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Efficacy of cell membrane protective agents in preventing hydrogen peroxide cytotoxicity in chick embryo cardiac myocytes

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Efficacy of cell membrane protective agents in preventing hydrogen peroxide cytotoxicity in chick embryo cardiac myocytes
Creator:
Wallner, Juliann S
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English
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x, 50 leaves : illustrations (some color) ; 29 cm

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Subjects / Keywords:
Antibody-dependent cell cytotoxicity ( lcsh )
Active oxygen ( lcsh )
Chicks ( lcsh )
Active oxygen ( fast )
Antibody-dependent cell cytotoxicity ( fast )
Chicks ( fast )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Includes bibliographical references (leaves 44-50).
General Note:
Submitted in partial fulfillment of the requirements for the degree, Master of Arts, Department of Biology
Statement of Responsibility:
by Juliann S. Wallner.

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University of Colorado Denver
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ocm30839170
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Full Text
EFFICACY OF CELL MEMBRANE PROTECTIVE AGENTS
IN PREVENTING HYDROGEN PEROXIDE CYTOTOXICITY
IN CHICK EMBRYO CARDIAC MYOCYTES
by
Juliann S. Wallner
B.S., Colorado State University, 1989
A thesis submitted to the
Faculty of the Graduate School of the
University of Colorado at Denver
in partial fulfillment
of the requirements for the degree of
Master of Arts
Biology
1993


This thesis for the Master of Arts
degree by
Juliann S. Wallner
has been approved for the
Department of
Biology
by
Gerald Audesirk
Date


Juliann S. Wallner (M.A., Biology)
Efficacy of Cell Membrane Protective Agents in Preventing Hydrogen
Peroxide Cytotoxicity in Chick Embryo Cardiac Myocytes
Thesis directed by Associate Professor Gerald Audesirk
The return of molecular oxygen to ischemic tissues at the time of
reperfusion leads to the production of reactive oxygen species which
cause cellular damage. This is injury beyond that incurred during ischemia
and is termed reperfusion injury. Reactive oxygen species, such as are
released during ischemia and reperfusion, have the myocardial cell
membrane as a major target. Membrane lipids are particularly vulnerable
to lipid peroxidation. Therefore, protection of the membrane lipids by a
suitable pharmacological agent is proposed as a means of preventing
injury due to reactive oxygen species. Agents which stabilize the cell
membrane or prevent lipid peroxidation are potentially useful in preventing
or attenuating reperfusion injury. To assess this possibility, we exposed
chick embryo cardiac myocytes cultured for 4 days to 1.5mM hydrogen
peroxide (H2O2) and evaluated the efficacy of several membrane
protective agents in preventing cytotoxicity. Release of an intracellular
enzyme, lactate dehydrogenase (LDH), was measured as an index of cell
injury relative to 100% release when cells were lysed with 1% Triton X-
100. Exposure of untreated cells to 1.5mM H2O2 for 20 hrs resulted in
68% LDH release. However, exposure to H2O2 in cells treated with a
membrane protective 21-aminosteroid, U74500, resulted in a
iii


concentration-dependent reduction in LDH release (49% LDH release at a
concentration of 50pM, 39% at 100|iM, 30% at 200pM and 22% at
400pM). U74500 did not scavenge H2O2 in vitro. Lidocaine, propranolol,
diltiazem, probucol and Trolox C, all nonscavenger agents with known
membrane protective properties, failed to attenuate LDH release due to
H2O2. Thus, U74500 protected cardiac myocytes against H2O2 by a
nonscavenging mechanism, whereas other membrane active agents were
ineffective. The partially protective effect of U74500 was probably due to
inhibition of lipid peroxidation.
This abstract accurately represents the content of the candidates thesis. I
recommend its publication.
SignedJ
Gerald Audesirk


ACKNOWLEDGEMENTS
I would like to thank Dr. Lawrence Horwitz for his support and guidance
throughout this project and his valuable suggestions regarding this
manuscript, and Dr. Gerald Audesirk for his time and direction leading to
the completion of this project. I would also like to thank Dr. Stephen E.
Buxser at The Upjohn Company for providing us with the 21-aminosteroid
U74500 and measuring its membrane incorporation. This research was
supported by a grant from the National Institutes of Health.
v


CONTENTS
Figures..................................................viii
Tables...................................................x
CHAPTER
1. INTRODUCTION ...............................................1
Oxygen-Derived Free Radicals ............................1
Myocardial Ischemia and the Concept
of Reperfusion Injury ...................................2
The Role of Oxygen-Derived Free Radicals
In Reperfusion Injury ...................................5
Lipid Peroxidation.......................................9
Rationale and Experimental Hypothesis ..................11
Experimental Model......................................12
Agents Which prevent Lipid Peroxidation..............13
Agents With Non-specific Membrane
Protective Effects ..................................14
LDH Release as an Indicator of Cellular
Integrity ...........................................15
2. EXPERIMENTAL PROCEDURES ...............................16
Cell Culture............................................16
Experimental Protocols .................................17
LDH Measurement ........................................18
Measurement of Incorporated U74500 .................... 19
vi


Immunohistochemical Staining ...........................20
Statistical Analysis ...................................21
3. EXPERIMENTAL RESULTS.......................................22
Effects of Lipid Soluble Agents on
H2O2- mediated Cytotoxicity ............................22
Incorporation of U74500 Emulsion Into
the Cell Membrane.......................................23
Purity of Chick Embryo Cardiac
Myocyte Monolayer.......................................24
4. DISCUSSION ................................................37
APPENDIX
A. Structural Formulas of Lipid-soluble Agents ...............42
B. Structural Formulas of Nonlipid-soluble Agents.............43
LITERATURE CITED
44


25
26
27
28
29
30
31
32
33
34
35
Figure
3.1.
FIGURES
Cell Injury Index versus incubation time for
U74500 emulsion .....................................
3.2. Cell Injury Index versus incubation time for 30 min.
pre-exposure of U74500 ...........................
3.3. Cell Injury Index versus incubation time for
U74500 in DMSO .............................
3.4. Cell Injury Index versus incubation time for
probucol ............................................
3.5. Cell Injury Index versus incubation time for
Trolox C ............................................
3.6. Cell Injury Index versus incubation time for
propranolol .........................................
3.7. Cell Injury Index versus incubation time for
diltiazem ...........................................
3.8. Cell Injury Index versus incubation time for
lidocaine ...........................................
3.9. Bovine fibroblasts immunohistochemically stained for
vimentin using the monoclonal antibody for vimentin;
positive control ....................................
3.10. Bovine fibroblasts immunohistochemically stained for
vimentin without using the monoclonal antibody for
vimentin; negative control ..........................
3.11. Chick embryo cardiac myocytes immunohistochemically
stained for vimentin using the monoclonal antibody
for vimentin ........................................
Vlll


3.12. Chick embryo cardiac myocytes immunohistochemically
stained for vimentin without using the monoclonal
antibody for vimentin ....................................... 36
IX


TABLES
Table
3.1. Cell Injury Index data for U74500 emulsion....................25
3.2. Cell Injury Index data for 30 min. pre-exposure
of U74500 emulsion ........................................26
3.3. Cell Injury Index data for U74500 in DMSO ................... 27
3.4. Cell Injury Index data for probucol ......................... 28
3.5. Cell Injury Index data for Trolox C ......................... 29
3.6. Cell Injury Index data for propranolol ...................... 30
3.7. Cell Injury Index data for diltiazem ........................ 31
3.8. Cell Injury Index data for lidocaine......................... 32
x


