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The role of poly (ADP-ribose) polymerase-1 inhibitors

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Title:
The role of poly (ADP-ribose) polymerase-1 inhibitors prevention of non glutathione-dependent carbon tetrachloride-induced hepatotoxicity
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Book
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English
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Grivas, Paul Christopher
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University of South Florida
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Tampa, Fla
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Subjects / Keywords:
PARP
Theophylline
Necrosis
Liver
Free radical
Dissertations, Academic -- Public Health -- Doctoral -- USF   ( lcsh )
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bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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Abstract:
ABSTRACT: Carbon tetrachloride (CCl4) is a hepatotoxicant known to elevate alanine aminotransferase (ALT) and other liver enzyme levels, and cause lipid peroxidation, as well as centrilobular necrosis. A number of poly ADP-ribose polymerase (PARP) inhibitors were administered via intraperitoneal (i.p.) injections to male ICR mice as cotreatments at various time intervals relative to the CCl4. Aminophylline, a water soluble complex consisting of two molecules of theophylline bridged by ethylene diamine, was administered one-half hour, one hour and two hours after CCl4. The levels of ALT in the serum, as well as malondialdehyde and its equivalent markers of oxidative damage in the liver, were significantly reduced by aminophylline, relative to those in mice receiving only CCl4. The hepatoprotective effects of aminophylline were confirmed via the examination of histopathologic samples from the livers of mice receiving aminophylline in conjunction with CCl4 as opposed to those administered CCl4 alone. The potential benefit to society as a result of this research is that aminophylline, which has already been approved by the Food and Drug Administration (FDA), could potentially be administered in the event of an overexposure to CCl4 or similar halocarbons to minimize the free radical-mediated hepatotoxicity resulting from overexposure.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2007.
Bibliography:
Includes bibliographical references.
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by Paul Christopher Grivas.
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Title from PDF of title page.
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Document formatted into pages; contains 141 pages.
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Includes vita.

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oclc - 174146364
usfldc doi - E14-SFE0001953
usfldc handle - e14.1953
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ABSTRACT: Carbon tetrachloride (CCl4) is a hepatotoxicant known to elevate alanine aminotransferase (ALT) and other liver enzyme levels, and cause lipid peroxidation, as well as centrilobular necrosis. A number of poly ADP-ribose polymerase (PARP) inhibitors were administered via intraperitoneal (i.p.) injections to male ICR mice as cotreatments at various time intervals relative to the CCl4. Aminophylline, a water soluble complex consisting of two molecules of theophylline bridged by ethylene diamine, was administered one-half hour, one hour and two hours after CCl4. The levels of ALT in the serum, as well as malondialdehyde and its equivalent markers of oxidative damage in the liver, were significantly reduced by aminophylline, relative to those in mice receiving only CCl4. The hepatoprotective effects of aminophylline were confirmed via the examination of histopathologic samples from the livers of mice receiving aminophylline in conjunction with CCl4 as opposed to those administered CCl4 alone. The potential benefit to society as a result of this research is that aminophylline, which has already been approved by the Food and Drug Administration (FDA), could potentially be administered in the event of an overexposure to CCl4 or similar halocarbons to minimize the free radical-mediated hepatotoxicity resulting from overexposure.
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The Role of Poly (ADP-Ribose) Polymerase-1 Inhibitors: Prevention of Non GlutathioneDependent Carbon Tetrachloride-Induced Hepatotoxicity by Paul Christopher Grivas A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Environmental and Occupational Health College of Public Health University of South Florida Major Professor: Raymond D. Harbison, Ph.D. Carlos A. Muro-Cacho, M.D., Ph.D. Ira S. Richards, Ph.D. Andrea M. Spehar, D.V.M., J.D. Date of Approval: March 19, 2007 Keywords: PARP, theophylline, necrosis, liver, free radical Copyright 2007, Paul Christopher Grivas

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Dedication I dedicate this dissertation to my Mother, Nancy Grivas, and my Father, Jack Grivas. My Mother provided invaluable moral support, and had faith in me when I had scant little in myself. She also ingrained in me at a young age, the importance of a solid work ethic and a commitment to one’s goals. My completion of this dissertation is a testament to her encouragement as much as it is to my own efforts. My Father, through his own struggles with stomach cancer, displayed the type of courage, about which I had only read prior to witnessing in him. This dissertation is also dedicated to the memory of my grandparents, Lawrence and Marion Tatum, James Grivas and Margaret Jaeger. Although they have departed, their desire for a better life for generations that followed them will never die.

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Acknowledgements I would like to express my appreciation to my major professor, Dr. Raymond D. Harbison, for his advice, support and tutelage, which made everything possible. I am also extremely grateful for my other committee members, both past and present. Dr. Philip Roets has also proven invaluable, not only as a committee member, but also with regards to improving my teaching skills. Dr. Ira Richards, through his helpful input and refreshing humor, has proven quite helpful. Dr. Carlos Muro-Cacho has helped immensely in my understanding of pathology. Dr. M. Rony Francois has inspired me through his work ethic and his insatiable thirst for knowledge. Last, but certainly not least, I am grateful for Dr. Andrea Spehar’s meticulous attention to detail. My hosts at Kyoto University also deserve my deepest appreciation. Dr. Kunihiro Ueda and Dr. Seigo Tanaka sped me through the steep learning curve of ADPribosylation. Dr. Masenori Takehashi, Dr. Liping Chen, Dr. Shinya Iida, Dr. Mikio Takano, Jumpei Takagi and Chizu Yamada have also assisted me in ways too numerous to list. Lastly, I am grateful for the support and collaboration of numerous friends and colleagues. Among these are Dr. Melissa Derby, Dr. David Johnson, Dr. Marek Banasik, Dr. Richard Taylor, Kelly Hall, Robin DeHate, Mirtha Whaley, Angela Clem, Michael Martinez, J. Vladimir Mabout, G. Scott Dotson and P. Jill Maxwell.

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i Table of Contents List of Tables ii List of Figures iv Abstract vii Chapter One: Introduction 1 Statement of the Problem 1 Specific Objectives 1 Objective 1 1 Objective 2 2 Chapter Two: Literature Review 3 Carbon Tetrachloride-Induced Hepatotoxicity 3 PARP Inhibition 11 Chapter Three: Materials and Methods 21 In Vitro Methodologies 21 Cytotoxicity Assays 22 PARP Activity Assays 23 In Vivo Methodologies 25 Serum ALT/AST Assays 28 TBARS Assays 29 Histopathologic Staining / Immunohistochemistry 30 Chapter Four: Results 33 In Vitro Results 34 Cytotoxicity Assays 36 PARP Activity Assays 43 In Vitro Conclusions 53 In Vivo Results 53 Serum ALT/AST Assays 54 TBARS Assays 93 Histopathologic Staining / Immunohistochemistry 98 In Vivo Conclusions 113 Summary of Findings 114 Chapter Five: Discussion 117 Explanation of Experimental Findings 117 Impact on Public Health 123 Possible Future Studies 126 Conclusion 131 References Cited 132 About the Author End Page

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ii List of Tables Table 1 Percent Cytotoxicity at Varied PARP Inhibitor Levels 37 Table 2 Percent Cytotoxicity with Varied Carbon Tetrachloride Levels 41 Table 3 PARP Activity as a Function of Duration of CCl4 Exposure 45 Table 4 PARP Activity With and Without 6( 5H )-Phenanthridinone Cotreatment 48 Table 5 PARP Activity With MNNG, Carbon Tetrachloride and 6( 5H )Phenanthridinone. 51 Table 6 Serum AST Levels at Varied Intervals After Intraperitoneal Carbon Tetrachloride Administration 55 Table 7 Serum ALT Levels With and Without 0.2 ml/kg Carbon Tetrachloride 59 Table 8 Serum ALT Levels With Carbon Tetrachloride and Varying 6( 5H )-Phenanthridinone Co-treatments 63 Table 9 Serum ALT Levels with Carbon Tetrachloride and Varied Numbers of 6( 5H )-Phenanthridinone Treatments 66 Table 10 Serum ALT Levels With Delayed 6( 5H )-Phenanthridinone Administration 69 Table 11 Serum ALT Levels With Nicotinamide and Carbon Tetrachloride 72 Table 12 Serum ALT Levels at Two Different Dosages of Nicotinamide 75 Table 13 Serum ALT Levels Using Carbon Tetrachloride, Aminophylline and Nicotinamide 79 Table 14 72-Hour Serum ALT Levels With Aminophylline and Carbon Tetrachloride 82

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iii Table 15 Serum ALT Levels With Three Administrations of Aminophylline 85 Table 16 Serum ALT Levels With Two Administrations of Aminophylline 88 Table 17 Serum ALT Levels with Three Co-Treatments With Aminophylline 91 Table 18 Standards for TBARS Assay 95 Table 19 Hepatic Lipid Peroxidation Levels With Two Dosages of Aminophylline 96 Table 20 Results of Histopathological Assessments for CCl4 and Aminophylline Treatments 100

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iv List of Figures Figure 1. Mechanism of Carbon Tetrachloride-Induced Lipid Peroxidation 5 Figure 2. Pathways of Theophylline Metabolism 20 Figure 3. Percent Cytotoxicity at Varied PARP Inhibitor Levels 38 Figure 4. Percent Cytotoxicity with Varied Carbon Tetrachloride Levels 42 Figure 5. PARP Activity as a Function of Duration of CCl4 Exposure 46 Figure 6. PARP Activity With and Without 6( 5H )-Phenanthridinone Cotreatment 49 Figure 7. PARP Activity With MNNG, Carbon Tetrachloride and 6( 5H )Phenanthridinone. 52 Figure 8. Serum AST Levels at Varied Intervals After Intraperitoneal Carbon Tetrachloride Administration 56 Figure 9. Serum ALT Levels With and Without 0.2 ml/kg Carbon Tetrachloride 60 Figure 10. Serum ALT Levels With Carbon Tetrachloride and Varying 6( 5H )Phenanthridinone Co-treatments 64 Figure 11. Serum ALT Levels with Carbon Tetrachloride and Varied Numbers of 6( 5H )-Phenanthridinone Treatments 67 Figure 12. Serum ALT Levels With Delayed 6( 5H )-Phenanthridinone Administration 70 Figure 13. Serum ALT Levels With Nicotinamide and Carbon Tetrachloride 73 Figure 14. Serum ALT Levels at Two Different Dosages of Nicotinamide 77 Figure 15. Serum ALT Levels Using Carbon Tetrachloride, Aminophylline and Nicotinamide 80

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v Figure 16. 72-Hour Serum ALT Levels With Aminophylline and Carbon Tetrachloride 83 Figure 17. Serum ALT Levels With Three Administrations of Aminophylline 86 Figure 18. Serum ALT Levels With Two Administrations of Aminophylline 89 Figure 19. Serum ALT Levels with Three Co-Treatments With Aminophylline 92 Figure 20. Standard Curve for TBARS Assay 95 Figure 21. Hepatic Lipid Peroxidation Levels With Two Dosages of Aminophylline 97 Figure 22. Hematoxylin and Eosin Staining Results: Control Treatment 101 Figure 23. Hematoxylin and Eosin Staining Results: Aminophylline Treatment 102 Figure 24. Hematoxylin and Eosin Staining Results: Aminophylline and Carbon Tetrachloride Treatment 103 Figure 25. Hematoxylin and Eosin Staining Results: Carbon Tetrachloride Treatment 104 Figure 26. TUNEL Staining Results: Control Treatment 105 Figure 27. TUNEL Staining Results: Aminophylline Treatment 106 Figure 28. TUNEL Staining Results: Aminophylline and Carbon Tetrachloride Treatment 107 Figure 29. TUNEL Staining Results: Aminophylline Treatment 108 Figure 30. Cleaved Caspase-3 Immunohistochemistry Results: Control Treatment 109 Figure 31. Cleaved Caspase-3 Immunohistochemistry Results: Aminophylline Treatment 110 Figure 32. Cleaved Caspase-3 Immunohistochemistry Results: Aminophylline and Carbon Tetrachloride Treatment 111

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vi Figure 33. Cleaved Caspase-3 Immunohistochemistry Results: Carbon Tetrachloride Treatment 112 Figure 34. Summary of Experimental Findings 116

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vii The Role of Poly (ADP-Ribose) Polymerase-1 Inhibitors: Prevention Of Non Glutathione-Dependent Carbon Tetrachloride-Induced Hepatotoxicity Paul Christopher Grivas ABSTRACT Carbon tetrachloride (CCl4) is a hepatotoxicant known to elevate alanine aminotransferase (ALT) and other liver enzyme levels, and cause lipid peroxidation, as well as centrilobular necrosis. A number of poly ADP-ribose polymerase (PARP) inhibitors were administered via intraperitoneal (i.p.) injections to male ICR mice as cotreatments at various time intervals relative to the CCl4. Aminophylline, a water soluble complex consisting of two molecules of theophylline bridged by ethylene diamine, was administered one-half hour, one hour and two hours after CCl4. The levels of ALT in the serum, as well as malondialdehyde and its equivalent markers of oxidative damage in the liver, were significantly reduced by aminophylline, relative to those in mice receiving only CCl4. The hepatoprotective effects of aminophylline were confirmed via the examination of histopathologic samples from the livers of mice receiving aminophylline in conjunction with CCl4 as opposed to those administered CCl4 alone. The potential benefit to society as a result of this research is that aminophylline, which has already been approved by the Food and Drug Administration (FDA), could potentially be administered in the event of an overexposure to CCl4 or similar halocarbons to minimize

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viii the free radical-mediated hepatotoxicity resulting from overexposure.

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1 Chapter One Introduction Statement of the Problem Carbon tetrachloride (CCl4) has been shown to cause hepatic centrilobular necrosis in humans and laboratory animals and the known mechanism of action for CCl4 is thought to be free radical mediated and glutathione independent. It is hypothesized that the acute hepatotoxic effects of a wide variety of environmental pollutants and chemicals used in the workplace can be mediated by modulating the rate of poly ADP ribosylation. Co-treatment or prior treatment of laboratory animals with a PARP inhibitor ( e.g. 6( 5H )-phenanthridinone or aminophylline) may reduce the acute hepatotoxicity produced by a variety of agents, particularly those with mechanisms of action that require free radical production. Specific Objectives Objective 1 : To determine whether the co-administration of aminophylline, a PARP inhibitor, significantly decreases the carbon tetrachloride-induced hepatotoxicity. Carbon tetrachloride was selected to be studied in this research because of the compound’s ability to be used as a model for a number of compounds that have similar mechanisms of action.

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2 The specific aims of this objective include the following: Specific Aim 1 : To determine whether CCl4 induces liver damage using serum assays for alanine aminotransferase (ALT/GPT), aspartate aminotransferase (AST/GOT) and the thiobarbituric acid reactive substances (TBARS) hepatic tissue malondialdehyde assay. This will allow for the testing of the hypothesis that the co-administration of aminophylline reduces the acute toxicity of CCl4. Specific Aim 2 : To confirm the presence of CCl4-induced hepatotoxic effects via histopathologic examination techniques used on the livers extracted from mice, including hematoxylin and eosin (H&E), terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL), and cleaved caspase 3. These staining methods and evaluations are used in order to determine the extent of cellular damage. Objective 2: To determine whether the co-treatment of aminophylline subsequent to CCl4 exposure significantly decreases the acute hepatotoxic effects of the model compound.

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3 Chapter Two Literature Review Carbon Tetrachloride-Induced Hepatotoxicity Carbon tetrachloride (CCl4) is produced via the chlorination of a number of low molecular weight hydrocarbons. For years, it was used as a dry-cleaning solvent, as well as in a number of consumer products for household cleaning and other purposes (ATSDR, 2005). Since the gradual ban of CCl4, first from consumer products in the 1990’s, then from use as a dry-cleaning solvent, production has been dramatically reduced (ATSDR, 2005). Because it is believed to be capable of depleting the ozone layer, CCl4 was one of the compounds to be phased out by 1996 as part of the Montreal Protocol, in which the United States and numerous other countries participated (den Elzen et al., 1992). Prior to this phase out date, the United States Environmental Protection Agency divided the end product limits between the eight firms that were producing CCl4 in 1989. Even with its elimination from consumer products, carbon tetrachloride is persistent in the environment, resulting in systemically absorbed dosages of approximately 0.1 g/kg/day in a typical 70 kg adult who inhales 20 cubic meters of air per day. (ATSDR, 2005). Ironically enough, CCl4 is still being produced as a byproduct in the manufacture of perchlorethylene (tetrachloroethylene, Cl2C=CCl2), the

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4 replacement of CCl4 as a dry cleaning solvent. CCl4 is also released into the atmosphere as a product of the environmental decomposition of perchlorethylene (Singh et al ., 1975). The primary toxicological effects of carbon tetrachloride are that two of its reductive metabolites produced by the CYP2E1 isoform of cytochrome P-450, the trichloromethyl radical ( € CCl3) and the chlorine radical ( € Cl) cause free radical damage to lipids, primarily in the cell and organelle membranes of hepatocytes. (ATSDR, 2005). (Figure 1). The former of these, € CCl3, is of particular concern, because of its rapid incorporation of oxygen to form the trichloromethyl peroxide radical ( € OOCCl3). The trichloromethyl peroxide radical can then interact with lipids (L), such as the phospholipids found in membranes, resulting in the formation of lipid peroxide radicals (LOO € ), which can cause a chain reaction until it is terminated by binding to another radical, pairing their lone electrons. This process of lipid peroxidation can significantly damage hepatic plasma membranes, that ultimately results in a necrotic response. When trichloromethyl and chloride radicals interact with water they stabilize, forming chloroform (HCCl3) and hydrochloric acid (HCl), respectively (Recknagel, 1967). However, the residual product in both cases is € OOH (peroxy radical), a very reactive and harmful compound in its own right. Other possible effects are damage to mitochondria and catecholamine-induced fatty liver and necrosis (Recknagel, 1967).

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5Cl Cl Cl Cl ClC Cl Cl Cl Cl C Cl Cl OO Cl O O Cl Cl Cl Cl Cl O O Cl Cl Cl O O H C C OO O O P450 + + A. B. C. + R + R R R D. R R + R R A. Reductive free radical formation B. Trichloromethyl peroxide radical formation C. Lipid radical formation D. Lipid peroxide radical formation. Figure 1: Mechanism of Carbon Tetrachloride-Induced Lipid Peroxidation. Carbon tetrachloride is dehalogenated by cytochrome P450, resulting in two free radicals, the trichloromethyl radical and the chloride radical. In the presence of oxygen, the trichloromethyl radical will bind and form a trichloromethylperoxy radical. This radical and others can remove an electron from lipids, forming lipid free radicals. These free radicals can then become peroxy radicals when in the presence of oxygen. The lipid peroxy radicals can cause chain reactions until they are terminated by forming stable dimers with other free radicals. Malondialdehyde (MDA), a lipid peroxy radical dimer, is a unit of measurement for the thiobarbituric acid reactive substances (TBARS) assay. Lipid peroxidation results in damage to cell and organ membranes.

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6 There have been a number of in vitro studies that have associated CCl4 exposure with specific hepatocellular pathologic changes. Two fairly recent studies have evaluated gene expression and other changes in response to CCl4 exposure (Harries et al., 2001, Holden et al., 2000). Dai and Cederbaum (1995) determined that dosing HepG2 cells with 2mM CCl4 yielded a 30-50% decrease in cytochrome P4502E1 activity and levels. Beddowes et al. (2003) found elevated 8-oxodeoxyguanosine (8-oxodG) and malondialdehyde guanosine (M1dG) adducts and DNA strand breaks in HepG2 cells dosed with 4mM CCl4 and butathione sulfoxamine (BSO), which depletes reduced glutathione (GSH) levels. Reduction of oxygen in the environment of male Wistar rats from 21% to 7% for the 24-hour interval commencing three hours prior to intraperitoneal administration of 0.1 ml/kg CCl4 resulted in increases in the elevations of ALT and aspartate aminotransferase (AST) in the rats (Shibayama, 1986). Hypoxia and CCl4 were each found to decrease arachidonic acid levels in the livers of male Sprague-Dawley rats, while palmitic, oleic and linoleic acid levels increased, suggesting that hypoxia potentiates CCl4 -induced hepatotoxicity, based on similar effects (Frank et al, 1987). Increased binding of CCl4 metabolites to hepatic microsomal proteins and lipids occurred at 12% oxygen versus the normal 21% in male rats (Shen et al, 1982). Costa and Trudell (1989) strongly suggest that since the primary zone of CCl4-induced hepatic necrosis, the centrilobular area, has the lowest levels of oxygen, it is reasonable to assume that the low oxygen levels are a factor in this localization. These studies run contrary to findings that singlet oxygen radicals were at lower levels in rats subjected to the oxygen equivalent of 4,400 m

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7 altitude, as measured using in situ liver chemiluminescence (Costa et al, 1993). However, rats exposed to elevated concentrations of oxygen in conjunction with CCl4 have increased survivability and diminished hepatotoxicity (Burkhart et al, 1991). The role of the sympathetic nervous system in the exacerbation of CCl4 -mediated hepatotoxicity has been thoroughly studied, and is believed to be a function of thermoregulation. The reduction of the hepatotoxic effects due to CCl4 following spinal cord transection has been shown to vary based on the extent of the transsection, but only when the rats were kept at ambient temperature, and not incubated (Larson and Plaa, 1963). The degree of hepatoprotection decreased as the location of the spinal cord transsection was lowered from the 7th cervical vertebra to the 4th lumbar vertebra, and cervical cordotomy offered no protection against the early phases (3 hours) of oral dosage with 1.25 ml/kg CCl4 (Larson et al, 1964). Cordotomized rats were found to develop lesions due to CCl4 much later (55-65 hours) than non-cordotomized rats, possibly due to the diminution by over 50% of oxygen consumption (Larson and Plaa, 1965). Immunological sympathectomy using antisympathetic nerve growth factor was ineffective against the hepatotoxic effects of CCl4 (Larson et al, 1964). The hepatotoxic effects of CCl4 have also been documented following human exposures. Although the most common route of exposure to CCl4 in humans is via inhalation, transdermal and oral routes of exposure may occur (ATSDR, 2005). Despite efforts to phase out the use of CCl4 as a result of the Montreal Protocol, the National Institute for Occupational Safety and Health (NIOSH) has determined that approximately 58,000 people in the United States st ill work with the hepatotoxicant (ATSDR, 2005).

