ANTIOXIDANT AND PRO-OXIDANT NATURE OF CATECHOLAMINES

Arno Garakhanian Siraki

A thesis submitted in confomiity with the requirements for the degree of Master of Science

Graduate Department of Pharmacology University of Toronto

O Copyright by Amo Garakhanian Siraki 2000

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ANTIOXIDANT AND PRO-OXLDANT NATURE OF CATECHOLAMINES Master of Science, 2000 Arno Garakhanian S i f i Department of Pharmacology University of Toronto

ABSTRACT

This study compares the antioxidant versus pro-oxidant potential of catecholamines.

Firstly, catecholamines scavenged superoxide, and also prevented hypoxia-reoxygenation injury.

The femc complexes of catecholarnines were much more effective and cytoprotective. This

could prove useful in stroke therapy. Secondly, catecholamines were shown to mediate the

oxidation of ascorbate and NADH, which was directly related to their O-quinone half-life.

Glutathione prevented NADH oxidation, and glutathione-conjugates were formed, indicating that

O-quinones were the metabolites responsibie for the oxidation. Lastly, dopamine cytotoxicity

was potentiated by catalytic manganese(Iï) concentrations which resulted in toxic dopamine O-

quinone formation, offering insight into the Parkinson's-like disorder found in manganese

miners. Dopamine was metabolized by hepatocyte P450 peroxygenase to a cytotoxic product

(possibly dopamine O-quinone), utilizing H202 formed by monoamine oxidase, and possibly

relevant to idiopathic Parkinson's disease. In concIusion, the balance of catecholamines as anti-

or pro-oxidant wiii depend on dose and arnbient cellular conditions.

ACKNOWLEDGMENTS

1 wish to thank my parents, Thomas and Alvart, for their support in my acadernic endeavors,

without which this degree, or my previous one, would not have been possible. My family,

including my grandparents in Toronto, Arsham and Zaghik, my aunt Rima, and my siblings,

Arby and Anita, were supportive and understanding of my objectives. Aiso, the latter two didn't

get in my way, so 1 actually could get some work done.

1 hold the highest respect and admiration for m y supervisor, Dr. Peter J. O'Brien, who

gave me a chance to show my capabilities in a research environment. It's no secret that 1 didn't

achieve high marks in our systematic grading system, but Dr. O'Brien's guidance facilitated my

development in scientific research. 1 regard him as a wizard and myself as a wizard's apprentice,

in the tradition of leaming by example and leadership, and not simply by words, but by actions.

Furthemore, he kept his promise for the duration of rny M.Sc. as a part-time student-I did

finish on time, as we agreed in 1998. His support and undying enthusiasm is inspirational and

motivational, which facilitated progress in al1 aspects of lab activities. Indeed, it's with his

support and encouragement that 1 currentiy wish to pursue a Ph.D.-something I was opposed to

a year ago-for what 1 believe are the right reasons: the need to know and to let others know.

Dr. O'Brien, 1 salute you.

1 wish to thank my defense cornmittee, who accepted the responsibilities that 1 requested.

My interna1 appraiser, Dr. Denise Tomkins, came through on short notice in my time of need.

My extemal appraiser, Dr. Peter Pennefather, was available for consultation long before this

event. 1 could discuss topics related and unrelated to my research area. His insight and

ingenuity was remarkable and always of interest. Cheers! My thanks to Dr. Jose Nobrega, who

participated as the additionai voting member. Also thanks to Dr. Aiian Okey for volunteering to

iii

chair the defense. I thank everyone for their understanding and Bexibiiity. September is a

difficult month for both professors as well as M.Sc. students since the students want to avoid

paying tuition and professors are busy with gants. In this respect, I am really appreciative of the

efforts of my defense committee.

1 wish to mention the members of my lab whom I got dong with ... al1 of them. Itys quite

surprising that such a large and diverse group of people could get dong without making plans to

poison one another. Specifically, I'd like to thank Dr. Majid Moridani for his help and

suggestions in research and academic choices. My most sincere "whauap" to everyone in the

lab. Although these fine peopie are my coileagues, I also regard them as my friends. Speaking

of which, 1 wish to mention ail my fnends across the spectnim. Actually. 1 will not mention

them in verbatim. They know who they are, as do 1. 1 think a mere mention doesn't do justice to

such a bond. If one of you is reading this, and if you are indeed a true fnend, then you know

what I am thinking and what 1 mean, (and no, I'm not BS-ing).

My sincerest thanks to Ms. Angela Moy who hired me at GlaxoWellcome Canada, and

allowed a flexible working schedule with my studies as the fmt priority. By giving me a chance

to prove my capabilities, I became part of a cohesive working team that brought out my best

qualities. It goes without saying, that 1 have Dr. Hira Kazarians to thank for sponsoring me as a

summer snident at GW. Also, thanks to Mr. Naresh Persaud who allowed me to continue

balancing my studies with work. My heilo's to everyone in the QC & QA teams at GW.

Also 1 wish to thank Dr. Tigran V. Chalikian who offered his guidance and help at any

time. Even though our research areas were different, he offered guidance in research and career

objectives.

1 have made the decision to pursue a Ph.D. In case things don't go my way, however, 1

have the foilowing request: that this research be continued, particularly the section of dopamine

metabolic activation by P450 peroxygenase activity. It is my instinct, with the direct aid and

supervision of Dr. O'Brien. that this system is somehow involved in the etiology of idiopathic

Parkinson's disease. No one has thought of this. Therefore, if 1 cannot pursue it, 1 request that

the reader of this work take into account this uncharted territory and let people know about it.

Nothing is more noble than the search for knowledge and tmth.

"Ail good is knowledge. AU evil is ignorance" - Socrates.

TABLE OF CONTENTS

Abstract

Acknowledgements

Ab breviations

List of Tables

List of Figures

List of Schemes

List of Publications, Abstracts, and Posters

Page

. . 11

iii

viii

ix

X

xi

xii

Generd Introduction 1

Chapter 1 : Superoxide radical scavenging and attenuation of 13

hypoxia-reoxygenation injury by femc complexes in isolated rat hepatocytes.

Abstract

Introduction

Materials & Methods

Resul ts

Discussion

Chapter 2: Catecholamine O-quinones mediate ascorbic acid and

NADH oxidation, which is prevented by GSH: the relationship

between O-quinone stabiiity and catecholamine cyclization.

Abstract

Introduction

MateriaIs & Methods

R e d ts

Discussion

Chapter 3: Dopamine metabolic activation by P450 peroxygenase

activity versus manganese (II):

DA O-quinone as the mediator of cytotoxicity

Abstract

Introduction

Materials & Methods

Discussion

General Conclusions and Future Expenments

Re ferences

C.4

DA

DOPA

DOPAC

ErnA

EPI

GSH

GSSG

H202

HRP

H x

HVA

MAO

NAcDA

~ a . 1 0 ~

NE

NQO ROS

SOD

ozb XO

XTr

catec holamine

dopamine

3,4-dihydroxyphenyldanine

3,4-dihydroxyphen y lacetic acid

ethylenediaminetetraacetic acid

epinephnne

glutathione

glutathione disulfide (oxidized)

hydrogen peroxide

horse radish peroxidase

hypoxanthine

homovanillic acid

monoamine oxidase

N-ace tyldopamine

sodium periodate

norepinephnne

NAD(P)H:Quinone Oxidoreductase

reactive oxygen species

superoxide dismutase

superoxide

xanthine oxidase

2 , 3 - b i s [ 2 - m e t h o x y - 4 - n i t r o - 5 - s u l f o p h e n y p

viii

LIST OF TABLES

Table 1 . 1 ICso values for OzC scavenging activity of neurotransrnitters

and neurotransmitter-iron(m) complexes in the HX/XO system.

Table 1.3 Cytotoxicity of hepatocytes upon hypoxia-reoxygenation by

neurotransmitters and neurotransmitter: iron(iII) complexes.

Table 1.3 O2 uptake with 2: 1 neurotransmitter:metaI complexes.

Table 2.1 Cyclization rates and O-quinone half-life of catecholamines.

Table 2.2 Ascorbate CO-oxidation with Substrates Using WM202 &

Tyrosinase.

Table 2.3 NADH Oxidation In The Presence Of Ascorbate or GSH

by HRP/H20t.

Table 2.4 GSH depletion in microsornai preparation.

TabIe 3.1 IntracelluIar [GSH] and [GSSG] after incubation with DA or

tyramine.

LIST OF FIGURES

Figure 1. The Catecholamine Biosynthetic Cascade.

Figure II. Proposed Oxidative Pathways for DOPA and CAS.

Fig III. Structures of compounds used in this study.

Figure 2.1 Percent NADH Oxidized by Catechol(amine)s by

HRP/H202 and the Inhibitory Effect of Ascorbate or GSH.

Figure 2.2 Products Found by Mass Spectroscopy with NAcDA + Tyrosinase

or HRP/H202 + GSH.

Figure 3.1 Cornparison of Dopamine Cytotoxicity Cataiyzed by Different

Metals.

Figure 3.2 Cytoprotection Against DA:M~" by GSH, Ascorbate, and Xylitol.

Figure 3.3 NQO Inhibition Promotes DA/M~'+ toxicity.

Figure 3.4 MAO Inhibitors Prevent DA Cytotoxicity.

Figure 3.5 Involvement of H202 in DA Metabolic Activation.

Figure 3.6 CYP2E1 is hvolved in the Peroxygenase-mediated Activation of DA. 63

Figure 3.7 Tyramine, unlike DA Cytotoxicity, is Inhibited by a MAO Inhibitor 64

and a ROS scavenger, but not by a CYP 2E1 Inhibitor.

LIST OF SCHEMES

Scheme 2.1 Biochemical pathways involved in the oxidation of catechols to 42

O-quinones and the mechanisrns of ascorbate or NADH oxidation and GSH

conjugation.

Scheme 2.2 Reduction of a CA O-quinone and aminochrome. 49

Scheme 3.1 Proposed mechanisrn of ~n'+-catalyzed DA cytotoxicity. 59

Scheme 3.2 Mechanism of manganese-catalyzed DA O-quinone formation. 67

Scheme 3.3 Proposed pathway for DA rnetabolic activation by P450 68

peroxygenase activity/H202.

LIST OF PUBLICATIONS, ABSTRACTS, AND POSTERS

Pub iicatiolis:

1. Siraki A.G., Smythies J*, O'Brien P.J. Superoxide radical scavenging and attenuation of

hypoxia-reoxygenation injury by femc complexes. Submitted to Neuroscience Letters.

Abstracts:

1. Chan T.S., Galatai G., Mondani M.Y., Siraki A.G., Scobie H., Beard K., Eghbd M.A., and

O'Brien P.J. Hydrogen peroxide supports P450 catalyzed xenobiotic/dmg metabolism to

form cytotoxic reactive intermediates. Presented at the Biological Reactive Intermediates

Sixth International Symposium, Iuly 16-20,2000, Pans, France.

2. Siraki AG., Chan T.S., O'Brien L., Parekh K., and O'Brien P.J. Manganese (II) as a

selective catalyst for dopamine toxicity: possible rnechanism for metal-induced Parkinson's

disease. Presented at the 43* annual meeting of the Canadian Federation of Biological

Societies, Ottawa Congress Centre, Ottawa, June 22-25,2000.

Posters:

1. Siraki A.G. and O'Brien P.J. Antioxidant activity of catecholic neurotransmitters:

superoxide radical scavenging by ferric complexes. Presented at Visions in Phamacology,

St. Michael's College, University of Toronto, June 4, 1999.

2. Siraki AG., Chan T.S., and O'Brien P.J. Dopamine metabolic activation by rnanganese (IL')

versus P450 peroxygenase activity. Presented at Visions in Phamacology, Hart House,

University of Toronto, May 26,2000.

3. Siraki A G . and O'Brien P.J. Manganese (II) as a selective catalyst for dopamine toxicity:

possible mechanism for metal-induced Parkinson's disease. Presented at the first annual

meeting of the Oxidative Stress Consortium. May 12- 14,2000, Hamilton, Ontario.

xii

GENERAL INTRODUCTION

Oxidative stress has been irnplicated in many disease conditions, including central

nervous system disorders such as Parkinson's and Alzheimer's disease, as well as acute

phenomena like ischemia-reperfhion injury or stroke (1-5). Antioxidants are essential

components of a living system for defense against such long- and short-term oxidative insults.

For exarnple, ascorbate and glutathione (GSH) are the rnost abundant low molecular weight

antioxidants in the central nervous system, with concentrations of lOmM and 2.5mM in neurons,

respectively (6, 7). Since the central nervous system does not regenerate, it is of paramount

importance to maintain the longevity of these cells for normal function. Free radicais such as

superoxide (023 and hydrogen peroxide (H202), cm be lethal for the cell, especially if the latter

reacts with trace amounts of iron or copper, which yield the toxic hydroxyl radical (8). Although

the role of oxygen free radicais is well documented, the focus of this study was to charactenze

the role that catecholamines could play in contributing to the balance of anti- versus pro-oxidant

forces.

