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Lutein Activates the Transcription Factor Nrf2 in Human Retinal Pigment Epithelial Cells Katja Frede, Franziska Ebert, Anna Kipp, Tanja Schwerdtle, and Susanne Baldermann J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b01929 • Publication Date (Web): 30 Jun 2017 Downloaded from http://pubs.acs.org on June 30, 2017

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Journal of Agricultural and Food Chemistry

Title: Lutein Activates the Transcription Factor Nrf2 in Human Retinal Pigment Epithelial Cells

Short title: Nrf2 Activation in ARPE-19 Cells by Lutein

Authors: Katja Fredeab*, ([email protected]); Franziska Ebertb, ([email protected]); Anna P. Kippc, ([email protected]); Tanja Schwerdtleb, ([email protected]); Susanne Baldermannab, ([email protected])

Affiliations: a

Leibniz-Institute of Vegetable and Ornamental Crops Großbeeren/Erfurt e.V., Plant Quality and

Food Security, Theodor-Echtermeyer-Weg 1, 14979 Großbeeren, Germany b

University of Potsdam, Institute of Nutritional Science, Department of Food Chemistry, Arthur-

Scheunert-Allee 114-116, 14558 Nuthetal, Germany c

Friedrich Schiller University Jena, Institute of Nutrition, Dornburger Straße 24, 07743 Jena,

Germany

* Corresponding author: Katja Frede - Leibniz-Institute of Vegetable and Ornamental Crops Großbeeren/Erfurt e.V., Plant Quality and Food Security, Theodor-Echtermeyer-Weg 1, 14979 Großbeeren, Germany. Tel.: +49 (0) 33701-78313; Fax. +49 (0) 33701-55391; Email: [email protected]

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Abstract

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The degeneration of the retinal pigment epithelium caused by oxidative damage is a stage of

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development in age-related macular degeneration (AMD). The carotenoid lutein is a major

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macular pigment that may reduce the incidence and progression of AMD, but the underlying

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mechanism is currently still not fully understood. Carotenoids are known to be direct

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antioxidants. However, carotenoids can also activate cellular pathways resulting in indirect

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antioxidant effects. Here, we investigate the influence of lutein on the activation of nuclear factor

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erythroid 2-related factor 2 (Nrf2) target genes in human retinal pigment epithelial cells (ARPE-

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19 cells) using lutein-loaded Tween40 micelles. The micelles were identified as a suitable

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delivery system since they were non-toxic in APRE-19 cells up to 0.04% Tween40 and led to a

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cellular lutein accumulation of 62 µM ± 14 µM after 24 h. Lutein significantly enhanced Nrf2

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translocation to the nucleus 1.5 ± 0.4-fold compared to unloaded micelles after 4 h. Furthermore,

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lutein treatment for 24 h significantly increased the transcripts of NAD(P)H:quinone

14

oxidoreductase 1 (NQO1) by 1.7 ± 0.1-fold, glutamate-cysteine ligase regulatory subunit

15

(GCLm) by 1.4 ± 0.1-fold, and heme oxygenase-1 (HO-1) by 1.8 ± 0.3-fold. Moreover, we

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observed a significant enhancement of NQO1 activity by 1.2 ± 0.1-fold. Collectively, this study

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indicates that lutein not only serves as a direct antioxidant, but also activates Nrf2 in ARPE-19

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cells.

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Keywords: lutein, Nrf2, ARPE-19 cells, AMD, Tween40 micelles

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Introduction

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Carotenoids are natural yellow, orange, and red pigments that are synthesized by plants, algae,

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bacteria, and fungi.1 In plants, they are required for photosynthesis, are attractants for pollinators

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and seed dispersers, and serve as substrates in the biosynthesis of carotenoid-derived

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phytohormones and plant volatile scents.1 Animals and humans cannot synthesize carotenoids,

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and therefore, they can only obtain these compounds from their diet.1 Carotenoids are of special

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interest because they promote health in animals and humans since they have the potential to

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protect against cardiovascular diseases, certain types of cancer, eye-related diseases, and light-

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induced skin damage.2

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For the carotenoid lutein, there is evidence that it reduces the incidence and progression of age-

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related macular degeneration (AMD).3-5 AMD is an age-related, progressive degeneration of

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photoreceptors in the macular region of the retina,6 which is becoming a global burden.7 In detail,

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it is estimated that the number of people with AMD will be 196 million in 2020 and 288 million

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in 2040. Therefore, preventing the development of this disease is of great interest. The

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carotenoids lutein, zeaxanthin, and meso-zeaxanthin are the only carotenoids found in the macula

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of the retina. These macular carotenoids are important for optimal visual performance because of

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their ability to filter blue light and consequently, they can attenuate both chromatic aberration and

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light scatter.8 Moreover, these carotenoids protect against AMD by reducing oxidative stress in

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the retina via different mechanisms. For example, the above-mentioned filter ability decreases the

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amount of energy-rich light reaching the photoreceptors.2 Furthermore, carotenoids serve as

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direct antioxidants and can activate cellular pathways resulting in indirect antioxidant effects.9

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The cellular pathways activated by lutein remain to be fully determined to advance our current

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understanding of the cellular signalling function of lutein in the eye.



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The initial pathogenesis of AMD involves the degeneration of the underlying retinal pigment

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epithelium (RPE) caused by oxidative damage.6 Moreover, nuclear factor erythroid 2-related

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factor 2 (Nrf2) signaling is impaired in the aging RPE, thereby rendering it more vulnerable to

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oxidative stress.10 The transcription factor Nrf2 regulates genes encoding antioxidative and phase

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II enzymes that are involved in the maintenance of the cellular redox status as well as in the

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detoxification of xenobiotics.11,12 Nrf2 activates genes by binding to antioxidant response

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elements (AREs).13 Under unstressed conditions, the Kelch-like ECH-associated protein 1

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(Keap1) suppresses Nrf2 transcriptional activity, which subsequently leads to the proteasomal

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degradation of Nrf2.14,15 Upon activation of the Nrf2 pathway, Nrf2 translocates to the nucleus,

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forms a heterodimer with a small musculoaponeurotic fibrosarcoma (Maf) protein that then binds

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to the ARE and activates genes under the control of the AREs.13,14 Enzymes regulated by Nrf2

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include - amongst others - NAD(P)H:quinone oxidoreductase 1 (NQO1), a quinone reductase that

