Free Radical Biomedicine: Principles, Clinical ... - Bentham Science

benthamscience.com

Free Radical Biomedicine: Principles, Clinical ... - Bentham Science

Free Radical Biomedicine:

Principles, Clinical Correlations, and

Methodologies

Author

Y. Robert Li

Virginia Tech Corporate Research Center

Blacksburg, Virginia

USA


eBooks End User License Agreement

Please read this license agreement carefully before using this eBook. Your use of this eBook/chapter constitutes your agreement

to the terms and conditions set forth in this License Agreement. Bentham Science Publishers agrees to grant the user of this

eBook/chapter, a non-exclusive, nontransferable license to download and use this eBook/chapter under the following terms and

conditions:

1. This eBook/chapter may be downloaded and used by one user on one computer. The user may make one back-up copy of this

publication to avoid losing it. The user may not give copies of this publication to others, or make it available for others to copy or

download. For a multi-user license contact permission@benthamscience.org

2. All rights reserved: All content in this publication is copyrighted and Bentham Science Publishers own the copyright. You may

not copy, reproduce, modify, remove, delete, augment, add to, publish, transmit, sell, resell, create derivative works from, or in

any way exploit any of this publication’s content, in any form by any means, in whole or in part, without the prior written

permission from Bentham Science Publishers.

3. The user may print one or more copies/pages of this eBook/chapter for their personal use. The user may not print pages from

this eBook/chapter or the entire printed eBook/chapter for general distribution, for promotion, for creating new works, or for

resale. Specific permission must be obtained from the publisher for such requirements. Requests must be sent to the permissions

department at E-mail: permission@benthamscience.org

4. The unauthorized use or distribution of copyrighted or other proprietary content is illegal and could subject the purchaser to

substantial money damages. The purchaser will be liable for any damage resulting from misuse of this publication or any

violation of this License Agreement, including any infringement of copyrights or proprietary rights.

Warranty Disclaimer: The publisher does not guarantee that the information in this publication is error-free, or warrants that it

will meet the users’ requirements or that the operation of the publication will be uninterrupted or error-free. This publication is

provided "as is" without warranty of any kind, either express or implied or statutory, including, without limitation, implied

warranties of merchantability and fitness for a particular purpose. The entire risk as to the results and performance of this

publication is assumed by the user. In no event will the publisher be liable for any damages, including, without limitation,

incidental and consequential damages and damages for lost data or profits arising out of the use or inability to use the publication.

The entire liability of the publisher shall be limited to the amount actually paid by the user for the eBook or eBook license

agreement.

Limitation of Liability: Under no circumstances shall Bentham Science Publishers, its staff, editors and authors, be liable for

any special or consequential damages that result from the use of, or the inability to use, the materials in this site.

eBook Product Disclaimer: No responsibility is assumed by Bentham Science Publishers, its staff or members of the editorial

board for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any

use or operation of any methods, products instruction, advertisements or ideas contained in the publication purchased or read by

the user(s). Any dispute will be governed exclusively by the laws of the U.A.E. and will be settled exclusively by the competent

Court at the city of Dubai, U.A.E.

You (the user) acknowledge that you have read this Agreement, and agree to be bound by its terms and conditions.

Permission for Use of Material and Reproduction

Photocopying Information for Users Outside the USA: Bentham Science Publishers grants authorization for individuals to

photocopy copyright material for private research use, on the sole basis that requests for such use are referred directly to the

requestor's local Reproduction Rights Organization (RRO). The copyright fee is US $25.00 per copy per article exclusive of any

charge or fee levied. In order to contact your local RRO, please contact the International Federation of Reproduction Rights

Organisations (IFRRO), Rue du Prince Royal 87, B-I050 Brussels, Belgium; Tel: +32 2 551 08 99; Fax: +32 2 551 08 95; E-mail:

secretariat@ifrro.org; url: www.ifrro.org This authorization does not extend to any other kind of copying by any means, in any

form, and for any purpose other than private research use.

Photocopying Information for Users in the USA: Authorization to photocopy items for internal or personal use, or the internal

or personal use of specific clients, is granted by Bentham Science Publishers for libraries and other users registered with the

Copyright Clearance Center (CCC) Transactional Reporting Services, provided that the appropriate fee of US $25.00 per copy

per chapter is paid directly to Copyright Clearance Center, 222 Rosewood Drive, Danvers MA 01923, USA. Refer also to

www.copyright.com


CONTENTS

About the Book i

About the Author ii

Preface iii

CHAPTERS

PART I: FUNDAMENTALS OF FREE RADICAL BIOMEDICINE

1. Introduction to Free Radical Biomedicine 3

2. Free Radicals and Related Reactive Species 10

3. Antioxidants 40

PART II: FREE RADICALS AND DISEASES

4. Free Radicals and Related Reactive Species in Cardiovascular Diseases 94

5. Free Radicals and Related Reactive Species in Diabetes Mellitus and Metabolic

Syndrome 113

6. Free Radicals and Related Reactive Species in Neurological Diseases 127

7. Free Radicals and Related Reactive Species in Pulmonary Diseases 153

8. Free Radicals and Related Reactive Species in Hepatic and Gastrointestinal Diseases 173

9. Free Radicals and Related Reactive Species in Renal Diseases 202

10. Free Radicals and Related Reactive Species in Cancer 217

11. Free Radicals and Related Reactive Species in Aging 232

12. Free Radicals and Related Reactive Species in Other Diseases and Conditions 242

PART III: METHODOLOGIES IN FREE RADICAL RESEARCH

13. Detection of Free Radicals and Related Reactive Species 270

14. Detection of Damage of Biomolecules by Free Radicals and Related Reactive Species 295

15. Detection and Measurement of Cellular and Tissue Antioxidants 307

Index 326


About the Book

Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies is a new book that takes

a unique approach to integrating knowledge in free radical biomedicine from essentials to advances, from

basic research to clinical correlations, and from theories to methodologies. The book presents scientific

knowledge in free radical biomedicine in an organized, cogent, and in-depth manner. The consistent format

of writing, the full-color illustrations, and the comprehensive list of references make the book accessible

and engaging. Besides its value as a textbook for undergraduate and graduate students in life science, Free

Radical Biomedicine: Principles, Clinical Correlations, and Methodologies will also be a useful reference

for biomedical research scientists, clinicians, and other health professionals.

i


ii

About the Author

Y. Robert Li, MD, MPH, PhD, is a Professor and Chair of Department of Pharmacology of EVCOM at

Virginia Tech Corporate Research Center, USA. He is an Adjunct Professor of Department of Biomedical

Sciences and Pathobiology at Virginia Polytechnic Institute and State University, and an Affiliate Professor

at Virginia Tech-Wake Forest University School of Biomedical Engineering and Sciences. He currently

serves as Co-Editor-in-Chief for Toxicology Letters and on the editiorial boards of Cardiovascular

Toxicology, Experimental Biology and Medicine, Molecular and Cellular Biochemistry, Neurochemical

Research, and Spinal Cord. Dr. Li is an active researcher in the areas of free radicals, antioxidants, and

drug discovery, and the author of over 100 peer-reviewed publications and the monograph, Antioxidants in

Biology and Medicine. The research in his laboratories has been funded by the United States National

Cancer Institute (NCI), National Heart, Lung and Blood Institute (NHLBI), National Institute of Diabetes

and Digestive and Kidney Diseases (NIDDK), American Institute for Cancer Research (AICR), and Harvey

W. Peters Research Center Foundation.


PREFACE

The last two to three decades have witnessed the remarkable advances in our knowledge about

free radicals and related reactive species, especially reactive oxygen and nitrogen species

(ROS/RNS) in biomedicine. It has been increasingly recognized that ROS/RNS play an important

role in the pathophysiology of diverse diseases, including cardiovascular diseases, diabetes and

metabolic syndrome, neurological disorders, and cancer among many others. In agreement with

this notion, substantial studies have demonstrated the effectiveness of various antioxidant-based

modalities in disease intervention. New knowledge in free radical biomedicine will certainly

further increase our ability to develop more effective mechanistically-based strategies to combat

human diseases that involve a free radical-mediated pathophysiological component.

This book integrates knowledge in free radical biomedicine from essentials to advances, and from

basic research to clinical correlations. It also covers basic and advanced methodologies in free

radical research. It is hoped that the book will provide the reader a unique approach to

understanding the rapidly evolving field of free radical biomedicine.

This book would not have been possible without the assistance of my son Jason Z. Li who drew

all the chemical structures for the whole book, and my wife H. Zhu, MD for her critical review of

the entire book manuscript. I am grateful to those (over 100 scientists worldwide) who provided

me reprints of their publications and/or expertly reviewed part of the book manuscript. I am

thankful for the time and effort made by the editorial personnel, especially Ms. Sarah Khan at

Bentham Science Publishers, and the constructive suggestions resulting from the outside peerreview

of the entire book manuscript sponsored by Bentham Science Publishers.

iii

Y. Robert Li, MD, MPH, PhD

Blacksburg, Virginia

December 2011


PART I: FUNDAMENTALS OF FREE RADICAL BIOMEDICINE


Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies, 2012, 3-9 3

Introduction to Free Radical Biomedicine

Y. Robert Li

All rights reserved - © 2012 Bentham Science Publishers

CHAPTER 1

Abstract: The term free radical refers to any chemical species capable of independent existence that

contains one or more unpaired electrons. Free radicals and related reactive species are formed in

biological systems, and can cause detrimental as well as beneficial biological effects. Free radical

biomedicine deals with the biological effects of free radicals and related reactive species as well as

antioxidants with an emphasis on the involvement of these reactive species and antioxidants in heath

and disease. This chapter provides a brief historical overview of this rapidly evolving field and

introduces common terms and concepts in free radical biomedicine. These include reactive oxygen and

nitrogen species (ROS/RNS), antioxidants, oxidative stress, redox signaling, and free radical paradigm.

The aims and layout of the book are also described in this chapter.

Keywords: Antioxidants, Free radical biomedicine, Free radical paradigm, Free radicals, Nitrative stress,

Nitrosative stress, Oxidative stress, Reactive nitrogen species, Reactive oxygen species, Redox signaling.

CHAPTER AT A GLANCE

1. HISTORICAL OVERVIEW

2. DEFINITIONS AND CONCEPTS

2.1. Free Radicals and Related Reactive Species

2.2. Antioxidants

2.3. Oxidative Stress and Related Terms

2.4. Redox Signaling

2.5. Free Radical Paradigm

3. AIMS AND LAYOUT OF THE BOOK

3.1. Aims

3.2. Layout

4. REFERENCES

1. HISTORICAL OVERVIEW

Molecular oxygen is an essential element of aerobic life, yet incomplete reduction or excitation of

molecular oxygen during aerobic metabolism generates oxygen-containing reactive species that pose a

serious threat to aerobic organisms. Although more than two centuries ago Joseph Priestley noticed that

“oxygen might not be so proper for use in the usual healthy state of the body…” insights into the

mechanisms underlying oxygen toxicity were provided in several articles published during 1950’s and

1960’s. In 1954 R. Gerschman and coworkers published an article in Science, hypothesizing that oxygen

poisoning and radiation injury have at least one common basis of action, possibly through the formation of

oxidizing free radicals [1]. Another important event in the investigation of oxygen toxicity was the finding

by J.M. McCord and I. Fridovich in 1969 of an enzymatic function of a protein containing both copper and

zinc, which then was known alternatively as erythrocuprein, hepatocuprein, or cerebrocuprein [2]. The


4 Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies Y. Robert Li

.-

function of this enzyme is the catalysis of the dismutation of superoxide anion radical (O2 ) to produce

hydrogen peroxide and molecular oxygen. This discovery has triggered extensive investigation into the free

radical mechanisms of oxygen toxicity. Accumulating evidence over the last several decades demonstrates

a critical involvement of free radicals and related reactive species in the pathophysiology of a variety of

diseases. While free radicals and related reactive species produce deleterious effects in biological systems,

it is important to bear in mind that these reactive species also play important physiological roles, such as

antimicrobial activity [3] and participation in cell signal transduction [4]. The term biological system is

often used, but typically ill-defined. In this book, a biological system refers to a system consisting of

biological entities or processes, such as organs, tissues, cells, or biomolecules, or combinations of them. A

biomolecule is a molecule produced by living cells, e.g., a protein, carbohydrate, lipid, or nucleic acid.

2. DEFINITIONS AND CONCEPTS

Biomedicine refers to the application of the principles of the natural sciences, especially biology and

physiology, to clinical medicine. Free radical biomedicine deals with the biological effects of free radicals

and related reactive species as well as antioxidants with an emphasis on the involvement of these reactive

species and antioxidants in health and disease. To help understand this rapid evolving field, this section

provides definitions of commonly used terms and introduces some basic concepts in free radical

biomedicine. These terms and concepts include (1) free radicals and related reactive species, (2)

antioxidants, (3) oxidative stress and related terms, (4) redox signaling, and (5) free radical paradigm.

2.1. Free Radicals and Related Reactive Species

Free radical is a commonly encountered term in biology and medicine. It refers to any chemical species

capable of independent existence that contains one or more unpaired electrons. An unpaired electron refers

.-

to the one that occupies an atomic or molecular orbital by itself. The molecule O2 mentioned above is a

free radical. It is called superoxide anion radical because of its negative charge. The superscript dot

indicates the unpaired electron. In general, free radicals are reactive species in biological systems.

However, reactive species are not necessarily free radicals. Below are some commonly used terms to

describe various types of biologically relevant reactive species.

Reactive oxygen species (ROS) is a term frequently used in free radical biomedicine. This term can be simply

.-

defined as oxygen-containing reactive species. It is a collective term to include superoxide (O2 ), hydrogen

peroxide (H2O2), hydroxyl radical ( . OH), singlet oxygen ( 1 O2), peroxyl radical (LOO . ), alkoxyl radical (LO . ),

lipid hydroperoxide (LOOH), peroxynitrite anion (ONOO - ), hypochlorous acid (HOCl), and ozone (O3) [5].

Similarly, the term reactive nitrogen species (RNS) has been coined to include nitric oxide ( . NO), peroxynitrite

(ONOO - .

), nitrogen dioxide radical (NO2 ), and other oxides of nitrogen or nitrogen-containing reactive species.

The term reactive chlorine species (RCS) has also been coined to refer to chlorine-containing reactive species,

with hypocholorous acid (HOCl) as a typical member. As compared with ROS and RNS, the term RCS is less

frequently used in free radical biomedicine. As indicated above, the RCS hypochlorous acid and the RNS

peroxynitrite anion are also classified into ROS. Indeed, ROS comprise the most commonly encountered free

radicals and related species in biomedicine. Due to the increasingly recognized biological effects of nitric oxide

and related nitrogen-containing species, the term RNS has been becoming widely used in biomedicine. As such,

this book focuses on discussing the biological effects of both ROS and RNS, and the compound term

ROS/RNS is used throughout the book. Fig. (1.1) illustrates the formation of some common ROS from

excitation and univalent reduction of molecular oxygen in biological systems.

Although ROS is a widely used term in free radical biomedicine to describe oxygen-containing reactive

species, other exchangeable terms also exist in the literature, including reactive oxygen metabolites

(ROMs), reactive oxygen intermediates (ROIs), as well as reactive oxygen (RO) for short. For consistency,

these exchangeable terms are not used in this book.

Among the ROS listed above, some contain unpaired electrons, and thus belong to free radicals. They are

also called oxygen free radicals or oxyradicals. Examples of oxygen free radicals include superoxide anion


Introduction to Free Radical Biomedicine Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies 5

radical, hydroxyl radical, peroxyl radical, and alkoxyl radical. Some ROS do not contain any unpaired

electrons, and as such they are not free radicals. Examples of the non-radical ROS include hydrogen

peroxide, peroxynitrite anion, hypochlorous acid, and ozone. As shown in Fig. (1.1) and also discussed in

Chapter 2, singlet oxygen can exist in two states: the delta state ( 1 g) and sigma state ( 1 g + ). ( 1 g + ) 1 O2 is a

free radical because it contains unpaired electrons. One the other hand, ( 1 g) 1 O2 is a non-radical. Due to its

highly unstable nature, ( 1 g + ) 1 O2 is generally regarded to lack biological significance. Thus, in free radical

biomedicine, singlet oxygen usually refers to the 1 g state.

( 1g) 1O2 .. ..

O O

.. ..

..

..

Singlet

Oxygen

( 1 .. ..

O O

.. ..

+

g ) 1O2 . .

..

.. ..

O O

.. ..

(O2) Molecular

Oxygen

. .

..

.. ..

O O

.. ..

(O .‐

2 )

Superoxide

Anion Radical

.

..

..

.. ..

O O

.. ..

(HO .

2 )

Perhydroxyl

Radical

. H

‐ .. ..

H H . ..

e ‐ e ‐ e ‐ e ‐

pKa = 4.8

..

..

O O

.. ..

(H2O2) Hydrogen

Peroxide

..

..

..

( . O H

..

OH)

Hydroxyl

Radical

H

..

O

..

