Abstracts from the Division of Chemical Toxicology for the American Chemical Society National Meeting in Boston, August, 2010.
NIEHS Director's Perspective: Opportunities and Challenges
Linda S. Birnbaum(1), [email protected], PO Box 12233, Mail Drop B2-01, Research Triangle Park North Carolina 27709, United States . (1) National Institute of Environmental Health Sciences, NIH, Research Triangle Park North Carolina 27709, United States
In science, opportunity and challenge are often two sides of the same coin. Current challenges for environmental health include increased awareness of changing patterns of exposure and disease, and emerging exposures. Opportunities for environmental health include the ability to advance the research framework by promoting integrated scientific solutions, building on scientific developments, adding life-cycle analysis to environmental health research, and capitalizing on technological advances to find new solutions. Additional opportunities include new, expanded federal partnerships with non-traditional public health partners, such as the Departments of Transportation, Interior, and Housing and Urban Development. But we must remember to “close the loop” by changing how we think about environmental health, encouraging new paradigms, and translating basic science into human health protection. NIEHS is notably positioned to tackle these challenges and opportunities given our unique programs, including the Superfund Research Program and the National Toxicology Program, and the addition of new staff.
1. Structure and stability of duplex DNA bearing an aristolactam II-dA adduct: Insights into lesion mutagenesis and repair
Carlos R De los Santos(1), [email protected], BST 7-147, Stony Brook New York 11794-8651, United States ; Mark Lukin(1); Tanya Zaliznyak(1). (1) Pharmacology, Stony Brook University, Stony Brook NY 11794, United States
Aristolochic acids cause a type of renal fibrosis known as Balkan Endemic Nephropathy and are involved in transitional cell carcinoma of the upper urinary tract. Drug metabolizing enzymes transform aristolochic acids to the reactive aristolactam (AL) intermediates that can add to the peripheral amino groups of purine residues, forming stable AL-dG and AL-dA adducts in DNA. Aristolactam adducts block DNA replication and are mutagenic. We have incorporated a single ALII- dA adduct in oligomeric DNA and determined its stability and solution conformation. The damaged duplex is a regular right-handed helix stabilized by Watson-Crick alignments across all canonical base pairs. The AL moiety intercalates between bases of the opposing strand, displacing the thymine counter residue out of the helix. Duplex stability is slightly reduced, indicating strong stabilization of the duplex by hydrophobic interactions. We will discuss the implications of these results toward the mechanisms of AAs mutagenesis and repair.
2. Gold nanoparticles can reduce cell proliferation by altering plating surfaces
Athena M. Keene(1), [email protected], 6211 Forest Mill Ln, Laurel MD 20707, United States ; Katherine M. Tyner(1). (1) Office of Testing and Research, Food and Drug Administration, Silver Spring MD 20993, United States
Gold nanoparticles are being investigated for biomedical applications, but reports vary on their toxicity. Primary particles and self-associated structures, which were shown to enter cells by confocal microscopy, were tested for their abilities to alter cell proliferation, modify the cell cycle, and cause apoptosis. In adherent Caco2 and HepG2 cells, 0.1 g/L of any gold structure decreased proliferation by ~50%. Neither cell line experienced a cell cycle change or increased apoptosis. To determine if gold debris on the cell plating surface affected proliferation, assays were run using suspension H211 cells and adherent cells that were trypsinized and re-plated after incubation with the gold structures. After re-plating, confocal microscopy showed the gold structures were present in the cells, but the debris layer was removed. Proliferation was unaffected in these experiments. The gold debris layer appears to prevent proliferation without causing cellular toxicity, showing caution is necessary when interpreting nanoparticle toxicity assays.
3. Formation and in-vivo replication studies of bulky DNA lesions induced by reactive oxygen species
Yinsheng Wang(1), [email protected], 900 University Ave, Riverside CA 92521-0403, United States ; Jin Wang(1); Bifeng Yuan(1); Candace Guerrero(1); Jianshuang Wang(1). (1) Department of Chemistry, University of California Riverside, Riverside CA 92521-0403, United States
Endogenously and exogenously induced reactive oxygen species can result in damage to DNA. These DNA lesions have been implicated in the pathogenesis of cancer and neurodegeneration as well as in the natural processes of aging. We employed LC-MS/MS with the standard isotope dilution technique and quantified the levels of a group of ROS-induced bulky lesions, which encompass the (5'R) and (5'S) diastereomers of 8,5'-cyclo-2'-deoxyadenosine and 8,5'-cyclo-2'-deoxyguanosine, and intrastrand cross-link lesions in rat tissues. We found that the purine cyclonucleosides and the G[8- 5m]T intrastrand cross-link lesions exhibited different age-dependent distributions in different types of tissues. In addition, in-vivo replication studies with the use of shuttle vector technology revealed that these lesions could perturb both the efficiency and fidelity of DNA replication in cells. The results from these studies offered molecule-level understanding of the implications of these lesions in aging and aging-related pathological conditions.
4. LASSO-ing potential pregnane X receptor agonists
Sean Ekins(1), [email protected], 601 Runnymede Avenue, Jenkintown PA 19046, United States ; Antony J Williams(2); Zsolt Zsoldos(3); Aniko Simon(3); Orr Ravitz(3); Valery Tkachenko(4). (1) Collaborations in Chemistry, Jenkintown PA 19046, United States (2) ChemSpider, Royal Society of Chemistry, Wake Forest NC 27587, United States (3) SimBioSys, Inc, Toronto ONT M9W 6V1, Canada (4) ChemSpider, Royal Society of Chemistry, Rockville MD 20850, United States
The Pregnane X receptor (PXR) is a transcriptional regulator of cytochrome P450 3A4, P-glycoprotein and many other genes involved in metabolism and excretion. Studies have used machine learning methods to predict PXR agonist activity for drugs but none have been made publically available. We utilized 203 PXR agonists (EC50 for < 10 μM) with the Ligand Activity by Surface Similarity Order (LASSO) method, a ligand-based tool focused on similarity of biomolecular activity rather than structural similarity. Twenty five models were built using 8 -128 agonists. The models were tested using identification of agonists not in the training set or using 3k, 8k and 24k drug-like decoys including PXR inactive compounds (N=228). We found 64-128 actives provided acceptable enrichment factors of 10% in the top 1% of compounds screened. The best model will be deployed in ChemSpider.com, enabling prediction of PXR agonist activity for almost 20 million compounds in the database.
5. In vivo formation and repair of 1,3-butadiene-induced DNA-DNA cross-links Natalia Tretyakova(1), [email protected], 760E MCRB Masonic Cancer Center, 806 Mayo, 420 Delaware St. SE, Minneapolis MN 55455, United States . (1) Department of Medicinal Chemistry, University of Minnesota, Minneapolis MN 55455, United States
1,3-butadiene (BD) is an important industrial chemical and environmental pollutant present in urban air and classified as a human carcinogen. BD is metabolically activated to 1,2,3,4-diepoxybutane (DEB), which is believed to be the ultimate carcinogenic species of BD based on its potent mutagenicity and genotoxicity. We have developed quantitative HPLC-ESI-MS/MS methods for DEB- specific DNA-DNA cross-links, 1,4-bis-(guan-7-yl)-2,3-butanediol (bis-N7G-BD) and 1-(guan-7-yl)-4- (aden-1-yl)-2,3-butanediol (N7G-N1A-BD), and exocyclic DEB-dA adducts, 1,N6-(1-hydroxymethyl-2- hydroxypropan-1,3-diyl)-2'-deoxyadenosine (1,N6-α-HMHP-dA) and 1,N6-(2-hydroxy-3- hydroxymethylpropan-1,3-diyl)-2'-deoxyadenosine (1,N6-γ-HMHP-dA). DEB-specific DNA-DNA cross- links were quantified in tissues of laboratory rodents exposed to BD by inhalation, and their persistence and repair in vivo was investigated. We found that while bis-N7G-BD cross-links are 6 spontaneously lost by depurination (t 1/2 = 50 h), N7G-N1A-BD and 1,N - HMHP-dA lesions persist in vivo and are not repaired by BER (methyl purine glycosylase) or XPA-dependent nucleotide excision repair. Our results indicate that specific types of bifunctional DEB-DNA adducts accumulate in tissues following BD exposure and are likley to contribute to BD-mediated mutagenesis and cancer initiation.
6. Find a needle in a haystack: Recent studies on drug-induced testicular toxicity
Minghu Song(1), [email protected], Eastern Point Road, Groton CT 06340, United States ; Nigel Greene(1); Robert E Robert Chapin2(2). (1) Compound Safety Prediction Group, Worldwide Medicinal Chemistry, Pfizer Global Research and Development, Groton CT 06340, United States (2) Developmental & Reproductive Toxicology CoE, Drug Safety R&D, Pfizer Global Research and Development, Groton CT 06340, United States
Safety issues have been recognized as one of today's leading causes of drug failure. Extensive research has already been devoted to studying various drug-induced organ injuries, such as hepatotoxicity, cardiotoxicity and nephrotoxicity. Unlike the above, there are very few reviews on adverse effects of drugs on sexual organ functions, e.g. testicular dysfunction, despite the increasing number of reports of testicular damage due to certain drugs and food additives. We compiled a preliminary set of testicular toxicants through literature searches and identified several substructures that may be associated with testicular toxicity. Starting from this sparse data set, we explored relevant biological interaction networks and potential MOA through data and text mining of publicly available information. Such literature-curated knowledge in turn led us to propose novel MOA or structural alerts as well as expand current mechanistic understanding of safety risks associated with those chemotypes. This presentation also outlines our recent efforts to utilize other public data sources such as ToxCastTM to explore potential in vitro assay signatures associated with reproductive toxicity.
7. Analysis of 4-hydroxy-1-(3-pyridyl)-1-butanone-releasing DNA adducts in human exfoliated oral mucosa cells by liquid chromatography-electrospray ionization-tandem mass spectrometry
Irina Stepanov(1), [email protected], 420 Delaware St SE - MMC 806, Minneapolis MN 55455, United States ; John Muzic(1); Joni Jensen(1); Dorothy Hatsukami(1); Stephen S Hecht(1). (1) Masonic Cancer Center, University of Minnesota, Minneapolis MN 55455, United States
The tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'- nitrosonornicotine (NNN) are believed to play an important role in tobacco-induced cancers. Metabolic activation of both NNN and NNK leads to the formation of pyridyloxobutyl-DNA adducts, a substantial portion of which are unstable to DNA hydrolysis conditions and release 4-hydroxy-1-(3- pyridyl)-1-butanone (HPB). We developed a liquid chromatography-tandem mass spectrometry method to measure HPB-releasing DNA adducts in human oral cells. The adducts were detected in 9 out of 18 mouthwash samples from smokers and in 3 out of 15 samples from nonsmokers, averaging 33.6 pmol/mg DNA and 1 pmol/mg DNA, respectively. Preliminary data indicate that buccal cells and mitochondria could be the major sources of HPB-releasing DNA adducts measured in mouthwash samples. The adduct levels varied largely among smokers, suggesting inter-individual differences in NNK and/or NNN metabolic activation. HPB-releasing DNA adducts in oral cells could serve as a biomarker of susceptibility to tobacco-induced cancers in smokers.
8. Prediction of mixture interactions via cellular stress response
Jonathan W Boyd(1), [email protected], PO Box 6045, Morgantown WV 26506, United States ; Xueli Gao(1); Holly Williams(1); Nicholas Rubenstein(1). (1) C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown WV 26506, United States
While exposure to chemical mixtures is an everyday reality, an understanding of their combined effects, and any potential prediction thereof, is extremely limited due to the lack of temporal and spatial mechanisms for the individual constituents. To this end my laboratory has been developing in vitro assays to enable a high-throughput means of defining general toxicodynamic response pathways which have the potential to identify and predict possible interactions. A central aspect of our assays elucidates altered activity of cell signaling cascades which allows for the identification of both common and disparate dose-response pathways. Results will be presented with data gathered from hepatocytes exposed to deguelin, potassium cyanide, staurosporine and SB202190, alone and in combination, for 24 hours. In this study, we demonstrate the potential of a new in vitro approach for the prediction of toxic mixtures interactions that is fundamentally driven by the interdependence of energy metabolism, signal transduction, and cell survival.
9. TP53 mutational signature of aristolochic acid
Arthur P Grollman(1), [email protected], BST-8, 160, Stony Brook NY 11794-8651, United States ; Bojan Jelaković(2); Masaaki Moriya(1). (1) Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook NY 11794-8651, United States (2) Department of Internal Medicine, University of Zagreb, Zagreb 10000, Croatia
Aristolochic acid (AA), a powerful nephrotoxin and human carcinogen, reacts with DNA to form covalent deoxyadenosine-aristolactam adducts (dA-AL). These adducts persist in renal tissues, where they serve as robust biomarkers of exposure to AA. In humans, dA-AL adducts induce base substitutions in the TP-53 gene. The mutational spectrum of AA-induced urothelial carcinomas is dominated by A:T→T:A transversions located almost exclusively on the non-transcribed DNA strand. The molecular mechanism underlying this marked strand bias may account for the remarkable persistence of these adducts in human tissues. AA, thereby, joins vinyl chloride and aflatoxin as an established human chemical carcinogen with a definitive mutational signature in the TP-53 gene. This information, coupled with the use of dA-AL adducts as biomarkers of exposure, is being used to establish the etiologic role of AA in populations with a high prevalence of both urothelial cancer and chronic renal disease. (Supported by NIEHS grant ES04068)
10. Cytochrome P450 3A mediated thalidomide hydroxylation and formation of a glutathione conjugate
Goutam Chowdhury(1), [email protected], 638 Robinson Research Building, 2200 Pierce Avenue, Nashville TN 37232, United States ; Norie Murayama(2); Yusuke Okada(2); Yasuhiro Uno(3); Makiko Shimizu(2); Noria Shibata(4); F Peter Guengerich(1); Hiroshi Yamazaki(2). (1) Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University, Nashville TN 37232, United States (2) Laboratory of Drug Metabolism and Pharmacokinetics, Showa Pharmaceutical University, Machida Tokyo, Japan (3) Shin Nippon Biomedical Laboratories,, Japan (4) Nagoya Institute of Technology, Japan
Thalidomide [α-(N-phthalimido)glutarimide] was withdrawn from clinical use because of its teratogenicity but has been approved for the treatment of refractory multiple myeloma. The CYP2C19 genotype has been reported to be associated with cancer treatment outcome using thalidomide. Although two hydroxylated metabolites of thalidomide are formed at very low concentrations, little information regarding metabolism by P450s is available. The purpose of this study was to characterize thalidomide metabolism by human P450 enzymes and to better understand the activation of thalidomide and any interactions with GSH. Thalidomide was oxidized to hydroxythalidomide by liver microsomes and human P450s 3A4 and 3A5. P450 3A4 also oxidized 5- hydroxythalidomide to an epoxide or dihydroxy compound. Liquid chromatography-mass spectrometry analysis revealed formation of a glutathione conjugate by P450 3A4. These results suggest that human P450 3A4 mediated thalidomide hydroxylation lead to a glutathione conjugate, which may be of relevance in the pharmacological and toxicological actions of thalidomide.
11. Post-synthetic generation of FaPy lesions in oligonucleotides
Mark A Lukin(1), [email protected], Basic Sciences Tower 7-147, Stony Brook NY 11794- 8651, United States ; Conceicao A.S.A. Minetti(2); David P Remeta(2); Sivaprasad Attaluri(1); Francis Johnson(1); Kenneth J. Breslauer(2); de los Santos Carlos(1). (1) Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook NY 11794-8651, United States (2) Department of Chemistry and Chemical Biology, The State University of New Jersey, Piscataway New Jersey 08854, United States
A method for the generation of dG FaPy lesions in synthetic oligonucleotides has been developed. The procedure includes preparation of the synthetic oligonucleotide precursor containing 5- nitropyrimidine moiety. After deprotection and purification of the oligonucleotide by standard procedures, the nitro group is reduced by catalytic hydrogenation followed by formylation of the newly formed amino group with formyl imidazole in water-organic solution. The method allows a large-scale preparation of oligonucleotides containing an equilibrium mixture of alpha and beta FaPy anomers. The anomeric oligonucleotides interconvert very slowly in neutral conditions and can be chromatographically separated. Spectroscopic proterties, melting behaviour and interconversion of the FaPy containing oligonucleotides are discussed.
12. Mechanisms in chemical toxicology: Letting the molecules point the way
Richard N. Loeppky(1), [email protected], 125 Chemistry, Columbia MO 65211, United States . (1) Department of Chemistry, University of Missouri, Columbia MO 65211, United States
In prior work we have shown that N-nitrosodiethanolamine (NDELA) produces two characteristic DNA guanine adducts in vivo in rat liver; O6-hydroxyethyl-dG and 1-N2-glyoxal-dG (gdG). While the former adduct arises from expected nitrosamine metabolism pathways, gdG does not. The glyoxal equivalent arises from both prior β-oxidation of the nitrosamine and the P450-mediated oxidation of the glycolaldehyde produced from the α-hydroxylation of the nitrosamine. Consideration of these pathways and recent publications implicating the possible importance of glyoxal derived adducts as markers in diabetes, inflammation related oxidative damage, and other diseases, raises the question of what role DNA and protein adducts may play in the etiology of pathogenic processes other than cancer. The value and importance of chemical mechanisms in this kind of research is stressed in this brief prospective introduction to the symposium. 13. Recent studies on chemical mechanisms of carcinogenicity: N-Nitrosamines, aldehydes, and polycyclic aromatic hydrocarbons
Stephen S Hecht(1), [email protected], 420 Delaware St SE - MMC 806, Minneapolis MN 55455, United States ; Mingyao Wang(1); Guang Cheng(1); Yan Zhong(1); Siyi Zhang(1); Steven G Carmella(1). (1) Masonic Cancer Center, University of Minnesota, Minneapolis MN 55455, United States
The chemical classes of N-nitrosamines and polycyclic aromatic hydrocarbons (PAH) include strong carcinogens, some of which occur in the human environment, while aldehydes, generally less carcinogenic or inactive, often also occur endogenously in humans. N-Nitrosamines and PAH require metabolic activation for DNA binding and carcinogenesis whereas aldehydes do not. We have used mass spectrometric methods to investigate the metabolism and DNA binding of N-nitrosamines, aldehydes, and PAH in humans, with a goal of uncovering new mechanisms of carcinogenesis and accounting for inter-individual differences in cancer susceptibility. Recent results include the identification of previously unknown 7-(2'-carboxyethyl)guanine adducts in human liver DNA, quantitation of acrolein adducts in human white cell DNA from smokers and non-smokers, and characterization of human PAH metabolism in subjects dosed with [D10]phenanthrene orally or by inhalation. The results of these studies provide some new insights on the potential role of environmental chemicals in carcinogenesis.
14. Alkylanilines: Complexities in chemistry, toxicity, mutagenicity, and carcinogenesis
Steven R Tannenbaum(1), [email protected], 77 Massachusetts Avenue, Room 56-731A, Cambridge MA 02139, United States ; Paul L Skipper(1); Gerald N Wogan(1); Cui Liang(1); Laura Trudel(1); Wenjie Ye(1). (1) Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge MA 02139, United States
2,6-Dimethylamine (2,6-DMA), 3,5-dimethylamine (3,5-DMA) and 3-ethylamine (3-EA) exposures have all been identified as highly correlated with bladder cancer, and the National Toxicology Program has listed more than a dozen other alkylanilines as environmental contaminants that have not been evaluated for biological activity. The metabolism of this class of compounds is fairly well known, but the actual mechanisms of toxicity and mutagenesis remain problematic because multiple pathways form a variety of potentially electrophilic structures that may bind to DNA. The key pathway for toxicity begins with N-oxidation, but then branches with further oxidation to the aminophenol which can be oxidized to the quinoneamine which is also a potential quinonemethide. The toxicity, mutagenicity, and DNA reactions of these structures have been evaluated, and the results will be analyzed as a step toward predicting the activity of other members of the chemical class.
15. Chemistry and biochemistry of bis-electrophiles, thiols, and peptides related to DNA blockage and miscoding
F. Peter Guengerich(1), [email protected], 638 Robinson Research Building, 2200 Pierce Avenue, Nashville Tennessee 37232-0146, United States . (1) Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville Tennessee 37232-1046, United States
DNA damage is caused primarily by electrophiles and radicals, but the number of compounds and the detailed mechanisms are numerous and still expanding. bis-Electrophiles can either produce “internal” linkages (e.g. new rings) or crosslinks to other DNA or peptides. Along-standing focus of our own investigation has been thiol dependent reactions, developing with the ethylene dibromide/glutathione (GSH) paradigm. Subsequently this GSH transferase-dependent mechanism was shown to activate dihalomethanes. GSH transferase conjugation of butadiene diepoxide generates a relatively stable product that causes base pair mutations. The basic thiol chemistry shown for GSH is used in the crosslinking of a DNA repair protein, O6-alkylguanine transferase (AGT), to DNA and enhancement of mutation by bis-electrophiles. Mass spectrometry has been used to characterize these latter reactions and their selectivity. (Supported in part by USPHS R01 ES010546 and P30 ES000267.)
16. Reactive metabolites in the biotransformation of furan-containing molecules
Lisa A. Peterson(1), [email protected], 420 Delaware St SE Mayo Mail Code 806, Minneapolis MN 55125, United States . (1) Division of Environmental Health Sciences and Masonic Cancer Center, University of Minnesota, Minneapolis MN 55455, United States
Many xenobiotics containing a furan ring are toxic and/or carcinogenic. The harmful effects of these compounds require biotransformation of the furan ring. Oxidation of furan can generate an epoxide intermediate which, in some cases, will rapidly rearrange to a 1,4-unsaturated dicarbonyl compound. Either intermediate can react with cellular nucleophiles such as protein or DNA to trigger toxicity. Our studies with furan indicate that reaction of the 1,4-unsaturated dicarbonyl metabolite with glutathione (GSH) represents an additional pathway for adduct formation. P450 catalyzed oxidation of furan results in the crosslinking of GSH to a variety of cellular amines including amino acids, polyamines and proteins. The relative contribution of these reactive intermediates to the toxicological effects of furan-containing compounds will be determined by the nature of the furan ring substituents.
17. Chemistry and biology of DNA damage by structurally interesting heterocyclic compounds
Kent S Gates(1), [email protected], 125 Chemistry Bldg, Columbia MO 65211, United States . (1) Department of Chemistry, University of Missouri, Columbia MO 65211, United States
DNA serves as the molecular blueprint that directs all cellular operations. Accordingly, chemical modification of cellular DNA can have profound biological consequences. For example, the medicinal activity of many clinically-used anticancer drugs arises from their ability to cause DNA damage that kills rapidly dividing cancer cells. Anticancer therapy may be advanced through a fundamental understanding of the DNA damage chemistry that underlies the biological responses. We will describe our studies of several structurally interesting heterocyclic DNA-damaging anticancer agents. The relationships between the chemistry and biology of these agents will be considered.
18. Synthesis and quantitation of carboxymethylated DNA Lesions induced by diazoacetate
Jianshuang Wang(1), [email protected], 501 Big Springs Rd., Riverside CA 92507, United States ; Yinsheng Wang(1). (1) Department of Chemistry, University of California, Riverside, Riverside CA 92507, United States
Humans are exposed to both endogenous and exogenous N-nitroso compounds (NOCs). The exposure to endogenous NOCs (approximately 45-75% of the total NOC exposure) was found to be significantly associated with the risk of developing non-cardia gastric cancer in an epidemiological study. Many N-nitroso compounds can be metabolically activated to give diazoacetate which can result in the carboxymethylation of DNA. Unraveling the implications of the carboxymethylation chemistry of DNA in the etiology of gastric cancers requires the understanding of the formation, replication and repair of these carboxymethylated derivatives of DNA. Here we report the syntheses of authentic N3-carboxymethylthymidine (N3-CMdT), O4-carboxymethylthymidine (O4-CMdT), N4- carboxymethyl-2'-deoxycytosine (N4-CMdC), N6-carboxymethyl-2'-deoxyadenosine (N6-CMdA) and O6-carboxymethyl-2'-deoxyguanosine (O6-CMdG), site-specific incorporation of them into oligodeoxynucleotides, and quantification of these lesions in pancreatic cells by capillary HPLC- tandem mass spectrometry with stable isotope dilution method.
