July 2017

    Nicholas Geacintov
    Nicholas Geacintov
    Message from Chair

    As usual at this time of the year, many TOXI members are preparing to attend the annual meeting of our Division of Chemical Toxicology held in conjunction with the 254th American Chemical Society National Meeting in Washington, D.C. August 20-24, 2017. This year, Dr. Tom Spratt, our Program Chair, has assembled an exciting program that includes 108 presentations. If you are planning to attend the meeting and have not yet made hotel reservations, we urge you to do so immediately, since the choices for finding well located hotels at a reasonable price are dwindling rapidly as the meeting date approaches.

    The meeting begins on Sunday morning (August 20) with the Chemical Research in Toxicology Young Investigator Symposium. This year the Chemical Research in Toxicology Young Investigator Award winner is Dr. Huiwang Ai, Assistant Professor of Chemistry at the University of California Riverside. Professor Ai is well known for his outstanding and highly cited papers on the development of fluorescent probes for biological applications, including fluorescent biosensors for redox signaling and for evaluating oxidative stress. The Founders’ Award Symposium will be held as usual on Sunday afternoon. This year’s winner of the Founders’ Award is Dr. Ian Blair, A. N. Richards Professor of Pharmacology at the Perelman School of Medicine and Vice-Chair, Department of Systems Pharmacology and Translational Therapeutics at the University of Pennsylvania. Dr. Blair has published close to 400 papers that are widely cited because of his innovative application of mass spectrometry to biomedical problems. These include the development of stable-isotope labeling assays and ultra-sensitive techniques for detecting various biomarkers of oxidative stress. Ian is well known within the TOXI Division since he served several terms on the Editorial Board of our journal, Chemical Research in Toxicology. Both Award symposia will feature lectures by the awardees and other distinguished researchers in their respective areas of investigation.

    The Monday morning symposium (August 21) is dedicated to TOXI Young Investigators. This year, ten graduate students and post-docs will be describing the highlights of their research, and we look forward to hearing about the activities of this group of young and dynamic investigators.  Dr. Judy Bolton has organized a symposium on Biological Targets of Botanical Supplements that will be presented on Monday afternoon (August 21). This is a great topic that brings high-level chemistry-based research to the important area of natural product drug interactions that are relevant to human health.

    Drs. Yinsheng Wang and Orlando Schärer have organized a symposium on Cross-link DNA Repair on Tuesday morning (August 22). This is currently a hot area of research in the field of DNA repair because DNA cross-links are very difficult to remove by normal human DNA repair systems. On Tuesday afternoon, the topic of the symposium organized by  Drs. Fred Guengerich, Nick Meanwell and Griff Humphreys is Toxicological Considerations in Antibody Drug Conjugate Design and Development. This is another hot topic at the interface of chemistry and human health that ranges from basic research to translational research. We have been reading about this area in the popular press for a while, and it will be great to hear from experts on the subject about their most recent findings in the field of antibody-drug conjugate design and development.

    Another general interest highlight of the meeting is this year’s Keynote Lecture by Dr. Paul Watkins on late Tuesday afternoon (August 22). The topic of the lecture is Understanding hepatoxicity: man to mouse to computer. Dr. Watkins is a recipient of numerous honors and awards and is Professor of Medicine, Professor of Toxicology, and Professor of Experimental Therapeutics at the University of North Carolina, Chapel Hill. He also serves as the director of The Hamner-UNC Institute for Drug Safety Sciences, located at Research Triangle Park, and is also the Howard Q. Ferguson Distinguished Professor of Pharmacy, as well as the Director of the Institute for Drug Safety Sciences at the UNC Eshelman School of Pharmacy. Dr. Watkins is an expert on drug-induced liver disease, spanning the full spectrum from basic to clinical research, to clinical trials and regulatory affairs. We look forward to an interesting and stimulating keynote lecture that spans overlapping areas of basic and translational research.

    Tuesday evening is devoted to our General Poster Session (56 posters will be presented). Our Division will also present awards honoring platform and poster presentations. A light buffet dinner will be offered, as usual, during the poster session. Following the presentation of awards on Tuesday evening, there will be a general business meeting to which all TOXI members and meeting attendees are invited. The Tuesday evening session is a great opportunity for meeting and socializing with other TOXI members, discussing posters, and discussing science in general. This has been a popular event since we first started it when I was the Program Chair many years ago!  I am looking forward to seeing you there!

    One general shortcoming of the Tuesday evening session is that there is not enough time to see all of the posters. Please be reminded that many (20), if not all, of the posters displayed on Tuesday evening are also presented at the Sci-Mix session on Monday evening.  This event is yet another opportunity to see these great posters. Please get a head start on the posters on Monday evening, and then attend the Tuesday evening business meeting, poster, and dinner session with all of the TOXI members.  I am looking forward to seeing you there!

    Wednesday morning (August 23) is dedicated to senior investigators who are reporting their latest findings from their laboratories. This year we will have 11 such presentations (General Oral papers). The final symposium of the meeting on Wednesday afternoon (Advanced mass spectrometric techniques in toxicology) was organized by  Drs. Silvia Balbo and Peter Villalta. This session is based on exciting new applications of mass spectrometric techniques to the field of chemical toxicology by five experts in this area of research. This symposium is thematically related to the Founders Award Symposium Sunday afternoon, and is an appropriate final session of this meeting.

    I would like to alert you to another event associated with the mass spectrometry theme of this year’s meeting.  ACS Science Tuesdays is a weekly Q&A session, better known as AMA (Ask Me Anything) on Reddit.   Each week, ACS experts are featured on one of Reddit’s most popular communities, Reddit Science, to discuss various chemistry topics. Ian Blair, our 2017 Founders’ Award winner has agreed to host an AMA on August 15. The ACS chose this date as a lead-in to the Founders’ Award Symposium.  This is a wonderful opportunity to highlight our mass spectrometry and other symposia this year. For those who are not familiar with Reddit, it is an American social news aggregation, web content rating, and discussion website. (https://en.wikipedia.org/wiki/Reddit)

    Finally, I would like to thank Tom Spratt, and those who assisted him, for the wonderful job of organizing this outstanding TOXI 2017 program. I also would like to thank our Treasurer Dr. Natalia Tretyakova for securing yet another NIEHS Scientific Meeting Grant, which will help to cover some of the costs incurred by graduate students who will be presenting their work at the meeting.  Finally, I would like to thank Silva Balbo who secured funding from ThermoFisher.

    As a final note, I would like to urge anyone who is able to stay at the meeting through the last day.  It is important for us to attend the talks of our colleagues who made great efforts to prepare their presentations and travel to Washington to share their results with us!

    With best wishes to all,

    Nick Geacintov

    Chair

    Abstracts, 2017

    TOXI 1

    Targeted quantitative proteomic approaches for interrogating the human kinome

    Weili Miao, Yongsheng Xiao, Lei Guo, Yinsheng Wang, yinsheng.wang@ucr.edu. Univ of California Riverside, Riverside, California, United States

    Kinases play crucial roles in cell signaling, and aberrant kinase signaling has been observed in many human diseases including cancer. Kinases have also been actively pursued as molecular targets for anti-cancer therapy, where more than 100 distinct kinase inhibitors are under various stages of clinical trials for anti-cancer therapy. Understanding cell signaling and uncovering kinase targets for anti-cancer therapy call for high-throughput methods for interrogating quantitatively the human kinome. In this presentation, we will discuss our recent efforts toward the development of targeted quantitative proteomic approaches for assessing the human kinome in cancer cells and tissues. In addition, the application of the kinome profiling methods for uncovering novel kinase targets of clinically used kinase inhibitors, for revealing kinases involved in the promotion and suppression of cancer metastasis, and for discovering the alterations of signaling pathways arising from exposure to toxic chemicals will be described. Together, we hope to demonstrate that our targeted quantitative proteomic methods offer sensitive, accurate, reproducible and high-throughput quantitative analysis of the human kinome.

    TOXI 2

    Sequence-specific covalent capture for detection of disease-derived nucleic acid sequences

    Kent S. Gates1, gatesk@missouri.edu, Andrew Gu2, Maryam Imani Nejad1, Ruicheng Shi2, Xinyue Zhang2. (1) Department of Chemistry, University of Missouri, Columbia, Missouri, United States (2) Department of Bioengineering and Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri, United States

    Methods for the detection of DNA and RNA sequence are important in research and medicine. Logically, many strategies for the detection of nucleic acid sequence rely on Watson-Crick hybridization of a probe strand to target DNA or RNA in the sample. However, all hybridization-based approaches for sequence detection suffer from a common limitation: the non-covalent, inherently reversible nature of nucleic acid hybridization means that signal can be compromised by partial denaturation of the probe-target duplex during analysis (e.g. washing). Long probes provide a more durable signal, but lack sensitivity to mismatches, while short probes provide sequence specificity but do not yield a durable signal. Here we explore sequence-specific covalent capture of target nucleic acid sequences as a means to overcome these challenges. Sequence-selective capture reactions employed in fluorescence, electrophoretic, and protein nanopore-based detection of a cancer-driving T→A mutation at position 1799 of the human BRAF kinase gene sequence and other disease-relevant sequences will be described.

    TOXI 3

    Dynamic visualization of signaling molecules in living cells

    Jin Zhang, jzhang32@ucsd.edu. Pharmacology, University of California, San Diego, La Jolla, California, United States

    In this talk, I will first discuss several fluorescent biosensors that we recently developed to monitor intracellular signaling molecules. I will then present studies where we combined genetically encoded fluorescent biosensors, superresolution imaging, mathematic modeling and targeted biochemical perturbations to probe the spatiotemporal regulation of signal transduction.

    TOXI 4

    Seeing is believing: Fluorescent biosensors for redox signaling and oxidative stress

    Huiwang Ai, huiwang.ai@gmail.com. Department of Chemistry, University of California Riverside, Riverside, California, United States

    It is widely accepted that cells need to maintain reduction-oxidation (redox) homeostasis for proper functions. Redox chemical reactions have also been identified for broad uses in and between cells to generate and relay signals. Despite the progress made in the past decades, many aspects of redox regulation and signaling remain elusive. Research in this field is greatly hindered by the lack of available methods to detect redox-signaling molecules and protein redox states in cells and cell subdomains. Our laboratory uses protein engineering techniques to convert fluorescent proteins into fluorescent biosensors that can actively respond to redox changes. In this talk, I will cover several novel fluorescent biosensors for reactive oxygen species and redox modifiers.

    TOXI 5

    Biochemical and toxicological applications of mass spectrometry

    F P. Guengerich, f.guengerich@vanderbilt.edu. Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, United States

    Mass spectrometry (MS) has been utilized in a variety of new applications in this laboratory. Small proteins react with electrophiles, and the kinetic analysis of unstable protein adducts can be measured, e.g. trichloroethylene oxide. MS can be used to determine sequences of DNA primers elongated past DNA adducts on templates, particularly in mixtures, to evaluate the nature of miscoding. MS can also be used for the sequence analysis of DNA adducts, as demonstrated for polycyclic aromatic hydrocarbons and C4′ oxidation products. High-resolution (HR) MS can distinguish 2H from (natural abundance) 13C and was invaluable in elucidating the catalytic mechanism of P450 19A1, the steroid aromatase, as well as P450 11A1, the cholesterol side chain cleavage enzyme. Metabolomics-type approaches with HRMS have identified substrates for “orphan” P450s (e.g., 2W1, 2U1, 4X1), and HRMS has been useful in defining the location of the orphan P450 27C1 in skin. (Supported in part by NIH R01 ES02205, R01 ES010546, R01 ES026955, R37 CA090426, and R01 GM118122.)

    TOXI 6

    Human aldo-keto reductases and aryl hydrocarbon activation

    Trevor M. Penning, penning@upenn.edu. Center of Excellence in Environmental Toxicology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, United States

    Aldo-Keto Reductases (AKRs) are a superfamily of monomeric 37kDa NAD(P)(H) dependent oxidoreductases that act as reductases in vivo to convert ketones and aldehydes to secondary and primary alcohols, respectively for subsequent conjugation reactions. In humans 15 AKRs exist and details on their kinetic and catalytic mechanism and structural biology exist. AKR1D1 is the only human steroid 5β-reductase and reduces double bonds while AKR1C members exhibit nitroreductase activity expanding the AKR substrate repertoire. Our work has focused on the AKR1C subfamily and their roles in the metabolic activation of aryl hydrocarbons using LC/MS. Aryl hydrocarbons are suspect human carcinogens that pollute the environment represented by the pyrogenic polycyclic aromatic hydrocarbons (PAH; e.g. benzo[a]pyrene); the alkylated and oxygenated petrogenic PAH that come from oil-spills (e.g. C1-C4 phenanthrenes and C1-C2 chrysenes); and the nitro-PAH that come from diesel exhaust. AKR1C enzymes compete with the formation of diol-epoxides by converting PAH-trans-dihydrodiols to electrophilc and redox-active o-quinones; these are delivered by the aryl hydrocarbon receptor to the nucleus, generate 8-oxo-dGuo which mutates the p53 tumor suppressor gene to impact lung cancer initiation. Less is known about the metabolic activation of alkylated-PAH, and some cannot form diol-epoxides due to alkylation. Common metabolites formed include bis-electrophiles that may act as cross-linking agents as well as catechol-conjugates that can be used as urinary biomarkers for risk assessment of oil exposure in humans. Nitro-PAH can be reduced by AKR1C subfamily members to the corresponding hydroxylamino-derivatives for activation by sulfonation. The human AKR1C genes are among the most highly induced by the Nrf2-Keap1 system by the electrophiles and reactive-oxygen species that they generate suggesting that a functional consequence of AKR upregulation is enhanced aryl hydrocarbon activation. These findings suggest that the chemopreventive roles of Nrf2 activators is context dependent [Supported by P30-ES013508 to TMP].

    TOXI 7

    Chemical biology of DNA damage by α,β-unsaturated aldehydes

    Lawrence J. Marnett, larry.marnett@vanderbilt.edu. Vanderbilt University, Nashville, Tennessee, United States

    α,β-Unsaturated aldehydes react with nucleic acid bases to form exocyclic adducts that block Watson-Crick base-pairing and impede replication by DNA polymerases. Exocyclic adducts are bypassed by translesion polymerases. a process that induces base-pair substitutions and frameshift mutations. The adducts are repaired to differing extents by nucleotide excision repair and base excision repair. Certain exocyclic adducts retain electrophilic character while present in DNA and participate in cross-linking and hydrolytic ring-opening. For example, the pyrimidopurinone adduct, M1dG, derived from the reaction of base propenals with deoxyguanosine residues, ring-opens to N2-oxopropenyl-deoxyguanosine when positioned opposite deoxycytidine residues in DNA. M1dG also undergoes enzymatic oxidation by AlkB to form deoxyguanosine thereby directly repairing the lesion. In addition, M1dG has been found to be enzymatically oxidized in genomic DNA in a range of human cell lines to 6-oxo-M1dG. M1dG present in mitochondrial DNA is not oxidized to 6-oxo-M1dG. DNA adducts derived from a,β-unsaturated aldehydes are quantitatively significant endogenous lesions in humans and rodents and they exhibit a range of chemical reactions within the genome. This chemical reactivity leads to a diversity of biological outcomes that modulate mutagenesis and repair.

    TOXI 8

    S-Nitrosation is a systems-wide regulatory process

    Steven R. Tannenbaum, srt@mit.edu. Biological Engineering and Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States

    It is well established that NO, and especially S-nitrosation of cellular proteins play a key role in several toxicities and pathologies. Recently, several groups have developed high throughput mass spectrometric approaches for analysis of the SNO-proteome in different tissues, that has revealed hundreds of proteins associated with a toxic or pathological state. In particular, our method (SNOTRAP, SNO trapping by TriAryl Phosphine) directly traps a SNO-molecule without primary denitrosation, and in addition to mass spec proteomics allows imaging of cells and tissues in fresh, frozen, or fixed states. The discovery that there are hundreds of SNO-proteins in any physiological state has allowed analysis of the role of SNO on a systems level. What is remarkable is that there are major differences for the cognate proteins between organs and tissues, or even between different tissue localizations. Even more remarkable is the complete re-programming of the SNO-proteome in the same tissue localization for different toxicities or pathologies. The large number of proteins identified is conducive to analysis in the Gene Ontology system, providing a visualization of shifting biological processes , protein networks, and biochemical pathways induced by mutant proteins or toxic responses. Examples of mutations in the brain and arsenic in drinking water will be presented.

    TOXI 9

    Systems pharmacology approach to the study of mitochondrial dysfunction

    Ian A. Blair1, ianblair@upenn.edu, Qingqing Wang1, Lili Guo1, Liwei Weng1, Ashkan Salimatipour1, Wei-Ting Hwang3, David Lynch2, Clementina Mesaros1. (1) Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, Philadelphia, Pennsylvania, United States (2) Neurology, The Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, United States (3) Epidemiology and Biostatistics , University of Pennsylvania, Philadelphia, Pennsylvania, United States

    Selective cell death in neurodegenerative diseases such Parkinson’s disease and Friedreich’s ataxia (FRDA) is thought to involve mitochondrial complex 1 dysfunction. Coenzyme A (CoA) and CoA thioester derivatives are central players in numerous mitochondrial metabolic pathways. Much of the research on biomarkers of mitochondrial dysfunction has focused on products derived from the generation of reactive oxygen species and reactive nitrogen species. We have developed stable isotope labeling by essential nutrients in cell culture (SILEC) methodology to generate stable isotope labeled CoA and acyl-CoA thioester analogs for use as internal standards in LC-MS-based assays. Using stable isotope dilution liquid chromatography-mass spectrometry (LC-MS), we have now shown that inhibition of mitochondrial complex 1 causes a dramatic decrease in levels of succinyl-CoA with only a modest effect on acetyl-CoA. By using [13C]-labeling and isotopologue analysis, we have also characterized the metabolic derangement in lipid metabolism that occurs. The significant reduction in succinyl-CoA when complex I is inhibited suggests that lysine succinylation of important mitochondrial proteins is inhibited with the potential for increased lysine acetylation of the de-succinylated residues. The possibility that the mitochondrial repair protein high mobility group box-1 (HMGB)-1 is inactivated through acetylation is currently being explored. These methods have been elaborated in order to monitor metabolic dysfunction in vivo using isotopically labeled precursors in freshly isolated human platelets. This has led to the analysis of serum apolipoprotein A-I (ApoA-I) and the finding that concentrations were reduced in FRDA (129.4 mg/dL serum, n=50) when compared with controls (166.6 mg/dl serum; n=50). Knockdown of the FXN gene in HepG2 cells caused a significant decrease in ApoA-I biosynthesis. Overall, our systems pharmacology approach has facilitated the discovery of in vivo biomarkers for FRDA, a disease of mitochondrial dysfunction. Supported by NIH grants R21NS087343, P42ES023720, P30ES0135 and Penn Medicine/CHOP Friedreich’s Ataxia Center of Excellence.

    TOXI 10

    Effect of statins on HMG-CoA reductase pathway and apolipoprotein A-I production in Friedreich’s ataxia

    Lili Guo1, guolili@mail.med.upenn.edu, QingQing Wang1, Cassandra J. Strawser2, Lauren A. Hauser2, Wei-Ting Hwang1, David Lynch2, Clementina Mesaros1, Ian A. Blair1. (1) Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, Philadelphia, Pennsylvania, United States (2) Departments of Neurology & Pediatrics, The Children’s Hospital of Philadelphia and the University of Pennsylvania, Philadelphia, Pennsylvania, United States

    Statins can lower the level of chlesterol in blood by inhibiting the activity of HMG-CoA reductase in liver. HMG-CoA reductase/mevalonate pathway is not only used to synthesize cholestrol and many other biomolecules, it also regulates the production of Apolipoprotein A1 (ApoA-I) from liver, which is the major protein component of HDL particles in plasma. Friedreich’s ataxia (FRDA) is caused by genetic mutation that leads to reduced expression of mitochondrial protein frataxin. Increasing evidence has shown the defective mitochondrial metabolism in frataxin deficient cells. Through [13C]-labeling and isotopologue analysis, we discovered a significant decrease of HMG-CoA labeling from [13C6]-glucose and a marked increase of HMG-CoA from [13C16]-palmitate in FRDA platelets compared to those in control’s. Interestingly, using a highly specific and accurate LC-MS assay. we showed the ApoA-I level in serum was decreased in FRDA patients, which is linked to the higher risk of cardiovascular diseases of FRDA. Based on these observations, we examined the effect of statins on metabolism, and explored whether statin treatment has the potential in raising ApoA-I level in FRDA patients. Interestingly, ApoA-I released into culture medium was reduced in HepG2 cells with frataxin knockdown. Using this cell culture model, we examined the time and dose effects of different statins on ApoA-I production and metabolites in HMG-CoA reductase pathway. Statin treatment significantly elevated the ApoA-I levels from FXN knockdown cells, even more significant than the increase from control cells. This led to a pilot study with 10 FA patients using low dose of crestor. Our results shed light on the effect of statin on cell metabolism with mitochondrial difficiency. This study also suggested a potential treatment for FRDA patients in preventing cardiovascular diseases.

    TOXI 11

    Mechanism of bioactivation of the cooked meat carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in human prostate

    Madjda Bellamri1,2, bellamri@umn.edu, Robert J. Turesky1,2. (1) Masonic Cancer Center, Minneapolis, Minnesota, United States (2) Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota, United States

    Prostate cancer (PC) is the second most frequently diagnosed cancer in men and the second leading cause of cancer deaths in men in the United States. Epidemiologic studies have linked frequent consumption of red meat with PC incidence; however, the mechanism(s) by which a meat diet leads to PC has not been established. Cooked meat containing carcinogenic heterocyclic aromatic amines (HAA) may contribute to PC risk. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), an abundant HAA formed in cooked meat, is a rodent prostate carcinogen and classified as a possible human carcinogen (Group 2B) by IARC. Epidemiological studies have reported an association between frequent consumption of well-done cooked meats containing PhIP and PC risk. PhIP undergoes metabolic activation by cytochrome P450s, to form 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine (HONH-PhIP), which undergoes esterification by sulfotransferase (SULTs) or N-acetyltransferase (NATs) to form reactive intermediates that bind to DNA. Our research group recently identified DNA adducts of PhIP, but not adducts of other cooked meat HAA mutagens, including 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-9H-pyrido[2,3-b]indole (AαC) in prostate biopsy samples of PC patients by liquid chromatography–mass spectrometry. We sought to understand why PhIP but not the other cooked meat mutagens induce DNA damage in human prostate. The cytotoxicity and DNA adducts of the genotoxic HONH-PhIP, HONH-MeIQx, HONH-IQ, and HONH-AaC metabolites were measured in the human prostate (LNCaP) cell line. HNOH-PhIP, but not other HONH-HAA, induced a concentration-dependent cytotoxicity in LNCaP cells. Moreover, PhIP DNA adduct formation was 20-fold greater than those adducts formed with other HONH-HAA. Chemical inhibitors showed that SULT1A1 accounted for about 40% of the PhIP-DNA binding, whereas NAT2 and NAT1 accounted for 40 and 20%, respectively. Our data provide support to the epidemiological observations implicating PhIP formed in well-done cooked meats as a DNA-damaging agent that may contribute to the etiology of prostate cancer

    TOXI 12

    Novel class of hydroxyl radical scavenging antioxidants prevents oxidative DNA damage in fibroblast cells exposed to trivalent arsenic

    Safnas Farwin Abdul Salam, abdulssf@mail.uc.edu, Edward J. Merino, Haizhou Zhu, Purujit N. Gurjar. Chemistry, University of Cincinnati, Cincinnati, Ohio, United States

    Oxidative stress is a condition caused by disruption of balance between reactive oxygen species (ROS) and antioxidants in living system. Macromolecular damage caused by this stress condition has been identified as the underlying mechanism of several diseases and toxic environmental exposures. Antioxidant therapy is shown to be promising to overcome oxidative stress pathology, however with some limitations. One main reason for this limitation is due to lack of selectivity of antioxidants between toxic and benign forms of ROS. Hydrogen peroxide and superoxide play important role in cell signaling, but they interconvert into aggressive ROS forms such as hydroxyl radical in the presence of metal ions. Hydroxyl radical has sufficient oxidizing potential to abstract electrons from guanine base leading to cellular DNA damage. We designed a novel class of antioxidants that can selectively capture low abundant, toxic ROS such as hydroxyl radical. By HPLC studies, we show that the lead agent in this series (1e), selectively oxidizes in the presence of hydroxyl radical. Gel electrophoretic studies show that 1e prevents nicking of plasmid DNA in the presence of Fenton system. To test the antioxidant activity of these molecules within the cells, we treated human fibroblast cells with trivalent arsenic to induce oxidative damage. Arsenic trioxide is a common environmental toxin found in drinking water, soil and air. Exposure to As(III) is shown to elevate oxidative stress by increasing mitochondrial electron leakage. Treatment with 1e significantly increase cell viability and decrease DCF fluorescence of the fibroblast cells exposed to low concentration of As(III). To further prove that 1e prevents toxic effect of hydroxyl radical, we quantified hyperoxidized guanine lesions in cells treated with As(III) with and without 1e, using HPLC. Data shows that As treatment elevate guanine oxidation and treatment with 1e prevents this oxidative damage.

    TOXI 13

    Replicative bypass and mutagenic properties of alkylphosphotriester lesions in Escherichia coli

    Jiabin Wu3, jwu058@ucr.edu, Pengcheng Wang2, Yinsheng Wang1. (1) Univ of California Riverside, Riverside, California, United States (2) University of California Riverside, Riverside, California, United States (3) Environmental Toxicology, University of California Riverside, Riverside, California, United States

    Replicative Bypass and Mutagenic Properties of Alkylphosphotriester Lesions in Escherichia coli

    Jiabin Wu1, Pengcheng Wang2 and Yinsheng Wang1,2

    1Environmental Toxicology Graduate Program, 2Department of Chemistry, University of California, Riverside, CA92521

    Endogenous metabolism, environmental exposure, and treatment with some cancer chemotherapeutic agents can give rise to DNA alkylation, which can occur on the phosphate backbone, forming alkylphosphotriester (PTE). Among the alkylated DNA lesions, PTEs were previously shown to display the longest life-time in cells, suggesting that they could be used as biomarkers for monitoring the long-term exposure to alkylating reagents and they are resistant to DNA repair. However, not much is known about how the PTE lesions compromise the efficiency and fidelity of cellular DNA replication. In the present study, we synthesized oligodeoxyribonucleotides (ODNs) containing a site-specifically inserted alkylphosphotriester, with the alkyl group being a methyl, ethyl, n-propyl and n-butyl. We further characterized the identities of these ODNs by using LC-MS/MS analysis. Moreover, we ligated these ODNs into single-stranded M13 vectors and assessed how these lesions inhibit DNA replication and induce mutations in Escherichia coli cells.

    TOXI 14

    Abasic and oxidized abasic lesion bypass by DNA polymerase theta yields one- and two-nucleotide deletions

    Daniel J. Laverty1, dlavert1@jhu.edu, Marc M. Greenberg2. (1) Chemistry , Johns Hopkins University , Baltimore, Maryland, United States (2) Dept of Chemistry NCB RM 313, Johns Hopkins University, Baltimore, Maryland, United States

    DNA polymerase theta (Pol θ) is implicated in various cellular processes including double strand break repair, base excision repair, and apurinic/apyrimidinic (AP) site bypass. Pol θ expression correlates with poor cancer prognosis, so the ability of Pol θ to bypass the C4’-oxidized abasic site (C4-AP) and 2-deoxyribonolactone (L), which are generated by ionizing radiation and some antitumor antibiotics, is of interest. Translesion synthesis and subsequent extension by Pol θ were examined on primer-templates containing C4-AP and L, as well as an AP site or its tetrahydrofuran analogue (F). Pol θ conducts translesion synthesis on templates containing AP and F with similar efficiencies and inserts nucleotides in the order A > G > T. Translesion synthesis on templates containing C4-AP and L is less efficient, and the preference for A insertion is reduced for L and absent for C4-AP. Extension past all abasic lesions (AP, F, C4-AP, and L) resulted in deletions caused by the base 1 or 2 nucleotides downstream from the lesion acting as a template. Sequencing of AP bypass products revealed a strong preference for formation of deletions compared to full-length products. Furthermore, 2-nucleotide deletions were overwhelmingly preferred on a template sequence where 1- and 2-nucleotide deletions were possible. These results suggest that bypass of abasic lesions by Pol θ is highly mutagenic.

