Biomarkers of Organophosphate-Adducted Proteins

Summary

Principal Investigator: CHARLES MARK THOMPSON
Abstract: The short-term goals of this application are to identify adducted and altered protein biomarkers that correlate with organophosphate (OP) insecticide exposure and to use these biomarkers to generate antibodies to validate the detection of these biomarkers in biological fluids. Over 100 million pounds of OP insecticides are used each year but lead to over 25,000 reports to poison control centers each year including 10,000 involving children under age six. These reports only account for acute poisoning events and do not include low dose or chronic exposures. In addition to general exposure and food contamination, the safety of OPs is a large public health concern because OPs share chemical traits, structure and biological mechanism with nerve gas agents. These concerns are elevated by an array of neurologic and non-neurologic sequelae that have been reported in connection with exposure to OPs. To combat, monitor and provide therapeutic intervention, a blood cholinesterase test (BCT) has been conducted for decades. However, the test is limited and inadequate to assess OP exposure and its shortcomings identified by an EPA report questioned the merit of the BCT and suggested the need to examine true biomarkers of exposure. At this time, there is a serious void in effective tests of OP exposure. New tests are needed that are sensitive, selective, operate in real time and are based on reliable, well-characterized OP-biomarkers derived from exacting molecular events that correlate with cellular, tissue, organ or systemic toxicity. The long range goal of our research is to identify OP-adducted and/or OP-altered protein biomarkers that are associated with OP-induced sequelae and to develop diagnostic methods and tests that assess the human health risk and aid therapeutic action. For the proposed grant period, we will address the following specific aims: SA 1. Show that OP's result in highly specific OP-AChE adducts that can be identified and differentiated by antibodies. SA2. Identify and characterize OP-adducted protein biomarkers in SH-SY5Y neuroblastoma cells using OP-reporter molecules. SA3. Identify and characterize OP-protein biomarkers from human saliva, human serum and human erythrocytes and SA4. Prepare customized antibodies that recognize OP-adducted proteins identified in SA-II and SA-III and develop efficient diagnostic tests to identify and quantify OP-protein adducts.
Funding Period: ----------------2007 - ---------------2011-
more information: NIH RePORT

Top Publications

  1. pmc Mass spectrometry identifies covalent binding of soman, sarin, chlorpyrifos oxon, diisopropyl fluorophosphate, and FP-biotin to tyrosines on tubulin: a potential mechanism of long term toxicity by organophosphorus agents
    Hasmik Grigoryan
    Eppley Institute, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198 6805, USA
    Chem Biol Interact 175:180-6. 2008
  2. pmc Fluorescent probes of the isoxazole-dihydropyridine scaffold: MDR-1 binding and homology model
    Monika I Szabon-Watola
    Department of Chemistry, University of Idaho, Moscow, ID 83843, United States
    Bioorg Med Chem Lett 24:117-21. 2014
  3. pmc Oxidative stress resulting from exposure of a human salivary gland cells to paraoxon: an in vitro model for organophosphate oral exposure
    John M Prins
    Department of Biomedical and Pharmaceutical Sciences, College of Health Professions and Biomedical Sciences, The University of Montana, Missoula, MT 59812, United States
    Toxicol In Vitro 28:715-21. 2014
  4. pmc Five tyrosines and two serines in human albumin are labeled by the organophosphorus agent FP-biotin
    Shi Jian Ding
    Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA
    Chem Res Toxicol 21:1787-94. 2008
  5. pmc Diastereomers (R(C),S(P))- and (R(C),R(P))-S-methyl P-(3-azidopropyl)-N-[(1R)-1-phenylethyl]phosphonamidothioate
    Lilu Guo
    Department of Chemistry, University of Montana, Missoula, MT 59812, USA
    Acta Crystallogr C 65:o179-82. 2009
  6. pmc Antimicrobial, antimalarial, and antileishmanial activities of mono- and bis-quaternary pyridinium compounds
    Sandip B Bharate
    NIH COBRE Center for Structural and Functional Neuroscience, Department of Biomedical and Pharmaceutical Sciences, The University of Montana, Missoula, MT 59812, USA
    Chem Biol Drug Des 76:546-51. 2010
  7. pmc Paraoxon-induced protein expression changes to SH-SY5Y cells
    John M Prins
    The Center for Structural and Functional Neuroscience, Department of Biomedical and Pharmaceutical Sciences, The University of Montana, Missoula, Montana 59812, USA
    Chem Res Toxicol 23:1656-62. 2010
  8. pmc Subacute developmental exposure of zebrafish to the organophosphate pesticide metabolite, chlorpyrifos-oxon, results in defects in Rohon-Beard sensory neuron development
    Saskia M Jacobson
    Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, MT 59812, USA
    Aquat Toxicol 100:101-11. 2010
  9. pmc Thionate versus Oxon: comparison of stability, uptake, and cell toxicity of ((14)CH(3)O)(2)-labeled methyl parathion and methyl paraoxon with SH-SY5Y cells
    Sandip B Bharate
    Department of Biomedical and Pharmaceutical Sciences, The University of Montana, Missoula, MT 59812, USA
    J Agric Food Chem 58:8460-6. 2010
  10. pmc Organophosphorus pesticides decrease M2 muscarinic receptor function in guinea pig airway nerves via indirect mechanisms
    Becky J Proskocil
    Division of Pulmonary and Critical Care Medicine, Oregon Health and Science University, Portland, Oregon, United States of America
    PLoS ONE 5:e10562. 2010

