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INACTIVATION OF GLUTATHIONE S TRANSFERASE ZETA BY
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
This document is dedicated to my parents, and my husband, for their love and support
during these years.
I would like to thank my advisor Dr. Margaret James, without whose guidance and
encouragement this dissertation would not have been possible. I thank her for supporting
me, and for the several helpful discussions which greatly improved my understanding of
the subject. I would also like to extend my gratitude to my committee members, Dr. Peter
Stacpoole, Dr.Laszlo Prokai, and Dr.Nancy Denslow. Their direction and help were
essential to the success of this thesis. I would also like to thank Dr.George Henderson for
constantly advising me, and guiding me in this project. His expertise on the subject was
very helpful to me on several occasions.
I would also like to thank Dr.Xu Guo with whom I worked on several projects.
Laura Faux and Dr. Li-Quan Wang deserve special mention for patiently extending their
help numerous times.
I also thank fellow graduate students in our lab, James Sacco, Leah Stuchal and
Betty Nyagode, who have been very good friends all thorough these years. I hope these
friendships will continue for many years to come.
I will always be grateful to my parents, Suresh and Anita Dixit, for their
unwavering love and support. Their blessings and encouragement helped in my academic
career. Finally I thank my husband, Achint Srivastava for constantly encouraging me and
always being there for me.
TABLE OF CONTENTS
A C K N O W L E D G M E N T S ......... .................................................................................... iii
LIST OF TABLES ........ ........ ................................... ................. ............ vi
LIST OF FIGURES ......... ........................... .......... ....... vii
LIST OF ABBREV IA TION S ........... ...................................................... .............. ix
ABSTRACT ........ .............. ............. ...... ...................... xi
INTRODUCTION .................................. .. ... .... .......................
Pharm acokinetics of D C A .............................................................................. 2
M etabolism of DCA .................. ............................ ....... ................ .3
G lutathione S Transferase zeta............................................ ........................... 4
Possible Mechanisms of Dechlorination....................................... ..............8
Inactivation of G ST z ................................................................. ........................ 9
Specific Aim s............... ................... ...................... ... .... 17
M A TERIA L S A N D M ETH O D S............................................................ .....................19
Chem icals........... ........................................................................ 19
Animal Dosing Protocol for NTBC Studies..................................................20
Animal Dosing Protocol for GSTz Recovery and Low Dose Studies ...................21
Preparation of Cytosol and Microsomes from Livers ...........................................23
In-vitro GSTz Activity in Rat Liver Cytosol ................................. ...............23
H P L C A n aly sis ................................................. ................. 24
W western Blots............................................................. ...24
G C -M S A analysis of U rine.......................................................................... ....... 25
R ecom binant hG STz- c ......... .................................. .........................................26
Purification of Rabbit Anti-Human GSTz c-c................................................26
Immunoprecipitation of GSTz from Rat Liver Cytosol.......................................27
D CA M modified G STz-lc ......... .................... ......... .................................27
HPLC Analysis of Modified GSTz ..............................................28
Enzyme Digests of hGSTz-lc ............................................. ................... 29
MALDI-TOF Analysis of Intact GSTz ..................... ....................................30
M ALD I-TOF A analysis of D igests....................................... ......................... 30
LC/M S Analysis of Intact GSTz Ic-lc .............. ........... ......... ............... .31
L C -M S/M S of D igests ........................................... .................. ............... 31
R E SU L T S O F N TB C STU D IE S ............................................................ .....................33
Prelim inary Studies ............... ..... ...... ............. ... ...............33
Effect of NTBC on GSTz Activity and Expression ...........................................34
RESULTS OF GSTz RECOVERY AND LOW DOSE STUDIES ..................................38
Inactivation and Recovery of Hepatic GSTz ......... ............................................38
Drinking Water Consumption, Liver and Body Weight .......................................39
Effect of Low Doses of DCA on Hepatic GSTz .................................................41
Urinary Excretion of DCA and Maleylacetone.............. .... .................43
ADDUCTS OF HUMAN GSTz WITH GSH AND DCA...............................................45
HPLC Separation and Identification of Adduct ......... ........................ .................. 45
Mass Spectral Analysis of Intact Modified and Unmodified GSTz........................46
Tryptic and Lys-c Digests of Unmodified GSTz Ic-lc .......................................48
Tryptic Digest of GSH Modified GSTz Ic-lc. .......................................................53
Tryptic Digests of DCA Modified GSTz Ic-lc ....... .......................................54
Binding of C14 Labeled D CA to G STzlc-lc................................. ...... ...............63
Purification of Rabbit Anti-hGSTz Ic-lc Antibody. .............................................65
D IS C U S S IO N ................................ .....................................................6 7
L IST O F R EFER EN C E S ......... .................................... ........................... ............... 79
BIO GRA PH ICAL SK ETCH .................................................. ............................... 86
LIST OF TABLES
1-1 Concentration of haloacetic acids formed by chlorination
of drinking water ........... ................. ......... .......... ............
1-2 Specific activities of som e substrates of GSTz. ........................................ .................6
1-3 Key residues involved in G STz activity ..................... ....................... ............ ... 15
2-1 Dose and duration of NTBC and DCA treatment......................................................21
5-1 Theoretical masses of y and b ions of SSCSWR using Protein Prospector. ...............49
5-2 Peptides identified by Lys-c D igest....................................... .......................... 51
5-3 Peptides identified by tryptic digest ........................................ ........................ 51
5-4 Theoretical and observed ions of SSCSW R. ........................................ ............... 52
5-5 Theoretical and observed mass of y and b fragment ions in GSH modified
SSCSW R. ......................................................................... ........ ...... 56
5-6 y and b ions observed in the MS/MS of GSH and glyoxylate
m modified SSC SW R .................. ...................................... .. ........ .... 56
5-7 y and b ions in glyoxylate modified SSCSW R.............. ...........................................58
LIST OF FIGURES
1-1 Metabolism of Dichloroacetate (James et.al. 1998) ............................................. 5
1-3 In-vitro inhibition of GSTz in human liver cytosol by maleylacetone.....................10
1-4 Tyrosine catabolic pathw ay ......... ................. .........................................................11
1-5 Dose dependent inhibition of GSTz specific activity by DCA. ..................................13
1-6 W western blot of G STz protein from D CA ........................................ ..................... 14
3-1 R results from first prelim inary study ........................................ ........................ 34
3-4 Relative quantification of GSTz .................. ................. ................. 37
3-5 Representative western blot of rat liver cytosol. ................................. ............... 37
4-1 Inactivation and recovery of GSTz activity .................................... ............... 39
4-2 Loss and recovery of GSTz protein after treatment. ................................................40
4-3 Representative Western blot of control and DCA treated ...................... ...............41
4-4 Specific activity of GSTz in rats treated with
2.5 and 250 g/kg/d of D CA ........................................................ ............... 42
4-5 Expression of GSTz in rats treated with 2.5 and 250 ag/kg/d of DCA......................42
4-6 Representative western showing GSTz expression ........................... .....................43
4-7 Recovery of GSTz activity in rats treated with low doses of DCA.............................44
4-8 Restoration of GSTz protein in rats treated with low doses of DCA.........................44
5-1 Separation of DCA modified GSTz from unmodified GSTz...............................46
5-2 MALDI of undigested GSTz Ic-lc in
unm odified and modified form s. ........................................ .......................... 47
5-3 Fragmention pattern of peptides to give N and C terminal ions ..............................49
5-4 LC-MS/MS full scan of unmodified hGSTzlc-c ............................................... 50
5-5 MALDI of unmodified GSTz showing tryptic peptides ................ ......... ..........50
5-6 M S/M S of active site peptide showing daughter ions........................... ............... 53
5-7 M S/M S of glutathione modified active site peptide..............................................55
5-8 MS/MS of GSH and glyoxylate modified active site peptide ...................................57
5-9 M S/M S of glyoxylate modified active site peptide ...............................................59
5-10 MS/MS of glyoxylate modified DGGQQFFSK.................... ...............60
5-11 M S/M S of glyoxylate modified DFQALNPMK .................................................. 61
5-12 MS/MS of doubly charged glyoxylate modified DFQALNPMK. ............................62
5-13 Theoretical y and b ions of MISDLIAGGIQPLQNLSVLK
generated by Protein Prospector........................................ ........................... 63
5-14 Binding of C14 D CA to G STzlc-lc ........................................ ........ ............... 63
5-16 Western blot of rat liver cytosol using purified
rabbit-anti hum an G STz. ............................................... ............................... 66
5-17 Western blot of recombinant GSTz Ic-lc using purified rabbit anti-human GSTz as
prim ary antibody. ....................... ......... .. .. ..... .. .............. 66
6-1 Proposed reaction between Serine and Glyoxylate. ............................................. 77
6-2 Reaction of glyoxylate and aspartic acid to form anhydride................................77
LIST OF ABBREVIATIONS.
1. DCA: Dichloroacetate.
2. DTT: Dithiothreotal.
3. 6 ALA: delta aminolevulinate
4. CFA: Chlorofluoroacetate.
5. ECL: Enhanced chemiluminescence.
6. EDTA: Ethylene diamino tetraacetic acid.
7. EPA: Environment protection agency.
8. ESI: Electrospray ionization.
9. FA: Fumarylacetone.
10. FAA: Fumarylacetoacetate.
11. GC-MS: Gas chromatography mass spectrometry.
12. GSH: Glutathione.
13. GST; Glutathione S transferase.
14. GSTz: Glutathione S transferase zeta.
15. HEPES: 4-(2-hydroxyethyl)- 1-piperazineethanesulfonic acid
16. HPLC: High performance liquid chromatogrpahy.
17. LC-MS: Liquid chromatography coupled with mass spectrometry.
18. MAAI: Maleylacetoacetate isomerase.
19. MA: Maleylacetone.
20. MAA: Maleylacetoacetate.
21. MALDI: Matrix assisted laser desorption ionization.
22. MALDI-TOF: Matrix assisted laser desorption ionization-Time of flight.
23. NTBC: 2-(2-Nitro-4-trifluoromethylbenzoyyl)-cyclohexane-1, 3-dione.
24. SA: Succinyl Acetone
25. SDS-PAGE: Sodium dodecyl sulphate- polyacrylamide gel electrophoresis.
26. T-TBS: Tween tris buffered saline.
27. TOF: Time of flight
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
INACTIVATION OF GLUTATHIONE S TRANSFERASE ZETA BY
Chair: Margaret O. James
Major Department: Medicinal Chemistry.
The environmental contaminant dichloroacetate (DCA) is considered hazardous
by the US environment protection agency (EPA) but is also an important drug in the
clinical management of lactic acidosis. DCA has been shown to be carcinogenic in
rats and causes peripheral neuropathy in humans. It is metabolized by glutathione S
transferase zeta (GSTz) and inhibits its own metabolism by inactivating GSTz. This
aspect of DCA metabolism is also important because GSTz also metabolizes
endogenous substrates, namely maleylacetoacetate (MAA) and maleylacetone (MA),
which are intermediates in tyrosine catabolism.
The objectives of this research were to study the time course of inactivation and
recovery of GSTz following exposure to DCA at both clinical and environmentally
relevant doses and to identify possibly adducts of DCA with GSTz. Research
presented here also examined the in-vivo role of MA in inactivating GSTz.
Experiments conducted showed that DCA completely inactivated GSTz after one
week of exposure and that enzyme recovery was not as rapid as inactivation. It took
more than two weeks for the enzyme activity to return to control levels and enzyme
expression remained below control levels even after eight weeks of withdrawing
DCA treatment. These studies showed that enzyme recovery was slow and that the
protein needed to be re-synthesized for activity to be restored. Another important
finding was that environmental levels of DCA similar to those present in drinking
water inactivated GSTz. Exposure of rats to DCA in drinking water at levels of 2.5
and 250 [tg/kg/day significantly inhibited GSTz activity and decreased GSTz
expression. Longer duration of treatment had a more prominent effect suggesting a
cumulative effect. Adduct studies with recombinant hGSTz Ic-lc showed the
presence of adducts of GSTz with glutathione (GSH), and glyoxalate which is the
primary metabolite of DCA. Another important finding of this study was that the
reactive endogenous substrate for GSTz, maleylacetone, did not inhibit GSTz activity
or expression in-vivo. Previous studies with MA have shown that it inhibits GSTz in-
vitro but this is the first study, to our knowledge, which showed that it was not an
inhibitor of GSTz in-vivo.
Dichloroacetate (DCA) is a haloacetic acid formed in drinking water as a by-
product of chlorination. Its concentration in chlorinated drinking water has been
reported to be as high as 100tg/L in some samples (Gonzalez-Leon et.al. 1997). DCA
is also found in ground water in polluted sites as it is a metabolite of industrial
chemicals such as trichloroethylene. Table 1-1 shows the concentration of some
haloacetic acids in ground water. DCA is a biotransformation product of some
pharmaceuticals such as chloral hydrate (Henderson et.al. 1997). DCA derived from
trichloroethylene was found in seminal fluid of human subjects (Forkert et.al. 2002).
As well as being an environmental pollutant, it is also an investigational drug used in
the treatment of several metabolic disorders. DCA is the only available treatment for
congenital lactic acidosis, and lactic acidosis associated with conditions such as
diabetes and malaria (Stacpoole et.al. 1989). Daily human exposure ranges from
4[tg/kg in drinking water to about 50 mg/kg for therapeutic purpose. Reversible
peripheral neuropathy has been observed after prolonged administration of DCA to
humans. It also exhibits anxiolytic or sedative effects in adults given repeated doses
of DCA (Stacpoole et.al. 1998b).
Studies in rodents have shown that DCA is a non-genotoxic carcinogen, (Chang
et.al. 1992) and a peroxisome proliferator, (DeAngelo et.al. 1989). It also causes Ras-
oncogene activation in B6C3F1 mice (Ferreira-Gonzalez et.al. 1995).
