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Attenuation of bromobenzene-induced hepatotoxicity by poly(adp-ribose) polymerase inhibitors

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Title:
Attenuation of bromobenzene-induced hepatotoxicity by poly(adp-ribose) polymerase inhibitors
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English
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Hall, Kelly Waggoner
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Poly(adp-ribose)polymerase
Nicotinamide
6(5h)-phenanthridone
Bromobenzene
Hepatotoxicity
Dissertations, Academic -- Public Health -- Doctoral -- USF   ( lcsh )
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government publication (state, provincial, terriorial, dependent)   ( marcgt )
bibliography   ( marcgt )
theses   ( marcgt )
non-fiction   ( marcgt )

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ABSTRACT: Previous studies have shown extensive cellular damage can activate poly(ADP-ribose) polymerase-1 (PARP-1) and cause a rapid decrease in the levels of NAD+ and ATP, thereby preventing apoptosis and promoting necrosis and inflammation. The purpose of this study was to extend previous observations that inhibitors of PARP-1 could alter acetaminophen and carbon tetrachloride-induced hepatotoxicity. Bromobenzene (BB) a glutathione dependent hepatotoxicant was tested. Groups of male mice were treated with a single dosage of 112mg/kg (0.075 ml/kg) BB by the intraperitoneal (ip) route. All animals were maintained in a controlled environment and provided food and water ad libitum. This dosage of BB resulted in hepatotoxicity as measured by an increase in serum alanine transferase (ALT). BB treatment resulted in a 5-fold increase in ALT. Moderate hepatotoxicity was detected with this treatment regime.Subsequently, another group of mice were treated with three treatments of nicotinamide at 0.5, 1 and 2 hours following BB treatment. Serum ALT elevations were reduced by 90% at 24 hours following BB and nicotinamide treatments. BB-induced liver pathology was also blocked by nicotinamide. Mortality among BB treated animals was also significantly reduced by nicotinamide treatment. Mortality among mice treated with BB and nicotinamide was near control. The model was verified with a more potent and specific inhibitor, Phen. BB treatment was keep at the same level as in the previous study, and Phen was administered concomitantly. Serum ALT elevations were reduced by 75%. Phen also blocked BB-induced liver pathology. Mortality among mice treated with BB and Phen was reduced 75%. PARP-1 inhibitors appear to alter chemical-induced hepatotoxicity that has either a glutathione dependent or independent mechanism.
Thesis:
Thesis (Ph.D.)--University of South Florida, 2005.
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Includes bibliographical references.
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by Kelly Waggoner Hall.
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Title from PDF of title page.
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Document formatted into pages; contains 87 pages.
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Includes vita.

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usfldc doi - E14-SFE0001189
usfldc handle - e14.1189
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Attenuation of Bromobenzene-Induced Hepato toxicity by Poly(ADP-ribose) Polymerase Inhibitors by Kelly Waggoner Hall 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. Ira S. Richards, Ph.D. Philip P. Roets, Sc.D. Skai W.Schwartz, Ph.D. Date of Approval: July 15, 2005 Keywords: poly(adp-ribose)polymerase, nicotinamide, 6(5H)-phenanthridone, Bromobenzene, hepatotoxicity Copyright 2005 Kelly Waggoner Hall

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DEDICATION In memory of my mother, Earliene Waggoner, who died of a degenerative neurological disorder. Research will find the cause and cure, so others will not have to suffer.

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ACKNOWLEDGEMENTS I would like to thank each of my committee members for their support through this endeavor. First I would like to thank my major profe ssor, Dr. Raymond D. Harbison, for his guidance and motivation throughout this pr oject. Without his support, this project would never have seen a successful end. I f eel fortunate to have had such a dedicated individual as my major professor and mentor. I would like to thank Dr. Ira Richards. His tough questions and courses have led me to study areas that have helped me understa nd what I needed to. Many thanks to Dr. Phil Roets for his guidance through my masters degree and for being a great professor in many courses that I took. Thanks also go to Dr. Skai Schwartz for helping me understand Epidemiology, and learning to think outside the box. Many other individuals have helped me th roughout this project. I would like to thank Dr. Carlos Muro-Cacho for taking time out of his very busy schedule to assess my pathology slides. To Drs. Vasyl Sava a nd Adriana Velasquez, thank you lending your equipment and expertise in fluorescence work. I would also like to thank my fellow doctoral candidates and stude nts in the EOH department for their support and encouragement: Paul Grivas, Robin DeHate, Scott Dotson and Marilyn Williams. Lastly, I would like to thank my fami ly, Tom Hall, Jerry and Jan Waggoner and Karen Straus for all of the encouraging words th at have helped me make it to this point. Thank you for being there for me throughout this endeavor.

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i TABLE OF CONTENTS LIST OF TABLES..............................................................................................................ii LIST OF FIGURES...........................................................................................................iii LIST OF ABBREVIATIONS............................................................................................vi ABSTRACT.....................................................................................................................viii CHAPTER 1 INTRODUCTION.........................................................................................1 CHAPTER 2 MATERIALS AND METHODS................................................................12 CHAPTER 3 RESULTS....................................................................................................17 CHAPTER 4 DISCUSSION..............................................................................................62 REFERENCES..................................................................................................................70 ABOUT THE AUTHOR.......................................................................................End Page

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ii LIST OF TABLES Table 1 Experimental Design for Non Specific Inhibitor.............................................13 Table 2 Experimental Desi gn for Specific Inhibitor.....................................................13 Table 3 Screening of Inhibitors.....................................................................................17 Table 2 Histopathology Scores for Nicotinamide.........................................................32 Table 3 Histopathology Scores for Phen.......................................................................48

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iii LIST OF FIGURES Figure 1 Structure of Bromobenzene...............................................................................2 Figure 2 Metabolism of Bromobenzene...........................................................................3 Figure 3 Structure of PARP-1..........................................................................................6 Figure 4 Structure of Nicotinamide..................................................................................8 Figure 5 Structure of Phen................................................................................................8 Figure 6 Overview of PARP-1 Activation.....................................................................10 Figure 7 ALT Measurements at 12 hour intervals.........................................................19 Figure 8 Bromobenzene and Nicotinamide administered Concomitantly ....................20 Figure 9 Nicotinamide Administra tion 1 hour after Bromobenzene..............................21 Figure 10 Nicotinamide Administration 3 hours after Bromobenzene ...........................22 Figure 11 Nicotinamide Administration 1 and 2 hours after Bromobenzene.............23 Figure 12 Confirmation of 3 dose study...........................................................................24 Figure 13 72 hour Study for Bromobenzene and Nicotinamide......................................26 Figure 14 7 Day Mortality For Bromobenzene and Nicotinamide..................................27 Figure 15 Macroscopic View of Liver Administered Saline...........................................29 Figure 16 Macroscopic View of Li ver Administered Bromobenzene.............................30 Figure 17 Macroscopic View of Liver Administered Bromobenzene and Nicotinamide.............................................................................................31 Figure 18 H&E Staining of Saline Control......................................................................33 Figure 19 H&E Staining of Live r Administered Bromobenzene.....................................34

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iv Figure 20 H&E Staining of Live r Administered Bromobenzene and Nicotinamide.............................................................................................35 Figure 21 TUNEL Staining of Saline Control..................................................................36 Figure 22 TUNEL Staining of Live r Administered Bromobenzene................................37 Figure 23 H&E Staining of Live r Administered Bromobenzene and Nicotinamide.............................................................................................38 Figure 24 Cleaved Caspase 3 Staining of Saline Control................................................39 Figure 25 Cleaved Caspase 3 Staining of Liver Administered Bromobenzene..............40 Figure 26 Cleaved Caspase 3 Staining of Liver Administered Bromobenzene and Nicotinamide.............................................................................................41 Figure 27 Phen Administered Concomitantly with Bromobenzene at 10 mg/ml.............................................................................43 Figure 28 Phen Administered Concomitantly with Bromobenzene at 20 and 40 mg/ml.................................................................44 Figure 29 Confirmation of Phen Administered Concomitantly with Bromobenzene at 40 mg/ml.....................................................................45 Figure 30 7 day Mortality for BB, BB/Phen, BB/DMSO and DMSO.............................47 Figure 31 Macroscopic View of Li vers Administered Phen, DMSO And BB/Phen...................................................................................................49 Figure 32 H&E Staining of Live r Administered Bromobenzene And Phen..........................................................................................................50 Figure 33 H&E Staining of Live r Administered Bromobenzene And DMSO......................................................................................................51 Figure 34 H&E Staining of Liver Administered DMSO.................................................52 Figure 35 TUNEL Staining of Live r Administered Bromobenzene And Phen..........................................................................................................53

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v Figure 36 TUNEL Staining of Live r Administered Bromobenzene And DMSO......................................................................................................54 Figure 37 TUNEL Staining of Liver Administered DMSO.............................................55 Figure 38 Cleaved Caspase 3 Staining of Liver Administered Bromobenzene And Phen..........................................................................................................56 Figure 39 Cleaved Caspase 3 Staining of Liver Administered Bromobenzene And DMSO......................................................................................................57 Figure 40 Cleaved Caspase 3 Staining of Liver Administered DMSO............................58 Figure 41 Total Glutathione.............................................................................................60 Figure 42 Lipid Peroxidation...........................................................................................61

