Pathology: Oxidative Stress and Dietary Antioxidants

Zealand

Preface

Victor R. Preedy

Pathology is the study and science of disease and usually encompasses specific subfields from cells to the whole body. In the clinical setting, pathologists are specialists who may focus on one or more areas. For example, The Royal College of Pathologists has over 23 subspecialties, including histopathology. neuropathology, cellular pathology, molecular pathology, cytopathology, pediatric pathology, forensic pathology, clinical biochemistry (chemical pathology), hematology immunology, medical microbiology, virology, clinical cytogenetics, molecular genetics, oral-maxillofacial pathology, toxicology, and clinical embryology. Some areas of pathology overlap and the affected individual may have more than one manifestation of the disease, requiring attention by more than one practitioner.

Very often, oxidative stress is a feature of pathologies and affects different cells and organs in a number of ways. These can be manifested as changes in biomarker profiles (clinical biochemistry) or stained tissue sections of biopsies pre-or postmortem (histopathology). Oxidative stress can also arise due to nutritional imbalance before the onset of disease, at the point of diagnosis, and as a result of therapy. There is thus a fundamental need to understand the processes inherent in the oxidative stress of disease. This is important as oxidative stress can be ameliorated with pharmacological, nutraceutical, or natural agents. Food-based antioxidants, however, circumvent the side effects associated with pharmacological antioxidants or supplements. While pathologists and clinical workers understand the processes in disease, they are less conversant in the science of nutrition and dietetics. On the other hand, nutritionists and dietitians are less conversant with the detailed clinical background and science of pathology. Thus pathologists, healthcare workers, food scientists, and nutritionists are separated by divergent skills and professional disciplines that need to be bridged in order to advance medical science. Hitherto, this transdisciplinary divide has been difficult to bridge.

Pathology: Oxidative Stress and Dietary Antioxidants aims to cover, in a single volume, the science of oxidative stress in disease and the potentially therapeutic usage of antioxidants in the diet, food matrix, plants, or otherwise. However, in this book the science of oxidative stress is not described in isolation but in concert with other processes such as apoptosis, cell signaling, receptor mediated responses, and so on. This approach recognizes that diseases are often multifactorial and oxidative stress is a single component of this. In the book, each chapter has a section that describes other areas of pathology so that there is holistic coverage and applicability of the information.

The book imparts holistic information with a structured format.

Part I: Oxidative Stress and Pathology

Part II: Antioxidants and Pathology

Part III: Techniques and Resources

Each chapter has a section called Applications to Other Areas of Pathology.

Part I, Oxidative Stress and Pathology, covers free radicals (what they are), various disease, and cellular biochemistry. In Part II, Antioxidants and Pathology, we describe numerous agents and their actions. The caveat of the chapters in Part II is that there needs to be further in-depth analysis of these components in terms of safety and efficacy as some material is exploratory or preclinical. A cautionary and critical approach is needed. Nevertheless, the material in Part II can provide the framework for further in-depth analysis or studies. This would be via well-designed clinical trials or via the analysis of pathways, mechanisms, and components in order to devise new therapeutic strategies. Part III, Techniques and Resources, is more practically based.

The book is designed for nutritionists, dietitians, food scientists, pathologists, pathology residents and professionals, physicians, medical students, healthcare workers, the healthcare industry, and research scientists. The academic level ranges from students, undergraduate, post graduates, lecturers, and professors. Contributions are from leading national and international experts, including those from world-renowned institutions.

Part I

Oxidative Stress and Pathology

Outline

Chapter 1 Free radicals: what they are and what they do

Chapter 2 Oxidative stress in pathologies of male reproductive disorders

Chapter 3 Oxidative stress and diabetic retinopathy

Chapter 4 Pathological bases of oxidative stress in the development of cardiovascular diseases

Chapter 5 Pathological association between oxidative stress and chronic obstructive pulmonary disease

Chapter 6 Oxidative stress in sickle cell disease and emerging roles for antioxidants in treatment strategies

Chapter 7 Nrf2 and oxidative stress

Chapter 8 Redox signaling and antioxidant defense in pathogenic microorganisms: a link to disease and putative therapy

Chapter 9 Paraoxonase 1 as antioxidant enzyme in children

Chapter 10 The regulation of intracellular redox homeostasis in cancer progression and its therapy

Chapter 1

Free radicals: what they are and what they do

Eva Tvrdá and Filip Benko,    Department of Animal Physiology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture, Nitra, Slovakia

Abstract

Free radicals (FRs) have gained significant attention in the field of biology and medicine due to their double-edged sword roles in numerous physiological processes as well as their implications in a wide range of pathologies. FRs comprising reactive oxygen species (ROS) and reactive nitrogen species (RNS), are derived from endogenous and exogenous sources. Because of their unique characteristics, FRs have the ability to adversely affect vital biomolecules such as nucleic acids, lipids, and proteins, thereby altering the normal intracellular redox state leading to oxidative stress (OS) development. FR overproduction and the resulting OS have been implicated in a number of diseases such as diabetes mellitus, neurodegenerative and cardiovascular diseases, autoimmune disorders, and various cancers. This chapter focuses on the chemistry, properties, formation, and sources of FRs, followed by a discussion on their molecular targets. The chapter also provides a brief overview of the general roles of ROS and RNS in health and disease.

