Role of free radicals in ocular diseases: An overview

 

Preeti K. Suresh*, Abhishek K. Sah and Sanjay J. Daharwal

University Institute of Pharmacy, Pt. Ravishankar Shukla University, Raipur – 492010 (C.G.) India

 

ABSTRACT:

The aim of the present review is to focus on the potential consequences of free radicals in human health and disease with special reference to ocular disorders. The balance between the free radicals and antioxidants is crucial for good health. The risk of free radicals in health has been increasing gradually. Many of the degenerative disease like atherosclerosis, cancer inflammatory joint disease, asthma, diabetes, dementia and degenerative eye disorder are the result of imbalance of free radicals and antioxidants. In case of eye, free radicals are responsible for the pathogenesis of the majority of serious ocular diseases such as cataract, primary open angle glaucoma and age-related macular de­generation. The process of biological ageing might also have a free radical basis. Most free radical damage to cells involves oxygen free radicals or reactive oxygen species (ROS) which include non-radical species such as singlet oxygen and hydrogen peroxide as well as free radicals. The ROS can damage genetic material, cause lipid peroxidation in cell membranes and inactivate membrane-bound enzymes. Humans are well endowed with antioxidant defenses against ROS. These antioxidants include ascorbic acid (vitamin C), α-tocopherol (vitamin E), β-carotene, coenzyme Q10, enzymes such as catalase and superoxide dismutase, and trace elements including selenium and zinc. This review is an attempt to compile and document the information on various implications of free radicals in the ocular disorders.

 

 

 

 

INTRODUCTION:

In the present scenario there has been growing interest in deciphering the impact of oxidative reactions in human disease. Oxygen free radicals play major role in many physiological and pathological conditions associated with human health and disease including age related diseases as a biological phenomenon. Various cellular functions are altered by the release of oxygen free radicals, like DNA expression¹ and mitochondrial energy production². The over production of free radicals or an impairment of the oxidative/antioxidative balance, has been reported to be involved in the cell differentiation process, activation of specific metabolic pathways, and liver regeneration^3-5.

 

A free radical is any atom or molecule that has a single unpaired electron in an outer shell⁶ whereas normally electrons exist in pairs in specific orbitals in atoms or molecules⁷. It includes molecular oxygen, since ground-state oxygen has two unpaired electrons. The free radical nature of oxygen renders it high reactivity. The rates of oxygen reactions are extremely slow, even though they are thermodynamically favorable. A free radical procures electrons from other free radicals or molecules like reducing agents, so as to 'pair up' its unpaired electrons. Free radicals include oxygen, superoxide radical, some transition metal ions such as iron (II), copper (II), carbon and sulphur centred species^8,9.

 

Development of free radicals in the human

The free radicals along with reactive oxygen species (ROS) are developed from normal metabolic processes in the human body associated with external sources like exposure to ultra- violet and x-rays, ozone, smoking, air pollution and chemicals from industries¹⁰. Free radicals are generated continuously in the cells as a result of both enzymatic and non-enzymatic reactions. Various enzymatic reactions acting as a precursor of free radicals, include those involved in the respiratory chain, phagocytosis, prostaglandin synthesis and in the cytochrome P-450 system¹¹. Non-enzymatic reactions of oxygen with organic compounds along with ionizing reactions can generate free radicals. Sources for internal generation of free radicals include mitochondria, xanthine oxidase, peroxisomes, inflammation, phagocytosis, arachidonate pathways, exercise, ischemia/ reperfusion injury. Some externally generated sources of free radicals include cigarette smoke, environmental pollution, radiation, drugs, pesticides, industrial chemicals and ozone¹².

 

Mechanism for the formation of free radicals

A radical might donate its unpaired electron to other molecule. It might take electron from other molecule in order to pair or it might simply join to the molecule. A radical gives one electron or takes one electron or simply adds on to the anion to become a radical. Thus the future of the reactions that usually proceed as chain reaction is such that one radical begets another¹³.

 

Free radicals can be formed by three ways –

1. By homolytic cleavage of covalent bond of normal molecule, with each fragment retaining one of paired electrons:

                A : B                                       A* + B*

2. By the loss of single electron from normal molecule:

                A : B                                       A⁺ + B^-

3. By addition of single electron to normal molecule:

                A + ^e-                                      A

 

 

Fig 1: Internal sources of free radical generation¹²

 

Fig 2: External sources of free radical generation¹²  

 

TYPES OF FREE RADICALS AND THEIR DERIVATIVES IN LIVING ORGANISMS:

A. Reactive Oxygen Species:

ROS develop when superoxide anion is generated due to univalent reduction of triplet-state molecular oxygen (³O₂). These steps are directed by enzymes such as NADPH oxidases along with xanthine oxidase or nonenzymically by redoxreactive compounds such as compound of semi-ubiquinone in mitochondrial electron transport chain (Fig. 3). Superoxide dismutase convert superoxide enzymically into hydrogen peroxide^14,15. In tissues, superoxide is converted nonenzymically into the nonradical species of hydrogen peroxide and singlet oxygen (¹O₂) ¹⁶. Due to availability of reduced transition metals (like ferrous, cuprous ions), hydrogen peroxide can be converted directly into the highly reactive hydroxyl radical (^.OH) ². Hydrogen peroxide may be converted into water by the enzymes catalase or glutathione peroxidase (Fig. 3). In glutathione peroxidase reaction glutathione is oxidized to glutathione disulfide, which can be converted back to glutathione by glutathione reductase in an NADPH-consuming process. Superoxide and NO are converted by enzymes or nonenzymic chemical reactions into reactive nonradical species such as singlet oxygen (¹O₂), hydrogen peroxide, or peroxynitrite (ONOO^-), i.e., species which can in turn give rise to new radicals. Most of the regulatory effects are certainly not directly mediated by superoxide but slightly by its reactive oxygen species (ROS) derivatives. Frequently, different reactive species coexist in the reactive environment and make it difficult to identify unequivocally which agent is responsible for a given biological effect.