19. Differential expression of proteins in response to arsenic stress in Chlamydomonas reinhardtii
Chamari Walliwalagedara(1), [email protected], 2121, Euclid Avenue, Cleveland Ohio 44115, United States ; Harry van Keulen(2); Belinda Willard(3); Robert Wei(1). (1) Department of Chemistry, Cleveland State University, Cleveland Ohio 44115, United States (2) Department of Biological Geological and Environmental Sciences, Cleveland State University, Cleveland Ohio 44115, United States (3) Proteomics Core, Learner Research Institute, Cleveland Clinic, Cleveland Ohio 44195, United States
The aim of this study was to identify proteins that are differentially expressed in response to arsenate [As(V)] in the fresh water unicellular algae Chlamidomonas reinhardtti . The cells were exposed to different concentrations of As (v) ranging from 100-400 mM. When they were exposed to 200 mM As(v), their inhibition of growth was started to exhibit at the second day and remained until the experiments were terminated on the 6th day. First, we found that the conventional approaches for extracting soluble proteins with trichloroacetic acid in combination with acetone were not effective in the removal of chlorophyll and other materials, which contributed to loss of protein and the streakiness of the two dimensional (2D) gels. It was therefore necessary to develop an alternative protocol for extracting the soluble proteins from the cells. A protocol which employs two consecutive precipitation steps of ammonium sulfate ((NH4)2SO4 ) yielded high resolution mapping for proteomic studies. The 40% salt treatment removed chlorophyll and other viscous materials and the second precipitation step [60% (NH4)2SO4 ] recovered >90% of the soluble proteins. The shape of protein spots were much more discrete and the protein distribution patterns were highly reproducible. Seventeen (17) protein spot that are expressed in the 200 mM As(v) treated samples were selected to determine their identity by LC-MS/MS analysis. The expression of these proteins suggested impairment of photosystem, energy production, some aspects of carbohydrate metabolism, and antioxidative stress.
20. Nucleosome effect on deamination rate of 5-methyl cytosine in cyclobutane pyrimidine dimer DNA photoproduct
Qian Song(1), [email protected], One Brookings Drive, St Louis Missouri 63130, United States ; John Stephen Taylor(1). (1) Department of Chemistry, Washington University in St Louis, St Louis Missouri 63130, United States
Spontaneous deamination of cytosine to uracil in DNA is a ubiquitous source of C to T mutations, but occurs with a half-life of ～ 50,000 years. In contrast, cytosine within sunlight-induced cyclobutane dipyrimidine dimers (CPD's), deaminate within hours to days. Methylation of C increases the frequency of CPD formation at PyCG sites which correlates with C to T mutation hotspots in skin cancers. Whereas studies have shown that CPD formation and repair is modulated by its phase relative to the nucleosome, the effect of phase on the deamination rate of a CPD remains unknown. We have reconsituted chicken nucleosome core particles with labeled DNA and determined that a T=mC photodimer facing in towards the nucleosome deaminates 5 times slower compared to native DNA. Preliminary results indicate, however, that the dimer facing out deaminates a much more rapid rate. The implications of these results to UV mutagenesis will be discussed. 21. Small-molecule screening for suppressors of beta-cell apoptosis induced by inflammatory cytokines
Danny Hung-Chieh Chou(1)(2), [email protected], 12, Oxford St, Cambridge MA 02138, United States ; Patrick W Faloon(2); Hyman A Carrinski(2); Nicole E Bodycombe(2); Paul A Clemons(2); Stuart L Schreiber(1)(2); Bridget K Wagner(2). (1) Department of Chemistry and Chemical Biology, Harvard University, Cambridge MA 02138, United States (2) Chemical Biology Program, Broad Institute, Cambridge MA 02138, United States
Inflammatory cytokines are shown to mediate apoptosis of beta cells and reduce functional beta-cell mass in type 1 diabetes (T1D) models. Small molecules capable of suppressing cytokine-induced beta-cell apoptosis could lead to potential avenues for therapeutic intervention. We developed a set of phenotypic cell-based assays and screened for ~30,000 diverse small molecules to identify such small-molecule suppressors. We identified 33 small molecules capable of restoring cell viability in the presence of inflammatory cytokines. Among them, alsterpaullone, a GSK-3beta inhibitor, was able to prevent beta-cell apoptosis and restore glucose-stimulated insulin secretion. We further demonstrated that gene-silencing of GSK-3b via small-interfering RNA could prevent beta-cell apoptosis. Another compound synthesized by diversity-oriented synthesis showed effects to increase beta-cell viability. Only one of the total eight stereoisomers has this effect. Analogs were also synthesized to study the structure-activity relationship and identify the cellular products by proteomics-based SILAC experiments. These suppressors may be good candidates for therapeutic intervention in early stages of T1D.
22. Exocyclic deoxyadenosine adducts of 1,2,3,4-diepoxybutane: Synthesis and effects on DNA structure
Uthpala I Seneviratne(1)(2), [email protected], 790 MCRB, Tretyakova Lab, Mayo Mail code 806, 420 Delaware St SE, Minneapolis MN 55455-5001, United States ; Natalia Y Tretyakova(3)(2). (1) Departments of Chemistry, University of Minnesota, Minneapolis MN 55455, United States (2) Masonic Cancer Center, University of Minnesota, Minneapolis MN 55455, United States (3) Medicinal Chemistry, University of Minnesota, Minneapolis MN 55455, United States
1,2,3,4-Diepoxybutane (DEB) is the ultimate carcinogenic metabolite of 1,3-butadiene (BD), an important industrial and environmental chemical present in urban air and in cigarette smoke. Three exocyclic adenine lesions of DEB, N6,N6-DHB-dA (1), 1,N6-γ-HMHP-dA (2), and 1,N6-α-HMHP-dA (3), have been recently identified in our laboratory (Seneviratne et al Chem. Res.Toxicol. 2010, 23, 118- 133). A postoligomerization approach starting with 6-chloropurine was developed to generate DNA strands containing site and stereospecific adducts 1,2 and 3. We found that both lesions lower the thermodynamic stability of DNA when paired with dT. However, the stability of the DNA duplex containing 2 was increased when adenine residue was placed opposite the lesion, suggesting that DEB-dA adducts preferentially pair with A, potentially leading to A → T transversions. Solution state NMR studies and site specific mutagenesis were conducted to further test the role of exocyclic DEB- dA adducts in mutagenesis.
23. Analysis of r-7,t-8,9,c-10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene in human urine: A biomarker for directly assessing carcinogenic polycyclic aromatic hydrocarbon metabolic activation
Yan Zhong(1), [email protected], 425 East River Road, CCRB Room760, Minneapolis MN, United States ; Stephen S. Hecht(1); Steven G. Carmella(1); J. Bradley Hochalter(1). (1) Masonic Cancer Center, University of Minnesota, Minneapolis MN 55414, United States Polycyclic aromatic hydrocarbons (PAH) are believed to be causative agents for various types of human cancer. Benzo[a]pyrene (BaP) is a prototypic carcinogenic PAH, and requires metabolic activation to elicit its detrimental effects. The major end product of its metabolic activation pathway is r-7,t-8,9,c-10-tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaP-T). We have demonstrated a reliable method for quantitation of BaP-T in human urine. The urine was treated with β-glucuronidase and sulfatase, and purified by reverse phase polymeric and phenylboronic acid solid-phase extraction. The sample was silylated and analyzed by gas chromatography-negative ion chemical ionization- tandem mass spectrometry. BaP-T was readily detected in all urine samples so far including 33 exposed creosote workers (27+16 fmol/mL of urine) and 10 smokers (0.61+0.33 fmol/mL of urine). In addition, BaP-T in 33 creosote workers strongly correlated with two other PAH biomarkers, trans-anti- phenanthrene-tetrol and 1-hydroxypyrene, with correlation coefficients r = 0.86 and 0.82 respectively. This highly sensitive and selective method for quantitation of BaP-T may be important as a direct phenotyping approach to assess individual differences in susceptibility to PAH-related cancer.
24. Repair of the hydantoin lesions guanidinohydantoin (Gh) and spiroiminodihydantoin (Sp) by bacterial nucleotide excision repair
Paige L. McKibbin(1), [email protected], One Shields Avenue, Davis California 95616, United States ; Bennett Van Houten(2); Sheila S. David(1). (1) Department of Chemistry, University of California, Davis, Davis California 95616, United States (2) Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh Pennsylvania 15213, United States
The low redox potential of guanine makes it susceptible to oxidative damage, commonly forming 7,8- dihydro-8-oxoguanine (8-OxoG). Interestingly, 8-OxoG can be further oxidized to the hydantoin lesions, guanidinohydantion (Gh) and spiroiminodihydantoin (Sp). These hydantoin lesions are interesting due to their shape and mutagenic capacity. They are helix destabilizing, potent replication blocking lesions, substrates for base excision repair (BER) in vitro, and have been detected in cells. The nucleotide repair pathway (NER) is known to respond to bulky DNA adducts and lesions that can stall replication forks. In vitro single turnover kinetics was performed using recombinant UvrABC enzymes to measure the rate of excision of 8-OxoG, Gh, and Sp lesions from a 50 mer oligonucleotide duplex. Experiments revealed very modest excision for 8-OxoG and robust activity for Gh and Sp lesions. This suggests that hydantoin repair, at least in part, can be attributed to both the NER and BER pathways.
25. Structural characterization of formaldehyde-induced DNA-protein cross-links
Kun Lu(1), [email protected], Campus Box 7431, Chapel Hill NC 27599, United States ; Wenjie Ye(1); Li Zhou(2); Leonard B. Collins(1); Xian Chen(2); Avram Gold(1); Louise M Ball(1); James A. Swenberg(1). (1) Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill NC 27599, United States (2) Department of Biochemistry, University of North Carolina, Chapel Hill NC 27599, United States
Exposure to formaldehyde results in the formation of DNA-protein cross-links (DPCs) as a primary genotoxic effect. However, the structures of these cross-links have not yet been elucidated. We have characterized formaldehyde-induced cross-links of Lys, Cys, His, and Trp with dG, dA, and dC. Reaction of formaldehyde with Lys and dG gave the highest yield of cross-linked products, followed by reaction with Cys and dG. Yields from the other coupling reactions were lower by a factor of 10 or more. Detailed structural examination by NMR and mass spectrometry established that the cross- links between amino acids and deoxynucleosides and their oligomers involve a formaldehyde-derived methylene bridge. These structures will provide a basis for investigation of the characteristics and properties of DPCs formed in vivo and will be helpful in identifying biomarkers for the evaluation of formaldehyde exposure both at the site of contact and at distant sites. 26. Reactive carbonyl species derived from omega-3 and omega-6 fatty acids
Yu Wang(1), [email protected], 77 Massachusetts Ave. 56-731, Cambridge MA 02139, United States ; John S. Wishnok(1); Steven R. Tannenbaum(1)(2). (1) Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge MA 02139, United States (2) Department of Chemistry, Massachusetts Institute of Technology, Cambridge MA 02139, United States
Inflammation-related reactive oxygen species (ROS) and reactive nitrogen species (RNS) are associated with the development of cancer. ROS and RNS can directly damage bio-macromolecules such as proteins, DNA and lipids. Lipid peroxidation, however, can result in reactive carbonyl species (RCS) that can also modify proteins and DNA. In contrast to an extensive literature on the modification of proteins and DNA from omega-6 fatty acids, there are few studies on RCS generation from different fatty acids. Therefore, we have begun a comparison between omega-3 and omega-6 fatty acids. LC/MS/MS analysis of carbonyl-dinitrophenylhydrazine (DNPH) standards yielded characteristic fragment ions at m/z 151, 167, and 163. Linolenic acid and linoleic acid autoxidation products were then derivatized with DNPH and analyzed by LC/MS/MS in the precursor-ion mode using these three ions, revealing products common to both fatty acids as well some unique to each. We are in the process of characterizing these substances.
27. Beas2B cells as a model of polycyclic aromatic hydrocarbon metabolism in noncancerous human lung: Metabolic consequences of [3H]-B[a]P and [3H]-B[a]P-7,8- dihydrodiol treatment
Mary E Kushman(1)(2), [email protected], 135 John Morgan, 3620 Hamilton Walk, Philadelphia PA 19104, United States ; Stacy L Gelhaus(2)(3); Trevor M Penning(1)(2). (1) Center of Excellence in Environmental Toxicology, University of Pennsylvania, Philadelphia PA 19104, United States (2) Department of Pharmacology, University of Pennsylvania, Philadelphia PA 19104, United States (3) Center for Cancer Pharmacology, University of Pennsylvania, Philadelphia PA 19104, United States
Beas2B cells, an immortal but noncancerous human lung cell line, serve as a model in which to study PAH metabolism in non-transformed lung epithelium. Consistent with P450 expression profiling and detection of functional enzyme activity, Beas2B cells are competent in [3H]-B[a]P and [3H]-B[a]P-7,8- dihydrodiol metabolism. However, the metabolic profile obtained in this cell line was not significantly different between non-induced and TCDD-induced cells even though induction of P4501A1 and 1B1 mRNA was observed. Pretreatment of cells with TCDD for 24 hours also failed to eliminate the lag phase of PAH metabolism, but consumption of B[a]P was slightly enhanced. Metabolism of 3H-B[a]P yielded B[a]P-9,10-dihydrodiol as the predominant metabolite, while metabolism of 3H-B[a]P-7,8- dihydrodiol yielded B[a]P-tetrol 1 predominant. B[a]P-3,6-dione was detected. All metabolite identity was confirmed by LC/MS analysis. Taken together, these results suggest involvement of P450s and/or peroxidases in PAH metabolism in this cell line other than P4501A1/1B1. (Supported by 1R01- ES015857 to TMP).
28. Effect of O2-arylated diazeniumdiolates on cellular glutathione status
Ryan J Holland(1), [email protected], Fort Detrick, building 538, Frederick Maryland 21771, United States ; Joseph E Saavedra(2); Larry K Keefer(1); Anna E Maciag(2); Harinath Chakrapani(1). (1) Chemistry Section, Laboratory of Comparative Carcinogenesis, National Cancer Institute at Frederick, United States (2) Basic Research Program, SAIC-Frederick, National Cancer Institute at Frederick, United States Nitric oxide (NO) is central player in a variety of normal physiological processes and disease states, including cancer, culminating in the pursuit of nitric oxide prodrugs as potential drug candidates. Diazeniumdiolate-based nitric oxide (NO)–releasing prodrugs are a growing class of promising NO- based therapeutics. Of particular interest is a sub-class of O2-arylated diazeniumdiolates, as they are activated by nucleophiles, in particular glutathione, thus liberating their NO payload. Such compounds have been demonstrated to inhibit cell growth as well as stimulate pro-apoptotic pathways. This study highlights features of the chemical/biological cross-talk of the O2-arylated diazeniumdiolate signaling cascade. Acting as both electrophiles and inducers of nitrosative stress, the introduction of these compounds into the cellular environment can result in a distinct alteration in the cellular redox status as well as extensive protein glutathionylation. In the case of O2-arylated (bis)diazeniumdiolates, protein glutathionylation proceeds via two independent pathways resulting in structurally distinct modifications.
29. Stereoselective formation of N2-dG:N2-dG carbinolamine interstrand cross-link arising from trans-4-Hydroxynonenal derived exocyclic (6S,8R,11S) 1,N2-dG adduct
Hai Huang(1)(2)(3), [email protected], Vanderbilt University, Nashville TN 37205, United States ; Ivan D. Kozekov(1)(2)(3); Albena Kozekova(1)(2)(3); Surajit Banerjee(1)(2)(3); Carmelo J. Rizzo(1)(2)(3); Michael P. Stone(1)(2)(3). (1) Department of Chemistry, Vanderbilt University, Nashville TN 37205, United States (2) Center in Molecular Toxicology, Vanderbilt University, Nashville TN 37205, United States (3) Center for Structural Biology, Vanderbilt University, Nashville TN 37205, United States trans-4-Hydroxynonenal (HNE) is a peroxidation product of w-6 polyunsaturated fatty acids. Michael addition of HNE to deoxyguanosine yields four diastereomeric exocyclic 1,N2-dG adducts. The (6S,8R,11S) 1,N2-dG adduct opens to a N2-dG aldehydic adduct when placed opposite dC and forms a N2-dG:N2-dG interstrand cross-link in the 5'-CpG-3' sequence. The interstrand cross-link exists exclusively as a carbinolamine linkage with (6S,8R,11S)-configuration. The cross-linked duplex maintains an overall B-DNA geometry with minimal perturbations located at the cross-linking region. Hydrogen bonds between the HNE hydroxyl groups and cytosine O2 play crucial roles in cross-link formation. They are proposed to stabilize the cross-link, account for the stereoselectivity, and result in the higher yield of DNA cross-linking by the HNE adduct as compared to the corresponding acrolein and crotonaldehyde adducts. The presence of this interstrand cross-link in vivo is anticipated to interfere with DNA replication and transcription, thereby contributing to the etiology of human disease. Funded by NIH grant PO1 ES-05355 (I.D.K., C.J.R., and M.P.S.).
30. Sequence-dependent oxidation of 8-oxoguanine in DNA
Kok Seong Lim(1), [email protected], 77 Massachusetts Avenue, NE 47-296, Cambridge MA 02139, United States ; Liang Cui(1); Koli Taghizadeh(2); John S. Wishnok(1)(2); Steven R. Tannenbaum(1)(2)(3); Peter C. Dedon(1)(2). (1) Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge MA 02139, United States (2) Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge MA 02139, United States (3) Department of Chemistry, Massachusetts Institute of Technology, Cambridge MA 02139, United States
Emerging evidence suggests that DNA sequence context affects the chemistry of DNA damage. To address this problem, we recently developed a quadrupole/time-of-flight (QTOF) mass spectrometric method to define the spectrum of products arising from 8-oxoguanine oxidation in different 3- nucleotide DNA sequence contexts. We observed no sequence-dependence in the spectrum of products induced by nitrosoperoxycarbonate, including dehydroguanidinohydantoin (DGh), guanidinohydantoin, spiroiminodihydantoin, imidazolone (Iz), oxaluric acid and oxazolone (Oz). However, parallel studies with riboflavin-mediated photooxidation revealed a strong sequence- dependent effect on reactivity in the following order: CXA = AXG > GXG > TXC > AXC. DGh was the most abundant product in all sequence contexts, except -AXG- and -GXG- in which Iz appeared to be as abundant as DGh immediately after oxidation. The kinetics of conversion of Iz to Oz will be monitored using the QTOF method. Sequence-dependent variations in DNA damage chemistry likely play a role in determining damage and mutational hotspots in the human genome.
31. Redox-dependent anti-inflammatory lipid mediator generation and actions
Bruce Freeman(1), [email protected], 1356 BST, Lothrop Street, Pittsburgh PA 15261, United States . (1) Dept Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh PA 15261, United States
The posttranslational modification of proteins (PTPM), allows cells to dynamically regulate metabolism, growth, differentiation and immune responses. PTPM is a critical component of NO and redox signaling. In addition to activating guanylate cyclase via heme-iron coordination, NO reacts with reactive oxygen intermediates to yield species such as peroxynitrite and nitrogen dioxide, accelerating the chemical reaction rate and breadth of reactions that transduce redox signaling. One class of byproducts of these reactions, electrophilic unsaturated fatty acids, induce PTPM by reacting with protein thiols and other nucleophilic amino acids such as histidine. In particular, multiple transcriptional regulatory factors have prominent amino acids that are electrophile-reactive, providing cells with a capability for stress-related adaptive signaling reactions. This lecture addresses mechanisms of formation, molecular targets, cell signaling responses and physiological actions induced by low concentrations of electrophilic fatty acids and supports the concept electrophile- mediated PTPM reactions link cell function with inflammatory and metabolic status.
32. Identifying and quantifying cysteome modifications in response to NO donor and quinone donor drugs
Gregory R. J. Thatcher(1), [email protected], 833 S. Wood St., Chicago IL 60612, United States ; R. Esala P. Chandrasena(1); Vaishali Sinha(1); Gihani T. Wijewickrama(1); Tareisha Dunlap(1); Sujeewa C. Piyankarage(1); Hua Xu(1); Judy Bolton(1); Isaac T. Schiefer(1); Praneeth D. Edirisinghe(1); Yueting Wang(1). (1) University of Illinois at Chicago, United States
Reversible reactions of cysteine with oxidants and reactive nitrogen species derived from NO appear to mediate both pharmacologic and toxicologic regulatory mechanisms involving protein thiols (the cysteome). NO donating non-steroidal anti-inflammatory drugs (NO-NSAIDs) induce a variety of stress response pathways in cell culture; cysteome modification was compared for NO-NSAIDs and other classes of NO donor drugs identifying target proteins and quantifying modification using novel LC-MS/MS proteomics approaches. One family of NO-NSAIDs was bioactivated to a quinone that itself modified the cysteome; other drugs bioactivated to quinones were found to reversibly modify the cysteome. LC-MS/MS approaches to identification of quinone targets will be presented in addition to preliminary attempts to link biological activity and toxicity to the protein targets and type of cysteome modification elicited by these diverse electrophiles. Understanding the reactivity-activity relationship is essential to tune the balance between toxicity and cytoprotection pathways for new drug design.
33. Systems analysis of electrophile-induced gene expression: Role of HSF1-affected genes in cellular viability
Aaron T Jacobs(1), [email protected], 34 Rainbow Dr, Hilo HI 96720, United States . (1) Department of Pharmaceutical Sciences, University of Hawaii at Hilo, College of Pharmacy, Hilo HI 96720, United States The electrophilic lipid peroxidation end-product, 4-hydroxynonenal (HNE) has profound effects on gene expression in affected cells. We have taken a systems approach toward identifying and correlating altered signaling pathways; impacts on global gene expression; and consequences for cellular viability caused by HNE treatment. This approach identified a major role for the transcription factor, heat shock factor 1 (HSF1) in protecting cells from electrophile-induced apoptosis. Notably, HSF1 silencing by RNAi reduces levels of anti-apoptotic proteins Bcl-X(L), Mcl-1, and Bcl-2. This effect appears to be mediated by the Hsp70 co-chaperone, BAG3 (Bcl-2-associated athanogene domain 3), as silencing BAG3 causes a similar reduction in anti-apoptotic Bcl-2 family proteins. The available data indicate that BAG3 is HSF1 inducible and points to the interplay of HSF1, chaperones and Bcl-2 family proteins in mitigating apoptosis induced by lipid electrophiles.
34. Apoptosis signal-regulating kinase 1 (ASK1), a sensor-trigger for proapoptotic signaling in electrophile stress
Daniel C Liebler(1), [email protected], U1213 MRBIII, 465 21st Avenue South, Nashville TN 37232-6350, United States . (1) Department of Biochemistry, Vanderbilt University School of Medicine, Nashville TN 37232, United States
The mitogen activated protein kinase (MAPK) pathway is activated in diverse disease processes and chemical toxicities. Activation of the c-jun-N-terminal kinases (JNKs) is a critical event in stress signaling and is regulated by the MAP3K enzyme apoptosis signal-regulating kinase 1 (ASK1). This pathway is activated by electrophile probes, which activated the ASK1 substrate MKK4 and phosphorylation of the downstream substrates p38 MAPK and JNK in a concentration-dependent manner in HEK293 cells. We mapped adduction sites on ASK1 exposed to electrophiles in vitro and in intact cells. Treatment of ASK1 with electophiles did not inhibit the activity of the kinase, but dissociated the ASK1-TRX1 complex in cells, which results in ASK1 activation. Disruption of the complex was caused by ASK1 adduction, rather than TRX1 adduction. The results indicate that ASK1 serves as both a sensor of electrophile damage and a trigger for activation of the MAPK pathway.
