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Modulation of macrophage glutathione synthesis by oxidized LDL and the effects of glutathione content on cell-mediated LDL oxidation

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Modulation of macrophage glutathione synthesis by oxidized LDL and the effects of glutathione content on cell-mediated LDL oxidation

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MODULATION OF MACROPHAGE GLUTATHIONE SYNTHESIS
BY OXIDIZED LDL AND
THE EFFECTS OF GLUTATHIONE CONTENT ON
CELL-MEDIATED LDL OXIDATION
by
Lijiang Shen
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(Molecular Pharmacology & Toxicology)
December 2000
Copyright 2000 Lijiang Shen
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UMI Number: 3041523
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UNIVERSITY OF SOUTHERN CALIFORNIA
The Graduate School
University Park
LOS ANGELES, CALIFORNIA 90089 1695
This dissertation, w ritten b y
Under th e direction o f Ai.s— D issertation
Com m ittee, and approved b y a ll its members,
has been presented to and accepted b y The
Graduate School, in partial fu lfillm en t o f
requirem ents fo r th e degree o f
Li.iianq Shen
DOCTOR OF PHILOSOPHY
D ate December 18, 2000
DISSERJA TIO N COM M ITTEE
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DEDICATION
To my wife, Anna
To my parents
And to my dreams
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ACKNOWLEDGMENTS
First I like to thank my mentor and advisor, Dr. Alex Sevanian, for his
guidance, encouragement, and patience. This dissertation would not have been
possible without his support.
I would like to acknowledge my dissertation committee members, Drs.
Enrique Cadenas, Roger Duncan. Howard Neil Hodis, Florence M. Hofman. and
Alex Sevanian, for their time and insightful comment.
I would especially like to acknowledge Dr. Henry Jay Forman and his former
lab researchers, Drs. Rui-Ming Liu, Jinah Choi and Lin Gao for their expert advice
on GSH measurement.
I would also like to express sincere gratitude to my colleagues, Hazel
Peterson, Dr. Juliana Hwang, Dr. Ouliana Ziouzenkova, Dr. Liana Asatryan, and
many others for their assistance and advice.
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iv
TABLE OF CONTENTS
DEDICATION “
ACKNOWLEDGMENTS U i
LIST OF ABBREVIATIONS vii
LIST OF TABLES AND FIGURES x
ABSTRACT xii
CHAPTER 1 1
INTRODUCTION
1.1 Oxidative hypothesis of atherosclerosis 1
Composition of LDL and LDL oxidation 1
Initiation of LDL oxidation by vascular cells and 3
proatherogenic properties of oxLDL
Scavenger receptors-mediated oxLDL uptake 6
1.2 GSH and y-glutamyl cycle enzymes in antioxidant defense 8
Biological functions of GSH 8
The y-glutamyl cycle 1 1
GSH synthesis and y-glutamylcysteine synthetase 15
1.3 GSH changes in response to oxLDL-induced oxidative stress 17
CHAPTER 2 21
GENERAL EXPERIMENTAL DESIGN
2.1 Hypothesis 21
2.2 Specific Aims 21
2.3 Materials & Methods 22
Materials 22
Cell culture 22
Isolation of LDL from human plasma 23
LDL modification 23
LDL' separation 24
Measurement of H2O2 metabolism 25
Lipid peroxide measurement 25
TBAR (MDA) measurement 25
In vitro GSH depletion assay 26
LDL uptake by macrophages 26
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V
GPx activity assay 27
GSSG-reductase activity assay 27
Catalase activity assay 28
Measurement of intracellular GSH 28
Western blot analysis 29
MTT cytotoxicity assay 30
Statistical analysis 30
CHAPTER 3 31
ADAPTATION TO OXIDATIVE STRESS: MECHANISMS OF
OXLDL-INDUCED GSH DEPLETION AND REPLETION
3.1 Introduction 31
3.2 Results 34
OxLDL induced an initial depletion followed by an adaptive 34
increase in macrophage GSH content
OxLDL induced GSH-HS protein expression 38
Ebselen pretreatment reduced oxLDL-associated lipid 38
hydroperoxides
OxLDL-associated lipid hydroperoxide affects GSH content 41
OxLDL induced macrophage ROS production 44
3.3 Discussion 47
CHAPTER 4 51
THE EFFECTS OF SELENIUM SUPPLEMENTATION ON
PEROXIDE-INDUCED y-GCS INDUCTION AND GSH
SYNTHESIS
4.1 Introduction 51
4.2 Results 53
Effects of selenium supplementation on macrophage glutathione 53
peroxidase activities and protein expression
GSH depletion and peroxide metabolism in Se (+) cells 55
Peroxide-resistance of Se (+) cells 57
OxLDL induced GSH and y-GCS-HS changes in Se (+) cells 57
4.3 Discussion 60
CHAPTER 5 63
THE ROLE OF GSH CONTENT IN CELL-MEDIATED LDL
OXIDATION
5.1 Introduction 63
5.2 Results 66
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vi
Characterization of macrophage-mediated LDL oxidation 66
Modulation of macrophage GSH content 68
Macrophage GSH content affects the cell ability of oxidizing 71
LDL in culture
5.3 Discussion 74
CHAPTER 6 76
CONCLUSION
6.1 Conclusion 76
6.2 Proposed future work 79
6.3 Implications 81
REFERENCES 84
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LIST OF ABBREVIATIONS
AcLDL, acetylated LDL
AP-1, activator protein-1
ARE, antioxidant response element
BSO, L-buthionine S,/?-sulfoximine
CAT, catalase
cGPx, cytosolic glutathione peroxidase
DMNQ, 2,3-dimethoxy-l ,4-naphthoquinone
DMSO, dimethylsulfoximine
DNB, 2,4- fluorodinitrobenzene
DTP A, Diethylenetriaminepentaacetic acid Free Acid
DCFH-DA, 2,4 dinitrofluorobenzene & 2’,7’- Dichlorofluorescin diacetate
Ebselen, 2-phenyl-1,2-benzisoselenazol-3(2H)-one
EpRE, electrophile responsive element
ER, endoplasmic reticulum
FBS, fetal bovine serum
y-GC, y-glutamylcysteine
GCS, y-glutamylcysteine synthetase
GCS-HS, y-glutamylcysteine synthetase heavy (catalytic) subunit
GCS-LS, y-glutamylcysteine synthetase light (regulatory) subunit
GGT, y-glutamyltranspeptidase
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GI-GPx, gastrointestinal glutathione peroxidase
GS, glutathione synthetase
GSH, glutathione
GSSG, glutathione disulfide
GST, glutathione S-transferase
GRD, GSSG reductase
4HNE, 4-hydroxynonenal
HETE, hydroperoxyeicosatetraenoic acids
HPLC, high performance liquid chromatography
H2O2, hydrogen peroxide
HO, hydroxyl radical
LOX-1, Lectin-like oxLDL receptor-1
LDL, low-density lipoprotein
LDL', electronegatively charged LDL
LOO peroxyl radical
LOOH lipid hydroperoxide
MCP-1, monocyte chemotactic protein-1
MCSF, macrophage colony-stimulating factor
mm-LDL, minimally modified LDL
MTT, 3-(4,5-dimethylthiazoI-2-yl)-2,5-diphenyl-tetrazolium bromide
MDA, malonaldehyde
NF-kB, nuclear factor kappa B
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NAC, N-acetylcysteine
OTC, L-2-oxothiazolidine-4-carboxylic acid
PBS, phosphate buffered saline
PLA2, Phospholipase A2
PHGPx, phospholipid hydroperoxide glutathione peroxidase
pGPx, plasma GPx
PUFA, polyunsaturated fatty acids
ROS, reactive oxygen species
REM, relative eletrophoretic mobility
SDS, sodium dodecyl sulfate
SR-A, scavenger receptor
TBHQ, tert-butylhydroquinone
tBOOH, fert-butylhydroperoxide
TNFa, tumor necrosis factor alpha
TRE, PMA-responsive element
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X
LIST OF TABLES AND FIGURES
Table
Table 4.1 Effects of selenium supplementation on antioxidant enzyme 55
activities, protein contents and GSH levels in J774 macrophage
Figures
Fig. 1.1 Proatherogenic properties of oxLDL 5
Fig. 1.2 The structure of glutathione 9
Fig. 1.3 The mercapturic pathway 10
Fig. 1.4 Antioxidant functions of GSH 12
Fig. 1.5 y-Glutamyl Cycle 14
Fig. 1.6 Cellular redox regulated formation of y-GCS intersubunit 17
disulfide bond
Fig. 3.1 Ebselen structure and its glutathione peroxidase like activity 32
Fig. 3.2 Time course of macrophage GSH changes following LDL 35
treatments
Fig. 3.3 Dose-dependent effect of oxLDL on GSH depletion and adaptive 36
increase
Fig. 3.4 OxLDL-induced cytotoxicity 37
Fig. 3.5 OxLDL induced y-GCS-HS protein expression 39
Fig. 3.6 The effects of ebselen pretreatment on oxLDL and the uptake of 40
oxLDL by macrophages
Fig. 3.7 Effects of ebselen treated oxLDL on macrophage GSH status 42
Fig. 3.8 Correlation between oxLDL-associated LOOH and the extent of 43
GSH depletion
Fig. 3.9 GCS-HS protein expression at 10 hr incubation 45
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xi
Fig. 3.10 LDL induced ROS production in J774 A.1 macrophages 46
Fig. 4.1 Selenium supplementation induced increase of GPx activity and 54
protein expressions
Fig. 4.2 The in vitro GSH depletion, H2O2 metabolism 56
Fig. 4.3 MTT assay for peroxide-induced cytotoxicity 58
Fig. 4.4 Effects of oxLDL on Se (+) cell GSH and y-GCS-HS protein 59
induction
Fig. 5.1 Generation of reactive oxygen species by TBHQ 65
Fig. 5.2 Representative chromatogram of the HPLC fractions of cell- 67
modified LDL
Fig. 5.3 Time course studies of cell-mediated LDL oxidation (REM, 69
MDA)
Fig. 5.4 Modulating GSH levels by using BSO, tBHQ, NAC and OTC 70
Fig. 5.5 GSH monoester induced macrophage GSH increase 72
Fig. 5.6 Effects of BSO and TBHQ treatments on cell-mediated LDL 73
oxidation
Fig. 6.1 OxLDL-associated LOOH on macrophage GSH status 77
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ABSTRACT
MODULATION OF MACROPHAGE GLUTATHIONE SYNTHESIS BY
OXIDIZED LDL AND THE EFFECTS OF GLUTATHIONE CONTENT ON
CELL-MEDIATED LDL OXIDATION
Oxidized LDL (oxLDL) produced a rapid depletion of intracellular
glutathione (GSH) followed by an adaptive increase in J774 macrophages. OxLDL
also induced a transient increase in the levels of y-glutamylcysteine synthetase heavy
subunit (y-GCS-HS), representing the catalytic subunit of the rate-limiting enzyme
for GSH de novo synthesis. The induction took place within 3hr with maximum
levels observed by 10 hr of treatment. Pretreatment of oxLDL with ebselen inhibited
GSH depletion and attenuated the y-GCS-HS induction. OxLDL-associated lipid
hydroperoxides and their decomposition product aldehydes are two major
components thought to account for GSH depletion in macrophages. Ebselen
pretreatment had only a minor effect on aldehyde (MDA) levels, whereas peroxide
content was essentially abolished, suggesting that oxLDL-associated hydroperoxides
may mediate both GSH depletion and y-GCS-HS induction. Acetylated LDL
(AcLDL) also caused a moderate induction y-GCS-HS protein, suggesting a minor
involvement of scavenger receptor-mediated signaling of GSH synthesis.
Macrophages with higher glutathione peroxidase (GPx) activity experienced a more
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xiii
rapid and extensive depletion of GSH when treated with oxLDL under similar
conditions along with greater resistance to oxLDL or peroxide-induced cytotoxicity.
Change of macrophage GSH levels also appeared to affect the ability of cells
to modify LDL in culture. L-buthionine S,/?-sulfoximine (BSO) treatment depleted
80% of the cell GSH, that corresponded to a 70% increase in malonaldehyde
(MDA) and 40% increase in LDL' levels, representing the lipid peroxidation and
protein modification of LDL. Tert-butylhydroquinone (TBHQ) treatment caused a 3-
4 fold increase in macrophage GSH content and cells containing high GSH levels
mediated significantly less LDL oxidation.
We conclude that oxLDL-associated peroxides are primarily responsible for
GSH depletion, creating an oxidizing environment required for y-GCS induction and
compensatory GSH synthesis. This is facilitated in cells expressing high GPx activity
through a rapid depletion of GSH in the face of a peroxide-induced oxidant
challenge. Decreased intracellular GSH may further facilitate the oxidation of LDL
by vascular cells, this may contribute to the accumulation of atherogenic LDL
species. An increase in macrophage GSH status may also have a protective and
therapeutic effect by attenuating the oxidative stress that is thought to be involved in
early atherogenesis.
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1
CHAPTER 1
INTRODUCTION
1.1 Oxidative hypothesis of atherosclerosis
Atherosclerosis, the disease that underlines the formation of thrombi in
arteries of heart and brain, represents the leading cause of death in western
industrialized countries. Although the pathologic appearance and clinical
implications of atherosclerosis have been known for at least a century, an
understanding of the cellular and molecular mechanisms has largely emerged over
the past twenty years. Steinberg et al. (145) have reviewed some of the principal
findings pertaining to mechanisms underlying atherogenesis and proposed the
‘‘oxidative hypothesis of atherosclerosis”. It suggests that an important event during
the development of early atherosclerotic lesions is the oxidation of lipids contained
in low-density lipoprotein (LDL). This hypothesis is supported by a number of
studies demonstrating the atherogenic properties of oxidized LDL (oxLDL), the
existence of oxidatively modified LDL in atherosclerotic lesions, and the reduction
of atherosclerotic progression by antioxidants.
