Abstracts, American Chemical Society Division of Chemical

Chem. Res. Toxicol. 2006, 19, 1677-1701

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Abstracts, American Chemical Society Division of Chemical Toxicology, 232nd ACS National Meeting, San Francisco CA, September 10-September 14, 2006 Trevor M. Penning*,† Department of Pharmacology, Center of Excellence in EnVironmental Toxicology, UniVersity of PennsylVania School of Medicine, 130C John Morgan Building, 3620 Hamilton Walk, Philadelphia, PennsylVania 19104-6084 ReceiVed October 25, 2006

1. Strategies for the Efficient Chemical Modification of Cellular DNA. Kent S. Gates. Department of Chemistry, University of Missouri, 125 Chemistry Building 601 S. College Avenue, Columbia, Missouri 65211. Historically DNA-targeted natural products have proven a rich source of anticancer drugs. In addition, structurally unusual natural products that possess potent biological activity have the potential to reveal novel chemical and biological mechanisms of action. In the context of leinamycin and other natural products, we will discuss chemical strategies for the efficient covalent modification of cellular DNA. 2. DNA Adducts of Mitomycins: Structure-Activity Relationships. Maria Tomasz. Chemistry Department, Hunter College, City University of New York, 695 Park Avenue, New York City, New York 10021. Ashis K. Basu. Department of Chemistry, University of Connecticut, 55 North Eagleville Road, Storrs, Connecticut 06269. Treatment of tumor cells with the natural antitumor agent mitomycin C (MC) results in the formation of six major DNA adducts: DNA interstrand and intrastrand cross-links and four DNA monoadducts. The same six DNA adducts can be generated in cell-free systems. The DNA interstrand cross-link (ICL) has been thought to be the critical DNA adduct determining the cytotoxicity of MC. However, in the case of other cytotoxic antitumor agents, DNA monoadducts or intrastrand cross-links have also been implicated as critical DNA damage. We use several different approaches to classify the individual adducts of MC in terms of lethality: observation of the cytotoxicity of MC derivatives, testing the mutagenicity and lethality of individual adducts incorporated in plasmids, and biochemical processing of site-specific adducts in synthetic oligonucleotides in Vitro. Several remarkable results obtained recently will be presented. 3. Acylfulvenes: Probing Chemical Pathways for Antitumor Activity. Shana J. Sturla. Department of Medicinal Chemistry and the Cancer Center, University of Minnesota, 420 Delaware Street SE-MMC 806, Minneapolis, Minnesota 55455. Small molecules that bind to DNA are the most widely used form of cancer therapy but suffer from low selectivity and high toxicity. Motivated by the need to overcome these limitations, we are investigating mechanisms by which DNA-alkylating natural products and their derivatives affect tumor cell death. Acylfulvenes, semi-synthetic derivatives of toxic mushroomderived natural products, demonstrate promising anticancer activity. Biomolecular targets in cells, including proteins and DNA, are covalently modified by AFs. While this class of compounds is directly chemically reactive, AFs are efficiently * Corresponding author. Tel: 215-898-9445. Fax: 215-898-7180. E-mail: [email protected]. † Program Chair.

reductively bioactivated, producing unstable intermediates that may also alkylate biomolecular targets. Studies aimed at identifying corresponding reactions and products to establish a link between these pathways and cellular sensitivity will be discussed. 4. Mechanistic Studies on the DNA Cross-Linking Agent Azinomycin B. Robert S. Coleman. Department of Chemistry, The Ohio State University, 100 West 18th Avenue, Columbus, Ohio 43210. Azinomycin B is a naturally occurring antitumor agent that acts by the formation in interstrand DNA cross-links. Mechanistically, the agent appears to interact with DNA via the reactive epoxide and aziridine moieties. Studies on the mechanism of action of azinomycin B and synthetic partial structures will be presented. The stereochemical dependence of the sequence selective DNA alkylation and in Vitro cytotoxicity have been examined, and yeast gene expression and cellular localization studies provide evidence for the DNA damaging effects of the native agent in ViVo.

5. Mismatch Repair Proteins Participate in an Error-Free Processing of DNA Interstrand Cross-Links in Human Cells. Karen M. Vasquez and Qi Wu. Department of Carcinogenesis, University of Texas M.D. Anderson Cancer Center, 1808 Park Road 1-C, Smithville, Texas 78957. DNA interstrand cross-links (ICLs) present formidable blocks to DNA metabolic processes and must be repaired for cell survival. ICLs are induced in DNA by intercalating compounds such as the widely used chemotherapeutic agent psoralen. In bacteria, both nucleotide excision repair (NER) and homologous recombination are required for the repair of ICLs. The processing of ICLs in mammalian cells is not yet clearly defined. However, it is known that NER proteins assist in processing psoralen ICLs and can result in error-generating mutagenic repair. We show that proteins from mismatch repair (MMR) are also involved in eliminating psoralen ICLs in human cells in what appears to be an error-free process. Using psoralenconjugated triplex-forming oligonucleotides (TFOs) to direct ICLs to a specific site in targeted DNA, we demonstrate that the human recombinant protein complex MutSb specifically recognizes these lesions. MSH2 deficiency renders human cells hypersensitive to psoralen ICLs, suggesting a role for MSH2

10.1021/tx6002886 CCC: $33.50 © 2006 American Chemical Society Published on Web 11/23/2006

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in ICL processing. A reduced level of psoralen ICL processing in human MSH2-deficient cell extracts indicates that the human MSH2 protein is involved in the efficient processing of triplextargeted psoralen ICLs. Lack of MSH2 does not reduce the level of psoralen ICL-induced mutagenesis in human cells, indicating that MSH2 does not contribute to the error-generating repair of psoralen ICLs. Interestingly, MLH1 deficiency renders human cells resistant to psoralen ICLs and increases the level of psoralen ICL-induced mutagenesis in human cells. These data suggest that both MSH2 and MLH1 proteins play critical roles in the error-free repair of psoralen ICLs and, therefore, may represent a novel error-free mechanism for repairing ICLs. Thus, enhancement of MMR relative to NER may reduce the mutagenesis caused by DNA ICLs in humans. 6. Design of DNA Alkylating Agents That Block DNA Repair and Disrupt Cancer Specific Cellular Signaling Programs. John M. Essigmann and Robert G. Croy. Department of Chemistry and Biological Engineering Division, Massachusetts Institute of Technology, Room 56-669, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139. An approach to anticancer drug design is described in which a DNA damaging nitrogen mustard is tethered to a ligand for a steroid hormone receptor. The mustard forms DNA adducts that recruit the receptors, which are often expressed at high levels in tumors. One consequence of the binding of a receptor to the DNA adduct is that the adduct becomes shielded from DNA repair enzymes. Thus, the adduct persists and shows enhanced toxicity in steroid receptor positive tumors. The new DNA damaging agents also disrupt signaling pathways in malignant cells. The steroid receptors are transcription factors that control the pathways of growth and maintenance of secondary sexual characteristics. The compounds we designed form DNA adducts to which the androgen and estrogen receptors bind tightly. The transcription-promoting properties of the receptors are, therefore, effectively “hijacked” by the adducts. As a consequence, cancer related gene expression is disrupted. 7. Yatakemycin: Synthetic and Mechanistic Studies Defining Its DNA Alkylation Properties. Dale Boger. Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037. Studies of the DNA alkylation selectivity, efficiency, rate, and reversibility of natural (+)-yatakemycin, its synthetic unnatural enantiomer ent-(-)-yatakemycin, and key partial structures and analogues will be presented. 8. Chemical Modification of Protein Thiols in Diabetes. John W. Baynes, Norma Frizzell, Ryoji Nagai, Nathan L. Alderson, Yuping Wang, Nadja Alt, Matthew Blatnik, and Suzanne R. Thorpe. Department of Chemistry and Biochemistry, University of South Carolina, Graduate Science Research Center, 631 Sumter Street, Columbia, South Carolina 29208. Lipid and carbohydrate-derived reactive carbonyl compounds contribute to chemical modification of proteins in aging and age-related diseases, such as diabetes and atherosclerosis. Protein thiol groups, the most reactive nucleophiles in intracellular proteins, are modified by dicarbonyl compounds such as glyoxal, forming S-(carboxymethyl)cysteine, which is increased in muscle proteins in diabetes. We have identified another thiol modification, S-(2-succinyl)cysteine (2SC), which is formed by a Michael addition reaction of fumarate with thiol groups in proteins. 2SC was detected in human plasma albumin and skin collagen and in rat skeletal muscle proteins. 2SC increases in skin collagen with age and in the muscle protein of diabetic versus control rats. 2SC is the first example of spontaneous chemical modification of a protein by a metabolic intermediate

Penning

in the Krebs cycle. These and other observations identify fumarate as an endogenous electrophile and implicate fumarate in the regulation of metabolism in response to metabolic and oxidative stress. 9. Electrophile Responsive Proteome and Its Biological Effects in Endothelial Cells. Victor Darley-Usmar. Department of Pathology and Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294. Cellular redox signaling is mediated by the post-translational modification of proteins by reactive oxygen/nitrogen species or the products derived from their reactions. Initially, it was thought that lipid oxidation products have limited specificity in their ability to elicit a biological response, but it is now becoming clear that oxidized lipids are potent mediators of physiological and pathological processes. In the case of the class of oxidized lipids known as the lipid electrophiles, several receptor-dependent and independent mechanisms are now emerging. The formation of lipid oxidation products through either enzymatic or nonspecific lipid peroxidation generates a broad spectrum of structurally diverse compounds. These lipids form covalent adducts with proteins due to the presence of reactive groups such as an electrophilic carbon center and/or an aldehyde group. In order to characterize the interactions of these reactive lipids with proteins, we conjugated an electrophilic lipid (15-deoxy-prostaglandin J2) and an oxidizable lipid (arachidonic acid) with either a biotin or fluorescent tag. We then determined the nature of the interaction of these reactive lipids in Vitro using albumin as a model protein and also with proteins in intact endothelial cells. Interestingly, the 15-deoxyprostaglandin J2 reacted primarily with reduced thiols on albumin and with cellular proteins, whereas oxidized arachidonic acid formed adducts irrespective of thiol status. Experiments with the fluorescently tagged lipids also revealed that 15-deoxyprostaglandin J2 co-localized with mitochondria and that this localization was dependent on the electrophilic carbon. Oxidized arachidonic acid, however, did not localize to the mitochondria. Using a proteomics approach with tagged electrophilic lipids, we have identified a subset of proteins that we have termed the electrophile responsive proteome. The identity of these proteins will be discussed in the context of our increasing understanding of redox signaling and the response of the cell to oxidative stress. 10. Proteome Damage by Reactive Electrophiles. Daniel C. Liebler. Department of Biochemistry, Vanderbilt University Medical Center, 9264 Medical Research Building III, 465 21st Avenue South, Nashville, Tennessee 37212. Protein thiols are targets for alkylation damage by reactive electrophiles associated with inflammation, degenerative diseases, and chemical toxicity. Analysis of human cytoplasmic and nuclear protein targets of two different thiol-reactive electrophiles identified 897 cysteine targets, which mapped to 539 proteins. Targeting was selective and reproducible yet differed markedly between electrophile structures. A core group of 125 cysteines (14% of the total) was consistently modified by both electrophiles. We hypothesize that these thiol targets represent systems that are simultaneously responsive to redox regulation and susceptible to damage. Analyses of the kinetics of protein modification reveals differences in the rates of competing adduction reactions on the same protein and provides a basis for understanding how protein and electrophile structures confer target selectivity. Studies of mitochondrial and endoplasmic reticulum targets indicate similar targeting properties for thiol-reactive nucleophiles and may provide insights into ER stress and apoptosis-related damage responses in toxicity and disease.

Abstracts, ACS DiVision of Chemical Toxicology

11. Nitric Oxide Mediated Post-Translational Modifications. Harry Ischiropoulos. Departments of Pediatrics and Pharmacology, The University of Pennsylvania, Stokes Research Institute-Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania 19104. S-Nitrosylation, the selective modification of cysteine residues in proteins to form S-nitrosocysteine, is a major emerging mechanism by which nitric oxide acts as a signaling molecule. We employed a proteomic approach using selective peptide capturing and site-specific adduct mapping to identify the targets of S-nitrosylation in human aortic smooth muscle cells upon exposure to S-nitrosocysteine and propylamine NONOate. This strategy identified 20 unique S-nitrosocysteine-containing peptides belonging to 18 proteins, including cytoskeletal proteins, chaperones, and proteins of the translational machinery, vesicular transport, and signaling. Sequence analysis of the S-nitrosocysteine-containing peptides revealed the presence of acid-base motifs as well as hydrophobic motifs surrounding the identified cysteine residues. High-resolution immunogold electron microscopy supported the cellular localization of several of these proteins. Interestingly, seven of the 18 proteins identified are localized within the ER/Golgi complex, suggesting a role for S-nitrosylation in membrane trafficking and ER stress response in vascular smooth muscle. 12. GAPDH as a Sensor of NO Stress: S-Nitrosylation of GAPDH. Akira Sawa. Psychiatry, Johns Hopkins University, 600 N. Wolfe Street, CMSC 8-117, Baltimore, Maryland 21287. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a classic glycolytic enzyme, but its role as a mediator for cell death has recently been highlighted. Many groups reported that a pool of GAPDH translocates to the nucleus under a variety of stressors, most of which are associated with oxidative stress. Here, I report its sequential molecular mechanism as follows: first, a catalytic cysteine in GAPDH (C150 in rat GAPDH) is S-nitrosylated by nitric oxide (NO) that is generated from inducible nitric oxide synthase (iNOS) and/or neuronal NOS (nNOS); second, the modified GAPDH becomes capable of binding with Siah1, an E3 ubiquitin ligase, and stabilizes it; third, the GAPDH-Siah protein complex translocates to the nucleus, dependent on Siah1’s nuclear localization signal, and degrades Siah1’s substrates in the nucleus, which results in cytotoxicity. I will further discuss a possible role of GAPDH in neurodegenerative disorders. 13. Regulation of Gene Expression by Oxidative Stress. Cecil Pickett. Schering-Plough Research Institute, ScheringPlough Research Corporation, 2000 Galloping Hill Road, Kenilworth, New Jersey 07033-0530. Abstract text not available. 14. DNA Damage and Mutagenesis. Ashis K. Basu1, Laureen Colis1, Danielle Watt1, M. Abul Kalam1, Alvin Altamirano1, and Marc M. Greenberg2. (1) Department of Chemistry, University of Connecticut, 55 North Eagleville Road, Storrs, Connecticut 06269. (2) Department of Chemistry, Johns Hopkins University, 3400 N. Charles Street, Baltimore, Maryland 21218. Mutagenesis is one of the most deleterious consequences of DNA damage, and the relationship of the structure of the damage with its biological consequence has been the subject of numerous investigations by chemists, structural biologists, and biochemists. We have been studying the biological effects of the DNA adducts formed by the nitroaromatic carcinogens, 1-nitropyrene and dinitropyrenes. We have also explored the mutagenic potential of several oxidative DNA lesions, including thymine glycol, urea, 8-oxopurines, and the imidazole ring-opened formamidopyrimidines. The types and frequencies of mutagen-

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esis of these lesions depend on the structure of the lesion, the DNA sequence context, the type of cell, and the repair status of the latter. In addition, the presence of lesions near one another may influence its mutagenicity. Even though the identity of the DNA polymerase(s) and the repair proteins has been considered important for the mutagenic outcome, explanation of the cellular data poses significant challenges. What we learned from the results of these studies will be discussed. 15. Elucidating Structure-Function Relationships of DNA Adducts Using Computer Modeling. Suse Broyde. Department of Biology, New York University, 100 Washington Square East, New York, New York 10003. Bulky DNA lesions primarily block DNA polymerases, although some mutagenic bypass may occur as well. The blockage causes a switch to one or more bypass polymerases, which may transit the lesion in an error-free or error-prone manner. One goal of our work is to delineate structural features of adducts within polymerases that produce stalling in replicative polymerases or allow rare bypass. We also wish to define the structural properties that determine mutagenic or faithful nucleotide incorporation in bypass polymerases. We employ computer modeling with molecular dynamics simulations to produce ensembles of structures for detailed analyses. These results aid in the interpretation of experimental data and generate new hypotheses for further experimental testing. Dynamic structural features that can result in mutagenic translesion synthesis are being elucidated through these studies. 16. Cytochrome P450 and Chemical Toxicology. F. Peter Guengerich. Department of Biochemistry & Center in Molecular Toxicology, Vanderbilt University, 638 Robinson Research Bldg, 23rd and Pierce Avenues, Nashville, Tennessee 37232. Cytochrome P450 (P450) enzymes are the major catalysts involved in the metabolism of xenobiotic chemicals. The most general process is that of detoxication. Although activation of chemicals to reactive electrophilic species is less common, P450s are also the major catalysts, and many examples of toxicity of drugs and other chemicals are understood in this context. Roles of P450s in oxygen radical damage have been proposed but are less clear. Much is now known about the catalytic specificity of animal and human P450s, although a number of questions about the molecular basis of substrate specificity and catalysis are still unanswered, and a priori predictions are still difficult. Understanding the chemistry of oxidations by P450s has been important in rationalizing the biotransformation of chemicals to reactive and other products and in designing means to circumvent problems. A better understanding of the practical significance of P450 induction and inhibition has also developed. Supported in part by USPHS R37 CA090426, P30 ES000267. 17. Tobacco Carcinogen Biomarkers: Development and Application. Stephen S. Hecht, Pramod Upadhyaya, Mingyao Wang, Irina Stepanov, Yanbin Lao, Peter W. Villalta, and Steven G. Carmella. University of Minnesota Cancer Center, 420 Delaware Street SE-MMC 806, Minneapolis, Minnesota 55455. Although it has been known for more than 50 years that tobacco products cause cancer, the emergence of tobacco carcinogen biomarkers for assessing mechanisms of carcinogenesis in humans began only about 20 years ago, coinciding roughly with the beginning of Chemical Research in Toxicology. Tobacco carcinogen biomarkers include DNA adducts, hemoglobin adducts, and urinary metabolites. These quantitative measurements can be extremely informative with respect to the mechanisms and prevention of tobacco induced cancer. Our laboratory has focused on three classes of carcinogens in tobacco


products: tobacco-specific nitrosamines, polycyclic aromatic hydrocarbons (PAH), and aldehydes. Urinary metabolites of tobacco-specific nitrosamines such as 4-(methylnitrosamino)1-(3-pyridyl)-1-butanol (NNAL) and its glucuronides have provided important information about the uptake of the tobaccospecific lung carcinogen NNK in humans. This biomarker has been widely applied in studies of carcinogen uptake in smokers, smokeless tobacco users, and non-smokers exposed to environmental tobacco smoke. Methods for the analysis of other tobacco-specific nitrosamine metabolites in human urine and for their DNA adducts have also been developed. The uptake and metabolic activation of PAH differs widely among individuals, and we have developed phenanthrene metabolite ratios to probe these differences. The relationship between phenanthrene metabolite urinary phenotype and variants in PAH metabolizing genes has been investigated in smokers. Acetaldehyde and formaldehyde occur widely in the human environment including cigarette smoke. We have been investigating methods for the quantitation of their DNA adducts in laboratory animals and humans. Recent results from our research on the development and application of tobacco carcinogen biomarkers will be presented. 18. Quinoids Formed from Estrogens and Antiestrogens: Role in Carcinogenesis? Judy L. Bolton. Department of Medicinal Chemistry & Pharmacognosy, University of Illinois at Chicago, College of Pharmacy, 833 S. Wood Street M/C 781, Chicago, Illinois 60612. Estrogens and antiestrogens have been implicated in hormone dependent cancers; however, the carcinogenic mechanism(s) remains both controversial and elusive. One mechanism could involve the metabolism of these compounds to reactive intermediates, such as quinones, quinone methides, and di-quinone methides, which leads to oxidation and/or alkylation of DNA and proteins. We have previously shown that four SERMs, including tamoxifen, raloxifene, acolbifene, and arzoxifene, are all metabolized by P450 to quinone methides and in some cases o-quinones. In order to determine if these quinoids could cause protein modification, a novel raloxifene COATag (covert oxidatively activated tag) was synthesized in which the SERM was linked to biotin. This COATag facilitated the isolation and identification of covalently modified proteins following metabolic activation of the labeled raloxifene by rat liver microsomes. These data show that oxidative metabolism of raloxifene produces reactive intermediates of sufficient lifetime to covalently modify proteins in microsomes and that this would be an expected general feature of SERMs currently in use. These data suggest that cellular damage mediated by quinoids from estrogens or antiestrogens could contribute to the carcinogenic effects of these compounds in ViVo. Supported by CA73638, CA79870. 19. Environmental Genomics and Human Health. David Schwartz. Office of the Director, National Institute of Environmental Health Sciences, 79 T. W. Alexander Drive, Research Triangle Park, North Carolina 27709-2233. Abstract text not available. 20. A Role of Nitric Oxide in the Carcinogenic Process. Steven R. Tannenbaum. Biological Engineering, Massachusetts Institute of Technology, Room 56-731, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139. The paradox of NO is that one cannot simply determine the role of NO in toxicity and carcinogenicity. Experiments in cell culture demonstrate that there is a threshold dose for toxicity to exogenous NO. Experiments in mice demonstrate a protective role of NO in some models and a pathogenic role in others.