Catecholamine Synthesis & Metabolisrn

Catecholamines are spthesized from L-tyrosine either from diet or by enzymatic

hydroxylation of phenyldanine in select centml and penpheral neulons, including the adrenal

medulla. Tyrosine is actively transported from the blood into the adrenergic neuron ce11 bodies,

adrend chromaffin cells, as well as melanocytes (9, 10). The synthetic pathway was first

postulated by Blaschko in 1939, and later demonstrated in 1964 with the isolation of tyrosine

hydroxylase, (1 1- 13). Tyrosine is converted to L-3,4-dihydroxyphenylalaniae (L-DOPA) b y

tyrosine hydroxylase, which is the rate-limiting step in catecholamine (CA) synthesis. Dopa

decarboxylase (also referred to as aromatic amino acid decarboxylase) converts L-DOPA into

3,4-dihydroxyphenylethylamine (dopamine, DA), the fmt of the CAS in this biosynthetic

cascade. DA is converted to 3,4-dihydroxyphenylethanolamine (norepinephrine, NE) by

dopamine-ghydroxylase. The expression of this enzyme is restricted to the NE-containing

neurons of the central and perîpheral nervous system (14, 15). NE is converted to epinephrine

(EPI) by phenylethanolamine N-methyltransferase selectively present in the adrenal medulla and

in few neuronal groups in the lower brainstem (13). This sequence of reactions forms the CA

biosynthetic cascade, which requires the presence of various enzyme cofactors shown in figure 1.

CAS have specific enzymes present for their metabolism. DA, NE, and EPI can be

metabolized by monoamine oxidase, (MAO). This results in the formation of an aldehyde

intemediate, which is oxidized M e r to an acid, or reduced to an aicohol. The toxic byproduct

of this reaction, however, is H202 produced by MAO activity. Catechol-O-methyl tramferase is

also chiefly involved in CA metabolism. The enzyme catalyzes the methylation of one of the

hydroxyl groups on the catechol ring (usually position 3). The methylation can occur directly on

the parent compound (e.g., DA), but can also occur after deamination by MAO. This process cm

aiso be reversed, where methylation precedes deamination. These enzymes are responsible for

the metaboiic clearance of CAS, but other enzymes can also metabolize the latter. These are

dealt with in Chapter 3, where this process is referred to as metabolic activation, since they could

Iead to potentially cytotoxic products.

The oxidative metabolism of CAS is an alternative metabolic fate that is generaily not

discussed in the mainstream literature. CAS can form coloured oxidative endproducts, referred

to as aminochromes (16, 17). As shown in figure II, all the CAS can fonn amuiochromes, and

are named after their parent molecde, (e.g., adrenaline - adrenochrome, dopamine -

dopaminochrome, etc.). The formation of aminochromes in vivo is not widespread, but certain

observations conclusively demonstrate localized formation. In fact, this physiological process is

responsible for the building blocks of melanogenesis. In the skin, tyrosine is hydroxylated to

DOPA by tyrosinase, which is further oxidized to DOPA O-quinone, that rapidly cyclizes to the

dopachrome. This product polymerizes resulting in skin pigmentation known as melanin (18,

19). Although melanin is regarded as an extemal feature, some of the internal organs have also

been found to contain melanin. Occurrence of the latter has been reported in

pheochrornocytornas, (20). The substantia nigra is darkly coloured because of DA oxidation to

dopminochrome, which subsequently polymenzes to neuromelanin. In post-rnortem autopsy of

Parkinson's disease patients, the darkly pigmented substantia nigra loses its distinct, black

feature (21). A similar type of pigmentation is also seen in the NEtontaining locus ceruleus

(22). The role of neuromelanin is not fully understood as some think it may possess antioxidant

properties while othen consider it to result in neurodegeneration. Since melanins are thought to

contain 5,6-dihydroxyindoles (18), (formed by W e r oxidation of aminochromes), it is likely

that aminochromes must also be formed as their in vivo precursors.

Also used in this study, is the N-acetylated analog of DA, N-acetyldopamine (NAcDA).

It is likely that NAcDA rnay be an endogenous metabolite of DA in humans. Although not

detected in human caudate nucleus or mouse whole brain, it was found that NAcDA is most

likely formed peripherally rather than centraily (23). Even after injection of DA, NAcDA

formation (as glucoronide or methylated conjugates) was detected (24). This compound is tested

in Chapter 2, where its sipnificance of use will be elaborated thereupon.

Tyrosine

5rosin e Hydroxy kse

O2 (+ tetrahydrobiopterin, Oa ~ e ~ + )

L-DOPA

I DOPA Decarboxylase (+pyridoxal phosphate)

Dopamine

Dopamine-b Hydroxy lase (+asco rbic acid, eu2+)

Figure 1. The Catecholamine Biosynthetic Cascade.

Figure II. Proposed Oxidative Pathways for DOPA and CAS. 1, tyrosine; 2, DOPA; 3, DA; 4, NE; 5, EPI; 6, DOPA quinone; 7, leukodopachrome; 8, dopachrome; 9, 6-hydroxyDOPA; 10, 6-hydroxyDOPA O-quinone; 1 1, 6-hydroxyDOPA p-quinone; 12, DA O-quinone; 13, leukodopaminochrome; 14, dopaminochrome; 15,6-hydroxyDA; 16, 6-hydroxyDA O-quinone; 17, 6-hydroxyDA p-quinone; 18, NE O-quinone; 19, leukonoradrenochrorne; 20, noradrenochrome; 21, EPI O-quinone; 22, leukoadrenochrome; 23, adrenochrome; 24, dihydroxyindole derivatives; 25, indole quinone denvatives. The combination of 25 with other quinones may be the precursors for melanins (adapted fiom Ref. 17).

Function

CAS play an essential role in physiology. They regulate a vast number of processes

including cardiovascular functions such as heart rate and blood pressure, bronchial relaxation,

digestive activity, metabolisrn, and feeding behaviour (25-29). Since CAS have such a central

role in homeostasis, many synthetic CA analogs have been developed to either enhance or

attenuate the effects of CAS. In general, the effects of CA are mediated via interaction with

various adrenergic receptors (adrenoceptors) that are broadly classified as a or P, although DA is

a less potent agonist than NE or EPI. This classification was made on the basis of different

physiological effects of EPI, NE, and the synthetic CA, isoproterenol, (9). Since this general

classification, six subtypes of a adrenoceptors (al*, ale, ~ I C , a=, a z ~ , aZc) and three subtypes

of p adrenoceptors (Bi, Pz, P3) have been cloned so far. Each receptor is G-protein linked and

therefore stimulation by an agonist results in activation or inhibition of intracellular signaling

pathways, i.e., second messengers. The a2 adrenoceptors dl inhibit adenylate cyclase by

interacting with the Gi protein-bound receptor. Adrenoceptors, on the other hand, activate the

G, protein leading to increased inositol triphosphate and diacylglycerol levels, which can alter

the caicium flux of the cell.

In the autonornic nervous system, the key adrenergic effector is norepinephrine, (NE).

With few exceptions, most of the postganglionic sympathetic nerve endings secrete NE, whereas

al1 postganglionic parasympathetic nerves secrete acetylcholine. An action potential wiil

depoiarize the terminal fibres, increasing the membrane influx of ca2+, allowing this cation to

interact with the neurotransmitter storage vesicles adjacent to the membrane, causing them to

fuse with the latter and empty their contents into the extracellular space (30). NE is stored in

high concentrations in these vesicles in a complex with ATP. An active transport system exists

within the vesicles, essential for maintainhg a concentration gradient of up to 200-fold. h the

chrornafEn cells of the adrenal medulla, up to 80% of the NE leaves the storage vesicle to

become N-metbylated and form EPI (9).

In essence, CAS themselves can also be segregated: NE and EPI being more related to

each other, and DA having more unique properties. This distinction becomes clear in the centrai

nervous system more so than in the periphery, although DA does exhibit peripherai functions as

weIl(3 1). In the centrai nervous system, the adrenoceptors are also involved in behaviour, such

as food and water intake, in addition to vascular functions (32). DA is the weakest agonist for

the adrenoceptors, however, it has its own specific DA-receptors, which have prorninent central

effects. There are two classes of DA receptors: the D 1 family (which activate adenylate cyclase

and include the D5 type), and the D2 family, (al1 inhibit adenylate cyclase and include D3 and

D4) (32). The largest DA-ergic system is found in the ventral mesencephaiic system, which

includes the substantia nigra pars compacta and the ventral tegementd area. The latter is more

involved with behaviour and mood whereas the substantia nigra is the "Parkinsonian" area where

severe neurodegeneration results in the clinical effects of Parkinson's disease. The involvement

of D2 receptor in behaviour is highlighted by the efficacy of D2 antagonism in the treatment of

psychosis (32).

Catecholamines: the Double-Edged Sword

CAS are known to mediate toxicity penpherally by receptor-mediated and oxidative

mechanism. They may also be protective through their antioxidant properties. The balance of

this dual nature, therefore, is dictated by ambient conditions such as concentration, pH, oxygen

content, and localization. For the most part, however, the fiterature is currentiy inclined towards

CAS as mediaton or initiators of toxicity. Of the three endogenous CAS, DA stands out as being

either more cytoprotective or cytotoxic. DA was shown to inhibit lipid peroxidation, scavenge

02 and hydroxyi radicals more than a-tocopherol and much more than NE or EPI (33-35). On

the other hand, DA and L-DOPA can also cause protein binding or cell death in various neuronal

and non-neuronal ce11 cultures, either by formation of reactive oxygen species, semi- or ortho-

quinones (O-quinones), or by glutamate release (36-43). Glutamate release is thought to mediate

excitotoxicity by N-methyl-D-aspartate receptor binding, which leads to neuronal nitric oxide

synthase activation, producing nitric oxide toxicity (36,38). Furthemore, DA cm signal for ce11

death through the SAPWINK pathway in a process that requires the oxidation of DA to initiate

the apoptotic cascade (44). Oxidative mechanisms have gained much momentum as causes for

various disease conditions, and is a centrai theme to this thesis. However, they are not the only

mechanisms of toxicity.

The most widely documented toxicity of CAS in generai is their cardiotoxic effects.

Through adrenergic receptor stimulation, CAS can induce myocardial necrosis: hypoxia (a high

O2 demand and low supply), and ca2+ overioad from exaggerated P-adrenoceptor binding are

thought to be chiefly involved (45). Indeed, CAS can cause contractile failure and myocardial

necrosis even at non-lethai doses (46). Although these non-oxidative mechanisms have been

weiI documented, CA oxidation is also thought to be involved. When energy stores of neurons

are depleted (as in hypoxia), NE is likely to leave nerve terminais via the uptake carrier in the

reverse direction (47). Exogenous administration of NE has deleterious effects on acute regional

myocardial ischemia, even when a and P adrenoceptors are blocked, therefore, an oxidative

process is most likely occurring (48).

Adrenochrome, the oxidation end product of EPI, was shown to be toxic to myocardium,

but stiil less toxic than CAS themselves. Both reactive oxygen species and quinoid products are

thought to contribute to EPEinduced cardiac myocyte toxicity (48, 49). Adrenochrome being

less toxic than its precursor, EPI, suggests that the process leading to its formation produces

reactive oxygen species or that the intermediate products could confer toxicity. It could be a

combination of both.

Physiologicdly, where could excessive amounts of CAS be produced and released into

the circulation? Sympathetic dnve from the autonomie nervous system could release NE,

possibly in conditions of chronic stress. Indeed, NE is associated with post-traumatic stress

disorder (50). Hypertension is thought to involve excess CA content. One source could be the

adrenal medulla. This tissue is involved in the "fight-or-fiight" response to stresshl situations; it

is conceivable, therefore, that chronic stimulation of CA production and release could lead to

chronic hypertension. A neoplasm of the adrenal medulla, pheochromocytoma, is a CA-

producing tumor of the sympathetic nervous system that can be treated with phenoxybenzamine

(which irrevenibly blocks a adrenoceptors) and m-tyrosine (which inhibits tyrosine

hydroxylase) or adrenalectomy (5 1). hterestingly, oniy 0.1 to 0.3% of hypertensive patients

were diagnosed with pheochromocytoma (52).

CAS have also been associated with excessive hemolysis of human erythrocytes. The red

blood ceU is actively involved in the meiabolic clearance of CAS (particularly DA) since the red

blood ceus take up, metabolize, and release the latter. DA and adrenochrome also cause

excessive hemolysis when incubated with whole blood (53). CAS and L-DOPA also form

rheomelanin, a blood soluble melanin, which is hemolytic, especially the rheomelanin of DA

(54-57). Whether this occurs in vivo, however, is questionable. The erythrocyte is actually

involved in the clearance of CAS in the bloodstream. Studies show that erythrocyies

preferentiaüy take up DA, 10 fold more than NE (58). Phase II metabolism also occurs in

erythrocytes, including glucuronidation, methylation, and sulfation (59, 60). Interestingly, this

metabolism is more efficient with DA than NE or EPI.

