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Characterization of the hydrolysis of glutathione esters in rat liver lysates and their importance in multi-drug resistance

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Title:
Characterization of the hydrolysis of glutathione esters in rat liver lysates and their importance in multi-drug resistance
Creator:
Tucker, Douglas A
Publication Date:
Language:
English
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60 leaves : ; 28 cm

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Subjects / Keywords:
Glutathione ( lcsh )
Esters ( lcsh )
Drug resistance in cancer cells ( lcsh )
Drug resistance in cancer cells ( fast )
Esters ( fast )
Glutathione ( fast )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Bibliography:
Includes bibliographical references (leaves 54-60).
Thesis:
Department of Chemistry
Statement of Responsibility:
by Douglas A. Tucker.

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|University of Colorado Denver
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|Auraria Library
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ocm51821323
Classification:
LD1190.L46 2002m .T82 ( lcc )

Full Text
CHARACTERIZATION OF THE HYDROLYSIS OF GLUTATHIONE
ESTERS IN RAT LIVER LYSATES AND THEIR IMPORTANCE IN
MULTI-DRUG RESISTANCE
by
Douglas A. Tucker
B.S., Metropolitan State College of Denver, 1996
A thesis submitted to the
University of Colorado at Denver
in partial fulfillment
of the requirements for the degree of
Master of Science
Chemistry
2002


This thesis for the Master of Science
degree by
Douglas A. Tucker
has been approved
by
Date


Tucker, Douglas Arthur (M.S., Chemistry)
Characterization of the Hydrolysis of Glutathione Esters in Rat
Liver Lysates and Their Importance in Multi Drug Resistance
Thesis directed by Assistant Professor Ellen J. Levy
ABSTRACT
Selective modulation of glutathione, utilizing glutathione(mono)glycyl-
esters and buthionine sulfoximine may provide a novel approach to overcoming
multi-drug resistance in cancer. The characterization of the hydrolysis of
glutathione monoesters is the first step in the eventual identification of the
enzymes responsible for the hydrolysis of the glutathione esters. It was the goal
of this research to provide the initial characterization of the ethyl, propyl, n-
butyl, isobutyl, and benzyl esters in microsome and cytosol preparations. A
special emphasis on the n-butyl ester allowed for the determination of the
maximum velocity, and Michaelis constant for that esters hydrolysis. Insight
into the active sites of the responsible enzyme(s) may provide the basic
groundwork to begin the isolation of the hydrolytic activity and possibly open
avenues for the creation of novel chemotherapeutic approaches.
This abstract accurately represents the content of the candidates thesis. I
recommend its publication.
Signed_
Ellen J. Levy
m


DEDICATION
I wish to dedicate this thesis to my wife, Laura, for her intelligent observations,
loving support, and characteristic patience while I was writing this. I would
also like to dedicate this to my mother, for instilling in me the hunger for
knowledge that has defined much of my life.


ACKNOWLEDGEMENT
I wish to extend my thanks to my thesis advisor, Dr. Ellen Levy, for her
unerring direction and patience. I also wish to thank my fellow researcher
Mindi Hogan, without whom this thesis project would not have been possible.


CONTENTS
Figures...............................................................yin
Tables................................................................ix
Charts x
Chapter
1. Introduction......................................................01
1.1 Research Goal....................................................01.
1.2 The Characteristics of Glutathione................................01
1.3 Glutathione History...............................................03
1.4 Biochemistry of Glutathione......................................05
1.5 Glutathiones Redox Potential....................................08
1.6 Glutathiones Role in Biological Systems.........................08
1.7 Cancer...........................................................10
1.8 Radiotherapy and the Role of Glutathione.........................12
1.9 Chemotherapy Types and Mechanisms................................14
1.10 Multi Drug Resistance...........................................16
1.10.1 The Role of Glutathione in Multi-Drug Resistance...............20
1.11 Glutathione Transport...........................................22
1.12 Glutathione Repletion Strategies................................23
1.12.1 Glutathione Esters as Thiol Delivery Vehicles..................24
1.13 Glutathione Depletion Strategies................................27
1.13.1 Chemical Depletion.............................................27
1.13.2 Depletion Through Inhibition of Synthesis......................28
1.14 The Promise of Selective Modulation of Glutathione Levels.......29
vi


2. Experimental.................................................... 30
2.1 Data Generation and Handling....................................30
2.2 Equipment and Materials.........................................31
2.3 Buffer Compositions............................................ 32
2.4 Preparation of Rat Liver Microsome and Cytosol Lysates.......3.2
2.4.1 Evaluation of Cytosol and Microsomes for Total Protein Content..3.3
2.4.2 Evaluation of Cytosol and Microsomes for Esterase Activity......3.4
2.5 Determination of Percent Hydrolysis for the GSH-Monoesters....3.4
2.5.1 Tietze Assay................................................3.5.
2.5.1.1 Influences on the Tietze Assay.............................3.7
3. Results.........................................................3.8
3.1 Microsome Assay Results....................................3.8
3.2 Cytosol Assay Results...........................................41
3.3 Determination of VMax and Km for n-Butyl Ester in Cytosol.......44
4. Discussion .... .....................................4.9
References
54


FIGURES
Figure
1.1 Glutathione.......................................................02
1.2 Glutathione Disulfide.............................................03
1.3 GSH GSSG Redox Summary..........................................06
1.4 Central Role of the Glutathione Pool in Cellular
Resistance Mechanisms..........................................18
1.5 Glutathione Monoester............................................24
1.6 Glutathione Diester..............................................25
1.7 Buthionine Sulfoximine...........................................28
2.1 The Structure of DTNB............................................36
2.2 The structure of 2-nitro-5-thio benzoic acid 36


TABLES
j Tables
3.1 Free GSH Values for the Microsome Series Controls................38
3.2 Percent Hydrolysis of Glutathione Esters with Microsomes........39
i 3.3 Free GSH Values for the Cytosol Series Controls..................42
| 3.4 Percent Hydrolysis of Glutathione Esters with Cytosol...........42
j
| 3.5 n-Butyl VMax and Km Values......................................45
!
IX


CHARTS
Charts
3.1 Summary of the Microsomal Percent Hydrolysis as a Function of Time
for GSH Ethyl, Propyl, Isopropyl, n-Butyl, and Benzyl Esters..40
3.2 Summary of the Cytosolic Percent Hydrolysis as a Function of Time
for GSH Ethyl, Propyl, n-Butyl, and benzyl Esters.............43
3.3 Lineweaver-Burke; 1 / [S] vs. 1 /v..............................46
3.4 Eadie-Hofstee, v vs. v/[S.......................................47
3.5 Hanes; [S] vs. [S]/v.............................................48
x


1. Introduction
1.1 Research Goal
Glutathione esters have proven themselves efficient cellular thiol
delivery vehicles. Attempts at selective modulation of glutathione levels may
be potentially useful in overcoming multi-drug resistance in cancer.
Determination of the rates of hydrolysis of monoesters of glutathione is the first
step in the eventual identification of the enzymes responsible for the hydrolysis
of the GSH esters. Knowledge of the percent hydrolysis, the Michaelis
constant (Km), and the maximum velocity (VMax) for the hydrolytic reactions,
would allow for a characterization of the affinities of these enzymes toward the
differing glutathione monoesters. It was the goal of this research to provide the
basic characterization of the ethyl, propyl, butyl, isobutyl, and benzyl glycyl
esters in microsome and cytosol preparations. Additionally, maximum
velocity, and the Michaelis constant was determined for the n-butyl ester. From
this information, insight into the active sites of the responsible enzymes was
gained.
1.2 The Characteristics of Glutathione
Glutathione, although small, is nonetheless a peptide of legendary
proportions. Glutathione is a tripeptide thiol with important antioxidant ability.
It is made of the amino acids glutamic acid, cysteine, and glycine. Glutathione
is synonymously known as reduced glutathione, and gamma-L-glutamyl-L-
cysteinylglycine. It exists in two primary forms, the reduced glutathione (GSH)
form (see Figure 1.1) and its oxidized glutathione disulfide (GSSG) form (see
1


