novel insights into mechanism of antioxidant-mediated cellular protection through the quenching of intracellular reactive oxygen species

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© Sagun KC 2006

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AD) NOVEL INSIGHTS INTO MECHANISM OF ANTIOXIDANT-MEDIATED

CELLULAR PROTECTION THROUGH THE QUENCHING OF INTRACELLULAR REACTIVE OXYGEN SPECIES

Sagun KC, Ph.D Cornell University 2006

Reactive oxygen species (ROS), which are byproducts of intracellular

oxidative events, fulfill a wide variety of biological functions However, their

abnormal production and/or elevated intracellular accumulation is thought to be an important contributor in the pathogenesis of aging, degenerative diseases, and cancer

Mitochondria play a central role in oxidative metabolism and contribute

significantly towards intracellular ROS production Several antioxidants and

ROS-eliminating enzymes function in tandem to protect the mitochondrion from the damaging effects of ROS Among these is the water-soluble antioxidant vitamin C, which is present in mammalian mitochondria at measurable

concentrations However, the mechanism of mitochondrial uptake of vitamin C

and its mitochondrial functions are poorly understood We found that analogous

to its cellular uptake, vitamin C enters mitochondria in its oxidized form,

dehydroascorbic acid (DHA) through facilitative glucose transporter 1 (Glutt) Loading mitochondria with vitamin C via DHA treatment quenched mitochondrial

ROS, and conferred protection to the mitochondrial genome and membrane from oxidative injury

We also examined a putative cancer chemopreventive role of antioxidants

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found that ectopic over-expression of c-MYC resulted in the disruption of cellular redox state and a concomitant increase in oxidative DNA damage Loading cells with vitamin C prevented MYC-induced genomic damage The protective effect of a mitochondrially-targeted antioxidant (TPPB) indicated the involvement of mitochondrial ROS in MYC-elicited genomic damage Additionally, we discovered that deregulated MYC expression resulted in elevated production of intracellular superoxide (*O2), which when quenched by vitamin C or a selective ~°O2

quencher (Tiron), protected cells from MYC-induced cellular transformation Our

studies point to a putative cancer chemopreventive role of antioxidants, which are mediated via quenching of ROS

Lastly, we assessed a role of “°O2in oxidative DNA damage with the use

of an intracellular “*O2 generator, 2,3-dimethoxy-1-naphthoquinone (DMNQ) We

found that DMNQ treatment elicited intracellular oxidative stress and oxidative DNA damage, as assessed by levels of oxidized guanines (8-oxo-dG) and

apurinic/apyrimidic (AP)-sites in the genomic DNA Our studies point to the

involvement of hydroxyl radicals (°OH) in oxidative DNA damage, and indicate a

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BIOGRAPHICAL SKETCH

Sagun KC was born on November 14, 1976 in Kathmandu, Nepal He received his formative education at St Xavier’s School and Budhanilkantha School Upon

successful completion of Cambridge GCE Advanced-Level examinations in 1996, he received an academic scholarship to study at Bates College, where he performed his senior thesis in organic chemistry under the guidance of Dr David Ledlie In June 2000, he graduated from Bates College with a Bachelor of Science (BS) degree in Biological Chemistry In the same year, he matriculated

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ACKNOWLEDGEMENTS

| would like to thank my advisor, Dr David W Golde, for his outstanding

love and support during my tenure as a graduate student Thank you for serving as a perceptive mentor, and for encouraging me during the most difficult times Our thought-provoking discussions on religion, ethics, politics, and science have influenced me beyond the perceivable realms of your proper scientific tutelage

| am also very grateful to Dr Juan M Carcamo for his passion for science and for serving as a dedicated mentor following Dr Golde’s death in 2004 His timely stewardship of the laboratory during those uncertain periods provided a seamless transition for all members of the Golde laboratory, including myself

| would like to thank Dr Bill Bornmann of MD Anderson Cancer Center for scientific collaborations My special thanks to Dr David Araten, Ms Rachel Nepomusceno, Ms Alicia Pedraza, Mr George Stratis and Mr Tao Tong for their technical assistance

| am thankful to Dr Patricia Q Cortes of Mount Sinai School of Medicine,

Dr Garrett DeYulia Jr., and Ms Rashmi Shrestha for their editorial inputs

| would personally like to thank all members of Dr Golde’s laboratory for

their personal and professional contributions Thank you for your generosity, and your hospitality On a special note, | would like to thank Ms Anne DAlessandro for her vigilant love and care

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continuously providing support and guidance during my graduate tenure | am truly indebted to Dr Gross for taking me under his wing and serving as my

advisor during the difficult transition | would also like to express my gratitude

towards Dr Mark L Heaney for offering his scientific expertise and editorial inputs, and for representing the laboratory as a member of my thesis committee

! am also thankful to Dr Marcus M Reidenberg, who previously had

served as chair during my candidacy examinations, for agreeing to offer his valuable scientific expertise as the chairman of my thesis special committee Thank you to all the faculty, staff, and colleagues in the Department of Pharmacology for investing your time and effort towards cultivating better scientists of tomorrow

| am grateful for the financial contributions from the National Institutes of

Health (CA 30388), New York State Department of Health, and the Lebensfeld

and Schultz foundations, without which my graduate education would have been a pipedream

