Antioxidants in the retinal pigment epithelium

C H A P T E R 4

Antioxidants in the Retinal Pigment Epithelium

DAVID A . N E W S O M E , * t M I C H A E L V. M I C E L I , * t M A R K R. LILES,* D A V I D J . T A T E , JR* a n d P E T E R D. O L I V E R *~

*Sensory and Electrophysiology Research Unit, Touro Infirmary, 1401 Foucher Street, New Orleans, LA 70115, USA

tDepartments o f Ophthalmology and *Anatomy, Tulane University School o f Medicine, New Orleans, LA, USA

CONTENTS

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

2. Sources of Oxidative and Free Radical Damage to the Macula . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 2.1. Visible and UV Light . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 2.2. Drugs and Xenobiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 2.3. Iron and Melanin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104 2.4. Metabolic By-products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

3. Antioxidant Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106 3.1. The First Line of Defense . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106

3.1.2. Antioxidant enzymes in the RPE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107

4. Non-enzymatic Antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 4.1. Metallothionein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

4.1.1. Chemistry and induction of metallothionein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108 4.1.2. MT as an antioxidant . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

4.2. Zinc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109 4.2.1. MT as an indicator of zinc status . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 4.2.2. Age-related changes in tissue zinc and MT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

4.3. Other Antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 4.3.1. Ascorbate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 4.3.2. Vitamin E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 4.3.3.0-Carotene . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

5. Therapeutic Intervention . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114

6. Summary and Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117

1. INTRODUCTION

The retinal pigment epithelium (RPE), a neuroectodermally-derived monolayer, lies between a highly vascularized choroid and the neural retina. On its basal surface, the RPE adheres to Bruch's membrane, a complex extracellular matrix basement membrane with selective permeability and barrier properties (Bruch, 1844; Wolfrum, 1908; Nakaizumi, 1964;

Nakaizumi et al., 1964; Hogan, 1965; Kanwar and Farquhar, 1979; Kanwar et al., 1980; Pino and Essner, 1981; Pino et al., 1982). The luminal surface of the RPE lies in close association with the photoreceptor outer segments of the retina. Some of the essential functions of the RPE are summarized in Table 1.

This metabolically active complex exists in an oxygen-rich environment. Reactive oxygen inter- mediate species occur naturally in oxygen-contain-

Progress in Retinal and Eye Research Vol. 13, No. 1 © 1994 Pergamon Press Ltd Printed in Great Britain.

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102 D. A. NEWSC)ME el a[.

TABLE 1. Selected Functions o f the R P E

Contribution to interpbotoreceptor matrix and retinal attachment. Phagocytosis and digestion of shed rod and cone photoreceptor outer segments. Metabolism and recycling of vitamin A, polyunsaturated fatty acids from outer segment material, and other metabolites. Production of antioxidants. Production of trophic factors. Tight junctional barrier between choroid and sensory retina; pump functions. Production and maintenance of elements of Bruch's membrane.

(Bok, 1985; Sakuragawa and Kuwabara, 1976; LaVail etal., 1981; LaVail et al., 1985; Uehara etal., 1983; Berman, I991.)

ing environments and include derivatives of molecular oxygen, such as singlet oxygen, superoxide, hydrogen peroxide (H202) , and hydroxyl radicals, as well as other substances with the capacity to transfer single electrons (Pryor, 1978). Free radicals are highly reactive and can oxidize unsatu- rated fatty acids in cell membranes, produce DNA damage, depolymerize hyaluronate which is a constituent of Bruch's membrane, modulate nucleotide cyclase activity, oxidize proteins and influence the action and synthesis of prostaglandins and lipoperoxides (Bors et al., 1974; Rey- nolds and Moslen, 1980; Wiegand et al., 1984; Kashihara et al., 1992).

Since reactive oxygen species can be destructive, it is not surprising that a variety of intra- and extracellular free radical defense mechanisms have evolved and are present in numerous cells throughout the body, including the RPE (Handel- man and Dratz, 1986; Katz et al., 1982; Oberley and Beuttner, 1979; Fridovich, 1982; Michelson, 1982).

The three primary species of superoxide dismutase (SOD) show strong evolutionary conservation. All three species of SOD, glutathione peroxidase, and catalase are produced by human RPE cells (Handelman and Dratz, 1986; Newsome et al., 1990; Liles et al., 1991; Del Vecchio and Shaffer, 1991). With the exception of extracellular SOD (EC-SOD) (Marklund, 1984), most of these enzymes act intracellularly either in the cytoplasm or in specific organelles. For example, catalase is primarily localized in peroxisomes and is especially important in eliminating potentially harmful hydrogen peroxide (H202) generated as a by-product of the 0-oxidation of long-chain polyunsaturated fatty acids, which make up a great portion of the lipids in ingested photoreceptor outer segments (Robison and

Kuwabara, 1975; Osmundsen et al., 1991; Folz and Trobe, 1991). In experimental animal models, catalase has been shown to decrease with age in the brain, and may decline in zinc deficiency in an organ-specific manner (Taylor et al., 1988). Catalase is elevated in rats on food-restricted diets that result in extended lifespans (Rao et al., 1990; Koizumi et al., 1987).

Besides enzymes, the range of protective antioxidant substances extends to vitamins and pro- vitamins, including beta-carotene, a-tocopherol, and vitamin C. In addition, certain cysteine-rich molecules like glutathione (GSH) and metallothionein (MT) are thought to play an important antioxidant role due to the reactivity of their sulfhydryl groups. Trace elements, e.g. zinc and selenium, have been shown to be important factors in protection against oxidative stress. Some of these substances may function directly in metabolic cycles, as cofactors for enzymes, and possibly in gene regulation, e.g. zinc in zinc fingers.

The antioxidant capacity is important not only in maintenance of the integrity of the RPE and in controlling by-products of its natural metabolic load, but also may contribute to the durability of the RPE. This non-dividing monolayer must continue to function for a lifetime of vision.

In this chapter we will review and summarize the current state of knowledge of the sources of oxidative and free radical stress on the RPE, the protective antioxidant enzymes and non- enzymatic antioxidants. Scientific interest in the role of antioxidants in aging and the manipulation of these elements to promote wellness and longevity is increasing. We will briefly consider the current state of knowledge of therapeutic interventions utilizing antioxidants specifically to promote or maintain RPE function.

ANTIOXIDANTS IN THE RPE

We emphasize the focused nature of this review and remind the reader that although the biblio- graphy of this chapter is extensive, it by no means includes all of the important papers in this area. Antioxidant research in a number of organ systems, including brain, heart and liver, has been ongoing for many years, addressing a growing interest in cellular functions and DNA regulation in response to oxidative stress. In the present review, we have focused on what should become an exciting and expanding area of research in the RPE.

2. SOURCES OF OXIDATIVE A N D FREE RADICAL DAMAGE TO THE MACULA

The RPE, a highly energetic tissue, has numerous metabolic functions which necessitate a high oxygen uptake. Other metabolic functions directly increase the concentration of pro-oxidant metabolites such as the /l-oxidation of lipids, which can produce H202 as a by-product. Add to this the effects of high incident light and the possible occurrence of free radical reactions due to the presence of a high concentration of melanin and iron, and the retinal layers are at particular risk for deleterious free radical insults. Due to space limitations and an already existing literature concerning sources of oxidative and free radical stress in tissues, the reader is referred to an excellent review by Handelman and Dratz (1986). We will concentrate on more recent advances in our understanding of oxidative stress to the RPE. The reader is also referred to the chapter on retinal light damage by Organisciak and Winkler in this volume in which the effects of light are well covered. This present section will consider three categories of oxidative and free radical stress: external factors such as light and drugs, internal factors such as iron and melanin, and metabolic by-products such as superoxide anion, hydroxyl radical and H202.

