A reduced oxygen supply to cells, as occurs in ischemia or possibly aging, results in an alteration in the distribution of intracellular and extracellular ions across membranes. Furthermore, ischemia has been implicated in the pathogenesis of AMD.
31 Thus, the possibility exists that at later stages of AMD, free zinc, which may be present at very high concentrations within the photoreceptors and RPE, may be released into the extracellular space. If this process occurs, then the present data suggest that the elevated level of zinc has the potential to cause oxidative stress to photoreceptors and RPE cells. In addition, evidence is provided to show that antioxidants like trolox or metipranolol would be beneficial to such cells.
The role of zinc in AMD has been much discussed, especially in relation to the beneficial use of zinc supplementation, which has been generally supported by both laboratory and clinical data.
43 44 45 46 47 48 49 Also, low concentrations of zinc protect against oxidative stress of RPE cells.
25 50 Furthermore, important antioxidant RPE enzymes such as the metallothioneins are zinc-dependent, and their activities are affected by intake of dietary zinc.
51 However, the Eye Disease Case Control Study (EDCC)
52 and the Beaver Dam Eye Study (BDES)
53 found no significant relationship between serum zinc levels and the risk of AMD, in agreement with Stur et al.
45 We interpret these findings to suggest that in the very initial stages of AMD when perhaps nutrient deprivation plays a part, zinc supplementation may be beneficial, as this element has an intricate involvement in cellular antioxidant defense. However, as the disease progresses, zinc may be released into the extracellular space from the RPE and photoreceptors, and at this stage the positive effect of zinc supplementation may become redundant and even contribute to the disease process. This hypothesis would also predict that antioxidants are likely to have a positive effect at all stages of the AMD, provided they are present in sufficient concentrations.
In the present study it was clearly shown that zinc can act as an oxidant and can also induce death of RPE and photoreceptors. Evidence for the pro-oxidant property of zinc in comparison with SNP is documented by the lipid peroxidation studies (see
Fig. 1 ). The measurement of TBARS is generally considered to provide an index for lipid peroxidation. From these studies it can be concluded that zinc is a much weaker oxidant than SNP, but that it does work in a similar manner to promote lipid peroxidation. Previous studies have shown SNP to be as effective an oxidant in our system as iron/ascorbate.
34 35 EC
50 values for stimulation of TBARS in the present study for zinc and SNP were determined to be 100 and 10 μM, respectively. Moreover, maximum stimulation of TBARS formation for SNP was approximately twice that of zinc. The mechanisms by which zinc acts as an oxidant probably involves several events that eventually result in a generation of free radicals.
54 The effects on brain and retina-RPE membranes, shown in
Figure 1 , clearly support the view that zinc can directly interact with membrane constituents to generate free radicals. In addition, the effect of zinc on TBARS formation was, like SNP
(Table 1) , attenuated by metipranolol and trolox with other β-blockers used for the treatment of glaucoma having no effect.
Intravitreal injection of 150 μM zinc clearly caused mainly photoreceptor death with approximately 40% of the cells staining positively for TUNEL and caspase-3-/Bcl-2. Photoreceptor labeling was not uniform throughout the retina and, in this way, it resembled data previously reported that described the injection of SNP into the vitreous.
35 We suggest that this is related to the injection site. As found for SNP,
35 the maximum amount of photoreceptor apoptosis after intravitreal injection of zinc was detected after approximately 3 days. Moreover, the effect of zinc (150 μM vitreous concentration) on photoreceptor apoptosis was less than that of SNP (50 μM).
35 Thus, the effectiveness of zinc in comparison with SNP in inducing photoreceptor apoptosis in vivo resembled the in vitro lipid peroxidation studies on brain membranes. It is important to note that in preliminary experiments, zinc was injected into the vitreous humor at a concentration range of between 25 and 150 μM. It was clear from these studies that when zinc was present at the concentration of 150 μM, the appearance of photoreceptor apoptosis and its counteraction with metipranolol or trolox was optimal. Significantly, even when the concentration of zinc in the vitreous was at its greatest, no obvious detrimental effect appeared to be associated with the inner retina. Thus, we conclude that zinc, at an intravitreal concentration of 150 μM does not cause an acute, nonspecific lethal effect on the retina but rather that some photoreceptors are affected.
Quantification of the degree of photoreceptor death from analysis of retinal sections was difficult due to the variability associated with injection both in relation to the site and the depth into the vitreous humor. It was concluded that it would be necessary to analyze every section throughout a single retina to obtain meaningful results from these analyses. Quantification was therefore restricted to an analysis of Western blot data derived from whole retinal extracts. This showed that zinc clearly caused a reduction in rhodopsin kinase, an enzyme associated exclusively with photoreceptors in the eye.