 

Hydroperoxyl radical¹⁷:

It is also known as the perhydroxyl radical, and is the protonated form of superoxide with the chemical formula HO₂. Hydroperoxyl is formed through the transfer of a proton to an oxygen atom. HO₂ can act as an oxidant in a number of biologically important reactions, such as the abstraction of hydrogen atoms from tocopherol and polyunstaturated fatty acids in the lipid bilayer. As such, it may be an important initiator of lipid peroxidation.

 

Superoxide: Superoxide can act either as oxidant or reductant, it can oxidize sulphur, ascorbic acid or NADPH and it can reduce Cytochrome C and metal ions. A dismutation reaction leading to the formation of hydrogen peroxide and oxygen can occur spontaneously or is catalyzed by enzyme superoxide dismutase. In its protonated form (pKa 4.8) superoxide forms, because of its low concentration at physiological pH.

 

Hydrogen peroxide: The univalent reduction of superoxide produces hydrogen peroxide, which is not a free radical since all its electrons are paired. It readily permeates through the membranes and is therefore not compartmentalized in the cell. The main damages caused by this event are breaking up of DNA, resulting in single strand breaks and formation of DNA protein crosslink. Numerous enzymes (peroxidases) use hydrogen peroxide as a substrate in oxidation reactions involving the synthesis of complex organic molecule. This is an oxidizing agent but not specially reactive and its main significance lies in it being a source of hydroxyl radical in the presence of reactive transition metal ions.

 

Singlet oxygen: It is not a free radical but it can be formed in some radical reactions and can trigger off others. This arises from hydrogen peroxide molecules. Singlet oxygen on decomposition generates superoxide and hydroxyl radicals.

 

Triplet oxygen: Triplet oxygen can react with elements and ions to form oxides, but usually not with organic compounds, which are in singlet state. However, it reacts easily with free radical molecules produced by the action of other active radicals, radiations, ultra violet light, and heat or by complex formation with oxygen and transition metal to produce active peroxide radicals and trigger auto-oxidation of unsaturated fatty acids and others.       

 

B. Reactive Nitrogen Species

The nitrous oxide radical (NO^.) is produced by the oxidation of one of the terminal guanido-nitrogen atoms of L-arginine¹⁸. This reactive process is catalyzed by the enzyme nitric oxide synthase (NOS). Depending on the microenvironment, NO can be converted to different other reactive nitrogen species (RNS) such as nitrosonium cation (NO⁺), nitroxyl anion (NO^-) or peroxynitrite (ONOO^-)¹⁹. Some of the physiological effects may be mediated through the intermediate formation of S-nitroso-cysteine or S-nitroso-glutathione²⁰.

 

Fig 3: Pathways of reactive oxygen species (ROS) production and clearance.

(GSH, glutathione; GSSG, glutathione disulfide).

 

Fig 4: Different types of free radicals.

 

Table 1: Free radicals and their physiological functions

 

S. No.	Type of free radical	Source	Physiological function
1	Nitric oxide (NO˙)	Nitric oxide synthase	Smooth muscle relaxation and variousother cGMP dependent functions.
2	Superoxide (O2ˉ˙) and related ROS	NAD(P)H oxide	Control of ventilation, control of erythropoietin production and other hypoxia induced functions, smooth  muscle relaxation, Single transduction  from various membrane receptors and   enhancement of immunological function.
3	Superoxide (O2ˉ˙) and related ROS	Any source	Oxidative stress responses and homeostasis maintenance of redox

 

 

Most biologically-relevant free radicals are highly reactive. While a few free radicals such as melanin are not chemically reactive²¹, for most biological structures, free radical damage is closely associated with oxidative damage. Most free radical damage to cells involves oxygen free radicals and metabolites with chemically reactive, oxygen-containing groups. The eye is an organ with intense reactive oxygen species (ROS) activity, and it requires high levels of antioxidants to protect its unsaturated fatty acids²². Humans have an array of mechanisms that attempt to maintain the individual in a state of pro-oxidant-antioxidant balance. These antioxidants include ascorbic acid (vitamin C), α-tocopherol (vitamin E), β-carotene, coenzyme Q10, enzymes such as catalase and superoxide dismutase, and trace elements including selenium and zinc²³.

 

Fig 5: Various types of reactive oxygen species (ROS)

 

Fig 6: Free radicals, antioxidants, and macular degeneration²⁴

 

Fig 6: Role of antioxidants in stabilization of free radical

 

An imbalance between oxidants and antioxidants with respect to oxidants (oxidative stress) causes cell death and tissue damage and may potenti­ate ageing process. Oxidative stress is characterized by a redox imbalance between the generation of free radicals or other reactive species and antioxidant defenses, and it may be related to changes in mitochondrial function and protein clearance²⁵. Oxidative stress also favors potentially leading damage of tissue^26-31, lipids, pro­teins, enzymes, carbohydrates and DNA in cells, and can lead to cell death induced by RNA or DNA fragmentation and lipid peroxidation³².

 

The consequences of oxidative stress at the molecular level leads to the development of several ocular diseases like cataract, glaucoma, diabetic retinopathy, retinal degeneration, uveitis and age-related macular de­generation^33,34. Uveitis is one of the major sight threatening diseases in humans and horses and equine recurrent uveitis (ERU) is an incurable disease, which affects approximately 10% of the equine population³⁵.

 

Fig 7: Various nitrogen species and their biological function.