35. Role of lipoproteins on the biological activity and pharmacokinetics of hydrophobic drugs
Kishor M. Wasan(1), [email protected], 2146 East Mall, Vancouver BC, Canada . (1) Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver BC V6T 1Z3, Canada
In contrast to many traditional pharmaceutical agents which exhibit a high degree of aqueous solubility, new drug candidates are frequently highly lipophilic, poorly water soluble compounds. The aqueous milieu of the blood provides a thermodynamically unfavorable environment for the disposition of such hydrophobic drugs, a limitation which can be overcome by the association with circulating lipoproteins. Elucidation of the mechanisms that dictate drug-lipoprotein association and blood to tissue partitioning of lipoprotein-encapsulated drug may yield valuable insight into the factors governing the pharmacological activity and potential toxicity of these compounds. This talk will discuss the impact of hydrophobic drug-lipoprotein interactions on pharmacokinetics and biological activity of a variety of hydrophobic compounds and the importance of utilizing this information in drug discovery and development programs.
36. Interactions of dietary supplement mixtures and pure compounds on phase I drug metabolism
David J. Kroll(1), [email protected], 1801 Fayetteville Street, Durham NC 27707, United States . (1) North Carolina Central University, United States The US Food and Drug Administration regulates botanical supplements as foods but some exhibit pharmacological effects alone or interact with prescription pharmaceuticals. For example, hyperforin from St. John's wort increases cyclosporine and indinavir clearance via CYP3A4 induction. Interaction of botanicals and food products is particularly relevant for drugs exhibiting a narrow therapeutic range. Milk thistle (Silybum marianum) extract has been used historically as a hepatoprotectant. Reports of its CYP3A4 inhibition led us to collaborate with experts in pharmacognosy (N. Oberlies, UNC- Greensboro) and drug metabolism (M. Paine, UNC-Chapel Hill) to conduct supplemental studies on Dr. Paine's NIH project originally designed to investigate cranberry juice interactions with warfarin. Milk thistle extracts and pure compounds are being tested for inhibitory activity against CYP2C9, CYP2D6, and CYP3A4. Results to date indicate that select milk thistle compounds inhibit warfarin metabolism, providing the basis for cautionary information to be communicated to consumers and health care professionals.
37. Impact of food on the function of oral drug delivery systems
Rebecca L Carrier(1), [email protected], 342 Snell Engineering Center, 360 Huntington Avenue, Boston MA 02115, United States . (1) Department of Chemical Engineering, Northeastern University, Boston MA 02115, United States
The capability of food to affect the bioavailability of poorly water soluble drugs and the function of oral drug delivery systems, such as controlled release systems and solubilization technologies (e.g., cyclodextrins) is widely recognized. In particular, lipids have been considered a powerful tool for improving oral bioavailability and have been extensively studied as drug delivery agents, such as self- emulsifying drug delivery systems. However, there is an incomplete understanding of functional mechanisms of lipid-based drug delivery systems, and a lack of a general in vitro model that is able to predict a priori the in vivo performance of drug-lipid systems. The colloidal phases formed by lipid- based drug delivery systems are similar to and influenced by food and digestion products; the nature of these colloidal phases and their ultimate influence on the fate (dissolution kinetics, free and lipid- associated drug, absorption) of drug delivered to the GI tract are being studied.
38. Transporter-mediated drug-herb and drug-food interactions
Marilyn E. Morris(1), [email protected], 517 Hochstetter Hall, Amherst NY 14260, United States . (1) Department of Pharmaceutical Sciences, University at Buffalo, State University of New York, Amherst NY 14260, United States
Diet and dietary supplements can significantly impact drug disposition by interacting with drug transporters, due to the significant expression levels of drug transporters in organs important in drug absorption, distribution, and elimination. In this presentation, the interactions of diet/dietary supplements and some classes of nutrients rich in fruits and vegetables (e.g., flavonoids and isothiocyanates) with drug transporters will be discussed. The use of herbal preparations containing high doses of flavonoids for health maintenance has become very popular, raising the potential for interactions with drug therapies. Additionally, low doses of multiple flavonoids can have additive or synergistic effects on drug transport, representing a significant area of current research. This presentation will focus on three major members of the ATP Binding Cassette (ABC) efflux transporter family (namely P-glycoprotein, multidrug resistance proteins, and breast cancer resistance protein) and on members of the Solute Carrier (SLC) uptake transporters (including organic anion transporting polypeptide). 39. Mechanisms of cytochrome P450-mediated food-drug interactions: Mechanism-based inactivators and the grapefruit juice effect
Paul Hollenberg(1), [email protected] (e-mail), 2301 MSRB III, 1150 W. Medical Center Drive, Ann Arbor MI 48109, United States . (1) University of Michigan Medical School, United States
The cytochrome P450s (P450s) catalyze the biotransformation of many drugs. Various foods and herbs alter drug metabolism by P450s due to the presence of compounds which result in mechanism- based inactivation of the P450s. Grapefruit juice significantly increases the oral bioavailability of numerous drugs metabolized by P450s due to bergamottin (BG) and 6,7-dihydroxybergamottin, two furanocoumarins that are effective mechanism-based inactivators of human P450s 3A4, 3A5, and 2B6. BG inactivated these P450s in a reconstituted system consisting of purified human P450, P450 reductase, cytochrome b5, and phospholipids. The inactivation was time- and concentration- dependent and required BG metabolism. Loss of catalytic activity exhibited pseudo-first-order kinetics and kinact and KI values were calculated. Depending on the P450, the loss in activity was due to heme modification, protein modification, or both. The structures of the primary metabolites of BG and the reactive intermediates trapped with glutathione were determined.
40. Physicochemical and pharmacological properties as predictors of drug safety and success
James Empfield(1), [email protected], 1800 Concord Pike, PO Box 15437, Wilmington DE 19850, United States . (1) Chemistry, Astra-Zeneca Pharmaceuticals, Wilmington DE 19850, United States
The success rate of drug candidates progressing through R&D and reaching the market is low. Therefore understanding the major causes of drug attrition and developing approaches to address these is of prime importance to the pharmaceutical industry. Over the past decade toxicity has emerged as the major cause of attrition in early development. This talk will focus on approaches that medicinal chemists can take to reduce drug attrition. Specific emphasis will be placed on the link between physicochemical properties and toxicity as well as the use of pharmacological profiling s a means to manage the odds of success.
41. Aromatic ring count and compound developability
Simon J.F. Macdonald(1), [email protected], Gunnels Wood Road, Stevenage Hertfordshire SG1 2NY, United Kingdom ; Timothy J Ritchie(1); Rob Young(1); Alan Hill(1). (1) Respiratory CEDD, Medicines Research Centre, GlaxoSmithKline, Stevenage Hertfordshire SG1 2NY, United Kingdom
Aromatic rings are almost ubiquitous in drug molecules. There is now a plethora of simple and robust synthetic coupling methodologies alongside a very wide range of commercially available starting materials. But what are the consequences of including multiple aromatic rings in drug molecules ? Based on data from tens of thousands of molecules in the GSK corporate collection, we will present analyses on the impact of aromatic ring count and aromatic ring type on developability properties (hERG activity, solubility, p450, lipophilicity).
42. Molecular matched pairs derived QSAR for the optimization of ADMET properties
Stephen Johnson(1), [email protected], Province Line Road, Princeton New Jersey 08543, Albania ; Malcolm Davis(1); Shana Posy(1); Brian Claus(1). (1) CADD, Applied Biotechnology, Bristol- Myers Squibb Research and Development, Princeton New jersey 08543, United States QSAR models are often described as either “global” or “local” models based on the degree of chemical homogeneity of the training data. We present an alternate view of local QSAR in which the difference in activity between pairs of structurally similar compounds is related to differences in physicochemical properties. Since pairs of compounds are related by particular molecular transformations, the model is local even though the collection of training compounds may be quite diverse overall. We will demonstrate the approach using several ADME and toxicity endpoints considered during the discovery process.
43. Moving beyond rules: The development of a central nervous system multi-parameter optimization (CNS MPO) approach to enable alignment of drug-like properties
Travis T. Wager(1), [email protected], 558 Eastern Point Road, Groton CT 06340, United States ; Xinjun Hou(1); Patrick R. Verhoest(1); Anabella Villalobos(1). (1) Neuroscience Medicinal Chemistry, Pfizer Global Research and Development, Groton CT 06340, United States
The interplay among commonly used physicochemical properties in drug design was examined and utilized to create a prospective design tool focused on the alignment of key drug-like attributes. Using a set of six physicochemical parameters (ClogP, ClogD, MW, TPSA, HBD, and pKa), a drug-likeness Central Nervous System Multi-Parameter Optimization (CNS MPO) algorithm was built and applied to a set of marketed CNS drugs (N = 119) and Pfizer CNS candidates (N = 108) as well as to a large diversity set. A relationship between an increasing CNS MPO score and alignment of key in vitro attributes of drug discovery (permeability, P-gp efflux, metabolic stability, and safety) was seen in all three sets. The CNS MPO scoring function offers advantages over hard cut-offs or utilization of single parameters to optimize structure-activity relationships (SAR) by expanding medicinal chemistry design space through a holistic assessment approach.
44. Affinity purification of kidney enzymes that bind mercapturic acids
Erin Veldman(1), [email protected], New Science Complex, Stone Road, Guelph Ontario N1G 2W1, Canada ; P. David Josephy(1). (1) Department of Molecular and Cellular Biology, University of Guelph, Guelph Ontario N1G 2W1, Canada
Mercapturic acid (N-acetylcysteine S-conjugate) formation is a major route for the metabolism and elimination of glutathione conjugates, but the enzymes of the mercapturic acid pathway are still not completely characterized. We have developed an affinity purification strategy for identifying enzymes that act on mercapturic acids. Mono- and dimercapturates of dopamine were synthesized by an electrochemical method (Shen et al., Chem. Res. Toxicol. 9, 1117-1126, 1996), purified by HPLC, and covalently coupled to an agarose column (AminoLink™, Thermo Scientific) via the dopamine ethylamino substituent. Solubilized porcine kidney cortex microsomes were applied to the column. Bound proteins were eluted with dopamine mercapturate, trypsinized, and identified by LC-MS-MS. Identified proteins included flavin-containing monooxygenase, monoamine oxidase B, membrane metallo-endopeptidase, gamma-glutamyltranspeptidase 1, membrane alanine aminopeptidase, fatty acid amide hydrolase, etc. We are now examining the interactions of these proteins with mercapturic acids.
45. Translesion synthesis past the γ-radiation-induced guanine- thymine intrastrand crosslink G[8,5-Me]T
Paromita Raychaudhury(1), [email protected], 55 N Eagleville Rd, Storrs Connecticut 06269, United States ; Ashis K Basu(1). (1) Department of Chemistry, University of Connecticut, Storrs Connecticut 06269, United States γ-Radiation-induced intra-strand guanine-thymine cross-link, G[8,5-Me]T, was shown to be mutagenic in mammalian cells. The lesion is a strong block of replicative DNA polymerases, but Y-family DNA polymerases can bypass the lesion. In this study we compared the nucleotide insertion opposite the cross-linked bases as well as further extension by three Y-family DNA polymerases. We found that yeast DNA polymerase η (yPol η) preferably incorporated dAMP opposite the cross-linked guanine. Further extension was one-hundred-fold less efficient for the G*:A pair compared to the G*:C pair. Both Dpo4 and human DNA polymerase η (hPol η), incorporated dCMP opposite the cross-linked guanine and extension was more efficent for the G*:C pair compared to the G*:A pair. A plasmid- based DNA sequencing assay was developed to evaluate the nucleotide incorporation pattern of the full-length products by these enzymes. The ypol η was found to be more error prone than the other two enzymes. Targeted G→T was the major type of mutation by all three DNA polymerases.
46. Structural studies of a repair-resistant dibenzo[a,l]pyrene-derived DNA adduct in a nucleosome
Yuqin Cai(1), [email protected], Brown Building, Room 1009, 100 Washington Square East, New York N.Y. 10003, United States ; Nicholas E. Geacintov(2); Suse Broyde(1). (1) Department of Biology, New York University, New York N.Y. 10003, United States (2) Department of Chemistry, New York University, New York N.Y. 10003, United States
Nucleotide excision repair (NER) of DNA damage in eukaryotic cells requires access to the lesions that are packaged in nucleosomes within chromatin. However, the effect of the nucleosomal environment on properties of bulky adducts and the accessibility to NER proteins of such lesions remains unknown. As an example of a bulky adduct, we investigated the 14S (−)–trans–anti– DB[a,l]P–N6–dA adduct. This lesion is derived from the extremely potent tumorigen, dibenzo[a,l]pyrene, and is NER–resistant in a human HeLa cell–extract assay without nucleosomes. We investigated the dynamic structural properties of this adduct in a nucleosome core particle utilizing 50–ns MD simulations. Detailed analyses show that the DB[a,l]P ring system is well– intercalated on the 3´–side of the damaged adenine and well–stacked with adjacent base pairs, similar to the structure in duplex DNA without protein. These results suggest that this adduct would remain repair–resistant in the more cell–like environment of the histone–containing nucleosome.
47. Base flipping free energy profiles for damaged DNA
Han Zheng(1), [email protected], Brown Building, Room 1009, 100 Washington Sqaure East, New York New York 10003, United States ; Yuqin Cai(1); Yanzi Zhou(2); Nicholas E. Geacintov(2); Suse Broyde(1); Yingkai Zhang(2); Shenglong Wang(2). (1) Department of Biology, New York University, New York New York 10025, United States (2) Department of Chemistry, New York University, New York New York 10003, United States
Recognition of bulky DNA lesions by the nucleotide excision repair machinery is believed to be initiated by local thermodynamic destabilization; this facilitates β- hairpin intrusion at the damage site by the human XPC⁄HR23B recognition factor, accompanied by flipping of two partner bases to the lesion, as suggested by the crystal structure of the yeast orthologue (Min J.H. et al., 2007). To computationally investigate this proposed mechanism, we have employed the recently developed Repository Based Adaptive Umbrella Sampling method (Zheng H. et al., 2009) for computing base flipping free energies. This method makes feasible the challenging computational task of obtaining two dimensional free energy profiles, such as the flipping of two bases simultaneously. Flipping free energy profiles for unmodified and modified duplexes for which repair data with human HeLa cell extract assays are available will be presented. 48. Experimental approach to enhance parent ion fragmentation for metabolite identification studies: Application of stepped CID and HCD in an Orbitrap-XL
Upendra A Argikar(1), [email protected], 250 Massachusetts Avenue, 1B-204, Cambridge MA 02139, United States ; Jennifer L Bushee(1). (1) Metabolism and Pharmacokinetics, Novartis Institutes for Biomedical Research Inc., Cambridge MA 02139, United States
Poor parent compound fragmentation of new chemical entities is often a hurdle for conducting biotransformation studies. In order to empower structural elucidation, a high energy collisional dissociation(HCD)-MS/MS method was developed in conjuncture with a collision dissociation energy (CID)-MS. LC-MS experiments were carried out for several commercial compounds on an LTQ- Orbitrap-XL®. Full MS spectra, HCD MS/MS and data dependent CID-MS were carried out in an accurate mass mode. The HCD cell generated cleaner and richer MS/MS spectra and even led to identification of low mass fragments. SNCE enabled HCD-MS/MS and CID-MS data were qualitatively superior to regular CID MS. An LC-MS method with stepped HCD- and CID-enabled parent ion fragmentation resulted in comprehensive structural information and was remarkably better than a generic CID based LC-MS method. By improving the fragmentation efficiency, it is evident that such an acquisition procedure can enhance identification of metabolic soft spots.
49. Effects of pharmaceutical excipients on 4-methyl umbelliferone glucuronidation: An application for compounds with low solubility
Upendra A Argikar(1), [email protected], 250 Massachusetts Avenue, 1B-204, Cambridge MA 02139, United States ; Guiqing Liang(1); Jennifer L Bushee(1); Vinayak Hosagrahara(1); Wendy Lee(1); Lipa Shah(2); Sudhakar Garad(2). (1) Metabolism and Pharmacokinetics, Novartis Institutes for Biomedical Research Inc., Cambridge MA 02139, United States (2) Chemical and Pharmaceutical Profiling, Novartis Institutes for Biomedical Research Inc., Cambridge MA 02139, United States
The low amount of organic solvents (<1%) tolerated in microsomal incubations often is not sufficient to solubilize substrates and essential additives, such as alamethicin, thus affecting the accuracy of the data generated. The present study is aimed at studying the application of pharmaceutical excipients to aid solubilization of compounds under in vitro glucuronidation incubations without affecting the reaction kinetics. Kinetics of 4-MU glucuronidation in the presence of excipients were compared to that in control incubations lacking any excipients. In addition, IC75 values were calculated for each excipient. We conclude that hydroxy-propyl-b-cyclodextrin may be employed in in vitro glucuronidation incubations up to 0.5% w/v without affecting intrinsic clearance of 4-MU. Our studies show, for the first time, that pharmaceutical excipients can be successfully used to conduct in vitro glucuronidation studies of substrates with poor aqueous solubility.
50. Translesion synthesis past the 2,6-diamino-4-hydroxy-N5-(methyl)-formamido-pyrimidine (MeFAPy-dG) adduct in oligonucleotides by DNA polymerases and selective incision of the α- anomer of MeFAPy-dG by Escherichia coli Endo IV
Plamen P Christov(1), [email protected], Stevenson Center 7650, Nashville TN 37235, United States ; Carmelo J Rizzo(1). (1) Department of Chemistry, Vanderbilt University, Nashville TN 37235, United States
Methylating agents react with DNA to give a cationic N7-methyl deoxyguanosine adduct, which can undergo base-induced opening of the imidazole ring to form 2,6-diamino-4-hydroxy-N5-(methyl)- formamidopyrimidine (MeFAPy-dG). We have developed chemistry for site-specific synthesis of oligonucleotides containing the MeFAPy-dG lesion. The β-MeFAPy-dG lesion can undergo anomerization leading to the formation of α-anomers. We found that the α-MeFAPy-dG anomer can be selectively incised by Escherichia Coli Endo IV. We utilized the selective Endo IV incision in combination with in vitro polymerase extension assays and found that the α-MeFAPy-dG anomer is a strong block to replication catalyzed by Klenow fragment and yeast DNA Polymerase η; however, Sulfolobus solfataricus Dpo4, human Polymerases η and κ can bypass the α-MeFAPy-dG lesion.
51. Quinone-enhanced reduction of nitric oxide by xanthine/xanthine oxidase
Antonio E. Alegria(1), [email protected], 100 Road 908, Humacao Puerto Rico, Puerto Rico ; Pedro Sanchez-Cruz(1). (1) Department of Chemistry, University of Puerto Rico, Humacao Puerto Rico 00791, Puerto Rico
The redox potential of nitric oxide is ~-0.8 V. Thus, outer sphere reduction of NO would be very dificult, if not impossible, in biological systems. However, we have found that the quinones 1,4- naphthoquinone, methyl-1,4-naphthoquinone, tetramethyl-1,4-benzoquinone, 2,3-dimethoxy-5- methyl-1,4-benzoquinone, 2,6-dimethylbenzoquinone, 2,6-dimethoxybenzoquinone and 9,10- phenanthraquinone enhance the rate of nitric oxide reduction by xanthine/xanthine oxidase in nitrogen-saturated phosphate buffer (pH 7.4). Maximum initial rates of NO reduction (Vmax) and the amount of nitrous oxide produced after 5 minutes of reaction increase with quinone one- and two- electron redox potentials measured in acetonitrile. One of the most active quinones of those studied is 9,10-phenanthraquinone with a Vmax value 10 times larger than that corresponding to the absence of quinone, under the conditions of this work. The NO reduction mechanism should involve an inner sphere NO reduction step for this to be possible. Since NO production is enhanced under hypoxia and under certain pathological conditions, the observations obtained in this work are very relevant to such conditions.
52. Quantification of four oxidized guanine lesions from reaction of DNA with 3- morpholinosydnonimine
Liang Cui(1), [email protected], 77 Massachusetts Avenue, Room 56-731, Cambridge MA 02139, United States ; Erin G Prestwich(1); Laura J Trudel(1); John S Wishnok(1); Peter C Dedon(1); Steven R Tannenbaum(1). (1) Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge MA 02139, United States
Spiroiminodihydantoin (Sp), Guanidinohydantoin (Gh), Oxazolone (Ox) and Nitroimidazole (Ni) are four pro-mutagenic oxidation products of guanine and potential markers of DNA oxidation from inflammation and disease. CT-DNA was treated with 1mM SIN-1, and 13C15N-labled Sp, Gh and Ox were synthesized. SIN-1 oxidized CT-DNA enzymatically digested to nucleosides, and digested DNA was prepurified by HPLC. The oxidation products were analyzed by HPLC-tandem mass spectrometry in the multiple reaction monitoring (MRM) mode, and the detection limit was 10 fmol for Sp and Gh, and 20 fmol for Ox and Ni. All of the four products were formed from 40 to 100 times more in treated DNA than in control DNA. This method is used to detect in vivo lesions in DNA from cell and tissue samples with simultaneous measurement of 8-oxodG and incorporation of isotopomeric 8-oxodG in the assay procedure to account for artifactual formation of Sp, Gh, Ox and Ni.
53. Quantitative mapping of cysteome for the protein S-nitrosylation
R. Esala P. Chandrasena(1), [email protected], 833 S. Wood Street,, Chicago, IL, 60612, United States ; Gihani T. Wijewickrama(1); Vaishali Sinha(1); Hua Xu(1); Gregory R. J. Thatcher(1). (1) Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago IL 60612, United States Substantial evidence has been provided in the literature that unregulated protein S-nitrosylation cause neurological diseases in humans. The biotin switch technique combined with mass spectrometry has been used to identify S-nitrosylated proteins in complex biological samples; these techniques involved extensive sample preparation steps before the mass spectrometric analysis and the extent of protein S-nitrosylation was not investigated. We have developed a novel mass spectrometric technique named d-Switch to unambiguously identify and quantify the S-nitrosylated proteins in cells. We have treated the SH-SY5Y cells with CysNO and d-Switch was conducted to identify and quantify the S-nitrosylated proteins: after the NEM labeling, proteins were separated in SDS-PAGE and the gel bands were excised and subjected to in gel tryptic digestion. The digested peptides were analyzed by nLC-LTQ-FTICR and the data were searched using Massmatrix. We were able to identify and quantify more than fifty different proteins which nitrosylated with CysNO in different amounts. Some of these proteins are associates with the neurological disorders such as Alzheimer and Parkinsons. Therefore these findings provide potential insight into understanding the mechanistic basis of the neurological disorders.