CHAPTER 1
INTRODUCTION
Oxygen-Derived Free Radicals
An atom consists of a central nucleus (containing protons and
neutrons) and an outer shell of orbiting electrons. Electrons associate and
orbit in pairs around the nucleus. An atom with one or more unpaired
electrons is a free radical species, as is any molecule containing one or
more atoms with unpaired electrons. Since free electrons are unstable,
free radical species are generally more reactive than nonradicals. When
two molecules with unpaired electrons meet, the electrons join to form a
pair which stabilizes the two molecules. However, since most molecules
formed under physiologic conditions do not have unpaired electrons, free
radicals produced in vivo will most likely react with nonradicals. This
creates a chain reaction in which the free radical species steals an
electron (usually in the form of a hydrogen atom) from a nonradical in
order to stabilize itself, and in turn the nonradical becomes a free radical
species (Del Maestro et al., 1980; Lucchesi, 1990; Halliwell, 1991).
Under normal conditions of cellular metabolism, oxygen is capable of
accepting a total of four electrons. The addition of one electron results in
the formation of superoxide radical (-02-)- With the addition of two
electrons, hydrogen peroxide (H2O2) is formed. Hydroxyl radical (-OH) is


formed by the addition of a third electron. The presence of iron or other
transition metal ions is necessary for the production of OH (Starke and
Farber, 1985). Finally, the addition of a fourth electron forms water (H2O).
The following table demonstrates how these oxygen-derived free radicals
are formed (Lesnefsky et al., 1990):
The reduction of molecular oxygen to superoxide anion
O2 + e => -02"
Dismutation of superoxide anion
2-02" + 2H+ => H2O2 + O2
The Fenton reaction
02' + Fe+3 => Fe+2 + O2
Fe+2 + H2O2 => Fe+3 + HO + OH
Net: -02' + H2O2 => O2 + OH" + *0H
Myocardial Ischemia and the Concept of Reperfusion Injury
Myocardial ischemia is the reduction or cessation of blood flow to the
tissues of the heart. It usually results from vascular obstruction due to
clot, atherosclerosis or both. This often leads to tissue necrosis, a process
termed acute myocardial infarction. Although different tissues show
varying degrees of injury due to ischemia, all tissues will eventually
progress to cellular death if not reperfused. Contracting cardiac
myocytes typically have a high oxygen consumption and energy
expenditure, which make them heavily dependent on normal amounts of
blood flow in vivo. As a result, cardiac myocytes are especially sensitive
to ischemia. Reperfusion is the return of blood flow and consequently
2


the return of oxygen to the ischemic tissues. Recognition of the need to
reperfuse ischemic tissues has led to the development of interventions to
aid this process. These include pharmacologic agents which dissolve
blood clots that are obstructing vessels and mechanical methods of
physically removing or bypassing blood clots. The latter include
angioplasty, atherectomy and surgical coronary bypass, in which vessels
are grafted into the artery beyond the clot. It is now clearly established
that these interventions are effective in reducing mortality among patients
with acute myocardial infarction, and when applied within an appropriate
time frame, they will salvage tissues that have not already progressed to
irreversible injury (Rude et al., 1981).
Myocardial reperfusion injury refers to cellular damage resulting from
the return of molecular oxygen to the ischemic tissues at the time of
reperfusion. This is injury beyond that incurred during ischemia and
includes both reversible and irreversible injury to myocytes that were
viable until the moment of reperfusion. Beyersdorf et al. (1989) have
provided evidence that coronary occlusion of 6 hours, in the absence of
reperfusion, does not necessarily lead to cellular death. This suggests
that myocardial injury associated with ischemia < 6 hours is not solely due
to the lack of blood flow but to the return of blood flow. This is a
paradoxical situation, in that reperfusion of ischemic tissue is critical for the
survival of the tissue, but reperfusion itself results in tissue injury.
Reperfusion injury after brief periods of ischemia is reversible i.e.,
there is no permanent injury if the individual survives the acute event. This
3


reversible injury comprises myocardial stunning and reperfusion
arrhythmias. Myocardial stunning refers to a transient contractile
dysfunction of the heart which typically lasts from several hours to several
days after normal blood flow and oxygenation are restored (Bolli et al.,
1987). Usually this is limited to ischemia of less than 30 minutes, a period
of exposure which under most conditions does not result in myocardial
tissue necrosis either during ischemia or reperfusion. During the period of
dysfunction after normal oxygenation has been restored, there are
decreased myocardial energy stores evidenced as diminished adenosine
triphosphate or creatine phosphate. The other form of reversible
reperfusion injury consists of arrhythmias which tend to occur during the
first 45 minutes of reperfusion. They do not seem to depend on whether
or not myocardial necrosis has occurred, and they generally subside and
do not recur.
With periods of ischemia of 1 hour or longer followed by reperfusion,
myocardial necrosis usually occurs. Duration of ischemia greater than 6
hours results in virtually complete irreversible tissue necrosis (Beyersdorf,
1989). Consequently, irreversible reperfusion injury follows periods of
ischemia between 1 and 6 hours. During this time, mechanisms of injury
from both ischemia and reperfusion probably play a role. However,
distinguishing the precise proportion of irreversible injury due to ischemia
versus irreversible injury due to reperfusion has been difficult.
Since myocardial cells cannot regenerate in adults, the necrosis of
myocardial cells has serious consequences. Coronary endothelial cells
4


may also become injured or necrotic during reperfusion after 1 hour or
more of ischemia, but unlike myocardial cells, endothelial cells are capable
of regeneration (VanBenthuysen et al., 1987). Endothelial cells are
resistant to ischemia and are usually damaged only during reperfusion
(VanBenthuysen et al., 1987).
The Role of Oxygen-Derived Free Radicals in Reperfusion Injury
There is considerable evidence that supports the role of oxygen-
derived free radical injury during reperfusion (Guarnieri et al., 1980;
Romaschin et al., 1987; Kuzuya et al., 1990; Zweir et al., 1984; Carrea et
al.,1991a ). Free radical production increases 100-fold during reperfusion
with a peak at 2 to 4 minutes into reperfusion and continuing for 3 hours
(Bolli, 1988). Mitochondria, activated neutrophils and tissue containing the
enzyme xanthine oxidase are all capable of producing reactive species of
oxygen during reperfusion. Many mitochondria exist in cardiac myocytes
as would be expected in cells with high rates of oxygen consumption. The
major source of oxygen-derived free radicals in the mitochondria is the
cytochrome oxidase pathway in which four electrons are transferred from
reduced cytochrome c through the copper and heme prosthetic groups of
cytochrome oxidase, where molecular oxygen (O2) serves as the electron
acceptor (Boveris and Chance, 1973). During normal cellular respiration,
this process occurs without the introduction of reduced intermediates (i.e.,
capable of donating an electron) to the cytosol. However, under
conditions in which the mitochondrion has been damaged, or during
5


sudden reintroduction of high concentrations of molecular oxygen, such as
is encountered during reperfusion, the univalent (1 electron) reduction of
oxygen leads to the production of superoxide anion, or the bivalent (2
electron) reduction of oxygen leads to the production of hydrogen
peroxide. Oxygen-derived free radicals can also be generated in the
mitochondrion during the sequential reduction of mitochondrial
flavoproteins, ubiquinone, and cytochromes from substrate metabolism.
This occurs as a single electron transfer, consequently the intermediate
ubisemiquinone can react with molecular oxygen to form superoxide anion
(Cadenas et al., 1977).
Ubiquinone + 1e- => Ubisemiquinone + 02 => -02"
The polymorphonuclear neutophil possesses the capability of
producing reactive oxygen species under the influence of appropriate
stimuli. This event, termed the respiratory burst, produces superoxide
radical, hydrogen peroxide, and other reactive oxygen species (Babior,
1978). Superoxide radical results from the univalent reduction of oxygen,
catalyzed by the enzyme NADPH oxidase. The main function of the
respiratory burst is to provide a defense mechanism against invading
microorganisms. The killing of microorganisms by reactive oxygen
species occurs within the phagocytic vacuole of the neutrophil (Curnutte
et al., 1987). However, reactive oxygen species may also be released into
the extracellular environment, where they are capable of damaging
adjacent tissue.
6


