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Director: Robert J. Turesky, Ph.D. Executive SummaryIntroductionThe Division of Chemistry contributes to NCTR/FDA-directed research initiatives through investigations on sensor technology for rapid screening to assess food quality and detect volatile explosives, nuclear magnetic resonance (NMR)-based metabonomics, mass spectrometry (MS) applications in proteomics, biomarkers, analytical chemistry and counter-terrorism, Strategic Research Goal: Predictive Toxicology using computational chemistry and artificial intelligence, as well as analytical chemistry collaborations for the National Toxicology Programs (NTP). The Division has made investments in state-of-the-art equipment in MS and a high-field NMR instrument for the establishment of proteomics and metabonomics facilities. These facilities and new techniques will complement the newly created program centers in structural and functional genomics, toxicogenomics, the Heptatoxicity Lab, and other research divisions at NCTR to elucidate mechanisms of action of chemicals and products under question. FY 2002 AccomplishmentsThe newly created Mass Spectrometry Proteomics Laboratory has rapidly transformed from its conception in 2001 into an established laboratory in 2002. Two scientists with substantial experience in proteomics have been hired with a third position to be filled soon. The laboratory houses state-of-the-art MS equipment, a matrix-assisted laser desorption ionization–time-of-flight (MALDI-TOF), an electrospray ionization–quadropole time-of-flight (ESI-Q-TOF), and an ion trap with both MALDI and ESI capabilities, as well as a full line of robotics to aid in sample processing. Both ESI instruments are equipped with custom-designed nanoHPLC sources that allow high sensitivity required for most proteomic experiments. The Mass Spectrometry Counter Terrorism Program was initiated in 2002 with the purchase and installation of a MALDI-TOF and a pyrolysis metastable atom bombardment (MAB)-TOF MS. These instruments will be utilized to produce unique mass spectra of microorganisms, and in conjunction with pattern recognition, rapidly characterize bio-terror agents or bio-terror hoax materials such as talc or flour. In computational chemistry, pattern recognition-based methods for the non-invasive diagnosis of brain tumors were developed in collaboration with researchers at the University of Arkansas for Medical Sciences (UAMS). A patent was submitted for three-dimensional quantitative spectrometric data-activity relationship (3D-QSDAR) (E07126), where models are developed using the combination of NMR spectral information with internal structural connectivity information and relating this combined pattern of information to biological endpoints. Advanced Chemistry Development (ACD) in Toronto, Canada, has negotiated a license for the previous SDAR patent (E07068) and is expected to license the patent for 3D-QSDAR. Pharmacokinetic studies were done with the University of Pittsburgh and the UAMS on hyperforin (HF), a major active component of St. John's wort, which may affect drug-herbal interactions that diminish therapeutic efficacy of medications (E0705611). The methods developed to measure HF in plasma will aid to assess the effects of St John’s wort on human xenobiotic enzymes. In a caloric restriction study, a 10-percent reduction in calories significantly increased the life span of experimental animals (E692401), possibly through reduced free radical damage that may contribute to aging and disease since antioxidant potential increased. Moreover, antioxidant enzyme potential and mitochondrial function increased in obese women who have undergone gastric bypass surgery (E699101), suggesting that reduction in caloric intake (even small reductions) may be beneficial in humans. Rapid, chemical sensor technology (designated as Food Quality Indicator [FQI] to assess food quality (E0708001) was evaluated by the National Marine Fisheries and Canadian Center for Fisheries Innovation (CCFI), St. John, Newfoundland, Canada. The commercial version of Fresh Tag™ was more rapid, sensitive and rugged than other comparative