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1.1. OBSTRUCTIVE AIRWAY DISEASE

Obstructive airway disease is a category of respiratory problems which is

characterized by airway obstruction and chronic airway inflammation by decreasing

radii of the bronchioles (Sin et al., 2006). It includes mainly asthma and chronic

obstructive pulmonary disease (COPD). Asthma has limitation of airflow that is either

fully or partially reversible and progressively worsens over time due to a variety of

stimuli (Barnes, 1990; GINA, 2008) while in COPD, airflow limitation/obstruction is

irreversible or only partially reversible (ATS Statement, 1995). There has been a

marked increase in global prevalence, morbidity and mortality of obstructive airway

diseases that imposed a substantial economic burden on the society during last 40

years (GINA, 2010; GOLD, 2010). Obstructive airway diseases are the most common

causes of respiratory illness in all ages (Theisen and Bruckbauer, 2003; WHO, 2008).

1.2. OXIDATIVE STRESS: CLASSICAL VIEW

Oxygen is an essential element for aerobic life but paradoxically, it can be

toxic even at atmospheric concentrations. In aerobic cells, oxygen serves as an

electron acceptor in many enzymatic and non-enzymatic reactions; however, addition

of electrons to oxygen can result into formation of toxic reactive oxygen species

(ROS) (Halliwell and Gutteridge, 1989). All organisms have evolved with elaborated

cellular defences that are collectively termed as antioxidants to overcome ROS

induced toxicity. The imbalance between ROS and antioxidants is termed as oxidative

stress [Fig 1]. Thus, oxidative stress may be defined as a violation in the balance

between oxidants and antioxidants. Besides, oxidative stress occurs in many allergic

as well as immunologic disorders. However, the most of the research has focused on

the toxic effects of ROS but many reports advocate that ROS at physiologic

concentrations might play additional roles such as cell signalling and inflammatory

response. The theory of oxygen-free radicals has been known since a half century ago.

However, only within the last two decades, there was an explosive discovery about

their roles in the development of diseases as well as in the health protective effects of

antioxidants. However, normal metabolic processes in all cells are the major source of

ROS (Mak and Chan-Yeung, 2006). Hence, this study is very important in context of

medical science.

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(a) Normal

Antioxidants Reactive oxygen species

 

 

 

(b) Oxidative stress

Antioxidants

Reactive oxygen species

Fig. 1: An imbalance between ROS (oxidants) and antioxidants in elevation of

oxidative stress. (a) Normally, there are sufficient antioxidants in the body

to counter the production of a small amount of ROS (b) If either antioxidants

are diminished or production of ROS is increased (e.g. during an asthma and

COPD), the balance of antioxidants and ROS is tipped toward oxidative

stress (Bowler et al., 2004).

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1.2.1. Chemistry of ROS

A molecule with one or more unpaired electron in its outer shell is called a

free radical, which can be independently exist and remain so until and unless their

valence shells electrons get paired and attain stability. (Droge, 2002; Valko et al., 2004;

Halliwell and Gutteridge, 2007). ROS and reactive nitrogen species (RNS) are the

terms used which collectively includes free radicals and other non-radical reactive

derivatives that are also called oxidants. Radicals are highly unstable than non-radical

species, although they have stronger reactivity potential. These highly reactive species

involved in various diseases and continuously generated in human body during

normal metabolic processes (Halliwell and Gutteridge, 1989). Several mechanisms in

aerobic cells reportedly lead to the production of free radicals. These are formed from

parent molecules via the breakage of a chemical bond keeping one electron by each of

its fragment or by cleavage of a radical to generate another radical. Besides, such

molecules are also produced in redox reactions (Bahorun et al., 2006; Halliwell and

Gutteridge, 2007). Some of the common free radicals are hydroxyl (OH•), superoxide

(O2•¯), nitric oxide (NO•), nitrogen dioxide (NO2

•), peroxyl (ROO•) and lipid peroxyl

(LOO•). In addition, hydrogen peroxide (H2O2), ozone (O3), singlet oxygen (1O2),

hypochlorous acid (HOCl), nitrous acid (HNO2), peroxynitrite (ONOO¯), dinitrogen

trioxide (N2O3), lipid peroxide (LOOH) are not free radicals yet these are oxidants

and can easily lead to free radical reactions in living organisms (Genestra, 2007).

Biological free radicals are thus highly unstable molecules that have electrons

available to react with various biomolecules including lipids, proteins and DNA

[Fig 2].

The presence of an unpaired electron makes the species highly reactive. The

lifetime of various radicals is only a fraction of a sec but there is a considerable

difference among various radicals (Pryor, 1986). When a free radical reacts with a

non-radical molecule, the target molecule is converted into a radical, which may

further react with another molecule. The formation of most of primary ROS is the

reduction of molecular oxygen with the formation of O2•¯ (Fridovich, 1983). However,

the reactivity of O2•¯ is quite low but it is capable of initiating free-radical chain

reactions. O2•¯ undergoes a dismutation to form H2O2 spontaneously or enzymatically.

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Fig. 2: Molecular consequences of oxidative stress (Adapted from Kirkham and

Rahman, 2006).

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The spontaneous dismutation rate is slow whereas the reaction catalyzed by SOD

is 104 times much faster (Halliwell and Gutteridge, 1989). O2•¯ can also react with

nitric NO• to form ONOO¯ (Beckman et al., 1994).

O2•¯ + O2

•¯ + 2H+ H2O2 + O2

H2O2 is more stable than O2•¯. It can diffuse through the plasma membrane

and if not scavenged locally by catalase or glutathione peroxidase (GPx), can promote

chain radical reactions far from its origin (Halliwell and Gutteridge, 1989). As the

lifetime of OH• is extremely short, then it is considered to be the most reactive and

potentially harmful radical and expected to react at or close to its site of formation.

The OH• radical is generated from H2O2 through the Fenton reaction catalyzed by the

transition metals iron or copper (II)

or from O2•¯ and H2O2 through the Haber-Weiss reaction catalyzed by iron or copper

(III) (Halliwell and Gutteridge, 1989)

Metal

O2•¯ + H2O2 O2 + OH• + OH ¯

Catalyst

1.2.2. Formation of free radicals and oxidants

Free radicals are chemical species having unpaired electrons that are generated

in vitro as well as in vivo.  In living beings, these molecules can be formed by two

ways. The first way of generation is comprised of enzymatic reactions that include

various steps in different oxidation-reduction reactions of metabolic pathways; such

as oxidative phosphorylation in mitochondria, phagocytosis by cells of the immune

Fe2+ + H2O2 Fe3+ + OH• + OH ¯ 

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system and also during the inflammatory mechanism, the prostaglandin synthesis and

the cytochrome P450 system [Fig 3] (Willcox et al., 2004; Halliwell, 2007;

Valko et al., 2007). The O2•¯ is generated via several cellular oxidases such as

NADPH oxidase, xanthine oxidase (XO), peroxidase and after formation it

participates in several reactions yielding various ROS and RNS such as H2O2, OH•,

ONOO¯, HOCl etc. H2O2 (a non radical) is produced by the action of several oxidative

enzymes including amino acid oxidase and XO. OH• is the most reactive free radical

in vivo, formed by the reaction of O2•¯ with H2O2 in the presence of catalyst Fe2+ or

Cu+. This reaction is known as the Fenton reaction (Droge, 2002; Valko et al., 2004;

Genestra, 2007). HOCl is produced by neutrophil-derived enzyme, myeloperoxidase

(MPO) which oxidizes chloride ions (Cl¯) in the presence of H2O2. NO• is formed in

biological tissues from the oxidation of L-arginine to citrulline by nitric oxide

synthase (Willcox et al., 2004; Valko et al., 2007). The alternate way of production of

biological free radicals is from non-enzymatic reactions that include substrate level

oxidative phosphorylation in mitochondria.

Free radicals can also be produced from non-enzymatic reactions of oxygen

with organic compounds as well as those initiated by ionizing radiations such as O3

and ultraviolet radiation (Mustafa, 1990). Moreover, these are also generated from

excessive exercising, exposure to ultra violet and ionizing radiation, redox chemicals,

cigarette smoking, environmental pollution and during aging (Frei, 1994;

Parthasarathy et al., 1999; Valko et al., 2005, 2006). Exogenous sources include

pollution (air and water), heavy metals (lead, mercury, cadmium etc.), certain drugs

(gentamycin, cyclosporine), smoking and radiations etc. These agents after getting

into the body via different routes are decomposed or metabolized that consequently

generate various free radicals (Pham-Huy et al., 2008).