There are probably one or more chemoattractants produced to draw
the neutrophil to the ischemic area during reperfusion. It has been shown
that ischemic myocardium produces a tissue protease that activates the
complement cascade (Hill and Ward, 1971). Coincidently, activation of
complement during ischemia results in the migration of neutrophils into the
previously ischemic area (McManus et al., 1983; Pinckard et al., 1983;
Rossen et al., 1985). Leukotrienes and platelet activating factor (PAF) are
other potent neutrophil chemotactic agents. Leukotrienes are metabolites
of arachidonic acid via the lipoxygenase pathway. Leucotriene B4 (LTB4)
is known to be a chemotactic agent for neutrophils (Rowzer and Kargman,
1985). Platelet activating factor potentiates the adhesion of neutrophils to
vascular endothelium. Its formation is induced by thrombin in endothelial
cells (Zimmerman, et al. 1990).
During reperfusion, the neutrophil migrates into the previously ischemic
area where it attaches to the vascular endothelium and subsequently
produces and releases oygen-derived free radicals. (Harlan, 1985; 1987).
Neutrophils accumulate in the myocardium during the first 4 hours of
reperfusion following ischemia (Dreyer et al., 1991). Before neutrophils
can attack myocardium or endothelium, they must be activated to express
adhesion receptors. Ischemia and reperfusion results in increased levels
of neutrophil activation and expression of adhesion proteins such as
integrins and selectins (Entman et al., 1990). Upregulation of adhesion
proteins on endothelial and myocardial cells also occurs during ischemia
and reperfusion (Smith et al., 1988; 1989). There is substantial evidence
7


that polymorphonuclear neutrophils play a crucial role in irreversible
myocardial or reversible coronary endothelial injury. Antileukocyte
measures can reduce myocardial or coronary endothelial injury (Sheridan
et al., 1991; Simpson et al., 1990).
Under normal conditions, xanthine oxidase exists almost entirely as a
dehydrogenase in the heart. The dehydrogenase form hydroxylates
purines and by hydration/dehydrogenation, transfers electrons to NAD+.
During ischemia, xanthine dehydrogenase is rapidly converted to the
oxidase form which uses oxygen as the electron acceptor, producing
superoxide radical and hydrogen peroxide (Chambers et al., 1985). The
breakdown of ATP in the ischemic heart causes accumulation of
hypoxanthine, the substrate for xanthine oxidase, while reperfusion
provides the 02 (Eddy et al., 1987).
Xanthine oxidase is present in myocardium in widely varying amounts
among species. In humans and rabbits, the concentration is very low and
the relevance of this mechanism of reactive oxygen species production
has been unclear. However, even in the rabbit there is evidence that the
xanthine oxidase system is capable of producing physiologically significant
effects due to reactive oxygen species (Terada et al., 1991). In all species
there appear to be substantial concentrations of xanthine oxidase in
vascular endothelial cells which may be a source of -02" during
reperfusion. This might occur for both reversible and irreversible forms of
reperfusion injury.
8


Endogenous antioxidants (scavengers of oxygen free radicals) exist to
limit the toxic effects of these molecules. Superoxide dismutase (SOD)
converts superoxide to hydrogen peroxide, greatly increasing the rate at
which superoxide is removed.
SOD
2-02 + 2H+ => H2O2 + O2
The H2O2 resulting from the actions of SOD and from other sources is
removed by two other enzymes, catalase and glutathione peroxidase.
Catalase rapidly decomposes H2O2 into water and oxygen.
Catalase
2 H2O2 => 2H2O + O2
Glutathione peroxidase (GP) uses H2O2 to convert reduced glutathione
(GSH) to oxidized glutathione (GSSG). Glutathione reductase (GR)
reduces the GSSG back to GSH at the expense of oxidizing NADPH.
GP GR
2GSH + H2O2 => GSSG + H2O + NADPH => 2GSH + NADP+
When the endogenous antioxidants become overwhelmed by oxygen-
derived free radicals, tissue injury results.
Lipid Peroxidation
With reperfusion injury, the target sites of oxygen-derived free radicals
are most likely to be components of the cell membrane (Halliwell, 1991).
Two important sites of attack of these free radicals are the membrane
lipids and proteins. In the nucleus, DNA can also be a target of oxygen-
derived free radicals. This occurs during x-ray irradiation where oxygen
9


radicals are produced by excited water molecules throughout the cell
causing peroxidation or linking of DNA. This does not appear to be a
relevant factor in reperfusion injury where the associated formation of
oxygen radicals occurs outside of the nucleus.The lipids that constitute the
cell membrane, especially those lipids with unsaturated double bonds
(having available hydrogens), are susceptible to free radical attack leading
to the formation of lipid peroxides. A free radical (-R) with sufficient
energy to abstract a hydrogen atom from a carbon of an unsaturated fatty
acid (LH) can initiate a chain reaction in bulk lipid. The resulting carbon-
centered radical (-L) rapidly reacts with molecular oxygen to form a peroxy
radical (-L02), which itself can abstract a hydrogen atom from an
unsaturated fatty acid, leaving a carbon-centered radical and a lipid
hydroperoxide (LOOH). The free-radical chain reaction propagates until
two free radicals combine to terminate the chain (Gutteridge, 1987).
LH + -R => -L + RH Initiation
L+ O2 => -L02 Propagation
LH + LO2 => LOOH + L
L+L=* LL
LO2 + -L02 => LOOL + O2 Termination
LO2 + -L => LOOL
Lipids in the cell membrane exist as phospholipids, which provide the
backbone of membrane structure. Phospholipids are esters of two
molecules of fatty acid with one molecule of glycerol. Attached to the
10