DCA is metabolized primarily in the liver by glutathione S transferase zeta
(GSTz) to glyoxylate (James et.al. 1997,Tong et.al. 1998b). DCA inhibits its own
metabolism after repeated administration due to inactivation of its metabolizing
enzyme (James et.al. 1997, Cornett et.al. 1999). The mechanism of this inactivation is
not clear, and understanding this mechanism may provide further information about
Table 1-1 Concentration of haloacetic acids formed by chlorination of drinking water
(Adapted from Boorman 1999)
Haloacetic acids Concentration, [tg/lt
Dichloroacetic acid 15 74
Trichloroacetic acid 11 85
Bromochloroacetic acid 3.2 49
Monochloroacetic acid 1.3 5.8
Dibromoacetic acid <0.5 7.4
Monobromoacetic acid <0.5 1.7
Pharmacokinetics of DCA
At therapeutic doses, DCA is rapidly and almost completely absorbed orally
and peak plasma levels are achieved within 15-30 minutes of administration
(Stacpoole et.al. 1998a). A very interesting feature of DCA kinetics is that repeated
dosing of rats with clinical doses of DCA prolongs its half-life (James et.al. 1998). A
similar effect was also demonstrated in human subjects (Curry et.al. 1991). An
increase in plasma concentration of the drug and area under plasma concentration-
time curve was observed after repeated dose of DCA. Studies also showed that the
total body clearance of DCA was reduced in rats pretreated with DCA (Gonzalez-
Leon et.al. 1997). This increase in plasma tl/2 and decrease in clearance of DCA was
due to impaired metabolism. Pharmacokinetics of DCA in large animals was found to
be different than that in young animals. After two doses of DCA, 50mg/kg, peak
plasma concentration in large rats was five fold higher than young animals, and the
elimination half-life of DCA was slowed from 5.4 to 9.7 hrs (James et.al. 1998).
Schultz et.al. (2002) showed that DCA treatment in drinking water had no effect on
the elimination of the drug in 60-week old mice when compared to results obtained
from aged matched controls. This report suggests DCA treatment caused no
significant difference in the plasma tl/2 of the drug in aged animals whereas, in
younger animals (10 weeks old) the tl/2 was significantly longer in animals treated
with DCA. Another important finding of this study was that the capacity to excrete
DCA in 60-week old control mice was 25 % of that of the 10-week old mice (Schultz
Metabolism of DCA
The glutathione conjugation of DCA is thought to proceed via a nucleophilic
substitution reaction with the thiol group of glutathione acting as a nucleophile and
the chlorine of the haloacid being the leaving group. The known metabolites of DCA
(figurel-1) are glyoxylate, glycolate, oxalate, and carbon dioxide (C02) Fischer
344 rats (180-240 g) given single oral doses of 28.2 or 282 mg of [14C] DCA/kg
excreted 25-35% of the dose as CO2 and 12-35% in urine; 20-36% of the the
radioactivity from DCA was recovered in rat tissues (Lin et.al. 1993). The rats that
received 282 mg/kg excreted less of the 14C as CO2 and more in urine, and the
percentage of unmetabolizedDCA in urine ranged from 0.6% of the dose for the
28.2 mg/kg group to 20% of the dose for the 282 mg/kg group (Lin et.al. 1993).
James et.al (1998) showed that 20-44% of the radioactivity from DCA (50mg/kg) was
excreted as CO2 in 24 hours.
Glyoxylate can be routed through several different pathways. It can be
converted to oxalate or glycolate by lactate dehydrogenase. It can undergo
transamination by aminotransferases to form glycine which can enter several
pathways such as conjugation to exogenous carboxylic acids such as benzoic acid.
Alternatively glyoxylate can be oxidised to CO2 by ca-ketoglutarate-glyoxylate
carboligase. Radioactivity from C14 labeled DCA was found in glycine and serine
residues of plasma proteins. Glycine conjugates like benzoyl glycine (hippuric acid)
and phenylacetyl glycine were identified in the urine of DCA treated rats (James et.al.
Glutathione S Transferase zeta
GSTz belongs to a family of glutathione (GSH) conjugating phase 2
metabolism enzymes responsible for the detoxification of several xenobiotics. All
GSTs exist as either homodimers or heterodimers. These enzymes metabolize
electrophilic substrates by catalyzing the attack of sulfhydryl group of glutathione on
the electrophilic center of the substrate. The sulfhydryl group of GSH is typically
activated by either a serine or tyrosine residue in the GSH binding site of GSTs.
This residue binds to GSH by intermolecular hydrogen bonds and lowers its
pKa from 9.3 to 6.5-7.4 (Nieslanik et.al. 1998). GSTz isomerizes maleylacetone
(MA) to fumarylacetone (FA), through the nucleophilic attachment of GSH to the P-
carbon of the cis double bond in MA.
Cl -C -
GSII tz II II 0
C -OH GI Lactate II II
H C -C OH Dehydrogenase HO -C -C -OH
H -N -C -C
HO -C -C -OH
0 H O
-C -N -C -CH-OH
Fig 1-1 Metabolism of Dichloroacetate (James et.al. 1998)
Loss of GSH and reketonization results in the formation of the cis product. In
alpha, mu, and pi classes tyrosine activates the GSH whereas in zeta and theta classes
GSH is activated by serine. GSTz is a recently recognized addition to the GST family
and shares very little similarity to other classes of GST's. It has activity against t-
butyl and cumene hydroperoxides, but does not show activity against 1-chloro-2,4-
dinitrobenzene, a model substrate used to measure GST activity (Harada et.al. 1987)
Common substrates of GSTzl-1 include alpha-beta unsaturated carboxylic
compounds such as MA, and alpha halo acids such as DCA and chlorofluoroacetate
(CFA) (Table 1-2).
Table 1-2 Specific activities of some substrates of GSTz. Data are presented as means
+ SD (n = 3). ND, not detectable. (Adapted from Tong et.al. 1998a)
nmoles/min/mg purified protein
bromochloroacetic acid 1411 + 65
bromofluoroacetic acid 5028 + 150
chlorofluoroacetic acid 3883 + 43
dibromoacetic acid 155 + 4
dichloroacetic acid 1038 + 20
difluoroacetic acid ND
fluoroacetic acid 72 + 16
GSTz is a cytosolic enzyme found in a number of species ranging from
Caenorhabditis elegans to rats and humans. GSTz, is identical to maleylacetoacetate
isomerase (MAAI), an enzyme in the tyrosine catabolic pathway (Knox et.al. 1955).
Its importance in this pathway is demonstrated by the fact that MAAI knock out mice
that are given high protein diet have low survival rates (Ferandez-Canon et.al.
2002). These mice also showed renal and hepatic damage suggesting that absence of
this enzyme in a natural habitat, where a diet with high protein content is likely, may
lead to deleterious effects. Patients with tyrosinemia or other diseases of tyrosine
catabolic pathway exhibit liver damage. This has been attributed to the accumulation
of reactive tyrosine metabolites such as MA, fumarylacetone (FA) and
MAAI is a dimer with a subunit molecular weight of 25 kDa. The active site in
GSTz is more polar and smaller than that in other GSTs. The active site has an
electropositive region surrounding it, which attracts negatively charged compounds
like maleylacetoacetate (MAA) and DCA. The structure of the enzyme was recently
elucidated by Polekhina and co-workers. The crystal structure shows the presence of
a sulfate ion in the active site (Polekhina et.al. 2001). GSH and the sulfate ion are
located in a very deep crevice between the C-terminal and the N-terminal domain.
The crystal structure also suggests that a carboxylate group in the substrate is
essential. DCA is thus a good substrate and orients itself in the active site such that
the carboxylate moiety forms a salt bridge with Arg 175 (Polekhina et.al. 2001). This
salt bridge optimally orients DCA for GSH attack on the alpha carbon and loss of one
of the chlorine atoms. However, beta haloacids are not substrates since these
molecules cannot orient properly in the binding site for attack by GSH.
Immunohistochemical analysis of tissues from rat showed that GSTz was expressed
in hepatocytes and epithelial lining of gastrointestinal tract and other tissues (Lantum
et.al. 2002a). Northern blot of human RNA from different tissues showed that GSTz
mRNA was present in liver, heart, brain, skeletal muscle and kidneys (Fernandez-
Canon et.al 1999).
Four polymorphic variants of human GSTzl-1 have been identified and are
designated as la-la, lb-lb, Ic-lc, and Id-id (Blackburn et.al. 2001). The specific
activity of each of these variants is different for each substrate. GSTzla-la has the
highest activity towards DCA. It metabolizes DCA 3.6 times faster than other three
polymorphs, but has the lowest Vmax for isomerisation of MA to fumarylacetone
(FA) (Blackburn et.al. 2001). There was a 6-fold difference in the isomerase activity
of the four isoforms with Ic having the highest isomerase activity. The rate (Vmax)
of formation of FA was in the order: lb-lb Ic-lc > la-la -Id-ld (Lantum et.al.
2002b). Blackburn et.al (2001) reported that all the polymorphs had similar activity
for chlorofluoroacetate (CFA), but Lantum et al (2002) reported that the Vmax of the
variants with CFA as substrate was different. GSTla-la had the highest Vmax and
Km for CFA followed by lb-lb, Ic-lc and Id-id.
Possible Mechanisms of Dechlorination
Several theories exist for probable mechanisms of dechlorination of DCA. The
proposed pathway (Anderson et.al. 2002) suggests that the first step involves
conjugation with glutathione and loss of one chlorine to form a S-(a-
chlorocarboxymethyl glutathione), Fig 1-2, (1). This moiety may then lose the second
chlorine to give a zwitterionic intermediate (2), or it could react with the GSTz itself
to form an inactive adduct. This glutathione intermediate (1) is reactive and may
undergo an SN2 type reaction in the presence of water to form glyoxylate. Another
pathway suggests that the zwitterionic intermediate (2) itself could also react with the
enzyme to form an inactive adduct similar to that suggested in the alternate pathway.
The zwitterionic intermediate could also react with water in an SN1 type reaction to
form the metabolite glyoxylate.
A study with deuterated DCA (Wempe et.al. 1999), showed that the deuterium
label is retained in the glyoxylate formed from DCA. This study proposed a
mechanism in which the zwiterrionic intermediate (2) is formed, which then reacts
with water, loses the glutathione moiety to form glyoxylate.
CI\ /C1 GSH/GSTZ-I ci /SG -C -
/C\ \IH Coo- G_ _
H C -HC1 H /H OO\- H COO
CO- H CO-(2)
,HC1 GSTZ-1 SG
SN2 pathl a\\ /C SN
Fig 1-2 Proposed mechanism of dechlorination and inactivation
(Adapted from Anderson et.al. 2002).
Inactivation of GSTz
The mechanism of GSTz inactivation by DCA is not entirely clear. Two
hypotheses have been postulated for this inactivation. One is that DCA directly
inactivates the enzyme by forming inactive adducts with it. The second hypothesis
suggests that the enzyme is indirectly inactivated by its physiological substrates
maleylacetoacetate or maleyl acetone which may get accumulated due to chronic
DCA treatment. Since GSTz is identical to Maleylacetoacetate isomerase (MAAI), an
enzyme of tyrosine catabolic pathway, DCA also perturbs tyrosine catabolism (Fig 1-
3) by inhibiting this enzyme. MAAI isomerises MAA to fumarylacetoacetate (FAA)
and maleylacetone (MA) to fumarylacetone (FA). Inhibition of this enzyme will lead
to an accumulation of these physiological substrates, which are known alkylating
agents (Seltzer, 1973). Cytotoxic effects have been demonstrated for FAA, which
stimulates apoptosis in mouse hepatocytes and human HepG2 cells (Jorquera et.al.
1999). Accumulation of tyrosine metabolites could also be associated with toxic side-
effects of DCA. Inhibition of MAAI may cause an accumulation of succinyl acetone
(SA) which is an inhibitor of heme biosynthesis. A homozygous knock out mouse for
MAAI showed elevated levels of SA in the urine (Lim et.al. 2004). The concentration
of SA in the urine of these mice was 1.6 [tmol/L and those that were treated with
phenylalanine had 4.5 [tmol/L. SA inhibits the formation of porphobilinogen from 6
aminolevulinate (6 ALA), a key step in heme catabolism. This could lead to
accumulation of 6 ALA which is a neurotoxin.
Previous in-vitro studies with maleyl acetone have shown that when MA was
incubated with human hepatic cytosol (figure 1-4) it inhibited the amount of
glyoxylate formed from DCA, in a dose dependent manner (Cornett et.al. 1999).
Average IC50 0.108 mM
0.00 I I I I
-1.25 -1.00 -0.75 -0.50 -0.25
Log mM MA
Fig 1-3 In-vitro inhibition of GSTz in human liver cytosol by maleylacetone
HO t OOH
O 0 OH
HO OH HO- V -v O-
ntisate / Alcaptor
cy leads to
also known as C
;STz .' 002
Fumarylacetone* -OOC O COO-
Deficiency leads to Hereditary fumarylacetoacetate
TyrosinemiaType I and > hydrolase(FAH)
-OOCy OA 00
kylating Agents \ -_coo-
Fig 1-4 Tyrosine catabolic pathway
It was further shown by Ammini et.al. (2003) that activity of the enzyme was
only partially restored after extensive dialysis of the protein suggesting reversible and
Using chlorofluoroacetate (CFA) as substrate, Lantum et.al. showed that MA
and FA are mixed function inhibitors of GSTzl-1 activity. They further hypothesized
that MA and FA are non- mechanism based inactivators of GSTz polymorphs. Mass
spectral analysis of GSTzl-1 variants that had been previously incubated with MA
and FA showed that MA and FA alkylated the Cys 16 and Cys 205 residues of the
enzyme (Lantum et.al. 2002c). A similar experiment with succinylacetone (SA)
however, showed that SA did not inhibit the enzyme. Thus an alpha beta unsaturated
carbonyl in the substrate appeared to be the essential functionality for inhibition.
Various haloacetates were examined for possible inactivation of GSTz.
Administration of chlorofluoroacetic acid, bromofluoroacetic acid, 2- chloropropionic
acid and difluoroacetic acid had no effect on GSTz activity.
To study the inhibition of GSTz by MAA and MA in-vivo we employed an
inhibitor of 4 hydroxyphenyl pyruvate dioxygenase.
0 0 NO2
This compound 2-(2-Nitro-4-trifluoromethylbenzoyl)-cyclohexane-1, 3-dione
(NTBC) prevents the formation and accumulation of tyrosine metabolites (Lock et.al.
1998). Thus pre-administering this compound to rats helped us determine the in-vivo
role of MA and MAA in inactivation of GSTz. NTBC has been used in the treatment
of hereditary tyrosinemia type I (HTT1). HTT1 is a severe inherited disease caused
due to a deficiency of fumarylacetoacetate hydrolase. This deficiency leads to an
accumulation of toxic metabolites, MAA, FAA, and SA. These compounds are
thought to be the main cause of liver and kidney damage in HTT1 patients. Since
NTBC inhibits 4-hydroxyphenyl pyruvate dioxygenase, it halts tyrosine catabolism in
its second step and prevents formation or accumulation of the toxic metabolites
thought to be responsible for liver disease.