methods tested by CCFI. An interagency agreement was established with the Federal Aviation Administration (FAA) to develop similar rapid sensor methods to detect explosives for protection of the air transportation industry (E0708101). Solid phase and immunoaffinity procedures, followed by liquid chromatography electrospray ionization (LC-ESI) tandem MS methods, were developed to isolate and quantitate the dietary and tobacco carcinogens heterocyclic aromatic amines (HAAs) and their metabolites in human urine of population studies that are at elevated risks of cancer development. The Analytical Support and Mass Spectrometry Branches continued collaborations on chemicals under investigation by the NTP, including the Aloe barbadensis constituents, aloin A and malic acid, and several anti-HIV drugs. The Division collaborated with the Center for Veterinary Medicine (CVM) on analysis of the antibiotic erythromycin A (EA) in salmon (E06980) and provided guidance to CVM for inter-laboratory method trials for leucomalachite green and multi-sulfonamide methods. The Mass Spectrometry Branch also continued collaborations on NTP initiatives on isoflavones (E02138), fumonisins and ethinylestradiol in dose feed (E02138), and methods development of anti-HIV drugs (E02141). The branch also conducted characterization of HF degradation products (E07056) and validation of a LC-electrochemical method for EA in salmon (E06980). In collaboration with the Division of Microbiology, microbial or fungal metabolites of EA (E07075), daidzein (E07007), fluoroquinolones (E07052) and polycyclic aromatic hydrocarbons (E07075) were characterized by MS to understand mechanisms of antibiotic and antimicrobial resistance and biodegradation of environmental contaminants. FY 2003 PlansSeveral new research initiatives are planned for 2003. In Proteomics:
In Counter-Terrorism:
In Computational Chemistry:
In NMR-Based Metabonomics:
Public Health SignificanceThe new technologies in MS applications for proteomics, biomarker development and counter-terrorism, NMR-based metabonomics, and computational chemistry that are under development in the Division of Chemistry will aid in addressing many health and food safety issues. MS methods in proteomics will advance studies on mechanisms of antibiotic resistance, biochemical modifications of proteins associated with cellular metabolism and protein function, and signature proteins as biomakers of disease states that are under study at NCTR. MS is also a critical means to assess exposure to environmental toxins and an emerging technology to rapidly identify bioterror-related materials. NMR-based metabonomics is a non-invasive method that can measure the kinetic profile of onset, progression, and recovery from toxic events induced by drugs. The data obtained can aid in the interpretation of biological effects of drugs and in the establishment of FDA policies during an investigational new drug (IND) or new drug applications (NDA) submission to the FDA. The use of computational chemistry methods in examining patterns of diseases, such as breast cancer, may enable us to identify disease states at a much earlier juncture, perhaps before the onset of pathological abnormalities, and lead to earlier treatments. All of these programs are using exciting, state-of-the-art technologies to improve human health and safety. Research ProjectsPI: Ang, CatharinaTitle: ADDEND: Development of Analytical Methodologies for
Assessing Bioactive Herbal Ingredients in Functional Food Systems Objective(s):
Title: Influence of Hyperforin Concentration on Drug
Interactions Objective(s):
PI: Beger, RichardTitle: Producing Spectrometric Data Activity Relationship (SDAR)
Models for Compounds Binding to Receptors of Toxic Responses. Objective(s):
Title: Methods for Predicting Toxicological Properties of
Molecules from Their NMR Chemical Shifts Through-bond and Through-space Distance
Connectivity Patterns Objective(s):
PI: Billedeau, StanleyTitle: Development of Methods for Analysis and Confirmation of
Erythromycin A Residues in Tissue Samples from Terrestrial and Aquatic Farmed
Animals by Liquid Chromatography Objective(s):