Penning

The complexity is obvious when one considers that there are at least three isoforms of NO synthase, multiple possible intracellular localizations for these enzymes, multiple types of NOsignaling, multiple types of NO chemical reactions, and so forth. Can these complexities be resolved in a rational understanding of NO chemistry? We are not there yet, but we are moving in that direction. 21. Arsenic: The King of Poisons, the Poisons of Kings, and the Bane of Investigators. H. Vas Aposhian1, Mihaela D. Avram2, George Tsaprailis3, and Uttam K. Chowdhury2. (1) Department of Molecular and Cellular Biology, The University of Arizona, Life Sciences South Building, Tucson, Arizona 85721. (2) Department of Molecular and Cellular Biology, The University of Arizona, Life Sciences South 444, P.O. Box 210106, Tucson, Arizona 85721. (3) Department of Pharmacology and Toxicology, The University of Arizona, Center for Toxicology, P.O. Box 10207, Tucson, Arizona 85721. Arsenic-contaminated drinking water is the cause of the greatest public health calamity of the last 25 years. Yet, there is still a mystery as to the molecular mechanisms of inorganic arsenic toxicity. The human metabolizes inorganic arsenic via a number of methylation steps. Recently, the established pathways have been questioned, and new ones have been proposed by Hayakawa et al. (2005). Newer proteomics techniques such as differential in-gel electrophoresis (DIGE) have allowed the investigation of proteins whose syntheses are inhibited by arsenic species as well as proteins whose syntheses appear to increase and may be nontoxic reservoirs for arsenic. The relationships of many of these proteins with arsenic metabolism and toxicity are being identified for the first time by using DIGE coupled to LC-MS/MS technologies. Supported in part by NIEHS Grant ES-04940 and SWEHSC Grant ES06694. 22. Oxidative Stress Induced Macromolecule Modification and Cell Signaling. Koji Uchida. Laboratory of Food and Biodynamics, Nagoya University Graduate School of Bioagricultural Science, Furo-cho Chikusa-ku, Nagoya 464-8601, Japan. It is now recognized that acrolein is an endogenous electrophile that could be formed in cells via lipid peroxidation, amino acid oxidation, and polyamine metabolism. Its high reactivity indeed makes this aldehyde a dangerous substance for the living cell. A number of reports have appeared describing the damaging effects of acrolein on the tracheal ciliatory movement and the pulmonary wall. It has also been shown that acrolein initiates urinary bladder carcinogenesis in rats. We have investigated the reaction of protein with this carcinogenic aldehyde and identified a novel acrolein-lysine adduct, N-(3formyl-3,4-dehydropiperidino)lysine (FDP-lysine), as the major product. In our recent study, we have also shown that the FDP adduct is a thiol-reactive electrophile, which reacts with sulfhydryl groups to form thioether adducts. In this presentation, after a brief summary on our recent progress in lipid adduction of proteins, I will show you some of our latest data on the thiolation of FDP adducts generated in the oxidized low-density lipoptoteins. Furthermore, glutathione-dependent detoxification of FDP adducts in the oxidized LDL will also be discussed. 23. Role of Reactive Metabolites and Other Risk Factors in Determining Susceptibility to Drug-Induced Liver Disease. Lance R. Pohl. Molecular and Cellular Toxicology Section, NIH National Heart Lung and Blood Institute, Building 10, Room 8N110, Bethesda, Maryland 20892-1760. Drug-induced liver disease (DILD) is thought to be caused by both allergic and non-allergic mechanisms of pathology that are mediated by reactive metabolites of drugs and endogenous


biochemicals. Most cases are idiosyncratic and difficult to predict due in large part to insufficient knowledge of predisposing risk factors. Recent murine model studies, however, have led to the discovery of several potential susceptibility factors that may predispose individuals to DILD. For example, mice deficient in interleukin (IL)-4, IL-6, IL-10, IL-13, or cyclooxygenase-2 are more susceptible to DILD than wild-type mice, while deficiencies in other factors, including macrophage migration inhibitory factor and osteopontin, make mice less susceptible to DILD. Other potential risk factors for DILD have been discovered by proteomic and genomic analyses when protein and mRNA levels in livers of susceptible and resistant strains of mice were compared before and after drug treatment. These findings suggest that polymorphisms and/or environmental factors that affect the activities of both hepatoprotective and hepatotoxicant factors may contribute to the incidence of DILD. 24. Integrating Genomic and Proteomic Approaches to Identify Targets of Environmental Chemical-Induced Nephrocarcinogenicity. Serrine S. Lau. Department of Pharmacology and Toxicology, University of Arizona College of Pharmacy, Southwest Environmental Health Science Center, 1703 E. Mabel Street, Tucson, Arizona 85721-0207. Identification of genetic markers in individuals predisposed to tumor development after occupational or environmental exposure to potential carcinogens requires an understanding of how specific genes determine susceptibility to chemical-induced carcinogenesis. Knowledge of the proportion of susceptible individuals in the population and the relative cancer susceptibility of normal and predisposed groups will make it possible to estimate human risk from carcinogen exposure. We use the Eker rat (Tsc-2EK/+), which bears a mutation in one allele of the tuberous sclerosis-2 (Tsc-2) tumor suppressor gene and codes for the protein tuberin, as an animal model to investigate the molecular events that culminate in 2,3,5-tris-(glutathion-S-yl)hydroquinone (TGHQ)-induced renal cancer. We have shown that (i) cell proliferation is insufficient, but loss of tuberin is necessary for chemical-induced nephrocarcinogenicity in the Eker rat; (ii) reduced constitutive 8-oxoguanine-DNA glycosylase (OGG1) expression and impaired induction following oxidative DNA damage in the tuberin deficient Eker rat, suggesting that heterozygosity at the Tsc-2 locus predisposes to an increase in DNA mutations, even in the absence of a loss of the normal allele, indicating that tuberin is haploinsufficent for this putative DNA repair function; and (iii) tuberin modulates various signaling pathways including ERK and B-raf activities in transformed renal epithelial cells. We have developed mass spectrometry based methodology to determine changes in both global protein expression and in post-translational modifications during chemical-induced renal carcinogenesis. At present, our results indicate that the Tsc-2 gene influences constitutive OGG1 expression and the ability of OGG1 to respond to an oxidative stress. The mechanisms coupling tuberin expression to the regulation of OGG1 are not known and are currently under investigation. Gene and protein targets in response to TGHQ mediated nephrotoxicity will be discussed. (Supported by grants GM39338, GM070890, and ES06694. 25. Global Systems Biology and Statistical Spectroscopic Approaches to Biomarker Discovery. Jeremy K. Nicholson. Biological Chemistry, Imperial College, London University, Sir Alexander Fleming Building, Exhibition Road, London, United Kingdom. Current challenges in personalized healthcare and population phenotype screening involve the multiparametric measurement of biological features of individuals and groups of people that


relate to the risk of disease development or adverse drug reactions. Pharmacogenomic approaches have achieved limited success but ultimately are deficient in that gene-environment interactions that underpin most disease processes are not sampled using genomic or even proteomic methods. We have shown that metabolic spectroscopy (NMR and MS) linked to advanced chemometric methods can allow the statistical prediction of outcomes of drug intervention (pharmaco-metabonomics (Clayton, T. A., Nicholson, J. K., and et al. (2006) Pharmacometabonomic phenotyping and personalised drug treatment. Nature 440, 1073)) and also for recovery of latent biomarker information on disease processes. These “top-down” systems biology approaches using metabolic end-points and signatures and offer new ways of screening individuals for potentially toxic drug reactions and for understanding population susceptibility to disease. As these methods rely on minimally invasive spectroscopic measurements, they offer a novel approach to personalized healthcare solutions. 26. Solution Structures of the Food Mutagen 2-Amino3-methylimidazo[4,5-f]quinoline (IQ) in the Recognition Sequence of the NarI Restriction Enzyme: Sequence Context Effect. Feng Wang. Department of Chemistry, Vanderbilt University, Box 1822, Station B, Nashville, Tennessee 37235. C. Eric Elmquist. Biological Engineering Division, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Room NE47-293, Cambridge, Massachusetts 02139. James S. Stover. Department of Chemistry and Center in Molecular Toxicology, Vanderbilt University, VU Station B 351822, Vanderbilt University, Nashville, Tennessee 37235-1822. Carmelo J. Rizzo. Department of Chemistry, Center in Molecular Toxicology and Vanderbilt Institute of Chemical Biology, Vanderbilt University, P.O Box 1822 Station B, Vanderbilt University, Nashville, Tennessee 37235-1822. Micheal P. Stone. Department of Chemistry, Vanderbilt University, 1822 B, Nashville, Tennessee 37235. 2-Amino-3-methylimidazo[4,5-f]quinoline (IQ) is a mutagenic and carcinogenic heterocyclic amine found in cooked meats. NMR revealed sequence-specific differences in oligodeoxynucleotide duplexes containing [IQ]dG positioned in the C-[IQ]G1-G, G-[IQ]G2-C, and C-[IQ]G3-C contexts in the C-G1-G2C-G3-C-C recognition sequence of the NarI restriction enzyme, named NarIIQ1, NarIIQ2, and NarIIQ3. In NarIIQ3, IQ was intercalated. The adducted guanine was displaced into the major groove with its glycosyl torsion angle in the syn conformation. The complementary cytosine was displaced into the major groove. For NarIIQ1 and NarIIQ2, the modified guanines were also in the syn conformation with the complementary cytosines displaced into the major groove. In NarIIQ1, the IQ ring was in the minor groove oriented in the 5′-direction. The IQ ring in NarIIQ2 was partially intercalated; it extended into the minor groove in the 3′-direction. These sequence-dependent differences in structure may correlate with sequence-dependent differences in trans-lesion replication. Funded by CA-55678 (to M.P.S). 27. Characterization and Quantitation of 2′-Deoxyguanosine Adducts of the Dietary Carcinogen PhIP by Linear Quadrupole Ion Trap Mass Spectrometry. Angela K. Goodenough. Division of Environmental Disease Prevention, Wadsworth Center, NYS Department of Health, Empire State Plaza, P.O. Box 509, Albany, New York 12201-0509. Robert J. Turesky, Division of Environmental Disease Prevention, Wadsworth Center, NYS Department of Health, Empire State Plaza, P.O. Box 509, Albany, New York 12201-0509. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is an animal and potential human carcinogen that occurs in grilled


meats. DNA adducts of PhIP have been detected by 32Ppostlabeling, immunohistochemistry, and accelerator and gas chromatography-negative ion-chemical ionization mass spectrometry (MS) methods. Although these techniques are sensitive, they provide little structural information on the molecule. Moreover, the biochemical methods are not quantitative. Triple quadrupole MS has been used to quantitate DNA adducts, but the tandem MS scan mode provides limited structural information because generally only a single transition, from the loss of deoxyribose, is monitored. The linear quadrupole ion trap mass spectrometer is a powerful screening instrument because of its ability to perform MSn experiments, enabling both quantification and more extensive characterization of analytes. We have employed these enhanced scanning capabilities of ion trap MS to characterize and quantitate isomeric deoxyguanosine adducts of PhIP in animal and human DNA at levels g1 adduct in 108 base pairs. 28. Synthesis and Site-Specific Incorporation of the 2,6-Diamino-4-hydroxy-N5-(methyl)-formamidopyrimidine (Me-FAPy) Adduct into Oligonucleotides. Plamen P. Christov,1 Ivan D. Kozekov,1 Kyle L. Brown,2 Michael P. Stone,2 Thomas M. Harris,1 and Carmelo J. Rizzo1. (1) Department of Chemistry, Center in Molecular Toxicology and Vanderbilt Institute of Chemical Biology, Vanderbilt University, P.O. Box 1822, Nashville, Tennessee 37235. (2) Department of Chemistry and Center in Molecular Toxicology, Vanderbilt University, Nashville, Tennessee 37235. Alkyl halides, sulfonates, and methanesulfonates react with DNA to give cationic N7-alkyl guanine adducts. These unstable adducts can undergo loss of the base to give abasic sites or hydrolytic opening of the imidazole ring to form 2,6-diamino4-hydroxy-N5-(alkyl)-formamidopyrimidines (FAPy’s) (1). These important DNA lesions have received little attention because of the poor accessibility of site-specifically adducted oligodeoxynucleotides. We have developed a four-step method for the synthesis of phosphoramidite 2 in 24% overall yield. Phosphoramidite 2 has been used for the preparation of oligodeoxynucleotides, which will now allow the structure, repair, and miscoding properties of the Me-FAPy adduct to be assessed.

29. Benzo[a]pyrene-Mediated DNA Adduct Formation in Bronchoalveolar Cells. Stacy Lynn Gelhaus1, Qian Ruan2, Seon Hwa Lee3, Trevor M. Penning4, and Ian A. Blair2. (1) Department of Pharmacology, Center for Cancer Pharmacology, University of Pennsylvania, 421 Curie Boulevard, BRB II/III Room 841, Philadelphia, Pennsylvania 19104. (2) Center for Cancer Pharmacology, University of Pennsylvania, 848 Biomedical Research Building, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104. (3) Department of Pharmacology, Center for Cancer Pharmacology, University of Pennsylvania, 846 BRB II/III, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104. (4) Center of Excellence in Environmental Toxicology, Department of Pharmacology, University of Pennsylvania School of Medicine, 135 John Morgan Building, 3620 Hamilton Walk, Philadelphia, Pennsylvania.

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Activation of PAHs by P450 is one of the most widely accepted pathways of PAH metabolism. P450 1A1/1B1 oxidizes benzo[a]pyrene (B[a]P) to 7,8-dihydroxy-9,10-epoxy,7,8,9,10tetrahydrobenzo[a]pyrene (B[a]PDE), the ultimate carcinogen. B[a]PDE is able to form adducts with dGuo and dAdo in DNA. H358, human bronchoalveolar cells, do not constitutively express P450s 1A1/1B1. However, pretreatment of the cells with 2,3,7,8-tetrachlorobenzo-p-dioxin (TCDD) for 12 h induced these P450s. Treatment of the TCDD-induced cells with (()B[a]P-7,8-dihydro-7,8-diol (B[a]P-7,8-dihydrodiol) yielded B[a]PDE-DNA adducts that were quantified by a stable isotope dilution, liquid chromatography-multiple reaction monitoring/ mass spectrometry assay. An unexpected increase in B[a]PDEDNA adduct formation was observed in cells that had not been subjected to TCDD pretreatment. The adducts arose primarily from the (-)-enantiomer of B[a]P-7,8-dihydrodiol. Increased adduct formation may arise from induction of an unknown P450 or by an alternative oxidation pathway. Supported by NIH grants PO1 CA 92537 and P30 ES013508. 30. Translesion Synthesis by Sulfolobus solfataricus DNA Polymerase Dpo4 across Polycyclic Aromatic Hydrocarbon Diol Epoxide Adducts of Deoxyadenosine. Goutam Chowdhury1, Hong Zang1, Karen C. Angel1, Thomas M. Harris2, and F. Peter Guengerich1. (1) Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, 638 Robinson Research Building, 23rd and Pierce Avenues, Nashville, Tennessee 37232. (2) Department of Chemistry and Center in Molecular Toxicology, Vanderbilt University, 7332 Stevenson Center, Nashville, Tennessee 37235. Polycyclic aromatic hydrocarbons (PAHs) are potent environmental carcinogens, known for over hundred years. PAHs are metabolically activated to reactive diol epoxides, which react with DNA to form various covalent adducts. The mechanisms by which DNA adducts of PAH derivatives cause mutations have been of immense interest. Three different N6-adenyl PAHdiol epoxide oligonucleotide derivatives (dA-ATBA-1S, dAATBA-11S, and dA-STBP-10S) were studied with the archebacterial translesion DNA polymerase Sulfolobus solfataricus Dpo4. Steady-state kinetic analysis indicated the insertion of all four dNTPs opposite all three N6-adenyl PAH adducts, with only slightly varying misincorporation efficiencies. Full-length extension across the N6-adenyl PAH derivatives proceeded to apparent completion at 45 °C in the presence of added dimethylsulfoxide. Analysis of the products by LCMS/MS indicated the presence of mixtures of products corresponding to both error-free synthesis and mixtures of polymerization/ realignment steps. The results demonstrate the complexity of polymerization opposite these bulky N6-adenyl PAH adducts, even with a single polymerase.

31. Formation and Accumulation of Pyridyloxobutyl (POB)-DNA Adducts in F344 Rats Treated with TobaccoSpecific Carcinogens. Yanbin Lao, Nanxiong Yu, Fekadu Kassie, and Stephen S. Hecht. University of Minnesota Cancer Center, 420 Delaware Street SE-MMC 806, Minneapolis, Minnesota 55455.