Composition o f LDL and LDL oxidation
Human LDL is defined as a population of lipoprotein that can be isolated by
ultracentrifugation within a density range of 1.019 to 1.063 g/ml. LDL molecules are
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spherical particles with a diameter of 19-25 nm and mean molecular mass of 2.5
million Kd (52). LDL consists of a protein and lipid domain, with apolipoprotein B
(apoB) being embedded in the surface of the particle. Each LDL particle contains
about 1600 molecules of cholesterylester and 170 molecules of triglycerides that
form an inner lipophilic core. This core is surrounded by a monolayer of about 700
phospholipid molecules and 600 molecules of free cholesterol. The polar head
groups of the phospholipids are located at the surface of the LDL particle and
contribute to the stability and miscibility of LDL in an aqueous solution. The total
number of fatty acids bound to different lipid classes of LDL is about 2600, half of
them are polyunsaturated fatty acids (PUFA) such as linolieic (about 86%).
arachidonic (about 12%) and docosahexaenoic acid (about 2%). These PUFA are
extremely susceptible to oxidation and are protected by the presence of several
lipophilic antioxidants, such as a-tocopherol, y-tocopherol, a- carotene, P-carotene.
ubiquinol-10 etc.
LDL oxidation is a free-radical-driven lipid peroxidation chain reaction, for
which simplified elementary reactions are shown as below:
INITIATION LH + X * ■ > L' +XH
V + ■ O 2 ■ » LOO"
PROPAGATION LOO- + LH » LOOH + L‘
INHIBITION BY ANTIOXIDANT LOO- + AH ■ » LOOH + A
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The attack of a free racial X’ and the hydrogen abstraction from one of the
LDL lipid (LH) bound PUFA leads to a carbon-centered lipid radical (L ), which
rapidly forms a lipid peroxyl radical (LOO'). The formation of LOO' triggers a chain
propagation reaction, with the LOO' abstracting a hydrogen atom from an adjacent
lipid and forming an unstable lipid hydroperoxide (LOOH) and a new L'. The further
decomposition of LOOH leads to formation of end products such as aldehydes,
ketones, alcohols, epoxides and hydrocarbons. This lipid peroxidation reaction can
be inhibited by antioxidants (AH) in LDL such as a-tocopherol. a-tocopherol
scavenges LOO' radical and forms less reactive tocopheroxyl radical, thus terminates
lipid chain propagation reaction.
Initiation o f LDL oxidation by cells and proatherogenic properties o f oxLDL
LDL oxidation in the blood stream is minimal because of the presence of
very efficient antioxidant defense mechanisms, including antioxidant vitamins (C, E
and P-carotene) and antioxidant enzymes such as extracellular glutathione
peroxidase (144). However, in the microenvironment of the arterial intima, the levels
of antioxidants are lower than those in plasma. Therefore, LDL 'trapped” within the
intima may be especially susceptible to oxidative modification by cells. A variety of
vascular cells, including endothelial cells, smooth muscle cells, lymphocytes and
monocyte-macrophages, can oxidize LDL in culture (57). However, the contribution
of the individual cell types to the generation of oxidative stress and the mechanisms
by which cells initiated LDL oxidation in vivo are still unclear. Several possible
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4
mechanisms involve cellular enzyme such as 15-lipoxygenase (100, 125), NADPH-
oxidase (12, 13), and myeloperoxidase (40, 68), or reactive nitrogen species (119,
120), metal ions (70) and thiols (69).
The distinct proatherogenic properties of oxLDL are summarized in Figure
1.1. Formation of cell-derived oxidants, transfer of peroxides from cell membrane
and reduction of transition metals could initiate LDL lipid peroxidation which then
leads to formation of the minimally modified LDL (mm-LDL). In biochemical terms,
mmLDL refers as the LDL contains small amounts of LOOH (seed peroxides) and
peroxide decomposition products, and without apo B modifications (84). MmLDL
induces endothelial cells to express adhesion molecules, proatherogenic proteins
such as monocyte chemotactic protein-1 (MCP-1) and macrophage colony-
stimulating factor (MCSF), which stimulate monocyte recruitment and
differentiation to macrophages in the arterial walls (20). The macrophages further
stimulate peroxidation of LDL. As oxidation proceeds, mm-LDL becomes more
heavily oxidized due to the extensive decomposition of LOOH and oxidative
modification of apo B. The peroxide decomposition products, such as aldehydes,
account for the major biological properties of extensively oxidized LDL. OxLDL is
chemotactic for monocytes and inhibits macrophage migration (20). It also
stimulates smooth muscle collagen production (79), enhances smooth muscle cell
proliferation (112), impairs vasodilatation via inhibition of endothelial nitric oxide
synthase expression (90) and releases vasoconstrictors such as endothelin-1 (67).
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° 2 * ^
OH
ONOO
myeloperoxidase
(HOCI, tyrosyl)
>
LDL
Stimulates expression
of MCP-1 and MCSF
Induces expression of
adhesion molecules
Cytotoxic
Induces expression of
proinflammatory cytokines
Transform monocytes
to resident macrophages
( 15-lipoxygenase
thiols
transition metal
transfer of lipid
^ hydroperoxides
Initiation o f lipid peroxidation
Cholesterol & CE accumulation
Foam cell formation
Fig. 1.1 Proatherogenic properties of oxLDL
Chemotactic
Induces expression of
proinflammatory cytokines
Chain propagation & decomposition
of lipid peroxidation
Immunogenic
Impairs vasodilatation
Stimulate smooth muscle cell
proliferation and collagen production
Platelet aggregation
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Furthermore, oxLDL has been shown cytotoxic to a number of cell types (51, 72).
The lipid radicals and the lipid peroxidation end products attack apo B protein and
modify the protein through adduct formation, cross-linking and defragmentation.
Modification of positively charged lysine residues of apo B lipoprotein leads to
formation of more electronegatively charged LDL. This heavily oxidized LDL is
recognized by scavenger receptors on arterial macrophages (71). The uptake of
oxLDL by scavenger receptor pathway is not subject to feedback regulation, thus
results in massive uptake of oxLDL-associated cholesterol by macrophages.
Scavenger receptor-mediated oxLDL uptake
Scavenger receptors are characterized by their ability to interact with a broad
variety of ligands that include modified proteins, lipoproteins, and some polyanionic
polysaccharides. In addition, some of them also interact with senescent cells,
polyanionic phospholipids, and bacterial components (87). The scavenger receptors
which have been shown to interact with oxLDL including class A scavenger receptor
(SR-A), CD 36, SR-BI, macrosialin/CD68, LOX-1. The “acetyl LDL receptor" or
SR-A is the first macrophage scavenger receptor to be identified (86). There are two
types of SR-A: type I receptors contain a cysteine-rich domain at their extracellular
COOH terminus, whereas type II receptors do not. The collagen-like domain of SR-
A is thought to mediate ligand binding (47). SR-A is normally present on the surface
of macrophages, Kupffer cells and endothelial cells. Its expression can also be
induced in other cell types. Both types of SR-A mediate endocytosis of acetylated
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LDL (AcLDL) and oxLDL (87). CD36, a class B type of scavenger receptor, is an
88-kDa transmembrane glycoprotein. It is found on monocyte/macrophages (148),
platelets (149), microvascular endothelium (85, 115), and some other organs (1,61).
CD36 mediates endocytosis of oxLDL but not of AcLDL (50). The murine
scavenger receptor class B type I (SR-BI), a member of the CD36 family, binds to
oxLDL (3) and HDL (2). The major function of SR-BI is believed to involve
cholesterol transport, in particular, to mediate the uptake of HDL cholesterol ester by
the liver and steroidogenic tissue (129,150). CLA-1 is the human homologue of SR-
BI (28). Others receptors such as macrosialin/CD68 (38, 97), Lectin-like oxLDL
receptor-1 (LOX-1) (111) have also reported to bind oxLDL. It was found that the
SR-AI/II/apo E double-knockout mice develop smaller atherosclerotic lesions than
apoE knockout mice (146). Although it has not been determined whether this was
due to the decreased uptake of oxLDL or some other action of SR-AI/II. Human
monocytes genetically deficient in CD36 have shown a decreased capacity to take up
oxLDL (113). These findings implicate both SR-A and CD36 scavenger receptors in
the metabolism of modified lipoproteins and link SR-A to the pathogenesis of
atherosclerosis. Furthermore, binding of oxLDL particles to scavenger receptors such
as CD36 (99) and LOX-1 (35) have been shown to generate of reactive oxygen
species (ROS) which may facilitate further oxidation. Unregulated uptake of
modified LDL could account for the transformation of monocyte-derived
macrophages to foam cells in atherosclerotic lesions (144), and at the same time,
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8
could induce severe oxidative stress or cell death if cells antioxidant defenses are
overwhelmed (88).
1.2 GSH and y-glutamyl cycle enzymes in antioxidant defense
Biological functions o f GSH
Glutathione (L-y-glutamyl-L-cysteinylglycine, [GSH]) (Figure 1.2), the most
abundant antioxidant in cells, plays a major role in cellular defense against oxidative
stress. GSH is present at a concentration of 1 ~ 10 mM in most mammalian cells,
whereas the extracellular concentration is about only 1-10 uM. Glutathione exists
in the reduced form (GSH) and oxidized form (GSSG). Normally, total glutathione is
referred as GSH plus twice the GSSG levels. The majority of glutathione (90%)
resides in the cytosol and nucleus, about 10 % of the glutathione is located in the
mitochondria and a small percentage in the endoplasmic reticulum (ER). In the
cytosol and mitochondria, the GSH to GSSG exceed 10:1 (102), whereas in ER, the
ratio is 3:1 (77). Two of the vital functions of GSH are summarized as following:
I) Conjugation function o f GSH Conjugation of xenobiotics or their metabolites is
one of the major functions of GSH. A variety of xenobiotic eletrophilic compounds
form conjugates with GSH, including some compounds produced during lipid
peroxidation, such as 4-hydroxynonenal (HNE) (130). Although GSH reacts
spontaneously with some eletrophiles (165), most these reactions require catalysis by
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9
y-carboxyl linkage
y-glutamyl cysteinyl glycine
Fig. 1.2 The structure of glutathione.
The amino-terminal glutamate and cysteine are linked by the y-carboxyl group of
glutamate.
an enzyme known as GSH S-transferase (GST) (10). GSH conjugation is often
considered as a mechanism of detoxification, because the conjugates are normally
excreted from the cells. The metabolism of GSH conjugates (the mercapturic
pathway) is summarized in Figure 1J . It begins with cleavage of the y-glutamyl
moiety by y-glutamyltranspeptidase (GGT), leaving a cysteinyl-glycine conjugate.
The cysteinyl-glycine double bond is cleaved by dipeptidase, resulting in a cysteinyl
conjugate. Acetylation of the cysteinyl amino group then forms a mercapturic acid
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X + y-GIu-CySH-Gly
GST
y-Glu-CyS-Gly
X
GGT
CyS-Gly
X
Dipeptidase
CyS-X
N-acetylase
N-acetyl-Cys-X
(mercapturic acid)
Fig. 1.3 The mercapturic pathway.
X= electrophilic compound, GST = GSH S-transferase, GGT
glutamytranspeptides
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11
that can easily excreted in the urine. The conjugation reaction results in an
irreversible loss of the L-cysteine residue of intracellular GSH.
2) Antioxidant function o f GSH As a consequence of aerobic metabolism, all
aerobic organisms are subject to certain level of physiological oxidative stress. The
ROS, such as superoxide (O^-), hydrogen peroxide (H2O2) and hydroxyl radical
(HO), can cause lipid peroxidation, protein oxidation, and cell injury. The
endogenously produced hydrogen peroxides and lipid hydroperoxides form during
lipid peroxidation can be reduced by GSH through the action of glutathione
peroxidase, forming GSSG (Figure 1.4). GSSG can be reduced back to GSH by
GSSG reductase (GRD) at the expense of NADPH. In the mitochondria, GSH is
particularly important in detoxifying hydrogen peroxide due to the lack of catalase.
Severe oxidative stress may lead to the accumulation of GSSG in the cytosol. To
prevent shift in the redox status, GSSG can be actively exported from of the cell
through an ATP dependent transport pathway (138) or react with a protein sulfhydyrl
group to form mixed protein disulfide (22). As a result, severe oxidative stress leads
to intracellular GSH depletion.
The y-glutamyl cycle
Maintenance of reduced GSH pool may be important for general cell
function, especially when cells are under severe oxidative stress. Although a few
epithelial cell types can assimilate intact GSH from extracellular fluid (64,65,89),
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12
CAT
HA
Peroxisome
Mitochondria
ROOH
2 GSH 2 GSH
GRD GPx or GST GPx
GSSG NADPH GSSG
PSSG PSH
Fig. 1.4 Antioxidant functions of GSH
CAT = catalase, GPx = glutathione peroxidase, GST = glutathione S-transferase
GRD = glutathione reductase, PSH = protein sulfhydryl, PSSG = protein mixed
disulfide
most cells maintain GSH levels by de novo synthesis of GSH using constitutive
amino acids, glutamate, glycine, and cysteine. Among them, cysteine is found to be
the rate limiting amino acid for GSH synthesis due to its lower intracellular
concentration.
