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Abstracts, American Chemical Society Division of Chemical Toxicology, 226th ACS National Meeting, New York, New York, September 7-11, 2003 Peter C. Dedon, Program Chair Biological Engineering Division, Massachusetts Institute of Technology, NE47-277 at 500 Technology Square, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139 Received October 3, 2003

As Program Chair for the Division of Chemical Toxicology, I am delighted to present the Division abstracts from the ACS meeting in New York in this issue of Chemical Research in Toxicology. I would also like to take this opportunity to update readers about changes in the Programming activities of the Division of Chemical Toxicology, changes that are intended to increase attendance at the national ACS meetings. This year’s Division activities were tremendously successful. The five symposia, which were all attended, drew national and international media attention. Prof. Judy Bolton’s session on hormone replacement therapy, “Chemical Toxicology of Hormone Replacement Therapy: Perspectives on the Women’s Health Initiative 2002,” was covered in Nature Magazine’s on-line Science Update (http://www.nature.com/nsu/030908/030907-3.html) and in the October 6, 2003 issue of Chemical & Engineering News (volume 81, issue 40). The annual dinner and poster session was attended by an overflow crowd that was up 20% from pervious years. I would like to take this opportunity to welcome the Program Chair Elect, Prof. Trevor Penning, Professor of Pharmacology, Biochemistry, and Biophysics and Obstetrics and Gynecology at the University of Pennsylvania School of Medicine. Trevor and I are well under way with planning the program for next year’s ACS meeting in Philadelphia (August 22-24, 2004). As we look to the future, I would like to inform TOXI members and other readers of CRT about several changes in TOXI programming activities that will enhance the quality of our meetings and make them attractive to a wider audience. The first is a new effort to invite young scientists to become more involved in Division activities and programming. Those scientists who have recently embarked on their career in industry or academia represent the future of the Division, and it is imperative that they be given a role in shaping the Division. Our second new policy is to program at least one industrially oriented symposium each year, one that is either organized by an industrial member of TOXI or consists mainly of industrial speakers. We are eager to provide programming that is attractive to our industry members, since industry scientists comprise nearly 15% of TOXI membership and bring an important perspective on chemical toxicology that complements our many academic members. So, I hope you find the following abstracts useful and interesting. See you in Philadelphia!

1. DNA Damaging Agents that Hijack Transcription Factors. John Essigmann. Biological Engineering Division and Department of Chemistry, Massachusetts Institute of Technology, 56-669, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139. Fax: 617-2535445. E-mail: [email protected]. Binary toxicants consisting of a DNA-damaging aniline mustard tethered to ligands for selected steroid receptors have been synthesized. The compounds were designed to form unusual DNA adducts that attract transcription factors, hinder DNA repair, and disrupt cellular signaling pathways. The compounds potently inhibit the growth of breast and prostate tumors in vivo. The mechanism of action of these toxicants will be discussed, and novel analytical methodology will be presented that allows for sensitive adduct detection in the tissues, including tumor tissue, of tumor-bearing mice. 2. Biological Applications of Accelerator Mass Spectrometry (AMS). Steven R. Tannenbaum, Paul L. Skipper, and Rosa Lieberman. Biological Engineering Division, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Room 56-731A, Cambridge, Massachusetts 02139. Fax: 617-252-1787. E-mail: [email protected]. Accelerator mass spectrometry (AMS) has been an important tool for measurement of carbon-14 in biological samples, giving detection limits many orders of magnitude lower than is capable with liquid scintillation counting. We have developed a system for continuous detection of C-14 in CO2 that provides a significantly greater rate of sample processing than the traditional graphite procedure. Application of the system to drug metabolism and toxicology will be described. 3. Stable Isotope Labeling Mass Spectrometry to Map the Reactivity of DNA Bases toward Carcinogens and Antitumor Agents. Natalia Tretyakova. Department of Medicinal Chemistry and Cancer Center, University of Minnesota, 760E CCRB, 806 Mayo, 420 Delaware Street Southeast, Minneapolis, Minnesota 55455. Fax: 612-626-5135. E-mail: [email protected]. A stable isotope labeling HPLC-ESI-MS/MS approach was developed to map the formation of guanine adducts induced by tobacco carcinogens, e.g., 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene, within DNA sequences. 15N3-labeled guanine was incorporated at specific sites within DNA duplexes derived from the K-ras protooncogene and p53 tumor suppressor gene, followed by carcinogen treatment and HPLC-MS/MS analysis to determine adduct yields at the

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labeled position. Our results indicate that the reactivity of a given base toward activated tobacco carcinogens is affected by sequence context, endogenous cytosine methylation, and the nature of the DNA modifying agent. The applicability of this approach to structural studies of DNA cross-linking by bifunctional electrophiles is also demonstrated. 4. Formation and Properties of the Leinamycin2′-deoxyguanosine Adduct in Double-Stranded DNA. Kent S. Gates. Department of Chemistry, University of Missouri, 125 Chemistry Building, Columbia, Missouri 65211. Fax: 573-882-2754. E-mail: [email protected]. Leinamycin (1) is a structurally unique antitumor antibiotic that damages DNA by novel chemical mechanisms. Leinamycin is (relatively) stable in water; however, upon entering the thiol-rich environment of the cell, reaction with thiols initiates an intriguing cascade of chemical reactions that leads to oxidative stress and formation of covalent leinamycin-DNA adducts. The results of our efforts to elucidate the chemistry of DNA adduct formation and the biologically relevant chemical properties of leinamycin-DNA adducts will be presented.

Glaser, and Ming Qian. Department of Chemistry, University of Missouri-Columbia, Columbia, Missouri 65211. Fax: 573-882-2754. E-mail: [email protected] and [email protected]. Our theoretical studies revealed that pyrimidine ringopened 4-oxomethylene-5-cyanoimino-4,5-dihydroimidazole (1) is a key intermediate in nitrosative guanosine deamination. Nucleophilic addition to 1 is facile and leads to the 5-cyanoamino-imidazoles (2), and we are exploring the chemistry of 2. We have previously demonstrated experimentally that the chemistry of 2 can explain the formations of xanthosine and oxanosine. We now report experimental results that demonstrate the competence of 2 to form dG-to-dG cross-link 4. Reaction of cyanoamine 2 (R1 ) CH2-O-CH2-CH2-OH, X ) NH2) with guanosine 3 (R2 ) ribofuranosyl) affords 4. The standard 4 was synthesized independently adapting Hopkins’s palladium-catalyzed coupling method with elements of Johnson’s cross-link synthesis. LC-MSMS analysis shows the identity of the standard 4 and the cross-link formed between 2 and 3. Hence, we have demonstrated that the chemistry of 2 can explain the formation of all known guanine deamination products.

5. Novel Approach to the Synthesis of Malondialdehyde Adducts of Deoxyguanosine and Deoxyadenosine. Hao Wang, Lawrence J. Marnett, Thomas M. Harris, and Carmelo J. Rizzo. Department of Chemistry and Center in Molecular Toxicology, Vanderbilt University, Box 1822, Station B, Nashville, Tennessee 37235-1822. Fax: 615-322-2649. E-mail: hao.wang@ vanderbilt.edu. Malondialdehyde is a mutagenic product of lipid peroxidation and prostaglandin biosynthesis. It or its reactive equivalent also arise through oxidative damage to DNA. Malondialdehyde reacts with DNA to produce adducts of deoxyguanosine (1, M1G) and deoxyadenosine (2, M1A). An efficient new route based on organoselenium chemistry has been developed for preparation of 1 and 2, which significantly improves their availability. Oligonucleotides containing site specific adducts of M1G have been prepared using this chemistry in a postoligomerization synthetic strategy. Efforts are underway to extend the methodology to oligonucleotides containing M1A that cannot be prepared from the phosphoramidite of M1A. Supported by NIH grant P01-ES05355 and R01-ES11331.

7. Low pKa and Reactivity of CYS145 of O6Alkylguanine-DNA Alkyltransferase. F. Peter Guengerich,1 Qingming Fang,2 Liping Liu,2 and Anthony E. Pegg.2 (1) Department of Biochemistry & Center in Molecular Toxicology, Vanderbilt University, 638 Robinson Research Building, 23rd and Pierce Avenues, Nashville, Tennessee 37232. Fax: 615-322-3141. Email: [email protected]. (2) Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine. The active site Cys residue (145) of the human DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) was shown to be highly reactive with electrophiles (e.g., 1-chloro-2,4-dinitrobenzene) unrelated to normal DNA substrates. The pKa of the thiolate anion was estimated to be ∼4 by UV spectroscopy (A240 measurements) and the rates of reaction with 4,4′-dithiopyridine as a function of pH, similar to Cys25 of papain (4.6). The bacterial homologues ADA and OGT also had low pKa vaules, as judged by these criteria. Bound Zn2+ had previously been shown to be necessary for the activity of AGT in DNA repair. Zn2+ was shown to be necessary for the low pKa of Cys145; the pKa was 3.8 for Zn2+-fortified AGT and ∼8.7 for Zn2+-depleted AGT. The low pKa and high reactivity of AGT Cys145 are of significance in that AGT can react with many electrophiles and lose DNA repair capability. Supported in part by USPHS Grants R01 ES10546, R01 CA18137, and P30 ES00267. 8. Measurement of Free Concentration as a More Intrinsic Dose Parameter in an in Vitro Assay for Estrogenic Activity. Minne B. Heringa,1 Richard

6. Experimental Demonstration of a New Mechanism for dG-to-dG Cross-Link Formation. Rainer


Schreurs,1 Paul T. van der Saag,2 Bart van der Burg,3 and Joop L. M. Hermens.1 (1) IRAS, Utrecht Univeristy, Yalelaan 2, 3584 CL Utrecht, Netherlands. Fax: +3130-2535077. E-mail: [email protected]. (2) Hubrecht Laboratory, Netherlands Institute for Developmental Biology. (3) BioDetection Systems BV. Dose-response curves of in vitro assays are usually based on the nominal (i.e., added) concentration of test compound. However, various authors have shown that the response decreases as more protein is present in the assay medium. Some suggested that calculating the free concentration of the test compound would give improvement, but this is not straightforward. Instead, we have, for the first time, applied nd-SPME (negligible depletion solid phase microextraction) to measure free concentrations of [3H]estradiol during an in vitro estrogenicity assay, performed with different protein concentrations in the assay medium. As expected, the dose-response curves shifted to the right with increasing protein concentration when using nominal concentrations. However, plotting the free concentration instead resulted in one single dose-response curve. Calculated free concentrations did not correspond well with measured free concentrations. This confirms the importance of the free concentration in dose-response curves and shows that directly measuring is better than calculating it. 9. Nitrolic Acids: Possible Endogenous Toxicants, from the Nitrosation of Amino Acids. Richard N. Loeppky, Emma L. Teuten, Erqing Tang, and Nicholas Power. Department of Chemistry, University of Missouri, 125 Chemistry Building, Columbia, Missouri 65211. Fax: 573-882-2754. E-mail: [email protected] and [email protected]. In the course of investigating the aldehyde-mediated nitrosation of the phenylalanine 1, under conditions mimicking those present in the human stomach, we discovered the formation of an unknown substance having the formula C8H8N2O3, which had lost the carboxyl group. X-ray crystallographic analysis showed that this substance was 2-phenylethylnitrolic acid (2-phenyl-1oximino-1-nitroethane) 2. Reactions of other R-amino acids, alanine and leucine, demonstrated that nitrolic acids were also formed during their nitrous acid nitrosation and that the presence of an aldehyde in the nitrosating mixture was unimportant to this chemistry. The nitrosation of phenylalanine in buffered acetic acid at 22 °C was studied as a function of pH and [NO2-]. Typical product yields are shown in the equation (pH 3.9, 2.9 equiv of [NO2-]). HPLC analysis of reaction mixtures showed that the nitrolic acid could be detected with 1 equiv of [NO2-] but not with 0.5. The yield of the nitrolic acid decreased with pH and increased with [NO2-]. Several nitrolic acids have been reported to be mutagenic. Further investigation of their toxicity is underway. The products of these nitrosation reactions arise from the transient diazonium ion (or resulting R-lactone) produced from the nitrosation by nucleophilic displacement by H2O (to 3), AcO- (to 4), or NO2- (2 or 5). The latter process generates 2-nitro-3-phenylpropionic acid, which either C-nitrosates through its enol and decarboxylates to give 2 or decarboxylates to give 5. The nitrosation of the latter gives 2 but is very slow as compared to the generation of 2 under the reaction conditions. Nitrolic acids are reactive and undergo mild base-catalyzed conversion to unstable, electrophilic nitrile oxides.

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10. Toxicity QSARs for Halogenated Aliphatics Derived Using Metabolite Descriptors. Steven Trohalaki,1 Ruth Pachter,1 Kevin T. Geiss,2 and John M. Frazier.2 (1) Air Force Research Laboratory, Materials & Manufacturing Directorate, AFRL/MLPJ Building 651, 3005 P Street, Suite 1, Wright-Patterson Air Force Base, Ohio 45433-7702. Fax: 937-255-3377. E-mail: [email protected]. (2) Air Force Research Laboratory, Human Effectiveness Directorate. Halogenated aliphatic hydrocarbons (HAs), whose annual worldwide production is measured in the thousands of pounds, are common, widely used industrial and household solvents, fumigants, and intermediates in chemical syntheses. Human exposure to HAs may pose a serious health hazard for a variety of reasons. Several HAs have been shown to be carcinogenic or otherwise toxic while genetic damage has been demonstrated by metabolites of some HA species that bind to DNA. Other HAs interact with cellular structures, affecting chromosome segregation and thereby inducing genotoxicity. For P450-mediated hepatotoxicity, the biophysical mechanism involves dissociative attachment resulting in free radicals, which then react with oxygen to form peroxyl radicals. Using a set of six in vitro hepatotoxicity endpoints determined for 20 HAs, we derived two and three parameter QSARs using quantum descriptors calculated from the neutral HA species. QSARs of about the same statistical quality were also obtained using quantum descriptors calculated from either the free radical or the peroxyl radical metabolites. However, in most cases, QSARs derived using descriptors calculated from both neutral HAs and metabolites are statistically superior to those derived using either neutral HA descriptors or metabolite descriptors. 11. Hemoproteins and Biological Free Radicals. Paul R. Ortiz de Montellano, Olivier Lardinois, and Christophe Colas. Department of Pharmaceutical Chemistry, University of California, San Francisco, 600 16th Street, San Francisco, California 94143-2283. Fax: 415502-4728. E-mail: [email protected]. Free radicals have been implicated as causative or aggravating factors in a diversity of pathological situations, including major disease states such as inflammation, atherosclerosis, and cancer. Free radicals are formed as catalytic intermediates in the turnover of hemecontaining enzymes but are also formed as reactive byproducts in the adventitious reactions of hemoproteins with peroxides and other oxidizing agents. In the case of the mammalian peroxidases, radicals are involved in maturation of the proteins by promoting both covalent attachment of the heme group to the protein and the formation of specific amino acid cross-links. In these same peroxidases, and in other hemoproteins such as myoglobin, diffusible carbon-centered radicals are formed that can be transferred from one protein to another, resulting in protein-protein cross-links. Electron removal from tyrosine residues to give the corresponding radicals depends on both their distance from the oxidizing center


and the physical environment of the individual residues. Radicals represent a release from oxidative stress that can be used in a productive or destructive manner. This work was supported by grant GM32488. 12. Reversibility of DNA Alkylation by Quinone Methide Intermediates. Steven E. Rokita. Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742. Quinone methide and related electrophilic intermediates are generated in biological systems during metabolism of various food preservatives, drugs such as tamoxifen, and certain natural products. The apparent selectivity of these intermediates for weak rather than strong nucleophiles initially seemed counterintuitive. However, recent detection of transient and kinetic products of alkylation has helped to reconcile the apparent contradiction between the strength of nucleophiles in DNA and the profile of its alkylation products generated with a model quinone methide. The reversible nature of this alkylation adds an important perspective to the biological activity of quinone methides and has proved advantageous in the design of a gene specific reagent.

13. Chemical Mechanisms of Oxidative DNA Damage by the Antitumor Agent 3-Amino-1,2,4-benzotriazine 1,4-Di-N-oxide (Tirapazamine). Kent S. Gates. Department of Chemistry, University of MissouriColumbia, 601 South College Avenue, Columbia, Missouri 65211. Fax: 573-882-2754. E-mail: [email protected]. The compound 3-amino-1,2,4-benzotriazine 1,4-di-Noxide (tirapazamine, 1) is currently undergoing a variety of clinical trials as a potential antitumor agent. Following in vivo enzymatic activation, this drug selectively causes oxidative DNA damage in oxygen-poor (hypoxic) tumor cells via an unusual sequence of chemical reactions. We will discuss the chemical processes that are relevant to oxidative DNA damage engendered by tirapazamine and other heterocyclic N-oxides.

14. Deoxyribose Oxidation as a Source of Protein and DNA Adducts. Peter C. Dedon. Biological Engineering Division, Massachusetts Institute of Technology, 56-787, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139. Fax: 617-258-0225. E-mail: [email protected]. DNA oxidation by a variety of endogenous and exogenous agents has been implicated in the pathophysiology of cancer and the diseases of aging, with an estimation of thousands of oxidative insults per day per cell from oxygen radicals and related species that arise from normal metabolism and inflammation. While nucleobase damage has been the focus of most studies, there is emerging evidence that deoxyribose oxidation plays a critical role in the genetic toxicology of oxidative stress. Oxidation of each position in deoxyribose leads to the formation of a unique set of electrophilic and potentially


genotoxic products that react with DNA and proteins to form adducts. This presentation will cover recent progress in understanding this phenomenon, including evidence consistent with base propenals, derived from 4′-oxidation of deoxyribose, as an important source of the mutagenic M1G DNA adduct in cells and with modification of histone proteins by the 3′-formyl phosphate product of deoxyribose 5′-oxidation. 15. Oxidation of Guanosine via Singlet Oxygen vs One Electron Mechanisms. Cynthia J. Burrows, and James G. Muller. Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, Utah 84112. Guanine is the most susceptible of the four DNA bases to oxidation leading to a wide range of heterocyclic products. Curiously, oxidants as disparate as singlet oxygen and redox active transition metal complexes may lead to the same products although hydroxyl radical generates yet another set of lesions. In recent work, we have attempted to understand the reaction pathways of a family of reactive oxygen species in order to understand the oxidative DNA damage likely to be present in the genome. Lesions currently under study include spiroiminodihydantoin and guanidinohydantoin. The organic chemistry of their formation and their biochemical properties with polymerases and DNA repair enzymes will be presented. 16. Mechanisms of Lipid Peroxidation: Radical Clocks, Antioxidants, and Reactive Secondary Products. Ned A. Porter. Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37235. The autoxidation of polyunsaturated fatty acids and esters has been the focus of intense investigation because of its potential importance in biology and medicine. Mechanistic studies suggest that one key to understanding product distribution in diene fatty acid oxidation is the reversibility of oxygen addition to intermediate pentadienyl radicals. Thus, loss of oxygen from diene peroxyl radicals competes with hydrogen atom trapping of the peroxyls by phenolic antioxidants. The distribution of products derived from linoleate esters depends therefore on the rates of hydrogen atom transfer from ArOH to, and β-fragmentation of, intermediate peroxyls as well as on the partitioning of oxygen to the reactive sites on the delocalized radical intermediates. On the basis of this competition, we have developed (radical clocks) for timing critical rates involved in chain oxidation and inhibition. Experimental and theoretical approaches will be described that permit a rational discussion of products formed in lipid peroxidation. 17. Peroxide-Induced Iron Signaling and Apoptosis: The Effect of Nitric Oxide. Balaraman Kalyanaraman. Department of Biophysics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, Wisconsin 53226. E-mail: [email protected]. Endothelial cells exposed to hydroperoxides, oxidized low-density lipoprotein, or redox active xenobiotics undergo apoptotic cell death. The objective of this talk is to delineate the role of redox active iron in oxidant-induced apoptosis. Results from our studies show that the transferrin receptors (TfR) located on the surface of the endothelial cells play a key role in modulating the oxidant-induced apoptotic signaling mechanism. We show that the extracellular iron transported through TfR is responsible for initiating cellular oxidative damage leading to apoptosis. In the presence of TfR antibody that