DCA inhibited the activity and expression of its metabolizing enzyme and thus
impaired its own metabolism.
o0.0 I I I I r- I r--
Fig 1-5. Dose dependent inhibition of GSTz specific activity by DCA. ) ,
permission from Coett etal. 1999).
'I 'I E E E E
Dose and duration of DCAtreatment
Fig 1-5. Dose dependent inhibition of GSTz specific activity by DCA. *, **, ***
indicate that means are significantly different from the control (Reproduced with
permission from Cornett et.al. 1999).
The loss in the specific activity of the enzyme was proportional to the dose and
duration of treatment (figure 1-5). It was further shown that the amount of
immunoreactive GSTz protein (Fig 1-6) in rat liver cytosol was reduced after DCA
treatment. Cornett et.al. also studied the in-vitro inactivation of GSTz in rat and liver
cytosol by DCA. DCA inactivated rat cytosolic GSTz but did not show the same
effect on human cytosol. Later studies by Tzeng et.al. in 2000 showed that different
polymorphic variants of the enzyme have different inactivation half lives. The longest
half-life was exhibited by la-la (23+lmins) and the other variants had similar half
lives (9-10 mins).
Northern blots showed that the steady state levels of mRNA for the protein remained
unaltered after a five-day treatment of DCA. Since message was not altered, it
appears that DCA does not interfere with the transcription of GSTz. Above data
indicate that GSTz was inhibited during its reaction with DCA and that adducts
formed with DCA may lead to loss of immunoreactive protein.
Control 4mg/kg 12.5 mg/kg 50 mg/kg 200 mg/kg
1D 5D 1D 5D 1D 5D ID 5D
Fig 1-6 Western blot of GSTz protein from DCA treated rats showing a dose
dependent decrease in amount of immunoreactive protein. (Reproduced with
permission from Ammini et.al. 2003)
Recent mass spectral analysis of GSTz incubated with DCA in the presence of
GSH (Anderson et.al. 2002), suggested that DCA,or a reaction intermediate reacts
with a single nucleophilic site or residue on GSTz and thus inactivated the enzyme. It
was also proposed in this study that the nucleophilic site on the enzyme, which is
covalently modified, is a cysteine-16 residue present in the catalytic site, and that this
residue may form a disulphide bond with the thiol moiety in the reaction intermediate
(Fig 1-2) However, recombinant GSTz with a cys-16 deletion mutation was found to
retain both, isomerase, and glutathione conjugating properties. This observation
indicates that cys-16 residue may be present in the active site but it is not directly
involved in catalyzing GSH conjugation. It was further shown (Lantum et.al. 2002c)
that this cys-16 mutant showed some resistance to inactivation by MA. But this
mutant was not completely immune to inactivation suggesting that there is another
residue on the enzyme which may be involved in inactivation.
Table 1-3 Key residues involved in GSTz activity (Adapted from Board et.al. 2003)
hGSTzlc-lc 0.91+ 0.31
S14A 0.007 0.001
S15A 0.025 0.0004
R175A 0.216 0.003
R175K 0.68 + 0.04
Other residues are also implicated in inactivating the enzyme. Active site
series (position 14 and 15), arginine at 175 and also a cysteine at 205 may be
involved in the reaction with DCA. These residues have reactive functional groups
which may form adducts with DCA or a metabolite.
Table 1-3 stresses the importance of active site series for GSTz activity.
Mutating these residues dramatically decreased the activity of the enzyme. Replacing
the basic arginine with alanine caused a significant decrease in activity. This decrease
was not as profound when arginine is replaced with another basic amino acid, lysine.
Anderson et.al. (2004) studied the reactivity of glyoxylate with nucleophilic
amino acids. They found that when glyoxylate derived from the reaction of DCA with
GSTz and GSH was allowed to react with nucleophilic residues, it formed adducts
with these amino acids. Adducts containing one or two molecules of glyoxylate
covalently bound to reactive groups on the amino acids were observed. Glyoxylate
also reacted with N terminal amino group of the peptide to form an imine. This
adduct had a mass shift of 74 Da from the unmodified peptide. Another peptide
containing arginine, tryptophan, and cysteine formed an adduct with 2 molecules of
glyoxylate. It was found that cysteine was not involved in the reaction. The exact
chemical nature of modification could not be determined due to inadequate mass
spectral information. These observations suggested that glyoxylate is a reactive
metabolite which may react with amino acids in proteins. Its ability to react with
GSTz was not reported.
Inhibition of GSTz by DCA is well studied but the time required for the
enzyme to recover its activity after sub-chronic treatment, once the drug is
withdrawn, is not known. Since DCA treatment destroys the protein, it is clear that re-
synthesis of the enzyme is necessary to regain activity. In an experiment by Tzeng
et.al. (2000) it was observed that the activity of recombinant hGSTz la-la that was
inactivated by DCA could not be restored by an overnight dialysis of the inactivated
protein. This again indicates that synthesis of protein, not removal of the drug alone,
is necessary to recover activity. But the exact time course for its recovery hasn't been
completely studied at both environmentally, as well as clinically relevant doses.
1. To study the role of maleylacetone in destroying GSTz.
Inhibition of GSTz by DCA may lead to accumulation of the endogenous
substrates of the enzyme. These substrates, which are reactive, can alkylate the
enzyme, thus destroying the protein. The effect of such accumulation on GSTz
activity and expression needs to be elucidated. NTBC, an inhibitor of MA and
MAA formation is used to study the detrimental effects of these substrates on
GSTz. This compound when co-administered could prevent the formation and
subsequent accumulation of MA and MAA. The effect of DCA on GSTz can be
then studied in the absence of any interference from these substrates. This
approach will provide an insight to the in-vivo effects of MAAI's endogenous
substrates and their role in destroying the protein.
2. To study the time course of loss and post-treatment recovery of GSTz
following DCA administration.
Previous studies have proven that DCA administration results in the loss of
activity and expression of GSTz. It is also important to determine the time
required for the enzyme to regain its pre-treatment activity and expression after
cessation of DCA treatment. This information could prove useful in the
treatment and care of patients who are chronically administered the drug. In this
work we have determined via in-vivo experiments, the time required for the
immunoreactive protein to return to pre-treatment levels and the enzyme to
return to pre-treatment activity.
3. To test the hypothesis that environmental levels of DCA inactivate its
metabolizing enzyme, GSTz.
Dichloroacetate is considered an environmental contaminant by the EPA and is
a by-product of chlorination and metabolite of industrial solvents. Since DCA is
present in drinking water, it is a ubiquitous environmental pollutant affecting a
large populace. Human exposure to DCA occurs chronically at low tg/kg
levels. All studies done so far have been with clinical doses of DCA which are
several times higher (mg/kg) than the environmental dose. It was important to
determine the effect DCA has on GSTz activity at a dose to which majority of
people are exposed.
4. To identify and characterize, by mass spectrometry, the probable adduct(s)
of DCA and its metabolites with GSTz.
We hypothesize that dichloroacetate inhibits the enzyme by reacting with the
protein and thus destroys it. The mechanism of inhibition was studied by
identifying adducts of the protein with DCA or reaction intermediates. Amino
acid residues involved in the reaction with DCA and GSH were identified by
mass spectroscopic analysis of the enzyme after its reaction with DCA in the
presence of GSH. This experiment helped establish a method to prepare and
separate DCA modified GSTz and analyze the same by LC-MS and matrix
assisted laser desorption ionisation (MALDI).
MATERIALS AND METHODS
2-(2-Nitro-4-trifluoromethylbenzoyyl)-cyclohexane-1, 3-dione (NTBC) was
obtained from Apoteket laboratories, Sweden. C14 radiolabeled DCA (specific
activity 52mCi/mmole, 99% pure by thin layer chromatography) was purchased from
American Radiolabeled Chemicals, St. Louis, MO. C14 DCA was converted to its
sodium salt by the addition of equimolar sodium hydroxide and was diluted as
necessary with unlabelled NaDCA for use in assays. Unlabelled DCA for dosing in
rat studies was purchased from TCI America, Portland OR. Glutathione, HEPES,
DTT, P-mercaptoethanol, unlabeled sodium dichloroacetate were all purchased from
Sigma-Aldrich Chemical Company, St.Louis, MO. Zephyrhills mineral water was
purchased from local stores. ECL chemiluminescence detection kit, Hyperfilm ECL,
donkey anti-mouse and goat anti-rabbit secondary antibodies were purchased from
Amersham Biosciences, Piscataway, NJ. HPLC grade trifluoroacetic acid was bought
from Pierce. Methanol, acetonitrile, ammonium bicarbonate, potassium phosphate
monobasic, potassium phosphate dibasic, sodium chloride, potassium chloride,
glycerol, sucrose, EDTA, hydrochloric acid, were purchased from Fisher Scientific.
In Flow scintillant fluid was purchased from INUS, Tampa, Fl. Ecolume scintillation
cocktail was bought from ICN, Costa Mesa CA. Tetrabutylammonium hydrogen
sulfate (PICA) was purchased from Waters Corporation, Franklin, MA.
Polyacrylamide Ready Gels (12 %), high and low range molecular weight markers,
G250 coomassie Blue, tris-glycine SDS-PAGE buffers, Biorad mini Protean II Cell,
and power supplies were purchased from Biorad, Hercules, CA. All other chemicals
were of purest grade available and were bought from commercial suppliers.
Animal Dosing Protocol for NTBC Studies
Male Sprague Dawley rats, weighing between 180-200 gm were used in this
study. They were divided into various groups and were weighed each day prior to
dosing. Drugs were administered via oral gavage. They were housed in conventional
cages, kept on a 12 hour light and dark cycle and given free access to food and water.
Since NTBC is insoluble in water and in ethanol, its solution was prepared by
mixing NTBC with water and then adding ammonium hydroxide to the mixture to
raise its pH to 10.5. NTBC quickly goes into solution at alkaline pH. The pH was
then brought down to about 8.5 by adding hydrochloric acid. Solutions of lmg/ml and
4mg/ml NTBC were prepared in this alkaline medium. Sodium DCA is readily
soluble in water and 50mg/ml solution was made in deionized water.
Two preliminary studies were performed in which duration of NTBC treatment
was varied. In the first study, 10 male rats were divided into 3 groups. One group
(n=4) was given 50mg/kg DCA, 2nd group (n=3) was given NTBC (Img/kg) for for 5
days and DCA (50mg/kg) along with NTBC for the last two days, 3rd group was
given NTBC alone for 5 days. Animals were sacrificed after the last dose and livers
were removed. Cytosol from liver was used to assay GSTz activity.
In the second preliminary study 24 male rats were divided into 6 groups with 4
animals in each group. Animals were treated with 50mg/kg NaDCA for either one or
five days or were given 4mg/kg NTBC for 15 days. Some animals received NTBC for
15 days and NaDCA (50mg/kg) along with NTBC for the last one or five days of
treatment. Study also included an untreated control group. Animals were kept in
metabolic cages on the first and last day of treatment to collect urine.
Another study with 54 male animals was performed where a longer exposure to
2 different doses of NTBC was studied. Male Sprague Dawley rats were divided into
9 groups with 6 animals in each group. Animals were given 50mg/Kg NaDCA for
either one or five days (NOD1 and NOD5 respectively). Some animals received 14
days of NTBC treatment (Img/Kg or 4mg/Kg) and then DCA along with NTBC for
either one or five days (N1D5 and N4D5). The study also included a no-treatment
control (NODO), Img/Kg NTBC control (N1DO) and 4mg/Kg NTBC control (N4DO).
Table 2-1 Dose and duration of NTBC and DCA treatment
(50MG/KG) 0 1 4
0 NODO N1DO N4DO
1 DAY NOD1 N1D1 N4D1
5 DAYS NOD5 N1D5 N4D5
Animals were kept in metabolic cages for the collection of urine (24 hours) on
the 1st day of treatment, on the last day of NTBC treatment, on the 1st day of DCA
treatment and on the final day of treatment. All animals were sacrificed on the last
day of study by decapitation; their livers were removed for preparation of microsomes
Animal Dosing Protocol for GSTz Recovery and Low Dose Studies
Male Sprague Dawley rats were kept in individual cages, on a 12 hour light-
dark cycle and given free access to food and water. Their initial weight was between
170-200 G. As the study progressed the weight of some animals increased up to 500
gm. Animals were kept in metabolic cages for 24 hours prior to and on the last day of
both treatment and recovery, to collect urine. Urine was centrifuged to sediment food
or fecal matter and the supernatant was stored at -800C until analysis.
Since municipal drinking water contains [g/L levels of DCA, mineral water
(Zephyrhills) was used as drinking water in low dose, control and post treatment i.e.
recovery groups. This prevented the influence of any DCA other than the pre-
determined dose. Animals receiving high dose (50mg/kg) of NaDCA were given
municipal drinking water as [g/lt levels will not interfere at such high doses. Water
consumption and body weight was monitored thrice a week and dose was adjusted
based on changes in body weight and amount of water consumed by individual rat.
This ensured accurate dosing which was essential, especially in the pg/kg dose
To study time course of inhibition and recovery animals (n=6 per group) were
given 50mg/kg/day NaDCA in drinking water for 1, 4, or 8 weeks. Animals were
sacrificed at the end of treatment. Alternatively some groups were kept for an
additional 1, 4, or 8 weeks after they were taken off the drug. These animals were
sacrificed at the end of this recovery period. Each of the above groups had a
corresponding untreated control for comparison.
To study the effect of low dose of NaDCA, male Sprague Dawley rats were
given either 2.5kg/kg/day or 250kg/kg/day via drinking water for either 4, 8 or 12
weeks (n=6 per group). Enzyme recovery was studied after 8 weeks of treatment
following which DCA was withdrawn for either 1 or 8 weeks. These animals were
then sacrificed and livers were used to prepare cytosol and microsomes. About 1-3G
of liver from each rat was immediately frozen in liquid nitrogen and stored at -800C
for future use.
Preparation of Cytosol and Microsomes from Livers
The animals were euthanized by placing them in carbon dioxide chambers.
Once the animal showed no vital life signs such as breathing and heart beat, it was
dissected open and blood was collected from the vena cava in a heparinized syringe.
Blood samples were centrifuged to separate plasma. Red blood cells were washed
with saline twice and stored at -800C. Livers were removed quickly and placed in ice
cold homogenizing buffer (1.15% KC1 buffered to pH 7.4 with potassium phosphate).
