Selenomethionine (Se-Met) Induces the Cystine/Glutamate Exchanger

Article Selenomethionine (Se-Met) Induces the Cystine/Glutamate Exchanger SLC7A11 in Cultured Human Retinal Pigment Epithelial (RPE) Cells: Implications for Antioxidant Therapy in Aging Retina

Sudha Ananth 1, Seiji Miyauchi 1,†, Muthusamy Thangaraju 1, Ravirajsinh N. Jadeja 1,2 , Manuela Bartoli 2,3, Vadivel Ganapathy 4 and Pamela M. Martin 1,2,3,5,*

1 Department of Biochemistry and Molecular Biology, Augusta University, Augusta, GA 30912, USA; [email protected] (S.A.); [email protected] (S.M.); [email protected] (M.T.); [email protected] (R.N.J.) 2 Culver Vision Discovery Institute, Augusta University, Augusta, GA 30912, USA; [email protected] 3 Department of Ophthalmology, Augusta University, Augusta, GA 30912, USA 4 Department of Cell Biology and Biochemistry, Texas Tech Health Science Center, Lubbock, TX 79430, USA; [email protected] 5 Georgia Cancer Center, Augusta University, Augusta, GA 30912, USA * Correspondence: [email protected]; Tel.: +706-721-4220; Fax: +706-721-6608 † Present afﬁliation—Faculty of Pharmaceutical Sciences, Toho University, Funabashi, Chiba 274-8510, Japan.

Abstract: Oxidative damage has been identified as a major causative factor in degenerative diseases of the retina; retinal pigment epithelial (RPE) cells are at high risk. Hence, identifying novel strategies for increasing the antioxidant capacity of RPE cells, the purpose of this study, is important. Specifically, we evaluated the influence of selenium in the form of selenomethionine (Se-Met) in cultured RPE cells Citation: Ananth, S.; Miyauchi, S.; on system xc- expression and functional activity and on cellular levels of glutathione, a major cellular Thangaraju, M.; Jadeja, R.N.; Bartoli, antioxidant. ARPE-19 and mouse RPE cells were cultured with and without selenomethionine M.; Ganapathy, V.; Martin, P.M. (Se-Met), the principal form of selenium in the diet. Promoter activity assay, uptake assay, RT-PCR, Selenomethionine (Se-Met) Induces northern and western blots, and immunofluorescence were used to analyze the expression of xc-, the Cystine/Glutamate Exchanger Nrf2, and its target genes. Se-Met activated Nrf2 and induced the expression and function of xc- SLC7A11 in Cultured Human Retinal Pigment Epithelial (RPE) Cells: in RPE. Other target genes of Nrf2 were also induced. System xc- consists of two subunits, and Implications for Antioxidant Therapy Se-Met induced the subunit responsible for transport activity (SLC7A11). Selenocysteine also induced in Aging Retina. Antioxidants 2021, xc- but with less potency. The effect of Se-met on xc- was associated with an increase in maximal 10, 9. https://dx.doi.org/10.3390/ velocity and an increase in substrate affinity. Se-Met increased the cellular levels of glutathione in antiox10010009 the control, an oxidatively stressed RPE. The Se-Met effect was selective; under identical conditions, taurine transport was not affected and Na+-coupled glutamate transport was inhibited. This study Received: 1 December 2020 demonstrates that Se-Met enhances the antioxidant capacity of RPE by inducing the transporter xc- Accepted: 21 December 2020 with a consequent increase in glutathione. Published: 24 December 2020 Keywords: retina; retinal pigment epithelium (RPE); oxidative stress; age-related macular degenera- Publisher’s Note: MDPI stays neu- tion (AMD), antioxidants; selenium; selenomethionine (Se-Met); SLC7A11; xCT; system xc; glutathione tral with regard to jurisdictional claims in published maps and institutional affiliations.

1. Introduction Age-related macular degeneration (AMD) is the leading cause of blindness among Copyright: © 2020 by the authors. Li- persons age 60 and above in the U.S. and other highly developed nations. The repertoire censee MDPI, Basel, Switzerland. This of clinical and experimental studies implicating oxidative stress as a principal causative article is an open access article distributed factor and retinal pigment epithelium (RPE) as a primary site of pathology in the disease under the terms and conditions of the is quite large and steadily expanding [1–4]. RPE cells generate a substantial amount of Creative Commons Attribution (CC BY) reactive oxygen species (ROS) as a consequence of their normal physiologic functions license (https://creativecommons.org/ (i.e., light absorption/photo-oxidation, phagocytosis of lipid-rich outer segments, etc.) [5]. licenses/by/4.0/).

Antioxidants 2021, 10, 9. https://dx.doi.org/10.3390/antiox10010009 https://www.mdpi.com/journal/antioxidants Antioxidants 2021, 10, 9 2 of 17

Interestingly, they are at the same time equipped with cellular machinery to counter the potentially detrimental effects of ROS. The risk of oxidative damage to these cells is therefore influenced critically by the ratio of oxidant generation to the corresponding activity of endogenous cellular antioxidant defenses. Interestingly, increased age, the major risk factor for AMD, is associated with a progressive and irreversible accumulation of ROS in most major cell/tissue types, including RPE/retina, and a diminished capacity for protection against oxidative stress. This is compounded further by genetic and environmental factors that contribute to disruption of the delicate balance between pro- and antioxidant factors in favor of pro-oxidants/increased oxidative stress [6,7]. Given the high susceptibility of RPE to oxidant-induced damage, identification of novel strategies for protecting these cells and potentially, in turn, slowing the development/progression of this sight-debilitating disease is critically important. Glutathione (GSH) is the major antioxidant responsible for the protection of RPE against oxidant-induced damage/dysfunction [8]. The ability of cells to synthesize GSH depends largely upon the availability of cysteine, a factor that hinges critically upon the activity of the cystine-glutamate exchanger, also known as system xc- [9,10]. System xc- mediates the Na+-independent entry of cystine into cells coupled to the efflux of intracellular glutamate. Extracellularly, glutamate participates in neurotransmission and other signaling pathways, whereas cystine is rapidly reduced intracellularly to cysteine, the rate- limiting factor in GSH synthesis. Aside from those functions, the transport system has also been shown to play an essential role in the regulation of cell cycle progression [11]. System xc- is therefore a critical regulator of endogenous GSH synthesis, cellular redox balance, and ultimately cell fate [12,13]. As such, strategies to induce the expression and function of this transport system have a direct impact not only on the normal biology of RPE cells but also on the ability of these and other retinal cells to respond effectively to oxidative stress [12,14]. Thus, the potential therapeutic value of such strategies is high. In the present study, we evaluated selenomethionine (Se-Met), an organic form of selenium, for this very purpose. Specifically, we show that, in cultured human retinal pigment epithelial cells, selenium in the form of Se-Met induces nuclear factor erythroid 2-related factor 2 (Nrf2) expression; upregulates the expression of xCT, the functional subunit of the system xc- transport system; and therefore positively influences the production of GSH. Previous studies have demonstrated the natural antioxidant properties of selenium-containing compounds, a property thought to be related primarily to their incorporation into selenoproteins like the glutathione peroxidases (GPXs) and thioredoxin reductases (TXNRDs) [15,16]; however, our present findings provide a novel additional mechanism to explain the antioxidant feature of these compounds.

2. Materials and Methods 2.1. Materials Reagents were obtained from the following sources: unlabeled amino acids (Sigma- Aldrich; St. Louis, MO, USA); TRIzol and Lipofectamine 2000 (Invitrogen-Gibco; Carlsbad, CA, USA); GeneAmp RT-PCR kit (Applied Biosystems/Life Technologies; Carlsbad, CA, USA); Taq polymerase kit (TaKaRa; Mountain View, CA, USA); Power Block (Biogenex; San Ramon, CA, USA); GSH-Glo Glutathione Assay Kit, pUIIR3-EGFP and pGL3-Basic vec- tors (Promega; Madison, WI, USA); rabbit polyclonal anti-human Nrf2 antibody (AbCam; Cambridge, MA, USA); Fluoromount mounting media (Sigma, St. Louis, MO, USA); En- hanced ChemiLuminescence kit (PerkinElmer Life Sciences, Waltham, MA, USA); Bio-Rad RC DC protein assay kit (Bio-Rad; Hercules, CA, USA); Pierce Pinpoint Cell Surface Isola- tion kit (Thermoscientiﬁc; Rockford, IL, USA); and [3H]-glutamate and [3H]-taurine (Perkin Elmer Life Sciences; Boston, MA, USA). Rabbit polyclonal anti-xCT antibody was a gener- ous gift from Dr. Sylvia B. Smith [17]. Culture media and supplements, goat anti-rabbit Alexa Fluor 568 secondary antibody, and Hoechst 33342 stain were from Invitrogen-Gibco (Carlsbad, CA, USA). Antioxidants 2021, 10, 9 3 of 17

2.2. Animals and Cell Culture Normal C57BL/6J mice (males, aged 3 weeks) were obtained from the Jackson Labo- ratory (Bar Harbor, ME) and used to establish primary cultures of RPE per our previously published method [18–20]. All animals used in the study were maintained in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the Institutional Committee for Animal Use in Research and Education of the Augusta University (2009–0214). Human RPE cells (ARPE-19) were obtained from American Type Culture Collection (Manassas, VA, USA). Cell viability was evaluated using trypan blue exclusion assay. All experiments, other than immunofluorescence studies in which it was necessary to evaluate single cells (see below in Section 2.6. “Immunofluo- rescence Analysis”), were performed using completely confluent, highly differentiated ARPE-19 cultures, and the key findings were confirmed in primary mouse RPE.

2.3. RT (Reverse Transcription)-PCR RNA was isolated from ARPE-19 cells cultured in the presence or absence of Se- Met (1 mM, 16 h) and used for RT-PCR using the GeneAmp RT-PCR kit. System xc- is a heterodimeric protein made up of xCT, the transport subunit, and 4F2hc, the subunit responsible for trafficking of xCT to the plasma membrane. We analyzed the expressions of both xCT and 4F2hc. Primer sequences and expected product sizes for xCT and 4F2hc have been described [21]. Information specific to the other primers used in the study is provided in Supplementary Table S1. In all PCR reactions, 18 S ribosomal RNA served as the internal control. The molecular identity of the PCR products was confirmed by sequencing analysis.

