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Effect of doxorubicin-induced apoptosis on gender

University of Florida Institutional Repository

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EFFECT OF DOXORUBICIN-INDUCED APOPTOSIS ON GENDER By YOUNGMOK C. JANG A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN EXERCISE AND SPORT SCIENCES UNIVERSITY OF FLORIDA 2003

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Copyright 2003 by Youngmok C. Jang

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This thesis is dedicated to: Eun-ah Lee, for her love and support. Im extremely lucky to have her in my life.

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ACKNOWLEDGMENTS Many individuals contributed a great deal in helping to complete this project. First, I would like to thank my committee chair and mentor, Dr. Christiaan Leeuwenburgh. The guidance, support, and patience Dr. Leeuwenburgh has given me during this project are very much appreciated. Dr. Leeuwenburgh has shown me what it takes to be successful as a scientist and also in life. I will always be grateful. I would also like to thank the other members of my committee, Dr. Scott Powers and Dr. Stephen Dodd, for their input and expertise on this project. I would also like to thank my colleagues in the Biochemistry of Aging Laboratory, for their help and friendship. Special thanks go to Dr. Barry Drew, Dr. Suma Kendaiah and Tracey Phillips, for their help in collecting data. Finally, I would like to extend my gratitude to my family for their love, encouragement, and support throughout my life. Specifically, my parents, my brother Youngmin, my mother-in-law, sister-in-law Eun-Sun, Eun-Sook, and Eun-Young all deserve my deepest appreciation. iv

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TABLE OF CONTENTS Page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES............................................................................................................vii LIST OF FIGURES.........................................................................................................viii CHAPTER 1 INTRODUCTION........................................................................................................1 Introduction...................................................................................................................1 Specific Aims................................................................................................................4 2 REVIEW OF LITERATURE.......................................................................................5 Doxorubicin and Cardiotoxicity...................................................................................5 Doxorubicin and Oxidants............................................................................................6 Apoptosis......................................................................................................................6 Mitochondrial Mediated Pathway.........................................................................8 Death Receptor Mediated Pathway.......................................................................9 Regulators of Apoptosis........................................................................................9 Gender Related Differences in the Cardiovascular System........................................10 Gender Differences in Apoptosis................................................................................11 Summary.....................................................................................................................13 3 METHODS.................................................................................................................14 Animals and Experimental Design.............................................................................14 Tissue Harvesting.......................................................................................................14 Cellular Fractionation.................................................................................................15 Protein Concentration.................................................................................................15 Mitochondrial Functional Parameters.........................................................................15 Mitochondrial Respiratory Function...................................................................15 ATP Content and Production...............................................................................16 Mitochondrial Hydrogen Peroxide (H 2 O 2 ) Production.......................................16 Biochemical Assays....................................................................................................17 Estrogen (Estradiol).............................................................................................17 Cytosolic Monoand Oligo-nucleosomes...........................................................17 Western Blots Analysis.......................................................................................18 v

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Cytosolic Cytochrome C.....................................................................................18 Caspase-3 Activity...............................................................................................18 Caspase-3 Content...............................................................................................19 Caspase-8.............................................................................................................19 Inhibitors of Apoptosis (XIAP, FLIP, ARC).......................................................19 Bcl-2 and Bax......................................................................................................19 Antioxidant Enzyme Assay........................................................................................19 Statistical Analysis......................................................................................................19 4 RESULTS...................................................................................................................20 Morphological Characteristics....................................................................................20 Plasma Estrogen Levels in Male and Female Rats.....................................................21 Mitochondrial Function..............................................................................................22 Hydrogen Peroxide Production in Isolated Mitochondria..........................................23 Glutathione Peroxidase Activity.................................................................................24 Apoptosis Determined by Mono and Oligo-nucleosomes..........................................24 Caspase-3 Activity and Caspase-3 Content................................................................25 Mitochondrial Mediated Pathway of Apoptosis.........................................................28 Mitochondrial Regulators of Cytochrome C Release.................................................29 Receptor Mediated Pathway of Apoptosis.................................................................30 5 DISCUSSION.............................................................................................................33 Overview of Principle Findings..................................................................................33 Body Weight and Heart Weight.................................................................................34 Oxidant Production and Antioxidant Enzymes..........................................................34 Apoptosis Induced by Doxorubicin............................................................................35 Mitochondrial Mediated Pathway of Apoptosis.........................................................35 Receptor-Mediated Pathway of Apoptosis.................................................................36 Mitochondrial Function and Doxorubicin Treatment.................................................37 Limitations in Present Study.......................................................................................37 Conclusion and Future Direction................................................................................38 LIST OF REFERENCES...................................................................................................40 BIOGRAPHICAL SKETCH.............................................................................................46 vi

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LIST OF TABLES Table page 4-1 Body weight, heart weight, and heart weight to body weight ratio of male and female rats treated with doxorubicin or saline.........................................................21 4-2 Plasma 17estradiol level......................................................................................21 4-3 The effects of doxorubicin administration on mitochondrial function....................22 vii

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LIST OF FIGURES Figure page 1-1 Doxorubicin-induced mitochondrial damage and apoptosis......................................2 2-1 Overview of apoptotic pathways..............................................................................12 4-1 The effect of doxorubicin administration on heart mitochondrial oxidant production.................................................................................................................23 4-2 The effect of doxorubicin administration on heart mitochondrial glutathione peroxidase activity (GPX)........................................................................................24 4-3 The effect of doxorubicin administration (10mg/kg) on the content of monoand oligo-nucleosomes in the heart cytosol....................................................................25 4-4 The effect of doxorubicin administration (10mg/kg) on caspase-3 activity............26 4-5 The effect of doxorubicin administration on the caspase-3 content (A) and cleaved caspase-3 concentration (B) determined by Western blot analysis..........................27 4-6 The effect of doxorubicin administration (10mg/kg) on the amount of anti-apototic proteins XIAP measured in cytosolic fraction using Western method....................28 4-7 The effect of doxorubicin administration (10mg/kg) on cytochrome c concentration in the cytosol......................................................................................28 4-8 The effect of doxorubicin administration (10mg/kg) on the content of Bcl-2 (A) and Bax (B) measured in the cardiac mitochondria using Western Blot analysis...29 4-9 The effect of doxorubicin administration (10mg/kg) on the content (A) Procaspase-8 and (B) cleaved caspase-8...........................................................31 4-10 The effect of doxorubicin administration (10mg/kg) on the content of cFLIP measured in cytosolic fraction using Western blot method.....................................32 viii

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Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science in Exercise and Sport Sciences EFFECT OF DOXORUBICIN-INDUCED APOPTOSIS ON GENDER By Youngmok C. Jang August 2003 Chair: Christiaan Leeuwenbugh Major Department: Exercise and Sport Sciences Doxorubicin is a powerful anthracycline antibiotic used to treat a multitude of human neoplasms. However, doxorubicin causes severe cardiac toxicity, which compromises its clinical usefulness. Females are believed to be better protected against cardiovascular insults. This study examined the gender differences in doxorubicin-induced apoptosis. We administered doxorubicin at clinical levels (10mg/kg of body weight) to male and female rats. After one day and four days later, we measured the oxidant production and examined different apoptotic pathways. Females produced less oxidants in isolated mitochondria compared to males and were able to scavenge oxidant faster than male rats. We assessed the apoptotic index by measuring DNA fragmentation. Male rats had a significantly increased level of apoptosis one day after doxorubicin treatment, but no changes were seen in females rats in both one and four days after the treatment. The effectors of apoptosis, caspase-3 activity were significantly increased at day four, in both ix

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males and females. These data suggest that caspase-independent pathway might be involved in doxorubicin-induced apoptosis. Mitochondrial-mediated pathway was not involved in activating capase-3 at day four. Cytochrome c release was prevented by anti-apoptotic protein Bcl-2 at day four in males. No changes were detected in receptor-mediated pathway. The initiator caspase-8 and its inhibitor cFLIP did not change in response to doxorubicin administration. These findings suggest that doxorubicin induces apoptosis through other novel apoptotic pathways such as sarcoplasmic reticulum mediated pathway. A better understanding of gender difference in doxorubicin-induced proand anti-apoptotic signaling pathways in cancerous and non-cancerous cells may lead to new and improved therapeutic protocols for mitigating the toxic side effects of doxorubicin. x

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CHAPTER 1 INTRODUCTION Introduction The anthracycline antibiotic doxorubicin (adriamycin) is one of the most effective chemotherapeutic agents for treating human neoplasms such as leukemia, lymphomas, breast cancer, and many solid tumors. However, chronic use can become associated with acute and chronic cardiotoxicities (1). The cardiotoxicity is dose dependent and causes irreversible myocardial damage, resulting in dilated cardiomyopathy with fatal congestive heart failure (1, 2). The exact mechanism of doxorubicin-induced cardiomyopathy remains unclear, but most of the evidence indicates that reactive oxygen species (oxidants) are involved (3). It is believed that mitochondrial derived oxidants play a significant role in triggering this toxicity (4, 5). Isolated heart mitochondria have been shown to shuttle single electrons to doxorubicin, giving rise to oxygen radicals through the autoxidation of adriamycin semiquinones. Evidence suggests that NADH dehydrogenase associated with complex I of the electron transport chain is intrinsically involved in this one electron transfer to doxorubicin further generating free radicals (oxidants) (6). Reactive oxygen species have been reported to cause irreversible tissue damage by inactivating key proteins and enzymes present in cardiac sarcoplasmic reticulum and mitochondria, and they are also believed to induce apoptosis (7). Apoptosis is an evolutionary conserved form of cell suicide through which multicellular organisms eliminate redundant, damaged, or infected cells (8-10). The central component of this form of cell death is a proteolytic system involving a family of 1

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2 cysteine proteases called caspases (11). Interest in the control of apoptosis has grown significantly since the realization that disturbed apoptosis may contribute to cancer, degenerative diseases, and cardiomyopathies with the chronic use of doxorubicin (12, 13). Investigations in experimental animal models have demonstrated that apoptosis is one of the mechanisms of myocyte cell death with ischemic cardiac injury and heart failure (13). Further, apoptosis is now being implicated in anthracycline induced cardiotoxicity (Figure 1-1), which is one of the major limitations to the use of this otherwise highly efficacious antineoplastic drug (7). Oxidants GSH, ATP Bcl-XL Bcl-2 Complex II Caspase activation????? Nuclear DNADamageComplex IComplex IIICytochrome c release OxidizedAmino Acids? Apoptosis? Figure 1-1. Doxorubicin-induced mitochondrial damage and apoptosis. Oxidants, such as superoxide (O 2 ), hydrogen peroxide (H 2 O 2 ), hydroxyl radicals (HO ), and peroxynitrite (ONOO ) may be generated by doxorubicin toxicity. Oxidants will affect mitochondrial redox status and may cause extensive oxidative damage to proteins. Furthermore, the mitochondrial transition pore can open releasing cytochrome c and possibly affecting mitochondria function. Cytochrome c can activate cytosolic caspases to induce apoptosis. Doxorubicin may induce anti-apoptotic (Bcl-2, Bcl-X L ) and pro-apoptotic (Bax, Bad) proteins. Compelling evidence from several epidemiological and clinical studies indicates a substantially higher incidence of heart failure and cardiovascular diseases in men compared with age-matched women (14-16). The basis for these differences have been attributed in part, to the cardioprotective effects of estrogen (17). The assumption that

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3 female sex hormones are largely responsible for the low incidence of cardiovascular disease in pre-menopausal women arises from the absence of similar cardiovascular protection in post-menopausal women, an occurrence paralleled by declining estrogen levels (16). However, many other mechanisms may exist and this requires further investigation. The benefits of estrogen gradually shift from the vascular system to the myocardium (15). This view is supported by the fact that functional estrogen receptors have been detected in the myocardium (18). In addition, females have higher levels of telomerase activity after adulthood and therefore an enhanced ability to preserve cardiac myocytes viability following an injury. This may increase the potential for growth and could certainly become critical factor to ensure an longer life span (19). In recent years, several investigations have documented that female hearts are inherently protected by estrogen against apoptotic cell death (20). It is speculated that estrogen reduces the activity of ICE-like protease caspase-3, which is an effector / downstream mediator of apoptosis (18, 21). In addition, gender differences in myocardial activation of Akt/PKB can also subsequently inhibit apoptosis by phosphorylating the Bcl-2 family member Bad and caspase-9, which are key components of the intrinsic cell death machinery (22). Although much of the existing research has focused on trying to unravel the mechanisms responsible for doxorubicin induced cardiotoxicity and the numerous ways estrogen may afford protection to the cardiovascular system in females, no experiments have been carried out to determine if male and female hearts respond differently to an acute dose of doxorubicin. Therefore, we attempted to determine this possibility in these proposed experiments.

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4 Specific Aims This study will investigate whether there are sex differences following doxorubicin treatment in oxidant production, mitochondrial function, proand antiapoptotic proteins and the overall incidence of apoptosis in male and female Fischer 344 rats. Question 1 Are there sex differences in oxidant production, mitochondrial function and responses by antioxidant defenses one day and four days after doxorubicin administration? Hypothesis 1 Female rats will have less oxidant production, less mitochondrial dysfunction, and an enhanced antioxidant defense adaptation following doxorubicin treatment. Question 2 Are there differences in the overall incidence in apoptosis and caspase-3 activation between male and female rats following administration of doxorubicin? Hypothesis 2 Female hearts will have exhibited less apoptotic cell death and caspase-3 activation. Question 3 Are there differences in the adaptation of proand anti-apoptotic regulatory proteins in response to doxorubicin cardiotoxicity? Hypothesis 3 Female hearts will have higher expression of antiapoptotic proteins as compared to male hearts, which may partly explain differences in apoptosis.

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CHAPTER 2 REVIEW OF LITERATURE Doxorubicin and Cardiotoxicity Doxorubicin (Adriamycin) is a powerful anthracycline antibiotic, originally isolated from the fungus streptomyces peucetius. It is used to treat many human neoplasms, including acute leukemias, lymphomas, stomach, breast and ovarian cancers, Kaposis Sarcoma, and bone tumors (23). However, doxorubicin causes severe cardiac toxicity, which compromises its clinical usefulness (24). Chronic toxic effects of doxorubicin often develop after several weeks or months of treatment, and sometimes even 4 to 20 years after discontinuation of the treatment. Thus, the risk of developing heart failure in cancer patients treated with doxorubicin remains a life-long threat (2). Since the first report of doxorubicin-induced cardiomyopathy (25), extensive clinical as well as basic research efforts have been focused on understanding the pathophysiology of congestive heart failure caused by this drug. A number of mechanisms have been proposed to explain the development of doxorubicin-induced cardiomyopathy including direct DNA damage and interference with DNA repair (26), change in adrenergic function (27), abnormalities in the mitochondria (28), lysosomal dysfunction (2), altered sarcolemmal Ca 2+ transport, Na + -K + ATPase and Ca 2+ ATPase, imbalance in myocardial electrolytes (2), free radical formation (2, 28, 29), reduction in myocardial antioxidant enzyme activities (29), lipid peroxidation (30), and apoptosis (28, 29, 31). Furthermore, the cytotoxic action by doxorubicin involves the cytoskeleton of both tumor cells and cardiomyocytes (32). Cytoskeletal changes following doxorubicin 5

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6 administration include reduction in density of myofibrillar bundles (33), alterations on the Z-disc structure (33), and disarray and depolymerization of actin filaments (33, 34). This list demonstrates that the cause of doxorubicin-induced cardiomyopathy is probably multifactorial and the mechanism is complex. However, most of these changes have an underlying cause, which is highly likely to be damage from reactive oxygen species. Moreover, damage to either the mitochondria or the sarcoplasmic reticulum could create a favorable environment for the induction of apoptosis (28). Doxorubicin and Oxidants Heart mitochondria are thought to play an important role in mediating oxidative damage. The enzyme NADH dehydrogenase, a major player in transferring electrons to harmful oxidants interacts with doxorubicin. The quinone ring of doxorubicin, a part of its tetracyclic moiety, undergoes redox cycling between quinone and semiquinone. During this process, oxidizing agents, including oxygen, capture free generated electrons and then initiate a chain reaction that leads to the generation of superoxide anion production. The superoxide anion radical generated can undergo dismutation to hydrogen peroxide. Specifically, in the presence of redox active free metal ions, formation of the highly reactive hydroxyl radical could account for membrane damage (35). If subjected to oxidant insult, mitochondria can then be triggered to release cytochrome c and other apoptogenic factors that activate a cascade of initiator and effector caspases to induce apoptosis. Apoptosis Adult cardiomyocytes are post-mitotic cells that once destroyed, are slowly replaced. Consequently, their loss can contribute to the functional decline of the myocardium leading to heart disease (36). Until recently, the mode of cell death involved

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7 in cardiomyocyte loss was not clear and was attributed to necrosis (13). It is now believed that apoptosis is also involved in cardiomyocyte cell death, and it plays an important role (37). Necrosis is a rapid and irreversible process that occurs when cells are severely damaged. Necrosis involves swelling of the cell and its organelles, disruption of mitochondria, membrane rupture, and cell lysis (38). It is a destructive process, as release of cellular content into the surrounding environment can cause further damage or death to neighboring cells. Apoptosis, on the other hand, is a highly organized, energy dependent mechanism whereby a cell commits suicide without causing damage to surrounding tissue and it occurs normally during development, tissue turnover, and in the immune system (12). Apoptosis is characterized by cellular condensation while maintaining intra-organelle integrity, membrane blebbing, DNA fragmentation into oligoand mono-nucleosomes, destruction of the cytoskeleton, and formation of apoptotic bodies which are endocytosed by macrophages and neighboring cells (12). The apoptotic process is mediated by the activation of cysteine proteases known as caspases, which cleave each other and other proteins after an aspartate residue within a specified amino acid sequence (11). There are approximately 14 caspases identified that participate in the apoptotic process depending on the signaling pathways (11). Various stimuli such as oxidative stress (29, 39), mitochondrial dysfunction (28), elevated intracellular Ca 2+ excessive DNA damage and various cytokines can induce apoptosis, and different signaling pathways have been described in various cell types. The two most widely studied pathways in cardiac myocytes are the mitochondrial mediated and the death receptor mediated pathways.