54. Proteomic analysis of protein covalent modification by quinone methide-releasing drugs
Sujeewa C. Piyankarage(1), [email protected], 833 South Wood Street, Chicago IL 60612, United States ; Tareisha Dunlap(1); R. Esala P. Chandrasena(1); Gregory R. J. Thatcher(1). (1) Department of Medicinal Chemistry & Pharmacognosy, University of Illinois at Chicago, Chicago IL 60612, United States
Quinone methide (QM) is a short-lived reactive intermediate thought to have both cytotoxic and cytoprotective properties. The importance of QM formation from NO-donating nonsteroidal anti- inflammatory drugs such as the NO-aspirin, NCX 4040, has previously been discussed, highlighting the chemoprevention perspective. This study aims at identifying and analyzing protein modifications by the QM-forming drugs to better understand the underlying chemoprevention processes, molecular pathways, and targets. Drugs including NO-aspirins were reacted with the model protein, glutathione S-transferase P1 (GST P1), in the presence of enzymes, followed by tryptic digestion and LC-MS analysis to identify the QM modifications. The stability of the QM modification with respect to pH and temperature was considered. Cell experiments were performed to identify the proteins undergoing drug-induced-QM modifications and correlated with induction of cellular stress response pathways. The findings of this study aid in understanding the balance between cytotoxicity and cytoprotection in protein-QM modification and in designing colon cancer chemoprevention agents.
55. Genotoxicity screening validation using electro-optical and magnetic bioreactor arrays
Linlin Zhao(1), [email protected], 55 N. Eagleville Rd, Storrs CT 06269, United States ; Shenmin Pan(1); James F Rusling(1)(2); John B Schenkman(2). (1) Chemistry, University of Connecticut, Storrs CT 06269, United States (2) Cell Biology, University of Connecticut Health Center, Storrs CT 06269, United States
Formation of nucleic acid base adducts results from exposure to xenobiotics through various sources, such as environmental pollutants, drugs, and endogenous compounds. DNA adducts have proven to be accurate and reliable biomarkers for predicting genotoxicity of chemicals. We have developed a two-tier molecular-structure based genotoxicity screening approach involving electro-optical and magnetic bioreactor arrays. Both methods feature thin films of oxidative and conjugation enzymes and DNA. A metallopolymer is included in electro-optical arrays to produce increased light emission for damaged DNA. The ultimate goal is to establish a universal platform for genotoxicity screening of unknown compounds. To achieve this, we are developing standardized methodologies with low, medium and high concentrations for fifteen different chemicals including drugs and carcinogens with known genotoxic pathways, and find correlations of these data with each other and with animal toxicity metrics, in order to validate the gentoxicity screening platform.
56. Characterization of a deoxyguanosine-lysine crosslink induced by methylgyoxal
Katya V Petrova(1), [email protected], VU Station B 351822, Nashville TN 37235, United States ; Amy D Millsap(1); Carmelo J Rizzo(1). (1) Chemistry Department, Vanderbilt University, Nashville TN 37235, United States
A 2001 study offered evidence that methylglyoxal, a known endogenous and environmental mutagen, caused a stable DNA-protein crosslink with Klenow fragment. When dGuo was incubated with Nα- acetyllysine in the presence of methylglyoxal a crosslink product was found, but the structure was not reported. We have reexamined this reaction and a structure of that crosslink formation was determined. The modified protein was purified and characterized using NMR and LC-MS/MS, and confirmed by chemical synthesis. A mechanism for the formation of this crosslink is proposed.
57. Metabolic profiling of mitochondrial dysfunction
Qiuwei Xu(1), [email protected], 770 Sumneytown Pike, West Point PA 19486, United States ; Heather Vu(1); William H. Schaefer(1); Liping Liu(1); James Monroe(1); Chi-Sung Chiu(1). (1) Safety Assessment, Merck Research Laboratories, West Point PA 19486, United States
Mitochondrial dysfunction is debilitative given its critical roles in maintaining normal cell function. Mitochondrial diseases are devastating to energy-hungry organs, and drug induced mitochondrial toxicities often cause attrition of drug candidates. Studying mitochondrial impairment associated with disease or drug toxicity using metabolomic profiling can reveal detailed steps in the metabolic pathways that have been disrupted. Mitochondrial impairment often stems from disruption of the electron transport chain or oxidative phosphorylation. It diminishes oxidative respiration and produces scant amount of ATP insufficient to sustain normal cell energy requirements. Although it is difficult to track the electron transport chain at the cellular level, close coupling of the tricarboxylic acid cycle (TCA) with the electron transport chain provides a mechanism to monitor mitochondrial function by measuring endogenous metabolites in the TCA cycle. As a quantitative, non-destructive, and chemically-specific analytical tool, NMR provides a global view of endogenous metabolites that are either in the TCA cycle or in different metabolic pathways related to energy producing processes in mitochondria. This approach does not require isolation of mitochondria. Moreover, it provides a better understanding of mitochondrial function in the context of metabolic activities outside mitochondria. In this poster, we will present our quantitative NMR metabolic profiles of mitochondrial activities in cells and tissues.
58. Evaluation of 9,12-dioxo-10(E)-dodecenoic acid and 4-oxo-2(E)-nonenal derived lysine adducts as potential biomarkers of inflammation
Fengping Li(1), [email protected], 77 Massachusetts Avenue, Room 56-731A, Cambridge MA 02139, United States ; Elizabeth Iffrig(1); Charles Knutson(1); John S Wishnok(1); Steven R Tannenbaum(1). (1) Department of Biological Engineering and Chemistry, Massachusetts Institute of Technology, Cambridge MA 02139, United States Polyunsaturated fatty acids can be oxidized to 13-hydroperoxy-octadecadienoic acid (13-HPODE) enzymatically and nonenzymatically. Reactive α,β-unsaturated aldehydes, e.g. 9,12-dioxo-10(E)- dodecenoic acid (DODE) and 4-oxo-2(E)-nonenal (ONE), are decomposition products of 13-HPODE. Previous studies demonstrated that DODE and ONE can modify proteins and that lysine is one of the major modified amino acids. Our recent data suggest that signals from DODE-Lys and ONE-Lys adducts are higher in digested 13-HPODE treated human serum than in untreated human serum. The goals of this study were to characterize the structures of DODE-Lys and ONE-Lys and quantify them in digested human serum in order to evaluate these two adducts as potential biomarkers of inflammation and oxidative stress.
59. Utility of hepatocyte bioreactor proteomics for identification of drug-induced liver toxicity
Viral Brahmbhatt(1), [email protected], 77 Massachusetts Ave, Cambridge MA 02139, United States ; Ajit Dash(1); Samuel W Inman(1); Linda Griffith(1); Steven R Tannenbaum(1). (1) Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge MA 02139, United States
Drug-induced liver toxicity is a major cause of failures in clinical trials. Recent studies in animal models support a possible role of low-grade inflammation in precipitation of idiosyncratic drug reactions. We have developed a liver bioreactor culture system that retains in vivo hepatocyte function and architecture and can be formulated with hepatocytes alone or in combination with liver non-parenchymal cells (Dash et al 2010). In the present study, we have utilized lipopolysaccharide (LPS) to provide low-grade inflammation in both monoculture and co-culture bioreactor systems. Reactors were treated with either Ranitidine or Nefazodone, drugs that are known to cause idiosyncratic drug reactions, with or without LPS. Quantitative proteomic data was then collected from the bioreactor culture medium and analyzed for changes in secreted proteins. Preliminary studies show that out of 136 proteins identified, 44 proteins show a 2-fold change with Nefazodone and 14 proteins show a 2-fold change with Ranitidine.
60. Assembly and characterization of a system for delivering biologically relevant levels of nitric oxide and oxygen for studies of inflammation
Vasileios Dendroulakis(1), [email protected], 70 Pacific St Apt 716, Cambridge MA 02139, United States ; Peter C. Dedon(2); William M. Deen(1)(2); Brandon S. Russell(2); C. Eric Elmquist(2); Laura J. Trudel(2); Gerald N. Wogan(2). (1) Department of Chemical Engineering, Massachusetts Institute of Technology, Cambridge MA 02139, United States (2) Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge MA 02139, United States
Nitric oxide (NO) is a gaseous free radical involved in the regulation of blood pressure, neurotransmission and innate immunity in mammals. For example, to combat invading microorganisms, macrophages generate high levels of NO that reacts with oxygen to form the oxidant • nitrogen dioxide radical (NO2 ) and the potent nitrosating agent nitrous anhydride (N2O3) and with superoxide to form the strong oxidants peroxynitrite (ONOO-) and nitrosoperoxycarbonate - (ONOOCO2 ). These species also damage cellular biomolecules, causing cell death and mutation. To study the role of NO in biological systems, we constructed a system to deliver NO and O2 gases at constant, biologically relevant concentrations. Here we describe the components of the delivery system, its characterization for delivering NO and O2 into aqueous buffer and cell culture media and its application to studying deamination of RNA. Using a mass transfer model developed to predict concentration profiles of the gases in the various media, we determined that photo-sensitive reactions taking place in media, such as riboflavin-derived superoxide synthesis, did not affect the steady-state levels of NO or O2 in the device. This delivery system is readily constructed from commercially available components and provides the most rigorous means to deliver gaseous NO at well-controlled, biologically relevant concentrations.
61. Quantitative profiling of serum bile acids by LC/MRM during liver tumor promotion associated with chronic Helicobacter infection and nuclear receptor deficiency
Yu Zeng(1), [email protected], 56-722, 77 Mass ave, Cambridge MA 02139, United States ; Alexis Garcia(2); John S. Wishnok(1); James G. Fox(2); Steve R. Tannenbaum(1)(3). (1) Department of Biological Engineering, Massachusettes Institute of Technology, Cambridge MA 02139, United States (2) Division of Comparative Medicine, Massachusettes Institute of Technology, Cambridge MA 02139, United States (3) Department of Chemistry, Massachusettes Institute of Technology, Cambridge MA 02139, United States
In mice, chronic infection with Helicobacter hepaticus (Hh) is associated with hepatitis and liver cancer. The nuclear receptor constitutive androstane receptor (CAR) regulates the expression of enzymes involved in endobiotic and xenobiotic metabolism. To understand the role of chronic infection in liver cancer progression, we measured the serum concentrations of selected bile acids in a mouse model of tumor initiation by diethylnitrosamine (DEN) followed by chronic Hh infection as a tumor promoter. Eight groups of C3H/HeNCrl mice were stratified based on their genotype, tumor initiation and tumor promotion status. The serum concentration of 8 selected mono-, di-, and tri- hydroxylated bile acids were measured employing a assay based on LC/MRM. Increased serum concentration of chenodeoxycholic acid (P<0.01) was detected in CAR-/-+DEN+Hh mice compared to CAR+/++DEN+Hh mice. Since increased liver tumor promotion was observed in CAR-/-+DEN+Hh mice relative to CAR+/++DEN+Hh mice, our results support a role of chenodeoxycholic acid in tumor promotion.
62. Cytotoxicity study of nanomaterials based on bioimpedance analysis and confirmed by cellular uptake
Sanghyo Kim(1), [email protected], San 65, Bokjeong dong, Sujeong gu, Seongnam si Gyeonggi do 461701, Republic of Korea ; Karthikeyan Kandasamy(1); Mohana Marimuthu(1). (1) Bionanotechnology, Kyungwon University, Seongnam si Gyeonggi do 461701, Republic of Korea
Toxicity of nanomaterials is still a major issue of emerging nanotechnology. Bioimpedance system can be one of the candidates which possess great utility so far in many medical research fields. The present reports demonstrate the functional ability of ECIS system for in vitro cytotoxicity analysis of newly synthesized AuNPs using ascorbic acid as natural reducing agent under autoclave condition and SWCNT(commercially obtained). ECIS results were validated by conventional WST-1 assay and accompanied by TEM and FESEM to examine the cellular uptake of nanoparticles and CNT inside the VERO cells. Results evinced that higher concentration of CNT possesses toxic effect to cells whereas all concentration of AuNPs doesn't show any cytotoxicity because of natural reducing agent. The potential toxicity of CNT may be due to the interaction with intracellular proteins and DNA, which leads to the cell death. Bioimpedance method has higher degree of efficiency to analyze the cytotoxicity of nanomaterials.
63. DNA-protein cross-links mediated by hydrogen peroxide
Edward K. Hawkins(1), [email protected], VU Station B 351822, Nashville Tennessee 37235-1822, United States ; Carmelo J. Rizzo(1). (1) Departments of Chemistry and Biochemistry and Center in Molecular Toxicology, Vanderbilt University, Nashville Tennessee 37235- 1822, United States DNA-protein cross-links constitute an important type of DNA damage. While it has been known for some time that the nucleophilic amine of lysine side chains can form reversible imine cross-links with electrophilic DNA-aldehyde adducts such as acrolein, it seems likely that irreversible cross-links are involved in biological damage. A 2008 study offered evidence that hydrogen peroxide can oxidize the reversible imine cross-link to a stable amide cross-link between lysine and simple aldehydes such as hexanal. Our hypothesis is that hydrogen peroxide, which under conditions of oxidative stress is produced in tandem with α,β-unsaturated aldehydes, also mediates the formation of amide cross- links between aldehyde-adducted DNA and protein. The formation of amide bond cross-links has been demonstrated in model systems between acrolein adducts of deoxyadenosine or deoxyguanosine and Nα-acetyl lysine. The nucleoside-amino acid cross-links were isolated and characterized, and the structures confirmed by synthesis of chemical standards.
64. Metabolism of benzo[a]pyrene-7,8-dione in human lung cells
Meng Huang(1), [email protected], 135 John Morgan Building, 3620 Hamilton Walk, Philadelphia PA 19104-6084, United States ; Trevor M. Penning(1), [email protected], 130C John Morgan Bldg, 3620 Hamilton Walk, Philadelphia PA 19104-6084, United States ; Xiaojing Liu(1); Ian A. Blair(1). (1) Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia PA 19104, United States
Benzo[a]pyrene-7,8-dione (B[a]P-7,8-dione) is produced in human lung cells by the oxidation of B[a]P-7,8-trans-dihydrodiol catalyzed by aldo-keto reductases (AKRs). However, information relevant to the in vivo metabolism of B[a]P-7,8-dione in these cells is lacking. We studied the metabolic fate of 2.0 μM [3H]-B[a]P-7,8-dione in human bronchoalveolar H358 cells, human lung adenocarcinoma A549 cells, and immortalized human bronchial epithelial HBEC-KT cells. In these three cell lines, radioactivity was distributed in the organic phase and aqueous phase of medium, as well as in the cell lysate and cell pellets. After acidification of the medium, several metabolites of [3H]-B[a]P-7,8-dione were detected in the organic phase of the medium by HPLC-UV-RAM. The structures of B[a]P-7,8- dione metabolites varied in the cell lines and were identified as GSH-B[a]P-7,8-dione, N-acetyl-L- cysteine-B[a]P-7,8-dione, N-acetyl-L-cysteine-7,8-dihydroxy-B[a]P and o-methylated-7,8-dihydroxy- B[a]P by LC-MS/MS and were subsequently validated by comparison to authentic synthesized standards. (Supported by 1R01-CA39504 to TMP)
65. Effects of cytosine methylation on O6-alkylguanine DNA alkyltransferase repair of O6- (pyridyloxobutyl)guanine adducts in CG dinucleotides
Delshanee Kotandeniya(1), [email protected], Mayo Mail Code 806, 420 Delaware St SE, Minneapolis MN 55455, United States ; Natalia Y Tretyakova(1); Anthony E Pegg(2). (1) Department of Medicinal Chemistry and the Cancer Center, University of Minnesota, Minneapolis Minnesota 55455, United States (2) Department of Cellular and Molecular Physiology, Milton S. Hershey Medical Center, Pennsylvania State University College of Medicine, Hershey Pennsylvania 17033, United States
Promutagenic O6-[4-oxo-4-(3-pyridyl)butyl]guanine (O6-POB-G) and O6-methylguanine (O6-Me-G) lesions induced by tobacco specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), are removed by a specialized repair protein, O6-alkylguanine-DNA alkyltransferase (AGT). Our previous studies revealed that AGT repair of O6-Me-G lesions at CG dinucleotides can be inhibited by endogenous cytosine methylation. In the present work, the effects of cytosine methylation on the rate of repair of O6-POB-dG adducts by AGT protein in p53 derived sequences were investigated. A guanine residue of frequently mutated 5'-CG-3' or 5'-MeCG-3' dinucleotides of the p53 tumor suppressor gene was replaced with O6-POB-G, and the resulting DNA duplexes were incubated with recombinant human AGT, followed by rapid quenching, acid hydrolysis, and quantification of unrepaired O6-POB-dG by isotope dilution HPLC-ESI-MS/MS. Our results reveal that the presence of MeC reduces the rate of AGT-mediated repair of O6-POB-dG adducts, potentially contributing to smoking-induced mutagenesis in the p53 gene.
66. Detoxification of structurally diverse PAH o-quinones by human recombinant COMT via o-methylation of PAH catechins
Li Zhang(1), [email protected], 135 John Morgan Building, 3620 Hamilton Walk, Philadelphia PA 19104, United States ; Trevor M. Penning(1); Showket Bhat(2); Ian A. Blair(2). (1) Centers of Excellence in Environmental Toxicology, Department of Pharmacology, University of Pennsylvania, United States (2) Cancer Pharmacology, Department of Pharmacology, University of Pennsylvania, United States
Polycyclic aromatic hydrocarbons (PAH) are metabolically activated to intermediate trans-dihydrodiols which are oxidized by aldo-keto reductases (AKRs) to yield o-quinones that are redox-active and cause oxidative DNA damage in human lung cells. We investigated whether o-methylation by human recombinant COMT is a detoxification route of a panel of structurally diverse PAH catechols produced during the redox-cycling process. Several classes of PAH o-quinones produced by AKRs were employed in the studies. PAH o-quinones were reduced to corresponding catechols by dithiothreitol under anaerobic conditions and then further o-methylated by human COMT in the presence of S- adenosyl-L-methionine as a methyl group donor. The formation of the o-methylated catechol was detected by HPLC-RAM-UV and LC-MS-MS. Human COMT was able to catalyze o-methylation of most of PAH catechols and generate isomeric metabolites. It is concluded that human COMT may play a critical role in the detoxification of PAH o-quinones [Supported by 1R01-CA-39504 awarded to TMP].
67. Changes in the spectrum of tRNA secondary modifications as biomarker signatures of exposure to toxic stimuli
Clement T. Y. Chan(1), [email protected], 77 Massachusetts Institute of Technology, Rm NE47-293, Cambridge Massachusetts 02138, United States ; Peter C. Dedon(2); Madhu Dyavaiah(3); Koli Taghizadeh(4); Thomas J. Begley(3). (1) Department of Chemistry, Massachusetts Institute of Technology, Cambridge Massachusetts 02138, United States (2) Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge Massachusetts 02138, United States (3) Department of Biomedical Sciences, University at Albany, State University of New York, Rensselaer New York 12144, United States (4) Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge Massachusetts 02138, United States
Secondary modifications of the canonical nucleosides in DNA and RNA play critical roles in cell physiology. In contrast to the few modifications in DNA, there are >100 different chemically modified nucleobases in rRNA and tRNA among all living organisms. However, the broader physiological function of these modified nucleosides has eluded definition. Recent studies indicate that the yeast tRNA methyl transferase, Trm9p, confers resistance to exposure to an alkylating agent by catalyzing the formation of a tRNA nucleobase modification that enhances translation of critical stress response transcripts. To test the broader hypothesis that changes in the spectrum of tRNA base modifications serve as biomarker signatures of exposure to different chemical and biological agents, we developed a coupled liquid-chromatography, electrospray-ionization, triple quadrupole mass spectrometry method (LC/MS-MS) to quantify changes in the spectrum of the >25 RNA modifications in yeast exposed to different classes of chemical stressors, including hydrogen peroxide, methyl methanesulfonate, sodium hypochlorite, ionizing radiation and sodium arsenite. The results reveal unique agent- and dose-specific changes in the spectrum of tRNA modifications. To assess the role of modified nucleosides in cell survival, we quantified cytotoxicity for each agent in yeast strains lacking specific tRNA modifying enzymes, with the observation that many of the RNA secondary modifications are critical to the survival response. These and other results with a variety of chemical and physical agents will be discussed in terms of the chemical biology of RNA secondary modifications in states of both pathology and normal physiology.
68. Inhibition of 4-aminobiphenyl-induced DNA damage by Sulforaphane and 5,6- Dihydrocyclopenta[c]-dithiole-3(4H)-thione in bladder cells and tissues
Kristen L Randall(1), [email protected], 102 Hurtig Hall, 360 Huntington Ave, Boston Massachusetts 02115, United States ; Yi Ding(2); Joseph D Paonessa(2); Dayana Argoti(1); Rex Munday(3); Yuesheng Zhang(2); Paul Vouros(1). (1) Department of Chemistry and Chemical Biology, Northeastern University, Boston Massachusetts 02115, United States (2) Department of Cancer Prevention and Control, Roswell Park Cancer Institute, Buffalo New York 14263, United States (3) Department of AgResearch, Ruakura Research Center, Hamilton Waikato 3240, New Zealand
Isothiocyanates and dithiolethiones are well-known classes of chemopreventive agents, whose mechanisms of action include induction of carcinogen-detoxifying Phase 2 enzymes. The human bladder carcinogen 4-aminobiphenyl (4-ABP) is present in tobacco smoke and other environmental sources. Indeed, increased 4-ABP-DNA adduct levels are detected in the bladder tissues of tobacco smokers and a large percentage of human bladder tumors. Our laboratory's sensitive LC-MS/MS method for attomole detection and quantification of dG-C8-4-ABP has allowed us to use dG-C8-4- ABP as a biomarker in the search for agents that protect cells against 4-ABP. We demonstrate herein that both sulforaphane (SF, an isothiocyanate) and 5,6 Dihydrocyclopenta[c]-dithiole-3(4H)-thione (CPDT, a dithiolethione) significantly inhibited 4-ABP-induced DNA damage in both human bladder cells in vitro and mouse bladder tissues in vivo. Further studies are underway to understand the mechanisms by which CPDT and SF inhibit 4-ABP-induced DNA adduct formation.
69. Oxidative stress-induced protein modification: Application of clickable linoleic acid analogs
Peter Slade(1), [email protected], 29851 Willow Creek Rd, Eugene OR 97402, United States ; Robbert Aggeler(1); Tamara Nyberg(1); Chad Pikens(1); Upinder Singh(1); Brian Agnew(1). (1) Life Technologies-Molecular Probes, Eugene OR 97402, United States
Cellular oxidative stress can result in protein-carbonyl modification through the oxidation of linoleic acid followed by electrophilic additions. The identification of linoleic acid-derived protein modifications is challenging. This study demonstrates a novel click chemistry-based method to label and identify these modified proteins. Macrophages were metabolically labeled with linoleic acid -azide or -alkyne clickable analogs and treated with hemin to induce oxidative stress. Fixed macrophages, or isolated macrophage proteins, were click-labeled with fluorescent dyes and analyzed by fluorescence microscopy, or SDS-PAGE, respectively. In each case, there was a dramatic increase in fluorescence signal upon hemin treatment. Azide modified proteins were enriched on alkyne resin. After stringent washing, bound proteins were digested off the resin and analyzed by mass spectrometry. The resulting peptide pools demonstrate unprecedented selectivity and purity of labeled samples versus controls. Preliminary results have identified >50 modified proteins and iTRAQ® kit quantification studies are in progress.