binds to the TfR and blocks the transferrin iron uptake, oxidant-induced intracellular oxidative damage and apoptosis were completely inhibited. We suggest that oxidantinduced iron signaling mechanism is primarily responsible for endothelial apoptosis induced by extracellular hydroperoxides and intracellular redox active compounds. 18. Introduction and Summary of Joachim Liehr’s Unique Contributions to the Field of Estrogen Carcinogenesis. Judy L. Bolton. Department of Medicinal Chemistry & Pharmacognosy, University of Illinois College of Pharmacy, 833 South Wood Street, Chicago, Illimois 60612-7231. Fax: 312-996-7107. Email: [email protected]. There is a clear association between excessive exposure to synthetic and endogenous estrogens and the development of cancer in several tissues. The central theme of this symposium is that excessive binding of estrogens to the estrogen receptor and/or the formation of redox active/ electrophilic quinoids are important mechanisms of carcinogenesis for endogenous estrogens and certain estrogens present in estrogen replacement formulations. The significance of this symposium was compounded by the recently released data from the Women’s Health Initiative Study, which was terminated after 5.3 years primarily due to an increase in hormone-dependent cancers observed in women receiving Prempro. This symposium will bring leaders in the field of estrogen carcinogenesis together in order to discuss the findings of the Women’s Health Initiative from a chemical and biological perspective and to determine rational goals for future research efforts. In addition, Joachim Liehr’s (1942-2003) unique contributions to this field will be highlighted. 19. Estrogen Plus Progestin and Cancer: The Women’s Health Initiative Clinical Trial. Sylvia Smoller. Department of Epidemiology and Population Health, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. E-mail: smoller@ aecom.yu.edu. The Women’s Health Initiative (WHI) clinical trial of estrogen plus progestin, sponsored by the National Institutes of Health, randomized 16 608 women in 40 clinical centers in the U.S., who were ages 50-79, who had an intact uterus, to either 0.625 mg of conjugated equine estrogen plus 2.5 mg of medroxyprogesterone daily or placebo. The primary objectives were to see if this preparation would prevent coronary heart disease and osteoporosis and to determine if it would cause breast cancer. The trial was stopped three years earlier than planned, after 5.6 years, because it was found that there was a significant increase in breast cancer, as well as increases in heart disease, stroke, and pulmonary embolism, and the overall harm outweighed the benefit. Estrogen plus progestin resulted in a 26% increase in breast cancer, as well as a 37% reduction in colorectal cancer. Quality of life data indicated no clinically significant benefits of estrogen plus progestin. 20. Catechol-O-methyltransferase (COMT) and Catechol Estrogens in Breast Cancer. Anne E. Sullivan,1 Julie E. Goodman,2 and James D. Yager.1 (1) Bloomberg School of Public Health, Johns Hopkins University, 615 North Wolfe Street, Baltimore, Maryland 21205. Fax: 410-955-0116. E-mail: [email protected] and [email protected]. (2) Laboratory of Human Carcinogenesis, National Cancer Institute. Catechol estrogen detoxification occurs through methylation by COMT. Inhibition of COMT causes increased


oxidative DNA damage. A polymorphism encoding a low activity COMT is associated with breast cancer risk. COMT low (COMTL) and high (COMTH) do not differ kinetically. A 3-4-fold difference in COMT activity was reported in cytosols from human cells and tissues from subjects with COMTLL and COMTHH genotypes, although activity was not corrected for COMT protein levels. Using cytosolic fractions from human hepatocytes, we observed that COMT activity directly correlates with COMT protein levels. That is, hepatocyte cytosols from subjects with a COMTLL genotype had less COMT protein and activity as compared to hepatocyte cytosols from COMTHH subjects. In addition, COMT in cytosolic fractions of COMTLL breast epithelial cell lines is more thermolabile than in COMTHH lines, which is consistent with observations of others using human erythrocytes and other tissues. This suggests the hypothesis that the decrease in COMT protein levels in cytosols of subjects with the COMTLL genotype may be due to increased turnover of the low activity enzyme. Studies are underway to test this hypothesis. 21. Catechol Estrogen-DNA Adducts in the Initiation of Breast and Other Cancers. Eleanor G. Rogan, and Ercole L. Cavalieri. Eppley Cancer Institute, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, Nebraska 681986805. Fax: 402-559-8068. E-mail: [email protected]. Estrogens are inducers of human breast cancer. The metabolite 4-hydroxyestradiol (4-OHE2) is carcinogenic in animals, whereas 2-OHE2 is borderline. Oxidation of these catechol estrogens produces E2-3,4-quinone (Q) and E2-2,3-Q, which react with DNA to form predominantly depurinating adducts that generate apurinic sites. E23,4-Q forms >99.9% adducts that depurinate from DNA, whereas E2-2,3-Q forms much lower levels of a depurinating adduct. When mixtures of E2-3,4-Q and E2-2,3-Q react with DNA, E2-3,4-Q is preferentially bound, forming abundant depurinating adducts. This also occurs when mixtures of 4-OHE2 and 2-OHE2 are enzymically activated to quinones and reacted with DNA. We hypothesize that imbalances in estrogen metabolism lead to reaction of E2-3,4-Q with DNA, resulting in oncogenic mutations. In human breast tissue, levels of 4-OHE2 and quinone-glutathione conjugates are significantly higher in women with breast carcinoma as compared to women without. These results support the role of depurinating 4-OHE2-DNA adducts in initiation of breast and other cancers. USPHS Grants P01 CA49210 and R01 CA49917. 22. DNA Damage Induced by an Equine Estrogen Metabolite (4-OHEN) in Solution and the Characteristics of Site Specifically Modified Oligodeoxynucleotide Model Systems Containing Single, Stable Covalent Adducts. Nicholas E. Geacintov,1 Alexander Kolbanovskiy,1 Kuzmin Kuzmin,1 Minsun Chang,2 and Judy L. Bolton.2 (1) Chemistry Department, New York University, 31 Washington Place, New York, New York 10003-5180. E-mail: [email protected]. (2) College of Pharmacy, Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago. The equine estrogen equilenin is one of the important components of premarin, a formulation widely prescribed as a hormone replacement treatment for postmenopausal women. Equilenin is metabolized by mammalian P450 enzymes to the catechol 4-hydroxyequilenin (4-OHEN), which, in turn, is readily oxidized to the highly reactive 4-OHEN o-quinone derivative. The latter reacts readily


with C, G, and A in native DNA to form covalently modified bases (Bolton, J. L., Pisha, E., Zhang, F., and Qiu, S. (1998) Chem. Res. Toxicol. 11, 1113). We have reacted oligodeoxynucleotides with 4-OHEN in aerated aqueous buffer solution and found that numerous, stable, covalent adducts are formed with the same bases as well. Damage resulting from the reactions of reactive oxygen species with the nucleobases in the same oligonucleotides has also been identified. The different covalently modified reaction products can be easily separated from one another and purified by reversed phase HPLC techniques. The adducts and their positions within the oligonucleotides are identified by a combination of mass spectrometry and exonuclease digestion techniques. The circular dichroism spectra of the modified nucleosides, especially those involving dC residues (Bolton, J. L., Pisha, E., Zhang, F., and Qiu, S. (1998) Chem. Res. Toxicol. 11, 1113), indicate that at least two types of stereoisomeric adducts are formed, exhibiting CD spectra of opposite sign but of similar shape. The oligonucleotide duplexes containing single 4-OHEN-modified bases are thermally less stable than the identical unmodified duplexes. The lowering in the duplex melting points, Tm, depends on the position of the modified base within the double-stranded oligonucleotides. These site specifically modified oligonucleotides containing single, defined lesions derived from the reaction of 4-OHEN with different oligonucleotides are suitable for site specific mutagenesis and DNA repair studies in in vitro model systems. 23. Standardization and Structural Annotation of Public Toxicity Databases: Improving SAR Capabilities and Linkage to ‘Omics Data. Ann M. Richard,1 ClarLynda R. Williams,1 and Jamie M. Burch.2 (1) U.S. EPA, MD B143-06, Research Triangle Park, North Carolina 27711. Fax: 919-685-3263. E-mail: [email protected]. (2) EPA/NC Central University Student COOP. A major impediment to the greater participation of chemists in exploring structure-activity relationships (SAR) in toxicology is the lack of chemical structure annotation to much historical public toxicity information. Chemical structures and the chemistry underlying toxicity in biological systems provide a natural common metric for SAR exploration of these diverse data as well as for linking these data more broadly to the data being generated in the emerging ‘omics fields. The DSSTox (Distributed Structure-Searchable Toxicity) data network is a new public initiative whose objectives are to (i) promote a standard SDF format and minimal level of chemical structure annotation to public toxicity data; (ii) provide a central registry for publishing and making these data files accessible to all; and (iii) involve the toxicological experts and sources most familiar with these data in providing minimal description and documentation for these data sets. This abstract does not represent EPA policy. 24. Learning from Data to Improve Predictive Power of the Toxicity Models. Chihae Yang, Paul Blower, Kevin P. Cross, and Glenn Myatt. LeadScope, Inc., 1245 Kinnear Road, Columbus, Ohio 43212. Email: [email protected]. The ability to accurately predict toxicity is increasingly becoming more important as pharmaceutical and chemical industries move toward efficient up-front screening to reduce late stage attrition. Understanding chemically induced toxicity is also important for addressing national


security issues. Although several computational methodologies are available in the current predictive toxicology arena, it is well-accepted that these methods still require much improvement. One of the serious issues is the lack of access to the data from which the black box “models” or “predictions” are derived. Large amounts of toxicity information are publicly available; however, most of these databases are not optimized for building structure-toxicity relationships. Quite often, a new focused data set has to be constructed to develop end point relationships with structural features. Understanding the data space of structural diversity, mechanistic complexity, and data distribution is the first step to learning from the data. Characterizing the focused data sets before building any predictive models is critical. Once the data set is selected from this structure-data space, structural rules for specific toxicity can be learned by extracting scaffolds. This paper will demonstrate how learning from data and applying the knowledge back to the data set will improve the predictive power of the resulting models. 25. Predicting Human and Environmental Toxicity of Chemicals Based on Their Shape and Electrostatic Features. William J. Welsh,1 Randy Zauhar,2 Vladyslav Kholodovych,3 Karthigeyan Nagarajan,1 and Ni Ai.1 (1) Department of Pharmacology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, 661 Hoes Lane, Picataway, New Jersey 08854. Fax: 732-235-3475. E-mail: welshwj@ umdnj.edu. (2) Department of Chemistry and Biochemistry, University of The Sciences. (3) Pharmacology, UMDNJ-RWJMS. Computational molecular modeling has demonstrated utility in a wide range of applications, including drug discovery, toxicology, and environmental risk assessment. Computational toxicology (CompTox) imposes special demands on such approaches for several reasons. First, measured toxicological data are often sparse, imprecise, and sometimes nonexistent. Second, data sets of toxic chemicals often fail to follow a logical sequence or congeneric series, even for compounds that obey the same mechanism of action. Third, the relationship between the measured toxicological endpoint (e.g., LD50) and the calculated molecular features is often nonlinear rather than linear. Last, “false negatives” can often lead to dire consequences. Here, we present two computational approaches that address these issues. First, the Polynomial Neural Network (PNN) is a powerful iterative neural network algorithm that automatically yields both linear and nonlinear quantitative structure-activity relationship (QSAR) regression models in parametric form. The PNN thus integrates the best features of artificial neural networks (i.e., inherent nonlinearity) and multivariate regression analysis (i.e., parametric equation) within a single entity. Second, the “Shape Signatures” algorithm is a novel tool for rapidly screening large numbers of small molecules against each other (or against a receptor pocket) based on shape and electrostatic similarity (complementarity). We will demonstrate the utility of both computational tools in applications of significance to human toxicology and environmental risk assessment. 26. Structure-Function Relationships in Carcinogen-Damaged DNA through Computer Modeling. Suse Broyde,1 Rebecca Perlow,1 Min Wu,2 Shixiang Yan,2 and Nicholas E. Geacintov.2 (1) Department of Biology, New York University, 100 Washington Square East, Room 1009 Silver, New York, New York 10003.


E-mail: [email protected]. (2) Department of Chemistry, New York University. We are employing molecular dynamics simulations using AMBER to investigate structures of DNA damaged through covalent modification with carcinogenic and mutagenic bay and fjord region metabolites of polycyclic aromatic hydrocarbons, environmental pollutants present in automobile exhaust, and tobacco smoke. Simulations are being carried out both for modified DNA duplexes in solution and for modified primer/template complexes with polymerase enzymes. These studies are beginning to elucidate on a molecular level the origins of stereoisomer effects, bay and fjord region effects, and base sequence context effects observed in nucleotide excision repair experiments and in primer extension experiments. 27. High-Performance Computing, Computational Chemistry, and Toxicology. David A. Dixon. Fundamental Sciences Directorate, Pacific Northwest National Laboratory, P.O. Box 999, Richland, Washington 99352. Fax: 509-375-6776. E-mail: david.dixon@ pnl.gov. Chang-Guo Zhan. W. R. Wiley Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory. The development of new theoretical methods, new algorithms, and new software that incorporates these advances on modern high-performance massively parallel computer architectures promises to lead to new understanding of the behavior of chemicals and to the use of simulation methods to replace expensive experiments. These new developments promise to revolutionize the size and types of systems that can be treated. We will describe applications of computational chemistry to toxicology studies for the chromogenic behavior of neurotoxins, the chemistry of perfluorinated compounds, and the molecular dynamics simulations of molecules in cell signaling pathways. In addition, we will describe the use of new computational approaches for the study of complex signaling pathways. 28. Comparison of Translesion Bypass of GuanineN2 Monoadducts of Mitomycin C and Guanine-N7 Monoadducts of 2,7-Diaminomitosene by DNA Polymerases. Cristina C. Clement, and Maria Tomasz. Chemistry Department, Hunter College, City University of New York, 695 Park Avenue, New York City, New York 10021. E-mail: [email protected]. The guanine (G)-N2 DNA monoadduct of mitomycin C (MC), a cytotoxic anticancer drug, inhibits translesion bypass by DNA polymerases as shown previously. The noncytotoxic MC metabolite 2,7-diaminomitosene (2,7DAM) forms a G-N7 DNA monoadduct in vitro and in vivo. We tested a potential correlation between the relative ease of bypass of the two adducts and the lack of cytotoxicity of 2,7-DAM. 24-mer and 27-mer DNA templates, adducted at a single guanine either with MC or with 2,7-DAM, and 15-mer primers served as substrates for extension by T7 exo- and Klenow fragment DNA polymerases. In these systems, the G-N7-2,7-DAM adduct was bypassed, resulting in the production of fully extended primer, although at a slower rate than the control nonalkylated template. In sharp contrast, the G-N2-MC monoadduct was not bypassed beyond the adduct position. These results indicate a positive correlation between inhibition of DNA synthesis by the monoadducts of MC and 2,7-DAM and the cytotoxicities of these drugs.


29. Formation and Mass Spectrometric Analysis of DNA and Nucleoside Adducts by S-(1-Acetoxymethyl)glutathione and by Glutathione-S-transferase Mediated Activation of Dihaloalkanes. F. Peter Guengerich. Department of Biochemistry & Center in Molecular Toxicology, Vanderbilt University, 638 Robinson Research Building, 23rd and Pierce Avenues, Nashville, Tennessee 37232. Fax: 615-322-3141. Email: [email protected]. Glenn A. Marsch. Department of Chemistry and Physics, Union University, UU 1881, 1050 Union University Drive, Jackson, Tennessee 38305. Fax: 731-661-5175. E-mail: gmarsch@ uu.edu. Sisir Botta. Center in Molecular Toxicology, Vanderbilt University. To characterize DNA damage by dihaloalkanes, a rapid DNA digest under acidic conditions was developed to identify, by LC-ESI-MS, alkaline labile DNA-dihaloalkane adducts digested to nucleosides. DNA was then covalently modified by S-(1-acetoxymethyl)glutathione (GSCH2OAc), a reagent modeling activated dihaloalkanes. Adducts to G, A, and T were detected at high ratios of [GSCH2OAc]/[DNA]. The relative efficacy of adduct formation was G > T > A. The four DNA nucleosides were then reacted with dihaloalkanes CH2Cl2 and CH2Br2 in the presence of glutathione and glutathione-S-transferases from bacteria (DM-11), rat (GST 5-5), and human (h-T1). All enzymes formed adducts to all four nucleosides, with dGuo most readily modified. CH2Cl2 and CH2Br2 appeared to form adducts equally well. The relative efficiency of adduct formation by GSTs was rat 5-5 > human T1 > DM-11, showing that human GST formed adducts efficiently under the conditions used in this work. Supported in part by USPHS R35CA44353 and P30ES00267. 30. Hydroxyl Radical Oxidation of Cytochrome c by Aerobic Radiolysis. Gang Sun,1 Benedicta N. Nukuna,2 and Vernon E. Anderson.1 (1) Department of Biochemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106-4935. E-mail: [email protected]. (2) Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation. Cytochrome c, a small globular protein associated with the inner mitochondrial membrane, is an electron transporter between cytochrome c reductase and cytochrome c oxidase. Its oxidative damage could result in the activity decline of respiratory chain and serve as a marker for mitochondrial generated oxidation. In this study, to investigate possible pathways of cytochrome c oxidation in vivo, horse heart cytochrome c was exposed to radiolytically generated ‚OH. Spectrometric analysis of irradiated cytochrome c demonstrates the oxidation of Met80, which is confirmed by MALDI-TOF and ESI tandem mass spectrometric analysis of tryptic peptides of irradiated cytochrome c. Additionally, analysis of oxidized cytochrome c by mass spectrometry showed that no fragmentation of cytochrome c was detected, confirming that the ‚OH attacked side chain rather than backbone to generate hydroxylated products in the uncleaved protein. Using solutions containing 50 atom % 18O, our study also provides a novel method of determining the source of oxygen during ‚OH mediated oxidation of proteins. We have also validated the high susceptibility of the solvent accessible sulfur containing amino acids and the aromatic amino acids toward ‚OH oxidation. In addition, two tryptic peptides with multiple oxidized sites were characterized by their unique retention times and


fragmentation patterns. A secondary oxidation event was observed in the methionine containing peptide, MIFAGIK, on resulting in a prominent M + 32 peak due to methionine being oxidized to the sulfone. There was partial correlation between the reactivity of ‚OH with protein residues and their solvent accessibilities in crystallography based surface structures implying that this method could serve to supplement probes of macromolecular structures. 31. Identification and Characterization of Novel Oxidative Cross-Link Lesions. Yinsheng Wang, Qibin Zhang, and Zhenjiu Liu. Department of Chemistry, University of California at Riverside, 900 University Avenue, Riverside, California 92521-0403. Fax: 909-7874713. E-mail: [email protected]. DNA damage induced by reactive oxygen species (ROS) has been implicated in the pathogenesis of a number of human diseases including cancer and aging. Although a multitude of single base lesions induced by ROS have been identified and characterized, only a small number of cross-link lesions that involve two adjacent DNA bases have been characterized. The latter lesions may have implications in the CC f TT and mCG f TT tandem double mutations observed by Loeb and Pfeifer and their co-workers. In our laboratory, we have been using two different experimental approaches to identify new oxidative cross-link lesions. Through one electron photooxidation by using 2-methyl-1,4-naphthoquinone as a photosensitizer, we were able to isolate two novel cross-link lesions formed between two adjacent cytosines. In addition, we have synthesized a photolabile precursor of a reactive intermediate of 5-methylcytosine, incorporated it into dinucleside monophosphates and oligodeoxynucleotides, irradiated with 254 nm light, and identified a novel cross-link lesion between adjacent 5-methylcytosine and guanine. 1H NMR and mass spectrometry were used as major tools for the structure characterization of those lesions. The structure determination of those cross-link lesions provides the first step toward the understanding of the biological implcations of the cross-link lesions in the reported tandem double mutations. 32. Multiple DNA Adducts and Formaldehyde Cross-Links in DNA Reacted with Chemically Activated Tobacco Specific Nitrosamines. Stephen S. Hecht, Pramod Upadhyaya, Guang Cheng, Yongli Shi, Peter W. Villalta, Shana J. Sturla, and Mingyao Wang. University of Minnesota Cancer Center, 420 Delaware Street SE-MMC 806, Minneapolis, Minnesota 55455. Fax: 612-626-5135. E-mail: [email protected]. The tobacco specific nitrosamine 4-(methylnitrosamino)1-(3-pyridyl)-1-butanone (NNK) and its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) are considered to play a significant role as causes of cancer in people who use tobacco products. DNA adducts are central to the carcinogenic process. Previous studies have demonstrated that NNK and NNAL produce methyl and pyridyloxobutyl (POB) adducts in DNA. The methyl adducts have been characterized as 7-methylguanine, O6methylguanine, and O4-methylthymidine, and their role in tumorigenesis has been partially established. The previously characterized POB adducts include O6-POBguanine and 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB)releasing adducts. We have now found that a wide variety of POB and formaldehyde adducts are also formed upon methyl hydroxylation of NNK and NNAL, which produces alkylating agents plus formaldehyde. These include


7-POB-deoxyguanosine (7-POB-dG), N2-POB-dG, O2POB-deoxycytidine (O2-POB-dC), 7-pyridylhydroxybutyldG (7-PHB-dG), N2-PHB-dG, O6-PHB-dG, O2-PHB-dC, x-POB-thymidine, x-PHB-thymidine, x-POB-deoxyadenosine (x-POB-dA), x-PHB-dA, N2-hydroxymethyl-dG, N6-hydroxymethyl-dA, di(N2-deoxyguanosyl)methane, di(N6-deoxyadenosyl)methane, and (N2-deoxyguanosyl-N6deoxyadenosyl)methane. Thus, DNA damage by NNK and NNAL may be far more diverse than previously realized. 33. Pyrimidine Ring Opening in Nitrosative Guanosine Deamination: Experimental Evidence from 18O-Labeling Studies. Rainer Glaser, Sundeep Rayat, and Papiya Majumdar. Department of Chemistry, University of Missouri-Columbia, Columbia, Missouri 65211. Fax: 573 882 2754. E-mail: [email protected] and [email protected]. Deamination of guanosine leads to xanthosine and oxanosine. Dediazoniation of guaninediazonium ion occurs in concert with pyrimidine ring opening and deprotonation to form 1. The functional groups in 1 are extremely susceptible to water addition, and 2-4 are possible hydrolysis products. Oxanosine can be formed from 2-4 and xanthosine from 3 and 4. We studied the deamination of guanosine in 18O-labeled water and of 18O(6) guanosine in water. An improved enzymatic synthesis was developed to prepare 18O(6) guanosine. The MS and 13C NMR analyses of the labeling experiments exclude 4 as a precursor to xanthosine, and 4 also is unlikely as a precursor of oxanosine because the intramolecular cyclizations of 2 or 3 should be faster than the addition of a second water. The experiments show that some of the oxanosine must be formed via 3, and this result demonstrates the very existence of 3. Because of the existence of 3 and because of its chemical competence to form xanthosine, this result suggests that xanthosine may be formed by way of a pyrimidine ring opening and reclosure mechanism.