Figure 1.2). Glutathione is omnipresent, being found in the aqueous cellular
environs of plants, animals, and bacteria. It is essential not only to the
maintained health and longevity of cells and organisms including humans, but
indeed is an absolute necessity for survival. Glutathione is an important bio-
molecule that wears many hats. It is involved in such diverse biological
processes as protein synthesis (through use of its amino acid components),
maintenance of protein thiol moieties and antioxidants reducing power (like
ascorbate), the reduction of ribonucleotides to form the deoxyribonucleotide
precursors of DNA, and in enzyme catalysis (acting as cofactor for many
enzymes). It also acts to moderate the redox balance of the cell, largely through
the regulation of 2, R-SH
-> R-S-S-R, this helps
regulate transmembrane
transport (the transport Ca-
ATPases), and it moderates
eight enzymes involved in
glucose metabolism. In
addition, metallothioneins
(proteins that bind heavy
metals and sulfhydryl
poisons to aid in their
removal) are induced, their levels augmented, by the oxidative challenge of the
intracellular environment, which is moderated by GSH. Tubulin (a protein that
makes up microtubules, structural components of cells and cell bodies), and
thioredoxins (thioredoxin system is responsible for Calvin cycle regulation and
to some extent glycolytic regulation at phosphofructokinase), are regulated by
glutathione through -SH groups at or near their active sites. Without
2


glutathione or with glutathione levels too low, an organism will die. Indeed,
experimental depletion of glutathione has been shown to induce apoptosis
(programmed cell death). Given such a central role, a long history of
glutathione study and understanding would be expected. To be sure, the roots
of our current knowledge of glutathione reach back over a hundred years. 786>
11, 13, 14
1.3 Glutathione History
The discovery and
characterization of
glutathione began in the
late 1800s. J. De Rey-
Pailhade performed
experiments demonstrating
that hydrogen sulfide is
formed when yeast is
smashed in the presence of
elemental sulfur, indicating
the presence of a reducing
agent. His research led
him to report that the
substance responsible for
this conversion was found
in many tissues. He
showed it bleached certain
dyes, and reacted with


i
!
some halides. This new substance was named philothion by him, drawn from
the Greek words for love and sulfur. In 1921, philothion was renamed
glutathione, after the discovery that it contained both glutamine and cysteine.
Relatively soon thereafter, the structure of glutathione was determined through
synthesis and chemical studies, and its tri-peptide structure was known by 1927.
Awareness of the importance and ubiquitous nature of glutathione was
increased in the early 1980s with a virtual explosion of research interest in the
tripeptide. This research has led to a good understanding of glutathiones
'j
primary roles and metabolism in biological systems.
I
4


1.4 Biochemistry of Glutathione
As mentioned above, glutathione exists in two primary forms. The first
and most abundant is its reduced form (see Figure 1.1). The second oxidized or
binary form is termed glutathione disulfide (GSSG, see Figure 1.2).
Glutathione is oxidized to glutathione disulfide via several mechanisms; these
mechanisms are reasonably well understood. The first of these mechanisms is
via peroxidases, which utilize GSH to reduce peroxides. Equation 1.1 is an
example of this with hydrogen peroxide.
2GSH + H202 GSHPerox-ses > GSSG + 2H20 (Equation 1.1)
Another route of oxidation of GSH to GSSG is via free radicals. These
reactions are non-enzymatically catalyzed. They occur with a high frequency in
the mitochondria, where reactive oxygen species are regularly created. An
example of this is the reaction of GSH with the especially damaging hydroxyl
radical.
2GSH + 2HO* -> GSSG + 2H20 (Equation 1.2)
Glutathione transhydrogenase can also, among other reactions, oxidize or
reduce the pair by interconverting them between GSH and GSSG.
2GSH < GSH Transhydrogenase^ QggQ (Equation 1.3)
5


The rejuvenation of glutathione disulfide to glutathione is achieved via
glutathione disulfide reductase utilizing NADPH and H+, as given in Equation
1.4.
GSSG + NADPH + H+ -GSSG R**Sg-> 2GSH + NADP+ (Equation 1.4)
A redox summary of the cyclic oxidation and reduction of GSH and GSSG is
given in Figure 1.3.'
The synthesis and degradation pathways of glutathione are also
reasonably well understood. Glutathione is enzymatically synthesized in cells
by y-glutamylcysteine synthetase, which constructs y-glutamylcysteine from
glutamic acid (utilizing ATP, see Equation 1.5). The addition of glycine by
glutathione synthetase (also utilizing ATP) immediately ensues (Equation 1.6).
The process is feedback inhibited at the first step by glutathione. '
6


Cys + Glu + ATP
y-glutamylcysteine synthetase
* y-Glu-CysH + ADP + P, (Equation 1.5)
Y-Glu-CysH+ ATP Glutathl0ne synlheta5e > y-Glu-CysH-Gly +ADP + P, (Equation 1.6)
The degradation of glutathione, glutathione disulfide, and sulfur
conjugates of glutathione is accomplished by y-glutamyl transpeptidase, a
membrane bound protein. y-Glutamyl amino acid and cysteinylglycine are
produced (see Equation 1.7).
y-Glu-Cys-Gly > Glu + CyS-Gly (Equation 1.7)
The cysteinylglycine thus formed is split by membrane-bound (and cytosolic)
dipeptidases in the final degradative step producing cysteine and glycine (see
Equation 1.8) that are fed back into the synthesis cycle or utilized elsewhere.
Cys-Gly --!raM'dase > Cys + Gly (Equation 1.8)
7


1.5 Glutathiones Redox Potential
The basis for glutathiones reducing power lies in the standard reduction
potential of the redox couple GSH / GSSG. The more positive the standard
reduction potential, the greater the driving force for reduction. The redox
potential of the GSH / GSSH couple is E0 = -0.330v. To put this in context,
compare this to the redox potential of the powerful and ubiquitous NADH /
NAD+ couple, which has a redox potential of E0 = -.320v. 459
1.6 Glutathiones Role in Biological Systems
The role of glutathione in biological systems is associated with its high
electron donating capacity, and therefore its thiol (-S-H) group (see Figure 1.1).
Glutathiones free radical scavenging ability, sulfhydryl donation capacity,
electron donating characteristics, and millimolar cellular concentrations are the
reasons that glutathione plays a large role in cells as an antioxidant and
reductive agent. In cells, it exists primarily as GSH. In fact, the glutathione
profile of healthy cells usually includes only about 1 %-10% GSSG. This ratio
is considered by many to be one of the paramount indicators of cellular health
and oxidative stress. 10
Of all mammalian cells and tissue types, GSH is most concentrated in
the liver, an organ with numerous roles including various
8


degradative and detoxification functions. The high liver concentration is
partially because it is used in conjunction with NADPH by the p450 phase II
enzymes. The cytochromes p450 are an extensive family of oxidative
detoxifying enzymes. These enzymes hydroxylate hydrophobic molecules to
more soluble products for excretion. GSH is needed in the liver for conjugation
with these modified toxins and for multitudes of other detoxifying and
oxidative challenges presented there. 5
The liver has two pools of glutathione. The first has a slow turnover,
with a half-life of about two to four hours. This population of GSH is found in
the cytosol of liver cells. Among other things, it is utilized as a cofactor for
GSH Transhydrogenases in detoxification pathways, after cytochromes p450
activity (GSH Transhydrogenases catalyze the conversion of GSH to differing
S-substituted addition products and interconverts GSH and GSSG (see Equation
1.3). This cytosolic pool is also utilized as a substrate for gamma-glutamyl
transpeptidases, which are enzymes located on cell surfaces and are responsible
for transferring the glutamine moiety from GSH to other amino acids for
subsequent uptake into the cell. The second pool of GSH has a much shorter
half-life, approximately 30 minutes. It is found in the mitochondria of cells.
This glutathione is used to fight the large numbers of highly reactive and
damaging oxygen species (reactive oxygen species, ROS) which include
superoxide, hydroxyl radical, and peroxides created during oxidative
phosphorylation. 611
Glutathiones role as antioxidant is of paramount importance. It is
estimated that two to five percent of all the electrons that pass through the
oxidative phosphorylation system are converted to superoxide and other oxygen
radicals. The oxidative phosphorylation system processes at least 95 percent of
9


