- Message from Chair
- Summary of the TOXI Program
- Simon Chan: 2018 Chemical Research in Toxicology Young Investigator
- John Essigmann. Keynote Lecture
- Founders’ Award Winner: Judy Bolton
- Call for Nominations for Founders’ Award
- Program for the 256th ACS Meeting
- Abstracts from the 256th ACS Meeting
- Amanda Bryant-Friedrich – ACS Fellow
In this issue:
Message from Chair
We just received the great news that Professor Amanda Bryant-Friedrich, a long-time member of the TOXI Division, is listed among the members of the 2018 Class of ACS Fellows!! Heartiest congratulations, Amanda!
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 254thAmerican Chemical Society National Meeting in Boston MA. August 19-23, 2018. This year, Dr. Thomas Spratt, our Program Chair, has assembled an exciting program that includes 105 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.
This year, Tom Spratt and I, with the agreement and advice of the Executive Committee, have sought to engage other members of TOXI to assist the Program Chair in organizing, and planning the themes and contents of our TOXI meetings. The Planning Committee consists of five appointed members who will select the themes and contents of future meetings and symposia, identify the most suitable candidates to chair these symposia, and help the chair to write and submit NIH or other formal grant applications. The functions of the second group (also appointed), the Program Development Committee, is to develop new ideas for symposia, submit funding proposals to potential industrial donors to support TOXI symposia and meetings, evaluate and organize incoming abstracts, assist in organizing and selecting the best student/post-doc oral and poster presentations, and assist the Chair when needed. Both committees are expected to communicate with one another and with the Program Chair, as necessary. The rosters for this year’s committees have been completed, but we will be looking to rotate some of the vacancies in the Program Development Committee next year and beyond. We are seeking interested and enthusiastic TOXI members at the senior graduate student or higher levels who would like to actively participate in one of the most important activities of our division.
The meeting begins on Sunday morning (August 19) with a symposium organized by Zucai Suo that examines how translesion DNA polymerases operate at the replication fork. The Founders’ Award Symposium will be held as usual on Sunday afternoon. This year the honoree is Dr Judy Bolton, Distinguished Professor and Head of the Medicinal Chemistry and Pharmacognosy Department at the University of Illinois at Chicago. Dr Bolton is known for her studies on the toxic effects of estrogens and antiestrogens.
The Monday morning symposium (August 20) is dedicated to TOXI Young Investigators. This year, eleven 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. In the afternoon, Dr. Silvia Balbo has organized a symposium on the Chemical Toxicology of Nanomaterials. This is a hot topic that aligns with the l theme of the American Chemical Society Meeting: Nanoscience, Nanotechnology & Beyond.
Dr. Barry Rosen has organized a symposium on Mechanisms of Binding, Transport & Biotransformation of Toxic Metals on Tuesday morning (August 21). On Tuesday afternoon,the Chemical Research in Toxicology Young Investigator Award Symposium will be held. This year’s Awardee is Simon Chan, Associate Professor in the Department of Chemistry at the Hong Kong University of Science and Technology. The Keynote speaker is John Essigmann, William R. (1956) and Betsy P. Leitch Professor in Residence of Chemistry in the MIT Department of Chemistry.
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!
On Wednesday morning, Drs. Fred Guengerich, Nick Meanwell and Griff Humphreys organized a symposium entitles ‘Nanomaterials in Drug Delivery: Efficacy & Toxicity.’ This is another hot topic at the interface of chemistry and human health and aligns with the theme of the ACS meeting. Our program closes with the Topics in Chemical Toxicology Symposium, in which ten investigators will discuss their work.
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 Boston to share their results with us!
With best wishes to all,
Chair, Division of Chemical Toxicology
256th ACS National Meeting & Exposition
Boston Convention and Exhibition Center
Aug 19, 2018 – Aug 23, 2018
The headliners for the Division of Chemical Toxicology’s Program at the National meetings are
- Founder’s Award Winner Judy Bolton,
- Chemical Research in Toxicology Young Investigator Simon Chan
- Keynote Speaker John Essigmann.
The Founders’ Award symposium will be held on Sunday afternoon. Judy Bolton has invited four scientists to discuss their research on cellular responses to electrophiles. Lisa Peterson will discuss the targets of furan metabolites, Natalia Tretyakova will examine DNA and protein adducts, Albena Dinkova-Kostova will talk about Keap1/Nrf2 signalling, and Yimon Aye will discuss using targeted adduct formation as drugs. The title of Judy Bolton’s Richard Loeppky Lecture will be “Botanicals electrophiles modify multiple targets.”
The Chemical Research in Toxicology Young Investigator Award symposium will be held on Tuesday afternoon. Simon Chan has invited three colleagues to discuss their research on the mechanisms by which electrophiles are toxic. Jetty Lee will talk about oxidized polyunsaturated fatty acids, Clement Chan will discuss nitrosative stress in microorganisms, and Rob Turesky will examine biomonitoring of human exposure to carcinogens by mass spectrometry. Simon Chan will present the award lecture on his work on the toxicity of aristolochic acids.
The Keynote Lecture will be held right after the Chemical Research in Toxicology Young Investigator Award symposium on Tuesday. John Essigmann’s lecture in entitled “Linking mutational spectra of chemical carcinogens to the mutational patterns
seen in human tumors.”
The theme for this year’s meeting is Nanoscience, nanotechnology and beyond. The TOXI Division has four symposia that aligns with this theme.
On Monday Morning, Silvia Balbo has organized a symposium entitles “Chemical Toxicology of Nanomaterials“. Five speakers from the US and Europe will discuss: the future of nanotoxicology research (Alison Elder), the toxicity of nano-lithium battery material (Vivian Feng), how size matters (Anna Shvedova), epigenetic changes caused by nano particles (Lode Godederis), and the mechanisms of how nanomaterials causes toxicity (Annette Kraegeloh).
On Wednesday Morning, Fred Guengerich, Griff Humphreys, and Nick Meanwell organized a symposium entitles “Nanomaterials in Drug Delivery: Efficacy & Toxicity Considerations.” While nanomaterials are being used as delivery devices, they present their own toxicological problems. Five speakers from academia and industry will discuss: why colloidal nanoparticle formulations are toxic (Murphy), targeting vs enhanced selectivity by nanodelivery devices (Darvari), expansile nanoparticles (Grinstaff), nanoparticle – biological interfases (Mahmoudi), and mast cell activation in the safe development of nanotechnologies (Brown)
On Sunday Morning Zucai Suo organized a symposium examining how nanotechnology is being used to study Translesion DNA Polymerases. Joe Loparo will discuss his research on single molecule lesion bypass in bacteria, Zucai Suo will examine his use of FRET to explore mechanisms of lesion bypass. Michael Trakselis will examine polymerase switch mechanism, and Zach Purscell will examine how DNA polymerase epsilon is involved in mismatch repair.
On Tuesday Morning, Barry Rosen has organized a symposium entitled “Mechanisms of Binding, Transport & Biotransformation of Toxic Metals.” Topics in this symposium include binding, transport & biotransformation of toxic metals (Vincent Pecoraro), degradation of environmental organoarsenicals (Barry Rosen), carbon-metal bond cleavage (James Omichinski), and copper transport proteins in the processing of platinum anticancer
drugs (Oleg Dmitriev).
The TOXI Young Investigator symposium, in which students and post-docs prevent their work will be held on Monday Morning.
The TOXI Reception and Poster Session will be held on Tuesday Evening. The Business Meeting and Award presentation to the top posters and oral presentations will follow.
The last symposium, on Wednesday afternoon is the The Current Topics in Chemical Toxicology symposium. This symposium is one of my favorites because established scientists give concise talks of their latest research.
Simon Chan wins the 2018 Chemical Research in Toxicology Young Investigator Award. Simon Chan is Associate Professor in the Department of Chemistry at the Hong Kong University of Science and Technology. Visit Lab website. Dr Chan will present a lecture on Tuesday afternoon, Aug 21, 2018 during the Division’s Program at the ACS National Meeting in Boston. Simon Chan joins a list of impressive young scientists who have won this award including Yinsheng Wang, Dean Naisbitt, Shana Sturla, Penny Beuning, Yimon Aye, and Huiwang Ai.
Dr Chan’s research focus is on the chemical toxicology of food safety. His most important paper, “Quantitation of Aristolochic Acids in Corn, Wheat Grain, and Soil Samples Collected in Serbia: Identifying a Novel Exposure Pathway in the Etiology of Balkan Endemic Nephropathy,” J Agric. Food Chem., 64:5928-5934. (2016), was named the “Research Article of the Year” in the Journal of Agriculture and Food Chemistry. Through the work of Arthur Grollman and colleagues, we know that aristolochic acids are the cause of Balkan Endemic Neuropathy. It was thought that contamination of wheat and corn with Aristolochia clematitis berries was the route of human exposure to aristolochic acids. However, Simon Chan and co-workers showed that humans can be exposed to aristolochic acids directly through corn and wheat because these plants can absorb aristolochic acids from the soil. This work will impact strategies that we use to decrease exposure to these harmful environmental toxicants.
John Essigmann will present the Keynote lecture on Tuesday afternoon, August 21, 2018 during the TOXI program at the American Chemical Society National Convention in Boston. John is the William R. (1956) and Betsy P. Leitch Professor in Residence of Chemistry in the MIT Department of Chemistry. He is also Professor of Toxicology and Biological Engineering in the MIT Department of Biological Engineering. In addition, he is Director of the MIT Center for Environmental Health Sciences.
John earned a BS in Chemistry at Northeastern University and a PhD from MIT under the direction of Gerald Wogan, a pioneer in the field of chemical toxicology.
The overarching theme in John’s research is to understand how DNA damage leads to cancer and cell death. In particular, he strives to understand how the chemical and physical properties of specific DNA adducts lead to mutagenesis or cell death. He then uses this knowledge to design antitumor drugs in a process described as Fatal Engineering.
John’s lab was the first to synthesize and insert a single DNA adduct at a specific site in DNA in a cell. This revolutionary process has lead to his lab and, many others, probe the effects of specific DNA adducts to mutagenesis and lethality. In addition, the DNA damage response to these adducts have been identified.
John’s has used the knowledge gained in his mechanistic studies to design programmable antitumor drugs through a process described as Fatal Engineering. The Essigmann lab exploits the presence of tumor specific proteins, so that repair of the DNA adduct derived from the drug will occur in normal cells but fail in tumor cells. Thus the programmable drug kills tumor but not normal cells.
Judy Bolton has been named the recipient of the 2018 Founders’ Award. Judy Bolton is currently Distinguished Professor and Head, Medicinal Chemistry and Pharmacognosy at the University of Illinois at Chicago. Dr Bolton’s research in chemical toxicology is primarily focused on post-menopausal women’s health. She studies the carcinogenic effects of estrogens and antiestrogens and investigates natural alternatives to hormone replacement therapy. She is interested in determining why women who are taking hormone replacement therapy or selective estrogen receptor modulators (SERMs) are at increased risk for developing hormone dependent cancers such as breast or endometrial cancers. By developing a good understanding of the mechanism of how these widely prescribe drugs lead to increased cancer risk, we will be able to design alternatives that maintain the beneficial properties of estrogens/SERMs without generating genotoxic side effects. Dr Bolton has been active in educating the next generation of breast cancer researchers and chemical biologists as she has mentored over 20 Ph.D. and 25 postdoctoral fellows. Dr Bolton will organize a symposium at the TOXI Division’s program this summer in Boston.
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
2018 Judy Bolton
A nomination document should include:
- Nomination letter containing an evaluation of the nominee’s accomplishments, innovative research into Chemical Toxicology.
- Two additional letters of support.
- The candidates CV with a list of publications and patents.
Nominations should be sent directly to Awards Committee Chair no later than Monday October 15th 2018 at email@example.com
TOXI Program at the 256th ACS Meeting
Translesion DNA Polymerases
8:30 Introductory Remarks
8:35 TOXI 1 . Mechanisms to coordinate multiple DNA polymerases for TLS . M.A. Trakselis
9:15 TOXI 2 . Explosive mutation accumulation triggered by heterozygous human Pol ε proofreading-deficiency is driven by suppression of mismatch repair . Z.F. Purcell
10:10 TOXI 3 . Finding their way: How error-prone polymerases gain access to the bacterial replisome . J.J. Loparo
10:50 TOXI 4 . Mechanistic basis for the bypass of a bulky DNA adduct catalyzed by a Y-family DNA polymerase . R. Vayas, G. Efthimiopoulos, J. Tokarsky, C. Malik, A.K. Basu, Z. Suo
1:00 Introductory Remarks
1:10 TOXI 5 . Biological targets of electrophilic furan metabolites . L.A. Peterson
1:50 TOXI 6 . Mass spectrometry studies of DNA and protein adducts of reactive electrophiles . N.Y. Tretyakova
2:30 TOXI 7 . Electrophilic targeting of Keap1/Nrf2 signalling for disease prevention and treatment . A. Dinkova-Kostova, T. Honda, A.Y. Abramov
3:25 TOXI 8 . Chasing rainbows? Targeted covalent ligand design guided by precision electrophile signaling technologies . Y. Aye
4:05 TOXI 9 . Botanicals electrophiles modify multiple targets . J.L. Bolton
8:00 TOXI 10 . Repair and processing of DNA lesions induced by a dynamic electrophile . S. Byrne, K. Yang, S. Rokita
8:20 TOXI 11 . Mechanisms of bioactivation of the tobacco carcinogens and 2-amino-9H-pyrido[2,3-b]indole (AαC) and 4-aminobiphenyl (4-ABP) in human bladder . M. Bellamri, L. Yao, R. Turesky
8:40 TOXI 12 . Development of a novel approach for measuring N’-nitrosnornicotine bioactivation in humans by using deuterium-labeled analogs . E. Carlson, A. Goode, V. Gurvich, I. Stepanov, V. Jain, P. Upadhyaya, S.S. Hecht
9:00 TOXI 13 . A scheduled LC-SRM method for targeted DNA adductome analysis . Y. Cui, P. Wang, Y. Wang
9:30 TOXI 14 . Significant impact of divalent metal ions on the fidelity, sugar selectivity, and drug incorporation efficiency of human PrimPol . J. Tokarsky, P. Wallenmeyer, K. Phi, Z. Suo
9:50 TOXI 15 . Incorporating histone H2A variants facilitates global excision of uracil residues in nucleosomes . C. Li, S. Delaney
10:10 TOXI 16 . Integrating multi-“omics”- mass spectrometry-based methods to characterize electronic cigarette exposure in humans . R.P. Dator, P.W. Villalta, C.J. Hooyman, L.A. Maertens, S. Balbo
10:30 TOXI 17 . The C’5-pseudouridinyl radical . I. Sappy
11:00 TOXI 18 . Transcriptional inhibition and repair mechanism of alkyl phosphotriester DNA adducts in mammalian cells . Y. Tan, J. Wu, Y. Wang
11:20 TOXI 19 . Mass spectroscopy-based metabolomics reveals new insights on the biological effects of copper oxide nanoparticles in a human colon carcinoma cell line . N.G. Chavez Soria, D.S. Aga, G. Atilla-Gokcumen
11:40 TOXI 20 . A sensitive method for quantitation of abasic sites in isolated and cellular DNA by electrospray ionization tandem mass spectrometry . H. Chen, C.J. Rizzo, R.J. Turesky
Chemical Toxicology of Nanomaterials
1:00 Introductory Remarks
1:05 TOXI 21 . The future of nanotoxicology research: Filling knowledge gaps to safeguard health . A. Elder
1:45 TOXI 22 . Investigation of toxicity mechanism of nano-scale lithium battery material NMC to bacterial models . V. Feng
2:25 TOXI 23 . The size, surface chemistry and reactivity – all matter as toxicity determinants of fibrous nanomaterials . A.A. Shvedova
3:20 TOXI 24 . DNA methylation alterations by nanoparticles . L. Godderis
4:00 TOXI 25 . Nanomaterial induced mechanisms: Focus on nano cell interactions . A. Kraegeloh
Mechanisms of Binding, Transport & Biotransformation of Toxic Metals
8:30 TOXI 26 . Control of metal geometries within de novo designed three-stranded coiled coils. T. Pinter, L. Ruckthong, c. ervin, V.L. Pecoraro
9:15 TOXI 27 . ArsI, a C-As lyase for degradation of environmental organoarsenicals . V.S. Nadar, M. Yoshinaga, B.P. Rosen
10:00 TOXI 28 . Understanding the mechanism of carbon-metal bond cleavage by the organomercurial lyase MerB . H. Wahba, M. Stevenson, D. Wilcox, J.G. Omichinski
10:45 TOXI 29 . Interplay of copper transport proteins in the processing of platinum anticancer drugs in the cell . N. Dolgova, C. Yu, O. Dmitriev
Chemical Research in Toxicology Young Investigator Award
1:00 Introductory Remarks
1:10 TOXI 30 . Oxidized polyunsaturated fatty acids: Are they toxic or bioactive? .
1:10 TOXI 30. Oxidized polyunsaturated fatty acids: Are they toxic or bioactive? J. Lee
1:50 TOXI 31 . Antibiotics induce nitrosative stress in microorganisms . C.T. Chan
2:45 TOXI 32 . Advances in human biomonitoring of carcinogens by ion trap and high resolution accurate mass spectrometry . R. Turesky
3:25 TOXI 33 . Chemical approaches to investigate the toxicity of aristolochic acids . W. Chan
4:30 Introductory Remarks
4:40 TOXI 34 . Linking mutational spectra of chemical carcinogens to the mutational patterns seen in human tumors . J. Essigmann, B.I. Fedeles
7:00 – 9:00 Posters
TOXI 36 . Toxic effects and molecular mechanism of silver nanoparticles to Daphnia magna . J. Hou
TOXI 37 . Modified 3-deaza-3-alkyl-adenosines as minor groove alkylation mimics in translesion DNA synthesis . L.J. Weselinski, V. Begoyan, G. Kenyon, M. Tanasova
TOXI 38 . Ultrasensitive high-resolution mass spectrometric analysis of methyl DNA phosphate adducts in human lung . B. Ma, P.W. Villalta, J.B. Hochalter, I. Stepanov, S.S. Hecht
TOXI 39 . Screening for DNA adducts in human colon by high-resolution nano-ESI UHPLC/MSn . D. Konorev, R. Turesky
TOXI 40 . Application of an in silico tool for the risk assessment of an industrial process compliant to ICH M7 guidelines . M. Burns, M. Ott, S.J. Webb
TOXI 41 . Methylation in human hemoglobin is associated with age as analyzed by liquid chromatography tandem mass spectrometry . H.C. Chen, S. Ip
TOXI 42 . Characterizing uracil DNA glycosylase processivity in nucleosome core particles . E. Kennedy, S. Delaney
TOXI 43 . Structural and dynamic impact of single ribonucleotide incorporation on nucleosome structure . I. Fu, D. Smith, S. Broyde
TOXI 44 . Identification of photo-degradation products of nitroguanidine and toxicological implications . L. Moores, A. Kennedy, K.A. Gust, M.K. Shukla, L.K. Rabalais, D.L. Henderson, S.J. Jones
TOXI 45 . Gallic acid derivatives inhibit DNA repair enzyme ALKBH2 . Q. Tang, F. Chen, H. Ma, K. Bian, D. Li
TOXI 46 . Conformation-specific replication block from bulky 4-aminobiphenyl-modified DNA lesions . A. Cai, K. Bian, F. Chen, D. Li, B. Cho
TOXI 48 . Efficiency of initiating base excision repair on nucleosome substrates . A. Garlow, S. Delaney
TOXI 49 . Replication studies of N3-methyladenine in Escherichia coli cells . J. Yuan, Y. Wang
TOXI 50 . Unmasking the role of protein modification in the observed toxicity of aristolochic acid . C. Chan, W. Chan
TOXI 51 . Adverse reactions induced by the antiepileptic drug oxcarbazepine may stem from its metabolic biotransformation to carbamazepine . I. Martins, C. Charneira, M. Marques, A. Antunes
TOXI 52 . Identification of 4-(methylnitroamino)-1-(3-pyridyl-1-oxide)-1-butanone, a novel metabolite of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in rat urine . L. von Weymarn, R. Dator, S. Balbo, S.E. Murphy
TOXI 53 . Initiation of repair of DNA nucleobase lesions in the nucleosome core particle . M.E. Tarantino, S. Delaney
TOXI 54 . Dual cell system for in vitro studies of toxic blood gases . W.G. Senanayake, I. Petrikovics, D.E. Thompson
TOXI 55 . Analysis of acrolein-derived 1,N2-propanodeoxyguanosine adducts in human lung DNA from smokers and nonsmokers . J. Yang, S. Balbo, P.W. Villalta, S.S. Hecht
TOXI 56 . High-resolution/accurate mass DNA adductomics to screen for doxorubicin-induced adducts as biomarkers of therapeutic efficacy . A. Stornetta, K. Walters, R.P. Dator, V. Guidolin, P.W. Villalta, S. Balbo
TOXI 57 . Molecular level studies of the impact of poly (oxonorbornenes) and their gold nanoparticles conjugates on D. rerio. embryos . J.N. Klutts, A. Laranang, Z. Zheng, J. Saar, K. Lienkamp, R. Brewster, Z. Rosenzweig
TOXI 58 . Oxidation and removal of cytosine derivatives in the nucleosome . P. Caffrey, S. Delaney
TOXI 59 . Determining the basis of E. coli DinB and human pol kappa DNA damage specificity . H. Stern, T.A. Coulther, J. Winters, C.L. Mills, M.J. Ondrechen, P. Beuning
TOXI 60 . Prediction of the interaction region between the Y-family polymerase DinB and the transcription-repair coupling factor Mfd in E. coli . S.K. Fields, P. Beuning
TOXI 61 . Machine learning models for predicting hepatic steatosis based on in vivo data . B. Zdrazil, S. Jain, S. Klinting, S. Escher, G.F. Ecker, U. Norinder
TOXI 62 . Predicting drug metabolites using bacterial-based models . P.C. Rosado, J.P. Cruz, M.C. Justino, M. Marques, G.C. Justino
TOXI 63 . Characterization of LexA-regulated protein YbfE in E. coli . A. Hotchkiss, C. Kramer, P. Beuning
TOXI 64 . DNA damage induced by oxidative stress and lipid peroxidation in leukocyte DNA from African-American and Caucasian smokers . C. Ruszczak, B. Ma, J. Jensen, D. Hatsukami, I. Stepanov
TOXI 65 . Analysis of the spectrum of DNA modifications in Pseudomonas aeruginosa . E.A. Carlson, N.C. Wamer, T.A. Dodson, E.G. Prestwich
TOXI 66 . Probing the conformational dynamics of the Beta sliding clamp in Escherichia coli . M.L. Liriano, B. Koleva, P. Beuning
TOXI 67 . Potential DNA oxidation adducts for disease biomarkers . N.C. Wamer, E.A. Carlson, T.A. Dodson, E.G. Prestwich
TOXI 68 . Rapid microplate assay for acellular reactive oxygen species generation induced by engineered nanomaterials in real-time . R. Coreas, W. Zhong
TOXI 69 . Identifying toxicology concepts in the replacement of mercury catalysts during the acetylene hydrochlorination of vinyl chloride monomers . L. Green, J. Marshall, A.S. Cannon
TOXI 70 . Petrogenic and pyrogenic polycyclic aromatic hydrocarbons in human urine: comparison of their levels between two geographic regions . C. Mesaros, M. Huang, L.C. Hackfeld, R.P. Hodge, I.A. Blair, T.M. Penning
TOXI 71 . Dissecting interactions between E. coli DNA polymerase III and single-stranded DNA binding protein to gain insights into polymerase management . J. McIsaac, m. ondrechen, P.J. Beuning
TOXI 72 . Release of lead (Pb) and formation of disinfection byproducts during drinking water disinfection in the water distribution system . J. Liu, V.K. Sharma, C.M. Sayes
TOXI 73 . Mineralogy dependent dissolution of inhaled uranium in simulated lung fluids in uranium mine lands, New Mexico . E. Hettiarachchi, S. Paul, D. Cadol, B. Frey, G. Rubasinghege
TOXI 74 . Reactive oxygen species (ROS)-dependent release of an inhibitor from an aptamer . G. Premnauth, E.J. Merino
TOXI 75 . Effect of surface charge on toxicity of AuNPs; Are cationic AuNPs toxic? .