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TABLE OF CONTENTS

CHAPTER ONE: GENERAL INTRODUCTION 1

1.1 Goal of this thesis 1

1.2 The biology of ROS 3

1.3 Intracellular sources of ROS 5

1.4 The chemistry of ROS 8

1.5 ROS-induced genotoxicity 11

1.6 ROS-induced modulation of protein function 12

1.7 ROS in signaling 14

1.8 Downstream cascades in ROS-mediated signaling 15

1.9 Cellular defense against ROS 16

1.10 Antioxidant-mediated protection of the genome 26

1.11 References 27

CHAPTER TWO: VITAMIN C ENTERS MITOCHONDRIA VIA FACILITATIVEGLUCOSE TRANSPORTER 1 (GLUT1) AND CONFERS PROTECTION AGAINST OXIDATIVE INJURY 2.1 — Introduction 40 2.2 Materials and Methods 42 2.3 Results 51 2.4 Discussion 72 2.5 References 78

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3.5 References 120

CHAPTER FOUR: SUPEROXIDE-MEDIATED OXIDATIVE

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Figure 2.1 Figure 2.2 Figure 2.3 Figure 2.4 Figure 2.5 Figure 2.6 Figure 2.7 Figure 2.8 Figure 2.9 Figure 2.10 Figure 2.11 Figure 2.12 Figure 2.13 Figure 2.14 LIST OF FIGURES

Mitochondria transport vitamin Cin its oxidized form, dehydroascorbic acid (DHA), which is reduced and mitochondrially accumulated as ascorbic acid (AA)

Mitochondrial transport of vitamin C occurs through a facilitative

glucose transporter

Facilitative glucose transporter 1 (Glut1) has the highest probability of mitochondrial localization based on computational prediction Facilitative glucose transporter 1 (Glut1) localizes to mitochondria

in human kidney 293T cells

Facilitative glucose transporter 1 localizes to mitochondria in mammaiian cells

Natively expressed Glut1 localizes to mitochondria in murine NIH/3T3 cells

In vitro synthesized Glut1 is imported into mitochondria purified from human kidney 293T cells

Mitochondrial AA (mtAA) quenches ROS in control and oxidatively

stressed mitochondria

Vitamin C attenuates H2O2 generated as a result of superoxide

overproduction in purified mitochondria

Mitochondrial AA (mtAA) protects mitochondrial DNA (mtDNA) from oxidative mutagenesis

Mitochondrial AA protects mitochondrial DNA from oxidative damage

Generality of mitochondrial uptake of vitamin C and its role in

protection of the mitochondrial DNA in human liver cells

Generality of mitochondrial uptake of vitamin C and its role in protection of the mitochondrial DNA in human myeloid cells

Vitamin C protects the mitochondrial membrane from oxidative

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Figure 3.1 Figure 3.2 Figure 3.3 Figure 3.4 Figure 3.5 Figure 3.6 Figure 3.7 Figure 3.8 Figure 3.9 Figure 3.10 Figure 3.11 Figure 3.12 Figure 4.1 Figure 4.2 Figure 4.3

Transient excess of MYC expression in 293T cells induces oxidative DNA damage

Vitamin C prevents DNA damage induced by transient excess of MYC

Constitutive expression of MYC elicits DNA damage in HeLa cells Elevated level of oxidative DNA damage in MYC-positive HeLa cells is not due to MYC-induced apoptosis

Vitamin C protects HeLa cells against DNA damage elicited by

constitutive MYC expression

Increased genomic damage in MYC-positive HeLa cells is due to

intracellular oxidative stress

Intracellular oxidative stress elicited by the transactivation of MYC is inhibited by vitamin C

MYC transactivation is sufficient to induce DNA damage in rodent fibroblasts, which is inhibited by loading cells with vitamin C

A mitochondrially-targeted antioxidant (TPPB) inhibits MYC-elicited oxidative DNA damage

Elevated superoxide levels in MYC _ over-expressing murine fibroblasts are quenched by antioxidants

Role of superoxide in MYC-induced cellular transformation

Vitamin C inhibits oncogenic transformation of rodent fibroblasts

Vitamin C attenuates intracellular ROS in HL-60 cells treated with an oxidative stressor

Vitamin C protects HL-60 cells against oxidative DNA damage

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lllustration 1.1 Illustration 1.2 Illustration 1.3 Illustration 2.1 Illustration 2.2 (Illustration 3.1 Illustration 4.1 LIST OF ILLUSTRATIONS

Etiology and chemistry of reactive oxygen species (ROS) generated in a eukaryotic cell

Intracellular metabolism of vitamin C

Parallel mechanism of transport and intracellular trapping of glucose and vitamin C in eukaryotic cells

Commonly used fluorometric probes for the study of ROS Schematic illustration of vitamin C uptake and recycling in the cell

By quenching intracellular ROS, antioxidants inhibit MYC- elicited oxidative DNA damage and cellular transformation Mechanism of ROS generation by an intracellular redox cycling agent (DMNQ) with an outline of reactions involved in

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LIST OF ABBREVIATIONS 8-oxo-dG 8-oxo-7,8-dihydro-2'-deoxyguanosine 40HT 4-hydroxytamoxifen Am mitochondrial membrane potential AA ascorbic acid AP1 activator protein 1 AP apurinic/apyrimidic ATP adenosine triphosphate Bcl2 B cell leukemia 2 CA constitutively active

