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Metabolism of dichloroacetate in the Sprague Dawley rat and humans, involvement of the tyrosine catabolic pathway

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Metabolism of dichloroacetate in the Sprague Dawley rat and humans, involvement of the tyrosine catabolic pathway
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Cornett, Rachel, 1969-
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English
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xv, 94 leaves : ill. ; 29 cm.

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Cytosol ( jstor )
Dosage ( jstor )
Enzymes ( jstor )
Glyoxylates ( jstor )
In vitro fertilization ( jstor )
Incubation ( jstor )
Liver ( jstor )
Metabolism ( jstor )
Metabolites ( jstor )
Rats ( jstor )
Department of Medicinal Chemistry thesis Ph. D ( mesh )
Dichloroacetate -- metabolism ( mesh )
Dissertations, Academic -- College of Pharmacy -- Department of Medicinal Chemistry -- UF ( mesh )
Dissertations, Academic -- Medicinal Chemistry -- UF
Glutathione -- metabolism ( mesh )
Glyoxylates -- metabolism ( mesh )
Hippurates -- metabolism ( mesh )
Liver -- physiology ( mesh )
Medicinal Chemistry thesis, Ph. D
Rats, Sprague-Dawley ( mesh )
Research ( mesh )
Tyrosine -- metabolism ( mesh )
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 2000.
Bibliography:
Bibliography: leaves 89-93.
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Typescript.
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Vita.
Statement of Responsibility:
by Rachel Cornett.

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Full Text
METABOLISM OF DICHLOROACETATE IN THE SPRAGUE DAWLEY RAT
AND HUMANS, INVOLVEMENT OF THE TYROSINE CATABOLIC PATHWAY
By
RACHEL CORNETT
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 2000




Copyright 2000
by
Rachel Comrnett




Dedicated to my parents John and Sally Comrnett




Acknowledgements
I would like to thank my major professor, Dr. M.O. James, for her patience,
guidance, sharing her immense knowledge, and support. I would like to thank members of my committee; Dr. Steven Roberts, for his toxicology expertise, Dr. Peter W. Stacpoole, for his knowlege of every aspect of DCA research and writing advice, Dr. Kenneth Sloan, for his help with chemical mechanisms and synthetic procedures, and Dr. John Perrin, for his pharmacokinetic and physical chemistry knowledge and always having the latest "scoop." I would like to thank Dr. G.N. Henderson for his scientific input and all the GC-MS data found in this work, which should also include thanks to his lab members Ximeng Yan, Albert L. Shroads, and Jang Cheung. I would also like to thank my fellow lab members; Laura Faux, Zeen Tong, and Donald Sikazwe,for their help over the years, specifically for showing me the way around in the lab when I began as a pharmacist who had forgotten most of the laboratory techniques learned in undergraduate courses. Laura should receive special thanks for her perfectionist example and excellent bench technique. Dr. Bernard Gadagbui also deserves a special mention for his willingness to assist all lab members and his expertise in protein chemistry. Alicia Ally, Dr. Peter van den Hurk, and Zhen Lou have always been helpful, cheerful, and easy to work with. I would also like to thank my husband, Dr. Ralf Mueller, for his help not only with organic chemistry but also in putting up with me while I was writing my "Doktorarbeit." I would like to thank my mother, Sally Cornett, for her support in all
iv




endeavors, and my father, John Comrnett, for calling me crazy for going back to school, which gave me an additional impetus to return.
V




TABLE OF CONTENTS
p ae
A CKN OW LED GEM EN TS ............................................................................................. iv
LIST OF TABLES ..................................................................................................... ix
LIST OF FIGURES ................................................................................................ x
ABBREVIATION S ................................................................................................. xiii
ABSTRA CT ........................................................................................................... xiv
CHAPTERS
1 INTRODU CTION ......................................................................................... 1
M echanism of A ction .................................................................................... 1
Glutathione-S-Transferase Zeta ..................................................................... 3
G lutathione-S-Transferases ........................................................................... 6
Potential M echanism s of D CA Dechlorination ............................................. 7
M etabolites of D CA .................................................................................... 11
Involvem ent of the Tyrosine Catabolic Pathw ay ........................................ 12
2 M A TERIALS AND M ETH OD S ............................................................... 15
Chem icals and Equipm ent .......................................................................... 15
Anim als ...................................................................................................... 16
In V ivo Studies .............................................................................. 16
Effect of In Vivo DCA Pre-Treatment on its In V itro M etabolism ................................................................... 17
Dose Response Studies .................................................................. 17
Preparation of Rat Liver Cytosol ............................................................... 18
Preparation of Hum an Liver Cytosol ......................................................... 18
A ssays of D CA M etabolism ....................................................................... 19
General ............................................................................................ 19
Cytosol D ialysis .............................................................................. 19
Reducing Agent Dependence Incubations ...................................... 20
Linear Range of Glyoxylate Production in Rat Hepatic Cytosol ..... 21
vi




Rat Apparent KM and Vmax Determination Incubations .................. 22
Linear Range of Glyoxylate Production in Human
H epatic C ytosol ................................................................................22
Apparent KM and Vmax in Dialyzed Human Liver Cytosol
Incubations ...................................................................................... 24
In Vitro DCA Inhibition Incubations ...............................................24
DCA Pre-Incubation Comparison between Dialyzed Rat and Human
C ytosol .............................................................................................25
DCA Binding Study in Dialyzed Rat and Human Hepatic Cytosol ......26
In Vitro GSTz Activity in DCA Dosed Animals .............................27
A nalyses ....................................................................................................... 27
Ion Pair H PLC .................................................................................27
Strong Anion Exchange HPLC .......................................................28
13C N M R A nalysis ..........................................................................28
Fluorescence Analysis ....................................................................29
GC-MS Confirmation of Glyoxylate ..............................................30
GC-MS Analysis of Maleylacetone ............................................... 31
Synthesis of Maleylacetone ........................................................... 32
M aleylacetone M ethods ......................................................................... 35
Maleylacetone IC50 Studies ........................................................... 35
Pre-Incubation Studies ................................................................... 36
Maleylacetone Ki Studies ............................................................... 36
Pre-Treatment of Dialyzed Rat and Human Hepatic
Cytosol with Maleylacetone .............................................37
3 IN VIVO STUDIES' RESULTS ................................................................... 38
Ion Pair HPLC Analysis of Urine ................................................................. 38
13C NMR Analysis of Rat Urine ................................................................... 38
M aleylacetone in U rine ................................................................................. 42
4 IN VITRO STUDIES' RESULTS ................................................................ 44
Identification of Glyoxylate as a Product of DCA Metabolism ................... 44
Development of a Fluorescence Assay of Glyoxylate ..........................46
Strong Anion Exchange HPLC Analysis of Incubations .......................47
Reducing Agent Dependence Study ............................................................. 48
Effect of DCA Treatment on Activity of GSTz ............................................ 48
Linear Range of Glyoxylate Production in Rat Hepatic Cytosol .................. 50
Rat Apparent KM and Vmax of GSH Determinations ..................................... 55
vii




Rat Apparent KM and Vmax of DCA Determinations ..................................... 56
Linear Range of Glyoxylate Production in Human Hepatic Cytosol ............ 58
Human Hepatic Cytosol Apparent KM and Vmax Results ............................... 59
In Vitro Inhibition Studies' Results ............................................................. 66
D CA B inding Study ..................................................................71
In Vitro GSTz Activity in DCA Dosed Animals ........................................... 71
5 RESULTS OF INHIBITION STUDIES WITH MALEYLACETONE .............. 74
Maleylacetone IC50 Studies ........................................................................... 74
K i of M aleylacetone ....................................................................................... 74
Pre-Treatment of Dialyzed Rat and Human Hepatic Cytosol with
M aleyalcetone .........................................................................80
6 D ISC U SSIO N ..................................................................................................... 82
R EFEREN CE S ............................................... ............................................................... 89
BIOGRAPHICAL SKETCH .......................................................................................... 94
viii




LIST OF TABLES
Table page
1. Alpha-beta unsaturated carboxylic acids metabolized by GSTz ............................... 5
2. Haloacids metabolized by GSTz ............................................................................. 10
3. Reducing agent dependence study ........................................................................... 49
4. Apparent KM (mM) and Vmax (nmoles/minute/mg protein) of
GSH in dialyzed rat cytosol .................................................................. 55
5. Apparent KM (mM) and Vmax (nmoles/minute/mg protein) of DCA in
dialyzed rat cytosol ............................................................................... 57
6. GSTz activity in DCA treated and control animals..................................................73
ix




LIST OF FIGURES
Figure page
1. Mechanism of action of DCA on pyruvate dehydrogenase ....................................... 2
2. Tyrosine catabolism ................................................................................................... 4
3. Possible mechanism for the conversion of DCA to glyoxylate .................................. 7
4. Possible mechanism accounting for retention of deuterium in glyoxylate from
D C A dechlorination ........................................................................................... 8
5. Proposed mechanism for removal of fluorine avoiding the zwitterion
interm ediate........................................................................................................ 9
6. Metabolites of DCA as understood in 1995................................................................13
7. Possible routes of CO2 formation .............................................................................. 14
8. Formation of 2,2', 4,4'-tetrahydroxydiphenylacetic acid lactone ............................... 29
9. Synthesis of m aleylacetone ........................................................................................ 33
10. 'H-NMR of maleylacetone ...................................................................................... 34
11. R at 18, 0-3 hour urine sam ple .................................................................................. 39
12. 13C N M R of rat urine ............................................................................................... 40
13. GC-MS of methylated urine ..................................................................................... 41
14. Urinary excretion of maleylacetone in rats treated with 200 mg/kg DCA .............. 43
15. GC-MS of methylated rat liver cytosol incubated with 13C labeled
D C A .............................................................................................. 45
16. Separation of 14C DCA and 14C glyoxylate by strong anion exchange HPLC ..........47
17. Time course studies in control and DCA treated dialyzed rat hepatic cytosol .......... 51
x




18. Glyoxylate formed (nmoles) from a range of protein concentrations ....................... 52
19. Glyoxylate formed (nmoles) from various DCA concentrations in
rat hepatic cytosol ................................................................................................. 53
20. Glyoxylate produced (nmoles) from a range of GSH concentrations in rat
hepatic cytosol.................................................................................. 54
21. Apparent KM of GSH in representative dialyzed rat cytosol..................................... 56
22. Apparent KM of DCA in dialyzed rat cytosol ........................................................... 58
23. Time course study in human hepatic cytosol ............................................................ 60
24. Glyoxylate formed in dialyzed human hepatic cytosol from a range of protein
concentrations ................................................................................... 61
25. Glyoxylate formed in dialyzed human hepatic cytosol from various DCA
concentrations ................................................................................62
26. Glyoxylate formed from a range of GSH concentrations in dialyzed human hepatic
cytosol....................................................................................... .. 63
27. Lineweaver-Burk determination of the KM and Vmax of DCA at
0.024 m M G SH ...................................................................................................... 64
28. KM of GSH in dialyzed human cytosol at 0.025 mM DCA ....................................... 65
29. Activity of dialyzed hepatic cytosol after pre-incubation with
0.2 m M D C A ......................................................................................................... 68
30. Residual GSTz specific activity at 30, 45, and 60 minutes in dialyzed
human cytosol pre-treated with 0, 0.5, 0.75, or 1 mM DCA .................................. 69
31. Comparison of the effect of DCA pre-treatment on GSTz specific
activity between rat and human dialyzed hepatic cytosol ..................................... 70
32. Specific activity of rat hepatic cytosol from rats dosed with a broad
range of D CA concentrations ................................................................................ 72
xi




33. Initial incubation of DCA with maleylacetone ......................................................... 75
34. IC50 of MA in rat hepatic cytosol .............................................................................. 76
35. IC50 of MA in human hepatic cytosol ....................................................................... 77
36. Maleylacetone pre-incubation comparison ............................................... 78
37. Lineweaver-Burk Ki determination of MA against
D C A m etabolism .................................................................................................... 79
38. Effect of maleylacetone pre-treatment in dialyzed rat and human hepatic cytosol ... 81 39. DCA metabolic pathways known at the end of this study ...............................84
xii




ABBREVIATIONS
ANOVA analysis of variance DCA dichloroacetate DTT dithiothreitol (threo-1,4-dimercapto-2,3-butanediol) EPA environmental protection agency FAA fumarylacetoacetate FA fumarylacetone GSH reduced glutathione GSSG oxidized glutathione GST glutathione-S-transferase HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] HPLC high pressure liquid chromatography NADH nicotinamide adenine dinucleotide, reduced NADPH nicotinamide adenine dinucleotide phosphate, reduced NMR nuclear magnetic resonance SAX strong anion exchange SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
Xiii




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
METABOLISM OF DICHLOROACETATE IN THE SPRAGUE DAWLEY RAT AND HUMANS, INVOLVEMENT OF THE TYROSINE CATABOLIC PATHWAY
By
Rachel Cornett
May, 2000
Chair: Dr. Margaret O. James
Major Department: Medicinal Chemistry
Dichloroacetate (DCA) is considered a hazardous environmental contaminant by the EPA, but is also an investigational drug for the treatment of lactic acidosis in children with inborn errors of metabolism. Chronic administration of DCA leads to toxicity including peripheral neuropathy and ocular opacities. DCA also inhibits its own metabolism, which can be seen as early as after the first dose. The aims of the research conducted over the past four years have been to uncover the overall metabolic pathways of the metabolism of DCA and to examine hypotheses related to the mechanism of its inhibition of its own metabolism. Research into DCA metabolism has included in vivo and in vitro experiments in rats, and in vitro investigations with human liver cytosol. There were several important findings in the metabolism of DCA including the
xiv




confirmation of glyoxylate as an intermediate, which had not previously been unequivocally identified, the requirement of glutathione (GSH) for DCA metabolism to glyoxylate, and the discovery of hippurate and other glycine conjugates as metabolites of DCA. Advancements in uncovering the mechanism of DCA auto-inhibition include demonstrating a decrease in the rate of in vitro DCA metabolism in hepatic cytosol prepared from DCA treated rats, a direct inhibitory effect in dialyzed rat hepatic cytosol but not dialyzed human hepatic cytosol incubated with DCA, inhibition of DCA metabolism by maleylacetone, a native substrate of GSTz, and an increase in maleylacetone levels in DCA treated rats, suggesting an interaction between DCA and the tyrosine catabolic pathway. Uncovering the interaction between DCA and the tyrosine catabolic pathway may lead to improvements in the treatment of children with lactic acidosis and insights into the mechanisms of DCA's toxicity and rodent hepatocarcinogenicity.
xv




CHAPTER ONE
INTRODUCTION
Dichloroacetate (DCA) is considered by the EPA to be an environmental
contaminant resulting from both the chlorination of drinking water and the breakdown of trichloroethylene, but is also used as a therapeutic agent in the treatment of lactic acidosis in several clinical settings. DCA is the only available treatment for congenital lactic acidosis, which if left untreated is fatal. It can also be used to treat lactic acidosis in patients undergoing liver transplantation, acute malaria, and cardiovascular disease.' A confounding property of DCA is that it inhibits its own metabolism. An increase in the plasma elimination half-life of the drug can be seen as early as the second dose.2 Prolonged administration of DCA causes toxicity including possible ocular toxicity and peripheral neuropathy.3 Uncovering the pathways of metabolism may yield insight into the mechanisms of DCA's toxicity and auto-inhibition. This may ultimately lead to changes in treatment that improve the quality of life and survival rates of children with congenital lactic acidosis.
Mechanism of Action
DCA lowers lactate levels through stimulation of pyruvate dehydrogenase indirectly by inhibition of phosphate dehydrogenase kinase, preventing its
1




phosphorylation and subsequent inactivation. This increases the amount of pyruvate
converted to acetyl-CoA, resulting in a reduction of lactate and alanine levels which are
precursors of pyruvate in gluconeogenesis (Figure 1).3
Alanine Pyruvate
S Aminotransferase QQ Dehydrogenase Q
H2N--C-OH H3C-C-C-OH 2 H3C-C-S-CoA
CH3 (PDH) Acetyl CoA
Alanine Pyruvate Lactate
Dehydrogenase
H,o
H3C-C-C-OH
OH
Lactate
PDH-P
Inhibition Inactive
DCA Phosphptase
PDH
Active ..'
Figure 1
Mechanism of Action of DCA on pyruvate dehydrogenase.




3
Glutathione-S-Transferase Zeta
At the time the present study was begun, the known metabolites of DCA were
oxalate, CO2, and glycine. Glyoxylate and glycolate were postulated as intermediates, as was monochloroacetate (MCA).4 During the course of this study, it was shown definitively that DCA was converted to glyoxylate by liver cytosol, in a glutathionedependent pathway. Recently it has been shown that the dechlorinating enzyme is GSTz, which has been isolated from hepatic rat cytosol.6 GSTz was thought to be a novel enzyme but is in fact a glutathione dependent isomerase involved in the catabolism of tyrosine, first isolated as maleylacetoacetate isomerase 1955 by Knox and Edwards (Figure 2). Its endogenous ligands, maleylacetoacetate, and the decarboxylation product of maleylacetoacetate, maleylacetone, are known alkylating agents and scavengers of glutathione.8 The enzyme utilizes GSH as a catalyst in the isomerization of maleylacetoacetate (cis) to fumarylacetoacetate (FAA) (trans) through the nucleophilic attachment of GSH to the P-carbon of the cis double bond. Following reketonization and loss of GSH, the more stable trans form results. Additional alpha-beta unsaturated acids which are isomerized by GSTz are listed in Table 1. Maleylacetoacetate is similar in structure to maleic acid, which can be toxic. It also forms GSH adducts.8 Maleylacetoacetate, FAA, and succinylacetone, a reduction product of FAA, are thought to be responsible for the toxic effects seen in hereditary tyrosinemia type I, which is due




to the deficiency of fumarylacetoacetate hydrolase, the step following maleylacetoacetate
isomerase in the breakdown of tyrosine.9
H NH0
-CH-COOH
Phenylalanlne
Deficiency 4-phenylalnine leads to PKU monooxygenase
C-CH-COOH
H NH
HO" Tyrosine
tyrosine
aminotransferase
HO
HO- -COOHO H
4-Hydroxyphenylpyruvate
Inhibited by NTBC 4-hydroxyphenylpyruvate Inhibited y NTBC- dioxygenase
HO HO 0
H
Homogentisate
IDeliciency leads to 1,2-homogentisate % Alcaptonuria, dioxygenase Black Urine Disease
0 0
-OCDecarboxylase c -0 0 ryC OOC OO Reduction OCOO0 0 /
0
Maleylacetone 4-Maleylacetoacetate* (cis) Succinylacetoacetate 4-maleylacetoacetate
isomerase (GSTz), SH CO2 MAAI iM.AAI CS oSH ooDCA T, 1
OOC OOC COO OOC C000
0 0 Succinylacetone Fumarylacetone 4-Faumarylacetoacetate* (trans)
Deficiency leads to Hereditary fumarylac toacetate d-Ainoevulimte* inhibition TyrosinemrniaType I and hydrolase(FAH) succinylacetone formation Porphobilinogen
*Alkylating Agents OOC / 0- CO0 *NurotxinCOO"
**Neurotoxin Fumarate COO Acetoacetate Figure 2
Tyrosine catabolism




5
Table 1
Alpha-beta unsaturated carboxylic acids metabolized by GSTz.10
0
-OOC "OOC CH3 -0C CH3
0 COO- 0 HO
Maleylacetoacetate Z,Z -Maleylacetone, ketoenol Z,E -Maleylacetone, ketoenol
OOC CH3 "OOC CH3 0C CH3
0 0
O cis-p-acetylacrylate O
Maleylacetone, diketo Z,E-6,-keto-2,4-heptadienoate
-00C 0 CH3 00C /=\-/Coo- 0
OOC- CH3 "OOCCOO- -OOC OCH3 Z,Z-muconate
E,Z -6,-keto-2,4-heptadienoate Z,Z-muconate monomethyl ester
-OOC /= OCH3
O
Z0E-muconate monomethyl ester
Z,E-muconate monomethyl ester




6
The toxic effects of hereditary tyrosinemia type I include liver failure, cirrhosis, hepatocellular carcinoma, glomerulosclerosis, and peripheral neuropathy.9 A direct cause and effect relationship between maleylacetoacetate and the toxic effects of hereditary tyrosinemia type I has not been conclusively determined, due to the instability of maleylacetoacetate. 11
Glutathione-S-Transferases
The enzymes of the GST family can be found in both the microsomal and the
cytosolic fractions of cells. The cytosolic enzymes in mammals include six classes; alpha, mu, pi, kappa (a recently discovered mitochondrial species12), theta, and zeta, which are selective rather than specific in their metabolism of electrophilic compounds. All known members exist as either homodimers or heterodimers.13 The enzymes are responsible for the detoxification (and occasional activation) of many endogenous and xenobiotic electrophiles through the nucleophilic attack of electrophilic centers by the sulfhydryl group of the enzyme activated tripeptide, glutathione. This activation of glutathione is accomplished through the presence of a catalytic serine or tyrosine at the glutathione binding site of the enzyme, which hydrogen bonds to the thiol of glutathione and lowers its pKa from 9.3 to 6.5-7.4.14 The activated GSH then attacks the electrophile in an SN2 like reaction.14 Tyrosine is found in the catalytic site of GST alpha, mu, and pi, whereas serine is the catalytic amino acid for the theta and zeta classes. Zeta is a recent addition to




the GST family of enzymes, shares little similarity to the alpha, mu, and pi classes, and is only slightly similar to GST theta. GSTz (GST zeta) like other GST's is a dimer of 24.2 kDa subunits, and has activity against t-butyl and cumene hydroperoxides, but is not strongly active against 1-chloro-2,4-dinitrobenzene, a substrate used to measure GST activity in cytosol preparations.15 GSTz has been found in a number of species including carnations, Caenorhabditis elegans, rats, and humans.16 GSTz is interesting in its ability to both isomerize alpha, beta-unsaturated carboxylates and to dehalogenate certain haloacids (Tables 1 and 2).
Potential Mechanisms of DCA Dechlorination
One postulated pathway for the metabolism of DCA to glyoxylate by GSTz is through the displacement of one chlorine atom by glutathione, hydrolysis of the second chlorine to give S-(ot-hydroxycarboxymethyl)glutathione, and elimination of GSH to give glyoxylate (Figure 3). 17
HO C10 H20 HO 00
11 GST zeta l H0 + GH
Cl--C-OH G GS-C-COH GS---OH H-C-C-OH + GSH
C1 GSH H HCI OH Figure 3
Possible mechanism for the conversion of DCA to glyoxylate.




The mechanism of dechlorination of DCA has recently been investigated using deuterated DCA.18 Deuterium was retained in the conversion of DCA to glyoxylate, suggesting a mechanism in which the initial glutathione adduct loses the remaining chloride ion through neighboring group participation leaving a resonance stabilized zwitterionic intermediate, GS+=CHCOO- which can react with water to form a hemithioacetal and lose glutathione to form glyoxylate. The deuterium is retained in this mechanism (Figure 4).
H20
Cl -C GS _. O -Cl- or GSTz Cl O- +GSH/GSTz C1 O_
Deuterated GS + O- GS ODichloroacetate
D 0 -GSH 4 GSTz OO O' GS O GS ODeuterated InactivatedGSTz Glyoxylate
Figure 4
Possible mechanism accounting for retention of deuterium in glyoxylate from DCA dechlorination.