E. Lee, Y. Kwon
TOXI 76 . Nanomaterials in marine environment: toxicity to Artemia salina with and without the presence of Phe and Cd2+ . J. Lu, X. Lv, Z. Chen, X. Zhu
TOXI 77 . Molecular characterization of alcohol-induced DNA damage for cancer prevention . V. Guidolin, A. Carra’, P.W. Villalta, E. Carlson, S. Balbo
TOXI 78 . EB-Fapy-dG adducts of 1,3-butadiene: Synthesis, structural identification, and detection in human cells . S.S. Pujari, A. Groehler, D. Najjar, N.Y. Tretyakova
TOXI 79 . Inter-individual differences in metabolism of 1,3-butadiene . A. Degner, G. Madugundu, R. Arora, L.A. Peterson, N.Y. Tretyakova
TOXI 80 . 2, 2’, 3, 5’, 6 polychlorinated biphenyls (PCB-95) induce behavioral and GABAgenic neurotransmitter changes in zebrafish at early developmental exposure . P. Ranasinghe, C.M. Lee
TOXI 81 . Investigation of the effect of 2-phenethyl isothiocyanate (PEITC) on the levels of 4-hydroxy-1-(3-pyridyl)-1-butanone-releasing DNA adducts in oral cells of smokers . A. Jain, G. Yakovlev, B. Ma, I. Stepanov
TOXI 82 . Smoking and inflammation mediated epigenetic changes in a mouse model of lung cancer . J. Fernandez, C. Seiler, Q. Han, N.Y. Tretyakova
TOXI 83 . Independent synthesis and fate of DNA lesions generated from oxidative damage at the C-3′ and C-5′ position of deoxyribonucleotides . M. Bedi, A.C. Bryant-Friedrich
TOXI 84 . Ecotoxicology of nano-perovskites in aquatic environment . T. Zhou, W. Fan
TOXI 85 . Thermodynamic exposure reduction by amendment techniques to limit bioaccumulation during ongoing depositional input – a sediment mesocosm study with three organisms . A.P. Wang
TOXI 86 . MegaTox for predicting compound liabilities . K.M. Zorn, T. Lane, D.P. Russo, A. Clark, S. Ekins
TOXI 87 . Information-derived adverse outcome pathways with a case study on structural cardiotoxicity . A. Bender
TOXI 88 . Morphology-dependent cytotoxicity of SiC nanomaterials to human mesenchymal stem cells . F. Chen, G. Li, E. Zhao, J.V. Jokerst
TOXI 89 . Nanotoxicity predictive modeling: A case study on metal oxides nanoparticles . B. Rasulev
TOXI 90 . Surface-modified gold nanoparticles and their long-term impact on cellular pathways . P. Falagan Lotsch, E. Grzincic, C.J. Murphy
TOXI 91 . Noninvasive measurement of bladder carcinogen DNA adducts in human urinary cells by liquid chromatography-tandem mass spectrometry . B. Yun, M. Bellamri, S. Krishnamachari, R. Turesky
Nanomaterials in Drug Delivery: Efficacy & Toxicity Considerations
8:00 Introductory Remarks
8:05 TOXI 92 . What exactly is toxic about colloidal nanoparticle formulations? Results from the molecular level and the cellular level . C.J. Murphy
8:45 TOXI 93 . Targeting or enhanced selectivity: Toxicological considerations of nanoparticle therapeutics . R. Darvari
9:25 TOXI 94 . Expansile nanoparticles for the treatment of intraperitoneal mesothelioma . M.W. Grinstaff
10:20 TOXI 95 . Debugging nano–bio interfaces . M. Mahmoudi
11:00 TOXI 96 . Understanding mast cell activation in the safe development of nanotechnologies . J. Brown
Topics in Chemical Toxicology
1:00 TOXI 97 . Base and nucleotide excision repair of site-specific oxidatively generated guanine lesions in DNA substrates transfected into human cells . V. Shafirovich, K. Kropachev, M. Kolbanovskiy, N.E. Geacintov
1:20 TOXI 98 . Withdrawn
1:40 TOXI 99 . Site-specific production of hydroxyl radicals and synergistic DNA damage induced by the non-enzymatic activation of the anti-tuberculosis drug isoniazid by Cu(II) . B. Zhu
2:00 TOXI 100 . Insights into the molecular mechanism of alkylation- and platination-induced mutagenesis . S. Lee, Y. Kou
2:20 TOXI 101 . Kinetic basis of DNA synthesis by human DNA polymerase/primase PrimPol . L. Zhao
3:00 TOXI 102 . Levels of glyoxal-induced hemoglobin modifications correlate with DNA cross-links in human blood as determined by mass spectrometry . H.C. Chen, C. Liu
3:20 TOXI 103 . High mobility group box 1: A re-evaluation of its role in cancer . I.A. Blair, L. Weng, L. Guo, A. Vachani, C. Mesaros
3:40 TOXI 104 . Determining associations between transcriptomics and toxicity using co-expression network methods . B. Alexander-Dann, T. James, A. Bender
4:00 TOXI 105 . Inhibitors of the mitochondrial respiratory complex – Structure-based prediction of toxicity . G.F. Ecker, F. Troger, S. Jain, B. Zdrazil
4:20 TOXI 106 . Configurational and conformational equilibria of the N6-(2-Deoxy-D-erythro-pentofuranoysl-)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine (MeFapy-dG) lesion in DNA . M.P. Stone, S.N. Bamberger, C.K. Malik, T.L. Johnson-Salyard, S.K. Brown, H. Pan, C.J. Rizzo, M.W. Voehler
4:40 TOXI 107 . Using open bioactivity data for developing machine-learning prediction models for chemical modulators of the retinoid X receptor (RXR) signaling pathway . S. Kim
Abstracts for the 256th ACS Meeting
Mechanisms to coordinate multiple DNA polymerases for TLS
Duplication of the genome requires efficient, rapid, and coordinated activities of many enzymes that interact within a larger replisome complex. However, whether those interactions are stable or stochastic is currently being debated. These enzymatic abilities include high processivities, fidelities and plasticities to ensure faithful genome maintenance in spite of many obstacles to DNA synthesis. Many multisubunit enzyme subcomplexes have been identified within the replisome and most stabilize enzyme complexes on DNA for continued and processive enzymatic action over long stretches of the genome. One of the most well characterized subcomplexes is the elongation complex minimally consisting of the DNA polymerase holoenzyme which includes accessory proteins known to stabilize the association of the polymerase and direct repeated catalysis. In particular, the stability of the DNA polymerase holoenzyme is sensitive to stable secondary DNA structures, protein/DNA complexes, and various DNA damage. High fidelity (HiFi) DNA polymerases in complex with their accessory proteins are responsible for the bulk of genome synthesis, however, most organisms encode multiple separate specialized DNA polymerases to aid in overcoming genomic obstacles with translesion synthesis (TLS). The presence of anywhere from 2 to 18 separate DNA polymerases (depending on the organism) all with identical specificities for a 3’-OH primer/template DNA creates a complex multiequilibrium within a cell. Moreover, many of these DNA polymerases have been shown to directly or indirectly interact to enhance catalysis or coordinate synthesis across DNA lesions. However, multiequilibrium exchange processes can also predominate, allowing for a dynamic access to the DNA template. DNA polymerase active site coordination will be shown using a variety of kinetic and thermodynamic assays. These and other DNA polymerase interactions and equilibria properties will be put into context and compared across different domains of life to illustrate DNA synthesis mechanisms that maintain a high degree of fidelity and plasticity in spite of a multitude of genomic obstacles.
Explosive mutation accumulation triggered by heterozygous human Pol ε proofreading-deficiency is driven by suppression of mismatch repair
Tumors defective for DNA polymerase (Pol) ε proofreading have an incredibly high tumor mutation burden, ranging from 10 to over 500 mutations per megabase. This is among the highest mutation burden of any tumor type. A major unanswered question is whether loss of Pol ε proofreading by itself is sufficient to drive this mutagenesis, or whether additional factors are necessary. To address this, we used a combination of in vitro biochemistry using recombinant enzyme and next generation sequencing on human cell lines engineered to have defects in Pol ε proofreading and mismatch repair. Absent mismatch repair, monoallelic Pol ε proofreading deficiency causes a large and rapid increase in a unique mutation signature, similar to that observed in tumors from patients with biallelic mismatch repair deficiency and heterozygous Pol ε mutations. Restoration of mismatch repair is sufficient to suppress the explosive overall mutation accumulation. These results strongly suggest that concomitant suppression of mismatch repair, a hallmark of colorectal and other aggressive cancers, is a critical force for driving the explosive mutagenesis seen in tumors expressing exonuclease-deficient Pol ε
Finding their way: How error-prone polymerases gain access to the bacterial replisome
DNA damage can potently block the DNA replication machinery. Error prone polymerases can alleviate this block by gaining access to the replisome and synthesizing past the lesion in a process known as translesion synthesis (TLS). Given their low fidelity, the access of TLS polymerases to the replication fork must be tightly regulated in order to minimize error-prone DNA synthesis. Using a combination of in vitro and cell-based single-molecule assays along with biochemical and genetic approaches, we are determining how TLS polymerases are recruited to the bacterial replisome and gain access to the primer template junction. While the TLS polymerase, Pol IV, can simultaneously bind the β2 processivity clamp with the replicative polymerase, Pol III, in minimal in vitro reconstitutions, we find that it is largely restricted from replisomes in cells in the absence of DNA damage. Interactions between Pol III and the β2 clamp act as a gatekeeper to minimize TLS polymerase access; strengthening these interactions can dramatically suppress TLS in vitro and in cells. DNA damage dramatically enriches Pol IV near replisomes in cells with previously unknown interactions with the replisome being the major determinant of Pol IV recruitment to stalled forks.
Mechanistic basis for the bypass of a bulky DNA adduct catalyzed by a Y-family DNA polymerase
1-Nitropyrene (1-NP), an environmental pollutant, induces DNA damage in vivo and is considered to be carcinogenic. The DNA adducts formed by the 1-NP metabolites stall replicative DNA polymerases but are presumably bypassed by error-prone Y-family DNA polymerases at the expense of replication fidelity and efficiency in vivo. Our running start assays confirmed that a site-specifically placed 8-(deoxyguanosin-N2-yl)-1-aminopyrene (dG1,8), one of the DNA adducts derived from 1-NP, can be bypassed by Sulfolobus solfataricus DNA polymerase IV (Dpo4), although this representative Y-family enzyme was paused strongly by the lesion. Pre-steady-state kinetic assays were employed to determine the low nucleotide incorporation fidelity and establish a minimal kinetic mechanism for the dG1,8 bypass by Dpo4. To reveal a structural basis for dCTP incorporation opposite dG1,8, we solved the crystal structures of the complexes of Dpo4 and DNA containing a templating dG1,8 lesion in the absence or presence of dCTP. The Dpo4-DNA-dG1,8 binary structure shows that the aminopyrene moiety of the lesion stacks against the primer/template junction pair, while its dG moiety projected into the cleft between the Finger and Little Finger domains of Dpo4. In the Dpo4-DNA-dG1,8-dCTP ternary structure, the aminopyrene moiety of the dG1,8 lesion, is sandwiched between the nascent and junction base pairs, while its base is present in the major groove. Moreover, dCTP forms a Watson–Crick base pair with dG, two nucleotides upstream from the dG1,8 site, creating a complex for “-2” frameshift mutation. Mechanistically, these crystal structures provide additional insight into the aforementioned minimal kinetic mechanism.
Biological targets of electrophilic furan metabolites
Furan is a potent liver toxicant and carcinogen in rodents. Humans are exposed to furan through a variety of sources including heat-treated foods, tobacco and wood smoke and air pollution. Furan’s toxic effects requires metabolism. Oxidation by cytochrome P450 2E1 catalysis leads to the formation of a reactive α,β-unsaturated dialdehyde, cis-2-butene-1,4-dial (BDA). BDA is toxic and mutagenic to cells and consequently is considered responsible for the toxic effects of furan. In vitro, this compound reacts readily with protein and DNA nucleophiles. Studies in hepatocytes demonstrated that the reaction of BDA with glutathione (GSH) generates a different spectrum of reaction products in which BDA crosslinks GSH to wide variety of cellular amines including protein lysine residues, amino acids and polyamines. The relative amounts of these two reactive compounds is determined in part by the concentration of GSH present in the cell. Analysis of urinary metabolites provides evidence that both electrophilic metabolites are formed in vivo. The role of these two reactive metabolites in the overall toxic and carcinogenic effects of furan requires further study. Understanding the contributions of each metabolite to the overall toxic and carcinogenic properties of furan will assist the assessment of risk associated human furan exposure.
Mass spectrometry studies of DNA and protein adducts of reactive electrophiles
DNA and protein adducts are formed when electrophilic compounds react with nucleophilic sites in cellular biomolecules. The formation of covalent adducts with proteins and DNA is associated with increased risk for cancer and other chronic diseases. Our laboratory employs mass spectrometry based methodologies to identify novel DNA modifications, to elucidate their biological impacts, and to develop ultra-sensitive methodologies for their detection in humans. This presentation will discuss our recent efforts to characterize novel DNA adducts induced by human carcinogen 1,3-butadiene and to develop human DNA based biomarkers of exposure. We will also show recent data identifying unknown N-terminal amino groups of hemoglobin adducts in human blood as a lesion induced by quinone methides (QMs). The structural assignments for DNA and protein adducts were confirmed using high resolution mass spectrometry (HRMS) and comparisons with reference adducts synthesized in our laboratory. These investigations of DNA adducts are important because they help link human diseases such as cancer to specific environmental, dietary, and lifestyle factors that induce genetic and epigenetic damage. Accurate measurements of DNA adducts in humans can evaluate individual’s exposure to carcinogens and help identify individuals at greatest risk.
Electrophilic targeting of Keap1/Nrf2 signalling for disease prevention and treatment
The transcription factor Nrf2 regulates large networks of proteins that protect against damage by oxidants, electrophiles, and pro-inflammatory agents, allowing adaptation and survival under conditions of stress. Pharmacological activation of Nrf2 with small molecules (termed inducers) holds promise as a potential strategy for the prevention and treatment of chronic disease. Many inducers, such as the isothiocyanates and cyanoenones, are electrophiles and activate Nrf2 by targeting discreet highly reactive sensor cysteines within Kelch-like ECH-associated protein 1 (Keap1), the main negative regulator of Nrf2. At low concentrations, representatives of these two inducer classes chemically modify C151 in Keap1 impairing its repressor function, and allowing for Nrf2 accumulation and enhanced transcription of its target genes. However, at higher inducer concentrations, Nrf2 accumulation proceeds in the absence of C151, albeit at a lower magnitude. This flexibility in the sensing mechanism ensures a robust cytoprotective response. The Nrf2 regulatory network includes drug metabolizing, antioxidant, anti-inflammatory and metabolic enzymes, and affects mitochondrial function. Abnormal mitochondrial function is seen in patients with various neurological conditions, such as epilepsy, frontotemporal dementia, and Parkinson’s disease, and pharmacological Nrf2 activation with electrophilic inducers, some of which are in clinical trials, shows beneficial effects in cell culture and animal models of these conditions.
Chasing rainbows? Targeted covalent ligand design guided by precision electrophile signaling technologies
Precisely timed and spatially regulated electrophilic chemical signals are the essence of the non-conventional (and largely non-enzyme-assisted) biological signaling paradigm broadly known as redox signaling. However, defining the precise consequences of localized signals that engage with specific protein targets under physiologic conditions has proven to be highly challenging. Our laboratory has begun to address this missing link between specific low-occupancy electrophilic modifications upstream and dominant cellular responses downstream.
This talk will highlight how the fundamental knowledge garnered from these mechanistic and basic science studies provides a new avenue toward the design and development of precision covalent medicine.
Botanicals electrophiles modify multiple targets
Botanical dietary supplements with 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 preventive effects linked to multiple biological targets including hormonal, metabolic, inflammatory, and/or epigenetic pathways. For example, hops, licorice, and red clover extracts contain weak electrophiles which can activate the Keap1-nrf2 pathway and turn on the synthesis of detoxification enzymes such as quinone reductase and GST. 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 and their electrophilic metabolites used for women’s health, will ultimately lead to standardized botanical products with higher efficacy, safety, and chemopreventive properties.
Repair and processing of DNA lesions induced by a dynamic electrophile
Quinone methides (QMs) are a class of transient electrophilic intermediates that are formed in vivo from the metabolism of various compounds such as mitomycin C, tamoxifen, and butylated hydroxytoluene. Bifunctional QMs, capable of crosslinking DNA due to the presence of two electrophiles, alkylate DNA by forming both reversible and irreversible adducts. The formation of reversible adducts extends the effective lifetime of QMs. A bifunctional QM conjugated to the intercalator acridine (bisQMAc) was observed to migrate along duplex DNA by means of its dynamic alkylation. This process was observed for naked DNA that did not contain the histone proteins that package DNA in cells. For DNA to fit within the confines of a cell nucleus, it must be wrapped around octamers of histone proteins to form nucleosome core particles (NCPs). The compaction in NCPs has now been found to protect DNA against reaction of bisQMAc. Conversely, DNA alkylation by bisQMAc impedes assembly of DNA into NCPs. The histone proteins also serve as a target for QM alkylation, as bisQMAc reacts to form DNA- protein crosslinks within NCPs. BisQMAc was observed to alkylate a limited number of sites on the histones, primarily in their hydrophobic regions. BisQMAc’s intrinsic reversible chemistry permitted its transfer from a covalent adduct on DNA to a covalent adduct on the histones. Transfer of bisQMAc between DNA and protein, along with its dynamic migration in DNA, may pose challenges for cells as they attempt to repair the adducts formed by QMs. Preliminary evidence suggests that a bisQMAc DNA crosslink does not inhibit DNA polymerases as they process damaged DNA. Future investigations will seek to further characterize primer extension of damaged DNA by polymerases, as well examine repair of bisQMAc by the nucleotide excision repair enzymes.
Mechanisms of bioactivation of the tobacco carcinogens and 2-amino-9H–pyrido[2,3-b]indole (AαC) and 4-aminobiphenyl (4-ABP) in human bladder
Tobacco smoke is a major risk factor for lung cancer but also for liver, bladder, and gastrointestinal tract cancers. The combustion of tobacco produces many classes of carcinogens, including polycyclic aromatic hydrocarbons, N-nitrosamines, aromatic amines (AAs), and heterocyclic aromatic amines (HAAs). 2-Amino-9H–pyrido[2,3-b]indole (AαC) is the most abundant HAA formed in tobacco smoke with levels ranging from 37 to 258 ng/cigarette. These amounts are 25−100-fold higher than those levels of 4-aminobiphenyl (4-ABP) and 2-naphthylamine, two AAs implicated in bladder cancer of smokers. AαC is a liver carcinogen, a transgene colon mutagen, and an inducer of colonic aberrant crypt foci, an early biomarker of colon neoplasia, in mice. The carcinogenic potential of AαC is unknown in humans. However, AαC damages DNA in human lymphocytes and lymphoblastic cells, based on the Comet assay. Moreover, AαC is bioactivated in human hepatocytes and T lymphocytes with greater efficiency than other HAAs or 4-ABP, leading to the formation of high levels of DNA adducts. AαC is bioactivated by cytochrome P450s (CYPs), to form 2-hydroxyamino-9H-pyrido[2,3-b]indole (HONH-AαC), which undergoes esterification by sulfotransferase (SULTs) or N-acetyltransferase (NATs) to form reactive intermediates that bind to DNA. We examined the capacity of 4-ABP, AαC, but also 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-3,8-dimethylmidazo[4,5-f]quinoxaline (MeIQx), carcinogenic HAAs formed also formed in tobacco smoke or cooked meats, to undergo bioactivation in human bladder. DNA adducts levels were measured is RT4 cells, an epithelial human bladder cell line. AαC and 4-ABP form DNA adducts in RT4 cells, whereas PhIP and MeIQx do not. Thus, bladder cells can directly bioactivate AαC and 4-ABP and prior hepatic N-oxidation is not required. The inhibition of CYP1 activity, by α-naphthoflavone, resulted in a strong decrease DNA adduct formation of AαC but had no effect of the level of 4-ABP adducts, indicating the involvement of other CYPs or oxidases in the bioactivation of 4-ABP. Our current work is to identify the major CYPs involved in the bioactivation of 4-ABP and the role of NATs and UDP-Glucuronosyl-Transferases (UGTs) in the metabolism of 4-ABP and AαC in bladder cells. Our data provide support to the epidemiological observations implicating AAs and possibly AαC in tobacco smoke as risk factors for bladder cancer.
Development of a novel approach for measuring N’-nitrosnornicotine bioactivation in humans by using deuterium-labeled analogs
N’-Nitrosonornicotine (NNN) is a critical component of smokeless tobacco due to its relatively high abundance and ability to cause tumors in the esophagus and oral cavity of laboratory animals. For these reasons, NNN is considered a likely cause of tobacco-related cancers. To exhibit these effects, NNN must be bioactivated by cytochrome P450 oxidation, implying that individuals who extensively activate NNN may have a higher risk for cancer. P450 oxidation generates highly reactive α-hydroxyNNN species that can bind to DNA. These species also hydrolyze and are further oxidized to 4-oxo-4-(3-pyridyl)butanoic acid (keto acid) or 4-hydroxy-4-(3-pyridyl)butanoic acid (hydroxy acid), which are rapidly excreted in urine and have potential as human biomarkers for NNN activation; however, these products are also minor metabolites of nicotine. Because nicotine is >10,000 fold higher than NNN in tobacco, it is unclear what amount of keto acid and hydroxy acid result from NNN bioactivation. To overcome this issue, we will measure NNN activation in humans who use smokeless tobacco containing [pyridine-D4]NNN (0.2 µg/g tobacco). After a week of acclimation, 24-hour urine will be collected for three days and analyzed by an established assay for the sum of both metabolites. Because [pyridine-D4]NNN is 4 amu heavier, NNN-derived metabolites will be completely distinct from nicotine-derived metabolites. In pursuit of this goal, our lab has developed a spray-based method for enriching tobacco with [pyridine-D4]NNN, gained full FDA and IRB approval, optimized the GMP process for manufacturing [pyridine-D4]NNN-enriched smokeless tobacco, and tested our assay on [pyridine-D4]NNN-treated rats. Ongoing work includes recruiting subjects and analyzing urine samples as they become available. This study will provide the first data on NNN bioactivation in humans and identify inter-individual differences in NNN bioactivation.