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8 As exposure to CCl4 via consumer products has greatly diminished, studies of occupational exposures and their impacts on the workers serve as a vital source of human toxicity data. The relative hepatotoxic effects of CCl4 in humans is greater than for similar haloalkanes, and is associated with necrotic, cirrhotic and steatotic hepatic lesions (Zimmerman and Lewis, 1995). A 34-year-old man, who accidentally ingested “half a mouthful” of CCl4 recovered liver function, as determined by enzyme assays, within 15 days, but his kidneys were unable to reabsorb water after 139 days (Alston, 1970). Increased radiographic density was found in the gastrointestinal tract of a 22-year-old man, who ingested approximately 12 ounces of CCl4 with the same amount of water, in a failed suicide attempt (Bagnasco et al, 1978). A seaman who used CCl4 to clean shipboard machinery was admitted to a hospital with nausea, vomiting, headaches and lightheadedness, elevated heart rate and blood pressure; returning to normal liver function after artificial ventilation, peritoneal dialysis and other inpatient care for two weeks (Hadi and El Mikatti, 1981). The use of CCl4 as a carpet cleaning solvent led to hepatoma in the resident five days later, with the patient’s reports of nausea, vomiting and anuria (Tracey and Sherlock, 1968). Workers exposed to concentrations of CCl4 above 1 ppm for periods ranging from less than 1 year to 5 years had significantly elevated levels of hepatic enzymes, but those exposed to lesser concentrations did not (Tomenson et al ., 1995). Chronic exposures to CCl4 may cause micronodular cirrhosis and hypertension of the portal system, which can arise decades after the exposure, as Gitlin (1980) found in an aircraft repairman exposed during the 1940’s.

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9 A number of factors have been shown to exacerbate the hepatotoxic effects of CCl4 in laboratory animals, among them other xenobiotics and hypoxia. Male and female ICR mice orally administered 2,5-hexanedione (15 mmol/kg) 18 hours prior to intraperitoneal administration of CCl4 (10 ul/kg) had 22-fold and 300-fold elevations in serum ALT relative to those lacking the pretreatment, respectively (Jernigan and Harbison, 1982). Also, the 48-hour mean lethal dosage of CCl4 was decreased 67-fold in chlordecone-treated (10 ppm in diet) rats, versus 1.6-fold for phenobarbitol-treated (225 ppm in diet) counterparts (Mehendale, 1984). Kodavanti et al (1990) found that hepatic ATP levels in rats given intraperitoneal injections 100 l/kg did not decrease significantly. The same dosage in rats fed 10 ppm chlordecone resulted in reductions of 31% and 81% at one and six hours, respectively. Two catecholamines, epinephrine and norepinephrine, were found to potentiate CCl4-induced hepatotoxicity in mice, as evidenced by elevated ALT levels, whether administered concomitantly with, or six hours before or after CCl4 (Schwetz and Plaa, 1969). Perhaps not surprisingly, intraperitoneal co-treatment of male ICR mice with 200 mg/kg phenylpropanolamine, which produces effects similar to catecholamines, potentiated the effects of intraperitoneal CCl4 dosages of 20-200 l/kg (Roberts et al, 1991). Concomitant intraperitoneal co-administration of rats with methamphetamine along with CCl4 significantly increased the extent of liver injury, based on elevated ALT results, as well as histopathologic differences (Roberts et al, 1995b). The mechanism for this potentiation was studied in mice, and is not believed to be linked to binding of CCl4 to proteins and lipids, as it was not observed when methamphetamine dosage occurred

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10 three hours following CCl4 (Roberts et al, 1995a). Furthermore, the potentiation of CCl4induced hepatotoxicity was found not to be due to increased concentrations of chloroform (Roberts et al, 1994). There is reason to believe that ethanol may exacerbate the hepatic damage caused by CCl4, either through the increased activity of one or more metabolic enzymes or consumption of oxygen. Wei et al (1971a, 1971b) found that ethanol, with or without induction of cold shock, can potentiate the hepatotoxicity of CCl4, as evidenced by elevated ALT levels. Hasumura et al (1974) attributed the potentiation by ethanol to increased ornithine carbamyl transferase and ALT activity. Co-treatment with ethanol is believed to exacerbate the hepatotoxic effects of CCl4, at least in part, as a result of decreased hepatic oxygen tension (Sato et al, 1983). This decreased oxygen tension may be a result of the oxidative metabolism of ethanol, into acetaldehyde and ultimately into acetic acid. Deer et al (1987) assessed the concentrations of CCl4 resulting from grain fumigation, finding that all time-weighted average (TWA) exposures were well below 2 ppm, averaging from 0.002 to 0.1 ppm, varying as a function on the type of task being performed. These airborne concentrations of CCl4 were far below the Occupational Health and Safety Administration (OSHA) permissible exposure limit (PEL) TWA of 10 ppm, and its ceiling of 25 ppm. The usage of CCl4 as a fumigant was to be phased out in the United States pursuant the Montreal Protocol, due to its potential to deplete the ozone layer (den Elzen, 1992). Despite this, CCl4 can be still used to create more complex compounds, since the Montreal Protocol was primarily focused on the end use or disposal

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11 of these compounds. As such, people working in proximity to chemical processes utilizing CCl4 may still be at some risk of exposure. PARP Inhibition Poly (ADP-Ribose) Polymerase-1 (PARP-1) (Enzyme Commission Number 2.4.2.30) is an enzyme located in the nucleus, and becomes active in the presence of DNA damage. PARP-1 binds nicotinamide adenosine dinucleotide (NAD+), and then affixes the adenosine diphosphate–ribose (ADP-ribose) moiety of NAD+ to a target protein or DNA. Unlike Mono (ADP-ribosyl) Transferase (Enzyme Commission Number 2.4.2.31), which only adds one ADP-ribose moiety to the site of damage, PARP1 adds a chain of ADP-ribose moieties, with occasional side branches. This process of polymerizing ADP-ribose onto a double strand DNA terminus can deplete the cell of NAD+. As a result of this depletion, adenosine triphosphate (ATP), a critical energy storage compound, can become depleted as a result of diminished NAD+, possibly resulting in necrosis. The activity of PARP-1 has been found to increase in the presence of DNA strand termini, responding to the likely double strand breaks. N-methyl-N’-nitro-Nnitrosoguanidine (MNNG) is a compound that has been found to activate PARP-1, due to the ability of MNNG to cause DNA strand breaks, and serves as a positive control in PARP assays. It is hypothesized that the severity of acute hepatotoxic effects of a variety of environmental pollutants and chemicals in the workplace can be mediated by modulating

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12 the rate of poly ADP ribosylation. A number of compounds have been found to inhibit Poly (ADP-Ribose) Polymerase (PARP) (Banasik et al., 1992). Ueda et al. (1995) reported that inhibition of PARP may play a role in initiating cellular differentiation. It is hypothesized that the co-treatment or prior treatment of laboratory animals with a PARP inhibitor ( e.g. 6( 5H )-phenanthridinone) would reduce the acute hepatotoxicity of a number of compounds, particularly those with mechanisms of action that require free radical production. A similar effect, as reported by Ueda, has been reported when PARP inhibitors were administered following cardiac reperfusion, to minimize the free radicalmediated oxidative damage that often results. Similarly, Ueda reported in a seminar (2003) that zonal necrosis often occurs at the site of myocardial infarctions (heart attacks) and could be minimized by the timely administration of a PARP inhibitor. Poly (ADP-ribose) polymerase, also known as PARP, poly (ADP-ribose) synthetase (PARS), is a critical enzyme that responds to DNA single-strand breakage. Although it can benefit cells at normal activity levels, excessive induction of PARP can deplete cells of the primary substrate for PARP, nicotinamide adenine diphosphate (NAD+) (Soriano et al., 2001). NAD+ is necessary for ATP production, specifically for glycolysis and the electron transport chain. In the absence of NAD+ and ATP, cells often undergo necrosis. PARP is also believed to modulate nuclear factor kappa B activity and induce genes for inducible nitric oxide synthetase and intercellular adhesion molecule 1 (Soriano et al., 2001). As a result of these PARP-mediated changes, an inflammatory response ensues. From their studies of PARP-1 knockout mice, Shall and de Murcia (2000) found

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13 that PARP appears to play a critical role in the inflammatory response, as well as in the oxidative damage associated with reperfusion. Mota-Felipe et al. (2002) used a battery of tests to evaluate the impact of the PARP inhibitor, 5-aminoisoquinolinone (5-AIQ), on reperfusion injury in male Wistar rats. They found significantly lower increases in serum gamma-glutamyl transferase, lactate dehydrogenase, transaminases, as well as significantly lower amounts of liver malondialdehyde, which is a measurement of lipid peroxidation, in rats treated with 5-AIQ. Another group of enzymes, mono (ADP-ribosyl) transferases, is similar to PARP in that they both utilize NAD+ and transfer ADP-ribose. However, not only do they transfer a different number of ADP-ribosyl moieties, but they also target different amino acids and proteins (Banasik and Ueda, 1994). Banasik et al. (1992) evaluated the effectiveness of a number of PARP inhibitors. They found four highly effective inhibito rs, 4-amino-1,8-naphthalimide, 6(5H)and 2nitro-6( 5H )-phenanthridinones, and 1,5-dihydroxyisoquinoline, which had 50% inhibitory concentrations ranging from 0.18 to 0.39 mol/l. These concentrations were roughly two orders of magnitude greater than the comparable concentration for 3aminobenzamide. Benzamide, a PARP inhibitor, has been shown to diminish the production of oxygen radicals by tumor promoter-activated neutrophils (Troll et al., 1990). Other compounds and intracellular conditions may have lesser impacts on PARP activity. Banasik and Ueda (1999) found that dimethyl sulfoxide is a biphasic inhibitor

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14 of PARP at 37 C, and is monophasic at 25 C. As with many other enzymes, the activity of PARP may be modulated by changes in pH, osmolarity, temperature and other factors. A number of PARP inhibitors have been shown to induce cell differentiation in murine teratocarcinoma EC cells, with a nearly complete change within one week (Ueda et al., 1995). This ability to promote differentiation in an immortalized, nondifferentiated cell line may encourage further research on the anticancer potential of PARP inhibitors. Rappeneau et al. (2000) exposed a human bronchial epithelial cell line (16HBE14o-) to mechlorethamine, a nitrogen mustard, to test the effectiveness of 6( 5H )phenanthridinone and other PARP inhibitors. These inhibitors were found to prevent the metabolic dysfunction found in cells only exposed to mechlorethamine. A study by Cole and Perez-Polo (2002) using PC12 cells, which model sympathetic neurons, showed that treatment with 3-aminobenzamide, a PARP inhibitor, significantly reduced the percentage of cells that died after hydrogen peroxide exposure. Pacher et al. (2002) tested the efficacy of PJ34, a phenanthridinone-derivative PARP inhibitor, to reduce the cardiotoxic effects of Escherichia coli endotoxin (lipopolysaccharide). Mice and rats that received both the endotoxin (55mg/kg) and the PARP inhibitor had increased survival and cardiac function than those that received the endotoxin alone. Pacher et al. (2002) also tested the ability of PJ34 to reduce the cardiotoxicity of doxorubicin in BALB/c mice, finding improved cardiac function and survival, along with diminished serum creatine kinase lactate dehydrogenase activity levels. Jagtap et al. (2002) performed a series of ex vivo analyses of the protective effects of PJ34 against oxidative damage associated with splanchic occlusion and

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15 reperfusion in Balb/cmice. Also administered was E. coli lipopolysaccharide (10 mg/kg) via intraperitoneal injection to Wistar rats. PJ34 was found to reduce the production of inflammatory cytokines and chemokines in a dose-dependent fashion. Liaudet et al. (2002) also used the same PARP inhibitor to determine whether E. coli lipopolysaccharide-induced lung inflammation could be minimized. The BALB/c mice that received both lipopolysaccharide and PJ34 had lower levels of cytokines, chemokines, nitric oxide production, lipid peroxidation, neutrophil accumulation in alveoli, and excess lung permeability than mice that only received the lipopolysaccharide. The efficacy of two PARP-1 inhibitors, 6( 5H )-phenanthridinone and benzamide, to reduce the development of experimental allergic encephalitis (EAE) in myelinimmunized rats was evaluated by Chiarugi (2002). These PARP inhibitors significantly reduced clinical score, neuroimmune infiltration, nitric oxide synthase, interleukin-1beta and -2, cyclooxygenase-2, tumor necrosis factor-alpha and interferon-gamma in the spinal cord, which are all associated with the development and progression of EAE. Also there was no evidence of PARP-1 production, nor that of NAD(+) or ATP depletion, which can occur as a result of the substantial induction of PARP activity in these rats (Chiarugi, 2002). Aminophylline is a compound consisting of two molecules of theophylline, bridged by ethylene diamine. Aminophylline is of medical importance, as it is watersoluble, which allows for more versatile modes of administration, primarily via injection. Once aminophylline comes in contact with biological fluids, it disassociates into its constituents, two molecules of theophylline and one molecule of ethylene diamine. The

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16 Food and Drug Administration (FDA) has issued a fact sheet on aminophylline regarding its potential for adverse effects from its use in thigh creams, which they attribute to reports of allergic responses to ethylene diamine, a component in aminophylline. Aminophylline has a low therapeutic index, as is the case with theophylline. As such, the doses which have been found to be efficacious anti-asthma remedies are not much lower than those associated with adverse health effects. Fortunately, with the advent of aminophylline, it has become easier to titrate the dosage of aminophylline to one that is efficacious, yet lacking in adverse response. Theophylline (CAS # 58-55-9) is a naturally occurring methylxanthine alkaloid found in tea and chocolate, and is structurally similar to theobromine and caffeine (NTP, 1998). It was first isolated from tea by Albrecht Kossel in 1888, and was initially synthesized by Wilhelm Traube twelve years later. Theophylline is administered to asthmatics to relax the smooth musculature of the bronchi, resulting in bronchial dilation and diuresis (NTP, 1998). Theophylline can also stimulate the heart and the central nervous system via positive inotrophic (increased contractile force) as well as through positive chronotrophic (increased pulse) actions, resulting in elevated blood pressure (NTP, 1998). Theophylline, a non-selective phosphodiesterase inhibitor, has been proven to be a highly effective inhibitor of the release of histamine from human basophils (Weston et al ., 1997). Given the common use of theophylline, and its similarity in structure to a number of other pharmaceuticals, the National Toxicology Program administered an array of tests to determine the potential for toxicological and/or carcinogenic outcomes in mice and rats (NTP, 1998). There were no reports of

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17 theophylline-induced carcinogenicity in humans (NTP, 1998). Although there were some lesser findings ( e.g. increased rates of uterine hyperplasia in female rats), no proof of carcinogenic action was seen as a result of theophylline in male and female F344/N rats administered up to 75 mg/kg of theophylline, nor in male or female B6C3F1 mice administered up to150 mg/kg and 75 mg/kg of theophylline, respectively (NTP, 1998). There have been many discoveries about the mechanism of action for theophylline, which is the functional unit and biologically active component of aminophylline. Its structure resembles that of DNA and RNA purine bases (Cornish, 1957). As a result of this similarity, Bruns (1980) found that theophylline competes with adenosine for the nucleoside’s receptors, and results in ATP, ADP and AMP-induced increases in cyclic AMP (cAMP) levels. This is important because of cAMP’s role in cell signaling, potentially leading to apoptotic or necrotic responses. Theophylline also functions as a non-specific phosphodiesterase inhibitor, distinguishing itself from newer phosphodiesterase-4 inhibitors (Boswell-Smith et al., 2006). Another important mechanism of action associated with theophylline entails reversing the oxidation of histone deacetylase as a result of oxidative damage (Ito et al., 2002). Theophylline activates histone deacetylases, which then suppress gene transcription, including those for inflammatory gene products (Barnes, 2005). Theophylline works in conjunction with corticosteroids, which activate the glucocorticoid receptors, which are also critical to the suppression of inflammatory gene product transcription (Barnes, 2005). This antioxidative mechanism may be a factor in preventing lipid peroxidation. Yano et al ., (2006) found that theophylline inhibits the conversion of pulmonary fibroblasts to

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18 myofibroblasts by TGF-beta in COPD and asthma patients via cAMP and Protein Kinase A. Theophylline also inhibits the production of collagen by suppressing its messenger RNA. (Yano et al ., 2006). Huber et al ., (2006) reported that theophylline, 200 mg administered via intravenous fluids 30 minutes prior to 100 ml of iodinated contrast medium, protected the kidneys, as evidenced by significantly lower serum creatinine levels, likely due to its adenosine receptor antagonism. Last, but not least, theophylline (0.2, 1, 10 mM) significantly reduced the depletion of NAD+ caused by hydrogen peroxide (300 M) in human lung epithelial cells(Moonen et al 2005). This is attributed to the inhibition of PARP (Moonen et al ., 2005). Theophylline can be metabolized by four distinct mechanisms. Caffeine (1,3,7,trimethyl xanthine) can be demethylated in a reversible fashion to theophylline (1,3dimethylxanthine (Minton and Henry, 1996). Subsequent irreversible demethylations can then occur at the 1 or 3 positions, yielding 3-methylxanthine or 1-methylxanthine, with the latter rapidly metabolized to 1-methyluric acid (Minton and Henry, 1996). An alternate route of metabolism for theophylline involves its irreversible oxidation to 1,3 dimethyluric acid. Caffeine was the most common product of theophylline metabolism in explanted fetal livers, followed by 1,3-dimethyluric acid and 3-methylxanthine (Aranda, 1979). The kinetics of metabolism is reported to be first-order at therapeutic levels, then becomes zero-order at higher levels once the enzymes become saturated (Minton, 1996). Although this series of reactions is known to exist, additional factors can alter the metabolism of theophylline. Theophylline has been found to bind to proteins in serum, thereby altering the pharmacokinetics (Ogilvie, 1978). In an analysis of 47

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19 patients treated with theophylline, Jacobs et al. (1976) found that there was little correlation between oral dosage of theophylline and its concentration in serum. This makes it difficult for clinicians to titrate the appropriate dosage of theophylline, which is efficacious, yet does not cause any toxic effects. Theophylline has a low therapeutic index, in that the ratio between toxic dosages or levels and those that are efficacious is low (serum levels range from 10-20 g/ml (Minton, 1996)). However, Jacobs et al (1976) reported gastrointestional symptoms of toxicity, such as nausea and vomiting, at levels as low as 15 g/ml, which became more common at levels in excess of 25 g/ml. Non-gastrointestinal symptoms of theophylline toxicity included tremors at 26.4g/ml, agitation at 28.5g/ml, seizures at 39.9 g/ml, and two patients with tachycardia at 46.3 and 49.5 g/ml (Jacobs, 1976). A number of compounds can potentiate the toxic effects of theophylline. Adult male rats which were pretreated intravenously with caffeine required significantly less theophylline, also administered intravenously, to induce seizures (Yasuhara, 1988). This would appear self-explanatory, given the reversible conversion of theophylline to and from caffeine, with the presence of the latter shifting the equilibrium in favor of the former.

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20 Figure 2. Pathways of Theophylline Metabolism (Minton and Henry, 1996)

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21 Chapter Three Materials and Methods There were several experimental procedures utilized to assess the level of damage induced by CCl4 and inhibited by inhibitors of (poly ADP-ribose) polymerase-1 (PARP1). The differing nature of the in vitro and in vivo experiments allows for a division of this chapter into separate parts for in vitro and in vivo methodologies. In Vitro Methodologies All in vitro experiments were conducted using hepatocytes from a HepG2 cell line in the laboratory of Dr. Kunihiro Ueda at the Institute for Chemical Research at Kyoto University. These hepatocytes were acquired by Dr. Kunihiro Ueda and Dr. Seigo Tanaka from the Rikken Laboratories in Japan. The HepG2 cell line is derived from human primary hepatic cancer cells excised from a patient, and is widely utilized to assess the hepatotoxic effects of various compounds. The cell viability assay that was utilized to determine the proper dosage of 6( 5H )phenanthridinone to protect the hepatocytes was the lactate dehydrogenase (LDH) cytotoxicity assay. These cytotoxicity assays were used to determine whether 6( 5H )phenanthridinone and other PARP inhibitors were protective against CCl4-induced hepatotoxicity. LDH is an intracellular enzyme which, when found in the extracellular

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22 milieu, is indicative of a loss of plasma memb rane integrity. The two phospholipid layers of the plasma membrane are susceptible to oxidative damage as a result of the trichloromethyl peroxide radi cal and other metabolites of CCl4. The extent of cell mortality caused by a toxicant can be estimated by comparing the absorbance at 490 nm of the cell culture media for various treatment groups in comparison with a group treated with a lysis solution, which serves as a positive control. After showing a reduction in LDH leakage into the cell culture media, PARP activity assays were conducted to determine whether the observed hepatoprotective effect of 6( 5H )-phenanthridinone was related to the inhibition of PARP. Cytotoxicity Assays In order to determine whether the co-treatment with 6( 5H )-phenanthridinone is beneficial to the hepatocytes, LDH concentrations in cell media were measured using a colorimetric assay (Promega, Madison, WI, USA). Samples containing 2 x 104 HepG2 cells in 200 l of Eagle minimal essential media (1 x105 cells/ml) HepG2 cells were resuspended and transferred to each of 48-well plates in 200 l of Eagle minimal essential media per well. The media was treated with the appropriate concentrations of CCl4 ranging from five to twenty millimoles per liter (mM), and 6( 5H )-phenanthridinone ranging from ten to forty micromoles per liter (M). Each treatment combination was tested in triplicate on 48-well plates, with triplicate blanks, which contained treated media, but no HepG2 cells. The well plates were incubated at 37C for a period of twenty-four hours prior to the start of the assay. The positive control was established via the administration of 20l of the 10x cell lysis solution provided with the assay reagents

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23 two hours prior to the assay in wells with previously untreated cells. The supernatants were drawn from each of the wells and centrifuged at 250g for four minutes, and 50 l of each supernatant was placed in its corresponding well on a 96-well plate. Fifty microliters of a sample buffer containing NAD+ and lactate, substrates for lactate dehydrogenase, were added and then the well plate was incubated in a closed drawer for thirty minutes. After the incubation period expired, 50 l of the stop solution was added to each well, and the well plate analyzed. The absorbance readings were taken using a Bio-Rad Model 550 microplate reader (Hercules, CA, USA) at a wavelength of 490 nm. PARP Activity Assays In order to determine whether the activity of PARP-1 is modulated by the administration of CCl4 and/or 6( 5H )-phenanthridinone, an in vitro assay involving the incorporation of radiolabeled NAD+ into poly-ADP ribose was utilized. HepG2 cells were cultured to a minimum of 50% confluence on 60 mm glass cell culture dishes. Duplicate treatments with CCl4 and/or 6( 5H )-phenanthridinone were conducted via dilution in 5 ml of cell culture media, sonication, then replacement of the previous cell culture media with the appropriate treatment. Culture dishes were returned to the incubator following the change of media. Upon completion of the exposure interval, the cell culture dishes were removed from the incubator. After the cells were scraped from each of the culture dishes and pipetted into its own 50 ml centrifuge tube, each of these dishes was washed with 5 ml of phosphate buffered saline (PBS, pH 7.2), which was also pipetted into the appropriate centrifuge tube. These tubes (kept on ice) were

