Gonzalez iglesias et al, jaas, 2017

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Cite this: DOI: 10.1039/c6ja00431h

Quantitative distribution of Zn, Fe and Cu in the human lens and study of the Zn–metallothionein redox system in cultured lens epithelial cells by elemental MS†‡ He´ctor Gonza´lez-Iglesias, *ab Carson Petrash,a Sara Rodr´ıguez-Mene´ndez, a Montserrat Garc´ıa, ab Lydia Alvarez, Luis Ferna´ndez-Vega Cueto,ab ´ ac Beatriz Ferna´ndez, Rosario Pereiro, ac Alfredo Sanz-Medel c and Miguel Coca-Prados*ad

ac

The human lens is constantly subjected to exogenous and endogenous stressors, leading to oxidative cellular damage and, with time, to cataract formation. Metallothioneins (MTs) are a group of important enzymes that use metals (i.e., Zn and Cu) to protect tissues from the deleterious effects of free radicals associated with oxidative stress. This work combines elemental mass spectrometry with bio-analytical methodologies to determine (i) the total amount, the quantitative speciation and the cellular distribution of trace elements (i.e., Zn, Fe, and Cu) in human lenses and their corresponding capsules; and (ii) the effects of “exogenous” metal (i.e.,

68

ZnSO4, isotopically enriched in

68

Zn) and stressor (i.e., IL-1a) on the

zinc–MT redox system in cultured human lens epithelial cells (HLEsv) in vitro. Of all the elements analyzed, Zn was the most abundant, and it was equally present in both the capsule (9.7 2.5 mg g 1 tissue) and the lens (9.5 1.2 mg g 1 tissue). In contrast, Fe was found to be more than 6-fold more abundant in the capsule (1.6 0.4 mg g 1) than in the lens (0.2 0.1 mg g 1). Zinc in the lens is mainly associated with high molecular mass proteins, whereas in the capsule it is mostly bound to low and Received 30th November 2016 Accepted 7th June 2017

medium molecular mass proteins. The localization of Zn, Cu and MTs in the lens showed their preferential co-distribution in the lens epithelial cell layer, underneath the anterior capsule. Exogenous

DOI: 10.1039/c6ja00431h

Zn is capable of inducing a stoichiometric change in MT proteins from Zn3–MT to Zn7–MT within lens

rsc.li/jaas

epithelial cells in vitro, which may be related to their antioxidant capacity.

Introduction The lens is a transparent avascular tissue whose main function is to transmit and focus light onto the retina.1–3 It is comprised of two cell populations, the lens epithelial cells and the elongated ber cells, enclosed in a thin elastic capsule with asymmetric biconvex curvature (see Fig. 1). Throughout life, regenerative lens epithelial cells differentiate into tightly packed ber cells that form the bulk of the lens.4 The

transparency of the lens is made possible by the rapid degradation of all organelles during the latter stages of ber cell differentiation.5,6 The mammalian lens consists mainly of water (70% w/w) and protein (30% w/w).3 Crystallins (i.e., a, b and g)

a

Fundaci´ on de Investigaci´ on Oalmol´ogica, Instituto Universitario Fern´ andez-Vega, Instituto Oalmol´ ogico Fern´ andez-Vega, Universidad de Oviedo, Avda. Dres.

Fernandez-Vega, 34, 33012, Oviedo, Spain. E-mail: h.gonzalez@o.as; Fax: +34-985233-288; Tel: +34 985240141

b c

Instituto Oalmol´ ogico Fern´ andez-Vega, Oviedo, Spain

Department of Physical and Analytical Chemistry, University of Oviedo, Spain

d

Department of Ophthalmology and Visual Science, Yale University School of Medicine, 300 George St, 8100A, New Haven, CT 06510, USA † 5th themed issue devoted to Young Analytical Scientists. ‡ Electronic supplementary 10.1039/c6ja00431h

information

(ESI)

available.

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See

DOI:

Fig. 1 Schematic view of the human lens. The mammalian lens (enlarged image) is coated by a thin membrane, the capsule. The anterior capsule covers the epithelial cell layer.

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are the major structural proteins constituting up to 90% of total proteins in the human lens.7 The human lens is constantly subjected to oxidative stress from multiple sources, including daily exposure to ultraviolet light, chemical insults and a highly oxidative milieu.8 Oxidative damage plays a key role in age-related diseases of the ocular lens, most specically cataract formation.9,10 Age-related cataracts are characterized by extensive protein modication and aggregation within the lens bers, due to increased oxidative stress.11,12 The oxidative damage is normally minimized by the presence of a range of antioxidants,13 cellular repair systems,14 and metabolic enzymes, including superoxide dismutase, catalase, and glutathione peroxidase.13 All these enzymes require metals, i.e., Zn, Cu and Fe, and semi-metals, i.e., Se, to act as cofactors for their functions.11,15 Conversely, impaired metabolism of these transition metals may contribute to the formation of oxidative stress-inducing species.16–18 Therefore, the knowledge of metal sub-cellular partitioning in the lens is very useful to obtain biochemical information on metals and their homeostasis.

Recently, we have shown the presence of a novel antioxidant system associated with the zinc–metallothionein redox complex in the human eye.19,20 This antioxidant system captures and neutralizes free radicals through cysteine sulfur ligands present in metallothioneins (MTs) that serve as zinc-ion donors in a redox-dependent fashion.21,22 MTs are a group of cytosolic zincbinding proteins of low molecular mass (6 kDa to 7 kDa) consisting of multiple isoforms that are clustered into four groups (1 to 4), which share a high degree of homology at the nucleotide and amino acid levels,23 and are highly expressed throughout the ocular tissues, particularly in the lens. The higher levels of expression and diversity of MT isoforms may be related to the continuous subjection of the eye to environmental insults.19 The study of metals involved in the defense mechanism against oxidative stress in the eye and their role in biological functions and ocular disease is of great interest. In this work, we determined the concentration of Zn, Fe, Cu and Se in human lenses from post-mortem eye donors by isotope dilution (ID) analysis and elemental mass spectrometry (i.e., ICP-MS). The relevance of these trace elements in the physiology and pathophysiology of the eye depends not only on their levels, but also on their intracellular distribution. Therefore, we carried out the quantitative speciation of Zn, Fe, and Cu in the water-soluble protein fractions of human lenses (naked lenses) and their capsules with chromatographic separation of the proteins (based on molecular mass) coupled online to an elemental MS detector. Additionally, we studied the elemental distribution of Zn and Cu in cryogenic sections of human lenses by laser ablation (LA)-ICP-MS and compared these ndings with the molecular distribution of MTs in de-paraffinized lens sections.

