CHAPTER 2: REVIEW OF LITERATURE - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/5005/10/10_chapter2.pdf · The development of senile cataract is multifactorial in origin and

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CHAPTER 2: REVIEW OF LITERATURE

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The eye lens, which contains perhaps the highest concentration of proteins

compared to any other tissue (Heijtmancik et al., 1995), is the only organ

that has been extensively studied and being studied for over a century. As

early as 1833, Sir David Brewster deduced the fine structure of cod lens

and reported to have 5 million fibre cells, each measuring 4.8 µm. Mörner

(1894) initiated the studies on biochemistry of lens when he described high

concentration of heterogeneous structural proteins in the bovine lens, now

known as crystallins. Hence, the lens has served as a model system in

studying developmental and structural biology and also for understanding

age related diseases.

In this chapter, a review on development of lens and its general anatomy,

senile cataract with reference to their classification, prevalence, etiology,

reactive oxygen species and its role in cataractogenesis, role of antioxidant

enzymes, modulation of antioxidative enzyme and molecular genetic

aspects of superoxide dismutase gene has been attempted.

2.1. DEVELOPMENT OF THE LENS

The ocular lens is a transparent biconvex optical structure in the eye

suspended posterior to the iris and anterior to the vitreous body by zonular

ligaments. At birth, the lens has an equatorial diameter of 6.5 mm and an

anterior-posterior depth of 3.5 mm. This dimension increases to 9 mm and

5 mm respectively by adulthood (Lambert, 1997). The lens is an avascular

organ with a single cell type that follows a regular pattern of development

throughout life (Grainger, 1992). Its organogenesis begins in the fourth

week of gestation. The lens is derived from surface ectoderm which shows

a dish-shaped thickening in the region overlying optic vesicle to form the

epithelial cells of the lens placode. Invagination of the lens placode

produces the lens pit, which closes over to form the lens vesicle. This is

ultimately pinched off from the surface ectoderm (Snell and Lemp, 1989;

Cvekl and Piatigorsky, 1996).

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The posterior epithelial cells lining the lens vesicle lose their nuclei,

elongate and form the primary lens fibers by the seventh week of gestation.

These non-nucleated fiber cells gradually fill the lumen of the lens vesicle

at the center, creating a nearly spherical structure which will eventually

become the embryonic lens nucleus (Kuszak and Brown, 1991). Anteriorly,

the lens vesicle retains a monolayer of cuboidal epithelial cells which

persists throughout life. The cuboidal cells in the germinative zone of

equatorial region continue to divide and differentiate into spindle shaped

secondary fiber cells which are laid down over the primary lens fibers and

form the bulk of the lens (Peyman, 1987).

As the new secondary fiber cells arise, the earlier ones move towards the

nucleus and form more compact central fibers. Thus, a highly ordered

concentric shell of nucleated secondary fibers is arranged in lamellae of

varying refractive index around the nucleus. The density of cellular

organelle decreases and cells become pycnotic as the secondary fiber cell

move inwards and become metabolically inactive. Lines of optical

discontinuity or sutures occur at points where secondary lens fibers come

into opposition (Kuszak et al., 1984).

The lens is surrounded by a semipermeable elastic collagenous capsule that

develops from the deposition of basement membrane. It is first detectable at

5-6 weeks of gestation and continues to thicken throughout life (Kuszak,

1990). The major components of the lens capsule are type IV collagen,

laminin, entactin, heparin sulfate proteoglycan and fibronectin (Cammarata

et al.,1986). The zonular fibers develop from the non-pigmented epithelium

of the ciliary body during the fifth month of gestation (Lambert, 1997).

Successful lens development results in a transparent, biconvex lens that is

divided into the lens capsule, epithelium and the lens substance, that is,

outer cortex containing nucleated fiber cells and an inner nucleus with

compacted non-nucleated central fibers. The nucleus is composed of the

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outer adult nucleus laid down after birth and the inner embryonic and fetal

nuclei present at birth (Garland et al., 1996).

Ultrastructurally, the secondary fiber cells are rich in orgenelles and contain

large amounts of water-soluble protein viz., crystallins and cytoskeletal

proteins such as actin and vimentin. They are connected with each other by

gap junctions and are involved in metabolic, synthetic and transport

processes (Kistler and Bullivant, 1989; Goodenough et al., 1996).

2.2. CLASSIFICATION OF SENILE CATARACT

Cataract is an opacification of the ocular lens sufficient to impair vision.

The development of senile cataract is multifactorial in origin and increases

in incidence with aging. It is usually bilateral and begins either in the

superficial cortex or close to the nucleus of the lens. Based on the location

of the opacity, senile cataract can be divided into Nuclear, Cortical and

Posterior subcapsular cataracts. Pure forms of cataract (with only one type

of opacity present) are found more frequently in the early stages of the

disease, but as the cataract becomes more severe, several types of opacity

often co-exist in the same lens producing the so-called mixed type of

cataract (Ottonello et al., 2000). In terms of prevalence, cortical cataract is

the most common, followed by nuclear and posterior subcapsular cataract.

2.2.1. NUCLEAR CATARACT

Nuclear cataract is defined as the opacification of lens nucleus, which can

impair visual path. Normally nuclear cataract arises as a yellowing of the lens

nucleus, which can be termed as nuclear sclerosis. Some degree of nuclear

sclerosis and yellowing is considered physiologically normal in older adult

patients, and in general, this condition interferes only minimally with visual

function. An excessive amount of sclerosis and yellowing is called nuclear

cataract and cause a central opacity (Fig.2.1a). Nuclear cataracts tend to

progress slowly. They are usually bilateral but may be asymmetric. Nuclear

cataracts typically cause greater impairment of distance vision than of near

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vision. Commonly, in the early stages, the progressive hardening of the lens

nucleus causes an increase in the refractive index of the lens and thus a

myopic shift in refraction. In some cases, the myopic shift transiently enables

presbyopic individuals to read without spectacles, a condition referred to as

“second sight”. In very advanced cases, the lens nucleus becomes opaque and

brown and is called a brunescent nuclear cataract (Fig.2.1b). Upon

maturation these brunescent cataracts may develop in to black cataract.

2.2.2. CORTICAL CATARACT

Cortical cataract is defined as the opacification in the lens fiber regions,

which may results from changes in the ionic composition and subsequent

hydration of the lens fibers. Cortical cataracts also called cuneiform opacities

are usually bilateral but are often asymmetric (Fig. 2.1c). Their effect on

visual function varies greatly, depending on the location of the opacification

relative to the visual axis. A common symptom of cortical cataract is glare

from intense focal light sources, such as headlights of oncoming cars.

Cortical cataracts vary greatly in their rate of progression; some cortical

opacity remains unchanged for prolonged periods, while others progress

rapidly. As the lens continues to take up water, it may swell and is called an

intumescent cortical cataract. When the entire cortex from the capsule to the

nucleus becomes white and opaque the cataract is said to be mature

(Fig.2.1d). A hypermature cataract occurs when there is leakage of

degenerated cortical material through the lens capsule, leaving the capsule

wrinkled and shrunken (Fig.2.1e). A morganian cataract occurs when further

liquefaction of the cortex allows free movement of the nucleus within the

capsular bag.

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2.2.3. POSTERIOR SUBCAPSULAR CATARACT

Posterior subcapsular, or cupuliform, cataracts are often seen in patients

younger than those presenting with nuclear or cortical cataracts (Fig.2f).

Posterior subcapsular cataracts are located in the posterior cortical layer and

are usually axial. In later stages, granular opacities and a plaque like opacity

of the posterior subcapsular cortex are seen. A patient with a posterior

subcapsular cataract often complains of glare and poor vision under bright

lighting conditions; the posterior subcapsular cataract obscures more of the

papillary aperture when miosis is induced by bright lights, accommodation,

or miotics. In addition to being one of the main types of age-related cataract,

posterior subcapsular cataract can occur as a result of trauma, systemic and

topical corticosteroid use, inflammation, and exposure to ionizing radiation.

Posterior subcapsular cataract is associated with posterior migration of the

lens epithelial cells in the posterior subcapsular area, with aberrant

enlargement (Fig.2.1g).

Figure 2.1: Photographs of different types of senile cataract: a) Nuclear b) slit-lamp image of brunescent c) cortical d) mature e) hyper mature f) dense posterior subcapsular (PSC) g) retroillumination picture of PSC h) PSC with plaque.

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2.2.4. CATARACT LENS WITH OTHER ABNORMAL ENTITIES

2.2.4.1. Cataract with plaque

Plaque appeared as a diffuse area with an irregular thickened border or

small multiple island of thickened capsule, results from the migration of

lens epithelial cells through the cleavage either between anterior capsule

and cortical fibers or between posterior capsule and cortical fibers. The

migrated lens epithelial cell may settle themselves on the central or

peripheral capsule and then deposit extracellular matrix proteins and

produce fibrous plaque (Vasavada et al., 1997). The peculiar morphological

features were degeneration and liquefaction of lens cortex, formation of

morganian globules, posterior migration of epithelium, bladder cell

formation and focal calcification. The plaques can be subdivided into

anterior and posterior plaque based on its polarity and central or peripheral

based on its location. The presence of plaque was mostly observed in

posterior subcapsular cataracts (Fig.2h).