The liver was rinsed twice, patted dry, weighed, and placed in a volume of ice cold
buffer equal to 4 times the weight of the liver. It was then minced with scissors and
transferred to a homogenizing vessel of Potter-Elvejhem type, and homogenized by
motor driven Teflon pestle. The homogenate was poured in Sorvall centrifuge tubes
and centrifuged at 13,300 g for 20 min. The supernatant containing cytosol and
microsomes was transferred to polycarbonate ultracentrifuge tubes, and centrifuged at
170,000 g for 45 min. The supernatant was cytosol which was stored in aliquots at -
800C. The protein concentrations of cytosol was determined by the method of Lowry
In-vitro GSTz Activity in Rat Liver Cytosol
Assays were conducted under saturating conditions of DCA and GSH as
described in James et.al. 1997. Reaction mixture consisting of 0.1 M Hepes buffer,
pH 7.6, 1 mM glutathione (freshly prepared), 1 mg of rat liver cytosol, and water to
make a final volume of 0.25 ml was pre-incubated in a water bath at 370C for 2 min.
The reaction was started by adding 0.2mM C14 labeled NaDCA (11.86 mCi/mmole).
The mixture was vortex mixed and incubated for further 10 minutes. The reaction was
terminated by adding 0.5 ml of ice cold methanol. Blanks were samples in which
reaction was stopped at zero time. The whole mixture was vortex mixed and
centrifuged and supernatent was filtered through 0.45tlm nylon centrifuge filter to
remove particulate matter. The filtrate was then analyzed by HPLC.
Samples from DCA assay were analyzed at room temperature by isocratic
reversed phase HPLC (James et.al. 1997). An Isco (model 2350) pump with a manual
injection port was used to inject samples into a 50 x 4.6mm Beckman octadecylsilane
pre-column coupled to a 5a, 80A, 250 x 4.6mm Beckman octadecylsilane analytical
column. The mobile phase used was 0.005M-tetrabutylammonium hydrogen sulfate
(ion-pair reagent) in 30% methanol and the flow rate was Iml/min. The eluent was
analyzed first by UV detector (Dynamax, UV-1, Rainin instruments) at a wavelength
of 220 nm, and then by radiochemical detector (Flo one Beta, Radiomatic
Liver cytosolic protein (40 [tg) or recombinant hGSTzlc-lc (0.5-5 [tg) was first
denatured by heating at 900C for 5 minutes in a 2x SDS-PAGE sample buffer (0.5M
tris-HCL, glycerol, 10%w/v SDS, 2-0 mercaptoethanol, 0.05% bromophenol blue)
and then loaded on 12% polyacrylamide gels. These gels were electrophoresed for
Hour at 200V in Mini Protean apparatus. Then the protein was transferred to a
nitrocellulose membrane overnight at 40C at 30V.
The membrane was then blocked in 5% non-fat milk in 0.05% Tween-Tris
Buffered Saline (T-TBS) at room temperature for 1 hour after which it was rinsed
briefly twice with fresh changes of T-TBS, and then once for 15 minutes and twice
for 5 minutes. Primary antibody (chicken anti-mouse GSTz) was diluted 1:30,000 in
5% non-fat milk in 0.05% T-TBS. The membrane was then incubated in the primary
antibody for 2 hours at room temperature on a rotary shaker. It was then washed as in
the first step. The secondary antibody (donkey anti-chicken) was diluted 1:50,000 in
5% non-fat milk in 0.05% T-TBS. The membrane was incubated in the secondary
antibody for 1 hour at room temperature on a rotary shaker after which it was washed
as before. The membrane was then incubated for 1 minute in the chemiluminescence
solution. It was immediately exposed to X-ray film, which was then developed.
Western blots for quantitating GSTz in liver cytosol, had hGSTz Ic-lc as a
control or standard for comparison. Same amount of the recombinant protein was
loaded on each gel. The blots were developed, scanned and analysed using
ScanAnalysis software. Area covered by cytosolic GSTz was compared with the
standard hGSTz Ic-lc and the relative amount for each sample was calculated.
GC-MS Analysis of Urine
Tyrosine metabolites in urine from treated and control animals were analysed
by A.L Shroads in Dr. Stacpoole's laboratory using a previous published method
(Yan et.al. 1997). Briefly, urine samples were methylated by BF3 (12%) in methanol,
extracted into CH2C12 and analysed on a Hewlett Packard 5972 mass spectrometer.
The column used was HP-WAX, 30mm x 0.25mm, 0.15 [tm film thickness, helium
was used as carrier gas and GC temperatures were 400C for 2 min, then to 100C at
50C per minute, then to 2400C at 150C per minute, and held at 2400C for 5 min. 2-
oxohexanoic acid was used as an internal standard.
Recombinant 6X N-terminal His tagged GSTz-lc was expressed in E-coli cells
and purified on a nickel affinity column (Qiagen, Valencia, CA) according to the
manufacturers instructions. Construct for GSTz-lc and its expression was performed
by Dr. Xu Guo in Dr Peter Stacpoole's laboratory. Protein concentration was
measured by Biorad protein reagent. The purity of the expressed enzyme was
determined by SDS-PAGE followed by western blot. Enzyme activity was
determined using DCA as a substrate by the assay described earlier, but with 5 ag of
Purification of Rabbit Anti-Human GSTz Ic-lc Antibody from Anti-Sera.
Rabbit anti-serum was obtained from Cocalico Biologicals (Reamstown, PA),
batch # UF447 using recombinant hGSTz Ic-lc as antigen. This anti-serum was then
purified using Pierce protein A antibody purification kit. Anti-Sera was diluted 1:1
with binding buffer (provided in the kit, pH 8.0, contains EDTA), applied to the
protein A column and allowed to flow through. The column was then washed with the
binding buffer at least 4 times or until the washings showed no absorbance at 280nm.
The antibody was then eluted from the column with the elution buffer (pH 2.8
contains primary amine), fractions were collected and neutralized with 1M tris buffer
pH 8.0. Fractions with the highest absorbance contained the IgG and were pooled.
The antibody was then checked for cross reactivity with GSTz by using it as a
primary antibody in western blots of liver cytosol or recombinant hGSTz Ic-1c.
Immunoprecipitation of GSTz from Rat Liver Cytosol
Seized X protein A immunoprecipitation kit from Pierce was used for this
procedure. Purified antibody from rabbit anti-sera was first desalted using Pierce
desalting column (potassium phosphate buffer, pH 7.6) to remove primary amines
which interfere with immunoprecipitation. The antibody was first bound to Protein A
by mixing them and incubating for 30 minutes at room temperature. The bound
antibody was then crosslinked with protein A using 25[l disuccinimidyl suberate
(8mg reconstituted in 80 tl DMSO). After incubating the bound antibody with the
cross-linking reagent for 30 minutes excess reagent was washed off thoroughly using
wash buffer (0.14 M NaC1, 0.008 M NaPO4, 0.002 M K2PO4, and 0.01M KC1, pH
7.4). Rat liver cytosol (2-3ml) was then incubated with the crosslinked antibody
overnight at 40C. After this incubation protein A was washed to remove unbound
antigen. Immunoprecipitated antigen is eluted by washing this protein A with elution
buffer (pH 2.8 contains primary amine). The eluted antigen was then analyzed by
DCA Modified GSTz-lc
Reaction mixtures consisting of 0.1 M ammonium bicarbonate buffer, pH 7.6, 1
mM glutathione (freshly prepared), 20[tg-lmg of purified GSTz and 2-10mM DCA
were incubated in eppendorf tubes for 2 hours. At the end of incubation the tubes
were placed on ice to stop the reaction. In some experiments, incubation time was
varied to increase the amount of modified GSTz formed but never exceeded 24 hours.
Blanks were samples devoid of either GSTz, DCA or GSH. The reaction was
terminated by placing the tubes on ice.
Binding of DCA to GSTz was studied by incubating 50[g GSTz-lc with
1.8mM C14 sodium DCA (12ci) and ImM GSH in 0.1M ammonium bicarbonate for
24 hours. GSH was replenished once during the reaction. The reaction mixture was
then placed in a 3000MW cut-off dialysis cassette (Pierce) and the protein was
dialyzed overnight with 500 ml of 25mM ammonium bicarbonate. Dialysis buffer
was analyzed by ion-pair HPLC (described before) to determine the amount of
glyoxylate and unreacted DCA in the reaction.
Protein was denatured in SDS-PAGE sample buffer and electrophoresed on a
12% polyacrylamide gel for 1 hour at 200V. The gel was dried and autoradiographed
overnight on Instant Imager.
To determine the amount of radioactivity bound to GSTz protein, 4[l or 2.5ag
of the protein from incubate was placed in a scintillation vial. Ecolume scintillation
cocktail was added and the radioactivity was measured by liquid scintillation
spectrometry. The dialysis buffer was also counted on liquid scintillation counter to
determine the radioactivity not bound to the protein. Background counts were
deducted from the total dpm and the corrected values were then used to determine the
moles of DCA bound per mole of GSTz and the amount of unbound radioactivity.
HPLC Analysis of Modified GSTz
Incubation mixture containing modified GSTz was analyzed by HPLC on a
gradient reversed phase system. The samples were introduced by a manual injection
port into a Jupitor 5[t, 300A, 250 x 4.6mm C18 column. HPLC was controlled by
Beckman Gold Nouveau software. The analysis was started at 100% Solvent A
(40%acetonitrile with 0.1% trifluoroacetic acid) with a 40 minute linear gradient to
Solvent B (57% acetonitrile with 0.1% trifluoroacetic acid). The conditions were
maintained at 100% solvent B for 10 minutes before returning to initial conditions.
The eluate was first analyzed by a uv detector (Beckman instruments) at 214 nm and
then by a fluorescence detector (Schimadzu) with excitation: 220nm and emission:
300nm. For some experiments fractions of modified and unmodified enzyme were
collected. Fractions were then concentrated on a SpeedVac, and kept at 40C until
Enzyme Digests of hGSTz-lc
GSTz-lc (100[g-lmg) was incubated with 2-10mM DCA, and ImM GSH, in
0.1M ammonium bicarbonate for 24 hours at 370C. GSH was replenished once after
10 hours of incubation. In some reactions, GSTz-lc was incubated with 1mM GSH
alone under similar conditions.
Reaction mixtures were then placed in a 3000 MW cut-off filter centrifuge tube
(Microcon, Amersham Biosciences) and protein was recovered after 2 concentration
dilution cycles using 25mM ammonium bicarbonate, pH 7.6. Protein was digested
using trypsin, endoprotease lys-c, or Glu-c. Lyophillized sequencing grade trypsin,
lys-c, and Glu-C (Roche molecular biochemicals, IN) were reconstituted in buffers
according to manufacturers instructions. Enzyme to hGSTzlc-lc ratio was 1:25.
Digestion was done at 370C for 18 hours. Digests of GSTz-lc which had not been
incubated with GSH or DCA were also prepared. Lyophillized sequencing grade
trypsin and lysine (Roche molecular biochemicals, IN) were reconstituted in buffers
according to manufacturers instructions. Digests were concentrated with a SpeedVac
concentrator and stored at 40C until analysis.
MALDI-TOF Analysis of Intact GSTz
Intact samples of unmodified GSTz and GSTz that had been previously
incubated with 10mM DCA and ImM GSH for 24 hours were analyzed by MALDI.
Samples were first subjected to dialysis using a dialysis tube with IkD cutoff
membrane against deionized water at 40C for 24 hrs with 3 changes of water. These
samples were then concentrated on a SpeedVac concentrator to about 5-10 pmol/dl.
Samples were then spotted on the MALDI plate to which sinapinic acid (10mg/ml in
5% acetonitrile containing 0.1% trifluoroacetic acid) had been previously applied.
After drying the matrix was re-applied.
MALDI-TOF analysis was done on Voyager-DE mass spectrometer (Applied
Biosystems, Foster City, CA). MALDI-TOF mass spectroscopy was run in positive
and linear mode. Ion extraction delay, grid voltage, and laser intensity were adjusted
to achieve optimal resolution depending on sample mass.
Masses of modified and unmodified GSTz were measured to determine mass shift
after reaction with DCA.
MALDI-TOF Analysis of Digests
Tryptic peptides derived from modified and unmodified GSTz, were first
purified and desalted using C18 ZipTip (Millipore, Bedford, MA). Samples were
loaded on ZipTips that had been equilibrated according to manufacturer's
instructions. The ZipTip was washed with 0.1% TFA to remove salts and the peptides
were eluted with 50% acetonitrile with 0.1%TFA. Desalted samples were then
applied to MALDI plate using a-cyano-4-hydroxycinnamic acid in 50% acetonitrile
with 0.1%TFA as the matrix. Samples were analysed on a Voyager-DE Pro MALDI-
TOF mass spectrometer (Applied Biosystems, Foster City, CA). MALDI-TOF
spectrometry was run in positive, delayed extraction and reflectron mode. Ion
extraction delay, grid voltage, and laser intensity were adjusted to achieve optimal
resolution depending on sample mass.
LC/MS Analysis of Intact GSTz Ic-lc
50[g of GSTz that has been incubated with 10mM DCA, ImM GSH for 24
hours, 50tg GSTz which had been incubated with ImM GSH for 24 hours, and GSTz
alone were first ultrafiltered as described before. These samples were injected into a
Phenomenex Synergi 4t Hydro-RP 800 A pore, 4 [tm, 2x150mm column (Torrace,
CA) equipped with a C18 guard column (2mmx4mm) which was coupled to a
Thermofinnigan (San Jose, CA) LCQ with electrospray ionization (ESI). Proteins
were eluted with a linear gradient at a flow rate of 0.15ml/min. The starting mobile
phase was, 95% solvent A (0.5% formic acid and 2mM ammonium format in water)
and methanol was introduced in a linear gradient to 50% Solvent B (0.5% formic acid
in Methanol) over 15 mins. The amount of methanol was then increased to 95%
Solvent B over 80 minutes. Mobile phase was modified, post-column to assist
ionization of the analytes. The modifying solution used was 1% formic acid in
methanol (20ul/min) and was added via another pump. ESI-MS data was collected
over 2 ranges: m/z 100-450 and m/z 440-2000.
LC-MS/MS of Digests.
Digests of GSTzlc-lc which had been incubated with DCA and/or GSH were
prepared as described before. These samples were injected into a Phenomenex
Synergi 4[t Hydro-RP 800A pore, 4 [tm, 2x150mm column (Torrace, CA) equipped
with a C18 guard column (2mmx4mm) which was coupled to a Thermofinnigan (San
Jose, CA) LCQ with electrospray ionization (ESI). Proteins were eluted with a linear
gradient at a flow rate of 0.15ml/min. The gradient was started with 100% Solvent A
(0.5% formic acid and 2mM ammonium format in water) and a linear gradient to
95% Solvent B (0.5% formic acid in Methanol) was run in 80 mins. ESI-MS data was
collected over 2 ranges: m/z 100-450 and m/z 440-2000. Data dependent MS/MS was
done of the most intense ion in each scan. This data was analysed by Thermofinnigan
Xcalibur software (version 1.2). Theoretical protein masses, peptide and MS/MS
fragment ion masses were generated using Sequest Browser (Thermofinnigan San
Jose, CA), Swiss-Prot and Protein Prospector (http://prospector.ucsf.edu).
