1 GLUTATHIONE TRANSFERASE Z1 CATALYZED BIOTRANSFORMATION OF DICHLOROACETATE ROLE S OF MITOCHONDRION, SUBJECT AGE, GSTZ1 HAPLOTYPE AND CHLORIDE INTERACTION By WENJUN LI 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 2011
2 2011 Wenjun Li
3 To my mom and dad, and those who have loved and supported me
4 ACKNOWLEDGMENTS Five years o f graduate school have helped me mature as a researcher and as an individual First and foremost, I would like to deliver my most sincere thanks to Dr. Margaret James, my Ph.D. mentor and role model during these years. I deeply appreciate the continuous su pport, encouragement and enlightenment Dr. James has provided throughout my Ph.D. study Her dedication fairness and integrity to work and her kindness and respect to people have and will always guide me in my own research and doings My gratitude extend s to my collaborators and committee members. Dr. Peter Stacpoole has provided me with expert insights into dichloroacetate research and firsthand information from clinical studies both of which motivated me to dig deeper into the study and reminded me of our goal of helping people. Dr s Ronald Hines and Taimour Langaee have both given me generous assistance in various aspects of the study. I am thankful to Dr s Ken Sloan Raymond Booth and Raymond Bergeron for assisting with the dissertation and the ir supp ort during graduate study I would like to thank all of m y fellow students and colleagues for their support and encouragements In particular, Yuan Gu has made substantial contributions to our study and Sriram Ambadapadi h as been so kind to hold our regula r coffee break s Special thanks goes to my fianc Zhuming Sun, who truly knows me and cares for me. Together, we walk through the happiness and bitterness of research and life. Finally, I am most grateful to my parents for their forever love, support and u nderstandings They are f riends and teachers of my life
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TABLES ................................ ................................ ................................ ............ 7 LIST OF FIGURES ................................ ................................ ................................ .......... 8 LIST OF ABBREVIATIONS ................................ ................................ ........................... 10 ABSTRACT ................................ ................................ ................................ ................... 12 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 14 DCA as an Environmental Agent ................................ ................................ ............ 14 DCA as a Therapeutic Agent ................................ ................................ .................. 15 DCA Biotransformation ................................ ................................ ........................... 17 GSTZ1 as the Metabolizing Enzyme ................................ ................................ 17 DCA Induced Inactivation of GSTZ1 ................................ ................................ 19 Discrepancy in studies of GSTZ1 inactivation ................................ ............ 20 GSTZ1 Polymorphism ................................ ................................ ...................... 20 DCA Pharmacokinetics ................................ ................................ ........................... 22 Overview of DCA Absorption, Distribution, Metabolism and Elimination .......... 22 Subject Age and GSTZ1 Genotype i n Determining DCA Pharmacokinetics .... 23 DCA Pharmacotoxicology ................................ ................................ ....................... 25 Postulated Mechanisms of DCA Toxicity ................................ .......................... 25 Clinical Pharmacotoxicology of DCA ................................ ................................ 26 Hypotheses ................................ ................................ ................................ ............. 27 2 MITOCHONDRION AS A NOVEL SITE OF DICHL OROACETATE BIOTRANSFORMATION BY GLUTATHIONE TRANSFERASE Z1 ....................... 35 Specific Aim ................................ ................................ ................................ ............ 35 Materials and Methods ................................ ................................ ............................ 35 Subcellular Fractionation of Human and Rat Livers ................................ ......... 35 Subfractionation of Liver Mitochondria ................................ ............................. 36 Electrophoresis and Western Blots ................................ ................................ .. 37 GSTZ1 Activity ................................ ................................ ................................ 38 Immunoprecipitation of Mitochondrial GSTZ1 ................................ .................. 39 LC MS/MS ................................ ................................ ................................ ........ 40 Protein Search Algorithm ................................ ................................ .................. 40 Results ................................ ................................ ................................ .................... 41 Expression and Activity of GSTZ1 in Hepatic Mitochondria ............................. 41 Mitochondrial GSTZ1 Is Localized in the Matrix ................................ ............... 42
6 L C MS/MS Identification of the Mitochondrial GSTZ1 ................................ ...... 43 Kinetic Study of Cytosolic and Mitochondrial GSTZ1 ................................ ....... 44 Discussion ................................ ................................ ................................ .............. 44 3 ROLES OF SUBJECT AGE AND HAPLOTYPE ON GSTZ1 EXPRESSION AND ACTIVITY WITH DICHLOROACETATE IN HUMAN LIVER ........................... 59 Specific Aim ................................ ................................ ................................ ............ 59 Materials and Methods ................................ ................................ ............................ 59 Human Liver Samples ................................ ................................ ...................... 59 GSTZ1 Activity with DCA ................................ ................................ .................. 60 Western Blot Analysis of GSTZ1 Expression ................................ ................... 60 Genotyping ................................ ................................ ................................ ....... 61 Data Analysis ................................ ................................ ................................ ... 62 Results ................................ ................................ ................................ .................... 62 Developmental Pattern of GSTZ1 in Human Liver Cytosol ............................... 62 Impact of Z1A Variant ................................ ................................ ....................... 63 Correlation Analysis of GSTZ1 Activity and Expression ................................ ... 64 Role of Haplotype on GSTZ1 Expression and A ctivity ................................ ..... 64 Role of Haplotype on Mitochondrial GSTZ1 ................................ ..................... 65 Discussion ................................ ................................ ................................ .............. 66 4 CHLORIDE MODULATES GSTZ1 HAPLOTYPE DEPENDENT INACTIVATION BY DICHLOROACETATE ................................ ............................. 75 Specific Aim ................................ ................................ ................................ ............ 75 Materials and Methods ................................ ................................ ............................ 75 Human Liver Samples and GSTZ1 Genotype Information ............................... 75 Assay of DCA Induced Inactivation of GSTZ1 in Human Liver Cytosol ............ 76 Data Analysis ................................ ................................ ................................ ... 76 Results ................................ ................................ ................................ .................... 77 Chloride Protected GSTZ1 from DCA Inactivation ................................ ........... 77 Time Course of GSTZ1 Inactivation by DCA ................................ .................... 77 Effects of Other Anions ................................ ................................ ..................... 78 Discussion ................................ ................................ ................................ .............. 78 5 CONCLUSION ................................ ................................ ................................ ........ 87 LIST OF REFERENCES ................................ ................................ ............................... 89 BIOGRAPHICA L SKETCH ................................ ................................ ............................ 98
7 LIST OF TABLES Table page 1 1 Specific activities and inactivation half lives of polymorphic variants of expressed recombinant hGSTZ1. ................................ ................................ ....... 32 1 2 Plasma kinetics of DCA in children and adults after single and repeated doses ................................ ................................ ................................ .................. 33 1 3 Pharmacokinetics of 25 mg/kg 1, 2 13 C DCA after 1 and 5 Doses in healthy adult volunteers. ................................ ................................ ................................ 34 2 1 Expression and activity of GSTZ1 in the liver cytosol and mitochondria of human, control rat, and DCA rat. ................................ ................................ ........ 56 2 2 Characteristics of unique spectra of tryptic peptides of human and rat mitochondrial GSTZ1 identified by ESI QTOF with 95% probability. .................. 57 2 3 Michaelis Menten parameters of the glutathione dependent biotransformation of DCA in the dialyzed cytosol and mitochondria of rat livers. ............................ 58 3 1 GSTZ1 expression and activity with DCA in cytosol and mit ochondria of individuals with various haplotypes. ................................ ................................ .... 74 4 1 Summary of GSTZ1 control activity with DCA and chloride concentration to achieve 50% protection of GSTZ1 from DCA inactivation (EC50) ..................... 84 4 2 Summary of GSTZ1 inactivation half lives (t 1/2 ). ................................ ................. 85 4 3 Effects of various anions in modulating GSTZ1 inactivation by DCA. ................. 86
8 LIST OF FIGURES Figure page 1 1 Site and mechanism of DCA action. ................................ ................................ ... 28 1 2 GSTZ1 cata lyzed A) dechlorination of DCA to glyoxylate and B) isomerism of MAA and MA respectively to FAA and FA. ................................ ......................... 29 1 3 Metabolic pathways of DCA. ................................ ................................ ............... 30 1 4 Phenylalanine/tyrosine catabolic pathways. ................................ ....................... 31 2 1 Representative Western blot of immunoreactive GSTZ1 in the liver cytosol and mitochondria of human, control rat, and DCA rat. ................................ ........ 50 2 2 The expression of GSTZ1 and the cross reacting protein in the mitochondria and cytosol isolated from fresh livers of control rats, and in the cytosol isolated from frozen livers of control and DCA treated rats (500 mg DCA/kg/day for 8 weeks). ................................ ................................ ................... 51 2 3 Structure of the mitochondrion. ................................ ................................ ........... 52 2 4 Enrichment of GSTZ1 expression an d activity in the matrix of rat liver mitochondria.. ................................ ................................ ................................ ..... 53 2 5 LC ESI QTOF analysis of the tryptic peptides of human and rat mitochondrial GSTZ1. ................................ ................................ ................................ ............... 54 2 6 MS/MS fragmentation of the peptide 41 DGGQQFSK 48 of human mitochondrial GSTZ1. ................................ ................................ ......................... 54 2 7 Lineweaver Burk plots of the rate of DCA biotransformation (v) versus the concentrations of A) cosubstrate GSH and B) subs trate DCA in the liver cytosol and mitochondria. ................................ ................................ ................... 55 3 1 Western blot analysis of GSTZ1 in 0.55 17.6 ng purified hGSTZ1C 1C and 5 and 10 g of one human liver cytosol (HL cyt). ................................ ............... 69 3 2 Scatter plot analyses of GSTZ1 A) protein expression and B) activity with DCA as a function of age. ................................ ................................ ................... 70 3 3 Box and whisker plot analyses of GSTZ1 A) protein expression and B) activity with DCA as a function of age groups. ................................ ................... 71 3 4 Correlation analysis of GSTZ1 protein expression and activity with D CA.. ......... 72 3 5 Role of GSTZ1 haplotype on A) protein expression and B) the ratio of DCA metabolizing activity to protein expression.. ................................ ....................... 73
9 4 1 Chloride protected human liver cytosolic GSTZ1 from DCA inactivation in a [Cl ] and GSTZ1 haplotype dependent manner.. ................................ ............... 82 4 2 The time course of GSTZ1 inactivation by DCA in the A) abs ence and B) presence of 38 mM KCl.. ................................ ................................ .................... 83
10 LIST OF ABBREVIATION S ALA delta aminolevulinate ALDH1A1 aldehyde dehydrogenase 1A1 AUC area under plasma concentration time curve CypD Cyclophilin D CytC Cytochrome C DCA dichloroacetate DME drug metabolizing enzyme ESI QTOF electrospray ionization hybrid quadrupole time of f light FA fumarylacetone FAA fumarylacetoacetate GSH glutathione GSTZ1 glutathione transferase zeta 1 HPLC high performance liquid chromatography IM inner membrane IMS intermembrane space LOD limit of detection MA maleylacetone MAA maleylacetoacetate MAAI m aleylacetoacetate isomerase MCA monochloroacetate MELAS mitochondrial myopathy, encephalopathy, lactic acidosis and stroke like episodes MWCO molecular weight cutoff OM outer membrane PDC pyruvate dehydrogenase complex
11 S D Sprague Dawley SNP single nucleot ide polymorphism t 1/2 elimination half life
12 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 GLUTATHIONE TRANSFERA SE Z1 CATALYZED BIOTRANSFORMATION OF DICHLOROACETATE ROLE S OF MITOCHONDRION, SUBJECT AGE, GSTZ1 HAPLOTYPE AND CHLORIDE INTERACTION By Wenjun Li May 2011 Chair: Margaret O. James Major: Pharmaceutical Science s Medicinal Chemistry and Toxicology Dichl oroacetate (DCA) is a potential environmental hazard and an investigational drug. By inhibiting pyruvate dehydrogenase kinase in the mitochondria, DCA remo dels cellular energy metabolism and there fore is studied for treatments of lactic acidosis and, recen tly, solid tumors. DCA inactivates its own metabolizing enzyme glutathione transferase Z1 (GSTZ1) Thus repeated doses of DCA result in reduced drug clearance an effect shown to be influenced by subject age and GSTZ1 haplotype In this study we investig ate the roles of mitochondrion, subject age, GSTZ1 haplotype and chloride ion on GSTZ1 catalyzed biotransformation of DCA in human liver We demonstrate d that mitochondrion is a novel site of DCA biotransformation catalyzed by GSTZ1, an enzyme co localized in cytosol and mitochondrial matrix. GSTZ1 activity with DCA was 2.5 3 fold higher in cytosol than in whole mitochondria, being proportional to its protein content in the two compartments. Rat mitochondrial GSTZ1 had a 2.5 fold higher App K m for glutathi one than cytosolic GSTZ1, whereas the App K m s for DCA were identical. DCA treatment at 500 mg/kg/day for 8 weeks inactivated liver mitochondrial and cytosolic GSTZ1 to a similar extent (~10% of control) in rat s
13 By studying human liver cytosols from 10 week s of gestation to 74 years of life, we demonstrate d age as the major determinant of GSTZ1 protein expression and activity with DCA during human liver development. With very low levels in the fetus, GSTZ1 expression and activity increased in the neonatal pe riod, rose further over the course of children development and attained variable but similar levels between ages 7 and 74. GSTZ1 haplotype showed no effect on protein expression but affected enzyme activity with DCA Z1A carriers in general possessed a ~ 3 fold higher activity with DCA than noncarriers at a given level of expression. Chloride, a major electrolyte in the body, was shown by the current study to protect GSTZ1 from DCA inactivation in a Cl concentration and GSTZ1 haplotype dependent manner. To achieve 50% protection of cytosolic GSTZ1 in a 2 h incubation with 0.5 mM DCA, 33.6 mM Cl was required for Z1C / Z1A subjects compared to ~13 mM for Z1C and Z1D homozygo us subjects At physiological concentration of chloride (38 mM), t he inactivation ha lf life of GSTZ1 was 2 fold longer in Z1C and Z1D homozygous subjects (over 5 h ) than Z1C / Z1A subjects (2.5 h ).