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8 Mitochondrial Mediated Pathway Mitochondrial dysfunction is a well-known stimulus for mitochondrial-mediated apoptosis initiating the release of apoptogenic factors from the mitochondria. Release of cytochrome c, as well as other apoptogenic factors, from mitochondria to the cytosol initiates mitochondrial apoptosis (28). Cytochrome c then forms an apoptosis initiating complex with apoptosis protease-activating factor (Apaf-1), dATP and procaspase-9, resulting in the self-cleavage and activation of caspase-9 (28). The active caspase-9 then cleaves and activates procaspase-3. This, in turn, activates a cascade of caspases, internally breaking down the cell (11). Some of the targeted proteins of caspase-3 are procaspase-6 and procaspase-7, poly ADP ribose polymerase (PARP), inhibitors of DNA fragmentation factor (DFF) and caspase activated DNase (CAD) resulting in DNA fragmentation (11). The Bcl-2 family of proteins regulates the release of cytochrome c (40). These include Bax, Bad, and Bid, which are pro-apoptotic proteins that favor cytochrome c release and Bcl-2 and BclX L which are anti-apoptotic proteins that inhibit cytochrome c release (40, 41). The mechanisms of how Bcl-2 family proteins regulate release of cytochrome c and apoptogenic factors are under investigation. Some of the hypotheses include: 1) physical rupture of the outer membrane, 2) a channel formed by pro-apoptotic Bcl-2 family proteins such as Bax, and 3) opening of a pore via a membrane permeability transition pore (MPT) characterized by loss of the mitochondrial membrane potential (11, 41). The mitochondria also release other apoptogenic factors such as apoptosis inducing factor (AIF) which is a caspase-independent mitochondrial death effector (42). AIF can only be released upon MPT. These pro-apoptotic factors translocate to the nucleus where

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9 they induce DNA chromatin condensation and large-scale DNA fragmentation into approximately 180-200 kbp. Previous studies have confirmed this observation in a variety of cell types (40, 42). Death Receptor Mediated Pathway Various receptors can mediate apoptosis such as tumor necrosis factor receptor 1 TNFR1) and Fas/CD95. Ligand binding to TNFR1 or Fas can induce apoptosis in an effector cell by the activation of procaspase-8, which cleaves and activates procaspase-3 to initiate the caspase cascade (43). Alternatively, binding of ligand to TNFR1 can induce a pro-inflammatory/anti-apoptotic response mediated through the cytosolic transcription factor NF-B. The presence or recruitment of adaptor proteins to TNFR1 determines the outcome: caspase activation or NF-B activation. Thus, whether the cell chooses a survival pathway or a death pathway in response to TNFR activation depends on the interaction of various signaling pathways and regulators of these pathways (43). It has been shown that Fas antigens were overexpressed in myocytes of dilated-cardiac myopathies, chronic heart failure, and myocardial infarction (36). Nakamura et al. (44) observed apoptotic cell death via Fas-mediated pathway in adriamycin-induced cardiac myopathy rats. Regulators of Apoptosis Apoptosis is a sequential, multi step process made up of many different layers of regulation. This is of particular importance in post-mitotic cardiac myocytes in order to avoid unnecessary death of salvageable cells and to promote apoptosis in response to irreversible cellular damage, as opposed to necrosis, which could further harm the myocardium. The final steps of apoptotic death are highly conserved and likely to be

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10 mediated by a similar set of caspases. Various inhibitory regulatory mechanisms exist within cells that target caspases. These include cFLIP and the inhibitors of apoptosis proteins (IAP) family (40). These inhibitors are present in various cell types and may also play an important role in the heart (13). The known IAPs include XIAP, cIAP, and cIAP2 (40). It is believed that these IAPs bind to cleaved/activated caspases and inhibit their activity. XIAP is consired one of the most active inhibitors of capse-3. Alternatively cFLIP inhibits the activity of caspase-8 and is highly expressed in the heart under normal physiological conditions but is degraded after ischemia/reperfusion (45). Recently an inhibitor of apoptosis that is expressed almost exclusively in skeletal muscle and heart has been characterized. ARC (apoptosis repressor with caspase recruitment domain) was first shown to interact with caspase-8 and -2 and to attenuate apoptosis induced by stimulation of death receptors (46). More recently it was demonstrated that ARC inhibits cytochrome c release from mitochondria and protects against hypoxia-induced apoptosis (47). This study suggested that ARC can exert its effect at different levels in the apoptotic pathway and may be a key regulator of apoptosis in the heart. Gender Related Differences in the Cardiovascular System There are significant sex differences in the incidence of a variety of cardiovascular diseases and acute myocardial injuries (16). In recent years, several lines of evidence strongly suggest that loss of myocytes occur with heart failure, ischemia/reperfusion injury and aging. This loss of cardiac cells may occur because of both necrosis and apoptosis. This emerging concept of cardiac myocyte death by apoptosis may have important implications in terms of studying gender-based differences. It is well established that the female ovarian steroid hormone estrogen has strong cardio-protective

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11 properties (48). Possible mechanisms behind this protectection might include systemic effects such as improvement in the lipid profile, reduction in lipid peroxidation, and stimulation of endothelium dependent NO production (18). Grohe et al. (18) demonstrated through immunofluorescent assays that cardiac myocytes and fibroblasts express functional estrogen receptor proteins in both male and female rats. Biological effects of estrogen generally require the presence of estrogen receptor, a ligand dependent transcriptional factor that regulates the expression of genes transduced by estrogen. However, additional mechanisms, may exist to explain the increased tolerance of female rats to a myocardial insult (48). Gender Differences in Apoptosis There is evidence that estrogen plays an important role in modulating certain cell-death related signals to inhibit apoptosis. Using estrogen (17 estradiol) at physiological concentration has shown to inhibit apoptosis in cardiac myocytes (17). Pelzer et al.(17) studied apoptosis in cultured cardiac myocytes induced by staurosporine, a tyrosine kinase inhibitor and a potent pro-apoptotic agent. A significant reduction in apoptosis was observed in cells that were simultaneously treated with estrogen and staurosporine. 17-Estradiol has been found to reduce the activity of ICE-like protease caspase-3 an effector and downstream mediator of apoptosis. Moreover, Camper-Kirby and coworkers (22) recently reported a significant difference between sex in myocardial activation of Akt. Both localization of phospho Akt in the myocardial nuclei and cytosolic localization of phospho-forkhead, a downstream nuclear target of Akt, were found elevated in sexually mature female mice compared to that in male mice. Akt, also known as Protein Kinase B, is a serine/threonine kinase, which lies at the intersection of multiple cellular

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12 signaling pathways involved in the regulation of glucose metabolism, gene transcription, and cell survival (49). It is a down stream effector molecule for signal transduction initiated by survival factors such as IGF-1. Figure 2-1. Overview of apoptotic pathways. These factors bind to their respective cell surface receptors triggering the activation of several kinases including the PI3K. This pathway then activates Akt through phosphorylation which can subsequently inhibit apoptosis by phosphorylating Bcl-2 family member Bad and caspase-9, both of which are components of intrinsic cell death

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13 machinery and the forkhead transcription factor (FKHRL1). FKHRL1 in a dephosphorylated state translocates to the nucleus where it induces target genes such as Fas ligand and triggers apoptosis. Hence, estrogen, by bringing about Akt-dependent phosphorylation and inactivation of FKHRL1 (Figure 2-1), suppresses the transcription of death genes and promotes survival (50). Summary A great deal of effort has been expended in trying to prevent or mitigate the cardiotoxic side effect of doxorubicin. However, it is imperative that any method designed to minimize the cardiotoxic effect of doxorubicin also maintains its antineoplastic efficacy. Surprisingly, no studies have been conducted to examine if sex differences exist in oxidative stress and apoptosis following the use of this drug.

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CHAPTER 3 METHODS Animals and Experimental Design Male and female Fisher 344 rats (National Institute of Aging colony, Harlan Sprague Dawley, Indianapolis, IN) were used in this study. The animals were housed individually in a temperature (18-22C) and light-controlled environment with a 12-hour light/dark cycle and were provided with food and water ad libitum. Animals from both male and female groups were randomly assigned either to a control or to a doxorubicin-treated group. Doxorubicin hydrochloride (Sigma Chemical Co., St. Louis, MO) was dissolved in saline and administered by intraperitoneal injection at a dose of 10 mg/kg to the group receiving treatment. Male and female animals were sacrificed one day after doxorubicin injection (n= 12). In order to see if there were adaptations additional groups of male and female rats were sacrificed four days after the injection (n=12). The control group was injected with an equal volume of saline and they were sacrificed on day one (n=12) and day four (n=12). Tissue Harvesting Animals were anesthetized with an intraperitoneal injection of sodium pentobarbital (5 mg/100 g body weight). The chest was opened and blood removed directly by cardiac puncture. This was followed by severing the inferior vena cava and perfusion of the heart with 10 ml of ice-cold antioxidant buffer containing 100 M diethylenetriaminepentaacetic acid (DTPA), 1 mM butylated hydroxytoluene (BHT), 1% 14

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15 ethanol, 10 mM 3-aminotriazole, and 50 mM NaHPO 4 (pH 7.4). After perfusion, the entire heart was excised, rinsed in antioxidant buffer to remove any remaining blood, blotted dry, and weighed. Cellular Fractionation Isolation of Mitochondrial and Cytosolic Fractions. The atria of the heart was removed and stored at C until analysis. A 500mg portion of the left ventricle was used for isolation of mitochondria. Tissue was weighed and minced in 5 volumes of isolation buffer (0.225M mannitol, 0.075 M sucrose, 0.2% fatty acid free bovine serum albumin, pH 7.4). The tissue was homogenized in a Potter-Elvehjem glass homogenizer and centrifuged for 10 minutes at 700g. The resulting supernatants were centrifuged again for 10 minutes at 8,000g. The supernatant (cytosolic fraction) was aliquotted and stored at C. The mitochondrial pellet was resuspended in 5 mL of isolation buffer and centrifuged for 10 minutes at 8000g. The final mitochondrial pellet was resuspended in 1 mL of isolation buffer, aliquotted and stored for later analyses. Protein Concentration Cytosolic and mitochondrial protein concentrations was determined using the method developed by Bradford (51). Mitochondrial Functional Parameters Mitochondrial Respiratory Function To assess mitochondrial damage due to isolation procedures, we calculated the respiratory control ratio (state 3 respiration / state 4 respiration), which is commonly used as an index for mitochondrial function. Mitochondrial respiration was monitored at 37C in incubation buffer (145 mM KCL, 30 mM Hepes, 5 mM KH 2 PO 4 3 mM MgCl 2 0.1

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16 mM EGTA, 0.1 % fatty-acid free albumin, pH 7.4), 2.5 mM pyruvate, 2.5 mM malate, and 0.25 mg mitochondrial protein for a total volume of 500 l. State 4 respiration (no ADP) was read for 2 minutes and monitored using a chart recorder. State 3 respiration (with ADP) was measured in the presence of 500 M ADP for 10 minutes or until the oxygen pressure equals zero. Oxygen consumption was calculated as ng atom O 2 consumed/mg protein/minute. Respiratory measurements were completed within 3 hours after mitochondrial isolation and performed in duplicate. ATP Content and Production ATP production in isolated mitochondria was measured using a luminometer (model TD-20/20, Turner Designs, Sunnyvale, CA). The assay uses firefly luciferase, which fluoresces in proportion to the presence of ATP. Freshly isolated mitochondria were added to a cuvette containing 1 mM ADP, 1 mM pyruvate, 1 mM malate, and a Luciferin-Luciferase ATP monitoring reagent (ATP Determination Kit A-6608, Molecular Probes, Eugene, OR). A blank cuvette containing no metabolic substrate was assayed to account for nonspecific ATP production. A known ATP concentrations was used to establish a standard curve. Results were expressed as nmol ATP produced/mg protein/minute. The P/O ratio used as an index of mitochondrial efficiency was calculated by taking nmol ATP produced/mg protein/min divided by ng atoms of oxygen consumed/mg protein/min of state 3. The P/O ratio represents the number of ADP molecules phosphorylated per mole of oxygen atoms consumed. Mitochondrial Hydrogen Peroxide (H 2 O 2 ) Production The rate of mitochondrial H 2 O 2 production was measured according to Barja (52) using a fluorescent microplate reader (GeminiXS, Molecular Devices, Sunnyvale, CA).

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17 Incubation buffer (145 mM KCl, 3 mM MgCl 2 5 mM KH 2 PO 4 30 mM Hepes, 0.1 mM EGTA, 0.1% BSA, pH 7.4), mitochondria (0.25 mg/ml), horseradish peroxidase (5.7 U/ml), and homovanilic acid (0.1 mM) were added to test tubes. The reaction was initiated by the addition of pyruvate and malate (2.5 mM each) and the tubes are placed in a shaking water bath for 15 minutes at 37C. The reaction was stopped by placing tubes on ice and adding 0.5 ml of stopping solution (0.1 M glycine, 25 mM EDTA, pH 12.0). Fluorescence was determined using a fluorescent microplate reader and a standard curve was generated for each analysis using glucose-glucose oxidase. Biochemical Assays Estrogen (Estradiol) Plasma estradiol concentration was measured using radioimmunoassay (RIA) kit at Yerkes Endocrine Laboratory (Atlanta, GA). All samples and standards were measured in duplicates. Cytosolic Monoand Oligo-nucleosomes DNA fragmentation was quantified by measuring the content of cytosolic monoand oligonucleosomes (180 base pair nucleotides or multiples) using a Cell Death ELISA (Roche Molecular Biochemicals, Germany) according to instructions from the manufacturer. The assay is based on the quantitative sandwich-enzyme-immunoassayprinciple. Wells are coated with a monoclonal anti-histone antibody. Nucleosome in the sample binds to the antibody followed by the addition of anti-DNA-peroxidase, which reacts with the DNA, associated with the histones. The amount of peroxidase retained in the immunocomplexes is determined photometrically with ABTS (2.2 azino-di-[3-ethylbenzthiazoline sulfonate]) as a substrate. All samples were read in triplicates. Results were reported as arbitrary OD units/mg protein.

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18 Western Blots Analysis Proteins were separated on a 4-20% precast polyacrylamide gel (BMA, Rockland). Proteins were transferred onto a nitrocellulose membrane. Nitrocellulose membranes were blocked overnight using a blocking solution containing 0.05% Tween and 5.0% dry milk. Membranes were incubated for 90 minutes with the primary antibody at the desired dilution. Membranes were incubated for 90 minutes in anti-rabbit or mouse Ig horseradish peroxidase (Amersham Life Science) with an appropriate dilution. Blots were developed using ECL (Amersham Pharmacia Biotech, England). The protein bands were analyzed using Kodak Image Station 440CF (Eastman Kodak, Rochester, NY). Cytosolic Cytochrome C Cytosolic cytochrome c was quantified using an ELISA kit from R&D Systems (Minneapolis, MN) which employs the sandwich enzyme immunoassay technique. Data were reported as nmol/mg cytosolic protein. Caspase-3 Activity Caspase-3 activity was measured using the synthetic peptide n-acetyl-DEVD-AMC (BD PharMingen, San Diego, CA). This assay detects activated caspase-3 and, to a lesser extent, caspases -6, -7, and -8. Active caspases cleave the AMC from the peptide and the free AMC fluoresces. Standards of active caspase-3 were prepared. Briefly, 1 mL of assay buffer (20mM HEPES, 10% glycerol, 1 M DTT, and 14 L of Ac-DEVD-AMC/mL of buffer) and 50 L of sample will be added to a microcentrifuge tube and protected from the light. Samples were incubated at 37C for 60 minutes after which fluorescence will be measured on a spectrofluorometer with an excitation wavelength of 380 nm and an emission wavelength of 440 nm.

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19 Caspase-3 Content A Western blot protocol was used to detect the full length and the activated (cleaved) caspase-3. A polyclonal anti-caspase-3 antibody (Stressgen, Canada) was used. Caspase-8 A Western blot protocol was used to detect the full length and the activated (cleaved) caspase-8. A polyclonal anti-caspase-8 antibody (Steersmen, Canada) was used. Inhibitors of Apoptosis (XIAP, FLIP, ARC) Endogenous inhibitors of apoptosis were measured using Western blot. The cytosolic fraction was used to assess the content of these inhibitors. The following antibodies will be used: monoclonal XIAP (MBL, Watertown), polyclonal ARC (Ab-1) (Oncogene, Boston) and antiserum FLIP (Alexis, San Diego). Bcl-2 and Bax The content of Bax and Bcl-2 were measured using Western blots. The following antibodies were used: polyclonal Bax (Ab-1) and polyclonal Bcl-2 (Ab-4) (Santa Cruz Biotechnology). Antioxidant Enzyme Assay Glutathione Peroxidase Activity Selenium-dependent glutathione peroxidase activity was assayed according to Nakamura et al. (53) with modification, using H 2 O 2 as the substrate. Statistical Analysis Two-way ANOVA was used for comparisons between groups. Bonferroni post hoc test was performed if significance was found. A p-value of <0.05 were considered significant.