the oxygen used in the body, and therefore this radical generating mechanism
and its control are metabolically significant. If it is not countered (primarily by
GSH), the damage to DNA, proteins, and phospholipids (responsible for
membrane integrity) would be large and detrimental to the cell. The
accumulated effect of oxidative damage on cell systems has been linked to
degenerative diseases such as atherosclerosis and the progressive loss of organ
function we know as aging. 61 l3,14
Glutathione also plays a role as a systematic antitoxin. Normally, GSH
levels are low extracellularly, but there is an exception to this in the lungs. In
the lower regions of the lungs where pollutants and toxins can accumulate,
levels of extracellular GSH are higher. In this area, glutathione uses its
reductive abilities to protect the lung tissues from free radicals generated from
inhaled toxins (like tar from cigarettes) and from the by-products of active
phagocytic processes which often utilize peroxides to kill invading organisms.
Glutathione levels are also higher in the intestines and kidneys where exposure
to toxins is more frequent. Glutathione is certainly multifunctional, but it plays
a role in addition to those it carries out in the lungs, kidneys, liver, intestines,
and general cellular functions, this is the role it plays in disease. One of the
most important diseases in which glutathione acts out a role is cancer. 61314
1.7 Cancer
Cancer is not a single disease; it is a term that applies to more than one
hundred conditions of progressive and uncontrolled (neoplastic) growth whose
natural course is fatal. Cancer cells, unlike benign tumor cells, exhibit the
properties of invasion and metastasis (the transfer of disease from one organ or
system to another). Cancer is possible in nearly every tissue of the body; some
10


tissues can even yield several different types of malignancies. To add to the
complexities of the disease, each type of malignancy has its own specific
features and characteristics. Cancer includes the three large divisions of
carcinoma, sarcoma, and leukemia. These three divisions represent many
different types of malignant tumors. A carcinoma is a tumor that arises from
epithelium (tissue that covers internal and external surfaces of the body).
Adenocarcinoma is an example of a carcinoma; it arises from glandular
epithelium. Sarcomas are tumors that arise from connective tissue (bone,
blood, cartilage). Chondrosarcoma is an example of a tumor arising from
connective tissue (cartilage). The third major division, leukemia, is classified
as such primarily because of historical and clinical reasons, since in the
absolute broadest sense it is a sarcoma, originating from blood tissues. ,516,17
18, 19,20
Cancer cells arise because they ignore the growth regulation signals of
the body, derived from two primary gene types. They begin to proliferate in
accordance with their own internal agenda. Eventually, cancerous cells can
develop the ability to escape their current locale and migrate to different areas
of the body to setup malignancies there. These types of tumors become more
and more aggressive over time and they eventual disrupt the organs and
systems in which they inhabit, and can prove fatal. Traditionally, surgery was
the first and primary treatment for cancer of nearly all types and today it still
plays an important role. However, the treatment of cancer has become much
more complex and survival rates have increased dramatically through
technological improvements in radiotherapy, and new promising drugs and
biologicals capable of relieving the toxicities of anticancer agents. 16,718
11


1
1.8 Radiotherapy and the Role of Glutathione
Radiation has played a role in medicine since its discovery a century
ago. Modem radiotherapy is used to treat many types of cancer, and commonly
utilizes x-ray and y-radiation. When radiation interacts with matter, the energy
of the radiation is transferred to the matter in any of several mechanisms. The
mechanism that is of the highest importance in biological tissues is called
Compton scattering. 19
Compton scattering can be simply thought of as a collision between the
photon, or quantized energy of the radiation and an outer electron, somewhat
like one billiard ball striking another. The result is the transfer of a certain
amount of energy to the electron and a change in energy and direction of the
photon. If the energy transfer to the electron is of a high enough magnitude, the
electron will be freed of its atom, effectively ionizing the molecule. The
electron and photon will then continue to interact, and ionize the tissue until the
initial energy of the incoming photon is completely dissipated as ionizations
and other forms of energy. This cascade of energy transfers and ionizations has
a profound negative impact upon the integrity of the biomolecules and the cell.
19,33
The impact of these energy transfers occurs through two primary
mechanisms in the cell. The first, and traditionally considered the most
important mechanism, because of the proven high cytotoxicity associated with
DNA damage, is the direct interaction with DNA. This direct interaction with
DNA causes ionization of the DNA and thus, highly lethal double stranded
breaks in the DNA, as well as single stranded breaks, and cross-linking between
DNA strands and DNA and related proteins. I7l9'20
12


Energy depositions in a cell are, however, extremely random in nature
and can occur in any molecule of the cell. Since water is the most abundant
single molecule in any living cell, it is reasonable that much of the radio-
interaction will occur with water (and dissolved oxygen). Indeed, the second
mechanism involves the formation of the highly reactive hydroxyl radical
species (and other ROSs, especially in highly aerobic environments). Once
formed, the hydroxyl radical proceeds to interact rapidly with anything,
including DNA, that it comes in contact with, damaging it through an oxidative
process. Thus, damage to DNA and other portions of the cell, due to radiation
can be through direct interaction with the radiation or through radical
intermediates created as a secondary product of the radiation. I7,19
This damage is offset by free radical scavengers in the cell, which can
reduce the ionized DNA through donation of a hydrogen atom to the ionized
species. Most of these free radical scavengers have thiol moieties (like cysteine
or GSH). The balance between these two opposing forces is called the
competition model for radiation cell killing. (An example using glutathione is
given in Equations 1.9 and 1.10).
(DNA) ------- (DNA)* + e' (Equation 1.9)
2(DNA)* + 2GSH-2(DNA)H + 2GS* GSSG + 2(DNA)H (Equation 1.10)
This model may not do justice to the role of glutathione in preventing
cytotoxic DNA damage, since it ignores the effect of GSH on radicals before
they interact with DNA. In fact, it has been shown that under some conditions,
13


cells can be entirely protected from the damaging effects of radiation if
presented with an excess of an importable GSH source (GSH monoesters), and
many tumor lines exhibit a resistance to radiotherapy that can be reduced or
removed by inhibition of GSH content in the cell. Additionally, cells tend to
build up a radio-resistance over a period of treatments. Given this tendency,
and the potential danger to healthy tissues it is understandable that radiotherapy
usually goes hand in hand with other treatments, like chemotherapy. I2, l7,19,20,
33,34
1.9 Chemotherapy Types and Mechanisms
Chemotherapy is used at some point in the treatment of nearly all cancer
patients. Typical anticancer drugs can be classified into several categories and
function as inducers of apoptic pathways. The basic drug categories include the
antimetabolites and antimitotic drugs, the anti-tumor antibiotics, alkylating
agents, and platinum compounds. 15,17,20
Before a proliferating cell can divide, it must build up its active proteins
and duplicate its DNA. Antimetabolite and antimitotic drugs interfere with
these processes, yielding cells that are unable to duplicate their DNA or
continue with the cell cycle. This type of irregular interference in the cell cycle
induces apoptosis. Antimetabolite drugs can act as nucleic acid analogs and/or
are capable of deactivating vital enzymes. 5-Azacytidine is a well-known
leukemia drug that works via both these mechanisms. It interferes with nucleic
acid metabolism by its incorporation into nucleic acids (it is a close analog),
and by competitively inhibiting uridine kinase (involved in pyramidine
metabolism). Antimitotic drugs are chemicals whose action targets mitosis or
14


some specific phase of cell division. One such drug is Etoposide. Etoposide is
a semisynthetic compound that acts as a mitotic spindle poison, and is one of
the first-line drugs used against lung cancer. i7,64
The majority of anti-tumor antibiotics are attained from Streptomyces
species. These drugs bind to DNA extremely tightly preventing transcription
and can be the source of local DNA unwinding and damage. Bleomycin sulfate
is an example of an anti-tumor antibiotic currently in use against a variety of
malignancies. It is used as the primary pharmaceutical for the treatment and
cure of 75% of testicular cancer in the United States, and sees widespread use
in the treatment of Hodgkins disease and squamous cell carcinoma. It is
obtained from Streptomyces verticillus, a species of actinomycetes (similar to
fungi). Bleomycin functions because of its dual ended nature. It has a region
that binds DNA at one end, and at the other has an incorporated Fe2+. The Fe2+
moiety is rapidly oxidized to Fe3+ by dissolved molecular oxygen, creating
toxic oxygen radicals in the direct proximity of the bound DNA. In the absence
of adequate reductive species, the oxygen radicals can attack the DNA, cleaving
the sugar and releasing free bases. This DNA damage results in the initiation of
apoptic pathways, and the death of the cell. 1720
Alkylating agents are some of the oldest chemotherapy drugs in
existence. They function by covalently bonding to biomolecules, particularly
DNA. The most potent of these agents are called bifunctional since they are
capable of binding DNA or protein at each of their two ends and permanently
cross-linking the molecules. Nitrogen mustard (N,N-bis(2-
chloroethyl)methylamine) developed initially for its possibilities as a chemical
warfare agent, is one of these bifunctional compounds (two active, chloroethyl
groups).17
15


