division of Bioengineering & Environmental Health, Massachusetts Institute of Technology. (3) Biological Engineering Division, Mas-

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sachusetts Institute of Technology. (4) Massachusetts Institute of Technology. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP, 1) is a heterocyclic aromatic amine formed in cooked meat that has been found to induce cancer of the breast, colon, and prostate of rats. Evaluation of the carcinogenic role in humans would be enhanced by improvements in exposure assessment that could be achieved with appropriate biomarkers. PhIP is extensively metabolized in vivo, with formation of the presumed carcinogenic 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (2) being a major pathway. In this study, a phase II conjugate of the hydroxylamine was synthesized and the reaction with human serum albumin (HSA) was investigated. The acetoxy conjugate (3) was selected because it has been implicated in the formation of DNA adducts. A peptide adduct of the metabolite was isolated from the tryptic digest of HSA following reaction with the protein. Preliminary results suggest a structure consisting of four amino acids with 1 covalently bound to a homocysteinS-yl residue of methionine. The occurrence of a carcinogenic metabolite forming a homocysteine adduct has been previously reported. Results of experiments to characterize the adduct by mass spectrometry will be described.

87. Metal-Catalyzed Oxidation and Liquid Chromatography Mass Spectrometry for Detection of Amyloid-β Modifications. Koichi Inoue, Tomoyuki Oe, and Ian A. Blair. Center for Cancer Pharmacology, University of Pennsylvania, 856 BRB II/III, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104-6160. Fax: 215-573-9889. E-mail: [email protected]. It has been suggested that Alzheimer’s disease (AD) arises from the aggregation of amyloid-β (Aβ) in the brain. Aβ peptides are derived from the proteolytic processing of the amyloid precursor protein by a combination of β- and γ-secretase enzymes. Aβ is neurotoxic in the aggregated fibrillar form and plays a key role in the pathogenesis of AD. However, a causal relationship between the Aβ and the development of AD has not been conclusively demonstrated. Aβ precipitation and toxicity in AD is thought to be mediated through oxidative stress that is induced by environmental factors. There are three potential sites of oxidative modifications in the first 16 amino acid residues of Aβ1-40 and Aβ1-42, at H6, Y11, H13, and H14. Using a combination of trypsin digestion and tandem mass spectrometry, we have established the precise sites of oxidation that arise from metal-catalyzed oxidation of Aβ1-16, a truncated form of Aβ. The oxidized tryptic fragments that have been obtained will be useful for determining whether similar sites of oxidation can be observed in intact Aβ1-40 or Aβ1-42 obtained from the CSF of AD subjects. 88. LC/MS Identification of Gallic Acid and Procyanidins from Grape Seed Extract with Anticancer Activity in DU145 Human Prostate Carcinoma Cells. Ravikanth Veluri, John A. Thompson, Rajesh