1.2.3. Free radicals in health and diseases

The ROS and RNS generated in living systems collectively induce oxidative

and nitrosative stress respectively. All of them have dual roles in biological systems

that mean these are useful if released in small amount but elicit harmful effects if

generated beyond the threshold capacity of antioxidant defence system

(Baek et al., 2003; Valko et al., 2004).

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Fig. 3: Formation of free radicals.

(Adapted from: http://thehoghiehub.com/2012/02/26/tips-for-battling-free-

radicals-and-living-healthy-especially-in-china/)

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At low or moderate concentrations, ROS and RNS are necessary for the

maturation of cellular structures and can act as weapons for the host defence system.

The beneficial effects of ROS during the immune responses are respiratory burst that

destroy pathogens (e.g. O2•¯, ONOO¯), help in killing of microbes during

inflammation, detoxify xenobiotics or induce mitogenic response (Young and

Woodside, 2001; Valko et al., 2006; Pacher et al., 2007). Furthermore, the radicals

are also involved in various physiological and cellular signalling systems (Halliwell,

2007). Their production by nonphagocytic NADPH oxidase isoforms plays a

significant role in the regulation of intracellular signalling cascades in various types of

nonphagocytic cells including fibroblasts, endothelial cells, vascular smooth muscle

cells, cardiac myocytes and thyroid tissue. In addition, RNS also mediate a number of

important biological functions. For example, NO• is a prominent signal molecule for

many physiological processes like blood pressure regulation, homeostatic regulation,

glomerular and tubular function, cell proliferation, transcription, neurotransmission,

smooth muscle relaxation, energy metabolism and immune response (Koshland, 1992;

Bergendi et al., 1999; Alderton et al., 2001). Thus, ROS and RNS at low or moderate

levels are beneficial for human health.

Contrary to all these, when produced in excess these molecules can exert

damaging effects to cellular membranes, proteins, lipids and DNA up to various

extents depending upon the amount and reactivity of radicals (Young and Woodside,

2001; Droge, 2002; Willcox et al., 2004; Pacher et al., 2007). For example, OH• are

highly reactive while O2•¯ are less damaging by self but can initiate chain reactions

leading to the formation of other strong ROS (Halliwell , 1994; Wickens, 2001; Valko

et al., 2004). Hence, their check by endogenous and exogenous antioxidants under

normal levels is obligatory for healthy life in all aerobes. Oxidative stress can arise

when cells cannot adequately destroy the excess of formed free radicals [Fig 4].

Thus, oxidative stress results from an imbalance between formation and

neutralization of ROS/RNS. For example, OH• and ONOO¯ in excess can damage cell

membranes and lipoproteins by a process called lipid peroxidation (LPO) which is a

radical-mediated chain reaction initiated by abstraction of a hydrogen atom from a

polyunsaturated lipid and terminated by chain-breaking antioxidants such as

α-tocopherol (Halliwell and Gutteridge, 1989).

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Endogenous sources

Mitochondrial leak

Respiratory burst

Enzyme reactions

Auto-oxidation

reactions

Lipid peroxidation

Free radical

production

O2•¯, H2O2

Transition

Metals

Fe2+, Cu+

OH•

Modified DNA bases

Environmental sources

Cigarette smoke

Pollutants

UV light

Ionising radiation

Xenobiotics

Protein damage

             

 

 

Tissue damage

Fig. 4: Major sources of free radicals in the body and consequences of free

radical damage (Young and Woodside, 2001).

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This reaction leads to the formation of malondialdehyde (MDA) and conjugated

dienes compounds which are cytotoxic as well as mutagenic.

LPO occurs by a radical chain reactions that once started, it can spreads

rapidly and affects a large number of lipid molecules (Frei, 1994). Excessive

peroxidation of membrane lipids disrupts the bilayer arrangement, decreases

membrane fluidity, increases membrane permeability and modifies membrane-bound

proteins. Proteins may also be damaged by ROS/RNS leading to structural changes

and loss of enzyme activity (Frei, 1994; Halliwell, 2007). Oxidative damage to DNA

leads to the formation of different oxidative DNA lesions which can ultimately cause

mutations. The body has several mechanisms to counteract these attacks by using

DNA repair enzymes and/or antioxidants (Willcox et al., 2004; Genestra, 2007). In

addition, ROS can activate or inactivate proteins by oxidizing sulfhydryl groups and

modifying the amino acids (Davies and Delsignore, 1987). Regardless to their source

of generation, they can alter the behaviour of several cell types manifested by various

diseases and pathological conditions. Oxidative and nitrosative stress have been

manifested in general decline in the functions of various organs leading to several

diseases including diabetes mellitus, AIDS, cardiovascular disease, neurodegenerative

disease and various cancers (Ketterer and Coles, 1991; Pace and Leaf, 1995; Meyer

et al., 1998). Parallel to these literatures it is also reported that many inflammatory

lung diseases are caused by free radical mediated toxicity (Rahman et al., 1996a).

1.2.4. Role of antioxidants and oxidative stress biomarkers

Biological system exposed to oxidative challenge both endogenously as well

as exogenously are protected by well developed enzymatic and non-enzymatic

antioxidant defence systems (Halliwell, 1996; Knight, 2000). “Antioxidant is any

substance that, when present at low concentration, compared with those of an

oxidizable substrate, significantly delays or prevents the oxidation of that substrate”

(Halliwell, 1994; Halliwell and Gutteridge, 2007). In physiological conditions, these

defence mechanisms maintain a low steady-state concentration of free radicals in cells

and their activities are precisely regulated (Harris, 1992).

The biological system exhibits several antioxidant defence roles in order to

prevent injury (Gutteridge, 1995; Burdon, 1996). Some of these are as follows:

(i) Remove oxygen or decrease local oxygen concentrations.

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(ii) Remove catalytic metal ions such as iron and copper.

(iii) Remove key ROS such as O2•¯and H2O2.

(iv) Scavenge, initiating free radicals such as OH•, alkoxyl and ROO• species.

(v) Break chain of initiated sequence.

(vi) Quench or scavenge 1O2.

The positive effect of antioxidants and free radical scavengers for decreasing the

ROS/RNS induced toxicities in vivo confirms involvement of oxidative and

nitrosative stress (Rahman et al., 1996a; Young and Woodside, 2001). Hence,

decreasing such stress by any non-toxic agent or molecule can be a very suitable

solution to address clinical aspect of the toxicity [Fig 5].

1.2.5. Types of ROSs and RNSs

Superoxide anion (O2•¯): It is produced during the process of electron

transport chain. It cannot diffuse far away from the site of its own origin and

can generate other ROS. The electron gets attached to 1O2 and form O2, which

further oxidizes all molecules (Halliwell and Gutteridge, 1989).

O2¯+ e¯ O2

• (Superoxide radical)

Hydrogen peroxide (H2O2): Though not a free radical, while it is capable of

generating free radicals by reacting with transition metals. It can traverse

through the cell membrane. O2•¯ gets dissimulated to H2O2 by enzyme

superoxide dismutase (SOD).

Hydrogen radical (OH•): It is the most reactive species which attacks on

biological molecules (Halliwell and Gutteridge, 1989).

O2• + O2

• + 2H+ H2O2 + O2 SOD

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Fig. 5: Oxidative/nitrosative stress biomarkers used as in detection of disease

initiation and progression (Dalle-Donne et al., 2005).

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OH• is produced from H2O2 by the process of Fenton Reaction (Fenton, 1984) in the

presence of Fe2+ or copper.

H2O2 OH• (Hydroxyl radical) + OH ¯ (Hydroxyl ion)

Organic radicals: These are generated when O2•¯ or OH• radical indiscriminately

extract electrons from other molecules.

Oxides of nitrogen: NO• has a single electron and binds to other compounds

containing single electron. As a gaseous state, it diffuses through the cytosol

and lipid membranes into cells. At low concentration, it functions

physiologically as a neurotransmitter and also as a hormone assisting

compound in vasodilation. However, at high concentration it combines with

O2 or O2•¯ to form additional reactive toxic species, containing both N2 and O2

(Pryor et al., 2006; Pacher et al., 2007).