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vi LIST OF ABBREVIATIONS ALT Alanine aminotrasferase APAP Acetaminophen ATP Adenosine triphosphate BHQ 2-bromohydroquinone BB Bromobenzene DMSO Dimethyl sulfoxide DTNB 5-5-Dithiobis(2 -nitrobenzoic acid) GR Glutathione Reductase GSH Glutathione H&E Hematoxylin-eosin IACUC Institutional Animal Care and Use Committee IHC Immunochemistry IP Interperitoneal kDa Kilodaltons NAD Nicotinamide Adenine Dinucleotide NADPH -Nicotinamide Adenine Di nucleotide Phosphate NAPQI N-aceyl-p-benzoquionone imide NMN Nicotinamide Mononucleotide PARP-1 Poly(ADP-Ribose)polymerase-1

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vii Phe Phentolamine Phen 6( 5H )-Phenanthridinone TCA Trichloroacetic Acid TUNEL Terminal deoxynucleotidyl Transf erase Biotin-dUTP Nick End Labeling MW Molecular Weight

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viii Attenuation of Bromobenzene-Induced Hepato toxicity by Poly(ADP-ribose) Polymerase Inhibitors Kelly Waggoner Hall ABSTRACT Previous studies have shown extensive cel lular damage can activate poly(ADP-ribose) polymerase-1 (PARP-1) and cause a ra pid decrease in the levels of NAD + and ATP, thereby preventing apoptosis and promoting necrosis and inflammation. The purpose of this study was to extend previous observations that inhibitors of PARP-1 could alter acetaminophen and carbon tetrachloride-induced hepatotoxicity. Bromobenzene (BB) a glutathione dependent hepatotoxicant was tested. Groups of male mice were treated with a single dosage of 112mg/kg (0.075 ml/kg) BB by the intraperitoneal (ip) route. All animals were maintained in a controlled environment and provided food and water ad libitum. This dosage of BB resulted in hepato toxicity as measured by an increase in serum alanine transferase (ALT). BB treatment resulted in a 5-fold increase in ALT. Moderate hepatotoxicity was detected with th is treatment regime. Subsequently, another group of mice were treated w ith three treatments of ni cotinamide at 0.5, 1 and 2 hours following BB treatment. Serum ALT elevat ions were reduced by 90% at 24 hours following BB and nicotinamide treatments. BB-induced liver pathology was also blocked by nicotinamide. Mortality among BB treated animal s was also significantly

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ix reduced by nicotinamide treatment. Mo rtality among mice treated with BB and nicotinamide was near control. The model was verified with a more potent and specific inhibitor, Phen. BB treatment was keep at the same level as in the previous study, and Phen was administered concomitantly. Se rum ALT elevations were reduced by 75%. Phen also blocked BB-induced liver pathol ogy. Mortality among mi ce treated with BB and Phen was reduced 75%. PARP-1 inhibi tors appear to alter chemical-induced hepatotoxicity that has either a glutathione dependent or independent mechanism. PARP1 inhibitors may have pharmacological appl ication for modifying chemical-induced liver injury.

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1 CHAPTER 1 INTRODUCTION Previous studies investigating hydrocar bon-induced hepatotoxicity have provided a basis for the experimental hypothesis of th e current investigati on. Bromobenzene (BB) is a pungent smelling organic solvent that is used for large-scale crystallizations where the use of a heavy liquid is preferred. (PPRTV, 1999) The compound is a common component of motor oils and has been used as an intermediate in industrial chemical syntheses. These uses have led to occupa tional exposures, dermal and inhalation, and the release of BB into the environment as a c ontaminate (Van Vleet and Schnellmann, 2003). The liver, kidney and lung are identified histolog ically as the primary target organs. The liver is the most sensitive target organ following acute oral exposure (PPRTV, 1999). BB (Figure 1) has been studied extensivel y in the literature BB has been known to cause necrosis of the centrilobular parenchymal cells in the liver (Brodie et al. 1971). Jollow et al. (Jollow et al. 1974) determined that BB-induced hepatic necrosis was due to the formation of a reactive metabolite that ar ylates vital cellular macromolecules, thereby causing the hepatic damage. This metabol ite is BB 3,4-oxide which is formed by a cytochrome P-450 mixed function oxidase system (Monks et al. 1982).

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Br Figure 1. Structure of bromobenzene (BB). Many drugs and aromatic hydrocarbons are metabolized to reactive epoxide intermediates by the hepatic cytochrome P-450 mixed function oxidase system (Monks et al. 1984). Chemically reactive metabolites of BB, 2,3and 3,4-BB epoxides are formed via this system (Lau and Zannoni, 1981). The toxic reactive metabolite of BB, 3,4epoxide subsequently rearranges nonenzymatically to form p-bromophenol or is converted to the 3,4-dihydrodiol by epoxide hydrolase. Mo st of the 3,4-epoxide is detoxified to two glutathione conjugates by glutathione transfer ases in the liver. It has been postulated that o-bromophenol occurs from a nontoxic precursor, BB-2,3-oxide (Figure 2) (Monks et al. 1984). Human liver microsomes generate a much greater proportion of the hepatotoxic metabolite (3,4-epoxide) than the nonhepatotoxic metabolite (2,3-epoxide). Humans may experi ence a greater hepatotoxicity at lower doses of BB than do mice (Kerger et al. 1988c) 2

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Br Br Br H H O H H O Br OH Br OH Br OH GS H H H Br OH OH H Br H H SG OH Bromobenzene 2,3-epoxide Bromobenzene 3,4-epoxide o -Bromophenol p -Bromophenol Nonenzymatic Rearrangement Nonenzymatic Rearrangement 3,4-Dihydrodiol H20 Epoxide Hydrolase NADPH + O2 Epoxide Synthetase GSH S-transferase GSH S-transferase Figure 2. Bromobenzene is metabolized vi a the hepatic mixed function oxygenase system to reactive intermediates 2,3a nd 3,4-bromobenzene epoxides. The toxic metabolite, 3,4-epoxide rearranges nonenzymatically to form p-bromophenol or is converted to 3,4-dihydrodiol by epoxide hydrolase. Most of the 3,4-epoxide is detoxified to two glutathione conjugates by glutathione (GSH) transferases in the liver. It has been postulated that o-bromophenol is a nonenzymatic rearrangement of a nontoxic precursor, BB-2,3-oxide. 3

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4 Jollow et al. showed that administration of BB to rats resulted in a rapid and extensive depletion of glutathione from the liver. After approximately 5 hours about 15% of the initial level remains. Hepatic glutat hione remained depleted during the metabolism of BB (Jollow et al., 1974). This had led to the unde rstanding that BB forms conjugates with hepatic glutathione and that under these conditions the GSH level is decreased, making the liver cells more susceptible to th e development of lipid peroxidation (Casini et al. 1985). It has been show n that the magnitude of the BB induced hepatotoxicity is dependent on the hepatic gl utathione present during its liver detoxification (Jollow et al. 1974; Kerger et al. 1988a) BB has been used in various experime nts as a model compound inducing liver and kidney impairment (Szymanska, 1998). Syma nska stated that no lethal dose of BB could be found, and used a sta tistical method to determine the approximate lethal dose (ALD). The ALD dose for BB was set at 900 mg /kg. Doses used in this study were 200, 400 and 800 mg/kg. The %ALD was 22.2, 44.4 and 88.9. These doses were for outbred male Imp Balb/cJ mice, 23 30 g body weight. Mice used in the current study are male ICR mice, 25 30 g body weight. Past experime nts have used B6C3F1 mice at a lower dose of BB (0.05 ml/kg) due to the fact that th is strain had been used in a number of studies of hepatotoxic and carcinogenic agents (Kerger, 1988). Roberts et al. stated that BB toxicity occurs in both the inbred B6C3 F1 and outbred ICR mice, but the toxicity occurred at lower doses w ith the B6C3F1 mice (Roberts et al. 1997). This led to experimentation using a higher dose of BB to cause hepatotoxicity. Past studies have shown that BB hepatoto xicity could be reduced. Phentolamine (Kerger et al. 1988a; Kerger et al. 1988b) and cystamine (De Ferreyra et al. 1979) have

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5 been studied in the past. In all cases the conclusions were that BB hepatotoxicity could be reduced by treatment with these agents. A new novel method for reducing hepatotoxicity and centrilobular necrosis usi ng PARP-1 inhibitors is the focus of this current study. Current research of BB has studied the t oxicity of BB in mice and rats at the molecular level using transcriptomics. Transcriptomics is the determination of the expression of genes (Heijne et al. 2003). Bartosiewicz, et al administered male swisswebster mice at a level of 2.5g/kg BB. All mice died before the 48-hour time point. They found that there were 14 genes that were expressed in the liver. These genes were involved in DNA damage response, oxida tive stress and phase II metabolism (Bartosiewicz et al., 2001) Rat studies using male Wistar Rats receiving an i.p. injection of BB also analy zed gene expression. (Heijne et al. 2003). The gene expressions that Heijne et al. found to be significantly changed upon BB metabolism included those involved in glutathione conjugation. In 1963, the enzyme poly(ADP-ribose) was discovered by Dr. Paul Mandels (Chambon et al., 1963) laboratory in France. It was originally thought to be poly(A), a DNA dependent enzyme induced by nicotinamide mononucleotide (NMN). Poly(A) polymerase catalyzes the addition of ade nosine to the 3 end of RNA (Alberts et al. 2002). By 1966 it was ascertained that the enzyme was slightly structurally different than poly(A), it was a novel structur e now termed poly(ADP-ribose ). It was discovered that poly(ADP-ribose) was not a nucleic acid, be cause unlike DNA and RNA which have a phosphodiester bond joining th e polymer units, ther e is a ribose (1 2) ribosephosphate-phosphate backbone. The anomeric carbon of one ADP-Ribose molecule is

