Metallomics 2014 6 201 by Instituto Oftalmológico Fernández-Vega

Metallomics PERSPECTIVE

Cite this: Metallomics, 2014, 6, 201

Metallothioneins (MTs) in the human eye: a perspective article on the zinc–MT redox cycle a a a a He ´ctor Gonzalez-Iglesias, Lydia Alvarez, Montserrat Garcı´a, Carson Petrash, b ac Alfredo Sanz-Medel and Miguel Coca-Prados*

Metallothioneins (MTs) are zinc-ion-binding proteins with a wide range of functions, among which are neuroprotection, maintenance of cellular zinc homeostasis, and defense against oxidative damage and inflammation. The human eye is enriched in MTs, and multiple isoforms may contribute to distinct antioxidant defense mechanisms in various ocular tissues. Zinc is a main regulator of MT gene and protein expression, and we recently applied bioanalytical techniques to address key questions on its relationship with MTs, including the stoichiometry of zinc–MT, the fate of zinc tracers (natZn and

Zn) in

MTs during activation by exogenous zinc and cytokines, and the concentration of MTs in human ocular cells. We found that exogenously introduced zinc induced a potent de novo synthesis of MTs as well as a strong inhibition of pro-inflammatory cytokines. Zinc and cytokines also promote a stoichiometric transition of the MT complex from Zn6Cu1–MT to Zn7–MT, suggesting that MTs may interact more effectively with reactive oxygen species to decrease potential oxidative damage. Levels of MTs decrease Received 7th October 2013, Accepted 11th December 2013

with aging and disease, which may result in zinc release that is potentially cytotoxic. This state is also

DOI: 10.1039/c3mt00298e

of MTs has been impaired. In this review we propose a working model of the ‘‘zinc–metallothionein

observed with increased oxidative stress and inflammation, suggesting that the antioxidant function redox cycle’’ to regenerate and enhance the antioxidant function of MTs with the aim of combating the

www.rsc.org/metallomics

progression of these disease states.

1. Introduction 1.1 Update on metallothioneins: definition, classification and functions Metallothioneins (MTs) are low molecular mass proteins (6 to 7 kDa), cysteine rich (approximately 30% of their amino acids are cysteine) and present in all organisms studied.1–3 In mammals, MTs predominantly bind zinc, but under conditions of copper or cadmium overload, zinc can be readily displaced in favor of these other metals.4 MTs consist of two structural domains that form metal–thiolate clusters: the a-domain (32–61 residues), which can bind up to four zinc ions and contains eleven cysteinyl residues, and the b-domain (1–31 residues), which can bind up to three zinc ions and contains nine cysteinyl residues.5 Recent studies have shown the coexistence of partially-metalated intermediates of MT complexes along with the apo-protein and the

Fundacioń de Investigacioń Oftalmolo´gica, Instituto Oftalmolo´gico ńdez-Vega, Avda. Dres. Fernandez-Vega, 34, 33012, Oviedo, Spain. Ferna E-mail: miguel.coca-prados@fio.as; Fax: +34 985233288; Tel: +34 985240141 b Department of Physical and Analytical Chemistry, University of Oviedo, c/Julian Claverıá, 8, 33006, Oviedo, Spain c Department of Ophthalmology and Visual Science, Yale University School of Medicine, 300 George St, 8100A, New Haven, CT 06510, USA

This journal is © The Royal Society of Chemistry 2014

fully-metalated protein, which indicates a non-cooperative metalation mechanism.6,7 In humans, the MT family consists of multiple isoforms (and subisoforms) arranged into four groups, MT1 to MT4, which share a high degree of homology at the nucleotide and amino acid levels.8 MT1 and MT2 are abundantly expressed in almost all tissues, MT3 is expressed in the central nervous system (CNS) including the retina, and MT4 is found in stratified tissues.1 There are at least eight functional genes that encode each of the MT1 subisoforms (MT1A, MT1B, MT1E, MT1F, MT1G, MT1H, MT1M, and MT1X). Likewise distinct genes encode each of the other MT isoforms, namely MT2 (MT2A), MT3 and MT4.9 The reason for this high diversity of MT isoforms is currently unknown. To date, several intracellular functions have been ascribed to MTs, including regulation of intracellular zinc metabolism and storage, zinc donation to target apo-metalloproteins (especially zinc finger proteins and enzymes), metal detoxification (i.e., Cd, Hg, Zn, Cu, etc.),10 promotion of neuroprotection,11,12 contribution to the control of cellular proliferation and apoptosis, and defense against oxidative damage and inflammation.5 There are a growing number of studies reporting the extracellular activity of MTs. In the brain, it has been shown that MTs are released from astrocytes following CNS injury, and they

Metallomics, 2014, 6, 201--208 | 201

Perspective

interact directly with neurons promoting cell survival, neuroprotection, and enhancing regenerative growth.12–14 1.2