Scientific Experts

  • Charles Thompson
  • Hasmik Grigoryan
  • Lilu Guo
  • Saskia M Jacobson
  • Sandip B Bharate
  • John M Prins
  • Kathleen M George
  • Bin Li
  • Monika I Szabon-Watola
  • Becky J Proskocil
  • Tony E Reeves
  • Douglas M Cerasoli
  • DENISE A BIRKHOLZ
  • Steven H Hinrichs
  • Patrick Masson
  • Lawrence M Schopfer
  • Oksana Lockridge
  • Shi Jian Ding
  • Scott A Steiger
  • Christina D Hayes
  • Nicholas R Natale
  • Chih Kai Chao
  • Sarah V Ulatowski
  • Allison D Fryer
  • Donald A Bruun
  • Pamela J Lein
  • Kristin Bruk Artinger
  • EUGENIA C OLESNICKY KILLIAN
  • John Carr
  • James E Carlson
  • Florian Nachon
  • Yifeng Li
  • Oluwatoyin Asojo
  • Larry Tong
  • Weihua Xue

Detail Information

Publications18

  1. pmc Mass spectrometry identifies covalent binding of soman, sarin, chlorpyrifos oxon, diisopropyl fluorophosphate, and FP-biotin to tyrosines on tubulin: a potential mechanism of long term toxicity by organophosphorus agents
    Hasmik Grigoryan
    Eppley Institute, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Omaha, NE 68198 6805, USA
    Chem Biol Interact 175:180-6. 2008
    ..It is concluded that OP bind covalently to tubulin, and that this binding could explain cognitive impairment associated with OP exposure...
  2. pmc Fluorescent probes of the isoxazole-dihydropyridine scaffold: MDR-1 binding and homology model
    Monika I Szabon-Watola
    Department of Chemistry, University of Idaho, Moscow, ID 83843, United States
    Bioorg Med Chem Lett 24:117-21. 2014
    ..A homology model for IDHP binding at MDR-1 is presented which represents our current working hypothesis. ..
  3. pmc Oxidative stress resulting from exposure of a human salivary gland cells to paraoxon: an in vitro model for organophosphate oral exposure
    John M Prins
    Department of Biomedical and Pharmaceutical Sciences, College of Health Professions and Biomedical Sciences, The University of Montana, Missoula, MT 59812, United States
    Toxicol In Vitro 28:715-21. 2014
    ..The ROS genes gss, gstm2, gstt2 and sod2 were upregulated, and the presence of superoxide following 10 μM PX exposure was determined via dihydroethidium fluorescence studies further implicating PX-induced oxidative stress in HSG cells...
  4. pmc Five tyrosines and two serines in human albumin are labeled by the organophosphorus agent FP-biotin
    Shi Jian Ding
    Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, Nebraska 68198, USA
    Chem Res Toxicol 21:1787-94. 2008
    ..Diethoxyphosphate-labeled Tyr 411 was stable for months at pH 7.4. These results will be useful in the development of specific antibodies to detect OP exposure and to engineer albumin for use as an OP scavenger...
  5. pmc Diastereomers (R(C),S(P))- and (R(C),R(P))-S-methyl P-(3-azidopropyl)-N-[(1R)-1-phenylethyl]phosphonamidothioate
    Lilu Guo
    Department of Chemistry, University of Montana, Missoula, MT 59812, USA
    Acta Crystallogr C 65:o179-82. 2009
    ..O = 2.8609 (18) and 2.966 (3) A in (I) and (II), respectively], with C-H...pi interchain interactions of ca 3.5 A...
  6. pmc Antimicrobial, antimalarial, and antileishmanial activities of mono- and bis-quaternary pyridinium compounds
    Sandip B Bharate
    NIH COBRE Center for Structural and Functional Neuroscience, Department of Biomedical and Pharmaceutical Sciences, The University of Montana, Missoula, MT 59812, USA
    Chem Biol Drug Des 76:546-51. 2010
    ....
  7. pmc Paraoxon-induced protein expression changes to SH-SY5Y cells
    John M Prins
    The Center for Structural and Functional Neuroscience, Department of Biomedical and Pharmaceutical Sciences, The University of Montana, Missoula, Montana 59812, USA
    Chem Res Toxicol 23:1656-62. 2010
    ..3-fold), heterogeneous nuclear ribonucleoprotein C (+2.8-fold), and H(+) transporting ATP synthase beta chain (-3.1-fold). Western blot analysis conducted on total protein isolates confirmed the expression changes in these three proteins...
  8. pmc Subacute developmental exposure of zebrafish to the organophosphate pesticide metabolite, chlorpyrifos-oxon, results in defects in Rohon-Beard sensory neuron development
    Saskia M Jacobson
    Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, MT 59812, USA
    Aquat Toxicol 100:101-11. 2010
    ....
  9. pmc Thionate versus Oxon: comparison of stability, uptake, and cell toxicity of ((14)CH(3)O)(2)-labeled methyl parathion and methyl paraoxon with SH-SY5Y cells
    Sandip B Bharate