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13
GSH serves as a continuous source for cysteine via y-glutamyl cycle (101)
(Figure 1.5). Here, GSH is released from the cell by carrier-mediated transporter(s)
(93), and depends on GGT to transfer the y-glutamyl moiety of GSH to another
amino acid, forming y-glutamyl amino acids and cysteinylglycine. y-glutamyl amino
acids can then be transported back into the cell to complete the cycle. Once inside
the cell, y-glutamyl amino can be further metabolized by 5-oxoprolinase to release
the amino acid and 5-oxoproline, which can be converted to glutamate. Cystine is the
most active amino acid acceptor of the y-glutamyl group. As cystine can be readily
reduced to cysteine once inside the cell. This reaction of cystine in the GGT-
catalyzed reactions constitutes a major scavenger pathway for recovery of cysteine.
Cysteinylglycine is broken down by dipeptidase to generate cysteine and glycine,
which are transported back into cells by different carriers (42). Cysteine is
transported by the sodium dependent ASC system (17. 82), which is highly
stereospecific and pH sensitive (94). About 90% of plasma cysteine exist as cystine,
and cystine is transported into cells utilizing the X c * system, which is a cystine-
glutamate antiport (15). It also has been suggested that cystine can react with GSH to
form cysteine which is transported much efficiently into the cells (44). Some cells
respond to GSH depletion or oxidative stress by increasing the activities of these
transport systems and thereby the supplies of GSH by de novo synthesis (16, 43,
141). The essential role of y-glutamyl cycle is to provide a source of cysteine that is
not as abundant as the other two amino acids.
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14
amino acid
extracellular
y-g/u-amino acid
y-glu-cys-gty' G* 51
1
in mu ii 1 1 n 1 1 1 in in i in mm 1 1 n 11111 n | i hi iiimiii
amino
▼ acid
5-oxoproline
ATP
GS y-GCS
ADP ATP ADP ATP
Fig. 1.5 y-Glutamyl Cycle
GSH serves as a continuous source for cysteine via y-glutamyl cycle. GSH is
released from the cell by carrier-mediated transporters) and depends on GGT to
transfer the y-glutamyl moiety of GSH to another amino acid (the best acceptor is
cystine) forming y-glutamyl amino acids and cysteinylglycine. The y-glutamyl amino
acids are transported back into the cell, and further metabolized to release the amino
acid and 5-oxoproline, which can be converted to glutamate. Cysteinylglycine is
broken down by dipeptidase (DP) to generate cysteine and glycine. Cysteine is
transported by sodium dependent ASC system, whereas cystine utilizes the X c *
system to transport into cells.
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15
GSH synthesis and yglutamylcysteine synthetase
GSH is synthesized in two sequential ATP-dependent enzymatic reactions
that are catalyzed by y-glutamylcysteine synthetase (GCS) and glutathione
synthetase (GS) (101):
L-Glutamate + L-cysteine + MgATP
-> L-y-Glutamyl-L-cysteine + MgATP + Pi (1)
L-y-Glutamyl-L-cysteine + Glycine + MgATP
-> Glutathione + MgATP + Pi (2)
The intermediate L-y-Glutamyl-L-cysteine can be acted on either by GSH
synthetase forming GSH or by y-glutamylcyclotransferase forming 5-oxo-L-proline
and L-cysteine. Because the Km value of y-glutamylcyclotransferase for y-
glutamylcysteine is -12 fold higher than that of GSH synthetase, and total activity of
the y-glutamylcyclotransferase in cells is much lower, thus GSH synthesis is favored
(143).
The intracellular GSH levels are mainly regulated by activity of rate-limiting
enzyme y-GCS (162). y-GCS is a heterodimer consisting of a catalytic heavy subunit
(GCS-HS, Mr 73,000) containing all substrate binding sites and a regulatory subunit
(GCS-LS, Mr 31,000) that modulates the affinity of heavy subunit for substrate and
inhibitors. GCS-HS and GCS-LS are encoded by separate genes (154). The absolute
amounts and ratios of each subunit vary among different normal human tissues (59).
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16
In most cases, increases in GCS-HS levels have been shown to coincide with
elevation in GSH levels (107), co-ordinate up-regulation of regulatory subunit also
has been found under oxidative stress (55). However in Hela cells, overexpression of
the y-GCS-LS alone has been shown to be sufficient to increase GCS activity and
GSH levels (153), suggesting a role for both subunits in GSH synthesis. For rat or
human y-GCS, about 30-70% is stabilized by an intersubunit disulfide bond as well
as noncovalent bonds (136). The formation of this intersubunit disulfide bond may
be regulated by cellular redox status (75) (Figure 1.6). Oxidizing conditions that
result in the depletion of GSH promote the intersubunit disulfide bond formation.
This produces a conformational change increasing the affinity and specificity of the
substrate binding sites and optimizing y-GCS activity. On the other hand, reducing
conditions provided by physiological concentrations of GSH decrease intersubunit
disulfide formation, producing a conformational change that favors GSH feedback
inhibition (75).
Several factors control the rate of de novo GSH synthesis, including the rate-
limiting enzyme y-GCS, the availability of cysteine (6 , 32) and the feedback
inhibition of GCS activity by GSH. Approximately 80% of GCS is inactive when
GSH is present at the normal concentration for a given cell type (106, 128).
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17
s --- /
substrates
HS
t GSH
inhibitors
Fig. 1.6 Cellular redox regulated formation of y-GCS intersubunit disulfide
bond. (Modified from Huang et al. 1993)
Oxidizing conditions that result in the depletion of GSH or increase of GSSG
promote the intersubunit disulfide bond formation. This produces a conformational
change that increases the affinity and specificity of the substrate binding sites and
optimizes y-GCS activity. On the other hand, reducing conditions provided by
physiological concentrations of GSH decrease intersubunit disulfide formation,
producing a conformational change that favors GSH feedback inhibition. However,
the levels of GSH may only be reflective of the redox (thiol) status in cells that form
the disulfide (S-S) bond of GCS subunits.
1J Cellular GSH changes in response to oxLDL-induced oxidative stress
OxLDL contains high concentrations of lipid peroxidation productions such
as peroxides and aldehydes, which are known to be cytotoxic (51, 72). Since
vascular cells sequester this form of modified LDL through SR, thus are likely
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18
subjected to severe oxidative stress or cell death if the cellular antioxidant defenses
are overwhelmed. It is therefore interested to know how the cellular antioxidants,
such as GSH, response to the oxidative challenge induced by oxLDL.
Acute exposure of oxLDL decreases GSH pool at least transiently and then
followed by an elevation in GSH levels, which is considered as a protective response
against oxidative stress. OxLDL induced GSH depletion and repletion have been
found in a number of vascular cells, such as macrophages (39), smooth muscle cells
(141) and endothelial cells (33). Although the molecular mechanism by which this
takes place is unclear, the initial depletion of GSH might due to the detoxification of
oxLDL containing lipid hydroperoxides or aldehydes. The formation of GSSG or
GSH-conjugates and their exportation from the cells leads to a net loss of GSH (78).
Although GSH can be partially recovered due to the action of GRD following
oxidative stress, the adaptive increase of GSH is believed primary due to the de novo
synthesis. Recently, oxLDL-induced increases in the expression of y-GCS-HS
mRNA and GSH levels have been found in human vascular endothelial cells, and the
induction of y-GCS enzyme is mediated by transcription factor activator protein-1
(AP-1) (33).
AP-1 is a heterodimer composed of the c-Fos and c-Jun proteins, or a
homodimer of c-Jun proteins. AP-1 controls the expression of many genes, and
activates these genes by binding to TP A response elements (TRE)s in their promoter
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19
region (8 ). AP-l or AP-1 like-response elements have been identified in the 5’-
flanking promoter regions of both human GCS subunits (55, 108, 109). The
expression of y-GCS gene is under the control of the thiol redox state through two
major steps. The first step concerns the mechanism of activation of the AP-1, which
allows its redistribution into the nucleus. The second step deals with the binding of
AP-1 to the promoter region located in the 5’ end of the y-GCS gene. Fos and Jun
contain redox-sensitive cysteine residue in their DNA binding domain. These
cysteines are essential for the recognition of the binding site through electrostatic
interactions with specific DNA bases. AP-1-DNA binding is enhanced by a reducing
nucleus environment provided by thioredoxin and/or nuclear redox protein 1 (Ref-1)
(73), and is inhibited by GSSG accumulation probably due to the formation of the
disulfide bond of cysteine resides (56). However, oxidative stress imposed by
hydrogen peroxide treatment, UV irradiation, TNF a or depletion of intracellular
GSH using DL-buthionine-(SR)-sulfoximine also have shown to stimulate AP-1-
DNA binding (103, 123). The differences in biological response to these agents may
be cell-specific or may dependent on the extent of cytosolic/nucleus thiol redox
changes. Oxidant-induced severe redox changes could affect nucleus redox potential
and reduce the AP-1 DNA binding. Therefore, variations in the intracellular redox
state mediated by nonlethal oxidative stress can transiently modify the activity of
transcription factors and either upregulate or downregulate gene expression. The
GSH redox changes in response to physiological stimuli have been shown to be of
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20
sufficient magnitude to control the activity of some redox-sensitive proteins such as
GST and NADPH: quinone reductase. Although the correlation between GSH redox
change and GCS induction needs further study to be established (83).
In this study, we investigated major oxLDL-associated factors underlying
macrophage GSH changes and y-GCS induction in relation to oxLDL-induced
oxidative stress. We also studied the potential effects of GSH content on cell-
mediated LDL oxidation.
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21
CHAPTER 2
GENERAL EXPERIMENTAL DESIGN
2.1 Hypothesis
Depletion and compensatory increase in macrophage GSH levels following
uptake of oxLDL is primarily due to the LDL-associated lipid hydroperoxides.
Peroxide-mediated oxidation of GSH leads to the induction of y-GCS and the
increase of GSH synthesis. Depletion of macrophage GSH content will also affect
the ability of macrophages to oxidize LDL.
2.2 Specific Aims
The specific aims for this project were:
1. To characterize the changes in macrophage GSH contents and y-GCS protein
expression in response to oxLDL-induced oxidative stress.
2. To determine which oxLDL-associated factors induce GSH depletion in
macrophages.
3. To determine the extent of GSH changes and y-GCS induction in Se (+) ceils
with higher peroxide-detoxifying capacities.
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4. To test whether the changes in macrophage GSH content affect their ability to
oxidize LDL in culture.
2.3 Materials & Methods
Materials
J774A.1 macrophage cell line was purchased from ATCC (Manassas,
Virginia). Sodium selenite, GSH, GSSG, fucoidin, glutathione reductase, NADPH,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT), tert-butyl
hydroperoxide (t-BuOOH), Diethylenetriaminepentaacetic acid Free Acid (DTPA),
2.4 dinitrofluorobenzene and 2’,T - Dichlorofluorescin diacetate (DCFH-DA) were
obtained from Sigma (St. Louis, MO). y-Glutamyl-glutamic acid (GGA) was from
Bachem Bioscience (Torrance, CA). l,r-dioctadecyl-3,3,3\3'-
tetramethylindocarbocyanine perchlorate (Dil) was from Molecular Probes (Eugene.
Oregon).
Cell culture
J774A.1 macrophages were maintained in Dulbecco’s Modified Eagle’s
Medium (DMEM) supplemented with 10% (vol/vol) fetal bovine serum (FBS)
(Omega Scientific Inc., CA) and 0.05 mg/ml gentamicin (Omega Scientific Inc.,
CA), at 37°C in a humidified incubator (5% CO2, 95% air). Experiments were
performed when cells reached about 90% confluent
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23
Isolation of LDL from human plasma
Human LDL (i/1.019-1.063 g/ml) was prepared by ultracentrifugation using a
Beckman L8-55 ultracentrifuge equipped with SW-41 rotors as described previously
(74). The LDL fraction was dialyzed and concentrated using a centrifugal filter
device (Millipore Corporation, MA) with a molecular weight cut off at 30K. LDL at
1 mg protein/ml phosphate buffered saline (PBS) containing lOOpM EDTA was
sterilized by filtration through 0.2 pm syringe filter (Coming Corporation, NY) and
stored at 4°C until used for various experiments. Protein concentration was measured
by the Bio-Rad protein assay reagent (Bio-Rad, CA) using bovine serum albumin as
a standard.
LDL modification
Human LDL was diluted to 0.2 mg LDL protein/ml and incubated with 10
pM CUSO4 for 20 hr at 37°C. Oxidation was terminated by adding 100 pM EDTA
and cooling. LDL was dialyzed and reconcentrated to 1 mg protein/ml. sterilized and
stored at 4°C in PBS containing 100 pM EDTA. Acetylated LDL (AcLDL) was
prepared by chemical modification of LDL with acetic anhydride as described by
Basu et al. (18). In a typical preparation, 1 ml of PBS containing 2 mg LDL protein
was added to 1 ml of a saturated solution of sodium acetate with continuous stirring
at 4°C. After the addition of a total mass of acetic anhydride equal to 1.5 times the
mass of protein, the mixture was stirred for additional 30 min without further
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24
additions. The reaction solution was then dialyzed using a centrifugal filter device
(Millipore Corporation, MA) with a molecular weight cut off at 30K. The AcLDL
solution was then stored at lmg LDL protein/ml in PBS containing 100 (iM EDTA
under argon. LOOH levels for unmodified LDL (nLDL), AcLDL and oxLDL were
25.9 + 12.8,23.7 + 2.3 and 1528 + 6 6 nmol/mg LDL protein, respectively.