Deleterious effect of free radical in ocular disease:

The eye is a highly metabolically active structure, continually bathed in light with highly functional absorption and metabolism. Oxidative and particularly photo-oxidative processes are critical factors in ocular pathologic conditions. Oxidative stress is a key player in the mechanism of inflammation of eye diseases of conjunctiva, cornea and uvea³⁶. Cataract formation in the lens, retinal degeneration, optic nerve pathologic conditions, inflammation in optic neuritis and degenerative glaucoma can be attributed to oxidative stress which occurs throughout the eye and has been implicated in different types of tissue damage^37-39.

 

Cataract:

Oxidative stress and ROS are involved in many ocular diseases including cataract⁴⁰. Oxidative stress is an impor­tant factor in age-related processes in the body including senile cataract⁴¹. Production of ROS and reduction of endogenous antioxidants both contrib­ute to cataract formation⁴². UV-induced oxidation damage seems to play a major role in specific pathological conditions of cataract and retinal degeneration⁴³. The eye lens is also subjected to oxi­dative stress from radiation and others sources and this can damage the crystalline proteins, li­pids, polysaccharides and nucleic acids⁴¹. How­ever, it has several mechanisms to protect its com­ponents from oxidative stress and to maintain its redox state. These include enzymatic pathways and high concentration of ascorbate and reduced glutathione⁴⁴. However, with ageing, accumula­tion of oxidized lens components and decreased efficiency of repair mechanisms can contribute to the development of cataract⁴⁴. Chronic UV-induced ROS and oxidative stress-induced damage to lens gap junctions and consequent altered intercellular communication leads to cataract⁴⁴.

 

Glaucoma:

ROS play an important role in the pathogenesis of primary open angle glaucoma (POAG)⁴⁵. Oxidative DNA damage signifi­cantly increased in the trabecular meshwork (TM) of glaucomatous patients as compared to controls. Oxidative stress occurred, not only in the meshwork but also in retinal cells and appears to be involved in the neuronal death affecting the op­tic nerve in POAG⁴⁵. The perturbation of the oxidants versus antioxidant balance can lead to increased oxidative damage, es­pecially when the first line of antioxidant defense weakens with age⁴⁶. In glaucoma oxidative stress can cause chronic changes in the aqueous and vitreous humor, which may induce alterations in the trabecular meshwork and the optic nerve head⁴⁶. Zanon et al. observed that glaucomatous eyes had a significant increase in oxidative status and decreased antioxidant activity in the aqueous humor as compared to the cataract eyes and concluded that oxidative stress plays an important pathogenic role in POAG⁴⁷. Fernandez et al. reported an increase in the expression and enzymatic activity of nitric oxide syn­thase (NOS) isoenzymes and nitrotyrosine in the TM of patients with POAG. The increase correlated with visual field defects and it was concluded that the increased level of nitrotyrosine may serve as a vector of oxidative stress in the progression of cell death of the TM in patients with POAG⁴⁸.

 

Autoimmune uveitis (AIU):

Autoimmune and inflammatory uveitis are a group of potentially blinding diseases that arise without a known infectious trigger and are often associated with immunological responses to unique retinal proteins⁴⁹. Experimental studies have implicated free radicals in the pathogenesis of uveitis suggesting that free radicals and oxidative stress play a crucial role in the pathogenesis of the disease^49,50. Photoreceptor mito­chondrial oxidative stress has been considered to be the initial pathological event in experimental autoim­mune uveitis⁵¹. Determination of alterations in retinal mitochondrial levels in response to oxidative stress during the early phase of experimental autoimmune uveitis showed the presence of mitochondrial-specif­ic oxidative stress-related proteins in the retina along with down regulation of ATP synthase; providing evi­dence of stress related retinal damage⁵¹.

 

Pseudoexfoliation syndrome:

Pseudoexfoliation syndrome (PEX) is a common age-related fibrillopathy of unknown cause, recognized by chronic deposition of abnormal pseudoexfolia­tion material on the anterior segment structures of the eye⁵². Oxidative stress has been implicated in the development of this condition. Gartaganis et al., in­vestigated the oxidative status in lens epithelial cells of patients with PEX syndrome and found a decrease in the levels of glutathione (GSH) and glutathione disulphide (GSSG) compared with non-PEX lens epithelial cells, as well as increase in lipid peroxi­dation product malondialdehyde (MDA) levels⁵³. The increased MDA and decreased GSH levels indicate high oxidative stress. Also, GSSG usually increases in cases of high-oxidative stress, although this is not always the case as it may not always accumulate in cells⁵³. The authors suggested a role for oxidative stress in the pathogenesis and progression of PEX syndrome.

 

Macular degeneration:

Macular degeneration involves gradual loss in middle acuity and the presence of drusen (bumps) in the macula in the eye. The macula is located in the middle portion of the retina which is responsible for acute vision. The middle portion of the macula, in the innermost layers of the retina are displaced to one side allowing light to pass directly to the retina and offer high visual acuity, along with increased risk of radiation damage to the retina. The main cause of generation of reactive oxygen species is when UV and visible light radiation passes from retina to the photoreceptor (rod and cones) and pigmented epithelial cell (PE). The transformation of light into the nerve impulse through the photoreceptors develops free radicals. These free radicals include reactive oxygen species like hydrogen peroxide, superoxide and hydroxyl radicals. But the eye has many effective antioxidants which are capable of reducing these free radicals. If the electron is consumed from one of the lipids in photoreceptor, the lipid peroxide cascade through photoreceptor segment, and then these can be reduced by vitamin E or other lipid-soluble antioxidants, otherwise they can injure the integrity and fluidity of the membrane. In case of injury to photoreceptor, outer portion is sloughed off into the pigmented epithelial cell and new discs are generated. Pigmented epithelial cells are associated with phagocytization, digestion and recycling of these compounds under normal conditions. However, when these molecules are modified by oxidation of their unsaturated bonds, they are not easily digested by the lysosomal enzymes. This leads to the development of undigested molecule in the pigmented epithelial cell which is called as lipofuscin granules. If appropriate amount of lipofuscin gets accumulated, the pigmented epithelial cells deposit these granules resulting in a bump between the pigmented epithelial cells and choroid. This bump is called drusen. This sort of separation in physical and metabolic activity of the pigmented epithelial cell and the photoreceptors from its blood circulation in the choroid directly produce injury to the photoreceptors and cause macular degeneration (Fig 6) ^54,55.

