International Journal of Molecular Sciences

Article Free Radical Scavenging andand CellularCellular AntioxidantAntioxidant Properties of Astaxanthin

Janina DoseDose 11, Seiichi Matsugo 2,, Haruka Haruka Yokokawa Yokokawa 2,, Yutaro Yutaro Koshida Koshida 2,, Shigetoshi Shigetoshi Okazaki Okazaki 33, , Ulrike Seidel 1,, Manfred Eggersdorfer 4,, Gerald Gerald Rimbach Rimbach 11 andand Tuba Tuba Esatbeyoglu Esatbeyoglu 1,1,**

Received: 12 October 2015; Accepted: 8 January 2016; Published: January 14 January 2016 2016 Academic Editor: Esra Capanoglu

11 InstituteInstitute of of Human Human Nutrition Nutrition and and Food Food Science, Science, University University of of Kiel, Kiel, Hermann-Rodewald-Straße Hermann-Rodewald-Straße 6, 6, D-24118D-24118 Kiel, Kiel, Germany; Germany; dose@foodsci [email protected] (J.D.); (J.D.); seidel [email protected]@foodsci.uni-kiel.de (U.S.); (U.S.); [email protected]@foodsci.uni-kiel.de (G.R.) (G.R.) 2 SchoolSchool of of Natural Natural System, System, Kanazawa Kanazawa Univer University,sity, Kakuma-machi, Kakuma-machi, Kanazawa Kanazawa 920-1192, 920-1192, Japan; Japan; [email protected]@se.kanazawa-u.ac.jp (S.M.); (S.M.); haruside [email protected]@gmail.com (H.Y.); (H.Y.); [email protected] [email protected] (Y.K.) (Y.K.) 3 MedicalMedical Photonics Photonics Research Research Center, Center, Hamamatsu Hamamatsu Univ Universityersity School School of of Medicine, Medicine, Handamachi Handamachi 1-20-1, 1-20-1, Higashi-ku,Higashi-ku, Hamamatsu, Hamamatsu, Shizuoka Shizuoka 431- 431-3192,3192, Japan; Japan; [email protected] [email protected] 4 DSMDSM Nutritional Nutritional Products, Products, P.O. P.O. Box Box 2676, 2676, 4002 4002 Base Basel,l, Switzerland; Switzerland; [email protected] [email protected] ** Correspondence:Correspondence: esatbeyoglu@f [email protected];oodsci.uni-kiel.de; Tel.: Tel.: +49-431-880-5333 +49-431-880-5333

Abstract: Astaxanthin is a coloring agent which is used as a feed additive in aquaculture nutrition. Recently, potentialpotential healthhealth benefitsbenefits ofof astaxanthinastaxanthin have been discusseddiscussed which may be partly related to its its free free radical radical scavenging scavenging and and antioxidant antioxidant properties. properties. Our Our electron electron spin spin resonance resonance (ESR) (ESR) and andspin spintrapping trapping data suggest data suggest that synthetic that synthetic astaxanthi astaxanthinn is a potent is a free potent radical free scavenger radical scavenger in terms inof termsdiphenylpicryl-hydrazyl of diphenylpicryl-hydrazyl (DPPH) (DPPH)and galvinox and galvinoxylyl free freeradicals. radicals. Furthermore, Furthermore, astaxanthinastaxanthin dose-dependently quenched singlet oxygen as determined by photon counting. In addition to free radical scavenging and singlet oxygenoxygen quenchingquenching properties, astaxanthin induced the antioxidantantioxidant paroxoanase-1, enhanced enhanced glutathione glutathione conc concentrationsentrations and and prevented prevented lipid lipid peroxidation peroxidation in incultured cultured hepatocytes. hepatocytes. Present Present results results suggest suggest th that,at, beyond beyond its its coloring coloring properties, syntheticsynthetic astaxanthin exhibits free radicalradical scavenging,scavenging, singlet oxygen quenching,quenching, and antioxidantantioxidant activitiesactivities which couldcould probablyprobably positivelypositively affectaffect animalanimal andand humanhuman health.health.

Keywords: astaxanthin; free radical scavenging;scavenging; antioxidant; electron spinspin resonanceresonance spectroscopyspectroscopy

1. Introduction Introduction 1 1 1 Astaxanthin (3,3 ′-dihydroxy--dihydroxy-ββ,β′,β-carotene-4,4-carotene-4,4′-dione,-dione, for for chemical chemical structure structure see see Figure Figure 1)1 )is is a axanthophyll xanthophyll carotenoid carotenoid [1] [1 ]which which naturally naturally occurs occurs in in algae, algae, krill, krill, trout, trout, crayfish, crayfish, and and salmon. Astaxanthin isis widely usedused in aquaculture nutritionnutrition asas aa coloringcoloring agentagent [[2].2]. In addition to its coloring properties, astaxanthinastaxanthin maymay alsoalso affectaffect immuneimmune statusstatus andand reproductionreproduction [[3,4].3,4].