Keywords

Free radicals; reactive oxygen species; reactive nitrogen species; oxidative stress; redox state; sources; chemistry; properties

List of abbreviations

–SH sulfhydryl group

¹O2 singlet oxygen

¹Δg delta state

¹εg sigma state

4-HNA 4- hydroxynonenal

8-oxoG 7, 8-dihydro-8-oxo-guanosine

As arsenic

CAT catalase

Cd cadmium

CH2 methylene

Co cobalt

Cr chromium

Cu copper

Fe iron

FR free radical

G3PDH glyceraldehyde-3-phosphate dehydrogenase

GPx glutathione peroxidase

H2O2 hydrogen peroxide

Hg mercury

HOCl hypochlorous acid

L• lipid radical

LOO• lipid peroxyl radical

LPO lipid peroxidation

MDA malondialdehyde

Mn manganese

NADPH nicotinamide adenine dinucleotide phosphate

NO• nitric oxide

NOS nitric oxide synthases

NS nitrosative stress

O2•− superoxide radical

O3 ozone

OH• hydroxyl radical

ONOO− peroxynitrite

ONOOCO2− nitroso peroxo carboxylate

ONOOH peroxynitrous acid

OONO− peroxynitrite

OS oxidative stress

Pb lead

RNS reactive nitrogen species

RO• alkoxyl radical

ROO• peroxyl radical

ROOH lipoperoxide

ROS reactive oxygen species

SOD superoxide dismutase

Introduction

Oxygen is a crucial element for life on Earth as it plays a dominant role in the controlled oxidation of molecules containing carbon, subsequently leading to the generation of energy essential for the survival of all aerobic systems. Interestingly, its oxidative properties make oxygen exhibit conflicting behavior in that it is essential for life while it can also aggravate cellular damage by a variety of oxidative events.¹

Aerobic cells are persistently facing this so-called oxygen paradox: while oxygen is imperative to sustain aerobic life, at the same time, it may become highly toxic to cell survival.² This dark side of oxygen relates directly to the fact that each oxygen atom has one unpaired electron in its outer valence shell, while molecular oxygen has two unpaired electrons. The reductive properties of the cellular environment provide numerous opportunities for oxygen to undergo unexpected univalent reduction. As such, aerobic metabolism leads to the generation of a diversity of by-products called free radicals (FRs),¹–³ which, under normal circumstances, are necessary for a normal cell survival.⁴ On the other hand, if FR levels become too high, either because of their overproduction or due to a low antioxidant capacity, oxidative stress (OS) emerges that often has fatal consequences on cell function.²,⁵

Prior to the 1970s, FRs were widely overlooked either because of lack of evidence concerning their existence in a living system or because they were considered to be generally insignificant. Regardless of little attention received from the scientific community for decades, their existence, formation, and importance in a diversity of diseases is now widely accepted.⁵

Free radicals: general characteristics

A FR is defined as any atom, molecule, or a fragment of an atom or molecule that contains at least one unpaired electron and exhibits, to a certain extent, an independent existence.² FRs may be created by the (1) addition of a single electron to a neutral atom or molecule, or by the (2) loss of one electron from a neutral atom or molecule:

Additionally, FRs may also be created by the (3) homolysis of covalent bonds that may lead to the creation of intermediate reactive by-products. This process requires energy such as heat, UV light, or ionizing radiation. FRs may have positive, negative, or neutral charges.³,⁴

The presence of an unpaired electron results in certain common properties shared by most radicals. FRs are generally unstable and highly reactive. They can either donate an electron to, or accept an electron from, other molecules, therefore behaving as oxidants or reductants, respectively.²,⁵ Since they have an unpaired electron, they are electrophilic and attack sites of increased electron density, which are usually present in compounds with nitrogen atoms (proteins, nucleic acids) and carbon–carbon double bonds (phospholipids, polyunsaturated fatty acids (PUFAs)).⁴ During such an electron interchange, new radicals are formed from previously nonradical molecules and chain reactions may occur.