2.1. Visible and UV Light

Ultraviolet light less than 400 nm is a very important source of free radical damage in tissues such as skin and cornea which are directly exposed to this stress. Due to the absorption of virtually all

103

wavelengths less than 400 nm by the lens, the effect of UV light on the macula, except in aphakic eyes, is probably negligible (Rosen, 1985). Visible light, on the other hand, may have significant effects on the RPE. Rhodopsin absorbs light at 500 nm, and light toxicity at that wavelength may be associated with the death of rod cells (Noell, 1980). It has also been demonstrated that blue light of 430 nm can cause damage to the RPE, probably by interacting with hemopro- reins (Pautler et al., 1990). The mechanism of this damage is not clear but may be via a free radical mechanism involving 02- and the Fe 3+ of cytochrome a3. Light has been shown to initiate lipid peroxidation by singlet oxygen (Girotti, 1990). In rats, constant illumination has been shown to lead to accumulation of lipid peroxidation products in the retina (Wiegand et aL, 1983), and light exposure may increase retinal antioxidants (Penn et aL, 1987). The antioxidant ascorbate has been shown to reduce the degene- rative changes associated with intense green light of 490- 580 nm (Organisciak, 1985; Blanks et al., 1992). Both of these types of damage may be important in the aging retina and RPE.

Age-related macular degeneration is more prevalent in persons with blue irises and less prevalent among blacks (Hyman et al., 1983; Ferris, 1983). Since melanin content in the RPE is the same in these two groups, the screening effects of melanin in the iris may be the important difference, although no one has directly measured macular light flux in blue- versus brown-eyed people. More recent data suggest that iris color is not a risk factor for the neovascular complication of age-related macular degeneration, further complicating this issue (The Eye Disease Case- Control Study Group, 1992). It is also thought that above 500 nm the thermal effects of light dominate over photochemical effects, although there is no sharp demarcation between the two (Ham et al., 1986). It is apparently the intensity and duration of light exposure which determine the type of stress to the tissue (Newsome et al., 1986).

2.2. Drugs and Xenobiotics

In addition to the sources of oxidation which occur 'naturally', human tissues can be subjected

104 D. A. NEWSOME et al.

to a variety of stresses due to drugs, industrial chemicals and poisons. These include ethanol, cigarette smoke, herbicides such as paraquat and diaquat, toluene, certain chemotherapeutic agents and hyperbaric oxygen. With all of these substances except possibly oxygen, the main target organ is not the eye, and their contribution to retinal disease can only be estimated or inferred from epidemiological studies. It has been shown that a number of compounds, including ethanol, methamphetamine and toluene can cross the b lood-bra in barrier and cause oxidative damage in the brain (LeBel and Bondy, 1991; Jesberger and Richardson, 1991). This is indirect evidence that these compounds can increase oxidative damage to the retina and the RPE. Furthermore, ethanol depresses hepatic levels of vitamin A by interfering with retinol metabolism and exacer- bates the production of toxic radicals by inducing the microsomal cytochrome P-450 system (Lieber, 1991; McCay et al., 1992). Cigarette smoke has been shown to induce lipid peroxidation and changes in lipoproteins in human blood plasma in vitro (Frei et al., 1991). Tobacco smoking has been identified as a risk factor for age-related macular degeneration in some studies (Hyman et al., 1992; The Eye Disease Case-Control Study Group, 1992).

The herbicides, paraquat and diaquat, can cause toxicity within cells via a free radical mechanism. This effect has produced experimental cataracts in rabbits (Bhuyan et al., 1991). Other drugs which may be important sources of free radical stress are chemotherapeutic agents such as adriamycin, which causes cardiotoxicity to occur through a free radical mechanism involving iron (Villani et al., 1990). While a deleterious effect of adriamycin on the RPE has not been shown, the amount of circulating drug reaching the RPE during chemotherapy may be significant. Experimental uveitis can be induced in rats by injecting with lipopolysaccharide or retinal specific antigens (Hoekzema et al., 1992; Wu et al., 1992). The subsequent invasion of the inflamed ciliary body and choroid by polymorphonuclear ieukocytes has been shown to cause peroxidation of retinal lipids, possibly from superoxide produced from these professional

phagocytes (Wu et al., 1992). This may be an important source of free radical damage during ocular gram negative infections.

2.3. Iron and Melanin

The participation of iron in free radical reactions has been demonstrated in vitro and is probably important in vivo. Iron participates in the Fenton reaction by its ability to redox cycle, producing the very reactive hydroxyl radical:

F e 2+ + a ~ o 2 ~ Fe 3+ + .OH + OH-. The amount of iron found in the RPE is

considerable (Hunt and Davis, 1992) and iron is a component of melanin (Potts and Au, 1976; Ulshafer, 1990). However, the amount of catalytically available iron or other transition metals capable of redox cycling is not easily determined (Ryan and Aust, 1992).

The contribution of iron to free radical toxicity is becoming clearer. Ferric iron is obviously deleterious in vitro, contributing to lipid peroxidation of ROS lipids, especially in the presence of chemicals such as ascorbate, which can contribute to redox cycling (De La Paz and Anderson, 1992). Iron and copper have also been implicated in the oxidation of proteins (Stadtman, 1990). In ongoing nutrition studies, indirect evidence regarding the effect of iron on the macula suggests that persons who take multivitamins may be at increased risk for the development of age-related macular degeneration (Hyman et al., 1992). The culprit may be the iron contained in most supplements, although without definitive data, this is speculative.

Whether melanin acts as a pro-oxidant or an antioxidant is unclear. Melanin is probably photoprotective at some wavelengths. There is evidence from recent studies that melanin does not contribute to retinal phototoxicity by UV light at 360 nm (Rapp and Smith, 1992). Melanin may contribute to local heating and tissue damage under extreme light conditions, such as those involved in laser therapy, and iron bound to melanin may contribute to free radical generation (Sarna, 1992). We have performed experiments to determine whether melanin reduces or contributes to free radical damage in the RPE cell. In these

ANTIOXIDANTS IN THE RPE 105

experiments, we remelanized cultured RPE cells p m 2750

by feeding them isolated bovine melanin (Boulton o I 2600

and Marshall, 1985) and measured the effect of • s 2250

paraquat, a known free radical generator, on lipid r 2000 peroxidation and cell viability. The results ~ lr50 (Fig. 1A) clearly show that cells containing R 1500 melanin have significantly increased thiobarbi- s 1250

rn 1000 turic acid reactive substances (TBARS). Further- g more, the effect of paraquat on lipid peroxidation P 75o

r 5001 is increased dramatically in cells previously fed o t 250 melanin. Surprisingly, paraquat was less toxic to e o melanin-fed cells (Fig. IB). Melanin may bind , paraquat and increase the effect of the drug on neighboring lipids. However, by so doing, the toxicity of paraquat may be reduced by keeping the generated free radicals away from more % critical components of the electron transport v system of the mitochondria or labile nuclear DNA. i

In the eye, melanin may act in a similar fashion, e It may protect against iron or drug toxicity in the c same manner by binding free iron or drugs such as T paraquat and not allowing them to participate in 8 redox cycling. By the same mechanism, melanin may contribute to local free radical stress. Further studies using better models should determine whether melanin protects the RPE cell or contributes to deleterious effects. It is possible that the divalent and transition metal content of melanin may contribute to free radical production in the macula. This, along with the possibility that the metal content of melanin may depend upon nutrition, suggests that diet may be important in preventing or reducing the effects of free radical generation in the RPE.

