Graduate Theses and Dissertations Graduate College

2013

Green tea polyphenols prevent Parkinson's disease:

in vitro and in vivo studies
Dan Chen
Iowa State University

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Chen, Dan, "Green tea polyphenols prevent Parkinson's disease: in vitro and in vivo studies" (2013). Graduate Theses and Dissertations.
13626.
http://lib.dr.iastate.edu/etd/13626

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 Green tea polyphenols prevent Parkinsons disease:

in vitro and in vivo studies

by

Dan Chen

A dissertation submitted to the graduate faculty

in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

Major: Toxicology

Program of Study Committee:

Manju Reddy, Major Professor
Anumantha Kanthasamy
Richard Martin
Matthew Rowling
Peng Liu

Iowa State University

Ames, Iowa
2013
Copyright Dan Chen, 2013. All rights reserved.
 ii

TABLE OF CONTENTS

LIST OF FIGURES iv

LIST OF TABLES v

CHAPTER 1. GENERAL INTRODUCTION 1

Introduction 1
Thesis organization 4

CHAPTER 2. LITERATURE REVIEW 5

I. Parkinsons disease (PD) 5
A. Pathogenesis and risk factors of PD 6
B. Animal models of PD 8
C. Cell models of PD 9
D. Mechanism of 6-OHDA-induced neuron cell damage 10
II. Oxidative stress and neurodegeneration 11
A. Oxidative stress and mitochondrial dysfunction 13
B. Metal related neurodegeneration, with particular consideration of iron 14
C. Brain iron and neurodegeneration 15
D. Oxidative stress & neuroinflammation in PD 18
III. Treatment and prevention strategies of PD 22
A. Treatments 22
B. Prevention strategies 24
C. Green tea polyphenols properties & mechanisms of protection 30
IV. References 38

CHAPTER 3. EGCG PROTECTS AGAINST 6-OHDA

INDUCED NEUROTOXICITY IN A CELL CULTURE MODEL 59
Abstract 59
Introduction 60
Materials and methods 61
Results 67
Discussion 69
Literature Cited 73

CHAPTER 4. THE PROTECTION OF EGCG AGAINST

6-OHDA-INDUCED OXIDATIVE STRESS AND INFLAMMATION 86
Abstract 86
Introduction 87
Materials and methods 89
Results 92
Discussion 93
Literature Cited 96
 iii

CHAPTER 5. BENEFICIAL EFFECTS OF GREEN TEA

CONSUMPTION IN PARKINSONS DISEASE PATIENTS 105
Abstract 105
Introduction 106
Methods 108
Results 110
Discussion 112
References 115

CHAPTER 6. GENERAL CONCLUSION 126

References 128

ACKNOWLEDGEMENTS 130
 iv

LIST OF FIGURES

Chapter 2. Figure 1. The iron uptake into cell. 17

Chapter 2. Figure 2. Protective mechanism of nutrients in mitochondria. 30

Chapter 2. Figure 3. Structure of catechins in green tea. 32

Chapter 3. Figure 1. Effects of 6-OHDA and EGCG on cell death. 78

Chapter 3. Figure 2. Effects of 6-OHDA and EGCG on cell viability. 79

Chapter 3. Figure 3. Effects of 6-OHDA and EGCG on cell apoptosis. 80

Chapter 3. Figure 4. EGCG prevented TH+ neuronal loss by 6-OHDA. 81

Chapter 3. Figure 5. Effect of 6-OHDA and EGCG on iron-related

proteins expression. 82

Chapter 3. Figure 6. Effect of 6-OHDA and EGCG on cell iron uptake. 86

Chapter 4. Figure 1. EGCG attenuated 6-OHDA-induced

intracellular ROS generation in N27 cells. 101

Chapter 4. Figure 2. Effect of 6-OHDA and EGCG on lipid peroxidation. 102

Chapter 4. Figure 3. Effect of EGCG on PCC induced by 6-OHDA. 103

Chapter 4. Figure 4. Effects of 6-OHDA and EGCG on

Nrf2, HO-1 and PPAR protein expression. 104

Chapter 5. Figure 1. Antioxidant enzymes activities

in erythrocytes at baseline and after 3 mo green tea consumption. 122

Chapter 5. Figure 2. Lipid peroxidation and protein damage

at baseline and after 3 mo green consumption. 123
 v

LIST OF TABLES

Chapter 2. Table 1. Iron chelators and neuroprotection. 35

Chapter 3. Table 1. Summary of the primers for iron-related genes. 84

Chapter 3. Table 2. Effect of 6-OHDA and EGCG on the

iron-related genes expression. 85

Chapter 5. Table 1. PD rating scales in subjects at baseline

and after 3 mo green tea consumption. 120

Chapter 5. Table 2. Subject description and iron status at

baseline and after 3 mo green tea consumption. 121

Chapter 5. Appendix. PD Patients Demographic Data. 124

 1

CHAPTER 1. GENERAL INTRODUCTION

Parkinsons disease (PD) is associated with the degeneration of dopaminergic

neurons in the substantia nigra (SN). One of the pathological hallmarks of PD is the

presence of intracellular Lewy bodies (LBs). The soluble protein -synuclein is

expressed in presynaptic region and its aggregation is highly involved in the

formation of LBs. Other factors that may also contribute to the progression of PD

include: aging, genetic mutations, oxidative stress, environmental toxins, and

inflammation. Among these factors oxidative stress has been considered the main

factor in causing PD. Due to the high energy metabolism ratio and the structure of SN

in normal brain, there is relatively high levels of basal oxidative stress. Postmortem

studies and in vitro neuron studies demonstrate that elevated oxidative stress is linked

to PD.

Oxygen-generated free radical is the most important class which contributes to

ROS and oxidative stress. The enzyme or metal-catalyzed process can directly or

indirectly generate free radical. Current evidence shows the changes in the balance of

redox transition metals are involved in free radical generation. Iron is an important

trace element which participants in many physiological functions, such as

neurotransmission and neuromelination. Many iron transporters and regulators are

responsible for maintaining iron homeostasis. Iron can be up-taken via divalent metal

transporter (DMT1), transferrin, and transferrin receptors (TfR). Ferrtin is responsible

for iron chelation and storage; while ferroportin 1 (Fpn1) is used for iron exportation.

Other regulators such as hepcidin and iron regulatory proteins (IRP) are also

important in regulating the iron-related factors and maintaining the regular iron labile

pool. Various factors can regulate iron transporters and cause iron dyshomeostasis

including environmental toxins and genetic mutations. If iron dyshomeostasis occurs

 2

it could exaggerate the generation of free radicals. The excess free radicals such as

superoxides, can force enzymes to release free iron from iron-containing molecules.

The neurotoxins 6-hydroxydopamine (6-OHDA) and

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) act as the classic toxins to

induce PD, and even though the mechanisms remain unclear they are still widely used

in PD research. There is evidence that 6-OHDA releases iron from ferritin and the

released iron can generate highly reactive hydroxyl radical via the Fenton reaction,

consequently leading to neurodegeneration. Markers of oxidative stress in the SN

which are reflected by enhanced lipid peroxidation, protein carbonyls, are confirmed

in PD. In addition, the oxidative burst triggers inflammation, which is also involved in

neurodegeneration.

Several methods such as pharmacological treatments and surgical treatments can be

used to treat PD. However, since the PD symptoms represent more than 50% neuronal

cell death in SN, prevention methods are ideal ways for neuroprotection rather than

the therapeutic methods. Prevention methods such as, antioxidation, antiinflammation,

are considered as effective neuroprotection strategies. Attention is focused on the

metal-induced formation of free radicals and the protective role of antioxidants

(nuclear factor erythroid 2-related factor 2, Nrf2/antioxidant system),

anti-inflammatory system (such as peroxisome proliferator-activated receptor ,

PPAR) and flavonoids (catechins). The iron chelators, such as desferroxamine have

shown to be effective in preventing or delaying PD progression. Epidemiological data

shows the positive relationship between green tea consumption and the lowered risk

of PD in Asian countries. In vitro and animal studies showed epigallocatechin gallate

(EGCG) protected against 6-OHDA induced neurotoxicity. However, limited human

 3

study data shows green tea as well as its main component EGCG directly protected

against neurotoxicity.

EGCG may benefit neurological disorders from several aspects: iron chelation,

antioxidation, antiinflammation, and other pathway modulators. In this study we

assessed the effect on iron homeostasis with6-OHDA induced neurotoxicity and the

preventive effects of EGCG against neurotoxicity in N27 cells by attenuating reactive

oxygen species (ROS) derived-oxidation and inflammation. We also show the

beneficial effects of green tea consumption in PD patients. Our entire project contains

three sets of studies. In the first study, we hypothesized that 6-OHDA and EGCG

treatment would regulate iron related elements. We used dopaminergic N27 cells to

determine the 6-OHDA induced dyshomeostasis by regulating iron transporter

mRNAs and protein expressions, such as DMT1, Fpn1, and hepcidin. Meanwhile,

EGCG protected against the adverse effects of 6-OHDA by normalizing and

maintaining iron metabolism and homeostasis. In the second study, we showed the

direct evidence that EGCG maintained the ROS homeostasis by suppressing 6-OHDA

induced oxidative damage on lipids and proteins. We also conducted the preliminary

study examining the stabilization of Nrf2 antioxidant system and PPAR

anti-inflammatory system by EGCG in N27 cells. In the third study, our hypothesis

was to identify the beneficial effects of green tea consumption on PD patients,

including slowing down the PD progression by improving the antioxidant status and

reducing oxidative damage. We found out that the intervention might improve the

quality of life of PD patients based on PD symptom rating scales and other measures

as well as enhance the antioxidant status, reduce oxidative damage on lipids and

proteins. We also showed that green tea intervention would not affect the iron status.

All together these findings in this study reveal the importance of iron regulation and
 4

oxidative damage in PD progression; the neuroprotection of EGCG by its iron

chelation, antioxidant, and antiinflammation capabilities in vitro, as well as beneficial

effect of and green tea consumption in PD patients.

Dissertation Organization

This dissertation is organized into six chapters, including a general introduction, a

literature review, three chapters on research publications, and a final conclusion.

Chapter 1 is a general introduction. Chapter 2 is a literature review of nutritional

prevention on oxidative stress-associated PD. Chapter 3 and 4 are the manuscripts

prepared to be submitted to Journal of Nutrition. Chapter 5 is manuscript prepared to

be submitted to Movement Disorders Journal. Chapter 6 is the general conclusion of

the studies which include the general conclusions and future directions. The

references of this dissertation are formatted in the Journal of Nutrition requirement at

the end of chapter 1 to 4. All the tables, figures, and legends in chapter 3, 4 and 5 are

placed as close as possible to the original text references.

 5

CHAPTER 2. LITERATURE REVIEW

I. PARKINSONS DISEASE

Parkinsons disease (PD) is the second most common neurodegenerative disease

after AD, affecting approximately 1% of the population over 50 years old and causes

an approximate $25 billion economic burden annually (1). PD is characterized by

progressive loss of control over voluntary movement, which results primarily from the

progressive loss of dopaminergic neurons in the SN, located in the mid-brain (2). The

symptoms of PD include tremor, rigidity, bradykinesia, hypokinesia and akinesia. The

pathological hallmarks of PD are the loss of the nigrostriatal dopaminergic neurons

and the presence of intraneuronal proteinacious cytoplasmic inclusions, LBs. LBs are

spherical eosinophilic cytoplasmic protein aggregates composed of numerous proteins,

including -synuclein, parkin, ubiquitin, and neurofilaments (4, 5). LBs are more than

15 m in diameter and have an organized structure containing a dense hyaline core

surrounded by a clear halo. PD-associated loss of dopaminergic neurons is

concentrated in ventrolateral and caudal portions of the SN, whereas during normal

aging the dorsomedial aspect is affected. The striatal dopaminergic nerve terminals

are the primary target of the degenerative process. Neurodegeneration and LB

formations are found in noradrenergic, serotonergic, and cholinergic systems, as well

as in the cerebral cortex, olfactory bulb, and autonomic nervous system. Degeneration

of hippocampal structures and cholinergic cortical inputs contribute to the high rate of

dementia related to PD, particularly in older patients. The disease is also considered

as a tyrosine hydroxylase (TH) deficiency syndrome, since TH catalyzes the

formation of L-3,4-dihydroxyphenylalanie, the rate-limiting step in the biosynthesis

of DA in the striatum (6). In general, the appearance of disease symptoms is observed

 6

after loss of 80% of striatal DA and 50% of the TH-immunoreactive dopaminergic

neurons in the SN (7).

A. Pathogenesis and Risk Factors of PD

Major hypotheses related to the progressive loss of dopaminergic neurons are

misfolding, aggregation of proteins, mitochondrial dysfunction, and the consequent

oxidative stress, including toxic oxidized DA species. LBs consist of aggregates of the

presynaptic soluble protein, -synuclein. -synuclein decreases mitochondrial

complex I activity and increases ROS production (8). Oxidative damage can also

interact with -synuclein and enhance its misfolding. Moreover, multiple cell

death-related molecular pathways are activated during PD progression, such as

apoptosis and necrosis.

1. Age

Age has been considered a potential risk factor for PD because most patients

develop PD after 50 years of age. The incidence of PD increases markedly over age

55, with a 20/100,000 incidence at age 55 to a 120/100,000 incidence at age 70 (3).

Pathologically, aging is associated with a decline of pigmented neurons in the SN. It

is also suggested that the aging nigrostriatal system may be more vulnerable to

damage by exogenous and endogenous toxins since the neurotoxin MPTP causes

more severe pathological and neurochemical injury in older mice (9).

2. Genetic Susceptibility

Common sporadic PD shares similar phenotype with genetic forms of PD, and the

same pathogenic and genetic mechanisms help to clarify the key biochemical pathway
 7

of PD progression. These PD genes that have been identified and studied in some

detail: -synucelin, parkin, and ubiquitin c-terminal hydrolase L1 (UCHL1) appear to

participate in the ubiquitin-proteasome system (UPS), affecting protein clearance and

cause LBs protein aggregation, which is the characteristic of PD neuropathology. In

addition, familial PD genes, such as PTEN-induced putative kinase 1 (PINK-1), DJ-1

or dominant Leucine-Rich Repeat Kinase 2 (LRRK2) are closely related to the

impairment of mitochondrial complex I.

3. Environment

Several epidemiological studies have suggested that exposure to different

environmental agents including pesticides, insecticides, heavy metals, and microbial

toxins may increase the risk of PD (10, 11). A byproduct of an illicit narcotic drug,

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) as well as its metabolite MPP+,

has been shown to cause the same signs and symptoms as PD. As mitochondrial

poisons, rotenone, and paraquat also activate the progression of PD. Human

epidemiological studies have implicated a higher incidence of PD in residents of rural

environments with exposure to herbicides and pesticides (12). However, cigarette

smoking and coffee drinking are inversely associated with the risk for development of

PD (13), reinforcing the concept that some environmental factors do modify PD

susceptibility.

4. Inflammation

Inflammation has also been implicated in the pathogenesis of PD.

Pro-inflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis

factor- (TNF-) may induce microglia activation and cause microglia related to
 8

neuronal injury (14). The negative association between the low risk of PD and high

anti-inflammatory activities indicates the role of inflammation in PD progression, and

suggests anti-inflammatory therapy as a potential neuroprotective strategy.

5. Oxidative Stress

Although environmental, genetic, and inflammatory factors contributed to the

etiology of PD, oxidative stress has been confirmed to play the main role in PD

progression (14). Brain tissue is susceptible to oxidative stress since it is rich in

polyunsaturated fatty acids, which can easily undergo lipid peroxidation. In addition,

high levels of ATP consumption in the brain could cause high oxidative metabolism

as the electrons might leak to form oxygen free radicals. The features of enhanced

oxidative stress linked with PD are supported by both postmortem studies and by

studies showing oxidative stress inducing nigral cell degeneration (15, 16). The

factors, including an impairment of antioxidant status, such as reduced glutathione

(GSH), increased reactive species, augmented DA oxidation, and high iron levels,

which are associated with oxidative stress, have also been found in the SN in PD (17).

B. Animal Models of PD

6-OHDA administration is widely used to induce PD in animal models, especially

in rats (19-21). 6-OHDA impairs sympathetic neuron nerve terminals in the peripheral

nervous system when administered systemically. Due to its chemical similarity with

catecholamine, it has a high affinity for the catecholamine transporter and is able to

damage the catecholaminergic neurons (22). Different from systemic administration

of MPTP, 6-OHDA only partly crosses the blood brain barrier (BBB). Brain injection

into the SN is one main way to kill TH-positive neurons, causing a dose-dependent
 9

loss in DA in the central nerve system (CNS) (23). Mice with 6-OHDA lesions in the

medial forebrain bundle (MFB) indicate the dose-dependent manner of damage on

midbrain DA neurons (30). Lundblad (2004) reported the first 6-OHDA lesioned mice

developing dyskinesia, which was similar to those previously characterized in the rat

and in other animal models of PD (31) and PD patients (32, 33). Compared with the

corresponding rat model, MFB-lesioned mice show a much larger number of

TH-positive cell bodies in the striatum. However, the functional importance of these

neurons is yet to be clarified.

C. Cell Models of PD

Cell cultures, such as mesencephalic slice cultures, primary immature

dopaminergic neurons, and immortalized cell lines, either in a proliferating state or in

a differentiated state, are used for the screening of pathogenesis and drug candidates

in PD research (39). The loss of TH+ immunoreactivity is used as a marker in primary

neuron model cells, and neurotoxins, including 6-OHDA and MPP+, can cause

significant TH+ cell death (40-42). The difficulty in producing the primary

mesencephalic neuron cells in large quantities limits their usefulness even if they have

the physiological characteristic of neurons of the SN. The catecholaminergic

neuroblastoma cell lines, such as SH-SY5Y, have been used to clarify the proposed

mechanisms of 6-OHDA and MPTP neurodegeneration in PD, including oxidative

stress and inflammation (43-45). The SH-SY5Y cells are less sensitive to neurotoxins

since they are not neuronal cells. N27 cells are from an immortalized line derived

from the rat mesencephalon, and may be more physiologically similar to

dopaminergic neurons. N27 cells can express both tyrosine hydroxylase and the DA

transporter, and produce measurable amounts of DA (46). N27 cells are sensitive to
 10

6-OHDA and can help to clarify the 6-OHDA induced neurotoxicity. The MN9D cell

line, a fusion of embryonic ventral mesencephalic and neuroblastoma cells, is

extensively used as a model of DA neurons because they express tyrosine hydroxylase

and can synthesize and release DA. These cells are also used to understand the

mechanisms and to develop potential therapeutics to prevent DA neuronal loss in PD

research (47).