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bound to the adenosine moiety of the next via a 1 2 glycosidic linkage (Althaus and Richter, 1987). Poly (ADP-Ribose) polymerase (PARP-1) is a 113 kDa nuclear enzyme that catalyzes the transfer of the ADP-ribose moiety of NAD + to various nuclear acceptor proteins and then to the protein bound ADP-ribose(Ueda and Hayaishi, 1985). There are 3 main domains: the N-terminal DNA-bindi ng domain (DBD) containing two zinc fingers, the auto modification domain and the C-terminal domain (Figure 3) (Southan and Szab, 2003) A C D E F 859 908 988 1014 656 524 383-476 NLS F2 F1 N C DNA trading domain Zn fingers Automodification domain BRCT Catalytic domain B 1 Figure 3. The structure of PARP-1. Th e DNA-binding domain corresponds to regions A,B and C. Section A contai ns two zinc fingers (F1 and F2) that are involved in DNA strand break recognition. Section B is th e nuclear location signal (NLS). The NLS includes the caspase cleavage site. Region C has an unknown function. Region D consists of several glutamic acid residues, which are sites of automodification. Regions E and F encompass the catalytic doma in. Figure adapted from (de Murcia et al. 1991; de Murcia and Shall, 2000) 6

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7 The two zinc fingers (F1 and F2) in the amino (N) terminal end of PARP-1 are important for DNA binding and the enzyme activ ity of PARP-1. PARP-1 is activated by the presence of both single and double DNA strand breaks. F1, closed to the N terminal end, is essential for PARP-1 enzyme activity in response to nicked DNA or double strand breaks (Ikejima et al., 1990; Uchida and Miwa, 1994). Automodification leads to the forma tion of long, branched PARPs on target proteins, primarily itself, using NAD + as a substrate, which leads to a depletion of NAD + (Herceg and Wang, 2001). By manipulating the NAD concentration or the chemical nature of the poly(ADPribosyl)ation substrate, Alvarez-Gonzalez and Mendoza-Alvarez were ab le to dissect the C-terminal end of ADP-ribose polymerase into individual reac tions of initiation, elongation and branching (Alvarez-Gonzalez an d Mendoza-Alvarez, 19 95). Initiation is the attachment of an ADP-ribose moiety to an acceptor protein. Elongation is where additional ADP-ribose moieties are attach ed to protein-bound ADP-ribosyl residues. Branching is the introduction of an ADP-ribos e residue via a linkage that initiates a branch along the linear portion of the polymer Another component of the C terminal end is Abortive N A Dase, which cleaves NAD + into nicotinamide and free ADP-ribose (de Murcia and Shall, 2000). Figure 2 is a graphical represen tation of PARP-1. With excessive activation of PARP-1, its substrate NAD + is depleted and in efforts to resynthesize NAD + ATP is also depleted (Figure 6) (Ha and Snyder, 1999). PARP-1 is cleaved by caspases (primary caspase -3 and caspase-7) during apoptosis. This cleavage separates the DNA binding domain fr om the catalytic domain, resulting in the inactivation of PARP-1 (Affar et al., 2001).

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Figures 4 and 5 are the chemical structures of the inhibitors chosen for this study. Isoquinoline derivates (Figure 5) have polyaromatic hetero cyclics with a carboxyl group in the second ring. This and the third arom atic ring also appears to be critical for inhibition to occur (Weltin et al. 1997) N O NH2 Figure 4. Chemical structure of nicotin amide, a nonspecific PARP inhibitor. NH O Figure 5. Chemical structure of Phen. Phen is an isoquinoline derivative and is a very potent inhibitor. Inhibition of PARP-1 has been investigated due to the fact that PARP activation has been found to contribute to an energyconsuming cellular process, which leads to cellular NAD and ATP depletion, mitochond rial dysfunction and an overall cellular dysfunction. This process can lead to ce ll death through necrosis (Southan and Szab, 2003). PARP is involved in the regulation of many cellular processes such as DNA 8

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9 repair, gene transcription, cell cycle pr ogression, cell death, chromatin functions and genomic stability. (Figure 6) (Tentori et al, 2002). As of 2003, NIC is the only PARP-1 inhi bitor that has been administered to humans. Two clinical trials have utilized NIC, one was a pilot trial in osteoarthritic patients, the other was a trial in Type I diabetes. The diab etes trail showed promise in Phase II studies. Phase III trials did not s how efficacy. This was attributed to the concentration of the inhibitor. Any amount ove r 3 g/day to humans begins to exert varial toxic effects (Southan and Szab, 2003). This laboratory has conduc ted the only studies using Phen in vivo as a PARP-1 inhibitor. The first study was attenuation of CCl 4 -induced hepatotoxicity (Su et al., 2003; Banasik et al. 2004) and this current study showi ng the attenuation of BB-induced hepatotoxicity. Both BB and CCl 4 cause centrilobular (zone 3) necrosis.

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D N A D A M A G E Mild Unrepairable Excessive Activation of PARP DNA Repair Activation of PARP Overactivation of PARP Insufficient DNA Repair Apoptosis ATP NAD Necrosis Figure 6. Overview of PARP-1 activation to DNA damage. Inhibition of the overaction of PARP will change the pathway to the unrepairable pathway. 10

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11 Research Question It has been shown previously that BB causes centrilobular hepato toxicity. BB causes hepatotoxicity through glutathione deplet ion. The research hypothesis includes: A non-specific PARP-1 inhibitor can a ttenuate BB-induced hepatotoxicity A specific PARP-1 inhibitor can at tenuate BB-induced hepatotoxicity PARP-1 inhibitors can atte nuate hepatotoxicity inde pendent of BB metabolism.

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12 CHAPTER 2 METHODS Animals and Treatment: Male, ICR mice, 25-30 g, were obtained from Harlan Sprague-Dawley, Inc. (Indianapolis, IN). Animals were housed 4 per cage, had free access to food and water and were exposed to a 12-hour light/dark cycle. Animals were acclimated for 7 days prior to testing. Drugs and chemicals were administered by intraperitoneal injection (i .p.) at a volume of 0.05-ml/10 kg body weight. The animals were cared for in accordance with Guide to the Care and Use of Experimental Animals and the University of South Florida Institutional Animal Care and Use Committee (IACUC) approved this study. All test solu tions were prepared fresh immediately preceding animal treatment. BB, Nic and Ph en were obtained from Sigma Chemical Company (St. Louis, MO). All other reagen ts were of the best quality commercially available. BB was dissolved in corn oil pr ior to administration. Nic was dissolved in saline. 6(5-H)-Phen was dissolved in 100% DMSO. Groups for comparison were treated with either (1) corn oil ve hicle, (2) PARP-1 inhibitor, (3) BB or (4) BB and PARP inhibitor. Inhibitor Efficacy: PARP-1 inhibitors were screened for efficacy in reducing ALT. BB was administered to all mice with the exception of controls. PARP-1 inhibitors were administered subsequent to BB-administration.

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13 Experimental Design: Animals were random ized into groups as shown in Tables 1 and 2. BB was administered as a singl e, i.p. dosage at 112 mg/kg (0.075 mg/kg). Animals were randomly assigned to groups with the number of animals in each of the treated groups being approximately double that of the number in the control groups. The n for each treatment group ranged from 6 to 121 depending on the size of the experiment. Control groups ranged from 4 to 8 mice. Treatment Group Chemicals Volume Group I Vehicle only 50 l/10 g body weight Group II Bromobenzene (112mg/kg) 50 l/10 g body weight Group III NIC (PARP-1 inhibitor) 50 l/10 g body weight Group III BB + NIC 50 l/10 g body weight Table 1. Experimental Design for control and treatment group for the non-specific inhibitor study Treatment Group Chemicals Volume Group I Vehicle only 50 l/10 g body weight Group II Bromobenzene (112mg/kg) 50 l/10 g body weight Group III Phen (PARP-1 inhibitor) 50 l/10 g body weight Group IV DMSO (vehicle for Phen) 50 l/10 g body weight Group V BB + Phen 50 l/10 g body weight Group V BB + DMSO 50 l/10 g body weight Table 2. Experimental design for controls and treatment groups for specific inhibitor