4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and its metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), are both potent pulmonary carcinogens in rodents. (S)NNAL, similar to NNK, was more tumorigenic than (R)-NNAL in A/J mice. (S)-NNAL was preferentially retained in tissues and converted to NNK. Cytochrome P450-mediated R-methylhydroxylation of NNK, followed by DNA alkylation, results in the formation of pyridyloxobutyl(POB)-DNA adducts, that is, O6-POB-dGuo, 7-POB-Gua, O2-POB-Thy, and O2-POBCyt. In this study, F344 male rats were treated with 10 ppm of NNK, (R)-NNAL, or (S)-NNAL in drinking water. After 1, 2, 5, 10, 16, or 20-weeks treatment, POB-DNA adducts were quantitated by HPLC-ESI-MS/MS analysis of liver and lung DNA. Total adduct levels were higher in lung than liver for all three groups. The total adduct levels in the (S)-NNAL group were 0.6-1.4 times as great as those of the NNK group and 6-17 times higher than those of the (R)-NNAL group. This study supports the hypothesis that preferential retention in ViVo and conversion of (S)-NNAL to NNK may be responsible for its tumorigenicity. 32. Role of Electrostatic Interactions in the Unusual Selectivity of Nitrosoperoxycarbonate for Guanines with the Highest Ionization Potentials in DNA. Yelena Margolin1, Vladimir Shafirovich2, Nicholas E. Geacintov2, and Peter C. Dedon3. (1) Biological Engineering Division, NE47-277, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139. (2) Department of Chemistry, New York University, 31 Washington Place, New York, New York 10003. (3) Biological Engineering Division and Center for Environmental Health Sciences, Massachusetts Institute of Technology, NE47-277, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139. Nitrosoperoxycarbonate is a chemical mediator of inflammation that we have shown to react preferentially with guanines in 5′-GC-3′ sequence motifs associated with the highest sequence-specific guanine ionization potentials. This selectivity is in sharp contrast to that of other one-electron oxidants such as photoactivated riboflavin that target guanines with the lowest ionization potentials such as runs of G. We hypothesized that the negative charge of nitrosoperoxycarbonate may be one factor determining its unusual sequence selectivity because it may limit access of the oxidant for the negatively charged DNA, resulting in selective oxidation of the most solvent accessible guanines. To test this hypothesis, we have determined the sequence selectivity of guanine oxidation for a variety of charged and uncharged oxidants including Fe(II)-EDTA, Fe(II)/H2O2, and gamma-radiation. The results reveal important insights into the basis for the unusual sequence selectivity of nitrosoperoxycarbonate. 33. Reversible DNA Alkylation by Leinamycin. Sanjay Dutta1, Joseph Szekely1, Tony Nooner1, and Kent S. Gates2. (1) Department of Chemistry, University of Missouris Columbia, 601 South College Avenue, Columbia, Missouri 65211. (2) Departments of Chemistry and Biochemistry, University of MissourisColumbia, 601 S. College Avenue, Columbia, Missouri 65211. Leinamycin is a structurally novel Steptomyces-derived natural product with potent antitumor and antibiotic activity. It is a thiol-triggered DNA-damaging agent that generates an episulfonium ion that alkylates deoxyguanosine residues in double-strand DNA. Here in our work, we have shown that leinamycin is the first natural product that reversibly alkylates N7-deoxyguanosine residues in DNA under physiological conditions. The leinamycin adduct, either in duplex DNA or in


isolated nucleoside, decomposes to regenerate an episulfonium ion derived alkylating agent. Thus, leinamycin may react reversibly with DNA to locate thermodynamically favored alkylating sites on the double helix.

34. Kinetics of Carboplatin-DNA Binding in Genomic DNA and Bladder Cancer Cells as Determined by Accelerator Mass Spectrometry. Sang Soo Hah. Biosciences Directorate, Lawrence Livermore National Laboratory, 7000 East Avenue, L-441, Livermore, California 94551. Paul T. Henderson. Biosciences Directorate, Lawrence Livermore National Laboratory, Mail Code L-441, 7000 East Avenue, Livermore, California 94550-9698. We report the use of accelerator mass spectrometry (AMS) to measure both the kinetics of [14C]carboplatin-DNA adduct formation with genomic DNA and drug uptake and DNA binding in T24 human bladder cancer cells. The strength of this approach is that only carboplatin-DNA monoadducts contained radiocarbon in the adducted DNA, thus allowing for the calculation of kinetic rates and concentrations within the system. Using this direct measurement, the percent of radioactivity bound to DNA in the form of monoadducts was calculated as a function of time using salmon sperm DNA. Knowledge of both the starting concentration of the parent carboplatin and the concentration of radiocarbon in the DNA at a variety of time points allowed calculation of the rates of Pt-DNA monoadduct formation and conversion to toxic cross-links. Importantly, the rate of carboplatin-DNA monoadduct formation was observed to be approximately 100-fold slower than that reported for the more potent analogue cisplatin, which may explain the lower toxicity of the compound. T24 human bladder cancer cells were incubated with a subpharmacological dose of [14C]carboplatin, and the rate of accumulation of radiocarbon was measured by AMS in the cells and in nuclear DNA. The lowest concentration of radiocarbon measured in the DNA was approximately 1 attomole per 10 micrograms of DNA. This sensitivity may allow the method to be useful for clinical applications. Work was performed at the Research Resource for Biomedical AMS, operated at UC LLNL under the auspices of the U.S. DOE contract #W-7405-ENG-48 and partially supported by NIH/NCRR, Biomedical Technology Program grant #P41 RR13461 and DOE/LDRD grant 06-LW-023. 35. Understanding the Biological Activity of S-Deoxy Leinamycin: Thiol Triggered Release of H2S from the 1,2Dithiolan-3-one Heterocycle. Santhosh Sivaramakrishnan and Kent S. Gates. Department of Chemistry, University of Missouris Columbia, 125 Chemistry building, 601 S. College Avenue, Columbia, Missouri 65211. The DNA-damaging natural product leinamycin shows exceedingly potent anticancer activity (IC50 ) 14 nM against HeLa cells). Upon entering the thiol-rich interior of cells, the reaction of leinamycin with biological thiols leads to both DNA alkylation and the generation of reactive oxygen species (O2•-, H2O2, and HO•). All known chemical mechanisms of DNA damage by leinamycin absolutely depend on the S-oxide group highlighted in the Figure shown below. Therefore, it is interesting to note that S-deoxy leinamycin retains substantial biological


activity (IC50 ) 2.1 µM). Here, we show that the reaction of thiols with the 1,2-dithiolan-3-one heterocycle found in S-deoxy leinamycin leads to the generation of reactive oxygen species via a novel mechanism involving the release of hydrogen sulfide. Thus, the 1,2-dithiolan-3-one heterocycle may provide a means for the delivery of the known cell killing agent H2S to the interior of cells.

36. DNA-Protein Cross-Links Induced by Bis-Functional Electrophiles. Elisabeth M. Loecken. Department of Biochemistry, Vanderbilt University, Nashville, Tennessee 37235. Amy Joan L. Ham. Department of Biochemistry, Mass Spectrometry Research Center, Vanderbilt University, 9114C Medical Research Building III, 465 21st Avenue South, Nashville, Tennessee 37232-8575. D. C. Liebler. Mass Spectrometry Research Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232. F. Peter Guengerich. Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University, 638 Robinson Research Bldg, 23rd and Pierce Avenues, Nashville, Tennessee 37232. Mass spectral approaches have been used to screen human fibroblast nuclei for proteins with nucleophilic sites that may undergo reactions with bis-functional electrophiles to form DNA-protein cross-links. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was identified as a potential candidate for cross-linking to DNA through an active site cysteine residue (Cys246). Electrophoretic mobility shift assays confirm the formation of cross-links between GAPDH and oligonucleotides in the presence of ethylene dibromide, butadiene diepoxide, and dibromomethane. Active site cysteine (Cys 246) adducts were observed in the mass spectral analysis of ethylene dibromide and butadiene diepoxide-treated GAPDH. Proteomic screening is currently underway with human liver nuclei and bacterial cells to identify other proteins that cross-link to DNA in the presence of bis-functional electrophiles. Supported in part by USPHS R01 ES10546, T32 ES07028. 37. UVA-Induced Oxidation of 6-Thioguanine in Mammalian Cells. Chunang Gu. Environmental Toxicology Graduate Program, University of California at Riverside, Mail Drop 027, Riverside, California 92521-0403. Yinsheng Wang. Department of Chemistry, University of California at Riverside, Mail Drop 027, Riverside, California 92521-0403. Azathioprine (Aza), a widely used anti-cancer drug and immunosuppressive agent, could lead to the incorporation of 6-thioguanine (6-TG) into human DNA upon metabolic activation. However, an increased UVA photosensitivity was observed for aza-treated patients. Recently. it was found that UVA irradiation of 6-TG-bearing DNA could generate reactive oxygen species, further resulting in mutagenic oxidation products. Here. we found that the major UVA photooxidation product of 6-TG is guanine-6-sulfinic acid (Gua-6-SO2H). In addition, Gua-6SO2H could be converted to guanine-6-sulfonic acid (Gua-6SO3H) when exposed to air. Interestingly, the rate of this conversion is much slower in oligodeoxyribonucleotides than in mononucleoside. Further studies are being carried out to assess the formation of the two oxidation products of 6-TG in cultured human cells in ViVo and to examine the mutagenic

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properties of Gua-6-SO2H. We believe that the results offer a better understanding of the dramatic increase of skin cancer among patients treated with aza. 38. Amiodarone-Induced Cytotoxicity: Link to Mitochondrial Dysfunction and Labile Iron Involvement. Adrian C. Nicolescu1, Bruce C. Hill2, James F. Brien1, William J. Racz1, and Thomas E. Massey1. (1) Department of Pharmacology & Toxicology, Queen’s University, Kingston, ON K7L 3N6, Canada. (2) Department of Biochemistry, Queen’s University, Kingston, ON K7L 3N6, Canada, Kingston, ON K7L 3N6, Canada. Amiodarone (AM) is an effective drug for the treatment of life-threatening cardiac dysrhythmias. However, AM can produce serious hepatic and pulmonary toxicities. Several hypotheses have been proposed for the mechanism of AM-induced cytotoxicity, including altered inflammatory mediator release, mitochondrial dysfunction, and free radical formation. We investigated the toxic susceptibility and mitochondrial activity of a human peripheral lung epithelial cell line (HPL1A) exposed to AM. The effects of AM on mitochondrial free radical reducing activity in isolated mitochondria and on the free Fe2+induced free radical production were analyzed by electron paramagnetic resonance spectroscopy using the spin-trapping probes TEMPO and DMPO. AM cytotoxicity in HPL1A cells was concentration- and time-dependent. The free radical reducing activity of HPL1A cells was decreased 2-fold following exposure to low AM concentrations (5-10 µM). The cytoprotective agents, PBN and Trolox C, had a minimal effect on AMinduced cytotoxicity. Respiring mitochondria treated with AM had decreased free radical reducing activity. Additionally, in the presence of ethanol (a hydrogen atom donor), AM increased Fe2+-induced free radical production 2-fold compared to that of control. These data demonstrate that AM targets mitochondrial complex I and that AM can amplify free radical formation. Both of these processes may be involved in the multifaceted mechanism of AM-induced cytotoxicity. Supported by CIHR grant MOP-13257. 39. In Vitro Evidence for Mechanism Based Inactivation of P450 2E1 by 1,2-Epoxy-3-butene. Gunnar Boysen1, Nadia I. Georgieva1, Cameron O. Scarlett2, Brenda Temple3, Christoph H. Borchers2, and James Swenberg4. (1) Environmental Sciences and Engineering, University of North Carolina at Chapel Hill, CB# 7431, 180 Rosenau Hall, Chapel Hill, North Carolina 27599. (2) Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599. (3) R. L. Juliano Structural Bioinformatics Core, University of North Carolina at Chapel Hill, CB #7260, Chapel Hill, North Carolina 27599. (4) Laboratory of Molecular Carcinogenesis and Mutagenesis, University of North Carolina, CB# 7432, Room 253c Rosenau Hall, Chapel Hill, North Carolina 27599. 1,3-Butadiene (BD) is metabolized by cytochrome P450 2E1 to several epoxides that are considered toxic and carcinogenic. The first step of BD metabolism is oxidation to 1,2-epoxy-3butene (EB). It has been shown that P450s can be inactivated by the covalent binding of reactive metabolites to protein or heme. In this study, it was shown that EB binds to four histidine and two tyrosine residues in human P450 2E1. Protein modeling revealed that five of these modifications are at residues, which are considered crucial for enzyme activity. Further, it is shown that preincubation of P450 2E1 overexpressing microsomes with EB at a 1:1 ratio for 30 min reduces its activity for p-nitrophenol hydroxylation by 60%. These data support the hypothesis that EB can inactivate P450 2E1 by covalent modifications and thus add an additional regulatory factor of BD metabolism.


40. Toxicological and Structural Features of KIAA1363: A Novel Detoxifying Enzyme for Organophosphorus Nerve Poisons. Daniel K. Nomura1, Kathleen A. Durkin2, Kyle P. Chiang3, Gary B. Quistad4, Benjamin F. Cravatt3, and John E. Casida4. (1) Nutritional Sciences and Toxicology, Molecular Toxicology Graduate Group, University of California, Berkeley, Environmental Chemistry and Toxicology Laboratory, Berkeley, California 94720-3112. (2) Molecular Graphics Facility, College of Chemistry, University of California, Berkeley, Berkeley, California 94720-1460. (3) The Skaggs Institute for Chemical Biology and Departments of Chemistry and Cell Biology, The Scripps Research Institute, La Jolla, California 92037-1000. (4) Department of Environmental Sciences, Policy and Management, University of California, Berkeley, Environmental Chemistry and Toxicology Laboratory, Berkeley, California 947203112. Most organophosphorus (OP) toxicants such as the insecticide metabolite chlorpyrifos oxon (CPO) act by inhibiting acetylcholinesterase (AChE). The primary CPO-detoxifying enzyme is identified as KIAA1363 in mouse brain, spinal cord, kidney, heart, lung, testes, and muscle but not liver. KIAA1363 protects AChE and monoacylglycerol lipase from CPO inhibition. KIAA1363 and AChE are similar in sensitivity to seven OP insecticides and analogues, prompting structural comparisons of their active sites relative to OP potency and selectivity. Homology modeling places KIAA1363 in the hormone-sensitive lipase family with a catalytic triad of S191-D348-H378. OP structure optimization and CPO docking suggest that KIAA1363 has a much larger binding pocket than AChE, allowing excellent selectivity for KIAA1363 with long chain alkyl phosphates and phosphonates. KIAA1363 reactivates much faster than AChE, possibly because KIAA1363 lacks a mobile catalytic His, which is considered to be important in preventing AChE dephosphorylation. 41. Strategies to Reduce Drug Candidate Failure due to Off-Target Activities. Bruce D. Car. Discovery Toxicology Department, Bristol-Myers Squibb Co, Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey 085434000. To optimize safety profiling, a 14-year retrospective analysis of drug attrition due to toxicity was undertaken. Over 80% of liabilities were detected in toxicology studies 2 weeks in duration. Such studies complemented by screens for target selectivity and counterscreens for known liabilities would identify nearly all toxicity. Leading off-target causes of attrition included cardiovascular (34%), hepatic (13%), C/PNS (10%), and hematologic (10%) causes. Approximately 30% of these resulted from biotransformation-mediated rather than direct effects. Most cardiovascular liabilities resulted from drug and metabolite interactions with single or multiple cardiac ionchannels or GPCRs, including hERG, L/T-Ca++, Na+, 5HT2A, and others. Particular attention is given to avoiding the inappropriate termination of molecules or chemotypes based on isolated in Vitro results, with considerations of in ViVo corroborative findings, seriousness or reversibility of liabilities, and the risk-benefit of the drug indication all weighing into the consideration of a molecule’s advancement. 42. Structural Basis of Drug Binding to hERG K+ Channels. Michael C. Sanguinetti. Department of Physiology and Nora Eccles Harrison Cardiovascular Research & Training Institute, University of Utah, Salt Lake City, Utah 84112. Block of hERG K+ channels by a surprisingly large number of drugs prolongs the QT interval of the body surface electrocardiogram and increases the risk of cardiac arrhythmia. This


side effect is a major hurdle to the development of new drugs. The molecular determinants of hERG channel block have been defined using a site-directed mutagenesis approach. hERG channels are formed by the coassembly of 4 subunits, each containing 6 alpha-helical transmembrane domains. Two aromatic residues, Tyr652 and Phe656, located in the S6 domain of hERG and two polar residues, Thr623 and Ser624, are critical for interaction with structurally diverse drugs. By contrast, hERG channel agonists bind to specific residues located between the S5 and S6 domains. Blocker potency is well correlated with the hydrophobicity of Phe656 and an aromatic side group at position 652, suggesting the importance of a cation-pi interaction between Tyr652 and a basic N of the drug. 43. Properties of the hERG Potassium Channel That Promote Promiscuous Binding of Small Organic Molecules. Robert Pearlstein. Novartis Institutes for BioMedical Research, 250 Mass Avenue, Cambridge, Massachusetts 02139. Ramy Farid, Schro¨dinger, Inc., 120 W. 45th Street, 29th Floor, New York, New York 10036. Tyler Day. Schrodinger, Tower 45, 120 West 45th Street, New York, New York 10036-4041. Richard Friesner. Department of Chemistry, Columbia University, 116th and Broadway, New York, New York 10027. The hERG potassium channel is a key cardiac ion channel that terminates the plateau phase of ventricular repolarization. The inactivated form of hERG promiscuously binds small organic compounds in the ion conduction pathway, leading to adverse effects. In the absence of cocrystal structures, overall binding mode(s), bound conformation(s), and interactions have yet to be determined and the basis for promiscuity explained. We created a homology model of the homo-tetrameric pore domain of hERG and docked a set of known blockers. Our calculations suggest that the symmetry of the pore domain and the multiplicity of key side chains identified from mutagenesis are responsible for the propensity of the cavity to bind compounds containing aromatic and basic groups. Predicted binding geometries and interactions will be described for representative blockers, together with general insights learned from the analysis of our homology model. 44. The hERG Channel: Tuned In? Chemical Strategies for Dialing Out an Off-Target Source of Candidate Failure. Brian Springhthorpe. Department of Chemistry, AstraZeneca R&D Charnwood, Bakewell Road, Loughborough, LE11 5RH, England. The rapid component of the delayed rectifier potassium current (IKr) is carried by channels encoded by the human ethera-go-go-related gene (hERG) and plays a key role in cardiac repolaristion. Pharmacological inhibition of this channel can lead to prolongation of the QT interval, which is associated with a potentially fatal arrhythmia called Torsades de Pointes (TdP). Drug-induced TdP has led to the withdrawal of a number of drugs and has resulted in regulatory guidelines aimed at ensuring that the clinical risk of a compound causing QT interval prolongation is assessed. This presentation will briefly introduce the terms hERG, QT, and TdP and describe chemical approaches that have been shown to reduce activity at the hERG channel, including correlations with physicochemical parameters. The presentation will also briefly discuss the overall risk assessment of pre-clinical compounds and recommend a safety margin that may de-risk the possibility of QT interval prolongation in man. 45. Mutagenic Nucleotide Incorporation and Hindered Translocation by a Food Carcinogen C8-dG Adduct in Sulfolobus solfataricus P2 DNA Polymerase IV (Dpo4): Modeling and Dynamics Studies. Ling Zhang1, Olga Rechkoblit2, Lihua Wang3, Dinshaw J. Patel2, Robert Shapiro1, and