RESULTS OF NTBC STUDIES
Tyrosine metabolism was blocked by NTBC to determine the effects of MA
and MAA on GSTz activity and expression. Preliminary studies were conducted to
determine appropriate dose of NTBC, duration of treatment, and to obtain preliminary
information about the effects of NTBC on GSTz. etc.
Results of the short preliminary study described in chapter 2 showed that rats
given NTBC for 3 days prior to DCA (2 days) had a higher GSTz activity than
animals that received DCA alone (Fig 3-1). But this result was not consistent across
the group. GSTz activity in one rat was significantly lower than other 3 in the group
(n=4). Another rat had a very high GSTz activity compared to others in the group.
Due to these outliers this study was not conclusive and required further investigation.
In the second preliminary study duration of NTBC pre-treatment was increased,
to help understand in more detail the effects of NTBC on GSTz inactivation. As
expected, GSTz activity of rats treated with DCA alone was significantly different
than the untreated control (Fig 3-2). When NTBC was given prior to DCA, there was
an increase in GSTz activity compared to its activity in rats given DCA alone. But
this increase was not statistically significant due to high standard deviation in group
given NTBC and DCA. This was a similar issue observed in the earlier study and
hence data from the second study was not considered conclusive.
DC1 DC2 DC3 DC4 ND1 ND2 ND3 ND4 NC1 NC2
DCA 50mglkg NTBC4mg/kg NTBC 4mg/kg
Fig 3-1 Results from first preliminary study. Preliminary study with 10 animals, given
DCA (n=4), 50mg/kg shown as DC1-4, NTBC 4mg/kg and DCA (n=4), 50mg/kg
shown as ND1-4, NTBC alone (n=2) 4mg/kg shown as NC1-2. Units of specific
activity are nmoles/min/mg.
There was no difference between the GSTz activities of rats given DCA alone
for one day and rats given NTBC prior to DCA (one day). Since these results did not
completely coincide with those obtained from the 1st study, further research in this
area was necessary.
Effect of NTBC on GSTz Activity and Expression
A final study was then performed to conclusively determine the effects of
NTBC on GSTz activity and expression. In this study a low dose (Img/kg) and a high
dose (4mg/kg) of NTBC were given for 14 days prior to DCA administration. These
doses were chosen to study dose dependency of response. GSTz activity was
measured by determining the amount of glyoxylate formed during in-vitro
metabolism of DCA by GSTz. The results are shown in fig 3-3.
.. NTBC +DCA 5 days
I B B DCA 1 day
1 m NTBC+DCA 1 day
Fig 3-2 Results from second preliminary study. Animals were given 4mg/kg NTBC
(15 days) and 50mg/kg DCA (1 or 5 days). n=4, error bars show standard deviation
and units of specific activity are nmoles/min/mg. indicates means are significantly
different than controls (p<0.001)
NOD1- DCA alone 1 day.
T NOD5- DCA alone days.
l N1DO- NTBC 1mg/kg.
S-. N1D1-NTBC 1mg/kg 14days+DCA
-* _1T Iday.
.E N1D5- NTBC 1mg/kg 14 days+
.~- DCA days.
oZ- N4DO- NTBC 4mg/kg.
_.. N4D1- NTBC 4mg/kg 14 days+
"o DCA Iday.
S* N4D5- NTBC 4mg/kg 14 days+
0 1 1 DCA 5 days.
Fig 3-3 Comparison of GSTz activity in NTBC studies. Error bars show standard
deviation. n=6, indicates that means are significantly different from untreated
DCA treatment in the absence of NTBC i.e. groups NOD1 and NOD5 for 1 and
5 days respectively significantly reduces GSTz activity. Loss of activity was more
pronounced after 5 days of treatment, which was consistent with previously published
reports. NTBC treatment prior to administering DCA (groups N1DI, N4D1, N1D5
and N4D5) did not have any significant effect on GSTz activity. GSTz activity in
N4D5 group was not significantly different than NOD5 group and enzyme activity in
N1D5 did not differ from the activity in NOD5 group. But the enzyme showed
significantly higher activity in N4D1 group than in N1DI group suggesting that the
higher dose of NTBC was probably the reason for increase in activity.
On the contrary when DCA treatment was increased from 1 day to 5 days there
was no such change in activity i.e. N1D5 is not significantly different than N4D5.
Interestingly, the NTBC controls (N4DO and N1DO) showed a significantly lower
activity than the no treatment control. Thus it appears that NTBC treatment itself is
probably decreasing the activity. Western blots of hepatic cytosol showed that there is
no change in the amount of GSTz expressed when NTBC is given prior to DCA
treatment compared to GSTz in rats given DCA alone. DCA treatment however
reduces the amount of protein expressed in all animals.
Western blots (Fig 3-4) also shows a significant reduction in GSTz expressed in
animals treated with NTBC alone. This is in accordance with enzyme activity studies
which showed a reduction in activity when rats were treated with NTBC alone.
However, this decrease in enzyme expression was not dose related.
1.2 NODO- Control
NOD1- DCA alone 1 day.
1 NOD5- DCA alone days.
N1DO- NTBC 1mg/kg.
S0.8 N1D1-NTBC 1mg/kg 14days+DCA
0.6 N1D5- NTBC 1mg/kg 14 days+
Z DCA days.
| 4 N4DO- NTBC 4mg/kg.
So.4- N4D1- NTBC 4mg/kg 14 days+
0.2 DCA iday.
0.2 N4D5- NTBC 4mg/kg 14 days+
SDCA 5 days.
Fig 3-4 Relative quantification of GSTz from hepatic cytosol by western blot. n=6,
error bars indicate standard deviation, indicate means are significantly different than
control i.e. NODO (p<0.005).
Fig 3-5 Representative western blot of rat liver cytosol. Labels represent 50mg/kg
DCA for 5 days (NOD5), control (NODO), NTBC Img/kg + DCA 50mg/kg (N1D5),
NTBC 4mg/kg + DCA 50mg/kg (N4D5).
RESULTS OF GSTZ RECOVERY AND LOW DOSE STUDIES
Inactivation and Recovery of Hepatic GSTz
Time course of inactivation and restoration of GSTz activity and expression
were studied by analyzing cytosolic GSTz from animals treated sub-chronically with
50mg/kg/d DCA in drinking water.
Data from activity studies shows that GSTz activity is significantly (p<0.0001)
lost after one week's treatment with 50mg/kg DCA. Over the 8 week treatment period
DCA activity remained at very low levels which are significantly lower than
corresponding controls. After 8 weeks of treatment animals were taken off the drug to
study recovery. Enzyme activity was studied after 1, 2, 4 and 8 weeks of recovery.
Figure 4-1 shows that although loss in activity was rapid, the recovery was slow.
After 1 week of recovery the activity was significantly lower than the control. Only
54% of control activity was present after one week of withdrawing the drug. Even
after 2 weeks of recovery the enzyme activity (84% of matched control) did not reach
the same level and was significantly lower (p<0.05) than the corresponding control.
Enzyme activity was restored to control levels only after 4 weeks of withdrawing the
Western blots of cytosolic protein also showed a loss of immunoreactive
protein expressed. Amount of immunoreactive protein from western blots was
expressed as amounts relative to 0.01ltg of GSTzlc-lc.
Fig 4-1 Inactivation and recovery of GSTz activity after treatment with 50mg/kg
DCA in drinking water. Error bars indicate standard deviation, n=6. indicate that
means are significantly different than control.
One week of treatment with 50mg/kg/day of DCA resulted in 95% decrease in
protein expression (p<0.0001) and this remained low over 8 week exposure (Fig 4-2).
After DCA withdrawal, GSTz expression increased gradually, but unlike enzyme
activity, it remained significantly lower than controls after 8 weeks of withdrawing
Drinking Water Consumption, Liver and Body Weight
Drinking water and body weight of each rat was monitored in order to adjust
the amount of DCA given each day accordingly. Water consumption generally did
not vary significantly within individuals or treatment groups depending on weight or
dose. One exception was the 50mg/kg group treated for one week. This group
Rcco\ cir pIhase
0 0b 0b '
consumed 133.5+10 ml/kg/d and the matched control group drank 111.1+9 ml/kg/d.
Individual animal was weighed thrice a week in order to maintain an accurate dose
over the treatment period. There was no significant difference in the body weight
between treatment groups during the course of study. Livers of each animal were
weighed and liver to body weight ratio was calculated to determine the change in
liver weight due to DCA. This ratio was not significantly changed in animals treated
with low doses of the drug. The 50mg/kg/d DCA dose significantly increased the
liver to body weight ratio after 8 weeks of treatment when compared to matched
untreated control (p<0.005). This index returned to control levels 8 weeks after
withdrawing the drug.
1.8 Treatment phase
1.6 Recovery phase
B C E F G J L O Q R S T U X
1 week 4 weeks 8 weeks 8+1 8+2 8+4 8+8
weeks week weeks weeks
] Control 50mg/Akg
Fig 4-2 Loss and recovery of GSTz protein after treatment with 50mg/kg DCA.
Values are expressed relative to 0.01 tg of hGSTz Ic-lc. Error bars indicate standard
deviation, n=6. indicate that means are significantly different than control.
25kDa 4-- 5 6
Fig 4-3 Representative Western blot of control and DCA treated (50mg/kg, 1 week)
rat liver cytosol. Lanes 1-3 were loaded with control and lanes 4-6 were loaded with
DCA treated liver cytosol.
Effect of Low Doses of DCA on Hepatic GSTz
In this study, animals were treated with 2.5 and 250 [tg/kg/d of NaDCA for 4, 8
and 12 weeks. GSTz activity and expression was studied after this sub-chronic
exposure to environmentally relevant levels of the drug. The specific activity (Fig 4-
4) of the enzyme was significantly decreased after 8 weeks of exposure to both 2.5
and 250 ag/kg/d of DCA (p<0.001 in both groups). This effect was more pronounced
in the 12 week treatment group where there was a suggestion of dose dependent
effect. Recovery of activity was studied after the 8 week treatment. After 8 weeks of
exposure to 2.5 and 250 [tg/kg/d doses GSTz expression decreased 20% (p<0.05) and
30% (p<0.01) respectively (Fig 4-5). Exposure to these low doses of DCA for 12
weeks also significantly depleted GSTz protein. These observations show that DCA
depletes GSTz activity and expression at environmentally relevant concentrations.
Recovery of enzyme activity and expression were also studied after 8 weeks of
treatment at these low doses. After one week of withdrawing the drug GSTz activity
returned to control levels in both 2.5 and 250 tg/kg treatment groups (Fig 4-7).
However enzyme expression (Fig 4-8) was not restored after one week of recovery. It
returned to control levels in 2.5 tg/kg group after 8 weeks of recovery but remained
significantly lower (p<0.05) than control in animals treated with 250 [tg/kg of DCA.
Fig 4-4 Specific activity of GSTz in rats treated with 2.5 and 250 [g/kg/d of DCA in
drinking water. Error bars indicate standard deviation (n=6) and indicate that means
are significantly different than controls.
4 weeks 8 weeks 12 weeks
M control ] 2.5pg/g/day F- 250pg/kg/day
Fig 4-5 Expression of GSTz in rats treated with 2.5 and 250 [g/kg/d of DCA in
drinking water. Values are expressed relative to 0.01 g of hGSTz Ic-lc. Error bars
indicate standard deviation (n=6) and indicate that means are significantly different
1 2 3 4 5 6
25 kDa %w qI
Fig 4-6 Representative western showing GSTz expression in control (lanes 1-2)
250g/kg (lanes 3-4),and 2.5 pg/kg (lanes 5-6). Rats were exposed to these doses of
DCA for 8 weeks.
Urinary Excretion of DCA and Maleylacetone
Rats excreted DCA in urine at the rate of 4.5 mg/kg/24h after one week of
treatment with 50mg/kg DCA. This rate increased to 5.5 mg/kg/24 h after 8 weeks of
treatment with the same dose. DCA excretion dropped dramatically (less than 10
[g/kg/24 h) after week of withdrawing the drug. No DCA was detected in urine of
rats treated with 2.5 and 250 pg/kg/day of the drug. Urinary analysis showed that rats
excreted MA at the rate of 60-75 [g/kg/24 h during 8 weeks of treatment with the
high dose of DCA. MA could not be detected after one week of withdrawing the
drug. MA was not detected in the urine of rats treated with both 2.5 and 250
pg/kg/day of DCA.
Fig 4-7 Recovery of GSTz activity in rats treated with low doses of DCA. n=6, error
bars indicate standard deviation, indicate that means are significantly different than
control. Activity is restored after one week of withdrawing DCA.
8 week treatment
1 week recovery
8 week recovery
IH Control i 2.5 pg/kg/d 250 pg/kg/d
Fig 4-8 Restoration of GSTz protein in rats treated with low doses of DCA.Values are
expressed relative to 0.01 pg of hGSTz Ic-lc. n=6, error bars indicate standard
deviation, indicate that means are significantly different than control.
8 r 1 w re 8 w re T
... .. .
....:- .. .
M- Control Ii] 2.5 pg/kg/d 250 pg/kg/d
8 week treatment
1 week recovery
8 week recovery
ADDUCTS OF HUMAN GSTZ 1C-1C WITH DCA AND GSH.
HPLC Separation and Identification of Adduct
After its reaction with DCA in the presence of GSH, GSTz was analysed by
reverse phase HPLC coupled with a fluorescence detector. Under the mobile phase
conditions described, adduct of GSTz with DCA eluted between 30 and 33 minutes
(Fig 5-1) and the parent unreacted GSTz eluted at about 35 minutes. This was
confirmed by running purified and unreacted GSTz and blanks devoid of either
GSTz, DCA or GSH. Purified enzyme eluted at about 35 minutes. The samples
devoid of GSTz did not show any peaks in the spectra, and those devoid of either
DCA or GSH showed a peak at 35 minutes which corresponded to the retention time
of unreacted GSTz. This confirmed the identity of both peaks and showed that DCA
formed an adduct with the enzyme only in the presence of GSH. It was seen that the
amount of modified protein was dependent on the concentration of DCA. As the
concentration was increased from 2 mM up to 10mM the area of the peak
representing the modified protein increased and there was a corresponding decrease
in the area of the peak representing unmodified GSTz.