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promotes obligatory 2-electron reductions,16 glutamate-cysteine ligase regulatory subunit

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(GCLm), a subunit of GCL catalyzing the first, rate limiting step of glutathione synthesis,17 and

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heme oxygenase-1 (HO-1), an enzyme degrading heme to biliverdin.18 Several phytochemicals

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can increase Nrf2 target genes in RPE cells, e.g. the isothiocyanate sulforaphane, saponins, and

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phenolic compounds.19-24 Also carotenoids can activate the Nrf2 pathway in RPE cells. For

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example, zeaxanthin and astaxanthin both lead to an activation of Nrf2 in ARPE-19 cells.9,25,26

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However, the exact role of lutein relating to Nrf2 in the RPE still remains to be determined. It is

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important to separately evaluate different carotenoids in different cell lines because the degree of

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Nrf2 activation varies. For instance, zeaxanthin activates Nrf2 more strongly than astaxanthin in

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the ARPE-19 cells.25,26 On the one hand, lutein activates the Nrf2 pathway in mouse microglial

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BV-2 cells27 and in mice liver.28 On the other hand, lutein induces antioxidative enzymes in a

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neuronal cell line (PC12D) without activating the Nrf2 pathway.29 Since current data support a 4 ACS Paragon Plus Environment

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preventive role of lutein against AMD,3-5 it is of high interest to increase macular carotenoids by

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a higher dietary uptake of lutein using lutein-rich foods or lutein supplements. Clarifying the

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protective function of lutein in the eye is important to support the need of this lutein-rich diet.

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Thus, the aim of this study is to investigate for the first time whether lutein activates the Nrf2

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pathway in ARPE-19 cells, thereby leading to the subsequent expression of Nrf2 target genes.

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Materials and Methods

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Chemicals

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Lutein was isolated from Xanthophyll pastös (Tagetes erecta oleoresin, Roth, Karlsruhe,

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Germany) using column chromatography, followed by saponification with ethanolic KOH,

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extraction with diethyl ether and precipitation from methanol. Purity and identity were confirmed

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by HPLC-DAD, 1H-NMR, and

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FAD (FAD-Na2*2H2O), NADP (ß-NADP-Na2), as well as neutral red and tert-butyl methyl ether

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were purchased from Roth (Karlsruhe, Germany). ARPE-19 cells were purchased from LGC

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Standards (Teddington, UK). Dulbecco's Modified Eagle Medium (DMEM)/Ham's F-12 and L-

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glutamine were obtained from Biochrom (Berlin, Germany). Fetal calf serum (FCS) was obtained

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from PAA Laboratories (Pasching, Austria). Bovine serum albumin (BSA), dicoumarol (3,3´-

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Methylen-bis(4-hydroxycoumarin)), DMSO, EDTA, formaldehyde (36.5-38% in H2O), glucose-

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6-phosphate, glucose-6-phosphate-dehydrogenase, HEPES, hydrogen peroxide (25-35%), KCl,

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menadione,

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penicillin/streptomycin (5000 U penicillin and 5 mg streptomycin/mL), resazurin sodium salt,

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trypsin solution 10x, and Tween20 were bought from Sigma-Aldrich (St. Louis, Missouri, USA).

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The antibody anti-Nrf2 was purchased from Santa Cruz (Dallas, Texas, USA), anti-histone H2B

13

C-NMR spectroscopy (data not shown). Ammonium acetate,

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium


bromide,


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from Epitomics (Burlingame, California, USA), and anti-rabbit from Cell Signaling Technology

97

(Danvers, Massachusetts, USA). R,S-sulforaphane was obtained from Enzo Life Sciences

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(Farmingdale, New York, USA). The RNeasy Plus Mini Kit (50) and the RNase-free Dnase Set

99

were purchased from Qiagen (Hilden, Germany). Oligo (dT)12−18 primers and SuperScript III

100

reverse transcriptase were purchased from Thermo Fisher Scientific (Waltham, Massachusetts,

101

USA). SensiMix™ SYBR Low-ROX Kit was bought from Bioline (Luckenwalde, Germany).

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Methanol and Tween40 were purchased from Th. Geyer (Berlin, Germany). Dichloromethane,

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HCl, n-hexane, K2HPO4, KH2PO4, Na2HPO4, phosphatase inhibitor (sodium vanadate), 2-

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propanol, protease inhibitor cocktail set III (EDTA-free), and Triton X-100 were obtained from

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Merck (Darmstadt, Germany). Ethanol and acetic acid were bought from VWR International

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(Radnor, Pennsylvania, USA). Tris was obtained from AppliChem (Darmstadt, Germany).

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Protein Assay Dye Reagent Concentrate and Protein Assay Standard II were purchased from Bio-

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Rad (Hercules, California, USA). Non-fat dry milk powder was bought from TSI (Zeven,

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Germany). Finally, NaCl was purchased from AppliChem (Darmstadt, Germany).

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Preparation of lutein-loaded Tween40 micelles

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The micelles were prepared in darkened tubes according to a slightly modified method previously

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described by Lornejad-Schäfer, et al.30, which produces micelles with low cytotoxicity and good

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cellular uptake of carotenoids. A stock solution of a lutein standard of about 175 µM in

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dichloromethane was prepared, diluted in ethanol, and quantified spectrophotometrically (final

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concentration of dichloromethane ≤ 10%; specific absorption coefficient A1%,1cm = 2550 at

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445 nm in ethanol31). Aliquots were dried under a stream of N2 and stored at -80 °C until further

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use. Lutein aliquots were dissolved in dichloromethane/ethanol (1/20, v/v) to a final

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concentration of 70.5 µM and transferred with ethanol to a round bottom flask. The solvent was 6 ACS Paragon Plus Environment

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removed in a rotary evaporator. Tween40/ethanol (1/4029, v/v) was added to lutein in a ratio of

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20 µL Tween40/1 µmol lutein. After treatment in an ultrasonic bath, the solvent was evaporated

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again. Lutein was micellized by adding cell culture medium followed by sonication in an

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ultrasonic bath. The micelles were again diluted with cell culture medium before applying to the

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ARPE-19 cells. Cells were treated with concentrations ranging from 0.5 µM (0.001% Tween40)

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to 40 µM (0.08% Tween40) lutein. Finally, for the control, micelles without lutein were prepared

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in the exact same way.