MTs and zinc homeostasis

MTs are among the most important zinc-binding proteins in eukaryotic cells. Cytosolic zinc-binding proteins can buffer fluctuations in zinc concentration by direct binding to intracellular zinc, shuttling it into internal stores or removing it from the cells altogether. It is noteworthy that zinc–MT binding is thermodynamically stable, which gives to these proteins a key role in intracellular zinc homeostasis.15 Zinc is an essential element in the cell that serves as a catalytic cofactor to enzymes, a structural component of proteins, and is involved in many cellular processes including gene expression.16 Additionally, although zinc itself does not exhibit redox properties, it can exert important effects on the redox metabolism of the cell. However, a high concentration of exchangeable ‘‘free’’ zinc is toxic to cells, so it is imperative that zinc is appropriately buffered to limit its concentration to physiologically acceptable intracellular levels.17 In addition to MTs, cellular homeostasis of zinc is strictly regulated by zinc transporters, which are involved in zinc intake and output from cells and organelles. There are two families of zinc transporters: the ZnT and the ZIP. ZnTs usually transport zinc from the cytosol into the extracellular space or into intracellular organelles, while the ZIP family of zinc transporters promotes the transport of zinc from the extracellular environment or from cellular organelles into the cytoplasm.18 Significantly, the identified zinc transporters have not been shown to function in mitochondria and nuclei; however, MTs can be transported into these organelles. Hence, MTs provide the pathway for zinc to be available to mitochondria and nuclei and exert subsequent effects on mitochondrial respiratory function, gene regulation and control of cell proliferation and differentiation.19,20 1.3

Oxidative stress and MTs in the human eye

Oxidative stress is caused by an imbalance between free radical production and the cells’ ability to readily detoxify the reactive intermediates and repair the resulting damage. The human eye is subjected to oxidative stress from multiple sources, including daily exposure to sunlight (i.e., visible and UV light) and the special microenvironment with abundant photo-sensitizers and high-energy and high-oxygen-consuming tissues (which support highly oxidative milieu).21 Oxidative stress has been associated with many eye diseases, including cataracts,22–24 glaucoma,25–27 retinitis pigmentosa (RP),28,29 diabetic retinopathy (DR),30,31 and aged-related macular degeneration (AMD).32,33 Oxidative damage is normally minimized by the presence of a range of antioxidants and eﬃcient cellular repair systems. Ocular tissues are enriched in certain antioxidants in the form of metabolic enzymes (i.e., selenium-dependent peroxidases, glutathione peroxidases, dismutases, etc.) or small molecules. A lesser-known antioxidant system in the eye consists of MTs, which may mitigate cell destruction induced by oxidative stress by capturing and neutralizing free radicals through cysteine sulfur ligands.8,34,35 This property of MTs is likely linked to

202 | Metallomics, 2014, 6, 201--208

Metallomics

their ability to bind zinc and serve as zinc-ion donors in a redoxdependent fashion in cellular processes.36,37 The cluster structure of zinc–MT provides a chemical basis by which the cysteine sulfur ligands can be oxidized and reduced concomitantly with the release and binding of zinc. This oxido-reductive mechanism confers antioxidant activity on the zinc–MT complex.6,38–40

2. MT expression and tissue distribution in the human eye 2.1

Gene expression

In a recent study, Alvarez et al.41 examined the gene expression profiling of MT isoforms in normal human eye donors (cadavers) by microarray analysis, and compared their abundance between tissues [i.e., cornea, trabecular meshwork (TM), lens, iris, ciliary body (CB), retina, retinal pigment epithelium (RPE) and sclera]. Overall, the lens was the ocular tissue with the highest level of MT expression, followed by RPE, iris, cornea, CB, sclera, retina and TM. Furthermore, multiple MT isoforms are highly expressed throughout these ocular tissues (see Fig. 1). In particular, MT1A, MT2A and MT1X sub-isoforms were found highly expressed in all tissues, while MT1E, MT1F, MT1M and MT1G were expressed in much lower levels, with the exception of MT1G that was expressed abundantly in lens followed by the RPE. MT1H and MT3 isoforms were expressed in low levels, but restricted preferentially to the lens and the retina, respectively. Given that the cornea and the lens represent natural barriers to external environmental insults (i.e., UV light) and that they play important roles in combating free radicals, it is plausible that the higher levels of expression and diversity of MT isoforms found in these tissues may contribute to cellular defense against oxidative damage. For example, the elevated expression of MTs in the RPE may serve as a protective mechanism in the blood– retina barrier to combat the influx of toxic substances such as heavy metal ions into the retina. Moreover, since the RPE is particularly susceptible to oxidative damage, due to its high metabolic oxygen flux and its high phagocytic activity, MTs would protect the RPE cells against apoptosis and oxidative stress.42 Unsaturated fatty acids in the photoreceptors are particularly susceptible to attack by hydroxyl radicals, and MTs could play a role as a hydroxyl radical scavenger in these cells. Tate et al. have studied the expression of MTs in the human chorioretinal complex.43 By in situ hybridization they detected mRNA for diﬀerent MT isoforms in the RPE, and choroid, and in a lower level in the sclera. Expression was also detected in the developing photoreceptor layer, the outer nuclear layer and the inner nuclear layer of the retina. There is experimental evidence that the highest levels of zinc within the mammalian eye are present in the RPE–choroid complex.44,45 However, it remains unknown to what extent this zinc is bound to MTs and other zinc-binding proteins. For example, although pigmented tissues such as RPE and iris have quite similar gene expression (mRNA) levels of MTs, zinc concentration in RPE is much higher when compared to iris. This is likely due to the fact that within the RPE cells, zinc is preferentially associated with melanosomes, which is the main element bound to melanin and is