In response to infectious agents and other stimuli, phagocytic cells of the

immune system exhibit a rapid consumption of O2, called respiratory burst. It

is a major source of O2•¯, H2O2, OH• radical and HOCl. HOCl is a powerful

oxidant formed in body by the activated neutrophils. Free radicals are also

produced by neutrophils or macrophages during an inflammation, cause

injury.

1.2.5.1. Lipid peroxidation (LPO)

The best characterized biological damage is exhibited by OH• as its ability to

stimulate free radical chain reaction known as LPO. Membrane lipids are highly

susceptible to free radical damage which is highly detrimental for the functioning of

the cells (Devasagayam et al., 2003). The general scheme of LPO includes initiation,

propagation and termination which provide an outline of typical radical reaction

(Gutteridge, 1995). Membrane containing poly unsaturated fatty acids (PUFA) are

bathed on O2 rich metal ion, containing fluids that serve as good target for

peroxidation [Fig 6].

Fe2+ Fe3+

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O

OH

O

O

 

Fatty acid with three double bonds

-H Hydrogen abstraction by hydroxyl radical

Unstable carbon radical

Molecular

Rearrangement

Conjugated diene

Oxygen uptake

Peroxyl radical

+ H Hydrogen abstraction Chain reaction

Lipid hydroperoxide

Malondialdehyde,

4-hydroxynonenal,

ethane/pentane etc.

Fig. 6: Schematic representation of lipid peroxidation (Young and McEneny,

2001).

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The end products of LPO have been found more stable and are measured as an index

of oxidative stress (Halliwell and Gutteridge, 1989). LPO increases in a number of

diseases (Gutteridge, 1995). Oxidative stress induces peroxidation of membrane lipids

leads to alterations in the biological properties of the membrane including the degree

of fluidity. Thus, it can inactivate membrane bound receptors or enzymes which may

impair normal cellular functions and increases tissue permeability. Moreover, it may

contribute to amplify cellular damage, resulting into generation of oxidized products,

some of which are chemically reactive and lead to covalent modification in critical

macromolecules. Products of LPO have been commonly used as biomarkers of

oxidative stress/damage. It generates a variety of relatively stable end products (Dalle-

Donne et al., 2006). MDA is a physiological ketoaldehyde produced by peroxidation

of unsaturated lipids as a by-product of arachidonic acid metabolism. The excess of

MDA produced as a result of tissue injury can combine with the free amino groups of

proteins and produce MDA modified protein products. Moreover, 4-hydroxy-2-

isoprostanes is found another end product of LPO (Montuschi et al., 2000; Cracowski

et al., 2002). Compared with free radicals, the aldehydes are relatively more stable

and can easily diffuse within or even escape from the cell. Some of these aldehydes

have been shown to exhibit facile reactivity within various biomolecules including

proteins, DNA and phospholipids, generating stable products at the end of the series

of reactions that contribute to the pathogenesis of many diseases (Dalle-Donne et al.,

2006).

1.2.6. Types of Antioxidants

(a) Enzymatic antioxidants

Superoxide dismutase (SOD): SOD was discovered by McCord and

Frodovich in 1969. It acts as a primary defense against oxidative stress that

converts O2•¯ into H2O2 and O2 (Coursin et al., 1985).

2O2• H2O2

SOD

2H+ O2

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Thus, it is essential to maintain life in all aerobic organisms (McCord and

Frodovich, 1969). There are three forms of SOD present in human viz. cytosolic

copper, zinc SOD (Cu, Zn-SOD), mitochondrial Mn-SOD and an extracellular SOD

(Marklund, 1990; Landis and Tower, 2005). Cu, Zn-SOD contains two identical sub-

units with a molecular weight of 32 kDa. Copper takes part in the dismutation

reaction and zinc appears to stabilize the enzyme (Halliwell and Gutteridge, 1989).

O2•¯ is itself unstable and it decays spontaneously into O2 and H2O2 but SOD is able to

increase the rate of intracellular dismutation by a factor of 109 (Fridovich, 1978).

Catalase: It is a tetrameric protein (molecular weight 22-350 kDa), having

iron protophyrin III as a prosthetic group. It reduce H2O2 into H2O and O2 by

undergoing alternate divalent oxidation and reduction at its active site; thus

preventing the formation of OH• radical (Chance et al., 1979).

Catalase-Fe (II) + H2O2 Compound-1

Compound-1 + H2O2 Catalase-Fe (III) + 2H2O + O2

Glutathione peroxidase (GPx): It is a selenoprotein and one of the members

from the family of selenium enzymes that occurs mainly in cytosol and

mitochondria in eukaryotic cells (Zakowskii et al., 1978). It provides

protective role in prevention of free radical injury due to selenium. The

reactive sulfhydryl (-SH) groups reduce H2O2 to non toxic alcohols. Two

glutathione molecules are oxidized to form a single molecule of glutathione

disulfide.

ROOH + 2GSH ROH + H2O + GSSG

The kinetics of GPx is responsible for a very high rate of peroxide reduction

(Chaudiere et al., 1984; Flohe and Gunzler, 1984). The oxidized glutathione (GSSG)

is reduced back to -SH form by glutathione reductase which contains FAD and

catalyzes transfer of electrons from NADPH to the disulfide bond of GSSG.

GSSG + NADPH + H+ 2GSH + NADP+

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Late, NADPH is regenerated from the reaction catalysed by

glucose-6-phosphate dehydrogenase in the hexose monophosphate shunt.

(b) Non-enzymatic antioxidants

Vitamin E (α-tocopherol): It is a membrane bound antioxidant that terminates

chain reaction of LPO by scavenging LOO• (Bast et al., 1991; Davies, 1995;

Hensley et al., 2004).

Vitamin C (ascorbic acid): It regenerates α-tocopherol radicals and

lipoproteins in membranes (Kojo, 2004) and scavenges O2•¯ and OH•

(McCay, 1985).

Other non-enzymatic antioxidants including β-carotene (scavenger of O2•¯ and

LOO•), uric acid (scavenger of OH•, O2•¯ and LOO•), glucose (OH• scavenger),

billuribin (LOO• scavenger), taurine (HOCl quencher), transferrin (transition metal

binding), cysteine and cysteamine (donator of sulfhydryl groups) (Comhair and

Erzurum, 2002), melatonin (Rahimi et al., 2005) and flavanoids (Schroeter et al., 2002)

are supporting members of the overall antioxidant system in our body.

1.3. AIRWAY OXIDATIVE STRESS

The lung is constantly exposed to a high-oxygen environment with its large

surface area and a constant high blood supply. Hence, airways have unique feature in

both their exposure to high levels of environmental oxidants and unusually equipped

with high concentration of extracellular antioxidants. Lung cells are the first to

encounter inspired oxygen and the oxygen concentration in the lungs is relatively high

as compared to levels in other organs (Crapo, 1986). The generation of ROS is further

increased by inhaled toxic particles and gases. Evidences suggest that ROS produced

both intracellularly by lung parenchymal cells and extracellularly by lung

macrophages and infiltrating neutrophils, play a central role in the pathogenesis of

various lung diseases, such as adult respiratory distress syndrome (ARDS), asthma,

COPD, bronchopulmonary dysplasia (BPD), granulomatous lung diseases and

asbestos-induced diseases (Heffner and Repine, 1989; Barnes, 1990;

Sibille and Reynolds, 1990; Kamp et al., 1992; Kinnula et al., 1995).

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1.3.1. Sources of ROS/oxidants in lungs

The basic findings on ROS generation in lung cells have come mostly from

rabbit and rat lungs while the generation of ROS has also been investigated in human

alveolar macrophages (Kinnula et al., 1995). Production of ROS is increased as a

result of exposure to high oxygen concentrations as well as presence of exogenous

oxidants like O3, asbestos fibers, cigarette smoke, radiation and certain drugs in the

inspired gas (Sibille and Reynolds, 1990; Crystal, 1991; Kamp et al., 1992).

O2•¯ generation in the mitochondrial respiratory chain is associated with NADH

dehydrogenase and the ubiquinone cytochrome b complex (Freeman and Crapo,

1982). Findings of increased mitochondrial ROS generation after exposure to

hyperoxia suggest that mitochondria are an important site of ROS production in the

lungs (Turrens et al., 1982).

O2•¯ generation is highest when the respiratory chain carriers located in the

inner mitochondrial membrane are greatly reduced (Freeman and Crapo, 1982).