To better understand the role of the Zn–MTs redox system in the defense mechanism against oxidative damage and inammatory stress in the lens, we used an in vitro cellular model representative of the human lens epithelial cells (HLEsv). We studied the effects of metals (i.e., Zn in the form of ZnSO4, and isotopically enriched in 68Zn) and inammatory cytokines (i.e., IL-1a) on MT proteins in HLEsv cells by using enriched stable isotopes as tracers and ICP-MS detection.

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Experimental Instrumentation Two elemental mass spectrometry instruments were used for multielemental isotopic measurements: an Element 2 sector eld (SF)-ICP-MS unit (Thermo Fisher Scientic, Bremen, Germany) and an ORS-ICP-MS model Agilent 7500ce (Agilent Technologies, Tokyo, Japan) equipped with an octopole reaction system. For chromatographic separations (i.e., multielemental speciation), a HPLC system (Shimadzu, model LC20AD, Kyoto, Japan) and the corresponding column (i.e., size exclusion or anion exchange) were used. A scavenger column (25 0.5 mm id) packed with Kelex-100 (Schering, Germany) was used to remove metal ions present in the mobile phases.

For bioimaging studies, an LSX-213 laser ablation system (Teledyne Cetac Technologies, Omaha, NE, USA) was coupled to the Element 2 ICP-MS to obtain images of Zn and Cu distribution in lens sections. The commercial ablation cell from Teledyne Cetac was replaced by a novel Peltier-cooled ablation cell built in-house.24 The plasma operating conditions, acquisition parameters and chromatographic conditions are shown in Table S1 (see the ESI).‡

Enriched stable isotopes Stable isotope solutions enriched in 34S (99.61% abundance of 34 S), 67Zn (94.01% abundance of 67Zn), 54Fe (96.80% abundance of 54Fe), or 74Se (98.84% abundance of 74Se) were purchased from Cambridge Isotope Laboratories (Andover, MA, USA). 65Cu (90.03% abundance of 65Cu) was purchased from Spectrascan (Teknolab AS Dr¨ obak, Norway). 68Zn (99.23% abundance of 68 Zn) was purchased from Isoex (San Francisco, CA, USA). The isotopically enriched zinc sulphate solution (68ZnSO4) was prepared from elemental 68Zn (powder) by dissolving the metal in a minimum volume of supra-pure grade H2SO4 (96% v/v, Suprapur® Merck Millipore, Germany), and then diluting it with ultrapure water.

Collection of tissues from postmortem human eye donors A total of 20 eye globes from adult donors (cadavers), without relevant ocular pathologies affecting the lens, ranging in age from 45 to 75 years were obtained within 24 h of the post mortem through the Hospital Universitario Central de Asturias (HUCA, Oviedo, Spain). The procedures adhere to the tenets of the Declaration of Helsinki, and full ethical approval was obtained from the Clinical Research Ethics Committee at the HUCA. The lens from each eye was extracted, and further dissected into its capsule and the rest of the lens (i.e., naked lens or lens without capsule). Both tissues were stored at 80 C until further use. Additionally, for bioimaging studies by LAICP-MS, the dissected human lenses were directly frozen at 80 C without any further treatment. The lenses were embedded in an Optimal Cutting Temperature Compound, cut (100 mm thickness) in a Microm HM550 cryostat (Thermo Fisher Scientic, Walldorf, Germany), at 20 C, and mounted on microscope glass slides (Superfrost® Plus, Thermo Scientic).

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Total multielemental determination in lenses and capsules by isotope dilution-ICP-MS A total of ten lenses and their corresponding capsules were dissected and used for quantitative analysis of the total Zn, Fe, Cu and Se. Both naked lenses and capsules were mineralized in high purity grade 3 mL of concentrated HNO3 (65% v/v, Suprapur® Merck, Germany) + 1.5 mL of H2O2 (30% w/v, Aristar® BDH, UK) and 300 mL of HNO3 + 150 mL of H2O2, respectively, assisted by the use of an ultrasonic bath (Fisher Scientic, UK). The digested samples were diluted with ultrapure water (Milli-Q system, Millipore) to reach 2% (v/v) HNO3. Preparatory blank tubes (no tissue added) were similarly treated as samples, and used as background controls. The digested samples were analyzed by ID-ICP-MS following previous reports.19,25

Quantitative speciation of Zn, Fe and Cu in lenses and capsules by SEC-ID-ICP-MS Ten lenses and their corresponding capsules were used for quantitative speciation of Zn, Fe and Cu. For water-soluble cellular protein extraction, each dissected lens and capsule were separately placed in tubes and covered with 1–2 mL or 150 mL of cold 10 mM Tris-buffer (pH ¼ 7.4, metal free) respectively, under a N2 atmosphere. Tissue was homogenized on ice with a Miccra D-8 device and a homogenizing tool, DS-5/k (ARTmoderne Labortechnik e.k., Germany). The homogenized tissue was disrupted on ice by three 30 second pulses of ultrasonication (Sonopuls HD 2070 Bandelin, Germany), and centrifuged at 14 000g (4 C), for 30 min (Eppendorf centrifuge 5415R, Germany). The supernatant fractions obtained were aliquoted and stored at 80 C until use. Protein concentration was determined in each sample using the bicinchoninic acid assay (QuantiPro BCA Assay kit, MilliporeSigma, USA).26 A size exclusion column (SEC-Superdex 200 10/300 GL, Amersham Biosciences, UK) was employed for the separation of the water-soluble protein fraction of the lenses and capsules, based on their molecular mass, at a ow rate of 0.6 mL min 1 of 10 mM Tris–HCl base (pH ¼ 7.4). The quantication of Zn-, Feand Cu-binding proteins in the water-soluble protein extracts of the lenses and capsules was carried out by on-line post-column ID analysis, as described in previous articles (see the ESI‡ for further details).19,27,28

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MT distribution in human lenses by immunohistochemistry The cellular distribution of MT proteins in human lens sections was visualized by indirect immunouorescence, following conventional protocols described in the ESI.‡ Human lens cell line and common culture conditions A human lens epithelial cell line was used to study the effects of exogenous zinc and pro-inammatory cytokines such as interleukin-1a (IL-1a) on MT protein synthesis and gene expression.

HLEsv cells were cultured in T25 asks, in Dulbecco's modied eagle medium/nutrient mixture F-12 (DMEM/F12, Invitrogen, Carlsbad, CA, USA), containing 10% (v/v) fetal bovine serum (FBS) and 1% (v/v) penicillin/streptomycin, at 37 C and 5% CO2. The optimal seeding density and cell viability were determined using a CyQUANT® Cell Proliferation Assay Kit (Invitrogen) (see the ESI‡ for further details).