Scheduled LC-SRM method for targeted DNA adductome analysis
DNA adducts constantly arise from endogenous metabolism and environmental exposure. This usually results in an array of DNA lesions. For example, exposure to cigarette smoke is known to give rise to a plethora of alkylated DNA lesions. Therefore, a DNA adductomic approach is required to robustly quantify DNA adducts in cellular and tissue DNA.
Recent developments in scheduled selected-reaction monitoring (SRM) analysis of peptides and proteins have afforded highly sensitive and high-throughput analytical methods for samples in complicated matrices. In this respect, normalized retention time (iRT) has been widely used for scheduled SRM analyses of peptides, where iRT is empirically derived from the retention time of analyte of interest relative to that of the standard peptides. By using a similar method, we established the iRT values of approximately 20 synthetic modified nucleoside standards from the retention times of canonical 2′-deoxynucleosides and ribonucleosides on an nLC-nESI-MS/MS/MS system (LTQ XL) as well as on a high-resolution nLC-nESI-MS/MS system (Q Exactive Plus).
Since some DNA adducts are bulky and may not be released quantitatively during the enzymatic digestion process, release efficiencies of DNA adducts were evaluated by digesting calf thymus DNA spiked with different amount of adduct containing oligodeoxynucleotides (ODNs). We examined the release efficiencies of several DNA adducts, including the (5′S) diastereomer of 8,5′-cyclo-2′-deoxyadenosine (S-cdA) and 8,5′-cyclo-2′-deoxyguanosine (S-cdG). We found that the release of S-cdG from a lesion-containing oligodeoxynucleotide was nearly quantitative, whereas only 15% S-cdA could be released as a mononucleoside under the same digestion conditions. We are now in the process of employing this DNA adductomic approach for assessing the formation and repair of oxidatively induced and alkylated DNA lesions.
Significant impact of divalent metal ions on the fidelity, sugar selectivity, and drug incorporation efficiency of human PrimPol
Human PrimPol is a recently discovered bifunctional enzyme that displays template-directed primase activity and DNA polymerase activity. PrimPol has been implicated in nuclear and mitochondrial DNA replication fork progression and restart as well as DNA lesion bypass. Published evidence suggests that PrimPol is a Mn2+-dependent enzyme as it shows significantly improved primase and polymerase activities when coordinating Mn2+, rather than Mg2+, as a divalent metal ion cofactor. However, the abundance of cellular Mg2+ is much greater than that of Mn2+. At present, there is debate among those who study PrimPol as to which divalent metal ion cofactor is utilized by this unique protein. We experimentally studied PrimPol in the presence of both cofactors in an attempt to elucidate which of them is preferred. Our pre-steady-state kinetic analysis revealed that PrimPol incorporates correct nucleotides with 100-fold higher efficiency with Mn2+ than with Mg2+. Strikingly, the fidelity of PrimPol in the presence of Mn2+ was determined to be in the range of 3.4×10-2 to 3.8×10-1, indicating that PrimPol displays extremely unfaithful polymerase activity, which may not be biologically relevant. Additionally, we demonstrated a simple kinetic basis for discrimination by PrimPol to select for deoxyribonucleotides over ribonucleotides with both Mg2+ and Mn2+ likely arising from decreased nucleotide binding and subsequent incorporation. Furthermore, PrimPol also showed efficient incorporation of the anti-cancer drugs, cytarabine and gemcitabine, onto growing DNA strands revealing its potential role in cellular toxicities induced by these drugs.
Incorporating histone H2A variants facilitates global excision of uracil residues in nucleosomes
In eukaryotes, histone variants are proteins that displace their canonical counterpart in nucleosomes. Histone variant incorporation can lead to profound chromatin structure alterations thereby impacting multiple biological processes, including DNA repair, transcriptional regulation and genome stability. While the deposition of H2A.X and H2A.Z variants into chromatin has been well-defined for signaling double strand break and nucleotide excision repair and for promoting the recruitment of DNA repair machinery, roles of histone variants in base excision repair (BER) remain largely unknown. Here, we investigate the influence of histone variants from H2A family on global excision of uracil initiated by uracil DNA glycosylase (UDG) and single-strand selective monofunctional uracil DNA glycosylase (SMUG1). Specifically, we reconstitute a population of variant-containing nucleosome core particle (NCP) substrates, which bear uracil residues at a variety of translational and rotational positions, and evaluate the global profile of UDG and SMUG1 repair in packaged DNA. We find that rotational positioning of nucleobases correlates with UDG activity in canonical NCP substrate except in the dyad region. In contrast, repair initiated by SMUG1 is significantly suppressed. Notably, some variants are observed to facilitate uracil removal in defined regions of the NCP. The results will be discussed in terms of histone protein core composition, as well as structural features and dynamics of variant-containing NCPs. Our observations reveal potential functional properties of H2A variants in BER within the context of chromatin.
Integrating multi-“omics”- mass spectrometry-based methods to characterize electronic cigarette exposure in humans
Characterizing chemical exposures in humans remains a significant challenge. Our lab has been developing state-of-the-art high-resolution mass spectrometry-based methods to characterize the exposome and determine how it may influence overall health. Because it is challenging to capture the complexity of exposures, often at trace levels, improved tools are needed to move this field of research forward. We have recently developed a neutral loss (NL) screening and relative quantitation strategy for the targeted analysis of reactive carbonyls in biological fluids. In addition, a comprehensive DNA adductomics approach was developed to characterize covalent modifications in DNA generated from these exposures. Here, we have integrated these MS-based methods to characterize electronic cigarette exposure in humans, with the goal of identifying reactive carbonyls generated during vaping and the corresponding DNA adducts formed in the oral cavity. Human saliva and oral cell samples from e-cigarette users (n=10) and non-user controls (n=10) were obtained to screen reactive carbonyls and DNA adducts. Reactive carbonyls in saliva were derivatized with 2,4-dinitrophenylhydrazine to form hydrazones and analyzed by the NL screening method. Likewise, DNA from oral cells were isolated, hydrolyzed to nucleosides, and analyzed by both targeted and non-targeted DNA adductomics approaches. Using the NL loss screening strategy, increased levels of acrolein, methylglyoxal, and formaldehyde were observed after vaping, while the levels of acetaldehyde and glyoxal vary within subjects possibly due to variations in composition of the e-liquid used. This information was then used to develop targeted and non-targeted DNA adductomics approaches to monitor the corresponding DNA adducts in oral cells of e-cigarette users. Our results showed increased levels of acrolein-derived DNA adducts, in particular, gamma-OH-Acro-dG in e-cigarette users compared to non-users. We are currently investigating other DNA adducts that might be relevant to e-cigarette exposure using non-targeted DNA adductomics approaches and expanding our analysis to a larger sample size.
The C’5-pseudouridinyl radical
Pseudouridine, the 5-ribosyl isomer of uridine (U) is the most abundant nucleic acid modification found in all domains of life and all types of RNA. Studies have shown that, urinary levels of pseudouridine are higher in Alzheimer’s disease (AD) patients and that RNA oxidation is a major component in the pathogenesis of Alzheimer’s disease (AD) and other neurodegenerative diseases. These studies point to a correlation between higher urinary levels of pseudouridine and oxidative stress in AD. The goal of this project is to synthesize a C5’-radical precursor for pseudouridine to study the effect of oxidative stress on RNA at this site. The synthesized substrate will be used to determine the products derived from the C5’-radical and their effect on RNA structure and function. Hence, this will provide key information about the role of this nucleoside in RNA related processes and disease etiology
Transcriptional inhibition and repair mechanism of alkyl phosphotriester DNA adducts in mammalian cells
Most mammalian cells in nature are terminally differentiated and non-dividing but actively transcribing mRNAs to maintain normal physiological processes. While it’s well understood that DNA adduct formation is a critical step of carcinogenesis in proliferating cells, it’s also important to explore how transcriptional machinery responds toward DNA damage regardless of cell proliferating states. In addition to modifying nucleobase moieties, environmental and endogenous genotoxins can also attack non-carbon bonded oxygen atoms on backbone phosphate groups in DNA to produce phosphotriester adducts. Researches have shown that phosphotriester lesions are fairly common and persistent in physiological conditions, yet little is known whether they impede transcription, and whether and how they are repaired in mammalian cells. To address these questions, we employed previously reported competitive transcription and adduct bypass (CTAB) assay to assess the effects of site-specifically incorporated alkyl phosphotriester adducts, i.e. Sp and Rp diastereomers of Tp(Me)T, Tp(Et)T, Tp(nPr)T and Tp(nBu)T on the efficiency and fidelity of transcription in mammalian cells. Moreover, we demonstrated that the effect of these PTEs on transcription is modulated by transcription-coupled nucleotide-excision repair pathway. In particular, we observed genetic depletion of CSB and inactivation of XPA in cultured human cells led to marked diminution of transcription bypass efficiencies of the alkyl phosphotriester lesions. Together, we revealed, for the first time, the repair and transcriptional perturbations of alkyl phosphotriester lesions in human cells.
Mass spectroscopy-based metabolomics reveals new insights on the biological effects of copper oxide nanoparticles in a human colon carcinoma cell line
Engineered nanomaterials have unique properties compared to their bulk counterparts. Hence, they have been applied to consumer products such as electronics, as well as in medicine and agriculture. Copper oxide nanoparticles (CuO NPs) are one example of nanomaterials used in a wide range of consumer products due to their conductivity and biocidal properties. CuO NPs have shown to be toxic in various organisms, however the reasons for their toxicity is yet to be elucidated. As such, there is a growing need to determine the molecular effects of these nanoparticles to different biological systems. In this work, the toxicity of CuO NPs was evaluated in different cell lines, showing a dose-dependent toxicity. To investigate the underlying reasons for their toxicity, an untargeted metabolomics approach was employed in human colon carcinoma cells using liquid chromatography high resolution mass spectrometry-based metabolomics. Significant changes in the metabolome were observed after exposure to CuO NPs, in agreement with concentration-dependent toxicity, reflecting cellular stress induced by CuO NPs. Based on the changes in different metabolites classes induced by CuO NP exposure and previous studies from our laboratory, we propose that oxidative stress plays a role in CuO NP-induced toxicity. These result provide insights on the biological effects of CuO NPs in mammalian cells and their effect on cellular homeostasis.
Sensitive method for quantitation of abasic sites in isolated and cellular DNA by electrospray ionization tandem mass spectrometry
The depurination of DNA bases producing an abasic site is a frequent lesion in DNA. This process may occur spontaneously, induced by chemical carcinogens, radiation, oxidative stress, or as an intermediate of the aberrant base excision repair pathway. Several anti-tumor drugs used in chemotherapy also lead to abasic site formation. If not efficiently repaired, the chemically unstable abasic lesions can lead to DNA strand scission, DNA-DNA cross-links and induce mutations. Therefore, detection of abasic sites can provide information about DNA damage due to an array of reactive species. The most well-established method for measuring abasic sites is the use of a biotin-containing aldehyde-reactive probe (ARP) to react with the deoxyribose residue. Labeling with ARP is a semi-quantitative method that requires external standards. In addition, the labeling process is cumbersome and can produce artefactual abasic sites because the derivatization of the abasic site is performed after the isolation of DNA. Our lab previously developed an unambiguous method for quantification of abasic sites following derivatization with O-4-nitrobenzylhydroxylamine and employing liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The detection limit was 3 abasic sites per 107 nucleotides. In this work, we describe a new LC-MS method for labeling abasic sites. We incubated DNA with O-(pyridin-3-ylmethyl)hydroxylamine (PMOA) and used an organocatalyst to accelerate the derivatization reaction. To mitigate artifact formation, we directly treated PMOA with living cells or isolated nuclei and extracted DNA after removing excess labeling reagent. After addition of 13C-labeled internal standard, the DNA was digested with a cocktail of nucleases, followed by solid phase extraction. The level of labeled abasic sites were quantitated by LC-ESI-MS/MS. The labeling method and stability of the derivatized abasic site were established using single- and double-stranded oligonucleotide containing a specific amount of abasic site. The sensitivity of the method is dramatically improved by the use of pyridinyl functional group, with a limit of detection of 50 fg on column.
The future of nanotoxicology research: Filling knowledge gaps to safeguard health
Advances in nanotechnology have fueled the development of products and devices that address needs in medicine, engineering, and electronics. The great diversity in applications and nanomaterial physicochemistry, along with high production volumes in some cases, has led to concerns about adverse health outcomes that might result from unintentional exposures. Over the last decade, much has been learned about the mechanisms by which some classes of nanomaterials exert toxicity. There are, however, significant knowledge gaps that present challenges for fitting this mechanistic information into a comprehensive understanding of human and environmental health risk. First, there is a relative dearth of nanomaterial-specific exposure data amid a growing recognition that exposures are likely to be complex, particularly when the entire product lifecycle is considered. Secondly, little is understood about if and how nanomaterials are delivered to target tissues and if they undergo physicochemical changes as they are transported and accumulate in tissues. Thirdly, the dose that accumulates in target tissues is largely unknown. Lastly, while much has been learned about the physicochemical properties that are linked to adverse outcomes, there is a remaining need to determine if the relationships hold true across a wide range of diverse nanomaterials. Addressing these questions in the context of both realistic exposure conditions and what is known at present about response mechanisms is the immediate need for future research and will ensure the safer development of nanomaterial-containing products.
Investigation of toxicity mechanism of nano-scale lithium battery material NMC to bacterial models
As nanoscale lithium-intercalating complex metal oxides, such as lithium nickel manganese cobalt oxide (NMC), are more widely used as the cathode material in lithium-ion batteries, the environmental and health risk assessment of these materials are increasingly imperative. Bacteria, at the bottom of the food chain, are key environmental indicators in assessing the influence of the release of such material. In this study, we examined the impact of liquid suspensions of NMC to model bacteria, Bacillus subtilis and Shewnella oneidensis, observed that bacterial respiration and growth were both affected by the presence of NMC material. We further investigated the mechanism of toxicity by conducting ion dissolution studies, intracellular ROS and metal-uptake assays. Bacterial DNA double strands breakage was observed in both bacterial models by single-cell gel electrophoresis. A high-resolution mass spectrometry-based approach has also been applied to identify bacterial DNA adducts upon nanomaterial exposure. These results together suggest that DNA damage as a toxicity mechanism of NMC exposure to bacterial cells, likely via intracellular ROS generation and transition metal ion uptake.
Size, surface chemistry and reactivity – all matter as toxicity determinants of fibrous nanomaterials
During evolution, all living organisms have been exposed to and dealt with a variety of natural nanoparticles, from viruses and products of biological decay, to various fibrillar organic compounds, and minerals produced by the chemical weathering of rocks and volcanic eruptions. However, recent explosive technological revolution in the field of material science and nanotechnology yielded an unprecedented variety and large amount of engineered nanomaterials (EN) with remarkably broad applications in essentially all spheres of life. The limited understanding of how these nano-size products interact with molecular machineries of cells, subcellular organelles and tissues, possibly affecting human health and safety are a major public concern. Among different fibrous nanomaterials, Carbon Nanotubes (CNTs) and Nanocellulose (NC) are emerging as important objects of toxicological studies. Of particular interest, are likely toxic effects of CNTs, especially with respect to pulmonary and potential cancer outcomes. Recognition of EN by the cells of the immune system is the primary event that defines the meaning and the magnitude of complex immune reactions. Notably, immune-competent cells may respond to nanoparticles through mechanisms and pathways utilized for the detection of viruses and bacteria. Therefore, there may be complex relationships between the infection process and inflammatory responses to nanoparticles resulting in potent effects of nanoparticles on pulmonary clearance of different pathogens. We will present data demonstrating that NC and CNT in doses relevant to potential occupational exposures, may exert toxic effects in different types of human cells, lungs of exposed animals and human subjects. We will discuss the use of computational and structural modeling approaches for the identification of essential patterns in the nanoparticle-biomolecule interactions for prediction of their immunomodulatory effects. The different mechanisms of toxicity will be discussed in the context of risk assessment and regulatory measures and actions and their sufficiency in environmental and occupational settings.
DNA methylation alterations by nanoparticles
Introduction: Concerns about the carcinogenicity of carbon nanotubes (CNT) remain. While studies have addressed genotoxic effects of CNT, only limited are available on epigenetic effects. We investigated DNA methylation alterations in sub-cytotoxic and sub-genotoxic dose in in vitro, animals and humans.
Material and Methods: In vitro studies were performed in 16HBE and THP-1. BALB/c mice were administered intra-tracheally with single-wall CNT SWCNTs and multi-wall MWCNTs at high dose of 2.5 mg/kg and low dose of 0.25 mg/kg for 48 hrs. Finally, 24 workers exposed to aggregates of MWCNT of 500 nm–100 μm with concentrations of 4.6–42.6 μg/m3 and 43 unexposed referents were followed-up. Global DNA methylation and demethylation patterns were analysed by LC-MS/MS. Methylation of specific genes was measured by Pyromark 24 ® (Qiagen). Genome-wide assessment of DNA methylation was performed with Infinium HumanMethylation450 BeadChip Array.
Results: In general, we did not find global DNA methylation alteration for both CNTs. In 16HBE, respectively 2398 and 493 genes were hypomethylated after exposure to MWCNTs and SWCNTs. SWCNT exposure showed methylation changes on functionally important genes, such as SKI proto-oncogene (SKI), glutathione S-transferase pi 1 (GTSP1) and shroom family member 2 (SHROOM2) and neurofibromatosis type I (NF1). In THP-1 cells, CNTs induced promoter-specific altered methylation of genes involved in several signaling cascade, vascular endothelial growth factor and platelet activation pathways. In lungs of BALB/c mice CNTs affected methylation of Atm gene, despite no effects on oxidative stress and DNA damage, global DNA methylation and hydroxymethylation was observed. Finally, analysis of gene-specific DNA methylation showed significant changes for DNMT1, ATM, SKI, and HDAC4 promoter CpGs in MWCNT-exposed workers.
Conclusions: Epigenetic changes seem to occur at sub cyto-genotoxic concentrations. Alteration in DNA methylation pattern could be a natural reaction of cells but could also silence critical genes and reprogram cellular functions.
Nanomaterial induced mechanisms: Focus on nano cell interactions
The promising applications of nanomaterials – ranging from energy storage to biomedicine – are based on effects arising from the structure and properties of their building blocks. In contrast, the physico-chemical properties of nanomaterials constitute the basis for biological effects of nanomaterials. The latter are promoted by specific entry pathways and distribution within the body and tissues, by uptake into single cells, specific intracellular distribution as well as by interactions with functional macromolecules. In order to predict any toxic potential of nanomaterials, it is of paramount importance to understand the materials properties and behavior under relevant conditions as well as the chemical and molecular mechanisms behind biological responses.
Generally, it is assumed that the presence of nanomaterials close to or even within tissues or cells aggravates their effects. Here, “structural mechanisms” are addressed that contribute to nanomaterials effects. The detection and localization of nanomaterials in tissues or within cells are key for understanding their interaction mechanisms and effects.
Examples for the application of confocal laser scanning and super resolution STED microscopy in the analysis of nano cell interactions are given. The latter was used to quantify the internalization of silica nanoparticles into A549 cells, allowing for a comparison between extracellular and intracellular nanoparticle numbers. Using live cell imaging, the association of nanoparticles with various types of vesicles in these cells was shown. Using differentiated Caco-2 cells, it was shown that these cells did not internalize quantum dots and that intact quantum dots did not translocate across the cell layer. Furthermore, STED microscopy revealed agglomeration of 32 nm nanoparticles after penetration into the nuclei of Caco-2 cells.
Finally, the distribution of silica nanoparticles in 3D liver microtissues, prepared from HepG2 cells, was analyzed, giving insight into the penetration of nanoparticles into tissues.
Control of metal geometries within de novo designed three-stranded coiled coils
Long interspersed nuclear elements-1 (L1) is an active autonomous retrotransposon. L1 encodes two proteins in different open reading frames. The protein expressed by the first open reading frame (ORF1p) may be an RNA binding and chaperone protein. It contains a three-stranded coiled coil (3SCC) domain near the N-terminus that facilitates formation of the biologically active homotrimer. This 3SCC domain is composed of seven amino acid (labelled a,b,c,d,e,f,and g within the heptad) repeats and a stammer insertion of three extra residues that modifies the helical structure. Cysteine residues are located within the 3SCC at three hydrophobic positions (2 a- and 1 d-layer sites) within this domain. Based on sequence and crystallographic comparison to our de novo designed peptides that contain cysteine layers in the hydrophobic core of 3SCCs, we propose that ORF1p binds transition or heavy metals with relatively high affinities, a feature that has not been previously recognized. We first demonstrate the geometric similarities between the ORF1p and our de novo designed construct tris(cysteine) layers. We propose that due to the high degree of similarity, that the ORF1p binds metal ions in a similar fashion as TRI and Grand constructs. Thus, the metal binding properties of these tris(cysteine) environments will be reviewed and their potential application to ORF1p metal binding will be discussed. This has major implications for ORF1p coiled coil domain stability and dynamics, ultimately significantly impacting the resulting biological activity, and a largely unexplored avenue of the chemical biology of DNA mobile elements.
ArsI, a C-As lyase for degradation of environmental organoarsenicals
Organoarsenicals are introduced into the environment both from microbial biotransformation of inorganic arsenic or anthropogenically. Methylarsenicals such as monosodium methylarsonic acid (MSMA) have been extensively utilized as herbicides, and aromatic arsenicals such as roxarsone (Rox) are used as antimicrobial growth promoters for poultry and swine. The active and toxic forms are the trivalent arsenic-containing species. Microorganisms with an arsI gene degrade these toxic organoarsenicals to less inorganic arsenic. ArsI is a non-heme ferrous-dependent dioxygenase that catalyzes C-As bond cleavage. The crystal structure of TcArsI from the thermophilic bacterium Thermomonospora curvata was solved in the apo form, with metals Ni(II), Co(II) or Fe(III) or with substrates phenylarsenite (PhAs(III)) or trivalent Rox(III). Many dioygenases bind substrates directly to the active site. In contrast, the ArsI organoarsenical binding site is a vicinal cysteine pair in a flexible loop. The structure of a mutant protein (Y100H/V102F) was solved in two different crystal forms with two other orientations of the flexible loop. These results suggest that a loop-gating mechanism controls the catalytic reaction (Fig. 1). In the ligand-free open state, the loop is exposed to solvent, where it can bind trivalent organoarsenical. The loop moves toward the active site, where forms a closed state that orients the C-As bond for dioxygen addition and cleavage. Elucidation of the enzymatic mechanism of this novel C-As lyase reaction will enhance our understanding of recycling of environmental organoarsenicals.