2.2.4.2. Cataract with corticocapsular adhesions

Corticocapsular adhesions (CCA), is an adhesion formed between the lens

capsule and the adjacent cortical layer. CCA is also believed to be similar

to plaque in terms of its origin. The formation of CCA takes place when the

mitotically active equatorial lens epithelial cells proliferate and migrate

either to anterior or posterior region of the lens and accumulates its

secretary extracellular glycoproteins in its premises (Vasavada et al., 2003).

2.3. PREVALENCE OF SENILE CATARACT

According to a recent survey the number of blind in India is estimated to be

18.7 million of which 9.5 million were due to cataract (Dandona et al.,

2001). If there is no change in the current trend of blindness, the number of

blind people in India would increase to 31.6 million in 2020 (Dandona et

al., 2001). The prevalence of cataract in India was 43.32 % among the

older individuals aged over 50 and the prevalence increased with increasing

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age (Minassian and Mehra, 1990; Bachani et al., 2000). A study carried out

in three different districts of Tamilnadu in southern India reported that 69 -

72% of blindness was due to age related cataract (Thulsiraj et al., 2003;

Thulsiraj et al., 2002; Nirmalan et al., 2002). A similar study carried out by

Dandona et al., (2001a; 2002) has shown that the prevalence of blindness in

Andhrapradesh, southern India was to be 1.66% of which 40 - 44 % of

blindness was due to cataract. A population based survey done in Rajasthan

in western India showed that 67.5% of blindness was due to cataract

(Murthy et al., 2001). A rapid assessment of cataract blindness carried out

by Limburg et al., (1999) in an urban district of Gujarat, western India

showed that about 16.2 % of blindness was due to cataract. Chatterjee,

(1973) determined the prevalence of cataract in five different regions of

northern India, ranging from dry hot plains to the mountains in the

Himalayan region. It was seen that the overall prevalence was lower in the

mountains than in the plains, apparently indicating that people inhabiting

the plains develop cataract ten years earlier than those inhabiting the

mountains. A similar study conducted in Nepal also showed higher

prevalence of cataract in the plains (4.2%) than in the mountains (1.9%)

(Brilliant et al., 1985).

In general, approximately 25% of the population over 65 and about 50%

over 80 years has serious loss of vision because of cataract and it accounts

for 42% of all blindness worldwide (Kupfer et al., 1994). In cross-sectional

studies, the prevalence of cataracts is 50% in people between the ages of 65

and 74, increasing to 70% in those over the age of 75. Since the population

over 55 is most susceptible to lens opacification, the incidence is expected

to increase 4-fold and it is predicted that there will be 40 million blind due

to cataract by the year 2025 in all over world (Kupfer, 1984).

Blindness due to cataract presents an enormous problem in India not only in

terms of human morbidity but also in terms of economic loss and social

burden (Vajpayee et al., 1999). Since most of the blind people in the country

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are in rural areas where surgical service is least available, a large proportion

of patients in the rural areas continue to remain blind. This situation has

many social implications such as loss of productivity, breakdown of

interpersonal relationships, depressive manifestations, loss of self esteem and

most patients lead an isolated humiliating life (Angra et al., 1997). It was

estimated that the economic burden of blindness in India for the year 1997 is

Rs. 159 billion (US$ 4.4 billion), and the cumulative loss over life time of the

blind is Rs. 2,787 billion (US$ 77.4 billion). The cost of treating all cases of

blindness in India is Rs. 5.3 billion (US$ 0.15 billion) (Shamanna et al.,

1998). Even in developed countries cataract is the leading cause of low

vision, and about 1.3 million cataract operations are performed annually at a

cost of $3.5 billion, which accounts for approximately 60% of the Medicare

budget for vision (Ellwein and Urato, 2002).

In developing countries, since there is less number of surgeons to perform

cataract surgeries and also the cataract patients usually wait until they lose

significant visual outcome, a significant number of people are permanently

blind due to cataract. The combined effect of declining death rates, rapid

increase of older population due to increasing life expectancy and the limited

capacity to cover the increased demand for cataract surgical services cause an

increase in blindness from senile cataract (Foster and Johnson, 1993). It is

apparent that it will not be possible to eliminate the overall blindness caused

by cataract by increasing the number of surgeons and it may not be possible

to keep the total number of people with cataract worldwide from growing.

2.4. FACTORS ASSOCIATED WITH SENILE CATARACTS

Epidemiological studies have increased our knowledge of cataract in

various ways: first defining the size of the problem of cataract, usually

relative to other blinding conditions, then determining prevalence and

incidence in different regions and more recently in identifying risk factors

for cataract.

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Cataract has been suggested to be associated with many diseases such as

diabetes (Caird, 1973), hypertension (Kahn et al., 1977), myopia (Weale,

1980), renal failure (Hollowich et al., 1975; Harding and Crabbe, 1984),

obesity (Weintraub et al., 2002a), hypercholestremia, collagen vascular

disorders, atopy, skin disorders, diarrhea, and also with malnutrition and

poverty. Epidemiological and clinical studies implicating that the senile

cataract arises from exposure to sunlight (Zigman et al., 1979), ultraviolet

radiation (UVR) (Rosmini et al., 1994; Gittinger, 2001), cigarette (West et

al., 1995; Paik and Dillon, 2000) or wood smoke (Shalini et al. 1994), X-

ray (Lipman et al., 1988), infrared, microwave, ionizing radiation and nitic

oxide (Örnek et al., 2003). Some reports also suggested that autoimmunity,

abnormal cell division and oxidative stress could also be responsible for

senile cataract (Bhuyan and Bhuyan, 1978; Giblin et al., 1982; Spector,

1984; Padgoankar et al., 1999).

2.4.1. Human life style factors:

Diabetes was found associated with cataract and in vitro and in vivo

experiments support the view that diabetes is a cause of cataract. The

earlier epidemiological studies reviewed by Caird, 1973), concluded that

diabetes causes a more rapid maturation but may not affect initiation of

cataract. It seems that cataract is not induced by diabetes, but helps in

maturation. Olofsson et al (2005, 2009) has shown in an in vitro and in vivo

model that the diabetes complication enhances the severity of cataract in

superoxide dismutase null mice. The association between cataract and

hypertension was first noted in the Framingham study where the

individuals had elevated systolic blood pressure was found to have cataract

at later stages (Khan et al., 1977). Weale (1980) suggested that lenses of

myopes would be subject to excessive mechanical stress, which could lead

to cataract. Myopia could account for 7% of cataract, having the second

attributable risk after diabetes (van Heyningen and Harding, 1988; Harding

and van Heyningen, 1989). Severe diarrhoea - dehydration with associated

malnutrition, acidaemia, elevated urea and cyanate levels would lead to

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cataract (Harding and Rixon, 1980). Renal failure, another risk factor for

cataract, also causes high levels of blood urea. Both cyanide and

thiocynate concentrations are raised in blood of smokers and to a lesser

extent in renal failure patients (Cailleux et al., 1988). A strong association

has been found between nuclear cataract and smoking (Spector, 1995). The

attributable risk is about 20% and appears to be cumulative, although the

cessation of smoking is thought to substantially reduce the risk. Heavy beer

drinking was found to be associated with a two-fold increase of cataract in

a case-controlled study in Oxfordshire (Harding and van Heyningen, 1988).

Ritter et al. (1993) also reported that consumption of alcohol causes

significant lens opacities through his population based study conducted in

Beaver Dam.

2.4.2. Chemicals and drugs:

Many chemical substances and ions are found to be cataractogenic if they

reach the lens in large amounts. It has been suggested that quinones and

naphthalene may be responsible for a large variety of senile cataracts

(Ogino and Yasukura, 1957). Some exerts their action through oxidative

mechanisms and produce changes similar to those seen in nuclear cataracts.

Napthaquinone catalyzes the auto oxidation of ascorbic acid, leads to

accumulation of H2O2, which in turn may cause cataract. Naphthalene

induced cataracts are characterized by the formation of yellow and brown

proteins as seen in nuclear cataracts. Cataract formation has been reported

following administration of corticosteroids by several routes: systemic,

topical, and subconjuctival e.g., prolonged treatment of eyelid dermatitis

with topical corticosteroids cause cataract. In man, the cataract from patient

who has taken corticosteroids can have a variety of appearances but

posterior sub-capsular and nuclear cataract predominates. The occurrence

of posterior subcapsular cataract is related to dose and duration of

treatment, and individual susceptibility to corticosteroids. However, this

would vary between individuals. In one study of patients treated with oral

prednisone, 11% treated with 10 mg of prednisone daily developed

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cataracts, as did 30% of those receiving 10-15 mg daily, and 80% of those

receiving more than 15 mg per day. In a study of patients receiving topical

corticosteroids following keratoplasty, 505 of patients developed cataracts

after receiving 765 drops of 0.1% dexamethasone over 10.5 months.

Histopathologically and clinically, posterior subcapsular cataract formation

occurring subsequent to corticosteroid use cannot be distinguished from

senescent posterior subcapsular cataract changes. Steroids could induce

cataracts by elevating glucose levels and binding to lens proteins (Harding

and Crabbe, 1984). Glucocorticoid-protein adducts were identified by

radio-immuno assay in lenses of patients treated with steroids - mostly

prednisone (Manabe et al., 1984). Gaps can be found between lens

epithelial cells in the patients treated with corticosteroids (Karim et al.,

1989).

