Download SOD is an enzyme with four different types of metal

REVIEW
OF
LITERATURE
2.0 REVIEW OF LITERATURE
For years, scientists have been working to find out ways to boost human body’s primary
antioxidant enzyme: superoxide dismutase (SOD). This enzyme is present both intracellularly
and extracellularly. This antioxidant defense system plays the most crucial role in reducing the
oxidative stress and helps in combating various degenerative diseases and increases life span. We
all know that without oxygen, we could not survive. Various organelles within our cells utilizes
oxygen along with other molecules generates energy to carry out many biochemical processes.
However, during this process of metabolic reactions of nutrients with oxygen, certain oxygen
molecules are generated as by-products, known as free radicals and reactive oxygen species.
These unstable, highly reactive molecules play central role in all the cellular processes.
Free radicals are also known as reactive species having a single unpaired electron in their
outermost orbit (Riley 1994). This unstable configuration creates energy which is released
through reactions with neighboring molecules, such as proteins, lipids, carbohydrates, and
nucleic acids. The majority of free radicals damaging biological systems are oxygen-free
radicals, and these are generally known as “reactive oxygen species” (ROS). They are mainly
derived from oxygen and nitrogen, and are produced in our body by various endogenous
mechanisms, like exposure to physico-chemical and patho-physiological conditions. The term
reactive oxygen species is often used in the biomedical free radical literature is a collective term
that includes not only oxygen-centered radicals but also some non-radical derivatives of oxygen,
such as hydrogen peroxide, singlet oxygen and hypochlorous acid. These are the main byproducts found in the cells of aerobic organisms, and initiate auto-catalytic reactions within the
molecules to produce free radicals and to propagate the chain of damage. These Reactive oxygen
species can be (i) produced during metal catalyzed reactions (ii) generated UV rays, X-rays and
gamma rays (iii) are produced by
macrophages during inflammation, (iv) occur in the
atmosphere as pollutants and (iv) by-products of mitochondrial electron transport reactions, and
various other mechanisms (Cadenas 1989).. The ROS are produced both endogenously and
exogenously. The endogenous sources of ROS are mitochondria, peroxisomes, cytochrome P450
metabolism, and activation during inflammation processes (Inoue et al 2003).
An antioxidant molecule is stable enough to donate an electron to a highly reactive free radical,
neutralizes it, thus reducing its capacity to damage. These antioxidants inhibit cellular damage by
their free radical scavenging property (Halliwell, 1995). The low molecular antioxidants interact
with free radicals, terminate the chain reaction to prevent oxidative damage. Some naturally
occurring antioxidants include glutathione, ubiquinol, and uric acid, produced during normal
metabolism in the body. Other small molecular antioxidants are found in the diet. There are
several enzymes systems present within the body that neutralize these free radicals, the most
important are micronutrient (vitamins) antioxidants are vitamin E (α-tocopherol), vitamin C
(ascorbic acid), and B-carotene (Levine, 1994). The body cannot manufacture these micronutrients, so they have to be supplied in the diet. In living organisms, the level of these free
radicals and other ‘reactive oxygen species’ are controlled by a complex web of antioxidant
defenses, which minimize oxidation damage to bio-molecules. In human diseases, this ‘oxidant–
antioxidant’ balance is in favor of the reactive oxygen species increasing oxidative damage.
In scientific/biomedical literature the term ‘free radical’ includes related reactive species such as
‘excited states’ that lead to free radical generation or those species that results from free radical
reactions. In general, free radicals are short lived, having half-lives in milli-, micro- or
nanoseconds. Imbalance in Free radicals production is directly related to several human diseases
as well as ageing (Harman, 1958; Halliwell and Gutteridge, 2000) by adversely altering the
structure of lipids, proteins and DNA. Lipids are mostly prone to free radical damage which
leads to lipid peroxidation and thus adverse alterations. Free radical damage to proteins results in
complete loss of enzyme activity. Damage caused to DNA leads to mutagenesis and
carcinogenesis. ‘Antioxidants’ are substances that neutralize the delitirious effects of free
radicals and their reactions (Sies, 1997). Nature has antioxidant defense mechanisms- superoxide
dismutase (SOD), glutathione, glutathione peroxidises, reductase, catalase and vitamin E,
vitamin C etc., apart from the various dietary components. The antioxidant enzymes produced
within our bodies are complex protein structures incorporating minerals such as copper, zinc,
manganese, nickel etc. in their intricate structures. These antioxidant enzymes are the body’s
most potential defences against free radicals. There are epidemiological evidences related with
higher intake of components/foods with antioxidant abilities to reduce many pathological
disorders.
Plants and animals have a complex system of multiple types of antioxidants. These antioxidant
defenses fall into three different categories:
1. Lipid Soluble and Membrane associated antioxidants (Alpha Tocopherol and Carotenoids).
2. Water soluble reductants (Ascorbate and Glutathione).
3. Enzyme antioxidants (Peroxidase and Catalase, Glutathione reductase and Superoxide
dismutase).
Antioxidants act at different stages: prevention, interception and repair. Preventive antioxidants
attempt to stop the production of Reactive oxygen species. These include superoxide dismutase
enzyme (SOD). Primary antioxidants such as superoxide dismutase are our first and most
important line of defense against highly reactive, potentially destructive oxygen-derived free
radicals. Superoxide radicals are negatively charged atoms, produced when oxygen acquires an
extra electron. Superoxide dismutase is an oxido-reductase enzyme catalyzing reaction between
superoxide and hydrogen to yield molecular oxygen and hydrogen peroxide. This type of
transformation is known as dismutation, hence the enzyme’s name. This enzyme protects cell
against deleterious effects of superoxide. SOD catalyses the dismutation of super-oxide radical to
H2O2 and Catalase enzyme further breaks it down to water (Sies, 1997; Cadenas and Packer,
1996).
2.1 Free Radicals
Although oxygen (O2) is vital to sustain life but its toxic properties were discovered long back.
In as early as 1954, O2 toxicity was suggested to be mediated through free radicals or, more
generally, reactive oxygen species (ROS) (Gerschman, 2001). ROS are highly reactive oxygencontaining products as a byproduct of normal cellular metabolism that can lead to oxidative
stress by damaging cells. The deleterious effect of World War II (1939-1945) led to the birth of
free radical biochemistry. The two atom bombs (6th August 1945, Hiroshima and 9th August
1945, Nagasaki) led massive deaths to entire population, and the ones who survived had
shortened life-span. In 1954, Gershman and Gilbert had suggested that the lethal effects of
ionizing radiation lead to formation of reactive oxygen species (ROS). Since then free radicals
(atoms with an unpaired electron) such as ROS and reactive nitrogen species (RNS) have gained
notoriety (Gilbert et al., 1981). If not disposed of efficiently; ROS can cause cellular and genetic
damage leading to carcinogenesis, Senescence, and neurodegenerative disorders (Droge, 2007;
Finkel and Holbrook, 2000) Importance of ROS in biological systems was then established by a
series of discoveries, which indicated that living systems have not only adapted to protect
themselves from ROS, but have also evolved mechanisms for the advantageous use of ROS in
various physiological functions (Valko, et al., 2007).
It is important to note that ROS and RNS are produced in a cell in a regulated manner, maintains
homeostasis in normal healthy tissues and plays important role as signaling molecules.
According to cell environment superoxide (O2•-), hydrogen peroxide (H2O2) and nitric oxide
(NO) are produced. Hence, it is important to emphasize on the beneficial role of free radicals.
a) Generation of ATP (universal energy currency) from ADP in the mitochondria: oxidative
phosphorylation.
b) Detoxification of xenobiotics by Cytochrome P450 (oxidizing enzymes)
c) Apoptosis of effete or defective cells
d) Killing of micro-organisms and cancer cells by macrophages and cytotoxic lymphocytes
e) Oxygenases (eg. COX: cyclo-oxygenases, LOX: lipoxygenase) for the generation and
production of prostaglandins, which have many regulatory functions. In recent years, it
has become completely clear the ROS, such as O2•- and H2O2 may act as second
messengers.
Observations made some twenty years ago had suggested that ROS plays major role in
modulating cellular function. These include cancer (possibly triggered by free radical-induced
damage to cellular DNA) and inflammatory and degenerative diseases such as Alzheimer’s,
arthritis, atherosclerosis, and diabetes (Barouki et al., 2006; Morrow et al., 2005). Recently
scientists have proved that increased oxidative stress with age damages cellular structure and
function (Yu and Chung, 2006). An important example is ill effects of free radicals to collagen,
forming the skin’s “scaffolding.” Healthy collagen is responsible for the skin’s elasticity and, to
no small degree, its youthful appearance. As we age, internally generated reactive oxygen
species gradually damage the molecular structure of collagen, eventually producing outward
signs of aging such as skin wrinkling and sagging. The Danish researchers have reported that
SOD binds directly to collagen, which it protects from oxidation and reported in the Journal of
Biological Chemistry, that superoxide dismutase significantly protects type I collagen from
oxidative breakdown. Furthermore, they noted this interaction plays significant physiological
role in preventing fragmentation of collagen during oxidative stress (Petersen et al., 2004).
Studies done then revealed that exogenous H2O2 mimics the action of the insulin growth factor. It
has been understood that NO•, a free radical produced enzymatically plays a physiological role in
vasodialation and neurotransmission and this further supported the concept that ROS and RNS
can act as second messengers to modulate signaling pathways. This made tremendous impact in
the field of redox signaling (Yamamoto et al., 2000). There is extensive evidence to prove the
possible role of free radicals in the development of various degenerative diseases. Free radical
damage inside the cells leads to the pathological changes in the body associated with aging. They
are the major factors for progressive decline in the functioning of the immune system (Pike and
Chandra, 1995).
2.2 Antioxidants
Antioxidants’ are substances that neutralize free radicals and their actions (Sies, 1997). Nature
has provided each and every cell with protective mechanisms against ill effects of free radicals:
superoxide dismutase (SOD), glutathione reductase, glutathione peroxidase, thiols thioredoxin
and disulfide bonding are buffering systems in every cell. α-Tocopherol (vitamin E) is an