9
The formation of the zwitterionic intermediate is theorized to allow the attack of a nucleophilic site on GSTz, leading to inactivation. However, the mechanism seen in Figure 3 allows retention of the deuterium label without the formation of a zwitterionic intermediate. The observation that fluorinated haloacids (Table 2) do not lead to inactivation of GSTz is attributed to the decreased basicity of fluoride, which makes it a poor leaving group. The adducted glutathione leaves first, without the formation of a zwitterion (Figure 5). Other haloacids metabolized by GSTz are found in Table 2.
Cl o -cl- D S O OHHOD O -HD O
CI-HF D 0 F 0- +GSH/GSTz F O" -GSH F O O Deuterated
Deuterated Glyoxylate Dichloroacetate
Figure 5
Proposed mechanism for removal of fluorine avoiding the zwitterion intermediate.




10
Haloacids Metabolized by GSTz
BrO BrO CIO
I II I II I II
F-C-C-OH Cl-C-C-OH F-C-C-OH
I I I
H H H
Bromo fluoroacetic acid Bromochloroacetic acid Chlorofluoroacetic acid
5028 150 1411 65.2 3883 43.0
C10 HO HO
I II I II I II
Cl-C-C-OH H3C-C-C-OH H3C-C-C-OH
I I I
H I Cl
Dichloroacetic acid 2-lodopropionic acid (R)-2-Chloropropionic acid
1038 19.8 (R,S) 2532 42.9 318 14.0
HO Cl 0 HO
I II II I II
H3C-C-C-OH H3C-C-C-OH H3C-C-C-OH Br CCl
2-Bromopropionic acid (S)-2-Chloropropionic acid 2,2-Dichloropropionic acid
(R,S) 2142 107.9 1809 71.3 244 6.1
HO HO BrO
I II I II I II
F-C-C-OH H3C-C-C-OH Br-C-C-OH
I I I H Cl H
Fluoroacetic acid (R,S)-2-Chloropropionic acid Dibromoacetic acid
172 16.0 155 4.0 1655 66.3
Table 2
Haloacids found to be metabolized by GSTz. Difluoroacetic acid, (R,S)-2Fluoropropionic acid, and 3,3-Dichloropropionic acid were not found to be metabolized
7
by GSTz. Rates reported in nmoles product formed (from 0.5 mM substrate)/min/mg purified rat GSTz as means + SD, n=3.




11
Metabolites of DCA
DCA was known to be metabolized to CO2, glycine, and oxalate4, but glyoxylate had not been unequivocally identified; it was presumed a metabolite from its co-elution with standard on HPLC.19 The first step in the metabolism of DCA is its dechlorination to glyoxylate, which was been found to be catalyzed by GSTz. DCA's dechlorination product, glyoxylate, may be further metabolized to numerous products. Glycolate and the terminal metabolite oxalate, are formed via the cytosolic enzyme lactate dehydrogenase.20 Glyoxylate can also be decarboxylated in the mitochondria to CO2 via the enzyme otketoglutarate:glyoxylate carboligase.21 Transamination of glyoxylate to glycine occurs in the peroxisomes by the action of glyoxylate aminotransferase, or in the mitochondrial matrix by any of several enzymes (Figure 6).
DCA derived glycine becomes available for entry into many routes of
intermediary metabolism, such as the "1 carbon pool" after incorporation into N5, N10methylene tetrahydrofolate, glycolysis through mitochondrial serine hydroxymethyl transferase (Figure 7), or for conjugation with carboxylic acids via glycine Nacyltransferase.22




12
Involvement of the Tyrosine Catabolic Pathway in DCA Metabolism
It is possible that DCA and maleylacetoacetate/maleylacetone are inhibitors of GSTz, and that the auto-inhibition phenomena seen in DCA metabolism may in fact be due to the buildup of maleylacetoacetate or maleylacetone, which could alkylate the enzyme or the locally available glutathione. Alkylation of GSH is unlikely to affect DCA kinetics given the low KM for GSH.5 It is also possible that DCA induced inhibition of maleylacetoacetate degradation could account for some of the toxic effects presently attributed to DCA which are in common with those seen in hereditary hypertyrosinemia type I, such as peripheral neuropathy and hepatocellular carcinoma, since the effects of hereditary hypertyrosinemia type I are due to the buildup in cells of several compounds, including maleylacetoacetate.
In summary, the specific aims of this work have been to elucidate unknown
urinary metabolites of DCA, to uncover the metabolic pathways of DCA metabolism to glyoxylate, and to test two hypotheses concerning DCA's auto-inhibition. One hypothesis was that DCA directly inhibits its own metabolism through a "poisoning" effect on the enzyme. The other was that DCA could compete with the native substrates for GSTz, maleylacetoacetate and/or maleylacetone, which could lead to their buildup or alkylate the enzyme, which could render it inactive.




13
H 0 0 0 0O I II II II II II
Cl-C-C-OH Dechlorination H-C-C-OH Lactate HO-C-C-OH
cl GSTz* Dehydrogenase O-CCO
c1 t Cytosol Glyoxylate** Cytosol Oxalate
Dichloroacetic Cytosol s oAcid
Amin )transferases
a-Ketoglutarate:Glyoxylate (several) Lactate
Carboligase Mitochondria Dehydrogenase
Mitochondria Peroxisomes Cytosol
HO HO I II I II
H-N-C-C-OH HO-C-C-OH
Co2 H HH
Glycine Glycolate
Figure 6
Metabolites of DCA as understood in 1995. *The enzyme investigated in this study.
**Not unequivocally determined by 1995.




14
HO OO
I I-I Dechlorination II I a-Ketoglutarate:Glyoxylate *C
CI-C-C-OH HCC-H *~
I GST-z H-C-C--OH Carboligase Mitochondria
Cl Cytosol Glyoxylate
Dichloroacetic Aminotransferases
Acid (several)
HO
I II,
H-N-C-C-OH
I I
NAD+, H H
NAD THF Glycine Serine Hydroxymethyl transferase
**Glycine Synthase / Mitochondria
(Glycine Cleavage System
via formate) Mitochondria NH
HO-C-COOH
H H
H
H2N N Serine
'e+ X CO2, NH4+, NADH Serine Dehydratase Loss of 14labeled 3
0 H2C-N-R carbon ytosol
N 5,Nl-methylene THF 0
1-carbon donor in many reactions HzC-C-COOH Pyruvate
route fLgsyinefI14C aee
**Major route of glycine PDH 02 Loss of 14C labeled degradation in mammalian Mitochondria carbon tissues
H3C-CSCoA
Acetyl CoA
Figure 7
Possible Routes of CO2 Formation




CHAPTER TWO
MATERIALS AND METHODS
Chemicals and Equipment
14
C radiolabeled DCA was purchased from American Radiolabeled Chemicals, St. Louis, MO. The specific activity was 55.5 mCi/mmol and the reported and measured 14
radiochemical purity was >99.9%. C-Dichloroacetic acid was converted to its sodium salt by equimolar addition of NaOH. Na[1,2-13 C-]-DCA was purchased from Cambridge Isotope Laboratories, maleylacetone. Unlabeled NaDCA was obtained from TCI America, Portland, OR. GSH, NADPH, NADH, glyoxylate, oxalate, glycolate, KC1, isobutanol, resorcinol, trichloroacetic acid, and HEPES were all purchased from Sigma Chemical Company (St. Louis, MO). Methanol, KH2PO4 and K2HPO4, ascorbic acid, NaC1, hydrochloric acid, sodium chloride, and sodium acetate were purchased from Fisher Chemical Company (Fair Lawn, NJ). DTT, Biorad Protein Dye Reagent, sodium dodecyl sulfate were purchased from Biorad (Hercules, CA). Carbasorb and Permafluor cocktails for use with a Tricarb tissue oxidizer and Floscint II cocktail for the radiochemical detection on HPLC were purchased from Packard Instruments, Chicago, IL. Ecolume scintillation cocktail was purchased from ICN, Costa Mesa, CA. Anhydrous tetrahydrofuran, maleic anhydride, isopropenyl acetate, glycerol, anhydrous magnesium sulfate, and CH2C2, were purchased from Aldrich Chemical Company, St. Louis, MO. All other chemicals used were the purest grade available from Millipore, Woburn, MA, Fisher Scientific, Orlando, FL, Sigma Chemical Co., St. Louis, MO or Aldrich Chemical Co., St. Louis, MO.
15




16
Ready Gels, Biorad mini-Protean II Cell, Power Pac 3000, and Biorad Model
1000/500 power supplies were purchased from Biorad (Hercules, CA). Sepharose 4B gel was purchased from Pharmacia Biotech (Piscataway, NJ). The glass metabolism cage for the radiolabeled studies was purchased from Stanford Glass, Stanford, CA. The plastic cages were from Nalgene Metabolic Cages, Rochester, NY.
Animals
In Vivo Studies
Male Sprague Dawley rats were used in these studies. In the studies of urinary metabolites of DCA, five male Sprague Dawley rats with weights averaging 230 grams were cannulated at the jugular vein for blood sampling in the initial in vivo study according to the method of Harms and Ojeda.23 Rats were divided into two groups, single 14 13
and repeat dose. The single dose rats received one 1% C + 99% C labeled DCA dose of 50 mg/kg by oral gavage. The repeat dose rats received one 50 mg/kg dose of unlabeled DCA the first day, then the second day each received 50 mg/kg of DCA
14 13
labeled with 1% C + 99% C. Each rat was kept for 24 hours in the metabolism cage, which collected the rats' CO2, urine, and feces. Urine samples were collected at 3, 6, 9, 12 and 24 hours. At the end of sampling, rats were sacrificed and their organs removed for oxidation of 100 mg tissue samples on a Tri-Carb tissue oxidizer.




17
Effect of In Vivo DCA Pre-treatment on its In Vitro Metabolism
For the initial in vitro studies of DCA inhibition, cytosol was obtained from livers of 36 day old Harlan Sprague Dawley rats which received either 2 ml/kg water or unlabeled DCA at a dose of 50 mg/kg (2 ml/kg), by oral gavage as one daily dose for 2 days. The rats were sacrificed on the third day and hepatic cytosol prepared.24
Dose Response Studies
A similar study was conducted with 13C-DCA in plastic metabolism cages. Male Sprague Dawley rats (250 to 300g) were acclimatized for one week before experimentation. During dosing periods and control days the rats were kept in metabolism cages (Nalgene Metabolic Cages, Rochester, NY) and urine was collected over 24 hours during the control day (day 0) and on the study days. Neutralized NaDCA at doses of 4, 12.5, 50, 200 and 1000 mg/kg was administered by oral gavage in the morning (7:30AM) for one or five days. Twenty-four hours after the last dose, rats were sacrificed and their livers were removed for preparation of liver cytosol.24 Four animals were used for each dose investigated. Urine samples were analyzed for maleylacetone content using GC-MS described below. Hepatic cytosol was used to measure 14C-DCA metabolism.




18
Preparation of Rat Liver Cvtosol
Male Sprague Dawley rats were sacrificed by decapitation. Their livers were excised and cytosolic fractions prepared by differential centrifugation described previously. Livers were rinsed in 1.15 % KC1, 0.05 M potassium phosphate buffer, pH
7.4, three times, then minced before homogenization using four up and down strokes in a Potter-Elvehjem vessel. Homogenized samples were then centrifuged at 13,300 g for 30 minutes. The supernatant was transferred to polycarbonate tubes for centrifugation at 170,000 g for 50 minutes to separate cytosol and the microsomal pellet. The pellets were resuspended in buffer and resedimented for the preparation of washed microsomes.24 Preparation of Human Liver Cvtosol
Liver sections (10-15 g) were minced and homogenized in 25-30 ml
homogenizing buffer, 1.15% KC1, 0.05 M potassium phosphate, pH 7.4, 0.1 mM phenylmethylsulfonyl fluoride, with several up and down strokes in a Potter-Elvehjem homogenizing vessel. Cytosolic fractions were obtained through differential centrifugation as above.24 Remaining liver pieces were stored either in homogenizing buffer at -800C or frozen alone in aluminum foil at -800C. Protein concentrations of all cytosol samples were performed according to the method of Lowry et al.25




19
Assays of DCA Metabolism
General
All incubations were performed in duplicate in borosilicate tubes at 370C in a
shaking water bath. Tubes contained 0.1 M Hepes, pH 7.6, GSH (0-1 mM), cytosol (0-1.5 mg), and DCA (0-0.2 mM) in a final volume of 0.25 ml. In most studies DCA was added last and tubes were incubated for 0-60 minutes. Reactions were stopped by the addition of 0.5 ml ice-cold methanol. Tubes were centrifuged at 1500 rpm for 20 minutes and the supernatant transferred to microfuge tubes for storage at -200C until 14C-glyoxylate quantitation by HPLC with radioactivity detection, or the fluorescence assay of unlabeled glyoxylate (see below). The apparent KM, Ki, and Vmax were determined from the linear equation of the graph in the Graph Pad Prism program by the Lineweaver Burke or Hanes methods.2226 The student's t-test was used in the statistical analysis of the apparent KM and Vmax averages.
Cvtosol Dialysis
For the rat apparent KM studies, cytosol was dialyzed in four liters of a 1.15%
KC1, 0.05 M potassium phosphate pH 7.4 buffer (dialysis buffer), and in dialysis tubing with a molecular weight cutoff of 12,000 Daltons (molecular units). After 2-3 hours in a multi-dialyzer apparatus from Spectrum, the buffer was changed and the cytosol was dialyzed for 2 additional hours. For comparison of NADH, NADPH and GSH dependency, cytosol from four control rats and four DCA dosed rats was dialyzed as




20
above, then incubated at 370C for 15 minutes with either 2 mM NADPH to reduce any GSSG present to GSH, or with water as a control. This cytosol was then re-dialyzed for 2 hours on the multi-dialyzer and used immediately in the incubation. For the GSH apparent KM studies, the cytosol was first incubated at 370C for 15 minutes with 2 mM NADPH to reduce any GSSG present to GSH and to free any protein bound GSH, then dialyzed as above. In experiments when smaller samples of protein were needed, or fewer samples required, 3 ml aliquots were dialyzed in 250 ml dialysis buffer for one hour, then the buffer was changed and the samples dialyzed overnight.
For the in vitro DCA inhibition studies cytosol samples (10 ml) were dialyzed at 4oC in 50 x buffer volume with either 1.15% KC1, 0.05 M KH2PO4, pH 7.4, 0.1 mM dithiothreitol, or 0.1 M Hepes, pH 7.6 and 1 mM GSH, in dialysis tubing with a MW cutoff of 12,000 Daltons. After 1-2 hours the buffer was changed and the cytosol was dialyzed overnight. Protein concentrations of all cytosol samples were quantitated.26
Reducing Agent Dependence Incubations
Concentrations of 1 mM GSH, 2 mM NADH, and 2 mM NADPH were used
along with blanks without any reducing agent, blanks with reducing agent but no cytosol, and one zero time blank with 1 mM GSH.




21
Linear Range of Glyoxylate Production in Rat Hepatic Cvtosol
A time course of glyoxylate production was performed with 0.2 mM DCA, 1 mM GSH, 1 mg dialyzed rat hepatic cytosolic protein, and 0.1 M Hepes pH 7.6. Incubations were stopped by the addition of ice-cold methanol at time 0, 5, 10, 15, 30, 45 and 60 minutes. Tubes were centrifuged at 1500 rpm for 20 minutes and the supernatant transferred to microfuge tubes for storage at -200C until 14C-glyoxylate quantitation by radiation detection coupled to ion pair HPLC (see below).
A range of GSH concentrations including 0, 0.00625, 0.05, 0.1, 0.25, 0.5, 1 and 1.5 mM were used in 15 minute incubations at 370C with 0.2 mM DCA, 1 mg dialyzed rat hepatic cytosolic protein and 0.1 M Hepes pH 7.6. Reactions were stopped by the addition of 0.5 ml ice-cold methanol after 15 minutes. Tubes were centrifuged at 1500 rpm for 20 minutes and the supernatant transferred to microfuge tubes for storage at 200C until 14C-glyoxylate quantitation by radiation detection coupled to ion pair HPLC.
Different DCA concentrations were incubated with 1 mM GSH, 1 mg dialyzed rat hepatic cytosolic protein and 0.1 M Hepes pH 7.6. DCA concentrations included 0.05,
0.1, 0.2 and 0.5 mM DCA. Incubations were stopped by the addition of ice-cold methanol after 15 minutes. Tubes were centrifuged at 1500 rpm for 20 minutes and the supernatant transferred to microfuge tubes for storage at -200C until 14C-glyoxylate quantitation by radiation detection coupled to ion pair HPLC.
Protein concentrations were also varied to investigate the linear production of glyoxylate. Dialyzed hepatic rat cytosol was incubated at concentrations of 0, 0.05, 0.1, 0.15, 0.2 and 0.25 mg in a final incubation volume of 0.25 ml. Tubes also contained 0.2 mM DCA, 1 mM GSH and 0.1 M Hepes pH 7.6. Incubations were stopped by the




22
addition of ice-cold methanol after 15 minutes. Tubes were centrifuged at 1500 rpm for 20 minutes and the supernatant transferred to microfuge tubes for storage at -200C until 14C-glyoxylate quantitation by radiation detection coupled to ion pair HPLC.
Rat Apparent K and V Determination Incubations
For the apparent KM of DCA determination all tubes contained 1 mM GSH and either 0.1, 0.075, 0.05, 0.025, 0.01, 0.005, or 0.002 mM DCA. For the apparent KM of GSH determination all tubes contained 0.2 mM DCA and either 0.10, 0.075, 0.05, 0.025, or 0.01 mM GSH. All samples were incubated for 15 minutes and contained from 0.2-4 mg/ml cytosolic protein. Samples were analyzed by HPLC coupled to radiation detection. Percent 14C-glyoxylate produced was used to determine the specific activity as nmoles/minute/mg protein. KM and Vmax were determined through Lineweaver-Burk plots of 1/specific activity vs. 1/mM GSH or 1/mM DCA, with the x intercept being 1/KM and the y intercept being 1/Vmax. Values were determined using Graph Pad Prism software.
Linear Range of Glvoxvlate Production in Human Hepatic Cvtosol
A time course for glyoxylate production was performed with 0.2 mM DCA, 1 mM GSH, 1 mg dialyzed hepatic cytosolic protein from human liver, and 0.1 M Hepes pH 7.6. Incubations were stopped by the addition of 750 jl 5% TCA at time 0, 10, 14.25, 28.5, 42.75, 57, 90 and 120 minutes. Tubes were centrifuged at 1500 rpm for 20 minutes




23
to precipitate protein and the supernatant transferred to tubes containing resorcinol and hydrochloric acid for glyoxylate measurement as described in the fluorescence assay.
A range of GSH concentrations including 0, 0.01, 0.025, 0.05, 0.075, 0.1, 0.2 and
0.25 mM were used in 30 minute incubations at 370C with 0.2 mM DCA, 0.9 mg dialyzed human hepatic cytosolic protein, and 0.1 M Hepes pH 7.6. Incubations were stopped by the addition of 750 p.d 5% TCA. Tubes were centrifuged at 1500 rpm for 20 minutes to precipitate protein and the supernatant transferred to tubes containing resorcinol and hydrochloric acid for glyoxylate measurement as described in the fluorescence assay.
Different DCA concentrations were incubated with 1 mM GSH, 0.9 mg dialyzed hepatic cytosolic protein, and 0.1 M Hepes pH 7.6. DCA concentrations included 0.002, 0.005, 0.01, 0.025, and 0.05 mM DCA. Incubations were stopped after 30 minutes by the addition of 750 pl 5% TCA. Tubes were centrifuged at 1500 rpm for 20 minutes to precipitate protein and the supernatant transferred to tubes containing resorcinol and hydrochloric acid for glyoxylate measurement as described in the fluorescence assay.
Protein concentrations were also varied to investigate the linear production of glyoxylate. Dialyzed human hepatic cytosol was incubated at concentrations of 0, 0.25,
0.5, 0.75, 1, 1.5, 1.76 and 2 mg in a final incubation volume of 0.25 ml. Tubes also contained 0.2 mM DCA, 1 mM GSH, and 0.1 M Hepes pH 7.6. Incubations were stopped by the addition of ice-cold methanol after 60 minutes. Tubes were centrifuged at 1500 rpm for 20 minutes and the supernatant transferred to microfuge tubes for storage at
-200C until 14C-glyoxylate quantitation by radiation detection coupled to ion pair HPLC.




24
Apparent K and Vax in Dialyzed Human Liver Cvtosol Incubations
Various concentrations of both GSH and DCA were used simultaneously to
eliminate the possible substrate inhibition from either agent. DCA concentrations were
0.002, 0.005, 0.01, 0.025, and 0.05 mM. GSH concentrations were 0.01, 0.025, 0.05,
0.075, and 0.1 mM. Tubes were incubated for 10 minutes with a protein concentration of either 0.628 mg/ml (0.002 -0.01 mM DCA) or 1.568 mg/ml (0.025 and 0.05 mM DCA). Samples were analyzed as above with rat hepatic cytosol.
In Vitro DCA Inhibition Incubations
Tubes contained 0.1 M Hepes buffer pH 7.6, 1 mM GSH, and 0.2 mM DCA. The amount of cytosolic protein from human liver in the tubes for the time course incubations was approximately 4 mg/ml. DCA was added last in all incubations in 30-second intervals. Water replaced DCA in the control tubes. At time intervals of 0, 5, 10, 20 and 30 minutes, 250 tl were removed from all treatment tubes to assay glyoxylate production using the fluorescence assay. In all studies after the initial incubations, the remaining cytosol was placed on ice, then transferred to dialysis tubing for dialysis as described above. After the second dialysis, control and DCA treated cytosol were both incubated with 0.2 mM DCA, approximately 2 mg/ml protein, 0.1 M Hepes buffer pH 7.6, and 1 mM GSH at 0, 5, 10, 15, 20, and 30 minutes, then assayed for glyoxylate production as above.
For the inhibition studies with different DCA concentrations, treatment tubes contained either 0, 0.5, 0.75 or 1 mM DCA, and 0.1 M Hepes buffer, pH 7.6,




25
approximately 2 mg/ml protein from human liver, and 1 mM GSH. Initial incubations were performed for 30 minutes at 370C. After 30 minutes, 250 pl portions were removed from all tubes for activity measurement, and the remaining cytosol was put on ice, then dialyzed overnight. For the measurement of residual activity the concentrations of DCA in the treated group corresponded with the treatment concentrations, and the control cytosol was also incubated with either 0.5, 0.75 or 1 mM DCA, 0.1 M Hepes buffer pH
7.6, approximately 1.5 mg/ml protein, and 1 mM GSH. After 30 minutes, 250 pl aliquots were checked for activity using the fluorescence assay. To simplify the results from this experiment, another experiment was conducted in which the measurement of residual activity was performed with 0.2 mM DCA in all tubes to ease comparison between initial treatments with the three different concentrations of DCA and the control cytosol.
DCA Pre-Incubation Comparison between Dialyzed Rat and Human Cvtosol
To determine the effect of pre-incubation with DCA in rat (N=3) and human
(N=3) dialyzed cytosol, duplicate incubation tubes contained 0.1 M Hepes buffer pH 7.6, 1 mM GSH, 0.5 mM DCA, and approximately 4 mg/ml dialyzed hepatic cytosolic protein in a final volume of 10 ml. DCA was added last to all incubations. Water replaced DCA in the control tubes. At 10 min (rat cytosol) or 30 min (human cytosol), 250 pl portions were removed from all tubes to assay glyoxylate formation using the fluorescence assay described below. After the incubation with DCA or water for 30 min, the remaining samples (8.75 ml) were dialyzed as described above in 0.1 M Hepes pH 7.6, and 1 mM GSH. After the second dialysis, protein concentrations were determined again and control




26
and DCA treated cytosol samples (approximately 4 mg/ml) were separately incubated for 0-60 min with 0.2 mM DCA and 0.5 mM GSH in a final volume of 0.25 ml. The reactions were stopped by addition of 750 ptl ice-cold 5% trichloroacetic acid and were assayed for glyoxylate using the fluorescence assay.
DCA Binding Study in Dialyzed Rat and Human Hepatic Cytosol
To determine whether DCA binds cytosolic protein, duplicate samples of dialyzed rat and human hepatic cytosol were incubated with 0.5 mM 14C-DCA. Dialysates consisting of 4 mg protein, were incubated for ten minutes at 370C with 0.5 mM 14CDCA, 0.1 M Hepes pH 7.6, and water in a final volume of 1 ml. Glutathione was excluded to prevent metabolism of DCA. A ten microliter aliquot from each incubation sample was removed for counting on a scintillation counter. After ten minutes, the samples were ultrafiltered for ten minutes using a 0.45 jtm cutoff. Aliquots of ten microliters were removed from both the supernatants and filtrates from each incubation sample. Counts measured in disintegrations/min of aliquots from the samples before and after filtration were compared to determine the extent, if any, of DCA binding.