Mitochondrial H2O2 is mainly derived from the dismutation of O2•¯. Approximately

1 to 2% of the oxygen consumed by the mitochondria is estimated to be converted to

O2•¯ and H2O2. Cell cytoplasm is also an important site of ROS production. One non-

enzymatic source of ROS is the generation of OH• by iron-catalyzed reactions

(Fenton and Haber-Weiss reactions). In addition, ROS formed by intracellular

enzymes, i.e. XO is a cytoplasmic enzyme which is present in normal conditions in its

dehydrogenase form without any detectable ROS generation. In ischemic tissues, it is

converted to oxidase leading to the production of O2•¯and H2O2 during reoxygenation

(McCord, 1985). Because the level of XO in lung tissue is low, its role in ROS

production is probably not significant (Linder et al., 1999). Neutrophils contain MPO

which converts H2O2 to HOCl in the presence of halide ions, a relatively long-lived

oxidant (Halliwell and Gutteridge, 1989). Furthermore, during inflammation under the

influence of chemo-attractants, neutrophils migrate to lungs in large amount (Tkaczyk

and Vizek, 2007). Besides, eosinophils contain NADPH oxidase which generates O2•¯.

Other sources of ROS possibly relevant in the lungs are microsomal cytochrome

P-450 enzymes and peroxisomal oxidases. ROS play an important role in the host

defence against microbicidal agents. During phagocytosis activated inflammatory

cells generate ROS through membrane-bound NADPH oxidase, the phenomenon

called respiratory burst (Babior, 1984). Respiratory burst is induced by various

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endogenous and extracellular factors such as opsonized bacteria, endotoxin,

cytokines, fibrous material, phorbol esters and formyl methionyl leucylphenylalanine

(Rossi, 1986). Endothelial cells are a source of oxidative stress in lungs due to

oxidoreductase complex, NADPH oxidase (Jones et al., 1996) and formation of OH•

radicals by iron ions found in proximity to endothelial surface (Terada, 1996). It has

been reported that both smooth muscle cells as well as fibroblasts contains NADPH

oxidase which generates O2•¯ (Thannickal and Fanburg, 2000; Thabut et al., 2002).

Recent studies also have reported that Type II pneumocytes have enzymatic

properties that result in the generation of oxidants (Kinnula et al., 1991; Van Klaveren

et al., 1997).

Airway diseases such as asthma and COPD have evidence of increased

oxidative stress, suggesting that reactive oxygen and nitrogen species may overwhelm

antioxidant defences in these diseases (Bowler and Crapo, 2002; Fig 7). In the resting

state, the balance between antioxidants and oxidants is sufficient to prevent the

disruption of normal physiologic functions; however, either increases in oxidants or

decreases in antioxidants can disrupt this balance. There is now substantial evidence

that inflammatory lung diseases such as asthma and COPD are characterized by

systemic and local chronic inflammation and oxidative stress (MacNee, 2001;

Caramori and Papi, 2004; Guo and Ward, 2007; Hoshino and Mishima, 2008).

An oxidant/antioxidant imbalance in favour of oxidants can lead to lung injury due to

direct, unprotected oxidative damage to air space epithelial cells (Lannan et al., 1994).

ROS may result in remodelling of extracellular matrix, cause apoptosis and

mitochondrial respiration as well as in regulation of cell proliferation

(Richter et al., 1995; Gutteridge and Halliwell, 2000). Alveolar repair responses and

immune modulation in the lung may also be influenced by ROS (Richter et al., 1995;

Gutteridge and Halliwell, 2000).

Furthermore, high levels of ROS have been implicated in initiating

inflammatory responses in the lungs through the activation of transcription factors

such as nuclear factor- κ B (NF- κ B) and activator protein-1 (AP-1). Their activation

modulates signal transduction and gene expression of pro-inflammatory mediators

(Guyton et al., 1996; Rahman and MacNee, 1998). It is proposed that ROS produced

by phagocytes that have been recruited to sites of inflammation leads to major

damage resulting into many chronic inflammatory lung diseases including asthma and

COPD (Hatch, 1995; Rahman and MacNee, 1996, 1999, 2000; Dworski, 2000).

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Fig. 7: ROS and antioxidant enzymes in airways (A) environmental sources and

(B) enzymatic antioxidants (Bowler and Crapo, 2002).

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The composition of inflammatory cell types that invade tissues varies widely in

asthma and COPD. It is reported that there are few differences in the characteristics of

the ROS produced in these diseases (Jeffery, 1998; Saetta, 1999; Barnes, 2000).

1.3.2. Mechanism of oxidant mediated lung inflammation

A number of proposed pathologic mechanisms have been identified which can

induce development of airway obstruction (O’Byrne and Postma, 1999). Oxidative

injury due to exposure of oxidants from diverse sources resulting from a lack of

antioxidants in the body may be related to reduced forced expiratory volume (FEV)

and thus these factors contribute to airflow obstruction (MacNee, 2007). Inactivation

of anti-proteases, inflammation, infection, direct cell damage and disturbances in the

antioxidant defence are primary mechanisms that appear to be involved in it

(Li et al., 1994; Rahman et al., 1996a; Repine et al., 1997). Recruitment and

activation of neutrophils and other inflammatory mediators in lungs, may also involve

in production of IL-8 by alveolar macrophages, epithelial or other lung cells.

Inflammation not only causes airflow limitation but also leads to fibrosis, gland

hypertrophy and elevated smooth muscle tone. It amplifies airflow limitation by

deforming and narrowing the airway lumen (Matsuba et al., 1989).

1.3.3. Lung defence

Lung cells contain several antioxidant defence mechanisms to prevent injury. It

differs profoundly in their resistance to oxidant mediated stress that may be due to

differences in their antioxidant capacity or in the balance between oxidants and

antioxidants in the cells. However, prevention of ROS formation and repair of

oxidative damage are also essential for the survival of aerobic organisms.

Sequestration of transition metals like binding to storage and transport proteins

minimizes the “free” iron within the cells and prevents the formation of highly

reactive radicals (Fridovich and Freeman, 1986; Heffner and Repine, 1989). The

airway contains high molecular mucopolypeptide glycoproteins synthesized by

epithelial cells and glands which respond to inflammation. The epithelial lining fluid

contains antioxidant enzymes like catalase, SOD and GPx while ceruloplasmins,

transferring ascorbate, ferritin, bilirubin, immunoglobulins, anti-inflammatory cells

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22

derived from blood are the additional ones. Epithelial cells also take part in lung

defence (Comhair and Erzurum, 2002; Nicod, 2005).

1.4. Role of pulmonary function test in obstructive airway diseases

Udwadia (2007) commented that there is paucity of data and epidemiology on

obstructive airway disease in India. In mild obstruction, patient may not be aware that

his/her lung function is abnormal but during moderate due to chronic respiratory

symptoms or exacerbation, patients seek medical attention. The basic abnormality in

obstructive airway disease is airflow limitation. Thus, a global consensus on

diagnosis, classification and treatment protocol is required. Clinical severity of asthma

was determined using the criteria (appropriate clinical and lung function tests) defined

in GINA (Bateman et al., 2008) and diagnosis was established on the basis of

recurrent symptoms of breathlessness, chest tightness and wheezing with an

improvement of greater than 12% in forced expiratory volume in 1 sec (FEV1) after

inhalation of 200 μg of salbutamol from a nebulizer. It was sub-classified into

intermittent, mild persistent, moderate persistent and severe persistent according to

the frequency of symptoms, levels of FEV1 (% predicted), FEV1% variability and

treatment protocol (Yawn, 2008). The guideline of GOLD (updated, 2008), define

COPD based on spirometric criteria by using the FEV1 and its ratio to the forced vital

capacity (FVC). The main criterion for COPD is a FEV1/FVC ratio less than 70%.

Sub-classification into mild, moderate, severe, very severe is done by various levels

of FEV1 as percentage predicted value.

Lung function test interpretation is commonly based on comparison of

observed values with reference values based on healthy subjects as well as

comparisons with recognised diseases and/or values, previously measured in the same

patient. The normal values vary with age, sex, size and race.

1.5. ASTHMA

Asthma is a common airway disease and one of the major global health

problems which is characterized by chronic inflammation of the airways involving

variable and recurring symptoms including variable airflow obstruction and increased

airway responsiveness to a variety of stimuli (Barnes, 1990).