essential nutrient which functions as a chain-breaking antioxidant which prevents the
propagation of free radical reactions in cell membranes in the human body. Ascorbic acid
(vitamin C) is also part of the normal protecting mechanism. The non-enzymatic antioxidants
include flavonoids, carotenoids, α-lipoic acid, glutathione related polyphenols etc. A major
defense mechanism of cells against ROS was explored in 1969 by McCord and Fridovich
reporting the dismutation of superoxide radical (O2-) by erythrocuprein and suggested the name
as superoxide dismutase (SOD; EC 1.15.1.1) for it (Imlay et al., 2011). Erythrocuprein protein
was discovered around 30 years ago by Mann and Keilin as a bovine liver protein of unknown
function. SOD is found in almost all organisms exposed to oxygen and is responsible for
converting O2• into hydrogen peroxide (H2O2) and O2. H2O2 is converted into water by enzymes
like catalase and peroxidases.
2.2.1 Levels of Antioxidant Action
Antioxidants are substances that neutralize the actions of free radicals, acting at different stages.
They act at different levels of prevention, interception and repair. Preventive antioxidants stop
the formation of reactive oxygen species (ROS). These include superoxide dismutase (SOD)
catalysing the dismutation of superoxide to H2O2 and catalase that breaks it down to water (Sies,
1997; Cadenas and Packer, 1996). Free radical interception is done by radical scavenging.
Various antioxidants like vitamins E, vitamin C, carotenoids and flavonoids etc. help in
scavenging the free radicals. At repair and reconstitution level, repair enzymes are involved.
Among the various antioxidants produced inside our body, SOD plays the primary role. It
transforms the most reactive, and the most dangerous, free radicals into ions that are less
reactive. These less reactive ions are then transformed by Catalase and Gpx (peroxidases) to
water and oxygen. This transformation process is called dismutation, and thus name Superoxide
Dismutase. SOD "signals" other cells to increase the production of more SOD, preparing the
antioxidant defense system against free-radical attack.
2.3 Superoxide Dismutase (SOD)
Superoxide is a free radical (a compound with an unpaired electron) and an ion with a charge of 1. Its molecular formula is O2-. Since it has an unpaired electron, it is highly reactive. Certain
processes that cells use to extract energy from food create superoxide ions as a byproduct, so
cells need a way to quickly break down the superoxides before they do any serious damage.
Superoxide dismutase (SOD) is an enzyme that catalyses the dismutation of superoxide radicals
(Fridovich, 1978).
O2- + O2- + 2H+  H2O2 + O2
SOD is a ubiquitous enzyme discovered by Irwin Fridovich and Joe McCord in 1969. Irwin
Fridovich and his student, Joe McCord, made the landmark discovery of the SOD enzyme in
1968. They had developed the "superoxide theory of oxygen toxicity" which states the
superoxide radical inflicts major damage to the body, and SOD, eliminates the destructive
radical, the body's first line of defense. Several forms of SOD exist: these are the proteins cofactored with either copper and zinc, or manganese, iron, or nickel. Thus, there are three families
of superoxide dismutase, depending on the metal cofactor: Cu-Zn (binding both copper and
zinc), Fe and Mn types (binding either iron or manganese), and the Ni type, binding nickel.
Present inside and outside cell membranes, SOD is the body’s primary internal anti-oxidant
defense system, and plays a central role in reducing oxidative stress implicated in various lifethreatening diseases. Since medieval times, plants have been the source of medicines for the
treatment of diseases. Despite the accessibility of an abundance of engineered medications,
plants remain an essential piece of the wellbeing in different countries. WHO recognizes 80% of
world’s population survives on natural plant extracts as source of medicine. Scientists are
making constant attempts to procure naturally occurring plants extracts as rich source of
antioxidant enzyme, Superoxide dismutase.
In other words, SOD is an essential systemic enzyme found in every cell. SOD present within
our cells, control both an antioxidant and an anti-inflammatory in the body, neutralizing free
radicals that lead to aging.. SOD also helps the body use zinc, copper and manganese and has
been used to treat arthritis, inflammatory diseases, various cardiovascular disorders, prostate
problems, corneal ulcers, burn injuries and neurodegenerative disorders due to long term damage
from exposure to smoke and radiation, and to prevent side of effects of cancer drugs (Chopra et
al., 1997).
2.3.1 Discovery of SOD
Superoxide dismutase (SOD, EC 1.15.1.1) discovered by American biochemist Irwin Fridovich
and his graduate student Joe McCord in 1969 (McCord and Fridovich, 1969). According to Dr.
Culter, it is a factor that controls life spans. Therefore, research on SOD activity will be
important in understanding of various mechanisms of life.
SOD is present in most organisms, aerobic as well as anaerobic, and plays a central role in
cellular protection against oxidative stress conditions (Fridovich, 1995).SOD's were known as
metalloproteins with unknown function. For example, Cu-Zn SOD was initially known as
erythrocuprein and as an anti-inflammatory drug "Orgotein" (McCord and Fridovich, 1988).
SOD is an intracellular enzyme produced endogenously, present in each and every cell in the
body. SOD present in cells is represented by a group of metalloenzymes having different
prosthetic groups. The prevalent enzyme is copper-zinc Cu-Zn SOD, which is the most stable
dimeric protein (32,000 D). Free radicals, containing unstable oxygen molecules constantly
attack nearby organs and tissues, are produced during everyday activities like eating and
breathing. The body counters its negative effects through naturally produced antioxidants, such
as superoxide dismutase (SOD). However, several factors (for example, smoking, pollution,
drugs, UV light, pesticides) can disrupt the balance and overwhelm the body with too many free
radicals, thus resulting in heart disease, cancer, aging, and more than 50 other conditions.
Reducing the threat is possible by strengthening and reinforcing the body's natural levels of SOD
through nutritional supplements and a diet rich in antioxidants.
2.3.2 Distribution and Phylogeny of Superoxide Dismutase:
Figure 2.1 Neighbour-joining phylogenetic tree based on the amino acid sequences of
representative SOD enzymes (courtesy Bafana et al., 2011)
Different SOD classes have been colour-coded as: Cu,Zn SODs – grey italic, Mn SOD – black
italic, Fe SOD – black plain, and Ni SOD – grey plain letters. SWISS-PROT IDs are given in
parentheses after the names of source organisms (Courtsey Bafana et al., 2011).
SOD is an enzyme with four different types of metal ions, dividing this family into Cu,Zn-, Fe-,
Mn- and Ni-SODs. About 2 billion years ago during the oxygenation of biosphere the evolution
of SOD and other antioxidant enzymes was probably triggered by production of O2 by
photosynthetic organisms. Two major kinds of SOD appeared independently in prokaryotes
parallely, Cu-Zn SODs and Fe SODs/Mn SODs. Fe/Mn SODs then evolved into Fe and Mn
SODs by gene duplication (Fig. 2.1). This may be the reason why Fe and Mn SODs are closely
related with regard to three-dimensional structure and amino acid sequence. However, their
crystal structures and catalytic mechanism are completely different as compared to Cu, Zn SOD,
supporting the hypothesis of independent evolution (Smith et al., 1992).
The Cu-Zn SOD is found mainly in eukaryotes, chloroplast and bacteria. Animals have two
different forms of Cu-Zn SOD cytosolic SOD1 and extracellular EC SOD/SOD3. SOD3 is
distinct from SOD1 not only in terms of molecular weight but also on amino acid composition as
well. The evolutionary tree for Cu-Zn SOD, based on multiple sequence alignment and structural
superimpositions of crystal structures, shows that EC SOD diverged from SOD1 at an early stage
of evolution, even before the differentiation of fungi, plants, and metazoans (Zelko, et al., 2002).
Unlike other organisms, plants have been reported to possess multiple forms of Cu, Zn SOD,
which are encoded by more than one gene. Phylogenetically, they group with the eukaryotic CuZn SODs, and are clustered into two main subgroups of chloroplastic and cytosolic SODs,
indicating that these two subgroups diverged early in the evolution of plants R.C. Fink et
al.,2002. Mn SOD occurs in prokaryotes and mitochondria of the eukaryotes. In prokaryotes it is
encoded by the sodA gene, while in animal mitochondria it is known as SOD2. Similarly, Fe
SOD is found in prokaryotes (SodB) and chloroplasts. The mitochondrial and chloroplastic Mnand Fe SODs have been proposed to be prokaryotic in origin (Van et al., 1990) and their origin
probably lies with the endosymbiont. Apart from chloroplast and mitochondria, higher organisms
also accumulated SODs in other compartments where O2 could be activated (microsome,
peroxisome, glyoxysome, extracellular matrix and cytosol). This was important because
superoxide is membrane-impermeable and must be detoxified in the same compartment where it
is formed.
In the Fe- and Mn SOD group, Fe SOD is proposed to be more ancient because of an abundance
of Fe in soluble Fe (II) form on primitive earth. As the level of O2 in the primitive environment
increased, availability of Fe (II) decreased, probably causing a shift to the use of more available
Mn. A phylogenetic tree of Fe- and Mn SODs shows short distances separating Fe SODs from
Mn SODs, confirming a common phylogenetic origin for these two, and suggesting likely
frequent horizontal gene transfer (Schafer et al., 2003). Since archaea and eubacteria separated
much before the appearance of O2. Many of the archaeal Fe- and Mn SODs are extremely
thermostable. The high apparent Tm values of these enzymes (some about 40 ◦C above the
optimal growth temperature) suggest their evolutionary origin under the conditions of the early
earth, i.e. a hot environment. Surprisingly, these hyperthermophilic (Schafer et al., 2003;
Castellano et al., 2006; Pedersen et al., 2009). Ni-dependent enzymes (SodN) have only been
described from Streptomyces and cyanobacteria so far and hence, not much information is
available about them (Dupont et al. 2008). Dupont and his co-workers analyzed putative Ni SOD
sequences from public database and observed that most of them belonged to marine
environment.
Figure 2.2. Physico-chemical stability of SODs from certain sources (Courtesy Bafana et al.,
2011).
2.3.2 Types of superoxide dismutase
SODs were earlier known as a group of metalloproteins with unknown function; e.g. Cu-Zn SOD
was known as erythrocuprein as an anti-inflammatory drug "Orgotein" (McCord and Fridovich,
1988). Likewise, Brewer (1967) identified a protein that later became known as superoxide
dismutase as an indophenol oxidase by protein analysis of starch gels using the phenazinetetrazolium technique (Brewer, 1967).
There are different types of SOD according to the metal co-factored with copper and zinc,
or manganese, iron, or nickel. Thus, there are three major families of SOD, depending on the
metal cofactor: Cu-Zn (binding both copper and zinc), Fe and Mn types (binding either iron or
manganese), and the Ni type, binding nickel.