27
In Vitro GSTz Activity in DCA Dosed Animals
Duplicate hepatic cytosol samples from DCA treated animals containing
approximately 1 mg protein were incubated at 370C in a shaking water bath with 0.1 M Hepes, 1 mM GSH, 0.2 mM 14C-NaDCA and water for a final volume of 0.25 ml. After 5-15 min, reactions were stopped by adding 0.5 ml ice-cold methanol. Tubes were placed on ice and then centrifuged at 1500 rpm to precipitate protein. Supernatants were transferred to microfuge tubes and stored at -200C prior to HPLC analysis and radiochemical detection of 14C-glyoxylate produced from 14C-DCA.
Analyses
Ion Pair HPLC
Samples investigating the apparent KM and Vmax in rat and human cytosol samples were analyzed by HPLC. Raining filterfuge tubes with a 0.45 p.m cutoff were used to filter the incubation samples' supernatants before HPLC analysis. The mobile phase was 70:30 0.005 M aqueous tetrabutylammonium sulfate (low UV Pic A, Waters, MA):methanol. An Isco model 2350 pump was used with a Rainin Dynamax UV-1 detector, a Radiomatic Flo One B Radioactive Flow Detector, and a Beckman Ultrasphere
5 pm x 4.6 mm x 25 cm reverse phase ODS column coupled to a Beckman Ultrasphere 5 p.m x 4.6 mm x 4.5 cm pre-column. All retention times were compared with standards of DCA, oxalate and glyoxylate for confirmation.




28
Strong Anion Exchange HPLC Analysis
Rainin filterfuge tubes with a 0.45 pm cutoff were used to filter the samples
before HPLC analysis. The mobile phase was 0.1 M potassium phosphate at pH 3. An Isco model 2350 pump was used with a Rainin Dynamax UV-1 detector, a Radiomatic Flo One 13 Radioactive Flow Detector, and a Zorbax strong anion exchange column, 4.6 mm ID x 15 cm. All retention times were compared with standards of DCA (5.3 min) and glyoxylate (3.0 min) for confirmation. Percent conversion of 14C-DCA to 14C-glyoxylate was determined by radiochemical detection and used to calculate nmoles glyoxylate formed/min/mg protein.
13
1C NMR Analysis
One sample of urine collected from a rat dosed with 99% [1,2-13C]DCA and 1% [1-14C]DCA contained 300 pg of DCA equivalents (70.5% of the DCA derived 14C) as the unknown major urinary metabolite. This was diluted with 150 Pl of D20 to adjust magnetic field homogeneity and to maintain stable lock frequency. This was examined in a 5-mm tube. A trace of 3-(trimethylsilyl)propionate-2,2,3,3-d4 sodium salt was included as an internal standard of chemical shift reference. 13C NMR analysis was performed on a 300 MHz Varian Unity NMR spectrometer with a 5-mm broad band probe regulated at 250C, spinning at 20 Hz (Center for Structural Biology, University of Florida). Four spectra were acquired; one proton-coupled spectrum and three proton-decoupled spectra. One proton-decoupled spectra was acquired with continuous proton-decoupling. Ten thousand transients were accumulated over a 27.75 hour period. The free induction decay




29
was processed with an exponential line broadening of 1 Hz before Fourier transformation.
Fluorescence Analysis
For certain experiments it was favorable to use unlabeled DCA to avoid excessive contamination and to speed quantitation of unlabeled glyoxylate. The quantification of unlabeled glyoxylate through fluorimetry of its condensation product with resorcinol was modified from the methods of Zarambski and Hodgkinson (Figure 8).27
OH
H HO OH HC-C-OH -)NO
2 OH HOHOO Resorcinol Glyoxylate 2,2',4,4'-Tetrahydroxydiphenylacetic acid lactone Fluorescent
Figure 8
Formation of 2,2',4,4'-tetrahydroxydiphenylacetic acid lactone.
Glyoxylate standards and incubation samples were deproteinized with 750 pl 5% TCA. An aliquot of this mixture (750 pl) was transferred to marble covered tubes containing 25 ptl 5% resorcinol and 375 ptl concentrated HCI and heated for five minutes at 1000C. After cooling, samples were extracted by vortexing for eight seconds with 1.25 ml isobutanol. Tubes were centrifuged at 1500 rpm for ten minutes. The isobutanol layer




30
(1 ml) was transferred to tubes containing 2.5 ml 25% K2CO3. These tubes were mixed with a vortex for eight seconds, centrifuged, and 2 ml of the aqueous layer was transferred to tubes containing 250 pl 10% ascorbic acid. After the addition of 4 ml 0.1 M sodium carbonate-bicarbonate buffer pH 9.6, tubes were incubated at room temperature for thirty minutes. Fluorescence of 2,2',4,4'-tetrahydroxydiphenylacetic acid lactone was measured using an Fmax fluorescence plate reader, with an excitation of 485 nm and an emission X of 538 nm.
GC-MS Confirmation of Glvoxylate
Urinary metabolites were separated and identified using a Hewlett-Packard 5890 series II plus GC, a 5972A mass selective detector and a Vectra multimedia VL2 4/66 computer with ChemStation software.28 Samples were spiked with 4-chlorobutyric acid as an internal standard, and methylated by heating with an equal volume of a 14% solution of boron trifluoride in methanol at 1150C for 15 min, then extracted into methylene chloride. The methylene chloride extract was injected onto an HP-Wax column, 30 m x 0.25 mm with 0.15 pm film thickness, phase ratio 420 with helium carrier gas at a flow rate of 1.21 ml/min and an inlet pressure of 9 psi. The GC temperature was 350C for 4 min, followed by a linear gradient to 1000C at 30C/min then up to 2400C at 500C/min. The temperature was held at 2400C for 8 min. The retention time of the methyl esters of DCA and metabolites were as follows: DCA, 10.8 min; glyoxylate 11.2 min; oxalate 11.7 min; hippuric acid 20 min. The urinary metabolites were identified by their GC retention times and by matching the complete mass spectrum of the 13C-labeled metabolite to the mass spectrum of known 12C standards (performed in




31
Dr. G.N. Henderson's laboratory by Ximeng Yan, University of Florida, College of Medicine).
GC-MS Analysis of Maleylacetone
Urine (0.2 ml) was pipetted into a Coming culture tube with a Teflon -lined screw cap. BF3 (12%) in MeOH, 0.5 ml, and 50 jtl (960 pg/ml) of 2-oxohexanoic acid (internal standard) were added, heated at 1100C for 15 min and allowed to cool to room temperature. One ml of water and one ml of CH2C12 were added. The culture tube was vortex mixed for 1 min, placed on a shaker for 5 min and centifuged (100C) at 3000 rpm for 10 min. The lower CH2C12 layer was pipetted into a 2 ml autosampler vial that contained a 100 ul glass insert. Samples were analyzed on a Hewlett Packard 5972 mass spectrometer. The column used was Hewlett Packard HP-WAX (Cross linked Polyethylene Glycol) 30m x 0.25mm x 0.15 um film thickness. The flow rate of zero grade helium was 1.1 ml/min. The GC was temperature programmed as follows: at 400C for 2 min; then to 1000C at 50C per minute; then to 2400C at 150C per minute, and held for 5 min at 2400C. 2-Oxohexanoic acid and maleylacetone had retention times of 13.67 and 20.93 min, respectively. A single ion monitoring method was used for quantitation (performed in Dr. G.N. Henderson's laboratory by Jing Cheung and Larry Shroads, University of Florida, College of Medicine).




32
Synthesis of Maleylacetone
All steps were performed under positive nitrogen flow according to the methods of Fowler and Seltzer, at 1/3 literature scale (Figure 9).29 Aluminum chloride (20 g) was added to 150 ml methylene chloride while stirring. Maleic anhydride (6.67 g, 0.068 mol) was added and the mixture stirred at room temperature for 30 minutes prior to the addition of 7.34 ml (6.67 g, 0.067 mol) isopropenyl acetate over 5 minutes using a dripping funnel. After 5 hours, the mixture was slowly added to 167 ml ice-cold 2 N HC1. The organic phase was separated, and the aqueous layer extracted with 67 ml CH2C2. The organic phase was filtered through Celite to remove particulates, washed with 66 ml 5% Na2CO3 in three portions, then with saturated NaC1. The organic phase was dried with anhydrous MgSO4. The solvent was removed with a rotary evaporator and the residue was frozen overnight at -800C. The following day the crude material (770 mg) was applied to a flash column with 3:1 pentane:ethyl acetate as the mobile phase. Spotting of the crude material on silica TLC plates revealed three major spots. Fractions containing the spot which did not match the spots of the two starting materials and were the reported pale yellow color of the butenolide, were combined and their solvent removed using a rotary evaporator, then dried overnight under vacuum. The 'H-NMR in CDCl3 confirmed the structure of 4-acetonylidenebut-2-ene-4-olide, in accordance with published results (Figure 9).31 The 'H-NMR showed resonances at 8 2.58 (s, 3H, CH3), 5.58 (s, 1H, vinyl), 6.46 and 7.53 (q, 2H) (Figure 9). To 0.13 g butenolide was added 25 ml of 1 N NaOH; this was washed with 10 ml CH2C12. The aqueous (basic) layer was then acidified with concentrated HCI and extracted with 40 ml CH2C12 on ice. The organic phase was washed with 10 ml saturated NaCl solution, dried with anhydrous




33
MgSO4, and filtered through Celite to remove particulates. The solvent was removed at room temperature using a rotary evaporator and the oily product dried overnight under vacuum or under a constant stream of nitrogen for several hours. The 'H-NMR in CDC13 confirmed the structure of maleylacetone (Figure 10) in accordance with published results.31, 30 It displayed resonances at 8 2.3 (s, 3H, CH3), 2.9 (q, 2H, J=16 Hz), 6.12 and 7.35 (q, 2H, J=5.71 Hz), 5.79 (s, 1H, vinyl), 6.38 (s, 2H, vinyls), 2.24 (s, 3H, CH3), 5.79 (s, 1H, vinyl), 6.38 (s, 2H, vinyls). No broad OH signal was seen at 8 7-8 ppm, although a broad band was seen from 6 1-2 ppm due to the presence of water.
OCOCH3 HI O O + H2C AIC3 1. NaOH O- CH3 2. HQ N CH3 CH2CI2 0 Maleic Anhydride Isopropenyl acetate
0
4-acetonylidenebut-2-ene-4-olide
HO2C HO02C
CH3 m -"""" CH3
0 HO
0 0 Maleylacetone
Figure 9
Synthesis of maleylacetone.




34
2 3 2 3
HO2C / __5 7 H25a 7a H02C rCHz ? CH3 Keto form 0 Enol form 0 7a
7
2/3 keto
Sa
3, 5
8 7 5 5 4 3 2 1 0 pp.
Figure 10
HNMR of maleylacetone




35
Maleylacetone Methods
Maleylacetone ICs0 Studies
In a preliminary study, radiolabeled 1-14C-DCA (0.2 mM) was incubated with
maleylacetone at the following concentrations; 0.07, 0.1, 0.2 and 0.4 mM. HPLC coupled with radiation detection was used to monitor the production of radiolabeled glyoxylate from DCA to investigate any inhibition of DCA metabolism. Approximately 30 mg aliquots of maleylacetone were prepared by hydrolysis of the 4-acetonylidenebut-2-ene4-olide,8 and were accurately weighed and diluted in a known amount of THF. Aliquots were delivered into tubes to produce final concentrations ranging from 0-0.4 mM maleylacetone. The THF was evaporated under a constant stream of nitrogen, and the tubes were kept on ice until the aqueous incubation solution was added. Incubations consisted of 0.1 M Hepes pH 7.6, 0.1 mM GSH, approximately 1 mg human hepatic cytosolic protein, 0.2 mM 14C-DCA and water for a final volume of 0.25 ml. Tubes containing THF alone were evaporated under nitrogen to serve as blanks.
After the initial incubation found inhibition of DCA metabolism by
maleylacetone, rat and human (n=3) hepatic cytosol samples were incubated with maleylacetone in concentrations from 0-0.4 mM maleylacetone. THF was replaced with CH2C2 as the solvent vehicle for maleylacetone delivery due to the instability of THF. To avoid possible destruction of GSH and protein before adding DCA in these IC50 studies, 0.1 M Hepes pH 7.6, 0.2 mM 14C-NaDCA, and water were added first to the maleylacetone containing tubes. The reaction was initiated by adding 1 mg cytosol (human) or 0.5 mg cytosol (rat) combined with 1 mM GSH. Tubes were incubated for 0-




36
10 min (rat samples) or 0-30 min (human samples) at 370C in a shaking water bath. Reactions were stopped by adding 0.5 ml ice-cold methanol. Volumes of CH2C2 alone, identical to those used to deliver maleylacetone, were evaporated from tubes to serve as blanks. Tubes were centrifuged at 1500 rpm for 20 minutes to precipitate protein. Supernatants were transferred to microfuge tubes for storage at -200C before strong anion exchange HPLC analysis of 14C-glyoxylate produced from 14C-DCA.
Pre-Incubation Studies
Cytosol from rat and human samples (n=3) and all ingredients except for DCA
were incubated with maleylacetone for 30 minutes before the addition of DCA to initiate the reaction. Reactions were stopped and stored as described above.
Malevlacetone Ki Studies
Aliquots of maleylacetone were delivered into borosilicate tubes in a solution of THF which was evaporated with nitrogen. Concentrations of maleylacetone included
0.05, 0.07, 0.1, 0.2 and 0.4 mM. Various concentrations of DCA included 0.0025,
0.00375, 0.005, 0.015 and 0.025 mM. Tubes were incubated at 370C for 30 minutes with
1 mM GSH, 0.1 M Hepes pH 7.6, and 0.5-1.8 mg human hepatic cytosolic protein from human liver. Protein concentrations were varied to maintain glyoxylate production in the linear range. Tubes were pre-incubated for 2 minutes and DCA was added last to initiate the reaction. Reactions were stopped by the addition of 0.5 ml ice-cold methanol. Tubes were centrifuged at 1500 rpm for 20 minutes to precipitate protein. Supernatants were




37
transferred to microfuge tubes for storage at -200C before strong anion exchange HPLC analysis of 14C-glyoxylate produced from 14C-DCA.
Pre-Treatment of Dialyzed Rat and Human Hepatic Cvtosol with Malevlacetone
In an experiment similar to that performed with DCA on page 25, dialyzed rat and human cytosol was incubated for 30 minutes with maleylacetone, and then dialyzed again overnight to check for the persistence of maleylacetone inhibition. Incubation tubes contained 0.1 M Hepes pH 7.6, either 0 or 1 mM GSH, 0.4 mM maleylacetone, and approximately 5mg/ml (rats) or 8 mg/ml (humans) protein in a final volume of 2 ml. After 30 minutes duplicate aliquots were removed for initial activity measurement with DCA. From samples incubated with GSH, 245 pl aliquots were removed and added to tubes containing NaDCA for a final concentration of 0.2 mM. From samples incubated without GSH, 195 tl aliquots were removed and added to tubes containing GSH and DCA for final concentrations of 1 mM (GSH) and 0.2 mM (DCA). These samples were incubated for 30 more minutes and reactions were stopped by the addition of 750 [tl 5% TCA then assayed for glyoxylate production using the fluorescence assay.
Remaining maleylacetone incubation samples were dialyzed overnight in 0.1 M Hepes pH 7.6, with either 1 mM GSH (for GSH containing treatments) or 0.01 mM DTT (for treatments without GSH). To measure the final activity, the next day all samples were incubated with 0.2 mM DCA for 30 minutes. Reactions were stopped with TCA and assayed for glyoxylate production using the fluorescence assay.




CHAPTER THREE
IN VIVO STUDIES' RESULTS
Ion Pair HPLC Analysis of Urine
Monochloroacetic acid (MCA) and trichloroacetic acid (DCA contaminants) and the metabolites of DCA (oxalate, glyoxylate and glycine) were well separated from DCA by reversed phase HPLC using the ion pair reagent, PICA (tetrabutylammonium sulfate). MCA was not separated from oxalate, and glyoxylate was not separated from glycolate or glycine. A standard of hippuric acid had the same retention time (10.2 minutes) as an unknown metabolite found in all rat urine samples This major unknown metabolite was particularly prominent in the 3-6 hour urine sample of rat 21, and the 0-3 hour sample rat 18 (Figure 11). Phenacetyl glycine had a retention time of 14 minutes, identical to one of the other urinary metabolites observed in some samples.31
13
C NMR Analysis of Rat Urine
The 13C NMR spectrum from rat 21, 3-6 hour urine sample revealed a triplet at 8 180 ppm, a singlet at 8 167 ppm, four peaks in the aromatic region from 8 129 to 6 136 ppm, and CH2 bands from 8 46.3 to 8 47.1 ppm. These peaks suggest the structure
38




39
13 13 13
X- CH2- COOH derived from 1,2- C-DCA. The spectrum of sodium hippurate was similar (Figure 12).
This evidence was corroborated by GC-MS analysis of the methylated urine sample (Figure 13). The ratio of M + 2 : M peaks was approximately four times the natural abundance of 13C. The production of aC-radiolabeled hippuric acid from DCA administration indicates that DCA derived glyoxylate may be transaminated to glycine, which is known to undergo conjugation with benzoic acid, a common preservative and known end product of phenylalanine metabolism.32
o cpm
Gx
E D
H
Repeat dose, 180-265 g, fed, 0-3 h urine
Full scale y axis, 2800 cpm
Figure 11
Rat 18, 0-3 Hour Urine Sample; H, (hippuric acid retention 10.2 min); D, (DCA retention
7.0 min); Ox, (oxalate retention 4.2 min); Gx, (glyoxylate retention 3.2 min). The radiochemical detection scan is shown for clarity.




40
0 H 0 11 I II
4 C-- -N --C-OH
0 0
In II
HO-C-C-OH 4 2
5 5 2
15
3
220 266 I6 156 148 120 i1g so 60 46 20 0 PP.
Figure 12 13C-NMR of rat urine. The conditions are described in the methods section under 13C NMR analysis.




41
0..or *"O9 OS
" -&&S4--C-OCH3
I0,H 0
..or C-N-C-C-OCH3
*w 20.8 H
. ,Y, 9 o,
LDCC-N-C-C-OCH3
~, ~ Ob o 1 0' 12'M %4.M0 tW IB.0 000 "M0021.2 H i!
- Io
n00.0
0 M 0 0.
- eot- 140 1s 1 id1t
Figure 13
GC-MS of methylated urine. Rat was given one 50 mg/kg dose of a 99:1 mixture of I3C:14C-DCA. Top panel, GC trace, lower left panel, MS of the 20.8 minute peak, hippurate. Bottom right panel, MS of the 21.2 minute peak, phenylacetylglycine. Insets are expansions of the molecular ion peaks, showing M + 2 peaks arising from the 13 enrichment of glycine.31




42
Maleylacetone in Urine
The concentrations of maleylacetone in urine of rats treated with DCA doses of 50 mg/kg or more were measured by GC-MS from 24-hour samples collected before and after treatment. Maleylacetone excretion increased in a dose dependent manner. Figure 14 shows the changes in urinary maleylacetone levels for rats treated with five daily doses of 200 mg/kg of DCA. The mean SD (n=4) maleylacetone concentration before 200 mg/kg DCA treatment was 24.5 15.6 ptg/kg/day and increased to a maximum of 410.4 169.1 ptg/kg/day. Similar increases in maleylacetone excretion were observed for rats treated with 50 mg/kg (increasing to 298.4 32.0 jg/kg/day maximum) and in rats treated with lg/kg (increasing to 739.2 107.8 jig/kg/day maximum).




43
600
550
o 500450
l 400P" 350300
; 250200
" 15010050
0
day 0 day 1 day 2 day 3 day 4 day 5 DCA administration
Figure 14
Urinary excretion of maleylacetone in rats treated with 200 mg/kg DCA.




CHAPTER FOUR
IN VITRO STUDIES' RESULTS
Identification of Glyoxylate as a Product of DCA Metabolism
A standard of glyoxylate was found to have a retention time of 3.8 minutes using the ion pair HPLC method. This retention time agreed with the retention time of the principal unknown metabolite seen in DCA incubations (low amounts of oxalate were sometimes also produced). GC-MS of methylated incubation extracts was preformed and again the metabolite's retention time agreed with that of a methylated glyoxylate standard, 11.1 minutes. The mass spectra of the unknown peaks also matched that of methylated standard, with the most abundant fragment at m/z 75 corresponding to
-CH(OCH3)2. The fragments at m/z 75 and m/z 76 arise from 12C and 13C glyoxylate, and the enrichment at m/z 76 conclusiveley demonstrate that glyoxylate is a labeled metabolite of 1,2-13C-DCA (Figure 15).
44




45
abundance
2oooo A
0 Glyox 16000 1, 11
f60 HC-C-OH ocA
12000 Glyoxylate
8000
Oxal
4, 9"
me . ... 12
Abundance 7660 76
600 1(CH3O)2CHCOOCH,
* *
5000
4000 47
3000 2000 1000
33 80
,I 1o Is.. . o i , "I
25 30 s 40 4s so as so s 7o 75 So as so 9s loo
Figure 15
GC-MS of methylated rat liver cytosol incubated with 13C labeled DCA.




