Our Chapter

Microbial superoxide dismutase enzyme as therapeutic agent and future
gene therapy
Hatem M. El Shafey 1*, Saleh A. Bahashwan2, Abdulaziz A. Alghaithy3 and Samah Ghanem3,4
1
Munawara, Kingdom of Saudi Arabia.
Microbiology Department, National Center for Radiation Research and Technology, P.O. Box 29, Nasr City, Cairo, Egypt. Medical Laboratory Department, Faculty of Health Sciences, Taibah University, P.O. Box 3893 Al Madinah Al
2
Kingdom of Saudi Arabia. Pharmacology Department, Faculty of Health Sciences, Taibah University, P.O. Box 3893 Al Madinah Al Munawara,
3
Madinah Al-Munawara, Kingdom of Saudi Arabia. Medical Laboratory Technology Department, Faculty of Applied Medical Sciences, Taibah University, P.O. Box 344 Al-
4
*Corresponding author: e-mail: hatem.elshafey@laposte.net, Phone: +2 0102894001
All aerobically growing organisms come into contact with reactive oxygen species or free radicals, generated as a
byproduct of normal respiratory processes or from encounter with exogenous oxidants. To counter the damaging effect of
reactive oxygen species, cells have evolved anti-oxidant defense systems, whose expression is usually induced by reactive
oxygen species and/or oxidants. One of the most important antioxidant defense systems in nearly all cells exposed to
oxygen is the production of superoxide dismutase enzyme. Superoxide dismutases discovered by Irwin Fridovich and Joe
McCord, are class of metal cofactored enzymes that detoxify these free radicals by catalyzing the dismutation of
superoxide into oxygen and hydrogen peroxide. Seen to their continuous exposure to high oxidative stress during growth
and metabolism, aerobic microorganisms represent an excellent source for production of superoxide dismutases. Many
studies clarified the efficacy of superoxide dismutases as a therapy in the treatment of many diseases, in addition to their
inclusion in cosmetic products to reduce free radical damage to skin. Recently, superoxide dismutases became target for
gene therapeutic approaches. In the present chapter, the history of the discovery of superoxide dismutase enzymes, their
types, structure, therapeutic and cosmetic uses, in addition to their therapeutic potential in future gene therapy, have been
reviewed and discussed. Microbiology Department, Faculty of Science, Helwan University, Post code 11795, Ain Helwan, Helwan, Egypt.
Keywords superoxide dismutase; microorganisms; gene therapy
1. Introduction
An inevitable consequence of life in an oxygen-rich environment is the formation of reactive oxygen species (ROS),
which are unavoidable by-products of aerobic metabolism. ROS produced from metabolic events, such as respiration
(as a consequence of the incomplete reduction of oxygen) or fatty acid oxidation or due to environmental processes.
Oxidative stress occurs when the rate of generation of reactive compounds exceeds the detoxification capacity of the
cell [1]. If not disposed off efficiently, ROS can cause cellular and genetic damage leading to carcinogenesis,
senescence, and neurodegenerative disorders [2, 3]. Because cells under oxidative stress are at risk for lethal or
mutagenic damage, all aerobic organisms have evolved defense mechanisms to cope with ROS. Part of this response
involves the reprogramming of gene expression to increase levels of antioxidant enzymes such as superoxide dismutase
and catalase, which limit the levels of superoxide (O 2˙-) and hydrogen peroxide (H2O2), respectively.
2. Reactive oxygen species (ROS)
Molecular oxygen (O
oxygen increases when it accepts one, two, or three electrons to form superoxide anions (O
(H
known as reactive oxygen species (ROS). Reactive oxygen species (ROS) is a term which encompasses all highly
reactive, oxygen-containing molecules, including free radicals. Types of ROS include the hydroxyl radical, the
superoxide anion radical, hydrogen peroxide, singlet oxygen, nitric oxide radical, hypochlorite radical, and various lipid
peroxides. All are capable of reacting with membrane lipids, nucleic acids, proteins and enzymes, and other small
molecules, resulting in cellular damage [5]. ROS are generated by exposure to radiation, heavy metals, and redox active
compounds, and also occur under normal metabolism [6].