2.4. Northern Blot Analysis Northern blotting was performed to analyze the steady-state levels of xCT mRNA and 4F2hc mRNA in ARPE-19 cells cultured in the presence or absence of selenomethionine (1 mM, 16 h) following the method of Bridges et al. [10]. β-Actin mRNA was monitored as an internal control.

2.5. Luciferase Promoter Assay SLC7A11, the gene coding for human xCT, maps to chromosome 4, at 4q28-q32. The promoter region of this gene contains a putative ARE-Nrf2 binding site (Genbank ac- cession number NT_016354) [22]. To evaluate whether the effects of Se-Met on xCT expression are mediated via transcription, luciferase promoter assay was performed following our previously published method [23]. Two different SLC7A11-specific promoter constructs were designed; the first, which we dubbed SLC7A11 promoter-long, was designed such that the portion of the promoter region amplified included the purported ARE-Nrf2 binding site and was approximately 780 bp in length. The second, SLC7A11 promoter-short, was designed such that the region selected for amplification was located just downstream of the ARE-Nrf2 binding site, and in turn, was only approximately 400 bp in length. SLC7A11 promoter-long- and SLC7A11 promoter-short-luciferase constructs were generated by first subcloning the 0.7 and 0.4-kb promoter fragments (obtained by PCR using human genomic DNA as the template), respectively, into the pGEM-T Easy vector and then by cloning the XhoI/NcoI-digested promoter inserts into the pGL3-Basic luciferase vector. The primers used for PCR were 50-AACTCGAGGACTGTATTGCCTT-30 (forward, promoter-long), 50-AACTCGAGGCTGGAGGCTTCTC-30 (forward, promoter- short), and 50-TGACCATGGTCATAGTAGGGACA-30 (reverse, NcoI site/pGL3-Basic). All constructs were verified by sequencing. ARPE-19 cells (approximately 80% confluent) were transfected with the respective plasmids using Lipofectamine 2000 according to the manufacturer’s instructions (Invitrogen). Se-Met (1 mM) was added to the culture medium of “treated” cells 32 h post-transfection. Cells were collected, and lysates were prepared 48 h post-transfection. Luciferase activity was measured using a Tecan microplate reader (Tecan US, Inc., Morrisville, NC, USA) and was used as an indicator of promoter Antioxidants 2021, 10, 9 4 of 17

activation. Luciferase activity was normalized for protein levels and compared with empty vector-transfected control cells.

2.6. Immunofluorescence Analysis ARPE-19 cells were seeded on glass coverslips and cultured in the presence or absence of Se-Met (1 mM) for 16 h. Cells were then air-dried, fixed in 4% (w/v) paraformaldehyde, washed with 0.01 M PBS (pH 7.4), and blocked with 1× Power Block for 1 h at room temperature. Following overnight incubation at 4 ◦C with anti-xCT (1:500), anti-Nrf2 antibody (1:100), or PBS (negative controls), cells were again washed in PBS and incubated for 45 min at room temperature with goat anti-rabbit Alexa Fluor 568 secondary antibody at a dilution of 1:2000. Following a final PBS wash, cells were incubated at room temperature with Hoechst 33342 (1:10,000), a nuclear counterstain, and coverslips were mounted on standard glass microscope slides using Fluoromount mounting media. Epifluorescence vi- sualization of immunolabeled proteins was done using the Zeiss Axioplan-2 microscope, Axiovision software, and an HRM camera.

2.7. Western Blot Analysis Western blotting was performed to detect Nrf2 or xCT in protein lysates prepared from ARPE-19 cells cultured in the presence or absence of Se-Met (1 mM, 16 h). For analysis of Nrf2 expression, protein lysates were subjected to SDS-PAGE, then transferred to nitrocellulose membranes, and incubated with antibody against Nrf2 overnight at 4 ◦C. Membranes were then incubated with horseradish peroxidase-conjugated anti-rabbit IgG antibody and washed, and antibody-speciﬁc bands were detected using chemiluminescence. xCT protein localizes both intracellularly and on the plasma membrane; however, it is the protein present on the plasma membrane that is functionally active, responsible directly for the transport activity. As such, to evaluate the effects of Se-Met treatment on the plasma membrane versus cytosolic fraction of xCT protein, membrane proteins were biotinylated using the Pinpoint Cell Surface Isolation Kit according to the manufacturer’s instructions and the method of Mysona et al. [24]. In brief, cells were labeled with thiol-cleavable amine-reactive biotinylation reagent and then lysed in the presence of a mild detergent. Labeled membrane proteins were collected using a column containing immobilized NeutrAvidin gel; the intracellular protein fraction was present in the eluate. Membrane proteins were released by treatment of the column with SDS-polyacrylaminde gel sample buffer containing 50 mM DTT, protein concentrations were determined via Bio-Rad RC DC protein assay, and western blotting was performed. β-Actin served as the loading control.

2.8. Assay of System xc- Transport Activity System xc- is a Na+-independent transport system that, under physiologic conditions, mediates cellular entry of cystine in exchange for intracellular glutamate. However, the activity of this transporter is routinely measured by monitoring the cellular uptake of [3H]-glutamate rather than [3H]-cystine under Na+-free conditions, particularly in RPE5 [25]. This because, in addition to system xc-, RPE cells express a transport system known as b0,+ which can also contribute to the influx of cystine. However, when glutamate is used as the substrate, the uptake appears to be mediated exclusively by system xc-. Hence in the present study, the transport activity of system xc- was monitored as the influx of [3H]-glutamate in exchange for intracellular unlabeled glutamate. Specifically, we measured the uptake of [3H]-glutamate (25 µM) in ARPE-19 cells or primary mouse RPE cells cultured in the presence or absence of Se-Met, selenocysteine (Se-Cys), S-adenosyl methionine (SAM), glutamate (Glu), cystine (Cys), methionine (Met), cysteine (Cys), glutamine, phenylalanine, aspartic acid, arginine, lysine, valine, or alanine at 1 or 5 mM using a Na+-free uptake buffer [21]. Kinetic analysis of system xc- activity in cells cultured in the presence or absence of Se-Met was performed by measuring the transport activity over a wide range of glutamate concentrations (2.5–1000 µM) and by analyzing the data Antioxidants 2021, 10, 9 5 of 17

according to the Michaelis–Menten equation describing a single saturable transport system. Kt and Vmax were calculated using the nonlinear regression method and then conﬁrmed using the linear regression method. xc- is not the sole transport system responsible for mediating glutamate uptake in mammalian cells; members of the excitatory amino acid transporter (EAAT) family also contribute to the uptake. To determine whether the activity of EAATs (Na+-dependent glutamate transporters) is also inﬂuenced by Se-Met, uptake of [3H]-glutamate was also monitored in a Na+-containing buffer (25 mM Hepes/Tris (pH 7.5), 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4, and 5 mM glucose).

2.9. Hydrogen Peroxide (H2O2) Treatment and Glutathione Measurement ARPE-19 cells were seeded in 6-well plates (0.2 × 106 cells/well) and grown to approximately 80% conﬂuence. Cells were pretreated for 1 h with or without Se-Met (1 mM), followed by 2 h exposure to 500 µMH2O2. The concentration of H2O2 used in these studies was determined based upon prior experimentation [21]. Cellular glutathione levels were measured using the GSH Glo assay kit as previously described [21].

2.10. Data Analysis All cell treatments were performed at least in triplicate. All data are expressed as mean ± S.E.M. Student’s t-test (normally distributed data) or the Mann–Whitney U test (nonparametric data) were used to determine signiﬁcance. p < 0.05 was considered to be statistically signiﬁcant.

3. Results 3.1. Se-Met-Induced Upregulation of Nrf2 The antioxidant properties of selenium are well documented, particularly with respect to their usefulness in the treatment/prevention of cancer-related pathologies [26]. Such properties are thought to be a direct consequence of the induction of the Nrf2/phase II antioxidant response pathway. Interestingly, compounds capable of inducing Nrf2 and its target genes have been shown to be similarly protective of retinal cells, particularly RPE [27,28]; however, to our knowledge, whether selenium elicits a similar effect on Nrf2/phase II antioxidant responses in RPE has not been studied. Given our present interest in exploring selenium as a potential antioxidant therapy for preservation of RPE and prevention of retinal pathology, we exposed human RPE (ARPE-19) cells to Se-Met, the primary form of selenium in the diet and evaluated Nrf2 protein expression using immunofluorescence and western blotting. A positive signal (red fluorescence) indicative of Nrf2 protein expression was detectable in control, untreated cells (Figure1A). The intensity of this signal was increased significantly in Se-Met-treated cells. To confirm these findings, additional cells were cultured for 4–6 weeks to facilitate proper differentiation. The protein was then isolated from these highly differentiated cells cultured and used for western blotting; a similar increase in Nrf2 expression was detected using this method (Figure1B). Nrf2 induces several genes crucial to the cellular defense against oxidative stress, including xCT. As such, using RNA obtained from cells treated identically as described above, we evaluated the expression of xCT (SLC7A11) and 4F2hc, the catalytic and regulatory subunits of system xc-, respectively, by RT-PCR (Figure1C). xCT expression increased robustly in Se-Met-treated cells. The expression of 4F2hc was not affected. We evaluated also the expression of a number of other well-known Nrf2 target genes and/or selenoproteins including glutamate-cysteine ligase catalytic subunit (GCLC), glutamate-cysteine ligase regulatory subunit (GCLM), heme oxygenase 1 (HO-1), glutathione S-transferase A1 (GSTA1), glutathione S-transferase A2 (GSTA2), glutathione peroxidase 1 (GPX1), thioredoxin reductase (TXNRD), and NAD(P)H oxidase/quinone 1 (NQO1) (Figure1D). Indeed, we found the expression of a number of these genes to be upregulated also in Se-Met-treated cells. Antioxidants 2021, 10, x FOR PEER REVIEW 6 of 18

cysteine ligase regulatory subunit (GCLM), heme oxygenase 1 (HO-1), glutathione S- transferase A1 (GSTA1), glutathione S-transferase A2 (GSTA2), glutathione peroxidase 1 (GPX1), thioredoxin reductase (TXNRD), and NAD(P)H oxidase/quinone 1 (NQO1) (Fig- ure 1D). Indeed, we found the expression of a number of these genes to be upregulated Antioxidants 2021, 10, 9 6 of 17 also in Se-Met-treated cells.