PI: Buzatu, DanTitle: Comparison of Principal Components Analysis (PCA) and
Artificial Neural Networks (ANN) for Prediction of Qualitative and Quantitative
Biological End Points from Spectrometric Data Objective(s):
PI: Feuers, RitchieTitle: Influence of Dietary Restriction on Somatic Mutation and
Antioxidant Enzymes Induced by Exposure of Female and Male Fischer 344 Rats to
Bleomycin (BLM) Objective(s):
Title: Memphis Study: Evaluation of Calorically Restricted
Human Surgical Samples Received from Department of Surgery University of
Tennessee, Memphis Objective(s):
PI: Leakey, JulianTitle: Chronic Bioassay of Chloral Hydrate in Male B6C3F1 Mice
Using Idealized Body Weight Curves that are Normalized by Modulation of Caloric
Intake Objective(s):
Title: ADDEND: Dose Response to Chloral Hydrate in Dietary
Restricted Mice Objective(s):
PI: Miller, DwightTitle: Development of Devices/Methods for Determination of
Food/Seafood Quality Objective(s):
Title: Innovative Methods for Determining Food Quality:
Decomposition, Safety and/or Economic Fraud Objective(s):
Title: Rapid Screening Test for Food Quality Objective(s):
Title: Application of Solid Phase Detection Systems to
Explosives in Airplane Cargo Objective(s):
PI: Turesky, RobertTitle: Human Risk Assessment of Heterocyclic Aromatic Amines:
Exposure, Development of Novel Biomarkers of Cytochrome P450 1A2 Activity and
DNA Adduct Formation Objective(s):
Title: Toxicological Effects of Ochratoxin A Objective(s):
PI: Wilkes, JonTitle: First Phase Development of a Rapid Screening Method for
Identification of Complex Mixtures by Pyrolysis-Mass Spectrometry with
Computerized Pattern Recognition Objective(s):
Title: Combining MAB/MS with Pattern Recognition to Sub-type
Bacteria Objective(s):
PublicationsAng, C.Y. and Cui, Y., Modulation of Cytochrome P450 Enzymes by St. John's Wort Constituents Hyperforin and Hypericin in Primary Cultures of Human Hepatocytes, Proceedings 6th International ISSX Meeting. Accepted: 10/7/2001 Ang, C.Y., Cui, Y., Chang, H.C. and Heinze, T.M., Determination of St. John's Wort Components in Dietary Supplements and Functional Foods by Liquid Chromatography, J. AOAC International. Accepted: 7/3/2002 (E0705601) Beger, R. and Buzatu, D.A., Wilkes, J.G. and Lay, J.O., Comparative Structural Connectivity Spectra Analysis (CoSCSA) Models of Steroids Binding to the Corticosteroid Binding Globulin, J. Chem. Inf. Comp. Sci., 42(5):1123-1131. Accepted: 5/13/2002 (E0712601) Beger, R., Mcconnell, K., Arthanari, H., Young, M.A., Beveridge, D.L. and Bolton, P.H., Assessment of the Molecular Dynamics Structure of DNA in Solution Based on Calculated and Observed NMR NOESY Volumes, Biopolymers. Accepted: 7/20/2002 (N/A) Beger, R., Wilkes, J.G., Comparative Structural Connectivity Spectra Analysis (CoSCSA) Models of Steroids Binding to the Aromatase Enzyme, J. Molecular Recognition, 15:154-162. Accepted: 5/9/2002 (E0712601) Chang, H., Huffer, D.M., Chiarelli, M.P., Blankenship, L., Culp, S. and Cho, B.P., Characterization of DNA Adducts Derived from syn-Benzo[ghi]fluoranthene-3,4-Dihydrodiol-5,5a-Epoxide and Comparative DNA Binding Studies with Structurally-Related anti-Diolepoxides of Benzo[ghi]fluoranthene and Benzo[c]phenanthrene, Chemical Research in Toxicology, 15:198-208. Accepted: 12/11/2001 (S00179) Churchwell, M.I., Holder, C.L., Little, D., Preece, S.W., Smith, D.J. and Doerge, D.R., LC-ES/MS/MS Analysis of incurred ractopamine residues in livestock tissues, Rapid Communications in Mass Spectrometry, 16:1261-1265. Accepted: 5/7/2002 (E0694501) Cui, Y., Ang, C.Y., Gurley, B. and Leakey, J.E., Determination of Hyperforin in Human Plasma Using Solid Waste Phase Extraction and High-Performance Liquid Chromatography with Ultraviolet Detection, Journal of Chromatography B. Accepted: 6/28/2002 (P00436) Cui, Y., Ang, C.Y., Liu, F., Rankin, J.D. and Chang, H.C., Ultrasonic Extraction and Liquid Chromatographic Analysis of Dietary Supplement Products Containing St. John's Wort (Hypericum perforatum L.), Functional Food Ingredients: Trends and Prospects:, American Chemical Society. Accepted: 10/22/2001 (E0705601) Cui, Y. and Ang, C.Y., Supercritical Fluid Extraction