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23

1.5.1. Definition

Asthma is a chronic lung disease characterized by recurrent breathing

problems. This is a disorder defined by its clinical, physiological and pathological

characteristics. The Global Initiative for Asthma (GINA, 2008) defines; asthma is a

“chronic inflammatory disorder of the airways in which many cells and cellular

elements play a role. The chronic inflammation is associated with the airway hyper

responsiveness that leads to recurrent episodes of wheezing, breathlessness, chest

tightness and coughing, particularly at night or in the early morning. These episodes

are usually associated with widespread but variable, airflow obstruction within the

lung that is often reversible. Airway inflammation is the most proximate cause of the

recurrent episodes of airflow limitation in asthma.

This unifying definition of asthma highlights the clinical hallmarks of the

disease: (i) inflammatory process (ii) airway hyper-responsiveness (iii) obstruction in

the normal airflow (iv) increased airway remodelling. It is clear that asthma is a very

heterogeneous disease as it includes immunopathology, clinical different phenotypes,

non-uniform response to therapies and natural history (Holgate, 2008). Thus, asthma

considers a syndrome with different risk factors, prognosis and response to treatment

(Reed, 2006). These points are the necessities to overcome the historical

simplification that defined asthma as merely an inflammatory disease of the lung.

1.5.2. Asthma, a major health issue

There is a long history of changing concepts on asthma from the antiquity to the

modern times. In ancient times, asthma was already recognised in many cultures and

was known by different names including the Chinese, Egyptian, Hebrew, Mesoptamia

and Greco-Romans (Veith, 1966; Sakula, 1988; Saavedra-Delgado et al., 1991;

Cserhati, 2004; 2005). The term “asthma” originated from the Greek word

“asthmaino”, indicating gasping (laboured breathing), and the term was first used by

Hippocrates (460-377 BC) in the Corpus Hippocraticum (Diamant et al., 2007).

Asthma known as “tamka swasa” in the ancient Vedic texts, and has got a good

clinical description in Charaka Samhita, the first Ayurveda materia medica from India

(Jaggi, 1961). During the 17th and 18th centuries, physicians realised that asthma was

due to constriction of the bronchi. In the 1960´s physicians discovered that asthma is

an inflammatory disease.

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24

There is an enormous global burden of asthma with estimated 300 millions

individuals suffering from this disease. Its prevalence varies from 1-18% of the

population in different countries and increases globally by 50% every decade

(Masoli et al., 2004). Worldwide approximate 2.5 lakhs people die from asthma

yearly with a higher mortality in under developed countries. Most of the Asian

countries including India and China, although report a relatively lower prevalence

rates than those in the West, account for a huge burden in terms of absolute number of

patients (Wong and Chan-Yeung, 2005; Aggarwal et al., 2006; Jindal, 2007). The rate

of asthma also increases as communities adopt western lifestyles and become

urbanized. With the projected increase in the proportion of the world's population that

is urban from 45% to 59% by 2025, there is likely to be a marked increase in the

number of asthmatics worldwide in the coming two decades. It is estimated that there

may be an additional 100 million persons with asthma by 2025 (Masoli et al., 2004).

The World Health Organization has estimated that 15 million disability-adjusted life

years (DALYs) are lost annually due to asthma, representing 1% of the total global

disease burden which reflects the high prevalence and severity of asthma. The number

of DALYs lost due to asthma is similar to that for diabetes, cirrhosis of the liver or

schizophrenia. Therefore, asthma represents a substantial burden in terms of medical

costs (hospital and medication), economical costs (Masoli et al., 2004) and social

impact (reduced quality of life, premature deaths, absence from school)

(Thompson, 1984; Masoli et al., 2004).

1.5.3. Burden of disease in India

There is limited data on asthma epidemiology from the developing world

including India (Subbarao et al., 2009). Despite studies suffering from several

scientific drawbacks including lack of uniformity in methodology and analysis of

data, some meaningful attempts have been made. Asthma rates are officially low in

India, although some recent evidence indicates that the true prevalence of the disease

is higher (Aggarwal et al., 2006). The data on asthma available in India revealed that

there is a median prevalence of about 2.4% in adults of over 15 years of age

(Aggarwal et al., 2006) and the prevalence is higher in children. The total burden of

asthma in India at an overall prevalence of 3% is estimated at over 30 million patients.

Among adults over the age of 15, a median prevalence is 2.4% (Aggarwal et al. 2006).

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25

The population prevalence of asthma reported in different field studies is variable and

ranges from 2.4-6.4% [Table 1].

1.5.4. Risk factors and causes of asthma

The factors influencing the risk of asthma can broadly be divided into two

types: One of the factor causing its development and another that triggers its

symptoms that can be further sub-divided into: (i) host-dependent (genetic)

(ii) environmental factors (Barnes, 1995; Busse et al., 2001; Martinez, 2007).

1.5.4.1. Host factors

Asthma has a heritable component and it has been known that multiple

genes may be involved in the pathogenesis of asthma (Holloway et al., 1999;

Wiesch et al., 1999). Genes linked to the development of asthma has focused on four

major aspects: production of allergen specific IgE antibodies (atopy), expression of

airway hyper-responsiveness, generation of inflammatory mediators (cytokines,

chemokines and growth factors) and determination of the ratio between Th1 and Th2

immune responses (Strachan, 1989). Apart from these, obesity and certain mediators

such as leptins affect airway function and causes disease development

(Shore and Fredberg, 2005; Beuther et al., 2006). It showed that male sex is also a

risk factor for asthma in children and prior to the age of 14, the prevalence of asthma

is nearly twice (Horwood et al., 1985).

1.5.4.2. Environmental factors

Although indoor and outdoor allergens are well known to exacerbate

asthmatic conditions but their specific role in the development of asthma is still not

fully resolved. It has also been shown that during infancy a number of viruses have

been associated with the inception of the asthmatic phenotype. The ‘hygiene

hypothesis’ of asthma suggests that exposure of infections early in life influence the

development of a child immune system along with a non-allergic pathway, leading to

a reduced risk of asthma and other allergic diseases.

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26

Table 1: The data of incidence of asthma in urban and rural patients of India

(Source: Murthy and Sastry, 2005a)

Estimated number of patients with asthma (in lakhs)

Males Females Total

Year

Urban Rural Urban Rural Urban Rural

1996 24.18 92.18 22.56 83.42 46.73 175.60

2001 27.15 100.67 24.82 94.76 51.97 195.43

2006 30.76 114.34 28.30 108.34 59.05 222.68

2011 34.57 128.69 32.05 122.89 66.62 251.58

2016 37.30 139.50 35.97 137.99 73.27 277.49

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27

Over 300 substances have been associated with occupational asthma due to

occupational sensitizers which is defined as asthma caused by exposure to an agent

encountered in the work environment (Newman, 1995; Fabbri et al., 1997; Vanables

and Chan-Yeung, 1997; Malo et al., 2004). These are highly reactive small molecules

that may cause alterations in airway sensitivity. Tobacco smoking is also associated

with decline of lung function and increases severity of disease. Exposure of tobacco

smoke both prenatally as well as after birth are associated with greater risk of

developing asthma like symptoms in early childhood. Exposure of environmental

tobacco smoke (passive smoking) increases the risk of lower tract illness in infancy

and childhood (Nafstad et al., 1997). The role of outdoor air pollution in causing

asthma is controversial (ATS, 2000). Hitherto, children raised in a polluted

environment have been found to have diminished lung function (Gauderman et al.,

2004). Similar associations have been observed in indoor air pollution. Diet is also

influencing the asthma risk factor and data revealed that infants fed on cow’s milk or

soy proteins have a higher incidence of wheezing illness in early childhood compared

to those on breast milk (Friedman and Zeiger, 2005). Some data also indicate that

certain characteristics of western diets have increased asthma diseases (Devereux and

Seaton, 2005).

1.5.5. Classification

Asthma is clinically classified according to the frequency of symptoms, FEV1

and peak expiratory flow rate (Yawn, 2008). Asthma may also be classified as atopic

(extrinsic) or non-atopic (intrinsic) based on whether symptoms are precipitated by

allergens or not, respectively [Table 2].