Copper and Zinc- Most commonly used by eukaryotes. The cytosols of virtually
all eukaryotic cells contain an SOD enzyme with copper and zinc (Cu-Zn-SOD). For
example, Cu-Zn-SOD available commercially is normally purified from the bovine
erythrocytes The Cu-Zn enzyme is a homodimer of molecular weight 32,500. The bovine
Cu-Zn protein was the first SOD structure to be solved, in 1975. It is an 8-stranded structure
beta-barrel, with active site is located in between the barrel and two surface loops. The two
subunits are very tightly attached back-to-back, mainly by some electrostatic interactions
hydrophobic interactions. The ligands of the copper and zinc are six histidine and
one aspartate side-chains; one histidine is shared between the two metals (Tainer et al.,
1983).

Iron- Many bacteria and E. coli also contain a form of the enzyme with iron (Fe-SOD); some
bacteria contain Fe-SOD, others Mn-SOD, and some contain both. Fe-SOD can be found in
the plastids of plants. The 3-D structures of the homologous Mn and Fe superoxide
dismutases have the same arrangement of alpha-helices, and their active sites contain the
same type and arrangement of amino acid side-chains.

Manganese- Chicken liver, (and nearly all other) mitochondria, and many bacteria (such
as E. coli), contain a form with manganese (Mn-SOD): for example, the Mn-SOD found in
human mitochondria. The ligands of the manganese ions are 3 histidine side-chains,
an aspartate side-chain and a water molecule or hydroxy ligand, depending on the Mn
oxidation state (respectively II and III) (Borgstahl et al., 1992).

