Citation
Factors Modulating Dichloroacetate-Induced Glutathione Transferase Zeta 1 Inactivation in a Rat Model

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Title:
Factors Modulating Dichloroacetate-Induced Glutathione Transferase Zeta 1 Inactivation in a Rat Model
Creator:
Smeltz, Marci G
Publisher:
University of Florida
Publication Date:
Language:
English

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Pharmaceutical Sciences
Medicinal Chemistry
Committee Chair:
JAMES,MARGARET O
Committee Co-Chair:
DING,YOUSONG
Committee Members:
XING,CHENGGUO
MARTYNIUK,CHRISTOPHER
STACPOOLE,PETER W

Subjects

Subjects / Keywords:
dichloroacetate
gstz
mitochondria

Notes

General Note:
Dichloroacetate (DCA) is an investigational drug that also exists in the environment as a by-product from water chlorination. In the mitochondria, DCA reactivates energy metabolism and is undergoing investigation as an anticancer therapeutic. Chronic dosing of DCA leads to inactivation of the enzyme solely responsible for its biotransformation, glutathione transferase zeta 1 (GSTZ1). Numerous intrinsic factors can influence this drug-induced inactivation. In this study, we utilized a rodent model administered a single dose of DCA to investigate the time course of inactivation of liver cytosolic and mitochondrial GSTZ1 over 24 h. We measured enzyme expression and activity with DCA and its endogenous substrate, maleylacetone (MA), in 4- and 52-wk old rats, assessed the impacts of chloride (Cl-) on this drug-enzyme interaction, and investigated the expression and activity of GSTZ1 in extrahepatic sites. We demonstrated that rat hepatic mitochondrial GSTZ1 is more susceptible to DCA-induced GSTZ1 inactivation than that in the cytosol. In DCA-treated rats, hepatic GSTZ1 activity was reduced by more than 90% 4 h after a single dose, and mitochondrial GSTZ1 showed greater loss of than cytosolic GSTZ1. With MA as the substrate, enzyme activity was much greater, up to 116 times compared to when DCA was used as the substrate. Following DCA exposure, GSTZ1 activity with MA as the substrate reduced to a similar extent as when DCA was the substrate. Young rats recovered more activity than old rats by 24 h after DCA administration. More rapid GSTZ1 inactivation occurred in adult rats, with faster half-lives of loss of GSTZ1 activity in both the cytosol and mitochondria compared to young rats. In vitro studies showed that higher Cl- concentrations attenuated GSTZ1 inactivation, especially in young rats. Finally, despite much lower GSTZ1 enzyme expression, kidney, brain, and heart exhibited DCA metabolizing capabilities. Kidney homogenate had lower activity than expected from the measured GSTZ1 expression. It was later identified that gamma-glutamyl transpeptidase was responsible for rapid consumption of glutathione, leading to low GSTZ1 activity. In conclusion, this rodent model provided insight into factors that modulate DCA-induced GSTZ1 inactivation, including age, Cl-, and cellular location, and demonstrated extrahepatic GSTZ1 expression.

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5/31/2018

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1 FACTORS MODULATING DICHLOROACETATE INDUCED GLUTATHIONE TRANSFERASE ZETA 1 INACTIVATION IN A RAT MODEL By MARCI GAYLE SMELTZ A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2017

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2 2017 Marci Gayle Smeltz

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3 To everyone that ha s encouraged and helped me pursue my dreams

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4 ACKNOWLEDGMENTS Over the course of five years, I have matured as an independent scientist while completing my graduate education. It was one of the most challenging but rewarding experiences in my life. Most importantly I would like to thank my Ph.D. me ntor, Dr. Margaret O. James, for her encouragement and support through this entire process. She has helped me fully develop my scientific reasoning skills, patiently supported me through trying new laboratory techniques, and provided me numerous opportuni ties to interact with others around the world with diverse research interests and dreams. I am extremely grateful for her guidance throughout my academic career. I also want to exten d thanks to my collaborators and committee members. Dr. Peter W. Stacpoo le has provided me a plethora of detailed insights into dichloroacetate research and has reminded me that helping others is the ultimate goal with clinical research. I am also thankful for Dr. Yousoung Ding and Dr. Christopher Martyniuk for their willingness to assist with the development of my graduate research and encouragement while tackling these research projects To the other professors in the College of Pharmacy here at the University of Florida and m y former mentors from West Virginia University and Savannah River National Laboratory I am very grateful for your supervision and assistance Pursuing this degree would not have been possible without the unwavering support I received from my family and fr iends. Without the love, encouragement, and support of my parents, Ronald and Elaine Smeltz, none of this would have ever developed, even with the thousands of miles separating us. I also want t o thank the loyal friendships I developed during my undergra duate years at West Virginia University including Jessica Wilson, and those that are still flourishing here at the

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5 University of Florida, particularly Guo Zhong and Katherine Cisneros. Your assistance, hugs and laughter have left a huge imprint on my he art. Additionally, many thanks are necessary for the knowledge, help, and love I received from Laura Rowland Faux; you ha ve been the best second mom a girl could ask for! Finally, I want to thank my little sidekick, Darcy, for the infinite number of tail wags, kisses, and tennis ball sessions received daily, especially when I needed a little relief from the stress that graduate school can create. There are no words to describe my gratitude to each influential person I met during my long education al journ ey I will take your love with me as my adventure continues.

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6 TABLE OF CONTENTS P age ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 9 LIST OF FIGURES ................................ ................................ ................................ ........ 10 LIST OF ABBREVIATIONS ................................ ................................ ........................... 12 ABSTRACT ................................ ................................ ................................ ................... 14 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 16 Dichlo roacetate ................................ ................................ ................................ ....... 16 A Small Molecule ................................ ................................ .............................. 16 An Environmental Toxi ca n t ................................ ................................ ............... 16 A Therapeutic Agent ................................ ................................ ......................... 17 Glutathione Transferases ................................ ................................ ........................ 21 Glut athione Transferase Zeta 1 ................................ ................................ .............. 22 DCA Biotransformation by GSTZ1 and Pharmacokinetics ................................ ...... 25 GSTZ1 Inactivation Induced by DCA ................................ ................................ 26 Physiological Substrate Inhibition of MAAI/GSTZ1 ................................ ........... 29 Role of GSTZ1 in Mitochondria ................................ ................................ ............... 30 Factors Influencing DCA Pharmacokinetics ................................ ............................ 32 GSTZ1 Polymorphisms ................................ ................................ .................... 32 Age ................................ ................................ ................................ ................... 34 Intracellular Cl Concentration ................................ ................................ .......... 35 DCA Toxicity ................................ ................................ ................................ ........... 37 Peripheral Neuropathy ................................ ................................ ..................... 37 Errors in Phenylalanine and Tyrosine Catabolism ................................ ............ 38 Alkylating Agents ................................ ................................ .............................. 40 Significance and Specific Aims ................................ ................................ ............... 41 Rat Cytosolic and Mitochondrial GSTZ1 Properties with DCA and MA ............ 42 Rat Cytosolic and Mitochondrial GSTZ1 Inactivation Rates Pro tection by Cl .. 43 Influence of Extrahepatic GSTZ1 Expression on DCA Biotransformation ........ 43 2 DICHLOROACETATE INDUCED GLUTATHIONE TRANSFERASE ZETA 1 INACTIVATION DIFFERS IN RAT HEPATIC SUBCELLULAR FRACTIONS ......... 50 Specific Aims ................................ ................................ ................................ .......... 50 Materials and Methods ................................ ................................ ............................ 50 Chemicals and Reagents ................................ ................................ ................. 50 Animals and DCA Administration ................................ ................................ ..... 51

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7 Subcellular Fractionation of Rat Livers ................................ ............................. 51 Cytosolic and Mitochondrial GSTZ1 Activity Measurement .............................. 52 Western Blot Analysis of Cytosolic and Mitochondrial GSTZ1 Expression ....... 53 GSTZ1 Inactivation Half Life Determination in the Presence of Cl .................. 53 Attenuating DCA Induced GSTZ1 Inactivation by Cl ................................ ....... 54 Glutathione Levels in Hepatic Subcellular Fractions ................................ ........ 54 Data Analysis ................................ ................................ ................................ ... 55 Results ................................ ................................ ................................ .................... 56 DCA Induced Inacti vation of GSTZ1 Activity in Rat Subcellular Fractions ....... 56 In Vivo Half Life of Loss of GSTZ1 Activity ................................ ....................... 56 GSTZ1 Protein Expression in Cytosol and Mitochondria ................................ .. 57 Correlation Analysis of GSTZ1 Activity and Protein Expression ....................... 58 In Vitro Half Life of GSTZ1 Inactivation with Cl ................................ ................ 58 Cl Effect on Attenuating GSTZ1 Inactivati on by DCA ................................ ...... 59 GSH Determination in Rat Cytosol and Mitochondria ................................ ....... 59 Discussion ................................ ................................ ................................ .............. 60 3 LOSS OF MALEYLAC ETOACETATE ISOMERASE ACTIVITY IN RATS FOLLOWING DICHLOROACETATE TREATMENT ................................ ............... 80 Specific Aim ................................ ................................ ................................ ............ 80 Materials and Methods ................................ ................................ ............................ 80 Chemicals ................................ ................................ ................................ ......... 80 Animals, DCA Admin istration, and Hepatic Subcellular Fractionation .............. 81 Activity Measurement with DCA as Substrate ................................ .................. 81 Western Blot Analysis of Cytosolic and Mitochondrial GSTZ1 Expression ....... 81 Activity Measurement with MA as Substrate ................................ .................... 81 GSH Adduct Determination ................................ ................................ .............. 83 LC MS Monitoring of Adduct Formation ................................ ........................... 83 Statistical Analysis ................................ ................................ ............................ 85 Results ................................ ................................ ................................ .................... 85 Rat GSTZ1 Activity D etermination with MA as Substrate ................................ 85 Half Life of Loss of Cytosolic GSTZ1 Activity with MA as Substrate ................ 86 Correlation Analysis of Activity with DCA and MA as Substrates ..................... 86 Correlation Analysis of Activity with MA as Substrate and GSTZ1 Protein Expression ................................ ................................ ................................ .... 87 GSH Adduct Formation ................................ ................................ .................... 87 Discussion ................................ ................................ ................................ .............. 89 4 ROLE OF EXTRAHEPATIC GLUTHATHIONE TRANSFERASE ZETA 1 IN DICHLOROACETATE BIOTRANSFORMATION ................................ .................. 10 6 Specific Aim ................................ ................................ ................................ .......... 106 Materials and Methods ................................ ................................ .......................... 107 Rat Tissues ................................ ................................ ................................ .... 107 GSTZ1 Expression ................................ ................................ ......................... 107 GSTZ1 Acti vity and Kidney Inhibition Assays ................................ ................. 107

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8 GSH Determination ................................ ................................ ........................ 108 glutamyl transpeptidase (GGT) Activity ................................ ....................... 108 GGT Inhibition ................................ ................................ ................................ 108 Statistical Analysis ................................ ................................ .......................... 109 Results ................................ ................................ ................................ .................. 109 GSTZ1 Activity and Expression in Extrahepatic Tissues ................................ 109 GSTZ1 Inhibitory Factor Present in Kidney Mitochondria ............................... 110 GSH Determination in Rat Kidney Mitochondria ................................ ............. 111 GGT Activity Determination and Inhibition by Azaserine ................................ 111 Discussion ................................ ................................ ................................ ............ 111 5 CONCLUSION ................................ ................................ ................................ ...... 119 LIST OF REFERENCES ................................ ................................ ............................. 125 BIOGRAPH ICAL SKETCH ................................ ................................ .......................... 134

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9 LIST OF TABLES Table P age 1 1 DCA pharmacokinetics in humans and rats. ................................ ....................... 49 2 1 Average GSTZ1 activity measured in DCA treated rat cytosol and mitochondria. ................................ ................................ ................................ ...... 76 2 2 Average GSTZ1 relative expression levels in DCA treated rat cytosol and mitochondria. ................................ ................................ ................................ ...... 77 2 3 Measured Cl levels in rat liver cytosol and mitochondria. ................................ .. 78 2 4 Measured GSH and GSSG levels in rat liver cytosol and mitochondria. ............ 79 3 1 Optimized conditions for monitoring adduct formation between GSH and FA by LC MS in negative ion mode. ................................ ................................ ....... 102 3 2 Average MAAI activity with M A, GSTZ1 activity with DCA, and substrate activity ratio in DCA treated young and adult rat cytosol. ................................ 103 3 3 Average MAAI activit y with MA, GSTZ1 activity with DCA, and substrate activity ratio in DCA treated young and adult rat mitochondria. ........................ 104 3 4. Adduct formation between GSH and FA in adult rat cytosol and mitochondria. 105

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10 LIST OF FIGURES Figure P age 1 1 DCA interaction with the mitochondrial pyruvate dehydrogenase complex. ....... 44 1 2 Influence of mit ochondrial energy production in solid tumors. ............................ 45 1 3 GSTZ1 catalyzed reactions. ................................ ................................ ............... 46 1 4 DCA dechlorination by GSTZ1 to glyoxylate and other metabolites. .................. 47 1 5 Phenylalanine and tyrosine catabolism. ................................ ............................. 48 2 1 GSTZ1 activity remaining up to 24 h after a sing le dose of DCA was administered to rats. ................................ ................................ ........................... 68 2 2 In vivo half life of loss of rat GSTZ1 activity following treatment with DCA was d etermined. ................................ ................................ ................................ ......... 69 2 3 GSTZ1 protein loss with time after DCA treatment in young rats. ...................... 70 2 4 GSTZ1 expression remaining over a 24 h peri od after a single dose of DCA. ... 71 2 5 Relationship between GSTZ1 activity and relative protein expression. .............. 72 2 6 In vitro half life of rat GSTZ1 inactivation following incubation with 2 mM DCA was determined in the presence of physiologically relevant Cl ......................... 73 2 7 Effect of Cl concentration on DCA induced GSTZ1 inactivation in matching rat hepatic fractions with 2 mM DCA. ................................ ................................ 74 2 8 Cytosolic GSH measurements in control rats and DCA treated rats. ................. 75 3 1 Synthesis of maleylacetone (MA). ................................ ................................ ...... 95 3 2 GSTZ1 activity remaining after DCA treatment to rats with either DCA or MA as the substrate. ................................ ................................ ................................ 96 3 3 Positive relationship between GSTZ1 activity with MA as the substrate and DCA. ................................ ................................ ................................ ................... 97 3 4 Relationship between GSTZ1 activity with MA as the substrate and relative protein expression. ................................ ................................ ............................. 98 3 5 Addition product formation between GSH and FA by UV spectrometry ............. 99 3 6 Negative product ion spectrum of adduct formation between GSH and FA. ..... 100

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11 3 7 LC MS spectrum of non enzymatic adduct formation between GSH and FA. .. 101 4 1 Extrahepatic GSTZ1 expression and activity in rats ................................ ........ 115 4 2 Protein in rat kidney dramatically reduces GSTZ1 activity with DCA. ............... 116 4 3 Michaelis Menten curve of GSH consumpti on in rat kidney mitochondria. ....... 117 4 4 Effect of azaserine on the inactivation of GGT activity in rat kidney mito chondria. ................................ ................................ ................................ .... 118

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12 LIST OF ABBREVIATIONS ALA delta aminolevulinate ATP adenosine triphosphate CDNB 1 chloro 2,4 dinitrobenzene Cl chloride DCA dichloroacetate FA fumarylacetone FAA fumarylacetoacetate FADH2 flavin adenine dinucleotide, hydroquinone form FAH fumarylacetoacetate hydrolase GGT glutamyl transpeptidase GSH reduced glutathione GSSG oxidized glutathione GST glutathione transferase GSTZ1 glutathione transferase zeta 1 HTT1 hereditary tyrosinemia type 1 Km Michaelis constant MA maleylacetone MAA maleylacetoacetate MAAI maleylacetoacetate isomerase MAPEG membrane associated proteins in eicosanoid and glutathione metabolism MELAS mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke like episodes MTP mitochondrial transition pore NaDCA sodium dichloroacetate

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13 NADH nicotinamide adenine dinucleotide NTBC 2 (2 nitro 4 trifluoromethylbenzoyl) cyclohexane 1,3 dione PAH pulmonary arterial hypertension PDC pyruvate dehydrogenase complex PDK pyruvate dehydrogenase complex kinase ROS reactive oxygen species SA succinylacetone SD Sprague Dawley rats SNPs single nucleotide polymorphisms 14 SSC 16 serine 14, serine 15, cysteine 16 motif in GSTZ1 active site TCA trichloroacetic acid Vmax maximum enzyme rate Vmax/Km enzyme efficiency

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14 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy FACTORS MODULATING DICH LO R O ACETATE INDUCED GLUTATHIONE TRANSFERASE ZETA 1 INACTIVATION IN A RAT MODEL By Marci Gayle Smeltz May 2017 Chair: Margaret O. James Major: Pharmaceutical Sciences Dichloroacetate (DCA) is an investigational drug that also exists in the environment as a by product from water chlorination. In the mitochondria, DCA reactivates energy metabolism and is undergoing investigation as a n anticancer therap eutic Chronic dosing of DCA leads to inactivation of the enzyme solely responsible for its biotransformation, glutathione transferase zeta 1 (GSTZ1). Numerous intrinsic factors can influence this drug indu ced inactivation. In this study, we utilized a rodent model administered a single dose of DCA to investigate the time course of inactivation of liver cytosolic and mitochondrial GSTZ1 over 24 h. We measured enzyme expression and activity with DCA and its endogenous substrate, maleylacetone (MA ), in 4 and 52 wk old rats assessed the impacts of chloride (Cl ) on this drug enzyme interaction, and investigated the expression and activity of GSTZ1 in extrahepatic sites. We demonstrated that rat hepatic mito chondrial GSTZ1 is more susceptible to DCA induced GSTZ1 inactivation than that in the cytosol. In DCA treated rats, hepatic GSTZ1 activity was reduced by more than 90% 4 h after a single dose, and mitochondria l GSTZ1 showed greater loss of than cytosolic GSTZ1 With MA as the substrate, enzyme activity was much greater, up to 116 times compared to when DCA

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15 was used as the substrate. F ollowing DCA exposure, GSTZ1 a ctivity with MA as the substrate reduced to a similar extent as when DCA was the substrate Young rats recovered more activity than old rats by 24 h after DCA administration. More rapid GSTZ1 inactivation occurred in adult rats with faster half lives of loss of GSTZ1 activity in both the cytosol and mitochondria compared to young rats In vi tro studies showed that h igher Cl concentrations attenuated GSTZ1 inactivation, especially in young rats Finally, despite much lower GSTZ1 enzyme expression, kidney brain, and heart exhibit ed DCA metabolizing capabilities. Kidney homogenate had lower activity than expected from the measured GSTZ1 expression. It was later identified that gamma glutamyl transpeptidase was responsible for rapid consumption of glutathione leading to low GSTZ1 activity. In conclusion, this rod ent model provided insight into factors that modulate DCA induced GSTZ1 inactivation including age, Cl and cellular location, and demonstrated extrahepatic GSTZ1 expression.