Suse Broyde3. (1) Department of Chemistry, New York University, 100 Washington Square East, New York, New York 10003. (2) Cellular Biochemistry & Biophysics Program, Memorial Sloan-Kattering Cancer Center, 1275 York Avenue, New York, New York 10021. (3) Department of Biology, New York University, 100 Washington Square East, 1009 Silver Center, New York, New York 10003. Bulky carcinogen-DNA adducts commonly cause replicative polymerases to stall, leading to a switch to bypass polymerases. We have investigated nucleotide incorporation opposite the major adduct of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in the DinB family polymerase, Dpo4, using molecular modeling and molecular dynamics simulations. PhIP, the most prevalent heterocyclic aromatic amine formed by cooking of proteinaceous food, is mutagenic in mammalian cells and is implicated in mammary and colon tumors. Our results show that the dG-C8-PhIP adduct can be accommodated in the spacious major groove Dpo4 open pocket, with Dpo4 capable of incorporating dCTP, dTTP, or dATP opposite the adduct reasonably well. However, the PhIP ring system on the minor groove side would seriously disturb the active site, regardless of the presence and identity of dNTP in the active site. Furthermore, the simulations indicate that dATP and dTTP are better incorporated in the damaged system than in their respective mismatched but unmodified controls, suggesting that the PhIP-adduct enhances incorporation of these mismatches. Finally, bulky dG-C8 adducts, situated in the major groove, are likely to impede translocation in this polymerase (Rechkoblit et al. (2006), PLoS Biol., 4, e11). However, N2-dG adducts, which can reside on the minor groove side, appear to cause less hindrance. Supported by NIH CA75449 (to S.B.), CA46533 (to D.J.P.), and Ruth L. Kirschstein National Research Service Award F32 GM069152 (to O.R.). 46. Structural and Thermodynamic Studies of 4-Hydroxyequilenin-Derived Stereoisomeric Adducts to dC, dA, and dG in Duplex DNA. Shuang Ding1, Robert Shapiro1, Nicholas E. Geacintov1, and Suse Broyde2. (1) Department of Chemistry, New York University, 100 Washington Square East, Room 1001, New York, New York 10003. (2) Department of Biology, New York University, 100 Washington Square East, New York, New York 10003. Excessive exposure to estrogen through hormone replacement therapy (HRT) is linked to an increased risk of developing breast and endometrial cancer. Equilin and equilenin, the major components of the HRT drug Premarin, can be metabolized to the catechol 4-hydroxyequilenin (4-OHEN). The quinoids produced by 4-OHEN oxidation react with dC, dA, and dG to form unusual stable cyclic bulky adducts, with four stereoisomers identified for each base adduct. These adducts have been found in Vitro and in human breast tumor tissue. Experimental evidence from in Vitro primer extension studies has shown that the 4-OHEN-dC and 4-OHEN-dA adducts can be bypassed with infidelity in several error-prone bypass polymerases. The results varied with adduct stereochemistry. We have carried out molecular modeling and molecular dynamics simulations to investigate the structures and thermodynamics of the stereoisomeric dC, dA, and dG 4-OHEN adducts in DNA duplexes. These adducts are rigid lesions with obstructed Watson-Crick edges and near perpendicular ring systems. Our results show that the 4-OHEN-A and C adducts have similar ring systems and structural properties and can reside in the DNA major groove with a syn damaged base, or in the minor groove with an anti damaged base. However, the G adducts, whose ring systems differ, are positioned in the major groove for both syn

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and anti conformations. The adduct stereochemistry governs the specific orientation of each stereoisomer, and the stereoisomeric lesions distort the DNA duplexes differentially. These differences are likely to underlie the differential repair susceptibilities and mutagenic properties of the adducts. Supported by CA75449 (to S.B.) and CA112412 (to N.E.G.). 47. Characterization of a Novel 8-Oxo-guanine Derivative of 4-Hydroxyestradiol-1-N7-guanine. Leslie Machado, Alissa Rennie, Marlin D. Friesen, and John D. Groopman. Department of Environmental Health Sciences, Johns Hopkins University, 615 North Wolfe Street, Baltimore, Maryland 21205. Estrogen-DNA adduct formation is a putative contributor to the carcinogenic process meditated by the estrogens. There are numerous literature reports establishing the formation of a 4-hydroxyestradiol-1- N7-guanine adduct that is a consequence of depurination from DNA. In the course of studying the chemistry of this adduct by mass spectrometry, a novel MH+ ion was detected. This MH+ ion was 16 mass units larger than the 4-hydroxyestradiol-1- N7-guanine adduct. This new adduct could be quantitatively formed under ambient temperatures and neutral pH. Further MS/MS analysis revealed a 168 m/z fragment that is identical to 8-oxo-guanine. NMR data (400 MHz) confirm the structure of the 4-hydroxyestradiol-1- N7guanine adduct, and preliminary NMR studies support this new oxidation product. We are continuing to explore the formation and dosimetry of this new adduct to determine its potential biological relevance and toxicology. 48. Induced CD/19F NMR Characteristics as Novel Markers for Arylamine-Induced Conformational Heterogeneities. Fengting Liang, Nidhi Jain, Srinivasa Rao Meneni, and Bongsup Cho. Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, Kingston, Rhode Island 02881. The environmental carcinogen 2-nitrofluorene produces C8-dG-AF as the most persistent DNA adduct. This N-deacetylated lesion is known to exist in three prototype conformers depending on sequence context: B-type (B), stacked (S), and wedged (W). Here, we report novel ICD/19F NMR characteristics for two sets of model duplexes in which the base opposite (-CG*C-)(-GXG-) or 3′-flanking (-AG*X-)(-XAT-) the lesion is varied systematically (G* ) fluorinated AF adduct, X ) G, A, C, T). The W-conformer, which occurs exclusively in mispaired duplexes, is characterized by a strongly positive ICD290-360nm. The “wedged” AF-moiety between the walls of the minor groove is responsible for unusual duplex stabilities observed for the W-conformation. The nature of the 3′-flanking base to the lesion contributes significantly to the duplex stability of the W-conformer. Temperature-dependent ICD290-360nm is sequence-dependent and can be used as a simple marker for probing the AF-induced B-S-W conformational heterogeneities. Supported by NIH grants R01CA98296 and #P20 RR016457. 49. Probing the Aminofluorene-Induced Conformational Heterogeneity by 2-Aminopurine Fluorescence Studies. Nidhi Jain1, Yana Reshetnyak2, and Bongsup Cho1. (1) Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, 41 Lower College Road, Kingston, Rhode Island 02881. (2) Department of Physics, University of Rhode Island, Kingston, Rhode Island 02881. Aminofluorene-modified DNA duplexes exist in equilibrium between stacked (S) and B-type (B) conformers. In this study, the fluorescence probe 2-aminopurine (P) was incorporated into either the modified [(-TPG*NC-)(-GNCTA-)] or the complementary [(-TTG*NC-)(-GNCPA-)] strand of 12-mer duplexes, in which the base 3′-flanking the lesion is varied (G* ) adduct,


N ) G,A,C,T). The fluorescence emission results were analyzed in terms of the S/B population data obtained from 19F NMR in order to gain insight into the local base stacking interactions at the lesion site. Upon modification, the mostly S-conformeric duplexes (-TPG*NC-, N ) G,A) exhibited a large red shift (∼15 nm) along with a significant decrease in fluorescence emission. The P on the modified strand was found to experience a greater stacking than that on the opposite strand. The relative stacking or unstacking of P in the duplex can be attributed to the sequence-dependent AF-induced S/B conformational heterogeneity. Supported by NIH grants R01CA98296 and #P20 RR016457. 50. Base Sequence Effects on the Structural and Thermodynamic Properties of the 10S(+)-trans-anti-Benzo[a]pyrene diol epoxide-N2-dG Adduct. Yuqin Cai1, Nicholas E. Geacintov1, and Suse Broyde2. (1) Department of Chemistry, New York University, 100 Washington Square East, 1001, New York, New York 10003. (2) Department of Biology, New York University, 100 Washington Square East, New York, New York 10003. The influence of local base sequence effects on the structural and thermodynamic properties of a bulky 10S (+)-trans-antiB[a]PDE-N2-dG lesion in double stranded DNA has been investigated. The adduct is flanked either by cytidines (CG*C) or by thymidines (TG*T) in otherwise identical 11-mer duplexes. We employed molecular modeling, molecular dynamics simulations, and free energy calculations to elucidate possible structural differences. We find that the adduct is in the B-DNA minor groove, 5′-directed along the modified strand, in both cases. Base-displaced intercalated conformations are of higher energy. TG*T is locally more flexible. One hydrogen bond on the 5′-side of the lesion is disrupted 26% of the time, while in CG*C, the disruption occurs less than 3% of the time. This agrees with the experimental NMR observation that there is a loss of hydrogen bonding in the 5′-side neighboring base pairs in the TG*T sequence context (Xu et al., (1998) Biochemistry 37, 769-778). This produces greater flexibility in the timedependence of measured geometric properties, including minor groove dimensions, carcinogen-base orientation, Watson-Crick hydrogen bond distances and angles, solvent accessible surface area of the B[a]P moiety, and DNA duplex bend angle. These results are in line with nucleotide excision repair results, showing that the efficiency of incision by UvrABC proteins of the B[a]PDE modified oligonucleotide duplexes is higher by a factor of ∼1.5-2 at 37 °C in the TG*T sequence context than that in CG*C (Ruan et al., (2006), unpublished work). Supported by NIH grants CA28038 (to S.B.) and CA 099194 (to N.E.G.). 51. Conformation of a 14S(-)-trans-anti-Dibenzo[a,l]pyrene diol epoxide-dG Adduct in an 11-mer DNA Duplex Investigated by NMR Methods. Fabian A. Rodriguez1, Yijin Tang1, Jane M. Sayer2, Donald M. Jerina2, and Nicholas E. Geacintov1. (1) Department of Chemistry, New York University, 31 Washington Place, Brown Building, Room 453, New York, New York 10003. (2) NIDDK, NIH, Laboratory of Bioorganic Chemistry, Bethesda, Maryland 20892. The polycyclic aromatic hydrocarbon (PAH), dibenzo[a,l]pyrene (dB[a,l]P), is one of the most potent PAH carcinogens known in rodent model systems. Like other PAHs, it is metabolized in ViVo to fjord region dB[a,l]P 11,12-diol 13,14epoxides (DE) that bind to N2-dG and N6-dA in DNA. We studied the solution structure by NMR of a 14S(-)-trans-antidB[a,l]PDE-dG (G*) adduct opposite dC in an 11-mer duplex in a 5′-(...CG*C...)‚(...GCG...) sequence context. We found conformational heterogeneity and disruption of base-pairing at


G*-C and one to two 5′-flanking base pairs, depending on temperature, suggesting that the polycyclic aromatic DB[a,l]P residue is inserted into the helix 5′ to the modified nucleoside. Removal of the C opposite G* in the complementary strand (5′-(...CG*C...)‚(...G-G...) deletion duplex), changes the conformation to an intercalated one, as shown by two sharp, upfield shifted imino proton resonances. The susceptibilities of these two duplexes with different conformations to incision by nucleotide excision repair enzymes is being investigated to determine relationships between adduct structure and incision efficiency. Research was supported by NIH grant CA 099194. 52. LC/MS/MS Analysis of Benzo[a]pyrene DNA Adducts Derived from Diol Epoxide and Cation Radical Pathways. Mona I. Churchwell, Jian Yan, Qingsu Xia, Peter P. Fu, Frederick A. Beland, and Daniel R. Doerge. Division of Biochemical Toxicology, NCTR, 3900 NCTR Road, Jefferson, Arkansas 72079. Benzo[a]pyrene (BP) is metabolically activated by monooxygenation to give stable diol epoxide DNA adducts (BPDEdG) and one-electron oxidation to give unstable depurinating DNA adducts (e.g., BP-6-N7-Gua, BP-6-C8-Gua, and BP-6N1-Ade). The relative role of each pathway in BP-mediated carcinogenesis is controversial, in part because it has not been possible to measure both types of DNA adducts using a common analytical procedure. A modification of our previous LC-ES/ MS/MS procedure to quantify BPDE-dG (F.A. Beland et al., (2005) Chem. Res. Toxicol. 18, 1306) made it also possible to quantify BP-6-N7-Gua, BP-6-C8-Gua, and BP-6-N1-Ade at a level of approximately one adduct per 10(8) nucleotides when 100 µg of DNA samples were enzymatically hydrolyzed and then heated to 100 °C for 15 min. This high sensitivity and specificity for the analysis of DNA adducts from both diol epoxide and depurination pathways may be useful in determining their respective roles in BP-mediated carcinogenesis. 53. Efficiencies of Incorporation of Different Nucleotides Opposite (+)-trans-B[a]P-Gua Lesions Catalyzed by the Thermophilic Y-Family Polymerase, Dpo4. Lida Oum1, Pingna Xu2, Alexander Kolbanovskiy1, J. Krzeminiski3, S. Amin3, Suse Broyde2, and N. E. Geacintov1. (1) Department of Chemistry, New York University, 29 Washington Place, New York, New York 10003. (2) Department of Biology, New York University, 100 Washington Square East, New York, New York 10003. (3) Department of Pharmacology, Penn State College of Medicine, Hershey, Pennsylvania 17033. Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) is a thermostable archael enzyme and a member of the error-prone and lesions-bypass Y-family. Six different 43-mer oligonucleotide templates were synthesized, which were identical except for the two bases, X and Y, flanking a (+)-trans-antiB[a]P-N2-Gua adduct (G*). The 5′-flanking base X plays a role in lesion bypass by a mechanism involving slipped frameshift intermediates, whereas the 3′-flanking base Y affects the overall efficiencies of nucleotide insertion and extension. The insertion efficiencies of different single nucleotides depend on the temperature of the reactions, and the sequences of the fully extended primer strands are being analyzed to determine if these single nucleotide insertion experiments are reflected in the actual insertion of nucleotides when all four dNTPs are present. Computer modeling studies of ternary Dpo4 complexes show that translesion bypass can occur readily in the spacious active site of Dpo4 with G* in an anti or a syn glycosidic angle conformation. 54. Different Translesion Bypass of Guanine-N2 Monoadducts of Mitomycin C and Guanine-N7 Monoadducts of 2,7Diaminomitosene by DNA Polymerase eta. Cristina Clement.


Chemistry Department, Lehman College, City University of New York, 250 Bedford Park BLVD West, Bronx, New York City, New York 10468. Maria Tomasz. Chemistry Department, Hunter College, City University of New York, 695 Park Avenue, New York City, New York 10021. We were interested to probe the efficiency of translesion synthesis past two monoadducts of mitomycin C (MC), the guanine (G)-N2 DNA monoadduct of mitomycin C (MC) and the G-N7 DNA monoadduct of 2,7-diaminomitosene, which is a non-cytotoxic derivative of MC. 24-mer DNA templates, adducted at a single guanine either with MC or 2,7-DAM, were synthesized and submitted to extension of primers by errorprone DNA polymerase eta. In the 24-mer template/15-mer primer system, the G-N7-2,7-DAM adduct was bypassed by DNA polymerase eta, resulting in the production of a fully extended primer. However, the extension was at a slower rate compared with that of the control, non-alkylated template. In analogous studies of other bulky adducts, polymerase eta showed similarly low efficiencies of primer extension under such conditions (Wang, L. et al. (2005) Chem. Res. Toxicol. 18(3), 441-456). However, using a lower (2:1) or 3:1 substrate/ enzyme (S/E) ratio, we observed substantially more efficient translesion synthesis (TLS) and the extension to 23 nt length of the primer. In sharp contrast, the G-N2-MC monoadduct was not bypassed by DNA polymerase eta, and a 19-mer abortive product (1 nucleotide before lesion) accumulated at the same rate as the 24-mer product from the control template. This paper shows that DNA polymerase eta performs translesion synthesis of another bulky adduct, N7-dG-2,7-DAM, with less efficiency than T7 exo, and Klenow exo-DNA polymerases (Chem. Res. Toxicol. (2005), 18(2), 213-223). This new data on the error-prone TLS of this bulky adduct by DNA polymerase eta raises new questions about the relationship between the structure of the bulky adducts and the efficiency with which they are bypassed with fidelity by error-prone DNA polymerases. 55. DNA Adducts Derived from Carcinogenic Aristolochic Acids. Radha R. Bonala. Department of Pharmacological Sciences, State University of New York, Stony Brook, New York 11794. Sivaprasad Attaluri. Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794. Francis Johnson. Department of Pharmacological Sciences, SUNY-Stony Brook, Stony Brook, New York 11794-8651.

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We have developed new methods for the synthesis of the lactams (IIa and IIb) of the naturally occurring but deadly nephrotoxic and carcinogenic aristolochic acids (Ia and Ib). The conversion of the lactams to the intermediates suitable for conversion to the known DNA adducts (IIIa, IIIb, IVa, and IVb) will be described. Attempts to introduce these adducts synthetically into oligomeric DNA will also be discussed. 56. Detection of Multiple DNA Adducts in Human Lung and Esophagus Tissue by Liquid Chromatography Electrospray Ionization Tandem Mass Spectrometry (LC/ESI-MS/ MS). Robert A. Kanaly1, Saburo Matsui1, Tomoyuki Hanaoka2, and Tomonari Matsuda1. (1) Department of Technology and Ecology, Kyoto University, Kyoto, 606-8501, Japan. (2) Epidemiology and Prevention Division, National Cancer Center Research Institute, Tokyo, 104-0045, Japan. The development of strategies designed to detect inter-tissue variations in susceptibility to DNA damage may advance our understanding of the role of DNA adducts in cancer etiology and as biomarkers to exposure. An LC/ESI-MS/MS DNA adduct detection method designed to detect the neutral loss of 2′deoxyribose from positively ionized 2′-deoxynucleoside adducts transmitting the [M + H]+ > [M + H - 116]+ transition over a total of 374 transitions was applied to analyze esophageal and peripherally and centrally located lung tissue DNA taken from the same individual. The final analysis indicated that the largest adducts were distributed similarly across the samples (84% to 90% similarity; n ) 50) but also revealed that there were large differences in the relative amounts of some adducts detected in both lung and esophagus tissue DNA. Adduct identity confirmation by comparison with authentic adduct standards was also performed where possible. The potential of the method is discussed. 57. Novel Exocyclic dA Adducts of 1,2,3,4-Diepoxybutane. Sergey Antsypovich, Rebecca Guza, Danae Quirk Dorr, Crystal Pitts, Brock Matter, and Natalia Tretyakova. Department of Medicinal Chemistry and the Cancer Center, University of Minnesota, 425 East River Road, 790 CCRB, Minneapolis, Minnesota 55455. 1,2,3,4-Diepoxybutane (DEB) is the suspected ultimate carcinogenic metabolite of 1,3-butadiene, which is used in the manufacturing of styrene-butadiene rubber and is also present in automobile emissions and in cigarette smoke. DEB is a direct mutagen capable of inducing large deletions and point mutations. In the current study, the formation of novel exocyclic deoxyadenosine-DEB adducts was investigated. Compound 1 was prepared by coupling 6-chloropurine deoxyriboside with 1-amino3,4-epoxybutan-2-ol. The latter was generated in situ from N-Fmoc-1-amino-3,4-epoxybutan-2-ol, which was obtained by epoxidation of N-Fmoc-1-amino-3-buten-2-ol. Compound 2 was obtained as a side product upon the synthesis of compound 1, and compound 3 was observed following the incubation of compound 1 in water at room temperature at neutral pH. The novel deoxyadenosine lesions were isolated by reverse-phase HPLC. The structures of nucleoside adducts were determined by UV spectroscopy, capillary HPLC-ESI+-MS/MS, 1H, COSY, NOESY, HMQC, and HMBC NMR spectroscopy and assigned as N6-(2-hydroxy-3,4-epoxybutan-1-yl)-2′-deoxyadenosine (1), N6,N6-(2,3-dihydroxybutan-1,4-diyl)-2′-deoxyadenosine (2), and 1,N6-(2,3-dihydroxybutan-1,4-diyl)-2′-deoxyadenosine (3). HPLC-ESI-MS/MS studies indicate that these lesions are also present in DEB-treated calf thymus DNA. Site specifically modified oligodeoxynucleotides containing 2 and 3 are being prepared to investigate their potential role in the mutagenesis of DEB.