Figure 1. Selenomethionine (Se-Met)-induced upregulation of Nrf2 in ARPE-19 cells: (A) immunocy- Figuretochemical 1. Selenomethionine analysis of Nrf2 (Se protein-Met)- ininduced ARPE-19 upregulation cells cultured of Nrf2 in the in presenceARPE-19 (+Se-Met)cells: (A) immuno- or absence cytochemical analysis of Nrf2 protein in ARPE-19 cells cultured in the presence (+Se-Met) or ab- (−Se-Met) of selenomethionine (1 mM, 16 h), where cells in which PBS was substituted for the sence (−Se-Met) of selenomethionine (1 mM, 16 h), where cells in which PBS was substituted for primary antibody served as the negative control (Neg. Con); (B) western blot analysis of Nrf2 in the primary antibody served as the negative control (Neg. Con); (B) western blot analysis of Nrf2 incontrol control and and Se-Met-treated Se-Met-treated ARPE-19 ARPE-19 cells; cells; (C ()C RT-PCR) RT-PCR analysis analysis of mRNAof mRNA transcripts transcripts speciﬁc specific for for xCT xCT(SLC7A11) (SLC7A11 and) and 4F2hc 4F2hc in control in control (−Se-Met) (−Se-Met and) and Se-Met-treated Se-Met-treated ARPE-19 ARPE- cells;19 cells; and and (D) ( RT-PCRD) RT-PCR anal- analysisysis of Nrf2of Nrf2 target target genes: genes: glutamate-cysteine glutamate-cysteine ligase ligase catalytic catalytic subunit subunit (GCLC), (GCLC), glutamate-cysteine glutamate-cyste- ineligase ligase regulatory regulatory subunit subunit (GCLM), (GCLM heme), heme oxygenase-1 oxygenase (HO-1),-1 (HO- glutathione1), glutathione S-transferase S-transferase A1 (GSTA1),A1 (GSTA1glutathione), glutathione S-transferase S-transferase A2 (GSTA2), A2 ( thioredoxinGSTA2), thioredoxin reductase (TXNRD),reductase glutathione(TXNRD), glutathione peroxidase (GPX),pe- roxidaseand NAD(P)H (GPX), and oxidase: NAD quinone(P)H oxidase: 1 (NQO1), quinone in ARPE-19 1 (NQO1 cells), in ARPE cultured-19 incells the cultured presence in (+Se-Met) the pres- or enceabsence (+Se- (Met−Se-Met)) or absence of selenomethionine (−Se-Met) of selenomethionine (1 mM, 16 h). (1 mM, 16 h).

3.2.3.2. Se Se-Met-Induced-Met-Induced Upregulation Upregulation of of xCT xCT Expression Expression GivenGiven our our specific specific interest interest in inmechanisms mechanisms capable capable of stimulating of stimulating system system xc- and, xc- and, in turn,in turn, endogenous endogenous GSH GSH synthesis, synthesis, we sought we sought to explore to explore this phenomenon this phenomenon further. further. As statedAs stated above, above, xCT is xCT coded is coded for by for SLC7A11, by SLC7A11, an Nrf2 an target Nrf2 targetgene [29] gene. Though [29]. Though we show we increasedshow increased expressions expressions of both of Nrf2 both and Nrf2 xCT and in xCT conjunction in conjunction with with Se-Met Se-Met treatment treatment of ARPEof ARPE-19-19 cells, cells, we have we have not established not established the cause the cause-and-effect-and-effect relationship relationship between between the two. the Undertwo. Under unstressed unstressed conditions, conditions, Nrf2 Nrf2 resides resides in the in the cytoplasm, cytoplasm, complexed complexed to to Kelch Kelch-like-like ECHECH-associated-associated protein protein (Keap1 (Keap1);); this this is is its its inactive inactive state. state. This This complex complex is is disrupted disrupted by by oxidativeoxidative stress, stress, and and Nrf2 Nrf2 is is released released and and translocated translocated into into the the nucleus. nucleus. Nrf2 Nrf2 then then binds binds antioxidantantioxidant response response elements elements ( (ARE)ARE) in the promoters ofof itsits targettargetgenes, genes, thereby thereby inducing induc- ingtheir their expression. expression. The The presence presence of of an an AREARE bindingbinding site within the the promoter promoter region region of of SLC7A11 (xCT) has been reported [29,30]. To determine whether the Se-Met-induced upregulation of xCT observed in our experimental system is due to Nrf2-dependent induction of the SLC7A11 promoter activity, we designed two SLC7A11-specific promoter constructs: one containing the ARE-binding site (SLC7A11 promoter-long) and another in which the ARE-site was excluded (SLC7A11 promoter-short), each driving an EGFP or luciferase reporter. Luciferase activity in vector control cells was negligible irrespective of Se-Met exposure (Figure2A) but was increased significantly in ARPE-19 cells transfected with SLC7A11 promoter-long and exposed to Se-Met (p < 0.001). These data strongly support the Se-Met-induced activation of ARE/Nrf2 in the promoter region of xCT (SLC7A11). Antioxidants 2021, 10, x FOR PEER REVIEW 7 of 18

SLC7A11 (xCT) has been reported [29,30]. To determine whether the Se-Met-induced upregulation of xCT observed in our experimental system is due to Nrf2-dependent induction of the SLC7A11 promoter activity, we designed two SLC7A11-specific promoter constructs: one containing the ARE-binding site (SLC7A11 promoter-long) and another in which the ARE-site was excluded (SLC7A11 promoter-short), each driving an EGFP or luciferase reporter. Luciferase activity in vector control cells was negligible irrespective of Antioxidants 2021, 10, 9 Se-Met exposure (Figure 2A) but was increased significantly in ARPE-19 cells transfected7 of 17 with SLC7A11 promoter-long and exposed to Se-Met (p < 0.001). These data strongly support the Se-Met-induced activation of ARE/Nrf2 in the promoter region of xCT (SLC7A11). The RT-PCRRT-PCR resultsresults aboveabove showingshowing upregulatedupregulated xCTxCT expressionexpression uponupon Se-MetSe-Met treatmenttreatment (Figure(Figure1 1CC)) are are consistent consistent with with this this prediction; prediction; however, however, we we confirmed confirmed the the RT-PCR RT-PCR data data by bynorthern northern blot blot (Figure (Figure2B). To2B evaluate). To evaluate whether whether the Se-Met-induced the Se-Met-induced increase increase in xCT mRNAin xCT mRNAis associated is associated with a similar with a increase similar inincrease xCT protein, in xCT ARPE-19 protein, cellsARPE were-19 culturedcells were identically cultured identicallyas described as above described and xCT abo proteinve and was xCT analyzed protein by was immunofluorescence analyzed by immunofluorescence (Figure2C) and western(Figure 2C blotting) and western (Figure2 D).blotting Both methods(Figure 2D revealed). Both robustmethods increases revealed in robust xCT protein increases in the in presencexCT protein of Se-Met,in the presence and the of increase Se-Met, was and specificallythe increase found was specifically in the transporter found in density the trans- in porterthe plasma density membrane. in the plasma membrane.

Figure 2. SeSe-Met-induced-Met-induced expression of xCT (SLC7A11):(SLC7A11): ( A) luciferase reporter assay of Se-MetSe-Met (1(1 mM)-inducedmM)-induced SLC7A11 (xCT)(xCT) promoterpromoter activationactivation inin inin ARPE-19 ARPE-19 cells cells transfected transfected with with luciferase luciferase reporter reporter constructs constructs containing containing either either the the short short or longor long form form of SLC7A11 of SLC7A11 promoter promoter (* p < ( 0.001* p < compared0.001 compared to the controlto the (control−Se-Met) (−Se SLC7A11-Met) SLC7A11 promoter-long promoter cells);-long (B )cells northern); (B) blotnorthern analysis blot of analysis xCT and of 4F2hcxCT and expression 4F2hc expression in control andin control Se-Met-treated and Se-Met ARPE-19-treated cells; ARPE (C-)19 immunocytochemical cells; (C) immunocytochemical analysis of xCTanalysis protein of xCT in ARPE-19 protein in cells ARPE cultured-19 cells in thecultured presence in the (+Se-Met) presence or (+Se absence-Met) (or−Se-Met) absence of (−Se selenomethionine-Met) of selenomethionine (1 mM, 16 h),(1 wheremM, 16 cells h), where in which cells PBS in which was substituted PBS was substituted for primary for antibody primary served antibody as theserved negative as the control negative (Neg. control Con); (Neg. and Con (D)) west-; and ern(D) blotwestern analysis blot of analysis xCT protein of xC inT plasmaprotein membranein plasma versusmembrane cytosolic versus protein cytosolic fractions protein isolated fractions from isolated ARPE-19 from cells ARPE cultured-19 cells cultured in the presence or absence of Se-Met (1 mM, 16 h). in the presence or absence of Se-Met (1 mM, 16 h). 3.3. Characterization of Se-Met-Induced xc- Transport Activity 3.3. CharacterizationConfluent ARPE of- Se-Met-Induced19 cell cultures xc-were Transport exposed Activity to in Se-Met (1 mM) for 0–16 h, fol- lowedConfluent by the monitoring ARPE-19 cellof [3H] cultures-glutamate were exposeduptake under to in Na+ Se-Met-free (1 conditions, mM) for 0–16 a well h,- fol-es- tablishedlowed by read the- monitoringout of system of xc [3H]-glutamate- transport activity. uptake With under respect Na+-free to time conditions,-dependency, a well- the greatestestablished stimulation read-out in of xc system- transport xc- transport activity was activity. observed With in respect cells exposed to time-dependency, to Se-Met for the greatest stimulation in xc- transport activity was observed in cells exposed to Se-Met for 16 h (Figure3A). The dose-dependency of the effect of Se-Met was also evaluated. Significant stimulation of xc- transport activity was observed in the presence of Se-Met at concentrations as low as 100 µM and increased steadily with increasing doses of Se-Met (Figure3B). To confirm that the increase in system xc- transport activity observed in the presence of higher doses of Se-Met was not an artifact of cellular stress or toxicity, cell viability was confirmed via trypan blue exclusion assay (Figure3C). Indeed, the viability of the cells was not affected by Se-Met at any of the concentrations used in the present study. Antioxidants 2021, 10, x FOR PEER REVIEW 8 of 18