 

 

 

Fig 8: Implications of oxidative stress on different human organs.

Antioxidants and their role in the prevention of ocular disease:

Antioxidants are reducing agents, and limit oxidative damage to biological structures by rendering free radicals passive⁵⁶. Free radicals, the chemically reactive substances produced in the human body can damage cellular elements (particularly membrane lipids and genetic materials)⁵⁷. Antioxidants are scavengers of free radicals which prevent the damage of tissue by free radicals⁵⁸. Antioxidants defend and repair the biological system which causes detoxification of oxidants like hydrogen peroxide, superoxide and lipid hydroperoxides radicals. They prevent the destruction of free radicals and maintain their physiological homeostasis^59-61. Antioxidants present in the human body act as natural defense system and combat the free radicals by counteracting the effect of oxidants. These include some non-enzyme, low molecular weight compounds like ferritin, ascorbate and α-tocopherol, enzymes like catalase, glucose-6-phosphate, glutathione peroxide and superoxide dismutase⁶². The naturally occurring antioxidants in the body are referred to as endogenous antioxidants and those taken as nutrient supplements are called exogenous antioxidants (Fig. 9). The endogenous antioxidants can either be enzymatic or non-enzymatic. Enzyme antioxidants continuously neutralize the reactive oxygen species (ROS) and reactive nitrogen species (RNS) like superoxide dismutase (SOD), catalase (CAT), glutathione peroxide (GPO), glutathione reductase (GRT) ^63-69. Superoxide anion radicals (O₂•ˉ) are converted directly into hydrogen peroxide (H₂O₂) by the involvement of enzyme superoxide dismutase (SOD) and then into water molecule by catalase or glutathione peroxidase (GPO).

 

Fig 9: Classification of various antioxidants

The efficient use of antioxidant as a therapeutic agent can be significant in minimizing various degenerative processes^70-27.

 

Fig 10: Some prominent free radical scavengers.

 

Novel approaches to reduce toxicity of free radical in ocular damage:

Various studies have reported the effect of antioxidants in minimizing and preventing the free radical generated ocular disease (Table 2). Antioxidants play a direct role in the prevention of free radical generated ocular damage, like ascorbate (vitamin C), α-tocopherol (vitamin E), glutathione, lipoic acid also called thioctic acid. Lipoic acid contains both aqueous and lipid soluble portion and two thiol group allows lipoic acid to energize vitamin E, ascorbic acid⁷³ and glutathione⁷⁴.

 

Equine Ocular Diseases:

Lipid peroxidation action is supported by oxidative stress and is particularly active in polyunsaturated fatty acid (PUFA)-rich biomembranes. Terrasa et al., 2009, reported the effect of endogenous lipid antioxidants (α-tocopherol) on non-enzymatic lipid peroxidation oxidative stress of rod outer segment membranes⁷⁵. Lipid peroxidation of the equine rod outer segment was induced by Fe²+-ascorbate, and it was evaluated by using chemiluminescence with or without pre-treatment of α-tocopherol. With α-tocopherol pre-treatment, chemiluminescence values were significantly decreased. After 3 h incubation, 95% of total PUFAs were destroyed by peroxidation, whereas in α-tocopherol pre-treated rod outer segment the percentage was significantly decreased. The results show that rod outer segment was highly sensitive to oxidative damage, since their fatty acid composition was markedly modified during the lipid peroxidation process. The protective role of α-tocopherol as an antioxidant was evident and it could be used in the treatment of equine ocular diseases in which free radicals are involved.

 

Cataract:

Kailash et al., 1992 investigated in vitro an antihypertensive drug and free radical scavenger activity of 1-[(2s)-3-Mercapto-2-methylpropionyl]-L-proline (captopril), on the rabbit eye lens from peroxidative and oxidative damage induced diquat⁷⁶. For the investigation of anticataract efficacy of captopril, a group of five rabbits was treated with topical captopril (1% in 0.15 M NaCl, w/v), and 50 μl were instilled onto both eyes four times a day for 8 weeks. Adopting the same protocol, the eyes of five rabbits were treated with topical 0.15 M NaCl as a control for captopril treatment. At the end of the first week of treatment, a single intravitreal dose of 120 nmole diquat in 30μl of 0.15 M NaCl was injected into the right eye of each rabbit of both the groups. As a control for intravitreal diquat injection, the left eye of all the rabbits was injected with the diluent, 30μl per eye. The intravitreal diquat or its diluent injection was only for one time. Slit-lamp biomicroscopic observation of the diquat-injected cataract did not advance while in four of five rabbits treated with the captopril or diluent were normal. However, since the number of animals used for the in vivo studies was few, further confirmation of the anticataract effect of captopril is necessary. In diquat-injected right eyes of animals treated with captopril, the integrated rate of O₂⁻ production was about 50% less (p < .001) in the aqueous humor, vitreous humor, and lens, compared with O₂⁻, 33.49 ± 2.26 μM (mean ± SEM) in the aqueous humor, 17.12 ± 0.75 μM in the vitreous humor, and 31.44 ± 1.29 nmole/g wet weight in the lens of the diquat-injected right eyes treated with the diluent. Similar significant (p < .01) differences in the production of •OH and H₂O₂ in eye tissues were also observed. Captopril protected against lipid peroxidation and oxidation of the reduced form of glutathione (GSH) and protein -SH of the lens in diquat-induced cataract in rabbits. Above investigation shows that captopril, act as an antioxidant in vivo, and can maintain the diquat-induced altered biochemical parameters in the eye close to normal and retard the progression of oxygen free-radical-triggered cataract in rabbits.