and High-Performance Liquid Chromatographic Determination of Phloroglucinols in St. John's Wort (Hypercium perforatum L.), J. Agri. Food Chemistry, 50:2755-2759. Accepted: 3/11/2002 (E0705601) Delatour, T., Guy, P.A., Stadler, R.H. and Turesky, R., 3-Nitrotyrosine Butyl Ester: A Novel Derivative to Assess Tyrosine Nitration in Rat Plasma by Liquid Chromatography-Tandem Mass Spectrometry Detection, Analytical Biochemistry, 302:10-18. Accepted: 1/30/2002 (E0709401) Delatour, T., Richoz, J., Vouros, P. and Turesky, R., Simultaneous Determination of 3-nitrotyrosine and Tyrosine in Plasma Proteins of Rats and Assessment of Artifactual Tyrosine Nitration, Journal of Chromatography A. Accepted: 7/1/2002 (N/A) Doerge, D.R. and Chang, H.C., Inactivation of thyroid peroxidase by soy isoflavones, in vitro and in vivo, Journal of Chromatography. Accepted: 10/25/2001 (E0213201) Fu, P.P., Yang, Y., Xia, Q., Chou, M.W., Cui, Y. and Lin, G., Pyrrolizidine alkaloids-tumorigenic components in Chinese herbal medicines and dietary supplements, J. Food Drug Analys. Accepted: 8/28/2002 (E0710401) Gorlewska, K., Teitel, C.H., Lay, J.O., Roberts, D.W. and Kadlubar, F.F., The role of lactoperoxidase-catalyzed activation of aromatic and heterocyclic amines in relation to breast carcinogenesis, Chemical Research in Toxicology, in press. Accepted: 7/11/2002 Gurley, B., Gardner, S., Hubbard, M., Williams, D.K., Gentry, W.B., Cui, Y. and Ang, C.Y., Cytochrome P-450 Phenotypic Ratios for Predicting Herb-Drug Interactions in Humans, Clinical Pharmacology and Therapeutics. Accepted: 5/30/2002 Huber, W.W., Scharf, G., Rossmanith, W., Prustomersky, S., Grasl-Kraupp, B., Peter, B., Turesky, R. and Schulte-Hermann, R., The Coffee Components Kahweol and Cafestol Induce y-Glutamylcystein Synthetase, the Rate Limiting Enzyme of Chemoprotective Gluthione Synthesis, in Several Organs of the Rat, Arch. Toxicol., 75:685-694. Accepted: 10/15/2001 (E0709101) Huber, W.W., Prustomersky, S., Scharf, G., Turesky, R. and Schulte-Hermann, R., Enhancement of the chemoprotective enzymes glucuronosyl transferase and glutathione transferase in specific organs of the rat by the coffee components kahweol and cafestol, Arch.Toxicol., 76:209-217. Accepted: 1/11/2002 Hur, H., Beger, R., Heinze, T.M., Lay, J.O., Freeman, J.P. and Rafii, F., Isolation of an anaerobic intestinal bacterium capable of cleaving the C-ring of the isoflavonoid daidzein, Arch. Microbiol., 178:8-12. Accepted: 2/19/2002 (E0700701) Lewis, S.M., Mayhugh, M.A., Freni, S.C., Cardoso, S.S., Buffington, C.K., Jairaj, K., Turturro, A. and Feuers, R.J., Assessment and significance of 24-h Energy Intake Patterns Among Young and Aged Non-Affluent Southern U.S. Women, The Journal of Nutrition, Health & Aging. Accepted: 5/7/2002 (E0699801) Lewis, S.M., Mayhugh, M.A., Freni, S.C., Thorn, B.T., Cardoso, S.S., Buffington, C.K., Jairaj, K., Turturro, A. and Feuers, R.J., Assessment of Antioxidant Nutrient Intake of a Population of Southern U.S. African-American and Caucasian Women of Various Ages When Compared to Dietary Reference Intakes, The Journal of Nutrition, Health & Aging. Accepted: 5/7/2002 (E0699801) Luo, W., Ang, C.Y., Gehring, T.A., Heinze, T.M. and Mattia, A., Determination of Phenolic Compounds in Dietary Supplements and Tea Blends Containing Echinacea by Liquid Chromatography with Coulometric Electrochemical Detection, J. AOAC International. Accepted: 8/19/2002 (E0705601) Luo, W., Li, H., Ang, C.Y. and Zhang, Y., Rapid Method for the Determination of Total 5-Methyltetrahydrofolate in Blood by Liquid Chromatography with Fluorescence Detection, J. Chromatography B, 766:331-337. Accepted: 10/29/2001 Moody, J.D., Doerge, D.R., Freeman, J.P. and Cerniglia, C.E., Degradation of biphenyl by Mycobacterium sp. PYR-1, Applied Microbiology and Biotechnology, 58:364-369. Accepted: 10/19/2001 (E0690101) Moody, J.D., Freeman, J.P., Fu, P.P. and Cerniglia, C.E., Biotransformation of mirtazapine by Cunninghamella elegans, Drug Metabolism and Disposition. Accepted: 7/21/2002 (E0690101) Mottier, P., Gremaud, E., Guy, P.A. and Turesky, R., Comparison of Gas Chromatography-Mass Spectrometry and Liquid Chromatography-Tandem