14 CHAPTER 1 INTRODUCTION DCA as a n Environmental Agent Dichloroacetate (DCA) is a ubiquitous molecule in our biosphere. Human population s can b e exposed to DCA through consumption of chlorinated drinking water and food bathing or swimming in chlorinated pools, or in vivo metabolism of certain xenobiotics and drugs. DCA is g enerated as a byproduct during water chlorination and, is detected in U. S. municipal water supplies at concentrations of 30 160 g/ L (Mughal, 1992) P eople are estimated to be expos ed to a few g DCA / kg body weight daily by drinking municipal water DCA is also detected in swimming pool water and some raw and processed foods (WHO, 2005) ; it can be formed during the in vivo metabolism of certain pharmaceuticals, such as chloral hydrate (Henderson et al., 1997) as well as widely used industrial solvents, trichloroethylene and perchlo roethylene (Monster, 1986) both of which may be released into the atmosphere and contaminate surface water. However, as the rate and extent of DCA absorption via these routes ha ve not been determined, their contribution to DCA environmental exposure le vel remain s unclear To an environmental toxicologist, DCA is considered to be an environmental carcinogen based on its ability to induce liver tumors in i nbred strains of rodents at doses hundreds to thousands of times higher than levels encountered throu gh the environment (Bull, 2000) Several mechanisms have been postul ated for the carcinogenic effect of DCA: peroxisom e proliferation hypomethylation and mutagenicity of DNA, glycogen accumulation, and reparative hyperplasia following injury (Bull, 2000; WHO, 2004) However, the r elevance of the se findings in rodents to humans is lacking
15 So far, no evidence of DCA causing neoplasia in humans has been reported. The potential health risks of DCA at environmental levels have been recently reviewed and data do not support DCA to be a hazard to general populations at large (Stacpoole, 2011) DCA as a Therapeutic Agent As a therapeutic agent, DCA was first synthesized a s an ion complex with diisopropylammonium in 1952 by Krebs and Krebs during the exploration of Vitamin B15 (pangamic acid) (Stacpoole, 1969) Since then the pharmacological effects of DC A have been extensively stud ied and the therapeutic and investigational uses of DCA keep expanding. Administ ered as the diisopropylammonium salt during the 60s and as the sodium salt later on, DCA has been used clinically over 50 years for the treatment of several metabolic and cardi ovascular disorders, including diabetes mellitus, hyperlipoproteinemia, and lactic acidosis (Stacpoole, 1989) Recently, DCA was studied in several clinical trials for chronic treatment of genetic mitochondrial diseases, such as congeni tal lactic acidosis p yruvate dehydrogenase complex (PDC) deficiency, and mitochondrial myopathy, encephalopathy, lactic acidosis and stroke like episodes (MELAS) (Barshop et al., 2004; Berendzen et al., 2006; Kaufmann et al., 2006; Stac poole et al., 2006; Stacpoole et al., 2008) As a metabolic modulator, DCA is currently being investigated for anti proliferative and pro apoptotic effects in pulmonary arterial hypertension and cancer (McMurtry et al., 2004; Michelakis et al., 2008) The rationale for DCA therapy is based on its ability to efficiently stimulate the activity of pyruvate dehydrogenase complex (PDC). PDC is a multi enzyme complex that catalyzes the irreversible oxidation of pyruvate to acetyl coenzyme A (CoA) (Figure 1 1). Pyruvate is a pivotal molecule in the cellular carbon fuel and energy metabolism.
16 Produced from glucose by glycolysis in the cytosol, pyruvate is converted to lactate and alanine in cytosol, or is transported into m itochondria and oxidized to acetyl CoA. Under aerobic conditions, this equilibrium is determined by the activity of PDC, which is in turn regulated by reversible phosphorylation. PDC is phosphorylated to the inactive state by PDC kinase and dephosphorylate d to the active state by PDC phosphatase. DCA state, and increasing the oxidation of pyruvate to acetyl CoA. As a result, DCA promotes the flux of pyruvate into mitoch ondria to enter the Krebs cycle and simultaneously reduces the amount of alanine and lactate in the cytosol. Because reducing equivalents of NADH and FADH 2 are generated during PDC catalyzed reaction and Krebs cycle the respiratory chain is in turn stimu lated to generate reactive oxygen species and ATP. Such effects on restoring mitochondrial energy production and thus overcoming the Warburg effect contribute to the emerging roles of DCA as a metabolic modulator in cancer and pulmonary arterial hypertensi on (Archer et al., 2008; Michelakis et al., 2008) For therapeutic uses, sodium DCA is administered at 10 mg/kg to over 100 mg/kg intravenously or by mouth for a few days to over several years. Patients treated wit h DCA are reported to develop occasional asymptomatic hepatotoxicity and more frequently a peripheral neuropathy. T he reversible peri pheral sensory motor neuropathy is the major adverse effect that limits chronic DCA use and, o ccurs more frequently in adu lts than children (Kaufmann et al., 2006; Stacpoole et al., 2006; Stacpoole et al., 2008) Clinical manifestations include tingling of the fingers and toes and weakness of
17 the facial and distal muscles of the extre mities; however, the mechanism of this toxicity is still under investigation DCA Biotransformation GSTZ1 as the Metabolizing Enzyme The initial s tep of DCA biotransformation is the dechlorination to glyoxylate. This reaction is glutathione (GSH) dependent and catalyzed exclusively by glutathione transferase zeta 1 (GSTZ1), a member of the GST superfamily. GST superfamily consist s of two distinct and structurally unrelated groups: the soluble GSTs and the microsomal and membrane bound GSTs. GSTZ1 belongs to the soluble GST s which constitute up to ~10% of cellular protein and is a major family in Phase II drug metabolizing enzymes (DME s). To date, seven classes of soluble GSTs have been identified in mammals namely GST Alpha, Mu, Pi, Theta, Zeta, Omega and Sigma (Mannervik et al., 2005) Liver c ytosol is the principal location for the soluble GSTs. Recently, several members of soluble GSTs, e.g. GST A4, P1, M1 and O1 we re also found to be present in mitochondri a and/or nucleus (Hayes et al., 2005; Gallagher et al., 2006; Goto et al., 2009) Like other soluble GSTs, GST Z 1 is present in the cytosol and expressed most abundantly in the liver (Lantum et al., 2002a) GSTs are dimeric proteins in nature Both homodimers and heterodimers of different subunits within the same class have been observed in tissue So far, only one subunit of the zeta class, i.e. Z1, has been identified in ma mmalian species. Accordingly, only the homodim eri c form of this protein i.e. GST Z1 1, is known (Board et al., 1997; Polekhina et al., 2001) For convenience we use GSTZ1, which in essence refers to its homodimer in the remain der of the document.
18 Members of GSTs catalyze a variety of reactions, including conjugation, addition elimination and isomerism These reactions are dependent on GSH the cofactor of GSTs. C ommon substrates, such as 1 chloro 2,4 dinitrobenz ene and ethacrynic acid exist for GSTs. However, GSTZ1 has little or no activity with these prototypical substrates (Board et al., 1997) Instead, it catalyzes the dehalogenation of halo carboxylic acids (Tong et al., 1998a) For example, GSTZ1 converts dichloroacetate and chlorofluoroacetate to glyoxylate (Figure 1 2A ) This difference in substrate selectivity can be explained, in part, by the structural features of GSTZ1 active site. Reveal ed by the crystal structure, the hGSTZ1 active site is a lot smaller and composed of more polar residues than other GSTs, which may contribute to its preference for relatively small and polar substrates, such as short chain halo acids (Polekhina et al., 2001). Id entified by the bioinformatics approaches in the late 90s, GSTZ1 is proven identical to the long known maleylacetoacetate isomerase (MAAI) (Blackburn et al., 1998; Fernandez Canon and Penalva, 1998) It isomerizes m aleylacetoacetate (MAA) and its analog maleylacetone (MA) respectively to fumarylacetoacetat e (FAA) and fumarylacetone (FA) at the penultimate step of tyrosine/phenylalanine catabolism (Figure 1 2B ). H uman recombinant GSTZ1 possesses hundreds to thousands fold higher specific activities for the endobiotic substrate MA than for DCA and other xenobiotic substrates (Table 1 1) (Board and Anders, 2005) S pecies differences in DCA biotranformation are observed in that V max /K m for mouse liver cy tosol was 1.6 fold and 6.4 fold higher than those for rat and human, respectively (Tong et al., 1998a)
19 DCA I nduced Inactivation of GSTZ1 In vivo studies of DCA in rats demonstrate that DCA treatment reduces the protein expression and activity of liver cytosolic GSTZ1 in a do se and duration dependent manner (Anderson et al., 1999; Cornett et al., 1999) At low er doses (4 and 12.5 mg/kg /day ), 5 days of treatment were required to reduce GSTZ1 activity to ~50 80% of control level Wherea s 60% i nhibition of GSTZ1 was achieved by a single dose of 50 mg/kg DCA and over 90% inhibition by 1000 mg/kg DCA (Cornett et al., 1999) Restoration of GST Z1 activity occurred gradually over days to weeks after cessation of DCA treatment (Anderson et al., 1999; Guo et al., 2006) In all cases, changes in GST Z1 activity are positively correlated to its protein express ion without affecting mRNA level (Anderson et al., 1999; Ammini et al., 2003; Guo et al., 2006) demonstrated time and DCA concentration dependent inactivation of GST Z1 in vitro using recombinant enzymes and liver cytosols from human, rat and mouse. The reaction required GSH and could not be reversed by dialysis. The same lab reported a GSTZ1 protein adduct formed between the active site residue Cys16 and a proposed me tabolic intermediate of DCA (Anderson et al., 2002) This adducted GSTZ1 was suggested to be inactive and undergo rapid proteolysis in vivo based on the observed parallel decreases in protein activity and immunoreactivity after DCA administration (Anderson et al., 1999) Together with the unaffected level of mRNA, DCA induced inactivation of GSTZ1 appeared to be wholly a post transcriptional event, probably through covalent modification of GSTZ1 followed by rapid degradation.
20 Discrepancy in studies of GSTZ1 inactivation T he inactivation half life of human liver cytosol ic GSTZ1 was reported to be 22 min by incubating with 0.5 mM DCA and 5 mM GSH in potassium phosphate buffer (Tzeng et al., 2000) In stark contrast, a previous study from our lab found no loss of GSTZ1 activity at all in human liver cytosol after 30 min incubation with the same concentration of DCA and 1 mM GSH in Hepes buffer (Cornett et al., 1999) Careful examination of the assay conditions revealed the presence of chloride ion in our protein samples but which was ca used by the differen ce in dialysis buffer compositions Chloride is a major electrolyte in the human body. It is present at ~100 mM in the serum and ~ 38 mM in the liver for adults under normal physiological condition (Widdowson and Dickerson, 1960; Morrison, 1990) Defects in chloride homeostasis are implicated in a number of diseases, including but not limited to cystic fibrosis, kidney dysfunctions and neurological disorders (De Koninck, 2007; Edwards and Kahl, 2010) Thus, literature has largely been focused on the flux of chloride across membrane s and cellular events triggered following the flux with regard to chloride function (Edwards and Kahl, 2010) In addition chloride modulation of protein function ha s been documented. These include both positive and negative effects on enzyme activity (Garcia Espana et al., 1991; Meijer et al., 1992) drug re ceptor interaction (Tavoulari et al. 2009) and protein oligomerization (Kitani and Fujisawa, 1984; Sasaki et al., 2009) GSTZ1 Polymorphism Genetic p olymorphism of DMEs is well recognized as a major contributor to the inter individual differences in drug response. N on synonymous single nucleotide polymorphisms (SNPs) can occur in the coding or regulatory region of a gene and thus result in a n altered function and/ or expression of the gene product (protein) Several
21 classes of GST enzymes, e.g. A, M T and P, are encoded by genes with functional polymorphism s Most notably, individuals carrying null alleles of GST M1 and T 1 have been suggested to be associated with an increased risk of certain cancer s and some adverse health outcomes (Hayes et al., 2005) For example, a recent meta analysis of GST polymorphism and colorectal cancer risk showed an increased risk in GSTM1 and GSTT1 null alle le carriers in Caucasian populations (Economopoulos and Sergentanis, 2010) To date five non synonymous SNPs have been reported in the coding region of human GSTZ1 gene. These include four SNPs (T23C, Leu8Pro; G94A, Glu32Lys; G124A Gly42Arg; and C245T Thr82Met ) identified through analysis of expressed sequence tag database (Blackburn et al., 2000; Blackburn et al., 2001) and one newly discovered SNP (G 295 A Val99Met ) during human DCA pharmacokinetic study ( Shroads et al., 2011 ) Except for L8P GSTZ1 haplotypes bearing other SNPs ha ve been foun d in population s with varying frequency : EGT (Z1C ) is the most common variant (~50%), followed by KGT ( Z1B ) (~30%) and EGM (Z1D) (~15%), while KRT (Z1A) (~5%) and KGM (0.4%) are the rarest. The V 99M variant was identified just recently in one individual be aring EGM however, it s occurrence in population s has not been studied. The recombinant proteins of the four common GST Z 1 variants EGT, KGT, EGM and KRT, exhibit different catalytic activities with a range of substrates (Board and Anders, 2005) (Table 1 1 ). The wild type EGT allele ha d the highest activity with endogenous substrate MA followed by KGT while KRT and EGM ha d 80% lower activities. With chloroflu o roacetate, similar activities we re observed among variants.