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Cell culture

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The human retinal pigment epithelial cell line ARPE-19 (ATCC® CRL2302™) is a suitable

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model for RPE cells because it expresses typical RPE markers such as cellular retinaldehyde

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binding protein (CRALBP ) and RPE-specific 65 kDa protein (RPE65).32 Cells were cultivated in

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25 cm2 cell culture flasks (passages 8-32) and were maintained at 37 °C and 5% CO2 in 100%

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humidified air. The culture medium consisted of DMEM/Ham's F-12, 10% (v/v) FCS, 100 U/mL

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penicillin, 0.1 mg/mL streptomycin, and 2 mM L-glutamine. Cells were subcultured every 2-

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3 days. Unless otherwise stated, cells were seeded at a density of 40,000 cells/cm2 in all

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experiments.

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Neutral red- and resazurin-based cytotoxicity assays

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To determine cell viability after incubation with micelles, both a neutral red- and a resazurin-

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based assay were carried out. The reduction of resazurin to the red fluorescent resorufin indicates

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enzymatic and metabolic activity of cells, while the neutral red test measures lysosomal

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integrity.33,34



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ARPE-19 cells were seeded in 96-well plates at a density of 23,300 cells/cm2 and cultivated for

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48 h. Cells were treated with different micelles concentrations with and without lutein for 24 h.

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The neutral red-based assay was performed as described previously with neutral red-containing

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medium at a concentration of 230 µM.35 For the resazurin based-assay, cells were incubated with

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resazurin-containing medium at a concentration of 40 µM for 3 h at 37 °C. Fluorescence was

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determined in a plate reader (Infinite M200 Pro, Tecan Group Ltd., Zürich, Switzerland;

149

excitation: 530 nm, emission: 590 nm). Values of untreated control wells were set to 100%

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viability.

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In vitro test of cellular lutein uptake

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ARPE-19 cells were cultivated for 2 days after seeding and incubated with lutein-loaded micelles

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for 24 h. The final concentrations were 1 µM lutein (0.002% Tween40), 5 µM lutein (0.01%

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Tween40), and 10 µM lutein (0.02% Tween40). Afterwards, the cell monolayers were washed

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twice with warm (37 °C) PBS-A (pH 7.4, 100 mM NaCl, 4.5 mM KCl, 7 mM Na2HPO4, 3 mM

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KH2PO4). Cells were detached with 2 mL trypsin, suspended in 5 mL cold FCS/PBS-A (5/95,

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v/v), and put on ice. Cell number and cell volume were determined (CASY Model TTC 150 µm,

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Roche Diagnostics, Mannheim, Germany). The cell suspension was centrifuged (4 °C, 5 min,

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314 x g), resuspended in 1 mL cold PBS-A, and centrifuged again (4 °C, 5 min, 2380 x g). The

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cell pellet was stored at -80 °C under N2 until analysis of lutein content.

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LC-QToF-MS analysis of lutein

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The lutein from the cell pellets was extracted by adding 250 µL water, placing in an ultrasonic

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bath for 5 min, adding 500 µL ethanol and 700 µL n-hexane, and then shaking for 5 min at

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1400 rpm (modified after Schweigert, et al.36). After centrifugation for 10 min at 4000 x g the 8 ACS Paragon Plus Environment

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supernatant was transferred into a new tube. The extraction step was repeated twice using 700 µL

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n-hexane. After concentrating the combined supernatant under N2 to dryness, it was re-dissolved

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in 20 μL dichloromethane and 80 µL 2-propanol. The solution was filtered (0.2 μm, PTFE) and

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kept at 10 °C in the autosampler. Lutein content was analyzed as previously described.37

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Separation was performed on a C30-column (YMC Co. Ltd., Kyoto, Japan, YMC C30, 100 x

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2.1 mm, 3 µm) on an Agilent Technologies 1290 Infinity UHPLC. The column temperature was

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maintained at 20 °C. The mobile phases were (A) methanol//water (96/4, v/v) and (B)

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methanol/tert-butyl methyl ether/water (6/90/4, v/v/v). To increase the ionization, 20 mM

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ammonium acetate was added to the mobile phases. The flow rate was 0.2 mL/min. Elution was

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carried out with the following gradient: 100% A for 10 min, 100% A to 80% A in 7 min, 80% A

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for 28 min, 80% A to 0% A in 10 min, and 0% A to 100% A in 2 min. The samples were

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analyzed on an Agilent Technologies 6530 QTOF LC/MS equipped with an APCI ion source in

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positive ionization mode. The gas temperature was set to 300 °C at a flow rate of 8 L/min, the

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vaporizer to 350 °C, and the nebulizer pressure to 35 psig. The voltage was set to 3500 V and a

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fragmentor voltage of 175 V was applied at a corona current of 4 µA. A lutein standard was

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prepared and the concentration was determined spectrophotometrically as aforementioned in the

183

section micelles preparation. The lutein standard was used for external calibration. Lutein

184

quantification was performed at a detection wavelength of 450 nm.

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Western blot analysis of nuclear Nrf2 protein levels

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Cells were cultivated for 68 h before stimulation with 10 µM lutein (0.02% Tween40), unloaded

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micelles (0.02% Tween40), 10 µM sulforaphane (positive control for Nrf2 activation), or water

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for 4 h. Cells were washed twice with PBS-B (pH 7.4, 140 mM NaCl, 10 mM Na2HPO4, 3 mM

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KH2PO4), harvested with PBS-B, and centrifuged at 250 x g for 5 min at 4 °C. The supernatant 9 ACS Paragon Plus Environment


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was discarded. Cell pellets were resuspended in 350 µL hypotonic homogenization buffer

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(20 mM HEPES, 1 mM EDTA, pH 7.5, containing 1 µL/mL protease inhibitor cocktail set III,

193

EDTA-free, and 2 µL/mL phosphatase inhibitor (sodium vanadate)). After homogenization with

194

a Potter homogenizer (Potter S, Typ 8533024, B. Braun Biotech International, Melsungen,

195

Germany), the lysate was centrifuged at 720 x g for 15 min at 4 °C. The precipitated nuclei were

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washed in 200 µL homogenization buffer, resuspended in 100 µL homogenization buffer, and

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sonicated. The nuclear extract was centrifuged at 20,000 x g for 15 min at 4 °C. The protein

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content of the supernatant was determined according to Bradford.38

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Samples (10 µg protein) were separated on a gradient SDS-PAGE gel (7.5-15%) and transferred