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16 CHAPTER 1 INTRODUCTION Dichloroacetate A Small Molecule With a molecular weight of 129 Da, dichloroacetic acid, or more commonly referenced in literature as the anion, dichloroacetate (DCA), is also known as dichloroethanoic acid (Stacpoole, 1989). The chemical formula for DCA is Cl 2 CHCOO DCA is typically provided as a sodium salt formation (NaDCA), has a bitter taste, is odorless, and is stable in both solid and aqueous states. Physiochemic ally, the dissociation constant of the free acid is 5.50 x 10 2 equating to a pK a of 1.26. A unique property of DCA is the contrasting capacities of this small molecule, being present in the environment with possible adverse health effects as well as h aving therapeutic potential in numerous medical conditions for improved health outcome (Stacpoole, 2011). A n Environmental Toxi ca n t DCA has been documented in n umerous environmental sources over the last few decade s. Human exposure to DCA can resul t from consuming chlorinated drinking water and food and from in vivo metabolism of numerous xenobiotics and drugs. As a by product of water chlorination, DCA detection levels in municipal drinking water in the United States are as high as 160 g/L (Gonza lez Leon et al., 1997; Stacpoole et al., 1998a). Estimated daily human exposure to DCA is 2 4 g/kg body weight via drinking water and DCA can be present as a disinfection by product in meats and other food products ( Stacpoole et al., 1998a ). On the bas is of results from rodent toxicology studies the United States Environmental Protection Agency set a limit in drinking water for five halogenated acetic acids, including DCA, to be 60 g/mL (Villanueva et al.,

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17 2014). DCA can be produced in the human body as a metabolite from various industrial solvents, including tetrachloroethylene and trichloroethylene, from release into the atmosphere to contaminate surface water (Stacpoole, 2011) Additionally, DCA can result as a metabolite of therapeutic drugs a nd agents, like chloral hydrate Despite the available data that document relevant sources of DCA exposure to humans, quantifying the c ontribution of each source remai ns to be determined. A working group of the International Agency for Research on Cancer con cluded that DCA is a possible human carcinogen (Group 2B) based on animal model f indings of liver cancer in sev eral strains of inbred rodents given high levels of DCA ( IARC, 2006 ). Evidence also show ed that several organs are susceptible targets for DCA i n rodents and dogs, including the kidney and nervous system ( Bull 200 0 ). However, there is disagreement on the true classification of DCA as a possible human carcinogen since rodents are the basis for this finding and differ in certain aspects related to DCA and target protein interactions in particular with peroxisome proliferator activated receptor alpha Other studies and reviews have examined the health risks of DCA at environmental levels do not support the conclusion that DCA is a significant c arci nogenic hazard to humans (Stacpoole, 2011). A Therapeutic Agent Research over the last half century evaluated the pharmacological effects of DCA for use in treating several metabolic and cardiovascular diseases, initially investigating applications for diabetes, hyperlipoproteinemia, and lactic acidosis (Stacpoole, 1989). Administration of NaDCA to rodents and patients resulted in lowered glucose, lactate, and pyruvate conce ntrations, indicating the potential of DCA as an

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18 antidiabetic agent as well as for treating lactic acidosis (McAllister et al., 1973; Blackshear et al., 1974; Stacpoole et al., 1978; Stacpoole and Greene, 1992). Within the last two decades, DCA has been extensively examined in several clinical trials for genetic mitochondrial disea s e s, such as congenital lactic acidosis; pyruvate dehydrogenase complex deficiency; and mitochondrial myopathy, encephalopathy, lactic acidosis and stroke like symptoms (MELAS) (Barshop et al., 2004; Berendzen et al., 2006; Kaufmann et al., 2006; Stacpool e et al., 2006; Stacpoole et al., 2008a). DCA also recently emerged as a metabolic modulator S everal scientific studies and early clinical phase trials identified that DCA exhibit ed anti proliferative and pro apoptotic effects in pulmonary arterial hype rtension (PAH) and several different cancers, including glioblastoma, non small cell lung cancer, and breast cancer (McMurty et al., 2004; Bonnet et al., 2007; Michelakis et al., 2008; Michelakis et al., 2010; Dunbar et al., 2014; Kankotia and Stacpoole, 2 014). The mechanism of action of DCA in the treatment of such diseases involves the pyruvate dehydrogenase complex (PDC), which is required for fuel homeostasis. This multi enzyme complex is within the inner mitochondrial membrane and converts pyruvat e to acetyl CoA, essential compounds for glycolysis, the Krebs cycle, and cellular respiration (Stacpoole et al., 1998b). In the cytosol, pyruvate is converted to lactate and alanine. PDC regulation is by reversible phosphorylation through PDC kinase (PD K) and PDC phosphatase (Holness and Sugden, 2003). PDK is a mitochondrial kinase that controls aerobic oxidation of carbohydrate sources I n mammals, four different forms of PDK exist (PDK1, PDK2, PDK3, and PDK4) that regulate PDC in a tissue specific ma nner (Berendzen et al., 2006; Klyuyeva et al.,

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19 2007). PDK2 is present in all tissues, being the most sensitive to DCA (Sutendra and Michelakis, 2013). DCA acts as an inhibitor to PDK, maintaining PDC in a catalytically active un phosphorylated sta te (Figure 1 1). Inhibition of PDK by DCA occurs in a dose dependent fashion to stimulate PDC (Sutendra and Michelakis, 2013), and crystallographic data suggest this is a result of DCA occupying the pyruvate binding site of the regulatory domain of this m ulti enzyme complex (Klyuyeva et al., 2007). This leads to the irreversible oxidation of pyruvate to acetyl CoA to increase the delivery of pyruvate into the mitochondria for increased energy production (Stacpoole, 1989). In turn, this causes a reduction in lactate and alanine levels. Reducing equivalents of NADH and FADH 2 also form which stimulates the respiratory chain to generate A TP and reactive oxygen species ( ROS). A bnormalities in cellular energy metabolism may play major roles in carcinogenesis and tumor progression (Seyfried et al., 2014). Classically, cancer cells are characterized as having a glycolytic phenotype through maintaining bioenergetic requirements wit h increased release of lactate via dysfunctional cellular respiration and reduced glucose oxidation, known as the Warburg effect (Warburg et al., 1927; Michelakis et al., 2008). Inhibition of the PDC has been shown to decrease mitochondrial ROS production cause hyperpolarization of the mitochondrial membrane, close the mitochondrial transition pore (MTP), reduce apoptosis, and increase proliferation result ing from aerobic glycolysis (Figure 1 2) (Hsu and Sabatini, 2008; Wallace, 2012). Cell death is char acteristically associated with R O S generation and its harmful effects to the body. When abnormal ROS production results, it becomes an

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20 important factor for mediating apoptosis by overexpressing proteases, notably caspases, involved in this response (Glush akova et al., 2011). Such stress is a target in disease treatment, like cancer, for inducing ROS generation for improved outcome (Hu et al., 2005). DCA can reverse the Warburg effect to reactivate mitochondrial energy metabolism and restore apoptosis to cancer cells by decreasing lactate formation in several types of cancer (Bonnet et al., 2007; Michelakis et al., 2008; Wong et al., 2008; Madhok et al., 2010; Michelakis et al., 2010; Dunbar et al., 2014). Similarly, PAH is a disease resulting from right heart failure and elevated pulmonary vascular resistance and has benefited from DCA administration (McMurty et al., 2004). In rats, DCA prevented and reversed symptoms of PAH by inducing mitochondrial dependent apoptosis. Because of such action to resto re mitochondrial energy, investigation continues to identify potential DCA use for treatment in these two diseases (Michelakis et al., 2008; Piao et al., 2013 ; Kankotia and Stacpoole, 2014 ; James et al., 2017 ). Because of its small size and solubility in w ater, DCA has the ability to cross many cellular membranes, including the blood brain barrier (Stacpoole, 1989). Therapeutically, the dose of DCA provided to children and adults with acquired and inborn errors of metabolism can be thousands of times great er than exposure from the environment. Typical clinical administration of DCA is provided either intravenously or orally at a dose between 10 100 mg/kg body weight/day and has been used for patient treatment for several days to more than a decade (Stacpoo le et al., 2008a). DCA has nearly 100% absorption after oral administration and is metabolize d with little urinary recovery of the parent drug (James et al., 1998; Stacpoole et al., 1998b). T oxicity has

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21 been observed with chronic DCA administration. Thi s often presents as reversible elevation of liver transaminases and peripheral neuropathy ; the latter is the main adverse effect that limits use of DCA (Stacpoole et al., 2008b). This continues to be a major focus of current DCA research. Glutathione Tra nsferases Responsible for phase II reactions, glutathione transferases (GSTs) are essential for the metabolism and detoxification of both endogenous compounds and xenobiotics, like drugs and environmental contaminants (Nebert and Vasiliou, 2004). However, some interactions with molecules can lead to increased toxicity. GSTs catalyze phase II conjugation of reduced glutathione (GSH or glutamyl cysteinyl glycine) with a range of electrophilic and non polar substrates, forming a water soluble GSH conjugate that can be transported out of the cell through various efflux pumps (Hayes et al., 2005; Board and Menon, 2013). Common substrates for these GSH dependent reactions include 1 chloro 2 4 dinitrobenzene (CDNB) and ethacrynic acid. While a major role of GSTs is to catalyze conjugation reactions, these enzymes can also catalyze addition elimination and isomeri zation reactions and can reversibly bind several endogenous substrates (Hayes et al., 2005). The catalytic potential of GST activity results fr om its capacity to lower the pK a of the sulfhydryl moiety of GSH from 9.0 in aqueous solution to approximately 6.5 when GSH bonds to the active site of GSTs, producing a thiolate anion (GS ) near neutral pH (Nerbert and Vasiliou, 2004). Through the format ion of GS catalysis by GST results in the formation of a bond with electrophilic substrates. Two sites within an individual GST allow binding of GSH (G site) and hydrophobic substrates (H site), where the active enzyme exists as a dimer with subunits be tween 23 30 kDa (Board and Menon, 2013).

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22 Despite similarities across all forms of GSTs, the active site of this protein, including the G site and H site, demonstrate significant differences, le a ding to substrate selectivity and reaction variations. Within the GST superfamily, four structurally different gene families exist: cytosolic or soluble GSTs, mitochondrial GSTs, membrane associated proteins in eicosanoid and glutathione metabolism (MAPEG), and prokaryotic fosfomycin resistance proteins (Board and M enon, 2013). Literature places the m ost emphas is on the soluble GST family, which accoun ts for 1 0% of total cellular protein in the liver (Board and Menon, 2013). Based on the complete amino acid sequences deciphered by cDNA clones, seven distinct mammal ian cytosolic GSTs exist to date: alpha, mu, pi, sigma, theta, omega and zeta. Evidence has shown that several members of the cytosolic GSTs are also present in the mitochondria, including the recently identified enzyme, glutathione transferase zeta 1 (L i et al., 2011; Board and Menon, 2013). Glutathione Transferase Zeta 1 From the exploitation of bioinformatics and human expressed sequence tag databases, the zeta class of GSTs was uncovered within the last quarter century, positively identified in the cytosol and mitochondria of humans as well as numerous eukaryotic species plants, and fungi (Board et al., 1997; Board and Anders, 2005; Li et al., 2011). G lutathione transferase zeta 1 (GSTZ1) is a peptide of 216 residues and ha s a predicted mol ecular weight of 24.2 kDa. One gene has been identified for GSTZ1 in humans, located on chromosome 14q24.3 (Blackburn et al., 1998). To date, only one subunit of the zeta class exists so enzyme notation is as the homodimer (Polekhina et al., 2001). Imm unohistochemical detection revealed that GSTZ1 mainly is expressed in

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23 the liver for both humans and rats, with l ow er levels in the brain and other organs (Lantum et al., 2002a). Two published crystal structures for human GSTZ1 exist and provide important insight into the molecular interactions of this protein (Polekhina et al., 2001; Boone et al., 2014). Controversy continues to persist as to the exact human recombinant form in the Polekhina report; in the literature, hGSTZ1A is the cited crystal structure but data align more closely with hGSTZ1B. Recently, x ray crystallographic analysis identified a preliminary structure for hGSTZ1A and will be useful in further assessing characteristics of this enzyme (Boone et al., 2014). Lik e other cytosolic GSTs, GSTZ1 adopts a canonical fold that assembles as a dimer Deep within a crevice between the N and C terminals, the active site is present (Blackburn et al., 1998; Board et a l., 2003; Board and Anders, 2005 ). T he active site attrac ts negatively charged compounds, like DCA. Specifically, the two serine residues in the characteristic motif are believ e d to interact with GS for catalytic function. Serine 14 has the greatest effect on DCA activity because of its favorable orientation. Cysteine 16 also can bind to GSH, providing another essential anchor of this enzyme as modification of this residue leads to alterations in GSTZ1 function (Board et al., 2003). A third characteristic feature of GSTZ1 is the salt bridge that forms betwee n arginine haloacids, like DCA. This is critical for attack of GSH on carbon and halide displacement (Polekhina et al., 2001). Such features play e residues of the 14 SSC 16 motif.

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24 Unlike other GSTs, GSTZ1 does not catalyze the conjugation of common GST substrates, like CDNB (Tong et al., 1998a). Initial studies showed that DCA is convert ed to g lyoxylate in the presence of GSH in both rat and human l iver cytosol (Figure 1 3A) (Lipscomb et al., 1995; James et al., 1997). GSTZ1 catalyzes the haloacids, including DCA, bromochloroacetic acid, bromofluoroacetic acid, chlorofluoroacetic acid, dibromoacetic acid, 2 bromopropi onic acid, 2 chloropropionic acid, and 2 iodopropionic acid, but not haloacids that solely contain fluorine (Tong et al., 1998b). T his transformation is GSH dependent and solely catalyzed by GSTZ1. Research also indicated that GSTZ1 catalyzes impor tant homeostatic reactions, where GSTZ1 is identical to maleylacetoacetate isomerase (MAAI) (Blackburn et al., 1998). MAAI catalyzes the next to last step in the phenylalanine and t yrosine catabolic pathway, where p roducts of this pathway enter the citric acid cycle. Specifically, MAAI/GSTZ1 catalyzes the cis trans isomerization of maleylacetoacetate (MAA) to fumarylacetoacetate (FAA) and maleylacetone (MA) to fumarylacetone (FA) around a carbon double bond (Figure 1 3B and Figure 1 3C). This reaction re quires, but does not consume GSH. As with DCA metabolism, the mechanism of this isomerization involves enzyme catalyzed nucleophilic addition of GSH to MAA or MA to give an intermediate acid, then bond rotation and GSH elimination with formation of the trans double bond to yield FAA and FA, respectively (Board and Anders, 2005). Unfortunately, enzyme dysfunction within phenylalanine and tyrosine catabolism has been associated with many metabolic disorders, yet specific impairments of MAAI have not direc tly correlated with any known

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25 conditions (Blackburn et al., 1998; Stacpoole et al., 2008b). However, o ne recent report suggests that a deficiency in MAAI may pla y a role in hypersuccinylaceton emia (Yang et al., 2016). Elevated levels of succinylacetone ( SA), a by product of tyrosine catabolism, is a biomarker for hereditary tyrosinemia type 1, a severe autosomal disease from the deficiency of another enzyme in the degradation of tyrosine. In this study, an individual predict ed to possess low MAAI /GSTZ1 activity had the highest levels of SA, implying a relationship for hypersuccinylacetonemia development. Such results suggest that a deficiency in MAAI may produce liver or kidney failure, but additional clinical investigations are necessary for confirmin g these observations. Because of the selective natur e of GSTZ1, this enzyme only biotransform s MAA haloacids, including DCA (Tong et al., 1998b). Enzyme catalysis utilizes the carbon alpha to the carboxylic acid moie ty of these halogen containing acids, which must be unblocked for proper activity with GSH (Board and Anders, 2005 ). Although GSTZ1 has the ability to biotransform such halogen containing carboxylic acids, the efficiency of isomerization for the intermedi ates of the phenylalanine and tyrosine cat abolic pathway is much greater with specific activity hundreds to thousands of times larger than with DCA or other substrates (Board and Anders, 2005). DCA Biotransformation by GSTZ1 and Pharmacokinetics After oral and intravenous administration, GSTZ1 dechlorinates DCA in the liver to its primary and inactive metabolite, glyoxylate (Lipscomb et al., 1995; James et al., 1997; Board and Anders, 2005). DCA peak plasma concentrations often occur within 30 min after ad ministration (Stacpoole et al., 1998b). Tissue distribution of DCA is dose dependent, being found mainly in the rat liver and muscle with 16% and 23% of a

PAGE 26

26 radio labelled DCA dose, respectively (Lin et al., 1993; James et al., 1998). G lyoxylate can be subs equently transform ed into glycine and related conjugates by transamination and carbon dioxide by carboligase action (Figure 1 4 ) (Stacpoole, 1989; Lin et al., 1993; James et al., 1998 ; Tong et al., 1998a). Minor pathways are oxidation to oxalate and reduc tion to glycolate P lasma expired air, and urine serve as the primary collection and detection site of these metabolites Principally, exhalation of CO 2 and elimination in the urine are the two major excretion pathways (Lin et al., 1993). Less than 1% of the administered dose reduces to monochloroacetat e a potential neurotoxin, in the blood and only plays a minor role in the metabolism and toxicity of DCA (Shroads et al., 2008). Mechanistically, the first step in GSTZ1 catalyzed metabolism of DCA occurs by displacing one chlori n e of DCA with GSH via a S N 2 reaction to form S chlorocarboxymethyl) glutathione (Figure 1 4 ) (Polekhina et al., 2001; Anderson et al., 2002). The second chlorine of DCA is then lost to form a proposed unstable carbonium sulfonium intermediate Via S N 1 like hydrolysis, this unstable intermediate then decomposes to form glyoxylate and release GSH. Predictions also indicated that an inactivated complex with GSTZ1 could form from the interaction with the carbonium sulfoniu m complex, which may affect the ability of GSTZ1 to metabolize DCA (Anderson et al., 1999). GSTZ1 Inactivation Induced by DCA Not only is DCA a substrate for GSTZ1, it is also a mechanism based inactivator of GSTZ1. Early studies showed a progressive red uction in GSTZ1 activity with DCA exposure. Studies in rodents demonstrated a time and dose dependent effect on reducing the protein expression levels and activity of GSTZ1, by measuring the

PAGE 27

27 production of glyoxylate in liver cytosol (Anderson et al., 199 9; Cornett et al., 1999). Five days of administering DCA at low doses (4 and 12.5 mg/kg body weight/day) significantly decrease d GSTZ1 activity. A single dose of 50 mg/kg body weight to male Sprague Dawley (SD) rats resulted in more than 60% reduction of GSTZ1 activity 24 h later. In rats provided 1 g/kg body weight of DCA the formation of glyoxylate was decreased by 93% (Cornett et al., 1999). Recovery times of GSTZ1 activity seem dependent on the length and dose of DCA administered. In male Fischer 344 rats given DCA by intraperitoneal injection, GSTZ1 quickly degraded and did not return to initial activity measures for more than ten days, with a GSTZ1 synthesis half life of 3.3 days (Anderson et al., 1999). Male SD rats given 50 mg/kg body weight in drinking water for an extended period had much slower recovery of GSTZ1 compared to animals given DCA at an environmentally measured dose (Guo et al., 2006) It was proposed that recovery of GSTZ1 activity preceded protein expression in those given the higher dose of DCA yet further investigation is needed to evaluate the cause of this effect Despite known differences in the distribution of DCA and its metabolites in tissues, several studies reported that repeated DCA do sing prolongs the terminal elimination half life i n rat, human, and other species (Curry et al., 1991; James et al., 1998; Shroads et al., 2008; Maisenbacher et al., 2013). One study examined DCA kinetics of children diagnosed with congenital lactic acido sis, where the half life dramatically increased from 0.74 h at the initiation of the study to 7.47 h by month nine (Stacpoole et al., 1998b). A second study also showed a similar phenomenon with drug nave children having much higher plasma DCA concentrat ions after a six month

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28 regimen of DCA (Shroads et al., 2008). This prolong ed elimination half life is believed to result from the inactivation of GSTZ1 (Anderson et al., 1999; Tzeng et al., 2000). Other in vitro experiments confirmed the ability o f DCA to inhibit GSTZ1, in time and DCA concentration dependent manners in human, rat, and mouse liver cytosol (Tzeng et al., 2000). It appears that the inactivation of GSTZ1 activity by DCA depends on the species. For rat, mouse, and human, the in vitr o inactivation half life for GSTZ1 with DCA in the absence of chloride ( Cl ) was 5.44, 6.61, and 22 min, respectively, yet the half maximal inhibitory concentration did not differ between species (Tzeng et al., 2000). Although this study exhibited differe nces in the ability of GSTZ1 to biotransform DCA between species this does not completely describe DCA induced GSTZ1 inactivation. A concurrent study showed that little inactivation resulted in human and rat liver cytosol in the presence of Cl (Cornett et al., 1999). Subsequent investigations have revealed that Cl has a protective effect on GSTZ1 inactivation (Zhong et al., 2014) C ovalent modification of GSTZ1 and/or the production of a GSTZ1 protein adduct tivation in both humans and rats (Anderson et al., 2004). This adduct could form between the nucleophilic cysteine 16 residue of the active site of GSTZ1 and the unstable carbonium sulfonium intermediate of DCA, leading to a covalently modified and inacti vated enzyme that undergoes degradation to cause GSTZ1 inactivation (Figure 1 4 ) (Anderson et al., 1999; Tzeng et al., 2000; Polekhina et al., 2001). Additionally, reported adducts contain ed GSH and glyoxylate with serine 14 modification (Dixit, 2005). T o identify this mechanism of inactivation and subsequent protein loss more investigation is needed