2.4. Metabolic By-products

Most likely, the main source of oxidative damage in the RPE cell is a consequence of normal oxygen metabolism. Oxygen is toxic due to its ability to exist in a singlet or excited state (Cadenas, 1989). Furthermore, it has been estimated that as much as 2°70 of the cell's mitochondrial oxygen consumption results in the production of reactive oxygen species (Chance et al., 1979), which is substantial. Depending upon the culture conditions, cultured human RPE cells

El Control Cells

too 200 400 800 1000 uM PARAQUAT

(A)

120

110

100~

9O

8O

7O

60

5O

4O

3O

2O

10

0 - - o

O Co°,r:'C.,,. 1]

i't 100 200 400 800 1000

uM PARAQUAT

FIG. 1. (A) Effect of melanin feeding and paraquat treatment on concentration of TBARS in cultured human RPE cells. Cells were repigmented by feeding isolated bovine melanin and after one week treated with varying concentrations of paraquat for an additional five days. Error bars are _+ SEM, n = 3. (B) Effect of melanin feeding and paraquat treatment on cell viability after paraquat feeding. The same cells were used as described in (A) in a parallel experiment in which cell viability was measured after paraquat t reatment using a MTT assay.

utilize oxygen at the rate of 1 5 - 30 nmol /min / mg, the majority of which is mitochondrial (Miceli et al., 1990; Miceli and Newsome, 1991). Another source of reactive oxygen species that has not received much attention in the RPE is peroxisomal fl-oxidation of fatty acids. The RPE phagocytoses and degrades a large quantity of lipid in the spent rod and cone outer segment. It is estimated that each human RPE cell must phagocytose and degrade 100 million photoreceptor disks in an 80 year lifetime (Young, 1982). Although it is likely that some of this material is recycled, there is evidence that much of the lipid material is metabolized. We have shown that the feeding of

106 D.A. NEWSOME et al,

0 ~ U [ 'l I ~1

n t s ~ # , ~

- - RPE A u t o f l u o r e s c e n c e ~-~ O X Y B U R S T Labe led ROS 1 HR H O X Y B U R S T Labe led ROS 4 HR

Log Fluorescence

FIG. 2. Flow cytometric data showing RPE cellular autofluorescence and the increase in fluorescence of cells treated with H2DCF (OXYBURST)-labeled bovine ROS for 1 and 4 hr. The fluorescence of control cells treated with antioxidants during ROS uptake was 16% of the fluorescence shown at 4 hr, demonstrating that the increase in fluorescence was due to oxidation of the probe

during phagocytosis.

isolated bovine rod outer segments (ROS) to cultured human RPE cells increases the non- mitochondrial oxygen consumption to 10°70 of the total uptake (Miceli et al. , 1992, submitted). We speculate that this may be due to an increase in peroxisomal/J-oxidation of the ROS lipids. Very- long-chain fatty acids have been shown to be oxidized almost exclusively in peroxisomes in other cell systems and it is possible that this process is occurring in RPE cells (Singh, 1987; Jakobs and Wanders, 1991). Since peroxisomes do not possess electron transport machinery, H202 is produced as a consequence of peroxisomal /J-oxidation by the acyl CoA oxidase (van den Bosch et al. , 1992). Peroxisomes also contain other oxidases that contribute to formation of H202. RPE cells have high catalase activities (Boulton et al. , 1990; Unger et al. , 1991; Liles et al. , 1991), and it is presumed that the location is mainly in the peroxisome.

It has been shown that phagocytosis results in the production of reactive oxygen species by using the probe 2',7, dichlorodihydrofluorescein (H2DCF) (Ryan et al. , 1990). When H2DCF is covalently attached to isolated bovine ROS and fed to cultured RPE cells, the non-fluorescent probe is oxidized, becomes fluorescent, and can be quantitatively measured in a flow cytometer (Fig. 2). The results of these experiments show that

ROS phagocytosis produces reactive oxygen species, although the exact nature of the pro- oxidant species is not known. Experiments are currently underway to quantitate H202 and superoxide production in human RPE cells during ROS phagocytosis. Superoxide has been reported to be produced by cultured porcine RPE cells (Dorey et al. , 1989); however, our data indicate that this is not due to a macrophage-type respiratory burst. It is possible that the main oxidant formed during ROS phagocytosis is H202, which may indicate a role for H202 in age-related changes in the macula, especially in light of reports of decreased catalase activity in RPE from donors with macular degeneration (Liles et al. , 1991).

3. A N T I O X I D A N T E N Z Y M E S

3.1. The First Line of Defense

There are numerous cellular enzymatic systems involved in defense against free radical damage. A hierarchy exists among these systems, in which the pre-eminent role is thought to be that of the 'first line of defense', antioxidant enzymes that directly scavenge and detoxify free radicals. Without this antioxidant defense, free radicals can inhibit crucial enzymatic systems, peroxidize membranes, and damage DNA, contributing to the accumulation of non-metabolizable aging products and the development of disease (Machlin and Bendich, 1987; Meneghini, 1988; Olson, 1988). The repair of this free radical-mediated cellular injury is crucial, and is accomplished in part by DNA repair enzymes and by lysosomal enzymes which degrade oxidized cellular constituents (Davies and Lin, 1988; Rivett, 1985). The viability of aging cells may depend upon the successful interaction between these disparate antioxidant and repair enzymatic systems.

The conventional triad of antioxidant enzymes includes SOD, the glutathione redox cycle, and catalase (Halliwell et al. , 1992). Superoxide anion (O2-), a short-lived oxygen radical, is produced as a by-product of mitochondrial respiration by the NADPH oxidase respiratory burst of neutrophils


TABLE 2. Activity Levels o f SOD, Catalase, SeGPx in Adult and FetaI RPE In Vivo and In Vitro