In cells, one-electron modification of molecules can yield sulfur-, oxygen-, carbon-, and nitrogen-centered FRs.⁴ Furthermore, ions of transition metals have a radical nature (Cu²+, Fe²+, etc.).²

The majority of FRs have a very short half-life, which is why it is very difficult to synthesize, store, quantify, or use them in experimental or clinical settings.²

The most common and important FRs related to biology are oxygen-centered radicals, called reactive oxygen species (ROS) and nitrogen-centered molecules, called reactive nitrogen species (RNS).³

ROS and RNS can be further classified into two groups of compounds; radicals and nonradicals. Radicals are species that contain at least one unpaired electron in the outer shells around the nucleus and are capable of existing independently. Nonradical species are not FRs per se, but can easily lead to oxidative reactions in living systems.²–⁵ An overview of the most common ROS and RNS is provided in Table 1.1.

Table 1.1

Oxidative chain reactions

Much of the potency of FRs stems from their natural tendency to be involved in oxidative processes occurring during a chain reaction.

FR reactions have three distinct identifiable phases¹:

1. Initiation: initial creation of FRs. Usually it is a hemolytic cleavage with the assistance of heat, UV, or metal catalysts.

2. Propagation: FRs are generated and regenerated repeatedly as a result of the chain reaction.

3. Termination: two FRs react with each other to form a stable, nonradical product.

Selected free and nonfree radicals: characteristics and behavior

Superoxide radical (O2•−)

The recognition that living systems produce significant amounts of superoxide through normal metabolic pathways and that enzymes, particularly superoxide dismutase (SOD), help protect cells against the potential toxicity of this FR,⁶ represents the pillars of why FR-associated biology was considered to be superoxide-centric for decades. O2•− is the principal FR generated as a result of a monovalent reduction of oxygen and the addition of one electron⁴,⁵ by enzymatic processes, autooxidation, or by nonenzymatic electron transfers.²

O2•− is mostly produced within the mitochondria by xanthine oxidase,⁷ lipoxygenase, cyclooxygenase,⁸ or NADPH-dependent oxidase. Superoxide does not react with most molecules in aqueous solutions. Its reactivity with nonradical molecules is pH-dependent. The radical is a stronger reductant than oxidant.² As a reducing agent, O2•− reacts with iron complexes such as cytochrome and reduces Fe³+ to Fe²+:

As an oxidant, O2•− can oxidize transition metal ions (Cu³+, Fe⁴+, Mn³+), dopamine, adrenaline, or ascorbate, leading to the formation of hydrogen peroxide.²

Superoxide radicals react with another superoxide radical in a dismutation reaction, in which one radical is oxidized to oxygen while the other is reduced to hydrogen peroxide.⁹

The primary toxicity of O2•− is associated with its ability to initiate oxidative chain reactions, followed by the generation of other reactive metabolites such as hydrogen peroxide, hydroxyl radical, singlet oxygen, or peroxynitrite (ONOO−).²,⁵

Hydrogen peroxide (H2O2)

Hydrogen peroxide is a two-electron product of oxygen reduction that is able to participate in signal transduction, cell proliferation, differentiation, and response to stress.²

H2O2 is primarily formed in a dismutation reaction catalyzed by SOD, but it can also be produced by other enzymes such as xanthine, glucose monoamine, and D-amino acid oxidases, or during ascorbate and polyphenol oxidation.¹ The molecule has no unpaired electrons and, hence, is not a radical in nature,² but it can cause significant damage to cells even at low concentrations (10 μM). H2O2 is soluble in water and has the ability to easily penetrate through biological membranes.⁵ Furthermore, it can directly damage some enzymes such as glyceraldehyde-3-phosphate dehydrogenase (G3PDH) through oxidation of the sulfhydryl (–SH) group.¹⁰

Within the cell, H2O2 has the ability to accumulate within the cell and interact with iron and copper ions leading to significant damage to lipids, deoxyribonucleic acid (DNA), and proteins through the hydroxyl radical emerging from interactions between H2O2 and transition metals.²,⁵

Hydroxyl radical (OH•)

Hydroxyl radicals are defined as the neutral form of the hydroxide ion and are highly reactive FRs² that can aggressively react with organic and inorganic molecules and cause more severe damage to the cell than any other FR.¹⁰ OH• is created in the Fenton reaction as a result of interactions between H2O2 and metal ions (Fe+2 or Cu+) often bound in complex forms with different proteins such as ferritin or ceruloplasmin.¹¹ Under stress conditions, excessive O2•− may release these metal ions from their respective complexes. OH• may also be created by the Haber–Weiss reaction comprising O2•− and H2O2 interactions.¹²

Other sources of OH• are also known, such as reactions with hypochlorous acid, quinones, and semiquinones.⁵ Hydroxyl radicals are particularly dangerous because of their ability to reduce disulfide bonds in proteins, above all fibrinogen, resulting in their unfolding and scrambled refolding into abnormal spatial configurations. Consequences of such reactions may be translated into numerous disorders, such as atherosclerosis, cancer, and neurological pathologies.¹³

Alkoxyl (RO•) and peroxyl (ROO•) radicals

Alkoxyl and peroxyl radicals are products of lipid peroxidation (LPO) following the decomposition of lipoperoxides (ROOH) by UV and/or heat in the presence of transition metal ions.²