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29 Physiological Substrate Inhibition of MAAI /GSTZ1 Since GSTZ1 is identical to MAAI, perturbation of the phenylalanine and tyrosine catabolic pathway can result after DCA administration or from other intermediates in this pathway (Figure 1 5 ) MA is very active with MAAI and is a c ytotoxic metabolite of tyrosine (Seltzer and Lin, 1979; Lantum et al., 2002b; Lantum et al., 2003). MA forms after decarboxyl ation of labile MAA and is isomerized to trans FA by GSTZ1. Research implies that t hese products are linked to observed toxicities. Competition exists between DCA and MA, occurring in a mixed or non competitive fashion where DCA induced changes in MA ex cretion in a dose dependent manner (Cornett et al., 1999; Lantum et al., 2003). S tudies showed that incubation of rat and human hepatic cytosol with MA inhibited the formation of glyoxylate from DCA in a concentration dependent manner in vitro having a m ean IC 50 value of 264 M MA for rat liver cytosol and 125 M MA for human liver cytosol (Cornett et al., 1999). The concentration of MA in the urine of rats given DCA at a dose of 50 1000 mg/kg body weight increased in a dose dependent manner confirming that DCA exerts significant alterations in phenylalanine and tyrosine catabolism by inhibiting MAAI action. In vivo studies also indicated that DCA depletes MAAI activity, inhibiting isomerization of both MAA and MA. After five days of treating male SD rats at different DCA doses, a 37% reduction in activity occurred as well as a 2.5 fold decrease in protein abundance haloacids also have the ability to reduce GSTZ1 activity in rats, including 2,2 dichloropropionate and dib romoacetate (Anderson et al., 1999). Despite the changes in MAAI / GSTZ1 activity and protein expression, DCA does not affect steady state mRNA levels and such alterations in this protein are likely to occur post translationally from predicte d adduct formation

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30 (Anderson et al., 1999; Ammini et al., 2003; Guo et al., 2006). GSTZ1 activity recovery in humans chronically administered DCA is similar to rats, taking over three months in healthy individuals provided multiple doses of DCA (Curry et al., 1991). Likewise, MA and FA have the ability to inhibit the conversion of DCA to glyoxylate in human liver cytosolic GSTZ1 with chlorofluoroacetate as the substrate, and these two compounds act as non mechanism based inactivators of GSTZ1 (Lantum et al., 2002b; Lantum et al., 2002c). This is likely to result from alkylation and adduct binding to cysteine 16 and cysteine 205 of GSTZ1. Interestingly, GSH ha s a protective effect against MA and FA inactivation by blocking the binding of these compoun ds to cysteine 16 in the active site for GSTZ1 (Lantum et al., 2002b). Both of these studies demonstrate the ability for MA and FA to covalently modify and inactivate proteins containing cysteine residues. Collectively, these data show that physiological substrate interactions with this enzyme contribute to DCA induced toxicities and play a role in perturbing phenylalanine and tyrosine catabolism. Role of GSTZ1 in Mitochondria GSTZ1 has two cellular locations within the liver, positively identified in t he mitochondrial matrix and cytosol (Li et al., 2011). A pproximately 14% of total hepatic GSTZ1 is in the mitochondria (Li et al., 2011). The expression level in the mitochondria is much lower than in the cytosol (at least two fold lower), which is consi stent with lower GSTZ1 activity N o detection of GSTZ1 was observed in the hepatic microsomal fraction from SD rats. DCA is transported into the mitochondrial matrix through the monocarboxylate transporter system and a sodium coupled monocarboxylate trans porter to then gain access in to the mitochondria (James et al., 2017) As the mitochondria it is important to understand the

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31 critical role of mitochondrial GSTZ1 in DCA biotransformation and the therapeutic potentia l of DCA Several other classes of cytosolic GSTs are in the mitochondria, including alpha, mu, and pi isoforms (Raza, 2011). It is hypothesized that the presence of such GSTs in different compartments is an evolutionary response for cell protection to toxicity, GSH maintenance, and oxidative stress. In many of these GSTs having dual locations, similar molecular sizes and N terminal sequences exist between the cytosol ic and mitochondrial forms GSTZ1 also possesse s a similar molecular weight to other GSTs by the rate of migration by SDS PAGE (Li et al., 2011). Proteomic analysis for GSTZ1 also showed that the amino acid sequence of rat and human mitochondrial matrix GSTZ1 were identical to formerly published cytosolic sequences based on the identifica tion of three tryptic peptides, spanning 12% of the entire sequence (Li et al., 2011). In particular, the N terminus was blocked in human cytosolic and mitochondrial GSTZ1, while the C terminus shared the same identity in the mitochondrial form. This sug gests that a potential targeting sequence for GSTZ1 would exist within the mature protein. Kinetic studies in rats showed identical app K m values for DCA in cytosolic and mitochondrial GSTZ1 fractions, yet dissimilar app K m values for GSH (0.5 mM in mitochondria and 0.19 mM in cytosol) (Li et al., 2011). This implies that the binding site of GSH in GSTZ1 differs in these two fractions and may be a result of post translational modifications. However, cytosolic and mitochond rial GSH is present at concentrations (10 14 mM) far above that of the measured K m value (Mari et al., 2013), so the rate of DCA biotransformation should not be limited in either subcellular fraction at

PAGE 32

32 physiological conditions. For the mitochondrial form of GSTZ1, its higher app K m for GSH implies weaker binding access than the cytosolic form (Li et al., 2011). This kinetic data indicate that the two forms of GSTZ1 differ, which may be due to changes in the primary sequence or result from post translation al modifications of GSTZ1. The susceptibility of mitochondrial GSTZ1 to DCA inactivation remains an important unanswered question. Factors Influencing DCA Pharmacokinetics Since DCA inhibits its own metabolism, marked increases in area under the curve and elimination half life result after treatment with this small molecule, as previously described (Curry et al., 1991; James et al., 1998; Shroads et al., 2008). GSTZ1 polymor phisms, age, and intracellular Cl concentration appear to be the most influential determinants of DCA pharmacokinetics in humans (Stacpoole, 2011; Zhong et al., 2014). GSTZ1 Polymorphisms For many enzyme super families, the existence of genetic polymorphi sms occurs from non synonymous single nucleotide polymorphisms (SNPs) in the coding or regulatory region of a gene. Such changes in the genetic code lead to altered enzyme function, protein expression, or drug response. For GSTZ1, 17 SNPs occur in the co ding region of this enzyme (James et al., 2017) Four of these SNPs were reported by the expressed sequence tag database: Leu8Pro, Glu32Lys, Gly42Arg, and Thr82Met (Blackburn et al., 2000; Blackburn et al., 2001). T here was discovery of a n additional SNP but investigation of its occurrence remains incomplete (Shroads et al., 2012). Three of these SNPs are functionally important, leading to five major haplotypes (Blackburn et al., 2000). EGT, or GSTZ1C, is the most common haplotype, denoted as the wild type (50%). Decreasing order of the prevalence of the other haplotypes is KGT

PAGE 33

33 (1B; 28%), EGM (1D; 15%), KRT (1A; 7%), and KGM (1F; 0.4%), the rarest. One report of a mutation at nucleotide position 32 corresponds to the Leu8Pro mutation, denoted as GSTZ1 E, but no individuals with such a haplotype have been identified (Blackburn et al., 2001). Another report identified additional SNPs, but the occurrence of these haplotypes is low (Yang et al., 2016). Despite similar protein expression levels and cellul ar distributions, the four major variants of GSTZ1 influence catalytic activity (Blackburn et al., 2001; Li et al., 2012). A recent study confirmed the role of KRT in producing higher DCA metabolizing activity in vitro in liver cytosol, when paired with o r without the EGT haplotype (Li et al., 2012). Another study showed that KRT was more rapidly inactivated in the presence of Cl than EGT, KGM, and EGM in vitro (Zhong et al., 2014) I n the absence of Cl all four haplotypes were inactivated at similar rates A more recent examination of the significance of GSTZ1 haplotype on the kinetics and biotransformation of DCA in humans was completed in vivo (Shroads et al., 2012). Delayed clearance of DCA is apparent in individuals that are heterozy gous or homozygous for methionine substitution at amino acid position 88 (EGM, KGM), leading to slower rates of DCA kinetics and elevated plasma levels. On the other hand, EGT and KGT alleles exhibit rapid metabolism of DCA, and when KRT is paired with on e of these alleles, DCA pharmacokinetics is not altered. Yet, KRT homozygosity presents similar kinetic measures as individuals with at least one EGM or KGM allele. As a result, GSTZ1 haplotype variability can alter DCA kinetics and biotransformation as well as GSTZ1 function, thereby increasing the risk to certain populations to DCA and its precursors.

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34 Age A second important determinant of DCA pharmacokinetics is age. Age directly relate s to the clearance of DCA after chronic human exposure (Shroads e t al., 2008). Children diagnosed with congenital lactic acidosis (mean age of 5 y r ) and adults with MELAS (mean age of 24 y r ) were studied over six months with daily administration of 25 mg DCA/kg body weight. With increasing age, a dramatic decrease in DCA plasma clearance, a ten fold increase in elimination half life, and greater than 20 fold increase in area under the curve for the adult population resulted while children only had a three fold and four fold increase in the same pharmacokinetic paramet ers compared to the beginning of the clinical experiment (Table 1 1). This indicates that young er age confers greater tolerance to chronic DCA administration because of the faster elimination of DCA, while changes in DCA kinetics are more pronounced in ad ults with repeated dosing. Experiments investigating the relationship between age and DCA pharmacokinetics in rodents, on the other hand, have provided disparate responses. Using B6C3F1 mice, 60 wk old animals were unaffected by DCA treatment with similar toxicokinetic behavior as control mice, whereas 10 wk old mice were more sensitive to the changes induced by DCA administered in drinking water at concentrations from 0.05 g/L to 2.0 g/L (Schultz et al., 2002). Additionally tyrosine catabolism remained unaffected in these aged mice This study suggested that chronic administration of DCA to old (adult) mice compensated for GSTZ1 activity loss in the liver, while younger aged mice have more dynamic GSTZ1 activity inactivation and recovery. Therefore, yo ung age plays a larger role in toxicity

PAGE 35

35 In contrast a recent study that examined the effects of age in male SD rats had similar observations to those in humans (Shroads et al., 2008). T hree groups of different aged rats were used: young (5 7 wk), adul t (7 months), and old (15 months) (Table 1 1). Pharmacokinetic analysis showed that increasing age lead to a longer half life of elimination and increased area under the curve measurements. Likewise, the measured nave GSTZ1 activity was inversely relate d to age, where young rats had 1.4 fold higher activity than the old rats (1.60 v s 1.12 nmol/min/mg). However, a fter five days of DCA treatment at 50 mg/kg body weight, all three age groups exhibited almost a 90% decrease in GSTZ1 activity (0.1 0.2 nmol/m in/mg across all three age groups) and a reduction in protein expression. Lastly, studies have utilized human liver s from 10 w k gestation to 74 y r to examine the ontogeny of GSTZ1 (Li et al., 2012; Zhong and James unpublished). Age dependent changes in the activity and protein expression of GSTZ1 occurred in both the cytosol and mitochondria. GSTZ1 expression was undetectable in the fetus, but after birth began to increase with neonatal development. Chi ldren fr om 1 7 y r generally had lower GSTZ1 activity than those over the age of 7 yr (Li et al., 2012). C learance was significantly slower in adults with chronic DCA administration which may be due to the larger liver size compared to children. Despite extensiv e ontogeny analysis of GSTZ1, the cause of the apparently greater inactivation of GSTZ1 activity with age still requires further examination. Importantly, human GSTZ1 haplotypes and age may jointly play a significant role in understanding the pharmacokine tics of DCA Intracellular Cl Concentration Cl is the most abundant anion in organisms, and its electrochemical imbalance is necessary for numerous important physiological functions, including signaling (Duran et

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36 al., 2010). Cl channels are in both plasma membranes and intracellular organelles and provide a range of functions, such as pH, cell cycle, and cell volume (Jentsch et al., 2002). In human liver, Cl levels have been measured from 25.1 to 62.9 mM for adults under normal conditions, wi th mean concentrations of 42 mM (Widdowson and Dickerson, 1960; Jahn et al., 2015). Intracellular Cl concentration changes cause various inherited diseases, including Cystic Fibrosis, and i nfluenc e cell shape and volume in cancer through active transport of Cl along with other physiologically relevant cations (Duran et al., 2010; Cuddapah and Sontheimer, 2011). Physiological concentrations of anions, specifically Cl modulate the interaction between GSTZ1 and DCA (Zhong et al., 2014). Two former studies showed discrepancies in the inactivation of GSTZ1 after exposure to DCA in human and rat liver cytosol as described above (Cornett et al., 1999; Tzeng et al., 2000). T he presence of Cl prevented the inactivation of human GSTZ1 (Zhong et al., 2014 ). T he physiological concentration of Cl prolongs the half life of GSTZ1 inactivation after DCA exposure in vitro where Cl attenuated inactivation in a concentration and time dependent manner in human liver cytosol (Zhong et al., 2014). Bromide, iodi de, and sulfite also protected against DCA induced GSTZ1 inactivation in a concentration dependent manner, yet the concentration of these anions in the blood is orders of magnitude lower than Cl and only play minor roles (Zhong et al., 2014) Particularl y for Cl this protective effect occurs in a haplotype dependent manner in humans and may influence the rate of clearance. T he mechanism of the effect is not certain, but the nucleophilic properties of Cl may prevent adduct formation or even change GST Z1 protein conformation after anion binding to its active site.

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37 A recent study reported that Cl concentrations relate with age in humans (Jahn et al., 2015). Average c ytosolic and mitochondrial Cl concentration s in h uman live r samples from 1 day to 84 yr of age were 105.2 and 4.2 mM, respectively, when accounting for the relative volume of each compartment in the hepatocyte. For cytosolic Cl concentration, i ncreasing age was associated with decreased Cl concentration. Mitochondrial Cl concentration, on the other hand, exhibited a weak positive correlat ion with age. These findings indicate that the concentration of Cl dictates the rate of GSTZ1 inactivation induced after DCA exposure T he lower mitochondrial concentration predisposes to faster inactivation while higher cytosolic Cl concentration attenuates the inactivation induced by DCA. DCA Toxicity Peripheral Neuropathy Various tissues and organs are susceptible to DCA toxicity (Stacpoole et al., 1998a). Only the liver and n ervous system appear to be the most relevant organs applicable to human safety from studies with rodents. Chronic DCA administration can result in symptomatic, mild, but reversible elevation of hepatic transaminases (Stacpoole et al., 2008b). For the cen tral nervous system, a sensation of calmness and drowsiness results in some adults, having properties similar to anxiolytics. The most problematic effect from chronic DCA treatment in dogs, rats, and humans is reversible peripheral neuropathy and it is the major limiting factor in DCA administration (Stacpoole et al., 1998a). Patients exhibit p eripheral neuropathy as a tingling sensation in the extremities, muscle weakness as well as reduced nerve conduction velocity (Calcutt et al., 2009). It is beli eved that t he i nactivation of GSTZ1 by

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38 DCA influences this toxicity, leading to subsequent perturbations of the phenylalanine and tyrosine catabolic pathway. T he mechanism that causes peripheral neuropathy remains poorly understood Two major contributing factors exist (Stacpoole et al, 2008b). DCA induces reversible inhibition of the expression of myelin proteins in rat Schwann cells and dorsal root ganglia neurons in vitro (Felitsyn et al., 2007). This indicates that d e myelination results from DCA trea tment. Secondly, age as previously mentioned, is a significant factor for DCA toxicity Such neurotoxicity is age dependent with ch ronic treatment of DCA and often is observed during adulthood (Shroads et al., 2008; Stacpoole, 2011). The mechanism unde rlying the limiting consequence of DCA remains a critical area to study. Errors in Phenylalanine and Tyrosine Cataboli sm Numerous diseases related to inborn errors of metabolism involve the catabolism of phenylalanine and tyrosine (Figure 1 5 ). Five of these enzymes are associated with human genetic diseases, with MAAI being the only exception. The most common disorder is phenylketonuria, resulting from absence of phenylalanine hydroxylase activity and affecting almost 30,000 newborns each y ear (Fernandez Canon et al., 2002). Another disorder associated with this pathway is alkaptonuria, also known as black urine disease, where homogentisic acid accumulation leads to a dark color in the urine and damage to cartilage. The most severe type of metabolic disorder is hereditary tyrosinemia type 1 (HTT1), having a wo rldwide prevalence of 100,000, linked to a deficiency and loss of function mutation in the enzyme fumarylacetoacetate hydrolase (FAH) (Stacpoole, 2011). This condition results in dama ge to the hepatic, nervous, and renal systems. There is also a very high rate of hepatocellular carcinoma in those that possess the

PAGE 39

39 deficiency of FAH (Fernandez Canon et al., 2002). With this deficiency, accumulation of FAA results as well as other toxi c species like MAA and succinylacetone (SA). Often, neurological crises occur with disease progression, seen as peripheral neuropathy, porphyria, and ataxia (Gibbs et al., 1993). Accumulation of MAA, FAA, and SA is thought to result from a deficiency of MAAI, yet little clinical information exists. One study identified a link to elevated SA levels with MAAI deficiency with no apparent negative impacts to the liver or kidney (Yang et al., 2016). M ice generated with the deletion of MAAI have investigate d the severity of abnormalities with such a deficiency (Fernandez Canon et al., 2002; Ammini et al., 2003; Lim et al., 2004). MAAI knockout mice accumulated FAA and SA in urine, but otherwise appeared healthy (Fernandez Canon et al., 2002). Administration of substrates of this catabolic pathway to these genetically altered rodents caused severe organ damage and was lethal in a subset of these animals. Th ese data suggest that GSTZ1 is physiologically important, deficiency of MAAI /GSTZ1 is more mild than lo ss of FAH, and a deficiency of both MAAI /GSTZ1 and FAH would likely present as renal injury from accumulation of toxic compounds like FAA and MAA (Fernandez Canon et al., 2002) A second similar study with GSTZ1 knockout mice resulted in enlarged liver an d kidneys, major changes in the kidneys and accumulation of serum SA (Lim et al., 2004). Inte restingly, these knockout mice had higher abundance of several members of the alpha, mu, and pi class GSTs in the liver, indicating that met abolic adaptations oc cur with G S T Z1 deficiency. Additionally, DCA pharmacokinetics showed that DCA was unchanged and present at high concentrations in the plasma and urine with high levels of measured tyrosine catabolites in this mouse model (Ammini et al., 2003).