    TOXI 15

    Characterization of the 2,6-diamino-4-hydroxy-N5-(methyl)-formamidopyrimidine DNA lesion

    Stephanie Bamberger2, stephanie.n.bamberger@vanderbilt.edu, Hope Pan2, Ryan Bowen1, Chanchal Kumar Malik2, Tracy Johnson-Salyard2, Carmelo Rizzo2, Michael P. Stone2. (1) Vanderbilt University, Nashville, Tennessee, United States (2) Chemistry, Vanderbilt University, Nashville, Tennessee, United States

    The N7 imidazole nitrogen of guanine is the most susceptible site for DNA methylation, resulting in the formation of the cationic adduct 7-methylguanine (7MeG). Opening of the imidazole ring occurs upon exposure to hydroxide ion, producing the 2,6-diamino-4-hydroxy-N5-(methyl)-formamidopyrimidine (MeFapy-dG) lesion. This lesion is known to convert between α and β anomers, but the possibility of additional conformational isomers cannot be ruled out. We report progress towards characterizing this DNA adduct, including the synthesis and purification of a 12mer oligodeoxynucleotide in which the CH3 group of the MeFapy-dG is 13C-labeled in the sequence 5′-CATXATGACGCT-3′; X=13C-MeFapy-dG. The reverse phase HPLC chromatogram showed two equilibrating chromatographic peaks that were verified as the 13C-MeFapy-dG 12mer by MALDI mass spectrometry. It is hypothesized that the two equilibrating peaks are a result of anomerization of the MeFapy-dG adduct; such epimerization has been found to occur in other Fapy lesions. Investigation by NMR spectroscopy of the MeFapy-dG phosphoramidite has provided the first spectroscopic evidence that epimerization also occurs in the MeFapy-dG lesion. Eight unique spin systems of the deoxyribose ring were observed in a TOCSY experiment, indicative of eight isomers. NOE cross-peaks between the protons of the deoxyribose ring revealed that four of the species were the α anomer and four were the β anomer; the α anomer was the major component. Rotational isomers arising from rotation around the C5N5 bond were observed in the β anomer. HSQC experiments of the 13C-labeled MeFapy-dG 12mer indicated the methyl group of MeFapy-dG is in ten and six unique chemical environments in single strand and in duplex, respectively. The methyl peaks were spilt into two distinct groups. A series of temperature dependent HSQC experiments of the single strand indicated that the two groups are caused by anomers rather than rotamers. In the NMR spectra of the duplex the C1H1′-C1H6, C1H1′-A2H8, A2H1′-A2H8, and A2H1′-T3H6 NOE cross-peaks exhibited doubling, as did corresponding NOE cross-peaks in the deoxyribose H2′ and H2” region of the spectrum. Tripling of the T3H1′-T3H6 and C21H5-C21H6 NOE cross-peaks and corresponding NOE cross-peaks for T3 in the deoxyribose H2′ and H2” region of the spectrum was also observed. These NMR data indicated the presence of multiple distinct chemical environments for nucleotides near the lesion.

    TOXI 16

    Engineering a replicative DNA polymerase for specific damage bypass capability

    Timothy A. Coulther1, tcoulth@gmail.com, Mary J. Ondrechen1, Penny J. Beuning2. (1) Chemistry & Chemical Biology, Northeastern University, Boston, Massachusetts, United States (2) Chemistry and Chemical Biology, Northeastern University, Everett, Massachusetts, United States

    Enzyme engineering seeks to create an enzyme, either de novo or by modification of a known protein, with a new desired function. Biocatalysis has advantages over conventional catalytic processes, but there are not always natural enzymes with the desired properties and activity. For example, many DNA polymerases can replicate DNA accurately at high speeds but can be inhibited by bases that have been damaged by environmental mutagens, oxidative stress, or UV light. Depending on the type of damage, polymerases may not be able to insert nucleotides opposite a lesion or may do so with reduced accuracy. Specialized polymerases may be well suited to bypass the damage, but are typically less accurate and less efficient, even on undamaged DNA. This accuracy tradeoff allows genomic replication, and life, to continue, but can contribute to problems such as antibiotic resistance or oncogenesis.
    A hybrid DNA polymerase that retains the accuracy and speed of replicative polymerases, while incorporating specific lesion-bypass abilities, would be a useful biochemical tool. This research focuses on the common oxidative DNA lesion 8-oxoguanine, which is quite mutagenic despite the small size of the damage. The additional hydrogen bond donor allows formation of a Hoogsteen pair with adenosine when 8-oxo-dG resides in the syn conformation. Molecular dynamics and docking are being utilized to understand the lesion conformation and relative fidelity of model polymerases and engineered variants. Our computational methods, THEMATICS and POOL, identify functional residues and assess whether variants retain the electrostatic properties of the natural polymerase for catalysis. The combination of these computational methods is being used to identify specific variants for biochemical characterization, including thermal stability, catalytic activity, lesion bypass capability, and fidelity. Supported by NSF-MCB-1517290 and the National Institute of Justice.

    TOXI 17

    Mechanism of ribonucleotide incorporation by human DNA polymerase Eta

    YAN SU1, yan.su.1@vanderbilt.edu, Martin Egli1, F P. Guengerich1. (1) Biochemistry, Vanderbilt University, Nashville, Tennessee, United States (1) Vanderbilt University , Nashville, Tennessee, United States (1) Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, United States

    Ribonucleotides and 2′-deoxyribonucleotides are the basic units for RNA and DNA, respectively, and the only difference is the extra 2′-OH group on the ribonucleotide sugar. Cellular rNTP concentrations are much higher than those of dNTP. When copying DNA, DNA polymerases not only select the base of the incoming dNTP to form a Watson-Crick pair with the template base but also distinguish the sugar moiety. Some DNA polymerases use a steric gate residue to prevent rNTP incorporation by creating a clash with the 2′-OH group. Y-family human DNA polymerase eta (hpol eta) is of interest owing to its spacious active site (especially in the major groove) and tolerance of DNA lesions. Here, we show that hpol eta maintains base selectivity when incorporating rNTPs opposite undamaged DNA and the DNA lesions 7,8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG) and cyclobutane pyrimidine dimer (CPD) but with rates that are 103-fold lower than for inserting the corresponding dNTPs. X-ray crystal structures show that hpol eta scaffolds the incoming rNTP to pair with the template base (dG) or 8-oxodG with a significant propeller twist. As a result, the 2′-OH group avoids a clash with the steric gate, Phe-18, but the distance between primer end and Pa of the incoming rNTP increases by 1 Å, elevating the energy barrier and slowing polymerization compared with dNTP. In addition, Tyr-92 was identified as a second line of defense to maintain the position of Phe-18.

    TOXI 18

    Independent generation of 2′-deoxyadenosine-N6-yl radical and its reactivity in DNA

    Liwei Zheng1, whu.lwzheng@gmail.com, Markus Griesser2, Derek A. Pratt2, Marc M. Greenberg1. (1) Department of Chemistry, Johns Hopkins University, Baltimore, Maryland, United States (2) Department of Chemistry & Biomolecular Sciences, University of Ottawa, Ottawa, Ontario, Canada

    Neutral purine radicals are important reactive intermediates of oxidative damage of DNA and DNA charge transfer. However, the understanding of their reactivity is limited due to the lack of means to generate them in a controlled manner. Herein we descried an approach for the site specific, independent generation of the 2′-deoxyadenosine-N6-yl radical (dA●). The method involves sequential Norrish Type I photocleavage and β-fragmentation of the initially formed alkyl radical to form dA● in high fidelity and efficiency. The reactivity of dA● in DNA was investigated. dA● cannot initiate efficient hole migration in DNA, even the process is thermodynamically favorable. dA● degrades to deoxyinosine which is potentially mutagenic. A tandem lesion is formed in oligomers in the presence of oxygen. Experiments suggest the formation of this tandem lesion is initiated by the addition of dA● to 5’ adjacent thymidine following the formation of a thymidine peroxy radical analogue, which damages adjacent deoxyguanosine to give 8-oxo-deoxyguanosine.

    TOXI 19

    Investigation into the reactivity of a C5′-uridinyl radical

    Matthew Ellis2,1, Mellis5@rockets.utoledo.edu, Amanda C. Bryant-Friedrich2,1. (1) Medicinal & Biological Chemistry, The University of Toledo , Toledo, Ohio, United States (2) University of Toledo, Toledo, Ohio, United States

    Constituents of the endogenous exposome include reactive oxygen species, which cause oxidative damage to nucleic acids, proteins, and lipids. It has been shown that such damage to RNA plays a role in the initiation of neurodegenerative diseases, such as Alzheimer’s. To investigate this phenomenon from a molecular level, modified uridines were synthesized and subjected to photolytic activation, generating a C5′-uridinyl radical mimicking the hydrogen atom abstraction observed under conditions of oxidative stress. This was accomplished with a tert-butyl ketone at the C5′ position, which undergoes type I Norrish cleavage. Studies were performed under anaerobic and aerobic conditions in the presence or absence of reductant at varying pH to emulate the diversity of the cellular environment. Identification of damage lesions formed in the process may assist in the development of new assays based on biomarkers of oxidative damage.

    TOXI 20

    Arsenite binds to the RING finger domain of FANCL E3 ubiquitin ligase and inhibits DNA interstrand cross-link repair

    Yinsheng Wang2, Ji Jiang1, jjian009@ucr.edu. (1) CMDB, UC-Riverside, Riverside, California, United States (2) Univ of California Riverside, Riverside, California, United States

    Ji Jiang1, Marina Bellani2, Lin Li3, Pengcheng Wang4, Michael M. Seidman2, and Yinsheng Wang1,3,4*

    1Cell, Molecular and Developmental Biology Graduate Program, 3Department of Chemistry, 4Environmental Toxicology Graduate Program, University of California, Riverside, CA 92521-0403
    2Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224.

    Human exposure to arsenic in drinking water is known to be associated with the development of bladder, lung, kidney, and skin cancers. However, the molecular mechanisms underlying the carcinogenic effects of arsenic species remain incompletely understood. DNA interstrand cross-links (ICLs) are among the most cytotoxic type of DNA lesions that block DNA replication and transcription, and these lesions can be induced by endogenous metabolism and by exposure to exogenous agents. Fanconi anemia (FA) is a congenital disorder manifested with elevated sensitivity toward DNA interstrand cross-linking agents, and monoubiquitination of FANCD2 by FANCL is a crucial step in the FA repair pathway. Here we demonstrated that As3+ could bind to the PHD/RING finger domain of FANCL in vitro and in cells. This binding led to compromised ubiquitination of FANCD2 in cells and diminished recruitment of FANCD2 to chromatin and DNA damage sites induced by 4,5¢,8-trimethylpsoralen plus UVA irradiation. We also demonstrated, by employing immunoprecipitation with PTMScan® Ubiquitin Remnant Motif (K-ε-GG) Kit and LC-MS analysis, a diminished level of ubiquitination of FANCD2 in cells upon treatment with As3+. Furthermore, clonogenic survival assay results showed that arsenite co-exposure rendered cells more sensitive toward DNA interstrand cross-linking agents. Together, our study suggested that arsenite may confer its carcinogenic effect partly through compromising genomic stability via perturbation of the Fanconi anemia pathway

    TOXI 21

    Pharmacokinetic interactions between drugs and licorice botanical dietary supplements used by menopausal women

    Richard B. Van Breemen, breemen@uic.edu. Univ of Illinois, Chicago, Illinois, United States

    Concerns regarding increased risks of stroke and breast cancer have prompted many women to consider botanical dietary supplements instead of conventional hormone therapy for the management of menopausal symptoms such as hot flashes. Among botanical supplement alternatives to hormone therapy, licorice (Glycyrrhiza sp.) is an increasingly popular alternative and has a long history of use in Traditional Chinese Medicine as well as in Western pharmacopeias. To ensure the safe use of licorice dietary supplements, we investigated their potential for drug interactions. Standardized licorice extracts of Glycyrrhiza glabra, G. inflata, and G. uralensis, the three most common species used in dietary supplements, were studied for possible inhibition or induction of cytochrome P450 enzymes involved in drug metabolism. In studies using human liver microsomes and a cocktail probe substrate assay, all three licorice species showed inhibition of CYP2B6, CYP2C8, CYP2C9 and CYP2C19. G. inflata also inhibited CYP1A2, CYP2D6 and CYP3A4. Cryopreserved human hepatocytes were used to predict effects of the extracts and individual licorice compounds on cytochrome P450 enzyme induction. CYP3A4 was unaffected by all Glycyrrhiza sp, and CYP1A2 and CYP2B6 were induced in hepatocytes by G. uralensis and G. inflata. Due to these data, dietary supplements containing G. uralensis and G. inflata are more likely than G. glabra to cause clinically relevant drug interactions.

    TOXI 22

    Intestinal UGTs as targets for pharmacokinetic natural product-drug interactions

    Mary Paine, mary.paine@wsu.edu. Pharmaceutical Sciences, Washington State University, Spokane, Washington, United States

    Natural products (NPs), which encompass botanical dietary supplements, fruit juices, and teas, constitute an ever-growing share of consumer and health care markets worldwide. Myriad NPs are promoted to have medicinal properties, which may or may not be proven in clinical trials. Regardless, many patients supplement their prescribed drug regimens with these products to alleviate adverse effects and/or enhance therapeutic efficacy. However, some NP-drug combinations can lead to unwanted interactions. Pharmacokinetic mechanisms underlying these interactions include modulation of the pre-systemic and/or systemic metabolism of the object drug by one or more NP constituents (precipitants), leading to altered systemic or tissue drug exposure and suboptimal pharmacologic effect(s). NPs typically contain multiple polyphenolic constituents that undergo extensive metabolism by the UDP-glucuronosyltransferases (UGTs). Because NPs typically are taken orally, constituent concentrations are theoretically much higher in the intestine than in the liver. As such, UGTs in the intestine may represent pertinent targets for NP-drug interactions. Accurate assessment of these complex and understudied interactions, regardless of the target, requires a comprehensive understanding of NP chemical make-up, knowledge of candidate precipitant NP constituents, and established human-derived in vitro systems to recover robust parameters needed to populate mathematical models used to inform the need for clinical NP-drug interaction studies. Testing this multifaceted approach with exemplar NPs and clinically relevant object drug substrates would provide a framework for developing needed guidance for rigorous assessment of potential pharmacokinetic NP-drug interactions, the ultimate goal of which is to convey definitive information to both clinicians and patients about the risk of supplementing prescribed drug regimens with certain NPs.

    TOXI 23

    KEAP1 and done? Targeting the NRF2 pathway with sulforaphane

    Thomas Kensler1,2, tkensler@jhsph.edu. (1) Johns Hopkins University, Baltimore, Maryland, United States (2) University of Pittsburgh, Pittsburgh, Pennsylvania, United States

    Sulforaphane is a promising agent under evaluation in many settings of disease prevention. This bioactive phytochemical affects many molecular targets in cellular and animal models; however, amongst the most sensitive is KEAP1, a key sensor for the adaptive stress response system regulated through the transcription factor NRF2. KEAP1 is a sulfhydryl-rich protein that represses NRF2 signaling by facilitating the poly ubiquitination of NRF2 thereby enabling its subsequent proteasomal degradation. Interaction of sulforaphane with KEAP1 disrupts this function and allows for nuclear accumulation of NRF2 and activation of its transcriptional program. Enhanced transcription of NRF2 target genes provokes a strong cytoprotective response that enhances resistance to carcinogenesis and other diseases mediated by exposures to electrophiles and oxidants. The efficacy of sulforaphane has been broadly established in animal models. Clinical evaluation of sulforaphane has been largely conducted by utilizing preparations of broccoli or broccoli sprouts rich in either sulforaphane or its precursor form in plants, a stable β-thioglucose conjugate termed glucoraphanin. We have conducted a series of clinical trials in Qidong, China, a region where exposures to food- and air-borne carcinogens has been considerable, to evaluate the suitability of broccoli sprout beverages, rich in either glucoraphanin or sulforaphane or both for their bioavailability, tolerability and pharmacodynamic action in cost-effective, population-based interventions. Results from these clinical trials indicate that interventions with well characterized preparations of broccoli sprouts enhance the detoxication of aflatoxins and air-borne toxins, which may in turn attenuate their associated health risks, including cancer, in exposed individuals. Supported by NIH grants R35 CA197222 and R01 CA190610.

    TOXI 24

    Comparing general and specific biological targets for assessing sufficient similarity of related botanical dietary supplements

    Cynthia V. Rider, cynthia.rider@nih.gov, Stephanie Smith-Roe, Stephen S. Ferguson. NTP, NIEHS, Research Triangle Park, North Carolina, United States

    Botanical dietary supplements are complex mixtures that can be highly variable in composition, making safety evaluation difficult. Due to the large investment in comprehensive toxicological evaluation, a single sample is typically assessed with the assumption that it is representative of nominally-related products. However, methods for comparing across complex mixtures are required to determine whether the assumption of similarity is supported scientifically. We have developed three case studies with Ginkgo biloba extract (GBE), Echinacea purpurea extract (EPE), and black cohosh extract (BCE) to explore approaches for determining sufficient similarity. These botanicals display a range of biological activity in animal studies with GBE eliciting liver toxicity and carcinogenicity likely through a non-genotoxic mechanism, BCE acting through a genotoxic mechanism, and EPE displaying limited biological activity in immune system targets in short term studies. For each case study, a number of samples procured from different suppliers were assessed in in vitro primary human hepatocyte assays measuring activity of five receptors (AhR, CAR, PXR, FXR, and PPARα), which served as general indicators of biological activity. Results from these assays were compared to an evaluation of chemical similarity and, in the case of GBE, a subset of diverse samples were also evaluated in short term in vivo studies measuring liver weight and gene expression changes. For BCE, results from the hepatocyte assays were compared to an in vitro assay for genotoxicity. We found that for GBE, the specific markers known to be associated with hepatotoxicity (CAR/PXR) provided a better indication of similarity than general biological activity, while for BCE, genotoxicity was not as useful for distinguishing among samples. Evaluation of all three case studies will help to inform future application of sufficient similarity methods to botanicals and other complex mixtures.

    TOXI 25

    Botanicals modulate estrogen metabolism through multiple targets

    Judy L. Bolton, judy.bolton@uic.edu. Univ Illinois, Chicago, Illinois, United States

    Botanical dietary supplements with hormonal effects are increasingly popular for women’s health particularly for older women. These women tend to take botanical supplements as natural alternatives to traditional hormone therapy to relieve menopausal symptoms. Several of these botanicals could have additional breast cancer preventive effects linked to multiple biological targets including hormonal, metabolic, inflammatory, and/or epigenetic pathways. For example, hops, licorice, and red clover extracts could modulate estrogen receptor cross talk and/or arylhydrocarbon receptor (AhR) mediated P450 1B1 bioactivation of estrogens which could inhibit genotoxic quinone formation. Inflammation has been linked to induction of P450 1B1 and anti-inflammatory bioactive compounds in these botanicals could also inhibit estrogen carcinogenesis through NF-kB mediated pathways. Finally, although botanicals are perceived as natural safe remedies, it is important for women and their health care providers to realize that they have not been rigorously tested for potential toxic effects and/or drug/botanical interactions. Understanding the mechanism of action and biological targets of these supplements used for women’s health, will ultimately lead to standardized botanical products with higher efficacy, safety, and chemopreventive properties. Support for this work was provided by P50 AT00155

    TOXI 26

    Mechanisms of replication-coupled repair

    Johannes C. Walter, johannes_walter@hms.harvard.edu. Biological Chemistry and Moleuclar Pharmacology, Harvard Medical School, Boston, Massachusetts, United States

    Our genomes are continually damaged by exogenous and endogenous agents. Much of this damage can be repaired at any stage of the cell cycle. However, certain highly toxic lesions, including covalent DNA protein cross-links (DPCs) and DNA interstrand cross-links (ICLs) are repaired primarily in S phase, after collision with a DNA replication fork. We have recapitulated replication-coupled ICL and DPC repair in Xenopus egg extracts, allowing detailed mechanistic analysis. I will present our current understanding of these replication-coupled DNA repair processes

    TOXI 27

    Interstrand DNA cross-links derived from abasic sites in duplex DNA

    Kent S. Gates, gatesk@missouri.edu. Department of Chemistry, University of Missouri, Columbia, Missouri, United States

    Damage to cellular DNA is unavoidable. Endogenous DNA damage in mammalian cells may contribute to aging, neurodegeneration, mitochondrial dysfunction, mutagenesis, and cancer. To better understand the roles of endogenous DNA damage in human health and disease, it is critical to identify the exceptionally bioactive lesions that occur spontaneously in cellular DNA. Along these lines, we have identified a series of new interstrand cross-links arising from abasic sites that are commonly-occurring lesions in genomic DNA. This presentation will describe the formation, structure, properties, and biochemical processing of abasic site-derived, interstrand cross-links in duplex DNA.

    TOXI 28

    Replication and repair of DNA interstrand cross-link lesions in human cells

    Nathan Price1, Shuo Liu1, Kent S. Gates2, Yinsheng Wang1, yinsheng.wang@ucr.edu. (1) Univ of California Riverside, Riverside, California, United States (2) Univ of Missouri, Columbia, Missouri, United States

    Exposure to environmental agents and endogenous metabolites and cancer chemotherapy can give rise to DNA interstrand cross-link lesions. In this presentation, I will discuss our application of shuttle vector methods to examine the efficiency and fidelity with which DNA interstrand cross-link-containing plasmids are replicated in cultured human cells that are proficient in translesion synthesis or the isogenic cells deficient in individual translesion synthesis DNA polymerases. We will demonstrate that the shuttle vector-based studies provided important molecular level understanding about how DNA interstrand cross-link lesions impede DNA replication and induce mutagenesis in human cells as well as about how individual translesion synthesis DNA polymerases function in replication across these lesions. Additionally, we will describe the use of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantitative measurements of DNA interstrand cross-link lesions in mammalian cells and for assessing the repair of these lesions.

    TOXI 29

    Lesion proximal FANCD2 is required for replication independent repair of interstrand crosslinks

    Manikandan Paramasivam, Marina Bellani, Julia Gichimu, Himabindu Gali, Michael Seidman, seidmanm@grc.nia.nih.gov. LMG, National Institute on Aging/NIH, Baltimore, Maryland, United States

    Persistent DNA damage, a feature of repair deficiencies like Fanconi Anemia (FA), results in persistent induction of the DNA Damage Response (DDR) which activates pathways for repair, apoptosis, senescence, and inflammation. FA cells are diagnostically hypersensitive to DNA interstrand crosslinks (ICLs). ICL repair, and the role of FA proteins, has been studied most extensively in the context of DNA replication. In contrast, the DDR induced by ICLs, and the participation of FA proteins, independent of replication, is poorly characterized. We designed strategies to study the DDR in response to ICLs. We employed confocal microscopy to follow the recruitment of DDR factors, to laser localized, antigen tagged, ICLs in nuclei of living cells. We developed a modified Proximity Ligation Assay (PLA) to identify DDR proteins that were proximal or distal to the ICLs. This approach has greater resolution (30nm) than analysis of protein co-localization by conventional immunofluorescence (>200nm). We also measured ssDNA intermediates generated during repair of ICLs.
    We followed the involvement of the key Fanconi protein, FANCD2, in the DDR and ICL repair. Recruitment of FANCD2 was cell cycle independent, required the FA core complex and ubiquitination, and promoted the removal of ICLs in G1 cells. FANCD2 participated in two distinct DDR pathways. The recruitment of most of the FANCD2 was dependent on canonical DDR mediators such as γ-H2AX, MDC1, and 53BP1. A minor fraction entered a repair pathway dependent on NER sensing/processing factors (XPC, XPA, XPF), and was proximal to the ICLs. The association of FANCD2 with the canonical DDR factors was independent of ICL repair, while the ICL proximal recruitment was independent of the DDR proteins. The NER-dependent, ICL-proximal, fraction of FANCD2 was required for the generation of ssDNA during processing of the crosslink. SNM1A exonuclease interacted with FANCD2 and played a primary role in ssDNA formation during ICL repair in G1 cells. The FANCD2 associated nuclease FAN1, made a smaller contribution to ssDNA production. These results demonstrate that FA proteins, including FANCD2, can participate in removal of the ICLs from genomic DNA in replication independent pathways.

    TOXI 30

    Hydrogen peroxide activated DNA cross-linking agents and their biomedical application

    Xiaohua Peng1, pengx@uwm.edu, Wenbing Chen2, Yibin Wang1, Heli Fan1. (1) Chemistry, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin, United States (2) uwm, Milwaukee, Wisconsin, United States

    Many DNA cross-linking agents are used as anticancer agents. However the major concern for anticancer chemotherapeutic agents is their side-effects due to lack of specificity towards cancer cells. The development of anticancer prodrugs that are activated by unique features inside cancer cells is an attractive approach to achieve therapeutic activity and selectivity. Due to their fast growth and redox imbalance, most cancer cells have increased levels of reactive oxygen species (ROS) in comparison to their normal counterpart, such as superoxide, H2O2, and the hydroxyl radical. We exploit this distinctive feature of cancer cells to develop drugs that target tumors while cause less damage to surrounding healthy cells. In particular, we use boron chemistry for developing H2O2-activated prodrugs. A series of boron-based DNA cross-linking agents have been discovered to be activated by H2O2 that is considered as one of the most common ROS in cancer cells. These agents are not active by themselves but can selectively react with H2O2 to release quinone methides or nitrogen mustards which are capable of alkylating or cross-linking DNA. In vitro cytotoxicity assays showed that these agents were not toxic towards normal cells but greatly inhibited cancer cell growth. The efficacy and selectivity were further investigated in normal mice and Xenograft breast cancer models. The details of the chemistry and implications will be discussed.

    TOXI 31

    Constitutive role of Fanconi anemia D2 gene in protecting cell from crosslinking DNA damage

    Lei Li, leili@mdanderson.org. Experimental Radiation Oncology , MD Anderson Cancer Center, Houston, Texas, United States

    In response to DNA ICL formation, the FA core complex’s E3 ligase activity monoubiquitinates the FANCD2 protein and the ubiquitinated form of FANCD2 is shown to be critical for the recruitment of nuclease activities to repair ICL lesions. Given that all the FA gene products act coordinately in the monoubiquitination of FANCD2, it is unclear why loss of different classic FA gene functions creates varying degrees of sensitivities to crosslinking damage. The phenotypical deviations are also reflected by the skewed patient mutation characteristics among different FA complementation groups.
    To distinguish the functional importance of the FA core complex and FANCD2, we generated isogenic deletion mutants of FANCL and FANCD2. We found that FANCD2 deletion results higher levels of spontaneous damage and sensitivity to replication blocking reagents, compared to the loss of FANCL. This result suggests that in the absence of FANCL-mediated monoubiquitination, FANCD2 provides a basal level protection countering replication stress. Such function from unmodified FANCD2 is likely provided through optimized recruitment of nucleolytic activities for the processing and protection of stressed replication forks. Our results suggest a constitutive role of FANCD2 in dealing with steady state levels of endogenous replication stress.

    TOXI 32

    Antibody drug conjugates: Design considerations for improving efficacy and safety

    Pamela Trail, PTrail23@gmail.com. Regeneron, Tarrytown, New York, United States

    Antibody drug conjugates (ADCs) are a rapidly evolving therapeutic class that exploits the target-selectivity of monoclonal antibodies (MAbs) to deliver drugs to antigen-expressing cells. The regulatory approval of ADCs in both hematologic malignancies (brentuximab vedotin) and solid tumors (ado-trastuzumab emtansine) clearly demonstrates the clinical potential of ADCs. In general, ADCs consist of a targeting MAb covalently attached to a drug payload via a cleavable or non-cleavable linker. Although simple in concept, to achieve the design goal of improved therapeutic efficacy and improved safety, each of the components of an ADC; the MAb, linker and drug need to considered in the context of the targeted antigen, the selectivity of antigen expression and the biology of the tumor type on which the target antigen is expressed. There is now clinical information, of various levels of maturity, available on over 40 ADCs. This meeting will discuss the current state of ADC design and modifications that may improve both the efficacy and safety of this treatment modality.

    TOXI 33

    ADC linker immolation and cell killing activity

    Donglu Zhang, zhangd38@gene.com. Genentech, South San Francisco, California, United States

    For antibody-drug conjugates (ADCs) with a commonly used cleavable disulfide or peptide linker, immolation is often a necessary step after linker cleavage to exert cell killing activities. The payload delivery to the site of action by an ADC is limited by pharmacokinetic and dispositional characteristics of the antibody and is relatively slow compared to the delivery of a small molecule drug (SM) after oral administration. The ADC linker determines the mechanism and rate of payload release, which affects both payload exposure and cell killing activities, and is thus a critical part of ADC design. A linker will need to release the payload in tumors in a right form and at a proper rate to achieve a threshold concentration to inhibit cell growth. At the same time, it is desirable to prevent premature payload release into circulation and non-targeted organs. Examples will be given to demonstrate that proper immolation is required for both disulfide and peptide linkers for ADC biological activities.

    TOXI 34

    Development of next generation calicheamicin ADCs

    Omar K. Ahmad, omar.ahmad@pfizer.com. Pfizer, Providence, Rhode Island, United States

    The calicheamicins are potent DNA-damaging agents that are capable of promoting double-strand DNA breaks and have been used as payloads for Mylotarg® and inotuzumab ozogamicin. Harnessing the reactivity of these natural products has been critical for the design and development of calicheamicin-based ADCs. The structural complexity poses a significant challenge to understanding the chemistry of these molecules. For the development of next generation calicheamicin ADCs, our work has focused on refining the linker chemistry and the method and site of conjugation, as well as understanding the reactivity and SAR of the payload itself, with the goals of improving efflux, efficacy and more importantly, selectivity. These modifications have led to a more efficacious and safer conjugate that overcame hepatotoxicity observed with first and second generation calicheamicin ADCs.