H

(H2O) Water

Fig. (1.1). Excitation and univalent reduction of molecular oxygen to yield ROS in biological systems. As indicated,

ground state molecular oxygen is a free radical (a diradical) because it contains two unpaired electrons. Ground state

molecular oxygen is much less reactive than ROS due to spin restriction caused by the same spin direction of its two

unpaired electrons. Ground state molecular oxygen (O 2) can be excited to form singlet oxygen ( 1 O 2). There are two

states of singlet oxygen: delta and sigma. The sigma state singlet oxygen is a free radical, whereas the delta state is a

non-radical. One electron reduction of the ground state molecular oxygen gives rise to superoxide anion radical (O 2 .- ),

which then undergoes another one electron reduction to yield hydrogen peroxide (H2O 2). One electron reduction of

hydrogen peroxide generates hydroxyl radical ( . OH), which can then be reduced by one electron to form water.

Perhydroxyl radical (HO2 . ) is the protonated form of superoxide anion radical. See Chapter 2 for detailed discussion of

these ROS.

A number of other terms related to ROS/RNS are also frequently used in free radical biomedicine. These

include oxidation, reduction, redox, as well as oxidant and reductant. Oxidation refers to loss of one or

more electrons by an atom or molecule. Reduction refers to gain of one or more electrons by an atom or

molecule. If chemical A oxidizes chemical B (another way of saying this is that chemical B reduces

chemical A), then chemical A is an oxidant, chemical B a reductant for that particular reaction. The term

redox refers to reduction-oxidation.

ROS/RNS are reactive species capable of causing damage to biomolecules, including proteins, lipids, and

nucleic acids, leading to cell and tissue injury. Reactions of these reactive species with biomolecules also

generate a large array of secondary electrophiles, including ,-unsaturated aldehydes, -6 and -3

unsaturated fatty acids, as well as nitro-fatty acids. The term electrophile refers to an electron-deficient

species that undergoes covalent reactions by accepting an electron pair from an electron-rich biomolecule

(nucleophile). Electrophilic species can also be derived from biotransformation of xenobiotics. The term

xenobiotic can be defined as any substance that does not occur naturally in the human body. While high

levels of electrophiles, especially ,-unsaturated aldehydes are able to cause cell and tissue injury, under

certain conditions controlled formation of these electrophilic species is involved in redox signaling (see

Section 2.4 for discussion of redox signaling).

Due to the detrimental nature of the above biological reactive species, mammalian species including

humans have evolved a series of antioxidant defenses to protect vital biomolecules from ROS/RNS as well

as electrophile-mediated damage. In addition, a number of compounds derived from dietary sources,

especially fruits and vegetables also exhibit antioxidant activities in biological systems.

..

..

..


10 Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies, 2012, 10-39

Free Radicals and Related Reactive Species

Y. Robert Li

All rights reserved - © 2012 Bentham Science Publishers

CHAPTER 2

Abstract: Reactive oxygen and nitrogen species (ROS/RNS) are collective terms that refer to a number

of oxygen- or nitrogen-containing reactive species. They are produced from various cellular pathways

as well as exogenous sources. ROS/RNS are able to cause damage to a variety of biomolecules,

including lipids, proteins, and nucleic acids, leading to tissue injury and disease processes. Under

certain circumstances, the well-controlled production of ROS/RNS also fulfils important physiological

roles, including antimicrobial activity and participation in cell signaling. The biological effects of

ROS/RNS, including both beneficial and detrimental effects are dependent on the types of ROS/RNS

and their concentrations and duration of exposure, as well as the types of tissues/cells that the

ROS/RNS are produced from or act on. The causal or contributing role of ROS/RNS in various diseases

makes it necessary to devise strategies to mitigate the tissue injury and therefore protect against the

disease process. Use of exogenous compounds with antioxidant properties and upregulation of

endogenous cellular antioxidant enzymes by chemical inducers are among the potential approaches to

the intervention of diseases with augmented ROS/RNS as underlying mechanisms.

Keywords: Alkoxyl radical, Hydrogen disulfide, Hydrogen peroxide, Hydroxyl radical, Hypochlorous

acid, Nitric oxide, Ozone, Peroxyl radical, Peroxynitrite, Singlet oxygen, Superoxide.

CHAPTER AT A GLANCE

1. OVERVIEW

2. BASIC CHEMISTRY OF FREE RADICALS AND RELATED REACTIVE SPECIES

2.1. Free Radical Reactions

2.2. Reaction Rate Constant

2.3. Reduction Potential

3. COMMON TYPES OF FREE RADICALS AND RELATED REACTIVE SPECIES

3.1. Superoxide Anion Radical (O2 .- )

3.2. Hydrogen Peroxide (H2O2)

3.3. Hydroxyl Radical ( . OH)

3.4. Peroxynitrite Anion (ONOO - )

3.5. Nitric Oxide ( . NO) and Other Related Nitrogen Oxides

3.6. Peroxyl Radical (LOO . ) and Alkoxyl Radical (LO . )

3.7. Singlet Oxygen ( 1 O2)

3.8. Hypochlorous Acid (HOCl)

3.9. Other Related Species

4. SOURCES OF FREE RADICALS AND RELATED REACTIVE SPECIES


Free Radicals and Related Reactive Species Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies 11

4.1. Endogenous Sources

4.2. Exogenous Sources

5. BIOLOGICAL EFFECTS OF FREE RADICALS AND RELATED REACTIVE SPECIES

5.1. Molecular Targets

5.2. Detrimental Effects at Cellular Level

5.3. Physiological Effects

6. FREE RADICALS AND RELATED REACTIVE SPECIES IN TOXICOLOGY AND

PHARMACOLOGY

6.1. As Mediators of Adverse Effects of Xenobiotics Including Drugs

6.2. As Mediators of Pharmacological Effects of Drugs

7. ROLE OF FREE RADICALS AND RELATED REACTIVE SPECIES IN DISEASES

7.1. Establishment of Causal Relationship

7.2. Involvement in Disease Process: An Overview

8. INTERVENTION OF FREE RADICALS AND RELATED REACTIVE SPECIES

8.1. An Overview of the Potential Approaches

8.2. An Overview of Applications to Human Disease Intervention

9. REFERENCES

1. OVERVIEW

As stated in Chapter 1, biologically relevant reactive species include reactive oxygen species (ROS), reactive

nitrogen species (RNS), as well as other free radicals and non-free radical species. Among these reactive

species, ROS/RNS are most commonly encountered in biological systems and free radical biomedicine.

ROS/RNS are collective terms that refer to a number of oxygen- or nitrogen-containing reactive species. They

are produced from various cellular pathways as well as exogenous sources. ROS/RNS are able to cause damage

to a variety of biomolecules, leading to tissue injury and disease processes. Under certain conditions, ROS/RNS

also carry out important physiological functions. The biological effects of ROS/RNS are determined by

multiple factors, including the chemical properties of the individual ROS/RNS and effectiveness of the

available tissue antioxidant defenses, as well as the intrinsic susceptibility of the cells or tissues to injury. This

chapter begins with a description of free radical reactions and related basic chemistry. It then provides a

succinct discussion of the major chemical properties and biological activities of the most commonly

encountered ROS/RNS in biomedicine, followed by an overview of their sources and biological effects.

Considering the involvement of ROS/RNS in drug action and chemical toxicity, the role of these reactive

species in toxicology and pharmacology is also briefly discussed. An overview of the role of ROS/RNS in

diseases and a brief introduction to the general approaches, especially antioxidant-based strategies for disease

intervention are presented toward the end of the chapter. A detailed coverage of antioxidants is provided in the

subsequent Chapter 3.


12 Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies Y. Robert Li

2. BASIC CHEMISTRY OF FREE RADICALS AND RELATED REACTIVE SPECIES

2. 1. Free Radical Reactions

Many ROS/RNS are either free radicals or derived from free radical reactions. Free radical reactions are

also involved in ROS/RNS-mediated biological effects. Thus, it is imperative to understand the basic

chemistry of free radical reactions. Due to their high reactivity, free radicals usually do not react with great

selectivity. The inherent chemical complexity of the biological systems further complicates the study of

free radical-mediated biological reactions. However, three general classes of free radical reactions are

recognized. They are atom abstraction, free radical addition, and electron transfer.

2.1.1. Atom Abstraction

A free radical may abstract a hydrogen atom from a C-H bond of a biomolecule, such as a lipid molecule

(LH). As the hydrogen atom has only one electron, an unpaired electron must be left on the carbon after

removal of the hydrogen atom. This gives rise to the carbon-centered radical (L . ). For example, hydroxyl

radical ( . OH; see Section 3.3 below for more discussion) is able to abstract a hydrogen atom from a

hydrocarbon side chain of a polyunsaturated fatty acid (Reaction 1).

. OH + LH → H2O + L . (1)

In this reaction, the hydroxyl radical combines with the hydrogen atom to form water, and the

polyunsaturated fatty acid is converted to a carbon-centered radical. As discussed below, hydrogen atom

abstraction by hydroxyl radical in an important mechanism by which lipid peroxidation in biological

systems is initiated.

2.1.2. Free Radical Addition

There are two types of free radical addition reactions, one occurs between a free radical and a non-free

radical, and the other occurs between two free radicals.

2.1.2.1. Free Radical Addition between a Free Radical and a Non-Free Radical

A free radical (A . ) adds onto another non-free radical molecule (B) to form an adduct compound. Such an

adduct compound still has an unpaired electron initially associated with the free radical A . . The reaction is

depicted as: A . + B [A-B] . For example, hydroxyl radical ( . OH) reacts with the spin-trap 5,5dimethylpyrroline-N-oxide

(DMPO) to form a DMPO-hydroxyl adduct [DMPO . -OH] (Reaction 2).

. OH + DMPO → [DMPO . -OH] (2)

This adduct still contains an unpaired electron, and is thus a free radical species. This free radical adduct

has a much longer half-life than does the free hydroxyl radical, and as such can be detected by electron

paramagnetic resonance (EPR) technique (Chapter 13). Free radical addition is the basis for EPR spin

trapping detection of short-lived free radicals, including hydroxyl radical and superoxide anion radical.

Another example of free radical addition is the reaction between hydroxyl radical and the guanine base of

DNA. In the reaction, hydroxyl radical is added to the guanine (G) forming an intermediate free radical

adduct, which is then converted to 8-hydroxyguanine (Reaction 3).

. OH + G in DNA → [8-OH-G radical] → 8-hydroxyguanine (3)

8-Hydroxyguanine (8-OH-G) or 8-hydroxy-2’-deoxygunosine (8-OH-dG) is a commonly used biomarker

for oxidative DNA damage (Section 5.2 of Chapter 14).

2.1.2.2. Free Radical Addition between Two Free Radicals

A free radical (A . ) is added onto another free radical (B . ) or the same radical (A . ) to form a non-free radical

product. The reaction is depicted as: A . + B . AB or A . + A . AA. In the above reactions, the unpaired


40 Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies, 2012, 40-93

Antioxidants

Y. Robert Li

All rights reserved - © 2012 Bentham Science Publishers

CHAPTER 3

Abstract: Due to the detrimental nature of reactive oxygen and nitrogen species (ROS/RNS), a number

of antioxidants have been evolved to control these reactive species in mammals, including humans. The

endogenous antioxidative defenses include antioxidant enzymes/proteins and non-protein antioxidant

compounds synthesized by cells. Mammalian cells and tissues also contain antioxidant compounds

derived from the diet. In addition, a number of synthesized antioxidant compounds have become

available. This chapter provides a survey of the various types of antioxidants encountered in free radical

biomedicine. The chemical or biochemical properties, biological activities, and molecular regulation of

antioxidants are discussed in the context of their implications for disease intervention.

Keywords: Antioxidant vitamins, Antioxidants, carotenoids, Catalase, Dietary antioxidants, Glutaredoxin,

Glutathione system, Heme oxygenase, Methionine sulfoxide reductase, NAD(P)H:quinone oxidoreductase,

Non-protein antioxidants, Nrf2, Paraoxonase, Polyphenols, Superoxide dismutase, Thioredoxin system.

CHAPTER AT A GLANCE

1. OVERVIEW

1.1. Defining Antioxidants

1.2. Classification Schemes of Antioxidants

1.3. Phase 2 Proteins

1.4. Caveats

2. TYPES OF ANTIOXIDANTS

2.1. Superoxide Dismutase

2.2. Catalase

2.3. Glutathione and its Synthesizing Enzymes

2.4. Glutathione Peroxidase

2.5. Glutathione Reductase

2.6. Glutathione S-Transferase

2.7. NAD(P)H:Quinone Oxidoreductase

2.8. Heme Oxygenase

2.9. Thioredoxin

2.10. Peroxiredoxin

2.11. Thioredoxin Reductase

2.12. Glutaredoxin


Antioxidants Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies 41

2.13. Methionine Sulfoxide Reductase

2.14. Paraoxonase

2.15. Other Protein Antioxidants

2.16. Other Non-protein Antioxidants Synthesized by Cells

2.17. Non-protein Antioxidants Derived from the Diet

2.18. Synthetic Non-protein Antioxidants

3. MOLECULAR REGULATION OF ANTIOXIDANTS

3.1. Antioxidant Response Element (ARE) and Nrf2

3.2. Other Transcription Factors

3.3. Chemical Inducibility of Antioxidants and Chemoprotection

4. ANTIOXIDANT-BASED INTERVENTION

4.1. Overall Strategies

4.2. Animal Studies

4.3. Human Studies

5. REFERENCES

1. OVERVIEW

As stated in Chapter 2, free radicals and related reactive species, especially reactive oxygen and nitrogen

species (ROS/RNS) are able to cause damage to biomolecules, leading to cell and tissue injury in mammals,

including humans. Thus, a number of endogenous antioxidant defenses have been evolved in mammals to

protect against the damage caused by ROS/RNS. In addition, a variety of exogenous compounds from natural

sources, including foods exhibit antioxidant properties in biological systems. This chapter describes the various

types of endogenous antioxidants in mammals as well as exogenous antioxidant compounds frequently

encountered in free radical biomedicine. It should be borne in mind that antioxidant enzymes or proteins are

widely distributed in various animal species, microorganisms, as well as plants. This book focuses on the

discussion of knowledge on antioxidants obtained from studies in mammalian species, including humans

though knowledge obtained from studies in lower organisms is also of critical importance.

1.1. Defining Antioxidants

The term antioxidant is frequently used in biomedicine, and has been defined in various ways in the

literature. One way to define the term is that antioxidant is any substance that can prevent, reduce or repair

the ROS/RNS-induced damage of a target biomolecule. There are several potential modes of action by

which antioxidants protect biomolecules from ROS/RNS-induced damage. Fig. (3.1) illustrates these

potential modes of antioxidant protection.

1.1.1. Inhibition of ROS/RNS Formation

ROS/RNS are produced by a number of cellular sources, including mitochondrial electron transport chain

and NAD(P)H oxidases (also known as NOX enzymes). Antioxidants may act on these cellular sources,


42 Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies Y. Robert Li

inhibiting or preventing the formation of ROS/RNS. For example, mono-O-methylated flavanols inhibit

NAD(P)H oxidases in vascular cells, leading to decreased superoxide production. Flavanols are phenolic

compounds found in plants (Section 2.17.4).

Sources of

ROS/RNS

inhibition

ROS/RNS

Scavenging

Antioxidants

Fig. (3.1). The three modes of action of antioxidants. Antioxidants may inhibit the ROS/RNS formation, scavenge

ROS/RNS, or repair the ROS/RNS-damaged biomolecules. See text (Section 1.1) for detailed description. ROS/RNS,

reactive oxygen and nitrogen species.

1.1.2. Scavenging of ROS/RNS

Many endogenous and exogenous antioxidants can directly scavenge ROS/RNS. Scavenging of ROS/RNS

can occur through enzyme catalyzed reactions. For example, superoxide dismutase catalyzes the

dismutation of superoxide to form hydrogen peroxide and molecular oxygen (Section 2.1). Non-enzymatic

antioxidants can also scavenge ROS/RNS through direct chemical reactions. These reactions can lead to the

formation of secondary free radical species from the non-enzymatic antioxidants. For example, the

antioxidant -tocopherol scavenges lipid peroxyl radical by reducing the lipid peroxyl radical to lipid

hydroperoxide (Section 2.3 of Chapter 2). In the reaction, the -tocopherol is oxidized to -tocopherol

radical. The -tocopherol radical is much less reactive than the lipid peroxyl radical.

1.1.3. Removal or Repair of the ROS/RNS-Induced Damage

ROS/RNS can cause damage or modifications to biomolecules, including proteins, lipids and nucleic acids.

Mammalian cells are equipped with various mechanisms to remove or repair the ROS/RNS-damaged or

modified biomolecules. For example, ROS/RNS oxidize methionine residues in proteins, yielding

methionine sulfoxide. The enzyme methionine sulfoxide reductase reduces the methionine sulfoxide in

proteins back to the normal methionine (Section 2.13). This is an important repairing mechanism for

oxidative protein damage in mammals. Mammalian cells also contain enzymes for repairing oxidative

damage of lipids and nucleic acids.

1.2. Classification Schemes of Antioxidants

In free radical biomedicine, antioxidants are classified in various ways (Table 3.1).