200

to nitrocellulose (2 h, 1.2 mA/cm2, 4 °C). Blots were blocked in 5% non-fat dry milk in Tris-

201

buffered saline with 0.1% Tween20 at room temperature for 1 h. Western blot analysis was

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performed with a rabbit-anti-human-Nrf2 antibody (1:1000; Santa Cruz, sc-13032) and a rabbit-

203

anti-human-histone-H2B antibody (1:15000; Epitomics, 1810-1). A horseradish peroxidase-

204

conjugated anti-rabbit antibody (1:2000) was used as secondary antibody. Bands were visualized

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by chemiluminescence imaging (Intelligent Dark Box LAS 3000, FUJIFILM, Tokyo, Japan). The

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relevant band for Nrf2 is at 95-110 kDa.39

207 208

RT-qPCR analysis of Nrf2 target gene transcripts

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The cells were cultivated for 48 h or 68 h after seeding. The incubation with lutein-loaded

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micelles for 24 h (long-term effect) or 4 h (short-term effect) was performed with 10 µM lutein

211

(0.02% Tween40). Furthermore, cells were treated with micelles without lutein as control. Cells,

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which were grown for 48 h and treated with 10 µM sulforaphane for 24 h, served as positive

213

control for Nrf2 pathway activation. The control cells for the sulforaphane treatment were


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incubated with water. Cells were harvested as described in the section ‘In vitro test of cellular

215

lutein uptake’.

216

RNA was extracted using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) as described by

217

the

218

spectrophotometrically at 260 nm (Nanodrop ND1000, Thermo Fisher Scientific, Waltham,

219

Massachusetts, USA). The purity was checked using the ratios of absorbance 260/280 and

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260/230. cDNA was synthesized from 1 µg total RNA using SuperScript III reverse transcriptase

221

(Thermo Fisher Scientific, Waltham, Massachusetts, USA) with oligo (dT)12−18 primers according

222

to the manufacturer’s instructions. Each transcript quantification was carried out in triplicate. The

223

reaction mix per well consisted of 3 µL cDNA (diluted 1:10), 5 µL 2x SensiMix™ SYBR Low-

224

ROX, and 2 µL 2 µM primer (forward and reverse primer). The RT-qPCR was carried out in 96-

225

well reaction plates on a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Inc.,

226

Hercules, California, USA). The thermal cycling conditions were as follows: 50 °C for 2 min,

227

95 °C for 10 min, 39 cycles of 95 °C for 15 s, and 60 °C for 1 min. Afterwards, a melt curve

228

analysis was performed. The amplification efficiency (E) for each primer pair was determined

229

using a cDNA dilution series and used for calculation of gene expression. The expression was

230

calculated as n-fold induction of the gene of interest in treated compared to control cells by the

231

∆∆Cq method using the geometric mean of three reference genes.40,41 Reference genes were beta-

232

actin (βAct), hypoxanthine phosphoribosyl transferase 1 (HPRT1) and peptidylprolyl

233

isomerase A (PPIA). No-template controls were included. Primers and amplification efficiencies

234

were

235

CCTTAGGGCAGGTAGATTCAG

236

ATGTTGGGATACTGTGGGCTCTGG (forward) and CCCTTTCTGGCTTTTAGTCCTTCCT

237

(reverse),

manufacturer,

as

including

follows:

E

=

on-column

NQO1

100%;

HO-1

-

DNase

I

digestion.

RNA

TGGCTAGGTATCATTCAACTC (reverse),25

-

E

=

100%;

GCCAGCAACAAAGTGCAAGAT 11

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was

quantified

(forward) GCLm

(forward)

and -

and


(reverse),25

GGTAAGGAAGCCAGCCAAGAG

239

CCACACCTTCTACAATGAGC (forward) and GGTCTCAAACATGATCTGGG (reverse)25,

240

E = 96%;

241

CCTGACCAAGGAAAGCAAAG

242

AGACAAGGTCCCAAAGAC (forward) and ACCACCCTGACACATAAA (reverse),42 E =

243

100%.

-

=

100%;

βAct

238

HPRT1

E

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GACCAGTCAACAGGGGACAT (reverse),42

E

=

97%;

(forward) and

PPIA

-

and -

244 245

Measurement of NQO1 activity

246

ARPE-19 cells were seeded and cultivated for 48 h. Cells were treated with 10 µM lutein (0.02%

247

Tween40), unloaded micelles (0.02% Tween40), 10 µM sulforaphane, or water for 24 h.

248

Cells were washed twice with PBS-B (pH 7.4, 140 mM NaCl, 10 mM Na2HPO4, 3 mM

249

KH2PO4) and transferred to a new tube with 150 µL/well homogenization buffer (100 mM Tris-

250

HCl, 300 mM KCl, 0.01% Triton X-100, pH 7.6, containing 1 µL/mL protease inhibitor cocktail

251

set III, EDTA-free, and 2 µL/mL phosphatase inhibitor (sodium vanadate)). Cells were

252

homogenized with an ultrasonic homogenizer and centrifuged for 15 min at 21000 x g and 4 °C

253

before the protein contents of the supernatants were measured according to Bradford.38

254

NQO1 activity was determined as described previously.43 In principle, reduction of 3-(4,5-

255

dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) mediated by menadione was

256

measured on a microtiter plate absorbance reader at 590 nm (Synergy 2, Biotek Instruments, Bad

257

Friedrichshall, Germany). For the assay, 50 µL water and 190 µL reaction buffer (25 mM Tris-

258

HCl, pH 7.4, 0.665 mg/mL BSA, 0.01% Tween20, 5 µM FAD, 1 mM glucose-6-phosphate,

259

30 µM

260

dehydrogenase) were added to 10 µL lysate per well before measurement of the kinetics in the

261

microtiter plate absorbance reader. Another plate was measured with 50 µL NQO1 inhibitor

NADP,

0.72 mM

MTT,

50 µM

menadione,

0.3 U/mL


glucose-6-phosphate

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dicoumarol (0.3 mM dicoumarol, 0.5% DMSO, 5 mM potassium phosphate buffer (pH 7.4))

263

instead of water. NQO1 activity was calculated as the difference between the rates of MTT

264

reduction with and without dicoumarol (ɛreduced MTT = 11.961 mM-1 cm-1 at 590 nm).