    TOXI 35

    Potent antibody-based conjugates for cancer therapy: From early stage research to a clinically approved drug

    Peter D. Senter, psenter@seagen.com. Seattle Genetics Inc, Bothell, Washington, United States

    Monoclonal antibodies (mAbs) have played a major role in cancer medicine, with active drugs such as trastuzumab (Herceptin), cetuximab (Erbitux), bevacizumab (Avastin) and rituximab (Rituxan) in a wide range of therapeutic applications. The mechanisms of these agents involve such activities as direct signaling, interactions with Fcg receptors on effector cells, and complement fixation. Several approaches have been explored to improve antibody-based therapies for cancer treatment by optimizing such activities and by conscripting their selectivity profiles for the delivery of high potency cytotoxic drugs. The field has advanced significantly in the past few years, with the approval of brentuximab vedotin (ADCETRIS) for the treatment of relapsed Hodgkin and anaplastic large cell lymphomas. The antibody-drug conjugate (ADC) is comprised of a potent antimitotic agent, monomethyl auristatin E (MMAE), conjugated to an anti-CD30 mAb through a lysosomally cleavable dipeptide linker. This presentation will describe the discovery and development of this drug and will overview advancements in the field of ADCs for cancer therapy, with particular emphasis on parameters that impact activity, tolerability, and potency.

    TOXI 36

    Pentachlorophenol alters secretion of interleukin 6 (IL-6) from human immune cells

    Tamara Martin2, tmartin5911@comcast.net, Margaret Whalen1. (1) Chemistry, Tennessee State University, Nashville, Tennessee, United States (2) Biology, Tennessee State University, Nashville, Tennessee, United States

    Pentachlorophenol (PCP) is an environmental contaminant found in human blood. PCP has been used in fungicides, insecticides, herbicides, and antifouling paint. A number of cancers are associated with exposure to this contaminant. Previous studies in our lab show that PCP inhibits the lytic function of highly enriched human natural killer (NK) lymphocytes and decreases the expression of several surface proteins on NK cells. Interleukin 6 (IL-6) is a cytokine produced by lymphocytes and macrophages in response to infection. It mediates the acute phase response to injury by stimulating liver production of acute phase proteins and elevation in its levels has been associated with rheumatoid arthritis, systemic lupus erythematosus, psoriasis, Crohn’s disease, and tumor progression. An increase or decrease in the level of Il-6 may lead to immune dysfunction or increased potential for inflammatory disorders and tumors. The objective of this study was to examine if exposure of human immune cells to PCP alters the secretion of IL-6. Immune cells included Peripheral Blood mononuclear cells (PBMCS), monocyte-depleted (MD) PBMCs, and enriched NK cells. Cells were exposed to 5 µM to 0.05 µM PCP for 24 h, 48 h and 6 days. Results indicate that PCP alters secretion of IL-6 from each immune cell preparation. MD-PBMCs and PBMCs showed increased secretion of IL-6 after at least one length of exposure to at least one concentration of PCP. The concentrations and lengths of exposure at which these increases occurred varied among the donors. These results suggest that PCP-induced alterations in IL-6 secretion may have the potential to cause immune system dysregulation.

    TOXI 37

    Analysis of methylated and ethylated peptides in human hemoglobin by liquid chromatography mass spectrometry: Association with cigarette smoking

    Hauh-Jyun C. Chen, chehjc@ccu.edu.tw, Sun Wai Ip, Fu-Di Lin. Chem Biochem Dept, Natl Chung Cheng University, Chia-Yi, Taiwan

    Alkylating agents contained in cigarettes smoke might be related to cancer development. Post-translational protein methylation and ethylation may cause alteration of protein functions. Human hemoglobin (Hb) is a target for molecular dosimetry due to its easy accessibility. The goal of this study is to investigate the relationship between levels of methylation and ethylation at specific sites of Hb in smokers and in nonsmokers. Because of the low contents of modification in Hb isolated from blood, methylation and ethylation sites was identified in Hb incubated with the methylating agent (methyl methanesulfonate, MMS) or ethylating agent (ethyl methanesulfonate, EMS), respectively, by accurate mass measurement. After trypsin digestion, 7 methylation sites and 9 ethylation sites were identified by the nanoflow liquid chromatography-nanospray ionization coupled with a high resolution mass spectrometry. The relative extents of methylation and ethylation were measured in blood samples from 13 smokers and 22 nonsmokers using the selected reaction monitoring (SRM) mode. No statistically significant difference was found in all the methylated peptides. On the other hand, the extents of ethylation at α-His-50, β-Val-1, and β-Cys-93 of Hb are significantly higher in smokers than in nonsmokers (p < 0.05). Therefore, this assay should be helpful in measuring Hb ethylation level as a biomarker for assessing the exposure to cigarette smoking.

    TOXI 38

    Simultaneous determination of a major peroxidation-derived DNA adduct, M1dG and its oxidized metabolite 6-oxo-M1dG, in human leukocyte DNA by liquid chromatography nanoelectrospray-high resolution tandem mass spectrometry

    Bin Ma1, bma@umn.edu, Christopher Ruszczak1, Peter W. Villalta1, Orrette R. Wauchope2, Lawrence J. Marnett2, Irina Stepanov1. (1) Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, United States (2) Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee, United States

    3-(2-Deoxy-β-D-erythropentofuranosyl)pyrimido[1,2-α]purin-10(3H)-one (M1dG) is a DNA adduct formed from the lipid peroxidation-derived malondialdehyde or from the DNA peroxidation-derived base propenal. M1dG is mutagenic in both bacterial and mammalian cells and is present in the genomic DNA of healthy human beings. Recent in-vitro studies indicate that M1dG in genomic DNA can be oxidized to a single metabolite, 6-oxo-M1dG. We have developed a liquid chromatography (LC)-nanoelectrospray ionization (NSI)-high resolution tandem mass spectrometry (HRMS/MS) method for simultaneous analysis of M1dG and 6-oxo-M1dG in human leukocyte DNA. Internal standards [13C3]-M1dG and [15N5]-6-oxo-M1dG were added during enzymatic hydrolysis of DNA and the hydrolysate was then mixed with sodium borohydride to reduce M1dG and [13C3]M1dG to their 5,6-dihydro derivatives. The samples were purified by solid-phase extraction and column chromatography, and analyzed by LC-NSI-HRMS/MS. The analysis was performed in a pseudo MS3 mode by applying a source fragmentation voltage of 10 V, followed by higher energy collision dissociation (HCD) fragmentation, isolation widths of 1 amu and product ion spectra acquired at a resolution of 15,000. Quantitation was performed using accurate mass extracted ion chromatograms of m/z 153.0406 (m/z 306→190→) for 5,6-dihydro-M1dG, m/z 153.0406 (m/z 309→193→) for [13C3]-5,6-dihydro-M1dG, m/z 153.0406 (m/z 320→204→) for 6-oxo-M1dG, and m/z 157.0288 (m/z 325→209→) for [15N5]-6-oxo-M1dG with a mass tolerance of 5 ppm. Method accuracy and precision were characterized. The method was applied to the analysis of leukocyte DNA from smokers and nonsmokers. This unique HRMS/MS-based method can be used in future studies investigating the biological consequences of these peroxidation-derived DNA lesions in humans.

    TOXI 39

    Pyridylhydroxybutyl, pyridyloxobutyl and methyl DNA phosphate adduct formation in rats treated chronically with enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol

    Bin Ma, bma@umn.edu, Adam T. Zarth, Erik Carlson, Peter W. Villalta, Pramod Upadhyaya, Irina Stepanov, Stephen S. Hecht. Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, United States

    The tobacco-specific lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is converted to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in a reaction which is both stereoselective and reversible. NNAL is also a lung carcinogen. Both NNAL and NNK are metabolized to reactive intermediates which react with DNA to form DNA adducts, an important step in their mechanisms of carcinogenesis. We have previously characterized pyridylhydroxybutyl and pyridyloxobutyl DNA phosphate and base adducts in NNK-treated rats. In this study, we investigated the potential formation of these adducts rats treated chronically with 5 ppm of (R)-NNAL and (S)-NNAL in their drinking water for 10, 30, 50 and 70 weeks. The potential formation of methyl phosphate adducts in the same rats was also investigated. We report the formation and quantitation of pyridylhydroxybutyl, pyridyloxobutyl and methyl DNA phosphate adducts in (R)- and (S)-NNAL-treated rats, by using a novel liquid chromatography-nanoelectrospray ionization-high-resolution tandem mass spectrometry method. In the lung of rats treated with (R)-NNAL, the levels of pyridylhydroxybutyl and pyridyloxobutyl phosphate adducts were 4530–6920 and 46–175 fmol/mg DNA over the course of the study, respectively; while in (S)-NNAL-treated rats, the levels of these two types of adducts were 3480–4180 and 1180–4650 fmol/mg DNA, respectively. Quantitation of methyl phosphate adducts in these treated rats are underway. Some of the pyridylhydroxybutyl phosphate adducts persisted in the rat lung for over 70 weeks, suggesting that they could be potential biomarkers of chronic exposure to NNAL and NNK. This study provides important new information regarding DNA damage by NNAL and NNK, and could contribute to understanding the mechanism of tobacco-related carcinogenesis.

    TOXI 40

    Evidence for indole-3-methyl isothiocyanate formation upon human consumption of Brussels sprouts

    Pramod Upadhyaya, upadh003@umn.edu, Adam T. Zarth, Naomi Fujioka, Vincent Fritz, Stephen S. Hecht. Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, United States

    Glucobrassicin, a quantitatively significant constituent of Brassica vegetables, gives rise to indole-3-carbinol (I3C) and its dimer di-indolylmethane (DIM) when the vegetables are chewed, in a reaction catalyzed by the plant enzyme myrosinase, released during chewing. I3C and DIM have been extensively studied in regard to their cancer prevention and therapeutic properties. However, the presumed intermediate isothiocyanate on the pathway from glucobrassican to I3C, indole-3-methyl isothiocyanate (IMITC), has to our knowledge never been observed as an intermediate in this process. Therefore, in the study reported here, we investigated the formation and presence in human urine of an N-acetylcysteine conjugate of IMITC – IMITC-NAC- in order to gain a more complete understanding of the biochemical pathways leading to formation of I3C and DIM upon consumption of Brussels sprouts, a vegetable rich in glucobrassican. IMITC-NAC is the product of glutathione conjugation of IMITC, followed by normal cellular processing of glutathione conjugates to mercapturic acids. Standard IMITC-NAC was synthesized by reacting indole-3-methylamine with thiophosgene at -40 °C followed by addition of N-acetylcysteine or, for the internal standard, [13C,15N]N-acetylcysteine. The products were purified by HPLC and the structures confirmed by NMR and MS. Ten subjects consumed Brussels sprouts – containing 100 to 500 µmol glucobrassicin – once daily for 2 days. Urine was collected at intervals up to 24 h after vegetable consumption. Urinary IMITC-NAC was quantified by LC-ESI-MS/MS-SRM with [13C,15N]IMITC-NAC as internal standard. Levels of IMITC-NAC in the urine of these 10 subjects ranged from 0.2 to 30.2 pmol/mL urine. The majority of IMITC-NAC was excreted in the first 12 h after vegetable consumption. Levels of IMITC-NAC in urine were significantly lower than those of DIM. These results provide the first evidence for the presumed intermediacy of IMITC in the formation of I3C and DIM in humans who consumed Brussels sprouts as a source of glucobrassican.

    TOXI 41

    Qualitative analysis of the pyrolysis of cocaine and methamphetamine

    Stephen Raso1, sraso@mix.wvu.edu, Olivia Dodd1, Suzanne Bell2. (1) Chemistry, West Virginia University, Morgantown, West Virginia, United States (2) Forensic Analytical Chemistry, West Virginia University, Morgantown, West Virginia, United States

    Clandestine drugs are commonly ingested by heated vapor inhalation or smoking. Depending on temperature, heating/smoking (here, collectively referred to as pyrolysis) generates a complex mixture of degradation products that often includes metabolites. In the case of heroin as an example, literature reports have shown a strong correlation between chronic ingestion of heroin by smoking with development of toxic spongiform leukoencephalopathy. This suggests that thermal degradation produces compounds in sufficient quantities that could contribute to additional toxicity due to pyrolysis. The smoking process, especially of illicit substances, is varied and notoriously difficult to model in the laboratory. Methods that have been used range from heating samples in sealed capillary tubes to analytical pyroprobe accessories interfaced with GC-MS. While it is difficult to directly correlate what is produced in laboratory settings with what would actually be ingested, it is reasonable to expect more volatile compounds would be readily drawn into the lungs. Accordingly, an apparatus was developed with the goal of collecting the greatest number of thermal degradation products, including the more volatile ones. The design, operation, and temperature ranges associated with this device will be presented. Replicate samples of cocaine and methamphetamine were pyrolyzed in the apparatus and the results compared to previous literature and laboratory studies. Identifications were confirmed with reference standards where possible. Detailed findings will be presented along with implications and potential applications in the form of assays for markers of smoking.

    TOXI 42

    Drosophila melanogaster fatty acid amide production in the presence of Diminazene aceturate

    Gabriela Suarez, gsuarez@eckerd.edu, Karin C. Prins, Benjamin S. Meyer, Ryan L. Anderson, David J. Merkler. Chemistry, University of South Florida, Tampa, Florida, United States

    Arylalkylamine N-acytransferase-like 2 (AANATL2) has been shown to play a significant role in the biosynthesis of fatty acid amides by catalyzing the formation of long-chain N-acylserotonins and N-acyldopamines, in Drosophila melanogaster. The contribution of fatty acid amides to cell signaling remains poorly understood, partially due to a lack of information about the enzymes that synthesize and degrade them. In order to elucidate the full extent to which arylalkylamine N-acetyltransfersases affect the biosynthesis of certain fatty acid amides in a complete biological system, an inhibitor was introduced orally to wild type populations of fruit flies. Diminazene aceturate (Berenil) is the drug of choice in the treatment of trypanosomiasis (African sleeping sickness) in livestock. Berenil is considered to be a dirty drug because its mechanism of action is poorly understood, despite it having been on the market since 1955. Using Drosophila melanogaster as a model organism for the study of long-chain fatty acid amide production, endogenous levels of specific fatty acid amides were quantified with LC/QTOF-MS, and used as a basis for comparison for larvae exposed to Berenil. Over a long period of exposure to Berenil, it was shown that the production of long-chain N-acyldopamines and N-acylserotonins were significantly altered. Endogenous levels of N-acyldopamines showed a significant decrease while levels of endogenous N-acylethanolamine increased in exposed larvae. This suggests that inhibition of AANATL2 in Drosophila melanogaster may have been achieved by ingestion of Berenil. Through tracking the various stages of wild type flies, growth charts were also prepared and showed discrepancies among exposed and unexposed flies when major pupation and molting events occurred in their lifecycle. Despite there being no general consensus of insect arylalkylamine N-acetyltransferases (iAANAT) working as a timezyme in insects these findings suggest that iAANAT may play a role in the timing of major physiological events.

    TOXI 43

    Site-specific incorporation of N-(2′-deoxyguanosine-8-yl)-6-aminochrysene adduct in DNA and its replication in human cells

    Kimberly R. Rebello, Arindom Chatterjee, arindom294@gmail.com, Paritosh Pande, Ashis K. Basu. Department of Chemistry, University of Connecticut, Storrs Mansfield, Connecticut, United States

    The environmental pollutant 6-nitrochrysene (6-NC) is a potent mutagen and mammary carcinogen in rats. 6-NC is the most potent carcinogen ever tested in the newborn mouse assay. Upon metabolic activation, 6-nitrochrysene forms two major adducts with 2′-deoxyguanosine, one at the C8-position, N-(dG-8-yl)-6-AC, and the other at the N2-position, 5-(dG-N2-yl)-6-AC. Here, we report the total synthesis of site-specific oligonucleotides containing the 6-NC-derived C8 dG adduct, N-(dG-8-yl)-6-AC. Pd-catalyzed Buchwald-Hartwig cross coupling of 6-aminochrysene with protected C8-bromo-dG derivative served as the key reaction to furnish protected N-(dG-8-yl)-6-AC in 57% yield. The monomer for solid-phase DNA synthesis was prepared by its deprotection followed by conversion to the corresponding phosphoramidite, which was used to synthesize the site-specific oligonucleotides. The adduct-containing oligonucleotides were purified by reversed phase HPLC followed by denaturing polyacrylamide gel electrophoresis and characterized by mass spectrometry. An oligonucleotide containing N-(dG-8-yl)-6-AC was incorporated into a plasmid and replicated in human embryonic kidney 293T cells, which showed that the DNA adduct is mutagenic inducing largely G→T transversions. We also investigated the roles of several translesion synthesis DNA polymerases in its bypass using siRNA knockdown approach.

    TOXI 44

    Absolute quantification of plasma fibulin-3 as a biomarker for asbestos exposure by immunoprecipitation-high resolution mass spectrometry

    QingQing Wang, qingqing@mail.med.upenn.edu, Liwei Weng, Clementina Mesaros, Ian A. Blair. Penn SRP center and Center of Excellence in Environmental Toxicology, University of Pennsylvania, Philadelphia, Pennsylvania, United States

    Asbestos exposure is known to cause lung cancer and mesothelioma. The exceptionally long latency periods of most asbestos-related diseases have hampered its preventative treatment. New biomarkers are needed to detect asbestos exposure at an earlier stage and to individualize treatment. Fiblin-3 was recently reported as a new potential biomarker for pleural malignant mesothelioma. However, controversy results were reported due to unsatifactory bioanalytical methodology or biological variability. In this study, we developed an immunoprecipitation approach coupled with nanoLC-high resolution mass spectrometry (NanoLC-HRMS) method for quantifying fibulin-3 in human plasma as a biomarker for asbestos exposure and evaluated its prognostic value.
    Sheep Anti-fibulin-3 polyclonal antibody was selected out of five antibodies as it provides the highest specificity and pull-down efficiency. Dimethyl pimelimidate (DMP) was used to cross-link the antibody to protein A/G beads which enable fewer antibody contamination and less matrix effect on MS. Recombinant fibulin-3 was expressed from HEK293 cells using stable isotope labeling by amino acids in cell culture (SILAC) strategy, then it was spiked into plasma samples at initial step of sample preparation as an internal standard. Three signature peptides were selected from native fibulin-3 and SILAC-labeled fibulin-3. Parallel reaction monitoring (PRM) was used on Q Exactive HF (Thermo) for providing high selectivity and high sensitivity. Absolute quantification was based on the ratio of endogenous protein and SILAC-labeled protein. The low limit of quantification of current method reaches to attomole level in human plasma. This improved sensitivity and specificity was obtained by antibody-based immunoprecipitation step, as well as the use of HRMS under PRM mode. The enhanced sensitivity and specificity offered by the current method will allow for a more complete analysis of fibulin-3 and fibulin family, shedding light on previously unknown mechanisms of asbestos exposure.

    TOXI 45

    Synthesis and in vivo quantitation of 2′-deoxyadenosine adducts resulting from bioactivation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol

    Erik S. Carlson, carl3857@umn.edu, Pramod Upadhyaya, Stephen S. Hecht. Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, United States

    4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) are carcinogenic nitrosamines thought to play a pivotal role in initiating tobacco-related lung cancer. To exhibit their carcinogenicity, these must be activated by cytochrome P450 oxidation. This produces a-hydroxyNNK or a-hydroxyNNAL, which spontaneously decompose to diazohydroxides that bind to DNA in the form of pyridyloxobutyl (POB) or pyridylhydroxybutyl (PHB) adducts, respectively. Accumulation of these adducts can lead to mutations and cancer, if formed in critical onco-or tumor suppressor genes. To date, our lab has studied and quantified the POB and PHB adducts of guanine, cytosine, and thymine; however, adenine adducts are still uncharacterized. Based on literature and in vitro experiments, we hypothesize that in vivo alkylation of adenine will primarily form N6-POB/PHB-dA and N1-POB/PHB-dI adducts after Dimroth rearrangement or deamination, respectively. To test this hypothesis, we synthesized authentic standards of all four DNA adducts and quantified their levels in vivo. The syntheses of N6-POB-dA and N6-PHB-dA have previously been reported and identified in vivo. Presented here is the synthesis of N1-POB-dI and N1-PHB-dI followed by in vivo quantitation of all four adducts in F344 rats treated with NNK or NNAL. Both 2′-deoxyinosine adduct structures were confirmed by NMR and HRMS. In vivo quantitation was performed on liver and lung tissues using an isotope-dilution LC-ESI+-MS/MS method for detection. Preliminary work has identified the formation of all four adducts and ongoing work seeks to accurately measure their formation levels. Together, understanding the structure and formation level of NNK- and NNAL-derived adenine adducts will not only complete the profile of possible base adducts, but also provide a new set of biomarkers. These have possible application in individual cancer risk assessment while improving our understanding of NNK and NNAL carcinogenesis.

    TOXI 46

    Lesion recognition in nucleotide excision repair: Relationship between the structural properties of adducts and initial binding of XPC to the damaged site

    Hong Mu1, hm761@nyu.edu, Nicholas E. Geacintov2, Yingkai Zhang2, Suse Broyde1,2. (1) Biology, New York University, New York, New York, United States (2) Chemistry, New York University, New York, New York, United States

    Nucleotide excision repair (NER) recognizes a variety of DNA adducts derived from polycyclic aromatic chemicals. Lesion recognition for subsequent assembly of factors that are required for successful NER requires the xeroderma pigmentosum C protein complex (XPC). The crystal structure of Rad4 (yeast-XPC) with lesion-containing DNA shows that two bases opposite the lesion flip into yeast-XPC and the duplex opens through insertion of a beta-hairpin. These are essential features of productive binding for subsequent NER (productively bound open complex) [Min, J. H., and Pavletich, N. P. (2007) Nature 449, 570−575]. We have previously determined productive binding pathways and energetics of yeast-XPC to two well-repaired DNA lesions [Mu, H. et al. (2015) Biochemistry. 54(34):5263-7; Mu, H. et al. (2017) Chem. Res. Toxicol. ]; these studies have suggested that different pathways for productive binding to structurally different lesions can be adopted by yeast-XPC, and differences are manifested upon initial binding. In order to elucidate how a variety of adduct structures impact initial binding of XPC, we have carried out molecular dynamics simulations for a library of different lesions with varying chemical structures, conformations in duplex DNA, and relative NER efficiencies measured in human HeLa cell extracts. We investigated adducts derived from benzo[a]pyrene, dibenzo[a,l]pyrene, and the food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). Our results reveal that initial binding is distinctly affected by the structure of the adduct in duplex DNA. Conformational capture by yeast-XPC of an extruded partner base opposite the lesion facilitates initial binding; however, when the partner nucleotide is missing so that capture of a partner base is not possible, initial binding is impeded. Furthermore, an adduct that sterically blocks the interface between the damaged DNA and XPC hampers initial binding. We hypothesize that impeded initial binding hinders the subsequent binding stages that produce the productively bound open complex.

    TOXI 47

    Accurate quantification of serum protein mesothelioma biomarkers

    Liwei Weng, wengl@mail.med.upenn.edu, Clementina Mesaros, Ian Blair. Department of Parmacology, University of Pennsylvania, Philadelphia, Pennsylvania, United States

    High mobility group box-1 (HMGB1) is a non-histone chromosomal protein that is highly conserved in eukaryotic cells. It is known to play a regulatory role in inflammatory immune responses and has recently proved to be a potential novel therapeutic target in malignant mesothelioma (MM). HMGB1 normally locates in the nucleus. However, during cell necrosis due to asbestos fibers, HMGB1 undergoes acetylation followed by translocation from the nucleus to the cytoplasm, and then secreted to extracellular space, where it binds to and activates pro-inflammatory mediators. Given the role it plays in inflammatory processes, HMGB1 may hold promise as a biomarker of cell transformative processes and thus hold utility as an indicator of asbestos exposure. A recent report reveals that the serum levels of both HMGB1 and acetylated HMGB1 are elevated in MM patients using an HMBG1 ELISA kit. Herein, we developed a stable isotope dilution HPLC-MS method, which has higher sensitivity and specificity compared with currently available HMBG1 ELISAs, to accurately quantify the HMGB1 levels and the acetylated HMGB1 levels in serum. Stable isotopically labeled HMGB1 was expressed using Stable isotope labeling by amino acids in cell culture (SILAC) strategy and was added as the internal standard (ISTD) at the initial step of the sample preparation. An anti-acetylation antibody was utilized to immunoprecipitate acetylated HMGB1 from the serum. In addition, dimethyl pimilimidate (DMP) was used to cross-link the antibody with the magnetic beads, in order to enable the elution of the target. Following elution, Glu-C digestion of HMBG1 yields peptides including two nucleus localization signal (NLS) fragments. These two key peptides are highly acetylated, which prevents HMGB1 from reentering the nucleus. Absolute quantification was achieved by analyzing the ratio of these two peptides from endogenous form and ISTD. The serum actylated HMGB1 levels in mesothelioma patiens were compared with healthy controls as well as with individuals that were heavily exposed to asbestos. The improved accuracy and enhanced sensitivity of this assay provides a thorough quantification method for acetylated HMGB1 and further identifies if the acetylated form is a promising biomarker for mesothelioma patients.

    TOXI 48

    Nrf2-Keap1 signaling and implications for the metabolic activation of nitroarenes

    Jessica Murray1, jrmurray8@gmail.com, Meng Huang1, Clementina Mesaros1, Volker Arlt2, Karam El Bayoumy3, Ian A. Blair1, Trevor M. Penning1. (1) Center of Excellence in Environmental Toxicology, University of Pennsylvania, Philadelphia, Pennsylvania, United States (2) Analytical and Environmental Sciences, King’s College London, London, United Kingdom (3) Biochemistry and Molecular Biology, Penn State College of Medicine, Hershey, Pennsylvania, United States

    Diesel engine exhaust (DEE) is listed as a Group 1 Carcinogen by the International Agency for Research on Cancer and contributes to occupational and environmental causes of lung cancer. Nitrated-polycyclic aromatic hydrocarbons, or nitroarenes, are major constituents of DEE and are detected in ambient air pollution. Nitroarenes require metabolic activation to exert their mutagenic and tumorigenic effects. NQO1 is considered the primary nitroreductase in the metabolic activation of nitroarenes. However, we have previously shown that AKR1C1-1C3 catalyze the nitroreduction of 3-nitrobenzanthrone, a representative nitroarene, using discontinuous UV-HPLC assays and product identification by LC/MS/MS. AKR1C1-1C3 also display dihydrodiol dehydrogenase and nitroreductase activity towards 6-nitrochrysene-1,2-dihydrodiol. The nitroreduction of diverse nitroarenes by AKR1C enzymes suggest that they may play a role in the activation of these diesel engine exhaust carcinogens. Notably NQO1 and AKR1C genes are highly induced by Nrf2-Keap1-ARE signaling, suggesting that the antioxidant response may not be entirely protective in the context of DEE exposures. We have demonstrated that AKR1C1-1C3 are highly upregulated by Nrf2 inducers R-sulforaphane and 1[2-Cyano-3,12-dioxooleana-1,9(11)-dien-28-oyl]imidazole in immortalized human bronchial epithelial cells which raises the question of whether these potential chemopreventative agents may upregulate metabolic activation of nitroarenes. These genes are also upregulated by environmentally relevant mixtures (cigarette smoke condensate, etc), suggesting that bioreactive intermediates produced during the metabolic activation of polycyclic aromatic hydrocarbons and nitroarenes can further upregulate expression of antioxidant response genes and possibly induce their own genotoxicity via Nrf2-Keap1-ARE signaling. To determine whether Nrf2 modulation of AKR1C genes leads to increased toxication of 3-NBA, 15N-labelled 3-NBA-dGuo adducts will be synthesized and quantified by LC-MS/MS.

    TOXI 49

    Toward genome-wide mapping of O(6)-methylguanine damage and repair in a human cell line

    Maureen McKeague1, mmckeague@gmail.com, Ioannis A. Trantakis1, Janine Döhring2, Pablo Steinberg2, Shana J. Sturla1. (1) D-HEST, ETH Zurich, Zurich, Zurich, Switzerland (2) Institute for Food Toxicology and Analytical Chemistry, University of Veterinary Medicine Hannover, Hannover, Germany

    Chemical alkylation of DNA bases giving rise to DNA adducts is a basis of mutagenicity and carcinogenesis. Among the various types of DNA adducts that may be formed, O6-alkylguanine adducts are of biological relevance because of their high propensity for inducing mutations prevalent in cancer. The lack of strategies for defining the distribution of damage throughout the genome and its dynamic changes in the process of exposure limits our ability to understand the likelihood of key mutations arising or contributing to carcinogenesis. Our long-term goal is to simultaneously identify, localize, and quantify DNA lesions to improve our understanding of the causal relationship between adduct formation, mutagenesis, and tumorigenesis.

    This presentation will focus on our work devising a new method to generate a de novo map of the genome-wide distribution of O(6)-methylguanine. To test our method, we employed a human colon epithelial cell line with stable inhibition of O6-alkylguanine DNA alkyltransferase expression and activity. DNA was extracted from cells treated with N-Nitroso-N-methylurea, fragmented, and immunoprecipitated using an anti-O(6)-methylguanine antibody. Enrichment of alkylated regions by immunoprecipitation was quantified by determining O(6)-methylguanine levels by LCMS/MS. Knowledge and new tools established in this study provide insight on the potential and limitations in genome-wide alkylation mapping in human cells.