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14 Time Study: This study was performed to find the optimum time for BB toxicity. Mice were administered w ith BB and asphyxiated by carbon dioxide at 12-hour, 24-hour and 36-hour time frames. Blood was drawn by cardiac puncture. Serum Biochemistry: Serum Alanine Ami no Transferase (ALT) was determined by the method of Reitman et al.(Reitman and Frankel, 1957) using a commercially prepared reagent kit from TECO Diagnostics (Anaheim, CA). Whole blood was collected in micro centrifuge tubes and allowed to clot at room temperature for a minimum of 5 minutes. Serum was obtai ned after centrifuga tion at 6000 g for 10 minutes. ALT assays were obtained as directed in the diagnostics kit. Gross Pathology: Necropsy was performed on all mice and visual findings were recorded to correlate with ALT. Photogra phs were taken of random samples from BB, BB/NIC, BB/Phen and controls. Histopathology: Livers were preserved in 10% neutral buffered formalin (Fisher Scientific, Fair Lawn, NJ). 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 hemo toxylin/eosin to assess parenchymal histopathological changes. The methodology used to evaluate hepatotoxicity have been described previously (Price et al. 1999). Briefly, the parameters evaluated were proliferation, apoptosis, necrosis and fibrosis Proliferation was de termined according to the presence of four parameters: hepatocy te hyperplasia, hepatocyte hypertrophy, mitotic activity and binculeation. To determine prolif eration intensity, the su m of the individual intensity of each of the four parameters was used to reach a total score of no observable

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15 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 visualization of apoptotic bodices and also intact apoptotic nucle i. It is a useful method fo r detecting apoptosis at the earliest stages. A solid brown nuclear stai ning identified apoptotic hepatocytes. The number of apoptotic cells was expressed as a percentage of total number of cells (Apoptotic Index, AI). The apopt otic index was then converted to a numerical score. No observable apotosis is given a score of zero. This equates to AI 0%. Mild intensity, given a score of 1, represents and AI <25% Moderate intensity, given a score of 2, represents AI 25% to 50%. Prominent inte nsity, given a score of 3 represents AI 50%. Cleaved Caspase-3 histopathology was performed using Cleaved Caspase-3 (Asp175) (5A1) Rabbit Monoclonal Anti body from Cell Signaling Technology. The method of immunochemistry was used. Cleav ed caspase-3 (Asp175) detects levels of large fragments (17-19 kDa) of activated caspase 3 resulting from cleavage adjacent to Asp 175. Expression of cleaved caspase 3 stains brown. 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 3 is 51-75% expression and a 4 is 76-100% expression. Glutathione: Total glutathione (oxidized and reduced) was measured using a kinetic assay measured at wavelength 405 nm. Briefly, livers were removed and homogenized (1 in 10 w/v in 7.4 pH phosphate buffered saline). Supernate was separated by centrifugation of an aliquot of liver hom ogenate. The supernate was deprotienated

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16 with tricloroacetic acid (TCA ). After deproteination, the supernate was decanted into a microcentrifuge tube and DTNB and GR reag ents were added. Immediately before analysis, NADPH was added. GSH was measur ed for 3 minutes at intervals of 15 seconds. A standard curve was a plot of in crease in optical density (OD)/min as a function of the concentration of GSH. Values were calculated from the standard curve and normalized to the weight of the liver to report values as M GSH/mg liver. Lipid Peroxidation: Lipid Peroxidation wa s measured using a spectrophotometeric method. The method used is based on the reaction of malondial dehyde (MDA) with thiobarbituric acid. Livers were removed and homogenized (1 in 10 w/v in 7.4 pH phosphate buffered saline). Supernate was seperated by centrifugation of an aliquot of liver homogenate. Butylated hydr oxytoluene in methanol wa s added to an aliquot of supernate. 1M phosphoric acid and 2-thioba rituric acid were added. The sample was incubated for 60 minutes at 60 o C and analyzed. Values were calculated from the standard curve and ratioed to the weight of the liver to report the values as M MDA/mg liver. Statistical Analysis: Comparisons am ong groups were made using a one-way analysis of variance (ANOVA), with a Student Neuman-Keuls post test. In all cases statistical differences were considered significant with p 0.05. All statis tical analysis were performed with GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego, CA, www.graphpad.com).

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17 CHAPTER 3 RESULTS Mice were treated with BB with out w ithout a cotreatment with a PARP-1 inhibitor. Table 3 lists the PARP-1 inhibitors that were screened for efficacy. Nic and Phen were selected as the nonspecific and spec ific inhibitors for this study. BB treatment alone resulted in a 5-fold increase in ALT. Mice treated with Nic at time intervals of 1.2, 1 and 2 hours following BB decreased serum ALT activity by 90%. Mice treated concomitantly with BB and Phen showed a 75% reduction in serum ALT activity. Inhibitor IC50 ( M) Notes Benzamide 22 No positive findings 4-Aminobenzamide 1800 Solubility issues did not use Aminophylline 3.3 No reproducible results Nicotinamide 210 Inhibitor used in this study 6( 5H )-Phenanthridinone 0.30 Inhibi tor used in this study Table 3. Screening of inhibitors. Five inhibitors were screened for this study. Of those screened, only Nic and Phen had positive results. Benzamide showed no significant reduction in ALT. 4-Aminobenzamide wa s soluble in salin e upon heating, but precipitate was observed after 12 hours. Am inophylline was screened at concentrations from 1 mg/kg to 100 mg/kg with no reproducible results.

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18 Serum ALT activity was measured at 12hour intervals to find the point of maximum activity. At the 12-hour time poi nt, there was no statistically significant difference in serum ALT activity expressed by BB and controls. At the 24-hour time point, there was a 345% increase in serum ALT activity compared to the 12-hour time point for BB. Serum ALT activity decreased 22% between 24 hours and 36 hours. The maximum serum ALT activity was expressed at the 24-hour time point. All experimental data will be collected at the 24-hour ti me point for the dur ation of the study. BB and Nic were co-administered conc omitantly (Figure 8). There was no statistically significant difference in seru m ALT activity of BB with a concomitant administration of Nic when compared to ad ministration of BB alone. The timing of the subsequent administration of Nic in a single i.p. injection was varied from 1 hour to 3 hours. Administration of Nic at 1 hour afte r BB (Figure 9) reduced serum ALT activity by 53%. Administration of Nic at 3 hours af ter BB shows no statistically significant decrease in serum ALT activity. Administration of Nic at 3 time inte rvals, hour, 1 hour and 2 hours after BB showed a 90% reduction in serum ALT (Figur e 11). To confirm these findings, the experiment was repeated (Figure 12). There was an 85% reduction in serum ALT activity.

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1 2h r C 1 2hr BB 24 hr C 2 4-hr BB 3 6h r C 3 6hr BB 0 100 200 300ALT (IU/L) * Figure 7. ALT measurements (IU/L) measured at 12-hour intervals. Mi ce were sacrificed every twelve hours and ALT wa s measured to find the time point where the ALT for BB was at the maximum. Serum ALT level is expressed for controls (C) and BB (BB) as mean SEM. An represents a value that is st atistically different from the starting point at 12 hours. Statistical differences of ALT da ta were determined using one-way analysis of variance (p<0.05). 19

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Control BB NIC BB/NIC 0 50 100 150 200 250 300 350ALT (IU/L) Figure 8. BB and Nicotinamide administ ered concomitantly. Controls were administered corn oil. Nic was administ ered to a group of mice to verify that no elevation of ALT would result. Results are expressed as mean SEM Statistical differences of ALT data were determined us ing one-way analysis of variance (p<0.05). There is no significant lowering of A LT when BB and Nic are administered concomitantly. 20

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Co n t r ol BB NI C B B/N IC 0 50 100 150 200 250 300 350ALT (IU/L) Figure 9. Nicotinamide was administered 1 hour after BB dose. Serum ALT level is expressed for controls, bromoben zene (BB), nicotinamide (NIC) and bromobenzene/nicotinamide (BB/NIC) as mean SEM. An represents a value that is statistically different from BB. Statistical differences of ALT data were determined using one-way analysis of variance (p<0.05). 21

PAGE 34

Co n t rol BB N IC B B/NI C 3 hr 0 100 200 300ALT (IU/L) Figure 10. Nicotinamide treatment 3 hours af ter BB treatment. Serum ALT level is expressed for controls, bromoben zene (BB), nicotinamide (NIC) and bromobenzene/nicotinamide (BB/NIC) as mean SEM. There is no statistically significant difference in ALT is seen betw een BB and BB/NIC at 3-hour post treatment. 22

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Control B B NIC B B/Nic 3x 0 100 200 300 400ALT (IU/L) Figure 11. Nicotinamide (NIC) administrati on given at 1 and 2 hours. Serum ALT level is expressed for controls, brom obenzene (BB), nicotinamide (NIC) and bromobenzene/nicotinamide (BB/NIC) as mean SEM. An represents a value that is statistically different from the BB. A statis tical difference of ALT data was determined using one-way analysis of variance (p<0.05). 23

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Control BB BB/Nic 3x 0 100 200 300 400ALT (IU/L) Figure 12. Confirmation of BB and 3x dose, wher e 3x refers to Nic administered 1 and 2 hours after BB. Serum ALT level is expr essed for controls, bromobenzene (BB), nicotinamide (NIC) and bromobenzen e/nicotinamide (BB/NIC) as mean SEM. Results are expressed as mean SEM. An represents that BB/NI C is statistically different from BB. Statistical differences of ALT data was determined using one-way analysis of variance (p<0.05). 24

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25 Gross anatomical findings showed hemorrhagic and necrotic damage in mice treated with BB. When Nic was administered subsequent to BB, no pathological changes were observed. Visual observ ations were recorded on all mice that blood was drawn on. A correlation with ALT was performed. Findi ngs showed that for those mice having gross pathological changes, there was an incr ease in ALT. Because the mice selected were male ICR mice, there was some interspeci es variation. This is shown in graphs by error bars. As indicated by the data in figure 13, Nic administered in 3 subsequent administrations after BB, has the ability to reduce serum ALT activity for up to 72 hours. ALT activity was measured every 12 hours and compared to the ALT activity of BB at 24 hours, which is the time point at which the maximum ALT is expressed (Figure 7). The reduction in ALT was approximate ly 80% at every time point. Controls with corn oil and Nicotinamide are shown as a reference. All BB + Nic ALT responses were near that of controls. A seven day mortality study was performed for both mice administered BB, and mice that received multiple administrations of Nic subsequent to BB administration. Nic completely protected against mortality, while 56% of those mice administered BB alone died within the 7 day period (Figure 14).