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24 subsequently centrifuged at 3,000 revolutions per minute (rpm) for five minutes at 4 C. Digitonin, which is used to perforate the nuclear and plasma membranes of the HepG2 cells, was administered at 0.005% in PBS at 4 C. The pellets were resuspended in 1.00 ml of 0.005% digitonin, transferred to microcentrifuge tubes, and then kept on ice for 3 minutes. Following this interval, the microcentrifuge tubes were centrifuged at 6,000 rpm for 5 minutes at 4 C. After the supernatant was removed, the pellets were resuspended in 1.00 ml of PBS, which allowed for the cells to be counted. The numbers of cells were equalized by resuspending the cells, then removing the proportionate volume of PBS and cells. After this, the microcentrifuge tubes were spun once again at 6,000 rpm for 5 minutes at 4 C. The supernatants were removed from the microcentrifuge tubes, and the pellets were kept on ice. The reaction mixture for this assay was partially prepared in a 50 ml centrifuge tube, and consisted of 190 l of reaction buffer and 5 l of 10 mM NAD per sample, plus two additional volumes of each, one for the background reading and one to ensure the transfer of the entire volume from the centrifuge tube to each sample. The remaining 5 l of carbon-14 radiolabeled NAD (0.1 uCi Adenosine 14C NAD) per sample (with two additional aliquots) was added in the radioisotope laboratory. This reaction mixture was mixed and kept on ice until used. The samples were individually resuspended in the reaction mixture and were placed in a 30 C bath for 5 minutes, to allow for the formation of poly-ADP ribose. This kinetically based reaction was halted by the addition and pipette-based mixing of 800 l of ice-cold (0-4C) 25% trichloroacetic acid (TCA). The samples were placed on ice for 30 minutes. The samples were then vacuum-filtered using methylcellulose membranes and washed

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25 thrice with 3 ml of 5% TCA. The membranes were dried under heat lamps for 30 minutes. After that, they were placed in liquid scintillation vials containing 5 ml of scintillation fluid. These vials were counted for carbon-14 over 5 minute intervals. The resulting counts per minute for each sample were indicative of the activity of PARP. In Vivo Methodologies All of the animal experiments were performed in accordance with the animal welfare guidelines established by the Division of Comparative Medicine, University of South Florida Office of Research. The Division of Comparative Medicine has been fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). The National Research Council devised the Guide for the Care and Use of Laboratory Animals which has also been the basis of many of the policies, along with the FDA Good Laboratory Procedures. The male ICR mice were housed at the vivarium at the USF College of Public Health for a period of no fewer than seven days to allow for them to become acclimated. Prior to being sacrificed, the mice received tap water and food (Harlan Rodent Chow, 2018) ad libitum Male mice were selected exclusively for these experiments to eliminate the potential for variation as a result of murine ovulatory and hormonal cycles. Each of the mice was weighed prior to dosing to ensure the proper dosage was administered. Since the toxicant used in these experiments, CCl4, was dissolved in corn oil and the co-treatments; 6( 5H )-phenanthridinone, benzamide, nicotinamide and aminophylline, were dissolved in saline (with dimethyl sulfoxide in the case of 6( 5H )-

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26 phenanthridinone), two separate injections were required. Control mice were administered injections with both solvents, and mice receiving either the toxicant or the co-treatment were administered the solvent for the other treatment. This process of injecting the vehicle for mice not receiving the toxicant and/or co-treatment is performed to ensure that any difference in effects observed in the mice, their serum and tissue assays and the liver histopathology from the treatments would have to be a function of the chemicals themselves, and not from the injections. The injections have the potential to cause inflammatory responses to the peritoneal cavity and skin, as a result of the tearing of the skin by the needle and the introduction of bacteria and other flora/fauna from the fur and surface of the skin into the peritoneal cavity. This can be further exacerbated by the introduction of bacteria to the site of injection through scratching or biting by the mouse that was injected, or by other mice in the same cage. The impact of the injections were minimized by utilizing tuberculin syringes with 25 gauge needles and by replacing them frequently, to ensure that the edges the needles are sufficiently sharp. Following the intraperitoneal injection of the appropriate treatment regimen, the abdominal fur of each mouse was inspected to confirm that the entire dosage was delivered, and did not seep out of the site of the needle puncture. Upon the completion of the treatment duration, the mice were euthanized via simple asphyxiation with carbon dioxide. As a result of this process, the mice underwent respiratory arrest, during which time the blood became aggregated in the heart and the lungs. Blood was taken from mice via cardiac puncture with a 1 ml tuberculin syringe with a 25 gauge needle. Then, with the needle removed, the blood was slowly expelled

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27 from the syringe into a 1.5 ml microcentrifuge tube. Under optimal conditions, the volume of blood collected per mouse ranged from 0.7 to 1.0 ml. After waiting for a period of 10 minutes, the blood was centrifuged at 6,000 x g for 10 minutes. Following centrifugation, the supernatant (serum) from each microcentrifuge tube was transferred to its corresponding serum sample microcentrifuge tube using a 200 l micropipetter. Following the collection of the serum samples, they were stored at 4 C for less than two days prior to being assayed, to minimize the potential for degradation. In summation, Male ICR mice (Harlan, Indianapolis, IN) were housed and handled in accordance with AAALAC guidelines. Mice were randomized into four groups, receiving either CCl4 (0.025 ml/kg, Sigma-Aldrich, St. Louis, MO) in corn oil or corn oil via intraperitoneal (IP) injection, with total volumes of 150 l. At intervals of 30, 60, and in some experiments 120 minutes following the toxicant, mice were injected with 150 l of aminophylline (50 mg/kg, Sigma-Aldrich) in PBS or saline. Mice were sacrificed after 24 hours, so that the blood and liver tissue could be collected. Serum ALT was assayed with an endpoint colorimetric assay (Teco Diagnostics, Anaheim, CA). Liver samples were homogenized 10:1 in saline, and the homogenates were assayed for lipid peroxidation, in malondialdehyde equivalent concentrations, using the thiobarbituric acid reactive substances (TBARS) assay (Northwest Life Science Specialties, Vancouver, WA). Liver samples were also collected in formalin and encased in paraffin for histopathhologic analysis. The procedures used were hematoxylin and eosin H&E staining, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay and caspase-3 immunohistochemistry. One way ANOVA was used to assess the

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28 significance of differences, with p-values below 0.05 indicative of significant differences. Serum ALT/AST Assays Two of the most efficient methodologies available to evaluate the extent of the CCl4-induced hepatocellular damage are the colorimetric endpoint assays for ALT and AST. Other assays are available, however personal experience with some of the kinetic assays suggests that sera from mice with extensive hepatocellular damage may be difficult, if not impossible to use. The assay reagents (Teco Diagnostics, Anaheim, CA) were utilized to produce, an endpoint r eaction, which can be detected in the visible light spectrum, as previously reported by Reitman and Frankel (1957). The substrate solution consisted of 0.2 M L-alanine, 2.0 mM alpha-ketoglutaric acid, 100 mM phosphate buffer at pH 7.4 and 0.2% preservatives. A portion of the L-alanine from the substrate was converted to pyruvate, and the alpha-ketoglutaric acid was converted to glutamic acid. The resulting pyruvate was subsequently reacted with a color reagent consisting of 1.0 mM 2,4-dinitrophenylhydrazine in 1 N hydrochloric acid, to yield 2,4dinitrophenylhydrazone. This red-colored product absorbs visible light, with a maximum absorbance at 505 nm. The substrate solution was transferred in 250 l aliquots via micropipette into labeled microcentrifuge tubes. The serum samples were removed from storage at 4C and centrifuged at 6,000 x g for ten minutes at ambient temperature, to ensure that any erroneously transferred erythrocytes or other materials are relegated to the bottom of the microcentrifuge tubes prior to removal of aliquots for the assay. After warming the

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29 substrate in a 37 C water bath for three to five minutes, 50 l of serum was transferred to the corresponding tube for each sample. A reagent blank was also prepared, along with a standard, in which 50 l of a 70 IU/ml standard was used in lieu of serum. The original microcentrifuge tubes containing serum were returned to storage at 4 C, and those containing the substrate solution and serum or standard were mixed and incubated at 37C for a period of 30 minutes along with the reagent blank. Following this interval, 250 l of color reagent was added to each microcentrifuge tube, and the samples were once again mixed and returned to the 37 C water bath for a ten minute incubation period. After the ten minutes elapsed, 1.00 ml of the color developing solution was added to each microcentrifuge tube, which was subsequently mixed and returned to the water bath for five minutes. After this interval, the samples were transferred to disposable semi-micro cuvettes, which were placed into a UV/Visible spectrophotometer, and the absorbances were recorded at a wavelength of 505 nm. TBARS Assays After the mice were euthanized, their livers were removed and placed in 50ml centrifuge tubes on ice and subsequently frozen at -70 C until they could be homogenized and assayed. The livers were homogenized in a 1:10 weight to volume proportion in cold PBS (Fisher Scientific, Pittsburgh, PA) and returned to the -70 C freezer until the assays were conducted. The TBARS assay (Northwest Life Science Specialties, Vancouver, WA) provided a saline buffer for this purpose, but its volume would have been insufficient for the requisite dilutions, so the PBS from the above

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30 supplier was utilized in lieu of the one provided. Microcentrifuge tubes were labeled for the standards and the samples. Ten l of butylated hydroxytoluene (BHT) was added to each tube, followed by 250 l of the standard or liver homogenate, 250 l of 1 M phosphoric acid and 250 l of thiobarbituric acid (TBA) solution (TBA in 10.5 ml deionized water). The tubes were closed and mixed with a vortex mixer for five seconds, and then incubated at 60 C on a dry bath (Fisher Scientific, Pittsburgh, PA) for one hour. The tubes were then centrifuged at 10,000 x g for three minutes, and the absorbance readings were taken at 572 nm. The absorbance readings for the standard concentrations (in MDA equivalents) were used to create a standard curve, which was used to determine the levels of MDA equivalents for the homogenates. These results were then multiplied by 11, to account for the 1:10 dilution, to determine the final concentrations of MDA equivalents. Histopathologic Staining / Immunohistochemistry Livers were preserved in 10% neutral buffered formalin (Fisher Scientific, Pittsburgh, PA). The livers were sectioned (5 6 mm), dehydrated with ethyl alcohol, cleared with xylene and embedded in paraffin. Sections of 5to 6-mm were mounted, dried and stained with hemotoxylin/eosin to assess parenchymal histopathological changes. The methodology used to evaluate hepatotoxicity has been described previously (Price et al. 1999). Briefly, the parameters evaluated were proliferation, apoptosis, necrosis and fibrosis. Proliferation was determined according to the presence of four parameters: hepatocyte hyperplasia, hepatocyte hypertrophy, mitotic activity and

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31 binculeation. To determine proliferation intensity, the sum of the individual intensity of each of the four parameters was used to reach a total score of no observable pathological changes (0), mild intensity (1), moderate intensity (2) or prominent intensity (3). Apoptosis was evaluated by using ApopTag Plus In Situ Apoptosis Detection, according to a standard protocol. This method allows for visualization of apoptotic bodices and also intact apoptotic nuclei. It is a useful method for detecting apoptosis at the earliest stages. A solid brown nuclear staining identified apoptotic hepatocytes. The number of apoptotic cells was expressed as a percentage of total number of cells (Apoptotic Index, AI). The AI was then converted to a numerical score. No observable apotosis is given a score of zero, and this equates to AI 0%; mild intensity is given a score of 1, and represents AI <25%; moderate intensity is given a score of 2, and represents AI 25% to 50%; prominent intensity is given a score of 3, and represents AI 50%. Cleaved Caspase-3 histopathology was performed using Cleaved Caspase-3 (Asp175) (5A1) Rabbit Monoclonal Antibody from Cell Signaling Technology. The method of immunochemistry was used. Cleaved caspase-3 (Asp175) detects levels of large fragments (17-19 kDa) of activated caspase-3 resulting from cleavage adjacent to Asp175. Expression of cleaved caspase-3 stains a deep red to brown in color. The scale for expression is 0 to 4. A score of 0 is no expression of cleaved caspase-3. A score of 1 is <25% of the centrilobular region expressing cleaved caspase-3. A score of 2 is 26-50% expression. A score of 3 is 51-75% expression. A score of 4 is 76-100% expression. The cleavage of caspase-3 is a step in the apoptotic response which commits cells to

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32 programmed cell death.

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33 Chapter Four Results The results of these experiments strongly suggest that the administration of CCl4 causes hepatocellular damage. This is evidenced in the significant increases in LDH levels and PARP activity in a human cell line (HepG2), as well as in the increases in ALT, lipid peroxidation, and the histopathological findings of necrosis and apoptosis in ICR mice. The use of 6(5H)-phenanthridinone or aminophylline significantly reduced or blocked these adverse effects to the liver. This suggests that PARP inhibitors may play a role in protecting the liver against the effects of carbon tetrachloride, a free radicalmediated, glutathione-independent hepatotoxicant. The findings from these experiments are divided into the subcategories of in vitro and in vivo assay results and other relevant findings. The in vitro experiments conducted yielded the following assay results: lactate dehydrogenase (LDH) and poly (ADP-ribose) polymerase (PARP) activity. The in vivo experiments yielded results derived from assays for two analytes: alanine aminotransferase (ALT) and thiobarbituric acid reactive substances (TBARS). The in vivo experiments also yielded the following: histopathological stains using hematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL), and immunohistochemistry for the cleaved form of caspase-3. These tests were utilized in

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34 conjunction with one another, using the same mice. For example, by using the serum for ALT and divided liver for histopathology and TBARS, I was able to gather the maximum amount of phenomenological data per experiment. In Vitro Results Data from preliminary in vivo experiments conducted in the laboratory suggested that the hepatotoxicity induced by CCl4 in male ICR mice can be attenuated via the use of 6( 5H )-phenanthridinone, an inhibitor of poly(ADP-ribose) polymerase (PARP). The levels of glutamic oxalacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT), two hepatocellular enzymes, in the serum samples were significantly lower in mice receiving both carbon tetrachloride and 6( 5H )-phenanthridinone, than in those only treated with carbon tetrachloride. Also, there were dramatic histopathologic differences in liver samples from carbon tetrachloride-treated mice, between those receiving 6( 5H )phenanthridinone as a co-treatment, and those that did not. Hematoxylin and eosin staining revealed severe centrilobular necrosis in mice receiving only carbon tetrachloride, and normal morphology in those also administered 6( 5H )phenanthridinone. TUNEL staining, which was used to detect apoptosis, revealed numerous centrilobular hepatocytes undergoing apoptosis in mice treated solely with carbon tetrachloride, but not in mice receiving both treatments. As a result of these in vivo experiments, there was an interest in determining whether PARP is directly involved in the attenuation of this hepatotoxicity, or perhaps some other aspect of the co-treatment with 6( 5H )-phenanthridinone may play a role in its

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35 hepatoprotection. One possible alternative explanation for the reduction in carbon tetrachloride-induced hepatotoxicity is the inhibition of various cytochrome p450 CYP isoforms by substituted phenanthridinone compounds. Competitive binding assays using tritium-radiolabeled 2,3,7,8-tetrachlorodibenzo-p-dioxin as a substrate revealed 50% inhibition of cytochrome P450 activity at concentrations of 317 to 5870 nM for a variety of 2-substituted phenanthridinones (Liu et al., 1994). Also, dimethyl sulfoxide (DMSO) can have an inhibitory effect on the activity of various isoforms of cytochrome P450 (CYP), most notably an 89% inhibition of CYP2E1 at 1% DMSO (Easterbrook, Lu et al. 2001). CYP2E1 is the isoform of cytochrome P450, which is primarily responsible for the bioactivation of carbon tetrachloride to the trichloromethyl radical (•CCl3). This trichloromethyl radical can spontaneously combine with oxygen, yielding the trichloromethyl peroxy radical (•OOCCl3). This and subsequent radicals are believed to be responsible for the resulting lipid peroxidation and microsomal damage (Rao and Recknagel, 1968). The two assays performed on the HepG2 cells using 6( 5H )-phenanthridinone and benzamide as PARP inhibitors were utilized to determine whether there was damage to the hepatocytes, as well as whether PARP was involved. These tests involved the presence of LDH, an intracellular enzyme, in the cell culture media, and the activity of PARP, as evidenced by the conversion of carbon-14 radiolabeled NAD into poly(ADPribose).

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36 Ctyotoxicity Assays The cytotoxicity assay was used to determine the viability of the cells in general, and the structural integrity of the hepatic cell membranes in particular, following exposure to CCl4. Because LDH is an intracellular enzyme, when it is present in the cell culture media, it suggests that the free radical-mediated damage caused by CCl4 was sufficient to let this enzyme escape the cells. The following tables and charts suggest that PARP inhibitors can reduce this damage to the hepatocytes. These data also suggest that the extent of damage to the hepatocytes increases with the concentration of CCl4. The LDH levels were not, however, established as absolute concentrations and, since the numbers of cells may not have been identical between experiments, the results were only relative indications of the extent of loss of membrane integrity and/or death of the hepatocytes. The use of a positive control, cells exposed to the cell lysis solution, can help determine the extent of the cytotoxicity.

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37 Table 1: Percent Cytotoxicity at Varied PARP Inhibitor Levels. The results are expressed as percent cytotoxicity, as determined by the ratio of absorbances at 490 nm for the cell culture media for each sample in comparison to the mean result for the positive control group, in which the cells were exposed to the lysis solution. The data are presented in Figure 3. Percent Cytotoxicity, HepG2 Cells Sample 1 Sample 2 Sample 3 Average Std Dev Std Err Ttest Control 3.85164 22.5755 18.6104 15.013 9.8668 5.69662 0.2% DMSO 6.84736 3.02067 12.2829 5.3699 7.758 4.47910 DMSO+20uM Phen 6.84736 3.6566 3.84615 4.7834 1.79 1.03344 DMSO+40uM Phen 9.84308 2.54372 2.8536 5.0801 4.1277 2.38315 DMSO+1mM BA 12.2682 25.2782 5.58313 10.654 15.494 8.94537 DMSO + 10mM CCl4 51.3552 59.3005 50 53.552 5.0243 2.90079 DMSO + 10mM CCl4 + 20uM Phen 20.97 32.5914 21.7122 25.091 6.506 3.75622 0.00070 DMSO + 10mM CCl4 + 40uM Phen 6.84736 4.45151 11.5385 7.6124 3.6049 2.08128 0.00534 DMSO + 10mM CCl4 + 1mM BA 28.388 26.868 0 18.419 15.969 9.21979 0.02362

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38 0 10 20 30 40 50 60 Treatment Groups% Cytotoxicity DMSO Control 10mM CCl4 CCl4+20uM Phen CCl4+40uM Phen CCl4+1mM BzAm ** ** Figure 3: Percent Cytotoxicity at Varied PARP Inhibitor Levels. Cytotoxicity assay data for hepatocytes exposed to 10 mM CCl4 and 6( 5H )-phenanthridinone (10 mM CCl4+20 M Phen, 10 mM CCl4+40 M Phen) and 10 mM CCl4 and 1 mM benzamide (10 mM CCl4+1mM BzAm) compared to those exposed to 10 mM CCl4 only (10 mM CCl4). Results are expressed as mean SEM. One-tailed Paired T-tests were performed with indicating a significant difference at p<0.05 and ** indicating a significant difference at p<0.01.

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39 The results of this experiment address two critical issues, the adverse impact of CCl4 on the human liver cells, and the possibility of this deleterious impact being lessened by phenanthridinone, a specific PARP inhibitor. The addition of 10 mM CCl4 and 0.2% DMSO to the culture media resulted in nearly ten-fold elevations in the level of LDH in the culture media. The impact of this finding on the condition of the hepatocytes is important, because LDH is an intracellular enzyme that should not be found outside the cells, and elevated levels of LDH outside the cells are indicative of damage to the plasma membranes. The loss of plasma membrane integrity can be irreversible; as such the LDH assay is often used to assess the extent of cell viability. The extent of membrane damage to the hepatocytes can be estimated as a function of the findings for the positive control lysis buffer solution, which is believed to result in complete lysis of the hepatocytes. As such, if one assumes the linearity of the LDH assay, roughly fifty percent of the hepatocytes incurred plasma membrane damage as a result of the introduction of 10 mM CCl4 to the cell culture media. It appears that the co-administration of 6( 5H )phenanthridinone mitigates this cell membrane damage. The CCl4-mediated increase in LDH in the cell culture media was significantly reduced (p<0.01) by the addition of 20 or 40 M 6( 5H )-phenanthridinone, however in the case of the former, the LDH levels were still triple those found when only 0.2% DMSO was added to the media. Although it is unclear as to whether the resulting LDH levels in the cell culture media when 20 M 6( 5H )-phenanthridinone and 10 mM CCl4 were administered, were indicative of irreversible damage to the hepatocytes. However, one can assume that the presence of 6( 5H )-phenanthridinone in the media is hepatoprotective

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40 against CCl4–mediated hepatocellular damage.

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41 Table 2: Percent Cytotoxicity with Varied Carbon Tetrachloride Levels. The results are expressed as percent cytotoxicity, as determined by the ratio of absorbances at 490 nm for the cell culture media for each sample in comparison to the mean result for the positive control group, in which the cells were exposed to the lysis solution. These data, which are presented in Figure 4, suggest that these higher concentrations of carbon tetrachloride produce hepatocellular damage comparable to the lysis solution, which was used to maximize cell death. Percent Cytotoxicity, HepG2 Cells Sample 1 Sample 2 Sample 3 Average Std Dev Std Err T-Test Control 2.26077 2.06124 1.61544 1.97915 0.3304 0.19076 0.2% DMSO 12.6198 17.1946 12.7379 14.1841 2.60781 1.50562 0.2%DMSO + 20mM CCl4 138.339 103.922 132.65 124.97 18.4491 10.6516 0.00590 0.2%DMSO +40mM CCl4 75.2396 68.4766 86.3836 76.6999 9.04237 5.22061 0.00525 0.2%DMSO + 80mM CCl4 101.757 80.9955 109.37 97.3744 14.6864 8.47922 0.00698

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42 0 20 40 60 80 100 120 140 160 Treatment Groups% Cytotoxicity DMSO Control 20mM CCl4 40mM CCl4 80mM CCl4 ** ** ** Figure 4: Percent Cytotoxicity at Varied Carbon Tetrachloride Levels. Cytotoxicity assay data for hepatocytes exposed to 20 mM CCl4 40 mM CCl4, and 60 mM CCl4 compared to those exposed to the DMSO controls. Results are expressed as mean SEM. Onetailed Paired T-tests were performed with ** indicating a significant difference at p<0.01.