34. Quantifying DNA Base Deamination Lesions in TK6 and NH32 Cells Exposed to Biologically Relevant Levels of Nitric Oxide. Min Dong, and Peter C. Dedon. Biological Engineering Division, Massachusetts Institute of Technology, Building 56, Room 786, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139. Fax: 617-258-0225. E-mail: [email protected]. Nitric oxide (NO) is a physiologically important molecule that forms genotoxic derivatives associated with inflammation and cancer. In particular, nitrous anhydride and nitrosoperoxycarbonate cause nitrosative and oxidative DNA damage, respectively. To define the predominant chemistry in vivo, we initiated studies to quantify the different DNA lesions produced by reactive nitrogen species with a focus here on base deamination. A sensitive LC/MS technique was developed to quantify (fmol) deoxyxanthosine (dX) and deoxyoxanosine (dO) from dG; deoxyinosine (dI) from dA; and deoxyuridine (dU) from dC. TK6 and NH32 cells were exposed to NO using a novel delivery system that mimics inflammatory


conditions (steady state: 1 µM NO, 180 µM O2). We found a linear dose-response for dX, dI, and dU as a function of NO exposure, with no detectable formation of dO. Furthermore, we investigated the relationships among NO exposure, levels of DNA lesions, and biological indices of NO-induced toxicity. 35. Sequence Specificity of Repair of O6-Methylguanine by O6-Alkylguanine-DNA Alkyltransferase Is Due to Differences in the Rate of Methyl Transfer not Differences in Binding. Thomas E. Spratt, and Aviva S. Meyer. American Health Foundation Cancer Center, Institute for Cancer Prevention, One Dana Road, Valhalla, New York 10595. Fax: 914-7293344. E-mail: [email protected]. O6-Alkylguanine-DNA alkyltransferase (AGT) repairs O6-methyl guanine (O6mG) at different rates depending on the surrounding DNA sequence. We examined the mechanism underlying the sequence specificity by reacting an excess of AGT with oligodeoxynucleotide duplexes containing O6mG at different sites. The oligodeoxynucleotides (0.5 nM) 5′-CGT GGC GCT XYA GGC GTG AGC3′, in which X or Y was G or O6mG, annealed to its complementary strand, were reacted with an excess of protein at 25 °C. The reactions were quenched by the addition of 0.1 N NaOH at various times with a RQF-3 rapid quench instrument. The extents of reaction were monitored by ion exchange HPLC. The time courses followed first-order kinetics. The first-order rate constants were plotted against the initial concentration of AGT and fitted to the hyperbolic equation kobs ) kinact[AGT]0/(KS + [AGT]0). AGT from humans and the ada and ogt genes from Escherichia coli reacted with DNA in which X ) O6mG faster than Y ) O6mG due to an increased kinact. The KS values were found to be 50100 nM, approximately 100-fold lower than Kd values found in EMSA assays. These results indicate that the surrounding DNA sequence does not influence the affinity of AGT to O6mG but influences methyl transfer. 36. Site Selective Generation of Guanine Radicals by Carbonate Radical Anions and Their Further Transformation to Site Specific Spiroiminodihydantoin Lesions in DNA. Avrum Joffe,1 N. E. Geacintov,2 and Vladimir Shafirovich.1 (1) Department of Chemistry, New York University, 29 Washington Place, New York, New York 10003. Fax: 212-998-8421. E-mail: [email protected] and [email protected]. (2) Chemistry Department, New York University. Carbonate radical anions are potentially important oxidants of nucleic acids in physiological environments. One mechanism of generation of these radical anions involves the decomposition of peroxynitrite/carbon dioxide complexes. However, the mechanisms of action are poorly understood, and the end products of oxidation of DNA by carbonate radicals have not been characterized. These oxidation pathways were explored in this work, starting from the laser pulse-induced generation of the primary radical species to the identification of the stable oxidative modifications (lesions). The cascade of events was initiated by utilizing 308 nm XeCl excimer laser pulses to generate carbonate radical anions on submicrosecond time scales. This laser flash photolysis method involved the photodissociation of persulfate to sulfate radical anions and the one electron oxidation of bicarbonate anions by the sulfate radicals to yield the carbonate radical anions. The site selective oxidation of G and 8-oxo-G residues by carbonate radicals results in the


formation of unique end products, the diastereomeric spiroiminodihydantoin (Sp) lesions, the products of a four electron oxidation of guanine. These lesions, formed in high yields (40-60%), were isolated by reversed phase HPLC and identified by MALDI-TOF mass spectrometry. These assignments were supported by the characteristic circular dichroism spectra of opposite signs of the two lesions. The oxidation of guanine to Sp diastereomers occurs, at least in part, via the formation of 8-oxo-G lesions as intermediates. Consistent with this conclusion, small concentrations of 8-oxo-G lesions were detected during the course of oxidation of guanine bases in DNA by carbonate radical anions. The Sp lesions can be considered as the terminal products of the oxidation of G and 8-oxo-G in DNA by carbonate radical anions. The mechanistic aspects of these site selective reactions in DNA initiated by carbonate radicals are discussed. Supported by NIH Grant ES11589. 37. Spectroscopic Detection of Interstrand DNA Cross-Links by 1,N2-Deoxyguanosine Exocyclic Adducts of r,β-Unsaturated Aldehydes. Michael P. Stone,1 Young-Jin Cho,2 Hye-Young H. Kim,3 Markus Voehler,1 Jaison Jacob,4 and Thomas M. Harris.5 (1) Department of Chemistry, Center in Molecular Toxicology, and the Vanderbilt-Ingram Cancer Center, Vanderbilt University, VU Station B Box 351822, Nashville, Tennessee 37235. Fax: 615-322-7591. E-mail: stone@ toxicology.mc.vanderbilt.edu. (2) Department of Chemistry, Center in Molecular Toxicology, and VanderbiltIngram Cancer Center, Vanderbilt University. (3) Center for Cancer Pharmacology, University of Pennsylvania. (4) Department of Biochemistry, Center in Molecular Toxicology, and the Vanderbilt-Ingram Cancer Center, Vanderbilt University. (5) Department of Chemistry, Center in Molecular Toxicology, and the Vanderbilt-Ingram Cancer Center, Vanderbilt University. Acrolein and crotonaldehyde react with deoxyguanosine to form the stereoisomeric 1,N2-exocyclic adducts γ-OH-PdG and R-CH3-γ-OH-PdG, where PdG ) propanodeoxyguanosine. These equilibrate with the corresponding aldehydes N2-(γ-oxopropyl)-dG and N2-(R-CH3γ-oxopropyl)-dG. NMR spectroscopy enabled the chemistry of these adducts to be monitored in DNA, in situ. When the acrolein γ-OH-PdG adduct was placed opposite dC in duplex DNA at pH 7, the equilibrium shifted predominantly toward N2-(γ-oxopropyl)-dG. When the crotonaldehyde (R)R- and (R)S-R-CH3-γ-OH-PdG adducts were placed opposite dC in duplex DNA at pH 7, mixtures of the exocyclic adducts and their corresponding aldehydes were observed. Both the N2-(γ-oxopropyl)-dG and the N2-(R-CH3-γ-oxopropyl)-dG aldehydes equilibrated with their hydrates. In 5′-CpG-3′ sequences, the acrolein N2-(γ-oxopropyl)-dG and crotonaldehyde N2-(R(R)-CH3γ-oxopropyl)-dG aldehydes, but not the crotonaldehyde N2-(R(S)-CH3-γ-oxopropyl)-dG aldehyde, reacted with the deoxyguanosine N2 amino group of the 5′-neighbor base pair, to reversibly form interstrand dG-dG cross-links. These existed predominantly as carbinolamines and not the corresponding imines. Supported by NIH Grant ES05355. 38. The Major Metabolite of the Cancer Chemopreventitive Oltipraz Is a Phase 2 Enzyme Inducer of Comparable Potency and Initiates Nuclear Translocation of Transcription Factor nrf2. Jacobus P. Petzer,1 Mettachit Navamal,1 Mi-Kyoung Kwak,2 Jesse Johnson,2 Thomas W. Kensler,2 and James C. Fishbein.1


(1) Department of Chemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250. Fax: 410-455-2608. E-mail: jpetzer@ umbc.edu. (2) Department of Environmental Health Sciences, Johns Hopkins Bloomberg School of Public Health. The biochemical basis for cancer chemoprevention by dithiolethiones is becoming increasingly clear. Dithiolethiones such as oltipraz (1) raise the levels of many phase 2 xenobiotic metabolizing enzymes through enhanced transcription, which results in both an increase in the quenching of potentially carsinogenic electrophiles and an increase in export of xenobiotic metabolites through conjugation. We are concerned with the mechanism by which oltipraz initiates phase 2 enzyme induction and whether in vivo generated metabolites might be responsible for induction. Oltipraz is extensively metabolized, mainly to the methylated pyrrolpyrazine (2), which is itself further converted to a number of oxidized and glucuronylated forms. It has been demonstrated that 2 is not an inducer of phase 2 enzymes. The mechanism for formation of 2 is believed to proceed via the methylation of the putative intermediate 3, which in turn is generated from chemical or enzyme-activated reduction of oltipraz. Adopting a prodrug strategy, we show that metabolite 3 generated from synthetic precursors 4 and 5 induces phase 2 enzymes with potency comparable to that of oltipraz in cultured cells. We further show that both oltipraz and 4 stimulate nuclear translocation of the transcription factor Nrf2 and transactivate the antioxidant response element (ARE), both processes believed to be involved in phase 2 enzyme induction. Supported by RO1 CA91032.

39. Products from Oxidation of 8-oxo-7,8-Dihydroguanosine by Nitrogen Dioxide Radical. Hongbin Yu,1 John S. Wishnok,1 and Steven R. Tannenbaum.2 (1) Biological Engineering Division, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Room 56-731, Cambridge, Massachusetts 02139. Fax: 617-252-1787. E-mail: [email protected]. (2) Biological Engineering Division and Department of Chemistry, Massachusetts Institute of Technology. 8-Oxo-7,8-dihydroguanosine (8-oxo-dG) is a major and ubiquitous product from DNA oxidation. It is more reactive toward oxidation than guanosine, its precursor. Oxidation of 8-oxo-dG has been intensively studied because of its importance in understanding oxidative damage in DNA. We have been interested in the oxidation of DNA by reactive oxygen and nitrogen species. ‚NO2 may be generated via the decomposition of peroxynitrite or the oxidation of nitrite by peroxidase. Previously, other investigators studied the kinetics of the oxidation of 8-oxo-dG by ‚NO2 (Shafirvich et al. (2001)


Chem. Res. Toxicol. 14, 233-241). Here, we report products from the oxidation of 8-oxo-dG by ‚NO2. Using ‚NO2 generated from horseradish peroxidase (HRP)/H2O2/ NO2- or myeloperoxidase (MPO)/H2O2/NO2-, we have characterized the oxidation products of 8-oxo-dG. In the HRP/H2O2/NO2- system, two major products, spiroiminodihydantoin and oxazolone, are formed; in the MPO/ H2O2/NO2- system, only spiroiminodihydantoin is found to be the major product. Results from the oxidation of 8-oxo-dG in DNA by ‚NO2 will also be discussed. 40. Preparation of Deuterium-Labeled 4-oxo-2Nonenals for Mechanistic Studies of Oxidative Damage to Proteins. Jasbir S. Arora, Tomoyuki Oe, Seon Hwa Lee, and Ian A. Blair. Center for Cancer Pharmacology, University of Pennsylvania, 1246 BRB II/ III, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104-6160. Fax: 215-573-9889. Oxidative stress causes damage to cellular macromolecules such as proteins and DNA by reactive oxygen species (ROS) or ROS-derived lipid hydroperoxides breakdown products. Lipid hydroperoxides are also formed enzymatically during oxidative stress. Both 15-lipoxygenase and cyclooxygenase-2 convert linoleic acid into 13(S)-hydroperoxyoctadecadienoic acid (13-HPODE), the prototypic ω-6 polyunsaturated fatty acid hydroperoxide. We have shown that 4-hydroperoxy-2-nonenal, 4-oxo-2nonenal, 4-hydroxy-2-nonenal, and 4,5-epoxy-2-decenal are formed from 13-HPODE with a variety of initiators. The reaction of 4-oxo-2-nonenal with these species can generate several regioisomers that require extensive NMR studies to determine the correct structures. However, it is possible to determine the regioselectivity of addition by use of deuterium-labeled substrates coupled with mass spectrometry. We report the preparation and utilization of 2-[D1]-, 3-[D1]-, and 2,3-[D2]-4-oxo-2-nonenals for mechanistic studies of lipid hydroperoxidederived modifications to proteins. The deuterated nonenals were used to confirm the regioselectivity of addition to N-R-Ac-HAK to form cyclic peptides. Supported by NIH Grants CA91016 and CA95586. 41. r-Substituted N-Nitroso-morpholines: Chemistry, Reactivity, and Products. Hyun-Joong Kim, and James C. Fishbein. Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250. Fax: 410-455-2608. E-mail: [email protected]. We have carried out an 11 step synthesis of the R-acetoxy-N-nitrosomorpholine. The R-hydroxy compound was generated transiently from the R-hydroperoxy derivative, derived from the R-acetoxy precursor. The kinetics of decay of the R-hydroxy compound obey a three term rate law, with terms for hydrogen ion-dependent, pH-independent, and hydroxide ion-dependent reactions. The reaction of R-hydroxy-N-nitrosomorpholine in aqueous solution, 25 °C, pH 6.6, yields (2-hydroxy-ethoxy) acetaldehyde (62%), acetaldehyde (26%), and glycolaldehyde (26%), based on analysis of the dintrophenylhydrazine derivatives. The observation that the major product is (2-hydroxy-ethoxy) acetaldehyde suggests that novel protein and DNA lesions may be anticipated from Nnitrosomorpholine exposure. These results and further progress related to identifying nucleoside alkylation products will be presented. 42. Adamantyl Propargyl Ethers as Cytochrome P450 Inhibitors. Maryam Foroozesh, Tasha P. Smith, and Jasmine Mesbah. Department of Chemistry, Xavier


University of LA, 1 Drexel Drive, New Orleans, Louisiana 70125. E-mail: [email protected]. Cytochrome P450 enzymes are a superfamily of hemoproteins involved in the metabolism of drugs and environmental chemicals including chemical carcinogens, thus making them the target of many studies (1-3). In our previous research, we have determined that certain aryl and arylalkyl acetylenes act as inhibitors of these enzymes (4, 5). The degree and type of inhibition, as well as the selectivity toward different enzymes, greatly depend on the size and shape of the molecule and the placement of the triple bond. We recently reported the synthesis and characterization of a new family of acetylenes containing an adamantly ring system (6, 7). The four compounds, 1-adamantyl propargyl ether (1-APE), 2-adamantyl propargyl ether (2-APE), 1-adamantylmethyl propargyl ether (1-AMPE), and 1-adamantylethylpropargyl ether (1-AEPE), have been studied in vitro as potential inhibitors of cytochromes P450 2B1 and 2B6 in rats and humans. The results of these studies are presented here. 43. Analysis of Nitrated Proteins by Nitrotyrosine Specific Affinity Probes and Mass Spectrometry. Vadiraja Bhat, George N. Nikov, Christine Chiang, John S. Wishnok, and Steven R. Tannenbaum. Biological Engineering Division, Massachusetts Institute of Technology, 77 Massachusetts Avenue, 56-738A, Cambridge, Massachusetts 02139. Fax: 617-252-1787. E-mail: [email protected]. Tyrosine nitration is a well-established protein modification that occurs in vivo in disease states associated with oxidative stress and increased nitric oxide synthase activity. Nitration of specific tyrosine residues has been reported to affect protein structure and function, suggesting that 3-nitrotyrosine formation may not only be a disease marker but may also be involved in the pathogenesis of certain diseases. It is, however, difficult to identify sites of nitration using currently existing methods. We describe a new proteomics-based approach for the characterization of nitrated proteins. Our method combines specific isolation of nitrated proteins from complex protein mixtures with mass spectrometric determination of the amino acid sequence and the site of nitration of individual proteins. A complex protein mixture, e.g., serum or cell lysate, was enriched for nitrotyrosine-containing proteins by immunoprecipitation with anti-nitrotyrosine antibodies. The nitrotyrosines were then reduced to aminotyrosines with a strong reducing agent in parallel in-gel and in-solution procedures. In the present work, we have used nitrated human serum albumin as a model protein to develop the selective capture method. After reducing the disulfide bonds with dithiothreitol and alkylating the free sulfhydryl groups with iodoacetamide, all of the nitrotyrosines were reduced to aminotyrosines using sodium dithionite. Cleavable biotin tags that were specific to aminotyrosine at pH 5.0 were attached to aminotyrosines, and the albumin was subsequently digested with trypsin. To selectively capture the biotinylated peptides, extracted tryptic peptides were subjected to a biotin-avidin affinity chromatography column. Purified tryptic peptides were identified by MALDI-MS. Using this method, we can effectively enrich the nitrated peptides from a digest of nitrated human serum albumin. We have also purified nitrated human serum albumin from a complex protein mixture by spiking SJL mouse plasma with nitrated human serum


albumin and then subjecting the mixture to the procedure described above. The enriched protein was then digested with trypsin and confirmed as human serum albumin by peptide mass fingerprinting with the Internet software package, MASCOT. 44. Arginine Adducts as Endogeneous Inhibitors of nNOS: Analysis Using an LC/MS-Based Enzyme Assay. Susanne Goeters. Department of Pharmacology, University of Pennsylvania, 1257 BRB II/III, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104. Fax: 215573-9889. E-mail: [email protected]. Tomoyuki Oe, Center for Cancer Pharmacology, University of Pennsylvania. Ian A. Blair. Center for Cancer Pharmacology and Genomics Institute Proteomics Facility, University of Pennsylvania. Hydroperoxides of arachidonic acid and other polyunsaturated acids such as linolenic and linoleic acid are suspected to be involved in the generation of chronic and spontaneous diseases due to their ability to form reactive degradation products. The main lipid peroxidation (LPO) products are hydroxy acids and reactive aldehydes. Glyoxal and methylglyoxal, which have been detected as LPO products, are known to form adducts with Larginine. We have shown previously that 4-oxo-2-nonenal (another LPO product) also forms an adduct with arginine. However, adducts with 4-hydroxy-2-nonenal or malondialdehyde (two important products of LPO) have not been described. We report the characterization of L-arginine adducts formed from LPO products using LC/ MS/MS and NMR. We also report an investigation of their potency as potential endogenous nitric oxide synthase (NOS) inhibitors by use of an LC/MS/MS based nNOS enzyme assay. Supported by NIH RO1-CA95586. 45. Aryllithium-Mediated Carbon-Nitrogen Bond Formation with Nitroarenes. Tianle Yang, and Bongsup Cho. Department of Biomedical Sciences, University of Rhode Island, 41 Lower College Road, Kingston, Rhode Island 02881. Fax: 401-874-5048. E-mail: twan8812@ postoffice.uri.edu. Diarylamines belong to an important class of functional groups that are accessible mostly via metal-catalyzed cross-coupling C-N bond formation using aromatic amines as precursors (the Buchwald-Hartwig reaction). We have explored the utility of nitroarenes as starting materials for the preparation of diarylamines including arylamineDNA adducts. Reactions of 4-(N,N-dimethylamino)phenyllithium with a series of nitroarenes afforded the corresponding diarylamines in moderate yields. The use of at least 3 equiv of aryllithium is required to complete the reaction. The utility of the new reaction lies in its simplicity and complementarities to existing procedures. A “nitroso”-based mechanism for this novel nitroreductive N-arylation and its application to adduct synthesis will be discussed.