Platinum compounds have also found use as anticancer agents.
Cisplatin (cis-diaminedichloroplatinum (II)) and carboplatin (cis-(l,l-
cyclobutanedicarboxylato)diarnineplatinum(II)) are both widely used, generally
in combination with other agents. Inside the cell, positively charged platinum
complexes are formed that react with DNA. Interstrand and guanine-guanine
cross-linking appear to be the 0)40lethal events. 17
1.10 Multi Drug Resistance
The number of tumor types in which the use of chemotherapy alone can
bring about prolonged survival or cure is small. There is one primary reason
for this, the development of drug resistance in the tumor cells. This may be
resistance to a single agent (resultant from a mutation), or more importantly the
development of multi-drug resistance (MDR). MDR confers an impressive
resistance to a wide variety of chemically dissimilar agents to the tumor cell. In
the laboratory setting, many possible mechanisms have been found for the
development of MDR. The translation of this knowledge to higher survival
rates and better clinical outcomes has yet to be achieved and is one of the
greatest problems of modem cancer research. I9,2064
Drug resistance can be broken down into two broad categories. The
first is known as innate resistance and results from the initial failure of a tumor
to respond to a treatment. The second, and clinically more important type, is
known as acquired resistance. It arises when the tumor recurs some time after
the initial treatment and often displays resistance to drugs it has never before
encountered. There are several recognized mechanisms of acquired drug
resistance, these include the p-glycoprotein and related transporter mechanisms,
16


DNA repair pathways, and glutathione and glutathione related enzyme systems.
One can demonstrate at least a partial link to cytosolic glutathione levels in all
of these mechanisms. 19-21
The first recognized mechanism is that of the Pgp system.
P-glycoprotein, p-170 (Pgp) is a membrane-surface, energy-dependant,
transport glycoprotein. This protein increases the export of anti-cancer drugs
from the cell, lowering the effective intracellular concentration (see Figure 1.4).
It is expressed at levels thought to be physiologically significant in about 50%
of human cancers. 21 22,23
17


pool in Cellular Resistance Mechanisms.
18


Another transport-generated mechanism of MDR is multi-drug resistant
associated protein (MRP1). MRP1 is a membrane-bound transporter that offers
MDR by transporting GSH-drug conjugates out of the cell, away from drug-
sensitive cytosolic areas. The mechanism is similar to that of Pgp, but
functions independent of the expression of Pgp. I9'21
The second type of acquired MDR is DNA repair systems. The role of
DNA repair pathways, although incompletely understood, has also come to the
forefront of recent study in the development of drug resistance. The cancer cell
with the ability to overcome significant damage done to its genetic material is
much more likely to survive a chemotherapeutic series than is one that is forced
down an apoptic pathway due to cumulative DNA damage. The measurement
of certain repair proteins in DNA recovery pathways has been used to asses the
role of DNA repair as a source of drug resistance. Elevated levels of these
proteins have been found and associated with aggressive cancer types and poor
prognosis. I926
The third primary source of drug resistance is attributed to a
modification in the maintenance of GSH homeostasis, through the interaction of
a number of biochemical pathways. Increased levels of non-protein thiols,
especially GSH, and the modulation of several GSH associated enzymes have
20 ^ 1
been proven to play a part in MDR.
19


1.10.1 The Role of Glutathione
in Multi-Drug Resistance
Reduced glutathione contributes to MDR through its ability to
coordinate with charged drugs, and eliminate free radicals produced by some
anti-cancer pharmaceuticals. For example, GSH is capable of binding to the
positively charged electrophilic centers on the active groups of some alkylating
agents. This leaves the drug much more susceptible to modification by GSH
transhydrogenases and the cytochromes p450, which modify the drug for higher
10 ^fl 0 I
solubility and easier expulsion from the body in urine and bile.
GSH transhydrogenases (GSTs) play a role in MDR. GSTs are a group
of detoxification isoenzymes. GST-71 is the dominant GST isoenzyme sub-type
in many tumors, and has been shown to exist in a higher concentration in tumor
cell lines than in equivalent healthy tissues. This increased level of GST-71,
independent of other factors, has been correlated to a weaker response to
chemotherapy and overall survival. GST activity is directly related to GSH
levels within the cell, as GSH acts as cofactor for GSTs. 19'21'2731
Glutathione reductase and metallothioneins are also actors in the
development of MDR. Elevated levels of GSH reductase (see Figure 1.3 and
Equation 1.4) present in tumor cells allow for the adequate recycling of GSSG
when the cell is under chemotherapeutic challenge. Thus, GSSG reductase
levels are intimately entwined with GSH levels, and therefore all of the other
GSH related mechanisms of MDR. Enhanced levels of metallothioneins have
been associated with MDR. The proposed mechanism of this association is
20


through the interaction and neutralization of metallothioneins with toxic
electrophilic drugs and their metabolites. Glutathione plays an important, albeit
indirect, role in this mechanism of MDR. Metallothionein activation and
regulation is dependent upon the redox condition of the cell, which is related to
the cytosolic GSH / GSSG ratio. 19-2U 32
It is apparent that GSH homeostatic modulation is very important in the
generation of multi-drug resistant cells. GSH cytosolic levels appear to be
related in some way, to many of the major mechanisms of multi-drug resistance
in tumor cell lines. Cytosolic GSH levels are linked, in one way or another, to
the MDR mechanisms of electrophilic drug interception, free radical
scavenging, GST functionality and elimination, and MRP1 export processes.
Additionally, there are data that although incomplete, offer some evidence that
the Pgp transporter system may be associated with GSH concentrations. This
association is hypothesized to be through the redox potential of the cell (and
drug modification). Some researchers believe that the Pgp and GSH systems
should be considered in their entirety as one system, a general cell protective
shield against foreign toxins. Furthermore, an increase in GSH-based
detoxifying enzyme activity was shown to have a direct correlation to increased
DNA repair activity, and thus contributed to MDR. (See Figure 1.4 for a
summary of GSHs relationship to MDR mechanisms). 17,1921,27-32
Glutathione and its related systems play a monumental role in both
chemotherapeutic MDR and in the establishment of radio-insensitive tumor
lines. The ability to increase or decrease glutathione levels is thought to hold
an important promise in cancer treatment. To realize this possibility, an
understanding of the transport characteristics of glutathione in the body is
necessary.
21


1.11 Glutathione Transport
Oral intake of glutathione has been demonstrated to result in absorption
through the intestinal lumen, vessel endothelium, retinal pigmented epithelial
cells, and cells of the kidneys proximal tubule and is then transported via
facilitated diffusion into the blood. Further transport from the plasma into
erythrocytes as glutathione disulfide has also been evidenced. Oral intake has
been shown to influence plasma GSH levels, but the consensus is that the
plasma GSH system is so well buffered, that very little change is detected in
healthy unfasted subjects. 35,36,41
The cells of the body, other than those previously mentioned, however,
are incapable of absorbing glutathione as an intact tripeptide to any measurable
degree. Instead, glutathione is broken down outside of the cell and then
transported in and reassembled. This lack of specific GSH transport and
reliance on reassembly was demonstrated by the introduction of glutathione
extracellularly in the presence of a GSH synthesis enzyme inhibitor (y-
glutamylcysteine synthetase specifically inhibited by methionine sulfoximine)
and without. The cellular glutathione levels increased to a significant degree
only in the trials that were free of the inhibitor. This indicates that the
mechanism of increased cellular GSH level s relies on the assembly enzyme
rather than a direct GSH-transporter. 36-38
The majority of data on mitochondrial transport of glutathione has been
generated from the study of liver and kidney fractions. It is apparent that there
are different possible mechanisms in each of these tissue types. In liver, it
22