enzymatic cascades and pathological processes in many diseases by inducing oxidative stress [
causes widespread damage to DNA, RNA, protein, and lipids when the degree of oxidative stress exceeds the capacity
of the cell defense systems. There is growing evidence that ROS are important for the induction of apoptosis (the
process of programmed cell death: PCD) [9]. There is now universal agreement that free radicals are involved in the
physical, biochemical, and pathological changes associated with aging [10]. And there is growing evidence that free 2) is not reactive with most compounds due to its two unpaired electrons [4]. The activity of2˙-), hydrogen peroxide2O2), and the hydroxyl radicals (OH-), respectively. These mediators of oxidative stress (O2˙-, H2O2, and OH-) areROS affect normal cell functions by activating a number of7, 8]. Oxygen toxicity
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radicals are involved in the initiation of cellular injury observed in neurodegenerative diseases such as: Alzheimers’s
disease (AD), Huntington’s disease (HD), Parkinson’s disease (PD) [11]. In addition, ROS can act in all stages of
carcinogenesis leading to cancer development [12]. There is also increasing evidence that ROS plays a significant role
in the development of diabetes. To counter the damaging effect of reactive oxygen species, cells have evolved antioxidant
defense systems, whose expression is usually induced by reactive oxygen species and/or oxidants [13].
3. Antioxidants
The term “antioxidant” refers to any molecule capable of stabilizing or deactivating free radicals before they attack
cells. Humans have evolved highly complex antioxidant systems (enzymic and nonenzymic), which work
synergistically, and in combination with each other to protect the cells and organ systems of the body against free
radical damage. The antioxidants exist endogenously or obtained exogenously e.g., as a part of a diet or as dietary
supplements. Some dietary compounds that do not neutralize free radicals, but enhance endogenous activity may also be
classified as antioxidants [10]. Non-enzymatic antioxidants include uric acid, glutathione, bilirubin, thiols (glutathione,
thioredoxin and lipoic acid), albumin, melatonin, carotenoids, natural flavonoids and other compounds and nutritional
factors including vitamins such as E and C and phenols [14]. Enzymatic antioxidants include the superoxide dismutases,
the glutathione peroxidases [GSHPx] and catalase [15]. The most important source of antioxidants is provided by
nutrition (Exogenous antioxidant); many belonging to the phenol family. Nutritional antioxidants act through different
mechanisms and in different compartments, but are mainly free radical scavengers: 1) they directly neutralize free
radicals, 2) they reduce the peroxide concentrations and repair oxidized membranes, 3) they quench iron to decrease
reactive oxygen species production, 4) via lipid metabolism, short-chain free fatty acids and cholesteryl esters neutralize
reactive oxygen species [16].
4. The history of discovery of superoxide dismutase
Superoxide dismutases (SOD, EC 1.15.1.1) discovered by American biochemist Irwin Fridovich and his graduate
student Joe McCord in 1969 [17]. Interestingly, the original paper was cited 39 times in just the year 2009 - forty years
after original publication. PubMed lists over 49,000 papers published on superoxide dismutase since its discovery in
1965. Few other biochemists have had such an impact on the field of biochemistry. SOD has been found in most
organisms, aerobic and anaerobic, and plays a key role in cellular protection against oxidative stress conditions [18].
5. Function of superoxide dismutase
SODs enzymes were previously thought to be several metalloproteins with unknown function (for example, CuZnSOD
was known as erythrocuprein) [19]. But now it is proven that SOD catalyzes the conversion of superoxide anions to
dioxygen and hydrogen peroxide; the latter being broken in turn to water by catalase or peroxidase. SOD neutralizes
superoxide ions by going through successive oxidative and reductive cycles of transition metal ions at its active site
[20].
6. Sources of SODs
Most organisms, microorganisms, plants and animals have at least one superoxide dismutase enzyme [21]. While one of
the exceedingly rare exceptions is
[22 ,23]. 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 [24]. SODs were also produced efficiently by many
microbial species [25, 26]. Seen to their continuous exposure to high oxidative stress during growth and metabolism,
aerobic microorganisms represent an excellent source for production of superoxide dismutases.
Of many aerobic microorganisms considered as a potent source of superoxide dismutase,
glutamicum
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 [27, 28]. Cloning techniques reported
to be used successfully with many corynebacterial genes [29, 30, 31, 32]. 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. Lactobacillus plantarum and related lactobacilli, which use a different mechanismCorynebacterium, an industrial relevant producer of amino acids and vitamins, is considered as an excellent candidate for this
7. Types of SODs
SODs are distinguished into several types depending on the type of metal cofactors.
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(1) SOD containing either manganese (Mn-SOD) or iron (Fe-SOD) has been found in the human mitochondria in
addition to the cytoplasm of prokaryotic cells and have very similar sequences and structures [33, 34, 35, 27, 28].
(2) Copper and zinc-containing SOD (Cu,Zn-SOD) has been described in humans as cytosolic and extracellular
SOD. Until relatively recently, Cu,Zn-SOD was considered to be an almost exclusively eukaryotic enzyme, and its
presence in bacteria, originally identified in a very small number of microorganisms, was thought to be an exception
rather than a rule [36, 26]. Cu,Zn-SOD has been found in the periplasm of several Gram-negative pathogenic and
endosymbiotic bacteria and are evolutionarily unrelated to Mn,Fe-SODs [37, 38, 39, 40, 36, 41, 35].