Damaged

Biomolecules

1.2.1. Endogenous and Exogenous Antioxidants

Based on sources, antioxidants are classified into endogenous and exogenous antioxidants. Endogenous

antioxidants refer to those naturally occurring in cells or tissues. These include superoxide dismutase, catalase

and the reduced form of glutathione, to name a few. On the other hand, those derived from dietary sources or

synthesized in laboratories are called exogenous antioxidants. Vitamins C and E, and various synthetic

mimetics of endogenous antioxidant enzymes are examples of exogenous antioxidants (Sections 2.17 and 2.18).

1.2.2. Intracellular and Extracellular Antioxidants

Based on locations, antioxidants are classified into intracellular and extracellular antioxidants. Intracellular

antioxidants are those present inside cells, such as copper, zinc superoxide dismutase. Extracellular

Repair


94 Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies, 2012, 94-112

Y. Robert Li

All rights reserved - © 2012 Bentham Science Publishers

CHAPTER 4

Free Radicals and Related Reactive Species in Cardiovascular Diseases

Abstract: There is substantial evidence supporting that oxidative stress plays a causal role in the

pathophysiology of various cardiovascular diseases in animal models. These include hypertension,

atherosclerosis, myocardial ischemia-reperfusion injury, heart failure, cardiac arrhythmias, and

cardiotoxicity induced by certain drugs and environmental toxicants. A number of cellular sources of

reactive oxygen and nitrogen species (ROS/RNS) are identified in cardiovascular tissues, including

NAD(P)H oxidases, xanthine oxidoreductase, mitochondrial electron transport chain, uncoupled endothelial

nitric oxide synthase, lipoxygenases, and cytochrome P450 system. Augmented formation of ROS/RNS

from these sources results in cardiovascular injury via various mechanisms. Suppression of the augmented

formation of ROS/RNS by overexpression of endogenous antioxidants or administration of exogenous

antioxidants attenuates the severity of cardiovascular diseases in animal models. In contrast to animal

studies, large scale clinical trials on using antioxidant vitamin supplements (mainly vitamin E) in general

populations for the intervention of human cardiovascular diseases have been disappointing. Possible reasons

for the negative results include the doses and forms of the vitamins used, the time-window of intervention,

and the general populations included in the trials. In this context, multiple small clinical trials in selected

patients with unusual oxidative stress show the benefits for antioxidant therapies in human cardiovascular

diseases. Future studies should focus on the development of more effective antioxidants and testing their

effectiveness in well-designed clinical trials.

Keywords: Antioxidant intervention, Atherosclerosis, Cardiac arrhythmia, Cardiotoxicity, Cardiovascular

diseases, Heart failure, Hypertension, Myocardial ischemia-reperfusion injury, Oxidative stress, Reactive

nitrogen species, Reactive oxygen species.

CHAPTER AT A GLANCE

1. OVERVIEW

2. FREE RADICALS AND RELATED REACTIVE SPECIES IN HYPERTENSION

2.1. Introduction to Hypertension

2.2. Animal Studies

2.3. Human Studies and Clinical Correlations

3. FREE RADICALS AND RELATED REACTIVE SPECIES IN ATHEROSCLEROSIS

3.1. Introduction to Atherosclerosis

3.2. Animal Studies

3.3. Human Studies and Clinical Correlations

4. FREE RADICALS AND RELATED REACTIVE SPECIES IN MYOCARDIAL ISCHEMIA-

REPERFUSION INJURY

4.1. Introduction to Myocardial Ischemia-Reperfusion Injury

4.2. Animal Studies

4.3. Human Studies and Clinical Correlations


Cardiovascular Diseases Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies 95

5. FREE RADICALS AND RELATED REACTIVE SPECIES IN HEART FAILURE

5.1. Introduction to Heart Failure

5.2. Animal Studies

5.3. Human Studies and Clinical Correlations

6. FREE RADICALS AND RELATED REACTIVE SPECIES IN OTHER FORMS OF

CARDIOVASCULAR DISORDERS

6.1. Cardiac Arrhythmias

6.2. Drug/Xenobiotic-Induced Cardiovascular Toxicity

7. REFERENCES

1. OVERVIEW

1.1. Introduction to Cardiovascular Diseases

Cardiovascular diseases remain a leading cause of deaths worldwide. In the United States more than 80

million adults (more than one in three) have one or more types of cardiovascular diseases. Mortality data

show that cardiovascular diseases as the underlying causes of death accounted for 33.6% (813,804) of all

2,243,712 deaths in 2007, or one of every 2.9 deaths in the United States. The 2007 overall death rate from

cardiovascular diseases in the United States was 251.2 per 100,000. On the basis of 2007 mortality rate

data, more than 2,200 Americans die of cardiovascular diseases each day, an average of one death every 39

seconds. The total cost of cardiovascular diseases in the United States for 2007 is estimated to be $286

billion, which is more than that of any other diagnostic group.

Based on the 2011 Update from American Heart Association [1], the prevalence of various types of

cardiovascular diseases in the United States is as follows.

High blood pressure (hypertension): 76,400,000

Coronary heart disease: 16,300,000

Myocardial infarction: 7,900,000

Angina pectoris: 9,000,000

Heart failure: 5,700,000

Stroke: 7,000,000

Congenital cardiovascular defects: 650,000-1,300,000

Although the mortality and morbidity of cardiovascular diseases have been significantly reduced over the

past decades owning to availability of surgical procedures and drugs, cardiovascular diseases remain a

major public health issue in the United States as well as worldwide. Hence, it is of critical importance to

further develop more effective approaches to the prevention and treatment of cardiovascular diseases. In

this context, a better understanding of the pathophysiological processes of cardiovascular diseases

contributes to our increased ability to develop more effective mechanistically-based strategies for

intervention of these diseases. Studies over the last two decades have provided substantial evidence for a


96 Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies Y. Robert Li

critical involvement of free radicals and related reactive species, especially reactive oxygen and nitrogen

species (ROS/RNS) in the pathophysiology of various forms of cardiovascular diseases. This chapter

discusses the role of ROS/RNS in hypertension, atherosclerosis, myocardial ischemia-reperfusion injury,

heart failure, and other forms of cardiovascular disorders. Before discussion of these individual

cardiovascular disorders, it is necessary to provide an overview of the major sources of ROS/RNS in

cardiovascular tissues.

1.2. Sources of ROS/RNS in Cardiovascular Tissues

As to be discussed below, increased ROS/RNS levels are frequently observed in various cardiovascular

diseases. There are a number of cellular factors and pathways contributing to the ROS/RNS formation in

cardiovascular tissues. Among them NAD(P)H oxidases, xanthine oxidoreductase, mitochondrial electron

transport chain, and uncoupled endothelial nitric oxide synthase (eNOS) are of major importance. Under

certain circumstances, other sources, such as inducible nitric oxide synthase (iNOS), cytochrome P450

system and lipoxygenases, may also become significant. iNOS is expressed in infiltrated inflammatory cells

as well as cardiovascular cells under inflammation. In addition, myeloperoxidase derived from infiltrated

inflammatory cells contributes to the formation of hypochlorous acid. Table 4.1 lists the various sources of

ROS/RNS in cardiovascular tissues.

Table 4.1. Various sources of ROS/RNS in cardiovascular tissues.

Sources of ROS/RNS Primary ROS/RNS Products

NAD(P)H oxidases O 2 .-

Xanthine oxidoreductase O 2 .- , H2O 2

Mitochondrial electron transport chain O 2 .-

Uncoupled eNOS O 2 .- and . NO (O2 .- + . NO → ONOO - )

iNOS

. NO

Cytochrome P450 system O 2 .-

Lipoxygenases O 2 .-

Myeloperoxidase HOCl

Note: O2 .- , superoxide; . NO, nitric oxide; ONOO - , peroxynitrite; HOCl, hypochlorous acid; eNOS, endothelial nitric oxide synthase;

iNOS, inducible nitric oxide synthase.

2. FREE RADICALS AND RELATED REACTIVE SPECIES IN HYPERTENSION

2.1. Introduction to Hypertension

2.1.1. Definition and Epidemiology

Hypertension is defined as systolic pressure >140 mm Hg and/or diastolic pressure >90 mm Hg. Based on

JNC7 (the Seventh Report of the Joint National Committee on the Prevention, Detection, Evaluation, and

Treatment of High Blood Pressure), blood pressure is classified into 4 categories (Table 4.2). According to

JNC7, hypertension is further classified into two stages, i.e., stage 1 and stage 2. The term prehypertension is

also defined in JNC7 as systolic pressure between 120 and 139 or diastolic pressure between 80 and 89 mm Hg.

Table 4.2. JNC7 classification of blood pressure.

Blood Pressure

Classification

Systolic Blood Pressure

mm Hg

Normal 100

Diastolic Blood Pressure

mm Hg


Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies, 2012, 113-126 113

Y. Robert Li

All rights reserved - © 2012 Bentham Science Publishers

CHAPTER 5

Free Radicals and Related Reactive Species in Diabetes Mellitus and

Metabolic Syndrome

Abstract: Both diabetes mellitus (often simply referred to as diabetes) and metabolic syndrome are

prevalent metabolic disorders that pose significant impact on public health. Development of these metabolic

disorders involves the complex interactions between genes and environment, and the exact underlying

pathophysiology remains to be fully elucidated. Extensive studies in animal models provide ample evidence

for oxidative stress as an important pathophysiological component of both diabetes and metabolic

syndrome. In line with experimental studies, observational epidemiological studies and interventional

clinical trials suggest that oxidative stress may also play an important role in the pathophysiology of diabetes

and metabolic syndrome in humans. The increasing recognition of the involvement of oxidative stress in

diabetes and metabolic syndrome has prompted extensive research to develop antioxidant-based approaches

to the preventive and therapeutic intervention of these prevalent metabolic disorders.

Keywords: Antioxidant intervention, Diabetes, Metabolic syndrome, Obesity, Oxidative stress, Reactive

nitrogen species, Reactive oxygen species.

CHAPTER AT A GLANCE

1. OVERVIEW

1.1. Introduction to Diabetes Mellitus

1.2. Introduction to Metabolic Syndrome

2. FREE RADICALS AND RELATED REACTIVE SPECIES IN DIABETES MELLITUS

2.1. Animal Studies

2.2. Human Studies and Clinical Correlations

3. FREE RADICALS AND RELATED REACTIVE SPECIES IN METABOLIC SYNDROME

3.1. Animal Studies

3.2. Human Studies and Clinical Correlations

4. REFERENCES

1. OVERVIEW

Diabetes and metabolic syndrome are prevalent metabolic diseases of major public health concern in both

developed and developing countries. They are also important risk factors for cardiovascular diseases. This

section introduces diabetes and metabolic syndrome, including definition, clinical features, epidemiology,

and pathophysiology so as to set a stage for the subsequent discussion of the role of reactive oxygen and

nitrogen species (ROS/RNS) in the disease processes as well as antioxidant-based intervention.

1.1. Introduction to Diabetes Mellitus

1.1.1. Definition, Classification, and Diagnostic Criteria

Diabetes mellitus, often simply referred to as diabetes, is a group of metabolic diseases characterized by

hyperglycemia resulting from defects in insulin secretion, insulin action, or both. The chronic


114 Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies Y. Robert Li

hyperglycemia of diabetes is associated with long-term damage, dysfunction, and failure of different

organs, especially the eyes, kidneys, nerves, heart, and blood vessels.

The classification of diabetes includes four clinical classes (Table 5.1) [1].

Table 5.1. Classification of diabetes mellitus.

Class Description

Type 1 diabetes Results from -cell destruction, usually leading to absolute insulin deficiency; comprises ~10% of

people with diabetes

Type 2 diabetes Results from a progressive insulin secretory defect on the background of insulin resistance;

comprises ~90% of people with diabetes

Other specific

types of diabetes

Gestational

diabetes

Due to other causes, e.g., genetic defects in cell function, genetic defects in insulin action,

diseases of the exocrine pancreas (such as cystic fibrosis), and drug or chemical-induced diabetes

(such as in the treatment of AIDS or after organ transplantation)

Diabetes diagnosed during pregnancy

The current diagnostic criteria for diabetes are summarized in Table 5.2 [1].

Table 5.2. Criteria for the diagnosis of diabetes mellitus.

Criteria Description

1 A1C >6.5%. The test should be performed in a laboratory using a method that is NGSP certified

and standardized to the DCCT assay

OR

2 FPG >126 mg/dl (7.0 mM). Fasting is defined as no caloric intake for at least 8 hours

OR

3 2-h plasma glucose >200 mg/dl (11.1 mM) during an OGTT. The test should be performed as

described by the World Health Organization, using a glucose load containing the equivalent of 75 g

anhydrous glucose dissolved in water

OR

4 In a patient with classic symptoms of hyperglycemia or hyperglycemic crisis, a random plasma

glucose >200 mg/dl (11.1 mM)

Note to Table 5.2: In the absence of unequaivocal hyperglycemia, criteria 1-3 should be confirmed by repeated testing; The

glycosylated hemoglobin A1C (or HbA1C) is a widely used marker of chronic glycemia, reflecting average blood glucose levels over

a 2- to 3-month period of time. NGSP, National Glycohemoglobin Standardization Program; DCCT, Diabetes Control and

Complications Trial; FPG, fasting plasma glucose; OGTT stands for oral glucose tolerance test.

Table 5.3. Categories of increased risk for diabetes mellitus.

Categories Description

Fasting plasma glucose (FPG): 100-125 mg/dl (5.6-6.9 mM) This condition is referred to as impaired fasting

glucose (IFG)

2-h plasma glucose on the 75-g OGTT: 140-199 mg/dl (7.8-

11.0 mM)

A1C: 5.7–6.4%

This condition is referred to as impaired glucose

tolerance (IGT)

Note to Table 5.3: For all three tests, risk is continuous, extending below the lower limit of the range and becoming disproportionately

greater at higher ends of the range; Individuals with IFG and/or IGT have been referred to as having pre-diabetes.

In the 2010 Clinical Practice Recommendations of the American Diabetes Association, categories of

increased risk for diabetes have been included to refer to an intermediate group of individuals whose


Diabetes Mellitus and Metabolic Syndrome Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies 115

glucose or HbA1C levels, although not meeting criteria for diabetes, are nevertheless too high to be

considered normal. These individuals have relatively high risk for the future development of diabetes. The

categories of increased risk for diabetes are summarized in Table 5.3 [1].

1.1.2. Epidemiology and Related Issues

Based on 2009 World Health Organization (WHO) Diabetes Fact Sheet, more than 220 million people

worldwide have diabetes. In 2005 an estimated 1.1 million people worldwide died from diabetes. The

actual number is likely to be much larger because although people may live for years with diabetes, their

cause of death is often recorded as heart disease or kidney failure. WHO projects that deaths from diabetes

will double between 2005 and 2030. By 2030, an estimated 360 million people will be affected by diabetes.

Diabetes and its complications have a significant economic impact on individuals, families, health systems,

and countries. For example, WHO estimates that in the period 2006-2015, China will lose $558 billion in

foregone national income due to heart disease, stroke, and diabetes alone.

Based on data from the 2007 National Diabetes Fact Sheet (the most recent year for which data is

available), diabetes statistics in the United States is summarized as following.

23.6 million people in the United States (7.8% of the population) have diabetes: diagnosed

(17.9 million); undiagnosed (5.7 million).

Pre-diabetes: 57 million people.

New cases: 1.6 million new cases of diabetes are diagnosed in people aged 20 years and older

each year.

Death: Diabetes was the seventh leading cause of death listed on U.S. death certificates in

2006. This ranking is based on the 72,507 death certificates in 2006 in which diabetes was

listed as the underlying cause of death. According to death certificate reports, diabetes

contributed to a total of 233,619 deaths in 2005, the latest year for which data on contributing

causes of death are available.

Costs: The total costs of diagnosed diabetes in the United States in 2007 were $174 billion.

1.1.3. Clinical Features and Pathophysiology

1.1.3.1. Clinical Manifestations

Manifestations of marked hyperglycemia include polyuria, polydipsia, weight loss, sometimes with

polyphagia, and blurred vision. Acute, life-threatening consequences of uncontrolled diabetes are

hyperglycemia with ketoacidosis or the nonketotic hyperosmolar syndrome. Chronic hyperglycemia may

lead to the long-term complications of diabetes, which include the following (Fig. 5.1).

Foot Ulcers and

Limb Amputation

Diabetic

Neuropathy

Cardiovascular

Diseases

Diabetes

Diabetic

Nephropathy

Diabetic

Retinopathy

Fig. (5.1). Schematic illustration of diabetic complications. See text (Section 1.1.3.1) for detailed description.


Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies, 2012, 127-152 127

Y. Robert Li

All rights reserved - © 2012 Bentham Science Publishers

CHAPTER 6

Free Radicals and Related Reactive Species in Neurological Diseases

Abstract: Due to its high rate of oxygen utilization and richness in lipids containing polyunsaturated fatty

acids, the central nervous system is vulnerable to reactive oxygen and nitrogen species (ROS/RNS)mediated

injury. Indeed, increasing evidence shows oxidative stress as an important mechanism underlying

various neurological disorders. This chapter summarizes both experimental and clinical data supporting

oxidative stress as a critical pathophysiological component of such common neurological diseases as stroke,

Parkinson’s disease, and Alzheimer’s disease. The role of oxidative stress in other neurological disorders,

including amyotrophic lateral sclerosis, Huntington’s disease, traumatic brain injury, spinal cord injury, and

drug/xenobiotic-induced neurotoxicity is also discussed in this chapter.