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Agarwal, and Chapla Agarwal. Department of Pharmaceutical Sciences, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Box C238, Denver, Colorado 80262. Fax: 303-315-0274. E-mail: [email protected]. The anticancer efficacy of grape seed extract (GSE) against prostate cancer (PCA) in cells and animal models has recently been described. GSE is a complex mixture containing gallic acid (GA), catechin (C), epicatechin (EC), and procyanidins (oligomers of C and/or EC). An ethyl acetate extract of GSE was separated into fractions I-VIII by gel filtration chromatography, and each was analyzed by HPLC and negative ion LC/MS/MS. Gallic acid (I) was more effective than other fractions in causing growth inhibition and apoptotic death of human PCA DU145 cells. Fractions II and III containing C, EC, and procyanidins B1 (EC-C dimer), B2 (EC-EC dimer), and B3 (C-C dimer) were nearly ineffective, but V-VIII caused significant growth inhibition and apoptosis with the highest activity present in the later fractions containing procyanidin trimers and GA esters of dimers and trimers. These data provide insight into the active constituents of GSE against PCA. 89. Metabolic Activation of PAH trans-Dihydrodiols by Human Aldo-Keto Reductase 1B10. Amy M. Quinn and Trevor M. Penning. Department of Pharmacology, University of Pennsylvania School of Medicine, 135 John Morgan Building, 3620 Hamilton Walk, Philadelphia, Pennsylvania 19104-6084. Fax: 215-573-2236. E-mail: [email protected]. We report that members of the aldo-keto reductase AKR1B subfamily have dihydrodiol dehydrogenase activity in vitro. Recently, AKR1B10 was shown to be overexpressed in nonsmall cell lung carcinoma [Fukumoto et al. (2005) Clin. Cancer Res. 11, 1776-1785], raising the possibility that this enzyme may be able to convert polycyclic aromatic hydrocarbon (PAH) trans-dihydrodiols to generate reactive and redox-active o-quinones such as members of the AKR1C subfamily. Using recombinant AKR1B10, preliminary data suggest that this enzyme can convert the trans-dihydrodiol of benzo[a]pyrene (BP), a ubiquitous PAH and constituent of tobacco smoke, to BP7,8-dione but with the incorrect stereochemistry to be relevant for BP activation in vivo. However, this enzyme can activate both stereoisomers of benz[a]anthracene-3,4diol and 7,12-dimethylbenz[a]anthracene-3,4-diol. These studies implicate an important role for AKR1B10 in PAH activation and tobacco smoke carcinogenesis. Supported by Grants R01-CA39504 and P01-CA-092537 to T.M.P. 90. PAH o-Quinones Produced by the AKR Pathway Predominantly Generate Abasic Sites and 8-Oxo-dGuo via Reactive Oxygen. Jong Heum Park. Department of Pharmacology, University of Pennsylvania School of Medicine, 135 John Morgan Building, 3620 Hamilton Walk. Fax: 215-573-2236. E-mail: [email protected]. Trevor M. Penning. Department of Pharmacology, University of Pennsylvania, 130c John Morgan Building, 3620 Hamilton Walk. Fax: 215-573-2236. E-mail: penning@ pharm.med.upenn.edu. PAHs require metabolic activation to exert their deleterious effects. Among these pathways, only aldo-keto reductases activate PAH trans-dihydrodiols to their corresponding o-quinones, which oxidatively damage DNA. We employed the aldehyde reactive probe to detect the formation of abasic sites in calf thymus DNA exposed


