chapter 2: review of literature -...
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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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CHAPTER 2: REVIEW OF LITERATURE
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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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CHAPTER 2: REVIEW OF LITERATURE
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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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CHAPTER 2: REVIEW OF LITERATURE
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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CHAPTER 2: REVIEW OF LITERATURE
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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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CHAPTER 2: REVIEW OF LITERATURE
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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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CHAPTER 2: REVIEW OF LITERATURE
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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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CHAPTER 2: REVIEW OF LITERATURE
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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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CHAPTER 2: REVIEW OF LITERATURE
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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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CHAPTER 2: REVIEW OF LITERATURE
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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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CHAPTER 2: REVIEW OF LITERATURE
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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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CHAPTER 2: REVIEW OF LITERATURE
34
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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CHAPTER 2: REVIEW OF LITERATURE
35
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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CHAPTER 2: REVIEW OF LITERATURE
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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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CHAPTER 2: REVIEW OF LITERATURE
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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.