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40 These stud ies indicate that DCA exerts its effects solely on MAAI /GSTZ1 in vivo and, loss of this enzyme causes accumulation of reactive phenylalanine and tyrosine degradation intermediates linked with observed DCA toxicity. Alkylating Agents Many of the intermediates of the phenylalanine and tyrosine catabolic pathway have adverse effects related to GSTZ1, especially when accumulation of such species occurs. MAA and MA, its decarboxylated product, are well known alkylating agents that bind G SH and other thiols through Michael addition reactions, acting as competitive inhibitors that lead to enzyme inactivation and interference with GSH binding in the active site of GSTZ1 (Seltzer and Lin, 1979; Lantum et al., 2002b). B ecause of the ir reactiv ity these intermediates of tyrosine catabolism are believed to react with cellular proteins and may lead to the observed toxicity. FAA is also as a strong electr ophile that has mutagenic properties (Fernandez Canon et al., 2002). As a result MAA, MA, a nd FAA are active compounds of study to understand potential toxicity development that occurs with DCA induced GSTZ1 inactivation. Peripheral neuropathy associated with DCA exposure also could be linked to aminolevulina ALA ) SA is a competitive inhibitor ALA dehydratase (porphobilinogen synthase), an essential enzyme in heme ALA dehydratase increase s the ALA. Associated with several ty pes of neuropathies, including lead ALA is a neurotoxin that causes oxidation and disruptions to heme biosynthesis (Felitsyn et al., 2008). Chronic DCA administration can cause ALA are urinary biomarkers. Rats ALA dehydratase,

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41 through competitive inhibition from SA, MA, and/or FA (Lantum et al., 2003). Our lab measured elevated MA in male SD rat urine (Cornett et al., 1999) MA, in high concentrations, may have implicat ions for the pathophysiology of DCA and its possible cytotoxicity (Lantum et al., 2003). A second study with SD rats of three different ages indicated that excretion of MA increased with age and the degree of toxicity (Shroads et al., 2008). In humans wi th genetic mitochondrial diseases, age and GSTZ1 haplotype modulate s ALA in urine after DCA administration ALA in children chronically provided DCA were not di fferent from children diagnosed with HTT1. This information has assist ed in predicting DCA toxicokinetics Significance and Specific Aims Despite the continued use of DCA for medical treatment, toxicity limits chronic therapeutic administration of this agent. Repeated dosing of DCA causes GSTZ1 inactivation, increas es its half life, reduc es clearance, and exacerbates observed side effects. Several factors have been shown to modulate DCA pharmacokinetics, including age and GSTZ1 haplotypes additional factors have been identified that may also influence the interaction between GSTZ1 and DCA. First, GSTZ1 recently was identified in the liver mitochondria, and this localization is essential for the action of DCA The second factor is Cl a physiologically important anion that attenuate s GSTZ1 inactivation by DCA in vitro at appropriate physiological concentrations. Third extrahepatic GSTZ1 expression may influence DCA biotransfo rmation with notable expression in the brain, heart, and kidney in rats. In this doctoral research the properties of hepatic cytosolic and mitochondrial GSTZ1, the protective effect of Cl on GSTZ1 inactivation in the liver, and extrahepatic

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42 GSTZ1 expres sion with great interest in the kidney GSTZ1 activity and expression were examined by using a rat model after a single dose of DCA. Rat Cytosolic and M itochondrial G STZ1 Properties with DCA and MA Cytosolic and mitochondrial GSTZ1 properties were examined by measuring GSTZ1 activity with DCA and MA as substrates and GSTZ1 expression levels to test two hypotheses. First, mitochondrial GSTZ1 is more sensitive to inactivation by DCA compared to cytosolic GSTZ1 Second, GSTZ1 activity with MA reduces in concert with activity with DCA following inactivation and loss of GSTZ1 enzyme by DCA treatment It is known that GSTZ1 is present in both liver cytos ol and mitochondria and exhibit differing K m values for GSH (Li et al., 2011) T he concentration of DCA may be higher in the mitochondria from its ability to cross the cell and be taken up into the mitochondrial matrix. Recent findings in human liver have shown that Cl concentration in the mitochondria is much lower than in the cytosol (Jahn et al., 20 16), suggesting that Cl will have less of a protective effect for mitochondrial GSTZ1. T his could result in more rapid GSTZ1 inactivation in the mitochondria than in the cytosol. Secondly, as an endogenous substrate of GSTZ1, MA is a much more efficient substrate than DCA. Concentrations of MA become elevate d with DCA induced inactivation of GSTZ1, which subsequently perturb phenylalanine and tyrosine catabolism. This interaction between DCA and MA occurs in a competitive manner (Cornett et al., 1999). As a result, this may explain observed differences in recovery times of GSTZ1 activity and protein expression and possible toxicity development. Understanding the properties of mitochondrial GSTZ1 and the effect of DCA exposure on this cellular site of GSTZ1 expression will improve our knowledge of the clinical benefits and possible toxicity of DCA.

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43 R at C ytosolic and M itochondrial GSTZ1 I nactivation R ates Protection by Cl The attenuation of GSTZ1 inactivation by Cl was explored by in vivo and in vitro approaches to test the hypothesis that mitochondrial and cytosolic GSTZ1 exhibit differences in rates of inactivation by DCA in the presence of Cl Despite recent investigation that Cl influences the rate of GSTZ1 inactivation by DCA in a conce ntration and haplotype dependent manner in human liver and prolong s the half life of inactivation (Zhong et al., 2014) the mechanism of such attenuation remains un solved, and little exists on the in vitro interactions of mitochondrial GSTZ1 with Cl Fo rmation of a protein adduct is associated with DCA induced GSTZ1 inactivation, and this step is presumed to be modulated by Cl The studie s described in this dissertation will further our understanding of the role of Cl in stabilizing GSTZ1 from DCA ind uced inactivation. Influence of Extrahepatic GSTZ1 Expression on DCA Biotransformation Although GSTZ1 is mostly present in the liver, other tissues possess GSTZ1 expression, including the brain, heart, and kidneys (Lantum et al., 2002a). Preliminary studies suggest that the kidney plays an important role in GS TZ1 function, as this tissue had higher GSTZ1 protein expression compared with othe r extrahepatic organs An observed disparity in the correlation between rat kidney GSTZ1 activity and expression prompted our investigation of the hypothesis that a protein in the kidney inhibits GSTZ1 activity and function. Potential interaction of this unidentified protein in the kidney may impact the ability for DCA to be biotransformed, particularly after chronic dosing. Despite the lower levels of GSTZ1 in extrahepatic sites, the kidney, brain, and heart may influence the in vivo metabolism of DCA o r contribute to drug clearance This study will assist in optimizing DCA dosing based on numerous physiological factors.

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44 Figure 1 1. DCA inter a ction with the m itochondrial pyruvate d ehydrogenase c omplex.

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45 Figure 1 2. Influence of m itochondr ial e nergy p roduction in s olid t umors. MTP refers to mitochondrial transition pore, found in the inner mitochondrial membrane and is responsible for allowing the free passage of molecules of less than 1500 Da into the mitochondria.

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46 Figure 1 3. GSTZ1 c atalyzed reactions. A) Dechlorination of DCA to glyoxylate. B) Isomerization of m aleylacetoacetate to fumarylacetoacetate. C) Isomerization of m aleylacetone to fumarylacetone

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4 7 Figure 1 4 DCA d echlorination by GSTZ1 to g lyoxylate and other m etabolites.

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48 Figure 1 5 Phenylalanine and t yrosine c atabolism.

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49 Table 1 1. DCA p harmacokinetics in h umans and r ats. HUMAN RAT Children (mean age, 5 years; n=5) Adults (mean age, 24 years; n=4) Parameter First Dose Six Months after DCA First Dose Six Months after DCA Young (5 7 weeks) Adult (7 months) Old (15 months) t 1/2 (h) 2.5 6.4 2.1 21 4.5 5.8 7.0 AUC (g/mL h) 83 340 70 1500 370 290 700 Clearance (mL/h) 150 37 180 8.3 130 170 71 Humans were administered DCA for 6 months at 25 mg/kg/day, while 5 days of 50 mg/kg/day DCA given to rats Data shown as mean. (Adapted from Shroads et al., 2008)

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50 CHAPTER 2 DICHLOROACETATE INDUCED GLUTATHIONE TRANSFERASE ZETA 1 INACTIVATION DIFFERS IN RAT HEPATIC SUBCELLULAR FRACTIONS Specific Aims As DCA is taken up into the mitochondrial matrix (Stacpoole et al., 1998 a ) to exert its dynamic effects, mitochondrial GSTZ1 plays an important role in DCA biotransformation. With Cl levels much lower and the concentra tion of DCA potentially higher in the mitochondria than the cytosol, more rapid mitochondrial GSTZ1 inactivation could affect clinical DCA use. S tudies have identified that rats, mice, and humans show similar differences in the biotransformation of DCA wi th age, where more rapid metabolism of DCA occurs with young age and pharmacokinetic parameters in rats and humans were similar between related age groups (James et al., 1998; Schultz et al., 2002; Shroads et al., 2008). Current studies that examine the mechanism of DCA induced peripheral neuropathy are being conducted with female SD rats, since similarities in the fate of DCA between rats and humans ha ve been noted (Calcutt et al., 2009). W e tested the hypotheses that GSTZ1 protein would be lost more rapidly in the mitochondria than in the cytosol and that mitochondrial and cytosolic GSTZ1 have differing rates of inactiva tion by DCA in the presence of Cl Using female SD rats, we examined the differences in DCA induced GSTZ1 inactivation in liver cyt osol and mitochondria after a single administration of DCA over a 24 h time course. Materials and Methods Chemicals and Reagents 14 C DCA having a specific activity 56 mCi/mmol and 99% purity was obtained from American Radiolabeled Chemicals (St. Louis, M O), converted to its sodium salt by addition of NaHCO 3 and diluted with unlabeled Na DCA to make a 2 mM substrate

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51 Na DCA was from TCI America (Portland, OR). An E.coli expression vector (pET 21a) containing DCA for H is tagged rat GSTZ1 (NP_0011029515.1) was produced by Bio Basic Inc (Amherst, NY) and purified by Ni agarose affinity chromatography. 13 C GSH was purchased from Cambridge Isotope Laboratories, Inc. (Cambridge, MA). All other chem icals used in this study were of high purity and purchased from commercial suppliers. Animals and DCA Administration Based on previous studies of the dose response of DCA to GSTZ1 activity after 24 h of administration to male S D rats (Cornett et al., 1999), the selected dose was 100 mg/kg as this dose reduced GSTZ1 activity by 90% at 24 h. Na DCA (100 mg/kg) was administered by oral gavage to female SD rats (Charles River Laboratories, Wilmington, MA) aged 4 wk (young) and 52 wk (adult) to model child ren and adults, respectively. Housed two animals per cage, rats were acclimated for at least 1 wk in the designated facilities under a 12 h light/dark cycle with constant temperature and humidity conditions. T al Care and Use Committee approved these studies Animals had free access to water and rat chow during the 2, 4, 8, 12, and 24 h after dosing (n=6, per time point an d age). Control rats (n=6, per time point and age) dosed orally with sodium acetate (100 mg/kg), were subjected to the same dosing time course. Subcellular Fractionation of Rat Livers After sacrifice by CO 2 at specified time intervals, livers were removed and washed with 1.15% potassium chloride and 0.05 M pota ssium phosphate buffer (pH 7.4) to remove excess blood Differential centrifugation separated the livers into

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52 s ubcellular fractions, i.e. the cytosol, mitochondria, and microsomes as pr eviously reported (James et al., 1997). Samples were flushed with nitrogen and stored in aliquots at 80 o C until use. Dialysis of cytosol and washed mitochondria was completed b y ultrafiltration for four cycles at 5,000 g for 30 min with 10 kDa molecular weight cut off Amicon centrifugal filters (Millipore Corporation, Billerica, MA) against 1.15% potassium chloride and 0.05 M potassium phosphate buffer (pH 7.4) This washing process removed small molecules before use in assays, including sucrose that inhi bit s GSTZ1 activity (Li et al., 2011). Determination of p rotein concentration of each sample was by the bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific, Waltham, MA) with bovine serum albumin as the protein standard. Cytosolic and Mitocho ndrial GSTZ1 Activity Measurement GSTZ1 activity measurement was with 14 C DCA as the substrate. Assay products analysis was by HPLC coupled with radiochemical detection as formerly published (James et al., 1997). To determine the rate of transformation o f DCA to glyoxylate, the assay incubated 0.1 mg or 0.3 mg dialyzed protein (cytosol or mitochondria, respectively) with a saturating concentration of 14 C DCA of 0.2 mM, 1 mM or 5 mM GSH (cytosol or mitochondria), and 0.1 M Hepes NaOH (pH 7.6), in an assay volume of 0.1 mL. The reaction was initiated with the addition of protein, incubated at 37 o cold methanol. Performed in duplicate, activity was computed as nmol glyoxylate formed per min incubation per mg protein. Substrate consumption did not exceed 15%. The limit of quantitation was 0.23 nmol glyoxylate. Use of t he method of Tzeng et al (2000) calculate d the in vivo half life of loss of GSTZ1 activity from the linear portion of natural

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53 log linear plots of the ratio of mean GSTZ1 activities of treated cytosol and mitochondria to respective control activities at each time point and age. Western Blot Analysis of Cytosolic and Mitochondrial GSTZ1 Expression Rabbit polyclonal anti rat G STZ1 was custom produced using H is tagged rat GSTZ1 as the antigen (Cocalico Biologicals, Inc., Reamstown, PA). The antiserum was purified using the Pierce Protein A Antibody Purification Kit (Thermo Fisher Scientific, Waltham, MA) before use. Known amounts of protein (5 SDS electrophoresis using 12% polyacrylamide gels and transferred by electrophoresis onto nitrocellulose membranes (Bio Rad Laboratories, Hercules, CA) as previously documented (Li et al., 2012). The measure d GSTZ1 activity guided t he amount of protein used. The membrane was incubated with this custom GSTZ1 antibody diluted 1:1000 and then with horseradish peroxidase antibody, 1:5000 (GE Healthcare, Chalfont St. Giles, UK). Developed by ECL Plus Western Blo tting detection reagents (Thermo Fisher Scientific, Waltham, MA), protein signals were visualized on the ChemiDoc MP System (Bio Rad Laboratories, Hercules, CA). A standard curve using purified rat GSTZ1 was linear from 0.25 10 ng GSTZ1. To quantify sampl es, 5 included as a reference control on each blot. Linear regression calculated the GSTZ1 content with protein levels express ion The limit of quantiation was determined to be 0.66 ng GSTZ1. GSTZ1 Inactivation Half Life Determination in the Presence of Cl An established protocol quantitated Cl concentrations in rat samples by monitoring the conversion of pentafluorobenzyl bromi de to pentafluorobenzyl chloride via HPLC (Jahn et al., 2015). GSTZ1 inactivation was determined by incubating protein

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54 in the presence of physiologically measured KCl (n=3 per subcellular fraction and age), similar to a published procedure (Zhong et al., 2014). In short, dialyzed protein (0.5 1.8 mg) was incubated in the presence of physiologically relevant KCl concentrations (44 mM in young and adult cytosol, 1 mM in young mitochondria, and 2 mM in adult mitochondria) with 2 mM DCA, 5 mM GSH, and 0.1 M p otassium phosphate buffer (pH 7.4) in a final volume of 0.5 mL for 0 3 h. Recovered protein was assayed for GSTZ1 activity with 14 C DCA by HPLC analysis. Attenuating DCA Induced GSTZ1 Inactivation by Cl Like the methods for determining GSTZ1 inactivation half lives with Cl the effective Cl concentration (EC 50 ) where 50% protection of GSTZ1 activity from inactivation was identified after incubation with various KCl concentrations for 45 min. In order to get a clear understanding of this Cl effect betw een subcellular fractions, three young and adult rats were selected for comparative analysis. The reaction assay included d ialyzed protein (0.5 1 mg) with 2 mM DCA, 5 mM GSH, and various KCl concentrations (0 2 M in cytosol; 0 0.5 M in mitochondria) in a final volume of 0.5 mL. GSTZ1 activity was then determined with recovered protein and expressed as a percentage of GSTZ1 activity to the highest KCl concentration incubated for each analyzed sample. Glutathione Levels in Hepatic Subcellular Fract ions With r at liver tissue samples (75 100 mg), c ytosolic and mitochondrial fractions and whole liver (100 L ) were homogenized using conical pestles for 5 to 10 min with a 400 L solution containing 10% TCA, 1mM EDTA, and 1mM BHT in a 1.7 mL micro centrifuge tube. T he internal standard 13 C GSH (1 M ) were added to each tube. The tubes were centrifuged at 9000 relative centrifugal force for 10 min and 200

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55 L of the supernatant was removed into a micro centrifuge tube filter. The sam ples were centrifuged for an additional 10 min at 9000 relative centrifugal force and the filtrate was transferred to an amber 1.5 m L autosampler vial fitted with a 400 L glass insert. LC MS analysis was performed as previously described by Squellerio e t al. (2012) on a Thermo Scientifc TSQ Quantum Access Max mass spectrometer equipped with electrospray ionization and operated in the multiple reaction monitoring mode. GSH (308.049 Da) was ionized to produce 76.314, 84.235 and 161.90 Da. GSH IS (311 Da) produced the product ions 118.2 and 165 Da. Oxidized GSH ( GSSG ) (613.09 Da) produced the masses 234.6 and 355.1 Da. Collison energies were optimized for each transition. GSH, GSH IS, and GSSG were separated on a Phenomenex Luna 5m PFP(2) 150 x 3.00 mm column with a mobile phase of 99% 0.75 mM ammonium formate/ formic acid (pH=3.5) and 1% methanol (iso cratic) and a flow rate of 200 L /min. GSH and GSH IS coeluted (retention time 4.1 min) under these conditions, but unique product ions allowed for indep endent quantitation. GSSG eluted at 9.1 min. Data Analysis The in vivo and in vitro half life of GSTZ1 activity loss/inactivation and the Cl concentration that protected against 50% of DCA induced GSTZ1 inactivation were determined using previous methods (Zhong et al., 2014). The Cl EC 50 calculation was performed by normalizing GSTZ1 activity as a percent against the GSTZ1 activity determined with the highest KCl concentration. Means, standard deviations, standard errors of the mean, and statistical significance were calculated and analyzed by Excel (Microsoft Office, Redmond, WA)

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56 and/or GraphPad Prism v5 (GraphPad Software Inc., San Diego, CA). Comparisons between age groups an d subcellular fractions used either one way ANOVA with t test. A p value of less than 0.05 was considered to be statistically significant. Results DCA Induced Inactivation of GSTZ1 Activity in Rat Subcellular Fractions DCA treatment to both young and adult rats led to a dramatic and rapid depletion of both cytosolic and mitochondrial GSTZ1 activity over time. Activity in control rats was about ten fold higher in the cytosol than mitochondria ( Table 2 1). To examine the impact of DCA treatment on GSTZ1 activity, specific activit ies for each young and adult rat cytosol and mitochondria samples were expressed as the percentage of GSTZ1 activity remaining in DCA treated samples compared to averag e control activities at each time point and subcellular fraction (Figure 2 1). By 4 h after DCA administration there was more than 90% depletion of GSTZ1 activity in all treated rat cytosol and mitochondria, both young and adult. Some r ecovery of activi ty occurred by 24 h in the cytosol for both young and adult rats, and to a lesser extent in the mitochondria However, activity was well below pre dose values and control GSTZ1 activities. In Vivo Half Life of Loss of GSTZ1 Activity Log linear loss of G STZ1 activity occurs through 4 h in the cytosol and 2 h in the mitochondria based on percent GSTZ1 activity remaining. Activity data were transformed by computing the percent control activity based on the total average control activity within each age gro T he time at which 50% activity remain ed was determined by fitting the data to the equation

PAGE 57

57 ln(A/A o )= k obs t, where A is the measured activity and A o is the average control activity per age and subc ellular fraction at each time point with lines being forced through the origin (Figure 2 2). The half life (t 1/2 ) was determined from the equation t 1/2 =ln2/k obs The half lives of loss of GSTZ1 activity in young rat cytosol and mitochondria were determined as 1.17 0.34 h (mean SD) and 0.61 0.05 h, respectively. For the adult rat cytosol, the half life was determined to be 0.75 0.08 h, and the mitochondrial half life of loss of GSTZ1 activity was shorter, 0.46 0.09 h. The mitochondrial half life was shown to be significantly shorter than the cytosolic half life for both ages, ( p <0.01 in young and p <0.001 in adult). Adult rats had a shorter half life in the cytosol ( p <0.05) and mitochondria ( p <0.01) than in comparative young rat subcellu lar fractions. GSTZ1 Protein Expression in Cytosol and Mitochondria To semi quantitate the protein expression levels of rat GSTZ1, a standard curve was prepared with 0.25 to 10 ng of purified rat GSTZ1 and a young rat reference control cytosol sample, performed in triplicate for the expression of purified rat GSTZ1 and presented with an R 2 of 0.99, was acceptable for use in computing DCA treated and control rat cytosolic and mitochon drial GSTZ1 protein levels. Relative levels of GSTZ1 were at least five times higher in the cytosol than mitochondria for both DCA treated and control rats, p <0.0001 (Figure 2 3A and Table 2 2). After DCA treatment, cytosolic and mitochondrial GSTZ1 pro tein expression was quickly lost in both young and adult rats (Figure 2 3B and Figure 2 3C). Similar to the measured GSTZ1 activities, little protein expression was visible after 4 h in the cytosol and mitochondria, being at least 75% less than average qu antitated control GSTZ1 expression levels, in respective subcellular locations (F igure 2 4) Unlike the activity, no