Distribution of MTs in HLEsv cells by immunohistochemistry The cellular distribution of MT proteins in HLEsv cells was visualized by indirect immunouorescence, as seen in the ESI,‡ following conventional protocols.

Microarray analysis of HLEsv cells: cellular treatment and RNA extraction To study the effects of Zn and interleukin on gene expression, HLEsv cells were treated either under (i) control conditions (no treatment) or with (ii) 68ZnSO4 (100 mM); (iii) IL-1a (100 U mL 1) (Millipore, CA, USA); or (iv) 68ZnSO4 (100 mM) + IL-1a (100 U mL 1). Total RNA was isolated using the TRIzol® Reagent (Invitrogen, Carlsbad, CA). The RNA concentration and quality were determined using a bioanalyzer (Agilent 2100 Bioanalyzer, Agilent Technologies Inc., Santa Clara, USA). The global prole of gene expression was assayed using the Illumina BeadChip array platform (HumanHT-12 v4.0 Expression BeadChip Kit, Illumina, San Diego, CA). cRNA labeling and hybridization to the chip and array data analysis were carried out at the Genome Analysis Platform (CIC bioGUNE, Derio, Spain). Raw data from each of the microarrays were normalized and the background was subtracted.

Cellular treatments and MT protein extraction in HLEsv cells

Distribution of zinc and copper in human lenses by LA-ICPMS The study of the elemental distribution of Zn and Cu in human lens sections was carried out by the LA-ICP-MS technique.

Cryogenic conditions are preferable for metal determination in biological samples, both for the sample preparation step and the LA-ICP-MS analysis, because of the preservation of the native status of the sample, specically the diffusible ions.24 Therefore, the human lens sections were analysed at 20 C using a LA-ICP-MS system with a Peltier-cooled cell, which maintains the integrity of the sample (see the ESI‡ for the operating conditions).

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Twenty-four hours before each treatment, cells were washed twice with PBS, and the serum-containing medium was replaced by the serum-free medium EX-CELL™ Hybridoma (MilliporeSigma, USA). Then, HLEsv cells were treated for 24 h either with (1) 68ZnSO4 (25 mM); (2) 68ZnSO4 (50 mM); (3) 68ZnSO4 (100 mM); (4) IL-1a (100 U mL 1) (Millipore, CA, USA); (5) 68 ZnSO4 (25 mM) + IL-1a (100 U mL 1); (6) 68ZnSO4 (50 mM) + IL1a (100 U mL 1); or (7) 68ZnSO4 (100 mM) + IL-1a (100 U mL 1); or (8) in the absence of 68ZnSO4 and IL-1a (i.e., control). Each experiment was carried out in triplicate. The water-soluble cellular proteins, including MTs, were extracted as previously described (see the MT protein extraction in HLEsv cells section in the ESI‡).19,29 Total protein was quantied in the cytosolic

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fraction (water-soluble proteins) using the QuantiPro™ BCA Assay kit (Sigma-Aldrich, USA).

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Fractionation and quantication of zinc-binding proteins, including MTs, by isotope pattern deconvolution (IPD)-HPLCICP-MS The fractionation and quantication of the cytosolic watersoluble proteins from HLEsv cells were carried out exactly as described elsewhere.19 Thus, following the chromatographic separations, we proceeded to the quantication of S-, Cu- and Zn-binding proteins, including MTs, by ICP-MS detection and mathematical calculations based on Isotope Pattern Deconvolution (IPD).19 The quantication of S- and Cu-binding proteins was carried out similarly to the quantitative speciation previously described, while the quantication of Zn-binding proteins was carried out by IPD-HPLC-ICP-MS, with simultaneous S and Cu determination.

Results and discussion Total multielemental determination of human lenses and capsules The ID-ICP-MS methodology was used to determine, simultaneously, the total levels of Zn, Fe, Cu and Se in ten capsules and ten naked lenses (i.e., lenses without capsules) from ve postmortem donors (i.e., cadavers), as described in the Experimental section.30 Table 1 shows the concentrations of Zn, Fe, Cu and Se, expressed in micrograms per gram of wet tissue (the indicates the obtained standard deviation). Zinc was, by far, the most abundant of the analyzed elements, in both capsules (9.77 2.53 mg Zn per g wet tissue) and lenses (9.48 1.20 mg Zn per g wet tissue), followed by Fe (1.65 0.41 mg Fe per g wet capsule; 0.25 0.11 mg Fe per g wet lens), Cu (0.39 0.12 mg Cu per g wet capsule; 0.23 0.04 mg Cu per g wet lens) and Se (0.16 0.03 mg Se per g wet capsule; 0.26 0.08 mg Se per g wet lens). The high standard deviation observed is due to the heterogeneity of the biological samples from different post-mortem donors. When the distributions of these elements were compared, Fe was the only element with a statistically signicant difference between the capsule and the naked lens. It was found to be more than 6

times more concentrated in the capsule than in the naked lens, indicating that its distribution is non-homogeneous.

Earlier studies have shown a wide variability in the reported levels of these trace elements in human lenses. Hawse et al.31 analyzed the metal content of forty-ve decapsulated human lenses by ICP-emission spectroscopy, and found that Zn was more concentrated than Fe and Cu. The results of this study are consistent with the levels found in the present work, assuming 60–70% of water content.32 On the other hand, Shukla et al.33 determined the levels of Zn and Cu in healthy and cataractous human lenses using an air–acetylene ame atomic absorption spectrometer and found similar levels of Cu (0.3–0.7 mg g 1 dry tissue) to those found in our study, but the concentrations of Zn were 5 to 50 times higher (52–505 mg g 1 dry tissue) than our values.

Another published study showed that Zn levels found in senile cataractous lenses reached 27 mg g 1 dry tissue, which represents approximately 10 mg g 1 wet tissue,34 which is in line with our present results. Cekiç et al.35 compared in normal human lenses the levels of Zn (16.5 2.5 mg g 1 dry weight), Cu (0.53 0.08 mg g 1 dry weight) and Se (0.83 0.18 mg g 1 dry weight), and showed similar values to those obtained in this work, considering once again 60–70% water content. Garner et al.36 reported an Fe level of 7 ng mg 1 dry weight and Levi et al.37 reported 10 ng Fe per mg protein, in whole lenses, which is up to 8 times higher than that in the present study. In contrast, Fe levels in the eye lens have been reported to be between 0.18 and 9.6 mg g 1 wet weight.38 Current evidence suggests that iron levels in the lens decrease with aging, although the signicance of this observation is open to speculation.31 Previous work suggested that Fe was associated preferentially to the epithelial cell layer of the lens and not to the capsule membrane itself.25 While the above studies have examined trace elements in the human lens, to our knowledge this is the rst study showing a signicant differential distribution of Fe in the capsule (6-fold more abundant) when compared to that in the lens.