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43 The purpose of the previous study was to assess whether the hepatocellular plasma membrane damage caused by CCl4 was maximized at 10 mM, or whether higher concentrations could produce even more cell lysis. The extent of cell membrane damage is estimated by assaying for the presence of LDH in the cell culture media, as evidenced by the absorbance of visible light with a wavelength of 490 nm, which is due to the dehydrogenation of lactate to pyruvate and the reduction of NAD+ to NADH. When compared with the LDH assay results for wells where the lysis buffer was used as a positive control, the incorporation of higher concentrations of CCl4 into the cell culture media resulted in LDH levels comparable to those for the lysis buffer. The ratio of absorbance measurements at 490 nm for 10 mM CCl4 versus the lysis buffer in the previous experiment was roughly 0.5, whereas in this latter experiment, the higher concentrations of CCl4 in the cell culture media approximated a ratio of 1 to 1. It was unclear from subsequent experiments as to whether 6( 5H )-phenanthridinone could consistently and significantly protect against this more extensive hepatocellular damage. PARP Activity Assays PARP activity assays were conducted to determine if the protection provided by 6( 5H )-phenanthridinone is directly related to the inhibition of PARP, or whether some other mechanism is the cause. There is a direct correlation between the intensity of exposure to CCl4, in terms of concentration or duration of exposure, and PARP activity, given there are enough viable cells to proceed with the assay. N-Methyl-N'-Nitro-NNitrosoguanidine (MNNG), a known mutagen, was administered to serve as a strong

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44 positive control for PARP activity. Incubation in media treated only with DMSO, the solvent in which 6( 5H )-phenanthridinone is dissolved, was used as a baseline in the absence of PARP-inducing compounds. Although DMSO has been reported to inhibit PARP, the level of inhibition at this low concentration is assumed to be insignificant, thus it did not impact the results. These findings strongly suggest that the increase in PARP activity due to MNNG and CCl4 is largely attenuated by 6( 5H )-phenanthridinone, giving insight into the mechanism by which it protects the hepatocytes.

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45 Table 3: PARP Activity With and Without 6( 5H )-Phenanthridinone Co-treatment. 6( 5H )-phenanthridinone reduces increases in PARP activity caused by N-Methyl-N'Nitro-N-Nitrosoguanidine (MNNG), a known mutagen which serves as a positive control. PARP activity is assessed relative to the polymerization of carbon-14 radiolabeled NAD+ into poly (ADP-ribose). These results are expressed as multiples of the DMSO controls in Figure 5. PARP Activity Assay, HepG2 Cells 1.70x10^6 cells/sample MNNG 2 hr. exposure 5 min. count time Treatment 14C CPM #1 14C CPM #2 Blank CPM Avg. Diff. 0.2% DMSO, 4 hr. 2104.6 3122.2 900.8 1712.6 200 uM MNNG + 0.2% DMSO 37672.2 46513.6 900.8 41192.1 MNNG + DMSO + 40 uM Phen 4098 3674.8 900.8 2985.6

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46 0 2 4 6 8 10 12 14 16 18 20 Treatment GroupsMultiples of DMSO Controls 200 uM MNNG + 0.2% DMSO MNNG + DMSO + 40 uM Phen Figure 5: PARP Activity With and Without 6( 5H )-Phenanthridinone Co-treatment. Oneway Paired T-tests were performed between MNNG and MNNG + 6,( 5H )phenanthridinone. Results are expressed as mean SEM. One-tailed Paired T-tests were performed with indicating a significant difference at p<0.05. This proves the hypothesis that 6,( 5H )-phenanthridinone can protect against the overactivation of PARP caused by this known mutagen and PARP inducer.

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47 The results from this PARP activity assay indicate that 6( 5H )-phenanthridinone (40 M) can significantly reduce the effects of MNNG, a known mutagen. The PARP activity, as evidenced by the counts of carbon-14 radiolabeled NAD+ that were polymerized into poly ADP-ribose. Unlike the radiolabeled NAD+, which would have passed through the pores in the methylcellulose membranes during the sample washings, the polyADP-ribose would not have done so, and as such it remained present on the membranes and resulted in the elevated carbon-14 counts. The counts of carbon-14 were much higher in this experiment than in the previous one, most likely because a new aliquot of carbon-14 radiolabeled NAD+ was used in this experiment.

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48 Table 4: PARP Activity With 6( 5H )-Phenanthridinone and Carbon Tetrachloride. The increases in PARP activities associated with CCl4, as evidenced by increased carbon-14 activities from the incorporation of radiolabeled NAD into poly (ADP-ribose), were diminished by 40 M 6( 5H )-phenanthridinone. This indicates that 6( 5H )phenanthridinone prevents the over activation of PARP, which can result in cell death by necrosis, due to a lack of cellular energy. The results are expressed as multiples of the DMSO control in Figure 6. PARP Activity, HepG2 Cells, 1.255x10^6 cells/sample 5 min. count time Treatment 14C CPM #1 14C CPM #2 Blank CPM Avg. Diff. 0.2% DMSO, 4 hr. 977.8 832.8 161.6 743.7 0.2% DMSO + 40uM Phen, 4 hr. 302.6 1096.2 161.6 537.8 DMSO + 20mM CCl4, 1 hr. 2017.6 1741 161.6 1717.7 DMSO + 20mM CCl4 + Phen, 1 hr. 552.2 553.8 161.6 391.4 DMSO + 20mM CCl4, 2 hr. 2520.6 2534.6 161.6 2366 DMSO + mM CCl4 + Phen, 2 hr. 39.2 650.6 161.6 183.3

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49 0 0.5 1 1.5 2 2.5 3 3.5 Treatment GroupsMultiples of DMSO Controls DMSO + 20mM CCl4, 1 hr. DMSO + 20mM CCl4 + Phen, 1 hr. DMSO + 20mM CCl4, 2 hr. DMSO + mM CCl4 + Phen, 2 hr. ** Figure 6: PARP Activity Using 6( 5H )-Phenanthridinone and Carbon Tetrachloride. Oneway Paired T-tests were performed between CCl4 and CCl4 + Phen at one and two hours of exposure. Results are expressed as mean SEM. One-tailed Paired T-tests were performed with indicating a significant difference at p<0.05 and with ** indicating a significant difference at p<0.01.

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50 These PARP activity assay results support the hypothesis that MNNG and CCl4 increase the numbers of carbon-14 counts of NAD+ that were converted into poly ADPribose, and that 6( 5H )-phenanthridinone significantly diminished these increases in PARP activity. The overall carbon-14 count totals were lower in the second experiment than in the immediately previous experiment. This was most likely due in part to the lower concentration of cells in the cell culture dishes in this experiment than in the past.

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51 Table 5: PARP Activity With MNNG, Carbon Tetrachloride and 6( 5H )Phenanthridinone. The co-treatment with 6( 5H )-phenanthridinone once again appears to reduce the N-Methyl-N'-Nitro-N-Nitrosoguanidine (MNNG) and carbon tetrachlorideinduced increases in PARP activity, as evidenced by diminished carbon-14 counts for the insoluble ADP-ribose polymer. The results are expressed as multiples of the DMSO control in Figure 7. PARP Activity Assay, HepG2 Cells 1.175x10^6 cells/sample MNNG 2 hr. exposure 5 min. count time Treatment 14C CPM #1 14C CPM #2 Blank CPM Avg. Diff. 0.2% DMSO, 4 hr. 559.2 1123 44 797.1 0.2% DMSO + 40mM Phen, 4 hr. 541.2 524.4 44 488.8 200 uM MNNG + 0.2% DMSO 3649 6295.8 44 4928.4 MNNG + DMSO + 40 mM Phen 1099.4 844.4 44 927.9 DMSO + 20mM CCl4, 1 hr. 1406.6 1749 44 1533.8 DMSO + mM CCl4 + Phen, 1 hr. 623.6 335.6 44 435.6

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52 0 1 2 3 4 5 6 7 Treatment GroupsMultiples of DMSO Controls 200 uM MNNG + 0.2% DMSO MNNG + DMSO + 40 mM Phen DMSO + 20mM CCl4, 1 hr. DMSO + mM CCl4 + Phen, 1 hr. ** Figure 7: PARP Activity With MNNG, Carbon Tetrachloride and 6( 5H )Phenanthridinone. One-way Paired T-tests were performed between N-Methyl-N'-NitroN-Nitrosoguanidine (MNNG) and MNNG + Phen, and between CCl4 and CCl4 + Phen at one, two and four hours of exposure. Results are expressed as mean SEM. One-tailed Paired T-tests were performed with indicative of significant difference at p<0.05 and with ** indicative of significant difference at p<0.01.

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53 In Vitro Conclusions In summation, the in vitro experiments strongly suggest that 6( 5H )phenanthridinone, an inhibitor of PARP, can protect human hepatocytes from the HepG2 cell line from the free radical-mediated hepatotoxic effects of CCl4. The results of the LDH assays show that there is a relative increase in LDH in the serum when CCl4 is incorporated into the culture media and a relative decrease in LDH levels when 6( 5H )phenanthridinone was also incorporated into the cell culture media. Similar phenomena were observed with the PARP activity assays. The introduction of CCl4 into the culture media resulted in significantly elevated incorporation of NAD+ into the ADP-Ribose polymer. When 6( 5H )-phenanthridinone was incorporated in conjunction with CCl4 in the culture media, the relative increases in PARP activity were reduced. These results suggest that 6( 5H )-phenanthridinone and possibly other specific or non-specific PARP inhibitors, could protect the liver from CCl4–induced, non glutathione, free radical mediated hepatotoxicity. In Vivo Results The fact that carbon tetrachloride induced hepatocellular damage in the in vitro assays was strongly suggestive of comparable effects in vivo The extent of hepatic damage caused by carbon tetrachloride and protection provided by PARP inhibitors was determined via serum assays for ALT and AST, TBARS lipid peroxidation assays in liver tissue, histopathologic staining, and immunohistochemistry testing of the liver tissue.

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54 Serum ALT/AST Assays The dose response and time course characteristics of carbon tetrachloride-induced hepatotoxicity were determined using serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) assays, which are also constituent tests in human liver panels. The publications which addressed carbon tetrachloride had varied intervals between dosage and blood collection, but none of these reported using intervals in excess of twenty four (24) hours. These results strongly suggest that the ALT levels, which are indicative of hepatic cell membrane damage, reach their maximum levels at a time period of twenty four (24) hours after intraperitoneal administration of carbon tetrachloride. As such, this would be the optimal time to euthanize the male ICR mice for subsequent assays. The AST assays were also conducted to determine the optimal time after exposure for the determination of damage. As was the case for the ALT assays, the AST levels were greatest at a period of twenty four (24) hours after intraperitoneal administration of carbon tetrachloride.

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55 Table 6: Serum AST Levels at Varied Intervals After Intraperitoneal Carbon Tetrachloride Administration. Results are displayed graphically on the following page. AST Time Course at 600 mg/kg CCl4 Treatment Absorbance Conc. From Curve D.F. Concentration Average Std Dev Std Error Corn Oil 0.232 -6.857 10 -68.571 Corn Oil 0.276 8.857 10 88.571 Corn Oil 0.276 8.857 10 88.571 Corn Oil 0.24 -4.000 10 -40.000 Corn Oil 0.247 -1.500 10 -15.000 Corn Oil 0.264 4.571 10 45.714 Corn Oil 0.276 8.857 10 88.571 26.837 67.219 25.406 CCl4, 3 hrs. 0.394 51.000 10 510.000 CCl4, 3 hrs. 0.4 53.143 11 584.571 CCl4, 3 hrs. 0.372 43.143 10 431.429 CCl4, 3 hrs. 0.376 44.571 10 445.714 CCl4, 3 hrs. 0.327 27.071 10 270.714 CCl4, 3 hrs. 0.319 24.214 10 242.143 CCl4, 3 hrs. 0.404 54.571 10 545.714 CCl4, 3 hrs. 0.511 92.786 10 927.857 CCl4, 3 hrs. 0.41 56.714 10 567.143 502.810 201.107 67.036 CCl4, 6 hrs. 0.384 47.429 50 2371.429 CCl4, 6 hrs. 0.558 109.571 11 1205.286 CCl4, 6 hrs. 0.316 23.143 10 231.429 CCl4, 6 hrs. 0.456 73.143 10 731.429 CCl4, 6 hrs. 0.366 41.000 10 410.000 CCl4, 6 hrs. 0.368 41.714 10 417.143 CCl4, 6 hrs. 0.323 25.643 10 256.429 CCl4, 6 hrs. 0.362 39.571 10 395.714 CCl4, 6 hrs. 0.413 57.786 10 577.857 732.968 682.653 227.551 CCl4, 18 hrs. 0.321 24.929 10 249.286 CCl4, 18 hrs. 0.369 42.071 10 420.714 CCl4, 18 hrs. 0.398 52.429 10 524.286 CCl4, 18 hrs. 0.35 35.286 10 352.857 CCl4, 18 hrs. 0.308 20.286 10 202.857 CCl4, 18 hrs. 0.305 19.214 10 192.143 CCl4, 18 hrs. 0.322 25.286 10 252.857 CCl4, 18 hrs. 0.288 13.143 10 131.429 CCl4, 18 hrs. 0.516 94.571 50 4728.571 783.889 1484.371 494.790 CCl4, 18 hrs. 0.363 39.929 10 399.286 CCl4, 24 hrs. 0.348 34.571 11 380.286 CCl4, 24 hrs. 0.308 20.286 10 202.857 CCl4, 24 hrs. 0.341 32.071 10 320.714 CCl4, 24 hrs. 0.555 108.500 10 1085.000 CCl4, 24 hrs. 0.379 45.643 10 456.429 CCl4, 24 hrs. 0.585 119.214 50 5960.714 CCl4, 24 hrs. 0.387 48.500 10 485.000 CCl4, 24 hrs. 0.326 26.714 10 267.143 1061.937 1854.755 618.252

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56 0 200 400 600 800 1000 1200 1400 1600 1800 Treatment GroupsAST (IU/L) Corn Oil 600 mg/kg CCl4, 3 hours 600 mg/kg CCl4, 6 hours 600 mg/kg CCl4, 18 hours 600 mg/kg CCl4, 24 hours Figure 8: Serum AST Levels at Varied Intervals After Intraperitoneal Carbon Tetrachloride Administration. Adult male ICR mice with an average weight of 30 grams were sacrificed at the specified intervals following intraperitoneal administration of carbon tetrachloride. Results are expressed as mean SEM. These results suggest that the 24 hour interval is optimal for the collection of blood for serum transaminase assays

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57 This AST assay was conducted to determine the optimal time interval following intraperitoneal administration of 600 mg/kg CCl4 at which point the AST levels were the highest. This would be the preferred time interval following the injection of CCl4, that the mice should be euthanized and the blood should be drawn and centrifuged for the serum needed for this assay. The AST levels were the highest at a period of 24 hours following the intraperitoneal administration of this toxicant. As a result, it was selected for subsequent experiments. The 18-hour interval between intraperitoneal administration of CCl4 and euthanasia of the mice resulted in the second-highest serum AST levels. In subsequent serum transaminase assays, the 24 hour interval was used, despite the substantial elevation at 18 hours.. Once the optimal interval between carbon tetrachloride administration and euthanasia of the mice was established, the dose-response curve was investigated. The concern was that the administered dosage may not be high enough to result in significant damage to the hepatocytes. Also, with the kinetic NADH coupled ultraviolet assay used at the time, it was possible to exhaust all the substrate (alanine) before the completion of the assay, skewing results downward. This explained the lower observed ALT level with the highest dosage of carbon tetrachloride. An additional ALT assay was conducted at even lower concentrations of carbon tetrachloride. The results, which are not presented, clearly indicated the inadequacy of the kinetic assay method, as the samples one would expect to have the highest ALT levels had the lowest levels, likely due to premature exhaustion of the substrate. Although the assay method used changed, the lower toxicant level of 0.200 mg/kg carbon tetrachloride was utilized in subsequent experiments, as this

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58 level was known to generate ALT levels indicative of substantial hepatocellular damage.

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59 Table 7: Serum ALT Levels With and Without 0.2 ml/kg Carbon Tetrachloride. This experiment used a non-kinetic protocol, which did not allow for substrate exhaustion. The results of this experiment suggest that administration of 0.2 ml/kg of carbon tetrachloride caused a significant increase in ALT values. Elevated ALT levels in the serum are indicative of damage to the liver cells, as this assay is a component of the liver panel of diagnostic tests. Results are presented graphically in Figure 9. CCl4 Dosage Sample ID A505 Conc. From Std. D.F. Concentration Average Std Dev Std. Error Reagent Blank 0.443 Standard (70 IU) 0.713 0.2 ml/kg 1 1.567 291.407 1 291.407 0.2 ml/kg 2 1.558 289.074 1 289.074 0.2 ml/kg 3 1.577 294.000 1 294.000 0.2 ml/kg 4 1.592 297.889 1 297.889 0.2 ml/kg 5 1.565 290.889 1 290.889 0.2 ml/kg 6 1.572 292.704 1 292.704 0.2 ml/kg 7 1.55 287.000 1 287.000 0.2 ml/kg 8 1.538 283.889 1 283.889 290.856 4.3037 1.5216 Control 37 0.61 43.296 1 43.296 Control 38 0.574 33.963 1 33.963 38.630 6.5997 4.6667

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60 0 50 100 150 200 250 300 350 Treatment GroupsALT (IU/L) Control 0.2 ml/kg ** Figure 9: Serum ALT Levels With and Without 0.2 ml/kg Carbon Tetrachloride. Serum levels of ALT (IU/L) were assayed in mice administered 0.2 ml/kg of CCl4, in comparison to control mice given the vehicle, corn oil, alone. Adult male ICR mice with an average weight of 30 grams were sacrificed 24 hours following intraperitoneal administration of carbon tetrachloride. Results are expressed as mean SEM. Twosample unequal variance (heteroscedastic) one-way T-tests were performed with ** indicating a significant difference at p<0.01.

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61 In this experiment, the first to use a non-kinetic methodology for the ALT assay, the results confirmed that the intraperitoneal administration of 0.200 ml/kg of CCl4 could produce significant hepatocellular damage. The absorbance readings at 505 nm for this dosage of CCl4 were so high as to extend beyond the linear range of the assay methodology. Unfortunately, there was not enough serum remaining for subsequent dilutions of several of the samples. As a result, the serum ALT concentrations, which reflected a statistically significant (p<0.01) increase in serum ALT, if anything, were artificially low. The instructions that came with the assay kit suggested that at higher serum concentrations of ALT above the linear range, the absorbance of visible light at 505 nm did not increase proportionally with serum ALT levels. All the samples from treatment groups involving CCl4 were diluted, with the expectation that the resulting absorbances and serum concentrations of ALT would be in the linear range. This linear range includes the standard concentration of ALT, which was provided in conjunction with the assay reagents. As a result, the serum concentrations of ALT derived from the comparison with the standard concentration could be multiplied by the applicable dilution factors (DF) to produce the final concentrations. These should prove to be more accurate than those in which the undiluted samples yielded absorbance readings beyond the linear range of the curve. Given the in vitro results, there is reason to believe that inhibition of PARP may diminish the hepatotoxic effects associated with CCl4. The results of serum ALT and aspartate aminotransferase (AST) assays when PARP inhibitors were co-treatments with carbon tetrachloride confirm these beliefs, with some caveats. The co-administration of

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62 6( 5H )-phenanthridinone requires dissolution or suspension in dimethyl sulfoxide (DMSO), which poses its own health concerns. Apparently, solubility concerns may have precluded the proper dosing with 6( 5H )-phenanthridinone, as it was only soluble in pure DMSO. Attempts were made to solubilize 6( 5H )-phenanthridinone in corn oil, but perhaps due in part to the high viscosity of the corn oil, no amount of heating or mixing could result in a solution or stable suspension. This was evidenced by the white precipitate, which began to form beneath the corn oil. As a result of these solubility concerns, it was impossible to ensure that the proper dosage of 6( 5H )-phenanthridinone was injected into the peritoneal cavity of each of the mice. This makes water soluble PARP inhibitors, such as nicotinamide and aminophylline better choices for intraperitoneal administration. The other critical reason for selecting aminophylline as a co-treatment is that it has been approved by the Food and Drug Administration, albeit for other purposes. In contrast, many other PARP inhibitors have yet to be approved for any purpose. Nicotinamide is an essential nutrient, which would likely require less effort for FDA approval than most other compounds.

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63 Table 8: Serum ALT Levels With Carbon Tetrachloride and Varying 6( 5H )Phenanthridinone Co-treatments. The results are presented graphically in Figure 10. CCl4 6(5H)phenanthridinone Dosage Treatment Sample A505 Conc. D.F. Conc. Average Std Dev Std Err. Reagent Blank 0.478 Standard 0.726 70 IU/ml 0.2 ml/kg 0 mg/kg 1 1.628 324.597 5 1622.984 0.2 ml/kg 0 mg/kg 2 1.629 324.879 5 1624.395 0.2 ml/kg 0 mg/kg 3 1.568 307.661 5 1538.306 0.2 ml/kg 0 mg/kg 4 1.62 322.339 5 1611.694 0.2 ml/kg 0 mg/kg 5 1.639 327.702 5 1638.508 0.2 ml/kg 0 mg/kg 6 1.621 322.621 5 1613.105 0.2 ml/kg 0 mg/kg 7 1.6 316.694 5 1583.468 1604.6371 33.7728 12.7649 0.2 ml/kg 5 mg/kg, Concomitant 8 1.6 316.694 5 1583.468 0.2 ml/kg 5 mg/kg, Concomitant 9 1.616 321.210 5 1606.048 0.2 ml/kg 5 mg/kg, Concomitant 10 1.611 319.798 5 1598.992 0.2 ml/kg 5 mg/kg, Concomitant 11 1.629 324.879 5 1624.395 0.2 ml/kg 5 mg/kg, Concomitant 12 1.623 323.185 5 1615.927 0.2 ml/kg 5 mg/kg, Concomitant 13 1.653 331.653 5 1658.266 0.2 ml/kg 5 mg/kg, Concomitant 14 1.475 281.411 5 1407.056 1584.879 81.8346 30.9306 0.2 ml/kg 10 mg/kg, Concomitant 15 1.611 319.798 5 1598.992 0.2 ml/kg 10 mg/kg, Concomitant 16 1.626 324.032 5 1620.161 0.2 ml/kg 10 mg/kg, Concomitant 17 1.601 316.976 5 1584.879 0.2 ml/kg 10 mg/kg, Concomitant 18 1.607 318.669 5 1593.347 0.2 ml/kg 10 mg/kg, Concomitant 19 1.623 323.185 5 1615.927 0.2 ml/kg 10 mg/kg, Concomitant 20 1.609 319.234 5 1596.169 0.2 ml/kg 10 mg/kg, Concomitant 21 1.621 322.621 5 1613.105 1603.226 13.2140 4.9944 0.2 ml/kg 5 mg/kg, 3 & 6 hrs. Post 22 1.623 323.185 5 1615.927 0.2 ml/kg 5 mg/kg, 3 & 6 hrs. Post 23 1.61 319.516 5 1597.581 0.2 ml/kg 5 mg/kg, 3 & 6 hrs. Post 24 1.659 333.347 5 1666.734 0.2 ml/kg 5 mg/kg, 3 & 6 hrs. Post 25 1.6 316.694 5 1583.468 0.2 ml/kg 5 mg/kg, 3 & 6 hrs. Post 26 1.629 324.879 5 1624.395 0.2 ml/kg 5 mg/kg, 3 & 6 hrs. Post 27 1.526 295.806 5 1479.032 0.2 ml/kg 5 mg/kg, 3 & 6 hrs. Post 28 1.592 314.435 5 1572.177 1591.331 58.3989 22.0727 0 ml/kg 0 mg/kg 29 1.502 289.032 5 1445.161 red serum omit 0 ml/kg 0 mg/kg 30 0.477 -0.282 5 -1.411 0 ml/kg 10 mg/kg, Concomitant 31 0.984 142.823 5 714.113 red serum omit 0 ml/kg 10 mg/kg, Concomitant 32 0.482 1.129 5 5.645

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64 0 200 400 600 800 1000 1200 1400 1600 1800 Treatment GroupsALT (IU/L) Control 10 mg/kg Phen (concomitant) 0.2 ml/kg CCl4 0.2 ml/kg CCl4 +5 mg/kg Phen 0.2 ml/kg CCl4 +10 mg/kg Phen 0.2 ml/kg CCl4 +5 mg/kg 2x (3 and 6hr) Phen Figure 10: Serum ALT Levels With Carbon Tetrachloride and Varying 6( 5H )Phenanthridinone Co-treatments. Adult male ICR mice with an average weight of 30 grams were sacrificed 24 hours following intraperitoneal administration of carbon tetrachloride. Results are expressed as mean SEM. Two-sample unequal variance (heteroscedastic) one-way T-tests were performed. There were no significant reductions in the serum ALT levels as a result of the different co-treatment intervals for 6( 5H )phenanthridinone.