LDL' separation
LDL' (electronegatively charged LDL) was separated from human total
plasma LDL using ion exchange high pressure liquid chromatography (Perkin-
Elmer, CT) with an UNO Q1 column (Bio-Rad Instruments, CA) (74). 1 ml sample
(containing 0.1-0.2 mg LDL protein) was injected and eluted at 1 ml/minute with
0.01 M Tris-HCl buffer (Buffer A, pH 7.2) for 5 minutes, and then the gradient was
linearly increased over 15 minutes to 300 mM NaCl in 0.01 M Tris-HCl buffer
(Buffer B, pH=7.2). The eluent was monitored at k = 280 nm using a LC-90 UV
spectrophotometric detector (Perkin-Elmer, CT), the peaks were integrated with
Axxi-chrom 727 chromatography software (Axxiom-chromatography Inc., CA). For
cell culture treatment, LDL' fractions were purified and salts removed by centrifugal
dialysis using a 30K molecular weight cutoff filter (Millipore Corporation, MA). The
samples were then diluted in PBS and added to cultured cells. The LOOH levels for
LDL' were about 700-800 nmol/mg LDL protein.
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25
Measurement of H2O2 metabolism
H2O2 in cell culture medium was measured using a biological oxygen monitor
(Yellow Spring Instrument Co. Inc., Ohio) following the addition of excess catalase
(9). J774 macrophages (1-2 x 107 ) were plated in a 100 mm dish with 20 ml RPMI
medium 1640 (Life Technologies, Inc., MD). An initial concentration of 100 pM
H2O2 was added to medium and samples were taken every 5 min to measure the
remaining H2O2 concentration. A calibration curve was made with H2O2 reagent for
each experiment.
Lipid peroxide measurement
LOOH levels in LDL were determined using the modified method of
Auerbach et al. (11). Samples were added to a mixed solution containing
leucomethylene blue (LMB) and hemoglobin for 1 hr. Oxidation of LMB to
methylene blue was monitored at 650 nm using a microplate reader (Cambridge
Technology Inc., MA). /-butyl hydroperoxide (/-BuOOH) was used as calibration
standard.
TBAR measurement
This assay is a modified method of Buege (26). Samples (0.5 ml) were
mixed thoroughly with 1 ml TCA-TBA-HCL solution (15% w/v trichloroacetic acid,
0.375% w/v thiobarbituric acid and 0.25 N hydrochloric acid) and heated for 15 min.
After cooling, the floccuient precipitates were removed by centrifugation. The
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26
absorbance of the sample was determined at 535 nm against a blank containing all
reagents except the LDL samples. The malondialdehyde equivalent (MDA)
concentration was calculated using an extinction coefficient of 1.56 x 10s M'1cm'1 .
In vitro GSH depletion assay
This assay is modified from the method of Ursini et al. (132). Cells were
lyzed in PBS by ultrasonication and centrifuged at 2000 rpm for 5 min. NADPH
depletion rates were measured at 340 nm for 8 min using Beckman DU-650
spectrophotometer in a reaction mixture containing 0.2 mM NADPH, 0.1% TritonX-
100, 0.1 M Tris buffer containing 5 mM EDTA, 1 unit glutathione reductase, 3 mM
GSH and cell lysate (derived from 100 pg cell protein) to which 200 pM r-BuOOH
was added as a substrate. Basal rates of depletion were obtained using all reagents
except cell lysate. GSH depletion rates are calculated assuming that 1 mole NADPH
depletion is equivalent to 2 moles GSH.
LDL uptake by macrophages
Measurement of LDL uptake by J774 cells was accomplished using
fluorescently labeled LDL, referred to as Dil-LDL. Dil-LDL was prepared by
incubating 500 pg/ml LDL with Dil in DMSO to reach a final concentration of 50 ng
Dil/pg LDL. Cells (2-3 x 10s) were then incubated with 10 pg Dil-LDL/ml at 37°C
for 3 hr. The cell monolayer was washed twice with PBS, followed by addition of
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27
1.2 ml isopropyl alcohol. Plates were shaken gently at room temperature in the dark
for 30 min. The sample extracts were measured in a fluorometer (Hitachi, CA) at Ex
523 nm, Em 563 nm. The amount of uptake was corrected on the basis of cell
numbers.
Enzyme assays
GPx activity was determined by spectrophotometrically monitoring the
oxidation of NADPH at 340 nm. One unit of activity is defined as the amount of
enzyme catalyzing the oxidation of 1 nmol NADPH per minute. Assay mixtures (1
ml) contained 3 mM GSH, 0.2 mM NADPH, 1 unit glutathione reducatase (GRD),
0.5% Triton X-100 and 100 pg cell protein in 100 mM Tris buffer containing 5 mM
EDTA (pH = 7.4). /-BuOOH was used as a substrate. Since /-BuOOH is a common
substrate for both cytosolic glutathione peroxidase (cGPx) and Phospholipid
glutathione peroxidase (PHGPx), the results represent total GPx activity.
GSSG-reductase activity was determined by monitoring the decrease of
absorbance at 340 nm during the reduction of GSSG by NADPH (49). Reaction
mixtures contained 0.1 M potassium phosphate buffer (pH = 7.6), 1 mM GSSG, 0.1
mM NADPH and 100 pg cell protein.
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Catalase activity was determined by monitoring H2O2 consumption at 240
nm. Assay mixtures (0.5 ml) included 300 pi 0.05 M potassium phosphate buffer
containing lmM DTPA, 100 pg cell protein and 100 pi of 100 mM H2O2. Sample
absorbance was monitored every 0.2 second for 2 minutes. Results were plotted
semi-logarithmically against time fo-tonm = 43.6 M*1 cm 1 ). Catalase activity was
determined from the slope of the plot divided by cell protein (k’= s' 1 mg'1 ).
Measurement of intracellular GSH
Intracellular GSH and GSSG content were determined by HPLC according to
the method of Fariss and Reed (53). Briefly, cell samples following different
treatments were washed twice with ice-cold PBS and collected in 1 ml 10%
perchloric acid containing 2 mM EDTA and 7.5 nmol of GGA (Bachem Bioscience.,
CA) as an internal standard. After centrifugation, supernatants were collected and
stored at -20°C. Supernatants from each sample (0.45 ml) were mixed with 45 pi
iodoacetic acid (0.0186g/ml 0.2 mM cresol purple), and adjusted the pH to 8-9
(definitely purple) using 2 M K.OH/ 2.4 M KHCO3 solution. The mixtures were
incubated in the dark for 15 minutes, followed by addition of 0.45ml of 1%
dinitrofluorobenzene (DNB) per sample. The reaction mixtures were incubated
overnight at 4°C, and then terminated by adding 45 pi I M L-lysine. After an
additional 2-4 hr at 4°C (in the dark), then sample were briefly concentrated and the
supernatants injected into a Spheri-5 amino column. GSH, GSSG and GGA contents
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29
were analyzed using a Perkin Elmer HPLC with spectrophotometric detection at
365nm. The elution solvents were 80% methanol (solvent A) and 0.5 M sodium
acetate in 64% methanol (solvent B). The mobile phase started with 70% solvent A
and 30% solvent B for 5 minutes, followed by a 15 minutes liner increase to 100%
solvent B and maintained for another 30 minutes. The flow rate was 1.5 ml/min.
All standards were run under the same condition and the peak areas were
integrated. The peaks were integrated with Axxi-chrom 727 chromatography
software (Axxiom-chromatography Inc., CA). The GSH and GSSG contents were
normalized by sample protein and expressed as nmol/mg cell protein.
Western blot analysis
J774 A. 1 macrophages were washed twice with Dulbecco’s PBS, scraped
and collected by low speed centrifugation. The cell pellet was lysed in 150 pi GLB
(0.3% SDS, 50 mM Tris pH 8.0, 1% p-mercaptoethanol) and then treated with 10 pi
of RNase/DNase solution (1 mg/ml DNase, 0.5 mg/ml RNase A, 500 mM Tris pH
6 . 8 and 50 mM MgCh) for 1 min. The samples were mixed with 5X Laemmli
sample loading buffer (4:1), heated for 2 min at 100°C, and separated
electrophoretically on 12% PAGE (Bio-Rad Mini protein system). Proteins were
transferred to Immobilin P membranes (Millipore Corporation, MA) at 100 V for 1
hr. The membrane was blotted with 5% nonfat dry milk in TBS with Tween-20 for
1-2 hr. Rabbit anti-mouse cGPx or PHGPx antibody (provided by Dr. Fulvio Ursini)
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30
was diluted 1:1000 and incubated with membranes for 2 hr at 37°C. Rabbit anti-rat
GCS-HS antibody (provided by Dr. Henry Forman) was diluted 1:5000 and
incubated with membranes overnight at 4°C. The guidelines provided in the ECL
Western blotting kit for chemiluminescence detection with hyperfilm were followed.
Protein bands were quantified by densitometry.
MTT Cytotoxicity Assay
Cell viability was determined spectrophotometrically by measuring
mitochondrial dehydrogenase activity (45). Briefly, cells were plated at a 48-well
dish (Coming Incorporation, NY) and treated with different reagents. After
treatment, stock MTT solution (5 mg/ml) was added to each well at 10% of culture
medium volume, and incubated for 2 hours at 37°C. After the incubation, the original
medium was removed and dissolved in isopropanol for 30 min. Absorbance was
measured at 570 nm using microplate reader. The extent of cytotoxicity was
determined from the number of surviving cells as calculated from the amount of
formazan produced in control and treated cells.
Statistical Analysis
Data are expressed as mean ± S.E. unless indicated otherwise and evaluated
by Student’s t test. For all analyses, p < 0.05 is considered statistically significant.
The numbers of samples used in each experiment are indicated under “Results” or in
the figure legends.
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31
CHAPTER 3
ADAPTATION TO OXIDATIVE STRESS: MECHANISMS OF OXLDL-
INDUCED GSH DEPLETION AND REPLETION
3.1 Introduction
OxLDL has been shown to cause an initial decrease followed by an adaptive
increase of GSH in macrophages (39), and human vascular endothelial cells (33).
However, the specific components of the oxLDL particles that induce these changes
have not been determined. The major oxidative products of oxLDL include lipid
hydroperoxides and their degradation products such as MDA and HNE, all of which
could responsible for GSH depletion through either oxidation or conjugation
reactions. Our major goal is to determine the contribution of these components in
oxLDL-induced GSH changes. Our strategy is to selectively reduce the peroxide
content in the oxLDL by using ebselen (2-phenyl-l,2-benzisoselenazol-3(2H)-one).
By comparing the effect of oxLDL and peroxide-depleted (ebselen treated) oxLDL.
we will able to determine the contribution of oxLDL-associated peroxides in GSH
changes.
Ebselen is an organic seleno-compound with glutathione peroxidase-like
activity (95, 140) (Figure 3.1). The peroxide-reducing reactions proceed in three
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32
N
E-Cys-SeH + ROOH -> E-Cys-SeOH + ROH (1)
E-Cys-SeOH + GSH -> E-Cys-Se-SG + H2 0 (2)
E-Cys-Se-SG + GSH -► E-Cys-SeH + GSSG (3)
ROOH + 2GSH -► ROH + H2 0 + GSSG (4)
Fig. 3.1 Ebselen structure and its GPx like activity
main steps, involving the enzyme-bound selenocysteine, E-Cys-SeH, present as
selenol or selenolate. In reaction (1), the organic hydroperoxide, ROOH, reacts in a
diffusion-controlled reaction to yield the selenenic acid, E-Cys-SeOH and the
corresponding alcohol, ROH. The following two steps involving a sequential
reduction by GSH. Reaction (2) yields the selenodisulfide and water, and reaction (3)
gives the selenol and the GSSG. The overall reaction is just like that of glutathione
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33
peroxidase. Substrate specificity for ebselen ranges from hydrogen peroxide and low
molecular weight organic hydroperoxides to membrane-bound or oxLDL-associated
phospholipid and cholesterol hydroperoxides (13S). In addition to glutathione, the
thiol reductant cosubstrates also include dithioerythritol, N-acetylcysteine, or other
suitable thiol compounds. Besides the well-known peroxide-reducing ability, ebselen
also has been shown to scavenge free radical, peroxynitrite and singlet oxygen (140).
Ebselen pretreatment improves the resistance of leukemia cells to peroxide-induced
cytotoxicity (58), ebselen also reduce the cell apoptosis induced by oxidative stress
developed during open-heart surgery (98), all this can be explained by the
glutathione peroxidase like activity of ebselen. Since ebselen is considered as an
antioxidant in tissue, it may also have a potential pharmacological application.
In this chapter, we first characterized the oxLDL induced GSH changes and
y-GCS induction in J774 macrophages, and then we further studied the major
oxLDL-associated factors underlying GSH depletion and repletion in relation to
oxLDL-induced oxidative stress.