265 266

Statistical analyses

267

Data are presented as the means ± SD. For statistical analyses IBM SPSS Statistics 22 (IBM

268

Deutschland, Ehningen, Germany) was used. Results were analyzed using one-way ANOVA

269

followed by Tukey HSD post hoc test. For comparison of two samples, significant differences

270

were determined by Student’s t-test for unpaired samples. A p-value ≤ 0.05 was considered as

271

statistically significant.

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Results

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Examination of micelles cytotoxicity

276

To test the suitability of micelles for delivering lutein to ARPE-19 cells, the effect of Tween40

277

micelles with and without lutein on cell viability was tested in a neutral red- and a resazurin-

278

based assay (Figure 1). A 24 h treatment with Tween40 micelles ranging from 0.001% to 0.04%

279

neither reduced lysosomal integrity nor dehydrogenase activity, no matter whether they contained

280

lutein (0.5-20 µM) or not. At 0.08% Tween40, lysosomal integrity significantly decreased to 60%

281

± 20% without lutein and to 53% ± 24% with 40 µM lutein. The same concentration of Tween40

282

led to a dehydrogenase activity of 71% ± 17% without lutein and 75% ± 19% with 40 µM lutein.

283 284

Cellular lutein uptake



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Cellular lutein uptake using several non-toxic concentrations was tested. In this experiment, we

286

could not detect a significant reduction in cell number or cell volume by the micelles which

287

confirmed the results of the above-mentioned viability assays. We found that the used micelles

288

were a suitable delivery system for lutein to ARPE-19 cells. The cells were incubated with 1 µM

289

lutein (0.002% Tween40). After 24 h, the measured cellular lutein concentration was 31 µM

290

± 8 µM (Figure 2) which is equivalent to 102 pmol/1x106 cells ± 24 pmol/1x106 cells (Figure S1

291

in Supporting Information). The cellular lutein uptake increased in parallel to the increased lutein

292

concentration. After incubating the cells with 10 µM lutein (0.02% Tween40) for 24 h, the

293

cellular lutein concentration reached a plateau at 60 µM (200 pmol/1x106 cells). A concentration

294

of 10 µM lutein (0.02% Tween40) was defined as working concentration for the subsequent

295

experiments.

296 297

Determination of Nrf2 translocation by lutein

298

Western blot analysis of nuclear fractions showed that lutein at 10 µM (0.02% Tween40) for 4 h

299

enhanced the Nrf2 translocation 1.5 ± 0.4-fold compared to unloaded micelles (Figure 3). The

300

positive control sulforaphane (10 µM, 4 h) increased the nuclear translocation of Nrf2 by

301

4.9 ± 0.5-fold.

302 303

Induction of Nrf2 target genes by lutein in ARPE-19 cells

304

ARPE-19 cells were incubated with 10 µM lutein (0.02% Tween40) for 4 h or 24 h to analyze the

305

effect on Nrf2 target genes (Figure 4). After treatment with lutein-loaded micelles for 24 h,

306

1.7 ± 0.1-fold higher transcript levels of NQO1 compared to unloaded micelles were detected.

307

Treatment with lutein-loaded micelles increased GCLm-mRNA by 1.6 ± 0.3-fold after 4 h and by


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1.4 ± 0.1-fold after 24 h. In the presence of lutein-loaded micelles, HO-1 mRNA was induced

309

significantly 3.5 ± 0.7-fold after 4 h. After 24 h, the mRNA level was 1.8 ± 0.3-fold higher

310

compared to unloaded micelles. Similar results for the analyzed Nrf2 target genes were obtained

311

after treatment with only 5 µM lutein (0.01% Tween40) (Figure S2). A treatment with 10 µM

312

sulforaphane for 24 h served as positive control. As expected, the mRNA expression of NQO1

313

increased by 6.8 ± 1.3-fold, of GCLm by 2.5 ± 0.2-fold, and of HO-1 by 1.5 ± 0.2-fold in the cells

314

treated with sulforaphane.

315

In addition to transcript levels, we also investigated the effect of lutein on NQO1 activity

316

(Figure 5). Lutein (10 µM, 0.02% Tween40) led to a significant enhancement of activity by

317

1.2 ± 0.1-fold from 113.0 mU/mg protein ± 4.5 mU/mg protein in cells incubated with unloaded

318

micelles to 137.9 mU/mg protein ± 7.0 mU/mg protein in cells treated with lutein-loaded micelles

319

for 24 h. The positive control sulforaphane (10 µM, 24 h) showed a significant increase of

320

specific activity by 2.3 ± 0.03-fold from 99.9 mU/mg protein ± 4.9 mU/mg protein in untreated

321

cells to 225.7 mU/mg protein ± 3.3 mU/mg protein in sulforaphane-treated cells.

322 323

Discussion

324

The Nrf2 pathway regulates the transcription of phase II genes that process and eliminate

325

carcinogens and toxins as well as the transcription of many genes with direct or indirect

326

antioxidant effects such as enzymes related to glutathione or to the thioredoxin metabolism,

327

superoxide dismutase, NQO1, HO-1, and catalase.12,19 In our study, we focused on typical Nrf2

328

target genes with antioxidant effects: GCLm is a subunit of the glutamate-cysteine ligase that

329

catalyzes the rate-limiting step in glutathione synthesis. NQO1 and HO-1 degrade quinones and

330

heme, respectively. Quinones and heme both transfer electrons and are therefore direct sources of 15 ACS Paragon Plus Environment


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331

free radicals.12 The goal of our study was to investigate whether the macular pigment lutein is

332

able to activate Nrf2 and enhances the expression of Nrf2 target genes in ARPE-19 cells.