Phenothiazines, a major group of psychotropic medications, can cause

pigmented deposits in the anterior lens epithelium. These deposits appear to

be both dose and duration dependent. The lens changes associated with

phenothiazine use are generally visually insignificant. Although visually

significant cataracts have been reported relatively commonly in elderly

patients receiving topical anticholinesterases, progressive cataract

formation has not been reported in children treated with echothiophate for

accommodative esotropia. Long-acting anticholinesterases such as

echothiophate iodide and demecarium bromide can cause cataracts.

Usually, these cataracts first appear as small vacuoles with in and posterior

to anterior lens capsule and epithelium. The anticholinesterase cataract may

progress to posterior cortical and nuclear lens changes as well.

Amiodarone, an antiarrhythmic medication, has been reported to cause

stellate anterior axial pigment deposition. Noxious substances have

basically interfering with the optical clarity of the lens. These substances

may act at the lens surface to change the passive permeability

characteristics of the membranes. For example, long acting cholineesterase

inhibitors used in the treatment of glaucoma produce lens opacities

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(Axelsson, 1973). The drug tri-paranol, which is used to treat

hypercholesterolaemia, is highly cataractogenic. A wide range of drugs and

harmones are known to alter ion permeabilities. Any substance that

increases sodium permeability is likely to be potentially cataractogenic.

Certain drugs which are administered for some systemic and chronic

diseases found to reduce the proliferative activity of cells in the target

organ. Rajkumar et al. (2001) reported that the anticancer agents

Cleistanthins inhibit the proliferative activity of tumor cell lines. Thus

administration of such drugs would affect the normal cell viability by

increased cell death, which would ultimately lead to disturbance in cellular

homeostasis. Women taking the anticancer drug tamoxifen to prevent or

treat cancer have a higher risk of developing cataract especially posterior

subcapsular cataracts. It was reported that systemic and topical steroids are

found to be a significant risk factors for posterior subcapsular cataract

(Hodge et al., 1995).

Hypertension and diuretic consumption did not appear as risk factors in

Oxford but the graded properties of different diuretics did emerge and with

a similar sequence to that found in Edinburg (Harding and van Heyningen,

1988). Diuretics may raise urea levels and thus contribute to these

differences but when all diabetics and individuals receiving diuretics were

excluded a relationship between high plasma urea and cataract remained.

The only significant association of individual diuretics was an apparent

protective effect by cyclopenthiazide and a risk associated with

spironolactone, which is a steroid. There was no significant association of

particular sites of opacity with diuretic use (Cuthbert et al., 1987).

Ionic imbalance has been reported in many human cataracts. Sodium

(Harding and Crabbe, 1984), calcium (Bunce et al., 1984) and selenium

(Ostadalova et al., 1978) increase with increasing severity of cataract. The

increased calcium may activate proteolysis in the lens, as several lens

proteases require calcium. Fragmented crystalline and membrane proteins

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have been found in selenite induced cataract (Shearer et al., 1987, David et

al., 1988). One scenario described for selenite-induced cataract is that

calcium rises only in the nucleus (Shearer and David, 1983) leading to

activation of calpain II and thus nuclear cataract (David et al., 1987,

Hightower et al., 1987). Other data showed that calcium levels increased

rapidly in cortex as well as in nucleus (Hightower et al., 1987) almost to

concentrations required to activate calpain II (David and Shearer, 1986).

The calcium levels inside the human lenses rose about 9 mM from 0.5 mM

and concentrations above 2 mM were invariably associated with discrete

sub capsular opacification. In very advanced cataracts, calcium may

accumulate in sufficient amounts to form crystals of calcium oxalate (van

Heyningen, 1972). Indeed an elevated sodium level was found in 67%

cataracts, most of which had nuclear as well as cortical changes

(Marcantonio et al., 1980). The overall potassium level falls in complete

cortical cataract (Maraini and Mangili, 1973).

2.4.3. Radiation:

In geographic locations where the UVR components of sunlight are more

intense, cataracts occur at an incidence higher than that in locations where

UVR components are less dominant (Zigman et al., 1979).

UVR encompasses wavelength ranges between 1 and 400 nm. It is

commonly divided into four parts according to wavelength: the 1-100 nm

band is mostly described as ‘far UVR’ or extreme UVR’. The other bands

are UVC (100-280 nm), which is blocked by the ozone layer; UVB (280 –

320 nm), of which the portion from 290 nm and below is impeded by the

cornea and UVA (320 – 400 nm), which penetrates the cornea. Higher

wavelength penetrates much greater and lens absorbs maximal radiation

(Boettner and Wolter, 1962; Zigman, 1986). The absorbed radiation is an

important factor in causing lens damage through photooxidation and

subsequently leading to cataract formation.

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Photo-oxidation of a protein molecule can take place either through the

direct absorption of radiation by that molecule or through an indirect

process involving a photo sensitizer. In the direct route, light is absorbed

by chromophores in the proteins predominantly the aromatic amino acids,

tryptophan and tyrosine. These aromatic amino acids absorb light

predominantly at wavelengths below 300 nm. Since the cornea would

normally filter out most light below 300 nm, very little of these

wavelengths would reach the lens. Furthermore, the little that does pass the

cornea would be expected to absorb in the anterior epithelial layer of the

lens. The epithelium was disturbed and multiple epithelial layers formed,

and at later stages cells clumped together. So, a more likely route of protein

modification would be through photosensitized reactions involving near

UV light (300-450 nm). Light of these wavelengths is transmitted by the

cornea and is absorbed maximally by the lens where it could activate a

photosensitizer molecule like riboflavin.

In general, UV radiations causes cross linking of lens proteins (Dillon et

al., 1989). UVA irradiation, possibly induce elevation of active oxygen

species, concomitant with decreased activities of antioxidative enzymes. It

may result in gradual oxidation of membrane proteins and lipids, thereby

compromising membrane structure and function. Several publications have

reported elevation of hydrogen peroxide (H2O2) and singlet oxygen (O2.-) as

a result of UVA exposure (Linetsky et al., 1996; Linetsky and Ortwerth,

1996; Giangiacomo et al., 1996). UV radiation affects not only the

structural protein of the lens and other ocular tissues but affects important

enzymes that catalyses bio-chemical reaction and DNA in the ocular

tissues. Since UVA is suggested to traverse cornea and reach lens epithelia,

the lens epithelium might well harbor the first target of UVA damage that

later expands to inner lens components, possibly serving as a trigger

leading eventually to cataract formation. Sidjanin et al. (1993) monitored

cell survival following induction of cultured rabbit lens epithelial cells with

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18J/cm2 UVA exposure and found that exposure to UVA causes DNA

strand breaks, which triggers the cells to undergo death.

Several studies suggest that UVA radiation (320 to 400 nm) did not

develop opacities, whereas UVB radiation (290 to 320 nm) causes

opacities. But, Dillon et al. (1999) claimed that old human lens proteins

absorb two orders of magnitude more UVA and visible light than UVB.

Ecological investigations of individuals living in climates with varying

degrees of UVB radiation (285-315nm) indicate that a strong positive

association exists between the prevalence and severity of cataract and

ambient UVB exposure. Measurements of the exposure of individuals in

cohort and case-controlled studies have shown that UVB light is a major

risk factor for cortical cataract and to a lesser extent, posterior subcapsular

cataract. Though studies have been done for several decades to elucidate

the role of UVR in cataractogenesis, still limited data is available with

regard to the specific wavelengths responsible for cataract formation. It is

suggested that UVA traverses the cornea more efficiently than UVB and

may therefore contribute to a greater extend to the damage created in the

lens. On the other hand, UVB wavelength is shorter and more energetic and

therefore, a smaller amount of its radiation has been suggested as capable

of being harmful in initiating damage to the lens. UVB is considered to be

an important risk factor for cortical cataract (Hodge et al., 1995). However,

the controversy still exists regarding the wavelength responsible for

cataractogenesis.

Cataracts due to ionizing radiations have been found in human being

following accidental exposure or as a result of therapeutic administration of

radiation. Nowadays exposure of human being to ionizing radiation such as

X-ray is inevitable, since it has more importance in diagnostic field. X-rays

induce aggregation, insolubilization, deamination, and disulphide formation

in proteins and fragmentation of other macromolecules. It was found that

X-irradiation fragments epithelial cell nuclei and releases acid phosphatase

and presumably other lysosomal enzymes in the damaged cells (Brogli and

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Worgul et al., 1985). Fragmentation of epithelial cell nuclei and cell

damage leads to decreased mitosis, and loss of glutathione synthesis,

followed by loss of ATP, glutathione reductase and aldehyde

dehydrogenase. This cumulative effects then cause protein damage,

epithelial damage, swelling, cortical and nuclear opacification (Harding and

Crabbe, 1984). Opacities first appear in the equatorial sub capsular region,

then posterior sub capsular region followed by vacuolization of the cortex,

dense nuclear opacity and finally complete opacification (Lipman et al.,

1988). These changes could be due to direct effect of the radiation on the

protein or to an indirect mechanism. The indirect effects of ionizing

radiation on proteins are due to the reactive intermediates formed as a result

of the radiolysis of water. In many respects, radiation damage resembles

the effects of oxygen toxicity because of the involvement of oxidizing free

radicals.