(3) In addition, two novel SODs containing nickel (Ni-SOD) or both iron and zinc (FeZn-SOD) have been
characterized as cytoplasmic enzymes of
small metalloprotein with superoxide dismutase activity, unrelated to classical SODs, has also been recently identified
in the sulphate-reducing bacterium Streptomyces griseus and Streptomyces coelicolor [42, 43]. Another type ofDesulfovibrio [44].
8. Structure of SODs
In humans (as in all other mammals and most chordates), three forms of superoxide dismutase are present. SOD1,
SOD2 and SOD3.
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.
SOD2: It is the mitochondrial SOD having the Mn (manganese) in its reactive centre. Mn-SOD is a homotetramer
with a molecular weight of 96 kDa and contains one manganese atom per subunit [15], and it cycles from Mn(III) to
Mn(II), and back to Mn(III) during the two-step dismutation of superoxide.
SOD3: Extracellular superoxide dismutase contains Cu, Zn (copper and zinc), and is a tetramer (consists of four
subunits) [15].
Fig. 1. Structure of the monomeric unit of Mn-human mitochondrial superoxide dismutase (SOD2) [45].
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Fig. 2. Structure of the active site of Mn-human mitochondrial superoxide dismutase (SOD2) [45].
9. SOD as a pharmaceutical product
Superoxide dismutase, by scavenging of free oxygen radicals, might interrupt inflammatory cascades and thereby limit
further disease progression [46]. Numerous studies have established safety of superoxide dismutases drugs in animals
and man. Many studies reported using superoxide dismutases as an anti-inflammatory agent [47]. Superoxide
dismutases were reported for the treatment of not only systemic inflammatory diseases but also skin ulcer lesions,
especially due to burn and wounds, where liposomal-encapsulated SOD injection was effective [48]. Besides direct
inhibition of joint tissue destruction, the mechanism of action of SOD in reducing the severity of arthritic inflammation
includes limitation of existing positive feedback between secretion of ROS and inflammatory cytokine production [49].
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. In the course of such studies,
researchers discovered that SODs stimulate hair growth and decrease hair loss [50], ameliorate radiation-induced side
effects
for breast cancer [53, 54, 55] using Cu/Zn superoxide dismutase where no other effective therapy exists [56]. SOD
might be proposed as a potent antagonist of this major fibrogenic growth factor [53], and as a potential antifibrotic drug
for hepatitis C related fibrosis [57]. In addition SODs are used for preparation of many pharmaceutical compositions for
treatment of many diseases including myocardial ischemia [57, 58], Peyronie's Disease [46], multiple sclerosis [59],
colitis [60]; and in improving a clinical irradiation treatment of malignant diseases such as breast cancer [61]. There is
also increasing evidence that radical scavengers like superoxide dismutase may influence the outcome and progression
of diabetic retinopathy [62]. Dramatic improvement of clinical active Behcet's syndrome symptoms treated by CuZn
SOD was also reported [63].
Superoxide dismutase mimetics [64] such as tempol has also a beneficial effects in several experimental models of
hypertension and acute kidney injury [65]; while sodium thiopental and propofol are used to treat reperfusion injury and
traumatic brain injury [66]. Superoxide dismutase mimetics have also been reported to extent the mean life-span of the
wild-type worm
The topical application of SOD cream was concluded to be effective for rapidly improving of many cases such as
progressive systemic sclerosis (PSS), systemic lupus erythematosus (SLE), Behcet's disease, herpes simplex and burns,
even when the symptoms were stabilized for several weeks before the treatment [68]. Wound excision in conjunction
with SOD-treatment might be therapeutic in the management of severe burns suggesting that superoxide radicals may
play a critical role in the pathogenesis following thermal injury [69, 70]. [51] such as radiation-induced sclerosis [52] and radiation-induced fibrosis (radiofibrosis) following irradiationCaenorhabditis elegans by a mean of 67 percent increase [67].
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10. SOD as a future gene therapy
Gene therapy is the insertion of genes into an individual's cells and tissues to treat disease, such as cancer [71, 72],
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 [73, 74].
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 [75].
Viral-mediated gene-delivery systems are used as tools in gene therapy [76]. 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 non-viral options for gene delivery [77]. The
simplest method is the direct introduction of therapeutic DNA into target cells. This approach is limited in its
application 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 irradiationinduced
cystitis [78]. 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