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C 24-hr BB 24 -h r NIC 24-hr BB /NI C 12 h r BB/N IC 24 hr BB /N IC 36 hr BB/N I C 48 hr B B/NIC 60 hr BB/NIC 72 hr 0 50 100 150 200 250ALT (IU/L) * ** Figure 13. 72 hour time study for BB/NIC. Se rum ALT level is expressed for controls (C), bromobenzene (BB) and bromobenzene/ nicotinamide (BB/NI C) BB/NIC at 12, 24, 36, 48, 60 and 72-hours was near that of the c ontrol at 24 hours. Previous experiments (figure 6) showed that ALT for controls doe s not change significantly over time. All responses are reported as mean SEM. An represents a value that is statistically different from the BB at 24 hours. Statistical differences of ALT data was determined using one-way analysis of variance (p<0.05). 26

PAGE 39

BB BB/NIC 0 10 20 30 40 50 60% Mortality Figure 14. 7-day mortality for mice admini stered BB only and mice administered BB + Nic. BB alone showed a 56% mortality, wh ile BB + Nic showed a 0% mortality. NIC was protective agains t BB-induced mortality. 27

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28 Livers from mice treated with BB, with or without subsequent Nic treatment, were removed and sectioned for histopathol ogic examination. On mouse from each treatment group was selected for macroscopic examination (Figures 15 17). Figure 15 shows the liver treated with saline. There were no abnor malities in the tissue. Figure 16 shows the liver treated with BB. There was hemorrhagic damage on the outer edges of the liver. Figure 16B is a close up of the necrotic damage. Figure 17 is a macroscopic view of a liver treated with BB and Nic. No tissue abnormalities ar e seen. Macroscopic examination of liver samples were in accordance with the biochemical findings.

PAGE 41

Figure 15. Macroscopic view of liver from a mouse administered sa line only. Liver was removed immediately following necropsy. No tissue abnormalities were apparent. 29

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A B Figure 16. Macroscopic view of liver from a mouse administered bromobenzene. Liver was removed immediately following necropsy. Liver is hemorrhagic around edges with necrosis visible on upper surface (A). Clos e up of the necrotic damage on the upper surface (B). 30

PAGE 43

Figure 17. Macroscopic view liv er from a mouse treated with BB and Nic. Liver was removed immediately following necropsy. No tissue abnormalities are apparent. 31

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32 Table 4 lists the histopathologi cal evaluation of necrosis, apoptosis and cleaved caspase 3 (Caspase IHC). The values reported are the av erages of the findings. Figures 18 20 are hematoxylin-eosin stained to assess parenchym al histopathological ch anges. Figures 21 23 are TUNEL stained to assess apoptosis. Figures 24 26 are stained for cleaved caspase 3 to assess the evidence of expression. Administered Substance H&E TUNEL Cleaved Caspase 3 (Caspase IHC) Control 0 0 0 Nicotinamide 0 0 0 Bromobenzene 3+ 1+ necrosis 2-3 apoptosis 3+ 1+ Bromobenzene/ Nicotinamide 50% of samples showed no necrosis, no apoptosis reversible 1+ Focal 3+ reversible 0 Table 4. Histopathologic evaluation of H&E, TUNEL and Immunochemistry. H&E was graded using a scale 0 to 3 and is a sum of four parameters he patocyte hyperplasia, hepatocyte hypertrophy, mitotic activity and binculeation (n o expression of histological changes = 0; mild intensity = 1; moderate intensity = 2; prominent intensity = 3). TUNEL is expressed as the number of apoptotic cells as a percentage of total number of cells (Apoptotic Index or AI). AI is expre ssed on a scale of 0 to 4. (no expression of apoptosis = AI 0% or a score of 0; mild intensity = AI < 25% or a score of 1; moderate intensity = AI 25% to 50% or a score of 2; prominent intensity = AI > 50% or a score of 3). Immunochemistry was used to evaluate cas pase expression using a scale of 0 to 4 (0 = no immunochemistry expression; 1 < 25%; 2 = 26 50%; 3 = 51 %; 4 = 75 100%).

PAGE 45

Figure 18. H&E staining of saline contro l mouse liver. An arrow indicates a centrilobular vein. H&E was graded using a scale 0 to 3 and is a sum of four parameters hepatocyte hyperplasia, hepatocyte hypertrophy, mitotic act ivity and binculeation (no expression of histological changes = 0; mild intensity = 1; moderate intensity = 2; prominent intensity = 3). Histopathology expression was given a score of 0. 33

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Figure 19. H&E staining of liver from mouse administered bromobenzene. An arrow indicates a centrilobular vein. H&E was graded using a scale 0 to 3 and is a sum of four parameters hepatocyte hyperplasia, he patocyte hypertrophy, mitotic activity and binculeation (No expressioion of histological changes = 0; mild intensity = 1; moderate intensity = 2; prominent intensity = 3). Centr ilobular damage ( ) indicates swelling of the cells, termed ballooning. Histopathology expression was given a score of 3+. 34

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Figure 20. H&E stain of liver tissue from mouse administered with bromobenzene and nicotinamide. An arrow indicates a centril obular vein. H&E was graded using a scale 0 to 3 and is a sum of four parameters he patocyte hyperplasia, hepatocyte hypertrophy, mitotic activity and binculeation (No expres sion of histological changes = 0; mild intensity = 1; moderate intensity = 2; prominent intensity = 3). Histopathology expression was given a score of 1+. Fifty percent of samples showed no necrosis, no apoptosis and reversible hypertrophy. 35

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Figure 21. TUNEL staining of sa line control. An arrow indi cates a centrilobular vein. TUNEL is expressed as the number of apoptotic cells as a percentage of total number of cells (Apoptotic Index or AI). AI is expre ssed on a scale of 0 to 4. (no expression of apoptosis = AI 0% or a score of 0; mild expression = AI < 25% or a score of 1; moderate expression = AI 25% to 50% or a score of 2; prominent expr ession = AI > 50% or a score of 3). TUNEL expression was given a score of 0. 36

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Figure 22. TUNEL staining of BB induced damage. An arrow indicates a centrilobular vein. Apoptosis is indicated by ( or ). TUNEL is expressed as the number of apoptotic cells as a percentage of total numbe r of cells (Apoptotic Index or AI). AI is expressed on a scale of 0 to 4. (no expression of apoptosis = AI 0% or a score of 0; mild expression = AI < 25% or a score of 1; mode rate expression = AI 25% to 50% or a score of 2; prominent expression = AI > 50% or a score of 3). TUNEL expression was given a score of 3+. 37

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Figure 23. TUNEL stain of liver with a BB and NIC dose. An arrow indicates a centrilobular vein. TUNEL is expressed as the number of apoptotic cel ls as a percentage of total number of cells (Apoptotic Index or AI). AI is expres sed on a scale of 0 to 4. (no expression of apoptosis = AI 0% or a score of 0; mild expr ession = AI < 25% or a score of 1; moderate expression = AI 25% to 50% or a score of 2; prom inent expression = AI > 50% or a score of 3). TUNEL e xpression was given a score of 0. 38

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Figure 24. Cleaved Caspase 3 staining of a liv er from a mouse that was administered saline. Immunochemistry was used to evaluate caspase expression using a scale of 0 to 4 (0 = no immunochemistry expression; 1 < 25%; 2 = 26 50%; 3 = 51 %; 4 = 75 100%). Immunochemistry evaluation was negative (a score of 0). An arrow indicates the centrilobular vein. 39

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Figure 25. Cleaved Caspase 3 staining of a liv er from a mouse that was administered bromobenzene. Immunochemistry was used to evaluate caspase expression using a scale of 0 to 4 (0 = no immunochemistry expres sion; 1 < 25%; 2 = 26 50%; 3 = 51 %; 4 = 75 100%). Immunochemistry evaluation rated the damage as a 1+. An arrow indicates the centrilobular vein. Cleav ed caspase 3 stains brown. 40