Microwave radiation is non-ionizing and there is no incidence for

microwave-induced cataract. But in experimental system it has been proved

that repeated sub threshold dosage of microwave radiation could produce

opacification. The bio-chemical changes include decreased concentrations

of ascorbate and glutathione, decreased DNA synthesis and mitotic activity

followed by lowered Na, K-ATPase and consequent ionic imbalance

(Harding and Crabbe, 1984, Lipman et al., 1988). Pulse microwave

radiation as used in radar and communication systems is more hazardous

than continuous radiation. A few studies suggested that microwave and

infrared (glass blowers cataract) cause cataract (Bouchet and Marsol, 1967;

Zaret et al., 1976). Radioactive material like plutonium can also cause

cataract (Griffith et al., 1985).

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2.4.4. Genes and genetics:

Human lens consists of different types of structural proteins, which are

framed in a highly ordered arrangement to maintain lens transparency and

cellular house keeping proteins to maintain cellular homeostasis. The

normal functioning of these structural and house keeping proteins are

controlled by the genetic components in the human genome. When the

genetic components get disturbed or altered the whole molecular

architecture would be distorted, which can result in opacification of lens.

Since these alterations are considered to be age-related phenomena, the

normal synthesis of housekeeping proteins would be obstructed as the

individual ages.

It is likely that genetics would also have certain role in mechanism of age-

related cataractogenesis since it involves a well-established series of

developmental stages. Genetic studies of senile cataract are in their infancy

because most of the familial congenital cataracts are associated with the

single abnormal gene where as senile cataract is likely to be multifactorial

and therefore, determined by a number of different genes and

environmental factors. A small number of genes have been reported to be

associated with senile cataract in limited populations. However, advances

in molecular biology and genetics have greatly accelerated elucidation of

the genetic contribution to age-related cataract. Epidemiological studies

have documented tendencies for cataracts to occur more frequently in

relatives of cataract patients than in the general population. Genetic studies

have demonstrated contributory roles of some specific genes in age related

cataract in small population, and molecular studies have shown changes in

expression of specific genes in cataractous lenses (Heijtmancik and

Kantorow, 2004). Seland, (1974) reported that the up or down regulation of

extracellular glycoproteins such as collagen, oesteonectin (Norose et al.,

1998; Kantorow et al., 2000) are responsible for cataractogenesis. Besides,

over expression of vimentin (Li et al., 1995) and presenilin (Frederikse and

Zigler, 1998) are also found to be involved in senile cataractogenesis.

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23

Recently, a transversion mutation in lens integral membrane 1 gene has

been found to play role in age related cataract (Pras et al., 2002). Though

the genetic role of galactokinase (Okano et al., 2001) and glutathione S-

transferase (Sekine et al., 1995) in cataractogenesis have been reported in

Japan, its impact been modest, and these findings have generally not been

replicated in other populations (Alberti et al., 1996). A new locus for

autosomal recessive progressive and age-related cataract has been mapped

to chromosome 9q13-q22. Analysis of this locus would provide insight into

the cause of the more common sporadic form of age-related cataract (Heon

et al., 2001). Though known Mendelian-inherited forms of congenital

cataract provide several potential candidate genes for senile cataract

(Congdon, 2001) the variation in phenotype and age of onset cataract in

aged individual hinders the applicability of these candidate genes in senile

cataracts. A practical difficulty in studies of the genetics of cataract as with

all age-related diseases is that unaffected status at the time of examination

may mean that the individual is truly unaffected or that he or she has

simply not manifested the phenotype yet.

Congdon et al., (2004) made an attempt to evaluate the heritability of

nuclear cataract in a cohort of older sibships recruited through the Salisbury

Eye Examination (SEE) on Maryland’s Eastern Shore and identified that

genetics play a significant role in nuclear cataract. Moreover, this result

was consistent with previous population-based investigations (Heiba et al.,

1993) and twin studies (Hammond et al., 2000). Several independent lines

of investigation in different populations have supported the heritability of

nuclear cataract. Hammond et al. (2000) reported a heritability figure of

48% in their twin study of nuclear cataract, and Heiba et al. (1993)

estimated that a single major gene may account for 35% of nuclear cataract

variation in the Beaver Dam Eye Study population. Meanwhile, the

heritability factor for cortical cataract was accounted to 50 percent (Heiba

et al., 1995; Hammond et al., 2001). Recently, a new locus on chromosome

6p12-q12 for age-related cortical cataract has been identified. This new

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24

locus has been already implicated in congenital cataracts (Iyengar et al.,

2004).

2.4.5. Hormonal deficiency:

Growing knowledge in hormonal regulation of several age-related

processes widens the research focus to elucidate its exact role in prevention

of certain age-related/ gender specific diseases. Melatonin, a hormone,

which is secreted by pineal gland, has been reported to have antioxidant

property. Abe et al. (1994) showed a potent inhibitory effect of melatonin

on cataract formation in new-born rats. It clearly showed that melatonin

acts as cataract preventing agent by inhibiting the damaging effect of free

radicals. Thus age-related reduction in secretion of such hormones would

affect certain protective roles in human beings. It was reported that the

incidence of cataract in postmenopausal women is higher than in age-

matched men. This leads to the notion that the absence of estrogen may

contribute to the increased risk of cataract (Shibata et al., 1994).

Studies using tissue culture and animal models also suggest beneficial

effects of estrogen in lens. In a lens culture system estrogen protected

lenses against cataracts induced by transforming growth factor (TGF) -β

(Hales et al., 1997). In a recent study carried out by Wang et al. (2003)

showed protective effect of estrogen against oxidative stress.

2.4.6. Reactive oxygen species and oxidative stress:

One of the paradoxes in nature is that oxygen, which is necessary for the

survival of a respiring organism, is also toxic for it. Exposure of an

organism to oxygen tension in excess of those normally encountered,

results in oxygen poisoning. This is generally followed by death of the

organism. In early stages of development of the earth, life essentially

developed in the absence of oxygen. However, with the evolution of life

forms that produced oxygen, allowed gradual increase in content of oxygen

in the atmosphere and organisms either began to develop defenses against

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25

oxygen poisoning or sought environmental niches in which little oxygen

was present. However, there are some significant advantages of oxygen for

living being for generation of energy during synthetic and degradative

metabolic reactions. Meanwhile, the generation of reactive oxygen species

(ROS) as a result of such metabolic reaction (Lee et al., 1998) and damage

due to it should also be considered.

2.4.6.1. Generation of reactive oxygen species as normal cellular

counterparts and its function

Oxygen in its ground state (i.e., triplet 3O2) is unreactive and reduction of

oxygen takes place by addition of electrons and it leads to the formation of

hydroperoxide (HO2•), hydrogen peroxide (H2O2), hydroxyl radical (HO•),

superoxide anion (O2.-) and singlet oxygen (O.-) (Halliwell and Cross,

1994), which are termed as reactive oxygen species (ROS).

Super oxide is the anion formed from the ionization of hydroperoxide. The

hydroperoxide can convert into super oxide anion and vice versa. Super

oxide in the cell causes peroxidation of unsaturated fatty acids. Protonated

superoxide, i.e., HO2 is more reactive than O2.- causing peroxidation of

polyunsaturated fatty acids (Bielski et al., 1983; Aikens and Dix, 1991).

Fortunately, little HO2 is found at physiological pH. Having a short half-

life, O2.- must react at the site at which it is generated or a short distance

from its origin. It can also act as a reducing agent and a chain propagating

radical in a number of auto-oxidations.

O2 + e- + H+ → HO2•

HO2• + e- + H+ → H2O2

H2O2 + e- + H+ → OH• + H2O

OH• + e- + H+ → H2O

HO2•→ O2

•-

O2•- + O2

•- + 2H+ → H2O2 + O2

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26

Unlike superoxide, hydrogen peroxide is relatively stable, easily moves

through cell membrane and affects sites far from its origin. H2O2 is the least

reactive of the intermediates of oxygen reduction but it is a strong oxidizing

agent than O2.-, being able to oxidize thiols such as cysteine and sulphides

such as methionine. Certain key enzymes, which require thiols in their

active centers, can be inactivated by H2O2. A potential source of H2O2 is

through auto-oxidation of ascorbic acid. So, potentially toxic levels of H2O2

could be generated if there was a decrease in mechanisms for removing

H2O2.

Under conditions where H2O2 doesn't accumulate, it is likely that the toxic

effects of H2O2 are due to its ability to undergo the Haber-weiss

dismutation with O2.- to generate hydroxyl radicals (OH. ). It can very

readily abstract H+ from a variety of substances to generate reactive free

radicals. OH is a highly reactive species interacting with almost anything in

its immediate vicinity, thus, OH can react with lipids, sugars, proteins and

nucleic acids (Halliwell and Cross, 1994). When OH reacts with DNA, for

example, it attacks at the deoxyribose and produces a variety of products,

and some of which are mutagenic. It can also add on to an unsaturated bond

of the purine and pyrimidine bases forming radicals that may react with

oxygen to form peroxyl radicals. These reactions can cause severe damage

to the DNA leading to strand breaks and products, which cannot be

repaired. These radicals can then decay in several ways to form oxidation

products.

Hydroperoxide and hydroxyl radicals are considered to be highly reactive

than hydrogen peroxide and these intermediates are probably responsible

for the toxicity of oxygen. It has also been suggested that peroxinitrite,

arising from NO reacting with O2.- may decompose to give hydroxyl radical

(Beckman et al., 1990).