22 Nevertheless, the le s s frequently occurring variant KRT ha d 4 5 fold higher specific activity with DCA as substrate than the other three variants, for which the activities we re similar. Interestingly, different rates in DCA inactivation we re also observed between variants in t hat the inactivation half life was twice as long with KRT as the other s Thus, it was postulated that the higher specific activity of KRT for DCA may due to its slower rate of inactivation (Tzeng et al., 2000) Polymorphisms in the regula tory region which may modulate gene transcription and expression, have been shown to contribu t e to the inter individual variation s in drug response (Hines et al., 2008) Fang et al. (2006) identified 10 SNP s in the promo ter region of GSTZ 1 from a sampled population but found no significant regulatory effects. DCA Pharmacokinetics Overview of DCA Absorption, Distribution, Metabolism and Elimination A t ypical therapeutic dose range of DCA in human s is 25 100 mg/kg/day for short term and chronic treatment (Stacpoole et al., 1998) Due t o its high affinity for the monocarboxylate transport system that facilitates cellular uptake, DCA is quickly absorbed after oral administration and has a bioavailability approaching unity at therapeutic doses (Stacpoole et al., 1998) Stimulation of PDC activity generally occurs minutes after oral or parenteral DCA ad ministration, and DCA peak plasma level is reache d within 30 min p.o. (Stacpoole et al., 2003) Consistent with mitochondria being the site of action, DCA is a substrate for mitochondrial pyru vate transporter so that it competes with pyruvate for entry into mitochondria (Figure 1 1 ). Muscle and liver appear to be the major tissues of DCA distribution (James et al., 1998; Stacpoole et al., 1998) One hou r after a single oral dose of 50 mg/kg 14 C DCA to overnight fasted S D rats, muscle and liver accumulated 22.5% and 16.3% of the dose,
23 respectively, followed by gastrointestinal tract (9.26%), fat tissue (4.58%), and kidney (1.37%). After dosing for 24 h radioactivity decreased in all tissues, accompanied by marked increases of radioactivity in expired CO 2 (37.2%), urine (9.8%) and feces (1.39%) (James et al., 1998) The amount and distribution of DCA and its metabolites during excretion are shown to be dependent on the dosage and dosing schedule of DCA, as well as the species, age, and nutrition al status of the subject. Nevertheless, CO 2 is the major metabolite identified in vivo accounting for 19 44% of the DCA dose (Larson and Bull, 1992; Lin et al., 1993; James et al., 1998) DCA undergoes extensive metabolism after oral and intravenous administration so that very little parent DCA is recovered in the urine after a single dose (Larson and Bull, 1992; Jam es et al., 1998; Ammini et al., 2003; Guo et al., 2006) DCA is primarily dechlorinated to glyoxylate in the liver by GSTZ1 (Tong et al., 1998b) Glyoxylate can be further metabolized to glycine, glycine conjugate s, oxalate, glycolate, and CO 2 by various enzymes in the cytosol and mitochondria (Figure 1 3 ). Less than 1% of a DCA dose can be reductively dechlorinated to the potential neuro toxin, monochloroacetate (MCA) in the circulation through a not yet known mec hanism (Shroads et al., 2008) Interestingly, the ratio of DCA reductive dechlorination to MCA did not increase in GSTZ1 knock out mice even though the primary biotransformation pathway of DCA to glyoxylate was imp aired (Ammini et al., 2003) Therefore, the alternative biotransformation of DCA to MCA probably has a minor role in the metabolism and toxicity of DCA. Subject Age and GSTZ1 Genotype in Determining DCA Pharmacokinetics After two decades of research on DCA pharmacokinetics, it is now well recognized that DCA inhibits its own metabolism and elimination by inactivating the met abolizing
24 enzyme GSTZ1. Therefore, DCA exhibits marked increase s in area under plasma concentration time curve (AUC) and elimination half life ( t 1/2 ) after just one or multiple prior dos es a phenomenon shown in human s regardless of gender, age, ethnicity, state of health, or route of administration (Curry et al., 1991) Recently, subject age and GSTZ1 genotype emerge d as important determinants of DCA pharmacokinetics. S tudy of chronic DCA treatment in children wit h heterogeneous causes of congenital lactic acidosis (mean age 5.2 years at entry, n = 5) and adults with MELAS (mean age 24 years at entry, n=4) revealed a striking age dependent decrease in DCA plasma clearance. With the same dosing regime (12.5 mg/kg/12 h ) and similar kinetic indices after first DCA dose, adults exhibited 10 fold in crease in elimination t 1/2 and over 20 fold increase in AUC compared to the 3 and 4 fold increases, respectively, in children at the end of 6 months (Table 1 2). Treatment of S D rats with 25 mg/kg/day DCA for 1 and 5 days reproduced similar age related differences at DCA pharmacokinetics at day 5 (Shroads et al., 2008) In addition to age, Shroads et al. ( 2011 ) recently reported the use of GSTZ1 haplotype as a criterion to segregate individuals to fast and slow DCA metabolizers. After 5 days of 25 mg/kg/day DCA, healthy adult subjects carrying at least one copy of EGT exhibited 3 fold faster plasma clearance than EGT non carriers, esp ecially those harboring G42R and/or T82M SNPs (Table 1 3) Similarly, EGT carriers in children with genetic mitochondrial diseases exhibit faster clearance of DCA than their EGT non carrier counterparts after 12 months of treatment at 12.5 mg DCA/kg/12 h
25 DCA Pharmacotoxicology Postulated Mechanisms of DCA Toxicity Reversible peripheral neuropathy is the major adverse effect in chronic DCA treatment. Although the mechanism is not yet elucidated recent studies suggest that DCA induced inactivation of GSTZ1/ MAAI and subsequent disruption of phenylalanine/tyrosine catabolism may contribute to DCA toxicity. P henylalanine and tyrosine cataboli c pathway is involved in seve ral inborn errors of metabolism (Figure 1 4) Hereditary tyrosinemia type 1, caused by defi ciency in the ultimate enzyme fumarylacetoacetate hydrolase, is the most severe form with symptom s of severe damage in hepatic, renal and nervous system s (Russo et al., 2001) The p eripheral neuropathy that occurs both in patients with hereditary tyrosinemia type 1 and DCA treatment has been attributed in part to the elevated levels of neurotoxin delta aminolevulinate ( ALA) and alkylating agents MAA and MA due to loss of functions of fumarylacetoacetate hydrolase or GSTZ1. MAA and its decarboxylated product MA are known alkylating agents that can attack g lutathione and other thiols (Lantum et al., 2002b) C ytotoxicity of MA was suggested to involve oxidative stress a s shown in mouse hepatocytes and splenocytes (Lantum et al., 2003; Theodoratos et al., 2009) Additionally, accumulated MAA and FAA can be converted to succinylacetoacetate and succinylacetone. Succinylacetone is a potent inhibitor of ALA dehydratase (Kic ~0.15 M) that catalyzes the oxidation of ALA to porphobilinogen during an early step in heme biosysnthesis. ALA has been implicated as a neurotoxin in several neuropathies either through direct action as a pro oxidant or subsequ ent perturbation of heme biosynthesis (Felitsyn et al., 2008) As a
26 result of succinylacetone action the concentration of ALA is increased in DCA and tyrosinemia patients possibly contributing to the nerve toxicity. Clinical Pharmacotoxicology of DCA DCA associated peripheral neuropathy occurs frequently in adult patients treated for genetic mitochondrial diseases, gliob lastoma multiforme, and MELAS. St udy of adult MELAS patients (mean age of entry was 30 years) with 12.5 mg DCA/kg/12 h ended prematurely due to significant worsening of drug associated neuropathy in some individuals after a few weeks of treatment (Kaufmann et al., 2006) In contrast, children (mean age of entry was 5.6 years) with heterogeneous causes of congenital lactic acidosis showed no worsening of hepatotoxicity or neurotoxicity after 6 months of trea tment at the same DCA dose (Stacpoole et al., 2006) Urinary concentrations of MA and ALA have been used as the biochemical indicators of DCA toxicity due to their aforementioned properties. Although urinary concentrations of MA and ALA were increased in children with lactic acidosis at the end of 6 months treatment, the ir levels were lower than tho se found in tyrosinemia patients (Shroads et al., 2011 ) A s tudy of S D rats treated with DCA 25 mg/kg/day for 5 days reproduced a similar age dependent toxicity and demonstrated higher concentration s of MA in both urine and plasma of old r ats compared with young rats (Shroads et al., 2008) In addition to age, GSTZ1 genotype was also shown to modulate MA level in humans receiving DCA treatment After 5 days of 25 mg/kg DCA, urinary MA was not detect able in healthy volunteers carrying at least one EGT allele but appeared in 5 out of the 6 EGT non carriers (Shroads et al., 2011 )
27 Hypothes e s This study has been designed to test the following hypothes e s. Firstly, DCA is known to be actively taken up int o the mitochondrion, where it exerts its pharmacological action by inhibiting pyruvate dehydrogenase kinase. Several members of soluble GSTs have recently been shown to dual localize in cytosol and mitochondria. We test ed the hypothesis that GSTZ1 was also present in the mitochondria and the mitochondrial GSTZ1, if present, was capable of metabolizing DCA. Second ly, a ge and genetic polymorphism are two major determinants of DMEs that cause altered pharmacokinetics and toxicity. The human GSTZ1 gene is known to exhibit functional polymorphism s Given the observed difference s in DCA pharmacotoxicology between children and adults, and betwe en EGT carriers and noncarriers, w e test ed the hypothesis that the expression and activity of GSTZ1 were higher in children than adults and were subject to modulation by gene tic polymorphi sm Third ly, w ith chloride present in liver cytosol we failed to detect GSTZ1 inactivation under otherwise similar condition s as those reported by Tzeng et al. (2000). Chloride modulation o f protein structure and activity has been documented in the literature We thus test ed the hypothesis that chloride modulate d DCA induced inactivation of GSTZ1 and that the potency of chloride modulation varied among GSTZ1 haplotype s
28 Figure 1 1. Sit e and mechanism of DCA action.
29 A B Figure 1 2. GSTZ1 catalyzed A) dechlorination of DCA to glyoxylate and B) isomerism of MAA and MA respectively to FAA and FA
30 Figure 1 3 Metabolic pathway s of DCA.
31 Figure 1 4. Phenylalanine/t yrosine catabolic pathways.
32 T able 1 1. Specific activities and inactivation half lives of polymorphic variants of expressed recombinant hGSTZ1 GSTZ1 Haplotype Maleylacetone (mol/min/ mg) a Chlorofluroacetate (mol/min/mg) a DCA (mol/min/mg) a Half life of DCA inactivation (min) b KRT 318 91 1.35 0.05 1.61 0.02 23 1 KGT 1010 217 1.34 0.03 0.45 0.02 9.6 0.3 EGT 1856 716 1.29 0.05 0.45 0.03 10.1 0.5 EGM 464 215 1.27 0.08 0.30 0.025 9.5 0.3 a Data from Blackburn et al. (2001) b Data from Tzeng et al. (2000)
33 Table 1 2. Plasma kinetics of DCA in children and adults after single and repeated doses Parameter First dose Six months Children Adults C hildren Adults No. subjects 5 4 5 4 Age (yr) 5.2 1.8 24 10 5.7 1.8 24.5 10 Elimination t 1/2 ( h ) 2.5 0.4 2.1 1.5 6.4 3.4 21 5.8 C max (g/ml) 23 9.1 25 6.6 35 10 53 18 AUC (h*g/ml) 83 33 70 18 340 130 1500 700 Clearance 150 60 180 46 37 14 8.3 4.6 Data were mean SD of results obtained after the first DCA dose and after 6 months of daily exposure to 12.5 mg/kg DCA every 12 h (Adapted from Shroads et al., 2008)
34 Table 1 3 Pharmacokinetics of 25 mg/kg 1, 2 13 C DCA after 1 and 5 Doses in healthy adult volunteers. After 1st DCA Dose After 5th DCA Dose C max AUC t 1/2 CL C max AUC t 1/2 CL Subject Age/Sex/Race GSTz1/MAAI Genotype (g/ml) (g/ml min) (min) (ml/min) (g/ml) (g/ml min) (min) ( ml/min) EGT Carriers 1 24/F/White EGT/EGT 27.2 8211 79 3.04 36.1 21703 305 1.15 (2.6)* 2 25/F/White EGT/EGT 60.3 8644 97 2.89 27.9 11705 218 2.14 (1.4) 3 24/M/White EGT/EGT 19.4 1313 46 19.05 28.5 7899 122 3.17 (6.0) 4 23/M/Black EGT/EGT 41. 5 3689 37 6.78 32.7 12612 232 1.98 (3.4) 5 26/F/Asian EGT/KGT 20.4 2622 36 9.54 22.6 11783 310 2.12 (4.5) 6 23/F/Black EGT/KRT 18.1 1423 80 17.57 28.5 7934 126 3.15 (5.6) 7 25/F/White EGT/KRT 14.4 1538 102 16.25 22.8 13715 323 1.82 (8.9) EGT Non car riers 8 25/F/White KRT/KGT 31.8 3366 41 7.43 27 .0 11746 243 2.13 (3.5) 9 37/M/White KGT/KGT 24.7 4048 102 6.18 26.5 29214 727 0.86 (7.2) 10 33/F/White KRT/KRT 12.4 1493 96 16.74 33.1 79525 1592 0.31 (54.0) 11 21/M/White KRT/EGM 49.4 5301 47 4.7 2 35.3 98683 1774 0.25 (18.9) 12 26/M/White KGM/KGT 27.8 78165 1264 0.32 40.8 302977 5408 0.08 (4.0) Values in parentheses denote fold change in clearance ( CL ) between first and fifth doses. Adapted from Shroads et al., 2011
35 CHAPTER 2 MITOCHONDRION AS A NOVEL SITE OF DICHLOROACETATE BIOTRANSFORMATION BY GLUTATHI ONE TRANSFERASE Z 1 Specific Aim GSTZ1 is a member of the cytosolic GST superfamily 1 Like other GSTs, GSTZ1 is present in the cytosol and is most abundantly expressed in the liver (Lantum et al., 2002a) Over the past decade, increasing numbers of drug metabolizing enzymes have been shown to exist in multiple subcellular compartments. These include several cytosolic GSTs (GSTA1, GSTA2, GSTA4, GSTP1, and GSTM1) that were recently found to be co localized in the hepatic mitochondria (Raza et al., 2002; Gallagher et al., 2006) Because DCA is known to be taken up by the mitochondria, we tested the postulate that thi s organelle is also a site of DCA biotransformation by examining the expression and activity of GSTZ1 in liver mitochondria from humans and rats. Materials and Methods Subcellular F ractionation of Human and Rat L ivers Adult female Sprague Dawley (SD) rats were treated by gavage with tap water vehicle (n=9, control group) or 500 mg/kg/day DCA (n=11, DCA treated group) for 8 weeks. Neuropathy was confirmed by measuring sciatic motor nerve conduction velocity and paw thermal response latency exactly as describ ed elsewhere followed by sacrifice by decapitation (Calcutt et al., 2009) Studies were performed following approval by the local IACUC. De identified normal human liver samples, 1 2 g, were collected during surger y under a protocol approved by Institutional Review Board of Shands Hospital at 1 This work was published previously by Wenjun Li, Margaret O. James, Sarah C. McKenzie, Nigel A. Calcutt, Chen Liu, and Peter W. Stacpoole, Mitochondrion as a Novel Site of Dichloroacetate Biotransformation by Glutathione Transferase 1, J Pharmac ol Exp Ther 2011 336:87 94. Copyright 2011 by The American Society for Pharmacology and Experimental Therapeutics. Reprinted with permission of the American Society for Pharmacology and Experimental Therapeutics. All rights reserved.
