Duquesne University Duquesne Scholarship Collection Electronic Theses and Dissertations Spring 2011 Cyanate Formation Following 2-Chloroacrylonitrile Exposure and the Role of Cytochrome P-450 Paul Malichky Follow this and additional works at: https://dsc.duq.edu/etd Recommended Citation Malichky, P. (2011). Cyanate Formation Following 2-Chloroacrylonitrile Exposure and the Role of Cytochrome P-450 (Doctoral dissertation, Duquesne University). Retrieved from https://dsc.duq.edu/etd/860 This Immediate Access is brought to you for free and open access by Duquesne Scholarship Collection. It has been accepted for inclusion in Electronic Theses and Dissertations by an authorized administrator of Duquesne Scholarship Collection. For more information, please contact [email protected]. CYANATE FORMATION FOLLOWING 2-CHLOROACRYLONITRILE EXPOSURE AND THE ROLE OF CYTOCHROME P-450 A Dissertation Submitted to the Graduate School of Pharmaceutical Sciences Duquesne University In partial fulfillment of the requirements for the degree of Doctor of Philosophy By Paul Malichky March 2011 Copyright by Paul Malichky 2011 CYANATE FORMATION FOLLOWING 2-CHLOROACRYLONITRILE EXPOSURE AND THE ROLE OF CYTOCHROME P-450 By Paul Malichky Approved March 16, 2011 ________________________________ ________________________________ David A. Johnson, Ph.D. Paula Witt-Enderby, Ph.D. Dissertation Chairperson Professor of Pharmacology-Toxicology Associate Professor of Pharmacology- Graduate School of Pharmaceutical Toxicology Sciences Graduate School of Pharmaceutical Duquesne University, Pittsburgh, PA Sciences Duquesne University, Pittsburgh, PA ________________________________ ________________________________ Frederick W. Fochtman, Ph.D. Janet M. Mostowy, Ph.D. Director, Forensic Science and Law Vice President School of Natural and Environmental Product Safety & Regulatory Affairs Sciences Bayer Material Science, Pittsburgh, PA Duquesne University, Pittsburgh, PA ________________________________ ________________________________ J. Douglas Bricker, Ph.D. James K. Drennen, Ph.D. Dean, Mylan School of Pharmacy and the Associate Dean, Graduate Programs and Graduate School of Pharmaceutical Research Sciences Graduate School of Pharmaceutical Duquesne University, Pittsburgh, PA Sciences Duquesne University, Pittsburgh, PA iii ABSTRACT CYANATE FORMATION FOLLOWING 2-CHLOROACRYLONITRILE EXPOSURE AND THE ROLE OF CYTOCHROME P-450 By Paul Malichky May 2011 Dissertation supervised by David A. Johnson, Ph.D. 2-Chloroacrylonitrile (2-CAN) is a volatile, toxic chemical used as a raw material to manufacture pesticides. Although the toxic mechanism is not fully understood, the hypothesis in the literature is that 2-CAN may be metabolized to cyanide. An understanding of the mechanism of 2-CAN toxicity will permit more appropriate treatments in the event of an accidental exposure. Four groups of male Sprague-Dawley rats were given intraperitoneal (i.p.) injections. Group 1 was a control that received only saline. Group 2 received 40 mg/kg of 2-CAN. Group 3 received 100 mg/kg of 5-phenyl-1-pentyne (5P1P) an inhibitor of cytochrome P-450. Group 4 received 100 mg/kg of 5P1P one hour before 40 mg/kg of 2- CAN. Three hours following administration, the animals were euthanized via cardiac puncture. Cardiac blood was collected for cyanide and cyanate analysis. Liver and lung iv tissues were harvested and analyzed for glutathione. In a separate experiment, the time to onset of hind limb paralysis was measured in animals receiving 40 mg/kg 2-CAN and 100 mg/kg 5P1P one hour prior to 40 mg/kg 2-CAN. Blood cyanide levels were increased in the 2-CAN group, but did not exceed 0.06 µg/mL, a concentration well below the minimum for producing adverse health effects. Compared to control animals, the 2-CAN treatment group had depleted glutathione by 20% in the lung and 44% in the liver. When 5P1P was administered prior to 2-CAN, there was no significant change in glutathione concentrations. Plasma cyanate levels in the 2-CAN group were 436 nmol/L plasma, a 390% increase over control. When a 5P1P was administered prior to 2-CAN, cyanate levels were reduced by 55% to less than 195 nmol/L plasma. In addition to decreasing plasma cyanate levels, 5P1P 1 hour prior to 2-CAN delayed the onset of hind limb paralysis from 6 minutes to 16 minutes. Since cyanide was not generated in substantial concentrations following 2-CAN administration, the use of an antidote specific for cyanide for 2-CAN toxicity may not be effective. The findings of increased cyanate levels in 2-CAN, combined with the hind-limb paralysis data suggest that cyanate, not cyanide, is the toxic metabolic product resulting from acute 2-CAN exposure. v ACKNOWLEDGEMENT I would like to express my gratitude to my advisor, David A. Johnson, Ph.D. for his leadership, guidance, and input throughout the many years of pursuing this degree. I would like to thank my dissertation committee, J. Douglas Bricker, Ph.D., Frederick W. Fochtman, Ph.D., Janet Mostowy, Ph.D., and Paula Witt-Enderby, Ph.D. This group provided excellent input and direction for the research. PPG Industries Inc. and Bayer Corporation who financially supported this research and also provided flexible work hours. My loving family who endured and motivated me. Alec, Andrew, Adam, and my wife Janell. Thank you for your love and support. Jeremy Painter who helped with some animal work. Carl Thompson and MaryAnn Fuhry for chemistry support. Dave Hardesty for keeping the equipment running. vi TABLE OF CONTENTS Page Abstract .............................................................................................................................. iv Acknowledgement ............................................................................................................. vi List of Tables .......................................................................................................................x List of Figures .................................................................................................................... xi List of Abbreviations ........................................................................................................ xii I. Introduction ......................................................................................................................1 A. Statement of Problem .................................................................................................1 B. Literature Survey ........................................................................................................3 II. Materials and Methods .................................................................................................18 A. Materials and Equipment ........................................................................................18 1. Facilities ..............................................................................................................18 2. Animals ...............................................................................................................18 3. Chemicals ............................................................................................................19 4. Materials .............................................................................................................21 5. Equipment ...........................................................................................................23 6. Computer Software .............................................................................................24 B. Methodology and Procedures ..................................................................................26 1. Hypothesis ..........................................................................................................26 2. Specific Aims .....................................................................................................26 3. Experimental Design ..........................................................................................27 4. Sample Collection ..............................................................................................27 vii a. Tissue Glutathione, Blood Cyanide, and Plasma Cyanate Collection ......27 5. Tissue Glutathione, Blood Cyanide, and Plasma Cyanate Analysis ...................28 a. Plasma Cyanate Analysis .........................................................................28 b. Tissue Glutathione Analysis ....................................................................31 c. Blood Cyanide Analysis ...........................................................................33 6. Measurement of Hind-limb Paralysis .................................................................35 7. Statistical Analysis ..............................................................................................36 III. Results and Discussion ...............................................................................................37 A. Results .....................................................................................................................37 1. Gross Animal Observations ..............................................................................37 2. Tissue GSH Levels ...........................................................................................38 B. Discussion ...............................................................................................................53 C. Summary 2-CAN mechanism of action ..................................................................60