to BP-7,8-dione and DMBA-3,4-dione in the presence and absence of redox-cycling conditions. The formation of 8-oxo-dGuo was detected by coupling the assay with 8-oxoguanine N-glycosylase (hOGG1) and was independently validated by HPLC-ECD. In the absence of redoxcycling, a modest amount of aldehydic sites was detected indicating a low level of covalent quinone depurinating adducts. In the presence of NADPH and CuCl2, PAH o-quinones increased the formation of abasic sites and 8-oxo-dGuo and their formation was blocked by NaN3. This assay showed that the dominant source of aldehydic sites in the DNA were lesions caused by reactive oxygen and not by PAH o-quinone N7-guanine depurinating adducts. Supported by Grants R01-CA39504 and P01CA-092537 to T.M.P. 91. Measuring PAH-Induced Oxidative Stress in Human Lung Cells. Kirk A. Tacka. Department of Pharmacology, University of Pennsylvania School of Medicine, 135 John Morgan Building, 3620 Hamilton Walk. Fax: 215-573-2236. E-mail: tacka@ mail.med.upenn.edu. Trevor M. Penning. Department of Pharmacology, University of Pennsylvania, 130C John Morgan Building, 3620 Hamilton Walk. Fax: 215-8987180. E-mail: [email protected]. Polycyclic aromatic hydrocarbons (PAH) require metabolic activation to exert their genotoxic effects. PAH trans-dihydrodiols can be converted to PAH o-quinones by members of the aldo-keto reductase (AKR) superfamily. PAH o-quinones can undergo redox cycling to generate reactive oxygen species (ROS) causing oxidative stress. The ability of the PAH o-quinone benzo[a]pyrene7,8-dione (BPQ) to generate intracellular ROS in human lung cells (A549 and H358) was measured by fluorescent microscopy using the probe 2′,7-dichlorodihydrofluorescein diacetate (H2DCF-DA). Treatments with BPQ produced a dose-dependent increase in ROS generation in both cell lines. In addition, A549 cells (AKR1C1-3 and AKR1A1 overexpressing) when treated with the transdihydrodiol precursor of BPQ (BP-diol) generated ROS. By contrast, ROS generation was not observed in H358 cells (AKR-null) when treated with BP-diol, suggesting that PAH-induced ROS generation is AKR-dependent. The consequences of PAH-induced ROS generation on the redox state [GSSG/GSH and NAD(P)+/NAD(P)H] of lung cells will be presented. Supported by Grants R01CA39504 and P01-CA-092537 to T.M.P. 92. Evaluating the Scientific Evidence for Potential Reduced-Risk Tobacco Products. Amy M. Brownawell, Michael C. Falk, Keith Lenghaus, Kara D. Lewis, Paula M. Nixon, and Catherine St. Hilaire. Life Sciences Research Office, 9650 Rockville Pike, Bethesda, Maryland 20814. E-mail: [email protected]. The Life Sciences Research Office (LSRO), a nonprofit biomedical research organization, is evaluating the science base necessary to substantiate claims for potential reduced-risk tobacco products and is developing a detailed agenda to address current research gaps. If feasible, LSRO will also develop an evaluative process for the scientific assessment of potential reduced-risk tobacco products. This review is intended to build on the findings and recommendations of a Committee of the Institute of Medicine, which are detailed in the report, Clearing the Smoke: Assessing the Science Base for Tobacco Harm Reduction. LSRO has convened a multidisciplinary, advisory committee to guide the project and provide a risk characterization. Satellite committees will provide


independent analyses of the state-of-the-science in three areas: hazard identification and dose-response assessment, individual exposure assessment, and population exposure and behavior assessment. This project is sponsored by Philip Morris USA. 93. Mechanism-Based Inactivation of Human Cytochrome P450 2B6 by Substituted Phenyl Diaziridines. Satish Goud Puppali,1 John M. Rimoldi,1 Chitra Sridar,2 Yoshimasa Kobayashi,2 and Paul F. Hollenberg.2 (1) Department of Medicinal Chemistry, University of Mississippi, 417 Faser Hall, School of Pharmacy, University, Mississippi 38677. E-mail: [email protected]. (2) Department of Pharmacology, University of Michigan. The cytochrome P450 enzymes represent a superfamily of hemoproteins that catalyze a variety of endogenous and xenobiotic oxidation reactions. The development of mechanism-based inactivators for specific CYP450 isozymes is critical for studying structural and mechanistic information concerning the active site. A series of phenyl-substituted diaziridines were designed to serve as “phenylhydrazine isosteres” and examined for their ability to inactivate a set of six purified cytochrome P450s. Several of the diaziridines inhibited CYP450 2B6 in a reconstituted system in a time-, concentration-, and NADPH-dependent manner. Initial results suggest that inactivation is a consequence of protein modification and does not involve destruction of heme. The synthesis, kinetic analysis, and profiles of inactivation of CYP450 2B6 will be presented. The work is supported in part by NIH Grant CA16954, Daiichi Pharmaceutical Co., Ltd., and a grant from the Centers for Disease Control and Prevention.