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Figure 26. Cleaved Caspase 3 staining of a liv er from a mouse that was administered BB and Nic. Immunochemistry was used to evalua te caspase expression us ing a scale of 0 to 4 (0 = no immunochemistry expression; 1 < 25%; 2 = 26 50%; 3 = 51 %; 4 = 75 100%). Immunochemistry evaluation was negative (a score of 0). An arrow indicates the centrilobular vein. 41

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42 Histopathologic evaluation of the liver se ctions were in accordance with the biochemical findings. Mice administered only saline showed no expression of abnormalities, no expression of apoptosis and no expression of cleaved caspase-3. With the administration of BB, there is histopathol ogic expression of swelling of cells, or ballooning. Apoptosis is ev ident, and there is expre ssion of cleaved Caspase-3. Administration of Nic subsequent to BB attenuated the hepatotoxicity. BB and Phen (10 mg/ml) were administer ed concomitantly (Figure 27). Due to Phen being soluble in 100% DMSO, BB and DM SO were administered concomitantly to assess the inhibitory effect s of DMSO. Phen administered concomitantly with BB reduced serum ALT activity by 54%. DMSO administered concomitantly with BB reduced serum ALT activity by 40%. Reductions in ALT activity were statistically significant when compared to the activity expressed by BB. BB and Phen (20 mg/ml) administered concomitantly (Figure 28) decreased serum ALT activity by 58% compared to anim als treated with BB alone. BB and Phen (40 mg/ml) administered concomitantly (F igure 20) decreased serum ALT activity by 73% compared to animals treated with BB al one. Phen at a concentration of 40 mg/ml showed the largest reduction in serum ALT activity. This level of inhibitor was repeated (Figure 29) in a subsequent study to verify results. The reduction in serum ALT activity in the subsequent experiment was 74% co mpared to animals treated with BB alone.

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BB BB/10 Phen B B/DMSO 10 Phen DMS O 0 100 200 300 400ALT (IU/L) * Figure 27. Phen was administered concomita ntly with BB. Phen is soluble in 100% DMSO. The dose of BB/DMSO showed a statis tically significant lowering of ALT. The dose of 10 mg/ml also showed a statistically significant lowering of ALT. Results are expressed as mean SEM. An represents statistically significant difference when compared to BB. Statistical differences of ALT data was determined using one-way analysis of variance (p<0.05). 43

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BB BB/20 Phen BB/40 Phen 20 Ph en 4 0 Ph en DM SO 0 50 100 150 200 250 300 350ALT (IU/L) * Figure 28. Screening of 20, and 40 mg/ml concomitant administration of BB and Phen. Results are expressed as mean SEM. Results are expressed as mean SEM. An represents statistically significant differe nce when compared to BB. Statistical differences of ALT data was determined us ing one-way analysis of variance (p<0.05). Statistical differences of ALT data were de termined using one-way analysis of variance (p<0.05). 44

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Brom obenzene BB/40 m g/m l Phen 0 100 200 300IU/L Figure 29. Confirmation experiment to show repeatability at the BB/40 mg/mL Phen level. The reduction in ALT was comparable to the initial experiment at this level. An represents statistically significant differe nce when compared to BB. Statistical differences of ALT data was determined us ing one-way analysis of variance (p<0.05). 45

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46 A 7-day mortality study was conducted us ing four groups of mice. One group received only BB, one group received BB + Phen at a concentration of 40 mg/kg, one group received BB + DMSO and one group received DMSO only. The sample size for all groups was n = 8 (Figure 30). There was a 50% mortality in mice that were administered BB. This is comparable to the 56% seen in the previ ous experiment (Figure 14). There was 12.5% (1 in 8) mortality in mice administered BB + Phen, Phen only and DMSO only. Due to Phen solubility in 100% DMSO, mice were administered DMSO alone as a control.

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BB BB/Phen Phen DMSO 0 10 20 30 40 50% Mortalilty Figure 30. 7 day mortality for BB, BB/P hen, BB/DMSO and DMSO only. There was 50% mortality for BB only, and 12.5% for othe r groups. There was a 4-fold decrease in mortality. All groups with one death were administered DM SO, either alone, or as the vehicle for Phen. N=8 for all groups. 47

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48 Histopathology was performed for BB/Phen, Ph en and DMSO by a certified pathologist Table 5 lists the histopathologi cal evaluation of necrosis, apoptosis and cleaved caspases 3 (Caspase IHC). Macroscopic pathology was also performed (Figur e 31). Figures 32 34 are hematoxylin-eosin stained to assess parenchymal histopathological changes. Figures 35 37 are TUNEL stained to assess apoptosis. Figures 38 405 are stained for cleaved caspase 3 to assess the evidence of expression. No abnormalities were seen any of the pathology samples for this experiment. Administered Substance H&E TUNEL Cleaved Caspase 3 (Caspase IHC) Bromobenzene/Phen 0 0 0 Bromobenzene/DMSO 0 0 0 DMSO Control 0 0 0 Table 5. Histopathologic evaluation of H&E, TUNEL and Immunochemistry. H&E was graded using a scale 0 to 3 and is a sum of four parameters he patocyte hyperplasia, hepatocyte hypertrophy, mitotic activity and binculeation (n o expression of histological changes = 0; mild intensity = 1; moderate intensity = 2; prominent intensity = 3). TUNEL is expressed as the number of apoptotic cells as a percentage of total number of cells (Apoptotic Index or AI). AI is expre ssed on a scale of 0 to 4. (no expression of apoptosis = AI 0% or a score of 0; mild intensity = AI < 25% or a score of 1; moderate intensity = AI 25% to 50% or a score of 2; prominent intensity = AI > 50% or a score of 3). Immunochemistry was used to evaluate cas pase expression using a scale of 0 to 4 (0 = no immunochemistry expression; 1 < 25%; 2 = 26 50%; 3 = 51 %; 4 = 75 100%).

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A B C Figure 31. Macroscopic view of livers from mi ce administered Phen (A), DMSO (B) and BB/Phen (C). Liver was removed imme diately following necropsy. No tissue abnormalities were apparent. 49

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Figure 32. H&E stain of liver tissue from a m ouse administered bromobenzene and Phen concomitantly. An arrow indicates a centrilobular vein. H&E was graded using a scale 0 to 3 and is a sum of four parameters he patocyte hyperplasia, hepatocyte hypertrophy, mitotic activity and binculeation (No expres sion of histological changes = 0; mild intensity = 1; moderate intensity = 2; prominent intensity = 3). Histopathology was given a rating of 0. 50

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Figure 33. H&E stain of liver tissue from a mouse administered bromobenzene and DMSO concomitantly. An arrow indicates a centrilobular vein. H&E was graded using a scale 0 to 3 and is a sum of four parameters hepatocyte hyperplasia, hepatocyte hypertrophy, mitotic activity and binculeation (No expression of hist ological changes = 0; mild intensity = 1; moderate intensity = 2; prominent intensity = 3). Histopathology was given a rating of 0. 51

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Figure 34. H&E stain of liver tissue from a mouse administered DMSO. An arrow indicates a centrilobular vein. H&E was graded using a scale 0 to 3 and is a sum of four parameters hepatocyte hyperplasia, he patocyte hypertrophy, mitotic activity and binculeation (No expression of histological ch anges = 0; mild intensity = 1; moderate intensity = 2; prominent intensity = 3) Histopathology was given a rating of 0. 52

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Figure 35. TUNEL stain of liver tissue fr om a mouse administered bromobenzene and Phen concomitantly. An arrow indicates a centrilobular vein. T UNEL is expressed as the number of apoptotic cells as a percentage of total number of cells (Apoptotic Index or AI). AI is expressed on a scale of 0 to 4. (No expression of apoptosis = AI 0% or a score of 0; mild expression = AI < 25% or a score of 1; moderate expression= AI 25% to 50% or a score of 2; prominent expression = AI > 50% or a score of 3). TUNEL expression was given a score of 0. 53

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Figure 36. TUNEL stain of liver tissue from a mouse administered bromobenzene and DMSO concomitantly. An arrow indicates a centrilobular vein. T UNEL is expressed as the number of apoptotic cells as a percentage of total number of cells (Apoptotic Index or AI). AI is expressed on a scale of 0 to 4. (No expression of apoptosis = AI 0% or a score of 0; mild expression = AI < 25% or a score of 1; moderate expression= AI 25% to 50% or a score of 2; prominent expression = AI > 50% or a score of 3). TUNEL expression was given a score of 0. 54

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Figure 37. TUNEL stain of liver tissue from a mouse administered DMSO. An arrow indicates a centrilobular vein. TUNEL is expressed as the nu mber of apoptotic cells as a percentage of total number of cells (Apoptotic In dex or AI). AI is expressed on a scale of 0 to 4. (No expression of apoptosis = AI 0% or a score of 0; mild expression = AI < 25% or a score of 1; moderate expression= AI 25% to 50% or a score of 2; prominent expression = AI > 50% or a score of 3). TUNEL expression was given a score of 0. 55

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Figure 38. Cleaved Caspase 3 staining of a liv er from a mouse that was administered BB and Phen. Immunochemistry was used to eval uate caspase expressi on using a scale of 0 to 4 (0 = no immunochemistry expression; 1 < 25%; 2 = 26 50%; 3 = 51 %; 4 = 75 100%). Immunochemistry evaluation was nega tive (a score of 0). An arrow indicates the centrilobular vein. 56