16 h (Figure 3A). The dose-dependency of the effect of Se-Met was also evaluated. Signif- icant stimulation of xc- transport activity was observed in the presence of Se-Met at concentrations as low as 100 μM and increased steadily with increasing doses of Se-Met (Fig- ure 3B). To confirm that the increase in system xc- transport activity observed in the presence of higher doses of Se-Met was not an artifact of cellular stress or toxicity, cell viability Antioxidants 2021, 10, 9 was confirmed via trypan blue exclusion assay (Figure 3C). Indeed, the viability of 8the of 17 cells was not affected by Se-Met at any of the concentrations used in the present study.

Figure 3. Selenomethionine (Se-Met)-induced stimulation of xc- transport activity is time- and dose- Figuredependent. 3. Selenomethionine (A) Uptake of [3H]-glutamate (Se-Met)-induced (2.5 stimulationµM) was measured of xc- transport for 15 min activity in ARPE-19 is time cells- and cultured dosein the-dependent. presence or(A absence) Uptake of of Se-Met [3H]-glutamate (1 mM) for ( 0–162.5 μ h.M) Uptake was measured was monitored for 15 min at 37 in◦ CARPE in the-19 absence cells cultured in the presence or absence of Se-Met (1 mM) for 0–16 h. Uptake was monitored at 37 of Na+. (B) Na+-independent [3H]-glutamate (2.5 µM) uptake was measured for 15 min in ARPE-19 °C in the absence of Na+. (B) Na+-independent [3H]-glutamate (2.5 μM) uptake was measured for cells cultured in the presence or absence of varying concentrations (0–2500 µM) of selenomethionine 15 min in ARPE-19 cells cultured in the presence or absence of varying concentrations (0–2500 μM) of(Se-Met) selenomethionine or methionine (Se- (Met)Met) or for methionine 16 h. (* p < 0.05;(Met **) forp < 16 0.01; h. ( compared* p < 0.05; to** p corresponding < 0.01; compared concentration to cor- respondingof Met). (C concentration) Cell viability of was Met determined). (C) Cell viability by trypan was blue determined exclusion assay.by trypan blue exclusion assay. To conﬁrm the contribution of system xc- to the observed Na+-dependent uptake of [3H]-glutamate,To confirm the we contribution evaluated theof system substrate xc- selectivity to the observed of the Na+ uptake-dependent process. uptake Uptake of of [3H][3H]-glutamate-glutamate, we in evaluated the control the and substrate Se-Met-treated selectivity ARPE-19 of the uptake cells was process. evaluated Uptake in of the [3H]presence-glutamate of various in the control unlabeled and aminoSe-Met acids-treated at ARPE a concentration-19 cells was of evaluated 5 mM. The in the uptake pres- of ence[3H]-glutamate of various unlabeled was inhibited amino robustly acids at by a cystineconcentration and unlabeled of 5 mM. glutamate. The uptake Inhibition of [3H] by- glutamatecysteine waswas muchinhibited less. robustly Other aminoby cystine acids and examined unlabeled had glutamate. no or little Inhibition inhibitory by effectcys- teine(Figure was4 ).much System less. xc- Other is selective amino acids for cystine examined and had glutamate, no or little and inhibitory the observed effect substrate (Figure selectivity of the Se-Met-induced transport system responsible for [3H]-glutamate uptake 4). System xc- is selective for cystine and glutamate, and the observed substrate selectivity in ARPE-19 cells conﬁrms the identity of system xc-.Se-Met, though the major form of sele- of the Se-Met-induced transport system responsible for [3H]-glutamate uptake in ARPE- nium present in the diet is not the sole form attainable from dietary sources; selenocysteine 19 cells confirms the identity of system xc-.Se-Met, though the major form of selenium (Se-Cys) also represents a dietary source of selenium [31]. Selenoamino acids such as Se-Met present in the diet is not the sole form attainable from dietary sources; selenocysteine (Se- and Se-Cys can be incorporated directly into selenoproteins such as TXNRDs and GPXs or Cys) also represents a dietary source of selenium [31]. Selenoamino acids such as Se-Met alternately substituted for methionine (Met) or cysteine (Cys), respectively, in other bio- and Se-Cys can be incorporated directly into selenoproteins such as TXNRDs and GPXs logic reactions. In fact, the random substitution of selenoamino acids for sulfur-containing or alternately substituted for methionine (Met) or cysteine (Cys), respectively, in other amino acids is not thought to substantially alter protein structure or function [32]. As such, biologic reactions. In fact, the random substitution of selenoamino acids for sulfur-con- here, we evaluated the differential effects of Se-Met, Se-Cys, Cys, and Met on system xc- taining amino acids is not thought to substantially alter protein structure or function [32]. transport activity. ARPE-19 cells were cultured in the presence or absence of 1 mM Se-Met, Se-Cys, Cys, or Met (16 h incubation), and the Na+-independent uptake of [3H]-glutamate was monitored. As observed earlier (Figures3 and4), xc- activity almost doubled in the presence of Se-Met (Figure5A). Moderate stimulation was observed also in the presence of Se-Cys; however, Met and Cys had little to no effect on xc- transport activity. Antioxidants 2021, 10, x FOR PEER REVIEW 9 of 18

As such, here, we evaluated the differential effects of Se-Met, Se-Cys, Cys, and Met on system xc- transport activity. ARPE-19 cells were cultured in the presence or absence of 1 mM Se-Met, Se-Cys, Cys, or Met (16 h incubation), and the Na+-independent uptake of [3H]-glutamate was monitored. As observed earlier (Figures 3 and 4), xc- activity almost doubled in the presence of Se-Met (Figure 5A). Moderate stimulation was observed also Antioxidants 2021, 10, 9 in the presence of Se-Cys; however, Met and Cys had little to no effect on xc- transport9 of 17 activity.

FigureFigure 4. SuSubstratebstrate specificity speciﬁcity of system xc-xc- in control andand selenomethionineselenomethionine (Se-Met)-treated(Se-Met)-treated ARPE- ARPE19 cells:-19 uptake cells: uptake of [3H]-glutamate of [3H]-glutamate (2.5 µ M)(2.5 was μM measured) was measured in control in control and Se-Met-treated and Se-Met-treated ARPE-19 ◦ ARPEcells in-19 the cells absence in the ofabsence Na+ for of 15Na+ min for at 15 37 minC at and 37 in°C the and presence in the presence or absence or absence of excess of unlabeledexcess unlabeledamino acids, amino each acids, at a concentration each at a concentration of 5 mM (* ofp <5 mM 0.01; ( *** pp << 0.01; 0.001). ** p < 0.001).

Members of the excitatory amino acid transporter (EAAT) family also mediate glutamate uptake, but only in a Na+-dependent manner. Many members of the EAAT family are expressed in retina; RPE specifically expresses at least two subtypes: EAAT3 (EAAC1) and EAAT4 [33]. As such, in addition to evaluating the differential influence of Cys and Met versus Se-Met and Se-Cys on Na+- independent glutamate uptake/system xc- trans- port activity, we asked also whether these compounds influence the cellular uptake of glutamate in RPE accomplished by EAATs. ARPE-19 cells were cultured identically as described above, and the uptake of [3H]-glutamate was monitored in the presence of Na+ (Figure5B) . Under these conditions, glutamate uptake was again stimulated significantly in the presence of Se-Met and moderately by Se-Cys; Met and Cys had very little effect. It is important to note however that, when conducting uptake assays in the presence of Na+, both Na+-dependent and Na+-independent mechanisms of glutamate transport are at work; hence, glutamate uptake under these conditions is representative of the “total” glutamate uptake mediated by xc- and EAATs. The specific contribution of EAATs to glutamate uptake is determined by calculating Na+-coupled uptake (i.e., uptake in the presence of Na+ minus uptake in the absence of Na+). These calculations showed that the activity of EAATs was not influenced by Se-Met, Se-Cys, Met, or Cysteine (Figure5C). Antioxidants 2021, 10, 9 10 of 17 Antioxidants 2021, 10, x FOR PEER REVIEW 10 of 18

Figure 5. DifferentialFigure 5. Differential influence of influen selenoaminoce of selenoamino acids (Se-Met acids and ( Se-Cys)Se-Met and sulfurSe-Cys amino) and sulfur acids (Metamino and acids Cys) on system xc- transport(Met activity: and Cys ARPE-19) on system cells were xc- transport cultured inactivity: the presence ARPE- or19 absencecells were of selenomethioninecultured in the presence (Se-Met), or selenocysteine (Se-Cys), methionineabsence of (Met), selenomethionine and cysteine (Cys),(Se-Met each), selenocysteine at 1 mM, for 16(Se h.-Cys Uptake), methionine of [3H]-glutamate (Met), and (2.5 cysteineµM) was measured for 15 min at(Cys 37 )◦,C each inthe at 1 presence mM, for and16 h. absence Uptake ofof Na+:[3H]-glutamate (A) Na+-independent, (2.5 μM) was xc-measured -specific for glutamate 15 min at uptake; 37 °C (B) total glutamate uptake;in the presence and (C) Na+ and -dependent, absence of Na+: excitatory (A) Na+ amino-independent, acid transporter xc- -specific (EAAT)-specific glutamate glutamate uptake; (B uptake.) total (* p < 0.05, ** p < 0.01 comparedglutamate to uptake; control). and (C) Na+ -dependent, excitatory amino acid transporter (EAAT)-specific glutamate uptake. (* p < 0.05, ** p < 0.01 compared to control).