46
Development of a Fluorescence Assay of Glyoxylate
The assay for the fluorescence detection of glyoxylate proceeds through the
condensation of two equivalents of resorcinol with one equivalent of glyoxylate, to yield the fluorescent product; 2,2',4,4'-tetrahydroxydiphenylacetic acid lactone.27 Modifications of the protocol as published were made to increase reproducibility in the assay. Modifications included mixing samples for timed intervals with a vortex and heating in a dry bath at constant temperature for a timed period. These modifications improved reproducibility, bringing the r2 value of the standard curve to 0.997 from 0.878 as calculated by the ANOVA method in Excel software. This experiment allows the analysis of several incubation samples in approximately three hours, compared to the several days required by the HPLC/radioactivity detection assay described by James et al.5 The loss of sensitivity as compared to the HPLC method is generally outweighed by the decrease in time required for the assay. The HPLC method is much more expensive, requires 10 minute runs per sample and a one hour equilibration time, but has the advantages of greater reproducibility due to fewer steps before the final analysis and a lower detection limit compared to that of the fluorescence assay (0.1 tg glyoxylate = 1 nmol, vs. 0.001 nmol).




47
Strong Anion Exchange HPLC Analysis of Incubations
14C DCA and 14C labeled glyoxylate were well separated on the strong anion exchange column with retention times of 5.3 minutes and 3.0 minutes, respectively, as determined through radiation detection (Figure 16).
CPM
2000 DCA
1000
Glyoxylate
0
0'00 100 2'00 3'00 4'00 5'00 6'00 7'00 8'00 min
Figure 16
Separation of 14C DCA and 14C glyoxylate by strong anion exchange HPLC.




48
Reducing Agent Dependence Study
Incubation of the dialyzed cytosol with NADPH before the second dialysis
eliminated metabolism from NADH or NADPH. GSH was the only reducing agent to contribute to the metabolism of DCA (Table 3). The small amount of metabolism from NADH (maximum 1.06 % product) was not enough to be clearly distinguished from the baseline in the HPLC chromatogram. The metabolism from NADH and NADPH seen in single dialysis studies is now believed to be due to generation of GSH in the incubation mixture.33 Enzyme activity was stable in the presence of GSH, but declined rapidly if the dialyzed sample was heated to 37oC.
Effect of DCA Treatment on Activity of GSTz
Treatment of rats with DCA significantly lowered the metabolism of DCA in dialyzed hepatic cytosol samples used in subsequent in vitro incubations, also shown in Table 3.




49
Table 3
Reducing agent dependence study.
Control Rats DCA Treated Rats1 nmoles glyoxylate formed/minute/mg protein GSH 1 mM 1.43 0.13 0.45 0.10 NADPH 2 mM 0.02 0.02 <0.01 NADH 2 mM 0.14 0.17 <0.01
1
DCA treated rats received two daily doses of DCA (50 mg/kg) and were sacrificed on the third day. Incubations (15 minutes) contained 0.2 mM DCA, 1 mM GSH and 1 mg cytosolic protein. *DCA treated animals had significantly lower activities than controls, p<0.001




50
Linear Range of Glvoxylate Production in Rat Hepatic Cytosol
Time course studies in rats found glyoxylate production to be linear up to 30 minutes in both control and DCA treated cytosol (Figure 17). From this experiment 15 minutes was used as a standard time point for future kinetic incubations. Production of glyoxylate was found to be linear up to 1.5 mg protein (Figure 18). One milligram protein was used in most incubations, although protein concentration was sometimes decreased in KM and Vmax studies to bring the percent products below 20%.The incubation with a range of GSH concentrations found glyoxylate production to be linear up to 0.5 mM GSH. In subsequent determinations of the KM of DCA, 1 mM GSH was used to assure saturation of GSTz (Figure 19). Glyoxylate production was linear up to 0.1 mM DCA (Figure 20). In incubations to determine the KM of GSH in dialyzed rat cytosol, 0.2 mM DCA was used to assure a saturating concentration.




51
40
>1W
?20
10
0 20 40 60 80
Time, Min
Figure 17 Time course studies in control (W1) and DCA treated (Dl) dialyzed rat hepatic cytosol. The results shown are for individuals. Similar results were seen for the other cytosol samples.




52
40
30
20
10
0 II
0 0.5 1 1.5 2 mg Cytosolic Protein
Figure 18 Glyoxylate formed (nmol) from a range of protein concentrations. The results shown are for individuals. Similar results were seen for the other cytosol samples.




53
20
15
o 5 Z 10
* 5
0
0 0.5 1 1.5 2
mM GSH
Figure 19 Glyoxylate produced (nmol) from a range of GSH concentrations in rat hepatic cytosol. The results shown are from one rat. The study was repeated twice with similar results.




54
20
~15 ,
o +
10 o 5
0 5
0 0.2 0.4 0.6 mM DCA
Figure 20 Glyoxylate formed (nmol) from various DCA concentrations in rat hepatic cytosol. The results shown are for individuals. Similar results were seen for the other cytosol samples.




55
Rat Apparent KM and V ,x of GSH
The average apparent KM value for GSH (Figure 21) in control animals was 0.0688 mM, and 0.0528 mM in DCA treated animals, which were not significantly different (p>0.05). The average apparent Vmax value was 1.587 nmol/min/mg protein for control animals, and 0.674 nmoles/min/mg protein in the DCA treated animals, which were significantly different (p<0.05). The Vmax values of the control rats were greater than those of the DCA treated rats by an approximate factor of 2 (Table 4).
Table 4
Apparent KM (mM) and Vmax (nmol/min/mg protein) of GSH in dialyzed rat cytosol. values are means SD.
KM Vmax
Control 0.0688 0.0115 1.587 0.159 DCA Treated 0.0528 0.032 0.674 0.181




56
7.5- Apparent KM
. 5 0.0625 mM
>0
S 5.0*E
II I I I I
0.::= d2.5
-25 0 25 50 75 100 125 1/mM GSH
Figure 21
Apparent KM of GSH in dialyzed rat cytosol.
Rat Apparent Ku and Vma, of DCA Determinations
The average apparent KM value for DCA (Figure 22) was 0.00748 0.00187 mM, in control animals, and 0.00745 0.00191 mM, in DCA treated animals (not significantly different, p>0.05). The average apparent Vmax values were 0.548 0.153 nmol/min/mg protein for control rats, and 0.236 0.0288 nmol/min/mg protein for DCA treated rats (significantly different, p<0.05). The Vmax values of the control rats were again greater than the DCA treated rats values by an approximate factor of 2 (Table 5). However, the




57
apparent Vmax values for both groups were lower than those determined with a higher DCA concentration in the study of the GSH apparent KM (Table 4).
Table 5
Apparent KM (p~M) and Vmax (nmol/min/mg protein) of DCA in dialyzed rat cytosol. Values are means SD.
KM Vmax
Control 7.48 1. 87 0.548 0.153 DCA Treated 7.45 1.909 0.236 0.0288




58
Apparent KM
7.5- 0.00755 mM Co
0
ct 5.0uE
U) 0 2.5-200 -100 0 100 200 300 400 500 1/mM DCA
Figure 22
Apparent KM of DCA in dialyzed rat cytosol.
Linear Range of Glyoxylate Production in Human Hepatic Cytosol
Time course studies in human liver found glyoxylate production to be linear up to 60 minutes. From this experiment, 30 minutes was used as a standard time point for future kinetic incubations (Figure 23). Production of glyoxylate was found to be linear up to 1.5 mg protein (Figure 24). One milligram protein was used in most incubations, although protein concentration was sometimes decreased in KM and Vmax studies to bring the percent products below 20%. Glyoxylate production was linear up to 0.025 mM DCA




59
(Figure 25). In incubations to determine the KM of GSH in dialyzed human hepatic cytosol, 0.2 mM DCA was used to assure a saturating concentration. The incubation with a range of GSH concentrations found glyoxylate production to be linear up to 0.05 mM GSH. In subsequent determinations of the KM of DCA, 1 mM GSH was used to assure saturation of GSTz (Figure 26).
Human Hepatic Cytosol Apparent K and V axResults
Cytosolic incubations of dialyzed human liver cytosol revealed the average apparent KM of DCA to be 0.00487 mM and the average apparent Vmax to be 0.374 nmol/min/mg protein (Figure 27). The average apparent KM of GSH was found to be
0.0076 mM and the average apparent Vmax 0.262 nmol/min/mg protein. (Figure 28). The maximal velocities are higher in the control rats studied than in the human sample.




60
35
3 30 A
; 25 A S20
15
10 A
5 -A
0
0 50 100 150 Time, min
Figure 23
Time course study in human hepatic cytosol.




61
25
20
o0 15 *
l5
0 II
0 0.5 1 1.5 2 2.5
Cytosolic Protein, mg
Figure 24 Glyoxylate formed in dialyzed human hepatic cytosol from a range of protein concentrations




62
6
5 *
4 *
1
0 I I
0 0.01 0.02 0.03 0.04 0.05 0.06 mM DCA Figure 25
Glyoxylate formed in dialyzed human hepatic cytosol from various DCA concentrations.




63
10
O6
D 4-|
0
0
0 I I I
0 0.05 0.1 0.15 0.2 0.25 0.3 mM GSH
Figure 26 Glyoxylate produced from a range of GSH concentrations in dialyzed human hepatic cytosol.




64
50- Apparent KM a 0.00680 mM 4-1
I I I I I I I
-200 -100 0 100 200 300 400 500 600 1/mM DCA
Figure 27
Lineweaver-Burk determination of the KM and Vmax of DCA at 0.024 mM GSH in dialyzed human hepatic cytosol.




65
.e 0.4SApparent KM
5: 0.00642 mM
, 0.30.2
o.1
-0.025 0.000 0.025 0.050 0.075 0.100 0.125 mM GSH
Figure 28 Hames plot determination of the KM of GSH in dialyzed human cytosol at 0.025 mM DCA.




66
In Vitro Inhibition Study Results
These studies sought to determine if in vitro pre-incubation with DCA would reduce the GSTz activity of cytosol in subsequent incubations. The first time course inhibition incubation with 0.2 mM DCA was repeated because glyoxylate levels were below the detection limits of the fluorescence assay. The protein amount was increased, and no inhibition of DCA metabolism was observed. DCA treated cytosol had an average specific activity of 0.323 0.0691 nmol/min/mg protein, whereas the control cytosol after the incubation with DCA had a specific activity of 0.261 0.0486 nmol/min/mg protein (p<0.05) (Figure 29).
The unexpected result of the inhibition experiment led to the incubation with
higher concentrations of DCA. In this experiment the control cytosol incubations again had lower specific activity than those pre-treated with DCA. For the 0.5 mM DCA pretreatment, the average specific activity was 0.665 0.176 nmol/min/mg protein, and
0.601 0.315 nmol/minute/mg protein for the control. (p>0.05) The 0.75 mM DCA pretreatment had an average specific activity of 0.702 0.348 nmol/min/mg protein, and
0.483 0.0740 nmol/min/mg protein for the control. (p<0.05) The 1 mM DCA pretreatment had an average specific activity of 1.029 0.245 nmol/min/mg protein, and
0.531 0.137 nmol/minute/mg protein for the control (p<0.05)




67
In the final experiment with three concentrations of DCA in the initial incubation, then 0.2 mM for the second incubation to ease comparison, again no in vitro inhibition of DCA was seen (Figure 30). The control cytosol had a specific activity of 0.398 0.050 nmol/min/mg protein, compared to 0.472 0.0685 nmol/min/mg protein for 0.5 mM DCA treatment (p>0.05), 0.480 0.0623 nmol/min/mg protein for the 0.75 mM DCA treatment (p<0.05), and 0.596 0.0452 nmol/min/mg protein for the 0.1 mM DCA treatment (p<0.05).
In the absence of DCA pre-incubation, GSTz specific activity in cytosol obtained from rat livers was approximately four-fold higher than that measured in cytosol from human livers. Pre-incubation with 0.5 mM DCA failed to significantly alter enzyme specific activity in human liver but decreased that of rat liver 56 percent (p<0.001). Thus, the effect of prior exposure to DCA on hepatic GSTz specific activity, and the subsequent biotransformation of DCA to glyoxylate, appeared to be qualitatively different between these two species (Figure 31).




68
Pre-Treatment 10.0
--- No DCA
-&- 0.2 mM DCA e0l 7.55.0
-2.5
0.01 .,.
0 10 20 30 40 50 60 70 Time (min)
Figure 29 Activity of dialyzed human hepatic cytosol after pre-incubation with 0.2 mM DCA




69
Treatments E 0.5 mM DCA
0.75- = 0.75 mM DCA
1 ImM DCA
M control
0.50- T
0.00 ::"
30.0 45.0 60.0 Min
Figure 30 Residual GSTz specific activity at 30, 45, and 60 min in dialyzed human cytosol pretreated with 0, 0.5, 0.75, or 1 mM DCA.




70
1.5 Pre-treatment
E HnoDCA MHDCA
.41
410 R noDCA R DCA E 0.5
0
30 min Incubation Figure 31
Comparison of the effect of DCA pre-treatment on GSTz specific activity between rat and human dialyzed hepatic cytosol, values are means (n=3) + SD. H=human, R=rat.




71
DCA Binding Study in Dialyzed Rat and Human Hepatic Cytosol
Radiolabeled DCA was not found to bind hepatic cytosolic protein to any appreciable extent in either rat or human samples. Percent binding was found to be
4.45 % in the rat samples and 13.45 % in the human sample. This was not enough to account for the lack of a direct inhibitory effect of DCA seen in the pre-incubation dialysis studies with human hepatic cytosol samples.
In Vitro GSTz Activity in DCA Dosed Animals
The mean SD specific activity of GSTz in livers of rats not treated with DCA was 1.49 0.14 nmol glyoxylate formed/min/mg protein. Oral administration of DCA to rats inhibited hepatic GSTz specific activity in a dose dependent manner (Figure 32, Table 6). At the lowest doses (4 mg/kg and 12.5 mg/kg), five days of DCA administration was required to significantly decrease enzyme activity. At doses of 50 mg/kg or greater, however, DCA significantly inhibited GSTz after only a single exposure. A single 1 g/kg dose decreased glyoxylate formation by 93 percent.




0 Co < GSTz specific activity S 0 (nmol, glyoxylate
C D formed/mm/mg protein) 0 "- I I I m*
-.0
Control
0
4mg/kg 1 day
4mg/kg 5 days 0 12.5 mg/kgl day
S12.5 mg/kg 5 days o a
50 mg/kg 1 day
0
W 50 mg/kg 5 days
2
p: 200 mg/kg 1 day 200 mg/kg 5 days
0
0 1000 mg/kg 1 day
> 1000 mg/kg 5 days




73
Table 6
GSTz Activity in DCA treated and control animals. Values are reported in nmol/min/mg protein, means SD. Dose/kg =dose of DCA.
Dose/kg Average Specific Activity
Control 1.49 0.144 4mg 1 day 1.50 0.073 4 mg 5 days 1.18 0.072 12.5 mg 1 day 1.17 0.172 12.5 mg 5 days 0.72 0.174 50 mg 1 day 0.52 0.126 50 mg 5 days 0.28 0.174 200 mg 1 day 0.22 0.043 200 mg 5 days 0.088 0.006 1000 mg 1 day 0.047 0.019 1000 mg 5 days 0.029 0.002




CHAPTER FIVE
RESULTS OF INHIBITION STUDIES WITH MALEYLACETONE Malevylacetone ICs0 Studies
Maleylacetone inhibited DCA conversion to glyoxylate in a dose dependent manner. When rat or human hepatic cytosol was incubated with 0.05-0.4 mM maleylacetone (a natural substrate for GSTz), DCA biotransformation to glyoxylate was inhibited. The apparent IC50 of DCA metabolism by maleylacetone in the first study with human hepatic cytosol was between 0.1 and 0.2 mM (Figure 33). The mean SD IC50 value was 0.264 0.039 mM maleylacetone for the rat liver cytosol (n=3) (Figure 34), and 0.125 0.029 mM maleylacetone (n=4) for human liver cytosol (Figure 35). Preincubation for 30 minutes resulted in a greater increase in maleylacetone inhibition than was seen without pre-incubation in human and rat hepatic cytosol (Figure 36). This suggests different enzymatic binding sites for maleylacetone and DCA, and noncompetitive inhibition.
Ki of Maleylacetone
The results of the Ki of maleylacetone against DCA metabolism were not as reproducible as those used to find the IC50 values. The competition between maleylacetone and DCA appeared to be mixed, or non-competitive which would suggest 74




75
different active sites for the metabolism of the native maleylacetone and DCA (Figure 37). The average Ki for maleylacetone with the different DCA concentrations studied was
8.4 3 pM.
0.5
U Apparent IC50
0 0.40.152 mM
0.3
o0.2
0.1
0.0 , 1 1 ,
0.0 0.1 0.2 0.3 0.4 0.5 mM Maleylacetone
Figure 33 Initial incubation of DCA with maleylacetone.




76
A
A *0.200 Ri
- o.oa R1
*A R2
0.0 5 R3
-0.10-0.40I I I I
0.0 0.1 0.2 0.3 0.4
mM Maleylacetone
Figure 34 IC50 of maleylacetone in rat hepatic cytosol. R1 (rat one), R2 (rat two), R3 (rat three).




77
0.00
-0.252'
-o.5o
i -0.75-1.00-1.250-1.50-1.75-2.00 1 , ,
0.0 0.1 0.2 0.3 0.4 0.5 mM Maleylacetone
Figure 35 IC50so of maleylacetone in human hepatic cytosol. The four symbols represent four human liver samples.




78
30 Minute 0.1 mM MA Pre-Incubation Comparison, Rats
2- M Control
l [I MA Not Pre-Incubated = [ 1 30 minPI+ GSH
'z = 130 min PI-GSH
0
0
30 Minute 0.1 mM MA Pre-Incubation Comparison, Humans
0.2
M Control
=~ MA Not Pre-Incubated [22 30 min PI-GSH X E 1 CE330minPI+GSH
7a_'. o.- T40.1
0.0
Figure 36
Maleylacetone pre-incubation comparison. PI-GSH=pre-incubated without GSH, PI + GSH = pre-incubated with GSH.




79
50
Mixed Type 25Inhibition?
III I I
-200 -100 0 100 200 300
1/mM DCA
Figure 37 Lineweaver Burk Ki determination of maleylacetone against DCA metabolism. Symbols represent different maleylacetone concentrations.




80
Pre-Treatment of Dialyzed Rat and Human Hepatic Cvtosol with Maleylacetone
Pre-treatment with 0.4 mM maleylacetone led to inhibition of DCA metabolism which persisted through dialysis in both rat and human hepatic cytosol (p<0.01, rat samples with GSH, p<0.05 rat samples without GSH, p<0.001 human samples with GSH, no significant difference, human samples without GSH). The presence of 1 mM GSH with the initial maleylacetone treatments decreased maleylacetone's inhibition of DCA metabolism in both rat and human samples. This was expected due to the in vitro binding of maleylacetone and GSH.
In the final activity measurement the presence of GSH appeared to increase
maleylacetone's inhibition of DCA metabolism with a greater effect seen in the human samples, but the decreased level of significance seen in all samples which did not contain GSH may be due to their overall loss of activity (Figure 38).




81
Initial Activity Measurement after
0.4 mM MA Pre-treatment SControl + GSH o 1.00- I 0.4 mM MA+ GSH
' E O Control GSH
S 0.75- M 0.4 mM MA- GSH Q0 .0.500.25
0.00^
Rats Humans
Final Activity Measurement with
0.2 mM DCA I.E0 Control + GSH
1.00- E! O.4 mM MA + GSH
E0.75 Control GSH
0.75- 0.4 mM MA- GSH
0.50
0.25
0.00- ...
Rats Humans
Figure 38
Effect of maleylacetone pre-treatment in dialyzed rat and human cytosol.




CHAPTER SIX
DISCUSSION
Dichloroacetate is an important chemical for two reasons; it is a drug used to treat human disease in a clinical setting (lactic acidosis), and it is an environmental contaminant considered hazardous by the EPA. Dichloroacetate is used as an investigational drug to treat lactic acidosis in children with inborn errors of metabolism, lactic acidosis due to acute malarial infection, and also has the pharmacological properties of lowering blood glucose levels and cholesterol. DCA is found in chlorinated drinking water and in soil contaminated with trichloroethylene as a breakdown product. DCA's inhibition of its own metabolism, carcinogenicity in rodents, and its toxicity in chronic administration, including adverse effects on the nervous system, liver and other tissues, necessitate the understanding of its biotransformation in detail and the mechanism of its auto-inhibition. Over the last four years ongoing in vivo and in vitro studies have successfully elucidated new metabolites, new pathways of biotransformation, and an important interaction between DCA metabolism and metabolism of the amino acid tyrosine.36
In the in vivo studies using rats as models for human DCA metabolism, prolonged half lives were observed in the repeatedly dosed rats compared to the single dosed animals, in agreement with half-life prolongation seen upon administration of DCA to humans. Glyoxylate was confirmed as an intermediate in DCA metabolism by GC-MS.
82




83
This intermediate had only been identified previously by co-elution on HPLC.19 Three unknown urinary metabolites were also observed. Two of the new urinary metabolites were identified as hippuric acid and phenacetyl glycine, which are glycine conjugates produced from the metabolism of DCA to glyoxylate and its subsequent transamination to glycine.31 Hippurate, the conjugation product of glycine and benzoic acid, is formed by the enzyme benzoyl-CoA: glycine N-acyltransferase, and phenylacetylglycine is formed similarly by the enzyme phenylacetyl-CoA: glycine N-acyltransferase.34'35 This leads to a new pathway in the metabolism of DCA (compare Figure 6 to Figure 39).




84
HO OO O O I II II II II II
Cl-C-C-OH Dechlorination H-C-C-OH Lactate HO-C-C-OH
Cl GST-z Dehydrogenase Oxalate
CtslGlyoxylate Cytosol Oxalate Dichloroacetic Cytosol Cytosol Acid
Aminotransferases
ac-Ketoglutarate:Glyoxylate A (several) es Lactate
Carboligase Mitochondria Dehydrogenase
Mitochondria Peroxisomes Cytosol
H 0 HO I II I II
H-N-C-C-OH HO-C-C-OH
I I I CO2 HH H
Glycine Glycolate Benzoyl CoA Amino acid Phenylacetyl CoA Amino acid
N-acyltransferase N-acyltransferase
Mitochondria Mitochondria
o HO HO H O \/I/-\\I II I
C-N-C-C-OH -C C-N-C-C-OH
i- I( I
H H H HH Benzoylglycine Phenacetylglycine (Hippuric Acid)
Figure 39
DCA metabolic pathways known at the end of this study.