Understanding the mechanism of carbon-metal bond cleavage by the organomercurial lyase MerB
Mercury is introduced into the environment through both natural occurrences and as a result of numerous human activities. Once introduced into the environment, mercury can exist as elemental mercury (Hg0), ionic mercury (HgI and HgII) or methylmercury (MeHg), and there is a constant flux between these three forms as part of the natural biogeochemical cycle. Select bacterial strains survive in mercury-contaminated environments due to the presence of a transferable genetic element (the mer operon), which enables them to convert both HgII and MeHg to the less toxic Hg0 using two enzymes, the organomercurial lyase MerB and the mercuric ion reductase MerA. To identity features responsible for its unique catalytic activity at the atomic level, structural studies were performed with MerB in the presence of organomercurial, organotin or organolead compounds. The studies with organomercurial compounds identified three key catalytic residues (C96, D99 and C159) required for carbon-Hg bond cleavage and demonstrated that the HgII product remains bound in the active site. Studies with dimethyltin (DMT), triethyltin (TET) and trimethyllead (TML) revealed that these compound inhibit MerB using distinct mechanisms, but in all cases the initial binding event requires D99 (Fig. 1). In contrast, diethyltin (DET) and diethyllead (DEL) are substrates and the resulting metal ion products remain bound in the active site of MerB. Together, these studies demonstrate that D99 of MerB plays a key role in organometal substrate selectivity, carbon-metal bond cleavage as well as in transferring the metal ion product to MerA. Due to its unique activity, MerB represents an ideal model system for creating green chemistry to remediate MeHg-contaminated sites and these results provide important mechanistic insights into identifying new ways to exploit MerB for such purposes.
Interplay of copper transport proteins in the processing of platinum anticancer drugs in the cell
Cisplatin (cis-diamminedichloridoplatinum(II)) has been widely used against testicular, ovarian, cervical, bladder, head and neck cancers, melanoma and lymphomas for several decades. The predominant mechanism of anticancer activity of cisplatin is DNA cross-linking, which interferes with replication and transcription. Despite severe side effects and intrinsic and acquired tumor resistance, cisplatin remains a mainstay of anticancer chemotherapy along with the other platinum derivatives, such as carboplatin and oxaliplatin. Cancer cells acquire resistance to cisplatin by various mechanisms. Copper transport proteins Ctr1, Atox1, ATP7A and ATP7B have all been implicated in modulating cell sensitivity to platinum drugs. Cisplatin binds to the CxxC motifs in copper chaperone Atox1, and in the metal-binding domains of the ATP-driven copper transporters ATP7A and ATP7B, which normally bind copper. Elevated expression of ATP7A and ATP7B has been correlated with cancer resistance to cisplatin, while the role of Atox1 appears to be more complex. In the cell, Atox1 may transfer platinum to the metal binding domains of ATP7B, similar to its function in delivering copper to these proteins. At the same time, Atox1 has been reported to be a transcriptional regulator, suggesting an intriguing possibility that interaction of Atox1-Pt with DNA may potentiate pharmacological activity of cisplatin. Investigation of cisplatin reactions with Atox1 under the physiological redox conditions, and gene-specific DNA cross-link profiling in cancer cells led us to a model that places Atox1 is at the junction of cisplatin detoxification and target delivery pathways.
Oxidized polyunsaturated fatty acids: Are they toxic or bioactive?
Polyunsaturated fatty acids (PUFA) are essential in our body and sufficient level can only be achieved through our diet. Being conjugated to phospholipids, it is vulnerable to peroxidation in situ by oxidants and free radicals due to the multiple skipped diene units in the structure, and subsequently generate oxidized compounds known as isoprostanoids (IsoPs) and isofuranoids (IsoFs). Under homeostasis or normal oxygen tension, the release of IsoPs predominates whereas under extreme oxidative stress, IsoFs are released in a greater amount. In in vivo, it is hydrolyzed by phospholipase A2 and platelet activating factor acetylhydrolase into free form for circulation. IsoPs and IsoFs from n-6 PUFA, arachidonic acid is recognized as reliable biomarkers to assess oxidative stress in biological systems, and augmentation such as 15-F2t-Isoprostane is considered toxic and often related to diseases. However, other functional IsoPs and IsoFs are released through other PUFAs such as those derived from adrenic acid (n-6 PUFA), where it recently showed to have prognostic values for neurological diseases, and from docosahexaenoic acid (n-3 PUFA) where it displayed bioactive and protective values in vascular diseases. Methods of identification and application of IsoPs and IsoFs in disease models will be explained in this presentation and cautions in interpreting data related to oxidative stress damage through PUFA will be noted.
Antibiotics induce nitrosative stress in microorganisms
Antibiotics generate bacterial lethality via various cellular pathways, including alteration in cellular respiratory activities that promote reactive oxygen species (ROS) production and subsequently, damage a wide range of cellular components. Here, we used a quantitative approach to study the role of a specific ROS, peroxynitrite (ONOO–), in antibiotic-induced cell death. We demonstrated that antibiotic treatments led to increased 3-nitrotyrosine (NO2Y) generation in proteomes whose formation specifically requires ONOO– to serve as a nitrating agent. Additionally, decreasing cellular ONOO- levels by using chemical and genetic methods reduced NO2Y formation, as well as cell killing during antibiotic exposures. Furthermore, we observed that antibiotics elevated anaerobic respiratory activities which consumed nitrate to serve as an electron acceptor during the process. Elimination of nitrate consumption by nitrate reductase gene deletion imposed protective effect to the mutant against antibiotic stresses and reduced NO2Y formation, suggesting that the anaerobic respiratory pathway is involved in NO2Y formation. Together, our quantitative analyses support that ONOO- contributes to antibiotic-induced cell death through exerting nitrosative stress which led to formation of damaged products, such as NO2Y.
Advances in human biomonitoring of carcinogens by ion trap and high resolution accurate mass spectrometry
Protein and DNA adducts formed by hazardous chemicals in the environment and diet are key events in toxicity and chemical carcinogenesis. Robust biomarkers of toxic chemicals are required for risk assessment. Our research has been devoted to the development of long-term stable biomarkers of heterocyclic aromatic amines (HAAs) a class of probable human carcinogens formed in cooked meat and tobacco smoke, and Aristolochic acids (AA), upper urinary tract carcinogens found in Aristolochia herbs used for medicinal purposes. Using liquid chromatography with triple quadrupole (TQ), ion trap (IT) or Orbitrap high-resolution (HR)-multistage MSn scanning, we have characterized and measured HAAs, their urinary metabolites, protein, and DNA adducts in humans. For example, the HAA 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP) was measured in human hair and its metabolites were quantified in urine by TQ/MS2. By employing IT MSn, we characterized and measured human serum albumin adducts of PhIP. We used HR-MSn to show that PhIP can form DNA adducts in human prostrate while other HAAs do not. Thus, PhIP forms reactive intermediates capable of damaging macromolecules in humans; however, a signature mutation is still sought to link PhIP with cancer. Our investigations on AA conducted with Dr. A.P. Grollman and colleagues at Stony Brook University employing IT MSn showed that AA-DNA adducts are frequently detected in renal tissues of kidney cancer patients in Asia where traditional herbs are often used. AA-DNA adducts combined with the characteristic mutational spectrum induced by AA in P53 gene, provide compelling evidence for a role of AA in upper urothelial tract cancer. We are developing methods to screen for multiple exogenous and endogenous DNA adducts of genotoxicants by untargeted HR-MSn scanning. HR-MS instrumentation has greatly expanded our ability to measure biomarkers of carcinogens and can be employed to identify exposures and assess the etiological role of hazardous chemicals in human cancers.
Chemical approaches to investigate the toxicity of aristolochic acids
Aristolochic acids (AAs) are a family of phytotoxins produced naturally by Aristolochia plants and are among the most potent human carcinogens and nephrotoxins. Prolong exposure to AAs through mis-use of AAs-containing herbal medicine or via dietary intake of AAs-contaminated food were recognized as a major cause of nephropathy, kidney failure, and upper tract urothelial cancer – the so-called aristolochic acid nephropathy or Balkan endemic nephropathy. Although the use of AAs-containing herbs are banned worldwide, it was estimated that over 100 million people are at risk of AA poisoning. Despite decades of research, the pathophysiology underlying the toxicity of AAs, the molecular mechanism of renal interstitial fibrosis in particular, remain poorly understood. We have developed liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) with isotope-dilution methods for the rigorous quantification of two major oxidative stress-induced oxidation products: the 8-oxo-deoxyguanosine in DNA oxidation; and the N6-formyl-lysine formed as formaldehyde, generated from oxidative stress, reacted with lysine residues in proteins. The study revealed significantly elevated levels of the biomarkers in the target organs (e.g. kidney) but not in the non-target organs (e.g. liver), indicating oxidative stress could be one of the mechanisms in the pathogenic process for AAs. Further, we investigated the potential used of protein adducts of AAs as an alternative dosimeter to DNA-AA adducts for assessing the risk of exposure to AAs. Results showed for the first time significantly higher levels of protein adducts than DNA adducts, indicating the protein adducts may be a more sensitive biomarker in risk assessment of AAs in kidney failure and upper tract urothelial cancer. Because there is currently no available method for diagnosis of AAs poisoning, and the etiological pathways are difficult to be determined. It is our belief that quantitating the protein adducts in blood/tissue protein may provide a promising method to fill this gap in clinical testing and to identify high risk individuals.
Linking mutational spectra of chemical carcinogens to the mutational patterns seen in human tumors
Mutations are a permanent historical record of past chemical and biochemical events experienced by a cell. With every replication, the genome accumulates mutations in patterns that reflect: (a) the sequence context-dependent formation of DNA damage; (b) the differential activity of DNA repair proteins, which, depending on the type of lesion, can eliminate, ignore or intensify the mutagenic consequences of the lesion; and (c) the choice of replication machinery that synthesizes the nascent genomic copy. These three factors result in a richly contoured sequence context-dependent mutational spectrum that, from appearances, is distinct for most individual forms of DNA damage. Such a mutagenic pattern, if appropriately decoded can reveal the history of genome-altering events such as chemical exposures, pathogen-stimulated base deaminations, metabolic stress, and inflammation, which in turn can provide an indication of the underlying causes and mechanisms of genetic disease. Modern tools have positioned us to develop a deep mechanistic understanding of the cellular factors and pathways that modulate a mutational process and, in turn, provide opportunities for better diagnostic and prognostic biomarkers, better exposure risk assessment and even actionable therapeutic targets. The goal of this talk is to present a bottom-up, lesion-centric framework of mutagenesis that integrates the contributions of lesion replication, lesion repair and lesion formation to explain the complex mutational spectra that emerge in the genome following exposure to mutagens. The mutational spectra of the well-studied hepatocarcinogen aflatoxin B1 serve as specific examples, but the implications are meant to be generalizable
Prediction of carcinogenic behavior of hexacyclic polycyclic aromatic hydrocarbons using aromatic sextet theory and ionization potentials
Polycyclic aromatic hydrocarbons (PAHs) with hexacyclic rings of the isomer families C24H14, C26H14, and C28H14 are commonly identified in environmental combustion samples and many of them represent biological hazards. For instance, in the C24H14 group, eight out of thirteen PAHs are experimental proven carcinogenic molecules. In an effort to predict the carcinogenic conduct of the other five C24H14, nine C26H14, and eight C28H14 isomers, a method using the aromatic sextet theory and the ionization potential has been proposed. To the extent of my knowledge, it is the first time that the carcinogenic conducts of dibenzo[de,mn]naphthacene, naphtho[8,1,2-bcd]perylene, phenanthro[5,4,3,2-abcde]perylene, benzo[lmn]naphtho[2,1,8-qra]perylene, phenanthro[2,1,10,9,8,7-pqrstuv]pentaphene, benzo[cd]naphtho[3,2,1,8-pqra]perylene, and peri-naphthacenonaphthacene have been suggested. The results obtained are consistent with the predictions formerly made using ultraviolet-visible shifts of the spectral bands due to the formation of carcinogenic PAHs.
Toxic effects and molecular mechanism of silver nanoparticles to Daphnia magna
Silver nanoparticles (AgNPs) have been assessed to have a high exposure risk for humans and aquatic organisms. Toxicity varies considerably between different types of AgNPs. This study aimed to investigate the toxic effects of AgNPs with different particle sizes (40 and 110 nm) and different surface coatings (sodium citrate and polyvinylpyrrolidone, PVP) on Daphnia magna and their mechanisms of action. The results revealed that the citrate-coated AgNPs were more toxic than PVPcoated AgNPs and that the 40 nm AgNPs were more toxic than the 110 nm AgNPs. Transcriptome analysis further revealed that the toxic effects of AgNPs on D. magna were related to the mechanisms of ion binding and several metabolic pathways, such as the “RNA polymerase” pathway and the “protein digestion and absorption” pathway. Moreover, the principal component analysis (PAC) results found that surface coating was the major factor that determines the toxicities compared to particle size. These results could help us better understand the possible mechanism of AgNP toxicity in aquatic invertebrates at the transcriptome level and establish an important foundation for revealing the broad impacts of nanoparticles on aquatic environments.
Modified 3-deaza-3-alkyl-adenosines as minor groove alkylation mimics in translesion DNA synthesis
Among the variety of nucleic acid modifications, alkylation at the N3-site of purines such as adenosine, plays a critical role in the processing of genomic information. However, a direct evaluation of the outcomes of adenosine modification at the N3-site is compromised by N3-alkylation-driven depurination of DNA. Recently, we described a general strategy for the synthesis of 3-deaza-3-alkyl-adenine and adenosine derivatives. The resulting 3-deaza-3-alkyl-adenosine phosphoramidites were incorporated into oligonucleotides, and the influence of 3-alkyl group was investigated through DNA duplex thermal stability studies. We assessed the impact of DNA minor groove alkylation on the replication and transcription processes using primer extension assay with human DNA and RNA polymerases.
Ultrasensitive high-resolution mass spectrometric analysis of methyl DNA phosphate adducts in human lung
Genomic DNA can be modified by endogenous and exogenous methylating agents to form methyl DNA adducts, which could play a key role in subsequent biological processes. The formation of methyl DNA adducts is a critical step in carcinogenesis due to exposure to methylating carcinogens, while it also serves as a mechanism of therapeutic methylating agents. Methyl DNA phosphate adducts, formed by methylation of the oxygen atoms of the DNA phosphodiester linkages, have been detected in animals treated with methylating carcinogens. However, detection of these adducts in humans has not been reported. We developed an ultrasensitive liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry method for detecting methyl DNA phosphate adducts. The developed methodology had a limit of detection of 4 amol on-column of thymidylyl(3′-5′)thymidine methyl phosphotriester (TpMeT), one of the methyl DNA phosphate adducts. Nineteen structurally unique methyl DNA phosphate adducts were detected in human lung DNA. The adduct levels were measured in tumor and adjacent tissue pairs from 15 subjects, with DNA containing means of 9.3 and 12.8 adducts per 109 nucleotides, respectively. The detection of methyl DNA phosphate adducts in human lung in this study provides a novel methodology for applying these adducts as biomarkers to study human exposure to environmental and medicinal methylating agents.
Screening for DNA adducts in human colon by high-resolution nano-ESI UHPLC/MSn
The International Agency for Research on Cancer (IARC) has classified processed/cured meats as Group 1 carcinogens (carcinogenic to humans), and grilled meats as Group 2A carcinogens (possibly carcinogenic to humans), largely implicated in colorectal cancer. Cooked meats contain several classes of carcinogens including heterocyclic aromatic amines (HAAs) and polycyclic aromatic hydrocarbons (PAHs) in grilled red meats, and N-nitroso compounds (NOCs) in processed meats. These compounds undergo bioactivation by enzymes to produce reactive species that form DNA adducts, which can lead to cancer. 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most mass-abundant HAA formed in high-temperature cooked meats, and is a colorectal and prostate carcinogen in rodents. The prototypical PAH is benzo[a]pyrene, a recognized human lung and possible gastrointestinal tract carcinogen. NOCs are formed during the curing process of meat. Secondary amines in meat products also may undergo nitrosation in the GI tract to form endogenous NOC. In addition, ingested heme has been proposed to catalyze the formation of lipid peroxidation products in the lower GI tract and contribute to colonic DNA damage. Whereas HAAs and PAHs lead to bulky DNA adducts formed with 2′-deoxyguanosine (dG), NOCs produce small alkylation products adducts such as O6-methyl-2′-deoxyguanosine and O6-carboxylmethyl-2′-deoxyguanosine. However, specific mass spectrometry-based methods have rarely been employed to screen for DNA adducts in human colorectum. Recently, our laboratory identified PhIP-DNA adducts in the prostate biopsy samples of cancer patients. Identification of mutation-prone DNA adducts of meat mutagens in colorectum would strengthen the epidemiology paradigms for a role of these chemicals in the etiology of colorectal cancer. We are developing methods to measure DNA adducts of these different classes carcinogens in human colorectum. DNA is isolated from fresh-frozen tumor adjacent colon tissue and matching formalin-fixed paraffin-embedded (FFPE) tissue. DNA is then digested with a cocktail of enzymes to produce individual non-modified nucleosides and chemically-modified nucleosides. The DNA adducts are enriched from non-modified nucleosides either by offline solid-phase extraction or online trapping before analysis via high-resolution nanoESI-UHPLC-MS/MS. The limit of quantification for these DNA lesions is 2 – 5 adducts in 109 DNA bases.
Application of an in silico tool for the risk assessment of an industrial process compliant to ICH M7 guidelines
In the development of pharmaceutical agents the use of highly reactive reagents, though beneficial in synthesis, also results in an increased probability of the reagents leading to mutagenicity if they persist as an impurity. Controlling these potentially mutagenic impurities (PMIs) is a critical part of the development of any pharmaceutical product. The ICH M7 guidance outlines a series of control options that are aligned with risk-based principles. One control strategy, proposed by Teasdale et al, is explicitly referenced in ICH M7 and allows for the application of scientific understanding of the physicochemical properties of a PMI and the processes involved to justify its removal via a semi-quantitative scoring system. The approach relies on the expert knowledge of the risk assessor, however the approach can be further enhanced by the application of a standardised in silico system, namely Mirabilis, and the adherence to a consortium-driven framework.
The Mirabilis software has been developed as a standardised in silico tool for the prediction of purge factors of impurities according to the Teasdale approach, led by a cross-industry consortium. Herein we present a theoretical case study to highlight the process involved when performing a risk assessment aligned to control option 4 for regulatory disclosure using Mirabilis. When performed ahead of other control options, the approach can facilitate the identification and subsequent solution to problems associated with quantification of PMI levels in the final product.
Methylation in human hemoglobin is associated with age as analyzed by liquid chromatography tandem mass spectrometry
Methylation of biomolecules is involved in many biological processes, including signal transduction and regulation of transcription. The contributing methylating agents can originate from endogenous and exogenous sources (such as cigarette smoking). Human hemoglobin is easily accessible from blood and has been used as molecular dosimetry for chemical exposure. We recently developed a method for characterization and quantification of the extents of methylation and ethylation in hemoglobin by nanoflow liquid chromatography-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) under the selected reaction monitoring mode. Using this method, the relative extents of methylated and ethylated peptides in human hemoglobin were quantified in nonsmoking subjects with various ages. Among the nine methylation sites, we found that the extents of methylation were significantly higher in old subjects at the N-terminal and His-20 of α-globin, and at the N-terminal and Glu-26 of β-globin. Moreover, the extents of methylation at these sites were significantly correlated with the age of the subjects. On the other hand, no statistically significant difference was found in the ethylated peptides. Our results suggest that that age should be considered as a factor when studying methylated biomolecules as biomarkers in epidemiological studies.
Characterizing uracil DNA glycosylase processivity in nucleosome core particles
Our DNA is constantly exposed to both endogenous and exogenous sources of damage that necessitates multiple repair mechanisms to protect the genetic information it contains. For instance, the base excision repair (BER) pathway targets modified nucleobases, removes the nucleotide, and replaces it with the appropriate unmodified nucleotide in its place. BER is initiated by glycosylases, which recognize and remove modified nucleobases. Glycosylases have the challenge of locating specific lesions quickly and efficiently among a genome of undamaged DNA. Previous studies on free duplex have shown that this efficiency might be attributed to a glycosylase’s processivity, or its ability to locate and excise multiple modified nucleobases in a single binding event. There are two main mechanisms of processivity between lesion sites: DNA sliding and hopping. Sliding is characterized by the glycosylase’s close association to the DNA by following the phosphate backbone. Hopping is characterized by small dissociations from the DNA with a high probability of re-associating. The relative contribution of each mechanism depends on the location and distance between the lesion sites within the free duplex.
Thus far, the characterization of glycosylase processivity has only been on free duplex substrates. However, genomic DNA is packaged in an organized, compact manner to fit in the cell nucleus. Our goal is to determine how glycosylase processivity is influenced by DNA compaction. We use the first order of DNA packaging, the nucleosome core particle (NCP), to study the ability of uracil DNA glycosylase (UDG) to recognize and remove two site-specific uracil lesions within an NCP DNA substrate.
Structural and dynamic impact of single ribonucleotide incorporation on nucleosome structure
Ribonucleotides misincorporated by replicative DNA polymerases are by far the most common DNA lesion. The presence of ribonucleotides in DNA is associated with genome instability, including replication stress, chromosome instability, gross chromosomal rearrangement, and other mutagenic events. Nucleosome and chromatin assembly as well as nucleosome positioning are affected by the presence of ribonucleotides. It has been shown that nucleosome formation is reduced with only a single ribonucleotide present. Ribonucleotides are primarily removed from DNA by the ribonucleotide excision repair (RER) pathway via the RNase H2 enzyme; this enzyme is specialized to remove a single ribonucleotide within duplex DNA. While the structural implications of a single ribonucleotide in duplex DNA have been well studied, how a single ribonucleotide embedded in nucleosomal DNA impacts nucleosome structure and dynamics, and the impact of chromatin structure on RER, have not been explored. We have carried out three-microsecond molecular dynamics simulations of nucleosomes containing a single ribonucleotide incorporated at various translational and rotational positions. We find that the presence of the C2′-OH group on the ribose impacts the local conformation and dynamics of both the ribose-containing nucleotide as well as nearby DNA nucleotides; the nature of these alterations depends on the rotational and translational setting, including whether the ribose faces toward or away from the histones. Ultimately, the origin of the ribose’s effect stems from its preference for the C3′-endo conformation; however, the specific nature of the impact is governed by the local environment. Because RNase H2 interacts with the C2′-OH group in the C3′-endo conformation, we hypothesize that the recognition of this lesion by the enzyme is modulated by the stability and dynamics of the ribose C3′-endo conformation and its local concomitants that are revealed in our MD simulations.