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CHAPTER 4 RESULTS Morphological Characteristics We determined if there were sex differences in body weight, heart weight, heart weight-to-body weight ratio (Table 4-1), and estradiol levels (Table 4-2). The body weights of the male rats were significantly greater than in the female rats (p <0.05). Moreover, a significant gender difference was observed in heart weight. The hearts of males were (36%) greater then female hearts (p<0001). However, the female rats had a significantly higher heart weight-to-body weight ratio (p<0.001). Furthermore, body weight was not different one day after doxorubicin treatment in male or female rats. However, there was a significant decrease (10%) in body weight four days after doxorubicin treatment in the male rats (p=0.004), but not in female rats. Furthermore, the heart weights of the male and female rats remained unaltered 1 day following doxorubicin treatment. However, in both sexes, there was a significant decrease in heart weight four days after doxorubicin treatment. The males showed a 15% decline (p<0.001) and the females showed a slightly smaller (13%) decrease (p<0.05). The heart weight-to-body weight ratio in male and female rats was not affected after 1 day or 4 days after doxorubicin treatment (Table 4-1). 20

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21 Table 4-1. Body weight, heart weight, and heart weight to body weight ratio of male and female rats treated with doxorubicin or saline. Male Female Control Dox Day 1 Dox Day 4 Control Dox Day 1 Dox Day 4 Body weight 358.8.0 357.2.0 323.9.0 197.6.0 190.6.0 176.0.0 Heart weight 0.81.07 0.78.05 0.69.04 0.52.03 0.51.05 0.45.01 HW:BW 0.0022 0.0001 0.0021 0.0008 0.0023 0.0007 0.0026 0.0001 0.0027 0.0002 0.0026 0.0009 Body weight, heart weight, and the heart to body weight ratio (HW:BW) of male (n=12) and female rats (n=12) sacrificed one day and four days after administration of 10mg/kg doxorubicin or equal volume of saline (Mean SEM). Male (n=12) and female (n=12) animals were injected with equal volume saline on day1 (n=6) and day 4 (n=6). *Significant sex difference (p<0.05). Significantly different from saline injected rats (p<0.05). Units: gram. Plasma Estrogen Levels in Male and Female Rats 17-Beta estradiol was measured in the plasma using a radioimmunoassay (RIA) method to determine the differences of this sex hormone between the male and female rats. Moreover, since estradiol has cardio-protective effects we wanted to know the variability with each group. Surprisingly, there was only a 20-30% difference between young male and female rats (p<0.05). Furthermore, the variability between the individual female rats was only approximately (20-30%), with a maximum level of estradiol of 13.8 pg/mL and the minimum level of 5.4 pg/mL. Table 4-2. Plasma 17estradiol level. Control Dox Day 1 Dox Day4 Male 5.70.6 6.01.5 6.10.7 Female 8.42.7* 8.72.4* 7.01.4* Plasma estrogen (estradiol) in male and female rats sacrificed one day and four days after injection with 10mg/kg doxorubicin vs. controls (MeanSEM). Significant sex difference (p<0.05). Units: pg/ml

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22 Mitochondrial Function We determined the effect of doxorubicin treatment on mitochondrial function in male and female rats (Table 4-3). Mitochondrial oxygen consumption was measured in state 4 (no ADP) and during state 3 respiration (0.5 mM ADP). There were no sex differences between State 4 and State 3 measurements. Moreover, doxorubicin treatment following either 1 day or 4 days did not effect State 4 or State 3 respiration. The amount of ATP produced by the mitochondria was used as a parameter to determine sex differences in energy production and as a parameter to assess mitochondrial function following doxorubicin administration. There was a significant sex difference in ATP production; the female rats produced significantly less ATP per milligram of protein of freshly isolated mitochondria determined in State 3 (p=0.0032). However, rates of ATP production in isolated mitochondria one day or four days after doxorubicin treatment were not different from those of controls. The P/O ratio, which compares the amount of ATP that is phosphorylated with the amount of oxygen consumed, also did not differ between groups. Table 4-3. The effects of doxorubicin administration on mitochondrial function. Male Female Control Dox Day 1 Dox Day 4 Control Dox Day 1 Dox Day 4 State 4 VO 2 8.401.36 3.015.10 8.492.51 9.353.04 7.204.75 8.733.14 State 3 VO 2 42.2128.61 47.5126.1 34.6224.81 31.7822.46 25.9417.18 21.086.74 ATP 31.75.81 38.08.84 33.32.47 27.81.74 26.75.37 23.42.33 P/O Ratio 1.150.37 1.060.52 1.480.80 1.340.50 0.890.32 1.180.33 State 4 and State 3; nmol oxygen consumption/mg protein/minute; ATP production; nmol ATP/mg protein/minute. Values presented are mean SEM. *Significant gender difference. (p<0.05)

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23 Hydrogen Peroxide Production in Isolated Mitochondria In order to measure the rate of reactive oxygen species production by heart mitochondria, we measured hydrogen peroxide (H2O2) formation in freshly isolated mitochondria (Figure 4-1). Interestingly, the amount of H2O2 production was significantly different between the two sexes. Male rats produced significantly higher levels of hydrogen peroxide compared to female rats ( p =0.003). Doxorubicin did not change hydrogen peroxide production in the male rats after day 1. In striking contrast, there was a significant decrease in hydrogen peroxide production after day 1 in the female rats ( p< 0.05). Males did show a significant drop ( p <0.001) in hydrogen peroxide production after day 4, but females remained at approximately the same level of hydrogen peroxide production as compared to day 1. Figure 4-1. The effect of doxorubicin admi nistration on heart mitochondrial oxidant production. Mitochondria were isolated on day 1 and day 4 after the injection of doxorubicin 10mg/mL) or equal volume of saline in control animals. Values presented are mean SEM. Significantly different from the control group (* p <0.001) and (** p <0.05). Significant gender difference ( p <0.05). **

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24 Glutathione Peroxidase Activity To assess adaptations to mitochondrial hydrogen peroxide production, we determined mitochondrial glutathione peroxidase (GPX) activity (Figure 4-2). Male rats had significantly higher level of GPX activity compared to female rats (p=0.001). There was no significant difference in GPX activity detected after doxorubicin treatment on any of the days following doxorubicin treatment. Figure 4-2. The effect of doxorubicin administration on heart mitochondrial glutathione peroxidase activity (GPX). Values presented are SEM. Significant gender difference (p<0.05). Apoptosis Determined by Mono and Oligo-nucleosomes To assess the overall incidence of apoptotic cell death we determined the levels of monoand oligo-nucleosome contents in the isolated cytosolic fraction of the heart. Apoptosis is characterized by the formation of monoand oligo-nucleosome in the nucleus. These DNA fragments are transported to the cytosol for degradation. Levels of monoand oligo-nucleosome were similar in the male and female control animals.

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25 However, male rat hearts contained greater levels of monoand oligo-nucleosome content one day after doxorubicin treatment (p<0.01) and levels returned back to those seen in control animals after day 4. In contrast, female rats showed no increases in the levels of monoand oligo-nucleosome after one day or four days after doxorubicin administration, suggesting that female hearts were more resistant to apoptosis (Figure 4-3). Figure 4-3. The effect of doxorubicin administration (10mg/kg) on the content of monoand oligo-nucleosomes in the heart cytosol. Values presented are SEM. Significantly different from the control group (p<0.05). Caspase-3 Activity and Caspase-3 Content Caspase-3 is considered a central caspase of apoptosis, because most caspases are able to activate this caspase to initiate apoptosis. There were no differences in caspase-3 proteolytic enzyme activity between male and female control rats and 1 day after doxorubicin administration. However, four days after the administration of doxorubicin there were significant increases in both the male and female rats (p<0.01) (Figure 4-4).

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26 Figure 4-4. The effect of doxorubicin administration (10mg/kg) on caspase-3 activity. Caspase-3 activity measured using fluorescence ELISA method. Values presented are mean SEM. Significantly different from the control group (*p<0.05) and (**p<0.05) To determine if the rate of synthesis of caspase-3 was altered we measured the protein levels of the zymogen or pro-caspase-3 and the cleaved caspase-3 content (Figure 5). The total levels of the zymogen form of caspase-3 remained unaltered (Figure 4-5A), suggesting that the absolute protein levels of this caspase were not altered during the experimental phase in any of the groups. However, the content of the cleaved active caspase-3 was significantly increased after day four in both the male and female rats (Figure 4-5B).

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27 Figure 4-5. The effect of doxorubicin administration on the caspase-3 content (A) and cleaved caspase-3 concentration (B) determined by Western blot analysis. Values presented are mean SEM. Significantly different from the control group (*p<0.05) and (**p<0.05). We determined if gender differences and changes to doxorubicin treatment existed in the levels of caspase inhibitor, X-linked inhibitor apoptotic protein (XIAP) a repressor of caspase-3 activity (Figure 4-6). We found no differences due to sex or doxorubicin treatment in any of the groups.

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28 Figure 4-6. The effect of doxorubicin admini stration (10mg/kg) on the amount of antiapototic proteins XIAP measured in cytosolic fraction using Western method. Values presented are SEM. Mitochondrial Mediated Pathway of Apoptosis Apoptosis in cardiac myocytes is often asso ciated with the rele ase of cytochrome c from the mitochondria (Figure 4-7). Female co ntrol rats had significantly lower cytosolic cytochrome c levels compared to male rats ( p <0.05). The dose of 10mg/mL of doxorubicin did not result in increase d levels of cytosolic cytochrome c determined after 1 day or 4 days of doxorubicin treatment. Figure 4-7. The effect of doxorubicin ad ministration (10mg/kg) on cytochrome c concentration in the cytosol. Values presented are SEM. Significant gender difference ( p <0.05).

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29 Mitochondrial Regulators of Cytochrome C Release The levels of Bcl-2, an anti-apoptotic protein, and Bax, a pro-apoptotic protein were determined in isolated cardiac mitochondria (Figure 8). Figure 4-8. The effect of doxorubicin administration (10mg/kg) on the content of Bcl-2 (A) and Bax (B) measured in the cardiac mitochondria using Western Blot analysis. Values presented are SEM. Significantly different from control animals (*p<0.05).

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30 There were no differences in the levels of these proteins due to gender. However, there was an increase in the levels of Bcl-2 four days after doxorubicin treatment in the male and female, but these changes were only statistically significant in the male animals (Figure 8A). The levels of Bax were not significantly different between gender and doxorubicin had no significant effect on this pro-apoptotic protein (Figure 8B). Receptor Mediated Pathway of Apoptosis To investigate the possibility if receptor mediated cell death was involved following doxorubicin administration we determined the content of capase-8 (Figure 4-9 A&B). Pro-caspase-8 content was not different between male and female rats (Figure 4-9 A). Moreover, doxorubicin treatment did not result in a significant change in the inactive procaspase-8 concentration, suggesting no increase in the synthesis rate of this pro-caspase during the 4-day experiment phase. Moreover, the cleaved caspase-8 did not differ significantly between sexes (Figure 4-9B). Furthermore, doxorubicin treated rats did not show a statistically significant change in the levels of the cleaved form. We also measured cFLIP, which is a potent inhibitor of caspase-8 activation and found no differences in cFLIP due to gender or doxorubicin treatment (Figure 4-10).

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31 Figure 4-9 The effect of doxorubicin admi nistration (10mg/kg) on the content (A) Procaspase-8 and (B) cleaved caspase-8. Values presented are mean SEM.

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32 Figure 4-10. The effect of doxorubicin administration (10mg/kg) on the content of cFLIP measured in cytosolic fraction using Western blot method. Values presented are mean SEM.

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CHAPTER 5 DISCUSSION Overview of Principle Findings These are the first experiments to examine if gender differences exist in cardiotoxicity induced by doxorubicin as determined by mitochondrial function, oxidative stress, and apoptosis. No significant differences were observed in mitochondrial function following doxorubicin treatment in any of the groups. However, our data shows that female rats produce lower levels of hydrogen peroxide one day after doxorubicin treatment compared to male rats. Moreover, the apoptotic index determined by the levels of monoand oligo-nucleosomes was significantly increased in male rats, but not in female rats 1 day after doxorubicin treatment. In addition, caspase-3 activity and cleaved caspase-3 content were increased after 4 days in male rats as well as in female rats. Sex differences in mitochondrial oxidant production immediately after doxorubicin could have influenced the release of pro-apoptotic factors, such as cytochrome c, AIF, or Endonuclease G (54) and may explain the sex differences observed in DNA fragmentation pattern. However, levels of cytosolic cytochrome c remained unaffected in both the male and female rats, which could be explained by the increase of Bcl-2 content in the isolated mitochondria. Furthermore, these data show that the receptor mediated pathway is unlikely the major cause for the increased activation of caspase-3 in vivo. 33

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34 Body Weight and Heart Weight Doxorubicin administration caused a significant reduction in body mass in male rats, but not in female rats. Furthermore, four days after doxorubicin treatment the animals heart weights of both the male and female rats were significantly lower compared to those injected with saline. The loss in heart weight was probably due to 1) increased levels of apoptosis and 2) an inhibition of protein synthesis by doxorubicin (55). Other factors, such as dehydration and food consumption could have played a role in the loss of heart weight. Heart weight-to-body weight ratio was significantly higher in female rats, which could have reduced the absolute toxic effect of the drug (10 mg of doxorubicin/kg of body weight) on the female rats. However, drug metabolism and fat-to-lean-body mass ratio may have also played a role in the cardiotoxic effect of doxorubicin. Oxidant Production and Antioxidant Enzymes Female rats had significantly lower hydrogen peroxide (H 2 O 2 ) production compared to the male group, which supported our initial hypothesis. Moreover, in response to doxorubicin, male and female rats showed a different rate of oxidant production. Hydrogen peroxide (H 2 O 2 ) production was decreased one day after doxorubicin administration in females. A possible explanation for this observation might be that mitochondria could adapt to cytotoxic stress by enhanced electron coupling and a reduction in the free radical leak. In addition, it remains possible that mitochondrial biogenesis was significantly increased after doxorubicin treatment and that newly synthesized mitochondria adapted and produced less hydrogen peroxide. In response to the reduction in mitochondrial reactive oxygen species in the control female rats, we found a similar decrease in mitochondrial glutathione peroxidase (GPX)

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35 activity. Acute doxorubicin administration did not alter GPX activity in either male or female rats suggesting that GPX activity was not up regulated within the 1 or 4 day period after the oxidative stress insult. This result agrees with previous study by Li et al. (55) and recent data from our laboratory (29), which show that a single dose (20mg/Kg) of doxorubicin did not have any influence on the activity of GPX four days after administration. Apoptosis Induced by Doxorubicin The overall incidence of apoptosis in control animals measured by DNA fragmentation did not differ between sexes. However, in response to doxorubicin administration, male rats showed an increased concentration of cytosolic monoand oligonucleosomes on day 1 only, whereas in female rats, no change was seen in DNA fragmentation after day 1 and day 4. This data suggests that female rats might have been able to prevent the loss in heart cells due to adriamycin-induced cardiotoxicity. Surprisingly, the key effector caspase-3, showed significant increase in activity and content after four days of doxorubicin treatment in both male and female rats. Hence, the caspase-3 data does not correlate with the levels of apoptosis detected by the concentrations of monoand oligo-nucleosomes. Furthermore, we did not detect any changes in XIAP content due to gender or doxorubicin treatment to add an explanation to this finding. Therefore, we further investigated if the mitochondrial-mediated pathway of apoptosis was responsible for the differences seen in apoptosis and caspase-3 activation. Mitochondrial Mediated Pathway of Apoptosis To confirm which pathway may have been responsible for the activation of caspase-3 we examined the mitochondrial-mediated pathway. Cytosolic levels of cytochrome c in male and female rats showed a very similar response and therefore this

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36 pro-apoptotic protein does not appear to be responsible for the increased DNA fragmentation seen in the male rats. However, it remains possible that changes in cytochrome c levels occurred immediately after doxorubicin treatment and that the effects of apoptosis, such as caspase-9 activation or AIF release occurred before day 1. Moreover, it appeared that the mitochondrial anti-apoptotic protein, Bcl-2 was significantly increased in response to doxorubicin administration in both male and female animals four days after treatment. Hence, it is possible that other adaptive response might have occurred during the four-day experimental period. For example, other inhibitory proteins such as cytosolic ARC could have been sequestered by the mitochondria and affected the release of cytochrome c, AIF, and endonuclease G (54) differently. ARC is known to inhibit cytochome c release from mitochondria and protects against hypoxiainduced apoptosis in heart (56). Therefore, up-regulation of this protein could have altered the release of cytochrome c from mitochondria. Receptor-Mediated Pathway of Apoptosis We investigated if the receptor-mediated pathway was affected by doxorubicin treatment and if gender differences were present. Others have shown that Fas-mediated activation of caspse-8 in vitro with doxorubicin treatment causes apoptosis (44). Although there were gradual increases in the levels of cleaved caspase-8 after day one and day four, these changes were not statistically significant. Therefore, from the changes observed in this study, using an in vivo model, it appears that this pathway was not a major player in the activation of caspase-3. Furthermore, no significant changes were detected in the caspase-8 inhibitor cFLIP due to gender or doxorubicin treatment. Hence, it remains possible that the dose injected in these rats (10mg/kg) did not induce a

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37 sufficient stress response to significantly activate this pathway or this pathway does not play an essential role in inducing apoptosis. Mitochondrial Function and Doxorubicin Treatment Mitochondrial function and ATP production remained unaltered after acute doxorubicin treatment. This is in strong agreement with the current literature which suggest that mitochondrial DNA damage and damage to respiratory complexes needs to be excessive (exceed ~50% of its function) before significant decreases in ATP production are observed (57). Rossignal et al. (57) and several others (58, 59) have shown that it was possible to inhibit the activity of a respiratory chain complex (~30-50%), up to a critical level, without affecting the rate of mitochondrial respiration and ATP synthesis (57). The mitochondria functional data in our present study supports this hypothesis. Limitations in Present Study One of the potential limitations to this study is that we did not control for the estrous cycle. Although, there were only moderate fluctuations (20-30% change in estrogen between males and females and within each group), the variability in estrous cycle could have been a confounding variable. Levels of estrogen found in the plasma were in pg/mL level, which might be too low to have any significant effect on attenuating oxidative stress compared to lipid and water soluble antioxidant found in plasma (60). However, most studies suggest that estrogens protective effects may stem from binding to cell surface receptors and up-regulating a variety of cellular proteins, such as NOS (18, 61) and heat shock proteins (18, 62). Moreover, most studies show an anti-apoptotic effect of estrogen in cell culture models which might have very little relevance in a complex biological in vivo model, such as that used in this study (17, 18). In addition,

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38 estradiol levels of Fischer 344 rats are at their highest levels around the age of 9 to 12-months (63). Our rats were 6 months of age and might not have been mature enough to receive the full protection of estrogen. Therefore, the levels of estrogen in this study may not have been sufficient to be cardioprotective and it would be worthwhile to investigate rats with ovariectomy and to see if supplementing estradiol may have an effect on apoptotic signaling pathways in vivo. Conclusion and Future Direction To our knowledge, these were the first experiments that look at the effects of gender and doxorubicin in relation to oxidative stress and cell death. The principle findings from this study include: 1) there are gender differences in doxorubicin-induced oxidant production and apoptosis; 2) mitochondrial pathways may have been involved, but the rapid up-regulation of Bcl-2 may have prevented significant cell death by this pathway and 3) receptor mediated cell death appears to play a minor role, since no adaptations in cFLIP and significant increases in caspase-8 were observed following doxorubicin treatment. Other pathways, such as caspase-12, an endoplasmic reticulum-mediated pathway might be also partly responsible for the apoptosis observed in the male rats. The activation of the endoplasmic reticulum-mediated pathway causes the release of calcium and the activation of caspase-12. Indeed, numerous studies have reported that doxorubicin can alter Ca 2+ homeostasis (2, 7, 64). Therefore, more research is needed to further elucidate other possible mechanisms and to determine the rate of mitochondrial biogenesis, mitochondrial proteolytic degradation; and activation of autophagy through lysosomal pathways following doxorubicin-induced cardiotoxicity. A better understanding of gender difference in doxorubicin-induced proand anti-apoptotic

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39 signaling pathways in cancerous and non-cancerous cells may lead to new and improved therapeutic protocols for mitigating the toxic side effects of doxorubicin.