ROS is also considered to be responsible for both the arrest of growth and

the start of cell differentiation. Low levels of ROS may be beneficial or

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even indispensable in process such as intra cellular signalling (Schulze-

Osthoff et al., 1997), cell proliferation or apoptosis (Vogt et al., 1998),

immunity (Sun et al., 1998) and defense against microorganisms (Lee et

al., 1998). In contrast high doses and/or inadequate removal of ROS result

in oxidative stress (OS), which may cause severe metabolic malfunctions

and damage to biological macromolecules (Lledias et al., 1998).

OS can be defined as the state where the overall generation of ROS exceeds

the overall antioxidant defenses (Sies, 1985). When there is a significant

increase in ROS, the OS is recognized throughout the cell and then,

depending on the extent of the stress, the cell may recover, be seriously

modified, or die. With the weakening of the antioxidative defenses, OS

may increase and exacerbate the development of various diseases such as

cancer, hypertension, diabetes, atherosclerosis, inflammation, premature

ageing and cataract (Miller et al., 1993). The striking feature is that with

almost every type of cataract, oxidation is a relatively early event. This is

probably due to the age-dependant decline in the defenses against oxidative

stress as well as the increase in oxidative stress (Harding, 1970; Dovrat and

Gershon, 1981; Rathbun and Bovis, 1986a; 1986b).

Role of oxidative stress for the development of cataract was assessed by

repeated exposure of animals to hyperbaric oxygen. After prolonged

exposure to hyperbaric oxygen the animals developed opacity in lens and

mitotic abnormalities in their lens epithelial cells (Schocket et al., 1972).

The same phenomenon was observed in human beings where patients

treated with hyperbaric oxygen, for 2h at 200-250 kPa and 150-850

exposures, for persistent leg ulcers. The patients survive well but half of

those with clear lens nuclei before treatment developed nuclear cataract

with decreased visual acuity during treatment and most of the others

develop some turbidity (Palmquist et al., 1984). The changes seen

following exposure to hyperbaric oxygen appear to be due to OS by the

generation of ROS.

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ROS, such as H2O2 are capable of modifying proteins in lens by the process

of oxidation, when its concentration rose beyond normal level. It is

identified that the concentration of H2O2 required for such oxidation are

higher than those normally found in lens (0.02-0.06 mM in the vitreous and

aqueous humors) (Bhuyan and Bhuyan, 1977). Since the capsule is

permeable to H2O2 (Pirie, 1965, Fukui, 1976) it is possible that H2O2 could

enter the lens from the aqueous humour and cause oxidative changes

(Spector and Garner, 1981). The oxidative modifications of number of

cellular constituents are thought to result in lens opacification (Garadi et

al., 1987; Padgoankar et al., 1989, 1999; Reddy et al., 1980; 1984).

Hydroxyl radicals are also be involved in some of the oxidation in the lens.

A growing perception is that the changes induced by OS (Kinoshita, 1986),

hyperglycemia, and glycation (Lyons, 1992), in protein lead to an increased

susceptibility to oxidative damage (Baynes, 1991). This is manifested by

increased activity of aldose reductase by decreasing NADPH. NADPH is

found to disrupting the reductive reactions, which maintain key

antioxidative components such as glutathione (Kinoshita, 1986). Together

with the increased aldose reductase activity, sorbital dehydrogenase in

some tissues is stimulated, leading to increased levels of NADH. Increased

NADH levels are believed to stimulate prostaglandin H2 synthesis, leading

to free radical production (Smith, 1986). There is evidence that increased

glucose concentrations may inhibit antioxidant enzymes such as superoxide

dismutase (SOD), catalase and glutathione peroxidase (Giugliano and

Ceriello, 1996). If true, such inhibition would critically compromise the

antioxidative defense of the affected tissue. In normal lens the oxidants are

probably reduced to harmless levels by a variety of protective enzymes

involved in scavenging mechanisms. A decrease in the activity of one or

more of these enzymes could lead to increased concentrations of oxidants

and hence cataracts.

Since majority of etiological factors described above are associated

indirectly to the process of cataractogenesis and moreover the information

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are not sufficient to interrelate the risk factor to particular cataract, the

possible role of gene and genetics have to be elucidated in senile cataract.

However, the majority of etiological factors among systemic disorder,

drugs, radiation are found to play role in cataractogenesis via oxidation and

oxidative stress by generating reactive oxygen species, which in turn induce

antioxidants and antioxidative enzyme genes. It was suggested that the

candidate genes that contribute to senile cataract include not only those

capable of causing congenital cataracts but also may be genes encoding

enzymes that protect the lens from oxidation or other types of

environmental insults (Heijtmancik, 1998; Ottonello et al., 2000). A more

likely scenario is that genetic studies of cataract will eventually yield

knowledge of the protein pathways involved in lens opacity, so that

discovery of anti cataract agents may proceed in a rational fashion, rather

than through the current process of hit or miss. Besides, the challenge now

is to identify ‘senile cataract genes’ with the future possibility of targets for

intervention through gene therapy after a strong genetic component is

established. Thus, dissecting the role of antioxidative enzymes and its

genes may provide significant hint to determine the etiopathogeneis of

cataractogenesis.

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Figure 2.2: Flow chart of effects of reactive oxygen species

2.5. ANTIOXIDANT ENZYMES

With the advent of oxygen rich atmosphere, it was necessary for any

aerobic organism to develop defenses against the primary oxidative stress

components. Our nature has provided us with a wide array of non-

enzymatic and enzymatic anti-oxidants to serve as a defense system to

protect cells against reactive oxygen species and oxidative stress (Reddy et

al., 1980) by detoxifying the most prevalent oxidants. There are basically

two classes of free radical scavenging defense enzymes. Phase I enzymes

comprise of superoxide dismutase, catalase, glutathione peroxidase and

glutathione reductase and Phase II enzymes comprise of quinone reductase

and glutathione S-transferase. Since phase I enzymes are actively involved

in scavenging reactive oxygen species evolving during normal and

Regulate cell growth

cell differentiation death by apoptosis

and necrosis

Activation of signal transduction

cell proliferation

Decreased efficiency of DNA polymerase

DNA repair

Modulates stress induced proteins

and genes

Oxidative damage to

proteins Induce lipid peroxidation

Chemical changes in bases

Change in DNA conformation

Enhancement of “hot spot”

mutagenecity

Modified H-bonding

Block in replication

Inaccurate replication

Mutation

ROS

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pathological conditions, research in these enzymes holds much importance

than other enzymes.

2.5.1. Mechanism of phase I defense enzyme

Superoxide anion is degraded by superoxide dismutase (SOD) in a

dismutation reaction yielding H2O2 and O2. It is interesting that nature

chose to eliminate O2..- by a reaction in which the less reactive but

potentially dangerous H2O2 is formed. In animals, H2O2 is detoxified by

two different enzyme systems namely catalase and glutathione peroxidase.

Catalase protects cells from hydrogen peroxide generated within them and

it reacts with H2O2 to form water and molecular oxygen. Even though CAT

is not essential for some cell types under normal condition, it plays an

important role in the acquisition of tolerance to oxidative stress in the

adaptive response of cells (Hunt et al., 1998). Glutathione peroxidase

(GSHPx) has selenium in the active center and the enzyme is found

predominantly in the cytoplasm of the cell. Glutathione peroxidase

catalyzes the reduction of a variety of hydro peroxides (ROOH and H2O2)

using GSH, thereby, protecting mammalian cells against oxidative damage.

The enzyme effectively metabolizes H2O2 at low concentrations but

requires high concentrations of glutathione (GSH) for optimal activity. This

enzyme can be coupled with glutathione reductase system to regenerate

reduced glutathione. All these enzymes are concentrated in the epithelial

layer.

2.5.2. Antioxidant enzymes in lens

Lens epithelium plays an important role in the maintenance of normal

physiology, metabolic activity, homeostasis and protecting lens from

different diseases (Reddy, 1979, 1990; Ikebe et al., 1989; Giblin et al.,

1985, 1987). It is stated that a variety of etiological factors and

environmental insults can induce the generation of ROS, which in turn

threaten the reducing environment normally maintained in the lens and lens

epithelial cells by oxidative stress (Spector, 1995).

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In non-cataractous conditions highest activity of all defense enzymes in

lens epithelial layer were identified using animal models. However,

constant decrease in activity throughout the remainder of the lens was

observed with increase in age. The decline was thought to cause by damage

to enzymes, which could not be re-synthesized, especially in central parts

of the lens. Among all anti oxidant enzyme, superoxide dismutase forms

the first line of defense and followed by catalase and glutathione

peroxidase. It has been suggested that glutathione peroxidase provides the

major line of defense against endogenous H2O2 and catalase protects the

lens from exogenous H2O2 such as that generated by auto oxidation of

ascorbate in the aqueous humour in the lens (Pirie, 1965).

Superoxide dismutase is considered as a primary defense enzyme among all

antioxidant enzymes against reactive oxygen species. The enzyme has been

demonstrated in a number of ocular tissues from various species and also in

blood of different individuals between 50 and 93 years of age (Table 2.1).

Its activity in the lens is considerably lower than that found in other parts of

the eye such as the iris or ciliary body (Bhuyan and Bhuyan, 1978) and also

the level of this enzyme was found to decrease with age (Casado et al.,

1998). SOD activity was assayed in normal and cataractous lenses. In

normal whole human lenses, SOD showed no significant difference in

activity during aging. However, SOD activity in both nucleus and equator

decreased with increasing age. SOD activity was significantly lower in

human lenses with mature cataract than normal clear lenses (Ohrloff and

Hockwin, 1984).