46. ATR-FTIR Study of Organophosphorus Hydrolase Secondary Structure of Langmuir-Blodgett Films. Jiayin Zheng, Xihui Cao, Sarita V. Mello, and Roger M. Leblanc. Department of Chemistry, University


of Miami, 1301 Memorial Drive, Coral Gables, Florida 33146. E-mail: [email protected]. Organophosphorus (OP) compounds are widely used in the agriculture industry around the world as pesticides and insecticides. However, they are neurotoxic compounds, which will irreversibly inhibit the enzyme acetylcholinesterase, essential for the functioning of the central nervous system in human and insects. Organophosphorus hydrolase (OPH) based biosensor offered the method of determination as well as detoxification for OP compounds, so the surface chemistry and the spectroscopic properties of OPH Langmuir and LangmuirBlodgett films are of much importance. FTIR spectroscopy, especially in the attenuated total reflectance (ATR) mode, is well-suited for determining structural features of proteins. We describe here some of the ATR results of the OPH Langmuir-Blodgett films. The ATR spectra of the OPH mono- and multilayer are assigned to the ∫A Ä -helical and ∫A ˆ -sheet or turn conformation. The effects of the surface pressure of deposition and the number of layers on the ATR spectra will be discussed. 47. Bioactivation of the SERM, EM-652 to Quinoids. Ju Liu. Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612. Fax: 312-996-7107. E-mail: [email protected]. Gregory R. J. Thatcher. Department of Medicinal Chemistry & Pharmacognosy, University of Illinois at Chicago, College of Pharmacy. Judy L. Bolton. Department of Medicinal Chemistry & Pharmacognosy, University of Illinois at Chicago, College of Pharmacy. EM-652 (acolbifene), a fourth generation SERM (selective estrogen receptor modulator), is the active form of the ester prodrug EM-800. As a pure antagonist on breast tumor development and growth, it does not stimulate endometrial tissue and offers more potent protection against bone loss than tamoxifen. The potential for EM652 to be metabolized to cytotoxic electrophilic metabolites was investigated in this study. EM-652 could be chemically oxidized to its quinone methide, and its halflife was determined to be 32 s under physiological conditions. This quinone methide reacted with GSH (glutathione) to form three mono-GSH conjugates characterized by LC-MS/MS and NMR. The incubation of EM652 with rat liver microsomes and tyrosinase in the presence of GSH also showed formation of an EM-652 quinone methide and o-quinone GSH conjugates. These data imply that EM-652 could be oxidized to an electrophilic quinone methide and/or o-quinone, which could alkylate cellular macromolecules in vivo. 48. Biomarkers of Acrolein Exposure: Competitive ELISA for the Quantitation of AcroleinProtein Adducts. Hui Li, Jianling Wang, Bhupendra S. Kaphalia, Ghulam A. S. Ansari, and M. Firoze Khan. Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77555-0609. Acrolein is a toxic environmental pollutant. There is a need for a sensitive and specific method to monitor human acrolein exposure. Acrolein causes covalent modification of proteins leading to acrolein-protein adducts (APA). A competitive ELISA method for the quantitation of APA in biological samples was developed using a fixed dilution of anti-acrolein-KLH antibody and varying concentrations of acrolein-albumin adducts, which provided a linear detection range between 250 and 10 000 pg.


Potential of the method for use as a biomarker of acrolein exposure was evaluated by orally administering single or 7 doses of 9.2 mg/kg/day acrolein in rats and quantitating APA in the serum. Greater formation of APA was observed in acrolein-treated rats as compared to controls. Western blot analyses of APA in the sera from acroleintreated rats also showed protein bands with greater intensity. Results suggest that APA quantitation has a potential to be used as a biomarker of acrolein exposure and for risk assessment. 49. Quantification of Strand Breaks and Abasic Sites by Accelerator Mass Spectrometry. Xinfeng Zhou, and Peter C. Dedon. Biological Engineering Division, Massachusetts Institute of Technology, 56-786, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139. Fax: 617-258-0225. E-mail: [email protected]. We have developed a simple and rapid method to quantify abasic (AP) sites and strand breaks induced by many DNA-damaging agents. The method exploits the reaction of aldehyde- and ketone-containing deoxyribose oxidation products with 14C-methoxyamine to form stable oxime derivatives that are quantified by accelerator mass spectrometry (AMS). The method detects one lesion in 106 nt in 1 µg of DNA (14C specific activity 0.5 Ci/mol) and was validated using uracil-containing DNA as a standard for AP sites, with a >0.95 correlation between the quantities of AP sites and the methoxyamine labels. DNA oxidized by γ-radiation also showed a 1:1 correlation between deoxyribose oxidation events (strand breaks, AP sites) and methoxyamine labels. The method offers an index of total deoxyribose oxidation that allows comparison of different oxidants, and when combined with DNA glycosylases, the approach measures the proportions of base and deoxyribose lesions. Furthermore, the high membrane permeability of methoxyamine permits quantification of cellular AP sites and strand breaks. 50. Cancer Chemopreventive Oltipraz Generates Superoxide Radical. Murugesan Velayutham,1 Frederick A. Villamena,1 James C. Fishbein,2 and Jay L. Zweier.1 (1) Department of Internal Medicine, The Ohio State University, 124, TMRF, 420, West 12th Avenue, Columbus, Ohio 43210. Fax: 614-292-8454. E-mail: [email protected]. (2) Department of Chemistry and Biochemistry, University of Maryland, Baltimore County. Oltipraz (OLT) (1) is a cancer preventive drug currently in phase 2 clinical trials as a protective agent against environmentally induced hepatocellular carcinoma. It has been shown that oltipraz may exert its chemopreventive effects through radical generation (Kim, W., and Gates, K. S. (1997) Chem. Res. Toxicol. 10, 296-301). EPR spin trapping studies of OLT in phosphate buffer solution (10% acetonitrile) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) exhibited a 1:2:2:1 quartet spectrum with hyperfine coupling constants aN ) aH ) 14.9 G, characteristic of the hydroxyl radical adduct of DMPO. When the solution was bubbled with argon, there was no EPR signal. The EPR signal was quenched by the addition of the superoxide scavenging enzyme Cu,Zn-superoxide dismutase. In 80% dimethyl sulfoxide, OLT and DMPO exhibited a spectrum with hyperfine values of aN ) 12.7 G, aH1 ) 10.3 G, and aH2 ) 1.3 G, corresponding to the superoxide radical adduct of DMPO. These studies demonstrate that OLT reacts with oxygen with genera-


tion of superoxide free radical. Supported by RO1 CA91032-01 (J.C.F.).

51. Characterizing Hepatotoxicity in Collagen Sandwiches of Rat Hepatocytes. Dora Farkas, Brianna Crantz, Saraswathi Mandapati, and Steven Tannenbaum. Bioengineering Division, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Room 56-731, Cambridge, Massachusetts 02139. Fax: 617-252-1787. E-mail: [email protected]. Primary rat hepatocytes sandwiched between two layers of collagen maintain their viability, protein secretion, and P450 activity for several weeks. We have characterized the toxicity of several compounds in this system by measuring the secretions of urea and albumin and studying changes in cell morphology. The toxins that we have studied include aflatoxin B1, cadmium, methylmethanesulfonate (MMS), N-Methyl-N′-nitro-N-nitrosoguanidine (MNNG), and acetaminophen. We have found the collagen sandwich to be more suitable for characterizing the toxicity of compounds, which do not require metabolism, specifically cadmium and MMS. A 1 mM dose of MNNG or acetaminophen was not toxic, possibly because the cells do not have the metabolic capacity necessary to convert the compounds to toxic metabolites. We have also shown that aflatoxin B1 is toxic in nanomolar doses. Treatment with aflatoxin B1 significantly reduced the secretion of two acute phase proteins, R1-antitrypsin and R2-macroglobulin. PCR analysis showed that the mRNA for these two proteins is decreased in cells treated with aflatoxin B1, suggesting that the proteins are transcriptionally down-regulated. In summary, we have shown that collagen sandwiches are suitable for studying the toxicity of direct-acting compounds, cadmium and MMS, and aflatoxin B1 but not for certain compounds requiring metabolic activation such as MNNG and acetaminophen. 52. Chemical Characterization of Fluoro-Jade B, a High Affinity Histochemical Marker of ToxicantInduced Neuronal Degeneration. Lulu Xu,1 Thomas Heinze,2 Amy Pogge,1 William Slikker Jr.,1 and Larry Schmued.1 (1) Division of Neurotoxicology, NCTR/FDA, 53D-203G, 3900 NCTR Road, Jefferson, Arkansas 72079. Fax: 870-543-7636. E-mail: [email protected]. (2) Division of Chemistry, FDA/National Center for Toxicological Research. Fluoro-Jade B (FJB) is a very effective fluorescent dye used for detecting degenerating neurons in tissue sections. It has been previously synthesized as a mixture containing a number of structural analogues of FluoroJade. The present research focused on isolation, purification, and identification of the biologically active compound(s) of FJB. The reaction mechanism suggests eight possible products from the resorcinol and benzenetetracarboxylic dianhydride reaction. To separate each component, two analytical HPLC methods have been successfully developed, which consists of a reversed phase C18 column and either a pH gradient or an acetonitrile gradient, respectively. For each fraction collected during


the quantitative separation, the negative ion electrospray mass spectra showed the expected [M - H-] ions and reasonable fragmentation with in-source collision-induced dissociation (CID). The proton NMR spectra characterized each compound. All theoretically predicted compounds were detected, isolated, and characterized. Three of these compounds are highly fluorescent and will be evaluated as the possible active moiety of FJB. 53. Combinatorial Chemistry of Organophosphorus Hydrolase Binding Sites for Molecular Recognition of Organophosphorus Compounds. Jiayin Zheng, Xiaojun Ji, and Roger M. Leblanc. Department of Chemistry, University of Miami, 1301 Memorial Drive, Coral Gables, Florida 33146. E-mail: zhengjiayin@ yahoo.com. Organophosphorus hydrolase (OPH) is a bacterial enzyme that has been shown to degrade a wide range of neurotoxic organophosphate nerve agents. So the OPH based biosensors have shown a promising future in the detection of the organophosphorus compounds (OPs), which are widely used in agriculture as pesticides and insecticides. Several experimental techniques, including surface pressure-area isotherms, UV-vis absorption, and epifluorescence microscopy at the air-water interface, were used to study the properties of the three OPH libraries (OPH. LIB. A; OPH. LIB. B; OPH. LIB. C) form from the binding sites of OPH. On the basis of the experimental results, OPH. LIB. A used here seems to be a very stable and general method to design surfaceoriented enzymatic bioassays in order to meet different analytical purposes, in particular molecular recognition. 54. Defining the Source of M1G in Isolated DNA and Cells. Xinfeng Zhou, and Peter C. Dedon. Biological Engineering Division, Massachusetts Institute of Technology, 56-786, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139. Fax: 617-258-0225. E-mail: [email protected]. The mutagenic M1G adduct arises in reactions of DNA with malondialdeyde (MDA; lipid peroxidation) or base propenals (deoxyribose 4′-oxidation). We have begun to define the source of M1G in isolated DNA and in cells exposed to oxidizing agents. Purified DNA was treated with γ-radiation, Fe-EDTA, peroxynitrite, and bleomycin, and the levels of MDA, base propenals, and M1G were determined. Fe-EDTA and γ-radiation produced high levels of MDA but no detectable M1G or base propenals (A,T) despite high levels of deoxyribose oxidation. However, peroxynitrite and bleomycin caused high levels of M1G and base propenals but no detectable MDA. For cellular studies, Escherichia coli and human TK6 cells containing varying amounts of membrane linoleic acid (18:2; GC analysis) were exposed to peroxynitrite or SIN1, a peroxynitrite generator, respectively. In both models, the M1G level was inversely correlated with the quantity of membrane PUFA. These results have implications for the endogenous source of M1G. 55. Development and Application of an Improved Method for Determination of 1-Hydroxypyrene (1HOP) in Human Urine. Steven G. Camella,1 Ky-Anh Le,1 Dorothy K. Hatsukami,2 and Stephen S. Hecht.1 (1) University of Minnesota Cancer Center, 420 Delaware Street SE-MMC 806, Minneapolis, Minnesota 55455. Fax: 612-626-5135. E-mail: [email protected]. (2) Transdisciplinary Tobacco Use Research Center, University of Minnesota.


Urinary 1-HOP is a biomarker for human uptake of polycyclic aromatic hydrocarbons (PAH), many of which are potent carcinogens. PAH are likely causative factors for human cancers associated with occupational exposures and cigarette smoking. We have developed an improved method for analysis of 1-HOP in human urine. 1-Hydroxybenz[a]anthracene was used as internal standard. Enrichment of 1-HOP, freed from its conjugates by enzyme treatment, was accomplished with 96-well compatible solid phase extraction cartridges. Autoinjection HPLC fluorescence was used for quantitation. Precision and accuracy were excellent. Major advantages of the new method include speed of analysis, improved sensitivity, and use of an internal standard. The method was applied to determine levels of 1-HOP in the urine of smokers and nonsmokers, to assess the longitudinal stability of 1-HOP levels, and to determine the effects of reduction in cigarettes per day on 1-HOP levels in urine. The new method should be widely applicable in studies of human PAH uptake. 56. Development of a Sensitive Assay for the Detection of DNA Adducts Formed by cis-2-Butene1,4-dial. Michael C. Byrns. Division of Environmental and Occupational Health, University of Minnesota, Minneapolis, Minnesota 55455. E-mail: [email protected]. Lisa A. Peterson. Division of Environmental and Occupational Health and Cancer Center, University of Minnesota. Furan is an environmental compound that has been shown to be toxic and carcinogenic in rodents. Both genotoxic and nongenotoxic mechanisms have been proposed for the carcinogenic activity of furan. Furan is metabolized by P450 2E1 to the reactive metabolite cis2-butene-1,4-dial, which is thought to be responsible for the biological effects of furan exposure. cis-2-Butene-1,4dial reacts with N2 and N3 of dCyd, N1 and N6 of dAdo, and N1 and N2 of dGuo. The initial products of these reactions were previously characterized; however, the purine adducts undergo dehydration to form substituted etheno adducts. The nucleoside adducts can also be detected in enzyme hydrolysates of DNA treated with cis2-butene-1,4-dial by monitoring the neutral loss of 2′deoxyribose with LC-MS/MS. However, poor separation of the adducts from the unmodified nucleosides and polymerization of some of the adducts limited the sensitivity of this assay. Because each of the adducts retains an aldehyde functionality, they can be derivatized with O-alkylhydroxylamines. O-Methyl- and O-benzyl-hydroxylamine both react readily with the adducts to form the corresponding oximes. Derivatization with O-benzylhydroxylamine considerably improved the separation of the adducts from the unmodified nucleosides, thus enhancing the detection of these adducts in DNA treated with cis2-butene-1,4-dial. Supported by ES-10577. 57. Development of Tools to Distinguish Nitrosative and Oxidative DNA Damage at Sites of Inflammation. Min Dong, Yelena Margolin, Viengsai Vongchampa, and Peter C. Dedon. Biological Engineering Division, Massachusetts Institute of Technology, Building 56, Room 786, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139. Fax: 617-258-0225. E-mail: [email protected]. Activated macrophages produce nitric oxide (NO) that reacts to form DNA-damaging species such as nitrous anhydride (N2O3) and peroxynitrite (ONOO). N2O3 deaminates DNA bases to form xanthine (dX), hypoxanthine