Members3.4. of the Kinetic excitatory Analysis amino of Se-Met-Induced acid transporter System (EAAT xc-) Transport family also Activity mediate glutamate uptake, but onlyTo gainin a Na+ a better-dependent understanding manner. ofMany the members effects of of Se-Met the EAAT on the family transport system, are expressed inwe retina; performed RPE specifically additional expresses studies to at determineleast two subtypes: which of EAAT3 the kinetic (EAAC1 parameters) of the and EAAT4 [33]system. As such, (xc-)-mediated in addition transport to evaluating process the are differential affected by influence this compound of Cys (Figureand 6). Both ki- Met versus Senetic-Met parameters, and Se-Cys namely on Na+ the- Michaelisindependent constant glutamate (Kt)and uptake/system the maximal xc velocity- (Vmax), transport activity,were we affected. asked also Kt whether was reduced thesefrom compounds 353 ± 24influenceµM to 129the cellular± 24 µ Muptake in the presence of of glutamate inSe-Met RPE accomplished (1 mM) (p < 0.01);by EAATs. Vmax ARPE was increased-19 cells were from cultured 0.74 ± 0.03 identically nmol/106 as cells/min to described above,0.91 and± 0.07the uptake nmol/106 of [3H] cells/min-glutamate (p < was 0.05). monitored Eadie–Hofstee in the presence transformation of Na+ of the data is (Figure 5B). Underprovided these in conditions, the insetof glutamate Figure6. uptake These datawas demonstrateagain stimulated that significantly the increase in activity of in the presencesystem of Se-Met xc- asand a resultmoderately of treatment by Se- ofCys; the Met cells and with Cys Se-Met had very is associated little effect. with It an increase in is important tomaximal note however velocity that, (increase when conducting in Vmax) and uptake an increase assays in in the substrate presence afﬁnity of Na+, (decrease in Kt). both Na+-dependent and Na+-independent mechanisms of glutamate transport are at work; hence, glutamate uptake under these conditions is representative of the “total” glutamate uptake mediated by xc- and EAATs. The specific contribution of EAATs to glutamate uptake is determined by calculating Na+-coupled uptake (i.e., uptake in the presence of Na+ minus uptake in the absence of Na+). These calculations showed that the activity of EAATs was not influenced by Se-Met, Se-Cys, Met, or Cysteine (Figure 5C).

3.4. Kinetic Analysis of Se-Met-Induced System xc- Transport Activity To gain a better understanding of the effects of Se-Met on the transport system, we performed additional studies to determine which of the kinetic parameters of the system (xc-)-mediated transport process are affected by this compound (Figure 6). Both kinetic parameters, namely the Michaelis constant (Kt) and the maximal velocity (Vmax), were Antioxidants 2021, 10, x FOR PEER REVIEW 11 of 18

affected. Kt was reduced from 353 ± 24 μM to 129 ± 24 μM in the presence of Se-Met (1 mM) (p < 0.01); Vmax was increased from 0.74 ± 0.03 nmol/106 cells/min to 0.91 ± 0.07 nmol/106 cells/min (p < 0.05). Eadie–Hofstee transformation of the data is provided in the inset of Figure 6. These data demonstrate that the increase in activity of system xc- as a Antioxidants 2021, 10, 9 result of treatment of the cells with Se-Met is associated with an increase in maximal11 ofve- 17 locity (increase in Vmax) and an increase in substrate affinity (decrease in Kt).

Figure 6. Kinetic analysis of system xc- activity in the presence or absence of Se-Met in ARPE-19 Figure 6. Kinetic analysis of system xc- activity in the presence or absence of Se-Met in ARPE-19 cells: saturation kinetics of glutamate uptake in ARPE-19 cells cultured for 16 h in the presence cells: saturation kinetics of glutamate uptake in ARPE-19 cells cultured for 16 h in the presence or absenceor absence of 1 ofmM 1 mMselenomethionine selenomethionine (Se-Met (Se-Met)) was evaluated was evaluated by monitoring by monitoring the uptake the uptake of [3H] of- [3H]- glutamateglutamate in in Na+ Na+-free-free medium medium with with various various concentrations concentrations of ofunlabeled unlabeled glutamate glutamate (0– (0–20002000 μMµ).M). Inset:Inset: Eadie Eadie–Hofstee–Hofstee plot. plot. 3.5. Influence of Se-Met on Glutathione Levels in ARPE-19 Cells with and without Oxidative Stress 3.5. Influence of Se-Met on Glutathione Levels in ARPE-19 Cells with and without Oxidative StressWe have demonstrated the ability of Se-Met to induce Nrf2 and, as a result, increase the activity of the cystine transport system xc- in cultured human retinal pigment epithelial We have demonstrated the ability of Se-Met to induce Nrf2 and, as a result, increase cells. System xc- is critical for cellular delivery of cysteine in the form of cystine and, the activity of the cystine transport system xc- in cultured human retinal pigment epithe- hence, for the synthesis of glutathione. Indeed, others have shown that increasing system lial cells. System xc- is critical for cellular delivery of cysteine in the form of cystine and, (xc-)-mediated cystine transport can result in increased intracellular GSH [10,34]. Therefore, hence, for the synthesis of glutathione. Indeed, others have shown that increasing system we hypothesized the same to be true with respect to the Se-Met-induced increase in system (xc-xc-) transport-mediated activity cystine observedtransport in can our result experimental in increased system. intracellular We tested GSH the [10,34] validity. There- of this fore,prediction we hypothesized by culturing the ARPE-19 same to cellsbe true in thewith presence respect to or the absence Se-Met of-induced Se-Met (1 increase mM) and in system xc- transport activity observed in our experimental system. We tested the validity in the presence or absence of H2O2 (500 µM). GSH concentrations were then measured. ofCellular this prediction concentrations by culturing of GSH ARPE increased-19 cells significantly in the presence in cells or absence exposed of to Se Se-Met-Met (1 undermM) andnormal in the conditions presence (Figure or absence7). The of effectH2O2 was(500 evenμM). moreGSH pronouncedconcentrations when were the then cells meas- were ured.subjected Cellular to oxidative concentrations stress. Asof expected,GSH increased based significantly upon our previous in cells studies exposed [21 to], levelsSe-Met of under normal conditions (Figure 7). The effect was even more pronounced when the cells GSH were reduced significantly when the cells were exposed to H2O2-induced oxidative were subjected to oxidative stress. As expected, based upon our previous studies [21], stress. This is because GSH is consumed in the process of H2O2 detoxification. However, levels of GSH were reduced significantly when the cells were exposed to H2O2-induced the H2O2-induced depletion of GSH was attenuated significantly (approximately 85% inhibition) in the presence of Se-Met. These results suggest that Se-Met-induced augmentation of system xc- expression significantly enhances the ability of RPE cells to synthesize GSH and consequently to counter oxidative stress. Antioxidants 2021, 10, x FOR PEER REVIEW 12 of 18

oxidative stress. This is because GSH is consumed in the process of H2O2 detoxification. However, the H2O2-induced depletion of GSH was attenuated significantly (approxi- Antioxidants 2021, 10, 9 mately 85% inhibition) in the presence of Se-Met. These results suggest that Se-Met12- ofin- 17 duced augmentation of system xc- expression significantly enhances the ability of RPE cells to synthesize GSH and consequently to counter oxidative stress.

. Figure 7. Se-Met raises intracellular levels of glutathione (GSH) protecting ARPE-19 cells against FigureH2O2 -induced7. Se-Met GSH raises depletion. intracellular Oxidative levels stressof glutathione was induced (GSH in) ARPE-19protecting cells ARPE using-19 hydrogencells against peroxide (H O , 500 µM) in the presence or absence of Se-Met (1 mM). Intracellular levels of glutathione H2O2-induced2 2 GSH depletion. Oxidative stress was induced in ARPE-19 cells using hydrogen peroxide(GSH) (H were2O2, 500 then μ measuredM) in the presence using the or GSH-Glo absence Assayof Se-Met kit (Promega).(1 mM). Intracellular (* p < 0.01; levels ** p < of 0.001). glutathione (GSH) were then measured using the GSH-Glo Assay kit (Promega). (* p < 0.01; ** p < 0.001). 3.6. Se-Met Induces System xc- Activity also in Primary Cultures of Mouse RPE 3.6. SeThe-Met studies Induces described System xc- thus Activity far were also in carried Primary out Cultures with ARPE-19 of Mouse cells, RPE a “transformed” RPE cell line. To confirm that the effect of Se-Met on system xc- activity in ARPE-19 cells The studies described thus far were carried out with ARPE-19 cells, a “transformed” is not an artifact of a transformed cell line, we used primary cultures of mouse RPE cells. RPE cell line. To confirm that the effect of Se-Met on system xc- activity in ARPE-19 cells We isolated RPE cells from normal mouse eyes, cultured them in the presence or absence is not an artifact of a transformed cell line, we used primary cultures of mouse RPE cells. of Se-Met (identically as described for ARPE-19 cells), and monitored the uptake of [3H]- We isolated RPE cells from normal mouse eyes, cultured them in the presence or absence glutamate in the presence and absence of Na+. Indeed, the stimulatory effects of Se-Met of Se-Met (identically as described for ARPE-19 cells), and monitored the uptake of [3H]- on Na+-independent glutamate uptake indicative of system xc- activity were observed glutamate in the presence and absence of Na+. Indeed, the stimulatory effects of Se-Met also in these cells (Figure8A). Also as observed with ARPE-19 cells, the inductive effect on Na+-independent glutamate uptake indicative of system xc- activity were observed of Se-Met was specific to system xc-; EAAT activity was not increased in primary RPE also in these cells (Figure 8A). Also as observed with ARPE-19 cells, the inductive effect cells when exposed to Se-Met. In fact, the EAAT activity, measured as Na+-dependent ofglutamate Se-Met was uptake, specific was to reduced system inxc cells-; EAAT exposed activity to Se-Met was not (Figure increased8B). Thein primary specificity RPE of cellsthe effectwhen of exposed Se-Met onto Se system-Met. xc-In wasfact, addressedthe EAAT by activity, examining measured the activity as Na+ of an-dependent unrelated glutamatetransporter, uptake, namely was the reduced transport in cells system exposed responsible to Se-Met for the(Figure uptake 8B) of. The taurine. specificity Taurine, of thewith effect its robustof Se-Met antioxidative on system xc and- was other addressed protective by examini properties,ng the is crucial activity to of normal an unrelated retinal transporter,health and visualnamely function the transport [35,36]. system We found responsible that the uptakefor the ofuptake taurine of wastaurine. not alteredTaurine, in withprimary its robust RPE cells antioxidative when treated and withother Se-Met protective (Figure properties,8C). is crucial to normal retinal health and visual function [35,36]. We found that the uptake of taurine was not altered in primary RPE cells when treated with Se-Met (Figure 8C). AntioxidantsAntioxidants 20202121,, 1010,, x 9 FOR PEER REVIEW 1313 of of 18 17