Both organic radicals are relatively good oxidants¹⁰ with the ability to subtract hydrogen from other lipid molecules, leading to the branching of LPO. Such chain reactions may also proceed to generate new FRs (e.g., singlet oxygen) and can promote tumor development.¹⁴

Singlet oxygen (¹O2)

Singlet oxygen is a highly excited, metastable, nonradical state of molecular oxygen and a highly reactive and toxic ROS.² ¹O2 can exist in two states: (1) upon activation, the molecular oxygen is excited to the first delta state (¹Δg) and, then, (2) to an even higher excited singlet state, the sigma state (¹εg). The ¹Δg state is extremely reactive and is normally produced by the activation of neutrophils and eosinophils.¹⁵ It is also formed during selected enzymatic reactions catalyzed by enzymes such as lipoxygenases, dioxygenases, and lactoperoxidase.¹⁶

These observations suggest that ¹O2 may be a significant biochemical intermediate in a number of biological processes. Moreover, ¹O2 may be generated from ozone and cysteine, methionine, glutathione, albumin, ascorbic acid, and NAD(P)H.¹⁰,¹⁶

¹O2 is a highly powerful oxidizing agent that can cause DNA¹⁷ and tissue damage,¹⁵ particularly in skin and eyes. Exposure to light containing high levels of ¹O2 may lead to the accumulation of porphyrins in the skin thereby causing swelling, blistering, eruptions, and scarring.⁵,¹⁰,¹⁴

Ozone (O3)

Ozone is a powerful oxidant that may be formed by catalyzed water oxidation, which plays important roles in inflammation.¹⁸ O3 has the ability to generate other reactive intermediates by oxidizing specific molecules and functional groups such as lipids, nucleic acids, and amines, alcohols, aldehydes, and sulfhydryl present in proteins.¹⁹ Exposure to O3 may also cause chromosomal aberrations due to a direct attack by O3 or by other FRs generated as a result of its reactions.²,¹⁹

Hypochlorous acid (HOCl)

Hypochlorous acid is a major oxidant created from hydrogen peroxide and chloride within the activated neutrophils at the site of inflammation. The reaction is catalyzed by the enzyme myeloperoxidase.²,⁵

HOCl is a potent ROS involved in oxidation and chlorination processes. It can pass through membranes, causing damage to membrane proteins and lipids as well as intracellular biomolecules. It has the ability to oxidize thiols, urate, and ascorbate.²⁰ HOCl chlorinates amines to give chloramines; tyrosyl residues creating ring chlorinated products, cholesterol, and unsaturated lipids to form chlorohydrins.²⁰,²¹ It also chlorinates pyrimidine bases in DNA and tyrosine units in proteins, leading to protein and DNA fragmentation by a variety of mechanisms.¹⁰ Furthermore, HOCl produced by activated neutrophils may interact with superoxide, leading to the formation of OH•. Reactions between HOCl and H2O2 may lead to ¹O2 generation.²

Nitric oxide (nitrogen monoxide; NO•)

NO• is a colorless gas that is synthetized by various nitric oxide synthases (NOS) while converting L-arginine to L-citrulline.²² There are three types of NOS isoforms: neuronal NOS (nNOS, NOS1), inducible NOS (iNOS; NOS2), and endothelial NOS (eNOS, NOS3), all of which are involved in the formation of NO•.

The molecule is soluble in both water and lipids, which is why it is able to readily diffuse through the plasma membrane and cytoplasm.²³ Under physiological conditions NO• is an important intracellular second messenger that is involved in smooth muscle relaxation within blood vessels. NO• also plays important roles in the regulation of cellular redox states and enzymatic activities by protein nitrosylation.²³,²⁴ Moreover, it is involved in numerous other biological processes such as blood pressure and immune regulation, platelet aggregation, lung vasodilatation, as well as neurotransmission and defensive mechanisms.²⁴

To a certain extent NO• may be regarded as an FR scavenger as it interacts with hydroxyl, peroxyl, or thiyl radicals leading to the generation of nitric acid, nitrosoperoxyl, or nitrosothiol, respectively, with the latter being responsible for the inhibition of LPO by NO•. However, at high concentrations (>mmol/L), NO• may be involved in the pathology of septic shock, chronic inflammation, increased risk for oncogenesis, transplant rejection, asthma, epilepsy, and neurodegenerative diseases.¹⁰

Other reactive nitrogen species

Peroxynitrite (oxoperoxonitrate; OONO−) emerges from the reaction between O2•− and NO•. OONO− may diffuse to more distant places in the cell where lipids, proteins, or DNA can be oxidized and nitrated.²

The molecule is highly toxic²⁵ and can directly react with CO2 to form highly reactive nitroso peroxo carboxylate (ONOOCO2−) or peroxynitrous acid (ONOOH). ONOOH undergoes homolysis to form OH• and NO2, or rearranges to form NO3. OONO− may oxidize lipids, methionine, and tyrosine residues in proteins and DNA to form nitroguanine.²–⁵ Tyrosine nitration in proteins is generally used as a biomarker of peroxynitrite formation.