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34
3.2 Results
OxLDL induced an initial depletion followed by an adaptive increase in macrophage
GSH content
To study the effects of oxLDL on intracellular GSH, GSH levels were
measured over a 24 hr period in J774 A.l macrophages treated with 200 pg/ml
oxLDL, LDL\ AcLDL and nLDL. A marked decrease in GSH was observed within
3 hr after oxLDL treatment followed by a recovery to basal levels at 6 hr and an
adaptive increase by 24 hr (Figure 3.2). GSH continued to increase to 30 hr of
incubation and then gradually decreased to basal levels (data not shown). Mildly
oxidized LDL (LDL\ (74)) which is not a ligand for the scavenger receptor (14,41),
produced a small and non-significant initial decrease and subsequent increase in
GSH levels. In contrast, nLDL had no effect on intracellular GSH levels. AcLDL
caused no GSH depletion but elicited a transient 20% increase between 3-6 hr. Both
the initial depletion and adaptive increase of GSH were oxLDL dose dependent as
shown in Figure 3.3. A dose up to 50 pg/ml oxLDL showed no effect on
macrophage GSH content, only higher doses of oxLDL induced the changes in GSH
levels. This indicates that with lower level of oxidative stress, there is no net loss of
GSH through oxidation, probably due to the action of GSSG reductase system. The
changes in GSH were not due to compromised cell metabolism, as no severe toxicity
was found at this level of oxLDL treatment (Figure 3.4). OxLDL induced
cytotoxicity as determined by using MTT assay showed that surviving fractions after
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35
250
oxLDL
AcLDL
200
2
s
nLDL
<3 150
* 8
£
X 100
u
Time (hr)
Fig. 3.2 Time course of macrophage GSH changes following LDL treatments.
J774 A.l macrophages (2 x 106 cells/well) were treated with 200 |ig/ml oxLDL (■),
LDL‘ (♦), AcLDL (□) and nLDL (O) up to 24 hr in 10% FBS DMEM. Intracellular
GSH levels were determined according to Materials and Methods. The GSH content
is expressed as % of untreated control. Data are the mean ± S.E. (%) for at least three
independent experiments. LOOH levels in nLDL, AcLDL, LDL‘ and oxLDL were
25.9 + 12.8,23.7 ± 2.3,822 + 56, and 1528 + 6 6 nmol/mg LDL protein, respectively,
and MDA levels in nLDL and oxLDL were 1.3 + 0.12 and 12.5 + 3.6 nmol/mg LDL
protein,
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36
1 2 0
3 hr
100 n
a
O
o
0 50 100 150 2 0 0 250
240
24 hr
200
1 160
120
80
40
0
0 50 100 150 200 250
oxLDL (|ig/ml)
Fig. 33 Dose-dependent effect of oxLDL on GSH depletion and adaptive
increase. J774 A.l macrophages (2 x 106 cells/well) were treated with oxLDL at 0,
10, 50,100, 200 pg/ml for 3 hr or 24 hr. (A), oxLDL induced GSH decrease at 3 hr.
(B), oxLDL induced GSH increase at 24 hr. The GSH content is expressed as mean +
S.E. (%) of untreated controls for three independent analyses with duplicate
measurements at each time point
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37
100 r
J 80
60
40
20
0
200 400
oxLDL (jig/ml)
Fig. 3.4 oxLDL-induced cytotoxicity
J774 macrophages (2 x 105 /well in 24 well dish) were treated with oxLDL for 24 hr.
OxLDL induced cytotoxicity were determined by using MTT assay. Data were mean
+ SE % of untreated control.
oxLDL treatments were 86.7 + 0.3% and 73.7 + 0.8% for 200 pg/ml and 400 (ig/ml
oxLDL, respectively. Together these data suggest that only oxidatively modified
LDL is able to induce significant GSH depletion and adaptive increase in
macrophages.
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38
OxLDL induced GSH-HS protein expression
Intracellular GSH levels are mainly regulated by the activity of the rate-
limiting enzyme y-GCS (162). Western analysis of y-GCS-HS protein expression
was performed to determine whether the GSH increases produced by oxLDL were
due to y-GCS induction. When cells were treated with 200 pg/ml oxLDL, a increase
y-GCS-HS protein was observed as early as 3 hr, with a maximum 4-5 fold increase
by 10 hr followed by a sustained 2-fold protein induction at 24 hr post-treatment
(Figure 3.5). The time course for y-GCS-HS expression correlated with a delayed
increase in GSH after its initial depletion as shown in Figure 3.2. As expected,
nLDL treatment at the same dose caused no induction of y-GCS enzyme. These
results are paralleled with the recent finding in human vascular endothelial cells (33),
suggesting an oxLDL-induced de novo synthesis of GSH via induction of y-GCS
enzyme.
Ebselen pretreatment reduced oxLDL-associated lipid hydroperoxides
LDL lipid peroxidation leads to formation of LOOH and their degradation
products, aldehydes, such as MDA. We considered that these two components might
be primarily responsible for GSH depletion. Pretreatment of oxLDL with 50 pM
ebselen and 3 mM GSH for up to 30 min reduced the peroxide content by greater
than 90%, with only a minor effect on MDA levels (Figure 3.6 A). Ebselen
treatment did not affect oxLDL uptake by macrophages as shown in (Figure 3.6 B),
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39
y-GCS-HS protein
67kd
S 5
e
‘3
e
fc.
0
10 24
Time (hr)
600
oxLDL
500
nLDL
400
300
200
100
0
10 20 30
Time (hr)
Fig. 3.5 OxLDL induced y-GCS-HS protein expression.
J774 A. 1 macrophages (2-3 x 106/well) were treated with 200 (ig/ml nLDL, oxLDL
for the incubation periods indicated. y-GCS-HS protein levels were determined by
Western analysis, using anti-rat GCS-HS antibody. Data are expressed as % of
untreated controls (NC). * Significantly different from nLDL, P < 0.05.
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40
2
i m
e
©
U
140
120
MDA 100
80
60
40
LOOH
20
0
0 10 20 30 40
Time (min)
B
0.08
« • > 0.06
g 0.04
0.00
w /o Ebselen w Ebselen
Fig. 3.6 The effects of ebselen pretreatment on oxLDL and the uptake of oxLDL
by macrophages
(A). OxLDL was pretreated with 50 pM Ebselen plus 3 mM GSH for 30 min at 37
°C, Data are mean + S.E. (%) of three independent experiments.
(B). Cells (2-3 x 105 ) were then incubated with 10 pg Dil-LDL/ml at 37°C for 3 hr as
described in Materials and Methods. OxLDL uptake is expressed as mg oxLDL
protein per 105 cells in 3 hr. Data are the mean + S.E. for two independent
experiments with triplicate measurements for each sample.
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41
since a similar uptake of Dil oxLDL was found for ebselen-treated and untreated
oxLDL by macrophages. By using ebselen, we able to differentiate the
OxLDL-associated lipid hydroperoxide affects GSH content
Ebselen-treated oxLDL at a dose of up to 200 pg/ml caused neither the initial
GSH depletion nor the subsequent adaptive GSH increase (Figure 3.7). These effects
were not likely due to residual ebselen activity in the oxLDL, since the GPx-like
activity of potentially contaminating ebselen was less than 10 nmol/min/mg LDL,
less than 10% of the GPx activity in macrophages under normal culture condition.
Fucoidin (a SR-A blocker) (46), partially prevented the initial depletion but not the
adaptive increase in GSH, indicating that rapid uptake by SR-A may contribute, but
is not absolutely required for oxLDL-induced GSH changes. A strong correlation (R
= -0.869) has been found between LDL-associate LOOH and the extent of GSH
depletion using LDL contains different peroxide content (Figure 3.8), whereas a
relatively weak correlation (R = 0.427) was found between LOOH level and the
extent of GSH increase (data not shown). These LDL particles were either oxLDL or
AcLDL form different plasma pools and modified at same procedures, thus the rates
of uptake of these modified LDL were expected to be very similar. These findings
suggest that the uptake of oxLDL-associated lipid hydroperoxides is primarily
responsible for GSH depletion.
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x 1 0 0 '
Vi
u
50
0.0
3 hr 24 hr
Fig. 3.7 Effects of ebselen treated oxLDL on macrophage GSH status
J774 A. 1 macrophages (2-3 x 106/well) were treated with 200 pg/ml oxLDL (n=4)
and ebselen pretreated oxLDL (n=5) for 3 hr (white bar), or 24 hr (gray bar). To
block scavenger receptors, cells were pretreated with 50 pg/ml fucoidin for 3 hr prior
to the addition of oxLDL. Data are expressed as mean + S.E. (%) for three
independent experiments. * P < 0.05, comparison to 0 hr untreated control, **P <
0.05, comparison to 3hr oxLDL treatment, *** P < 0.05, comparison to 24 hr oxLDL
treatment.
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43
140
R = -0.869
n = 12
P< 0.0006
GSH decrease (3 hr)
120
100
200 300 400 600 0 100 500
LOOH GiM)
Fig. 3.8 Correlation between LDL-associated LOOH and the extent of GSH
depletion
LDLs isolated from different plasma pools were modified at same procedure for
more than five separate experiments. LOOH content for oxLDL ranges from 1300 ~
2800 nmol/mg LDL protein, with media value about 1600 nmol/mg LDL protein,
whereas only 23.7 + 2.3 nmol/mg LDL protein LOOH in AcLDL. GSH depletion (%
of NC) was plotted against LOOH concentrations that were equivalent to 200 pg/ml
oxLDL or AcLDL.
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44
AcLDL, and ebselen pretreated oxLDL also induced an increase in y-GCS-
HS levels, with similar but truncated patterns of expression over time that were
approximately 55% and 35% less, respectively, than oxLDL treatment at 10 hr
(Figure 3.9). These data suggest a peroxide-mediated de novo GSH synthesis via
induction of y-GCS after exposure to oxLDL. However, other alternative pathways
such as the scavenger receptor-mediated signaling events may also contribute to
enzyme induction.
OxLDL induced macrophage ROS production
It is well known that y-GCS activity and transcription can be affected by a
variety of factors, including oxidative stress which produces ROS (124, 139, 152).
To determine if oxLDL-induced ROS production in J774 macrophages corresponded
to the induction of GSH synthesis, cells were treated with 200 (ig/ml nLDL, AcLDL
and oxLDL for 3 hr, and then labeled with 100 (iM DCFH-DA for another 30 min,
followed by measurement of DCF absorbance at 502 nm. nLDL, AcLDL and oxLDL
treatment increased DCF absorbance by 106 + 5.2%, 125 ± 9.9%, and 136 ± 13.5%,
respectively, over the spontaneous rate of DCF oxidation in cells as shown in Figure
3.10. This suggests that ROS generation is associated with LDL-scavenger receptor
binding and this could also partially contribute to the induction of macrophage y-
GCS induction as shown in Figure 3.9.
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45
y-GCS-HS protein (10 hr incubation)
67kd
NC AcLDL Eb-oxLDL oxLDL
600
500
£
s
• M
4 )
400
o
S.
v i
300
X
t
VI
U
2 0 0
>-
1 0 0
0
NC AcLDL Eb-oxLDL oxLDL
Fig. 3.9 GCS-HS protein expression at 10 hr incubation
J774 macrophages (2-3 x 106/well) were treated with 200 pg/ml AcLDL (n=3),
oxLDL (n=4) and ebselen pretreated oxLDL (n=4) for 10 hr, then samples were
collected for Western bolt analysis using anti-rat GCS-HS antibody * Significantly
different from NC (P < 0.05). ** Significantly different from AcLDL (P < 0.05).
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46
200
160
I
s
e
120
u
*3
£
80
V
o
40
0
control nLDL AcLDL oxLDL
Fig. 3.10 LDL induced ROS production in J774 A. 1 macrophages
J774 A. 1 macrophages were pleated in 24 well dish (2-3 x 105/well) and treated with
200 pg/ml nLDL, AcLDL and oxLDL for 3hr, then labeled with DCFH-DA for 30
min according the Materials and Method. The absorbance of DCF at 502 nm was
measured spectrophotometrically, and the distribution of DCF in cells expressed as
% of untreated controls. * Significantly different from untreated controls (P < 0.05).
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47
3 3 Discussion
In this chapter, we demonstrated that oxLDL-induced macrophage GSH
increase is due to the de novo synthesis of GSH via induction of y-GCS protein.
OxLDL-associated peroxides are primarily responsible for GSH depletion, creating
an oxidizing environment required for y-GCS induction and compensatory GSH
synthesis.
OxLDL-induced GSH depletion and repletion are not specific to the heavily
oxidized LDL. LDL' (a mildly oxidized LDL that is isolated from human plasma)
shows a similar but less severe effect. However, LDL' is recognized mostly by nLDL
receptor (14, 41), thus the extent of LDL' uptake and amount of peroxide delivered
to macrophages is likely to be much less than oxLDL which is taken up by scavenger
receptors. Similarly, nLDL and AcLDL with low levels of oxidized lipid have little if
any effect on GSH levels. These data suggest that the oxidized lipids in LDL
particles are primarily responsible for depletion of cellular GSH and account
substantially for the induction of GSH synthesis. The extent of GSH changes are
correlated with the level of LDL lipid oxidation and the amount of LDL-associated
oxidized lipid taken up by cells.
OxLDL-mediated GSH depletion is thought to be mediated by the activity of
glutathione peroxidase (155) and GST (10, 76), all of which can metabolize oxLDL-
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48
associated lipid hydroperoxides and aldehydes at the expense of GSH. Although part
of the GSSG formed can be reduced back to GSH through the action of GRD (4), the
rate of GSH oxidation could exceed the rate of GSSG reduction especially when
GRD is inhibited by decreased supply of reducing equivalents. The resulting increase
in GSSG/GSH ratios may have at least two consequences (62): (I) a shift in the thiol
redox status may activate certain oxidant responsive transcriptional elements ( 1 1 0 ,
137); (2) GSSG can be preferentially secreted from cells, depleting the total non
protein thiol pool.