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58 apparent recovery of GSTZ1 expression occurred by 24 h in the cytosol or mitochondria for either age. Correlation Analysis of GSTZ1 Activi ty and Protein Expression Correlations were very strong between GSTZ1 activity and relative GSTZ1 protein expression level for each of the subcellular fractions and ages, where the analysis included both DCA treated and control rat measurements (Figure 2 5 ). Generally, with increasing GSTZ1 activity, there was an increase in the relative expression level of GSTZ1. The computed Pearson r values for the young rat cytosol and mitochondria were 0. 91 and 0. 8 2, respectively. Similar values were obtained for th e adult rat hepatic fractions, 0. 91 for cytosol and 0.8 4 for mitochondria. The slopes of the lines computed for linear regression were shown to be significantly different between ages for both subcellular fractions, p <0.0001 (young rat cytosol, 0.23; youn g rat mitochondria, 0.36; adult rat cytosol, 0.20; and adult rat mitochondria, 0.27). In Vitro Half Life of GSTZ1 Inactivation with Cl The Cl concentration was determined in rat cytosol and m itochondria (Table 2 3) The cytosolic Cl levels in young and adult rats were similar (about 44 mM) but f or the mitochondria, Cl levels were significantly different between the two age groups (0.8 and 1.8 mM, for young and adult rats) p <0.0001 Using these determined physiological concentrati ons, incubations of control rat samples for both age groups and subcellular fractions with 2 mM DCA determine d the in vitro half life of GSTZ1 inactivation. Previous reports have determined that the half life of GSTZ1 inactivation in the absence of Cl t o be 5.44 min in the cytosol (Tzeng et al, 2000). Our lab oratory similarly found the half life of GSTZ1 inactivation in rat liver cytosol and mitochondria to be very short within 10 min In the presence of physiologically relevant Cl concentrations,

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59 cy tosolic half lives of inactivation were determined to be significantly different between the two rat age groups, 1.74 0.06 h and 1.60 0.05 h (mean SD) for the young and adult rats, respectively ( p <0.05) (Figure 2 6 ). On the other hand, the inactivat ion half life in the mitochondria was similar between ages, 0.46 0.03 h in the young rats and 0.51 0.03 h in the adult rats. Cl Effect on Attenuating GSTZ1 Inactivation by DCA Cl was able to prevent GSTZ1 inactivation when incubated with 2 mM DCA f or 45 min (Figure 2 7 ). With KCl concentrations of at least 1 M and 0.3 M in the cytosol and mitochondria, respectively, full protection of GSTZ1 activity from inactivation by DCA occurred Over a large range of KCl concentrations, the cytosolic Cl EC 50 was similar for young and adult rats, 81.8 13.5 mM (mean SD) and 84.0 4.6 mM, respectively. The mitochondrial EC 50 was much lower for both age groups. For the young rat mitochondria, the EC 50 was determined as 52.9 3.3 mM ( p <0.05, compared to yo ung rat cytosol), and the EC 50 was 38.9 11.9 mM for the adult mitochondria ( p <0.01 compared to adult rat cytosol ). GSH Determination in Rat Cytosol and Mitochondria Oxidized and reduced GSH levels were determined for rat cytosol mitochondria and whole liver (Table 2 4) N o obvious trend related to time in DCA treated animals existed for GSH n or GSSG There were, however, some differences at specific time points between treated and control GSH and GSSG levels, including adult rat cytosol at 1 and 2 h, young rat cytosol at 24 h, and young rat mitochondria at 1 h (Figure 2 8) Generally, young rats had higher levels of GSH in the cytosol, whereas adult rats had higher levels of GSSG in both the cytosol and mitochondria. Expressed as a ratio of GSH to GSSG, DCA treated young rat cytosol had the highest ratio, whereas adult rat

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60 cytosol had a lower ratio ( p <0.0001). The mitochondrial GSH to GSSG ratio generally was at least four fold less than in the cytosol for both age groups, p <0.0001. Disc ussion Despite accounting for less than 20% of all hepatic GSTZ1, mitochondrial GSTZ1 is likely to be essential in the biotransformation and therapeutic benefits of DCA. Little examination on the features of the hepatic subcellular fractions of GSTZ1 exi sts to date In this study, rats were used as an experimental model to investigate the stability and time related properties of cytosolic and mitochondrial liver GSTZ1 after a single exposure to DCA to better understand the clinical benefits and developme nt of adverse effects of this orphan drug. One of the most significant and obvious observations from this study was the difference between cytosolic and mitochondrial GSTZ1 activity and protein expression after DCA administration. Like that of the propo rtion disparity in hepatic GSTZ1 expression control GSTZ1 activities and measured GSTZ1 relative protein expression were much greater in the cytosol than mitochondria (at least 80% of total GSTZ1 activity). After DCA treatment, though, GSTZ1 activity los s was apparent, rapidly decreasing in both subcellular fractions by 4 h after dosing. Age also was not a large factor in the rapid depletion of GSTZ1 activity after DCA treatment, which was similar to previous observation with various aged rats (Shroads e t al., 2008). W ith increasing time after dose, a very small recovery in enzyme activity was noticeable by 24 h after DCA treatment mainly in the cytosol. The slight recovery in cytosolic activity after 12 h could be due to increased physical activity dur ing the dark/active rat cycle with more frequent eating and drinking that enhances protein turnover (Saghir and Schultz, 2002). Similarly, r elative protein expression levels were reduced with time after DCA treatment,

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61 but protein expression showed little recovery by 24 h One former study indicated that rat GSTZ1 activity also significantly decreased with exposure time to DCA and remained low even after drug withdrawal, taking about two months to return to basal levels (Guo et al., 2006). Between subcel lular fractions, it was easily noticeable that the half life of loss of cytosolic and mitochondrial GSTZ1 activity was different after DCA treatment. Despite the rapid decline of activity in both fractions over the first 4 h, the loss of GSTZ1 activity in the mitochondria was greater, having a half life of loss of GSTZ1 activity almost half that in the cytosol. Evidence is also growing that the transport of DCA into the mitochondria may influence the ability for DCA to interact more readily and effectivel y with GSTZ1. Yet, the exact mechanism for this transport remains unclear, but various monocarboxylate transporters may be actively involved in enhancing DCA concentration for initiating mitochondrial oxidative metabolism ( James et al., 2017 ). This confi rms our hypothesis that less GSTZ1 would be present in the mitochondria after DCA administration and supports the theory that the mitochondrion is the pivotal site of DCA action and subsequent inactivation of GSTZ1. However, a number of other factors also influence the stability of GSTZ1 with DCA administration. One recently identified factor in understanding DCA induced GSTZ1 inactivation is physiological Cl concentration. Data ha ve indicated that Cl can prolong the half life of DCA induced GSTZ1 i nactivation in humans (Zhong et al., 2014). As a reference, human liver mitochondrial Cl concentration is about 25 fold lower than in the cytosol (Jahn et al., 2015). In rats, mitochondrial Cl levels were at least 20 times less than measured cytosolic Cl (Table 2 3) As a result, observed differences in GSTZ1 relative

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62 expression between subcellular fractions are likely to be a result of lower Cl particularly in rat mitochondria, making GSTZ1 more susceptible to inactivation. For in vitro stud ies with rats, the absence of Cl in cytosolic and mitochondrial samples led to a very short half life of only a few minutes; however, upon adding physiologically relevant concentrations to assay mixtures, this half life was at least six fold longer as has been similarly observed in humans (Zhong et al., 2014). Comparing these in vitro half lives to the estimated in vivo half lives of loss of GSTZ1 activity, mitochondrial values were quite similar. The cytosolic half lives were much longer with the additi on of Cl than determined in vivo particularly for the adult rat attenuating GSTZ1 inactivation. This suggests that additional Cl in the cytosol can provide better protection to prevent DCA induced GSTZ1 inactivation than in the mitochondria. L ower le vels of Cl in the mitochondria may have less of a protective effect to the inactivation induced by DCA Concurrently, these lower mitochondrial Cl levels may influence the more rapid loss of GSTZ1, as observed with the shorter h alf life of loss of GSTZ1 between hepatic fractions Yet, it is important to address that other inherent properties of GSTZ1 may be influential in the action of protecting against DCA induced inactivation besides Cl levels alone. One other explanation for these subcellular fraction differences in activity and expression may involve the co substrate of this interaction between DCA and GSTZ1. Li et al. (2011) noted the presence of GSTZ1 in the mitochondrial matrix of hepatic cells, having similar K m values for DCA in the cytosol and mitochondria, but a larger K m for GSH in the mitochondria. This implies that the binding site of GSH in GSTZ1 differs in these two fractions. However, cytosolic and mitochondrial GSH is present at

PAGE 63

63 concentrations far abov e that of the measured K m value (Mari et al., 2013), so the rate of DCA biotransformation should not be limited in either subcellular fraction at physiological conditions. The mitochondrial form of GSTZ1 possesses a higher K m for GSH, implying weaker bind ing access than the cytosolic form, which may be visible from the much larger cytosolic activity (Li et al., 2011). These kinetic data corroborate that the two forms of GSTZ1 differ, but further studies are necessary to confirm how this influences observe d differences between cytosolic and mitochondrial GSTZ1. In attempts to understand the importance of GSH levels for the action of GSTZ1, we recently identified that GSH consumption occurs at an excessive rate in mitochondria of some rat organs, particularl y the kidney and resulted in almost the complete loss of GSTZ1 activity. O ther proteins in these organs may influence the availability o f GSH for proper GSTZ1 catalysis If less GSH is present, inactivation of GSTZ1 could occur faster Physiologically measured GSH levels were lower in the rat liver mitochondria than the cytosol in this study, ad ding evidence to this theory. In rats, about 10% of total hepatic GSH is found in the mitochondria, with about 90% of that accounting for reduced GSH (Meredith and Reed, 1981). One study showed that rat mitochondrial GSH poo l is about 5 nmol/mg, while the cytosolic pool for GSH is more than 20 nmol/mg ( Comar et al., 2013). In our study, mitochondrial GSH levels were similar to former measurements, about 4 nmol/mg but were dramatically lower than observed cytosolic levels with average GSH levels in each age and fraction over 47 nmol/mg ( p <0.0001). It has been hypothesized that lower GSH levels are linked to greater toxicity, causing limited GSTZ1 functio n in mice (Theodoratos et al., 2012). Interestingly, pre treatment of these mice with DCA increased the expression of

PAGE 64

64 glutamate cysteine ligase, an enzyme of GSH synthesis. T herefore, it has been suggested that DCA can provide a protective role from hepa totoxicity produced by chemicals that cause oxidative stress or consume this thiol containing compound (Theodoratos et al., 2012). However, our results do not agree with this former finding, as we found no difference in rat GSH levels in controls or DCA t reat ed rats (Table 2 4) This may be a species specific response or controlled by other unknown factors. Additional ly, we explored the possibility that GSH could be degraded in rat liver mitochondria. After 15 min incubation at 37 o C no significant loss of GSH was observed suggesting that GSH consumption is not a major factor in GSTZ1 loss. This still begs the question of what other dissimilarities exist between cytosolic and mitochondrial GSTZ1 to result in such dramatic differences in DCA induced inac tivation. T here are inherently unique properties of these forms of GSTZ1 that are unknown One theory is that downstream effects, i.e. post translational modifications, could influence this loss in activity and protein expression. It also has been postu lated that covalent modification of GSTZ1 or the production of a GSTZ1 protein adduct may be the cause of such irreversible inactivation in both humans and rats (Anderson et al., 2004). This adduct is believed to form between a nucleophilic cysteine resid ue of the active site of GSTZ1 and the unstable intermediate of DCA, leading to a covalently modified and inactivated enzyme that undergoes degradation to cause the observed GSTZ1 inactivation (Anderson et al., 1999; Tzeng et al., 2000; Polekhina et al., 2 001). Additionally, adducts were reported to contain GSH and glyoxylate with serine modification. This potential adduction may be altered if GSH, Cl or DCA itself binds to the active site of GSTZ1, causing other changes in GSTZ1 function. No leads hav e

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65 been identified to date to confirm whether post translational modifications and/ or adduct formation are the primary cause s of GSTZ1 inactivation and warrants future investigations of such effects. Finally, age also appears to be a factor in observed diff erences in rat GSTZ1 inactivation by DCA. In this study, we utilized two groups of female SD rats, 4 and 52 wk, to model children and mid life adults, respectively. Obvious differences in the in vivo half life of loss of GSTZ1 activity in the cytosol and mitochondria, mitochondrial Cl levels, and the Cl EC 50 in the mitochondria were notable between young and adult rats. In humans, age is an important determinant of DCA pharmacokinetics, particularly for the clearance of DCA after chronic exposure in patients suffering from a variety of genetic mitochondrial diseases (Shroads et al., 2008). With increasing age, there is a dramatic decrease in DCA plasma clearance and a ten fold increase in the elimin ation half life for the adult population, while children only had a three fold and four fold increase in the same pharmacokinetic parameters after DCA administration. These findings most directly relate to the much shorter half life of loss of GSTZ1 activ ity and higher DCA plasma concentration in the cytosol in adults, implying GSTZ1 is lost quicker with age and may be deleterious to the function of this enzyme as DCA can build up faster. This implies that young age is more tolerant to DCA administration, while changes in DCA kinetics are more dramatic in adults (Shroads et al., 2008). The difference in recovery could possibly explain the variability of peripheral neuropathy severity with age, where the slower clearance of DCA from adults could be an unde rlying cause of this reversible neurologic effect.

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66 On the other hand, experiments investigating age with DCA pharmacokinetics in rodents have provided contrasting findings Using mice, 60 wk animals were unaffected by DCA treatment with similar toxicoki netic behavior as control mice of the same age, whereas 10 wk mice were more sensitive to the changes induced by DCA administration in drinking water (Schultz et al., 2002). From this study, chronic administration of DCA to older mice compensated for GSTZ 1 activity loss in the liver, while younger aged mice have more dynamic GSTZ1 activity inactivation and recovery. On the other hand, our data align more closely to a study that examined the effects of age in male SD rats, where the half life of DCA elimin ation increases with age after multiple doses of DCA were administered (Shroads et al., 2008). It is possible that the physiological substrates for GSTZ1 (i.e., intermediates of tyrosine degradation) may also be influential in the recovery of GSTZ1 after DCA treatment and side effect development (Lantum et al., 2002 a ). Additional investigation is required to examine this relationship. In conclusion, this study examined the time course loss of rat GSTZ1 after a single dose of DCA and showed that the rate of GSTZ1 inactivation differs after DCA administration in rat liver cytosol and mitochondria. Additional factors that influence DCA induced GSTZ1 inactivation are increasing age and low Cl concentrations, which may be linked to the more frequent ly o bserved adverse side effect development Other properties of GSTZ1 in the liver cytosol and mitochondria likely affect DCA biotransformation, like protein modifications. As a result, the in vivo examination of a single dose of DCA in young and adult rats has provided important observations on DCA induced GSTZ1 inactivation, has addressed differences in hepatic cytosol and

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67 mitochondrial GSTZ1 stability and the attenuating effect of Cl and has furthered our understanding of clinical applications of DCA in human disease treatment.

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68 Figure 2 1. GSTZ1 activity remaining up to 24 h after a single dose of DCA was administered to rats. A) Young rat GSTZ1 activity loss (cytosol, black circle; mitochondria, red open circle) B ) GSTZ1 a ctivity loss in adult rats (cytosol, blue square; mitochondria, open grey square). All values are shown as a percentage of the average control activity per time point in each subcellular fraction mean SEM, n=6.

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69 Figure 2 2. I n vivo half life of loss of rat GSTZ1 activity following treatment with DCA was determined. 50% of GSTZ1 act ivity remaining occurs when Ln( A/A o ) equals Ln (0.5), depicted by the black dashed line [Ln (A/A 0 )= 0.692] The half lives of loss were determined to be 1.17 h and 0.75 h in young (black circle) and adult (blue square) cytosol, respectively; for the mitochondria, the half lives were shorter than that determined for cytosolic half lives by age ( p <0.01), computed as 0.61 h and 0.46 h in young (red open circle) and adult (grey open square) rats.

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70 Figure 2 3. GSTZ1 protein los s with time after DCA treatment in young rats. A) D ifferences in GSTZ1 protein expression levels in control ra ts, 15 g protein loaded for both rat cytosol and mitochondria. B) Q ualitative loss of cytosolic GSTZ1 protein expression in a DCA treated young rat C) Q ualitative loss of mitochondrial GSTZ1 protein expression in a DCA treated young rat at each time point ) For illustration purposes, the mitochondrial loss of GSTZ1 (panel C) was exposed 4 times longer than the cytosolic loss of GSTZ1 (panel B) using the BioRad ChemiDoc system.

PAGE 71

71 Figure 2 4 GSTZ1 expression remaining over a 24 h period after a single dose of DCA. A) Young rat GSTZ1 expression loss (cytosol, black circle; mitochondria, red open circle) B ) GSTZ1 expression loss in adult rats (cytosol, blue square; mitochondria, open grey square). All values are shown as a percentage of the average control protein expression level per time point in each subcellular fraction mean, n=6.

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72 Figure 2 5 R elationship between GSTZ1 activity and relative protein expression A) Correla tion of activity and expression in young (black circle) and adult (blue square) rat cytosol B) Y oung (open red circle) and adult (ope n grey square) rat mitochondrial GSTZ1 activity and expression correlation All DCA treated and control rats were included in this analysis, n=89 for young rat cytosol and mitochondria; n=88 for adult rat cytosol and mitochondria. The slopes of the lines were significantly different between ages for both subcellular fractions, p <0.0001.

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73 Figure 2 6 I n vitro half life of rat GSTZ1 inactivation following incubation with 2 mM DCA was determined in the presence of physiologically relevant Cl 50% of GSTZ1 inactivation after DCA treatment occurs when Ln(A/A o ) equals Ln(0.5), depicted by the black dashed line. The inactivation half lives were determined to be 1.74 h and 1.60 h in young rat cytosol (black circle) and adult rat cytosol (blue square), respectively. The mitochondrial half lives were significantly shorter compared to cytosolic half lives for each ag e group ( p <0.0001), 0.46 h and 0.51 h in young (red open circle) and adult (grey open square) rats. Values shown as mean SEM, n=3 per age and subcellular fraction.

PAGE 74

74 Figure 2 7 E ffect of Cl concentration on DCA induced GSTZ1 inactivation in match ing rat hepatic fractions with 2 mM DCA. A) The cytosolic EC 50 were not different between young (black icons) and adult (blue icons) rats, 81.8 and 84.0 mM, respectively. B) M itochondrial EC 50 Cl determinations were much lower compared to the cytosol ( p <0.01), being 52.9 mM for young rats (red icons) and 38.9 mM for adult rats (grey icons). The concentration range of KCl was 0 2 M in the cytosol and 0 0.50 M in the mitochondria. The Cl EC 50 to protect against 50% of GSTZ1 inactivation was determined for young and adult cytosol and mitochondria (n=3 per age and subcellular fraction), where data points are shown as mean values of duplicate determinations for each sample and lines are curves fi t for each individual sample. The dashed black line represents when the Cl concentration prevented 50% of GSTZ1 activity from being lost from DCA treatment.

PAGE 75

75 Figure 2 8. Cytosolic GSH measurements in control rats and DCA treated rats A) Average y oung rat control (blue square) and DCA treated rat (red circle) GSH measurements were 82.0 17.1 nmol/mg protein and 79.9 18.9 nmol/mg protein, respectively. B) Adult rat control (blue square) and DCA treated rat (red circle) GSH measurements were lower than young rats, with averages of 47.9 12.5 nmol/mg protein and 57.4 11.5 nmol/mg protein, respectively. Between DCA treated and control adult rat cytosol at 1 and 2 h, the GSH concentration differed (*, p <0.05). Da ta are presented as mean SD, n=6 per treatment and time point (n=5 for 1h, 4h adult controls; n=4 for 8 h, 24 h young treated).

PAGE 76

76 Table 2 1. Average GSTZ1 a ctivity m easured in DCA treated r at c ytosol and mi tochondria. Values are presented as mean SEM, n=6 (8 h time point, n=5), for each DCA treatment time point and the average control activity (n=42) for each age and subcellular fraction. Data analyzed to test for age related differences by one way p <0.05, ** p <0.01, *** p <0.001.

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77 Table 2 2. Average GSTZ1 r elative e xpression l evels in DCA treated r at c ytosol and m itochondria. Values are presented as mean SEM, n=6 for each DCA treatment time point. Total average control also is presented for each age and subcellular fraction, n=42. Data analyzed to test for age related differences by one way ANOVA p <0.05, ** p <0.01, *** p <0.001.

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78 Table 2 3. Measured Cl levels in rat liver cytosol and mitochondria. Age Cl (mM) Cytosol **** Mitochondria Young 44.4 4.2 0.84 0.31 Adult 41.6 4.0 1.75 0.69 Values presented as mean SD, n=89 for young rat measurements and n=88 for adult rat measurements. Data analyzed to test for age related differences by subcellular fraction Cl concentration via t test. **** p <0.0001.