70. Paradoxical down-regulation of benzo[a]pyrene-mediated DNA-adduct formation and concurrent up-regulation of glutathione-adduct formation by 2,3,7,8-tetrachlorodibenzo-p- dioxin in human lung cells
Stacy L. Gelhaus(1), [email protected], 421 Curie Boulevard BRB II, Room 841, Philadelphia PA 19104, United States ; Trevor M. Penning(1); Ian A. Blair(1). (1) Centers of Cancer Pharmacology and Excellence in Environmental Toxicology, University of Pennsylvania, Philadelphia PA 19104, United States
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous, environmental pollutants. Benzo[a]pyrene (B[a]P), a prototypic PAH, is often used to elucidate the pathways of metabolic activation. Metabolism of B[a]P by cytochrome P450 (CYP) 1A1/1B1 and epoxide hydrolase results in the formation of the ultimate carcinogen, (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro-B[a]P (B[a]PDE). B[a]PDE is able to translocate to the nucleus where it forms DNA-adducts, primarily (+)-anti-trans-B[a]PDE-N2- dGuo (B[a]PDE-dGuo). Typically, pre-treatment of cells with 2,3,7,8-tetrachlorobenzo-p-dioxin (TCDD) will induce CYP1A1/1B1 expression through the aryl hydrocarbon receptor (AhR) pathway thus causing an increase in B[a]PDE-dGuo formation. Unexpectedly, TCDD induction caused a decrease in B[a]PDE-dGuo formation versus the untreated, control human bronchoalveolar, H358 cells. Further investigation revealed that the TCDD-mediated decrease in B[a]PDE-dGuo simultaneously occurs with an increase in B[a]P-glutathione-adduct formation. All adducts were monitored by stable isotope dilution liquid chromatography-mass spectrometry. This study reveals that the subtle balance between activation and detoxification of B[a]P in lung cells. Research supported by NIH 5F32ES016683.
71. Stable isotope dilution HPLC-ESI--MS/MS analysis of urinary butadiene metabolites as biomarkers of exposure to 1,3-butadiene
Srikanth Kotapati(1), [email protected], MMC 806, 420 Delaware St. S.E., 790 MCRB, Minneapolis Minnesota 55455, United States ; Brock A Matter(1); Dewakar Sangaraju(1); Natalia Y Tretyakova(1). (1) Department of Medicinal Chemistry and Masonic Cancer Center, University of Minnesota, Minneapolis Minnesota 55455, United States
Tobacco smoke contains over 60 known carcinogens, including 1,3-butadiene (BD). BD is bio- activated to three mutagenic epoxide metabolites: 1,2,3,4-diepoxybutane, 3,4-epoxy-1-butene, and 3,4-epoxy-1,2-butanediol. If not detoxified via epoxide hydrolysis or glutathione conjugation, these metabolites can react with nucleophilic sites in DNA and proteins to form covalent adducts. Glutathione conjugates of the above mentioned epoxides are further excreted in urine as mercapturic acids: 1-hydroxy 2-(N-acetylcysteinyl)-3-butene (MHBMA), 1,2-dihydroxy-4-(N-acetylcysteinyl)-butane (DHBMA), 1,2,3-trihydroxy-4-(N-acetylcysteinyl)-butane (THBMA) and 2,3-dihydroxy-1,4-(N- acetylcysteinyl)-butane (bis-BDMA) and can be used as biomarkers of BD exposure. A stable isotope dilution HPLC-ESI--MS/MS method was developed for quantitative analysis of the butadiene mercapturic acids in human urine. It will be applied in a future large scale study of a multi-ethnic cohort of African American and European American smokers to test our hypothesis whether ethnic/racial differences in metabolic activation and deactivation of BD and subsequently smoking- induced cancer incidence are caused by polymorphisms in the genes encoding xenobiotic metabolizing enzymes.
72. Sensitive liquid chromatography/mass spectrometry method for the quantification of low levels of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in urine of non-smokers
Showket H Bhat(1), [email protected], 841-850 Biomedical Research Bldg II/III, 421 Curie Blvd, Philadelphia PA 19104-6160, United States ; Stacy L Gelhaus(1); Clementina A Mesaros(1); Ian A Blair(1). (1) Centers of cancer Pharmacology and Excellence in Environmental Toxicology, University of Pennsylvania, Philadelphia Pennslvania 19104, United States
Total NNAL in urine has emerged as a candidate biomarker for determining the level of environmental tobacco smoke (ETS) exposure in non-smokers. Our aim is to develop a sensitive stable isotope dilution liquid chromatography electrospray ionization multiple reaction monitoring mass spectrometry method suitable for quantitation of low levels of total NNAL in non-smoker urine. Here, we describe a novel derivatization of urinary NNAL by alkylation at the pyridine nitrogen resulting in the formation of a quaternized/pre-ionized derivative leading to enhanced sensitivity. Samples were analyzed on a reversed-phase nano-LC system coupled to a triple quadruple mass spectrometer equipped with a nano-spray source. Standard curves were linear from 0.1 to 20 pg/mL of NNAL in urine with a typical r2 > 0.99. Among 50 non-smokers total NNAL concentrations ranged from undetectable to 25 pg/mL urine. The method met the criteria of ≤ 15% precision and accuracy between 85 % and 115 %.
73. Persistent ROS generation in H358 cells treated with PAH-o-quinones
Sankha S Basu(1), [email protected], 421 Curie Boulevard, BRB II/III Room 845, Philadelphia PA 19146, United States ; Stacy L Gelhaus(1); Meng Huang(1); Trevor Penning(1); Ian Blair(1). (1) Centers for Cancer Pharmacology and Excellence in Environmental Toxicology, University of Pennsylvania School of Medicine, Philadelphia PA 19104, United States
Metabolism of benzo[a]pyrene by aldo-keto-reductases (AKRs) results in the formation of B[a]P-7,8- dione (BPQ), which has been shown to undergo futile redox cycling resulting in reactive oxygen species (ROS) formation in lung cells. In this study, H358 cells were treated with BPQ for a range of doses and incubation times. Liquid chromatography-mass spectrometry was used to measure changes in glutathione/glutathione disulfide levels and to monitor BPQ levels in cells and media after treatment. Intracellular ROS generation was monitored using flow cytometry. We found that while BPQ rapidly disappeared from both media and cells, increased oxidative stress persisted in cells for 24 hours. Furthermore, a significant fraction of BPQ was associated with proteins and resulted in persistent autofluorescent products that were visualized by fluorescence microscopy and quantified by flow cytometry. These long-lived BPQ adducts represent a potential intracellular ROS-generating reservoir and may play a significant role in smoking-related oxidative stress and carcinogenicity.
74. Identification of tyrosine chlorination sites in human serum albumin for potential inflammatory disease biomarker discovery
Pilsoo Kang(1), [email protected], 77 Massachusetts Avenue, Rm 56-731, Cambridge MA 02139, United States ; Yu Zeng(1); John S. Wishnok(1); Steven R. Tannenbaum(1)(2). (1) Department of Biological Engineering, Massachusetts Institute of Technology, Boston MA 02139, United States (2) Department of Chemistry, Massachusetts Institute of Technology, Boston MA 02139, United States
Targeted quantification of specific serum protein adducts arising from oxidative-stress-related reactive chlorine can be exploited to discover novel biomarkers in inflammatory disease progression. During inflammation, reactive nitrogen and oxygen species can oxidatively damage proteins, resulting in a variety of adducts. Hypochlorous acid (HOCl) and other reactive chlorine species are also generated by neutrophils via myeloperoxidase (MPO)-catalyzed reaction of hydrogen peroxide and chloride, leading in turn to chlorinated amino acids, e.g, 3-chlorotyrosine. Although chlorination of tyrosine may not be a major product of oxidative stress, chlorinated tyrosine is a unique and stable marker for neutrophil (MPO) activity. Protein adducts in biological fluids are good targets for an assay of oxidative damage from inflammation. Proteomic analysis (LC-MS) of MPO-catalyzed chlorination of a target protein (human serum albumin) has identified specific target peptides that will be suitable for future biomarker investigations.
75. Total synthesis of Aristolochic acids I & II and their derivatives
SIVAPRASAD ATTALURI(1), [email protected], Graduate Chemistry Building, Room No. 610, Stony Brook NY 11794-3400, United States ; RADHA R BONALA(1); FRANCIS JOHNSON(1). (1) Department of Pharmacological Sciences, Stony Brook University, Stony Brook NY 11794, United States
Aristolochic acids of aristolochia species have been reported to be nephrotoxic and genotoxic, causing severe kidney fibrosis and subsequently urothelial tumors. In common with other nitroaromatic hydrocarbons these compounds are converted in the liver to the corresponding hydroxyl amines which are procarcinogens and lead to DNA adducts. Due to this aristolochic acids drawn much attention because of their detrimental effect on human health. We developed a novel methodology for the total synthesis of the aristolochic acids (I & II) and related congeners.
76. Quantification of the 2-deoxyribonolactone and nucleoside 5'-aldehyde products of 2- deoxyribose oxidation in DNA and cells by isotope-dilution gas chromatography mass spectrometry
Wan Chan(1), [email protected], 77 Massachusetts Avenue, Cambridge Massachusetts 02139, United States ; Peter Dedon(1)(2). (1) Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge Massachusetts 02139, United States (2) Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge Massachusetts 02139, United States
The oxidation of 2-deoxyribose in DNA has emerged as a critical determinant of the cellular toxicity of oxidative damage to DNA, with oxidation of each carbon producing a unique spectrum of electrophilic products. We have developed and validated an isotope-dilution gas chromatography-coupled mass spectrometry (GC-MS) method for the rigorous quantification of two major 2-deoxyribose oxidation products: the 2-deoxyribonolactone abasic site of 1'-oxidation and the nucleoside 5'-aldehyde of 5'- oxidation chemistry. The method entails elimination of these products as 5-methylene-2(5H)-furanone (5MF) and furfural, respectively, followed by derivatization with pentafluorophenylhydrazine (PFPH), addition of isotopically labeled PFPH derivatives as internal standards, extraction of the derivatives, and quantification by GC-MS analysis. The precision and accuracy of the method were validated with oligodeoxynucleotides containing the 2-deoxyribonolactone and nucleoside 5'-aldehyde lesions. Further, the well defined 2-deoxyribose oxidation chemistry of the enediyne antibiotics, I neocarzinostatin and calicheamicin g1 , was exploited in control studies.
77. Activity-based serum protease profiling for biomarkers of chronic inflammation
Ujjal Sarkar(1), [email protected], 77 Massachusetts Avenue, 56-738, Cambridge MA 02139-4307, United States ; Charles G. Knutson(1); John S. Wishnok(1); Steven R. Tannenbaum(1)(2). (1) Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge MA 02139, United States (2) Department of Chemistry, Massachusetts Institute of Technology, Cambridge MA 02139, United States
Chronic inflammation is associated with diseases such as heart disease, diabetes, and cancer. A variety of proteases, e.g.,serine, cysteine and metallo- proteases, are involved in chronic inflammation. Consequently, there is a need to analyze proteases as potential biomarkers by utilizing active-site-directed probes to probe the mechanism and monitor disease progression. We employed alpha-chymotrypsin (alpha-ChT), and trypsin enzymes as a platform for activity based protein profiling studies. Enzymatic reaction with a series of active-site directed and mechanism-based inhibitors were studied and characterized by ESI-TOF. Under our optimized reaction conditions, masses of native alpha-ChT and enzyme-inhibitor covalent adducts were observed. The adducts were characterized using tryptic digestion and subjected to analysis of the active site containing peptides. Furthermore, probe-modified peptides will be identified and quantified by mass spectrometry, with the expectation that this approach will lead to applications for diagnostics and therapeutics. Recent efforts toward these fronts will be briefly presented.
78. Mass spectrometric detection of hemoglobin adducts from the anti-HIV drug nevirapine and its metabolite 12-hydroxy-nevirapine in rats
M. Matilde Marques(1), [email protected], Av Rovisco Pais, Lisboa P1049-001, Portugal ; Sofia A. Pereira(2); Emília C. Monteiro(2); Frederick A. Beland(3); Alexandra M.M. Antunes(1); Ana L.A. Godinho(1); M. Conceição Oliveira(1). (1) Centro de Química Estrutural, Instituto Superior Técnico, Universidade Técnica de Lisboa, Lisboa P1049-001, Portugal (2) Departamento de Farmacologia, Faculdade de Ciências Médicas, Universidade Nova de Lisboa, Lisboa P1169-056, Portugal (3) Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson Arkansas 72079, United States
Nevirapine (NVP), a non-nucleoside reverse transcriptase inhibitor used against HIV-1, is associated with severe toxicities involving liver and skin. NVP metabolism to 12-hydroxy-NVP and subsequent sulfotransferase-mediated conjugation is thought to play a role in these processes. To obtain evidence for NVP activation via 12-hydroxy-NVP, we investigated the binding of NVP and 12- hydroxy-NVP to plasma proteins in Sprague-Dawley rats treated intraperitoneally with seven daily doses (130 μmol/kg bw) of NVP or 12-hydroxy-NVP. Following N-alkyl Edman degradation with phenyl isothiocyanate and HPLC-ESI-MS/MS analysis, the N-terminal valine adduct, 12-[5-isopropyl- 4-oxo-3-phenyl-2-thioxoimidazolidin-1-yl]-NVP, was identified in hemoglobin from both NVP- and 12- hydroxy-NVP-treated rats by comparison with a synthetic standard prepared from ethyl valinate and the model ester, 12-mesyloxy-NVP. These results confirm the involvement of 12-hydroxy-NVP in the metabolic activation of NVP and suggest that the N-terminal valine-NVP adducts can be used as biomarkers of NVP toxicity. Supported by FCT (PPCDT/QUI/56582/2004), FEDER, and the NTP.
79. Mechanistic studies on the biotransformation of BMS-690514, an ErbB/VEGF tyrosine kinase inhibitor, to metabolite M1
Haizheng Hong(1), [email protected], Route 206 and Province Line Rd, Princeton NJ 08543, United States ; Janet Caceres-Cortes(1); Hong Su(1); Xiaohua Huang(1); Samuel Bonacorsi(2); Yang Hong(2); Yuan Tian(2); Ramaswamy A. Iyer(1); William G. Humphreys(1); Lisa J. Christopher(1). (1) Department of Pharmaceutical Candidate Optimization, Bristol Myers Squibb Co., Princeton NJ 08543, United States (2) Department of Radiochemistry, Bristol Myer Squibb Co., Princeton NJ 08543, United States
BMS-690514 is an oral oncologic agent being developed for the treatment of advanced NSCLC and breast cancer. M1 and M37 were two of the primary hydroxylated metabolites with the hydroxyl group located at the same position of the molecules. Mass spectral and NMR analysis revealed that the pyrrolotriazine moiety of BMS-690514 remained intact in M37 while it rearranged into a pyridotriazine group for M1. In vitro experiments with deuterated or tritiated BMS-690514 indicated that the isotope label was retained at the site of hydroxylation for M1, while the label was lost in M37. The mechanism for the formation of M1 was proposed that BMS-690514 was first oxidized by CYP enzymes and then the pyrrolotriazine moiety opened, forming an aldehyde intermediate, which reacted with the secondary amine of the methoxyaniline group in the molecule and formed the pyridotriazine moiety of M1. Metabolite M37 may be formed through a mechanistically distinct pathway. 80. Systematic study of 2'-deoxyoxanosine formation during nitrosative deamination of 2'- deoxyguanosine under biologically relevant conditions
Pallavi Lonkar(1), [email protected], NE-47, 77 Massachusetts Avenue, Cambridge MA 02139, United States ; Jose L. McFaline(1); Peter C Dedon(1). (1) Biological Engineering, Massachusetts Institute of Technology, Cambridge MA 02139, United States
The link between chronic inflammation and cancer may involve infiltration of macrophages and neutrophils into tissues with subsequent production of reactive oxygen and nitrogen species that cause damage in surrounding host cells. The deamination of DNA bases occurs by both chemical and enzymatic mechanisms, including nitrosation reactions with derivatives of nitric oxide released from macrophages that have infiltrated the inflammed tissues. Numerous in vitro studies of the nitrosative deamination of 2'-deoxyguanosine (dG), have shown evidence for the formation of both 2'- deoxyxanthosine (dX) and 2'-deoxyoxanosine (dO) as damage products of dG deamination. We have been unable to detect dO at a level of >1 per 107 nt in isolated DNA exposed to biologically relevant levels of nitrous anhydride, an NO-derived nitrosating species, or in E. coli, cultured mammalian cells, or in tissues from mouse models of inflammation. To test a recent model of base-pairing influences on dO formation, we have used an NO/O2 delivery system and a sensitive LC/MS-MS method to quantify the formation of dX and dO from dG as a free nucleoside and in single- and double-stranded oligonucleotides. The results shed light on the biological relevance of dO as a product of nitrosative deamination of dG and offer clues as to whether it is predominantly formed in ss/ds DNA or in nucleotide pool.
81. NMR structure of duplex DNA having a β-carba-fapy-dG residue
Tanya Zaliznyak(1), [email protected], BST 7-147, Stony Brook New York 11794- 8651, United States ; Mark Lukin(1); Sivaprasad Attaluri(1); Radha R. Bonala(1); Francis Johnson(1); Carlos De los Santos(1). (1) Pharmacology, Stony Brook University, Stony Brook New York 11794- 8651, United States
Known for more than thirty years, formamido pyrimidine lesions (fapy) are among the most abundant adducts produced by oxidative stress. The structure of fapy-dG containing duplexes, however, is still unknown. In order to understand how fapy-dG affects DNA structure, we prepared and determined the solution structure of oligomeric DNA containing a β-carbocyclic fapy-dG residue. The carbocyclic analogue has the deoxyribose ring replaced by cyclopentane, a minor modification that suppresses the epimerization reaction. Our data show mostly normal NOE connectivities, which associate with a regular right-handed helix somehow distorted at the lesion site. Fapy-dG exists as two stable rotamers corresponding to the cis and trans orientations of formyl group. Both rotamers have fapy stacking inside the helix and forming Watson-Crick hydrogen bonds with the opposite cytosine residue. Restrained MD simulations generated structures for each rotameric form, which show the formyl group of fapy positioned in major groove of the duplex.
82. New mass spectrometry based method for quantifying protein thiol modification in response to nitrosative and oxidative stress
Vaishali Sinha(1), [email protected], 833 S. Wood St., Chicago IL 60612, United States ; Gihani T Wijewickrama(1); R. Esala P Chandrasena(1); Isaac T Schiefer(1); Gregory R.J. Thatcher(1). (1) Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago IL 60612, United States
Cysteine residues in proteins represent a target for electrophilic modification and oxidation. Such post translational modification of critical cysteine residues may lead to the regulation of protein function. It has been suggested that nitrosation of cysteine residues by NO is an important mechanism by which both physiological NO signaling and nitrosative stress can be transduced into a cellular response; protein nitrosylation has been argued to be a signaling mechanism rivaling protein phosphorylation in importance. However, nitrosylation is not the only chemical modification of protein thiols by NO: NO donors and endogenously produced NO can induce a variety of other modifications such as disulfide bond formation, sulfenic, sulfinic, sulfonic acids etc. Their differential chemistry is predicted to result in different mechanism of action and biological effects. In this study, oxidation & nitrosation are quantified in response to several families of NO donor drugs (nitrosothiol, nitrates, nitrites, NONOates etc). Using GSTP1-1 as a model protein, the popular biotin switch (BS) technique was modified coupled with immunoblotting and a new mass spectrometry technique developed for quantifying nitrosation and oxidation of protein cysteine residues. On applying the method to neuronal cell culture, quantifies nitrosative damage in protein targets for human neurodegerative diseases.
83. Conformational effects of C8-arylguanine substitution on B/Z-DNA in oligonucleotides with one modified base
Brian C. Train(1), [email protected], 1 Medical Center Drive, Morgantown West Virginia 26505- 9530, United States ; Vorasit Vongsutilers(1); Daniel J. Phillips(2); Peter M. Gannett(1). (1) Department of Pharmaceutical and Pharmacological Sciences, West Virginia University, Morgantown West Virginia 26505, United States (2) Department of Chemistry, Bethany College, Bethany West Virginia 26032, United States
We have shown that C8-arylguanine adducts bearing a para-substituent shift the B/Z-DNA equilibrium toward the Z form in oligonucleotide sequences with two modified bases. The para-substituent was found to have a significant and predictable effect on the B/Z-DNA equilibrium by altering base-pair stacking in the B form and modifying the minor groove widths of both the B and Z conformations. Here a series of C8-arylguanine adducts bearing para-substituents have been prepared in hairpin- turn oligonucleotide sequences containing only one modified base. The conformational effects of aryl substitution were determined by NMR spectroscopy, molecular modeling and circular dichroism (CD). CD was also used to quantify B/Z DNA ratios. Our results to date show that oligonucleotide sequences bearing only one C8-arylguanine modified base exhibit similar effects on B/Z DNA equilibrium as for oligonucleotides with two aryl substitutions. (Supported by WVEPSCoR STEM EPS2005-27)
84. Trapping of a catechol estrogen quinone by GSH is reversible and does not block estrogenic activity: Problematic detoxification of 4-hydroxyequilenin by GSH conjugate formation
Yue-Ting Wang(1), [email protected], 833 S Wood St, Chicago Illinois 60612, United States ; Kuan- Wei Peng(1); Minsun Chang(2); Zhican Wang(1); Zhihui Qin(1); Judy L. Bolton(1); Gregory R. J. Thatcher(1). (1) Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago Illinois 60612, United States (2) Division of Biological Sciences, College of Science, Sookmyung Women[apos]s University, Seoul 140-742, Republic of Korea
The major phase I metabolite of equine estrogens 4-hydroxyequilenin (4-OHEN) may elicit hormonal carcinogenesis at estrogen receptors, and chemical carcinogenesis via its autoxidation product 4- OHEN quinone. In cell culture, 4-OHEN quinone is anticipated to be trapped and detoxified by cellular GSH. However, formation of the quinone-GSH conjugate was observed to be reversible in the presence of NADPH. 4-OHEN was regenerated from mono-GSH quinone conjugate at a rate dependent on NADPH concentration. A SNAr-like addition-elimination mechanism is proposed for this reaction, providing a reasonable explanation for the observed inability of thiols to attenuate the estrogenic potency of 4-OHEN in MCF-7 cells. Thiol exchange of GSH in quinone-GSH conjugates occured in the presence of other thiols. Thiol exchange with protein thiols can be expected. These observations urge us to reconsider the conventional knowledge of GSH detoxification: trapping by cellular thiols may not terminate estrogen-dependent carcinogenesis attributed to 4-OHEN.
85. Fluorescent probe for direct detection of hypoxia
Anuruddha M.D. Rajapakse(1), [email protected], 125 Chemistry Building, 601 S. College Avenue, Columbia MO 65211, United States ; Ujjal Sarkar(1); Kent S. Gates(1). (1) chemistry, University of Missouri-Columbia, columbia MO 65211, United States
The low-oxygen conditions (hypoxia) found in solid tumors correlates with poor prognosis. Thus, detection of hypoxic regions may be useful in the clinical characterization of cancer. Here we describe 6-nitroquinoline as a direct-light-up fluorescent probe of hypoxia. Upon selective enzymatic reduction under hypoxic conditions, 6-nitroquinoline was expected to produce fluorescent compounds such as 6-aminoquinoline. We examined the ability of two different enzymatic reducing systems to drive the enzymatic reduction of 6-nitroquinoline under both anaerobic and aerobic conditions. The resulting metabolites were characterized by fluorescence spectroscopy, HPLC and mass spectrometry. We find that 6-nitroquinoline can, indeed, serve as a direct light-up probe for hypoxia under physiologically-relevant conditions. Interesting aspects of enzyme selectivity and the fluorescence of the metabolic intermediates, such as 6-hydroxylaminoquinoline, will be presented.
86. Estimating the interactions of signaling proteins in response to increasing dose
Holly N Williams(1), [email protected], PO Box 6045, Morgantown WV 26506, United States ; Xueli Gao(1); J Nicholas Rubenstein(1); Amber C Cook(1); Jonathan W Boyd(1). (1) Department of Chemistry, West Virginia University, Morgantown West Virginia 26506, United States
While understanding cellular signaling cascades in the temporal domain is key to determining the mechanisms of response, the importance of dose-response characteristics cannot be ignored. Interactions of signaling proteins in response to increasing dose offers a unique perspective into the function of biochemical networks, and holds the potential to aid in the eventual prediction of toxicity by identifying adaptive strategies for survival. Linking potential signaling cascades as they relate to functional consequences is examined via the phosphorylation activity of several key proteins (AKT, ERK1/2, JNK, p38MAPK, HSP27, IkBa, p53, and p90RSK) which are related to networks of both cellular survival and death in response to increasing dose. Hepatocytes were exposed to 4 different compounds (deguelin, staurosporine, SB202190, and SB202474) which all exhibit network response and induction of apoptosis within a 24 hour period, and relative phosphorylation states were examined and analyzed in order to explore the protein interactions.