CCCP carbonyl cyanide m-chloro- phenylhydrazone c-MYC cellular myelocytomatosis oncogene

COXVIII cytochrome oxidase VIII

Cu”/Cu” copper (I)/copper (II)

DCFDA 2',7'-dichlorofluorescin diacetate DHA dehydroascorbic acid

DHLA dihydrolipoic acid

DIOC6(3) 3,3'-dihexyloxacarbocyanine iodide DMNQ 2,3-dimethoxy-1-naphthoquinone DN dominant negative DNA deoxyribonucleic acid DPI diphenyleneiodinium EGFP enhanced green fluorescent protein ER estrogen receptor

ETC electron transport chain

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HepG2 human hepatocellular carcinoma cells HL60 human myeloid leukemia cells

hMTH1 human MutT homolog 1 hMYH human MutY homolog

hOGG1 human 8-oxoguanine DNA glycosylase H-ras Harvey rat sarcoma viral oncogene HRP horseradish peroxidase

HTLV human T cell leukemia virus IB incubation buffer

LA lipoic acid

MAPK mitogen-activated protein kinase Mcl1 myeloid cell leukemia 1

MnSOD manganese superoxide dismutase MPT membrane pore transition

mtAA mitochondrial ascorbic acid mtDNA mitochondrial DNA NF«B nuclear factor kappa B NO nitric oxide NOx NADPH oxidase “*O2 superoxide °OH hydroxyl radical ONOO peroxynitrite

PBS phosphate buffered saline pGcT plastidic glucose transporter

Raci Ras-related C3 botulinum toxin substrate 1 RNS reactive nitrogen species

ROS reactive oxygen species SH sulfhydryl group (thiol moiety)

SVCT sodium-dependent vitamin C transporter TBARS thiobarbituric acid reactive substances Tiron 4,5-dihydroxy-1,3-benzenedisulfonic acid TPPB triphenylphosphonium bromide

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CHAPTER ONE

GENERAL INTRODUCTION

1.1 Goal of this thesis:

Vitamin C is one of the most abundant cellular antioxidants It is universally transported into cells in its oxidized form, dehydroascorbic acid (DHA), via facilitative glucose transporters Although mitochondria isolated from

mammalian cells contain ascorbic acid (AA) at measurable concentrations, the

mechanism of its mitochondrial uptake and its mitochondrial functions are poorly understood Utilizing theoretical, functional, and physical studies, we discovered that a parallel mechanism exists for the mitochondrial transport of vitamin C We found that analogous to the cellular uptake, the oxidized form of vitamin C enters mitochondria via facilitative glucose transporter 1 (Glut1), and is reduced and

accumulated as mitochondrial ascorbic acid (mtAA) We initially performed in

vitro assays to assess the protective effect of mtAA using purified mitochondria, and subsequently assessed mitochondrial protection in the cellular setting Vitamin C effectively quenched mitochondrial ROS production under both scenarios To simulate oxidative disorders, we used a mitochondrial electron transport chain inhibitor and a redox cycling agent to educe mitochondrial ROS

Vitamin C attenuated the toxic effects of both agents, as well as inhibited

mitochondrial membrane depolarization in response to a protonophore (CCCP) The next step was to examine a role of ROS in cancer, and more

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c-MYC Although transient excess of MYC was known to induce oxidative stress

in fibroblasts, the mechanism of ROS over-production, and its involvement in

MYC-elicited genomic damage and cellular transformation was not properly understood Additionally, the cancer chemopreventive effects of antioxidants

such as vitamin C needed a thorough examination In a variety of cell-lines we

found that ectopic MYC over-expression resulted in elevation of intracellular

ROS, attenuation of intracellular antioxidants, and a concomitant elevation in

oxidative DNA damage Vitamin C and other antioxidants attenuated these effects of MYC With the use of a selective superoxide (*O2) scavenger, Tiron,

we identified a participatory role of “°O2 in MYC-elicited genomic damage and

cellular transformation, which parallels previous discovery on its involvement in

ras-mediated cellular transformation Our studies point to a putative cancer chemopreventive role of antioxidants, which are mediated, in part, via quenching

of ROS involved in oncogenic signaling

Lastly, we investigated a direct role of “°O2 in oxidative DNA damage and

examined a role of intracellular antioxidants in genomic protection “°O2 was

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generate °OH through Haber-Weiss reaction The inhibitory effects of AA and a- lipoic acid, which quench “°O2 but not H2O2, provide necessary evidence to our hypothesis on the mechanism of *OH generation in the cellular setting

It is the goal of this thesis to study the cytoprotective effects of antioxidants against oxidative stress elicited by a variety of chemical and/or genetic agents It is well known that steady state liberation of intracellular ROS is attributed to mitochondrial respiration Additionally, abnormal production of intracellular ROS due to genetic or age-associated defects in mitochondrial function is thought to be important contributor to the pathogenesis of aging, degenerative diseases, and cancer Our studies with mitochondrial antioxidants provide strategies for effectively contesting mitochondrial ROS production, and point to the preventive role of antioxidants against oxidative disorders