 

Kilic et al., 1999 reported protective effect of taurine in model in vitro diabetic cataract and investigated in isolated rat lenses⁷⁷. Isolated rat lenses were incubated in medium 199 in elevated glucose (55±6 mm) with taurine (5 mm). The concentration of taurine in the lenses was determined by amino acid analysis. Accumulative leakage of the intracellular enzyme lactate dehydrogenase (LDH) was used to estimate damage to the lens. In the clear lenses, prior to vacuole formation, after 1 or 2 days of incubation, the taurine and amino acids in lenses decreased progressively in concentration. In lenses incubated with 5 mm taurine, the level of taurine was higher than control lenses. In taurine-treated lenses LDH leakage was significantly decreased, and lens clarity was maintained, similar to that found previously for vitamin C and lipoic acid. To test whether taurine has similar antioxidant activity, its ability to decrease luminol luminescence generated by superoxide from hypoxanthine (xanthine oxidase) and peroxide from diluted glucose (glucose oxidase) was tested. For either superoxide or peroxide, the luminescence was decreased to zero, as a function of increasing taurine concentration, at 30 mm, approximately the physiological concentration of taurine in the lens. Spin trapping confirmed that taurine scavenged superoxide which was consistent with a role for taurine as an important antioxidant protecting the lens against oxidative insults. Amino acids also had antioxidant activity in this assay, and as a group, when all activities were summed, their loss also contributed significantly to the antioxidant loss. Taken in conjunction with Wolff and Crabbe's observation of increased free radical generation by glucose auto-oxidation in diabetes, this evidence suggest a push-pull mechanism for increased oxidative stress in diabetic cataract, involving both increased free radicals and decreased radical scavenging antioxidants.

 

Age-related cataractogenesis is a significant health problem worldwide. Oxidative stress has been suggested to be a common underlying mechanism of cataractogenesis and augmentation of the antioxidant defenses of the ocular lens has been shown to prevent or delay cataractogenesis⁷⁸. For these investigations the efficacy of curcumin (as antioxidant) in an in vitro rat model was tested. Rats were maintained on an AIN-76 diet (ICN Pharmaceuticals Inc. Cleveland) for 2 weeks, and then given a daily dose of corn oil alone or 75 mg curcumin/kg in corn oil for 14 days. Their eye lenses were removed and cultured for 72 h in vitro in the presence or absence of 100 µmol of 4-hydroxy-2-nonenal (4-HNE)/L, a highly electrophilic product of lipid peroxidation. These studies showed that 4-HNE caused opacifications of cultured lenses as indicated by the measurements of transmitted light intensity using digital image analysis. The lenses from curcumin-treated rats were much more resistant to 4-HNE-induced opacification than were lenses from control animals. Curcumin treatment caused a significant induction of the glutathione S-transferase (GST) isozyme rGSl8-8 in rat lens epithelium. Since rGST8-8 utilizes 4-HNE as a preferred substrate, it suggests that the protective effect of curcumin may be mediated through the induction of this GST isozyme. These findings suggest that curcumin may be potential protective agent against cataractogenesis induced by lipid peroxidation.

 

Zhang and Wang, 2009 reported the effects of the molecular weight (MW) and concentration of trimethyl chitosan (TMC) on the characteristics of Coenzyme Q10-loaded liposomes coated with trimethyl chitosan⁷⁹. The efficacy of the antioxidant Coenzyme Q10 in delaying selenite-induced cataract was assessed. The thick polymer layer on the surface of the liposomes nanoparticle was confirmed by transmission electron microscopy (TEM) and formulation also characterized for particle size distribution and zeta potential. The entrapment efficiency was almost the same as when the polymer was added to liposomes. The Draize test and histological analysis indicated the excellent ocular tolerance of TMC for topical administration. γ- scintigraphy showed that the drug elimination of polymer-coated liposomes is significantly slower than the radiolabelled solution used as a control. An almost 4.8-fold increase in the precorneal residence time was achieved in the presence of TMC with a higher MW as compared with the control group. Furthermore, the anti-cataract effect was evaluated by morphological examination and analysis of biochemical changes. Coenzyme Q10 exhibited a markedly anti-cataract effect with the percentage of lens opacity being about 53% at the final examination. The mean activities of superoxide dismutase and reduced glutathione were significantly higher in the Coenzyme Q10-treated group than in the cataract model group, while malondialdehyde was significantly lower. These studies concluded that physical properties and precorneal retention time of liposomes could be modified with TMC and ophthalmic instillation of Coenzyme Q10 was able to retard selenite-induced cataract formation.

 

Lead (Pb) is known to disrupt the pro-oxidant/anti-oxidant balance of tissues which leads to biochemical and physiological dysfunction. Neal et al., 1998 investigated the effects of exposure on the redox status of the lenses of Fisher 344 rats and examined whether antioxidant or chelator administration reversed these changes⁸⁰. Animals were given 5 weeks of 2000 ppm Pb exposure followed by 1 week of antioxidant, chelator or distilled water administration. Glutathione (GSH) and cysteine (CYS) levels decreased in the Pb-exposed group. N-acetylcysteine or 2,3-dimercaptopsuccinic acid (Succimer) supplementation following Pb intoxication resulted in increases in the GSH and CYS levels. Protein bound glutathione (PSSG) and cysteine (PSSC) increased following Pb exposure. In the Succimer-treated animals, the PSSG decreased significantly. The glutathione disulfide (GSSG) levels remained unchanged. Malondialdehyde (MDA) levels, a major lipid peroxidation byproduct, increased following Pb exposure and decreased following Succimer treatment. These findings suggested that antioxidant supplementation, as well as chelation, following Pb exposure may enhance the reductive status of lenses.