appears that the rapid translocation of GSH may be due to dicarboxylate / 2-
oxoglutarate, glutamate transporters, or by yet undefined means. Nevertheless,
it is clear that GSH is translocated rapidly and intact from the cytosol to the
mitochondria 42-46
1.12 Glutathione Repletion Strategies
Other compounds besides GSH itself positively influence cellular
glutathione levels. The sulfur-containing amino acid L-cysteine is the primary
limiting metabolite in the production of GSH. L-Cysteine, however, is toxic in
the body, capable of reacting with O2 at blood plasma levels, creating
dangerous reactive oxygen species, including hydroxyl radical. L-methionine is
also capable of altering glutathione levels. However, this essential amino acid
is metabolically upstream and must be first converted to cysteine, before it is
useful. The methionine to cysteine pathway requires many cofactors and may
be inactive in neonates and in certain adults. N-acetyl-cysteine (NAC) acts as a
cysteine precursor without the toxic effects of cysteine, but is only effective at
raising GSH levels when they are abnormally low. If GSH levels are normal,
NAC appears to have no effect. L-2-oxothiazolidine-4-carboxylate (OTC)
serves as a substrate for the enzyme, 5-oxoprolinase that catalyzes its
conversion to S-carboxy-cysteine. S-carboxy-cysteine is hydrolyzed to yield
cysteine. However, the needed enzyme is not found in all tissues in the body
and it is unclear if it is effective as a systematic booster to GSH levels. 36,40
23


1.12.1 Glutathione Esters as Thiol Delivery Vehicles
Given the lack of systemic cellular uptake of intact GSH and the
cytotoxic effects and reliance on similar enzymatic machinery of most of the
repletion vehicles, glutathione monoesters and more importantly, diesters, come
to the forefront as GSH delivery vehicles. The ethyl monoglycyl ester of
glutathione (glutathione ethyl ester or GEE, see Figure 1.5, where R is an ethyl
moiety) has been shown to survive and be absorbed intact through the mouse
gastrointestinal tract. Treatment with GEE increases GSH levels in many
human cell types even after severe depletion of GSH by buthionine sulfoximine
(BSO, see figure 1.7).
24


There have been reports of toxicities associated with the alcohols
produced when hydrolysis of glutathione esters take place in cells. Thus far,
these toxicities have been blamed on metal impurities in the preparations of the
esters. Glutathione diethyl ester (GDE, see Figure 1.6, where the R groups are
ethyl substituents) was found to be better at increasing thiol levels in
erythrocytes, lymphocytes, ovarian tumor cells, and fibroblasts than GEE. This
increased performance of GDE over GEE at increasing GSH levels is fairly
well established. GDE is rapidly transported into and out of human cells and is
hydrolyzed to GEE and then eventually to GSH. GDE transport into cells is
several times greater than GEE. Therefore, it appears that glutathione diesters
may be the best thiol delivery vehicles for human cells. They are absorbed
intact when given orally, they enter and exit cells efficiently and rapidly, and no
toxicities have yet been verified. 4047,48
25


Once in the cell, glutathione esters are enzymatically hydrolyzed to
yield the corresponding alcohol and glutathione. A comprehensive profile of
esterase activity is far from a reality, and the presence of non-specific esterase
activity is probable. Non-specific activity may be derived from the presence of
A esterases (as defined by the IUBMB) including the aryldialkylphosphatases,
diisopropylfluorophosphatases, and arylesterases, all of which are found at
significant levels in many mammals including humans. Additionally, specific
GSH esterase activity has been identified from human liver tissue in the
enzymes glyoxylase II, s-succinylglutathionehydrolase, and s-
formylglutathionehydrolase. Specific activity has also been identified in at
least four carboxylesterases, a type of B esterases, isolated from rat liver
microsomes. (These four carboxylesterases are each named differently by
different researchers and are uniformly referred to only by their isoelectric
point). Certainly, there are additional enzymes that may have GSH-ester
hydrolytic activity, some of which may even be currently unknown proteins.
This broad variability in possible activity, specificity, and affinity lies at the
heart of the potential of GSH esters as vehicles of MDR reversal. 54,55,65,6667
26


1.13 Glutathione Depletion Strategies
1.13.1 Chemical Depletion
Glutathione levels can be depleted either chemically or by inhibition of
glutathione synthesis. Chemical depletion can be accomplished by any of
several means. The first general types of chemical depleting agents are the
substrates of glutathione transhydrogenases. a, p-unsaturated carbonyl
compounds are weak electrophiles that react with GSH in the presence of the
transhydrogenases. Diethyl Maleate (DEM), in use since 1969, is the most
common agent used in this class. The second general type of depleting agent
includes the electrophilic species formed via the cytochrome p450-dependent
monooxygenase pathways. In these pathways, relatively inert compounds are
turned into reactive electrophiles that are capable of depleting GSH in vivo.
Representative inducers of the monooxygenase system are phenobarbital and 3-
methylcholanthrene. The third major division of glutathione depleters is the
thiol oxidants. These are simply oxidants that convert GSH to GSSG. They are
used mostly in cell preparations and include the diazenecarboxylic acid
derivatives. In addition to these three main types, various other compounds
have also been implemented as glutathione depletion methods and fasting has
proved to seriously deplete GSH levels. 49-53
27


1.13.2 Depletion Through Inhibition of Synthesis
The depletion of glutathione through inhibition of its biosynthesis is by
far the most useful method when considering possible clinical utility. Two
major approaches have
taken center stage in
this regard. The first is
through the use of 5-
oxo-L-prolinase
inhibitors, like 2-
imidazoladine-4-
carboxylate and others,
which competitively inhibit 5-oxoprolinase, allowing the buildup of 5-
oxoproline and preventing its conversion to glutamate by oxoprolinase. This
lack of glutamate inhibits the synthesis of GSH at a basic level. The second
and most promising method is the use of buthionine sulfoximine (BSO, see
Figure 1.7). BSO acts on the first step in the biosynthesis of glutathione. It
inhibits the enzyme y-glutamylcysteine synthetase. BSO works by tightly
binding to y-glutamylcysteine synthetase after it (BSO) has been
phosphorylated. This phosphorylated BSO resembles the natural intermediate,
and is irreversibly but not covalently, bound to the active site. 37,49
3 o'
\
CFU-S CHo C 0
/ V /
ch2-ch2 sIH CH2HC
/ \ +
h3c NH3
Figure 1.7: Buthionine Sulfoximine (BSO)
28


1.14 The Promise of Selective Modulation
of Glutathione Levels
With the ability and understanding to replete and deplete cells of their
GSH it becomes apparent that a method that allowed selective modulation
would hold an enormous promise for the treatment of cancer, through
chemotherapy and the reduction or removal of MDR. Depletion by BSO and
selective repletion could target cancer cells specifically while protecting normal
cells at a standard or even elevated level of protection. To attain this specificity
of repletion would require the use of a well-absorbed and rapidly transported
GSH delivery vehicle capable of sidestepping the enzymatic biosynthesis steps
blocked in the cell by BSO. Glutathione esters have proven to be such a
delivery system.
This selective modulation could be attained through several means.
Enzymatic profiles in cancer and healthy cells have been known to differ in
other contexts. It is reasonable, therefore, to expect that there may be
differences in the level of expression or particular active site affinity for
differing alcohol groups on the esters, in any or all of the various hydrolytic
enzymes responsible for the hydrolysis of the esters into their substituent
glutathione and alcohol products. 16
To be able to take advantage of the differing profiles and activity of the
enzymes responsible for the hydrolysis of glutathione esters, it is necessary to
first know and identify what those enzymes are. One way to begin this
enzymatic characterization is to establish a profile of the ester hydrolysis.
Additionally, by using differing alcohol groups on the GSH esters, one can
determine the natural affinity of the hydrolytic enzyme(s) and thus obtain an
29


insight into their active sites. A good deal of previous work has been done on
various esters and their hydrolysis in differing samples by Dr. E. J. Levy and
others in association with her. It is the goal of this research to expand that
research and to provide the further characterization of the ethyl, propyl, butyl,
isobutyl, and benzyl ester hydrolysis in both microsomal and cytosolic
preparations of rat liver. A special focus on n-butyl ester will allow for the
determination of its Km and VMax.
2. Experimental
2.1 Data Generation and Handling
Hydrolytic data were gathered in two basic steps after the glutathione
monoesters were formed and purified. The first step functioned as a
preparation for analysis by the second. In this first portion, all of the GSH
monoesters were allowed to independently react with rat liver based cell
preparations. The reaction was quenched at various time intervals, generating
the time-based profile of the hydrolysis of the ester by either the microsome
preparation or the cytosol preparations. These reactions were then analyzed in
the second step, by the Tietze assay. This assay generates time versus
absorption data for each of the quenched samples prepared earlier. From these
data, the percent hydrolysis, concentration of glutathione, and the reaction
velocity were determined. Further, more complete trials were run on the n-
butyl monoester of glutathione. The data from these n-butyl trials were used to
evaluate the VMax and Km using Lineweaver-Burke, Eadie-Hofsted, and Hanes /
Woolf linear approaches.