85
An important finding in this work was the unequivocal identification of
glyoxylate as a metabolite of DCA.5 This led to exploration into the mechanism of DCA dechlorination, and may lead to insights into its mechanism of auto-inhibition. From the studies of reducing agent dependence, glutathione was found to be the only reducing agent capable of supporting DCA metabolism in hepatic cytosol, which narrowed the choice of enzyme candidates for DCA metabolism. This enzyme was found to be the GSH dependent GSTz.
A decrease in the rate of DCA metabolism in vitro was seen in the experiments using cytosol from both DCA dosed rats and water dosed (control) rats. The KM of DCA was similar in control and DCA dosed rats, but the Vmax decreased by approximately one half.5 This is consistent with loss of enzyme, which could be due to down-regulation of the gene or degradation of the enzyme. The enzyme could be degraded by several mechanisms, including formation of a reactive metabolite of DCA (mechanism-based inhibition), or the buildup of the natural substrates for GSTz (maleylacetone and maleylacetoacetate), either of which could alkylate the enzyme and lead to its degradation.
In vitro incubations performed with dialyzed human and rat cytosol, to determine if the inhibition of DCA metabolism was due to a direct effect of DCA on the enzyme, found a lack of a direct inhibitory effect in human cytosol; but rat cytosol incubated with DCA suffered a significant loss of GSTz activity.36 This indicates different mechanisms are involved in the inhibition of DCA metabolism in the rat and the human. Although




Full Text

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METABOLISM OF DICHLOROACETATE IN THE SPRAGUE DAWLEY RAT AND HUMANS, INVOLVEMENT OF THE TYROSINE CATABOLIC PATHWAY By RACHEL CORNETT 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 2000

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Copyright 2000 by Rachel Cornett

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Dedicated to my parents John and Sally Cornett

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Acknowledgements I would like to thank my major professor, Dr. M.O. James, for her patience, guidance, sharing her immense knowledge, and support. I would like to thank members of my committee; Dr. Steven Roberts, for his toxicology expertise, Dr. Peter W. Stacpoole, for his knowlege of every aspect ofDCA research and writing advice, Dr. Kenneth Sloan, for his help with chemical mechanisms and synthetic procedures, and Dr. John Perrin, for his pharmacokinetic and physical chemistry knowledge and always having the latest "scoop." I would like to thank Dr. G.N. Henderson for his scientific input and all the GC-MS data found in this work, which should also include thanks to his lab members Ximeng Yan, Albert L. Shroads, and Jang Cheung. I would also like to thank my fellow lab members; Laura Faux, Zeen Tong, and Donald Sikazwe,for their help over the years, specifically for showing me the way around in the lab when I began as a pharmacist who had forgotten most of the laboratory techniques learned in undergraduate courses. Laura should receive special thanks for her perfectionist example and excellent bench technique Dr. Bernard Gadagbui also deserves a special mention for his willingness to assist all lab members and his expertise in protein chemistry. Alicia Ally, Dr. Peter van den Hurk, and Zhen Lou have always been helpful, cheerful, and easy to work with I would also like to thank my husband, Dr. Ralf Mueller, for his help not only with organic chemistry but also in putting up with me while I was writing my "Doktorarbeit." I would like to thank my mother, Sally Cornett, for her support in all iv

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endeavors, and my father, John Cornett, for calling me crazy for going back to school, which gave me an additional impetus to return. V

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TABLE OF CONTENTS ACKNOWLEDGEMENTS ........................ ............................................................. iv LIST OF TABLES ................................................................................................... .. ix LIST OF FIGURES ................................................................................................... x ABBREVIATIONS .................... ..... . ........ ..... .... ...... ... ......... ........................... xiii ABSTRACT ......... ................................................ ..... ............ .... ....... . .... . xiv CHAPTERS 1 INTRODUCTION ............................... ..... ..... ....... ...... .... .... .... ........ .... 1 Mechanism of Action ......................................... ......... ...... .... ..... ............. 1 Glutathione-S-Transferase Zeta ..................................................................... 3 Glutathione-S-Transferases .............................................. ............. ........ ... 6 Potential Mechanisms ofDCA Dechlorination .............................................. 7 Metabolites of DCA .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 11 Involvement of the Tyrosine Catabolic Pathway ......................................... 12 2 MATERIALS AND METHODS ................................................................ 15 Chemicals and Equipment .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 15 Animals ........ ...................... ................. ............. .... . .... .... . ........ ..... 16 In Vivo Studies ........ ....................................................................... 16 Effect ofln Vivo DCA Pre-Treatment on its In Vitro Metabolism .................................................................... 17 Dose Response Studies .................................................................. 1 7 Preparation of Rat Liver Cytosol ........ ........................................................ 18 Preparation of Human Liver Cytosol .......................................................... 18 Assays of DCA Metabolism ........................................................................ 19 General .......................................... ..... .... .... .... ............................ 19 Cytosol Dialysis ............................................................................... 19 Reducing Agent Dependence Incubations ....................................... 20 Linear Range of Glyoxylate Production in Rat Hepatic Cytosol ..... 21 VI

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Rat Apparent KM and V max Determination Incubations ..... .... ....... 22 Linear Range of Glyoxylate Production in Human Hepatic Cytosol ...................... .............. .... .... ........... ...... . .... ......... 22 Apparent KM and V max in Dialyzed Human Liver Cytosol Incubations .... . . ..... ............... ................................................... 24 In Vitro DCA Inhibition Incubations ................................................ 24 DCA Pre-Incubation Comparison between Dialyzed Rat and Human Cytosol ............ ... ............................................................................... 25 DCA Binding Study in Dialyzed Rat and Human Hepatic Cytosol ...... 26 In Vitro GSTz Activity in DCA Dosed Animals .............................. 27 Analyses ..................................................................................... .................. 27 Ion Pair HPLC .... .............................................................................. 27 Strong Anion Exchange HPLC ....................................................... .28 13C NMR Analysis ................................................ .......................... 28 Fluorescence Analysis .................. . .... ......... ... .. ... ...... ................... 29 GC-MS Confirmation of Glyoxylate ............................................... 30 GC-MS Analysis of Maleylacetone ................................................ 31 Synthesis of Maleylacetone ............................................................ 32 Maleylacetone Methods ......................................... .... ............................. 35 Maleylacetone IC50 Studies .............. ............................................. 35 Pre-Incubation Studies .............. ......... .............. .............................. 36 Maleylacetone Ki Studies ................................................................ 36 Pre-Treatment of Dialyzed Rat and Human Hepatic Cytosol with Maleylacetone .............................................. 37 3 IN VIVO STUDIES' RESULTS .................................................................... 38 Ion Pair HPLC Analysis of Urine .................................................................. 38 13C NMR Analysis of Rat Urine ........................................................... .. ....... 38 Maleylacetone in Urine .................................................................................. 42 4 IN VITRO STUDIES' RESULTS ................................................................. 44 Identification of Glyoxylate as a Product ofDCA Metabolism .................... 44 Development of a Fluorescence Assay of Glyoxylate ................... .. ... .46 Strong Anion Exchange HPLC Analysis oflncubations ....................... .47 Reducing Agent Dependence Study .............................................................. 48 Effect ofDCA Treatment on Activity of GSTz ............................................. 48 Linear Range of Glyoxylate Production in Rat Hepatic Cytosol ................... 50 Rat Apparent KM and V max of GSH Determinations ...................................... 55 Vll

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Rat Apparent KM and Vmax ofDCA Determinations ..... .............................. 56 Linear Range of Glyoxylate Production in Human Hepatic Cytosol .. ..... .. .. 58 Human Hepatic Cytosol Apparent KM and V max Results .... .. .......... ..... .......... 59 In Vitro Inhibition Studies' Results ............. ....... ..................... .... ............. 66 DCA Binding Study ...................................... .......................... .. 71 In Vitro GSTz Activity in DCA Dosed Animals .... ....... ....... ....... ..... ........ ... 71 5 RESULTS OF INHIBITION STUDIES WITH MALEYLACETONE ... ..... .. .... 74 Maleylacetone IC50 Studies ........ .............................................. ...... ............. 74 Ki of Maleylacetone .. ..... .. .. .. ....... ..... ....... ......... ............ ........... ...... .. .......... 7 4 Pre-Treatment of Dialyzed Rat and Human Hepatic Cytosol with Maleyalcetone .............. ............. .. .. ................... ............ ......... 80 6 DISCUSSION .... .. ... .. .. ......... .. .. .. ............ .. .. .. .. .. ...... ..... ........ .. .... .. ..... .. ..... ........... 82 REFERENCES ................................... ....... .. .. .......... ...... .. .... .... .... .. .... ..... .. .. ..... .... .. .. .. 89 BIOGRAPHICAL SKETCH .. .. .... ....... ......... ...... .. .. ..... ...... ........ ..... .. ................ ..... .. .... 94 Vlll

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LIST OF TABLES 1. Alpha-beta unsaturated carboxylic acids metabolized by GSTz .......... ...................... 5 2 Haloacids metabolized by GSTz .......... .. ..... .. .. .. .. .. .. ...... ... ........ ..... .. ..... .. .... .. ........ .. 10 3 Reducing agent dependence study .. ........... ......... .. ................................................. 49 4 Apparent KM (mM) and V max (nmoles / minute/mg protein) of GSH in dialyzed rat cytosol ............ .............................. ...... ....... ........ 55 5. Apparent KM (mM) and Ymax (nmoles / minute / mg protein) ofDCA in dialyzed rat cytosol .......... ......... .. .. ...... ......... ........ .. ..... .. .. .... ..... .. .. ..... .... 57 6. GSTz activity in DCA treated and control animals .. .......... .. .... ............... ..... .... ..... 73 ix

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LIST OF FIGURES Figure 1. Mechanism of action of DCA on pyruvate dehydrogenase ... .... ............ ....... ..... .... 2 2. Tyrosine catabolism ........................................................ ......... ........... . . .... . ... 4 3. Possible mechanism for the conversion ofDCA to glyoxylate ..................... .... ..... .... 7 4. Possible mechanism accounting for retention of deuterium in glyoxylate from DCA dechlorination ........... ........... ............... .................................................... 8 5. Proposed mechanism for removal of fluorine avoiding the zwitterion intermediate ........... ........................ .................................... ............... ............. 9 6 Metabolites ofDCA as understood in 1995 ................................................................ 13 7. Possible routes of CO2 formation .......................... ........................... ...... ............... 14 8. Formation of 2,2', 4,4'-tetrahydroxydiphenylacetic acid lactone ... ..... .... . . ......... 29 9. Synthesis of maleylacetone ... .... . ..... ..... ..... ..... ..... ....... ...... .... .... ........ ..... 33 10. 1H-NMR of maleylacetone ........................... ....... ....... . .... .... .... .... .... ..... ...... . 34 11. Rat 18, 0-3 hour urine sample ................................................................. .... .............. 39 12. 13C NMR of rat urine .... ..... ... ........... ........... ..... ............. ........... ........................ 40 13. GC-MS of methylated urine ................................... ..... ....... .... ...... ...... . .... ... 41 14. Urinary excretion of maleylacetone in rats treated with 200 mg/kg DCA .............. 43 15. GC-MS of methylated rat liver cytosol incubated with 13C labeled DCA ......... .............. .. ........................... ............................................... 45 16. Separation of 14C DCA and 14C glyoxylate by strong anion exchange HPLC ......... .47 17. Time course studies in control and DCA treated dialyzed rat hepatic cytosol .......... 51 X

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18. Glyoxylate formed (nmoles) from a range of protein concentrations ........................ 52 19. Glyoxylate formed (nmoles) from various DCA concentrations in rat hepatic cytosol .... ..... .......... ............ .... .. .. .. .. .. .... ........ ....................................... 53 20. Glyoxylate produced (nmoles) from a range of GSH concentrations in rat hepatic cytosol. ...................................................... .. ............... ........... 54 21. Apparent KM of GSH in representative dialyzed rat cytosol.. .... .... ........ .. ..... .... ... ..... 56 22. Apparent KM ofDCA in dialyzed rat cytosol .... .................................. ................ .. 58 23. Time course study in human hepatic cytosol ....................... .... .... .. ..... .. .................. 60 24. Glyoxylate formed in dialyzed human hepatic cytosol from a range of protein concentrations ............................................ ................ .... .. ........... .. .. 61 25. Glyoxylate formed in dialyzed human hepatic cytosol from various DCA concentrations .................................... ........................ .... ...................... 62 26 Glyoxylate formed from a range of GSH concentrations in dialyzed human hepatic cytosol. ............................................................................................. 63 27. Lineweaver-Burk determination of the KM and Vmax ofDCA at 0.024 mM GSH .................................... .................................................................. 64 28. KM of GSH in dialyzed human cytosol at 0 025 mM DCA .. ..................................... 65 29. Activity of dialyzed hepatic cytosol after pre-incubation with 0 2 mM DCA .. .. .. ....... ....... ............ ............... .............. ..................... .. ................ 68 30. Residual GSTz specific activity at 30, 45, and 60 minutes in dialyzed human cytosol pre-treated with 0, 0.5, 0.75, or 1 mM DCA ... ................... .. ....... ... 69 31. Comparison of the effect ofDCA pre-treatment on GSTz specific activity between rat and human dialyzed hepatic cytosol ............. ..... .. .. ..... ........ .. 70 32. Specific activity of rat hepatic cytosol from rats dosed with a broad range of DCA concentrations ....................................... .......... ................... .... ..... .. 72 XI

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33. Initial incubation of DCA with maleylacetone .......... ..... ......... ................................. 75 34 IC50 of MA in rat hepatic cytosol ............................... ........ ..... ..... .. ...... .. .... .. ..... .... ... 76 35. IC50 of MA in human hepatic cytosol ........................................................................ 77 36. Maleylacetone pre-incubation comparison ................................................ 78 37. Lineweaver-Burk Ki determination of MA against DCA metabolism ..................................................................................................... 79 38. Effect of maleylacetone pre-treatment in dialyzed rat and human hepatic cytosol ... 81 39. DCA metabolic pathways known at the end of this study ................................ 84 XU

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ANOVA DCA DTT EPA FAA FA GSH GSSG GST HEPES HPLC NADH NADPH NMR SAX SDS-PAGE ABBREVIATIONS analysis of variance dichloroacetate dithiothreitol (threo-1,4-dimercapto-2,3-butanediol) environmental protection agency fumarylacetoacetate fumary lacetone reduced glutathione oxidized glutathione glutathione-S-transferase [ 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid] high pressure liquid chromatography nicotinamide adenine dinucleotide, reduced nicotinamide adenine dinucleotide phosphate, reduced nuclear magnetic resonance strong anion exchange sodium dodecyl sulfate polyacrylamide gel electrophoresis xiii

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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 METABOLISM OF DICHLOROACETATE IN THE SPRAGUE DAWLEY RAT AND HUMANS, INVOLVEMENT OF THE TYROSINE CATABOLIC PATHWAY By Rachel Cornett May,2000 Chair: Dr. Margaret 0. James Major Department: Medicinal Chemistry Dichloroacetate (DCA) is considered a hazardous environmental contaminant by the EPA, but is also an investigational drug for the treatment of lactic acidosis in children with inborn errors of metabolism. Chronic administration of DCA leads to toxicity including peripheral neuropathy and ocular opacities. DCA also inhibits its own metabolism, which can be seen as early as after the first dose. The aims of the research conducted over the past four years have been to uncover the overall metabolic pathways of the metabolism ofDCA and to examine hypotheses related to the mechanism of its inhibition of its own metabolism Research into DCA metabolism has included in vivo and in vitro experiments in rats, and in vitro investigations with human liver cytosol. There were several important findings in the metabolism of DCA including the XIV

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confirmation of glyoxylate as an intermediate, which had not previously been unequivocally identified, the requirement of glutathione (GSH) for DCA metabolism to glyoxylate, and the discovery of hippurate and other glycine conjugates as metabolites of DCA. Advancements in uncovering the mechanism ofDCA auto-inhibition include demonstrating a decrease in the rate of in vitro DCA metabolism in hepatic cytosol prepared from DCA treated rats, a direct inhibitory effect in dialyzed rat hepatic cytosol but not dialyzed human hepatic cytosol incubated with DCA, inhibition of DCA metabolism by maleylacetone, a native substrate of GSTz, and an increase in maleylacetone levels in DCA treated rats, suggesting an interaction between DCA and the tyrosine catabolic pathway. Uncovering the interaction between DCA and the tyrosine catabolic pathway may lead to improvements in the treatment of children with lactic acidosis and insights into the mechanisms ofDCA's toxicity and rodent hepatocarcinogenicity. xv

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CHAPTER ONE INTRODUCTION Dichloroacetate (DCA) is considered by the EPA to be an environmental contaminant resulting from both the chlorination of drinking water and the breakdown of trichloroethylene, but is also used as a therapeutic agent in the treatment of lactic acidosis in several clinical settings DCA is the only available treatment for congenital lactic acidosis which if left untreated is fatal. It can also be used to treat lactic acidosis in patients undergoing liver transplantation, acute malaria, and cardiovascular disease. 1 A confounding property of DCA is that it inhibits its own metabolism. An increase in the plasma elimination half-life of the drug can be seen as early as the second dose .2 Prolonged administration of DCA causes toxicity including possible ocular toxicity and peripheral neuropathy.3 Uncovering the pathways of metabolism may yield insight into the mechanisms ofDCA's toxicity and auto-inhibition This may ultimately lead to changes in treatment that improve the quality of life and survival rates of children with congenital lactic acidosis. Mechanism of Action DCA lowers lactate levels through stimulation of pyruvate dehydrogenase indirectly by inhibition of phosphate dehydrogenase kinase, preventing its

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2 phosphorylation and subsequent inactivation. This increases the amount of pyruvate converted to acetyl-CoA, resulting in a reduction of lactate and alanine levels which are precursors of pyruvate in gluconeogenesis (Figure 1).3 lji 9 H2N-~-C-OH CH3 Alanine Alanine Aminotransferase ..... .. 00 H3C-C C-OH Pyruvate Pyruvate Dehydrogenase 0 ----~ H3C-C -S-CoA (PDH) Acetyl CoA Dehydrogle~~!~i t Inhibition DCA Kinase Figure 1 lji 9 H3C-~ C OH OH Lactate PDH-P Inactive PDH ....._ / Active ~ Mechanism of Action of DCA on pyruvate dehydrogenase.

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3 Glutathione-S-Transferase Zeta At the time the present study was begun, the known metabolites ofDCA were oxalate, CO2, and glycine. Glyoxylate and glycolate were postulated as intermediates, as was monochloroacetate (MCA) .4 During the course ofthis study, it was shown definitively that DCA was converted to glyoxylate by liver cytosol in a glutathione dependent pathway.5 Recently it has been shown that the dechlorinating enzyme is GSTz, which has been isolated from hepatic rat cytosol.6 GSTz was thought to be a novel enzyme but is in fact a glutathione dependent isomerase involved in the catabolism of tyrosine, first isolated as maleylacetoacetate isomerase 1955 by Knox and Edwards (Figure 2).7 Its endogenous ligands, maleylacetoacetate, and the decarboxylation product of maleylacetoacetate, maleylacetone, are known alkylating agents and scavengers of glutathione. 8 The enzyme utilizes GSH as a catalyst in the isomerization of maleylacetoacetate (cis) to fumarylacetoacetate (FAA) (trans) t hrough the nucleophilic attachment of GSH to the P-carbon of the cis double bond. Following reketonization and loss of GSH, the more stable trans form results Additional alpha-beta unsaturated acids which are isomerized by GSTz are listed in Table 1. Maleylacetoacetate is similar in structure to maleic acid which can be toxic. It also forms GSH adducts. 8 Maleylacetoacetate, FAA, and succinylacetone, a reduction product of FAA, are thought to be responsible for the toxic effects seen in hereditary tyrosinemia type I, which is due

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4 t o the deficiency of fumarylacetoacetate hydrolase, the step following maleylacetoacetate i somerase in the breakdown oftyrosine.9 Figure 2 Deficiency kads t o PK U r;m, o-~-CHCOO H Phen y lalanine J 4-phenylalnin c monooxyge nase t;JH2 ~ C CHCOO H ,o T~: .. ,_. H O ~~-i-coo 4-H y drox y phen y lp y ru v ate Inhib ited b y NTBC c:======:::) l 4 h y droxyphenylpyruvate dio xygenase HO \? ~ c c-o ~,ii OH Hom ogenttsa te 1 ,2-hom ogentisate d ioxygenase l Deficiency l ead s to :::====== Alcaptonuria, Black l.lr ine Discas~ 0 Decarboxylase ooc/=\o Jl.__ coo----ooc,./'\ Jl.coo fr Reduction -fr -Maleylacetone MAAI j GSH 0 oocy 4-Male y lacetoacetate* 4-maleylacetoacetate l isomerase (GSTz) GS H MAAI DCA c::===========~ ooc,h\ Jl. -Yr -coo 0 (cis) 0 Fumarylacetone 4 -F umar y lacetoacet ate* (trans) Deticienrv leads to H e redilJ.rV arylacetoacetate T}TOsinemiaType I and ====== o l ase(FAH) s w .. ~cinyla<.:cton~ formation ooc o *Alkylating Agents ~coo **Neurotoxin coo Fumarate Aceto acetate 0 Succinylacetoacetate t:co, oocy 0 Sucdnylacetone d-Anunolcvulinatc l inhibiti o n "{ Porphobilinogcn Tyrosine catabolism

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5 Ta ble 1 Alpha-beta unsaturated carboxylic acids metabolized by GSTz.10 -ooc~ -ooc?=ycH 0 ~CH3 HO 3 -ooc 0 COO-0 HO Maleylacetoacetate Z Z -Maleylacetone, ketoenol Z,E -Maleylacetone, ketoenol -ooc0CH ~CH -ooc~CH3 -ooc 3 0 3 0 0 cis-P-acetylacrylate 0 Maleylacetone diketo Z E-6,-keto-2,4-heptadienoate -ooc~ coo0 CH 3 -ooc~ -ooc~ocH3 Z,Z-muconate E Z -6 -keto-2 ,4heptadienoate Z,Z-muconate monomethyl ester -ooc~OCH, 0 Z,E-muconate monometh y l ester

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6 The toxic effects of hereditary tyrosinemia type I include liver failure, cirrhosis, hepatocellular carcinoma, glomerulosclerosis, and peripheral neuropathy.9 A direct cause and effect relationship between maleylacetoacetate and the toxic effects of hereditary tyrosinemia type I has not been conclusively determined, due to the instability of maleylacetoacetate. 11 Glutathione-S-Transferases The enzymes of the GST family can be found in both the microsomal and the cytosolic fractions of cells. The cytosolic enzymes in mammals include six classes; alpha, mu, pi, kappa (a recently discovered mitochondrial species12), theta, and zeta, which are selective rather than specific in their metabolism of electrophilic compounds. All known members exist as either homodimers or heterodimers.13 The enzymes are responsible for the detoxification (and occasional activation) of many endogenous and xenobiotic electrophiles through the nucleophilic attack of electrophilic centers by the sulfhydryl group of the enzyme activated tripeptide, glutathione. This activation of glutathione is accomplished through the presence of a catalytic serine or tyrosine at the glutathione binding site of the enzyme, which hydrogen bonds to the thiol of glutathione and lowers its pKa from 9.3 to 6.5-7.4.14 The activated GSH then attacks the electrophile in an SN2 like reaction.14 Tyrosine is found in the catalytic site of GST alpha, mu, and pi, whereas serine is the catalytic amino acid for the theta and zeta classes. Zeta is a recent addition to

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7 the GST family of enzymes, shares little similarity to the alpha, mu, and pi classes, and is only slightly similar to GST theta. GSTz (GST zeta) like other GS T's is a dimer of 24.2 kDa subunits, and has activity against t-butyl and cumene hydroperoxides, but is not strongly active against 1-chloro-2,4-dinitrobenzene, a substrate used to measure GST activity in cytosol preparations.15 GSTz has been found in a number of species including carnations, Caenorhabditis elegans, rats, and humans.16 GSTz is interesting in its ability to both isomerize alpha, beta-unsaturated carboxylates and to dehalogenate certain haloacids (Tables 1 and 2). Potential Mechanisms of DCA Dechlorination One postulated pathway for the metabolism ofDCA to glyoxylate by GSTz is through the displacement of one chlorine atom by glutathione, hydrolysis of the second chlorine to give S-( a-hydroxycarboxymethyl)glutathione, and elimination of GSH to give glyoxylate (Figure 3).17 HO GST ta ClO H20 J;I 9 Cl-C-C-OH ---1: GS-C-C-OH GS-C-C-OH Cl GSH H -HCl OH 00 II II H-C-C-OH + GSH Figure 3 Possible mechanism for the conversion of DCA to glyoxylate.