 

The ability of 2,6-dimethoxyquinone (DMQ) to impair 86Rb uptake by bovine lens epithelial cells was found to be independent of exogenous ascorbate in contrast to the impairment induced by Fe/Cu or riboflavin plus light. The cytotoxicity was associated with an electron spin resonance (ESR) detectable singlet radical (g = 2·0062) which also formed on incubation of DMQ with glutathione (GSH) or gamma-crystallin in vitro. Formation of the stable free radical appeared to require conjugation of DMQ by peptidyl thiol and required transition metal catalysis. Redox activity of quinone conjugates was suggested to be of relevance to an oxidative damage hypothesis of cataract⁸¹.

 

Mares et al., (2004) reported the effect of antioxidant in lowering the sequence of opacities of lens, and decrease the risk of cataract⁸². Retinal ischemia/reperfusion (I/R) results in neuronal death and generation of reactive oxygen species⁸³. The investigation consisted of the neuroprotective effect of manganese superoxide dismutase (SOD2) on retinal ganglion cells (RGCs) in an I/R-induced retinal injury model. One eye of each wistar rat was pretreated with recombinant adeno-associated virus containing the SOD2 gene (AAV-SOD2) or recombinant AAV containing the GFP gene (AAV-GFP) by intravitreal injection 21 days before initiation of I/R injury. Retinal ischemia/reperfusion I/R injury were induced by elevating intraocular pressure for 1 h, and reperfusion was established immediately afterward. The number of retinal ganglion cells RGCs and the inner plexiform layer (IPL) thickness were investigated by Fluorogold retrograde labeling and hematoxylin and eosin staining at 6 h, 24 h, 72 h, and 5 days after injury. Superoxide anion, the number of RGCs, IPL thickness, malondialdehyde (MDA) level, 8-hydroxy-2-deoxyguanosine (8-OHdG) level, MnSOD (manganese superoxide dismutase) activity, and nitrotyrosine level were measured by fluorescence staining, immunohistochemistry, and enzyme-linked immunosorbent analysis at 5 days after I/R injury. Severe RGC loss, reduced IPL thickness, reduced MnSOD activity, and increased superoxide ion, MDA, 8-OHdG, and nitrotyrosine production were observed after I/R injury. Administration of AAV-SOD2 significantly reduced the levels of superoxide ion, MDA, 8-OHdG, and nitrotyrosine and prevented the damage to RGCs and IPL. Delivery of the antioxidant gene inhibited I/R-induced RGC and IPL damage by reducing oxidative stress and nitrative stress, suggesting that MnSOD may be relevant for the neuroprotection of the inner retina from retinal ganglion cells I/R-related diseases.

Wang et al., (2011) investigated the effect of Crataegus pinnatifida (hawthorn tree) leaves extract in selenite-induced cataract in vivo and antioxidant effects in vitro⁸⁴. In vitro antioxidant assay of C. pinnatifida leaves extract on Nitrous oxide (NO) production inhibition, aldose reductase inhibition, and O₂ - radical scavenging activities gave the IC50 of 98.3, 89.7, and 5.98 μg/mL, respectively. C. pinnatifida leaves extract was characterized to have nine flavonoids via LC–MS/MS qualitative analysis. Based on in vitro screening results, C. pinnatifida leaves extract eye drops in 0.1% hydroxypropyl methyl cellulose solution were prepared and was administered thrice a day in rat pups with selenite-induced oxidative stress (SIOS). It was observed that there was a significant increase in serum superoxide dismutase (SOD) and catalase (CAT) activities, and it also tended to reduce MDA level as compared to control group. The antioxidant enzyme SOD, CAT, and GSH activities in lens showed a significant increase. These results indicate that C. pinnatifida leaves extract are a potential biomarker for prevention of cataracts.

 

Analogs of N,N-dimethyl-4-(pyrimidin-2-yl)- piperazine-1-sulfonamide possessing either a free radical scavenger group (FRS), chelating groups (CHL), or both (FRS+CHL) were synthesized⁸⁵. Electrospray ionization mass spectrometry studies indicated that select members of this series bind ions in the relative order of Cu¹⁺= Cu²⁺ > Fe²⁺= Fe³⁺>Zn²⁺ with no binding of Ca²⁺ or Mg²⁺ was observed. In vitro evaluation of these compounds in human lens epithelial, human retinal pigmented epithelial, and human hippocampal astrocyte cell lines indicates that all analogs possessing the FRS group as well as the water soluble vitamin E analog 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid protect these cells against decreased cell viability and glutathione levels induced by hydrogen peroxide. In addition, those compounds possessing CHL groups also protected these cells against hydroxyl radicals generated by the Fenton reaction. These compounds were found to be good candidates for the preventive treatment of cataract, age-related macular degeneration (AMD), and Alzheimer's dementia (AD).