Identification of photo-degradation products of nitroguanidine and toxicological implications
Insensitive munitions (IMs) provide increased soldier safety via decreasing the likelihood of sympathetic detonations during their manufacture, shipping, storage, and use. IMX-101 is a triple base, melt-cast explosive containing dinitroanisole (DNAN), nitrotriazolone (NTO), and nitroguanidine (NQ). The toxicity of the constituents (LC50 = 25, 1235, 1486, and 63 mg/L for DNAN, NTO, NQ, and the IMX-101 mixture, respectively in Daphnia pulex) are lower than the toxicity of trinitrotoluene (TNT, 4.03 mg/L in Daphnia magna), the compound IMX-101 is to replace. However, upon irradiation by intense laboratory UV light the toxicity of NQ is increased 100 to 1000 fold over the parent material, leading to challenges with wastewater discharge at manufacturing facilities requiring new treatment methods to be integrated. This has spurred research towards identifying which of the many products generated lead to this multiple order of magnitude increase in toxic responses to the tested organisms. Understanding the mechanisms of this degradation process and toxicity have been explored experimentally and computationally, finding inorganic (nitrite, nitrate, ammonia, cyanide) and organic (cyanamide, cyanoguanidine, nitrosoguanidine) chemicals as photo-degradation products, and various modes of oxidative stress leading which can result in organismal death. Interestingly, the onset of the 100-fold increase in toxicity to D. pulex occured when only a modest proportion of the parent NQ has been UV-transformed (~5%). Further, the UV-photolysis products are relatively stable where the increased toxicity in D. pulex was persistent after UV-treated solutions were allowed to age on a laboratory bench for 8 days. Work is currently underway to determine the toxicity of the identified products, with further planned research efforts focusing on the toxicology of NQ solutions degraded under natural sunlight to determine the veracity of the experiments conducted under intense UV irradiation.
Gallic acid derivatives inhibit DNA repair enzyme ALKBH2
Nuclear DNA is constantly exposed to stresses from environmental and endogenous processes to generate DNA damages. Living organisms have evolved a number of repair pathways to avoid the deleterious effects of DNA lesions. One such pathway involves the α-ketoglutarate/Fe(II)-dependent AlkB family enzymes that catalyze the direct removal of alkyl adducts. Recent studies have reported several inhibitors for the AlkB and other α-ketoglutarate/Fe(II)-dependent enzymes. Here, we demonstrate a class of natural products, gallic acid and its derivatives inhibit the ALKBH2 enzyme, a human homolog of AlkB, by chelating the Fe(II) ion in the catalytic cycle. In this study, we carry out biochemical reactions of ALKBH2 with gallic acid derivatives. We also investigate other possible competitors for α-ketoglutarate and ascorbic acid in order to explore the inhibitory mechanisms of gallic acid and its derivatives.
Conformation-specific replication block from bulky 4-aminobiphenyl-modified DNA lesions
Bulky DNA adducts often exist in multiple conformations that are slowly interconverted to one another. Different conformations have shown to lead to different kinetic and binding capacities, presumably resulting in different mutational consequences. Studies on conformation-specific replication block are lacking due to the complex dynamic nature of DNA replication. A bulky lesion-induced replication block is difficult to capture because existing assays usually detect a consequence of all conformers or the culmination of cellular behaviors. Recently, we have mapped all 16-possible DNA duplexes (CTTCTNG*NCCTC: N= A, G, C, T; G*=ABP) containing the human bladder carcinogen 4-aminobiphenyl (ABP). The results revealed a striking sequence-dependent mixture of B-type (B)/stacked (S) conformations (TG1*G2T [67%B:33%S] and TG1G2*T [100%B]). In this study, we attempted to correlate replication block to different conformations derived from the bulky ABP lesion. We utilized a combination of surface plasmon resonance (SPR) and steady-state kinetics as well as E. coli in-cell replication bypass assays to study the conformational differences in vitro and in the cell. The SPR results indicated that the bulky ABP lesion increases polymerase binding affinity and decreases nucleotide selectivity. The Lineweaver–Burke enzyme inhibition model suggested that the S-conformer may play as a replication blocker.
Mutagenicity of DNA-peptide crosslink in human cells
DNA-protein crosslinks (DPCs) are formed when proteins bind covalently to DNA following exposure to certain metal compounds, aldehydes, and free-radicals generated by antitumor drugs or ionizing radiations. These extremely bulky lesions can potentially disrupt vital cellular processes like chromatin packaging, replication, and DNA repair, thereby affecting the integrity of the genome. Despite their relevance to human health, however, the molecular basis of mutagenicity and genotoxicity of DPC is still poorly understood. During the repair process, certain DPCs are believed to undergo partial cleavage of the protein to generate a DNA-peptide crosslink (DpC). In this study, a 23-nucleotide long fragment of DpC was synthesized with an 11-mer peptide cross-linked to a modified nucleobase 5-formylcytosine (5fC). This DpC was ligated to a gapped plasmid to generate a DpC containing construct, which was replicated in human embryonic kidney (HEK) 293T cells. A comparative study of translesion synthesis (TLS) efficiency and mutagenesis of the DpC in a series of cell lines with knockouts or knockdowns of various TLS polymerases was performed. Mutation frequency was significantly lowered in Pol η/κ double knockout cells, suggesting that they play critical role in error prone bypass of this DpC. Furthermore, Pol ζ was found to play a key role in the error prone bypass, as determined by a substantial reduction in mutagenicity in Pol κ/ζ double knockout cells. However, viability of the DpC was not significantly affected in these cells, suggesting a role of replicative polymerases such as pol δ/ε to bypass the lesion. Hence, we propose that both replicative and TLS polymerases are involved in the bypass of DpC in human cells.
Efficiency of initiating base excision repair on nucleosome substrates
Our DNA is under the constant threat of damage from exogenous and endogenous sources leading to numerous abnormalities with deleterious effects on our physiology. The human body has mechanisms, such as base excision repair (BER), to rectify these DNA lesions and has been well studied on free duplex substrates. However, in vivo DNA is known to be packaged around an octamer of histone proteins in nucleosomes, the repeating unit of which is the nucleosome core particle (NCP). It remains unclear if BER is effective in NCP substrates and what governs its activity. It is also of interest to examine the role of post translational modifications (PTMs) on the histone proteins. The goal of this work is to quantify the repair ability of glycosylases in various rotational and translational positions in an NCP substrate with an analog of histone H3K56Ac.
Replication studies of N3-methyladenine in Escherichia coli cells
N3-methyl adenine (3-mA) is a cytotoxic lesion formed from the reaction of adenine in DNA with SN2-type methylationg agents. Toxicity of this lesion has been attributed to its ability to impede DNA replication since the N3-methyl group strongly blocks the interaction between polymerases and the minor groove of DNA. However, the detail mechanism has yet been identified because 3-mA has a short half-life in vitro and is readily converted to an abasic site. Here we synthesized oligodeoxyribonucleotides (ODNs) containing a site-specifically inserted stable 3-deaza analogue of 3-mA, 3-deaza-3-methyl adenine (3-dmA). We will incorporate the ODNs into single-stranded M13 plasmid and investigate how 3-dmA impedes DNA replication and induces mutations in E. coli cells. We will also define the identities of translesion synthesis DNA polymerases involved in bypassing the lesion in cells.
Unmasking the role of protein modification in the observed toxicity of aristolochic acid
With emerging evidence pointing to the presence of aristolochic acid (AA), a potent nephrotoxic and carcinogenic phytotoxin, in food crops and the detection of AA-DNA adducts in various patient tissues, AA has been proved to have play a crucial role in the development of Chinese herb nephropathy and Balkan endemic nephropathy (BEN). Previous studies also described the abilities of AA to induce oxidative stress both in vitro and in vivo. However, the toxicological mechanism remain poorly understood. We initiated the study by exposing Escherichia coli and Sprague Dawley rats to AA. Proteins were then extracted from E. coli cells and SD rat tissues (including kidneys, lung, liver and stomach). Biomarkers of oxidative stress-induced modifications to proteinic lysine and tyrosine, such as nitrated, halogenated, hydroxylated and formylated, were quantitated using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) coupled with stable isotope dilution method to measure their concentrations in E. coli cells and rat tissues. The levels of different biomarkers in rat tissues were further compared to reveal the potential organ-specific oxidative damages to human by AA. It is anticipated that results from our studies could put forward the knowledge of AA cytotoxicity associated with BEN.
Adverse reactions induced by the antiepileptic drug oxcarbazepine may stem from its metabolic biotransformation to carbamazepine
Oxcarbazepine (OXCBZ), a second-generation anticonvulsant used in the long-term treatment of epilepsy, was added to the set of treatment choices with the aim to overcome the most common adverse effects of carbamazepine (CBZ), a widely used first-generation antiepileptic. However, while eliciting milder symptoms, OXCBZ is associated with the same adverse reactions induced by CBZ.
Currently, little is known about the mechanisms underlying OXCBZ-induced toxic outcomes; nonetheless, drug bioactivation is anticipated to be a major cause of these events. This assumption led us to investigate the biologically plausible Phase II sulfonation of the reduced monohydroxy derivative (licarbazepine, MHD), which is the clinically relevant OXCBZ metabolite. Interestingly, all attempts to obtain the sulfonation product of MHD, either by standard chemical methods or under biologically relevant conditions, led to the formation of CBZ, stemming from an elimination process. CBZ formation, following MHD sulfonation, is consistent with the previously reported identification of CBZ in the plasma of patients on OXCBZ therapy, and represents a credible explanation for the similar but milder/delayed adverse reactions associated with OXCBZ as compared to CBZ.
Identification of 4-(methylnitroamino)-1-(3-pyridyl-1-oxide)-1-butanone, a novel metabolite of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in rat urine
The tobacco specific carcinogen NNK is considered a human lung carcinogen, and many studies have characterized the pathways of NNK metabolism in rats and humans. However, in a NNK metabolic profiling study of rats that were administered NNK and [pyridine-D4]NNK we identify a novel urinary metabolite. The accurate mass, m/z 240.0977, and the fragmentation pattern of this metabolite were consistent with an NNK-N-oxide derivative with one additional oxygen atom. The identity of this metabolite could not be confirmed from mass spectrometry fragmentation alone. Fortuitously , the NNK-N-oxide standard we used contained the same compound as a minor (1.5%) contaminant. Purification of the contaminant by HPLC, followed by LC-MS/MS, 1H-NMR and 1H-1H-COSY NMR analysis confirmed the identity of this compound as 4-(methylnitroamino)-1-(3-pyridyl-1-oxide)-1-butanone . A key identifying feature of the proton NMR spectrum of the new metabolite was loss of the E/Z characteristics observed in N-nitroso- as opposed to N-nitro- compounds. This metabolite was also generated in vitro from NNK in the presence of phenobarbital induced rat liver microsomes and rat P450 2B1 (Corning® Supersomes™). A related metabolite, m/z 242.1135, was also detected in rat urine and in vitro microsomal incubations. The accurate mass and fragmentation pattern of this metabolite suggests that it is the reduced form of the nitro amine metabolite, 4-(methylnitroamino)-1-(3-pyridyl-1-oxide)-1-butanol. The presence of this second novel metabolite is consistent with the abundance of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in the urine of NNK-treated rats. The possible presence of these metabolites in smokers’ urine is currently being investigated.
Initiation of repair of DNA nucleobase lesions in the nucleosome core particle
Our DNA is subject to damaging agents, resulting in the formation of several types of damage, including single-strand and double-strand breaks, crosslink formation, and nucleobase modifications. In particular, nucleobase lesions include deamination of cytosine and 5-methylcytosine to uracil and thymine, respectively, the oxidation of guanine to 8-oxo-7,8-dihydorguanine, and the formation of abasic sites. The base excision repair (BER) pathway removes and repairs damage to nucleobases, initiated by recognition and removal of the damage by a DNA glycosylase. The resulting abasic site is then further processed in the pathway: the backbone is nicked, the deoxyribose-phosphate removed, the gap filled, and finally the backbone sealed. Though the steps of BER have been characterized on free duplex substrate, nuclear DNA is packaged into chromatin. The most basic unit of chromatin is the nucleosome core particle (NCP) consisting of ~150 bp of DNA wrapped about an octameric core of histone proteins (two copies each of H2A, H2B, H3, and H4). We seek to determine how DNA packaging affects the initiation of BER on nucleobase lesions in the context of the nucleosome core particle.
Dual cell system for in vitro studies of toxic blood gases
Reactive blood gases such as hydrogen sulfide, methyl mercaptan, and hydrogen cyanide are naturally present in blood at trace levels. At higher concentrations these blood gases become toxic. Direct detection of the blood gases themselves is challenging because their signals are often masked by those of their reaction partners and the more abundant components of the complex matrix in which they reside. Here we overcome this challenge by separating the gas detection cell from the reaction cell. The two cells share a common well-sealed headspace. The volatile blood gases partition out of the reaction matrix into the headspace, and out of the headspace into the detection solution, where they undergo chemical reactions tailored for trapping and detecting the blood gas of interest. The kinetics of reactions can be followed by simultaneously monitoring spectral changes in both cells and by gas phase monitoring (for example by SERS) of the headspace. In preliminary tests of the system, we have generated mercaptans by reducing polysulfides with tris(2-carboxyethyl)phosphine (TCEP) in the reaction cell. Mercaptans are highly toxic: appropriate precautions were taken to ensure safety. We present data showing proof-of-principle of the system: the mercaptan gases generated in the sealed reaction cell successfully traverse the headspace and are trapped by reaction with Ellman’s reagent (DTNB) in the detection cell. The kinetics of this model reaction has been followed with UV visible spectrometry. By separating the detection and reaction chemistry, this sealed dual-cell provides a cost-effective and flexible apparatus for studying the kinetics of blood gas reactions: and trapping and detecting the blood gases involved.
Analysis of acrolein-derived 1,N2-propanodeoxyguanosine adducts in human lung DNA from smokers and nonsmokers
Acrolein, a highly reactive α,β-unsaturated aldehyde, is widely distributed in the environment arising from incomplete combustion, and also present endogenously from metabolism of amino acids and polyamines, and lipid peroxidation. It also occurs at high concentrations in cigarette smoke (~18-98 μg/cigarette), making cigarettes a major source of acrolein exposure for smokers. Acrolein reacts readily with dGuo in DNA to form two pairs of regioisomeric 1,N2-propanodeoxyguanosine adducts: (6R/S)-3-(2′-deoxyribos-1′-yl)-5,6,7,8,-tetrahydro-6-hydroxypyrimido[1,2-α]purine-10(3H)one (α-OH-Acr-dGuo) and (8R/S)-3-(2′-deoxyribos-1′-yl)-5,6,7,8,-tetrahydro-8-hydroxypyrimido[1,2-α]purine-10(3H)one (γ-OH-Acr-dGuo). A previous study has demonstrated that acrolein-DNA adducts are preferentially formed at p53 mutational hotspots in human lung cancer, which is contradictory to the earlier hypothesis that p53 mutations are due to DNA reactions with polycyclic aromatic hydrocarbon diol epoxides. These results raise the possibility that acrolein, which occurs in quantities up to 10,000 fold greater than benzo[a]pyrene in cigarette smoke, may be a major etiological agent for cigarette smoking-related lung cancer. To test this hypothesis, we developed a sensitive liquid chromatography-nanoelectrospray ionization-high resolution tandem mass spectrometry (LC-NSI-HRMS/MS) method to analyze Acr-dGuo adducts in small amounts of human lung DNA from smokers and nonsmokers, whose matched urine samples have been analyzed for tobacco biomarkers to confirm smoking status. The newly modified method utilizes glutathione (GSH) as an acrolein scavenger and extensive washing steps to remove unmodified dGuo before evaporation to minimize artifactual formation of Acr-dGuo during sample preparation. Both α-OH-Acr-dGuo (3.2 ± 0.8 adducts/109 nucleotides) and γ-OH-Acr-dGuo (10.1 ± 1.4 adducts/109 nucleotides) have been detected and quantified from only 30 μg of calf thymus DNA (CTDNA) with good reproducibility. We plan to analyze normal lung tissue DNA from lung cancer patients (smokers and nonsmokers) to investigate if acrolein is an important factor for cigarette smoking-related lung cancer.
High-resolution/accurate mass DNA adductomics to screen for doxorubicin-induced adducts as biomarkers of therapeutic efficacy
Patients with the same phenotypical cancer can have different responses to a given treatment regimen due to genetic alterations. This is also true of animals with cancer, such as dogs. Monitoring biomarkers of efficacy is a strategy for stratifying patients to predict outcome and allow for personalization of treatment regimens. The majority of the currently used chemotherapy agents act by interfering with cell replication, an overactive process in rapidly dividing cancer cells, through the formation of genotoxic DNA modifications (adducts). Adducts from DNA anticancer drugs are good candidate biomarkers due to their specificity and stability in DNA. We developed a high-resolution/accurate mass (HRAM) LC-MS3 targeted and untargeted DNA adductomic workflow to screen and monitor anticancer drug-induced adducts. The workflow is based on data-dependent triggered MS2 fragmentation events for the most abundant full scan ions, and confirmation of DNA adduct identities by triggered MS3 fragmentation by accurate mass monitoring of neutral losses of common features from DNA or the drug. Initial screening is performed in drug-exposed purified DNA, followed by confirmation of adducts in drug-exposed cells or biopsies from drug-treated patients. Relative quantitation of the detected adducts is achieved by using a mixture of isotope-labeled adducts as internal standards, obtained by exposing the drug to 15N-labeled DNA. We applied the workflow to study the DNA modifications induced by doxorubicin (DOX), a component of the first-line treatment for high-grade canine lymphoma. Exposure of purified DNA with DOX in the presence of formaldehyde revealed the formation of several DNA modifications, among them the previously reported monoadduct resulting from reaction of DOX and formaldehyde with the N2 position of deoxyguanosine. These DNA modifications will now be targeted in blood cells or lymph node samples of dogs undergoing therapy with DOX to investigate the relationship between DNA adduct levels and therapy response.
Molecular level studies of the impact of poly (oxonorbornenes) and their gold nanoparticles conjugates on D. rerio. embryos
Poly (oxonorbornenes) (PONs) are amphiphilic ligands which are known to possess antimicrobial properties that involves membrane penetration. These properties result from the two characteristic side chains of PONs: a hydrophobic alkyl and a charged amine. In this study, we elucidated the impact of the molecular structure of PONs on their activity and the activity of PONs-gold nanoparticles conjugates (PONs-AuNPs) on the model vertebrate organism, D. rerio. Specifically, we determined how changing the amine/alkyl ratio and the molecular weight of PONs affect the activity of PONs and PONs-AuNPs on this model organism. D. rerio. embryos were exposed for 96 hours to increasing concentrations of PONs of varying amine/alkyl ratios and molecular weights. The embryos were counted every 12 hours to determine their viability. Initial results indicate that the viability of D. rerio. embryos decreases with decreasing amine/alkyl ratio, or increasing hydrophobic character. Additionally, increasing the molecular weight of PONs increases their adverse impact on D. rerio. embryos for PONs of all amine/alkyl ratio. This suggests a stronger interaction due to increased amine charge density when large PONs associate with the D. rerio. embryos. Currently, we are investigating the impact of PONs-AuNPs on D. rerio. embryos to determine whether conjugating PONs to AuNPs further increases their activity as was previously shown for other model membranes and living organisms.
Oxidation and removal of cytosine derivatives in the nucleosome
Epigenetic regulation within an organism allows for the modulation of gene expression without alterations to the underlying genetic code. While there are a variety of methods for gene silencing, the main modification to nucleobases is the methylation of cytosine to 5-methylcytosine. The mechanism of reversing this methylation and allowing for gene expression within the context of eukaryotic DNA packaging is unknown. However, it has been observed that ten-eleven translocation (TET) enzymes can oxidize 5-methylcytosine. The fate and function of these oxidized derivatives is currently under investigation. Two oxidized derivatives, 5-formylcytosine and 5-carboxycytosine, are substrates within the base excision repair pathway. Specifically, an enzyme that initiates this repair pathway, known as glycosylase, has been shown to recognize these substrates. This glycosylase is known as Thymine DNA glycosylase (TDG). The ability of TDG and TET proteins to act on substrates within the context of the nucleosome is unknown. This poster will present the design and assembly of substrates that will allow for the elucidation of these enzyme’s activities in this biologically relevant context. Specifically, the experiments required to understand the variable effects of positioning of DNA nucleobases on enzyme activity in the nucleosome core particle (NCP) will be explored. Because the NCP is a complex substrate, a thorough investigation to determine the activity of any DNA enzyme in the NCP must investigate many sites through the NCP to make any definitive judgements.
Determining the basis of E. coli DinB and human pol kappa DNA damage specificity
DNA is constantly threatened by endogenous and exogenous sources of damage. Y family of DNA polymerases possess the specialized ability to copy damaged DNA, albeit at the potential risk of mutagenesis. Escherichia coli has two Y family polymerases that are specialized to bypass damage when copying DNA in a process called translesion synthesis (TLS). E. coli Y-family DNA polymerase DinB is involved in bypassing deoxyguanosine adducts at the N2 position. Humans have four Y family polymerases, including DNA polymerase kappa. E. coli DinB and human pol kappa both bypass minor groove adducts and are inhibited by major groove adducts. In order to probe the importance of particular residues in the activity and specificity of the polymerases, the computational tools Partial Order Optimum Likelihood (POOL) and Structurally Aligned Local Sites of Activity (SALSA) were utilized. POOL predicts catalytically important residues, including those outside of the active site that do not have direct contact with substrates. SALSA compares the spatial arrangements of the POOL-predicted residues across multiple structures of aligned proteins. Multiple structures of pol kappa were submitted to SALSA to understand the contribution of different residues in the selectivity for various DNA adducts. POOL predicted distal residues in differing regions of DinB and pol kappa, therefore, DinB and pol kappa variants with mutations at the predicted distal positions were constructed and are being subject to primer extension assays to study the contribution of these distal residues to polymerase activity. We have identified variants with a range of activity on undamaged and damaged DNA; in particular several mutations in the DinB little finger domain severely reduce activity. Mutations of residues at corresponding positions in DNA pol kappa are being assayed to determine their effects on catalysis, even though in some cases POOL did not rank them as highly important to catalysis. This work will reveal additional features of Y-family polymerases important for damage specificity and activity.
Prediction of the interaction region between the Y-family polymerase DinB and the transcription-repair coupling factor Mfd in E. coli
Accurate DNA replication and transcription are critical for the survival of all organisms. DNA damage can interfere with replication efficiency and fidelity. DNA damage can also cause the transcription elongation complex to stall on the DNA and prevent DNA repair from occurring. The transcription-repair coupling factor Mfd can rescue stalled transcription and recruit DNA damage repair proteins to repair the DNA lesion. It is hypothesized that the Y-family polymerase DinB which is involved in bypassing DNA damage is one of these proteins recruited by Mfd. The protein-protein docking server ClusPro 2.0 was used to generate potential docking poses which were then analyzed to determine the most likely interaction. Several possible configurations have been identified, which will now be probed biochemically using protein truncations and mutations at the predicted sites of interaction.
Machine learning models for predicting hepatic steatosis based on in vivo data
Hepatic steatosis (fatty liver) is a severe liver disease induced by the excessive accumulation of fatty acids in hepatocytes. Factors which may provoke the onset of steatosis are manifold: excessive alcohol consumption, obesity, diabetes, or mitochondrial dysfunction. Aggravation of the disease (persistence of provoking factors) typically leads to steatohepatitis, liver cirrhosis and liver cancer.
In this study we aim to build reliable in-silico models for predicting hepatic steatosis on basis of in vivo data. After careful data curation, the data set of 1121 compounds (131 steatosis positives, 990 steatosis negatives) was split into a training set (70 %) and an external test set (30 %) by keeping the initial distribution of actives/inactives of the original data set. We used different machine learning techniques such as Random Forest, Support vector machine, Artificial Neural Networks and NaiveBayes in combination with different sets of molecular descriptors (physiochemical properties, Toxprint fingerprints, RDKit fingerprints) in order to identify models with high predictive power. In addition, the influence of the incorporation of different sets of biological fingerprints – such as predictions from in vitro steatosis models – was systematically investigated. Further, we used techniques such as cost sensitive learning, stratified bagging and conformal prediction in order to handle the data imbalance.