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65 The results of this experiment were disconcerting, because 6( 5H )phenanthridinone appeared to be extremely effective against the hepatotoxic effects of CCl4 in the in vitro studies. The results of these in vitro studies strongly suggested that the addition of 6( 5H )-phenanthridinone to the cell culture media substantially, and in most cases, significantly reduced the hepatotoxic effects of CCl4, as evidenced by the reduction in LDH levels in the culture media, and diminished PARP activity. The primary issue preventing the use of 6( 5H )-phenanthridinone appears to be that of solubility. I was incapable of solubilizing 6( 5H )-phenanthridinone in corn oil or saline. It would have been possible to solubilize 6( 5H )-phenanthridinone in pure DMSO. However, Banasik and Ueda (1994) also reported that DMSO is a PARP inhibitor in its own right. Also, because of potential health concerns, specifically related to hepatic damage, DMSO may not make for the safest vehicle for otherwise insoluble drugs. Fortunately, Pacher et al (2002) have discovered water soluble phenanthridinone derivatives. In the following experiment, I again tried administering 6( 5H )phenanthridinone, only this time via multiple intraperitoneal injections at different intervals following the intraperitoneal administration of CCl4 with the hopes of finding it effective at one or more of these time points.

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66 Table 9: Serum ALT Levels with Carbon Tetrachloride and Varied Numbers of 6( 5H )Phenanthridinone Treatments. These results are displayed graphically in Figure 11. CCl4 Phen Dosage Treatment Sample A505 Conc. D.F. Concentration Avg. Std Dev Std Err Reagent Bl. 0.467 Standard 0.702 70 IU/l 0.1 ml/kg 0 mg/kg 1 1.63 346.426 5 1732.128 0.1 ml/kg 0 mg/kg 2 1.53 316.638 5 1583.191 0.1 ml/kg 0 mg/kg 3 1.506 309.489 5 1547.447 0.1 ml/kg 0 mg/kg 4 1.613 341.362 5 1706.809 0.1 ml/kg 0 mg/kg 5 1.663 356.255 5 1781.277 1670.1702 100.1899 44.8063 0.1 ml/kg 10 mg/kg, 45 min post 6 1.633 347.319 5 1736.596 0.1 ml/kg 10 mg/kg, 45 min post 7 1.625 344.936 5 1724.681 0.1 ml/kg 10 mg/kg, 45 min post 8 1.651 352.681 5 1763.404 0.1 ml/kg 10 mg/kg, 45 min post 9 1.645 350.894 5 1754.468 0.1 ml/kg 10 mg/kg, 45 min post 10 1.587 333.617 5 1668.085 1729.447 37.4775 16.7604 0.1 ml/kg 10 mg/kg, 45 & 90 min 11 1.596 336.298 5 1681.489 0.1 ml/kg 10 mg/kg, 45 & 90 min 12 1.641 349.702 5 1748.511 0.1 ml/kg 10 mg/kg, 45 & 90 min 13 1.389 274.638 5 1373.191 0.1 ml/kg 10 mg/kg, 45 & 90 min 14 1.527 315.745 5 1578.723 0.1 ml/kg 10 mg/kg, 45 & 90 min 15 1.3 248.128 5 1240.638 1524.511 212.8605 95.1941 0.1 ml/kg 10 mg/kg, 45, 90 & 135 min 16 1.574 329.745 5 1648.723 0.1 ml/kg 10 mg/kg, 45, 90 & 135 min 17 1.647 351.489 5 1757.447 0.1 ml/kg 10 mg/kg, 45, 90 & 135 min 18 1.195 216.851 5 1084.255 0.1 ml/kg 10 mg/kg, 45, 90 & 135 min 19 1.596 336.298 5 1681.489 0.1 ml/kg 10 mg/kg, 45, 90 & 135 min 20 1.589 334.213 5 1671.064 1568.596 273.8227 122.4572 0.1 ml/kg 10 mg/kg, 45, 90, 135, 180 21 1.639 349.106 5 1745.532 0.1 ml/kg 10 mg/kg, 45, 90, 135, 180 22 1.625 344.936 5 1724.681 0.1 ml/kg 10 mg/kg, 45, 90, 135, 180 23 1.645 350.894 5 1754.468 0.1 ml/kg 10 mg/kg, 45, 90, 135, 180 24 1.548 322.000 5 1610.000 0.1 ml/kg 10 mg/kg, 45, 90, 135, 180 25 1.04 170.681 5 853.404 1537.617 386.8566 173.0076 0 ml/kg 10 mg/kg, 45, 90, 135, 180 26 0.882 123.617 5 618.085 0 ml/kg 10 mg/kg, 45, 90, 135, 180 27 0.475 2.383 5 11.915 0 ml/kg 10 mg/kg, 45, 90, 135, 180 28 0.501 10.128 5 50.638 0 ml/kg 10 mg/kg, 45, 90, 135, 180 29 0.5 9.830 5 49.149 182.447 290.9775 145.4887 0 ml/kg 0 mg/kg 30 0.491 7.149 5 35.745 0 ml/kg 0 mg/kg 31 0.576 32.468 5 162.340 red serum 0 ml/kg 0 mg/kg 32 0.495 8.340 5 41.702 79.929 71.4325 41.2415

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67 0 200 400 600 800 1000 1200 1400 1600 1800 2000 Treatment GroupsALT (IU/L) Control 10 mg/kg Phen 4x 0.1 ml/kg CCl4 0.1 ml/kg CCl4 + 10mg/kg Phen" 0.1 ml/kg CCl4 + 10mg/kg Phen 2x" 0.1 ml/kg CCl4 + 10mg/kg Phen 3x" 0.1 ml/kg CCl4 + 10mg/kg Phen 4x" Figure 11: Serum ALT Levels with Carbon Tetrachloride and Varied Numbers of 6( 5H )Phenanthridinone Treatments. Adult male ICR mice with an average weight of 30 grams were sacrificed 24 hours following intraperitoneal administration of carbon tetrachloride. Results are expressed as mean SEM. Two-sample unequal variance (heteroscedastic) one-way T-tests were performed. There were no significant reductions in the serum ALT levels as a result of the different numbers of co-treatments with 6( 5H )-phenanthridinone.

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68 The results of this experiment strongly suggest that there still appears to be a solubility problem, because the thorough vortex mixing of 6( 5H )-phenanthridinone in saline merely produced a suspension. The particles of 6( 5H )-phenanthridinone that were suspended in saline may not have been effectively delivered through the 25 gauge needle into the peritoneal cavity. The next factor to test was whether, by administering the 6( 5H )-phenanthridinone to the mice at a later period following carbon tetrachloride exposure, more of the 6( 5H )phenanthridinone may be available to prevent hepatocellular damage. This experiment failed to yield any results indicative of a protective effect by 6( 5H )-phenanthridinone against carbon tetrachloride-induced hepatotoxicity. This suggests that delaying the administration of 6( 5H )-phenanthridinone does not protect the livers from carbon tetrachloride-induced hepatocellular damage. In the following experiment, I again tried administering 6( 5H )-phenanthridinone, only at different intervals following the intraperitoneal administration of CCl4 with the hopes of finding it effective at one of these time points.

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69 Table 10: Serum ALT Levels With Delayed 6( 5H )-Phenanthridinone Administration. The results are displayed in graphical form in Figure 12. CCl4 Phen Dosage Treatment Sample A505 Conc. D.F. Conc. Avg Std Dev Std Err Reagent Blank 0.488 Standard 0.721 70 IU/ml 0 ml/kg 0 mg/kg 1 0.512 7.210 5 36.052 0 ml/kg 0 mg/kg 2 0.458 -9.013 5 -45.064 0 ml/kg 0 mg/kg 3 0.588 30.043 5 150.215 47.0672 98.1044 56.6406 0 ml/kg 10 mg/kg, 3 & 6 hrs. post 4 0.476 -3.605 5 -18.026 0 ml/kg 10 mg/kg, 3 & 6 hrs. post 5 0.494 1.803 5 9.013 0 ml/kg 10 mg/kg, 3 & 6 hrs. post 6 0.559 21.330 5 106.652 0 ml/kg 10 mg/kg, 3 & 6 hrs. post 7 0.589 30.343 5 151.717 62.3391 80.1129 40.0564 0.1 ml/kg 10 mg/kg, 3 & 6 hrs. post 8 1.667 354.206 5 1771.030 0.1 ml/kg 10 mg/kg, 3 & 6 hrs. post 9 1.661 352.403 5 1762.017 0.1 ml/kg 10 mg/kg, 3 & 6 hrs. post 10 1.656 350.901 5 1754.506 0.1 ml/kg 10 mg/kg, 3 & 6 hrs. post 11 1.648 348.498 5 1742.489 0.1 ml/kg 10 mg/kg, 3 & 6 hrs. post 12 1.655 350.601 5 1753.004 0.1 ml/kg 10 mg/kg, 3 & 6 hrs. post 13 1.501 304.335 5 1521.674 0.1 ml/kg 10 mg/kg, 3 & 6 hrs. post 15 1.512 307.639 5 1538.197 1691.845 111.0495 41.9728 0.1 ml/kg 10 mg/kg, 3 hrs. post 16 1.566 323.863 5 1619.313 0.1 ml/kg 10 mg/kg, 3 hrs. post 17 1.622 340.687 5 1703.433 0.1 ml/kg 10 mg/kg, 3 hrs. post 18 1.619 339.785 5 1698.927 0.1 ml/kg 10 mg/kg, 3 hrs. post 19 1.64 346.094 5 1730.472 0.1 ml/kg 10 mg/kg, 3 hrs. post 20 1.65 349.099 5 1745.494 0.1 ml/kg 10 mg/kg, 3 hrs. post 21 1.601 334.378 5 1671.888 0.1 ml/kg 10 mg/kg, 3 hrs. post 22 1.646 347.897 5 1739.485 0.1 ml/kg 10 mg/kg, 3 hrs. post 23 1.634 344.292 5 1721.459 1703.809 41.7736 14.7692 0.1 ml/kg 0 mg/kg 24 1.666 353.906 5 1769.528 0.1 ml/kg 0 mg/kg 25 1.646 347.897 5 1739.485 0.1 ml/kg 0 mg/kg 26 1.627 342.189 5 1710.944 0.1 ml/kg 0 mg/kg 27 1.639 345.794 5 1728.970 0.1 ml/kg 0 mg/kg 28 1.636 344.893 5 1724.464 0.1 ml/kg 0 mg/kg 29 1.627 342.189 5 1710.944 0.1 ml/kg 0 mg/kg 30 1.629 342.790 5 1713.948 0.1 ml/kg 0 mg/kg 31 1.634 344.292 5 1721.459 1727.468 19.6020 6.9304

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70 0 200 400 600 800 1000 1200 1400 1600 1800 2000 Treatment GroupsALT (IU/L) Control 10mg/kg Phen 3, 6 hrs 0.1 ml/kg CCl4 0.1 ml/kg CCl4 + 10 mg/kg Phen 3 hrs 0.1 ml/kg CCl4 + 10 mg/kg Phen 3, 6 hrs Figure 12: Serum ALT Levels With Delayed 6( 5H )-Phenanthridinone Administration. Adult male ICR mice with an average weight of 30 grams were sacrificed 24 hours following intraperitoneal administration of carbon tetrachloride. The delayed administration of 6( 5H )-phenanthridinone did not significantly reduce the CCl4-induced elevation in serum ALT levels. Results are expressed as mean SEM. Two-sample unequal variance (heteroscedastic) one-way T-tests were performed. There were no significant reductions in the serum ALT levels as a result of the different numbers of delayed co-treatments with 6( 5H )-phenanthridinone.

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71 The results of this experiment strongly suggested that, shy of using high percentages of DMSO, in which 6( 5H )-phenanthridinone is soluble, there was no feasible means of delivering 6( 5H )-phenanthridinone effectively via intraperitoneal injection. As a result, I decided to try other PARP inhibitors that, despite their substantially greater effective concentrations, may be more effective against the hepatotoxic effects of CCl4, when administered via intraperitoneal injection. Following these experiments with 6( 5H )-phenanthridinone and 4aminobenzamide, it became critical to determine whether any PARP inhibitors could be proven effective at reducing the hepatotoxic effects of carbon tetrachloride. Nicotinamide (a.k.a. niacinamide) has been reportedly shown (Gibb and Brody, 1967) to reduce the hepatotoxic effects of carbon tetrachloride, suggesting it would be a good cotreatment to test in the ICR mice. The following results confirm the fact that nicotinamide, for reasons that may be related or unrelated to the inhibition of PARP-1, was capable of reducing ALT levels. Much higher dosages (250 and 500 mg/kg) of nicotinamide were used in the following experiments, due to its higher in vitro PARP-1 inhibition concentration (Banasik, 1994). These results suggest that when sufficiently high levels of nicotinamide are used, it may effectively protect the livers against the hepatotoxic effects of carbon tetrachloride.

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72 Table 11: Serum ALT Levels With Nicotinamide and Carbon Tetrachloride. Results are displayed in graphical format on Figure 13. CCl4 Nicotinamide Dosage Dosage A505 Conc. D.F. Conc. Avg Std Dev Std Err Rgnt. Blank 0.452 Standard 0.71 70 IU/ml 0 ml/kg 0 mg/kg 0.465 3.527 5 17.636 0 ml/kg 0 mg/kg 0.469 4.612 5 23.062 0 ml/kg 0 mg/kg 0.477 6.783 5 33.915 24.8708 8.2889 4.7856 0.025 ml/kg 0 mg/kg 1.38 251.783 5 1258.915 0.025 ml/kg 0 mg/kg 1.379 251.512 5 1257.558 0.025 ml/kg 0 mg/kg 1.227 210.271 5 1051.357 1189.2765 119.4441 68.9611 0 ml/kg 250 mg/kg, 1 hr. post CCl4 0.468 4.341 5 21.705 0 ml/kg 250 mg/kg, 1 hr. post CCl4 0.481 7.868 5 39.341 0 ml/kg 250 mg/kg, 1 hr. post CCl4 0.459 1.899 5 9.496 23.514 15.0045 8.6628 0.025 ml/kg 250 mg/kg, 1 hr. post CCl4 0.464 3.256 5 16.279 0.025 ml/kg 250 mg/kg, 1 hr. post CCl4 0.864 111.783 5 558.915 0.025 ml/kg 250 mg/kg, 1 hr. post CCl4 0.747 80.039 5 400.194 0.025 ml/kg 250 mg/kg, 1 hr. post CCl4 1.052 162.791 5 813.953 0.025 ml/kg 250 mg/kg, 1 hr. post CCl4 0.933 130.504 5 652.519 0.025 ml/kg 250 mg/kg, 1 hr. post CCl4 1.136 185.581 5 927.907 0.025 ml/kg 250 mg/kg, 1 hr. post CCl4 0.912 124.806 5 624.031 0.025 ml/kg 250 mg/kg, 1 hr. post CCl4 0.787 90.891 5 454.457 0.025 ml/kg 250 mg/kg, 1 hr. post CCl4 0.516 17.364 5 86.822 0.025 ml/kg 250 mg/kg, 1 hr. post CCl4 0.961 138.101 5 690.504 522.558 292.8270 92.6000

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73 0 200 400 600 800 1000 1200 1400 Treatment GroupsALT (IU/L) Control 250 mg/kg Nic 0.025 ml/kg CCl4 0.025 CCl4,250 Nic ** Figure 13: Serum ALT Levels With Nicotinamide and Carbon Tetrachloride. Adult male ICR mice with an average weight of 30 grams were sacrificed 24 hours following intraperitoneal administration of carbon tetrachloride. Results are expressed as mean SEM. Two-sample unequal variance (heteroscedastic) one-way T-tests were performed, with ** indicative of a significant difference at p<0.01. Nicotinamide appears to provide some measure of protection against CCl4-induced increases in serum ALT levels, at these lower dosages of carbon tetrachloride, when administered via intraperitoneal injection one hour following the toxicant.

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74 The results of this experiment showed some promise, as the intraperitoneal administration of 250 mg/kg of nicotinamide one hour following the intraperitoneal injection of CCl4 was capable of reducing the adverse effects of this hepatotoxicant. Although the hepatoprotective effect of 250 mg/kg nicotinamide against 0.025 ml/kg CCl4 was significant, there was still a substantial elevation in ALT levels over those for the controls, which is still likely indicative of substantial hepatocellular damage. The next experiment tested whether a higher dosage (500 mg/kg) of nicotinamide would be more hepatoprotective against CCl4, further reducing serum ALT levels closer to those of the control mice.

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75 Table 12: Serum ALT Levels at Two Different Dosages of Nicotinamide. Results are displayed graphically in Figure 14. ALT Treatment A505 Blank Diff. Std Diff/St-bl DF Conc Avg Std Dev Std Err Control 0.529 0.412 0.117 0.650 0.4916 1 34.412 Control 0.514 0.412 0.102 0.650 0.4286 1 30.000 Control 0.539 0.412 0.127 0.650 0.5336 1 37.353 Control 0.514 0.412 0.102 0.650 0.4286 1 30.000 Control 0.62 0.412 0.208 0.650 0.8739 1 61.176 Control 0.55 0.412 0.138 0.650 0.5798 1 40.588 Control 0.563 0.412 0.151 0.650 0.6345 1 44.412 Control 0.541 0.412 0.129 0.650 0.5420 1 37.941 39.4853 10.0613 3.5572 Nic 250 Control 0.571 0.424 0.147 0.669 0.6000 1 42.000 Nic 250 Control 0.631 0.424 0.207 0.669 0.8449 1 59.143 Nic 250 Control 0.592 0.424 0.168 0.669 0.6857 1 48.000 Nic 250 Control 0.527 0.424 0.103 0.669 0.4204 1 29.429 Nic 250 Control 0.612 0.424 0.188 0.669 0.7673 1 53.714 Nic 250 Control 0.389 0.412 0.023 0.650 -0.0966 1 -6.765 Nic 250 Control 0.507 0.412 0.095 0.650 0.3992 1 27.941 Nic 250 Control 0.555 0.412 0.143 0.650 0.6008 1 42.059 Nic 250 Control 0.593 0.412 0.181 0.650 0.7605 1 53.235 38.7507 20.0998 6.6999 Nic 500 Control 0.509 0.412 0.097 0.650 0.4076 1 28.529 Nic 500 Control 0.51 0.412 0.098 0.650 0.4118 1 28.824 Nic 500 Control 0.512 0.412 0.100 0.650 0.4202 1 29.412 Nic 500 Control 0.589 0.412 0.177 0.650 0.7437 1 52.059 Nic 500 Control 0.488 0.412 0.076 0.650 0.3193 1 22.353 Nic 500 Control 0.58 0.412 0.168 0.650 0.7059 1 49.412 Nic 500 Control 0.569 0.412 0.157 0.650 0.6597 1 46.176 Nic 500 Control 0.658 0.412 0.246 0.650 1.0336 1 72.353 Nic 500 Control 0.52 0.412 0.108 0.650 0.4538 1 31.765 40.0980 16.0725 5.3575 0.025 ml/kg CCl4 1.178 0.424 0.754 0.669 3.0776 5 1077.143 0.025 ml/kg CCl4 1.513 0.424 1.089 0.669 4.4449 5 1555.714 0.025 ml/kg CCl4 1.603 0.424 1.179 0.669 4.8122 5 1684.286 0.025 ml/kg CCl4 1.603 0.424 1.179 0.669 4.8122 5 1684.286 0.025 ml/kg CCl4 1.196 0.424 0.772 0.669 3.1510 5 1102.857 0.025 ml/kg CCl4 1.346 0.424 0.922 0.669 3.7633 5 1317.143 0.025 ml/kg CCl4 1.612 0.424 1.188 0.669 4.8490 5 1697.143 0.025 ml/kg CCl4 1.389 0.424 0.965 0.669 3.9388 5 1378.571 0.025 ml/kg CCl4 1.356 0.424 0.932 0.669 3.8041 5 1331.429 1425.4 243.1383 81.0461

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76 Table 12 (Continued) CCl4/Nic 250 1.244 0.424 0.820 0.669 3.3469 5 1171.429 CCl4/Nic 250 1.112 0.424 0.688 0.669 2.8082 5 982.857 CCl4/Nic 250 0.579 0.424 0.155 0.669 0.6327 5 221.429 CCl4/Nic 250 0.402 0.424 0.022 0.669 0.0898 5 -31.429 CCl4/Nic 250 1.213 0.424 0.789 0.669 3.2204 5 1127.143 CCl4/Nic 250 1.052 0.424 0.628 0.669 2.5633 5 897.143 CCl4/Nic 250 0.548 0.424 0.124 0.669 0.5061 5 177.143 CCl4/Nic 250 0.556 0.424 0.132 0.669 0.5388 5 188.571 CCl4/Nic 250 0.778 0.424 0.354 0.669 1.4449 5 505.714 CCl4/Nic 250 0.883 0.424 0.459 0.669 1.8735 5 655.714 CCl4/Nic 250 0.554 0.424 0.130 0.669 0.5306 5 185.714 CCl4/Nic 250 1.402 0.424 0.978 0.669 3.9918 5 1397.143 CCl4/Nic 250 0.458 0.424 0.034 0.669 0.1388 5 48.571 CCl4/Nic 250 0.881 0.424 0.457 0.669 1.8653 5 652.857 CCl4/Nic 250 1.389 0.424 0.965 0.669 3. 9388 5 1378.571 637.24 495.7453 128.0009 CCl4/Nic 500 0.474 0.424 0.050 0.669 0.2041 5 71.429 CCl4/Nic 500 0.486 0.424 0.062 0.669 0.2531 5 88.571 CCl4/Nic 500 0.569 0.424 0.145 0.669 0.5918 5 207.143 CCl4/Nic 500 0.829 0.424 0.405 0.669 1.6531 5 578.571 CCl4/Nic 500 0.627 0.424 0.203 0.669 0.8286 5 290.000 CCl4/Nic 500 0.803 0.424 0.379 0.669 1.5469 5 541.429 CCl4/Nic 500 0.772 0.424 0.348 0.669 1.4204 5 497.143 CCl4/Nic 500 0.829 0.424 0.405 0.669 1.6531 5 578.571 CCl4/Nic 500 0.435 0.424 0.011 0.669 0.0449 5 15.714 CCl4/Nic 500 0.413 0.424 0.011 0.669 0.0449 5 -15.714 CCl4/Nic 500 0.437 0.424 0.013 0.669 0.0531 5 18.571 CCl4/Nic 500 0.883 0.424 0.459 0.669 1.8735 5 655.714 CCl4/Nic 500 0.39 0.424 0.034 0.669 0.1388 5 -48.571 CCl4/Nic 500 0.681 0.424 0.257 0.669 1.0490 5 367.143 CCl4/Nic 500 1.075 0.424 0.651 0.669 2.6571 5 930.000 318.3810 300.2496 77.5241

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77 0 200 400 600 800 1000 1200 1400 1600 Treatment GroupsALT(IU/L) Control Nic 250 Control Nic 500 Control 0.025 ml/kg CCl4 CCl4/Nic 250 CCl4/Nic 500 ** ** Figure 14: Serum ALT Levels at Two Different Dosages of Nicotinamide. Adult male ICR mice with an average weight of 30 grams were sacrificed 24 hours following intraperitoneal administration of carbon tetrachloride. Results are expressed as mean SEM. Two-sample unequal variance (heteroscedastic) one-way T-tests were performed, with ** indicating a significant difference at p<0.01. Nicotinamide was found to significantly reduce the CCl4-induced increases in serum ALT levels in a dose-dependent fashion. This suggests that nicotinamide protects against the hepatotoxic effects of CCl4 in a dose-dependent fashion.