glycerol is a phosphorylated alcohol such as choline or serine. Many of
the fatty acids in phospholipids are polyunsaturated fatty acids, which,
because they are generally liquid at 37C, are essential in maintaining
membrane fluidity. The hydrophobic membrane interior not only maintains
membrane fluidity, it also facilitates movement of proteins and lipids.
Membrane proteins are involved in the transport of ions and the
maintenance of cellular ionic homeostasis.. Membrane proteins which
contain sulfhydryl groups, such as those with methionyl residues, are
especially susceptible to free radical attack (Lesnefsky et al., 1991). Lipid
peroxides formed by the action of free radicals on the membrane lipids
can also cause fragmentation and polymerization of cell proteins. It is
apparent, therefore, that an excessive accumulation of oxygen-derived
free radicals during reperfusion can lead to disruption of the cell
membrane and loss of its enzymatic activity.
Rationale and Experimental Hypothesis
We are proposing that reactive oxygen species, such as are released
during ischemia and reperfusion, have the myocardial cell membrane as a
major target. Membrane lipids are particularly vulnerable to lipid
peroxidation which can lead to protein fragmentation and further lipid
peroxidation. Therefore, protection of the cell membrane by a suitable
pharmacological agent is proposed as a means of preventing injury due to
reactive oxygen species. Agents which stabilize the cell membrane or
prevent lipid peroxidation are potentially useful in preventing or attenuating
11


reperfusion injury. To assess this possibility, we exposed cultured cardiac
myocytes to hydrogen peroxide. The specific hypotheses are as follows:
1. Reactive oxygen species produced during reperfusion result in
peroxidation of cardiac myocyte membrane lipids.
2. Lipid peroxidation results in cardiac myocyte injury manifested by
leakage of cellular proteins.
3. Agents which inhibit lipid peroxidation or nonspecifically protect
myocardial cell membranes will prevent leakage of cellular proteins.
Experimental Model
Application of hydrogen peroxide to chick embryo cardiac myocytes
was used to simulate oxygen-derived free radical reperfusion injury.
Cardiac myocyte injury was measured as an index of release of lactate
dehydrogenase (LDH). The protective effects of various agents that either
inhibit lipid peroxidation or nonspecifically protect myocardial cell
membranes, but do not substantially alter the generation or accumulation
of reactive oxygen species, were assessed. The agents tested were the
21-aminosteroid U74500, Trolox C and probucol, all lipid soluble inhibitors
of lipid peroxidation, and lidocaine, propranolol and diltiazem, which are
nonspecific membrane protective agents. Chick embryo cardiac myocytes
have been used extensively for studies of myocardial injury, metabolism
and biochemistry (Barry, et al., 1975; Barry and Smith, 1982; Werdan et
al., 1984; Hunter, et al., 1986). Results correlate well with those in
myocytes from adult mammalian species and have shown correlation with
12


in vivo experiments in adult dogs. Fertilized chick eggs are much less
expensive and chick embryo cardiac myocytes are more convenient to
obtain than alternative sources of myocytes and do not require anesthesia
or other interventions required in adult animals. At 10 days of incubation,
chick embryos are too immature to respond to noxious stimuli when they
are removed from the egg.
Hydrogen peroxide is produced by mitochondria, activated neutrophils
and tissue containing the enzyme xanthine oxidase. Its application
therefore represents an acceptable simulation of oxygen-derived free
radical injury during reperfusion. H2O2 is commercially available, easily
applied and readily enters cells. Superoxide radical can also be generated
in vitro but its ability to enter cells is channel dependent. Hydroxyl radical
is presumedly generated intracellularly from 02" and H2O2 in the
presence of iron or other transition metals by way of the Fenton reaction.
Agents Which Prevent Lipid Peroxidation. The 21-aminosteroid
U74500 (Braughler et al., 1987; Natale et al., 1988; Gibson et al., 1989;
Holzgrefe et al., 1990; Carrea et al., 1991b), Trolox C (Mickle et al., 1989;
Rubenstein et al., 1990; Wu et al., 1990; Massey and Burton, 1990), and
probucol (Kuzuya et al. 1991; Kuzuya and Kuzuya, 1993; Gotoh et al.,
1992) are inhibitors of lipid peroxidation. There is evidence in various
models which support the ability of all these agents to attenuate tissue
injury caused by reactive oxygen species.
U74500, although it contains a steriod moiety, is devoid of
glucocorticoid activities (it does not bind to glucocorticoid receptors).
13


However, it and other 21-aminosteroids are lipophillic and inhibit lipid
peroxidation (Braughler et al. 1987). The exact mechanism by which
U74500 inhibits lipid peroxidation is unknown.
Trolox C is a water soluble derivative of <=-tocopherol (vitamin E).
Trolox is the aromatized polar region of the c-tocopherol molecule, devoid
of the long hydrophobic tail (phytol). Endogenous c-tocopherol is an
antioxidant, localized in the hydrophobic interior of cell membranes
(Halliwell, 1991). It possesses a hydroxyl group whose hydrogen is easily
removed and consequently is preferentially oxidized over phospholipids in
the membrane. Intact c-tocopherol is not water soluble and therefore is
impossible to administer intravenously or to cells in culture. The only
successful method of c-tocopherol administration is orally and thus
localization into cell membranes would be delayed.
Probucol is a lipophilic compound that readily enters the cell
membrane. Parthasarathy et al., 1986, reported that probucol inhibited
both cell-mediated and copper-catalyzed (by way of the Fenton reaction)
oxidative modification of low density lipoprotein (LDL). It is thought that
the hydroxyl group on each of the two phenolic moieties of probucol are
preferentially oxidized, in a manner similar to that of c-tocopherol.
Agents With Non-specific Membrane Protective Effects.
Lidocaine, a cationic local anesthetic agent, diltiazem, a calcium channel
blocker with vasodilating activity, and propranolol, a fB-adrenergic blocker,
are all thought to exert a nonspecific membrane protective effect.
Lidocaine has been shown to modestly decrease infarct size in a dog
14


model of ischemia and reperfusion. This protective effect may be due to a
myocardial membrane stabilizing effect or to an imposed reduction in
release of reactive oxygen metabolites from neutrophils (Lesnefsky, E. et
al. 1989). The 13-adrenergic blocker, propranolol, is cationic, amphiphilic
and probably capable of forming complexes with phospholipids in
membranes. These complexes may be less susceptible to attack by
reactive oxygen species than uncomplexed phospholipids (Mak et al.,
1989). Diltiazem is soluble in membrane lipids and also contains an
aromatic unsaturated ring. The aromatic unsaturated ring is common to
most classic aromatic chain-breaking antioxidants, providing resonance
stabilization for trapped radicals. Both the chemical and lipophilic
properties of diltiazem suggest that it may act as a lipophilic chain-
breaking antioxidant (Mak et al., 1992).
LDH Release as an Indicator of Cellular Integrity. Lactate
dehydrogenase (LDH) is a high molecular weight protein (MW ~ 140,000)
and is found in almost all animal tissues. Since transport of LDH across
the cell membrane does not occur, release of LDH into the extracellular
environment indicates a loss of cell membrane integrity. Unlike certain
other enzymes such as creatine phosphokinase (CPK), LDH is not readily
denatured with exposure to reactive oxygen species. Therefore, LDH is a
reliable indicator of cellular integrity.
15