Non-dopaminergic cell lines, such as CHO-K1 and HEK293 are also used for

studying PD (48, 49). The PC12 cell line, a rat pheochromocytoma cell line, has

been extensively used as a tool for studying the function of neurotrophic factors and

neuronal differentiation, and is widely used as a model cell to study the environmental

effects, oxidative stress, and genetic manipulation on PD progression (50). Several

studies showed that 6-OHDA induced PC12 cell death by oxidative stress, cell

apoptosis, and necrosis in a pro-inflammatory manner (50, 51) .

There are still several other cell lines, such as peripheral T-lymphocytes, that can

be used to study the inflammatory and cell apoptotic pathways during PD progression

(52).

D. Mechanism of 6-OHDA-induced Neuron Cell Damage

Neurotoxins are shown to depolarize the mitochondrial membrane potential, disturb

the mitochondrial function, inhibit the ubiquitin proteasome system (UPS), and

decrease proteasomal function (53-55). Consequently, they cause ubiquitinated

protein accumulation in the mitochondria, such as -synuclein, resulting into a

reduced activity of mitochondrial complex I, and increase influx of Ca2+ into the cell,

causing an excitotoxic condition (56). 6-OHDA has been shown to reversibly inhibit

both mitochondrial complexes I and IV activities in rat brain with inhibition

 11

concentration (IC50) of 10 and 34 M (57-59). 6-OHDA is mechanistically similar to

DA, binding to DA receptors on neuropeptide substance P (SP)-containing striatal

projection neurons, releasing SP. SP can then potentiate both DA and glutamate

release to cause oxidative stress and glutamate excitotoxicity, contributing to

dopaminergic cell death (60). In addition, 6-OHDA is also subjected to auto-oxidation

to generate ROS, including quinines, hydrogen peroxide, superoxide radicals, and

hydroxyl radicals (61). It has been shown in vitro that DA reacts with Fe (II) in the

presence of hydrogen peroxide to generate 6-OHDA (62). 6-OHDA can then react

with ferritin to cause iron release and subsequent cellular damage (63). 6-OHDA also

induces NO and iNOS over-expression, cell apoptotic and necrotic cell death, and

pro-inflammatory gene (IL-1B, TNF-a, COX-2) activation (50, 51, 64).

The molecular pathways involved in 6-OHDA-induced cell death have also been

studied in vivo, however less is known about the mechanism of progressive

dopaminergic neuronal death by the striatal injection of 6-OHDA. 6-OHDA activates

NF-B, p53 and the c-jun N-terminal kinase (JNK) pathway, which has been

identified in dopaminergic cell death in both the MFB and the striatal model (65, 66).

In addition, the anti-inflammatory PPAR was shown to attenuate 6-OHDA toxicity

in a rat model, indicating the involvement of an inflammatory reaction. More recently,

6-OHDA has been shown to elevate the activation of an apoptosis signal regulating

kinase 1 (ASK1), indicating the oxidative stress and apoptotic pathways in PD

progression (47).

II. OXIDATIVE STRESS AND NEURODEGENERATION

Oxidative stress can result from a variety of conditions that represent either

increased ROS production or a decreased antioxidant defense. The superoxide radical

 12

is considered as the primary ROS, which further generates secondary ROS by

interacting with other molecules. For example, hydrogen peroxide can be generated

by a dismutase reaction. The superoxide dismutase (SOD) enzyme also works in

conjunction with other H2O2removing enzymes, such as catalase and glutathione

peroxidase (GPx). The production of H2O2 and O2 is tightly linked with the

participation of redox-active trace metals.

The redox states should be maintained within strict physiological limits. In vivo,

under stressed conditions, an excess of superoxide acts as an oxidant of [4Fe-4S]

cluster-containing enzymes, releasing free iron from iron-containing molecules. The

released Fe2+ can participate in the Fenton reaction, generating highly reactive

hydroxyl radicals (67). Cells of the immune system also produce both the superoxide

anion and nitric oxide during the oxidative burst triggered during inflammatory

processes. Under these conditions, nitric oxide and the superoxide anion may react

together to produce significant amounts of a much more oxidatively active molecule,

such as peroxynitrite anion (ONOO-) (68, 69).

The oxidation of cellular components by oxidative stress results in modifications of

DNA, proteins, lipids, and carbohydrates, which are associated with cell death either

by necrosis or by apoptosis. 4-hydroxy-2-nonenal (HNE) is the main aldehyde

by-product formed during lipid peroxidation, and considered to be one of the most

reliable markers of oxidative stress. Protein carbonylation is the ROS-induced

post-translational modification of amino acid side chains. The protein carbonyl is

widely used as marker of oxidative protein damage (70, 71). In addition, lipid

peroxidation can also exacerbate the oxidation of protein. Some protein carbonylation

from lipid-derived aldehydes is more prevalent than that formed via direct amino acid
 13

side chain oxidation (72). ROS can also attack DNA, generating DNA lesions and

resulting in mutagenesis and cell death, especially in the progression of DNA/RNA

polymerases.

Nigral dopaminergic neurons are particularly exposed to oxidative stress due to the

high monoamine oxidase (MAO) enzyme activity which yields H2O2 and other ROS.

H2O2 could also react with iron in the SN to produce highly toxic hydroxyl radicals.

The auto-oxidation of DA can also acerbate the ROS level (73). Due to the

significantly lower concentration of antioxidants in the brain compared with other

organs (74, 75), the brain is much more sensitive to oxidative stress. Previous work

has shown that neurodegeneration is closely related to increased ROS levels in brain.

Postmortem brains of PD patients have shown high levels of oxidation of lipids,

proteins, and nucleic acids in the SN. The loss of functional nicotinic acetylcholine

receptors (nAChRs) in neuron cells due to the intracellular ROS is like to have

significant implications for the early progression of PD (76-78), therefore oxidative

stress is directly involved in the loss of dopaminergic nigral neurons.

A. Oxidative Stress and Mitochondrial Dysfunction

There is considerable evidence for mitochondrial dysfunction in the brain of PD

patients (79, 80). The dramatic loss of function of mitochondrial complex I activity

may be the central tenet of sporadic PD, consequently promoting oxidative stress.

Damage to mitochondrial complex I by oxidative stress can be acerbated by trace

metals, environmental toxins, and familial Parkinson related genes. Epidemiological

studies showed MPTP is related to acute and irreversible Parkinsonism in human and

non-human primates (81). Different from 6-OHDA, MPTP is a lipophilic molecule

that can easily cross the BBB and be transferred to

 14

1-methyl-4-phenyl-2,3-dihydropyridinium (MPDP) in glial cells and metabolized to

the active toxic compound (MPP+, 1-methyl-4-phenylpyridinium) (82). MPP+ can be

taken up by DA neurons and concentrated in mitochondria, causing complex I

dysfunction and oxidative stress (83). MPTP is responsible for several downstream

apoptotic events in SN neurons, including up-regulation of inflammatory factors,

release of cytochrome c, and activation of caspase-3 activity. In addition to MPTP,

pesticides with related properties, such as rotenone, paraquat, dieldrin, and maneb

exhibit common features to inhibit the mitochondrial respiratory chain and produce

oxidative stress (84, 85).

Significant evidence suggests that accumulation of -synuclein in mitochondria

elevates oxidative stress, which is relevant to PD pathogenesis. Complex I defects

cause -synuclein aggregation and this aggregation would lead to further

mitochondrial dysfunction, such as decreased mitochondrial membrane potential, and

oxidation of mitochondrial proteins (86, 87). Further studies are needed to elucidate

the precise functions of -synuclein in the regulation of mitochondrial functions.

Additionally, other genes linked to familial PD have been implicated in the

mitochondrial function and oxidative stress response.

B. Metal Related Neurodegeneration, With Particular Consideration of Iron

Over the last decade, the redox ability of metal ions, such as copper, has been

shown to lead to radical formation, and reactive nitrogen and oxygen species,

contributing to various diseases. These metals can be coupled with proteins,

accelerating the electronic reactions. A number of recent studies showed that

-synuclein interacts with zinc, copper, and iron, consequently in the state of

aggregation (88). Therefore, it has become widely accepted that dyshomeostasis of

 15

these redox metal ions (manganese, copper, iron, lead, and aluminum) is accompanied

by oxidative stress, which is a key factor in a large number of neurodegenerative

diseases, such as PD, Alzheimers disease, Hungtingtons disease, multiple sclerosis

and Friedreichs ataxia (89). It has been shown that the concentration of zinc and iron

are increased and copper is decreased in the SN in PD. However, the mechanisms of

these changes remain unclear. Iron accumulation is closely related to

neurodegenerative diseases. The elevation of brain iron such as in the putamen, motor

cortex, sensory cortex and thalamus, are localized within H- and L-ferritin and

neuromelanin with no adverse effects (90). However, excessive iron in cellular

constituents, like the mitochondria, or specific regions, such as SN and lateral globus

pallidus, can lead to the generation of toxic free radicals which can damage cells (89).

Numerous studies have confirmed that there is an elevated iron level in the SN of PD

patients compared to age-matched controls (91, 92). The oxidative stress in brain

regions can cause peroxidation of polyunsaturated fatty acids in membrane

phospholipids, generating protein carbonyls and damaging neurons.

C. Brain Iron and Neurodegeneration

Iron is a necessary cofactor in many metabolic processes in the central nervous

system, including oxidative phosphorylation, myelin synthesis, nitric oxide

metabolism, and oxygen transport. It plays an important role in electron transfer and

affects numerous enzymes, including a number of key enzymes for neurotransmitter

biosynthesis in the brain, such as tyrosine hydroxylase, which is involved in the

metabolism of DA (93).

Serum iron is delivered to cells via transferrin (Tf). Endothelial cells can express

transferrin receptor (TfR) on the luminal side of the capillaries in the BBB, and serum
 16

iron can be delivered into the brain via the transferrin (Tf)-TfR-cell model, which is

regulated by astrocytes. The iron which was in a diferric form in transferrin (Fe2+-Tf)

binds to TfR, followed by receptor-mediated endocytosis, therefore iron can be

transported from the luminal to the abluminal sides of the blood capillary endothelial

cells (BCECs). Later, iron is released from transferrin within the endosome under the

acidic pH environment and the apotransferrin is recycled back to the plasma

membrane to bind to more iron. DMT1 is responsible for transporting iron in and out

of the endosome into the cytoplasm, and then metalloreductases can reduce Fe3+ to

Fe2+. Therefore, cytoplasmic iron can be transferred to the mitochondria for biological

usage, while the extra iron is stored in cytosolic ferritin (94, 95). Astrocyte cell

membranes contain ceruloplasmin (Cp) which is linked to glycophosphoinostitide

(GPI) (96). The ferroxidase activity of GPI-Cp is required for the stability of Fpn1 in

cells, facilitating the iron efflux from the cells (97). Fpn1 is present in neurons and

could therefore represent the mechanism by which iron is exported from such cells

(98). Additionally, another iron regulator, hepcidin, is responsible for regulation of

iron homeostasis. Hepcidin can bind to Fpn1 and down-regulate Fpn1 mRNA and

protein expression by inducing its internalization and degradation, promoting iron

retention (99). Hepcidin is expressed in several organs, such as liver and brain, and is

closely related to the increased iron. It is also considered as an oxidation indicator,

associated with oxidative stress.

Overall, iron metabolism and iron homeostasis at both the cellular and systemic

levels is mainly regulated by the iron-associated proteins, such as ferric reductase,

DMT1, the iron exporter Fpn1, and the membrane-bound ferroxidase. The translation

of these proteins is modulated by the IRP system, together with the other iron

homeostasis regulators, including hepcidin.

 17

Fig. 1 The iron uptake into cell. Holotransferrin (HOLO-TF) binds to transferrin receptors (TFR) at the
cell surface. The complexes localize to clathrin-coated pits, which initiate endocytosis. Specialized
endosomes form and are acidified by a proton pump. At acidic pH, iron is released from transferrin and
is co-transported with protons out of the endosomes by the divalent metal ion transporter DCT1.
Apotransferrin (APO-TF) bound to TFR is returned to the cell membrane, where, at neutral pH, they
dissociate to participate in further rounds of iron delivery. In non-erythroid cells, iron is stored as
ferritin and hemosiderin (89).

IRPs are the key factors to post-transcriptionally regulate the iron-associated

proteins involved in cellular iron metabolism. IRP1 and IRP2 also function as

cytosolic iron sensors. In conditions of iron deficiency, IRPs bind with IRE, which is

in the 5-untranslated regions of the mRNA, preventing initiation of translation of

ferritin and Fpn1. In contrast, in the case of transferrin receptor and DMT1 where the

IREs are in the 3-untranslated region, binding of the IRPs to the mRNAs protects

them against degradation by nucleases. Therefore, this leads to increased iron uptake

and reduced iron storage and export. In conditions of iron abundance, the IRPs are no

longer active in binding, allowing ferritin and Fpn1 mRNA to be translated and

inhibiting iron uptake by reducing transferrin receptor and DMT1 synthesis.

Disruption of iron transport proteins could cause excess iron in the brain. The cause

might be genetic or by neurotoxins. The interruption of iron homeostasis is involved

in a number of neurodegenerative disorders, including PD. The main adverse effect of

 18

excess iron is the production of highly reactive hydroxyl radicals via the Haber-Weiss

and Fenton reactions (100):

If the increased ROS levels exceed the capacity of the self-antioxidant defense

system, the cells will experience oxidative stress, membrane oxidation and protein

damage. Increased oxidative stress is one of the contributors to PD progression. In

addition, oxidative stress also exacerbates the intracellular free iron level, further

promoting the -synuclein aggregation to form LBs in PD brains (101).

Even if the iron dyshomeostasis is related to neurological diseases, it is still under

debate whether impaired iron homeostasis is the primary cause. Genetic

hemochromatosis and thalassemic patients with high circulating iron, transferrin

saturation, and high levels of low-molecular weight iron, do not exhibit increased

brain iron. Evidence shows that higher iron distribution in different brain regions was

associated with pathologic status.

D. Oxidative Stress and Neuroinflammation in PD

The immune responses within the brain are regulated by resident immune cells.

Microglia are the major resident immune cells in the brain, providing innate immunity

via the production of neurotoxic factors including cytokines, chemokines, and

prostaglandins. Those factors also contribute to the immune activities of surrounding

astrocytes and oligodendrocytes (102). Pro-inflammatory cytokines such as TNF-

and IL-1 act on astrocytes, inducing the adaptive immune response, while

chemokines such as monocyte chemotactic protein-1 (MCP-1)/Chemokine (C-C motif)

 19

ligand 2 (CCL2) recruit additional immune cells. These inflammatory responses may

potentiate neuronal cell damage through oxidative stress. Neuroinflammation and

oxidative stress share common linkages and influence each other. One of the

functions of proinflammatory cytokines is to produce oxidative stress, which can

subsequently shape the profile of cytokine release. Microglia are a major source of

ROS in the CNS. Under the pathologic condition where there is reduced antioxidant

level, oxidative stress causes more free radicals to be released into the cytosol,

inactivating proteins or interrupting intracellular signaling pathways. In addition, ROS

can initiate pro-inflammatory pathways in neuronal cells. Compared to cortical or

hippocampal neurons, midbrain DA neurons are more susceptible to

pro-inflammatory cytokines such as TNF (103). Therefore, future therapeutic

interventions to limit the early pro-inflammatory response would be beneficial in

reducing microglia activation and elevated oxidative stress (104).

Epidemiological, animal and human studies implicated the innate and peripheral

neuroinflammatory response contributing to the onset of PD. Increased immune cells,

such as CD4+ and CD8+ have been found in post-mortem human PD brains (105,

106). The use of positron emission tomography (PET) imaging has confirmed the

activated microglia in human PD brain (107). Elevated levels of pro-inflammatory

cytokines have been found in the SN of PD brains. Factors known to promote

neuroinflammatory response, including traumatic brain injury, and exposure to

viruses and infectious agents, have all been proposed as increased risk factors for

developing PD (108). In addition, 6-OHDA and MPTP are also commonly shown to

cause PD in animal models, and display features of neuroinflammation.

 20

1. Antioxidation

Peroxisome proliferator receptor (PPAR) is a family of ligand-dependent nuclear

hormone receptor transcription factors that play a key role in the regulation of

inflammatory responses, antioxidation, and brain function. PPARs stimulate gene

expression by binding to peroxisome-proliferator response elements (PPREs) of the

target genes (109). There are three structurally homologous iso-types: PPAR,

PPAR/, and PPAR. PPAR is expressed in several organs, including liver, kidney,

intestine, heart, skeletal muscle, and brain. PPAR is involved in acetylcholine

metabolism, excitatory neurotransmission, and oxidative stress defense. PPAR can

regulate lipid metabolism and energy homeostasis (110). PPAR/ is ubiquitously

expressed in all cell types, including immature oligodendrocytes, and promotes

differentiation and myelination in the CNS. PPAR/ also can regulate lipid

metabolism in the brain (111). PPAR can be detected in adipose tissue, intestinal

mucosa, retina, skeletal muscle, heart, liver, and lymphoid organs. PPAR has been

implicated in the pathology of numerous diseases including obesity, diabetes,

atherosclerosis, and cancer. PPAR is not only an anti-inflammatory factor, but also

has antioxidant properties, and is used as treatment of oxidative stress related diseases,

including PD, AD, and multiple sclerosis (112, 113). In these disorders, PPAR

agonists have the potential to modulate various signaling pathways, including matrix

metalloproteinase-9, mitogen-activated protein kinases (MAPK), signal transducer

and activator of transcription, mitochondrial uncoupling protein 2, and amyloid

precursor protein degradation (114). PPAR may directly modulate the expression of

several antioxidant genes in response to oxidative stress, including catalase, SOD,

GPx, the scavenger receptor CD362, and nitric oxide synthase (eNOS) (115-119).

PPAR can regulate heme oxygenase-1 (HO-1) expression, however this effect is still
 21

under debate (120, 121). It is obvious that PPAR is closely related to antioxidative

defense mechanisms. Recent evidence had confirmed that Nrf2 and PPAR were

linked by a positive feedback loop that sustained the expression of antioxidant genes

and antioxidant system under oxidative stress (119, 122). Therefore, antioxidation and

anti-inflammatory pathways might be simultaneously involved in the progression of

neurodegeneration and the clarification of the mechanisms on the interaction will be

needed to be researched.

2. Anti-inflammatory System

The neuroinflammatory diseases, including AD, and PD, present major challenges

to the health care system and impose substantial economic costs around the world

(123). Since evidence shows that PPAR is expressed in certain areas of the brain,

such as neurons and glial cells, it may possibly be involved in neuroinflammation and

subsequent neurodegeneration. As shown in a MPTP model, PPAR exerts both

anti-inflammatory and antioxidant effects by inhibiting glial activity, which may

account for the attenuation of dopaminergic cell loss in both the SN and striatum. In

the LPS-induced inflammatory model of PD, PPAR showed the ability to restore

striatal DA, mitochondria function, and significant dopaminergic neuroprotection by

directly inhibiting mitochondrial fatty acid metabolism and altering mitochondrial

uncoupling protein expression (124, 125).

