Melanin and Oxidative Reactions

involves the formation of highly reactive transient species that are potentially very toxic to the melanocyte.2 Therefore all key steps of melanogenesis take place in melanosomes—sub cellular organelles that limit the exposure of the cellular environment to melanogenic intermediates.3 As a result, melanin in melanocytes is present in the form of discernible units such as pigment granules whose size and geometry are determined by the phenotype of the melanosomes. Melanin granules in the human retinal pigment epithelium are typically elongated and relatively large (2–3 mm long and 1 mm wide), while such pigment granules in the human choroids are smaller and somewhat more spherical.4 In the human skin, the ultrastructure of melanosomes usually relates to the type of melanin they produce.5 Thus typical melanosomes that produce so-called eumelanin have ellipsoidal-lamellar structure with melanin deposited in a uniform pattern. On the other hand, melanosomes that form so-called pheomelanin, are usually round and granular with uneven deposition of pigment. Eumelanin originates from tyrosine or DOPA, and pheomelanin formation requires, in addition, the presence of cysteine or glutathione.6 It is believed that key intermediates in the biosynthetic pathway for eumelanin, are 5,6-dihydroxyindole and 5,6-dihydroxyindole-2-carboxylic acid, as well as their fully oxidized forms.7 In the biosynthetic pathway for pheomelanin, a similar role may be played by 1,4-bezothiazynylalanine, which is derived from cysteinyldopas.8 The understanding of the molecular structure of melanin has undergone a substantial evolution. Thus while previously melanin was viewed as very large molecule of hetero-polymeric structure,9 recent studies using advanced imaging techniques such as scanning electron, tunnelling and atomic force microscopies indicate that the actual building block of eumelanin is a relatively small planar oligomer with maximum dimension of 0.4 1.0 nm that is preferentially aggregated into fundamental aggregates of 3–4 -stocked oligomers.10–12 According to this view, the macroscopic morphology of eumelanin pigment granules is a result of hierarchical self-assembly, in which the building blocks of eumelanin assemble

into hundred-nanometre structures, which then aggregate to form the final pigment granule.13,14 Although the exact nature of the forces that are involved in the

assembly of nanoaggregates and of hundred-nanometer structures remain unknown, it can be speculated that Van der Waals, – and hydrophobic interactions play a key role.

One of the most characteristic and unique features of melanin is its paramagnetism. Melanins are the only biological material that both in vivo and in vitro contain a significant amount of persistent free radical centres that are easily detected by electron paramagnetic resonance (EPR) spectroscopy.15,16 Importantly, the EPR signals of melanin are specific for the two main types of melanin pigments. At standard EPR frequency (X-band), eumelanins have a single slightly asymmetric line 0.4–0.6 mT wide with a g-factor close to 2.004. The EPR spectrum of pheomelanin typically consists of three spectral features with an overall width of about 3.0 mT and g = 2.005. It must be stressed that even though the EPR signal of melanin is very persistent, the free radicals in

Figure 1.1 Univalent reduction of oxygen and univalent oxidation of nitric oxide (see page 2).

Figure 5.2 Immunohistochemical staining for SOD3 in the human cornea. A: Note a pronounced staining of the cell borders in the epithelium, and a stromal staining which is interleaved between the stromal collagen lamellae. The stromal staining is slightly weaker in the anterior, than in the posterior stroma. B: Detail of immunohistochemical staining for SOD3 in the human corneal epithelium. Note intense staining of the cell borders and intercellular space. C: Staining for SOD1 in the human corneal epithelium. Note the staining of the cytosol and nuclei (see page 59).

Figure 10.4 Retinal morphology and protein nitration during the course of EAU. Nitrated retinal proteins from days 0 (D0), 5 (D5), 10 (D10), 12 (D12), and 14 (D14) p.i. were immunoblotted with anti-nitrotyrosine (B), the relative intensities of nitrated proteins were quantified (C) and correlated with morphologic changes in EAU (A). Maximal intensities of tyrosine-nitration were seen in days 5 and 10, with well preserved retinal structures. Day 12 marked the onset of inflammation with arrival of inflammatory cells (see page 137 ).

Figure 10.5 Localization of tyrosine-nitrated proteins in the retina. Polyclonal nitrotyrosine antibody and anti-rabbit IgG conjugated with biotin were used for the detection. A: non-immunized control animals and B: EAU day 5 p.i. Note the intense localization of nitrated proteins seen only in the photoreceptor inner segments (B) (see page 138).

Figure 15.2 For tyrosinase immunocytochemistry, the RPE monolayer was prepared and exposed to ROS for 4 hrs. The expression of tyrosinase was investigated before feeding with ROS, as well as 5 and 24 hours afterwards. 2A Five hours after feeding with ROS, no staining was visible with anti-tyrosine hydroxylase antibodies. 2B Without feeding with ROS no staining was found with anti-tyrosinase antibodies. 2C Five hours after feeding with ROS faint staining was observed with anti-tyrosinase antibodies corresponding to DOPA positive vesicles in Fig. 1B, 2D. Twenty-four hours after feeding with ROS intense staining of lysosome-like organelles (arrows) was found with anti-tyrosinase antibodies. These organelles correspond to those shown in Fig. 1G, 1B (see page 203).

melanins are by no means chemically stable. Indeed it has been demonstrated that the concentration of melanin free radicals can be changed reversibly by almost two orders of magnitude.17 Among agents that can induce melanin free radicals are ultraviolet and visible radiation, high pH, redox systems and diamagnetic multivalent metal ions.18 It is believed that most of the changes in the free radicals induced by theses agents are due to changes in the so-called comproportionation equilibrium, i.e. the equilibrium between the fully reduced and oxidized melanin subunits, and their semi-reduced (semi-oxidized) states, as shown in the equation below:

Q þ QH2 $ 2SQ þ 2Hþ

The monomers are o-quinones, o-hyrdoquinones, and o-semiquinones in the case of eumelanin; corresponding units for pheomelanin are o-quinonimines, o-aminophenols, and o-semiquiononimines respectively.