(dI), and uracil (dU), while ONOO causes oxidation of dG. However, the relative contributions of these chemistries to inflammation-induced DNA damage are unknown. To this end, we identified DNA glycosylases that differentially recognize the two classes of DNA lesions and combined them with the DNA damage mapping technique, ligation-mediated PCR (LMPCR), to map the lesions in specific sequences in purified DNA and in cells. A mixture of Escherichia coli AlkA and uracil glycosylase recognized N2O3-exposed DNA (removal dX, dI, and dU) but not ONOO-damaged DNA. E. coli Fpg, which recognizes most ONOO-induced DNA lesions, reacted only with ONOO-damaged DNA. We applied this technique to study ONOO- and N2O3-induced DNA damage in different states of supercoiling and for DNA damage with ONOO delivered by rapid bolus injection and slow infusion. 58. Differential Modification of the Ligand Binding Domains of Human Estrogen Receptors r and β by the Equine Estrogen Metabolite, 4-Hydroxyequilenin. Minsun Chang,1 Barbara Calamini,2 Dejan Nikolic,1 Kiira Ratia,2 Richard B. van Breemen,1 Andrew D. Mesecar,2 and Judy L. Bolton.1 (1) Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612. Fax: 312-996-7107. E-mail: [email protected]. (2) Center for Pharmaceutical Biotechnology, College of Pharmacy, University of Illinois at Chicago. Estrogen receptors (ERs) R and β, which are nuclear transcription factos, are shown to play a role in estrogeninduced carcinogenesis, although it is not clear what the role of each receptor is in the mechanism of carcinogenesis. 4-Hydroxyequilenin (4-OHEN) is a redox active metabolite of equine estrogens present in Premarin, the widely prescribed estrogen replacement formulation. It has been shown that 4-OHEN was much more toxic and induced considerably more DNA damage in ER R positive breast cancer cells as compared to an ER R negative cell line. We hypothesize that 4-OHEN binds to the ERs, which carries it directly to estrogen sensitive genes, where DNA damage occurs. In our study, the potential for 4-OHEN to covalently bind to the LBDs of two ERs was investigated using ESI-MS analysis. ER R-LBD showed more sensitivity to 4-OHEN-o-quinone-mediated modification as compared to ER β-LBD. The presence of ligands for ERs such as estradiol, genistein, raloxifene, and 4-hydroxytamoxifen slightly protected the ER β-LBD but not the ER R-LBD from covalent modification. Our data implies that there are differential roles for the two ERs in 4-OHEN-induced DNA damage and carcinogenesis in vivo. 59. DNA Adduction and in Vitro Mutagenicity of Chlorophenols by Peroxidase Activation: Role of Ambident (O- vs C-) Phenoxyl Radicals. Jian Dai,1 Dean R. Lantero,2 Amy L. Sloat,1 Marissa Adams,2 Steven A. Akman,2 and Richard A. Manderville.1 (1) Department of Chemistry, Wake Forest University, Salem Hall, Winston-Salem, North Carolina 27109. Fax: 336-758-4656. E-mail: [email protected]. (2) Department of Cancer Biology, Wake Forest University School of Medicine. Chlorophenols have been widely used as biocides and are important environmental pollutants. The prooxidant properties of chlorophenols are thought to attribute to their toxic effects by facilitating oxidative stress, oxida-


tive DNA damage, and direct DNA adducts. In this study, the abilities of pentachlorophenol (PCP) and 2,4,6trichlorophenol (TCP) to react with calf thymus DNA in the presence of horseradish peroxidase (HRP)/H2O2 were investigated using LC/MS and NMR. These efforts follow from our recent findings that phenoxyl radicals of chlorophenols react at the C8-position of 2′-deoxyguanosine (dG) to yield O- and C-bound adducts. The major adduct from the PCP/HRP/H2O2 treatment was identified as the O-bonded C8-dG adduct indicating a major role for the PCP-phenoxyl radical in the DNA adduction process. In contrast, TCP reacts with dG to form a small amount of the O-bonded C8-dG adduct; the major species being a cyclic adduct resulting from attachment of dG to 2,6dichloro-1,4-benzoquinone. Assessment of the in vitro mutagenicity of PCP and TCP using the human mutation reporter plasmid pSP189 shows that both are directly genotoxic mutagens upon activation by HRP/H2O2. The implications of these findings to chlorophenol mutagenicity and carcinogenicity will be discussed. 60. DNA Damage and Cytotoxicity Induced by Minor Groove Binding Methyl Sulfonate Esters. Sridhar Varadarajan,1 Dharini Shah,2 Prasad Dande,3 Samuel Settles,4 and Barry Gold.1 (1) Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Box 986805, 600 South 42nd Street, Omaha, Nebraska 68198-6805. Fax: 402-559-4651. E-mail: [email protected] and [email protected]. (2) Eppley Institute and Department of Pharmaceutical Sciences, University of Nebraska Medical Center. (3) Medicinal Chemistry, ISIS Pharmaceuticals. (4) Eppley Institute for Research in Cancer and Department of Pharmaceutical Sciences, University of Nebraska Medical Center. Minor groove specific DNA equilibrium binding dipeptides (lex) based on N-methylpyrrole carboxamide and/ or N-methylimidazolecarboxamide subunits have been modified with an O-methyl sulfonate ester functionality in order to target DNA methylation in the minor groove at A/T and/or G/C rich sequences. Using HPLC and sequencing gel analyses, the Me-lex compounds all selectively react with DNA to afford N3-alkyladenine as the major adduct. The formation of the N3-alkyladenine lesions is sequence-dependent based on the equilibrium binding preferences of the different lex dipeptides. In addition to the reaction at adenine, the molecules designed to target G/C sequences also generate lesions at guanine; however, the methylation is not sequencedependent and takes places in the major groove at N7G. To determine how the level of the different DNA adducts and sequence selectivity for their formation affects cytotoxicity, the Me-lex analogues were tested in wild-type Escherichia coli and in mutant strains defective in base excision repair (tag and/or alkA glycosylases or apn endonuclease), nucleotide excision repair (uvrA), base and nucleotide excision repair (tag/alkA/uvrA), and O6alkylguanine-DNA alkyltransferase (ada/ogt). The results demonstrate the importance of 3-alkyladenine lesions in cellular toxicity and the dominant role of base excision repair. There is no evidence that the sequence specificity is related to toxicity. 61. DNA Interchain Cross-Link Formation by a Deoxyguanosine Adduct of the Epoxide of Vinyl Chloride. Ivan D. Kozekov, Angela K. Brock, Carmelo J. Rizzo, and Thomas M Harris. Department of Chemistry and Center in Molecular Toxicology, Vanderbilt University, Nashville, Tennessee 37235-1822. Fax: 615-


322-2649. E-mail: [email protected]. Vinyl chloride is an important industrial chemical, which is known to be a human carcinogen strongly associated with hepatic angiosarcoma. It is metabolized to chlorooxirane, a bifunctional electrophile, which can react with DNA. Proximal and distal hydroxyethano adducts arise from reaction with deoxyguanosine. Oligonucleotides containing these adducts have been synthesized. A slow dehydration of the distal adduct gives 1,N2etheno-dG, but the proximal does not dehydrate. In a CpG sequence context, the proximal adduct undergoes interchain cross-linking with deoxyguanosine, forming a carbinolamine. The cross-linking reaction is comparable to that of analogous proximal hydroxypropano adducts of enals. Supported by NIH Grant P01-ES05355.

62. Dockings of Structurally Related Diolepoxides of Benzo[ghi]fluoranthene and Benzo[c]phenanthrene with DNA: Sequence Dependence. Kenneth R. Overly,1 Bongsup P. Cho,2 Amy K. McIntyre,1 and Roland F. Ostapoff.1 (1) Department of Chemistry and Biochemistry, Providence College, 549 River Avenue, Providence, Rhode Island 02918. Fax: 401-865-1438. E-mail: [email protected]. (2) Department of Biomedical Sciences, University of Rhode Island. Metabolic activation of polycyclic aromatic hydrocarbons (PAHs) to reactive diolepoxides (DEs) followed by binding to DNA is a putative mechanism for causing DNA lesions. We proposed ((2002) Chem. Res. Toxicol. 15, 198-208) that the pseudo-diaxial diol conformation of DEs has more favorable electrostatic interactions in the minor groove of DNA than the pseudo-diequatorial conformation. To explore this hypothesis and examine the sequence dependence of DNA binding, anti- and syn-DEs of benzo[c]phenanthrene (B[c]P) and its planar analogue benzo[ghi]fluoranthene (B[ghi]F) were flexibly docked into different sequences of canonical B-DNA using Au-


toDock. Blind and restricted docking grids allow for evaluation of minor vs major groove binding preference. The effects of DE conformation and sequence-dependent variations in DNA geometry on binding affinity are discussed. 63. Effect of Bound Carcinogens on DNA Misincorporation Kinetics Catalyzed by DNA Polymerases. Hong Zang,1 F. P. Guengerich,1 and Thomas M. Harris.2 (1) Department of Biochemistry & Center in Molecular Toxicology, Vanderbilt University, 638 Robinson Research Building, 23rd and Pierce Avenues, Nashville, Tennessee 37232. E-mail: hongzang0114@ hotmail.com. (2) Department of Chemistry and Center in Molecular Toxicology, Vanderbilt University. We examined DNA polymerase reactions for several DNA adducts, with benz[a]anthracene-diol epoxide and benzo[a]pyrene-diol epoxide residues on the N6 of adenine, acrolein and crotonaldehyde adducts on guanine, epoxy butanediol, styrene epoxide, and benzo[a]pyrenediol epoxide residues on the N2 atom of guanine. These residues on the DNA templates strongly blocked primer extension catalyzed by bacteriophage T7 exo-DNA polymerase and HIV reverse transcriptase. In the cases of dG-acrolein, dG-crotonaldehyde, dG-benzo[a]pyrene, dAbenzo[a]pyrene, and dA-benz[a]anthracene adducts, dA was the most favored nucleotide incorporated opposite to the adducts. For the dG-epoxy butanediol and dGstyrene epoxide adducts, dT was favored. Such misincorporation would lead to potential mutagenic events. Steady state kinetics for these DNA substrates was determined for T7 exo-DNA polymerase. However, there were no significant incorporations by reverse transcriptase for all of the lesions studied. 64. Effect of Bulk at Guanine N2 on DNA Polymerase Blockage, Bypass, and Fidelity. Jeong-Yun Choi. Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, 642 Robinson Research Building, 23rd Avenue at Pierce. Fax: 615-322-3141. E-mail: choijyun@ toxicology.mc.vanderbilt.edu. F. P. Guengerich. Department of Biochemistry & Center in Molecular Toxicology, Vanderbilt University, 638 Robinson Research Building, 23rd and Pierce Avenues. Fax: 615-322-3141. E-mail: [email protected]. The N2 atom of guanine is susceptible to modifications by various potential carcinogens including styrene oxide and acetaldehyde. We prepared guanine (G) nucleosides with increased bulk at N2 and synthesized oligonucleotides to examine the effect of bulk at guanine N2 on blockage, bypass, and fidelity in DNA replication by the processive viral DNA polymerases HIV1-reverse transcriptase (RT) and phage T7- and a trans-lesion synthesis DNA polymerase, human Pol kappa. RT and T7- effectively bypassed only N2-methylG but were strongly blocked by N2-ethylG, N2-isobutylG, N2-CH2-benzylG, and N2-CH2-9-anthracenylG. In contrast, Pol kappa effectively bypassed N2-ethylG, N2-isobutylG, and N2CH2-benzylG in a relatively error-free manner. Proliferating cell nuclear antigen (PCNA) stimulated the activity of Pol kappa slightly. Pol kappa was unable to bypass N2-CH2-9-anthracenylG. These results suggest that human Pol kappa may play a role in the error-free bypass of a certain of guanine N2-adducts and demonstrate the role of bulk in lesions. Supported in part by USPHS R01 ES10375 and P30 ES00267.


65. Effects of Brief Exposure to High Concentrations of Nitric Oxide-Air Mixtures on Breathing in Awake Rats. Zengfa Gu, and Adolph J. Januskiewicz. Department of Respiratory Research, Walter Reed Army Institute of Research, 503 Robert Grant Avenue, Silver Spring, Maryland 20910. E-mail: zengfa.gu@ na.amedd.army.mil. In the presence of oxygen (O2), nitric oxide (NO) is rapidly converted to a potentially toxic gas, nitrogen dioxide (NO2). Brief exposure to high concentration of NO is of concern in military operations. This study investigated the changes in breathing resulting from 5 min exposures to mixtures of 3000, 6000, and 10 000 ppm NO with medical grade air (20% O2, 80% N2). Male SpragueDawley rats (250-380 g) were randomly grouped and placed in nose-only position into restrainers connected to a polyethylene exposure chamber. Respiration was recorded and analyzed using the DATAQ data acquisition system. In a 5 min stabilizing period immediately before exposure, none of animals showed apnea. During 5 min exposure, repetitive apnea occurred in each animal. Apneic events that lasted for longer than 2.0 s were counted. During 5 min exposure in 3000, 6000, and 10 000 ppm groups, apneic events were 14.0 ( 1.0, 17.5 ( 6.5, and 22.0 ( 4.0 (mean ( SD), respectively; apneic durations were 2.93 ( 0.12, 3.30 ( 0.20, and 3.33 ( 0.40 s (mean ( SD), respectively; and total apneic times were 41.1 ( 2.9, 56.5 ( 18.0, and 75.6 ( 4.4 s (mean ( SD), respectively. These results suggest that brief inhalation of an NO-air mixture may cause severe respiratory disorder involved by the toxicities of both NO and NO2. 66. Effects of Methylation and Temperature on Nucleotide Excision Repair of Stereoisomeric Benzo[a]pyrene Diol Epoxide-N2-dG Adducts in a 5-MeCG* p53 Codon 273 Sequence Context Catalyzed by UvrABC Proteins from B. caldotenax. Katharine C. Colgate,1 Xuanwei Huang,2 Aleksandr Kolbanovskiy,2 Milan Skorvaga,3 Bennett Van Houten,3 Shantu Amin,4 and Nicholas E. Geacintov.5 (1) Department of Chemistry, New York University, 31 Washington Place, New York, New York 10003. Fax: 212-998-8421. E-mail: [email protected]. (2) Chemistry Department, New York University. (3) NIEHS-NIH. (4) American Health Foundation, Valhalla, New York 10595. (5) Department of Chemistry, New York University. A disproportionate number of mutations are found in certain codons of the p53 gene, mostly at CpG dinucleotide sequences, which are highly methylated in human tissues. There is evidence that bulky carcinogens such as the diol epoxide r7,t8-dihydroxy-t9,10-epoxy-7,8,9,10tetrahydrobenzo[a]pyrene (B[a]PDE) with N2-guanine are enhanced when the C in CpG dinucleotide sequences in DNA is methylated. We have shown that there is a striking change in the conformation of the (-)-transB[a]P-N2-dG lesions (G*) in the CpG* sequence. In the case of the stereoisomeric (+)-trans-B[a]P-N2-dG adduct, there are no marked changes in adduct conformations, but the solvent accessibility of the pyrenyl residue is enhanced in the 5-MeCpG sequence context. Because differential DNA repair in methylated and nonmethylated sequences may affect the persistence and thus the mutagenic potentials of these lesions, it is important to evaluate the effects of methylation on the removal of such bulky adducts by nucleotide excision repair enzymes. Employing duplexes 54 base pairs long with a central sequence containing the p53 273 codon of exon 8, the


effect of methylation on the excision of (+)- and (-)-transB[a]P-N2-dG adducts by UvrABC proteins from the thermophilic protein B. caldotenax was investigated at 37 and 55 °C. In both stereoisomers at 37 °C, nucleotide excision repair is greater by factors of about 2′′C3 in the 5-MeCpG* sequence context than in the CpG* sequence context, but at 55 °C, this difference disappears. Our hypothesis is that at the higher temperature, the thermal disorder tends to reduce any differences in adduct conformations that exist at the lower temperature of 37 °C. Supported by NIH/NCI Grants CA20851 and CA76660. 67. Elucidating Nucleotide Incorporation Opposite a Bulky DNA Lesion by a Y-Family Bypass DNA Polymerase through Molecular Dynamics Simulations. Min Wu. Department of Chemistry, New York University, 100 Washington Square East, Room 1001, New York, New York 10003. E-mail: mw402@ nyu.edu. Shixiang Yan. Department of Chemistry, New York University, New York, New York 10003. E-mail: [email protected]. N. E. Geacintov, Chemistry Department, New York University. S. Broyde. Department of Biology, New York University. DNA damage by bulky adducts mostly blocks replication by classical replicative DNA polymerases. However, it has recently been proposed that Y-family DNA polymerases replace stalled replicative DNA polymerases since they can bypass a variety of bulky DNA lesions. Translesion synthesis by Y-family DNA polymerases can be error-prone and may result in mutations that lead to cancer initiation. To understand the molecular mechanism by which Y-family polymerases incorporate nucleotides opposite bulky DNA adducts, we have carried out a computational study of the intercalated 1S (-)-transanti-B[c]Ph-N6-dA adduct in the human proto-oncogene N-ras codon 61 sequence context at a Y-family DNA polymerase active site. Molecular modeling and molecular dynamics simulations were carried out for a representative Y-family DNA polymerase, Dpo4, complexed with the adducted DNA in the active site with either correct or mismatched incoming nucleotides. Our results showed that all four incoming nucleotides can be accommodated within the Dpo4 active site opposite the adduct. This suggests that this polymerase may allow nucleotide incorporation opposite this intercalated bulky DNA adduct in either an error-free or an error-prone fashion. Detailed structural analyses revealed that each nucleotide is accommodated by different protein interactions and with different structural stability opposite the damaged base. It is likely that the open and flexible features of the Dpo4 active site allow the accommodation of different bases opposite this bulky lesion. Our simulations may provide clues to the molecular mechanisms by which Y-family DNA polymerases accomplish translesion replication. 68. Elucidation of Estradiol Metabolism in Human Subjects by LC/MS. Xingpin Cui, Ye Tian, Seon Hwa Lee, Peter O’Dwyer, Peter Moate, Ray Boston, and Ian A. Blair. Center for Cancer Pharmacology, Department of Pharmacology, University of Pennsylvania, 1254 BRBII/III, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104-6160. Fax: 215-573-9889. E-mail: xingpin@ spirit.gcrc.upenn.edu. An ultrahigh sensitivity and specificity assay has been developed for the quantitation of estradiol (E2), estrone (E1), and their methoxy metabolites based on liquid chromatography (LC)/electron capture atmospheric pres-


sure chemical ionization (APCI)/tandem mass spectrometry (MS/MS). The estrogens in plasma were first extracted by ethyl acetate followed by C18 SPE. After they were derivatized with pentafluorobenzyl bromide (PFB), the samples were dried under nitrogen followed by redisolution in 2% 2-propanol/hexane; 45 µL of derivatized samples was injected into the HPLC column (250 mm × 4.6 mm i.d., 5 µm). The total run time for an assay was 35 min. A mobile phase gradient gave excellent separation between the six estrogens. The detection limit for the assay was 10 pg/mL for each estradiol and its five metabolites. The times to reach the maximum concentrations (tmax) were between 20 min to 1 h for E2, 4-10 h for E1, and 8-10 h for 2MeE1. Supported by NIH Grant CA82707. 69. Equine Estrogen Metabolite 4-Hydroxyequilenin (4-OHEN) Is a More Potent Inhibitor of the Variant Form of Catechol O-Methyltransferase (COMT). Yan Li,1 Jiaqin Yao,1 Minsun Chang,1 Linning Yu,2 James D. Yager,3 Andrew D. Mesecar,4 Richard B. van Breemen,1 and Judy Bolton.1 (1) Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 833 South Wood Street, Chicago, Illinois 60612. Fax: 312-996-7107. E-mail: [email protected]. (2) Department of Medicinal Chemistry & Pharmacognosy, University of Illinois at Chicago, College of Pharmacy. (3) School of Hygiene and Public Health, Johns Hopkins University. (4) Center for Pharmaceutical Biotechnology, College of Pharmacy, University of Illinois at Chicago, Illinois 60607. COMT catalyzes the methylation of catechols including 4-OHEN. It has been shown that COMT is genetically polymorphic with a wild type and variant form where Val108 has been substituted with methionine. It has been suggested that women who have the homozygous variant COMT are at increased risk for developing breast cancer. In the present study, Michaelis Menten analysis showed no difference between the relative ability of each isoform to methylate 4-OHEN. However, we found that variant COMT was more susceptible to 4-OHEN-mediated irreversible inactivation. Furthermore, thermotropic studies indicated that variant COMT had a lower melting temperature than wild-type COMT, which suggests that the Val to Met substitution changes the 3D structure of variant COMT making it more susceptible to inhibition. In support of this, variant COMT was more easily oxidized by 4-OHEN. These data suggest that 4-OHENmediated inhibition of variant COMT in vivo could affect the detoxification of catechol estrogens. 70. Evidence for the Role of a Sulfenate Ion in a Leinamycin-Type Rearrangement Reaction. Kripa Keerthi. Department of Chemistry, University of Missouri, Columbia, 125 Chemistry building, University of Missouri, Columbia, Missouri 65211. Fax: 573-882-2754. E-mail: [email protected]. Kent S. Gates. Departments of Chemistry and Biochemistry, University of Missouri-Columbia. Leinamycin (1) is a potent antitumor antibiotic that was isolated from the culture broth of Streptomyces sp. in 1989. DNA damage by leinamycin occurs by two major pathways, DNA oxidation and DNA alkylation at N-7 guanine. The alkylative pathway of the biomolecule involves the formation of episulfonium ion. It is thought that leinamycin sulfenate ion (RSO-) may be a key player leading to the generation of episulfonium ion. In this poster, we report a novel and interesting method


using a simple model system to generate and trap the leinamycin sulfenate ion intermediate. This helps us understand further the alkylative pathway of DNA damage by leinamycin.