Enzyme activity In situ In vitro (per mg protein) Adult Fetal Adult Fetal

SOD (Units) 412 ± 60 418 ± 47 264 ± 40 122 _ 11.5 Catalase (Units) 88 ± 13 19 ± 3 20 ± 5 9 ± 3 SeGPx (mUnits) 112 ± 20 271 ± 53 220 ± 29 249 ± 41

and macrophages (Babior, 1984) and by metal- catalyzed oxidations (Stadtman, 1990), among other sources. The dismutation of 0 2- to H 2 0 2 is catalyzed by SOD (Bannister et al., 1987), which is known to have three isoforms: (1) manganese- SOD, which is derived evolutionarily from the bacteria and found only in the mitochondria of the eukaryote, (2) coppe r - z inc -SOD, found only in eukaryotic cells and present throughout the cytosol, nucleus, and many organelles, and (3) extracellular SOD, a copper and zinc dependent enzyme present in plasma, lymph and synovial fluids (Marklund, 1984). H202 (produced by the action of SOD or other processes) is decomposed by both the glutathione redox cycle and catalase. The glutathione cycle consists of glutathione (GSH), glutathione reductase (GR), selenium- dependent glutathione peroxidase (GPx), and glutathione-S-transferase. The oxidation of GSH b y H 2 0 2 is catalyzed by GPx, and the oxidized glutathione (GSSG) is reduced back to GSH by GR and NADPH. Inhibition of glutathione synthesis with buthionine sulfoxamine leads to substantial cellular free radical damage (Martensson et al., 1989). Other studies have shown that glutathione peroxidase is an important H 2 0 2

scavenging system in prevention of lipid peroxidation (Simmons and Jamall, 1989; Hiraishi et al., 1991). However, the most important function of GPx may be its role in breaking down potentially harmful hydroperoxides (van Kuijk et al., 1987; Krinsky, 1992). An additional form of phospho- lipid GPx, found in liver, has not yet been studied in the RPE/retinal complex (Ursini et al., 1982; Krinsky, 1992).

Catalase, which decomposes H202, was the first enzyme known to science (Chance and Oshino, 1971). With the exception of the red blood cell (Scott et al., 1991), catalase is found primarily in

the peroxisome. In fact, the standard histochemical stain for peroxisomes detects catalase activity (Novikoff et al., 1972). It appears that catalase has a specialized role in decomposing H 2 0 2 in the peroxisome compared with the general role of the glutathione redox cycle in the cytoplasm.

3.1.2. ANTIOXIDANT ENZYMES IN THE RPE

The RPE is extremely rich in antioxidant enzymes. SOD, the glutathione enzymes, and catalase are ubiquitous in mammalian tissue (Marklund et al., 1982). The bovine retina is apparently an exception, as SOD activities from bovine retina have been reported to be completely inhibited by cyanide, suggesting an absence of the cyanide-insensitive Mn-SOD (Crouch etal . , 1978). Human RPE, however, is known to have both copper-zinc and manganous forms of SOD (New- some et al., 1990). The expression of RPE Mn- SOD has been shown to be selectively stimulated by treatment with bacterial lipopolysaccharide (Del Vecchio and Shaffer, 1991). Our measure- ments of adult and fetal antioxidant enzymes have shown that the activities of catalase and SOD are higher in adult RPE than in fetal RPE (Table 2). This would imply that the adult RPE is under considerably more oxidative stress than the fetal RPE due to light, phagocytic load, and surround- ing oxygen tension. When exposed to H202,

cultured adult RPE cells were 40% more resistant to cytotoxicity than fetal cells. Inhibition of catalase activity by treatment of cultured adult RPE cells with 3-amino-l,2,4-triazole (3AT) i n c r e a s e d H 2 0 2 cytotoxicity by 2-fold. The decreased Mn-SOD activity of cultured fetal RPE compared with cultured adult RPE may be the primary cause of increased fetal RPE mortality from paraquat treatment. Immunolocalization of

108 D. A. NEWSOME el al.

Mn-SOD to the mitochondria of primary cultures indicate that senescent changes in the RPE layer may occur at different rates within individual cells (Oliver and Newsome, 1992).

The RPE is rich in peroxisomes and catalase activity, as measured by immunohistochemical (Atalla et al., 1987) and biochemical (Liles et al., 1991) techniques. In the RPE, the microperoxisomes esterify and store vitamin A, and, through the process of peroxisomal /3-oxidation, degrade the long-chain fatty acids of the outer segments (Leuenberger and Novikoff, 1975). The phagocytosis and degradation of shed ROS and the subsequent recycling of certain components is critical to retinal performance (Bok, 1985), but is also a source of oxidative stress (Miceli et al., 1992, submitted). ROS are rich in long-chain fatty acids, the chain-shortening of which (by peroxisomal 0-oxidation) results in the production of H202 as a by-product (Osmundsen et al., 1991). As a defense against H 2 0 2 , the retina and ROS contain GPx and glutathione-S-transferase activities (Naash et al., 1988; Naash and Ander- son, 1989). Left unscavenged, H z O 2 c a n cause peroxidation of lipids (Logani and Davies, 1979) and inactivation of lysosomal enzymes (Kobayashi et al., 1982; Niwa et al., 1985).

We have shown that catalase activity in human donor RPE significantly declined from the fifth to the ninth decade of life. Furthermore, in donors with evidence of age-related macular degeneration, catalase activity was approximately 32% lower than in normal age-matched donors (Liles et al., 1991) (Fig. 3).

Age-related degeneration of the retina is often associated with morphological changes suggestive of free radical damage. Two striking examples of age-related changes in the RPE are the intracellular accumulation of the aging pigment lipofuscin and the formation of drusen, a lipoproteinaceous material, at the RPE basal surface (Wing et al., 1978; Sarks, 1976). The extent to which these aging products interfere with normal retinal function and the mechanism of their formation are important unanswered questions.

The consequences of a loss of catalase activity by the RPE can be seen in experiments in which we inhibited catalase activity of cultured RPE using the herbicide 3AT. When these cells were fed

140 r- . . . . . . . . . . . . . .::Z: -: :_ Macullr RPE

1001120 ~ 1 ] ~ Perlpher.I RPE .....

2o l ! ;,

!

o ' i ~ _ . . . . . . . NORMAL DISEASED N O R M A L

(n • 10) (n - 6) (n • 10) DISEASED

(n • 8)

F]o. 3. Catalase activity in macular (shaded bars) and peripheral (hatched bars) RPE freshly isolated from human donor eyes. The decrease in activity correlated

with disease status.

isolated bovine ROS, both autofluorescence and lipid peroxidation were increased significantly (Liles et al., 1992, manuscript in preparation). It is not clear whether the inhibition of catalase induced an increase in either lipofuscin or drusen, but our data suggests that catalase activity is necessary to prevent free radical damage to the polyunsaturated fatty acids of the ROS. Further studies are necessary to characterize the nature of 3AT-induced autofluorescence, as well as the efficacy of antioxidant supplementation in the prevention of free radical damage to the RPE.

4. NON-ENZYMATIC ANTIOXIDANTS

4.1. Metaliothionein

4 .1 .1 . CHEMISTRY AND INDUCTION OF METALLOTHIONEIN

Since the discovery of metallothionein (MT), there have been numerous reports regarding its physiochemical properties and biosynthesis. MT is no longer considered a protein with the primary function of detoxifying cadmium and other toxic metals. MT is induced by a variety of stimuli, including zinc, copper, infection and other cellular stresses, and is intimately involved in the trace element and antioxidant systems of cells and tissues (Bremner and Beattie, 1990). MT is found


in a wide range of tissues, most notably liver, kidney, pancreas, intestine (Kagi and Kojima, 1987) and recently in the RPE (Oliver et ai., 1992). MT occurs in vivo with its complement of bound metals (Cousins 1985). It is a single polypeptide with a molecular weight of about 6500, and cysteine residues account for 30°7o of its amino acid composition. As mentioned, one of the main features of MT is its high metal content; it can bind up to 7 g atoms of cadmium or zinc and up to 12 g atoms of copper per molecule. All of the cysteine residues of MT are involved in the binding of metals with a relative affinity of copper > cadmium > zinc (Bremner and Beattie, 1990). These differences may be relevant in metal - metal interactions (Bremner and Beattie, 1990). Fluctuations in the bioavailable trace element status may affect the stability and concentration of MT in vivo.