CHAPTER 2
EXPERIMENTAL PROCEDURES
Cell Culture
Monolayer cultures of spontaneously beating chick embryo cardiac
myocytes were prepared by minor modification of the methods of Barry et
al., 1975, and Barry and Smith, 1982. Hearts from 10 day old chick
embryos (White Leghorn) were dissected free using sterile technique.
Large vessels were removed and the isolated hearts were minced and
placed in calcium and magnesium free Hanks balanced salt solution
(HBSS). The tissue was trypsinized in 10 ml aliquots of 0.025% trypsin in
HBSS for 5 cycles at 7 min per cycle in a 37C shaking water bath. The
supernatants from cycles 2 through 5 containing dissociated myocytes
were placed in separate conical tubes containing iced trypsin inhibitor
solution (50% calf bovine serum and 50% HBSS) and centrifuged at 600 x
g for 10 min. The cell pellets were combined and resuspended in culture
medium containing 54% balanced salt solution (116 mM NaCI, 1.0 mM
NaH2P04, 0.8 mM MgSC>4, 1.18 mM KCI, 26.2 mM NaHCC>3, 0.87 mM
CaCl2, 5.6 mM glucose), 40% Medium 199 with Hanks salts, 6% heat
inactivated fetal bovine serum (Hyclone Laboratories, Logan, UT), and
10U penicillin/ml with 10pg streptomycin/ml. Cells were plated at 6 x 10^


cells/ml in 6 well plates (15 mm well diameter) at 3 ml/well for lactate
dehydrogenase (LDH) release experiments and U74500 incorporation
assays. Cultures were incubated in 5% C02 in ambient air at 37C.
Experimental Protocols
All studies were performed on spontaneously beating myocyte
monolayers after 4 days of primary culture. Spent medium was removed,
the cells gently washed with phosphate buffered saline (PBS: 0.137 M
NaCI, 2.683 mM KCI, 8.1 mM Na2HP04, 1.47 mM KH2PO4) and fresh
medium containing 6% calf bovine serum (without antibiotics) were added
at 2 ml/well. Some experiments were performed in modified Tyrodes
solution (118mM NaCI, 11.9mM NaHC03, 0.0004mM NaH2PC>4, 4.4mM
KCI, 1.0mM MgCl2, 0.9mM CaCl2-2H20, and 11mM D-glucose) instead of
medium. Cytotoxicity was measured by taking fifty pi aliquots of medium
at approximately 2, 4, 7 and 20 hrs of incubation and measuring the
release of lactate dehydrogenase (LDH) into the medium. All conditions
were replicated in 3 wells and all experiments were repeated at least once
to ensure reproducibility. Wells with medium alone, H2O2 alone, cell lysis
with 1% Triton X 100 and experiment-specific controls were included in
each experiment. U74500 was obtained from the Upjohn Company
(Kalamazoo, Ml) as an emulsion and as a dry solid. The solid form was
solubilized at 100X concentration in deionized H2O with 0.1 M DMSO. The
final concentration of DMSO on the cells was 1 x 10'3 M. Twenty mM
Trolox C (Aldrich Chemical Co., Milwaukee, Wl) was solubilized in medium
17


without serum by adding 1 N NaOH to pH 12 and then vortexing until the
compound was completely dissolved. The Trolox solution was then
immediately neutralized with 1 N HCI and 6% serum was added. The
probucol solution was prepared by first making a 10mM solution of
probucol in 99% ETOH. From this a 0.5mM solution was made in calf
bovine serum, which was used to make the final concentration of 50|nM
probucol in medium with 10% serum and 0.5% EtOH. For the preparation
of cells in which incorporated U74500 was to be measured, cells were
incubated for 4 hrs with the experiment-specific treatment, rinsed and
harvested using 0.021 mM trypsin/0.526 mM EDTA-4Na (Sigma Chemical
Co., St. Louis, MO). Samples pooled from three wells (triturated,
concentrated using low speed centrifugation, and resuspended in 500pl
PBS) were loaded onto 1 ml of 0.4 M sucrose and centrifuged at 10,000 x
g for 3 5 min to remove any U74500 that might be nonspecifically bound
to the cells. The supernatant and sucrose layers were removed, leaving
the cell pellet, and replaced with 200 pi 95% ethanol.
LDH Measurement
To assess cell damage, LDH activity was measured by minor
modification of the fluorometric technique of Green et al., 1984. This is
based on the reaction pyruvate + NADH + H+ LDH_> L-lactate +
NAD+. To the 50 pi aliquots of medium were added 50 pi 3.2 M
ammonium sulfate, 100 pi diluting medium (0.167 M imadizole, 0.083%
BSA; pH 7.0) and immediately before reading, 1 ml assay reagent (20 mM
18


imadizole, 0.02% BSA, 1 mM sodium pyruvate and 25 pM (3-NADH,
sodium salt). The disappearance of NADH was measured fluorometrically
at an exciting wavelength of 340 nm and an emitting wavelength of 465
nm. Release of LDH was standardized with a cell injury index defined as
(A-B) / (C-B) x 100 where A = LDH activity in the test sample, B = LDH
activity in samples from wells in which cells were exposed to medium
alone (0% control) and C = LDH activity in samples from wells in which
cells were lysed with 1% Triton X-100 (100% control). For all lipid
membrane protective agents tested, a dose response was done to
determine the maximum dose possible without causing cytotoxicity. In
addition, each agent tested was added to a known quantity of purified LDH
and none significantly altered fluorometric measurements of LDH activity.
Measurement of Incorporated U74500
General analytical methodology for measurement of intact U74006F (a
21-aminosteroid similar to U74500) in rat plasma has been described
elsewhere (Cox and Pullen, 1988). Briefly, the quantitation of U74500
was performed using HPLC analysis. The ethanol extracts (preparation
described in experimental methods) were loaded directly onto a
Supelcosil LC-CN reverse phase column which was eluted isocratically
in 40% (v/v) acetonitrile : 60% water buffered with 20mM sodium
phosphate at pH 7.0. U74500 was detected electrochemically using
Waters 460 electrochemical detectors at a voltage setting of 760 mV.
Tetrabutyl ammonium perchlorate was used as the ion pairing agent in the
19


mobile phase. Peak heights were measured and quantified by comparison
to standards.
Immunohistochemical Staining
Cultured chick embryo cardiac myocytes were stained with an
immunohistochemical stain specific for fibroblasts, smooth muscle and
endothelial cells to determine the percent contaminating cells. Chick
embryo cardiac myocytes were plated into 8 well chamber slides (Miles
Laboratories, Lab-Tek Division, Naperville, IL) at a concentration of 6.5 x
1()4 cells/ml; 0.3 ml/well. Just before the cells reached confluency, they
were fixed in methanol/acetic acid 3:1 V/V for 10 min. at 4C. Prior to
staining, the wells were quenched with 5% hydrogen peroxide in methanol
for 30 min. to remove any remaining endogenous peroxidase. The cells
were then stained for vimentin (an intermediate filament protein) using the
Vectastain ABC kit (Vector Labs, Burlingame, CA). The primary antibody
used was a monoclonal mouse anti-swine vimentin (DAKO-Vimentin V9;
DAKO, Glostrup, Denmark). DAKO-Vimentin V9 labels lymphoid cells,
endothelial cells, fibroblasts and smooth muscle cells, and shows a broad
interspecies reactivity, including chicken Osborn et al., 1984). The
substrate for horseradish peroxidase was 3,3-diaminobenzidine (DAB;
Vector Labs, SK-4100). Incubation in the DAB substrate solution was
done until a brown color developed, indicating a positive stain. Cells were
then counter-stained with the nuclear stain, hematoxylin (purple) and
sequentially dehydrated with graded alcohols (50,70,80,90,100% ethanol)
20


followed by xylene (50/50 100% ethanol) and then mounted in non-
aqueous mounting media. A monolayer of bovine fibroblasts was
concurrently cultured and stained as a positive control. In addition, chick
embryo cardiac myocytes and bovine fibroblasts were stained without
using the monoclonal antibody as negative controls.
Statistical Analysis
All statistical comparisons were by analysis of variance using the
Scheffe test of significance.
i
21
I