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79 Table 2 4. Measured GSH and GSSG levels in rat liver cytosol and mitochondria. Age/Fraction GSH ( nmol/mg ) GSSG ( nmol/mg ) **** GSH:GSSG **** YOUNG CYTOSOL Treated 8 2.0 17.1 **** 1.85 0.59 48. 7 1 5.1 Control 79.9 18.9 2.05 0 .54 40.5 11.2 ADULT CYTOSOL Treated 47.9 12.5 2.53 0. 36 19.3 5.9 Control 57.4 11.5 2.40 0. 44 24.9 7.6 YOUNG MITOCHONDRIA Treated 4.31 1.25* 0.40 0.09 10.5 2.6 Control 4.34 1.18 0.39 0.10 11.1 1.4 ADULT MITOCHONDRIA Treated 3.74 1.29 1.01 0.29 3.9 1.3 Control 3.84 0.89 0.94 0.25 4.3 1. 6 YOUNG WHOLE LIVER Treated 8.63 2.97 **** 0.23 0.11 44.9 25.8 Control 8.17 3.04 **** 0.19 0.07 51.0 34.3 ADULT WHOLE LIVER Treated 3.91 0.63 0.79 0.20 5.2 1.3 Control 3.95 0.64 0.80 0.22 5.2 1.1 Values presented as mean SD, n= 4 4 for young rat treated measurements n=42 for young rat control measurements, n=48 for adult rat treated measurements, a nd n= 40 for adult rat control measurement s. Data analyzed to test for age related differences between fraction treatments t test p <0.05, **** p <0.0001

PAGE 80

80 CHAPTER 3 LOSS OF MALEYLACETOACETATE ISOMERASE ACTIVITY IN RATS FOLLOWING DICHLOROACETATE TREATMENT Specific Aim GSTZ1 catalyzes important physiological rea ctions, where it was shown to be identical to maleylacetoacetate isomerase (MAAI), catalyzing the next to last step in tyrosine catabolism (Blackburn et al., 1998). Due to the selective nature of MAAI/GSTZ1, the efficiency of isomerization for the interme diates of the tyrosine catabolic pathway is much greater, having specific activity hundreds of times greater than with DCA or other substrates (Board and Anders, 2005). Perturbation of the tyrosine catabolic pathway occurs after DCA administration and/or from build up of intermediates in this pathway, including reactive maleylacetone (MA) and fumarylacetone (FA), leading to the development of neurologic and hepatic toxicities associated with inborn errors of metabolism (Seltzer and Lin, 1979; Lantum et al. 2003). As a result, this may explain observed differences in the recovery time of GSTZ1 activity and protein expression in humans and rats after chronic DCA administration and the development of toxic side effects. In this study, we investigated the hy pothesis that GSTZ1 activity with MA as the substrate reduces in concert with activity with DCA following inactivation and loss of MAAI/GSTZ1 protein by DCA treatment using a female SD rat model It was also determined if any loss of GSTZ1 activity with D CA and M A as substrates is associated to r educe GSTZ1 protein levels Materials and Methods Chemicals 14 C DCA (99% purity) was obtained from American Radiolabeled Chemicals (St. Louis, MO), and unlabeled clinical grade sodium DCA was purchased from TCI America

PAGE 81

81 (Portland, OR). MA and FA were synthesized by the method of Fowler and Seltzer (1970), where the butenolide intermediate was puri fied by column chromatography in a solvent system of heptane:ethyl acetate (3:1) (Figure 3 1) 1 H NMR and UV spectroscopy confirmed the identity of MA and FA (purity >90%). All other chemicals used were of reagent grade or higher and obtained from commer cial suppliers. Animals, DCA Administration, and Hepatic Subcellular Fractionation F emale SD rats of 4 and 52 wks of age (Charles River Laboratories, Wilmington, MA) were used to model children and adults, respectively Rats were administered 100 mg/kg body weight NaDCA by oral gavage at 8:00 AM and sacrificed at various times from 0.25 to 24 h Control rats received sodium acetate (100 mg/kg body weight) and were subjected to the same dosing and sacrifice time course. After sacrifice, livers were remo ved, and subcellular fractions were prepared by differential centrifugation. Activity Measurement with DCA as Substrate Rat cytosolic and mitochondrial GSTZ1 activity was measured by HPLC with 14 C DCA as the substrate as previously described (Chapter Two ) Western Blot Analysis of Cytosolic and Mitochondrial GSTZ1 Expression Known amounts of protein (5 electrophoresis using 12% polyacrylamide gels and transferred by electrophoresis onto nitrocellulose membranes (Bio Rad Lab oratories, Hercules, CA) A custom rabbit polyclonal anti rat GSTZ1 antibody was used (Cocalico Custom Antibody, Reamstown, PA) as previously described (Chapter Two) Activity Measurement with MA as Substrate Using HPLC, MAAI activity was determined by measuring the formation of FA from MA, with modifications to a previous method (Lantum et al., 2002b). Rat MAAI

PAGE 82

82 activities were determined at 25 o C using a reaction mixture of rat protein (0.1 2 mg) 1 mM GSH, 0.01 M potassium phosphate buffer pH 7.4 and 1 mM MA in acetonitrile (made fresh daily), to a final volume of 0.25 mL. Acetonitrile accounted for 0.0 2 5 mL of the reaction mixture volume. The isomerization reaction was initiated with 1 mM MA and quenched after 30 sec or 5 min (cytosol or mitoc hondria respectively ethoxycoumarin in acetonitrile the internal standard. Previous assessment of GSTZ1 activity with DCA as the substrate dictated t he amou nt of rat protein used in this assay For each DCA treatment time points (0.25, 0.5, 1, 2, 4, 8, 24 h) for both ages and subcellular fractions and three representative controls for each age and fraction activity with MA was determined. All samples were analyzed on a Shimadzu HPLC with a C 18 column (4.6 mm x cle size, Supelco Analytical/Sigma Aldrich). The column was eluted with a 0 100% acetonitrile gradient at a flow rate of 0.75 mL/min over 31 min Solvent A was water with 0.075% acetic acid, and solvent B was acetonitrile with 0.075% acetic acid. The mu lti step gradient of was 0% B to 15% B over 3 min before increasing to 45% B over 17 min then to 100% B over 4 min and held at 100% B for 3.5 min before returning to 0% B and equilibrated over 3 min. The absorbance of FA was monitored at 312 nm its known UV max at physiological pH (Fowler and Seltzer, 1970) The concentration of FA formed in reaction mixtures w as quantified with a standard curve prepared with known concentrations of FA in acetonitrile (0 25 nmol) plotted against the ratio of the peak ar ea of FA to 7 ethoxycoumarin. Peaks monitored included MA (8.9 min), FA (15.6 min), and 7 ethoxycoumarin (25.7 min). The non enzymatic rate of conversion of MA to FA was quantitated

PAGE 83

83 reaction mixture ( average of 10.13 0.77 n mol/min/mg protein, mean SD n=3 ). The rate of non enzymatic formation of FA was subtracted from each measured sample with rat protein to give a rate of nmol FA per min incubation per mg protein The limit of quantitation was calculated to be 6.72 nmol FA. To ensure that the use of acetonitrile did not inhibit GSTZ1 activity, activity with 14 C DCA as the substrate was completed, since this is the same enzyme in both assays (See Chapter Two methods). With the radiolabeled substrate, a cetonitrile was included in the reaction assay mixture, 10% v/v, as this amount was utilized in the assay with MA. The activity of adult rat liver control cytosol was inhibited by 10.24 2.57% (mean SD) n=3. As a result, a correction factor of 1.1024 was included in the measured activities with MA as the substrate to account for this loss. GSH Adduct Determination Using UV spectroscopy (UV 2550, Shimadzu Corporation, Kyoto, Japan), adduct formation between GSH and FA was monitored based on a former method (Selt zer, 1 973). Made in water, 0.05 m M FA and 0.50 mM GSH were combined and incubated at room temperature for 4 h before stopping the reaction with 0.1 M KOH to an alkaline pH T he absorption maxima of FA and the GSH adduct were then determined Subsequent studie s examined the impact of pH on the formation of this addition product. Over 48 h, absorbance was recorded in a reaction mixture of 0.05 m M FA and 0.5 mM GSH in potassium phosphate buffer, pH 7.4. LC MS Monitoring of Adduct Formation Formation of an add uct between GSH and FA was monitored by mass spectrometry. Following published methods (Dieckhaus et al., 2005), the preparative HPLC system consisted of pumps and photodiode array detector, while the MS analysis

PAGE 84

84 was conducted on a Sciex API 3200 mass spe ctrometer (AB/MDS Sciex, Toronto, directly infused at a anions were isolated, m/z 155 [M H] (FA), m/z 306 (GS ), and m/z 461.9 (adduct). Subsequent analysis of t he GSH adducts were analyzed by LC MS in negative ion mode. The analytes were eluted at a rate of 0.5 mL/min, where mobile phases consisted of water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). The gradient began at 0% B for 2 min before increasing to 50% B over 13 min, again increa sing to 100% B over 5 min, held at 100% B for 5 min, before returning to 0% B over 5 min and re equilibrated at 0% B for 10 min. The a dduct eluted at 14 min. To monitor the formation of this adduct, several product ion pairs were selected, including 461.7 306 for the fragmentation of GSH from the adduct, 306.0 127.9 for a fragment of GSH, and 461.7 254.0 as a confirmation of the adduct forming. Conditions were optimized for each product ion for specific and selective identification (Table 3 1). An ex cess of GSH improved the formation of this adduct. As a result, each incubation included 0.1 mM FA and 1 mM GSH in 0.01 M potassium phosphate buffer cytosol and mit ochondria after incubation at 37 o C for 1 h, in a reaction volume of 1 mL. The reaction was quenched with 1 mL of ice cold acetonitrile with 0.2% formic acid before centrifugation at 2000 g for 5 min to pellet the protein. The non enzymatic formation of th is adduct was also followed in the absence of protein. Summation of the peak areas for the product ion pairs, 306.0 127.9 and 461.7 306, were used as a

PAGE 85

85 quantitation tool, where the average peak area for the non enzymatic formation was subtracted from each sample containing protein. Statistical Analysis Comparisons between age groups and subcellular fractions used one way Pearson correlation analysis compared activities with MA and DCA as substrates or p rotein expression levels using GraphPad Prism v6 (GraphPad Software Inc., San Diego, CA). Data w ere considered significant if p <0.05. Results Rat GSTZ1 Activity Determination with MA as Substrate Similar to determining GSTZ1 activity with DCA as the substrate, DCA treatment to young and adult rats led to a rapid loss of cytosolic and mitochondrial activity with MA as the substrate with time. Mitochondrial activities were at least 25 times lower than cytosolic activities (Tables 3 2 and 3 3 ). With i ncreasing loss of activity and expression as formerly determined with DCA as the substrate ( Table 2 1 and Table 2 2 ), DCA treated rats required higher protein concentrations to account for this loss in both the cytosol and mitochondria. Time points after 4 h in both subcellular fractions and ages required up to 2 mg of protein for detectable formation of FA. Expressed as a percentage of the determined control activity when MA was the substrate (i.e., the amount of activity remaining), specific activities for each young and adult cytosolic and mitochondrial sample showed a dramatic reduction in activity with increasing time For rat cytosolic activities with MA as the substrate, less than 10% of activity compared to controls remained by 4 h, similar to wh en DCA w as the substrate (Figure 3 2 A and Figure 3 2 B). Similar responses were observed in the mitochondria,

PAGE 86

86 yet when MA was the substrate, activities were dramatically reduced even at time points within the first 2 h (Figure 3 2 C and Figure 3 2 D) T he p ercent mitochondrial activity remaining with DCA as the substrate was almost double that when MA was the substrate at 0.25 and 0.5 h. Half Life of Loss of Cytosolic GSTZ1 Activity with MA as S ubstrate The half life of loss of MAAI activity was computed from log linear plots of MAAI activity versus time, being linear through 4 h. Activities were transformed by computing the percent control activity, where the time when 50% remaining was determined. The young and adult rat hepatic cytosolic half lives we re 0.94 0.09 h (mean SD) and 0.64 0.03 h, respectively ( p < 0.01). This is similar to a trend observed with DCA as the substrate, having a shorter half life of loss of GSTZ1 activity in adult rats compared to young rats (1.5 fold difference Figure 2 2 ) Unfortunately, the half life of loss of activity with MA as the substrate was undetermined for the mitochondrial fraction, as more than 50% activity loss occurred by 0.25 h. Correlation Analysis of Activity with DCA and MA as Substrates Generally, with increasing loss of activity with DCA as the substrate, a similar reduction in activity occurred when MA was the substrate (Figure 3 3) A strong positive correlation was noted in both young and adult rat cytosolic and mitochondrial activities between DCA and MA, with Pearson correlation coefficients ( r ) greater than 0.93 for all studied ages and fractions. Between young and adult rats, the slopes of the lines computed for linear regression were significantly different for both subcellular fractions, p <0.0001.

PAGE 87

87 Correlation Analysis of Activity with MA as Substrate and GSTZ1 Protein Expression A strong correlation was noted when c omparing activity with MA as the substrate to the relative protein expression in rat cytosol; as activity with MA as the s ubstrate increased, the relative protein expression level also increased (Figure 3 4 A). When MA was the substrate, cytosolic activity for both age groups of rats had a strong relationship with protein expression, R 2 of 0.84 and 0.82 and Pearson r of 0.91 and 0.9 2 for young and adult rats respectively. However, mitochondrial activity with MA was not significantly related to measured GSTZ1 expression (Figure 3 4 B) This is primarily observed in adult rat mitochondria ( r= 0.69) In contrast the young rat mitochondrial MAAI activity was more strongly correlated with GSTZ1 protein expression (Pearson r 0.87). This lack of a strong relationship with expression levels and activity with MA may indicate that other interactions between substrates, products, and/ or co substrates are occurring to influence this disparity. Regardless, linear regression analysis indicated that the slopes were significantly different between ages for both subcellular fractions, p <0.0001. GSH Adduct Formation Using 100 g of control young rat mitochondrial protein, various concentrations of GSH ( 0.5 to 50 mM ) were used in an incubation mixture with 1 mM MA and 0.01 M potassium phosphate buffer and the formation of FA was monitored by HPLC With increasing GSH, there was a loss of FA formation. 50 mM GSH had about one order of magnitude less formation of FA compared to 0.5 mM (4.27 nmol and 0.40 nmol, respectively). This observation may result from the formation of an addition product

PAGE 88

88 be tween GSH and FA leading to decreased measurable isomerization as formerly investigated (Seltzer, 1973). Subsequently, i ncubation of 0.05 m M FA with 0.5 mM GSH in phosphate buffer pH 7.4 for 4 h at room temperature before stopping the reaction with KOH (pH ~13) resulted in the formation of an addition product. Maximum absorption of FA under alkaline conditions was 345 nm, while 299 nm was the maximum for the GSH adduct (Figure 3 5 A ). Additional s tudies were conducted to determine whether this adduct is visible under physiologically relevant conditions in order to explain the loss of formation of FA while monitoring the isomerization of MA to FA. Incubation of 0.05 mM FA and 0 .5 mM GSH over 48 h showed an increased absorbance around 317 nm (Figure 3 5 B ) This could indicate the presence of this addition product formerly cited (Seltzer, 1973). However, 1 mM FA has a similar wavelength of absorption (315 nm), so resolving these peaks effectively only results under alkaline conditions As a result, furthe r investigation was performed by LC MS in negative ion mode to monitor the formation of this adduct between GSH and FA. This adduct has a molecular weight of 463 Da ([M H] of 461.7) Combination of 0.1 mM FA and 1 mM GSH resulted in the formation of an adduct after incubating at 37 o C for 1 h (Figure 3 6). With optimized LC MS conditions, separation of FA, GSH, and the adduct was successful. The n on enzymatic formation of this adduct was quite rapid, with maximum formation by 1 hr (Figure 3 7). Peak ar eas for the product ion pair of 461.7 306.0 were larger than the areas for the ion pair of 30 6.0 127.9. The average summed peak area for the two monitored product ion pairs for the non enzymatic formation was 2,879,000 158,898 cps (mean SD) Then,

PAGE 89

89 rats, the formation of this adduct between GSH and FA was followed after a 1 h incubation to monitor differences between subcellular fractions (Table 3 4). The non enzymatic formation of this adduct between GSH and FA accounted for a majority of the formed addition product in the presence of protein. After subtracting out the non enzymatic formation, there was no difference between this addition product in adult liver cytosol and mitochondria (94,500 vs. 136,833 cp s, cytosol vs. mitochondria; p =0.44) Discussion Focus on the interaction of DCA with GSTZ1 has overshadowed the need to understand the physiological role of GSTZ1. As has been identified, GSTZ1 is identical to MAAI, the penultimate enzyme in tyrosi ne catabolism (Blackburn et al., 1998). It has been speculat ed that a pharmacologically induced deficiency of this enzyme may be associated with several of the multi organ toxic side effects in humans as well as rats and other species (Lantum et al., 2002 a). As a result, this study investigated the influence of DCA induced inactivation of GSTZ1 on the physiological function of this enzyme with MA to provide a possible explanation for these side effects. We showed that GSTZ1 activity with MA reduce s in co ncert with activity with DCA following inactivation and loss of MAAI/GSTZ1 protein by DCA treatment using a female SD rat model. Hepatic a ctivity with DCA as the substrate was reduced after in vivo treatment with DCA. Similar observations occurred in this study with MA as the substrate where longer exposure to DCA led to a more dramatic depletion of activity. By 8 h after a single dose of DCA, cytosolic and mitochondrial activities with MA as the substrate were less than 1% of controls.