87. Proteomic analysis of sera derived from patients with inflammatory bowel disease
Charles G Knutson(1), [email protected], 77 Massachusetts Avenue, 56-722, Cambridge MA 02139, United States ; Joshua R Korzenik(2); John S Wishnok(1); Steven R Tannenbaum(1). (1) Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge MA 02139, United States (2) Gastrointestinal Unit, Massachusetts General Hospital Crohn[apos]s and Colitis Center, Boston MA 02114, United States
Inflammatory bowel disease (IBD) is marked by intermittent and severe activation of the mucosal immune system in the gastrointestinal tract. Few biomarkers are available in the clinic and none of them predict the transition from remission to active disease. The availability of such markers would therefore be an asset for efficient diagnosis and treatment of IBD. Our laboratory is identifying new biomarkers of IBD present in the serum of patients with clinically diagnosed ulcerative colitis or Crohn's disease. In this study we performed a proteomic analysis of serum proteins obtained from patients exhibiting active ulcerative colitis or Crohn's disease in comparison to healthy human controls. The investigation revealed altered levels of 12 acute phase proteins in patients with IBD (p < 0.01) and 2 appear specific to ulcerative colitis. Further analysis of these samples will identify the types of lipid peroxidation-mediated chemical modifications found on IBD serum proteins.
88. Competitive removal of oxidatively generated guanine lesions in DNA by NER and BER in human cell extracts
Konstantin Y Kropachev(1), [email protected], 100 Washington Square East, Room 1001, New York NY 10003, United States ; Marina Kolbanovsky(1); Vladinmir Y Shafirovich(1); Nicholas E Geacintov(1). (1) Department of Chemistry, NYU, New York NY 10003, United States
We have studied the reaction pathways of guanine radicals in DNA in aqueous solution. Among the different lesions formed are the stereoisomeric Spiroiminodihydantoin (Sp) lesions and the recently discovered intrastrand cross-linked G*-T* and G*CT* lesions characterized by a covalent G(C8)- T(N3) bond (C. Crean et al, Nucl. Acids. Res. 36:742, 2008). We investigated the repair of these oxidatively generated lesions in cell-free extracts from human HeLa cells. All three are substrates of both nucleotide excision repair (NER) and base excision repair (BER). While both the Sp lesions, as well as the intrastrand cross-linked G*CT* lesion are medium to excellent substrates of NER, the strand with the G*T* cross-linked product is only weakly incised. Typically, the levels of BER excision products rise rapidly within the first ~ 5 – 10 min of incubation and then reach a constant level, while the levels of the typical NER 24-30-mer DNA fragments increase approximately linearly with time at least up to 40 min. Our hypothesis is that the NER system may function as a backup for BER. Supported by NIEHS Grants ES 011589, and by CA 099194.
89. Human NER recognition mechanisms involving the undamaged strand of the double helix
Ying Chen(1), [email protected], 100 Washington Square East, Room 1001, New York NY 10003, United States ; Konstantin Y Kropachev(1); Zhi Liu(1); Marina Kolbanovsky(1); Nicholas E Geacintov(1). (1) Department of Chemistry, NYU, New York NY 10003, United States
Nucleotide excision repair (NER) is one the most important cellular defense mechanisms which protects the genome by removing bulky base modifications from DNA. However, the recognition mechanism of NER is not clearly understood yet. It was proposed that NER containing bulky BPDE- N2-dG and other lesions in double-stranded DNA requires a flipped out base dC in the unmodified complementary strand opposite the lesion for efficient repair since deleting this dC completely abrogates NER (Buterin, Chem. Biol. 12, 913, 2005). We examined this hypothesis by placing either dC (normal duplex), or the mismatched bases dT, dA, dG, or a deletion site (missing nucleotide), opposite the (+)-cis-anti-BPDE-N2-guanine adduct (G*). Although all of the complementary strands, except the one with the deleted dC have the potential for extruding bases opposite G*, only the double strands with dC and dT are good substrates for NER, while those with dA and dG are resistant to NER in human cell extracts. These findings suggest that the recognition mechanism of NER is determined not necessarily by a flipped out base, but by the conformational features of these modified different duplexes which are being investigated by NMR and other physico-chemical techniques. Reaserch supported by NIH grant CA099194. 90. Solution conformations of the major (+)-trans-anti-BPDE-N2-guanine adduct opposite an abasic site in a DNA duplex
Zhi Liu(1), [email protected], 100 Washington Square East, Room 1001, New York NY 10003, United States ; Chin Lin(1); Lei Jia(1); Shuang Ding(2); Suse Broyde(2); Nicholas E. Geacintov(1); Adam Schwaid(1). (1) Department of Chemistry, NYU, New York NY 10003, United States (2) Department of Biology, NYU, New York NY 10003, United States
The solution conformation of the (+)-trans-anti-BPDE-N2-guanine adduct (G*) opposite its normal base dC and a deletion site have been studied earlier. Interestingly, the normal and full duplex is a substrate of the human nucleotide excision (NER) system, while the same lesion at a deletion site is completely resistant to NER. In order to elucidate the basis of this resistance to NER, we are investigating the NMR structure of G* opposite a tetrahydrofuran (abasic) site opposite the lesion. Here we report that the major solution structure of the (+)-trans-anti-BPDE-N2-guanine adduct opposite this abasic site (in an otherwise fully complementary DNA duplex) involves intercalation, as in the deletion duplex. In contrast, in the full duplex with dC opposite G*, the BPDE aromatic ring system is positioned in the minor groove. The differences in these adduct conformations will be described, and their impact on NER efficiencies will be discussed. Research supported by NIH grant CA099194
91. Using Saenger sequencing to analyze base-sequence effects on in vitro translesion bypass catalyzed by a Y-family polymerase: Dpo4
Yuan-Cho Lee(1), [email protected], 100 Washington Square East, Room 1001, New York NY 10003, United States ; Alexander Kolbanovsky(1); Suse Broyde(2); Nicholas E Geacintov(1). (1) Department of Chemistry, NYU, New York NY 10003, United States (2) Department of Biology, NYU, New York NY 10003, United States
Benzo[a]pyrene, an important pre-carcinogen and environmental contaminant, is metabolized to the diol epoxide BPDE in cellular environments that reacts with G in DNA to form predominantly BPDE- N2-dG adducts. It is well established that during DNA synthesis, high-fidelity DNA polymerases are stalled by this lesion, and that the lower fidelity Y-family polymerases perform translesion synthesis. In order to study the effects of base sequence context on translesion bypass in vitro, we selected the polymerase Dpo4 from Sulfolobus sulfataricus P2 and 43-mer DNA templates that contained the identical 10S (+)-trans-anti-BPDE-N2-dG adduct (G*) in the sequence context XG*Y. We used Saenger methods to sequence Dpo4 bypass products and found predominantly (~50%) -1 frameshift deletions in all sequence contexts. A significant preference for dATP insertion, and minor but variable extents of dGTP, CTP, and TTP incorporation in the TG*T, AG*T, TG*A, AG*A sequences were observed as well. The insertion of the incorrect nucleotide is sequence context-dependent and our sequencing results provide the evidence needed for interpreting these results by molecular modeling techniques. Supported by CA 099194.
92. DNA adducts formed from different metabolic pathways of 3,5-dimethylaniline
Wenjie Ye(1), [email protected], 25 Ames Street, 56-738, Cambridge MA 02139, United States ; Paul L. Skipper(1); Liang Cui(1); John S. Wishnok(1); Steven R. Tannenbaum(1). (1) Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge Massachusetts 02139, United States
Some environmental alkylanilines, e.g., 3,5-dimethylaniline (3,5-DMA) may play a role in the etiology of human bladder cancer. Animal models suggest that this might involve DNA adducts formed either by N-hydroxylation via nitrenium ions or by aminophenol formed via a quinine imine. We are investigating the structures of the 3,5-DMA-DNA adducts formed by both these pathways. Calf thymus DNA (ct-DNA) containing tritium-labeled deoxyguanosine was incubated with 3,5-DMA, several different liver S9s, and an NADPH regeneration system. Following enzymatic hydrolysis, adduct profiles were determined using HPLC with radioactivity & UV detection. A labeled product co- eluting with nitrenium-related N-(deoxyguanosin-8-yl)-3,5-dimethylaniline was observed. To determine the DNA adducts from the second pathway, ct-DNA was reacted with 3,5-dimethyl-p- quinone imine. Following the enzymatic hydrolysis of DNA, the products were analyzed by liquid chromatography with detection by time-of-flight and tandem quadrupole mass spectrometry. The exact mass and daughter ion mass spectra suggested that the major product was N-(deoxyadenosin- 8-yl)-2,6-dimethylaminophenol.
93. Lipoxygenase metabolites from cyclooxygenases
Xiaojing Liu(1), [email protected], 421 Curie Blvd. BRB II/III, room 841, Philadelphia PA 19104, United States ; Clementina Mesaros(1); Ian A. Blair(1). (1) Center of Excellence in Environmental Toxicology, University of Pennsylvania, Philadelphia PA 19104, United States
There has been enormous interest in cyclooxygenase (COX)-derived eicosanoids as mediators of physiological processes. However, eicosanoids derived from the lipoxygenase (LOX) activity of COXs have been largely ignored. Numerous studies of purified COX-1 and COX-2 enzymes have shown that the 11(R)-hydroxyeicosatetraenoic acid (HETE), 15(S)-HETE, and 15(R)-HETE metabolites are formed in relatively high amounts. They are formed by reduction of the corresponding hydroperoxyeicosatetraenoic acids (HPETES). However decomposition of the HPETEs before they are reduced could potentially result in the formation of reactive bifunctional electrophiles. We developed a chiral LC-MS for analysis of enantiomers derived from the 4-hydroperoxy-2(E)-nonenal and 4-hydroxy-2-(E)-nonenal and shown that they are formed with remarkable enantioselectivity from 15-HPETEs. However, homolytic decomposition of 11(R)-HPETE resulted in racemic products. This provided insight into mechanisms of HPETE decomposition pathways and suggest provided a means to monitor enantioselective formation of intracellular reactive bifunctional electrophiles-derived glutathione-adducts. Supported by NIH grants RO1 CA 91016
94. Acetyl cysteine adducts of eicosatetraenoic acids: Synthesis, characterization, and identification in smoker's urine
Jasbir S Arora(1), [email protected], 421 Curie Blvd., 841 BRB II/III, Philadelphia PA 19104, United States ; Suhong Zhang(1); Ian A Blair(1). (1) Center for Cancer Pharmacology, University of Pennsylvania, Philadelphia PA 19104, United States
GSH is involved in the formation of endogenous bioactive eicosanoids and is a source of reducing equivalents in a number of biosynthetic reactions. GSH acts as a co-factor in the reduction of reactive oxygen species and lipid hydroperoxides by glutathione peroxidases and glutathione-S transferases (GSTs) through the use of its nucleophilic sulfahydryl group. Subsequent cleaved and modified (by the action of enzymes like of gamma-glutamyl transpeptidase, cysteinyl glycinase and N-acetyl transferase) products of these adducts if characterized and identified can provide quantitative measure of enzymatic and non-enzymatic cellular oxidative stress. The present study has been carried out to detect the acetyl cysteinyl adducts of some of the arachidonic acid derived eicosatetraenoic acids (oxo-ETEs) in the smoker's urine. The labeled adducts of oxo-ETEs with acetyl cysteine were synthesized. The smoker's and non smoker's urine were spiked with the labeled adducts. The samples were analyzed by LC/MS to identify endogenous oxo-ETE adducts. 95. Quantification of urinary eicosanoids by liquid chromatography/mass spectrometry
Clementina A Mesaros(1), [email protected], 421 Curie Blvd, Philadelphia PA 19104, United States ; Ian A Blair(1). (1) Department of Pharmacology, Centers for Cancer Pharmacology and Excellence in Environmental Toxicology, University of Pennsylvania, Philadelphia PA 19104, United States
Cyclooxygenase (COX)-derived eicosanoids have important roles as autacoids involved in the regulation of cardiovascular function as well as certain stages of tumor progression. 20- hydroxyeicosatetraenoic acid (20-HETE) and thromboxane A are vasoconstrictors, but prostaglandins (PGs) PGE2 and 6-keto-PGF1α are vasodilators. 12-HETE and 5-HETE were reported to have tumor growth potential. Many of the eicosanoids are present in only trace amounts in biological fluids. The discovery of atmospheric pressure ionization-mass spectrometry methodology has revolutionized our ability to analyze endogenous eicosanoids. Here we describe targeted eicosanoid analyses using stable isotope dilution liquid chromatography multiple reaction monitoring MS methods suitable for low level quantitation of HETEs and PGs in urine. Two different derivatization techniques have been used to increase the sensitivity of the method. Urinary measurements represent a non-invasive approach to estimate eicosanoids biosynthesis in different disease states. Supported by NIH grant RO1 CA 91016 and NIEHS grant ES013508-04.
96. Nitrenium ion stability of aryl-amines
Lewis Whitehead(1), [email protected], 100 Technology Square, Cambridge MA 02139, United States ; Patrick McCarren(1); Gregory Bebernitz(1); Melissa Grondine(1); Louise Kirman(1); Herbert Schuster(1); Jacob Klickstein(1); Susanne Glowienke(2); Peter Gedeck(3). (1) Global Discovery Chemistry, Novartis Institutes for Biomedical Research, Cambridge Massachusetts 02139, United States (2) Novartis Pharma AG, Muttentz CH-4132, Switzerland (3) Novartis Pharma AG, Basel CH- 4056, Switzerland
Aryl-amines have the potential for an AMES toxicity liability. In-silico calculations can assess the energy required to form reactive intermediates of aryl-amines that may lead to DNA adducts, and therefore allow predictions to aid NIBR design non-toxic small molecules more efficiently.
97. Quantitation of aristolochic acid adducts in murine DNA
Kimberly A Conlon(1), [email protected], Chemistry Building, Stony Brook New York 11794-3400, United States ; Charles R Iden(1); Thomas A Rosenquist(1). (1) Department of Pharmacological Sciences, Stony Brook University, Stony Brook NY 11794-3400, United States
Aristolochic acids are components of herbal preparations containing Aristolochia species used medicinally by many cultures throughout the world. The herbs are given to treat a variety of human disorders, but the aristolochic acids are severely nephrotoxic and lead to renal interstitial fibrosis accompanied by urothelial carcinomas. Aristolochic acids I and II are activated by metabolic enzymes to produce N-hydroxy-aristolactams (AL-I, AL-II), which react with exocyclic amino groups in dG and dA forming DNA adducts. We have developed LC/MS/MS methods using selected reaction monitoring to quantify the dG and dA adducts of AL-I and AL-II. Monitoring the transition from the molecular ion to the BH+ ion was selected for the quantitation of each adduct using a triple quadrupole mass spectrometer. Standard curves for each adduct were created using a synthetic standard, and the method was applied to DNA isolated from the kidneys of mice treated with the aristolochic acids. 98. Sequence-context dependent recognition and repair of aristolochic acid-DNA adducts
Jung-Eun Yeo(1), [email protected], Stony Brook NY, United States ; Rhada R. Bonala(1); Sivaprasad Attaluri(1); Irina Izaitseva(1); Francis Johnson(1); Charles R. Iden(1); Orlando D. Scharer(1). (1) Department of Chemistry and Department of Pharmacological Sciences, Stony Brook University, Stony Brook New York 11790, United States
Aristolochic acids (AA) are associated with AA nephropathy and urothelial cancer. Upon metabolic activation, AA reacts with DNA forming the mutagenic dA-aristolactam (AL) and dG-AL adducts. dA- AL adducts are remarkably persistent in human cells, indicating that they are resistant to repair. Interestingly, mutations in the TP53 gene in human urothelial cancers induced by AA are limited to non-transcribed strand, suggesting that transcription coupled nucleotide excision repair (NER), but not global genome NER efficiently removed dA-AL adducts. We have generated plasmids containing a site-specific dA-AL or dG-AL lesion and have studied how these adducts are repaired by NER. Our studies indicate that the repair efficiency derived is dependent upon the sequence context in which these lesions are found and that dA-AL is entirely resistant to repair in certain contexts. Present studies are aimed at understanding how these differences are correlated with the binding of the damage recognition protein XPC-RAD23B.
99. Predicting mutagenicity of aromatic amines using hierarchical support vector regression approach
Max K. Leong(1)(2), [email protected]; Sheng-Wen Lin(1); Hong-Bin Chen(1). (1) Department of Chemistry, National Dong Hwa University, Shoufeng Hualien 97401, Taiwan Republic of China (2) Department of Life Science and Institute of Biotechnology, National Dong Hwa University, Shoufeng Hualien 97401, Taiwan Republic of China
Many aromatic amines, which commonly can be found in a wide variety of industries, foods, and environment, are potentially mutagenic or even carcinogenic in some cases. Therefore, it is of practical importance to develop an in silico mode to predict their mutagenicity. A predictive model was built using the novel hierarchical support vector regression scheme based on comprehensive data collection of a homogeneous set of mutagenicity data (TA98+S9). The predictions by this model are in good agreement with the experimental observations for those molecules in the training set, the test set, and the outlier set. Various model cross-comparisons were also conducted. The results indicate that this HSVR model is highly accurate, robust, and consistent. Thus, it can be employed as a tool for predicting mutagenicity of aromatic amines.
100. Trapping reversible ortho-quinone methide - nucleoside adducts
Michael P McCrane(1), [email protected], 0107 Chemistry Building, College Park MD 20742, United States ; Omer Ad(1); Emily E Weinert(1); Ying Ling(1); Yiu-Fai Lam(1); Steven E Rokita(1); Eugene P Mazzola(2). (1) Department of Chemistry and Biochemistry, University of Maryland, College Park MD 20742, United States (2) Department of Chemistry and Biochemistry, UMD-FDA Joint Institute, College Park MD 20742, United States
Electrophilic ortho-quinone methides (o-QM) are generated during xenobiotic metabolism of a variety of compounds and can alkylate DNA. From model studies based on nucleosides, o-QMs react most readily, but reversibly with strong nucleophiles. Their reaction is less efficient, but irreversible with weak nucleophiles. The hour time-scale of the reverse reactions complicates analysis of their products in DNA, which requires enzymatic digestion and chromatographic separation. Instead, a chemical trap utilizing bis[(trifluoroacetoxy)iodo]benzene has been developed to transform the reversible o-QM-DNA adducts into irreversible derivatives, capable of surviving such analysis. Preliminary studies, involving a model o-QM and 2'-deoxycytidine resulted in the isolation of a surprising derivative likely due to over-oxidation. A second model o-QM compound included an alkyl group to block over-oxidation. A surprising derivative was still generated from oxidation of its adduct, but the final derivative should be sufficient for identifying labile o-QM adducts in DNA.
101. Non-mutagenic bypass of a photodimer of T-methylC in DNA by yeast polymerase eta
Qian Song(1), [email protected], 5536 Pershing Ave, Apt 511, St Louis Missouri 63112, United States ; John-Stephen Taylor(1). (1) Department of Chemistry, Washington University in St Louis, St Louis MO 63130, United States
Cyclobutane pyrimidine dimers (CPDs) are largely responsible for sunlight induced 5-methycytosine (mC) to T mutations at PyCG sites observed in tumor suppressor genes in skin cancers. One mechanism for the mC to T mutations is that the CPD is excluded from the active site of a DNA polymerase and A is inserted by a non-templated mechanism. Another is that the mC tautomerizes to a tautomer that codes as a T. A third possibility is that the mC must first deaminate to T before trans- lesion synthesis. We have prepared a DNA template containing a site-specific T=mCG CPD and find that the trans photodimer polymerase eta inserts dGMP almost exclusively opposite the mC, whereas it inserts almost exclusively dAMP opposite the T resulting from deamination. The high fidelity of non- mutagenic insertion opposite the mC and its deamination product provides strong support for the deamination mechanism for sunlight induced mC to T mutations.
102. Generation and investigation of the C3'-deoxy-3'-thymidinyl radical in single-stranded oligonucleotides
Buthina Abdallah(1), [email protected], 2801 W. Bancroft Street, Toledo Ohio 43606, United States ; Amanda Bryant-Friedrich(1). (1) Department of Medicinal and Biological Chemistry, University of Toledo, Toledo Ohio 43606, United States
Oxidative damage to DNA through ionizing radiation (IR) is partially produced by the secondary particles created along the ionization track. Secondary low-energy electrons (LEEs, 1-20 eV) are the most abundant secondary species produced by primary IR. It is believed that carbon-centered radicals on the sugar moiety are a major reactive intermediate formed in the process of damage by LEE. One of these is the C3'-deoxy-3'-thymidinyl radical. The synthesis of C3'-deoxy-3'- (selenophenyl)thymidine as a precursor of this radical has been completed. The modified nucleoside was incorporated into oligonucleotides using reverse DNA synthesis. Photochemical generation of the C3'-deoxy-3'-thymidinyl radical within DNA was achieved using ultraviolet light (≥320nm). Identification of the damage products obtained from the generation of the C3' radical is currently in progress. The outcomes of these experiments will greatly add to our understanding of the mechanistic pathways of oxidative damage to DNA as it relates to LEEs.
103. Synthesis of a [3-15N]-5-methylcytosine cyclobutane pyrimidine dimer-containing DNA decamer for tautomerization and base pairing studies
Rongsheng E Wang(1), [email protected], 1 Brookings Drive, St Louis MO 63130, United States ; Jeffrey L Kao(1); John Stephen Taylor(1). (1) Department of Chemistry, Washington University in St Louis, St Louis MO 63130, United States
Cyclobutane pyrimidine dimers (CPDs) have been correlated with sunlight induced 5-methycytosine (mC) to T mutations at PymCG sites in tumor suppressor genes in skin cancers. One possible mechanism for the mC to T mutations is that the mC in a CPD tautomerizes to an E-imino tautomer and codes as though it were a T. Trans-photodimer DNA synthesis studies in our lab suggest that the mC remains in its amino tautomeric state during nucleotide insertion. To independently determine the tautomeric state and base pairing properties of the mC in a dimer we have synthesized a convertible building block for introducing N-15 into the N3 position of 5-methylcytosine in a dodecamer DNA. This dodecamer was then irradiated, and the cis-syn cyclobutane pyrimidine dimer containing product was isolated and investigated by 2D N-15 NMR. Preliminary studies suggest that it is indeed in its amino tautomeric form in aqueous solution.
104. Reactivity and stability of the 5'-aldehyde in DNA
Suaad A Abdallah(1), [email protected], 2801 West Bancroft Street, Toledo OH 43606, United States ; Amanda C Bryant-Friedrich(1). (1) [quot]Department of Medicinal & Biological Chemistry[quot], University of Toledo, Toledo OH 43606, United States
Oxidative stress can lead to the formation of reactive oxygen and nitrogen species (ROS/RNS) which interact with DNA, damaging the sugar phosphate backbone. This damage interferes with DNA repair, transcription and translation. When ROS abstract hydrogen atoms from the deoxyribose moiety of DNA, single and double strand breaks form. These strand breaks can be associated with the formation of electrophilic fragments which are capable of reaction with local nucleophiles. Since the 5'- hydrogen of deoxyribose in B-Form DNA is the most accessible to solvent, they are highly vulnerable to abstraction which leads to the formation of the 5'- radical. Under aerobic conditions the radical forms the electrophilic 5'-aldehyde. The half life of this lesion in single stranded DNA was determined to be ~96 hours under physiological conditions. Adduct formation between the damage derived α,β-unsaturated 5'-aldehyde and adenine was detected. The reactivity of this lesion under physiological conditions is under investigation.