The effect of complexing of diamagnetic multivalent metal ions on the melanin EPR signal is an important diagnostic test that can be used to determine the molecular nature of the subunits and, hence, the type of melanin studied.19 Thus, EPR spectroscopy is a unique physical method that enables non-destructive analysis and characterization of melanin pigments with good sensitivity and high accuracy.20

It seems generally accepted that melanin in the skin and eye acts as a natural sunscreen that by absorbing and scattering, hence attenuating, solar radiation, particularly the energetic UV and short wavelength visible photons, protects the pigmented tissue against adverse photo-reactions. Indeed a distinct correlation between the resistance of the human skin to UV-induced erythema and sunburn, and constitutive pigmentation of the skin is usually observed.21 Epidemiological data also suggest that the incidence of solar radiation-related skin cancer is higher in individuals with genetically-determined poor ability of the skin to tan and low pigmentation.22 In addition, skin susceptibility to socalled photo-ageing may inversely correlate with pigmentation of the skin.23 In

cultured melanocytes, melanin was shown to offer protection against induction of major DNA lesions by UVB24,25 and UVA-induced membrane damage.26 A

significant inverse correlation between baseline skin pigmentation and the extent of UV-induced DNA damage was also reported by an independent study.27

Although the role of chronic exposure of the human retina to solar radiation in the ethiology of AMD remains controversial,28,29 it is of interest to note

that AMD is more often found in individuals with lower content of the uveal melanin.30

The molecular and cellular mechanism of photoprotection offered by melanin is not fully understood. Of course, the ability of melanin pigments to absorb light with the efficiency that increses inversely with the light wavelength is intrinsically photoprotective, providing that the energy of the absorbed photons is rapidly and safely utilized in non-photochemical processes. Indeed recent studies confirm a very efficient nonradiative de-excitation of melanin, following

the absorption of ultraviolet and visible photons.31–33 The studies clearly show that melanin is a system in which a very efficient thermal relaxation occurs, this is to say that energy absorbed by melanin photons is rapidly converted into heat via very fast internal conversion. As a result, the risk of potentially damaging photochemical reactions, mediated by melanin, is significantly reduced.

Antioxidant Properties of Melanin

There is another mode of photoprotective action of melanin. It is related to its ability to act as an antioxidant, i.e., an agent that protects other molecules by neutralizing oxidizing free radicals and other so-called ‘‘reactive oxygen species’’, being present at lower concentration than the oxidisable substrate molecules. While photochemical oxidising reactions are typically accompanied by the formation of reactive oxygen species, the presence of redox-active metal ions, such as iron and copper, is believed to elevate the oxidative damage via Fenton-type processes.34 That’s why antioxidant action may also depend on sequestration of redox-active metal ions. In model systems of different complexity, synthetic and natural melanins have been shown to act as efficient scavengers of reactive free radicals, quenchers of singlet oxygen and excited triplet states of certain photosensitising dye molecules, and inhibitors of lipid peroxidation.35–43

Using pulse radiolysis as a direct method for generation of selected free radicals and for monitoring their lifetime in the absence and presence of syn-

thetic DOPA-melanin, apparent rate constants of the interaction of the radicals with melanin were obtained.41,44,45 The data, shown in Table 1, can be sum-

marized as follows: this synthetic eumelanin exhibits reactivity with both oxidizing and reducing radicals. The observable reactivity increases with the absolute value of the one-electron reduction potential of the radicals studied and with their intrinsic lifetime. The reactivity also depends on the electric charge of the radicals being higher for the positively charged species and lower for the negatively charged radicals. As expected, melanin interacted most rapidly with OH (one of the most oxidising free radicals) and with hydrated electron (the most reducing species known). However, this eumelanin also interacted quite efficiently with superoxide anion, which is a poor oxidant and only a mild reductant.46 In addition, melanin interacted with reasonably high rate constants with peroxyl and carbon-centred radicals that may be involved in peroxidation of lipids. The interaction of melanin with oxidizing and reducing radicals can be explained by the hydroquinone and quinone nature of the melanin subunits, which can act as efficient electron donors and acceptors, respectively.

Using steady-state photosensitised generation of singlet oxygen and EPRoximetry to monitor oxygen consumption, rate constant of the interaction of singlet oxygen with synthetic DOPA-melanin was measured.47 Again, the corresponding bimolecular rate constant was quite high (above 107 M 1s 1), indicating that melanin could be a good quencher of this important reactive oxygen species.

Table 1 Second-order Rate Constants for the Interaction of DOPA-melanin with Free Radicals and Singlet Oxygen

Note: Rates estimated for melanin monomers, assuming the molecular weight of a representative melanin monomer is 150.