Nickel- Prokaryotic. This has a hexameric structure built from right-handed 4-helix bundles,
each containing N-terminal hooks that chelate a Ni ion. The Ni-hook contains the motif HisCys-X-X-Pro-Cys-Gly-X-Tyr; it provides most of the interactions critical for metal binding
and catalysis and is, therefore, a likely diagnostic of NiSOD (Barondeau et al., 2004;
Wuerges et al., 2004).
Figure 2.3 Types of SOD (Courtesy Bafana et al., 2011).
2.3.3 Sources of superoxide dismutase
2.3.3.1 Superoxide Dismutase in Mammals
Superoxide dismutases are a ubiquitous family of enzymes that function to efficiently catalyze
the dismutation of superoxide anions. There unique and highly compartmentalized mammalian
superoxide dismutases have been biochemically and molecularly characterized till date. SOD1,
or Cu-Zn SOD (EC 1.15.1.1), was the first enzyme to be characterized and is a copper and zinccontaining homodimer that is found almost exclusively in intracellular cytoplasmic spaces.
SOD2, or Mn-SOD (EC 1.15.1.1), exists as a tetramer and is initially synthesized containing a
leader peptide, which targets this manganese-containing enzyme exclusively to the mitochondrial
spaces. SOD3, or EC-SOD (EC 1.15.1.1), is the most recently characterized SOD, exists as a
copper and zinc-containing tetramer, and is synthesized containing a signal peptide that directs
this enzyme exclusively to extracellular spaces.
In humans (as in all other mammals and most chordates), three forms of superoxide dismutase
are present: SOD1, SOD2 and SOD3.
a) SOD1: It is a dimer (consists of two units) SOD located in the cytoplasm, containing Cu,
Zn (copper and zinc) and has two identical subunits with a molecular weight of 32 kDa
and each of the subunit contains as the active site, a dinulcear metal cluster constituted by
copper and zinc ions, and it specifically catalyzes the dismutation of the superoxide anion
to oxygen and water (Chang et al., 1988; Keller et al., 1991; Crapo et al., 1992; Liou et
al., 1993).
b) SOD2: It is the mitochondrial SOD having the Mn (manganese) in its reactive centre and
has been localized in the mitochodria of aerobic cells (Weisiger and Fridovich, 1973).
Mn-SOD is a homotetramer with a molecular weight of 96 kDa,i.e., 23 kDa per subunit
(Barra et al., 1984) and contains one manganese atom per subunit (Mates et al,1999), and
it cycles from Mn(III) to Mn(II), and back to Mn(III) during the two-step dismutation of
superoxide. SOD2 has been shown to play a major role in promoting cellular
differentiation and tumorgenesis and in protecting against hyperoxia-induced pulmonary
toxicity (Wispe et al., 1992).
c) SOD3: Extracellular superoxide dismutase contains Cu, Zn (copper and zinc), and is a
tetramer (consists of four subunits) (Mates et al., 1999). The enzyme exists as a
homotetramer of molecular weight 135,000 Da with high affinity for heparin (Marklund,
1994) SOD3 was first detected in human plasma, lymph, ascites, and cerebrospinal fluids
(Marklund et al., 1984; Marklund et al., 1987). 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.
Thus, the numerous studies on the physiological function of SOD1 and SOD2 and their role in
protection against ROS are summarized in several excellent reviews (Fridovich, 1995; McCord
and Fridovich, 1988; Lam, 1979; Fridovich, 1997, 1989). However, the available information
related to SOD3 has not been reviewed in a comparative perspective along with the other two
isoforms. This review focuses on comparative characteristics of all three SOD genes, their
evolution and ontogeny, and their transcriptional regulation by various intra and extracellular
stimuli.
Figure 2.4 SOD 1 (Courtesy Bafana et al., 2011)
Figure 2.5 SOD 2 (Courtesy Bafana et al., 2011)
Figure 2.6 SOD 3 (Courtesy Bafana et al., 2011)
2.3.3.2 Superoxide Dismutase in Micro-organisms
Microorganisms represent an economic source for production of different superoxide dismutase.
Aerobic microorganisms with high oxygen demand such as Corynebacterium glutamicum are
suggested to have a hyper antioxidant defense system including production of abundance
superoxide dismutase enzyme, so special attention should be paid to ameliorate the superoxide
dismutase production capacity of those microorganisms. Recently, superoxide dismutase became
a promising genetically therapeutic target for reduction of oxidative stress involved in
pathophysiology aging and many disorders.
Most organisms, microorganisms, plants and animals have at least one superoxide dismutase
enzyme (Parkes et al., 1998).While one of the exceedingly rare exceptions is Lactobacillus
plantarum and related lactobacilli, which use a different mechanism (Davis et al., 2000). SODs
were also produced efficiently by many microbial species (Inoue et al., 2003). Seen to their
continuous exposure to high oxidative stress during growth and metabolism, aerobic
microorganisms represent an excellent source for production of superoxide dismutases.
An unusual targeting sequence of mitochondrial superoxide dismutase was done in Plasmodium
falciparum. The intra intra erythrocytic stages of Plasmodium falciparum are exposed to
oxidative stress and require functional antioxidant systems to survive. In addition to the
parasite’s known iron dependent superoxide dismutase PfSOD1, a second SOD gene (PfSOD2)
interrupted by 8 introns was identified on chromosome 6. Molecular modeling shows that the
structure of PfSOD2 is similar to other iron dependent SODs and phylogenetic analysis suggests
PfSOD1 and PfSOD2 are the result of ancestral gene duplication. Both SOD genes are
transcribed during the erythrocytic cycle with PfSOD1 mRNA levels up to 35 folds higher than
those of PfSOD2 (Sienkiewicz, 2004).
Among many aerobic microorganisms considered as a potent source of superoxide dismutase,
Corynebacterium glutamicum, an industrial relevant producer of amino acids and vitamins, is
considered as an excellent candidate for this purpose seen to its high need of oxygen during
amino acid production, nominating it to have a hyper antioxidant defense system including
production of abundance superoxide dismutase enzyme (Allen and Tresini, 2000; Kashiwagi et
al., 2005). Cloning techniques reported to be used successfully with many corynebacterial genes
(Kashiwagi et al., 2005; Lishnevskaia, 2004; Riedl et al., 2005; Zhang et al., 2002). Thus it
would be interesting to enhance superoxide dismutase production using cloning strategies. In
addition other microbial species should also be considered for extraction of different superoxide
dismutase types.
2.3.3.3 Superoxide Dismutase in Plants
Since ancient times plants have played the most important role in preventing and curing diseases.
There are several evidences in this context and Ayurveda, Unani, and homeopathic formulations
have always and still being used for disease treatment. Large amount of Plants have been used as
potential sources of SOD. Literature strongly supports the used of SOD extracted from plants
and their role in disease prevention. Extracted SOD from plants is the further studied on various
parameters to understand their impact on Human health. The physiochemical properties of super
oxide dismutase enzyme from green peas (Pisum sativum) were studied. Chloroplasts of
Nicotiana tabacum have two super oxide dismutases: a Fe and a Cu-Zn containing enzyme.
Different SOD enzymes are needed, particularly according to stress conditions (Kurepa et al.,
1997). Previously, it was thought that Fe-SOD was present in limited species of seed plants such
as Ginkgoaceae, Nymphaceae and Cruciferae. More recently Fe-SOD genes were isolated in
various angiosperm species lacking phylogenetic relationships to one another, Arabidopsis
thaliana, tobacco, tomato, soybean and rice.
Mitochondrial manganese super oxide dismutase (Mn-SOD) has been isolated and characterized
from Capsicum annuum L. Purification and properties of cytosolic Cu-Zn SOD from water
melon (Stimulus vulgaris) have been studied. Oral supplementation with melon superoxide
dismutase extract promotes antioxidant defenses in the brain and prevents stress-induced
impairment of spatial memory (Nakajima et al. 2009). Effect of dietary turmeric on iron-induced
lipid per oxidation was studied in the rat liver. Dietary turmeric lowers lipid per oxidation by
enhancing the activities of super oxide dismutase enzyme, catalases and glutathione peroxidases.
Curcumin and Turmerin are the two components of turmeric, which make turmeric an excellent
dietary anti-oxidant (Reddy et al., 1994).
Root extract of Withania somnifera was used for the regulation of lead –induced oxidative
damage in male mouse, it has been seen that root extract increased the activities of antioxidant
enzymes (Chaurasia et al., 2000). The antioxidant activity of Withania somnifera was assessed, it
was observed that this increased super oxide dismutase activity in rat brain (Bhattacharya et al.,
2001). Withania somnifera, popularly known as Ashwagandha is widely considered as the Indian
ginseng. In Ayurveda, it is classified as a rasayana (rejuvenation) and expected to promote
physical and mental health, rejuvenate the body in debilitated conditions and increase longevity.
Having wide range of activity, it is used to treat almost all disorders that affect the human health.
The pharmacological basis and the use of W. somnifera in various diseases like central nervous
system (CNS) disorders, particularly its indication in epilepsy, stress and neurodegenerative
diseases such as Parkinson's and Alzheimer's disorders, tardive dyskinesia, cerebral ischemia,