    TOXI 50

    Role of PARP-1 in the base excision repair of chromatin substates

    Yu Zeng, zyqshshl66@tamu.edu, Deb R. Banerjee, Charles Deckard, Jonathan T. Sczepanski. Chemistry, Texas A&M University, College Station, Texas, United States

    The apurinic/apyrimidinic (AP) site in genomic DNA arises through spontaneous hydrolytic cleavage of the glycosydic bond or base removal by DNA glycosylases. The base excision repair (BER) pathway is responsible for repairing AP sites, which are recognized and incised by AP endonuclease-1 (APE1). AP sites are also recognized by poly(ADP-ribose) polymerase-1 (PARP-1), an abundant nuclear protein that polymerizes long chains of poly(ADP-ribose) (PAR) onto itself and target proteins, thereby modulating their activities. Both APE1 and PARP-1 can bind to the same BER intermediate after APE1 incision at the AP site, and PARP-1 is able to stimulate APE1 strand activity. Furthermore, PARP-1 becomes activated for PAR synthesis following AP site incision by APE1, which enhances recruitment of additional repair factors to the damage site. Thus, it appears PARP-1 plays a significant role in BER. Our project aims to study the relationship between PARP-1 and BER within the context of chromatin. We have prepared both mononucleosomes and (for the first time) nucleosomal arrays containing precisely positioned AP sites. These models allow us to investigate how specific chromatin architectures influence BER, and the role of PARP-1 in this process. For example, we show that PARP-1’s ability to stimulate APE1-catalyzed strand incision depends on the rotational orientation and position of the AP site within chromatin, and that this stimulatory effect is abolished upon PARP-1 activation. Our recent progress in this area will be presented.

    TOXI 51

    DNA cross-linking by the anticancer prodrug PR-104A in oligonucleotides

    Sara Danielli1, dasara@student.ethz.ch, Alessia Stornetta3, Shana J. Sturla2. (1) ETH Zürich, Institute of Food, Nutrition and Health, Zürich, Switzerland (2) ETH Zurich, Zurich, Switzerland (3) Health Sciences and Technology, ETH Zurich, Zürich, Switzerland

    DNA cross-links are amongst the most severe forms of DNA damage. The experimental anticancer pre-prodrug PR-104 induces toxicity by forming DNA interstrand cross-links (ICLs) following systemic hydrolysis and reductive bioactivation by hypoxia-associated oxidoreductases or aldo-keto reductase 1C3 (AKR1C3). The goal of this study was to identify the chemical structures of DNA cross-links formed by PR-104A and by its metabolites. The experimental strategy involved evaluating reactions between 15-mer DNA duplexes, the prodrug, and AKR1C3 by tandem mass spectrometry. The results suggest the formation of 1) adenine-adenine intrastrand cross-links directly, and also interstrand by the corresponding hydroxylamine; 2) guanine-adenine ICLs directly and by the hydroxylamine; 3) guanine-guanine intra- and interstrand cross-links directly and when the 5-nitro group is reduced; and 4) distinct patterns of preferential 1,2- vs. 1,3-cross-links formation. The profile of cross-links provides a chemical basis for the relevance of hypoxia and AKR1C3 expression for affecting drug potency in cells.

    TOXI 52

    Investigation of the presence in human urine of mercapturic acids derived from phenanthrene

    Guang Cheng, cheng037@umn.edu, Adam T. Zarth, Pramod Upadhyaya, Peter W. Villalta, Silvia Balbo, Stephen S. Hecht. Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, United States

    One widely accepted pathway of metabolic activation of carcinogenic polycyclic aromatic hydrocarbons (PAH) proceeds through formation of “bay region” diol epoxides which are highly reactive with DNA and can cause mutations. Phenanthrene (Phe) is the simplest PAH with a bay region and an excellent model for the study of PAH metabolism. In previous studies in which [D10]Phe was administered to smokers, we observed higher levels of [D10]Phe-tetraols derived from [D10]Phe-diol epoxides in subjects who were null for the glutathione-S-transferase M1 (GSTM1) gene. We hypothesized that simple Phe-epoxides, the primary metabolites of Phe, were detoxified by glutathione conjugate formation, which would result ultimately in the excretion of the corresponding mercapturic acids in urine. We synthesized the four stereoisomeric mercapturic acids that would result from attack of glutathione on Phe-1,2-epoxide and Phe-3,4-epoxide, followed by normal processing of the conjugates (e.g. 2-hydroxy-1-(N-acetylcysteinyl)-1,2-dihydrophenanthrene (2-HOPhe-1-NAC), 1-HOPhe-2-NAC, 4-HOPhe-3-NAC, and 3-HOPhe-4-NAC, as well as the corresponding dehydrated metabolites (e.g. Phe-3-NAC) and their sulfoxides, and N-acetylcysteine conjugates of quinones, e.g. Phe-3,4-dione-1-NAC and Phe-1,2-dione-4-NAC. These standards were used in liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry analyses of urine samples from smokers and creosote workers, the latter exposed to unusually high levels of PAH. None of the compounds was detected in the urine of smokers. Only relatively low amounts of Phe-3-NAC (6.8-179 pmol/ml), Phe-3,4-dione-1-NAC (1.7-28 pmol/ml), and the sulfoxide derivatives were detected in the urine of creosote workers;. These results demonstrate some new pathways of PAH-mercapturic acid formation, but do not provide an explanation for the role of GSTM1 null status on Phe-tetraol formation.

    TOXI 53

    Arsenite binds to the zinc finger domains of TIP60 histone acetyltransferase and induces its degradation via the 26S proteasome

    Lok Ming Tam4, lok.tam@email.ucr.edu, Ji Jiang1, Pengcheng Wang3, Lin Li3, Yinsheng Wang2. (1) CMDB, UC-Riverside, Riverside, California, United States (2) Univ of California Riverside, Riverside, California, United States (3) University of California Riverside, Riverside, California, United States (4) ETOX, University of California Riverside, Riverside, California, United States

    Arsenic, a ubiquitous environmental contaminant with widespread public health concern, is abundant naturally in minerals from the Earth’s crust and derived anthropogenically due to industrial applications, and it affects almost 150 million people in over 70 countries. Epidemiological studies have revealed that daily human exposure to arsenic in drinking water is associated with the prevalence of skin, lung and bladder cancers. Furthermore, aberrant histone modifications (e.g. acetylation) were found to be associated with arsenic exposure in previous studies, arsenic carcinogenesis is thought to stem partly from the perturbation of epigenetic pathways. Considering that zinc finger motif is present in and is indispensable for the enzymatic activities of crucial histone modifying enzymes especially the MYST family of histone acetyltransferases (e.g. TIP60), we reasoned that As3+ may target the zinc finger motif of these enzymes, thereby disturbing their enzymatic activities and altering histone acetylation. Herein, we found that As3+could bind directly to the zinc finger domain of TIP60 in vitro and in cells. In addition, exposure to As3+ could lead to a dose-dependent decrease in the protein level of TIP60 via the ubiquitin-proteasome pathway. Together, the results from the present study revealed, for the first time, that arsenite may alter epigenetic signaling by targeting cysteine residues in the zinc finger domains of TIP60 histone acetyltransferase, thereby altering H4K16Ac histone epigenetic mark. Thus, our results shed important light on the mechanisms underlying the arsenic-induced epigenotoxicity and carcinogenesis in humans.

    TOXI 54

    Substituent effects of bifunctional agents on photo-induced DNA interstrand cross-link formation

    Heli Fan, helifan@uwm.edu, Xiaohua Peng. Chemistry, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin, United States

    A series of bifunctional benzene derivatives with various aromatic substituents and benzylic leaving groups were synthesized. Their photoreactivity towards DNA were studied by DNA cross-linking assay. Most of these compounds efficiently form DNA interstrand cross-links (ICL) upon 350 nm irradiation. The mechanism for DNA cross-linking was investigated by free radical and carbocation trapping reactions using 2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO) and methoxyamine, respectively. The DNA ICL formation was via a carbocation which was generated either through oxidation of a free radical or via direct heterolysis of a C-X bond. The free radical trapping products and/or cation trapping products obtained from the monomer trapping reactions provided further evidences for determining the path ways of DNA cross-linking. Both substitutions and leaving groups greatly affect the reactivity, ICL efficiency, and the mechanism for DNA cross-linking. The cross-linking sites were determined by studying the stability of the DNA ICL products upon heating in piperidine as well as by characterizing the adducts formed between these bifunctional benzene derivatives and four natural nucleosides upon photo irradiation. dG, dA, and dC were the major alkylation sites for these bifunctional benzene derivatives upon photo irradiation.

    TOXI 55

    Estrogenic activity of polycyclic aromatic hydrocarbon ortho-quinones in human endometrium

    Isabelle G. Lee, islee@upenn.edu, Clementina Mesaros, Trevor M. Penning. Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, Philadelphia, Pennsylvania, United States

    Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous byproducts of incomplete combustion that are converted to reactive genotoxicants. In addition they can act as ligands for the aryl hydrocarbon receptor (AhR), and some PAHs or their metabolites can activate estrogen receptors (ER), resulting in endocrine disruption. In one pathway of PAH activation, aldo-keto reductases (AKRs) convert PAH trans-dihydrodiols into PAH ortho (o)-quinones, which are then shuttled into the the nucleus by AhR to modulate AhR-target gene expression. Given the similarity between planar PAH o-quinones and estrogen o-quinones, we hypothesize that PAH o-quinones can bind and activate ERs in estrogen target tissues e.g. endometrium. This activation may modulate ER-target genes leading to cell proliferation. We tested the estrogenicity of 3 PAH o-quinones (benzo [a] pyrene-7,8-dione (BPQ), benz[a]anthracene-3,4-dione and 5-methyl-chrysene-1,2-dione) in endometrial cells. We used the inducible alkaline phosphatase activity in Ishikawa cells, a human endometrial adenocarcinoma cell-line, as the read-out for ER activation. We demonstrated that these compounds induce ER activity, and that this activation is inhibited by Fulvestrant, an ER antagonist. We now demonstrate that the representative PAH, benzo [a] pyrene (BaP), and its metabolite, benzo [a] pyrene-7,8- dihydrodiol (BPD), upregulate AKR 1C1 and 1C3 expression, both at the mRNA and protein level which would be responsible for BPQ formation. Using high performance liquid chromatography and APCI mass spectrometry in the selected reaction monitoring mode, we find that BaP and BPD can be metabolized to the estrogenic BPQ in Ishikawa cells. Low micromolar concentrations of BPQ increase Ishikawa cell proliferation to the same level observed with nanomolar concentrations of estrogen, 17-α-ethinyl estradiol. Western blot analysis show that Ishikawa cells express both ERα, and ERβ. Our work indicates that PAH o-quinones may play a role in the disruption of ER signaling in the endometrium most likely through ERα.

    TOXI 56

    Deep learning methods applied to physicochemical and toxicological endpoints

    Boris Sattarov4, bsattarov@yahoo.com, Alexandru Korotcov4, Valery Tkachenko2, Christopher Grulke3,1, Antony J. Williams1. (1) National Center for Computational Toxicology, Environmental Protection Agency, Wake Forest, North Carolina, United States (2) SCIENCE DATA SOFTWARE, LLC, Rockville, Maryland, United States (3) Zachary Piper Solutions, New Hill, North Carolina, United States (4) Science Data Experts, Rockville, Maryland, United States

    Chemical and pharmaceutical companies, and government agencies regulating both chemical and biological compounds, all strive to develop new methods to provide efficient prioritization, evaluation and safety assessments for the hundreds of new chemicals that enter the market annually. While there is a lot of historical data available within the various agencies, organizations and companies, significant gaps remain in both the quantity and quality of data available coupled with optimal predictive methods. Traditional QSAR methods are based on sets of features (fingerprints) which representing the functional characteristics of chemicals. Unfortunately, due to both data gaps and limitations in the development of QSAR models, read-across approaches have become a popular area of research. Successes in the application of Artificial Neural Networks, and specifically in Deep Learning Neural Networks, has delivered a new optimism that the lack of data and limited feature sets can be overcome by using Deep Learning methods. In this poster we will present a comparison of various machine learning methods applied to several toxicological and physicochemical parameter endpoints. This abstract does not reflect U.S. EPA policy.

    TOXI 57

    Total synthesis of site-specific oligonucleotides containing 2′-deoxyadenosine adduct formed by 6-nitrochrysene and their biological studies

    Brent V. Powell, brent.powell@uconn.edu, Ashis K. Basu. Department of Chemistry, University of Connecticut, Storrs Mansfield, Connecticut, United States

    The environmental pollutant 6-nitrochrysene (6-NC) is a potent mutagen in bacteria and it induces mammary carcinoma in rats. It is an exceptionally potent carcinogen in newborn mice. 6-NC is metabolically activated by two pathways, which include only nitroreduction and both nitroreduction and ring oxidation. The nitroreduction pathway generates dG-C8, dG-N2, and the dA-C8 adduct, N-(dA-8-yl)-6-AC. Here, we report the synthesis of the phosphoramidite monomer of the major C8-dA adduct of 6-NC from dA in 10 steps and its incorporation into oligonucleotides. The key step in the synthesis of oligonucleotides containing N-(dA-8-yl)-6-AC was palladium-catalyzed Buchwald-Hartwig cross coupling chemistry, which was achieved in 75% yield. The protected N-(dA-8-yl)-6-AC derivative was deprotected and then converted to appropriately protected phosphoramidite monomer for solid-phase DNA synthesis. The resulting phosphoramidite was used to synthesize a 12-mer and a 15-mer oligonucleotide. The adduct-containing oligonucleotides were purified by reversed phase HPLC followed by denaturing polyacrylamide gel electrophoresis and characterized by mass spectrometry. These modified oligonucleotides are now being used to study the mutagenic and repair efficiency of N-(dA-8-yl)-6-AC in a plasmid construct.

    TOXI 58

    Determination of heavy metal acceptable concentration using fixed monitoring benchmarks in river system and soil pore-water in S.Korea

    Buyun Jeong2, bjeong6@snu.ac.kr, jinsung an3, gihyeon yu1, Kyoungphile Nam2. (2) Seoul National University Civil Environ Eng, Seoul, Korea (the Republic of) (3) Seoul National University, Seoul, Korea (the Republic of)

    Heavy metal toxicity in water varies greatly as environmental factors including pH, cations and dissolved organic carbon (DOC) change, which means its toxicity would be different to different organisms according to the surrounding conditions even though the total concentration of the heavy metal remains the same. To consider those factors, the new model called biotic ligand model (BLM) was developed in 2001 predicting heavy metal concentration in water system using fish gill as a ligand where the metal binds to. The BLM has been used widely but its limitation is that it does not reflect the temporal variation of those environmental factors. The seasonal variations of the pH, DOC or ion concentrations are not taken into account when evaluating the heavy metal toxicity and to deal with this problem, fixed monitoring benchmarks (FMB) method was introduced by HydroQual in 2008. FMB determines site-specific permissible concentration using a distribution of toxic unit (TU) dividing BLM-predicted safe concentrations by the dissolved heavy metal concentrations in site water.
    In this study, the FMB was applied to local river system in Han River, Seoul, S.Korea to determine acceptable concentrations of heavy metal among varying instantaneous water quality criteria (IWQC) which is calculated through the BLM with Daphnia Magna. With the temporal variation of the BLM-predicted safe concentrations over 30months of sampling period, the FMB method yields a single number that could be a site-specific standard to the local water system. Five heavy metals including Cu, Zn, Pb, Cd and Ni in Han River were evaluated.
    With successful application of FMB to the river system, the idea of FMB could be expanded to determine the heavy metal acceptable concentration in soil using soil pore-water. The environmental factors in the pore-water also vary as time goes so such change influences the determination of site-specific heavy metal concentration and its acceptable concentration. To determine the IWQC values using the BLM, barley was chosen as it’s a sensitive species to toxicity and changing environment.
    By considering the changing environmental factors over time, the fixed-site criterion for targeted heavy metal could be developed in local river system in Seoul, South Korea and the concept of FMB was also applied to soil pore-water to develop the acceptable heavy metal concentrations.

    TOXI 59

    Determination of the ecotoxicological threshold concentration of Cu in soil pore water in Korea with biotic ligand model and species sensitivity distribution

    gihyeon yu1, gihyeon007@snu.ac.kr, Buyun Jeong3, Kyoungphile Nam2. (1) Seoul National University, Seoul, Korea (the Republic of) (2) Seoul National University Civil Environ Eng, Seoul, Korea (the Republic of) (3) Civil and Environmental Engineering, Seoul National University, Seoul, Korea (the Republic of)

    Biotic ligand model (BLM) and species sensitivity distribution (SSD) was used to determine the site-specific threshold concentration of Cu (5% hazardous concentration, HC5) in soil porewater in korea. This combination ensured the ecological stability of biological community in soil. Model parameters of Cu for BLM were collected for six plants, one collembola, and two earthworms in the several published literatures. 50% effective activity (EC50{Cu2+}) was calculated from BLM and the predicted EC50{Cu2+} varied from 2 nM to 251 μM according to the variation of the environmental factors (pH, major cation/anion concentrations) in soil porewater and kinds of species. The 5% hazardous activity (HA5) and HC5 calculated from SSD also varied from 0.076 to 0.4 μg/L and 0.4 to 83.4 μg/L, respectively. Among the various environmental factors of soil porewater, it was the pH which significantly changed HA5 and HC5. According to the increase of pH, these values decreased due to the decrease of H+ competition effect in the region of pH lower than 7. In the region above pH 7, HC5 increased due to the formation of complexes of Cu with inorganic ligands (i.e., CuOH+, CuCO3(aq), Cu(CO3)22-) with increasing pH. In the presence of dissolved organic carbon (DOC), Cu and DOC form a complex which decreases the activity of Cu2+ in the soil porewater, leading to an increase in HC5. Especially, many complexes of Cu with DOC were formed due to the deprotonation of DOC at the pH of neutral region, resulting in up to 292-fold increase in HC5 (0.48 to 140 μg/L).

    HA5 calculated from SSD using EC50{Cu2+} predicted using Cu-BLM and the corresponding HC5 as a function of pH

    TOXI 60

    Versatile method to construct model DNA-protein cross-links (DPCs)

    Suresh Pujari3,1, spujari@umn.edu, Mandy Zhang2, Shaofei Ji1,2, Mark D. Distefano2, Natalia Y. Tretyakova3,1. (1) Department of Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota, United States (2) Department of Chemistry, University of Minnesota, Minneapolis, Minnesota, United States (3) Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, United States

    Exposure to exogenous and endogenous agents can result in irreversible entrapment of cellular proteins on genomic DNA, giving rise to DNA-protein cross-links (DPCs). Owing to their unusually bulky size, DPC lesions can interfere with DNA transactions including replication, transcription, and repair. However, our understanding of the mechanisms of DPC formation and repair is incomplete due to the paucity of synthetic methodologies to generate site-specific model DNA-protein cross-links for biological studies. We have developed a convenient method for the construction of DNA strands containing model DPCs, which takes advantage of the ability of substituted hydroxylamines to form stable oxime conjugates with aldehydes under mild conditions. A structural analogue of lysine was prepared where the ε-CH2 group was replaced with oxygen (oxy-Lys) and incorporated into peptides using solid phase synthesis. oxy-Lys-containing peptides were linked with a VHH protein segment using sortase mediated ligation. DNA strands containing site-specific 5-formyl-dC and 7-oxoethyl-7-deaza-dG residues were prepared by phosphoramidite chemistry and conjugated to oxy-Lys containing peptides and proteins to obtain structurally defined DNA-peptide and DNA-protein cross-links, which were characterized by gel electrophoresis and mass spectrometry. The conjugation reaction was site-specific in respect to both protein and DNA, provided excellent reaction yields, and formed stable DPCs amenable to biological evaluation.

    TOXI 61

    Modified deaza-adenosine mimics ad DNA minor groove alkylation probes

    Lukasz J. Weselinski, lweselin@mtu.edu, Vagarshak Begoyan, Shuai Xia, Alexis Ferrier, Marina Tanasova. Chemistry, Michigan Technological University, Houghton, Michigan, United States

    3-Deaza-3-substituted adenosine derivatives as nucleoside analogs offer a broad potential for biochemical and medicinal applications including the basis for developing anticancer or antiviral agents. Furthermore, among the variety of nucleic acid modifications, alkylation at the N3-site of purines, particularly of adenosine, plays a critical role in the processing of genomic information. A direct evaluation of the outcomes of adenosine modification at the N3-site is, however, compromised by N3-alkylation-driven depurination of DNA. Accordingly, the directed synthesis of novel nucleosides with tailor-made functionalities is of high interest for biochemical and medicinal sciences.Accessing such probes has proven to be challenging due to the high functionalization of purines, requiring a tuning of their electronic properties to achieve the desired chemical reactivity. Here we describe a general strategy that allows the deliberate synthesis of various 3-deaza-3-substituted adenine derivatives through metal-catalyzed cross-coupling reactions of a common precursor. The resulting alkylated 3-deazaadenosine mimics were also tested as DNA alkylation probes to assess the impact of DNA minor groove alkylation on duplex stability. Overall, the development of this methodology is expected to facilitate biochemical studies on the impact of DNA minor groove alkylation, and promote synthesis of unnatural nucleosides for various biochemical and biomedical application.

    TOXI 62

    Development of rapid, high throughout labeling methods for measuring aldehydes from P450 reactions

    Amanda M. Hanson, AMHanson@uams.edu, Dustyn A. Barnette, Grover P. Miller. Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States

    N-Dealkylation is a common metabolic step carried out by cytochromes P450 toward drugs, pollutants, and dietary molecules; the oxidative reaction ultimately yields an amine and an aldehyde. The aldehydes are typically low molecular weight and thus not amenable to identification and quantitation by traditional analytical tools. The common strategy is to label aldehydes (or more generally, carbonyls) through reaction with 2,4-dinitrophenylhydrazine to form the corresponding hydrazones. This method relies on acidification with high levels of HCl, liquid-liquid extraction in glass reaction vials, and isolation of fractions containing hydrazones for dry down/resuspension for LC analysis using absorbance and/or MS detection. Nevertheless, this approach requires harsh acidic conditions, labor-intensive liquid-liquid extraction, and multiple other steps that make it unsuitable for processing large numbers of samples. As an alternative, we are developing high throughput labeling methods to identify and quantitate carbonyls including aldehydes using absorbance and fluorescence tags. We are currently optimizing the labeling of a diverse set of alkyl, allyl, and aromatic aldehydes as standards and products of reactions with dansyl hydrazine (DNH). In brief, samples are arranged in a 96 well plastic microplate. The addition of a 10-fold excess of acetonitrile precipitates proteins and buffer, which are then removed by centrifugation. Aldehydes are then labeled in 15 min upon DNH addition in the presence of acetic acid as a catalyst. After 15 min reaction, samples are dried down and then resuspended in mobile for LC analysis measuring fluorescence and MS (due to increase in molecular weight form the label). On-going studies have shown that DNH reacts almost stoichiometrically with aldehydes, so only low molar excess is necessary. Only catalytic amounts of acid needed but does lead to formation of acid hydrazone, which is resolvable chromatographically or by MS. Moreover, signal intensities for hydrazones depended on depended on aldehyde structure. Current efforts are establishing the limits of detection and quantification for this method before exploring labeling of other carbonyls and other labels. These seminal findings provide encouraging results for a more efficient, less costly yet sensitive strategy to identify and quantitate aldehydes such as those from P450 reactions.

    TOXI 63

    Bypass efficiency and mutagenesis assays of site-specific arylamine DNA adducts in cell

    Ke Bian1, kebian@my.uri.edu, Fangyi Chen1, Qi Tang1, Deyu Li2. (1) Biomedical pharmaceutical science, University of Rhode Island, Kingston, Rhode Island, United States (2) University of Rhode Island, Kingston, Rhode Island, United States

    Arylamine is one of the major bulky organic carcinogens for human cancers. The Arylaminemodified dG lesions, of which the C8 position is substituted by 4-amminobiphenyl (ABP) or 2- aminofluorene (AF), are the objects of our studies. These DNA-adducts adopt multiple conformations including “stacked” (S) and “B-type” (B) conformers in double-strand duplexes showing highly sequence-dependent characteristics. A simple swap of one nucleotide by other three bases or swap of the location of adducts within the sequences will cause a significant change of the mixture of conformers and also lead to different nucleotide excision repair efficiencies. Site-specific bypass efficiency and mutagenicity studies will be employed to test the effects of arylamine-adducts in cells.

    TOXI 64

    Comprehensive kinetic study of ALKBH2 and related family enzymes

    Michael Vittori1, michael_vittori@my.uri.edu, Ke Bian2, kebian@my.uri.edu, Fangyi Chen2, Qi Tang2, Deyu Li2. (1) University of Rhode Island, Kingston, Rhode Island, United States (2) Biomedical pharmaceutical science, University of Rhode Island, Kingston, Rhode Island, United States

    The human ABH2 protein, an α-Ketoglutarate/Fe(II)-dependent dioxygenase, repairs alkylation damage in DNA through oxidative demethylation, with relative specificity for 1-methyladenine (m1A) and 3-methylcytosine (m3C) lesions. In addition to understanding how these systems correct certain DNA defects, it is important to thoroughly analyze the factors that influence the functioning of these catalytic protein complexes and the conditions that allow these systems to function optimally. While several studies have already independently examined the factors associated with ABH2 enzyme functioning, a comprehensive analysis of these determinants has yet to be conducted.

    In this project we examined the factors that regulate ABH2 function. Here we have evaluated the effects that varying concentrations of α-ketoglutarate, iron (II), and ascorbic acid have on the ability of ABH2 to correct m1A and m3C lesions in DNA. Additionally, we have reported the impact of pH and temperature on the catalytic repair of m1A and m3C lesions by ABH2. We also investigated whether various reducing agents could be used in place of ascorbic acid in the oxidative demethylation reaction catalyzed by ABH2. The results indicate that the optimal operating conditions for ABH2 may differ from what has been previously reported in the literature. Furthermore, our findings indicate that m3C and m1A lesions are repaired more efficiently by ABH2 at pH conditions below the pKa values for these lesions.

    TOXI 65

    Expression of a fragment of DNA polymerase zeta from Dictyostelium discoideum

    Stanley K. Mauldin, smauldin@holyfamily.edu, Dian He. Holy Family University, Philadelphia, Pennsylvania, United States

    DNA polymerase zeta (Pol ζ) is an enzyme involved in translesion DNA synthesis (TLS) in which the enzyme will replicate over DNA damage at the replication fork. Pol-zeta mediated TLS is an important mechanism to prevent DNA replication fork collapse. Pol zeta is composed of two subunits REV3 and REV7. REV3 is the catalytic subunit, while REV7 is an regulatory subunit that stimulates REV3 activity. DNA pol zeta is categorized as a B family DNA polymerase in contrast to other translesion polymerases which are members of the Y-family. Dictyostelium discoideum is an excellent model system to study DNA repair. The organism is highly resistant to most known DNA damaging agents and is considered a repair extremophile. In many ways, D. discoideum mimics cancer cells that have become resistant to chemotherapeutic agents. The purpose of this project is to express in E. coli a 564 amino acid fragment of the D. discoideum Pol zeta and purify it for X-ray crystallography. The crystal structure would then be compared to computerized molecular modelling of the D. discoideum Pol zeta. The nucleotide sequence of the D. discoideum Pol zeta (1692 bp) was obtained from the genomic database (DictyBase.org). The DNA fragment was initially going to be made by Integrated DNA Technologies. However, D. discoideum is highly AT-rich and this created problems for the synthesis. Since the fragment is to be expressed in E. coli, approximately every third codon was changed to either a C or G at the third position without changing the amino acid. These changes allowed IDT to make the DNA fragment. For cloning a BamHI and an XhoI restriction site was added to the 5’ end and 3’ end respectively. The fragment was cloned into pGEX-4T-1 expression vector cut with the same restriction enzymes. This expression vector will tag the Pol zeta with glutathione-S-transferase (GST). The cloned fragment was then expressed in E. coli BL21 by induction with isopropyl-thiogalactoside (IPTG). An approximately 89 kilodalton protein was observed from two independent clones after SDS-PAGE. Future work will include: confirming the expression of GST-tagged Pol zeta using an antibody to the GST tag, purifying the Pol zeta using glutathione-Sepharose, and crystallization of the protein for X-ray structure determination.

    TOXI 66

    Replication and repair of 8-methoxypsoralen-derived DNA-DNA interstrand cross-links in human cells

    Nathan E. Price3, nathan.price@ucr.edu, Yinsheng Wang1, Kent S. Gates2. (1) Univ of California Riverside, Riverside, California, United States (2) Univ of Missouri, Columbia, Missouri, United States (3) Chemistry, University of California, Riverside, Riverside, California, United States

    Psoralen can induce DNA interstrand cross-link lesion formation upon exposure to UVA light. Interstrand cross-links are thought to prevent strand separation of the DNA double helix and, as a result, are strong impediments to replication and transcription. When an interstrand cross-link is encountered during DNA replication, complex repair processes are initiated. However, it is not well understood what specific machinery aids in the replication-coupled repair of psoralen-derived cross-links. Here we conducted shuttle vector-based replication studies by employing a double-stranded plasmid harboring a site-specifically incorporated 8-methoxypsoralen interstrand cross-link and human cells deficient in one or more TLS polymerases or structure-specific endonucleases; each of which may play an important role in the cross-link bypass and repair. The data revealed the relative bypass and mutation frequencies for the 8-methoxypsoralen-derived interstrand cross-link lesions in these cells, which help define the roles of individual proteins during this complex replication-coupled repair process in human cells.