Keywords: Alzheimer’s disease, Amyotrophic lateral disease, Antioxidant intervention, Huntington’s

disease, Neurological diseases, Neurotoxicity, Oxidative stress, Parkinson’s disease, Reactive nitrogen

species, Reactive oxygen species, Spinal cord injury, Stroke, Traumatic brain injury.

CHAPTER AT A GLANCE

1. OVERVIEW

2. FREE RADICALS AND RELATED REACTIVE SPECIES IN STROKE

2.1. Introduction to Stroke

2.2. Animal Studies

2.3. Human Studies and Clinical Correlations

3. FREE RADICALS AND RELATED REACTIVE SPECIES IN PARKINSON’S DISEASE

3.1. Introduction to Parkinson’s Disease

3.2. Animal Studies

3.3. Human Studies and Clinical Correlations

4. FREE RADICALS AND RELATED REACTIVE SPECIES IN ALZHEIMER’S DISEASE

4.1. Introduction to Alzheimer’s Disease

4.2. Animal Studies

4.3. Human Studies and Clinical Correlations

5. FREE RADICALS AND RELATED REACTIVE SPECIES IN OTHER FORMS OF

NEUROLOGICAL DISORDERS

5.1. Amyotrophic Lateral Sclerosis

5.2. Huntington’s Disease

5.3. Traumatic Brain Injury


128 Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies Y. Robert Li

5.4. Spinal Cord Injury

5.5. Drug/Xenobiotic-Induced Neurotoxicity

6. REFERENCES

1. OVERVIEW

The human nervous system is the site of consciousness, cognition, and behavior; as such it is the most intricate

structure known to exist. About one third of the genes encoded in the human genome are expressed in the

nervous system. It is estimated that each mature human brain consists approximately 100 billion neurons,

several million miles of axons and dendrites, and over 10 15 synapes. Neurons exist within a dense parenchyma

of multifunctional glial cells that synthesize myelin, preserve homeostasis, and regulate immunity and

inflammation. The extreme complex structure and functionality of the nervous system make it a vulnerable

target of both exogenous and endogenous pathophysiological factors. There are numerous types of neurological

diseases, and the common ones worldwide include (in alphabetic order) dementia (e.g., Alzheimer’s disease),

epilepsy, headache disorders, multiple sclerosis, neuroinfections, neurological disorders associated with

malnutrition, pain associated with neurological disorders, Parkinson’s disease, stroke, and traumatic brain

injury. Neurological diseases kill an estimated 6.8 million people each year, equating to 12% of global deaths.

Neurological diseases are also common and costly in the United States. An estimated 180 million Americans

suffer from a neurological disease, resulting in an annual cost of over 700 billion dollars.

As the global population ages, the impact of neurological diseases in both developed and developing countries

will increase. Hence, developing effective public health strategies and therapeutic modalities are essential for

combating these disorders of increasing global public health importance. Studies over the last several decades

have resulted in a great understanding of the pathophysiology of many neurological diseases, which provides a

basis for the development of drugs for both preventive and therapeutic intervention of these diseases. In this

context, accumulating evidence supports a critical role for free radicals and related reactive species in the

pathophysiological processes of many common and rare neurological diseases. This chapter summarizes the

key findings from both experimental models and human studies, highlighting oxidative stress as an important

pathophysiological component of various types of neurological diseases, including stroke, Alzheimer’s disease,

Parkinson’s disease, amyotrophic lateral sclerosis, Huntington’s disease, traumatic brain injury, spinal cord

injury, and drug/xenobiotic-induced neurotoxicity. The antioxidant-based intervention of these neurological

diseases is also discussed in the chapter.

2. FREE RADICALS AND RELATED REACTIVE SPECIES IN STROKE

2.1. Introduction to Stroke

2.1.1. Definition and Epidemiology

The term cerebral vascular disease refers to disorders of the vasculature of the central nervous system. A

stroke or cerebrovascular accident is defined as an abrupt onset of a neurological deficit that occurs as a

result of either inadequate blood flow (ischemic stroke) or hemorrhage into the brain tissue (parenchymal or

intracerebral hemorrhage) or surrounding subarachnoid space (subarachnoid hemorrhage). Focal ischemic

stroke is usually caused by thrombosis of the cerebral vessels themselves or by emboli from a proximal

arterial source or the heart. Focal ischemic stroke (often simply referred to as ischemic stroke) accounts for

87% of all cases. A generalized reduction in cerebral blood flow due to systemic hypotension (e.g.,

cardiogenic shock) usually produces syncope, and in more severe instances, results in hypoxic-ischemic

encephalopathy. This condition is, however, not classified as stroke. Another condition related to cerebral

ischemia is transient ischemic attack (TIA). A TIA is a transient episode of neurological dysfunction caused

by focal brain, spinal cord, or retinal ischemia, without acute infarction [1]. Patients with TIAs are at high

risk of early stroke. Table 6.1 lists the classification of stroke.


Neurological Diseases Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies 129

Table 6.1. Classification of stroke.

Type of Stroke % of All Cases Cause

Ischemic stroke 87 1) Cerebral thrombosis

2) Cerebral embolism

Hemorrhagic stroke 13 1) Intracerebral hemorrhage

2) Subarachnoid hemorrhage

Based on American Heart Association Heart Disease and Stroke Statistics-2011 Update [2], an estimated

7,000,000 Americans age of 20 and over have had a stroke, and each year, about 795,000 Americans

experience a new or recurrent stroke. Stroke mortality in 2007 was 135,952, and stroke is currently the third

leading cause of deaths immediately after heart disease and cancer. Worldwide, approximately 16 million

first-ever strokes occur annually, with a death toll of ~5.7 million people per year. The incidence of

cerebral vascular diseases increases with age and the number of strokes is projected to increase as the

elderly population grows, with a doubling in stroke deaths in the United States by 2030. Globally, the death

toll from stroke will reach 7.8 million per year by 2030 [3].

2.1.2. Pathophysiology

Acute occlusion of an intracranial vessel causes reduction of blood flow to the brain region that it supplies.

A complete blockage of blood flow to the region causes cell death within a few minutes. If blood flow is

restored before significant cell death occurs, the patients may experience only transient symptoms (i.e., a

transient ischemic attack). Conversely, infarction occurs upon prolonged occlusion. Tissue surrounding the

core region of infarction is ischemic but without irreversible cell injury, and is referred to as ischemic

penumbra. This region is functionally impaired but potentially salvageable. The penumbra may be detected

by magnetic resonance imaging. The ischemic penumbra will progress into infarction unless reperfusion is

improved or cells made relatively more resistant to injury. With time, the infarct core expands into the

entire ischemic penumbra, and the therapeutic opportunity to prevent cell death is lost. Therefore, detecting

a penumbra in patients can help identify those who might benefit most from acute treatments that restore

blood flow (e.g., thrombolytic therapy) or treatments for the future that render viable brain cells more

resistant to ischemic injury [4, 5].

Several modes of cell death occur during ischemia. In the area of severely limited blood supply, ATP

depletion leads to acidosis and loss of ionic homeostasis. As a consequence, cells swell and membrane

ruptures, and necrosis occurs. Within the ischemic penumbra, multiple mechanisms have been identified to

contribute to the irreversible injury of the cells, which usually die of apoptosis. These pathophysiological

mechanisms include excitotoxiciy, calcium dysregulation, mitochondrial dysfunction, formation of reactive

oxygen and nitrogen species (ROS/RNS), and inflammation [4, 5]. It should be mentioned that these

mechanisms are not mutually exclusive, and oxidative stress appears to play a central role as the other

mechanistic pathways all lead to the increased formation of ROS/RNS. Although restoration of blood flow

is essential for salvage of the ischemic penumbra, reperfusion also causes additional tissue injury beyond

that generated by ischemia alone. Reperfusion results in production of a much larger amount of ROS/RNS

than ischemia, and as such oxidative stress plays a more pronounced part in reperfusion injury. The

following two sections discuss the major findings on the causative involvement of oxidative stress in

ischemic stroke in both animal models and human subjects. Although being not a focus of discussion in this

chapter, evidence also supports an oxidative stress mechanism of hemorrhagic stroke.

2.2. Animal Studies

2.2.1. Evidence

Experimental animal models of stroke serve as an important tool to investigate the pathophysiological

mechanisms of ischemic stroke as well as develop anti-stroke drugs [6, 7]. Most of the stroke models are

carried out on rodents though some on nonhuman primates. The most commonly used stroke models are

focal cerebral ischemia models that consist of embolic stroke model and intraluminal suture middle cerebral


Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies, 2012, 153-172 153

Y. Robert Li

All rights reserved - © 2012 Bentham Science Publishers

CHAPTER 7

Free Radicals and Related Reactive Species in Pulmonary Diseases

Abstract: Pulmonary diseases are common clinical entities encountered by physicians and represent a

major issue of public health worldwide. Understanding of the detailed pathophysiological mechanisms

of pulmonary disorders is of critical importance for developing more effective therapeutic approaches to

combating these diseases. This chapter discusses experimental and clinical studies showing oxidative

stress as a crucial pathophysiological factor in diverse pulmonary diseases. These include chronic

obstructive pulmonary disease, asthma, cystic fibrosis, acute respiratory distress syndrome, pulmonary

ischemia-reperfusion injury, idiopathic pulmonary fibrosis, and drug/xenobiotic-induced pulmonary

toxicity. The development of antioxidant-based modalities for the intervention of these pulmonary

diseases is also covered in this chapter.

Keywords: Acute respiratory distress syndrome, Antioxidant intervention, Asthma, Chronic obstructive

pulmonary disease, Cystic fibrosis, Idiopathic pulmonary fibrosis, Oxidative stress, Pulmonary diseases,

Pulmonary ischemia-reperfusion injury, Pulmonary toxicity, Reactive nitrogen species, Reactive oxygen

species.

CHAPTER AT A GLANCE

1. OVERVIEW

2. FREE RADICALS AND RELATED REACTIVE SPECIES IN CHRONIC OBSTRUCTIVE

PULMONARY DISEASE

2.1. Introduction to Chronic Obstructive Pulmonary Disease

2.2. Animal Studies

2.3. Human Studies and Clinical Correlations

3. FREE RADICALS AND RELATED REACTIVE SPECIES IN ASTHMA

3.1. Introduction to Asthma

3.2. Animal Studies

3.3. Human Studies and Clinical Correlations

4. FREE RADICALS AND RELATED REACTIVE SPECIES IN OTHER FORMS OF PULMONARY

DISORDERS

4.1. Cystic Fibrosis

4.2. Acute Respiratory Distress Syndrome

4.3. Pulmonary Ischemia-Reperfusion Injury

4.4. Idiopathic Pulmonary Fibrosis

4.5. Drug/Xenobiotic-Induced Pulmonary Toxicity

5. REFERENCES


154 Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies Y. Robert Li

1. OVERVIEW

Pulmonary diseases in the adult are some of the most common clinical entities encountered by physicians.

At least four of the top ten causes of death by medical illnesses in the United States are related to

pulmonary dysfunction and injury in one way or another: (1) cancer, the second leading cause of death, of

which lung cancer is the most common cause of cancer-related death; (2) chronic obstructive pulmonary

disease (COPD), the fourth leading cause of death; (3) pneumonia, together with influenza being the eighth

leading cause of death; and (4) septicemia, the tenth leading cause of death, for which pulmonary injury is a

significant contributor.

Pulmonary diseases are usually grouped into those affecting (1) the airways (e.g., COPD, asthma, cystic

fibrosis), (2) the interstitium (e.g., idiopathic pulmonary fibrosis, drug-induced pulmonary fibrosis), and (3)

the pulmonary vascular system (e.g., primary pulmonary hypertension, pulmonary thromboembolism). This

classification into discrete compartments is, however, deceptively neat. In reality, disease in one

compartment is often accompanied by alterations in another. Pulmonary disorders also include acute

respiratory distress syndrome, ischemia-reperfusion injury, as well as cancer. This chapter discusses the

pulmonary disorders in which substantial evidence supports an important role for oxidative stress in disease

pathophysiology. The involvement of oxidative stress in cancer development is covered in chapter 10.

2. FREE RADICALS AND RELATED REACTIVE SPECIES IN CHRONIC OBSTRUCTIVE

PULMONARY DISEASE

2.1. Introduction to Chronic Obstructive Pulmonary Disease

2.1.1. Definition and Epidemiology

The obstructive pulmonary diseases are a group of disorders that cause dyspnea and are characterized by

airflow limitation on pulmonary function testing. These disorders are highly prevalent and include

emphysema, chronic bronchitis, bronchiolitis, brochiectasis, and asthma, among others. The disorders

emphysema, chronic bronchitis, and bronchiolitis have been grouped together in the clinical term chronic

obstructive pulmonary disease (COPD) as they often coexist in the same patient, share the same etiological

factors (e.g., cigarette smoking), and are similar in clinical manifestations as well as treatments. In general,

COPD is characterized by irreversible or poorly reversible airflow limitation, and this feature serves to

distinguish COPD from asthma, a chronic inflammatory disease of the airways characterized by airway

hyperactivity and reversible airflow limitation (see Section 3 for the discussion of asthma).

COPD affects more than 10% of the United States adult population and as mentioned above is the fourth

leading cause of death in this country. COPD also represents a growing global public health problem and is

predicted to become the third most common cause of death by 2020. Cigarette smoking is by far the most

important known cause of COPD (over 90%), but clinically significant disease develops in only a small

portion (~20%) of smokers. Other factors such as air pollution, occupational exposure to various types of

dusts and fumes, and infections contribute to the occurrence, severity, and progression of the disease [1].

Apart from the important preventive steps of smoking cessation, there are no other specific treatments for

COPD that are effective in reversing the condition [2].

2.1.2. Pathophysiology

COPD is characterized by progressive airflow obstruction of the peripheral airways, associated with lung

inflammation, emphysema, and mucus hypersecretion. Our understanding of the pathophysiology of COPD

has increased over the last two to three decades. Although the detailed molecular and cellular events

underlying COPD remain to be further elucidated, it is now apparent that the pathogenesis of COPD

involves multiple processes with inflammation and oxidative stress as two key components [3, 4]. Chronic

exposure to tobacco smoke or other air pollutants elicits inflammatory responses in the lung. The infiltrated

inflammatory cells release proteolytic enzymes (e.g., elastase) in excess to antiproteases, such as 1antitrypsin.

The disturbance of the balance between proteases and antiproteases results in the uncontrolled

digestion of the pulmonary elastic tissue, leading to emphysema. In addition to the above inflammatory


Pulmonary Diseases Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies 155

responses, tobacco smoke also presents a significant oxidant burden to the lung as cigarette smoke contains

many oxidizing species in large quantities, which may overwhelm the normal antioxidant defenses of the

lung tissue, leading to oxidative tissue injury. Moreover, oxidative stress also causes inhibition of 1antitrypsin,

thereby aggravating the protease/antiprotease imbalance. Indeed, as detailed below, multiple

lines of evidence support oxidative stress as an important mechanism of COPD.

2.2. Animal Studies

As stated above (Section 2.1.1), currently there is no specific therapy for reversing the disease condition of

COPD. Therefore, there is a great need to further understand the pathophysiological mechanisms that could

lead to new therapeutic strategies. The development of experimental animal models of human COPD has

greatly helped dissect the mechanisms at the cellular and molecular levels [5, 6]. These experimental

models are commonly based on the induction of COPD-like lesions in the lungs and airways using noxious

inhalants, such as tobacco smoke, nitrogen dioxide, or sulfur oxide. Depending on the duration and

intensity of exposure, these noxious stimuli induce signs of chronic inflammation, oxidative stress, and

airway remodeling. Emphysema can be induced by combining such exposure with instillation of tissuedigesting

enzymes (e.g., elastase) or instillation of the digestive enzymes alone. Other experimental

approaches are based on genetically-manipulated mice (e.g., mice lacking the tissue inhibitor of

metalloproteinase-3) which develop COPD-like lesions with emphysema. Studies using these various

experimental models have provided important insights into the oxidative stress mechanism of COPD. These

models also serve as important tools for developing antioxidant-based modalities for the intervention of the

disease processes. The sections below intend to first summarize the major evidence supporting the critical

involvement of oxidative stress in COPD and then describe the sources of ROS/RNS as well as the

mechanism of oxidative injury underlying COPD.

2.2.1. Evidence

There are multiple lines of evidence together supporting a causative role for oxidative stress in the initiation

and progression of COPD in animal models. They are described below.

1) Augmented formation of reactive oxygen and nitrogen species (ROS/RNS): As noted earlier in

Section 2.1.1, cigarette smoking is the most common cause of COPD. Tobacco smoke

contains thousands of chemical compounds, and both the tar and gas phases consist of

numerous free radicals and other oxidants, which are present in high concentrations. In

addition to the ROS/RNS directly present in cigarette smoke, pulmonary inflammation

caused by tobacco smoke or air pollutants also results in the production of large amounts of

ROS/RNS from activation of inflammatory cells (e.g., macrophages, neutrophils) [7].