94. Synthesis of Fenthion Sulfoxide and Fenoxon Sulfoxide Enantiomers: Effect of Sulfur Chirality on Acetylcholinesterase Activity. Rama Sarma Gadepalli,1 Mae Nillos,2 Jay Gan,2 Daniel Schlenk,2 Frank R. Fronczek,3 and John M. Rimoldi.1 (1) Department of Medicinal Chemistry, University of Mississippi, 417 Faser Hall, School of Pharmacy, University, Mississippi 38677. E-mail: [email protected]. (2) Department of Environmental Sciences, University of California Riverside. (3) Department of Chemistry, Louisiana State University. Flavin-containing monooxygenases (FMO) are responsible for catalyzing the sulfur oxidation of specific organophosphate insecticides. Recombinant FMO1 has been shown to metabolize fenthion to (+)-fenthion sulfoxide in a stereoselective fashion. To elucidate the absolute configuration of the sulfoxide metabolite, we established an efficient synthesis of both enantiomers of fenthion sulfoxide, which were subsequently transformed to chiral fenoxon sulfoxides using a two-step protocol. The use of a chiral oxidant (Davis oxaziridines) in separate reactions afforded chiral fenthion sulfoxides with high ee (>82%) from the parent sulfide, which were crystallized to >99% ee. The absolute configuration of the sulfoxide generated from fenthion metabolism by FMO1 was determined to be (R)-(+)-fenthion sulfoxide by X-ray analysis and by comparison of synthetic standards to enzymatically

1988


produced sulfoxides by HPLC analysis. The synthesis, characterization, and acetylcholinesterase activity of these chiral metabolites will be presented.

95. Interleukins 2 and 12 Do not Remediate Altered MAP kinases p38 and p44/42 Phosphorylation in Tributyltin-Exposed Human Natural Killer Cells. Aloice O. Aluoch. Department of Biological Sciences, Tennessee State University, 3500 John A. Merritt Boulevard, Nashville, Tennessee 37209. E-mail: aorodo@ juno.com. Margaret M. Whalen. Department of Chemistry, Tennessee State University. Tributyltin (TBT) is a toxic chemical known to inhibit NK cell cytotoxicity irreversibly with accompanying alteration of MAPKs p38 and p44/42 activation (phosphorylation) states. Both interleukins 2 and 12 can reverse TBT-inhibited cytotoxicity in NK cells. We examined whether IL-2 and IL-12 can remediate TBTaltered phosphorylation of p38 and p44/42. Neither IL-2 nor IL-12 remediated TBT-altered phosphorylation of p38 and p44/42 after either 24 or 48 h. Furthermore, no net phosphorylation of either p38 or p44/42 was induced in non-TBT-exposed cells after 48 h with either IL-2 or IL12 even though IL-2 activated both p38 and p44/42 after 24 h. These data suggest that IL-2 and IL-12 do not remediate TBT-induced altered phosphorylation of both p38 and p44/42 after both 24 and 48 h in TBT-free media. 96. Anorexic Region in Tissue by Theory of Krogh in Cartesian Coordinates. Kal Renganathan Sharma. Formerly Principal, Mathur Post, Anna Universitys Sakthi Engineering College, Mount, Ramapuram (opp. MGR Gardens), Chennai 602105, India. Fax: 2526019. E-mail: [email protected]. A microscopic view of capillaries in tissue indicates a repetitive arrangement of capillaries surrounded by a cylindrical layer of tissue. The residence time of the blood in the capillary is in the order of 1 s. The wave diffusion and relaxation time are comparable in magnitude to the residence time of the blood. Krogh (1919) showed that the cylindrical capillary tissue model can be used to study the supply of oxygen to muscle. The tissue space surrounding the capillary is considered a continuous phase albeit it consists of discrete cells. An effective diffusivity DT can be used to represent the diffusion process in the tissue. The driving force for the diffusion is driven by the consumption of the solute by the cells within the tissue space. The Michaelis-Menten equation can be used to describe the metabolic consumption of the solute in the tissue space. The governing equation for diffusion at steady state was written in Cartesian coordinates and the critical distance beyond which no diffusion can be expected was calculated. The quadratric equation is then