    TOXI 67

    Polymerase bypass of DNA-protein and DNA-peptide cross-links

    Shaofei Ji1, jixxx199@umn.edu, Orlando Scharer4, Natalia Y. Tretyakova2,3. (1) Chemistry, University of Minnesota, Minneapolis, Minnesota, United States (2) Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota, United States (3) Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, United States (4) Chemistry and Pharmacological Sciences, Stony Brook University, Stony Brook, New York, United States

    DNA-protein cross-links (DPCs) are commonly formed upon exposure to endogenous, environmental and chemotherapeutic agents such as α,β unsaturated carbonyls, diepoxides, nitrogen mustards and platinum compounds. Because of their considerable size and helix-distorting nature, DPCs can interfere with accurate DNA replication and transcription, leading to toxicity and mutations. In this present work, the effects of model DPCs on the fidelity and efficiency of replication and transcription was investigated. Structurally defined DNA-protein and DNA-peptide conjugates were generated via reductive amination approach developed in our laboratory and subjected to in vitro DNA replication and transcription experiments in the presence of lesion bypass DNA polymerases and T7 RNA polymerase, respectively. While full size DNA-protein cross-links completely blocked DNA and RNA polymerases, DNA-peptide cross-links were bypassed. Replication bypass of an 11-mer peptide cross-linked to the C5 position of C in the presence of hPol η and hPol κ induced targeted C→T transitions and -1 deletions. However, polymerase bypass of the same peptide conjugated to C7 position of 7-deaza-G was error-free. Transcriptional mutagenesis of DNA-peptide cross-links in the presence of T7 RNA polymerase was investigated by RT-PCR in combination with LC-MS/MS. DNA-peptide cross-links conjugated to C5 position of C induced large amounts of C → T mutations and deletions, while the corresponding guanine lesions were not mutagenic.

    TOXI 68

    Conformational and configurational equilibra of a 2′-deoxyribosylurea adduct in single strand and duplex DNA

    Andrew H. Kellum2, kellumah@gmail.com, Michael P. Stone3, Ashis K. Basu1, Jasti Vijay1. (1) Department of Chemistry, University of Connecticut, Storrs Mansfield, Connecticut, United States (2) Vanderbilt University, Nashville, Tennessee, United States (3) Chemistry, Vanderbilt University, Nashville, Tennessee, United States

    2′-Deoxyribosylurea (urea) lesions form within DNA as a result of the cleavage of thymine from hydroxyl radicals resulting from ionizing radiation. In addition, urea lesions are able to form from 8-oxoguanine. Previous NMR studies in the Meunier lab of the urea lesion on the nucleoside level showed that the 2′-deoxyribose ring equilibrated between the alpha (α) and beta (β) configurations. In this study we examined the urea lesion in the single strand oligodeoxyribonucleotide 5′-(CTXA)-3′ and in duplex DNA 5′-GCGXGCG-3′:5′-CGCACGC-3′ (X=urea). Reverse phased HPLC revealed a ratio of 1:1 between two equilibrating species in single strand DNA. In addition, the presence of two equilibrating species was present in duplex DNA. Trapping the different species in basic environment slowed the equilibration supporting the hypothesis that the species are anomers. NMR spectroscopy was used to definitely identify the two different species in single strand DNA. NOESY and TOCSY spectra were used to assign the resonances of the different nucleotides. NOESY spectra revealed the identity of the two different species as α and β anomers of the urea lesion. TOCSY and one-dimensional NMR spectroscopy showed that the urea lesion was in the anti conformation regardless of the configuration the 2′-deoxyribose ring. The presence of the α anomer in single strand DNA is a possible explanation as to why the lesion acts a strong block to replication. Future studies will isolate the individual anomers and study the structural effects of each anomer in duplex DNA by NMR spectroscopy.

    TOXI 69

    Terbinafine bioactivation pathways to liver toxicity assessed using predictive modeling and experimental approaches

    Dustyn A. Barnette2, DABarnette@uams.edu, Lena Dang3, Tyler Hughes3, S Joshua Swamidass3, Grover P. Miller1. (1) Dept of Biochem and Mol Biol, Little Rock, Arkansas, United States (2) Biochemistry, University of AR for Medical Sciences, Little Rock, Arkansas, United States (3) Washington University, Saint Louis, Missouri, United States

    Lamisil (terbinafine) is widely prescribed and effective antifungal drug that causes idiosyncratic liver toxicity. Terbinafine undergoes extensive metabolism including N-dealkylation to yield 6,6-dimethyl-2-hepten-4-ynal (TBF-A). This reactive allylic aldehyde was proposed to transiently form a glutathione conjugate that enables off target damage of the bile duct leading to liver toxicity. Nevertheless, TBF-A toxicological relevance remains unclear due to a lack of identification of pathways leading to and competing with TBF-A formation. We propose terbinafine undergoes rapid N-demethylation to form desmethyl-terbinafine (dTBF) followed by subsequent N-dealkylation to yield TBF-A and naphthylmethylamine (NMA). This pathway is the most probable one based on modeling terbinafine N-dealkylation using deep learning neural networks. We are experimentally testing the hypothesis and model using in vitro reactions with human liver microsomes. Based on LC-MS analysis, we identified primary and secondary metabolites corresponding to N-dealkylated and hydroxylated products of terbinafine. Aldehyde metabolites from N-dealkylation were not observed directly, and they were trapped with dansylhydrazine labeling. Our identification of dTBF and its hydroxylated derivatives from terbinafine reactions suggest a rapid initial N-demethylation. Furthermore, we identified NMA and oxidized derivatives from dTBF reactions. These compounds are co-metabolites of TBF-A, allowing us to infer its formation even though we were not able to directly identify it directly or trap it with glutathione. Together, these data support the proposed two-step pathway for TBF-A formation from terbinafine under biological conditions. On-going kinetic studies will reveal the efficiency of subsequent TBF-A formation and competing detoxification pathways. Moreover, inhibitor phenotyping revealed CYP1A2 and 2C9 oxidize terbinafine and CYP1A2, 2B6, 2C9, and 3A4 oxidize dTBF in pathways competing with TBF-A formation. Overall, we have identified terbinafine metabolic pathways including preferred bioactivation pathways for TBF-A and competing oxidation pathways. Knowledge of these mechanistic details will confirm the most probable model prediction for TBF-A formation and experimentally demonstrate whether the efficiency of the pathway could contribute to reported liver toxicity.

    TOXI 70

    Sequence-dependent repair of 1, N6-ethenoadenine by the AlkB family DNA repair enzymes

    Qi Tang, qitang@my.uri.edu, Fangyi Chen, Ke Bian, Deyu Li. Biomedical and Pharmaceutical Sciences, University of Rhode Island, Kingston, Rhode Island, United States

    DNA adducts are constantly generated from exogenous and endogenous processes, causing genome instability and eventually lead to cancer and other genetic diseases. To avoid the damages from DNA lesions, organisms have evolved an array of DNA repair strategies to protect cells against the adverse effect. One of the repair proteins, the adaptive response enzyme AlkB of E.coli, has been reported to repair various adducts including m1A, m3C and eA. The repair efficiency is depending on the sequence context, especially the neighbor bases from 3’ and 5’ ends. In this project, we are aiming to test the repair of AlkB both in vitro and in vivo by applying enzymatic reactions and REAP&CRAB assays. We are also planning to study the AlkB mammalian homologs ABH2 and ABH3. The information obtained from here may provide explanation for the mutational spectra of etheno adducts and the interconnection with repair enzymes.

    TOXI 71

    Independent generation of neutral purine radicals involved in DNA damage

    Liwei Zheng, whu.lwzheng@gmail.com, Marc M. Greenberg. Department of Chemistry, Johns Hopkins University, Baltimore, Maryland, United States

    Formal hydrogen atom abstraction from the nitrogen-hydrogen bonds in purine nucleosides produces reactive intermediates that are important in nucleic acid oxidation. However, little is known about the relativities of these radicals due to the lack of controlled means to generate them. We developed two general strategies for independently generating neutral purine radicals, 2′-deoxyadenosine-N6-yl radical and 2′-deoxyguanosine-N2-yl radical. The first approach utilizes photoinduced homocleavage of N-N bonds of the corresponding diphenylhydrazines. The second strategy consists of Norrish Type I photocleavage and β-fragmentation cascade to form neutral purine radicals and acetone. The effectiveness of both strategies is supported by product analysis. These photochemical methods promise to be useful for elucidating the reactivity of neutral purine radicals in nucleic acid oligomers via site-specific production.

    TOXI 72

    Importance of the glutathione and its degradation by γ-glutamyl transferase in lung tumor development

    Rosalind B. Penney1,4, Nicholas S. Kowalkowski1, Eric R. Siegel3, Gunnar Boysen2, gboysen@uams.edu. (1) Environmental and Occupational Health, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States (2) College of Public Health, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States (3) Biostatistics, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States (4) The Winthrop P. Rockefeller Cancer Institute, University of Arkansas for Medical Sciences, Little Rock, Arkansas, United States

    Metabolomics studies in lymph node aspirates demonstrate that lung tumor types can be diagnosed based on their metabolite profiles. Follow up studies show that 30% of lung tumors excrete high amounts of glutathione and from γ-glutamyl-amino acids. Subsequent validation experiments in lung tumor cell lines confirmed the increased excretion of glutathione and formation of γ-glutamyl-amino acids. GSH concentrations in lung tumors have been known to be as high as 10 mM, while formation of γ-glutamyl-amino acids has not been reported previously. About 50% of lung tumors express active γ-glutamyl transferase, an enzyme that hydrolyzes glutathione or transfers the γ-glutamyl from glutathione to free amino acids or dipeptides, if present at high concentrations. γ-glutamyl transferase is a key enzyme of the γ-glutamyl cycle, a biochemical pathway that had long been considered to be physiologically irrelevant because of the high km-values of the enzymes involved. However, the function and biological significance of high glutathione in lung tumors has been unanswered. We show herein that lung tumors make γ-glutamyl-amino acids, suggesting an active γ-glutamyl cycle in lung tumors. The observed extracellular glutathione and γ-glutamyl-glutamine led us to hypothesize that they participate in cell-cell interaction. Using a standard transformation assay, it is shown that γ-glutamyl-glutamine induces transformation in normal NIH-3T3 cells and small airway epithelial cells. In contrast, glutathione induces transformations only in GGT positive small airway epithelial cells. Based on these findings we conclude that glutathione is a universal amino acid checkpoint. When sufficient amino acids are available glutathione is metabolized to γ-glutamyl-glutamine, which subsequently triggers proliferation of tumor cells and transformation of normal epithelia cells.

    TOXI 73

    Mitochondrial M1dG levels linked to oxidative stress and mitochondrial dysfunction in disease

    Orrette R. Wauchope2, Michelle M. Mitchener1, michelle.m.mitchener@vanderbilt.edu, William N. Beavers1, James Galligan2, Philip Kingsley2, Ha-Na Shim3,4, Thomas Blackwell3,4, Thong Luong3,4, Mark deCaestecker5,6, Joshua P. Fessel3,4, Lawrence J. Marnett2,1. (1) Chemistry, Vanderbilt University, Nashville, Tennessee, United States (2) Biochemistry, Vanderbilt University, Nashville, Tennessee, United States (3) Cancer Biology, Vanderbilt University, Nashville, Tennessee, United States (4) Pharmacology, Vanderbilt University, Nashville, Tennessee, United States (5) Cell and Developmental Biology, Vanderbilt University, Nashville, Tennessee, United States (6) Medicine, Vanderbilt University, Nashville, Tennessee, United States

    Lipid and DNA peroxidation give rise to DNA adducts, the most abundant of which is 3-(2-deoxy-β-D-erythro-pentofuranosyl)pyrimido[1,2-α]purin-10(3H)-one (M1dG). While previous studies have reported higher levels of M1dG in mitochondrial versus genomic DNA, perhaps not surprisingly given the relative dearth of repair pathways in the mitochondrion compared to the nucleus, little research effort has been dedicated to understanding mitochondrial M1dG formation or its potential role in disease pathogenesis. Herein we present data confirming that levels of M1dG are indeed higher basally in the mitochondrion than in the nucleus, and that these levels are elevated upon exogenous electrophile stimulation in multiple human cell lines. Notably, mitochondrial levels were particularly high in RAW264.7 macrophages, leading us to hypothesize a correlation between oxidative stress and mitochondrial M1dG levels. To test this theory, cells were treated with rotenone, which inhibits Complex I of the electron transport chain. Rotenone treatment led to increased levels of M1dG, an increase that could be reversed by the concomitant addition of a mitochondria-targeted free radical scavenger mitoTEMPO. Furthermore, mitoTEMPO treatment reduced basal mitochondrial M1dG levels by about two-fold, while the ubiquitously distributed free radical scavenger TEMPOL had no effect on either untreated or rotenone-challenged cells. To assess whether this observed correlation between oxidative stress and mitochondrial M1dG levels applied in an animal model of disease, we isolated pulmonary microvascular endothelial cells from transgenic mice bearing a mutant BMPR2 gene, which predisposes them to develop pulmonary arterial hypertension, a reported mitochondria-linked disease state. Indeed, levels of mitochondrial M1dG were elevated in the BMPR2 mutant mice compared to their wild-type counterparts. Collectively, our data provide evidence of a strong correlation between oxidative stress and mitochondrial M1dG levels and suggest that this adduct may be an ideal biomarker for monitoring disease progression in conditions characterized by mitochondrial dysfunction.

    TOXI 74

    Mechanisms of recognition of bulky DNA lesions by the DNA damaging sensor XPC

    Katie M. Feher, katie.feher@nyu.edu, Kadeem D. Walsh, Nicholas E. Geacintov. Chemistry, New York University , New York, New York, United States

    The mammalian Nucleotide Excision Repair (NER), the DNA lesion is first recognized by the NER DNA damage sensing XPC-RAD23B factor (XPC) that binds to the site of the damage, followed by a verification step carried out by the subsequent binding of the multiple-protein factor TFIIH. Earlier work in our laboratory has shown that the bulky DNA adducts derived from the binding of (+)-7R,8S-dihydrodiol-7S,8R-epoxy-benzo[a]pyrene (B[a]PDE) to the exocyclic amino groups of guanine yield (+)-cis- and (+)-trans-addition products at the C10 position(G*). Translesion synthesis (TLS) past these lesions by polymerases can occur in an error-free manner by inserting a C opposite G*, or by an error-prone manner by inserting X = A, or G, or bypassing G* entirely via the formation of a frameshift intermediate. These TLS mechanisms can yield the following mismatched duplexes I(X) and/or the Deletion duplex I(Del) (Figure). In the case of the normal duplexes with X = C, the NER efficiency of the stereoisomeric (+)-cis-G* adducts is five time greater than in the case of the (+)-trans-adduct, but the XPC binding efficiency is similar in both cases, suggesting that the verification step determines the differences in NER efficiencies. However, in the case of the X = A, G, and I(Del), the same G* adducts are fully NER-resistant, and the XPC binding efficiency is strongly diminished. In these cases the NER efficiency is abrogated by a decrease in the binding of the damage-sensing XPC factor. These results suggest that the NER efficiencies of bulky DNA lesions may depend on either the first or the second bipartite step.

    TOXI 75

    Mitochondrial DNA adducts of lipid peroxidation products with rotenone

    Kevin P. Gillespie1, gillespiekevinp@gmail.com, Ian A. Blair2. (1) Systems Pharmacology and Translational Therapeutics, University of Pennsylvania, Philadelphia, Pennsylvania, United States (2) Upenn Ctr For Cancer Pharm, Philadelphia, Pennsylvania, United States

    Environmental exposure to the pesticide rotenone is positively associated with Parkinson’s disease incidence. .Although rotenone-induced oxidative stress is well-documented, there is also evidence for mitochondria-selective injury in rotenone toxicity. Mitochondrial lipid peroxidation is one potential component of rotenone toxicity. ROS-induced lipid peroxidation generates electrophilic aldehydes, including 4-hydroxynonenal (HNE) and 4-oxononenal (ONE). These compounds generate nuclear DNA and protein adducts, but their mtDNA adducts are first explicitly reported here with a developed liquid chromatography tandem mass spectrometry method. This finding suggests that mitochondrial protein and mtDNA adducts of lipid peroxidation products may be biomarkers of selective oxidative injury of mitochondria in neurodegenerative disease

    TOXI 76

    Temporal impact of toxic exposures on cellular recovery

    Julia A. Mouch1, jmouch@bethanywv.edu, Alice Han1, Julie V. Miller2, Nicole Prince1, Maren S. Prediger1, Jonathan W. Boyd1. (1) C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, West Virginia, United States (2) CDC/NIOSH, Morgantown, West Virginia, United States

    In response to a toxic exposure, the pharmacodynamic response of cells is primarily regulated by a coordinated signaling network of phosphorylation/dephosphorylation series mediated by kinases and phosphates. The time course, however, from initial exposure to a measurable end point (e.g. cell death) can encompass a large time ranging from minutes to hours. By monitoring phosphorylation, early signaling may foreshadow the “point of no return” long before the final steps of cell death. In this study, we address the concept of cell recovery by exposing HepG2 cells to a GSK-3α/β inhibitor (10, 20, 30, 40, 50, 100 µM) or a MEK1 inhibitor (1, 5, 10, 20, 50 µM) treatment. The inhibitor was then removed after various durations of exposure (1, 5, 10, 20 min and 24 h). Dead cells were measured with an ethidium homodimer-1 probe every 20 minutes for 24 hours following the initial exposure to monitor plasma membrane degradation. Significant cell death did not transpire until at least 40 minutes after exposure (for 20-50 µM MEK1 and 30-100 µM GSK-3α/β doses). Cell death was observed after all the exposure durations (for 5-50 µM MEK1 doses and 20-100 µM GSK-3α/β doses) at 24 h post-treatment. The results indicate an exposure time as low as 1 minute was sufficient enough for kinase inhibitors to initiate cell mechanisms that commit the cell to death. Furthermore, because there were minimal significant differences in relative cell death at 24 hours across the varying exposure durations, the removal of the toxicant could not reverse the initial steps of cell death and, as such, there may be no benefit in removing the toxicant earlier. The findings of this study highlight the cell’s inability to recover, predetermined by cellular pharmacodynamics events that occur at early time points.

    TOXI 77

    Resolving metabolic switches in cellular energy demand

    Maren S. Prediger1,2, maren_prediger@web.de. (1) C. Eugene Bennett Department of Chemistry, West Virginia University, Morgantown, West Virginia, United States (2) Fakultät für Chemie und Pharmazie, Julius-Maximilians Universität Würzburg, Würzburg, BY, Germany

    The phenomenon of hormesis has experienced increasing interest by the scientific community, especially finding compounds that exert beneficial low-exposure stress-protective effects. Elucidating the underlying cellular signaling pathways that lead to a new homeostatic state can potentially provide crucial information about new therapeutic targets to prevent and/or treat diseases. Resveratrol, a polyphenol commonly found in peanuts, grapes, and subsequently in wine, is known for a variety of effects – beneficial and detrimental – depending on the investigated cell type. In human hepatic carcinoma cells, HepG2, an increase in proliferation has been observed at doses between 10 µM to 100 µM resveratrol after 16 hours, while higher doses decrease cell viability markedly eventually leading to cell death. Preliminary cell viability and plasma membrane degradation experiments after 72 hours of exposure show a statistically significant (p≤ 0.05) increase in cell proliferation associated with a dose of 100 µM. In order to better characterize hormesis, we have further explored the bioenergetic responses of HepG2 cells exposed to resveratrol. As described previously, we have used a series of real-time in vitro assays to determine early time points of changes associated with exposure. Additional experiments at these critical time points will allow identification of increased or decreased signaling network activity, with a focus on deviations of autophagic flux and activation of caspases. This research helps to further elucidate the mechanism of action that enables hormesis, and offers unique insight into the switches in metabolic states at early time points that can provide important information for its applicability to disease prevention and treatment.

    TOXI 78

    Characterization of a domoic acid-producing diatom

    Min-Ying Wang2, mywang@dragon.nchu.edu.tw, Su-Yuan Lai1, Ping-Ting Lin2, Hsing-Li Lai2. (1) Food Science and Technology, Central Taiwan University of Science and Technology, Taichung, Taiwan (2) National Chung Hsing University, Taichung, N/A, Taiwan

    Diatom is a major group of unicellular algae in nature. Here, we described the characterization of a domoic acid (DA)-producing diatom, Halamphora sp. NCHU MYW AQ4, collected from marine waters at Kenting, Taiwan. The morphology of Halamphora sp. NCHU MYW AQ4 displayed patterns consistent with the generic characteristics of the genus Halamphora. The size (about 20 µm), the dorsiventrality of the cells in valve view, the protracted, rostate valve ends and the seemingly central helictoglossa sitting on a raphe ledge placed NCHU MYW AQ4 among the Halamphora clade. The 18S rDNA molecular analysis was also consistent with our morphological analysis. Using NJ, MP and ML evolutionary tree constructs, our strains were found to be in the same branch with Halamphora sp. with bootstrap support; thus, we assign Halamphora sp. NCHU MYW AQ4 to this DA-producing strain.

    TOXI 79

    Size matters: Mesoporous silica nanoparticles size governs the vascular angiogenesis process, proliferation, and nanotoxicity

    Magdiel Inggrid Setyawati, chemis@nus.edu.sg, David Leong. National University of Singapore, Singapore, Singapore

    Recent advancements in synthesis and the ability to rationally manipulate the physicochemical properties of nanomaterials (NM) open up new horizons for clinically relevant nanomedicine applications. While the vasculature is not the common target of interest by these nanomedicine, due to the intravenous injection as a popular route of entry for this nanomedicine, has inevitably made the vasculature as the main organ of tissue where there are unintended effects of those nanomedicine formulations. Despite its obvious importance for any intravenously introduced nanomedicine, testing for side effects or toxicity of nanomedicine on endothelial cells, which line the vasculature, are often overlooked. Here, we studied the effect one of the common nanomedicine vehicles, mesoporous silica nanoparticle (MSN), to the endothelial cells. We observed MSN could inhibit cell migration and invasion in size dependent manner. This inhibition on the cell migration and invasion resulted with impediment of blood vessel formation process, angiogenesis. In addition, MSN was also found to curbed endothelial cell proliferation and induced nanotoxicity on the cells. Through our mechanistic study, we found that reactive oxygen species (ROS) played modulatory role on the observed size dependent MSN toxicity and angiogenesis inhibition. Overall, our finding show case that MSN may pose unintended risk in human patient. Identifying this potential risk of NM-based application and the underlining mechanism behind its nanotoxicity would enable nanotechnologist to re-design their nanomedicine to negate unintended adverse effects.

    TOXI 80

    Development of a threshold of toxicological concern framework based on chemoinformatics

    Mitchell Cheeseman, mcheeseman@steptoe.com. Steptoe Johnson LLP, Washington, District of Columbia, United States

    The threshold of toxicological concern (TTC) is a tool developed originally to support the safety assessment of over 3,000 food flavors. Currently, the TTC framework is based on the so-called Cramer-Hall decision tree and establishes three safe human exposure thresholds based on three broad Cramer classes of chemicals. We have applied chemoinformatics to an enlarged toxicological data set to identify a larger number of chemotypes for which more precise safe human exposure thresholds can be established. This revised TTC analysis and framework provide a more flexible less static basis for chemical risk management. We present information on the database expansion and quality control, chemoinformatics analysis and development of the resulting decision framework. We will also explore the utility of the new framework in the prioritization of safety testing for antimicrobial chemicals.

    TOXI 81

    Evidence of bioactivation of the anti-HIV drug etravirine to reactive metabolites in vitro and in vivo

    Ana L. Godinho1, Cristina C. Jacob1,2, Sofia A. Pereira3, M S. Marques1, matilde.marques@tecnico.ulisboa.pt, Alexandra Antunes1. (1) Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal (2) National Center for Toxicological Research, Jefferson, Arkansas, United States (3) NOVA Medical School, Faculdade de Ciências Médicas, Universidade NOVA de Lisboa, Lisboa, Portugal

    Etravirine (ETV) is a 2nd-generation Non-Nucleoside Reverse Transcriptase Inhibitor (NNRTI) used against HIV-1. Although ETV addition to the set of treatment choices aimed to overcome the most common adverse effects of 1st generation NNRTIs, post-marketing reports of severe ETV-induced skin rash and hypersensitivity reactions have compelled the U.S. FDA to issue a safety alert. Since patients who start ETV-based therapies are typically debilitated by a history of virus resistance and previous drug-induced side effects, administration of a potentially (geno)toxic drug must be considered carefully. The development of reliable prognostic tools for early risk/benefit estimations is therefore urgent.
    Currently, little is known about the mechanisms underlying ETV-induced toxic outcomes; nonetheless, drug bioactivation is anticipated as a major cause of these events. To search for potential bioactivation pathways we performed ETV incubations with rat and human S9 fractions in the presence of bionucleophiles, and analyzed urines of HIV patients on ETV therapy. Using liquid chromatography coupled to quadrupole-time-of-flight (Q-ToF) mass spectrometry we obtained evidence of multiple bioactivation pathways leading to the formation of covalent adducts with glutathione and N-acetyl-cysteine. These results suggest that similar reactions can occur with cysteine residues of proteins, supporting a role for bioactivation at the onset of the toxic effects elicited by ETV.

    TOXI 82

    Chemistry of independently generated thymidine radical cation: DNA hole transfer and other competing processes

    Huabing Sun, sunhuabing1985@hotmail.com, Marc M. Greenberg. Dept of Chemistry, Johns Hopkins University, Baltimore, Maryland, United States

    The pyrimidine/purine radical cations (“holes”) are important reactive intermediates resulting from the direct effect of ionizing radiation. Their transfer along DNA and other reactions, such as nucleophilic trapping are important DNA damage processes. To improve our understanding of the direct ionization of DNA, we developed the first photolabile nucleosides to independently generate the thymidine radical cation upon irradiation at 350 nm. Experiments were carried out on nucleosides and chemically synthesized oligonucleotides containing the precursors at defined sites. 18O-Labelling and trapping experiments involving nucleosides verified the radical cation formation and reaction pathways. We determined that deprotonation at the N3 position of thymidine radical cation (pKa 3.5) competes with nucleophilic trapping and other processes. Charge transfer was detected in DNA using a GGG sequence to localize the hole and measure transfer efficiency. Hole transfer efficiency is sequence-dependent. Independent generation of the thymidine radical cation facilitates additional examination of the reactivity of this important intermediate.

    TOXI 83

    Histone protein tails inhibit depurination of N7-methylated deoxyguanosine and form DNA-protein crosslinks with alkylated DNA in nucleosome core particles

    Kun Yang1, kyang37@jhu.edu, Marc M. Greenberg2. (1) Johns Hopkins University, Baltimore, Maryland, United States (2) Dept of Chemistry NCB RM 313, Johns Hopkins University, Baltimore, Maryland, United States

    Alkylating agents are commonly used as anticancer drugs. DNA methylation predominantly occurs at the N7 position of guanine (N7mdG). N7mdG hydrolyzes to yield an abasic site (AP) with a half life of ~102-190 h in naked DNA. However, N7mdG reactivity in nucleosome core particles (NCPs) has not been reported. We reconstituted NCPs containing N7mdG at specific positions. N7mdG depurination in NCPs is ~1.3-5.7 fold slower than in naked DNA. Larger decreases in depurination rates were observed when N7mdG is proximal to the histone tails. Experiments using mutated proteins indicate that the histone tails are responsible for the majority of the decreased depurination rates. Importantly, we discovered that the histone protein tails form DNA-protein crosslinks (DPCs) with N7mdG, and the yields of DPCs are comparable to the amounts of AP. To our knowledge, DPCs have never been reported from monoalkylation agents in chromatin. The formation of DPCs from N7mdG suggests an alternative mechanism for the cytotoxicity of monofunctional DNA alkylating agents.

    TOXI 84

    Degradation from C5′ oxidation and its adducts as potential biomarkers

    Shin H. Cho, shin.cho@rockets.utoledo.edu, Amanda C. Bryant-Friedrich. Medicinal and Biological Chemistry, University of Toledo, Toledo, Ohio, United States

    Endogenous exposome is any exposure from the internal environment within the body which can have the propensity for developing various diseases throughout lifetime. It includes metabolism of byproducts from mitochondria, infection, inflammation, oxidative stress from reactive oxygen species, and so on. Oxidative stress can have an impact on DNA, resulting in the strand breakage by the abstraction of hydrogen at various carbon centers in deoxyribose moiety. Among the 5 carbons in this moiety, the hydrogen at the 5′ carbon is more susceptible to abstraction compared to others. Nonetheless, the exact mechanism to understand how these are degraded are yet to be investigated. Here we present the investigation of oxidative damage products resulting from 5′-hydrogen abstraction. The goal of this project is to prove whether the degradation of the nucleoside modified at C5′ position can be used as potential biomarkers to monitor oxidative stress. Such task can be initiated by identifying the degradation products derived from C5′ oxidation of DNA. This involves the multi-step synthesis of C5′ modified nucleoside followed by incubation studies using analytical techniques such as HPLC. Similar studies will be conducted in oligonucleotides.