This journal is © The Royal Society of Chemistry 2014

Metallomics

Perspective

Fig. 1 Gene expression profiling of metallothionein isoforms [i.e., MT1 (MT1A, MT1B, MT1E, MT1F, MT1G, MT1H, MT1M AND MT1X), MT2 (MT2A) and MT3] in multiple ocular tissues [cornea, trabecular meshwork (TM), iris, lens, ciliary body (CB), retina, retinal pigment epithelium (RPE), and sclera] of the human eye. The indicated tissues were dissected, and their RNA was isolated and analyzed on the Illumina microarray platform HumanHT-12 v4.0. The relative hybridization signal obtained for each MT isoform was normalized with internal controls and expressed as arbitrary units (AU), on a logarithmic scale. In the center of the figure a schematic picture of the structure of the human eye is illustrated.

required for melanin synthesis.46â&#x20AC;&#x201C;48 However, MT affinity for zinc ions differs for each of the seven zinc ions ranging from log K = 11.8 to 7.7, while synthetic melanin affinity for zinc ranges from log K = 5.8 to 3.7 (situation in vivo is predictable to be lower). Therefore, it should be expected that zinc bioavailability from MT is greater than from melanin.15,49 2.2

Tissue localization

Fig. 2 shows the cellular distribution determined by indirect immunofluorescence of MT1/2 isoforms in cornea and lens and MT3 in retina and iris. In human cornea, MT1/2 isoforms are confined in the cytoplasm of the epithelium and endothelium, reflecting a combination of the multiple MT1 sub-isoforms expressed by these cells.41 In the human lens there is a spatial and isoform-specific cell distribution of MT1/2 along the epithelium and lens fibers, in line with data presented by Oppermann et al.50 In the retina, MT1/2 isoforms have been immunolocalized in the inner plexiform and inner nuclear layers. In the RPE, the presence of intracellular melanin granules causes a false positive signal upon MT1/2 immunolocalization, as already noted in other studies.42 However, Nishimura et al. have reported the localization of MT1/2 in RPE of rats.51 In the latter study MT1/2 immunostaining was also observed in the corneal epithelium and endothelium, in the epithelium and equatorial fibers of the lens and in the

This journal is ÂŠ The Royal Society of Chemistry 2014

retinal nerve fibers and inner plexiform layers, which is in agreement with our results. In contrast, MT3 antibodies preferentially labeled retinal ganglion cells (RGCs) with a cellular staining of the nerve fiber layer (NFL) and the cytoplasm, whereas in iris MT3 is confined in the cytoplasm of the stromal cells. The cell-restricted and isoform-specific MT3 detection in RGCs may reflect biochemical and/or physiological diďŹ&#x20AC;erences with other MT isoforms. RGCs are the first retinal cells to undergo cell death in eye diseases such as glaucoma, and they are highly sensitive to damage during oxidative stress caused by intense visible and UV light exposure. Recently, Tsuruma et al.52 investigated the functional role of MT3 in light-induced retinal damage and suggested that MT3 may be an important neuroprotective substance to prevent retinal degeneration. Moreover, it has been shown that RGCs in vivo rapidly internalize MTs secreted by astrocytes, promoting axonal regeneration.12

3. Mechanisms of MT regulation Little is known about MT regulation in the eye. We recently used a human corneal cell line (HCEsv) as an in vitro model to examine the mechanism(s) regulating MT gene expression and

Metallomics, 2014, 6, 201--208 | 203

Perspective

Metallomics

Fig. 2 Cellular distribution of MT1/2 isoforms in human cornea and lens, and MT3 in human retina and iris. FPE sections (5 mm thick) were stained with the monoclonal antibody to MT1/2 or MT3 and analyzed using a fluorescent microscope (Axioskop; Carl Zeiss, Go ¨ ttingen, Germany). Panels A, C, E, and G are phase contrast micrographs of B, D, F, and H. Panels B, D, F, and H are merged images of DAPI staining of cell nuclei (blue) and rhodamine-labeled (red) micrographs. MT1/2 antisera labeled the corneal epithelium and endothelium (panel B), while in lens it labeled lens epithelium (panel D). MT3 antisera labeled the nerve fiber layer and retinal ganglion cells (panel F), while in iris it labeled the cytoplasm of the stromal cells.

Fig. 3 Mechanism of MT regulation and oxidative stress responses in an in vitro cell model. Zinc exerted a strong up-regulation on MT1/2 expression and an inhibition on cytokines IL6 and IL8 expression, in HCEsv cells. ZnT-1 (zinc exporter) is up-regulated, whereas Zip1 is down-regulated at the expression level.

protein synthesis.41 The predominant eﬀects exerted by exogenous zinc on HCEsv cells are represented in Fig. 3 and 4. Zinc exerted a dual but antagonistic eﬀect on HCEsv cells (Fig. 3). On one hand, it induced a robust up-regulation of MT expression, and on the other hand, an inhibition of cytokine expression (i.e., IL6 and IL8). Because MTs are highly recognized for their antioxidant properties, it is reasonable to suggest that zinc may represent a key cellular factor in oxidative stress by establishing crosstalk communication between MTs and inflammatory cytokines. This is relevant because a number of ocular diseases including AMD and glaucoma are associated with cell-mediated immune responses.53,54