1.5.6. Pathophysiology of Asthma

Asthma is an inflammatory disorder in which inflamed airways and broncho-

constriction results in airway narrowing. Bronchial inflammation or airway

inflammation is a multicellular process involving mainly Th2 lymphocytes,

eosinophils, activated mast cells, neutrophils, macrophages and basophils. Besides, it

also causes narrowing due to edema and also leads to swelling because of an immune

response to allergens (Burke et al., 2003). Airflow limitation in asthma is recurrent

and caused by a variety of changes in the airway.

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28

Table 2: Clinical classification of severity in asthma

Severity Symptom frequency

Night-time symptoms

FEV1% predicted

FEV1% Variability

Use of short-acting β2 agonist for symptom control

Intermittent <1 per week ≤2 per month

≥80% <20% ≤2 days per week

Mild persistent

>1 per week but <1 per day

>2 per month

≥80% 20-30% >2 days/week

but not daily

Moderate persistent

Daily >1 per week

60–80% >30% Daily

Severe persistent

Daily Frequent <60% >30% Several times per day

(Adapted by Yawn, 2008)

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29

Bronchoconstriction: In asthma, the dominant physiological event leading to

clinical symptoms is airway narrowing and a subsequent interference with airflow. In

acute exacerbations of asthma, bronchial smooth muscle contraction

(bronchoconstriction) occurs quickly to narrow the airways in response to exposure to

a variety of stimuli including allergens or irritants. Allergen-induced acute

bronchoconstriction results from an IgE-dependent release of mediators from mast

cells that includes histamine, tryptase, leukotrienes as well as prostaglandins that

directly contract airway smooth muscle. Aspirin and other nonsteroidal

anti-inflammatory drugs can also cause acute airflow obstruction in some patients.

Evidences indicate that non IgE-dependent responses involve mediator release from

airway cells (Stevenson and Szczeklik, 2006). In addition, other stimuli including

exercise, cold air and irritants mediate acute airflow obstruction. The mechanisms

regulating the airway response to these factors are not well defined but the intensity of

the response appears related to underlying airway inflammation. Stress may also play

a key role in precipitating asthma exacerbations. The mechanisms involved have yet

to be established and may include enhanced generation of pro-inflammatory cytokines

(Barnes, 2002).

Airway edema: Disease becomes more persistent with advance stage of

inflammation leading to limit airflow. Furthermore, the additional factors include

edema, inflammation, mucus hyper secretion and the formation of inspissated mucus

plugs as well as structural changes including hypertrophy and hyperplasia of the

airway smooth muscle play contributive role in airway obstruction.

Airway hyper-responsiveness: Airway hyper-responsiveness is linked to

both airway inflammation and remodeling, partially reversible by bronchodilators that

relax the airway smooth muscle. It is an exaggerated bronchoconstrictor response to a

wide variety of stimuli. The degree to which airway hyper-responsiveness can be

defined by contractile responses to challenges with methacholine correlates with the

clinical severity of asthma. The mechanisms influencing airway hyper-responsiveness

are multiple and include inflammation, dysfunctional neuro-regulation and structural

changes.

Airway remodelling: Airway remodelling in asthma includes thickening of

reticular basement membrane, epithelium fragility, hypertrophy of mucus secreting

glands, hypertrophy and hyperplasia of airway smooth muscle and increased

deposition of extracellular matrix. Permanent structural changes can occur in the

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30

airway associated with a progressive loss of lung function that is not prevented or

fully reversed by any of the current therapies. Airway remodelling involves an

activation of many of the structural cells with consequent permanent changes in the

airway that increase airflow obstruction and airway responsiveness. These structural

changes can include thickening of the sub-basement membrane, subepithelial fibrosis,

airway smooth muscle hypertrophy and hyperplasia, blood vessel proliferation and

dilation, mucous gland hyperplasia and hypersecretion (Chung, 2000). Regulation of

the repair and remodelling process is not well established but both the process of

repair and its regulation are likely to be key events in explaining persistent nature of

the disease limiting to a therapeutic response.

1.5.7. Oxidative stress and antioxidants in asthma

Many observations suggest that oxidative stress plays an important role in the

pathogenesis of asthma. Although, it is difficult to get direct measurements of ROS in

asthmatic patients yet recent studies of exhaled gases from asthmatic patients have

shown increased H2O2 (Emelyanov et al., 2001; Ichinose et al., 2000; Ganas et al., 2001)

and NO levels (Kharitonov et al., 1995b). Furthermore, an increase in ROS production

has been found to be inversely correlated with FEV1 (Jarjour and Calhoun, 1994).

Airway inflammatory cells are likely to be the source of these increases. For instance,

airway macrophages from asthmatic patients produce more O2•¯ than those from

control subjects (Calhoun et al., 1992). Besides, antigen challenge increases

spontaneous ROS from airway eosinophils in patients with asthma

(Sanders et al., 1995) while IFN-γ blunts this response in allergic patients, however

circulating inflammatory cells might also be a source. Studies advocate that peripheral

blood monocytes are activated to secrete O2•¯ when IgE binds to membrane receptors

(Demoly et al., 1994). It was also reported that eosinophils isolated from antigen

challenged asthmatic patients after 24 h produce more H2O2 upon challenging with

antigen (Evans et al., 1996). Blood eosinophils and monocytes also produce more

ROS in asthmatic patients as compared to control subjects (Calhoun et al., 1992;

Vachier et al., 1992, 1994). Thus both airway and intravascular inflammatory cells

contribute to elevated oxidative stress in asthma. Multiple investigators have shown

that increases in ROS occur during asthma and are associated with damage to a wide

range of biologic molecules in the lung. Enhanced level of airway isoprostanes

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31

(Montuschi et al., 1999), ethane (Paredi et al., 2000) and urinary isoprostanes

(Dworski et al., 2001) suggest that oxidative stress occurs both at epithelial cell as

well as in endothelial cell membranes. Elevated nitrotyrosine (Hanazawa et al., 2000)

and chlorotyrosine (Wu et al., 2000; MacPherson et al., 2001) levels from airway

lavage samples suggest that proteins are also damaged. Although the consequences of

oxidative modifications to proteins are not well studied; several investigators have

demonstrated diminished activity of proteins such as α1-protease inhibitor

(Gaillard et al., 1992). In addition, steroid therapy has been able to attenuate the level

of H2O2, nitrotyrosine and ethane formation suggesting a correlation between

inflammation and oxidative stress. The increase in ROS during asthma exacerbation

might overwhelm endogenous antioxidant defenses. Although airway glutathione is

increased in asthmatic patients, the ratio of oxidized to reduced glutathione also

increases (Kelly et al., 1999). This increase in reduced glutathione suggests an

adaptive response; however, other airway antioxidants, such as ascorbate and α-

tocopherol, are decreased (Kelly et al., 1999) while SOD activity is diminished in

cells from lavage and brushing samples of patients with asthma (Smith et al., 1997).

Several studies reveal that low blood or dietary antioxidants might be a risk factor for

asthma (Olusi et al., 1979; Powell et al., 1994; Vural and Uzun, 2000), however,

dietary supplementation with vitamins, such as ascorbate, has not been found to be

beneficial (Ting et al., 1983; Kaur et al., 2001).

Thus, oxidative stress has been increasingly recognized as one of the major

factors contributing to the chronic inflammatory process (Barnes, 1990). There is

increased generation of oxidants and lipid peroxidation products in asthma patients

including O2•, H2O2, HOCl and OH• (Bowler, 2004; Caramori and Papi, 2004; Psarras

et al., 2005). The enhanced oxidative stress in asthma is reflected by increased

production of protein carbonyls and isoprostanes in plasma (Rahman, et al., 1996a;

Wood et al., 2000) as well as raised level of NO in exhaled air (Kharitonov et al.,

1994) besides reduction of reduced glutathione in bronchoalveolar lavage (BAL) fluid

(Kelly et al., 1999). Moreover, it was also reported that protein sulfhydryls and total

antioxidant status were found to be decrease in plasma concomitant with increased

SOD activity in BAL cells (Pearson et al., 1991).

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32

1.6. CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD)

COPD has probably always existed but has been called by different names since

ancient times. With advancement in the scientific methods and research techniques,

much work has been conducted to study the nature and cause of the disease. Further,

the quest for knowledge continues to this day and it is still a burning research topic in

the area of medical research. The term ‘COPD’ was firstly coined by Mitchell in the

United States of America (Mitchell et al., 1964). Terms like chronic obstructive

airway disease (COAD), chronic obstructive respiratory disease (CORD), chronic

airway obstruction (CAO) and chronic airflow limitation (CAL) are generally used

and are considered as synonyms of COPD.