2.2 Equipment and Materials
The Varian Cary IE spectrophotometer was used for enzyme assays.
Centrifugation was performed using the Beckman L5-50E centrifuge. Rat
livers were obtained through Harlan Bioproducts; male Sprague-Dawley rats
were fasted overnight before harvesting and N2(i)-flash freezing. They were
shipped in dry ice and stored at -70 degrees C upon receipt. 5,5dithiobis(2-
nitrobenzoic acid) (DTNB), glycerol, p-nitrophenyl acetate (PnpAc), 5-
sulfosalicylic acid dehydrate (SSA), and all
alcohols except methyl alcohol, were obtained from Aldrich. Glutathione was
purchased through Calbiochem. Acetic acid was from Mallincrodt. Prepared
Dulbeccos phosphate buffered saline (PBS) was from Gibco/BRL. Methanol
and Whatman DE53 (diethylaminoethyl) cellulose anion-exchange resin were
from Fisher Scientific. Reduced p-nicotinamide adenine di-nucleotide
phosphate (NADPH), dithiothreitol, ethylenediaminetetraacetic acid (EDTA),
and glutathione reductase were all obtained through Sigma. Acetonitrile and
barium chloride were from J.T. Baker. Protein assay solutions and standards
were all supplied by Bio-Rad. Tris(hydroxymethyl)aminomethane (Tris
acetate) was obtained from Boehringer-Mannheim. Glutathione monoesters
were prepared using the protocol of Anderson et al (1994). All other equipment
and reagents used, that have not been listed, were of the highest quality and
purity. 56
31


2.3 Buffer Compositions
Several buffer solutions were used repeatedly throughout the
experimental processes. Stock Tietze Buffer was 143mM sodium phosphate,
6.3mM EDTA (pH 7.4). Daily Buffer was 0.248mg/ml NADPH in Stock
Tietze Buffer. Pyrophosphate Buffer was 0.1M pyrophosphate (pH 7.4), 20%
(w/v) glycerol, ImM EDTA and 0.1 mM DTT. Pig Liver Esterase Buffer (PLE
Buffer/Buffer E) was 50mM sodium pyrophosphate (pH 7.0) and 0.1M NaCl.
Buffer A was 0.1M Tris Acetate (pH 7.4), O.lmM DTT, and ImM EDTA.
Buffer B was 0.1M Tris Acetate (pH 7.4), 20% (w/v) glycerol, O.lmM DTT
and ImM EDTA.
2.4 Preparation of Rat Liver Microsome
and Cytosol Lysates
Both rat liver microsome and cytosol preparations were used.
Microsomes are made when the endoplasmic reticulum (ER) of a cell is
mechanically disrupted and pieces reform into smaller vesicles. Rough
microsomes are made from the rough ER and contain the enzymes associated
with that organelle. However, the benefit of a microsome preparation to this
research was the inclusion of a great number of smooth vesicles referred to as
smooth microsomes. These smooth microsomes are made of smooth ER but,
more importantly, they also include a great number of vesicles made of the
plasma membrane, Golgi apparatus, endosomes, and mitochondria (with
32


membrane proteins intact). The interiors of these vesicles are consistent with
the cytosol or lumen of their respective organelles and the membrane proteins
are functionally preserved in the vesicle membrane. This may be of importance
if any of the hydrolytic activity is membrane-bound. The cytosol preparations
do not include, in functional order, the membrane bound activity of the liver
cells. However, they do contain far higher protein concentrations than does a
microsome preparation. For these reasons, both sample types were utilized in
this research.
The procedure by which the rat liver microsomes and cytosol was
isolated was put forth by Juris Ozols in 1990. All steps were done at 4 degrees
C. The rat livers were minced, rinsed and homogenized at (lg/5ml) in buffer A.
The sample was then centrifuged and the resulting pellet discarded. The sample
was centrifuged again and the cytosolic fraction (supernatant) was removed and
dialyzed overnight with one change of buffer B, to remove glutathione. The
pellet was resuspended in pyrophosphate buffer and was again centrifuged.
This pellet, containing the microsomal fraction, was resuspended in buffer B.
Both of the samples were portioned into .5ml to 1ml aliquots. They were then
flash frozen in liquid nitrogen and stored at -70 degrees C thereafter, which has
been demonstrated to be an adequate storage method without loss of activity.
58. 59
2.4.1 Evaluation of Cytosol and Microsomes
for Total Protein Content
The total protein concentration value of 103.5 mg/ml for the cytosolic
supernatant and 20mg/ml for the microsome preparation was generated using
the Bio-Rad total protein assay (phosphoric acid assay). Bovine gamma
globulin standards, prepared from frozen stocks, were used to generate a
33


calibration curve. Samples and standards were read after 15 minutes but not
after 60 minutes. Once generated, samples were diluted to within the range of
the curve, and using the absorbance of the sample at 595nm and the linear
regression equation for the calibration curve, the protein concentration was
calculated.
2.4.2 Evaluation of Cytosol and Microsomes
for Esterase Activity
Cytosol and microsomal esterase activity was established by monitoring
hydrolysis of p-nitrophenyl acetate (PnpAc) at 412nm. 600pl of cytosol /
microsomes was added to 900pl of 10% SSA. A 50:50 mixture of buffer A and
buffer B was used to dilute lOOpl of this sample to 1ml. 50pl of 60mM PnpAc
in acetonitrile was added to 200pl of sample in 950pl of PLE buffer. The
hydrolysis of the PnpAc results in a color change and thus a change in the
absorbance. The cytosol had an esterase activity of 0.761 Absorbance/min/mg
protein, and the microsomes had an activity of 4.735 Absorbance/min/mg
protein.
2.5 Determination of Percent Hydrolysis
for the GSH-Monoesters
Glutathione ester solutions were prepared in PLE buffer. The solutions
were vortexed gently until completely dissolved. This was especially difficult
with the benzyl ester, whose solubility is quite low in the aqueous buffer. The
benzyl ester was therefore intermittently warmed to 30 degrees C (< 1 minute
warming) and vortexed to accommodate the reductase. Once prepared, the
esters were kept on ice until used. Reaction tubes were created for each of the
34


ester and microsome/cytosol combinations, ester controls (at each
concentration), buffer controls, and microsome or cytosol controls. The
reaction tubes were created by combining 750pl of glutathione ester with 250pl
of cell lysate (cytosol or microsomes). The final ester concentrations were 8,
12, or 16mM. Buffer controls were prepared using 750pl of PLE and 250pl of
buffer B. Cell lysate controls were prepared using 750pi of PLE buffer and
250pl of cell lysate. Reaction tubes were incubated at 37 degrees C for 15, 30,
45, and 60 minutes. At each time point, aliquots were removed and quenched
by adding the sample to SSA at a final concentration of 3.33%. The quenched
tubes were then stored at -20 degrees C for the Tietze assay.
2.5.1 Tietze Assay
The quenched tubes from the sampling preparation were analyzed using
the Tietze assay. The Tietze assay is an indirect-continuous or recycling
assay. The Tietze assay is termed indirect and continuous because this type of
assay involves carrying out the manipulations necessary to detect (indirectly)
product formation in such a way as to allow the change to be followed
continuously as it occurs. This type of assay is less prone to error than
sampling assays due to this continuous nature. Free GSH gives a signal
indirectly through its interaction with DTNB, a dicyclic compound linked by a
reducible disulfide bond (See Figure 2.1). Free GSH (from the hydrolysis of
the ester) cleaves this bond by acting like a nucleophile, attacking the disulfide
bond and forming a disulfide bond with one-half of the DTNB, and reducing
the other half. The other half of the DTNB or 2-nitro-5-thiobenzoic acid (TNB)
is released into the mixture (see Figure 2.2 and Equation 2.1). TNB is a yellow
35


colored compound that absorbs at 412nm. Therefore, for each free GSH
molecule, one absorptive compound is released (TNB). This is a 1:1, free GSH
to signal ratio. Glutathione reductase (and included NADPH) is used to recycle
GSSG back to GSH and let the reaction continue (see Equation 2.2). This
allows for the determination of a slope from a linear regression based on the
absorbance vs.
time data.
Since the
concentration of
GSH is never
changed, this
slope is
proportional to,
and was used to
determine the
glutathione
concentration in
the sample by
using a standard
curve, generated
from the slopes
of known GSH
standards.
The Tietze assay was performed by adding 25 pi of each sample to
700pl of Daily buffer, lOOpl of 6mM DTNB, and 175pl of DI water in a
disposable cuvette. Stock
36