71. Evidence in Escherichia coli that N3-Methyladenine Lesions and Cytotoxicity Induced by a Minor Groove Binding Methyl Sulfonate Ester Can Be Modulated in Vivo by Netropsin. Dharini Shah,1 Sridhar Varadarajan,2 and Barry Gold.1 (1) Eppley Institute and Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Box 986805, 600 South 42nd Street, Omaha, Nebraska 68198-6805. Fax: 402-559-4651. E-mail: [email protected] and bgold@ unmc.edu. (2) Eppley Institute for Research in Cancer, University of Nebraska Medical Center. The use of DNA equilibrium binding molecules to sequence and site selectively transfer alkyl groups to DNA is an approach to generate cytotoxic DNA damage while avoiding the formation of promutagenic lesions that increase the risk for the development of secondary cancer. We have previously reported that a neutral DNA equilibrium binding agent based on an N-methylpyrrole carboxamide dipeptide (lex) and modified with an Omethyl sulfonate ester functionality (Me-lex) selectively affords N3-methyladenine lesions in almost 99% yield relative to the formation of other adducts. While in vitro interactions between the lex dipeptide and DNA have been thoroughly studied, in vivo interactions are more difficult to elucidate. We report herein the relationship between the in vivo formation of N3-methyladenine and the toxicity in wild-type and repair mutant Escherichia coli. In addition, the effect of netropsin, a potent competitive inhibitor of lex binding to DNA, on DNA damage and cytotoxicity in E. coli is reported. The results show a clear relationship between the levels of N3-methyladenine and the toxicity in an alkA/tag glycosylase mutant that cannot remove the adduct from its genome. The coaddition of netropsin has a dramatic inhibitory effect on both DNA methylation and cytotoxicity. A similar relationship between the formation of N3 methyladenine and toxicity is also observed with methyl methanesulfonate, which does not selectively methylate DNA at the same A/T rich regions as Me-lex. However, netropsin affects neither the level of N3-methyladenine nor its toxicity in E. coli. 72. Human Liver Microsomal Reduction of Pyrrolizidine Alkaloid N-Oxides to Form the Corresponding Carcinogenic Parent Compounds. Yuping Wang, Qingsu Xia, Ya-Chen Yang, Jian Yan, Peter P. Fu, and Ming W. Chou. Division of Biochemical Toxicology, National Center for Toxicological Research, 3900 NCTR Road, Jefferson, Arkansas 72079. Fax: 870-5437136. E-mail: [email protected]. The metabolic activation of pyrrolizidine alkaloid (PAs) N-oxides by rat or human liver microsomes was investigated under either oxidative or hypoxic conditions. The results show that the 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) and the corresponding parent PAs are the major metabolites. Under the oxidative


conditions, reduction of the N-oxide to PA is inhibited, while under hypoxic conditions, DHP formation is dramatically decreased. The level of human liver microsomal enzyme activity is between uninduced and dexamethasone-induced rat liver microsomes. DHP-derived DNA adducts were detected and quantified by 32P-postlabeling/HPLC analysis. The same DHP-derived DNA adducts were also found in liver DNA of F344 rats fed PA N-oxides (1.0 mg/kg). Our results indicate that PA N-oxides may be hepatocarcinogenic to rats through a genotoxic mechanism via the conversion of the N-oxides to their corresponding parent PAs and that this may be relevant to humans. 73. Human Liver Microsomal Testosterone Hydroxylation and Its Interaction Patterns with Acetaminophen. Man Ho Choi, Paul L. Skipper, John S. Wishnok, and Steven R. Tannenbaum. Biological Engineering Division, MIT, 77 Massachusetts Avenue, Room 56-738, Cambridge, Massachusetts 02139. Fax: 617-2521787. E-mail: [email protected]. The main pathways of testosterone oxidative metabolism by human liver microsomes are 1β-, 2R-/β-, 6β-, 15β-, and 16β-hydroxylation, which are mainly catalyzed by cytochrome P450s 2C9, 2C19, and 3A4. In this study, however, testosterone 11β-hydroxylation activity was characterized from human liver microsomes. The altered pattern of cytochrome P450-dependent testosterone hydroxylation was also identified in human liver microsomes treated with acetaminophen (APAP), which is capable of inducing liver damage in human. Because APAP is widely used, its potential interaction with testosterone metabolism would be of great interest. The structures of five hydroxylated metabolites of testosterone (2β, 6β, 11β, 15β, and 16β-OHT) and another oxidative metabolite at the C-17 position, androstenedione, formed by these microsomes, were identified by liquid chromatography with UV detection at 240 nm and liquid chromatography-tandem mass spectrometry. All metabolite peaks were subsequently confirmed with accelerator mass spectrometry following incubations of 10 pCi [4-14C]testosterone in human liver microsomes. 6β-Hydroxylation was the dominant metabolic pathway in all systems investigated, but 15β, 2β, and 16β-OHT and androstenedione were also detected. A previously undetected hydroxytestosterone, 11β-OHT, was found to be a minor metabolite formed by human liver microsomal enzymes. It is formed more readily by CYP3A4 as compared to CYP2C9 and CYP2C19. At the concentration of 1 mM APAP, testosterone 6β- and 16β-hydroxylase activities were increased 18 and 31%, respectively, while 11βhydroxylase activity was decreased 38%. The formation of androstenedione was also increased 33% by 10 mM APAP. These findings indicate that human extraadrenal enzymes catalyze the 11β-hydroxylation of testosterone, and testosterone hydroxylase activities are affected by APAP exposure. 74. Identification of a Pyridyloxobutylated Deoxycytidine Adduct of 4-(Acetoxymethylnitrosamino)1-(3-pyridyl)-1-butanone (NNKCH2OAc), a Chemically Activated Form of Tobacco Specific Nitrosamines. Mingyao Wang, Guang Cheng, Yongli Shi, Peter W. Villalta, Shana J. Sturla, Pramod Upadhyaya, and Stephen S. Hecht. University of Minnesota Cancer Center, 420 Delaware Street Southeast-MMC 806, Minneapolis, Minnesota 55455. Fax: 612-626-5135. E-mail: [email protected].


The tobacco specific nitrosamines 4-(methylnitrosamino)1-(3-pyridyl)-1-butanone (NNK) and N′-nitrosonornicotine (NNN) may cause certain tobacco-induced cancers in humans. Metabolic activation of NNK (by methyl hydroxylation) and NNN (by 2′-hydroxylation) yields a pyridyloxobutylating intermediate (POB-NdN-OH) that reacts with DNA to give thermally unstable 4-hydroxy1-(3-pyridyl)-1-butanone (HPB)-releasing adducts, which have been detected in human lung. The structures of HPB-releasing adducts eluded characterization, but recently, we determined that one of these is 7-pyridyloxobutyldeoxyguanosine (7-POB-dG). We further investigated reactions of DNA with NNKCH2OAc, which produces POB-NdN-OH upon hydrolysis. Using LCMS, we identified a deoxycytidine (dC) adduct, which is thermally unstable, giving HPB and a POB-cytosine. The amount of this material is at least as great as 7-POBdG, based on MS peak areas. Treatment with NaBH4 yields material with the same chromatographic properties as O2-pyridylhydroxybutyl-dC, independently prepared. These results indicate that O2-POB-dC is a quantitatively significant HPB-releasing DNA adduct, which can be formed in the metabolic activation of NNK and NNN. 75. Influences of Nucleotide and Base Excision Repair Defects on the Lethality and Mutagenicity Induced by Me-lex, a Sequence Selective N3Adenine Methylating Agent. Paola Monti,1 Raffaella Iannone,1 Paolo Campomenosi,2 Yari Ciribilli,1 Alberto Inga,1 Sridhar Varadarajan,3 Dharini Shah,3 Paolo Menichini,1 Gilberto Fronza,1 and Barry Gold.3 (1) Mutagenesis Laboratory, National Cancer Research Institute (IST), L.go R. Benzi, 10, 16132 Genova, Italy. (2) Human Genetics Laboratory, University of Insubria. (3) Eppley Institute for Research in Cancer, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, Nebraska 68198-6805. Fax: 402-559-4651. E-mail: bgold@ unmc.edu. Using a yeast shuttle vector system, we have previously reported on the toxicity and mutagenicity of Melex, a compound that selectively generates 3-methyladenine (3-MeA). We observed that a mutant strain defective in Mag1, the glycosylase that excises 3-MeA in the initial step of base excision repair (BER), is significantly more sensitive to the toxicity of Me-lex but shows only a marginal increase in mutagenicity. A strain defective in AP endonuclease activity (apn1/apn2), also required for functional BER, is equally sensitive to the toxicity as the mag1 mutant but showed a high mutant frequency. In the current work, we have expanded our analysis to strains that are defective in nucleotide excision repair (NER) by deleting Rad14 and in both BER/NER (mag1/ rad14). The results indicate that in yeast both BER and NER can play a protective role against 3-MeA-mediated toxicity and mutagenicity, which may involve a common abasic site intermediate, although the role of the latter is appreciable only in a BER background. 76. Insights into Nucleotide Incorporation Opposite a Benzo[a]pyrene Diol Epoxide DNA Adduct by a Y-Family DNA Polymerase through Molecular Modeling and Molecular Dynamics Simulations. Rebecca A. Perlow,1 Ilya Likhterov,1 David A. Scicchitano,1 Nicholas E. Geacintov,2 and Suse Broyde.1 (1) Department of Biology, New York University, 100 Washington Square East, Room 1009 Silver, Mail Code 5181, New York, New York 10003. Fax: 212-995-4015. E-


mail: [email protected]. (2) Department of Chemistry, New York University. The Y-family of DNA polymerases has been implicated in the bypass of DNA damage that poses blocks to their replicative high fidelity relatives. While replicative DNA polymerases employ a number of mechanisms to ensure fidelity, Y-family DNA polymerases appear to lack such mechanisms, resulting in higher error rates during replication of undamaged DNA templates and the ability to bypass aberrant bases. Such aberrant bases include those damaged by metabolically activated environmental carcinogens, including the polycyclic aromatic hydrocarbons (PAHs). Benzo[a]pyrene (BP) is a common PAH that is metabolized into a number of reactive intermediates, including the tumorigenic (+)-anti-benzo[a]pyrene diol epoxide (BPDE); (+)-anti-BPDE primarily reacts with DNA to form the mutagenic 10S(+)-trans-anti-N2-[BP]dG ((+)-ta[BP]G) adduct. Induction of the SOS response in Escherichia coli, which induces expression of Y-family DNA polymerases, increases bypass of 10S(+)-ta[BP]G, with a significant increase in both error-free and errorprone bypass events. Little is known about the mechanism by which Y-family DNA polymerases bypass bulky lesions such as 10S(+)-ta[BP]G more readily than their replicative counterparts. The Y-family DNA polymerase Dpo4 from S. solfataricus P2 provides a unique opportunity to investigate the structure-function relationship involved in adduct bypass. We carried out primerextension assays in conjunction with molecular modeling and dynamics studies in order to elucidate the structurefunction relationship involved in bypass of 10S(+)ta[BP]G by Dpo4. Running- and standing-start primerextension assays were carried out to determine the propensity of Dpo4 to bypass 10S(+)-ta[BP]G and the nucleotide most often incorporated opposite the adduct, respectively. Molecular modeling and dynamics simulations were then utilized to elucidate the accommodation of each possible dNTP opposite the adduct within the active site of Dpo4. The modified guanine can be accommodated in either the syn or anti conformation due to the limited protein-DNA contacts and high degree of solvent exposure of the primer template and nascent base pair. In addition, Dpo4 can incorporate each of the four dNTPs opposite 10S(+)-ta[BP]G during primer-extension assays, which is likely the result of a flexible and spacious active site region. Understanding the consequences of bulky adduct on low fidelity DNA polymerases can help shed light into the manner in which these lesions are processed in vivo. 77. Investigating a Food Carcinogen-Induced Frameshift Mutation: Polymerase Models. Ling Zhang,1 Robert Shapiro,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. A current hypothesis proposes that bulky carcinogenDNA adducts may cause frameshift mutations through a polymerase switch model, a mechanism that may involve incorporation of the normal partner opposite the adduct in a replicative polymerase, with possible blockage, followed by adduct bypass with slippage in an errorprone Y family polymerase. We have carried out a computer modeling and molecular dynamics study to elucidate on a molecular level the feasibility of this mechanism. PhIP, 2-amino-1-methyl-6-phenylimidazo-


[4,5-b]pyridine, is the most abundant of the carcinogenic heterocyclic aromatic amines in the human diet. We have investigated the major adduct of PhIP, to C8 of guanine, in a -1 deletion hotspot sequence, in DNA polymerase ternary complexes. The crystal structures of ternary complexes for a pol R family polymerase (Franklin, M. C. (2001) Cell 105, 657-667) and a Y-family bypass polymerase (Ling, H. (2001) Cell 107, 91-102) served as the initial polymerase structural models. These were remodeled to incorporate the PhIP adduct in the active sites and then subjected to molecular dynamics simulations with AMBER. Our results show that the adduct can reside on the major groove side of the modified DNA template opposite incoming dCTP in the high fidelity pol R family polymerase. We also find that a slipped mutagenic intermediate structure can be comfortably accommodated in the Y-family polymerase active site. Supported by NIH. 78. Investigation of C-H Bond Cleavage by Cytochrome P450 3A4. Joel A. Krauser, and F. Peter Guengerich. Department of Biochemistry & Center in Molecular Toxicology, Vanderbilt University, 638 Robinson Research Building, 23rd and Pierce Avenues, Nashville, Tennessee 37232-0146. A detailed mechanistic understanding of human cytochrome P450 3A4 oxidation of toxicants, drugs, and steroids is crucial for elucidating toxicant-drug and drug-drug interactions. The central hypothesis of our model is that C-H bond breaking step is at least partially rate-limiting in the hydroxylation of testosterone. The 6βhydroxylation of testosterone is the most rapid reaction known for P450 3A4 and generally considered to be rather prototypic. We have synthesized 2,2,4,6,6-d5testosterone and 6,6-d2-testosterone, and found these compounds have noncompetitive kinetic deuterium isotope effects of DV ) 1.3 and 1.8 and D(V/K) of 1.6 and 2.1, respectively. Both deuterated testosterone derivatives exhibited a primary deuterium kinetic isotope, which may indicate that the C-H vs C-D bond cleavage step is at least partially rate limiting. Supported in part by USPHS Grants R01 CA90426 and P30 ES00267. 79. Isolation of Rodent Airway Epithelial Cell Proteins Facilitates in Vivo Proteomics Studies of Lung Toxicity. Åsa M. Wheelock,1 Lu Zhang,2 Mai-Uyen Tran,3 Dexter Morin,1 Sharron Penn,2 Alan Buckpitt,1 and Charles Plopper.3 (1) Departments of Molecular Biosciences, University of California Davis, 1311 Haring Hall, Davis, California 95616. E-mail: amkarlsson@ ucdavis.edu. (2) Advanced Research Team, Amersham Biosciences. (3) Department of Anatomy, Physiology and Cell Biology, University of California Davis. Recent developments in genomics, proteomics, and metabolomics hold substantial promise for understanding cellular responses to toxicants. Gene expression profiling is now considered standard procedure, but numerous publications reporting a lack of correlation between mRNA and protein expression emphasize the importance of conducting parallel proteomics studies. The cellular complexity of the lung presents great challenges for in vivo proteomics, and improved isolation methods for proteins from specific lung cell phenotypes are required. To address this issue, we have developed a novel method for isolation of rodent airway epithelial cell proteins, which facilitates in vivo proteomics studies of two target cell phenotypes of the lung, Clara cells and ciliated cells. The airway epithelial cell proteins are reproducibly


solubilized, leaving the underlying basement membrane and smooth muscle intact as shown by histopathological analyses. The method yields epithelial cell specific proteins in 5-fold higher concentrations and reduces the yield of nonepithelial cell proteins 13-fold in comparison to samples from microdissected airways. In addition, 36% more protein spots were detectable by two-dimensional electrophoresis. 80. LC-MS-MS and CE Analyses of Ultrathin DNA Films for Toxicity Screening after Neutral Thermal Hydrolysis. Maricar C. Tarun, and James F. Rusling. Department of Chemistry, University of Connecticut, 55 North Eagleville Road, Storrs, Connecticut 06269. Fax: 860-486-2981. E-mail: [email protected]. Ultrathin films of the structure PSS/(PDDA/DNA)2 were constructed layer-by-layer on carbon cloth. Incubation of these films in styrene oxide (SO), a suspected carcinogen, causes damage to the DNA on the film. Such damage can be detected by square wave voltammetry (SWV) using catalytic oxidation with Ru(bpy)32+ (bpy ) 2,2′-bipyridine). These same films were analyzed for the formation of DNA-SO adducts using LC-MS-MS and CE. Films incubated with SO were hydrolyzed by neutral thermal hydrolysis, a procedure that selectively releases guanine adducts alkylated at N-7 and adenine adducts alkylated at N-3. LC-MS-MS analyses revealed two peaks corresponding to a guanine-styrene oxide adduct, possibly isomers, and an adenine adduct. CE analysis of the neutral hydrolysate gave a peak that corresponds to a guanine adduct. Comparative DNA damage rate studies of the voltammetric sensors and the LC-MS-MS and CE analyses will be discussed. 81. Lipid Hydroperoxide-Derived Modifications to the Repeat Hexamer Motif of r-Synuclein. Seon Hwa Lee, Tomoyuki Oe, and Ian A. Blair. Center for Cancer Pharmacology, University of Pennsylvania, 1246 Biomedical Research Building II/III, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104-6160. Fax: 215573-9889. E-mail: [email protected]. Oxidative stress has been implicated as an important mechanism in neurodegeneration, particularly in Alzheimer’s disease (AD). A major consequence of oxidative stress is lipid peroxidation. We have embarked on the characterization of covalent modifications to R-synuclein resulting from reactions with lipid hydroperoxide-derived bifunctional electrophiles. The R-synuclein molecule is extremely complex. Therefore, initial studies have focused on the undecapeptide Ac-A27EAAGKTKEGV37, which contains the repeat hexamer motif (KTKEGV) that is an important characteristic of the N-terminal amphipathic lipid-binding R-helical domains of R-synuclein and other apolipoproteins. The prototypic lipid hydroperoxide, 13-hydroperoxyoctadecadienoic acid (13-HPODE), was allowed to undergo vitamin C-mediated homolytic decomposition in the presence of Ac-A27EAAGKTKEGV37. Modified peptides were derived from 11-carboxy-4-oxo2-undecenal, 4-oxo-2-nonenal (4-ONE), and 4-hydroxy2-nonenal. The 4-ONE peptide was 16 times more abundant than the next most abundant modified peptide. Supported by University of Pennsylvania ADCC Pilot Grant Program. 82. Lipid Hydroperoxide-Mediated Protein Modifications. Tomoyuki Oe, Jasbir S. Arora, Seon Hwa Lee, and Ian A. Blair. Center for Cancer Pharmacology, University of Pennsylvania, 1246 BRB II/III, 421 Curie