204 | Metallomics, 2014, 6, 201--208

The inhibition of pro-inflammatory cytokine gene expression by zinc may be mediated by either preventing the dimerization of Stat3 proteins, or by activating NF-kB with IkB.55 Conversely, whether cytokines trigger the release of zinc from vesicles into the intracellular space or the uptake of zinc is at present unknown. It has been suggested that intracellular zinc ions, when not bound to proteins, may be stored in vesicles and then released by the action of membrane-bound zinc-transporter proteins.15 Zinc also exerted a distinct effect on two zinc transporters: the up-regulation of SLC30A1 (ZnT-1), and the down-regulation of SLC39A1 (Zip1). Interestingly, out of these transporters ZnT-1 exerts efflux whereas Zip1 exerts influx of zinc. This result is consistent with a protective response of cultured cells to an excess of external zinc. The effects elicited by zinc and pro-inflammatory cytokines on MT protein synthesis have also been studied. Fig. 4 shows the concentration of MTs (expressed in micrograms per 106 cells) in HCEsv cells after treatment with zinc at 24 h, 48 h and 72 h. Using a new bioanalytical and methodological approach (IPD-ICP-MS), it was possible to quantitate the intracellular concentrations of MTs under steady-state conditions from MTs induced by extracellular zinc as a result of ‘‘de novo’’ synthesis. Furthermore, the stoichiometric transition of the Zn–MT complex in HCEsv cells before and after zinc treatment was also estimated. The findings indicated that zinc or cytokines changed the elemental composition of MTs from Zn6Cu1–MT to Zn7–MT. This suggests that new MTs are saturated by zinc ions. Thus, cells may adapt to metal overloads by inducing a robust

This journal is © The Royal Society of Chemistry 2014

Metallomics

Perspective

Fig. 4 Micrograms of cellular MTs per 106 cells in HCEsv cells labeled with natZn and 68Zn, at 24, 48, and 72 h in the absence (control) or presence of: (i) 68ZnSO4 (100 mM) alone; (ii) IL1a (100 U ml 1), and (iii) a combination of 68ZnSO4 (100 mM) and IL1a (100 U ml 1). The concentration of MTs containing, natural (natZn) and ‘‘exogenous’’ zinc (68Zn), was determined by IPD-HLPC-ICP-MS. The Zn–MT stoichiometry is also indicated.

stimulation of MT synthesis to increase the metal binding capacity, and the molecules themselves may permit occupation of their seventh zinc-binding site. In contrast and according to recent studies, in the absence of an excess of zinc ions, the last metal binding site in MTs might not be occupied, leading to partially metalated species, i.e., Zn6–MTs.6 Previous studies have reported that Zn7–MT is able to release one zinc atom to other zinc-binding proteins in an energetically favorable fashion.5,9 In this way, it can be gathered that the Zn7–MT species has a greater antioxidant capacity than Znx–MT (where x ranges from 0 to 6). Thus, in a state of zinc excess (but below cytotoxic levels) MTs may interact more effectively with reactive oxygen species (ROS) to decrease the potential oxidative damage. Our prediction is that Zn7–MT species will render the cell or tissue more resistant to oxidative stress insults.

4. The zinc–MT redox cycle and its potential implication in AMD Age-related macular degeneration (AMD) is the leading cause of irreversible blindness among persons 65 years or older. At least 60 million people worldwide are aﬀected, and the number will triple in thirteen years.56 Risk factors that may contribute to the development of AMD include genetics, inflammation, smoking, and oxidative stress associated with metal dyshomeostasis.57,58 AMD is clinically characterized by the progressive and irreversible central vision loss. There are two forms of end-stage AMD: geographic atrophy (‘‘dry’’ AMD, 85–90% of cases) and neovascular (‘‘wet’’ AMD, 10–15% of cases).59 In both forms, the affected area is the macula and there is a decline in sharp central vision as a consequence of photoreceptor degeneration

and loss. In geographic atrophy AMD, the RPE cells slowly degenerate and may atrophy completely, which eventually causes blindness; in neovascular AMD, the growth of new blood vessels from the choroid through Bruch’s membranes and the RPE, under the macula, leads rapidly to blindness.60 The hallmark of AMD is the build-up of deposits in the subretinal space between the RPE and Bruch’s membranes, called sub-RPE deposits or drusen.58 The composition of sub-RPE deposits is very complex,61 containing proteins (such as vitronectin, apolipoproteins, crystallins, etc.), lipids, and anomalous accumulation of zinc that can reach millimolar levels, some of which is in the exchangeable (ionic or loosely protein bound) form.62 In line with this, the Age-Related Eye Disease Study (AREDS, 2001) showed that treatment of AMD with zinc supplements or antioxidants plus zinc significantly reduced the progression of the neovascular form of AMD in patients at intermediate and late stages.63 However, no apparent eﬀect of lower-dose zinc on progression to advanced AMD was found in the latter AREDS2 study,64 and current evidence on zinc intake for the prevention of AMD is inconclusive.65 How zinc supplementation may help to slow down the progression of AMD is not quite understood, and an increase of zinc availability would have multiple eﬀects. For instance, at intermediate and late stages of AMD, cellular zinc concentration could be lower as a result of the formation of a higher number of sub-RPE deposits and zinc supplementation might trigger uptake of zinc into the RPE–choroid complex,66 helping to maintain the retinal functions. Moreover, the increased plasma zinc level may also boost the immune system67 and provide better protection against inflammation.68 A current line of research in AMD suggests that its progression could be reduced or stopped if sub-RPE deposit formation is modulated. In this regard, a key question to be addressed is