Of key importance, and centrai to this thesis, is the potential of anti- or pro-oxidant CA

activity in the brain. Of specific interest is DA, and its role in the overall well being of its host

cell. The DAergic neurons of the susbtantia nigra pars compacta die (hence fail to produce DA)

due to unknown causes in idiopathic Parkinson's disease. These cells are darkly pigmented with

the DA oxidation polymer called neuromelanin. This differs from peripheral pigments since

those melanins are DOPA derived. Neurornelanin is positively correlated with susceptibility of

DA-ergic neurons to degeneration (6 1,62). However, in a study of PC 12 ce11 apoptosis, DA was

much more toxic than its corresponding melanin (63). The exact structure of neuromelanin is not

known, but is thought to contain cysteinyl-DA, dihydroxyindoles, and possibly transition metals

(18). The paradox lies in the fact that DA. the very substance that is deficient in Parkinson's

disease, may play a part in the etiology of the disease.

Relevance for Study

CAS are physiologically essential, without which normal embryogenesis and

development could not occur (13, 64). Although they possess some secondary antioxidant

properties, this is balanced (or overbalanced) by their inherent toxicity. This is the focus of this

thesis: presenting CAS as a doubleedged sword. Various CAS were chosen for this study dong

side CA-like moIecules, mg. III). Chapter 1 deals with antioxidant characteristics of the CAS.

Chapter 2 aims at distinguishing the characteristics of CAS that make one potentiaily more toxic

than the other. Chapter 3 focuses on CAS, particularly DA as a mediator of cytotoxicity in vitro. -

The potential ciinical implications of this research apply to CA-induced toxicity at the cardiac

and CNS level.

toH gvk. / NH2

K) 0" Tyrosine DOPA a-mcthy IDOPA

p N H 2 HO ~ N b HO HO / /NH

DA NE EPI

HO /

HVA DOPAC NAcDA O

Fig m. Structures of compounds used in this study.

Chapter 1

Superoxide radical scavenging and attenuation of hypoxia-reoxygeaation injury by ratechdamine ferric complexes in isolated rat hepatocfles.

Reactive oxygen species have been implicated in the pathogenesis of hypoxia-reoxygenation

injury. Previously, it was demonstrated that 2: 1 catecholic iron complexes were more effective

than uncomplexed catechois at (a) scavenging superoxide radicals generated enzymatically, and

@) protecting hepatocytes against hypoxia-reoxygenation injuy (65). Based on these findinps.

we sought to demonstrate similar effects using CA neurotransmitters. Various CA-iron

complexes were shown to be more effective than uncomplexed CAS at scavenging O?'- radicds

and could be used to protect cells from hypoxia-reoxygenation injury. a-MethylDOPA

complexed with femc ion (2: 1) showed the greatest 0;- scavenging potency amongst the CA-

iron complexes. The uncomplexed CAS were much less effective at scavenging O<- radicds

than the CA-iron complexes. DA was the most effective OzC scavenger among the

uncomplexed CAS. The 0 2 - scavenging effectiveness of the latter seemed to correlate with

their reduction potentials, but not directly to their pKp values. Furthemore, DA:iron(m)

complex protected isolated hepatocytes against hypoxia-reoxygenation injury at concentrations

four fold lower than that required for protection by DA alone.

KEY WORDS: superoxide dismutase mimics; iron; hypoxia-reoxygenation injury;

catecholarnines; antioxidants; neurotransmittea; isolated hepatocytes.

The damaging effects of ischemia-reperfusion are weH known and have been extensively

documented, and although some controversy exists regarding the specific mechanism leading to

tissue damage, reactive oxygen species (ROS) are thought to be involved. in hypoxia,

hypoxanthine accumulates as a result of ATP catabolism and xanthine dehydrogenase is

proteolytically cleaved or oxidized to f o m xanthine oxidase (66, 67). On reoxygenation, ROS

formation occurs as a result of the action of this enzyme (66-68). Furthemore, reoxygenation of

anoxic isolated mitochondria also increase mitochondnal ROS formation which impairs its own

functions and contributes to cytotoxicity (69). Transgenic mice overexpressing human copper-

zinc superoxide dismutase (SOD) show significant protection in cerebral ischemia in rnice (70).

Also, polyethylene glycol- or liposome entrapped-SOD has been shown to protect blood-brain

barrier in mice (7 1,72).

Recently some or the classical monoamine neurotransmitters, including DA, have been

shown to inhibit lipid peroxidation (33,34,73), particularly if catalyzed by iron (35). Catechols

are very effective at chelating iron to form tris(catecho1ato)ferrate and may act as a cellular iron

transporter (as enterobactin) in some bactena (74). Catechol-iron complexes have high stability

constants and low reduction potentials and cm, at a physiological pH, predominantiy form a 2: 1

catechol-femc complex (75-77).

Previously, it was shown that catechol-femc complexes are much more effective than

uncomplexed catechols at scavenging and protecting hepatocpes fiom hypoxia-

reoxygenation injury (65). Based on these findings, we sought to compare the O*- scavenging

activity and the cytoprotective effectiveness of uncomplexed CA neurotransmitters and their iron

complexes. CA or phenolic neurotrmsrnitters studied include DA, NE, DOPA, a-methylDOPA,

and EPI, as well as serotonin, tyrosine, and metabolites of DA and serotonin. Serotonin, and its

metabolite 5-hydroxyindoleacetic acid, was tested in order to compare a hydroxyindoles to

catecholamines. It was found that the 0; scavenging activity of most of these catecholic

neurotransrnitters were markedly enhanced when complexed with iron, and a rank-order for the

02C~cavenging ability of the catecholic neurotransmitters was deterrnined.

MATERIALS & METHODS

Catecholic neurotransmitters, FeC13, CuS04, hypoxanthine (HX), ethylenediarnine-

tetracetic acid (EDTA), 2,3-bis[2-methoxy-4-nitro-5-sulfophenyfl--

carboxanilide (m), and xanthine oxidase (EC 1.1.3.22; XO) were purchased from Sigma

Chernical Co., (Oakville, Ont., Canada). Neurotransmitters were resuspended in ~ i l l i ~ @ water

or O.1M HCl, to make 5m.M stock solutions. Femc complexes were prepared from the

neurotransmitter stock solutions and a fresh solution of 5mM FeC13, in a 2:l ratio of

neurotransmi tter:~e~+.

Solutions of XTT ( 2 0 , HX (35pM), three concentrations of neurotransrnitter:~e"*

complex (10,25, and SOpM), and XO (25mU/mL) were added to a ImL cuvette. A O.1M Tris-

HCl buffer (pH 7.4) was used at room temperature. The rest of the solutions were kept on ice.

The solutions were added in the following sequence: Tris-HCl buffer, XïT, HX,

neurotransmitter or iron complex, and XO, which was added last to initiate the reaction. A DW-

2000 split-beam spectrophotometer (SLM Instruments Inc., Urbana, IL) was used to follow the

reduction of X T î by Oz'- at )I = 470n.m. Data points of the time-based single wavelength scan

were plotted and ICso values (for inhibithg IUT reduction by 027 were calculated with

regression analysis. Methods were adapted from previous work carried out in our laboratory

(65). Nitro blue tetrazoliurn was however, replaced with XTï-a new tetrazoliurn salt-for the

measurement of Oz' radical formation, which reportedly does not interfere with XO activity

(78). Furthemore, to assess whether or not the iron complexes could interfere with XO activity,

uric acid production (k293nm) was measured in the presence and absence of the cornplex.

Hepatocytes were isolated from Sprague-Dawley rats (275-300g) by collagenase

pemision of the liver, as previously described (65). Isolated hepatocytes (106 cellslml) were

suspended in Krebs-Henseleit buffer (pH 7.4) containing HEPES (12.5rnM) in continuously

rotating, 50mL round bottom flasks using a Rotavapor rotary evaporator (Buchi, Switzerland) in

a water bath heated to 37 O C . The cells were first exposed to 5%COz/1O%O2/85%N2 atmosphere

for 30 min. Hypoxia was initiated by changing the atmosphere to 5%C02/95%Nz.

Reoxygenation with 1 %02/5%C02/94%N2 occurred at 70 minutes. C ytotoxicity was determined

by withdrawing a 100 pL aliquot to which was added 100 pL of trypan blue dye (0.1 %w/v) to

quanti@ ce11 viability (trypan blue exclusion assay).

Oxygen consumption by the neurotransmitter:iron complexes was measured using a

Clarke type electrode. ImM neurotransrnitter (e.g., DA) was pipetted into the oxygen electrode

charnber containing O.1M Tris-HC1 buffer (pH 7.4) at 20 OC and oxygen uptake was followed

after the addition of 0.5rn.M FeC13 or CuS04. Tris-HCI buffer was used as phosphate buffers

have metal contaminants, and could lead to enoneous results in this metal-sensitive system.

Statistical significance of the difference between control and experimental groups was

detemiined by Student's t-test.

RESULTS

As shown in Table 1.1, the phenolic/catecholic neurotransmitters showed varying degrees

of O?' scavenging activity. DA was the strongest scavenger and EPI the weakest. The order of

decreasing effectiveness was: DA > NE > tyrosine > serotonin > 5-hydroxyindoleacetic acid >

a-methylDOPA > 3,4-dihydroxyphenylacetic acid (DOPAC) > DOPA > EPI. Homovanillic

acid ( W A ) however, showed no detectable Oz' scavenging activity. A marked increase in

SOD-mimicking ability was however found when the phenolic neurotransmitters were

cornplexed with femc iron. Serotonin showed only marginal activity, and HVA showed no

detectable OzC scavenging activity when complexed with femc ions. Femc iron alone showed

no detectable O; scavenging activity. The m k order for the OzC scavenging of the

neurotransmitter-~e'+ complexes was: a-methyiDOPA > NE > 3.4-dihydroxyphenylacetic acid >

EPI > DA > DOPA > 5-hydroxyindole acetic acid > tyrosine > serotonin. It is interesting to note

that some of the weakest Oc scavenging substrates became much more active when complexed

with iron. Any direct inhibition of XO was mled out since the iron complexes did not affect uric

acid production even at much higher concentrations.

As shown in Table 1.2, SOD, DA, and serotonin protected hepatocytes from hypoxia-

reoxygenation injury. Furthemiore, when the DA was complexed with femc iron, 40pM of the

D A : F ~ ~ + was as effective as 150pM DA in protecting the hepatocytes from hypoxia-

reoxygenation injury. DA was much more cytoprotective as a femc iron complex yet ferric iron

alone was not cytoprotective. A similar enhancement of the cytoprotective effectiveness of NE

and serotonin was also found when these catecholic/phenoLic neurotmsrnitters were complexed

with ferric iron.

The iron complexes formed a purple colour upon addition of FeC13,

by the addition of excess EDTA to reform DA with no evidence of

which was reversed

quinone formation.

Furthemore, very little oxygen uptake was observed over a 1-hour incubation period when 1 mM

D A - F ~ ~ + was incubated in contrat to DA-CU", (Table 3). The copper complexes however,

gradually fomed a deep yellow coloured product, which was not reversed by EDTA.

DISCUSSION

The results descnbed show that femc iron complexes of catecholic neurotransmitters

were much more effective at scavenging Oz' radicals than the uncomplexed neurotransmitter.

The femc iron catecholic complexes most potent at scavenging 02- radicals were a-

r n e t h y ~ 0 ~ ~ - ~ e 3 + (IC50 - ISpM), NE-F~)+ (& - 4pM), DOPAC-F~~+ (1Cso - 15pM), EPE

~ e ~ + (ICSo - 17CLM), and DA-F~)' (IC 50 - 19p.M). Serotonin-Fe '+ was the least potent 0;-

scavenger because hydroxyindoles have a much lower affinity for l?e3+ than catechols (79).

For the uncomplexed neurotransmittes, DA and NE were the most potent Oz*

scavengers (IcSa - 55p.M and - 1 IOpM, respectively). The Oz* scavenging ability of DA and

NE may be refiected by their low reduction potential, however this does not inàicate why

serotonin is not an equal, if not a better scavenger by virtue of its reduction potential (Table 1).

It is likely that the catechol moiety yields a more potent 0; scavenging activity, since serotonin

is an indoleamine containing a phenol group. Although the reduction potentials (E/mV) partly

correlate to 02 scavenging, pK. values did not show such a direct correlation (Table 1). The

les t effective scavenger was EPI whereas HVA, a methylated metaboiite of DOPAC, w u

ineffective probably because of its lack of a catechol moiety.

Hepatocytes in our hypoxia-reoxygenation model were significantly protected against

reoxygenation injury by the femc complexes. The DA-F~~ ' complex conferred the most potent

cytoprotection, equivalent to approximately 4 times DA alone. Interestingly, serotonin was more

potent in vitro in cornparison to the enzymatic 0;- scavenging systern, indicating that sorne

other mode of cytoprotection may be provided. XO inhibition can be ruled out since no

enzymatic interference was observed with the femc complexes. However, it has been reported

that DA-F~~ ' complex at 1mM can undergo autoxidation if 5mM cysteine is present (80).

However, we have found that DA-F~'+ was not cytotoxic to isolated hepatocytes even at

concentrations 20-fold higher than those used here (see Chapter 3, Fig. 3.1 for details).

Ischernic brain injury following stroke ensues over a penod of hours, during which a

cascade of cellular and biochemical events inevitably Ieads to destruction of brain tissue.

Synthetic SODkatalase mimetics such as saien-manganese complexes (known as EUK-8 and

EUK-134), showed substantial neuroprotective effects in a rat stroke model (4). Although the

involvement of ROS in ischemic brain injury is not the only mechanism of damage, it is one of

the primary elements. Thus, it would be interesting to investigate whether DA-iron is more

effective than DA at protecting dopaminergic neurons against OzC mediated reoxygenation

injury.