The intracellular GSH levels are mainly regulated by activity of rate-limiting
enzyme y-GCS (162). In this study, we demonstrated that the oxLDL-induced GSH
adaptive increase was associated with a transient increase in y-GCS-HS protein
expression that coincided with increased de novo GSH synthesis, representing a
cellular protective response against oxLDL-induced oxidative stress. The decrease in
y-GCS-HS protein content at 24 hr may reflect a feedback inhibition arising from the
elevated GSH.
Ebselen pretreatment of oxLDL essentially abolished the peroxides but not
the aldehydes in oxLDL. Peroxide-depleted oxLDL did not have significant effects
on GSH levels. Moreover, a strong correlation also found between the extent of GSH
depletion and oxLDL-associated LOOH, which further emphasized the important
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49
role of peroxides in oxLDL-induced GSH depletion. Under these conditions,
oxLDL-associated aldehydes alone might not be sufficient to induce GSH depletion
and adaptive response. 4-hydroxynonenal (HNE) has been reported to induce
depletion of GSH followed by an adaptive increase in THP-1 cells (39). Estimation
of the HNE levels in oxLDL based on the content of MDA suggests that the levels of
reactive aldehydes were insufficient to induce the GSH depletion by HNE as shown
in previous studies. Since oxLDL-associated HNE is taken up by scavenger
receptors, using pure HNE reagent may not represent the same exposure conditions
as produced by oxLDL.
Ebselen-treated oxLDL and AcLDL, both of which contain very low
peroxide levels, induced a small to moderate increase in y-GCS-HS protein
expression that was associated with small changes in GSH levels, and neither
ebselen-treated oxLDL and AcLDL caused a significant depletion of GSH. It is
possible that the y-GCS induction could very sensitive to peroxide-mediated GSH
(83). However, the extent of GSH de novo synthesis may also determined by
peroxide-mediated formation of the intersubunit disulfide bond of y-GCS and
diminish of the feedback inhibition of GSH.
The promoter region of the y-GCS-HS gene contains binding sites for
transcription factors such as NF-kB, AP-1 (109). The oxLDL induced y-GCS-HS
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50
mRNA expression is mediated by the AP-l-DNA binding (33).
hydroperoxyeicosatetraenoic acids (HETE) derivatives of arachidonic acid also has
been shown to increase AP-1 expression and DNA binding (126). The lipid
hydroperoxides from oxLDL as well as peroxides generated inside cells (either
through lipid peroxidation or by H2O2 formation) may stimulate AP-1 activation,
especially if they persist in cells after exposure to oxidant challenge. It is plausible
that oxLDL-associated hydroperoxides induce an elevation in intracellular GSSG
levels due to the GSH-GPx mediated reduction of these peroxides. Elevations in
intracellular GSSG have also been shown to cause AP-1 activation (56). This
peroxide-mediated activation of transcription factor could lead to an increase of y-
GCS expression, although the detail relationship between GSH oxidation and y-GCS
induction needs to be further investigated.
The rate of de novo GSH synthesis is also determined by the availability of
substrates. Cysteine, the rate limiting substrate, is taken up by cells as cystine
through Na* independent X c ' system, and once inside the cell, is rapidly reduced
back to cysteine. OxLDL has been reported to increase cystine transport and GSH
levels in human umbilical artery smooth muscle cells (HUASMC) (141). Peroxides
such as f-BuOOH and H2O2 also show similar effects on cystine transport in human
erythrocytes (128) and human umbilical vein endothelial cells (HUVEC) (104). It is,
therefore, plausible that oxLDL-associated hydroperoxides could also affect cystine
transport and substrates availability for de novo GSH synthesis.
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51
CHAPTER 4
THE EFFECTS OF SELENIUM SUPPLEMENTATION ON PEROXIDE-
INDUCED y-GCS INDUCTION AND GSH SYNTHESIS
4.1 Introduction
Reactive oxygen species such as 0 { and H2O2 are continuously produced in
vivo as intermediates of normal biochemical processes. Cells protect themselves
against these ROS through a complex defense system, including a number of
antioxidant enzymes. Among them, Superoxide dismutase (SOD) catalyzes the
conversion of O2’ into H2O2 and O2. H2O2, in turn, can be detoxified by catalase or
glutathione peroxidase to H2O and O2. The reduction of GSSG to GSH is catalyzed
by the widely distributed flavoprotein glutathione reductase, which uses NADPH as
a co-substrate. In this chapter, we further confirmed the roles of peroxide in GSH
depletion and y-GCS induction using selenium supplemented cells with higher
glutathione peroxidase activities.
The family of glutathione peroxidases is comprised of at least four distinct
mammalian selenoproteins. The classical or cytosolic glutathione peroxidase (cGPx)
was the first mammalian selenoprotein to be identified (54). The phospholipid
hydroperoxide glutathione peroxidase (PHGPx) was later described in 1982 (157).
Among these intracellular glutathione peroxidases, cGPx is located mostly in the
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52
cytosol and mitochondrial matrix, whereas PHGPx is found in both cytosol and
membrane bound forms. The extracellular or plasma GPx (pGPx) (147) and the
gastrointestinal glutathione peroxidase (GI-GPx) (34) were also identified. cGPx
reduces only soluble hydroperoxides, such as H2O2, fatty acid hydroperoxides and t-
BuOOH. PHGPx (156), and to some extent pGPx (161), also reduce hydroperoxides
of more complex lipids such as phospholipid and cholesterol hydroperoxides in cell
membrane (96, 151) and oxLDL (135). The substrate specificity for GI-GPx appears
similar to that of cGPx (34). cGPx, pGPx and GI-GPx are homotetramers, whereas
PHGPx is a monomer with a molecular size smaller than the subunits of the other
GPx (24). The small size and its hydrophobic surface have been implicated in its
ability to react with complex lipids in membranes. The most common biological role
of these glutathione peroxidases is to defense against oxidative stress, but the
unusual tissue distribution, hormone-dependent expression, and activity changes
during differentiation seen in the case of PHGPx, may suggest more specific
functions for the glutathione peroxidase family (23).
Biosynthesis of selenoperoxidase depends on the availability of selenium.
Cellular glutathione peroxidase activities can be manipulated by reducing serum
levels or via selenium supplementation in culture medium (58, 134). Similar effects
can also be achieved in vivo by dietary selenium control (131). Since the primary
function of glutathione peroxidase is to detoxify peroxides at the expense of GSH,
the metabolism of peroxides can affect GSH levels through the catalytic action of
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53
selenoperoxidases. Thus, selenium supplemented cells (Se (+) cells), was used to
further study the effects of oxLDL-associated peroxides on macrophage GSH status
and y-GCS induction.
4.2 Results
Effects o f selenium supplementation on macrophage glutathione peroxidase
activities and protein expression
100 nM Na2Se0 3 supplementation in normal culture medium gradually
increased macrophage GPx activity, which reached maximal 6 -fold incrase by 6-7
days (Figure 4.1 A). Selenium supplementation up to 2 weeks did not further
increase the glutathione peroxidase activity. Since r-BuOOH was used as a substrate
in this assay, the results represent the total glutathione peroxidase activity. The
increase of glutathione peroxidase activity was correlated with a 4-6 fold increase in
cGPx protein and less than 2-fold increase in PHGPx protein (Figure 4.1 B). The
effects of selenium supplementation on glutathione peroxidase as well as other
antioxidant enzymes were quantified as shown in Table 4.1. Selenium
supplementation only selectively increased glutathione peroxidase activity and
protein expressions, and showed no effects on other antioxidant enzymes such as
GRD and catalase (CAT). The intracellular GSH content was also unaffected by
selenium supplementation.
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54
8 0 0
700
600
e
u
5 0 0
400
w
at
E
—
= - 300
M
a.
U
200
100
Time (day)
B Western bolt
PHGPx
►
it.
Control cells Se cells
Fig. 4.1 Selenium supplementation induced increase of GPx activity and protein
expression
(A) J774 A.1 macrophages were supplemented with 100 nM Na2SeC>3 in 10 % FBS +
DMEM for up to 16 days. Samples were collected at the time indicated, total GPx
activity were determined using t-BuOOH as substrate. Data was expressed as mean ±
S.E.
(B) Western bolt analysis of cGPx and PHGPx protein expression of control cells
and Se (+) cells (selenium supplementation for 7 days).
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55
Table 4.1 Effects of selenium supplementation on antioxidant enzyme activities,
protein contents and GSH levels in J774 macrophage
Enzyme Activity
(U/mg cell protein) (1 x mg'1 protein x s'1 )
Protein Content GSH
(relative density) ( nmol/mg cell protein)
Cells GPx GRD CAT cGPx PHGPx GSH
Control cells 116 + 42 6 .1 ± 0.6 0.059 ± 0.003 9.4 ±3.1 10.6 + 2.4 26.7 + 2.4
Se (+) cells 656 + 16 6.4 ± 0.1 0.054 + 0.012 30 + 3.5 17.3 + 1.3 28.5 + 2.5
J774 A.1 macrophages were supplemented with 100 aM Na2SeC>3 in 10 % FBS +
DMEM for a week. Data represent the mean + S.E. of three or more independent
experiments. GRD = glutathione reductase, CAT = catalase.
GSH depletion and peroxide metabolism in Se (+) cells
To characterize the detoxification capacity in Se (+) cells with higher GPx
levels, we measured GSH consumption following peroxide addition to cell lysates.
The GSH consumption rates for Se (+) cells was about 5 times faster than control
cells after addition of 200 pM r-BuOOH (Figure 4.2 A). The faster GSH depletion
rates in Se (+) cells were also associated with 20% greater peroxide (H2O2)
consumption rates than control cells (Figure 4.2 B). Since H2O2 is substrate for both
catalase and glutathione peroxidase, a much substantial difference may be expected
for peroxide consumption if the substrates were limited specifically to glutathione
peroxidase.
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56
4 0 0
A
300
OOOOOOOOOOOO
a
u
*
►
a 200
» ■
u
S
S 100
0 4 6 8 10
B
□ Control cells
♦ S e (+ ) cells
100
80
20
0 10 20 30
Time (min)
Fig. 4.2 The in vitro GSH depletion, H2O2 metabolism
(A) GSH depletion was measured using cell lysates from Se (+) cells (♦), and
control cells (□). Baseline (O) represents all reagents except cell lysate. Arrow
indicates the time of r-BuOOH (200 pM) addition.
(B) H2O2 metabolism. 1-2 x 107 J774 macrophages were plated in a 100 mm dish
with 20 ml RPMI medium. An initial concentration of 100 pM H2O2 was added to
the medium, samples were taken every 5 min to measure remaining H2O2 levels.
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57
Peroxide-resistance ofSe (+) cells
The peroxide-induced cytotoxicity in Se (+) cells vs control cells was
compared. Macrophages were treated with 200 pM H2O2 in PBS for 30 minutes
followed by medium change and then incubated for additional 24 hr. Cell viability
was determined using the MTT assay. Figure 4 J shows that Se (+) cells were more
resistant to peroxide-induced challenge and the extent of resistance to oxidative
stress was directly correlated to the glutathione peroxidase activity and the rate of
peroxide metabolism.
OxLDL induced GSH and y-GCS-HS changes in Se (+) cells
In Chapter 3, the potential importance of oxLDL-associated lipid
hydroperoxides on GSH content and y-GCS induction were described. To determine
the extent of oxLDL-induced GSH depletion and repletion in Se (+) cells with an
enhanced peroxide reducing capacity, Se (+) cells were treated with 200 pg/ml
oxLDL for up to 24 hr. OxLDL treatment caused a more rapid initial decrease but a
less extensive adaptive increase of GSH levels in Se (+) cells. The rapid depletion of
GSH after 3 hr treatment of Se (+) cells with oxLDL was accompanied by a 2-fold
increase in GSSG/GSH ratios, whereas only a 30% increase was found in control
cells (Figure 4.4 A). The basal levels of GSSG/GSH ratio in Se (+) cells and control
cells are about 5.2% and 4%, respectively. The extent of GSSG/GSH increase was
strongly correlated with the cellular GPx activity (R = 0.77) (data not shown).
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58
100
control cells Se (+) cells
Fig. 4J MTT assay for peroxide-induced cytotoxicity
J774 A. 1 Macrophages (2-3xlOs/well in 48-well dish) were treated with H2O2 (200
pM) for 30 min in PBS followed by medium changes to 10% FBS DMEM for
addition 24 hr. Data are expressed as % of untreated control (n=3). White bar,
control cells, black bar, Se (+) cells * Significantly different from control cells (P <
0.05).
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59
a
U
9
$
250
■ Control cells
♦ Se (+) cells
200
50
j o a o
100
200.0
a 100.0
50
0.0
0
1 2 18
Time (hr)
24
10 hr incubation
C o n tr o l c e lls Se ( + ) c e lls
Fig. 4.4 Effects of oxLDL on Se (+) ceil GSH and y-GCS-HS protein induction.
(A) Se (+) cells (♦ ) (n=3) and control cells (■) (n=4) (2 x 106/well) were treated
with 200 (ig/ml oxLDL for up to 24 hr, The GSH content is expressed as % of
untreated controls (NC). Figure insert presents the GSSG/GSH ratios (as % of NC)
after 3 hr oxLDL treatment. The basal levels of GSSG/GSH ratio in Se (+) cells and
control cells are about 5.2% and 4%, respectively. * P < 0.05, compare to 0 hr NC.
Data are expressed as mean + S.E. (%).