Metallomics, 2014, 6, 201--208 | 205

Perspective

the origin of zinc accumulation in these deposits.44 Cell injury induced in RPE cells by oxidative stress and aging could result in breakdown and/or damage to these cells, and this may trigger the release of zinc from MTs and melanosomes into the extracellular space where deposit formation occurs.62,69,70 This sub-RPE deposits’ growth may occur through oligomerization of proteins including complement proteins (i.e., complement factor H), which also leads to uncontrolled inflammation71,72 and impairment of mitochondrial function.38 Intracellular zinc depletion may also induce apoptosis of RPE and retinal cells, impair cellular metal homeostasis by altering RPE antioxidant metalloenzymes and redox cycle interactions and enhancing oxidative stress and cell damage.62 Sub-RPE deposit formation and RPE cell injury progression may be delayed by avoiding the depletion of intracellular zinc, where MTs play an important role in cytosolic zinc buﬀering and muﬄing. Based on current knowledge, an imbalance in the cellular redox state begins with ageing. The extent and consequences of cellular oxidation caused by this imbalance depend upon the antioxidant defense capability of the RPE and neighboring tissues.73 As previously mentioned, there is an antioxidant system that could be of fundamental importance in the RPE and retina: the Zn–metallothionein/thionein system. There is evidence that implicates the imbalance of this antioxidant defense system with the development of AMD.74 Tate et al. reported that MTs declined as a function of age in the macular-RPE of patients with AMD, with proportionately greater loss in the macula than in the periphery.75 Furthermore, it was determined that the macular region contains less zinc and MTs than the peripheral RPE. Besides, a decrease was observed in the MT mRNA expression levels in simian retina with early AMD compared to healthy subjects.76 Erie et al. showed that the zinc level in RPE–choroid complexes decreased in AMD donors compared to donors not affected by AMD.77 Newsome et al. established that zinc levels are reduced in human eyes with signs of AMD.78 All these findings suggest that MT expression and intracellular zinc levels decrease with aging and oxidative stress in the RPE, and most dramatically in the macular region, which may result in an alteration of the MTs zinc-binding capacity. Oxidative stress is deeply correlated with the degeneration of the retina and RPE, and MTs may protect against retinal and RPE damage by acting as endogenous antioxidants. Thus, reactivating the antioxidant capacity of the system may reverse or reduce this imbalance. We hypothesized that the zinc–metallothionein redox cycle may have potential implications in AMD. The scheme of Fig. 5 depicts the reactions of the zinc–MT redox cycle based on previous publications.5,38,79–81 Under physiological oxidative conditions, zinc bound to MT (Zn–MT) is released through the thiolate cluster and MT-disulfide (thionin) is formed. Zinc (i.e., free zinc ions) released from MTs may either accumulate in secretory vesicles called ‘‘zincosomes’’ or in mitochondria. It may also interact with the metal regulatory transcription factor 1 (MTF-1), which translocates from the cytoplasm into the nucleus and up-regulates the production of MT,6 or be donated to other zinc-binding proteins. This latter process is facilitated in the presence of nitric oxide, ROS and oxidized glutathione (GSSG).

206 | Metallomics, 2014, 6, 201--208

Metallomics

Fig. 5 Zinc–metallothionein redox cycle. Zinc bound to MT (Zn–MT) is released to zinc stores and zinc-binding proteins under physiological oxidative conditions, forming MT-disulfide (thionin). This process is enhanced in the presence of free radicals such as nitric oxide (NO), reactive oxygen species (ROS) and oxidized glutathione (GSSG). MTdisulfide may be degraded or, in a reduced environment, reduced to MT-thiol (thionein). This reduction is facilitated in the presence of a selenium-derived catalyst (i.e., selenocystamine). MT-thiol binds zinc to form Zn–MT, thermodynamically stable.

MT-disulfide (thionin) is unstable and prone to degradation. However, in a more reduced cellular environment, for example with glutathione (GSH), and in the presence of a seleniumderived catalyst (*Se, i.e., selenocystamine), it will reduce thionin into thionein. Zinc–MT will be then quickly reconstituted in the reduced form. This premise is consistent, as stated above, with the view that MT levels likely decrease with aging, as well as their ability and of melanosomes to bind zinc,81–83 increasing the concentration of unbound zinc in RPE cells in the macular region.75 This situation will lead to increased oxidative stress, cytotoxicity by free zinc, and inflammation. In this working hypothesis, a reduction in the concentration of MTs and/or loss of zinc-binding activity by MTs will lead over time to increased cellular toxicity of free-zinc, and therefore to an impaired zinc–MT redox cycle. In line with our hypothesis, we predict that the regeneration of thionein through the GSH–GSSG system (gray arrow in the scheme of Fig. 5), and/or the induction of MT synthesis (dark circle in Fig. 5), will restore the RPE redox imbalance, which may prevent zinc release and slow or stop the formation of subRPE deposits and consequently uncontrolled inflammation. This assumption, of course, needs to be tested experimentally, and if validated, it will require the development of Zn–MT stimulating molecules to study their effects on animal models of AMD.