105. Exploring the link between urban aerosols and DNA adducts using capillary nanoLC- ESI-MS/MS
Joshua J Klaene(1), [email protected], 360 Huntington Ave., 102 Hurtig Hall, Boston MA 02115, United States ; James Glick(1); Euripides G Stephanou(2); Paul Vouros(1). (1) Department of Chemistry and Chemical Biology and the Barnett Institute, Northeastern University, Boston Massachusetts 02115, United States (2) Department of Chemistry, Environmental Chemical Processes Laboratory (ECPL), University of Crete, Heraklion Crete EL-71003, Greece
Urban aerosols contain a variety of organic compounds including PAH's. Ingesting these compounds is a direct hazard to human health. In this study we use mass spectrometry to study the link between exposure to urban aerosols and DNA adducts and investigate potential competitive/preferential metabolism upon simultaneous exposure to multiple procarcinogens. Air particles with an aerodynamic diameter < 2.5 μm, from an urban location in the Eastern Mediterranean, were collected and dosed into metabolically competent MCL-5 human lymphoblast cells. DNA was isolated and digested, enriched and analyzed using capillary RPLC-nanoESI coupled to one of three mass analyzers; a QqQ, a quadrupole ion trap, and a Q-Tof. We have determined that B(a)P, present in the particulate phase at roughly 0.02 ng/m3, is being metabolized and reacting with deoxyguanosine to form BPDE-dG adducts. Our dosing scheme closely simulates real-world exposures to particle- associated PAH's.
106. Biotransformation of epo-folate conjugate (BMS-753493)
Xiaomei Gu(1), [email protected], 206 & Provinceline Road, Princeton NJ 08543, United States ; Yueping Zhang(2); Celia Darienzo(3); Yuan Tian(4); Yang Hong(4); Samuel Bonacorsi(4); W. Griffith Humphreys(1); Ramaswamy A Iyer(1). (1) Department of Biotransformation, Bristol-Myers Squibb, Princeton NJ 08543, United States (2) Department of Metabolism and Pharmacokinetics, Bristol- Myers Squibb, Princeton NJ 08543, United States (3) Department of Bioanalytical Sciences, Bristol- Myers Squibb, Princeton NJ 08543, United States (4) Department of Chemistry, Bristol-Myers Squibb, Princeton NJ 08543, United States
BMS-753493, an epothilone-folate conjugate was designed to target folate receptor alpha (FRα) expressing tumor cells. The pharmacologically active moiety, BMS-748285, is conjugated to folic acid by a cleavable disulfide linker and a solubilizing peptide. In hepatocytes and microsomes, BMS- 753493 was metabolized to generate the active moiety BMS-748285. In addition, BMS-753493 underwent disulfide exchange reactions with glutathione and cysteine to give mixed disulfides. BMS- 748285 itself undergoes hydrolysis (M1, BMS-767100), mono-oxidations, and conversion to a P+2 metabolite, which can be further oxidized or hydrolysed. Mixed disulfides of the conjugate with glutathione, cysteine and with plasma protein were observed in systemic circulation after administration of BMS-753493. In a [3H] BMS-753493 BDC rat study, the recovery of the radioactive dose in bile and urine was 20.1% and 53.5%, respectively. Urinary excretion was the predominant route of elimination with BMS-753493 accounting for 43% of the dose. The major metabolic pathway was hydrolysis.
107. Structural studies of exocyclic deoxyadenosine adducts of 1,2,3,4-diepoxybutane
Ewa A Kowal(1), [email protected], Room SC 7300 Stevenson Science Center, 24th Avenue and Garland Ave., Nashville TN 37235, United States ; Michael P Stone(1); Uthpala Seneviratne(2); Natalia Tretyakova(2). (1) Department of Chemistry, Center in Molecular Toxicology, Vanderbilt University, Nashville Tennessee 37235, United States (2) Department of Medicinal Chemistry and Masonic Cancer Center and Department of Chemistry, University of Minnesota, Minneapolis Minnesota 55455, United States
Novel exocyclic S,S- and R,R-N6,N6-(2,3-dihydroxybutan-1,4-diyl)-2'-deoxyadenosine (N6,N6-DHB- dA) adducts arising from 1,2,3,4-diepoxybutane (DEB), a metabolite of 1,3-butadiene, have been identified. These bis-alkylation products at N6-dA prevent Watson-Crick base pair formation. Thermal melting studies show both diastereomers destabilize DNA. Nuclear Magnetic Resonance (NMR) spectroscopy reveals that the imino protons of thymines that are opposite the adducts are not observed. The H6/H8-H1' region of the NOESY spectrum for both isomers is similar compared to unmodified duplex suggesting a localized structural changes at the lesion site. No break in the sequential connectivities was observed. The biggest chemical shift differences were observed for the modified bases, 5' and 3' neighbors and thymine which is opposite the adduct. The exocyclic ring in the adduct is located in the major groove of DNA. Supported by NIH grant RO1 ES-05509 (M.P.S.).
108. High-throughput screening of organophosphate antidotes in a zebrafish model
Shan Jin(1)(2), [email protected], 149, 13th Street, 4th floor, CVRC, Charlestown MA 02129- 2060, United States ; Randall Peterson(1)(2). (1) Medicine, Harvard Medical School, Charlestown MA 02129, United States (2) Cardiovascular Research Center, Massachusetts General Hospital, Charlestown MA 02129, United States
Organophosphates are widely used as pesticides and nerve agents and represent important potential chemical threats. More and better antidotes for organophosphate toxicity are needed. We seek to build an antidote library of a variety of chemicals with different mechanisms for counteracting organophosphates. A model has been developed in which exposure of zebrafish to an organophosphate results in high mortality. By subjecting the model to high-throughput screening (HTS), several novle small molecule countermeasures have been identified. Further studies of dose response, pharmacology, structure-activity relationship (SAR), and mechanism of action are under way. 109. Phenotyping human livers for repair of O6-methylguanine and O6-[4-oxo-4-(3- pyridyl)butyl]guanine adducts by O6-Alkylguanine-DNA alkyl-transferase
Janel K Warmka(1), [email protected], MMC 806, 420 Delaware St. SE, Minneapolis MN 55455, United States ; Qingming Fang(2); Anna M Urban(1); Anthony E Pegg(2); Lisa A Peterson(1). (1) Masonic Cancer Center, University of Minnesota, Minneapolis MN 55455, United States (2) Department of Cellular and Molecular Physiology, Pennsylvania State University, Hershey PA 17033, United States
The tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a human carcinogen, is metabolized to DNA methylating and pyridyloxobutylating compounds. DNA adducts formed include O6-methylguanine (O 6-meG) and O 6-[4-oxo-4-(3-pyridyl)butyl]guanine (O 6-pobG). O6- Alkylguanine-DNA alkyl-transferase (AGT) protects the cell by repairing O 6-alkylguanine. Epidemiological data suggest that genetic variation in AGT could be involved in increased cancer risk. Therefore, we investigated if individuals with different AGT genotypes vary in their ability to repair O 6- pobG relative to O 6-meG. Our data show that independent of AGT genotype, human liver homogenates have similar abilities to repair O 6-pobG relative to O 6-meG in one sequence context. Interestingly, O 6-pobG is not completely repaired even in the presence of excess levels of AGT. Studies are under way to investigate the mechanism behind incomplete O 6-pobG repair. [Supported by CA-115309].
110. Role of pyridyloxobutyl DNA adducts in nitrosamine-induced cancers
Anna M Urban(1)(2), [email protected], 420 Delaware Street SE, MMC 806, Minneapolis MN 55455, United States ; Pramod Upadhyaya(2); Lisa A Peterson(1)(2). (1) Division of Environmental Health Sciences, University of Minnesota, Minneapolis MN 55455, United States (2) Masonic Cancer Center, University of Minnesota, Minneapolis MN 55455, United States
The tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N'- nitrosonornicotine (NNN) generate pyridyloxobutyl DNA adducts. Data indicate that O6-[4-3-(pyridyl)- 4-oxobut-1-yl]deoxyguanosine (O6-pobdG) and O2-[4-3-(pyridyl)-4-oxobuty-1-yl]deoxythymidine (O2pobdT) contribute to the mutagenic properties of the pyridyloxobutylation pathway. O6- Alkylguanine-DNA alkyltransferase (AGT) is known to repair O6-pobdG, while nucleotide excision repair has been implicated as a pathway for O2pobdT repair. To determine the role of DNA adduct formation and repair in the tumorigenic properties of pyridyloxobutylating nitrosamines, A/J mice were treated with the model pyridyloxobutylating agent 4-(acetoxymethyl-nitrosamino)-1-(3-pyridyl)-1- butanone (NNKOAc) in single and chronic dosing regimens. DNA adduct levels and tumor yield were measured in the lung. Our findings indicate that O2pobdT is not repaired following a single or chronic dose of NNKOAc, however O6-pobdG is repaired even when AGT is depleted by co-administration of O6-benzylguanine, an AGT depletion agent. Studies are underway to correlate these adducts to lung tumor formation. [Supported by CA-115309]
111. Investigation of cytotoxic and mutagenic effects of O2-methylthymine, a minor-groove DNA adduct
Nisana Andersen(1), [email protected], 501 Big Springs Rd, Riverside CA 92521, United States ; Jianshuang Wang(1); Yinsheng Wang(1). (1) Department of Chemistry, University of California at Riverside, Riverside CA 92521, United States
Human genomic DNA is susceptible to modifications induced by alkylating agents, which have the ability to transfer an alkyl group to all oxygen atoms in nucleobases in DNA. Little work has been done to assess the cytotoxic and mutagenic properties of the O2-alkyl derivatives of thymine, where the modification occurs in the minor groove of DNA. Previous studies revealed that DinB DNA polymerases, e.g. pol IV in E. coli and pol k in humans, are capable of accurately and efficiently bypassing some minor-groove N2-modified dG derivatives. Whether these DinB DNA polymerases are capable of accurately bypassing O2-alkylthymines has yet to be explored. Initial experiments show human pol k-mediated nucleotide incorporation opposite O2-methylthymine is neither accurate nor efficient. We are now in the process of investigating how O2-methylthymine perturbs the efficiency and fidelity of DNA replication in cells and which translesion synthesis DNA polymerase(s) is(are) involved in bypassing this lesion in cells.
112. Quantification of 8-5'-cyclo-2'-deoxyguanosine and 8-5'-cyclo-2'-deoxyadensosine in calf thymus DNA treated with Fenton Reagents
Candace R Guerrero(1), [email protected], 501 Big Springs Rd, Riverside California 92521, United States ; Yinsheng Wang(1). (1) Department of Chemistry, University of California- Riverside, Riverside California 92521, United States
Purine cyclonucleosides, which include 8-5'-cyclo-2'-deoxyguanosine (cyclo-dG) and 8-5'-cyclo-2'- deoxyadenosine (cyclo-dA), are known to be induced in human cells by ionizing radiation. The formation of these lesions may bear significant implications in cancer and neurological diseases. It has yet to be explored how effectively these cyclopurine lesions can be induced by Fenton reagents, which may represent the major endogenous source of reactive oxygen species (ROS), in comparison to commonly observed single-nucleobase lesions. Here, we employed LC-MS/MS with the stable isotope dilution techniques, to quantify the levels of both diastereomers of cyclo-dG and cyclo-dA in calf thymus DNA treated with a transition metal-driven Fenton reaction. Initial results revealed the dose-responsive formation of cyclopurine lesions and single-base lesions in isolated DNA upon 2+ treatment with Cu /H2O2/ascorbate.
113. Role of carcinogen-induced conformational heterogeneity in translesion synthesis
Vaidyanathan Ganesan(1), [email protected], 41 Lower College Road, Kingston Rhode Island 02881, United States ; Bongsup P. Cho(1). (1) Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, Kingston Rhode Island 02881, United States
The environmental mutagen 2-aminofluorene forms an AF adduct at the C8 position of guanine. This adduct persists in vivo and is known to adopt three unique conformations (B, S, and W) depending on neighboring sequence contexts. We hypothesized that AF-induced heterogeneity at the replication fork influences the thermodynamics and kinetics of polymerase binding affinity. Here, we conducted NMR/CD and primer extension studies on two flanking sequence contexts (-CG*A-, –TG*A-) with a fluorine-labeled AF lesion to study the influence of the S/B/W conformational heterogeneity on translesion synthesis. We observed significant differences between the sequences, both in full length extension as well as in running start experiments. Steady-state kinetics experiments were carried out around the lesion site. The same sequences were incorporated into a 67-mer template with a primer containing dideoxynucleobase at the 3'-position to form a replication fork on a gold surface and treated with KF exo- and DPO4. The results from these experiments provided insight on the molecular interactions between the lesion and polymerases, as well as the role of conformational heterogeneity in arylamine mutagenesis. 114. Spectroscopic and thermodynamic insights into sequence effects of 2- acetylaminofluorene-induced conformational heterogeneity
Satyakam Patnaik(1), [email protected], 41 Lower College Road, Kingston Rhode Island 02881, United States ; Bongsup P. Cho(1). (1) department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, Kingston Rhode Island 02881, United States
2-Acetylaminofuorene is one of the most extensively studied model arylamine carcinogens. Upon activation in vivo, it reacts with cellular DNA to produce two major bulky adducts at C8 of guanine (AF and AAF). AAF differs from AF in that the nitrogen at the adduct linkage is acetylated, contributing to the differences in their mutational and repair properties. Although AAF-induced conformational heterogeneity has been recognized, little is known about its sequence dependence and the nature of minor conformations. This is remarkable, given the extensive functional data available on AAF in the literature. In the present study, we conducted a systematic spectroscopic study on a series of 12-mer NG*N DNA duplexes (d[CTTCTNG*NCCTC], N=G, A, C, T), in which G* was modified with a fluorine labeled AAF. 19F NMR/CD and calorimetric data were obtained to probe the sequence dependence of AAF-induced conformational heterogeneity in terms of the three plausible arylamine-induced conformations: major (B) groove binding, minor (W) groove binding, and stacked (S) conformers. The results indicate a strong flanking sequence dependence of AAF-induced conformational heterogeneity, which may provide insight into the mechanisms of AAF mutagenesis.
115. Long-range sequence effect on the arylamine-induced conformational heterogeneity
Vipin Jain(1), [email protected], 412 Lower College Road, Kingston Rhode Island 02881, United States ; Bongsup P. Cho(1); Satyakam Patnaik(1); Vaidyanathan Ganesan(1); Eva Darian(2); Alexander D. MacKerell(2). (1) Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island, Kingston Rhode Island 02881, United States (2) Department of Pharmaceutical Sciences, University of Maryland, Baltimore Maryland 21201, United States
Arylamine-modified DNA exists in one or two of three distinct conformers, B-type (B), stacked (S) or wedge (W), depending on sequence contexts. We previously reported that 4'-fluoro-4-aminobiphenyl (FABP)-modified duplexes (CCATCG*CTACC) exhibited a 2:3 mixture of B- and S-conformers, whereas the isomeric duplex with the G*CA context exclusively produced the B-conformer. To better understand this dramatic sequence effect, we have expanded our lesion repertoire to include 7-fluoro- 2-aminofluorene (FAF) and 7-fluoro-2-acetylaminofluorene (FAAF). In addition, we have conducted a systematic study involving simulated translesion synthesis of a 19-mer template oligonucleotide (5'- CTTACCATCG*CXACCATTC -3', X=T or A) annealed with different lengths of primers (n-1, n, n+3, and n+9). The structural and conformational origin of the G*CT effect was probed by dynamic 19F NMR, circular dichroism (CD), and molecular dynamics simulations. Primer extension assays (standing and running starts) revealed that extension slows down in these sequence, but at different rates. The mutational consequences of this unique sequence effect will be discussed in terms of the S/B conformational heterogeneity.
116. Defining the role of RNA secondary modifications in the response of microbial pathogens to chemical mediators of inflammation
Brandon S Russell(1), [email protected], 77 Massachusetts Avenue, Building NE47-295, Cambridge MA 02139, United States ; Kok-Seong Lim(1); Tsz Yan Clement Chan(1); James G Fox(1)(2)(3); Peter C Dedon(1)(3). (1) Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge MA 02139, United States (2) Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge MA 02139, United States (3) Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge MA 02139, United States In addition to the canonical ribonucleosides, there are >100 enzymatically modified ribonucleosides in tRNA and rRNA in all domains of life, with 15-25 in any given organism. Recent studies in yeast and human cells have revealed a critical role for ribonucleoside secondary modifications in the translational response of cells to toxic exposures. Toward the goal of understanding bacterial responses to the human innate immune system in chronic inflammation, we have developed an LC- ESI-QTOF method to characterize and quantify the complete spectrum of ribonucleosides in microbial pathogens, beginning with Helicobacter pylori. Initial studies to define the complete spectrum of ribonucleosides have resulted in identification of several previously unreported species. After establishing dose-response relationships for exposure to microbicidal chemical mediators of inflammation, exposure-induced changes in the spectrum of ribonucleoside secondary modifications were quantified by LC-ESI-QTOF. The results provide insights into the role of RNA secondary modifications in bacterial responses to inflammation.
117. Alterations in the serum metabolome induced by chronic inflammation
Erin G. Prestwich(1), [email protected], 77 Massachusetts Ave; NE47-295, Cambridge MA 02139, United States ; Peter C. Dedon(1)(2); Koli Taghizadeh(2); I. Ramesh Babu(1). (1) Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge MA 02139, United States (2) Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge MA 02139, United States
Chronic inflammation is mechanistically linked to human disease through the release of chemical and biological mediators, such as cytokines and reactive oxygen and nitrogen species, by immune cells at sites of inflammation. We studied inflammation-promoted changes in the serum metabolome in the SJL mouse model of nitric oxide over-production, which simulates human inflammatory processes. Deproteinized serum was analyzed using LC-MS and LC-MS/MS methods and the resulting spectra were comparatively analyzed on the basis of m/z values and retention time, targeting metabolites that changed two-fold or more. Significant differences in levels of several lipids were detected, but of special interest were both novel and expected changes in molecules present in the arginine and proline metabolic pathways. Additionally, a subset of animals fed the nitric oxide synthase inhibitor N- methyl-arginine exhibited altered metabolite levels. The potential application of these and other metabolites as biomarkers of inflammation will be discussed.
118. Antioxidant activity of some wines produced in Moldova and their related ability to bind N-nitrosamine precursors
Diana Porubin(1), [email protected], 60, Mateevici Street, Chisinau Chisinau MD-2000, Republic of Moldova . (1) Department of Ecologic and Industrial Chemistry, Moldova State University, Chisinau Chisinau MD-2000, Republic of Moldova
Epidemiological data illustrated that red wine can reduce the rate of mortality caused by heart disease, and also possesses a great variety of other beneficial biological activities. Since wine is known to contain relatively high amounts of polyphenols, and phenolic compounds are potent blocking agents of nitrosation, it is possible that wine can have cancer chemopreventive properties via the reduction of human exposure to endogenously formed carcinogenic N-nitroso compounds. The goal of this study was to evaluate antioxidant activity of grape extract and some wines produced in the Republic of Moldova, and to assess their related ability to bind nitrite, the precursor of nitrosating species. Red wine from the North of Moldova showed the highest degree of nitrite reduction in the (99, 64%), as compared to other tested wine samples. This type of wine also had the highest content of total polyphenols and possessed the highest antioxidant activity. Overall there was a positive correlation between the content of polyphenols, the antioxidant activity, and the ability to reduce nitrosating agents. 119. Differences in detailed chemistries among cigars and cigarettes and their potential relevance to the toxicological properties of mainstream cigar smoke
John H. Lauterbach(1), [email protected], 211 Old Club Court, Macon GA 31210, United States ; Deborah A. Grimm(2). (1) Chemistry & Toxicology, Lauterbach & Associates, LLC, Macon GA 31210, United States (2) Coordinated Instrumentation Facility, Tulane University, New Orleans LA 70118, United States
Several experts on smoking-related diseases have noted the rapid increase in the sales of certain types of cigars at the same time cigarette sales have been declining. Unlike cigarette tobaccos and cigarette smoke, whose chemical and toxicological properties have been well characterized, relatively little is known about cigars, particularly the newer types of machine-made cigars. Such contemporary products often have flavored tobacco fillers, and use reconstituted tobacco wrappers and binders as opposed to leaf tobacco used on premium cigars. Thus, detailed analyses of the fillers, binders, and wrappers would likely provide more information on product chemistries than would analyses on mainstream cigar smoke. We used two GC-MS scan techniques [Direct Silylation scan (in situ extraction and silylation with DMF/BSTFA) and Hexafluoroisopropanol scan (in situ extraction with HFP)] to characterize several brand-styles of contemporary cigars. The results from the two scans provided very good discrimination of tobacco fillers, binders, and wrappers.
120. Genetic toxicity of Fe3O4 nanoparticles
Xiao-Lin Bi(1), [email protected], 19B Yuquan Road, Wuzilou, A310, Beijing Beijing 100049, China ; Bing Wang(1); Xiangyu Meng(1); Wei Yue(1). (1) Key Laboratory for Biologicals Effects of Nanomaterials and Nanosafety, Chinese Academy of Sciences, Beijing 100049, China
Fe3O4 is widely used in biomedical applications, such as imaging, diagnosing etc. The potential toxicity could limit its in vivo use when it is nanosized. We used the animal model Drosophila Melanogaster to investigate its genetic toxicity. Drosophila Melangaster has been used in research for about 100 years, it is a powerful tool for genetic and developmental study. We feed Fe3O4 nanoparticles to male/female adult flies. We found the nanosized Fe3O4 led to reduced fertility in a dosage-dependent manner. Nanosized Fe3O4 caused the redistribution of Ca, Zn, Cu, Mn in embryos, and cellular apoptosis happened in embryos coordinate with redistribution of these metals. Nanosized Fe3O4 caused damages not only in treated adults, also in their untreated descendants.
121. Assessment of non-occupational exposure to polycyclic aromatic hydrocarbons through personal air sampling and urinary biomonitoring
Zheng Li(1), [email protected], 4770 Buford Highway, F-53, Atlanta GA 30341, United States ; James A Mulholland(2); Lovisa C Romanoff(1); Erin N Pittman(1); Debra A Trinidad(1); Andreas Sjodin(1). (1) National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta GA 30341, United States (2) School of Environmental and Civil Engineering, Georgia Institute of Technology, Atlanta GA 30332, United States
Non-occupational exposure to polycyclic aromatic hydrocarbons (PAHs) was studied in 8 nonsmoking volunteers. Personal air samples were collected continuously from the subjects at home, work and during commute, while all individual urine samples were collected during the 8-day period. Naphthalene was found at higher concentrations in indoor air at residences, whereas concentrations of larger PAHs (≥4 rings) were higher in air samples collected while commuting. Urinary PAH metabolite levels increased following reported high inhalation and/or dietary exposure. On days with a low-PAH diet, total amounts of inhaled naphthalene and fluorene over 24-hour periods were well correlated with their excreted urinary metabolite levels. During days with a high dietary intake, only naphthalene was significantly correlated with its metabolites. Our findings suggest that these non- occupationally exposed subjects were exposed to naphthalene primarily through indoor air inhalation and to other PAHs such as pyrene mainly through ingestion and traffic-related outdoor air inhalation.