    TOXI 85

    Functional characterization of glutathione S-transferases by photoreactive and mechanism-based activity-based probes

    Ethan Stoddard, ethan.stoddard@pnnl.gov, Bryan Killinger, Reji N. Nair, Natalie Sadler, Jordan Smith, Richard Corley, Aaron T. Wright. Biological Sciences, Pacific Northwest National Laboratory, Richland, Washington, United States

    Glutathione S-transferases comprise a highly diverse family of phase II drug metabolizing enzymes whose shared function is the conjugation of reduced glutathione to various endo and xenobiotics. Although the total activity of these enzymes can be measured by colorimetric assays, measurement of the individual contribution from specific isoforms and their contribution to the detoxification of xenobiotics in complex biological samples has not been possible. For this reason, we have developed two activity based probes that characterize active glutathione transferases in mammalian tissues. The GST active site is comprised of a glutathione binding “G site” and a distinct substrate binding “H site”. Therefore, we developed (1) a glutathione-based ABP (GSH-ABP) hypothesized to show activity for the “G site” and (2) a probe designed to mimic a substrate molecule and show “H site” activity (GST-ABP). The GSH-ABP features a photoreactive moiety for UV-induced covalent binding to GSTs and glutathione-binding enzymes. Quantitative proteomic analysis of GSH-ABP enriched enzyme targets finds labeling of several GST isoforms, the most active being GSTA4, GSTM1, and GSTM2. The GST-ABP is a derivative of a known mechanism-based inhibitor of GSTs that binds cysteine(s) within the GST active site and inhibits GST activity. Quantitative proteomic analysis of GST-ABP enriched targets reveals selective targeting of GSTT2. Validation of both probes and the corresponding confident GST targets was carried out by competition experiments. We show the ability of both probes to characterize GSTs in several mammalian tissues. We also show the utility of the probes in detecting changes to GST activity incurred due to obesity.

    TOXI 86

    Using medaka embryos coupled with a whole sediment exposure strategy to assess copper bioavailability and toxicity in sediment

    Wei Li, r04623017@ntu.edu.tw, Pei-Jen Chen. Department of Agricultural Chemistry, National Taiwan University, Taipei, Taiwan

    Sediment pollution has been an important environmental issue for decades. When contaminants, especially heavy metals, enter aquatic environments such as rivers, lakes or ocean, most metals would settle down with water matrices (e.g., dissolved matters or suspended particles) into sediment. If the polluted sediment is disturbed or changed its chemical condition, the sediment heavy metals could be released to the water column again.
    Copper (Cu) is a commonly detected sediment contaminants, and its concentration can reach to hundreds to thousands mg/L in sediment. Aquatic organisms are very sensitive to Cu ion, which at μg/L level can cause lethal effect to some species. In this study, we aim to investigate the dissolution, mobility and bioavailability of different Cu-contaminated environmental sediments and their association to toxicity using a whole sediment exposure strategy with embryos of medaka fish (Oryzias latipes) as a model organism. The Cu ion in overlying and pore water and Cu speciation in sediment were quantified with filtration, Chelex 100 or sequential extraction, respectively. The correlation of Cu bioaccumulation in treated embryos with sediment properties and different Cu species was performed to understand bioavailability and subsequent bioaccumulation of Cu from sediment to embryos. Our results demonstrated that sediment with lower levels of pH values, cation exchange capacity or organic matter tended to release Cu ion in pore and overlaying water, thus causing higher copper bioavailability (e.g., copper accumulation in embryos) and toxicity (e.g., embryos mortality). Either dissolved Cu in overlaying water and pore water or Chelex 100 extractable Cu showed a good correlation with Cu bioaccumulation and embryos mortality. Our results demonstrated that the established embryos exposure system can successfully assess the copper bioavailability and toxicity from sediment exposures.

    TOXI 87

    Histones are targets for modification by the glycolytic by-product methylglyoxal

    James Galligan3, James.J.Galligan@Vanderbilt.edu, James A. Wepy4, Matthew Streeter2, Philip Kingsley3, Michelle M. Mitchener4, Orrette R. Wauchope5, William N. Beavers3, Kristie Rose1, Tina Wang2, David A. Spiegel2, Lawrence J. Marnett3. (1) Vanderbilt University, Nashville, Tennessee, United States (2) Dept of Chemistry, Yale University, New Haven, Connecticut, United States (3) Biochemistry, Vanderbilt University, Nashville, Tennessee, United States (4) Chemistry, Vanderbilt University, Nashville, Tennessee, United States

    Histone post-translational modifications (PTMs) regulate chromatin dynamics, DNA accessibility, and transcription to expand the genetic code. Many of these PTMs are produced through cellular metabolism to offer both feedback and feedforward regulation. This is perhaps most evident with glycolysis, which is one of the most tightly controlled cellular processes. Despite tight glycolytic regulation, secondary intermediates are still produced, often with deleterious consequences on cellular homeostasis. In an effort to identify novel histone PTMs derived from glycolytic intermediates, we used mass spectrometry (MS) and developed an untargeted data-dependent acquisition method to quantify Lys and Arg PTMs. Using SILAC cell lines treated with either low glucose (5mM) or high glucose (25mM), we identify both Lys and Arg modifications stemming from the glycolytic by-product methylglyoxal (MGO). This α-oxoaldehyde is produced in μM quantities in diabetic patients and is capable of covalently adducting proteins (Lys and Arg) and DNA. Using an LC-MS/MS method developed by our laboratory (QuARKMod), we show that the levels of MGO-derived Arg adducts are on the order of other canonical histone PTMs (e.g. methylation). Further, using a CRISPR-Cas9 knockout of the major MGO detoxification enzyme, glyoxalase 1 (GLO1), we show substantial increases in both soluble MGO as well as MGO-derived Arg and Lys adducts on both chromatin and isolated histones. To identify sites of modification, high-resolution mass spectrometry (MS) was utilized, revealing MGO-derived Lys and Arg adducts on all four-core histones. We hypothesize that this adduction may offer a novel feedback mechanism for bouts of hyperglycemia. The mechanisms by which these adducts may alter gene expression is currently under investigation.

    TOXI 88

    Wide selected ion monitoring (SIM)/MS2 data independent acquisition method for DNA adduct omics analysis

    Jingshu Guo1,2, guoj@umn.edu, Peter W. Villalta1, Robert J. Turesky1,2. (1) Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, United States (2) Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota, United States

    Long-term exposures to environmental toxicants and endogenous electrophiles are causative factors for human diseases including cancer. The formation of DNA adducts reflects the internal exposure to these chemicals and they can serve as biomarkers for risk assessment. Liquid chromatography-multistage mass spectrometry (LC-MSn) is the most common method for biomonitoring DNA adducts of carcinogens. This “targeted” approach is often used to biomonitor a single exposure and a single adduct, and generally offers limited evidence for a role of a chemical in cancer risk. In the era of the “exposome”, an “untargeted” method that simultaneously detects many DNA adducts present in the genome provides an “adductomics” signature reflecting global chemical exposures that can lead to mutations and cancer. We have developed a Wide-SIM-MS2 screening method utilizing ultra-performance LC nanoelectrospray ionization high resolution Orbitrap MSn with on-line enrichment of hydrophobic DNA adducts, where ten consecutive SIM scan windows of 30 m/z were performed to detect the precursors of modified nucleosides. After each SIM scan event, the same mass range is subjected to MS/MS fragmentation. The SIM and MS/MS data are analyzed for the presence of co-eluting chromatographic peaks with masses differing by -116.0473 Da, the mass of the neutral loss of deoxyribose. We have compared our method with published adductomics methods using multiple reaction monitoring via triple quadrupole MS, and by constant neutral loss triggering MS3 by ion trap MS. Wide-SIM-MS2 is superior in sensitivity, specificity, and breadth of adduct coverage to the other methods. It can be used in a “targeted” manner by extracting adducts of interest, or in an “untargeted” fashion where a chromatographic peak-picking algorithm is applied to retrieve putative DNA adducts. Wide-SIM-MS2 was successfully used to detect DNA adducts derived from the diet, environment and endogenous electrophiles in human prostate and bladder specimens.

    TOXI 89

    Investigation of environmental fate and toxic mechanisms of monovalent and trivalent thallium

    Ching-Hsin Yang, b00603039@ntu.edu.tw, Pei-Jen Chen. Department of Agricultural Chemistry, National Taiwan University, Taipei, Taiwan

    Thallium (Tl) is a trace element with high toxicity to mammals. It belongs to a category of technology-critical elements that are key components for the new or development technologies. The anthropic processes of Tl entering the aquatic environment include mining, coal combustion and cement production that release significant amount of Tl. Tl is a priority pollutant suggested by the U.S. EPA and its environmental fate and hazardous risk to aquatic life and human health is gaining serious attention recently. Tl exists in the environment as monovalent Tl(I) and trivalent Tl(III) states. Tl(I) is thought to be more thermodynamically stable than Tl(III); however, recent studies indicate that Tl(III) may exist in surface water, and seems much more toxic than Tl(I) to aquatic invertebrates. However, environmental transformation between Tl(I) and Tl(II) and their toxicity to higher tropic levels of aquatic organisms such as fish remain unclear. In this study, we aim to understand the stability of Tl(I) and Tl(III) in water under environmental relevant conditions. The toxic effect of different Tl species and associated modes of toxic action are investigated using medaka fish (Oryzias latipes) as a model organism. Our results showed that Tl(I) appeared to induce higher acute lethality to medaka larvae than Tl(III) at the tested concentration ranges and water conditions. To reveal toxic mechanism of Tl, we compared the transcriptome in medaka larvae from Tl(I)-treated and control groups using next generation sequencing (NGS). Differential gene expression (DGE) profiles showed that Tl(I) significantly induced alteration in cholesterol synthesis, steroid hormone metabolism and phase I metabolism related genes. These Tl-induced transcriptional alteration was confirmed by qPCR analysis and related enzymatic assays. The correlation between Ti speciation and toxicity in water is under further investigation. Based on our results, we imply that Tl has significant ecotoxicological impact to the aquatic ecosystem.

    TOXI 90

    Biological uptake, distribution and depuration of radio-labeled graphene in adult zebrafish

    Liang Mao, lmaouga@gmail.com. School of the Environment, Nanjing University, Nanjing Jiangsu, China

    The exciting commercial application potential of graphene materials may inevitably lead to their increasing release into the environment where they may pose ecological risks. This study focused on using carbon-14 labeled few-layer graphene (FLG) to determine whether the size of graphene plays a role in its uptake, depuration and biodistribution in adult zebrafish. After 48 h exposure to larger FLG (L-FLG) at 250 μg/L, the amount of graphene in the organism was close to 48 mg/kg fish dry mass, which was more than 170-fold greater than the body burden of those exposed to the same concentration of smaller FLG (S-FLG). The amount of uptake for both L-FLG and S-FLG increased by a factor of 2.5 and 16, respectively, when natural organic matter (NOM) was added in the exposure suspension. While the L-FLG mainly accumulated in the gut of adult zebrafish, the S-FLG was found in both the gut and liver after exposure with or without NOM. Strikingly, the S-FLG was able to pass through the intestinal wall and enter the intestinal epithelial cells and blood. The presence of NOM increased the quantity of S-FLG in these cells. Exposure to L-FLG or S-FLG also had a significantly different impact on the intestinal microbial community structure.

    TOXI 91

    Bringing it all together: A web-based database for chemical and biological data to support environmental toxicology

    Antony J. Williams, tony27587@gmail.com, Christopher Grulke, Jennifer Smith, Sean Watford, Rebecca Jolley, Jeremy Dunne, Elizabeth Edmiston, Jeff Edwards. National Center for Computational Toxicology, Environmental Protection Agency, Wake Forest, North Carolina, United States

    The EPA Comptox Chemsitry Dashboard is a web-based application providing access to a set of data resources provided by the National Center of Computational Toxicology. Sitting on a foundation of chemistry data for ~750,000 chemical substances the application integrates bioassay screening results, physicochemical and toxicological endpoints (both experimental and predicted) and consumer product and functional use data. Integrating to a series of other EPA and public databases (e.g. ECOTOX, the EPA Substance Registry Service and EPA’s ChemView), the dashboard is a hub for connecting to other releases of value to environmental scientists and toxicologists. The underpinning architecture has been developed in a manner allowing for the release of the dashboard as a public resource as well as for delivering an application to be used inside EPA’s confidential business information environment where the application is being assessed for potential applications to support risk assessment. This presentation will provide an overview of the dashboard focusing specifically on the most recent functionality supporting deeper integration to the ToxCast bioassay data and exposure data and predictions available via the dashboard. This abstract does not reflect U.S. EPA policy.

    TOXI 92

    Understanding hepatoxicity: Man to mouse to computer

    Paul B. Watkins, pwatkins@email.unc.edu. School of Pharmacy, Medicine and Public Health, University of North Carolina, Chapel Hill, North Carolina, United States

    Drug Induced Liver Injury (DILI) remains a major adverse drug event that leads to termination of clinical development programs, or regulatory actions including failure to approve for marketing, restricted indications, and withdrawal from the marketplace. Current preclinical models, even “humanized” ones, do not reliably identify molecules that cause DILI in man and conversely, current models can predict liver safety liabilities for molecules that are in fact safe for patients. The University of North Carolina Institute for Drug Safety Sciences (IDSS) has built research programs that are designed to bridge the gap in knowledge between preclinical models and patients. These approaches include bioprinted, organotypic liver cultures and use of the Collaborative Cross strains of mice to identify pathways that underlie DILI susceptibility. In addition, the Institute founded the DILIsim Initiative (http://www.dilisym.com) which is a major public-private partnership that has involved scientists from 16 major pharmaceutical companies, academia, and the FDA. This initiative has developed an in silico model incorporating mathematical equations that simulate the interacting pathways that underlie patient susceptibility to DILI. The mechanistic insights gained by these integrated approaches generate hypotheses regarding genetic or serum biomarkers that have been successfully tested in tissue banks obtained from clinical trials or from the Drug-Induced Liver Injury Network (DILIN). Although the past focus of IDSS research has been on legacy drugs that have significant liver safety liabilities, we are now utilizing our approaches to improve DILI risk identification in new drug candidates and to identify personalized risk management strategies to “save” some drugs currently in development.

    TOXI 93

    Chemistry and biology of N5-alkyl-fapy-dG damage in DNA

    Michael P. Stone2, michael.p.stone@vanderbilt.edu, Martin Egli1, R. S. Lloyd3, Amanda Mc Cullough3, Carmelo Rizzo4, Robert J. Turesky5. (1) Biochemistry, Vanderbilt University, Nashville, Tennessee, United States (2) Chemistry, Vanderbilt University, Nashville, Tennessee, United States (3) Oregon Institute of Occupational Health Sciences, Oregon Health & Science University, Portland, Oregon, United States (5) Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota, United States

    Formamidopyrimidine (Fapy-dG) DNA lesions arise following exposures to a variety of genotoxins. These are rearrangement products formed subsequent to N7 alkylation of dG; their chemical biology is complex. Fapy-dG lesions equilibrate between α and β anomers, and undergo synanti rotation about the glycosyl bond. Also, there is potential for E-Z geometrical isomers about the formyl bond and atropisomers about the C5N5 bond. Recent results on a series of Fapy-dG lesions will be discussed. We have utilized site-specific incorporation of NMR-active isotope-labels to follow interconversions of Fapy-dG lesions in situ. Fapy-dG adducts are often substrates for base excision repair (BER). Recent results on BER of AFB1-Fapy-dG lesions will be presented. If not repaired, error-prone bypass of Fapy-dG lesions in primate cells reveals predominantly G to T transversions. Studies in vitro reveal the mutation spectra differ depending on the identity of polymerase and local sequence. DNA pol ζ incorporates dATP opposite AFB1-Fapy-dG and extends from this mismatch. Upon challenge with AFB1, survival of murine cells deficient in pol ζ (Rev3L-/-) was reduced relative to Rev3L+/- cells or Rev3L-/- cells complemented through expression of the wild-type human REV3L. Following AFB1 exposure cell-cycle progression of Rev3L-/- mouse embryo fibroblasts (MEFs) was arrested in late S/G2 phase. These cells showed increases in replication-dependent formation of γ-H2AX foci, micronuclei, and chromosomal aberrations (chromatid breaks and radials) relative to Rev3L+/- cells. Thus, pol ζ seems essential for processing AFB1-induced Fapy-dG damage. Using crystallographic approaches, we have determined active-site configurations of ternary insertion-step complexes on DNA template:primers modified site-specifically with N5-alkyl-Fapy-dG lesions.

    TOXI 94

    Aldehydes increase the tumorigenic properties of tobacco specific nitrosamines in rodent tumor models

    Lisa A. Peterson1, peter431@umn.edu, Marissa K. Oram1, Monica Flavin1, Donna Seabloom2, William E. Smith1, Ingrid Cornax1, M. Gerard O’Sullivan1, Pramod Upadhyaya1, Lin Zhang3, Stephen S. Hecht1, Silvia Balbo1, Timothy S. Wiedmann2. (1) Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, United States (1) Mayo Mail Code 806, U of MN Masonic Cancer Center, Minneapolis, Minnesota, United States (2) College of Pharmacy, University of Minnesota, Minneapolis, Minnesota, United States (3) School of Public Health, University of Minnesota, Minneapolis, Minnesota, United States

    Tobacco smoke is a complex mixture of toxic and carcinogenic chemicals. Toxicological studies have focused primarily on either single chemical exposures or the more complex mixture of tobacco smoke or its fractions. Fewer studies investigate the interaction between specific tobacco chemicals. Aldehydes in tobacco smoke may enhance the carcinogenic properties of 4-methylnitrosamino-1-(3-pyridyl)-1-butanone (NNK) or N-nitrosonornicotine (NNN) through a variety of mechanisms. This hypothesis was tested in two established rodent tumor models, the NNK-induced A/J mouse lung tumor model and the NNN-induced rat esophageal tumor model. In the first model, A/J mice were exposed to NNK (i.p, 0, 2.5, or 7.5 µmol in saline) in the presence or absence of acetaldehyde (0 or 360 ppmv) or formaldehyde (0 or 15 ppmv) for 3 hours in a nose-only inhalation chamber. Lung tumors were counted 16 weeks later. Neither aldehyde by itself induced lung tumors. However, mice receiving both NNK and acetaldehyde or formaldehyde had an increased number of adenomas with dysplasia or progression than those receiving only NNK, suggesting that aldehydes may increase dysplasia in tumors initiated by NNK. The aldehyde co-exposure did not affect the levels of NNK-derived methyl DNA adduct levels post-exposure. In the second model, rats were exposed to 4 or 8 ppm NNN in the drinking water in the presence or absence of 3000 ppm acetaldehyde for up to 100 weeks. The number of esophageal papillomas per rat were doubled in animals receiving both acetaldehyde and 8 ppm NNN (0.5 versus 1.15 tumors/rat, respectively). Acetaldehyde alone did not cause esophageal tumors. DNA adduct levels will be measured to determine if acetaldehyde co-exposure alters the formation and/or repair of NNN-derived DNA damage. These studies support the hypothesis that the aldehydes present in the tobacco mixture interact to enhance the carcinogenic potency of the tobacco specific nitrosamines.

    TOXI 95

    Unwinding kinetics of carcinogenic adducts: Correlation with processing by nucleotide excision repair machinery

    Vladimir Shafirovich1, vs5@nyu.edu, Axel Y. Epie1, Vincent Zheng1, Marina Kolbanovskiy1, Nicholas E. Geacintov2. (1) New York University, New York, New York, United States (2) chemistry, New York University, Mountainside, New Jersey, United States

    Helicases are molecular motor proteins that unwind the DNA double helix by an ATP-driven mechanism. The transcription initiation and nucleotide excision repair (NER) factors (TFIIH) contain two DNA helicases, XPB and XPD. The latter have opposite polarities and extend the open and strand-separated DNA configuration around the lesion site that plays a crucial role in damage verification during the nucleotide excision repair (NER) process. We were therefore interested in elucidating the efficiencies of unwinding of forked DNA substrates that harbor a series of related carcinogen-DNA adducts that are either excellent substrates of NER, or are resistant to NER. The adducts were derived from the binding of the bay region benzo[a]pyrene-, or dibenzo[a,l]pyrene-diol epoxides to the exocyclic amino groups of adenine or guanine in the double-stranded region of the forked DNA. The effects of these different DNA adducts on the rates of unwinding catalyzed by the recombinant E. coli RecQ DNA helicase were determined. The relative rate constants of DNA unwinding were measured in real time by a fluorescence method. The key observations are that the rates of unwinding are highly sensitive to the adduct stereochemistry and were correlated with the efficiency of their repair by NER pathways in extracts from human cells.

    TOXI 96

    Structural insights into the post-chemistry steps of nucleotide incorporation catalyzed by a DNA polymerase

    Zucai Suo, suo.3@osu.edu. Dept of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio, United States

    DNA polymerases are essential enzymes that faithfully and efficiently replicate genomic information.1−3 The mechanism of nucleotide incorporation by DNA polymerases has been extensively studied structurally and kinetically, but several key steps following phosphodiester bond formation remain structurally uncharacterized due to utilization of natural nucleotides. It is thought that the release of pyrophosphate (PPi ) triggers reverse conformational changes in a polymerase in order to complete a full catalytic cycle as well as prepare for DNA translocation and subsequent incorporation events. Here, by using the triphosphates of chain-terminating antiviral drugs lamivudine ((-)3TC-TP) and emtricitabine ((-)FTC-TP), we structurally reveal the correct sequence of post-chemistry steps during nucleotide incorporation by human DNA polymerase β (hPolβ) and provide a structural basis for PPi release. These post-catalytic structures reveal hPolβ in an open conformation with PPi bound in the active site, thereby strongly suggesting that the reverse conformational changes occur prior to PPi release. The results also help to refine the role of the newly discovered third divalent metal ion for DNA polymerase-catalyzed nucleotide incorporation. Furthermore, a post-chemistry structure of hPolβ in the open conformation, following incorporation of (-)3TCMP, with a second (-)3TC-TP molecule bound to the active site in the absence of PPi , suggests that nucleotide binding stimulates PPi dissociation and occurs before polymerase translocation. Our structural characterization defines the order of the elusive post-chemistry steps in the canonical mechanism of a DNA polymerase.

    TOXI 97

    Central role of PCNA in promoting replication of damaged DNA

    George-Lucian Moldovan, gmoldovan@pennstatehealth.psu.edu. Penn State College of Medicine, Hershey, Pennsylvania, United States

    Understanding how cells orchestrate the response to DNA damage during S-phase is critical. DNA damage can stall the replication machinery and delay the cellular replication program. The restart of stalled replication forks is mediated by the replication protein PCNA, a ring-shaped molecule that encircles DNA and acts as a processivity factor for DNA polymerases. Post-translational modification of PCNA with ubiquitin at stalled replication forks has been proposed to promote Translesion Synthesis-mediated lesion bypass. In order to understand these events at the molecular level, we generated PCNA ubiquitination-deficient cell lines using CRISPR/Cas9 technology. Our studies identified an essential role of PCNA ubiquitination for DNA replication and replication-associated mutagenesis. Moreover, we identified the ubiquitin ligase HUWE1 as a novel mediator of PCNA-dependent fork restart.

    TOXI 98

    Lucidin-dervied N2-guanine DNA lesion is not a major contributor to the mutagenicity of lucidin

    Linlin Zhao, fredlinzhao@gmail.com. Department of Chemistry and Biochemistry, Central Michigan University, Mount Pleasant, Michigan, United States

    Lucidin-3-O-primeveroside (LuP) is an anthraquinone derivative present in madder root, which is used as colorant. LuP can be metabolically converted to genotoxic compound lucidin, and subsequently form adenine and guanine DNA lesions. In rats, lucidin generates G to T and A to T transversions and A to G transitions. However, questions remain regarding how lucidin-derived DNA adducts correlate to these mutations. In this work, a structural analogue of lucidin-derived 2′-deoxyguanosine DNA adduct, N2-methyl-(1,3-dimethoxyanthraquinone)-deoxyguanosine (LdG), is used to understand the mutagenic mechanisms of the lucidin-induced DNA damage during translesion DNA synthesis (TLS), a major source of DNA damage-induced mutations. Nucleotide incorporation efficiency and fidelity were determined for several relevant human recombinant TLS DNA polymerases during LdG DNA damage bypass. Observed from enzyme kinetic analysis and LC-MS-based bypass product sequencing, DNA polymerase κ is highly efficient and accurate in replicating past the LdG adduct, which is consistent with the role of DNA polymerase κ in bypassing N2-deoxyguanosine adducts. Other three DNA polymerases were error-prone and inefficient in bypassing LdG adduct. X-ray crystal structures of DNA polymerase κ with LdG-containing duplex and an incoming nucleotide revealed the structural mechanism of the error-free bypass. Together, our results suggest that lucidin-dervied N2-guanine DNA lesion is not a major contributor to the mutagenicity of lucidin in DNA polymerase κ proficient cells.

    TOXI 99

    Spore photoproduct within DNA is a surprisingly poor substrate for its designated repair enzyme: The spore photoproduct lyase

    Lei Li1,3, lilei@iupui.edu, Linlin Yang1, Setlow Peter2. (1) Department of Chemistry & Chemical Biology, Indiana University Purdue University Indianapolis, Indianapolis, Indiana, United States (2) Department of Molecular Biology and Biophysics, UConn Health, Farmington, Connecticut, United States (3) Department of Dermatology, Indiana University School of Medicine, Indianapolis, Indiana, United States

    UV radiation of DNA generates three major types of pyrimidine dimers, cyclobutane pyrimidine dimers (CPDs), pyrimidine (6-4) pyrimidone photoproducts (6-4PPs) and 5-thyminyl-5,6-dihydrothymine, i.e. the spore photoproduct (SP). All these dimers can be repaired by direct reversal enzymes where CPDs and 6-4PPs are repaired by the respective photolyase and SP is repaired by spore photoproduct lyase (SPL). The CPD photolyase was shown to exhibit ~ 100,000-fold higher affinity toward CPD in CPD-containing DNA than undamaged DNA, explaining the highly efficient CPD repair in duplex DNA by the enzyme. It was widely expected that SPL may exhibit a similarly high affinity toward SP in duplex DNA leading to an efficient SP repair. Surprisingly, our latest studies using SP-containing short oligonucleotides, pUC 18 plasmid DNA, and E. coli genomic DNA found that they are all poor substrates for SPL in general, exhibiting turnover numbers of 0.01-0.2 min-1. The faster turnover numbers are reached under single turnover conditions, and SPL activity is lower with oligonucleotide substrates at higher concentrations. Moreover, SP-containing oligonucleotides do not go past one turnover. In contrast, the dinucleotide SP TpT exhibits a turnover number of 0.3 ~ 0.4 min-1, and the reaction may reach up to 10 turnovers. These observations distinguish SPL from DNA photolyases. To the best of our knowledge, SPL represents an unprecedented example of a major DNA repair enzyme that cannot effectively repair its substrate lesion within the normal DNA conformation adopted in growing cells. Factors such as other DNA binding proteins may have to cooperate with SPL to enable the efficient SP repair in germinating spores. Moreover, if SP is produced in growing cells, the SP repair may be slow and potentially problematic.

    TOXI 100

    Real-time prediction of physicochemical and toxicological endpoints using the web-based CompTox Chemistry Dashboard

    Antony J. Williams1, tony27587@gmail.com, Todd Martin3, Valery Tkachenko2, Christopher Grulke1, Kamel Mansouri4. (1) National Center for Computational Toxicology, Environmental Protection Agency, Wake Forest, North Carolina, United States (2) SCIENCE DATA SOFTWARE, LLC, Rockville, Maryland, United States (3) US EPA, Cincinnati, Ohio, United States (4) National Center for Computational toxicology, US EPA / ORISE, Research Triangle Park, North Carolina, United States

    The EPA CompTox Chemistry Dashboard developed by the National Center for Computational Toxicology (NCCT) provides access to data for ~750,000 chemical substances. The data include experimental and predicted data for physicochemical, environmental fate and transport and toxicological endpoints. Predicted data have been harvested from open data sources as well as generated using available online web services (for example EPI Suite), our EPA-NCCT OPERA models and using the EPA Toxicity Estimation Software Tool (T.E.S.T). Both OPERA and T.E.S.T utilize QSAR (Quantitative Structure Activity Relationship) models based on 2D molecular descriptors. While this offers access to a rich aggregation of data these are limited to pre-predicted data and limited to the chemicals included on the dashboard. This presentation will provide an overview of our efforts to provide real-time prediction (for single chemicals or chemical sets), using publicly available web services and our own open prediction models from the EPA. This will provide the community access to predictions for physicochemical, fate and transport and toxicological endpoints that can be used for the purpose of hazard, risk and exposure assessment and to support read -across applications. This abstract does not reflect U.S. EPA policy.

    TOXI 101

    Reaction of the antiepileptic drug carbamazepine with bionucleophiles: Bioactivation is not required

    Inês L. Martins, João P. Telo, M S. Marques, matilde.marques@tecnico.ulisboa.pt, Alexandra Antunes. Centro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, Lisboa, Portugal

    Carbamazepine (CBZ, 1) is an antiepileptic drug used in the long-term treatment of epilepsy, and also in the management of chronic pain, bipolar disorders, and schizophrenia. Despite its widespread use, CBZ is associated with central nervous system toxicity and severe hypersensitivity reactions, which raises concerns about its chronic administration. While the precise mechanisms of CBZ-induced toxic events are still unclear, the metabolic activation to its major metabolite, carbamazepine-10,11-epoxide (CBZE), has been thought to play a significant role in the toxic responses elicited by the drug.
    We report herein evidence of direct covalent binding between CBZ and sulphur-based bionucleophiles (eg. N-acetyl-L-cysteine, glutathione) yielding multiple products, namely the same adducts obtained upon ring-opening of CBZE. Interestingly, when compared with CBZE, CBZ reacts faster and more efficiently, particularly in the presence of oxygen. These results suggest that the bioavalibility of CBZ may be compromised under hypoxic conditions. Furthermore, the observed reaction with sulphur bionucleophiles suggests that similar reactions with cysteine residues of proteins can occur. These data support a direct role for CBZ, without the need for bioactivation, at the onset of the toxic events elicited by this antiepileptic drug.