Sources of free radicals

FRs have long been proposed to play only negative roles in organisms, particularly when produced in large amounts, at the wrong place, or when the defense mechanisms of the organism designed to counteract their toxic effect fail.²⁶

FRs may originate from endogenous or exogenous sources. The most prominent endogenous sources include organelles that have high requirements for oxygen and are illustrated in Fig. 1.1.

Figure 1.1 Typical endogenous sources of free radicals.

Mitochondria

Mitochondria are the main source of intracellular FRs. Superoxide as a by-product of normal mitochondrial metabolism is produced at two major sites within the electron transport chain, specifically complex I (NADH dehydrogenase) and complex III (ubiquinone cytochrome c reductase). The generation of superoxide is nonenzymatic and increases with the metabolic rate.²⁷ O2•− is subsequently converted to H2O2 by the mitochondrial SOD. H2O2 can be further detoxified by catalase (CAT) and glutathione peroxidase (GPx).

Other mitochondrial components that may contribute to FR generation include monoamino oxidase, α-ketoglutarate dehydrogenase, glycerol phosphate dehydrogenase, and p66shc.²,⁵,²⁶,²⁷

Peroxisomes

The peroxisomal respiratory pathway involves the transfer of electrons from various metabolites, primarily fatty acids, to oxygen and resulting in the formation of H2O2.²⁸ However, energy is released in the form of heat. Other FRs produced in peroxisomes include O2•− OH• and NO•. Different peroxisomal enzymes such as acyl CoA oxidases, D-amino acid oxidase, L-α-hydroxy oxidase, urate oxidase, xanthine oxidase, and D-aspartate oxidase have also been shown to produce different FRs.⁵,²⁸

Endoplasmic reticulum

Various enzymes such as cytochrome p-450, b5, and diamine oxidase contribute to FR generation within the endoplasmic reticulum. A considerate proportion of H2O2 is created by the action of the important thiol oxidase enzyme, Erop1p, which catalyzes the transfer of electrons from dithiols to molecular oxygen.⁵,²⁹

Other relevant endogenous sources of FRs include metal-catalyzed reactions, prostaglandin synthesis, autoxidation of adrenalin, reduced riboflavin, FMNH2, FADH2, xanthine oxidase, cascade of arachidonic acid, immune cells, stress, excessive exercise, cancer, aging, ischemia-reperfusion, atherogenesis, hemodialysis, and so forth.¹–³

On the other hand, FRs may enter biological systems from diverse exogenous sources,¹–⁵,²⁶ among which the most notable ones are outlined in Fig. 1.2.

Figure 1.2 Most common exogenous sources of free radicals.

Oxidative stress

A living organism is a well-organized and sophisticated system where FRs act under strict control and at specific locations only.

If FRs have already been generated and/or have caused damage, the organism’s intricate system designed to protect it against the toxic effects of reactive metabolites readily recognizes damaged molecules and quickly reacts to remove any structures that had been irreparably altered. However, if one of the protective mechanisms of the organism against FRs fails, their action becomes uncontrollable, causing damage to molecules and cells, thereby leading to the death of the organism.²,¹⁴,²⁶

When there is an imbalance between the FR production and/or concentration (ROS/RNS) and antioxidant defense systems, the former will be produced at higher levels leading to OS and/or nitrosative stress (NS). Since FRs are highly reactive, their targets comprise all important classes of biological molecules including proteins, lipids, and nucleic acids.¹⁰,¹⁴ The impact of OS/NS depends on the type of oxidant, the location and intensity of its production, the composition and activities of relevant antioxidants, and the ability of repair systems.²,⁵ A summary of causes and consequences of FR and/or ROS overproduction are provided in Fig. 1.3.

Figure 1.3 Circumstances leading to free radical/ROS overproduction and their implications on the living system.

Proteins

Protein oxidation may be caused by a variety of radical species such as O2•−, OH•, RO•, ROO•, and nonradical species such as H2O2, O3, HOCl, ¹O2, and OONO−. FR oxidize different amino acids, causing formation of protein cross-links, protein denaturation, and the loss of their functionality, decreased enzymatic activity, and altered function of receptors and transport proteins.³⁰ Amino acids containing sulfur such as methionine and cysteine are more susceptible to oxidation and may be easily converted to disulfides and methionine sulfoxide.³⁰,³¹