1.2 The biology of ROS

Aerobic organisms living in an oxygen-rich atmosphere rely on controlled oxidation of metabolic substrates to generate energy It is essentially comprised

of four-electron reduction of O2 to H2O within the mitochondrial electron transport

chain (ETC), resulting in the formation of a high-energy phosphate bond of ATP

Additionally, metabolic pathways involved in biosynthesis or degradation are

often comprised of a series of oxidative reactions, which liberate pro-oxidants

into the cellular milieu The byproducts of these intracellular oxidative events are partially reduced metabolites of molecular Oz, collectively known as reactive

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anion (*O2), hydroxyl radical (°OH), and hydrogen peroxide (H202) In addition,

another free radical molecule of biological origin, nitric oxide (NO°) reacts with superoxide to form a highly toxic pro-oxidant, peroxynitrite (ONOO’), which can

damage cellular proteins and lipids via nitrosylation (Huie and Padmaja 1993; Hogg, Joseph, and Kalyanaraman 1994; Ischiropoulos and al-Mehdi 1995) In a similar manner, oxidized metabolites of membrane lipids, organic

hydroperoxides, and secondary lipoxidation products comprise of an important

class of highly reactive lipid-based pro-oxidants

ROS are adversely and non-specifically reactive against almost all biological macromolecules Nucleic acids, proteins, and lipids constitute three

most important classes of biological macromolecules that are highly susceptible

to the damaging effects of ROS (Halliwell and Gutteridge 1989) Therefore, cells have principally developed two types of defense mechanisms to minimize the damaging effects of ROS The first line of defense is associated with curbing ROS production, and ensuring that it is properly compartmentalized into discrete cellular areas, such as vesicles Antioxidants (vitamin C and E), low molecular

weight thiols (glutathione), and ROS-eliminating enzymes (superoxide

dismutase, catalase, peroxidase) provide this first line of defense As a second

line of defense, cells have also developed a wide variety of repair mechanisms to

ensure genomic and cellular integrity in the event of an oxidative insult These

include enzymatic reduction of reversibly oxidized proteins, degradation of

irreversibly modified proteins (Davies and Goldberg 1987), and activation of

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1.3 Intracellular sources of ROS

Several enzymatic and non-enzymatic processes contribute towards intracellular ROS production (Illustration 1.1A) Basal production of ROS is

mostly attributable to mitochondrial respiration because of the central role it plays

in oxidative metabolism (Chance, Sies, and Boveris 1979) Side reactions of the various mitochondrial electron transport chain (ETC) complexes (complex |, Il and III) with molecular oxygen results in the formation of superoxide (®O2) as an oxidative byproduct It has been estimated that nearly 2% percent of all the

electrons traveling down the ETC never make it to the end, and thus contribute

towards ~“°O2 production (Cadenas and Davies 2000) In addition, metabolic enzymes in the mitochondrial membrane, such as monoamine oxidase, also lead to the production of ROS in the vicinity of the mitochondria (Lai and Yu 1997) Manganese superoxide dismutase (MnSOD), an important ROS-eliminating enzyme in mitochondrial membrane, is responsible for the dismutation of mitochondrial “°Oz2 into H2O2 and Oz (Wispe et al 1989)

The endoplasmic reticulum (ER) contains enzymes that generate ROS as byproducts of hydroxylation and oxidation reactions involved in protein folding (Tu and Weissman 2002) The ER also expresses several isoforms of NAD(P)H

oxidase (specifically, NOx-1 and NOx-4), which generate ROS and contribute to

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Relative Energy 0 1 2 3 4

Equivalent Reductions ("e)

illustration 1.1 Etiology and chemistry of reactive oxygen species (ROS) generated in a eukaryotic cell (A) Sources of intracellular ROS — 1

Mitochondria 2 Mitochondrial outer membrane 3 Endoplasmic Reticulum 4 Plasma membrane 5 Cytoplasm 6 Lysosomes/peroxisomes (B) Path of

electrons involved in the four-electron reduction of molecular O2 in the mitochondrial electron transport chain The red arrows depict Fenton reactions involved in the generation of hydroxyl radicals (°OH) The blue arrows depict enzymatic reductions of deleterious ROS into less reactive species such as H202 and Oz Enzymes listed: CAT — catalase, PDX — peroxidase, SOD — superoxide

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degradation of xenobiotics (Bondy and Naderi 1994; Rashba-Step and Cederbaum 1994)

Membrane-associated oxidases appear to contribute significantly to the

production of ROS in response to intrinsic or extrinsic signaling pathways One of the most well characterized membrane oxidase is the phagocytic NAD(P)H oxidase (NOx), a multi-protein flavo-enzyme complex that is assembled and

activated in the plasma membrane and rapidly releases “*Oz into the phagocytic vacuoles or the extracellular milieu (Babior 1999) Since several components of NOx have been successfully cloned in non-phagocytic cells (Cheng et al 2001), it appears that these membrane oxidases are not only important for host defense, but also have other secondary functions This hypothesis was verified with the discovery that ROS generated by various NOx isoforms play a critical role in both intracellular and intercellular signaling events (Finkel 1998) Other ubiquitously expressed membrane-bound oxidases such as xanthine oxidase (XO) are thought to be important contributors to vascular dysfunction in vivo (Guzik et al 2000; McNally et al 2003) XO produces '*O;¿ or HạO; as byproducts of purine metabolism