Although the results of the reviewed studies are consistent with melanin being an efficient scavenger of reactive free radicals and a quencher of singlet oxygen, it seems rather unlikely that these properties of melanin play a critical role in the antioxidant action of this pigment. This is because of the limited lifetime of randomly generated reactive species. These species would interact with many constituents of the pigmented cell before having a chance to diffuse to the proximity of the melanosomes (or pigment granules), where they would then need to penetrate the melanosome surface and their membrane, in order to interact with the melanin active groups. Of course, the free radical scavenging and singlet oxygen quenching abilities of melanin may be of importance if the formation of such reactive species is ‘‘site-specific’’, i.e. their generation predominantly occurs within the melanin granule or in its proximity.48

It can be postulated that melanin principally exerts its antioxidant action by binding of redox-active metal ions and photosensitising dye molecules. It has been demonstrated that iron and copper ions that are bound to melanin are inefficient generators of free hydroxyl radicals.49,50 Even though melanin complexes with ferrous and cuprous ions are readily oxidized by molecular oxygen and hydrogen peroxide, very few OH radicals leak out of the melanin

structure.50 Ferric and cupric ions, on the other hand, after binding to melanin, become significantly more difficult to reduce by mild reductants. Sequestration of iron ions has been identified as a major mechanism for the inhibitory effects of melanin on lipid peroxidation.51,52

Free radical and singlet oxygen-induced oxidation of lipids can schematically be written as:

R is an initiating free radical; LH is unsaturated lipid; LOO , LO , LOOH are the lipid peroxyl and alkoxyl radical, and hydroperoxide, respectively; S, 1S, and 3S are a photosensitising dye molecule in its ground state, singlet and triplet excited states, respectively; 3O2, 1O2 are ground triplet and excited singlet states of molecular oxygen.

The inhibitory effect of melanin on oxidation of lipids is described by the following reactions responsible for scavenging of radicals and binding of iron ions:

Mel þ R. !. Mel RðMelox þ RHÞ

Mel þ LOO ! Mel LOOðMelox þ LOOHÞ

Mel þ1O2 ! Melox

Mel þ Fe2þ ! Mel Fe2þ

Mel Fe2þ þ H2O2 ! site specific formation and decay of hydroxyl radicals Mel þ Fe3þ ! Mel Fe3þðdifficult to reduce by biological electron donorsÞ Mel; Melox are melanin in its initial and oxidised state; respectively

Similar mechanism for antioxidant action of melanin can be considered in systems where photo-oxidation reactions are sensitised by positively charged dye molecules. Using laser flash photolysis and EPR-oximetry it has been shown that binding by melanin of two cationic porphyrins dramatically reduced the photosensitising efficiency of the dyes, i.e. their abilities to photo-generate singlet oxygen and free radicals.53 The mechanism of the quenching of excited sates of the dyes bound to melanin was recently determined by femto-second absorption and pico-second emission spectroscopies.54 It has been concluded that such a binding facilitates an ultrafast energy transfer from the excited porphyrin molecule to melanin. The excited energy is then rapidly converted into heat. Because of its speed, the process involves only singlet excited states. No triplet states are formed and, consequently, no photochemistry occurs:

Mel þ Sþ ! Mel Sþ

Mel Sþ þ hv ! 1ðMel SþÞ ! Mel Sþ þ heat

Sþ is the ground state of a positively charged photosensitising dye molecule:

MELANIN PROPERTIES RELEVANT FOR ITS REPORTED

PRO-OXIDANT AND PHOTOTOXIC ACTION

There is ample literature indicating that melanotic systems may act as prooxidizing agents. Thus, it has been reported that: melanogenic intermediates photo-initiated DNA damage in vitro,55 in human melanocytes from dark skin, UVA induced 40 times more DNA single strand breaks than in melanocytes from light skin,56 UV-irradiated melanin, particularly pheomelanin, sensitised adjacent cells to capsase-3 independent apoptosis,57 and pheomelanin synthesis sensitised melanocytes to oxidative DNA damage by UVA.58 RPE melanin and synthetic DOPA melanin mediated photooxidation of ascorbic acid,59,60 synthetic eumelanin sensitised isolated DNA to induction of the oxidative DNA base damage by UVA irradiation61 and, under certain experimental conditions, isolated RPE melanosomes were apparently able to photosensitise peroxidation of lipids.62

In view of the reported data, it should be explained how melanin, which usually behaves as a good antioxidant, may become a pro-oxidant. It appears that certain experimental conditions can stimulate pro-oxidant properties of melanin. Thus in the presence of photosensitising dye molecules that do not bind to melanin53 or high concentration of electron donors,60 as a result of an overload with redox-active metal ions,40 and after aerobic exposure of melanin to high intensity UV-vis radiation, melanin may behave as an oxidizing system. It was postulated that melanotic system could act as a pro-oxidant if the pigment contained high percentage of the pheomelanin component or was present in a very low aggregation state.63 In addition, results of a recent study suggest that also in vivo melanin may loose its antioxidant efficiency and even become a pro-oxidant.64

As a redox-system, melanin can engage in a number of electron-transfer reactions. It has been demonstrated that melanin mediates aerobic oxidation of ascorbic acid and NADH, and that such electron-transfer reactions are greatly accelerated by irradiation with short-wavelength visible light.59,60 Even in the dark, melanin was shown to reduce ferric complexes with EDTA or other strong chelators.49 Under such conditions, by acting as an electron donor, melanin may drive the Fenton-reaction that generates highly damaging free hydroxyl radicals or other strongly oxidizing species. This is in a striking contrast to melanotic systems, in which the iron ions are tightly bound to the melanin. As discussed above, in the latter, due to site-specific formation and decay of reactive species, virtually no free hydroxyl radicals are generated and very little or no oxidation of other substrates occurs.