36 the University of Florida (Gainesville, FL) for use in these studies. Livers were quickly removed, snap frozen in liquid nitrogen and stored at 80C until use. Frozen liver w as thawed and rinsed in ice cold homogenizing buffer (0.25 M sucrose, 0.02 M Hepes NaOH pH 7.4 and 0.1 mM phenylmethanesulfonyl fluoride). Rinsed livers were minced and homogenized in 5 volumes of homogenizing buffer with a motor driven Teflon pestle for 4 complete strokes. After sedimenting the nuclei and cell debris at 600 X g, mitochondria were pelleted by centrifuging the supernatant at 13,000 X g for 20 min. The 13,000 x g supernatant was further subjected to differential centrifugation to isolate cyto sol and washed microsomes (James et al., 1976) The mitochondrial pellet was resuspended and washed twice before being taken up in the resuspension buffer (0.25 M sucrose, 0.01 M Hepes NaOH pH 7.4, 0.1 mM dithiothr eitol, 0.1 mM EDTA, 0.1 mM phenylmethanesulfonyl fluoride and 5% glycerol) in a volume equal to the liver weight. The washed mitochondria and cytosol were stored in aliquots at 80C until use. All procedures were performed at 4C or on ice. Cytosol and mi tochondria were dialyzed with 10 kD MWCO Slide A Lyzer Dialysis Cassettes (Thermo Scientific, Rockford, IL) against 1.15% KCl and 0.05 M potassium phosphate buffer pH 7.4 before use for assays. Protein concentrations were determined by the Bradford method (Bio Rad, Hercules, CA) using bovine serum albumin (BSA; Sigma Aldrich, St. Louis, MO) as protein standard. Subfractionation of Liver M itochondria Three 9 week old male SD rats were killed by decapitation and their livers were quickly isolated and rinsed in ice cold homogenizing buffer to remove blood. The washed mitochondria were immediately isolated by procedures described above and subfractionat ed as follows. The suspension of washed mitochondria was diluted with
37 swelling buffer (0.01 M Tris HCl pH 7.4) to a final sucrose concentration of ~0.05 M and gently mixed with a magnetic stirrer at 4C for 15 min. Shrinking buffer (2 M sucrose, 100 mM Tr is HCl pH 7.4) was then added to the swelled mitochondria to give a final sucrose concentration of ~0.3 M. Afte r another 15 min stirring, the swollen and shrunk (shocked) mitochondria were centrifuged at 20,000 X g for 20 min. The supernatant yielded the intermembrane space (IMS) proteins. The pellet of shocked mitochondria was washed once resuspended in homogeniz ing buffer and stored at 80C. After 3 cycles of freezing and thawing, the shocked mitochondria were further subjected to 3 strokes of homogenization by a Dounce homogenizer and 5 cycles of 5 sec sonication at 25 sec intervals. The shocked mitochondria we re then centrifuged at 125,000 X g for 60 min to sediment the total mitochondrial membrane with the mitochondrial matrix remaining in the supernatant. The pellet containing the total mitochondrial membrane was resuspended in resuspension buffer. The IMS pr otein and matrix protein were concentrated by filtering through Amicon Ultra 15 ml filters of 10 kD MWCO (Millipore, Billerica, MA). Electrophoresis and Western B lots Known amounts of denatured protein were separated on 4 15% SDS PAGEs (Tris HCl Gel, Bio Rad) and subsequently electrotransferred onto polyvinylidene fluoride membrane ( PVDF, Millipore). Purified recombinant human GSTZ1 C 1 C was obtained as previously described (Guo et al., 2006) and used as positive c ontrol. After blocking, the membrane was incubated overnight at 4C with primary antibodies 1:2000 rabbit polyclonal anti hGSTZ1 C 1 C (Cocalico Biologicals, Inc., Reamstown, PA), 10.2 g/ml mouse monoclonal MitoProfile Membrane Integrity WB Antibody C ocktail (MitoSciences, Eugene, OR) or 1:2000 rabbit monoclonal anti ALDH1A1 (Abcam,
38 Cambridge, MA). The membrane was then washed and incubated with the corresponding secondary antibodies, horseradish peroxidase conjugated donkey anti rabbit IgG, 1:2000 or sheep anti 2 1:5000 (GE healthcare, Piscataway, NJ). P rotein signal was developed by Pierce ECL substrate (Thermo Scientific) on For quantitative analysis, the resulting hyperfilm was digitized by scanning and the density of bands was quantitated by ImageJ software (version 1.41o, National Institute of Health, USA). GSTZ1 A ctivity The specific activity of GSTZ1 was measured using [1 14 C ] DCA ( American Radiolabeled Chemicals, Inc., St. Louis, MO) as substrate and assay products were measured by a n HPLC method coupled with radiochemical detection (James et al., 1997) Assay conditions were optimized to achieve linearity of product formation with incubation time and protein concentration; substrate consumptio n did not exceed 15%. To determine the maximum reaction rate, assay tubes containing 0.2 mM 14 C DCA in 0.1 M Hepes NaOH pH 7.6 were incubated with either cytosolic protein and 2 mM GSH or mitochondrial protein and 5 mM GSH in a volume of 0.1 ml. After a 2 min pre incubation at 37C, the reaction was started by adding 14 C DCA. Tubes were incubated at 37 o C with gentle shaking for 15 min, after which the reaction was stopped by adding 0.1 ml ice cold methanol. For the sub mitochondrial experiment, enzyme speci fic activity was measured with 0.2 mM DCA and 2 mM GSH over 30 min incubation for all fractions. For kinetic studies cytosol and mitochondria were incubated with varying concentrations of GSH and DCA over adjusted incubation time s as specified in the resu lts. The kinetic parameters (V max and K m ) were derived with the software GraphPad
39 Prism version 4.03 (San Diego, CA) by fitting the data to the Michaelis Menten equation, V = V max [S]/(K m + [S]). G oodness of fit to the Michaelis Mention equation was determi ned by the software. Immunoprecipitation of M itochondrial GSTZ1 Soluble mitochondrial protein of human liver and mitochondrial matrix protein of rat liver were used for immunoprecipitation of GSTZ1. To obtain the soluble mitochondrial protein of human live r, the washed human liver mitochondria were frozen and thawed three times, and then diluted to about 2 mg/ml in homogenizing buffer. After sonication and homogenization, t he disrupted mitochondria were centrifuged at 125,000 X g for 60 min to pellet the to tal mitochondrial membrane. The soluble mitochondrial protein was collected from the supernatant and further concentrated using Amicon Ultra 15 ml filters of 10 kD MWCO. Immunoprecipitation of mitochondrial GSTZ1 was carried out using the Pierce Direct IP kit (Thermo Scientific) following the 10 g of hGSTZ1 C 1 C antibody was coupled to 30 l of a 50% slurry of AminoLink Plus coupling resin. Mitochondrial protein, 500 g, was incubated with the antibody coupled resin overnight at 4C. After centrifuging the mixture to remove unbound proteins, the resin was washed with IP lysis/wash buffer supplied by the kit. The antigen was then eluted with the primary amine containing, pH 2.8 elution buffer supplied by the kit. Eluants were neutralized with 1 M Tris HCl pH 9.5 in a 10:1 ratio. For each species, eluants from three columns were pooled and concentrated through Amicon Ultra 0.5 ml filters of 10 kD MWCO (Millipore). The concentrated eluants were denatured and separ ated on 4 15% SDS PAGEs. Protein bands from the rat or human samples that
40 migrated to the same position as the positive control, purified hGSTZ1 C were excised and digested with trypsin (Sheffield et al., 2006) for LC MS/MS protein identification. LC MS/MS The trypsin digested samples were inj ected onto a capillary trap (LC Packings PepMap; Dionex Corporation, Sunnyvale, CA) and washed for 5 min with a flow rate 10 l/min of 0.1% v/v acetic acid. The samples were loaded onto a LC Packings C18 PepMap HPLC column (Dionex Corporation, Sunnyvale, CA). The elution gradient of the HPLC column started at 97% solvent A (0.1% v/v acetic acid, 3% v/v acetonitrile and 96.9% v/v water), 3% solvent B (0.1% v/v acetic acid, 96.9% v/v acetonitrile and 3% v/v water) and finished at 40% solvent A, 60% solvent B for 60 min. LC MS/MS analysis was carried out on a hybrid quadrupole TOF mass spectrometer (QSTAR, Applied Biosystems, Framingham, MA). The focusing potential and ion spray voltage were set to 2 75 V and 2600 V, respectively. The information dependent acq uisition (IDA) mode of operation was employed in which a survey scan from m/z 400 1200 was acquired followed by collision induced dissociation of the three most intense ions. Survey and MS/MS spectra for each IDA cycle were accumulated for 1 and 3 sec, res pectively. Protein Search Algorithm T andem mass spectra were extracted by ABI Analyst version 1.1. All MS/MS samples were analyzed using Mascot (Matrix Science, London, UK; version 2.2.2). Mascot was set up to search the International Protein Index (IPI) d atabase with trypsin as the digestive enzyme. Mascot was searched with a fragment ion mass tolerance of 0.30 Da and a parent ion tolerance of 0.30 Da. Iodoacetamide derivatization of Cys, deamidation of Asn and Gln, oxidation of Met were specified in Masco t as variable modifications. Scaffold 2 (version Scaffold 02.03.01, Proteome Software Inc., Portland,
41 OR) was used to validate MS/MS based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater th an 95.0% probability, as specified by the Peptide Prophet algorithm (Keller et al., 2002) Protein identifications were accepted if they could be established at greate r than 99.0% probability and contained at least 2 identified unique peptides. Protein probabilities were assigned by the Protein Prophet algorithm (Nesvizhskii et al., 2003) Results Expression and A ctivity of GSTZ1 in H epatic Mitochondria In liver mitochondria of both human and rat, our hGSTZ1 antibody cross reacted with a protei n that migrated similarly to the purified hGSTZ1 and the cytosolic GSTZ1 at ~24 kD (Fig ure 2 1 ). GSTZ1 immunoreactivity was more intense in cytosol than in mitochondria for a given amount of protein, indicating a more abundant expression of GSTZ1 in cytoso l. To assure that the detection of mitochondrial immunoreactive GSTZ1 was not due to cytosolic contamination, mitochondrial purity was established by demonstrating minimal detection of the cytosolic marker aldehyde dehydrogenase 1A1 (ALDH1A1). Cytosolic ex pression of GSTZ1 was 2.4 fold and 3.9 fold higher than mitochondrial in human and rat livers, resp ectively, on a per milligram of protein basis (Table 2 1). This is consistent with the 2.5 3 fold higher activity of GSTZ1 in cytosol than in mitochondria, as determined using 14 C DCA as substrate. About 86% [human: 40.1/(40.1+6.78); rat: 0.92/(0.92+0.14)] of cellular GSTZ1 was located in cytosol and 14% was located in mitochondria, based on the higher yield of protein from cytosol than mitochondria and assu ming that 30 mg mitochondria were present per gram liver (Fleischer et al., 1979)
42 GSTZ1 is known to be inactivated by DCA after repeated exposure (Cornett et al., 1999) This was confirmed by the marked reduc tion in both the expression and activity of cytosolic GSTZ1 in rats treated with DCA 500 mg/kg/day for 8 weeks (Figure 2 1 and Table 2 1). The expression of mitochondrial GSTZ1 in these rats was also reduced to ~10% of control levels and the specific activ ity was reduced to below the detection limit (Table 2 1). Coincidentally, we observed a strong induction of cytosolic marker ALDH1A1 in the DCA treated rats compared to control rats. Our GSTZ1 antibody cross reacted with an unknown protein in rat mitochond ria that appeared 1 2 kD larger than GSTZ1 on SDS PAGE (Fig ure 2 1). The cytosolic expression of this cross reacting protein changed from not detectable in fresh control livers to barely detectable in frozen control livers to readily detect able in fr ozen DCA treated livers (Figure 2 2). This pattern of expression is similar to those of mitochondrial matrix protein cyclophilin D (CypD) and intermembrane space protein cytochrome C (CytC). Preliminary investigation of this protein by immunoprecipitation and LC MS/MS protein identification suggested that it was a mitochondrial matrix protein with less than 3% sequence identity to hGSTZ1 (NCBI Blastp). This result was further supported by its submitochondrial localizatio n in the matrix (see below, Figure 2 4 A) Mitochondrial G STZ1 I s L ocalized in the M atrix The mitochondrion has a double membrane structure that divides the organelle into four compartments: the outer membrane (OM), the intermembrane space (IMS), the inner membrane (IM) and the matrix (Figure 2 3) To investigate if DCA biotransformation occurs in the same mitochondrial compartment as its pharmacodynamic action, we examined GSTZ1 expression and activity in the washed mitochondria and three sub mitochondrial fractions: IMS, the matrix and the membr an es
43 (including OM and IM) (F igure 2 4 ). GSTZ1 expression and catalytic activity were greatest in the matrix, being nearly 3 times higher than in washed mitochondria. Low levels of GSTZ1 expression and activity were found in the membrane fraction, which migh t be due to incomplete release of matrix protein from the shocked mitochondria during fractionation. However, neither expression nor activity was detectable in the IMS. We also confirmed that GSTZ1 exists in cytosol, but not microsomes Similar levels of G STZ1 expression were detected in cytosol and mitochondrial matrix. However, the cytosolic marker ALDH1A1 was only detected in cytosol but not in mitochondrial matrix. This also indicated that the presence of GSTZ1 in mitochondria was not due to cytosolic c ontamination. LC MS/MS I dentification of the M itochondrial GSTZ1 The identity of the mitochondrial GSTZ1 was verified by immunoprecipitating the antibody reactive proteins from human and rat liver mitochondria and analyzing the tryptic pepti de sequences by LC ESI QTOF We used the matrix protein of rat mitochondria and the soluble protein of human mitochondria for immunoprecipitation. The immunoprecipitated GSTZ1 of human liver mitochondria was identified with 3 unique peptides in 4 unique spectra, covering 12% (27/216) of the hGSTZ1 protein sequence ( Figure 2 5 and Table 2 2). Although the coverage was relatively low, a clear ladder of fragmentation was shown in the peptide, 41 DGGQQFSK 48 (Figure 2 6 ), which increased our confidence of the protein identifica tion. Of the immunoprecipitated rat GSTZ1, 2 unique peptides covering 13% (28/216) of the rat GSTZ1 protei n sequence were identified (Figure 2 5 and Table 2 2).