1.6.1. World Health Organization (WHO) data

WHO recognises that COPD is one of major health issues and carries much

importance in the matters of public health (WHO, 2008). It is a life threatening

disease, leading to obstruction in normal breathing. The data reveal that

Nearly 210 million people suffer from COPD; out of this, 80 million suffer

with moderate to severe COPD.

COPD is responsible for 5% of total global mortality.

Total deaths from COPD have been projected to increase by more than

30% in the coming years.

It is projected that COPD would be the third leading cause of deaths

globally by 2030 (Lopez and Murray, 1998; Skrepnek and Skrepnek,

2004).

1.6.2. The disease burden in India

The relevant data on COPD in India is inadequate till now. However, it is

assumed that the chronic diseases were responsible for 53% of the total deaths and

also for 44% of DALY lost in India during 2005. Out of this, chronic respiratory

disease account for 7% of deaths and 3% of loss in form of DALYs (Reddy et al., 2005).

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33

The prevalent rates vary between 2 to 22% in men and from 1.2 to 19% in women

(Reddy and Gupta, 2004). A median rate of 5% in men and 2.7% in women has also

been reported (Jindal et al., 2001).

Table 3 summarizes the important field studies published in India during the

last 30 years and table 4 give a data pertaining to the estimated number in lakhs of

patients suffering with COPD, and also severity of the disease, placed under three

categories, (i) mild (ii) moderate and (iii) severe

1.6.3. Definition of COPD

CIBA Guest Symposium published a report highlighting the first attempt to

arrive a consensus definition of the disorders associated with chronic airflow

obstruction (CAO-CIBA foundation guest symposium, 1959). According to global

obstructive lung diseases, COPD is a disease characterized by airflow

limitation/obstruction that is either irreversible or only partially reversible. The

airflow dysfunction is usually progressive, associated with an abnormal inflammatory

response of the lungs to noxious particles or gases. It is a preventable and treatable

disease with some significant extra-pulmonary effects that may contribute to severity

(GOLD, 2010).

It is now  termed ‘syndrome’ by many expert as it is composed of chronic

bronchitis, small  airway disease (bronchiolitis) and emphysema that vary in

proportions between affected individuals. The British Thoracic Society defines COPD

as a chronic, slowly progressive disorder characterized by airflow obstruction

(reduced FEV1 and FEV1/FVC ratio) that does not change markedly over several

months. Most of the lung function impairment is fixed although some reversibility can

be produced by bronchodilator or other therapy (BTS guidelines, 1997). According to

the American Thoracic Society, COPD is a disease state characterized by the presence

of airflow obstruction due to chronic bronchitis or emphysema; the airflow

obstruction is generally progressive, may be accompanied by airway hyper reactivity

and may be partially reversible (ATS, 1995). European Respiratory Society defines

COPD as a disorder characterized by reduced maximum expiratory flow and slow

forced emptying of the lungs which do not change markedly over several months.

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34

Table 3: A summary of important field studies from India on COPD prevalence

Sex COPD

Prevalence (%)

Population

Group

Age

Distribution

(Years) Male Female Male Female

Authors

U.P. (Rural) 30-70+ 629 511 6.7 4.5

Bhattacharya et

al., 1975

Chennai

(Urban)

5-60+ 408 409 1.9 1.2 Thiruvengadam

et al., 1977

Delhi

(Urban) 5-94

552 441 8 4.3 Vishwanathan

and Singh,

1977

New Delhi

(Urban)

3-60+ 1087 1011 8.1 4.6 Radha et al.,

1977

U.P. (Rural) 20-70+ 775 649 9 4.5 Nigam et al.,

1982

Chandigarh

(Urban)

15-65+ 2121 2251 5.5 2.9 Malik, 1986

Punjab

(Urban)

15-70+ 1475 1329 5 2.7

Jindal, 1993

Tamil Nadu

(Rural)

30+ 4857 1329 4.1 2.5 Ray et al., 1995

Bangalore,

Delhi,

Kanpur

>35 18217 17078 5 3.2 Jindal et al.,

2006

(Adapted by Murthy and Sastry, 2005b)

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Table 4: Estimated numbers of patients with COPD (in lakhs) according to

population projection for India

YEAR MILD MODERATE SEVERE TOTAL

1996 75.67 33.28 21.06 130.11

2001 89.92 38.23 24.19 149.35

2006 99.04 43.57 27.57 170.18

2011 112.52 49.49 31.32 193.33

2016 129.30 56.87 35.99 222.16

(Source: Murthy and Sastry, 2005b)

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36

Most of the airflow limitation is slowly progressive and irreversible. This airflow

limitation is due to varying combinations of airways disease and emphysema, and the

relative contribution of the two processes is difficult to define in vivo (Siafakas et al.,

1995). These various definitions are meant to help in understanding the disease

burden, the treatment costs as well as the mortality and morbidity of COPD.

1.6.4. Classification of severity of COPD

The clinical severity of COPD is clinically classified according to the

frequency of symptoms and respiratory function tests (GOLD guidelines, 2008)

[Table 5].

1.6.5. Risk factors of COPD

The major risk factors causes to COPD can be divided into (i) external and (ii)

internal factors.

1.6.5.1. External factors

The most common risk factor contributing to COPD is chronic tobacco smoke

(Rahman et al., 1996a; MacNee and Donaldson, 2002; Buist et al., 2008; GOLD,

2010). The likelihood of developing COPD increases with age and cumulative smoke

exposure. A key mechanism in the pathogenesis of COPD is thought to be an

abnormal inflammatory response in the lungs to inhalation of toxic particles and gases

derived from tobacco smoke, air pollution and/or occupational exposures (Yoshida

and Tuder, 2007). Tobacco smoke which is a mixture of over 4000 chemical

constituents and one of the most important cause, leading to development of COPD.

Amongst males, tobacco smoking is responsible for more than 80% of patients (Jindal

et al., 2001). Both cigarette and bidi smoking is equally responsible (Khan, 2002). A

passive exposure to cigarette-smoke known as environmental tobacco smoke may also

contribute to respiratory symptoms and development of COPD (Smith, 2000; Jaakkola

and Jaakkola, 2002). It adds to an increase total burden of inhaled irritants, organic

dusts and gases in the lungs (Buist and Vollmer, 1994; Dayal et al., 1994;

Leuenberger et al., 1994).

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Table 5: Clinical classification of severity for COPD according to GOLD criteria

Stage Description Spirometry Characteristics

0 At risk Normal FEV1

Normal FEV1/FVC

Chronic respiratory symptoms (e.g. cough sputum production etc.)

I Mild COPD FEV1 ≥80% predicted

FEV1/FVC <70%

With or without chronic cough and/or sputum production

II Moderate COPD 50%≤ FEV1 <80% predicted FEV1/FVC <70%

With or without chronic cough and/or sputum production and shortness of breath; clinical intervention

III Severe COPD 30%≤ FEV1 <50% predicted FEV1/FVC <70%

Continued progression of symptoms and worsening of airflow

IV Very severe COPD

FEV1 <30% predicted

FEV1 <50% predicted with chronic respiratory failure FEV1/FVC <70%

Severe limitation of airflow or the presence of long term respiratory failure

(Source: GOLD, 2008)

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38

Recently, the analysis of the tobacco company research reveals that second hand

cigarette smoke appeared to exhibit greater toxicity and tumorigenicity than the main

stream smoke (Schik et al., 2005; Schik and Glantz, 2006). Hence, for the

pathophysiology of COPD both active and passive smoking is responsible (Fisner et

al., 2005; Jindal et al., 2006; Marsh et al., 2006). It has been reported that at least

50% of smokers develop to COPD (Lundback et al., 2003). The crucial factor seems

to be the amount of smoke and also the extent of inhalation (Lange et al., 1989). The

correlation is so strong and positive that the term ‘Tobaccosis’ and ‘Smoker’s Lung’

are used as alternative names of the disease (Brandt et al., 1997; Cabrera and Perez,

2004).

COPD is also caused by prolonged exposure to occupational dusts and chemicals

(vapours, irritants and fumes) (Krzyzanowski et al., 1986; Humerfelt et al., 1993).