It is important to realise that even though melanin very efficiently converts energy of the absorbed photons into heat, the residual aerobic photochemistry of melanin may lead to significant changes of the pigment granules that will lower their ability to sequester metal ions and scavenge reactive oxygen species; particularly, if melanin is exposed to high fluxes of intense UV or short-wavelength visible radiation. The aerobic photochemistry of melanin is responsible for generation of superoxide anion and hydrogen peroxide, which, in the presence of

adventitious redox-active metal ions, will lead to the formation of hydroxyl radicals or other strongly oxidizing species. The mechanism of melanin-induced photo-formation of superoxide anion and hydrogen peroxide is believed to involve the interaction of transient radicals generated in melanin by light with molecular oxygen. Interestingly, simple orto-semiquinones, generated by pulse radiolysis, did not show any significant reactivity with molecular oxygen.65 The higher efficiency of the melanin ortho-semiquinone centres to interact with oxygen may be due to their longer lifetime compared to that of the free orto-semiquinones generated in solution. The light induced oxygen reduction by melanin can be described by the following reactions:

MelfðmÞQ; ðnÞQH2g þ hv ! Melfðm kÞQ; ðn kÞQH2; ð2kÞSQg

Melfðm kÞQ; ðn kÞQH2; ð2kÞSQg þ O2 !

Melfðm þ kÞQ; ðn kÞQH2g þ ð2kÞO:2

Q, QH2 and SQ are the quinone, hydroquinone and semiquinone functional groups of eumelanin, and indices m, n, k denote the number of the corresponding groups in the melanin. For simplicity, in the notation used above, the intrinsic melanin free radicals are ignored and only the extrinsic, i.e. the inducible melanin radicals are indicated.

Melanin of the human retinal pigment epithelium (RPE) is unique in that it is formed early during fetal development and serves its biological role(s) for the entire life of an individual.66 This is because melanin in these post-mitotic nondividing cells shows very little, if any, metabolic turnover.67 Using EPR spectroscopy for unambigous detection and quantification of melanin, we have recently shown that the amount of human RPE melanin decreases with age of the donors.64 While the exact molecular and cellular mechanisms of this phenomenon are not clearly understood, it can be postulated that the RPE melanin undergoes in situ photobleaching. Considering the exposure of the outer retina to high accumulative doses of visible light and oxygen concentration, this is not an impossible scenario, particularly if the normal antioxidant defense systems are compromised. It can further be postulated that the decreased amount of melanin in the ageing RPE may reduce the efficiency of melanin to protect the RPE, as well as the entire retina, against oxidative stress. Importantly, chronic oxidative stress to the retina has been suggested to be a contributing factor in the development of age-related macular degeneration.68

Interestingly, not only the amount of melanin decreases in the aging human RPE, also the photoreactivity of the remaining melanin granules increases with age.69 Using EPR-oximetry and EPR-spin trapping, we have shown that purified RPE melanosomes, isolated from human donors of different age, exhibit a distinct age-dependent increase in their efficiency to photo-consume oxygen and to photogenerate superoxide anion. It appears that melanin in the senescent human RPE becomes substantially modified and that this chemical modification increases its aerobic photoreactivity. It can be postulated that, in part, this is due

to an accumulation of photosensitising component(s) in the aging RPE melanin granule. Cosidering that the aging RPE also accumulates high amounts of lipofuscin, a powerful photogenerator of reactive oxygen species,70 it can be speculated that the risk of oxidative stress in the human retinal is greatly elevated in senescence.

ACKNOWLEDGMENT

The authors thank the Ministry of Science and Information Technology (KBN), and NIH (R01 EY013722) for providing financial support.

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64.Sarna T, Burke JM, Korytowski W, et al. Loss of melanin from human RPE with aging: possible role of melanin photooxidation. Exp Eye Res 2003; 76:89–98.

65.Kalyanaraman B, Hintz P, Sealy RC. An electron spin resonance study of free radicals from catechol estrogens. Fed Proc 1986; 45:2477–2484.

66.Marmor MF. Structure, function and disease of the retinal pigment epithelium. In: Marmor MF, Wolfensberger TJ, eds. The Retinal Pigment Epithelium. Function and Disease. New York: Oxford University, 1998:3–9.

67.Boulton M. Melanin and the retinal pigment epithelium. In: Marmor MF, Wolfensberger TJ, eds. The Retinal Pigment Epithelium. Function and Disease. New York: Oxford University, 1998:68–85.

68.Beatty S, Koh H, Phil M, et al. The role of oxidative stress in the pathogenesis of age-related macular degeneration. Surv Ophthalmol 2000; 45:115–134.

69.Ro´zanowska M, Korytowski W, Ro´zanowski B, et al. Photoreactivity of aged human RPE melanosomes: a comparison with lipofuscin. Invest Ophthalmol Vis Sci 2002; 43:2088–2096.

70.Ro´zanowska M, Jarvis-Evans J, Korytowski W, et al. Blue light-induced reactivity of retinal age pigment. In vitro generation of oxygen-reactive species. J Biol Chem 1995; 270:18825–18830.

It has been repeatedly suggested that oxidative stress may play an important role in the pathogenesis of late diabetic complications,1 though it is not clear yet whether increased oxidative stress has a primary role in the pathogenesis of diabetic complications, or if it is simply the consequence of the presence of complications.2 In diabetes, oxidative stress seems to be caused by both an increased production of free radicals and a sharp reduction in antioxidant defences.3

DIABETIC RETINOPATHY AND ANTIOXIDANTS

Diabetic retinopathy is the major cause of adult blindness in developed countries. It has been recently reviewed that one third of the diabetic patients will have some degree of retinopathy within the first ten years after the onset of diabetes; twenty years after diabetes onset, 100% of patients with type 1 diabetes and 60% of patients with type 2, will develop diabetic retinopathy; 30% out of them will develop proliferative diabetic retinopathy.4

Retina is the neurosensorial tissue of the eye and is extremely rich in membranes with polyunsaturated lipids. This feature makes it especially sensitive to oxygen free radicals and to lipid peroxidation. There is substantial evidence from animal and clinical studies for both impaired antioxidant defences and increased oxidative damage in the retina of diabetic subjects that may be, in the case of animal studies, reversible with antioxidant supplementation. The antioxidants used in animal models of diabetic retinopathy, include: ascorbic acid, trolox, alpha-tocopherol acetate, N-acetyl cysteine, beta-carotene, selenium, vitamin C, alpha-lipoic acid, pycnogenol, green tea, zinc, ebselen, lutein, taurine, etc.5–14 In humans clinical trials with antioxidants have failed to reverse these effects or have shown contradictory results.15

Recently Ceriello16 suggested that new compounds which act as SOD or catalase mimics or superoxide scavengers, may be adequate tools in the prevention of diabetic vascular complications. This author suggested that the hyperglycemia-induced process of overproduction of superoxide seems to be the key event in the activation of all other pathways involved in the pathogenesis of diabetic complications.