44 Kinetic S tudy of C ytosolic and M itochondrial GSTZ1 Rat mitochondrial GSTZ1 had a 2.5 fold highe r App K m for GSH than the cytosolic GSTZ1, whereas the App K m s for DCA were identical (Table 2 3). With either GSH or DCA as the variable substrate, the App V m ax values of cytosolic GSTZ1 were 3 times those of mitochondrial GSTZ1, in good accordance with the 3.9 fold higher expression of cytosolic GSTZ1 per mg protein (Table 2 1). Lineweaver Burk plots of cytosolic GSTZ1 and mitochondrial GSTZ1 with GSH and DCA as substrates were shown for one represent ative rat in Figure 2 7 Discussion The mitochondrion is t However, its role in DCA biotransformation has been largely overshadowed by the prominence of cytosol and endoplasmic reticulum in drug metabolism. In this study we demonstrate the mitochondrion to be a seco nd site of DCA biotransformation in a reaction catalyzed by GSTZ1, an enzyme co localized in the mitochondrial matrix and cytosol. Furthermore, GSTZ1 expression and activity in the liver mitochondria are susceptible to DCA inactivation, as occurs with the cytosolic form of GSTZ1. We also verified partial sequences of mitochondrial GSTZ1 by LC MS/MS and compared the kinetics between cytosolic and mitochondrial GSTZ1 using DCA and GSH as substrates. The level of GSTZ1 expression is similar in cytosol and mito chondrial matrix, whereas it is about 70% less in intact mitochondria on a per milligram of protein basis. Although a low level of cytosolic contamination was to be expected in the liver mitochondria isolated by differential centrifugation, we confirmed th e authenticity of mitochondrial GSTZ1 by demonstrating minimal co detection of cytosolic marker
45 ALDH1A1. Moreover, we would expect to detect any cytosol originated GSTZ1 in the IMS fraction, which was obtained from the first supernatant of osmotically shoc ked mitochondria. In fact, GSTZ1 was detected in the mitochondrial matrix at an intensity similar to that in cytosol, but without detectable ALDH1A1 (Fig ure 2 4 ). This evidence firmly established the mitochondrial origin of GSTZ1. In light of this finding, we searched the literature for GSTZ1 identification from studies of mitochondrial proteome. Indeed, GSTZ1 was identified in the mitochondrial proteome of mouse liver, heart, and kidney (Mootha et al., 2003) Consistent with our finding, GSTZ1 was shown in the proteome of mitochondrial matrix but not int er membrane space of rat liver (Forner et al., 2006) Proteins targeted to the mitochondrial matrix usually possess a 15 40 amino acid long presequence at the N terminal that forms an amphipathic helix to inter act with mitochondrial transport machinery during translocation and the presequence is often cleaved off upon import. A few matrix targeting proteins with non cleaved presequences or C terminal presequences have also been identified (Pfanner and Geissler, 2001) All GSTs (GSTA1, GSTA2, GSTA4, GSTP1 and GSTM1) that are currently known to co localize in cytosol and mitochondrial matrix have similar molecular sizes and identical N terminals in both compartments (Raza et al., 2002; Gallagher et al., 2006) Mitochondrial import studies of GSTA4 and GSTM1 suggest non cleaved internal targeting sequences at the C terminal and N terminal, respectively, of the mature GST proteins (Robin et al., 2003; Goto et al., 2009) Furthermore, protein kinase A (PKA) and protein kinase C (PKC) catalyzed phosphorylation has been shown to facilitate GSTA4 translocation to the matrix (Robin et al., 2003) The present study of cytosolic and mitochondrial GSTZ1 also suggests their similar molecular weights, as shown by
46 the indistinguishable rates of migration on SDS PAGEs. To obtain more insight into protein structure, we attempted direct N terminal sequencing for human mitochondrial GSTZ1 immobilized on PVDF membrane. However, the study failed due to the blocked N terminus, which was also observed for rat cytosolic GSTZ1 (Tong e t al., 1998b) On the other hand, one tryptic peptide of human mitochondrial GSTZ1 identified by LC MS/MS shared the same identity as the C terminal amino acids of cytosolic GSTZ1, suggesting that the C termin us of GSTZ1 remained intact after mitochondria l import. Therefore, if a mitochondrial targeting sequence of GSTZ1 exists, it may reside within the mature protein and not undergo proteolysis upon import. GSTZ1 of both cytosol and mitochondria catalyzes GSH dependent dechlorination of DCA, exhibiting t he same App K m s (0.033 mM) for DCA, but a 2.5 fold difference in App K m s for GSH (0.50 mM mitochondria vs 0.19 mM cytosol). Of cytosolic GSTZ1, the App K m for GSH obtained currently using female SD rats is 2.5 fold higher than we previously reported using mal e SD rats (James et al., 1997) and is 3.2 fold higher than f old higher App K m for DCA was also found (Tong et al., 1998a) The higher App K m for GSH observed with mitochondrial GSTZ1 suggests that it has a weakened access or binding to GSH compared to its cytosolic counterpart. Inspection of the crystal structure of hGSTZ1 A 1 A with GSH revealed that GSH was bound in a deep crevice with close interaction with the active site residues Ser14 Ser15 Cys16 near the N terminus (Polekhina et al., 2001) Cys16 was found to be particularly important in mai ntaining proper binding and orientation with GSH. Mutation of Cys16 to Ala caused dramatic increases in K m s for GSH with various substrates (Board et al., 2003; Ricci et al., 2004) As demonstrated in mouse GSTA4,
47 the mitochondrial form is more heavily phosphorylated than the cytosolic form while possessing the same primary protein sequence (Robin et al., 2003) If post translational modification is involved in the GSTZ1 tra nslocation to mitochondria, modifications of residues around the GSH binding pocket may alter the conformation of this region in mitochondrial GSTZ1 and thus contribute to the reduced GSH affinity. Nevertheless, GSH is present at concentrations (2 10 mM) w ell above the K m values in cytosol and mitochondria (Hansen et al., 2006) ; therefore, the difference in K m for GSH should not affect or limit the rates of DCA dechlorination in either compartment under physiological conditions. Inspection of the DCA m etabolic pathway reveals that all but one of the enzymes involved in secondary biotransformation are located in the mitochondria ( Figure 1 3 ). Our study establishes a novel role of the mitochondrion in DCA primary biotransformation, which may allow efficie nt degradation of glyoxylate in mitoc hondria and therefore explain the fact that a large majority of DCA metabolites observed in vivo are derived from mitochondrial pathways of secondary biotransformation (Lin et al ., 1993; James et al., 1998) However, generation of glyoxylate in the mitochondria may perturb the mitochondrial redox homeostasis because glyoxylate is an electrophile and may react with cellular macromolecules ( Anderson et al., 2004) DCA induced inactivation of GSTZ1 was observed in both cytosol and mitochondria of rats treated with 500 mg/kg/day of DCA for 8 weeks. However, the question remains as to whether or not lower environmental and therapeutic doses of DCA would affect GSTZ1 similarly in the two compartments.
48 We unexpectedly observed a marked induction of cytosolic ALDH1A1 in the livers of DCA treated rats, which we speculate to be caused by an increased level of oxidative stress in the livers due to DC A treatment. ALDH1A1 is a cytosolic and inducible isoform of the aldehyde dehydrogenase family that catalyzes the oxidation of medium chain aliphatic aldehydes, including 4 hydroxynonenal and malondialdehyde (Alnouti and Klaassen, 2008) This is noteworthy because elevated levels of these indices of lipid peroxidation have recently been found in the sciatic nerve s of rats treated with the same dose of DCA as used in the present study (Calcutt et al., 2009) Furthermore, increased production of reactive oxygen species and lipid peroxidation have been demonstrated in the livers of mic e treated with high doses of DCA (Larson and Bull, 1992; Hassoun et al., 2010) Overexpression of ALDH1A1 has been shown to be an adaptive response to oxidative stress (Chou dhary et al., 2005; Leonard et al., 2006) Therefore, we tentatively attribute the induced expression of cytosolic ALDH1A1 to be a secondary response to DCA exposure. The GSTZ1 antibody cross reacting protein was shown to be a rat mitochondrial matrix pro tein but exhibited varying degrees of expression in the cytosol depending on the liver condition. Its appearance in the cytosol of frozen control liver could be due to mitochondrial membrane lesions caused by freezing the liver and thawing it on ice (Pallotti and Lenaz, 2007) It is notable that the expression of this protein was further increased in the DCA treated cytosol. We speculate that this altered expression is a combined result of freez e thawing the liver a nd further leakage of mitochondrial protein due to DCA treatment DCA can induce oxidative stress (Larson and Bull, 1992;
49 Hassoun et al., 2010) a condition detrimental to mitochondrial membrane integrity (Fulda et al., 2010) In conclusion, we demonstrate that the mitochondrion known to be the site of DCA pharmacological action on PDC is also a site of DCA biotransformation. The reaction i s catalyzed by GSTZ1, an enzyme co localized in the cytosol and the mitochondrial matrix. GSTZ1 of both compartments is inactivated by high doses of DCA and exhibits the same App K m s for DCA, but different App K m s for GSH. The discovery of this organelle as a second site of DCA biotransformation provides a new perspective on understanding DCA metabolism
50 Figure 2 1 Representative Western blot of immunoreactive GSTZ1 in the liver cytosol and mitochondria of human, control rat, and DCA rat. Shown are cyto sol and mitochondria of one individual from each group. The DCA rat was exposed to 500 mg/kg/day DCA for 8 weeks before preparation of subcellular fractions. A rabbit polyclonal antibody against human GSTZ1C 1C was used. Aldehyde dehydrogenase 1A1 (ALDH1A1 ) was used as a cytosolic marker to monitor mitochondria purity. hGSTZ1, purified recombinant human GSTZ1C 1C. Protein loading of each sample is indicated on the figure.
51 Figure 2 2 The expression of GSTZ1 and the cross reacting protein in the mitocho ndria (lanes 1 and 2) and cytosol (lanes 3 and 4) isolated from fresh livers of control rats, and in the cytosol isolated from frozen livers of control (lanes 5 and 6) and DCA treated (lanes 7 and 8) rats (500 mg DCA/kg/day for 8 weeks). The lower film was exposed for a prolonged period of time to show the change in the expression of the cross reacting protein. Complex V subunit a (CVa) and Complex III subunit Core 1 are protein markers for mitochondrial inner membrane (IM), Porin for mitochondrial outer me mbrane (OM), Cyclophilin D (CypD) for mitochondrial matrix, and Cytochrome C (CytC) for mitochondrial intermembrane space (IMS). Results from two rats are shown for each group. Each lane contains equal amounts of protein.
52 Figure 2 3. Structure of the mitoc hondrion. (Copyright by Molecular Expressions Reproduced with permission. )
53 A B Figure 2 4 Enrichment of GSTZ1 expression and activity in the matrix of rat liver mitochondria. A) Representative Western blot of GSTZ1 expression in t he subcellular and sub mitochondrial fractions of rat liver. Each fraction was loaded with equal amounts of protein and the fraction identities were confirmed by the predominant expression of respective marker proteins: ALDH1A1 for cytosol (Cyt), Complex V subunit a (CVa) and Complex III subunit Core 1 for mitochondrial inner membrane (IM), Porin for mitochondrial outer membrane (OM), Cyclophilin D (CypD) for mitochondrial matrix, and Cytochrome C (CytC) for mitochondrial intermembrane space (IMS). Mito., w ashed mitochondria; Memb., mitochondrial membranes ; Mics., microsomes. B) GSTZ1 activity in the cytosol and sub mitochondrial fractions of rat livers measured with 0.2 mM 14C DCA as substrate. Data are shown as mean S.D. of 3 rats. N.D., not detectable.
54 A B Figure 2 5 LC ESI QTOF analysis of the tryptic peptides of human and rat mitochondrial GSTZ1. A) Amino acid sequence of human GSTZ1 ( NCBI accession no. NP_665877). B) Amino acid sequence of rat GSTZ1 (NCBI accession no. NP_001102915). Peptide f ragments identified by MS are highlighted in yellow and modified amino acids are highlighted in green. Figure 2 6 MS/MS fragmentation of the peptide 41 DGGQQFSK 48 of human mitochondrial GSTZ1.