Quantitative speciation of Zn, Fe and Cu in human lenses and capsules by SEC-ID-ICP-MS We set out to examine the distribution of Zn, Fe and Cu according to the molecules to which they are associated, in

Table 1 Total concentrations of Zn, Fe, Cu and Se (in micrograms of the element per grams of wet tissue) determined in human capsules and lenses from 10 eyes (5 postmortem donors) by ID-ICP-MS. Data are shown as the mean of three analytical replicates per sample. The indicates the standard deviation

Zn (mg Zn per g wet tissue)

Fe (mg Fe per g wet tissue)

Cu (mg Cu per g wet tissue)

Se (mg Se per g wet tissue)

Sample

Capsule

Lens

Capsule

Lens

Capsule

Lens

Capsule

Lens

Donor #1 Donor #2 Donor #3 Donor #4 Donor #5 Average

8.16 0.02 8.81 0.03 7.94 0.04 9.86 0.02 14.1 0.1 9.77 2.53

7.57 0.09 9.3 0.1 9.48 0.09 10.66 0.04 10.3 0.1 9.48 1.20

1.092 0.004 1.335 0.004 1.84 0.03 1.909 0.007 2.057 0.006 1.65 0.41

0.38 0.01 0.362 0.003 0.183 0.003 0.182 0.001 0.162 0.001 0.25 0.11

0.282 0.002 0.278 0.002 0.361 0.008 0.552 0.003 0.478 0.002 0.39 0.12

0.189 0.001 0.199 0.002 0.232 0.001 0.266 0.002 0.276 0.002 0.23 0.04

0.132 0.004 0.140 0.003 0.132 0.002 0.177 0.002 0.212 0.002 0.16 0.03

0.180 0.002 0.217 0.005 0.395 0.004 0.232 0.001 0.265 0.003 0.26 0.08

a

a

Average of le and right eyes.

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Fig. 2 Zn, Fe and Cu multielemental chromatographic distribution profiles in the water-soluble protein fraction of the capsule and lens, respectively, obtained by SEC-ICP-MS.

human decapsulated lenses and in capsules with lens epithelial cells attached. Fig. 2 shows examples of the SEC-ICP-MS chromatogram proles of Zn, Fe and Cu distribution in a capsule (top) and lens (bottom). Considering the SEC calibration formula, different metal-binding biomolecules can be distinguished according to their molecular mass: (i) >600 kDa; (ii) >450 kDa; (iii) 600–250 kDa; (iv) 250–100 kDa; (v) 100–40 kDa; (vi) 30–10 kDa; and (vii) <10 kDa. As indicated in the Experimental section, quantitative speciation was carried out by on-line post-column SEC-ID-ICPMS analysis (species-unspecic spiking mode).30 Table 2 shows the micrograms of each element (i.e., Zn, Fe and Cu) per

gram of wet tissue in the water-soluble protein fraction of the lens and capsule (the indicates the obtained standard deviation), arranged according to the molecular mass of the metalloproteins to which they were bound.39–41 The detection limit for Se is higher compared to those for Zn and Cu, so the quantitative speciation of Se is not shown.42 Overall, when the quantitative speciation (Table 2) of Zn, Fe and Cu was compared to their total concentrations (Table 1), it was evident that in the capsule 30% of Zn, 63% of Fe and 64% of Cu were associated with the water-soluble protein fractions.

Similarly, in the lens 41% of Zn and the majority of Fe and Cu were associated with the water-soluble protein fractions.

Zn distribution prole. Zinc was found mainly associated with four binding fractions, both in the capsule and lens (see Fig. 2). Interestingly, Zn in the capsule is mostly associated with proteins of low and medium molecular mass (LMM, 30–10 kDa and MMM, 100–40 kDa, respectively), whereas in the lens Zn is mainly distributed in high molecular mass proteins (HMM, >450 kDa). Specically, about 70% of zinc binds to HMM proteins in the lens (2.663 0.619 mg g 1 tissue), while 70% of zinc binds to LMM and MMM proteins in the capsule (100–10 kDa; 2.058 1.345 mg g 1 tissue). In the capsule, Zn mainly binds to 30–10 kDa proteins (1.134 0.709 mg g 1 tissue), representing 40% of the total zinc (see Table 2).

Fe distribution prole. Both in the lens and capsule, Fe was found mainly associated with ve protein fractions (Fig. 2). In the lens, Fe mainly eluted bound to HMM proteins (>600 kDa; 0.138 0.049 mg g 1 tissue, representing 55% of the total Fe), while in the capsule Fe is bound to both HMM (>600 kDa; 0.412 0.169 mg g 1 tissue, representing 40% of the total Fe) and MMM proteins (600–250 kDa; 0.425 0.154 mg g 1 tissue, representing 42% of the total Fe). Strikingly, in the capsule Febinding to 600–250 kDa proteins is similar to Fe-binding to >600 kDa proteins, while in the lens Fe-binding to 600–250 kDa proteins is lower compared to Fe-binding to >600 kDa proteins (see Table 2). Similarly to total iron levels (Table 1), the concentration of Fe in the water-soluble protein fraction was over 3 times higher in the capsule, compared to the lens.

Cu distribution prole. Copper was found in all ve of the eluted fractions, both in the lens and capsule (see Fig. 2). The

Multielemental quantitative speciation of Zn, Fe and Cu bound to proteins (in micrograms of the element per gram of wet tissue), in the water-soluble protein fraction of the lens and capsule, obtained by ID-HPLC-ICP-MS. The indicates the standard deviation

Table 2

Zn (mg Zn per g tissue)

Fe (mg Fe per g tissue)

Cu (mg Cu per g tissue)

Molecular mass

Capsule

Lens

Capsule

Lens

Capsule

Total concentrationa >600 KDa >450 KDa 600–250 KDa 250–100 KDa 100–40 KDa 30–10 KDa <10 KDa

2.971 — 0.523 — 0.369 0.924 1.134 —

3.929 1.197 — 2.663 0.619 — 0.450 0.112 0.410 0.145 0.365 0.170 —

1.048 0.412 — 0.425 0.079 0.031 0.070 —

0.335 0.087 0.138 0.049 — 0.079 0.046 0.039 0.009 0.023 0.008 0.017 0.006 —

0.250 — 0.031 — 0.015 0.058 0.056 0.073

1.122 0.364 0.225 0.636 0.709

0.151 0.169 0.154 0.026 0.022 0.028

Lens 0.146 0.016 0.010 0.027 0.018 0.070

0.259 0.093 — 0.064 0.028 — 0.027 0.009 0.045 0.017 0.058 0.021 0.035 0.021

a Total concentration determined by Flow Injection Analysis (FIA)-ID-ICP-MS in the water-soluble protein fraction of the capsule and lens. n ¼ 10 capsules and 10 lenses from 5 post-mortem donors. Each sample was analyzed in triplicate.