PPAR could reduce the infiltration of immune cells to the BBB and down-regulate

pro-inflammatory genes such as COX-2, iNOS, and various cytokines, which were

associated with inflammation-induced neurodegeneration (126-128). The

neuroprotection of PPAR activation was also based on antiinflammation through

inhibiting IL-6, TNF expression, and toll-like receptor (129, 130).

 22

Additionally, epidemiological studies showed that 7% of PD patients have type II

diabetes or insulin desensitization. They suggest that diabetes accelerated the

progression of the motor and cognitive symptoms of PD, and insulin in the

nigrostriatal system is related to PD (131). Drugs used to treat PD, such as L-dopa or

bromocriptine alter insulin signaling and sensitivity. PPAR had been associated with

insulin sensitivity alteration and inhibition of inflammation. Therefore, PPAR

activation may offer a new clinical treatment approach to neuroinflammation and PD

(132).

III. TREATMENT AND PREVENTION STRATEGIES OF PD

Since it is impossible to cure PD, preventing and controlling the symptoms of PD

and minimizing side effects are the goals of anti-Parkinsonian therapy. Data is lacking

in many areas of PD treatment and prevention, therefore, there is currently wide

variation in the management of PD.

A. Treatments

1. Pharmacological Treatment

L-dopa has been used as the main pharmacological treatment of PD, and peripheral

decarboxylase inhibitor is used to limit metabolism of L-dopa. Even if L-dopa is the

most effective drug in the treatment of PD, long-term use can cause motor

fluctuations and dyskinesias (133). There is still debate over how to use L-dopa as a

neurological treatment, such as dosage and time. L-dopa has been shown to be toxic

to neurons in vitro, but these findings have not been substantiated in humans (134,

135).
 23

DA agonists are frequently used as adjunct therapy with L-dopa in early PD, and

they include bromocriptines, pergolide, lisuride, cabergoline, pramipexole and

ropinirole. The mechanism of action of DA agonists depends on their DA releasing,

inhibiting reuptake and DA receptor and stimulating capabilities. Monoamine oxidase

B (MAO-B) inhibitors can be used as dopaminergic therapy in patients with early PD

because they can inhibit DA catabolism and increase nigrostriatal DA levels. The

inhibition of catechol O-methyl transferase (COMT) can increase the bioavailability

and prolong the action of DA and is also considered as a therapy for neuroprotection.

In addition, anticholinergic medication methods are also used to treat PD, such as

benztropine and procyclidine. However, they are typically used in younger PD

patients, instead of patients older than 65.

2. Surgical Treatment

Recognition of the limitations of pharmacological therapy and improvement in

surgical technologies has led to a resurgence of surgery on PD patients in recent years.

Even though surgical treatment may raise concerns regarding safety, it may reduce

drug-induced dyskinesias and dystonias in PD, including ablative procedures (globus

pallidus or thalamus), deep brain stimulation and tissue transplant (136). Thalamic

stimulation has equal efficacy for tremor suppression, but with fewer adverse effects

compared to thalamotomy (137). Transplantation has shown promising benefits, but

studies are questioning its efficacy and safety, which has impeded this treatment

procedure (138).
 24

B. Prevention Strategies

Due to the efficacy and safety concerns about the treatment therapies of PD, the use

of antioxidants, anti-inflammatory drugs, and specific stress signal inhibitors as

prevention methods have provided potential beneficial effects for common

neurodegeneration.

1. Antioxidants

Selegiline, vitamin E, and rasagiline have been used in clinical trials due to their

antioxidant properties (139, 140). Selegiline reduces DA oxidation by inhibiting

MAO-B and has been used as an antioxidant drug. Although there exist concerns

about the efficacy of vitamin E in the prevention of PD, long-term, high dose vitamin

E dietary supplementation (e.g. 1600-2000 IU d--tocopheryl succinate) was shown

to be a successful therapeutic strategy for the prevention of PD (143, 144). Rasagiline

is a newer MAO-B inhibitor and its metabolite has potential antioxidant property. A

clinical study showed rasagiline reduced the oxidative effect of symptomatic efficacy.

Coenzyme Q10 (CoQ10) is a cofactor in the electron transport chain in mitochondria

metabolism and has been shown to reduce dopaminergic neurodegeneration in mouse

PD models (145). However, CoQ10 showed less or no significant protection on PD

progression based on different study designs, initiating concerns on the stability of its

usefulness (146-148). Even if none of these clinical studies have convincingly shown

that these antioxidant therapies slow disease progression, several of these trials

indicate that selegiline, rasagiline, and CoQ10 may be promising. In addition,

compared to current PD treatments, all of the antioxidant approaches are relatively

safer in neuroprotection. Epidemiological studies identified uric acid as a potential

 25

neuroprotective agent in PD, which acts as an antioxidant by scavenging ROS and

NOS.

2. Receptor antagonists

Epidemiological studies have shown that caffeine may reduce the incidence of PD,

leading to adenosine receptor antagonists as potential neuroprotective agents (13, 153).

Adenosine receptor antagonist A2a can improve symptoms in PD and be

neuroprotective, which was shown in several in vivo studies (154, 155). A2a can also

exert synergistically effects with L-dopa (156).

DA receptor agonists have been proposed as potentially neuroprotective by acting

at D2 autoreceptors found on dopaminergic SN terminals to suppress DA release and

thus reduce oxidative stress in vitro and in animal studies (157-160). Certain agonists,

such as pramipexole, may also act as direct antioxidants due to their hydroxylated

benzyl ring structure. However, it is still difficult to establish long-term systems for

neuroprotection of the receptor antagonists neuroprotection.

3. Anti-inflammatory agents

The use of anti-inflammatory drugs has been investigated in various diseases. In

PD, antiinflammation has been considered as a potential neuroprotective strategy in

drug development (161). Anti-inflammatory agents, including non-steroidal

anti-inflammatory drug (NSAIDs) and minocycline have been used to treat PD in

tissue culture and in animal models. NSAIDs, such as aspirin, have anti-inflammatory

effects, including reduction of TNF, nitric oxide, and superoxide production by

microglia (162). Statins may also act to show neuroprotection due to their

anti-inflammatory ability beyond scavenging free radicals (163). Epidemiological

 26

studies showed that statin use, particularly simvastatin, was associated with reduced

PD incidence (164, 165). Simvastatin had been shown to reduce DA loss in MPTP

animal models (162). Minocycline has anti-inflammatory effects independent of its

antimicrobial activity and protects against dopaminergic cell loss in both MPTP and

6-OHDA animal models (166, 167). Minocycline blocks microglial activation and

may also have anti-apoptotic activity in vitro (168).

4. -synuclein-directed therapies

Since protein aggregations, like -synuclein, are related to PD progression, the

disruption of -synuclein aggregation has been the focus of research to develop novel

therapies against PD. Augmentation of parkin or ubiquitin carboxy-terminal

hydrolase-L1 (UCH-L1) activity could promote the clearance of -synuclein and

other aggregated proteins (169, 170). Activation of lysosomal degradation could also

clear -synuclein. Therefore, the lysosomal enzyme cathepsin D could inhibit

-synuclein-induced neurotoxicity in cell culture and animal models (171). Because

oxidative modification and phosphorylation of -synuclein both promote protein

aggregation, antioxidants and kinase inhibitors could reduce -synuclein aggregation

and toxicity (172-174). In addition, direct blockers of -synuclein aggregation could

be a therapeutic strategy for PD. Increasing the expression of chaperone-like proteins,

such as 14-3-3, has been demonstrated to reduce -synuclein aggregation and could

serve as a basis for therapeutic intervention (175, 176). The chaperone protein Hsp70

reduced insoluble -synuclein aggregates in vitro and in vivo (177).

The COMT inhibitors entacapone and tolcapone are able to block fibril formation

of -synuclein. However, a strong limitation for the use of these compounds is that

they have a wide range of side effects, such as vomiting, nausea and hypotension, or
 27

in the case of tolcapone, hepatotoxicity (178). In addition, cholesterol reducing agents

have been shown to be effective in blocking -synuclein aggregation in vitro and in

vivo.

5. Anti-apoptotic agents

Several anti-apoptotic agents have been examined in controlled clinical trials.

Vasoactive intestinal peptide (VIP) can be considered as a good anti-apoptotic

candidate agent for 6-OHDA induced neurotoxicity and the treatment of PD (179).

Tauroursodeoxycholic acid (TUDCA) acts as an anti-apoptotic agent in MPTP mouse

PD models, used as a modulator of neurodegeneration in PD (180). The

propargylamine TCH346 is an anti-apoptotic factor and was shown in both 6-OHDA

and MPTP animal models to reduce dopaminergic cell loss (181, 182). Overall,

anti-apoptotic agents would be a perspective treatment for PD progression.

6. Kinase inhibitors

The activation of kinase pathways has been shown to be involved in the

progression of PD in in vitro and in vivo studies. The most common genetic cause of

PD to date is mutation in the gene LRRK2, which is associated with increased kinase

activity. Therefore, kinase inhibitors seems to be new neuroprotective strategies

against the progression of PD. Kinase inhibitors, such as

Z-Asp(Ome)-Ile-Pro-Asp(OMe)-FMK can block PKC cleavage and reduce

caspase-3 related neurotoxicity. It can also rescue TH+ neurons from MPP+ and

6-OHDA-induced toxicity in primary mesencephalic cultures (183).

 28

7. Polyphenols

Polyphenols are a class of highly bioactive compounds, and can be categorized into

flavonoids, stilbenes, phenolic acids, phenolic alcohols, and lignans. A number of in

vitro and in vivo studies indicate that these small molecules are able to modulate

neurological progression pathways due to their strong antioxidant and

anti-inflammatory effects. A natural polyphenol resveratrol can directly scavenge the

hydroxyl radical, inhibit H2O2 or lipid peroxide-dependent peroxidation (190, 191),

suppress COX-2-induced neuroinflammation (192), and reduce dopamine-induced

cell death in vitro (193, 194). In vivo, resveratrol attenuated 6-OHDA-induced DA

depletion and loss of dopaminergic neurons in rats (195).

It is important to note that many in vitro studies have focused on the

neuroprotective properties of flavonoids that differ from their forms in vivo. For

example, natural forms of flavonoids are hydrolyzed and conjugated by gut and liver

enzymes before entering the circulation (196), resulting in reduction of their

antioxidative activity (197, 198). Both intact and derivative forms of flavonoids such

as tea catechins and naringenin have been found to pass through the BBB (199). The

degree of permeability is governed by several factors, including compound

lipophilicity and the action of specific transporters on the BBB (200). Therefore,

elevating the bioavailability of flavonoids in CNS would benefit PD drug

development.

8. Nutritional strategies

Oxidative stress causes the loss of dopamine neuron within the central nervous

system. Therefore, an antioxidative has to potential to impart neuroprotection. A diet

containing high levels of antioxidants, such as complex phenols, vitamin E, and

 29

carotenoids, acts as a preventative measure for the disease. Higher intake of

vegetables, legumes, fruits, cereals, unsaturated fatty acids, and fish may add more

antioxidants, while a diet rich in dairy and animal fat products results in a higher risk

of developing PD. Foods which cause high plasma urate may reduce PD risk because

of urates ability to scavenge peroxynitrite and hydroxyl radicals resulting in

decreased oxidative stress.

Oxidative modification can cause mitochondrial decay, resulting in

neurodegeneration. Supplementation with mitochondrial nutrients may prevent

mitochondrial decay and the possible mechanisms may include: a. preventing

mitochondria from oxidative stress, with nutrients such as lipoic acid, vitamin E and

CoQ; b. repairing mitochondrial membranes, with nutrients such as carnitine; c.

functionally repairing and preventing mitochondrial damage, such as substrates or

coenzymes, or precursors of mitochondrial enzymes (quinine reductase and

glutathione-S-transferase), with nutrients such as lipoic acid, niacin, pantothenic acid,

riboflavinthiamine, biotin, and carnitine; d. inducing phase-2 enzymes to enhance

antioxidant defenses in cytosol as an indirect protection to mitochondria, with

nutrients such as lipoic acid.

The combination of two mitochondrial nutrients may have more effective

antioxidant capability than each has individually. It is reported that the combination of

R-alpha-lipoic acid (LA) and acetyl-L-carnitine (ALC) has a synergistic action against

rotenone-induced mitochondrial dysfunction, oxidative damage and a-synuclein

accumulation at lower concentrations then they have individually (100-1000-fold

lower).
 30

Figure 2. Possible protective mechanisms of nutrients in mitochondria. B1, thiamine, B2,

riboflavin; B3, niacin; B5, pantothenic acid; B6, pyridoxine; B12, cobalamin; CoQ, coenzyme
Q10; Cyt C, cytochrome c, LA, alpha-lipoic acid; PDH, pyruvate dehydrogenase; alpha-KGDH,
alpha-ketoglutarate dehydrogenase; BC-KADH, branched chain ketoacid dehydrogenase, CAT,
carnitine acyltransferases; Carboxilase includes pyruvate carboxylase, acetyl-CoA carboxylase,
and propionyl-CoA carboxylase, and THF-related enzymes includes methionine synthease,
methylene tetrahydrofolate reductase.

C. Green Tea Polyphenol Properties & Mechanisms of Protection

Green tea catechines have been shown to have many biological actions, including

antioxidant, free radical-scavenging, antiatherosclerotic, cardioprotective,

anti-inflammatory, and anticarcinogenic effects. There is still controversy on the

cancer-preventive, cardiovascular protective, or neuroprotective roles of green tea

intake due to the limitation of data. Most of the results have come from studies using

observational designs, case studies, case-control studies or cohort studies that lack the

rigor of clinical trials. Detailed information on intake quantities and cumulative

exposure to green tea (years of drinking) is lacking. However, human epidemiological

studies suggest that tea consumption is inversely correlated with incidence of

dementia, AD, and PD. In a cross-sectional study aimed at investigating an

association between consumption of green tea and cognitive function in elderly

 31

Japanese subjects, it was found that higher consumption of green tea is associated

with lower prevalence of cognitive impairment (201). In a case-controlled study in the

US, it was found that people that consumed 2 cups/day or more of tea presented a

decreased risk of PD (202). In consensus, a recent prospective 13-year study of nearly

30,000 Finnish adults, aged 25-74 years, demonstrated that drinking 3 or more cups of

tea per day is associated with a reduced risk of PD (203). These findings emphasize

the importance of well-designed controlled studies to assess a risk reduction of PD

and AD in consumers of green tea. The beneficial effects of green tea are attributed to

its polyphenolic compounds, particularly the catechins, approximately composing

30% of the dry weight of green tea leaves. There are higher quantities of catechins in

green tea than black or oolong tea due to the different processing steps. For green tea,

the polyphenol oxidase enzymes are inactivated and maintained in the monomeric

forms. Catechins account for 2535% of the dry green tea extract and consist of eight

polyphenolic flavonoid-type compounds, namely, (+)-catechin (C), ()-epicatechin

(EC), (+)-gallocatechin (GC), ()-epigallocatechin (EGC), (+)-catechingallate (CG),

()-epicatechin gallate (ECG), (+)-gallocatechin gallate (GCG) and EGCG. The most

abundant catechin is EGCG, which accounts for 65% of the total catechin content in

green tea. A cup of green tea (2.5 g of green tea leaves/200 ml of water) may contain

90 mg of EGCG and thus is thought to particularly contribute to the beneficial effects

attributed to green tea, such as its neuroprotective properties. Green tea and EGCG

have been used in many different cells and animal models to investigate mechanisms

underlying the various actions of green tea catechins.

 32

Fig.3. Structures of the major polyphenolic catechins present in green tea.

EGCG has been shown to improve age-related cognitive decline and protect against

cerebral injuries and brain inflammation and neuronal damage in experimental

autoimmune encephalomyelitis (204). Long-term administration of EGCG was

demonstrated to improve spatial cognition learning ability in rats (206). In line with

the in vivo findings, cell culture studies had demonstrated that EGCG prevented

neuronal cell death caused by the neurotoxins 6-OHDA and MPP+ in several cell

lines, such as human neuroblastoma SH-SY5Y cells and rat pheochromocytoma

(PC12) cells. EGCG not only has cancer chemopreventive, anti-angiogenic and

anti-mutagenic properties, but also has anti-diabetic properties in an insulin animal

model and anti-bacterial, anti-HIV, anti-aging, and anti-inflammatory activity. EGCG

has a catechol-like structure, which inhibits DA uptake, blocks DA competitors,

exerting neuroprotective effects. EGCG also inhibits the activity of the enzyme

COMT to block the metabolism of DA and related catecholamines. In addition,

EGCG is able to regulate the proteolytic processing of a-synuclein and reassemble the

structure of a-synuclein to convert it into a less toxic form. Beyond this, EGCG can

also promote the non-amyloidogenic a-secretase pathways of APP to soluble APP,

against AD.
 33

Low concentrations of EGCG are responsible for the antiapoptotic/neuroprotective

effects, whereas high doses account for its antiproliferative, antiangiogenic and

pro-apoptotic actions (207). For example, low concentrations of EGCG decreased the

expression of pro-apoptotic genes bax, bad, caspases1 and 6, cyclin-dependent kinase

inhibitor p21, fas-ligand and tumor necrosis factor-related neuronal cells, while high

concentrations induced apoptosis. Most of the effects of EGCG in cell culture systems

have been observed with relatively higher concentrations than those in the plasma or

tissues of animals or in human plasma after administration of green tea or EGCG. The

reason may be that EGCG is subjected to form a dimer, and auto-oxidizes under cell

culture conditions, reducing its half-life. It is not clear whether the activities observed

with high concentrations of EGCG in cell lines can be observed in vivo. However, it is

possible that increased consumption of green tea by humans could result in the

buildup of higher concentrations of EGCG in the plasma.

Bioavailability is an important factor for considering the beneficial effects of

EGCG use in vivo. EGCG has only limited systemic availability. There have been

some recent efforts to enhance its bioavailability by delivering EGCG using lipid

nanocapsules and liposome encapsulation. Indeed, there has been successful in vitro

and in vivo testing of the delivery of EGCG in polylactic acid-polyethylene glycol

nanoparticles to inhibit angiogenesis and induce apoptosis. It is shown that nanolipid

EGCG particles can significantly improve the neuronal -secretase and have more

than two-fold greater oral bioavailability than normal EGCG treatment on AD. Based

on the current findings, additional large, long-term cohort studies and clinical trials

should be performed to investigate the efficacy, pharmacology, safety, and

side-effects of standardized preparations of green tea and EGCG.

 34

1. EGCG Antioxidant Activity

Exogenous antioxidants also play a major role in maintaining cellular redox

balance. These include direct scavenging of HO, singlet oxygen, etc. Tea catechins

are powerful hydrogen-donating antioxidants and free radical scavengers of ROS and

NOS in vitro. The anti-oxidant property of catechins is due to the presence of the

phenolic hydroxyl groups on the B-ring in ungalloylated catechins (EC and EGC) and

in the B- and D-rings of the galloylated catechins (ECG and EGCG) (208, 209). The

presence of the 3, 4, 5-trihydroxy B-ring is important for antioxidant and

radical-scavenging activity.