AXc2 + BXc + C ) 0

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where A ) -(Ro/2DAB), B ) +(xc + tm)Ro/DAB, and C ) C0 + RozAT/VA + K0xc RoAT/2A - (Ro/2DAB)xc + (tm)2 + Ro(xc + tm)/DAB and A, B, and C are given in terms of the thickness of the capillary, capillary diameter, tissue diameter, binary diffusivity, entrance concentration, and area of the tissue. When the solution of the quadratic expression for the critical distance in the tissue is real and found to be less than the thickness of the tissue, then the onset of zero concentration will occur prior to the periphery of the tissue. This zone can be seen as the anorexic or oxygen depleted regions in the tissue. 97. Withdrawn. 98. Detoxification of Hg2+, As3+, and Pb2+ Metal Ions from Wastewater by Biosorption using Modified and Unmodified Coconut Fiber: pH, Temperature, and Particle Size Effects. Jude C. Igwe,1 Augustus A. Abia,2 Stephinie I. Okenwa,3 Benedict C. Gbaruko,4 and Emmanuel C. Nwokennaya.3 (1) Industrial Chemistry, Abia State University, Uturu, Nigeria, 60 St. Mary’s Avenue, Okigwe, Imo State, Okigwe 234, Nigeria. E-mail: [email protected]. (2) Department of Pure and Industrial Chemistry, University of Portharcourt River State. (3) Industrial Chemistry, Abia State University Uturu. (4) Industrial Chemistry, Abia State University. The pollution of the environment with toxic metals is a result of human activities such as minning and metallurgy. The effects of these metals on the ecosystems are of large economic and public health significance. Bioremediation consists of a group of applications, which involve the detoxification of hazardous substances instead of transferring them from one medium to another by means of microbes and plants. The pH, temperature, and particle size effects on the detoxification of Hg2+, As3+, and Pb 2+ metal ions using modified and unmodified coconut fiber were studied in this paper. The pH significantly affected the adsorption with a maximum at pH of 4 and minimum at pH of 6-8. The adsorption increased as the temperature increased until at 40 °C. The smaller particle size showed greater adsorption. Modification also increased the adsorption capacity. These results will serve as parameters for design of treatment plants for heavy metal detoxification using agricultural byproducts. 99. Levels of Toxic Elements in Soils of Abandoned Waste Dump Site. Nnenna E. Okoronkwo,1 A. O. Ano,2 Jude C. Igwe,1 E. C. Onwuchekwa,3 and I. Nnorom.4 (1) Industrial Chemistry, Abia State University, Uturu, 31 Pound Road, Aba, Aba 234, Nigeria. E-mail: [email protected]. (2) Soil Science, National Root Group Research Institute, Umudike. (3) Microbiology, Abia State University, Uturu. (4) Industrial Chemistry, Abia State University. The soils of an abandoned waste dumps sites, which have been presently used for crop cultivation, were investigated. Three points on the location were selected, and three pits of 100 cm depth were dug at selected points. Soil samples were collected at an interval of 10 cm from each pits. The physicochemical properties and toxic elements (As, Cd, Cr, Ni, and Pb) levels were determined. The results showed that the mean percentage of sand, silt, and clay was 75.01 ( 2.31, 12.87 ( 0.93, and 10.45 ( 1.47, respectively, while the mean pH was 6.89 ( 0.01. The mean percentage organic matter content was high and was 3.47 ( 0.41. The mean exchangeable Ca, Mg, K, and Na in Mequiv/100 g of soil was 4.20 ( 0.42, 3.41 ( 0.32, 0.31 ( 0.004, and 0.47 ( 0.006,