122. Bridging health effects of chlorine exposures and therapy with chemistry: Toward a mechanism-oriented therapy for chlorine exposures
Giuseppe L Squadrito(1)(2), [email protected], RPHB 530, 1665 University Boulevard, Birmingham Alabama 35294, United States ; Edward M Postlethwait(1)(2); Sadis Matalon(3)(2). (1) Department of Environmental Health Sciences, School of Medicine, University of Alabama at Birmingham, Birmingham Alabama 35294, United States (2) Centers for Pulmonary Injury and Repair and Free Radical Biology, University of Alabama at Birmingham, Birmingham Alabama 35294, United States (3) Department of Anesthesiology, School of Medicine, University of Alabama at Birmingham, Birmingham Alabama 35294, United States
For many years the toxicology of chlorine (Cl2) has been of much interest because of human exposures due to industrial, household, recreational, and transport accidents as well as deliberate acts resulting in the release of Cl2. Paradoxically, at present, therapy for Cl2 inhalation injury consists in alleviating pulmonary symptoms. Only recently, a chemical mechanism-oriented approach to prophylactic and post-exposure therapy has been employed wherein antioxidant replenishment, as well as various compounds to restore compromised alveolar function, are used to counteract Cl2 toxicity and to decrease morbidity. We here describe chemical and physical properties of Cl2 that are relevant to its chemical reactivity with water and biological molecules that are present in the lining fluid layers covering the airways and alveolar spaces, and conduct an axial Cl2 penetration analysis across the surface of the lung. This theoretical analysis is then used to assess the potential benefits of adjuvant antioxidant therapy and is compared to experimental results.
123. Biological oxidant damage of engineered nanomaterials: Relationship with physicochemical properties
Dhimiter Bello(1)(2), [email protected], One University Ave., Lowell MA 01854, United States ; Daniel Schmidt(3)(2); Anoop Kumar Pal(4); Eugene Rogers(5)(2); ShuFeng Hsieh(5)(4). (1) Work Environment, University of Massachusetts Lowell, Lowell MA 01854, United States (2) Center for High Rate Nanomanufacturing, University of Massachusetts Lowell, Lowell MA 01854, United States (3) Plastics Engineering, University of Massachusetts Lowell, Lowell MA 01854, United States (4) Biomedical Engineering & Biotechnology, University of Massachusetts Lowell, Lowell MA 01854, United States (5) Clinical Laboratory and Nutritional Sciences, University of Massachusetts Lowell, Lowell MA 01854, United States
Oxidative stress elicited by engineered nanomaterials (ENMs) is an important mechanism of toxicity of inhaled particles and holds promise as a novel metric for exposure assessment and toxicity screening. A Ferric Reducing Ability of Serum (FRAS) assay was used to assess biological oxidant damage (BOD) elicited by ENMs when in contact with human serum. Sixty ENMs, representing major ENM classes and subclasses, were tested by FRAS. The physicochemical properties of ENMs, including specific surface area (SSA), total and water-soluble transition metals and organic carbon, were also characterized. We report here on the observed range, the between- and within ENM class- variability in BOD and its relationship with several physicochemical properties. This study demonstrates tremendous within- (e.g. CNTs) and between-class variability in BOD of ENMs. BOD was strongly associated with SSA and several transition metals. We also report on the range and differences in the intrinsic surface activity index (BOD/m2) of ENMs. 124. Biosimulation of acetaminophen-induced liver injury using the Entelos Physiolab platform
Harvey J Clewell(1), [email protected], PO Box 12137, Research Triangle Park NC 27709, United States ; Brett Howell(1); Scott Siler(2); Richard Ho(2); Alison Harrel(1); Melvin Andersen(1); Paul Watkins(1). (1) The Hamner Institutes for Health Sciences, Research Triangle Park North Carolina 27709, United States (2) Entelos, Inc., Foster City California 94404, United States
A biosimulation model is being developed to provide a quantitative description of the key processes involved in acetaminophen-induced liver injury: acetaminophen metabolism, reactive metabolite formation, glutathione depletion and resynthesis, ROS formation, energy homeostasis, mitochondrial dysfunction, necrosis, apoptosis, and tissue regeneration. The current stage of the platform includes a whole-body physiologically based pharmacokinetic (PBPK) model for acetaminophen and its metabolites in the mouse, rat, and human. The PBPK model is linked to a pharmacodynamic (PD) model describing the reaction of acetaminophen-derived N-acetyl-p-benzoquinone imine (NAPQI) with glutathione and the resulting depletion and resynthesis of glutathione. The formation of NAPQI- adducts with cellular proteins and increased oxidative stress resulting from glutathione depletion are linked to cellular damage and response. This PBPK/PD model of acetaminophen metabolism and glutathione depletion has been used to investigate the extent to which differences in metabolism and glutathione homeostasis explain interspecies differences in susceptibility to acetaminophen toxicity.
125. Nuclear magnetic resonance- (NMR-) based study of post-mortem urines of opioid overdosed patients: Scope and limitations of the technique
Gloria Proni(1), [email protected], 445 West 59th Street #4405N, New York NY 10019, United States ; Nixon Tan(1); Donna K. Wilson(1); Elise Champeil(1); Barry S. Levine(2). (1) Science, John Jay College, New York NY 10019, United States (2) Department of Health and Mental Hygiene, Office of the Chief Medical Examiner, Baltimore MD 21201-1020, United States
Testing for substances of abuse in urine has great toxicological relevance. Testing is used in a forensic context to, among other things, identify drugs of abuse, to support or deny a person's statement regarding drug use, or to determine which drug may have caused an overdose. In the following presentation, the advantages and limitations of using nuclear magnetic resonance (NMR) spectroscopy for the identification of substances of abuse in urine is explored. Opioids were chosen for analysis since federal regulations regarding drug testing in urine mandate a higher cutoff level for opiooids than for other substances of abuse thereby making NMR spectroscopy more feasible when used for these drugs. NMR spectroscopy for the analysis of opioids has many advantages: 1) it allows for positive identifications of chemically different species (very similar substrates can be usually identified); 2) very little sample preparation or operator training is needed; 3) spectra could be gathered relatively quickly. NMR spectroscopy has proven to be a novel, feasible and useful technique for the study of opioids in urine samples and in fact can even be used to detect and quantify opioids in urine collected post-mortem from patients who have overdosed.
126. Role of lipid oxyl and peroxyl radicals in oxidative damage to guanine and 8- oxoguanine bases in DNA
Vladimir Shafirovich(1), [email protected], 29 Washington Place, New York New York 212-998-8421, United States ; Jie Shao(1); Nicholas E Geacintov(1). (1) Department of Chemistry, New York University, New York New York 212-998-8421, United States
Reactive intermediates derived from lipid peroxidation under conditions of oxidative stress (free radicals and electrophilic aldehydes and expoxides) induce permanent alterations and genetic instabilities that increase the risk of development of human cancers. Guanine is a primary target of free radical attack. We have developed a photochemical method for generation of oxyl and peroxyl radicals for studying in real time the reactions of these reactive intermediates with G and 8-oxoG bases in DNA. This approach reveals that oxyl radicals induce fast one-electron oxidation of G and 8- oxoG bases to form the corresponding neutral radicals. In contrast, peroxyl radicals do not react with observable rates with these substrates. The major pathways for formation of the end products of G and 8-oxoG oxidation are the combination of the nucleobase radicals with peroxyl or superoxide radicals to form the highly mutagenic 2,5-diamino-4H-imidazolone and dehydroguanidinohydantoin chemical end products. Supported by NIEHS Grant 5R01 ES011589-08.
127. Acute toxicity, biochemical responses and gene expression responses in Eisenia fetida exposed to polycyclic musks
Chun Chen(1), [email protected], 94 Weijin Road, Tianjin 300071, China ; Qixing Zhou(2). (1) Nankai University, China (2) Chinese Academy of Sciences, China
AHTN and HHCB are recognized as ubiquitous contaminants in the environment and have potentially adverse impacts on the soil animals. In this study, lethal toxicity on earthworm (E.fetdia) using the -2 filter paper contact test indicated that the 24 h-, 48 h-LC50 values of HHCB (32.60, 11.87 μg•cm ), was about 2-fold more toxic than AHTN (63.41, 20.76 μg•cm-2). The significant enhanced lipid peroxidation (LPO) levels in a time- and dose-dependent manner leads to reactive oxygen species (ROS)-induced cellular oxidative injury. In addition, the significantly up-regulation of SOD and CAT mRNA expression at low exposure doses of 0.6 μg•cm-2 AHTN, 0.3 μg•cm-2 HHCB over time, and down-regulation at 48 h exposure of high doses of 6.0 μg•cm-2 AHTN, 3.0 μg•cm-2 HHCB were observed. Overall, these results indicate that dermal contact of AHTN and HHCB results in pronounced physiological responses in earthworm and suggest that transcriptional level changes in antioxidant genes could be critical molecular biomarkers for stress levels.
128. Investigation of the non-covalent binding mode of leinamycin with double-stranded DNA
Mostafa I. Fekry(1), [email protected], 601 S. College Avenue, 125 Chemistry Building, Columbia MO 65211, United States ; Kent S. Gates(1). (1) University of Missouri, Columbia, United States
Non-covalent DNA binding drives the structurally novel antitumor antibiotic leinamycin to alkylate the N7-position of guanine residue in the DNA major groove. Interestingly, leinamycin does not contain classical non-covalent DNA-binding motifs. Here we will summarize efforts toward characterization of non-covalent DNA binding by leinamycin. Activated leinamycin alkylates single-stranded DNA poorly relative to double stranded DNA. Intercalators that block access to the N7-position of deoxyguanosine residues in the duplex inhibit DNA alkylation by leinamycin and we have measured the IC50 for this process. We have also examined the effects of 5-methyl-2'-deoxycytosine residues surrounding leinamycin alkylation sites. Finally, alkylation yields of duplexes containing 5'-CG and 5'-GC alkylation sites have been examined. The data is consistent with a model in which the thiazole-dienone fragment of leinamycin intercalates into DNA in a manner that facilitates alkylation of guanine in the major groove of DNA.
129. Chemical biology of NO in mechanisms of inflammation
David Wink(1), [email protected], Bldg. 10, Room B3-B69, Bethesda MD 20892, United States ; Christopher Switzer(1); Wilmarie Flores-Santana(1); Sharon Glynn(1); Lisa Ridnour(1); Robert Cheng(1). (1) National Cancer Institute, National Institutes of Health, United States Nitrogen oxide (NO) is a critical component of inflammation response. Nitric oxide and its congenitors are cytotoxic participants in the antitumoral and anti-pathogen mechanisms of the immune system, as well as being implicated as causative agents in tissue injury. At the same time, NO possesses beneficial properties protecting tissue against ischemia reperfusion injury and in immunosuppression. A clue to this dichotomy is that the biological properties of NO are determined by its chemistry with the molecular target, referred to as the chemical biology of NO. Here, the role of chemistry of NO in the inflammation process will be discussed. Critical determinants of NO behavior in inflammation are its concentration and the specific cellular pathways affected. The pro and anti-inflammatory pathways correlate with distinct concentrations of NO (ranging from nM-µM) that determine the outcome.
130. Oxidation of DNA bases leading to mutagenic lesions
Cynthia J. Burrows(1), [email protected]; Aaron M. Fleming(1); Sucharita Kundu(1). (1) Chemistry, University of Utah, Salt Lake City UT 84112-0850, United States
Reactive oxygen and nitrogen species generated by inflammatory responses can lead to molecular changes in DNA bases resulting in mutagenic lesions. Guanine is particularly susceptible to oxidation leading to a suite of damage products, many of which are highly mutagenic. The guanine oxidation pathway will be reviewed with particular emphasis on recent results on the participation of amine nucleophiles that yield a variety of hydantoin structures. Excision of these lesions with hNEIL1 will also be presented.
131. DNA damage and biomarkers of inflammation
Laura J Niedernhofer(1), [email protected], 5117 Centre Avenue, Hillman Cancer Center, 2.6, Pittsburgh PA 15213-1863, United States ; Andria R Robinson(1); Siobhan Gregg(1); Jeremy S Tilstra(1); Peter Wipf(2); George A Garinis(3); Paul D Robbins(1). (1) Department of Microbiology and Molecular Genetics, University of Pittsburgh, Pittsburgh PA 15260, United States (2) Department of Chemistry, University of Pittsburgh, Pittsburgh PA 15260, United States (3) Department of Cell Biology and Genetics, Institute for Molecular Biology and Biotechnology, Heraklion Crete GR 711 10, Greece
ERCC1-XPF is a nuclease that participates in the excision of multiple types of DNA damage. Mutations in XPF cause a progeroid syndrome, a disease of accelerated aging. Our lab uses a mouse model of this human progeria to investigate the molecular basis of aging. NF-kB is a transcription factor that is activated in response to stress including inflammatory and genotoxic stress. Using a transgenic reporter, we detected increased NF-kB activation in numerous tissues of progeroid and old normal mice. Either genetic or pharmacologic inhibition of NF-kB activation significantly delayed the onset of degenerative diseases in the progeroid mice. In addition, ERCC1- XPF deficient mice have increased oxidative DNA damage, lipid hydroperoxides and oxidized thiols compared to wild-type littermates. And chronically exposing the mice to a mitochondrial-targeted radical scavenger delayed aging. These data support the conclusion that NF-kB and oxidative stress are not only biomarkers, but also key drivers, of aging.
132. Lipid oxidation and inflammation
Ian Blair(1), [email protected], 854 Biomedical Research Building II/III, 421 Curie Boulevard, Philadelphia PA 19104, United States . (1) University of Pennsylvania School of Medicine, United States
Lipid oxidation products derived from the lipoxygenase activity of cyclooxygenases (COXs) have been largely ignored as potential anti-inflammatory agents in spite of their abundance as products of arachidonic acid (AA) metabolism. We showed previously that 11(R)-hydroxy-5,8,12,14-(Z,Z,E,Z)- eicosatetraenoic acid (11(R)-HETE) is a major oxidized lipid formed from arachidonic acid (AA) metabolism in rat intestinal epithelial cells that stably express COX-2 (RIES cells). 11(R)-HETE arises from peroxidase-mediated reduction of initially formed 11-hydroperoxy-5,8,12,14-(Z,Z,E,Z)- eicosatetraenoic acid (11(R)-HPETE). Surprisingly, 11(R)-HETE was an excellent substrate for 15- hydroxyprostaglandin dehydrogenase (PGDH), with a catalytic efficiency similar to that observed for 15(S)-HETE. The resulting metabolite was 11-oxo-5,8,12,14-(Z,Z,E,Z)-eicosatetraenoic acid (11-oxo- ETE). 11-Oxo-ETE and 15-oxo-ETE were secreted in nM concentrations when AA was added to RIES and human Caco-2 cells. 11-Oxo-ETE was 500-fold more potent than 15-deoxy-D12,14- prostaglandin J2 (15d-PGJ2) at inhibiting endothelial cell proliferation suggesting that it is the endogenous oxidized lipid responsible for the anti-inflammatory activity ascribed to 15-dPGJ2. Supported by RO1CA091016.
133. Chelation: The mechanism of action of AGE/ALE inhibitors
John Baynes(1), [email protected], 631 Sumter Street, Columbia SC 29208, United States . (1) University of South Carolina, United States
Glycoxidation and lipoxidation reactions produce irreversible adducts and crosslinks in protein, known as advanced glycation and lipoxidation end-products (AGE/ALEs). AGE/ALEs increase in long-lived proteins with age, and their rate of accumulation is increased in diabetes, nephropathy and inflammatory diseases. AGE/ALE inhibitors include a range of amino and thiol compounds that are proposed to act by trapping carbonyl intermediates in AGE/ALE formation. However, some of the newer generation of AGE/ALE inhibitors lack reactive functional groups, indicating an alternative mechanism of action. We propose that AGE/ALE inhibitors function primarily as chelators, inhibiting metal-catalyzed oxidation reactions. Indeed, simple chelators, such as trientine and citrate, inhibit AGE formation and development of complications in animal models of diabetes. The AGE-inhibitory activity of ACE inhibitors and ARBs probably also results from their chelating activity. Mild chelators should prove useful in treatment of diabetes, atherosclerosis, and other age-related diseases characterized by oxidative stress and inflammation.
134. Chemical pathology of phagocyte activation
Jay Heinecke(1), [email protected], 1959 NE Pacific Street, UW Mailbox 356426, Seattle WA 98195, United States ; Baohai Shao(1). (1) University of Washington School of Medicine, United States
High density lipoprotein (HDL) protects against cardiovascular disease by removing cholesterol from macrophages in the artery wall. This pathway is critically dependent upon two proteins: ABCA1, which promotes cellular cholesterol efflux, and lecithin cholesterol acyltransferase (LCAT), which rapidly esterifies free cholesterol. We have proposed that reactive intermediates generated by myeloperoxidase (MPO) convert HDL into a dysfunctional form. We used mass spectrometry (MS) to demonstrate that HDL isolated from heart disease patients contains elevated levels of 3- chlorotyrosine and 3-nitrotyrosine, two characteristic products of MPO. Oxidized apolipoprotein A-I (apoA-I), the major HDL protein, lost its ability to promote cellular cholesterol efflux by ABCA1 and LCAT. Tandem MS analysis of apoA-I isolated from human atherosclerotic lesions, together with biochemical studies of mutant forms of apoA-I, implicated oxidation of specific tyrosine and methionine residues in the loss of ABCA1 and LCAT activity. Our observations raise the possibility that MPO generates dysfunctional HDL in humans. 135. Proteomic analysis of inflammatory bowel disease
Steven R. Tannenbaum(1), [email protected], 77 Massachusetts Ave., Cambridge MA 02139, United States ; Yu Zeng(1); Charle Knutson(1); Arek Raczynski(1); John S. Wishnok(1); Joshua Korzenik(2). (1) Massachusetts Institute of Technology, United States (2) Massachusetts General Hospital, United States
Inflammation is a common component of many diseases, and is characterized by formation of large quantities of pro- and anti-inflammatory cytokines which are capable of signaling the cells of the innate immune system, both lymphocytes and granulocytes, as well as many types of epithelial cells. Often overlooked is the role of macrophages and neutrophils to produce very reactive chemicals which are capable of nonenzymatic modification of all of the constituents of a biological system, including proteins, nucleic acids, lipids, and glycans. glycomics). In this talk I will present our recent results on analysis of serum cytokines, serum protein changes, and serum and tissue protein modifications in one or more human diseases. The modifications include oxidation, halogenation, and carbonylation of protein side chains. These “biomarkers” may be used to examine the status of disease, evaluate therapeutic intervention, or even predict future course.
136. Hepatitis and liver disease
John Groopman(1), [email protected], 615 N. Wolfe Street, Baltimore MD 21205, United States . (1) The Johns Hopkins University, United States
A number of investigations have found that chronic carriers of HBV, as indicated by A number of investigations have revealed that chronic carriers of HBV, as indicated by sequential hepatitis B surface antigen (HBsAg) positivity at six month intervals, were at increased risk of developing liver cancer. The overwhelmingly significant etiological factors associated with development of liver cancer in the economically developing world are infection in early life with HBV and lifetime exposure to high levels of aflatoxin B1 (AFB1) in the diet. Indeed, the multiplicative interaction between HBV and AFB1 has been documented in two separate cohorts at high risk for HCC. Over the past 20 years, an appreciation for the role of the hepatitis C virus (HCV) has also emerged. HCV is contributing to HCC being the most rapidly rising solid tumor in the US and Japan. Knowledge of these progressive mechanisms and etiologic factors will permit the development of translational and preventive interventions.
137. Inflammation and asbestos-associated diseases
Brooke Mossman(1), [email protected], Pathology E203 Given, 89 Beaumont Drive, Burlington VT 05405, United States ; Jedd Hillegass(1); Arti Shukla(1); Maximilian MacPherson(1); Stacie Beuschel(1); Sherrill Lathrop(1); Timothy Perkins(1); Chad Steele(2). (1) University of Vermont College of Medicine, United States (2) University of Alabama School of Medicine, United States
Inflammation is an early response of rodents to inhalation of asbestos, and is observed in lung tissues and lavages from patients with asbestos-associated diseases. The mechanism of inflammation by asbestos fibers and its causality in lung or pleural disease are critical to prevention and therapy. We recently reported that crocidolite asbestos activates the Nalp3 inflammasome in human monocytes, that leads to secretion of IL-1B (Science: 320:674-677,2008). Much to our surprise, normal human mesothelial cells secrete a number of inflammatory (including IL-1B), proliferative and angiogenic cytokines in vitro that are similar to patterns in pleural lavage fluids after intraperitoneal injection of human mesothelioma cells into a SCID mouse xenograft model. Profound neutrophila , elevations in IL-6, IL-8 and IL-12 as well as G-CSF, VEGF, PDGF-BB and bFGF were produced prior to the development of MMs. We are currently investigating IL-1R antagonists in this model. 138. Bioactive chlorinated lipids derived from plasmalemmal plasmalogens
David Ford(1), [email protected], DuBourg Hall, Room 119, 1100 S. Grand Blvd, St. Louis MO 63104, United States . (1) St. Louis University School of Medicine, United States
Plasmalogens are a predominant plasmalemmal phospholipid possessing a vinyl ether bond that is vulnerable to attack by reactive chlorinating species resulting in the release of an a-chloro fatty aldehyde. a-Chloro fatty aldehyde is the precursor of a family of chlorinated lipids including a-chloro fatty acid and a-chloro fatty alcohol. a-Chloro fatty acid is incorporated into complex lipids and is catabolized in the liver to dicarboxylic acids that are excreted in urine. Chlorinated lipids are released from activated neutrophils and accumulate in both bronchial alveolar lavage fluid and plasma during lung injury. In addition to lung injury, these chlorinated lipids are elevated in both human atherosclerotic lesions and rat infarcted myocardium in comparisons to normal tissues. Pro- inflammatory properties of chlorinated lipids include chemoattraction, activation of NFkB, and alterations in normal cellular lipid metabolism. These novel chlorinated lipids may be critical molecular mediators of and provide new biomarkers for pro-inflammatory diseases.
139. Cyclooxygenase-based anti-inflammatory drugs
Lawrence Marnett(1), [email protected], 23rd Avenue South & Pierce, Room 850 RRB, Nashville TN 37232, United States ; M. Jashim Uddin(1); Brenda C. Crews(1); Philip J. Kingsley(1); Anna L. Blobaum(1); David W. Piston(1). (1) Vanderbilt University, School of Medicine, United States
Cyclooxygenase-2 (COX-2) plays a critical role in the synthesis of eicosanoids that contribute to inflammation and cancer. It is a highly validated target for the treatment of acute and chronic inflammation, cancer prevention, and adjuvant cancer therapy. The side effect profiles of non- steroidal anti-inflammatory drugs (NSAIDs) and COX-2-selective inhibitors have reduced their use in therapeutic applications. However, the availability of new clinical data suggest a more complex approach to therapy be adopted that takes into account risk and potential benefit. The structure of COX-2 makes it an ideal candidate for targeting, either for therapy or for imaging. Proof-of-concept experiments are described that demonstrate COX-2-targeted fluorescent imaging agents are useful for the detection of inflammatory and premalignant lesions. The results suggest novel strategies to exploit COX-2 for detection and treatment of a range of inflammation-related diseases.
140. Nitric oxide-based drug development
Larry Keefer(1), [email protected], Building 538, Room 205F, NCI-Frederick, Frederick MD 21702, United States . (1) National Cancer Institute, United States
Nitric oxide (NO) is a central player in the inflammatory process. To investigate its many roles in normal as well as pathophysiology, caged NO donors of the diazeniumdiolate class (also known as NONOates) have come into wide use as research tools, offering a range of reliable half-lives from 2 seconds to 20 hours for spontaneously generating authentic NO into aqueous media at physiological temperature and pH. Current work is aimed at exploring the utility of this caged NO chemistry in designing improved drugs and biomedical devices. My presentation will highlight a selection of recent advances in this area.