antidotal mechanisms for hydrogen sulfide toxicity

81 Pages·2000·3.58 MB·English

ANTIDOTAL MECHANISMS FOR
HYDROGEN SULFIDE TOXICITY
ALEKSANDRA MIHAJLOVIC
A thesis submitted in the conformity with the requirements
for the degree of Master of Science
Department of Phannaceutical Sciences
Faculty of Phannacy
University of Toronto
O Copyright by Aleksandra Mihajlovie 1999
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DEDICATION
This thesis is dedicated to:
My Father who taught me how to enjoy life,
My Mother who taught me how to fight Me,
My sister Maja who was there thmugh thick and thin,
My friends for being there.
Thank you al1 for al1 your unending love, help and support!
Tata znam da bi bio ponoson na mene!
Andjelki i Maji posveèqjern ovu tezu jer su verovale u mene., a baki i deki
Sto su ih &vali.
V i a S aga
ANTIDOTAL MECHAIIQTSMS FOR HVlbROGEN SULFIDE TOXICITY
M. Sc., 1999
Aleksandra Mihajlovic
Graduate Department of Pharmaceutical Sciences
Faculty of Pharmacy
University of Toronto
Hydrogen sulfide (H2S)t oxicity mechanisms are still not well understood
and currently used antidotes are ineffective. H2S was found to be not
only toxic per se but was metabolically activated to reactive metabolites
which can react with GSH or protein thiols to form persulfides. The
activation was probably catalyzed by cytochrome P450 CYP 3A and 2B as
cytotoxicity and GSH de pletion was prevented by metyrapone or SKF-
525A and markedly increased by hydroperoxides. Cytochrome c
prevented H2S cytotoxicity, a process in which cytochrome c was
reduced. Methemoglobin prevented H2S cytotoxicity by trapping H2S to
fom sulfmethemoglobin which catalyzed H2S autoxidation. Molybdenum,
barium and lead prevented cytotoxicity by fonning sulfide complexes
without H2S autoxidation while copper, cobalt, nickel and iron fomed
active complexes that catalyzed H2S autoxidation. Hydroxocobalatnin
was highly effective as an antidote in preventing H2S cytotoxicity in Mtro
and in vivo, where it was more potent and effective than currently used
nitrite.
1 sincerely thank my research supe~sor,D r. Peter O'Brien for
facilitating the completion of my research project by providing much
guidance and encouragement and for sharing his enthusiasm for science.
1 would also like to express my gratitude to my advisory cornmittee
member, Dr. Peter Pennefather, for his thoughtful suggestions and
criticai reading of rny thesis as well as my examiners, Dr. Rebecca
Prokipcak and Dr. Rena Bendayan for their suggestions and insightful
comments.
A special thanks goes to Dr. Sumsullah Khan for all his scientific,
technical and moral support through the rainy days of the research.
Endless gratitude to often forgotten help through Scyllas and
Charybds of administration to Merrylee Greenan, Graduate Secretary.
1 shail dearly remember the camaraderie and help of my peers -
Sylvia, Sophia, Reza, Jalal and Bin.
The investigations described in this thesis were financially
supported by a research grant from the Natural Science and Engineering
Research Council of Canada. The investigations were carrieci out in
Professor P. J. O'Brien's laboratory in the Faculty of Pharmacy,
University of Toronto, 19 Russell St., Toronto, Ontario, Canada, M5S
2S2.
Aleksandra Mihajlovic was fmancialiy supported by the University
of Toronto Open Fellowship (1997/98) and Ontario College of
Pharmacists Bursary (1996). Attendance of the 37a Annual Meeting of
Society of Toxicology (1998),S eattle, WA, USA, was partially supported
by the Faculty of Pharmacy, University of Toronto.
TABLE OF CONTEWTS
Title 1
Abstract II
Dedications III
Acknowledgements IV
v
Acknowledgement of Financial Support
Table of Contents VI
Summary of Abbreviations VI1
Ix
Summary of Tables
Summary of Figures X
Chapter 1 Introduction
Chapter 2 Materials and Methods
Chapter 3 Hemoproteins as Antidotes and Metabolic
Activators of Hydrogen Sulfide Cytotoxicity
Chapter 4 Metals as Antidotes and Activators
Chapter 5 Summary and Conclusions
References
B vitamin B (cyanocobalarnin)
12 12
B 12a vitamin B 12a (hydroxocobaiamin)
CHP cumene hydroperoxide
CYP cytochrome P450
Qt P450 cytochrome P450
Cyt. a, b or c cytochrome a, b or c
GSH reduced glutathione
GSSG oxidized glut athione
Hb hemoglobin
HEPES 4-(2-hydroxyethy1)1- - piperazine-
ethanesulfonic acid
HPLC high performance liquid
chrornatography
intraperitoneal
lethal dose that U s6 0 or 95%
animals respectively
Met-Hb methemoglobin
NADH nicotinamide adenine dinucleotide
(reduced)
nicoharnide adenine dinucleotide
phosphate (reduced)
hydroxoco balamin
standard deviation
2-dimethylaminoet hyl-S,2-diphenyl-
-n-pentanoate
-
TRIS-HCI Trizma base (Tris [hydroxymet hyl]
-amino methane)
UV ultra violet
Chaptef 3 Hemoprotehs as antidotes and rnetaboîic
activators of hydrogen sdde cytotoxicity
-
Table 3 1: Antidotal effect of hemoproteins towards H2S
cytotoxicity
Table 3-2: H2S modulated respiration in isolated hepatocytes
Table 3-3: H2S autoxidation catalyzed by hemoproteins
Table 3-4: Effect of P4SO inhibitors and activators on H2S
induced cytotoxicty
Metaïs as antidotes and activatorr
Table 4- 1: Antidotal effect of metal salts/complexes towards H2S
cytot oxicity
Table 4-2: Modulation of HB autoxidation by various metal sdts
Table 4-3: Effect of Fe, Cu and Ca chelating agents on H2S
induced cytotoxicity
Table 4-4: Modulation of H2S autoxidation by metal chelating
agents
Table 4-5: Antidotal properties of vitamin Bi2 versus vitamin Bila
against H2S cytotoxicity