In summary, we achieved reliable computational models for predicting hepatic steatosis based on in vivo data.
Predicting drug metabolites using bacterial-based models
The design of new drugs is focused on the druggability of the target, and essentially aims to obtain pharmaceutically active molecules. However, a significant number of drugs are associated with side effects of varying degree, ranging from minor to serious and lethal. Many of these toxic effects are triggered by the metabolic fate of drugs, particularly phase I and phase II metabolism. Phase I reactions, mediated by cytochrome P450 (CYP) systems, lead to the formation of oxidation products, while phase II reactions include sulfonation of either the drug or its phase I metabolites. Both of these types of drug metabolites have the potential to react with bionucleophiles, such as protein and DNA, forming covalent adducts that may lead to the impairment of the cellular function of those molecules.
Although experimental systems of metabolic prediction based on eukaryotic cells are available, they tend to be highly sensitive to a large number of interferences and are much more expensive to maintain than simpler bacterial models. Bacteria-bases models are, however, more difficult to implement due to the absence of the protein processing pathways characteristic of eukaryotic cells.
With this in mind, we are testing the expression of various cytochrome P450 and sulfotransferase isoforms in Escherichia coli lines in order to obtain a biomimetic system that replicates the known metabolic fate of selected model compounds as a first step to produce a reactor-like biomimetic bacterial model.
His-tagged human recombinant sulfotransferase isoform SULT1B1 and CYP isoforms 2A13, 2C8, 2D6, and 3A4 expressed in Escherichia coli have been shown to maintain their described metabolic activity isolated kinetic assays using model substrates, namely acetaminophen and the anti-retrovirals efavirenz and nevirapine.
Current work is aimed at immobilizing the proteins on a solid matrix to develop a flow-through reactor in which drugs and co-factors can be loaded and metabolites eluted in two simple sequential steps. The ultimate goal is to obtain a larger model of the pathways of drug metabolism using various isoforms of various enzymes simultaneously and recover the drug metabolites, validating it with well-studied model substrates and paving the way to a simple and effective drug prediction system.
Characterization of LexA-regulated protein YbfE in E. coli
DNA is constantly exposed to damage-causing agents. The SOS DNA damage response in Escherichia coli is regulated by the LexA protein, which controls the expression of more than 50 different genes. One of the many genes regulated by the SOS response encodes the protein of unknown function YbfE. Previous work in our lab has shown ybfE is essential to cell survival after exposure to the alkylating agents styrene oxide and chloroacetaldehyde. YbfE has been shown to cause a striking decrease in cell survival upon overexpression. To identify genetic interactions, a series of knockout strains have been investigated to determine whether deletion of any one gene alleviates the lethality associated with overexpression of ybfE. To date, assays in strains harboring deletions of mutS, uvrC, and uvrA proteins have not suppressed the lethal overexpression phenotype. However, advancements have been made in overcoming this lethality as work continues to obtain purified protein for further characterization. In addition, sensitivity studies with hydroxyurea, which inhibits DNA replication without inducing DNA damage, have been conducted. HU interacts with ribonucleotide reductase preventing the conversion of ribonucleotides to deoxyribonucleotides, leading to a depletion of available dNTPs. This depletion halts replication and initiates a series of cellular responses resulting in an increase in the production of reactive oxygen species. Deletion of the ybfE gene confers resistance to HU, suggesting a role for YbfE in DNA metabolism. Further investigation into protein interactions and the role of YbfE in the SOS response are ongoing. These studies will provide further insights into the role ybfE plays in the SOS response to DNA damage.
DNA damage induced by oxidative stress and lipid peroxidation in leukocyte DNA from African-American and Caucasian smokers
Smoking is a well-established source of oxidative stress and inflammation. The resulting oxidation and peroxidation processes can induce DNA damage and lead to genomic instability, which has been linked to the etiology of chronic diseases, including cancer, cardiovascular disease, and type II diabetes. Epidemiological studies have shown consistent ethnic disparities in the overall risk for such diseases, with African American smokers being at higher risk compared to other ethnic groups. Assessment of the oxidative stress- and lipid peroxidation-induced DNA damage can provide important insights into the underlying mechanisms of these differences. We applied our recently developed mass spectrometry-based methodologies to analyze 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) and 3-(2-deoxy-β-d-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine (M1dG) in leukocyte DNA of African-American and Caucasian smokers recruited for a study of tobacco carcinogen metabolism and lung cancer risk. The levels of 8-oxo-dG were measured in 48 smokers and ranged from 16.4 – 504.5 adducts/106 nucleotides. The average levels of adducts were higher in African-American than in Caucasian smokers, with the difference being larger between female smokers: 105.5 vs. 94.6 adducts/106 nucleotides, respectively. The levels of M1dG were measured in 18 smokers, demonstrating significant differences between African-American and Caucasian smokers: 1.94 vs. 0.33 adducts/108 nucleotides, respectively (p = 0.03), after adjustment for age, gender, and total nicotine intake. Analysis of both adducts is underway in a larger cohort. The results from this study will provide important insights on the role of oxidative stress-induced DNA damage in tobacco-associated diseases, and contribute to better understanding ethnic and gender differences of disease risks in smokers.
Analysis of the spectrum of DNA modifications in Pseudomonas aeruginosa
Pseudomonas aeruginosa is an ubiquitous gram negative, opportunistic, pathogenic bacteria that is the leading cause of death in patients with cystic fibrosis, and a common cause of nosocomial infections in hospitals. Previous studies have shown that modifications to tRNA in P. aeruginosa aid in the control of translation from mRNA to protein. In addition to enzymatic modifications to RNA, enzymatic modifications to DNA also exist in bacteria, including P. aeruginosa. Since unnecessary biological functions are shed through bacterial evolution, we hypothesize that DNA modifications in P. aeruginosa may function as additional regulatory elements. Indeed, it was recently discovered that modifications to DNA in Mycobacterium tuberculosis function to regulate genes and can improve the organism’s ability to withstand stress. As, the immune system combats P. aeruginosa through oxidative mechanisms, we are studying the ability of wild type or putative DNA methyltransferase mutants to survive oxidative stress. We have established liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods to analyze a variety of known and unknown modified DNA nucleosides, and are applying these methods to analyze the DNA of P. aeruginosa. We are investigating the spectrum of modified nucleosides in the wild type and mutant organisms to determine the genes that are responsible for the modifications in the organism with the eventual goal of determining their function in this pathogenic organism.
Probing the conformational dynamics of the Beta sliding clamp in Escherichia coli
In Escherichia coli, the DNA polymerase III (Pol III) holoenzyme, which consists of ten proteins, accounts for most of the DNA replication activity in the cell. Biochemical data has shown that in the presence of the DNA Pol III subcomponent, the β sliding clamp, replication progresses at a rate of >50 kb per association event, relative to 10 nucleotides per event in the absence of the sliding clamp. Thus, β is essential for efficient and highly processive DNA replication. Crystal structures of the β sliding clamp protein confirms that it is composed of two crescent-shaped units, or protomers, with each unit made up of three domains. The two protomers combine in a head-to- tail orientation to form a homodimer ring structure. A second multiprotein subunit of DNA Pol III, the clamp loading complex δδ’γ3, is needed to load the β sliding clamp ring onto the double-stranded DNA. One mechanistic model proposes that ATP binding at two γ domain interfaces induces a conformational change in the clamp loading complex facilitating the N-terminal end of the δ subunit to bind to the C-terminal face of the β sliding clamp. In a wrench-like action, the δ subunit opens the β clamp by dissociating Domain I from Domain III and then it loads onto the primed DNA duplex. With the β sliding clamp loaded, ATP hydrolysis is stimulated, causing the clamp to close, restoring the dimeric clamp ring structure and ejecting the loader complex. Our previous work showed that the β sliding clamp is not a rigid, closed structure in solution but rather that it is dynamic and likely able to open spontaneously. More recently, we have made mutations at the dimer interface of the β sliding clamp that in some cases do not interfere with its biochemical or cellular functions. Currently, we are designing second generation mutations to further characterize the exact role clamp dynamics and the clamp loader complex play in opening the β ring structure and loading it onto double-stranded DNA.
Potential DNA oxidation adducts for disease biomarkers
Reactive oxygen and nitrogen species occur endogenously at sites of inflammation and can cause damage to cellular proteins, lipids, and nucleic acids. This biomolecule damage can act as a sign of diseases associated with inflammation and oxidative stress, including cancer, atherosclerosis, and Alzheimer’s disease. We hypothesize that DNA damage products can act as a biomarker of disease, and seek to develop methods to further this aim. Guanine is the most easily oxidized of the nucleosides, leading to much study of the oxidative product, 8-oxo-dG as well as further oxidized products. However, since it is easily created as an artifact of DNA isolation, it is desired to determine whether other oxidized nucleosides are more suitable biomarkers. We aim to identify oxidative products of DNA resulting from treatment with peroxynitrite (ONOO−), SIN-1, and hydrogen peroxide (H2O2). These oxidants mimic the formation of radicals formed within the body’s immune response via macrophages and other pathways. It is also hypothesized that the damaged nucleic acids may cross-react with other biomolecules including glutathione, proteins, lipids, and other nucleic acids. These cross-linked species are being investigated as additional biomarkers for the identification of oxidative damage. We have developed a liquid chromatography-coupled tandem mass spectrometry (LC-MS/MS) method to identify and quantify known and novel oxidative products to explore the spectrum of DNA damage resulting from reactive oxygen and nitrogen species.
Rapid microplate assay for acellular reactive oxygen species generation induced by engineered nanomaterials in real-time
High levels of reactive oxygen species (ROS) can impede cellular homeostasis by increasing oxidative stress which results in lipid peroxidation, protein oxidation, and DNA damage. Recent investigations have associated the redox potential of engineered nanomaterials (ENMs) with ROS induction. These studies used plate readers and DCF, an indicating fluorescent dye, to measure ENM production of ROS. While several findings have confirmed the production of ROS by some ENMs, some data is questionable and there are contradicting results between different groups. These studies used ENMs that were either sonicated in a working solution of DCF leading to early auto-oxidation of the dye or they were prepared in a DCF-horseradish peroxidase (HRP) solution. HRP can catalyze the formation of ROS in the presence of H2O2 without ENMs.
We have developed a system in which we measure the induction of ROS in real time devoid of HRP in the system or the use of a DCF solution as an ENM dispersant. Using a Promega plate reader with a dual injector system we are able to measure the fluorescence of DCF to indicate ROS induction by several ENMs with environmentally relevant doses. The injector system introduces the light-sensitive dye into wells containing ENMs without exposing it to ambient light and a second injector shoots H2O2 into the system. Through these injections, absorption of the dye ono the ENM is minimized and the oxidative reactivity of the ENM can be measured in real time. The materials were synthesized by the Engineered Nanomaterials Resource and Coordination Core (ERCC) and the physiochemical properties (i.e.: diameter, hydrodynamic diameter, zeta potential) were characterized following a standardized and meticulous protocol developed for collaborative research. Preliminary results show that metal, graphene and metal-oxide nanoparticles, ranging in diameter size and core material, result in statistically significant induction of ROS through the oxidation of DCF. Using this data, the protein corona effect on ROS induction by the nanomaterials will be compared and correlations between protein composition and cytotoxicity will be established.
Identifying toxicology concepts in the replacement of mercury catalysts during the acetylene hydrochlorination of vinyl chloride monomers
Polyvinyl chloride (PVC) is the world’s third most produced synthetic plastic polymer and is an important precursor to products in many industries such as construction, healthcare, and manufacturing. PVC is a polymerized chain of vinyl chloride monomers (VCMs). China synthesizes the majority of the world’s VCMs through the hydrochlorination of acetylene derived from coal, which is catalyzed by mercury (II) chloride. Mercury(II) chloride is identified as a toxin due to its mutagenicity, corrosivity, and reproductive toxicity. Mercury is also very volatile at the reaction temperature of 180 °C and leaches into the environment as toxic fumes. The inorganic mercuric chloride is very soluble in water causing contamination to aquatic organisms and possible bioaccumulation. As part of the goal to promote the adoption of green chemistry, the Beyond Benign’s Toxicology Working Group is aligning toxicology concepts to chemistry concepts that can be taught in lectures and labs. The mechanism of toxicity and effectiveness were evaluated for mercury(II) chloride and an alternative, gold, to serve as a learning opportunity to discuss how to choose greener catalysts that do not have harmful impacts on environmental health.
Petrogenic and pyrogenic polycyclic aromatic hydrocarbons in human urine: comparison of their levels between two geographic regions
The human health hazard most associated with oil spills is exposure to petrogenic polycyclic aromatic hydrocabons (PAH). Petrogenic PAH are unique to oil spills because they are heavily alkylated and or oxygenated and are different from pyrogenic PAH that arise due to incomplete combustion of fossil fuels e.g. benzo[a]pyrene (B[a]P). B[a]P is a known human carcinogen and must be metabolically activated to exert its carcinogenic effects but little is known about the metabolism of petrogenic PAH.
Gulf Coast Health Alliance; health Risks related to the Macondo oil Spill (GC-HARMS) is a community-based participatory research project in which gulf-communities have continued concern about seafood safety due to its possible contamination with petrogenic PAHs. As part of the GC-HARMS consortium we elucidated the metabolic fate of some petrogenic PAHs in human hepatoma (HepG2) cells and human small intestine (CaCO2) cells to mimic pathways of exposure due to ingestion. We identified a number of unique metabolites, which could act as signatures of human exposure to oil spills.
We conducted a study to evaluate these biomarkers, in the urine of 40 exposed individuals from the gulf region cohort compared with 40 healthy controls from the Philadelphia area using liquid chromatography-high resolution mass spectrometry (LC-HRMS). The method used a long LC gradient and employed targeted and untargeted MS scans, in negative and positive mode, that allowed us to get a full picture of exposure to PAHs. Petrogenic and pyrogenic PAHs have been quantified in the two cohorts. Assessment of exposure to PAHs is important due to the widespread presence of PAHs in the environment and their toxicological relevance. Our findings support the usefulness of PAHs as potential biomarkers of non-occupational exposure.
Dissecting interactions between E. coli DNA polymerase III and single-stranded DNA binding protein to gain insights into polymerase management
The goal of this project is to understand how interactions between E. coli DNA polymerase III (pol III) and single-stranded DNA binding protein (SSB) contribute to polymerase management. Pol III is a high-fidelity polymerase responsible for producing exact copies of the template strand during replication and is composed of 10 subunits forming the holoenzyme, while the polymerase core contains three subunits. The individual components of the core complex are the alpha (polymerase), epsilon (proofreading) and theta (undetermined) subunits. SSB can inhibit the alpha subunit or the core complex unless the holoenzyme is assembled, suggesting a role in polymerase regulation. SSB has been found to interact with multiple proteins including two other polymerases, raising the possibility that SSB plays a role in polymerase switching at the replication fork. Computational methods including model construction, protein-protein docking, inter and intra-protein residue coevolution analysis as well as multisequence alignments were employed to predict regions of interaction between pol III core and SSB. Several thousand docking predictions were narrowed to under one thousand by incorporating a rational residue model which relied on the data provided from coevolution analysis and sequence alignments. The in silico results allow for a more focused experimental approach leading to the construction of protein variants with changes in predicted key residues as well as truncations, which are used to probe interaction between pol III and SSB. To probe the extent of the change in interaction between variants, experiments to identify deviation in DNA binding, substrate specificity, and fidelity are being pursued.
Release of lead (Pb) and formation of disinfection byproducts during drinking water disinfection in the water distribution system
Disinfection is an essential process in drinking water treatment for eliminating waterborne diseases. To prevent the regrowth of microorganisms, a certain amount of disinfectant residual is maintained in the water distribution system. Chlorine and monochloramine are common drinking water disinfectants, which can react with natural organic matter (NOM) and bromide in source water to generate brominated and chlorinated organic disinfection byproducts (DBPs). These DBPs have been demonstrated to be cytotoxic, genotoxic, mutagenic, and carcinogenic. Monochloramine was reported to generate lower concentrations of brominated and chlorinated DBPs than chlorine. Besides, lead (Pb)-containing plumbing materials are widely present in water distribution pipelines in the United States. Lead is a potentially toxic heavy metal to human. It is reported that chlorine can oxidize the lead pipeline material to PbO2, which can form an insoluble layer on the inside pipeline surface to protect underlying pipes. Comparatively, monochloramine oxidizes the lead material to Pb(II), mainly soluble Pb2+ present in final tap water. Thus, it is still controversial whether chlorination or chloramination generates safer drinking water. In this study, we compared the lead release, disinfectant residual, and halogenated DBP formation during chlorination and chloramination of simulated drinking water with and without the presence of PbO2. Results indicate that the lead release in the chloraminated samples were higher than those in the chlorinated samples; and the disinfectant residual and DBP formation in the water samples with PbO2 were different from those in the water samples without PbO2. The toxicity of disinfected drinking water depends on the combinatorial effect of disinfectant residual, lead, and DBPs.
Mineralogy dependent dissolution of inhaled uranium in simulated lung fluids in uranium mine lands, New Mexico
The accumulation of toxic heavy metals in mining environments has potential health hazards for humans. One such hazard is inhalation of accumulated heavy metals in fine dust. This study investigates the fate of uranium containing compounds in dusts and sediments from Grant Mineral District, NM, upon inhalation using two simulated lung fluids (SLFs) and PHREEQC3.3.8 modeling software. The Gamble’s solution mimics the human lungs’ interstitial environment whereas Artificial Lysosomal Fluid simulates the lung conditions at phagocytosis. The collected dusts and sediments were agitated in SLFs in custom-built glass reactors maintained at 37°C. The samples were withdrawn periodically and analyzed using an Inductively Coupled Plasma-Mass Spectrometer (ICP-MS). The results indicate uranium dissolution in human lungs depends on mineralogy and mode of transport. The dusts and sediments transported via wind demonstrate higher dissolution in interstitial conditions and those of collected around mine pits had to be phagocytized to obtain a considerable dissolution.
Reactive oxygen species (ROS)-dependent release of an inhibitor from an aptamer
Targeted therapy reduces off-target inhibitor effects by releasing payloads specifically at the site of disease like tumors or cancer microenvironments. A limiting step in release are the types of chemistry available to release the inhibitor. Current strategies include reduction, enzyme-assisted release, and other very stable linkers that require internalization. In this research, I am designing release strategies that depend on reactive oxygen, oxidative stress, and inflammation. These conditions are common at cancer microenvironment sites and thus can lead to selective release of an inhibitor. To begin my design, I have selected an aptamer that targets leukemia cells and an inhibitor that can tune the sensitivity of DNA damaging agents since these are common in leukemia treatments. I synthesized a model compound that mimics the functional group in the inhibitor. After synthesis, the model was exposed to oxidative stress including oxidase action, fenton conditions, superoxide, and hydrogen peroxide. I was able to tune the model inhibitor release such that the half-life was 51 minutes and the rate constant 0.81 h-1 in the presence of reactive oxygen species. An available alkene on the linker will then be used to attach a thiol-modified oligomer to the scaffold via click chemistry, followed by exposure to the same oxidative stress conditions to confirm the model drug release. The same studies will then be carried out using a PARP inhibitor linked to a leukemia-specific aptamer.
Effect of surface charge on toxicity of AuNPs; Are cationic AuNPs toxic?
Unique physicochemical properties of gold nanoparticles (AuNPs) make them prospective materials in biomedical applications such as chemical sensing, bio-imaging, and drug delivery. However, we still know a little about the properties of AuNPs that can interact efficiently with biological systems, especially with cells. It is important to understand and predict the biological effects of modified AuNPs with regard to their physicochemical properties for transition into the clinical setting. Among many variables, here we focused on the surface charges and the structures of ligands. For a systematic approach, we prepared fifteen different mono-dispersed AuNPs based on a sequential ligand exchange method. The prepared AuNPs carried various functional groups derived from amino acids and exhibited a series of surface charges ranging from -42.8±11.8 to +41.8±3.8 mV. Biological assays were performed to gauge the effects of charged AuNPs on various cellular functions and cell viability using mammalian cell lines. The cellular uptake of AuNPs and the subsequent changes in cytoskeletal structures and cell motility were also monitored. These studies showed that the cationic AuNPs showed mixed effects ranging from non-toxic to severely toxic while cytotoxicity of anionic and neutral AuNPs were negligible. The cytotoxic cationic AuNPs inhibited the formation of cytoskeletal structure, DNA replication and, consequently, proliferation of mammalian cells. The oxidative damage on genomic DNA was also observed. We suggested that the toxicity was originated from the ligand structure that can cause lytic. This results suggest that the structure of the ligand, but not the charge of the AuNPs, was an important factor determining the biological effects of AuNPs.
Nanomaterials in marine environment: toxicity to Artemia salina with and without the presence of Phe and Cd2+
Given the increasing potential of nanomaterials entering marine environments, it is imperative to assess their risks on marine ecosystem, including the direct toxicity to marine organisms and indirect toxicity brought by co-existing aquatic pollutants. The impact of manufactured nanoparticles on the toxicity of co-existing pollutants in aquatic environments has raised increasing concerns. However, the toxicity of polycyclic aromatic hydrocarbons or metal ions in the presence of nanomaterials to marine zooplankton has been rarely reported.
In the present study, the impacts of nTiO2, GO on the toxicity of phenanthrene (Phe) and cadium (Cd2+) to Artemia salina, a model marine zooplankton, were investigated. Although nTiO2 alone exerted a limited toxicity to A. salina within 48 h of exposure, nTiO2 strongly altered the toxicity of Phe and Cd2+ to A. salina. Compared with the individual toxicities of pollutants to A. salina, the toxicities of Phe and Cd2+ increased by 2.0% and 12.2%, respectively, in the presence of 5 mg/L nTiO2, but decreased by 24.5% and 57.1%, respectively, in the presence of 400 mg/L nTiO2. These concentration-dependent impacts of nTiO2 on the toxicity of Phe or Cd2+ might be attributed to the concurrent functions of several interrelated factors including the adsorption of pollutants on nTiO2, the nTiO2-faciliated bioaccumulation of pollutants, the limited gut volume in organisms, and the aggregation and sedimentation behaviors of nTiO2.
Although the lethal effects of GO alone to A. salina only appeared at high GO dose (500 mg/L), its sublethal toxicity (growth inhibition) at concentrations as low as 1 mg/L was observed by microscopy, which was likely closely related to the GO-induced oxidative stress in A. salina. Compared with the toxicity of Phe alone, GO-Phe exhibited a synergistic effect to A. salina at a high GO concentration. For GO-Cd2+, the toxicity was positively correlated with both GO dose and Cd2+ dose. The increased toxicity of GO-Phe or GO-Cd2+ at high doses might be attributed to the promoted bioaccumulation of toxicants by GO. These results presented in the study could help understand the effects of manufactured nanomaterials in marine environments.