and even in the management of drug addiction are reported.
The tannoid principles of the fruits of Emblica officinalis have been reported to exhibit
antioxidant activity in vitro and in vivo. This study confirms antioxidant effects of Emblica
officinalis and indicates the fruits of plant may have cardioprotective effect. Four aqueous
extracts from different medicinal plants were examined for their potential as anti-oxidants. The
anti -oxidant activity of these extracts were tested by studying inhibition of radiation induced
lipid per oxidation in rat liver microsomes at different doses, Momardica charantia, Glycyrrhiza
glabra, Acacia catechu and Terminalia chebula restore super oxide dismutase enzyme.
Terminalia chebula is the best antioxidant among the four (Naik et al., 2003). Determination of
antioxidant properties of aromatic herbs, olives and fresh fruit was performed using super oxide
dismutase as enzymatic sensor (Campanella et al., 2003).
The anti-oxidant activities of the various fractions from the herbs of Artemisia apiacea were
investigated.The antioxidant role of garlic oil in myocardial infraction in rats was studied. It was
observed that Garlic oil exerts its effects by modulating lipid per oxidation and enhancing
antioxidant and detoxifying enzyme systems (Saravanan et al., 2004). Studies were performed on
aqueous extract of Terminalia chebula as a potent antioxidant and a probable radio protector
(Naik et al., 2004).
Bacopa monniera is a nerve tonic used extensively in traditional Indian medicinal system “
Ayurveda”. Reports regarding its various antioxidative, adatogenic and memory enhancing roles
have already appeared in the last few decades. The neuroprotective role of Bacopa monniera was
studied and it was seen that Bacopa extract during aluminum treatment significantly prevented
the aluminum decrease in SOD activity as well as increased oxidative damage to lipids and
proteins. Protective effect was also observed at microscopic level. Purification and partial
characterization of a low temperature responsive Mn-SOD was obtained from Camellia sinensis.
Important progress has been made in the past five years concerning the effects of green and black
tea on health. Experimentation with new and accurate tools provide useful information about the
metabolism of tea components in the body, their mode of action as antioxidants at the cellular
level and their protective role in the development of cancer, cardiovascular disease and other
pathologies. The use of tea components as nutraceuticals and functional foods are also
reported.The manganese containing super oxide dismutase (Mn-SOD) was purified from a tea
clone, TEENALI, which showed lowest period of winter dormancy. Protein was purified using
leaves of tea by ammonium sulphate precipitation, followed by column chromatography using
DEAE-cellulose, and silica based size exclusion chromatography on HPLC system. The enzyme
had a native molecular weight of about 169 kDa and pH optima 8.0 (Vyas et al., 2005). C.
aromaticus had significant antioxidant activity and free radical-scavenging activity. The free
radical-scavenging property may be one of the mechanisms by which this drug is useful as a
foodstuff as well as a traditional medicine (Kumaran et al., 2005).
Barley roots contain super oxide dismutase enzyme. There is a significant increase in activities
of SOD in NaCl stressed Barley roots (Kim et al., 2005). The leaves of Allium species were
examined for the activities of antioxidant enzymes (catalase, peroxidase, super oxidedismutase,
glutathione peroxidase). The results showed that all Allium species had strong anti-oxidative
properties due to their high concentration of total flavonoids, high content of caretenoids and
chlorophylls, and very low concentrations of toxic oxygen radicals (Stajner et al., 2006). Studies
reveal the presence of super oxide dismutase enzyme in Tomato (Lycopersicon esculentum),
when tomato seedlings were grown in four different Cd concentrations; It has been studied that
Cd stress induces an oxidative stress response in tomato plants (Dong et al., 2006).
Superoxide dismutase was isolated and characterized from wheat seedlings. Two major
superoxide dismutases (SODs; SODs I and II) were found in the crude enzyme extract of wheat
seedlings (Lai et al., 2008). The antioxidant activity of methanol extracts of five plants from the
genus Phyllanthus was evaluated by various antioxidant assays (Kumaran et al., 2005). A
superoxide dismutase (SOD) with the molecular weight of 31,079 has been purified as a
homodimer from Panax ginseng by employing neutral pH buffer extraction, ammonium sulfate
precipitation, isoelectric point precipitation and ion exchange methods (Li et al., 2010).
In higher plants, superoxide dismutase enzymes (SODs) act as antioxidants and protect cellular
components from being oxidized by reactive oxygen species (ROS) (Alscher et al., 2002). ROS
can form as a result of drought, injury, herbicides and pesticides, ozone, plant metabolic activity,
nutrient deficiencies, photoinhibition, temperature above and below ground, toxic metals, and
UV or gamma rays (Smirnoff and Nicholas, 1993; Raychaudhuri et al., 2008). Specifically,
molecular O2 is reduced to O2- (an ROS called superoxide) when it absorbs an excited electron
released from compounds of the electron transport chain. Superoxide is known to denature
enzymes, oxidize lipids, and fragment DNA (Smirnoff and Nicholas, 1993). SODs catalyze the
production of O2 and H2O2 from superoxide (O2-), which results in less harmful reactants.
When acclimating to increased levels of oxidative stress, SOD concentrations typically increase
with the degree of stress conditions. The compartmentalization of different forms of SOD
throughout the plant makes them counteract stress very effectively. There are three well-known
and studied classes of SOD metallic coenzymes that exist in plants. First, Fe SODs consist of two
species, one homodimer (containing 1-2 g Fe) and one tetramer (containing 2-4 g Fe). They are
thought to be the most ancient SOD metalloenzymes and are found within both prokaryotes and
eukaryotes. Fe SODs are most abundantly localized inside plant chloroplasts, where are they are
indigenous. Second, Mn-SODs consist of a homodimer and homotetramer species each
containing a single Mn(III) atom per subunit. They are predominantly found in mitochondrion
and peroxisomes. Third, Cu-Zn SODs have electrical properties very different from the other two
classes. These are concentrated in the chloroplast, cytosol, and in some cases the extracellular
space. Note that Cu-Zn SODs provide less protection than Fe SODs when localized in the
chloroplast (Alscher et al., 2002, Smirnoff and Nicholas, 1993, Raychaudhuri et al., 2008).
The purified Cu-Zn SOD protein from Piper betle leaf was purified and characterized.It appeared
to be monomeric and its conversion into its dimeric form increased enzymatic activity in betel
nut oral extract. The enhanced activity of the SOD dimer was responsible for the continuous
production of hydrogen peroxide in the oral cavity. Thus, SOD may contribute to oral
carcinogenesis through the continuous formation of hydrogen peroxide in the oral cavity, in spite
of its protective role against cancer in vivo. (Liu et al., 2015)
It is widely accepted that a plant-based diet with high intake of fruits, vegetables, and other
nutrient-rich plant foods may reduce the risk of oxidative stress-related diseases (Rosenfeld et
al., 1996). SOD from natural plant extract can be easily delivered intact to the intestines and
absorbed into the bloodstream, thus effectively enhancing the body’s own primary defense
system. Once in circulation in the bloodstream, these powerful antioxidants go to work
detoxifying potentially harmful substances and reducing oxidative stress that might otherwise
contribute to aging and crippling diseases such as atherosclerosis, stroke, and arthritis. By
strengthening the body’s primary antioxidant systems, novel SOD-boosting supplements may
offer the most powerful free radical protection available today. The present study has been
focused on screening, isolation and purification of plant extract providing the best quantity of
Superoxide dismutase and Black Cardamom (Amomum subulatum) is a powerful anti-oxidant
and also a potent anti- cancerous agent.
Amomum subulatum (Black Cardamom) is very well known as the ‘Queen of Spices’. It is a
small herb with strong aromatic fragrances. It belongs to family Zingiberaceae, and is an
important economic crop in the Eastern Himalayas. It is found on slopes of hills where there is
plenty of well-drained water available, preferably in the north slopes of under the shade of trees.
Stems grow up to 5ft tall and leaves are found on the upper portion of the stem. It is an evergreen
plant with the old stems dying down after a few years. The rhizomes are a dull red in color. The
post harvest technology continues to be largely traditional. Large volume of crude Black
Cardamom is traded to the Indian market in Siliguri, which is ultimately sold as a spice. The oil
extracted after processing can be used in Ayurvedic medicine.
Black cardamom also known as “hill cardamom” is widely used in cooking for its unique taste
and powerful flavor. The oil extracted from the seeds of this herb is known as one of the most
effective essential oils and is widely used in aromatherapy. Apart from using it as a natural
remedy this spice offers a number of medicinal benefits, some of them are discussed as follows:

Gastro-Intestinal Health: Black cardamom has high positive impact on the gastrointestinal tract. It stimulates the gastric and intestinal glands to secret essential juices with
the help of its simulative properties. It also helps in regulating the procedure of juice
secretion in order to keep the amount of stomach acids under control. As a result, the
chances of developing gastric ulcers or other digestive disorders go down significantly.
This spice is helpful in curing heart burn and stomach cramps, the two most common
symptoms of gastro-intestinal disorders. Digestive properties of this substance make it
very important to heal chronic constipation and improve appetite. It provides relief from
abdominal gas and helps to get rid of indigestion as well as flatulence.

Cardiovascular Health: Black cardamom influences cardiac health to a great extent. It
helps in controlling cardiac rhythm and eventually keeps blood pressure under control.
Heart remains healthy by regular intake of black cardamom. It reduces the probabilities
of blood clot and is very effective in protecting from heat stroke/ sun stroke during
scorching summer.

Respiratory Health: It is very effective against serious respiratory problems. A number of
respiratory disorders including asthma, whooping cough, lung congestions, bronchitis,
pulmonary tuberculosis etc. can be treated successfully with this little spice. It warms up
respiratory tract so that the air circulation through the lungs becomes easy. Moreover,
black cardamom works as an expectorant and helps you stay away from cough, cold, sore
throat etc. by alleviating mucous membrane and normalizing the flow of mucous through
the respiratory tract.

Oral Health: Several dental disorders, such as teeth infection, gum infection etc. can be
treated with black cardamom. Furthermore, its strong aroma can help in curing halitosis
or bad breath.

Urinary Health: Being an effective diuretic, it facilitates urination and keeps renal system
healthy.

Anti-Carcinogenic Properties: There are two antioxidants named 3’-Diindolylmethane
(DIM) and Indole-3-Carbinol (I3C) in black cardamom, which combat breast, colon,
prostate and ovarian cancer. The anti-carcinogenic properties of the spice raises the
amount of glutathione (an antioxidant) in the body and prevent the generation and growth
of cancerous cells.

Detoxification: Studies have revealed that black cardamom is a good detoxifier for your
body. It is capable of eliminating caffeine from the blood so as to stay safe from the
adverse effects of the alkaloid.

Anesthetic Properties: The oil extracted from black cardamom is highly anesthetic and
sedative. It can curb acute pain like headache and provide immediate relief. The essential
oil prepared from the spice is also used in eliminating stress and fatigue.

Antiseptic and Antibacterial Properties: It has been seen that black cardamom can
destroy microbes of almost 14 different species. Hence, its intake boosts your immunity
and provides you protection against bacterial or viral infections.

Improves blood circulation: Black cardamom is full of the antioxidants, vitamin C and
the essential mineral potassium. Hence, regular consumption of the spice can keep your
internal system free from toxic materials, thereby improving the circulation of blood
throughout the skin surface and keeping it healthy and gives a firm, toned and youthful
look.

Anti-ageing: Black cardamom not only keeps your ageing at bay, but it also helps you in
getting a fairer skin complexion.

Anti-bacterial properties: It is used as a natural remedy for ‘contact dermatitis’ or skin
allergies. It provides nourishment to your scalp and hair strands. As a result making hair
strong, thick and shiny and prevents irritation and infection on scalp.
2.3.3.4 Superoxide dismutases and their impact upon human health
Superoxide dismutases (SOD), a group of metal-containing enzymes, have a vital anti-oxidant
role in human health, conferred by their scavenging of one of the reactive oxygen species,
superoxide anion. Three types of SODs are known in humans, with the most abundant being
cytosolic SOD1, identified by its Cu-Zn-containing prosthetic group. The presence of these
metals and the coordination to certain amino acids are essential for function. SODs are among
the first line of defense in the detoxification of products resulting from oxidative stress. Here, we
describe the importance of SOD function, and the need for coordination with other ROSscavenging enzymes in this pathway of detoxification. The impact of metal-deficient diets
(copper or zinc) or incorrect metal ion incorporation (copper chaperone for SOD) onto
nascent SOD,
are
also
examined.
Finally,
human
pathologies
associated
with
either SOD dysfunction or decreased activity are discussed with current progress on the
development of novel therapies (Johnson and Giulivi 2005).
It has been observed that marine organisms have the ability to cope with oxidative stress. The
antioxidative defense system includes enzymatic and non-enzymatic components. Among the
enzymatic system, superoxide dismutases are the first and most important of the antioxidant
metalloenzymes. Its use was earlier limited to non-drug applications in humans (include:
cosmetic, food, agriculture, and chemical industries) and drug applications in animals. A review
has been prepared focusing recent literatures on sources of marine SODs, the need for SOD and
different applications in industry (Zeinali, et al., 2015).
2.3.4 Effect of pH and various salts upon the activity of superoxide dismutase
The Cu-Zn superoxide dismutase (SODs) from ox, sheep, pig, yeast were investigated by pulse
radiolysis in order to evaluate the role of electrostatic interaction between O2- and SOD proteins
and the mechanism of action of the SOD enzymes (Bannister et al., 1987). The protein net
charge in this series varies, as evaluated by the protein pI values spanning over a large range of
pH: 8.0 (sheep), 6.5 (pig), 5.2 (ox), and 4.6 (yeast) (Bertini et al., 1981). The dependence upon
ionic strength also depends on the salt used. The dual and concerted dependence of the activities
of different SODs on pH and salt concentration is explained with the encounter of O2- with the
active-site copper being governed by the protonation of two positively charged groups in the
vicinity of the active site. The gradient between these localized charges and the rest of the
protein explains the different activities of the mammalian protein at lower pH (Davies et al.,
1988). Numerous studies have been investigated to study the influence of abiotic stress factors
on plants. Due to their direct or indirect presence they may affect the development, growth, basic
metabolisms in plants or any other living organisms. Each kind of these, such as heavy metals,
salt stress, chilling, drought or UV-B radiation, might induce the formation or the overproduction
of reactive oxygen species (ROS). Plant SODs, their characteristics of and the difference or the
similarity in the effects of different abiotic stress factors on SOD activity plays an important role
in plant physiology (Szőllősi et al., 2014).
2.3.5 Regulation of superoxide dismutase genes
Numerous short-lived and highly reactive oxygen species (ROS) such as superoxide (O2-),
hydroxyl radical, and hydrogen peroxide are continuously generated in vivo. Depending upon
concentration, location, and intracellular conditions, ROS can cause toxicity or act as signaling
molecules. The cellular levels of ROS are controlled by antioxidant enzymes and small-molecule
antioxidants. As major antioxidant enzymes, superoxide dismutases (SODs), including copperzinc superoxide dismutase (Cu-Zn SOD), manganese superoxide dismutase, and extracellular
superoxide dismutase, play a crucial role in scavenging O2-. This review focuses on the
regulation of the sod genes coding for these enzymes, with an emphasis on the human genes.
Studies to date on genes coding for Cu-Zn SOD (sod1) are mostly focused on alterations in the
coding region and their associations with amyotrophic lateral sclerosis. Evaluation of nucleotide
sequences reveals that regulatory elements of the sod2 gene reside in both the noncoding and the
coding region. Changes associated with sod2 lead to alterations in expression levels as well as
protein function. We also discuss the structural basis for the changes in SOD expression
associated with pathological conditions and where more work is needed to establish the
relationship between SODs and diseases (Miao and Clair, 2009).
Sod1 gene: Superoxide dismutase (Cu-Zn) also known as superoxide dismutase 1 or SOD1 is
an enzyme that in humans is encoded by the SOD1gene. SOD1 is one of three human superoxide
dismutases (Patterson et al., 1993). SOD1 binds copper and zinc ions and is one of three
superoxide
dismutases
responsible
for
destroying
free superoxide radicals
that
solublize cytoplasmic and mitochondrial intermembrane space protein, acting as a homodimer to
convert naturally occurring, but harmful, superoxide radicals to molecular oxygen and hydrogen
peroxide .
Sod3 gene: Extracellular superoxide dismutase (Cu-Zn SOD) is an enzyme that in humans is
encoded by the SOD3 gene. This gene encodes a member of the superoxide dismutase (SOD)
protein
family.
SODs
are
antioxidant enzymes that
catalyze
the
dismutation
of
two superoxide radicals into hydrogen peroxide and oxygen. The product of this gene is thought
to protect brain, lungs, and other tissues from oxidative stress. The protein is secreted into
the space and forms a glycosylated homotetramer that is anchored to the extracellular
matrix (ECM)
and
cell
surfaces
through
an
interaction
with heparan
sulfate proteoglycan and collagen. A fraction of the protein is cleaved near the C-terminus before
secretion to generate circulating tetramers that do not interact with the ECM (Mariani et al.,
2003).
2.3.6 Pharmacological activity of SOD enzyme