58. Structural Characterization of DNA Duplexes Having Exocyclic 2′-Deoxyguanosine Adducts. Carlos de los Santos, Tanya Zaliznyak, Radha R. Bonala, Francis Johnson, and Mark Lukin. Department of Pharmacological Sciences, State University of New York, Stony Brook, New York 11794. Exocyclic adducts are mutagenic DNA lesions that have been associated with the processes of aging and carcinogenesis. Bifunctional alkylating agents, such as vinyl chloride and acrolein, react with deoxyguanosine producing 1,N2-etheno-dG (1,N2-edG) and isomeric R-OH-1,N2-propano-dG (R-OH-PdG) and γ-OH-1,N2-propano-dG (γ-OH-PdG) adducts, respectively. Lipid peroxidation products also form these lesions. In spite of their chemical similarity, 1,N2-edG, R-OH-PdG and γ-OH-PdG have different mutagenic properties and different repair mechanisms. We have prepared several DNA duplexes containing these lesions opposite dC and dA residues and determined their structure in solution using high-resolution NMR spectroscopy. Our studies show that these lesions are readily accommodated in right-handed duplexes, where they cause only local structural perturbations. In spite of this similarity, stabilization of the adduct-containing base pairs is remarkably different for each lesion. We will discuss the implications of these structures for processes of mutagenesis and DNA repair.

59. Synthesis and Detection of the 2,6-Diamino-4-hydroxyN5-(2-oxoethyl)-formamidopyrimidine (FAPy) Adduct. Plamen P. Christov, Ivan D. Kozekov, Thomas M. Harris, and Carmelo J. Rizzo. Department of Chemistry, Center in Molecular Toxicology and Vanderbilt Institute of Chemical Biology, Vanderbilt University, P.O. Box 1822, Nashville, Tennessee 37235.

Vinyl chloride is an important industrial monomer and an established human carcinogen. The carcinogenic species is chlorooxirane, which arises from the in ViVo oxidation of vinyl chloride. The major adduct from the reaction of chlorooxirane with deoxyguanosine is the cationic N7-(2-oxoethyl) adduct; however, this species has been shown to be non-mutagenic. We hypothesize that the cationic adduct hydrolyzes to form the 2,6-


diamino-4-hydroxy-N5-(2-oxoethyl)-formamidopyrimidine (FAPy) adduct, which contributes to the carcinogenicity of vinyl chloride and related species. We have identified this FAPy adduct from the reaction of acetoxyoxirane with deoxyguanosine and DNA by a combination of chemical synthesis, HPLC, and mass spectral analysis. 60. Formation of an Intrachain Cross-Link by the Minor Deoxyguanosine Adduct of Acrolein. Ivan D. Kozekov, Constance M. Harris, Thomas M. Harris, and Carmelo J. Rizzo. Department of Chemistry, Center in Molecular Toxicology and Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, Tennessee 37235-1822. An ssDNA containing 6-hydroxy-5,6,7,8-tetrahydro[1,2-a]pyrimidopurin-10-one (1) in a 5′-GGG context was found to form an intrachain cross-link with the exocyclic amino group of the Gua in the same strand. A cross-linked species formed slowly and reversibly, reaching a level of 50% after 24 h in single strand DNA and about 35% in the duplex. LC-MS analysis of the enzymatic digestion of the cross-linked DNA showed only one diastereoisomer of bis-nucleoside 2. Reduction of the cross-linked DNA with NaCNBH3 followed by enzymatic digestion gives 3. The structure of 3 was unambiguously established by the synthesis of an authentic sample.

61. Interstrand Cross-Links Generated by Abasic Sites in Duplex DNA. Sanjay Dutta. Department of Chemistry, University of MissourisColumbia, 601 South College Avenue, Columbia, Missouri 65211. Kent S. Gates. Departments of Chemistry and Biochemistry, University of MissourisColumbia, 601 S. College Avenue, Columbia, Missouri 65211. Abasic sites are one of the most common lesions in cellular DNA. They can be generated by a variety of routes, including spontaneous depurination or exposure to DNA-alkylating drugs and mutagens. If left unrepaired, abasic sites are known to block DNA replication and transcription and are classified as cytotoxic lesions. Here, we have used synthetic 32P-labeled 2′-deoxyoligonucleotides to characterize interstrand cross-links generated by an abasic site in duplex DNA.

62. Stereoselective Synthesis of a cis-syn Thymine Dimer Building Block via an N3-Alkyl Auxiliary. Ajay Khstetry, Yimin Wang, and John Stephen Taylor. Department of Chemistry, Washington University in St. Louis, St. Louis, Missouri 63141. Exposure of DNA to UV light induces DNA photoproducts that cause mutations linked to skin cancer. Cyclobutane pyrimidine dimers are the major photoproduct formed in sunlight and have been the subject of many repair and mutagenesis studies. Though a cis-syn thymine dimer phosphoramidite building block was first synthesized about two decades ago, the route is inefficient and laborious to undertake because of a


lack of stereochemical control of the photodimerization reaction and the difficulty in separating the stereoisomers. We have recently developed a new synthetic route for the stereoselective synthesis of cis-syn thymine dimers that is based on our observation that N3 methylated thymidine dinucleotides have much higher preference for cis-syn thymine dimer formation. We then investigated the use of the base removable 2-(pnitrophenylphenyl sulfonyl) ethyl (TSE group) auxiliary for controlling stereoselectivity of DMT-Tp(CE)T photodimerization and found that one phosphodiester diastereomer of the 5′modified dinucleotide led to an almost exclusive formation of the cis-syn stereoisomer. Unfortunately, the TSE group could not be easily removed by standard ammonia deprotection conditions. We then changed the auxiliary to the pivaloyloxymethyl (POM) group, which gave a similar stereoselectivity and could be completely removed during the ammonia deprotection step, thereby affording a short and efficient route to cis-syn thymine dimers.

63. Correlation between Hemoglobin Adducts and DNA Adducts Following Acrylamide and Glycidamide Exposures in Fischer 344 Rats and B6C3F1 Mice. Eden Tareke, Mona I. Churchwell, L. Patrice McDaniel, Nathan C. Twaddle, and Daniel R. Doerge. Division of Biochemical Toxicology, NCTR, 3900 NCTR Road, Jefferson, Arkansas 72079. Acrylamide is a neurotoxic, mutagenic, and carcinogenic industrial chemical and food contaminant. We have assessed the relationships between serum levels of acrylamide and its genotoxic metabolite glycidamide and the levels of hemoglobin and DNA adducts in mice and rats administered single or repeated doses of acrylamide or glycidamide. There were significant linear relationships between the area under the serum time-concentration curve (AUC) for glycidamide and both liver glycidamide DNA adducts and glycidamide hemoglobin adducts in rodents administered either acrylamide or glycidamide (0.1 mg/kg bw). There was no relationship between either the AUC for acylamide or acylamide hemoglobin adducts and liver glycidamide DNA adducts. There was also a significant linear relationship between steady-state levels of glycidamide hemoglobin adducts and liver glycidamide DNA adducts for repeat dosing with acrylamide (1-2 mg/kg bw/d). These results suggest that glycidamide hemoglobin adduct levels can predict glycidamide DNA adduct levels when DNA samples are not available. 64. Lipid Peroxidation-Derived DNA Adducts Dominate the Spectrum of DNA Lesion Chemistry in Tissues from the SJL Mouse Model of Nitric Oxide Over-Production. Bo Pang1, Xinfeng Zhou, Hongbin Yu1, Min Dong1, Steven R. Tannenbaum2, and Peter C. Dedon3. (1) Biological Engineering Division, Massachusetts Institute of Technology, Biological Engineering Division, NE47, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139. (2) Biological Engineering Divi-

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sion and Department of Chemistry, Massachusetts Institute of Technology, Room 56-731A, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139. (3) Biological Engineering Division and Center for Environmental Health Sciences, Massachusetts Institute of Technology, NE47-277, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139. Inflammation is a major cause of disease, and there is growing appreciation for the complex chemistry of reactive species arising at sites of inflammation. These reactions can affect DNA directly or by generating DNA-reactive electrophiles in reactions with proteins, lipids, carbohydrates, and DNA itself. To define the genotoxic chemistry at sites of inflammation, we quantified (LC-MS/MS) a spectrum of DNA lesions arising in tissues from the SJL mouse model of inflammation, in which macrophages are activated to produce large quantities of nitric oxide (NO), nitrosoperoxycarbonate (ONOOCO2-), N2O3, and NO2. We surveyed DNA biomarkers from specific classes of inflammation chemistry: unsubstituted etheno-dA and- dG from lipid peroxidation; deoxynucleosides of xanthine (X), hypoxanthine (I), uracil (U), and oxanine (O) from nitrosative deamination; M1dG from DNA oxidation-derived base propenals; and 8-oxodG. We observed insignificant increases (320 nm. Through hydrogen trapping experiments using physiologically relevant H-atom donors and their derivatives, the stereoselectivity of repair, product distribution upon degradation of the radical of interest, and the rate of strand cleavage have been determined. 66. In Vitro and in ViWo Metabolism of the DNA Base Adduct, M1G. Charles G. Knutson1, Brenda C. Crews1, Donald F. Stec2, Markus Voehler2, and Lawrence J. Marnett3. (1) Department of Biochemistry, Vanderbilt Institute of Chemical Biology, Center in Molecular Toxicology, Vanderbilt University, School of Medicine, 850 Robinson Research Building, Nashville, Tennessee 37232-0146. (2) Department of Chemistry, Vanderbilt Institute of Chemical Biology, Vanderbilt University, 7300 Stevenson Center Drive, Nashville, Tennessee 37235. (3) Departments of Biochemistry and Chemistry, Vanderbilt Institute of Chemical Biology, Center in Molecular Toxicology, Vanderbilt University, School of Medicine, 850A Robinson Research Building, Nashville, Tennessee 37232-0146. Oxidative damage is considered a major contributing factor to genetic diseases including cancer. Our laboratory is evaluating endogenously formed DNA adducts as genomic biomarkers of oxidative injury. Our efforts have focused on factors affecting the detection of adducts in urine. Here, we demonstrate the base adduct, M1G, undergoes oxidative metabolism in Vitro in rat liver cytosol (KM ) 79 µM and Vmax ) 84 pmol/min/mg) and in ViVo when administered intravenously to Sprague Dawley rats. LC-MS/MS analysis revealed two metabolites containing successive additions of 16 mass units. One- and two-dimensional NMR experiments identified oxidation first at the 6-postion of the pyrimidopurinone ring forming 6-oxo-M1G and further oxidation at the 2-position of the imidazole ring, yielding a 2,6dioxo-M1G. In rat liver cytosols, allopurinol inhibited M1G metabolism, suggesting the involvement of xanthine oxidase. Indeed, purified bovine xanthine oxidase metabolized M1G to 6-oxo-M1G and 2,6-dioxo-M1G. 67. Fasicularin-Derived Aziridinium Ion: Theoretical Study of the Formation of the Putative Reactive Nitrogen Species. Papiya Majumdar1, Sanjay Dutta1, Rainer Glaser1, and Kent S. Gates2. (1) Department of Chemistry, University of MissourisColumbia, Columbia, Missouri 65211. (2) Departments of Chemistry and Biochemistry, University of Missouris Columbia, 601 S. College Avenue, Columbia, Missouri 65211.

Fasicularin 1h (R ) n-hexyl), a recently discovered marine alkaloid, has been shown to react with DNA by way of N7deoxyguanosine alkylation involving an aziridinium ion intermediate. The equilibrium structures of the fasicularin model 1m (R ) Me) and the aziridinium ion 9 have been investigated using density functional theory (B3LYP) with the fully polarized basis set 6-311G** including SCRF-PCM modeling of aqueous solvation. It is found that the chair-1m conformer is preferred over the boat-1m by 6.7 kcal/mol, and there is a barrier of 7.4


kcal/mol for the N-inversion chair-1m f boat-1m. An activation barrier of 14.1 kcal/mol was determined for the reaction boat1m f 9 + SCN-. The overall activation energy of 20.7 kcal/ mol provides for a well-balanced reactivity that suggests that fasicularin presents the first natural product with the ability to form a DNA-alkylating aziridinium ion by displacing a thiocyanate leaving group. 68. Bioactivation of the Antitumor Agent Tirapazamine by Thiols and Dithiols. Venkatraman Junnotula1, Delshanee Kotandeniya1, Goutam Chowdhury1, and Kent S. Gates2. (1) Department of Chemistry, University of MissourisColumbia, 601 S. College Avenue, Columbia, Missouri 65211. (2) Departments of Chemistry and Biochemistry, University of MissourisColumbia, 601 S. College Avenue, Columbia, Missouri 65211. Tirapazamine (TPZ) is a bioreductively activated, hypoxiaselective antitumor agent, currently undergoing clinical trials in combination with radiotherapy and cisplatin based chemotherapy. Hypoxia selective cell killing by TPZ relies upon the generation of the oxygen-sensitive intermediate, TPZ. In our studies, we examined the ability of the biological thiol, glutathione (GSH), and dithiothreitol (DTT) to activate TPZ. Our data shows that thiols and dithiols activate TPZ to yield the key oxygen sensitive intermediate, TPZ. The activation of TPZ by thiols is metal mediated. Currently, we are examining the possible cellular role of copper-containing SOD in activation of TPZ by thiols and dithiols.

69. Production of Reactive Oxygen Species by Redox Cycling of 1-Hydroxyphenazine: Toward a Molecular Understanding of the Bacterial Virulence Factor 1-Hydroxyphenazine. Sarmistha Sinha and Kent S. Gates. Department of Chemistry, University of MissourisColumbia, 601 S. College Avenue, Columbia, Missouri 65211.

1-Hydroxyphenazine is a secondary metabolite of P. aeroginosa, which infects the airways of the patients of cystic fibrosis and causes progressive tissue damage. Early studies suggested that 1-hydroxyphenazine is a bacterial virulence factor that plays an important role in such tissue damage. But the mechanisms underlying the biological properties of 1-hydroxyphenazine are


not well studied. We report chemical properties of 1-hydroxyphenazine, which might help to explain its biological activities. We used a DNA-damage assay to show that 1-hydroxyphenazine undergoes one-electron redox activation in the presence of the NADPH/cytochrome P450 reductase system and produces a superoxide radical, which leads to the production of reactive oxygen species (O2•-, H2O2, and HO•). This chemical process may directly damage lung tissue and may also trigger deleterious proinflammatory host immune responses such as the expression of IL-8 and ICAM-1. 70. Design and Synthesis of DNA Minor Groove Methylating Compounds That Target Pancreatic β-Cells. Andrew L. McIver, Andreas Linke, and Sridhar Varadarajan. Department of Chemistry and Biochemistry, University of North Carolina, Wilmington, 601 South College Road, Wilmington, North Carolina 28403. The design of compounds that form cytotoxic, non-mutagenic 3-methyladenine adducts in pancreatic β-cells is being studied in this project for potential applications in the treatment of diseases such as cancer and diabetes. These compounds are composed of three components: (1) a cell-targeting moiety, glucosamine, which targets the insulin producing pancreatic β-cells by way of the GLUT-2 transporters present on these cells; (2) a site-specific DNA methylating agent, Me-Lex, which has been shown to selectively produce cytotoxic, non-mutagenic N3-methyladenine adducts; and (3) a linker component that connects the two other components together. The linker is a critical component because it has to be such that the celltargeting and DNA-methylating properties of the two functional components are maintained. A synthetic route was developed, which enables the easy introduction of various linkers into the molecules. The design features, the synthesis, and the initial studies with these compounds will be described. 71. Disturbances in Purine Metabolism Lead to Substantial Incorporation of Hypoxanthine into DNA and RNA. Bo Pang1, C. Eric Elmquist1, Min Dong1, Richard P. Cunningham2, and Peter C. Dedon3. (1) Biological Engineering Division, Massachusetts Institute of Technology, Biological Engineering Division, NE47, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139. (2) Department of Biological Sciences, State University of New York, 1400 Washington Avenue, Albany, New York 12222. (3) Biological Engineering Division and Center for Environmental Health Sciences, Massachusetts Institute of Technology, NE47-277, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139. Deamination of nucleobases generates xanthine (X), hypoxanthine (I), oxanine (O), and uracil (U) and arises from a range of unrelated mechanisms, including hydrolysis, nitrosation, and deaminases. We present evidence for a fourth mechanism: incorporation of I and X into DNA and RNA as a result of defects in purine metabolism. We approached this problem using E. coli with defects in purine metabolism in conjunction with LC-MS/MS methods to quantify X, I, U, and O in DNA and RNA. We observed large (100-fold) increases in the levels of I in both DNA and RNA from E. coli missing genes for the conversion of IMP to AMP (purA; adenylosuccinate synthase), for the removal of I-containing nucleotide triphosphates (rdgB; dITPase), and for the repair of X and I in DNA (nfi; endoV). Similar mutations in X metabolism did not cause increases of X in DNA or RNA. The results are consistent with (1) differential increases in nucleotide triphosphates; (2) polymerase selectivity; or (3) differential repair of dX and dI in DNA. These observations suggest that disturbances in purine metabolism could act synergistically with inflammation to increase the

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mutagenic burden of nucleobase deamination products and to alter gene expression by interfering with RNA function.