The relevant antioxidant contribution of the substrates tested may be significant in the

light of neurotransmitter concentrations found in various brain regions. For exarnple, the DA

concentration in a dopaminergic nerve terminal has been reported to be approximately 50mM.

although mostly stored in vesicles (81, 82). Furthemore, it has been reported that astrocyte

miiochondria sequester redox-active iron in nigral astroglia (83).

In summary, neurotransmitter-iron(III) complexes were shown to be potent ozk radical

scavengers, and DA-iron was shown to convey potent cytoprotection when hepatocytes were

challenged with hypoxia-reoxygenation injury. Our model could reflect an in vivo mechanism

since hypoxia-reoxygenation has been shown to be as damaging as ischemia-repemision to the

liver (68). Further research is required to determine whether catecholamine-iron complexes are

also cytoprotective in a stroke model and could have therapeutic potential.

22

Table 1.1 ICso values for 0 1 scavenging activity of neurotransmitters and neurotransrnitter- iron(m) complexes

Neuro transmi tt e r

Ligand

DA

DOPAC

HVA

Serotonin

5-Hydrox yindole- acetic acid

Tyrosine

DOPA

a-methylDOPA

NE

EPI

in the HXXO system.

O?* scavenging was assessed spectrophotornetncaUy by following formation of the reduction product of XTT at h = 470nm as descnbed above. * Ref. (84); ** Refs. (85), (74).

Table 1.2 Cytotoxicity of hepatocytes upon hypoxia-reoxygenation by neurotransmitters and iron(III) complexes.

--

Neurotransmîtter % Cytotoxicity (70 min)

Control

SOD (lûûU/mL)

serotonin ( 1SOp.M) 35.0 & 3.6*

serotonin (40p.M) 59.3 + 4.0

serotonin-~e)+ (40:20FLM) 48.3 k 3.5* Isolated hepatocytes (10' cellslml) were incubated under a hypoxic atmosphere of 5%C02/95%N2 with neurotransrnitter/ion complexes followed by reoxygenation at 70 minutes with 1 %02/5%COt/94%N2 as described above. Cytotoxicity represents the percentage of dead cells assessed by %an blue uptake at 70 min. Results are the means of three separate expenments (I SEM). * p < 0.05

Table 1.3 O2 uptake with 2: 1 neurotransrnitter:metal complexes.

DA

a-methylDOPA

NE

DOPAC

EPI 1

Neuro transmitter

1 I

Oxygen uptake was measured using a Clarke-type O2 electrode in a 2m.L charnber containing O.1M Tris-HCl buffer pH 7.4 and neurotransrnitter (ImM) at 20 OC. The reaction was initiated by the addition of either OSmM femc or cupnc metals. Concentrations shown are finai in a 2mL volume.

O2 uptake (nmol O f i n ) FeC13 CuSO4

Chapter 2

Ascorbic acid, glutathione, and NADH oxidation by catecholarnine o-quinones: the relationship between O-quinone stability and catecholamine cyclization.

ABSTRACT

The toxicity of various CAS has been suggested as contributing to neurodegenerative

diseases, adrenal carcinogenesis, or repemision injury. In this study, the ability of oxidized CAS,

specifically CA O-quinones, to oxidize and/or deplete ascorbic acid, GSH, and NADH. have been

compared. The half-life of their respective O-quinones, before forming the irreversible

cyclization products, aminochromes, have been measured. It was found that the various CAS

were less likely to oxidize ascorbate and NADH if they have a short O-quinone half-life as a

result of rapid cyclization. even though quinoid end-products were formed. The results of this

study suggest that CA O-quinones mediate the oxidation of ascorbate or NADH, and depletion of

GSH catalyzed by microsornesMADPH. Therefore, CA O-quinones c m be regarded as

mediators of CA-related disease States.

KEY WORDS: catecholamines; quinones; ascorbic acid; NADH, aminochrome; peroxidase;

tyroshase.

CAS have been implicated in cardio- and neurotoxicity. The mechanisrns involved c m be

divided into two main tenets. The fmt tenet includes functional effects, such as receptor over-

stimulation (86), neuromodulation of adjacent neurons to induce subsequent toxicity (36). and

alterations in merid tone resulting in myocardial necrosis (46). The second tenet suggests that

the CAS are oxidized to reactive toxic metabolites (48, 87-89). The CA oxidation products that

are thought to mediate this damage include O-semiquinones. O-quinones, and their quinoid

oxidation endproduct, the aminochromes, (for details, see General Introduction, Fig. II). The CA

semiquinone is relatively short-lived, and is produced by a one-electron oxidation followed by

spontaneous disproportionation to an O-quinone and 0;- (90). L-DOPA and DA were proposed

to fom O-semiquinones that deplete GSH (91). Furthermore, it was suggested that DA o-

semiquinone produced from DA oquinone by NADPH cytochrome P450 reductase was

responsible for the cytotoxicity that occurred when Chinese hamster ovary cells were incubated

with DA (92). The unique property of the oxidative cyclization of CAS to form aminochromes

has lead researchen to consider that these oxidation end-products may possess some

toxicological relevance. Aminochromes deplete GSH by forming GSH conjugates catalyzed by

glutathione-S-tramferase (93). Furthemore, adrenochrome (EPI end-product) produced damage

to isolated and pemised rat hearts (94). A mechanistic hypothesis for aminochrome toxicity has

been explained regarding its cardiotoxic effects, but neurotoxic rnechanisms have been met by

controveny (16). However, the aminochromes for different CA have different properties as do

their precurson.

The question is, do CAS, specificaily DA, require metabolic activation for toxicity to

occur and if so, is it via transition metal catalyzed autoxidation (21) or does it involve enzymes

such as peroxidase, prostaglandin H synthase, XO, tyrosinase, lactoperoxidase, or cytochrome

P450? (95-99). DA, when oxidized, binds to protein ( IO), DNA (101), and inactivates enzymes

e.g. tyrosine hydroxylase ( 102, 103). The presence of GSH, dithiothreitol, cysteine, or ascorbate

prevented enzyme inactivation suggesting that the toxic reactive intermediates are scavenged by

these compounds. DA toxicity in PC12 cells was also prevented by GSH, N-acetylcysteine, or

dithiothreitol (5). The role of thiol-containing antioxidants in preventing catechol(amine)

induced cytotoxicity is, therefore, an important requirement for ce11 viability. Furthermore, the

involvement of quinone intermediates seems to play a part. However, certain molecular

explanations are lacking as to why one CA would possess more toxic potential than another. For

example, NAcDA was more effective than DA or DOPA at inactivating tyrosine hydroxylase

(102), whereas DA was at least 10 fold more effective than EPI at inducing cytotoxicity in

cortical neurons (104).

We hypothesize that the cytotoxic activity of CAS cm be attributed to the O-quinone

metabolite rather than the aminochrome metabolite proposed by other investigators (48, 54, 55,

93). The ring-closed products of CAS, the aminochromes, are fonned by an intramolecular

Michael l&addition of the unstable O-quinone as a result of the appropriately positioned intemal

nucleophile (arnino nitrogen) (17, 105). Thus, O-quinones are intermediates in aminochrome

formation. Because ascorbate and GSH or other thiol-containing molecules c m prevent the toxic

effects of catechol(amine)s or their comsponding oxidation products, we have investigated the

possibility that the CA O-quinone intermediates are responsible for toxicity. Accordingly, the

haif-life and reactivity CA oguinones as well as their effectiveness at oxidizing ascorbate,

NADH, or depleting GSH to form GSH conjugates have been rneasured.

MATERIALS & METHODS

AU chernicals and enzymes were purchased commercially from a local supplier. (Sigma-

Aidnch, Oakville, Ont.). Mushroom tyrosinase (monophenol rnonooxygenase:oxygen

oxidoreductase, EC 1.14.18.1) and peroxidase (from horseradish) (donor: hydrogen-peroxide

oxidoreductase; EC 1.1 1.1.7; HRP) were the enzymes used in this study.

Catecholamine Cyclization Assay

Cyclization was observed spectrophotometricall y (UV-240, Shimadzu, Japan) b y

oxidizing each substrate with NaIQ since this method would not lead to oxidation products other

than the aminochromes, (106). Phosphate buffer (SOrnM pH 4.0) was used because cyclization

occurs much faster at an aikaline pH. 250pM of each cornpound was added to a 2 mL cuvette

containing the buffer. The same concentration of NaIO4 was added to the cuvette to initiate the

reaction, upon which measurements were recorded irnrnediately. Rates were caiculaied

assurning that al1 of the CA had been oxidized to its corresponding aminochrome end product,

when the reaction was stopped. Before comrnencing this expenment, the spectra of each

compound were recorded to find the aminochrome maxima (&) at pH 4, at room temperature.

Ascorbate Oxidation using HRP/H2 4 and Tyrosinase

Kinetic scans were recorded spectrophotometricaily at 266nm. the maximum absorbance

of ascorbate. To eliminate the trace metal-catalyzed depletion of ascorbate, a 0.1M Tris-HCI pH

7.4 bufTer was used containing 2mM DETAPAC to chelate any trace metal contaminants.

Ascorbate (50p.M) was added to the cuvette containing the buffer, and then. l0pM of

catechol(amine) was added. For the peroxidative oxidation of substrates, 1Op.M HzOz was added

and the reaction was initiated by the addition of O.1p.M HRP. For direct O-quinone formation.

70UlrnL tyrosinase was added last to initiate the reaction. The specific reaction rate was

calculated using the kinetic equation to determine k,

k = l In a - - t a-x

where a represents the moles of reactant initially present, and x is the number of moles that have

reacted after t h e t, leaving a-x unreacted (107).

Ortho-quinone halflife of catechol(amine)s

Before formation of the aminochrome end-product (cyclization assay), the CA must first

form an O-quinone product (which is unstable at pH 7.4). The CA (250pM) was added to a 2 mL

cuvette containing phosphate buffer (0.05M pH 4.0), and 250p.M of NalOl was added to fom

the O-quinone. The half life of the O-quinone at 390-4Wnm was calculated first by obtaining k

(Eq. l), and then by applying the following:

NADH ondation assay

Since O-quinones carry out a two electron oxidation of NADH (1081, a correlation of

their half-life with NADH CO-oxidation would confirm that this intermediate is responsible for

NADH oxidation. A 0.1M Tris-HC1 pH 7.4 buffer containing 2mM DETAPAC was used since

NADH hydrolyzes at low pH and trace metd contaminam could cause ascorbate autoxidation.

10pM of catechol(amine) was added to a 2 mL cuvette, fokiowed by lOpM H202 and O.1p.M

HRP. Three scans were performed: a) NADH alone, b) ascorbate added before NADH, and c)

GSH added before NADH. Part b) and c) would show which moiety-the semiquinone radical

or O-quinone-is preferentiaiiy formed by the substrate and responsible for NADH oxidation.

NAcDA-GSH Conjugate Identification

Both tyrosinase and HRP were used to oxidize NAcDA in order to form a GSH

conjugate. The reaction was carried out in water since buffer salts would cause interference for

mass spectrometry. In order to determine the products formed, LmM GSH was added before and

after the reaction was initiated. The 0.lp.M HRP or 20 U of tyrosinase were added Iast to initiate

the reaction. For HRP oxidation, 1mM Hz& was added with NAcDA. Only different 6-

hydroxylated products were formed by the addition of 200p.M NaOH, due to the use of water

(instead of buffer) required for mass spectroscopy analysis. It must be noted that the use of

water (instead of pH 7.4 buffer) caused a reduction of pH to 5.5, which explains the rationale for

using NaOH.

Microsomal GSH deplet ion assay

NADPH was added to ImL Tris buffer (O. 1, pH 7.4 containing DETAPAC lrnM), GSH,

microsornes and the test compound. The mixture was pre-incubated for 1 hour at 37 O C from

which 250pL was added to 25p.L trichloroacetic acid (30% wlv), vortexed, left for 5 minutes and

centrifuged. lOOjL of the supernatant was then added to a mixture of 25pL DTNB (2mg/mL)

and 875p.L Tris buffer (O. 1M pH 8.94 containing ImM DETAPAC) and vortexed. The

absorbante of the solution was monitored at 412nm. The standard curve for GSH measwements

gave a regression coefficient of greater than 0.99 over the range of 5-500pM GSH

concentrations. If a P450 inhibitor or SOD was used, it was incubated for 5 minutes with

microsornes before addition of the test compound. NADPH was added last to initiate the

reac tion.

Catecholamine Cyclization Rates

The rates of CA cyclization were determined by calculating the rate of aminochrome

formation for each cornpound, (Table 2.1). The cyclization order fiom fastest to slowest found

wax EPI, a-methylDOPA. DOPA, NE, DA. Only catechols containing a terminal amino group

on the end of the dkyl chah have the ability to form cyclized aminochromes. therefore.

compounds such as DOPAC or NAcDA are not shown since they were oxidized to relatively

stable O-quinones.