The neuroprotective effect of green tea polyphenols in vivo may also involve the

regulation of antioxidant protective enzymes through the Nrf2/ARE system. EGCG

was found to elevate the activity of two major oxygen-radical species metabolizing

enzymes, SOD and catalase, in mice striatum (206). In peripheral tissue it has been

shown that EGCG at low concentrations activates the expression of some

stress-response genes, such as phase II detoxification enzyme,

glutathione-s-transferase and HO-1.

2. EGCG & Iron Chelation

The 3,4,5-trihydroxy B-ring of the catechins (shown in Fig. 3) have been shown to

be important for antioxidant and radical scavenging activity. EGCG was shown to be

more efficient as a radical scavenger due to the presence of the trihydroxyl group on

the B- and D-rings and the gallate moiety at the 3 position in the C ring. The green

tea catechins have been shown to be more effective antioxidants than vitamin C and E,

and the radical scavenging capability order is ECG>EGCG>EGC>EC>catechin.

There are two points of attachment of transition metal ions to flavonoid molecules:
 35

the o-diphenolic groups in the 3,4-dihydroxy positions in the B ring, and the keto

structure 4-keto, 3-hydroxy or 4-keto and 5-hydroxy in the C ring of the flavonols.

Therefore, the metal-chelating properties of green tea catechins are also important

contributors to their antioxidative activity. The chelation capability of EGCG has

major significance for the treatment of neurodegenerative diseases.

Desferal/desferrioxamine (DFO) was shown to be neuroprotective against 6-OHDA

and MPTP-induced neurotoxicity in rat and mouse models (212, 213). However, the

hydrophilic nature and large molecular size of DFO limited its absorption across the

gastrointestinal tract and prevents it from penetrating the BBB. EGCG has the

capability to cross the BBB and possess relatively potent metal-chelating properties

due to the gallate moiety present in the C-ring. EGCG and R-apomorphine prevented

iron-dependent -synuclein aggregation in the SN of MPTP-treated mice and restored

the nigro-striatal DA neurons (214). The chelation of iron by EGCG also activates

hypoxia signal transduction pathways, such as HIF-1, and regulates compensatory

factors, including prolyl-4-hydroxylase, chaperones, and the immunoglobulin-heavy

chain-binding protein, HSP90. EGCG can reduce the iron pool and activate IRP

expression (215, 216). The regulation of IRP is coordinated with iron-related proteins,

maintaining cellular iron hoemeostasis, survival, and proliferation, including ferritin,

TfR and Fpn1, etc.

Table 1. Iron chelators and neuroprotection

Iron Chelators PD prevention

Desferrioxamine Higher binding affinity to Fe3+ than Fe2+.

Protects dopamine in rat PD model. Also inhibits iron-promoted radical
damage in dopaminergic cell line in 6-OHDA.
Unable to cross the BBB when orally administrated due to the particle size
and hydrophilic feature. It has adverse effects.
 36

Table 1. Iron chelators and neuroprotection (Continued)

Clioquinol Reduces iron level in substantia nigra of mice.
(5-chloro-7-iodo-8- Pre-treatment prevents mice against MPTP toxicity.
hydroxyquinone) Produces unintended effects due to no iron selective, by reducing the S-
adenosylmethionine level, resulting into vitamin B12 deficiency.
Can be used orally.
HLA20 Protects P19 cells (mouse embryonal carcinoma cell line) against 6-OHDA.
Strong ferric chelator, can also inhibit MAO-B activity.
VK-28 Penetrates mitochondrial membrane.
(5-[4-(2-hydroxyeth Protects dopaminergic neuron against 6-OHDA in rat.
yl)piperazine-1-ylme
thyl]-quinoline-8-o1)
M30 Both iron chelator and MAO-B inhibitor. Similar to HLA20.
[5-(N-methyl-N-pro More effective MAO-B inhibitor than HLA20 and VK-28 in vivo.
pargyaminomethyl)-
8-hydroxyquinoline
Aroylhydrazones Charged hydrazone can cross membrane and remove iron more effectively.
PCIH
(2-pyridylcarboxald
ehyde isonicotinoyl
hydrazone)
H2O2-triggered H2O2 can trigger for the transformation of prochelator SIH-B into the active
prochelator chelator SIH, which in turn will form an inert complex with iron or copper
SIH-B and inhibit the Fenton reaction. Decrease the adverse effect of iron
(2-borobenzalh chelation. Experiment test in vivo is underway.
dehyde isocotinoyl
hydrazone)

Green tea Inhibits cytochrome p450, GST, lipid peroixdation in vitro and in vivo
polyphenols studies. Strong antioxidant at lower concentration, while prooxidant at high
EGCG concentration.
Quercetin Inhibits Fenton reaction in vitro. Strong iron chelator in vivo. Protects
mesencephalic dopamine neurons from apoptosis induced by MPP+.

Curcumin Decreases the iron level in vitro against neurotoxin, even if not as
effectively as DFO. Can protect dopamine level in 6-OHDA injected rat.
 37

3. EGCG & Protein Kinase Signaling Pathways

The neuroprotection of EGCG is shown to be involved in regulating intracellular

signaling pathways, such as protein kinase C (PKC) in the SH-SY5Y cells (207, 217).

To some extent, the PKC inhibitor can diminish the protection of EGCG. Therefore,

interventions to modulate specific PKC activity or PKC mediated signal transduction

pathways may constitute a potential therapeutic tool for neuroprotection. PKC

activation agonist was shown to be essential for neuroprotection against oxygen or

glucose deprivation in vitro. EGCG has been shown to phosphorylate and activate

PKC and protect against several neurotoxins, such as A and 6-OHDA (207). EGCG

can also relocate the isoform PKC to the membrane compartment in PC12 cells. An

animal study showed that two weeks of oral EGCG consumption prevented the

depletion of PKC and against Bax protein in the striatum and SN of mice with

MPTP administration (218). In addition to PKC, other cell signaling pathways are

implicated in the action of EGCG, such as the MAPK, phosphatidylinostitide 3-OH

kinase (PI3K)/Akt, and PKA signaling cascades and cell calcium influx regulation

(219). In general, green tea catechin can activate MAPK in both neuronal and

extraneuronal cultures to increase the extracellular signal-regulated kinase (ERK1/2)

activity, which was suppressed by oxidative stress (220). Epicatechin was shown to

stimulate phosphorylation of cAMP-response element binding protein (CREB) to

regulate neuronal viability and synaptic plasticity activity via ERK1/2 and Akt in

primary cortical neurons (221). Although EGCG had no effect on ERK1/2

phosphorylative levels, it was able to counteract the decline in ERK1/2 induced by

6-OHDA in neuroblastoma cells (207).

 38

4. EGCG & Inflammation

Since neuroinflammation reactions are closely related to PD, the inhibition of

inflammation can be a preventive strategy for neurological disorders. EGCG is shown

to be involved in the regulation of anti-inflammatory activity. The administration of

EGCG reduced expression of IL1, TNF, and TGF in C57BL/6 mice (222). EGCG

can also indirectly regulate the inflammatory effect through down-regulation of

NF-B and up-regulation of PPAR (223). Interestingly, it was found that higher dose

of dietary supplement of EGCG can promote inflammation reaction in C57BL/6 mice,

including TNF-a, IL-6, different from the protection effects of low dose (224).

However, the mechanism by which EGCG regulates anti-inflammatory action is still

unclear. Further studies are needed to clarify the involvement of EGCG in the

inflammatory pathways.

In summary, EGCG may prevent against PD progression by its antioxidant,

anti-carcinogenic, and anti-inflammatory properties, and by regulating iron

homeostasis. Further studies are required to clarify the mechanism in vivo. In addition,

the treatment methods, concentration, and structure modification, which are all related

to EGCG bioavailability, need to be investigated in animal models. Since limited data

is available to show the protection of EGG in humans, newly designed human clinical

trials are required to directly demonstrate the preventive effects of EGCG.

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CHAPTER 3. EGCG PROTECTS AGAINST 6-OHDA INDUCED

NEUROTOXICITY IN A CELL CULTURE MODEL

To be submitted to Journal of Nutrition

Dan Chen1, Anumantha G. Kanthasamy2, Manju B. Reddy1

1
Department of Food Science and Human Nutrition, 2Department of Biomedical

Sciences, Iowa State University, Ames, IA 50010

Abstract: Parkinsons disease (PD) is a progressive neurodegenerative disease that

causes severe DA depletion in the striatum of the brain and induces clinical signs such

as tremor, rigidity, akinesia, immobility and frequent falls. Disruption of iron

metabolism may be involved in the progression of PD. Our objective was to test

whether the protective effect of (-)-epigallocatechin-3-gallate (EGCG) against

6-hydroxydopamine (6-OHDA) induced neurotoxicity was by regulating iron

homeostasis in N27 cells. The protection of EGCG was assessed by SYTOX Green

assay, MTT and caspase-3 activity. Pretreatment of EGCG with 6-OHDA

significantly increased (p<0.0001) TH+ cell count (~ 3-fold) and neurite length (~

12-fold) compared to 6-OHDA alone in primary mesencephalic neurons. 6-OHDA

treatment significantly increased divalent metal transporter-1 (DMT1), hepcidin, and

decreased ferroportin-1 (Fpn1) expression, while pretreatment of EGCG significantly

55
altered these adverse effects of 6-OHDA. The increased ~2-fold (p<0.001) Fe cell

uptake by 6-OHDA confirmed iron burden associated with neurotoxicity.

Pretreatment of EGCG protected against the neurotoxicity by reducing the iron uptake

and retention compared to 6-OHDA. In conclusion, pretreatment of EGCG (not

co-treatment) protected against 6-OHDA induced neurotoxicity by regulating genes

 60

and proteins involved in brain iron homeostasis. However, further studies are

warranted to determine the mechanism of that regulation.

Key words: EGCG, Iron, 6-OHDA, Parkinsons disease

Introduction

Iron plays a crucial role in many physiological functions (1) including DNA

synthesis, oxygen transport, and mitochondrial respiration (2, 3). Being a cofactor of

tyrosine hydroxylase, iron is also involved in brain functions, such as

neurotransmission and myelination (4, 5). It is important to regulate intracellular iron

uptake, transport and storage. Iron homeostasis can be interrupted by dysfunction of

iron related proteins, leading to the increased iron accumulation. The accumulation of

unbound free iron in the cell further induces oxidative damage on cellular organelles

by producing free radicals via the Fenton reaction (6), resulting in chronic

neurological disorder diseases, including PD (7, 8).

Iron homeostasis and metabolism are regulated by iron-related proteins. The

divalent metal transporter 1 (DMT1) is one of the proteins responsible for intracellular

iron uptake. DMT1 can be upregulated by neurotoxin, such as 6-hydroxydopamine

(6-OHDA), resulting in intracellular iron accumulation, free radical formation and

neurodegeneration (9-11). However, it is still unclear whether iron influx and

subsequent free radical formation is a primary or secondary event in the

neurodegenerative process. Hepcidin is a key iron regulatory protein that is

responsible for normal iron homeostasis. Hepcidin is expressed in several organs,

such as the liver, brain, spinal cord and is closely related to intracellular iron

concentration. Its expression is also influenced by oxidative stress (12). Ferroportin 1

 61

(Fpn1) is the iron exporter which exports intracellular iron. Hepcidin can bind to Fpn1,

causing its internalization and degradation, promoting iron retention in the cell (13).

It has been confirmed that 6-OHDA can induce oxidative stress and cause severe

neuron cell death. 6-OHDA can release iron from ferritin (14, 15), induce depletion of

PKC kinases, stimulate inflammatory factors and cause cell membrane potential

hyperpolarization, etc. However, the detailed mechanisms remain unclear. Since

6-OHDA is shown to alter DMT1 expression and oxidative stress (9), there is a

possibility that 6-OHDA may exert its neurotoxicity by interrupting iron homeostasis

and subsequent oxidative damage.

Green tea polyphenol has been shown to provide beneficial effects against cancer,

inflammation, neurological disorders, including PD. (-)-Epigallocatechin-3-Gallate

(EGCG) is the most abundant polyphenol in green tea, and due to its capability of

scavenging free radicals and chelating iron, it has been shown to protect against

neurotoxins in several cell culture models of PD (16, 17). EGCG is reported to have

antioxidant activity to reduce oxidative stress. It also can affect protein kinase and cell

apoptosis. Several in vitro studies have shown EGCG reduces 6-OHDA neurotoxicity,

but the mechanism of the protection is not clear. We hypothesized that 6-OHDA and

EGCG administration would affect the regulation of iron via iron-related proteins. We

used immortalized rat mesencephalic dopaminergic neuronal cell line (N27 cells) to

determine whether 6-OHDA disrupted iron metabolism by regulating iron transporters,

such as DMT1, Fpn1 and hepcidin, etc. We also determined whether EGCG protected

against the adverse effects of 6-OHDA by normalizing and maintaining iron

homeostasis.
 62

Materials and Methods

Reagents. EGCG, 6-OHDA, and thiazolyl blue tetrazolium bromide (MTT) were

purchased from SigmaAldrich (St. Louis, MO). SYTOX Green Nucleic Acid Stain

was from Molecular Probes (Eugene, OR). Substrate for caspase-3,

Acetyl-Asp-Glu-Val-Asp-AFC (Ac-DEVD-AFC), was obtained from MP

Biomedicals (Solon, OH). The mouse TH+ antibody was obtained from Millipore

(Temecula, CA); Alexa 680-conjugated anti-mouse secondary antibodies, RPMI-1640

medium, fetal bovine serum, l-glutamine, penicillin, streptomycin and neurobasal

medium, B27 supplement, and Dulbeccos modified Eagles medium (DMEM) were

obtained from Invitrogen (Carlsbad, CA). Rabbit anti-rat-DMT1 polyclonal antibody,

rabbit anti-rat-MTP1 polyclonal antibody and rabbit anti-rat-hepcidin polyclonal

antibody were purchased from Alpha Diagnostic International (San Antonio, TX),

-actin mouse-anti-mouse-monoclonal antibody was from were from Sigma

Chemicals (St. Louis, MO). Secondary antibody Goat-anti-rabbit-IgG-HRP and

Goat-anti-mouse-IgG-HRP were obtained from Santa Cruz Biotechnology (Dallas,

TX). Supersignal west femoto chemiluminescent substrate for western blotting was
55
from Thermo Scientific (Rockford, IL). Fe was purchased from Perkinelmer

(Waltham, MA).

Cell culture. N27 cells were grown in RPMI-1640 medium containing 10% fetal

bovine serum, 2 mmol/L l-glutamine, 50 U penicillin and 50 g/mL streptomycin and

maintained at 37C in a humidified atmosphere containing 5% CO2 as described in

previous studies (18). We also used primary mesencephalic neuronal cultures to

determine the neuroprotective effect of EGCG. Primary mesencephalic neuronal

cultures were prepared from the ventral mesencephalon of gestational 14-day-old C57

black mice embryos as described previously (19). Mesencephalic tissues were

 63

dissected and kept in ice-cold Ca2+-free Hanks balanced salt solution. Cells then were

dissociated in Hans balanced salt solution containing trypsin, 0.25% EDTA for 30

min at 37C. Cells were suspended in neurobasal medium with 2% neurobasal

supplement (B27) after 10% FBS inactivating enzyme activity in DMEM medium.

The cells were then plated at density of 0.5 106 cells on 12-mm coverslips precoated

with 1 mg/ml poly-D-lysine. Cell cultures were maintained in 500 M l-glutamine,

100 IU/ ml penicillin, and 100 units streptomycin neurobasal medium, incubated in a

humidified CO2 incubator (5% CO2 and 37C). Medium was replaced half every 2

days and assays were conducted using cultures at 7 days old. Primary mesencephalic

dopaminergic neuronal cells were exposed to 6-OHDA (25 M) for 24 h in the

presence or absence of 2 h pretreatment of EGCG (1, 10 and 100 M). Cells were

then fixed for immunocytochemical analysis.

Sytox green assay. Equal numbers of N27 cells grown in 24-well plates with 500

l RPMI medium co-incubated with 1:1000 Sytox green dye. To determine the effects

of 6-OHDA and EGCG and protective effect of dosage from EGCG, N27 cells were

pretreated with EGCG at concentrations of 1, 10, 50 and 100 M for 2 h, and then

followed by 6-OHDA (100 M) treatment for 6 h. The assessment of cell death was

conducted using Sytox green nucleic stain as described previously (19). After each

treatment, Sytox green signal was detected using a fluorescence microplate reader

(Bio-Tek microplate reader) at an excitation wavelength of 485 nm and an emission

wavelength of 538 nm. The intensity of fluorescence was directly proportional to the

number of dead cells. To quantify cell death, fluorescence intensity was monitored

after the experiments were conducted and fluorescence pictures were taken using a

Nikon inverted fluorescence microscope equipped with a SPOT digital camera

(Diagnostic Instruments, Sterling Heights, MI).

 64

Cell viability. The N27 cells were pretreated and cotreated with EGCG (100 M),

followed by 6-OHDA treatment for 6 h, the cell viability was measured by MTT

assay as described previously (20). Briefly, after treatments, cells were washed with

PBS and then incubated with serum free RPMI medium containing 0.25 mg/mL MTT

reagent for 3 h at 37C. IsopropanolHCl (300 l) solution was added to dissolve

intracellular purple formazan and absorbance was read at 570 nm with a reference

wavelength of 630 nm using a microplate reader (Molecular Devices, Sunnyvale,

CA).

Caspase-3 activity. Caspase-3 activity was measured to assess cell apoptosis as

previously described (21). Briefly, N27 cells was pretreated with EGCG for 2 h,

followed by 6-OHDA treatment for 6 h, then the cells were resuspended in lysis

buffer (50 mM Tris-HCl, 1 mM EDTA, and 10 mM EGTA) containing 10 mM

digitonin for 20 min at 37C. Supernatants were treated with the fluorogenic

substrates Ac-DEVD-AFC for 1 h at 37C and fluorescence was measured at

excitation at 400 nm and emission at 505 nm using a Gemini XS fluorescence plate

reader (Molecular Devices Inc). The activity was measured as fluorescent units

(FU)/mg protein.

Immunocytochemistry. After treatments, the primary cultures were processed for

immunocytochemical analysis. Briefly, the cells were fixed with 4%

paraformaldehyde and permeabilized, and nonspecific sites were blocked with 5%

normal goat serum containing 0.4% BSA and 0.2% Triton-X 100 in PBS for 20 min.