The effects of copper and zinc on MT synthesis in the liver have been extensively reviewed (Cousins, 1985). Zinc, cadmium and iron induce MT in RPE cells (Oliver et al., 1992; Tate et al., 1992, unpublished data). The mechanism by which iron interacts with MT is not fully understood, and the stimulation of MT by iron may be an indirect effect due to increase in oxidative stress from the participation of iron in the Fenton reaction (Walkes and Klaassen, 1985; McCormick, 1987; Gunther et al., 1991).

The induction of MT by non-metal factors may be through direct or indirect mechanisms. MT is induced by glucocorticoids (Failla and Cousins, 1978; Etzel et al., 1979; Karin and Herschman, 1979; Karin et al., 1980), interleukins 1 and 6 (Cousins and Lenart, 1988; Karin et al., 1985; Herrlich et al., 1986; Schroeder and Cousins, 1990), la,25-dihydroxyvitamin D 3 (Karasawa et al., 1987; Shiraishi et al., 1983), oxidative stress (Sato et al., 1989), UV irradiation (Angel et al., 1986), infection (Subocinski et al., 1978), and CCL (Chvapil et al., 1973; Oh et al., 1978). In RPE cells, MT is induced by dexamethasone, la,25-dihydroxy vitamin D3, interleukin 6, oxidative stress, and retinoic acid (Oliver et al., 1992; Tate et al., 1992, unpublished data). The induction may be through a common pathway associated with metabolic and oxidative stress in the RPE and retina.

4.1.2. MT AS AN ANTIOXIDANT

There have been many studies suggesting MT may act as a free radical scavenger due to the abundance of thiol groups present on MT (Thornally and Vasak, 1985; Thomas et al., 1986; Hidalgo et al., 1988). Zinc thiolate clusters in MT have been reported to scavenge hydroxyl radicals (Thornally and Vasak, 1985), which is in agreement with other studies that thiol-containing compounds react with hydroxyl radicals. Pro- tection of cultured rat hepatocytes from chemical- induced free radical damage was observed by the addition of zinc to the medium. This addition increases cellular MT levels, suggesting MT may be one of the factors scavenging free radicals and reducing membrane damage (Hidalgo et al., 1988). With the exception of zinc, pretreatment with metals known to induce MT does not inhibit metal toxicity or the formation of peroxidized lipids in lung (Satoh et al., 1992; Coppen et al., 1988). The toxic effects of cadmium can be inhibited by increasing MT synthesis by exposure to zinc. This treatment does not affect cadmium uptake which is bound by the newly synthesized MT which reduces cadmium toxicity (Herrlich et al., 1986). The mechanism of zinc and MT protection against reactive oxygen intermediates may be through a zinc- MT complex rather than by zinc or MT alone. A zinc - MT complex may be attacked by reactive oxygen intermediates (ROI), causing a release of zinc that may protect against oxidative damage to biological structures (Fliss and Menard, 1992; Krezoski et al., 1984), especially membranes, and other critical cellular functions (Kondo et al., 1992; Dunn and Cousins, 1989). The significance of MT in protecting the RPE from toxic metals and reactive oxygen intermediates is under active investigation.

4.2. Zinc

Zinc is an essential element required for the maximal function of over 300 enzymes in DNA and RNA synthesis, proteolysis, and numerous other critical cellular functions. Zinc is also involved in protein-protein interactions and protein-DNA binding of hundreds of nuclear


regulatory proteins with zinc finger-like moieties. Zinc has the capacity to interact with up to four sutfhydryl groups on amino acids, a property which makes it a crucial element in three- dimensional structure and function of numerous proteins (Vallee and Auld, 1990; Coleman, 1992).

In the eye, the pigmented tissues have been shown to have high zinc content, primarily in the melanin granules. In monkeys, high levels of zinc in pigmented tissue appear to correlate with a high relative zinc uptake (Newsome et al., 1992). The amount of zinc in the melanin granules of the RPE is evident when one compares the pigmented and non-pigmented RPE of bovine eyes. Using flame atomic absorption spectroscopy, we found 1.15 and 0.2 ~g zinc/mg protein in the pigmented and non-pigmented bovine RPE, respectively. In the pigmented tissue, this represents approximately 2 ng zinc/106 granules or 18 × 10 6 molecules of zinc/granule.

In humans, we found a significant difference in the zinc content of macular compared with peripheral RPE, 0.89 and 1.73 ~g/mg protein, respectively (n = 12, p =0.001). This could be due to differences in pigmentation of the RPE, or it could indicate an altered zinc economy in the macular RPE. The greater amount of zinc in the periphery appears to correlate with our data showing higher MT levels in peripheral RPE (Tate et al., 1993).

While it is not clear whether pigment granules act as a storage site for zinc, an initial report in zinc-deficient pigs showed a decline in ocular melanosome zinc content (Samuelson et al., 1990). Initial studies in our laboratory using human RPE organ culture have shown that zinc-65 taken up by cells accumulates in pigment granules. Induction of MT, which increases zinc uptake in the RPE (Oliver et al., 1992), also results in increased movement of zinc into the pigment granules (Oliver et al., manuscript in preparation). In an in vitro system, human RPE cells, repigmented by feeding bovine melanin granules, took up approximately four times as much zinc-65 as did their non-pigmented controls. Induction of MT in these repigmented RPE cells resulted in a significant (1.7-fold) increase of zinc-65 uptake into both the cytoplasmic and pigment granule fractions of these cells. Thus, it appears that MT

may act as a shuttle for the intracellular distribution of zinc in human RPE.

Zinc deficiency has dramatic effects on rapidly dividing tissues, such as testis, and leads to dwarfism in humans (Prasad, 1991). Zinc is also important in mitosis and numerous other cellular functions. Many studies have shown that zinc deficiency can cause night blindness or changes in dark adaptation which can be reversed by administration of zinc (reviewed by McClain et al., 1985). Zinc deficiency in rats has been associated with ultrastructural changes in the retina and RPE, including accumulation of osmiophilic cytoplasmic granules. These changes may be related to alterations in retinoid metabolism (Shingwekar et al., 1979; Leure-duPree and McClain, 1982). In a separate study of zinc- deficient rats, it was demonstrated that lowered dietary zinc resulted in a reduction of retinol/ alcohol dehydrogenase of the retina (Huber and Gershoff, 1975). In the rainbow trout, zinc deficiency has been shown to be associated with cataractogenesis (Ketola, 1979). Considering the numerous functions of zinc, zinc deficiency in the RPE and retina could have deleterious effects at a number of levels, including retinol processing enzymes and retinol-binding proteins. Zinc deficiency has been shown to reduce circulating plasma retinol-binding proteins (McClain et al., 1985). One isolated report describes a study of ocular tissue from a person with retinitis pigmentosa with undetectable levels of interstitial retinol-binding protein, although the authors cautioned that this could be secondary to lost photoreceptors (Bridges et al., 1985). In a study of patients under parenteral nutrition, photopic a-wave-implicit times decreased with decreasing plasma zinc (Vinton et al., 1990).