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LIST OF REFERENCES 1. Singal, P.K., and Iliskovic, N. 1998. Doxorubicin-induced cardiomyopathy N Engl J Med 339:900-905. 2. Singal, P.K., Li, T., Kumar, D., Danelisen, I., and Iliskovic, N. 2000. Adriamycin-induced heart failure: mechanism and modulation. Mol Cell Biochem 207:77-86. 3. Raju, J., Coralie, P., and Hung-Yi, W. 1997. Molecular mechanisms of doxrubicin-induced cardiomyopathy: Selective suppression of Reiske iron-sulfer protein, ADP/ATP translocate, and phopho-fructokinase gene is associated with ATP depletion in rat cardiomyocyte. J Biol Chem 272:5828-5832. 4. Yen, H.C., Oberley, T.D., Vichitbandha, S., Ho, Y.S., and St Clair, D.K. 1996. The protective role of manganese superoxide dismutase against adriamycin-induced acute cardiac toxicity in transgenic mice [published erratum appears in J Clin Invest 1997 Mar 1;99(5):1141]. J Clin Invest 98:1253-1260. 5. Kang, Y.J., Chen, Y., Yu, A., Voss-McCowan, M., and Epstein, P.N. 1997. Overexpression of metallothionein in the heart of transgenic mice suppresses doxorubicin cardiotoxicity. J Clin Invest 100:1501-1506. 6. Nohl, H., Gille, L., and Staniek, K. 1998. The exogenous NADH dehydrogenase of heart mitochondria is the key enzyme responsible for selective cardiotoxicity of anthracyclines. Z Naturforsch [C] 53:279-285. 7. Kalyanaraman, B., Joseph, J., Kalivendi, S., Wang, S., Konorev, E., and Kotamraju, S. 2002. Doxorubicin-induced apoptosis: Implications in cardiotoxicity. Molecular and Cellular Biochemistry 234/235:119-124. 8. Steller, H. 1995. Mechanisms and genes of cellular suicide. Science 267:1445-1449. 9. Vaux, D.L., Haecker, G., and Strasser, A. 1994. An evolutionary perspective on apoptosis. Cell 76:777-779. 10. Kerr, J.F., Wyllie, A.H., and Currie, A.R. 1972. Apoptosis: A basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26:239-257. 40

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41 11. Green, D., and Kroemer, G. 1998. The central executioners of apoptosis: Caspases or mitochondria? Trends Cell Biol 8:267-271. 12. Duke, R.C., Ojcius, D.M., and Young, J.D. 1996. Cell suicide in health and disease. Sci Am 275:80-87. 13. Gill, C., Mestril, R., and Samali, A. 2002. Losing Heart: The role of apoptosis in heart disease-a novel therapeutic target? FASEB 20:135-146. 14. Mallat, Z. 2001. Age and gender effects on cardiomyocyte apoptosis in the human heart. Journal of Gerontolgy: MEDICAL SCIENCES 56A:M719-M723. 15. Babiker, F.A., De Windt, L.J., van Eickels, M., Grohe, C., Meyer, R., and Doevendans, P.A. 2002. Estrogenic hormone action in the heart: Regulatory network and function. Cardio Res 53:709-719. 16. Hayward, C.S., Kelly, R.P., and Collins, P. 2000. The role of gender, the menopause and hormone replacement on cardiovascular function. Cardio Res 46:28-49. 17. Pelzer, T., Schumann, M., Neumann, M., deJager, T., Stimpel, M., Serfling, E., and Neyses, L. 2000. 17 -Estradiol prevents programmed cell death in cardiac myocytes. Biochemical and Biophysical Research Communications 268:192-200. 18. Grohe, C., Meyer, R., and Vetter, H. 2002. Estrogen and the prevention of cardiac apoptosis. In Apoptosis methods in pharmacology and toxicology : Approaches to measurement and quantification. M.A. Davis, editor. Totowa, N.J.: Humana. 19. Leri, A., Malhotra, A., Liew C., Kajstura, J., and Anversa, P.. 2000. Telomerase Activity in Rat Cardiac Myocytes is Age and Gender Dependent. J Mol Cell Cardiol 32:385-390. 20. Leri, A., Malhotra, A., Liew, C.C., Kajstura, J., and Anversa, P. 2000. Telomerase Activity in Rat Cardiac Myocytes is Age and Gender Dependent. J Moll Cell Cardiol 32:385-390. 21. Wu, W., Lee, W.L., Wu, Y.Y., Chen, D., Liu, T.J., Jang, A., Sharma, P.M., and Wang, P.H. 2000. Expression of constitutively active phosphatidylinositol3-kinase inhibits activation of caspase-3 and apoptosis in cardiac muscle cells. J Biol Chem 275:40113-40119. 22. Camper-Kirby, D., Welch, S., Walker, A., Shiraishi, I., Setchell, K.D., Schaefer, E., Kajstura, J., Anversa, P., and Sussman, M.A. 2001. Myocardial Akt activation and gender: Increased nuclear activity in females versus males. Circ Res 88:1020-1027.

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42 23. Shan, K., Lincoff, A.M., and Young, J.B. 1996. Anthracycline-induced cardiotoxicity Ann Intern Med 125:47-58. 24. Mott, M.G. 1997. Anthracycline cardiotoxicity and its prevention. Ann N Y Acad Sci 824:221-228. 25. Bonadonna, G., Monfardin, S., and Beretta, G. 1970. Phase I and Preliminary phase II evaluation of adriamycin. Cancer Research 30:2572-2582. 26. Arena, E., and Gerbasi, F. 1974. DNA, RNA and protein synthesis in heart, liver, and brain of mice treated with daunorubicin and adriamycin. Int Res Commun Systemic Med Sci 2:1053-1061. 27. Tong, J., Ganguly, P.K., and Singal, P.K. 1991. Myocardial adrenergic changes at two stages of heart failure due to adriamycin treatment in rats. Am J Physiol 260:H909-H916. 28. Green, P., and Leeuwenburgh, C. 2002. Mitochondrial dysfunction is an early indicator of doxorubicin-induced apoptosis. Biochimica et Biophysica Acta 1588:94-101. 29. Childs, A., Phaneuf, S., Dirks, A., Phillips, T., and Leeuwenburgh, C. 2002. Doxorubicin treatment in vivo causes cytochrome c release and cardiomyocyte apoptosis, as well as increased mitochondrial efficiency, superoxide dismutase activity and Bcl-2:Bax ratio. Cancer Research 62:4592-4598. 30. Myers, C.E., McGuire, W.P., Liss, R.H., Ifrim, I., Grotzinger, K., and Young, R.C. 1977. Adriamycin: The role of lipid peroxidation in cardiac toxicity and tumor response. Science 197:165-167. 31. Zhang, J., Clarke, J., Herman, E., and Ferrans, V. 1996. Doxorubicin-induced apoptosis in spontaneous hypertensive rats: Differential effects in heart, liver, and intestine, and inhibition by ICRF-187. J Moll Cell Cardiol 28:1931-1943. 32. Arola, O.J., Saraste, A., Pulkki, K., Kallajoki, M., Parvinen, M., and Voipio-Pulkki, L.M. 2000. Acute doxorubicin cardiotoxicity involves cardiomyocyte apoptosis. Cancer Res 60:1789-1792. 33. Jaenke, R.S. 1974. Anthracycline antibiotic induced cardiomyopathy in rabbits. Lab Investigations 30:292-304. 34. Lewis, G., and Gonzales, B. 1986. Anthracyline effects on actin in rat mycardial cell. Lab Investigations 54. 35. Nohl, H. 1988. Identification of the site of adriamycin-activation in the heart cell. Biochem Pharmacol 37:2633-2637.

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43 36. Olivetti, G., Abbi, R., and Quaini, F. 1997. Apoptosis in the failing human heart. N Engl J Med 336:1131-1141. 37. James, T.N. 1999. Apoptosis in cardiac disease. Am. J. Med. 107:606-620. 38. Searle, J., Kerr, J.F., and Bishop, C.J. 1982. Necrosis and apoptosis: Distinct modes of cell death and fundamentally different significance. Pathol. Ann 17:229-259. 39. Von Harsdorf, R., Li, P.F., and Dietz, R. 1999. Signaling pathway in ROS-induced Cardiomyocyte Apoptosis. Circulation 99:2934-2941. 40. Green, D.R. 2000. Apoptotic Pathways: Paper wraps stone blunts scissors. Cell 102:1-4. 41. Reed, J.C. 1997. Bcl-2 family proteins: Regulators of apoptosis and chemoresistance in hematologic malignancies. Semin Hematol 34:9-19. 42. Penniger, J., and Kroemer, G. 2003. Mitochondria, AIF and caspasesrivaling for death execution. Nature Cell Biology 5:97-99. 43. Sun, X., MacFarlane, M., Zhuang, J., Wolf, B., Green, D.R., and Cohen, G.M. 1999. Distinct caspase cascades are initiated in receptor mediated and chemical induced apoptosis. J Biol Chem 274:5053-5060. 44. Nakamura, T., Ueda, Y., Juan, Y., Katsuda, S., Takahashi, H., and Koh, E. 2000. Fas-mediated apoptosis in adriamycin-induced cardiomyopathy in rats: In vivo study. Circulation 102:572-578. 45. Rasper, D.M., and Nicholson, D.M. 1998. Cell death attentuation by 'Usurpin' a mammalian DED-caspase homologue that precludes caspase-8 recruitment and activation by the CD-95(Fas, APO-1) receptor complex. Cell Death Differ 5:271-288. 46. Koseki, T., Inohara, N., Chen, S., and Nunez, G. 1998. ARC, inhibitor of apoptosis expressed in skeletal muscle and heart that interacts selectivity with caspases. Proc. Natl Acad Sci USA 95:5156-5160. 47. Ekhterae, D., Lin, Z., Lundberg, M.S., Crow, M.T., Brosius, F.C., and Nunez, G. 1999. ARC inhibits cytochrome c release from mitochondria and protects against hypoxia-induced apoptosis in herat derived H9c2 cells. Circulation Research 85:70-77. 48. Barp, J., Araujo, A.S.R., Fernandes, T.R.G., Rigatto, K.V., Llesuy, S., Bello-Klein, A., and Singal, P.K. 2002. Myocardial antioxidant and oxidative stress

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44 changes due to sex hormones. Brazilian Journal of Medical and Biological Research 35:1075-1081. 49. Kandel, E.S., and Hay, N. 1999. The regulation and the activities of the multifunctional serine/theronine kinase Akt/PKB. Exp Cell Res. 253:210-229. 50. Sugden, P., and Clerk, A. 2001. Akt like a women : Gender differences in susceptibility to cardiovascular disease. Circulation Research 88:957-977. 51. Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254. 52. Barja, G., and Herrero, A. 2000. Oxidative damage to mitochondrial DNA is inversely related to maximum life span in the heart and brain of mammals. Faseb J 14:312-318. 53. W. Nakamura, S.H.a.K.H. 1974. Purification and properties of rat liver glutathione peroxidase. Biochimica et Biophysica Acta 358:251-261. 54. Li, L.Y., Luo, X., and Wang, X. 2001. Endonuclease G is an apoptotic DNAse when released from mitochondria. Nature 412:95-99. 55. Li, T., Danelisen, I., Bello-Klein, A., and Singal, P.K. 2000. Effects of probucol on changes of antioxidant enzymes in adriamycininduced cardiomyopathy in rats. Cardiovasc Res 46:523-530. 56. Ekhterae, D., Lin, D., Lundberg, M.S., Crow, M.T., and Brosius, F.C. 1999. ARC inhibits cytochrome c release from mitochondria and protects against hypoxia-induced apoptosis in heart-derived H9c2 cells. Circulation Research 85:70-77. 57. Rossignol, R., Fauston, B., Rocher, C., Malgat, M., Mazat, J., and Letellier, T. 2003. Mitochondrial Threshold Effect. Biochem. J. 370:751-762. 58. Davey, G.P., Canevari, L., and Clark, J.B. 1997. Threshold effect in synaptosomal and nonsynaptosomal mitochondria from hippocampal CA1 and paramedian neocortex brain region. Neurochem 69:2564-2570. 59. Davey, G.P., Peuchen, S., and Clark, J.B. 1998. Energy threshold in brain mitochondria. J Biol Chem 273:12753-12757. 60. McHugh, N.A., Merrill, G.F., and Powell, S.R. 1998. Estrogen diminishes postischemic hydroxyl radical production. Am J Physiol (Heart Circ. Physiol. ) 274:H1950-H1954.

PAGE 55

45 61. Haynes, M.P., Sinha, D., Russell, K.S., Collinge, M., Fulton, D., Sessa, W.C., and Bender, J.R. 2000. Membrane Estrogen Receptor Engagement Activates eNOS via PI3K-Akt pathway in human endothelial cells. Circulation Research 87:677-682. 62. Li, C.Y., Lee, J.S., Ko, Y.G., Kim, J.I., and Seo, J.S. 2000. Heat shock protein 70 inhibits apoptosis downstream of cytochrome c and upstream of caspase-3 activation. J Biol Chem 275:25665-25671. 63. Kacew, S., Ruben, Z., and McConnell, R.F. 2000. Strain as a determinant factor in the differential responsiveness of rats to chemicals. CEJOEM 6:235-256. 64. Burke BE, Olson RD, Cusack BJ, Gambliel HA, and WH., D. 2003. Anthracycline cardiotoxicity in transgenic mice overexpressing SR Ca2+-ATPase. Biochem Biophys Res Commun. 303:504-507.

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BIOGRAPHICAL SKETCH Youngmok C. Jang was born in Seoul, South Korea, where he lived until moving to New York, in 1984. Youngmok returned to Korea in 1988 where he graduated from Korea University, Seoul, Korea, with a Bachelor of Science degree in physical education. In August 2001, Youngmok began his graduate studies in exercise physiology at the University of Florida where he taught for Human Physiology Laboratory and also worked as a research assistant under Dr. Christiaan Leeuwenburgh. After earning a Master of Science in Exercise and Sport Sciences degree, Youngmok plans to pursue his doctoral degree in the same field at the University of Florida. 46


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EFFECT OF DOXORUBICIN-INDUCED APOPTOSIS ON GENDER


By

YOUNGMOK C. JANG













A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE IN EXERCISE AND SPORT SCIENCES

UNIVERSITY OF FLORIDA


2003

































Copyright 2003

by

Youngmok C. Jang















This thesis is dedicated to:






Eun-ah Lee, for her love and support. I'm extremely lucky to have her in my life.















ACKNOWLEDGMENTS

Many individuals contributed a great deal in helping to complete this project. First,

I would like to thank my committee chair and mentor, Dr. Christiaan Leeuwenburgh.

The guidance, support, and patience Dr. Leeuwenburgh has given me during this project

are very much appreciated. Dr. Leeuwenburgh has shown me what it takes to be

successful as a scientist and also in life. I will always be grateful.

I would also like to thank the other members of my committee, Dr. Scott Powers

and Dr. Stephen Dodd, for their input and expertise on this project.

I would also like to thank my colleagues in the Biochemistry of Aging Laboratory,

for their help and friendship. Special thanks go to Dr. Barry Drew, Dr. Suma Kendaiah

and Tracey Phillips, for their help in collecting data.

Finally, I would like to extend my gratitude to my family for their love,

encouragement, and support throughout my life. Specifically, my parents, my brother

Youngmin, my mother-in-law, sister-in-law Eun-Sun, Eun-Sook, and Eun-Young all

deserve my deepest appreciation.
