Peroxynitrite Scavengers

The product of the reaction between nitric oxide ( NO), which is also increased in diabetes, and superoxide is peroxynitrite, a potent prooxidant. Therefore, we suggest that the use of peroxynitrite scavengers may also be a good option in the prevention of diabetic complications, and in particular of diabetic retinopathy.

These changes in superoxide and peroxynitrite production and the subsequent damage to DNA can also occur in nervous cells. Neural cell apoptosis in human tissues has been demonstrated in diabetic retina17 and more recently in neuropathy18 and the hippocampus of type 1 diabetic rats.19 It seems that neurons begin to die soon after the onset of experimental diabetes in rats. Peroxynitrite has

Table 2 GPx Activity (%) in the Eyes of Different Groups of Mice Treated with CR-6 During Two or Three Weeks Compared with the Control Groups

Figure 1 MDA concentration (mM) in the different groups of mice (*p <0,05 vs control and diabeticþcr6 two and three weeks; **p <0,05 vs. control and diabeticþcr6 three weeks).

After two or three weeks of diabetes, MDA levels in retina of diabetic mice (Figure 1) are increased when compared to controls, confirming the role of lipid peroxidation in diabetes. CR-6 is able to prevent this effect. We demonstrate herein in an experimental model of diabetic retinopathy that an antioxidant, like CR-6 which is a peroxynitrite scavenger and that is also able to inhibit photoreceptor apoptosis,23 can reverse the biochemical changes induced by

oxidative stress in diabetic retina. These results are similar to those reported by our group with lutein and ebselen11,12 and lead us to conclude that oxidative stress is

an early event in diabetic retinopathy.

Electroretinogram and Its Utility

The electroretinogram (ERG) has been used for decades to study the mechanisms of retinal physiology. In diabetes electrophysiological changes have been shown

to occur before clinical evident retinopathy.25,26 A reduction in the a-wave amplitude has been demonstrated in patients with type 1 diabetes25,27 and it has

been repeatedly reported that b-wave has reduced amplitude in diabetes in humans and also in animal models of diabetes11,12,25,28 (Figure 2).

already one week after the induction of diabetes11 in a similar animal model, and it is known that most diabetic rodent models develop changes such as pericyte loss, capillary dilation and increased basal membrane thickening, even after a year of diabetes.33 We agree with emergent theories which affirm that changes in neuronal and glial cell function often occur prior to vascular abnormalities, indeed it is possible that changes in retinal neurons and glia contribute to the development of the vascular changes.

In Figure 3 we can observe the decreased amplitude of the b-wave of the electroretinogram of diabetic mice only after two and three weeks of the induction of diabetes. Treatment with antioxidants, such as CR-6, restored b-wave amplitude, as well the biochemical changes-related to oxidative stress previously reported, without modifying glucose levels in blood mice. Although we have not noticed any changes in latency times of the electroretinogram, other studies have reported alterations in latency but at later times.34

CONCLUSION

Our data are consistent with those of Ceriello’s group, that in a recent review proposed antioxidant therapy for diabetic complications. We suggest antioxidants with peroxynitrite scavenger capacity such as CR-6, ebselen or lutein may also be adequate for the treatment of diabetic complications. CR-6 and ebselen are synthetic antioxidants but lutein35 is a natural antioxidant that can not be synthesized within the body and is provided by dietary intake. Other promising molecules are sulforaphanes.36 Sulforaphanes produce induction of phase 2 genes and result in the elevation of proteins that exert antioxidant activities, including the glutathione synthesis enzymes. We have also demonstrated that GSH metabolism is altered in diabetic retinopathy.

Further studies on ebselen, lutein or CR-6 as adequate adjuvant diabetes therapies must be performed to confirm the exact mechanism of action of these antioxidants.

ACKNOWLEDGMENTS

This work was supported by projects PI03/1710 from the Fondo de Investigacio´n Sanitaria to FB-M, and PRUCH04/30 to FJR.

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2.Baynes JW, Thorpe SR. Role of oxidative stress in diabetic complications. A new perspective on an old paradigm. Diabetes 1999; 48:1–9.

3.Giugliano D, Ceriello A, Paolisso G. Diabetes mellitus, hypertension and cardiovascular disease: which role for oxidative stress? Metabolism 1995; 44:363–368.

4.Aiello LP, Gardner TW, King GL, et al. Diabetic retinopathy. Diabetes Care 1998; 21:143–156.

5.Kowluru RA, Koppolu P, Chakrabarti S, et al. Diabetes-induced activation of nuclear transcriptional factor in the retina, and its inhibition by antioxidants. Free Radic Res 2003; 37:1169–1180.

6.Dene BA, Maritim AC, Sanders RA, et al. Effects of antioxidant treatment on normal and diabetic rat retinal enzyme activities. J Ocul Pharmacol Ther 2005; 21:28–35.