. Figure 8. Induction of xc- by selenomethionine (Se-Met) in mouse primary RPE cells: Primary mouse FigureRPE cells 8. Induction were cultured of xc- inby theselenomethionine presence or absence (Se-Met of) selenomethioninein mouse primary (Se-Met,RPE cells: 1 primary mM) for 16 h ◦ mousefollowed RPE by cells measurement were cultured of [3H]-glutamate in the presence (2.5 orµ absenceM) or [3H]-taurine of selenomet (25hionine nM) uptake (Se-Met, (15 1 min, mM 37) forC) 16in h the followed presence by and measurement absence of Na+.of [3H] (A-glutamate) Na+-independent, (2.5 μM) or (xc-)-specific [3H]-taurine glutamate (25 nM) uptake;uptake ( (15B) min, Na+- 37dependent, °C) in the presence excitatory and amino absence acid of transporter Na+. (A) Na+ (EAAT)-specific-independent, ( glutamatexc-)-specific uptake; glutamate and uptake; (C) Na+- (Bdependent) Na+-dependent, taurine (TAUT)excitatory uptake. amino (* acidp < 0.01)transporter. (EAAT)-specific glutamate uptake; and (C) Na+-dependent taurine (TAUT) uptake. (* p < 0.01). 4. Discussion 4. DiscussionRPE cells are vital to normal retinal health and visual function, performing such criticalRPE functions cells are byvital maintaining to normal outerretinal blood–retinal health and visual barrier function, properties, performing by nourishing such crit- and icalsupporting function photoreceptors by maintaining neurons, outer andblood by–retinal regulating barrier inflammation/immunity. properties, by nourishing As such,and supportingprotecting thesephotoreceptor cells from neurons, damage isand important. by regulating RPE has inflammation/immunity. a formidable antioxidant As defense such, protectingsystem, of these which cells GSH from is a major damage component. is important. The critical RPE has importance a formidable of GSH antioxidant in retina/RPE de- fenseis supported system, of by which its particularly GSH is a major high concentration component. The in this critical tissue/cell importance type of under GSH normalin retina/RPEphysiologic is supported conditions by [its37, 38particularly] and further high evidenced concentration by consequences in this tissue/cell associated type under with its decline in aging and in AMD [39,40]. GSH synthesis is largely dependent upon the normal physiologic conditions [37,38] and further evidenced by consequences associated availability of cysteine, a factor critically dependent, in turn, upon the activity of system with its decline in aging and in AMD [39,40]. GSH synthesis is largely dependent upon xc-. Therefore, strategies to enhance the expression/activity of this transport system may the availability of cysteine, a factor critically dependent, in turn, upon the activity of sys- be of benefit in the protection of retinal cells against oxidant-induced injury. Here, we show tem xc-. Therefore, strategies to enhance the expression/activity of this transport system Se-Met, an organic form of selenium, to be very effective in this regard. Specifically, may be of benefit in the protection of retinal cells against oxidant-induced injury. Here, we demonstrate the induction of Nrf2, a critical regulator of the cytoprotective response we show Se-Met, an organic form of selenium, to be very effective in this regard. Specifi- during oxidative stress, in association with exposure of cultured human RPE cells to Se-Met. cally, we demonstrate the induction of Nrf2, a critical regulator of the cytoprotective re- Though we did not directly evaluate the effect of Se-Met on the interaction between Nrf2 sponse during oxidative stress, in association with exposure of cultured human RPE cells and Keap 1, it is well documented that Nrf2 resides in the cytoplasm in association with to Se-Met. Though we did not directly evaluate the effect of Se-Met on the interaction Keap 1 when there is no oxidative stress, but the complex dissociates during oxidative stress between Nrf2 and Keap 1, it is well documented that Nrf2 resides in the cytoplasm in to release Nrf2, which then translocates into the nucleus to induce its target genes [41,42]. associatioInterestingly,n with Se-Met Keap may 1 when not only there facilitate is no oxidative the dissociation stress, but of Nrf2 the fromcomplex Keap dissociates 1 but also duringenhance oxidative its expression. stress to Our release studies Nrf2, show which that then the total translocates cellular levelsinto the of nucleus Nrf2 are to increased induce itsupon target exposure genes of[41,42] the cells. Interestingly, to Se-Met. If Se the-Met effect may of Se-Metnot only involves facilitate only the dissociation dissociation of theof Nrf2Nrf2/Keap from Keap 1 complex, 1 but also the enhance total levels its expression. of Nrf2 are Our expected studies to remainshow that the the same. total SLC7A11, cellular levelsthe gene of Nrf2 coding are for increased xCT, the upon functional expos subuniture of the of thecells xc- to transporter,Se-Met. If the is aeffect direct of target Se-Met for involvesNrf2 asevidenced only dissociation from SLC7A11-specific of the Nrf2/Keap promoter-reporter 1 complex, the total assays. levels PCR of Nrf2 and are northern expected blot tostudies remain strongly the same. support SLC7A11, the conclusion the gene that coding the activityfor xCT, of the system functional xc- is increased subunit of by the Se-Met xc- Antioxidants 2021, 10, 9 14 of 17

at least partly via enhanced transcription of SLC7A11. This is supported by the increase in density of the transporter protein in the plasma membrane. This however does not appear to be the only mode of action of Se-Met. The observed increase in substrate affinity in Se-Met-treated cells is intriguing. The increase in maximal velocity can be explained on the basis of the increased transporter density in the plasma membrane, but the change in substrate affinity was unexpected. This could only be due to some unknown changes in the transporter protein itself. One possibility is that the transporter activity is modulated by posttranslational changes such as thiol/disulfide interchange. Since the cellular levels of GSH are altered by Se-Met, it is possible that the xCT protein is subjected to thiol/disulfide interchange, which might influence substrate affinity. Additional studies are needed to confirm this speculation. The antioxidant properties of selenium-containing compounds like Se-Met are thought to be due primarily to the role of selenium as a constitutive metal cofactor in selenoproteins like the GPXs and TXNRDs. In addition, Se-Met induces the expression of GPX1 and TXNRD1 in ARPE-19 cells (Figure1D). Our present findings unravel another interesting mechanism for the antioxidant property of Se-Met: it upregulates Nrf2, an important transcription factor that is associated with cellular antioxidant machinery. We also evaluated the consequences of Se-Met-induced upregulation of the transport system xc- in terms of cellular glutathione status. Even under conditions not associated with oxidative stress, Se-Met increases the cellular levels of GSH, but the effect is much greater under conditions of oxidative stress. We do not know whether the former property, the Se-Met- induced upregulation of selenoprotein expression, is related to or directly impacts the latter, the Se-Met-induced stimulation of system xc- activity; however, in sum, our findings are highly supportive of the robust antioxidant properties of Se-Met in RPE and the potential usefulness of compounds of this nature in retina. Though at present antioxidant therapy is the standard recommendation for patients with dry AMD, there are conflicting opinions with regard to the true benefit, or lack thereof, of such therapy in terms of slowing/preventing progression of AMD. Furthermore, among those supportive of exogenous antioxidant vitamin/mineral supplementation, con- fusion exists as to what should be the appropriate makeup of the cocktail. At present, there is no selenium, in any form, present in Age-related Eye Disease Study (AREDS I and AREDS II) formulations [43], and to our present knowledge, there is no plan to incorporate selenium as such [44]. This is not because of any concrete evidence of a lack of gain in terms of antioxidant benefit or the presence of unwanted detrimental effects but rather is likely due in part to the large number of inconsistencies across the various studies conducted to date [40,45,46]. There are a large number of reports, clinical and experimental, touting the beneficial effects of selenium therapy in other diseases in which oxidative stress and resultant cellular damage are majorly involved (e.g., various types of cancer, HIV, and Alzheimer’s, etc.) [47–49]. Mechanisms provided to explain the beneficial effects of selenium in these paradigms include induction of Nrf2/phase II antioxidant signaling, regulation of p53-ERK1/2 activation, and attenuation of amyloid beta production and resultant cell death [50–53]. Many of these processes are congruent with our present findings in RPE and would be of substantial benefit in AMD to protect against oxidant-induced injury to RPE. Indeed, the critical biologic importance of selenium is without question, evidenced not only by the positive or beneficial effects associated with its supplemental use but also by the clinical consequences associated with its deficiency. Alternately, however, too much selenium can be toxic [54]. Selenium is available in a variety of different formulations; however, not all seleno-compounds are “created equal” with respect not only to their ability to raise serum selenium concentrations but also to their antioxidant and toxicological properties. This is supported by our present demonstration of the differential effects of Se-Met and Se-Cys on system xc- activity in RPE; Se-Met is much more potent than Se-Cys in stimulating xc- activity. Additionally, in comparison to other seleno-compounds, many of which have been shown to be cytotoxic at micromolar concentrations, clinical and experimental studies indicate that Se-Met can be used effectively at high doses with little Antioxidants 2021, 10, 9 15 of 17

associated toxicity [53,55,56]. In fact, in the present study, we exposed cultured human RPE cells to Se-Met at concentrations ranging from 100 µM up to 2.5 mM; signiﬁcant stimulatory effects on system xc- were observed at all concentrations tested, and cell viability was not signiﬁcantly affected even at higher doses (Figure3C). Collectively, these factors increase the attractiveness of Se-Met as a potential therapeutic drug for use in retina. Studies by others provide added support for the use of organic selenium for this very purpose [57–60]. However, additional studies focusing on the in vivo effects of Se-Met in retina/RPE and the optimal dosages for such are required before this compound can be included either alone or in combination with other antioxidant therapies in human patients.