CHAPTER 3
EXPERIMENTAL RESULTS
Effects of Lipid Soluble Agents on H202-mediated Cytotoxicity
The 21-aminosteroid U74500 proved to be the only effective agent
tested in attenuating cell injury caused by H2O2 in the cultured chick
embryo cardiac myocyte model- Exposure of untreated cells to 1.5mM
H2O2 for 20 hrs resulted in 68% LDH release. However, exposure to
H2O2 in cells treated with U74500 resulted in a concentration dependent
reduction in LDH release (49% LDH release at a concentration of 50pM,
39% at lOOpM, 30% at 200pM and 22% at 400pM) Figure 3.1. The
emulsion in which the U74500 was solubilized gave a slight reduction in
cell injury caused by H2O2 (Table 3.1).
Preincubation of 400pM U74500 in emulsion for 30 min. followed by
rinsing the cells and then application of 0.75mM H2O2 showed no
protection when compared to continuous incubation with 0.75mM H2O2
alone at 22hrs (Figure 3.2). Continuous incubation of 400(iM U74500 did
not completely protect against 0.75mM H2O2, resulting in 22% LDH
release at 22 and 48hrs (Figure 3.2).
Solid U74500 at a concentration of 200(i.M solubilized in DMSO
showed less of a protective effect than U74500 in emulsion; 50% LDH


release with 200pM U74500 in DMSO compared to 30% LDH release with
200pM U74500 in emulsion, when incubated simultaneously with 1.5mM
H2O2 (Table 3.1, Table 3.3). There was no difference between the
protection given by 100|iM and 200|iM U74500 in DMSO against H2O2
injury (Figure 3.3).
None of the other agents tested (Trolox C, lidocaine, propranolol,
probucol or diltiazem) showed any significant protection against
cytotoxicity due to H2O2 as shown in Figures 3.4 through 3.8.
Incorporation of U74500 Emulsion into the Cell Membrane
The incorporation of U74500 in the myocyte cell membrane was
measured at various time points of exposure to 1.5mM H2O2 to ensure
that it remained in the membrane.
Conditions prior to processing
200|iM 4hrs
400|iM 30 min.
400pM 30 min., rinse then H2O2 4hrs
400uM 30 min., rinse then H2O2 8hrs
U74500 nmoles
3.4
4.3
2.2
2.7
The incorporation of U74500 was concentration dependent, with more
U74500 being incorporated with the higher incubation concentration,
independent of incubation time. Detectable levels of U74500 remained in
the membrane up to 8 hrs of incubation with 1.5mM H2O2. There was no
23


change in the amount of U74500 incorporated into the membrane at 4
and 8 hrs of incubation with 1.5mM H2O2.
Purity of Chick Embryo Cardiac Myocyte Monolayer
The chick embryo cardiac myocyte monolayer had 2% of total cells that
stained positive for vimentin. This indicates a monolayer of 98% cardiac
myocytes and 2% either fibroblast, endothelial or smooth muscle cells. In
control experiments, all of the cells in a bovine fibroblast monolayer, in
which the antibody for vimentin was used, stained positive for vimentin.
None of the cells in a bovine fibroblast monolayer, in which the antibody
for vimentin was not used, stained positive for vimentin.
24


80
Cell
Injury
Index
Time (hrs)
H202
U74500 50 uM
+ H202
U74500 100 uM
+ H202
U74500 200uM
+ H202
U74500 400 uM
+ H202
Medium
Figure 3.1. Cell Injury Index versus incubation time from data in Table 3.1. Values are
mean SEM. The (*) indicates points significantly different from H202 alone (p<0.01,
n=6).
Cell Injury index 1 Standard Error
U74500 emulsion 2hr 4hr 7hr 23hr
Medium -0.10.7 2.4+1.7 -5.32.0 3.0+2.5
H202 1.5mM 11,93.0 33.54.1 57.6+2.1 68.04.4
400uM -12.52.1 -7.43+0.9 -7.85.6 16.9+2.3
400uM + H202 -8.4+1.3 1,02.3 4.21.8 22.111.4
200uM + H202 -0.31.0 12.92.4 14.03.0 30.013.0
100uM + H202 1.5+2.0 16.61.5 27.3+2.4 38.813.4
50uM + H202 12.52.1 19.1+1.6 35.82.1 49.113.0
Emulsion -0.41.5 0.7+1.1 -1.2+0.7 -6.112.5
Emulsion + H202 1.70.3 13.7+1.3 28.71.6 53.810.3
Table 3.1. Cell Injury Index data from myocytes exposed for 23hrs to varying concentrations
of U74500 emulsion, with or without H202, in medium with 6% serum.
25


Emulsion 30' pre-exp.
wash, then H202
H202
o 400uM 30' pre-exp.
wash, then H202
400uM + H202
Tyrode's
Figure 3.2. Cell Injury Index versus incubation time from data in Table 3.2. Values are
mean SEM. The (*) indicates points significantly different from H202 alone (p<0.01,
n=6).
Cell Injury Index Standard Error
U74500 emulsion 4hr 8hr 22hr 48 hr
Tyrode's -5.2+1.0 -2.2+1.7 3.42.1 4.02.0
H202 0.75mM -3.10.5 16.4+2.7 43.51.3 53.812.2
400uM continuous -4.32.4 1,81.3 6.20.4 25.312.1
400uM + H202 continuous 0.30.7 8.21.1 22.81.5 22.2+1.6
Emulsion + H202 continuous 0.60.3 15.2+3.9 35.52.6 49.412.0
400uM pre-exp. 30', wash then H202 4.5+1.9 14.8+0.9 41,72.6 49.912.2
Emulsion pre-exp. 30', wash then H202 2.84.5 21.12.4 50.73.0 53.712.0
Table 3.2. Cell Injury Index data from myocytes either pre-exposed to 400uM U74500 in
Tyrode's for 30 min., rinsed and then exposed to 0.75mM H202 in Tyrode's for 48hrs or
continuous exposure to 400uM U74500 and 0.75mM H202 in Tyrode's for 48hrs. The
dilution of the emulsion was the same as that to obtain 400uM of the U74500 in emulsion.
26


80
60
Cell 40
Injury
Index 20
0
-20
J____.___I__i___I I
5 10 15 20
Time (hrs)
H202
U74500100uM
+ H202
U74500 200uM
+ H202
Medium
U74500 200uM
_l
25
Figure 3.3. Cell Injury Index versus incubation time from data in Table 3.3. Values are
mean SEM. The (*) indicates points significantly different from H202 alone (p<0.01,
n=6).
Cell Injury Index Standard Error
U74500 in 1mM DMSO 2hr 4hr 7hr 21 hr
Medium (10% CBS) -5.7+0.4 -2.5+0.9 1.710.4 6.511.0
H202 1.5mM 2.61.3 29.41.6 65.1+1.9 67.3+2.4
200uM -5.0+0.5 0.9+0.6 0.910.9 4.511.2
200uM + H202 -3.00.9 21.511.2 38.911.4 49.811.8
100uM + H202 -2.61.2 22.911.0 65.111.9 67.312.4
Table 3.3. Cell Injury Index data from myocytes exposed to either 100uM or 200uM U74500
solubilized in 1mM DMSO, with or without 1.5mM H202, in medium with 6% serum.
27