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observed prole is similar in both tissues with the exception that the concentration of Cu bound to HMM proteins (>450 kDa) in the lens is 2-fold higher (0.064 0.028 mg g 1 tissue) than that in the capsule (0.031 0.016 mg g 1 tissue). Interestingly, Cu also binds to proteins or other molecules of molecular mass <10 kDa (capsule, 0.073 0.070 mg g 1 tissue; lens, 0.035 0.021 mg g 1 tissue), which was not seen in the Zn and Fe chromatographic proles.

Although extensive proteomic studies have been carried out to identify the proteins of the human lens tissue, including lens epithelial cells, cortex and nuclear regions,43–48 no studies have been conducted, so far, to identify the metal-binding proteins in the human lens. In the present study, we have addressed this issue by determining the quantitative speciation of metals associated with water-soluble lens proteins. Strikingly, iron is associated with HMM proteins both in the capsule and lens.

This HMM protein is probably ferritin, the iron storage protein ( 450 kDa), which is concentrated in the human lens epithelium and diminishes throughout the cortex and nucleus.49 The specic distribution of ferritin in the human lens may explain the high levels of Fe found in the capsules (containing lens epithelial cells) when compared to those in the decapsulated lenses (ber cells), with differences ranging from 6- to 3-fold (Tables 1 and 2, respectively). Furthermore, Fe in the lens capsule binds to MMM proteins, likely transferrin and ceruloplasmin, which are synthesized and secreted by lens epithelial cells.50,51 On the other hand, zinc, iron and copper bind mainly to HMM proteins in the decapsulated lens, probably crystallins. It is known that native a-crystallins bind Cu2+ and Zn2+.52,53 Noncovalent multicrystallin complexes are present in the watersoluble HMM protein fractions of normal human lenses; however, covalent multimers of crystallins are present in the water-insoluble proteins of aging human lenses.54 One possible conclusion from this result is that zinc in whole lenses is mainly associated with non-water soluble proteins most likely from the extracellular matrix. Conversely, iron and copper are associated most likely with water-soluble proteins in the lens, and considerably less in the capsule. Actually, copper and zinc bind mainly to MMM and LMM proteins in the capsule, likely superoxide dismutase, albumin45,46 and metallothioneins.

Surprisingly, MT proteins have not been identied so far in lenses by molecular MS technologies, because, in contrast to most cell proteins, MTs remain intact (undigested) upon being treated with trypsin. To overcome this drawback, samples should be denatured by the addition of EDTA, which strips heavy metals from MTs and renders them susceptible to tryptic digestion.55 Signicantly, 40% of the total zinc binds to 30–10 kDa proteins in the capsule, corresponding to the region of metallothioneins.

Bio-imaging by LA-ICP-MS: distribution of Zn and Cu in the human lens To verify that Zn and Cu in the lens capsule are associated with lens epithelial cells and not with the collagenous material of the capsule itself, we measured their elemental distribution in

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Elemental images of zinc (64Zn) and copper (63Cu) in frozen sections of the human lens (100 mm thickness) obtained by LA-ICPMS. Panel (A): microscope image of the lens section. The dotted white box shows the analyzed region. Panel (B): superposition of the zinc image (intensity in cps). Panel (C): superposition of the copper image (intensity in cps). Scale bar 500 mm.

Fig. 3

whole lens sections (see Fig. 3) by LA-ICP-MS. Frozen sections of the human lens were ablated line-by-line (25 mm spot diameter) and two-dimensional images of Zn and Cu distribution were convoluted using ImageJ-Fiji soware. Panel A of Fig. 3 shows a microscope image of the analyzed human lens cryo-section, where the anterior capsule, the localization of epithelial cells, and the equator are indicated. Panels (B) and (C) show the superposition of the qualitative elemental distribution of 64Zn+ and 63Cu+ obtained by LA-ICP-MS analysis. Preferential accumulation of both Zn and Cu can be observed underneath the anterior capsule, corresponding to the epithelial cell region.

Although this nding has been described previously,25 this observation corroborates that the metals found in the capsule arise most likely from the lens epithelial cells and not from the acellular components (i.e., collagen) of the capsule. Additionally, the limits of detection (LODs) for Zn and Cu were calculated according to the 3s criterion of the IUPAC using synthetic spiked gelatin standards. The LODs determined by LA-ICP-MS were found to be 86 ng g 1 for Zn and 30 ng g 1 for Cu under the experimental conditions used. As can be observed, these values are sufficiently low for the determination of the trace metals in the lens capsule.

Immunohistochemistry: localization of MTs in the human lens Since the elemental distribution of Zn and Cu, obtained by LAICP-MS, showed preferential localization in the epithelial cells of the lens located under the anterior capsule, we proceeded to study the cellular distribution of MTs (i.e., Zn/Cu-containing-proteins) in whole lens sections by indirect immunouorescence. The results are shown in Fig. 4. MT1/2 antibodies labeled most

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strongly the lens epithelial cells and stained the outer cortical lens bers with lower intensity. This result is consistent with previous observations by Oppermann et al.56 Epithelial lens cells are considered one of the rst sites of stress-induced damage caused by external insults.8 The specic MT localization in lens epithelium cells may be related to the role of this tissue as a natural barrier to environmental insults (i.e., UV light), where MTs act as an antioxidant system to combat free radicals.19,20 Thus, MTs are cytosolic zinc-binding proteins that promote defense against oxidative damage and inammation, and they are highly expressed throughout eye tissues, especially the lens.19 We also sought to verify that the lens epithelial cells, which are intimately associated with the anterior capsule, remain attached to the capsule during its dissection. To this end, sections from the lens before (whole lens) and aer dissection (lens without capsule) were stained with toluidine and examined by light microscopy. The lens epithelial cells were intensely stained with toluidine and remained attached to the capsule aer its dissection (see Fig. S1 in the ESI‡).