Molecular characterization of alcohol-induced DNA damage for cancer prevention
Acetaldehyde is the major metabolite of ethanol and has recently been classified by IARC as a Group-1 human carcinogen. Despite a clear association, the underlying mechanisms of ethanol-induced carcinogenesis remain unclear. Acetaldehyde is a DNA-modifying compound. If not eliminated or repaired, these modifications can result in miscoding events that lead to mutations, and ultimately, cancer initiation. To gain new advances on ethanol and acetaldehyde-induced DNA damage and devise preventive measures, an innovative mass spectrometry method able to simultaneously detect all acetaldehyde-derived DNA adducts was developed. Our method is based on a gas phase fractionation-adductomic approach, which monitors the constant neutral loss of features common to all DNA adducts: the 2’-deoxyribose and/or one of the four nucleobases. By exposing DNA samples to 12C2– and 13C2-acetaldehyde and performing the data analysis through in-house software, we were able to identify 20 new DNA adducts, as well as all previously observed DNA adducts associated with acetaldehyde exposure. The reduced product of Schiff bases/imines were found in samples reduced with NaBH3CN, meanwhile DNA crosslinks and Mannich products were observed in non-reduced samples. N6-ethyl-dAdo and N4-ethyl-dCyd were identified as novel adducts. Because of the biological relevance of DNA methylation and their structural similarity to methyl-dAdo and methyl-dCyd, characterization of N6-ethyl-dAdo and N4-ethyl-dCyd was achieved by synthetizing isotopically-labeled standards. N6-Ethyl-dAdo, N4-Ethyl-dCyd, D5-N6-Ethyl-dAdo and D5-N4-Ethyl-dCyd were synthesized, purified, and structurally characterized by one- and two-dimensional NMR and HRMS analyses. A dose-response relationship was observed between the three primary DNA adducts and acetaldehyde exposure. Finally, the presence of these adducts was confirmed in oral DNA samples from humans exposed to alcohol. Ultimately, we confirmed that our approach can characterize ethanol- and acetaldehyde-induced DNA damage and that both dAdo and dCyd adducts are potential markers of these exposures.
EB-Fapy-dG adducts of 1,3-butadiene: Synthesis, structural identification, and detection in human cells
1,3-Butadiene (BD) is an environmental and occupational toxicant classified as a human carcinogen. BD is metabolically activated by cytochrome P450 monooxygenases to 1,2-epoxy-3-butene (EB), which alkylates DNA to form a range of nucleobase adducts. Among these, the most abundant are the hydrolytically labile N7-guanine adducts such as N7-hydroxybuten-1-yl-dG (N7-EB-dG). We now report that N7-EB-dG can be converted to the corresponding ring open N6-(2-deoxy-D-erythro-pentofuranosyl)-2,6-diamino-3,4-dihydro-4-oxo-5-N-(2-hydroxybut-3-en-1-yl)-formamidopyrimidine (EB-Fapy-dG) adducts. We for the first time report the synthesis of EB-Fapy-dG and the EB-Fapy-dG lesions were detected in EB-treated calf thymus DNA and in EB-treated mammalian cells using quantitative isotope dilution nano HPLC-NSI-MS/MS. EB-Fapy-dG adduct formation in EB-treated calf thymus DNA was concentration dependent and was greatly accelerated at increased pH. EB-FAPy-dG adduct amounts were 2-fold higher in BER deficient NEIL1-/- mouse embryonic fibroblasts (MEF) as compared to isogenic controls (NEIL1+/+), suggesting that this lesion may be a substrate for NEIL1. However, NEIL1-/- cells were only slightly sensitized to EB as compared to NEIL1+/+ fibroblasts, suggesting that EB-FAPy-dG lesions are not the main cytotoxic species. To elucidate the biological effects of EB-FAPy lesions, they were site-specifically incorporated into DNA oligomers. Overall, our results indicate that ring open EB-FAPy-dG adducts do form under physiological conditions, prompting future studies to determine their contributions to genotoxicity and mutagenicity of 1,3-butadiene.
Inter-individual differences in metabolism of 1,3-butadiene
1,3-butadiene is an abundant carcinogen found in cigarette smoke and urban air which is bioactivated to form the reactive metabolite 3,4-epoxy-1-butene (EB). If not detoxified, this reactive epoxide can form DNA adducts by alkylating the N7 position of guanine, forming the monoadduct N7-(1-hydroxy-3-buten-2-yl) guanine (EB-GII). These DNA adducts can interfere with DNA replication and lead to cancer-inducing mutations if they are not repaired. EB-GII can be removed from DNA and excreted in urine, making it a promising biomarker candidate for butadiene exposure.
In order to determine the validity of urinary EB-GII as a biomarker for butadiene exposure, we investigated its temporal stability in smokers over 12 months and its association with smoking using a smoking cessation study. Using nano-LC-HRMS3 methodology to quantify these adducts in urine from 22 smokers over 12 months, we found that EB-GII adduct levels are stable. Additionally, we analyzed the urine from 17 smokers before and after smoking cessation. Immediately after smoking cessation, a sharp drop in urinary EB-GII levels was observed which plateaued at ~50% decrease of pre-cessation levels.
Additionally, we investigated whether variation in GSTT1 expression among ethnic groups leads to differences in butadiene metabolism, potentially affecting lung cancer risk. GSTT1 can catalyze the conjugation of glutathione (GSH) to EB, which is a major route of EB detoxification in humans. We quantified the levels of EB-GSH in HapMap human cells with different expression levels of GSTT1 after treatment with EB.
2, 2’, 3, 5’, 6 polychlorinated biphenyls (PCB-95) induce behavioral and GABAgenic neurotransmitter changes in zebrafish at early developmental exposure
Developmental exposure to neurotoxic chemicals showcases significant health concerns due to the vulnerability of the developing central nervous system (CNS) and the immature brain barrier. Non–dioxin-like (NDL) PCBs are long linked to neuropsychological dysfunctions but underlying mechanisms are not fully elucidated. Previous studies with embryonic PCB-95exposure, one such NDL-PCB congener, have shown altered larval behaviors on swimming speed, thigmotaxis and in Danio rerio (zebrafish). Behavior of an organism has strong correlation with the neurotransmitter profile that especially depends on the balance between the excitatory and inhibitory processes. Therefore, we determined to study the alteration of γ -aminobutyric acid (GABA) profile with embryonic PCB-95 exposure. Embryos at 2 cell stage with intact chorions were exposed to different concentrations of PCB-95 (0.5, 0.75, 1, 3 ppm) with two controls (E2 solution and the DMSO). Exposure time was at day 3 with 7 day incubation period. At day seven (7dpf), larvae were euthanized and analyzed for GABA using a liquid chromatography/tandem mass spectrometry (LC-MS/MS). We observed, low GABA concentrations for PCB-95 treated groups, compared to the controls. Dose dependent decrease was observed in treated group. These results were further validated with zebrafish brain sections, comparing the numeric dominance of early GABA- positive cells in day 7 zebrafish larvae with treated groups to controls. In conclusion, we suggest that the prenatal PCB-95 exposure alters developmental GABA expression. Together with our previous behavioral studies, these suggest that PCB-95-induced GABA expression may alter neuronal cell pattering and migration; thus resulting in neurodevelopment disorders.
Investigation of the effect of 2-phenethyl isothiocyanate (PEITC) on the levels of 4-hydroxy-1-(3-pyridyl)-1-butanone-releasing DNA adducts in oral cells of smokers
Tobacco-specific nitrosamines 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N’-nitrosonornicotine (NNN) are believed to play a key role in the lung, oral, and esophageal carcinogenesis in smokers. To exert their carcinogenicity, both compounds require metabolic activation by P450 enzymes, leading to the formation of 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing DNA adducts. Our recent study demonstrated that the levels of these adducts are significantly higher in oral cells of smokers with head and neck cancer compared to cancer-free smokers. Therefore, inhibition of HPB-releasing adduct formation in smokers could be pursued as a preventive measure to reduce the incidence of tobacco-induced cancers. We applied our recently developed high-resolution mass spectrometry-based method to investigate the effect of 2-phenethyl isothiocyanate (PEITC), a natural chemopreventive agent, on the levels of HPB-releasing DNA adducts in oral cells of smokers. The study participants ingested 40 mg of PEITC or placebo (olive oil) for 5 days in a randomized within-subject cross-over study design, with the two treatments being separated by a washout period. Analysis of oral samples from 22 study participants shows that the average levels of HPB-releasing DNA adducts are lower during PEITC than placebo treatment: 0.15 vs 0.21 pmol/mg DNA, respectively. Analysis of data by the order of treatment (PEITC-placebo or placebo-PEITC) indicates that the decrease of HPB-releasing DNA adducts in oral cells continues after the termination of PEITC treatment. These preliminary findings are consistent with the urinary biomarker data in the same subjects and provide a basis for further investigation of PEITC as an inhibitor of tobacco induced carcinogenesis in smokers.
Smoking and inflammation mediated epigenetic changes in a mouse model of lung cancer
5-Methylcytosine (mC) is a stable epigenetic modification of DNA that plays a key role in controlling gene expression. Epigenetic alterations such as aberrant cytosine methylation and changes in gene expression patterns are increasingly recognized as critical events in the development of cancer. The removal of DNA methylation marks is mediated by ten eleven translocation (Tet) dioxygenases which iteratively oxidize the methyl group to give 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), and 5-carboxylcytosine (caC). In addition to serving as demethylation intermediates, hmC, fC, and caC may function as additional epigenetic marks that fine-tune the levels of gene expression.
Smoking-induced lung tumors exhibit profound epigenetic alterations, aberrant patterns of gene expression, and genetic instability. Chronic obstructive pulmonary disease (COPD) is a major risk factor or lung cancer development in smokers. Cigarette smoke contains endotoxin lipopolysaccharide (LPS), which causes COPD-like symptoms in laboratory animals. Work in our laboratory has shown that inhalation exposure to LPS induces early global and loci-specific hydroxymethylation changes in Type II alveolar epithelial cells analogous to those observed in lung tumors. Thus, we hypothesize that chronic inflammation leads to changes in epigenetic marks in DNA and aberrant gene expression patterns, contributing to cancer development.
In the present work, we investigated the epigenetic effects of smoking and inflammation in the A/J mouse model of lung cancer. Mice were exposed to cigarette smoke, inflammatory agent LPS, or tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Global levels of DNA methylation, hydroxymethylation, formylation, and carboxylation were quantified using mass spectrometry, while loci-specific methylation was studied by RRBS, and gene expression levels were assessed by RNA seq. Our results suggest that the exposure to inflammatory agents present in cigarette smoke leads to early epigenetic changes which collaborate with genetic changes to drive the development of lung cancer.
Independent synthesis and fate of DNA lesions generated from oxidative damage at the C-3′ and C-5′ position of deoxyribonucleotides
When the 2′-deoxyribose moiety of DNA undergoes oxidative damage, an initial hydrogen abstraction occurs, leading to the formation of a carbon centered radical that reacts with cellular oxygen to form a peroxyl radical. Ultimately strand breaks and abasic sites can be generated with a variety of oxydized lesions specific to the position of the original Hydrogen abstraction. In this study, lesions emanating from oxidation at the C-3′ and C-5′ of the sugar have been independently synthesized and their ability to form adducts with biologically relevant thiols containing biomolecules have been studied. Those adducts were then investigated as potential biomarkers for oxidative stress in biological systems
Ecotoxicology of nano-perovskites in aquatic environment
Perovskite nanomaterials (PNMs) provide significant application potential, however the fate and potential ecological effects of PNM release into aquatic environments are completely unknown. To investigate this, five representative PNMs (LaFeO3, YFeO3, BiFeO3, LaMnO3 and LaCoO3) were synthesized and their physicochemical characteristics were compared to determine toxic mechanisms based on chronic exposure to the aquatic invertebrate Daphnia magna (D. magna). Results show that, LaCoO3 significantly inhibited daphnid growth and induced 100% mortality after 21d,in contrast, no detrimental effects were observed in D. magna exposed to the other PNMs tested, Synchrotron radiation X-ray fluorescence (SR-XRF) confirmed the distinctive distribution of LaCoO3, compared with the other PNMs, with oxygen vacancies playing a significant role in the toxicity of LaCoO3.
We than focus on the toxicity mechanism of LaCoO3, three concentrations(0, 5 and 50ppm) of LaCoO3 were exposed to D. magna for 48h, the bioaccumulation and biomarker responses of several antioxidant biomarkers such as reactive oxygen species (ROS), total superoxide dismutase (SOD), sodium-potassium ATPase (Na/K ATPase) in D. magna were measured during 48h. Result showed that large amount of LaCoO3 were accumulated in D. magna and located mainly in the gut and thoracic limbs areas. However, the speciation of ingested LaCoO3 did not changed during 48h, which is confirmed by the result of synchrotron near-edge X-ray absorption spectroscopy (XANES). In addition, higher concentration (50ppm) of LaCoO3 induced ROS and triggered oxidative stress, leading to a reduction in Na/K ATPase and SOD activity. Based on the combined dissolution experiment and toxicity results, it can be concluded that the toxicological effects of LaCoO3 nanoparticles can be largely attributed to the dissolved fraction rather than the nanoparticles or initially formed aggregates.
Thermodynamic exposure reduction by amendment techniques to limit bioaccumulation during ongoing depositional input – a sediment mesocosm study with three organisms
Polychlorinated biphenyl (PCB) contaminated sediments pose a risk to human health and ecological integrity. These bioaccumulate in organisms that inhabit contaminated waters. Sediments often continue to receive ongoing contaminant inputs after remedies are put in place due to incomplete source identification or poor source control. Studies have found correlation between porewater concentration and bioavailability. In this study, bioaccumulation was examined in 3 organisms with different associations with the sediment in laboratory mesocosms. Three replicate mesocosms received clean input sediment and three replicate mesocosms received contaminated input sediment (spiked with PCB 13, 54 and 173). This design allowed comprehensive testing of remediation efficacy of sand capping, mixed sand and activated carbon (AC) capping, and the combination of mixed capping and AC addition to surface. Organisms were analysed for lipid-normalized concentrations of PCBs and polyethylene passive samplers were deployed to determine sediment porewater concentrations. Percent reduction in sediment porewater and organism tissue concentration between uncapped and sand capped sediment was examined. Sand Capping showed efficacy in reducing total PCB porewater concentration and tissue concentration relative to control conditions. Total porewater concentration was reduced 69% and 80% in clean and spiked input mesocosms respectively. This was lower than in AC amended experiments which showed 100% reduction in porewater concentration in clean and spiked mesocosms. Fish tissue was reduced by 65% and 80% and in AC amendment 96% and 76% in clean and spiked mesocosms respectively. Sand capping, with the exception of congener 54 in worm tissue, showed decreased concentration of all input congeners in all organsims and in porewater relative to control conditions. AC amendement showed increases in tissue concentrations of input congeners across all three organisms in both spiked and clean input mesocosms relative to control. The parameters of these experiments, including relatively short duration (90 d) and lack of turbulence, present some limitaiton in extrapolation of results to field applications. AC reduces thermodynamic exposure to organisms and sand capping creates a physical barrier. Over time, AC will continue to sorb. This is not likely the case with the sand cap which may degrade overtime. Presentation will also include data from mixed cap and mixed cap+ AC experiments.
MegaTox for predicting compound liabilities
The life cycle of drug discovery unsurprisingly starts with demonstrating the compound’s in vitro potency at a target of interest or through a phenotypic screen. However, the majority of drugs fail later on in clinical trials, illustrating that anticipating undesired activity early is arguably just as important as potency to maximize the investment of drug development. A preliminary solution is implementing machine learning methods to predict adverse effects, but toxicity testing is usually performed on a small subset of promising lead compounds, so data availability is limited. However, this is not the primary problem as there are many sources of public databases and literature, as well as government efforts for data transparency. Rather, the problem is that little has been done to transform this data into models or provide a simple, reliable means of identifying toxic features of novel compounds.
We offer a solution to this problem with MegaTox, a toxicology-centric subset of Assay Central, a collection of Bayesian models of structure-activity relationship (SAR) data delivered in a self-contained executable program. SAR datasets are collated and stored using the code-management system Git, from which extended-connectivity fingerprints are calculated as contributing to activity the target of interest. Prediction scores of activity at undesired host targets (i.e. hERG and estrogen receptors) can be immediately generated by users, and visualization of active molecular features compliment human intuition to generate novel, nontoxic compounds. This technology ultimately aims to streamline drug design and reduce the superfluous synthesis of compounds that are more likely to fail due to common in vivo liabilities.
We will describe the workflow of using publicly-available SAR datasets to create predictive Bayesian models to drive our own drug discovery efforts. Models of acute oral rat toxicity, cell cytotoxicity, hERG, estrogen receptors, and other targets at multiple thresholds will be discussed. Receiver Operator Characteristic scores range from 0.81 to 0.96 and Matthews Correlation Coefficients ranged from 0.34 to 0.84. Additionally, estrogen receptor models generated with alternative algorithms outside of Assay Central are presented for comparison.
Information-derived adverse outcome pathways with a case study on structural cardiotoxicity
Adverse events resulting from drug therapy can be a cause of drug withdrawal, reduced and or restricted clinical use, as well as a major economic burden for society. To increase the safety of new drugs, there is a need to better understand the mechanisms causing the adverse events. One way to derive new mechanistic hypotheses is by linking data on drug adverse events with the drugs’ biological targets. In this study we have used datamining techniques and mutual information statistical approaches to find associations between reported adverse events collected from the FDA Adverse Event Reporting System and assay outcomes from ToxCast, with the aim to generate mechanistic hypotheses related to structural cardiotoxicity. Our workflow identified 22 adverse event-assay outcome associations. From these associations 10 implicated targets could be substantiated with evidence from previous studies reported in the literature. For two of the identified targets, we also describe a more detailed mechanism, forming putative adverse outcome pathways (AOPs) associated with structural cardiotoxicity. Our study also highlights the difficulties deriving these type of associations from the very limited amount of data available. The methods presented here are applicable across different types of data and toxicological endpoints, and we will explore further associations in the future.
Morphology-dependent cytotoxicity of SiC nanomaterials to human mesenchymal stem cells
SiC nanomaterials are promising for cell tracking due to their stable and strong luminescence. SiC nanowires (SiCNWs) and nanoparticles (SiCNPs) are biocompatible for multiple cell lineages. However, upon labeling human mesenchymal stem cells (hMSCs) with SiCNWs, we noted negative effects on cell adhesion, proliferation, migration, and pluripotency. We also studied the cytotoxicity of SiCNPs with an average size of 79 and 603 nm. The results show that the proliferation rate of SiCNW-labeled hMSCs is 123% lower than unlabeled hMSCs on day 6 (Figure 1A). Approximately 67%, 56%, 64%, and 31% of unlabeled, SiCNP80-, SiCNP600-, and SiCNW-labeled hMSCs adhered to the flask 24 hours after seeding (Figure 1B). Migration assay shows the cell density in the non-scratched area of SiCNWs-labeled hMSCs decreased from about 5,000 cells/cm2 to approximately 3,055 cells/cm2, while the other groups all increased to around 6,400 cells/cm2. In scratched area, cell density of unlabeled and SiCNPs-labeled hMSCs were around 5,400 cells/cm2 and it was only about 1,800 cells/cm2 for SiCNWs-labeled (Figure 1C). While SiCNP600- and SiCNP80-labeled cells differentiated into osteogenic and adipogenic lineages like unlabeled hMSCs, SiCNWs-labeled hMSCs failed to differentiate into osteocytes or adipocytes after treatment with differentiation induction media (Figure 1D). In confirmation of the prior literature, we noted that the SiCNWs do not affect the viability of cancer cells. Figure 1E shows the SiCNWs-labeled hMSCs achieved 100 % confluence similar to unlabeled hMSCs.
Nanotoxicity predictive modeling: A case study on metal oxides nanoparticles
The regulatory agencies should fulfil the data gap in toxicity for new chemicals including nano-sized compounds, including metal oxide nanoparticles (MeOx NPs) according to the registration, evaluation, authorisation and restriction of chemicals (REACH) legislation policy. This work demonstrates the possibility to predict cytotoxicity of MeOx NPs for a wide range of metal oxides. In addition, we demonstrate
an inter-species correlations in toxicity for metal oxide nanoparticles. The models are represented in context of five OECD principles of validation models accepted for regulatory use. The methodology is expected to be useful for potential hazard assessment of MeOx NPs and prioritisation for further testing and risk assessment.
Surface-modified gold nanoparticles and their long-term impact on cellular pathways
Gold nanoparticles (AuNPs) are promising nanomaterials in biomedicine and their presence in consumer products such as cosmetics is growing over time. With the increased exposure of engineered NPs to biological systems, regulatory agencies are raising concerns regarding NP adverse effects on human health and the environment. The mechanistic understanding of the impact of chemicals/nanomaterials on cellular pathways and networks is a new vision for toxicity assessment according to the 21st century Toxicity Testing (NRC). Despite the ever-widening range of knowledge into the effects of NPs on cells, the adverse outcome pathways triggered by AuNPs in human cells are still largely unknown. Moreover, the AuNPs hazard assessment mostly focuses on the acute stress response, usually at high doses, rather far from a realistic scenario of human exposure. Data on the AuNPs long-term low-dose exposure effects are still very sparse. Herein, we report the first study focused on the in vitro long-term effects of low dose of AuNPs (0.1nM) with varying shapes (spheres and rods) and surface coatings (citrate, poly(acrylic acid) (PAA) and poly(ethylene glycol) (PEG)) in cellular pathways related to stress and toxicity as well as in the global miRNA expression profile in non-cancerous human dermal fibroblast (HDF). Cells were exposed to NPs under both “non-chronic” (acute cell exposure, followed by 20 weeks in a NP-free cell media) and chronic (exposure to AuNPs continuously over 20 weeks) ways. The expression levels of 84 genes related to stress and toxicity pathways were evaluated by qPCR whereas the miRNA expression profile was assessed by miRNA-sequencing. Both chronic and non-chronic Au NPs exposures at low dose induce modifications at the gene level after long periods. Genes related to oxidative stress, cell cycle regulation, and inflammation are among those presenting differential expression levels. The cell stress response was not reversible over time upon removal of NPs, which is a concern. However, continual exposure (chronic condition) does lead to cellular adaptation to NPs at some level. Moreover, we showed that PEG, commonly used in nanoparticle surface-coating, is not as a benign surface chemistry as is generally supposed since PEG-coated nanoparticles induced high levels of genes related to inflammation and oxidative stress pathways. The miRNA expression profile also showed different patterns depending on the exposure condition and AuNP type.
Noninvasive measurement of bladder carcinogen DNA adducts in human urinary cells by liquid chromatography-tandem mass spectrometry
Bladder cancer (BC) is the sixth most common cancer affecting approximately 79,000 adults annually in the United States. Tobacco smoking and occupational exposures to carcinogens, such as aromatic amines, are the most important risk factors of BC. Several epidemiological studies have reported that the consumption of well-done meat or nitrite-cured meats is risk factors of BC; however, the data are inconsistent. Endogenous by-products of lipid peroxidation are other possible agents that damage DNA of the bladder. However, the identities of chemical agents that damage DNA and initiate BC remain largely unknown.
