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Proteomic Analysis of Diaminochlorotriazine Adducts in Wister Rat Pituitary Glands and LβT2 Rat Pituitary Cells

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Department of Environmental and Radiological Health Sciences, Department of Chemical and Biological Engineering, Proteomics and Metabolomics Facility, and Department of Biomedical Sciences, Anatomy and Neurobiology Section, Colorado State University, Fort Collins, Colorado 80523
* To whom correspondence should be addressed: Colorado State University, 1680 Campus Delivery, 132 Physiology Building, Fort Collins, CO 80523. Telephone: 970-491-8635 . Fax: 970-491-7569. E-mail: [email protected]
†Department of Environmental and Radiological Health Sciences.
‡Department of Chemical and Biological Engineering.
§Proteomics and Metabolomics Facility.
∥Department of Biomedical Sciences.
Cite this: Chem. Res. Toxicol. 2008, 21, 4, 844–851
Publication Date (Web):March 28, 2008
https://doi.org/10.1021/tx700386f
Copyright © 2008 American Chemical Society
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Abstract

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Atrazine (ATRA) is the most commonly applied herbicide in the United States and is frequently detected in drinking water at significant levels. After oral exposure, ATRA metabolism yields diaminochlorotriazine (DACT), an electrophilic molecule that has been shown to form covalent protein adducts. This research was designed to identify ATRA-induced protein adducts formed in the pituitary gland of ATRA-exposed rats and in DACT-exposed LβT2 rat pituitary cells. Immunohistochemistry showed diffuse cytoplasmic and nuclear staining in both pituitary sections and LβT2 cells indicating the formation of DACT protein adducts. Protein targets from both rat pituitaries and LβT2 cell culture were identified following two-dimensional electrophoresis (2DE), immunodetection, and matrix-assisted laser desorption ionization−time of flight mass spectrometry analysis. Western blots from both exposed rats and LβT2 cells revealed over 30 DACT-modified spots that were not present in control animals. Protein spots were matched to concurrently run 2DE gels stained with Sypro Ruby, excised, and in-gel-digested with trypsin. Mass spectrometry analysis of digest peptides resulted in the identification of 19 spots and 8 unique proteins in the rats and 21 spots and 19 unique proteins in LβT2 cells. The identified proteins present in both sample types included proteasome activator complex subunit 1, ubiquitin carboxyl-terminal hydrolase isozyme L1, tropomyosin, ERp57, and RNA-binding proteins. Each of these proteins contains active-site or solvent-exposed cysteine residues, making them viable targets for covalent modification by DACT.

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2DE of Sypro Ruby-stained water-soluble proteins from the pituitary of a female Wistar rat controls for 4 days (Supplemental Information 1), 2DE Western blot of water-soluble proteins from the pituitary of a female Wistar rat controls for 4 days (Supplemental Information 2), 2DE of Sypro Ruby-stained water-soluble proteins from LβT2 rat pituitary cells exposed to DMSO control for 24 h (Supplemental Information 3), 2DE Western blot of water-soluble proteins from LβT2 rat pituitary cells exposed to DMSO control for 24 h (Supplemental Information 4), DACT-adducted proteins in the pituitary of Wistar rats exposed to 200 mg/kg ATRA identified with MALDI−TOF/TOF MS (Supplemental Information 5), and DACT-adducted proteins in LβT2 cells exposed to 300 µM DACT identified with LC−MS/MS (Supplemental Data 6). This material is available free of charge via the Internet at http://pubs.acs.org.

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This article is cited by 20 publications.

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  10. Gregory P. Dooley, Ronald B. Tjalkens, William H. Hanneman. The atrazine metabolite diaminochlorotriazine suppresses LH release from murine LβT2 cells by suppressing GnRH-induced intracellular calcium transients. Toxicology Research 2013, 2 (3) , 180. https://doi.org/10.1039/c3tx20088d
  11. Jean-Paul Lasserre, Fred Fack, Tommaso Serchi, Dominique Revets, Sébastien Planchon, Jenny Renaut, Lucien Hoffmann, Arno C. Gutleb, Claude P. Muller, Torsten Bohn. Atrazine and PCB 153 and their effects on the proteome of subcellular fractions of human MCF-7 cells. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 2012, 1824 (6) , 833-841. https://doi.org/10.1016/j.bbapap.2012.03.014
  12. S. Moro, J. K. Chipman, P. Antczak, N. Turan, W. Dekant, F. Falciani, A. Mally. Identification and Pathway Mapping of Furan Target Proteins Reveal Mitochondrial Energy Production and Redox Regulation as Critical Targets of Furan Toxicity. Toxicological Sciences 2012, 126 (2) , 336-352. https://doi.org/10.1093/toxsci/kfs005
  13. Zhoumeng Lin, Jeffrey W. Fisher, Matthew K. Ross, Nikolay M. Filipov. A physiologically based pharmacokinetic model for atrazine and its main metabolites in the adult male C57BL/6 mouse. Toxicology and Applied Pharmacology 2011, 251 (1) , 16-31. https://doi.org/10.1016/j.taap.2010.11.009
  14. Gregory P. Dooley, Amanda K. Ashley, Marie E. Legare, Robert J. Handa, William H. Hanneman. Proteomic analysis of diaminochlorotriazine (DACT) adducts in three brain regions of Wistar Rats. Toxicology Letters 2010, 199 (1) , 17-21. https://doi.org/10.1016/j.toxlet.2010.07.014
  15. Jasmine George, Richa Singh, Zafar Mahmood, Yogeshwer Shukla. Toxicoproteomics: New paradigms in toxicology research. Toxicology Mechanisms and Methods 2010, 20 (7) , 415-423. https://doi.org/10.3109/15376511003667842
  16. Ian N.M. Day, Rod J. Thompson. UCHL1 (PGP 9.5): Neuronal biomarker and ubiquitin system protein. Progress in Neurobiology 2010, 90 (3) , 327-362. https://doi.org/10.1016/j.pneurobio.2009.10.020
  17. Su-wei Qi, Qing X. Li. Proteomics in Pesticide Toxicology. 2010,,, 603-626. https://doi.org/10.1016/B978-0-12-374367-1.00021-5
  18. Shengnan Xie, Colby Moya, Betul Bilgin, Arul Jayaraman, S Patrick Walton. Emerging affinity-based techniques in proteomics. Expert Review of Proteomics 2009, 6 (5) , 573-583. https://doi.org/10.1586/epr.09.74
  19. Chad D. Foradori, Laura R. Hinds, William H. Hanneman, Marie E. Legare, Colin M. Clay, Robert J. Handa. Atrazine Inhibits Pulsatile Luteinizing Hormone Release Without Altering Pituitary Sensitivity to a Gonadotropin-Releasing Hormone Receptor Agonist in Female Wistar Rats1. Biology of Reproduction 2009, 81 (1) , 40-45. https://doi.org/10.1095/biolreprod.108.075713
  20. Matthew K. Ross, Toni L. Jones, Nikolay M. Filipov. Disposition of the Herbicide 2-Chloro-4-(ethylamino)-6-(isopropylamino)- s -triazine (Atrazine) and Its Major Metabolites in Mice: A Liquid Chromatography/Mass Spectrometry Analysis of Urine, Plasma, and Tissue Levels. Drug Metabolism and Disposition 2009, 37 (4) , 776-786. https://doi.org/10.1124/dmd.108.024927

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