2) Depletion of antioxidants and increased biomarkers of oxidative damage: Both protein and

non-protein antioxidants are present in lung tissue. Notably, micromolar concentrations of the

antioxidants glutathione, vitamin C, uric acid, and -tocopherol are found in lung epithelial

lining fluid. Exposure to cigarette smoke or air pollutants causes rapid depletion of these

antioxidant compounds. Protein antioxidants, including superoxide dismutase, catalase,

glutathione peroxidase, heme oxygenase, and thioredoxin are also decreased in COPD though

a transient upregulation of certain antioxidant genes may occur during early phase of

exposure to noxious COPD-inducing inhalants in experimental animals. The early induction

of certain antioxidant genes may represent a compensative response to oxidative stress in

lung tissue. The overall decline of pulmonary antioxidant defenses in COPD may be

associated with the downregulation of Nrf2 signaling, a central regulating pathway

underlying antioxidant gene expression. It has been shown that decline of Nrf2-regulated

antioxidants in COPD is due to loss of DJ-1, a positive regulator of Nrf2 [8].

In line with the compromised antioxidant defenses in lung tissue, markers of oxidative

damage, such as lipid peroxidation products are detected in both exhaled breath condensates

and lung tissue as well as in plasma of animal models of COPD [9, 10]. Detection of these


Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies, 2012, 173-201 173

Y. Robert Li

All rights reserved - © 2012 Bentham Science Publishers

CHAPTER 8

Free Radicals and Related Reactive Species in Hepatic and

Gastrointestinal Diseases

Abstract: Due to their essential roles in diverse physiological processes, the liver and the

gastrointestinal system are the major sites of many common diseases. Multiple pathophysiological

mechanisms have been identified to underlie various hepatic and gastrointestinal disorders. This chapter

discusses the oxidative stress mechanism in the disease processes of hepatic and gastrointestinal

systems and introduces antioxidant-based modalities for the disease intervention. The disease entities

covered in the chapter include alcoholic fatty liver disease, nonalcoholic fatty liver disease, Wilson’s

disease, liver ischemia-reperfusion injury, drug/xenobiotic-induced hepatotoxcity, inflammatory bowel

disease, peptic ulcer disease and Helicobacter pylori infection, and gastroesophageal reflux disease.

Keywords: Alcoholic fatty liver disease, Antioxidant intervention, Gastroesophageal reflux disease,

Gastrointestinal diseases, Helicobacter pylori, Hepatic diseases, Hepatotoxicity, Inflammatory bowel

disease, Liver ischemia-reperfusion injury, Nonalcoholic fatty liver disease, Oxidative stress, Peptic ulcer

disease, Reactive nitrogen species, Reactive oxygen species, Wilson’s disease.

CHAPTER AT A GLANCE

1. OVERVIEW

2. FREE RADICALS AND RELATED REACTIVE SPECIES IN ALCOHOLIC LIVER DISEASE

2.1. Introduction to Alcoholic Liver Disease

2.2. Animal Studies

2.3. Human Studies and Clinical Correlations

3. FREE RADICALS AND RELATED REACTIVE SPECIES IN NONALCOHOLIC FATTY LIVER

DISEASE

3.1. Introduction to Nonalcoholic Liver Disease

3.2. Animal Studies

3.3. Human Studies and Clinical Correlations

4. FREE RADICALS AND RELATED REACTIVE SPECIES IN OTHER FORMS OF HEPATIC

DISORDERS

4.1. Wilson’s Disease

4.2. Liver Ischemia-Reperfusion Injury

4.3. Drug/Xenobiotic-Induced Hepatotoxicity

5. FREE RADICALS AND RELATED REACTIVE SPECIES IN INFLAMMATORY BOWEL DISEASE

5.1. Introduction to Inflammatory Bowel Disease

5.2. Animal Studies


174 Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies Y. Robert Li

5.3. Human Studies and Clinical Correlations

6. FREE RADICALS AND RELATED REACTIVE SPECIES IN OTHER FORMS OF

GASTROINTESTINAL DISORDERS

6.1. Peptic Ulcer Disease and Helicobacter pylori Infection

6.2. Gastroesophageal Reflux Disease

7. REFERENCES

1. OVERVIEW

The liver is the largest internal organ in the body and plays a central role in many important physiological

processes, including glucose homeostasis, plasma protein synthesis, synthesis and metabolism of lipids and

lipoproteins, bile acid synthesis, and vitamin storage. In addition, liver is vital in biotransformation,

detoxification, and excretion of both endogenous and exogenous compounds. Thus, it is not surprising that

liver is vulnerable to a variety of metabolic, toxic, microbial, and circulatory insults. Indeed, liver disorders

are prevalent, and chronic liver disease and cirrhosis are among the top 15 leading causes of death in the

United States, responsible for 29,165 deaths in 2007. This chapter discusses the common liver disorders in

which oxidative stress plays an important role. These include alcoholic liver disease, nonalcoholic fatty

liver disease, hepatic ischemia-reperfusion injury, and drug/xenobiotic-induced hepatotoxicity. This chapter

also discusses the role of oxidative stress in common gastrointestinal disorders, including inflammatory

bowel disease, peptic ulcer disease and Helicobacter pylori infection, and gastroesophageal reflux disease.

2. FREE RADICALS AND RELATED REACTIVE SPECIES IN ALCOHOLIC LIVER DISEASE

2.1. Introduction to Alcoholic Liver Disease

2.1.1. Definition and Epidemiology

Although alcohol is a general term for alcoholic compounds, here in this chapter alcohol refers specifically

to ethyl alcohol or ethanol. Alcohol has been a part of human culture since the beginning of recorded

history. Excessive use of alcohol contributes substantially to the global burden of disease (4% of the total

mortality) and is thus one of the largest avoidable risk factors [1]. In the United States, alcoholism is also a

major public health problem, and it is estimated that over 100 million Americans are alcoholic. Alcohol

abuse accounts for 100,000 to 200,000 deaths annually in the United States, of which 20,000 are

attributable directly to end-stage hepatic cirrhosis and many more are the result of automobile accidents.

Although excessive alcohol consumption is associated with a variety of disorders, alcoholic liver disease

(ALD) is of the great health impact. ALD encompasses a spectrum of hepatic disorders including: (1) fatty

liver (also known as steatosis, due to abnormal accumulation of lipids in hepatic tissue), (2) alcoholic

hepatitis (due to inflammation and necrosis), and (3) cirrhosis (due to excessive fibrosis) [2]. These are not

necessarily distinct stages of evolution of the disease, but rather multiple stages that may be present

simultaneously in a given individual. Over 90% of heavy drinkers develop fatty liver, and of those, 10-35%

develop alcoholic hepatitis. About 8-20% of chronic alcoholics develop liver cirrhosis.

2.1.2. Pathophysiology

Multiple pathways are involved in the genesis and progression of alcoholic fatty liver, alcoholic hepatitis,

and cirrhosis. They are summarized below.

1) Alcoholic fatty liver: Alcoholic fatty liver results from excessive accumulation of

triglycerides in hepatocytes. Alcohol is metabolized in hepatocytes through oxidation to

acetaldehyde, and subsequently from acetaldehyde to acetate (Fig. 8.1). The oxidative

metabolism of alcohol generates an excess of NADH, resulting in an increased ratio of


Hepatic and Gastrointestinal Diseases Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies 175

NADH to NAD + in hepatocytes. This altered NADH/NAD + ratio in hepatocytes leads to

inhibition of fatty acid oxidation and promotion of lipogenesis. In addition, alcohol promotes

lipid metabolism through inhibition of peroxisome proliferator-activated receptor (PPAR)

and AMP kinase and stimulation of sterol regulatory element-binding protein 1, a membranebound

transcription factor [3]. In combination, these effects result in a fat-storing metabolic

remodeling of the liver, which is manifested as excessive accumulation of lipids in

hepatocytes. Although fatty liver resolves with abstinence, it predisposes the individuals who

continue to drink to more serious forms of ALD, i.e., alcoholic hepatitis and cirrhosis.

Ethanol

(CH 3CH 2OH)

ALD Acetaldehyde

(CH3CHO) ADD

NAD + NADH NAD + NADH

Acetate

(CH 3COOH)

Fig. (8.1). Sequential metabolism of ethanol to form acetaldehyde and acetate. ALD, alcohol dehydrogenase; ADD,

aldehyde dehydrogenase.

2) Alcoholic hepatitis and cirrhosis: Alcoholic hepatitis is characterized by inflammation and

necrosis [4]. It is believed that alcohol compromises the intestinal barrier, which leads to

increased permeability and the subsequent presence of gut bacteria-derived

lipopolysaccharide (LPS) in the portal blood. LPS stimulates the Kupffer cells and possibly

other types of cells in the liver, resulting in excessive release of inflammatory cytokines and

other reactive species (e.g., ROS/RNS). The dysregulated inflammatory responses are

believed to primarily contribute to the hepatocyte injury and tissue necrosis. In addition,

accumulation of lipids also promotes inflammation. Progression of these detrimental

processes may eventually cause excessive accumulation of collagen in extracellular matrix,

resulting in liver fibrosis and cirrhosis. The crucial role of inflammation in ALD is supported

by the demonstrated therapeutic efficacy of classical anti-inflammatory drugs (e.g.,

glucocorticosteroids) in animal models and certain patient subgroups [4]. In addition to

dysregulated inflammation, recent work demonstrates that oxidative stress also plays an

important role in the development of alcoholic hepatitis and cirrhosis as well as fatty liver.

The sections below are intended to summarize the key findings from both animal

experiments and human studies that support oxidative stress as an important

pathophysiological component of ALD.

2.2. Animal Studies

Studies using animal models have contributed to our understanding of how ALD develops and how the

severity of liver injury is influenced by factors other than alcohol, such as nutrition, oxygen deprivation (as

occurs with sleep apnea and smoking), and gene regulation. In this regard, the intragastric feeding model

results in liver lesions that mimic human ALD, and has been widely used [5, 6]. Animal experiments have

provided important information on the pathophysiology of ALD including the causative involvement of

oxidative stress and also contributed to the development of new therapeutic approaches.

2.2.1. Evidence

1) Increased formation of reactive oxygen and nitrogen species (ROS/RNS): The ability of acute

and chronic alcohol treatment to increase the production of ROS/RNS as well as other free

radical species (e.g., 1-hydroxyethyl radical) has been demonstrated in a variety of systems,

including cell cultures and experimental animals, as well as in human subjects (see Section

2.3 for the discussion of human evidence). The detailed cellular sources and mechanisms of

ROS/RNS formation are described below in Section 2.2.2.

2) Depletion of antioxidants and increased biomarkers of oxidative damage: Although acute

exposure to alcohol may cause transient induction of certain antioxidant genes, excessive


202 Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies, 2012, 202-216

Free Radicals and Related Reactive Species in Renal Diseases

Y. Robert Li

All rights reserved - © 2012 Bentham Science Publishers

CHAPTER 9

Abstract: Kidney diseases are broadly classified into acute kidney injury and chronic kidney disease.

They collectively affect approximately 10% of the adult population in the United States and worldwide,

and represent a major issue of public health. As our understanding of the pathophysiology of kidney

diseases becomes more advanced, various new mechanistically-based therapeutic strategies for treating

these diseases have been developed. This chapter summarizes experimental and clinical evidence

supporting a causative role of oxidative stress in the pathophysiology of both acute kidney injury and

chronic kidney disease. It also discusses the recent development of antioxidant-based intervention of

kidney disorders.

Keywords: Acute kidney injury, Antioxidant intervention, Chronic kidney disease, Nephrotoxicity,

Oxidative stress, Reactive nitrogen species, Reactive oxygen species, Renal diseases.

CHAPTER AT A GLANCE

1. OVERVIEW

2. FREE RADICALS AND RELATED REACTIVE SPECIES IN ISCHEMIC ACUTE KIDNEY INJURY

2.1. Introduction to Acute Kidney Injury and Ischemic Acute Kidney Injury

2.1.1. Definition and Epidemiology

2.1.2. Pathophysiology

2.2. Animal Studies

2.2.1. Evidence

2.2.2. Sources of ROS/RNS

2.2.3. Mechanisms

2.2.4. Conclusion

2.3. Human Studies and Clinical Correlations

2.3.1. Evidence and Antioxidant Intervention

2.3.2. Conclusion and Perspectives

3. FREE RADICALS AND RELATED REACTIVE SPECIES IN NEPHROTOXIC ACUTE KIDNEY

INJURY

3.1. Introduction

3.2. Pathophysiology and Oxidative Stress

3.3. Antioxidant Intervention


Renal Diseases Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies 203

4. FREE RADICALS AND RELATED REACTIVE SPECIES IN CHRONIC KIDNEY DISEASE

4.1. Introduction to Chronic Kidney Disease

4.1.1. Definition and Epidemiology

4.1.2. Pathophysiology

4.2. Animal Studies

4.2.1. Evidence

4.2.2. Mechanisms

4.2.3. Sources of ROS/RNS

4.2.4. Conclusion

4.3. Human Studies and Clinical Correlations

5. REFERENCES

1. OVERVIEW

4.3.1. Evidence and Antioxidant Intervention

4.3.2. Conclusion and Perspectives

The kidney is one of the most highly differentiated organs in the body that has evolved to carry out a

number of important physiological activities. These include (1) excretion of waste products of metabolism,

(2) removal of metabolites of drugs and other xenobiotics, (3) regulation of body water and salt, (4)

regulation of blood pressure, (5) maintenance of acid-base balance, and (6) endocrine functions (e.g.,

release of hormones). The complex structure and diverse functions of the kidney make it a susceptible

organ of many diseases. Indeed, diseases of the kidney are very common and as complex as its structure

and physiological functions. It is estimated that kidney diseases affect approximately 10% of the adult

population in the United States and worldwide.

Kidney diseases are classified in various ways. One classification scheme divides kidney diseases into those

that affect the four basic structural components of the organ: glomeruli, tubules, interstitium, and blood

vessels. Kidney diseases are also classified broadly into acute kidney injury and chronic kidney disease.

The above two classification schemes are not mutually exclusive. In fact, both acute kidney injury and

chronic kidney disease can result from the diseases that affect the above four basic structural components.

Although detailed mechanisms of kidney diseases remain partially understood, studies over the past

decades have revealed various pathophysiological pathways, including apoptosis, autoimmunity,

inflammation, and oxidative stress. This chapter discusses the involvement of oxidative stress in the

pathogenesis of common kidney diseases, including acute kidney injury induced by ischemia-reperfusion,

acute kidney injury caused by nephrotoxicants, and chronic kidney disease. This chapter also describes the

experimental and clinical studies regarding the antioxidant-based intervention of the above kidney

disorders. It is hoped that a profound understanding of the oxidative stress mechanism of kidney diseases

and continued clinical research on antioxidant therapies will eventually lead to the development of effective

strategies to combat human kidney diseases.


204 Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies Y. Robert Li

2. FREE RADICALS AND RELATED REACTIVE SPECIES IN ISCHEMIC ACUTE KIDNEY

INJURY

2.1. Introduction to Acute Kidney Injury and Ischemic Acute Kidney Injury

2.1.1. Definition and Epidemiology

As mentioned above, kidney diseases may be acute or chronic. Acute kidney failure is a syndrome that can

be broadly defined as an abrupt decrease in glomerular filtration rate sufficient to result in retention of

nitrogenous waste products (i.e., urea nitrogen and creatinine) in the blood and perturbation of extracellular

fluid volume and electrolyte and acid-base homeostasis [1]. Retention of the nitrogenous wastes in blood is

referred to as azotemia. As there is no universally accepted definition for acute renal failure, recently the

term acute kidney injury has been proposed to reflect the entire spectrum of the syndrome causing acute

worsening of the renal function. The term acute kidney injury has been widely adopted in the literature and

refers to an abrupt (within 48 hours) reduction in kidney function currently defined as: an absolute increase

in serum creatinine of either >0.3 mg/dl, or a percentage increase of >50%, or a reduction in urine output

(documented oliguria of 6 hours) [2].

Acute kidney injury is a devastating and common problem in clinical medicine. It affects ~5% of

hospitalized patients and 30-50% of patients in intensive care unit, and is associated with high morbidity

and mortality. The incidence of acute kidney injury has been increasing both in the United States and

worldwide and becoming a major global health issue.

2.1.2. Pathophysiology

Causes of acute kidney injury are usually divided into three major categories: (1) diseases that cause renal

hypoperfusion, resulting in decreased function without frank parenchymal damage (known as prerenal

acute kidney injury or prerenal azotemia; accounting for ~55% of all cases), (2) diseases that directly

involve the renal parenchyma (known as intrinsic acute kidney injury; accounting for ~40% of all cases),

and (3) diseases associated with urinary tract obstruction (known as postrenal acute kidney injury or

postrenal azotemia; accounting for ~5% of all cases). These three categories and the inciting conditions are

listed in Table 9.1.

Table 9.1. Classification and etiology of acute kidney injury.