Tietze buffer was used to prepare a 1:4 dilution of GSSG reductase. The
diluted enzyme was added to the cuvette and was mixed after the cuvette had
been warmed at 30 degrees C in a water bath for 15 minutes. The absorbance
was measured at 412nm for 3 minutes. 63
2GSH + 2DTNB -> GSSG + 2TNB (Equation 2.1)
GSSG +2NADPH (!SSG Redmla > 2NADP+ +2GSH (Equation 2.2)
2.5.1.1 Influences on the Tietze Assay
Three primary things can adversely affect the Tietze results; these
include metal ions or proteins in the mixture, and incorrect pH (destabilizes the
reductase). EDTA, a metal chelating agent was used in to help reduce any
potential metal contamination. The mixtures were acidified and centrifuged
prior to running the Tietze to remove any functional proteins and the pH was
rigidly held below 7.6 for maximum operational efficiency of the GSSG
reductase. 60-62
37


3. Results
3.1 Microsome Assay Results
The experimental data for the free GSH content of all microsome series
controls and the percent hydrolysis of the five glutathione mono(glycyl)esters
are summarized hereafter in tables 3.1 and 3.2. The percent hydrolysis of the
GSH esters is presented graphically in Chart 3.1.
Table 3.1: Free GSH Values for the
Microsome Series Controls
Free GSH (mM)
Time Microsome Buffer Ethyl Propyl Isobutyl Butyl Benzyl
(min.) Control Control Ester Ester Ester Ester Ester
0 -0.018 -0.056 0.095 0.058 0.035 0.035 0.053
15 -0.015 -0.062 0.098 0.061 0.033 0.040 0.054
30 -0.019 -0.044 0.097 0.061 0.038 0.045 0.053
45 -0.013 -0.057 0.097 0.063 0.038 0.039 0.060
60 -0.017 -0.041 0.092 0.063 0.042 0.048 0.062
Control values are listed as mM concentrations of free glutathione, detected using the Tietze
assay.________________________________________________________________________________________
38


i
Table 3.2: Percent Hydrolysis of Glutathione
Esters with Microsomes.
Percent Hydrolysis of GSH Esters with Microsomes
Time Ethyl Propyl Isobutyl n-Butyl Benzyl
(min.) Ester Ester Ester Ester Ester
0 4.8% 3.3% 2.3% 2.4% 4.3%
15 6.1% 5.2% 5.9% 5.1% 23.5%
30 6.4% 6.7% 9.6% 7.8% 34.9%
45 6.6% 7.7% 10.5% 10.3% 46.4%
60 7.0% 9.1% 12.1% 10.9%
Percent hydrolysis values were determined from the initial calculated mM concentration
values and the final concentrations determined using the Tietze assay.___________________
i
i
i
39


Percentage Hydrolysis
Chart 3.1: Summary of Microsomal Percent Hydrolysis as a Function of Time for GSH Ethyl,
Propyl, Isobutyl, N-Butyl, and Benzyl Esters
0 10 20 30 40 50 60 70
Time, in Minutes
GSH Ethyl Ester GSH Propyl Ester GSH Isobutyl Ester ' GSH N-Butyl Ester x GSH Benzyl Ester


In summary, from table 3.1 the controls and GSH esters are not a
significant source of free GSH on their own. However, when the assay is run
and the microsomes and esters are combined, significant hydrolysis occurs. It
is interesting to note that hydrolysis increases as the overall size and bulk of the
ester grows. For the microsome data presented above, it is obvious that the
benzyl ester is by far the most hydrolyzed in a given amount of time. Whether
this is a result of its increased size, aromaticity, or both, is unknown. The
isobutyl ester and n-butyl ester are also illuminating in that, though very similar
in composition, their percent hydrolysis rates are different.
3.2 Cytosol Assay Results
The experimental data for the free GSH content of all cytosol series
controls and the percent hydrolysis of the five glutathione mono(glycyl)esters
are summarized hereafter in tables 3.3 and 3.4. The percent hydrolysis of the
GSH esters is presented graphically in Chart 3.2.
41


Table 3.3: Free GSH Values for the
Cytosol Series Controls.
Free Glutathione Concentration (mM)
Time Cytosol Buffer Ethyl Propyl n-Butyl Benzyl
(min.) Control Controls Ester Ester Ester Ester
0 0.140 -0.008 0.056 0.011 0.025 0.030
15 0.171 -0.007 0.048 0.014 0.025 0034
30 0.239 -0.011 0.042 0.018 0.028 0.034
45 0.219 -0.006 0.038 0.017 0.036 0.038
60 0.180 -0.008 0.035 0.018 0.038 0.036
Control values are listed as mM concentrations of free glutathione, detected using the
Tietze assay.
Table 3.4: Percent Hydrolysis of Glutathione
Esters with Cytosol.
Percent Hydrolysis of GSH Esters with Cytosol
Time Ethyl Propyl n-Butyl Benzyl
(minutes) Ester Ester Ester Ester
0 8.7% 5.6 % 7.0 % 7.5 %
15 11.2% 9.6 % 16.4 % 39.7 %
30 11.6% 15.3% 24.3 % 58.0 %
45 13.1 % 22.1 % 30.9 % 65.0 %
60 8.1 % 25.5 % 37.8 % 76.9 %
Percent hydrolysis values were determined from the initial calculated mM concentration values
and the final concentrations determined using the Tietze assay.
42


Percentage Hydrolysis
Chart 3.2: Percent Hydrolysis as a Function of Time for GSH: Ethyl, Propyl, N-Butyl and
Benzyl esters with Cytosol
90.000
80.000
70.000
60.000
50.000
40.000
30.000
20.000
10.000
0.000
10 20 30 40 50
Time, in Minutes
GSH Ethyl Ester GSH Propyl Ester GSH N-Butyl Ester x GSH Benzyl Ester
0
60
70


From table 3.3 the controls and GSH esters are shown not to be a
significant source of free GSH. When the assay is run and the cytosol and
esters are combined, significant hydrolysis occurs. Hydrolysis increases as the
overall size and bulk of the ester grows. For the cytosol data presented above,
it is obvious that the benzyl ester is by far the most hydrolyzed in a given
amount of time, nearly doubling in value from the n-butyl.
3.3 Determination of Viviax and Km
for n-Butyl Ester in Cytosol
The GSH concentration and time values were used to generate velocities
(v) for the n-butyl trials in cytosol. The data were used to plot a double
reciprocal or Lineweaver-Burke plot. In this type of graph, the y-intercept is
the reciprocal of the VMax and the x-intercept is the negative reciprocal of the
Michaelis constant, Km- This type of plot is widely used, but suffers from an
inherent flaw; it gives a grossly misleading impression of the experimental
error. Small errors in v lead to enormous error in 1/v. This type of plot has a
tendency therefore to launder poor data. It is included here, however, due to
its wide utilization in the research literature, its usefulness as an illustration of
the approximate magnitude of the Km and VMax and for comparison to the
Eadie-Hofstee and Hanes plots as a qualitative indicator of relative
experimental error. The Eadie-Hofstee plot is also utilized to illustrate Km and
VMax* In this type of plot, the slope is -Km and the y-intercept is VMax- It is a
less forgiving mechanism for treating the data and considered more accurate. It
too, however, has a flaw. Since it is a plot of v vs. v/[S], errors in v become
more significant because they are propagated on each axis. The Hanes or
j
44