Responses of HLEsv cell gene expression to zinc and proinammatory interleukin Due to the abundant expression of MT isoforms in the human lens,19,20 the preferential localization of MT1/2, Zn and Cu in the lens epithelial cells (Fig. 3 and 4) and the role of zinc–MTs in the defense mechanism against oxidative damage and inammatory stress, we have further studied the regulation of the Zn–MT system in an in vitro model representative of the human lens epithelial cells (HLEsv), and examined the effect of exogenous zinc (68ZnSO4) and/or interleukin treatments. To this end, we rst established a human lens epithelial cell line as described in the Experimental section. We optimized the seeding of the HLEsv cells with a cell proliferation assay kit, at a density of

Fig. 4 Cellular distribution of MT1/2 in the human lens by indirect

immunofluorescence. The MT1/2 antibody labeled the lens epithelium (see panel (C)). (A) Phase contrast micrograph. (B) DAPI staining of cell nuclei micrograph. (C) Rhodamine-labeled micrograph. (D) Merged image of (B) and (C). Scale bar 10 mm.

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10 000 cells per well in a 96-well plate, and then assessed the cell viability aer 24 h following the addition of Zn at different concentrations (i.e., 1, 10, 25, 75 and 100 mM), using the CyQUANT assay. The optimal Zn concentration was established between 25 and 50 mM, with a survival rate above 80% (Fig. S2, ESI‡). Additionally, 100 mM of zinc was selected to compare the obtained data with our previous study carried out on the cornea.19 The HLEsv cells exhibited many of the genotypic properties of lens epithelial cells in vivo, including the expression of MTs. We examined the cellular distribution of MTs in HLEsv cells by indirect immunouorescence using MT1/2 antibodies. The staining was detected in both the cytoplasm and nuclei by confocal microscopy (see Fig. S3, ESI‡).

We studied the effects of exogenously added zinc, in the form of 68ZnSO4 and/or pro-inammatory interleukin (IL-1a), on HLEsv cells by determining their impact on MTs and cytokine gene expression. Fig. 5 shows the hierarchical clustering and heat map of MT isoforms and cytokines in HLEsv cells, under steady-state conditions and upon 68ZnSO4 and/or interleukin IL1a treatments. The results can be summarized as follows: (i) under steady state conditions, HLEsv cells abundantly expressed MT isoforms in the following order: MT1A > MT2A > MT1E > MT1F > MT1X > MT1G > MT1M; (ii) 68ZnSO4 elicited a robust up-regulation of the gene expression of MT isoforms, ranging from 2.3-fold in MT1A to 44-fold in MT1H; (iii) 68ZnSO4 did not alter signicantly the expression of either pro- or antiinammatory cytokines, including IL1A, IL24, IL1B, IL6, IL32, IL10 and IL18, with the exception of IL8, which was approximately 1.53-fold up-regulated; (iv) 68ZnSO4 added in combination with IL-1a showed an additive but not synergistic effect on the expression of MT isoforms, when compared to Zn alone and interleukin alone; and (v) IL-1a added alone enhanced the expression of other cytokines (i.e., 39-fold in IL8) and MT

Fig. 5 Unsupervised hierarchical cluster and heat map of MT isoforms and cytokines expressed in cultured HLEsv cells either not treated (control) or exposed for 48 h to (i) ZnSO4 alone; (ii) IL-1a; or (iii) ZnSO4 + IL-1a. The color scale of the heat map represents the relative expression level of a gene (i.e., red bar, increased; blue bar, decreased) according to the log2 scale, in arbitrary units, depicted at the bottom.

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isoforms (i.e., approximately 2.7-fold in MT1G and MT2A and 2fold in MT1A and MT1E) (see Table S2, ESI‡).

Hawse et al.31 studied the induction of MTs and a-crystallins by toxic metals, including zinc, in human lens. In agreement with our data, Zn2+ induced the expression of MTs, while crystallins were not altered. In contrast, Cu2+ up-regulated the expression of crystallins but not MTs. Interestingly, both MTs and crystallins are able to bind Cu and Zn.52,53,57 Therefore, the induction of MTs and crystallins by metals suggests a metalspecic regulatory pathway in the lens.

Concentration of zinc (in mM) in MTs (dark gray bars), in proteins/molecules other than MTs (mid gray bars), and in all zincbinding proteins/molecules (light gray bars) in the water-soluble protein fraction of HLEsv cells, by IPD-HPLC-ICP-MS. HLEsv cells were either not treated (control) or exposed to 68ZnSO4 (25, 50 or 100 mM) and IL-1a (100 U mL 1) in combination or separately for 24 h.

Fig. 6

Quantication of zinc-binding proteins, including MTs, following exogenous zinc (68ZnSO4) and IL-1a treatments in HLEsv cells To understand how Zn regulates MT protein synthesis, we quantied the levels of Zn bound to MTs and to proteins/ molecules other than MTs in the water-soluble protein fraction of HLEsv cells aer treatments with 68ZnSO4 and IL-1a for 24 h by IPD-HPLC-ICP-MS, as described previously.19 By means of SEC coupled to ICP-MS we obtained the zinc elemental prole. Under the control conditions, Zn was distributed into three main and well-differentiated chromatographic peaks, based on the molecular mass of the metal-binding proteins, including (i) a main chromatographic peak with retention time between 24.8 and 27.8 min, matching with the predicted molecular size of MT proteins (7–14 kDa), and further identied; (ii) a second peak with a retention time of 10–13 min, likely corresponding to crystallin aggregates with a molecular mass higher than 600 kDa; and (iii) a small third peak, with a retention time between 28 and 30 min with a low molecular mass, suggesting bioligands/biomolecules <7 kDa (data not shown).

Zinc was also bound to a group of unresolved coeluting species in the 450–30 kDa region. For comparison purposes, the chromatographic peaks corresponding to >600 kDa, 450–30 kDa and <7 kDa were pooled and labeled zinc-binding proteins other than MTs, whereas the peak corresponding to MTs was labeled zinc-binding MT proteins. These results were conrmed by anion exchange-based chromatographic analysis.

Following each of the treatments described in the Experimental section, we applied an analytical approach based on SEC-ICP-MS and the use of mathematical calculations (i.e., IPD) to quantitate the absolute concentration of zinc-binding proteins. Fig. 6 shows the concentrations of Zn bound to MTs, to proteins/molecules other than MTs and the sum of both in the cytosolic fraction of HLEsv cells under each of the cellular treatments. This approach permitted us to distinguish, in cell extracts, the endogenous zinc (natZn) from the tracer or the exogenously added zinc (68Zn in the form of 68ZnSO4). Table S3 (see the ESI‡) shows the levels of natural (natZn), exogenous (68Zn) and total zinc (natZn + 68Zn) found in all the zinc bindingproteins and biomolecules detected in HLEsv extracts, i.e., MT proteins and proteins other than MTs, in the cytosolic fraction.

Cell numbers were determined and total protein levels were quantied in the obtained cytosolic fractions, with values ranging from 0.43 to 0.59 mg mL 1.