Figure 1. Chemical structure of astaxanthin.

Astaxanthin has has two two chiral chiral centers centers at positions at positions 3 and 3 3and'. The 3'. astaxanthin The astaxanthin stereoisomer stereoisomer -3S,3S1- is-3S the,3S main′- is formthe main found form in wild found salmon in wild [5]. salmon Most astaxanthin [5]. Most asta usedxanthin in aquaculture used in aquaculture nutrition is produced nutrition syntheticallyis produced whichsynthetically yields three which different yields stereoisomers, three different including stereoisomers,3S, 31S; 3R including, 31S; and 3R 3S,,3 13R′S[1; ].3R In, 1981,3′S; and Widmer 3R, 3et′R al. [1].[6] In 1981, Widmer et al. [6] synthesized astaxanthin from the educt 6-oxo-isophorone

Int. J. Mol. Sci. 2016, 17, 103; doi:10.3390/ijms17010103 www.mdpi.com/journal/ijmswww.mdpi.com/journal/ijms Int. J. Mol. Sci. 2016, 17, 103 2 of 14 synthesized astaxanthin from the educt 6-oxo-isophorone (3,5,5-trimethyl-2-cyclohexene-1,4-dione) via a Int. J. Mol. Sci. 2016, 17, 103 2 of 13 seven-step synthesis. Over a Wittig reaction of two equivalents C15-phosphonium salt with C10-dialdehyde, astaxanthin(3,5,5-trimethyl-2-cyclohexene-1,4-dione) was obtained with yields up to 50% via yieldsa seve [n-step6]. synthesis. Over a Wittig reaction of two Astaxanthinequivalents C15-phosphonium has also significant salt with applications C10-dialde inhyde, the nutraceuticalastaxanthin was industry obtained[ 7with]. Various yields up health benefits,to 50% including yields [6]. cardiovascular disease and cataract prevention, have been associated with astaxanthinAstaxanthin consumption has also [3,8 significant–10]. applications in the nutraceutical industry [7]. Various health Althoughbenefits, including it has been cardiovascular suggested thatdisease potential and cataract health prevention, benefits of astaxanthinhave been associated are, at least with partly, astaxanthin consumption [3,8–10]. mediated by its free radical scavenging [11], antioxidant [12,13], and -regulatory properties [14,15], Although it has been suggested that potential health benefits of astaxanthin are, at least partly, systematicmediated studies by its are free scarce. radical scavenging [11], antioxidant [12,13], and gene-regulatory properties In[14,15], previous systematic studies, studies free radicalare scarce. scavenging activity of astaxanthin has been determined by FRAP (ferric reducingIn previous antioxidant studies, free power) radical [16 scavenging], TEAC (trolox activity equivalent of astaxanthin antioxidant has been capacity)determined [17 by], and ORACFRAP (oxygen (ferric radical reducing absorbance antioxidant capacity) power) [16], [18] assays,TEAC (trolox respectively. equivalent Furthermore, antioxidant thecapacity) decolorization [17], of theand 2,2-diphenyl-1-picrylhydrazyl ORAC (oxygen radical absorbance (DPPH) radicalcapacity) may [18] be assays, used to respectively. assess the free Furthermore, radical scavenging the activitydecolorization of antioxidants, of the taking 2,2-diphenyl-1-picrylhydrazyl into account that alcohols (DPPH) as solvents radical may may be lead used to to an assess overestimation the free of its freeradical radical scavenging scavenging activity activity of antioxidants, [19–21]. The taking different into account antioxidant that alcohols test systems, as solvents as describedmay lead to in the literature,an overestimation differ in terms of ofits underlyingfree radical methodology,scavenging activity reagents, [19–21]. wavelength, The different pH, antioxidantetc. It is suggested test systems, as described in the literature, differ in terms of underlying methodology, reagents, that a combination of various assays should be used in assessing antioxidant activities in vitro [22]. wavelength, pH, etc. It is suggested that a combination of various assays should be used in assessing Inantioxidant addition activities to these ratherin vitro indirect [22]. methods, electron spin resonance spectroscopy (ESR) combined with spinIn trapping addition has to beenthese establishedrather indirect as amethods, robust method electron for spin the resonance direct assessment spectroscopy of free (ESR) radical reactionscombined [23]. with Therefore, spin trapping in this has study, been weestablished applied as ESR, a robust as wellmethod as photonfor the direct counting assessment methods, of to determinefree radical the free reactions radical [23]. scavenging Therefore, and in singletthis study, oxygen we applied quenching ESR, activity as well ofas astaxanthin.photon counting Furthermore,methods, to determine we studied the the free cellular radical antioxidant scavenging activityand singlet of astaxanthin oxygen quenching in cultured activity hepatocytes. of We addressedastaxanthin. the question, if and to what extend astaxanthin may induce enzymatic antioxidant defense mechanisms,Furthermore, includingwe studied paraoxoanase-1. the cellular antioxidant Furthermore, activity we determinedof astaxanthin cellular in cultured glutathione levelshepatocytes. in response We to addressed an astaxanthin the question, treatment, if and since to what glutathione extend astaxanthin is the most may important induce enzymatic endogenous antioxidant defense mechanisms, including paraoxoanase-1. Furthermore, we determined cellular cytosolic antioxidant centrally involved in redox signaling and stress response. glutathione levels in response to an astaxanthin treatment, since glutathione is the most important Overall,endogenous this cytosolic manuscript antioxidant aims at centrally providing invo comprehensivelved in redox signaling information and stress in terms response. of the free radical scavenging,Overall, antioxidant, this manuscript and cell aims signaling at providing modifying comprehensive properties ofinformation astaxanthin. in terms of the free radical scavenging, antioxidant, and cell signaling modifying properties of astaxanthin. 2. Results and Discussion We2. Results applied and ESR Discussion and spin trapping in order to directly quantify the free radical scavenging activity ofWe astaxanthin. applied ESR Astaxanthin and spin trapping dose-dependently in order to directly scavenged quantify DPPH the (Figurefree radical2A) andscavenging galvinoxyl (Figureactivity2B) free of astaxanthin. radicals. However, Astaxanthin astaxanthin dose-dependently did not scavengescavenged superoxideDPPH (Figure anion 2A) and free galvinoxyl radicals (data not shown).(Figure 2B) Accordingly, free radicals. astaxanthin However, didastaxanthin not inhibit did xanthinenot scavenge oxidase, superoxide which anion generates free radicals superoxide anion(data free radicalsnot shown). (Figure Accordingly,2C). When astaxanthin singlet oxygen did not is inhibit relaxed to the ground oxidase, state, which oxygen generates photon superoxide anion free radicals (Figure 2C). When singlet oxygen is relaxed to the ground state, emission is observed. Singlet oxygen quenching activity of astaxanthin as a function of emission oxygen photon emission is observed. Singlet oxygen quenching activity of astaxanthin as a function spectrum,of emission concentration, spectrum, andconcentration, decay curve and isdecay given curv ine Figure is given2D. in AsFigure shown 2D. As in Figureshown 2inD, Figure astaxanthin 2D, dose-dependentlyastaxanthin dose-dependently quenched singlet quenched oxygen singlet as determined oxygen as determined by photon by counting. photon counting.