The SOD activity of the human lens is about twice that of the rabbit and

calf lens (Bhuyan and Bhuyan, 1978). The cortex of rabbit and calf

contains about 1 unit/mg protein and the nucleus about 0.2 unit/mg protein

(Fecando and Augustein, unpublished). These levels might reflect a greater

requirement for protection against superoxide ion in the human lens than in

the lenses of other species. A precipitous decrease (70%) in SOD activity in

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lens and two-fold increase in the blood of nuclear cataract patients was

found (Delcourt et al., 1999). This is especially marked in the nucleus

where SOD activity drops to about 0.03 units/mg protein in posterior

subcapsular cataracts and also 25% decreases in cortical region. Cu/Zn-

SOD null mouse lenses showed a doubled basal superoxide concentration

and were more prone to develop photochemical cataract with more

opacities and more hydration than lenses from wild type mice. Therefore

Cu/Zn-SOD is an important superoxide scavenger in the lens, and it may

have a protective role against cataract formation (Behndig et al., 2001).

Though SOD activity is lost mainly from the nucleus the enzyme antigen

persists in some mature cataractous lenses (Scharf and Dovrat, 1986). This

indicates that the enzyme has become inactivated but not removed. Since

many of the proteinases identified in other tissues are perhaps absent from

the lens nucleus, it is not been removed. This supports the hypothesis that

alteration in genes due to mutations may influence the expression of active

product.

The enzymes of glutathione metabolism and changes in their activities have

been studied extensively in the human lens (Rathbun and Bovis, 1986a).

Glutathione peroxidase (GPx) levels were very low in the young lens,

increase to a peak at about 15 years of age and declined slowly thereafter.

However, a less degree of decrease in glutathione peroxidase activity in the

nuclear region of lens was found at the onset of nuclear cataract. Man has

considerably higher levels of glutathione reductase than most other species

(Harding, 1973) and the levels remain almost constant throughout adult

life. It was observed that glutathione reductase is not decreased with aging

in human lens nor in most human cataracts (Harding, 1973; Rogers and

Augusteyn, 1978). In another comparative study between nucleus and

cortical region of different cataracts, it was observed that glutathione

reductase was significantly lower in nucleus of cortical and posterior

subcapsular cataractous lenses. Glutahione S-transferase also maintains a

constant level through out adult life (Rathbun et al., 1986b). The activities

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of catalase have been measured in the cortical and nuclear sections of

human cataractous lenses and no changes were observed in the activity of

catalase with the progressive development of cataract. Insofar as these three

enzymes of glutathione metabolism and catalase form part of a protective

system in the lens there does not appear to be any significant weakening of

these defenses with age.

It is suggested that the inactivation or modulation in activity of these

antioxidant enzymes may result in elevation of the H2O2 and O2.- levels in

the lens and that this may be responsible for the oxidative modification of

the lens proteins observed in the cataracts. Since the cataract results from

the oxidation of protein it is assumed that the antioxidative enzymes are

inhibited at the level of either transcription or after translation. There are

some evidences for the inhibition of enzymes after translational process,

but no studies have been done at the transcriptional levels. Any mutation in

antioxidant enzyme genes will affect the normal synthesis of enzymes and

in turn it increases the level of ROS in the lenticular area, which in turn

oxidize the proteins of lens.

“Before deciding that any of the losses of enzymatic activity are important

in the aetiology of cataract, it is helpful to know if they occur in all

cataracts and at what stage of cataractogenesis” – Harding J.J..

Through several in vitro and in vivo studies, it was found that SOD enzyme

activities are reduced in most of the cataractous lenses. Thus being the first

and most important line of antioxidant enzyme, SOD would be the most

probable candidate for analysing the genetics of cataracts by its molecular

modulation in different age related cataracts.

2. 6. MOLECULAR MODULATION OF SUPEROXIDE

DISMUTASE

Superoxide dismutase was previously known as indolephenol oxidase. The

appearance of SOD (EC 1.15.1.1) enzymes was triggered by the

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proliferation of photosynthetic organisms that began to produce oxygen

about 2 billion years ago. A variety of antioxidant enzymes evolved to

neutralise the toxic effects of subproducts of oxygen utilisation.

Three unique and highly compartmentalised mammalian superoxide

dismutases have been biochemically and molecularly characterised to date,

namely cytosolic Cu/Zn-SOD (SOD1), mitochondrial Mn-SOD (SOD2)

and extra cellular EC-SOD (SOD3) (Majima et al., 1998) and their

genomic structure, cDNA, and proteins have been described. Other types of

SODs have also been reported, based on the requirement of the metal

species at the active site, iron-containing superoxide dismutase (Fe SOD).

Two isoforms of SOD have Cu- and Zn- in their catalytic center and are

localized to either intracellular cytoplasmic compartment (Cu/Zn-SOD or

SOD1) or to extracellular elements (EC-SOD or SOD3). The evolutionary

tree for Cu, Zn containing SOD, based on multiple sequence alignments

with structural superimposition of crystal structures, shows that

extracellular SOD diverged from the cytosolic form at early stages of

evolution, before the differentiation of fungi, plants and metazoa (Bordo et

al., 1994). The phylogenetic analysis of all known vertebrate SOD genes

showed close similarities between SOD1 and SOD 3 with very low

homology to SOD2 (Fig. 2.3). The structural core of SOD1 exists as a

Greek key β-barrels (Getzoff et al., 1989). The amino acid substitutions, as

well as deletions and insertions occur mostly outside of the structural motif.

These data support the theory that Cu/Zn-SOD evolution involved gene

duplication and fusion with subsequent addition of exons 1 and 3.

Interestingly, the evolutionary rates of Cu/Zn- and Mn-SOD differed

considerably during the last billion years. While Mn-SOD proteins have

evolved at a relatively constant rate, SOD1 evolved unusually slowly at the

beginning and erratically quickly in the most recent 100 million years

(Smith and Doolittle, 1992; Rodriguez-Trelles et al., 2001). Why such an

abnormal evolutionary rate took place remains unclear; one possible

explanation is that Cu, Zn containing SOD was caught in “folding-block”

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36

when most changes in amino acid composition were deleterious (Smith and

Doolittle, 1992). The accumulation of silent mutations finally led to an

escape from this “evolutionary hibernation” and a return to the faster

evolutionary rate. While the plausibility of this theory remains

questionable, the existence of aerobic life on Earth proves that SOD

successfully evolved as a potent protective enzyme against oxygen toxicity.

Figure 2.3: Genomic organization of human SOD gene family

homology

% identity

intron

exon

152

1800 450

70 118

2000

97

3500

63%

420

63%

41% 46%

SOD 1

CuZn-SOD

(human)

572

5 84

1336 3819

SOD 3

EC-SOD

(human)

97

280 4390

SOD 2

Mn-SOD

(human)

117

3085

180 523 203

2210

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2.6.1. SOD1 or Cu/Zn-SOD

SOD1, or SOD1, was the first enzyme to be characterised and is a copper

and zinc-containing homodimer. This enzyme is found almost exclusively

in intracellular cytoplasmic spaces and also in nuclear compartments, and

lysosomes of mammalian cells and in wide range of organisms, including

yeast, spinach, chicken liver and bovine blood (Chang et al., 1988; Crapo et

al., 1992; Liou et al., 1993). It is conserved throughout evolution, which

usually has two identical subunits of about 32 kDa, each containing a metal

cluster, the active site, constituted by a copper and a zinc atom bridged by a

common ligand: His 61 (Banci et al., 1998). The subunits of this enzyme

are stabilized by an intrachain disulfide bond, but associated by non-

covalent forces. This enzyme requires Cu and Zn for its biological activity,

and the loss of Cu results in its complete inactivation, leading in many

cases to the development of human diseases (Brown and Besinger, 1998).

SOD 1 is believed to play a major role in the first line of antioxidant

defense by catalyzing the dismutation of superoxide anion radicals, to form

hydrogen peroxide and molecular oxygen. SOD1, a cytosolic enzyme,

accounts for nearly 90% of total SOD.

SOD1 has great physiological significance and therapeutic potential. The

role of this enzyme has been investigated in various specific red blood cell

(RBC) disorders, such as iron deficiency anemia, oxidative hemolytic

anemia, thalassemia, sickle cell anemia, molecular dystrophy and cystic

fibrosis (Pan Chenko et al., 1979; Mavelli et al., 1984; Mizuno, 1984). In

recent studies, this enzyme has also been shown to be associated with

dengue fever, post cholecystectomy pain syndrome, malignant breast

disease, steroid sensitive nephritic syndrome and amyotrophic lateral

sclerosis (Gonzales et al., 1984; Steiber et al., 2000).

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2.6.2. SOD2 or Mn-SOD

SOD2 or Mn-SOD is a homotetramer (96 KDa) containing one manganese

atom per sub unit that cycle from Mn (III) to Mn (II) and back to Mn (III)

during two-step dismutation of superoxide. It exists as a homotetramer with

an individual subunit molecular weight of about 23,000 Da (Barra et al.,

1984). This enzyme has been exclusively localized to mitochondria of

aerobic cells (Mn-SOD or SOD2) (Weisiger and Fridovich, 1973). Mn-

SOD is a nuclear-encoded primary antioxidant enzyme, function to remove

the super oxide radicals of mitochondrial respiratory chains (Guan et al.,

1998). Initially, the synthesized enzyme has a leader peptide, which targets

this manganese-containing enzyme exclusively to the mitochondrial spaces.