Ortho-Quinone Half Life

The rank order for O-quinone half-life (Table 2.1) from longest to shortest was: NAcDA,

DOPAC, DA, NE, DOPA, a-methylDOPA, and EPI. This order was found to be inversely

related to cyclization rate. That is, the shorter the half-life for an O-quinone, the faster it would

cyclize to form its corresponding aminochrome. This relation is best illustrated by EPI, since its

O-quinone half-life was undetectable presumably because it is the most rapidly cyclizing CA

(690 nmolhin).

Ascorbate oxidation by catechol(amine)s

As shown in Table 2.2, with the HRP/H202 system, NAcDA caused the fastest rate of

ascorbate oxidation, followed by DOPAC, NE, DA, a-methylDOPA. DOPA or EPI. Ascorbate

oxidation by the tyrosinase system resulted in a slightiy different rank order with NAcDA still

the fastest, foilowed by DOPAC, DA, DOPA, a-methyIDOPA, NE, EPI. Adrenochrome, (the

cyclized O-quinone of EPI) was added in the absence of either enzyme, and did not oxidize

ascorbate at these low concentrations.

Prevention of NADH Oxidation with GSH versus Ascorbate

Ascorbate and GSH prevented the oxidation of NADH by HRP/H20-oxidized

catechol(amine)s. GSH, however, was far more effective than ascorbate (Table 2.3, Figure 2.2).

There was a direct correlation of oquinone half-life to NADH oxidation: the greater the o-

quinone haif-life (Table 2 4 , the greater the NADH oxidation rate. The order for NADH

oxidation was: NAcDA, DOPAC, DA, NE, and DOPA. EPI and a-methylDOPA are not shown

because NADH oxidation by these CAS was not detectable. Interestingiy, GSH was more

effective in preventing NADH oxidation by oxidized DOPAC, DA, and NE (- 30 fold

prevention), than NAcDA (- 7 fold prevention). Adrenochrome did not oxidize NADH under

these reaction conditions.

NAcDA-GSH Products Found

Mass spectrometry of the NAcDA-GSH products formed revealed the following masses:

195, 209, 500, and 516. These values correspond to NAcDA, 6-OH-p-quinone-NAcDA or 6-

hydroxy-O-quinone NAcDA, NAcDA-mono-GSH, and 6-hydroxy-NAcDA-mono-GSH,

respectively. As shown in Figure 3, a similar range of products were formed with tyrosinase as

with peroxidase.

Microsoma1 Catalyzed GSH Depletion by Catechol (arnine)~

As shown in Table 2.4, ai i of the catechol(arnines) depleted GSH when incubated with rat

liver microsomes. The order of CA effectiveness at depleting GSH found was: EPI>NAcDA>

DOPAC>DA>DOPA>NE. Interestingly, phenylimidazole (cytochrome P450 2E I inhibitor) or

SOD completely prevented GSH depletion. With the exception of EPI, this order of CA

effectiveness at depleting GSH was exactly the same as that found for ascorbate oxidation

catalyzed by the tyrosinase system, implying that the same reactive intermediate (O-quinone) was

involved.

Table 2.1 Cyclization rates and O-quinone half-life of catecholamines

Substrate (250pM) 1 h, (nm) 1 Rate of cyclization 1 O-quinone tn (min)

NAcDA DOPAC DA NE DOPA a-methy lDOP A EPI

1 (nmoVmin) N/ A 1 N/A 246.8

Conditions: 250pM substrate was added to a 2mL quartz cuvette containing 50mM phosphate buffer pH 4.0. 250pM Nd04 was added Iast to initiate the reaction. The kinetics of the reaction were followed at the specific aminochrome wavelength (L) for each substrate. The O-quinone half-life was measured kineticaily between 390400 nm. *Not detectable.

N/ A 470 485 475 475 487

N/ A 11.7 27.5 71 24 1 690

39.6 13.2 5 -9 5.1 3.2 -*

Table 2.2 Ascorbate Co-oxidation with Substrates Using KRi?/H20 & Tyrosinase.

Substrate ( 1 0

NAcDA DOPAC NE DA a-methylDOPA DOE4 EPI Adrenochrome alone* Conditions: 0.1M Tris-HCl pH 7.4 with 2mM DETAPAC was added f i t to a 2 mL cuvette.

Rate of Ascorbate oxidaüon, k (mine1)

50pM of ascorbaie was then added and the absorbante was recorded. 10pM substrate and H202

W/H207 0.665

were added and 0.lpM HRP was added to initiate the reaction. For tyrosinase-mediated oxidation, 70U/mL was added to initiate the reaction. Ascorbate oxidation was followed at k266 nm.

Tyrosinase 1.1

* Adrenochrome was added in the absence of either enzyme or H2O2.

Figure 2.1 Percent NADH Oxidized by Catechol(amine)s by HRP/H202 and the Inhibitory Effect of Ascorbate or GSH. 50pM substrate and 50pM Hz02 was added to a 2 mL quartz cuvette containing 0.1M Tris-HC1 buffer pH 7.4 with 2mM DETAPAC. 50p.M of NADH, with or without 50pM ascorbate or GSH, was added followed by O.1pM HRP to initiate the reaction. NADH oxidation (solid bars), in the presence of ascorbate (diagonal bars) or GSH (clear bars) was measured at a fixed wavelength (h = 340nm). Bars represent the average percent of NADH

Table 23 NADH Oxidation In The f resence Of Ascorbate or GSH bv HRP/H,O?. Substrate (50p.M)

NAcDA DOPAC DA NE DOPA EPI

I - - NADH oxidized in 10 minutes

NADH 48.1 f 5.2 41.6 1: 3.8 21.2 f 1.5 12.4 + 0.9 1.9 f 0.2 ,*

Ascorbate, W H 23.1 f 1.2 31.8f 1.7 15.8 I 1.0 8.8 10.3 0.8 =t 0.04 -*

GSH, NADH 6.7 k 0.5 1.3 f 0.08 0.7 f 0.04 0.4 I 0.03 0.3 f 0.01 -*

50p.M substrate and 50pM H202 was added to a 2 rnL quartz cuvette containing 0.1M Tris-HC1 buffer pH 7.4 with 2mM DETAPAC. 50pM of NADH, with or without 50pM ascorbate or GSH, was added and the absorption was followed kinetically at h340nm. O . 1 p M HRP was added last to initiate the reaction. Results are the mean of three separate experiments (IS.D.). * Not detectable. ** Adrenochrorne was added in the absence of HRP/&û2.

NAcDA

HO

û-HO-NAcDA quinone (orlho or pan,)

NAcDA

quinone

GSH 308

O 1 O0 200 300 400 500 600

mhc

Figure 2.2 Products Found by Mass Spectroscopy with NAcDA + Tyrosinase or HRPM202 + GSH. Conditions: ImM of NAcDA was added to a 1.5mL en end off via1 contalning purified water. 1mM GSH was added before or afier initiating the reaction. 20 U/mL of tyrosinase or O. 1pM HRPllmM H202 were added to initiate the reaction. Formation of the 6-hydroxylated NAcDA products was only accomplished by adding 200pM NaOH just before injection into the mass spectrometer.

Table 2.4 GSH depletion in microsomal preparation

% GSH depletion (rnicrosomes)

NAcDA

15 minutes

+ pheny Iimidazole + SOD

30 minutes

DOPAC

+ pheny limidazole + SOD

+ pheny Iimidazole -c SOD

DOPA

+ phenylimidazole + SOD

-

-

-

-

O I O Conditions: Microsorne = lmg/mL, NADPH = lmM, GSH = 200ph4, phenylimidazole =

300pM, and test compound = ImM in a total volume of 1mL (0.1 M Tris-HCl ImM DETAPAC). Percentages are derived from a standard c w e for GSH over the concentration range used at 412nrn (TNB peak absorbance). Phenylirnidazole or SOD was preincubated 5 minutes before initiating the reaction.

EPI

+ phenyIimidrrzole + SOD

+ phenyIimidazoIe + SOD

DISCUSSION

Our findings show that the CA-mediated ascorbate and NADH oxidation catalyzed by

HRP/H202 or tyrosinase is fastest with catechol(amine)s that form stable O-quinones and

suggests that the O-quinone metabolites mediate ascorbate or NADH oxidation and GSH

depletion in microsomesMADPH. The order for ascorbate oxidation by HRP/H202 ( N A c D b

DOPAC>NE>Dba-methylDOPA>DOPA=EPI) was aimost identical to the order found for

NADH oxidation (NAcDA>DOPAC>DbNE>DOPA>EPI=a-methylDOPA). Furthemore,

CAS with microsomes~ADPH depleted GSH-with one exception-in a similar order, (EPb

NAcDPUDOPAC>DA>DOPA>NE). The stability of O-quinones at pH 4 (NAcDA>DOPAC>

DA>NE>DOPba-methyDOPA>EPI) seems to be inversely related to the cyciization rate

(EPba-methylDOPA>DOPA>NE>DA). EPI O-quinone was undetectable presumably because

of its very rapid cyclization rate. Our findings for CA cyclization is in agreement with a

previous study using DA, NE, and EPI at an alkaline pH using periodate (17). NAcDA O-

quinone or DOPAC were the most stable O-quinones as they did not cyclize, and were most

effective at oxidizing ascorbate or NADH.

The cyclization property of the CAS causes them to form aminochrome end-products with

characteristic absorption spectra. As described in the General introduction (see Fig. LI), in order

to form these end-products, the CA must h t f o m its corresponding O-quinone. Our findings

show a clear inverse order between the stability of the O-quinone versus the cyclization rate

(aminochrome formation). If the O-quinone of a given CA has a long half-life, it will cyclize to

its minochrome more slowly than a CA O-quinone with a relatively short half-Me. Since this

cyclization occurs readily at physiological pH, an acidic buffer was used to study this

relationship for the CAS. NAcDA, therefore, was useful as a mode1 of a stable DA O-quinone, as

it did not form an aminochrome because it lacks the amino terminus present in CAS. Therefore,

in Eqn. 3, NAcDA O-quinone could represent a long-lived CA O-quinone intermediate, without

the event of forming its aminochrome end-product:

[O1 ~ a s t at T p~ CA CA oquinone b Arninoc hrome

Slow at 4 pH

Ascorbate is an important antioxidant in the brain that has a relatively high concentration.

In both rat and human striatum, ascorbate concentration is 1 to 2 mM (109, 1 IO), and

approximately 10 rnM in isolated nerve terminais (1 11). In order to determine what reactive CA

metabolite is involved in oxidizing ascorbate, Le., CA O-quinone or aminochrome, two enzyme

systems were used. HRP is a good mode1 peroxidase for endogenous prostaglandin H synthase.

another peroxidase (100). HRP catdyzes the 1-electron oxidation of a catechol substrate to a

serni-quinone intermediate. This semiquinone then disproportionates spontaneously to form an

O-quinone and OzC, as depicted in Eqn. 4:

HzOz + CA CA semi-quinone + H20 O2 + CA O-quinone + 4' (4)

The other enzyme used to oxidize CAS was tyrosinase. The latter uses O? as a cofactor and

catalyzes the 2 electron oxidation of a catecholamine to an O-quinone (Eqn. S), without the semi-

quinone intermediate:

Recent evidence suggests that tyrosinase could be expressed in the brain (1 12, 1 13), but others

disagree ( 1 8).

Dehydroascorbate r-

Asc

Scheme 2.1 Biochemical pathways involved in the oxidation of catechols to O-quinones and the mechanisms of ascorbate or NADH oxidation and GSH conjugation. (See text for details).

In order to determine whether CA O-quinone or aminochrome was responsible for the oxidation

of ascorbate, it was necessary to correlate O-quinone stability or cyclization rate with the

ascorbate oxidation rate. If the cyclization rate was to directly conelate with ascorbate oxidation

rate, this would indicate that the less stable a CA O-quinone is, the more tikely it is to oxidize

ascorbate (rnost probably due to the formation of its aminochrome endproduct). On the contrary,

we found that the O-quinone half-iife was directly related to the oxidation rate of ascorbate (the

longer O-quinone half-life would yield the faster rate of ascorbate oxidation). This implies that

the aminochrome end-product is not responsible for the oxidation of ascorbate. Indeed, it was

shown that adrenochrome, the EPI oxidation end-product, did not oxidize ascorbate, even though

it contains a quinoid structure. However, in another reaction system, 40pM adrenochrome and

20mM ascorbate showed that under these conditions, adrenochrome reduction (therefore

ascorbate oxidation) occurred ( 1 14). Therefore, it cannot be categorically concluded that

adrenochrome doesn't oxidize ascorbate; it simply occun at much higher concentration than for

CA oquinones.

Since we used two enzymatic systems to f ~ s t oxidize the catechol(amine) which then

oxidized ascorbate, the factor of substrate specificity was unavoidable. In the HRPR1202 system

for ascorbate oxidation, NE had a faster rate of ascorbate oxidation than DA. If O-quinone half-

Me is to correlate with ascorbate oxidation, then DA should have a higher rate. NE is oxidized

by HRP almost 5 fold faster than DA (98). The next discrepancy in this series was that

a-methylDOPA oxidized ascorbate faster than DOPA. HRP also catalyzed the oxidation of DA

five-fold higher faster than DOPA. One study showed that a charged amino group couid inhibit

access to the peroxidase heme group, (1 15). Possibly, the presence of a methyl group could give

a-methylDOPA easier access to the peroxidase active site. In the tyrosinase series, DOPA

oxidized ascorbate better than NE. Since DOPA cyclizes faster than NE, it should oxidize

ascorbate at a slower rate than NE. In fact, DOPA is about a 4 fold better tyrosinase substrate

than NE, which could explain this observation (98). Furthemore, the potency of NAcDA and

DOPAC in CO-oxidizing ascorbate could be explained by the presence of an acetyl group on the

alkyl c h a h N-acetyltyrosine was a 19 times better HRP substrate than tyrosine (1 15).