72. Defining the Spectrum of DNA, RNA, and Protein Adducts Arising from Lipid Peroxidation: A Comprehensive Approach. C. Eric Elmquist1, Jimmy Flarakos1, Rosa G. Liberman1, Paul L. Skipper1, Steven R. Tannenbaum2, and Peter C. Dedon3. (1) Biological Engineering Division, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Room NE47293, Cambridge, Massachusetts 02139. (2) Biological Engineering Division and Department of Chemistry, Massachusetts Institute of Technology, Room 56-731A, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139. (3) Biological Engineering Division and Center for Environmental Health Sciences, Massachusetts Institute of Technology, NE47-277, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139. Inflammatory processes are known to cause lipid peroxidation (LPO), which leads to the generation of electrophilic species capable of reacting with the nucleophilic components of DNA, RNA, and proteins. Although the resulting adducts have the potential for critical roles as both biomarkers of and participants in the pathophysiology of inflammation, we know little about the quantitative chemical spectrum of lipid peroxidation products and the adducts they form in cells and tissues. To illustrate this point, the two most frequently studied lipid peroxidation products, malondialdehyde (MDA) and trans-4-hydroxy-2nonenal (HNE), have been proposed to be the source of the mutagenic M1dG and etheno adducts, respectively, yet recent studies reveal base propenals as the major source of M1dG. To begin to unravel the complexity of the spectrum of products and adducts arising from lipid peroxidation, we have adopted a general approach in which human cell membranes are selectively labeled with 14C-labeled polyunsaturated fatty acids (PUFA) and subjected to oxidative stress, after which the products representing 14C-containing protein, DNA, and RNA adducts are quantified and chemically characterized by several applications of accelerator mass spectrometry (AMS). This approach has been applied to human HCT116 colon carcinoma cells labeled with 14C-labeled linoleic acid, the simplest and most abundant PUFA in human cells. GC/MS studies revealed that the labeling conditions result in a large increase of linoleate in the membranes, of which ∼90% was incorporated into phospholipids. AMS analysis was then applied to quantify 14Clabeling of purified protein, DNA, and RNA fractions from cells treated with various oxidative stresses, including ionizing radiation, peroxynitrite, and hydrogen peroxide. This type of approach will provide novel insights into the biologically relevant chemistry of cellular insults such as oxidative stress


and will establish a truly quantitative spectrum of adduct damage to cellular macromolecules. 73. Covalent Adducts Arising from the Reaction of 9,12Dioxo-10(E)-dodecenoic Acid with a Series of Proteins. Michelle V. Williams1, John S. Wishnok1, and Steven R. Tannenbaum2. (1) Biological Engineering Division, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139. (2) Biological Engineering Division and Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139. Polyunsaturated fatty acids can be converted to lipid hydroperoxides through nonenzymatic and enzymatic pathways. The prototypic ω6-lipid hydroperoxide, 13-HPODE, decomposes homolytically to highly reactive R,β-unsaturated aldehydes, such as 9,12-dioxo-10(E)-dodecenoic acid, 4-oxo-2(E)-nonenal, 4,5epoxy-2(E)-decenal, and 4-hydroxy-2(E)-nonenal. These products have previously been shown to form covalent DNA adducts. It has also been shown that both 4-oxo-2(E)-nonenal and 4-hydroxy-2(E)-nonenal can modify proteins. We have recently found that 9,12-dioxo-10(E)-dodecenoic acid will also modify proteins, with the major adduct from it is reaction with cytochrome C being analogous to the most abundant adduct resulting from the decomposition of 13-HPODE in the presence of cytochrome C. Protein modifications such as these are potential biomarkers of lipid hydroperoxide-mediated macromolecule damage. The goal of this study was to identify and characterize the modifications from the reaction of 9,12-dioxo10(E)-dodecenoic acid with a series of proteins in order to assess the value of these products as candidate biomarkers of oxidative damage. 74. AKR1C9 Oxidizes the Fjord-Region Benzo[g]chrysene11,12-dihydrodiol with a High Turnover Number and the Resultant Dione Forms Bis Conjugates. Carol A. Shultz1, Nisha T. Palackal1, Dipti Mangal2, Ronald G. Harvey3, Ian A. Blair4, and Trevor M. Penning5. (1) Department of Biochemistry and Biophysics, University of Pennsylvania, 3620 Hamilton Walk, 135 John Morgan Building, Philadelphia, Pennsylvania 19104. (2) Department of Pharmacology, Center for Cancer Pharmacology, University of Pennsylvania, 421 Curie Boulevard, 846, Biomedical Research Building, Philadelphia, Pennsylvania 19104. (3) Ben May Institute for Cancer Research, University of Chicago, 5841 South Maryland Avenue, MC6027, Chicago, Illinois 60637. (4) Center for Cancer Pharmacology, University of Pennsylvania, 854 BRB II/III, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104-6160. (5) Center of Excellence in Environmental Toxicology, Department of Pharmacology, University of Pennsylvania School of Medicine, 135 John Morgan Building, 3620 Hamilton Walk, Philadelphia, Pennsylvania 19104. Rat aldo-keto reductase 1C9 (AKR1C9) exhibits dihydrodiol dehydrogenase activity in the metabolic activation of polycyclic aromatic hydrocarbons (PAH). Fjord-region PAH are among the most potent carcinogens known. We find that (()-11,12dihydroxy-11,12-dihydrobenzo[g]chrysene (b[g]c-11,12-dihydrodiol), a proximate carcinogen derived from the fjord-region parent hydrocarbon, is oxidized by AKR1C9 with a specific activity a 100 times faster than that observed with the commonly studied benzo[a]pyrene-7,8-dihydrodiol. Although both the (+)and (-)-isomers of the b[g]c-11,12-dihydrodiol are formed in ViVo, AKR1C9 only oxidizes the (+)-stereoisomer. The product of the reaction, b[g]c-11,12-dione, was trapped as a thiol-ether conjugate with 2-mercaptoethanol and characterized by LC/MS versus an authentic synthetic standard. Evidence was obtained


for the formation of mono and bis conjugates, indicating that both a 1,6- and 1,4-Michael addition had occurred to the dione. Dimethylbenz[a]anthracene-3,4-dione also forms mono and bis conjugates with thiols, suggesting that hard nucleophiles preferentially alkylate hindered bay-region o-quinones to give more than one product. Supported by RO1 CA39504 and P30ES013508 awarded to T.M.P. 75. Conversion of Benzo[a]pyrene-7,8-dihydrodiol to Benzo[a]pyrene-7,8-dione by AKR1C Isoforms Causes Oxidative Stress in A549 Lung Adenocarcinoma Cells. Kirk A. Tacka1, Amy M. Quinn1, and Trevor Martin Penning2. (1) Department of Pharmacology, University of Pennsylvania School of Medicine, 135 John Morgan Building, 3620 Hamilton Walk, Philadelphia, Pennsylvania 19104-6084. (2) Center of Excellence in Environmental Toxicology, Department of Pharmacology, University of Pennsylvania, 3620 Hamilton Walk, Philadelphia, Pennsylvania 19104-6084. Polycyclic aromatic hydrocarbons (PAH) require metabolic activation to exert their genotoxic effects. PAH trans-dihydrodiols can be converted to PAH o-quinones by members of the aldo-keto reductase (AKR) superfamily. PAH o-quinones can undergo redox cycling to generate reactive oxygen species (ROS) and can cause oxidative stress and oxidative DNA damage. Here, we show that benzo[a]pyrene-7,8-dihydrodiol (BP-7,8-diol) is converted to benzo[a]pyrene-7,8-dione (BPQ) in the cellular extracts of A549 lung adenocarcinoma cells that constitutively overexpress AKR1C isoforms. Treatment of intact A549 cells with BP-7,8-diol and BPQ generated intracellular ROS and oxidative stress in a time-dependent manner. Conversely, benzo[a]pyrene-4,5-diol, (a non-AKR substrate) and anti-benzo[a]pyrene-diol-epoxide (product of the P450 pathway of PAH activation) did not produce these effects. Taken together, these findings suggest that PAH-induced ROS formation and oxidative stress result directly from the conversion BP-7,8-diol to BPQ by AKR1C isoforms in A549 cells. Supported by P01 CA 092357, P30 ES 013508, and R01 CA 39504 awarded to T.M.P. 76. Oxidative DNA Damage in Benzo[a]pyrene-7,8-dioneTreated Human Bronchoalveolar Cells. Dipti Mangal. Department of Pharmacology, Center for Cancer Pharmacology, University of Pennsylvania, 421 Curie Boulevard, 846, Biomedical Research Building, Philadelphia, Pennsylvania 19104. Seon Hwa Lee. Department of Pharmacology, Center for Cancer Pharmacology, University of Pennsylvania, 846 BRB II/III, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104. Trevor M. Penning. Department of Pharmacology, Center of Excellence in Environmental Toxicology, University of Pennsylvania, 130c John Morgan Building, 3620 Hamilton Walk, Philadelphia, Pennsylvania 19104-6084. Ian A. Blair. Center for Cancer Pharmacology, University of Pennsylvania, 854 BRB II/III, 421 Curie Boulevard, Philadelphia, Pennsylvania 19104-6160. We have proposed that DNA damage can result from benzo[a]pyrene (B[a]P) by a pathway in which the initial B[a]P-7,8dihydrodiol metabolite is converted to a catechol metabolite by aldo-keto reductases (AKRs). The catechol is then oxidized by two sequential 1-electron oxidations to a quinone metabolite with the concomitant generation of reactive oxygen species (ROS). A futile redox cycle produces ROS that causes oxidative damage to DNA. A stable isotope dilution liquid chromatography/electrospray ionization/selected reaction monitoring/mass spectrometry was used to provide maximal specificity and sensitivity. Human bronchoalveolar H358 cells transfected with AKR1A1 were then treated with B[a]P-7,8-dione and 8-oxodGuo levels determined. Levels of 8-oxo-dGuo were 3.07 (


1.12 adducts/106 dGuo at the start of the incubation and 4.56 ( 0.72 adducts/106 dGuo after 9 h. When the cells were pretreated with a catechol-O-methyl transferase inhibitor, 8-oxodGuo levels increased to 7.56 ( 1.93 adducts/106 dGuo after 9 h. Supported by NIH P01CA 92537 and P30 ES013508. 77. Novel Agents for the Redox Regulation of Protein Tyrosine Phosphatases. Jason LaButti1, Thomas Reilly2, Goutam Chowdhury3, and Kent S. Gates1. (1) Department of Chemistry, University of MissourisColumbia, 125 Chemistry Building, Columbia, Missouri 65211. (2) Department of Veterinary Pathobiology, University of MissourisColumbia, W203 Veterinary Medicine Building, Columbia, Missouri 65211. (3) Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, 638 Robinson Research Building, 23rd and Pierce Avenues, Nashville, Tennessee 37232. The cellular function of many proteins is regulated by the addition and removal of phosphoryl groups on tyrosine residues. This process is controlled by the coordinated action of protein tyrosine kinases that add the phosphoryl groups and protein tyrosine phosphatases that remove them. Growing evidence indicates that endogenously produced hydrogen peroxide serves as an intracellular signaling molecule that acts (in part) by inactivating protein tyrosine phosphatases via oxidation of the active site cysteine residue to a sulfenic acid. Here, we describe small molecules that efficiently inactivate protein tyrosine phosphatases via oxidation of the active site cysteine residue. These agents could prove useful as research tools for the study of cellular signal transduction or as lead compounds for the development of new phosphatase-targeted drugs.

78. Chemical Reactions Underlying the Redox Regulation of Protein Tyrosine Phosphatase 1B (PTP 1B). Santhosh Sivaramakrishnan and Kent S Gates. Department of Chemistry, University of MissourisColumbia, 125 Chemistry building, 601 S College Avenue, Columbia, Missouri 65211. The function of many proteins can be switched off or on by the oxidation of catalytic thiol residues to the sulfenic acid oxidation state (RSOH). This process is reversible because reaction of the sulfenic acid with cellular thiols readily converts the protein back to its native thiol form. However, further oxidation of the sulfenic acid to sulfinic acid (RSO2H) typically constitutes an irreversible inactivation of the protein. Here, we report chemical studies designed to model the redox regulation of the enzyme protein tyrosine phosphatase 1B (PTP1B). Oxidation of PTP1B’s catalytic cysteine to a sulfenic acid leads to the formation of an isothiazolidinone heterocycle at the enzyme active site. The results obtained with a small organic model of PTP1B suggest that the formation of this protein isothiazolidinone protects PTP1B against irreversible “overoxidation”. Further oxidation of the inactivated PTP1B yields a isothiazolidinone 1-oxide heterocycle that is easily converted back to the native enzyme upon reaction with thiols.

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79. Teucrin A-Induced Changes in Rat Liver Proteomes and Identification of Protein Targets of Teucrin A by MALDI-TOF MS and LC/MS/MS. Alexandra Druckova1, Raymond L. Mernaugh2, Amy Joan L. Ham3, David B. Friedman3, and Lawrence J. Marnett4. (1) Department of Chemistry, Vanderbilt University, 7300 Stevenson Center, Nashville, Tennessee 37235. (2) Molecular Recognition Facility, Vanderbilt University, Nashville, Tennessee 37232. (3) Department of Biochemistry, Mass Spectrometry Research Center, Vanderbilt University, 9114C Medical Research Building III, 465 21st Avenue South, Nashville, Tennessee 37232-8575. (4) Departments of Chemistry and Biochemistry, Vanderbilt Institute of Chemical Biology, Center in Molecular Toxicology, Vanderbilt University, 23rd Avenue at Pierce, RRB 850A, Nashville, Tennessee 37232. The toxicity of germander, a herb used to treat obesity, is attributed to cytochrome P450 activation of the furan ring of its major diterpenoid component (teucrin A) into a reactive metabolite capable of adducting proteins. We synthesized peptide conjugates of the 1,4-enedial derivative of teucrin A that served as epitopes for the selection of ScFv monoclonal antibodies. An ScFv was selected, which generated high sensitivity and selectivity for teucrin A-adducted peptides. Immunoprecipitation of rat liver homogenates following administration of a toxic dose of teucrin A afforded teucrin A-adducted proteins that were identified by LC/MS/MS. 2DDifference gel electrophoresis and MALDI-TOF MS were used to characterize changes in the liver proteome. Covalently modified proteins involved in heat shock, ER stress, and nutrient deprivation responses correlated with their decreased levels in the liver. 80. Identification of Nitrated Tryptic Peptides in Complex Biological Systems Using a Specific Immunoprecipitation Method Coupled with Mass Spectrometry. Jennifer R. Seal, John S. Wishnok, and Steven R. Tannenbaum. Biological Engineering Division, MIT, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139. Tyrosine nitration is an important post-translational modification that is associated with oxidative stress and activation of nitric oxide synthases, leading to increased production of nitric oxide. Described here is a method that combines the specific isolation and enrichment of nitrated peptides through immunoprecipitation with mass spectrometric identification of the peptide sequences. Using nitrated human serum albumin as a model system, the major nitrated peptides were enriched and positively identified by mass spectrometric methods, including confirmation of structure by peptide mass fingerprinting and LC/MS/MS, with data analysis by MASCOT and Spectrum Mill. Additionally, this method was applied to the analysis of hepatocytes that had been activated to produce nitric oxide, which, through the formation of other reactive nitrogen species, leads to tyrosine nitration. The inducible nitric oxide synthase (iNOS) was stimulated by a mixture containing an endotoxin (lipopolysaccharide) and cytokines (interferon J, interleukin-1, and tumor necrosis factor). Identified nitrated proteins will be presented. 81. Protein Adducts Generated during Nonenzymatic Oxidation of Linoleic Acid. De Lin, Xiaochun Zhu, and Lawrence M. Sayre. Department of Chemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106. Autoxidation of linoleic acid (LA) catalyzed by Fe(II)/ ascorbate results in unsaturated hydroperoxides, which undergo further oxidative evolution to generate a mixture of electrophiles,


including epoxyenones and dienones with an intact C-18 chain as well as cleavage products, such as 4-hydroxy-2-nonenal (HNE), 4-oxo-2-nonenal (ONE), and the analogous functionalities containing the carboxy rather than the methyl terminus of LA. Mass spectrometric studies have been performed following the incubation of two model proteins, apomyoglobin and beta-lactoglobulin, in the presence of LA, Fe(II), and ascorbate. A number of adducts found represent those previously observed when treating protein with the pure electrophilic modifier, including HNE-His Michael, ONE-Lys ketoamide, and the ONE-imidazolylpyrrole His-Lys cross-link. Nevertheless, an 18 h incubation of apomyoglobin, LA, and Fe(II)/ascorbate results in selective modification of H24 and H113 by various isomers of epoxyketooctadecenoic acid (EKODA) and ketooctadecadienoic acid (KODDA). The EKODA modifications were confirmed through modifications carried out with the four independently synthesized regio/stereoisomers of EKODA. 82. Global Metabolic Profiling Analysis with Parallel GCMS-AMS. Jimmy Flarakos1, Rosa G. Liberman1, Paul L. Skipper1, and Steven R. Tannenbaum2. (1) Biological Engineering Division, Massachusetts Institute of Technology, Building 56, Room 731, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139. (2) Biological Engineering Division and Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139. Isotopomeric methods have been applied successfully to elucidate novel metabolic pathways and fluxes in in ViVo and in Vitro systems. Incorporation of stable isotopes such as 13C enabled the profiling of differentially labeled metabolites to be determined with isotopomer ratios. However, determination of these ratios is complex due in part to the natural abundance of 13C. A rare isotope such as 14C (half-life or T1/2 ) 5730 y) allows for lower background levels and better signal-to-noise measurements. Accelerator mass spectrometry (AMS) has permitted the detection of 14C at levels of several orders of magnitude lower than that of isotope ratio and liquid scintillation counting methods. Our group has developed a parallel, GCMSD, AMS interface simultaneously determining the 12C and 14C levels from the effluent of a single GC column. This system was used to investigate metabolic fluxes of several classes of biomolecules, organic acids, amino acids, and fatty acids from primary cultured rat hepatocyte medium in an effort to identify biomarkers in hepatotoxic cells. 83. Structure-Activity-Bioactivation Relationship Studies on Arzoxifene Analogues. Zhihui Qin1, Hong Liu2, Cassia R. Overk1, Ping Yao1, Judy L. Bolton1, and Gregory R. J. Thatcher1. (1) Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, 833 S Wood Street MC 781, Building 924, Room 539, Chicago, Illinois 60612. (2) Drug Metabolism Department, Abbott Laboratories, 100 Abbott Park Road, R46V, GPRD, AP9, Abbott Park, Illinois 60064. The selective estrogen receptor modulators (SERMs), raloxifene and arzoxifene, are benzothiophene SERMs for clinical use in osteoporosis and breast cancer chemoprevention. Both raloxifene and DMA, the active metabolite of arzoxifene, are metabolized to electrophilic diquinone methides that are potentially toxic, electrophilic metabolites. The arzoxifene analogue, 4′F-DMA, has been shown to have similar antiestrogenic activity to DMA and raloxifene but with improved metabolic stability and attenuated formation of electrophilic metabolites. To further investigate structure-activity-bioactivation relationships in benzothiophene SERMs, a family of arzoxifene analogues was developed using an efficient novel synthetic


route. Ligand binding to ERRa´ and ERβaˆ and activity in ERRa´positive Ishikawa cells measured estrogenic or antiestrogenic activity. Metabolism and bioactivation was measured in rat liver microsomal incubations in the presence of GSH, in incubations with mushroom tyrosinase in the presence of GSH, and in human intestinal microsomes in the presence of UDPGA. The resulting data demonstrated a broad variety of oxidative chemistry, bioactivation, and metabolism profiles. These data show clearly that the development of benzothiophene SERMs with significantly attenuated bioactivation properties, although maintaining antiestrogenic activity, can be affected by minor structural modifications. 84. Formation and Fate of N-Nitrosamines from Pharmaceuticals at Physiological pH. Jong Sung Kim1, Martha G. Rhoades1, and Patrick J. Shea2. (1) Center for Environmental Toxicology, University of Nebraska, Lincoln, Nebraska 685830915. (2) School of Natural Resources, University of Nebraskas Lincoln, Lincoln, Nebraska 68583-0915. A number of factors leading to the acceleration or inhibition of N-nitrosation reactions may play an important role in the formation of N-nitroso compounds in foods, environmental matrices, and mammalian systems. Nitrosation can be influenced by pH and depends on the relative concentrations of substrates, catalysts, and inhibitors. Our objectives were to identify nitrosamine derivatives of nitrosatable pharmaceuticals and determine the effect of pH, dissolved metals, and redox potential on nitrosamine formation in the presence of nitrite in Vitro, emphasizing conditions simulating the pH of the human digestive tract. We synthesized N-nitrosamine derivatives from pharmaceuticals using a 4:1 molar excess of sodium nitrite at pH 2 to 5. N-nitrosamine derivatives were identified by LC-MS and quantified by HPLC. In the case of the antibiotic ethambutol (ETB), two N-nitrosoethambutol derivatives (NEBs) were detected in aqueous samples: mono-N-nitrosoethambutol (MNEB) and di-N-nitrosoethambutol (DNEB). The pH maximum for nitrosation of ETB is 3.5, and more DNEB was produced under anaerobic conditions. To determine the effects of dissolved metals on NEB formation, the reaction was carried out in the presence of sulfate salts of Al, Cu, and Fe. Although Al and Cu had little effect on DNEB formation compared to ETB alone, the presence of Fe resulted in much less DNEB. This indicates that Fe influences nitrosamine formation or at least the formation of DNEB. We investigated the effect of Fe concentration on NEB formation using different ETB/Fe ratios. ETB can form a chelate with Fe, and the formation of this metal-ETB complex may be limiting nitrosation. The amount of DNEB produced decreased as Fe concentration increased. The fate and bioavailablity of nitrosated pharmaceuticals and their complexes are important in estimating potential effects on human health, and further studies are in progress. 85. Toward the Development of Real-Time Detection of Breath Biomarkers from CBNR Exposure. Stephen T. Hobson, Todd E. Mlsna, Sanjay V. Patel, and Sabina Cemalovic. Seacoast Science, Inc., 2151 Las Palmas Drive, Suite C, Carlsbad, California 92011-1575. Seacoast Science is developing a microelectromechanical systems (MEMS) based rapid, non-invasive bio-dosimeter (MRBD) that allows for the real-time detection of biomarkers in exhaled breath. The system addresses the need for an instrument to assist in the rapid triage of a mass casualty event following suspected chemical, biological, radiation (“dirty bomb”), or nuclear (CBNR) exposure. The system includes a single-chip chemical sensor array and a small, lightweight, and low-power design for long-term and hand-held operation. Our