7.Yatoh S, Mizutani M, Yokoo T, et al. Antioxidants and an inhibitor of advanced glycation ameliorate death of retinal microvascular cells in diabetic retinopathy. Diabetes Metab Res Rev 2006; 22:38–45.

8.Mustata GT, Rosca M, Biemel KM, et al. Paradoxical effects of green tea (Camellia sinensis) and antioxidant vitamins in diabetic rats: improved retinopathy and renal mitochondrial defects but deterioration of collagen matrix glycoxidation and crosslinking. Diabetes 2005; 54:517–526.

9.Kowluru RA, Odenbach S. Effect of long-term administration of alpha-lipoic acid on retinal capillary cell death and the development of retinopathy in diabetic rats. Diabetes 2004; 53:3233–3238.

10.Moustafa SA. Zinc might protect oxidative changes in the retina and pancreas at the early stage of diabetic rats. Toxicol Appl Pharmacol 2004; 201:149–155.

11.Miranda M, Muriach M, Johnsen S, et al. Oxidative stress in a model for experimental diabetic retinopathy: treatment with antioxidants. Arch Soc Esp Oftalmol 2004; 79:289–294.

12.Miranda M, Muriach M, Roma J, et al. Oxidative stress in a model for experimental diabetic retinopathy II: the utility of peroxynitrite scavengers. Arch Soc Esp Oftalmol 2006; 81:27–32.

13.Di Leo MA, Ghirlanda G, Gentiloni Silveri N, et al. Potential therapeutic effect of antioxidants in experimental diabetic retina: a comparison between chronic taurine and vitamin E plus selenium supplementations. Free Radic Res 2003; 37:323–330.

14.Abiko T, Abiko A, Clermont AC, et al. Characterization of retinal leukostasis and hemodynamics in insulin resistance and diabetes: role of oxidants and protein kinase-C activation. Diabetes 2003; 52:829–837.

15.Marchioli R, Schweiger C, Levantesi G, et al. Antioxidant vitamins and prevention of cardiovascular disease: epidemiological and clinical trial data. Lipids 2001; 36(suppl):S53–S63.

16.Ceriello A. New insights on oxidative stress and diabetic complications may lead to a ‘‘causal’’ antioxidant therapy. Diabetes Care 2003; 26:1589–1596.

17.Barber AJ, Lieth E, Khin SA, et al. Neural apoptosis in the retina during experimental and human diabetes. Early onset and effect of insulin. J Clin Invest 1998; 102:783–791.

18.Cellek S, Qu W, Schmidt AM, et al. Synergistic action of advanced glycation end products and endogenous nitric oxide leads to neuronal apoptosis in vitro: a new insight into selective nitrergic neuropathy in diabetes. Diabetologia 2004; 47: 331–339.

19.Li ZG, Zhang W, Grunberger G, et al. Hippocampal neuronal apoptosis in type 1 diabetes. Brain Res 2002; 946:221–231.

20.Cowell RM, Russell JW. Nitrosative injury and antioxidant therapy in the management of diabetic neuropathy. J Investig Med 2004; 52:33–44.

21.Irurre J Jr., Casas J, Ramos I, et al. Inhibition of rat liver microsomal lipid peroxidation elicited by 2,2-dimethylchromenes and chromans containing fluorinated moieties resistant to cytochrome P-450 metabolism. Bioorg Med Chem 1993; 1:219–225.

22.Montoliu C, Llansola M, Sa´ez R, et al. Prevention of glutamate neurotoxicity in cultured neurons by 3,4-dihydro-6-hydroxy-7-methoxy-2,2-dimethyl-1(2H)-benzopyran (CR-6), a scavenger of nitric oxide. Biochem Pharmacol 1999; 58:255–261.

23.Sanvicens N, Go´mez-Vicente V, Masip I, et al. Oxidative stress-induced apoptosis in retinal photoreceptor cells is mediated by calpains and caspases and blocked by the oxygen radical scavenger CR-6. J Biol Chem 2004; 279:39268–39278.

24.Halliwell B. Oxidative stress markers in human disease: application to diabetes and to evaluation of the effects of antioxidants. In: Packer L, Ro¨sen P, Tritschler HJ, King GL, Azzi A, eds. Antioxidants in Diabetes Management. New York: Marcel Dekker, 2000:33–62.

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36.Gao X, Talalay P. Induction of phase 2 genes by sulforaphane protects retinal pigment epithelial cells against photooxidative damage. Proc Natl Acad Sci U S A 2004; 101:10446–10451.

RPE atrophy.6,7 Wet AMD describes any of the following findings in the macular area: choroidal neovascularization (CNV), hard exudates, fibrous scar, RPE and sensory retinal detachment (with or without hemorrhage). Dry AMD is the most common form of AMD affecting around 85% of AMD sufferers but while wet AMD is less common (*15%) it is the most severe form of the condition and can result in an acute loss of central vision.