Mass Spectrometry Methods to Quantify a-Tocopherol and a-Tocopheroluinone Levels in Human Plasma, Analytical Biochemistry, 301:128-135. Accepted: 10/8/2001 (E0709401) Paehler, A., Richoz, J., Soglia, J.R., Vouros, P. and Turesky, R., Analysis and Quantification of DNA Adducts of 2-Amino-3,8-dimethylimidazo[4,5-f]quanoxaline in Liver of Rats by Liquid Chromatography/Electrospray Tandem Mass Spectrometry, Chemical Research in Toxicology, 15(4):551-561. Accepted: 1/18/2002 (E0709101) Parshikov, I., Moody, J.D., Heinze, T.M., Freeman, J.P., Williams, A.J. and Sutherland, J.B., Transformation of cinoxacin by Beauveria bassiana, FEMS Microbiology Letters, 214:133-136. Accepted: 7/13/2002 (E0705201) Popke, J., Patton, R.E., Newport, G.D., Rushing, L.G., Fogle, C.M., Allen, R.R., Pearson, E., Hammond, T. and Paule, M.G., Assessing the potential toxicity of MK-801 and remacemide Chronic exposure in juvenile rhesus monkeys, Neurotoxicology and Teratology. Accepted: 1/29/2002 (E0280001) Ritter, C.L., Culp, S., Freeman, J.P., Marques, M.M., Beland, F.A. and Malejka-Giganti, D., DNA Adducts from Nitroreduction of 2,7-Dinitrofluorene, a Mammary Gland Carcinogen, Catalyzed by Rat Liver or Mammary Gland Cytosol, Chem. Res. Tox., 15:536-544. Accepted: 2/15/2002 (S00198) Shvartsburg, A.A., Wilkes, J.G., Siu, M.K. and Lay, J.O., Fragmentation and Charge Transfer in Gas-Phase Complexes of Divalent Metal Ions with Acetonitrile, Chemical Physics Letters, 350:216. Accepted: 12/15/2001 (N/A) Shvartsburg, A.A. and Wilkes, J.G., Fragmentation Chemistry of DMSO Complexes of Metal Dications, Journal of Physical Chemistry, 106:4543-4551. Accepted: 3/14/2002 (E0712001) Shvartsburg, A.A., Acetonitrile Complexes of Triply Charged Metal Ions: Are Ligated trications Intrinsically More Prone to Charge Reduction Than Dications?, Chemical Physics Letters, 360:479. Accepted: 4/15/2002 (E0702001) Shvartsburg, A.A., DMSO Complexes of Trivalent Metal Ions:First Microsolvated Trications Outside of Group 3, JACS. Accepted: 8/19/2002 (E0712001) Shvartsburg, A.A., Gas-Phase Metal Trications in Protic Solvent Complexes, J. American Chemical Society, 124:7910. Accepted: 6/3/2002 (E0712001) Shvartsburg, A.A., Isomer-Resolved Ion Spectroscopy, Physical Review Letters, 89. Accepted: 7/15/2002 (E0712001) Tong, B., Chang, S., Carpenter, E.E., O'Connor, C.J., Lay, J.O. and Norman, R.E., Di-u-halo-bis{tris(2-pyridyomethy)amine-K4N]nickel(II)}bis(triethylammonium)tetraperchlorate: Magnetostructural Studies, Inorganica Chimica Acta. Accepted: 10/1/2001 (E0700501) Turesky, R., Guengerich, F.P., Guillouzo, A. and Langouet, S., Metabolism of Heterocyclic Aromatic Amines by Human Hepatocytes and Cytochrome P4501A2, Mutation Research. Accepted: 7/25/2002 (E0709101) Turesky, R., Heterocyclic Aromatic Amine Metabolism, DNA Adduct Formation, Mutagenesis, and Carcinogenesis, Drug Metabolism Reviews, 34(3):625-650. Accepted: 8/1/2002 (E0709101) Wilkes, J.G., Glover, K.L., Holcomb, M., Rafii, F., Cao, X., Sutherland, J.B., Letarte, S. and Bertrand, M.J., Defining and Using Microbial Spectral Databases, Journal of the American Society of Mass Spectrometry, 13:875-887. Accepted: 3/21/2002 (E0693101) Concept PapersPI: Beger, RichardTitle: Concept - NMR-Based Metabonomics of Drugs Suspected of
Liver and Kidney Toxicity Objective(s):
PI: Buzatu, DanTitle: Concept - 13C Magnetic Resonance Breast Cancer
Diagnostic Models Objective(s):
Title: The Development of Dynamic Mass Spectral/Pattern
Recognition Based Methods for the Rapid Identification of Bioterror Agents Objective(s):
PI: Feuers, RitchieTitle: Concept - Development of Techniques in Proteomics for
Use in Chemistry Studies Objective(s):
PI: Turesky, RobertTitle: Dietary Factors in the Etiology of Human Cancer,
Biomonitoring of Heterocyclic Aromatic Amines – CRADA-funded portion of E0709101
Objective(s):
Title: Rapid Analysis of Protein Oxidation Markers by
ImmunoDetection and Mass Spectrometry Objective(s):
PI: Wilkes, JonTitle: Evaluation of Pyrolysis MAB/Tof MS and MALDI/Tof MS for
Rapid Characterization of Presumptive Bio-terror Agent Samples Objective(s):
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