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78 The results of this experiment confirmed the expectation that the hepatoprotective effects of nicotinamide were dose-dependent in nature. Although both the 250 and 500 mg/kg dosages of nicotinamide produced statistically significant (p < 0.01) reductions in serum ALT levels, the higher dosage was able to approach the levels of the control mice. The 250 and 500 mg/kg nicotinamide co-treatments reduced the CCl4–induced serum ALT elevations by 55% and 78%, respectively. Since the purpose of this line of research was to determine if as a class of drugs, PARP inhibitors, could potentially protect against CCl4–induced free radical mediated hepatotoxicity, it would be best to determine whether additional PARP inhibitors could mitigate this hepatic damage. The use of another PARP inhibitor may also help indirectly determine if the hepatoprotective effects of nicotinamide were merely a function of nicotinamide being a component of NAD+, the substrate for PARP. The following experiment compared the efficacies of nicotinamide and aminophylline for protection of hepatocytes against carbon tetrachloride-induced toxic effects. Although both co-treatments offered some measure of protection against carbon tetrachlorideinduced hepatotoxicity, aminophylline appeared to be more effective than nicotinamide.

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79 Table 13: Serum ALT Levels Using Carbon Tetrachloride, Aminophylline and Nicotinamide. Results of this experiment are displayed graphically in Figure 15. Treatment A505 Blank SampBl Stdbl R/(stbl) *70 D F Conc Avg. Std.Dev Std.Err control 0.533 0.46 0.073 0.174 0.420 29.368 1 29.368 control 0.531 0.46 0.071 0.174 0.408 28.563 1 28.563 control 0.594 0.46 0.134 0.174 0.770 53.908 1 53.908 control 0.519 0.46 0.059 0.174 0. 339 23.736 1 23.736 33.894 13.573 6.786 50 mg/kg amino 0.621 0.46 0.161 0.174 0.925 64.770 1 64.770 50 mg/kg amino 0.531 0.46 0.071 0.174 0.408 28.563 1 28.563 50 mg/kg amino 0.51 0.46 0.05 0.174 0.287 20.115 1 20.115 50 mg/kg amino 0.477 0.46 0.017 0.174 0.098 6.839 1 6.839 30.072 24.800 12.400 500 mg/kg nic 0.62 0.46 0.16 0.174 0.920 64.368 1 64.368 500 mg/kg nic 0.585 0.46 0.125 0.174 0.718 50.287 1 50.287 500 mg/kg nic 0.605 0.46 0.145 0.174 0.833 58.333 1 58.333 500 mg/kg nic 0.622 0.46 0.162 0.174 0.931 65.172 1 65.172 500 mg/kg nic 0.611 0.46 0.151 0.174 0.868 60.747 1 60.747 59.782 5.985 2.676 ccl4/amino 0.803 0.46 0.343 0.174 1.971 137.989 5 689.943 ccl4/amino 0.626 0.46 0.166 0.174 0.954 66.782 5 333.908 ccl4/amino 0.783 0.46 0.323 0.174 1.856 129.943 5 649.713 ccl4/amino 0.765 0.46 0.305 0.174 1.753 122.701 5 613.506 ccl4/amino 0.737 0.46 0.277 0.174 1.592 111.437 5 557.184 568.851 140.116 62.662 ccl4/nic 0.693 0.46 0.233 0.174 1.339 93.736 5 468.678 ccl4/nic 0.75 0.46 0.29 0.174 1.667 116.667 5 583.333 ccl4/nic 0.791 0.46 0.331 0.174 1.902 133.161 5 665.805 ccl4/nic 0.923 0.46 0.463 0.174 2. 661 186.264 5 931.322 662.284 196.732 87.981 0.025 ml/kg CCl4 1.081 0.46 0.621 0.174 3.569 249.828 5 1249.138 0.025 ml/kg CCl4 0.92 0.46 0.46 0.174 2.644 185.057 5 925.287 0.025 ml/kg CCl4 1.175 0.46 0.715 0.174 4.109 287.644 5 1438.218 0.025 ml/kg CCl4 1.098 0.46 0.638 0.174 3.667 256.667 5 1283.333 0.025 ml/kg CCl4 1.068 0.46 0.608 0.174 3.494 244.598 5 1222.989 1223.793 186.596 83.448

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80 0 200 400 600 800 1000 1200 1400 Treatment GroupsALT (IU/L) control 50 mg/kg amino 500 mg/kg nic 0.025 ml/kg CCl4 ccl4/amino ccl4/nic ** ** Figure 15: Serum ALT Levels Using Carbon Tetrachloride, Aminophylline and Nicotinamide. Adult male ICR mice with an average weight of 30 grams were sacrificed 24 hours following intraperitoneal administration of carbon tetrachloride. Results are expressed as mean SEM. Two-sample unequal variance (heteroscedastic) one-way Ttests were performed, with ** indicating a significant difference at p<0.01. These results suggest that both aminophylline and nicotinamide are capable of protecting the liver cells from CCl4-induced hepatocellular damage.

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81 The results of this experiment showed that both non-specific PARP inhibitors significantly (p < 0.01) reduced the CCl4-induced increases in serum ALT, although neither co-treatment resulted in ALT levels comparable to those of the control groups. The reductions in serum ALT concentrations as a result of the nicotinamide (500 mg/kg) and aminophylline (50 mg/kg) were 46% and 54%, respectively. The next experiment varied the dosages of the co-treatment, aminophylline, with the goal of determining what could be the optimal dosage, reducing the CCl4–induced increase in serum ALT the most, without causing any adverse effects. The highest of these dosages of aminophylline most likely produced serum theophylline levels in excess of the normal therapeutic concentrations, so the mice had to be observed closely to determine if they were showing any signs of theophylline overdose. The question arose whether aminophylline reduced the hepatotoxic effects of carbon tetrachloride, or simply delayed them. This question was answered by conducting a seventy-two (72) hour study for mice treated with carbon tetrachloride and aminophylline, with mice euthanized in twelve (12) hour intervals beyond 24 hours for collection of blood for the serum assays. There does not appear to be a significant delayed toxic effect when aminophylline is administered as a co-treatment. Therefore, aminophylline appears to be protective against carbon tetrachloride, and not just a mechanism to delay the onset of hepatotoxic effects.

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82 Table 14: 72-Hour Serum ALT Levels With Aminophylline and Carbon Tetrachloride. Results of this experiment are provided in bar graph format on the following page. Treatment A505 Blank SampBl Stdbl R/(st-bl) *70 D F Conc Avg. Std.Dev Std.Err 36 hr CCl4 0.514 0.47 0.044 0.256 0.172 12.031 5 60.156 36 hr CCl4 0.611 0.47 0.141 0.256 0.551 38.555 5 192.773 36 hr CCl4 0.651 0.47 0.181 0.256 0.707 49.492 5 247.461 36 hr CCl4 0.546 0.47 0.076 0.256 0.297 20.781 5 103.906 151.07 84.696 42.348 36-hr CCl4/amino 0.465 0.47 -0.005 0.256 -0.02 -1.367 5 -6.836 36-hr CCl4/amino 0.509 0.47 0.039 0.256 0.152 10.664 5 53.32 36-hr CCl4/amino 0.464 0.47 -0.006 0.256 -0.023 -1.641 5 -8.203 36-hr CCl4/amino 0.484 0.47 0.014 0.256 0.055 3.828 5 19.141 14.355 28.862 14.431 48-hr CCl4/amino 0.53 0.47 0.06 0.256 0.234 16.406 5 82.031 48-hr CCl4/amino 0.461 0.47 -0.009 0.256 -0.035 -2.461 5 -12.305 48-hr CCl4/amino 0.469 0.47 -0.001 0.256 -0.004 -0.273 5 -1.367 48-hr CCl4/amino 0.483 0.47 0.013 0.256 0.051 3.555 5 17.773 21.533 42.204 21.102 60-hr CCl4/amino 0.486 0.47 0.016 0.256 0.063 4.375 5 21.875 60-hr CCl4/amino 0.5 0.47 0.03 0.256 0.117 8.203 5 41.016 60-hr CCl4/amino 0.495 0.47 0.025 0.256 0.098 6.836 5 34.18 60-hr CCl4/amino 0.506 0.47 0.036 0.256 0.141 9.844 5 49.219 36.572 11.567 5.784 72-hr CCl4/amino 0.495 0.47 0.025 0.256 0.098 6.836 5 34.18 72-hr CCl4/amino 0.489 0.47 0.019 0.256 0.074 5.195 5 25.977 72-hr CCl4/amino 0.443 0.47 -0.027 0.256 -0.105 -7.383 5 -36.914 72-hr CCl4/amino 0.477 0.47 0.007 0.256 0.027 1.914 5 9.57 8.203 31.771 15.885

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83 0 50 100 150 200 250 Treatment GroupsALT (IU/L) 36 hr CCl4 36-hr CCl4/amino 48-hr CCl4/amino 60-hr CCl4/amino 72-hr CCl4/amino * Figure 16: 72-Hour Serum ALT Levels With Aminophylline and Carbon Tetrachloride. Adult male ICR mice with an average weight of 30 grams were sacrificed 24 hours following intraperitoneal administration of carbon tetrachloride. Results are expressed as mean SEM. Two-sample unequal variance (heteroscedastic) one-way T-tests were performed, with indicative of significant difference at p<0.05. This time course experiment was performed to determine whether intraperitoneal administration of aminophylline (50 mg/kg) one hour following the CCl4 (0.025 ml/kg) injection, was capable of reducing the CCl4-induced increase in serum ALT levels, or whether it merely delayed the metabolism of CCl4, and the resulting peak serum ALT concentration.

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84 This experiment confirmed what was expected, that aminophylline actually reduced the hepatotoxicity of CCl4, as evidenced by diminished serum ALT levels, rather than merely postponing the adverse effect. Had the hepatotoxic effects of CCl4 been merely delayed, there would have been an increase in serum ALT levels at some point after 24 hours. One may assume that, since the relative reduction in serum ALT levels was less at 12 hours than was observed at 24 hours in previous studies, that there may be a lag period of more than 12 hours before aminophylline begins to provide substantial hepatoprotection against CCl4. The next step in this series of experiments with aminophylline was to determine whether higher levels of this co-treatment, administered three times, at thirty (30), sixty (60) and one hundred twenty (120) minutes following the carbon tetrachloride were more beneficial or were potentially harmful to the mice. The use of multiple injections allowed for the gradual increase of aminophylline, and ultimately theophylline, in the blood stream and tissues. Unfortunately, mice administered 0.025 ml/kg carbon tetrachloride and either 100 or 200 mg/kg aminophylline three times did not survive, eliminating those higher co-treatments from further study. As a result, future studies focused on the results of administering 50 mg/kg of aminophylline at various intervals following the injection of CCl4.

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85 Table 15: Serum ALT Levels With Three Administrations of Aminophylline. The results of this experiment are provided in a graph in Figure 17. Treatment A505 Blank A-Bl Std-Bl (R)/Cal DF Conc Avg Std Dev Std Err Control 0.374 0.367 0.007 0.289 0.0242 1 1.696 Control 0.393 0.367 0.026 0.289 0.0900 1 6.298 Control 0.375 0.367 0.008 0.289 0.0277 1 1.938 Control 0.439 0.367 0.072 0.289 0.2491 1 17.439 6.8426 7.3743 3.6871 50 Animophylline x3 0.479 0.367 0.112 0.289 0.3875 1 27.128 50 Animophylline x3 0.388 0.367 0.021 0.289 0.0727 1 5.087 50 Animophylline x3 0.431 0.367 0.064 0.289 0.2215 1 15.502 50 Animophylline x3 0.437 0.367 0.07 0.289 0.2422 1 16.955 16.1678 9.0182 4.5091 0.025 ml/kg CCl4 0.874 0.367 0.507 0.289 1.7543 5 614.014 0.025 ml/kg CCl4 0.872 0.367 0.505 0.289 1.7474 5 611.592 0.025 ml/kg CCl4 0.63 0.367 0.263 0.289 0.9100 5 318.512 0.025 ml/kg CCl4 0.899 0.367 0.532 0.289 1.8408 5 644.291 0.025 ml/kg CCl4 0.581 0.367 0.214 0.289 0.7405 5 259.170 0.025 ml/kg CCl4 0.535 0.367 0.168 0.289 0.5813 5 203.460 0.025 ml/kg CCl4 1.012 0.367 0.645 0.289 2.2318 5 781.142 0.025 ml/kg CCl4 0.954 0.367 0.587 0.289 2.0311 5 710.900 517.8849 222.3591 78.6158 50 Amino + CCl4 0.449 0.367 0.082 0.289 0.2837 5 99.308 50 Amino + CCl4 0.461 0.367 0.094 0.289 0.3253 5 113.841 50 Amino + CCl4 0.382 0.367 0.015 0.289 0.0519 5 18.166 50 Amino + CCl4 0.453 0.367 0.086 0.289 0.2976 5 104.152 50 Amino + CCl4 0.586 0.367 0.219 0.289 0.7578 5 265.225 50 Amino + CCl4 0.376 0.367 0.009 0.289 0.0311 5 10.900 50 Amino + CCl4 0.506 0.367 0.139 0.289 0.4810 5 168.339 50 Amino + CCl4 0.388 0.367 0.021 0.289 0.0727 5 25.433 100.6704 86.4962 30.5810

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86 0 100 200 300 400 500 600 700 Treatment GroupsALT (IU/L) Control 50 Aminophylline x3 0.025 ml/kg CCl4 50 Amino x3 + CCl4 ** Figure 17: Serum ALT Levels With Three Administrations of Aminophylline. Adult male ICR mice with an average weight of 30 grams were sacrificed 24 hours following intraperitoneal administration of carbon tetrachloride. Results are expressed as mean SEM. Two-sample unequal variance (heteroscedastic) one-way T-tests were performed, with ** indicating a significant difference at p<0.01. The three administrations of 50 mg/kg aminophylline via intraperitoneal injection at 30, 60 and 120 minutes following a single intraperitoneal dosage of CCl4 (0.025 ml/kg) reduced the CCl4-induced increases in ALT levels over eighty percent.

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87 The serum ALT assay results for this experiment strongly suggest that the thrice administration of aminophylline (50 mg/kg) at 30, 60 and 120 minutes following the intraperitoneal administration of 0.025 ml/kg CCl4 protects against the hepatotoxic ffects of CCl4. The 81% reduction in serum ALT levels as a result of the co-treatment with aminophylline was statistically significant (p < 0.01). There was, however, a concern that one of the mice given both CCl4 and aminophylline exhibited a potential sign of theophylline toxicity (twitching) as a result of the three dosages of aminophylline, perhaps exacerbated in some fashion by the prior administration of CCl4. In response to the prior results, the next experiment further addressed potential concerns about aminophylline by opting for only two intraperitoneal administrations of 50 mg/kg aminophylline, at 30 and 60 minutes following the carbon tetrachloride, in lieu of three dosages. Although there was a significant hepatoprotective effect, the positive effects were substantially lower than when aminophylline was administered three times.

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88 Table 16: Serum ALT Levels With Two Administrations of Aminophylline. The results of this experiment are provided in a bar chart in Figure 18. CCl4+Amino ALT No Treatment A505 Blank A505-Bl StdBl (R)/Cal DF Conc. Avg Std Dev Std Err 25 Control 0.359 0.302 0.057 0.344 0.1657 1 11.599 26 Control 0.314 0.302 0.012 0.344 0.0349 1 2.442 27 Control 0.412 0.302 0.11 0.344 0.3198 1 22.384 28 Control 0.314 0.302 0.012 0.344 0.0349 1 2.442 29 Control 0.356 0.302 0.054 0.344 0.1570 1 10.988 30 Control 0.369 0.302 0.067 0.344 0.1948 1 13.634 31 Control 0.304 0.302 0.002 0.344 0.0058 1 0.407 9.1279 7.8639 2.9723 1 50 Aminophy lline x2 0.371 0.302 0.069 0.344 0.2006 1 14.041 2 50 Aminophy lline x2 0.362 0.302 0.06 0.344 0.1744 1 12.209 3 50 Aminophy lline x2 0.379 0.302 0.077 0.344 0.2238 1 15.669 4 50 Aminophy lline x2 0.361 0.302 0.059 0.344 0.1715 1 12.006 5 50 Aminophy lline x2 0.309 0.302 0.007 0.344 0.0203 1 1.424 6 50 Aminophy lline x2 0.403 0.302 0.101 0.344 0.2936 1 20.552 7 50 Aminophy lline x2 0.498 0.302 0.196 0.344 0.5698 1 39.884 8 50 Aminophy lline x2 0.406 0.302 0.104 0.344 0.3023 1 21.163 17.1185 11.0487 3.9063 9 50 Amino + CCl4 0.565 0.302 0.263 0.344 0.7645 5 267.587 10 50 Amino + CCl4 0.422 0.302 0.12 0.344 0.3488 5 122.093 11 50 Amino + CCl4 0.489 0.302 0.187 0.344 0.5436 5 190.262 12 50 Amino + CCl4 0.578 0.302 0.276 0.344 0.8023 5 280.814 13 50 Amino + CCl4 0.347 0.302 0.045 0.344 0.1308 5 45.785 14 50 Amino + CCl4 0.522 0.302 0.22 0.344 0.6395 5 223.837 15 50 Amino + CCl4 0.384 0.302 0.082 0.344 0.2384 5 83.430 16 50 Amino + CCl4 0.591 0.302 0.289 0.344 0.8401 5 294.041 188.4811 94.8566 33.5369 17 0.025 ml/kg CCl4 0.846 0.302 0.544 0.344 1.5814 5 553.488 18 0.025 ml/kg CCl4 0.673 0.302 0.371 0.344 1.0785 5 377.471 19 0.025 ml/kg CCl4 0.71 0.302 0.408 0.344 1.1860 5 415.116 20 0.025 ml/kg CCl4 0.492 0.302 0.19 0.344 0.5523 5 193.314 21 0.025 ml/kg CCl4 0.797 0.302 0.495 0.344 1.4390 5 503.634 22 0.025 ml/kg CCl4 0.507 0.302 0.205 0.344 0.5959 5 208.576 23 0.025 ml/kg CCl4 1.025 0.302 0.723 0.344 2.1017 5 735.610 24 0.025 ml/kg CCl4 0.857 0.302 0.555 0.344 1.6134 5 564.680 443.9862 184.6513 65.2841

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89 0 100 200 300 400 500 600 Treatment GroupALT (IU/L) Control 50 x2 Amino CCl4 Amino + CCl4 ** Figure 18: Serum ALT Levels with Two Administrations of Aminophylline. Adult male ICR mice with an average weight of 30 grams were sacrificed 24 hours following intraperitoneal administration of carbon tetrachloride. Results are expressed as mean SEM. Two-sample unequal variance (heteroscedastic) one-way T-tests were performed, with ** indicating a significant difference at p<0.01. Administration of 50 mg/kg aminophylline at 30 and 60 minutes reduced the CCl4-induced increase in ALT levels greater than fifty percent.

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90 This experiment showed that it was possible for two intraperitoneal administrations of aminophylline (50 mg/kg) at 30 and 60 minute intervals following the intraperitoneal administration of CCl4 to significantly reduce (p < 0.01) the serum ALT levels. Although the CCl4-induced elevation in serum ALT was diminished by 58%, the resulting levels were still indicative of some hepatocellular damage. The belief that there may have been some hepatocellular damage may be supported by the relatively low ALT levels for the group administered only CCl4. These results may have been lower than in the previous experiments, because the ALT standard solution may have degraded somewhat. Even though the ALT standard had not yet expired, a new standard would be used in subsequent experiments. Fortunately, ALT elevations are not as indicative of irreparable damage as other findings ( e.g LDH). Although these serum ALT assay results do not reflect a complete reversion to those of the control groups, they do suggest that aminophylline, when administered twice is significantly hepatoprotective against CCl4. The subsequent experiment tested whether the hepatoprotective results from the prior experiment, in which 50 mg/kg of aminophylline was administered thrice, at 30, 60 and 90 minute intervals following intraperitoneal administration of 0.025 ml/kg of CCl4, were repeatable. The reason for this is that these initial ALT assay results suggested that there was nearly a complete reversion to the control levels when both CCl4.and aminophylline were administered, and such a dosing regimen could be considered completely hepatoprotective.