Aside from their role in the synthesis of second messengers, such as arachodonic acid and nitric oxide, cytosolic enzymes of the lipoxygenase and

nitric oxide synthase (NOS) family can generate '°O; in the presence of

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elicit nitrosative stress and play a major role in the onset of Parkinson’s disease and diabetes in humans (Ebadi and Sharma 2003; Pacher et al 2005) Similarly, activation of the enzymes of the lipoxygenase/cyclooxygenase family results in the generation of “*O2 and H202 along with the synthesis of downstream effector molecules, hydroxyeicosatetraenoic acids (HETEs) and prostaglandins (PG), respectively (Baud et al 1983)

Peroxisomes compartmentalize various oxidative reactions and contain HạOz-producing flavin oxidases as basic enzymatic constituents Xanthine oxidase in the matrix and the electron transport components of the peroxisomal

membrane are possibly responsible for peroxisomal “*O2 production (del Rio et al 1992) Recently, a 20kDa peroxisomal membrane protein has also been implicated in the generation of oxygen radicals in peroxisomes and is thought to

play a role in the pathogenesis of renal diseases (Zwacka et al 1994)

1.4 The chemistry of ROS

The various enzymatic (e.g CYP450) and non-enzymatic (mitochondrial and peroxisomal ETC leakage) systems described above generate “°O2 as the initial byproduct of oxidative metabolism However, because of the unstable

nature of “°O2 at physiological pH, it undergoes a relatively slow dismutation

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Physiologically, this reaction is catalyzed by SOD enzymes (Illustration

1.1B), with a second-order rate constant of 2 x 10° M's” for Cu/Zn SOD and 1 x

10° M's” for MnSOD (Cadenas and Davies 2000) However, in the presence of

NO in the cellular milieu, SOD-mediated elimination of “°O2 is easily out-

competed by NO to form ONOO™ (Huie and Padmaja 1993), which can then

undergo various additive reactions to generate reactive nitrogen species (RNS)

such as ONO® and NOs (Pryor and Squadrito 1995; Kissner et al 1997)

"*O; + NO* -> ONOO' (4) k=6.9 x 109 M's” ONOO’ + H* -> ONOOH -> ONO® + °OH (5)

ONOOH + R -> R=O + ~NO2 + H” and/or R°OH + ONO® (6)

At low concentrations, the biology of ONOO is similar to that of NO® and it may even function as a donor of NO® in the cellular setting However, it is highly

reactive and can irreversibly damage proteins (Ischiropoulos and al-Mehdi 1995),

DNA (Douki and Cadet 1996) and lipids (Radi et al 1991) at higher concentrations Since ONOO' is optimally synthesized with equimolar concentrations of “°O2 and NO®, antioxidant-mediated quenching of “*O2 can be an effective way to contest intracellular ONOO production

H202 generated via dismutation of “°Oz2 Is relatively stable at physiological

pH and is considered to be less toxic than other pro-oxidants In the presence of catalase or peroxidases, H2O2 can be further reduced into harmless metabolites,

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role in intercellular signaling at low concentrations, and to elicit extracellular oxidative stress and facilitate cell death at higher concentrations (Upham et al 1997; Reznikov et al 2000)

H2O2 + H2O2 -> 2H2O and Oz (5) k=2 x 10°M's" Fe**/Cu** + “°O2 -> Fe**/Cu* + O2 (6)

Fe?'/Cu† + HaO¿ -> Fe?*/Cu?” + °OH + “OH (7)

H2Oz is only weakly reactive against proteins and DNA, its toxic effects are

generally attributed to the hydroxyl radical (°OH), which are formed via a transition metal (e.g Cu?” or Fe*")-catalyzed reaction Equation 7 is also known as the Fenton reaction and has a second order rate constant of 76 M's" for Fe”*

and 4700 M's" for Cu?* Alternatively, °OH may be generated through the

Haber-Weiss reaction when ~“°O2 spontaneously combines with H2O2 to form singlet oxygen ('O2) and ‘OH (Khan and Kasha 1994)

HạO; + '*O¿ -> 'O; + 'OH + °OH (8)

Interestingly, when antioxidants (AH') are administered in the presence of

transition metals, it presents a potential scenario for the generation of *OH

through a reduced transition metal intermediate Whether this reaction occurs in

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conditions One such potential scenario for in vitro artifacts of antioxidants is

through the Fenton chemistry in tissue culture media (Halliwell 2003)

AH' + FeŸ”/Cu”” -> *A + Fe**/Cu* + H* (9) kaa =10* M's"

HạO; + Fe?'/Cu'” -> Fe”"/Cu”” + *OH + 'OH (7)

1.5 ROS-induced genotoxicity

The hydroxyl radical (°OH) is considered to be the most reactive and

deleterious species involved in the oxidative modification of DNA (Rao, Goldstein, and Czapski 1991) It has been shown to cause DNA strand breaks, sites of base loss (abasic sites) and mutagenesis of all four bases of DNA (Epe

1991) The high electrophilicity, high thermokinetic reactivity, and a mechanism of production at the site of the DNA make °OH a prime species involved in

oxidative genotoxicity (Liu et al 2004)