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As shown Fig. 6 (and Table S3‡), under the control conditions the concentration of zinc bound to MTs represented approximately 45% of the total zinc (natZn + 68Zn) detected in all zinc-binding proteins in the water-soluble fraction. We also found that upon exposure of HLEsv cells for 24 h to 25, 50 and 100 mM of 68ZnSO4, the concentration of zinc (natZn + 68Zn) bound to MTs increased 1.51-, 4.64-, and 7.58-fold, respectively, when compared to that of the control. The combination of 68 ZnSO4 (25, 50 and 100 mM) and IL-1a (100 U mL 1) further increased the concentration of zinc in MTs by 3.75-, 5.71- and 8.60-fold, respectively, when compared to that of the control.

Finally, the cytokine IL-1a when added alone to HLEsv cells increased the concentration of zinc (natZn) by 3.39-fold.

Remarkably, the exposure of cells to 25 and 50 mM 68ZnSO4, alone or in combination with IL-1a, decreased the concentration of Zn bound to proteins/molecules other than MTs in the range of 0.67- to 0.99-fold; however exposure to 100 mM 68Zn + IL-1a resulted in a 1.14-fold increase. Interestingly, upon exposure to 25 and 50 mM 68ZnSO4 alone or together with interleukin, the concentration of natural Zn in all zinc-binding proteins/molecules decreased by 50–65% when compared to that of the control. This decrease is greater than that observed with 100 mM 68Zn exposure alone (22%) or with interleukin (13%).

From Table S3,‡ we estimated the percentage of 68Zn incorporated into proteins/molecules upon exposure to 68ZnSO4 alone or in combination with IL-1a (100 U mL 1). The percentage of zinc bound to MTs reached 80–90% of the total zinc, while zinc in proteins other than MTs ranged from 45 to 58%, suggesting that there is a preferential binding site in MTs. In overall terms, the percentage of 68Zn bound to proteins steadily increased with cellular treatments. Specically, the increases were found to be as follows: 67% upon exposure to 25 mM 68ZnSO4; 81% with 25 mM 68ZnSO4 + IL-1a (100 U mL 1); 84% with 50 mM 68ZnSO4; 85% with 50 mM 68ZnSO4 + IL-1a (100 U mL 1); 80% with 100 mM 68ZnSO4; and 84% with 100 mM 68 ZnSO4 + IL-1a (100 U mL 1).

As stated in the Experimental section, we additionally determined the levels of S and Cu bound to proteins in the

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cytosolic fraction of HLEsv cells. As previously described,19 the multiplex determination of Zn, Cu and S permits the absolute quantication of intracellular MTs under steady state conditions and upon treatment with 68ZnSO4, alone or in combination with interleukin. Moreover, the use of the mathematical tool based on IPD allows the differentiation of both natural (natZn–MTs) and exogenous (68Zn–MTs) metallothioneins. Fig. 7 and Table S4 (see the ESI‡) show the concentration of MTs in the water-soluble protein fraction of HLEsv cells containing natural (natZn) and/or exogenous zinc (68Zn), under the control conditions (no treatment) and aer exposure to zinc and/or IL1a. We found that the exposure of HLEsv cells to 68ZnSO4 for 24 h, alone or in combination with IL-1a, increased the total concentration of MTs (natZn–MTs + 68Zn–MTs) compared to that of the control, as follows: 1.63-fold with 25 mM 68ZnSO4; 1.85fold with 25 mM 68ZnSO4 + IL-1a (100 U mL 1); 1.96-fold with 50 mM 68ZnSO4; 2.40-fold with 50 mM 68ZnSO4 + IL-1a (100 U mL 1); 3.25-fold with 100 mM 68ZnSO4; 3.74-fold with 100 mM 68ZnSO4 + IL-1a (100 U mL 1); and 1.67-fold with IL-1a (100 U mL 1).

According to Table S4,‡ we estimated that during stimulation with 68ZnSO4, alone or in combination with interleukin, approximately 80–91% of the intracellular pool of MTs was loaded with exogenous 68Zn ions, and the remaining 9–20% contained natZn. However, these percentages do not correspond exclusively to newly synthesized MTs since the exchange of 68Zn with natZn in the MT proteins is occurring at the same time.

Considering this, the percentage of the newly synthesized MTs signicantly increased according to the treatment, as follows: 39% increase of the new MTs upon exposure to 25 mM 68ZnSO4; 46% with 25 mM 68ZnSO4 + IL-1a (100 U mL 1); 49% with 50 mM 68 ZnSO4; 58% with 50 mM 68ZnSO4 + IL-1a (100 U mL 1); 69% with 100 mM 68ZnSO4; and 73% with 100 mM 68ZnSO4 + IL-1a (100 U mL 1).

Our results support the observation that the increase induced by 68ZnSO4 in the gene expression of MTs in HLEsv cells correlated with signicant increases in MT proteins and bound zinc. We previously studied the effects of exogenous zinc, i.e., 100 mM 68ZnSO4, and IL-1a (100 U mL 1) on MT protein synthesis and metal binding in human corneal epithelial cells in vitro.19 Comparing this with our current study, it is noted that

Fig. 7 Concentration of MTs (in mM) in HLEsv cells labeled with natZn or 68Zn, determined by IPD-HPLC-ICP-MS. Cells were either not treated (control) or exposed to 68ZnSO4 (25, 50 or 100 mM) and IL-1a (100 U mL 1) in combination or separately for 24 h.

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the concentration of zinc in all zinc-binding species in corneal cells is 2-fold higher than that in lens cells, and similar levels were found upon zinc and interleukin exposure. However, it is noteworthy that MT protein levels in HLEsv cells are similar to those found in corneal cells, under steady state conditions and upon zinc and/or interleukin treatments.

We further calculated the elemental stoichiometric composition of MTs by determining the sulfur to metal (Zn and Cu) ratio, i.e., Zn : Cu : MT (see Table S4, ESI‡), considering that the number of sulfur atoms per MT molecule is known to be 21. The stoichiometry of MTs under the control conditions was Zn3Cu0.2MT. Exposure to 25 mM 68ZnSO4 reduced the relative amount of copper to Zn3Cu0.1MT. However, exposure to IL-1a (100 U mL 1) alone or with 25 mM 68ZnSO4 increased the relative amount of zinc bound to MTs to Zn6Cu0.1MT. Following exposure to 50 mM 68ZnSO4, 50 mM 68ZnSO4 + IL-1a (100 U mL 1), 100 mM 68ZnSO4, and 100 mM 68ZnSO4 + IL-1a (100 U mL 1) we observed the saturation of zinc binding sites in MTs, i.e., Zn7Cu0.1MT, Zn7Cu0.1MT, Zn7Cu0.2MT, and Zn7Cu0.1MT, respectively.