TABLE OF CONTENTS
Page

A C K N O W L E D G M E N T S ................................................................................................. iv

LIST O F TA B LE S ......... ............... ......... ..................... .. .............. .. vii

L IST O F F IG U R E S ......... ............................................................... ..... viii

CHAPTER

1 IN TR OD U CTION ............................................... .. ......................... ..

In tro d u ctio n .................................................................................. 1
Specific A im s........................................................ 4

2 REVIEW OF LITERATURE ......................................................... .............. 5

D oxorubicin and Cardiotoxicity ............................................................................5
D oxorubicin and O xidants .......................................................................... ...... 6
A poptosis ................................................................... . 6
M itochondrial M ediated Pathw ay ........................................ ...... ............... 8
D death R eceptor M edited Pathw ay ............................................ .....................9
R regulators of A poptosis ...................... ................................. .......... ...............9
Gender Related Differences in the Cardiovascular System......................................10
G ender D differences in A poptosis............................................. .............. ............... 11
S u m m a ry ................................ .....................................................1 3

3 M E TH O D S ..................................................................................... .. ................. 14

Animals and Experimental Design ............. ................... ..................... 14
Tissue Harvesting .................................... .. ......... ............... 14
Cellular Fractionation ............. .................................... ........................ 15
Protein Concentration .......... .. .............................. ........ .. ..................... 15
M itochondrial Functional Param eters...................................... ........ ............... 15
M mitochondrial Respiratory Function ....................................... ............... 15
A TP Content and Production ........................................................................16
Mitochondrial Hydrogen Peroxide (H202) Production ......................................16
B iochem ical A ssay s............ ............................................................ .......... ....... 17
E estrogen (E stradiol) ................................................. .................... 17
Cytosolic Mono- and Oligo-nucleosomes................................................17
W western B lots A analysis ............................................... ............................ 18


v









Cytosolic Cytochrom e C .................................. .....................................18
Caspase-3 A activity ....... .. .............. .. .. ................... .. ...... 18
Caspase-3 Content ....... ........ .. ........... .......... ...... ........ .. 19
C a sp a se -8 ............................................................................................................. 1 9
Inhibitors of Apoptosis (XIAP, FLIP, ARC).....................................................19
B c l-2 a n d B ax ................................................................................................ 1 9
A ntioxidant Enzym e A ssay ............................................... ............................. 19
Statistical A nalysis................................................... 19

4 R E S U L T S .............................................................................2 0

M orphological Characteristics............................................ ............................ 20
Plasma Estrogen Levels in Male and Female Rats................. ............................21
M mitochondrial Function ................................... .... ........................................... 22
Hydrogen Peroxide Production in Isolated Mitochondria........................................23
G lutathione Peroxidase A activity ...................................................... .... ........... 24
Apoptosis Determined by Mono and Oligo-nucleosomes........................................24
Caspase-3 Activity and Caspase-3 Content......................................................25
Mitochondrial Mediated Pathway of Apoptosis .....................................................28
Mitochondrial Regulators of Cytochrome C Release.............................................29
Receptor Mediated Pathway of Apoptosis ...................................... ...............30

5 DISCUSSION ............... ............. ........ ..........33

Overview of Principle Findings.... ... ...............................................................33
Body W eight and H eart W eight ........................................ ........................... 34
Oxidant Production and Antioxidant Enzymes .................................. ...............34
Apoptosis Induced by Doxorubicin...... ..............................................35
Mitochondrial Mediated Pathway of Apoptosis .....................................................35
Receptor-Mediated Pathway of Apoptosis.............................................................36
Mitochondrial Function and Doxorubicin Treatment............... ....... ............... 37
L im stations in Present Study......... ................. .................................. ............... 37
Conclusion and Future D irection........................................... .......... ............... 38

LIST OF REFEREN CES ................................................................... ............... 40

BIO GRAPH ICAL SK ETCH .................................................. ............................... 46
















LIST OF TABLES


Table p

4-1 Body weight, heart weight, and heart weight to body weight ratio of male and
female rats treated with doxorubicin or saline. .............................. ......... ...... .21

4-2 Plasm a 17-P estradiol level. .......................................................... .....................2 1

4-3 The effects of doxorubicin administration on mitochondrial function. ..................22















LIST OF FIGURES


Figure pge

1-1 Doxorubicin-induced mitochondrial damage and apoptosis ....................................2

2-1 Overview of apoptotic pathways ................................................................. ....... 12

4-1 The effect of doxorubicin administration on heart mitochondrial oxidant
production..................................... ................................. ......... 23

4-2 The effect of doxorubicin administration on heart mitochondrial glutathione
peroxidase activity (G PX ) ............................................... ............................. 24

4-3 The effect of doxorubicin administration (10mg/kg) on the content of mono- and
oligo-nucleosomes in the heart cytosol. ...................................... ............... 25

4-4 The effect of doxorubicin administration (10mg/kg) on caspase-3 activity. ...........26

4-5 The effect of doxorubicin administration on the caspase-3 content (A) and cleaved
caspase-3 concentration (B) determined by Western blot analysis.......................27

4-6 The effect of doxorubicin administration (10mg/kg) on the amount of anti-apototic
proteins XIAP measured in cytosolic fraction using Western method. ..................28

4-7 The effect of doxorubicin administration (10mg/kg) on cytochrome c
concentration in the cytosol........................................................... ............... 28

4-8 The effect of doxorubicin administration (10mg/kg) on the content of Bcl-2 (A)
and Bax (B) measured in the cardiac mitochondria using Western Blot analysis. ..29

4-9 The effect of doxorubicin administration (10mg/kg) on the content
(A) Procaspase-8 and (B) cleaved caspase-8. .................................. .................31

4-10 The effect of doxorubicin administration (10mg/kg) on the content of cFLIP
measured in cytosolic fraction using Western blot method. .............. ................32















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science in Exercise and Sport Sciences

EFFECT OF DOXORUBICIN-INDUCED APOPTOSIS ON GENDER

By

Youngmok C. Jang

August 2003

Chair: Christiaan Leeuwenbugh
Major Department: Exercise and Sport Sciences

Doxorubicin is a powerful anthracycline antibiotic used to treat a multitude of

human neoplasms. However, doxorubicin causes severe cardiac toxicity, which

compromises its clinical usefulness. Females are believed to be better protected against

cardiovascular insults. This study examined the gender differences in doxorubicin-

induced apoptosis. We administered doxorubicin at clinical levels (10mg/kg of body

weight) to male and female rats. After one day and four days later, we measured the

oxidant production and examined different apoptotic pathways. Females produced less

oxidants in isolated mitochondria compared to males and were able to scavenge oxidant

faster than male rats.

We assessed the apoptotic index by measuring DNA fragmentation. Male rats had

a significantly increased level of apoptosis one day after doxorubicin treatment, but no

changes were seen in females rats in both one and four days after the treatment. The

effectors of apoptosis, caspase-3 activity were significantly increased at day four, in both









males and females. These data suggest that caspase-independent pathway might be

involved in doxorubicin-induced apoptosis.

Mitochondrial-mediated pathway was not involved in activating capase-3 at day

four. Cytochrome c release was prevented by anti-apoptotic protein Bcl-2 at day four in

males. No changes were detected in receptor-mediated pathway. The initiator caspase-8

and its inhibitor cFLIP did not change in response to doxorubicin administration. These

findings suggest that doxorubicin induces apoptosis through other novel apoptotic

pathways such as sarcoplasmic reticulum mediated pathway.

A better understanding of gender difference in doxorubicin-induced pro- and anti-

apoptotic signaling pathways in cancerous and non-cancerous cells may lead to new and

improved therapeutic protocols for mitigating the toxic side effects of doxorubicin.














CHAPTER 1
INTRODUCTION

Introduction

The anthracycline antibiotic doxorubicin (adriamycin) is one of the most effective

chemotherapeutic agents for treating human neoplasms such as leukemia, lymphomas,

breast cancer, and many solid tumors. However, chronic use can become associated with

acute and chronic cardiotoxicities (1). The cardiotoxicity is dose dependent and causes

irreversible myocardial damage, resulting in dilated cardiomyopathy with fatal congestive

heart failure (1, 2). The exact mechanism of doxorubicin-induced cardiomyopathy

remains unclear, but most of the evidence indicates that reactive oxygen species

oxidantss) are involved (3). It is believed that mitochondrial derived oxidants play a

significant role in triggering this toxicity (4, 5). Isolated heart mitochondria have been

shown to shuttle single electrons to doxorubicin, giving rise to oxygen radicals through

the autoxidation of adriamycin semiquinones. Evidence suggests that NADH

dehydrogenase associated with complex I of the electron transport chain is intrinsically

involved in this one electron transfer to doxorubicin further generating free radicals

oxidantss) (6). Reactive oxygen species have been reported to cause irreversible tissue

damage by inactivating key proteins and enzymes present in cardiac sarcoplasmic

reticulum and mitochondria, and they are also believed to induce apoptosis (7).

Apoptosis is an evolutionary conserved form of cell suicide through which

multicellular organisms eliminate redundant, damaged, or infected cells (8-10). The

central component of this form of cell death is a proteolytic system involving a family of









cysteine proteases called caspases (11). Interest in the control of apoptosis has grown

significantly since the realization that disturbed apoptosis may contribute to cancer,

degenerative diseases, and cardiomyopathies with the chronic use of doxorubicin (12,

13). Investigations in experimental animal models have demonstrated that apoptosis is

one of the mechanisms of myocyte cell death with ischemic cardiac injury and heart

failure (13). Further, apoptosis is now being implicated in anthracycline induced

cardiotoxicity (Figure 1-1), which is one of the major limitations to the use of this

otherwise highly efficacious antineoplastic drug (7).






xidanls

Doxorubicin




Nuclear DNA Cytochrome c release
Damage |?
Caspase activation Apoptosis
Figure 1-1. Doxorubicin-induced mitochondrial damage and apoptosis.

Oxidants, such as superoxide (02'-), hydrogen peroxide (H202), hydroxyl radicals (HO'),
and peroxynitrite (ONOO-) may be generated by doxorubicin toxicity. Oxidants will
affect mitochondrial redox status and may cause extensive oxidative damage to proteins.
Furthermore, the mitochondrial transition pore can open releasing cytochrome c and
possibly affecting mitochondria function. Cytochrome c can activate cytosolic caspases
to induce apoptosis. Doxorubicin may induce anti-apoptotic (Bcl-2, Bcl-XL) and pro-
apoptotic (Bax, Bad) proteins.
Compelling evidence from several epidemiological and clinical studies indicates a

substantially higher incidence of heart failure and cardiovascular diseases in men

compared with age-matched women (14-16). The basis for these differences have been

attributed in part, to the cardioprotective effects of estrogen (17). The assumption that









female sex hormones are largely responsible for the low incidence of cardiovascular

disease in pre-menopausal women arises from the absence of similar cardiovascular

protection in post-menopausal women, an occurrence paralleled by declining estrogen

levels (16). However, many other mechanisms may exist and this requires further

investigation. The benefits of estrogen gradually shift from the vascular system to the

myocardium (15). This view is supported by the fact that functional estrogen receptors

have been detected in the myocardium (18). In addition, females have higher levels of

telomerase activity after adulthood and therefore an enhanced ability to preserve cardiac

myocytes viability following an injury. This may increase the potential for growth and

could certainly become critical factor to ensure an longer life span (19). In recent years,

several investigations have documented that female hearts are inherently protected by

estrogen against apoptotic cell death (20). It is speculated that estrogen reduces the

activity of ICE-like protease caspase-3, which is an effector / downstream mediator of

apoptosis (18, 21). In addition, gender differences in myocardial activation of Akt/PKB

can also subsequently inhibit apoptosis by phosphorylating the Bcl-2 family member Bad

and caspase-9, which are key components of the intrinsic cell death machinery (22).

Although much of the existing research has focused on trying to unravel the

mechanisms responsible for doxorubicin induced cardiotoxicity and the numerous ways

estrogen may afford protection to the cardiovascular system in females, no experiments

have been carried out to determine if male and female hearts respond differently to an

acute dose of doxorubicin. Therefore, we attempted to determine this possibility in these

proposed experiments.









Specific Aims

This study will investigate whether there are sex differences following doxorubicin

treatment in oxidant production, mitochondrial function, pro- and anti- apoptotic proteins

and the overall incidence of apoptosis in male and female Fischer 344 rats.

Question 1
Are there sex differences in oxidant production, mitochondrial function and

responses by antioxidant defenses one day and four days after doxorubicin

administration?

Hypothesis 1
Female rats will have less oxidant production, less mitochondrial dysfunction, and

an enhanced antioxidant defense adaptation following doxorubicin treatment.

Question 2
Are there differences in the overall incidence in apoptosis and caspase-3 activation

between male and female rats following administration of doxorubicin?

Hypothesis 2
Female hearts will have exhibited less apoptotic cell death and caspase-3 activation.

Question 3
Are there differences in the adaptation of pro- and anti-apoptotic regulatory

proteins in response to doxorubicin cardiotoxicity?

Hypothesis 3
Female hearts will have higher expression of anti- apoptotic proteins as compared

to male hearts, which may partly explain differences in apoptosis.














CHAPTER 2
REVIEW OF LITERATURE

Doxorubicin and Cardiotoxicity

Doxorubicin (Adriamycin) is a powerful anthracycline antibiotic, originally

isolated from the fungus streptomycespeucetius. It is used to treat many human

neoplasms, including acute leukemias, lymphomas, stomach, breast and ovarian cancers,

Kaposi's Sarcoma, and bone tumors (23). However, doxorubicin causes severe cardiac

toxicity, which compromises its clinical usefulness (24). Chronic toxic effects of

doxorubicin often develop after several weeks or months of treatment, and sometimes

even 4 to 20 years after discontinuation of the treatment. Thus, the risk of developing

heart failure in cancer patients treated with doxorubicin remains a life-long threat (2).

Since the first report of doxorubicin-induced cardiomyopathy (25), extensive

clinical as well as basic research efforts have been focused on understanding the

pathophysiology of congestive heart failure caused by this drug. A number of

mechanisms have been proposed to explain the development of doxorubicin-induced

cardiomyopathy including direct DNA damage and interference with DNA repair (26),

change in adrenergic function (27), abnormalities in the mitochondria (28), lysosomal

dysfunction (2), altered sarcolemmal Ca transport, Na-K+ ATPase and Ca2+ATPase,

imbalance in myocardial electrolytes (2), free radical formation (2, 28, 29), reduction in

myocardial antioxidant enzyme activities (29), lipid peroxidation (30), and apoptosis (28,

29, 31). Furthermore, the cytotoxic action by doxorubicin involves the cytoskeleton of

both tumor cells and cardiomyocytes (32). Cytoskeletal changes following doxorubicin









administration include reduction in density of myofibrillar bundles (33), alterations on the

Z-disc structure (33), and disarray and depolymerization of actin filaments (33, 34). This

list demonstrates that the cause of doxorubicin-induced cardiomyopathy is probably

multifactorial and the mechanism is complex. However, most of these changes have an

underlying cause, which is highly likely to be damage from reactive oxygen species.

Moreover, damage to either the mitochondria or the sarcoplasmic reticulum could create

a favorable environment for the induction of apoptosis (28).

Doxorubicin and Oxidants

Heart mitochondria are thought to play an important role in mediating oxidative

damage. The enzyme NADH dehydrogenase, a major player in transferring electrons to

harmful oxidants interacts with doxorubicin. The quinone ring of doxorubicin, a part of

its tetracyclic moiety, undergoes redox cycling between quinone and semiquinone.

During this process, oxidizing agents, including oxygen, capture free generated electrons

and then initiate a chain reaction that leads to the generation of superoxide anion

production. The superoxide anion radical generated can undergo dismutation to

hydrogen peroxide. Specifically, in the presence of redox active free metal ions,

formation of the highly reactive hydroxyl radical could account for membrane damage

(35). If subjected to oxidant insult, mitochondria can then be triggered to release

cytochrome c and other apoptogenic factors that activate a cascade of initiator and

effector caspases to induce apoptosis.

Apoptosis

Adult cardiomyocytes are post-mitotic cells that once destroyed, are slowly

replaced. Consequently, their loss can contribute to the functional decline of the

myocardium leading to heart disease (36). Until recently, the mode of cell death involved









in cardiomyocyte loss was not clear and was attributed to necrosis (13). It is now

believed that apoptosis is also involved in cardiomyocyte cell death, and it plays an

important role (37).

Necrosis is a rapid and irreversible process that occurs when cells are severely

damaged. Necrosis involves swelling of the cell and its organelles, disruption of

mitochondria, membrane rupture, and cell lysis (38). It is a destructive process, as

release of cellular content into the surrounding environment can cause further damage or

death to neighboring cells. Apoptosis, on the other hand, is a highly organized, energy

dependent mechanism whereby a cell commits suicide without causing damage to

surrounding tissue and it occurs normally during development, tissue turnover, and in the

immune system (12). Apoptosis is characterized by cellular condensation while

maintaining intra-organelle integrity, membrane blebbing, DNA fragmentation into oligo-

and mono-nucleosomes, destruction of the cytoskeleton, and formation of apoptotic

bodies which are endocytosed by macrophages and neighboring cells (12). The apoptotic

process is mediated by the activation of cysteine proteases known as caspases, which

cleave each other and other proteins after an aspartate residue within a specified amino

acid sequence (11). There are approximately 14 caspases identified that participate in the

apoptotic process depending on the signaling pathways (11).

Various stimuli such as oxidative stress (29, 39), mitochondrial dysfunction (28),

elevated intracellular Ca2+, excessive DNA damage and various cytokines can induce

apoptosis, and different signaling pathways have been described in various cell types.

The two most widely studied pathways in cardiac myocytes are the mitochondrial

mediated and the death receptor mediated pathways.









Mitochondrial Mediated Pathway

Mitochondrial dysfunction is a well-known stimulus for mitochondrial-mediated

apoptosis initiating the release of apoptogenic factors from the mitochondria. Release of

cytochrome c, as well as other apoptogenic factors, from mitochondria to the cytosol

initiates mitochondrial apoptosis (28). Cytochrome c then forms an apoptosis initiating

complex with apoptosis protease-activating factor (Apaf-1), dATP and procaspase-9,

resulting in the self-cleavage and activation of caspase-9 (28). The active caspase-9 then

cleaves and activates procaspase-3. This, in turn, activates a cascade of caspases,

internally breaking down the cell (11). Some of the targeted proteins of caspase-3 are

procaspase-6 and procaspase-7, poly ADP ribose polymerase (PARP), inhibitors of DNA

fragmentation factor (DFF) and caspase activated DNase (CAD) resulting in DNA

fragmentation (11).