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and Cohen 1992) Oxidative DNA damage coupled with compromised repair machinery can result in genomic destabilization and is readily evident in cancer cells in the form of increased DNA mutagenesis, chromosomal deletions, and translocations (Wiseman, Kaur, and Halliwell 1995; Beckman and Ames 1997) 1.6 ROS-mediated modulation of protein function

Although many studies have implicated a role of ROS in_ signal

transduction, relatively little is Known regarding the specific mechanism through

which oxidants modulate intracellular signaling A central paradox of ROS- mediated signaling involves specificity - how does a short-lived, highly reactive species maintain any order of specificity? Studies suggest that oxidants have

direct protein targets, which undergo functional modulation through a shift in

redox environment The effect of this redox modulation is usually a change in enzyme activity and/or modification of protein-protein interactions (Finkel 2000)

Proteins are highly susceptible targets of intracellular ROS Although ROS

may irreversibly damage all proteins at a supra-high concentration, it is generally

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dependent reduction, which are then fully reduced to active thiol moieties with the

reductive action of glutaredoxin (Grx) Additionally, S-glutathionylation, which involves conjugation of glutathione into active cysteine residues of protein kinases, has also been implicated in the modulation of protein function lron- sulfur containing clusters (4Fe-4S) of proteins are yet another viable targets for ROS-mediated modulation (Sweeney and Rabinowitz 1980) Redox-dependent modulation of proteins containing 4Fe-4S clusters has been evidenced in the aconitase family of proteins in both bacteria and mammals (Gardner and

Fridovich 1991, 1992; Gardner et al 1995) Recent proteomic studies have

similarly identified a biological role of protein S-nitrosylation in signal transduction and its potential involvement in the pathogenesis of oxidative and inflammatory disorders (Jaffrey et al 2001; Aulak et al 2001)

One of best examples of a redox-dependent feedback loop is evidenced by the transcriptional regulation of antioxidant genes in bacteria In E coli, the

OxyR transcription factor upregulates synthesis of a set of antioxidant genes and is activated through the formation of a disulfide bond Interestingly, OxyR is deactivated when reduced by glutaredoxin 1 (Grx1), one of the gene products of

the OxyR transcriptional machinery (Zheng, Aslund, and Storz 1998) A similar mechanism of redox regulation has been observed in the Fe-S cluster-containing

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through an activation shunt involving H2O2-mediated oxidation and subsequent

dimerization of two Prx homodimers (Wood, Poole, and Karplus 2003)

1.7 ROS in signaling

H;O; has been known to modulate cellular signaling molecules, such as kinases and phosphatases, via redox-dependent mechanisms (Sundaresan et al 1995; Hardwick and Sefton 1995) In particular, receptors possessing intrinsic

kinase domains, such as the EGF receptor (EGFR) and platelet-derived growth

factor receptor (PDGFR) have been shown to signal by means of ROS-mediated mechanisms Since signaling was abolished by catalase, the identity of the species involved in signaling was inferred to be H2Oz2 (Bae et al 1997; Bae et al 2000), a cell-permeable molecule that is generated directly as a result of receptor-ligand interaction and is capable of activating downstream signaling cascades (DeYulia et al 2005) Studies aimed at identifying signaling molecules upstream to H2O2 currently point towards the involvement of “°Oz in the activation of intracellular signaling (Dhar-Mascareno, personal communication)

Kinetic studies have shown that “°O2 could be one of the most potent pro-

oxidants involved in reversible modulation of protein function A direct role of “°O2 in the inhibition of PTP1B, and its potential implications in cell signaling has led to the identification of “°O2as an intracellular second messenger (Barrett, DeGnore, Keng et al 1999; Barrett, DeGnore, Konig et al 1999) It was recently reported

that ROS generated via receptor-ligand interaction triggers intracellular signaling in the receptor tyrosine kinase (RTK) family of proteins via inactivation of PTP1B

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the redox modulation of key signaling proteins such as protein kinase C (PKC) and soluble guanylyl cyclase (sGC) (Mittal and Murad 1977; Gopalakrishna and Anderson 1989; Brune, Schmidt, and Ullrich 1990) The potential role of “Oz in cellular transformation was previously reported with studies on “°O2-dependent mitogenic stimulation of human peripheral blood lymphocytes (Gallagher and Curtis 1984) and neoplastic transformation of epidermal cells (Nakamura,

Colburn, and Gindhart 1985) Additional evidence on the role of “*Oz in

intracellular signaling was provided by studies demonstrating its ability to transform cells in vitro (Suh et al 1999) Its involvement and functional

requirement in Ras-mediated transformation was found to be mediated via upregulation of NAD(P)H oxidase homologs (Irani et al 1997; Pennisi 1997; Arnold et al 2001; Mitsushita, Lambeth, and Kamata 2004)

1.8 Downstream cascades in ROS-mediated signaling

An accepted paradigm for neoplastic transformation is a multi-step

requirement for carcinogenesis, involving initiation, promotion, and progression of

cancer It is thought that ROS-mediated signaling and genomic damage may

facilitate one or more of these steps (Cerutti 1985; Cerutti et al 1992) Previous studies have revealed that ROS, which are generated in large quantities in cancer cells (Szatrowski and Nathan 1991), can induce cell proliferation (Burdon 1995) However, recent molecular approaches towards understanding ROS- mediated activation of mitogenic cascades have generated a diverse array of