Zinc is undoubtedly important for the antioxidant capacity of RPE cells, although the mechanism of action is not well understood. In rats, zinc deficiency results in increased lipid peroxidation in subcellular membranes of the liver (Burke and Fenton, 1985; Sullivan et al., 1980). Zinc also protects liver cells against carbon tetrachloride-induced liver injury in rats (Chvapil et al., 1973). However, in rats, bioavailable zinc has been shown to have profound effects on MT levels in hepatocytes (McCormick et al., 1981;

ANTIOXIDANTS IN THE R P E

Bremner and Davies, 1975; Sato et al., 1984). The protective effect of zinc may be related to levels of zinc- MT complexes within the cells, and not to zinc alone. In vitro, zinc protects cells against lipid peroxidation and helps stabilize cell membranes (Bettger et al., 1978; Bettger and O'Dell, 1981; Chvapil et al., 1973, 1979; Girotti et al., 1985; Coppen et al., 1988; Thomas et al., 1986; Thornally and Vasak, 1985). It has been suggested that zinc stabilization of cellular membranes may be especially important under high oxidative stress (Bray and Bettger, 1990). In addition, zinc may indirectly protect cells by inhibiting certain NADPH-oxidases that generate ROI (Chvapil et al., 1976; Ludwig et al., 1980; Kleiner and von Jagow, 1972; Jeffrey, 1983).

One important function of zinc may be to stabilize proteins by protecting functional -SH from undergoing oxidative damage (Gibbs et al., 1985). Zinc, with its high affinity for protein-SH groups, may inhibit ROI from interacting with proteins at these sites. The binding of zinc to protein-SH groups may displace Fe 2+ and Cu 2+ which could interact with H202 in a Fenton-type reaction and generate highly reactive hydroxyl molecules (Stadtman, 1990). In experiments with cultured hepatocytes, cells with elevated Cu 2+ were found to be more susceptible to paraquat and ascorbate mediated decreases in cellular glutathione levels (Zer et al., 1991). Since ROS contain iron and copper (McCormick, 1985), the release of Cu + or Fe 2+ from ingested photoreceptors, coupled with intracellular formation of H202, could be a possible cause of oxidative damage in intracellular organelles. Zinc and other antioxidants may be crucial for maintaining the functional integrity of RPE membranes.

In general, zinc deficiency does not appear to seriously reduce antioxidant enzyme activities in rats on a zinc-deficient diet (Bray and Bettger, 1990). In marginal zinc deficiency, certain cellular functions may be more sensitive to changes in zinc concentrations than others. Recent data from our laboratory showed that culturing RPE cells in medium with reduced zinc concentrations resulted in reduction of MT (Tate et al., 1993) and catalase (Oliver et al., manuscript in preparation). Other RPE cellular functions were significantly reduced when cultured in the low zinc medium, including

111

a-mannosidase and alkaline phosphatase. Enzyme activities which were maintained in low zinc included acid phosphatase and GPx. Zinc- dependent peptidases and hydrolases are most likely crucial for the metabolism of ingested ROS. Preliminary studies in our laboratory suggest that synthesis of proteoglycans by cultured RPE cells can also be affected by zinc levels. However, it is yet to be determined whether zinc affects the synthetic pathway, interactions with other macromolecules, or turnover and degradation. Proteoglycans and other extracellular matrix components may interact directly with reactive oxygen intermediates (Kashihara et aL, 1992). Matrix metalloproteinases that depend on zinc at their active catalytic site (Sapolsky, 1976; Spring- man, 1990; Kashihara et aL, 1992) deserve further study as these enzymes may be important in the remodeling and turnover of Bruch's membrane, which thickens and becomes brittle with age (Fig. 4).

4.2 .1 . MT AS AN INDICATOR OF ZINC STATUS

A close relationship exists between MT and zinc status in certain tissues. When we reduced the zinc concentration in RPE cell culture medium by Chelex-100 treatment (Tate et al., 1993), we observed a reduction in RPE proliferation and protein content. MT content in RPE also decreased under these conditions. Zinc measure- ments are usually obtained from hair, urine or plasma using atomic absorption spectrophoto- metry, or from antibody-mediated immune response or platelet aggregation assays (Hambidge et aL, 1986). Plasma zinc decreases in normal individuals when fed a zinc-deficient diet (Ham- bidge et al., 1986); however, plasma zinc can also be depressed by infection, disease, and pregnancy. This reduction may be due to activation of the MT gene (Cousins, 1985; Cousins and Lenart, 1988). Therefore, the decrease of plasma zinc may not indicate low zinc status, but rather a redistribution to tissues such as liver and bone marrow for functions related to host defense (Cousins and Lenart, 1988; Dunn and Cousins, 1989; Hambidge et al., 1986). Erythrocyte MT is less responsive to

1 12 D . A . NEWSOMt~ et al.

To Retina

\ / \

Phagosome

?

J GSH

Zn++

ZnM'[

Nucleus - DNA Membranes Proteins:

- metalloenzymes - structural - transcription factors

Facillitated

-'l I / \

FIG. 4. Interactions o f MT and zinc in RPE. Zinc taken up by RPE cells, if retained, associates with other molecules. MT may be critical for sequestering free zinc for temporary storage and distribution to macromolecules in various organelles and throughout the cytoplasm, and in retaining and recycling protein-bound zinc released during protein degradation. Release of zinc from Z n - M T can be initiated by interaction with superoxide or hydroxyl radicals, which in turn may accelerate the proteolytic degradation of MT. Glutathione may be involved in the reduction of oxidized MT (MTox),

thereby reconstituting its capacity to bind zinc.

stress and infection than plasma MT or plasma zinc and may be a more stable indicator of zinc status (Grider et al., 1990).

4.2 .2 . AGE-RELATED CHANGES IN TISSUE ZINC AND MT

Evidence has been published showing that correction of a marginal zinc deficiency in the elderly with dietary zinc supplementation may improve age-associated loss of immune responses (Lindeman et al., 1971; Duchateau et al., 1981). Zinc induces MT in the thymus and is associated with circulating thymic hormone (Olafson, 1985). Zinc is a cofactor in thymidine kinase, DNA and RNA polymerases. Thymic MT and zinc content is depressed in older mice, indicating that zinc and

MT are coordinately regulated in the thymus (Olafson, 1985).

We have shown that there is a decrease in MT with increasing donor age in human macular RPE. This decrease was greater in samples 70 years of age and older (Tate et al., 1993) and may reflect a decrease in available intracellular zinc. It is likely that certain zinc-dependent RPE-retinal functions undergo changes with age, which may contribute to a decrease in retinal function and to development of macular degeneration. The challenge is to identify which processes may be compromised by decreased bioavailability of zinc. Further study is necessary to learn more about the antioxidant and membrane stabilization roles of zinc, and whether there are critical levels of intracellular zinc that optimize crucial cellular functions.