With respect to ascorbate oxidation, CA cyclization may be considered as a pathway ihat

would prevent ascorbate oxidation. However, the aminochromes themselves may participate in

other biochemicaily relevant pathways not studied here. NADH is a vital cofactor in various

enzyrnatic reactions. NAD(P)H:quinone oxidoreductase, which reduces quinones back to their

parent dihydroxy molecule, uses NADH preferentially as a cofactor (1 16). In fact, semi-

dehydroascorbate reductase. responsible for the reduction of the ascorbyl radical, also uses

NADH as an electron donor (117). and NADH dehydrogenase is a key component of the

mitochondrial respiratory chah that utilizes NADH ( 1 18). Interestingly, the quinones formed in

this study affect both ascorbate and NADH. NADH oxidation was studied to determine if a

sirnilar trend would be seen as with ascorbate oxidation, i.e., direct involvement of the o-

quinone. NADH undergoes a two-electron oxidation by quinones with a 1 : 1 stoichiometry (Eqn.

6) (108), therefore without forming a serni-quinone intermediate:

CA o-quinone + NADH + H) -+ CA + NAD+ (0)

The catalytic activity of the various catechol(amine)s for oxidiYng NADH in the W&02

system was identical to the stability of their respective oquinones with the most rapid oxidation

of NADH occuming with the most stable O-quinones. By definition, therefore, the CAS with the

fastest cyclization rate would be the least effective at oxidizing NADH. Adrenochrome again

did not oxidize NADH Qust as with ascorbate above). In both ascorbate and NADH oxidation,

the lack of evidence for the oxidation of either compound by adrenochrome most likely reflects

its low redox potential (E = -0.253) compared with its precursor, EPI O-quinone (E = 0.38) (1 19).

Furthemore, both ascorbate and GSH prevented the oxidation of NADH, implicating the o-

quinone as the oxidiYng agent. It must be noted, however, that GSH provided substantially

more prevention of NADH oxidation than ascorbate. This probably reflects the different

mechanism of quinone reduction as depicted in Scheme 2.1. The "futile cycle" could occur with

ascorbate because of its two-step reduction of the O-quinone. Since quinones readily form GSH

conjugates (120), we have identified, for the first time, the NAcDA-GSH conjugate.

HRP and tyrosinase both produced GSH conjugates of NAcDA that were identified by

mass spectroscopy. Although NAcDA has only been found in the periphery (23), this result

could be extrapolated for the formation of the potent neurotoxin. 6-hydroxy-DA, since 6-

hydroxylated NAcDA was identified. Our finding could mean that the formation of 6-hydroxy-

DA could occur by two steps: an initial oxidation reaction foliowed by nucleophilic addition of

hydroxyl (water) to the 6-position of the benzene ring. The formation of 6-hydroxy-NAcDA was

demonstrated biochemically in the W/H202 system. however excess HzOl was required to

carry out the reaction (106). Although the 6-hydroxy NAcDA products were formed at an

aikaline pH, the 6-hydroxylation also occured at a physiologicai pH (106). Although

adrenochrome was shown to be relatively non-reactive in our system, one group studied which

glutathione-S-transferases would best conjugate GSH with the aminochromes themselves, but

did not study GSH conjugation to the correspondhg O-quinone precursors (93). It was shown

that the specific isofom of glutathione-S-transferase (Ml-1) that cataiyzed GSH conjugate

formation ffom adrenochrome was different fiom the isoforms that catalyzed GSH conjugate

fonnation with other aminochromes (93). Since this specific isofom is expressed in the rat Liver,

it could account for the GSH depletion observed by EPI (121). In our study, however, the GSH

conjugate of NAcDA was formed in the absence of glutathione-S-transferase.

Our assay for GSH depletion by CAS catalyzed by microsomesMADPH was similar to

the CA order for ascorbate oxidation catalyzed by tyrosinase system. Since tyrosinase produced

an O-quinone metabolite directly, it is most probable that this same O-quinone metabolite was

formed and caused the observed GSH depletion. Al1 catechol(arnine)s depleted GSH in an order

directly related to their O-quinone half-lives. One important exception. however, was EPI which

caused the most GSH depletion, although it was the least effective at oxidizing ascorbate. Since

EPI is the fastest CA to form its aminochrome (adrenochrome), and has a very short-lived

o-quinone, it is unlikely that EPI would deplete GSH catalyzed by microsornes via its EPI O-

quinone metabolite. It is therefore likely, that microsorna1 glutathione-S-transferase (122)

catalyzed adrenochrome:GSH conjugate formation.

The role of CYP 2El in generating O?' (Eqn. 7) that catalyzes the autoxidation of the

e' RH-(F~*C)-O~ --+ R H - ( F ~ ~ - O Z ' - ~ RH-(FelC)-02" + 02' 2, CA O-quinone (7)

2 8 + 202' SoD O2 + HZ02 (8)

CAS to form the CA O-quinone (Eqn. 8) was demonstrated since phenylirnidazole (CYP 2E1

inhibitor) and SOD both completely prevented the depletion of GSH by al1 compounds tested.

Oxidation of CAS have been shown to be mediated by 02- generated by P-450 or XO (43, 97,

123, 124). It must be noted, however, that OzC c m disproportionate to H202, which can also

oxidize GSH, but at a slower rate if not accompanied by GSH peroxidase catalysis. The

prevention of GSH depletion with SOD, therefore, niles out GSH oxidation by HzOz.

In addition to other cpotoxic events, we have established that catechol(amine) o-

quinones have the potential to oxidize ascorbate and NADH, as well as deplete GSH in a

microsorne/N ADPH catal yzed reac tion. Furthermore, as a mode1 arninoc home, adrenochrome

was shown to be relatively benign in this system. If the O-quinone, therefore, is to be considered

as a cellular hazard, how cm the ceii prevent oxidative injury? Various reductants exist in the

cell, but this discussion will focus on the enzymatic processes that can provide detoxification.

Quinones cm be reduced by one- or two-electron transfer enzymes. NADPH-cytochrome P-450

reductase catdyzes a one-electron reduction of a quinone to a semi-quinone. However, as was

shown above with the example of the ascorbate "fittile cycle," the semi-quinones are unstable

and will disproportionate to reform the quinone, concomitantly producing O?' (125).

Another enzyme that would offer better elirnination of quinones is NAD(P)H:Quinone

Oxidoreductase (NQO, EC 1.6.5.5.; also referred to as DT-diaphorase and quinone reductase).

This enzyme cataiyzes a two-electron reduction of the quinone to form its hydroquinone (126).

NQO would catalyze the reduction of CA O-quinone to its parent CA. In fact, one group has

performed extensive research on the aminochromes of DOPA, DA, and NE, to which end they

found that if NQO catalyzed the reduction of any one of these aminochromes to its hydroquinone

variety (referred to as leucoaminochrome), this molecuie would quickly autoxidize to reform the

aminochrome and form (127-129). This could represent a more downstream "futile cycle"

than that described above, in that the redox cycle is occumng with the aminochrome rather than

with its precursor (Scheme 2.2). Therefore, caution must be exercised in order to determine the

possible outcome of quinone reduction. The non-cyclized CA O-quinones have not yet been

shown to be substrates for NQO, although it is iikely that they are.

In summary, our findings show that CA O-quinones oxidize ascorbate, or NADH. They

aiso deplete GSH by forming GSH conjugates. The CA cyclization rate is inversely related to

the O-quinone half-life and the pro-oxidant activity of the O-quinone. Further work should be

canied out to study the in vitro and in vivo possibility of O-quinone toxicity contributing to the

pathological consequences of Parkinson's disease, as weli as the cardiotoxicity induced by CA

administration.

R1

HO O 0 (relatively slow autoxidation) t

HO

I /NH R2 /NH

CA

R2

CA o-quinone

NADH NAD+

Leucoaminochrome Aminoc hrome or reduced aminochrome (relatively stable) (unstable)

. L

O2

Scheme 2.2 Reduction of a CA oquinone and aminochrome. Although two-electron quinone reduction is generaiiy thought to be a protective event for the ce11 (as with the reduction of the CA O-quinone), reduction of the aminochrome results in the formation of an unstable hydroquinone that WU spontaneously re-oxidue to its CA precursor with concomitant Oz' production. If this "futile cycle" occurs repeatedly, it could Iead to oxidative stress.

Chapter 3

Dopamine rnetabolic activation by Pa50 peroxygenase activity versus manganese (II): DA O-quinone as the mediator of cytotoxicity.

ABSTRACT

DA depletion in the dopaminergic neurons of the substantia Riga is the biochemical basis

for Parkinson's disease. The incidence of a Parkinson's-type disease is markediy increased in

manganese minen. The purpose of this study was to compare the cytotoxic mechanisms of

manganesesatalyzed DA oxidation, versus normal cellular DA oxidation. Isolated hepatocytes

from Sprague-Dawley rats were used as a mode1 ce11 system since they are the ce11 choice for

studying dnig metabolism. Cytotoxicity for 700p.M DA:25pM ~ n ' + was 79.7 f 4.0 % compared

to 700 pM DA aione (- 1 %), and was prevented by GSH, ascorbate, and xylitol. The antioxidant

enzymes SOD, cataiase, and the antioxidants Trolox, Tempol, or butylated hydroxy toluene

(BHT) had no effect, indicating that ROS were not involved. Cytotoxicity was markedly

potentiated by the NQO inhibitor, dicumarol. It is concluded that DA O-quinone is responsible

for the cytotoxicity of DA:M~? The next set of experiments was aimed at determining the

cellular metabolic activation mechanism of DA in the absence of ~ n ' + . Al1 three MAO

inhibitors showed complete protection against DA induced cytotoxicity. Phenylimidazole

(CYPZE I inhibitor) d so prevented DA toxicity. However, dicumarol (NQO inhibitor) and azide

(catalase inhibitor) potentiated the toxicity of DA alone. The results of this study suggest that in

the hepatocyte DA is oxidized to the O-quinone via P450 peroxygenase activity which utilizes the

H202 generated by DA metabolism by MAO.

KEY WORDS: dopamine; manganese; Cytochrome P-450 CYP2E1; quinones; hydrogen

peroxide

The degeneration of DAergic cells in the substantia nigra results in the clinical

symptoms charactenzed by Parkinson's disease. Although the etiology of the disease remains

elusive, many possible factors have been identified. The most comrnon example is MPTP, a

byproduct of illicit mependine synthesis which rapidly induced Parkinson's disease in users.

Once injected, MPTP exerts its toxicity by selectively damaging DA-ergic neurons of the

substantia nigra (130). It has proved to be a useN tool for animal rnodels of Parkinson's

disease. This finding suggested that the possible etiology of the disease could originate from

exogenous sources. One distinct similarity is the effect of manganese intoxication. First

described by Couper in 1837, "manganism" has been an intriguing subject of study. It tesembles

the clinical effect of Parkinson's disease, manifesting the characteristics of rigidity, tremor, and

akinesa, although dystonia is reported as manganese related (131). Morphologically, the

autopsied brains of both manganese-intoxicated and Parkinson's disease patients reveal a loss of

neuromelanin, the dark polymer of the substantia nigra. One group has shown DA to form

cysteinyl conjugates in the presence of manganese, implying an oxidation reaction catalyzed by

the latter (80). Interestingly, manganese in the absence of DA has antioxidant properties (132).

Therefore, an interaction must exist between DA and manganese that initiates the production of

toxic products. The focus of this study was to determine what products mediate the toxicity in

isolated hepatocytes as a mode1 for manganese intoxication. DA, however, possesses its own

cytotoxic and cytoprotective properties. Some authors have found that DA and L-DOPA c m

induce cytotoxicity and apoptosis (87, 133). Others insist that DA and related compounds or

metabolites are antioxidants (33,34).

In this study, we have compared two mechanisms of DA cytotoxicity: one originating

exogenously, i.e., manganese, and the other occurrhg endogenously. We sought to determine

the mechanism whereby manganese (II) (Mn2+) acts as a catalyst for the oxidation of DA to toxic

products-namely, the DA O-quinone. It is already known that Mn2+ catalyzes the formation of

dopaminochrome-the endproduct of DA oxidation before melanization (2 1). Before formation

of dopaminochrome, however, the DA O-quinone must be formed. The latter is suggested by

Lloyd, who detected the presence of a DA semi-quinone radical by ESR in the presence of ~ n "

(134).

Surprisingly, the metabolic activation mechanism for DA is not known. We have

therefore investigated the cytotoxic rnechanism of DA resulting from its endogenous oxidative

metabolisrn in the absence of ~ n ~ + . No cellular studies have been reported before, although in

vitro enzyme studies have suggested that dopaminochrome toxicity may arise from oxygen

activation following reduction by NQO (128).