55 A B Fig ure 2 7 Lineweaver Burk plots of t he rate of DCA biotransformation (v) versus the concentrations of A) cosubstrate GSH and B) subst rate DCA in the liver cytosol ( ) and mitochondria ( ).The lines were constructed using K m and V max value s obtained from Michaelis Menten equation, and intercept 1/V max on Y axis and 1/K m on X axis. Data are shown as mean SEM of assay duplicates from one representative rat.
56 Table 2 1. Expression and activity of GSTZ1 in the liver cytosol and mitochondri a of human, control rat, and DCA rat. GSTZ1 expression per gram liver a GSTZ1 expression per mg protein a GSTZ1 activity (nmol/min/mg) c Cytosol b Mitochondria b Cytosol Mitochondria Cytosol Mitochondria Human (n=4) 4 0. 1 11 .8 6 78 1 11 0 54 0. 10 0. 23 0.0 4 0.49 0.08 0.20 0.07 Control rat (n=4) 0.92 0.06 0.14 0.01 0.94 0.07 0.24 0.02 1.47 0.09 0.48 0.02 DCA rat (n=4) 0.11 0.03 0.012 0.003 0.10 0.02 0.021 0.006 0.11 0.06 d N.D. a GSTZ1 expression was analyzed using 5 g human cytosol, 5 g mitochondria, 40 g rat cytosol and 100 g rat mitochondria on Western blots probed with hGSTZ1 polyclonal antibody. Band intensity was quantitated by ImageJ software. For human samples, the amounts of GSTZ 1 were quantitated by comparing the immuno intensity to a standard curve constructed with pure hGSTZ1. Data are shown as g hGSTZ1 / g liver or g hGSTZ1 / mg protein. For rat samples, individual GSTZ1 expression was normalized as a fraction of one control cytosol that had h ighest level of GSTZ1 expression. Data are shown as relative GSTZ1 expression / g liver or relative GSTZ1 expression / mg protein. b Cytosolic yield was calculated based on the total cytosolic protein recovered experimentally. Mitochondrial yield was stan dardized to 30 mg mitochondrial protein per gram liver to correct for loss of mitochondria during differential centrifugation (Fleischer et al., 1979) c Samples were dialyzed in 1.15% KCl, 0.05 M potassium phosphate buffer pH 7.4 before being assayed with 0.2 mM 14 C DCA as substrate. d Of the cytosol of f our DCA rats, only two showed detectable activities. The limit of detection was 0.067 nmol/min/mg. Data are shown as mean S.D. N.D., not detectable.
57 Table 2 2 Characteristics of unique spectra of tryptic peptides of human and rat mitochondrial GSTZ1 i dentified by ESI QTOF with 95% probability a Peptide sequence b Modifications identified by spectrum Mascot ion score Observed m/z Actual peptide mass (AMU) Spectrum charge Actual minus calculated peptide mass (AMU) Human 41 DGGQQFSK 48 56.8 433.6 4 865.27 2 0.12 49 DFQALNPMK 57 Oxidation (+16) 43.2 540.12 1,078.23 2 0.28 207 QPDTPTELRA 216 Pyro Glu c ( 17) 49.0 555.71 1,109.39 2 0.14 207 QPDTPTELRA 216 42.5 564.24 1,126.46 2 0.10 Rat 41 GGQQFSEEFQTLNPMK 57 Oxidation (+16) 37.9 657.8 7 1970.60 3 0.27 134 AITSGFNALEK 144 57.1 575.73 1149.44 2 0.16 a Probability score was calculated by Scaffold 2 software. b Underlined amino acids were identified by MS with modifications. c Pyroglutamate (Pyro Glu) formed from cyclization of N termi nal glutamine.
58 Table 2 3. Michaelis Menten parameters of the glutathione dependent biotransformation of DCA in the dialyzed cytosol and mitochondria of rat livers. GSH b DCA c App K m App V max App K m App V max (mM) (nmol/min/mg) (mM) (nmol/min/mg) Rat m itochondria a 0.50 0.12 0.56 0.12 0.033 0.002 0.57 0.01 Rat cytosol a 0.19 0.04** 1.67 0.14 0.033 0.005 1.66 0.15 a Rat liver mitochondria and cytosol were dialyzed at 4C overnight with three changes of 1.15% KCl, 0.05 M potassium phosph ate buffer pH 7.4. b With 0.2 mM DCA as substrate, cytosol was incubated with 0.0 5 2.5 mM GSH for 10 min and mitochondria were incubated with 0.1 7.5 mM GSH for 15 min. c With DCA concentration range of 0.005 0.2 mM, cytosol was incubated with 2 mM GSH for 5 min and mitochondria were incubated with 5 mM GSH for 10 min. ** p < 0.01 compared to App K m of mitochondria for GSH, analyzed by one tailed t test. Data are shown as mean S.D. of 3 rats.
59 CHAPTER 3 ROLES OF SUBJECT AGE AND HAPLOTYPE ON GSTZ1 EXPRESSION AND ACTIVITY WITH DI CHLOROACETATE IN HUM AN LIVER Specific Aim Developmental changes and genetic polymorphisms of drug metabolizing enzymes are respectively recognized as the major contributing factors to age related and inter individual differ ences in the pharmacotoxicology of many drugs. Several classes of GSTs (GSTA, GSTM, and GSTP) have been shown to exhibit distinct patterns of changes in their hepatic expression during human development (Strange et al., 1989) Studies using recombinant human GSTZ 1 have revealed that the Z1A ( KRT ) variant possessed different kinetic properties and rates of inactivation with DCA as substrate and inhibitor respectively, compared with other haplotypes. Given the observed differences in pharmacokinetics and toxicity b etween children and adults, and between Z1A and Z1D carriers and non carriers, we investigated the role of subject age and haplotype in determining the hepatic expression and activity of GSTZ1 in human liver cytosol samples ranging from 10 weeks of gestati on to 74 years of life. Materials and Methods Human L iver S amples A total of 2 49 human liver samples ranging from 10 weeks of gestation to 74 years of life w ere used in the current study. Human liver cytosol including 6 1 pre natal samples and 167 post nata l samples was obtained from a liver bank at the Medical College of Wisconsin (Milwaukee, WI). Donor characteristics and subcellular fraction preparations have been described previously (Koukouritaki et al., 2002) The other 21 post natal samples were de identified human livers, of which 14 were obtained from Vanderbilt University (Nashville, TN) and 7 were collected during surgery at Shands
60 Hospital at University of Florida (Gainesville, FL). Liver cytosol and mitoc hondria of these samples was prepared and dialyzed exactly as described in Chapter 2 The pellet of cell nuclei was used to isolate DNA for genotyping and haplotype analysis. Protein concentration was determined by BCA Protein Assay (Thermo Fisher Scientif ic, Waltham, MA). GSTZ1 A ctivity with DCA The specific activity of GSTZ1 was measured with [1 14 C]DCA as substrate and assay products were analyzed by HPLC coupled radio activity flow detector as previousl y described (James et al., 1997) Stock [1 14 C]DCA (55 mCi/mmol with 99% purity; American Radiolabeled Chemicals, Inc., St. Louis, MO) was converted to the sodium sa lt by addition of NaHCO 3 and diluted with unlabeled DCA (TCI America, Portland, OR). Human liver cytosol, 0.2 to 0.6 mg, was incubated with 1 mM glutathione, 0.1 M Hepes NaOH pH 7.6 and a saturating concentration of DCA at 0.2 mM in an assay volume of 0.1 ml. The reaction was started by adding DCA, allowed to proceed for 15 min at 37C with gentle shaking and stopped by adding 0.1 ml ice cold methanol. All assays were performed in duplicates; substrate consumption did not exceed 15%. The specific activity o f GSTZ1 was expressed as nmol glyoxylate formed/min/mg protein. The limit of detection ( LOD ) was 0.0147 nmol/min/mg. Samples with activity below LOD were imputed a value equal to the LOD divided by the square root of 2, i.e. 0.010 nmol/min/mg. Western B lo t A nalysis of GSTZ1 E xpression Known amounts of protein (5 to 60 g) were separated on 4 15% SDS PAGEs (Tris HCl Gel; Bio Rad Laboratories, Hercules, CA) and subsequently electrotransferred onto polyvinylidene fluoride membrane (Millipore Corporation, Bill erica, MA). The
61 amount of cytosol used was guided by GSTZ1 activity found in DCA assay. After blocking, the membrane was incubated with rabbit polyclonal anti hGSTZ1C 1C 1:5000 (Cocalico Biologicals, Inc., Reamstown, PA) then with horseradish peroxidase c onjugated donkey anti rabbit IgG 1:2000 (GE Healthcare, Piscataway, NJ). Protein signal was developed by ECL Plus Western Blotting detection reagents (Thermo Fisher Scientific) on Amersham Hyperfilm ECL (GE Healthcare). Films were digitized by scanning and the density of bands was analyzed by QuantiScan Software (Biosoft, Cambridge, UK). A GSTZ1 standard curve was constructed using 0.55 17.6 ng of purified recombinant hGSTZ1C 1C (Guo et al., 2006) A designated huma n liver cytosol, 5 g, was used as a reference control on each blot. The signal of each individual test sample was normalized against the reference control for analysis. The GSTZ1 content of samples was calculated by fitting data to the GSTZ1 standard curv e and expressed as g GSTZ1 per mg cytosolic or mitochondrial protein. Data were analyzed using Microsoft Office Excel 2007. The LOD was 0.0092 g GSTZ1/mg protein. Samples with expression below LOD were imputed a value equal to the LOD divided by the squa re root of 2, i.e. 0.0065 ng GSTZ1/g protein. Genotyping DNA from selected liver samples was subjected to PCR followed by pyrosequencing (Langaee and Ronaghi, 2005) targeting the thre e known GSTZ1 non synonymous single nucleotide polymorphisms (SNPs): G94>A, Glu Lys at amino acid position 32; G124>A, Gly Arg at position 42; and C245>T, Thr Met at position 82. Genotyping analysis was carried out using a PSQ HS 96 System (Biotage, Uppsal a, Sweden). Haplotypes were inferred from the unphased data by computational methods (PHASE software version 2.0.2) (Stephens et al., 2001)
62 Data Analysis Age breakpoints were determined by results obtained from regression tree analysis that gave the least deviation within groups and maximum variance betwe en groups ( CART v6, Salford Systems, San Diego, CA ). Outliers were defined as having specific contents 1.5 fold interquartile range away from the lower or upper quartiles. Statistical comparisons between age groups were performed using Kruskal Wallis nonpa rametric test, followed by stepwise step down comparisons ( IBM SPSS Statistics 19, Chicago, IL) A p value < 0.05 was accepted as significant. Data analyses and Loess curve fit were performed on IBM S PSS Statistics 19 Correlation analysis between GSTZ1 ac tivity and expression included samples with specific contents above the LODs and was determined v4.03, San Diego, CA). Results Developmental P attern of GSTZ1 in H uman L iver C ytosol To quantitate GSTZ1 protein ex pression, we compared GSTZ1 immunoreactivity in individual liver cytosol to a standard curve constructed with purified recombinant hGSTZ1C ( Figure 3 1 ). The activity of GSTZ1 was evaluated using the probe substrate DCA at a saturating concentration of 0.2 mM. Overall, GSTZ1 protein expression increased with age after birth and was paralleled by the increas e in its activity with DCA (Figure 3 2 ). Scatter plot analyses assisted with Loess curve fitting (not shown) suggested four phases of GSTZ1 development: 1 ) undetectable or very low levels in the fetus; 2) a rapid rise after birth to near half of adult levels by age 1; 3) the increase continued gradually over the course of children development; and 4) GSTZ1 expression
63 and activity were sustained at a stable level from late adolescen ce to age 74, the oldest sample studied. During pregnancy, fetal liver exhibited undetectable to very low levels of cytosolic GSTZ1 expression (<0.05 g GSTZ1/mg protein) and activity (<0.1 nmol glyoxylate/min/mg). A slight increa se was shown for GSTZ1 activity from 2 nd trimester (13 28 wk gestation) to 3 rd trimester (28 40 wk) (median 0.023 nmol/min/mg, n = 37 vs 0.042 nmol/min/mg, n = 11; p = 0.012, Mann Whitney U test) but not for expression. GSTZ1 developmental changes bet ween age groups were quantitated by dividing samples into 5 age brackets based on results of regression tree analysis. Steady and significant increases in both GSTZ1 protein expression (Figure 3 3A) and DCA metabolizing activity (Figure 3 3B) were observed from prenatal samples to samples of >7 74 Y In particular, a 3 fold increase in mean values was shown from the age bracket of >0 1 M to >1M 1Y. L ess than 2 fold increases were shown from >1M 1Y to >1 7Y and from >1 7Y to >7 74Y. Comparison o f samples between o lder children (>7 21Y) and adults (>21 74Y) showed no further change with age for either expression or activity. Impact of Z1A V ariant Several outliers were observed in the box and whisker plot analyses of GSTZ1 expression and activ ity. Of particular interest, 6 out of the 7 activity outliers in the >7Y 74Y bracket carried one allele of Z1A (Figure 3 3B) Plotting all known Z1A carriers in the scatter plots demonstrated a major impact of this allele on possessing high DCA metaboliz ing activity but not protein expression especially in samples aged 7 and above (Figure 3 2) No specificity was observed for the outliers of GSTZ1 expression.