333

In cell culture, it is often a problem to apply lipophilic carotenoids to the cells. We used a system

334

for delivering lipophilic phytochemicals to ARPE-19 cells which was previously described by

335

Lornejad-Schäfer, et al.30. Lutein-loaded micelles were produced by dissolving lutein in ethanol

336

and using Tween40 as a non-toxic surfactant. In their study, they showed that a Tween40

337

concentration of 0.1% in the culture medium reduces cell viability in a MTT assay, whereas

338

0.01% Tween40 has no damaging effect. Our results confirmed this study. With up to 0.04%

339

Tween40, we observed no reduction in lysosomal integrity or dehydrogenase activity. We next

340

evaluated the cellular lutein uptake to confirm that the used micelles are a suitable delivery

341

system of lutein to the cells. The cellular lutein concentration in the ARPE-19 cells ranged from

342

31 µM ± 8 µM to 62 µM ± 14 µM after incubating the cells with 1-10 µM lutein for 24 h. The

343

incubation concentrations were higher than the usual serum concentrations which can reach

344

2.7 µM after supplementation with 30 mg lutein per day.44 However, the cellular lutein

345

concentration is more crucial for the activation of Nrf2. The concentration of carotenoids in the

346

macular region of the retina is estimated to range between 0.3 mM and 1.3 mM.4 Studies on the

347

carotenoid distribution in the eye described a lower amount of lutein in the underlying RPE. In

348

detail, Sommerburg, et al.45 detected about a sixth and Bernstein, et al.46 about an eighteenth of

349

the amount of lutein in the submacular RPE compared to the macular retina (Sommerburg, et

350

al.45: 0.85 ng/mm2 in the macular retina, 0.15 ng/mm2 in the submacular RPE; Bernstein, et al.46:

351

0.70 ng/mm2 in the macular retina, 0.04 ng/mm2 in the submacular RPE). Thus, the cellular lutein

352

levels determined in our study did not yet reach the highest possible concentrations in the RPE.

353

Nevertheless, the measured cellular lutein concentrations were higher than the applied

354

concentrations indicating an accumulation mechanism. We assume that carotenoid-binding 16 ACS Paragon Plus Environment

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355

proteins are involved in this process. It is known that in the retina glutathione S-transferase pi

356

(GSTP1) serves as a zeaxanthin-binding protein and the steroidogenic acute regulatory domain

357

protein 3 (StARD3) as a lutein-binding protein.47,48 Tubulin is a less specific carotenoid-binding

358

protein of the retina.47 It still has to be determined which carotenoid-binding proteins may play a

359

role in the RPE. Since there is a higher diversity of carotenoids present in the RPE than in the

360

retina,46,49 other carotenoid binding proteins might be involved. Furthermore, our data indicate

361

that lutein uptake could be saturable in ARPE-19 cells, which might be a result of a protein-

362

mediated uptake. It is known that ARPE-19 cells absorb lutein and zeaxanthin via the scavenger

363

receptor class B1 (SR-B1) and the LDL receptor.50-52 In the study of During, et al.52, Tween40

364

micelles were used to deliver zeaxanthin to ARPE-19 cells. Knockdown of SR-B1 led to a

365

decreased uptake of zeaxanthin from the micelles. Thus, it seems that SR-B1 also plays a role

366

when Tween40 micelles are used for delivery.

367

Importantly, this is the first study to show the activation of Nrf2 by lutein in ARPE-19 cells. In

368

addition, we observed an induction of three Nrf2 target genes, namely NQO1, GCLm, and HO-1.

369

Thus, our results broaden our current knowledge about possible mechanisms by which lutein

370

modulates signaling pathways in the RPE. Other studies show that lutein has similar effects in

371

other tissues. For example, lutein led to an activation of Nrf2 in mouse microglial BV-2 cells.

372

The nuclear Nrf2 increased by 1.7-fold and the protein levels of NQO1 were upregulated 4-fold

373

and of HO-1 5-fold after incubation with 50 µM lutein for 4 h.27 Another study showed a nuclear

374

Nrf2 accumulation in mice liver after treatment with 40 mg/kg lutein for 5 weeks leading to a

375

1.6- and 1.4-fold enhancement of NQO1 and HO-1 mRNA, respectively.28 These values are

376

similar to our findings in RPE cells. Other carotenoids are also able to activate Nrf2 in the RPE.

377

Astaxanthin led to an increase (2.3-fold) of nuclear Nrf2 after incubation of ARPE-19 cells with

378

20 µM astaxanthin for 24 h.26 This carotenoid also induced transcript levels of NQO1 by 1.3-fold, 17 ACS Paragon Plus Environment


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of GCLm by 1.9-fold, and of HO-1 by 2-fold.26 Zeaxanthin (10 µM, 6 h) was also observed to

380

increase nuclear Nrf2 in ARPE-19 cells and upregulated Nrf2 target genes even more strongly.

381

The transcript levels of NQO1 increased by 3.7-fold, of GCLm by 3.2-fold, and of HO-1 by 4.5-

382

fold.25 The same study also showed that zeaxanthin induces Nrf2 target genes and GSH levels in

383

rats and reduces the oxidative damage in the retina of these animals.25

384

It was shown that carotenoid degradation products can activate Nrf2.53 Carotenoid degradation

385

products can be formed from chemical or enzymatic degradation. However, initial experiments

386

performed by our group could not detect any degradation of lutein in the medium under our cell

387

culture conditions for 24 h. Therefore, we assume that lutein is not degraded chemically in our

388

experiments. Concerning enzymatic degradation, the enzyme β,β-carotene-9′,10′-dioxygenase

389

(BCO2) is able to cleave lutein. However, Li, et al.54 showed that BCO2 is expressed in the

390

human retina, but the enzyme is inactive. A study with ARPE-19 cells could neither detect the

391

BCO2 protein nor any cleavage products.50

392

The goal of our study was to test whether lutein can activate Nrf2 in a RPE cell line thereby

393

protecting the cells against oxidative stress. Oxidative stress in the RPE plays a major role in the

394

development of AMD in older people.6 Reduction of oxidative damage in the RPE could thus be

395

an important mechanism to protect the overlying retina. How important it is to activate the Nrf2

396

pathway in the RPE in older people by applying phytochemicals like lutein is illustrated by the

397

following studies. Nrf2-deficient mice were observed to develop AMD-like retinal pathology

398

with aging.55 In another study, the induction of Nrf2 target genes, such as NQO1, GCLm, and

399

HO-1, in the RPE following oxidative stress induced with sodium iodate was impaired in older

400

compared to younger mice.10 Yet another study indicated that the HO-1 expression in the RPE

401

seems to decline with aging.56 Consistently, the glutathione redox system is affected by age as the

402

GSH level in the blood decreases.57 Consequently, older people seem to be more susceptible to 18 ACS Paragon Plus Environment

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403

oxidative stress because their defense system is impaired. Thus, it is of considerable interest to

404

increase cellular resistance against oxidative damage by upregulating Nrf2 target genes with

405

antioxidant properties. Of note is that there is evidence that lutein is protective against AMD. In

406

the Age-Related Eye Disease Study 2, participants with low dietary intake of lutein and

407

zeaxanthin prior to the start of the study, but who took lutein and zeaxanthin in the study, showed

408

a lower probability to develop advanced AMD compared to participants with similar dietary