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activate transcription factors such as activator protein 1 (AP-1) and nuclear factor kappa B (NF«B), resulting in the stimulation of downstream MAPK cascades Antioxidant-mediated quenching of ROS was found to inhibit transcription factor

activity and attenuate cell proliferation (Gupta, Rosenberger, and Bowden 1999) Studies have shown that elevated MnSOD expression can inhibit Jun N-terminal kinase (JNK)/AP-1 pathways and suppress tumor formation in a multistage skin

carcinogenesis model (Zhao et al 2001) On the other hand, ROS-mediated activation of stress-activated protein kinase (SAPK), whose primary function is to

promote apoptotic cell death, also needs to be considered (Storz, Tartaglia, and

Ames 1990) Since ROS can activate these divergent signaling pathways, it is hypothesized that there are two sides to ROS-mediated signaling and its implications in cancer Under optimal growth conditions, metabolically elevated ROS levels may confer a growth advantage to tumor cells via induction of growth

competent genes and other mitogenic signals However, prolonged exposure to

ROS can cause a reversal of cell-fate, leading to the induction of apoptotic and/or necrotic pathways (Benhar, Engelberg, and Levitzki 2002) Oxidative stress may also impart appropriate selection pressures towards conferring growth advantage, resistance, and malignant phenotype to otherwise normal cells

1.9 Cellular defense against ROS

Since ROS are generated normally via oxidative metabolism, and fulfill

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intracellular sequestration, leading to what is described as “oxidative stress” To prevent the toxic and mutagenic effects of ROS, cells have developed several types of defense mechanisms These include ROS-eliminating enzymes,

antioxidants, repair pathways, and other small molecules that serve as co-factors

and protect the cell and its genome from the detrimental effects of ROS 1 ROS eliminating enzymes

Enzymes of the superoxide dismutase (SOD) family constitute an important level of antioxidant defense against ROS These enzymes are comprised of a catalytic core containing a divalent transition metal, which is required for enzyme functionality Cytosolic and extracellular SODs usually have

Cu?* or Zn** bound to them, while mitochondrial SODs have Mn** in their

catalytic core MnSOD is critical for survival, as evidenced by embryonic lethality

in null mice (Li et al 1995), whereas Cu/Zn SOD knockouts, while viable, are

increasingly susceptible to neuronal death following axonal injury (Reaume et al

1996) Mutations in the Cu/Zn SOD gene have been linked with the pathogenesis

of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease in humans (Rosen et al 1993) The enzymatic function of superoxide dismutase is

to reduce and detoxify ˆ°O; into less reactive H2O2, which can be further

detoxified by catalase (CAT) or glutathione peroxidase (GPx)

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H2O2 GPx is a selenoprotein that depends on the reductive potential of

glutathione (GSH) to detoxify H2O2 and lipid organic hydroperoxides (Rotruck et

al 1973) Mitochondrial GPx are thought to play an important role in detoxification of HzO2 generated in mitochondria, especially in tissues lacking mitochondrial catalase (Sies and Moss 1978; Zakowski and Tappel 1978) Additionally, members of the newly identified peroxiredoxin (Prx) family of

proteins play a functionally redundant role in the detoxification of H2zO2 and

organic peroxides (Chae et al 1994) 2 Antioxidants

The cellular antioxidant system is principally comprised of small molecules that utilize their reductive potential to quench ROS GSH is the most abundant antioxidant of the cell, and its measure usually provides an indication of the cellular redox state (Hayes and McLellan 1999) It is a peptide composed of covalently bonded glutamate, cysteine, and glycine (y-L-glutamyl-L- cysteinylglycine), with the sulfhydryl group of cysteine critical for its antioxidant function GSH is synthesized from the intracellular amino acid pool by the dual

action of yGluCys synthase and glutathione synthase It is abundant cytosolically, often at millimolar concentrations, and can scavenge free radicals directly and/or

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Vitamin C is another abundant hydrophilic antioxidant involved in quenching ROS in both extracellular and intracellular milieu It is very stable in its

reduced form, ascorbic acid (AA), and can be regenerated intracellularly from its

oxidized from, dehydroascorbate (DHA), via enzymatic and non-enzymatic reductions (Wells et al 1990; Xu and Wells 1996; Rodriguez-Manzaneque et al 2002) It is synthesized from glucose in a multi-step biosynthetic pathway (IIlustration 1.2), which is intact in plants and most animals Although humans

and primates are deficient in one of the key enzymes involved in its biosynthesis

(L-glunonololactone oxidase), the bodily need for vitamin C can nevertheless be fulfilled by an antioxidant rich diet (Burns, Moltz, and Peyser 1956; Burns 1957)

In specialized cells, such as renal epithelial cells, osteoblasts,

adrenomedullary chromafin, and intestinal brush boarder vesicles, vitamin C is directly transported as ascorbic acid (AA) via sodium-dependent vitamin C transporters (SVCT) (Tsukaguchi et al 1999; Daruwala et al 1999) However, most cells transport vitamin C in its oxidized form, dehydroascorbic acid (DHA), via universally-expressed facilitative glucose transporters (Glut) (Vera et al 1993) Once inside cells, DHA is reduced and accumulated as ascorbic acid (AA) (Vera et al 1994) (Illustration 1.3) The in vivo intracellular concentration of AA is widely variable in humans, but concentrations as high as 4 mM have been reported in certain mononuclear cells (Levine et al 1996) In highly metabolic