5. Conclusions The current study demonstrates that selenomethionine, the major form of dietary selenium in humans, protects RPE cells from pro-oxidant injury via a multimodal Nrf2- related mechanism. Oxidative stress is a major component of pathology development in retina consequent to aging and degenerative diseases such as age-related macular degeneration and diabetic retinopathy. RPE is prominently impacted in these conditions. Thus, future studies to validate the collective beneﬁt of this compound in preclinical models of aging, RPE, and outer retinal dysfunction and/or degeneration are highly warranted given the potential broad utility of selenomethionine therapy.

Supplementary Materials: The following are available online at https://www.mdpi.com/2076-392 1/10/1/9/s1, Table S1: Primer sequences used for RT-PCR assays: human GCLC-NM_001197115.2, human GCLM-NM_001308253.2, human HO-1-NM_002133.3, human GSTA1-NM_145740.5; human GSTA2-NM_000846.5, human GPX1-NM_000581.3, human TXNRD1-NM_182742.3, and human NQO1-NM_000903.3. Author Contributions: V.G and P.M.M. are responsible for conceptualization of this study. Method- ology, analyses, and validation were done by S.A., S.M., and M.T., R.N.J. and M.B. contributed along with V.G. and P.M.M. to data interpretation, review, and manuscript preparation and editing. All authors have read and agreed to the published version of the manuscript. Funding: We would like to acknowledge the funding support for these studies from National Eye Institute grants NIH EY029113 and NIH EY022416 to P.M.M. Institutional Review Board Statement: The study was conducted according to the guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and approved by the Institutional Review Board of Augusta University (AUP: 2009-0214). Informed Consent Statement: Not applicable. Data Availability Statement: All datasets generated for this study are included in the article/Supple- mentary Materials. Conﬂicts of Interest: The authors declare no conﬂict of interest.

References 1. van Lookeren Campagne, M.; LeCouter, J.; Yaspan, B.L.; Ye, W. Mechanisms of age-related macular degeneration and therapeutic opportunities. J. Pathol. 2014, 232, 151–164. [CrossRef][PubMed] 2. Ambati, J.; Fowler, B.J. Mechanisms of age-related macular degeneration. Neuron 2012, 75, 26–39. [CrossRef][PubMed] 3. Scimone, C.; Alibrandi, S.; Scalinci, S.Z.; Battagliola, E.T.; D’Angelo, R.; Sidoti, A.; Donato, L. Expression of Pro-Angiogenic Markers Is Enhanced by Blue Light in Human RPE Cells. Antioxidants (Basel) 2020, 9, 1154. [CrossRef][PubMed] 4. Donato, L.; D’Angelo, R.; Alibrandi, S.; Rinaldi, C.; Sidoti, A.; Scimone, C. Effects of A2E-Induced Oxidative Stress on Retinal Epithelial Cells: New Insights on Differential Gene Response and Retinal Dystrophies. Antioxidants (Basel) 2020, 9, 307. [CrossRef] 5. Sparrow, J.R.; Hicks, D.; Hamel, C.P.The retinal pigment epithelium in health and disease. Curr. Mol. Med. 2010, 10, 802–823. [CrossRef] 6. Plafker, S.M.; O’Mealey, G.B.; Szweda, L.I. Mechanisms for countering oxidative stress and damage in retinal pigment epithelium. Int. Rev. Cell Mol. Biol. 2012, 298, 135–177. [CrossRef] 7. Cai, J.; Nelson, K.C.; Wu, M.; Sternberg, P., Jr.; Jones, D.P. Oxidative damage and protection of the RPE. Prog. Retin. Eye Res. 2000, 19, 205–221. [CrossRef] 8. Tokarz, P.; Kaarniranta, K.; Blasiak, J. Role of antioxidant enzymes and small molecular weight antioxidants in the pathogenesis of age-related macular degeneration (AMD). Biogerontology 2013, 14, 461–482. [CrossRef] Antioxidants 2021, 10, 9 16 of 17

9. Martis, R.M.; Knight, L.J.; Donaldson, P.J.; Lim, J.C. Identification, Expression, and Roles of the Cystine/Glutamate Antiporter in Ocular Tissues. Oxid. Med. Cell. Longev. 2020, 2020, 4594606. [CrossRef] 10. Bridges, C.C.; Kekuda, R.; Wang, H.; Prasad, P.D.; Mehta, P.; Huang, W.; Smith, S.B.; Ganapathy, V. Structure, function, and regulation of human cystine/glutamate transporter in retinal pigment epithelial cells. Investig. Ophthalmol. Vis. Sci. 2001, 42, 47–54. 11. Reddy, N.M.; Kleeberger, S.R.; Bream, J.H.; Fallon, P.G.; Kensler, T.W.; Yamamoto, M.; Reddy, S.P. Genetic disruption of the Nrf2 compromises cell-cycle progression by impairing GSH-induced redox signaling. Oncogene 2008, 27, 5821–5832. [CrossRef] [PubMed] 12. Lewerenz, J.; Hewett, S.J.; Huang, Y.; Lambros, M.; Gout, P.W.; Kalivas, P.W.; Massie, A.; Smolders, I.; Methner, A.; Pergande, M.; et al. The cystine/glutamate antiporter system x(c)(-) in health and disease: From molecular mechanisms to novel therapeutic opportunities. Antioxid. Redox Signal. 2013, 18, 522–555. [CrossRef][PubMed] 13. Banjac, A.; Perisic, T.; Sato, H.; Seiler, A.; Bannai, S.; Weiss, N.; Kolle, P.; Tschoep, K.; Issels, R.D.; Daniel, P.T.; et al. The cystine/cysteine cycle: A redox cycle regulating susceptibility versus resistance to cell death. Oncogene 2008, 27, 1618–1628. [CrossRef][PubMed] 14. Aragones, G.; Rowan, S.; Francisco, S.G.; Yang, W.; Weinberg, J.; Taylor, A.; Bejarano, E. Glyoxalase System as a Therapeutic Target against Diabetic Retinopathy. Antioxidants (Basel) 2020, 9, 1062. [CrossRef][PubMed] 15. Rahmanto, A.S.; Pattison, D.I.; Davies, M.J. Photo-oxidation-induced inactivation of the selenium-containing protective enzymes thioredoxin reductase and glutathione peroxidase. Free Radic. Biol. Med. 2012, 53, 1308–1316. [CrossRef] 16. Lane, H.W.; Strength, R.; Johnson, J.; White, M. Effect of chemical form of selenium on tissue glutathione peroxidase activity in developing rats. J. Nutr. 1991, 121, 80–86. [CrossRef] 17. Dun, Y.; Mysona, B.; Van Ells, T.; Amarnath, L.; Ola, M.S.; Ganapathy, V.; Smith, S.B. Expression of the cystine-glutamate exchanger (xc-) in retinal ganglion cells and regulation by nitric oxide and oxidative stress. Cell Tissue Res. 2006, 324, 189–202. [CrossRef] 18. Jadeja, R.N.; Powell, F.L.; Jones, M.A.; Fuller, J.; Joseph, E.; Thounaojam, M.C.; Bartoli, M.; Martin, P.M. Loss of NAMPT in aging retinal pigment epithelium reduces NAD(+) availability and promotes cellular senescence. Aging (Albany NY) 2018, 10, 1306–1323. [CrossRef] 19. Gambhir, D.; Ananth, S.; Veeranan-Karmegam, R.; Elangovan, S.; Hester, S.; Jennings, E.; Offermanns, S.; Nussbaum, J.J.; Smith, S.B.; Thangaraju, M.; et al. GPR109A as an anti-inflammatory receptor in retinal pigment epithelial cells and its relevance to diabetic retinopathy. Invest. Ophthalmol. Vis. Sci. 2012, 53, 2208–2217. [CrossRef] 20. Martin, P.M.; Ananth, S.; Cresci, G.; Roon, P.; Smith, S.; Ganapathy, V. Expression and localization of GPR109A (PUMA-G/HM74A) mRNA and protein in mammalian retinal pigment epithelium. Mol. Vis. 2009, 15, 362–372. 21. Gnana-Prakasam, J.P.; Thangaraju, M.; Liu, K.; Ha, Y.; Martin, P.M.; Smith, S.B.; Ganapathy, V. Absence of iron-regulatory protein Hfe results in hyperproliferation of retinal pigment epithelium: Role of cystine/glutamate exchanger. Biochem. J. 2009, 424, 243–252. [CrossRef] [PubMed] 22. Sato, H.; Nomura, S.; Maebara, K.; Sato, K.; Tamba, M.; Bannai, S. Transcriptional control of cystine/glutamate transporter gene by amino acid deprivation. Biochem. Biophys. Res. Commun. 2004, 325, 109–116. [CrossRef][PubMed] 23. Thangaraju, M.; Ananth, S.; Martin, P.M.; Roon, P.; Smith, S.B.; Sterneck, E.; Prasad, P.D.; Ganapathy, V. c/ebpdelta Null mouse as a model for the double knock-out of slc5a8 and slc5a12 in kidney. J. Biol. Chem. 2006, 281, 26769–26773. [CrossRef] 24. Mysona, B.; Dun, Y.; Duplantier, J.; Ganapathy, V.; Smith, S.B. Effects of hyperglycemia and oxidative stress on the glutamate transporters GLAST and system xc- in mouse retinal Muller glial cells. Cell Tissue Res. 2009, 335, 477–488. [CrossRef][PubMed] 25. Bridges, C.C.; Hu, H.; Miyauchi, S.; Siddaramappa, U.N.; Ganapathy, M.E.; Ignatowicz, L.; Maddox, D.M.; Smith, S.B.; Ganapathy, V. Induction of cystine-glutamate transporter xc- by human immunodeficiency virus type 1 transactivator protein tat in retinal pigment epithelium. Invest. Ophthalmol. Vis. Sci. 2004, 45, 2906–2914. [CrossRef] 26. Kursvietiene, L.; Mongirdiene, A.; Bernatoniene, J.; Sulinskiene, J.; Staneviciene, I. Selenium Anticancer Properties and Impact on Cellular Redox Status. Antioxidants (Basel) 2020, 9, 80. [CrossRef][PubMed] 27. Batliwala, S.; Xavier, C.; Liu, Y.; Wu, H.; Pang, I.H. Involvement of Nrf2 in Ocular Diseases. Oxid. Med. Cell. Longev. 2017, 2017, 1703810. [CrossRef] 28. Promsote, W.; Powell, F.L.; Veean, S.; Thounaojam, M.; Markand, S.; Saul, A.; Gutsaeva, D.; Bartoli, M.; Smith, S.B.; Ganapathy, V.; et al. Oral Monomethyl Fumarate Therapy Ameliorates Retinopathy in a Humanized Mouse Model of Sickle Cell Disease. Antioxid. Redox Signal. 2016, 25, 921–935. [CrossRef] 29. Habib, E.; Linher-Melville, K.; Lin, H.X.; Singh, G. Expression of xCT and activity of system xc(-) are regulated by NRF2 in human breast cancer cells in response to oxidative stress. Redox Biol. 2015, 5, 33–42. [CrossRef] 30. Bridges, R.J.; Natale, N.R.; Patel, S.A. System xc(-) cystine/glutamate antiporter: An update on molecular pharmacology and roles within the CNS. Br. J. Pharmacol. 2012, 165, 20–34. [CrossRef] 31. Pons, D.G.; Moran, C.; Alorda-Clara, M.; Oliver, J.; Roca, P.; Sastre-Serra, J. Micronutrients Selenomethionine and Selenocysteine Modulate the Redox Status of MCF-7 Breast Cancer Cells. Nutrients 2020, 12, 865. [CrossRef][PubMed] 32. Labunskyy, V.M.; Hatfield, D.L.; Gladyshev, V.N. Selenoproteins: Molecular pathways and physiological roles. Physiol. Rev. 2014, 94, 739–777. [CrossRef] [PubMed] 33. Maenpaa, H.; Gegelashvili, G.; Tahti, H. Expression of glutamate transporter subtypes in cultured retinal pigment epithelial and retinoblastoma cells. Curr. Eye Res. 2004, 28, 159–165. [CrossRef][PubMed] 34. Albano, R.; Raddatz, N.J.; Hjelmhaug, J.; Baker, D.A.; Lobner, D. Regulation of System xc(-) by Pharmacological Manipulation of Cellular Thiols. Oxid. Med. Cell. Longev. 2015, 2015, 269371. [CrossRef] 35. Ripps, H.; Shen, W. Review: Taurine: A “very essential” amino acid. Mol. Vis. 2012, 18, 2673–2686. Antioxidants 2021, 10, 9 17 of 17