respectively. The mean exchangeable acidity (EA) and exchangeable AI were 0.23 ( 0.002 and 0.69 ( 0.004, respectively. Of all of the five toxic elements studied, the highest mean concentration (mg/kg) of 133.74 ( 10.60 was recorded for Pb followed by Cr (22.27 ( 3.03), Ni (8.14 ( 0.33), and As (5.97 ( 0.32) while the least mean concentration of 1.64 ( 0.11 was recorded for Cd. The toxic elements were examined for dependency upon some soil factors through the use of correlation analysis, sand, organic matter, and effective cation exchange capacity (ECEC) correlated significantly and positively with Cr, Pb indicating that these factors largely control the concentration of these elements in the soils. 100. Integrated 2-Bed Adsorption System and Biofiltration for Treatment of Waste Gas Streams. Daekeun Kim. Civil and Environmental Engineering, University of Cincinnati, P.O. Box 210071, Cincinnati, Ohio 45221-0071. Fax: 513-556-2599. E-mail: kimdn@ email.uc.edu. Zhangli Cai. Department of Civil & Environmental Engineering, University of Cincinnati. George A. Sorial, Civil & Environmental Engineering Department, University of Cincinnati. Biological air treatment, i.e., biofiltration, is best operated at a steady load of contaminants; however, variations in contaminant load are common in real applications. This study evaluated the effectiveness of a 2-bed adsorption system for attenuating load fluctuation to a biofilter. The 2-bed adsorption system was designed for operating in a 2-step cycle, i.e., feeding (adsorption) and purging (desorption) in a fixed bed of adsorbents using gas pressure variation as the principal operating parameter. Regeneration of adsorbents was combined in the same process. The observed results showed that the net effect of the 2-bed adsorption was a reduction of the VOC concentration that makes it amenable for biofiltration. It was concluded from this study that the 2-step cycle in the 2-bed adsorption served successfully as a polishing unit to abate the initial acclimation for the biofilter and as a buffering unit to dampen the biofilter performance during fluctuating contaminant loading. 101. Restricted Adsorbent PSD Hindered Oligomerization of Phenolic Compounds. Qiuli Lu. Department of Civil & Environmental Engineering, University of Cincinnati. Fax: 513-556-2599. E-mail: luq@ email.uc.edu. George A. Sorial. Civil & Environmental Engineering Department, University of Cincinnati. Fax: 513-556-2599. E-mail: [email protected]. Phenolic compounds have been known to oligomerize on the surface of granular activated carbon (GAC) in the presence of molecular oxygen in the experimental environment. GAC has wide pore size distribution (PSD). On the other hand, activated carbon fibers (ACFs) are wellknown for their narrow PSD. The impact of adsorbate properties and adsorbent PSD was evaluated in this study. Anoxic (absence of molecular oxygen) and oxic (presence of molecular oxygen) single solute adsorption (2-methylphenol, 2-chlorophenol, and 2-nitrophenol) and multicomponent (binary and ternary) adsorption on GAC F400 and two ACFs (ACC-10 and ACC-15) were determined. For single solute adsorption, 2-methylphenol and 2-chlorophenol showed higher adsorptive capacity under oxic conditions than anoxic. The increase of adsorptive capacity under oxic conditions as compared to anoxic ones was related to the PSD of the adsorbent. 2-Nitrophenol showed no difference between anoxic and oxic adsorption