Oxidative insults to amino acids may result in the formation of different oxidation products such as nitrotryptophan, kynurenine, 2-, 3-, and 4-hydroxyphenylalanine; tyrosine-tyrosine cross-links, 2-oxohistidine, asparagine, aspartic acid, glutamic semialdehyde, and 2-amino-3-ketobutyric acid.³¹ Moreover, oxidative damage to amino acid residues such as lysine, proline, threonine, and arginine leads to carbonyl derivatives. The presence of protein carbonyls has been proposed as a marker of FR-mediated protein oxidation.³² Other specific markers of protein oxidation comprise O-tyrosine (a marker for OH•) and 3-nitrotyrosine (a marker for RNS). Increased levels of protein carbonyls may be observed in a number of pathological conditions such as Alzheimer’s disease and Parkinson’s disease, muscular dystrophy, cataracts, diabetes, and atherosclerosis.³²–³⁵

Deoxyribonucleic acid

ROS and RNS can oxidatively damage nucleic acids. Generally speaking, mitochondrial DNA is more susceptible to oxidative insults than nuclear DNA as it is located in close proximity to the primary source of the FR. ROS, most importantly OH•, directly interacts with all the components of DNA, causing a number of alternations including single- and double-stranded breaks. The radical is able to extract hydrogen to create a number of modified purine and pyrimidine by-products as well as DNA-protein cross-links. The most commonly observed pyrimidine adducts comprise thymine glycol, uracil glycol, and hydantoin. The purine adducts frequently observed under OS/NS conditions include 8-hydroxydeoxy guanosine, 8-hydroxy deoxy adenosine, and 2,6-diamino-4-hydroxy-5-formamidopyrimidine. Predominant adducts from the sugar moiety in DNA include glycolic acid, 2-deoxytetrodialdose, and erythrose.³⁶ 8-hydroxy deoxyguanosine is considered to be the leading biomarker of oxidative DNA damage and is frequently involved in mutagenesis, carcinogenesis, and ageing.³⁷

On the other hand, RNS, most importantly OONO−, interacts with guanine to create nitrative and oxidative DNA lesions such as 8-nitroguanine and 8-oxodeoxyguanosine, respectively.⁵,³⁶,³⁷ Accordingly, 8-nitroguanine is believed to be a mutagenic DNA lesion involved in carcinogenesis.

Ribonucleic acid

Ribonucleic acid (RNA) is more prone to oxidative damage than DNA due to its single-stranded structure and lack of effective repair mechanisms and protection. Furthermore, cytoplasmic RNAs are located in close proximity to the mitochondria where significant amounts of ROS are produced.³⁸ 7, 8-dihydro-8-oxo-guanosine (8-oxoG) is the most extensively studied RNA damage product and its levels are elevated in numerous pathological conditions including Alzheimer’s disease, Parkinson’s disease, hemochromastosis, atherosclerosis, and myopathies.³⁹–⁴¹

Lipids

Membrane lipids, particularly PUFA residues of phospholipids, are the substrate of choice for oxidation. LPO results in the loss of membrane functionality that is often accompanied by decreased fluidity and the inactivation of membrane-bound enzymes and receptors.²,⁵ LPO is initiated when FRs abstract hydrogen from a methylene group (CH2) in a fatty acid, which results in the formation of a carbon-centered lipid radical (L•). The lipid radical can easily interact with molecular oxygen to create a lipid peroxyl radical (LOO•). Subsequently, LOO• undergoes cyclic rearrangements to create endoperoxides that form malondialdehyde (MDA) and 4-hydroxyl nonenal (4-HNA), which may cause damage to DNA and proteins. Such lipid radicals may further propagate the peroxidation process by abstracting hydrogen from other lipid molecules.⁴²

Free radicals in health and disease

FR overproduction and the subsequent OS have been associated with a wide variety of pathologies, such as diabetes mellitus⁴³ and metabolic syndrome⁴⁴; atherosclerosis and cardiovascular diseases⁴⁵; skin and tumor diseases⁴⁶,⁴⁷; neurodegenerative diseases, including Alzheimer’s and Parkinson’s⁴⁸; autoimmune disorders⁴⁹; male and female infertility⁵⁰,⁵¹; as well as psychological impairments such as bipolar disorder, schizophrenia, or attention deficit hyperactivity disorder.⁵² The most prominent pathologies triggered or accompanied by OS are provided in Fig. 1.4.

Figure 1.4 Most common disease states triggered, or accompanied, by free radical overproduction.

Nevertheless, FRs and reactive intermediates are necessary for a number of cell functions and their deleterious effects come into place only when produced in excess. As such, FRs as well as their antioxidant counterparts may play various roles in the living system, some being beneficial while others are harmful. It must be emphasized that a complete suppression of FR formation is not desirable, which is why, under physiological conditions, controlled and moderate FR production is required.²,¹⁴,²⁶,⁵³,⁵⁴ A short summary of the most prominent physiological roles of FRs is provided in Table 1.2.