Acute Kidney Injury Inciting Conditions

Prerenal 1) Hypovolemia (hemorrhage, burns, dehydration)

2) Altered renal hemodynamics resulting in hypoperfusion (congestive heart failure,

sepsis, anaphylaxis, hepatorenal syndrome)

Intrinsic 1) Renovascular obstruction (atherosclerosis plaque, embolism, vasculitis)

2) Acute glomerulonephritis

3) Acute interstitial nephritis

4) Acute tubular necrosis (ischemia of prerenal cause if severe enough,

nephrotoxicants, infection)

Postrenal 1) Ureteric obstruction

2) Bladder neck obstruction

3) Urethral obstruction

The most common intrinsic renal disease that leads to acute kidney injury is an entity known as acute

tubular necrosis, which is a clinical syndrome characterized by an abrupt and sustained decline in

glomerular filtration rate occurring within minutes or days in response to an acute ischemic or nephrotoxic

insult. It should be noted that although it is called acute tubular necrosis, sublethal injury instead of necrosis

is often a major pathophysiological feature of the syndrome. Cell injury, apoptosis and necrosis,

inflammation, and oxidative stress are among the major pathophysiological components of acute kidney

injury caused by ischemia and nephrotoxicants [3, 4]. The role of oxidative stress in ischemic acute kidney


Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies, 2012, 217-231 217

Free Radicals and Related Reactive Species in Cancer

Y. Robert Li

All rights reserved - © 2012 Bentham Science Publishers

CHAPTER 10

Abstract: Cancer remains a leading cause of death in the human population worldwide. Both genetic

and environmental factors are involved in the development of cancer. Recent work also demonstrates a

critical role for reactive oxygen and nitrogen species (ROS/RNS) in multistage carcinogenesis. This

chapter examines the oxidative stress mechanism of carcinogenesis as well as antioxidant-based

modalities for cancer intervention. The chapter also discusses ROS as a potential tumor killing modality

in cancer therapy.

Keywords: Antioxidant intervention, Cancer, Carcinogens, Initiation, Multistage carcinogenesis, Oxidative

stress, Progression, Promotion, Reactive nitrogen species, Reactive oxygen species.

CHAPTER AT A GLANCE

1. OVERVIEW

2. CARCINOGENIC AGENTS

3. MULTISTAGE NATURE OF CARCINOGENESIS

3.1. Initiation

3.2. Promotion

3.3. Progression

4. MECHANISTIC ASPECTS OF FREE RADICALS AND RELATED REACTIVE SPECIES IN

MULTISTAGE CARCINOGENESIS

4.1. Sources of Free Radicals and Related Reactive Species

4.2. Molecular Mechanisms

5. EVIDENCE FOR A CAUSATIVE ROLE OF FREE RADICALS AND RELATED REACTIVE

SPECIES IN CANCER DEVELOPMENT

5.1. Cell Studies

5.2. Animal Studies

5.3. Human Studies and Clinical Correlations

6. FREE RADICALS AND RELATED REACTIVE SPECIES IN CANCER THERAPY

6.1. Free Radicals and Related Reactive Species as Mediators of Anticancer Drug Action

6.2. Targeting Cancer Cells by Free Radicals and Related Reactive Species

6.3. High-Dose Vitamin C Therapy of Human Cancer

7. REFERENCES


218 Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies Y. Robert Li

1. OVERVIEW

Cancer remains a leading cause of morbidity and mortality in the human population worldwide and the

costs to society of this dreaded disease are prodigious. In the United States, approximately 1.5 million new

cancer cases are diagnosed each year, and this estimate does not include carcinoma in situ (noninvasive

cancer) of any site except urinary bladder, and does not include basal and squamous cell skin cancer, which

are not required to be reported to cancer registries. Cancer is the second most common cause of death in the

United States, exceeded only by heart disease. More than half million Americans die of cancer each year,

accounting for nearly one of every four deaths.

Cancer is a group of diseases in which there is an uncontrolled proliferation of cells that express varying

degree of fidelity to their precursor cell of origin. It has been proposed that cancer has six hallmarks [1]: (1)

self-sufficiency in growth signals, (2) insensitivity to antigrowth signals, (3) evasion of apoptosis, (4) tissue

invasion and metastasis, (5) sustained angiogenesis, and (6) limitless replicative potential. The induction of

cancer, also known as carcinogenesis, is a multistage process that occurs over a long period of time,

decades in humans. These stages have been experimentally defined as initiation, promotion, and

progression. The causes of most human cancers remain unidentified; however, considerable evidence

suggests that environmental and life-style factors are important contributors. In addition, endogenous

chemicals, such as estrogens and androgens have also been shown to contribute to the development of

certain types of human cancer. Moreover, biologic agents, including certain bacteria and viruses are known

to cause cancer in humans. Thus, understanding the molecular and cellular process underlying multistage

carcinogenesis is of critical importance for cancer risk assessment as well as the development of

mechanistically-based chemopreventive and therapeutic strategies for the management of human cancer.

This chapter begins with a brief description of the multistage nature of carcinogenesis, followed by a

detailed discussion of the causal role of free radicals and related reactive species in multistage

carcinogenesis. The involvement of free radicals and related reactive species in cancer therapy is also

covered in the chapter.

2. CARCINOGENIC AGENTS

As noted above, cancer can be induced by exposure to various agents, including both endogenous and

exogenous substances. A carcinogen may be defined as an agent whose administration to previously

untreated animals leads to a statistically significant increased incidence of cancer as compared with that in

appropriate untreated control animals. Carcinogens can be chemicals, physical agents (such as ultraviolet

radiation and X- and gamma-radiation), and biologic agents (such as bacteria and viruses).

Based on the evidence of carcinogenicity in animals and humans, carcinogens may be classified as animal

carcinogens and/or human carcinogens. In the evaluation of carcinogenicity for humans, the International

Association for Research on Cancer (IARC) of the World Health Organization has categorized carcinogens

into five groups: (1) Group 1 (carcinogenic to humans), agents in this group have sufficient evidence of

carcinogenicity in humans, and as such are called known human carcinogens. Currently, there are over 100

group 1 agents. Aflatoxin B1, arsenic, asbestos, benzene, benzo[a]pyrene, chromium, cyclophosphamide,

cyclosporine, estrogens, ethanol, nickel, vinyl chloride, ultraviolet radiation, X- and gamma-radiation,

hepatitis B virus, hepatitis C virus, papilloma virus, and Helicobacter pylori belong to this group; (2) Group

2A (probably carcinogenic to humans), agents in this group possess sufficient evidence of carcinogenicity

in experimental animals, but have limit evidence of carcinogenicity in humans. These agents are thus

probably carcinogenic to humans. Within this group are acrylamide, adriamycin (doxorubicin), androgenic

(anabolic) steroids, dibenz[a,h]anthracence, nitrate, nitrite, polychlorinated biphenols, and styrene-7,8oxide;

(3) Group 2B (possibly carcinogenic to humans), agents in this group possess either limited evidence

of carcinogenicity in humans, or have sufficient evidence of carcinogenicity in experimental animals and

inadequate evidence of carcinogenicity in humans. Class 2B agents are possibly carcinogenic to humans.

Examples of Group 2B agents are styrene, urethane, and cell phone use; (4) Group 3, agents in this group

are not classifiable as to their carcinogenicity to humans. Examples include acrolein, caffeine, toluene, and

trichloroacetic acid; (5) Group 4 (probably not carcinogenic to humans), agents in this group possess


Cancer Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies 219

inadequate evidence of carcinogenicity in both experimental animals and humans, and thus are probably

not carcinogenic to humans. Table 10.1 lists some known human carcinogens.

Table 10.1. Some known human carcinogens (group 1 carcinogens).

Class Examples

Physical agents Solar radiation, ultraviolet radiation, X- and gamma-radiation

Chemical agents Aflatoxin B1, arsenic, asbestos, azathioprine, benzene, benzidine, benzo[a]pyrene, busulfan,

chromium, cyclophosphamide, cyclosporine, estrogens, ethanol, nickel, tamoxifen, vinyl

chloride

Biologic agents Epstein-Barr virus, hepatitis B virus, hepatitis C virus, papilloma virus, Helicobacter pylori

3. MULTISTAGE NATURE OF CARCINOGENESIS

It is recognized that the process of carcinogenesis involves a variety of biological changes which, to a great

extent, reflect the structural and functional alterations in the genome of the affected cell. As stated above,

the process of carcinogenesis consists of three experimentally defined stages beginning with initiation

followed by the intermediate step of promotion, from which evolves the stage of progression (Fig. 10.1).

Fig. (10.1). Schematic illustration of multistage carcinogenesis. See text (Section 3) for detailed description.

3.1. Initiation

Initiating

Agents

Normal Cell Initiated Cell

Initiation

•Results from DNA

mutations

•Mutations may occur in

oncogenes, protooncogenes,

or tumor suppressor genes

•Initiated cells may remain

dormant

•Initiated cells rarely

become neoplasticcells

without undergoing

promotion and progression

•Irreversible

Promoting

Agents

Preneoplastic Cells

Promotion

•Results from clonal

expansion of initiated cells

•Cell signaling and gene

expression are altered,

leading to increased cell

mitogenesis and/or reduced

apoptosis

•Leads to the formation of

preneoplasticcells

•Long duration and

reversible, especially at

earlier stage

Progressor

Agents

Neoplasticcells

Progression

•Results from additional

genomic structural

alterations due to karyotypic

instability

•Leads to the formation of

benign or malignant

neoplasms

•Cells may acquire malignant

phenotypes, such as

invasiveness, angiogenesis

and/or metastasis

•Irreversible

Initiation is a phenomenon of gene alteration, which may result from the interaction of ultimate carcinogens

with DNA in the target cells. Substances capable of initiating cells are called initiating agents. Initiation without

the following steps, promotion and progression, rarely yields malignant neoplasms. The term neoplasma refers

to an abnormal mass of tissue as a result of dysregulated proliferation of cells. The following steps are critically

involved in the initiation process by carcinogenic chemicals [2, 3]: (1) conversion of a chemical to a DNAreactive

metabolite (an ultimate carcinogen), (2) interaction of the ultimate carcinogen with DNA, leading to

DNA structural alteration, (3) DNA repair that may reverse the structural damage, and (4) cell proliferation

leading to the fixation of the DNA damage. Mutations of protooncogenes or oncogenes, such as the ras gene


232 Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies, 2012, 232-241

Free Radicals and Related Reactive Species in Aging

Y. Robert Li

All rights reserved - © 2012 Bentham Science Publishers

CHAPTER 11

Abstract: Aging is a universal, complicated biological process characterized by the progressive

accumulation in an organism of diverse, deleterious changes with time that increase the chance of

disease and death. Although the molecular mechanisms of aging remain elusive, increasing evidence

suggests a critical role for free radicals and related reactive species, especially reactive oxygen species

in the process of aging. This chapter provides an overview of the evidence from both experimental

models and human subjects that supports an oxidative stress mechanism of aging.

Keywords: Aging, Antioxidant intervention, Catalase, Lifespan, Longevity, Nrf2, Oxidative stress, p66 SHC ,

Paraoxonase 1, Reactive oxygen species, Sirtuins.

CHAPTER AT A GLANCE

1. OVERVIEW

2. CELL STUDIES

3. ANIMAL STUDIES

3.1. Increased Oxidative Damage and Decreased Antioxidant Defenses during Aging

3.2. Extension of Lifespan by Antioxidants

3.3. Extension of Lifespan by Inhibition of ROS Generation

3.4. Shortening of Lifespan by Antioxidant Gene Knockout

3.5. Controversies and Other Considerations

4. HUMAN STUDIES AND CLINICAL CORRELATIONS

4.1. Changes in Oxidative Stress Biomarkers and Antioxidants during Aging

4.2. Antioxidant Gene Polymorphisms and Longevity

4.3. Antioxidant Supplementation and Human Longevity

5. REFERENCES

1. OVERVIEW

Aging may be defined as a universal, complicated biological process characterized by the progressive

accumulation in an organism of diverse, deleterious changes with time that increase the chance of disease

and death. Aging is a major risk factor of many human diseases, including cardiovascular disorders,

neurodegenerative diseases, and cancer. Hence, understanding the molecular and cellular basis of aging

process is of importance for devising anti-aging strategies to promote the health conditions and extend the

longevity of the human population. Numerous theories have been proposed to explain the process of aging,

but none of them appears to be fully satisfactory. Among the various theories on aging, the free radical

theory has received much attention over the last several decades [1, 2].


Aging Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies 233

The origin of the free radical theory of aging dates back to the 1950’s, when D. Harman proposed that the

reaction of active free radicals, normally produced in the organisms, with cellular constituents initiates the

changes associated with aging [3]. Given that mitochondria are the major source of cellular free radicals

and reactive oxygen species (ROS), this theory has largely become known as mitochondrial free radical

theory of aging [4-6] (Fig. 11.1). It is also known as oxidative stress theory of aging in recent literature.

This chapter is intended to provide an overview of the evidence from cell, animal, and human studies that

supports the free radical mechanism of aging.

Fig. (11.1). Mitochondrial ROS theory of aging. ROS, reactive oxygen species.

2. CELL STUDIES

ROS

Aging

The pioneering work of L. Hayflick and P.S. Moorhead in the 1960’s demonstrated that primary cells

isolated from tissues and grown in cultures possess a finite capacity to undergo replication [7, 8]. Once

reaching their replicative limit, such cells were termed as senescent and viewed as aged cells. Subsequently,

senescent cells have been considered as a cellular model of organismal aging [9]. As originally defined,

cellular senescence is the inability of normal diploid cells to undergo further replication. However, it is now

recognized that in addition to cell-cycle arrest, senescence also confers altered responses to apoptotic

stimuli, morphological transformation, and altered gene expression.

Extensive studies over the past decade have provided multiple lines of evidence establishing a causative

involvement of oxidative stress in cellular senescence [10-12]. These are listed below.

A mild increase in oxygen tension (~40%) triggers cellular senescence, whereas decreasing

oxygen tension from the customary 21% oxygen to a more physiological level (3% oxygen)

leads to an increase in cell doublings before senescence.

Exposure of cells to moderate levels of oxidative stressors, such as redox cycling compounds

or ROS-generating enzymes induces cellular senescence.

Treatment of cells with various exogenous antioxidant compounds suppresses cellular

senescence. Similarly, overexpression of antioxidant enzymes delays cellular senescence.

Knockdown or deletion of cellular antioxidant genes, such as Cu,Zn superoxide dismutase

triggers early cellular senescence.

Cellular senescence is caused by telomere shortening. Oxidative stress promotes telomere

shortening. Telomerase, the enzyme catalyzing the elongation of telomeres is also sensitive to

oxidative stress-mediated inhibition.


234 Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies Y. Robert Li

3. ANIMAL STUDIES

In line with a causative role of oxidative stress in cellular senescence, animal studies also provide

substantial evidence supporting an important role of oxidative stress in aging process. Commonly used

animal models in aging studies include invertebrates (e.g., Caenorhabditis elegans, Drosophila

melanogaster) and small mammals (e.g., mice). The gold standard for determining whether aging is altered

is lifespan. Outlined below are the major experimental findings in this area.

3.1. Increased Oxidative Damage and Decreased Antioxidant Defenses during Aging

Substantial correlative studies demonstrate that increased levels of oxidative damage to cellular

constituents, including proteins, lipids, and nucleic acids, and decreased antioxidant defenses occur during

aging. Mitochondria isolated from old animals release more ROS than those from young counterparts.

Comparative biology studies show that in general the production of mitochondrial ROS is correlated

inversely with the lifespan of the animal species [13]. Although these correlative and comparative biology

studies do not establish a cause-and-effect relationship between oxidative stress and aging, the findings are

consistent with the free radical theory of aging.

3.2. Extension of Lifespan by Antioxidants

3.2.1. Exogenous Antioxidants

Different from the correlative and comparative biology studies, interventional studies using antioxidants

provide much more compelling evidence for a causative role of oxidative stress in aging. A number of

structurally-unrelated compounds with antioxidant properties, including glutathione precursors, dietary

polyphenols, antioxidant enzyme mimetics, spin traps, and antioxidant nanomaterials are shown to prolong

lifespan in various animal species. For example, treatment of mice with a carboxyfullerene superoxide

dismutase mimetic is found to not only reduce age-associated oxidative stress and mitochondrial free

radical production, but also improve cognition and significantly extends lifespan [14]. Carboxyfullerene is

a derivative of the nanomaterial fullerene. Other antioxidant nanomaterials, including cerium oxide and

platinium nanoparticles have also been demonstrated to prolong the lifespan of the invertebrate animals,

including Drosophila melanogaster and Caenorhabditis elegans [15, 16]. More recently, 4,4’diaminodiphenysulfone,

a human drug capable of inducing endogenous antioxidants, is shown to augment

the antioxidative stress capacity of Caenorhabditis elegans, improve mitochondrial function, and increase

the longevity of the organism [17].