 

The aging eye appears to be at considerable risk from oxidative stress⁸⁶. Lipid peroxidation (LPO) is one of the mechanisms of cataractogenesis, initiated by enhanced promotion of oxygen free radicals in the eye fluids and tissues and impaired enzymatic and non-enzymatic antioxidant defenses of the crystalline lens. Babizhayev (2011) proposed that mitochondria is one of the major sources of reactive oxygen species (ROS) in mammalian and human lens epithelial cells and the therapies that protect mitochondria in lens epithelial cells from damage and reduce damaging ROS generation may potentially ameliorate the effects of free radical-induced oxidation that occur in aging ocular tissues and in human cataract diseases⁸⁶. It has been found that rather than complete removal of oxidants by the high levels of protective enzyme activities such as superoxide dismutase (SOD), catalase, lipid peroxidases in transparent lenses, the lens conversely, possess a balance between peroxidants and antioxidants in a way that normal lens tends to generate oxidants diffusing from lenticular tissues, shifting the redox status of the lens to become more oxidizing during both morphogenesis and aging. Release of the oxidants O₂^-·, H₂O₂ , OH·, and lipid hydroperoxides by the intact lenses in the absence of respiratory inhibitors indicates that these metabolites are normal physiological products inversely related to the lens life-span potential (maturity of cataract) generated through the metal-ion catalyzed redox-coupled pro-oxidant activation of the lens reductants (ascorbic acid, glutathione). The membrane-bound phospholipid (PL) hydroperoxides escape detoxification by the lens enzymatic reduction. The lens cells containing these species would be vulnerable to peroxidative attack which trigger the PL hydroperoxide-dependent chain propagation of LPO and other damages in membrane (lipid and protein alterations). The increased concentrations of primary LPO products (diene conjugates, lipid hydroperoxides) and end fluorescent LPO products were detected in the lipid moiety of the aqueous humor samples obtained from patients with cataract as compared to normal donors. Since LPO is clinically important in many of the pathological effects and aging, new therapeutic modalities, such as patented N-acetylcarnosine prodrug lubricant eye drops, should treat the incessant infliction of damage to the lens cells and biomolecules by reactive lipid peroxides and oxygen species and "refashion" the affected lens membranes in the lack of important metabolic detoxification of PL peroxides. Combined in ophthalmic formulations with N-acetylcarnosine, mitochondria-targeted antioxidants are promising to become investigated as a potential tool for treating a number of ROS-related ocular diseases, including human cataracts.

 

Age Related Macular Degeneration (ARMD):

The neovascular age-related macular degeneration is related to oxidative stress involving the macular retinal pigment epithelium. In a study, the function of age, levels of enzymes which defend tissues against oxidative stress in the macular retinal pigment epithelium of human eyes with this disease was investigated⁸⁷. The surgical specimens of macular choroidal neovascular membranes from eyes with age-related macular degeneration and the macular regions of whole donor eyes with neovascular age-related macular degeneration or without evident ocular disease were studied by quantitative electron microscopic immunocyto-chemistry with colloidal gold–labeled second antibodies. Relative levels in retinal pigment epithelium cell cytoplasm and lysosomes were determined of five enzymes believed to protect cells from oxidative stress, as well as levels of the retinal pigment epithelium marker cytoplasmic retinaldehyde-binding protein, for comparison with the enzymes. Copper, zinc superoxide dismutase immune reactivity increased and catalase immune reactivity decreased with age in cytoplasm and lysosomes from macular retinal pigment epithelium cells of normal eyes and eyes with age-related macular degeneration. Cytoplasmic retinaldehyde-binding protein immunoreactivity showed no significant relationship to age or the presence of neovascular age-related macular degeneration. Glutathione peroxidase immunoreactivity was absent from human retinal pigment epithelium cells. Both heme oxygenase-1 and heme oxygenase-2 had highly significantly greater immunoreactivity in retinal pigment epithelium cell lysosomes than in cytoplasm, differing from the much greater cytoplasmic immunoreactivity of the other proteins studied. This immunoreactivity decreased with age, particularly in the lysosomes of retinal pigment epithelium cells from eyes with neovascular age-related macular degeneration. These decreases were of borderline significance (P = 0.067 for heme oxygenase-1; P = 0.12 for heme oxygenase-2) when eyes with age-related macular degeneration were compared with normal eyes by multivariable logistic regression. The study concluded that the high heme oxygenase-1 and heme oxygenase-2 lysosomal antigen levels in macular retinal pigment epithelium cells of eyes with neovascular age-related macular degeneration suggest that oxidative stress causes a pathologic up regulation of these enzymes. Increased lysosomal disposal may indicate that the reparative functions of these enzymes are accompanied by deleterious effects, necessitating their rapid removal from the cell. The much higher heme oxygenase-1 and heme oxygenase-2 antigen levels in macular retinal pigment epithelium cells from younger individuals suggest that protective mechanisms against oxidation and, hence, presumably to the development of age-related macular degeneration, decrease with age.

 

Proliferative Vitreoretinopathy:

Proliferative vitreoretinopathy (PVR) is a complication that develops in 5-10% of patients who undergo surgery to correct a detached retina⁸⁸. The only treatment option for PVR is surgical intervention, which has a limited success rate that diminishes in patients with recurring PVR. It was observed that antioxidants prevented intracellular signaling events that were essential for experimental PVR. The purpose of this study was to test whether N-acetyl-cysteine (NAC), an antioxidant used in a variety of clinical settings, was capable of protecting rabbits from PVR. Vitreous-driven activation of platelet derived growth factor receptor (PDGFR) and cellular responses intrinsic to PVR (contraction of collagen gels and cell proliferation) were blocked by concentrations of NAC that were well below the maximum tolerated dose. Furthermore, intra vitreal injection of NAC effectively protected rabbits from developing retinal detachment, which is the sight-robbing phase of PVR. Finally, these observations with an animal model appear relevant to clinical PVR because NAC prevented human PVR vitreous-induced contraction of primary RPE cells derived from a human PVR membrane. It was demonstrated that antioxidants significantly inhibited experimental PVR, and thus antioxidants have the potential to function as a PVR prophylactic in patients undergoing retinal surgery to repair a detached retina.