Our ndings suggest that, in the in vitro model of human lens epithelial cells, zinc or pro-inammatory cytokines changed the elemental composition of MTs from Zn3Cu0.2MT to Zn7Cu0.1MT or Zn7Cu0.2MT, indicating that the new MTs are saturated by zinc ions. These HLEsv cells appear to adapt quickly to metal overloads by inducing a strong stimulation of MT synthesis, simultaneously increasing their metal binding capacity and lling all of their zinc-binding sites.

The MT affinity for zinc ions differs depending on the formation of the thermodynamically stable system, Zn0–7MT. Earlier studies stated that four of the zinc ions are bound tightly, two are bound with intermediate strength, and one is bound relatively weakly, which implies that Zn7MT is able to release one zinc atom to other zinc-binding proteins in an energetically favorable fashion.23,58,59 We previously suggested that Zn7MT species have a greater antioxidant capacity than ZnxMT species (where x ranges from 0 to 6 zinc atoms).19,20 In view of our data, in the presence of a tolerable excess of zinc, MTs may act against oxidative stress, decreasing the potential cellular damage and rendering the cell or tissue more resistant to these insults, even though this hypothesis should be demonstrated.

It should be noted that, with in vitro models, under the control conditions we found Zn3Cu0.2MT species in the lens epithelial cells and Zn6Cu1MT species in corneal epithelial cells.

This implies that MT proteins in the lens are in a different redox state compared to those in the cornea.19 This observation may be related to the roles of the cornea and lens as natural barriers combating free radicals.8 The cornea is the initial barrier of the eye protecting the inner ocular tissues against external insults, including UV light, while the lens is the second barrier whose primary function is refraction of light. Our data suggest that the rst protective barrier, i.e., corneal epithelial cells, have a stronger Zn–MT antioxidant system, compared to the lens epithelium, probably due to the higher oxidative milieu in the cornea relative to the lens.

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Moreover, it is noteworthy that the use of interleukin in combination with 68ZnSO4 elicited a synergistic effect on the synthesis of MTs when compared with zinc sulphate or interleukin alone. Indeed, upon exposure to low zinc concentrations, i.e., 25 mM 68ZnSO4, we observed that the elemental stoichiometry of MTs (Zn3Cu0.2MT) remains unchanged compared to that of the control. However, upon exposure to the same zinc concentration but in combination with interleukin, i.e., 25 mM 68 ZnSO4 + IL-1a (100 U mL 1) the MTs lled almost all of their binding sites to reach Zn6Cu0.1MT, similar to the cells exposed to IL-1a (100 U mL 1) alone. Therefore the presence of interleukin induces the synthesis of MTs and promotes their saturation with zinc. This observation may suggest that the interleukin triggers an inammatory process, which causes cellular stress that subsequently induces MT proteins to ll all their binding sites with zinc in order to overcome this insult.

A previous study suggested that zinc represents a key cellular factor in oxidative stress by establishing crosstalk communication between MTs and inammatory cytokines.20 While this much is supported by the current study, the role of MTs in providing protection against inammation and oxidative stress, including the mechanistic effect of the saturation of MTs with zinc, remains to be investigated.60,61

Conclusions This work represents a multidisciplinary study addressing the speciation of Zn, Fe and Cu in the human lens and the use of an in vitro model of epithelial lens cells to investigate the system zinc–MTs. The combination of elemental mass spectrometry with bio-analytical methodologies, including immunohistochemistry, gene expression and cultured cells, is of great interest for understanding the roles of metals and metalbinding proteins in the human lens.

Zinc is highly concentrated in naked (i.e., decapsulated) human lenses and in capsules (with attached lens epithelial cells), followed to a lesser extent by iron, copper, and selenium.

The levels of Zn, Cu and Se are similar in both lenses and capsules, whereas Fe is 6 times more concentrated in the lens capsule than in the naked lens. The association of Zn, Fe and Cu with proteins has been studied in the water-soluble fraction of capsules and decapsulated lenses. Zn in the capsule is mostly associated with proteins of LMM and MMM, likely MTs, whereas in the lens Zn mainly binds to HMM proteins, probably crystallins. Elemental Zn and Cu are preferentially distributed underneath the capsule, probably in the epithelial cell layer of the lens. MT proteins provide defense against oxidative damage and inammation and are highly expressed in the lens, specically in the epithelial cells, which are the rst site of stress-induced damage caused by external insults.

We have studied the mechanisms regulating the zinc–MT system and MT gene expression by investigating the effects of zinc and interleukin in an in vitro model representative of the human lens epithelium cells. Zinc and interleukin induced the expression of MTs, while crystallins were not altered, suggesting a metal-specic regulatory pathway in the lens. We further quantied the levels of zinc bound to MTs and to proteins/

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molecules other than MTs in the water-soluble protein fraction of HLEsv cells. The percentage of zinc bound to MTs reached 80–90% of the total zinc, suggesting that there is a preferential binding site in these proteins. Finally, the use of a mathematical tool based on IPD allowed the quantication and stoichiometric determination of MTs. Zinc and a proinammatory cytokine increased the concentration of MTs and induced a stoichiometric change from Zn3–MT to Zn7–MT, increasing their metal binding capacity to ll all their zincbinding sites, and potentially rendering lens cells more resistant to oxidative stress insults and inammatory processes, which will be addressed in future studies.

Acknowledgements The authors would like to acknowledge the contribution of the COST Action TD1304, the network for the biology of zinc (Zincnet) (http://zinc-net.com). The Instituto Oalmol´ ogico Fern´ andez-Vega acknowledges nancial support from the “Plan de Ciencia, Tecnolog´ıa e Innovaci´ on” (PCTI) of Gobierno del Principado de Asturias, European FEDER co-nancing, and FICYT managing institution, through the project IE14-030. M. C. P. “Catedr´ atico Rafael del Pino en Oalmolog´ıa”, H. G. I., C. P., L. A., M. G., and M. C. P acknowledge nancial support from the “Fundaci´ on Rafael del Pino” and the “Fundaci´ on Ma Cristina Masaveu Peterson”. S. R. M., B. F., R. P. and A. S. M. acknowledge support from PCTI-FICYT through the Project GRUPIN14-092. M. G. acknowledges Torres Quevedo Fellowship (PTQ-12-05444) from the Spanish Ministry of Economy and Competitiveness. B. F. acknowledges her research contract RYC2014-14985 from the Spanish Ministry of Economy and Competitiveness through the Ram´ on y Cajal Program. The authors thank Enol Artime for technical support.

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