55 Moreover, the active form of caspase-3, the cleaved form of PARP, and Bcl-2 were all increased. The increase in the cleaved forms of both caspase-3 and PARP were consistent with what is expected to occur in apoptosis, but this was not the case for Bcl-2, where it would be anticipated to decrease rather than increase. One possible explanation is that those photoreceptors that did not label positively for TUNEL were mildly injured, resulting in an increase in their Bcl-2 protein levels, in an attempt to prevent cell death. This being the case, then the overall retinal Bcl-2 level would appear to be elevated with respect to untreated samples. Significantly, zinc effects were counteracted when metipranolol or trolox were co-injected. These in vivo studies on photoreceptor apoptosis were therefore consistent with the in vitro lipid peroxidation studies conducted on brain membranes.
In all experimental cases, retinal regions that showed no photoreceptor damage or apoptosis remained in contact with the RPE and appeared normal when analyzed by light microscopy. Retinal detachment was observed only at sites of photoreceptor damage. Furthermore, the RPE cell layer generally appeared normal throughout the eye, even in areas where photoreceptors were significantly damaged. Moreover, the effect of zinc injection on photoreceptors was localized in an obvious manner that reflected the distance from the injection site. Because there was a clear delineation in the toxic effect of zinc on retinal photoreceptors, and because no RPE cells appeared to have been induced to die by this ion after 3 days, in situ, at least when analyzed at the light microscopic level, then we could only conclude that retinal detachment occurred as a result of photoreceptor damage and did not precede this event. The fact that RPE cells appeared at a gross level to be unaffected by intraocular injections of zinc, in situ, at the time points analyzed in this study, implied that this ion did not penetrate as far as this epithelial monolayer in sufficient quantities to induce apoptosis. It is possible and, indeed, likely that zinc affected RPE cells in a less obvious, or sublethal manner and that this may have led to the death of these cells after longer periods of time (i.e., >3 days). This would especially be the case after photoreceptor loss and subsequent retinal detachment, because penetration of the ion into these cells would therefore have been greatly increased under these circumstances. Our studies, however, stopped short of describing the effects of intraocular injections of zinc on RPE cells in situ—merely concentrating on the neural retina. This is the reason that analysis of the RPE was restricted to cells in culture, since these remained a pure and homogeneous population, uncontaminated by other cell types.
Studies of RPE cells revealed that zinc above a certain concentration also induced apoptotic cell death that was counteracted by metipranolol or trolox but not by other ophthalmic β-adrenergic receptor blockers. The question that arises from these observations is why RPE cells and photoreceptors are more prone to the oxidant effect of zinc than are other retinal cell-types. The answer may relate to common membrane characteristics of the two cell types. It is known that photoreceptors are particularly susceptible to free radical damage
56 because of their high content of polyunsaturated fatty acids (e.g., DHA).
57 58 Whether RPE membranes contain such membrane components is unknown. The specific effect of zinc on photoreceptors after injection may also relate to its penetrance into the retina. Thus, even after injection of a vitreous concentration of 150 μM zinc, retinal levels may remain much lower, which could be sufficient only to cause death of photoreceptors because of their high DHA content. This notion would be compatible with the finding that 10 μM zinc has little effect on RPE cells as well as causing negligible lipid peroxidation to brain membranes.
In a prior study, we suggested that the reason that only metipranolol, of the β-blockers used to treat glaucoma, acts as an antioxidant relates to its structure,
35 as is the case with other nonophthalmic β-adrenoceptor antagonists.
59 60 61 Given that evidence exists from animal studies to show that topically applied drugs can reach the retina,
62 63 the possibility of employing metipranolol in this way to attenuate photoreceptor/RPE death in AMD is worthy of consideration. Metipranolol is completely converted in vivo to its active metabolite, desacetylmetipranolol,
64 and this is an even more potent antioxidant than metipranolol
(Table 1) . It is possible that the half-life of desacetylmetipranolol is greater than antioxidants such as vitamin E. If this is the case, then desacetylmetipranolol may accumulate in the eye with constant topical use, allowing for a more sustained antioxidant effect. It is theoretically possible that metipranolol merely binds to zinc and prevents its destructive action. However, the clear antioxidant activity of metipranolol and the fact that it will prevent photoreceptor destruction induced by other insults (e.g., nitric oxide).
35
In summary, the present study shows that above a certain concentration, zinc can cause apoptotic death of RPE cells in culture, as well as retinal photoreceptors after intravitreal injection. Furthermore, the nonselective β-blocker, metipranolol and its active metabolite, desacetylmetipranolol, can both partially attenuate zinc-induced lipid peroxidation, in a manner similar to trolox, and prevent zinc-induced retinal cell death. Given the proposed role of free radicals in the pathogenic destruction of RPE cells and photoreceptors in AMD, the remarkable antioxidant activity of metipranolol and desacetylmetipranolol may be of clinical importance.
The authors thank Nigel Swietalski for his expert technical assistance.