4.3. Other Antioxidants

4.3.1. ASCORBATE

Ascorbate occurs in high concentrations in ocular tissues. Studies on guinea pigs have shown 22 mg/dl wet tissue in retina and 7 mg/dl wet tissue in RPE (Woodford et al., 1983). Ascorbate, found predominantly in reduced form in the retina, exists mainly in its oxidized form, dehydroxyascorbate (DHA), in the RPE. During mild photic damage, the levels of reduced ascorbate in the retina and RPE decline with a concomitant increase in DHA. These findings were also extended to baboons (Tso et al., 1983). The high levels of DHA could be explained, at least in part, an ascorbate peroxidase activity in the RPE (Kaul et al., 1988). In this study, it was demonstrated that extracts of RPE could use ascorbate as a reducing agent in the peroxidative degradation of H202. Since the pH optimum (4.5) of this reaction was considerably lower than that of myeloperoxidase and glutathione peroxidase, the authors concluded that it was a distinct enzyme with peroxidative activity. The high ratio of DHA to ascorbate in the RPE after exposure to light indicates an active intracellular oxidative process, and that ascorbate is involved in protection of the RPE. Since ascorbate is also involved in the regeneration of oxidized a-tocopherol, an increase in DHA may indirectly reflect scavenging activity of a-tocopherol. A deficiency in ascorbate may have multiple effects on the RPE antioxidant economy.

Light and oxygen-mediated free radical damage to the retina and the importance of free radical scavengers have been topics of discussion for many years (Feeney and Berman, 1976). Light damage is thought to be mediated by oxidative free radicals, and ascorbate may be especially important to ocular tissues subjected to light. In rats, ascorbate ameliorated extensive damage to both the retina and the RPE (Blanks et al., 1992; Organisciak et al., 1985; Zong-Yi et al., 1985). However, it is not clear from these studies whether the protective effect of ascorbate is a direct result of protection of the RPE or from protection of the photoreceptors. Ascorbate has been shown to protect the lens from light damage


(Varma et al., 1979, 1982). Vitamin C has been shown to prevent formation of cataracts in experimental ani~aal models, and it was concluded from these studies that cataract formation was directly linked to oxidative damage (Devamano- haran et al., 1991). The ascorbate/DHA redox couple can help to maintain a proper cellular redox balance by influencing glucose utilization and favorable NADP/NADPH ratios (Varma et al., 1987). Recently, it has been suggested that high levels of ascorbate in the vitreous and aqueous ( 1 - 2 mM) remove oxygen and thus provide protection for the lens (Eaton, 1991). The reaction involves the metal catalyzed (Fe 2+, Cu +) oxidation of ascorbate by 02. The glutathione redox cycle then reduces the oxidized DHA to ascorbate, which leads to generation of H202. For maximum protection, this interaction of oxygen with ascorbate would require an effective glutathione redox cycle to recycle DHA and other antioxidant activity to remove H202.

4.3.2. VITAMIN E

Vitamin E includes a class of tocopherol compounds, with a-tocopherol being the most common. Its lipophilic properties and association with cell membranes makes vitamin E one of the primary non-enzymatic defenses against lipid peroxidation, and protects against rupture of cell membranes and release of destructive lysosomal enzymes (Chow, 1991). The lysosomal system of RPE cells is active in the digestion of phagocytosed ROS which are rich in polyunsaturated fatty acids (PUFAs). Reaction of ROI with PUFAs can initiate a chain reaction leading to lipid peroxide end-products. It has been demonstrated that vitamin E-deficient animals have increased levels of lipid peroxidation, an effect further enhanced by restriction of selenium in the diet which reduces glutathione peroxidase (Chow, 1991; Hollis et al., 1992). The maximum efficacy of the antioxidant functions of vitamin E depends on the presence of adequate amounts of ascorbate, glutathione peroxidase activity, and reduced glutathione (GSH). Vitamin E deficiency in rats is associated with disruption of ROS and an increase in RPE lipofuscin (Robison et ai., 1979). When vitamin E-

114

deficient animals were fed reduced amounts of vitamin A, a loss of photoreceptor cells was also observed. It was concluded that vitamin E protects ROS from oxidative damage (lipid peroxidation), and the increased autofluorescence in the RPE of vitamin E-deficient animals was due to accumulation of lipid breakdown products. The authors further suggested that vitamin E may have prevented peroxidative loss of vitamin A. In a separate study, rats on an antioxidant-deficient diet (i.e. reduced a- tocopherol, selenium, sulfur-containing amino acids, and chromium) showed a dramatic increase in RPE autofluorescence (Katz et al . , 1978). These animals had reduced levels of glutathione peroxidase activity.

Alpha-tocopherol may have a protective effect on RPE/retinal function not directly related to its action as an antioxidant (Handelman and Dratz, 1986). Inhibition of vitamin A esterase by a- tocopherol inhibits hydrolysis of vitamin A esters, thereby preventing a rise in tissue retinol. Since free vitamin A alcohol has a lytic effect on cell membranes, it has been suggested that part of the retinal damage due to vitamin E deficiency might be mediated by vitamin A release, causing damage to lysosomal membranes and ROS. Blocking lysosomal proteinase activity with leupeptin caused an accumulation of autofluorescent material in the RPE, most pronounced when rats were fed vitamin A-sufficient diets (Katz and Norberg, 1992).

D. A. NEWSOMIi el al.

disease, cataracts, aging and carcinogenesis (Krinsky, 1989, 1991; Rousseau et al. , 1992).

An initial study by Ham et al. (1984) suggested that /3-carotene ameliorates photic damage in monkey retina. Further studies by Tso and his coworkers demonstrated that /3-carotene prevented severe light damage to the retina and RPE in an experimental rat model (Tso, 1989). The ameliorative action of antioxidants against photic damage strongly suggests that oxidative stress plays an important part in photic retinopathy. Risk for age-related macular degeneration is negatively correlated with a diet rich in fruits and vegetables with high/3-carotene content (Jacques and Chylak, 1991).

In the RPE, light damage may be direct, or it may be indirect by increasing the concentration of light-damaged outer segments which must be phagocytosed. /3-carotene may act as an antioxidant in the retina and RPE by preventing the formation of lipid peroxidative products in microsomal membranes, presumably by scavenging peroxyl radicals (Palozza and Krinsky, 1991). In these reactions, /3-carotene may be the primary defense against peroxidation chain reactions at low oxygen tensions, while a-tocopherol may be more operative at ambient oxygen levels (Burton and Ingold, 1984) (Fig. 5).

5. THERAPEUTIC INTERVENTION

4.3.3. O-CAROTENE

//-Carotene is a precursor to vitamin A, and vitamin A itself may somehow exacerbate the effects of light damage to the retina (Noell, 1979; Carter-Dawson et al . , 1981).//-Carotene has many cellular functions, including that of an antioxidant capable of scavenging superoxide radicals, lipid autoxidation intermediates, and Fenton-generated radicals. /3-Carotene is an effective scavenger of free radicals and may provide protection in numerous disease processes associated with oxidative stress, including Alzheimer's disease, ataxia telangiectasia, atherosclerosis, Parkinson's

Macular degeneration associated with age and driisen has no known cause. As with many common diseases of unknown cause, there have been a number of proposed etiologies (Young, 1987) along with suggestions for therapeutic intervention. To date, very few scientifically solid clinical trials have been conducted. The purpose of this section is to review what we know about factors influencing macular degeneration and the role of antioxidants and their therapeutic manipulation for this disease.