Table 1.2

Depending on the type and concentration of FR, exposure time, and activation of antioxidant defense mechanisms and repair systems, cells exposed to oxidative insults may respond in three ways:

1. Adaptation: cells will upregulate their antioxidant defense systems.

2. Tissue injury: increased OS may cause damage to all molecular targets. The exact point of attack may often be unclear as injury mechanisms tend to overlap. Cells may respond in different ways; increased proliferation, halted cell cycle and senescence are often observed.

3. Cell death: this can occur by two mechanisms, necrosis and apoptosis. In necrotic cell death, the cell swells, ruptures, and releases its contents into the surrounding area significantly affecting neighboring cells. The intracellular content may comprise antioxidants such as catalase or glutathione, as well as oxidants such as copper and iron ions. Even if a cell enters necrosis by other than FR-mediated mechanisms, necrotic cell death usually leads to further oxidative damage to the adjacent tissue. In apoptosis, the cell’s intrinsic suicide mechanisms are activated; apoptotic cells do not release their contents and, thus, apoptosis does not, in general, cause further damage to surrounding cells.²,³,⁵,¹⁰,¹⁴

Based on the currently available information on FRs and the redox state of organisms, we may say that generated FRs and antioxidants play major roles in the cellular oxidative–reduction processes, thereby constantly changing the redox state of the cells. Such changes stimulate or inhibit the activities of communication molecules and regulatory proteins, leading to changes in the ability of signal pathways to control cellular fate. While an oxidative environment is more suitable for cell death by apoptosis or necrosis, a reducing milieu supports cell survival.²,⁵,⁵⁴

Conclusion

In conclusion, the oxidative state of a cell cannot be perceived as just black or white. FRs as well as antioxidants have regulatory functions in diverse physiological and pathological processes, precisely via a continuous modulation of the redox state of the organism. Nevertheless, currently we do not know where the exact border between their beneficial and harmful effects is. What such effects are depend on how we may benefit from modulating the effects of FRs, reactive metabolites, and antioxidants by regulating such determinants and their balance within the cellular redox state.

Applications to other areas of pathology

Understanding the chemistry and behavior of FRs has illuminated the nature of OS and its consequences for the organism. This, in turn, has already revealed possibilities to improve human health either by avoiding FR overproduction or by using antioxidants. Unraveling the specific nature of the cellular redox balance may offer new clues to define the fine line dividing the effects of FRs into positive and negative consequences. Antimicrobial and cytotoxic effects of FRs may be exploited particularly in anticancer therapies such as radiotherapy, chemotherapy, and immunotherapy. Since tumor cells contain fewer FR scavengers, it might be of interest to look for a way to produce large amounts of FRs in tumor tissues for future anticancer therapy. Furthermore, elucidation on the susceptibility of specific FRs toward antioxidant mechanisms of action may lead to the discovery of more disease-specific, target-directed, highly bioavailable antioxidants applicable for the management and/or treatment of a variety of diseases associated with OS.

Summary points

• Because of its unique characteristics, oxygen exhibits a double-edged behavior in aerobic cells.

• FRs are by-products of normal aerobic metabolism.

• FRs play important roles in fertilization, immune response, and cell signaling.

• An imbalance between FR production and antioxidant defense mechanisms leads to OS with detrimental effects on cell function and survival.

• It is crucial to understand the exact boundary between positive and negative effects of FRs in health and disease.

Acknowledgment

This chapter was supported by the Slovak Research and Development Agency grant no. APVV-15–0544 and by the KEGA grant no. 009SPU-4/2018.

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Chapter 2

Oxidative stress in pathologies of male reproductive disorders

Ashok Agarwal¹, Kristian Leisegang² and Pallav Sengupta³,    ¹American Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH, United States,    ²School of Natural Medicine, University of the Western Cape, Cape Town, South Africa,    ³Department of Physiology, Faculty of Medicine, Bioscience and Nursing, MAHSA University, Selangor, Malaysia

Abstract

The etiopathology of male infertility involves the complex interplay of numerous factors. Environmental and lifestyle factors, diet, exposure to radiation and several other factors may affect the male reproductive system. It is evident that most of these factors contribute to male infertility via induction of oxidative stress (OS). Excess generation of reactive oxygen species (ROS) in the male reproductive tract via endogenous or exogenous sources above the antioxidant capacity leads to OS. OS is responsible for altered sperm morphology and functions through lipid peroxidation, deoxyribose nucleic acid fragmentation, and germ cell apoptosis, which are reflected in poor semen parameters and fertilizing capability, resulting in male subfertility or infertility. This chapter confers the generation of ROS in the male reproductive tract, the role of ROS in pathogenesis of various male reproductive disorders, mechanisms by which OS affects male fertility, and an assessment of ROS and the use of antioxidants in the management of OS-induced male infertility.