The Bcl-2 family of proteins regulates the release of cytochrome c (40). These

include Bax, Bad, and Bid, which are pro-apoptotic proteins that favor cytochrome c

release and Bcl-2 and Bcl-xL which are anti-apoptotic proteins that inhibit cytochrome c

release (40, 41). The mechanisms of how Bcl-2 family proteins regulate release of

cytochrome c and apoptogenic factors are under investigation. Some of the hypotheses

include: 1) physical rupture of the outer membrane, 2) a channel formed by pro-apoptotic

Bcl-2 family proteins such as Bax, and 3) opening of a pore via a membrane permeability

transition pore (MPT) characterized by loss of the mitochondrial membrane potential (11,

41).

The mitochondria also release other apoptogenic factors such as apoptosis inducing

factor (AIF) which is a caspase-independent mitochondrial death effector (42). AIF can

only be released upon MPT. These pro-apoptotic factors translocate to the nucleus where









they induce DNA chromatin condensation and large-scale DNA fragmentation into

approximately 180-200 kbp. Previous studies have confirmed this observation in a

variety of cell types (40, 42).

Death Receptor Mediated Pathway

Various receptors can mediate apoptosis such as tumor necrosis factor receptor 1

TNFR1) and Fas/CD95. Ligand binding to TNFR1 or Fas can induce apoptosis in an

effector cell by the activation of procaspase-8, which cleaves and activates procaspase-3

to initiate the caspase cascade (43). Alternatively, binding of ligand to TNFR1 can

induce a pro-inflammatory/anti-apoptotic response mediated through the cytosolic

transcription factor NF-KB. The presence or recruitment of adaptor proteins to TNFR1

determines the outcome: caspase activation or NF-KB activation. Thus, whether the cell

chooses a survival pathway or a death pathway in response to TNFR activation depends

on the interaction of various signaling pathways and regulators of these pathways (43).

It has been shown that Fas antigens were overexpressed in myocytes of dilated-

cardiac myopathies, chronic heart failure, and myocardial infarction (36). Nakamura et

al. (44) observed apoptotic cell death via Fas-mediated pathway in adriamycin-induced

cardiac myopathy rats.

Regulators of Apoptosis

Apoptosis is a sequential, multi step process made up of many different layers of

regulation. This is of particular importance in post-mitotic cardiac myocytes in order to

avoid unnecessary death of salvageable cells and to promote apoptosis in response to

irreversible cellular damage, as opposed to necrosis, which could further harm the

myocardium. The final steps of apoptotic death are highly conserved and likely to be









mediated by a similar set of caspases. Various inhibitory regulatory mechanisms exist

within cells that target caspases. These include cFLIP and the inhibitors of apoptosis

proteins (IAP) family (40). These inhibitors are present in various cell types and may

also play an important role in the heart (13). The known IAPs include XIAP, clAP, and

cIAP2 (40). It is believed that these IAPs bind to cleaved/activated caspases and inhibit

their activity. XIAP is consired one of the most active inhibitors of capse-3.

Alternatively cFLIP inhibits the activity of caspase-8 and is highly expressed in the heart

under normal physiological conditions but is degraded after ischemia/reperfusion (45).

Recently an inhibitor of apoptosis that is expressed almost exclusively in skeletal

muscle and heart has been characterized. ARC (apoptosis repressor with caspase

recruitment domain) was first shown to interact with caspase-8 and -2 and to attenuate

apoptosis induced by stimulation of death receptors (46). More recently it was

demonstrated that ARC inhibits cytochrome c release from mitochondria and protects

against hypoxia-induced apoptosis (47). This study suggested that ARC can exert its

effect at different levels in the apoptotic pathway and may be a key regulator of apoptosis

in the heart.

Gender Related Differences in the Cardiovascular System

There are significant sex differences in the incidence of a variety of cardiovascular

diseases and acute myocardial injuries (16). In recent years, several lines of evidence

strongly suggest that loss of myocytes occur with heart failure, ischemia/reperfusion

injury and aging. This loss of cardiac cells may occur because of both necrosis and

apoptosis. This emerging concept of cardiac myocyte death by apoptosis may have

important implications in terms of studying gender-based differences. It is well

established that the female ovarian steroid hormone estrogen has strong cardio-protective









properties (48). Possible mechanisms behind this protectection might include systemic

effects such as improvement in the lipid profile, reduction in lipid peroxidation, and

stimulation of endothelium dependent NO production (18). Grohe et al. (18)

demonstrated through immunofluorescent assays that cardiac myocytes and fibroblasts

express functional estrogen receptor proteins in both male and female rats. Biological

effects of estrogen generally require the presence of estrogen receptor, a ligand dependent

transcriptional factor that regulates the expression of genes transduced by estrogen.

However, additional mechanisms, may exist to explain the increased tolerance of female

rats to a myocardial insult (48).

Gender Differences in Apoptosis

There is evidence that estrogen plays an important role in modulating certain cell-

death related signals to inhibit apoptosis. Using estrogen (173 estradiol) at physiological

concentration has shown to inhibit apoptosis in cardiac myocytes (17). Pelzer et al.(17)

studied apoptosis in cultured cardiac myocytes induced by staurosporine, a tyrosine

kinase inhibitor and a potent pro-apoptotic agent. A significant reduction in apoptosis

was observed in cells that were simultaneously treated with estrogen and staurosporine.

17p-Estradiol has been found to reduce the activity of ICE-like protease caspase-3 an

effector and downstream mediator of apoptosis. Moreover, Camper-Kirby and coworkers

(22) recently reported a significant difference between sex in myocardial activation of

Akt. Both localization of phospho Akt in the myocardial nuclei and cytosolic localization

of phospho-forkhead, a downstream nuclear target of Akt, were found elevated in

sexually mature female mice compared to that in male mice. Akt, also known as Protein

Kinase B, is a serine/threonine kinase, which lies at the intersection of multiple cellular









signaling pathways involved in the regulation of glucose metabolism, gene transcription,

and cell survival (49). It is a down stream effector molecule for signal transduction

initiated by survival factors such as IGF-1.

Death
Receptor


FA ER

FADD ROS I IGFR1
cFLIP +
SP13K
Bid Akt
BcIBid 2 ,--- Bax
S BCI-XL
Procaspase-8 ARC Bad



Cytochrome C

Apaf-1
ARC dATPProcaspase-9

IAP
Procaspase-3


APOPTOSIS
IAP

Figure 2-1. Overview of apoptotic pathways.

These factors bind to their respective cell surface receptors triggering the activation of

several kinases including the PI3K. This pathway then activates Akt through

phosphorylation which can subsequently inhibit apoptosis by phosphorylating Bcl-2

family member Bad and caspase-9, both of which are components of intrinsic cell death









machinery and the forkhead transcription factor (FKHRL1). FKHRL1 in a

dephosphorylated state translocates to the nucleus where it induces target genes such as

Fas ligand and triggers apoptosis. Hence, estrogen, by bringing about Akt-dependent

phosphorylation and inactivation of FKHRL1 (Figure 2-1), suppresses the transcription

of death genes and promotes survival (50).

Summary

A great deal of effort has been expended in trying to prevent or mitigate the

cardiotoxic side effect of doxorubicin. However, it is imperative that any method

designed to minimize the cardiotoxic effect of doxorubicin also maintains its

antineoplastic efficacy. Surprisingly, no studies have been conducted to examine if sex

differences exist in oxidative stress and apoptosis following the use of this drug.















CHAPTER 3
METHODS

Animals and Experimental Design

Male and female Fisher 344 rats (National Institute of Aging colony, Harlan

Sprague Dawley, Indianapolis, IN) were used in this study. The animals were housed

individually in a temperature (18-220C) and light-controlled environment with a 12-hour

light/dark cycle and were provided with food and water ad libitum. Animals from both

male and female groups were randomly assigned either to a control or to a doxorubicin-

treated group. Doxorubicin hydrochloride (Sigma Chemical Co., St. Louis, MO) was

dissolved in saline and administered by intraperitoneal injection at a dose of 10 mg/kg to

the group receiving treatment. Male and female animals were sacrificed one day after

doxorubicin injection (n= 12). In order to see if there were adaptations additional groups

of male and female rats were sacrificed four days after the injection (n=12). The control

group was injected with an equal volume of saline and they were sacrificed on day one

(n= 12) and day four (n= 12).

Tissue Harvesting

Animals were anesthetized with an intraperitoneal injection of sodium

pentobarbital (5 mg/100 g body weight). The chest was opened and blood removed

directly by cardiac puncture. This was followed by severing the inferior vena cava and

perfusion of the heart with 10 ml of ice-cold antioxidant buffer containing 100 [tM

diethylenetriaminepentaacetic acid (DTPA), 1 mM butylated hydroxytoluene (BHT), 1%









ethanol, 10 mM 3-aminotriazole, and 50 mM NaHPO4 (pH 7.4). After perfusion, the

entire heart was excised, rinsed in antioxidant buffer to remove any remaining blood,

blotted dry, and weighed.

Cellular Fractionation

Isolation of Mitochondrial and Cytosolic Fractions.

The atria of the heart was removed and stored at -800C until analysis. A 500mg

portion of the left ventricle was used for isolation of mitochondria. Tissue was weighed

and minced in 5 volumes of isolation buffer (0.225M mannitol, 0.075 M sucrose, 0.2%

fatty acid free bovine serum albumin, pH 7.4). The tissue was homogenized in a Potter-

Elvehjem glass homogenizer and centrifuged for 10 minutes at 700g. The resulting

supernatants were centrifuged again for 10 minutes at 8,000g. The supernatant (cytosolic

fraction) was aliquotted and stored at -800C. The mitochondrial pellet was resuspended

in 5 mL of isolation buffer and centrifuged for 10 minutes at 8000g. The final

mitochondrial pellet was resuspended in 1 mL of isolation buffer, aliquotted and stored

for later analyses.

Protein Concentration

Cytosolic and mitochondrial protein concentrations was determined using the

method developed by Bradford (51).

Mitochondrial Functional Parameters

Mitochondrial Respiratory Function

To assess mitochondrial damage due to isolation procedures, we calculated the

respiratory control ratio (state 3 respiration / state 4 respiration), which is commonly used

as an index for mitochondrial function. Mitochondrial respiration was monitored at 370C

in incubation buffer (145 mM KCL, 30 mM Hepes, 5 mM KH2PO4, 3 mM MgC12, 0.1









mM EGTA, 0.1 % fatty-acid free albumin, pH 7.4), 2.5 mM pyruvate, 2.5 mM malate,

and 0.25 mg mitochondrial protein for a total volume of 500 [tl. State 4 respiration (no

ADP) was read for 2 minutes and monitored using a chart recorder. State 3 respiration

(with ADP) was measured in the presence of 500 [tM ADP for 10 minutes or until the

oxygen pressure equals zero. Oxygen consumption was calculated as ng atom 02

consumed/mg protein/minute. Respiratory measurements were completed within 3 hours

after mitochondrial isolation and performed in duplicate.

ATP Content and Production

ATP production in isolated mitochondria was measured using a luminometer

(model TD-20/20, Turner Designs, Sunnyvale, CA). The assay uses firefly luciferase,

which fluoresces in proportion to the presence of ATP. Freshly isolated mitochondria

were added to a cuvette containing 1 mM ADP, 1 mM pyruvate, 1 mM malate, and a

Luciferin-Luciferase ATP monitoring reagent (ATP Determination Kit A-6608,

Molecular Probes, Eugene, OR). A blank cuvette containing no metabolic substrate was

assayed to account for nonspecific ATP production. A known ATP concentrations was

used to establish a standard curve. Results were expressed as nmol ATP produced/mg

protein/minute. The P/O ratio used as an index of mitochondrial efficiency was

calculated by taking nmol ATP produced/mg protein/min divided by ng atoms of oxygen

consumed/mg protein/min of state 3. The P/O ratio represents the number of ADP

molecules phosphorylated per mole of oxygen atoms consumed.

Mitochondrial Hydrogen Peroxide (H202) Production

The rate of mitochondrial H202 production was measured according to Barja (52)

using a fluorescent microplate reader (GeminiXS, Molecular Devices, Sunnyvale, CA).









Incubation buffer (145 mM KC1, 3 mM MgC12, 5 mM KH2PO4, 30 mM Hepes, 0.1 mM

EGTA, 0.1% BSA, pH 7.4), mitochondria (0.25 mg/ml), horseradish peroxidase (5.7

U/ml), and homovanilic acid (0.1 mM) were added to test tubes. The reaction was

initiated by the addition of pyruvate and malate (2.5 mM each) and the tubes are placed

in a shaking water bath for 15 minutes at 370C. The reaction was stopped by placing

tubes on ice and adding 0.5 ml of stopping solution (0.1 M glycine, 25 mM EDTA, pH

12.0). Fluorescence was determined using a fluorescent microplate reader and a standard

curve was generated for each analysis using glucose-glucose oxidase.

Biochemical Assays

Estrogen (Estradiol)

Plasma estradiol concentration was measured using radioimmunoassay (RIA) kit at

Yerkes Endocrine Laboratory (Atlanta, GA). All samples and standards were measured

in duplicates.

Cytosolic Mono- and Oligo-nucleosomes

DNA fragmentation was quantified by measuring the content of cytosolic mono-

and oligonucleosomes (180 base pair nucleotides or multiples) using a Cell Death ELISA

(Roche Molecular Biochemicals, Germany) according to instructions from the

manufacturer. The assay is based on the quantitative sandwich-enzyme-immunoassay-

principle. Wells are coated with a monoclonal anti-histone antibody. Nucleosome in the

sample binds to the antibody followed by the addition of anti-DNA-peroxidase, which

reacts with the DNA, associated with the histones. The amount of peroxidase retained in

the immunocomplexes is determined photometrically with ABTS (2.2' -azino-di-[3-

ethylbenzthiazoline sulfonate]) as a substrate. All samples were read in triplicates.

Results were reported as arbitrary OD units/mg protein.









Western Blots Analysis

Proteins were separated on a 4-20% precast polyacrylamide gel (BMA, Rockland).

Proteins were transferred onto a nitrocellulose membrane. Nitrocellulose membranes

were blocked overnight using a blocking solution containing 0.05% Tween and 5.0% dry

milk. Membranes were incubated for 90 minutes with the primary antibody at the desired

dilution. Membranes were incubated for 90 minutes in anti-rabbit or mouse Ig

horseradish peroxidase (Amersham Life Science) with an appropriate dilution. Blots

were developed using ECL (Amersham Pharmacia Biotech, England). The protein bands

were analyzed using Kodak Image Station 440CF (Eastman Kodak, Rochester, NY).

Cytosolic Cytochrome C

Cytosolic cytochrome c was quantified using an ELISA kit from R&D Systems

(Minneapolis, MN) which employs the sandwich enzyme immunoassay technique. Data

were reported as nmol/mg cytosolic protein.

Caspase-3 Activity

Caspase-3 activity was measured using the synthetic peptide n-acetyl-DEVD-AMC

(BD PharMingen, San Diego, CA). This assay detects activated caspase-3 and, to a lesser

extent, caspases -6, -7, and -8. Active caspases cleave the AMC from the peptide and the

free AMC fluoresces. Standards of active caspase-3 were prepared. Briefly, 1 mL of

assay buffer (20mM HEPES, 10% glycerol, 1 M DTT, and 14 tiL of Ac-DEVD-

AMC/mL of buffer) and 50 [L of sample will be added to a microcentrifuge tube and

protected from the light. Samples were incubated at 370C for 60 minutes after which

fluorescence will be measured on a spectrofluorometer with an excitation wavelength of

380 nm and an emission wavelength of 440 nm.









Caspase-3 Content

A Western blot protocol was used to detect the full length and the activated

(cleaved) caspase-3. A polyclonal anti-caspase-3 antibody (Stressgen, Canada) was used.

Caspase-8

A Western blot protocol was used to detect the full length and the activated

(cleaved) caspase-8. A polyclonal anti-caspase-8 antibody (Steersmen, Canada) was

used.

Inhibitors of Apoptosis (XIAP, FLIP, ARC)

Endogenous inhibitors of apoptosis were measured using Western blot. The

cytosolic fraction was used to assess the content of these inhibitors. The following

antibodies will be used: monoclonal XIAP (MBL, Watertown), polyclonal ARC (Ab-1)

(Oncogene, Boston) and antiserum FLIP (Alexis, San Diego).

Bcl-2 and Bax

The content of Bax and Bcl-2 were measured using Western blots. The following

antibodies were used: polyclonal Bax (Ab-1) and polyclonal Bcl-2 (Ab-4) (Santa Cruz

Biotechnology).

Antioxidant Enzyme Assay

Glutathione Peroxidase Activity

Selenium-dependent glutathione peroxidase activity was assayed according to

Nakamura et al. (53) with modification, using H202 as the substrate.

Statistical Analysis

Two-way ANOVA was used for comparisons between groups. Bonferroni post

hoc test was performed if significance was found. Ap-value of <0.05 were considered

significant.














CHAPTER 4
RESULTS

Morphological Characteristics

We determined if there were sex differences in body weight, heart weight, heart

weight-to-body weight ratio (Table 4-1), and estradiol levels (Table 4-2). The body

weights of the male rats were significantly greater than in the female rats (p <0.05).

Moreover, a significant gender difference was observed in heart weight. The hearts of

males were (36%) greater then female hearts (p<0001). However, the female rats had a

significantly higher heart weight-to-body weight ratio (p<0.001). Furthermore, body

weight was not different one day after doxorubicin treatment in male or female rats.

However, there was a significant decrease (10%) in body weight four days after

doxorubicin treatment in the male rats (p=0.004), but not in female rats. Furthermore, the

heart weights of the male and female rats remained unaltered 1 day following

doxorubicin treatment. However, in both sexes, there was a significant decrease in heart

weight four days after doxorubicin treatment. The males showed a 15% decline

(p<0.001) and the females showed a slightly smaller (13%) decrease (p<0.05). The heart

weight-to-body weight ratio in male and female rats was not affected after 1 day or 4

days after doxorubicin treatment (Table 4-1).