    Direct reaction of CBZ with N-acetyl-L-cysteine (NAC).

    TOXI 102

    Programed release of nitric oxide, via oxidative metabolism, in animals and humans from clinical candidate MK-8150

    Kaushik Mitra, kaushik_mitra@merck.com. Merck and Co., Inc., Kenilworth, New Jersey, United States

    A novel genre of diazoniumdiolate molecules, characterized by controlled release of nitric oxide (NO), was developed for the treatment of hypertension. This presentation will describe the in vitro and in vivo metabolism studies that enabled the build of the unique biotransformation-triggered time-delayed mechanism of NO release from these diazoniumdiolate molecules, culminating in the identification of MK-8150 as a clinical candidate. Additionally, the presentation will discuss the non-traditional PK/PD approach used to translate preclinical data to humans and the resulting outcome from human studies with MK-8150.

    TOXI 103

    Can pipe tobaccos be characterized for regulatory purposes without a puff of pipe smoke?

    John H. Lauterbach, john@lauterbachandassociates.com. Lauterbach Associates, LLC, Macon, Georgia, United States

    In May 2016, the US FDA issued the so-called “Deeming Regs” to include tobacco products other than cigarettes, roll-your-own (RYO) and make-your own (MYO) tobaccos, moist/dry snuff, snus, and chewing tobacco, which were included in 2009 legislation. Thus, pipe tobacco is subject to regulations that could, when issued for pipes and pipe tobacco, require the same types of testing as is required for cigarette RYO/MYO. However, the protocols for smoking pipes and selecting pipes to be used in regulatory testing are not as established as are the protocols for making and smoking tobaccos with smoking articles fabricated with RYO and MYO components where ISO standards exist. Moreover, pipe smokers are a very small percentage of US tobacco users and risks of adverse health effects from exclusive pipe smoking are reported to be less than for cigarette smoking. Furthermore, there are numerous brand-styles of pipe tobacco on the US market, some of which are made by small manufacturers, which cannot afford expensive testing requirements and remain in business. If numerous brand-styles had to be tested in pipe smoke for the FDA list of harmful and potentially harmful constituents (HPHC), it would take at least several years to develop such smoking protocols, especially if more than one “standard” pipe had to be used and evaluate them with a wide variety of pipe tobaccos. Consequently, a better approach is needed. This approach is to focus only on the pipe tobaccos. Moreover, this approach uses only simple AutoAnalyzer-type chemical determinations and simple LC methods to distinguish among brand-styles of pipe tobacco, and more importantly, distinguishing between expensive traditional products that are generally only available from tobacconists (or their Internet stores) and much lower cost products that appeal to a wider range of pipe smokers, especially beginning ones. Moreover, the value of chromatographic similarity indices obtained from routine LC data in terms of differentiating closely related brand-styles will be shown. Furthermore, techniques for differentiating “real” pipe tobacco from “faux” pipe tobacco (used for RYO/MYO) will be described.

    TOXI 104

    Advances in mass spectrometry techniques for metabolism, pharmacology and toxicology

    Jonathan Josephs, jonathan.josephs@thermofisher.com. Chromatography & Mass Spectrometry Division, ThermoFisher Scientific, San Jose, California, United States

    The advent of electrospray ionization heralded a new era in mass spectrometry in the early 90’s. Liquid chromatography-tandem mass spectrometry transformed the sciences of metabolism, pharmacology and toxicology. The adoption of high resolution accurate mass (HRAM) instrumentation over the last decade has resulted in another surge in analytical capabilities allowing for continued advances in our understanding of biological systems and disease states.
    Orbitrap technology has provided researchers with instrumentation that allows extreme resolution and mass accuracy in instrumentation that is robust, highly flexible and easy to use. The application of HRAM in combination with tandem mass spectrometry and sophisticated data dependent acquisition techniques supports a diverse range of applications that allow researchers to probe biological systems with great sensitivity, specificity and speed. Quantitative proteomic techniques are used to identify processes that are up and down regulated in an unbiased manner to determine protein biomarkers. Metabolomics studies can be used to monitor disease states and identify on target and off target toxicities. Highly sensitive biomarker assays may be developed to identify disease and monitor the efficacy of drugs to treat disease states. HRAM techniques are used at all stages of drug discovery and development to monitor target engagement, optimize drug candidates, determine PK/PD relationships and identify toxicities.
    Recent examples of how the latest HRAM technologies are applied to drug and biomarker quantitation, structural elucidation, ADME studies, toxicology and translational medicine will be demonstrated.

    TOXI 105

    Capillary electrophoresis for trace-level detection: Metabolites and proteins

    Peter Nemes2, petern@email.gwu.edu, Rosemary M. Onjiko2, Camille Lombard-Banek1. (1) Chemistry, George Washington University, Washington, District of Columbia, United States (2) Chemistry, The George Washington University, Washington, District of Columbia, United States

    Detection of trace amounts of molecules empowers the molecular characterization of a systems’ state during health and stress, such as exposure to toxins or drug molecules. Liquid chromatography (LC) mass spectrometry (MS) is the technology of choice for the measurement of metabolites and proteins from large amounts of starting materials. Here, we describe a custom-designed microanalytical capillary electrophoresis (CE) interface for electrospray ionization (ESI) tandem MS to also enable metabolic and proteomic characterization in trace amounts of samples. Our microanalytical CE-ESI-MS platform is compatible with 1–100 nL sample volume and is able to detect low attomoles of metabolite or hundreds of zeptomoles of protein standards, providing sufficient sensitivity to detect endogenous molecules in biological systems. As an example, we demonstrate the utility of the platform for characterizing the metabolic and proteomic activity state of identified single cells in the vertebrate embryo of the South African clawed frog (Xenopus laevis), which is a powerful cell/developmental biological model and has been used in toxicological studies. We detected hundreds of metabolite signals and ~2,000 protein groups in cells that are fated to give rise to nervous, epidermal, and hindgut tissues in the embryo. Furthermore, CE-ESI-MS was found quantitative over 3–5 log-order dynamic range, raising a potential to also quantify a system’s molecular activity state. As a proof-of-principle experiment, we manually dissected and capillary-sampled identified cells in the embryo and quantified the abundance of glutathione (GSH) and oxidized glutathione (GSSG). Dissected cells were found to yield significantly lower GSH/GSSG ratios, which suggests that these cells were exposed to higher oxidative stress. With label-free and ultrasensitive detection, CE-ESI-MS presents attractive alternative to traditional LC-MS in toxicological studies.

    TOXI 106

    Exposing the exposome: Utilizing global metabolomics to characterize toxicant exposure and effect

    Benedikt Warth1,2, benedikt.warth@univie.ac.at. (1) Center for Metabolomics, The Scripps Research Institute, La Jolla, California, United States (2) Department of Food Chemistry and Toxicology, University of Vienna, Vienna, Austria

    Chemical exposures can play a pivotal role in the etiology of human disease and it is estimated that hundreds of thousands of environmental chemicals may be linked to a large share of chronic disease. However, little is known about the complex interplay between environmental exposures, genetic susceptibility and potential adverse health outcomes. While we are constantly facing a mixture of exposures through the environment, diet, and endogenous processes, exposure and risk assessment is typically still based on the evaluation of single compounds. To address this challenge, high-resolution mass spectrometry (HR-MS) based metabolomics has been proposed as a future key technology.

    In this contribution, an optimized untargeted metabolomics workflow is presented which provides a solution to the two key tasks in current environmental health research of (1) assessing multiple exposures simultaneously and (2) evaluating their biological/toxicological effect. Employing HR-MS and the XCMS/METLIN data processing platform, thousands of metabolic features originating from endogenous metabolites, toxicants, other xenobiotics and their biotransformation products can now be assessed simultaneously using one analytical technique. The applicability of this workflow was demonstrated in human biofluids and a cell-based model. More specifically, xenoestrogens, which are suspected to may cause endocrine disruption, have been detected at low nanomolar concentrations. Moreover, their effect on cellular metabolism was evaluated using metabolic pathway analysis. This workflow enables, for the first time, the combination of holistic, untargeted exposure assessment with changes in endogenous metabolism for deciphering specific pathways affected by individual or mixtures of toxicants.

    The presented novel technology has many potential applications in the areas of toxicology, public health and beyond and may influence how we address the challenges in the new era of exposomics.

    TOXI 107

    Probing stress-induced effects on RNA and posttranscriptional modifications by LC-MS

    Balasubrahmanyam Addepalli, balasual@ucmail.uc.edu, Congliang Sun, Patrick Limbach. Chemistry, University of Cincinnati, Cincinnati, Ohio, United States

    Identification of damage inflicted on cellular biomolecules is the first step in understanding and facilitating the cellular stress tolerance responses. Although stress induced damage to proteins and DNA is well documented, information about RNA damage is largely extrapolated from studies on DNA. Our initial goal is to document the profile of UVR-induced RNA photoproducts and understand their characteristic production by LC-MS analysis, so that a library of RNA-photoproducts can be developed. Such a library will be helpful for conducting the comparative analysis of differential generation of RNA photoproducts especially when RNA is exposed to UVR with altered genetic background. Systematic studies indicating the appearance of significant amounts of RNA photoproducts, and loss of a specific class of modified nucleosides upon UVR exposure to RNA outside the cell will be presented. These changes will be compared against those observed in RNA under intracellular conditions.

    TOXI 108

    Advances in human biomonitoring of heterocyclic aromatic amines by high resolution accurate mass spectrometry

    Jingshu Guo2,5, guoj@umn.edu, Shun Xiao4,5, Yi Wang5,3, Byeong Hwa Yun2,5, Paari Murugan6, Christopher J. Weight7, Kami K. White8, Lynne R. Wilkens8, Loic Le Marchand8, Karen Dingley9, Michael A. Malfatti9, Kenneth Turteltaub9, Peter W. Villalta2, Robert J. Turesky2,1. (1) Department of Medicinal Chemistry and Masonic Cancer Center, Minneapolis, Minnesota, United States (2) Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota, United States (3) Medicinal Chemistry, Medical Science Building, University of Florida, Gainesville, Florida, United States (4) Masonic Cancer Center, University of Minnesota, Hangzhou, China (5) Medicinal Chemistry, University of Minnesota, Minneapolis, Minnesota, United States (6) Laboratory Medicine , University of Minnesota, Minneapolis, Minnesota, United States (7) Urology, University of Minnesota, Minneapolis, Minnesota, United States (8) Cancer Center, University of Hawaii, Honolulu, Hawaii, United States (9) Biosciences and Biotechnology Division, Center for Accelerator Mass Spectrometry, Lawrence Livermore National Laboratory, Livermore, California, United States

    Heterocyclic aromatic amines (HAAs) formed in tobacco smoke or cooked meat have been linked to human cancers. We studied the biotransformation of these carcinogens, and characterized DNA and protein adducts with low resolution mass spectrometry (MS), such as triple quadrupole (TQ) or ion trap (IT) MS. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is an abundant HAA formed in cooked meat. PhIP is a rodent prostate and possible human carcinogen. We have shown that TQ-MS can be employed to quantify PhIP in human hair and urine, and that IT multistage-MS (MSn) can measure DNA-PhIP adducts in animals and serum albumin (SA)-PhIP adducts ex vivo. However, our attempts to migrate such analytical methods to human samples were often unsuccessful because of the insufficient selectivity of the instruments. For example, the detection of PhIP in hair treated with permanent dye was impeded by isobaric interferences in the dye matrix. In response to these analytical challenges, we developed highly sensitive methods by coupling ultra-performance liquid chromatography to Orbitrap high-resolution (HR)-MSn. Hitherto, we have successfully measured PhIP in hair of healthy volunteers using hair dye and in patients with colorectal adenoma. Similarly, the low levels of adduct formation and the paucity of tissue limited our ability to measure adducts of carcinogens in humans by TQ- or IT-MS. We measured acid-labile SA-PhIP adducts in human volunteers administered a dietary-relevant dose of 14C-PhIP or a semi-controlled diet of well-done cooked meat. We identified DNA adducts of PhIP, but not other HAAs, in fresh-frozen prostatectomy samples and matching formalin-fixed tissues from prostate cancer patients, using HR-MSn. We are developing methods to screen for multiple exogenous and endogenous DNA adducts by untargeted MSn scanning. HR-MS instrumentation has greatly expanded our ability to measure biomarkers of dietary carcinogens, and can be employed to understand exposures and the etiological role of HAAs in human cancers.

    TOXI Program at 254th ACS Meeting

    SUNDAY MORNING

    Chemical Research in Toxicology Young Investigator Award

    H. Ai, Organizer; H. Ai, Presiding; S. S. Hecht, Presiding; T. M. Penning

    8.00.  Introductory Remarks , Huiwang   Al

    8:10.   TOXI 1.
    Targeted quantitative proteomic approaches for interrogating the human kinome
    Weili Miao, Yongsheng Xiao, Lei Guo, Yinsheng Wang
     ABSTRACT

    8:55. TOXI 2.
    Sequence-specific covalent capture for detection of disease-derived nucleic acid sequences
    Kent S. Gates,  Andrew Gu, Maryam Imani Nejad, Ruicheng Shi, Xinyue Zhang.
    ABSTRACT

    9:40  Intermission.

    9:55.  TOXI 3.
    Dynamic visualization of signaling molecules in living cells
    Jin Zhang.
    ABSTRACT

    10:40  Presentation of the Chemical Research in Toxicology Young Investigators Award.  Stephen Hecht and Trevor Penning

    10:50 TOXI 4.
    Seeing is believing: Fluorescent biosensors for redox signaling and oxidative stress
    Huiwang Ai
    ABSTRACT

    SUNDAY AFTERNOON

    Founders Award

    I. A. Blair, Organizer; I. A. Blair

    1:00. Presentation of Founders’ Award , Huiwang Al

    1:10. Introductory Remarks , Ian Blair

    1:15 TOXI 5.
    Biochemical and toxicological applications of mass spectrometry
    F P. Guengerich,
    ABSTRACT

    1:55 TOXI 6.
    Human aldo-keto reductases and aryl hydrocarbon activation
    Trevor M. Penning
    ABSTRACT

    2:35 TOXI 7.
    Chemical biology of DNA damage by α,β-unsaturated aldehydes
    Lawrence J. Marnett, larry.marnett@vanderbilt.edu.
    ABSTRACT

    3:15 Intermission.

    3:30 TOXI 8.
    S-Nitrosation is a systems-wide regulatory process
    Steven R. Tannenbaum
    ABSTRACT

    TOXI 9.
    Systems pharmacology approach to the study of mitochondrial dysfunction
    4:10 Ian A. Blair, Qingqing Wang, Lili Guo, Liwei Weng, Ashkan Salimatipour, Wei-Ting Hwang, David Lynch, Clementina Mesaros.
    ABSTRACT

    MONDAY MORNING

    TOXI Young Investigators

    Organizer; T. Spratt, Presiding; U. Sarkar and B. Ma,

    8:00 TOXI 10.
    Effect of statins on HMG-CoA reductase pathway and apolipoprotein A-I production in Friedreich’s ataxia
    Lili Guo, QingQing Wang, Cassandra J. Strawser, Lauren A. Hauser, Wei-Ting Hwang, David Lynch, Clementina Mesaros, Ian A. Blair.
    ABSTRACT

    8:20 TOXI 11.
    Mechanism of bioactivation of the cooked meat carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in human prostate
    Madjda Bellamri, Robert J. Turesky.
    ABSTRACT

    8:40 TOXI 12.
    Novel class of hydroxyl radical scavenging antioxidants prevents oxidative DNA damage in fibroblast cells exposed to trivalent arsenic
    Safnas Farwin Abdul Salam,  Edward J. Merino, Haizhou Zhu, Purujit N. Gurjar.
    ABSTRACT

    9:00 TOXI 13.
    Replicative bypass and mutagenic properties of alkylphosphotriester lesions in Escherichia coli
    Jiabin Wu, Pengcheng Wang, Yinsheng Wang.
    ABSTRACT

    9:20 TOXI 14.
    Abasic and oxidized abasic lesion bypass by DNA polymerase theta yields one- and two-nucleotide deletions
    Daniel J. Laverty,  Marc M. Greenberg.
    ABSTRACT

    9:40 Intermission.

    10:00 TOXI 15.
    Characterization of the 2,6-diamino-4-hydroxy-N5-(methyl)-formamidopyrimidine DNA lesion
    Stephanie Bamberger, Hope Pan, Ryan Bowen, Chanchal Kumar Malik, Tracy Johnson-Salyard, Carmelo Rizzo, Michael P. Stone.
    ABSTRACT

    10:20 TOXI 16.
    Engineering a replicative DNA polymerase for specific damage bypass capability
    Timothy A. Coulther,  Mary J. Ondrechen, Penny J. Beuning.
    ABSTRACT

    10:40 TOXI 17.
    Mechanism of ribonucleotide incorporation by human DNA polymerase Eta
    YAN SU,  Martin Egli, F P. Guengerich.
    ABSTRACT

    11:00 TOXI 18.
    Independent generation of 2′-deoxyadenosine-N6-yl radical and its reactivity in DNA
    Liwei Zheng,  Markus Griesser, Derek A. Pratt, Marc M. Greenberg.
    ABSTRACT

    11:20 TOXI 19.
    Investigation into the reactivity of a C5′-uridinyl radical
    Matthew Ellis,  Amanda C. Bryant-Friedrich.
    ABSTRACT

    11:40 TOXI 20.
    Arsenite binds to the RING finger domain of FANCL E3 ubiquitin ligase and inhibits DNA interstrand cross-link repair
    Yinsheng Wang, Ji Jiang
    ABSTRACT

    MONDAY AFTERNOON

    Biological Targets of Botanical Supplements

    Presiding, J. L. Bolton, Organizer; J. L. Bolton

    1:30 Introductory Remarks
    Judy Bolton

    1:35 TOXI 21.
    Pharmacokinetic interactions between drugs and licorice botanical dietary supplements used by menopausal women
    Richard B. Van Breemen
    ABSTRACT

    2:15 TOXI 22.
    Intestinal UGTs as targets for pharmacokinetic natural product-drug interactions
    Mary Paine
    ABSTRACT

    2:55 TOXI 23.
    KEAP1 and done? Targeting the NRF2 pathway with sulforaphane
    Thomas Kensler
    ABSTRACT

    3:35 Intermission.

    3:50 TOXI 24.
    Comparing general and specific biological targets for assessing sufficient similarity of related botanical dietary supplements
    Cynthia V. Rider,  Stephanie Smith-Roe, Stephen S. Ferguson.
    ABSTRACT

    4:30 TOXI 25.
    Botanicals modulate estrogen metabolism through multiple targets
    Judy L. Bolton
    ABSTRACT

    MONDAY EVENING

    Sci-Mix

    Organizer;T. Spratt

    TOXI 84.
    Degradation from C5′ oxidation and its adducts as potential biomarkers
    Shin H. Cho,Amanda C. Bryant-Friedrich.
    ABSTRACT

    TOXI 41.
    Qualitative analysis of the pyrolysis of cocaine and methamphetamine
    Stephen Raso, Olivia Dodd, Suzanne Bell.
    ABSTRACT

    TOXI 43.
    Site-specific incorporation of N-(2′-deoxyguanosine-8-yl)-6-aminochrysene adduct in DNA and its replication in human cells
    Kimberly R. Rebello, Arindom Chatterjee, Paritosh Pande, Ashis K. Basu.
    ABSTRACT

    TOXI 45.
    Synthesis and in vivo quantitation of 2′-deoxyadenosine adducts resulting from bioactivation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol
    Erik S. Carlson, Pramod Upadhyaya, Stephen S. Hecht.
    ABSTRACT

    TOXI 48.
    Nrf2-Keap1 signaling and implications for the metabolic activation of nitroarenes
    Jessica Murray,Meng Huang, Clementina Mesaros, Volker Arlt, Karam El Bayoumy, Ian A. Blair, Trevor M. Penning.
    ABSTRACT

    TOXI 54.
    Substituent effects of bifunctional agents on photo-induced DNA interstrand cross-link formation
    Heli Fan, Xiaohua Peng.
    ABSTRACT

    TOXI 55.
    Estrogenic activity of polycyclic aromatic hydrocarbon ortho-quinones in human endometrium
    Isabelle G. Lee,Clementina Mesaros, Trevor M. Penning.
    ABSTRACT

    TOXI 63.
    Bypass efficiency and mutagenesis assays of site-specific arylamine DNA adducts in cell
    Ke Bian,  Fangyi Chen, Qi Tang, Deyu Li.
    ABSTRACT

    TOXI 68.
    Conformational and configurational equilibra of a 2′-deoxyribosylurea adduct in single strand and duplex DNA
    Andrew H. Kellum, Michael P. Stone, Ashis K. Basu, Jasti Vijay.
    ABSTRACT

    TOXI 70.
    Sequence-dependent repair of 1, N6-ethenoadenine by the AlkB family DNA repair enzymes
    Qi Tang, Fangyi Chen, Ke Bian, Deyu Li.
    ABSTRACT

    TOXI 18.
    Independent generation of 2′-deoxyadenosine-N6-yl radical and its reactivity in DNA
    Liwei Zheng, Markus Griesser, Derek A. Pratt, Marc M. Greenberg.
    ABSTRACT

    TOXI 73.
    Mitochondrial M1dG levels linked to oxidative stress and mitochondrial dysfunction in disease
    Orrette R. Wauchope, Michelle M. Mitchener, William N. Beavers, James Galligan, Philip Kingsley, Ha-Na Shim, Thomas Blackwell, Thong Luong, Mark deCaestecker, Joshua P. Fessel, Lawrence J. Marnett.
    ABSTRACT

    TOXI 74.
    Mechanisms of recognition of bulky DNA lesions by the DNA damaging sensor XPC
    Katie M. Feher, Kadeem D. Walsh, Nicholas E. Geacintov.
    ABSTRACT

    TOXI 75.
    Mitochondrial DNA adducts of lipid peroxidation products with rotenone
    Kevin P. Gillespie, Ian A. Blair.
    ABSTRACT

    TOXI 77.
    Resolving metabolic switches in cellular energy demand
    Maren S. Prediger
    ABSTRACT

    TOXI 82.
    Chemistry of independently generated thymidine radical cation: DNA hole transfer and other competing processes
    Huabing Sun, Marc M. Greenberg.
    ABSTRACT

    TOXI 46.
    Lesion recognition in nucleotide excision repair: Relationship between the structural properties of adducts and initial binding of XPC to the damaged site
    Hong Mu,  Nicholas E. Geacintov, Yingkai Zhang, Suse Broyde.
    ABSTRACT

    TOXI 49.
    Toward genome-wide mapping of O(6)-methylguanine damage and repair in a human cell line
    Maureen McKeague, Ioannis A. Trantakis, Janine Döhring, Pablo Steinberg, Shana J. Sturla.
    ABSTRACT

    TOXI 50.
    Role of PARP-1 in the base excision repair of chromatin substates
    Yu Zeng,  Deb R. Banerjee, Charles Deckard, Jonathan T. Sczepanski.
    ABSTRACT

    TOXI 61.
    Modified deaza-adenosine mimics ad DNA minor groove alkylation probes
    Lukasz J. Weselinski,  Vagarshak Begoyan, Shuai Xia, Alexis Ferrier, Marina Tanasova.
    ABSTRACT

    TUESDAY MORNING

    Crosslink DNA Repair

    Organizer, O. Scharer, Y. Wang,; Presiding, Y. Wang, ; O. Scharer

    8:00 Introductory Remarks

    8:05 TOXI 26.
    Mechanisms of replication-coupled repair
    Johannes C. Walter
    ABSTRACT

    8:40 TOXI 27.
    Interstrand DNA cross-links derived from abasic sites in duplex DNA
    Kent S. Gates
    ABSTRACT

    9:15 TOXI 28.
    Replication and repair of DNA interstrand cross-link lesions in human cells
    Nathan Price, Shuo Liu, Kent S. Gates, Yinsheng Wang
    ABSTRACT

    9:50 Intermission.

    10:05 TOXI 29.
    Lesion proximal FANCD2 is required for replication independent repair of interstrand crosslinks
    Manikandan Paramasivam, Marina Bellani, Julia Gichimu, Himabindu Gali, Michael Seidman
    ABSTRACT

    10:40 TOXI 30.
    Hydrogen peroxide activated DNA cross-linking agents and their biomedical application
    Xiaohua Peng Wenbing Chen, Yibin Wang, Heli Fan.
    ABSTRACT

    11:15 TOXI 31.
    Constitutive role of Fanconi anemia D2 gene in protecting cell from crosslinking DNA damage
    Lei Li
    ABSTRACT

    TUESDAY AFTERNOON

    Toxicological Considerations in Antibody Drug Conjugate Design & Development

    F. Guengerich, Organizer; W. G. Humphreys, Organizer; N. A. Meanwell, Organizer; F. Guengerich, Presiding; N. A. Meanwell, Presiding; W. G. Humphreys

    1:00 Introductory Remarks

    1:10 TOXI 32.
    Antibody drug conjugates: Design considerations for improving efficacy and safety
    Pamela Trail
    ABSTRACT

    1:55 TOXI 33.
    ADC linker immolation and cell killing activity
    Donglu Zhang
    ABSTRACT

    2:40 Intermission.