Epidemiological studies to identify risk factors have been fundamental to our current understanding of the pathobiology of this debilitating condition. However, despite all the studies, the scientific community remains divided as to whether AMD is predominantly of environmental or genetic origin. It seems most likely that AMD is a multigenic condition in which variable combinations of gene defects make the individual more susceptible to environmental insults. Mutations associated with AMD have been located and include the bestrophin, fibulin-3, ELOVLA4, ABCR4, Ccl2, RPE65, superoxide dismutase, APOE, complement factor H genes and HTRA1.8,9 Epidemiological studies such as the

Beaver Dam Eye Study, Blue Mountain Eye Study, Chesapeake Bay Study and Rotterdam Study have identified a number of risk factors.10,11 Of these the two

most prominent are aging and smoking.12 Cumulative exposure to visible light is widely believed to contribute to AMD although the evidence is equivocal probably due to sampling technique. Other risk factors include pigmentation, diet, pharmacological agents and hypertension.12

PHOTOREACTIVITY OF THE RETINA

The retina is particularly susceptible to oxidative stress because of its high consumption of oxygen, its high proportion of polyunsaturated fatty acids, and its constant exposure to visible light. Furthermore, environmental risk factors such as diet (reduced intake of antioxidants, eg, carotenoids, vitamin E and vitamin C), smoking, photochemical reactions (e.g. light exposure of the retina) and mutations affecting the antioxidant status provide strong support that the generation of reactive oxygen species (ROS) plays a key role in cumulative oxidative damage to the retina.1,13

It is thought by many that AMD represents an extension of the normal aging process and when this passes a critical point some or all of the symptoms we associate with AMD are manifest. Susceptibility to ROS damage will be dependent on antioxidant potential, efficiency of repair processes and genetic susceptibility. These varied parameters and variations in gene penetrance help explain the variable age of onset of AMD ranging from 50 through to 100þ years.

It has long been considered that exposure to visible light, in particular the more energetic ‘‘blue light’’ component of the visible spectrum, makes a major

contribution to retinal ageing, RPE dysfunction and the pathogenesis of AMD.1,14,15 The retina contains numerous light absorbing species. These include

(a) the visual pigment rhodopsin and its retinal metabolites which collectively absorb across the whole of the visible spectrum, (b) the macular pigment

carotenoids (zeaxanthin and lutein) which strongly absorb between 400 and 530 nm, (c) flavins and flavoproteins which absorb blue light with maximum at about 430 nm, (d) hemoglobin and other proteins (e.g., mitochondrial enzymes such as cytochrome c) containing porphyrin which absorb strongly around

400 nm and (e) the broad band absorbers lipofuscin and melanin which have been implicated in blue light damage to the retina.16,17 It is these latter broad

band absorbers which will be considered in detail in this review.

PHOTOREACTIVITY OF LIPOFUSCIN

Lipofuscin is predominantly a lipid aggregate that accumulates with increasing age within metabolically active, post mitotic cells in a variety of tissues throughout the body. Lipofuscin, or age pigment as it is often referred to, is generated within the lysosomal system and is considered to represent the incomplete degradation of intracellular or extracellular substrates. The common substrate for lipofuscin for-

mation in most postmitotic tissues is autophagy of spent organelles such as mitochondria, Golgi bodies and endoplasmic reticulum.16,18,19 However, the

accumulation of lipofuscin within the retinal pigment epithelium (RPE) is unique in that in this cell type the major substrate for lipofuscin appears to be photoreceptor outer segment tips.16 There is support for this from biochemical analyses of lipofuscin.16,20–22 The predominant chloroform-soluble lipofuscin fluorophores are di-retinal conjugate A2E and other retinal metabolites. However, protein may also represents a component of lipofuscin granules ranging from estimates of 0–75% protein per granule. Proteins have been identified and show diverse origin ranging from photoreceptors to intracellular organelles and enzymes. Furthermore, the proteins exhibit extensive protein modifications, advanced glycation end products and lipid-protein adducts. However, the composition reported to date has to be treated with caution as the lipofuscin preparations are contaminated with cellular debris and a recent study by our group indicates that lipofuscin contains minimal if not zero protein.23 Notwithstanding, lipofuscin is a broad band absorber and contains a variety of chromophores with potential for the generation of reactive oxygen species (ROS).16

RPE lipofuscin granules exhibit a strong broad band emission spectrum with a peak at 600 nm and subsidiary shoulders located at 470 and 550 nm.25 We and others have demonstrated that RPE lipofuscin granules photogenerate the superoxide anion, singlet oxygen and hydrogen peroxide and that production increases with age and at the shorter wavelengths of the visible spectrum.26 Lipofuscin-photosensitized lipid peroxidation was also confirmed by the generation of lipid hydroperoxides and malondialdehyde.26 The reactive oxygen species generated not only oxidise intragranular and extragranular lipids but can also oxidise proteins and carbohydrates plus cause damage to nucleic acids. Analysis of the blue light photoreactivity of isolated human RPE cells demonstrates that the rate of photo-inducible oxygen uptake increases with donor age, and that oxygen uptake is predominantly due to endogenous lipofuscin.27

Furthermore, the photogeneration of ROS is strongly wavelength dependent with, for example, efficiency increasing with wavelength by a factor of 10 when excitations of 520 and 420 nm are compared.28 This suggests that lipofuscin may make a major contribution to the ‘‘blue light hazard’’ associated with retinal photodamage.29

The biological significance of the photogeneration of ROS by lipofuscin granules is confirmed by the oxidation of lipids and the inactivation of antioxidant (catalase) and lysosomal (acid phosphatase) enzymes.30 This suggests that RPE cells containing significant levels of lipofuscin and exposed to visible light could be compromised by oxidative damage to critical biomolecules.

To confirm the phototoxic potential of lipofuscin in a biological system cultured RPE cells containing lipofuscin granules were exposed to ‘‘blue’’ (400–550 nm) and ‘‘amber’’ (550–800 nm) light with an irradiance of 2.8 mW/cm2 for periods up to 48 hours.31,32 Exposure of lipofuscin containing cells to ‘‘blue’’ light caused lipid peroxidation (increased levels of malondialdehyde and 4-hydroxy-nononal), protein oxidation of integral proteins (protein carbonyl formation), an increase in lysosomal pH and loss of lysosomal integrity, cytoplasmic vacuolation and membrane blebbing culminating in cell death by 48 hours exposure. By contrast, cells exposed to amber light or maintained in the dark showed no adverse effect. Interestingly, exposure of RPE cells to blue light in the absence of lipofuscin resulted in mitochondrial DNA damage due to the generation of ROS within the mitochondrion which could be blocked by mitochondrial specific antioxidants.32 However, the exposure of RPE cells containing lipofuscin to blue light resulted in nuclear DNA damage in addition to the light-alone effect onmitochondrial DNA (mtDNA) suggesting that lipofuscin was able to generate longer life time lipid peroxides which are able to reach the nucleus.32

The most well characterized photosensitizer of RPE lipofuscin is A2E.