Cells were then incubated with antibodies directed against TH (1:500) overnight at

4C followed by incubation with Cy3-conjugated (1:1000) secondary antibody for 1 h

at room temperature. Secondary antibody treatments were followed by incubation

with 10 g/ml Hoechst 33342 for 3 min at room temperature to stain the nucleus.
 65

Then the coverslips containing cells were washed with PBS, mounted on a slide, and

viewed under a Nikon inverted fluorescence microscope (Model TE-2000U) and

images were captured with a SPOT digital camera (Diagnostic Instruments, Sterling

Heights, MI). TH+ cell count and neurite processes were measured as described (18).

Briefly, the total number of TH+ cells averaged 14/coverslip in untreated

mesencephalic cultures. For measurement of TH+ cell count, Metamorph Image

analysis software was used. The images were first thresholded, and then neuronal

count and volume were measured using the Integrated Morphometry Analysis (IMA)

function. For measurement of neuronal processes, the lengths of the processes were

marked by applying the region and length measurement function in the IMA.

Quantitative real-time PCR analyses. The mRNA expression levels of DMT1,

Fpn1, hepcidin, transferring receptor (TfR) and H-ferritin were quantified using

real-time PCR as described previously (22, 23). Briefly, N27 cells were pretreated

with EGCG and followed by 6-OHDA for 24 h. Total RNA was isolated from the

cells using absolutely RNA miniprep kit (Stratagene, Santa Clara, CA) and reverse

transcripted by High Capacity cDNA Reverse Transcription Kits (Applied Biosystems,

Foster City, CA). Quantitative real-time RT-PCR was performed using Brilliant

SYBR Green QPCR Master Mix kit and the Mx3000P QPCR system (Stratagene,

Santa Clara, CA). The 25 l PCR reaction mixture included 1 l of cDNA (produced

by 100 ng of RNA), 12.5 l of 2 master mix, and 100 nM each primer (listed in

Table 1). Cycling conditions contained an initial denaturation at 95C for 10 min,

followed by 40 cycles of amplification 95C for 30 s for denaturation, 60C for 30 s

for annealing, and 72C for 30 s for extension. Fluorescence was detected during the

annealing/extension step of each cycle. Dissociation curves were used to verify the

specificity of the PCR products. GAPDH gene was used as internal control. A
 66

comparative threshold cycle method was used to analyze the data. All reactions were

performed in triplicate.

Western blot analyses. DMT1, hepcidin and Fpn1 expression were evaluated by

Western blot assay. N27 cells were pretreated with EGCG and followed by 6-OHDA

for 24 h. Then the cells were washed with cold PBS and homogenized in RIPA buffer

containing 1% Triton X-100, 0.1% SDS, 1 mM phenylmethanesulfonyl fluoride

(PMSF) and protease inhibitors (pepstatin 1 mg/ml, aprotinin 1 mg/ml, leupeptin 1

mg/ml), and then sonicated on ice. After a centrifugation at 13,000 rpm for 15 min at

4C, the supernatant was collected. Aliquots of supernatants were assayed for protein

concentrations. Forty g of protein extract was mixed with an equal volume of 5

sample buffer (0.35 M TrisHCl, 10% SDS, 30% glycerol, 0.012% bromophenol

blue), and loaded onto a 12 % or 15 % SDS-polyacrylamide gel or Urine-gel,

electrophoresed, and then transferred to a nitrocellulose membrane (Bio-Rad,

Hercules, CA). The membrane was blocked with 5% nonfat dry milk in TBST and

then incubated with rabbit anti-rat DMT1 with IRE polyclonal antibody (1:200),

rabbit anti-rat Fpn1 polyclonal antibody (1:500) or rabbit anti-rat hepcidin polyclonal

antibody (1:500), followed by horseradish peroxidase-conjugated goat anti-rat IgG

antibodies (1:3000). To ensure even loading of the protein samples, -actin (1:2000)

was used as internal control, with the incubation of the corresponding

goat-anti-mouse-IgG-HRP secondary antibody (1:3000). All western blot assays were

performed triplicately in two separate experiments.

Cellular iron uptake. N27 cells were grown in 12-well plates. After 2 h

pretreatment with EGCG, N27 cells were exposed to 6-OHDA, and ferric chloride
55
(0.5 Ci/ml Fe) was also added to the media and incubated at 37C for 24 h. Cell

were then washed in cold 1 PBS twice and lysed in 250 l of lysis buffer (RIPA
 67

buffer). After centrifuging at 13,000 rpm for 10 min at 4C, 50 l of supernatant was

used to measure the 55Fe radioactivity in a scintillation counter mixed with the 10 ml

cocktail scintillation fluid. The 55Fe uptake was calculated by dividing the number of

counts per minute (CPM) and the values were normalized to cell protein concentration.

The experiments were performed in duplicate.

Statistics analyses. Data were analyzed with Prism 4.0 software (Graph Software,

San Diego, CA). All values are expressed as mean SEM and represented as

percentage of the respective controls. ANOVA with Tukeys multiple comparison test

was used to detect the differences among treatments. Student t-test was used to

compare the difference between 6-OHDA with EGCG pretreatment and 6-OHDA

alone groups in iron related genes expression. The mean differences were considered

significant at p<0.05.

Results

Effects of 6-OHDA and EGCG on cell death

The Sytox green assay showed the dose response of EGCG protected against

6-OHDA induced cell death (Fig. 1). 6-OHDA significantly increased cell death by

196% (p<0.0001) compared to control. Pretreatment of EGCG at a concentration of

50 and 100 M for 2 h decreased the cell death by 31 % (p<0.0001) and 55%

(p<0.0001) compared to 6-OHDA alone group. There was no significant decrease in

EGCG pretreatment at the concentration of 1 and 10 M.

EGCG protected against 6-OHDA induced decreased cell viability

As showed in Fig 2., cell viability was decreased to 59% (p<0.001) after 6 h

treatment with 6-OHDA, but EGCG pretreatment for 2 h partially protected against

this toxicity significantly by 21% (p<0.01). In contrast, only 10% (p>0.05) increase in
 68

cell viability in EGCG co-treatment group, suggesting pretreatment of EGCG shows

the protective effect.

EGCG protected against 6-OHDA induced cell apoptosis

Caspase-3 activity was used to determine the cell apoptosis (Fig. 4). Compared to

control, 6-OHDA significantly increased caspase-3 activity by ~ 12-fold (p<0.0001),

while pretreatment of EGCG for 2 h decreased the caspase-3 activity by 49%

(p<0.0001) compared to 6-OHDA. There was no change in caspase-3 activity in

EGCG treatment alone group, suggesting EGCG pretreatment protected against cell

apoptosis.

EGCG decreased TH+ neuronal loss from 6-OHDA in primary mesencephalic

cultures

Treatment with 6-OHDA for 24 h induced ~ 80% (p<0.0001) loss of TH+ cell count

(Fig. 5A), compared to control. Pretreatment of EGCG (1, 10, 100 M) for 2 h

significantly increased TH+ cell count by 155% (p<0.01), 205% (p<0.0001) and 290%

(p<0.0001), respectively (Fig. 5B). The average lengths of TH+ neuronal processes in

EGCG pretreated cells were significantly longer than the processes of neurons with

only 6-OHDA treated. We also found that 6-OHDA significantly decreased the TH+

neurite length by 93% (p<0.0001), while pretreated with EGCG (1, 10, 100 M) for 2

h significantly increased the neurite length by ~ 10-fold (p<0.0001), 12-fold

(p<0.0001), and 12-fold (p<0.0001), respectively (Fig. 5C). In addition, 6-OHDA

significantly decreased Hoechst activity, while pretreatment of EGCG (1, 10, or 100

M) increased Hoechst staining (Data not shown), suggesting the increased cell

availability. Overall, EGCG protected against 6-OHDA toxicity in primary cultures.

EGCG altered the 6-OHDA regulation on the mRNA expression of iron-related genes
 69

As shown in the Table 2, 6-OHDA significantly increased the mRNA expressions

of DMT1+IRE, hepcidin, TfR2, H-ferritin and decreased Fpn1 and TfR1. However,

EGCG altered the effects by decreasing DMT1+IRE by 60% (p<0.001), hepcidin by

54%, (p<0.05), H-ferritin by 53%, (p<0.05), TfR2 by 27%, (p<0.05), while increasing

Fpn1 by 70%, (p<0.01) and TfR1 by 96%, (p<0.01), compared to 6-OHDA, may

indicate the reduced iron burden in the cell.

EGCG altered the 6-OHDA regulation on the iron-related protein expressions

The regulation on iron related proteins DMT1+IRE, hepcidin and Fpn1 was

assessed by western blot. We found that 6-OHDA significantly increased DMT1+IRE

expression by 201% (p<0.05), hepcidin by 177% (p<0.05) and decreased Fpn1 by

61% (p<0.0001) compared to the control. Pretreatment of EGCG altered these effects

by decreasing DMT1+IRE by 66% (p<0.01), hepcidin by 43% (p<0.05) and

increasing Fpn1 by 82% (p<0.01) compared to 6-OHDA alone.

EGCG reduced iron burden induced by 6-OHDA

As shown in Fig. 7, 6-OHDA significantly increased 55Fe uptake to 196% (p<0.01).

55
Pretreatment of EGCG significantly decreased Fe in the cell by 27% (p<0.05)

compared to 6-OHDA, supporting the results of and mRNA and protein expression.

Discussion

Neurotoxins, such as 6-OHDA, have been widely used to establish PD model both

in vitro and in vivo studies (9, 24, 25). Due to the abilities to induce oxidative stress,

mitochondrial dysfunction and iron dyshomeostasis, 6-OHDA has been reported to

cause significant neuronal cell death, including SH-SY5Y, PC12. High dose of

6-OHDA treatment causes severe insult to cells and is effectively used for short-term

and acute pathological research. It was shown that the treatment of 6-OHDA at 100
 70

M for 6 h caused a complete loss of cell viabilities (26). In our study, we also found

that 6-OHDA at the same condition significantly increased dopaminergic N27 cell

death approximately 3-fold, cell apoptosis ~ 12-fold and decreased cell viability by

~40%. Relative lower concentration of 6-OHDA induced a smaller, but significant

decrease in cell viability and increase in apoptosis, which benefits the clarification of

mechanism of toxin induced neurodegeneration (27). In our study, we used a lower

dose of 6-OHDA (25 M) to determine the regulation on iron-related mRNA and

protein expression, as well as the intracellular iron uptake. Although higher amounts

of 6-OHDA (200 M) has been reported to induced rat primary nigral culture death

(28), lower concentrations (<50 M) are used by most primary neuron studies due to

sensitivity of neuron (29, 30). In our study, we used 10 M 6-OHDA to show the

significant TH+ cell loss after 24 h. Overall, the dose of 6-OHDA used in the study

depends on the cell types and the study aims.

Although the mechanisms of 6-OHDA induced neurotoxicity remain unclear,

several pathways have already been proposed, such as mitochondrial dysfunction,

inflammation, etc. It has been reported that 6-OHDA is able to release iron from

ferritin and alter DMT1 expression, suggesting that 6-OHDA might exert its toxicity

by interrupting iron homeostasis (31, 32). We found the disruption of iron metabolism

was closely related to the alteration of iron transporters and iron regulatory proteins.

In our study, we showed 6-OHDA up-regulated DMT1+IRE, hepcidin and decreased

Fpn1 expression, which might result in an excess iron condition. The alteration on

other iron-related factors, such as ferritin, TfR1, TfR2 by 6-OHDA also indicated an
55
intracellular iron excess status. Our Fe uptake data further confirmed 6-OHDA

caused high intracellular iron (~ 2-fold, p<0.01).

 71

It was shown that the up-regulation of DMT1 by 6-OHDA occurred in an

IRE/IRP-dependent manner (33, 34), indicating 6-OHDA may affect the activity of

IRPs, altering the translation and stabilization of DMT1 mRNA. In our study,

6-OHDA did not alter the expression of DMT1 without IRE (data not shown), also

suggesting the regulation on DMT1 via IRPs. The increase of DMT1 caused iron

influx, leading to oxidative stress. However, further studies are required to clarify

whether there is causal-effect relationship between DMT1 and oxidative stress.

Hepcidin is mainly involved in regulating iron homeostasis and can be regulated by

iron influx and iron overloading (35, 36), oxidative stress and inflammation. In our

study, we showed 6-OHDA significantly increased hepcidin expression in N27 cells.

The regulation on hepcidin may depend on the excess intracellular iron, which was
55
supported by the increased ferritin and Fe uptake. It was shown that TfR2 was

positively associated with hepcidin expression and may be another hepdicin regulator

(37, 38). Our data confirmed that increased TfR2 expression was accompanied by

increased hepcidin level. Since hepcidin can bind to Fpn1 and induce its

internalization and degradation, the increased hepcidin suppressed Fpn1 expression,

resulting in iron retention. 6-OHDA can cause not only the decreased Fpn1 shown in

our study, but also in primary mesencephalic neuron, MES23.5 cell (39, 40). The iron

overloading can also decrease Fpn1 in PC12 (41). Other studies showed toxin can

increase Fpn1 in primary cultured astrocytes, SH-SY5Y (40, 42). The difference of

Fpn1 regulation may imply the significance of different cell type in neuronal iron

homeostasis.

Since the alteration of iron level in the brain is linked to several neurological

disorders, maintaining normal iron homeostasis seems to be an ideal strategy for

neuroprotection (43). As iron chelator and antioxidant, EGCG has been reported to
 72

protect against neurotoxicity in numerous studies (44-46). In this study, we found that

EGCG protected N27 cell against 6-OHDA induced cell death. The EGCG

pretreatment method was considered more effective than co-treatment. We also found

that EGCG (10-100 M) exerted protection both in N27 cell and primary cultures and

the concentration at 100 M showed the best protective effects, whereas higher dose

(> 200 M) was found to be toxic (supplement data). This finding matched the

bell-shaped pattern of EGCG as a typical of antioxidants, showing neuroprotection at

low concentrations, and being pro-oxidant at high concentration (16, 17). We also

confirmed that EGCG (1 M -100 M) protected TH+ cells against 6-OHDA toxicity

in primary cultures. Different dosages of EGCG were also reported in other primary

cultures studies. It showed lower dose (<10 M) of EGCG protected neurons against

6-OHDA (47, 48), while other studies reported higher (25 to 400 M) EGCG

protected SH-SY5Y cell apoptosis induced by 6-OHDA (24).

Several proposed mechanisms may attribute to the neuroprotection of EGCG. First,

it may regulate the iron-related proteins, maintain normal iron homeostasis (49). In

this study, we showed the pretreatment of EGCG counteracted the adverse effect on

iron-related proteins by 6-OHDA, leading to less intracellular iron accumulation. The

55
decreased iron accumulation by EGCG pretreatment was supported by our Fe

uptake data, showing 27% (p<0.05) decrease in iron burden against 6-OHDA. Second,

EGCG may chelate the iron and the complex form of iron cannot be used by the cell,

reducing the iron induced ROS generation. We have shown the decreased 55Fe uptake

and decreased ROS in another study (data not shown). Third, EGCG may also

enhance the antioxidant system by up-regulating antioxidant enzyme (16, 50), and

scavenging the free radicals directly. The decreased oxidative stress may also affect

the iron homeostasis directly or indirectly. In this way, EGCG causes less iron and
 73

maintains the iron homeostasis. In addition, since the inflammation is related to

antioxidation and EGCG was shown to reduce inflammatory activity, it may indirectly

affect antioxidation and iron related protein. Further studies are needed to investigate

how EGCG regulates iron metabolism in neural cells.

In summary, 6-OHDA induced neurodegeneration may be via affecting iron

metabolism. The increased DMT1 expression significantly caused iron influx into cell

body, while decreased Fpn1 expression might have exacerbated iron accumulation.

The increased hepcidin, ferritin and 55Fe suggest the increased free iron concentration,

resulting into oxidative stress. EGCG exerted the protective effects against 6-OHDA

by decreasing DMT1, hepcidin, increasing Fpn1 and reducing cell iron uptake to

maintain normal iron level. EGCG may protect against neurotoxicity by modulating

iron homeostasis. Further studies are needed to elucidate the mechanisms of

protection by EGCG in vivo.

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 78

Figures and Tables

*
400 *
b b
b
300
(% of control)
Cell death

c
200
a
a
100

EGCG (M) - 0 1 10 50 100

6OHDA (M) - + + + + +

FIGURE 1 Effects of 6-OHDA and EGCG on cell death. The value are expressed as
mean SEM, n=8. Labeled means without a common letter differ, * P<0.001.
6-OHDA, 100 M.
 79

*
100
b
80 a

(% of control)
Cell Viability
a
60

40

20

0
A

A
A
D

D
D
H

H
H

O
O

O
6-

6-
6-

+
t+

t
-tr
tr
e-

Co
Pr

FIGURE 2 Effects of 6-OHDA and EGCG on cell viability. Cell viability was
evaluated by MTT assay and the values are expressed as mean SEM, n=6. Data are
represented as % of control (no treatment). Labeled means without a common letter
differ. 6-OHDA, 100 M for 6 h; EGCG 100 M. Pre-trt, EGCG pretreatment 2 h,
Co-trt, EGCG cotreatment with 6-OHDA. *P<0.01. **P<0.001
 80

*
1500
b

Caspase 3 activity
(% of control)
1000

c
500

a a
0

A
A

CG
l
ro

D
D
nt

EG
H
H
Co

O
O

6-
6-

+
CG
EG

FIGURE 3 Effects of 6-OHDA and EGCG on cell apoptosis. Cell apoptosis was
measured as caspase-3 activity. The values are expressed as mean SEM, n=3.
Labeled means without a common letter differ. 6-OHDA, 100 M for 6 h; EGCG 100
M. *P<0.001.
 81

150 150
** **
a a *
(% of control)

Neurite length
(% of control)
a a
TH+ nueron

100 * a 100
c
c c
50 50
b
b
0 0
l

A
A

A
l

A
A
ro

ro
D

D
D

D
D
D
nt

nt
H

H
H

H
H
H
Co

Co
O

O
O

O
O
O

6-

6-
6-

6-

6-

6-
6-
6-

+
+

+
+
E1

00
0

E1

00
0
E1

E1
E1

E1
B C

FIGURE 4 EGCG prevented TH+ neuronal loss by 6-OHDA. Immunochemistry

analysis was processed and measured with TH+ cell count (Green color) and neurite
length. Hoechst nuclear staining (Blue color) was used to identify the live cells (A).
Quantification data on TH+ number (B) and neurite length (C). The values are
expressed as mean SEM, n=3. Labeled means without a common letter differ.
6-OHDA, 10 M for 24 h; EGCG 1, 10, 100 M pretreatment 2 h. *P<0.01, *P<0.001.
 82

DMT1 Hepcidin Fpn1

Actin

A B C

**
**
250 250 * *
b ** 150 a
b

Hepcidin band density

a
DMT1 band density

200 200

(% of control)
(% of control)

Fpn1 band density

a

(% of control)
150 150 100
a a a
a b
100 a 100
a 50
50 50

0 0
0

CG
A
l

A
A

CG
A
l

ro
ro

A
A

CG
l
D

D
D

ro
nt
nt

D
D
EG
EG

H
H

nt
Co
Co

EG
H
H
O

O
O
O

Co

6O
O
6-

6-
6-
6-

6-
+
+

+
CG
CG

CG
EG
EG

EG
D E F

FIGURE 5 Effect of 6-OHDA and EGCG on iron-related proteins expression.