 

Uveitis:

Bilgiha et al., (1995) reported that melanin pigment has antioxidant effect against excess of dispersed light⁸⁹. To investigate whether it has a similiar effect in ocular inflammations, albino and pigmented guinea pigs were used and retinal glutathione peroxidase activities and lipid peroxide levels (expressed as thiobarbituric acid reactive substances) were measured in a model of lens induced uveitis. Although the increase in the levels of the retinal lipid peroxides were higher in the albino group (24 %, p<0.05), the decrease in the activities of glutathione peroxidase were higher in pigmented guinea pigs (26%, p<0.005). The results of the study suggested that non-pigmented animals are more sensitive to the ocular inflammations, and ocular melanin pigment may act as an endogenous antioxidant in lens induced uveitis.

 

In addition to the inhibition of xanthine oxidase, allopurinol is known to act, dependent on the dose, as a free radical scavenger, an antioxidant, and a "scavenger" of hypochlorous acid. This activity was investigated using a model of lens-induced uveitis⁹⁰. In this investigation lipid peroxides (LPO) were determined in aqueous humor and in retinal tissue. Reduced and oxidized glutathione (GSH and GSSG) of the aqueous humor and myeloperoxidase (MPO) activity in the iris-ciliary body complex were analyzed. Allopurinol and oxypurinol concentrations were determined by high-performance liquid chromatography in aqueous humor and retinal tissue of both control eyes and eyes with uveitis. These measurements were performed 6 hours after intravenous application of allopurinol. In lens-induced uveitis, LPO were significantly elevated, GSH was reduced, and GSSG and MPO were increased. A xanthine oxidase inhibition dose (<10 mg/kg body weight) of allopurinol showed no effects on oxidative tissue damage in the model used in this study. Higher doses, however, were able to reduce the oxidative damage. Allopurinol (20 mg/kg body weight) had slight effects on GSH and GSSG. All parameters improved using a dose of 50 mg/kg body weight; a dose of 100 mg/kg body weight only showed additional improvement in GSH and GSSG. There was no further change in the other parameters. Allopurinol and oxypurinol concentrations in aqueous humor and retinal tissue showed a dose dependency reaching scavenger concentrations after application of 50 mg/kg body weight of allopurinol. These results suggested that the xanthine oxidase mechanism plays a minor role in the oxidative tissue damage due to lens-induced uveitis. Free radicals and oxidants were generated by activated leukocytes; therefore, the effect of higher doses of allopurinol was due to its free radical scavenging and antioxidative activity.

 

Balci et al. investigated the effects of mobile-phone-emitted radiation on the oxidant/antioxidant balance in corneal and lens tissues and to observe protective effects of vitamin C⁹¹. For the study forty female albino wistar rats were taken and assigned to one of four groups containing 10 rats each. One group received a standardized daily dose of mobile phone radiation for 4 weeks. The second group received this same treatment along with a daily oral dose of vitamin C (250 mg/kg). The third group received this dose of vitamin C alone, while the fourth group received standard laboratory care and served as a control. In corneal and lens tissues, malondialdehyde (MDA) levels and activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) were measured with spectrophotometric methods. In corneal tissues, MDA level and CAT activity significantly increased in the mobile phone group compared with the mobile phone plus vitamin C group and the control group (p < 0.05), whereas SOD activity was significantly decreased (p < 0.05). In the lens tissues, only the MDA level significantly increased in the mobile phone group relative to mobile phone plus vitamin C group and the control groups (p < 0.05). In lens tissue, significant differences were not found between the groups in terms of SOD, GSH-Px, or CAT (p>0.05). The results of this study suggested that mobile telephone radiation leads to oxidative stress in corneal and lens tissues and antioxidants like vitamin C can help to prevent these effects.

 

 

Table 2: Types of antioxidants used in free radical induced ocular disease.

Antioxidant	Disease	Model	Reference
α – tocopherol (Endogenous lipid antioxidant)	Equine ocular disease	In vitro	[75]
Crantaegus pinnatifida	Cataract	In vitro	[84]
Manganese superoxide dismutase (SOD2)	Cataract	In vivo	[82]
N,N-dimethyl-4-(pyrimidin-2-yl)- piperazine-1-sulfonamide analog	Cataract, Age related macular degeneration, Alzheimer’s dementia	In vitro	[85]
Melanin pigment (Endogenous antioxidant)	Uveitis	In vivo	[89]
N- acetylcarnosine (Mitochondria targeted antioxidant)	Cataract	Clinical	[86]
Vitamin C	Oxidant/antioxidant balance  in corneal and lens	In vivo	[91]
Allopurinol, oxypurinol	Uveitis	In vivo	[90]
N-acetyl-cysteine (NAC),	Proliferative vitreoretinopathy (retinal detachment)	Clinical	[88]
Heme oxygenase-1 and heme oxygenase-2 lysosomal antigen	Neovascular age-related macular degeneration	Surgical	[87]
2,6-dimethoxyquinone	Cataract	in vitro	[81]
Coenzyme Q10	cataract	in vitro	[79]
Curcumin (as antioxidant)	Cataractogenesis induced by lipid peroxidation	in vitro	[78]
Taurine	Diabetic cataract	model in vitro	[77]
1-[(2s)-3-Mercapto-2-methylpropionyl]-L-proline (captopril), an antihypertensive drug	Cataract	in vitro	[76]

 

 

 

CONCLUSION:

There is ample evidence to implicate free radicals in ocular disease and toxicity of a great number of pathological conditions. Consequently, a better knowledge of these very reactive metabolites and their role in the organism, would allow the use of more suitable therapeutics to cure such disorders, based on the reduction of free radical concentrations and/or prevention of their reactions.

 

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Received on 03.09.2014          Modified on 10.09.2014

Accepted on 07.10.2014          © RJPT All right reserved