Macular degeneration is a chronic disease and usually appears in eyes that have previously given decades of good vision. There is often a strong family history, although the exact role of heredity remains uncertain. Once the earliest signs of


i ROS

z Mitochondria "~ - ~ / ~ Scavengers:

, / ~ \ ~ ~ ~ GSH, A, aT, BC, MT Phagolysoso~.~.~l~.._...~. ( \ \ X~ ) y x x /

/ / ' / / / / / / . /A FA \ \ ~ j~Cu++ ~ - / +++ O : | . "lPermanent Damage • 2 / ~ Pr°temSrRep air

~/ / / / / / / . / /~ Oxidases/ Cu+ ~ . ~ OH \ DNA Ji~gra~tion v ~ / / / Z / - . ,' Fe++ I ~

/ VLC~'~A * 0 ~ ~" " ~ J " ~Zn++ Mn SOD ? . . . .

CuZn ~¢ ' J ? RO (ReacUve hp,ds) // - ~..~ c~ ~_ \ H2 02 NADP.~, ~ ,,,=GSH , aT.~, ~..DH_A

eroxisome

\ . NADPH GSSG ~ V raT" a t~atalase ,~ ROH

r~2o

BM FIG. 5. Summary of reactive oxygen species and antioxidants in the RPE. Reactive oxygen species are generated by normal metabolic activity in numerous organelles, including mitochondria, phagolysomes and peroxisomes. Oxidative phosphorylation and numerous oxidases contribute to generation of superoxide anions and H20> Dismutation of superoxide anions by Mn- and CuZn-SOD is an additional source of H202, which can react with redox active metals to produce highly reactive OH./J-Oxidation of ingested fatty acids in peroxisomes also produces H202 as a by-product which is removed by the presence of catalase. The reactive superoxide anions and hydroxyl radicals may be neutralized by various non-enzymatic antioxidants capable of scavenging free radicals. Reactive lipid products may be inactivated enzymatically by glutathione dependent GPx or non-enzymatically by g-tocopherol. GSH, glutathione; A, ascorbate; aT, g-tocopherol; BC, beta carotene; MT, metallothionein; FA, fatty acids; VLCFA, very-long-chain fatty acid; GR,

glutathione reductase; DHA, dehydroascorbate.

115

macular degeneration appear, as long as a decade may pass before clinically significant reduction of vision occurs. At least 85°70 of macular degeneration cases are the chronic deteriorative or so- called 'dry' type. In some eyes, the course of the disease is complicated by subretinal neovasculari- zation which usually leads to a more rapid and severe loss of vision. The laser is useful in retarding vision loss in only some cases, and it should be remembered that the laser does not treat the underlying problem of macular degeneration.

What do we know about the antioxidant status of older individuals? As noted previously in this chapter, there is a general decline that occurs with aging of various antioxidant functions in many organ systems, including the RPE. Because antioxidant systems are dependent on a variety of nutritionally derived cofactors, it is reasonable to conclude that a declining bioavailability of these

cofactors could exacerbate the overall age-related reduction in antioxidant capability. Several well designed scientific studies of nutrition among older adults have consistently shown a significant reduction in intake of important antioxidant

TABLE 3. Mineral Levels o f Persons aged 60 to 65 Compared to RDAs or ESADDIs

Women Men mg/day mg/day

Copper ESADDI 2.00 - 3.00 2.00 - 3.00 Intake 0.86 1.17 Adequacy low low

Zinc RDA 15.00- 15.00 Intake 8.51 12.64 (% RDA) 56.70 84.30

(Pennington et al., 1986.)


cofactors, including trace elements (Carroll et al. , 1983) (Table 3).

When deficient intake is coupled with reduced gastrointestinal absorption, as is common in older persons, the stage is set for inadequate bioavailability of important nutrients. It is well known that stomach acid is reduced in older persons. This reduces the amount of trace elements released from ingested foodstuffs and hence decreases bioavailability. It has been shown in a number of studies that absorption of ingested vitamins and minerals support, in the most basic way, the use of oral supplementation but tell nothing about its efficacy (English-Westcott et al. , 1991; Newsome et al. , 1992; Prasad et al. , 1993; August et al. , 1989; Bunker et al. , 1987; Champagne, 1989; Craig et al. , 1990; Turnlund et al. , 1986).

There is growing evidence from a number of studies that maintaining higher blood levels of antioxidants reduces the risk of many serious diseases including cancer (Das, 1989; Dorgan and Schatzkin, 1991; Santamaria and Bianchi- Santamaria, 1991; Stahelin et al. , 1989). A recent analysis of the National Health and Nutrition Survey data indicates that individuals with a higher /3-carotene blood level experienced a significantly reduced incidence of the subretinal neovascular complication of macular degeneration. A placebo-controlled clinical trial of oral zinc supplementation showed a significant retar- dation of vision loss in those who received zinc (Newsome et al. , 1988).

Because nutritional supplementation should encompass the broader range of antioxidants, the more recently published studies of Mamalis and Pomerance (Bruce et al., 1991), showing an approximate 35070 protective effect of orally ingested broad spectrum antioxidant supplements, are significant. This protective effect matches the degree of protection conferred by higher amounts of antioxidant ingestion on cataract development reported by the National Cataract Study Group (Jacques et al. , 1988; Leske et al. , 1991).

Additional prospective placebo-controlled clinical trials are needed; there are at least two such studies in progress as this chapter goes to press.

While the results of these studies are eagerly awaited, it now seems reasonable to recommend broad spectrum antioxidant oral supplementation,

particularly in early cases of macular degeneration. The risks of this supplementation in clinical practice have proven to be extremely low. In our practice with over 1000 patients using a broad spectrum vitamin mineral antioxidant oral supplement, we have found a non-use rate of about 2°70, the majority of which is either due to gastrointestinal intolerance or to the simple ina- bility 'to take pills'. Thus, the risk/benefit ratio appears extremely favorable and is supported by widely appreciated anecdotal evidence of some vision improvement in patients who receive supplementation.

Future research will not only complete our understanding of the antioxidant cycles in the RPE but allow more effective intervention to help maintain good vision throughout life.

6. SUMMARY AND CONCLUSIONS

The RPE, important to maintenance of normal vision, exists in an environment associated with a high degree of oxidative stress. The RPE contains a number of protective systems including antioxidant enzymes and materials with antioxidant properties. Maintenance of a normally function- ing oxidative stress protection system requires a balance between substances such as catalase synthesized within the RPE cell and externally derived, conserved and recycled substances such as zinc, vitamin C and/3-carotene. Clinicopatho- logic correlative studies have associated abnor- malities of the RPE and Bruch's membrane with simple aging and with age-related diseases that severely reduce vision such as macular degeneration. Recently published biochemical pathologic correlative studies have shown age-related declines in various substances including catalase, glutathione peroxidase and metallothionein, all important to the protective anti-'wear and tear' enzyme activities in macular tissue. In many of these substances, additional studies have shown exag- gerated declines in the eyes of persons who have macular degeneration. Since the RPE is a non- dividing monolayer that must function and maintain itself throughout life, a clear understanding of antioxidant systems should expand our knowledge of a number of basic aging processes


and may open the door to manipulation of these processes to promote better vision throughout life.

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BLANKS, J. C., PICKFORD, M. S. and ORGANISCIAK, D. T. (1992) Ascorbate treatment prevents accumulation of phagosomes in RPE in light damage. Invest. Ophthalmol. Vis. Sci. 33" 2814-2821.

BOK, D. (1985) Retinal photoreceptor-pigment epithelium interactions. Invest. Ophthalmol. Vis. Sci. 26: 1659- 1693.

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Antioxidants in the retinal pigment epithelium

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