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0 *\ Ht 4 De + 2e7 Ascorbic Acid (AA) Dehydroascorbic Acid (DHA) Synthesis Degradation a ON LOH H 0 H HO Z9 X + H OH HO — OH = H OHOH H HO O HO OH

Glucose Oxalate Threonate

lilustration 1.2 Intracellular metabolism of vitamin C Vitamin C exists in equilibrium between its reduced form, ascorbic acid (AA) and its oxidized form, dehydroascorbic acid (DHA) DHA is universally transported into cells via facilitative glucose transporters (Glut), and it is reduced cytosolically to AA AA is converted back to DHA through the quenching of intracellular ROS Since

humans and primates lack the enzymes necessary for de novo synthesis of AA,

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Aside from its antioxidant functions, vitamin C is an important enzyme co- factor involved in the synthesis of collagen (Padh 1991) and carnitine (Rebouche 1991), and is postulated to be involved in the mitochondrial reduction of a- tocopherol (Packer, Slater, and Willson 1979) and ferricytochrome c (Myer et al 1980) Vitamin C also helps to maintain cellular redox state by sustaining the redox turnover of other antioxidants such as vitamin E and GSH, scavenging non-radical pro-oxidants, inhibiting lipid peroxidation, and maintaining the

reduced states of intracellular Fe** and Cu" (Frei, England, and Ames 1989)

Vitamin E is an important class of antioxidant because of its lipophilic property Natural vitamin E exists in eight different isomers, four tocopherols and tocotrienols, which consist of a chromanol ring, and a hydroxyl group that is capable of donating a hydrogen atom for the reduction of free radicals The hydrophobic side chain allows for insertion into lipids The biological activity of each isoform of vitamin E is widely variable with a-tocopherol being recognized as the most active form of vitamin E in humans Vitamin E is a powerful scavenger of “°O2 (Factor et al 2000) and mitochondrial lipid peroxides

(Paraidathathu, Palamanda, and Kehrer 1994) In addition, vitamin E may also

reduce basal “°Oz levels by disrupting the assembly of NOx (Cachia et al 1998) The thiol antioxidant a-lipoic acid [HSCH2zCH2CH(SH)C,HgCOOH] is an

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dinydrolipoic acid (DHLA) Both LA and DHLA can effectively donate electrons to quench reactive oxygen species such as superoxide radicals, hydroxyl radicals, hypochlorous acid (HOCI), peroxyl radicals, and singlet oxygen ('O2) (Packer, Witt, and Tritschler 1995) LA also protects membranes by interacting with vitamin C and glutathione, which may in turn recycle vitamin E (Packer, Witt, and Tritschler 1996)

3 Other small molecule antioxidants

Natural phytochemicals found in fruits, vegetable, spices, and herbs have been shown to display potent antioxidant properties Among these, polyphenols

and carotenoids stand out as the two most important groups of natural

antioxidants (Fuhrman and Aviram 2001) Carotenoids, including xanthophyils, are naturally colored compounds that exhibit potent inhibitory effect against lipid peroxidation and singlet oxygen (Deming et al 2001) Polyphenolics, which is an inclusive term that is used to cover different subgroups of the phenolic acids and flavinoids, are useful antioxidants because of their high redox potentials In

addition to serving as ROS-quenchers, they have metal-chelating properties that

allow inhibition of transition metal-catalyzed generation of hydroxyl radicals

(Rice-Evans et al 1995) Although collectively these phytochemicals are argued to be protective against metabolic disorders ranging from aging, degenerative

diseases, cardiovascular diseases, and cancer, the roles and effects of individual

compounds are often not well Known

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al 1994; Brigelius-Flohe et al 2001; Brash and Havre 2002) have proven greatly

effective in cancer chemoprevention studies Selenium is known to serve as a

co-factor in many ROS-eliminating enzymes Another trace lipophilic antioxidant, the mitochondrial coenzyme Q,)/Qio, is routinely used as an antioxidant

supplement It has been suggested as an effective chemoprotectant in cancer

chemotherapy studies (Kishi, Watanabe, and Folkers 1976; Matthews et al 1998)

4 Repair of oxidatively damaged macromolecules

Repair of oxidized proteins-Since protein thiols are highly susceptible to oxidation, cells have developed a system of enzymes that are intricately involved

in reducing oxidized residues of proteins (usually SH groups) These proteins consist of a CXXC active site motif, and require a coupling system (comprised of

GSH, NADPH, and a reductase) to reduce disulfides of proteins and other substrates Thioredoxin (Trx) (Larsson and Thelander 1965; Tsang and Weatherbee 1981) and glutaredoxin (Grx) (Holmgren 1976) are two important members of this protein superfamily, and are involved in the enzymatic

resuscitation of a broad range of oxidized proteins (Luthman et al 1979)

Repair of oxidized DNA bases-To protect the cells from the damaging effects of oxidized bases, organisms have developed three different sets of repair pathways, which are believed to work in concert to preserve the integrity of the

genome In general, the oxidized base lesions are removed from DNA by the