due to its high critical oxidation potential. For binary solute adsorption on ACC-10 and ternary adsorption on ACC-15, the narrow ACFs PSD was effective in hampering oligomerization of adsorbates. For binary adsorption on ACC-15 that involves the presence of 2-nitrophenol, no difference was noticed between oxic and anoxic isotherms. Significant differences between oxic and anoxic isotherms were noticed for other multicomponent adsorption systems. 102. Applying Global Gene Expression Data to Mechanisms of Hepatocarcinogenesis. Lois D. Lehman-McKeeman. Discovery Toxicology, Bristol Myers Squibb Pharmaceutical Research Institute, Route 206 and Province Line Road, P.O. Box 4000 MS H24-03, Princeton, New Jersey 08543. Fax: 609-252-6601. Email: [email protected]. A variety of agents are hepatocarcinogenic in rodents through mechanisms that do not involve reactive intermediates or direct DNA damage. Many of these compounds interact with nuclear hormone receptors to activate gene transcription, and microarray analyses provide a useful platform for evaluating gene expression patterns in response to these compounds. However, the early events associated with nuclear hormone receptor activation may or may not contribute to the more chronic consequence of tumor progression. This presentation will summarize the assessment of nongenotoxic rat liver carcinogens through the application of global gene expression patterns and illustrate how such data can be applied to deduce mechanisms of hepatocarcinogenicity. Transcriptomic evaluation of relevant transgenic and knockout models provide additional mechanistic insight, and the application of gene expression patterns to predicting liver tumor development and progression will be summarized. 103. Incorporation of Toxicogenomics into the Regulatory Process. John Leighton. Center for Drug Evaluation and Research, Food and Drug Administration, Rockville, Maryland 20852. Fax: 301-827-2823. E-mail: [email protected]. FDA and the pharmaceutical industry have recognized the importance of pharmacogenomics in decision-making, identifying novel biomarkers, and improving the safety assessment of new pharmaceuticals. To gain expertise in this field and develop a framework for accepting and reviewing data, voluntary submissions of genomic data have been encouraged as tools that help regulators gain experience and a contextual basis for reviewing and analyzing pharmacogenomic data. Unresolved issues such as cross-platform evaluation, data presentation, pathway interpretation, validation of signatures, and submission standards are being addressed. Out of internal and external interactions, a draft guidance document titled “Guidance for Industry: Pharmacogenomic Data Submissions” was developed, which will soon be finalized. This presentation will discuss FDA’s current thinking of the role of pharmacogenomics in preclinical safety assessment and provide an update on the pharmacogenomic guidance. 104. Toxicogenomic Approaches to Biomarker Discovery and Predictive Toxicology. James L. Stevens. Investigative Toxicology, Lilly Research Laboratories, 2001 West Main Street, Greeenfield, Indiana 46140. Fax: 317-276-1070. E-mail: stevens_james_l@ lilly.com.

1990


Recent progress illustrates the value of expression profiling in predictive toxicology. Few companies appear to be engaged in widespread expression profiling of clinical candidates, but expression profiling can add mechanistic information early in the development pipeline. Expression profiling is also providing value in areas such as biomarker discovery and mechanistic investigations. For example, recent work from two groups has identified novel biomarkers for gut toxicity associated with inhibition of γ-secretase, a key enzyme in processing of β-amyloid precursor proteins and a target for therapeutic intervention in Alzheimer’s disease. Another common toxicity for which biomarkers are lacking is bile duct hyperplasia. Recent work has also uncovered a novel biomarker candidate for BDH and provides insights into mechanisms underlying the toxicity. These examples will be discussed as case studies. 105. Use of Transcriptional Profiles to Classify Toxicants. Chris Bradfield. McArdle Laboratory for Cancer Research, University of Wisconsin, 1400 University Avenue, Madison, Wisconsin 53706. Fax: 608-2622824. E-mail: [email protected]. Modern genomic technology is helping us to classify chemicals and understand their mechanism of action. Central to the emerging field of toxicogenomics has been the use of microarray technology to define transcriptional profiles that result from particular chemical exposures. The vast amount of data that results from these studies has led to the development of bioinformatic and statistical approaches that help us to organize, analyze, and interpret this information. Here, we review the field of toxicogenomics from the perspective of one laboratory. Particular emphasis will be placed on toxicant classification, database development, and the informatics necessary to understand how profiles relate to the mechanism of action. 106. Defining the Complexities of Aryl Hydrocarbon Receptor-Regulated Signaling Pathways: Building a Road Map toward Defining the Risk of Dioxins for Humans. Tom Gasiewicz. Environmental Medicine, University of Rochester, 601 Elmwood Avenue, Rochester, New York 14642. Fax: 585-256-2591. Email: [email protected]. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is considered to be one of the most toxic chemicals known. Since the middle of the 20th century, contamination with this and related dioxin-like chemicals has been widespread and persistent. Because of their ubiquity, persistence, and extreme toxicity in laboratory animals, there has been considerable concern regarding their presence in the food chain and in human tissue. The fact that these chemicals have been shown to cause cancer, immune system disorders, reproductive and developmental abnormalities, and neurological and endocrine system alterations in animals at very low doses has further fueled this concern. There remains much controversy about the actual risk of these chemicals to human populations, and part of this is due to questions of how one extrapolates animal data to humans. Over the past two decades, much has been added to our understanding of the mechanisms by which these chemicals act. Of particular importance has been a large body of literature consistent with the hypothesis that TCDD produces its biologic and toxic effects by binding to a gene regulatory protein, the aryl hydrocarbon receptor (AhR). The mo-