3.2.2. Endogenous Antioxidants

In line with the lifespan-extending effects of exogenous antioxidants described above, targeted

overexpression of catalase in mitochondria results in extension of lifespan in mice [18]. These mice also

exhibit decreased oxidative stress, attenuated mitochondrial deletion, and delayed development of cardiac

pathology and cataract. Cardiac-specific overexpression of catalase in mice also prolongs lifespan and

attenuates aging-associated cardiomyocyte contractile dysfunction and protein damage [19]. These findings

suggest that catalase may be a longevity-determining enzyme [20] (Fig. 11.2). In addition to catalase,

cardiac overexpression of another antioxidant protein, metallothionein in mice has been shown to blunt

aging-associated superoxide production and oxidative damage, and increase the lifespan by 14% [21], an

extension comparable to that associated with overexpression of catalase in mitochondria [18]. In

Drosophila melanogaster, overexpression of Cu,Zn superoxide dismutase (Cu,ZnSOD) and catalase or

methionine sulfoxide reductase reduces oxidative stress and increases the longevity of the flies [22-24].

Recently, evidence also suggests a potential involvement of the antioxidant gene regulator, Nrf2 and related

Cap’n’collar transcription factors in protecting against senescence and promoting longevity [25-29] (Fig.

11.3). Collectively, the above experimental data point to the effectiveness of antioxidants in blunting agingassociated

oxidative stress and in extending lifespan in animal models.


242 Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies, 2012, 242-269

Y. Robert Li

All rights reserved - © 2012 Bentham Science Publishers

CHAPTER 12

Free Radicals and Related Reactive Species in Other Diseases and

Conditions

Abstract: The causal involvement of reactive oxygen and nitrogen species (ROS/RNS) in the

pathophysiology of common human diseases has been discussed in the preceding chapters. This chapter

summaries experimental and clinical evidence that shows a causative involvement of ROS/RNS in other

disease processes and related conditions. These include skin and eye disorders, diseases of reproductive

system, arthritic disorders and sepsis, as well as exercise, regenerative medicine, and nanomedicine.

Keywords: Antioxidant intervention, Arthritis, Erectile dysfunction, Exercise, Eye diseases, Infertility,

Nanomedicine, Oxidative stress, Reactive nitrogen species, Reactive oxygen species, Regenerative

medicine, Sepsis, Skin diseases.

CHAPTER AT A GLANCE

1. OVERVIEW

2. FREE RADICALS AND RELATED REACTIVE SPECIES IN SKIN DISEASES

2.1. Introduction

2.2. General Considerations on Oxidative Stress and Skin Diseases

2.3. Inflammatory Skin Diseases

2.4. Other Skin Diseases and Related Conditions

3. FREE RADICALS AND RELATED REACTIVE SPECIES IN EYE DISEASES

3.1. Introduction

3.2. Age-Related Macular Degeneration

3.3. Cataract

4. FREE RADICALS AND RELATED REACTIVE SPECIES IN DISEASES OF REPRODUCTIVE

SYSTEM

4.1. Introduction

4.2. Erectile Dysfunction

4.3. Infertility

5. FREE RADICALS AND RELATED REACTIVE SPECIES IN ARTHRITIC DISEASES

5.1. Introduction

5.2. Rheumatoid Arthritis

6. FREE RADICALS AND RELATED REACTIVE SPECIES IN SEPSIS


Other Diseases and Conditions Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies 243

6.1. Introduction

6.2. Animal Studies

6.3. Human Studies and Clinical Correlations

7. FREE RADICALS AND RELATED REACTIVE SPECIES IN EXERCISE

7.1. Exercise: Definition, Classification, and Recommendations

7.2. Exercise and Oxidative Stress

8. FREE RADICALS AND RELATED REACTIVE SPECIES IN REGENERATIVE MEDICINE

8.1. Introduction

8.2. ROS and Stem Cells

8.3. ROS and Skin Wound Healing

9. FREE RADICALS AND RELATED REACTIVE SPECIES IN NANOMEDICINE

9.1. Introduction

9.2. Nanomaterials and Antioxidant Activities: Two Scenarios

9.3. Scavenging of ROS/RNS by Nanomaterials via Surface Redox Chemistry

9.4. Antioxidant Nanomaterials as Disease-Protecting Modalities

9.5. Nanotoxicology

10. REFERENCES

1. OVERVIEW

The preceding chapters have discussed the causative and/or contributing role of oxidative stress in common

diseases, including cardiovascular diseases, diabetes and metabolic syndrome, neurological diseases, pulmonary

diseases, gastrointestinal and liver diseases, kidney diseases, and cancer, as well as in aging process. In addition

to the above diseases and aging, evidence also exists suggesting an important role for reactive oxygen and

nitrogen species (ROS/RNS) in many other disease processes and related conditions. This chapter summarizes

the experimental and clinical findings on the involvement of ROS/RNS in skin disorders, eye diseases,

disorders of reproductive system, arthritic diseases, and sepsis. It also discusses the significance of free radicals

and related reactive species in exercise, regenerative medicine, and nanomedicine.

2. FREE RADICALS AND RELATED REACTIVE SPECIES IN SKIN DISEASES

2.1. Introduction

The skin is the largest organ in the body. It is composed of an outer epidermis of ectodermal origin and an

underlying dermis of mesenchymal origin. Skin fulfills many critical functions, including (1) protection against

exogenous insults, (2) thermoregulation, (3) regulation of immunological response, (4) sensation, (5) secretion

of wastes, and (6) protection against water loss. As a complicated organ involved in the regulation of many

important physiological activities, skin is also a common site of diseases. Many skin diseases are intrinsic to the

skin, but some are manifestations of systemic diseases. Among this latter group are systemic lupus


244 Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies Y. Robert Li

erythematosus, acquired immunodeficiency syndrome (e.g., Kaposi sarcoma), and cutaneous T cell lymphoma.

This section focuses on the diseases that are primarily limited to the skin tissue.

Skin diseases are categorized in different ways. As summarized in Table 12.1, one classification scheme

divides skin diseases into six categories: (1) acute inflammatory skin diseases, (2) chronic inflammatory

skin diseases, (3) blistering (bullous) skin diseases, (4) infectious skin diseases, (5) tumors of the skin, and

(6) other skin diseases.

Table 12.1. Classification of skin diseases.

Group Subgroup and/or Examples of Diseases

Acute inflammatory skin diseases Urticaria

Acute eczematous dermatitis

1) Atopic dermatitis

2) Allergic contact dermatitis

3) Irritant contact dermatitis

Erythema multiforme

Chronic inflammatory skin diseases Psoriasis

Lichen planus

Lichen simplex chronicus

Blistering (bullous) skin diseases Pemphigus (vulgaris and foliaceus)

Bullous pemphigoid

Dermatitis herpetiformis

Infectious skin diseases Bacterial infection

Fungal infection

Viral infection

Neoplasms of the skin Keratosis

Squamous cell carcinoma

Basal cell carcinoma

Melanoma

Others Acne vulgaris

Vitiligo

Alopecia

Hair graying

Scleroderma

Ultraviolet radiation-induced skin injury

The pathophysiology of skin diseases is complex, involving many molecular and cellular events, including

inflammation, autoimmunity, direct injury, as well as oxidative stress and dysregulated redox signaling.

Indeed, studies over the last two decades have provided substantial evidence for an important role of

oxidative stress in the pathophysiological processes of such skin diseases as atopic dermatitis, allergic and

irritant contact dermatitis, psoriasis, acne vulgaris, vitiligo, alopecia and hair graying, scleroderma, and

ultraviolet radiation-induced skin injury. This section is intended to summarize the major findings from

both animal and human studies on the oxidative stress mechanisms of these diverse skin disorders. A

detailed discussion of the role of oxidative stress in cancer is provided in Chapter 10.

2.2. General Considerations on Oxidative Stress in Skin Diseases

2.2.1. Sources of ROS/RNS

There are many sources of ROS/RNS formation in skin diseases (Fig. 12.1). As many skin diseases are

inflammatory disorders in nature, activation of the infiltrated inflammatory cells as well as the pre-existing

inflammatory cells in skin tissue results in the formation of large amounts of ROS/RNS from NAD(P)H oxidase


270 Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies, 2012, 270-294

Detection of Free Radicals and Related Reactive Species

Y. Robert Li

All rights reserved - © 2012 Bentham Science Publishers

CHAPTER 13

Abstract: Detection and quantification of free radicals and related reactive species in biological

systems are a critical step in understanding the pathophysiological role of these reactive species in

disease processes. In this regard, a variety of methods and techniques have been developed over the past

two to three decades to detect free radicals and related reactive species, including the commonly

encountered reactive oxygen and nitrogen species (ROS/RNS) in mammalian cells or tissues. This

chapter provides an overview of the major technical approaches to detecting biological ROS/RNS and

describes some well-established ROS/RNS-detecting assays with an emphasis on their principles,

advantages, and potential limitations. The commonly detected ROS/RNS include superoxide, hydrogen

peroxide, hydroxyl radical, peroxynitrite, and nitric oxide. In addition, assays for detecting singlet

oxygen, hypochlorous acid, and molecular oxygen are also discussed in this chapter.

Keywords: Alkoxyl radical, Detection, Free radicals, Hydrogen peroxide, Hydroxyl radical, Hypochlorous

acid, Methodologies, Nitric oxide, Peroxynitrite, Reactive nitrogen species, Reactive oxygen species,

Singlet oxygen, Superoxide.

CHAPTER AT A GLANCE

1. OVERVIEW

2. MAJOR TECHNICAL APPROACHES TO DETECTING FREE RADICALS AND RELATED

REACTIVE SPECIES

2.1. UV/Vis Spectrophotometry

2.2. Fluorescence

2.3. Chemiluminescence

2.4. Electron Paramagnetic Resonance

2.5. Others

3. DETECTION OF SUPEROXIDE

3.1. Introduction

3.2. Some Commonly Used Superoxide-Detecting Assays

4. DETECTION OF HYDROGEN PEROXIDE

4.1. Introduction

4.2. Some Commonly Used Hydrogen Peroxide-Detecting Assays

4.3. Other Hydrogen Peroxide-Detecting Assays

5. DETECTION OF HYDROXYL RADICAL

5.1. Introduction

5.2. Some Commonly Used Hydroxyl Radical-Detecting Assays


Detection of Free Radicals Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies 271

6. DETECTION OF PEROXYNITRITE

6.1. Introduction

6.2. Some Commonly Used Peroxynitrite-Detecting Assays

7. DETECTION OF NITRIC OXIDE

7.1. Introduction

7.2. Some Commonly Used Nitric Oxide-Detecting Assays

8. DETECTION OF OVERALL CELLULAR OXIDATIVE STRESS

8.1. Introduction

8.2. 2’,7’-Dichlorofluorescein Assay

8.3. Other Assays for Detecting Overall Cellular Oxidative Stress

9. DETECTION OF OTHER RELATED SPECIES

9.1. Detection of Singlet Oxygen

9.2. Detection of Hypochlorous Acid

9.3. Detection of Molecular Oxygen

10. REFERENCES

1. OVERVIEW

As discussed in the preceding chapters, a growing body of evidence points to that free radicals and related

reactive species play a significant role in the pathophysiology of diverse human diseases. As such,

antioxidant-based modalities have been increasingly recognized as an important approach to disease

intervention. Continued basic and clinical research in free radical biomedicine will undoubtedly further

increase our ability to develop more effective mechanistically-based strategies to combat human diseases

that involve an oxidative stress mechanism. In this context, sensitive and reliable detection of reactive

oxygen and nitrogen species (ROS/RNS) as well as other related reactive species is a prerequisite for better

understanding of the pathophysiological involvement of oxidative stress in disease processes and the

development of antioxidant-based therapies. Hence, this chapter is intended to provide an overview of the

commonly used and well-established methods to detect ROS/RNS with an emphasis on the principles of the

techniques as well as the advantages and limitations of the assays. When applicable, newly developed

ROS/RNS-detecting techniques and methods are also covered in this chapter. The chapter begins with a

brief introduction to the major technical approaches to detecting biological ROS/RNS, followed by

description of some widely accepted methods for detecting the commonly encountered ROS/RNS and

related species in biomedicine. These include superoxide, hydrogen peroxide, hydroxyl radical,

peroxynitrite, and nitric oxide. The methods for detecting singlet oxygen, hypochlorous acid, and molecular

oxygen are also discussed. In addition, this chapter also describes commonly used assays for detecting

overall cellular oxidative stress.


272 Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies Y. Robert Li

2. MAJOR TECHNICAL APPROACHES TO DETECTING FREE RADICALS AND RELATED

REACTIVE SPECIES

2.1. Ultraviolet/Visible (UV/Vis) Spectrophotometry

UV/Vis spectrophotometry is employed to measure the amount of light that a sample absorbs. The

instrument operates by passing a beam of light through a sample and measuring the intensity of light

reaching a detector. The Beer-Lambert law states that the absorbance of a solution is directly proportional

to the concentration of the absorbing species in the solution and the path length. Thus, for a fixed path

length, UV/Vis Spectrophotometry can be used to determine the concentration of the absorber in a solution.

For detecting ROS/RNS by UV/Vis spectrophotometry, the detecting probe is incubated with the

ROS/RNS-generating system (e.g., enzymes, cells, tissues), and the reaction product is detected at a

specific absorption wavelength. The ideal ROS/RNS-detecting probes are substances with optical

properties that change markedly after reaction with ROS/RNS and show high specificity for different

ROS/RNS. Simplicity is a major advantage of the UV/Vis spectrophotometry-based ROS/RNS-detecting

assays. The principal disadvantage is the limited sensitivity as compared with other techniques, such as

fluorescence-based methods.

2.2. Fluorescence

Fluorescence is the emission of light that occurs within nanoseconds after the absorption of light (i.e.,

excitation) that is typically of shorter wavelength [1]. The maximum emission wavelength is denoted as

em and the maximum excitation wavelength as ex. em and ex vary with different fluorescent

molecules, which serve as the basis for detecting fluorescent substances. Fluorescence can be detected by

fluorescence spectrophotometry (also known as fluorometry) or visualized by fluorescence microscopy

(i.e., fluorescence imaging). The technique involves using a beam of light (ultraviolet or visible light) that

excites the fluorescent molecules and causes them to emit light of a lower energy, typically, but not

necessarily, visible light. For detecting ROS/RNS by fluorescence spectrophotometry or microscopy, a

fluorescence-based detecting probe is incubated with the ROS/RNS-generating system (e.g., enzymes,

cells, tissues), and the fluorescence of the reaction product is detected to determine the amounts of

ROS/RNS. In general, most of the available fluorescence-based ROS/RNS-detecting probes are nonfluorescent

or weakly fluorescent, but yield fluorescent products upon reaction with ROS/RNS. For

example, the fluorescence probe 2’,7’-dichlorodihydrofluorescein (non-fluorescent) is converted to the

fluorescent product 2’,7’-dichlorofluorescein after reaction with certain ROS/RNS. On the other hand,

some probes are fluorescent compounds and become non-fluorescent following reaction with ROS/RNS.

Fluorescence-based probes permit ROS/RNS detection with much higher sensitivity than UV/Vis

spectrophotometry. In addition, fluorescence microscopy-based imaging allows in situ visualization of

ROS/RNS formation in cells or tissue slices. Limited selectivity for different reactive species is often a

major disadvantage of the fluorescence techniques in detecting ROS/RNS production in biological systems.

2.3. Chemiluminescence

Chemiluminescence is the emission of light with limited emission of heat as a result of a chemical reaction.

Given reactants A and B, with an excited intermediate “E*”, the light-emitting reaction can be written as

follows (Reaction 1).

[A] + [B] → [E*] → [Products] + light (1)

For example, if [A] is luminol and [B] is hydrogen peroxide in the presence of a suitable catalyst (e.g.,

myeloperoxidase or horseradish peroxidase), Reaction 2 occurs, where 3-APA is 3-aminophthalate, and 3-

APA* is the excited state. The decay of 3-APA* to a lower energy level causes photon emission (also see

Section 4.2.2).

Luminol + H2O2 → [3-APA*] → 3-APA + light (2)


Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies, 2012, 295-306 295

Y. Robert Li

All rights reserved - © 2012 Bentham Science Publishers

CHAPTER 14

Detection of Damage of Biomolecules by Free Radicals and Related

Reactive Species

Abstract: Lipids, proteins, and nucleic acids are major cellular targets of reactive oxygen and nitrogen

species (ROS/RNS). Oxidative damage to these cellular constituents has been increasingly recognized as a

significant pathophysiological event leading to many disease processes. Thus, determination of ROS/RNSelicited

damage to lipids, proteins, and nuclear acids is crucial for investigating the oxidative mechanisms of

human diseases and assessing the efficacy of antioxidant-based therapies. This chapter first introduces the

general experimental approaches to assessing the effects of ROS/RNS on cellular biomolecules and then

describes the commonly used methods to determine ROS/RNS-mediated biological damage, with an

emphasis on the assays that detect lipid peroxidation, protein oxidation, and oxidative DNA base

modifications. The chapter ends with a discussion of biomarkers of oxidative stress and their value in

assessing disease pathophysiology as well as the efficacy of antioxidant intervention.

Keywords: Biomarkers, DNA base modifications, DNA damage, F2-isoprostanes, Lipid peroxidation,

Methodologies, Oxidative stress, Protein oxidation, Reactive nitrogen species, Reactive oxygen species.