MEMS technology utilizes an array of surface micromachined capacitors coated with chemoselective polymers and sol-gel based functionalized materials optimized for volatile organic compounds (VOCs), such as alkyl alcohols alkanes and straight chain aldehydes. These compounds have been reported as indicative of reactive oxygen species and oxidative stress. Its exceptionally low-power consumption allows battery-powered operation. Operationally, these detector systems will be small and inexpensive enough for personal issue to medical personnel and other first responders. 86. Molecular Polarizability as a Tool for Understanding the Congener-Specific Toxicities of Polychlorinated Naphthalenes. Gang Zheng. School of Environmental Science and Engineering, Huazhong University of Science and Technology, 1037 Luoyu Road, Wuhan 430074, China. Ting Yu. Environmental Science and Engineering, Huazhong University of Science and Technology, 1037 Luoyu Road, Wuhan 430074, China. Xiaohua Lu. Environmental Science Research Institute, Huazhong University of Science and Technology, 1037 Luoyu Road, Wuhan 430074, China. The molecular polarizabilities and molecular polarizability tensor components of polychlorinated naphthalenes (PCNs) have been calculated using semiempirical methods. The influence of chlorine substitutions on the molecular polarizability tensor components of PCNs has been investigated in comparison with polychlorinated dibenzo-p-dioxin (PCDD), and it has been found that none of the molecular polarizability tensor components of PCNs is strictly controlled by a specific chlorinesubstitution pattern due to the delocalization of p electrons over the whole PCN molecule. The molecular polarizabilities and molecular polarizability tensor components have been correlated with the dioxin-like toxicities (relative potencies) of 20 PCNs, and the results show that the dioxin-like toxicities of PCNs increase with the increase of their molecular polarizabilities.

87. Cytotoxicity and Mutagenicity of Potential Metabolites of 2,6- and 3,5-Dimethylaniline and 3-Ethylaniline. Hyun Gyung Jang1, Laura J. Trudel1, John S. Wishnok1, Steven R. Tannenbaum2, and Gerald N. Wogan1. (1) Biological Engineering Division, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139. (2) Biological Engineering Division and Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Room 56-731A, Cambridge, Massachusetts 02139. Substituted anilines are chemical intermediates used principally in the production of dyes but may also occur in tobacco smoke and as degradation products of aniline-based pesticides. We have recently demonstrated high levels of hemoglobin adducts of 2,6-dimethylanliline (2,6-DMA), 3,5-dimethylaniline (3,5-DMA), and 3-ethylaniline (3-EA) in the blood of non-

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smokers. Although 2,6-DMA has been shown to be clearly carcinogenic for the nasal cavity of rats and mutagenic for S. typhimurium, little information is available on its mechanism of action or on the genotoxicity of 3,5-DMA and 3-EA. We are testing the hypothesis that their toxic effects are attributable to hydroxylamine and/or aminophenol metabolites. We have synthesized and evaluated the cytotoxicity and mutagenicity of N-hydroxy-2,6-DMA, N-hydroxy-3,5-DMA, N-hydroxy-3-EA, 2,6-dimethylaminophenol, and 3,5-dimethylaminophenol in human lymphoblastoid TK6 and HCT116 colon cancer cells. All were cytotoxic to these cells, N-hydroxy-3-EA being the least potent; survival rates decreased linearly at doses in the range of 100-250 µM for N-hydroxy derivatives of 2,6-DMA and 3,5-DMA. The latter were also found to be mutagenic in human lymphoblastoid TK6 cells, with N-hydroxy-3,5-DMA being more active than N-hydroxy-2,6-DMA. Additional studies are in progress. 88. Investigations into the Role of a Radical Mechanism Employed by Limonene Hydroperoxides in Allergic Contact Dermatitis. Timothy M. Altamore, Anna Bo¨rje, and AnnTherese Karlberg. Dermatochemistry and Skin Allergy, Department of Chemistry, Go¨teborg University, Kemivagen 10, Go¨teborg, SE-41296, Sweden. (R)-Limonene is a monoterpene commonly used in fragrances and scented household products. Although not allergenic in itself, (R)-limonene is known to auto-oxidize on air exposure to give a variety of oxygenated compounds, of which the hydroperoxide derivatives (1) have been found to be potent allergens. The majority of haptens identified to date are electrophiles, which, once they have traversed the skin, can react with nucleophilic amino acid residues forming antigens that can activate the immune system and thereby cause contact allergy. However, it has also been suggested that within the epidermis, hydroperoxides can form free radicals (alkoxy-, peroxy- or carbon-centered), which also react with amino acid residues leading to the formation of antigens. This presentation will discuss the role of the radical mechanism in model systems when applied to (R)-limonene derived hydroperoxides.

89. Metal Oxide Microspheres and Nanoparticles: Preparation and Toxicity Evaluation. Won Hyuk Suh1, Ah Ram Jang2, Yoo-Hun Suh2, and Kenneth S. Suslick3. (1) School of Chemical Sciences, University of Illinois at Urbana-Champaign, 600 S. Mathews Avenue, Urbana, Illinois 61801. (2) Department of Phamacology, College of Medicine, Seoul National University, National Creative Research Initiative for Alzheimer’s Dementia and Neuroscience Research Institute, MRC, 28 Yeongeon-dong, Jongno-gu, Seoul, 110-799, South Korea. (3) School of Chemical Sciences Department of Chemistry, University of Illinois at Urbana-Champaign, 600 S. Mathews, Urbana, Illinois 61801. Metal oxide (i.e., titania and silica) microspheres (1 micron) were synthesized using an inexpensive ultrasonic generator (household humidifier; ultrasonic spray pyrolysis). Morphology and pore size were controlled by the silica to Ti(IV) ratio and silica particle size. Depending on these factors, sub-50 nm oxide nanoparticles and porous/hollow microspheres can be synthesized. In order to test the toxic effects of these engineered nanomaterials, we conducted cellular level toxicology experi-


ments using a colorimetric assay. These nanomaterials are taken up into the cytoplasm (but not the nucleus) of several mammalian cell lines and depending upon their composition show very little or distinct levels of cell toxicity (cytotoxicity). Small molecules like rhodamine and DHED (dehydroevodiamine HCl; Alzheimer’s disease therapeutic) can be delivered along with them, demonstrating that adsorption of certain chemicals could either have beneficial effects as drug carriers or have detrimental consequences to animals and humans if these nanomaterials were to reach the environment without proper control. Characterization methods used were cell viability assay, SEM, (S)TEM, optical/confocal microscopy, XRD, EDS, SAED, zeta potential, and BET. 90. Mechanisms of Sensitization to Hospital Chemical Disinfectants. Chris R. French. Biocides Research & Development, Advanced Sterilization Products, a Johnson and Johnson Company, 33 Technology Drive, Irvine, California 92618. Because of the widespread use of chemical disinfectants in hospital settings with varying degrees of engineering control, incidences of chemical sensitization are not uncommon. The root cause of allergic reactions that result from contact with biocides is related to the principal that many reactive antimicrobials draw their efficacy from the same mechanism that induces sensitization: the reaction of molecules with proteins to form covalently linked conjugates. With recent advances in modeling reactivity using quantitative structure-activity relationships (QSAR) and with improved methods for quanitfing sensitization potential such as local lymph node assay (LLNA), it is possible to begin to synthesize a unified theory that relates biocide molecular physics with both antimicrobial activity and sensitization potential. Such predictive ability is useful for both screening new biocide molecules and for occupational hazard assessments. The current work unifies the aforementioned characteristics drawing on structural attributes, protein reactivity, antimicrobial efficacy, and LLNA data. 91. Fire in Dancing Club with 194 Deaths Caused by Toxic Gas Inhalation. Osvaldo H. Curci, Roberto Cohen, Alejandra Parrini, Mariano Javier Gotelli, Alfredo Lo Balbo, and Carlos A. Gotelli. Centro de Investigaciones Toxicologicas, Juan B. Alberdi 2986, Buenos Aires 1406, Argentina. Medical-forensic and toxicological aspects are presented from a retrospective point of view of the event that resulted in the death of 194. The medical-forensic strategy was as follows: victim identification; cause of death determination; death mechanism determination; connected conditions investigation; and toxicological, radiological, anatomic-pathological and biochemical studies. The time at which the event took place as well as the number of rescuers and victims is described. From a total of 194 dead people, with an average age of 21 years, 192 necroscopic studies were performed. The origin of the fire was the deflagration of a firework, which caused the incomplete combustion of the ceiling elements, starting the fire and the emission of toxic gas and irritant fumes. The action mechanisms of carbon monoxide, hydrogen cyanide, and other gases are also described. 92. Characterization of Adenine Adducts from Amino Acid Derived Nitrolic Acids. Richard N. Loeppky1, Ujjal Sarkar1, Kent S. Gates1, Yinan Li1, Charles L. Barnes2, and Nicholas P. Power1. (1) Department of Chemistry, University of Missouri, 125 Chemistry Building, Columbia, Missouri 65211. (2) Department of Chemistry, University of Missouris Columbia, 601 S. College Avenue, Columbia, Missouri 65211.


Reactive, but isolable, nitrolic acids (RC(dNOH)NO2) are produced from the nitrosation of dietary amino acids under simulated gastric conditions. The possible DNA damage that could arise from nitrolic acids was probed by examining the reaction of propyl nitrolic acid (R ) Et) 1 with deoxyadenosine (dA), adenosine (Ado), and adenine (Ade). The major product is the novel pyrimidine-opened oxadiazole 2. Other reaction products are Z/E isomers of 1-propanoximylinosine 3. Structure elucidation involved both NMR and X-ray crystallography. HPLC tracking of the reaction of 1 (3 equiv) with dA at 50 °C showed that a 40% yield of 2 was produced within 2 h. Both 2 and 3 are perceived to arise from initial oximinylation of N-1 of the adenine by either 1 or a nitrile oxide derived from it. Nitrous acid, a decomposition product of 1, does not deaminate the adenine derivatives under the reaction conditions. The independent synthesis of 2, its stability, and mechanism of formation will be discussed.

93. Structure and Stability of Duplex DNA Containing a 3-(Deoxyguanosin-N2-yl)-2-acetylaminofluorene (dG(N2)AAF) Lesion. Carlos de los Santos, Tanya Zaliznyak, Radha R. Bonala, and Francis Johnson. Department of Pharmacological Sciences, State University of New York, Stony Brook, New York 11794. The carcinogenic pollutant 2-nitrofluorene produces several DNA adducts including the 3-(deoxyguanosin-N2-yl)-2-acetylaminofluorene (dG(N2)-AAF) lesion, a minor adduct that persists for a long time in rat tissue DNA after carcinogen administration. We present here the solution structure of a DNA duplex containing a dG(N2)-AAF residue, as determined by NMR spectroscopy and RMD. Spectroscopic data establish a right-handed duplex conformation with Watson-Crick base pair alignments. The AAF moiety resides in the minor grove of the helix, where it is directed toward the 5′-end of the modified strand. RMD shows that the duplex structure adjusts locally to the presence of the lesion, reducing the exposure of AAF to water. Analysis of UV melting profiles shows that the lesion increases the thermal and thermodynamic stability of DNA, an effect that is driven mainly by favorable entropy. The structure and stability of the dG(N2)-AAF duplex may have important implications for the recognition of bulky lesions by the NER system.


94. Unraveling the Aflatoxin-FAPY Conundrum: Structural Basis for Differential Replicative Processing of Isomeric Forms of the Formamidopyrimidine-Type DNA Adduct of Aflatoxin B1. Thomas M. Harris1, Kyle L. Brown2, James Z. Deng3, Rajkumar S. Iyer4, Lalitha G. Iyer4, Markus W. Voehler2, Michael P. Stone2, and Constance M. Harris1. (1) Department of Chemistry, Center in Molecular Toxicology and Vanderbilt Institute of Chemical Biology, Vanderbilt University, P.O. Box 1822, Station B, Nashville, Tennessee 37235. (2) Department of Chemistry and Center in Molecular Toxicology, Vanderbilt University, Nashville, Tennessee 37235. (3) Merck Research Laboratories, West Point, Pennsylvania 19486. (4) Gen-Probe, Inc., 110120 Genetic Center Drive, San Diego, California 92121. The formamidopyrimidine (FAPY) derived from the guanine N7 adduct of aflatoxin B1 (AFB) epoxide is a highly persistent lesion in DNA and is probably the basis of the potent mutagenicity and carcinogenicity of this mycotoxin. It exists as two separable but interconvertible forms that have been assigned by various workers as functional, positional, or conformational isomers. One of them is potently mutagenic and the other a complete block to replication. Structural studies carried out on the AFB-FAPY base, nucleoside, and oligonucleotides define equilibria involving geometrical isomers of the formamide, diastereomers at the congested pyrimidine C5-N5 bond, and anomers of the deoxyribose. In DNA, the chromatographically separable species are assigned as anomers. The beta anomer, which is the exclusive form in duplex DNA, is the mutagenic species. In single-stranded environments, both anomers are present with the alpha anomer being the predominant one. The alpha form is the replication block. 95. Analysis of Crotonaldehyde- and AcetaldehydeDerived 1,N2-Propanodeoxyguanosine Adducts in DNA from Human Tissues Using Liquid Chromatography-Electrospray Ionization-Tandem Mass Spectrometry. Siyi Zhang1, Peter W. Villalta2, Mingyao Wang2, and Stephen S. Hecht2. (1) Department of Medicinal Chemistry and The Cancer Center, University of Minnesota, 420 Delaware Street SE-MMC 806, Minneapolis, Minnesota 55455. (2) University of Minnesota Cancer Center, 420 Delaware Street SE-MMC 806, Minneapolis, Minnesota 55455. Crotonaldehyde, a mutagen and carcinogen, reacts with deoxyguanosine (dGuo) to generate diastereomeric 1,N2-propanodeoxyguanosine (Cro-dG) adducts. They can also be formed through the consecutive reaction of two acetaldehyde molecules with dGuo. Considering the importance of DNA adducts in carcinogenesis, we have developed a sensitive and specific LC-ESI-MS/MS method to explore the presence of Cro-dG adducts in DNA from various human tissues. DNA isolated from human tissues was enzymatically hydrolyzed in the presence of [15N5]Cro-dG as an internal standard. The Cro-dG adducts were enriched from the hydrolysate by solid-phase extraction and analyzed by LC-ESI-MS/MS, using selected reaction monitoring. This method allows the quantitation of the Cro-dG adducts at concentrations of 4 fmol/µmol dGuo starting with 1 mg of DNA. Using this method, DNA from the human liver, lung, and blood were analyzed. The Cro-dG adducts were detected more frequently in human lung DNA than in liver DNA. The results from this study demonstrated the presence of these adducts in human tissues, and the differences between liver and lung DNA require further study. 96. Use of the Rozman Scale in Tobacco Carcinogenesis. John H. Lauterbach. Lauterbach & Associates, LLC, 211 Old Club Court, Macon, Georgia 31210-4708.