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91 Table 17: Serum ALT Levels with Three Co-Treatments With Aminophylline. The results of this experiment are displayed graphically in Figure 19 on the following page. ALT No Treatment A505 Blank A-Bl Std-Bl(R)/Cal DFConc Avg Std Dev Std Err 1 Control 0.447 0.373 0.0740.2230.3318123.229 2 Control 0.414 0.373 0.0410.2230.1839112.870 3 Control 0.401 0.373 0.0280.2230.125618.789 4 Control 0.374 0.373 0.0010.2230.004510.314 5 Control 0.408 0.373 0.0350.2230.1570110.987 6 Control 0.376 0.373 0.0030.2230.013510.942 7 Control 0.394 0.373 0.0210.2230.094216.592 8 Control 0.404 0.373 0.0310.2230.139019.7319.18167.24942.5631 9 50 Animophylline x3 0.397 0.373 0.0240.2230.107617.534 10 50 Animophylline x3 0.38 0.373 0.0070.2230.031412.197 11 50 Animophylline x3 0.437 0.373 0.0640.2230.2870120.090 12 50 Animophylline x3 0.391 0.373 0.0180.2230.080715.650 13 50 Animophylline x3 0.427 0.373 0.0540.2230.2422116.951 14 50 Animophylline x3 0.391 0.373 0.0180.2230.080715.650 15 50 Animophylline x3 0.443 0.373 0.070.2230.3139121.973 16 50 Animophylline x3 0.404 0.373 0.0310.2230.139019.73111.22207.42972.6268 17 50 Amino + CCl4 0.482 0.373 0.1090.2230.48885171.076 18 50 Amino + CCl4 0.598 0.373 0.2250.2231.00905353.139 19 50 Amino + CCl4 0.489 0.373 0.1160.2230.52025182.063 20 50 Amino + CCl4 0.418 0.373 0.0450.2230.2018570.628 21 50 Amino + CCl4 0.507 0.373 0.1340.2230.60095210.314 22 50 Amino + CCl4 0.445 0.373 0.0720.2230.32295113.004 23 50 Amino + CCl4 0.61 0.373 0.2370.2231.06285371.973 24 50 Amino + CCl4 0.507 0.373 0.1340.2230.60095210.314210.3139105.459437.2855 25 0.025 ml/kg CCl4 0.862 0.373 0.4890.2232.19285767.489 26 0.025 ml/kg CCl4 0.782 0.373 0.4090.2231.83415641.928 27 0.025 ml/kg CCl4 1.198 0.373 0.8250.2233.699651294.843 28 0.025 ml/kg CCl4 1.047 0.373 0.6740.2233.022451057.848 29 0.025 ml/kg CCl4 1.227 0.373 0.8540.2233.829651340.359 30 0.025 ml/kg CCl4 0.803 0.373 0.430.2231.92835674.888 31 0.025 ml/kg CCl4 1.292 0.373 0.9190.2234.121151442.377 32 0.025 ml/kg CCl4 1.21 0.373 0.8370.2233.753451313.6771066.676327.8986115.9297

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92 0 200 400 600 800 1000 1200 Treatment GroupsALT (IU/L) Control 50 Amino x3 0.025 CCl4 Amino + CCl4 ** Figure 19: Serum ALT Levels with Three Co-Treatments With Aminophylline. Adult male ICR mice with an average weight of 30 grams were sacrificed 24 hours following intraperitoneal administration of carbon tetrachloride. Results are expressed as mean SEM. Two-sample unequal variance (heteroscedastic) one-way T-tests were performed, with ** indicating a significant difference at p<0.01. Aminophylline (50 mg/kg) was administered via intraperitoneal injection 30, 60 and 120 minutes following intraperitoneal administration of CCl4. The three administrations of aminophylline reduced the CCl4-induced increase in serum ALT levels by roughly eighty percent.

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93 This experiment suggests that the hepatoprotective effects of aminophylline observed in previous experiments were repeatable. The relative diminution of CCl4induced increases in serum ALT was comparable in this experiment (81%) as was the case in the prior study with three administrations of aminophylline (also 81%). There may, however be valid concerns about the safety of aminophylline, as a result of the observation of a potentially adverse effect in a prior experiment, although none were observed in the latest one. The serum ALT assay results strongly suggest that the intraperitoneal administration of aminophylline is extremely protective against the free radical-mediated hepatotoxicity caused by carbon tetrachloride. The increase in serum ALT levels as a result of 0.025 ml/kg intraperitoneal administration of carbon tetrachloride is roughly 100-fold. The reason for this is that structurally intact and viable hepatocytes do not normally release this intracellular enzyme into the interstitial fluid. This protective effect of aminophylline appears to be dose-dependent, as the three administrations of 50 mg/kg aminophylline are much more effective than two administrations, abstaining from the two-hour post-toxicant interval, with average reductions in ALT of approximately eighty percent and fifty percent, respectively. By administering lesser concentrations three times, it may reduce the potential for any adverse effects due to aminophylline. TBARS Assay The results from the experiment, in which three 50 mg/kg intraperitoneal dosages of aminophylline reduced the serum ALT levels elevated due to CCl4.by 80 percent,

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94 raised questions as to the mechanism by which this occurred. In the following experiment, the thiobarbituric acid reactive substances (TBARS) assay was conducted to determine the extent of lipid peroxidation caused by 0.025 ml/kg carbon tetrachloride and prevented by two 50 mg/kg dosages of aminophylline. The controls utilized in this experiment were also utilized from a similar study, in which the dilution in phosphate buffered saline was one part liver tissue in ten parts total, or one part in nine parts saline. The remaining samples were diluted one part to ten, which resulted in a dilution factor of eleven, compared to ten for the controls. The samples from mice treated with carbon tetrachloride and aminophylline yielded MDA equivalent levels comparable to the controls, whereas carbon tetrachloride elevated MDA equivalent levels three-fold. This indicates that aminophylline may protect against the production of lipid peroxides as a result of carbon tetrachloride.

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95 Table 18: Standards for TBARS Assay. Results of this portion of the TBARS assay are listed in the standard curve below. TBARS Calibration Curve Sample MDA Equiv. A532 A 0 0.003 B 1 0.032 C 2 0.079 D 3 0.176 E 4 0.215 TBARS Curve 6/3/05y = 0.0516x + 0.003 R2 = 0.9518 0.001 0.01 0.1 1 00.511.522.533.544.5 MDA EquivalentsA532 Figure 20: Standard Curve for TBARS Assay. This standard curve was established to determine the extent of lipid peroxidation, in MDA equivalents, of the samples. The TBARS results for these samples are on the following page.

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96 Table 19: Hepatic Lipid Peroxidation Levels With Two Dosages of Aminophylline. The results of this TBARS assay are displayed graphically in Figure 21 on the following page. TBARS No Treatment A532 Conc. DF MDA Avg Std Dev Std Err C1 Control 0.258 4.942 10 49.419 C2 Control 0.292 5.601 10 56.008 52.713 4.659 3.295 1 50 Animo x2 0.291 5.581 11 61.395 2 50 Animo x2 0.195 3.721 11 40.930 3 50 Animo x2 0.294 5.640 11 62.035 4 50 Animo x2 0.227 4.341 11 47.752 5 50 Animo x2 0.317 6.085 11 66.938 6 50 Animo x2 0.313 6.008 11 66.085 7 50 Animo x2 0.282 5.407 11 59.477 8 50 Animo x2 0.191 3.643 11 40.078 55.586 10.995 3.887 9 CCl4+Amino 0.282 5.407 11 59.477 10 CCl4+Amino 0.268 5.136 11 56.492 11 CCl4+Amino 0.224 4.283 11 47.112 12 CCl4+Amino 0.32 6.143 11 67.578 13 CCl4+Amino 0.22 4.205 11 46.260 14 CCl4+Amino 0.173 3.295 11 36.240 15 CCl4+Amino 0.27 5.174 11 56.919 16 CCl4+Amino 0.254 4.864 11 53.508 52.948 9.586 3.389 17 0.025 CCl4 0.789 15.233 11 167.558 18 0.025 CCl4 1.215 23.488 11 258.372 19 0.025 CCl4 0.959 18.527 11 203.798 20 0.025 CCl4 0.787 15.194 11 167.132 21 0.025 CCl4 0.834 16.105 11 177.151 22 0.025 CCl4 0.696 13.430 11 147.733 23 0.025 CCl4 0.74 14.283 11 157.112 24 0.025 CCl4 0.554 10.678 11 117.461 174.540 41.875 14.805

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97 0 20 40 60 80 100 120 140 160 180 200 Treatment GroupsMDA Equivalents (uM) Control 50 Animo x2 0.025 CCl4 CCl4+Amino ** Figure 21: Hepatic Lipid Peroxidation Levels With Two Dosages of Aminophylline. Adult male ICR mice with an average weight of 30 grams were sacrificed 24 hours following intraperitoneal administration of carbon tetrachloride. Results are expressed as mean SEM. Two-sample unequal variance (heteroscedastic) one-way T-tests were performed, with ** indicating a significant difference at p<0.01. The administration of 50 mg/kg aminophylline at 30 and 60 minutes following CCl4 reduced the CCl4-induced increases in MDA equivalents to levels comparable to the controls.

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98 The results of this hepatic tissue TBARS assay showed that aminophylline, administered in two 50 mg/kg dosages was able to significantly (p < 0.01) reduce the free radical damage caused by CCl4. The results of this experiment beg the question as to whether PARP inhibition may be the primary hepatoprotective mechanism of aminophylline. Perhaps it could entail phosphodiesterase inhibition or some other means of preventing lipid peroxidation. Regardless of which of the multitude of mechanisms of action attributed to aminophylline is the major cause of its hepatoprotective effects, it is clear that the extent of free radical hepatocellular damage caused by CCl4 is significantly reduced. Histopathological Staining / Immunohistochemistry The results of the histopathology strongly suggest that aminophylline protects against both the necrotic and apoptotic responses of the hepatocytes to carbon tetrachloride. The hematoxylin and eosin staining, along with the TUNEL and cleaved caspase-3 slides for two of the three random samples from mice, which were administered intraperitoneal injections of carbon tetrachloride (0.025 ml/kg) and the three administrations of aminophylline, were indistinguishable from the control samples. The samples from one of the mice, which was treated with carbon tetrachloride and aminophylline, did reflect minimal damage. However the extent of this was not comparable to that observed in mice that received carbon tetrachloride alone. The mice who were administered only carbon tetrachloride displayed widespread necrotic zones in the centrilobular regions, along with minimal positive results for the TUNEL (one

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99 response was strong) and cleaved caspase-3 immunohistochemistry. As was expected, there were no detectable histopathological damages to the livers of mice receiving only aminophylline

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100 Table 20: Results of Histopathological Assessments for CCl4 and Aminophylline Treatments. Grading of histopathologic staining /immunohistochemistry was as follows: H&E: presence or absence of necrosis and its location ( e.g. centrilobular, periportal) TUNEL: Apoptotic Index based on the percentage/intensity of apoptotic cells, from 0-3 0%/none = 0; 1-25%/mild = 1; 26-50%/moderate = 2; >50%/prominent = 3 Cleaved caspase-3: Percent of cells testing positive for cleaved (activated) caspase-3 using immunohistochemistry, from 0-4. 0% = 0; 1-25% = 1; 26-50% = 2; 51-75% = 3; >75% = 4. Histopathology Grading ID # Treatment Group Hematoxylin & Eosin TUNEL Cleaved Caspase-3 2 Control Normal 0 0 5 Control Normal 0 0 7 Control Normal 0 0 11 Aminophylline Normal 0 0 12 Aminophylline Normal 0 0 14 Aminophylline Normal 0 0 18 CCl4 & Aminophylline Normal 0 0 22 CCl4 & Aminophylline Normal 0 0 23 CCl4 & Aminophylline Centrilobular Necrosis 1 1 28 CCl4 Centrilobular Necrosis 1 1 29 CCl4 Centrilobular Necrosis 1 1 31 CCl4 Centrilobular Necrosis 3 1

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101 Figure 22: Hematoxylin and Eosin Staining Results: Control Treatment. Adult male ICR mice with an average weight of 30 grams were sacrificed 24 hours following intraperitoneal administration of carbon tetrachloride. Hematoxylin is the blue stain for positively charged molecules ( e.g. DNA). Eosin is the pink counterstain for negatively charged molecules ( e.g. protein). There were no necrotic zones found in this photographed slide, or for the other samples from control mice receiving only corn oil and saline, that were visually confirmed under the microscope, but not photographed due to resource constraints. These findings confirm the low ALT values in serum from mice in this treatment group, in that there was no damage to the liver.

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102 Figure 23: Hematoxylin and Eosin Staining Results: Aminophylline Treatment. Adult male ICR mice with an average weight of 30 grams were sacrificed 24 hours following intraperitoneal administration of carbon tetrachloride. Hematoxylin is the blue stain for positively charged molecules ( e.g. DNA). Eosin is the pink counterstain for negatively charged molecules ( e.g. protein). There were no necrotic zones found in this photographed slide, or for the other samples from mice receiving only corn oil and aminophylline, that were visually confirmed under the microscope, but not photographed due to resource constraints. These findings confirm the low ALT values in serum from mice in this treatment group, comparable to those of the controls, meaning that aminophylline does not cause liver damage.

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103 Figure 24: Hematoxylin and Eosin Staining Results: Aminophylline and Carbon Tetrachloride Treatment. Adult male ICR mice with an average weight of 30 grams were sacrificed 24 hours following intraperitoneal administration of carbon tetrachloride. Hematoxylin is the blue stain for positively charged molecules ( e.g. DNA). Eosin is the pink counterstain for negatively charged molecules ( e.g. protein). The majority of the samples from mice that were administered carbon tetrachloride and aminophylline displayed no indication of a necrotic response, as indicated in this slide. These results were comparable to those of the controls.

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104 Figure 25: Hematoxylin and Eosin Staining Results: Carbon Tetrachloride Treatment. Adult male ICR mice with an average weight of 30 grams were sacrificed 24 hours following intraperitoneal administration of carbon tetrachloride. Hematoxylin is the blue stain for positively charged molecules ( e.g. DNA). Eosin is the pink counterstain for negatively charged molecules ( e.g. protein). This group of mice received only carbon tetrachloride and saline, without aminophylline or any other PARP inhibitor. There appears to be a substantial necrotic response surrounding the central vein, as is reflected by the absence of intact cells. This slide confirms that elevations in serum ALT levels observed for this treatment group were indicative of irreversible hepatocellular damage.

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105 Figure 26: TUNEL Staining Results: Control Treatment. Adult male ICR mice with an average weight of 30 grams were sacrificed 24 hours following intraperitoneal administration of carbon tetrachloride. TUNEL stains for the presence of DNA double strand cleavage which is specifically associated with apoptosis, a form of controlled cell death. Grading of histopathologic staining was as follows: TUNEL: Apoptotic Index based on the percentage/intensity of apoptotic cells, from 0-3 0%/none = 0; 1-25%/mild = 1; 26-50%/moderate = 2; >50%/prominent = 3 The Apoptotic Index of this sample is 0. As was the case with the correspondingly low ALT levels for this treatment group, there was no evidence of hepatocellular injury.

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106 Figure 27: TUNEL Staining Results: Aminophylline Treatment. Adult male ICR mice with an average weight of 30 grams were sacrificed 24 hours following intraperitoneal administration of carbon tetrachloride. TUNEL stains for the presence of DNA double strand cleavage which is specifically associated with apoptosis. This slide is representative of the group that received corn oil and aminophylline, and no carbon tetrachloride. Grading of histopathologic staining was as follows: TUNEL: Apoptotic Index based on the percentage/intensity of apoptotic cells, from 0-3 0%/none = 0; 1-25%/mild = 1; 26-50%/moderate = 2; >50%/prominent = 3 The Apoptotic Index of this sample is 0. As was the case with the correspondingly low ALT levels for this treatment group, there was no evidence of hepatocellular injury.

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107 Figure 28: TUNEL Staining Results: Aminophylline and Carbon Tetrachloride Treatment. Adult male ICR mice with an average weight of 30 grams were sacrificed 24 hours following intraperitoneal administration of carbon tetrachloride. TUNEL stains for the presence of DNA double strand cleavage which is specifically associated with apoptosis. Grading of histopathologic staining was as follows: TUNEL: Apoptotic Index based on the percentage/intensity of apoptotic cells, from 0-3 0%/none = 0; 1-25%/mild = 1; 26-50%/moderate = 2; >50%/prominent = 3 The Apoptotic Index of this sample is 0.The majority of the samples from mice that were administered carbon tetrachloride and aminophylline displayed no positive cells. There was no evidence of hepatocellular injury in the majority of mice in this treatment group.

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108 Figure 29: TUNEL Staining Results: Carbon Tetrachloride Treatment. Adult male ICR mice with an average weight of 30 grams were sacrificed 24 hours following intraperitoneal administration of carbon tetrachloride. TUNEL stains for the presence of DNA double strand cleavage which is specifically associated with apoptosis, for the group receiving only carbon tetrachloride and saline. Grading was as follows: TUNEL: Apoptotic Index based on the percentage/intensity of apoptotic cells, from 0-3 0%/none = 0; 1-25%/mild = 1; 26-50%/moderate = 2; >50%/prominent = 3 The Apoptotic Index of this sample is 3, which is reflective of the strong or prominent apoptotic response, which is indicative of irreversible hepatocellular damage that corresponds with the elevated ALT levels for the mice in this treatment group.

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109 Figure 30: Cleaved Caspase-3 Immunohistochemistry Results: Control Treatment. Adult male ICR mice with an average weight of 30 grams were sacrificed 24 hours following intraperitoneal administration of carbon tetrachloride. The histopathology results for this immunohistochemical stain are reflected in this picture. The cleavage and activation of caspase-3 is a committed step toward apoptosis. Grading of the immunohistochemistry was as follows: Cleaved caspase-3: Percent of cells testing positive for cleaved (activated) caspase-3 using immunohistochemistry, from 0-4. 0% = 0; 1-25% = 1; 26-50% = 2; 51-75% = 3; >75% = 4. This sample received a grade of 0, indicating the lack of hepatic damage among controls.

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110 Figure 31: Cleaved Caspase-3 Immunohistochemistry Results: Aminophylline Treatment. Adult male ICR mice with an average weight of 30 grams were sacrificed 24 hours following intraperitoneal administration of carbon tetrachloride. The histopathology results for this immunohistochemical stain are reflected in this picture. The cleavage and activation of caspase-3 is a committed step toward apoptosis. Grading was as follows: Cleaved caspase-3: Percent of cells testing positive for cleaved (activated) caspase-3 using immunohistochemistry, from 0-4. 0% = 0; 1-25% = 1; 26-50% = 2; 51-75% = 3; >75% = 4. This sample received a grade of 0, indicative of the lack of hepatic damage in mice administered aminophylline.

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111 Figure 32: Cleaved Caspase-3 Immunohistochemistry Results: Aminophylline and Carbon Tetrachloride Treatment. Adult male ICR mice with an average weight of 30 grams were sacrificed 24 hours following intraperitoneal administration of carbon tetrachloride. The cleavage and activation of caspase-3 is a committed step toward apoptosis. Histopathologic grading for this immunohistochemistry was as follows: Cleaved caspase-3: Percent of cells testing positive for cleaved (activated) caspase-3 using immunohistochemistry, from 0-4. This sample received a grade of 0. 0% = 0; 1-25% = 1; 26-50% = 2; 51-75% = 3; >75% = 4. The majority of the samples from mice from this group displayed no positive cells, confirming that aminophylline is an effective co-treatment against carbon tetrachloride.

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112 Figure 33: Cleaved Caspase-3 Immunohistochemistry Results: Carbon Tetrachloride Treatment. Adult male ICR mice with an average weight of 30 grams were sacrificed 24 hours following intraperitoneal administration of carbon tetrachloride. The cleavage and activation of caspase-3 is a committed step toward apoptosis. Histopathologic grading for this immunohistochemistry was as follows: Cleaved caspase-3: Percent of cells testing positive for cleaved (activated) caspase-3 using immunohistochemistry, from 0-4. 0% = 0; 1-25% = 1; 26-50% = 2; 51-75% = 3; >75% = 4. This sample received a grade of 1. As expected, mice administered carbon tetrachloride had evidence of apoptosis.

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113 In one of the mice receiving carbon tetrachloride and aminophylline, there were unexpected results. This single mouse showed necrosis and apoptosis. There are a number of possible explanations for the occurrence of necrosis and apoptosis in one of the mice receiving carbon tetrachloride and aminophylline. The primary reason is most likely related to the use of outbred ICR mice. In inbred mice, the genetic relatedness is higher than normal littermates (0.5, or 50% of genes are common), to the point where it approaches 1 (100% common genes), which is indicative of identical twins or clones. Outbred mice in the numbers used in these experiments were of such numbers, that they could not have originated from the same litter. The significance of this is that unlike inbred mice, outbred mice may not always yield the same results as a result of differing genes. Therefore it would be expected that there would be some variation in the response to the toxicant and/or the co-treatment. In Vivo Conclusions The in vivo results, although independent from their in vitro counterparts, similarly suggest that PARP inhibitors protect against carbon tetrachloride-induced hepatotoxity. The serum ALT assays show that the intraperitoneal administration of CCl4 significantly increases the levels of ALT in the serum, strongly suggestive of hepatocellular damage. When aminophylline, a non-specific PARP inhibitor, was administered via intraperitoneal injection after the toxicant, the 100-fold increases in ALT were reduced by eighty percent. The administration of CCl4 by intraperitoneal injection significantly increased lipid peroxidation three-fold, as evidenced by the

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114 TBARS assay results. The co-treatment with aminophylline significantly reduced the extent of lipid peroxidation. Administration of CCl4 resulted in histopathology findings indicative of centrilobular necrosis and apoptosis. Histopathology findings for the majority of the mice administered CCl4 followed by aminophylline were negative for necrosis and apoptosis. The results complement each other in that the histopathology expands upon the nature and extent of damage caused by CCl4 and mitigated by aminophylline, as evidenced in the ALT assays. This is because elevated ALT levels can be caused by sources other than from the liver ( e.g. kidney, pancreas, heart and skeletal muscle) and may merely reflect reversible damage. In contrast, the histopathology indicates the presence of non-reversible damage specifically in the liver. Increases in ALT values may be indicative of temporary loss of hepatocellular membrane integrity. The indicators of apoptosis, DNA fragmentation (TUNEL) and cleavage of caspase-3 (immunohistochemistry), are indicative of committed steps toward programmed cell death. Similarly, the necrosis observed in the hematoxylin and eosin staining is irreversible. Because of these results, one can conclude that aminophylline, a nonspecific PARP inhibitor, can effectively protect the liver against the free radical, non glutathione-mediated hepatotoxic effects of CCl4. In short, aminophylline may be an effective treatment against exposure to carbon tetrachloride. Summary of Findings The in vitro and in vivo results independently associate CCl4 with hepatic damage, and suggest that the use of PARP inhibitors mitigates that damage. Although these

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115 results can not be directly correlated to one another, they each serve a purpose in that they provide information as to the mechanism of action of PARP inhibitors, as well as the phenomenological data critical to the use of PARP inhibitors to prevent hepatic damage. Ideally, the same assays would have been used for both the in vitro and in vivo assays. In the case of the latter, there was a concern that there would not be enough serum to conduct both ALT and LDH assays. Since earlier experiments using ALT have shown elevations as a result of administration of CCl4, there was no question as to the sensitivity of this test, thus the serum was reserved for ALT tests, and no further LDH assays were conducted. Although the in vitro and in vivo tests were not the identical assays, they all yielded similar results, in that the use of CCl4, either in the culture media or through intraperitoneal injections, resulted in substantial findings of hepatic damage. Likewise the use of a PARP inhibitor with the CCl4 significantly reduced the extent of the carbon tetrachloride-induced damage. In conclusion, these experiments strongly indicate that the use of PARP inhibitors, such as 6( 5H )-phenanthridinone and aminophylline can protect against the damage caused by carbon tetrachloride. This research is significant in that it may provide a means to protect against other free radical-mediated and glutathioneindependent hepatotoxicants in humans.