Western blot analysis on (A) DMT1 (+IRE), (B) Hepcidin, (C) Fpn1 proteins
expression which were normalized by -actin in N27 cells. Quantificative data are
shown in the bottom panels D, E, F, respectively. The values are expressed as mean
SEM, n=3. Labeled means without a common letter differ. 6-OHDA, 25 M for 24 h;
EGCG 100 M pretreatment 2 h. *P<0.01, *P<0.001.
 83

*
**
250
b
200 a

(% of control)
Fe uptake
150 a
a
100
55

50

CG
A

A
l
ro

D
nt

EG
H

H
Co

6O
O
6-

+
CG
EG

FIGURE 6 Iron uptake were the amount of 55Fe taken by the cell, which was
calculated by count per minute (CPM) normalized with protein concentration. The
values are expressed as mean SEM, n=3. Labeled means without a common letter
differ. 6-OHDA, 25 M for 24 h; EGCG 100 M. *P<0.05, **P<0.01.
 84

TABLE 1 Summary of the primers for iron-related gene expression

Hep Forward GAA GGC AAG ATG GCA CTA AGC A

Hep Reverse TCT CGT CTG TTG CCG GAG ATA G

Fp1 Forward CCA CCT GTG CCT CCC AGA T

Fp1 Reverse CCC ATG CCA GCC AAA AAT AC

DMT1+IRE Forward CAG TGC TCT GTA CGT AAC CTG TAA GC

DMT1+IRE Reverse CGC AGA AGA ACG AGG ACC AA

H-fer Forward GCC CTG AAG AAC TTT GCC AAA T

H-fer Reverse TGC AGG AAG ATT CGT CCA CCT

TfR1 Forward CTA GTA TCT TGA GGT GGG AGG AAG AG

TfR1 Reverse GAG AAT CCC AGT GAG GGT CAG A

TFR2 Forward AGC TGG GAC GGA GGT GAC TT

TFR2 Reverse TCC AGG CTC ACA TAC ACG ACA G

GAPDH Forward CCT GGA GAA ACC TGC CAA GTA T

GAPDH Reverse AGC CCA GGA TGC CCT TTA GT

All the primers were synthesized from Integrated DNA Technologies.

 85

TABLE 2 Effect of 6-OHDA and EGCG on the iron-related genes expression

Mean

6-OHDA EGCG+6-OHDA *

DMT1+IRE 2.30.2* 0.90.0ns <0.001

Hepcidin 1.70.1* 0.80.2ns <0.01

Fpn1 0.70.0* 1.20.2ns <0.05

H-ferritin 2.40.4* 1.10.2ns <0.05

TfR1 0.70.0* 1.30.1ns <0.01

TfR2 1.50.1* 1.10.1ns <0.05

Data were presented as mean SEM, n=3-6. Values represent the fold changes of the
ratio of mRNAs/GAPDH in treatments compared with control. Symbol * indicated
difference between 6-OHDA group and EGCG pretreatment group, and was
determined using student t-test. * p<0.05; ns, no significance.
 86

CHAPTER 4. THE PROTECTION OF EGCG AGAINST 6-OHDA-INDUCED

OXIDATIVE STRESS AND INFLAMMATION

To be submitted to Journal of Nutrition

Dan Chen1, Anumantha G. Kanthasamy2, Manju B. Reddy1

1
Department of Food Science and Human Nutrition, 2Department of Biomedical

Sciences, Iowa State University, Ames, IA 50010

Abstract

6-hydroxydopamine (6-OHDA) is classic neurotoxin being used for Parkinsons

disease research. 6-OHDA can increase intracellular reactive oxygen species (ROS)

and cause cell damage, which can be attenuated by (-)-Epigallocatechin-3-gallate

(EGCG) treatment. However, the mechanism by which EGCG protects against

6-OHDA toxicity remains unclear. In this study, we used CM-H2DCFDA probe to

determine the ROS level in N27 cells. A significant increase in ROS generation after

6-OHDA (25 M) treatment was found, while EGCG (100 M) attenuated the ROS

generation. We evaluated the oxidative damage by determining thobartituric acid

reactive substances (TBARS) and protein carbonyl content. 6-OHDA significantly

increased TBARS by 82.7% (p<0.05) and protein carbonyl content by 47.8 (p<0.05),

compared to the control. Pretreatment of EGCG decreased lipid peroxidation and

protein carbonyls by 36.4% (p<0.001) and 27.7% (p<0.05), respectively, compared to

6-OHDA. We also used western blot to test the E2-related factor 2 (Nrf2), heme

oxygenase-1(HO-1) and peroxisome-proliferator activator receptor (PPAR)

expression to evaluate the antioxidant and antiinflammation. 6-OHDA increased Nrf2

expression by 69.6% (p<0.001), HO-1 by 173.3% (p<0.001) and PPAR by 122.7%

 87

(p<0.001), compared to the control. Pretreatment of EGCG significantly normalized

these alterations induced by 6-OHDA. In conclusion, our results suggested that the

neurotoxicity of 6-OHDA in N27 cells was associated with ROS and inflammation

pathways, whereas pretreatment of EGCG suppressed the ROS generation, resulting

into the inactivation of Nrf2/HO-1 and PPAR pathway.

Introduction

6-hydroxydopamine (6-OHDA) can induce neurological damage, which is similar

to Parkinsons disease (PD). One of the main features of the neurotoxicity of

6-OHDA is involving in reactive oxygen species (ROS) generation. An increase in

intracellular ROS production would disrupt redox homeostasis, cause oxidative stress

and result into the irreversible oxidative modification on DNA, lipids and proteins (1).

The presence of excess intracellular Fe (II) and Fe (III) may promote the generation of

hydroperoxyl radical (HO) through the Fenton reaction. Our previous in vitro study

showed 6-OHDA induced the intracellular iron burden in immortalized rat

mesencephalic dopaminergic neuronal cell line (N27) cells. Therefore, the excess iron

may be involved in ROS generation and PD progression.

The transcription factor nuclear factor E2-related factor 2 (Nrf2) and antioxidant

response element (ARE) (Nrf2/ARE system) in cells can scavenge ROS and maintain

the redox homeostasis. Nrf2 is the main factor to activate antioxidant enzymes, phase

II detoxification enzymes (2, 3), such as heme oxygenase-1(HO-1). The activation of

detoxification enzymes directly scavenge free radicals and decrease the toxicity (4).

There are several ways to trigger Nrf2/ARE system to increase antioxidant enzyme

activity. For example, oxidative stress and electrophiles are the major activators on

Nrf2. 6-OHDA has been shown to induce Nrf2 expression due to the induced
 88

oxidative stress. In addition, the activation of Nrf2 can also be positively managed by

itself (5).

Inflammation has a major impact on toxin-induced pathogenesis for PD (6, 7). The

increasing evidence showed the positive correlationship between dopaminergic

neurodegeneration and neuroinflammation (8). It has been shown that microglia

inflammation was activated in 6-OHDA treated rats, indicating the critical role of

inflammation in PD progression (9). The intracellular anti-inflammatory system, such

as the peroxisome-proliferator activator receptors (PPAR), is responsible for

suppressing inflammation reaction and maintaining normal redox status. One of the

isoforms, PPAR has the anti-inflammatory abilities and its agonists can used to treat

inflammation-related diseases, including PD (10). In addition, PPAR can also

interact with Nrf2/ARE antioxidant system (11-16). It has been reported that PPAR

is involved in regulating HO-1 expression (17, 18). The relationship between Nrf2

and PPAR suggests that oxidative stress and inflammation are interacted. Nrf2 can

regulate PPAR expression under oxidative stress, thereby affecting

anti-inflammatory activity (14, 19). Therefore, antioxidation and anti-inflammatory

pathways might be simultaneously involved in neurodegeneration. However, detailed

mechanisms of the interaction between Nrf2 and PPAR are not clear at this time.

Since iron burden and ROS are attributed to neuronal damage, iron chelation and

antioxidative strategies are useful to protect neurodegeneration.

(-)-Epigallocatechin-3-gallate (EGCG) is an antioxidant used to counteract neurotoxin

and oxidative damage due to its iron chelation and free radical scavenging capabilities.

EGCG can induce the expression of several antioxidant enzymes, and eliminate ROS

and electrophile generation in the progression of neurodegeneration. EGCG has also

been shown to inhibit the inflammatory reactions against cancer and neurological
 89

diseases (20). It has been shown that EGCG treatment can prevent against

environmental toxin, such as 6-OHDA induced neurodegeneration both in vitro and in

vivo. However, the mechanism by which EGCG exerts its neuroprotection remains

unclear. In this study, we hypothesized that 6-OHDA increased ROS levels and

inflammation reaction, and cause oxidative damage in N27 cells, and EGCG

treatment may prevent the adverse effects by stabilizing the Nrf2, PPAR system, and

thus reducing the oxidative damage on lipids and proteins.

Materials and Methods

Reagents. EGCG, 6-OHDA, Hanks Balanced Salts (HBSS) used for cell culture

were purchased from Sigma (St. Louis, MO). RPMI-1640 medium, fetal bovine

serum, L-glutamine, penicillin, streptomycin and CM-H2DCFDA oxidative indicator

probe were obtained from Invitrogen (Carlsbad, CA). Trichloroacetic Acid (TCA)

was purchased from Fisher Scientific (Pittsburgh, PA). 1, 1, 3, 3-tetramethoxypropane,

2-Thiobarbituric Acid (TBA) were purchased from Sigma (St. Louis, MO). Protein

carbonyl was assessed by commercial kit from Cayman Chemical Company (Ann

Arbor, MI). Protein concentration was determined by Pierce BCA protein assay kit

(Rockford, IL). Rabbit anti-rat-Nrf2 polyclonal antibody, rabbit anti-rat-HO-1

polyclonal antibody and rabbit anti-rat-PPAR antibody, secondary antibody

goat-anti-rabbit-IgG-HRP and goat-anti-mouse-IgG-HRP were purchased from Santa

Cruz Biotechnology (Santa Cruz, CA). actin mouse-anti-mouse-monoclonal

antibody was purchased from Sigma (St. Louis, MO).

Cell culture. N27 cells were grown in RPMI-1640 medium containing 10% fetal

bovine serum, 2 mmol/L l-glutamine, 50 U penicillin and 50 g/mL streptomycin and

 90

maintained at 37C in a humidified atmosphere containing 5% CO2 as described

previously (21).

Treatments. N27 cells were pretreated with EGCG (100 M) for 2 h, followed by

6-OHDA (25 M) treatment for 24 h. Untreated cells were used as controls. After

treatments, cell lysate were collected used for the following assays.

Measurement of intracellular ROS generation. The formation of intracellular

ROS was measured by using a fluorescent probe, 2, 7-dichlorofluorescein diacetate as

described previously (22, 23). The N27 cells were plated as the density of 2104

cells/well on the 96-well plate. Medium was changed to HBSS before each treatment,

and CM-H2DCFDA was added to final concentration with 10 M to each well. The

fluorescence intensity of dichlorofluorescein (DCF) was continuously measured using

fluorescence spectrophotometer with excitation wavelength of 485 nm and emission

wavelength of 535 nm with 30 min interval for 8 h.

Thobartituric acid reactive substances (TBARS) assay. The TBARS assay was

used to determine the lipid peroxidation and performed fluorometrically with a

modified protocol (24). Briefly, cell lysates (200 l) from different treatments were

combined with ice cold 20% TCA (200 l), and then incubated on ice for 5 min.

Samples were centrifuged at 14,000 rpm at 4C for 5 min. Then 200 l supernatant

was mixed with 200 l 0.67% (w/v) TBA reagent, and incubated at 95C for 60 min.

Then 400 l butanol/pyridine (15:1) was added to each sample and centrifuged at

5,000 rpm for 5 min. The fluorescence intensity of malondialdehyde (MDA) in the

upper butanol/pyridine phase was quantified with an excitation wavelength of 520 nm

and emission wavelength of 550 nm on a black 96-well plate. 1, 1, 3,

3-tetramethoxypropane was used as a standard to calculate the final sample

concentration.
 91

Protein Carbonyl Content (PCC) assay. The protein oxidation in the cell lysates

samples were determined by measuring the PCC by using the DNPH

(2,4-dinitrophenylhydrazine). According to the protocol provided by Caymans

protein carbonyl assay kit (Ann Arbor, MI), the amount of protein-hydrozone product

was quantified spectrophotometrically at wavelength of 363nm on 96-well plate

reader. Total protein content of cell lysates was determined by Pierce BCA protein

assay kit (Rockford, IL). The PCC was calculated and normalized to the total protein

content.

Western blot analyses. After treatments described above, the N27 cells were

washed with ice-cold PBS, then was homogenized in RIPA buffer (containing 1%

Triton X-100, 0.1% SDS, 1 mM phenylmethanesulfonyl fluoride (PMSF) and

protease inhibitors, including pepstatin 1 mg/ml, aprotinin 1 mg/ml, leupeptin 1

mg/ml) and sonicated on ice 2 10 s. The supernatant was collected after maximum

centrifugation at 13,000 rpm for 15 min at 4C. Aliquots of supernatants were used to

measure the protein concentrations. Forty g of protein was mixed with 5 sample

buffer (0.35 M TrisCl, 10% SDS, 30% glycerol, 0.012% bromophenol blue), and

loaded onto a 12% or 15% SDS-polyacrylamide gel, electrophoresed and then

transferred to a nitrocellulose membrane (Bio-Rad). The membrane was blocked with

5% non-fat milk in TBST and then incubated with rabbit anti-rat Nrf2 polyclonal

antibody (1:500), rabbit anti-rat HO-1 polyclonal antibody (1:500) or rabbit anti-rat

PPAR antibody (1:500), respectively. Horseradish peroxidase-conjugated goat

anti-rat IgG antibody (1:3000, Santa Cruz, CA) was used as a secondary antibody. To

ensure even loading of the protein samples, -actin (1:1000) was used as internal

control, with the incubation of the corresponding goat-anti-mouse-IgG-HRP

secondary antibody (1:5000). All western blot assays were performed in triplicates.
 92

Statistics analyses. Data were analyzed with Prism 4.0 software (Graph Software,

San Diego, CA). All the measurements were normalized to the respective controls in

each experiment. All values were expressed as mean SEM as percentage of the

controls. One-way ANOVA with Tukeys multiple comparison test was used to

detect the differences among treatments and P < 0.05 was considered as significant.

Results

EGCG attenuated ROS generation induced by 6-OHDA

Intracellular ROS level was determined and shown in Figure 1. 6-OHDA increased

intracellular ROS level by 19 % (p<0.001) after 6 h treatment, and by 33.1 %

(p<0.001) after 8 h, compared to control group. Pretreatment of EGCG for 2 h

following with 6-OHDA treatment reduced ROS by 24.9 % (p<0.001) at 6 h, and by

31 % (p<0.001) at 8 h, compared to 6-OHDA treatment group; and reduced ROS by

10.6% (p<0.05) at 6 h, and by 8.1% (p>0.05) at 8 h, compared to control. There was a

decrease in ROS (14.3%, p<0.01) at 6 h, and (18.7%, p<0.001) at 8 h with EGCG

alone compared with control group. Overall, EGCG pretreatment attenuated 6-OHDA

induced ROS generation.

EGCG protected against 6-OHDA-induced lipid peroxidation

6-OHDA resulted in a marked increase (82.7%, p<0.001) in MDA concentration

after 24 h treatment (Fig 2). Pretreatment of EGCG for 2 h decreased lipid

peroxidation by 36.4% (p<0.001) compared to the 6-OHDA treatment. Although

EGCG alone had lower (by 30.3%, p<0.01) MDA value than 6-OHDA + EGCG

group, the value was not significant different compared to control.

 93

EGCG protected against 6-OHDA-induced PCC

As shown in Fig. 3, 6-OHDA increased the PCC by 47.8% (p<0.05) after 24 h

treatment compared to the control. Pretreatment of EGCG for 2 h decreased the PCC

by 27.7% (p<0.05), compared to 6-OHDA group. No difference between control and

EGCG alone suggested there was no effect of EGCG on PCC.

EGCG regulated antioxidant (Nrf2 and HO-1) and anti-inflammatory (PPAR)

system induced by 6-OHDA

The activities of Nrf2, HO-1 and PPAR were assessed and shown in Fig 4.

6-OHDA increased Nrf2 by 69.6% (p<0.001), HO-1 by 173.3% (p<0.001) and

PPAR by 122.7% (p<0.001) after 24 h treatment, compared to control group.

Pretreatment of EGCG for 2 h significantly decreased Nrf2 (by 36.7%, p<0.001),

HO-1 (by 49.3%, p<0.001) and PPAR (by 41.1%, p<0.01), compared to 6-OHDA

group. Pretreatment of lower concentration of EGCG (10 M) showed no decrease on

HO-1 and PPAR expression, but only significant decrease (by 18.9%, p<0.05) in

Nrf2, compared to 6-OHDA group. Therefore, these results suggested high

concentration of EGCG showed antioxidant and antiinflammation effects.

Discussion

6-OHDA is used as classical neurotoxin to induce neurodegneration and to

understand the treatment of PD. The neurotoxicity of 6-OHDA has been proposed by

several mechanisms, including free radicals damage, mitochondrial dysfunction, and

oxidative stress (25). Since iron is involved in PD due to its pro-oxidant nature, the

interruption of iron homeostasis by 6-OHDA causes neuronal damage. 6-OHDA can

alter the expression of iron-related protein, resulting into iron dyshomeostasis (26, 27).
 94

6-OHDA can cause iron release from cytosolic ferritin and increase intracellular free

iron concentration (28). The excess free iron could generate ROS via Fenton reaction,

causing oxidative stress and cell death, especially in the substantia nigra (29, 30). In

our previous study, we showed that 6-OHDA regulated iron-related protein

expression, increasing cellular iron uptake and retention in N27 cells. Here, we

confirmed that 6-OHDA significantly increased the intracellular ROS level and

oxidative damage to lipids and proteins in N27 cells. Therefore, iron dyshomeostasis

is critical to cause neuronal cell death and maintaining normal iron level is important

to prevent PD progression.

In addition to oxidative stress, inflammation also plays a critical role in PD. It had

been shown that neuroinflammation was significantly higher in PD brains, including

numerous inflammation-related enzymes and cytokines within the SN, striatum, and

cerebral spinal fluid. For example, tumor necrosis factors (TNF) and nitric oxide

synthase (iNOS) were found significantly elevated in PD patients, compared to

age-matched control (31). One of the proposed mechanisms of 6-OHDA toxicity is

the induction of inflammation (32, 33). Inflammatory factors, such as TNF could

directly activate cell apoptotic pathway or indirectly suppress the mitochondrial

function. Considerable evidence from in vitro and in vivo studies suggests that there is

an association between inflammation and ROS in PD (34, 35), and increased ROS

level could augment the inflammation process (36, 37), thereby exacerbating neuronal

damage.