Mitochondria are especially sensitive to oxidative damage, and it has been

reported that mitochondrial damage induced by oxidants can cause release

of calcium (Farber et al., 1990), protein oxidation (Bindoli, 1990), loss of

electron transport capacity (Zhang et al., 1990) and mitochondrial DNA

damage (Shay and Werbin, 1987). Oxidative stress can cause damage to

mitochondrial function by decreasing mitochondria-derived products,

which can result in damage to cell function and cause cell death (Liu and

Keefe, 2000). It is suggested that Mn SOD may have a protective effect on

mitochondria against H2O2 induced stress. Because Mn SOD is located near

and surrounding the nucleus, it may protect nuclear DNA against H2O2

induced stress, indirectly.

SOD2 has been shown to play a major role in promoting cellular

differentiation and tumorigenesis (St.Clair et al., 1994) and in protecting

against hyperoxia-induced pulmonary toxicity (Wispe et al., 1992). The

expression of Mn-SOD is essential for the survival of aerobic life and the

development of cellular resistance to oxygen radical-mediated toxicity.

Matsui et al. (2003) reported that the cells with elevated levels of Mn-SOD

protected against DNA strand breaks induced by H2O2.

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2.6.3. SOD3 or EC-SOD

SOD3, or ECSOD, is the most recently discovered and least characterised

SOD, exists as a copper and zinc containing homotetramer of molecular

weight 135,000 Da (Marklund, 1982). The synthesized enzyme has a signal

peptide that directs this enzyme exclusively to extracellular spaces. It is a

secretory glycoprotein with high affinity for certain glycosaminoglycans

such as heparin and heparan sulfate. It is found in the interstitial spaces of

tissues and also in extracellular fluids, accounting for the majority of the

SOD activity of plasma, lymph, ascites, cerebrospinal fluids and synovial

fluid (Marklund, 1982; Marklund et al., 1986; Adachi and Wang et al.,

1998). EC-SOD is not induced by its substrate or other oxidants and its

regulation in mammalian tissues primarily occurs in a manner co-ordinated

by cytokines, rather than as a response of individual cells to oxidants

(Buschfort et al., 1997). The expression pattern of SOD3 is highly

restricted to the specific cell type and tissues where its activity can exceed

that of SOD1 and SOD2.

What role(s) these SODs play in both normal and diseased states is only

slowly beginning to be understood. A molecular understanding of each of

these genes has proven useful towards deciphering their biological roles.

For example, a variety of single amino acid mutations in SOD1 have been

linked to familial amyotrophic lateral sclerosis. The mice bred after

knocking out SOD2 gene results in a lethal cardiomyopathy. A single

amino acid mutation in human SOD3 is associated with 10 to 30 fold

increase in serum SOD3 levels. As more information is obtained, further

insights will be gained.

It is necessary to study the comparative characteristics of all three SOD

genes, their evolution and ontogeny, and their transcriptional regulation by

various intra and extracellular stimuli to understand the disease profile.

This is essential to emphasize the importance of the study of all the

regulatory mechanisms of antioxidant enzymes at molecular level.

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2.7. MOLECULAR ORGANISATION OF SOD GENE

The SOD1, SOD2 and SOD3 genes have been localized to chromosomes

21q22 (Levanon et al., 1985), 6q25 (Church et al., 1992) and 4p-q21

(Hendrickson et al., 1990), respectively. The genomic structure and

organisation of all human SOD gene; SOD1 (Levanon et al., 1985), SOD2

(Church et al., 1992; Wan et al., 1994) and SOD3 (Folz and Crapo, 1994)

has been identified. There is striking similarity between SOD genes among

mammalian species. The SOD1 gene is present in a single copy per haploid

genome and spans 11 kb of chromosomal DNA. The coding region of

SOD1 and SOD2 gene contains five exons interrupted by four introns,

whereas SOD3 gene has only three exons. Genomic southern blotting

supports the existence of one SOD2 gene for human (Wan et al., 1994).

The SOD3 gene shares 40 – 60% similarities with SOD2. The TATA and

CCAAT boxes, as well as several highly conserved GC-rich regions have

been localized in the proximal promoter region of SOD1 gene. The 3’ end

of SOD1 gene possesses several poly (A) signal sequences that terminate

the mRNA species with different lengths. The promoter region of the

human SOD1 gene has been studied and several putative binding sites for

NF1, Sp1, AP1, AP2, GRE, HSF, and NF-kB transcription factors have

been found (Kim et al., 1994). The role of Sp1 and Egr1 transcription

factors in basal and inducible expression of human SOD1 has been

confirmed (Minc et al., 1999). The promoter regions of SOD2 gene share

common feature among all four species (rat, mouse, bovine and human).

Both SOD2 and SOD3 apparently lack classical TATA or CCAAT box.

However, GC-rich regions are present in SOD2 gene of all four species.

Such features can be typical of “house keeping” genes (Jones et al., 1988;

Dynan, 1986). The human SOD2 and SOD3 gene contain putative

transcriptional regulatory element as like that of SOD1 gene. The putative

NF-kB regulatory element for SOD2 gene is located in the 3’ flanking

region of the gene (Wan et al., 1994) while in the other species it is located

in the 5’flanking region (Jones et al., 1995). Also multiple copies of Ap-2

and Sp-1 consensus sequences are present in the promoter region of SOD2

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gene in all four species. The putative transcriptional response elements of

SOD3 gene include a metal regulatory element, an AP-1 site as well as two

potential antioxidant response elements (Folz and Crapo, 1994).

8. FACTORS CONTROLLING SOD EXPRESSION

2.8.1. TRANSCRIPTIONAL REGULATION

Transcriptional regulation of all three isoforms of superoxide dismutase is

highly controlled based on extra-and intracellular conditions.

2.8.1.1. SOD1

SOD1 was found to have a widespread distribution in a variety of cells

(Crapo et al., 1992). The expression of cytoplasmic SOD 1 is stable and its

activity is often considered as an internal control for SOD1 gene

expression.

2.8.1.1.1. Stimuli up regulating SOD1 expression

Despite the fact that SOD1 is considered as constitutively expressed gene,

its mRNA levels can be dramatically regulated by various physiological

conditions. SOD1 mRNA levels can be elevated by a wide array of

mechanical stimuli such as heat shock (Hass and Massaro, 1988; Yoo et al.,

1999a), shear stress (Inoue et al., 1996; Dimmeler et al., 1999), UVB-and

X-irradiation (Isoherranen et al., 1997; Leccia et al., 2001; Yamaoka et al.,

1994). Chemical agents such as heavy metals (Yoo et al., 1999b), hydrogen

peroxide (Yoo et al., 1999a), ozone (Rahman et al., 1991), nitric oxide

(Frank et al., 2000), arachidonic acid (Yoo et al., 1999c) and

xenochemicals and also many biological messengers are also involved in

over expression of SOD1 mRNA. SOD1 expression can also be triggered

by ginseng saponins through activation of the AP2 transcription factor

(Kim et al., 1996). Metal ions are a potent source for the large-scale

catalysis and production of ROS inside cells. In order to neutralise their

harmful effects, the cells increases the synthesis of SOD1 through the metal

responsive element located in the 5’-flanking region (Yoo et al., 1999b).

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2.8.1.1.2. Stimuli down regulating SOD1 expression

A down regulation of SOD1 has been shown in alveolar type II epithelial

cells and lung fibroblasts after exposure to hypoxia (Jackson et al., 1996).

The anticancer drug, mitomycin C also represses the transcription of SOD1

gene in human hepatoma HepG2 cells (Cho et al., 1997). Several steroidal

drugs such as dexamethasone, prednisolone is also found to have influence

in SOD1 mRNA expression (Sugino et al., 1998).

2.8.1.2. SOD2

Despite the fact that SOD2 is expressed in many cell types and tissues at

relatively high levels and also highly regulated by a variety of intracellular

and environmental cues. Characterization of the 5’ flanking genomic region

from rat (Kuo et al., 1999), bovine (Meyrick and Mangnuson, 1994), and

human (Wan et al., 1994; Zhang, 1996; Yeh et al., 1998) indicates that

SOD2 promoter is TATA and CAAT less but contains GC-rich sequences

immediately upstream from the transcription initiation site. Computer

analysis and foot-printing assays reveal a number of putative binding sites

for Sp1 and AP2 transcription factors in the proximal promoter of human

SOD2. The two proteins have opposite effects on SOD2 expression: while

the Sp1 element positively promotes transcription, the AP2 proteins

significantly repress the promoter activity (Zhu et al., 2001).

2.8.1.2.1. Stimuli up-regulating SOD2 expression:

A wide variety of compounds induce transcription of SOD2. Cytokines

such as interleukin (IL)-1, IL-4, IL-6 (Dougall and Nick, 1991), TNF-α

(Visner et al., 1992; Wong and Goeddel, 1988), lipopolysaccharide (LPS)

(Visner et al., 1990), and IFN-γ (Harris et al., 1991) are potent activators of

SOD2 in different tissues and cell types. The cytokine inducible enhancer

has been localized to the 236 bp sequence within intron 2 of murine (Jones

et al., 1997), rat and human (Rogers et al., 2000) SOD2 genes. The

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cytokine inducible enhancer regions contain binding sites for NF-kB,

C/EBP, and NF-1 transcription factors.