36. Militante, J.D.; Lombardini, J.B. Taurine: Evidence of physiological function in the retina. Nutr. Neurosci. 2002, 5, 75–90. [CrossRef] 37. Bellezza, I. Oxidative Stress in Age-Related Macular Degeneration: Nrf2 as Therapeutic Target. Front. Pharmacol. 2018, 9, 1280. [CrossRef] 38. Ganea, E.; Harding, J.J. Glutathione-related enzymes and the eye. Curr. Eye Res. 2006, 31, 1–11. [CrossRef] 39. Sun, Y.; Zheng, Y.; Wang, C.; Liu, Y. Glutathione depletion induces ferroptosis, autophagy, and premature cell senescence in retinal pigment epithelial cells. Cell Death Dis. 2018, 9, 753. [CrossRef] 40. Plestina-Borjan, I.; Katusic, D.; Medvidovic-Grubisic, M.; Supe-Domic, D.; Bucan, K.; Tandara, L.; Rogosic, V. Association of age-related macular degeneration with erythrocyte antioxidant enzymes activity and serum total antioxidant status. Oxid. Med. Cell. Longev. 2015, 2015, 804054. [CrossRef] 41. Lu, M.C.; Ji, J.A.; Jiang, Z.Y.; You, Q.D. The Keap1-Nrf2-ARE Pathway As a Potential Preventive and Therapeutic Target: An Update. Med. Res. Rev. 2016, 36, 924–963. [CrossRef][PubMed] 42. Hybertson, B.M.; Gao, B. Role of the Nrf2 signaling system in health and disease. Clin. Genet. 2014, 86, 447–452. [CrossRef] [PubMed] 43. Age-Related Eye Disease Study Research Group. A randomized, placebo-controlled, clinical trial of high-dose supplementation with vitamins C and E, beta carotene, and zinc for age-related macular degeneration and vision loss: AREDS report no. 8. Arch. Ophthalmol. 2001, 119, 1417–1436. [CrossRef] 44. Group, A.R.; Chew, E.Y.; Clemons, T.; SanGiovanni, J.P.; Danis, R.; Domalpally, A.; McBee, W.; Sperduto, R.; Ferris, F.L. The Age-Related Eye Disease Study 2 (AREDS2): Study design and baseline characteristics (AREDS2 report number 1). Ophthalmology 2012, 119, 2282–2289. [CrossRef] 45. Sheck, L.; Davies, J.; Wilson, G. Selenium and ocular health in New Zealand. N. Z. Med. J. 2010, 123, 85–94. 46. Flohe, L. Selenium, selenoproteins and vision. Dev. Ophthalmol. 2005, 38, 89–102. [CrossRef] 47. Gandin, V.; Khalkar, P.; Braude, J.; Fernandes, A.P. Organic selenium compounds as potential chemotherapeutic agents for improved cancer treatment. Free Radic. Biol. Med. 2018, 127, 80–97. [CrossRef] 48. Loef, M.; Schrauzer, G.N.; Walach, H. Selenium and Alzheimer’s disease: A systematic review. J. Alzheimers Dis. 2011, 26, 81–104. [CrossRef] 49. Stone, C.A.; Kawai, K.; Kupka, R.; Fawzi, W.W. Role of selenium in HIV infection. Nutr. Rev. 2010, 68, 671–681. [CrossRef] 50. Dridi, S.; Hirano, Y.; Tarallo, V.; Kim, Y.; Fowler, B.J.; Ambati, B.K.; Bogdanovich, S.; Chiodo, V.A.; Hauswirth, W.W.; Kugel, J.F.; et al. ERK1/2 activation is a therapeutic target in age-related macular degeneration. Proc. Natl. Acad. Sci. USA 2012, 109, 13781–13786. [CrossRef] 51. Filomeni, G.; Piccirillo, S.; Rotilio, G.; Ciriolo, M.R. p38(MAPK) and ERK1/2 dictate cell death/survival response to different pro-oxidant stimuli via p53 and Nrf2 in neuroblastoma cells SH-SY5Y. Biochem. Pharmacol. 2012, 83, 1349–1357. [CrossRef] [PubMed] 52. Xiong, S.; Markesbery, W.R.; Shao, C.; Lovell, M.A. Seleno-L-methionine protects against beta-amyloid and iron/hydrogen peroxide-mediated neuron death. Antioxid. Redox Signal. 2007, 9, 457–467. [CrossRef][PubMed] 53. Seo, Y.R.; Kelley, M.R.; Smith, M.L. Selenomethionine regulation of p53 by a ref1-dependent redox mechanism. Proc. Natl. Acad. Sci. USA 2002, 99, 14548–14553. [CrossRef] 54. MacFarquhar, J.K.; Broussard, D.L.; Melstrom, P.; Hutchinson, R.; Wolkin, A.; Martin, C.; Burk, R.F.; Dunn, J.R.; Green, A.L.; Hammond, R.; et al. Acute selenium toxicity associated with a dietary supplement. Arch. Intern. Med. 2010, 170, 256–261. [CrossRef][PubMed] 55. Nicastro, H.L.; Dunn, B.K. Selenium and prostate cancer prevention: Insights from the selenium and vitamin E cancer prevention trial (SELECT). Nutrients 2013, 5, 1122–1148. [CrossRef][PubMed] 56. Hoeﬁg, C.S.; Renko, K.; Kohrle, J.; Birringer, M.; Schomburg, L. Comparison of different selenocompounds with respect to nutritional value vs. toxicity using liver cells in culture. J. Nutr. Biochem. 2011, 22, 945–955. [CrossRef][PubMed] 57. Yazici, A.; Sogutlu-Sari, E.; Yay, A.; Aksit, H.; Kilic, A.; Aksit, D.; Yildiz, O.; Ermis, S.S. The protective effect of selenium in cisplatin-related retinotoxicity. Cutan. Ocul. Toxicol. 2014, 33, 327–332. [CrossRef][PubMed] 58. McCarty, M.F. The putative therapeutic value of high-dose selenium in proliferative retinopathies may reﬂect down-regulation of VEGF production by the hypoxic retina. Med. Hypotheses 2005, 64, 159–161. [CrossRef][PubMed] 59. Di Leo, M.A.; Ghirlanda, G.; Silveri, N.G.; Giardina, B.; Franconi, F.; Santini, S.A. Potential therapeutic effect of antioxidants in experimental diabetic retina: A comparison between chronic taurine and vitamin E plus selenium supplementations. Free Radic. Res. 2003, 37, 323–330. [CrossRef] 60. Head, K.A. Natural therapies for ocular disorders, part one: Diseases of the retina. Altern. Med. Rev. 1999, 4, 342–359.