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8 The mechanism of dechlorination of DCA has recently been in v estigated using deuterated DCA.1 8 Deuterium was retained in the conversion ofDCA to glyoxylate, suggesting a mechanism in which the initial glutathione adduct loses the remaining chloride ion through neighboring group participation leaving a resonance stabilized zwitterionic intermediate GS + =CHCOOwhich can react with water to form a hemithioacetal and lose glutathione to form glyoxylate The deuterium is retained in this mechanism (Fi g ure 4). H20 D 0 -C l D 0 -Cr [ orGSTz\ c1+-{ GS~ Cl 0+GSH/GST z Cl 0-D}-f D)--{O Deut e r a t e d GS+ 0GS 0Dichloroacetat e / DHO -GSH ,,p 0 D O HO~ GST z--f--{ 0 0-Go 0GS o Deuterated Inactivated GSTz Glyoxylate Figure 4 Possible mechanism accounting for retention of deuterium in glyoxylate from DCA dechlorination

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9 The formation of the zwitterionic intermediate is theorized to allow the attack of a nucleophilic site on GSTz, leading to inactivation. However, the mechanism seen in Figure 3 allows retention of the deuterium label without the formation of a zwitterionic intermediate. The observation that fluorinated haloacids (Table 2) do not lead to inactivation of GSTz is attributed to the decreased basicity of fluoride, which makes it a poor leaving group. The adducted glutathione leaves first, without the formation of a zwitterion (Figure 5). Other haloacids metabolized by GSTz are found in Table 2 ~o -Cl -~o +H 2 0 H D 0 -HF }-{ Cl GS (g~ F o +GSH/GSTz F ff -GSH i) ff Deuterated Deuterated Glyoxylate Dichloroacetate Figure 5 Proposed mechanism for removal of fluorine avoiding the zwitterion intermediate.

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BrO I II F-C-C-OH I H Bromo fluoroacetic acid 5028 150 ClO I II Cl-C-C-OH I H Di c hloroacetic acid 1038 19.8 HO I II H3C-y-C-OH Br 2-Bromopropionic acid (R ,S) 2142 107 9 HO I II F-C-C-OH I H Fluoroacetic acid 172 16.0 Table 2 10 Haloacids Metabolized by GSTz BrO I II Cl-C-C-OH I H Bromochloroacetic acid 1411 65. 2 HO I II H3C-y-C-OH I 2-Iodopropionic acid (R ,S) 2532 42.9 Cl 0 I II H C-C-C-OH 3 I Cl 2 2-Dichloropropionic acid 244 6 1 H 0 I II H3C-y-C-OH Cl (R S)-2-Chloropropionic acid 1655 66. 3 ClO I II F-C-C-OH I H Chlorofluoroacetic acid 3883 43.0 HO I II H3C-y-C-OH Cl (R)-2-Chloropropionic acid 318 14.0 HO I II H3C-y-C-OH Cl (S)-2-Chloropropionic acid 1809 71.3 BrO I II Br-C-C-OH I H Dibromoacetic acid 155 4 0 Haloacids found to be metabolized by GSTz. Difluoroacetic acid, (R,S)-2Fluoropro~ionic acid and 3 3-Dichloropropionic acid were not found to be metabolized by GSTz. 7 Rates reported in nmoles product formed (from 0.5 mM substrate) / min/mg purified rat GSTz as means SD, n=3.18

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11 Metabolites of DCA DCA was known to be metabolized to CO2 glycine, and oxalate 4, but glyoxylate had not been unequivocally identified; it was presumed a metabolite from its co-elution with standard on HPLC.1 9 The first step in the metabolism of DCA is its dechlorination to glyoxylate, which was been found to be catalyzed by GSTz. DCA's dechlorination product, glyoxylate, may be further metabolized to numerous products. Glycolate and the terminal metabolite oxalate, are formed via the cytosolic enzyme lactate dehydrogenase 20 Glyoxylate can also be decarboxylated in the mitochondria to CO2 via the enzyme aketoglutarate:glyoxylate carboligase. 21 Transamination of glyoxylate to glycine occurs in the peroxisomes by the action of glyoxylate aminotransferase, or in the mitochondrial matrix by any of several enzymes (Figure 6) DCA derived glycine becomes available for entry into many routes of intermediary metabolism, such as the "1 carbon pool" after incorporation into N5, N10methylene tetrahydrofolate, glycolysis through mitochondrial serine hydroxymethyl transferase (Figure 7), or for conjugation with carboxylic acids via glycine N acyltransferase. 22

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12 Involvement of the Tyrosine Catabolic Pathway in DCA Metabolism It is possible that DCA and maleylacetoacetate/maleylacetone are inhibitors of GSTz, and that the auto-inhibition phenomena seen in DCA metabolism may in fact be due to the buildup of maleylacetoacetate or maleylacetone, which could alkylate the enzyme or the locally available glutathione. Alkylation of GSH is unlikely to affect DCA kinetics given the low KM for GSH.5 It is also possible that DCA induced inhibition of maleylacetoacetate degradation could account for some of the toxic effects presently attributed to DCA which are in common with those seen in hereditary hypertyrosinemia type I, such as peripheral neuropathy and hepatocellular carcinoma, since the effects of hereditary hypertyrosinemia type I are due to the buildup in cells of several compounds, including maleylacetoacetate. In summary, the specific aims of this work have been to elucidate unknown urinary metabolites of DCA, to uncover the metabolic pathways of DCA metabolism to glyoxylate, and to test two hypotheses concerning DCA's auto-inhibition. One hypothesis was that DCA directly inhibits its own metabolism through a "poisoning" effect on the enzyme. The other was that DCA could compete with the native substrates for GSTz, maleylacetoacetate and/or maleylacetone, which could lead to their buildup or alkylate the enzyme, which could render it inactive.

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H 0 I II CJ-C-C-OH I C J Dichloroacetic Acid Dechlorination GSTz* Cytosol a-Ketoglutarate : Gl y o xy late Carboligase Figure 6 13 0 0 0 0 II 11 H-C C-OH L_a_c_ta_te _______ Dehydrogenase Glyoxylate** Cytosol II II HO-C-C-OH Amin transferases (seve ral ) Mitochondria Peroxisomes fl H-N C C-OH I I H H Glycine Oxalate Lactate Dehydrogenase Cytosol H 0 I II HO-C-C-OH I H Glycolate Metabolites ofDCA as understood in 1995. *The enzyme investigated in this study. **Not unequivocally determined by 1995

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H 0 I 11. Dechlorination CI-C-C-OH -------~I GST-z 14 00 II II H-C-C.1..0H _""'a-,...K..,.e_to,_,.g~lu_ta_ra_t_e_:G:-'.ly~o_x...,y_la_te-:-:-___ .. ~_ co, Carboligase Mitochondria Cl Dichloroacetic Acid Cytosol Glyoxylate IAminotransferases t (several) HO I 11. H-N-C-C-OH I I NAO+ THE H H :) Glycine **Glycine Synthase y (Glycine Cleavage System via formate) Mitochondria H H2NfJ~\ + CO2, NH4 +, NADH Loss of 14C labeled 0 H2C-N-R carbon N5, N1 0-methylene THF I-carbon donor in many reactions **Major route of glycine degradation in mammalian tissues Figure 7 Possible Routes of CO2 Formation Serine Hydroxymethyl transferase Mitochondria l;I J>;IH Ho-c-c-t:ooH i'ii'i Serine Serine Dehydratase -NH3 Cytoso l 0 H3C-C-*COOH Pyruvate PDH l"""-+'Co2 Loss of 14C labeled Mitochondria f carbon 0 H3C-CSC0A Acetyl CoA

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CHAPTER TWO MATERIALS AND METHODS Chemicals and Eguipment 14C radiolabeled DCA was purchased from American Radiolabeled Chemicals, St. Louis, MO. The specific activity was 55. 5 mCi/mmol and the reported and measured 14 radiochemical purity was >99.9%. C-Dichloroacetic acid was converted to its sodium 13 salt by equimolar addition ofNaOH. Na[l,2-C-]-DCA was purchased from Cambridge Isotope Laboratories, maleylacetone. Unlabeled NaDCA was obtained from TCI America, Portland, OR. GSH, NADPH, NADH, glyoxylate, oxalate, glycolate, KCl, isobutanol, resorcinol, trichloroacetic acid, and HEPES were all purchased from Sigma Chemical Company (St. Louis, MO) Methanol, KH2P04 and K2HP04, ascorbic acid, NaCl, hydrochloric acid, sodium chloride, and sodium acetate were purchased from Fisher Chemical Company (Fair Lawn, NJ). DTT, Biorad Protein Dye Reagent, sodium dodecyl sulfate were purchased from Biorad (Hercules, CA). Carbasorb and Permafluor cocktails for use with a Tricarb tissue oxidizer and Floscint II cocktail for the radiochemical detection on HPLC were purchased from Packard Instruments, Chicago, IL. Ecolume scintillation cocktail was purchased from ICN, Costa Mesa, CA. Anhydrous tetrahydrofuran, maleic anhydride, isopropenyl acetate, glycerol, anhydrous magnesium sulfate, and CH2Cli, were purchased from Aldrich Chemical Company, St. Louis, MO. All other chemicals used were the purest grade available from Millipore, Woburn, MA, Fisher Scientific, Orlando, FL, Sigma Chemical Co., St. Louis, MO or Aldrich Chemical Co., St. Louis, MO. 15

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16 Ready Gels, Biorad mini-Protean II Cell, Power Pac 3000, and Biorad Model 1000 / 500 power supplies were purchased from Biorad (Hercules, CA). Sepharose 4B gel was purchased from Pharmacia Biotech (Piscataway, NJ) The glass metabolism cage for the radiolabeled studies was purchased from Stanford Glass, Stanford, CA. The plastic cages were from Nalgene Metabolic Cages, Rochester, NY. Animals In Vivo Studies Male Sprague Dawley rats were used in these studies. In the studies of urinary metabolites ofDCA, five male Sprague Dawley rats with weights averaging 230 grams were cannulated at the jugular vein for blood sampling in the initial in vivo study according to the method of Harms and Ojeda.23 Rats were divided into two groups, single 14 13 and repeat dose. The single dose rats received one 1 % C + 99% C labeled DCA dose of 50 mg/kg by oral gavage. The repeat dose rats received one 50 mg/kg dose of unlabeled DCA the first day, then the second day each received 50 mg/kg ofDCA 14 13 labeled with 1 % C + 99% C. Each rat was kept for 24 hours m the metabolism cage, which collected the rats' CO2, urine, and feces. Urine samples were collected at 3 6, 9, 12 and 24 hours. At the end of sampling, rats were sacrificed and their organs removed for oxidation of 100 mg tissue samples on a Tri-Carb tissue oxidizer.

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17 Effect ofln Vivo DCA Pre-treatment on its In Vitro Metabolism For the initial in vitro studies of DCA inhibition, cytosol was obtained from livers of 36 day old Harlan Sprague Dawley rats which received either 2 ml/kg water or unlabeled DCA at a dose of 50 mg/kg (2 ml/kg), by oral gavage as one daily dose for 2 days The rats were sacrificed on the third day and hepatic cytosol prepared. 2 4 Dose Response Studies A similar study was conducted with 13C-DCA in plastic metabolism cages. Male Sprague Dawley rats (250 to 300g) were acclimatized for one week before experimentation. During dosing periods and control days the rats were kept in metabolism cages (Nalgene Metabolic Cages, Rochester, NY) and urine was collected over 24 hours during the control day (day 0) and on the study days Neutralized NaDCA at doses of 4 12. 5, 50,200 and 1000 mg/kg was administered by oral gavage in the morning (7:30AM) for one or five days. Twenty-four hours after the last dose, rats were sacrificed and their livers were removed for preparation ofliver cytosol.24 Four animals were used for each dose investigated. Urine samples were analyzed for maleylacetone content using GC-MS described below Hepatic cytosol was used to measure 14C-DCA metabolism

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18 Preparation of Rat Liver Cytosol Male Sprague Dawley rats were sacrificed by decapitation. Their livers were excised and cytosolic fractions prepared by differential centrifugation described previously. Livers were rinsed in 1.15 % KCl, 0.05 M potassium phosphate buffer, pH 7.4, three times, then minced before homogenization using four up and down strokes in a Potter-Elvehjem vessel. Homogenized samples were then centrifuged at 13,300 g for 30 minutes. The supernatant was transferred to polycarbonate tubes for centrifugation at 170,000 g for 50 minutes to separate cytosol and the microsomal pellet. The pellets were resuspended in buffer and resedimented for the preparation of washed microsomes.24 Preparation of Human Liver Cytosol Liver sections (10-15 g) were minced and homogenized in 25-30 ml homogenizing buffer, 1.15% KCl, 0.05 M potassium phosphate, pH 7.4, 0.1 mM phenylmethylsulfonyl fluoride, with several up and down strokes in a Potter-Elvehjem homogenizing vessel. Cytosolic fractions were obtained through differential centrifugation as above.24 Remaining liver pieces were stored either in homogenizing buffer at -so0c or frozen alone in aluminum foil at -so0c. Protein concentrations of all cytosol samples were performed according to the method of Lowry et al.25

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19 Assays of DCA Metabolism General All incubations were performed in duplicate in borosilicate tubes at 3 7C in a shaking water bath. Tubes contained 0.1 M Hepes, pH 7.6, GSH (0-1 mM), cytosol (0-1.5 mg), and DCA (0-0 2 mM) in a final volume of 0.25 ml. In most studies DCA was added last and tubes were incubated for 0-60 minutes. Reactions were stopped by the addition of 0.5 ml ice-cold methanol. Tubes were centrifuged at 1500 rpm for 20 minutes and the supernatant transferred to microfuge tubes for storage at -20C until 14C-glyoxylate quantitation by HPLC with radioactivity detection, or the fluorescence assay of unlabeled glyoxylate (see below). The apparent KM, Ki, and V max were determined from the linear equation of the graph in the Graph Pad Prism program by the Lineweaver Burke or Hanes methods.22 26 The student's t-test was used in the statistical analysis of the apparent KM and V max averages. Cytosol Dialysis For the rat apparent KM studies, cytosol was dialyzed in four liters of a 1.15% KCl, 0.05 M potassium phosphate pH 7.4 buffer (dialysis buffer), and in dialysis tubing with a molecular weight cutoff of 12,000 Daltons (molecular units). After 2-3 hours in a multi-dialyzer apparatus from Spectrum, the buffer was changed and the cytosol was dialyzed for 2 additional hours. For comparison ofNADH, NADPH and GSH dependency, cytosol from four control rats and four DCA dosed rats was dialyzed as

PAGE 35

20 above, then incubated at 37C for 15 minutes with either 2 mM NADPH to reduce any GSSG present to GSH, or with water as a control. This cytosol was then re-dialyzed for 2 hours on the multi-dialyzer and used immediately in the incubation. For the GSH apparent KM studies, the cytosol was first incubated at 37C for 15 minutes with 2 mM NADPH to reduce any GSSG present to GSH and to free any protein bound GSH, then dialyzed as above. In experiments when smaller samples of protein were needed, or fewer samples required, 3 ml aliquots were dialyzed in 250 ml dialysis buffer for one hour, then the buffer was changed and the samples dialyzed overnight. For the in vitro DCA inhibition studies cytosol samples (10 ml) were dialyzed at 4C in 50 x buffer volume with either 1.15% KCl, 0.05 M KH2P04 pH 7.4, 0 1 mM dithiothreitol, or 0.1 M Hepes, pH 7 6 and 1 mM GSH, in dialysis tubing with a MW cutoff of 12,000 Daltons. After 1-2 hours the buffer was changed and the cytosol was dialyzed overnight. Protein concentrations of all cytosol samples were quantitated.26 Reducing Agent Dependence Incubations Concentrations of 1 mM GSH, 2 mM NADH, and 2 mM NADPH were used along with blanks without any reducing agent, blanks with reducing agent but no cytosol, and one zero time blank with 1 mM GSH.

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21 Linear Range of Glyoxylate Production in Rat Hepatic Cytosol A time course of glyoxylate production was performed with 0.2 mM DCA, 1 mM GSH 1 mg dialyzed rat hepatic cytosolic protein and 0 1 M Hepes pH 7 6 Incubations were stopped by the addition of ice-cold methanol at time 0 5 10, 15, 30 45 and 60 minutes. Tubes were centrifuged at 1500 rpm for 20 minutes and the supernatant transferred to microfuge tubes for storage at -20C until 14C-glyoxylate quantitation by radiation detection coupled to ion pair HPLC (see below). A range ofGSH concentrations including 0, 0.00625, 0.05, 0 .1, 0.25, 0 5, 1 and 1.5 mM were used in 15 minute incubations at 37C with 0 2 mM DCA, 1 mg dialyzed rat hepatic cytosolic protein and 0.1 M Hepes pH 7.6. Reactions were stopped by the addition of 0 5 ml ice-cold methanol after 15 minutes. Tubes were centrifuged at 1500 rpm for 20 minutes and the supernatant transferred to microfuge tubes for storage at 20C until 14C-glyoxylate quantitation by radiation detection coupled to ion pair HPLC Different DCA concentrations were incubated with 1 mM GSH, 1 mg dialyzed rat hepatic cytosolic protein and 0.1 M Hepes pH 7.6. DCA concentrations included 0.05, 0.1, 0.2 and 0.5 mM DCA. Incubations were stopped by the addition of ice-cold methanol after 15 minutes Tubes were centrifuged at 1500 rpm for 20 minutes and the supernatant transferred to microfuge tubes for storage at -20C until 14C-glyoxylate quantitation by radiation detection coupled to ion pair HPLC. Protein concentrations were also varied to investigate the linear production of glyoxylate. Dialyzed hepatic rat cytosol was incubated at concentrations of 0, 0 05, 0.1, 0.15 0.2 and 0.25 mg in a final incubation volume of0.25 ml. Tubes also contained 0.2 mM DCA, 1 mM GSH and 0.1 M Hepes pH 7.6. Incubations were stopped by the

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22 addition of ice-cold methanol after 15 minutes. Tubes were centrifuged at 1500 rpm for 20 minutes and the supernatant transferred to microfuge tubes for storage at -20C until 14C-glyoxylate quantitation by radiation detection coupled to ion pair HPLC. Rat Apparent KM and V Determination Incubations __M For the apparent KM ofDCA determination all tubes contained 1 mM GSH and either 0.1, 0.075, 0 .05, 0.025, 0 01, 0 005, or 0 002 mM DCA. For the apparent KM of GSH determination all tubes contained 0.2 mM DCA and either 0.10, 0.075, 0.05, 0.025, or 0.01 mM GSH. All samples were incubated for 15 minutes and contained from 0.2-4 mg / ml cytosolic protein. Samples were analyzed by HPLC coupled to radiation detection. Percent 14C-glyoxylate produced was used to determine the specific activity as nmoles/ minute/mg protein. KM and V max were determined through Lineweaver-Burk plots of I/specific activity vs. 1/mM GSH or 1/mM DCA, with the x intercept being 1/KM and the y intercept being IN max Values were determined using Graph Pad Prism software Linear Range of Glyoxylate Production in Human Hepatic Cytosol A time course for glyoxylate production was performed with 0.2 mM DCA, 1 mM GSH, 1 mg dialyzed hepatic cytosolic protein from human liver, and 0 1 M Hepes pH 7 .6. Incubations were stopped by the addition of 750 % TCA at time 0, 10, 14.25, 28.5, 42 75, 57, 90 and 120 minutes. Tubes were centrifuged at 1500 rpm for 20 minutes

PAGE 38

23 to precipitate protein and the supernatant transferred to tubes containing resorcinol and hydrochloric acid for glyoxylate measurement as described in the fluorescence assay. A range of GSH concentrations including 0, 0.01, 0.025, 0 .05, 0.075 0.1, 0 2 and 0.25 mM were used in 30 minute incubations at 37C with 0.2 mM DCA 0.9 mg dialyzed human hepatic cytosolic protein, and 0.1 M Hepes pH 7.6. Incubations were stopped by the addition of 750 l 5% TCA. Tubes were centrifuged at 1500 rpm for 20 minutes to precipitate protein and the supernatant transferred to tubes containing resorcinol and hydrochloric acid for glyoxylate measurement as described in the fluorescence assay. Different DCA concentrations were incubated with 1 mM GSH 0 9 mg dialyzed hepatic cytosolic protein and 0.1 M Hepes pH 7.6. DCA concentrations included 0.002 0 005 0.01 0.025, and 0.05 mM DCA. Incubations were stopped after 30 minutes by the addition of 750 l 5 % TCA. Tubes were centrifuged at 1500 rpm for 20 minutes to precipitate protein and the supernatant transferred to tubes containing resorcinol and hydrochloric acid for glyoxylate measurement as described in the fluorescence assay. Protein concentrations were also varied to investigate the linear production of glyo x ylate. Dialyzed human hepatic cytosol was incubated at concentrations of 0 0.25 0 .5, 0.75 1 1.5, 1.76 and 2 mg in a final incubation volume of 0.25 ml. Tubes also contained 0.2 mM DCA 1 mM GSH and 0 1 M Hepes pH 7.6 Incubations were stopped by the addition of ice-cold methanol after 60 minutes. Tubes were centrifuged at 1500 rpm for 20 minutes and the supernatant transferred to microfuge tubes for storage at -20 C until 14C-glyoxylate quantitation by radiation detection coupled to ion pair HPLC.