5. Conclusion Metallothioneins are distributed ubiquitously throughout tissues of the human eye. The relative tissue-specific abundance of MT isoforms could reflect their potential role in protective mechanisms against oxidative stress and inflammation. However, the biological and biochemical significance of the MT isoforms within the eye is at present unknown. There is substantial evidence that zinc ions mediate a crosstalk between MTs and cytokines to modulate their expression, which provides clues to explore their role in

Metallomics

pro-inflammatory events and immune-regulated processes associated with eye diseases including AMD. Extensive evidence supports the hypothesis that with aging and oxidative stress, the Zn–MT redox cycle becomes impaired. Under oxidative stress, zinc is released from MTs, and higher, potentially cytotoxic, zinc-ion concentrations occur in the RPE. We suggest that the reactivation of the zinc–metallothionein redox cycle might restore the redox imbalance and prevent zinc release during AMD pathogenesis. The proof of concept of this hypothesis could open a window to the management of this devastating disease.

Acknowledgements This study has been supported in part by the CENIT-CeyeC research grant CEN-20091021 from the Spanish Ministry of ń de Investigacio ń Innovation and Development, the ‘‘Fundacio ´gica Ferna ńdez-Vega’’ (http://fio.fernandez-vega.com), Oftalmolo ń Rafael del Pino’’ (http://www.frdelpino.es), and the ‘‘Fundacio ń Ma Cristina Masaveu Peterson’’ (http://www.funda the ‘‘Fundacio ´tico cioncristinamasaveu.com). Miguel Coca-Prados is ‘‘Catedra ń de InvestigaRafael del Pino en Oftalmologıá’’ in the ‘‘Fundacio ń Oftalmolo ´gica, Instituto Oftalmolo ´gico Ferna ńdez-Vega’’ cio ń and Enol Artime, Oviedo, Spain. We thank Manuel Chaco ń de Investigacio ń Oftalmolo ´gica’’, for their from the ‘‘Fundacio technical support.

References ´k and G. Meloni, J. Biol. Inorg. Chem., 2011, 16(7), 1 M. Vasa 1067–1078. 2 K. Zangger and I. M. Armitage, Metallothioneins, in Handbook of Metalloproteins, ed. A. Messerschmidt, W. Bode and M. Cygler, 2004, John Wiley & Sons, Chichester, UK, vol. 3, pp. 353–364. 3 D. E. K. Sutherland and M. J. Stillman, Metallomics, 2011, 3, 444–463. 4 J. H. Kagi, Methods Enzymol., 1991, 205, 613–626. 5 S. G. Bell and B. L. Vallee, ChemBioChem, 2009, 10(1), 55–62. 6 D. E. K. Sutherland, K. L. Summers and M. J. Stillman, Biochemistry, 2012, 51(33), 6690–6700. 7 T. T. Ngu and M. J. Stillman, Dalton Trans., 2009, 5425–5433. ´k, J. Trace Elem. Med. Biol., 2005, 19(1), 13–17. 8 M. Vasa 9 Y. Li and W. Maret, J. Anal. At. Spectrom., 2008, 23, 1055–1062. 10 S. R. Davs and R. J. Cousins, J. Nutr., 2000, 130(5), 1085–1088. 11 M. Thiersch, W. Raﬀelsberger, R. Frigg, M. Samardzija, A. Wenzel, O. Poch and C. Grimm, BMC Genomics, 2008, 9, 73. 12 R. S. Chung, J. Hidalgo and A. K. West, J. Neurochem., 2008, 104, 14–20. 13 A. K. West, J. Hidalgo, D. Eddins, E. D. Levin and M. Aschner, Neurotoxicology, 2008, 29(3), 488–502. 14 R. S. Chung, M. Penkowa, J. Dittmann, C. E. King, C. Bartlett, J. W. Asmussen, J. Hidalgo, J. Carrasco, Y. K. Leung, A. K. Walker, S. J. Fung, S. A. Dunlop, M. Fitzgerald, L. D. Beazley, M. I. Chuah, J. C. Vickers and A. K. West, J. Biol. Chem., 2008, 283(22), 15349–15358.