Upon photoexcitation with blue light A2E is able to generate singlet oxygen and to promote an apoptotic form of cell death.33–35 However, A2E is only weakly

photoreactive in comparison with the hydrophobic components of RPE lipofuscin.16 It is likely that photooxidative forms of A2E such as A2E epoxides are more photoreactive than A2E itself, and it is these which make a significant contribution to lipofuscin phototoxicity. However, it has recently been reported that a considerable proportion of lipofuscin photoreactivity is present at the interface between the chloroform and methanol phases of Folch extracted material (A2E is in the chloroform phase).36 It is an increase in this chloroform insoluble phase which is responsible for the increase photoreactivity of lipofuscin granules with increasing age. Thus the role of A2E within lipofuscin remains equivocal. However, since A2E is a lysosomotrophic agent it may cause RPE dysfunction by destabilizing the lysosomal proton pump and impairing proteolytic degradation.37,38 Thus it is possible that A2E is formed initially to detoxify the highly photoreactive trans-retinal generated during the phototransduction process and is then irreversibly sequestered within lipofuscin to protect the RPE from further damage.

MELANOSOME PHOTOXICITY

Melanosomes are prominent within the young RPE however their number per cell decreases with increasing age.39 Furthermore, with age there is an increase in melanosome/lipofuscin complexes. The role of melanosomes within the

human RPE is equivocal but may include absorption of stray light, scavenging of ROS, sequestration of metal ions and the binding of xenobiotics.39,40 Notably the

photophysical properties of melanosomes change with increasing age. Blue light photoexcitation of melanosomes results in an age-related increase in both oxygen uptake and the accumulation of superoxide anion spin adducts.41 We have recently confirmed the phototoxicity of aged human melanosomes in an vitro model.42 Cell viability of RPE cells containing aged melanosomes was reduced by 60% after exposure to blue light for 48 hours as compared to 10% for young human melanosomes. This confirmed that the ROS generated by aged melanosomes can contribute to RPE dysfunction and that reduced metal binding by aged melanosomes may also contribute to oxidative stress in RPE cells by making more iron available for the Fenton reaction.

ANTIOXIDANTS

Although the RPE is constantly exposed to oxidative stress it is able to reduce oxidative damage through an efficient antioxidant system and active repair of oxidatively damaged biomolecules. The RPE relies on both enzymatic (superoxide dismutase, catalase and glutathione peroxidase) and non-enzymatic, dietary (ascorbate, a-tocopherol, lutein, zeaxanthin) antioxidants to negate the action of ROS.13,43 While the antioxidant system functions efficiently in the young there appear to be a decreased overall antioxidant activity with increasing age. It would further appear that the RPE is able to reverse nuclear DNA (nDNA) damage, protein oxidation and lipid peroxidation through efficient repair and replacement mechanisms. However, mtDNA repair, as for most other cell types, is poor. This, together with an age-related increase in ROS generators, such as lipofuscin, in the elderly results in the RPE being exposed to ever increasing levels of oxidative stress. Since oxidative damage is thought to play a fundamental role in the aging process it is likely that oxidative stress in the retina will be a major contributor to retinal aging and the pathogenesis of AMD.1,13

SUSCEPTIBILITY AND ADAPTATION OF THE

RPE TO REACTIVE OXYGEN SPECIES

Recent studies from this laboratory indicate that the RPE has a greater resistance to a diverse range of ROS compared to other cell types localised to tissues similarly exposed to high levels of oxidative stress.44 For instance, the RPE demonstrated a significantly greater resistance to hydrogen peroxide, tertbutylhydroperoxide, Paraquat and sodium arsenite than did liver hepatocytes, alveolar cells or corneal fibroblasts. It would appear that this increased resistance of the RPE to oxidative damage is, at least in part, influenced by the local

environment. Prior exposure of RPE cells to sublethal hydrogen peroxide demonstrated an adaptive response resulting in a greater resistance to subsequent toxic exposures compared to non-adapted RPE.45 This was associated with a greater catalase, CuZn-superoxide dismutase and glutathione peroxidase enzymatic activity and increased nuclear DNA protection. Interestingly, there was no adaptive benefit for mitochondrial DNA protection or repair in response to sublethal oxidative stress. This identifies the mitochondrion as a potential weak link in the otherwise efficient oxidative stress defenses of the RPE and that this may contribute to retinal aging and age-related disease.

CONCLUSION

The retina, due to its high oxygen environment, high concentration of unsaturated fatty acids and regular exposure to visible light is an ideal environment for the generation of ROS. Oxidative stress is further increased by the age-related accumulation of photoinducible-generators of ROS (e.g. lipofuscin). There is a wealth of literature supporting a relationship between oxidative stress and agerelated macular degeneration. While genetic and inflammatory components (e.g. complement factor H) are currently in vogue and clearly contribute to the pathobiology of AMD the recent findings that antioxidant therapy has a protective effect46 reinforces the importance of oxidative stress in the aetiology of AMD. Greater research is now required to determine the best dose regime in terms of combinations of antioxidants and their concentration.

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