Woolf plot is considered by many to be the preferable treatment for most
purposes. It is a plot of [S] vs. [S]/v and is similar to the Eadie-Hofstee plot for
that reason. The slope of the line is equal to the reciprocal of VMax and the x-
intercept is equal to -Km- It is more susceptible to error in the [S], since that
error is propagated on both axes. The information from this plot taken together
with the Eadie-Hofstee treatment generates a relatively good qualitative idea of
the experimental error. Table 3.9 summarizes the findings for Km and Vm3x- 61
Table 3.5: n-Butyl Vmsx and Km Values.
V Max Km
(mM/min) (mM)
Lineweaver-Burke 0.179 14.14
Eadie-Hofstee 0.147 9.42
Hanes / Woolf 0.165 12.34
45


Mv as (Min/mM)
Chart 3.3: Lineweaver-Burke; 1 / [S] vs. 1 tv,
y = 79.157x + 5.5931
R2 = 0.9249
r
25
20
!
|
|
15

10


5
-0.
15
-0.1
........ 0
-0.05 0
0.05 0.1 0.15 0.2
...........-5
0.25
1/[S] as (1/mM)


Velocity in mM / Min
-0.002
Chart 3.4: Eadie-Hofstee, v vs. v/[S] * = '94617x 0 1464
R2 = 0.5743
-------------- .. ...- .. ...... ~ ~~ "
0.02
v/[S) in mMA2 / Min


4^
oo
E
4
15
10
5
Chart 3.5: Hanes; [S] vs. [S]/v y = 6 0617x + 74 816
R2 = 0.9059
200
180
160
140
120
100
80
60
40
20
0
0 5 10 15 2)0
------20-------------.........................----------------------------------
[S], mM


4. Discussion
The lack of any meaningful hydrolysis in the control samples compared
to significant hydrolysis in the microsome and cytosol provides clear proof that
the hydrolysis of these esters is enzymatically catalyzed. The hydrolytic
activity, as suspected, seems to indicate multiple enzymes involved in the
catalysis. This belief is supported by the fact that hydrolysis did occur in the
microsomal fraction as well as the cytosolic fraction. In the microsomal
fraction, the enzymes are bound in the membranes of the microsomes, with
their active portions on either the interior or exterior of the vesicles. It is
unlikely that the same enzymes would be also be found in the cytosol, given
that membrane-bound proteins display long hydrophobic stretches that would
be unwelcome and conformationally disrupted in a strong aqueous environment
like the cytosol of a cell.
If you compare the difference in percent hydrolysis of the esters per
milligram of protein present in the sample per minute, it becomes apparent
which fraction (cytosolic or microsomal) has the greater activity. The
microsomal hydrolysis of the benzyl ester has a value of 4.24% hydrolysis per
mg total protein per minute. Compare this to the value of the cytosolic
hydrolysis of the benzyl ester which has a value of 1.24% hydrolysis per mg of
total protein per minute.
49


!
Comparing these two values shows that the microsomal fraction has greater
activity per total protein content than that of the cytosol. This indicates that a
good portion of the GSH ester hydrolytic activity may be membrane bound, and
present in the membranes of the endoplasmic reticulum and mitochondria.
The hydrolysis follows a general trend, both in microsome and cytosol
fractions. This trend does seem to suggest that there may be fewer, more
specific enzymes with similar active site characteristics, rather than large
numbers of non-specific enzymes with vastly differing active sites. It appears
that as the carbon chain of the ester gets bigger, so too does the affinity of the
enzyme(s) for the esters. There also appears to be a preference for branched
chains over unbranched ones. This is illustrated by the higher rates of
hydrolysis for the isobutyl as compared to the n-butyl ester. Additionally, the
largest hydrolysis percentages were determined to be generated with the benzyl
ester. This indicates a possible specific preference for bulky aromatic
substrates. This gives a small glimpse of the active site characteristics, for at
least the primary enzyme(s) involved in the hydrolysis. It suggests that the
active site(s) prefer large hydrophobic substrate molecules.
It has been long known that serine proteases are capable of catalyzing
the cleavage of esters, and thus can be termed esterases in that respect. All
serine proteases are characterized by a catalytic triad present in their active site.
Phenylmethylsulfonyl fluoride (PMSF) is an excellent inhibitor of serine
proteases. It acts by inactivating the catalytic triad. A brief inhibition side
experiment involving the cytosolic hydrolysis of PnpAc (a known good
substrate of the serine proteases, and the cytosolic fractions) was conducted
(data unpublished). This exploratory experiment showed that approximately
60% of the hydrolytic activity in the cytosolic fraction was inhibited by the
addition of PMSF. Even though this information is far from conclusive, when
50


examined in conjunction with the higher rates for larger aromatic structures
(benzyl ester) and what is known of serine proteases, it suggests a possible
catalytic triad-type active site, similar to that of chymotrypsins.
The hydrolytic data allowed for a preliminary determination of Km and
VMax- The data for the n-butyl ester hydrolysis was examined via three
variations on the Michaelis-Menton equation. The first method is the
Lineweaver-Burke double reciprocal plot, which returned the highest values for
both the Vm3x and Km, 0.179mM/min and 14.14mM respectively. This
however, is by far the least reliable of the three methods due to its large
propagation of error in velocity values as the reciprocal is taken. This nature of
the Lineweaver-Burke makes data appear better. This is demonstrated by
comparing the data scatter of all three graphical types and comparing their
correlation coefficients. The second method is the Eadie-Hofstee plot, which
returned values of 0.147mM/min and 9.42mM for the Vm3x and Km- These
values are much more reliable, but are still a bit suspect since errors in v are
cast into both axes. The third method is known as the Hanes or Woolf plot.
The Hanes plot yielded a Vmbx of 0.165mM/min and a Km of 12.34. This plot
too has a particular weakness. Since substrate concentration is involved in both
axes, it is more vulnerable to error in these values. 61
By looking at both the values generated by the Eadie-Hofstee and Hanes
plots it can be seen that the true values probably lie somewhere in the range
between the derived values. In addition, one can get a qualitative idea of where
the majority of error lies, and see that the Hanes values are probably closer by
comparing the correlation coefficients of the plots.
The worth of these determined values of Km is most apparent when the
reaction is examined from the aspect of the enzyme, substrate, and product
relationships as given in reaction 4.1.
51


k, k2
E + S ^^ ES--------------------- E+P (Equation 4.1)
k.i
In equation 4.1 kx values represent the rate constants associated with
each portion of the overall equation. The Michaelis constant can be described
in relation to these rate constants as is shown in equation 4.2 and 4.3. 60,61
Km = k.]/ki + k2/ki (Equation 4.2)
Km = Ks + k2/ki (Equation 4.3)
Ks is the dissociation constant of the Michaelis complex, as Ks
decreases, the enzymes affinity for substrate increases. Therefore, the relatively
large value for Km of between 9 and 12 mM for the n-butyl ester indicates a
rather low affinity of enzyme for substrate. This corresponds well with the
relatively low percent-hydrolysis for the non-aromatic esters that were tested,
and further supports the hypothesis of a serine protease-like active site. 5l8,60,
61
The next logical step in the investigation of this hydrolytic activity is the
isolation of the enzyme(s) responsible. It may be useful to begin looking for
proteins with molecular weights and characteristics similar to the serine
proteases. Indeed, the use of the large database on chymotrypsin may allow for
the construction of immunoaffinity columns or even columns that utilize
catalytic-triad active ligands. Additionally, further study of novel glutathione
esters modeled after compounds with groups found to be highly reactive with
the serine proteases catalytic triad, may also be useful.
52


In summary, it can be said that the hydrolytic activity appears to be
enzymatically catalyzed. The Km for the hydrolysis of the n-butyl ester
indicates a low affinity of the enzyme for the ester, and in conjunction with the
hydrolytic data, supports the conclusion that bulkier and / or aromatic esters
may provide better substrates for hydrolysis. The research also provides
helpful insight into the hydrolytic activity and suggests a catalytic triad similar
to the serine proteases.
The consequences of a selective glutathione modulation ability based on
BSO depletion and further research of this topic is widespread. Glutathione has
been indicated as a player in health issues ranging from general aging and viral
infections to the development of MDR in cancer chemotherapy. This research
is still in its infancy, but the magnitude of promise that it holds warrants the
further study and understanding of glutathione and selective modulation.
!
i
53
i


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