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24 Apparent KM and V in Dialyzed Human Liver Cytosol Incubations ---1!! Various concentrations of both GSH and DCA were used simultaneously to eliminate the possible substrate inhibition from either agent. DCA concentrations were 0 002, 0.005, 0.01, 0.025, and 0.05 mM. GSH concentrations were 0.01, 0.025, 0.05, 0.075, and 0.1 mM. Tubes were incubated for 10 minutes with a protein concentration of either 0.628 mg/ml (0.002 -0.01 mM DCA) or 1.568 mg/ml (0.025 and 0.05 mM DCA). Samples were analyzed as above with rat hepatic cytosol. In Vitro DCA Inhibition Incubations Tubes contained 0.1 M Hepes buffer pH 7.6, 1 mM GSH, and 0.2 mM DCA. The amount of cytosolic protein from human liver in the tubes for the time course incubations was approximately 4 mg/ml. DCA was added last in all incubations in 30-second intervals. Water replaced DCA in the control tubes. At time intervals of 0, 5, 10, 20 and 30 minutes, 250 l were removed from all treatment tubes to assay glyoxylate production using the fluorescence assay. In all studies after the initial incubations, the remaining cytosol was placed on ice, then transferred to dialysis tubing for dialysis as described above. After the second dialysis, control and DCA treated cytosol were both incubated with 0.2 mM DCA, approximately 2 mg/ml protein, 0.1 M Hepes buffer pH 7 .6, and 1 mM GSH at 0, 5, 10, 15, 20, and 30 minutes, then assayed for glyoxylate production as above. For the inhibition studies with different DCA concentrations, treatment tubes contained either 0, 0.5, 0.75 or 1 mM DCA, and 0.1 M Hepes buffer, pH 7.6,

PAGE 40

25 approximately 2 mg/ml protein from human liver, and 1 mM GSH. Initial incubations were performed for 30 minutes at 37C. After 30 minutes, 250 portions were removed from all tubes for activity measurement, and the remaining cytosol was put on ice, then dialyzed overnight. For the measurement ofresidual activity the concentrations ofDCA in the treated group corresponded with the treatment concentrations, and the control cytosol was also incubated with either 0 .5, 0.75 or 1 mM DCA, 0.1 M Hepes buffer pH 7.6, approximately 1.5 mg/ml protein, and 1 mM GSH. After 30 minutes, 250 aliquots were checked for activity using the fluorescence assay. To simplify the results from this experiment, another experiment was conducted in which the measurement of residual activity was performed with 0.2 mM DCA in all tubes to ease comparison between initial treatments with the three different concentrations ofDCA and the control cytosol. DCA Pre-Incubation Comparison between Dialyzed Rat and Human Cytosol To determine the effect of pre-incubation with DCA in rat (N=3) and human (N=3) dialyzed cytosol, duplicate incubation tubes contained 0 1 M Hepes buffer pH 7 .6, 1 mM GSH, 0.5 mM DCA, and approximately 4 mg/ml dialyzed hepatic cytosolic protein in a final volume of 10 ml. DCA was added last to all incubations. Water replaced DCA in the control tubes. At 10 min (rat cytosol) or 30 min (human cytosol), 250 l portions were removed from all tubes to assay glyoxylate formation using the fluorescence assay described below. After the incubation with DCA or water for 30 min, the remaining samples (8.75 ml) were dialyzed as described above in 0.1 M Hepes pH 7.6, and 1 mM GSH. After the second dialysis, protein concentrations were determined again and control

PAGE 41

26 and DCA treated cytosol samples (approximately 4 mg/ml) were separately incubated for 0-60 min with 0.2 mM DCA and 0 5 mM GSH in a final volume of 0.25 ml. The reactions were stopped by addition of 750 lice-cold 5% trichloroacetic acid and were assayed for glyoxylate using the fluorescence assay. DCA Binding Study in Dialyzed Rat and Human Hepatic Cytosol To determine whether DCA binds cytosolic protein, duplicate samples of dialyzed rat and human hepatic cytosol were incubated with 0.5 mM 14C-DCA. Dialysates consisting of 4 mg protein, were incubated for ten minutes at 37C with 0.5 mM 14C DCA, 0 1 M Hepes pH 7.6, and water in a final volume of 1 ml. Glutathione was excluded to prevent metabolism ofDCA. A ten microliter aliquot from each incubation sample was removed for counting on a scintillation counter. After ten minutes, the samples were ultrafiltered for ten minutes using a 0.45 m cutoff. Aliquots of ten microliters were removed from both the supematants and filtrates from each incubation sample. Counts measured in disintegrations / min of aliquots from the samples before and after filtration were compared to determine the extent, if any, ofDCA binding

PAGE 42

27 In Vitro GSTz Activity in DCA Dosed Animals Duplicate hepatic cytosol samples from DCA treated animals containing approximately 1 mg protein were incubated at 37C in a shaking water bath with 0.1 M Hepes, 1 mM GSH, 0.2 mM 14C-NaDCA and water for a final volume of 0.25 ml. After 5-15 min, reactions were stopped by adding 0 5 ml ice-cold methanol. Tubes were placed on ice and then centrifuged at 1500 rpm to precipitate protein Supematants were transferred to microfuge tubes and stored at -20C prior to HPLC analysis and radiochemical detection of 14C-glyoxylate produced from 14C-DCA. Analyses Ion Pair HPLC Samples investigating the apparent KM and V in rat and human cytosol samples max were analyzed by HPLC. Rainin filterfuge tubes with a 0.45 m cutoff were used to filter the incubation samples' supematants before HPLC analysis. The mobile phase was 70:30 0 005 M aqueous tetrabutylammonium sulfate (low UV Pie A, Waters, MA):methanol. An Isco model 2350 pump was used with a Rainin Dynamax UV-1 detector, a Radiomatic Flo One B Radioactive Flow Detector, and a Beckman Ultrasphere 5 m x 4 6 mm x 25 cm reverse phase ODS column coupled to a Beckman Ultrasphere 5 m x 4 6 mm x 4 5 cm pre-column. All retention times were compared with standards ofDCA, oxalate and glyoxylate for confirmation.

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28 Strong Anion Exchange HPLC Analysis Rainin filterfuge tubes with a 0.45 m cutoff were used to filter the samples before HPLC analysis. The mobile phase was 0.1 M potassium phosphate at pH 3. An Isco model 2350 pump was used with a Rainin Dynamax UV-1 detector, a Radiomatic Flo One J3 Radioactive Flow Detector, and a Zorbax strong anion exchange column, 4 6 mm ID x 15 cm. All retention times were compared with standards ofDCA (5. 3 min) and glyoxylate (3.0 min) for confirmation. Percent conversion of 14C-DCA to 14C-glyoxylate was determined by radiochemical detection and used to calculate nmoles glyoxylate formed/min/mg protein. 13 C NMR Analysis One sample of urine collected from a rat dosed with 99% [1,2-13C]DCA and 1 % [1-14C]DCA contained 300 g ofDCA equivalents (70 5% of the DCA derived 14C) as the unknown major urinary metabolite. This was diluted with 150 ofD20 to adjust magnetic field homogeneity and to maintain stable lock frequency. This was examined in a 5-mm tube A trace of 3-(trimethylsilyl)propionate-2,2,3,3-d4 sodium salt was included as an internal standard of chemical shift reference. 13C NMR analysis was performed on a 300 MHz Varian Unity NMR spectrometer with a 5-mm broad band probe regulated at 25C, spinning at 20 Hz (Center for Structural Biology, University of Florida). Four spectra were acquired; one proton-coupled spectrum and three proton-decoupled spectra. One proton-decoupled spectra was acquired with continuous proton-decoupling Ten thousand transients were accumulated over a 27.75 hour period. The free induction decay

PAGE 44

29 was processed with an exponential line broadening of 1 Hz before Fourier transformation. Fluorescence Analysis For certain experiments it was favorable to use unlabeled DCA to avoid excessive contamination and to speed quantitation of unlabeled glyoxylate The quantification of unlabeled glyoxylate through fluorimetry of its condensation product with resorcinol was modified from the methods of Zarambski and Hodgkinson (Figure 8) .2 7 OH 20 + ~OH Resorcinol Figure 8 00 HC-C-OH ..... Glyoxylate HO OH 2 ,2', 4 4'-Tetrahydroxydiphenylacetic acid lactone Fluorescent Formation of 2,2',4,4'-tetrahydroxydiphenylacetic acid lactone. Glyoxylate standards and incubation samples were deproteinized with 750 l 5% TCA. An aliquot of this mixture (750 l) was transferred to marble covered tubes containing 25 l 5% resorcinol and 375 l concentrated HCl and heated for five minutes at 100C. After cooling, samples were extracted by vortexing for eight seconds with 1.25 ml isobutanol. Tubes were centrifuged at 1500 rpm for ten minutes. The isobutanol layer

PAGE 45

30 (1 ml) was transferred to tubes containing 2.5 ml 25% K2C03 These tubes were mixed with a vortex for eight seconds, centrifuged, and 2 ml of the aqueous layer was transferred to tubes containing 250 l 10% ascorbic acid After the addition of 4 ml 0.1 M sodium carbonate-bicarbonate buffer pH 9.6, tubes were incubated at room temperature for thirty minutes. Fluorescence of 2,2',4,4'-tetrahydroxydiphenylacetic acid lactone was measured using an Fmax fluorescence plate reader, with an excitation 'A, of 485 nm and an emission 'A, of 538 nm. GC-MS Confirmation of Glyoxylate Urinary metabolites were separated and identified using a Hewlett-Packard 5890 series II plus GC, a 5972A mass selective detector and a Vectra multimedia VL2 4/66 computer with ChemStation software .28 Samples were spiked with 4-chlorobutyric acid as an internal standard, and methylated by heating with an equal volume of a 14% solution of boron trifluoride in methanol at 115C for 15 min, then extracted into methylene chloride. The methylene chloride extract was injected onto an HP-Wax column, 30 m x 0.25 mm with 0.15 m film thickness, phase ratio 420 with helium carrier gas at a flow rate of 1.21 ml/min and an inlet pressure of 9 psi. The GC temperature was 35C for 4 min, followed by a linear gradient to 100C at 3C/min then up to 240C at 50C/min. The temperature was held at 240C for 8 min. The retention time of the methyl esters of DCA and metabolites were as follows : DCA, 10 8 min; glyoxylate 11.2 min; oxalate 11. 7 min; hippuric acid 20 min. The urinary metabolites were identified by their GC retention times and by matching the complete mass spectrum 13 12 of the C-labeled metabolite to the mass spectrum of known C standards (performed in

PAGE 46

31 Dr. G.N. Henderson's laboratory by Ximeng Yan, University of Florida, College of Medicine) GC-MS Analysis ofMaleylacetone Urine (0.2 ml) was pipetted into a Coming culture tube with a Teflon -lined screw cap BF3 (12 % ) in MeOH, 0.5 ml, and 50 l (960 g/ml) of 2-oxohexanoic acid (internal standard) were added, heated at 110C for 15 min and allowed to cool to room temperature. One ml of water and one ml of CH2Ch were added. The culture tube was vortex mixed for 1 min, placed on a shaker for 5 min and centifuged (10C) at 3000 rpm for 10 min. The lower CH2Ch layer was pipetted into a 2 ml autosampler vial that contained a 100 ul glass insert Samples were analyzed on a Hewlett Packard 5972 mass spectrometer. The column used was Hewlett Packard HP-WAX (Cross linked Polyethylene Glycol) 30m x 0.25mm x 0.15 um film thickness. The flow rate of zero grade helium was 1 1 ml/min. The GC was temperature programmed as follows: at 40C for 2 min; then to 100C at 5C per minute; then to 240C at 15C per minute and held for 5 min at 240C. 2-0xohexanoic acid and maleylacetone had retention times of 13. 67 and 20 .93 min, respectively A single ion monitoring method was used for quantitation (performed in Dr. G.N. Henderson s laboratory by Jing Cheung and Larry Shroads, University of Florida, College of Medicine).

PAGE 47

32 Synthesis of Maleylacetone All steps were performed under positive nitrogen flow according to the methods of Fowler and Seltzer, at 1/3 literature scale (Figure 9).29 Aluminum chloride (20 g) was added to 150 ml methylene chloride while stirring. Maleic anhydride (6.67 g, 0.068 mol) was added and the mixture stirred at room temperature for 30 minutes prior to the addition of 7.34 ml (6.67 g, 0.067 mol) isopropenyl acetate over 5 minutes using a dripping funnel. After 5 hours, the mixture was slowly added to 167 ml ice-cold 2 N HCl. The organic phase was separated, and the aqueous layer extracted with 67 ml CH2Ch. The organic phase was filtered through Celite to remove particulates, washed with 66 ml 5% Na2C03 in three portions, then with saturated NaCl. The organic phase was dried with anhydrous MgS04 The solvent was removed with a rotary evaporator and the residue was frozen overnight at -80C. The following day the crude material (770 mg) was applied to a flash column with 3: 1 pentane:ethyl acetate as the mobile phase. Spotting of the crude material on silica TLC plates revealed three major spots. Fractions containing the spot which did not match the spots of the two starting materials and were the reported pale yellow color of the butenolide, were combined and their solvent removed using a rotary evaporator, then dried overnight under vacuum. The 1H-NMR in CDCh confirmed the structure of 4-acetonylidenebut-2-ene-4-olide, in accordance with published results (Figure 9).31 The 1H-NMR showed resonances at o 2.58 (s, 3H, CH3), 5.58 (s, lH, vinyl), 6.46 and 7.53 (q, 2H) (Figure 9). To 0.13 g butenolide was added 25 ml of 1 N NaOH; this was washed with 10 ml CH 2Ch. The aqueous (basic) layer was then acidified with concentrated HCl and extracted with 40 ml CH2Ch on ice. The organic phase was washed with 10 ml saturated NaCl solution, dried with anhydrous

PAGE 48

33 MgS04 and filtered through Celite to remove particulates. The solvent was removed at room temperature using a rotary evaporator and the oily product dried overnight under vacuum or under a constant stream of nitrogen for several hours. The 1H-NMR in CDCh confirmed the structure of maleylacetone (Figure 10) in accordance with published results.31 30 It displayed resonances at 8 2.3 (s, 3H, CH3), 2.9 (q, 2H, J=16 Hz), 6.12 and 7.35 (q, 2H, J=5.71 Hz), 5 79 (s, lH, vinyl), 6.38 (s, 2H, vinyls), 2.24 (s, 3H, CH3), 5.79 (s, lH, vinyl), 6 38 (s, 2H, vinyls). No broad OH signal was seen at 8 7-8 ppm, although a broad band was seen from 8 1-2 ppm due to the presence of water. + H,C~COCH, AICI, CH3 CH2Cl2 Maleic Anhydride lsopropenyl acetate H02C Maleylacetone Figure 9 Synthesis of maleylacetone. 0 4-acetonylidenebut-2-ene-4-olide CH3 1. NaOH 2, UC!

PAGE 49

2 3 Keto fonn 0 2/3 keto 5a 3 2 34 2 3 HO Enolfonn 5 7al I 1 I 0 -.~. 0 P PIII Figure 10 HNMR of maleylacetone

PAGE 50

35 Maleylacetone Methods Maleylacetone IC50 Studies 14 In a preliminary study, radiolabeled 1-C-DCA (0.2 mM) was incubated with maleylacetone at the following concentrations; 0.07, 0.1, 0.2 and 0.4 mM. HPLC coupled with radiation detection was used to monitor the production ofradiolabeled glyoxylate from DCA to investigate any inhibition of DCA metabolism. Approximately 30 mg aliquots of maleylacetone were prepared by hydrolysis of the 4-acetonylidenebut-2-ene4-olide, 8 and were accurately weighed and diluted in a known amount ofTHF. Aliquots were delivered into tubes to produce final concentrations ranging from 0-0.4 mM maleylacetone. The THF was evaporated under a constant stream of nitrogen, and the tubes were kept on ice until the aqueous incubation solution was added. Incubations consisted ofO.l M Hepes pH 7.6, 0.1 mM GSH, approximately 1 mg human hepatic cytosolic protein, 0.2 mM 14C-DCA and water for a final volume of 0.25 ml. Tubes containing THF alone were evaporated under nitrogen to serve as blanks. After the initial incubation found inhibition of DCA metabolism by maleylacetone, rat and human (n=3) hepatic cytosol samples were incubated with maleylacetone in concentrations from 0-0.4 mM maleylacetone. THF was replaced with CH2Cli as the solvent vehicle for maleylacetone delivery due to the instability of THF. To avoid possible destruction of GSH and protein before adding DCA in these IC50 studies, 0.1 M Hepes pH 7.6, 0.2 mM 14C-NaDCA, and water were added first to the maleylacetone containing tubes. The reaction was initiated by adding 1 mg cytosol (human) or 0.5 mg cytosol (rat) combined with 1 mM GSH. Tubes were incubated for 0-

PAGE 51

36 10 min (rat samples) or 0-30 min (human samples) at 37C in a shaking water bath. Reactions were stopped by adding 0 5 ml ice-cold methanol. Volumes of CH2Clz alone, identical to those used to deliver maleylacetone, were evaporated from tubes to serve as blanks. Tubes were centrifuged at 1500 rpm for 20 minutes to precipitate protein. Supematants were transferred to microfuge tubes for storage at -20C before strong anion exchange HPLC analysis of 14C-glyoxylate produced from 14C-DCA. Pre-Incubation Studies Cytosol from rat and human samples (n=3) and all ingredients except for DCA were incubated with maleylacetone for 30 minutes before the addition ofDCA to initiate the reaction. Reactions were stopped and stored as described above. Maleylacetone Ki Studies Aliquots of maleylacetone were delivered into borosilicate tubes in a solution of THF which was evaporated with nitrogen. Concentrations of maleylacetone included 0.05, 0 07, 0.1, 0.2 and 0.4 mM. Various concentrations ofDCA included 0.0025, 0.00375, 0 005, 0.015 and 0.025 mM. Tubes were incubated at 37C for 30 minutes with 1 mM GSH, 0 1 M Hepes pH 7.6, and 0.5-1 8 mg human hepatic cytosolic protein from human liver. Protein concentrations were varied to maintain glyoxylate production in the linear range. Tubes were pre-incubated for 2 minutes and DCA was added last to initiate the reaction. Reactions were stopped by the addition of 0.5 ml ice-cold methanol. Tubes were centrifuged at 1500 rpm for 20 minutes to precipitate protein Supematants were

PAGE 52

37 transferred to microfuge tubes for storage at -20C before strong anion exchange HPLC analysis of 14C-glyoxylate produced from 14C-DCA. Pre-Treatment of Dialyzed Rat and Human Hepatic Cytosol with Maleylacetone In an experiment similar to that performed with DCA on page 25, dialyzed rat and human cytosol was incubated for 30 minutes with maleylacetone, and then dialyzed again overnight to check for the persistence of maleylacetone inhibition. Incubation tubes contained 0.1 M Hepes pH 7.6, either O or 1 mM GSH, 0.4 mM maleylacetone, and approximately 5mg/ml (rats) or 8 mg/ml (humans) protein in a final volume of 2 ml. After 30 minutes duplicate aliquots were removed for initial activity measurement with DCA. From samples incubated with GSH, 245 aliquots were removed and added to tubes containing NaDCA for a final concentration of 0.2 mM. From samples incubated without GSH, 195 aliquots were removed and added to tubes containing GSH and DCA for final concentrations of 1 mM (GSH) and 0.2 mM (DCA). These samples were incubated for 30 more minutes and reactions were stopped by the addition of 750 5% TCA then assayed for glyoxylate production using the fluorescence assay. Remaining maleylacetone incubation samples were dialyzed overnight in 0.1 M Hepes pH 7.6, with either 1 mM GSH (for GSH containing treatments) or 0.01 mM DTT (for treatments without GSH). To measure the final activity, the next day all samples were incubated with 0.2 mM DCA for 30 minutes Reactions were stopped with TCA and assayed for glyoxylate production using the fluorescence assay.

PAGE 53

CHAPTER THREE IN VIVO STUDIES' RESULTS Ion Pair HPLC Analysis of Urine Monochloroacetic acid (MCA) and trichloroacetic acid (DCA contaminants) and the metabolites ofDCA (oxalate, glyoxylate and glycine) were well separated from DCA by reversed phase HPLC using the ion pair reagent, PICA (tetrabutylammonium sulfate). MCA was not separated from oxalate, and glyoxylate was not separated from glycolate or glycine. A standard ofhippuric acid had the same retention time (10.2 minutes) as an unknown metabolite found in all rat urine samples This major unknown metabolite was particularly prominent in the 3-6 hour urine sample ofrat 21, and the 0-3 hour sample rat 18 (Figure 11 ). Phenacetyl glycine had a retention time of 14 minutes, identical to one of the other urinary metabolites observed in some samples.31 13 C NMR Analysis of Rat Unne 13 ,: The C NMR spectrum from rat 21, 3-6 hour urine sample revealed a tnplet at u 180 ppm, a singlet at 8 167 ppm, four peaks in the aromatic region from 8 129 to 8 136 ppm, and CH2 bands from 8 46.3 to 8 47.1 ppm. These peaks suggest the structure 38

PAGE 54

39 13 13 13 XCH2COOH denved from 1,2C-DCA. The spectrum of sodium hippurate was similar (Figure 12). This evidence was corroborated by GC-MS analysis of the methylated urine sample (Figure 13). The ratio ofM + 2: M peaks was approximately four times the 13 13 . natural abundance of C. The production of C-rad1olabeled hippunc acid from DCA administration indicates that DCA derived glyoxylate may be transaminated to glycine, which is known to undergo conjugation with benzoic acid, a common preservative and known end product of phenylalanine metabolism.32 Figure 11 1~ cpm 0 ... !: ......c: Gx C s Ox E F D J O H S 1 Repeat dose, 180-265 g, fed 0-3 h urine Full scale y axis 2800 cpm Rat 18, 0-3 Hour Urine Sample; H, (hippuric acid retention 10.2 min); D, (DCA retention 7 0 min); Ox, (oxalate retention 4.2 min); Gx, (glyoxylate retention 3.2 min). The radiochemical detection scan is shown for clarity.

PAGE 55

0 H 0 II I II ("' c-N-9-c-OH 4 ~ r 3 H 2 1 221 Figure 12 0 0 II II HO-C-COH 5 5 1 5 3 211 11Q 1H 40 4 2 141 120 111 oa IO 41 21 0 ... 13C-NMR ofrat urine. The conditions are described in the methods section under 13C NMR analysis

PAGE 56

-, ... .01 'f'nw,-O 1 00 10. 00 11.00 U 00 .. """""" '"""' """"" """"" """"" 1000000 ,..,_>o ,,. Figure 13 103 0 I I .~ ,., 41 O'i1~ 'i1~ C C-N -C-C-OCH 212 fi Pf fi J -3'0000 200000 '"""" ,.c o """"' I 1~.0 '"""" 120000 IOOOO lOl)I) .~ ,,...,_,,o ~ 14d Hid tho 200 lid GC-MS of methylated urine. Rat was given one 50 mg/kg dose of a 99: 1 mixture of 13C:14C-DCA. Top panel GC trace, lower left panel MS of the 20.8 minute peak, hippurate Bottom right panel, MS of the 21.2 minute peak, phenylacetylglycine Insets are expansions of the molecular ion peaks sho wing M + 2 peaks arising from the 13C enrichment of glycine. 31

PAGE 57

42 Maleylacetone in Urine The concentrations of maleylacetone in urine of rats treated with DCA doses of 50 mg/kg or more were measured by GC-MS from 24-hour samples collected before and after treatment. Maleylacetone excretion increased in a dose dependent manner. Figure 14 shows the changes in urinary maleylacetone levels for rats treated with five daily doses of 200 mg/kg ofDCA. The mean SD (n=4) maleylacetone concentration before 200 mg/kg DCA treatment was 24.5 15. 6 g/kg/day and increased to a maximum of 410.4 169.1 g/kg / day. Similar increases in maleylacetone excretion were observed for rats treated with 50 mg/kg (increasing to 298.4 32.0 g/kg/day maximum) and in rats treated with lg/kg (increasing to 739.2 107.8 g/kg / day maximum).