For further mass spectral experiments it was important to have maximum
amount of modified protein and as little contamination from the unmodified protein
as possible. To achieve this, reaction was done with 10mM DCA and for 24 hours.
After this incubation period about 85% of the protein was in its modified form.
c:vaiahalMmnrxcat eChannd B
I) ii 10
Fig 5-1 Separation ofDCA modified GSTz from unmodified GSTz by gradient
reversed phase HPLC. GSTz was incubated with 6mM DCA and ImM GSH in 0.1M
Hepes and analysed by gradient HPLC described in methods. Adduct has a retention
time of 32.9 minutes and the unreacted GSTz Ic-lc eluted at 35.2 minutes.
Mass Spectral Analysis of Intact (undigested) Modified and Unmodified GSTz
To determine the mass of unmodified GSTz protein and that of the enzyme
modified by both GSH and DCA the reaction incubates were purified by
ultrafiltration or Ziptip and analyzed by matrix assisted laser desorption ionization
(MALDI) and electrospray ionization (ESI). MALDI of unmodified and DCA
modified protein showed a mass difference of about 278 mass units between the two
proteins (Fig 5-2).
16000 21000 26000 m/
Fig 5-2 MALDI of undigested GSTz Ic-lc in unmodified and modified forms. GSTz
was reacted with 10mM DCA and ImM GSH for 24 hours. The samples were
desalted and analysed by MALDI as described in Methods. Singly and doubly charged
masses are shown.
Data from MALDI was however could not be conclusive, since at such high
masses MALDI is not completely accurate. An expanded view of the MALDI spectra
(data not shown) shows that the mass given by the software is merely the highest
peak from several peaks typically observed in MALDI spectra of high molecular
weight proteins. Thus this mass is an estimate rather than an exact number. But
MALDI analysis proved that following reaction with DCA, GSTzlc-lc is converted
to an adduct with a mass higher than the parent protein.
ESI of intact protein gave a mass spectrum from which mass of the unmodified
protein was calculated as 25484 Kda. This is in close agreement to published mass
and the mass obtained from MALDI. ESI of samples in which GSTz had been
incubated with GSH alone yielded at least 3 possible proteins suggesting 3 possible
adducts with GSH. Calculated masses showed that these peptides had adducted up to
3 GSH molecules. They had masses of 25,789.6 +/- 8.1 u; addition of 1 GSH,
26,091.6 +/- 3.0 u; addition of 2 GSH's, and 26,398.5 +/- 4.0 u; addition of 3 GSH's.
ESI of DCA modified GSTz generated a mass spectrum so complex that it was
not possible to calculate an accurate mass of the modified protein. It was concluded
that digests of GSTz using endoproteinases would be the best possible method to
identify modification of GSTzlc-lc by DCA.
Tryptic and Lys-c Digests of Unmodified GSTz Ic-lc
Trypsin and Lys-c are endoproteinases that cut a particular protein at specific
internal residues. Trypsin cuts proteins at lysine (K) and arginine (R) residues
whereas Lys-c makes cuts only at the residue lysine. Fragments formed from such
digests can be identified from their masses.
For further confirmation of peptides, MS/MS of these fragments is performed which
results in daughter ions. Energy applied during MS/MS typically fragments peptides
along the backbone. Each residue of the peptide chain successively fragments off,
both in the N to C terminal and C to N terminal direction (Fig 5-3). Depending on the
location that the fragmentation occurs, and the nature of the ion remaining, MS/MS
may result in, a, b, c (N terminal) and x, y, or z (C terminal) ions (Cole, 1997). y ion
formation is the most likely to happen, and y ions are the ones most frequently seen. a
and b ions are also observed, but large a ions are rarer than small ones. c and x ions
are rarely seen and the existence of z ions is considered doubtful (Cole 1997).
Peptides that have been modified generate fragment ions which have a mass
equal to the sum of masses of unmodified ion and the modification.
A ion X ion_
B ion Y ion
C ion __Z ion__
R O R' O R" R'"'
I II I I I I II
H H H H H HH
Fig 5-3 Fragmention pattern of peptides to give N and C terminal ions. Most common
ions are y and b ions which are used for peptide identification.
Since each of these ions represents a certain amino acid residue, one can identify the
site of modification by determining the mass of modified y and b ions.
Theoretical fragment ions can be generated using software programs such as Protein
Prospector which are available on the internet. Using this program MS/MS ions of the
active site peptide SSCSWR were generated (Table 5-1).
Table 5-1 Theoretical masses ofy and b ions of SSCSWR using Protein Prospector.
b-H2 ions --- 157.06 260.07 347.10 533.18 --
bions --- 175.07 278.08 365.11 551.19 --
1 2 3 4 5 6
H- S S C S W R -OH
6 5 4 3 2 1
--- 638.27 551.24 448.23 361.20 175.12 yions
Trypsin and Lys-c generated several peptide fragments of GSTz which were
then identified by both MALDI and ESI as described in Methods. About 50-65% of
the protein sequence was covered by these digests. Digests with endoproteinase Glu-
C did not generate peptides that could be identified; hence this enzyme was not used
for further experiments.
6-2124 RT 014-5992 AV 636 NL 231E5
S [11. 9 127 4 136 1. l
LLJ.ILQ .... I 1 .1. ,. 1.1. II.
- S --r S,-J- I X m
.LL. 1 17173 2 1852 ,9O
1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
Fig 5-4 LC-MS/MS full scan of unmodified hGSTzlc-lc showing observed peptides.
The sequence corresponding to each of the observed is shown in table 5-2
'C ~:1 j216923
Figure 5-5 MALDI of unmodified GSTz showing tryptic peptides.
h I L IL~Y .I L.kI~i Lm ,
Details of the sequence of GSTz covered, and masses of the peptides by
Trypsin and Lys-c are shown below:
Table 5-2 Peptides identified by Lys-c Digest. a indicates ions observed by ESI. b
indicates doubly charged ions, C indicates triply charged ions.
Sequence covered Theoretical mass Observed ion Amino acids
PILYSYFRSSCSWRVRIALALK 2628.43 2629ac 6 -27
DFQALNPMK 1063.52 1063a 49-57
QVPTLK 685.42 685" 58-64
RASVRMISDLIAGGIQPLQNLSVLK 2678.72 2679ac 96-120
FKVDLTPYPTISSINK 1823.42 1823",b 176-191
RLLVLEAFQVSHPCRQPDTP TELRA 2875.5 2876ac 192-216
Table 5-3 Peptides identified by tryptic digest in ESI and MALDI spectra. a observed
by ESI, b indicates observed by MALDI, C indicates doubly charged ion.
Sequence covered Theoretical M+H ion Amino acids
SSCSWR 725.15 725a 14-19
IALALK 628.43 628ab 22-27
GIDYKTVPINLIK 1473.8 1475a,b 28-40
DGGQQFSK 866.4 866ab 41-48
DFQALNPMK 1063.52 1063ab 49-57
QVPTLKI 685.42 685ab 58-64
IDGITIHQ SLAIIEYLEETRPTPR 2765.48 2766ab 65-87
LLPQDPK 810.47 810.5ab 88-94
MISDLIAGGIQPLQNLSVLK 2110.19 2110a,b,c 101-120
FKVDLTPYPTISSINKR 1823.42 1823ab 176-192
LLVLEAFQVSHPCR 1611.86 1611a,b 193-206
QPDTPTELR 1056.53 1056ab 207-215
The peptide containing the active site (SSCSWR) was identified by MALDI
and LC-MS/MS of tryptic digests. The intensity of this peak was very low in MALDI,
hence confirmation of this peptide was not possible by MALDI alone. But LC-MS
gave a strong signal, and MS/MS identified y fragment ions of this peptide. Under the
HPLC conditions described before this peptide eluted at 15.84 minutes. This peptide
was not observed in the MALDI ofDCA-modified GSTzlc-lc.
Table 5- 4 Theoretical and observed ions of SSCSWR.
Ion type Theoretical mass Observed mass
(M+H) b ions y ions b ions y ions
88 175.1 ND ND
175.1 361.2 ND 361.4
278.1 448.2 ND 448.4
365.1 551.2 ND ND
551.2 638.3 533.1 533.1
(M+H) 533.2 533.2
Peptide containing the active site was also found in lys-c digests. But this
peptide (PILYSYFRSSCSWRVRIALALK) was too large and hence detecting
modifications of this peptide would be difficult. Hence further experiments focused
on modifications of SSCSWR obtained by tryptic digests.
Both digests did not cover the region in the protein from residues 121-175. This
region does not have a lysine or an arginine and hence could not be cut by either of
the enzymes. The mass of this peptide is about 5942 Da which is too large to be
identified as a singly or doubly charged peptide by ESI. It can only be observed if it is
triply (or higher) charged. Hence this region of the protein was not identified by
digests. The protein was then digested with endoproteinase Glu-C in order to obtain
smaller fragments of this peptide. Digests with Glu-C did not yield peptides which
could be identified by Sequest browser. Hence Glu-C digests were not investigated in
SEQ-3943-01 #580 RT 1584 AV 1 NL 3 77E4
F + c sid=2 00 d Full ms2 725 15@37 50 [185 00-1465 00]
10 298 0 3443 361 4
5 2289 247 0 2741 3262
0 I I I I
200 250 300 350 400 450 500 550 600 650 700
Fig 5-6 MS/MS of active site peptide (SSCSWR) showing daughter ions.
Tryptic Digest of GSH Modified GSTz Ic-lc.
GSTz Ic-lc that had been previously incubated with GSH was digested with
trypsin and the resulting peptides were analysed by MALDI and LC/MS/MS.
MALDI of tryptic digests identified a peptide whose mass was equal to the mass of
GSH adducted peptide 193-206 (LLVLEAFQVSHPC205R). Its mass was 1917 i.e.
1611+307-2H. Theoretical mass of this modified peptide is 1916.93. This peptide was
not identified by ESI of tryptic digests. LC-MS of tryptic digests identified an adduct
of GSH with the active site peptide (SSCSWR). The mass of this peptide is 1030
which is 305 mass units higher than the active site peptide. This peptide was not
I, I L
6074 638 4
1 5 1 601 9 6386
4826 5184 J1582 5 I
Jill J!it 1411 0 J
observed in MALDI of digests A doubly charged ion of this peptide was also
observed at 515.7 mass units. This peptide was eluted at 16.00 minutes which is
similar to the retention time of unmodified SSCSWR. Most abundant ion in the
spectra was 451.2 (doubly charged), resulting from a loss of y-glutamate from the
parent ion. Several y and b fragment ions (Fig 5-7) were identified which aided in
assigning modified amino acid residues. Masses of modified y and b ions are shown
in table 5-5.
The third adduct of GSH which was identified by ESI of GSH modified GSTz
was not observed in the LC-MS or MALDI of tryptic or lys-c derived peptides. This
adduct may be formed in the region from 121-175 amino acids. This region of the
protein has cysteine residues which may form a mixed disulphide with the thiol of
GSH. Since peptides from this region were not observed in the digests we were not
able to identify and characterize this adduct.
Tryptic Digests of DCA Modified GSTz Ic-lc
hGSTzlc-lc that had been incubated with DCA in the presence of GSH was
digested with trypsin and analysed by LC/MS. Spectra were searched for
modifications of the active site and for other possible modifications of other peptides.
Spectra showed the presence of active site peptide, but its abundance and hence the
intensity of signal was low. Since the abundance of this peptide was low the
instrument which was set for data dependent MS/MS did not perform MS/MS of this
ion. This is an important observation, since it suggests that this peptide has undergone
a reaction and has been modified.
Tryptic digest of DCA modified GSTz showed a peptide with a mass of 1104.5
which corresponds to the mass of active site peptide+glutathione+glyoxylate. This
peptide eluted at 16.24 minutes which is similar to the elution time of unmodified
active site peptide.
SEQ-3944-01 #573 RT 1583 AV 1 NL 1 10E4
T + c sid=l 00 d Full ms2 515 72@37 50 [130 00-1045 00]
35 4521 b5
25 vl/b2 5097
20 2882 2,4878
15 61 3729 4358
1: 1751 35
0 .,.... .....I ... ... .. ., ..--,. ,. .. ----., .. ., .
51 0 5832
I I I I I
S 200 300 400 500 600 700 800 900 1000
Fig 5-7 MS/MS of glutathione modified active site peptide (SSCSWR). y and b ions
refer to fragment ions observed in the modified peptide. This peptide was observed in
MALDI and ESI spectra of GSTz Ic-lc that had been incubated with GSH.
Masses of unmodified y and b ions of SSCSWR are shown in table 5-3 and
those of modified ions are shown in table 5-6. These ions have been modified by 74
(glyoxylate) and/or GSH to give corresponding modified ions.
MS/MS fragment ions (fig 5-8, table 5-7) show glyoxylate modified b2 and b3
ions suggesting that the one of the first three residues of the peptide (SSCSWR) have
been modified. b2 is modified by 74 i.e. glyoxylate suggesting that the serine at
position 14 or 15 (SSCSWR) may be covalently modified by glyoxylate.
Table 5-5 Theoretical and observed mass ofy and b fragment ions in GSH
modified SSCSWR. (active site peptide) M+H ion has a mass of 1030.4 Doubly
charged ion had a mass of 515.7.
Ion type Theoretical mass Actual mass
yl 175.1 175.1
y2 361.2 361
y3 448.2 448.4
y4 856.2 856.4
y5 943.2 ND
b2 175.1 175.1
b3 583.1 583.2
b4 670.1 670.4
b5 856.1 856.4
y4-y Glu 727.2 727.3
H20 709.3 709.1
Glu 451.2 451.2
Table 5-6 y and b ions observed in the MS/MS of GSH and glyoxylate modified
SSCSWR. M+H ion has a mass of 1104.5. This ion was observed in DCA modified
Further analysis of ions showed that yl, y2, y3 are not modified but y4, y5 and
y6 are adducted. Modified y4 ions has a mass of 856 which the mass of unmodified
y4 (551) + mass of GSH (307)-2H. Previous results show that the cysl6 is covalently
modified by GSH which is further confirmed in this mass spectrum. Adducted y5 ion
has a mass of 943 which corresponds to the mass of unmodified y5 (638) + mass of
1 ~ . .I ~thftnf tr,*rW~t *
9 1 9303
I ] (b5)
200 300 400 500 600 700 800 900 1000
Fig 5-8 MS/MS of GSH and glyoxylate modified active site peptide (SSCSWR).y and
b fragment ions are shown.