64 Correlation A nalysis of GSTZ1 A ctivity and E xpression A fair correlation of r 2 = 0.51 was obtain ed between GSTZ1 expression and activity with DCA when all postnatal samples with specific contents above the LODs were analyzed However, a few individuals clearly segregated from the main body by exhibiting 2 to 3 fold higher activity with DCA than othe rs at si milar levels of expression (Figure 3 4 ). In accord with the observed impact of the Z1A variant those high activity individuals carrie d at least one allele of Z1A Subsequent grouping of the genotyped individuals into Z1A carriers or noncarriers im proved the correlations to r 2 = 0.90 and 0.68, respectively. Therefore, GSTZ1 activity with DCA is dependent on both the level of protein expression and the haplotype of GSTZ1. Role of H aplotype o n GSTZ1 E xpression and A ctivity We further analyzed the rol e of each haplotype in determining GSTZ1 activity and expression. Individuals carrying only Z1C ( EGT ) and/or Z1B ( KGT ) alleles were grouped together due to their lack of difference in activity or expression (analysis not shown). Z1A ( KRT ) carrier, Z1 D ( EGM ) carrier and Z1A / Z1 D heterozygo te were grouped separately due to a recent study indicat ing low in vivo activity by carriers of Z1 D and Z1A alleles (Shroads et al., 2011) an d the current study showing high in vitro activity by Z1A allele In liver cyto sol of individuals aged 3 to 74, GSTZ1 haplotype had no effect on protein expression as shown by the similar medians of different haplotype groups (Figure 3 5A) On the other h and, Z1A allele when paired with Z1C allele clearly conferred a larger ratio of GSTZ1 activity to expression compared to other alleles demonstrating higher DCA metabolizing activity at a given level of prote in expression (Figure 3 5B). Two samples in our liver bank were homozygous for Z1A, however, both
65 were age d less than 1 M. These two 1A/1A homozygotes like 1C/1A heterozygotes, also exhibited relatively large ratios of activity to expression (ratio = 2.9 and 3.8). The two 1D/1A heterozygotes, although having similar ratios of activity to expression, exhibited distinct levels of expression: one was near the medians of other haplotypes and the other was expressed at a very low level. In addition to the low expression 1D/1A heterozygote, two individuals of 1B/1B and 1B/1C haplotype also possessed very low GSTZ1 content for their ages (5 11 Y ) (Figure 3 5A) A rare haplotype, KRT/KGT was identified in an individual of 17 days old. This individual, however, had essentially no expression nor activity of GST Z1 in that GSTZ1 immunoreactivity was invisible on its Western blot and the activity in assay was identical to blank, neither of which changed by assaying increased amou nts of protein. Resequencing the GSTZ1 gene of these individuals for the newly identifi ed V99M SNP, however, did not find the mutation. Role of Haplotype on Mitochondrial GSTZ1 In Chapter 2, w e reported the presence of GSTZ1 in liver mitochondria. Examination of GSTZ1 expression in cytosol and mitochondria showed similar distribution of the enzyme between the two compartments, independent of the allelic differences (Table 3 1). As with cytosolic GSTZ1, mitochondrial GSTZ1 of 1A/1C variant exhibited ~3 fold higher activity with DCA than Z1A noncarriers at a given level of expression. Therefor e, haplotype influenced mitochondrial and cytosolic GSTZ1 similarly for DCA metabolizing activity, but had no effect on protein expression or subcellular distribution of the enzyme
66 Discussion By studying human liver cytosols from 10 weeks of gestation to 74 years of life, we demonstrate d age as the major determinant of GSTZ1 protein expression and activity with DCA during human liver development. Similar to many of the DMEs studied to date, GSTZ1 was minimal ly expressed in the fetus and started increasing in the neonatal period The neonatal onset of GSTZ1 expression was also observed in the mRNA level in mouse livers, which reached adult level just 10 days after birth (Cui et al., 2010) Both transcriptional activation by members of the proline and acidic amino acid rich (PAR) family and epigenetic regulation of DNA and histone have been suggested to contribute to the postnatal onset of DMEs (Hines, 2008) Indeed, postnatal enrichment of histone H3 lysine 4 dimethylation has been shown in mouse Gstz1 gene (Cui et al., 2010) GSTZ1 haplotype had no effect on protein expression or subcellular distribution of the enzyme, but had a major impact on enzymatic activity with DCA. Consistent to results obtained with the polymorphic variants of recombinant hGSTZ1 (Blackburn et al., 2001) the Z1A allele paired with or without the Z1C allele, conferred higher DCA metabolizing activity for a gi ven level of expression in human liver samples. However, it was inconclusive whether the effect of the Z1A allele was dominant over or additive with the Z1C allele due to small number of sample s For the same reason, it remained unknown if Z1A conferred a similar effect on activity when paired with the Z1D allele, or rendered the enzyme unstable by the rare allelic combination. It is also noteworthy that although the Z1A allele associated high ratio of activity/expression was observed in individuals as early as 20 days postnatal its impact in the population was not prominent
67 until adolescence or is a result of low levels of protein expression in young KRT carriers. Unexplained by the current knowledge on GSTZ1 polymorphism several individuals in our liver bank exhibited very low GSTZ1 expression and activity for their age s A novel V al 99M et SNP was recently identified in a KGM/KGT individual with extraordinarily slow rate of DCA clearance (Shroads et al., 2011) However, none of the individuals with low GSTZ1 function in our study carried this mutation Leu 8 Pro is another less studied SNP that was suggested to be very poorly express ed (Blackburn et al., 2001) but its occurrence has not yet been reported in the population Therefore, it may be of interest to investigate the possible role s of L8P as well as other genetic or environmental factors that may contribute to the low functionality of GSTZ1. Human pharmacokinetics of DCA has been shown with age and GSTZ1 haplotype dependent differences (Shroads et al., 2008; Shroads et al., 2011) T he current finding of o lder children and adults (7 74 Y) having higher GSTZ1 activity than young children (1 7 Y) however, does not explain the slower clearance of DCA in adult s after chronic treatment (see Table 1 2) (Shroads et al. 2008) On the other hand, Z1A allele associated higher activity with DCA is consistent with the faster drug clearance by Z1A carriers after the 1 st DCA dose (see Table 1 3) (Shroads et al., 2011) No evidence of low GSTZ1 function was observed for Z1D allele. Thus, the current study provided no explanation for the greater reduction in DCA clearance in Z1A/Z1A and Z1A/Z1D individuals after repeated doses. We speculate that th is altered effect of Z1A allele in clearance between the initial and repeated doses may be related to a haplotype dependent susceptibility to DCA inactivation (see Chapter 4 ) Similarly, the
68 apparent lack of correlation between age related decrease in DC A clearance and increase in GSTZ1 activity may be related to DCA induced inactivation of GSTZ1 rather than t he initial enzym atic activity GSTZ1/MAAI has the important physiological function of isomerizing maleylacetoacetate at the penultimate step of phen ylalanine/tyrosine catabolism. In the fetal liver, this pathway is rate limited by tyrosine aminotransferase that has been shown to possess very low activity in the fetus and increase rapidly after birth (Delvalle a nd Greengard, 1977; Andersson et al., 1980; Ohisalo et al., 1982) Therefore, in fetal liver maleylacetoacetate presumably exists at a low concentration so that nonenzymatic conversion to fumarylacetoacetate c ould be sufficient for its degradation (Fer nandez Canon et al., 2002) As with tyrosine aminotransferase, the rapid onset of GSTZ1/MAAI in neonates may be regarded as a coordinated and adaptive response to the increased intake and thus degradation of amino acids as required for infant growth. In conclusion, we reported a neonatal onset and an age related increase in GSTZ1 protein expression during human liver development. GSTZ1 activity with DCA is directly correlated to the level of protein expression and dependent on GSTZ1 haplotype. The GSTZ1A (KRT) allele paired with or without the Z1C allele, confers a ~3 fold higher activity with DCA at a given level of protein expression than other haplotypes that possess similar activities with DCA.
69 A B Figure 3 1. Western blot analysis of GSTZ1 in 0.55 17.6 ng purified hGSTZ1C 1C and 5 and 10 g of one human liver cytosol (HL cyt). A) Western blot. B) Standard curve of 0.55 17.6 ng GSTZ1 expression analyzed by linear regression.
70 Figure 3 2 Scatter plot analyses of GSTZ1 A) protein expressio n and B) activity with DCA as a function of age. Axis breaks are set at birth and 1 year. Age of prenatal samples is shown as gestational age. Samples from individuals that carried at least one allele of GSTZ1A (KRT) are shown as filled triangle ( ) and all the others are shown as open circle ( )
71 Figure 3 3 Box and whisker plot analyses of GSTZ1 A) protein expression and B) activity with DCA as a function of age groups. The horizontal bar, box and whiskers denote the median, interquartile range and the lowest to highest values of the group, respectively. Outliers ( ) were defined as having specific contents 1.5 fold interquartile range away from the lower or upper quartile s. Mean and SD were reported and outliers were excluded from the analyses. Statistical significances ( p < 0.05) were obtained between age groups as analyzed by Kr u skal Wallis nonparametric test followed by step wise step down test. The number of samples ha ving specific contents below the LOD s was reported (
72 Figure 3 4 Correlation analysis of GSTZ1 protein expression and activity with DCA. R squared values were determined by Pearson correlation. The correlations were significa nt for all four groups (p < 0.0001). Postnatal samples with both activity and expression above the LO D s were included in the analysis. Most samples above and selective sample below age 3 were genotyped for GSTZ1.
73 Figure 3 5 Role of GSTZ1 haplotype on A) protein expression and B) the ratio of DCA metabolizing activity to protein expression Samples of age 3 and above with expression and activity above the LOD s were included in the analysis. The number of samples of each haplotype is given in parenthesi s. The h orizontal bar denotes the median.
74 Table 3 1. GSTZ1 expression and activity with DCA in cytosol and mitochondria of individuals with various haplotypes. Age (year) GSTZ1 Haplotype Cytosol Mitochondria Activity Expression Ratio of Act to Exp Activity Expression Ratio of Act to Exp nmol/min/mg g GSTZ1/ mg cytosol nmol/min/mg g GSTZ1/mg m itochondria HL735 74 1C / 1C 0.39 0.44 0.88 0.14 0.20 0.69 HL733 70 1C / 1B 0.53 0.49 1.07 0.21 0.19 1.09 HL750 10 1C / 1B 0.5 7 0.68 0.84 0.30 0.27 1.11 HL20 67 1D / 1D 0.47 0.54 0.87 0.16 0.24 0.67 Mean SD (n=4) 0.92 0.10 0.89 0.24 HL10 23 1C / 1A 1.24 0.45 2.76 0.87 0.30 2.87 HL97 21 1C / 1A 1.46 0.55 2.65 0.46 0.22 2.11 HL742 48 1C / 1A 1.50 0.60 2.49 0.51 0.24 2.17 Mean SD (n=3) 2.63 0.14 2.38 0.42
75 CHAPTER 4 CHLORIDE MODULAT ES GSTZ1 HAPLOTYPE DEPENDENT INACTIVATION BY DICH LOROACETATE Specific Aim To date, four major polymorphic variants of GSTZ1, i.e., EGT, KGT, EGM and KRT, have been studied in some detail both in vitro and in vivo. Study of recombinant enzymes showed that the KRT variant possesse d 4 5 fold higher specific activity with DCA as substrate and 2 fold slower rate of inactivation by DCA compared to the other variants which grouped together for activity and rate of inactivation (Tzeng et al., 2000; Blackburn et al., 2001) Th at th e KRT allele confer red higher activity with DCA was confirmed with human liver samples by our work (Chapter 3) Nevertheless, pharmacokinetic studies showed that KRT/KRT and KRT/EGM individuals exhibited a greater reduction in DCA clearance after repeated doses compared to EGT carriers ( Shroads et al., 2011 ). O ur previous study (Cornett et al., 1999) fa iled to reproduce Tzeng et al. (2000) result of DCA inactivation of human cytosolic GSTZ1 in vitro, which we speculate to be a result of the presence of chloride in our samples. To investigate these discrepancies, we use d human liver cytosols of differen t GSTZ1 haplotypes to examine 1) DCA induced inactivation of GSTZ1 and 2) the role of chloride in modulating GSTZ1 activity. Materials and Methods Human Liver Samples and GSTZ1 Genotype Information De identified human livers homozygous for EGT and EGM, and heterozygous of EGT/KRT were used in the current study. The sources of the livers and GSTZ1 genotyping procedures are described in Materials and Methods section of Chapter 3
76 Assay of DCA I nduced Inactivation of GSTZ1 in Human Liver Cytosol Human liver c ytosol was isolated exactly as described in Chapter 2 A liquots of cytosol were dialyzed against 0.1 M potassium phosphate pH 7.4 using Slide A Lyzer Dialysis cassette (10kD MWCO; Thermo Fisher Scientific ) with three changes of buffer to ensure complete r emoval of small molecules DCA induced inactivation of GSTZ1 was assayed by adapting the method of Tzeng et al (2000) In the presence or absence of KCl or other salts dialyzed cytosol (0.6 mg/ml) was incubated with 0.5 mM unlabelled DCA, 5 mM glutathione and 0.1 M potassium phosphate in an assay volume of 500 l a t 37C for 2 h To determine the EC50 of chloride in protecting GSTZ1 from DCA inactivation, samples were incubated with 0.77 308 mM KCl; the effects of other anions as the ir sodium or potassium salt s were tested at a concentration of 38 mM in assay s ; c o ntrol incubation s lacked DCA or additive salts To study the time course of GSTZ1 inactivation assay mixtures were incubated for 0 14 h with 38.3 mM KCl or 0 90 min without KCl. At the end of incubation, unbound substrate and product were removed by u ltrafiltration through Amicon Ultra 0.5 ml filters of 10 kD MWCO (Millipore Corporation ) following 3 concentration dilution cycles with 0.1 M potassium phosphate pH 7.4. Protein recovered by the filter was assayed for concentration and then activity with 0.2 mM [ 14 C]DCA as described in Chapter 3 Protein concentration was determined using Bio Rad Prot ein Assay (Bio Rad Laboratories ) with bovi ne serum albumin (Sigma Aldrich ) as protein standard. Data Analysis To determine the EC50 of chloride in protecting GSTZ1 from DCA inactivation, assay activity was normalized as a percent of control and plotted against the log KCl concentration EC50 was obtained by fitting the data to a dose response sigmoidal