PAGE 58

43 600 QJ 550 = 500 0 ..... QJ 450 u != -= 400 ....... "11:t 350 aJ N -!f 300 e_ ....... OJ) 250 ::t 200 = i:: 150 100 50 0 day 0 day 1 day 2 day 3 day 4 day 5 DCA administration Figure 14 Urinary excretion of maleylacetone in rats treated with 200 mg/kg DCA.

PAGE 59

CHAPTER FOUR IN VITRO STUDIES RESULTS Identification of Glyoxylate as a Product of DCA Metabolism A standard of glyoxylate was found to have a retention time of 3 8 minutes using the ion pair HPLC method This retention time agreed with the retention time of the principal unknown metabolite seen in DCA incubations (low amounts of oxalate were sometimes also produced). GC-MS of methylated incubation extracts was preformed and again the metabolite's retention time agreed with that of a methylated glyoxylate standard 11.1 minutes. The mass spectra of the unknown peaks also matched that of methylated standard with the most abundant fragment at m/z 75 corresponding to -cH(OCH3)2. The fragments at m/z 75 and m/z 76 arise from 1 2C and 13C glyoxylate, and the enrichment at m/z 76 conclusiveley demonstrate that glyoxylate is a labeled metabolite of 1,2-13C-DCA (Figure 15) 44

PAGE 60

4bundnc 20000 A 18000 12000 8000 ,, Time-> Abundnce 6000 6000 4000 3000 2000 F ig ure 15 7 45 00 Glyox II 11 HC-C-OH DCA Glyoxylate 8 9 10 11 12 76 41 7 GC-MS of methylated rat liver cytosol incubated with 13C l a beled DCA.

PAGE 61

46 Development of a Fluorescence Assay of Glyoxylate The assay for the fluorescence detection of glyoxylate proceeds through the condensation of two equivalents ofresorcinol with one equivalent of glyoxylate, to yield the fluorescent product; 2 2',4,4'-tetrahydroxydiphenylacetic acid lactone .27 Modifications of the protocol as published were made to increase reproducibility in the assay. Modifications included mixing samples for timed intervals with a vortex and heating in a dry bath at constant temperature for a timed period. These modifications improved reproducibility, bringing the r2 value of the standard curve to 0.997 from 0.878 as calculated by the ANOV A method in Excel software This experiment allows the analysis of several incubation samples in approximately three hours, compared to the several days required by the HPLC / radioactivity detection assay described by James et al.5 The loss of sensitivity as compared to the HPLC method is generally outweighed by the decrease in time required for the assay The HPLC method is much more expensive, requires 10 minute runs per sample and a one hour equilibration time but has the advantages of greater reproducibility due to fewer steps before the final analysis and a lower detection limit compared to that of the fluorescence assay (0.1 g glyoxylate 1 nmol, vs. 0.001 nmol).

PAGE 62

47 Strong Anion Exchange HPLC Analysis of Incubations 14C DCA and 14C labeled glyoxylate were well separated on the strong anion exchange column with retention times of 5.3 minutes and 3.0 minutes, respectively, as determined through radiation detection (Figure 16). 1000 Glyoxylate 0 0'00 1'00 2'00 3'00 4'00 5'00 6'00 7 '00 8'00 min Figure 16 Separation of 14C DCA and 14C glyoxylate by strong anion exchange HPLC.

PAGE 63

48 Reducing Agent Dependence Study Incubation of the dialyzed cytosol with NADPH before the second d i alysis eliminated metabolism from NADH or NADPH. GSH was the only reducing agent to contribute to the metabolism ofDCA (Table 3). The small amount of metabolism from NADH (maximum 1.06 % product) was not enough to be clearly distinguished from the baseline in the HPLC chromatogram The metabolism from NADH and NADPH seen in single dialysis studies is now believed to be due to generation of GSH in the incubation mixture 33 Enzyme activity was stable in the presence of GSH, but declined rapidly if the dialyzed sample was heated to 37C. Effect ofDCA Treatment on Activity ofGSTz Treatment ofrats with DCA significantly lowered the metabolism ofDCA in dialyzed hepatic cytosol samples used in subsequent in vitro incubations also shown in Table 3

PAGE 64

49 Table 3 Reducing agent dependence study. Control Rats DCA Treated Rats1 nmoles glyoxylate formed/minute/mg protein GSH lmM 1.43.13 0.45 0 .10 NADPH 2mM 0.02 0 02 <0.01 NADH 2mM 0.14 0.17 <0.01 1 DCA treated rats received two daily doses ofDCA (50 mg/kg) and were sacrificed on the third day. Incubations (15 minutes) contained 0.2 mM DCA, 1 mM GSH and 1 mg cytosolic protein. *DCA treated animals had significantly lower activities than controls, p < 0.001

PAGE 65

50 Linear Range of Glyoxylate Production in Rat Hepatic Cytosol Time course studies in rats found glyoxylate production to be linear up to 30 minutes in both control and DCA treated cytosol (Figure 17). From this experiment 15 minutes was used as a standard time point for future kinetic incubations. Production of glyoxylate was found to be linear up to 1 5 mg protein (Figure 18). One milligram protein was used in most incubations, although protein concentration was sometimes decreased in KM and V max studies to bring the percent products below 20%.The incubation with a range of GSH concentrations found glyoxylate production to be linear up to 0 5 mM GSH. In subsequent determinations of the KM of DCA, 1 mM GSH was used to assure saturation of GSTz (Figure 19). Glyoxylate production was linear up to 0.1 mM DCA (Figure 20). In incubations to determine the KM of GSH in dialyzed rat cytosol, 0.2 mM DCA was used to assure a saturating concentration.

PAGE 66

51 Cl) 40 ro ">.. 30 [;;] >< 0 .o 20 c., 1 0 10 0 0 20 40 60 80 Time Min Figure 17 Time course studies in control (Wl) and DCA treated (Dl) dialyzed rat hepatic cytosol. The results shown are for individuals. Similar results were seen for the other cytosol samples.

PAGE 67

52 40 ., >, 30 >< 0 >, 20 G 0 IO _. 0 0 0 5 1.5 2 mg Cytosolic Protein Figure 18 Glyoxylate formed (nmol) from a range of protein concentrations The results shown are for individuals. Similar results were seen for the other cytosol samples.

PAGE 68

53 20 II) 15 0 >, 10 6 0 5 s 0 0 0.5 1 1.5 2 mM GSH Figure 19 Glyoxylate produced (nmol) from a range of GSH concentrations in rat hepatic cytosol. The results shown are from one rat. The study was repeated twice with similar results

PAGE 69

54 20 Q) ..... ro ...... 15 >-, >-, 10 -...... t) ...... 0 5 0 I I 0 0.2 0.4 0.6 mMDCA Figure 20 Glyoxylate formed (nmol) from various DCA concentrations in rat hepatic cytosol. The results shown are for individuals. Similar results were seen for the other cytosol samples

PAGE 70

55 Rat Apparent KM and V max of GSH The average apparent KM value for GSH (Figure 21) in control animals was 0.0688 mM, and 0.0528 mM in DCA treated animals, which were not significantly different (p>0 05). The average apparent V max value was 1.587 nmol/min/mg protein for control animals, and 0.674 nmoles/min/mg protein in the DCA treated animals, which were significantly different (p<0.05). The V values of the control rats were greater max than those of the DCA treated rats by an approximate factor of 2 (Table 4). Table 4 Apparent KM (mM) and V max (nmol/min/mg protein) of GSH in dialyzed rat cytosol. values are means SD. Vmax Control 0.0688 0.0115 1.587 0.159 DCA Treated 0.0528 0.032 0.674 0.181

PAGE 71

C: >,.'-Q) > 0 .... t, a. 0.05). The average apparent V values were 0.548 0.153 nmol/min/mg max protein for control rats, and 0.236 0.0288 nmol/min/mg protein for DCA treated rats (significantly different, p<0.05). The V values of the control rats were again greater max than the DCA treated rats values by an approximate factor of 2 (Table 5). However, the

PAGE 72

57 apparent V m a x values for both groups were lower than those determined with a higher DCA concentration in the study of the GSH apparent KM (Table 4) Table 5 Apparent KM (M) and Ymax (nmol/min/mg protein) ofDCA in dialyzed rat cytosol. Values are means SD. KM Vmax Control 7.48 1. 87 0.548 0.153 DCA Treated 7.45 1.909 0.236 0.0288

PAGE 73

C: >,'--Q) --~ u C. c( C) CJ E .,,,_ ;i:: C: CJ Q) E C.::::, "' 0 ;:: E C: -200 Figure 22 7.5 -100 58 0 100 200 1/mM DCA 300 400 Apparent KM 0.00755 mM 500 Apparent KM ofDCA in dialyzed rat cytosol. Linear Range of Glyoxylate Production in Human Hepatic Cytosol Time course studies in human liver found glyoxylate production to be linear up to 60 minutes. From this experiment, 30 minutes was used as a standard time point for future kinetic incubations (Figure 23). Production of glyoxylate was found to be linear up to 1.5 mg protein (Figure 24). One milligram protein was used in most incubations, although protein concentration was sometimes decreased in KM and V max studies to bring the percent products below 20%. Glyoxylate production was linear up to 0.025 mM DCA

PAGE 74

59 (Figure 25) In incubations to determine the KM of GSH in dialyzed human hepatic cytosol, 0.2 rnM DCA was used to assure a saturating concentration. The incubation with a range of GSH concentrations found glyoxylate production to be linear up to 0 .05 rnM GSH In subsequent determinations of the KM of DCA, 1 rnM GSH was used to assure saturation of GSTz (Figure 26) Human Hepatic Cytosol Apparent KM and V ~Results Cytosolic incubations of dialyzed human liver cytosol revealed the average apparent KM ofDCA to be 0.00487 rnM and the average apparent V to be 0.374 max nmol/min/mg protein (Figure 27). The average apparent KM of GSH was found to be 0.0076 rnM and the average apparent V m a x 0.262 nmol/min/mg protein. (Figure 28). The maximal velocities are higher in the control rats studied than in the human sample.

PAGE 75

60 35 ---.--------------------, 30 '>-. 25 :>< 20 c5 15 11~ 0------....--------,---------1 0 50 100 150 Time, min Figure 23 Time course study in human hepatic cytosol.

PAGE 76

61 25 Q) i ..... 20 c:,:S >. :,<: 15 0 >. C 10 0 s 5 0 I 0 0.5 1 1.5 2 2.5 Cytosolic Protein mg Figure 24 Glyoxylate formed in dialyzed human hepatic cytosol from a range of protein concentrations

PAGE 77

6 5 11) 4 1ii 0 Q3 0 ,.Q 2 C 1 0 0. 0 Figure 25 I I I 0.01 0 02 62 I 0.03 mMDCA 0.04 0 05 0.06 Glyoxylate formed in dialyzed human hepatic cytosol from various DCA concentrations.

PAGE 78

63 10 (1) 8 I >< 6 0 I I 6 4 I 0 2 II 0 I I I I 0 0 05 0.1 0.15 0.2 0.25 0.3 mMGSH Figure 26 Glyoxylate produced from a range of GSH concentrations in dialyzed human hepatic cytosol.

PAGE 79

50 -200 -100 0 Figure 27 64 100 200 300 400 500 600 1/mMDCA Apparent KM 0.00680 mM Lineweaver-Burk determination of the KM and Ymax ofDCA at 0.024 mM GSH in dialyzed human hepatic cytosol.

PAGE 80

s:: 0.4 ..... -0 025 0 000 0.025 F i gure 28 65 0.050 rnMGSH 0 075 0.100 0.125 Apparent KM 0.00642 rnM Hames plot determination of the KM of GSH in dialyzed human cytosol at 0.025 mM DCA.

PAGE 81

66 In Vitro Inhibition Study Results These studies sought to determine if in vitro pre-incubation with DCA would reduce the GSTz activity of cytosol in subsequent incubations. The first time course inhibition incubation with 0 2 mM DCA was repeated because glyoxylate levels were below the detection limits of the fluorescence assay The protein amount was increased and no inhibition ofDCA metabolism was observed. DCA treated cytosol had an a v erage specific activity of 0 323 0 0691 nmol/min/mg protein, whereas the control cytosol after the incubation with DCA had a specific activity of 0.261 0.0486 nmol/min/mg protein (p < 0.05) (Figure 29). The unexpected result of the inhibition experiment led to the incubation with higher concentrations of DCA. In this experiment the control cytosol incubations again had lower specific activity than those pre-treated with DCA. For the 0.5 mM DCA pre treatment the average specific activity was 0.665 0.176 nmol/min/mg protein, and 0.601 0.315 nmol/minute /mg protein for the control. (p > 0 05) The 0.75 mM DCA pre treatment had an average specific activity of 0 702 0.348 nmol/min/mg protein and 0.483 0 0740 nmol/min/mg protein for the control. (p < 0.05) The 1 mM DCA pre treatment had an average specific activity of 1.029 .245 nmol/min/mg protein, and 0.531 0.137 nmol/minute / mg protein for the control (p < 0 05)

PAGE 82

67 In the final experiment with three concentrations of DCA in the initial incubation, then 0.2 mM for the second incubation to ease comparison, again no in vitro inhibition of DCA was seen (Figure 30). The control cytosol had a specific activity of 0.398 .050 nmol/min/mg protein, compared to 0.472 .0685 nmol/min/mg protein for 0.5 mM DCA treatment (p>0.05), 0.480 .0623 nmol/min/mg protein for the 0.75 mM DCA treatment (p<0.05), and 0.596 0.0452 nmol/min/mg protein for the 0.1 mM DCA treatment (p<0.05). In the absence of DCA pre-incubation, GSTz specific activity in cytosol obtained from rat livers was approximately four-fold higher than that measured in cytosol from human livers. Pre-incubation with 0.5 mM DCA failed to significantly alter enzyme specific activity in human liver but decreased that of rat liver 56 percent (p<0 001). Thus, the effect of prior exposure to DCA on hepatic GSTz specific activity, and the subsequent biotransformation ofDCA to glyoxylate, appeared to be qualitatively different between these two species (Figure 31 ).

PAGE 83

68 Pre-Treatment 10. 0 -~ -NoDCA :s e -11:-0.2 mM DCA -M Q 7.5 eQ ee< QJ Q u r.= Q 5 0 "'O QJ Q CJ >, = "'O 2.5 c, e C. o.o~~-----~~..--~--r-~----,...---~-.--~---.-~----. 0 10 20 30 40 50 60 70 Time (min) Figure 29 Activity of dialyzed human hepatic cytosol after pre-incubation with 0.2 mM DCA

PAGE 84

0.75 30 0 Figure 30 69 45.0 Min 60.0 Treatments E::J0.5 mMDCA t //10.75 mMDCA ~lmMDCA -control Residual GSTz specific activity at 30, 45, and 60 min in dialyzed human cytosol pre treated with 0, 0.5, 0.75, or 1 mM DCA.

PAGE 85

70 1.5 Pre-treatment El H no DCA BIH DCA I IDRnoDCA ~RDCA 0.5 0 30 min Incubation Figure 31 Comparison of the effect ofDCA pre-treatment on GSTz specific activity between rat and human dialyzed hepatic cytosol, values are means (n=3) SD. H=human, R=rat.

PAGE 86

71 DCA Binding Study in Dialyzed Rat and Human Hepatic Cytosol Radiolabeled DCA was not found to bind hepatic cytosolic protein to any appreciable extent in either rat or human samples. Percent binding was found to be 4.45 % in the rat samples and 13.45 % in the human sample. This was not enough to account for the lack of a direct inhibitory effect of DCA seen in the pre-incubation dialysis studies with human hepatic cytosol samples. In Vitro GSTz Activity in DCA Dosed Animals The mean SD specific activity of GSTz in livers of rats not treated with DCA was 1.49 .14 nmol glyoxylate formed/min/mg protein. Oral administration ofDCA to rats inhibited hepatic GSTz specific activity in a dose dependent manner (Figure 32, Table 6). At the lowest doses (4 mg/kg and 12.5 mg/kg), five days ofDCA administration was required to significantly decrease enzyme activity. At doses of 50 mg/kg or greater, however, DCA significantly inhibited GSTz after only a single exposure A single 1 g/kg dose decreased glyoxylate formation by 93 percent.

PAGE 87

(") C/) 0 'O :::s (1) (") (") (1) ..... :::s =i .:t (") a~ ..... (") 0 =-:::s < en ..... '< 0 >-+i "'1 a ::r .g a c=; (") '$. 0 en 3 Pl en 0.. 0 en (1) 0.. : (IQ (1) 0 >-+i tj n > >Tj (JQ. i:: w N Control 0 Q GSTz specific activity (nmol glyoxylate formed/mm/mg protein) 0 .... (/'I 0 (/'I 4mg/kg 1 day r -t:l > 4mg/kg 5 days 12.5 mg/kgl day ................. ..................... ........................... ... .............................................................. .......... ................................................. ...... c:i. 12.5 mg/kg 5 days 1111111111111,,,,,,,,,, 50 mg/kg 1 day c:i. c:i. :; 50 mg/kg 5 days = ..... 0 = 200 mg/kg 1 day 200 mg/kg 5 days 1000 mg/kg 1 day 1000 mg/kg 5 days -..J N

PAGE 88

73 Table 6 GSTz Activity in DCA treated and control animals. Values are reported in nmol/min/mg protein means SD. Dose/kg =dose ofDCA. Dose/kg Average Specific Activity Control 1.49 0 144 4 mg 1 day 1.50 0 073 4 mg 5 days 1.18. 072 12.5 mg 1 day 1.17 0.172 12. 5 mg 5 days 0 72 0 174 50 mg 1 day 0.52 0.126 50 mg 5 days 0.28 0.174 200 mg 1 day 0.22 .043 200 mg 5 days 0 088 0.006 1000 mg 1 day 0.047 0.019 1000 mg 5 days 0.029 0.002

PAGE 89

CHAPTER FIVE RESULTS OF INHIBITION STUDIES WITH MALEYLACETONE Maleylacetone IC50 Studies Maleylacetone inhibited DCA conversion to glyoxylate in a dose dependent manner. When rat or human hepatic cytosol was incubated with 0 05-0.4 mM maleylacetone (a natural substrate for GSTz), DCA biotransformation to glyoxylate was inhibited. The apparent IC50 ofDCA metabolism by maleylacetone in the first study with human hepatic cytosol was between 0.1 and 0.2 mM (Figure 33). The mean SD IC5 0 value was 0.264 .039 mM maleylacetone for the rat liver cytosol (n=3) (Figure 34) and 0.125 0.029 mM maleylacetone (n=4) for human liver cytosol (Figure 35). Preincubation for 30 minutes resulted in a greater increase in maleylacetone inhibition than was seen without pre-incubation in human and rat hepatic cytosol (Figure 36). This suggests different enzymatic binding sites for maleylacetone and DCA, and non competitive inhibition. Ki of Maleylacetone The results of the Ki of maleylacetone against DCA metabolism were not as reproducible as those used to find the IC50 values. The competition between maleylacetone and DCA appeared to be mixed, or non-competitive which would suggest 74

PAGE 90

75 different active sites for the metabolism of the native maleylacetone and DCA (Figure 37). The average K i for maleylacetone with the different DCA concentrations studied was 8.4 M 0.5 s::= (1) (1) 0 0.4 ...... 0.. >.. bI) 8 0.3 bs= bI) ...... 8 o 0 2 s c.8 0.1 Apparent IC50 0.152 mM O.O-+-~~~.....-~~~---~~~...--~~~---~~---. 0.0 0.1 0.2 0.3 0.4 0.5 mM Maleylacetone Figure 33 Initial incubation of DCA with maleylacetone.

PAGE 91

A 0 20 0.05 -0 .10 -0 .25 -0 40 0 0 Fi g ure 34 76 0 1 0 2 0 3 mM Maleylacetone 0.4 Rl R2 ... R3 IC50 of maleylacetone in rat hepatic cytosol. Rl (rat one) R2 (rat two) R3 (rat three).

PAGE 92

77 0.00 ;>-. -0 25 +-' -~ -bl) -0.50 +-' < s -0.75 u c.= s -1.00 --u-Cl) 0 ~ -1.25 bl) -1. 50 0 ....:l -1.75 -2.00 0.0 0.1 0.2 0.3 0.4 0.5 mM Maleylacetone Figure 35 IC50 ofmaleylacetone in human hepatic cytosol. The four symbols represent four human liver samples.

PAGE 93

Figure 36 78 30 Minute 0.1 mM MA Pre-Incubation Comparison Rats 2 0 0.2 0.0 30 Minute 0.1 mM MA Pre-Incubation Comparison, Humans -Control c::::::J MA Not Pre-Incubated i""'"'i 30 min PI+ GSH c::J 30 min PI-GSH -Control EJ MA Not Pre-Incubated 1 "'"'" 130 min Pl -GSH c:::::J 30 min PI+ GSH Maleylacetone pre-incubation comparison. PI-GSH=pre-incubated without GSH, PI+ GSH = pre-incubated with GSH.

PAGE 94

79 50 Mixed Type Inhibition? \ -200 -100 0 100 200 300 1 /mMDCA Figure 37 Lineweaver Burk Ki determination ofmaleylacetone against DCA metabolism. Symbols represent different maleylacetone concentrations.

PAGE 95

80 Pre-Treatment of Dialyzed Rat and Human Hepatic Cytosol with Maleylacetone Pre-treatment with 0.4 mM maleylacetone led to inhibition ofDCA metabolism which persisted through dialysis in both rat and human hepatic cytosol (p<0.01, rat samples with GSH, p<0.05 rat samples without GSH, p<0.001 human samples with GSH, no significant difference, human samples without GSH). The presence of 1 mM GSH with the initial maleylacetone treatments decreased maleylacetone's inhibition of DCA metabolism in both rat and human samples. This was expected due to the in vitro binding of maleylacetone and GSH.8 In the final activity measurement the presence of GSH appeared to increase maleylacetone's inhibition ofDCA metabolism with a greater effect seen in the human samples, but the decreased level of significance seen in all samples which did not contain GSH may be due to their overall loss of activity (Figure 38).

PAGE 96

>, 1.00 ..... : ~ ..... 0 .7 5 (.) s 0.50 (.) .._,.,~--S~ Roberts, I certify that I have read this study and t acceptable standards of scholarly presentation an Professor of Veterinary Medicine y opinion it conforms to ly ade ate, in scope and quality, as a dissertation for the degree of Doctor of Philosop Professor of I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in sc and quality, as a dissertation for the degree of Doctor of Philosophy. oan Professor of Medicinal Chemistry This dissertation was submitted to the Graduate Faculty of the College of Pharmacy and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. May 2000 Dean, Graduate School