409

intake who did not take additional lutein and zeaxanthin.5 However, by which mechanism lutein

410

protects the retina and how important the activation of Nrf2 is are both issues that remain to be

411

evaluated. In this context, there are already some evidences that lutein can reduce oxidative

412

damage and inflammation in the RPE. For example, lutein was observed to protect the

413

proteasome against photooxidative damage and to modulate the expression of inflammation-

414

related genes like IL-8 in ARPE-19 cells.58 Treatment of human D407 RPE cells with lutein

415

reduced hydrogen peroxide-induced cytotoxicity, ROS generation, and lipid peroxidation. Lutein

416

also reduced hydrogen peroxide-induced decrease in GSH levels and in the activity of superoxide

417

dismutase and glutathione peroxidase.59 However, lutein alone was not able to enhance GSH

418

levels and the aforementioned enzyme activities in unstressed conditions.59 The activation of

419

Nrf2 in RPE cells shown in our study could be another mechanism by which lutein reduces

420

oxidative damage in the RPE, thereby preventing the development of AMD.

421

In addition to carotenoids, other phytochemicals are known to activate Nrf2 in ARPE-19 cells.

422

Our study confirmed that the isothiocyanate sulforaphane upregulates Nrf2 target genes in

423

ARPE-19 cells as also shown in an earlier study of Gao and Talalay20. Moreover, escin (a

424

mixture of triterpene-saponines) was reported to increase nuclear Nrf2 levels and induce NQO1

425

mRNA 9-fold and HO-1 mRNA 17-fold after incubation of ARPE-19 cells with 10 µM escin for

426

2 h. Thus, escin is a stronger inducer than lutein.21 Further, different phenolic compounds are 19 ACS Paragon Plus Environment


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427

known to enhance Nrf2 target genes in the RPE. For example, treatment of ARPE-19 cells with

428

50 µM canolol for 24 h led to a 1.5-fold induction of HO-1 mRNA.22 Incubation of ARPE-19

429

cells with 5 µM 4-acetoxyphenol for 24 h increased nuclear Nrf2 and upregulated NQO1 mRNA

430

3.3-fold and HO-1 mRNA 2.5-fold.23 Finally, the flavonoid eriodictyol induced the nuclear

431

translocation of Nrf2 and enhanced HO-1 protein levels, NQO1 protein levels and glutathione

432

after incubating ARPE-19 cells with eriodictyol at a concentration of 0-100 μM for 2-24 h.24

433

Collectively, these studies show that the Nrf2 pathway is upregulated by various phytochemicals.

434

Further studies are now needed to determine the contribution of individual phytochemicals as

435

well as their synergistic or antagonistic effects.

436

In conclusion, our study demonstrates that lutein activates the Nrf2 pathway in ARPE-19 cells

437

and upregulates the antioxidant system, thereby leading to an enhancement of cellular resistance

438

of RPE cells against oxidative damage. We propose that better understanding of this mechanism

439

may lead to new therapeutic strategies as regards AMD.

440 441

Abbreviations used

442

AMD, age-related macular degeneration; ARE, antioxidant response element; βAct, beta-actin;

443

BCO2, β,β-carotene-9′,10′-dioxygenase; BSA, bovine serum albumin; CRALBP, cellular

444

retinaldehyde binding protein; DMEM, Dulbecco's Modified Eagle Medium; E, amplification

445

efficiency; FCS, fetal calf serum; GCLm, glutamate-cysteine ligase regulatory subunit; GSTP1,

446

glutathione S-transferase P 1; HO-1, heme oxygenase-1; HPRT1, hypoxanthine phosphoribosyl

447

transferase 1; Keap1, Kelch-like ECH-associated protein 1; LDL, low-density lipoprotein; Maf,

448

musculoaponeurotic fibrosarcoma; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

449

bromide; NQO1, NAD(P)H:quinone oxidoreductase 1; Nrf2, nuclear factor erythroid 2-related

450

factor 2; PPIA, peptidylprolyl isomerase A; Prdx1, peroxiredoxin 1; RPE, retinal pigment 20 ACS Paragon Plus Environment

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451

epithelium; RPE65, RPE-specific 65 kDa protein; SOD, superoxide dismutase; SR-B1, scavenger

452

receptor class B1; StARD3, steroidogenic acute regulatory domain protein 3

453 454

Acknowledgements

455

The authors would like to thank the Department of Food Chemistry, S. Deubel, and the

456

Department of Plant Quality and Food Security for their assistance.

457 458

Supporting Information

459

-

Figure S1. Cellular lutein uptake of ARPE-19 cells in pmol/1x106 cells.

460

-

Figure S2. Effect of lutein (5 μM lutein (0.01% Tween40)) on transcript levels of Nrf2

461

target genes.



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(42) Liu, X.; Xie, J.; Liu, Z.; Gong, Q.; Tian, R.; Su, G. Identification and validation of reference

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genes for quantitative RT-PCR analysis of retinal pigment epithelium cells under hypoxia and/or

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hyperglycemia. Gene 2016, 580, 41-46. 26 ACS Paragon Plus Environment

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(43) Müller, M.; Banning, A.; Brigelius-Flohe, R.; Kipp, A. Nrf2 target genes are induced under

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marginal selenium-deficiency. Genes Nutr. 2010, 5, 297-307.

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(44) Bone, R. A.; Landrum, J. T.; Guerra, L. H.; Ruiz, C. A. Lutein and zeaxanthin dietary

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supplements raise macular pigment density and serum concentrations of these carotenoids in

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humans. J Nutr. 2003, 133, 992-998.

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(45) Sommerburg, O.; Siems, W. G.; Hurst, J. S.; Lewis, J. W.; Kliger, D. S.; van Kuijk, F. J. G.

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M. Lutein and zeaxanthin are associated with photoreceptors in the human retina. Curr. Eye Res.

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1999, 19, 491-495.

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(46) Bernstein, P. S.; Khachik, F.; Carvalho, L. S.; Muir, G. J.; Zhao, D. Y.; Katz, N. B.

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Identification and quantitation of carotenoids and their metabolites in the tissues of the human

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eye. Exp. Eye Res. 2001, 72, 215-223.

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(47) Li, B.; Vachali, P.; Bernstein, P. S. Human ocular carotenoid-binding proteins. Photochem.

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Photobiol. Sci. 2010, 9, 1418-1425.