    2:55 TOXI 34.
    Development of next generation calicheamicin ADCs
    Omar K. Ahmad
    ABSTRACT

    3:40 TOXI 35.
    Potent antibody-based conjugates for cancer therapy: From early stage research to a clinically approved drug
    Peter D. Senter
    ABSTRACT

    TUESDAY EVENING

    Keynote Lecture

    N. E. Geacintov

    5:00 TOXI 92.
    Understanding hepatoxicity: Man to mouse to computer
    Paul B. Watkins
    ABSTRACT

    TUESDAY EVENING

    General Posters

    T. Spratt

    TOXI 36.
    Pentachlorophenol alters secretion of interleukin 6 (IL-6) from human immune cells
    Tamara Martin, Margaret Whalen.
    ABSTRACT

    TOXI 37.
    Analysis of methylated and ethylated peptides in human hemoglobin by liquid chromatography mass spectrometry: Association with cigarette smoking
    Hauh-Jyun C. Chen, Sun Wai Ip, Fu-Di Lin.
    ABSTRACT

    TOXI 38.
    Simultaneous determination of a major peroxidation-derived DNA adduct, M1dG and its oxidized metabolite 6-oxo-M1dG, in human leukocyte DNA by liquid chromatography nanoelectrospray-high resolution tandem mass spectrometry
    Bin Ma, Christopher Ruszczak, Peter W. Villalta, Orrette R. Wauchope, Lawrence J. Marnett, Irina Stepanov.
    ABSTRACT

    TOXI 39.
    Pyridylhydroxybutyl, pyridyloxobutyl and methyl DNA phosphate adduct formation in rats treated chronically with enantiomers of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol
    Bin Ma, Adam T. Zarth, Erik Carlson, Peter W. Villalta, Pramod Upadhyaya, Irina Stepanov, Stephen S. Hecht.
    ABSTRACT

    TOXI 40.
    Evidence for indole-3-methyl isothiocyanate formation upon human consumption of Brussels sprouts
    Pramod Upadhyaya, Adam T. Zarth, Naomi Fujioka, Vincent Fritz, Stephen S. Hecht.
    ABSTRACT

    TOXI 41.
    Qualitative analysis of the pyrolysis of cocaine and methamphetamine
    Stephen Raso, Olivia Dodd, Suzanne Bell.
    ABSTRACT

    TOXI 42.
    Drosophila melanogaster fatty acid amide production in the presence of Diminazene aceturate
    Gabriela Suarez, Karin C. Prins, Benjamin S. Meyer, Ryan L. Anderson, David J. Merkler.
    ABSTRACT

    TOXI 43.
    Site-specific incorporation of N-(2′-deoxyguanosine-8-yl)-6-aminochrysene adduct in DNA and its replication in human cells
    Kimberly R. Rebello, Arindom Chatterjee, Paritosh Pande, Ashis K. Basu.
    ABSTRACT

    TOXI 44.
    Absolute quantification of plasma fibulin-3 as a biomarker for asbestos exposure by immunoprecipitation-high resolution mass spectrometry
    QingQing Wang, Liwei Weng, Clementina Mesaros, Ian A. Blair.
    ABSTRACT

    TOXI 45.
    Synthesis and in vivo quantitation of 2′-deoxyadenosine adducts resulting from bioactivation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol
    Erik S. Carlson, Pramod Upadhyaya, Stephen S. Hecht.
    ABSTRACT

    TOXI 46.
    Lesion recognition in nucleotide excision repair: Relationship between the structural properties of adducts and initial binding of XPC to the damaged site
    Hong Mu, Nicholas E. Geacintov, Yingkai Zhang, Suse Broyde.
    ABSTRACT

    TOXI 47.
    Accurate quantification of serum protein mesothelioma biomarkers
    Liwei Weng, Clementina Mesaros, Ian Blair.
    ABSTRACT

    TOXI 48.
    Nrf2-Keap1 signaling and implications for the metabolic activation of nitroarenes
    Jessica Murray, Meng Huang, Clementina Mesaros, Volker Arlt, Karam El Bayoumy, Ian A. Blair, Trevor M. Penning.
    ABSTRACT

    TOXI 49.
    Toward genome-wide mapping of O(6)-methylguanine damage and repair in a human cell line
    Maureen McKeague, Ioannis A. Trantakis, Janine Döhring, Pablo Steinberg, Shana J. Sturla.
    ABSTRACT

    TOXI 50.
    Role of PARP-1 in the base excision repair of chromatin substates
    Yu Zeng, Deb R. Banerjee, Charles Deckard, Jonathan T. Sczepanski.
    ABSTRACT

    TOXI 51.
    DNA cross-linking by the anticancer prodrug PR-104A in oligonucleotides
    Sara Danielli,  Alessia Stornetta, Shana J. Sturla.
    ABSTRACT

    TOXI 52.
    Investigation of the presence in human urine of mercapturic acids derived from phenanthrene
    Guang Cheng, Adam T. Zarth, Pramod Upadhyaya, Peter W. Villalta, Silvia Balbo, Stephen S. Hecht.
    ABSTRACT

    TOXI 53.
    Arsenite binds to the zinc finger domains of TIP60 histone acetyltransferase and induces its degradation via the 26S proteasome
    Lok Ming Tam,  Ji Jiang, Pengcheng Wang, Lin Li, Yinsheng Wang.
    ABSTRACT

    TOXI 54.
    Substituent effects of bifunctional agents on photo-induced DNA interstrand cross-link formation
    Heli Fan, Xiaohua Peng.
    ABSTRACT

    TOXI 55.
    Estrogenic activity of polycyclic aromatic hydrocarbon ortho-quinones in human endometrium
    Isabelle G. Lee, Clementina Mesaros, Trevor M. Penning.
    ABSTRACT

    TOXI 56.
    Deep learning methods applied to physicochemical and toxicological endpoints
    Boris Sattarov, Alexandru Korotcov, Valery Tkachenko, Christopher Grulke, Antony J. Williams.
    ABSTRACT

    TOXI 57.
    Total synthesis of site-specific oligonucleotides containing 2′-deoxyadenosine adduct formed by 6-nitrochrysene and their biological studies
    Brent V. Powell, Ashis K. Basu.
    ABSTRACT

    TOXI 58.
    Determination of heavy metal acceptable concentration using fixed monitoring benchmarks in river system and soil pore-water in S.Korea
    Buyun Jeong,  jinsung an, gihyeon yu, Kyoungphile Nam.
    ABSTRACT

    TOXI 59.
    Determination of the ecotoxicological threshold concentration of Cu in soil pore water in Korea with biotic ligand model and species sensitivity distribution
    gihyeon yu,  Buyun Jeong, Kyoungphile Nam.
    ABSTRACT

    TOXI 60.
    Versatile method to construct model DNA-protein cross-links (DPCs)
    Suresh Pujari, Mandy Zhang, Shaofei Ji, Mark D. Distefano, Natalia Y. Tretyakova.
    ABSTRACT

    TOXI 61.
    Modified deaza-adenosine mimics ad DNA minor groove alkylation probes
    Lukasz J. Weselinski,  Vagarshak Begoyan, Shuai Xia, Alexis Ferrier, Marina Tanasova.
    ABSTRACT

    TOXI 62.
    Development of rapid, high throughout labeling methods for measuring aldehydes from P450 reactions
    Amanda M. Hanson,  Dustyn A. Barnette, Grover P. Miller.
    ABSTRACT

    TOXI 63.
    Bypass efficiency and mutagenesis assays of site-specific arylamine DNA adducts in cell
    Ke Bian, kebian@my.uri.edu, Fangyi Chen, Qi Tang, Deyu Li.
    ABSTRACT

    TOXI 64.
    Comprehensive kinetic study of ALKBH2 and related family enzymes
    Michael VittoriKe Bian, kebian@my.uri.edu, Fangyi Chen, Qi Tang, Deyu Li.
    ABSTRACT

    TOXI 65.
    Expression of a fragment of DNA polymerase zeta from Dictyostelium discoideum
    Stanley K. Mauldin,  Dian He.
    ABSTRACT

    TOXI 66.
    Replication and repair of 8-methoxypsoralen-derived DNA-DNA interstrand cross-links in human cells
    Nathan E. Price, Yinsheng Wang, Kent S. Gates.
    ABSTRACT

    TOXI 67.
    Polymerase bypass of DNA-protein and DNA-peptide cross-links
    Shaofei Ji, Orlando Scharer, Natalia Y. Tretyakova.
    ABSTRACT

    TOXI 68.
    Conformational and configurational equilibra of a 2′-deoxyribosylurea adduct in single strand and duplex DNA
    Andrew H. Kellum, Michael P. Stone, Ashis K. Basu, Jasti Vijay.
    ABSTRACT

    TOXI 69.
    Terbinafine bioactivation pathways to liver toxicity assessed using predictive modeling and experimental approaches
    Dustyn A. Barnette, Lena Dang, Tyler Hughes, S Joshua Swamidass, Grover P. Miller.
    ABSTRACT

    TOXI 70.
    Sequence-dependent repair of 1, N6-ethenoadenine by the AlkB family DNA repair enzymes
    Qi Tang,  Fangyi Chen, Ke Bian, Deyu Li.
    ABSTRACT

    TOXI 71.
    Independent generation of neutral purine radicals involved in DNA damage
    Liwei Zheng, Marc M. Greenberg.
    ABSTRACT

    TOXI 72.
    Importance of the glutathione and its degradation by γ-glutamyl transferase in lung tumor development
    Rosalind B. Penney, Nicholas S. Kowalkowski, Eric R. Siegel, Gunnar Boysen
    ABSTRACT

    TOXI 73.
    Mitochondrial M1dG levels linked to oxidative stress and mitochondrial dysfunction in disease
    Orrette R. Wauchope, Michelle M. Mitchener, William N. Beavers, James Galligan, Philip Kingsley, Ha-Na Shim, Thomas Blackwell, Thong Luong, Mark deCaestecker, Joshua P. Fessel, Lawrence J. Marnett.
    ABSTRACT

    TOXI 74.
    Mechanisms of recognition of bulky DNA lesions by the DNA damaging sensor XPC
    Katie M. Feher,Kadeem D. Walsh, Nicholas E. Geacintov.
    ABSTRACT

    TOXI 75.
    Mitochondrial DNA adducts of lipid peroxidation products with rotenone
    Kevin P. Gillespie, Ian A. Blair.
    ABSTRACT

    TOXI 76.
    Temporal impact of toxic exposures on cellular recovery
    Julia A. Mouch, jmouch@bethanywv.edu, Alice Han, Julie V. Miller, Nicole Prince, Maren S. Prediger, Jonathan W. Boyd.
    ABSTRACT

    TOXI 77.
    Resolving metabolic switches in cellular energy demand
    Maren S. Prediger
    ABSTRACT

    TOXI 78.
    Characterization of a domoic acid-producing diatom
    Min-Ying Wang, Su-Yuan Lai, Ping-Ting Lin, Hsing-Li Lai.
    ABSTRACT

    TOXI 79.
    Size matters: Mesoporous silica nanoparticles size governs the vascular angiogenesis process, proliferation, and nanotoxicity
    Magdiel Inggrid Setyawati, David Leong.
    ABSTRACT

    TOXI 80.
    Development of a threshold of toxicological concern framework based on chemoinformatics
    Mitchell Cheeseman
    ABSTRACT

    TOXI 81.
    Evidence of bioactivation of the anti-HIV drug etravirine to reactive metabolites in vitro and in vivo
    Ana L. Godinho, Cristina C. Jacob, Sofia A. Pereira, M S. Marques, Alexandra Antunes.
    ABSTRACT

    TOXI 82.
    Chemistry of independently generated thymidine radical cation: DNA hole transfer and other competing processes
    Huabing Sun,  Marc M. Greenberg.
    ABSTRACT

    TOXI 83.
    Histone protein tails inhibit depurination of N7-methylated deoxyguanosine and form DNA-protein crosslinks with alkylated DNA in nucleosome core particles
    Kun Yang,  Marc M. Greenberg.
    ABSTRACT

    TOXI 84.
    Degradation from C5′ oxidation and its adducts as potential biomarkers
    Shin H. Cho,  Amanda C. Bryant-Friedrich.
    ABSTRACT

    TOXI 85.
    Functional characterization of glutathione S-transferases by photoreactive and mechanism-based activity-based probes
    Ethan Stoddard, Bryan Killinger, Reji N. Nair, Natalie Sadler, Jordan Smith, Richard Corley, Aaron T. Wright.
    ABSTRACT

    TOXI 86.
    Using medaka embryos coupled with a whole sediment exposure strategy to assess copper bioavailability and toxicity in sediment
    Wei Li,  Pei-Jen Chen.
    ABSTRACT

    TOXI 87.
    Histones are targets for modification by the glycolytic by-product methylglyoxal
    James Galligan,  James A. Wepy, Matthew Streeter, Philip Kingsley, Michelle M. Mitchener, Orrette R. Wauchope, William N. Beavers, Kristie Rose, Tina Wang, David A. Spiegel, Lawrence J. Marnett.
    ABSTRACT

    TOXI 88.
    Wide selected ion monitoring (SIM)/MS2 data independent acquisition method for DNA adduct omics analysis
    Jingshu Guo,  Peter W. Villalta, Robert J. Turesky.
    ABSTRACT

    TOXI 89.
    Investigation of environmental fate and toxic mechanisms of monovalent and trivalent thallium
    Ching-Hsin Yang,  Pei-Jen Chen.
    ABSTRACT

    TOXI 90.
    Biological uptake, distribution and depuration of radio-labeled graphene in adult zebrafish
    Liang Mao
    ABSTRACT

    TOXI 91.
    Bringing it all together: A web-based database for chemical and biological data to support environmental toxicology
    Antony J. Williams, Christopher Grulke, Jennifer Smith, Sean Watford, Rebecca Jolley, Jeremy Dunne, Elizabeth Edmiston, Jeff Edwards.
    ABSTRACT

    WEDNESDAY MORNING

    General Papers

    T. Spratt, Presiding;G. P. Miller, L. Zhao

    8:00 TOXI 93.
    Chemistry and biology of N5-alkyl-fapy-dG damage in DNA
    Michael P. Stone,  Martin Egli, R. S. Lloyd, Amanda Mc Cullough, Carmelo Rizzo, Robert J. Turesky.
    ABSTRACT

    8:20 TOXI 94.
    Aldehydes increase the tumorigenic properties of tobacco specific nitrosamines in rodent tumor models
    Lisa A. Peterson,  Marissa K. Oram, Monica Flavin, Donna Seabloom, William E. Smith, Ingrid Cornax, M. Gerard O’Sullivan, Pramod Upadhyaya, Lin Zhang, Stephen S. Hecht, Silvia Balbo, Timothy S. Wiedmann.
    ABSTRACT

    8:40 TOXI 95.
    Unwinding kinetics of carcinogenic adducts: Correlation with processing by nucleotide excision repair machinery
    Vladimir Shafirovich,  Axel Y. Epie, Vincent Zheng, Marina Kolbanovskiy, Nicholas E. Geacintov.
    ABSTRACT

    9:00 Intermission

    9:10 TOXI 96.
    Structural insights into the post-chemistry steps of nucleotide incorporation catalyzed by a DNA polymerase
    Zucai Suo
    ABSTRACT

    9:30 TOXI 97.
    Central role of PCNA in promoting replication of damaged DNA
    George-Lucian Moldovan
    ABSTRACT

    9:50 TOXI 98.
    Lucidin-dervied N2-guanine DNA lesion is not a major contributor to the mutagenicity of lucidin
    Linlin Zhao
    ABSTRACT

    10:10 TOXI 99.
    Spore photoproduct within DNA is a surprisingly poor substrate for its designated repair enzyme: The spore photoproduct lyase
    Lei Li,  Linlin Yang, Setlow Peter.
    ABSTRACT

    10:10 Intermission

    10:40 TOXI 100.
    Real-time prediction of physicochemical and toxicological endpoints using the web-based CompTox Chemistry Dashboard
    Antony J. Williams, Todd Martin, Valery Tkachenko, Christopher Grulke, Kamel Mansouri.
    ABSTRACT

    11:00 TOXI 101.
    Reaction of the antiepileptic drug carbamazepine with bionucleophiles: Bioactivation is not required
    Inês L. Martins, João P. Telo, M S. Marques, Alexandra Antunes.
    ABSTRACT

    11:20 TOXI 102.
    Programed release of nitric oxide, via oxidative metabolism, in animals and humans from clinical candidate MK-8150
    Kaushik Mitra.
    ABSTRACT

    11:40 TOXI 103.
    Can pipe tobaccos be characterized for regulatory purposes without a puff of pipe smoke?
    John H. Lauterbach.
    ABSTRACT

    WEDNESDAY AFTERNOON

    Advanced Mass Spectrometric Techniques in Toxicology

    S. Balbo, P. Villalta

    1:00 Introductory Remarks

    1:05 TOXI 104.
    Advances in mass spectrometry techniques for metabolism, pharmacology and toxicology
    Jonathan Josephs.
    ABSTRACT

    1:14 TOXI 105.
    Capillary electrophoresis for trace-level detection: Metabolites and proteins
    Peter Nemes, Rosemary M. Onjiko, Camille Lombard-Banek.
    ABSTRACT

    2:25 TOXI 106.
    Exposing the exposome: Utilizing global metabolomics to characterize toxicant exposure and effect
    Benedikt Warth.
    ABSTRACT

    3:05 Intermission

    3:20 TOXI 107.
    Probing stress-induced effects on RNA and posttranscriptional modifications by LC-MS
    Balasubrahmanyam Addepalli, Congliang Sun, Patrick Limbach.
    ABSTRACT

    4:00 TOXI 108.
    Advances in human biomonitoring of heterocyclic aromatic amines by high resolution accurate mass spectrometry
    Jingshu Guo, Shun Xiao, Yi Wang, Byeong Hwa Yun, Paari Murugan, Christopher J. Weight, Kami K. White, Lynne R. Wilkens, Loic Le Marchand, Karen Dingley, Michael A. Malfatti, Kenneth Turteltaub, Peter W. Villalta, Robert J. Turesky.
    ABSTRACT


    Ian Blair, 2017 Founders Award Winner

    Ian A. Blair has won the 2017 Founders Award from the Division of Chemical Toxicology of the American Chemical Society.  Dr. Blair has made outstanding contributions to the field of Chemical Toxicology.  He has pioneered the use of mass spectrometry to identify biomarkers for carcinogenesis, cardiovascular disease, and neurodegeneration.  He developed electron capture atmospheric pressure chemical ionization, which makes it possible to conduct quantitative analyses on chiral biomolecules. You can see his current research interests on his lab website.  Also, consider reading an interview with Dr. Blair by Francesca Lake in Future Science OA.

    ACS Science Tuesdays on Reddit.  You can learn more about Dr. Blair’s research by participating in the ACS AMA Reddit session that will be held on August 15th.

    Dr. Blair will receive the Founders Award during a Symposium in his honor on August 20 at the American Chemicals Society’s National Meeting.  The symposium will comprise talks from Fred Guengerich, Trevor Penning, Larry Marnett, and Steve Tannenbaum, after which he will present the Richard Loeppky Lecture.

    Previous Founders Award Winners include:

    2008  Lawrence J Marnett
    2009  Stephen S Hecht
    2010  Richard Loeppky
    2011  F. Peter Guengerich
    2012  Thomas Baillie
    2013  Steve Tannenbaum
    2014  Paul Hollenberg
    2015  Arthur Grollman
    2016  Nicholas Geacintov and Suse Broyde

    Chemical Research in Toxicology, Young Investigator Award.

    Huiwang Ai, of the University of California, Riverside has won the sixth annual Chemical Research in Toxicology -Young Investigators Award.  He joins past winners Yinsheng Wang,  Dean Naisbitt, Shana Sturla, Penny Beuning, and Yimon Aye.

    Huiwang Ai
    Huiwang Ai

    Huiwang Ai’s  research  focuses on the engineering of novel molecular probes to peer into cells and brains to understand their communications. He uses a collection of innovative techniques, such as protein engineering and fluorescence and bioluminescence imaging, to dissect signaling pathways involving redox-active molecules, neuromodulators, and protein post-translational modifications.

    Huiwang will receive his award at a symposium during the Division of Chemical Toxicology Program at the American Chemical Society’s National Meeting on Sunday morning, August 20,in Washington DC.

    Call for Symposia Proposals

    The Program Committee of the Division of Chemical Toxicology requests proposals for future symposia to be held during the annual fall American Chemical Society Meeting.

    The proposals will be evaluated based on their ability to attract and engage a large audience, timeliness, and appropriateness for the Division of Chemical Toxicology.  We seek proposals that span the interests, and fully reflect the diversity, of the membership in the Division. Therefore, the selection of speakers should be balanced when considering gender, groups historically underrepresented in the profession, research area, employment sector, and career stage.

    The theme for the ACS Meeting in Boston in 2018 is Nanotechnology.  Proposals based on this theme are especially welcome

    The proposal should contain:

    • A symposium title.
    • A paragraph stating the idea behind the proposal, why it is timely, how this symposium will differ from those recently given at TOXI, and why TOXI is the appropriate forum for this symposium. (It should relate to the chemical mechanisms of toxicology)
    • Indicate your willingness to organize this symposium at a different (later) meeting than originally proposed.
    • A list of potential speakers

    E-mail proposals to Thomas E. Spratt at tes13@psu.edu, speak to him by phone (717-531-4623), or at the ACS meeting in Washington D.C. this August.

    Nominations for Founders Award

    The Executive Committee of the Division of Chemical Toxicology asks for nominations for the Founders’ Award. The award was established in honor of the founders of the Division and recognizes scientists whose work exemplifies the founders’ vision of excellence in the field of chemical toxicology. Nominees should be members of the Division of Chemical Toxicology whose scientific activities have emphasized innovative research in the general field of chemical toxicology.Previous winners were:

    2008  Lawrence J  Marnett
    2009:  Stephen S Hecht
    2010  Richard Loeppky
    2011  F. Peter Guengerich
    2012  Thomas Baillie
    2013  Steve Tannenbaum
    2014  Paul Hollenberg
    2015  Arthur Grollman
    2016  Nicholas Geacintov and Suse Broyde
    2017 Ian Blair

    A nomination document should include:

    1. Nomination letter containing an evaluation of the nominee’s accomplishments, innovative research into Chemical Toxicology.
    2. Two additional letters of support.
    3. The candidates CV with a list of publications and patents.

    Nominations should be submitted as a single PDF file (Name the PDF file with candidate’s name, for example: LawrenceMarnett_FoundersNomination.pdf)

    Nominations should be sent directly to Awards Committee Chair no later than Monday October 16TH 2017 at penning@upenn.edu

    Nominations for TOXI Officers

    The nominations committee, chaired by Kent Gates, will be putting together a slate of candidates for new TOXI officers as follows:

    Secretary
    His/her term will be January 1, 2018 through Dec 31, 2019.
    Executive Committee Members-at-Large
    One new member for a three-year term beginning January 1, 2018.
    Nomination Committee
    One new position for a three-year term beginning January 1, 2018.
    Counselor and Alternate Counselor
    The positions are for a three-year term beginning January 1, 2018.

    Contact Kent (GatesK@missouri.edu) if you want to nominate a member for a position.  Self nominations are welcome.

    Congratulations to TOXI members who had articles published in Chemical Research in Toxicology between March and June of 2017!

    March 2017
    Soft Cysteine Signaling Network: The Functional Significance of Cysteine in Protein Function and the Soft Acids/Bases Thiol Chemistry That Facilitates Cysteine Modification
    Ryan S. Wible and Thomas R. Sutter. Chem Res Toxicol. 2017, 30: 729-762. DOI: 10.1021/acs.chemrestox.6b00428

    Modulation of Cytotoxicity by Transcription-Coupled Nucleotide Excision Repair Is Independent of the Requirement for Bioactivation of Acylfulvene
    Claudia Otto, Graciela Spivak, Claudia M.N. Aloisi, Mirco Menigatti, Hanspeter Naegeli, Philip C. Hanawalt, Marina Tanasova, and Shana J. Sturla. Chem Res Toxicol. 2017, 30: 769-776. DOI: 10.1021/acs.chemrestox.6b00240

    DNA Product Formation in Female Sprague-Dawley Rats Following Polyhalogenated Aromatic Hydrocarbon (PHAH) Exposure
    Lina Gao, Esra Mutlu, Leonard B. Collins, Nigel J. Walker, Hadley J. Hartwell, James R. Olson, Wei Sun, Avram Gold, Louise M. Ball, and James A. Swenberg. Chem Res Toxicol. 2017, 30: 794-803. DOI: 10.1021/acs.chemrestox.6b00368

    DNA Adduct Profiles Predict in Vitro Cell Viability after Treatment with the Experimental Anticancer Prodrug PR104A
    Alessia Stornetta, Peter W. Villalta, Frederike Gossner, William R. Wilson, Silvia Balbo, and Shana J. Sturla. Chem Res Toxicol. 2017, 30: 830-839. DOI: 10.1021/acs.chemrestox.6b00412

    April 2017
    Systems Toxicology II: A Special Issue
    Thomas Hartung, Robert Kavlock, and Shana J. Sturla. Chem Res Toxicol. 2017, 30: 869. DOI: 10.1021/acs.chemrestox.7b00038

    Systems Toxicology: Real World Applications and Opportunities
    Thomas Hartung, Rex E. FitzGerald, Paul Jennings, Gary R. Mirams, Manuel C. Peitsch, Amin Rostami-Hodjegan, Imran Shah, Martin F. Wilks, and Shana J. Sturla. Chem Res Toxicol. 2017, 30: 870-882. DOI: 10.1021/acs.chemrestox.7b00003

    Toward the Design of Less Hazardous Chemicals: Exploring Comparative Oxidative Stress in Two Common Animal Models
    Jone Corrales, Lauren A. Kristofco, W. Baylor Steele, Gavin N. Saari, Jakub Kostal, E. Spencer Williams, Margaret Mills, Evan P. Gallagher, Terrance J. Kavanagh, Nancy Simcox, Longzhu Q. Shen, Fjodor Melnikov, Julie B. Zimmerman, Adelina M. Voutchkova-Kostal, Paul T. Anastas, and Bryan W. Brooks. Chem Res Toxicol. 2017, 30: 893-904. DOI: 10.1021/acs.chemrestox.6b00246

    Stem Cell Transcriptome Responses and Corresponding Biomarkers That Indicate the Transition from Adaptive Responses to Cytotoxicity
    Tanja Waldmann, Marianna Grinberg, André König, Eugen Rempel, Stefan Schildknecht, Margit Henry, Anna-Katharina Holzer, Nadine Dreser, Vaibhav Shinde, Agapios Sachinidis, Jörg Rahnenführer, Jan G. Hengstler, and Marcel Leist. Chem Res Toxicol. 2017, 30: 905-922. DOI: 10.1021/acs.chemrestox.6b00259

    Crowd-Sourced Verification of Computational Methods and Data in Systems Toxicology: A Case Study with a Heat-Not-Burn Candidate Modified Risk Tobacco Product
    Carine Poussin, Vincenzo Belcastro, Florian Martin, Stéphanie Boué, Manuel C. Peitsch, and Julia Hoeng. Chem Res Toxicol. 2017, 30: 934-945. DOI: 10.1021/acs.chemrestox.6b00345

    Mass Spectrometry Based Proteomics Study of Cisplatin-Induced DNA-Protein Cross-Linking in Human Fibrosarcoma (HT1080) Cells
    Xun Ming, Arnold Groehler IV, Erin D. Michaelson-Richie, Peter W. Villalta, Colin Campbell, and Natalia Y. Tretyakova. Chem Res Toxicol. 2017, 30: 980-995. DOI: 10.1021/acs.chemrestox.6b00389

    SILAC-Based Quantitative Proteomic Analysis Unveils Arsenite-Induced Perturbation of Multiple Pathways in Human Skin Fibroblast Cells
    Fan Zhang, Yongsheng Xiao, and Yinsheng Wang. Chem Res Toxicol. 2017, 30: 1006-1014. DOI: 10.1021/acs.chemrestox.6b00416

    Computational Approach to Structural Alerts: Furans, Phenols, Nitroaromatics, and Thiophenes
    Na Le Dang, Tyler B. Hughes, Grover P. Miller, and S. Joshua Swamidass. Chem Res Toxicol. 2017, 30: 1046-1059. DOI: 10.1021/acs.chemrestox.6b00336

    Potential Metabolic Activation of a Representative C4-Alkylated Polycyclic Aromatic Hydrocarbon Retene (1-Methyl-7-isopropyl-phenanthrene) Associated with the Deepwater Horizon Oil Spill in Human Hepatoma (HepG2) Cells
    Meng Huang, Clementina Mesaros, Linda C. Hackfeld, Richard P. Hodge, Tianzhu Zang, Ian A. Blair, and Trevor M. Penning. Chem Res Toxicol. 2017, 30: 1093-1101. DOI: 10.1021/acs.chemrestox.6b00457

    Oncometabolites D– and L-2-Hydroxyglutarate Inhibit the AlkB Family DNA Repair Enzymes under Physiological Conditions
    Fangyi Chen, Ke Bian, Qi Tang, Bogdan I. Fedeles, Vipender Singh, Zachary T. Humulock, John M. Essigmann, and Deyu Li. Chem Res Toxicol. 2017, 30: 1102-1110. DOI: 10.1021/acs.chemrestox.7b00009

    May 2017
    The Glutathione Conundrum: Stoichiometric Disconnect between Its Formation and Oxidative Stress
    Gunnar Boysen. Chem Res Toxicol. 2017, 30: 1113-1116. DOI: 10.1021/acs.chemrestox.7b00018

    Replicative Bypass Studies of α-Anomeric Lesions of 2’-Deoxyribonucleosides in Vitro
    Nicole L. Williams, Nicholas J. Amato, and Yinsheng Wang. Chem Res Toxicol. 2017, 30: 1127-1133. DOI: 10.1021/acs.chemrestox.6b00439

    Mutagenic Replication of N2-Deoxyguanosine Benzo[a]pyrene Adducts by Escherichia coli DNA Polymerase I and Sulfolobus solfataricus DNA Polymerase IV
    A. S. Prakasha Gowda, Jacek Krzeminski, Shantu Amin, Zucai Suo, and Thomas E. Spratt. Chem Res Toxicol. 2017, 30: 1168-1176. DOI: 10.1021/acs.chemrestox.6b00466

    Formation of S-[2-(N6-Deoxyadenosinyl)ethyl]glutathione in DNA and Replication Past the Adduct by Translesion DNA Polymerases
    Carl A. Sedgeman, Yan Su, and F. Peter Guengerich. Chem Res Toxicol. 2017, 30: 1188-1196. DOI: 10.1021/acs.chemrestox.7b00022

    Modeling Exposure in the Tox21 in Vitro Bioassays
    Fabian C. Fischer, Luise Henneberger, Maria König, Kai Bittermann, Lukas Linden, Kai-Uwe Goss, and Beate I. Escher. Chem Res Toxicol. 2017, 30: 1197-1208. DOI: 10.1021/acs.chemrestox.7b00023

    1,3-Butadiene-Induced Adenine DNA Adducts Are Genotoxic but Only Weakly Mutagenic When Replicated in Escherichia coli of Various Repair and Replication Backgrounds
    Shiou-chi Chang, Uthpala I. Seneviratne, Jie Wu, Natalia Tretyakova, and John M. Essigmann. Chem Res Toxicol. 2017, 30: 1230-1239. DOI: 10.1021/acs.chemrestox.7b00064

    Noncatalytic, N-terminal Domains of DNA Polymerase Lambda Affect Its Cellular Localization and DNA Damage Response
    Anthony A. Stephenson, David J. Taggart, and Zucai Suo. Chem Res Toxicol. 2017, 30: 1240-1249. DOI: 10.1021/acs.chemrestox.7b00067

    June 2017
    Quantification of Hemoglobin and White Blood Cell DNA Adducts of the Tobacco Carcinogens 2-Amino-9H-pyrido[2,3-b]indole and 4-Aminobiphenyl Formed in Humans by Nanoflow Liquid Chromatography/Ion Trap Multistage Mass Spectrometry
    Tingting Cai, Medjda Bellamri, Xun Ming, Woon-Puay Koh, Mimi C. Yu, and Robert J. Turesky. Chem Res Toxicol. 2017, 30: 1333-1343. DOI: 10.1021/acs.chemrestox.7b00072

    Nucleotide Excision Repair Lesion-Recognition Protein Rad4 Captures a Pre-Flipped Partner Base in a Benzo[a]pyrene-Derived DNA Lesion: How Structure Impacts the Binding Pathway
    Hong Mu, Nicholas E. Geacintov, Jung-Hyun Min, Yingkai Zhang, and Suse Broyde. Chem Res Toxicol. 2017, 30: 1344-1354. DOI: 10.1021/acs.chemrestox.7b00074

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