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Tobacco smoke, environmental tobacco smoke, and oral tobacco products have been reported to contain animal carcinogens. Depending on tobacco product type and conditions of use or exposure, doses of these substances received by an individual can vary. However, there is evidence that some carcinogens are much more potent than other ones. Besides potency, there is the threshold issue. Thresholds were estimated for several tobacco carcinogens using Waddell’s approach for flavors that are rodent carcinogens ((2002) Toxicol. Sci. 68, 275-279). For example, the estimated threshold for 1,3-butadiene on the basis of data for female B6C3F1 mice was 10E18 molecules/kg/day, whereas for males, it was 10E19.7 molecules/kg/day. On the basis of the mainstream 1,3-butadiene delivery for the KY1R4F cigarette under Health Canada conditions and 60 cigarettes per day, the dosage for a 70-kg smoker is about 10E18 molecules/ kg/day. Similar examples will also be given for carcinogens in ETS and oral tobacco products. 97. Base Sequence Effects on Guanine Oxidation by Carbonate Radical Anions: Kinetics and Gel Electrophoresis Studies. Young Ae Lee1, Byeong Hwa Yun1, Seog K. Kim2, Yelena Margolin3, Peter C. Dedon4, Nicholas E. Geacintov5, and Vladimir Shafirovich5. (1) Chemistry, New York University, 29 Washington Place, New York, New York 10003. (2) Chemistry Department, Yeongnam University, 214-1 Dea-dong, Gyongsan, 712-749, South Korea. (3) Biological Engineering Division, NE47-277, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139. (4) Division of Bioengineering and Environmental Health, M.I.T, 56-787, Cambridge, Massachusetts 02139. (5) Department of Chemistry, New York University, 31 Washington Place, Brown Building, Room 453, New York, New York 10003. Carbonate radicals arise from the decomposition of nitrosoperoxycarbonate, a chemical mediator of inflammation. This radical oxidizes guanine in DNA by electron abstraction reactions that culminate in the formation of guanine oxidation products. However, the mechanisms of action are poorly understood, and the effects of base sequence on the oxidation of the DNA bases have not been characterized. These effects were explored in this work, starting from the kinetics of DNA oxidation, to the formation of chemical products that were detected as alkali-labile lesions. The cascade of events was initiated by utilizing 308 nm XeCl excimer laser pulses to generate carbonate radicals. It has been shown that the base sequence dependence of the initial electron transfer step and the subsequent formation of guanine oxidation products are different from one another. The mechanistic aspects and biological implications of these oxidation reactions in DNA initiated by carbonate radicals will be discussed. Supported by NIH Grant 1 R01 CA110261. 98. Distribution of Structurally Defined Oxidative Guanine Lesions along K-ras and p53 Derived DNA Sequences. Natalia Tretyakova. Cancer Center, University of Minnesota, 425 E. River Road, Minneapolis, Minnesota 55455. Brock Matter. Department of Medicinal Chemistry and Cancer Center, University of Minnesota, 425 East River Road, 790 CCRB, Minneapolis, Minnesota 55455. Oxidative degradation of DNA in the presence of reactive oxygen species gives rise to a complex mixture of oxidative products, including 8-oxo-7,8-dihydro-2′-deoxy-guanosine (8oxo-dG), spiroiminodihydantoin, guanidinohydantoin Gh), 2,2diamino-4-[(2-deoxy-β-D-erythro-pentofuranosyl)amino]-4Himidazol-4-one (imidazolone), and its hydrolysis product, 2,2diamino-4-[(2-deoxy-β-D-erythro-pentofuranosyl)amino]-2,5dihydrooxazol-5-one (oxazolone). These oxidized bases are


strongly mispairing and are likely to contribute to mutagenesis and carcinogenesis. In the present work, stable isotope labeling in combination with liquid chromatography-electrospray ionization mass spectrometry was employed to monitor the formation of 8-oxo-dG and oxazolone at specific sites within DNA sequences derived from the K-ras protooncogene and the p53 tumor suppressor gene. A series of DNA duplexes representing K-ras and p53 mutational “hotspots” and surrounding sequences was prepared containing the 15N3, 13N1 stable isotope tag at a different guanine base. Following photooxidation in the presence of riboflavin, the distribution of 8-oxo-dG and oxazolone adducts along each duplex was calculated from isotope ratios determined by HPLC-ESI-MS/MS. We found that both oxidative lesions were formed nonrandomly. Oxazolone lesions were overproduced at the 5′-guanines within GG dinucleotides, including codons 245 and 248 corresponding to major p53 mutational “hot spots”. Unlike the commonly employed gel electrophoresisbased approach, our methodology provides structural information for oxidative guanine lesions, revealing sequence dependence of product distribution. 99. Structural Studies of a cis-5R,6S-Thymine Glycol Lesion in Duplex DNA by NMR. Kyle L. Brown. Department of Chemistry and Center in Molecular Toxicology, Vanderbilt University, Nashville, Tennessee 37235. Ashis K. Basu. Department of Chemistry, University of Connecticut, 55 North Eagleville Road, Storrs, Connecticut 06269. Michael P. Stone. Department of Chemistry and Center in Molecular Toxicology, Vanderbilt University, VU Station B Box 182235, Nashville, Tennessee 37235. The cis-5,6-dihydroxy-5,6-dihydrothymine lesion arising from the oxidation of thymine bases by reactive oxygen species was incorporated into the oligodeoxynucleotide sequence 5′GTGCGtgGTTTGT-3′. This sequence contains codon 273 of the p53 tumor suppressor gene and was annealed with its complement for structural analysis by NMR. Molecular restraints were obtained by NMR for use in restrained molecular dynamics simulations. NMR relaxation experiments and simulation results are indicative of multiple conformations of the lesion. This could play a role in reduced stability of thymine glycol modified DNA and increase the likelihood of repair by cellular mechanisms. This work was funded by NIH grant CA-55678 and NIEHS grant ES013324. 100. New Developments on the Study of Oxidative Intrastrand Cross-Link Lesions. Yinsheng Wang1, Haizheng Hong2, and Chunang Gu2. (1) Department of Chemistry, University of California at Riverside, Mail Drop 027, Riverside, California 92521-0403. (2) Environmental Toxicology Graduate Program, University of California at Riverside, 900 University Avenue, Riverside, California 92521 Reactive oxygen species (ROS), which can be formed from both endogenous and exogenous processes, can induce damage to DNA, which has been implicated in the pathogenesis of a number of human diseases including cancer and aging. Others and we have characterized the structures of a number of intrastrand cross-link lesions induced by reactive oxygen species, and some of these lesions can be induced in duplex DNA by gamma irradiation or Fenton reaction. Here, we will show that this type of lesion can also be induced in ViVo in Hela cells upon gamma irradiation. In this respect, we employed LC-MS/MS by using stable isotope-labeled internal standards and quantified the amounts of these lesions as well as the oxidative single-base lesions at several irradiation doses. In addition, we will show that the oxidative intrastrand cross-link lesions can be subjected to repair by nucleotide excision repair


enzymes. We are now in the process of examining the mutagenic properties of this type of lesions in ViVo. 101. One-Electron versus Two-Electron Processes in Metal-Mediated DNA Oxidation. Cynthia J. Burrows. Department of Chemistry, University of Utah, 315 S. 1400 East, Salt Lake City, Utah 84112-0850. Redox active transition metals modify nucleic acid structure through both binding interactions and oxidation chemistry. This presentation will summarize recent results in the characterization of guanine oxidation products in DNA including one-electron processes leading to either C5 or C8 oxidation products mediated by Ni(II) complexes as well as two-electron oxidation yielding 8-oxo-dG from Pt(IV) compounds. 102. Oxidative DNA Lesions from Chromate Exposure. Kent D. Sugden. Department of Chemistry, University of Montana, 32 Campus Drive, Missoula, Montana 59812. Cancer formation in humans following exposure to the hexavalent oxidation state of chromium has been recognized for over 100 years, but its mechanism of initiation is still poorly understood. Two putative pathways, an oxidative and binding pathway, have been proposed to account for the mechanism of cancer induction by chromium. The research in our lab focuses on understanding the oxidative pathway of chromium-induced DNA damage as it relates to the formation of base-specific lesions. We have found that oxidation of DNA by Cr(VI) forms a series of “further oxidized” lesions of guanine identified as spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh). These lesions have been identified arising from chromium oxidation both in Vitro and in cellular systems. The relative levels of these “further oxidized” lesions of guanine were found to be significantly greater than the “ubiquitous” 8-oxo-dG lesion. The formation of Sp and Gh lesions in these systems has implications for the carcinogenicity of chromate because they are significantly more mutagenic than the 8-oxo-dG lesion. 103. Role of Metabolism in the Toxicity and Carcinogenicity of Arsenic. David J. Thomas. Experimental Toxicology Division/Pharmacokinetic Branch, U.S. Environmental Protection Agency, MD B143-01, 109 T. W. Alexander Drive, Research Triangle Park, North Carolina 27711. A striking feature of inorganic arsenic metabolism is its conversion to methylated arsenicals. Intermediates and products formed in this pathway are likely to mediate some toxic and carcinogenic effects, which are ascribed to inorganic arsenic. Elucidating this pathway involves the development of analytical techniques needed to identify each arsenical and an examination of biological processes involved in metabolite formation. For example, one enzyme, arsenic (+3 oxidation state) methyltransferase (AS3MT), catalyzes a series of coupled reactions in which arsenicals are oxidatively methylated and reduced, forming a pathway from arsenite to its ultimate metabolite, trimethylarsine. AS3MT’s catalytic activity depends on cellular reductants (e.g., thioredoxin and glutathione) and is affected by common polymorphisms. Understanding the roles of cellular reductants and genetic variation in the control of AS3MT activity provides a more complete picture of linkage between the metabolism of arsenicals and their toxic and carcinogenic effects. (Abstract does not reflect US EPA policy). 104. Metal-Induced Carcinogenesis: The Role of Ascorbate. Konstantin Salnikow. Laboratory of Comparative Carcinogenesis, National Cancer Institute, NIH, P.O. Box B, Building 538, Room 206 E, Frederick, Maryland 21702. Ascorbate is a well-known intracellular antioxidant. In addition to scavenging free radicals, it serves to maintain iron in the Fe(II) state in numerous iron-containing dioxygenases


(IDs). Among them is a family of enzymes involved in posttranslational hydroxylation of cellular proteins, including collagens I-XXVII and HIF-1alpha and 2alpha proteins. A number of carcinogenic metals, including cobalt(II), nickel(II), and chromium(VI), can facilitate ascorbate oxidation to recyclable forms of either ascorbate free radical or dehydroascorbate. Besides ascorbate oxidation, exposure of cells to metals also affects ascorbate uptake via sodium-dependent transporter SVCT2. As a result, exposure to metals substantially depletes intracellular ascorbate stores. The decrease in intracellular levels of ascorbate inhibits ID’s activity and causes the loss of protein hydroxylation. The loss of collagen hydroxylation may affect extracellular matrix formation. The loss of HIF-1alpha protein hydroxylation leads to the accumulation of this protein and activation of HIF-1-dependent transcription. Thus, exposure to metals will result in disorganization of the extracellular matrix and shift cellular metabolism to a glycolytic pathway, producing typical features of the tumor phenotype. 105. Arsenic as a Co-Carcinogen. Toby G. Rossman, Ahmed N. Uddin, and Fredric J. Burns. Nelson Institute of Environmental Medicine, New York University School of Medicine, 57 Old Forge Road, Tuxedo, New York 10987. Arsenic in drinking water is associated with skin and other cancers. Arsenic in drinking water does not cause skin cancers in mice but enhances the skin tumorigenicity of solar UV irradiation. Arsenic compounds do not react with DNA and are not directly mutagenic, but arsenite is a comutagen and causes genomic instability (delayed mutagenesis). Other co-carcinogenic mechanisms might include effects on DNA repair, DNA methylation, aneuploidy, and signaling changes. Arsenic and selenium are mutually antagonistic. Low selenium levels may exacerbate effects of arsenic in some parts of the world. Selenium enhances the biliary excretion of arsenic through the formation of a diglutathione compound [(GS)2AsSe]-. Organoselenium compounds blocked arsenite-induced delayed mutagenesis. A synthetic selenium compound p-XSC prevented arsenite’s co-carcinogenesis. Selenium may protect via the antioxidant action of selenoproteins, increasing biliary excretion of arsenic or other effects on arsenic metabolism. Our results suggest that arsenic needs a carcinogenic partner. 106. Aberrant Repair of Cr-DNA Adducts as a Road to Genomic Instability in Chromate Carcinogenesis. Anatoly Zhitkovich. Center for Genomics and Proteomics, Department of Pathology & Laboratory Medicine, Brown University, Providence, Rhode Island 02912. Carcinogenic chromium(VI) induces numerous Cr-DNA adducts that are mutagenic and genotoxic in human cells. However, Cr adducts do not block replication in Vitro or cause major structural distortions in DNA duplex. We found that mismatch repair (MMR) proteins were responsible for the activation of Cr(VI) toxicity in human cells. The absence of MMR suppressed Cr(VI)-induced apoptosis and the formation of secondary toxic DNA lesions detected as foci of gammaH2AX. Replication of Cr-containing plasmids in MLH1+ and MLH1-/- cells confirmed that cellular MMR was specifically required for the genotoxic activity of Cr-DNA adducts. MSH2MSH6 heterodimer played a key role in the recognition of Cr-DNA damage and the initiation of the toxic responses. We propose that high frequency of microsatellite instability in lung cancers among chromate workers could result from the selective growth advantage of MMR- cells during chronic exposure to toxic doses of Cr(VI). 107. Transcriptional Activation and Silencing in Response to Chromium and Arsenic. Aaron Barchowsky1, Kimberley

Penning

O’Hara1, Antonia A. Nemec1, Jawed Alam2, and Linda R. Klei1. (1) Department of Environmental and Occupational Health, University of Pittsburgh Graduate School of Public Health, Bridgeside Point, Suite 350, 100 Technology Drive, Pittsburgh, Pennsylvania 15219. (2) Department of Molecular Genetics, Ochsner Medical Foundation. Airway epithelial cells are primary targets for metal-induced lung disease and cancers. However, overt changes in these cells in response to environmental or occupational exposures to metals or metal mixtures are often subtle. Instead, we hypothesized that Cr(VI) promotes lung pathogenesis by silencing cytoprotective genes such as heme oxygenase-1 (HO-1). Cr(VI) reduced HO-1 mRNA levels in ViVo and enhanced As(III)-stimulated apoptosis in cultured human bronchial epithelial (BEAS-2B) cells. Cr(VI) inhibited As(III)-induced BEAS-2B HO-1 expression by preventing Nrf2 DNA binding and transactivation of ARE cis-elements in the ho-1E1 enhancer region. In contrast, Cr(VI) induced a subset of inhibitory interferon response genes by stimulating signal transducer and activator of transcription1 (STAT1). This induction required histone deacetylase activity, which argues against previous theories of Cr(VI) silencing of genes by closing chromatin structure. These data suggest that Cr(VI) promotes injury in lung epithelium by stimulating inhibitory cell signaling. Supported by NIEHS RO1 ES10638. 108. Recent Advances in Molecular Toxicology Using Accelerator Mass Spectrometry. Paul T. Henderson, Sang Soo Hah, and Janna M. Mundt. Biosciences Directorate, Lawrence Livermore National Laboratory, 7000 East Avenue, L-441, Livermore, California 94551. Accelerator mass spectrometry (AMS) is a technique for the detection of extremely low-abundance isotopes such as radiocarbon. AMS was initially developed for radiocarbon dating but has recently become a powerful tool for biological analysis in which small (microgram) samples are combusted to carbon dioxide and reduced to graphite for subsequent quantitation. We report progress on quantifying the incorporation of oxidized nucleotides into DNA and RNA from the cytoplasm of MCF-7 cells. Such events can lead to mutations and altered protein function. Additionally, we report the determination of the kinetics of DNA adduct formation by the chemotherapeutic compound carboplatin. The kinetics as measured by AMS match literature values for the reaction of radiocarbon-labeled carboplatin with purified DNA. In bladder cancer cells exposed to carboplatin, AMS allowed a measurement sensitivity of 1 attomole per 10 micrograms of DNA. Such measurement sensitivity and precision may have clinical applications for individualized therapeutics. 109. Toxicogenomics: A Five-Year Perspective. Michael E. Burczynski. Pharmacogenomic Biomarkers, Wyeth Research, Biomarker Laboratory, 500 Arcola Road, Collegeville, Pennsylvania 19426. The pace and complexity of toxicogenomics research has accelerated tremendously since the inception of this area of biomedical inquiry less than a decade ago. Initial results demonstrated the existence of transcriptional differences following exposure of in Vitro and in ViVo model systems to classes of chemicals exhibiting known toxic mechanisms. On the basis of these findings, researchers have utilized increasingly complex strategies to investigate both the molecular basis of toxicity in mechanistic toxicogenomic studies and to identify toxicity at earlier time points during drug development using predictive toxicogenomic approaches. This presentation reviews the key initial observations that justified further explorations of the utility of toxicogenomic approaches, surveys the growing number of


molecular assays currently employed by researchers to answer questions in this field, and anticipates additional innovations on the horizon that will maintain the field of toxicogenomics as a frontier in chemical toxicology for many years to come. 110. Mouse Metabolomics. Frank J. Gonzalez. Laboratory of Metabolism, National Cancer Institute, Bethesda, Massachusetts 20892. Jeffrey R. Idle. Institute of Pharmacology, 1st Faculty of Medicine, Charles University, Prague, Czech Republic. Metabolomics is the study of changes in small molecules in cells, tissues, and biological fluids such as serum and urine. Metabolomics has tremendous potential in the search for biomarkers and for metabolite profiling. In an attempt to find biomarkers for cytochromes P450 expression, P450-null and humanized mice are compared. Urine is collected and subjected to ultra performance liquid chromatography coupled time-offlight mass spectrometry (UPLC-TOFMS). The UPLC-TOFMS data are deconvoluted by principle component analysis software in order to find compounds that correlate with the presence or absence of a specific P450 form. To study xenobiotic metabolism, drugs or other compounds are administered to mice, and serum and urine collected and examined for the presence of metabolites that are not found in untreated mice. Metabolites derived from the drug, and endogenous metabolites that reflect the drug’s biological activity or toxicity can be resolved. Recent results will be discussed. 111. Genetic Variants in the Quinone Reductase NQO1: Implications for Chemoprotection and Chemotherapy. David Ross. Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado at Denver and Health Sciences Center, 4200 East Ninth Avenue, Denver, Colorado 80262. NAD(P)H/quinone oxidoreductase 1 (NQO1) plays an important role in the detoxification of many environmental quinones including benzoquinones derived from the leukemogen benzene. NQO1 is also expressed at high levels throughout many human solid tumors, which has led to the design of antitumor quinones that can be bioactivated by NQO1. The NQO1*2 single nucleotide polymorphism is a C609T change in the cDNA that codes for a proline to serine change in the protein and has marked phenotypic consequences. No detectable or only trace levels of NQO1 protein are associated with the NQO1*2/*2 genotype because of rapid proteasomal degradation of the mutant NQO1*2 protein. The NQO1*2 polymorphism increased the relative risk for benzene poisoning in occupationally exposed individuals and has been associated with an increased risk of therapy-related and de-novo leukemias. The NQO1*2 polymorphism must also be considered in the use of chemotherapeutic agents designed to be activated by the high levels of NQO1 in human solid tumors. The NQO1*2 polymorphism has also been employed as a molecular tool to demonstrate proof of principle


for novel NQO1-directed antitumor agents in pre-clinical studies. In summary, the NQO1*2 polymorphism has a well-characterized phenotype and has implications for both chemoprotection and chemotherapy. Supported by NIH Grants ES09554 and CA51210. 112. Complex Recovery Networks Induced by DNA Damaging Agents. Leona Samson. Biological Engineering Division and Center for Environmental Health Sciences, MIT, 77 Massachusetts Avenue, 56-235, Cambridge, Massachusetts 02139. Exposure to toxic DNA damaging agents has long been known to induce increased transcription of various DNA repair genes and to activate cell cycle checkpoints, and the role of DNA repair and cell cycle checkpoints in protecting cells, tissues, and whole animals against the deleterious effects of such agents is well understood. However, recent genome-wide studies have uncovered the fact that a wide range of other cellular pathways play equally important roles in protecting cells against toxicity induced by a broad range of toxic agents, in particular alkylating agents. Global transcriptional profiling and genomic phenotyping of the yeast Saccharomyces cereVisiae together show that pathways involved in protein metabolism, RNA metabolism, and chromatin remodeling (to name but a few) play critical roles in the response of eukaryotic cells to alkylating agents. How these pathways collaborate together to form complex networks for cellular recovery from toxic damage exposure will be discussed. 113. Using Mouse ENU Mutagenesis to Define Gene Function and Model Human Disease. Monica J. Justice. Departments of Molecular and Human Genetics and Molecular Physiology and Biophysics, Baylor College of Medicine, One Baylor Plaza S413, Houston, Texas 77030. Now that the mouse and human genome sequences are complete, biologists need systematic approaches to determine the function of each gene. A powerful way to discover gene function is to determine the consequence of mutations in living organisms. Large-scale production of mouse mutations with the point mutagen N-ethyl-N-nitrosourea (ENU) is a key strategy for analyzing the human genome because mouse mutants will reveal functions unique to mammals, and many may model human diseases. Using the mouse as an experimental model, we carried out two high-efficiency ENU mutagenesis screens using balancer chromosomes to identify mutations on mouse Chromosomes 11 and 4. The unbiased isolation of lethal mutations shows that the fraction of essential genes differs greatly among regions and is unusually high for Chromosome 11. This high proportion of essential genes coincides with a high degree of linkage conservation among mammals. New mutants are described at www.mouse-genome.bcm.tmc.edu. TX6002886

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