Perspective

15 R. A. Colvin, W. R. Holmes, C. P. Fontaine and W. Maret, Metallomics, 2010, 2, 306–317. 16 B. L. Vallee and K. H. Falchuk, Physiol. Rev., 1993, 73(1), 79–118. 17 Q. Hao and W. Maret, J. Alzheimer’s Dis., 2005, 8(2), 161–170. 18 J. P. Liuzzi and R. J. Cousins, Annu. Rev. Nutr., 2004, 24, 151–172. 19 B. Ye, W. Maret and B. L. Vallee, Proc. Natl. Acad. Sci. U. S. A., 2001, 98, 2317–2322. 20 M. G. Cherian and M. D. Apostolova, Cell. Mol. Biol., 2000, 46, 347–353. 21 Y. Chen, G. Mehta and V. Vasiliou, Ocul. Surf., 2009, 7, 176–185. 22 A. Spector, FASEB J., 1995, 9, 1173–1182. 23 J. A. Vinson, Pathophysiology, 2006, 13, 151–162. 24 M. C. Ho, Y. J. Peng, S. J. Chen and S. H. Chiou, J. Clin. Gerontol. Geriatr., 2010, 1, 17–21. `, Mutat. Res., 2006, 612, 25 A. Izzotti, A. Bagnis and S. C. Sacca 105–114. 26 V. Zanon-Moreno, P. Marco-Ventura, A. Lleo-Perez, S. Pons-Vazquez, J. J. Garcia-Medina, I. Vinuesa-Silva, M. A. Moreno-Nadal and M. D. Pinazo-Duran, J. Glaucoma, 2008, 17, 263–268. 27 G. Tezel, Exp. Eye Res., 2011, 93(2), 78–186. 28 K. Komeima, B. S. Rogers and P. A. Campochiaro, J. Cell. Physiol., 2007, 213, 809–815. 29 S. Usui, B. C. Ovesson, S. Y. Lee, Y. J. Jo, T. Yoshida, A. Miki, K. Miki, T. Iwase, L. Lu and P. A. Compochiaro, J. Neurochem., 2009, 110, 1028–1037. 30 R. A. Kowluru and S. N. Abbas, Invest. Ophthalmol. Visual Sci., 2003, 44, 5327–5334. 31 J. W. Baynes, Diabetes, 1991, 40, 405–412. 32 J. G. Hollyfield, V. L. Bonilha, M. E. Rayborn, X. Yang, K. G. Shadrach, L. Lu, R. L. Ufret, R. G. Salomon and V. L. Perez, Nat. Med., 2008, 14, 194–198. 33 S. G. Jarret and M. E. Boulton, Mol. Aspects Med., 2012, 33, 399–417. 34 K. E. R. Duncan and M. J. Stillman, J. Inorg. Biochem., 2006, 100(12), 2101–2107. 35 N. Romero-Isart and M. Vasˇ´ ak, J. Inorg. Biochem., 2002, 88(3–4), 388–396. 36 W. Maret and A. Krezel, J. Mol. Med., 2007, 13(7–8), 371–375. 37 A. Krezel, Q. Hao and W. Maret, Arch. Biochem. Biophys., 2007, 463(2), 188–200. 38 Y. J. Kang, Exp. Biol. Med., 2006, 231, 1459–1467. 39 W. Maret and B. L. Vallee, Proc. Natl. Acad. Sci. U. S. A., 1998, 95, 3478–3482. 40 W. Maret, J. Nutr., 2000, 130(5S), 1455S–1458S. 41 L. Alvarez, H. Gonzalez-Iglesias, M. Garcia, S. Ghosh, A. Sanz-Medel and M. Coca-Prados, J. Biol. Chem., 2012, 287(34), 28456–28469. 42 H. Lu, D. M. Hunt, R. Ganti, A. Davis, K. Dutt, J. Alam and R. C. Hunt, Exp. Eye Res., 2002, 74(1), 83–92. 43 D. J. Tate, M. V. Miceli and D. A. Newsome, Curr. Eye Res., 2002, 24(1), 12–25. 44 N. Berzegar-Befroei, S. Cahyadi, A. Gango, T. Peto and I. Lengyel, Zinc and Eye Diseases, in Zinc in Human Health, ed. L. Rink, 2011, pp. 530–553.