Exfoliated urinary cells are a promising, noninvasive biospecimen to screen for DNA adducts formed by genotoxic chemicals in the genitourinary system. We have established an in vitro model system of exfoliated urinary cells to investigate the stability of DNA adducts in the urine matrix and to develop a quantitative method of retrieval, identification, and measurement of DNA adducts, using aristolochic acid (AA-I and 4-aminobiphenyl (4-ABP) as prototypical genitourinary human carcinogens. The RT4, a well-differentiated human bladder cancer cell line that largely retains transitional epithelial structure, has been reported to be a reliable model cell line to study aromatic amine metabolism, DNA adduct formation, and cytotoxicity. Thus, this cell line is ideal for our in vitro model system.
RT4 cells were treated with 4-ABP and AA-I for 24 hours. Afterward, one million treated cells were spiked into 40 mL of freshly voided human urine and incubated for up to 24 hours at either 4 °C or ambient temperature. The cell viability exceeded 80% over time and the recovery of DNA was comparable at both temperatures. The DNA adducts of 4-ABP and AA-I formed in RT4 cells were stable in the urine of three subjects up to 24 hours of incubation at both temperatures. The quantitation of DNA adducts was performed by UPLC-ion trap MS3. Our preliminary data are highly promising and suggest that DNA adducts of other genitourinary carcinogens can be screened in human urinary cells. With further optimization of the bioanalytical method, we will examine for DNA adducts in exfoliated urinary cells of tobacco smokers to identify major DNA damaging agents in tobacco smoke.
What exactly is toxic about colloidal nanoparticle formulations? Results from the molecular level and the cellular level
The subfield of nanotoxicology, now featured in numerous journals, is full of dose-response experiments of nanoparticles on some sort of cell. Many good things are learned in these experiments; but even for the most innocuous particles known, there is always some response to be had. In this talk I will discuss specific case studies of colloidal gold nanoparticles, and how the molecular precursors used to control their size and shape can be quantitatively the cause of cytotoxicity (and not the nanoparticle per se). In addition, long-term cytotoxic studies are rare in the literature; on this regard, I will show data that suggests that living cells, and even communities of living cells, adapt to nanoparticles and therefore self-mitigate any potential toxicity.
Targeting or enhanced selectivity: Toxicological considerations of nanoparticle therapeutics
The last decade has witnessed a surge in unleashing the potential of nanoparticles as tissue and cellular “targeted” therapeutic modalities. The learning from the growing number of such modalities in the development pipelines has necessitated an earlier-than-usual assessment of the toxicological understanding with respect to the nanoparticle design and characterization. Innovations in early toxicology study designs and selection of translatable preclinical models could positively impact the development timelines, and increase confidence in relevance of the results of such studies to the clinical outcomes. Study of nanoparticles biodistribution is critical in clarification of perceived targeting feature versus enhanced selectivity, and determination of the impact on efficacy and safety. A critical review of the field and gap analysis would enable educated risk taking and improve the quality of decisions in advancing the development of nanoparticle-based therapeutics.
Expansile nanoparticles for the treatment of intraperitoneal mesothelioma
The treatment outcomes for malignant peritoneal mesothelioma are poor and associated with high co-morbidities due to suboptimal drug delivery. Thus, there is an unmet need for new approaches that concentrate drug at the tumor for a prolonged period of time yielding improved antitumor efficacy and improved metrics of treatment success. A paclitaxel-loaded pH-responsive expansile nanoparticle (PTX-eNP) system is described that addresses these unique challenges to improve the outcomes for peritoneal mesothelioma. The synthesis of the eNPs is described followed by several particle characterization techniques, including qNano, DLS, SEM, and TEM that measure particle size as a function of pH and swelling time. The rate of tumoral uptake of eNPs is rapid and, subsequent disruption of autophagosomal trafficking leads to prolonged intracellular retention. Following intraperitoneal administration, eNPs rapidly and specifically localize to tumors within 4 hr of injection with persistent intratumoral retention for >14 days. The high tumor-specificity of PTX-eNPs leads to delivery of >100 times higher concentrations of drug in tumors compared to PTX alone. As a result, overall survival of animals with established mesothelioma more than doubled when animals were treated with multiple doses of PTX-eNPs compared to an equivalent dose of PTX (standard of care) or non-responsive PTX-loaded nanoparticles.
Debugging nano–bio interfaces
Nanoparticles (NPs) are becoming increasingly promising tools for medical diagnostics and therapeutics. Despite the advances in their biomedical applications and numerous publications, fewer than expected NPs have made it to clinical trials and even fewer have reached clinical practice. This wide gap between bench discoveries and clinical applications is mainly because of our limited understanding of the nanobiointerfaces. Although extensive studies have been conducted to enhance our understanding of the nanobiointerfaces, the literature remains unclear and contains conflicting information, even for seemingly identical NPs. The main goal of this talk is to introduce some of the existing “hidden” factors at the nanobiointerfaces to determine – unambiguously and reproducibly – the biological fate of NPs both in vitro and in vivo. Deeper understanding of the nanobiointerfaces, using the hidden factors, may accelerate clinical translation of nanobiotechnologies
Understanding mast cell activation in the safe development of nanotechnologies
Engineered nanomaterials (ENMs) have unique physicochemical properties with potential to impact diverse aspects of society. While the research and proposed applications of ENMs continue to grow rapidly, the health and safety of ENMs still remains a major concern to the public as well as to policy makers and funding agencies. With a fast-growth in the number of new ENMs, new inter disciplinary research approaches are necessary now, more than ever, to address safety concerns in light of the vast potential for the use of ENMs in food, cosmetics, medicine, construction, bioimaging, as well as the potential exposures during manufacturing or research use. While there has been considerable investigation into the properties of ENMs that elicit toxicity, little work has focused on the ability of ENMs to promote or exacerbate allergic disease. Mast cell activation, which is central to development of allergic disease, has been an impediment to a number of pharmaceuticals and will likely represent a challenge for nanomedicines. Consequently, research on the health and safety implications of ENMs must address the potential for nanomedicines, nano-based consumer products or occupational exposures and the impact on initiation of allergic disease or exacerbation of underlying allergic diseases such as asthma. We have established that certain ENMs elicit mast cell activation leading to adverse pulmonary and cardiovascular outcomes. This presentation will present data on the role of physicochemical properties in mast cell activation as well as novel mechanisms of mast cell activation. Lastly, data will be presented demonstrating a significant genetic component in modulating mast cell activation following exposure to nanoparticles.
Base and nucleotide excision repair of site-specific oxidatively generated guanine lesions in DNA substrates transfected into human cells
The interplay between different repair mechanisms in human cells has long been a subject of significant interest. We provide a direct demonstration that in intact human cells the non-bulky oxidatively generated 5-guanidinohydantoin (Gh) and diastereomeric spiroiminodihydantoin (S,R-Sp) lesions, which are known to be repaired by base excision repair (BER), are also substrates of nucleotide excision repair (NER). The 32P-internally labeled double-stranded DNA substrates harboring the lesions were transfected into fibroblasts or HeLa cells. After 2-8 h incubation, the transfected cells were lysed and the 32P-labeled BER and NER early incision and dual excision repair products, respectively, were recovered and quantified by electrophoretic gel autoradiography methods. A bulky benzo[a]pyrene-derived guanine adduct (positive control for NER) yielded similar yields of NER products, but no BER products, as expected. After 4 h incubation periods, the NER yields in the case of the Sp and Gh lesions were ~ 4% and the ratios of [BER]/[NER] incision yields were ~ 5 (Gh) and ~10 (Sp). In human fibroblasts deficient in the key NER factor, XPA, the Sp and Gh lesions were repaired almost exclusively by BER pathways, and the NER ladders of dual excision products were not observed. This approach provides a convenient method for a real-time evaluation of the DNA repair capacities of human cells with different repair backgrounds by simultaneously monitoring the early BER and NER incision events.
Reduction pathway-dependent cytotoxicity of reduced graphene oxide
The environmental transformation of graphene oxide (GO) can significantly change its physicochemical properties, thus altering its toxicity. Here, we demonstrated how exposure to simulated sunlight or reduction with ferrous iron (Fe2+, an environmentally abundant and mild reductant) significantly impacts the cytotoxicity of GO. GOs were reduced under both sets of conditions, but the Fe2+-reduced GO showed dramatically lower cytotoxicity than the light-reduced GO. A highly reactive graphene-based radical was generated when GO was exposed to light, and this radical contributed significantly to the oxidation of cell membranes. Moreover, light-induced reduction promoted the breakage of the graphene sheet, decreasing the lateral size and increasing the ability of the material to disrupt the membrane, which consequently enhanced cellular uptake. Interestingly, we observed a loss of C-O-C/C-O groups but an increase in C=O/O-C=O groups during Fe2+ reduction, which improved the radical quenching capacity of the material due to the restored sp2 structure and the increase in electron-shuttering groups. The reduction-induced aggregation significantly decreased cellular uptake relative to the internalization of the parent GO. These results emphasize that environmental transformation-induced property changes should be given adequate consideration in the risk assessment of GO.
Site-specific production of hydroxyl radicals and synergistic DNA damage induced by the non-enzymatic activation of the anti-tuberculosis drug isoniazid by Cu(II)
Isoniazid (INH) has been recognized as a frontline anti-tuberculosis drug against Mycobacterium tuberculosis (Mtb), which is readily activated by Mtb catalase peroxidase enzyme to produce the reactive isonicotinic acyl radical intermediate responsible for its anti-tuberculosis activity. However, it is not clear whether INH can be activated non-enzymatically by Cu(II). Here we found that INH and Cu(II) together could induce synergistic DNA damage, including DNA strand breakage and 8-oxodG formation, while neither of them alone has any effect. DNA damage induced by INH/Cu(II) could be inhibited by Cu(I)-specific chelator and catalase, but not by SOD and the typical hydroxyl radical scavengers. Interestingly, hydroxyl, hydrogen, C- and N-centered radicals were found to be produced during Cu(II)-catalyzed oxidation of INH by ESR spin-trapping method. The C-centered radical was further unequivocally identified by ESR and ESI-Q-TOF-MS as isonicotinic acyl radical, which can react with nicotinamide coenzyme NADH to form the critical isonicotinic acyl-NAD adducts. Low temperature ESR studies showed that Cu(II) was reduced to Cu(I) by INH. We proposed that the synergistic DNA damage induced by INH/Cu(II) might be due to the synergistic and site-specific production of hydroxyl radical near the binding site of copper and DNA. This study provided a new insight on non-enzymatic activation of INH by Cu(II), which may have important biological implications for future research on INH.
Insights into the molecular mechanism of alkylation- and platination-induced mutagenesis
The genomic DNA is persistently attacked by endogenous and exogenous alkylating agents (e.g., S-adenosyl methionine, cisplatin), which gives rise to a wide variety of alkylated DNA lesions such as N7-MeG, N3-MeA, O6-MeG, and cisplatin-GG intrastrand cross-links. These lesions, if not removed by DNA repair pathways, can be bypassed by error-prone translesion synthesis DNA polymerases, which could cause mutations and cancers. The structural basis for promutagenic replication past these lesions remains elusive. To elucidate the alkylation-induced mutagenesis mechanism, we conducted kinetic and structural studies of the bypass of these lesions by various human DNA polymerases. N7-MeG formed a Watson-Crick-like base pair, rather than a wobble base pair, with thymine, suggesting promutagenicity of N7-MeG. N3-MeA formed a Watson-Crick base pair with thymine but strongly deterred nucleotide incorporation opposite the lesion. O6-MeG formed a Watson-Crick-like base pair with thymine, which was consistent with the reported high mutagenicity and carcinogenicity of O6-MeG. Cisplatin-GG intrastrand cross-links engage in favorable interactions with adenine in non-instructional fashion, suggesting that the predominant cisplatin-induced G to T mutations may follow an “A-rule”. Taken together, these studies revealed the features of promutagenic base pairings of N7-MeG, O6-MeG and cisplatin-GG cross-links, thereby providing new insights into the molecular mechanisms of alkylated-induced mutagenesis and carcinogenesis.
Kinetic basis of DNA synthesis by human DNA polymerase/primase PrimPol
PrimPol is the most recently discovered human DNA polymerase/primase, which belongs to archaeo-eukaryotic primase (AEP) superfamily. PrimPol is important for nuclear and mitochondrial genomic maintenance particularly when the replication fork is stalled by DNA damage or secondary structures. Such role in genomic maintenance is likely to be attributed to PrimPol’s DNA lesion bypass and de novo DNA synthesis (re-priming) activities. Despite the previous biochemical characterizations, it remains unclear concerning the kinetic basis of PrimPol-catalyzed nucleotide incorporation. Such knowledge is essential for further the understanding of PrimPol-catalyzed DNA synthesis, as well as for the develop novel approaches to regulate the enzymatic activity of PrimPol. In the present study, we performed detailed kinetic analysis and computer simulations to better understand the mechanism by which PrimPol catalyzes the nucleotidyl-transfer reaction. Our experiments revealed that PrimPol-catalyzed nucleotide insertion entails a rate-limiting step prior to the chemistry step. We assigned this step as the conformational change of PrimPol from a non-productive to a productive conformation for nucleotidyl transfer. The partition between these conformations is dependent on the length of single-stranded DNA of the DNA substrate, consistent with PrimPol’s single-stranded DNA binding and primase activities. The computer simulated rate of the conformational change is comparable to the experimentally measured kcat, confirming that this conformational change is rate-limiting during the nucleotidyl transfer. Collectively, these data provide a kinetic framework for further understanding the catalytic and regulatory mechanisms of PrimPol.
Levels of glyoxal-induced hemoglobin modifications correlate with DNA cross-links in human blood as determined by mass spectrometry
Glyoxal is an oxoaldehyde derived from the degradation of glucose-protein conjugates and from lipid peroxidation in foods and in vivo, and it is also present in the environment. The plasma concentrations of glyoxal is higher in diabetic patients compared to nondiabetics. Glyoxal is known to covalently modify DNA and proteins. We previously developed sensitive assays based on nanoflow liquid chromatography-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) for quantification of DNA and hemoglobin adducts derived from glyoxal. In this study, we isolated and measured both leukocyte DNA and hemoglobin from the blood of type 2 diabetes mellitus (T2DM) patients and compared the DNA and hemoglobin adduct levels with those from nondiabetic subjects using the modified assays. The results showed that the levels of both glyoxal-induced DNA cross-linked adducts dG-gx-dC and dG-gx-dA in blood leukocyte DNA were statistically higher in T2DM patients than those in healthy individuals. The extents of glyoxal-induced hemoglobin modifications on α-Lys-11, α-Arg-92, β-Lys-17, and β-Lys-66 were statistically higher in T2DM patients compared to nondiabetics. Moreover, the levels of dG-gx-dC in leukocyte DNA correlated with α-Arg-92, and β-Lys-17, while levels of dG-gx-dA correlated with α-Lys-11, α-Lys-16, α-Arg-92, and β-Lys-66. To the best of our knowledge, this is one of the few reports of positive correlation between DNA and protein adducts of the same compound (glyoxal) in the blood from the same subjects. Because of the high abundance of hemoglobin in blood, the results suggest that quantification of modified peptides in hemoglobin might serve as a practical biomarker for assessing DNA and protein damages induced by glyoxal.
High mobility group box 1: A re-evaluation of its role in cancer
High mobility group box 1 (HMGB1), is a non-histone chromosomal protein, which can be secreted through a variety of pathways and bind to pattern recognition receptors to release pro-inflammatory cytokines. Previous studies have shown that serum HMGB1 is up-regulated in various cancers including breast, laryngeal, pancreatic, non-small cell lung cancer, pleural mesothelioma, prostate, and renal, and that it could be a biomarker for such diseases. However, these studies used immunoassay-based methods to analyze serum HMGB1 and the concentrations reported for healthy controls (0.10 to 39.7 ng/mL) and cases (0.15 to 58.6 ng/mL) both varied widely. Autoantibodies to HMGB1 in serum are found in healthy control subjects as well as in patients with cancer. HMGB1 also binds to haptoglobin, a highly abundant plasma protein. This means that antibodies used in immunoassays must compete with binding of HMGB1 to endogenous serum HMGB1 autoantibodies and haptoglobin. To overcome these potential problems, we developed and validated a specific and sensitive assay based on stable isotope dilution and immunopurification to quantify HMGB1 in plasma and serum using two-dimensional-nano-ultra high-performance liquid chromatography-parallel reaction monitoring/high resolution mass spectrometry. Using this assay, we found that serum HMGB1 in 24 healthy control subjects fell into a narrow range of 6.0 ± 2.1 ng/mL when compared with control values reported previously (0.1 to 39.7 ng/mL) where the analyses were conducted using immunoassay methodology. The concentration of HMGB1 in citrated and EDTA plasma from the same healthy control subjects was below the limit of detection of our assay (1 ng/mL) confirming that serum HMGB1 arises when blood is allowed to clot and that it cannot be a circulating cancer biomarker. In light of our finding, the role of HMGB1 in cancer will have to be re-evaluated. Furthermore, such studies should be conducted using plasma rather than serum.
Determining associations between transcriptomics and toxicity using co-expression network methods
The advent of largescale toxicogenomics databases based on in vivo studies allows direct association of histopathological phenotypes with gene expression across a range of time-points and doses. This association is not currently clear but is crucial in helping to understand potential mechanisms of toxicities in order to reduce late stage drug development failure due to unacceptable toxicity.
In this work, histopathological signatures were generated from the DrugMatrix database, using the histopathology ontology, HPATH, to group similar assays. These distinct signatures provided the basis for generating consensus toxic networks that were investigated using weighted gene co-expression network analysis (WGCNA). As a case study, cellular infiltrate in the liver was selected, 25 instances with the same signature defined as the toxic set and a consensus co-expression network was generated. Analysis of modules, defined as highly correlated genes, revealed five modules that were not conserved between the toxic and the control cases. The OPEN TG-GATES database was used to validate the conservation of these modules and showed one to be highly conserved and annotated with an appropriate histopathology signature. This module was significantly enriched in genes involved in cytokine and drug response pathways. This was confirmed by annotating with the Comparative Toxicogenomics Database, revealing the consistency with current knowledge and allowing previously unstudied compounds to be classified. This work shows associations between highly correlating genes and toxicities. This provides the potential to identify early stage biomarkers for toxicity that would otherwise be found from late stage histopathology.
Inhibitors of the mitochondrial respiratory complex – Structure-based prediction of toxicity
High throughput structural genomics such as pursued by the structural genomics consortium leads to a constantly increasing number of high quality structures of proteins. Besides supporting drug discovery and development, this also allows to consider structure-based approaches for in silico toxicology. Here we present our modelling efforts for mitochondrial respiratory complexes I, II, and III designed to gain insights into the ligand binding events as well as to learn what triggers ligand-complex selectivity. The basis for the study is a comprehensive data set of respiratory complex I, II, and III inhibitors collected in the framework of the EU-ToxRisk project (http://www.eu-toxrisk.eu).
While for human complex II a high resolution structure has not been resolved up to now, available structures of complex I and III allow to utilize structure-based approaches. In case of complex I, aligning the structure of Rhodobacter capsulatus with the available cyro-EM structure of the human analog allowed to define a proper binding site for complex I inhibitors. Docking of 5 compounds into this site followed by common scaffold clustering of all poses identified two clusters where rotenone and deguelin share the same binding mode. In case of human complex III, we face the advantage that the analogous bovine structure is available in a conformation bound to antimycin A. Aligning the bovine structure with the human one allowed to reproduce the antimycin A binding mode in the human protein.
Analysis of the binding modes aids in the understanding of the molecular basis triggering interaction and, ultimately, will lead to better prediction of mitochondrial toxicity of compounds.
Configurational and conformational equilibria of the N6-(2-Deoxy-D-erythro-pentofuranoysl-)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine (MeFapy-dG) lesion in DNA
Formamidopyrimidine (Fapy) lesions arise from N7-methylation of dG by chemotherapeutic agents such as temazolamide, forming N6-(2-Deoxy-D-erythro-pentofuranoysl-)-2,6-diamino-3,4-dihydro-4-oxo-5-N-methylformamidopyrimidine (MeFapy-dG). A 13C-MeFapy-dG lesion, in which the CH3 group of the MeFapy-dG was isotopically labeled with 13C, was incorporated into the trimer 5′-TXT-3′ and the dodecamer 5′-C1 A2 T3 X4 A5 T6 G7 A8 C9 G10C11T12-3’ (X = 13C-MeFapy-dG). We probed the equilibrium chemistry of the MeFapy-dG lesion in DNA. Our studies reveal that the MeFapy-dG lesion exists in single strand DNA as ten configurationally and conformationally discrete species, eight of which may be unequivocally assigned. MeFapy-dG lesions undergo epimerization to produce either α or β anomers. In the trimer single strand DNA, for each deoxyribose anomer, atropisomerism occurs around the C5–N5 bond to produce Ra and Sa atropisomers. Each atropisomer also exhibits geometrical isomerism about the formyl bond yielding E and Z conformations. The relative abundances of these species have been determined. In duplex DNA, the MeFapy-dG lesion existed as six configurationally and conformationally discrete species. These species may be differentially recognized during repair and replication processing, providing a mechanistic basis for the generation of sequence-specific repair and replication events in damaged DNA.
Using open bioactivity data for developing machine-learning prediction models for chemical modulators of the retinoid X receptor (RXR) signaling pathway
The retinoid X receptor (RXR) is a nuclear hormone receptor that functions as a transcription factor with roles in development, cell differentiation, metabolism, and cell death. Chemicals that interfere the RXR signaling pathway may cause adverse effects on human health. In this study, open bioactivity data available at PubChem (https://pubchem.ncbi.nlm.nih.gov) were used to develop prediction models for chemical modulators of RXR-alpha, which is a subtype of RXR that plays a role in metabolic signaling pathways, dermal cysts, cardiac development, insulin sensitization, etc. The models were constructed from quantitative high-throughput screening (qHTS) data from the Tox21 project, using various supervised machine learning methods (including support vector machine, random forest, neural network, k-nearest neighbors, decision tree, and naïve Bayes). The performance of the models was evaluated with an external data set containing bioactivity data submitted by ChEMBL and the NCATS Chemical Genomics Center (NCGC). This study showcases how open data in the public domain can be used to develop prediction models for chemical toxicity.
Amanda Bryant-Friedrich – ACS Fellow
Amanda Bryant-Friedrich received her PhD at Ruprecht Karls Universität, Heidelberg, Germany, with Professor Richard Neidlein, and was a post-doctoral fellow at Universität Basel, Basel, Switzerland, with Professor Bernd Giese. Her first faculty position was at Oakland University in Rochester Hills, Mich. She subsequently joined the University of Toledo and is currently Dean, College of Graduate Studies, Professor of Medicinal Chemistry, and Director, Shimadzu Laboratory for Pharmaceutical Research Excellence .
Amanda’s laboratory studies the mechanisms of oxidative damage to nucleic acids and the subsequent biological response. He lab uses a chemical approach by synthesizing organic molecules capable of generation of free radical species in and around DNA and RNA. The mechanisms by which nucleic acids are oxidized can be determined and the fate of their subsequent damage products investigated.
Amanda has played a large role in the Division of Chemical of Toxicology. She was Division Councillor, Program Chair, and is currently our representative on the American Chemical Society’s Multidisciplinary Program Planning Group. She also played a leadership role in the ACS’s Womens Chemists Committee and is on Large Range Planning Committee for the Division of Medicinal Chemistry