Boulevard, Philadelphia, Pennsylvania 19104-6160. Fax: 215-573-9889. Lipid hydroperoxides have been proposed to play a role in cancer by inducing covalent modifications to DNA. However, little is known about lipid hydroperoxidederived protein modifications. We have shown that lipid hydroperoxide-derived 4-oxo-2-nonenal can react with lysine and arginine to give stable adducts. This suggested that 4-oxo-2-nonenal could also induce covalent modifications to proteins. We report the characterization of a 4-oxo-nonenal-derived posttranslational modification to histone H4, an important component of nucleosomes where DNA is wrapped around histone octamers. Most of the posttranslational modifications to histone occur in the first 20 polar amino acids located at the amino terminus. However, using [D]- or [13C]-4-oxo-2-nonenal and synthetic peptides, we have discovered that 4-oxo2-nonenal reacts with a different region (H75AK77) of histone H4. Using a model tripeptide (Na-Ac-HAK), the chemical structure was identified by NMR as a unique cyclic peptide. Current studies are focused on detecting the lesion in vivo. Supported by NIH RO-1 CA95586. 83. Metabolism of the SERM Desmethylated Arzoxifene to Quinoids. Hong Liu, Yanan Yang, Linning Yu, Richard B. Van Breenmen, Gregory R. J. Thatcher, and Judy L. Bolton. Department of Medicinal Chemistry & Pharmacognosy, University of Illinois at Chicago, College of Pharmacy, 833 South Wood Street M/C 781, Chicago, Illinois 60612. E-mail: [email protected]. Selective estrogen receptor modulators (SERMs), such as tamoxifen, are playing very important roles in the treatment and prevention of breast cancer. However, tamoxifen has been associated with an increased risk of endometrial cancer possibly due to metabolism to electrophilic quinoids. The SERM, arzoxifene, is currently in clinical trials against breast cancer; it is critical to explore potential cytotoxic mechanisms of arzoxifene. In this study, the active form of arzoxifene in vivo, desmethylated arzoxifene (DMA), was synthesized and further oxidized to DMA quinone methide. The half-life of DMA quinone methide under physiological conditions was approximately 15 s. The DMA quinone methide is therefore considerably more reactive than that from 4-hydroxytamoxifen. LC-MS/MS and NMR analysis showed that DMA quinone methide reacted with GSH to give GSH conjugates. In addition, enzymatic oxidation of DMA in the presence of GSH gave DMA quinone methide and o-quinone GSH conjugates. These preliminary results suggest that DMA could be metabolized to electrophilic quinoids, which have the potential to cause toxicity in vivo. Supported by NIH Grant No. CA 798700. 84. Modification and Cross-Linking of Proteins and DNA by Lipid Peroxidation Products. Lawrence M. Sayre, and Quan Yuan. Department of Chemistry, Case Western Reserve University, Cleveland, Ohio 44106. Fax: 216-368-3006. E-mail: [email protected]. Bifunctional aldehydes that are derived from lipid peroxidation during oxidative stress are well-known to modify proteins and DNA, and this modification contributes to the pathophysiology of many diseases such as cancer and neurodegeneration. Among those aldehydes, 4-hydroxy-2-nonenal (HNE) has been studied widely, and recently, 4-oxo-2-nonenal (ONE) has also aroused interest because of its higher reactivity. Our research has shown that ONE, HNE, and trans,trans-2,4-decadienal (DDE),


all generated from lipid peroxidation, can readily modify and cross-link proteins as well as form DNA-protein cross-links, which may result in genetic damage. Carbonyl generation on proteins by adduction of the unsaturated aldehydes as well as their protein-protein and protein-DNA cross-linking potential was compared. To elucidate the chemistry underlying these observations, various model studies were carried out, using HPLC, NMR, and liquid chromatography-mass spectrometry. 85. Molecular Dynamics Simulations to Elucidate Unfaithful Nucleotide Incorporation Opposite a Carcinogen-Damaged Template in a DNA Polymerase Ternary Complex. Shixiang Yan,1 Min Wu,1 Nicholas E. Geacintov,1 and Suse Broyde.2 (1) Department of Chemistry, New York University, New York, New York 10003. E-mail: [email protected] and [email protected]. (2) Department of Biology, New York University. High fidelity DNA polymerases, such as T7 DNA polymerase, an A-family DNA polymerase, are predominantly blocked by bulky chemical lesions on the template strand during DNA replication. However, some mutagenic bypass can occur, which may lead to carcinogenesis. It has been shown that a DNA covalent adduct to adenine, derived from the chemical carcinogen benzo[a]pyrene (BP), primarily blocks T7 DNA polymerase; however, a mismatched dATP can be inserted opposite the damaged templating adenine base within the active site of the polymerase (Chary, and Lloyd (1995) Nucleic Acids Res. 23, 1398-1405). We have carried out molecular modeling and molecular dynamics (MD) simulations for the T7 DNA polymerase primer-template ternary complex containing an adducted templating adenine opposite all four incoming dNTPs within the polymerase active site using AMBER 6.0, to interpret these experimental findings on a molecular level. The starting model was a high-resolution X-ray crystal structure of the T7 DNA polymerase ternary complex (Doublis et al. (1998) Nature 391, 251-258). Enlargement of the dNTP base binding pocket due to steric hindrance between the BP ring system and the primer-template DNA permitted comfortable accommodation of the mismatched incoming dATP opposite the modified adenine templating base; the other nucleotides were less well-accommodated. This research is supported by NIH. 86. Noncovalent Binding Drives DNA Alkylation by the Antitumor Antibiotic Leinamycin. Sanjay Dutta, Hong Zang, Leonid Breydo, Tony Nooner, and Kent S. Gates. Department of Chemistry, University of Missouri-Columbia, 601 South College Avenue, Columbia, Missouri 65211. E-mail: [email protected]. Leinamycin (LM) is a natural product with potent anticancer activity. Leinamycin efficiently alkylates the N7-position of guanine residues in DNA. However, leinamycin does not alkylate single-stranded DNA, bulged DNA, or monomeric 2′-deoxyguanosine under physiological conditions. This suggests that efficient DNA alkylation by leinamycin is driven by noncovalent binding of the antibiotic. Here, we report that DNA alkylation by leinamycin follows Michaelis-Menten type (saturation) kinetics consistent with equilibrium noncovalent binding prior to reaction with N7 guanine of DNA. Our works show that noncovalent interactions are required for efficient DNA alkylation by leinamycin. We also will


consider the structural features required for noncovalent association of this antibiotic with DNA.

87. Nonoxidative Metabolism of Ethanol in HepG2 Cells: Contribution of Alcohol Dehydrogenase and Cytochrome P450 2E1. Hai Wu,1 M. Firoze Khan,1 Dahn L. Clemens,2 Thomas R. Jerrells,2 Ghulam A. S. Ansari,1 and Bhupendra S. Kaphalia.1 (1) Department of Pathology, University of Texas Medical Branch, Galveston, Texas 77555. (2) Pathology and Microbiology, University of Nebraska Medical Center. Oxidative metabolism of ethanol, catalyzed by hepatic alcohol dehydrogenase (ADH), is the major pathway for its disposition and is generally impaired during chronic alcoholism. Under these conditions, the contribution of hepatic cytochrome P-450 2E1 in the oxidative metabolism of ethanol is increased. Inhibition of hepatic ADH activity significantly increases nonoxidative metabolism of ethanol to fatty acid ethyl esters (FAEEs). FAEEs cause pancreatic toxicity in vitro and in vivo. In this study, we investigated the nonoxidative metabolism of ethanol in hepatocellularcarcinoma (HepG2) cells transfected with or without ADH and/or cytochrome P-450 2E1. The total FAEE formation was found significantly higher in HepG2 cells (44.7 nmol/25 × 106 cells) than those in ADH (15.4 nmol), and ADH and P-450 2E1 (15.8 nmol) transfected cells after 6 h of incubation at 800 mg% ethanol. These results indicate that lack of hepatic ADH activity only significantly enhances nonoxidative metabolism of ethanol to FAEEs. 88. Novel Medium Formulation for the Perfusion and Plating of Primary Hepatocytes to Improve Drug Metabolism Capacity in Vitro. Kevin Leach,1 Ajit Dash,1 Anand Sivaraman,1 Linda Griffith,2 and S. R. Tannenbaum.3 (1) Biological Engineering, MIT, 77 Massachusetts Ave Building 56-731, Cambridge, Massachusetts 02139. E-mail: [email protected]. (2) Division of Biological Engineering, Department of Chemical Engineering, Biotechnology Process Engineering Center, Massachusetts Institute of Technology. (3) Division of Biological Engineering, MIT. Primary hepatocyte cell culture is an important tool for liver biology, toxicology, and pharmaceutical research. A major drawback of traditional hepatocyte cell culture is the rapid loss of hepatocyte phenotype. Our approach to increasing hepatocyte function in cell culture has been to scavenge or prevent the generation of reactive oxygen or nitrogen in isolated rat hepatocytes. Rat hepatocytes were isolated by the two-step collagenase perfusion technique and plated in a collagen gel sandwich configuration. Antioxidants were added at the time of perfusion or at the time of plating and were removed 24 h after addition. Antioxidants proved effective in increasing some aspects of hepatocyte cell function, including: albumin secretion, urea synthesis, activity, induction, and basal expression of cytochrome P450 enzymes, 1A1, 2B1/2, 2E1, and 3A2. We studied different classes of oxidants that


are produced in the isolated hepatocyte. To scavenge the hydroxyl radical, mannitol (2 mM) was used and was effective in increasing inducibility of the p450 enzymes 1A1, 2B1/2, and 3A2 in cell culture as compared to untreated cultures. Tocopherol succinate (25 µM) was used as a peroxynitrite scavenger and an inhibitor of lipid peroxidation. Tocopherol succinate was effective in increasing the basal activity and inducibility of p450 1A1, as well as increased metabolism of testosterone and diazepam. 2-oxo-4-Thiazolidine carboxylic acid (OTZ) was used to increase intracellular glutathione levels. OTZ (250 µM) was effective in increasing both the basal levels and the inducibility of P450 1A1, 2B1/2, 3A2, as well as increasing the amount of albumin secretion and urea synthesis from the hepatocyte. Similar to tocopherol succinate, OTZ was effective at increasing the metabolism of testosterone and diazepam. Transcriptional profiling by microarray analysis was used to confirm the induction of oxidant responsive genes in the isolated hepatocytes and to characterize the effect of antioxidants on gene expression in cultured primary hepatocytes. Antioxidant treated hepatocytes maintain a differentiated state in culture for longer periods of time as compared to untreated hepatocytes and may represent a more predictive model for drug metabolism and toxicity studies. 89. Nucleoside Cross-Links Formed by Glyoxal. Angela K. Brock, Ivan D. Kozekov, Carmelo J. Rizzo, and Thomas M Harris. Department of Chemistry and Center in Molecular Toxicology, Vanderbilt University, Nashville, Tennessee 37235. Fax: 615-322-7591. E-mail: [email protected]. Glyoxal is a widely dispersed mutagen that is found in foods and beverages and in cigarette smoke. It arises by oxidation of nucleic acids, sugars, and lipids and by metabolism of a number of nitrosamines. Glyoxal reacts preferentially with 2′-deoxyguanosine to form an adduct with an exocyclic five-membered ring bearing two hydroxyl groups (gdG). Cross-links of gdG with dA, dC, and dG have previously been observed, but the bis-nucleosides were formed in low yields and the structures were not established. We have improved on the reaction conditions to obtain faster reactions and significantly improved yields. The structures of the bis-nucleosides have been elucidated using NMR and mass spectroscopy coupled with reduction by sodium borohydride to give products for which the structures have been established by independent synthesis. Supported by NIH Grant P01ES05355.

90. Nucleotide Excision Repair of Site Specific Cytidine Adducts Derived from the Equine Estrogen Metabolite 4-OHEN in DNA by UvrABC Proteins from Escherichia coli. Dandan Chen,1 Tongming Liu,1 Qian Ruan,1 Yue Zou,2 Vladimir Kuzmin,1 Alexander Kolbanovskiy,1 Minsun Chang,3 Judy L. Bolton,4 and Nicholas E. Geacintov.5 (1) Chemistry Department, New York University, 29 Washington Place, Room 453,


New York, New York 10003. Fax: 212-998-8421. Email: [email protected]. (2) East Tennessee State University. (3) Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, College of Pharmacy. (4) Department of Medicinal Chemistry & Pharmacognosy, University of Illinois at Chicago, College of Pharmacy. (5) Department of Chemistry, New York University. Equilenin, an equine estrogen, is a component of premarin, a formulation widely prescribed as a hormone replacement treatment for postmenopausal women. It is metabolized by mammalian P450 enzymes to the catechol 4-hydroxyequilenin (4-OHEN), which, in turn, is readily oxidized to the highly reactive 4-OHEN o-quinone derivative. The latter is known to react with all DNA bases except T to form covalent adducts (Bolton, J. L., Pisha, E., Zhang, F., and Qiu, S. (1998) Chem. Res. Toxicol. 11, 1113). We have reacted several 11-mer oligodeoxynucleotides with 4-OHEN in aerated aqueous buffer solution and found a number of different covalently modified oligonucleotides that can be separated (and purified) by reversed-phase HPLC mehods, followed by high resolution gel electrophoresis. We have extensively studied the properties of oligonucleotides with single modified cytidines that were characterized by a combination of exonuclease digestion, MALDI-TOF mass spectrometry, and circular dichroism techniques. The circular dichroism spectra indicate that different types of stereoisomeric adducts are formed, exhibiting CD spectra of opposite sign but of similar shape. The melting points of duplexes containing single site specific 4-OHEN-dC adducts are significantly lower than those of the unmodified oligonucleotide duplexes. The susceptibilities of the different stereoisomeric 4-OHEN-dC adducts to nucleotide excision repair were investigated using UvrABC enzymes from E. coli and will be described. Research supported by NIH Grant CA 73638 (J.L.B., P.I.). 91. Ocular Toxicity of Cyclopentadiene Vapor. Bruce H. Bailey. Industrial Hygiene Group, Mecklenburg York Research, 1836 Tamworth Drive, Charlotte, North Carolina 28210. E-mail: [email protected]. The utility of 1,3-cyclopentadiene for stereospecific synthesis in Diels-Alder reactions has made it a ubiquitous reagent in academic and industrial laboratories. The adduct from which it is usually prepared, dicyclopentadiene, has a benign toxicological profile. Its vapor, however, presents an acute ocular hazard to workers and students because of its paralyzing and destructive effect on the ciliary body, which contains the muscles responsible for lens deformation accommodating near and far vision. The usual means of 1,3-cyclopentadiene preparation, the reflux distillation of dicyclopentadiene, must be carefully monitored for the escape of vapors into breathing atmosphere as a result of a malfunctioning fume hood or the re-entrainment of fume hood exhausts. 92. Phenomenal Impact of Antioxidants on Nonenzymatic Protein Glycation: Design and Study of a Novel Carbonyl Group Trapping, Metal Ion Chelating, and Radical Trapping Pyridoxamine Derivative. Sean M. Culbertson,1 Ekaterina I. Vassilenko,1 Lisa D. Morrison,2 and Keith U. Ingold.1 (1) Steacie Institute for Molecular Sciences, National Research Council Canada, 100 Sussex Drive, Ottawa, ON K1A 0R6, Canada. Fax: 613-952-0068. E-mail: [email protected]. (2) Institute for Biological Sciences, National Research Council Canada.


Nonenzymatic protein glycation by reducing sugars, such as glucose or ribose, (Maillard reaction) leads to irreversible protein modifications collectively called advanced glycation end products (AGE), which are implicated in the pathogenesis of diabetic complications, atherosclerosis, and Alzheimer’s disease. This work introduces a novel multifunctional AGE inhibitor, 6-dimethylaminopyridoxamine, designed to act as a carbonyl group trap, metal ion chelator, and radical trapping antioxidant. We present unexpected results that provide mechanistic insight into inhibiting AGE formation by comparing three different classes of AGE inhibitors: carbonyl traps, chelators, and radical trapping antioxidants. Overall, our findings show a phenomenal impact of radical trapping antioxidants and challenge the currently generally accepted ideas that (i) effective AGE inhibitors must decrease formation of specific AGE products, (ii) transition metal ions are required for oxidative formation of AGE, and (iii) screening AGE inhibitors only in systems containing transition metal ions represents a valid estimate of potential in vivo mechanisms. 93. Proteomics Analysis of the Role of Oxidative Stress in Human Breast Cancer Development and Treatment. Yuan Yan,1 Valerie M. Weaver,1 and Ian A. Blair.2 (1) Center for Cancer Pharmacology, University of Pennsylvania, 1246 BRB II/III, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104-6160. Fax: 215-5739889. E-mail: [email protected]. (2) Center for Cancer Pharmacology and Genomics Institute Proteomics Facility, University of Pennsylvania. Reactive oxygen species (ROS) have been implicated in the pathogenesis of human breast cancer. Defining the molecular pathways linking oxidative stress to cellular dysfunction could therefore help to develop novel treatment and prevention strategies. In this study, we applied a proteomics approach to characterize quantitative and kinetic differences in the oxidative stress response of nonmalignant (S1) and malignant (T4-2) human mammary epithelial cell lines. S1 and T4-2 cells were harvested after 0, 1, 2, 4, and 6 h of oxidant treatment (hydrogen peroxide), and their global protein expression patterns were compared using differential gel electrophoresis (DIGE). On average, we were able to resolve over 1000 protein spots per gel. Using MALDI/TOF or LC/ ESI/MS/MS, we identified differentially expressed proteins, both novel and previously associated with the oxidative stress response. Additional experiments are underway to explore the relevance of these observations to breast cancer prevention and treatment. Supported by NIH Grant CA95586. 94. Quantitative Comparison of the Deoxyribose Oxidation Product Phosphoglycolaldehyde Induced by Radiation, Fe-EDTA, and Peroxynitrite. Christiane Collins, Xinfeng Zhou, and Peter C. Dedon. Biological Engineering Division, Massachusetts Institute of Technology, Building 56 Room 786, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139. Fax: 617-2580225. E-mail: [email protected]. Deoxyribose oxidation plays an important role in radical-mediated DNA damage, with a unique spectrum of toxic products derived from each position in the sugar. The chemistry of deoxyribose oxidation is further complicated by the observation that while bleomycin and γ-radiation both oxidize the 4′-position in deoxyribose, bleomycin produces base propenals while γ-radiation does


not. We have undertaken the development of analytical techniques to quantify and characterize deoxyribose oxidation chemistry, starting with a GC/MS method for 3′-phosphoglycolaldehyde residues arising from 3′-oxidation of deoxyribose. The results of DNA exposure to FeEDTA, γ- and R-radiation, and peroxynitrite under biological conditions reveal differences in the efficiency of 3-phosphoglycolaldehyde formation by these oxidants and suggest weaknesses in models relating DNA structure to chemical reactivity of DNA. These results provide important insights into the basic chemical mechanisms of DNA damage involved in the pathophysiology of human diseases such as cancer. 95. Redox-Activated DNA Cleavage by the Antibiotic Myxin and Its Methyl Analogue. Goutam Chowdhury. Department of Chemistry, University of Missouri-Columbia, 601 South College Avenue, Chemistry Building, Columbia, Missouri 65211. E-mail: gcad6@ mizzou.edu. Tarra E. Fuchs. Department of Chemistry, University of Missouri-Columbia. Kent S. Gates. Departments of Chemistry and Biochemistry, University of Missouri-Columbia. Myxin (1) is a broad spectrum antibiotic isolated from a Sorangium species. It is active against a wide variety of microorganisms including Gram-positive and Gramnegative bacteria, fungi, and yeast. DNA is an important target for this agent, although the mechanism of DNA cleavage remains unknown. Here, we will present the mechanism of in vitro DNA damage by myxin and methylmyxin (2). We will show that both 1 and 2 induce DNA single strand breaks upon bioreductive activation. Various mechanisms of reductive activation will be considered. Effects of oxygen concentration and noncovalent association with duplex DNA on DNA damage will be discussed. The results provide a molecular basis for understanding the antimicrobial properties of these wide spectrum antibiotic agents.

96. Removal of Arsenic from Drinking Water by Molecular Sieve Adsorption. Kal Renganathan Sharma, Principal, Mathur Post, and Anna University. Sakthi Engineering College, Department of Chemical & Biotechnology, Oragadam, Kancheepuram, Chennai 602105, India. Fax: 2526019. E-mail: [email protected]. Arsenic, a identified carcinogen at 50 ppb level in drinking water, needs to be reduced to the 10 ppb level according to EPA regulations. A zeolite molecular sieve with a pore size in between that of arsenic and water in a packed bed configuration is expored in this study to achieve that stated objective. The hyperbolic damped wave finite speed damped wave diffusion equation is used to model the solute concentration profile in the adsorbate and adsorbent phases in a fixed bed configuration. The governing equation in the adsorbate is obtained by including the effects of bulk convective flow by a Peclet number that is the ratio of the bulk flow velocity to the velocity of mass diffusion. Thus, the governing equation is

Uxx - Pe Ux - k* U ) Utt + Pe Uxt + Ut (1 + k*)


The steady state and transient state portions of the solution are obtained separately. Analysis of the hyperbolic PDE reveals that a certain minimum adsorber depth has to be obtained to avoid the subcritical damped oscillatory manifestation in concentration. This depth is given by

L(min) ) Pi Sqrt(c′ Dtr/e)/(1 - k*) where k* ) klatr/e, e ) voidage, tr ) relaxation time, and D ) diffusivity of arsenic. The method of Laplace transforms is used to obtain a limiting solution at infinite Peclet number for the average concentration in the bed. At the limit of zero Peclet number, the method of separation of variables is used to obtain the exact solution for the solute concentration in the adsorbent phase also. 97. Searching for the Active Agents in Cancer Chemoprevention by the Dithiolethione Oltipraz. Mettachit Navamal, Jacobus P. Petzer, Colleen McGrath, Jennifer Stewart, and James C. Fishbein. Department of Chemistry and Biochemistry, University of Maryland Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250. E-mail: [email protected]. Dithiolethiones, like oltipraz (1), are a class of compounds known to possess cancer chemopreventive properties. The chemopreventive activity observed in animal models has been postulated to be the result of enhanced detoxification by the elevated levels of phase 2 enzymes. The molecular basis, or mechanism, by which dithiolethiones manifest the increases in levels of phase 2 enzymes is unknown. Oltipraz is predominantly metabolized to 2, which is inactive in inducing phase 2 enzymes. We assumed this occurs via intermediate 3 and are interested in the biological properties of 3. Experimental evidence that 3 is indeed the major metabolite of oltipraz will be presented. Additionally, the chemistry of alternative precursors (4 and 5) to 3 will be presented. This permits a test, via 4 and 5, of whether 3 in fact mediates the cancer chemopreventive properties of oltipraz. Supported by RO1 CA91032.