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116 In Vitro Findings • Introduction of CCl4 to the culture media for HepG2 cells significantly increases relative LDH levels in the culture media. • Co-treatment of HepG2 cells with 6( 5H )-phenanthridinone significantly reduces the CCl4-induced increases in relative LDH levels in the culture media. • Introduction of CCl4 into the culture media with HepG2 cells significantly increases PARP Activity. • Co-treatment of HepG2 cells with 6( 5H )-phenanthridinone significantly reduces the CCl4-induced increases in PARP activity. In Vivo Findings • Intraperitoneal administration of ICR mice with CCl4 significantly increases serum ALT levels. • Subsequent intraperitoneal administration of ICR mice with aminophylline significantly reduced the CCl4–mediated elevations in serum ALT levels. • Intraperitoneal administration of ICR mice with CCl4 significantly increases lipid peroxidation in the liver. • Subsequent intraperitoneal administration of ICR mice with aminophylline significantly reduced the CCl4–mediated elevations in lipid peroxidation. • Intraperitoneal administration of ICR mice with CCl4 results in substantial centrilobular necrotic and apoptotic responses in liver tissue. • Subsequent intraperitoneal administration of ICR mice with aminophylline eliminated or reduced the presence of necrosis and apoptosis in the liver. Figure 34: Summary of Experimental Findings.

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117 Chapter Five Discussion The results of the previously discussed experiments make a strong case for the protective role of PARP inhibitors in general, and specifically aminophylline, against carbon tetrachloride-induced hepatotoxicity. The issue of correlation between the in vitro and in vivo experimental results also warrants some discussion. Aside from a general discussion and summation of the experimental results, there are other matters of importance. Two major themes, which warrant further discussion in light of the experiments reported in this dissertation, are the impact of this research on public health and the avenues for future study. Lastly, the conclusion summarizes and stresses the importance of the experimental findings. Explanation of Experimental Findings The following is a summary of the experimental results. These results are addressed in this section in the same general order as the materials and methods section and the results. The in vitro portion of these experiments is further divided into the LDH assay results and PARP activity assay results. The in vivo portion of these experiments is subdivided into ALT assay results, TBARS assay results and histopathology findings. The primary PARP inhibitor used in the in vitro portion of these experiments is 6( 5H )-

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118 phenanthridinone, and the main PARP inhibitor used in the in vivo experiments is aminophylline. The in vitro findings using 6( 5H )-phenanthridinone as a co-treatment show that, at concentrations below 40 M, this PARP inhibitor can protect against carbon tetrachloride-induced hepatotoxicity, without adversely affecting the metabolic activity of the hepatocytes. The protection against hepatotoxic effects is evidenced by the significant diminution in lactate dehydrogenase (LDH) activity in the culture media for cells receiving both treatments versus those exposed only to carbon tetrachloride. Further studies measuring the activity of PARP resulted in significant reductions in PARP activity when 6( 5H )-phenanthridinone was co-administered compared to when carbon tetrachloride was the only treatment. This diminution in PARP activity is critical, as excessive activity can lead to NAD and ATP depletion, which can result in necrosis. Lactate dehydrogenase is an intracellular enzyme, that if found in the culture medium, is associated with the loss of membrane integrity. As was displayed in Figure 1, lipids are common targets of the free radicals generated as a result of the dehalogenation of carbon tetrachloride. The phospholipids that constitute cell membranes are no exception to this rule. The relative LDH levels were significantly elevated as a result of the exposure to carbon tetrachloride in the culture media, which suggests that carbon tetrachloride damages the hepatocytes, resulting in the release of LDH, an intracellular enzyme, into the culture media. The co-administration of the cell culture media with 6( 5H )-phenanthridinone resulted in the reduction of the relative LDH levels by fifty percent.

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119 The fact that the LDH activity was only reduced roughly fifty percent may be as much a function of the solubility of 6( 5H )-phenanthridinone as any other factor. In order to completely solubilize 6( 5H )-phenanthridinone, one has to use pure dimethyl sulfoxide (DMSO) as a solvent. In my experiments, the 6( 5H )-phenanthridinone was solubilized in pure DMSO, and then a small amount of this solution was added to the media, resulting in a colloidal mixture. The use of DMSO was minimized because it is known to inhibit PARP by itself. Furthermore, the United States Food and Drug Administration (FDA) has reported hepatotoxic effects of topical administration of DMSO. The ultimate test as to whether PARP plays a role in the reduction in carbon tetrachloride-induced hepatotoxicity by 6( 5H )-phenanthridinone is the PARP activity assay. The incorporation of soluble carbon-14 radiolabeled NAD into insoluble poly ADP-ribose is used to assess the activity of PARP. The significant reduction in PARP activity by 6( 5H )-phenanthridinone strongly suggests that its hepatoprotective effects are directly related to PARP inhibition. The presence of identical DMSO concentrations in the cell media for all treatments eliminates it as a variable. Given these in vitro results, there is reason to believe that the co-administration of one or more PARP inhibitors could minimize the free radical-mediated hepatotoxic effects of carbon tetrachloride. The prior in vitro results confirming the hepatoprotective effects of 6( 5H )phenanthridinone would lead one to believe that it would be effective against the hepatotoxic effects of carbon tetrachloride in vivo Although it should be possible to inject a solution of 6( 5H )-phenanthridinone in pure DMSO, colloidal suspensions created by diluting these solutions in saline or deionized water do not allow for reliable

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120 administration. These difficulties have been resolved by Pacher et al (2002), who ut ilized PJ34, a water soluble derivative of 6( 5H )-phenanthridinone, to inhibit PARP. Nicotinamide (niacinamide) was also found to reduce the hepatotoxic effects of carbon tetrachloride in male ICR mice, however these results merely confirmed previous findings, likely made before nicotinamide was found to inhibit PARP. Although it would be intriguing to determine the primary mechanism by which nicotinamide is hepatoprotective, whether it is through PARP inhibition, its incorporation into additional NAD used by PARP, or some antioxidant capability, the fact that it has been previously shown to be efficacious was sufficient knowledge for those who reported the initial findings, as it was here. There were numerous other PARP inhibitors, which could be administered to test their hepatoprotective effects, among them aminophylline. Aminophylline has been reported to inhibit the activity of poly (ADP-ribose) polymerase-1 (PARP-1), and is currently used as an anti-asthmatic medication. Increases in PARP-1 activity have been associated with chemical-induced cellular damage. The purpose of this study was to determine whether aminophylline can protect against free radical-mediated hepatotoxicity, as is caused by carbon tetrachloride (CCl4). Male ICR mice were administered 0.025ml/kg of CCl4. CCl4-treated mice developed hepatotoxicity, as evidenced by a 100–fold increase in serum alanine aminotransferase (ALT) levels. Also, a significant increase in hepatic malondialdehyde (MDA) equivalents was measured. These effects were corroborated by histopathological findings of minimal to strong necrosis and apoptosis, the former as evidenced via hematoxylin and eosin (H&E) staining, and the latter by terminal deoxynucleotidyl transferase biotin-dUTP nick

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121 end labeling (TUNEL) and cleaved caspase-3 immunohistochemistry. Administration of aminophylline reduced ALT levels roughly eighty percent, MDA levels were decreased by seventy percent and necrosis was eliminated. Also treatment with aminophylline resulted in only slight changes in hepatocyte cytoplasm and maximum changes were seen at twenty-four hours following aminophylline treatment. These studies indicate that aminophylline treatment can inhibit CCl4-induced hepatotoxicity. Aminophylline, when administered via intraperitoneal injection thrice at thirty, sixty and one hundred twenty minutes following a single carbon tetrachloride injection, was proven to reduce the hepatotoxic effects of carbon tetrachloride. This is evident from the ALT results and the examination of histopathology samples. The hepatoprotective effect of aminophylline appears to work in a dose-response fashion, because, despite the statistically significant reduction in ALT with two intraperitoneal administrations of 50 mg/kg aminophylline, the percentage of the reduction was less than with three doses. The TBARS assays yielded significant reductions in malondialdehyde equivalents when aminophylline was administered twice. It may have done the same for three administrations, except that difficulty in establishing a calibration curve on the latter occasion made this difficult to confirm. The reduction in malondialdehyde equivalents in the TBARS assay is reflective of a reduction in lipid peroxidation. Collectively, these results strongly suggest that aminophylline, administered via intraperitoneal injection, is protective against the hepatotoxic effects of carbon tetrachloride in male ICR mice, even when this co-treatment is administered after the toxicant.

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122 Aminophylline was an intriguing choice as a co-treatment, as it has been approved by the FDA for the treatment of asthmatic patients. Although it may be difficult to titrate the dosage and assess the efficacious and/or adverse effects of aminophylline, it is possible to do so in a clinical setting. Since aminophylline is water soluble, it became much easier to administer a consistent and reliable dosage, which was not the case for other PARP inhibitors, such as 6( 5H )-phenanthridinone, benzamide and the various aminobenzamides. Aminophylline disassociates readily in the bloodstream into its constituents, two molecules of theophylline and one molecule of ethylene diamine. Since aminophylline and theophylline are well known to emergency room physicians, there is also likely to be a general understanding as to how it can be administered safely. This makes it all the more likely to be accepted as an antidote in the event of an accidental overexposure, if other studies can confirm its efficacy. When aminophylline or theophylline is administered to alleviate acute asthmatic symptoms, the dosage often has to be titrated to the appropriate level. Therefore, the selection of time periods for the intraperitoneal administration of aminophylline, thirty, sixty and one hundred twenty minutes following the intraperitoneal administration of carbon tetrachloride were done in part to mimic an emergency room response. Had aminophylline only been effective if administered prior to, or concomitant with, carbon tetrachloride, it would have been inconsistent with emergency room care. Furthermore, it would be unreasonable to keep workers on prophylactic medications, particularly those with the potential for adverse effects of their own. The reason for this is that such pharmacological measures that are supposed to be protective of employees should pose

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123 any undue risks to them in and of themselves. In summation, CCl4 is a model hepatotoxicant that produces free radical-mediated hepatic damage, as evidenced via elevated serum transaminases, lipid peroxidation and histopathologic changes. Aminophylline (50 mg/kg), administered via intraperitoneal injection, at intervals of thirty, sixty and ninety minutes following intraperitoneal administration of CCl4 (0.025 ml/kg), significantly reduced the hepatotoxic effects of the toxicant in male ICR mice. This is confirmed in this study, in the form of significantly reduced ALT levels in the serum, significantly fewer malondialdehyde equivalent concentrations, and differences in H&E, TUNEL and cleaved caspase-3 immunohistochemical analyses. These findings suggest that aminophylline may be similarly efficacious against other free radical-mediated hepatotoxicants. Impact on Public Health The results of these experiments strongly suggest that aminophylline has the potential to block the free radical-mediated hepatotoxicity of carbon tetrachloride. Some might argue that, since the end use of carbon tetrachloride, particularly as a fumigant, has been dramatically reduced by the United States and other parties as part of the Montreal Protocol for possible ozone depleting characteristics, that there is no real issue of impact on public health. There are three reasons, however why the results of these experiments still have the potential for a positive impact on public health; (1) the likelihood for exposure during remediation of contaminated sites, (2) that carbon tetrachloride is still used as an intermediate in the production of other compounds, and (3) that carbon

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124 tetrachloride is a model hepatotoxicant, with a similar mechanism of action to other free radical-mediated, non-glutathione dependent toxicants. There is still a significant likelihood of human contact with carbon tetrachloride. The presence of a specific chemical at waste sites is based on two factors, the extensiveness of its use and disposal, and the kinetics of its degradation. The Environmental Protection Agency has reported the presence of carbon tetrachloride in at least 423 of the 1,636 sites on the National Priority List. As a result, there is a significant potential for exposure among workers cleaning up the waste sites, which can only be prevented by the proper use of personal protective equipment. Carbon tetrachloride was widely used in the United States as a dry cleaning solvent, and in other commercial applications. Although many countries have largely curtailed the use of carbon tetrachloride, many of the developing countries which were not signatories to the Montreal Protocol may continue to use it. The theory that CCl4 depletes the ozone layer seem to defy logic, given the physical properties of this chemical. CCl4 has a vapor pressure (91.3 mm Hg at 20 C) which allows for significant volatilization, with a molecular weight of 153.8 and a vapor density of 5.32, suggesting that CCl4 vapors would be expected to remain at low altitudes, however some scientists must assume it can reach the upper atmosphere. In the event that carbon tetrachloride volatilizes, it is fairly persistent, with estimated presence in the trophosphere or stratosphere from 30 to 100 years (Galbally, 1976; Lovelock, 1977; Simmonds et al. 1983, 1988; Singh et al. 1979). As such, environmentalists fear the potential for depletion of the ozone layer as a result of carbon tetrachloride releases.

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125 The phasing out of CCl4 as an end use product does not preclude the potential for human exposure. The synthesis of complex organic compounds is based on the ability to use components with leaving groups, and attach them to each other, creating new end use products. Ironically, one of these end use compounds, perchlorethylene, has replaced CCl4 as the principal dry cleaning solvent. As a result, carbon tetrachloride is still a critical compound, albeit indirectly, to the dry cleaning industry. Carbon tetrachloride was also commonly used to produce chlorofluorocarbon refrigerants, such as trichlorofluoromethane and dichlorodifluromethane, (HSDB, 2003). As such a substantial amount of CCl4 is still being manufactured. The two remaining American facilities producing carbon tetrachloride in Louisiana and Kansas have annual capacities of 90 million and 20 million pounds, respectively (HSDB. 2003). Carbon tetrachloride is referred to as a model hepatotoxicant because its mechanism of action is comparable to that of similar compounds. There are a number of halocarbons, the metabolites of which can potentially cause free radical damage. Also some alcohols ( e.g. allyl alcohol) have the potential to cause free radical-mediated hepatotoxicity, and can in some cases ( e.g. alcohol and acetaminophen) alter the mechanism of toxicity, so that free radicals are generated. Of this wide array of chemicals, carbon tetrachloride has arguably the most potential for adverse effects at a given dosage, because the trichloromethyl radical generated through the reductive effects of cytochrome P-450 (specifically CYP2E1) is particularly effective in causing free radical damage. This may be largely due to the significant partial positivity of the carbon atom, which is caused by the electronegativity of the three chloride molecules, with

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126 which the carbon atom shares six of the seven electrons. By using model toxicants, such as carbon tetrachloride, it may be possible to focus on finding a number of antidotes for entire classes of compounds based on their mechanisms of action. The experiments in this dissertation were performed with the purpose of finding out whether one or more PARP inhibitors are capable of protecting the liver from the free radical-mediated, nonglutathione dependent hepatotoxic effects of carbon tetrachloride, with the potential to protect against additional compounds with the same or similar mechanisms of hepatotoxicity. In summation, carbon tetrachloride is still a chemical of concern at a number of sites, and poses significant health risks. Environmental Defense lists carbon tetrachloride as one of the eight hazardous air pollutants, which collectively contribute to 99% of total hazardous air pollutant-associated cancer risks (scorecard.org). Carbon tetrachloride is the third highest compound contributing to cancer risks, with an added cancer risk contribution of 5%. Given the former widespread use of carbon tetrachloride, and the persistence and hepatotoxicity of carbon tetrachloride, any effort to minimize its hazardous effects has the potential to benefit mankind. Possible Future Studies The experiments conducted in this dissertation establish the foundation for a number of opportunities for further study. These involve the use of different types of experimental animals, alternate forms of dosage or routes of exposure, expanded in vitro experimentation, different types of free radical-mediated hepatotoxicants and the testing

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127 of compounds with completely different mechanisms of toxicity ( e.g. Aryl Hydrocarbon receptor binding, glutathione oxidation or conjugation). In order to confidently assume that, prior to testing in humans, a compound would be both efficacious and safe at efficacious doses, experimentation in additional species and subspecies of animals is required. Although much is known about the toxicity of aminophylline and theophylline in humans, the efficacious dosage will have to be estimated, at least until anecdotal reports of their usage for this purpose emerge. The exclusive use of male ICR mice in these experiments was based on one overlying factor, their relatively low cost. The fact that these mice are outbred and are genetically different from each other may explain the variation in responses. Ideally, experiments like these would have been conducted using inbred mice, such as BALB/c and C57BL/6, to eliminate one or more of the potential variables, such as genetic differences in P-450 activity. Further experiments could be conducted using additional strains of mice and rats, among other laboratory animals, to ensure that the findings using the ICR mice were not aberrations. Another avenue for further experiments entails the route of exposure to carbon tetrachloride. The most common route of exposure to carbon tetrachloride in humans is via inhalation. There have also been a number of oral exposures, either accidental or intentional, as well as dermal exposures. As a matter of ensuring the delivery of the carbon tetrachloride, as well as minimizing the inhalation exposure of researchers and animal facility workers, the route of the dose used in the experiments was via intraperitoneal injection. If research facilities were capable to consistently deliver a fixed

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128 air concentration of carbon tetrachloride, and allow for the ability to maintain this level during administration of the PARP inhibitors as concomitant or delayed co-treatments, without exposing researchers in the process, the appropriate modification of previous studies for inhalation would be appropriate. Dermal studies would best be served by selecting experimental animals which have less hair than ICR mice, because shaving mice may result in breakage of the skin, which may result in direct toxicant-blood contact. Likewise, were ingestion experiments to be conducted in the future, they should be performed in experimental animals ( e.g. rats) that are incapable of vomiting the toxicant, because regurgitation would result in diminished dosages. In the alternative, if standardization of the concentrations of CCl4 in drinking water is deemed sufficient enough to control dosage, any laboratory animals could be given drinking water containing these fixed concentrations ad libitum If one were interested in further understanding the details of the mechanism by which aminophylline attenuates the hepatotoxic effects of carbon tetrachloride and other free radical-mediated toxicants, in vitro studies may prove quite useful. As was mentioned, the limited experiments using 6( 5H )-phenanthridinone as a co-treatment with carbon tetrachloride in HepG2 cells strongly suggest that the mechanism involves the inhibition of PARP-1. In conjunction with the inhibition of PARP-1, there may be an underlying inhibition of cytochrome P-450, specifically the CYP2E1 isoform of this enzyme, which is reported to generate trichloromethyl radicals via its reductive modification of carbon tetrachloride. There may be some issues of concern that HepG2 has limited cytochrome P-450 activity, specifically that of the CYP2E1 isoform.

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129 However, there are cell lines of HepG2 cells that have been modified to have significantly elevated CYP2E1 activity. Modified HepG2 cell lines have reportedly been produced, in which genes encoding for one or more P450 CYP isoforms have been artificially inserted into the genome. Unfortunately, these cells could not be acquired in a timely fashion to allow for sufficient growth for the required experiments. Future in vitro studies should incorporate the use of these cell lines in lieu of or in conjunction with HepG2 cells. Also, the mechanism of action for the hepatoprotective effects of aminophylline should be further investigated in order to confirm that PARP is inhibited by aminophylline. Not only is aminophylline an inhibitor of PARP, it is also a nonselective phosphodiesterase inhibitor, which is believed to protect spinal neuron cells against damage caused by glutamate and reactive oxygen species (Nakamizo et al 2003). Furthermore, the results of the TBARS assays suggest that aminophylline reduced the extent of CCl4-induced lipid peroxidation. Therefore, PARP activity assays should be conducted to confirm that the hepatoprotective effects provided by aminophylline are directly related to its inhibition of PARP, instead of via some other mechanism. However informative these possible cell culture experiments may be, they are no substitute for in vivo testing, because complex organisms are more than just a sum of their multitude of parts. One critical example of this is that collections of cells from the same organ are not subject to the impacts of hormones, chemokines and other biological compounds arising from other organs or organ systems. Also of importance is that there is no lag period between the time of dosage and exposure to the target cells in the case of in vitro studies,

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130 whereas for in vivo experiments, toxicants have to be carried to their respective target organ by blood circulation or some other biological process. Although carbon tetrachloride is considered a model toxicant, it should not be taken as gospel that aminophylline is equally protective against all free radical-mediated hepatotoxicants. Ideally, each of the toxicants would be administered with and without aminophylline to multiple varieties of multiple species of animals. Halogenated hydrocarbons of specific concern would most likely include dichloromethane, methyl bromide, formaldehyde, chloroform and others which are still in widespread use. The use of methyl bromide as a fumigant was supposed to be phased out years ago, however in the absence of acceptable alternatives, this was significantly delayed. The EPA has begun its phase-out in January of 2005, still allowing for quarantine and critical use, for which no other fumigant is effective (EPA, 2005). As such, there is st ill the potential for fumigators to be exposed to methyl bromide, at least in the near future. Formaldehyde is commonly used in resins, which are necessary for the construction and furnishing of buildings and mobile homes. Many of the remainder of these compounds are used as solvents, degreasers and other products, which allow for possible overexposures, and aminophylline may prove to protect against their adverse effects. Not all compounds have the same mechanism of action, so it must not be assumed that aminophylline would be capable of protecting against every hepatotoxicant. Preliminary unpublished experiments conducted in Dr. Harbison’s laboratory suggest that aminophylline is not protective against bromobenzene-induced hepatotoxicity, the mechanism of which involves conjugation with glutathione. This lack of protection may

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131 not be indicative for all compounds, which involve glutathione conjugation, but that would require further experimentation. Other possible mechanisms of toxicity include bile acid –induced hepatocytes apoptosis, inflammatory liver injury, glutathione depletion, protein adduct formation/ peroxynitrite release, and induced mitochondrial dysfunction (Jaeschke et al, 2002). As such, there are numerous opportunities for further studies, with the potential to significantly benefit mankind. Conclusion Carbon tetrachloride is a model hepatotoxicant that has a free radical-mediated, non-glutathione dependant mechanism of action. When added to the culture media for HepG2 human hepatocytes or administered by intraperitoneal injection to male ICR mice, carbon tetrachloride produced a series of hepatotoxic effects. These effects included elevated relative LDH levels and PARP activity in the case of the HepG2 cells, as well as abnormally high serum ALT levels, increased lipid peroxidation and histopathology findings indicative of necrosis and apoptosis in the ICR mice. The co-administration of PARP inhibitors, among them 6( 5H )-phenanthridinone in the cell culture studies and aminophylline and nicotinamide in the animal studies, was capable of significantly protecting against these hepatotoxic effects associated with carbon tetrachloride. These results strongly suggest that PARP inhibitors may effectively protect against the free radical-mediated non-glutathione dependent hepatotoxic effects of carbon tetrachloride and other compounds with similar mechanisms of action in humans.

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About the Author Paul Grivas is a Wisconsin native who moved to Florida in 1984. He performed undergraduate research at the University of South Florida under Dr. Dean Martin, wrote his honors thesis under Dr. Jay Wolfson and worked in various laboratories. He entered the Masters program in Toxicology at the College of Public Health in 2000, successfully completing the program in 2001. In 2002, Mr. Grivas entered the Ph.D. program in Toxicology. His research has led to two intervals as a visiting researcher in the Institute for Chemical Studies at Kyoto University and a Student Travel Award from the Society of Toxicology. Mr. Grivas taught undergraduate courses, and prepared graduate course materials and deliverables related to arsenic and cyanobacteria toxin-induced health risks for the Florida Department of Health. He will also receive a Juris Doctor from Florida State University in April, 2007 and take the Florida Bar Examination in July, 2007.