PAGE 90

90 Activity for the is omerization of MA is much greater than dechlorination of DCA for GSTZ1 (Board and Anders, 2005). Estimates suggested that it was 1000 fold haloacids. Yet, we did not observe such dramatic differences between these two enzymatic functions of MAAI/GSTZ1 compared to other published reports. Looking at the control animals, c ytosolic GSTZ1 enzymatic activity ratio of MA to DCA as a substrate was 69 times greater in young rats and was 1 09 fold higher in the adult cytosol. On the other hand, mitochondrial substrate ratio was dramatically lower, being 1 7 and 1 9 times greater with MA as a substrate compared to DCA in young and adult rats, respectively Such variability in GSTZ1 expr ession may also explain the lower enzymatic activity ratio of MA to DCA in the mitochondria compared to the cytosol. These hepatic subcellular fraction differences in GSTZ1 activity with MA implies that DCA inactivation of GSTZ1 is also involved in pertur bing tyrosine catabolism One concern with determining GSTZ1 activity with the substrate MA was the presence of 10% acetonitrile v/v S tudies have determined that aprotic solvents can have a substantial effect on enzyme activity when concentrations exceed 1% v/v particularly in hepatic microsomes for monooxygenase reactions in vitro (Sakalli et al., 2015). However, several aprotic solvents, including acetonitrile, had differing effects on the inhibition of GST activity (Aitio and Bend, 1979). When the c oncentration of acetonitrile was 3.3% v/v, activity with styrene oxide was inhibited by 21%. In our study, we identified that 10% v/v acetonitrile inhibited GSTZ1 activity by about 10%. A correction factor was then applied to account for this loss

PAGE 91

91 DCA is a mechanism based inactivator of GSTZ1, which also results in lowered MAAI/GSTZ1 activity in rats and humans. Administration of DCA can lead to toxicity, perhaps related to increased accumulation of metabolites of phenylalanine and tyrosine catabolism. may occur and could perturb heme homeostasis or alter the activity of other cysteine dependent enzymes ( Cornett et al., 1999; Lantum et al., 2003). Some have also noted that despi te the lack of a known deficiency of MAAI/GSTZ1, alteration of this enzyme is possible in two manners (Fernandez Canon et al., 2002). I t could be pharmacologically induced, through compounds like DCA. In addition depletion of p hysiologically relevant GSH concentrations could influence enzyme dysfunction An enzyme independent pathway also could influence MAAI/GSTZ1 activity. With increasing concentrations of GSH, up to 10 mM, this activity increased (Fernandez Canon et al., 2002) As a result, under standing how GSH interacts with MAA or its derivatives is essential in preventing the side effects of DCA treatment, especially hepatic injuries. In our study, increasing GSH concentrations resulted in lower FA formation in mitochondrial rat samples compared to standard assay conditions Former investigations postulated that the formation of an addition product between GSH and FA results (Seltzer, 1973). We confirmed that both MA and FA have maximum absorbance near 312 nm at physiological pH. Owing to the rapid formation of isomerization reactions, FA reacts rapidly with thiols at neutral pH. With increasing time up to 48 h, we observed greater absorbance of a peak near 317 nm under physiological conditions, which may account for the formation of t his adduct. However, the reaction of GSH with both FA and MA is quite rapid and thus, it is difficult to monitor the isomerization of MA

PAGE 92

92 to FA under these same UV conditions as well (Seltzer, 1973). However, u nder alkaline conditions, t his GSH adduct is more evident near 300 nm and is clearly resolved from the isomerization reaction substrate and product. GSH adds across the carbon double bond, altering the conjugation of both the substrate and product for MAAI activity and enhancing the nucleophilic att ack of this thiol. It is clear that th is addition product is forming and may influence the intrinsic properties of GSTZ1 with MA as a substrate after DCA treatment Due to the difficulty of identifying this adduct between GSH and FA under physiological c onditions, additional studies using negative ion mass spectrometry were conducted. Rapid formation of this addition product was observed, even in the absence of protein. This aligns with former findings indicating that FA can react quickly with thiols at neutral pH (Seltzer, 1973). It has been suggested that an excess of GSH lead s to a rapid half life, of about 1 min affecting the observed amount of FA formed in reactions to monitor GSTZ1 isomerization. In fact, it appears that this addition product oc curs chemically when GSH and FA are present. This is confirmed by the small increase in summed peak areas from LC MS analysis of specific product ion pairs in the As a result, formation of this adduc t between GSH and FA is predicted to impact enzyme function. Studies have suggested that FA is a more potent inactivator of GSTZ1 than MA, interacting with specific cysteine residues in the active site of this enzyme to result in Michael addition adducts ( Lantum et al., 2002b). These differences may be related to the reactivity of MA and FA with thiols. Such observed effects may be due to the non enzymatic formation of FA from MA during enzyme incubations. MA and FA are likely

PAGE 93

93 to contribute to the toxici ty associated with DCA administration. F urther investigation is necessary to understand how this interaction occurs, the role GSH plays in this process, and how this could influence features associated with the physiological features of this unique phase II enzyme. This leads to the question of whether the reduction of GSTZ1 activity and expression induced by DCA exposure disrupts tyrosine catabolism by reducing the conversion of MA (MAA) to FA (FAA). As both MA and MAA are chemically reactive intermediates of tyros ine catabolism, they are known to form adducts with proteins as well as GSH (Lantum et al., 2002b). It could be possible that the formation of a covalently modified enzyme with these intermediates may to catalyze this cis tr ans isomerization or biotransform any other substrates This could possibly explain the slightly higher activities with MA as the substrate seen in adult control rats in this study In conclusion, our study examined the cytosolic and mitochondrial properties of GSTZ1 with MA as the substrate after administration of a single dose of DCA over a 24 h time course to two age groups of female SD rats. Our HPLC method to monitor the isomeri zation improved the resolution of MA and FA compared to former methods, making this protocol novel and useful. Similar to activity with DCA, increasing loss of GSTZ1 protein expression leads to lower activity with MA. Mitochondrial activities were dramat ically lower than cytosolic measures, as less GSTZ1 exists within this hepatic subcellular compartment. Additionally, the rapid and chemically formed adduct between GSH and FA (and possibly MA) may affect GSTZ1 function and explain the rapid loss of activ ity when MA is the substrate. It was found that GSTZ1 activity with MA reduced in

PAGE 94

94 parallel with activity with DCA. This loss of activity and expression after DCA administration may be associated with the known toxicities of this small compound, particula rly effects that may occur with alterations in tyrosine catabolism.

PAGE 95

95 Figure 3 1. Synthesis of maleylacetone (MA). A) Preparation of MA and its trans isomer (fumarylacetone) result after synthesizing and purifying the intermediate 4 acetonylid enebutene 4 olide from maleic anhydride and isopropenyl acetate. B) MA was more than 90% pure with a 3:1 ratio of the pseudo acid and open acid forms with only minor impurities by 1 H NMR

PAGE 96

96 Figure 3 2 GSTZ1 activity remaining after DCA treatment to rats with either DCA or MA as the substrate. A) Y oung rat cytosol activity loss (DCA as substrate, open green diamond; MA as substrate, black circle) B) A ctivity loss in adult cytosol (DCA, open green tri angle; MA, blue square) C) Mitochondrial activity loss for young mitochondria (DCA, open green diamond; MA, open red circle) D ) A dult rat mitochondria l activities with DCA and MA as substrates (DCA, open green triangle; MA, open grey square). More tha n 90% of activity was lost with either DCA or MA as a substrate by 4 h after DCA dosing in all ages and subcellular fractions. Expressed as a percentage of the average control activity per time point for each subcellular fraction, values shown are mean SEM, n=3

PAGE 97

97 Figure 3 3 P ositive relationship between GSTZ1 activity with MA as the substrate and DCA A) Correlation in young (black circle) and adult (blue square) rat cytosol B) Y oung (open red circle) and adult (open grey square) rat mit ochondria l activity correlation All DCA treated and control rats were included in this analysis, n=24, for each age and subcellular fraction. The slopes of the lines were significantly different between ages in the cytosol only p <0.0001. A strong corr elation was noted for both ages and subcellular fractions with Pearson r of 0.98 and 0.97 for young and adult rat cytosol, while young and adult mitochondria were 0.94 and 0.93, respectively.

PAGE 98

98 Figure 3 4 R elationship between GSTZ1 activity with MA as the substrate and relative protein expression A) Correlation in young (black circle) and adult (blue square) rat cytosol B) Y oung (open red circle) and adult (open grey square) rat mitochondria l correlation All DCA treated and control rats were included in this analysis, n=24, for each age and subcellular fraction. The slopes of the lines were significantly different between ages for both subcellular fractions, p <0.0001. A stronger correlation was seen in the cytosol than mitochondria for youn g and adult rats with Pearson r of 0.92 and 0.91 for young and adult rat cytosol, while young and adult mitochondria were 0.87 and 0.69, respectively.

PAGE 99

99 Figure 3 5 A ddition product formation between GSH and FA by UV spectrometry. A) UV spectrum of 0.05 mM FA in 0.1 mM KOH ( blue ), 0.5 mM GSH in 0.1 mM KOH ( red ), and 0.05 mM FA plus 0.5 mM GSH at neutral pH after standing for 4 h before addition of 0.1 M KOH to make it basic (pH ~13) ( green ). B ) Under physiological conditions, UV spec trum of 0.05 mM FA plus 0.5 mM GSH over time (reaction monitored at 0.25 h [ blue ], 0.5 h [red], 1 h [green], 3 h [ purple ], 24 h [ light blue ], and 48 h [ orange ]).

PAGE 100

100 Figure 3 6. Negative product ion spectrum of adduct formation between GSH and FA. GSH and FA were combined in a 10:1 ratio in phosphate buffer and incubated for 1 h at 37 o C Molecular weights of interest, FA (155.0 Da), GSH (306.0 DA), and adduct (461.7 Da).

PAGE 101

101 Figure 3 7. LC MS spectrum of non enzymatic adduct formation between GSH and FA. Multiple reaction monitoring was used to follow the formation of this adduct, having a retention time of 14 min. Product ions followed were 155.0 68.8 (blue; FA), 306.0 143.0 (red; GS ), 461.7 306.0 (green; product io n of the adduct to GSH); 306.0 127.9 (black; product ion of GSH); 461.7 254.0 (grey; unique product ion of the adduct, used for confirmation).

PAGE 102

102 Table 3 1 Optimized conditions for monitoring adduct formation between GSH and FA by LC MS in negative ion mode Compound Product Ion Pair Declustering Potential (eV) Entrance Potential (eV) Collision Energy (eV) Collision Cell Exit Potential (eV) Fumarylacetone 155.0 68.8 20 9 22 6 Glutathione 306.0 143.0 65 5 26 3 Adduct 461.7 306.0 8 5 4.5 20 2 461.7 254.0 95 4.5 30 4 306.0 127.9 65 5 24 2

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103 Table 3 2 Average MAAI activity with MA, GSTZ1 activity with DCA, and substrate activity ratio in DCA treated young and adult rat cytosol. Time Young Cytosol Adult Cytosol Activity with MA Activity with DCA MA:DCA Substrate Ratio Activity with MA Activity with DCA MA:DCA Substrate Ratio 0.25 h 1 36 9 .9 *** 1.95 0.16 *** 70 10 1 83 14.5 1.54 0.36 1 22 17 0.5 h 1 28 18.0 *** 1.61 0.07 *** 80 9 1 63 20.4 1.04 0.13 1 56 1 2 1 h 74.2 7.0 1.01 0.27 75 2 3 83.1 15.6 0.98 0.23 86 9 2 h 38.9 7.2 0.67 0.07 ** 5 8 7 28.8 5. 9 0.43 0.10 6 7 1 4 h 8.69 1.67 ** 0.20 0.06 ** 4 5 4 3.65 2.20 0.07 0.02 49 20 8 h 1. 90 0. 19 0.11 0.02 19 4 2.07 0.1 5 0.06 0.02 3 6 9 24 h 26.5 1. 6 *** 0.44 0.08 ** 62 1 2 7.67 3. 65 0.14 0.09 5 7 7 Control 1 65 6. 9 *** 2.40 0.08 6 9 1 2 44 10 .3 2.24 0.04 1 09 6

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104 Table 3 3 Average MAAI activity with MA, GSTZ1 activity with DCA, and substrate activity ratio in DCA treated young and adult rat mitochondria. Time Young Mitochondria Adult Mitochondria Activity with MA Activity with DCA MA:DCA Substrate Ratio Activity with MA Activity with DCA MA:DCA Substrate Ratio 0.25 h 1. 41 0.2 2 *** 0.13 0.01 *** 1 1 2 2. 33 0.3 4 0.25 0.02 9 1 0.5 h 0. 90 0. 12 ** 0.12 0.01 *** 8 1 1. 35 0.1 3 0.22 0.01 6 1 1 h 0. 60 0.03 *** 0.07 0.01 *** 9 1 0. 50 0.03 0.12 0.02 4 1 2 h 0.2 8 0.01 *** 0.04 0.01 7 1 0. 50 0.0 8 0.03 0.01 1 7 2 4 h 0.1 8 0.04 *** 0.02 0.01 1 2 2 0.3 8 0.0 3 0.01 0.01 30 2 8 h 0.1 3 0.01 0.01 0.01 21 3 0.0 9 0.04 0.01 0.02 7 3 24 h 0.3 9 0.0 5 *** 0.03 0.01 1 3 3 0.1 7 0.0 4 0.02 0.01 9 3 Control 5.09 1.0 0 *** 0.30 0.01 *** 1 7 3 9.32 0.27 0.49 0.01 1 9 1 MAAI activity denoted as nmol FA per min incubation per mg protein; GSTZ1 activity is expressed as nmol glyoxylate per min incubation per mg protein. Values are mean SD, n=3 for each DCA treatment time point; total average control, n=3, for each age and fraction. Activity data analyzed to test for age related differences. p <0.05, ** p <0.01, *** p <0.001.

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105 Table 3 4. Adduct formation between GSH and FA in adult rat cytosol and mitochondria. Fraction Summed Peak Area of Product Ions (cps) Summed Peak Area of Product Ions Non enzymatic (cps) Non Enzymatic 2,879,000 158,898 Cytosol 2,973,500 73,555 94,500 73,555 Mitochondria 3,015,833 47,174 136,833 47,174 Values are mean SD, n=3 per fraction. Adduct formation was monitored with 0.05 mM FA and 0.5 mM GSH. Areas of product ions 461.7 306.0 and 306.0 127.9 were used to quantitate the relative adduct formation.

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106 CHAPTER 4 ROLE OF EXTRAHEPATIC G LUTHATHIONE TRANSFERASE ZETA 1 IN D ICHLOROACETATE BIOTRANSFORMATION Specific Aim GSTZ1 expression is not confined to just the liver. E xtrahepatic enzyme expression has been previously reported in male rats (175 20 0 g) using a polyclonal antibody ( Lantum et al., 2002a). S ixteen different tissues showed GSTZ1 protein expression through immunostaining, including the heart, brain, muscle, intestine, and kidney, but with differing intensities. Similarly, tissue dependent differences in activities with MA and DCA reflected the various patterns of GSTZ1 expression. E xtrahepatic expression of GSTZ1 may be influential in the biotransformation of DCA. The multi organ toxicity of DCA may correlate with lower expression and activity of GSTZ1 in impacted tissues, where prolonged treatment with DCA c ould lead to DCA induced GSTZ1 inactivation (Lantum et al., 2002a). T his earlier study only utilized young aged rats, but subsequent investigations revealed that age is an important factor in DCA kinetics and GSTZ1 expression after DCA treatment (Shroads et al., 2008). It is possible that with chronic DCA administration, there will be a larger contribution of these extrahepatic sites in DCA biotransformation, clearance, and GSTZ1 inactivation. We, therefore, examined the role of GSTZ1 expression and acti vity in rat extrahepatic tissues Preliminary investigation identified that the kidneys have similar GSTZ1 expression levels compared to the liver, but much lower GSTZ1 activity with DCA was measured. As a result, we hypothesized that a protein in the ki dney inhibits GSTZ1 activity and function.

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107 Materials and Methods Rat Tissues Organs were obtained from female SD rats (Charles River Laboratories, Wilmington, MA), aged 4 and 52 wk 0.5 a n d 24 h after Na DCA or sodium acetate treatment. From selected rats removal and processing of the b rain, heart, kidney, and skeletal muscle resulted in cell free homogenates In subsequent studies, kidney was fractionated into mitochondria and cytosol. Kidney mitochondrial subcellular fractions were also isolated (Li e t al., 2011) into the intermembrane space (IMS), mitochondrial membrane, and mitochondrial matrix. Briefly, mitochondria were swollen in buffer (0.01 M Tris HCl pH 7.4) before exposure to shrinking buffer (2 M sucrose and 0.1 mM Tris HCl pH 7.4) and then centrifuged at 20,000 g for 20 min to give the IMS as the supernatant. The pellet was frozen and thawed three times prior to centrifugation at 125,000 g for 60 min, where the supernatant included the mitochondrial matrix proteins and the pellet contained th e mitochondrial membrane proteins. GSTZ1 Expression Samples were run on SDS PAGE gels, transferred to nitrocellulose membranes and immunoblotted for GSTZ1 expression as described previously (Chapter Two) GSTZ1 Activity and Kidney Inhibition Assays GSTZ1 activity was measured with 14 C DCA by HPLC. Because of an initial observation that kidney GSTZ1 activity did not correlate with measured GSTZ1 protein expression, subsequent examination of this tissue was necessary Dialyzed liver cytosol was pre incubat ed with or without dialyzed kidney cell free homogenates at 37 o C for 15 min. Protein ratio ranged from 1:2 to 1:4, using a standard protein

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108 concentration of 350 g for kidney fractions. Only liver cytosol served as control groups. A ctivities are presented as a percentage of control activity with liver cytosol. GSH Determination Determination of GSH was performed by the method of Hissin and Hilf (1976). W ashed rat kidney mitochondria in 1.15% KCl and 0.05 M potassium phosphate pH 7.4 was incubated in 0.1 M sodium phosphate with 0.005 M EDTA pH 8 and freshly prepared GSH concentrations (0 10 mM) to a final volume of 0.2 mL for 15 min at 37 o C. Diluted samples were incubated at room temperature with a methanol solution o p hthalaldehyde for 30 min before fluorescence was determined at 420 nm (emission) and 350 nm (excitation). glutamyl transpeptidase (GGT) Activity Kid ney GGT activity was measured through the conversion of L glutamyl p nitroanilide to p nitroaniline (Sig ma Aldrich, St. Louis, MO) through a colorimetric reaction (Tate and Meister, 19 77 ). Using UV spectroscopy (UV 2550, Shimadzu Corporation, Kyoto, Japan), 1 mM L glutamyl p nitroanilide, 20 mM glycylglycine, 50 M Tris HCl pH 8 were incubated at 37 o C, and the reaction was monitored at 410 nm. Absorbance was linear from 1 to 4 min after addition of all reagents. E xpressed as nanomoles p nitroaniline formed per min reaction time per m g protein the known molar abs orption coefficient was used to calculate GGT activity ( 8800M 1 cm 1 ) GGT Inhibition GGT activity measurements in washed rat kidney mitochondria in the presence of a known GGT inhibitor, azaserine (Sigma Aldrich, St. Louis, MO) were used to positively identify this kidney protein Like determining nave GGT activity, 20 mM

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109 HCl pH 8 were mixed with 10 mM azaserine and pre incubated at 37 o C for 15 min as azaserine is a known m echanism based inhibitor of GGT (Tate and Meister, 197 7 ). Reaction initiation occurred with 1 mM L glutamyl p nitroanilide addition and the formation of p nitroanilide was monitored over time. Statistical Analysis Comparisons between age groups and t tests. Michaelis Menten kinetics for GSH consumption was completed by GraphPad Prism v6 (GraphPad Software Inc., San Diego, CA). Data w ere considered significant if p <0.05. Results GSTZ1 Activity and Expression i n Extrahepatic Tissues The brain, heart, and kidneys from young and adult rats at 0.5 and 24 h after DCA dosing were processed to make cell free homogenates (600 g ), as well as a sample of skeletal muscle in 24 h rats. Immunoblots of GSTZ1 in these tissue s showed detectable levels of enzyme in all control tissues, with the exception of skeletal muscle (Figure 4 1A). While the kidney showed the highest expression in both age groups, the brain had higher expression than the heart in young rats, while the op posite trend was true in the adult rats. GSTZ1 levels 24 h after DCA administration strikingly decreased in all tissues ( p <0.0001) GSTZ1 activity in brain and heart closely mirrored changes in enzyme expression after DCA exposure (Figure 4 1B and Figur e 4 1 C). Brain tissue from young rats showed higher activity levels than did brain tissue from adult animals. On the other hand, heart tissue showed the opposite relationship, with increasing activity with age.

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110 Age had a negative relationship in measure d GSTZ1 activity in the kidneys ( p <0.01). Like that of GSTZ1 expression, activity in the brain, heart, and kidney reduced with longer exposure to DCA, having less than 15% of controls. Interestingly, there was a lack of correlation between kidney GSTZ1 expression and activity (Figure 4 1). When GSTZ1 activity and expression measurements from the same tissue were combined, it was found that cytosolic and mitochondrial liver GSTZ1, from both young and adult rats, had an enzyme activity of ~ 2 nmol glyoxyla te formed/min/mg of GSTZ1 protein (Table 2 1). Kidney GSTZ1 activity was at least 100 fold less than in the liver. This disparity required additional investigation as extrahepatic tissues might be influential in the metabolic fate of DCA. GSTZ1 Inhibitor y Factor Present in Kidney Mitochondria In an effort to determine the cause of the low GSTZ1 enzyme activities observed in the kidney, a combination of 100 g liver cytosol protein and 350 g kidney cell free homogenate from an adult rat was measured for G STZ1 activity (Figure 4 2). The activity of the liver cytosol represents 100% activity, while a mixture of liver cytosol with dialyzed cell free kidney homogenates had little activity (Figure 4 2A). Yet, this inhibition could be ablated by pre boiling th e kidney homogenate or by the addition of an excess of GSH suggesting modulation by a protein Further processing the kidney into individual subcellular fractions led to positive identification of the source of GSTZ1 inhibition. Addition of kidney cytos olic protein to liver cytosol dramatically increased GSTZ1 activity, while addition of kidney mitochondrial protein showed the same degree of enzyme inhibition previously measured with kidney cell free homogenates (Figure 4 2B). Subsequent fractionation i nto preparations of mitochondrial membrane, intermembrane space, and matrix

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111 proteins showed that the inhibitory factor is present in the membrane and intermembrane space (Figure 4 2C). GSH Determination in Rat Kidney Mitochondria With this inhibitory pro tein in the kidney mitochondria and activity recovery glutamyl transpeptidase (GGT) could be influential in this process (Tate and Meister, 1985). GSH levels were quantitated in rat kidney mitochond ria via a fluorescent agent, where a standard curve of GSH was utilized to quantitate the nmol of GSH in kidney mitochondria and was linear up to 7.5 nmol GSH. Up to 10 mM GSH was incubated with three different rat kidney mitochondrial samples (Figure 4 3 ). Michaelis Menten kinetics identified an average K m of 0.58 0.14 mM for GSH in rat kidney mitochondria. GGT Activity Determination and Inhibition by Azaserine GGT activity was monitored through a colorimetric reaction forming p nitroaniline at 410 nm (Figure 4 4). Kidney mitochondrial GGT in control rats had 0.97 0.20 units of GGT activity per m g protein (mean SD) Addition of 10 mM azaserine resulted in GGT activity inhibition, having an activity of 434 nmol p nitroaniline per min per mg pro tein. These data demonstrate a 55% inhibition of GGT activity by azaserine. Liver mitochondrial GGT activity was negligible similar to findings confirming the lower expression of GGT in tissue from healthy rats This confirms the presence of GGT in rat kidney mitochondria which could explain the liver GSTZ1 inhibitory effect and disparity in activity and expression levels in the kidney. Discussion DCA treatment leads to the inactivation of GSTZ1 and a subsequent decrease in expression in humans and rod ents (Cornett et al., 1999). The majority of GSTZ1 is in

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112 the liver, but extrahepatic expression may be a factor modulating DCA biotransformation and clearance. Our understanding of factors affecting DCA metabolism in the liver has advanced in recent year s, but the functional role of GSTZ1 in other tissues remains un investigated. This study extends early work by others (Lantum et al., 2002a) using a more sensitive, species specific antibody probe for rat GSTZ1 to demonstrate notable GSTZ1 expression in br ain, heart, and kidneys of female SD rats of two ages. Age played a significant role in GSTZ1 expression of the brain and heart, where young rats had higher brain expression and adult rats showed greater expression in the heart. This expression decrease d with DCA treatment, but showed a lesser decrease than the liver in these same animals (Table 2 2). Kidney tissues possessed high GSTZ1 expression, roughly equivalent to observed liver mitochondrial expression in rats. Like in the liver, GSTZ1 activity in these tissues largely mirrored the expression data. The notable exception was low activity but high expression in the kidneys. In the presence of liver cytosol, rat kidney cell free homogenates were able to totally abolish GSTZ1 activity Subcellula r fractionation located this factor to the mitochondria, specifically the IMS and mitochondrial membrane. This inhibition diminished by pre boiling the kidney sample, strongly suggesting that the inhibitory factor is a protein. In addition, excess GSH re scued GSTZ1 activity from this kidney inhibitory effect. Thus, we postulated that an unidentified protein exists in the kidney that either can bind to GSTZ1 near the GSH binding site or actively utilizes GSH for its own physiological function.