Since oxidative stress and inflammation links to the progression of

neurodegeneration, antioxidation and antiinflammation would be useful as

neuroprevention strategies against PD. Nrf2/ARE system is considered a self-defense

antioxidant system to counteract the increased ROS and oxidative stress. Increased
 95

ROS levels and oxidative stress can trigger Nrf2 expression and Nrf2-associated

antioxidant enzymes expression (38, 39). In the present study, we showed that

6-OHDA increased not only ROS level, but also Nrf2 (by ~70%, p<0.001) and HO-1

expression (by ~ 173%, p<0.001) in N27 cells. However, the increased antioxidant

enzymes may not be enough to counteract the 6-OHDA toxicity, since we found

elevated oxidative damage to lipid (by ~83%, p<0.001) and protein (by ~48%,

p<0.05). Meanwhile, PPAR was associated with the anti-inflammatory system to

inhibit inflammatory reaction, increase DA, alter mitochondrial bioenergetics, reduce

oxidative stress and impede PD progression (10). In our study, we found the 6-OHDA

significantly increased PPAR expression, which may indicate the increased

inflammatory activity along with the increased ROS level. Similar to Nrf2, the

increased PPAR could not completely counteract against 6-OHDA toxicity, which

may indicate the inadequate anti-inflammatory effect. Although the Nrf2/ARE

antioxidant system is closely related to PPAR antiinflammation system (40), whether

there is a causal relationship remains unknown. Therefore, further research is needed

to explore the relationship between Nrf2 and PPAR.

EGCG has been considered as an antioxidant to decrease oxidative stress and

maintain the redox balance due to its high reduced potential and radical scavenging

ability. As showed in Figure 1, EGCG significantly decreased ROS level even

compared to the control group. EGCG is also considered as an iron chelator, therefore,

it can also decrease the ROS by binding free iron without affecting the iron

bioavailability. Several in vitro and in vivo studies have shown that EGCG treatment

can prevent cancer, vascular diseases and neurological disorders (41, 42). We have

previously shown that EGCG decreased iron uptake and retention in N27 cells and

prevented against 6-OHDA neurotoxicity, which was supported by our results showed
 96

that EGCG significantly reduced intracellular ROS, lipid peroxidation and protein

carbonyls, which maybe attributed through iron accumulation. Several studies

indicate EGCG can activate Nrf2 expression, which can induce antioxidant enzymes

expression, improve antioxidant defense ability, such as HO-1(42-44). However, we

were not able to show that EGCG increased either Nrf2 expression or PPAR activity

in our study. Both lower (10 M) and higher (100 M) concentration of EGCG

showed no effects on Nrf2, HO-1 and PPAR expression. One reason might be that

EGCG directly decreased the ROS level by scavenging the free radicals or chelating

excess iron. Therefore, the reduced ROS level and balanced redox potential would not

activate Nrf2/ARE system and PPAR activity. Another reason for the discrepancy of

our results might be related to the difference of cell types. We used N27 cells which

were much more sensitive to oxidative stress and complicated regulation pathways

compared to non-neuronal cells.

In conclusion, our in vitro data suggested that the neurotoxicity of 6-OHDA was

closely related to ROS, oxidative stress and inflammation. Pretreatment of EGCG

protected against 6-OHDA toxicity by inhibiting the ROS, consequently inactivating

Nrf2, HO-1 and PPAR expression. To our knowledge, this is the first in vitro study

of EGCG on Nrf2 regulation to prevent 6-OHDA induced neurotoxicity. Further

studies in vivo are needed to clarify the antioxidant and anti-inflammatory

mechanisms of EGCG protection in PD.

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 101

Figures and Tables

400
Control
* 6-OHDA
ROS (% of control)
300
* E100+6-OHDA
* E100
200

100

0
0 2 4 6 8 10
Time (h)

FIGURE 1 EGCG attenuated 6-OHDA-induced intracellular ROS generation in N27

cells. The fluorescence density showed in the time course of 6-OHDA increased ROS
levels. The values are expressed as mean SEM, n=3. 6-OHDA (25 M) was treated
for 24 h, pretreatment of EGCG (100 M) for 2 h. * p<0.001.
 102

**
**
200 b

MDA M/mg protein

150
*

(% of control)
a
ac
100 c

50

CG
A
l
ro

D
nt

EG
H

H
Co

-O
O
6-

+6
CG
EG
FIGURE 2 Effect of 6-OHDA and EGCG on lipid peroxidation. The values are
expressed as mean SEM, n=2 from 3 individual measurements. Means with letters
indicated comparison with control treatment. Symbols represented the comparison
between treatments. 6-OHDA, 25 M for 24 h, EGCG, 100 M pretreatment for 2 h.
* p<0.01, ** p<0.001.
 103

200 *
b

Protein carbonyl content

nmol/mg protein
150

(% of control)
a a
a
100

50

CG
A
l
ro

D
D
nt

EG
H
H
Co

6O
O
6-

+
CG
EG
FIGURE 3 Effect of EGCG on PCC induced by 6-OHDA. Cells were pretreated with
EGCG, followed by 6-OHDA treatment. The values are expressed as mean SEM
from duplicate measurements for 3 individual experiments. Labeled means without a
common letter differ. 6-OHDA, 25 M for 24 h, EGCG, 100 M pretreatment for 2 h.
* p<0.05.
 104

Nrf2 HO-1 PPAR

Actin

*** * 0.8 ***

0.6 *** 0.3 **
b * b ** ***
b b b
c 0.6

HO-1/actin

PPAR/actin
0.4 0.2
Nrf2/actin

a
a 0.4 a a
a
a
0.2 0.1
0.2

0.0 0.0 0.0

A
l

A
ro

A
l

A
A

A
l

ro
D

D
ro

D
nt
D

nt
H

H
nt

H
H

Co

Co
O
Co

-O
O
O

6-

6-

6-

6-
6-

6-

+6

+6
+6

0+

0+
0+

00

00
00

E1

E1
E1

E1

E1
E1

FIGURE 4 Effects of 6-OHDA and EGCG on Nrf2, HO-1 and PPAR protein
expression. Cells were pretreated with EGCG, followed by 6-OHDA treatment.
Western blot analysis (A) for Nrf2, HO-1and PPAR protein expression (normalized
with -actin in N27 cells), and quantification analysis (B). The values were expressed
as mean SEM from 4-8 individual measurements. Means with letters indicated
comparison with control treatment. Symbols represented the comparison between
treatments. 6-OHDA, 25 M for 24 h, EGCG, 10, 100 M pretreatment for 2 h.
*p<0.05, **p<0.01, ***p<0.001.

,
 105

CHAPTER 5. BENEFICAL EFFECTS OF GREEN TEA

CONSUMPTION IN PARKINSONS DISEASE PATIENTS

To be submitted to Journal of Movement Disorders

Dan Chen, MSc,1 Ying Zhou, MSc,1 Kelly Lyons, PhD,2 Rajesh Pahwa, MD,2 Manju

Reddy, PhD,1
1
Department of Food Science and Human Nutrition, Iowa State University, Ames, IA
2
Department of Neurology, University of Kansas Medical Center, Kansas City, KS

ABSTRACT: Antioxidants may offer protection on oxidative stress-associated

Parkinson's disease (PD). The objective of this study was to assess the beneficial

effect of green tea consumption (3 cups daily for 3 months) on quality of life, iron

status, antioxidant status, and oxidative damage in PD patients. Fourteen subjects

(51-79y) who were within the first five years of PD, on stable medication and not

regular green tea consumers were recruited. PD rating scales were used to evaluate

disease symptoms. Hemoglobin, serum iron, iron saturation and ferritin

concentrations were used to assess the iron status. Antioxidant enzymes including

catalase, superoxide dismutase (SOD), and glutathione peroxidase (GPx) were

measured to determine the antioxidant status. Lipid peroxidation and protein

carbonyls were used as oxidative damage markers. Wilcoxon matched pairs t-test and

student t-test were used for statistical analysis between baseline and 3 month

intervention data. Among the PD rating scales, we found a significant increase in

Mini-Mental State Examination (MMSE) (p<0.05) and a decrease in United

Parkinson's Disease Rating Scale (UPDRS) motor scores (p<0.05) after the

intervention, suggesting an improvement in cognitive and motor status. No changes

were found in iron status. Catalase and SOD activities were increased significantly
 106

(28%, p<0.05 and 37%, p<0.01, respectively), indicating the improvement of

antioxidant status. Both lipid peroxidation and protein carbonyls decreased by ~ 52%

(p<0.01) after intervention. In conclusion, green tea consumption may slow down PD

progression by improving the antioxidant status and reducing oxidative damages

without affecting iron status.

Key Words: Parkinsons disease; antioxidant enzymes; oxidative stress; green tea

consumption

Parkinsons disease (PD) is now considered the second most common

neurodegenerative disorder after Alzheimers disease, affecting approximately 1

percent of the population over 50 y.o. PD patients show classical motor symptoms

including bradykinesia, rigidity, and resting tremors due to the depletion of dopamine

in brain. Several factors, such as ageing, genetics, environment, oxidative stress, and

inflammation are involved in PD progression. Among these factors, oxidative stress is

one of the major causes in initiating and promoting neurodegeneration. Since brain

tissue has high levels of polyunsaturated fatty acids, it is prone to oxidative damage.

Excess iron accumulation in the brain leads to free radical formation and reactive

oxygen species (ROS) generation via the Fenton reaction, contributing to oxidation

damage of lipids and protein, and promoting cell death. Significant elevation of iron

was found in the substantia nigra (SN) of PD patients compared to the age-matched

control,1, 2 indicating the critical role of iron in PD progression.

Antioxidants counteracting the detrimental effects of ROS offer a promising

approach to protect neuronal cells. The antioxidant defense system is responsible for

removing free radicals, scavenging ROS or their precursors, maintaining redox

homeostasis, and decreasing oxidative damage.3 The antioxidant defense system can
 107

be classified as exogenous (natural or synthetic), such as ascorbic acid, lipoic acids,

polyphenols, and carotenoids, or endogenous, such as catalase, SOD, glutathione

reductase (GSH), and glutathione peroxidase (GPx). Evidence has shown the

beneficial effects of antioxidants in cell culture models.4-7 In vivo, higher levels of

antioxidants are required to result in protective effects on the central nerve system

(CNS), which is extremely sensitive to redox changes and oxidative dyshomeostasis

due to the high level of oxidative metabolism. Insufficient antioxidants and

antioxidant enzyme activities may not be able to offset the oxidative modifications

and damages. Several studies indicate that increased oxidative damage and reduced

antioxidant activities might be responsible for the onset and progression of PD.8-10

Therefore, the activation of antioxidant enzymes is considered to be a necessary

strategy to counteract the detrimental effects of ROS and restore the cellular redox

balance.11

Many antioxidants such as vitamin E, vitamin C, carotenoids, and flavonoids can

improve antioxidant status and decrease oxidative stress, therefore preventing

neurological disorders.12-14 Iron chelator, (i.e., desferrioxamine (DFO)) has shown

neuroprotective effects in in vitro and in vivo studies.15, 16 However, the side effects of

DFO limit its usefulness. Catechin polyphenols can act as antioxidants by scavenging

free radicals and chelating excess metal ions.17, 18 They may also indirectly reducing

oxidative stress inhibiting redox-sensitive transcription factors, nuclear factor-B, and

pro-oxidant enzymes, such as inducible nitric oxide synthase, and inducing phase II

antioxidant enzymes, such as glutathione S-transferases and SOD.

(-)-Epigallocatechin-3-Gallate (EGCG) is the most abundant polyphenol and iron

chelator present in green tea,19 which has been shown to be an antioxidant in vitro by

trapping peroxyl radicals and inhibiting lipid peroxidation, contributing to the

 108

neuroprotective effects of green tea in several PD model cells.20 EGCG can better

represent the antioxidant function of green tea in vivo, due to its bioavailability and

metabolism. Higher green tea consumption is related to the reduced risk of PD21-23

because it may offer a promising dietary source of neuroprotection. However, data

showing whether or not green tea directly or indirectly improves the PD patients

status is limited. The purpose of this study is to identify the beneficial effect of green

tea consumption in PD patients. We hypothesized that green tea consumption may

improve quality of life of PD patients by improving antioxidant status, thus, reducing

oxidative damage to lipids and proteins.

Patients and Methods

Study Design and Participants

Patients (n=15) (9 males, 6 females) were recruited from Parkinsons disease and

Movement Disorder Center at University of Kansas Medical Center (KUMC). The

inclusion criteria included patients within the first five years of PD, aged between 50

to 80 y, willing to drink green tea and were on stable medication regiment with no

changes in medication throughout the study. The exclusion criteria included

premenopausal women, Parkinsonism resulting from other causes including toxicity,

drugs and head trauma, and patients with uncontrolled chronic diseases, smokers, and

current green tea drinkers (> 3 cups/day). Subjects were provided with tea bags

(Lipton 100% Natural Green Tea) in batches at each visit and requested to drink 3

cups of green tea per day (1 green tea bag steeped in 1 cup of boiling water for 4

minutes).
 109

Assessment of PD Status at Baseline and Post-intervention

At baseline and at the 3 month visit, the United Parkinson's Disease Rating Scale

(UPDRS) was completed to assess the PD-related disability and impairment. 39-item

Parkinsons disease questionnaire (PDQ-39) was used to assess the daily quality of

life. Various other measures were used to evaluate PD symptoms, such as depression

(Beck Depression Inventory), anxiety (Beck Anxiety Inventory), sleepiness (Epworth

Sleepiness Scale), mental status (Mini Mental State Examination) and fatigue (Fatigue

Severity Scale). Hoehn and Yahr (H&Y) scale was used to assess the stage of PD and

Schwab and England Activities of Daily Living Scale (S&E) was used to test severity

of the disease. All the questionnaires were included in the appendix 2. Blood was

collected at baseline and 3 month intervention. The antioxidant enzyme activities in

erythrocytes, including catalase, SOD, and GPx were determined by using

commercial assay kits (Cayman Chemical Company, Ann Arbor, MI, USA). Serum

ferritin was measured with RIA kit (Ramco Laboratories, Houston, TX, USA).

Hemoglobin, serum iron saturation, and serum iron were determined by the

commercial clinical laboratory (LabCrop, Kansas city, MO, USA). Serum

malondialdehyde (MDA) concentration was estimated by thiobarbituric acid reactive

substances (TBARS) assay and used to determine lipid peroxidation. Protein

carbonyls in plasma were measured by using the commercial kits (Cayman Chemical

Laboratories, Houston, TX, USA).

 110

Statistical Analysis

Wilcoxon matched pairs t-test was used to compare the changes in PD rating scales

and student t-test was used to compare the changes in antioxidant enzymes, TBARS,

protein carbonyls, and iron status between baseline and post-intervention. The mean

differences between baseline and post-intervention were considered significant at

p<0.05.

Results

Subject Description

A total of 15 subjects were included in the study. One subject was withdrawn

during the study period. The median of age of the14 subjects was 61y.o. with a range

of 51 to 79y.o. Median BMI at baseline was 28.2 kg/m2 (overweight) with a BMI

range of 18 kg/m2 to 41.8 kg/m2. At 3 months intervention, median BMI was 28.6

kg/m2 (overweight) with a range of 18.5 kg/m2 to 45.6 kg/m2. Six of the subjects were

classified as obese (BMI > 30 kg/m2) and the remaining 8 participants had a BMI

between 18.5 kg/m2 to 28.7 kg/m2 following green tea consumption, classifying them

as normal to overweight (BMI 18.5 kg/m2 to 29.9 kg/m2 ).

Changes in PD Rating Scales

The data from PD rating scales for all subjects are listed in Table 1. Values are

expressed as mean + SD. Mean MMSE increased significantly (p<0.05) from

29.5+0.7 to 29.9+0.4, indicating improved mental status following green tea

consumption. The UPDRS motor exam score decreased significantly (p<0.05) from

21.2+7.5 to 18.1+9.2, indicating an improvement in motor status. On the contrary,

total activities of daily living (ADL) score from the UPDRS and the mobility score
 111

from the PDQ-39 increased significantly (p<0.05) from 9.2+5.7 to 9.9+5.7, and from

13.9+15 to 23.2+26.9 respectively, showing a decline in ability to perform ADL as

well as mobility status. There were no changes in total UPDRS and total PDQ-39

scores, suggesting the absence of change in PD symptoms and quality of life. There

were also no changes in the H&Y scale score, S&E scale score, indicating no stage or

severity changes in PD after green tea consumption.

Changes in Iron Status

Details of iron status are shown in Table 2. The values are expressed as mean + SD.

No significant changes were found in hemoglobin, serum iron, iron saturation, and

serum ferritin at baseline or post-intervention. At baseline and post-intervention,

average hemoglobin concentration was 14.3+1.5 g/dL and 14.2+1.5 g/dL, average

serum iron concentration was 85.9+28.5g/dL and 87.5+38.2 g/dL, average iron

saturation was 25.1+8.7%, 26.5+12.6 %, and average ferritin concentration was

61+50 ng/mL and 59+46 ng/mL. Overall, iron status was not affected by green tea

consumption.

Changes in Antioxidant Enzymes

Compared to baseline, the mean activity of SOD in erythrocytes increased by 37%

(p=0.0025) (Fig. 1A) post-intervention. Additionally, mean catalase activity increased

by 28% (p=0.0483) (Fig. 1B). Although there was a 13% increase in the mean activity

of GPx after green tea consumption, the change was not statistically significant

(p>0.05) (Fig. 1C). Overall, antioxidant enzymes activities were improved following

green tea consumption.

 112

Changes in Oxidative Damage

The mean value of MDA concentration decreased significantly (p<0.01) from

2.5+0.9 M to 1.2+0.8 M after green tea consumption (Fig. 2A), indicating a

decrease in lipid peroxidation. Protein carbonyl concentration also decreased

significantly (p<0.01) from 0.82+0.21 nmol/mg to 0.39+0.27 nmol/mg (Fig. 2B),

suggesting a decrease in protein damage.

Discussion

PD rating scales have been developed to measure the impact of PD on patients.