98. Stereoisomeric Base Adducts to 4-Hydroxyequilenin: Conformational Studies Using Quantum Mechanical Calculations. 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 drug Premarin (Wyeth), employed for estrogen replacement therapy, contains the equine estrogens equilin and equilenin, both of which can be metabolized to 4-hydroxyequilenin (4-OHEN). This catechol estrogen


autoxidizes to potent cytotoxic quinoids that can react with dG, dA, and dC to form a number of stereoisomeric cyclic adducts (Bolton, J. L., et al. (1998) Chem. Res. Toxicol. 11, 1113-1127). Increased risk of breast cancer associated with premarin treatment may be related to the formation of these bulky adducts, whose mutagenic potential may differ in a structure-dependent manner. We have computed conformations of stereoisomeric guanine, adenine, and cytosine base adducts using density functional theory. Our results reveal near mirror image conformations in stereoisomer adduct pairs for each modified base and suggest opposite orientations when the pairs are incorporated into duplex DNA. Differential response to DNA replication and repair enzymes may result when stereoisomeric lesions are processed in the cell. 99. Structural and Stereoisomer Effects of Model Estrogen-DNA Adducts. Lihua Wang. Biology Department, New York University, 1009 Main Building, 100 Washington Sqare East, New York, New York 10003. Fax: 212-995-4015. E-mail: [email protected]. Brian E. Hingerty, Life Sciences Division, Oak Ridge National Laboratory. Robert Shapiro, Department of Chemistry, New York University. Suse Broyde, Department of Biology, New York University. An extensive conformational analysis has been carried out for two diastereoisomeric pairs of model estrogenDNA adducts, N2-(2-hydroxyestron-6(R,β)-yl)-2′-deoxyguanosine (2-OHE1-6(R,β)-N2-dG) and N6-(2-hydroxyestron-6(R,β)-yl)-2′-deoxyadenosine (2-OHE1-6(R,β)-N6dA), in a B-DNA duplex and at a primer-template junction in DNA polymerase R. In vitro primer extension studies in pol R (Terashima et al. (1998) Biochemistry 37, 13807-13815) have shown that the dA adducts can incorporate dT, together with a small proportion of the incorrect base dC opposite the lesion, and they block less strongly than the dG adducts. Our searches revealed that the four-ring nonplanar 2-OHE1 moiety strongly prefers to reside in the minor groove of the dG adducts or the major groove of the dA adducts. No low-energy conformations involving intercalation of the 2-OHE1 moiety were located in the search. This stems from the largely nonplanar, nonaromatic nature of the 2-hydroxy estrone ring system and implies that the proclivity for such bulky, nonplanar adducts to reside at the DNA helix exterior is a plausible conformational feature of other structurally similar estrogen-DNA adducts. Possible structures of these adducts in pol R were also investigated through molecular modeling. The estrone rings in the dA adducts can be comfortably accommodated on the major groove side of the templating base within the catalytically competent closed conformation of the polymerase, permitting Watson-Crick base pairing with incoming dTTP and maintaining a distance suitable for reaction between the R phosphate and the primer terminus. However, the dG adducts cannot fit on the crowded minor groove side. These results can rationalize the observed greater proclivity for blockage by the dG adducts and the misincorporation of dC opposite the dA adducts. More broadly, our findings suggest how bulky estrogen-derived stable adducts may be accommodated within a model high fidelity polymerase for possible translesional synthesis or cause blockage that may lead to bypass by a low fidelity Y family polymerase. Different orientations of R and β stereoisomers found in both G and A adducts are intrinsic to the linkage site differences. These stereoiso-


mer-dependent orientational differences could be expected to produce different biochemical effects, as has been observed in the stereochemistry-dependent differential treatment of adducts derived from polycyclic aromatic hydrocarbons. 100. Studies of PAH Metabolic Activation Pathways by LC/MS. Hye-Young Kim,1 Ronald G. Harvey,2 Trevor M. Penning,1 and Ian Blair.1 (1) Center for Cancer Pharmacology, University of Pennsylvania, 1246 Biomedical Research Building II/III, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104-6160. Fax: 215-5739889. E-mail: [email protected]. (2) Ben May Institute for Cancer Research, University of Chicago. Polycyclic aromatic hydrocarbons(PAHs) are known to be carcinogenic and tumorigenic to humans through metabolic activation to the ultimate carcinogens. Three metabolic pathways have been proposed to activate PAHs including diol-epoxides by the combination of CYP450 and epoxide hydrolase, radical cation by CYP peroxidase, and redox-active o-quinines by members of aldo-keto reductase (AKR) family. Redox cycling of o-quinones results in the generation of reactive oxygen species (ROS) that can potentially damage DNA directly or induce lipid hydroperoxide-mediated damage. The role of PAH-diolepoxides in carcinogenesis has been studied extensively. However, the o-quinone pathway has not been fully investigated. Stable isotope LC/MS methods have been developed to quantify the PAH-derived 2°--deoxyguanosine adducts that result from the interaction of PAH-diol epoxides and o-quinones with calf thymus DNA. This will make it possible to assess the elative importance of these pathways in causing DNA damage. Supported by NIH Grant CA92537 101. Studies on the DNA-Damaging Properties of the Endogenous Mutagen Fecapentaene-12. Joseph Szekely,1 Juri Pospisil,1 and Kent S. Gates.2 (1) Department of Chemistry, University of Missouri, Columbia, 125 Chemistry Building, University of Missouri, Columbia, Missouri 65211. Fax: 573-882-2754. E-mail: js13a@ mizzou.edu. (2) Departments of Chemistry and Biochemistry, University of Missouri-Columbia. Fecapentaene-12 (1) has been isolated from the human gastrointestinal tract. Studies suggest that the compound may be an endogenous mutagen that contributes to the etiology of colon cancer. The molecular mechanisms of its mutagenecity are still not understood in detail. In this poster, we will present results of our recent investigations into the mechanisms of oxidative DNA damage and possible DNA adduct formation by the fecapentaenes.

102. Synthesis of 6-Substituted Di- and Tetrahydrobenzo[a]pyrene Derivatives. Mercy O. Ilori, Olawale J. Olushi, and Barbara Zajc. Department of Chemistry, City College of CUNY, 138th Street at Convent Avenue, New York, New York 10031. E-mail: [email protected]. Benzo[a]pyrene is a known environmental pollutant. Upon metabolism, four isomeric 9,10-epoxy-7,8-dihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene derivatives are formed. These can promote mutagenic events by binding to DNA via epoxide ring opening and attachment of the incipient carbocation to the nucleobases at the C-10


position of the hydrocarbon. Introduction of a substituent into benzo[a]pyrene ring can alter its biological activity. To study the electronic effect of substituents on the C-10 carbocation stability and its effect on DNA binding, synthesis of a series of 6-substituted 7,8-dihydro as well as 9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene derivatives has been undertaken. 6-Bromo and 6-chloro derivatives were synthesized via bromination and chlorination of 7-hydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene, respectively. Other substituted derivatives were produced through a common intermediate, the 6-bromo-7,8-dihydrobenzo[a]pyrene. Substituent electronic effects on the C-10 and H-10 resonances can now be evaluated in the dihydro and tetrahydrobenzo[a]pyrene derivatives.


with pore size in between that of arsenic and water in a packed bed configuration is expored in this study to achieve that stated objectives. The hyperbolic damped wave finite speed damped wave diffusion equation is used to model the solute concentration profile in the adsorbate and adsorbent phases in a fixed bed configuration. The governing equation in the adsorbate is obtained by including the effects of bulk convective flow by a Peclet number that is the ratio of the bulk flow velocity to the velocity of mass diffusion. Thus, the governing equation is

Uxx - Pe Ux - k* U ) Utt + Pe Uxt + Ut (1 + k*) The steady state and transient state portions of the solution are obtained separately. At zero Peclet number, the exact solution can be obtained by the method of separation of variables. This can be be the solute profile in the adsorbent also. Earlier studies of the problem such as Taitel (1972) and Barletta and Zanchini (1997) on a analogous finite slab heat transfer problem have raised some issues of second law of thermodynamic violations. The time domain solution is

V ) c1 exp(-t/2 - sqrt(1/4 - ln∧2) + c2 exp(-t/2 + sqrt(1/4 - ln∧2) From the initial condition at t ) 0, V ) 1 103. Triple Helix-Forming TRIPside Molecules that Target Mixed Purine/Pyrimidine DNA Sequences. Jiansen Li,1 Ronald A. Shikiya,2 Luis A. Marky,2 and Barry Gold.3 (1) Eppley Cancer Institute, University of Nebraska Medical Center, 6001 Dodge Street, Omaha, Nebraska 68198-6805. Fax: 402-5593339. E-mail: [email protected]. (2) Department of Pharmaceutical Sciences, University of Nebraska Medical Center. (3) Eppley Institute for Research in Cancer and Allied Diseases and Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska 68198-6805. Fax: 402-559-4651. E-mail: bgold@ unmc.edu. A new strategy to form stable and sequence specific triple helical DNA structures at mixed purine/pyrimidine sequences using a combination of four C-glycosides (TRIPsides) has been described (Li, et al. (2003) J. Am. Chem. Soc. 125, 2084). The partial realization of the approach is demonstrated using oligomers that can potentially fold into intramolecular triplexes that contain one or two major groove cross-overs of the purine Hoogsteen H-bond information. Using temperature-dependent electronic and fluorescence spectroscopy and differential scanning calorimetry, it is demonstrated that stable triplexes form at physiological conditions at nonhomopurine targets. In addition, triplexes using the TRIPsides form in a highly sequence specific manner. 104. Where Is the Second Law Violation in Hyperbolic Damped Wave Finite Diffusion Equation Solution? Kal Renganathan Sharma, Principal, Mathur Post, and Anna University. Sakthi Engineering College, Department of Chemical & Biotechnology, Oragadam, Kancheepuram, Chennai 602105, India. Fax: 2526019. E-mail: [email protected]. Arsenic, an identified carcinogen at 50 ppb level in drinking water, needs to be reduced to a 10 ppb level according to EPA regulations. A zeolite molecular sieve

c1 + c2 ) 1 or

V ) exp(-t/2 - sqrt(1/4 - ln∧2) + c2 exp(-t/2)Sinh + sqrt(1/4 - ln∧2) It can be reasoned that c2 needs to be set to zero. Not doing this may have lead the previous investigators to conclude that the second law of thermodynamics was violated. For ln∧2 > 1/4, c2 can be seen to be zero from the fact that at steady state V reaches zero. For values of ln∧2 < 1/4, for the expression to be valid for all values of ln∧2, c2 needs to be set to zero. For instance, when ln∧2 is zero, c2 alone will remain at steady state. The exp(-t/2)Sinh(sqrt(1/4 - ln∧2)) expression was examined for range, extremamas. Thus, the exact solution that is bifurcated is obtained. The bifurcation depends on the depth of the bed and the diffusivity and relaxation time values. 105. Measurements of Contents of Sulfurous Anhydride in Gas Medium. Nurliboy S. Hamraev, Ziyadulla N. Normuradov, and Dilorom Kurbanova. Department of Chemistry, Samarkand State University, 15 Universitetskiy Boulevard, Samarkand City, Uzbekistan. Fax: 998-662-312199. E-mail: [email protected]. To measure the concentration of sulfurous anhydride in different gas medium, the electroanalytical methods are widely used. The main elements of the electrochemical sensor for determination of contents of sulfurous anhydride in gas mixtures are solid electrodes. In this method, the investigating electrodes are used in the form of wires, disks, nets, and plates. For functioning the sensor in galvanic mode, we have chosen an accessory electrode. Taking into account of the advantages of electrochemical methods, we carried out investigations on the development of electrochemical sensors of sul-


furous anhydride. Additionally, we have studied the electrochemical behaviors of sulfurous anhydride on solid electrodes in water and water-organic solutions. These experiments have revealed the metrological and exploitation characteristics of the developed sensor. This electrochemical sensor can be used for continuous monitoring of the ecological situation in and around industrial enterprises. 106. The Determination of Carbon Oxide in Gas Media. Ziyadulla N. Normuradov, and Nurliboy S. Hamraev. Department of Chemistry, Samarkand State University, 15 Universitetskiy Blvd, Samarkand City, Uzbekistan. Fax: 998-662-312199. E-mail: ziyadulla@ yahoo.com. An enormous amount of toxic gases enter into the atmosphere annually. In polluting the atmosphere, the toxic gases often inflict irreparable damage not only on the environment but also on humans. One of these gases is carbon oxide, found in large quantities in automobile exhaust. Here, we present the development of a method for the determination of carbon oxide. The supplied purpose is reached by consecutively passing the analyzed gas mixture on a heated surface of incandescent threads (from metals) with an active catalyst between them. The difference of heat conductance of mixtures before and after passing on the surfaces of metal threads and selective catalyst is measured and represents a quantitative measure of the concentration of carbon oxide. The results show that this method allows one to determine the content of carbon oxide in gas mixtures from 0.01 to 5% volume. The designed method is the most efficient and possesses many possible applications, such as use in monitoring the environment. 107. Quantification of Posttranslational Modifications in Proteomics. Fred E. Regnier, Erin Seeley, Samir Julka, Hamid Mirzaei, Cathy Sioma, and Larry Riggs. Department of Chemistry, Purdue University, 560 Oval Drive, West Lafayette, Indiana 47907-2084. Fax: 765-494-0359. E-mail: [email protected]. Most proteins are modified in some way after leaving the ribosome. These posttranslational modifications often play a role in the biological activity of a protein. When these modifications are involved in cell signaling, regulation, and disease progression, they become of importance in human health. Understanding disease mechanisms, developing new therapeutics, and fabrication of diagnostics can be intimately related to posttranslational modifications. A critical component in correlating posttranslational modifications with disease is the ability to recognize the protein(s) involved and quantify changes in their structure. This presentation will focus on quantifying changes in phosphorylation, glycosylation, and oxidative damage. The first step in the process that we use involves an affinity selection approach that isolates all proteins with the particular type of posttranslational modification of interest. The second step is to recognize those proteins that have changed in the modification of interest through stable isotope coding. The final step is the actual quantification by isotope ratio analysis. The presentation will close with several examples of posttranslational modification pathologies and the role proteomics is playing in the study of those diseases. 108. Proteomics: Some Potential Pitfalls and Their Solution. Mark W. Duncan. Proteomics Facility,


University of Colorado Health Sciences Center, 4200 East Ninth Avenue C238, Denver, CO 80212. Fax: 303-3150274. E-mail: [email protected]. The potential of proteomics to contribute to toxicology, or for that matter, any area of the biological sciences, is compromised by limitations of the current strategies. Methods for making quantitative comparisons are imprecise and inaccurate and misleading results are common. There are also qualitative limitations. Existing algorithms for protein identification can frequently yield an unacceptably high rate of false positive and false negative identifications. Hence, laborious review of all findings is essential because without this, erroneous findings can redirect future studies and seriously compromise efficiency. We are focused on developing more accurate, precise, and efficient approaches to both qualitative and quantitative proteomics, and an important component of this exercise is defining the specific in vivo form(s) of each protein that we identify. The importance of 2D gel electrophoresis in quantitative comparisons will be demonstrated, and our efforts to develop improved algorithms for protein identification will be presented. 109. Proteomic Analysis of Stress Responses to Protein Alkylating Agents. Daniel C. Liebler, Linda L. Manza, Sheryl L. Stamer, and Darrin L. Smith. Southwest Environmental Health Sciences Center, University of Arizona, College of Pharmacy, P.O. Box 210207, Tucson, Arizona 85721-0207. E-mail: liebler@ pharmacy.arizona.edu. A common feature of many stress-inducing stimuli that contribute to cancer and chemical toxicity is the generation of reactive electrophiles that modify specific protein targets. Recent work indicates that protein-modifying stresses also trigger changes in posttranslational modification by the ubiquitination and small ubiquitin-like modifier (sumoylation) pathways. These pathways have emerged as key regulators of protein turnover, proteinprotein interactions, and transcription factors. We hypothesize that alkylation-induced perturbations in protein ubiquitination and sumoylation disrupt control of vital cellular functions. Western blot analyses of lysates from HEK 293 cells reveal a redistribution of SUMO-1 and SUMO-2/3 to different protein substrates upon treatment with the prototypical endogenous electrophile 4-hydroxy-2-nonenal (HNE). Expression of HA-epitopetagged SUMO-1 and SUMO-2/3 allows affinity capture of sumoylated proteins and subsequent analysis by tandem LC (ion exchange/reverse phase) coupled to tandem MS. MS-MS spectra are correlated with database protein sequences with Sequest. We have employed a new database system called Complete Hierarchical Integration of Protein Searches (CHIPS) to facilitate evaluation of Sequest search outputs. This approach allows a definition of the sumoylated portion of the proteome prior to and after treatment with HNE. Proteins identified include previously known and novel SUMO substrates. Moreover, the results indicate different sets of protein targets of SUMO-1 and SUMO-2/3. The application of affinity capture and tandem LC-tandem MS approaches enables the proteome-wide analysis of stress responses to alkylating agents. Supported by NIH Grants ES11811 and ES06694. 110. Enzyme-Catalyzed O-18 Labeling for Comparative Proteomics and Applications to Acquired Drug Resistance. Catherine Fenselau, and Kristy Reynolds. Department of Chemistry & Biochemistry, Uni-


versity of Maryland, College of Life Sciences, College Park, Maryland 20742. Fax: 301-405-8615. E-mail: [email protected]. A method has been developed in this laboratory to introduce two atoms of O-18 during protein proteolysis. Now we report the evaluation and application of a second generation method, in which O-18 is catalytically introduced into peptide products in a separate step from the initial proteolysis. A preliminary report has appeared (Yao, Afonso, and Fenselau (2003) J. Proteome Res.). Chief among the advantages of this two step strategy is the omission of the requirement to dry down proteins for resolubilization in O-18 enrichd water. Rather, more malleable peptides are dried and resolubilized. Analytical figures of merit will be presented. This approach is compatible with LC-LC-MS-MS analysis, also called mudpit and shotgun. We will report the use of shotgun analysis and catalytic labeling for quantitative comparisons of cytosolic protein levels in MCF7 human breast cancer cells with those in an MCF7 strain that is resistant to doxirubicin. Both up- and down regulated proteins have been identified. 111. Proteomics of Oxidative Stress. Ian A. Blair. Center for Cancer Pharmacology and Genomics Institute Proteomics Facility, University of Pennsylvania, 1254 BRB II/III, 421 Curie Boulevard, Philadelphia, Pennsyl-


vania 19104. Fax: 215-573-9889. E-mail: ian@ spirit.gcrc.upenn.edu. Oxidative stress results in the formation of reactive oxygen species (ROS), which can damage cellular macromolecules such as DNA and proteins. Lipid hydroperoxides are also formed enzymatically during oxidative stress. Both 15-lipoxygeanse and cyclooxygenase-2 convert linoleic acid into 13(S)-hydroperoxyoctadecadienoic acid (13-HPODE), the prototypic -6 polyunsaturated fatty acid (PUFA) hydroperoxide. 13-HPODE decomposes to 4-hydroperoxy-2-nonenal, 4-oxo-2-nonenal, 4-hydroxy2-nonenal, and 4,5-epoxy-2-decenal. Our studies have focused on their interaction with histone proteins. Using MS-based methodology, 87% amino acid coverage of the protease fragments from histone H4 was identified. Treatment of histone H4 with 4-oxo-2-nonenal resulted in decreased intensity of 66D-77K by more than an order of magnitude (as determined by LC/ESI/MS) when compared with the unmodified protein. This indicated that modification had occurred on 75H76A77K. Using a combination of deuterium isotope labeling and LC/MS/ MS, it was demonstrated that a cyclic peptide had been formed on the HAK motif. Supported by NIH RO-1 CA95586 and RO-191016. TX034204F

Abstracts, American Chemical Society Division of Chemical

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