Protein kinase C stimulating agents such as TPA induce human SOD2

expression via activation of a CREB-1/ATF-1 like factor, but not via NF-

kB or AP1 (Kim et al., 1999). Interestingly, the microtubule-active

anticancer drugs, vinblastin, taxol, and vincristine also induce SOD2

expression via activation of protein kinase C (Das et al., 1998). Manganese

ions, which at high concentrations are toxic to the cells, induce expression

of SOD2 in human breast cancer (Thongphasuk et al., 1999). Platelet-

derived growth factor induces the expression of the SOD2 gene in NIH3T3

cells, and its induction is associated with activation of Egr-1 transcription

factor (Maehara et al., 2001).

2.8.1.2.2. Stimuli down regulating SOD2:

The expression of SOD2 in many cancers is decreased due to methylation

of particular sequences in the intronic region (Huang et al., 1997; 1999)

and elevated levels of AP2 transcription factor, which interacts with the 5’

flanking sequences of SOD2 gene (Zhu et al., 2001).

2.8.1.2.3. Post translational regulation of SOD2:

SOD2 expression is regulated not only at the level of transcription, but also

at the level of translation by a RNA-binding protein. The 41 bp region,

located in the 3’-untranslated part of SOD2 mRNA binds the specific

protein that increases its translation efficiency (Chung et al., 1998). When

this cis-element was positioned after the coding region of chloramphenicol

acetyl transferase, it considerably increased the translation efficiency and

enzymatic activity of the reporter gene (Knirsch and Clerch, 2000). While

the identity of RNA-binding protein has not been determined, recent work

shows that SOD2 binding protein is phosphorylated by tyrosine kinase and

dephosphorylation is required for its binding activity (Knirsch and Clerch,

2001).

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In contrast to intracellular SOD1 and SOD2, the expression of SOD3

appears restricted to only a few cell types in several tissues. High levels of

SOD3 expression have been documented for alveolar type II cells (Folz et

al., 1997), proximal renal tubular cells (Folz R.J., unpublished

observation), vascular smooth muscular cells (Stralin et al., 1995), lung

macrophages (Loenders et al., 1998) and few cultured fibroblast cell lines

(Marklund, 1990). The features regulating such highly specific expression

are not yet known, but analysis of the 5’ flanking region of human SOD3

reveals several potential regulatory sequences such as glucocorticoid

response element, xenobiotic response element, and an antioxidant

response element (Folz and Crapo, 1994). The promoter regionof SOD3

lacks typical TATA or CAAT boxes but possess purine-rich sequences.

2.8.1.3. SOD3:

2.8.1.3.1. Stimuli up regulating SOD3:

In human fibroblasts, the level of SOD3 was elevated by IFN-γ and IL-1α,

while other cytokines such as IL-2, IL-3, IL-4, IL-6 and IL-8 demonstrated

no effect on its expression (Marklund, 1992).Similar results were reported

for the induction of SOD3 in rat sertoli cells, except IFN-γ has no effect on

the enzyme expression (Mruk et al., 1998). TNF-α and IFN-γ appear to be

a potent combination for the induction of SOD3 expression in rat alveolar

type II pneumocytes through NF-kB activation (Brady et al., 1997).

Because SOD3 exerts an important protective role in the vascular wall, the

vasoactive factors such as histamine, vasopressin, oxitocyn, endothelin-1,

serotonin, and heparin markedly increased enzyme level in the cultured

arterial smooth muscle cells (Stralin and Marklund, 2001). Further, exercise

training increases production of nitric oxide in mouse vessel endothelial

cells, which in turn upregulates expression of SOD3 in adjacent smooth

muscel cells (Fukai et al., 2000). Thus increased concentration of SOD3

prevents the degradation of NO by oxygen radicals. Angiotensin II strongly

induces SOD3 activity in mouse aortas (Fukai et al., 1999) and in cultured

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human smooth muscle cells (Stralin and Marklund, 2001) through

transcriptional activation and stabilization of mRNA. Interestingly, the

effect of angiotensin II on SOD3 expression is due to activation of p42/44

MAP kinase pathway, while nitric oxide exerts its effect through MAP

kinase p38. There are contradictory data on regulation of SOD3 expression

by cyclic nucleotides. The exposure of rat glioma cells to cAMP increases

SOD3 production while in mouse aortas it has no effect (Fukai et al., 2000;

Nicolai et al., 1996). Variation in expression between different tissues

clearly demonstrates that EC-SOD is not a house-keeping gene but is

strongly induced in certain physiological environments.

2.8.1.3.2. Stimuli down regulating SOD3:

The expression of SOD3 is repressed by different types of growth factors.

Transforming growth factor-β in human fibroblasts (Marklund, 1992) and

platelet-derived growth factors and fibroblast growth factor in vascular

smooth muscle cells (Stralin and Marklund, 2001) markedly down regulate

expression and excretion of SOD3. These responses are slow and develop

over several days.

2.8.2. MUTATION IN SOD GENE

More than 100 different mutations in the SOD1, at least 3 heterozygous

mutations in the proximal promoter of SOD2 and only one homozygous

missense mutation in SOD3 (Folz et al., 1994) gene have been identified.

The mutations in SOD1 gene are mainly found to be associated with

amyotrophic lateral sclerosis (ALS), whereas mutation in SOD2 is linked to

reduced transcriptional activity (Xu et al., 1999). Although only 2% of

patients with ALS and 10-15% with familial ALS have mutations in the

SOD1 gene, the discovery of these mutations by Rosen et al. (1993)

provided the first molecular insight into the pathogenesis of this disease. It

is hypothesised that mutations in the SOD1 gene may impair the

antioxidant enzyme activity that in turn could lead to accumulation of toxic

superoxide anions. This theory was dismissed experimentally when SOD1

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bearing the G93A mutation was over expressed in mice, resulting in motor

neuron disease despite the elevated SOD1 activity (Gurney et al., 1994).

Moreover, complete depletion of SOD1 in “knock-out” mice does not cause

any motor neuron abnormalities (Reaume et al., 1996), although they

exhibit increasing embryonic lethality and reduced fertility in females (Ho

et al., 1998). The opposite gain-of-function due to mutation can be

explained that mutations in the SOD1 gene would change the affinity of

enzyme to the natural and abnormal substrates (Wiedau-Pazos et al., 1996).

This change would in turn impair the ability of enzyme to bind zinc

(Estevez et al., 1999) or increase the enzyme aggregation (Bruijn et al.,

1998; Chou et al., 1996). Either way, the dominant mutations in SOD1 play

a key role in pathogenesis of familial ALS. Surprisingly only one mutation

and very few polymorphisms have been reported for SOD3 gene. The

reported mutation has been found to be in the heparin-binding domain that

is located in the centre of the carboxyl-terminal cluster of positively

charged amino acid residues. This mutation replaced arginine in position

213 by glycine, which causes an 8-15 fold increase in concentration of

plasma SOD3 levels (Folz and Crapo, 1994; Yamada et al., 1995;

Sandstrom et al., 1994). The effect of this SOD3 mutation is not entirely

clear but early studies suggest that this amino acid mutation impairs affinity

for heparin and endothelial cell surface and may reduce susceptibility to

trypsin-like proteases. This mutation has been found in 4% Swedish

(Marklund et al., 1997), 3% Australian (Adachi and Wang, 1998) and 6%

Japanese (Yamada et al., 1995) subjects. Two additional polymorphisms

have been identified in the human SOD3 gene; a transition mutation of A to

G at position 241 resulting in a Thr to Ala (T40A) substitution and a silent

transition mutation of C to T at position 280 (Yamada et al, 1997). While

the substitution of nucleotide A to G at position 241 creates a new BssHII

restriction site, the T40A amino acid change does not seem to affect

heparin binding capacity or the specific activity of EC-SOD (Yamada et al.,

1997).

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Multiple lines of evidence from cell culture and transgenic animal models

also indicate that the mutations in SOD1 gene cause the enzyme to acquire

toxic property (Price et al., 1998). Kruman et al (1999) have employed a

mouse model of ALS in order to test the toxicity of mutated SOD1 enzyme.

In this model over expression of a mutant familial ALS-linked SOD1 leads

to progressive motor neuron (MN) loss and a clinical phenotype

remarkably similar to that of human ALS patient. In contrast, mice

deficient in or over expressing wild type SOD1 does not develop motor

neuron disease. These findings suggest that mutant SOD1 is somehow toxic

to neurons. Although the mechanism of toxicity is not clearly established, it

has been shown that mutant SOD1 can form intra neuronal aggregates and

induce oxidative stress. Ratovitsky et al., (1999) have shown that a

mutation in the gene for SOD1 causes a form of familial amyotrophic

lateral sclerosis (FALS). In another study related to ALS, Vukosavic et al.,

(1999) have proposed that a mutation in the gene for SOD1, the only

proven cause of ALS, induces the disease by a toxic property that promotes

apoptosis. Other studies related to ALS have confirmed that ALS is caused

by a mutation in the gene for SOD1 (Pedersen and Mattson, 1999) also a

subset of familial cases of ALS is linked to a missense mutation in the gene

for SOD1 (Facchinett et al., 1999). The mutation has been associated with

the substitution of glycine for the conserved amino acid arginine at codon

85 (G58R). Different mutant enzymes show a remarkable degree of

variations with respect to activity, polypeptide half-life, and resistance to

proteolysis.

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