MATERIALS & METHODS

DA, tyramine, MnC12, CuS04, FeC13, GSH. ascorbic acid, xylitol, TEMPOL, TROLOX,

TEMPO, SOD (EC. 1.15.1. l), catalase, clorgyline, pargyline, phenelzine, sodium aide,

dicumarol. glucose, glucose oxidase (EC.1.1.3.4), and phenylimidazole were obtained

commercially, (Sigma Aldrich, Oakville, Ont.). Al1 chernicals were resuspended in ~ i l l i ~ @

purified water.

Hepatocyte Isolation and Preparation

Adult male Sprague-Dawley rats, 250-300 g, were obtained from Charles River Canada

Laboratories (Montreal, P.Q.), fed ad libitum and were allowed to acclimatize for 1 week on clay

chip bedding. Freshly isolated hepatocytes were prepared by collagenase perfusion of the Lver

as described by Moldeus et al., (135). Damaged cens, debris, and Kupffer cells were removed

by centrifugation with Percoll(136). The cells were preincubated in Krebs-Hensleit bicarbonate

buffer (pH 7.4) supplemented with 12.5mM HEPES for 30 minutes in a carbogen atmosphere, in

continuously rotating 50ml round bottom flasks at 37 O C before addition of chemicals.

Hepatocyte viability was assessed by the trypan blue (O. 1 % w/v) exclusion assay.

For statistical cornparison. at least n = 3 (flasks) was used for each condition tested. The

paired r-test was used since the experimental conditions for control and test flasks were identicai.

DA :~n'+ cytotoxicity experimen ts

M e r a 30 minute incubation period, DA was added to the cells immediately followed by

~n", (or other metals). Substrates added to modulate cytotoxicity. e.g., ascorbate or dicumarol,

were preincubated 30 minutes pnor to the addition of DAM&

DA Metabo lic Activation by CYP2 E I Peroxygenase Activiiy

Enzyme inhibitors or antioxidants were added as descnbed in the previous section, (prior

to the addition of DA alone). Since DA is rnetabolized by MAO-A in the rat (137), only

clorgyline was used to specifically inhibit that enzyme. The non-specific or irrevenible MAO

inhibitors pargyiine and phenelzine, respectively, were used for comparative purposes.

Similariy, both phenylirnidazole and metyrapone were used to inhibit different cytochrome P-

450 isoforms, 2E 1 and 2B 1, respectively.

lntracellular GSH and GSSG measurements

The total amount of GSH and oxidized glutathione (GSSG) in isolated hepatocytes were

measured by the HPLC analysis of deproteinized samples (5% meta phosphoric acid) after

derivatization with iodoacetic acid and fluoro-2,4-dinitrobenzene ( 13 8), using a Waters HPLC

system (model 510 pumps, WISP 710B auto injecter, and model 410 UVfvisible detector)

equipped with a Water rn Bondpack NH2 (10mM) 3.9 x 300 mm column. These methods were

used previously in our lab, and were repeated for our study, (1 39).

RESULTS

DA toxiciiy is potentiared by iWn2+, cu2+, but not ~ e ' +

The presence of 25p.M ~n'+ markedly enhanced DA cytotoxicity (approximately 80-

fold) (see Fig. 3.1). In the absence of ~n", a 21nM DA concentration was required to cause a

similar degree of cytotoxicity. The same concentration of CU" had a similar cataiytic effect and

enhanced DA cytotoxicity 46 fold. However, ~ e ~ + had no such effect on DA cytotoxicity, and

showed control cytotoxicity levels. Interestingly, ~ n " alone showed no cytotoxicity up to

ImM. However, 25p.M CU" alone caused some cytotoxicity in our system ( 140).

Cytoprotectiun against D A : M ~ ~ + by GSH, Ascorbate, and Xy litol

700p.M DA:SOpM ~ n ' + was used to cause 100% ce11 death since it was found to be a

lethal dosage. SOD, catalase, or ROS scavengers (TROLOX, TEMPOL, BHT) were ineffective

in preventing cytotoxicity, (Fig. 3.2). Only 1mM GSH, 1OmM ascorbate, or lOmM xylitol were

effective in preventing cytotoxicity. Ascorbate attenuated cytotoxicity presumably by reducing

the DA O-quinone to the serni-quinone, which subsequently reacts with ascorbyl radical,

resulting in the re-forming the parent compound. GSH is known to potentiy conjugate reactive

O-quinones such as DA O-quinone. Xylitol is a glycolytic substrate, which is oxidized to form

xylulose, using NAD' as an electron donor and producing NADH (141). The latter is the

cofactor for NQO and is utilized in quinone reduction.

NQO Inhibition and GSH Depletion Promo te DA: ~ n ~ + toxicity

Dicumarol, an inhibitor of NQO, potentiated DA:M~'+ toxicity (Fig. 3.3). Taken together

with Fig. 3 2, a potential mechanism of ~n'+-catal~zed DA toxicity is shown in Scheme 3.1.

Figure 3.1 Cornparison of Dopamine Cytotoxicity Catalyzed by Different Metals. Rasks containhg lOmL of hepatocyte suspension (106 cells/ml) were acclimated to a carbogen atmosphere before addition of corn ounds. In each case, DA was added first, followed by the metal (exeept with ImM Mn'+ alone). See text for details. * indicates significant difference from 7ûûp.M DA alone by paired t-test (p < 0.005).

Figure 3.2 Cytoprotection Against DA:M~'+ by GSH, Ascorbate, and Xylitol. Antioxidants or antioxidant enzymes were preincubated for 30 minutes before the addition of 7OO @A DA 150 pM Mn(II). GSH (ImM), ascorbate (IOmM), and xylitol(1OrnM) were the only antioxidants to significantly prevent ce11 death induced by 700m DA5Op.M ~ n " . * Indicates significant difference from 700m DA:SOpM hAn2+ by paired t-test @ c 0.005)

DA: Mn2+ 500: 10pM

Figure 3.3 NQO Inhibition Promotes DAIM^" toxicity. Dicumarol was preincubated for 30 minutes before the addition of DA / ~ n " to the hepatoc ytes. * Indicates significant difference from 500pM DA I 10pM ~ n ~ + by paired t-test @ < 0.005)

M n2+

NQO GS-DA

- P Y

NAD+ JT\ii iiicumarot A m NAD+

Xylulose Xylitol

Scheme 3.1 Proposed rnechanism of ~n~'-catal~zed DA cytotoxicity. See text for further details. DA-sQ, DA semi-quinone radical; DA-oQ, DA O-quinone; NQO, NAD(P)H:Quinone Oxidoreductase; GS-DA, DA-GSH conjugate; AA, ascorbic acid; A h , ascorbyl radical; DHAA, dehydro ascorbic acid.

M A O In hibitors Preven t DA Cyto toxicity

2mM DA alone is sufficient to cause significant ce11 death in 2 hours. The use of 20@l MAO-A

selective (clorgyline) and non-selective MAO inhibitors (pargyline, phenelzine) were effective at

protecting the ceils from DA-induced cytotoxicity, (Fig. 3.4).

H2 O2 is Involved in DA Metabolie Activation

IrnM DA alone does not affect hepatocyte viability, (Fig. 3.5). However, DA was highly

cpotoxic to NQO inhibited hepatocytes (with 20pM dicumarol). Inactivation of the MAO

activity of these hepatocytes prevented DA cytotoxicity. However, inactivation of catalase with

4mM azide rnarkedly increased DA cytotoxicity (100% ce11 death in 2 hours). At this

concentration, &de did not affect hepatocyte respiration or ce11 viability.

CYPZEI Oxidizes DA-not Tyramine-by &O2.- P450 Peroxygenase Activity

In the absence of ~n'+, 2mM DA was required to cause extensive ceIl death. As shown

in Fig. 3.6, the ROS scavengen (TEMPOL, TROLOX, TEMPO) did not confer any protection

against DA. Only 300pM phenylimidazole, a CYP2El inhibitor, was cytoprotective. The result

implicates the combined contribution of Hz02 and CYP2E1 in the peroxygenase-mediated

oxidation of DA. To contrast this finding with a sirnilar MAO substrate, 2mM tyramine was

used together with 300pM phenylimidazole and TEMPOL, (Fig. 3.7). There was no protective

effect of phenylirnidazole, but TEMPOL (ROS scavenger) significantly protected hepatocytes

from ce11 death, thus indicating a difierent mechanisrn of toxicity with this substrate.

Intracellular Levels of GSH and GSSG Refect Reactive Metabolites Formed

A cytotoxic concentration of DA almost completely depleted the hepatocytes of GSH,

with some GSSG formation (Table 3.1). Tyramine also caused the depletion of GSH, as a result

of GSH oxidation to GSSG. The DA O-quinone readily formed a GSH-conjugate (with GSH).

Figure 3.4 MAO Inhibitors Prevent DA Cytotoxicity. The MAO inhibitors (20pM) were preincubated for 30 minutes pnor to the addition of DA (2mM). * Indicates significant difference from 2mM DA alone, paired t-test @ < 0.005).

Figure 3.6 CYP2E 1 is hvolved in the Peroxygenase-mediated Activation of DA. The antioxidants and cytochrome P450 inhibiton, phenylirnidazole (CYP 2E 1 inhibitor), and metyrapone (CYP 2B 1 inhibitor), were preicubated for 30 minutes before the addition of DA. * Indicates significant difference from 2mM DA alone, paired t-test @ c 0.005).

Tyrarnine +Pargyline +Phenylimidazole +TEMPOL 2mM 20pM 3ww 3ûûpM

Figure 3.7 Tyramine, uniike DA Cytotoxicity, is Inhibited by a MAO Inhibitor and a ROS scavenger, but not by a CYP 2E 1 Inhibitor. Pargyline, phenylirnidazole or TEMPOL were preincubated for 30 minutes before the addition of tyramine. * Indicates significant ciifference from ImM DA alone, paired t-test @ < 0.05).

Table 3.1 Intracellular [GSH] and [GSSG] after incubation with DA or tyrarnine.

2mM Tyramine il Percentages are compared to control values. Values shown are the result of three separate experiments (S.E.M.). Isolated hepatocytes were allowed to acclimatize for 30 minutes in rotating round-bottom flasks at 37 O C . Either DA or tyramine was added to the flask, and at 3 hours incubation, GSH or GSSG concentrations were determined by HPLC. See text for details.

27.3 + 3.0 % 144.3 f 16.2 %

DISCUSSION

Our study was composed of two main parts: a) bln2+ catdyzed DA cytotoxicity, and b)

P450 peroxygenase activation of DA to a cytotoxic metabolite. Although the two approaches

focus on different etiology, i.e., exogenous/environmentai vs. endogenous, we believe that both

meet at the same juncture: the DA O-quinone, (Scheme 3.2,3.3).

Several theories have been presented to outline a mechanism for the Parkinson's-like

syndrome among manganese rninea. Manganese toxicity exhibits similar behavioural effects

seen in Parkinson's disease patients, with the exception of dystonia being a manganism

associated effect (13 1). One of the latesi reviews proposed a hypothesis for the mechanism of

~ n " toxicity in a scheme where the higher manganese oxidation states are thought to cataiyze

DA autoxidation and form H202, where higher rnanganese oxidation states are considered to be

the cytotoxic mediator (142). Another group proposed that DA could form toxic products,

possibly 6-hydroxy-DA via interaction of DA with free radicals produced by ~ n " (21). These

theories address the cataiyst, manganese, as the cytotoxic reactive species but the evidence

provided here shows that cytotoxichy was much more dependent on the DA concentration than

the ~ n " concentration. Furthemore, high concentrations of MnClz in the absence of DA were

not cytotoxic. In fact, manganese alone has relevant cytoprotective properties, such as ROS

scavenging and prevention of lipid peroxidation ( 132).

Previously, using electron spin resonance experiments. Mn2+ was shown to form a suong

but highly reactive complex with DA, which produced DA O-quinone and released Mn2+ (134).

Note that the manganese is in the same valence at the end of the ceaction, inaicating that only a

smail, hence catalytic, arnount of would be needed to oxidize DA (scheme 3.2). The

marked increase in DA induced cytotoxicity if hepatocyte NQO (EC L .6.5.5.) was inactivated

Scheme 3.2 Mechanism of manganesesatalyzed DA O-quinone formation, (adapted from Lloyd (1995), see ref. 129).

MAOl's (e.g., pargyIIne)

CYTOTOXICITY

Scheme 3.3 Proposed pathway for DA metabolic activation by P450 peroxygenase ac tivity/H202.

with dicumarol beforehand suggests that DA quinoid metabolites and not manganese higher

oxidation States, were responsible for the cytotoxicity.

Further evidence suggesting DA O-quinone involvement is the prevention of DA

cytotoxicity by xylitol. The latter is a sugar alcohol that is oxidized by xylitol dehydrogenase

(EC 1.1.1.9) to D-xylulose. The cofactor NAD+ is utilized as the electron acceptor in this

reaction, which forms NADH (141). We believe that NQO in the hepatocyte utilized the NADH

in order to reduce the quinone of DA. Microsomal NQO was recently characterized in the rat

liver and compared with its cytosolic counterpart. It was found to be more resistant to dicumarol

than the cytosolic NQO (1 16), indicating that the DA O-quinone is formed in the cytosol.