Another important fragment ion, b3 is modified by 379, which corresponds to
the mass of glyoxylate + GSH. The final C terminal i.e. y6 (14SSCSWR19) shows
modification by both GSH and glyoxylate, thus suggesting that serine 14 has been
modified by glyoxylate. Above data indicate that serine 14 in the GSH binding site
, II I I
has been covalently modified by glyoxylate and the glutathione forms a disulphide
bond with the sulfhydryl of cysteine 16. The hydroxyl of the serine may act as a
nucleophile (Fig 6-1) and attack the carbonyl carbon of glyoxylate and thus form an
adduct with the active site.
Glyoxylate modified SSCSWR was also observed in the spectra of DCA
modified GSTz. This peptide had a mass of 799.6 (fig 5-9, table 5-7)
which is equivalent to mass of the active site peptide (725) + glyoxylate (74).
Table 5-7 y and b ions in glyoxylate modified SSCSWR. M+H ion has a mass
Ion type mass Actual mass
yl 175.1 175.1
y2 361.2 361.4
y3 448.2 448.7
y4 551.2 551
y5 638.2 638.4
b2 249.1 249.3
b3 352.1 352.1
b4 439.1 439.8
b5 625.2 625.1
H20 781.3 781.6
Modification of either one of the series at position 14 or 15 was determined by
b2 ion. Unmodified b2 ion has a mass of 175 which when modified by glyoxylate
(74) gives a mass of 249. Other fragment ions also are diagnostic for modification of
serine residue. Since modification is near the N terminal of the peptide all the b ions
are modified. All the y ions of the peptide are I unadducted form except y6 ie.
(14SSCSWR19).This again suggested that serine 14 is modified by glyoxylate.
ESI of trypsin digest of the DCA modified GSTz yielded other peptides which
are different from the peptides observed in GSH modified GSTz and unmodified
GSTz. The peptide with the sequence DGGQQFFSK was modified by 56 mass units.
the mass of unmodified peptide was 866 and that of the modified was 922.4 (Fig 5-
448 7 507 4 y4
221 I 605
200 300 400 500 600 700 800 900 1000
Fig 5-9 MS/MS of glyoxylate modified active site peptide. GSTzlc-lc was incubated
with ImM GSH and 10mM DCA, digested with trypsin and analyzed by LC/MS as
described in methods.
MS/MS shows all the y ions intact suggesting that the modification has
occurred at the N-terminal end of the peptide. All the b ions of the peptide have been
modified by 56 mass units. This suggests that the residue modified is the N-terminal
end residue, "D" i.e. aspartic acid. Literature search for a shift in 56 mass units did
' "'" q
not offer any reasonable explanation. This modification could be a result of a reaction
with glyoxylic acid (Fig 6-1).The free carboxylic group in the aspartic acid could
form an anhydride with the glyoxylate. The mass of this adduct would be 56 mass
units greater than the unmodified peptide.
100 (M+H)-CO2 878
50 6 y6 3795
40 b3 4140 b6 751 4
35 5421 694
30 3 9053
25 68 3
20 28 6954
15 763 4 8612
3699 2 6002
S3812 4923 5431 645 3 906 5
S4807 6776 143
279 3142 I 5 67 1 7643 319 96
300 400 500 600 700 800 900 1000
Fig 5-10 MS/MS of glyoxylate modified DGGQQFFSK. b2ND, b3:286.0 (230.08),
b4:414.0 (358.14), b5:542.1 (486.19), b6:689.3 (633.26), b7: 776ND (720.3), ylND, y2:
234.15N, y3:381.2 (381.2), y4: (509.27), y5ND, y6: 694.3 (694.4), y7:751.4 (751.37),
ND indicate not detected. Masses in parentheses are those of theoretical, unmodified
A second peptide with the sequence, DFQALNPMK (1063.5) was also
similarly modified by 56 mass units to yield a peptide with the mass 1119.3 (Fig 5-
11), and its doubly charged fragment with mass 560.66 (Fig 5-12). All the b fragment
ions have been modified but none of the y ions have been modified. It can thus be
concluded that a residue at the N terminal of the peptide has been modified. This
peptide also has an aspartic acid residue at its N terminal end which is covalently
modified by glyoxylate.
Fig 5-11 MS/MS of glyoxylate modified DFQALNPMK. Mass of this peptide is
1119.3 which is 56 Da higher than parent peptide. Modification is at N terminal
aspartic acid. b2ND, b3:447.2 (391.16), b4:518.3 (462.2), b5:631.2 (575.28), b6:745.4
(689.33), b7:842.5 (786.38), b8:973.6 (917.42), ylND, y2ND, y3:375.4 (375.21),
y4:489.4 (489.25), y5:602.5 (602.33), y6:673.5 (673.37), y7:801.5 (801.43), y8:948.7
(948.5). ND indicates not detected and masses in parentheses are those for
unmodified theoretical ions.
A third peptide which was modified had the sequence,
MISDLIAGGIQPLQNLSVLK. The monoisotopic mass of the unmodified peptide is
2110. A doubly charged peptide with mass 1054.6 was observed in the LC/MS of
unmodified GSTz which corresponds to the mass of the doubly charged unmodified
peptide. LC/MS of DCA modified GSTz shows that this peptide was modified by 56
mass units. A doubly charged modified peptide with the mass 1084.2 (Fig 5-15) was
447 0489 6
2269 2911 3802 3980
602 3 631 3
1 995 9
S i I 1 . I' . . . I ; a I
200 300 400 500 600 700 800 900 1000 1100
Fig 5-12 MS/MS of doubly charged glyoxylate modified DFQALNPMK. y and b
ions same as those present in Fig 5-11 are observed
MS/MS of the peptide showed ions which were diagnostic for the identification of
the modified residue. Modified b4 ion (503.2) suggested that the modification is at or
before the 4th (N terminal) residue in the peptide MISDLIAGGIQPLQNLSVLK. This
residue (aspartic acid) was found to be covalently modified in other peptides
discussed earlier. It was thus concluded that the aspartic acid was covalently modified
by glyoxylate by 56 Da.
I I I I I
Fig 5-13 Theoretical y and b ions of MISDLIAGGIQPLQNLSVLK generated by
Binding of C14 Labeled DCA to GSTzlc-lc
GSTzlc-lc was reacted with C14 DCA in the presence and absence of GSH analyzed
by SDS-PAGE and autoradiography. GSTzlc-lc bound C14DCA in GSH dependent
manner as seen in Fig 5-13.
1 2 3 4
Fig 5-14 Binding of C14 DCA to GSTzlc-lc. Autoradiography of dried SDS PAGE
70 b9 blO yl0
55 1 11398
45 b6 y13
40 b5 b7/y7 y12 41
30 7295 1369
25 I 6165 8014 b12 14389 b16
2 9165 1409 3bl5
15 5032 L4 9 1
10 6 4 8866 295 1259 17209 18898
5 4466 95885 7 8i907
,3591 46 ,.588 67 I 1551 1 16078 238
400 600 800 1000 1200 1400 1600 1800 2000
Fig 5-15 MS/MS of doubly charged, glyoxylate modified
MISDLIAGGIQPLQNLSVLK. Mass of this peptide is 1084.2 Modification of 56 Da
is observed at aspartic acid. The following modified ions were detected b2ND, b3ND,
b4:503.2, b5:616.5, b6: 729.5, b7: 801.4, b8ND, b9: 914.5, bl0:1027.4, bllND, bl2:
1252.9, b13ND, bl4ND, b15:1607.8, b16:1720.9, bl7ND, bl8ND, b19:1011.4
(M+2H)2+..ND indicates not detected. Unmodified y and b ions are shown in
Figure 5-14 shows that the enzyme is labeled with radioactivity from DCA
only in the presence of GSH. GSTzlc-lc binds C14 DCA in GSH dependent manner
confirming the fact that DCA is a mechanism based inhibitor of GSTz. The
autoradiograph also shows a band at high molecular weight near the wells where
protein was loaded. It is not known if this band is that of a high molecular weight
protein or adduct or simply an artifact of the reaction.
Binding of DCA to GSTz was determined by liquid scintillation counting. It
was found that 5.68% of the radioactivity remains bound to the enzyme after
overnight dialysis of the protein to remove any unbound substrate or product. After
converting the dpm bound to the protein to moles, 5.44 moles of DCA were bound
per mole of enzyme.
Dialysis buffer was analysed by HPLC to determine the amount of unreacted
DCA in the unbound fraction. HPLC showed that 85% of DCA is converted to
glyoxylate after 24 hours of incubation with the enzyme and 15% was unreacted
Purification of Rabbit Anti-hGSTz Ic-lc Antibody.
As described in Chapter 2, serum from rabbits which had been given GSTz Ic-
Ic as an antigen was purified by protein A. This purified antibody was then used as a
primary antibody against cytosolic as well as recombinant GSTz to determine its
purity and cross reactivity. Western blots of recombinant GSTz Ic-lc showed that
this antibody shows strong cross-reactivity with the protein. It is a very sensitive
antibody and can detect as low as 10 ng of the recombinant purified protein (Fig 5-
16). It could detect cytosolic GSTz even in 5 ug of cytosolic protein (Fig 5-17).
This antibody was used to immunoprecipitate GSTz from liver cytosol. If
successful the protein thus obtained could then be used in MS experiments. The same
technique could also be used to isolate adducts of GSTz with DCA. These in vivo
adducts when analysed by MS could provide important information about the
mechanism of DCA's metabolism and inactivation of GSTz.
Attempts to immunoprecipitate GSTz from cytosol using the method described
in chapter two were not successful. The eluate from the protein A column cross-
linked to the antibody, when analysed by western blotting did not show the presence
of GSTz. The elution buffer used had a low pH (2.8) which may have destroyed the
protein. A gentle elution buffer, which does not have a low acidic pH, from Pierce
was used to ensure that GSTz was not destroyed during elution. But even with this
elution system no immunoprecipitated GSTz was recovered. This could be due to
inefficient binding of the antibody to GSTz or even due to too strong antigen-
48 kDa -- O
1 2 3 4 5
Fig 5-16 Western blot of rat liver cytosol using purified rabbit-anti human GSTz.
Lanes 1 to 5 were loaded with 40, 20, 10, 5 and 1 tg of cytosolic protein. Antibody
cross reacted with GSTz in all except the 5th lane. In lanes 1 and 2 a high molecular
weight band can be seen. This could be another protein or a dimer of GSTz.
1 2 3 4
Fig 5-17 Western blot of recombinant GSTz Ic-lc using purified rabbit anti-human
GSTz as primary antibody. Lanes 1 to 7 were loaded with 10, 15, 20, 25, 50, 80, and
100 ng of GSTz Ic-lc. The antibody can detect as low as 10 ng of the protein. In
lanes 5-7 another high molecular weight band is observed which may be the dimer of
Dichloroacetate is an important environmental chemical which is used clinically for
the treatment of metabolic disorders such as congenital lactic acidosis. It is also used to
treat acquired lactic acidosis due to malaria and other diseases. DCA facilitates lactate
removal by activating pyruvate dehydrogenase complex (PDH) by inhibiting PDH-kinase
(Reviewed in Stacpoole et.al 1998a,b). DCA is found in drinking water as a by-product
of chlorination and is a metabolite of important industrial solvents such as
trichloroethylene and tetrachloroethylene. Haloacetates are ubiquitous in the environment
and have been found in fog, rain, ponds, lakes etc (Rompe et.al. 2001, Karlsson et.al.
2000). DCA treatment (0.2-2.0 g/1 in drinking water for 2 weeks) reduced serum insulin
levels (Lingohr et.al. 2001). The decrease persisted for at least 8 weeks. Repeated
administration with DCA prolongs its plasma half-life in humans and rodents several
fold. This change in pharmacokinetic properties is due to inhibition of its metabolism as
shown by James et.al 1997, 1998, Ammini et.al. 2003 and in this work. DCA inhibits
GSTz, its metabolizing enzyme in vitro and in vivo in a dose dependent manner. Loss of
activity is accompanied with a loss of immunoreactive protein.
GSTz is an important enzyme in tyrosine catabolism and its inhibition may lead to
accumulation of tyrosine metabolites such as MAA and FAA. These are reactive
metabolites which may react with the enzyme leading to further inactivation. To
examine the role of these metabolites in GSTz inactivation, we blocked tyrosine
catabolism by NTBC, prior to MAA formation. NTBC inhibits the second enzyme in
tyrosine catabolism pathway (Fig 1-3); 4 hydroxyphenyl pyruvate dehydrogenase. This
in turn inhibits the formation of reactive tyrosine metabolites such as MAA and MA
downstream in the pathway. Inhibiting the formation of these metabolites may help
protect GSTz. MAAI knock out mice develop hepatic and kidney toxicity when given
high tyrosine diet (Fernandez-Canon et.al. 2002). This phenotype was reversed by
administering NTBC demonstrating the potentially harmful effects of tyrosine
In the present study NTBC was administered prior to DCA and enzyme activity
and expression were studied. But in this study no protective effect was observed at the
dose and treatment period studied. We conclude that effect of DCA on GSTz is the
predominant factor in inactivation and subsequent destruction of the enzyme. The in-
vivo role of MA in inhibiting the enzyme might be small or perhaps MA does not
accumulate to a concentration at which it can have serious effects on the enzyme. This is
in accordance with other reports which show that incubation of cytosolic or recombinant
GSTz with DCA in vitro inhibits the enzyme. Furthermore recent studies have shown
that in-vitro inhibition of cytosolic GSTz by MA is partially reversed by GSH (Ammini
et. al. 2003). Inactivation of polymorphic variants of GSTz by MA and FA was studied
by Lantum and co-workers. They found that all the variants were inhibited by both MA
and FA in the absence of GSH. But when similar experiments were done in the presence
of saturating concentrations of GSH (ImM) the enzymes retained their activities, which
suggested that GSH plays a protective role in this interaction. The mechanism for this
protective action is not known.
This is an important factor in the present context since the cellular concentration of
GSH (5mM) is saturating for GSTz because of its low Km for GSH (0.075mM, James et
al. 1997) Thus inhibition of GSTz by MA and FA in the body is only possible if the
cellular levels of GSH fall below the Km of GSH. Certain diseases, such as hereditary
tyrosinemia type-I, are associated with low hepatic glutathione concentrations (Lloyd
et.al. 1995). Many xenobiotics form glutathione-conjugates and, thereby, reduce cellular
glutathione concentrations (Lambert et.al 1976). But even in these cases the possibility
of the GSH concentrations falling below the Km is rare. This information supports the
conclusion that inhibition of GSTz by MA does not play a major role in inactivating the
enzyme and inhibition is related DCA.