(B) Maximum induction of y-GCS-HS protein in Se (+) cells and control cells by
oxLDL after lOhr of treatment y-GCS-HS protein levels were determined by
Western analysis
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60
Moreover, oxLDL induced a moderate increase in y-GCS-HS protein levels in Se (+)
cells, achieving maximum levels that were about 25 % lower than that found in
control cells (Figure 4.4 B). Taken together, these findings show that Se (+) cells
bearing higher GPx activities indeed detoxify peroxides and utilize GSH more
rapidly, but this does not determine the extent of peroxide-mediated y-GCS-HS
protein induction and GSH increase.
4 J Discussion
Glutathione peroxidases are considered as major cellular antioxidant
enzymes. Two variables determine the catalytic capacity of the glutathione
peroxidase: 1) the availability of GSH, and 2) the availability of selenium to support
glutathione peroxidase synthesis. Under most cell culture conditions, the availability
of selenium is very limited, since serum supplements generally contain low selenium
(134). Consequently, cells grown in 10% serum containing media have a much lower
supply of selenium than equivalent cells in the living organism.
Selenium is incorporated into polypeptide chains as selenocysteine, a UGA
codon in the mRNA corresponds to selenocysteine in the protein (27). Four unique
gene products are required for the biosynthesis of selenoprotein: (I) the tRNA[S < rIS e c
that carries the anticodon for UGA, (2) selenocysteine synthesis, (3) SELD, which
produces selenophosphate from selenide and ATP, and (4) SELB, a translation factor
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61
which binds to tRNA[S e r,S e c and directs the selenocysteine loaded tRNA to UGA
codon of the mRNA. A stem-loop structure (also called selenocysteine insertion
sequence (SECIS) in eukarytes) recognized by SELB is also required for the UGA to
specify selenocysteine incorporation instead of termination. Thus, the regulation of
glutathione peroxidase appears to occur at the translation level via message or
protein stabilization (15S), although transcriptional regulation has also been
implicated (31). Inefficient incorporation of selenocysteine at the UGA codon during
translation augments the nonsense codon-mediated decay of cytosolic glutathione
peroxidase mRNA. Under conditions of selenium deprivation, the selenocysteine
codon reduces the abundance of cytoplasmic glutathione peroxidase mRNA by a
translation dependent mechanism (105). However, the dependence of mRNA
stability on selenium is characteristic for each selenoprotein. Under selenium
restriction, the selenoproteins incorporate the trace element according to a fixed
hierarchy (23), probably reflects the relative importance of the individual proteins.
Among the two intracellular glutathione peroxidases, selenium supplementation or
deprivation has a much more potent effect on cGPx than PHGPx.
Se (+) cells with higher GPx activities exhibit a rapid GSH depletion, a faster
peroxide consumption and a higher GSSG accumulation after oxLDL challenge. The
initial decreases in total glutathione ([GSH] + 2[GSSG]) after a 3 hr treatment with
oxLDL was accompanied by an increased GSSG/GSH ratio that was positively
correlated with cellular GPx levels, and the rate/extent of GSH depletion. These
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62
further indicated peroxide-mediated GSH oxidation is primary responsible for GSH
depletion. Current methods for measuring [GSSG]j„ may underestimate the amount
of GSSG produced due to the efflux of GSSG, and the actual amount of GSSG
formed in response to oxidative stress may have been greater than indicated.
The redox changes of GSSG/GSH in response to physiological stimuli such
as differentiation and enzyme inducers have been shown to be of sufficient
magnitude to control the activity of some redox-sensitive proteins such as GST and
NADPH: quinone reductase, although the correlation between GSH redox change
and GCS induction needs further study to be established (83). The faster
accumulation of GSSG in Se (+) cells may trigger a swift cellular responses for the
induction of redox-sensitive antioxidant enzymes, thus providing Se (+) cells more
protection against oxidative stress. It is also possible that in Se (+) cells the rapid
reduction of peroxides and greater capacity to metabolize administered or internally
generated peroxides would result in lower concentrations of residual peroxides and
affect the degree of oxidative signaling of y-GCS expression. However, since the
majority of the oxLDL-associated lipid hydroperoxides, such as phospholipid and
cholesterol hydroperoxides, are substrates for PHGPx, they may become substrates
for cGPx only after the action of phospholipase A2 (PLA2) that release free fatty acid
LOOH. Since selenium supplementation has a greater effect on cGPx than PHGPx in
terms of enzyme activity and protein induction (Table 4.1), the extent of peroxide
reduction in Se cells may be limited.
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63
CHAPTERS
THE ROLE OF GSH CONTENT IN CELL-MEDIATED LDL OXIDATION
5.1 Introduction
The LDL oxidation hypothesis of atherosclerosis provides a new approach
for preventing or delaying the process of atherosclerotic lesion formation. Indeed,
antioxidant supplementation can reduce LDL oxidation and attenuate atherosclerosis
as shown by treatment of hypercholesterolemic rabbits with probucol (30), butylated
hydroxytoluene (BHT) (21) and vitamin E (160). Endothelial dysfunction and
reduced nitric oxide (NO) activity is associated with atherosclerosis and its clinical
manifestation such as unstable angina. Thiol supplementation with GSH selectively
improves human endothelial dysfunction by enhancing NO activity (122).
Because GSH has a critical role in the detoxification of electrophiles and
oxidative stress agents, strategies for pharmacologically maintaining or increasing
GSH levels have received considerable attention. A number of compounds were
shown to increase intracellular GSH content: including GSH esters, N-acetylcysteine
(NAC), L-2-oxothiazolidine-4-carboxylic acid (OTC) and tert-butylhydroquinone
(TBHQ). These compounds increase GSH content through different mechanisms.
Among them, GSH esters are readily taken up by the cells and de-esterified to
release GSH (7). NAC and OTC are considered as cysteine precursors. NAC
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64
increases intracellular GSH level through two mechanisms: it can be taken up into
cells and deacetylated to produce free cysteine, or act as an extracellular reducing
agent and reduce cystine to the more easily transported cysteine (118). OTC is an
alternative substrate for 5-oxo-L-prolinase, a widely distributed intracellular enzyme
that forms S-carboxy-L-cysteine and spontaneously loses CO2 to form L-cysteine (5,
7). TBHQ can be oxidized to a semiquinone and then to 2-rm-butyl (1,4)
paraquinone (TBQ) either enzymatically or by autooxidation, producing reactive
oxygen species (Figure 5.1). TBQ as well as other “redox-cycling quinones”, such
as menadione and DMNQ, can be reduced by one electron transfer reductases or
NAD(P)H-quinone reductase (DT-Diaphorase) to seminquinone radicals or
hydroquinones, at the expense of NAD(P)H. TBQ can also form conjugate with GSH
(81, 116, 117). Increased GCS subunit and GGT mRNA content, in addition to the
enzyme activity, have also been found after TBHQ treatment (91, 92). Whether this
is due to the secondary effects of ROS production/GSH depletion or direct
interaction between TBHQ and the enzyme is unclear.
Macrophage GSH content can also be reduced by L-buthionine-SR-
sulfoximine (L-SR-BSO), a specific inhibitor of glutathione synthesis (63). The L-
buthionine-S-sulfoximine (L-S-BSO) is subjected to ATP-dependent, enzyme
catalyzed phosphorylation on the sulfoximine nitrogen to form L-buthionine-S-
sulfoximine phosphate (L-S-BSO-P) (29). In the presence of MgATP, that product is
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65
OH
0 ,
0 ,
C(CH3 )3
T B H Q
OH
O
T B Q
Y
GSH
OH
SG
NAD(P)+
Cytochrome P460
Reductase
NAD(P)H
OH
Fig. 5.1Generation of reactive oxygen species by TBHQ
(Adapted from Liu RM et al. 1998)
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66
bound very tightly to the y-GCS L-cysteine binding site. Because of the presence of
MgATP in the cells, BSO is generally regarded as an irreversible inhibitor when
used in vivo or with cells.
In this chapter, a characterization of the cell-mediated LDL oxidation is
provided followed by studies on the effects of modulating GSH content on cell-
mediated LDL oxidation.
5.2 Results
Characterization o f macrophage-mediated LDL oxidation
The extent of macrophage-mediated LDL oxidation was determined by
measuring LDL’ formation and relative electrophoretic mobility (REM) and MDA
formation in cell modified LDL. J774 macrophages (2xl06 cells/well) were
incubated with 100 pg/ml LDL for up to 20 hr in Ham’s F-10 medium (containing 3
pM iron and 0.01 pM copper ions), then media were collected and concentrated
using microfilter. LDL' fraction was separated from the cell oxidized LDL using ion
exchange HPLC with an UNO Q1 column. Figure 5.2 shows a series of
chromatographs representing the time-course for changes in oxidized LDL fractions.
As shown in the figure, the majority of unmodified LDL (0 hr) is native LDL
(nLDL), with only very small amount of LDL’ (< 5%) and almost no heavily
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67
6000
>
s
5000 .
4000 .
3000
2000 •
1000
10 hr
0 hr
/ / /
nLDL LDL' LDL2 '
Fig. 5.2 Representative chromatogram of the HPLC fractions of cell-modified
LDL
J774 macrophages (2xl06 cells/well) were incubated with 100 (ig/ml LDL protein in
Ham’s F-10 medium for 0, 10, and 20 hr. cell modified LDL samples were collected
and concentrated for LDL' measurement using HPLC. Plasma LDL (0 hr) was
fractionated into unmodified native LDL (nLDL), mildly oxidized LDL (LDL') and
even more oxidized LDL (LDL2 * ) peaks. Macrophages modification lead to a
graduate decrease of nLDL peak and increase in LDL' and LDL2 ' peaks.
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68
oxidized LDL (LDL2 "). The two oxidized forms of LDL, LDL' and LDL2 * , represent
the major forms of modified LDL found in human plasma (74) as well as in cell-
modified LDL. LDL2 ' is the more electronegative and peroxide-enriched species that
recognized by scavenger receptors (unpublished data). Macrophages induced a
gradual increase in LDL* and later LDL2 ' formation after 10 and 20 hr incubation
with LDL.
The REM of both cell-oxidized LDL and LDL in cell-free medium were
determined. There were progressive increases in LDL electrophoretic mobility in
samples incubated with cells for 0, 4, 10, and 20 hr. A similar pattern of REM
increase was found in LDL modified in cell free system but with less extent (Figure
S 3 A). MDA formation, which represents the extent of lipid peroxidation in LDL,
further confirmed the results from LDL* and REM measurements (Figure 53 B). At
the end of 20 hr incubation, the MDA values were 51.5 + 3.9 vs 16.5 + 0.7 nmol/mg
LDL protein for LDL from cell culture system and cell-free system, respectively.
Modulation o f macrophage GSH content
A number of reagents were selected to modulate the levels of intracellular
GSH (Figure 5.4). BSO was used to reduce GSH content by inhibiting the GSH
synthesis. Pretreatment of cells with 50 [iM BSO for 20 hr depleted 80% of the GSH
content. Increasing BSO concentration to 100 jiM did not further decrease the GSH
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69
A Time (hr) 0 4 10 20
Cells
(+)
H
1 2 3 4 5 6 7
2000
1600
1200
800
400
0
0 5 10 20 25 15
Time (hr)
Fig. 5.3 Time course studies of cell-mediated LDL oxidation (REM, MDA)
J774 macrophages (2xl06 cells/well) were incubated with 100 pg/ml LDL protein in
F-10 medium for 0,4,10, and 20 hr.
(A) Agarose gel electrophoresis of cell-modified LDL following various intervals
oxidation. Lane 1 (0 hr) was nLDL, lane 2, 4, and 6 were LDL modified by cells.
Lane 3, 5, and 7 were LDL oxidized in cell-free system at corresponding times.
(B) MDA formation of cell-modified LDL. Cell-modified LDL (O), cell-free control
(■). Data were expressed as mean + S.E. * significantly different from cell-free
control (P < 0.05).
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70
Fig. 5.4 Modulating GSH levels by using BSO, tBHQ, NAC and OTC
J774 macrophages (2xl06/well) were pretreated with BSO, tBHQ, BSO + tBHQ (50
pM), NAC and OTC at the doses indicated for 20 hr, and then samples were
collected for GSH measurement. Data were expressed as mean ± S.E. * significantly
different from untreated control (NC) (P < 0.05).
levels. Pretreatment with TBHQ, 50 pM and 100 pM, increased GSH level up to 3.4
and 4 fold over basal level, respectively. Co-incubation of 50 pM TBHQ and BSO
did not increase GSH level, indicating that the effect of TBHQ on GSH levels was
mediated by induction of de novo synthesis. NAC 5 mM and OTC 2 mM
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71
pretreatment for 20 hr had no significant effects on GSH content. Pretreatment of
cells with 5 mM GSH ester caused a fast but transient increase in GSH with maximal
2-fold increase around 4-6 hr of incubation (Figure 5.5). None of the above
treatments caused severe cytotoxicity or cell morphology changes at the doses
indicated. Based on the above, the BSO and TBHQ models were selected to deplete
or increase GSH, respectively.