100 r
80
60
Cell
Injury 40
Index
20
0
-20
j_______I_______i_______I_______i______I_______i_______L
5 10 15 20
Time (hrs)

o
25
H202
Probucol 50uM
+ H202
Probucol 50 uM
0.5% EtOH
Medium
Figure 3.4. Cell Injury Index versus incubation time from data in Table 3.4. Values are
mean l SEM (n=6).
Cell Iniurv Index Standard Error
Probucol 2hr 4hr 7hr 23hr
Medium (10% CBS) -5.70.8 -4.012.3 4.712.2 5.113.6
H202 1.5mM -1.211.0 22.310.9 60.015.8 76.914.6
50uM 2.012.4 4.710.8 1.811.5 8.812.3
50uM + H202 -0.914.1 19.212.4 46.314.2 70.116.2
0.5% EtOH -1.311.3 3.112.3 3.210.9 7.211.0
Table 3.4. Cell Injury Index data from myocytes exposed for 23hrs to 50uM probucol in 0.5%
EtOH, with or without 1.5mM H202, in medium with 10% serum.
28


H202
Trolox 10mM
+ H202
Trolox 20mM
Medium
Trolox 10mM
Figure 3.5. Cell Injury Index versus incubation time from data in Table 3.5. Values are
mean SEM (n=6).
Cell Injury Index Standard Error
Trolox 2hr 4hr 7hr 21 hr
Medium -5.70.4 -2.5+0.9 1.70.4 6.51.0
H202 1,5mM 2.61.3 29.41.6 65.1 1.9 67.32.4
10mM 1 hr pre-exposure -4.80.6 1.10.7 0.71.0 1,81.6
10mM 1 hr pre-exposure then H202 4.0+2.2 37.7+3.7 60.7+1.1 65.9+1.0
20mM 1 hr pre-exposure 00.5 4.6+0.5 7.5+1.7 35.6+1.9
Table 3.5. Cell Injury Index data from myocytes exposed for 21hrs to 10mM or 20mM
Trolox C, with or without 1.5mM H202, in medium with 6% serum.
29




Propranolol
500uM
Propranolol
250uM + H202
H202
Propranolol
25 0 u M
Medium
Figure 3.6. Cell Injury Index versus incubation time from data in Table 3.6. Values are
mean SEM (n=6).
Cell Injury Index Standard Error
ProDranolol 2hr 4hr 7hr 2?hr
Medium -3.41.2 0.7+1.1 -4.21.5 6.91.4
H202 1,5mM 8.9+2.0 37.52.5 58.71.6 67.40.8
250uM -0.31.9 1.42.1 -1.82.4 16.1+1.4
250uM + H202 20.4+4.8 59.32.7 63.5+1.6 71,72.8
500uM 3.81.2 8.2+0.6 20.8+2.5 97.7+3.7
Table 3.6. Cell Injury Index from myocytes exposed for 23hrs to 250uM or 500uM
propranolol, with or without 1.5mM H202, in medium with 6% serum.
30


1 Diltiazem
500uM + H202
H202
Diltiazem
1 mM
Diltiazem
500uM
Medium
Figure 3.7. Cell Injury Index versus incubation time from data in Table 3.7. Values are
mean SEM (n=6).
Cell Iniurv Index Standard Error
Diltiazem 2hr 4hr 7hr 23hr
Medium -3.41.2 0.71.2 -4.21.5 6.91.4
H202 1.5mM 8.92.0 37.52.5 58.71.6 67.40.8
500uM 2.70.7 9.90.7 8.60.5 17.0+0.5
500uM + H202 14.4+1.2 41.63.5 61.12.2 74.4+4.4
1mM -1.4+2.4 0.3+1.2 5.11.3 34.0+0.9
Table 3.7. Cell Injury Index from myocytes exposed for 23hrs to 500uM or 1mM diltiazem,
with or without 1.5mM H202, in medium with 6% serum.
31


0 LidocainelmM
+ H202
H202
Lidocaine 2mM
LidocainelmM
o Medium
Figure 3.8. Cell Injury Index versus incubation time from data in Table 3.8. Values are
mean 1 SEM (n=6).
Cell Injury Index Standard Error
Lidocaine 2hr 4hr 7hr 23hr
Medium -2.71.5 3.40.9 -1.411.0 0.711.8
H202 1.5mM 2.51.2 32.71.1 59.012.0 67.010.9
1mM 2.41.8 2.00.8 3.212.2 3.212.1
1mM + H202 10.1+2.1 34.60.7 60.711.2 72.713.3
2mM 6.60.9 4.510.7 7.410.8 20.410.5
Table 3.8. Cell Injury Index data from myocytes exposed for 23hrs to 1mM or 2mM
lidocaine, with or without 1.5mM H202, in medium with 6% serum.
32


Figure 3.9. Bovine fibroblasts immunohistochemically stained for vimentin using the
monoclonal antibody for vimentin; positive control. Note the brown vimentin stain in
the cytoplasm. The fibroblasts were counter-stained with the nuclear stain hematoxylin
(purple). 25X magnification.
33


\
* §
Vr J * # V
* I \ *
A a* -ft **
^
1 f
#
/ v-

- .- #

* h
* 1)



v #
11 ?
/ w

-

-
%
9)
% %
* %
* #* *
' a jft
*.*


* %



#
> t
.* ;
r #
( i
^ g^.
# -* %% %
/*
% %
i *k
w k ^ *-.
*

* ##

t <
4
* *
mm
%% % **
%.
Figure 3.10. Bovine fibroblasts immunohistochemically stained for vimentin without using
the monoclonal antibody for vimentin; negative control. Note the absence of the brown
vimentin stain in the cytoplasm. The fibroblasts were counter-stained with the nuclear
stain hematoxylin (purple). 25X magnification.
34


Figure 3.11. Chick embryo cardiac myocytes immunohistochemically stained for vimentin
using the monoclonal antibody for vimentin. Note the absence of the brown vimentin stain
in all but a few of the cells' cytoplasm. The myocytes were counter-stained with the
nuclear stain hematoxylin (purple). 25X magnification.
35


Figure 3.12. Chick embryo cardiac myocytes immunohistochemically stained for vimentin
without using the monoclonal antibody for vimentin. Note the absence of the brown vimentin
stain in the cytoplasm. The myocytes were counter-stained with the nuclear stain hema-
toxylin (purple). 25X magnification.
36