SOD has powerful anti-inflammatory activity. For example, SOD is highly effective in
treatment
of
colonic
inflammation in
experimental colitis.Treatment
with
SOD
decreases reactive oxygen species generation and oxidative stress and, thus, inhibits
endothelial activation and indicate that modulation of factors that govern adhesion
molecule expression and leukocyte-endothelial interactions. Therefore, such as antioxidants
may be important new therapies for the treatment of inflammatory bowel disease (Yasui et
al., 2006). SOD has multiple pharmacological activities. E.g., it ameliorates cis-platinuminduced nephrotoxicity in rodents (Ginness et al., 1978). As "Orgotein" or "ontosein", a
pharmacologically-active purified bovine liver SOD, it is effective in the treatment of
urinary tract inflammatory diseases in men (Marberger et al., 1974). For a time, bovine
liver SOD even had regulatory approval in several European countries for such use. This
was
truncated,
apparently
by
concerns
about prion
disease.
An SOD-
mimetic agent, TEMPOL, is currently in clinical trials for radioprotection and to prevent
radiation-induced hair-loss. TEMPOL and similar SOD-mimetic nitroxides exhibit a
multiplicity of actions in diseases involving oxidative stress. It has been seen that Cysteine
residues play a key role in Cu-Zn SODs to understand toxicity in Caenorhabditis
elegans model of amyotrophic lateral sclerosis (Ogawa et al., 2015).
2.3.7 SOD in therapeutics
The greatest excitement in recent years in the area of free radical biology research has been
focused on the pathological production of oxygen- derived free radicals, and on the possibilities
of anti-radical and anti-oxidant approaches to therapy and prevention of diseases. There are large
numbers of disease caused due to over production of free radicals or Reactive Oxygen species
(ROS). Superoxide dismutase (SOD) has played an invaluable role in exploring the mechanisms
of disease by virtue of its absolute specificity for its substrate; due to the fact that it is an enzyme,
whereas the non-enzymatic radicals interfere in any other number of processes. SODs are used
for preparation of many pharmaceutical compositions for treatment of many diseases including
myocardial ischemia (Emerit et al., 2006; Downey et al., 1991) multiple sclerosis (Namaki et al.,
2009), colitis and in improving a clinical irradiation treatment of malignant diseases such as
breast cancer (Pajovic et al., 2003). Superoxide dismutase mimetics (Warner et al., 2004) such as
tempol has also beneficial effects in several experimental models of hypertension and acute
kidney injury (Quiroz et al., 2009). SOD mimetics have also been reported to extend the mean
life-span of the wild-type worm Caenorhabditis elegans by a mean of 67 percent increase
(Melov et al., 2000). SOD in its pharmaceutical form "Orgotein" is a potent anti-inflammatory
agent approved in many countries, and because of their effectiveness in the general treatment of
inflammatory and degenerative diseases, researchers are constantly looking at SODs and related
agents in experimental animal models of disease (Marberger et al., 1974). Numerous studies
have proved the safety of superoxide dismutase drugs in animals and man and studies prove
SOD as a strong anti-inflammatory agent (Zhang et al., 2002). Apart from invaluable health
benefits, significant contributions towards various diseases are discussed below.

Cardiovascular disorder
Atherosclerosis is the root cause of heart attacks, strokes, and peripheral vascular disease all
together known as "cardiovascular diseases". Atherosclerosis development depends on the
balance
between
proinflammatory;
anti
inflammatory,
and
antioxidative
defense
mechanisms.The significant role of oxygen-derived free radicals in myocardial reperfusion
injury is very well documented (Bolli et al., 1988). The involvement of SOD in lipid
peroxidation was analyzed using reperfused heart. The toxicity of high-dose superoxide
dismutase suggests that superoxide can both initiate and terminate lipid peroxidation (Nelson et
al., 1994). Role of SOD, oxidants and free radicals in reperfusion injury (a unique form of
myocardial damage) has studied (Zweier and Talukder et al., 2006).

Neurological disorders
As we are aware of the fact that Superoxide dismutase enzyme, plays an important role in
protecting tissues from superoxide anions, which are normally produced in all aerobic cells. In
brain tissue, aerobic metabolism increases with maturation and an increased level of SOD for
anti-oxidant defense is therefore required. Animal experiments demonstrate that the activity of
SOD in brain remains at a constant low level during fetal life and increases in the postnatal
period (Mavelli et al., 1982). Various strategies have been carried out in order to understand the
mechanisms involved in central nervous system. (Shukla, 1984). Free radical injury plays a
significant role in traumatic brain injury and SOD activity has been visualized in rat brain by
histochemical localization (Okabe et al., 1998).

Diabetes
Oxidative stress plays an important role in the development of diabetes complications, both
microvascular and cardiovascular. The outcome and progression of diabetic retinopathy is highly
influenced by SOD and its overexpression in transgenic diabetic mice prevents diabetic
retinopathy, nephropathy, and cardiomyopathy (Giacco and Michael 2010).

Aging
All cells are hypothesized to originate from stem cells. These stem cells mature as they divide
and eventually reach a fully differentiated cell, which cannot divide. Aging is caused by the loss
of stem cells, either due to cell death or terminal differentiation, or by eventual death of fully
differentiated cells; both brought about by oxygen radicals. Young people naturally produce the
antioxidant enzymes superoxide dismutase (SOD) and catalase as a defence against destructive
free radicals. Unfortunately, the levels of catalase and SOD continuously decline with age.
Recent research suggests that SOD boosts immunity, quenches the free radicals and promotes
longevity. Over the past 60 years, numerous studies have been performed to demonstrate the role
of SOD and its contribution to aging. SOD serves as an important antioxidant defense
mechanism in organisms, and any deletion or alteration of these genes shortens the life span as
proved in experiments on budding yeast Saccharomyces cerevisiae (Muid et al., 2014).

Hepatopathololical diseases
Many hepatic disorders are related to free radical formation and Superoxide dismutase, plays an
important role in curing various hepatopathological disorders. Liver damage due to alcoholinduced liver injury is associated with an increase in oxidants from a variety of possible sources;
SOD is used as a potential anti-fibrotic drug for hepatitis C related fibrosis (Emerit et al., 2006).
Superoxide dismutase extracted from fruits and vegetables has been potentially used for
curing various diseases. It has been found that an SOD rich melon extract “Extramel”
prevents aortic lipids and liver steatosis in diet-induced model of atherosclerosis. The
findings support the view that good quantity intake of melon juice extract rich in SOD has
potential beneficial effects for the treatment of
atherosclerosis and liver steatosis,
emphasizing its use as potential dietary therapy (Intes et al., 2012).

Renal disorders
Kidney is the only organ in human body which excretes toxins and harmful wastes and plays an
important role in filtration of body. Superoxide dismutase activity was investigated in Human
Glomerulonephritis. The study showed that renal function deterioration is directly proportional to
SOD activity in patients. Lower level of SOD activity, a decreases the scavenger reaction of
superoxide, thus making the tissue more prone to oxidative stress (Kashem et al., 1996). Novel
approaches to therapy have been explored including SOD gene therapy and SOD targeting to the
lung. In the future, new drugs interacting with superoxide may provide significant advances in
the treatment of lung diseases (Demiryurek et al., 1999). Radiation-mediated lung injury is one
of the limiting factors for radiation therapy. SOD-TAT is a fusion protein of hCuZn-superoxide
dismutase (SOD) and HIV-1 Tat protein transduction domain, it is useful in the prevention and
treatment of skin damage of guinea pigs by UVB radiation (Pan et al., 2012).

Arthritis
Arthritis can be cured and its potential is analyzed by using various animal models. Study was
performed by a rat model of arthritis. Evaluation of the therapeutic efficacy was performed by
SOD transdermal delivery using Tfs. The amelioration of disease symptoms on animal treated
with SOD–Tfs showed that epicutaneous application of SOD in especially developed mixed lipid
vesicles play a significant role in the reduction of inflammation, in the adjuvant arthritis model
(Simoes et al., 2005).

Inflammatory diseases
SOD potentially decreases ROS and oxidative stress related diseases by interrupting the
inflammatory cascades and scavenging free radicals (Riedl et al., 2005). It is highly effective in
treatment of colonic inflammation and to generate new therapies for the treatment
of inflammatory bowel diseases (Bhattacharjee et al., 2015). It possesses steroid-like antiinflammatory activity; repeatedly injecting low doses of SOD maintained the suppressed state of
edema (Oyanagui et al., 1998). Liposomal-encapsulated SOD injections were used to treat
systemic inflammatory diseases and skin ulcer lesions (Niwa et al., 1989). Catalase and
superoxide dismutase mimics (Conjugation of a metal ion chelator to aromatic amino acids
generates a series of novel metal-binding anti-oxidant enzyme) have been used for the treatment
of inflammatory diseases (Fisher et al., 2003).