CHAPTER AT A GLANCE

1. OVERVIEW

2. MAJOR EXPERIMENTAL APPROACHES TO ASSESSING THE EFFECTS OF ROS/RNS ON

BIOMOLECULES

3. DETECTION OF LIPID PEROXIDATION

3.1. Introduction

3.2. Detection of Intermediates: Lipid-Derived Radicals and Diene Conjugates

3.3. Detection of Primary End-Products: Lipid Hydroperoxides

3.4. Detection of Secondary End-Products: Aldehydes

3.5. Detection of Secondary End-Products: Ethane and Pentane

3.6. Detection of Secondary End-Products: Isoprostanes

4. DETECTION OF PROTEIN DAMAGE BY ROS/RNS

4.1. Introduction

4.2. Detection of Protein Carbonyls

4.3. Detection of Oxidation, Nitration, and Halogenations of Tyrosine

4.4. Detection of Protein Sulfhydryl Oxidation

4.5. Others

5. DETECTION OF DNA DAMAGE BY ROS/RNS


296 Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies Y. Robert Li

5.1. Introduction

5.2. Detection of 8-Hydroxy-2’-deoxyguanosine

5.3. Simultaneous Detection of Oxidative Modifications of Multiple DNA Bases

5.4. Detection of DNA Strand Breaks

6. BIOMARKERS OF OXIDATIVE DAMAGE

6.1. Definition of Biomarkers

6.2. Biomarkers of Oxidative Damage and Human Diseases

7. REFERENCES

1. OVERVIEW

Due to their high reactivity, free radicals and related reactive species readily cause oxidative damage to

biomolecules, including lipids, proteins, and nucleic acids in mammalian systems. Oxidative damage to

critical cellular constituents is the basis of oxidative stress-mediated disease processes. Hence, detection of

such oxidative damage by reactive oxygen and nitrogen species (ROS/RNS) is of importance for

understanding the oxidative stress mechanisms of disease pathophysiology and devising reliable

biomarkers to assess disease genesis and progression as well as the efficacy of antioxidant-based strategies

for disease intervention. This chapter begins with a brief introduction to the major experimental approaches

which are used to assess oxidative damage in experimental models and human subjects. It then examines

the commonly used methodologies for assessing the oxidative damage by ROS/RNS to cellular lipids,

proteins, and nucleic acids. Lastly, the chapter defines biomarkers and discusses the applications of

oxidative stress biomarkers in biomedicine.

2. MAJOR EXPERIMENTAL APPROACHES TO ASSESSING THE EFFECTS OF ROS/RNS ON

BIOMOLECULES

Depending on the levels and duration of exposure, ROS/RNS may cause two types of oxidative alterations:

(1) overt oxidative damage to cellular constituents, leading to destruction or dysfunction of biomolecues

(e.g., lipid peroxidation, enzyme inhibition, DNA strand cleavage), and (2) reversible oxidative

modifications of cell signaling molecules (e.g., transcription factors, protein kinases), leading to altered cell

signal transduction and gene expression.

The experimental approaches to assessing ovet oxidative damage to cellular constituents include (1)

detection of lipid peroxidation (e.g., formation of lipid peroxidation-derived aldehydes), (2) detection of

protein oxidation (e.g., formation of protein carbonyls), and (3) detection of oxidative DNA damage (e.g.,

DNA bases modifications, DNA cleavage). In addition to the above major cellular biomolecules,

carbohydrates can also be damaged by ROS/RNS.

Assessment of reversible oxidative modifications of cell signaling molecules by ROS/RNS can be achieved

by detecting the altered activities of transcription factors and protein kinases as well as changes in gene

expression. In this context, a number of transcription factors (e.g., NF-B, Nrf2, AP-1) and protein kinase

cascades (e.g., mitogen-activated protein kinases, protein kinase C) are subject to redox modulation by

ROS/RNS.

In general, overt oxidative damage to cellular constituents is the molecular basis of cell injury and cell

death, whereas reversible oxidative modifications of cell signaling molecules usually cause altered cell

proliferation and differentiation. In addition, ROS/RNS-mediated activation of cell signaling molecules,


Detection of Damage of Biomolecules Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies 297

such as Nrf2 may lead to the upregulation of antioxidants and other cytoprotective factors, which can be

viewed as an adaptive mechanism. Hence, reversible modifications of cell signaling pathways by

ROS/RNS (usually at low or moderate levels) do not necessarily always cause detrimental effects. The

subsequent sections focus on discussing the assessment of overt oxidative damage. It should be noted that

although oxidative damage is the most frequent form of deleterious effects of ROS/RNS, RNS also cause

nitrative stress, leading to nitration of biomolecules. Nitrative stress is also a significant mechanism of

disease pathogenesis. As such, RNS-mediated nitration is also discussed in this chapter.

3. DETECTION OF LIPID PEROXIDATION

3.1. Introduction

As discussed in Chapter 2, polyunsaturated fatty acids are susceptible to free radical attack with abstraction

of an allylic hydrogen atom from a methylene group to form a carbon-centered radical. This is followed by

bond rearrangement, the formation of a conjugated diene and reaction with oxygen to form a peroxyl

radical, which can then abstract a further hydrogen atom from another polyunsaturated fatty acid with the

formation of lipid hydroperoxides. Lipid hydroperoxides decompose to form alkoxyl and peroxyl radicals,

which participate in chain propagation reactions, or give rise to a variety of secondary end-products,

including aldehydes (e.g., malondialdehyde, acrolein, 4-hydroxy-2-nonenal) and alkanes (e.g., ethane,

pentane). In addition, endocyclization of the peroxyl radical followed by further attack by oxygen has been

identified as a novel pathway by which a group of compounds known as isoprostanes are formed [1].

Hence, assessment of lipid peroxidation in biological systems involves the detection of diverse

intermediates and end-products (Fig. 14.1).

R

Polyunsaturated Fatty Acid

Free Radicals

OH

O

H

Carbon‐centered Radical

Lipid Hydroperoxide

COOH

Oxidation and

Decomposition

Aldehydes

Other Species

Fig. (14.1). Schematic illustration of lipid peroxidation to yield diverse intermediates and end-products that can be

measured by various methods. Measurement of these products serves as a basis for assessing lipid peroxidation.

H

3.2. Detection of Intermediates: Lipid-Derived Radicals and Diene Conjugates

3.2.1. Detection of Lipid-Derived Radicals

Attack of polyunsaturated fatty acids by free radicals results in the formation of carbon-centered radicals as

well as the subsequent generation of alkoxyl and peroxyl radicals. These lipid-derived radicals can be

R

Conjugated Diene

O

.

O

O 2

Lipid Peroxyl Radical

Endoperoxide

Intermediate

Isoprostanes

COOH

Endocyclization

and Oxidation


Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies, 2012, 307-325 307

Y. Robert Li

All rights reserved - © 2012 Bentham Science Publishers

CHAPTER 15

Detection and Measurement of Cellular and Tissue Antioxidants

Abstract: The detrimental effects of reactive oxygen and nitrogen species (ROS/RNS) in mammalian

systems are determined not only by the amounts and reactivity of the ROS/RNS formed, but also by the

status of cellular and tissue antioxidant defenses. Thus, detection and measurement of endogenous

antioxidants are an important part of investigation into the mechanism of oxidative stress in human

disease development. This chapter provides an overview of the general experimental approaches to

assessing antioxidant defenses and describes the basic methodologies for measuring the activities and

levels of the major cellular and tissue antioxidants.

Keywords: Antioxidants, Catalase, Glutaredoxin, Glutathione system, Heme oxygenase, Methionine

sulfoxide reductase, Methodologies, NAD(P)H:quinone oxidoreductase, Paraoxonase, Superoxide

dismutase, Thioredoxin system, Total antioxidant capacity.

CHAPTER AT A GLANCE

1. OVERVIEW

2. MAJOR EXPERIMENTAL APPROACHES TO ANALYZING ANTIOXIDANTS

2.1. Measurement of Enzyme Activities

2.2. Analysis of Protein Expression

2.3. Determination of mRNA Levels

2.4. Analysis of Gene Transcription

2.5. Analysis of Transcription Factor Activities

2.6. Detection of Total Antioxidant Capacity

3. MEASUREMENT OF SUPEROXIDE DISMUTASE, CATALASE, AND GLUTATHIONE

PEROXIDASE ACTIVITIES

3.1. Introduction

3.2. Sample Preparation

3.3. Superoxide Dismutase

3.4. Catalase

3.5. Glutathione Peroxidase

4. MEASUREMENT OF GLUTATHIONE CONTENT AND -GLUTAMYLCYSTEINE LIGASE AND

GLUTATHIONE REDUCTASE ACTIVITIES

4.1. Introduction

4.2. Glutathione


308 Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies Y. Robert Li

4.3. -Glutamylcysteine Ligase

4.4. Glutathione Reductase

5. MEASUREMENT OF GLUTATHIONE S-TRANSFERASE AND NAD(P)H:QUINONE

OXIDOREDUCTASE 1 ACTIVITIES

5.1 Introduction

5.2. Glutathione S-Transferase

5.3. NAD(P)H:Quinone Oxidoreductase 1

6. MEASUREMENT OF THIOREDOXIN, PEROXIREDOXIN, AND THIOREDOXIN REDUCTASE

ACTIVITIES

6.1. Introduction

6.2. Thioredoxin

6.3. Peroxiredoxin

6.4. Thioredoxin Reductase

7. MEASUREMENT OF HEME OXYGENASE ACTIVITY

7.1. Introduction

7.2. Heme Oxygenase

8. MEASUREMENT OF OTHER ANTIOXIDANT ENZYME ACTIVITIES

8.1. Glutaredoxin

8.2. Methionine Sulfoxide Reductase

8.3. Paraoxonase

9. REFERENCES

1. OVERVIEW

Oxidative stress results from the imbalance between the formation of ROS/RNS and the functioning of

antioxidant defenses. The extent and outcome of oxidative damage to cellular constituents are determined

not only by the amounts and production rates of ROS/RNS, but also by the levels and activities of the

antioxidant defenses, including both nonprotein antioxidants (e.g., glutathione) and antioxidant enzymes

(e.g., superoxide dismutase, catalase, glutathione peroxidase). Hence, detection of cellular and tissue

antioxidants provides important information regarding the mechanisms underlying oxidative stress and

disease pathophysiology, and serves as a basis for devising strategies for disease intervention. In addition,

sensitive and reliable quantification of antioxidants in biological systems is also an important part of studies

aiming to develop novel pharmacological agents capable of inducing endogenous antioxidant defenses.

This last chapter of the book is intended to first provide an overview of the general approaches to analyzing

antioxidant defenses and then describe the routinely used methods for measuring the levels or activities of


Detection of Antioxidants Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies 309

common cellular antioxidants with an emphasis on the principles and major procedures of the assays for

measuring antioxidant enzymes.

2. MAJOR EXPERIMENTAL APPROACHES TO ANALYZING ANTIOXIDANTS

As stated in Chapter 3, mammalian cells and tissues are equipped with a range of antioxidants, including

both protein and non-protein antioxidants synthesized by cells. In addition, mammalian antioxidant

defenses also comprise the antioxidant compounds (e.g., vitamin C, vitamin E, carotenoids, and

polyphenols) derived from the diet. Although the proper functionality of mammalian cells depends on the

coordinated actions of these diverse dietary antioxidants, endogenous antioxidant enzymes seem to play a

more vital role in protecting against oxidative stress. This is evidenced by the essentiality of some

antioxidant enzymes in embryonic development as well as the disease phenotypes caused by deletion of

each of the many cellular antioxidant genes in animal models. Hence, methodologies have been developed

to specifically detect and measure cellular and tissue antioxidant enzymes and proteins. Antioxidant

enzymes and proteins can be assessed by a number of major experimental approaches, as summarized

below (Fig. 15.1).

TF

Protein

mRNA

Gene Transcription

Transcription Factors

Enzyme Activity Assays

Immunoblot Analysis

Immmunohistochemistry

Proteomics

Real‐Time PCR Analysis

RT‐PCR Analysis

Gene Array Profiling

Nuclear Run‐On Assay

Reporter Gene Assays

Immunoblot Analysis

Gel Shift Assay

Fig. (15.1). Major experimental approaches to analyzing cellular and tissue antioxidant defenses. See text (Section 2)

for detailed description. TF, transcription factor; RT-PCR, reverse transcriptase-polymerase chain reaction.

2.1. Measurement of Enzyme Activities

This is frequently done by using biochemical assays based on the chemical reactions catalyzed specifically

by the antioxidant enzymes. The rest of the chapter primarily describes the commonly used assays for

measuring the activities of cellular and tissue antioxidant enzymes that are commonly encountered in

biomedicine.

2.2. Analysis of Protein Expression

In addition to assaying the enzyme activities, the protein levels of cellular antioxidant enzymes can be

determined by immunoblot analysis via using specific antibodies. Immunoblot analysis is considered

semiquantitative. A concomitant standard curve utilizing purified enzymes may be carried out to estimate

the absolute amounts of the antioxidant proteins in the samples. Immunohistochemistry staining with the

use of specific antibodies can be carried out to visualize the protein expression of antioxidant enzymes in

individual cells of tissue slices. The recently developed proteomic techniques are powerful for large-scale

studies of cellular proteins, including antioxidant enzymes and other cytoprotective proteins.

2.3. Determination of mRNA Levels

Increased activities of cellular antioxidants usually result from increased protein expression, which in turn

frequently stems from increased levels of the mRNA. The mRNA levels can be analyzed via various


326 Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies, 2012, 326-342

12-O-tetracecanoylphorbol-13-acetate

1-chloro-2,4-dinitrobenzene

1-hydroxy-3-carboxy-pyrrolidine

1-hydroxyethyl radical

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine

1-methyl-4-phenylpyridinum

2,3,7,8-tetrachlorodibenzo-p-dioxin

2,3-diaminonaphthalene

2,3-dihysroxybenzoic acid

2,4,6-trinitrobenzenesulfonic acid

2,4-dinitrophenylhydrazine

2,4-disulfophenyl-N-tert-butylnitrone

2,5-dihydroxybenzoic acid

2,6-dichlorophenolindophenol

2,6-dicloroindophenol

2’,7’-dichlorodihydrofluorescein

2’,7’-dichlorofluorescein

20S proteasome

2-D proteomic gels

2-hydroxyethidium

2-hydroxyterephthalic acid

3,4-hydroxybenzoic acid

3-aminophthalate

3-nitropropionic acid

3-nitrotyrosine

4,4’-diaminodiphenysulfone

4,5-diaminofluorescein-2

4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl

4-hydroxy-2-nonenal

4-hydroxybenzoic acid

5,5’-dithiobis-(2-nitrobenzoic acid)

5,5-dimethyl-1-pyrroline N-oxide

5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide

5-ethoxycarbonyl-5-methyl-1-pyrroline N-oxide

5-thio-2-nitrobenzoic acid

5-thiobutyl butyrolactone

6-carboxy-2’,7’-dichlorodihydrofluorescein

6-hydroxydopamine

7,12-dimethybenz[a]anthracene

8-hydroxy-2’-deoxyguanosine

8-hydroxyguanine

8-oxo-2’-deoxyguanosine

9,10-dimethylanthracene

9,10-diphenylanthracene

A

acetaminophen

acetonitrile

acidosis

acne vulgaris

aconitase

Index

Y. Robert Li

All rights reserved - © 2012 Bentham Science Publishers


Index Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies 327

acrolein

acrylesterase

acute kidney injury

acute renal failure

acute respiratory distress syndrome

acute tubular necrosis

adhesion molecules

adipocyte

adipokines

adiponectin

adiposity

adult treatment panel III

advanced glycation end products

AEOL

aflatoxin B1

age-related macular degeneration

AGI-1067

air pollutants

airway inflammation

alcohol

alcoholic liver disease

alcoholic hepatitis

alcoholic liver injury

alcoholism

aldehyde

aldehyde dehydrogenase

alkaline elusion

alkanes

alkoxyl radical

allantoin

allergic rhinitis

alloxan

alopecia

Alzheimer’s disease

amenorrhea

aminotransferase

aminotriazole

amiodarone

AMP-activated protein kinase

amphetamines

Amplex Red

amputation

amyloid precursor protein

amyotrophic lateral sclerosis

androgens

anemia

angina pectoris

angiogenesis

angiotensin II

anovulation

anthocyanidins

anthracycline

anti-8-OH-dG antibody

antiangiogenesis


328 Free Radical Biomedicine: Principles, Clinical Correlations, and Methodologies Y. Robert Li

anticancer drugs

antioxidant

antioxidant enzyme mimetics

antioxidant gene regulation

antioxidant response element

antiplatelet

anxiety

AP-1

AP-2

APC min mice

ApoB

apoceruloplasmin

apocynin

ApoE

apoptosis

arachidonic acid

arginine

arthritis

asbestos

ascorbate

ascorbate radical

ascorbic acid

aspirin

astaxanthin

asthma

astrocytes

ataxia telangiectasia

ataxia with vitamin E deficiency

atherogenesis

atherosclerosis

atopic dermatitis

ATP7B

autoimmune encephalomyelitis

autophage

azotemia

B

basal cell carcinoma

benzene

benzo[a]pyrene

benzoyl peroxide

bilirubin

biliverdin

biliverdin reductase

biomarkers

biotransformation

biphenols

bleomycin

blindness

blood

blood pressure

bone marrow

bovine serum albumin

brain cancer

More magazines by this user
Similar magazines