Macrophage GSH content affects the cell ability o f oxidizing LDL in culture
i l l A macrophages (2x 106 cells/well) were pretreated with 50pM BSO, 50
pM TBHQ, DMSO (vehicle for TBHQ) or no pretreatment (untreated control) for 20
hr in 10% FBS + DMEM, followed by a medium change to Ham’s F-10 medium
containing 100 pg/ml LDL for another 20 hr. Samples were collected and
concentrated for LDL' and MDA assays. LDL' levels (representing LDL protein
modification) in BSO and TBHQ treated cells were 174 .4 + 12.6 % and 78. 9 + 8.6
% of NC. DMSO treatment showed no effect (98% of NC) (Figure 5.6 A). The same
samples were also used for MDA assay. MDA levels in LDL modified by BSO and
TBHQ treated cells were 142.8 + 2.7 % and 64.1+ 7.8 % of NC, respectively (Figure
5.6 B).
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72
.s
2
2
a
1 5
w
o n
I
E
7 0
60
50
40
30
20
10
0
25 20 10 15 0 5
Time (hr)
Fig. 5.5 GSH monester induced macrophage GSH increase
J774 macrophages (2xl06 /well) were pretreated with 5 mM GSH monoester, and
samples were collected at the time indicated for GSH measurement. Data were
expressed as mean + S.E.
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73
A 200
160
140
U
Z 100
©
< 60
a
S 40
20
.0
£
Fig. 5.6 Effects of BSO and TBHQ treatments on cell-mediated LDL oxidation
J774 macrophages (2xl06 /well) were plated in 10% FBS + DMEM and pretreated
with BSO, tBHQ, DMSO (vehicle for TBHQ) for 20 hr, then changed to F-10
medium and coincubated with 100 (jg/ml LDL for additional 20 hr. samples were
collected for LDL‘ (A) and MDA (B) measurements. Data were expressed as mean +
S.E. * significantly different from untreated control (NC) (P < 0.05).
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74
53 Discussion
In this study, the relationship between the intracellular GSH status and the
extent of LDL oxidation by macrophages was investigated. Iron-rich F-10 medium
was used in these studies. The trace amount of copper or iron is thought to be
involved during LDL oxidation in the arterial wall, as such transition metal ions have
been found in atherosclerotic lesion (142). Moreover, cerruloplasmin derived copper
ions are available to the arterial wall under certain pathological conditions (48).
Although other catalysts may have a role in LDL oxidation, it is generally agreed
that cultured cells produce oxidized LDL more efficiently in the presence of
transition metals.
BSO treatment decreased GSH to 20% of basal level, and this extent of GSH
depletion has been shown accompanied by a increase of ROS production and cell
lipid peroxidation (80, 163). Although decrease in cytosolic and mitochondrial GSH
may not have a direct effect on mitochondrial H2O1 production (60), ROS generated
from other sources such as NADPH oxidase, lipoxygenase, cycio-oxygenase and
xanthine oxidase pathways are possible (37). Severe GSH depletion to 10-20% of
basal level may compromise the ability of glutathione peroxidase to detoxify
peroxides (164), which can further increase ROS accumulation and cell lipid
peroxidation. Depletion of GSH by GCS antisense oligomers resulted in increased
cellular ROS production and calcium-mediated cell death, suggesting the important
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75
role of GSH in intracellular Ca2 + homeostasis (80). A disturbance in intracellular
Ca2 + homeostasis can result in activation of various of calcium-dependent proteases,
which can damage cytoskeletal and membrane proteins, and phospholipases, such as
PLA2 that catalyze hydrolysis of oxidized membrane phospholipids (127). Release
of these oxidized lipids could promote LDL oxidation by providing free oxidized
lipids that seed lipoprotein particles (145).
L-cysteine precursors, such as NAC and OTC, failed to increase intracellular
GSH levels in this cell system. This may due to the initial high cysteine/cystine
levels in our culture medium, which precludes further enhancement of substrates.
The transient increase of GSH by GSH monoesters could due to its efflux from the
cell (23). TBHQ induced about a 3-4 fold increase in GSH, at least in part due to the
GSH conjugation and ROS induced adaptive increase in GSH. An increase in y-GCS
and GGT mRNA levels has also been reported in TBHQ treated cells (91). Our
results show that cells with enhanced intracellular GSH levels inhibited LDL lipid
peroxidation and protein modification (in terms of LDL* production). This could due
to the inhibition of macrophage oxygenases and/or scavenging of free radicals by
excess cellular GSH. Thus, selective enhancement of cellular GSH may have
potential antioxidant effects, measured in terms of delayed or reduced atherosclerotic
lesion formation.
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76
CHPAPTER6
CONCLUSIONS
6.1 Conclusions
OxLDL induced an initial GSH depletion followed by an adaptive increase in
macrophages. OxLDL-associated LOOH was found to be primarily responsible for
the initial GSH depletion. Although the ROS generation via oxLDL-scavenger
receptor binding may also contribute to the GSH depletion and later y-GCS
induction, the effect was found to be minor compared to oxLDL-associated LOOH.
It is concluded that a pro-oxidant state is determined by LOOH through reactions
with cell components, decomposition reactions and radical chain propagation, and by
the utilization of GSH pools, the later being rapidly depleted through the action of
glutathione peroxidase.
Macrophages responded to oxLDL-induced oxidative stress by an elevation
in GSH levels via de novo synthesis. The possible roles of oxLDL-associated LOOH
in GSH synthesis are shown in Figure 6.1:
1) Increased y-GCS activity is postulated via the loss of negative feedback of
GSH and/or by promotion of disulfide bond formation between y-GCS subunits (75).
The activities of a great number of enzymes are dependent on the state of specific
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77
LDL
PLA: activation
^ (LOOH release)
GSH
(redistribution)
F o s , J u n
e x p r e s s i o n
F o s / J u n
U ® (ap-i)
Cystine
oxLDL
M i t o c h o n d r i a
SH SH
# o ^ C 5
N A D P H
o x i d a s e
iGSH
tGSSG
• o T y - G C S a c t i v i t y
L
r
R e f - 1
T h i o r e d o x i n
—JL
. m *
y - G C S g e n e ~ ~ s C ^
t r a n s c r i p t i o n \
—►
y - G C S
Fig. 6.1 OxLDL-associated LOOH on macrophage GSH status
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78
cysteine thiols in a relative reduced or oxidized state. Intracellular GSH is thought to
be the primary thiol that serves as a redox buffer and maintains the cell redox state.
The increase in GSSG/GSH ratio by oxLDL-associated LOOH may promote
formation of y-GCS disulfide bonds, and lead to a conformational changes that
increase y-GCS-HS substrate binding.
2) OxLDL-induced y-GCS-HS protein expression was demonstrated in this
study. OxLDL-induced the GSH depletion or GSSG accumulation may disturb the
cellular thiol redox status and provide a signal for AP-1 activation and translocation
due to the induction of stress activated c-Jun N-terminal protein kinase (JNK)
pathway (159). A reduced environment in cell nuclear provided by thioredoxin, the
nuclear redox protein, Ref-1 (73) or the possible redistribution of GSH (25, 158) may
facilitate AP-l binding to its TRE consensus regions, triggering y-GCS gene
transcription.
3) Peroxide or oxLDL-induced increase in cystine transport has been
demonstrated in a number of cells (114, 141). The induction of X * c transport system
under oxidative stress is strongly correlated with increases in intracellular GSH
levels and concomitant protection against further oxidative damage. An increase in y-
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79
GGT activity by oxLDL is also possible, which leads to increase degradation of
extracellular GSH and provides more substrates for GSH synthesis.
The adaptive increase in GSH represents a defense response that not only
protects the cell from oxidative damage, but also reduces the extent of further LDL
oxidation. However, unregulated uptake of oxLDL via scavenger receptor leads to
lipid loading inside macrophages, that may eventually compromise cell defense
responses and lead to extensive GSH depletion, in a manner similar to the BSO
treatment. Perturbation of intracellular Ca2 + homeostasis and ROS accumulation
could increase ceil membrane lipid perxidation. Transfer of oxidized cellular lipids
to LDL through cell membrane-LDL contact is thought to be one of the mechanisms
of initiating LDL oxidation-often referred to as “seeding” (145). Thus, the changes
of intracellular GSH could affect the extent of LDL oxidation.
6.2 Proposed future work
It has been shown in this thesis that sublethal oxLDL-induced oxidative stress
moderately decreases GSH followed by a sustained elevation of GSH. The elevation
of GSH appears to be due to induced synthesis as evidenced by increased y-GCS-HS
protein levels. OxLDL-associated lipid hydroperoxides were mainly responsible for
the prooxidant state that triggered the GCS induction. According to a recent paper by
Kirlin et al. (83), a minor redox change (12 mV of oxidation of GSH pool) in benzyl
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80
isothiocyanate (BIT) treated HT29 cells corresponded to an increase in GCS-HS
mRNA. The time course and extent of GSH changes was very similar to the oxLDL-
induced changes found in our study. However, a high level of redox change (60 mV
of oxidation of GSH pool) achieved in BIT treated sodium butyrate differentiated
cells showed less or no increase in GCS-HS mRNA induction. This implies that
either an increased mRNA is not necessarily associated with an increased GSH
synthesis rate or that a “bell shape” type of relationship exists between GSH redox
potential changes and GCS induction.
A future direction for study would be to further investigate the possible
effects of GSH redox potential changes in GCS induction. The GSH redox change
induced by 200 pg/ml oxLDL treatment is relatively small (less than 45% of GSH
decrease) and may not encompass the range of GSSG:GSH redox changes needed to
examine this relationship to GCS induction. In order to induce high levels of GSH
oxidation, oxLDL could be “preconditioned” with increased concentrations of
oxidized linoleic acid (13-hydroperoxyoctadecadienoic, 13-HPODE). The uptake of
these oxLDL-associated lipid hydroperoxides is expected to increase through
scavenger receptor-mediated pathway and achieve more GSH oxidation. However,
the amount of oxLDL-associated lipid hydroperoxides delivered to cells may also be
limited by the rate of macrophage oxLDL uptake. Thus, pure oxidized linoleic acid
can also be used at to induced high levels of GSH oxidation, although a different
time course of GSH changes and GCS induction may be expected due to faster
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81
transport of lipid hydroperoxides through cell membranes. The oxLDL-induced
redox potential Eh value can be calculated base on the changes of GSH and GSSG
(83), with the induction of GCS-HS mRNA and protein determined by Northern
hybridization analysis and Western analysis.
The regulation of GSH synthesis under oxidative stress is through multiple
components, the induction of light subunit of y-GCS (y-GCS-LS) under oxLDL-
induced oxidative stress remains to be identified. The possible effects of other y-
glutamyl cycle enzymes, such as y-GGT, in regulating GSH metabolism under such
oxidative stress also needs further study.
6J Implications
The therapeutic potential of thiol antioxidants in the clinical setting has been
widely discussed. Oxidative stress and reactive oxygen species are implicated in a
number of diseases and conditions, from the acquired immunodeficiency syndrome
(AIDS) to aging. There are many situations in which increasing cell and tissue levels
of GSH and other thiols might be beneficial. A wide variety of thiol-related
compounds have been used for these purposes. These include thiols such as GSH and
its derivatives, cysteine and NAC, dithiols such as lipoic acid, and "prothiol”
compounds such as OTC. Because of the differences in basal thiol levels in the
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82
various cell and tissue compartments, as well as the differences in the ability of
different thiols to cross cell membrane barriers, the way of delivery of thiols to the
desired site becomes quite complex.
Administration of NAC has historically been as a mucolytic agent in variety
of respiratory diseases. However, it also appears to have beneficial effects in
conditions characterized by decreased GSH or oxidative stress, such as HIV
infection, cancer, heart disease, influenza and cigarette smoking. NAC is currently
used as an antidote for acetaminophen poisoning, which involves depletion of GSH
levels and inhibition of GST activity (36). However, the side effects for NAC
treatment are relatively common, including vomiting, diarrhea, skin reactions, and
headache (19). OTC may be a better candidate for increase intracellular or tissue
GSH in vivo. Because it has low toxicity and shown to increase intracellular GSH
without significantly increasing plasma GSH in human volunteers (121). High levels
of GSH in plasma may be a * ‘prooxidant”. Changes in plasma pH lead to thiolate
anion (RS~) formation, in the present of O2 and trace amount of metals, it generates
thyil radical (RS) and 0 2 ', which are toxic to vascular cells, and has been shown to
promote LDL oxidation in cell cultures (69).
Our results have shown that macrophage GSH status affects the extent of cell
mediated LDL oxidation. This is consistent with other in vivo animal studies (131)
that showed a reduction of atherosclerotic lesion formation after increased tissue
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83
GSH-GPx levels in apo lipoprotein E-deficient mice. Besides the ability to inhibit
LDL oxidation, GSH has also been shown to affect vascular tone, which also play an
important role in early atherosclerotic development. Rush et al. found that addition
of GSH to endarterectomy specimens increased the prostacyclin (PGI2) in plaques to
levels found in normal arteries (133). PGh, a major vasodilator, appears to be
reduced in the arterial wall from atherosclerotic plaques compared to adjacent
normal intima. PGI2 synthetase is inhibited by lipid peroxides, such as 15-HPETE
(66), a site-specific peroxide-reducing effect by GSH and glutathione peroxides may
be responsible for restoring the PGI2 levels in arterial wall. All these findings
suggested that the thiol supplementation could have a great therapeutic potential in
attenuating atherosclerotic process, and its efficacy should be increased by selecting
compounds and methods of delivery that will minimize perturbations in the thiol
status of regions adjacent to the target areas.
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84
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Creator Shen, Lijiang (author)

Core Title Modulation of macrophage glutathione synthesis by oxidized LDL and the effects of glutathione content on cell-mediated LDL oxidation

Degree Doctor of Philosophy

Degree Program Molecular Pharmacology and Toxicology

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, cell,chemistry, biochemistry,Health Sciences, Pharmacology,Health Sciences, Toxicology,OAI-PMH Harvest

Language English

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Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-135643

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Rights Shen, Lijiang

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

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