Cancer
SOD is the key enzyme in the first metabolic step in elimination of superoxide, deficiency in
SOD or inhibition of the enzyme activity causes severe accumulation of O2 • - in cells and leads
to cell death. Studies reveal that SOD treatments of rodents before and after irradiation result in
increased survival and cellularity of the bone marrow and reduced skin reactions, pneumonitis
and pulmonary fibrosis (Schwartz et al., 1996). Targeting SOD is a most reliable approach
towards the selective killing of cancer cells. Studies have been performed using rat glioma cell
line in culture to understand the change of superoxide dismutase (SOD) in tumor cells by
hypoxia and hypoxia-normoxia exposure. The results obtained suggest that SOD in glioma cells
can be activated to compensate the damage from free radicals during hypoxic stress (Chang et
al., 1997).

SOD as a future gene therapy and Anti-Cancer Agent
Gene therapy is a novel therapeutic branch of modern medicine. It emerged during introduction
of recombinant DNA methodology in the 1970s. Many reports showed that SOD gene therapy
has been shown to attenuate tissue damages and improve the recovery of the tissue injuries
(Laurila et al., 2009); to provide the heart with protection against myocardial stunning (Li et al.,
1998); to ameliorate delayed diabetic wound healing (Luo et al.,
2004); to stent-induced
vascular injury (Brasen et al., 2007); for the attenuation of ischemia-reperfusion injury of testes
in rats (Palffy et al.,
2006); to attenuates ischemia-reperfusion injury in the rat kidney if
delivered to the kidney by intravenous injection (Yin et al., 2001). Reports demonstrate that
MnSOD gene therapy can be used in combination with other therapies as well, for example with
chemotherapy it can be used as dual therapy achieving synergetic action in suppressing the
malignant growth of colorectal cancer (Zhang et al., 2006); another example is in the rabbit
model of vein graft disease, combination extracellular SOD gene therapy, anti-inflammatory, and
anti-proliferative genes was found to be effective in decreasing neointimal formation (Turunen et
al., 2006).
Gene therapy is the insertion of genes into an individual's cells and tissues to treat disease, such
as cancer (Curiel and Douglas, 2004; Schaffer and Zhou, 2009), where deleterious mutant alleles
are replaced with functional ones. Although the technology of gene therapy is still in its infancy,
it has been used with some success. Nowadays, in vivo gene therapy is considered as an
attractive therapy for the treatment of many diseases (Panno, 2004; Herzog, 2010). Gene therapy
may be classified into the two following types: the first is the germ line gene therapy: in this
case, germ cells, i.e., sperms or eggs, are modified by the introduction of functional genes, which
are ordinarily integrated into their genomes. Therefore, the change due to therapy would be
heritable and would be passed on to later generations. This new approach, theoretically, should
be highly effective in counteracting genetic disorders and hereditary diseases. However, many
jurisdictions prohibit this for application in human beings, at least for the present, for a variety of
technical and ethical reasons. The second is the somatic gene therapy: in this case, the
therapeutic genes are transferred into the somatic cells of a patient. Any modifications and
effects will be restricted to the individual patient only, and will not be inherited by the patient's
offspring or later generations (Templeton, 2008). Viral-mediated gene-delivery systems are used
as tools in gene therapy (Curiel and Douglas, 2002). Some types of viruses physically insert their
genes into the host's genome, thus they were employed in gene therapeutic uses, such as
retroviruses, adenoviruses, adeno-associated viruses (from the parvovirus family), envelope
protein pseudo-typing of viral vectors, replicating vectors, cis and trans-acting elements, and
herpes simplex viruses. Besides virus-mediated gene-delivery systems, there are several nonviral options for gene delivery (Schaffer and Zhou, 2009). The simplest method is the direct
introduction of therapeutic DNA into target cells. This approach is limited in itsapplication
because it can be used only with certain tissues and requires large amounts of DNA. Another
non-viral approach involves the creation of an artificial lipid sphere with an aqueous core (the
liposome). This liposome, which carries the therapeutic DNA, is capable of passing the DNA
through the target cell's membrane. Researchers also are experimenting with introducing a 47th
(artificial human chromosome) into target cells. A problem with this potential method is the
difficulty in delivering such a large molecule to the nucleus of a target cell. Due to presence of
shortcomings for each gene transfer method, some hybrid methods developed combining two or
more techniques; virosomes are an example of combining liposomes with an inactivated HIV or
influenza virus. Gene therapeutic strategies were reported to be used successfully in reduction of
symptoms generated by radiotherapy. The total allowable radiotherapeutic dose is always limited
by the potential for developing irradiation induced cystitis (Schellhammer et al., 1986).
Successful reduction and protection against irradiation therapy side effects such as cystitis was
reported using manganese superoxide dismutase gene therapy. Induction of manganese
superoxide dismutase by gene therapy using plasmid/liposome complex (MnSOD-PL) contained
the complete human manganese SOD (MnSOD) transgene did not prevent disruption of barrier
function by irradiation but led to rapid regeneration of the urothelium and recovery of barrier
function. It is also reported that overexpression of a transgene for human (MnSOD-PL) delivered
by plasmid-liposome, or adenovirus to the mice lungs, before irradiation exposure, demonstrated
to decrease the late effects of whole lung irradiation (Epperly, 2002). It is also found that
inhalation radioprotective gene therapy using (MnSOD-PL) provided a practical and effective
method for delivery of lung-specific radioprotection during fractionated radiotherapy protocols
(Carpenter et al., 2005). Radioprotective gene therapy through retroviral expression of
manganese superoxide dismutase may be applicable to the haematopoietic compartment,
enabling radiation dose escalation in cancer therapy (Southgate et al., 2006).Ionizing irradiationinduced murine mucosal cell cycling and apoptosis was found to be decreased using intraoral
(MnSOD-PL) radioprotective gene therapy (Epperly et al., 2004). Previous data provided a
rational basis for the design of gene therapy approaches to organ protection from irradiation
damage. Many reports showed that SOD gene therapy has been shown to attenuate tissue
damages and improve the recovery of the tissue injuries (Laurila et al., 2009); to provide the
heart with substantial protection against myocardial stunning without the need for concomitant
administration of catalase (Li et al., 1998); to ameliorate delayed diabetic wound healing (Luo et
al., 2004) ; to stent-induced vascular injury (Brasen et al., 2007); to attenuates ischemiareperfusion injury of testes in (Palffy et al., 2006) rats ; to attenuates ischemia-reperfusion injury
in the rat kidney if delivered to the kidney by intravenous injection (Yin et al., 2001); to reduce
portal pressure in CCl4 cirrhotic rats with portal hypertension (Lavina et al., 2009); to reduce
restenosis and may be useful for the prevention of intimal hyperplasia after vascular
manipulations (Laukkanen et al., 2002); and to reduce oxidative stress in erectile dysfunction
(Deng et al., 2010). Results also suggested that if MnSOD gene could be effectively delivered to
a tumor in vivo using the adenovirus paradigm, effective tumor growth suppression could be
observed in hamster (Lam et al., 2006). Superoxide dismutases has been very well known to
play an essential role in stress-tolerance of higher plants. A full-length cDNA encoding Cu/Zn
SOD (BcCSD1) was isolated from young seedlings of non-heading Chinese cabbage (Brassica
campestris ssp. chinensis) by rapid amplification of cDNA ends (RACE).The study focuses to
provide useful information for further understanding the role of BcCSD1 resistant to abiotic
stress in Brassica campestris in the future (Cui et al., 2015).
Reports also demonstrated that MnSOD gene therapy could be applied in combination with other
therapies, for example with chemotherapy as a dual therapy achieving synergetic action in
suppressing the tumor growth of colorectal cancer (Zhang et al., 2006); another example is in the
rabbit model of vein graft disease, combination extracellular SOD gene therapy,
antiinflammatory, and antiproliferative genes was found to be effective in decreasing neointimal
formation (Turunnen et al., 2006).
Thus, SOD mimics have been shown to exert anti-cancer effects through pathways that appear
different from the action of common anticancer drugs that target critical cellular proteins.
Different and opposing explanations have been offered for both enzymes and their mimics: antioxidant vs. pro-oxidant actions have been suggested. All these studies indicate the immense
importance of SOD in various sectors with great future prospects as lead candidate in pharma
sector.