Keywords

Antioxidants; male infertility; lipid peroxidation; oxidative stress; sperm DNA fragmentation

List of abbreviations

3βHSD 3β-hydroxysteroid dehydrogenase

4-HNE 4-hydroxynonenal

AR acrosome reaction

ATP adenosine triphosphate

BMI body mass index

CAT catalase

CVD cardiovascular disease

CYP17 17α-hydroxylase/17, 20-lyase

DNA deoxyribose nucleic acid

ED erectile dysfunction

FR free radical

GnRH gonadotropin releasing hormone

GPx glutathione peroxidase

H2O2 hydrogen peroxide

HIV human immunodeficiency virus

HPG hypothalamic-pituitary-gonadal

IFN-Ɣ interferon-gamma

IL-1β interleukin 1-beta

JNK c-Jun N-terminal kinase

LPO lipid peroxidation

LDL low density lipoprotein

MDA malondialdehyde

MMP mitochondrial membrane potential

MNDs micronutrient deficiencies

mtDNA mitochondrial DNA

MTHFR methyl tetrahydrofolatereductase

NADPH nicotinamide adenine dinucleotide phosphate

NO nitric oxide

NOS nitric oxide synthase

O2 oxygen

O2− superoxide radical

ORP oxidation-reduction potential

OS oxidative stress

P450scc P450 cholesterol side-chain cleavage

PUFA polyunsaturated fatty acid

Redox reduction–oxidation reaction

ROS reactive oxygen species

SDF sperm DNA fragmentation

SOD superoxide dismutase

StAR steroidogenic acute regulatory

T2DM type 2 diabetes mellitus

TAC total antioxidant capacity

TBARS thiobarbituric acid assays

TH-1 T-helper lymphocyte type 1

TH-2 T-helper lymphocyte type 2

TNF-α tumor necrosis factor-alpha

WHO World Health Organization

Introduction

Male factor infertility contributes to almost half of the global prevalence of infertility.¹ The etiology of male infertility is still not completely understood. Some of the most deleterious factors that affect male fecundity include environmental, physiological, genetic, and epigenetic changes, dietary habits, and lifestyle factors. A common underlying mechanism by which most of these factors disrupt male fertility is by disturbing the intricate balance between the production and scavenging of reactive oxygen species (ROS). This imbalance of increased ROS generation and reduced antioxidant capacity leads to oxidative stress (OS).²

ROS, the toxic derivates of oxygen metabolism, mediate some important male reproductive functions such as sperm capacitation, hyperactivation, and acrosome reactions.³ However, excess generation of ROS impairs redox balance in the reproductive tract, leading to cellular and molecular damage.⁴ Spermatozoa are not adequately equipped for cell repair and thus are excessively vulnerable to OS due to the unique structure of spermatozoa with very little cytoplasmic content.⁵ They are readily affected by OS-induced lipid peroxidation (LPO) due to their high polyunsaturated fatty acid (PUFA) content in the sperm membrane.⁶ This culminates in the overall deterioration of semen quality, rendering men subfertile or infertile.⁷

Redox biology in male reproduction

O2−). Through regulatory phosphorylation/dephosphorylation (redox) switches, ROS have important cellular signaling mechanisms, mediated through adaption to physiological concentrations.⁸ This is further regulated through endogenous defense mechanisms called antioxidants that evolved to regulate increased ROS production through the rise of aerobic respiration and the mitochondria. Some of the important antioxidants include the superoxide dismutase (SOD) family, glutathione peroxidase (GPx), and catalase (CAT) peroxiredoxin and thioredoxin.⁹ A state of OS drives cellular injury and molecular pathophysiology similarly to the general adaption syndrome of cellular stressors.¹⁰ This results in an accumulation of cellular injury associated with ageing, and non-communicable chronic diseases including obesity and metabolic syndrome, cardiovascular diseases (CVDs), type II diabetes mellitus, numerous malignancies, and neurodegeneration.¹¹

With the discovery of ROS in the ejaculate, it was considered to be toxic to the spermatozoa. However, ROS have critical important physiological and regulatory functions in male reproduction¹² and have been shown to be critical in male fertility, including regulation of spermatogenesis.¹³ Through Sertoli cell and macrophage mediated early mitotic divisions of germ cells, subsequent meiotic divisions produce spermatids. These cells undergo chromatin condensation and associated membrane remodeling as part of their transformation into spermatozoa. Germ cells are a significant source of ROS that are critical for the sperm maturation process alongside other signaling functions.¹⁴ Following spermatogenesis, ROS are important in epididymal transport and maturation associated mostly with motility. ROS further mediate postejaculation maturation in the female reproductive tract, in which case redox switches are required for capacitation, acrosome reaction, sperm-zona binding, and oocyte fusion for successful fertilization.¹³ ROS are generated from spermatids and mature spermatozoa directly through a mechanism of ROS-induced ROS generation. H2O2 has been investigated as a significant mediator of redox biology and switches relevant to spermatogenesis and oocyte binding.¹⁵ These have been summarized in Fig.