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113 Literatu glutamyl transpeptidase (GGT) may be the culprit for this inhibitory effect. Found in membranes and cells with active absorptive glutamyl compounds (Tate and Meister, 1977). GGT cata lyzes the removal and transfer of glutamate from such compounds to water through hydrolysis or to an amino acid/peptide by transpeptidation. Implicated in e sis, and cardiovascular complica tions and GGT is used as an indicator of liver dysfunction (Hanigan, 2014; Wada et al., 2008), GGT has the greatest expression in kidney with much lower activity and expression in the pancreas and liver (Tate and Meister, 1985). As a result, we investiga ted the potential of GGT as the inhibitory protein that caused the discrepancy in GSTZ1 activity and expression in the kidney. GGT activity in the rat kidney mitochondria was similar to previous measures in nave rats (Hanigan et al., 1994). The average G GT activity in our female SD rats was 973 nmol p nitroaniline formed per min per mg protein. Further confirmation of the presence of GGT in the kidney mitochondria was conducted with a known inhibitor of GGT. We utilized azaserine ( O dia z oacetyl L serine ), a mechanism based inactivator of GGT (Wada et al., 2008). Addition of azaserine to the reaction assay mixture for measuring GGT activity reduced this activity by 55%, showing moderate inhibition of GGT but positively confirming its identity. In con clusion, these data provide insight into factors beyond hepatic GSTZ1 expression in in vivo DCA metabolism and clearance. Although the liver is the predominant site of DCA metabolism, other tissues and organs can provide meaningful activity, up to 15% of all GSTZ1 enzyme activity in young DCA treated rats. This may

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114 explain the faster clearance of DCA with young age after chronic DCA treatment (Shroads et al., 2008). Of these extrahepatic organs, the kidney was the most fascinating. During our investigati on, we identified that an inhibitory protein in the kidney was negatively modulating GSTZ1 activity. Later confirmation identified this protein to be GGT localized in the mitochondrial membrane of the kidney GGT may be influential in the import of cyto solic GSH or GSTZ1 into mitochondrial matrix or its presence could affect the contribution of DCA biotransformation in kidney GSTZ1 (Tate and Meister, 1985 ; Kisslov et al., 2014) As a result, the identification of kidney mitochondrial GGT identifies that the contribution of kidney GSTZ1 to DCA biotransformation is not accurately assessed in a cell free homogenate, as previously conducted (Lantum et al., 2002a), but rather must be studied in the individual subcellular fractions of this extra hepatic organ. This could have potential implications in the metabolism of substrates for GSTZ1 and should be an additional consideration in the in vivo fate of DCA

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115 Figure 4 1. Extrahepatic GSTZ1 e xpression and a ctivity in r ats. A) Western blots of rat GSTZ1 in 600 g supernatant fractions (45 g) of kidney, heart, brain, and skeletal muscle from rats treated with 100 mg/kg Na DCA (T) and 100 mg/kg control sodium acetate (C) at 0.5 and 24 h. B) M easured GSTZ1 activity in cell free homogenates of young and adult rats after 0.5 h of DCA treatment. C) GSTZ1 activity in cell free homogenates of young and adult rats after 24 h of DCA treatment.

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116 Figure 4 2. Protein in rat kidney dramatically reduce s GSTZ1 activity with DCA. A) GSTZ1 activity in liver with the addition of 600 g kidney homogenate, boiled kidney homogenate, and kidney homogenate in the presence of 100 mM GSH. B) Inhibition of rat GSTZ1 activity result ed only from proteins in kidney mitochondria. C ) P roteins in the IMS and mitochondrial membrane of rat kidney mitochondria are responsible for the inhibition of GSTZ1 activity. Protein used in all studies was 100 g liver cytosol and 350 g kidney fracti ons.

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117 Figure 4 3. Michaelis Menten curve of GSH consumption in rat kidney mitochondria. V max and K m computed as 39.54 2.52 nmol/mg/min and 0.58 0.14 mM, respectively. Data shown as mean SD, n=3.

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118 Figure 4 4. Effect of azaserine on the inactivation of GGT activity in rat kidney mitochondria. Addition of 10 mM azaserine to rat kidney mitochondria result ed in 55% inhibition compared to control activity (GGT control activity shown in blue; GGT activity with the addition of azaserine displayed in red). No GGT activity was detected in rat liver mitochondria (green). Data shown were linear based on measured a bsorbance with blank subtraction from 1 to 4 min.

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119 CHAPTER 5 CONCLUSION DCA has been extensively used for treating patients with a variety of disorders, including acquired and inborn errors of metabolism and cancer. However, toxicity limits chroni c dosing of DCA, caused by GSTZ1 inactivation. Extensive research uncovered numerous factors that influence DCA pharmacokinetics, particularly GSTZ1 haplotype in humans, age, and Cl In this dissertation, we utilized a rat model to examine several facto rs that influence DCA induced GSTZ1 inactivation, specifically GSTZ1 expression in the cytosol and mitochondria of the liver and other tissues, age, and Cl Rats were administered a single dose of DCA and followed over a 24 h time course. GSTZ1 expressi on is mainly in the liver, with more than 80% in hepatic cytosol. It is believed that th e small fraction of GSTZ1 in hepatic mitochondria is a factor for the dynamic pharmacological effects DCA exerts on the PDC and DCA biotransformation. Concurrently, l ow Cl in this subcellular fraction modulates the rate of inactivation by DCA on GSTZ1. With our rodent model, increasing time after DCA exposure resulted in reduced GSTZ1 in both hepatic fractions, but mitochondrial GSTZ1 was more sensitive to inactivati on by DCA. The in vivo half life of loss of GSTZ1 activity in the mitochondria was nearly two times as fast as in the cytosol. Age also influenced DCA induced GSTZ1 inactivation, where adult rats had shorter half lives compared to young rats. As in huma ns Cl levels were at least 20 fold lower in the mitochondria than the cytosol. Cl concentration in the cytosol attenuated GSTZ1 inactivation more significantly than in the mitochondria ( in vitro inactivation half lives of 1 .74 h in young cytosol and 0.46 h in young mitochondria).

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120 Although extensive studies have documented the interaction between DCA and GSTZ1, this study showed that MA, an endogenous substrate of GSTZ1 is much more readily metabolized; GSTZ1 activitie s with MA as the substrate are more than 100 times greater than when DCA is the substrate Elevated concentrations of the endogenous substrates perturb tyrosine catabolism after DCA induced GSTZ1 inactivation and may explain the origin of toxicity Like GSTZ1 activity with DCA as the substrate, rat GSTZ1 activity with MA as a substrate reduced dramatically after DCA administration, with mitochondrial activities being at least 25 times lower than cytosolic activities. In the mitochondria, a positive corre lation between activities with MA as the substrate and GSTZ1 expression result ed, but was not strong as in the cytosol This could be due to the formation of GSH adducts with FA and MA near the site of action of DCA, further inactivating GSTZ1. GSTZ1 is present in organs other than the liver, but to a much lower extent. Former studies have identified GSTZ1 in brain, heart, and kidneys among others Using our rodent model, we examined extrahepatic sites of GSTZ1 expression to determine their role in the metabolism of DCA and other endogenous substrates of GSTZ1. We confirmed that the brain, heart, and kidneys, but not muscle expressed GSTZ1 protein and catalytic activity, and showed that DCA treatment reduced both measures. The kidneys showed a maj or disparity in GSTZ1 activity and expression, with high expression but low activity in the kidney homogenate This prompted further examination of this site of GSTZ1, which revealed that GGT in the kidney mitochondria resulted in extensive consumption of GSH which is required for the catalytic activity of

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121 GSTZ1 GGT is likely the cause of low measured GSTZ1 activity in the kidney homogenate From these studies, we answered several questions related to the interaction between GSTZ1 and DCA using a fema le SD rat model. We first showed that mitochondrial GSTZ1 is much more sensitive to DCA exposure, where hepatic mitochondrial GSTZ1 activity loss occurs more quickly after DCA treatment to rats than for cytosolic GSTZ1. Secondly, Cl is important in prev enting GSTZ1 inactivation, playing a concurrent role in GSTZ1 function with hepatic subcellular fraction and age. The physiological function of GSTZ1 in isomerizing MA is also impacted after DCA treatment, as was observed over the 24 h time course with DC A treatment. Finally, other sites expressing GSTZ1 may play critical roles in DCA metabolism, especially in the kidneys. Even with further identifying factors that are involved in DCA induced GSTZ1 inactivation, our understanding of this interaction is s till not complete. As a result, these findings are stimulat ing additional investigations of this interaction. Several areas are of particular interest. Understanding the mechanism for Cl protection to GSTZ1 inactivation is urgent. Second the impacts of other sites of DCA biotransformation, like the kidneys, after chronic DCA treatment, will assist in selecting a dose for safely administering DCA to children and adults. Another area needing to be examined is the impacts to the physiolog ical function of GSTZ1 and its role in toxicity. The following experiments are ongoing or proposed to evaluate these topics. One of the most critical areas to be elucidated is the mechanism of Cl in attenuating GSTZ1 inactivation with DCA exposure. Usin g the different human GSTZ1

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122 haplotypes, we plan to investigate this interaction with selected ani ons ( Cl bromide, and iodide ) and substrates for GSTZ1, like DCA and GSH. Our laboratory was able to obtain a crystal structure for hGSTZ1A, but were unable to resolve amino acid residues from 40 to 60. Obtaining high resolution crystal structures for hGSTZ1A and hGSTZ1B will assist in our understanding of known haplotype differences in GSTZ1 catalytic activity with DCA related to position 42 (arginine versus glycine). We are currently optimizing conditions for these crystallography experiments. Next, i t is theorized that incubating different GSTZ1 haplotypes with anions, like Cl will allow us to identify where anions bind to influence GSTZ1 stability. W e will also be able to investigate the binding of substrates, like DCA, to further our knowledge of the inactivation that results through possible adduct formation. Such experiments will advance our knowledge on this unclear area of the interaction between DCA and GSTZ1. From this doctoral research, it was identified that age has a significant effect on GSTZ1 function, and other sites besides the liver may be essential in chronic DCA treatment. Our laboratory is currently undergoing investigation of the su b chronic administration of DCA in female SD rats for eight days and the ability of age to influence GSTZ1 activity and expression in sites other than the liver, particularly the brain, heart, kidneys, and intestine. We postulate that with younger age, th ere is a greater retention of activity and expression compared to adult rats after sub chronic DCA treatment. Additionally, this study will address the question of how extrahepatic sites contribute to GSTZ1 expression with DCA treatment and shed light on a possible cause for age related differences in DCA clearance and toxicity.

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123 A third area of DCA research that requires attention is the resulting toxicity with DCA treatment. P erturbation of tyrosine catabolism with DCA treatment may be associated with several of these toxicities to the liver and kidney. Yet, the specific interaction of these reactive intermediates has only had limited investigation. Related, the most problematic effect of DCA is the resulting peripheral neuropathy that occurs through a yet known mechanism. Studies are beginning to examine this adverse effect using both in vitro and in vivo methods. Therefore, looking into the role of reactive species of tyrosine catabolism with GSTZ1 inactivation and the mechanism that causes periph eral neuropathy would provide answers for improving dosing regimens and limiting toxicity. This research addressed several questions that have further clarified variable s that affect DCA metabolism. These are important and will have an impact on the use of DCA as a drug in treating numerous metabolic diseases and cancer. In particular these findings identified the greater susceptibility of mitochondrial hepatic GSTZ1 to DCA treatment age plays a major role in the different responses to this therapeutic Cl is an important modulator in GSTZ1 inactivation prevention, and extrahepatic GSTZ1 contributes to DCA metabolism. The use of this female SD rats assisted in our und erstanding of DCA induced GSTZ1 inactivation, allowing this animal model to become a standard in vivo technique for future investigations. From these findings, patient DCA dosing regimens can be improved when taking factors like age, Cl and physiologica l substrate levels into account Such influences may help in preventing toxicity that often occurs with chronic treatment. This doctoral research has opened additional doors for subsequent investigation s into the mechanisms underlying many of

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124 these inter actions between DCA and GSTZ1. In conclusion, this work along with previous and future studies will expand our knowledge on the variability in DCA response in people to enhance safe and effective dosing for disease treatment.

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125 LIST OF REFERENCES Aitio A and Bend JR (1979) Inhibition of Rat Liver Glutathione S Transferase Activity by Aprotic Solvents. FEBS Lett 101 :187 190. Ammini CV, Fernandez Canon J, Shroads AL, Cornett R, Cheung J, James MO, Henderson GN, Grompe M, and Stacpoole PW (2003) Pharmacologic or Genetic Ablation of Maleylacetoacetate Isomerase Increases Levels of Toxic Tyrosine Catabolites in Rodents. Biochem Pharmacol 66 : 2029 2038. Anderson WB, Board PG, and Anders MW (2004) Glutathione Transferase Zeta Catalyzed Bioactivation of Dichloroacetic Acid: Reaction of Glyoxylate with Amino Acid Nucleophiles. Chem Res Toxicol 17 :650 662. Anderson WB, Board PG, Gargano B, and Anders MW (1999 ) Inactivation of Glutathione Transferase Zeta by Dichloroacetic Acid and Other Fluorine Lacking Haloalkanoic Acids. Chem Res Toxicol 12 : 1144 1149. Anderson WB, Liebler DC, Board PG, and Anders MW (2002) Mass Spectral Characterization of Dichloroacetic A cid Modified Human Glutathione Transferase Zeta. Chem Res Toxicol 15 : 1387 1397. Barshop BA, Naviaux RK, McGowan KA, Levine F, Nyhan WL, Loupis Geller A, and Haas RH (2004) Chronic Treatment of Mitochondrial Disease Patients with Dichloroacetate. Mol Genet Metab 83 : 138 149. Berendzen K, Theriaque DW, Shuster J, and Stacpoole PW (2006) Therapeutic Potential of Dichloroacetate for Pyruvate Dehydrogenase Complex Deficiency. Metab 6 : 126 135. Blackburn AC, Coggan M, Tzeng H, Lantum H, Polekhina G, Parker MW, Ande rs MW, and Board PG (2001) GSTZ1d: A New Allele of Glutathione Transferase Zeta and Maleylacetoacetate Isomerase. Pharmacogenetics 11 :871 878. Blackburn AC, Tzeng H, Anders MW, and Board PG (2000) Discovery of a Functional Polymorphism in Human Glutathione Transferase Zeta by Expressed Sequence Tag Database Analysis. Pharmacogenetics 10 :49 57. Blackburn AC, Woollatt E, Sutherland GR, and Board PG (1998) Characterization and Chromosome Location of the Gene GSTZ1 Encoding the Human Zeta Class Glutathione Tran sferase and Maleylacetoacetate Isomerase. Cytogenet Cell Genet 83 : 109 114. Blackshear PJ, Holloway PAH, and Alberti KGMM (1974) The Metabolic Effects of Sodium Dichloroacetate in the Starved Rat. Biochem J 142 : 279 286. Board PG and Anders MW (2005) Human G lutathione Transferase Zeta. Methods Enzymol 401 : 61 77.

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133 Tzeng H, Blackburn AC, Board PG, and Anders MW (2000) Polymorphism and Species Dependent Inactivation of Glutathione Transferase Zeta by Dichloroacetate. Chem Res Toxicol 13 : 231 236 Villanueva CM Kogevinas M, Cordier S, Templeton MR, Vermeulen R, Nuckols JR, Nieuwenhuijsen MJ, and Levallois P (2014) Assessing Exposure and Health Consequences of Chemicals in Drinking Water: Current State of Knowledge and Research Needs. Environ Health Perspect 122 : 213 221. Wada K, Hiratake J, Irie M, Okada T, Yamada C, Kumagai H, Suzuki H, and Fukuyama K (2008) Crystal Structures of Escherichia coli Glutamyltranspeptidase in Complex with Azaserine and Acivicin: Novel Mechanistic Implication for Inhibition by Glut amine Antagonists. J Mol Biol 380 :361 372. Wallace DC (2012) Mitochondria and Cancer. Nat Rev Cancer 12 : 119 133. Warburg O, Wind F, and Negelein E (1927) The Metabolism of Tumors in the Body. J Gen Physiol 8 : 519 530. Widdowson EM and Dickerson JWT (1960) The Effect of Growth and Function on the Chemical Composition of Soft Tissues. Biochem J 77 : 30 43. Wong JYY, Huggins GS, Debidda M, Munshi NC, and De Vivo I (2008) Dichloroacetate Induces Apoptosis in Endometrial Cancer Cells. Gyencol Oncol 109 : 394 402. Ya ng H, Al Hertani W, Cyr D, Laframboise R, Parizeault G, Wang SP, Rossignol F, Berthier MT, Gigu re Y, Waters PJ, and Mitchell GA (2016) Hypersuccinylacetonaemia and Normal Liver Function in Maleylacetoacetate Isomerase Deficiency. J Med Genet 0 :1 7. Zhong G, Li W, Gu Y, Langaee T, Stacpoole PW, and James MO (2014) Chloride and Other Anions Inhibit Dichloroacetate Induced Inactivation of Human Liver GSTZ1 in a Haplotype Dependent Manner. Chem Biol Interact 215 : 33 39.

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134 BI OGRAPHICAL SKETCH Marci Smeltz was born in Harrisburg, Pennsylvania in 1989, the only child of Ronald and Elaine Smeltz. Her interests in the sciences developed early, finding particular intrigue to the investigative nature of forensic science and leaving an indelible impression on her young mind. Her fascination in the natural sciences, especially chemistry, influenced her to study forensic and investigative sciences, with a focus in forensic and drug chemistry, at West Virginia University. In 2011, she obtained dual BS degrees in chemistry and forensic science. While at West Virginia University Marci developed a strong interest in conducting academic research in an analytical forensic chemistry lab and afforded her many opportunities including being one of eleven students nationally recognized in 2009 for the Department of Homeland Security scholars program. This award allowed her exposure to government affiliated laboratory training at Savannah River National Laboratory in South Carolina for one year after completing her undergraduate studies. Yet, her thirst for knowledge motivated her to enter the Pharmaceutical Sciences graduate program offered by the College of Pharmacy at the University of Florida in f all 2012. Under the mentorship of Dr. Margaret O. James, Marci Smeltz received her doctorate in pharmaceutical sciences with concentrations in medicinal chemistry and toxicology in May 2017.