Metallomics, 2014, 6, 201--208 | 207

Perspective

45 M. Ugarte, G. W. Grime, G. Lord, K. Geraki, J. F. Collingwood, M. E. Finnegan, H. Farnfield, M. Merchant, M. J. Bailey, N. I. Ward, P. J. Foster, P. N. Bishop and N. Osborne, Metallomics, 2012, 4, 1245–1254. 46 J. Horcicko, J. Borovansky, J. Duchon and B. Prochazkova, Hoppe-Seyler’s Z. Physiol. Chem., 1973, 354, 203–204. 47 D. Kokkinou, H. U. Kasper, K. U. Bartz-Schmidt and U. Schraermeyer, Pigment Cell Res., 2004, 17, 515–518. 48 M. Furumura, F. Solano, N. Matsunaga, C. Sakai, R. A. Spritz and V. J. Hearing, Biochem. Biophys. Res. Commun., 1998, 242, 579–585. 49 E. Buszman, B. Kwaniak and A. Bogacz, Stud. Biophys., 1988, 12, 143–153. 50 B. Oppermann, W. Zhang, K. Magabo and M. Kantorow, Invest. Ophthalmol. Visual Sci., 2001, 42, 188–193. 51 H. Nishimura, N. Nishimura, S. Kobayashi and C. Tohyama, Histochemistry, 1991, 95, 535–539. 52 K. Tsuruma, H. Shimazaki, Y. Ohno, A. Honda, S. Imai, J. Lee, M. Shimazawa, M. Satoh and H. Hara, Invest. Ophthalmol. Visual Sci., 2012, 53, 7896–7903. 53 D. H. Anderson, R. F. Mullins, G. S. Hageman and L. V. Johnson, Am. J. Ophthalmol., 2002, 134, 411–431. 54 M. B. Wax and G. Tezel, Exp. Eye Res., 2009, 88(4), 825–830. 55 C. Kitabayashi, T. Fukada, M. Kanamoto, W. Ohashi, S. Hojyo, T. Atsumi, N. Ueda, L. Azuma, H. Hirota, M. Murakami and T. Hirano, Int. Immunol., 2010, 22(5), 375–386. 56 A. Taylor, Mol. Aspects Med., 2012, 33, 291–294. 57 L. S. Lim, P. Mitchell, J. M. Seddon, F. G. Holz and T. Y. Wong, Lancet, 2012, 379(9827), 1728–1738. 58 R. D. Jager, W. F. Mieler and J. W. Miller, N. Engl. J. Med., 2008, 358, 2606–2617. 59 P. S. Mettu, A. R. Wielgus, S. S. Ong and S. W. Cousins, Mol. Aspects Med., 2012, 33, 376–398. 60 I. Bhutto and G. Lutty, Mol. Aspects Med., 2012, 33, 295–317. 61 J. W. Crabb, M. Miyagi, X. Gu, K. Shadrach, K. A. West, H. Sakaguchi, M. Kamei, A. Hasan, L. Yan, M. E. Rayborn, R. G. Salomon and J. G. Hollyfield, Proc. Natl. Acad. Sci. U. S. A., 2002, 99, 14682–14687. 62 I. Lengyel, J. M. Flinn, T. Peto, D. H. Linkous, K. Cano, A. C. Bird, A. Lanzirotti, C. J. Frederickson and F. J. Van Kuijk, Exp. Eye Res., 2007, 84, 772–780. 63 Age-Related Eye Disease Study Research Group, Arch. Ophthalmol., 2001, 119, 1417–1436.

208 | Metallomics, 2014, 6, 201--208

Metallomics

64 Age-Related Eye Disease Study 2 (AREDS2) Research Group, JAMA, 2013, 309(19), 2005–2015. 65 R. Vishwanathan, M. Chung and E. J. Johnson, Invest. Ophthalmol. Visual Sci., 2013, 54(6), 3985–3998. 66 D. A. Newsome, P. D. Oliver, D. M. Dupree, M. V. Miceli and J. G. Diamond, Curr. Eye Res., 1992, 11, 213–217. 67 L. Rink and P. Gabriel, Proc. Nutr. Soc., 2000, 59, 541–552. 68 G. S. Hageman, P. J. Luthert, N. H. Victor Chong, L. V. Johnson, D. H. Anderson and R. F. Mullins, Prog. Retinal Eye Res., 2001, 20, 705–732. 69 A. C. Bird, Eye, 2003, 17, 457–466. 70 S. J. Perkins, R. Nan, A. I. Okemefuna, K. Li, S. Khan and A. Miller, Adv. Exp. Med. Biol., 2010, 703, 25–47. 71 R. Nan, I. Farabella, F. F. Schumacher, A. Miller, J. Gor, A. C. R. Martin, D. T. Jones, I. Lengyel and S. J. Perkins, J. Mol. Biol., 2011, 408, 714–735. 72 R. Nan, J. Gor, I. Lengyel and S. J. Perkins, J. Mol. Biol., 2008, 384, 1341–1352. 73 K. P. Ng, B. Gugiu, K. Renganathan, M. W. Davies, X. Gu, J. S. Crabb, S. R. Kim, M. B. Rozanoswska, V. L. Bonilha, M. E. Rayborn, R. G. Salomon, J. R. Sparrow, M. E. Boulton, J. G. Hollyfield and J. W. Crabb, Mol. Cell. Proteomics, 2008, 7, 1397–1405. 74 Y. Ito, H. Tanaka and H. Hara, Curr. Pharm. Biotechnol., 2013, 14, 400–407. 75 D. J. Tate, D. A. Newsome and P. D. Oliver, Invest. Ophthalmol. Visual Sci., 1993, 34(7), 2348–2351. 76 M. G. Nicolas, K. Fujiki, K. Murayama, M. T. Suzuki, N. Shindo, Y. Hotta, F. Iwata, T. Fujimura, Y. Yoshikawa, F. Cho and A. Kanai, Exp. Eye Res., 1996, 62(4), 399–408. 77 J. C. Erie, J. A. Good, J. A. Butz and J. S. Pulido, Am. J. Ophthalmol., 2009, 147, 276–282. 78 D. A. Newsome, M. V. Miceli, D. J. Tate Jr., N. W. Alcock and P. D. Oliver, J. Trace Elem. Exp. Med., 1996, 8(4), 193–199. 79 Y. Chen and W. Maret, Eur. J. Biochem., 2001, 268, 3346–3353. 80 A. Krezel and W. Maret, Biochem. J., 2007, 402, 551–558. 81 W. Maret, Biometals, 2009, 22, 149–157. 82 G. Musci, T. Persichini, M. Casadei, V. Mazzone, G. Venturini, F. Polticelli and M. Colasanti, Mech. Ageing Dev., 2006, 127(6), 544–551. 83 S. Julien, A. Biesemeier, D. Kokkinou, O. Eibl and U. Scharermeyer, PLoS One, 2011, 6(12), e29245.