It was also observed in this study that GSTz activity and protein levels were lower
in rats treated with NTBC alone compared to control. Thus NTBC itself appeared to
have an effect on the enzyme. This was unusual since NTBC itself is not known to
inhibit GSTz. We postulate that this depletion of GSTz may be related to depletion of
MAA and MA, the endogenous substrates of the enzyme. Reduced availability of its
biological substrates may lead to decreased expression of this enzyme. This may be due
to either decreased transcription of message or a decrease in translation of the message.
Future studies could address this issue by performing northern blots on liver mRNA
from rats treated with NTBC. These experiments may provide information on GSTz
message levels in NTBC treated and control animals.
Although inactivation of GSTz by DCA has been studied before, recovery of
enzyme activity and protein after long term administration has not been investigated.
Determining the time required to restore GSTz activity after chronic dose is important
for understanding DCA metabolism and pharmacokinetics. To study the recovery of
activity and GSTz protein we used male Sprague Dawley rats as models. These animals
were exposed sub-chronically to clinically relevant dose of DCA (50mg/kg/day) and
restoration of GSTz was studied. Treatment with DCA decreased enzyme activity and
expression after one week of exposure and remained suppressed during the 8 week
treatment period. Once the drug was withdrawn enzyme activity increased gradually.
Activity remained below control levels after 2 weeks of withdrawing the drug. It took up
to 4 weeks for the enzyme activity to return to control levels. The loss of activity of
GSTz due to DCA treatment correlated with the loss of immuno-reactive protein
observed in the westerns. This suggested that inactivated GSTz was degraded by
proteolysis. Recovery of activity and protein followed different patterns. Protein
expression did not return to control levels after 4 weeks of recovery. In fact amount of
immunoreactive protein remained significantly lower than control even after 8 weeks of
withdrawing DCA. Partial restoration (approximately 65% of controls) of DCA
elimination capacity and hepatic GST-zeta activity occurred after 48 h recovery from 14
d 2.0 g/1 DCA drinking water treatments in B6C3F1 mice (Schultz et.al. 2002).
Pharmacokinetic studies in humans have shown that a wash-out period of several weeks
is necessary before the kinetics return to pre-treatment levels (Stacpoole et.al. 1998).
This indicates a species dependent difference in the rate of protein re-synthesis.
Anderson et. al. (1999) studied the turnover of GSTz protein after a single dose of DCA
in Fisher rats. They reported the tl/2 of protein recovery to be 3 days. According to this
study GSTz activity and expression returned to pre-treatment levels 10-12 days after the
exposure. However our studies show that activity does not return to initial levels even
after 2 weeks of withdrawing the drug. We see a return to control levels only after 4
weeks of recovery. The protein expression does not return to pre-exposure levels even
after 8 weeks of recovery. The report by Anderson and co-workers also studied the
recovery of GSTz protein which paralleled the recovery of GSTz activity. This
discrepancy in our data and the data published by Anderson et.al. may be a function of
the prolonged exposure period we used in this study. After 8 weeks of exposure the
enzyme activity and expression are at very low levels and hence a longer recovery
period is necessary for the enzyme to regain its activity. Schultz et. al. (2002) reported
that interrupting protein re-synthesis by actinomycin-D blocked the recovery of GSTz
activity. These studies show that, not just removal of the inhibitor but re-synthesis of
protein is essential to regain the activity of the protein. This study further showed that
effects of DCA are long lasting and that re-synthesis of the enzyme is gradual. The
reasons for this slow synthesis of protein are currently unknown. Since DCA is excreted
unchanged in the urine, it is unlikely that it lingers in the body and continues to
inactivate the enzyme. It is possible that DCA or its metabolites may interfere with
pathways of protein synthesis.
DCA treatment resulted in decreased maternal weight gain and increase in liver
weight in pregnant rats (Smith et.al. 1992). This increase in liver weight after DCA
exposure was attributed to possible hyperplasia or hypertrophy. Our studies show an
increase in liver to body weight ratio in rats administered 50mg/kg DCA. But this ratio
returned to control levels once DCA was withdrawn. It is not known whether increase in
liver weight was due to hypertrophy related to DCA exposure. Histopathology on liver
tissue from these studies may be useful in determining whether DCA treatment led to
High doses of DCA (50mg/kg) led to accumulation of MA in the liver which was
excreted in the urine. Cornett et. al. (1999) had also shown a similar excretion of MA in
the urine of rats treated with 200mg/kg DCA. This indicated DCA treatment perturbed
tyrosine and led to accumulation of tyrosine metabolites. However MA was not detected
in the urine after one week of withdrawing the drug. This is an interesting observation
since GSTz which metabolizes MA was not completely recovered after one week of
recovery. About 55% of GSTz activity and only 30% of GSTz protein recovered after
one week of withdrawing DCA. This suggests that even low levels of GSTz are perhaps
sufficient to metabolize endogenous MA and MAA resulting from tyrosine catabolism.
Another reason for not detecting MA in the urine samples is probably the stability of this
compound. We have observed that synthetic MA is not a stable compound and can
isomerize to FA under aqueous conditions or degrade to other products. Thus it is
possible that urinary MA may have degraded and hence its concentration was below the
detection limit of our method. Urinary DCA levels increased during the 8 week
treatment period. This increase in DCA levels showed that loss in GSTz activity
inhibited the metabolism of DCA. The levels of monochloroacetate (MCA) were very
low suggesting that conversion to MCA was not the predominant pathway of
metabolism and that dechlorination to glyoxylate is the major route of
Exposure to DCA is mainly due to its presence in municipal drinking water or by
ground water contamination, as observed at certain superfund sites. Daily exposure to
DCA in non clinical setting is typically around 4tg/kg but may be exceeded at some
sites. Previous studies with DCA have predominantly used a clinical dose of the drug
(25mg/kg to 50mg/kg), which is several times higher than its concentration in
environment. Since human exposure to DCA is widespread at ptg/kg/day dose, it is
important to determine the effects of DCA at this environmental dose. The present study
has addressed these important aspects of DCA metabolism. In these studies exposure to
DCA in the environment was mimicked by exposing rats to DCA in drinking water.
Commercially available mineral water (Zephyrhills) was used to prevent the influence of
DCA present in municipal drinking water. Analysis of this water showed that DCA
concentrations in the water were below our method detection limits (25-1000 pg/ml, Jia
et.al. 2003). It was observed that low doses of DCA significantly inhibited GSTz activity
and expression. This effect was more pronounced in rats treated for longer duration
suggesting a cumulative effect of exposure to DCA. This finding suggests that DCA
concentrations found in some municipal drinking water alters hepatic GSTz. This is a
significant discovery since it has important implications on human health. GSTz is an
important enzyme involved in tyrosine catabolism and its inhibition may lead to
accumulation of reactive tyrosine metabolites and toxicity related to such metabolites.
Finally we also looked at identifying possible adducts of DCA with recombinant
hGSTzlc-lc which was expressed in E-coli. Adduct of the enzyme with DCA was
identified and separated from unadducted enzyme by HPLC. This adduct was further
characterized using various mass spectrometry methods. At least 3 adducts of the protein
with GSH were identified and two of them were further characterized by MS/MS. In
both cases thiol of GSH formed a covalent disulphide bond with the thiol of a cysteine
residue. Enzyme modeling studies showed that the cysteine 16 residue in the active site
is located 2.8 A from GSH (Polekhina et.al. 2001). Although this distance is not close
enough to form a disulphide linkage we demonstrate the formation of a disulphide
adduct of the thiol of GSH with the cysteinyl sulphur of cysteine 16. Anderson et. al.
(2002) also showed the presence of similar adduct. A disulphide adduct of GSH was also
identified with Cys 205 by MALDI-TOF. Cys 205 has been shown to react with MA and
FA and cause inactivation of the enzyme (Lantum et.al. 2002b). This cysteine was also
shown to bind to DTT when this compound was modeled in the crystal structure
(Polekhina et.al. 2001). Catalytic significance of these adducts is unknown. Glutathione
adducts have physiological importance since GSH may bind to the enzyme under
cellular conditions. Similar adducts of GSH with GSTO1, GSTM4 and PmGSTB1 has
also been reported (Board et.al. 2000, Rossjohn et.al. 1998, Cheng et.al. 2001)
Another interesting adduct was that of glyoxylate with the active site serine. The
mechanism of this reaction may involve the hydroxyl of the serine which may act as a
weak nucleophile and attack the carbonyl carbon of the acid. The resulting adduct has a
hemiacetal like structure. GST proteins have a serine or a tyrosine residue in their active
sites. These residues bind GSH and thus activate the thiol for reaction with xenobiotic
substrates. Any modification of this residue may lead to inactivation and subsequent
degradation of the protein. This adduct is also important because it demonstrates the
ability of glyoxylate to react with protein nucleophiles. Reaction of glyoxylate with
cellular proteins could possibly lead to toxicity.
It was surprising that glyoxylate did not react with cysteine since the cysteinyl
thiol has a lower pKa than the hydroxyl group and is a stronger nucleophile. This result
was consistent with a previous report in which glyoxylate was reacted with cysteine
containing peptides. Glyoxylate did not react with the cysteine but reacted with other
amino acid nucleophiles (Anderson et.al. 2004). Active sites of GST are characterized
by the presence of serine and cysteine residues. The crystal structure of GSTz showed
that serine at position 15 is oriented more favorably for interaction with GSH and
xenobiotic substrates and that the hydroxyl of serine 14 points away from the active site
(Polekhina et.al. 2001). But site directed mutagenesis showed that both the series are
essential for isomerase and GSH conjugation activities (Board et.al. 2003). Alignment of
the hGSTZ1 sequence with other GSTs suggested that Ser-14 aligned with the active-
site Ser-11 in the Theta-class GSTs and Ser-9 in the insect Delta class (Board et.al.
1997). The active site of GSTz isolated from Arabidopsis thaliana shows series at
positions 17 and 18 and a cysteine at position 19 (Thom et.al. 2001). The mutation of
Ser-17 in the A. thaliana Zeta-class GST (equivalent to Ser-14 in hGSTZ1) reduces its
activity to <2% and <6% of the wild-type activity with DCA and MA respectively.
Above information suggests that zeta class of GSTs have a conserved SSC motif in their
active site. All these residues play a role in either binding GSH or stabilizing the thiolate
of GSH. Thus any modification of these residues, chemical or genetic may lead to
inactivation of the enzyme.
Serine residues present in active sites have been known to form hemiacetal like
tetrahedral adducts with enzyme inhibitors. Peptide aldehydes which are substrates for
serine proteases react with the hydroxyl group of active site serine to form hemiacetals
(Kahayaoglu et.al. 1997). Aldehyde type peptide inhibitors react with serine residues in
pseudomonas carboxyl proteinase and hemiacetal type linkages and thus inactivate the
enzyme (Oyama et.al. 2002). In our study also we find a similar reaction between the
aldehyde of glyoxylate and the hydroxyl of serine to form a hemiacetal. Patil et.al (1989)
demonstrated the formation of glyoxylate adducts with the active site of threonine
dehydratase. It was proposed that this covalent interaction with the active site residues
inactivates the enzyme.
Glyoxylic acid and its esters used in organic chemistry are stored as their hydrates
and hemiacetal like compounds to avoid the rapid polymerization of the aldehyde.
Glyoxylates are susceptible to nucleophilic attack by heteronucleophiles which results in
the formation of the corresponding acetal like compounds (Meester et.al. 2003).
Other adducts of the enzyme with reaction intermediates were also observed. But
these were not formed with the active site residues. Reactions occurred with aspartic
acid residues of the protein. This may have been a reaction between glyoxylate and the
carboxylic acid moiety of the aspartic acid. The two carboxylic acid groups may form an
anhydride which will result in the mass increase of 57 Da. These findings suggest that
glyoxylate is a reactive metabolite and is involved in covalent interactions with the
enzyme. It is also clear that there are several sites on the enzyme which can be modified.
Importance of these modifications in inactivating the enzyme is unknown. These adducts
may target the enzyme for proteolysis.
/ + HO H-
0 0 NH
Fig 6-1 Proposed reaction between Serine and Glyoxylate. This reaction results in
addition of 74 Da to the protein
-I OH HO H
,AYo + )/
NH 0 / \
NH 0o o
Fig 6-2 Reaction of glyoxylate and aspartic acid to form anhydride. This covalent
interaction leads to mass increase of 56 Da
This study did not however identify any adducts of the active site peptide with
DCA itself or with a reaction intermediate. Anderson et.al. (2002) identified an adduct
of Cys 16 in the active site with S (chlorocarboxymethyl glutathione) which they
suggested may be the reason for inactivation of the enzyme. This study did not observe
this adduct. It either may be a low abundance adduct or an unstable intermediate due to
which is difficult to identify by the methods used in these studies. Future research could
address isolating and purifying this enzyme and any adducts with DCA or its metabolites
from cytosol of control and treated rats. This protein could then be characterized by
mass spectroscopy. This will help identify the exact mechanisms of DCA's inhibition of
Biological significance of adducts of glyoxylate with amino acids is not known.
The toxicity of ethylene glycol has been attributed to its metabolite, glyoxylate
(Richardson 1973). Glyoxylate caused partial inactivation glucose 6 phosphate
dehydrogenase (Anderson et.al.2004). Aldehyde containing xenobiotics are known to
play a role in toxicity. Croton aldehyde derived from crotyl alcohol was found to
contribute to toxicity by forming adducts with cellular biomolecules (Fontaine et.al.
2002). A range of rat liver cytosolic proteins showed C14 label derived from C14DCA
(Anderson et.al. 2004).This raises the possibility that metabolism of DCA by GSTz may
generate a bioactive metabolites which may react with biomolecules.
Toxicology and inactivation of GSTz by DCA is a complex mechanism which
warrants further research. The involvement of MAAI in DCA toxicity is still unclear.
Exposure to DCA and subsequent inactivation of MAAI may trigger other pathways
leading to toxicity.
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Vaishali Dixit, daughter of Suresh and Anita Dixit, was born and raised in
Mumbai (formerly Bombay), India. She completed her schooling at Gokhale High
School, and went to Pune college of Pharmacy, Pune, India, where she received
bachelor's degree in phamacy. After graduation she worked as an intern at Glaxo
Pharmaceuticals, India for a year. In January of 2000 she was accepted at the
University of Florida for a doctoral degree in medicinal chemistry. After graduation
she plans to work as a drug metabolism scientist in a drug company or continue her
training with a postdoctoral position.