CHAPTER 4
DISCUSSION
There is considerable evidence supporting the role of reactive oxygen
species in reperfusion injury. H2O2 is a reactive oxygen species produced
by neutrophils, mitochondria and tissue reactions involving the enzyme
xanthine oxidase. Antileukocyte measures have been shown to reduce
myocardial or coronary endothelial injury (Simpson et al., 1990; Sheridan
et al., 1991). Also, there is increased xanthine oxidase in coronary
endothelial cells which could contribute to generation of reactive oxygen
species and high numbers of mitochondria in the myocardium due to this
tissues high energy demand. Sudden reintroduction of high
concentrations of oxygen, such as is encountered during reperfusion,
increases the production of H2O2 in the mitochondrion. Therefore, our
model of applying H2O2 to cultured cardiac myocytes is potentially useful
in measuring on a cellular basis whether protection is obtained by the
application of various agents.
Exposure to H2O2 for -20 hrs caused substantial release of LDH in
untreated chick embryo cardiac myocytes. In typical experiments, the
release of LDH during exposure to 1.5mM H2O2 for ~20hrs was 60-70%


of the total LDH, measured by cell lysis with 1% Triton X-100. Most of the
release occurred between 4 and 8 hrs of exposure.
In previous work, we and others have demonstrated substantial
protection in cell culture or in vivo with either scavengers of H2O2
(catalase, dimethylthiourea and N-(2-mercaptopropionyl)-glycine) or the
iron chelator, deferoxamine (Jolly et al., 1984; Lesnefsky et al., 1990;
Carrea et al., 1991; Byler and Horwitz, 1991). Despite the promising
results with these agents, there are certain drawbacks to their clinical use.
Limited ability to quickly enter myocardial cells is a problem with agents
such as catalase and deferoxamine. Toxicity may limit usage of some
scavenger agents.
We sought to explore an alternative approach to protection against
oxidant injury by H2O2. Since the cell membrane is the principal target,
we examined agents which protect the cell membrane without scavenging
H2O2. Two groups of agents were examined. Lipid-soluble agents for
which there is previous evidence of inhibition or prevention of membrane
lipid peroxidation and nonlipid-soluble agents for which there is previous
evidence of nonspecific membrane stabilization.
Of the lipid soluble agents, the 21-aminosteroid, U74500, was the
only one to be effective at reducing cell injury due to H2O2. However, cell
injury was not completely eliminated by U74500. There are two
possibilities for this. The first is that U74500 does not completely protect
against membrane lipid peroxidation. The second is that other
mechanisms such as peroxidation of membrane protein sulfhydryls play an
38


important role in reperfusion injury. Pre-incubation and then removal of
U74500 with subsequent exposure to H2O2 resulted in no reduction in cell
injury. However, U74500 was retained in the cell membrane for the first 8
hrs of exposure, the time during which most of the injury occurred.
Evidence exists suggesting that U74500 chelates iron (Braughler et al.,
1987). Concordantly, it is possible that U74500 reduces myocardial injury
caused by H2O2 by limiting iron-mediated production of oxygen radicals.
It is likely that the mechanism of action is inhibition of lipid peroxidation as
shown by others (Braughler et al., 1987) either by a direct mechanism or
indirectly by scavenging H2O2 or by chelating iron. U74500s
ineffectiveness following pre-exposure can be explained by its proposed
indirect mechanisms or perhaps U74500 is somehow metabolized in the
cell membrane making it ineffective.
Alpha-tocopherol is an endogenous antioxidant (Halliwell, 1991). The
aromatized polar region (Trolox C) is thought to be involved in the
inhibition of lipid peroxidation, while the long hydrophobic tail (phytol)
anchors the c-tocopherol compound to the cell membrane. Trolox C itself
is lipophilic without the phytol tail. Previous studies have shown Trolox C
to delay cellular necrosis caused by reactive oxygen species (Mickle et al.,
1989; Wu et al., 1990; Wu et al., 1991). We found no reduction in cell
injury over 20hrs as a result of treatment with Trolox C. It likely that Trolox
C delays but does not reduce injury caused by reactive oxygen species.
A problem encountered with all of the lipid soluble agents is solubility in
medium. This was a particular problem with probucol in which the highest
39


concentration we could attain was 50|iM. This concentration may not
have been high enough to be effective in inhibiting lipid peroxidation. In
some previous in vivo studies, animals were fed probucol (Kita et al.,
1987; Dage et al., 1991). This allows a higher concentration to be reached
in the membrane. The effectiveness of both probucol and Trolox C could
have been limited in our preparation by their low solubility in aqueous
solutions.
None of the nonlipid-soluble agents (diltiazem, propranolol and
lidocaine) provided any protection against H2O2 cytotoxicity. These
agents are thought to provide nonspecific membrane stabilization. They
all appeared to have some effectiveness in different preparations by others
in which there was exposure to hypoxia and reoxygenation or to
superoxide radical. Perhaps, by stabilizing the membrane, they affect the
chloride channels in the membrane and thus limit the transport of
superoxide radical (Lynch and Fridovich, 1978. This would be irrelevant in
our model in which only H2O2 was applied because H2O2 freely diffuses
into cells. It is also possible that the positive effects of these agents in vivo
is due to an imposed reduction in release of reactive oxygen species from
neutrophils which would only be relevant in vivo.
Lipid peroxidation appears to be an important factor in reperfusion
injury as evidenced by the protective effects of U74500. Other forms of
membrane protection with nonlipid soluble agents were not successful in
our hands. Such agents may be more protective against some oxidants
than others. To assess the importance of lipid peroxidation and the
40


mechanism by which U74500 is protective, measurement of lipid
peroxides in cells treated with U74500 and exposed to H2O2 could be
compared to lipid peroxides measured in cells exposed to H2O2 alone.
41


APPENDIX A
Structural Formulas of Lipid-soluble Agents
CH,
CHj
NCHjCHj
n-ch,ch,
XXj £
U74500
C(CH3)3
PROBUCOL
COOH
TROLOX C
42


APPENDIX B
Structural Formulas of Nonlipid-soluble Agents
OH
PROPRANOLOL
S
H
CH2CH2N(CH3)2
DILTIAZEM
CH
nhcoch2n(c2h5)2
LIDOCAINE
43


LITERATURE CITED
Babior, B. (1978). Oxygen dependent microbial killing by phagocytes. N
Eng J Med. 298: 659-668.
Barry, W. and Smith, T. (1982). Mechanisms of transmembrane calcium
movement in cultured chick embryo ventricular cells. J Physiol. 325:
243-260.
Barry, W. et al. (1975). Inotropic effects of different calcium ion
concentrations on embryonic chick ventricle: comparison of single
cultured cells and intact muscle strips. Circ Research. 36: 727-734.
Beyersdorf, F. et al. (1989). Studies on prolonged acute regional ischemia:
evidence for preserved cellular viability after 6 hours of coronary
occlusion. J Thorac Cardiovas Surg. 98: 112-126.
Bolli, R. (1988). Oxygen-derived free radicals and post-ischemic
myocardial dysfunction (stunned myocardium). J Am Col Cardio.
12: 239-249.
Bolli, R. et al. (1987). Attenuation of dysfunction in the post-ischemic
stunned myocardium by dimethylthiourea. Circ. 76: 458-468.
Boveris, A. and Chance, B. (1973). The mitochondrial generation of
hydrogen peroxide: general properties and effect of hyperbaric
oxygen. Biochem J. 134: 707-716.
Braughler, J. et al. (1987). Novel 21-amino steroids as potent inhibitors of
iron-dependent lipid peroxidation. J Biol Chem. 262: 10438-10440.
Byler, R. and Horwitz, L. (1991). Cardiac myocyte injury due to reactive
oxygen metabolites is oxygen dependent. Clin Res. 39: 371 A.
Cadenas, E. et al. (1977). Production of superoxide radicals and hydrogen
peroxide by NADH-ubiquinone reductase and ubiquinol-cytochrome
C reductase from beef heart mitochondria. Arch Biochem Biophys.
180: 248-257.
44


Carrea, F. et al. (1991a). Reduction of canine myocardial infarct size by a
diffusible reactive oxygen metabolite scavenger.Circ Res. 68: 1652-
1659.
Carrea, F. et al. (1991b). U74006F decreases necrosis and enhances
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