77 curve with the top constrained to 100% and the bottom to 0 %. To determine the inactivation half life (t 1/2 ) of GSTZ1 the natural log of assay activity (A) divided by control activity (A 0 tubes assayed at time 0) was plotted against incubation time. Linear regression analysis was performed to fit data to the equ ation ln(A/A 0 ) = k obs t with lines forced to go through (0, 0). The inactivation t 1/2 was determined from the equation t 1/2 = ln2/k obs Data analysis was performed on GraphPad Prism v4.03 (San Diego, CA). t test was performed on Microsoft Office Excel 2007. Results Chloride P rotect ed GSTZ1 from DCA I nactivation GSTZ1 by DCA, we conducted several preliminary experiments and identified chloride to be a modulating fa ctor. Chloride protected human cytosolic GSTZ1 from DCA induced inactivation in a chloride concentration and GSTZ1 haplotype dependent manner (Figure 4 1) To achieve 50% protection of GSTZ1 from DCA inactivation a significantly higher (2.5X) concentrati on of chloride was required for EGT/KRT individuals (EC50 33.6 2.3 mM; mean SD, n=3) compared to EGT /EGT (12.3 2.7 mM, n =3) and EGM /EGM (13.8 mM, n=1) individuals (Table 4 1). Chloride is reported to be present at 38.3 mM in human livers (Widdowson and Dickerson, 1960) At 38 mM chloride, EGT/KRT individuals were inactivated to a larger extent tha n other variants after 2 h incubation. Time Course of GSTZ1 Inactivation by DCA W e next examined the time course of cytosolic GSTZ1 inactivation by 0.5 mM DCA in the absence or presence of physiological concentration (38 mM) of chloride (Figure 4 2) In th e absence of chloride DCA rapidly reduced GSTZ1 activity to 50% of
78 control level wi thin 30 min for all individuals, with a slight ly longer t 1/2 in EGT homozyotes (Table 4 2) With addition of 38 mM chloride, however, the rate of GSTZ1 inactivation by DCA was substantially reduced. Furthermore, a 2 fold difference in GSTZ1 inactivation t 1/2 was observed between KRT carriers and non carriers: over 5 h for EGT and EGM homozygo tes and 2.5 h for EGT/KRT heterozygo tes Effects of Other Anions We further screened some other anions for their influence on DCA induced inactivation of GSTZ1 by using a salt concentration of 38 mM over a 2 h incubation (Table 4 3). Of the halide series, Br and I exhibited a more potent protection for GSTZ1 than Cl while F had no ap parent effect. Among other anions tested only SO 3 2 showed p rotection for GSTZ1 with a potency stronger than that of Cl Discussion The current study showed that the rate of DCA induced inactivation of GSTZ1 was dependent on GSTZ1 haplotype and the prese nce of certain anions, such as chloride bromide, iodi d e and sulfite. Chloride, a major physiological electrolyte dose dependently attenuated DCA induced inactivation of GSTZ1 At a normal liver concentration of chloride (38 mM), cytosolic GSTZ1 of EGT/KR T individuals was inactivated > 2X faster by DCA than that of EGM and EGT homozygotes. Th e finding of chloride protection explains (1999) failure to observe DCA inactivation in human liver cytosol In that study, cytosols of EGT homozygotes were dialyzed in a buffer con taining 1.15% KCl which resulted in ~30 mM chloride in assay and effectively prevented GSTZ1 inactivation af ter an incubation of 30 min. Contradictory to the knowledge gained from the recombinant enzymes (Tzeng et al., 2000) the human cytosolic form of the KRT variant was more susceptible to DCA
79 inactivation regardless the presence of chloride. Meanwhile it is noteworthy that the recombinant enzymes were stored in a chloride containi ng buffer. Although the exact reason for this discrepancy is unknown, we suspect various factors contribute, including inconsistent concentration s of chloride in assay s and structural variation in the N terminus where the recombinant enzyme was constructed with the His tag near the GSH binding site KRT variant differs from other variants by the mutation of Gly to Arg at residue 42 which is located at a loop area on the top of GSH binding site. This loop region was suggested to be highly mobile and functi on as a gateway to the binding site of the enzyme. Therefore, conformation al changes in the loop region w ere previously proposed to occur upon uptake or r elease of substrate and/or product and between variants carrying Gly or Arg (Blackburn et al., 2001) Given that Arg has a long and positively charged side chain it may also exhibit ionic interaction s with the negatively charged substrate (DCA) product (glyoxylate) and/or currently identified modulating anions ( h alides and sulfite ) that do not apply to the variant s with Gly at position 42. In addition to chloride, some other anions (Br I and SO 3 2 ) showed similar protective effects for GSTZ1. On the other hand, s ulfate ion (SO 4 2 ) which co crystallized with the KRT variant of the recombinant hGSTZ1 in the proposed active site (Polekhina et al., 2001) afforded no protection in the current study We speculate that either the active site or an allosteric site of GSTZ1 may be involved in the binding of anions D epending on the property of the anion and its interaction with GSTZ1 and /or DCA, the anion binding may trigger conformational change and thus modulat e the interaction between DCA and the GSTZ1 enzyme. Interestingly, Tzeng et al. (2000) also
80 noted some prot ective effect by N acetyl L cysteine but not KCN. T he mechanism of action they proposed was that N acetyl L cysteine block ed the electrophilic intermediate of DCA from attacking GSTZ1. However, this mechanism is unlikely to explain the protection afforded by halides or sulfite. Analyses of previous studies suggest that chloride protection applies to rat GSTZ1 and has pharmacological significance The inactivation t 1/2 of rat GSTZ1 by 0.5 mM DCA increased from 5 min in chloride free cytosol of Fisher 344 rat s (Tzeng et al., 2000) to about 30 min in chloride containing cytosol of S D rat s (Cornett et al., 1999) I.p. injection of 0.3 mmol (i.e. 45 mg ) of DCA /kg to Fisher 344 rats reduced the activity and expression of liver cytosolic GSTZ 1 to 50 60% of control levels 1.5 h after treatment (Anderson et al., 1999) Since DCA is rapidly distributed to the liver after i.p. injection, the hepatic GSTZ1 should have been reduced more rapidly if not for the protection of modulating factors, such as Cl and other anions. Chloride is known to be present at 38 .3 mM in the liver of adults and slightly higher in infants (4 7 months old; 42.8 mM) and newborn s (55.8 mM) (Widdowson and Dickerson, 1960) Although little is known about the concentrations of other anions in the liver, bromide and iodi d e have been detected at ~26 M and ~ 0.34 M, respectively, in the human whole blood (Zhang et al., 2010) and sulfite at ~1.2 M in the serum (Mitsuhashi et al., 2004) Additionally, sulfite was reported to reduce oxalate production from glyoxylate in vitro and from rats i v infused with DCA (Sharma and Schwille, 1993) Together, current results strongly suggest that chloride and perhaps other anions protect GSTZ1 from in vivo inactivation by DCA an d may therefore be a factor modulating the rate of DCA clearance after repeated doses.
81 In conclusion, we demonstrated that chloride attenuated DCA induced inactivation of GSTZ1 in a chloride concentration and GSTZ1 haplotype dependent manner. In contrast to results obtained with the recombinant enzyme, the human cytosolic form of the KRT variant was more rapidly inactivated by DCA. At a physiological concentration of chloride and 0.5 mM DCA, GSTZ1 inactivation t 1/2 was 2 3 h shorter for KRT/EGT individuals than EGT and EGM homozygous individuals. This may contribute to the greater reduction in DCA clearance observed in KRT/KRT and KRT/EGM individuals after repeated doses.
82 Figure 4 1. Chloride protect ed human liver cytosolic GSTZ1 from DCA inactivation in a [Cl ] and GSTZ1 haplotype dependent manner. Dialyzed human liver cytosol, 0.6 mg/ml, was incubated with 0.5 mM DCA, 5 mM GSH and 0.77 308 mM KCl in 0.1 M K phosphate buffer pH 7.4 at 37 o C for 2 h Data were analyzed as described in Materials and Me thods. The control activity and EC50 of chloride protection for each sample were summarized in Table 4 1.
83 Figure 4 2. The time course of GSTZ1 inactivation by DCA in the A) absence and B) presence of 38 mM KCl. Human liver cytosol, 0.6 mg/ml, was inc ubated with 0.5 mM DCA, 5 mM GSH in 0.1 M K phosphate buffer pH 7.4 with or without 38.3 mM KCl at 37 o C for the indicated period of time. The i nactivation t 1/2 of GSTZ1 was determined as described in Materials and Methods, and summarized in Table 4 2.
84 Tab le 4 1. Summary of GSTZ1 control activity with DCA and chloride concentration to achieve 50% protection of GSTZ1 from DCA inactivation (EC50) obtained from Figure 4 1. EGT/EGT EGT/KRT EGM/EGM ID Control activity EC50 ID Control activity EC50 ID Contr ol activity EC50 nmol/min/mg mM nmol/min/mg mM nmol/min/mg mM HL100 0.61 15.0 HL10 0.85 36.3 HL20 0.35 13.8 HL113 0.73 9.6 HL97 1.27 32.2 HL735 0.29 12.2 HL742 1.06 32.3 Mean SD (n=3) 12.3 2.7 33.6 2.3 *** ***, p < 0.005 compared to EC50 of EGT/ EGT individuals, analyzed by two tailed t test assuming equal variances
85 Table 4 2. Summary of GSTZ1 inactivation half lives (t 1/2 ) obtained from Figure 4 2. GSTZ1 Haplotype Inactivation t 1/2 ( h ) without Cl with Cl EGT/EGT (n =2) 0.5 3, 0.49 5. 02, 5.73 EGT/KRT (n=2) 0.39, 0.38 2.37, 2.67 EGM/EGM (n=1) Not determined 5.55
86 Table 4 3 Effects of various anions in modulating GSTZ1 inactivation by DCA. Sodium or potassium salt of various anions at 38 mM was incubated with HL113 (EGT/EGT) liver cytosol, 0.5 mM DCA and 5 mM GSH at 37 o C for 2hr. Activity was shown as a percent of control, which was preincubated without DCA or additive salts DCA only F Cl Br I SO 4 2 SO 3 2 CO 3 2 CN % of control 12% 13% 80% 105% 102% 15% 100% 9% 3%
87 CHAPTER 5 CONCLUSION In this dissertation, we studied the roles of mitochondrion, subject age, enzyme haplotype and chloride interaction in GSTZ1 catalyzed biotransformation of DCA. is a second site of DCA biotransformation. The reaction is catalyzed by a mitochondrial pool of GSTZ1 localized in the matrix The identity of mitochondrial GSTZ1 was confirmed by partial protein sequences obtained from LC MS/MS analysis. Similar to the cytosolic form, mitochondrial GSTZ1 possessed catalytic activity with DCA and was inactivated by DCA treatment. W ith DCA as substrate, rat mitochondrial GSTZ1 exhibited a higher apparent K m for GSH than cytosolic GSTZ1, suggesting possible modification of the mitochondrial form relative to the cytosolic form. GSTZ1 haplotype had no effect on the relative distribution of the enzyme b etween cytosol and mitochondria ; but the Z1A (KRT) allele clearly conferred a ~3 fold higher activity with DCA in GSTZ1 of both compartments. Age is shown by the current study to be a major determinant of GSTZ1 expression and activity during human liver development. GSTZ1 was present at undetectable to very low levels in the fetal liver. Following birth, GSTZ1 level rose rapidly a nd reache d nearly half of that of adults by one month of age The increase continue d until adolescen ce when GSTZ1 reache d a stable level and maintain ed so throughout adulthood The developmental change in GSTZ1 activity with DCA paralleled that of its prot ein expression Therefore, the age related increase in DCA metabolizing activity did not explain the age dependent decrease in DCA clearance observed in vivo after chronic treatment.
88 GSTZ1 haplotype had no effect on protein expression but did affect enzym atic activity with DCA. Z1A carriers possessed higher DCA metabolizing activity than carriers of other alleles (Z1B, Z1C and Z1D) that had similar activity and expression. Accordingly Z1A carriers exhibited a major impact in GSTZ1 activity in populations of o lder children and adults The higher activity conferred by the Z1A allele was consistent to the generally faster clearance of DCA in its carriers after the 1 st dose of 25 mg/kg DCA; however, this did not explain the greater reduction in plasma clearanc e after the 5 th DCA dose as observed in Z1A/Z1A and Z1A/Z1D individuals We speculate a haplotype dependent susceptibility to DCA inactivation may exist and thus contribute to the in vivo differences after repeated doses. Study of GSTZ1 inactivation using liver cytosol reveal ed that chloride attenuated DCA induced inactivation of GSTZ1 in a chloride concentration and GSTZ1 haplotype dependent manner. In the absence of chloride, DCA rapidly reduced GSTZ1 activity to 50% of control level within 30 min for al l individuals. At a physiological concentration of chloride in the liver (38 mM), cytosolic GSTZ1 inactivation t 1/2 s were prolonged to 2.5 h for 1C/1A individuals and over 5 h for 1C and 1D homozygous individuals. In addition to chloride, other anions such as bromide, iodide and sulfite were shown to protect GSTZ1 from DCA inactivation at 38 mM. The current finding of faster inactivation of GSTZ1 carrying the Z1A allele may explain at least in part the more marked reduction in DCA clearance after repeated doses in 1A/1A and 1A/1D individuals, compared with those carrying other haplotypes.
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98 BIOGRAPHICAL SKETCH Wenjun Li was born in 1984 in Guangzhou, China, being the only child of Jianguo Li and X iangling Jian. She spent the first 18 years with her parents living in Shenzhen, a young and vigorous city that she loves so much and cherishes some of her best memories with. After graduati on from Shenzhen Experimental School in 2002, she was admi tted to Sun Yat s en University, spending the first two years in beautiful Zhuhai Campus and the latter two years in the historical Medical Campus in Guangzhou She wanted to study medicine but was eventually accepted to major in p harmaceutical s cience s During und ergraduate, she enjoyed learning chemistry but found herself more apt in doing biological experiments. The desire to combine the knowledge of chemistry to the practice of biology inspired and eventually motivated her to pursue her doctoral degree in the De partment of Medicinal Chemistry, University of Florida in 2006. After graduation, she wishes to obtain a postdoctoral training in Europe. Her goal is to establish a professional career in biomedical research and step on the soil of as many countries as she can