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(48) Li, B.; Vachali, P.; Frederick, J. M.; Bernstein, P. S. Identification of StARD3 as a lutein-

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binding protein in the macula of the primate retina. Biochemistry 2011, 50, 2541-2549.

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(49) Khachik, F.; de Moura, F. F.; Zhao, D. Y.; Aebischer, C.-P.; Bernstein, P. S.

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Transformations of selected carotenoids in plasma, liver, and ocular tissues of humans and in

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nonprimate animal models. Invest. Ophthalmol. Visual Sci. 2002, 43, 3383-3392.

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(50) Thomas, S. E.; Harrison, E. H. Mechanisms of selective delivery of xanthophylls to retinal

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pigment epithelial cells by human lipoproteins. J. Lipid Res. 2016, 57, 1865-1878.

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(51) Sato, Y.; Kondo, Y.; Sumi, M.; Takekuma, Y.; Sugawara, M. Intracellular uptake

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mechanism of lutein in retinal pigment epithelial cells. J. Pharm. Pharm. Sci. 2013, 16, 494-501.



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(52) During, A.; Doraiswamy, S.; Harrison, E. H. Xanthophylls are preferentially taken up

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compared with beta-carotene by retinal cells via a SRBI-dependent mechanism. J. Lipid Res.

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2008, 49, 1715-1724.

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(53) Gerhäuser, C.; Klimo, K.; Hummer, W.; Holzer, J.; Petermann, A.; Garreta-Rufas, A.;

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Bohmer, F. D.; Schreier, P. Identification of 3-hydroxy-beta-damascone and related carotenoid-

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derived aroma compounds as novel potent inducers of Nrf2-mediated phase 2 response with

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concomitant anti-inflammatory activity. Mol. Nutr. Food Res. 2009, 53, 1237-1244.

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(54) Li, B.; Vachali, P. P.; Gorusupudi, A.; Shen, Z.; Sharifzadeh, H.; Besch, B. M.; Nelson, K.;

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Horvath, M. M.; Frederick, J. M.; Baehr, W.; Bernstein, P. S. Inactivity of human beta,beta-

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carotene-9',10'-dioxygenase (BCO2) underlies retinal accumulation of the human macular

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carotenoid pigment. Proc. Natl. Acad. Sci. U. S. A. 2014, 111, 10173-10178.

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(55) Zhao, Z.; Chen, Y.; Wang, J.; Sternberg, P.; Freeman, M. L.; Grossniklaus, H. E.; Cai, J.

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Age-related retinopathy in NRF2-deficient mice. PLoS One 2011, 6, e19456.

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(56) Miyamura, N.; Ogawa, T.; Boylan, S.; Morse, L. S.; Handa, J. T.; Hjelmeland, L. M.

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Topographic and age-dependent expression of heme oxygenase-1 and catalase in the human

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retinal pigment epithelium. Invest. Ophthalmol. Visual Sci. 2004, 45, 1562-1565.

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(57) Erden-Inal, M.; Sunal, E.; Kanbak, G. Age-related changes in the glutathione redox system.

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Cell Biochem. Funct. 2002, 20, 61-66.

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Gierhart, D.; Shang, F. Lutein and zeaxanthin supplementation reduces photooxidative damage

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(59) Pintea, A.; Rugină, D. O.; Pop, R.; Bunea, A.; Socaciu, C. Xanthophylls protect against

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2011, 24, 830-836.

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Figure captions

628 629

Figure 1. Effect of Tween40 micelles without (T40) and with (L) lutein on (I) lysosomal

630

integrity and (II) dehydrogenase activity. ARPE-19 cells were treated with different micelles

631

concentrations for 24 h. Cell viability was measured using (I) neutral red- and (II) resazurin-

632

based assays. Lysosomal integrity and dehydrogenase activity of untreated control cells (C) were

633

set at 100% (dashed line). Values (%) are expressed as mean ± SD (n = 5: untreated cells, 0.001-

634

0.02% Tween40; n = 4: 0.04-0.08% Tween40). Significant difference (p ≤ 0.05) was determined

635

compared to untreated control: * micelles without lutein, # micelles with lutein.

636 637

Figure 2. Cellular lutein uptake of ARPE-19 cells. Cells were incubated with lutein-loaded

638

micelles for 24 h. Lutein was extracted from cells and quantified by LC-ToF-MS. Values are

639

presented as mean ± SD (n = 3).

640 641

Figure 3. Effect of lutein on nuclear Nrf2 protein levels. ARPE-19 cells were treated with (I)

642

10 µM lutein (0.02% Tween40) (L) or (II) 10 µM sulforaphane (SFN) for 4 h. Cells which were

643

treated with unloaded micelles (T40) or water-treated (C) served as controls. Nuclear cell

644

fractions were isolated. Nrf2 protein content was analyzed and normalized to histone H2B by

645

western blot analysis. The control cells were set at 1. Values are expressed as mean ± SD (I: n =

646

6; II: n = 3). Significant difference (* p ≤ 0.05, ** p ≤ 0.01) was determined compared to control

647

cells.

648 649

Figure 4. Effect of lutein on transcript levels of Nrf2 target genes. ARPE-19 cells were incubated

650

with 10 µM lutein (0.02% Tween40) (L) for 4 h and 24 h or with 10 µM sulforaphane (SFN) for 30 ACS Paragon Plus Environment

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651

24 h. The mRNA levels of (I) NQO1, (II) GCLm and (III) HO-1 were analyzed by RT-qPCR. All

652

values were normalized to the expression of the gene of interest in control cells (C, represented

653

by a dashed line): with unloaded micelle-treated cells for lutein-loaded micelles, water-treated

654

control cells for sulforaphane. Data are shown as mean ± SD (n = 3). Significant difference (* p

655

≤ 0.05, ** p ≤ 0.01) was determined compared to control cells.

656 657

Figure 5. Effect of lutein on relative NQO1 activity. ARPE-19 cells were treated with (I) 10 µM

658

lutein (0.02% Tween40) (L) or (II) 10 µM sulforaphane (SFN) for 24 h. Cells that were treated

659

with unloaded micelles (T40) or water (C) served as controls. NQO1 activity was analyzed by

660

measuring MTT reduction. Activities were determined as specific activities (mU/mg protein) and

661

normalized to control cells. Data are shown as mean ± SD (n = 3). Significant difference (** p

662

≤ 0.01, *** p ≤ 0.001) was determined compared to control cells.

663 664



Figure graphics

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