Table 4-1. Body weight, heart weight, and heart weight to body weight ratio of male and
female rats treated with doxorubicin or saline.
Male Female
Control Dox Day 1 Dox Day 4 Control Dox Day 1 Dox Day 4
Body
Body 358.8+31.0 357.224.0 323.925.01 197.610.0 190.614.0 176.0+7.0 *
weight
Heart
weit 0.81+0.07 0.78+0.05 0.69+0.04T 0.520.03 0.51+0.05 0.45+0.011 *
weight
0.0022 0.0021 0.0023 0.0026 0.0027 0.0026
HW:BW
0.0001 0.0008 0.0007 0.0001 0.0002* 0.0009*


Body weight, heart weight, and the heart to body weight ratio (HW:BW) of male (n=12)
and female rats (n=12) sacrificed one day and four days after administration of 10mg/kg
doxorubicin or equal volume of saline (Mean SEM). Male (n=12) and female (n=12)
animals were injected with equal volume saline on dayl (n=6) and day 4 (n=6).
*Significant sex difference (p<0.05). tSignificantly different from saline injected rats
(p<0.05). Units: gram.
Plasma Estrogen Levels in Male and Female Rats

17-Beta estradiol was measured in the plasma using a radioimmunoassay (RIA)

method to determine the differences of this sex hormone between the male and female

rats. Moreover, since estradiol has cardio-protective effects we wanted to know the

variability with each group. Surprisingly, there was only a 20-30% difference between

young male and female rats (p<0.05). Furthermore, the variability between the individual

female rats was only approximately (20-30%), with a maximum level of estradiol of 13.8

pg/mL and the minimum level of 5.4 pg/mL.

Table 4-2. Plasma 17-P estradiol level.

Control Dox Day 1 Dox Day4

Male 5.70.6 6.01.5 6.10.7

Female 8.42.7* 8.72.4* 7.01.4*


Plasma estrogen (estradiol) in male and female rats sacrificed one day and four days after
injection with 10mg/kg doxorubicin vs. controls (MeanSEM). Significant sex
difference (p<0.05). Units: pg/ml









Mitochondrial Function

We determined the effect of doxorubicin treatment on mitochondrial function in

male and female rats (Table 4-3). Mitochondrial oxygen consumption was measured in

state 4 (no ADP) and during state 3 respiration (0.5 mM ADP). There were no sex

differences between State 4 and State 3 measurements. Moreover, doxorubicin treatment

following either 1 day or 4 days did not effect State 4 or State 3 respiration. The amount

of ATP produced by the mitochondria was used as a parameter to determine sex

differences in energy production and as a parameter to assess mitochondrial function

following doxorubicin administration. There was a significant sex difference in ATP

production; the female rats produced significantly less ATP per milligram of protein of

freshly isolated mitochondria determined in State 3 (p=0.0032). However, rates of ATP

production in isolated mitochondria one day or four days after doxorubicin treatment

were not different from those of controls. The P/O ratio, which compares the amount of

ATP that is phosphorylated with the amount of oxygen consumed, also did not differ

between groups.

Table 4-3. The effects of doxorubicin administration on mitochondrial function.
Male Female
Control Dox Day 1 Dox Day 4 Control Dox Day 1 Dox Day 4
State 4
S 8.401.36 3.01+5.10 8.492.51 9.353.04 7.204.75 8.733.14
VO2
State 3
S 42.21+28.61 47.51+26.1 34.6224.81 31.7822.46 25.9417.18 21.086.74
VO2

ATP 31.758.81 38.086.84 33.328.47 27.81+8.74 26.7512.37 23.426.33 *

P/O
o 1.150.37 1.060.52 1.480.80 1.340.50 0.890.32 1.180.33
Ratio

State 4 and State 3; nmol oxygen consumption/mg protein/minute; ATP production; nmol
ATP/mg protein/minute. Values presented are mean + SEM. *Significant gender
difference. (p<0.05)









Hydrogen Peroxide Production in Isolated Mitochondria

In order to measure the rate of reactive oxygen species production by heart

mitochondria, we measured hydrogen peroxide (H202) formation in freshly isolated

mitochondria (Figure 4-1). Interestingly, the amount of H202 production was

significantly different between the two sexes. Male rats produced significantly higher

levels of hydrogen peroxide compared to female rats (p=0.003). Doxorubicin did not

change hydrogen peroxide production in the male rats after day 1. In striking contrast,

there was a significant decrease in hydrogen peroxide production after day 1 in the

female rats (p<0.05). Males did show a significant drop (p<0.001) in hydrogen peroxide

production after day 4, but females remained at approximately the same level of

hydrogen peroxide production as compared to day 1.







S 0.75-
OE


0
E 0.25

0.00

Male Female


Figure 4-1. The effect of doxorubicin administration on heart mitochondrial oxidant
production.

Mitochondria were isolated on day 1 and day 4 after the injection of doxorubicin
10mg/mL) or equal volume of saline in control animals. Values presented are mean +
SEM. Significantly different from the control group (*p<0.001) and (**p<0.05).
Significant gender difference (p<0.05).









Glutathione Peroxidase Activity

To assess adaptations to mitochondrial hydrogen peroxide production, we

determined mitochondrial glutathione peroxidase (GPX) activity (Figure 4-2). Male rats

had significantly higher level of GPX activity compared to female rats (p=0.001). There

was no significant difference in GPX activity detected after doxorubicin treatment on any

of the days following doxorubicin treatment.


1.5
M Control
Xe
'i I I Dox Day 1
1.0- I Dox Day 4
"uE
o c-
SE 0.5
E

0.0

Male Female



Figure 4-2. The effect of doxorubicin administration on heart mitochondrial glutathione
peroxidase activity (GPX).

Values presented are SEM. Significant gender difference (p<0.05).

Apoptosis Determined by Mono and Oligo-nucleosomes

To assess the overall incidence of apoptotic cell death we determined the levels of

mono- and oligo-nucleosome contents in the isolated cytosolic fraction of the heart.

Apoptosis is characterized by the formation of mono- and oligo-nucleosome in the

nucleus. These DNA fragments are transported to the cytosol for degradation. Levels of

mono- and oligo-nucleosome were similar in the male and female control animals.









However, male rat hearts contained greater levels of mono- and oligo-nucleosome

content one day after doxorubicin treatment (p<0.01) and levels returned back to those

seen in control animals after day 4. In contrast, female rats showed no increases in the

levels of mono- and oligo-nucleosome after one day or four days after doxorubicin

administration, suggesting that female hearts were more resistant to apoptosis (Figure 4-

3).



0.35 Control
0.30
x L =I Dox Day I
S0.25- M Dox Day 4
-E 0.20
0.15
S 0o.10
0.05
0.00

Male Female


Figure 4-3. The effect of doxorubicin administration (10mg/kg) on the content of mono-
and oligo-nucleosomes in the heart cytosol.

Values presented are SEM. Significantly different from the control group (p<0.05).

Caspase-3 Activity and Caspase-3 Content

Caspase-3 is considered a central caspase of apoptosis, because most caspases are

able to activate this caspase to initiate apoptosis. There were no differences in caspase-3

proteolytic enzyme activity between male and female control rats and 1 day after

doxorubicin administration. However, four days after the administration of doxorubicin

there were significant increases in both the male and female rats (p<0.01) (Figure 4-4).












12500-


uD -.
S' *10000-

IQ
7500-

7L '5000-

E 2500-

0-


Male


M Control
= Dox Day 1
EM Dox Day 4


Female


Figure 4-4. The effect of doxorubicin administration (10mg/kg) on caspase-3 activity.

Caspase-3 activity measured using fluorescence ELISA method. Values presented are
mean + SEM. Significantly different from the control group (*p<0.05) and (**p<0.05)


To determine if the rate of synthesis of caspase-3 was altered we measured the

protein levels of the zymogen or pro-caspase-3 and the cleaved caspase-3 content (Figure

5). The total levels of the zymogen form of caspase-3 remained unaltered (Figure 4-5A),

suggesting that the absolute protein levels of this caspase were not altered during the

experimental phase in any of the groups. However, the content of the cleaved active

caspase-3 was significantly increased after day four in both the male and female rats

(Figure 4-5B).











250000-

200000-
a Q
S>150000-
rn(
S 100000-

50000-

0-


Control
S Dox Day 1
MM Dox Day 4


Male Female


300000-


200000-


100000-


M Contol
I Dox Day 1
M Dox Day 4


Male Female


Figure 4-5. The effect of doxorubicin administration on the caspase-3 content (A) and
cleaved caspase-3 concentration (B) determined by Western blot analysis.

Values presented are mean + SEM. Significantly different from the control group
(*p<0.05) and (**p<0.05).


We determined if gender differences and changes to doxorubicin treatment existed

in the levels of caspase inhibitor, X-linked inhibitor apoptotic protein (XIAP) a repressor

of caspase-3 activity (Figure 4-6). We found no differences due to sex or doxorubicin

treatment in any of the groups.


CL,
(U



5 >








650000-



a-
5O000-
Q
0 4010000-
S? 300000-
S200oo000-
100000-
0-


i Control
mDox Day 1
i lDox Day 4


Ile
Male


Female


Figure 4-6. The effect of doxorubicin administration (10mg/kg) on the amount of anti-
apototic proteins XIAP measured in cytosolic fraction using Western method.
Values presented are SEM.
Mitochondrial Mediated Pathway of Apoptosis
Apoptosis in cardiac myocytes is often associated with the release of cytochrome c

from the mitochondria (Figure 4-7). Female control rats had significantly lower cytosolic

cytochrome c levels compared to male rats (p<0.05). The dose of 10mg/mL of

doxorubicin did not result in increased levels of cytosolic cytochrome c determined after

1 day or 4 days of doxorubicin treatment.

3000 h nh-ll


or

S 10O0L -


0-


I I


/ %.rUo I IU Li1
CI Dox Day 1
M Dox Day 4


Male Female

Figure 4-7. The effect of doxorubicin administration (10mg/kg) on cytochrome c
concentration in the cytosol.
Values presented are SEM. Significant gender difference (p<0.05).


IIB








Mitochondrial Regulators of Cytochrome C Release
The levels of Bcl-2, an anti-apoptotic protein, and Bax, a pro-apoptotic protein

were determined in isolated cardiac mitochondria (Figure 8).


125000-


100000-
0
c 75000-
S50000-

25000-


Male


0
> 1
CO
mm
E.
In


70000-
60000-
50000-
40000-
30000-
20000-
10000-
0-


Male


Control
I Dox Day 1
s Dox Day 4




1


Female


M Control
I Dox Day 1
M Dox Day 4


Female


Figure 4-8. The effect of doxorubicin administration (10mg/kg) on the content of Bcl-2
(A) and Bax (B) measured in the cardiac mitochondria using Western Blot
analysis.
Values presented are SEM. Significantly different from control animals (*p<0.05).


I I


,I


I









There were no differences in the levels of these proteins due to gender. However, there

was an increase in the levels of Bcl-2 four days after doxorubicin treatment in the male

and female, but these changes were only statistically significant in the male animals

(Figure 8A). The levels of Bax were not significantly different between gender and

doxorubicin had no significant effect on this pro-apoptotic protein (Figure 8B).

Receptor Mediated Pathway of Apoptosis

To investigate the possibility if receptor mediated cell death was involved

following doxorubicin administration we determined the content of capase-8 (Figure 4-9

A&B). Pro-caspase-8 content was not different between male and female rats (Figure 4-9

A). Moreover, doxorubicin treatment did not result in a significant change in the inactive

procaspase-8 concentration, suggesting no increase in the synthesis rate of this pro-

caspase during the 4-day experiment phase. Moreover, the cleaved caspase-8 did not

differ significantly between sexes (Figure 4-9B). Furthermore, doxorubicin treated rats

did not show a statistically significant change in the levels of the cleaved form. We also

measured cFLIP, which is a potent inhibitor of caspase-8 activation and found no

differences in cFLIP due to gender or doxorubicin treatment (Figure 4-10).












oP
U.
00

CiLQ:


600000
TnOO-



200l00.-


0-


Male


30iXDOO


8 4oaooa-
S3?00ri0-
S"4'"00000
-. 100000 -
o-

0-
-


B


Male


- Control
= Dox Day 1
l Dox Day 4


Female


SControl
" Do< Day 1
111 Dox Day 4


Female


Figure 4-9 The effect of doxorubicin administration (10mg/kg) on the content (A)
Procaspase-8 and (B) cleaved caspase-8.


Values presented are mean + SEM.





rl









150000 M Control
Dox Day 1
o M Dox Day 4
0 100000
a-
I[-
LL L
50000


0
I I
Male Female



Figure 4-10. The effect of doxorubicin administration (10mg/kg) on the content of cFLIP
measured in cytosolic fraction using Western blot method.


Values presented are mean + SEM.














CHAPTER 5
DISCUSSION

Overview of Principle Findings

These are the first experiments to examine if gender differences exist in

cardiotoxicity induced by doxorubicin as determined by mitochondrial function,

oxidative stress, and apoptosis. No significant differences were observed in

mitochondrial function following doxorubicin treatment in any of the groups. However,

our data shows that female rats produce lower levels of hydrogen peroxide one day after

doxorubicin treatment compared to male rats. Moreover, the apoptotic index determined

by the levels of mono- and oligo-nucleosomes was significantly increased in male rats,

but not in female rats 1 day after doxorubicin treatment. In addition, caspase-3 activity

and cleaved caspase-3 content were increased after 4 days in male rats as well as in

female rats. Sex differences in mitochondrial oxidant production immediately after

doxorubicin could have influenced the release of pro-apoptotic factors, such as

cytochrome c, AIF, or Endonuclease G (54) and may explain the sex differences

observed in DNA fragmentation pattern. However, levels of cytosolic cytochrome c

remained unaffected in both the male and female rats, which could be explained by the

increase of Bcl-2 content in the isolated mitochondria. Furthermore, these data show that

the receptor mediated pathway is unlikely the major cause for the increased activation of

caspase-3 in vivo.









Body Weight and Heart Weight

Doxorubicin administration caused a significant reduction in body mass in male

rats, but not in female rats. Furthermore, four days after doxorubicin treatment the

animals heart weights of both the male and female rats were significantly lower

compared to those injected with saline. The loss in heart weight was probably due to 1)

increased levels of apoptosis and 2) an inhibition of protein synthesis by doxorubicin (55).

Other factors, such as dehydration and food consumption could have played a role in the

loss of heart weight. Heart weight-to-body weight ratio was significantly higher in

female rats, which could have reduced the absolute toxic effect of the drug (10 mg of

doxorubicin/kg of body weight) on the female rats. However, drug metabolism and fat-

to-lean-body mass ratio may have also played a role in the cardiotoxic effect of

doxorubicin.

Oxidant Production and Antioxidant Enzymes

Female rats had significantly lower hydrogen peroxide (H202) production

compared to the male group, which supported our initial hypothesis. Moreover, in

response to doxorubicin, male and female rats showed a different rate of oxidant

production. Hydrogen peroxide (H202) production was decreased one day after

doxorubicin administration in females. A possible explanation for this observation might

be that mitochondria could adapt to cytotoxic stress by enhanced electron coupling and a

reduction in the free radical leak. In addition, it remains possible that mitochondrial

biogenesis was significantly increased after doxorubicin treatment and that newly

synthesized mitochondria adapted and produced less hydrogen peroxide.

In response to the reduction in mitochondrial reactive oxygen species in the control

female rats, we found a similar decrease in mitochondrial glutathione peroxidase (GPX)









activity. Acute doxorubicin administration did not alter GPX activity in either male or

female rats suggesting that GPX activity was not up regulated within the 1 or 4 day

period after the oxidative stress insult. This result agrees with previous study by Li et al.

(55) and recent data from our laboratory (29), which show that a single dose (20mg/Kg)

of doxorubicin did not have any influence on the activity of GPX four days after

administration.

Apoptosis Induced by Doxorubicin

The overall incidence of apoptosis in control animals measured by DNA

fragmentation did not differ between sexes. However, in response to doxorubicin

administration, male rats showed an increased concentration of cytosolic mono- and

oligo- nucleosomes on day 1 only, whereas in female rats, no change was seen in DNA

fragmentation after day 1 and day 4. This data suggests that female rats might have been

able to prevent the loss in heart cells due to adriamycin-induced cardiotoxicity.

Surprisingly, the key effector caspase-3, showed significant increase in activity and

content after four days of doxorubicin treatment in both male and female rats. Hence, the

caspase-3 data does not correlate with the levels of apoptosis detected by the

concentrations of mono- and oligo-nucleosomes. Furthermore, we did not detect any

changes in XIAP content due to gender or doxorubicin treatment to add an explanation to

this finding. Therefore, we further investigated if the mitochondrial-mediated pathway of

apoptosis was responsible for the differences seen in apoptosis and caspase-3 activation.

Mitochondrial Mediated Pathway of Apoptosis

To confirm which pathway may have been responsible for the activation of

caspase-3 we examined the mitochondrial-mediated pathway. Cytosolic levels of

cytochrome c in male and female rats showed a very similar response and therefore this









pro-apoptotic protein does not appear to be responsible for the increased DNA

fragmentation seen in the male rats. However, it remains possible that changes in

cytochrome c levels occurred immediately after doxorubicin treatment and that the

effects of apoptosis, such as caspase-9 activation or AIF release occurred before day 1.

Moreover, it appeared that the mitochondrial anti-apoptotic protein, Bcl-2 was

significantly increased in response to doxorubicin administration in both male and female

animals four days after treatment. Hence, it is possible that other adaptive response

might have occurred during the four-day experimental period. For example, other

inhibitory proteins such as cytosolic ARC could have been sequestered by the

mitochondria and affected the release of cytochrome c, AIF, and endonuclease G (54)

differently. ARC is known to inhibit cytochome c release from mitochondria and

protects against hypoxia-induced apoptosis in heart (56). Therefore, up-regulation of this

protein could have altered the release of cytochrome c from mitochondria.

Receptor-Mediated Pathway of Apoptosis

We investigated if the receptor-mediated pathway was affected by doxorubicin

treatment and if gender differences were present. Others have shown that Fas-mediated

activation of caspse-8 in vitro with doxorubicin treatment causes apoptosis (44).