    Department of Biomedical and Pharmaceutical Sciences, The University of Montana, Missoula, MT 59812, USA
    J Agric Food Chem 58:8460-6. 2010
    ..19 and 1.76 nM/10(6) cells for MPS and MPO, respectively. The amounts of MPS and MPO detected in the cytosol after 48 h of exposure time were 0.54 and 0.37 nM/10(6) cells, respectively...
  10. pmc Organophosphorus pesticides decrease M2 muscarinic receptor function in guinea pig airway nerves via indirect mechanisms
    Becky J Proskocil
    Division of Pulmonary and Critical Care Medicine, Oregon Health and Science University, Portland, Oregon, United States of America
    PLoS ONE 5:e10562. 2010
    ....
  11. pmc Mass spectrometric analyses of organophosphate insecticide oxon protein adducts
    Charles M Thompson
    Center for Structural and Functional Neuroscience, Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, Montana 59812, USA
    Environ Health Perspect 118:11-9. 2010
    ..Our aim was to review the literature covering the current state of the art in mass spectrometry (MS) used to identify OP protein biomarkers...
  12. pmc Tyrosines of human and mouse transferrin covalently labeled by organophosphorus agents: a new motif for binding to proteins that have no active site serine
    Bin Li
    Eppley Institute, University of Nebraska Medical Center, Omaha, Nebraska 68198 6805, USA
    Toxicol Sci 107:144-55. 2009
    ..The extent to which tyrosine modification by OP can occur in vivo and the toxicological implications of such modifications require further investigation...
  13. pmc Bisquaternary pyridinium oximes: Comparison of in vitro reactivation potency of compounds bearing aliphatic linkers and heteroaromatic linkers for paraoxon-inhibited electric eel and recombinant human acetylcholinesterase
    Sandip B Bharate
    NIH COBRE Center for Structural and Functional Neuroscience, Department of Biomedical and Pharmaceutical Sciences, The University of Montana, Missoula, MT 59812, USA
    Bioorg Med Chem 18:787-94. 2010
    ..042min(-1), which is better than obidoxime (3) for paraoxon-inhibited EeAChE. Compound 31 showed a k(r) 0.0041min(-1) that is near equal to pralidoxime (1) for paraoxon-inhibited rHuAChE...
  14. pmc Inactivation of acetylcholinesterase by various fluorophores
    Lilu Guo
    Department of Biomedical and Pharmaceutical Sciences, Center for Structural and Functional Neuroscience, The University of Montana, Missoula, MT 59812, USA
    J Enzyme Inhib Med Chem 25:116-20. 2010
    ..7 microM (competitive) and Ki' = 24.9 microM (noncompetitive), suggesting that Lissamine binds to free enzyme and enzyme-substrate complex...
  15. pmc Prdm1a is necessary for posterior pharyngeal arch development in zebrafish
    DENISE A BIRKHOLZ
    Department of Biomedical and Pharmaceutical Sciences, University of Montana, Center for Structural and Functional Neuroscience, Missoula, Montana, USA
    Dev Dyn 238:2575-87. 2009
    ..Together, these results indicate an essential role for prdm1a in the development of the zebrafish craniofacial skeleton...
  16. pmc New series of monoquaternary pyridinium oximes: Synthesis and reactivation potency for paraoxon-inhibited electric eel and recombinant human acetylcholinesterase
    Sandip B Bharate
    NIH COBRE Center for Structural and Functional Neuroscience, Department of Biomedical and Pharmaceutical Sciences, The University of Montana, Missoula, MT 59812, USA
    Bioorg Med Chem Lett 19:5101-4. 2009
    ..The N-pyridyl-CH(2)COOH analog 8 reactivated EeAChE (36%) and rHuAChE (15%) at 10(-4)M with a k(r) value better than 2-pyridine aldoxime methiodide (2-PAM) for rHuAChE...
  17. pmc Mass spectrometry identifies multiple organophosphorylated sites on tubulin
    Hasmik Grigoryan
    University of Nebraska Medical Center, Eppley Institute for Cancer Research, 986805 Nebraska Medical Center, Omaha, NE 68198 6805, USA
    Toxicol Appl Pharmacol 240:149-58. 2009
    ..In conclusion seventeen tyrosines in tubulin have the potential to covalently bind chlorpyrifos oxon. These results will be useful when searching for OP-labeled tubulin in live animals...
  18. pmc Inhibition of acetylcholinesterase by chromophore-linked fluorophosphonates
    Lilu Guo
    Department of Chemistry, The University of Montana, Missoula, MT 59812, United States
    Bioorg Med Chem Lett 20:1194-7. 2010
    ..Covalent attachment of the dabsyl-FPC analog to rMoAChE was demonstrated using size exclusion chromatography and spectroscopic analysis, and visualized using molecular modeling...