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Figure 39. Cleaved Caspase 3 staining of a liv er from a mouse that was administered bromobenzene and DMSO. Immunochemistry was used to evaluate caspase expression using a scale of 0 to 4 (0 = no immunochem istry expression; 1 < 25%; 2 = 26 50%; 3 = 51 %; 4 = 75 100%). Immunochemistry eval uation was negative (a score of 0). An arrow indicates the centrilobular vein. 57

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Figure 40. Cleaved Caspase 3 staining of a liv er from a mouse that was administered DMSO. Immunochemistry was used to evaluate caspase expression using a scale of 0 to 4 (0 = no immunochemistry evaluation; 1 < 25%; 2 = 26 50%; 3 = 51 %; 4 = 75 100%). Immunochemistry evaluation was negative (a score of 0). An arrow indicates the centrilobular vein. 58

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59 Total glutathione (reduced and oxidized) wa s measured in liver samples that were extracted 24 hours after BB administration (Figure 41). Livers were stored at C until analysis. Administration of BB alone resulted in an 81% reduction in GSH. The reduction in GSH is in accordance with the bi ochemical and histopathological findings. Administration of BB and subsequent injecti ons of Nic resulted in a 36% reduction in GSH. A concomitant administration of Ph en and BB resulted in a 91% reduction in GSH. This confirms that PARP-1 inhibition of BB-induced hepatotoxicity is independent of the metabolism of BB. Lipid peroxidation in the liver was assessed (Figure 42). Lipid peroxidation is used as an indirect measure of liver injury. The method used was a measurement of malondialdehyde (MDA) accumulation (TBARS). There was no statistically significant difference in any of the groups when compared to either the controls, which received no treatment, or BB alone. This shows that ei ther BB administration at 112 mg/kg is at a concentration that is insufficient to cause lip id peroxidation or reduc tions in GSH are not correlated to lipid peroxidation.

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CONTROL BB BB/NI C BB/PHEN 0 5 10 15 20 25GSH (uM)/g liver * Figure 41. Total glutathione (uM GSH/mg liver) measured for BB, BB/NIC and BB/Phen. Livers were extracted 24 hours afte r BB was administered. Livers were kept at C until analysis. An represents a st atistically significant depression of GSH as compared to the level measured in the control. In all groups there was a significant depression of glutathione as measured by one -way ANOVA. The level of significance is p 0.05. 60

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B B BB/NIC BB/PHE N BB / DMSO CONTR OL 0 1 2 3 4 5 6 7 8 9 10 11uM MDAg liver Figure 42. Lipid Peroxidation (TBARS) measured for BB, BB/NIC, BB/Phen BB/DMSO. Values reported are in uM MDA/mg liver. There was no statistical significant between any groups as measured by one-way ANOVA. Results are expressed as mean SEM. Significance was set at p 0.05. 61

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62 CHAPTER 4 DISCUSSION This experiment has shown that nicotinamide attenuates BB-induced hepatotoxicity. Because nicotinamide is a non-specific inhibitor, this experiment was repeated using a potent specific inhibitor to ve rify that the results could be replicated. The inhibitor selected was Phen. Phen is not soluble in saline, but was found to be soluble in 100% DMSO. Dime thyl sulfoxide (DMSO) has be neficial properties as an anti-inflammatory agent and as a treatment for ischemia. It has been shown to protect the liver of rats from BB when given up to 24 hours after oral administration (Lind and Gandolfi, 1999b). In a follow-up study, Lind and Gandolfi determined that DMSO looses its effectiveness if administered af ter the twenty four hour time point (Lind and Gandolfi, 1999a). Past evidence has shown that DMSO (IC 50 = 4.791 M) is capable of inhibiting PARP-1 (Banasik et al. 2004). In this experiment Phen, a potent PARP-1 inhibitor, was dissolved in 100% DMSO. This laboratory has shown that Phen was protective against carbo n tetrachloride (CCl 4 ) hepatotoxicity (Banasik et al. 2004). Animals were administered CCl 4 and Phen concomitantly. CCl 4 is bioactivated mainly by cytochrome P450 2E1 via a reductive dehalo genation to form the trichloromethyl free radical (Cl3C ) (Banasik et al., 2004). CCl 4 and BB cause centrilobular necrosis by different pathways. The CCl 4 experiment described in Banasi k, et al. was repeated using BB as the toxicant (Figure 27). The concen tration of the inhib itor used in the CCl 4

PAGE 75

63 experiment, 10 mg/kg was used in this study. BB and Phen were administered concomitantly and showed a significant decrease in serum ALT. The experiment was repeated using concentrations of 20 mg/kg a nd 40 mg/kg (Figure 28) to test for a further decrease in ALT. A concentration of 20 mg/kg showed a 2.6 fold decrease in serum ALT, a concentration of 40 mg/kg showed a 4-fold decrease. The experiment was repeated at 40 mg/kg concentrati on for confirmation (Figure 29). The first studies concentrated on nicotinam ide as the PARP-1 inhibitor. Work involving nicotinamide has been focuse d on acetaminophen. Acetaminophen (APAP) overdose causes hepatic and renal failure. The use of nicotinamide alone or in combination with N-acetylcystein, an antioxidant, or L-methione, an amino acid, suppressed hepatotoxicity (Krger et al. 1996; Krger et al. 1997). Nicotinamide was shown to prevent APAP-induced lipid per oxidation, DNA damage and the associated apoptotic and necrotic cell deaths (Ray et al., 2001). One function of PARP-1 is to participate in cellular recovery from DNA damage (Durkacz et al. 1980). At the site of a DNA strand break, PARP-1 catalyzes the transfer of the ADP-ribose moiety from its substrate NAD+ to either a protei n acceptor, or to the enzyme itself (automodification) (de Murcia and Menissier-de Murcia, 1994). PARP-1 activation has been found to contribute to an energy-consuming cellular process, which leads to NAD and ATP depletion, mitochond rial dysfunction and an overall cellular dysfunction. This process can eventually lead to cell death (Southan and Szab, 2003). PARP-1 inhibition by chemicals can prevent a drop in NAD. Preserving cellular energy level appears to be the main effect that PARP inhibitors exhibit in reducing necrotic cell death (Zhang and Li, 2000). BB, the toxicant used in this study, is a well-

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64 known and model chemical for causing hepati c centrilobular necrosis. The purpose of this study was to show BB hepatotoxicity was present and to show that PARP-1 inhibitors could attenuate that hepatotoxicity. The first i nhibitor chosen was NIC, a nonspecific inhibitor. The sec ond inhibitor chosen was Phen, a specific and potent inhibitor. Serum ALT is a measure of liver damage NIC resulted in a 90% reduction in serum ALT, PHEN resulted in a 75% reduction. To confirm that th e reduction in ALT was a measure of attenuation of hepatotoxi city, histopathology was analyzed. H&E, TUNEL and cleaved caspase 3 were assesse d and graded by a cer tified pathologist. PARP is cleaved by caspases, predomin antly caspase-3, during apoptosis (Affar et al. 2001). The pathology findings showed that BB induced damage including ballooning of cells, hyperplasia and apoptosis. Cleaved cas pase 3 was expressed. Saline and DMSO were analyzed controls. NIC is soluble in saline and PHEN is so luble in DMSO. The control samples showed no abnormal pathol ogy. BB/NIC and BB/PHEN were assessed for the same parameters as BB. Because DMSO has some inhibitory properties, BB/DMSO was also assessed. The pathologi cal evaluation showed that apoptosis was not present in any of the samples that contained inhibitors, including the BB/NIC, BB/PHEN and BB/DMSO. TUNEL and cleav ed caspase 3 were not expressed. Most of the toxic metabolism of BB is by way of the formation of the 3,4epoxide. Detoxification of the epoxide is by conjugation with GSH under the influence of glutathione-S-transferase. When the rate of formation of the 3,4-epoxide exceeds the rate of the detoxifying reaction and the availa ble GSH is depleted, the epoxide begins to bind covalently to tissue macromolecules a nd thereby cause necros is(Zimmerman, 1999). Glutathione is most concentrat ed in the liver. GSH levels were significantly reduced for

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65 BB, BB/NIC and BB/Phen. This finding showed that although GSH levels were reduced, the PARP-1 inhibitor treated animals had re duced ALT and no abnormal histopathology. This leads to a conclusion that the attenua tion of BB-induced hepato toxicity is totally independent of metabolism. Bromobenzene forms conjugates with hepatic GSH. This reduces GSH levels and the liver cells are made more susceptible to the development of lipid peroxidation (Comporti, 1985). In primary cultures of hepa tocytes, BB induced a rapid depletion of GSH followed by the appearance of lipid peroxidation (Casini et al. 1982). The methodology used to measure lipid peroxidation was a thiobarbituric aci d test. It is a spectrophotometeric quantitation of the pink co mplex formed after reaction of MDA with two molecules of TBA (Botsoglou et al. 1994). There was no st atistical difference in level of MDA between BB alone and BB when ad ministered with NIC, PHEN or DMSO. The findings for no lipid peroxidation are contrary to the work of Casini, et. al. The work done by Casini, et. al. was in vitro. This would lead to a conc lusion that either this work is not reproducible in vivo, or that the le vel of BB used was insufficient to produce lipid peroxidation. When a PARP-1 inhibitor is used, it would be expected that lipid peroxidation would not be expressed. Further study in the area of BB-induced in vivo lipid peroxidation using a higher concentra tion may show that lipid peroxidation may occur. The major pharmaceutical uses of PARP-1 inhibitors have focused on acetaminophen (APAP). APAP is a common analgesi c that is safe at therapeutic doses. Overdoses of APAP result in fulminant hepatic and renal tubular necrosis (Mitchell et al. 1973a). The toxicity results from the formation an arylating metabolite of APAP