Although there were gradual increases in the levels of cleaved caspase-8 after day one

and day four, these changes were not statistically significant. Therefore, from the

changes observed in this study, using an in vivo model, it appears that this pathway was

not a major player in the activation of caspase-3. Furthermore, no significant changes

were detected in the caspase-8 inhibitor cFLIP due to gender or doxorubicin treatment.

Hence, it remains possible that the dose injected in these rats (10mg/kg) did not induce a









sufficient stress response to significantly activate this pathway or this pathway does not

play an essential role in inducing apoptosis.

Mitochondrial Function and Doxorubicin Treatment

Mitochondrial function and ATP production remained unaltered after acute

doxorubicin treatment. This is in strong agreement with the current literature which

suggest that mitochondrial DNA damage and damage to respiratory complexes needs to

be excessive (exceed -50% of it's function) before significant decreases in ATP

production are observed (57). Rossignal et al. (57) and several others (58, 59) have

shown that it was possible to inhibit the activity of a respiratory chain complex (-30-

50%), up to a critical level, without affecting the rate of mitochondrial respiration and

ATP synthesis (57). The mitochondria functional data in our present study supports this

hypothesis.

Limitations in Present Study

One of the potential limitations to this study is that we did not control for the

estrous cycle. Although, there were only moderate fluctuations (20-30% change in

estrogen between males and females and within each group), the variability in estrous

cycle could have been a confounding variable. Levels of estrogen found in the plasma

were in pg/mL level, which might be too low to have any significant effect on attenuating

oxidative stress compared to lipid and water soluble antioxidant found in plasma (60).

However, most studies suggest that estrogen's protective effects may stem from binding

to cell surface receptors and up-regulating a variety of cellular proteins, such as NOS (18,

61) and heat shock proteins (18, 62). Moreover, most studies show an anti-apoptotic

effect of estrogen in cell culture models which might have very little relevance in a

complex biological in vivo model, such as that used in this study (17, 18). In addition,









estradiol levels of Fischer 344 rats are at their highest levels around the age of 9 to 12-

months (63). Our rats were 6 months of age and might not have been mature enough to

receive the full protection of estrogen. Therefore, the levels of estrogen in this study may

not have been sufficient to be cardioprotective and it would be worthwhile to investigate

rats with ovariectomy and to see if supplementing estradiol may have an effect on

apoptotic signaling pathways in vivo.

Conclusion and Future Direction

To our knowledge, these were the first experiments that look at the effects of

gender and doxorubicin in relation to oxidative stress and cell death. The principle

findings from this study include: 1) there are gender differences in doxorubicin-induced

oxidant production and apoptosis; 2) mitochondrial pathways may have been involved,

but the rapid up-regulation of Bcl-2 may have prevented significant cell death by this

pathway and 3) receptor mediated cell death appears to play a minor role, since no

adaptations in cFLIP and significant increases in caspase-8 were observed following

doxorubicin treatment.

Other pathways, such as caspase-12, an endoplasmic reticulum-mediated pathway

might be also partly responsible for the apoptosis observed in the male rats. The

activation of the endoplasmic reticulum-mediated pathway causes the release of calcium

and the activation of caspase-12. Indeed, numerous studies have reported that

doxorubicin can alter Ca2+ homeostasis (2, 7, 64). Therefore, more research is needed to

further elucidate other possible mechanisms and to determine the rate of mitochondrial

biogenesis, mitochondrial proteolytic degradation; and activation of autophagy through

lysosomal pathways following doxorubicin-induced cardiotoxicity. A better

understanding of gender difference in doxorubicin-induced pro- and anti-apoptotic






39


signaling pathways in cancerous and non-cancerous cells may lead to new and improved

therapeutic protocols for mitigating the toxic side effects of doxorubicin.















LIST OF REFERENCES


1. Singal, P.K., and Iliskovic, N. 1998. Doxorubicin-induced cardiomyopathy N
EnglJMed 339:900-905.

2. Singal, P.K., Li, T., Kumar, D., Danelisen, I., and Iliskovic, N. 2000. Adriamycin-
induced heart failure: mechanism and modulation. Mol Cell Biochem 207:77-86.

3. Raju, J., Coralie, P., and Hung-Yi, W. 1997. Molecular mechanisms of
doxrubicin-induced cardiomyopathy: Selective suppression of Reiske iron-sulfer
protein, ADP/ATP translocate, and phopho-fructokinase gene is associated with
ATP depletion in rat cardiomyocyte. JBiol Chem 272:5828-5832.

4. Yen, H.C., Oberley, T.D., Vichitbandha, S., Ho, Y.S., and St Clair, D.K. 1996.
The protective role of manganese superoxide dismutase against adriamycin-
induced acute cardiac toxicity in transgenic mice [published erratum appears in J
Clin Invest 1997 Mar 1;99(5):1141]. J Clin Invest 98:1253-1260.

5. Kang, Y.J., Chen, Y., Yu, A., Voss-McCowan, M., and Epstein, P.N. 1997.
Overexpression of metallothionein in the heart of transgenic mice suppresses
doxorubicin cardiotoxicity. J Clin Invest 100:1501-1506.

6. Nohl, H., Gille, L., and Staniek, K. 1998. The exogenous NADH dehydrogenase
of heart mitochondria is the key enzyme responsible for selective cardiotoxicity of
anthracyclines. ZNaturforsch [C] 53:279-285.

7. Kalyanaraman, B., Joseph, J., Kalivendi, S., Wang, S., Konorev, E., and
Kotamraju, S. 2002. Doxorubicin-induced apoptosis: Implications in
cardiotoxicity. Molecular and Cellular Biochemistry 234/235:119-124.

8. Steller, H. 1995. Mechanisms and genes of cellular suicide. Science 267:1445-
1449.

9. Vaux, D.L., Haecker, G., and Strasser, A. 1994. An evolutionary perspective on
apoptosis. Cell 76:777-779.

10. Kerr, J.F., Wyllie, A.H., and Currie, A.R. 1972. Apoptosis: A basic biological
phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer
26:239-257.









11. Green, D., and Kroemer, G. 1998. The central executioners of apoptosis:
Caspases or mitochondria? Trends Cell Biol 8:267-271.

12. Duke, R.C., Ojcius, D.M., and Young, J.D. 1996. Cell suicide in health and
disease. SciAm 275:80-87.

13. Gill, C., Mestril, R., and Samali, A. 2002. Losing Heart: The role of apoptosis in
heart disease-a novel therapeutic target? FASEB 20:135-146.

14. Mallat, Z. 2001. Age and gender effects on cardiomyocyte apoptosis in the human
heart. Journal of Gerontolgy: MEDICAL SCIENCES 56A:M719-M723.

15. Babiker, F.A., De Windt, L.J., van Eickels, M., Grohe, C., Meyer, R., and
Doevendans, P.A. 2002. Estrogenic hormone action in the heart: Regulatory
network and function. Cardio Res 53:709-719.

16. Hayward, C.S., Kelly, R.P., and Collins, P. 2000. The role of gender, the
menopause and hormone replacement on cardiovascular function. Cardio Res
46:28-49.

17. Pelzer, T., Schumann, M., Neumann, M., deJager, T., Stimpel, M., Serfling, E.,
and Neyses, L. 2000. 17 B-Estradiol prevents programmed cell death in cardiac
myocytes. Biochemical and Biophysical Research Communications 268:192-200.

18. Grohe, C., Meyer, R., and Vetter, H. 2002. Estrogen and the prevention of cardiac
apoptosis. In Apoptosis methods in pharmacology and toxicology : Approaches to
measurement and quantification. M.A. Davis, editor. Totowa, N.J.: Humana.

19. Leri, A., Malhotra, A., Liew C., Kajstura, J., and Anversa, P.. 2000. Telomerase
Activity in Rat Cardiac Myocytes is Age and Gender Dependent. JMol Cell
Cardiol 32:385-390.

20. Leri, A., Malhotra, A., Liew, C.C., Kajstura, J., and Anversa, P. 2000. Telomerase
Activity in Rat Cardiac Myocytes is Age and Gender Dependent. JMoll Cell
Cardiol 32:385-390.

21. Wu, W., Lee, W.L., Wu, Y.Y., Chen, D., Liu, T.J., Jang, A., Sharma, P.M., and
Wang, P.H. 2000. Expression of constitutively active phosphatidylinositol3-
kinase inhibits activation of caspase-3 and apoptosis in cardiac muscle cells. J
Biol Chem 275:40113-40119.

22. Camper-Kirby, D., Welch, S., Walker, A., Shiraishi, I., Setchell, K.D., Schaefer,
E., Kajstura, J., Anversa, P., and Sussman, M.A. 2001. Myocardial Akt activation
and gender: Increased nuclear activity in females versus males. Circ Res 88:1020-
1027.









23. Shan, K., Lincoff, A.M., and Young, J.B. 1996. Anthracycline-induced
cardiotoxicity Ann Intern Med 125:47-58.

24. Mott, M.G. 1997. Anthracycline cardiotoxicity and its prevention. Ann N YAcad
Sci 824:221-228.

25. Bonadonna, G., Monfardin, S., and Beretta, G. 1970. Phase I and Preliminary
phase II evaluation of adriamycin. Cancer Research 30:2572-2582.

26. Arena, E., and Gerbasi, F. 1974. DNA, RNA and protein synthesis in heart, liver,
and brain of mice treated with daunorubicin and adriamycin. Int Res Commun
Systemic Med Sci 2:1053-1061.

27. Tong, J., Ganguly, P.K., and Singal, P.K. 1991. Myocardial adrenergic changes at
two stages of heart failure due to adriamycin treatment in rats. Am JPhysiol
260:H909-H916.

28. Green, P., and Leeuwenburgh, C. 2002. Mitochondrial dysfunction is an early
indicator of doxorubicin-induced apoptosis. Biochimica et Biophysica Acta
1588:94-101.

29. Childs, A., Phaneuf, S., Dirks, A., Phillips, T., and Leeuwenburgh, C. 2002.
Doxorubicin treatment in vivo causes cytochrome c release and cardiomyocyte
apoptosis, as well as increased mitochondrial efficiency, superoxide dismutase
activity and Bcl-2:Bax ratio. Cancer Research 62:4592-4598.

30. Myers, C.E., McGuire, W.P., Liss, R.H., Ifrim, I., Grotzinger, K., and Young,
R.C. 1977. Adriamycin: The role of lipid peroxidation in cardiac toxicity and
tumor response. Science 197:165-167.

31. Zhang, J., Clarke, J., Herman, E., and Ferrans, V. 1996. Doxorubicin-induced
apoptosis in spontaneous hypertensive rats: Differential effects in heart, liver, and
intestine, and inhibition by ICRF-187. JMoll Cell Cardiol 28:1931-1943.

32. Arola, O.J., Saraste, A., Pulkki, K., Kallajoki, M., Parvinen, M., and Voipio-
Pulkki, L.M. 2000. Acute doxorubicin cardiotoxicity involves cardiomyocyte
apoptosis. Cancer Res 60:1789-1792.

33. Jaenke, R.S. 1974. Anthracycline antibiotic induced cardiomyopathy in rabbits.
Lab Investigations 30:292-304.

34. Lewis, G., and Gonzales, B. 1986. Anthracyline effects on actin in rat mycardial
cell. Lab Investigations 54.

35. Nohl, H. 1988. Identification of the site of adriamycin-activation in the heart cell.
Biochem Pharmacol 37:2633-2637.










36. Olivetti, G., Abbi, R., and Quaini, F. 1997. Apoptosis in the failing human heart.
NEnglJMed 336:1131-1141.

37. James, T.N. 1999. Apoptosis in cardiac disease. Am. J. Med. 107:606-620.

38. Searle, J., Kerr, J.F., and Bishop, C.J. 1982. Necrosis and apoptosis: Distinct
modes of cell death and fundamentally different significance. Pathol. Ann 17:229-
259.

39. Von Harsdorf, R., Li, P.F., and Dietz, R. 1999. Signaling pathway in ROS-
induced Cardiomyocyte Apoptosis. Circulation 99:2934-2941.

40. Green, D.R. 2000. Apoptotic Pathways: Paper wraps stone blunts scissors. Cell
102:1-4.

41. Reed, J.C. 1997. Bcl-2 family proteins: Regulators of apoptosis and
chemoresistance in hematologic malignancies. Semin Hematol 34:9-19.

42. Penniger, J., and Kroemer, G. 2003. Mitochondria, AIF and caspases- rivaling for
death execution. Nature Cell Biology 5:97-99.

43. Sun, X., MacFarlane, M., Zhuang, J., Wolf, B., Green, D.R., and Cohen, G.M.
1999. Distinct caspase cascades are initiated in receptor mediated and chemical
induced apoptosis. JBiol Chem 274:5053-5060.

44. Nakamura, T., Ueda, Y., Juan, Y., Katsuda, S., Takahashi, H., and Koh, E. 2000.
Fas-mediated apoptosis in adriamycin-induced cardiomyopathy in rats: In vivo
study. Circulation 102:572-578.

45. Rasper, D.M., and Nicholson, D.M. 1998. Cell death attentuation by 'Usurpin', a
mammalian DED-caspase homologue that precludes caspase-8 recruitment and
activation by the CD-95(Fas, APO-1) receptor complex. Cell Death Differ 5:271-
288.

46. Koseki, T., Inohara, N., Chen, S., and Nunez, G. 1998. ARC, inhibitor of
apoptosis expressed in skeletal muscle and heart that interacts selectivity with
caspases. Proc. NatlAcadSci USA 95:5156-5160.

47. Ekhterae, D., Lin, Z., Lundberg, M.S., Crow, M.T., Brosius, F.C., and Nunez, G.
1999. ARC inhibits cytochrome c release from mitochondria and protects against
hypoxia-induced apoptosis in herat derived H9c2 cells. Circulation Research
85:70-77.

48. Barp, J., Araujo, A.S.R., Fernandes, T.R.G., Rigatto, K.V., Llesuy, S., Bello-
Klein, A., and Singal, P.K. 2002. Myocardial antioxidant and oxidative stress









changes due to sex hormones. Brazilian Journal of Medical and Biological
Research 35:1075-1081.

49. Kandel, E.S., and Hay, N. 1999. The regulation and the activities of the
multifunctional serine/theronine kinase Akt/PKB. Exp CellRes. 253:210-229.

50. Sugden, P., and Clerk, A. 2001. Akt like a women : Gender differences in
susceptibility to cardiovascular disease. Circulation Research 88:957-977.

51. Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of
microgram quantities of protein utilizing the principle of protein-dye binding.
Anal Biochem 72:248-254.

52. Barja, G., and Herrero, A. 2000. Oxidative damage to mitochondrial DNA is
inversely related to maximum life span in the heart and brain of mammals. Faseb
J 14:312-318.

53. W. Nakamura, S.H.a.K.H. 1974. Purification and properties of rat liver
glutathione peroxidase. Biochimica et Biophysica Acta 358:251-261.

54. Li, L.Y., Luo, X., and Wang, X. 2001. Endonuclease G is an apoptotic DNAse
when released from mitochondria. Nature 412:95-99.

55. Li, T., Danelisen, I., Bello-Klein, A., and Singal, P.K. 2000. Effects of probucol
on changes of antioxidant enzymes in adriamycin- induced cardiomyopathy in
rats. Cardiovasc Res 46:523-530.

56. Ekhterae, D., Lin, D., Lundberg, M.S., Crow, M.T., and Brosius, F.C. 1999. ARC
inhibits cytochrome c release from mitochondria and protects against hypoxia-
induced apoptosis in heart-derived H9c2 cells. Circulation Research 85:70-77.

57. Rossignol, R., Fauston, B., Rocher, C., Malgat, M., Mazat, J., and Letellier, T.
2003. Mitochondrial Threshold Effect. Biochem. J 370:751-762.

58. Davey, G.P., Canevari, L., and Clark, J.B. 1997. Threshold effect in synaptosomal
and nonsynaptosomal mitochondria from hippocampal CA1 and paramedian
neocortex brain region. Neurochem 69:2564-2570.

59. Davey, G.P., Peuchen, S., and Clark, J.B. 1998. Energy threshold in brain
mitochondria. JBiol Chem 273:12753-12757.

60. McHugh, N.A., Merrill, G.F., and Powell, S.R. 1998. Estrogen diminishes
postischemic hydroxyl radical production. Am JPhysiol (Heart Circ. Physiol.)
274:H1950-H1954.









61. Haynes, M.P., Sinha, D., Russell, K.S., Collinge, M., Fulton, D., Sessa, W.C., and
Bender, J.R. 2000. Membrane Estrogen Receptor Engagement Activates eNOS
via PI3K-Akt pathway in human endothelial cells. Circulation Research 87:677-
682.

62. Li, C.Y., Lee, J.S., Ko, Y.G., Kim, J.I., and Seo, J.S. 2000. Heat shock protein 70
inhibits apoptosis downstream of cytochrome c and upstream of caspase-3
activation. JBiol Chem 275:25665-25671.

63. Kacew, S., Ruben, Z., and McConnell, R.F. 2000. Strain as a determinant factor
in the differential responsiveness of rats to chemicals. CEJOEM 6:235-256.

64. Burke BE, Olson RD, Cusack BJ, Gambliel HA, and WH., D. 2003.
Anthracycline cardiotoxicity in transgenic mice overexpressing SR Ca2+-ATPase.
Biochem Biophys Res Commun. 303:504-507.















BIOGRAPHICAL SKETCH

Youngmok C. Jang was born in Seoul, South Korea, where he lived until moving to

New York, in 1984. Youngmok returned to Korea in 1988 where he graduated from

Korea University, Seoul, Korea, with a Bachelor of Science degree in physical education.

In August 2001, Youngmok began his graduate studies in exercise physiology at the

University of Florida where he taught for Human Physiology Laboratory and also worked

as a research assistant under Dr. Christiaan Leeuwenburgh. After earning a Master of

Science in Exercise and Sport Sciences degree, Youngmok plans to pursue his doctoral

degree in the same field at the University of Florida.