Though independent of cigarette smoking, these can increase the risk of disease along

with indoor air pollution (Kauffman et al., 1979; Amelia, 1998). Experimental models

of in vitro cell culture or explanted trachea have provided proof that the airway

epithelium is capable of taking up diesel exhaust particles by endocytosis (Boland et

al., 1999). Verily, such particles can translocate through epithelial layer into the

underlying submucosa with capability to induce fibrosis in the airway wall (Churg

and Wright, 2002). O3 and NO2 will induce release of a variety of inflammatory

cytokines from the airway epithelium leading to increase in the epithelial

permeability (Bosson et al., 2003). In many developing countries indoor air

pollution from cooking fire smoke is a common cause of COPD, especially among

women (Kennedy et al., 2007). The existing evidence is indicative of the fact that a

poor socio-economic status also constitutes a risk factor for the development of

COPD (GOLD, 2010). Though, the role of this factor is not clearly defined yet this

pattern probably reflects exposure to various causative factors including indoor air

pollution, poor nutrition and/or other factors related to a low socio-economic status

(Higgins et al., 1977; Prescott and Vestbo, 1999). Further, the incidence and

prevalence of bacterial infections have been found to be positively correlated with an

accelerated decline in pulmonary functions in COPD (Donaldson et al., 2002).

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39

1.6.5.2. The internal factors

COPD is a polygenic disease in which deficiency of α-1 anti-trypsin (ATT)

constitutes a risk factor. It is primarily synthesized in liver. Its major function

constitutes protection of the lung such as elastin from neutrophil that mediates

destruction by irreversibly binding and inhibiting elastase (Travis and Salasen, 1989).

Smokers with congenital deficiency of ATT have been found susceptible to

emphysema (Tobin et al., 1983; Sandford and Pare, 2000; Barnes, 2004; Stoller and

Aboussouan, 2005). Genetic factors have also been linked to the development of COPD

that further directed to gender-based differences in obstructive airway diseases which

might be due to the interaction of sex-dependent genetic factors and socio-cultural

differences (Kauffmann and Becklalke, 2000; Blanc, 2003; Wai and Tarlo, 2003). The

airway of patients suffering from COPD secretes an excessive amount of mucus that

contains a complex dilute aqueous solution of lipids and glycoproteins (Reid, 1954).

Salts, enzymes, anti-enzymes, oxidants and antioxidants, antibacterial secretions,

cells/plasma derived mediators and various proteins are also included in its

constituents. All these components collectively act as a protective barrier and neutralize

the inhaled toxic gases. This mucus-mediated clearance is impaired in the patients with

COPD (Wanner et al., 1996). In addition, infections (viral and bacterial) may contribute

to the pathogenesis and progression of COPD (Retamales et al., 2001), besides the

bacterial colonization associated with the airway inflammation (Sethi et al., 2006)

and it may also play a significant role in exacerbations (Seemungal et al., 2001).

1.6.6. Oxidative stress and antioxidants in COPD

An imbalance between oxidants and antioxidants is known to play an

important role in the pathogenesis of COPD (Rahman et al., 1996a;

Repine et al., 1997; Drost et al., 2005; MacNee, 2005; 2007). The sources of the

increased oxidative stress derive from an increased burden of inhaled oxidants and

from the increased amounts of ROS generated by several inflammatory, immune and

structural cells of the airways.  It is well known that the biological systems of lung

airways are continuously exposed to oxidants and ROS are generated either

endogenously by metabolic reactions (mitochondrial electron transport during

respiration, activation of phagocytes) or exogenously (air pollutants, cigarette smoke).

Abundant production of ROS has been directly linked to oxidation of proteins, DNA

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40

and lipids resulting into lung injury or induction of variety of cellular responses

through the generation of secondary metabolic reactive species. ROS may result in

remodelling of extracellular matrix consequently triggering apoptosis, regulation of

mitochondrial respiration and regulate cell proliferation (Richter et al., 1995;

Gutteridge, 2000). Parallel to these, alveolar repair responses and immune modulation

in the lung may also be influenced by ROS. Furthermore, high levels of ROS have

been implicated in initiating inflammatory responses in the lungs through the

activation of transcription factors such as nuclear factor- κ B (NF- κ B) and activator

protein-1 (AP-1); their activation leads to signal transduction and gene expression of

pro-inflammatory mediators (Guyton et al., 1996; Rahman and MacNee, 1998;

Rahman, 2002). It is well proposed that ROS produced by phagocytes that have been

recruited to sites of inflammation are a major cause of the cell and tissue damage

associated with many chronic inflammatory lung diseases including COPD (Hatch,

1995; Dworski, 2000; Rahman et al., 1996a; 1997; 2000).

Some earlier studies on oxidative stress in COPD patients reported a wide

range of antioxidant disturbance. Taylor et al. (1986) reported a disturbance in plasma

antioxidant and FEV1/FVC ratio in patients with COPD. Lipid peroxidation products

measured as thiobarbituric acid reacting substances (TBARS) were reported to

increase in plasma and sputum of healthy smokers and smokers with COPD

(Lapenna et al., 1995; MacNee, 2005). TBARS in plasma have also been shown to

inversely correlate with FEV1 (% predicted) in a population based study, indicating

that lipid peroxidation is associated with airflow limitation (Schunemann et al., 1997).

A number of antioxidant disturbances have been reported in COPD patients but a

notably inconsistency exist. Further, the findings are difficult to compare because of the

different designs of the various diseases (Repine et al., 1997). Some studies report an

increase while others depict a decrease in antioxidant enzymes. Duthie et al. (1991)

reported a decrease GPx activity in smokers and concluded that they were more

susceptible to the LPO. It was also reported that decrease in GPx and SOD activity in

children of parents having a smoking history compared to children of non-smokers.

Conversely, an increase in SOD and catalase activity was also reported in young

smokers (McCusker and Hoidal, 1990). Kondo and co-workers (1994) reported that

an increased superoxide generation by alveolar macrophages in elderly smokers and

was found associated with decrease in antioxidant enzyme activity. It was previously

reported that a remarkable decrease in exhaled NO levels was found in chronic

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41

smokers (Kharitonov et al., 1995a).Certain cigarette smokers had increased GPx

activity in their epithelial lining fluid (Kathawalla et al., 1996). It was reported that

SOD, catalase, glutathione and vitamin E levels were significantly lower in COPD

patients as compared to control (Colak et al., 1995). Rahman et al. (1996a)

demonstrated a low level of plasma antioxidant status in patients with exacerbation of

COPD.

The composition of inflammatory cell types that invade tissues varies widely

in asthma and COPD. It has been suggested that there are differences in the

characteristics of the ROS produced in these diseases (Jeffery, 1998; Saetta, 1999;

Barnes, 2000). This chapter reviews the evidence for the role of ROS in pathogenesis

of asthma and COPD with discussing patho-physiological consequences of increased

ROS released in these conditions. Moreover, it also highlights the antioxidant in place

to protect against damaging effects of ROS.

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42

OBJECTIVES OF THE PRESENT WORK

• Both asthma and COPD are obstructive airway diseases and among one of the

major global health problem with increasing prevalence. Biomarkers of

oxidative stress and airway inflammation is a characteristic and important

hallmark in both diseases and therefore, in the past, investigations focused

strongly on the biochemical aspect of these disorders. Oxidative stress has

been increasingly implicated in the pathogenesis of lung diseases. Antioxidant

mechanisms and their regulation are poorly characterized in human lung, but

these may be important in the development and progression of pulmonary

diseases. Because its role on the pathogenesis and severity of these respiratory

diseases, the present study has been selected.

• In the first part (Chapter II) of this thesis, an effort has been made to evaluate

the level of oxidative stress biomarkers and antioxidants in patients with

asthma and their correlation with severity of disease has also been

investigated. Moreover, the relationship between oxidative variables with

obstruction in airways has also been established.

• In the second part (Chapter III) of this thesis, the level of oxidants and

antioxidants has been studied in COPD patients and an attempt has been done

to correlate their association with the degree of airflow obstruction

(FEV1% predicted). Herein we have also elucidated the role of oxidant-

antioxidant imbalance in disease progression and evaluated their relation with

severity of the disease.

• In the third part (Chapter IV) of this thesis, levels of carbon monoxide and

nitric oxide in exhaled air has been examined for the quantification of lung

oxidative stress in patients with asthma and COPD. The relationship between

these markers with airflow obstruction has also been investigated. In addition,

the effects of cigarette smoke on the oxidant stress by measuring these

biomarkers have also been made.