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66 (Mitchell et al. 1973b). The metabolite is N-aceyl-p-benzoquionone imide (NAPQI). Cytochromes 2E1, 1A2, 4A4 and 2A6 have been reported to oxidize APAP to NAPQI (James et al. 2003). APAP is a classic example of a compound de toxified by GSH (James et al. 1993). PARP-1 inhibition of acetaminophen has been studied using both specific and non-specific inhibitors. The i nhibitors studied include nicotinamide, benzamide, caffeine, theophylline, thymidine, L-tryptophan (Krger et al. 1996) Nacetylcysteine, L-Methionine (Krger et al. 1997), chlorpromazine and 4aminobenzamide (Ray et al., 2001). Of the inhibitors used in APAP studies nicotinamide, benzamide, theophylline (in the form of aminophylline hydrate) and 4-aminobenzamide were assessed. Aminophylline is 78% theophylline with th e addition of ethylenediamine for saline solubility. No reproducible re sults were obtained with this inhibitor. Benzamide gave no positive findings. 4-aminobenzamide had solubility problems in saline. It is soluble in hot saline with constant stirring (Ray, pe rsonal communication). Solubility was obtained at the boiling point of saline. Upon sitting, over a period of approximately 12 hours, precipitate was observed. This inhibitor was not used as the heating to an elevated temperature may have reduced the efficacy or changed the chemical structure of the inhibitor. This laboratory has conducted research in the area of PARP -1 inhibition of an environmental chemical, CCl 4 Because of its propensity to cause hepatotoxicity, CCl4 is routinely used as a model compound for gene rating centrilobular necrosis. Su et al. tested for polymer formation. The study found that there were significant quantities of PARP-1 in areas of sever necros is in the centrilobular region (Su et al., 2003). The

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67 conclusion of this study was that the necrogenic effects of CCl 4 were in part explained by a rapid decrease in cellular energy due to ove ractivation of PARP1. A follow-on study assessed the usage of a PARP-1 inhibitor to attenuate the CCl 4 -induced hepatotoxicity. The PARP-1 inhibitor selected for this study was Phen. Phen was dissolved in 5.5% DMSO and concomitantly administered with CCl 4 Serum ALT was reduced significantly for CCl 4 and Phen. There was no signicant difference in ALT for those treated with CCl 4 and those treated with CCl 4 + DMSO (5.5%) (Banasik et al., 2004). Animals treated with CCl 4 or CCl 4 + DMSO exhibited sever necrotic centrilobular lesions with pronounced areas of apoptosis (unpublished results). In the current experiment, Phen was dissolved in 100% DMSO. There was a statistically significant difference in ALT for BB, BB + Phen and BB + DMSO. The decrease in ALT for BB + DMSO is due to the concentration of DMSO A concentration of 5.5% was insufficient to dissolve Phen in the current study. After vortexing, centrifugation and sonication, a slurry was formed. No positive results were observed using this standard at this concentration. It was concl uded that the Phen was not being administered, but had precipitated. A solubility study confirmed that Phen was soluble in 100% DMSO. When a PARP-1 inhibitor is administered with BB, there was a reduction of ALT, no histopathological findings, gl utathione depletion and no lipi d peroxidation. This leads to a conclusion that the atte nuation of BB-induced hepatotoxi city is independent of its metabolism. It is also evidence that PA RP-1 inhibitors, both specific and non-specific can attenuate BB-induced hepatotoxicity. Two areas of further study of PARP-1 inhibition have b een identified. The first is nephrotoxicity. A model compound for nephrotoxicity is 2-bromohydroquinone (BHQ).

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68 BHQ is a metabolite of BB. BB is metabolized to the 3,4-oxide a nd further metabolized to o-bromophenol. In the liver o-bromophenol has been shown to form toxic metabolites which are then transported to the kidney (Lau et al. 1984). BHQ forms a diglutathione conjugate which has been shown to be nephrotoxic (Monks et al. 1985). This laboratory has shown that Phentolamine (Phe), an adrenergic antagonist, could modulate BHQinduced nephrotoxicity (Harbison et al. 2000). Phe was shown in the past to attenuate the hepatotoxicity of BB (Kerger et al. 1988a). Ethylene dibromide (EDB) is a model component for eliciting hepatoand nephrotoxicity. Conjugation with GSH has been shown to play a role in the bioactivation of EDB. This laboratory has shown that Phe attenuate d the nephrotoxicity of EDB (Harbison et al. 2003). PARP-1 inhibition attenuated the hepatoto xicity of BB. Based on past research using Phe in BB-induced hepatotoxicity, it is conjectured that PARP-1 can attenuate nephrotoxicity. Another area of research is to test the hypothesis that PARP -1 inhibitors can attenuate hepatotoxicity in the midzonal region of the liver. A model hepatotoxicant that causes apoptosis in this region is cocaine. Apoptosis contributes si gnificantly to cocaineinduced liver damage (Price et al. 1999). In the current study, PARP-1 attenuated apoptosis in the centrilob ular region of the liver. The results of this study extend previous observations of PARP-1 attenuation of CCl 4 -induced hepatotoxicity (Banasik et al., 2004). The role of PARP-1 attenuation of BB-induced hepatotoxicity has been describe d. The importance of these findings have important public health implications becau se there are pharmaceu ticals and industrial

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69 chemicals that cause hepatotoxicity. This study has shown that a non-specific PARP-1 inhibitor attenuates BB-induced hepatotoxicity at a concentration of approximately half of the toxic level to humans. A specific inhi bitor attenuates BB-induced hepatotoxicity at a concentration of 3,000% less than that of a non-specific PARP-1 inhibitor.

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74 Zimmerman, H. J. (1999). Hepatotoxicity, The Adverse Effects of Drugs and Other Chemicals on the Liver Lippincott Williams & Wilkins, Philadelphia.

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ABOUT THE AUTHOR Kelly W. Hall received her B.S. in Chemistr y from the University of Florida in 1991. She was employed at Honeywell, Inc. in St. Petersburg, FL. She then went to work at Environmental Science and Engineering in Gainesville, FL, where she worked as a Senior Staff Laboratory Scientist. She joined Bausch & Lomb in Tampa, FL in 1997 as a Chemist. While employed at Bausch & Lomb, she completed her MPH in Safety Management at the University of South Flor ida. After completing her MPH, she entered the Ph.D. program in Toxicology at the University of South Florida. She completed her Ph.D. while continuing to work full time at Bausch & Lomb. Bausch & Lomb generously provided tuition assistance for both the MPH and Ph.D. programs.


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Attenuation of bromobenzene-induced hepatotoxicity by poly(adp-ribose) polymerase inhibitors
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ABSTRACT: Previous studies have shown extensive cellular damage can activate poly(ADP-ribose) polymerase-1 (PARP-1) and cause a rapid decrease in the levels of NAD+ and ATP, thereby preventing apoptosis and promoting necrosis and inflammation. The purpose of this study was to extend previous observations that inhibitors of PARP-1 could alter acetaminophen and carbon tetrachloride-induced hepatotoxicity. Bromobenzene (BB) a glutathione dependent hepatotoxicant was tested. Groups of male mice were treated with a single dosage of 112mg/kg (0.075 ml/kg) BB by the intraperitoneal (ip) route. All animals were maintained in a controlled environment and provided food and water ad libitum. This dosage of BB resulted in hepatotoxicity as measured by an increase in serum alanine transferase (ALT). BB treatment resulted in a 5-fold increase in ALT. Moderate hepatotoxicity was detected with this treatment regime.Subsequently, another group of mice were treated with three treatments of nicotinamide at 0.5, 1 and 2 hours following BB treatment. Serum ALT elevations were reduced by 90% at 24 hours following BB and nicotinamide treatments. BB-induced liver pathology was also blocked by nicotinamide. Mortality among BB treated animals was also significantly reduced by nicotinamide treatment. Mortality among mice treated with BB and nicotinamide was near control. The model was verified with a more potent and specific inhibitor, Phen. BB treatment was keep at the same level as in the previous study, and Phen was administered concomitantly. Serum ALT elevations were reduced by 75%. Phen also blocked BB-induced liver pathology. Mortality among mice treated with BB and Phen was reduced 75%. PARP-1 inhibitors appear to alter chemical-induced hepatotoxicity that has either a glutathione dependent or independent mechanism.
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Poly(adp-ribose)polymerase.
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Hepatotoxicity.
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