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lecular changes initiated by this binding are the first in a series of events leading to numerous biochemical, cellular, and tissue changes that result in the toxic effects observed. The experimental data available support the biologic plausibility of similar end points to occur in human populations at some level of exposure. While clearly uncovering of these processes is a major advancement, we are also realizing that the molecular and cellular pathways that regulate species-, genetic-, cell-, and developmental-stage specific patterns of susceptibility to TCDD are very complex. Understanding these complexities may eventually help us to more specifically define human risk assessment and the dosage that may be associated with particular toxic end points, as well as the subpopulations may be most susceptible. 107. Toxicology of Dioxin and Related Compounds. James R. Olson. Pharmacology and Toxicology, University at Buffalo, 102 Farber Hall, Buffalo, New York 14214. Fax: 716-829-2801. E-mail: jolson@ acsu.buffalo.edu. Dioxin or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the most potent congener of a group of persistent halogenated aromatic hydrocarbons (HAHs), which includes polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), and biphenyls (PCBs). PCDDs and PCDFs are unwanted contaminants almost exclusively produced by industrial processes, including incineration, chlorine bleaching of paper and pulp, and the manufacture of chlorinated organics. These persistent compounds are highly lipophilic, resistant to biotransformation, and bioaccumulate in the environment. The toxicological concern regarding these compounds is in part due to their biological persistence, where biological half-lives can often be several years in humans. These compounds exhibit specific structure-activity relationships, where affinity for the cytosolic aryl hydrocarbon receptor (AhR) is required for a range of dioxin-like toxicological activities, including cancer, tumor promotion, endocrine disruption, immune suppression, reproductive and developmental toxicity, and other metabolic disorders. Toxic equivalency factors (TEFs) have been assigned to dioxinlike compounds based on their relative potency as compared to TCDD, which is assigned a TEF ) 1.0. Real world, complex mixtures of dioxin-like compounds are assigned total dioxin toxic equivalents (TEQs), which are calculated as the sum of the products of the concentrations of each congener and its respective TEF value. Thus, TEQs are used to represent the biological activity of a complex mixture as if it only contained TCDD. The continuing challenge is to identify the dose or TEQ concentration that will produce a given adverse effect. 108. Dioxin Risks in Perspective. Lesa L. Aylward. Exponent, Inc., 1800 Diagonal Road, Suite 300, Alexandria, Virginia 22314. Fax: 571-227-7299. E-mail: [email protected]. The risk of adverse health effects from exposure to dioxins or any other chemical or drug is related to both the intrinsic potential for harm from the chemical and to the amount of exposure. For dioxins, emissions, environmental levels, and general population exposure levels have declined dramatically in the United States over the past three decades as a result of numerous regulatory actions taken beginning in the 1970s. Animal species vary widely in their sensitivity to effects from dioxins, and available mechanistic data and studies of highly exposed human populations suggest that humans


are not the most sensitive species for dioxin exposure. Concern regarding potential adverse effects from current population exposures or small incremental exposures to dioxin is derived primarily from extrapolation from the most sensitive laboratory species and may not be war-


ranted given the large declines in exposures observed over the past 30 years. TX0502894

Abstracts, American Chemical Society Division of Chemical

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