These scales not only identify and monitor PD symptoms, but also assess severity and

disease progression. Additionally, PD rating scales are useful for determining a

patients eligibility for participation in research trials. UPDRS is the most commonly

used scale to assess motor disability, motor impairment, mental dysfunction, and

motor or non-motor complications. H&Y and S&E scales are commonly used to

determine the PD stage and severity.9 PDQ-39 and other PD symptom measures, such

as the MMSE, are also widely used PD rating scales. Our data indicated no change in

total UPDRS score, total PDQ-39 score, and PD symptoms measures, (i.e., BDI, BAI),

suggesting no disease progression following green tea consumption. We found that

there was approximately a 20% (p<0.05) decrease in motor examination scores and a

significant (p<0.05) increase in MMSE, representing an improvement in motor

abilities. There was no change in H&Y scale scores, as well as in S&E scale scores,

suggesting no significant change in the stage or severity of PD. However, changes in

total daily living score of UPDRS (~8%, p<0.05) and mobility score (~ 67%, p<0.05)

of PDQ-39 may indicate a worsening of PD status. Our results conflict with various

qualitative questionnaires used to assess PD status. In a recent study, motor

 113

assessment was quantitatively evaluated by full-body motion capture data with a deep

brain stimulator.24 This method enables us to devise more effective ways to track the

progression of neurodegenerative movement disorders other than the currently used

PD rating scales. Therefore, revising and designing new appropriate measurement

scales can improve the evaluation on PD.25

Iron plays a critical role in CNS functioning and is closely related to the

progression of neurodegenerative diseases. Excess iron can induce the ROS

generation and cause oxidative damage once it overrides the biological detoxification

ability. Significantly higher amount of iron in the brain were reported in PD patients

compared to age-matched controls.2, 26 On the other hand, low circulating iron levels

have also been reported in PD,27 suggesting iron deficiency or problems with iron

storage in tissues such as liver and brain, thus, contributing to PD progression. In a

study conducted by Qureshi (2006), it was shown that there was no change in serum

iron levels, but a significantly higher level of iron in cerebrospinal fluid in PD patients,
28
compared to a healthy control. In our study, iron status values at the baseline were

in the normal range and none of the values indicated iron deficiency anemia or iron

excess. Although there is a concern that polyphenols can inhibit iron absorption and

alter overall iron status, no changes in iron status after 3 months of green tea

consumption was observed in our study. Our iron status results are similar to the iron

status results reported in the previous study.29 Therefore, the iron relocation within

specific regions in brain might become an important issue to trigger the progression of

PD rather than affecting overall body iron status.

Oxidative stress can cause dopaminergic cell death and neurodegeneration.

Increased oxidative stress and decreased antioxidant enzymes have been shown in

several human studies with PD patients.8, 30, 31 Since PD is related to oxidative stress
 114

and iron accumulation in brain, maintaining normal iron homeostasis and enhancing

antioxidant capabilities seem to be the ideal strategies to prevent PD. Green tea

polyphenols are shown to prevent not only neurological diseases, but also cancer and

inflammatory processes.32-34 EGCG is the main catechin component, contributing to

the beneficial effects of green tea due to its iron chelation, antioxidant, and

antiinflammation capabilities.35 EGCG has shown to protect neurotoxin-induced

dopaminergic cell death in both in vitro and in vivo models.36, 37 EGCG is also found

to enhance the activities of antioxidant enzymes, catalase, and SOD in the striatum of

mice in a neurotoxin induced PD model.38 Our results support the previous studies,

since green tea polyphenols demonstrated a protective effect in PD patients. Although

iron status remained unaltered, there was a significant increase in catalase and SOD

activity and a decrease in oxidative damage in lipids and proteins, suggesting that

green tea polyphenols could potentially be used as therapeutic supplements. We

believe that the beneficial effects are due to green tea consumption since patients did

not change their medication use during the study and there was no indication of

changes in dietary consumption during the 3 month trial period.

Although epidemiological studies have shown a negative correlation between green

tea consumption and the risk of neurological disorders including PD,23, 39

limited

human data directly shows its effects against PD progression. A cross-sectional study

indicated that inverse relationship between green tea consumption and cognitive

impairment, but not cognitive decline, may be due to a small number of participants in

the study.39 Chan (2009) showed that a green tea polyphenol (0.4 g, 0.8 g, and 1.2 g

daily) intervention for early PD patients over a span of 6 months can improve UPDRS

scores.40 In our study, participants consumed 3 cups of green tea daily for 3 months,

with a total polyphenol concentration of approximately 550 mg per 3 tea bags . This
 115

dosage resulted in an improvement in antioxidant status and reduced oxidative

damage on PD patients after the 3-month intervention.

One limitation of our study was the lack of an age-matched control group without

PD and/or a control group including PD patients who didnt consume green tea.

During the 3-month green tea intervention, we did not find significant changes in the

total UPDRS score, PDQ-39 score, and most of the PD measure scales, suggesting the

need for a long-term study and a large group of PD patients. While we used a realistic,

dietary approach to green tea consumption, using a purified EGCG supplement may

prove to be more accurate for determining the beneficial effects on Parkinsons

disease patients in a future clinical study. Although our study had a few limitations, it

also had several strengths. Our 3-month green tea intervention significantly improved

antioxidant status and reduced oxidative damage in early PD patients without

affecting their iron status. Based on this pilot study, a future study including a large

number of subjects, the use of an EGCG supplement, and age-matched control group

is needed to clarify the effectiveness of EGCG in preventing the progression of PD.

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 120

Figures and Tables

TABLE 1. PD rating scales in subjects at baseline and after 3 mo green tea consumption
Mean (SD) Median (Range)

B P B P
PDQ-39

Mobility *
13.9 (15.0) 23.2 (26.9) 10 (0-45) 13.75 (0-67.5)
Activity of Daily Living
17.6 (15.1) 20.2 (16.2) 16.7 (0-33.3) 22.9 (0-45.8)
Emotional well-being
14.9 (11.2) 18.5 (21.8) 10.4 (0-33.3) 10.4 (0-66.7)
Stigma
20.5 (18.4) 22.8 (23.3) 18.8 (0-50) 18.8 (0-68.8)
Social support
10.1 (15.4) 11.9 (19.0) 0 (41.7) 0 (0-58.3)
Cognition
15.2 (12.7) 14.7 (12.9) 12.5 (0-18.8) 12.5 (0-37.5)
Communication
12.5 (15.2) 11.9 (17.2) 8.3 (0-41.7) 0 (0-50)
Bodily discomfort
32.1 (19.0) 25.6 (23.2) 25 (8.3-75) 20.8 (0-91.7)

PDQ Total Score 16.4(11.5) 19.6(18.4) 15.7 (2.6-42.9) 15.4 (0.64-44.4)

UPDRS

Mentation, Mood, and Behavior 0.7 (1.2) 1.1 (1.6) 0 (0-4) 0 (0-5)
Activities of Daily Living * 9.2(5.7) 9.9(5.7) 9.5 (0-22) 10.5 (1-22)
Motor Examination *
21.2(7.5) 18.1(9.2) 20 (8-38) 16 (6-38)
Total Score
31.1(12.6) 29(15) 31.5 (8-58) 26.5 (7-65)

Beck Depression Inventory 7.1(5.8) 7.9(7.9) 5.5 (0-21) 5.5 (0-24)

Beck anxiety inventory 10.3(6.7) 9.3(7.8) 9.5 (1-20) 9 (0-13)

Epworth Sleepiness Scale 7.8(4.4) 7.7(5.1) 6.5 (3-17) 7 (4-16)

Mini Mental State Examination 29.5(0.7) 29.9(0.4) 30 (28-30) 30 (29-30)

Fatigue Severity Scale 28.4(13.0) 32.1(14.3) 27 (13-55) 30.5 (12-55)

Wilcoxon matched pairs t-test is used to compare the changes on PD rating scales between baseline
and post-intervention. Values are mean (SD) and median (range), n=14, * p<0.05. B, baseline; P,
post-intervention.
 121

TABLE 2. Subject description and iron status at baseline and after 3 mo green tea consumption
Subject Height Weight BMI Hemoglobin Serum Iron Iron Saturation Serum Ferritin

# (M) (Kg) (Kg/M2) (g/dL) (g/dL) (%) (ng/ml)

B P B P B P B P B P B P

Mean 1.7 87 87.5 28.9 29.1 14.3 14.2 85.9 87.5 25.1 26.5 60.8 59.5

+ (SD) (0.1) (20.5) (22.5) (6.0) (6.7) (1.5) (1.5) (28.5) (38.2) (8.7) (12.6) (49.8) (46.3)

The height, weight, BMI and iron status values are expressed as mean + (SD), n=14. No significant changes were found in
hemoglobin, serum iron, iron saturation and serum ferritin at baseline and post-intervention. B, baseline; P, post-intervention.

121
 122

200
** 2000 * 150

150

catalase activity
SOD activity 1500

(nmol/min/ml)

(nmol/min/ml)
GPx activity
(U/ml) 100
100 1000

50
50 500

0 0 0
Baseline Post-intervention Baseline Post-intervention Baseline Post-intervention

A B C

FIG. 1. Antioxidant enzymes activities in erythrocytes at baseline and after 3 mo

green tea consumption. (A) SOD, (B) catalase, (C) GPx. n=14, * p<0.05, ** p<0.01.
 123

* *
3 1.0

MDA concentration 0.8

protein carbonyl
2

(nmol/mg)
0.6
(M)

0.4
1
0.2

0 0.0
Baseline Post-intervention Baseline Post-intervention

A B

FIG. 2. Lipid peroxidation (A, measured as MDA concentration in serum) and protein
damage (B, measured as protein carbonyl concentration in plasma) at baseline and
after 3 mo green consumption. n=14, *p<0.01.
 124

Appendix. PD Patients Demographic Data

Subject Age (Y) Gender PD Meds Con Meds

1 53 F Sinemet 25/100 qid, Sinemet CR 50/200 qhs, Flexeril, Lorazepam, Cymbalta, Protonix, Benadryl, Melatonin

Requip 3 mg tid

2 68 M Stalevo 150 qid, Sinemet 25/100 qid, Mirapex 0.25 Zocor, MVI, ASA, Tramadol, Caltrate, Cal-Mag-Zn liquid, Vit D3,

mg qhs Magnesium

3 55 F Azilect 1 mg qd, Mirapex 0.25 mg qd - bid, Stalevo MVI, Fish Oil, Flax seed, Calcium, Niacin, ASA, Glycolax powder,

50 qid Glucosamine, CoQ10,

4 66 M Sinemet 25/100 5.5 tabs/day Aricept, Wellbutrin, Alprazolam, Flomax, Advil, Tylenol

5 57 M Azilect 1 mg qd, Requip 3 mg tid, Sinemet 25/100 ASA

tid

6 51 M None Zocor, MVI, Vit D, ASA,CoQ10, Curcumin, Gingko Biloba, Fish Oil

7 61 M Sinemet 25/100 2 tabs qid, Requip 3mg tid Evoxac, Synthroid, Simvastatin, Clonazepam, Zantac, MVI, Vit E, Fish

Oil, Saw Palmetto

8 67 F Sinemet 25/100 tid Lipitor, Toprol SL, HCTZ, Enalapril, Celebrex, Omeprazole,

Gemfibrozil, Sertraline, Omega 3, Calcium, ASA, Neurontin

 125

Appendix. PD Patients Demographic Data (Continued)

Subject Age (Y) Gender PD Meds Con Meds

9 66 F Azilect 1mg qd, Requip XL 8mg qd Simvastatin, Atenolol, Calcium +D, Fosamax

10 54 F C/L 25/100 bid, Azilect 1 mg qd Altace, Prempro, Synthroid, Zoloft, Vytorin, Oxaprozin, Fish Oil,

Women's MVI

11 79 M Sinemet CR 50/200mg bid, Azilect 1 mg qd, Oxycodone, Hydrocodone, HCTZ, Optiva, Lidoderm Patch, Detrol LA,

Mirapex .5mg bid Tylenol, Motrin

12 56 M Sinemet 25/100mg qid, Mirapex 0.5mg tid Buspar, Remeron, Benicar, Azasite,

13 59 M Sinemet 25/100mg tid, Requip 2mg tid, Azilect Amlodipine, Pravastatin, Losartran HCTZ

1mg qd

14 66 F Azilect 1mg qd Claritin 24HR, Fosamax, Aleve, Simvastatin, ASA, Fish Oil, B12,

Cranberry, Cal/mg/Zinc with D, Glucosamine CH, Multi Vit & Mineral,

Biotin, D3

15 66 M Selegiline 5 mg bid Actos, Multi-Vit, Fish Oil, Tylenol

Median 61

(range) (51~79)
 126

CHAPTER 6. GENERAL CONCLUSION

Parkinsons disease (PD) is a progressive neurodegenerative disease causing severe

dopamine depletion in the striatum. Numerous evidences indicated that iron played an

important role in PD. Both in vitro and in vivo studies showed that excess iron was

closely associated with neuronal cell damage. Interestingly, several neurotoxins, such

as 6-OHDA can disrupt iron homeostasis by altering iron-related gene and protein

expression, consequently induce iron accumulation, ROS generation, redox

dyshomeostasis and oxidative stress. We also confirmed the irreversible oxidative

damage to lipids and proteins. Therefore, maintaining iron homeostasis is a promising

strategy to prevent PD.

Results from epidemiological, cell culture, animal and clinical studies supported

green tea polyphenols as well as the main constituent EGCG as neuroprevention

methods to counteract neurodegeneration. In vitro study, pretreatment of EGCG

showed more effective protection on neuronal cell against neurotoxin-induced cell

damage than co-treatment, suggesting the preventive method is an effective protection

on PD. It is also important to maintain the effective dosage of EGCG to show the

neuroprotection. In this study, we showed EGCG at a concentration range of 10 M

-100 M could protect neuron against 6-OHDA-induced cell apoptosis and cell death,

whereas higher dosage (200 M) was toxic to N27 cells and primary mesencephalic

neuron. EGCG protected against 6-OHDA toxicity by decreasing DMT1, hepcidin,

increasing Fpn1 expression and reducing cell iron uptake to maintain normal iron

level. The pretreatment of EGCG suppressed the ROS generation and oxidative stress,

attenuated lipid peroxdation and protein carbonyl, and decreased cell apoptosis and

death.
 127

Green tea consumption may offer a prospective dietary source of neuroprotection

on PD patients. However, limited data showed green tea directly or indirectly

improves the PD patients status. In our study, we have measured the PD patients

blood EGCG concentration around 1.3 M after consuming one bag of green tea with

the bioavailability less than 1%. The participants daily consumed 3 cups of green tea

for consecutive 3 months and this amount of green tea consumption showed an

improved antioxidant status, reduced oxidative damage to lipid and protein, and a

slow-down progression of PD without affecting the iron status. The poor

bioavailability of EGCG is the concern for the effective concentration. Human study

showed oral intake 20mg/kg body weight EGCG can cause plasma maximum

concentration (Cmax) at 78 ng/ml (1). Animal studies showed that rat has only 1.6%

bioavailability of EGCG, mice has relative higher, 26.5% (2, 3). The concentration is

far below the micromolar concentration usually required in vitro activity. Therefore,

modification of EGCG structure ((-)-EGCG octaacetate) is an effective way to

improve the bioavailability, which has already been shown to inhibit breast tumor

growth and prostate cancer in animal studies (4, 5).

Future studies are still needed to clarify the mechanism on neuroprotection. In the

first study, we showed EGCG reversed the adverse effects from 6-OHDA on iron

related gene and protein expression. However, whether the oxidative stress affected

the iron related factors, leading to the iron influx, or the influx of iron caused

oxidative stress remains unclear. Also, whether the protection effect of EGCG directly

depended on reducing oxidative stress, regulating the iron related factors, resulting in

the decreased iron intake is still unclear. In the second study, we confirmed that

Nrf2/ARE pathway, including HO-1 expression and PPAR anti-inflammatory

pathway were involved in the 6-OHDA induced neurotoxicity. We also showed that
 128

Nrf2 positively related to PPAR in neurodegeneration. However, whether there is a

cause-effect relation remains unclear. In the third study, although we showed the

beneficial effects of green tea consumption on PD patients, the improvement of health

care, modern quality of life and other medication may also contribute to the changes

and improve the PD status. Therefore, age-matched control is required in future

human study.

In conclusion, we showed the EGCG protection against 6-OHDA by normalizing

its adverse effect on iron homeostasis in vitro. We also figure out the EGCG

decreased the oxidative stress and inflammatory reaction which derived by 6-OHDA.

These results are preliminary data for future in vivo study. The human study showed

the beneficial effects of green tea consumption on PD patients, and this pilot human

study added supportive data for future study, long-term and higher dosage, with

modification on EGCG supplement will help us to clarify the therapeutic way to

prevent PD.

References

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Chan TH. The challenge of developing green tea polyphenols as therapeutic agents.
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2. Lambert JD, Lee MJ, Lu H, Meng X, Hong JJ, Seril DN, Sturgill MG, Yang CS.
Epigallocatechin-3-gallate is absorbed but extensively glucuronidated following
oral administration to mice. J Nutr. 2003;133:4172-7.

3. Chen L, Lee MJ, Li H, Yang CS. Absorption, distribution, elimination of tea

polyphenols in rats. Drug Metab Dispos. 1997;25:1045-50.

4. Lee SC, Chan WK, Lee TW, Lam WH, Wang X, Chan TH, Wong YC. Effect of a
prodrug of the green tea polyphenol (-)-epigallocatechin-3-gallate on the growth of
androgen-independent prostate cancer in vivo. Nutr Cancer. 2008;60:483-91.
 129

5. Landis-Piwowar KR, Huo C, Chen D, Milacic V, Shi G, Chan TH, Dou QP. A
novel prodrug of the green tea polyphenol (-)-epigallocatechin-3-gallate as a
potential anticancer agent. Cancer Res.2007;67:4303-10.
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ACKNOWLEDGEMENTS

I feel so blessed that I have a good mentor in my Ph.D. life. I would like to express

my sincere appreciation to my major professor, Dr. Manju Reddy for her continuous

guidance and support. I would also like to thank the members of my program of study

committee, Dr. Anumantha Kanthasamy, Dr. Richard Martin, Dr. Matthew Rowling

and Dr. Peng Liu for their professional opinions, great advices, and generous help

through my graduate studies.

I want to thank my colleagues from our lab: Seth Armah, Alyssa Beavers, Dr.

Martin Mutambuka, Ying Zhou and Xu Qi for their help and support during my

graduate study; from Dr. Kanthasamys lab: Dr. Huajun Jin for his methods and

advices on experiments; from Dr. Hargroves lab: Xiaoguang Wang for the western

blot imaging; from Dr. Scotts lab: Dr. Scott and Dr. Moran for training me with

RT-PCR techniques. From Dr. Franciss lab: Amber Noterman, Lindsay Macnab for

helping me with editing the thesis; From University of Kansas: Drs. Kelly Lyons and

Rajesh Pahwa for coordinating the human study; from Rutgers University: Dr. Chung

Yang for analyzing the EGCG; From Department of Food Science and Human

Nutrition: Jeanne Stewart, Jean Tilley, Erica Burkheimer, Brenda Emery,

Interdepartmental Toxicology: Linda Wild. Their constant assistance, valuable

suggestions and hand-on help were precious and unforgettable.

Last, but not least, I would like to thank my family. To my Mom and Dad, Thank

you for supporting me spiritually and guiding me through all the difficulties to make

my dreams come true.