DA:M~'+ cytotoxicity was dso prevented by ascorbate or GSH. The mechanism shown in

scheme 3.1 iilustrates the roles of ascorbate and GSH in detoxiQing the DA O-quinone.

However, SOD, catalase, or hydroxyl radical scavengen did not protect against DA cytotoxicity.

Taken together, these results suggest that the interaction of ~ n " with DA results in the

formation of a toxic DA O-quinone that is mainly responsible for ce11 death.

As a cornparison of the mechanism of DA:M~'+-induced cytotoxicity, we investigated a

possible pathway in which DA in the absence of exogenous agents (e.g., metals) could cause a

similar event. We found that DA induced cytotoxicity was prevented by MAO inhibitors, and

by a cytochrome P450 2E1 inhibitor, phenylimidazole, but not by a CYP 2Bl inhibitor

(metyrapone). Furthemore, DA-induced cytotoxicity was potentiated by NQO inhibition (by

dicumarol) and by catalase inhibition @y azide). ROS scavengers were not protective in this

system. This result is in conflict with other investigators who c l a h that DA cytotoxicity is due

to Hz02 formation resulting from MAO metabolism (143). Therefore, we investigated the

possibility that P450 peroxygenase catalyzed the metabolic activation of DA to a reactive

metabolite.

To assess the role of HzOz produced endogenously by MAO, we preincubated isolated

hepatocytes with clorgyline (MAO-A inhibitor) and pargyline or phenelzine (MAO-A and B

inhibitors) and added DA to these cells. We showed that al1 of the MAO inhibitors were

successful in preventing the toxicity exerted by DA. Presumably, the HzOz produced normally

by DA turnover could not cause ceii death by either ROS or P450 peroxygenase activity. This

result is in conflict with previous reports which assumed that DA or DOPA cytotoxicity is due to

ROS formation as a result of DA or DOPA autoxidation (87, 143). Since the MAO is inhibited

in our system, this leaves DA unmetabolized and subject to supposed autoxidation. However,

little cytotoxicity was detected when MAO was inhibited. The prevention of DA cytotoxicity

seen with clorgyline, a MAO-A inhibitor, is due to the preferentiai metabolism of DA by this

isoform in the rat, whereas in humans MAO-B is preferred (137, 144). To investigate the role of

Hz02 formed by MAO-rnediated DA deamination, we preincubated isolated hepatocytes with

dicumarol, with and without pargyline, and with azide (catalase inhibitor). Dicumarol greatly

potentiated an othenvise non-toxic concentration of DA. indicating that NQO detoxification

provides significant cytoprotection.

The presence of pargyline with dicumarol significantly reduced the cytotoxicity seen with

dicumarol alone, indicating that MAO metabolism is stdl required for the toxicity of DA in NQO

inactivated hepatocytes. Azide with dicumarol were synergistic in increasing DA cytotoxicity,

indicating that the presence of H202 in combination with DA initiates a cytotoxic reaction. Since

the concentration of DA used was non-toxic to hepatocytes, these results suggest that Hz02 is

involved in DA oxidative activation.

CYP 2E1 inhibited hepatocytes were resistant to DA, indicating that CYP2El is chiefly

involved in the bioactivation of DA. The CYP2Bl inhibitor, metyrapone, was not

cytoprotective. These results in combination lead us to believe that CYP2E1 peroxygenase

activity is involved in the metabolic activation of DA to a cytotoxic O-quinone. Hydroperoxides

are beiieved to bypass the rate limiting step of the monooxygenase system of P450, Le., femc

P450 reduction by NADPHP450 reductase (145). Previously, tert-butyl-hydroperoxide or H a 2

was shown to enhance the cytotoxicity and metabolic activation of a variety of phenolic

xenobiotics which was prevented by the CYP2El inhibitor, phenylirnidazolc. It was concluded

that physiological hydroperoxides can be used by P450 to support the bioactivation of these

xenobiotics (139, 146).

The only difference between tyramine and DA is the presence of a 3-hydroxy group on

the benzene ring, hence DA is also referred to as 3-hydroxytyramine. Oxidation of tyramine

would not therefore be able to fonn a quinone. Phenylimidazole did not protect isolated

hepatocytes against a toxic dose of tyramine, indicating that tyramine was not oxidized to a

cytotoxic species or that tyramine was not a substrate for P450 peroxygenase. The ROS

scavenger TEMPOL or MAO inhibitor, pargyline, however prevented tyramine cytotoxicity

suggesting that the cytotoxicity was caused by H202 generated by tyramine metabolism by

MAO.

An analysis of intracellular GSH and GSSG level in isolated hepatocytes exposed to

tyramine or DA shows that GSH is depleted by almost 100% when incubated with DA, whereas

tyramine incubation resuited in GSN oxidation to GSSG, probably by the HzOr generated by the

MAO catalyzed oxidation of tyramine. These data further provides evidence that a reactive DA

O-quinone is formed since quinones readily react with GSH to form a covalent GSH conjugate

(120).

In conclusion, what similarities and contrasts can be drawn between the manganese

catalyzed oxidation of DA versus its P450 peroxygenase bioactivation? Firstly, the keystone of

both mechanisms hinges on the formation of the DA O-quinone, (Scherne 3.1, 3.2, and 3.3).

Manganese acts as a tnie catalyst in that it fonns DA O-quinone and is retumed to its same

valence state (2+). The presence of oxygen is required in order to activate the reactive DA:M~'+

complex. The P450 peroxygenase metabolism of DA, however, requires H202 Presumably, a

hydroperoxide could substitute for MO2, but this was not studied here since the endogenous

production of the latter is physiologically linked to DA.

The hepatocyte has proved to be a very usehl mode1 ce11 to study CA cytotoxic

mechanisms, since it contains the relevant biotransforming enzymes, namely MAO and CYP2E 1

(147, 148). Hepatocytes aiso lack CA synthesizing ability, which allowed us to know

beforehand how much DA is present in the system to start with. However, it is now important to

study these CA cytotoxic mechanisms in cultured neuronal cells of substantia n i p . CYP2E1 is

localized in dopaminergic neurons and is inducible (149, 150). Also, these celk contain NQO

(DT-diaphorase), presumably to detoxiQ DA O-quinone that could be formed (151). Further

research in this area could reveal a new mechanism of Parkinson's disease and provide further

evidence for the therapeutic efficacy of MAO inhibition.

GENERAL CONCLUSIONS

Based on the duaiity of their nature, the CAS can be regarded as agents that possess both

antioxidant and pro-oxidant properties. This cornes as no surprise, since many antioxidant

substances also have their toxic components. The widely used antioxidant, ascorbate, for

example, has been shown to be both mutagenic and toxic to Chinese hamster ovary cells (152).

and has been used in chernotherapy to kill tumor cells (153). GSH is also responsible for the

metabolic activation of 1,2-dibromoethane to a reactive intermediate responsible for DNA

damage ( 154).

In Chapter 1, CAS and related compounds were shown to be highly effective at

scavenging 02* and furthemore, they prevented hepatocyte hypoxia-reoxygenation injury. It

was shown for the fmt time that the femc complexes of these compounds were in fact, much

more efficacious than CAS alone.

In chapter 2, CAS were shown to fom O-quinones that could mediate the depletion of

ascorbate, GSH, and NADH. Since the depletion of antioxidants is regarded as a toxic event, the

implications of such findings are that the CAS also have pro-oxidant activity. An interesting

finding was the rate of CA O-quinone cyclization was inversely proportional to the rate of

ascorbateMADH oxidation. We therefore propose that CA cyclization could be an inherent

antioxidant mechanism built into the chemistry of CAS. This holds for EPI, as the fastest

cyclizing CA, whereas DA was a slow cyclizer, implying that it had more toxic potential.

In Chapter 3, DA cytotoxicity was shown to be mediated by the O-quinone catalyzed by

endogenous P-450 peroxygenase activity, utilizing H202 generated by MAO rnetabolism of DA.

However, the addition of cataiytic amounts of MI?+ increased DA cytotoxicity more than 2-fold.

This suggests that M.n2+ in the environment could catalyze DA autoxidation and thereby activate

CAS.

Altogether, the results of this thesis have demonstrated that CAS can act as both

antioxidants and pro-oxidants. A key factor in differentiating cytotoxicity from cytoprotection

was the concentration of CA used. Cytoprotection by CAS in the ischemia-repemision injury

model occurred at micromolar concentration, whereas approximately 10-fold this concentration

was required to induce cytotoxicity. Their biphasic nature, therefore, depends on their

concentration.

F'UTUm EXPERLMENTS

L. As mentioned in Chapter 1, it would be of interest to inject a DA-Fe(Q complex

intrathecally in vivo to investigate whether it is protective in a rat stroke model. The two

questions to be asked regarding the CA-Fe(m) complexes are: a) would they confer similar

protection in a neuronal culture, and b) do they possess any therapeutic advantage in an in

vivo study? The complexes would be injected intrathecdly, since they may not pass the

blood brain barrier, so it's partition coefficient would need to be calculated. It should also be

tested for its stability in the blood. Also, it would be useful to find out the redox potential of

the iron complexes versus their CA ligand alone. This may shed light on the potential for

cytoprotection versus cytotoxicity of the given CA-Fe(Q complex, and allow for

predictability. For comparison, they shodd be tested with the synthetic manganese-sden

SOD-mimic complexes in a rat stroke model, as a standard for comparison of efficacy (4).

2. There should be more in vitro work perfonned in comparing the cytotoxic mechanisms and

effectiveness of CA O-quinones with their aminochrome metaboiites. Preliminary resuits

with isolated rat hepatocytes suggests that dicumarol (the NQO inhibitor), potentiates EPI

toxicity (presumably because the EPI O-quinone is not being reduced), but prevents

adrenoc hrome toxicity .

Compound % Cpotoxicity at 3 hrs

2mM EPI 8.4

2mM EPI + 20pM Dicumarol 70.7

2m.M Adrenochrome 56.0

2rn.M Adrenochrome + 20p.M Dicumarol 34.5

(Pilot data. See text for explanation)

This is contrary to the belief that NQO is a cytoprotective quinone reductase.

Adrenochrome, the most stable quinone intermediate formed dunng EPI metabolsim was

found to be actually activated by NQO, (see Chapter 2, Scheme 2.2). Segura-Aguilar's group

showed that NQO in vitro catalyzed the two-electron reduction of aminochromes to their

reduced aminochromes which caused oxygen activation. However, a cellular mode1 of this

concept has not been tested. Of coune, one must address the issue of whether adrenochrome

foms in vivo. Perhaps noradrenochrome (since the locus ceruieus is pigmented) or

doparninochrome (precuaor of neuromelanin of substantia nigra) would be more relevant for

study of cytotoxic mechanism.

3. The P-450 peroxygenase activation of DA (Chapter 3) should be investigated in

dopaminergic neurons as they contain CYP 2El (149, 150), NQO (15 l), and MAO (155).

To accomplish this objective, it is necessary to isolate and culture neurons of the substantia

nigra or striatai neurons.

4. Genetic polymorphisms may provide additional insights into the etiology of Parkinson's

disease. Genetic polymorphisms have been associated with the poor metabolizer phenotype

of CYP2D6 and CYPlAl in relation to Parkinson's disease. The data compiled by

Checkoway et al., shows that while some researchea have found an increased risk of

Parkinson's disease linked with these genetic polymorphisms, others have not found such an

association ( 156). Furthemore, researchers have investigated genetic pol ymorphisms in

MAO leading to less enzymatic activity. Although previous studies showed a specific

genetic polymorphism (intron 2, GT repeat) not to be associated with Parkinson's disease, a

recent study shows the opposite result with Chinese patients (157). Other genetic

polymorphisms have also been identified, but differ in the exact ailelic variant (156). MAO-

B knockouts were found to be resistant to MPTP (137, 158), corroborating previous findings

of MAO-B inhibition when challenged with this Parkinson's disease-simulating neurotoxin.

It would be useful to study DA-ergic toxicity in the MAO-B knockout mouse since DA is

preferentiaily metabolized by MAO-B in the mouse (and human). Since the system used in

Chapter 3 shows that MAO metaboiism of DA is a prerequisite for cytotoxicity, the MAO-B

knockout wouid allow for investigation of other pathways of DA toxicity.

5. The apparent role of NQO in detoxification of the DA O-quinone shows that in the case of

manganese toxicity andlor high DA concentration, this enzyme would be a critical reductase.

In fact, a polymorphism exists for NQO (159). if other genetic factors corne into play, it may

be possible to put together the ailele combination that could predispose an individual to a

higher nsk of Parkinson's disease.

6. Many researchea beiieve mitochondnal dysfunction to be involved in Parkinson's disease,

as Complex 1 (NADK-Q reductase) bas been shown to be markedly reduced in Parkinson's

disease (160, 161). Genetic polymorphisms have been identified in Complex 1, but more

research is required to provide evidence that wodd predispose an individual to Parkinson's

disease (156). Although there is some progress in our understanding of the genetic bais for

Parkinson's disease, many factors seem to contribute to this neurodegenerative disease

thereby making one specific cause difficult to isolate. The ideal combination for research

could involve a union of biochemical and genetic mechanisms to better our knowledge of the

etiology of Parkinson's disease in hopes for treatment.

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