140 DPPH radical A Control 120 25

50 100 100

80 150

200 60 250 40 300

20 400 ESR signal intensity (% (% control) of intensity signal ESR 500 0 600 Control0.00 0.03 25 0.0550 0.10100 0.15150 0.20200 0.25250 0.30300 0.40400 0.50500 0.60600 0.75750 750 Astaxanthin [µM] Astaxanthin [µM]

Figure 2. Cont. Figure 2. Cont. Int. J. Mol. Sci. 2016, 17, 103 3 of 14 Int. J. Mol. Sci. 2016, 17, 103 3 of 13

140 Galvinoxyl radical B Control 120 5

100 10

80 25

60 50

40 100

20 150 ESR signalintensity (% of control)

0 200 Control0 0.005 5 0.0110 0.025 25 0.0550 100 0.1 0.15150 200 0.2 0.25250 250 Astaxanthin [µM] Astaxanthin [µM] 100 C 90 80 70 60 50 40 30 20

Xanthine oxidase inhibition[%] 10 0 0 0.12 0.24 0.49 0.98 1.95 3.91 7.81 16 31 62.5 125 0 108 432 866 1731 3463

Allopurinol Astaxanthin [µM] [µM] 3 35x10 2 D Astaxanthin Astaxanthin 0 M 0M 30 -7 -7 1000 5.4*10 M 5.4*10 M 8 -6 -6 7 1.1*10 M 1.1*10 M -6 25 -6 6 2.1*10 M 2.1*10 M 5 -6 -6 4.2*10 M 4.2*10 M 4 -6 -6 8.1*10 M 20 8.1*10 M 3

2 15 Intensity unit][arb. Intensity [arb. unit] [arb. Intensity 10 100 8 7 6 5 5 4 0 3

1100 1150 1200 1250 1300 1350 1400 0 20 40 60 80 6 Wavelength [nm] Time [sec x 10 ]

Figure 2. Scavenging effects of astaxanthin on DPPH radical (A), galvinoxyl radical (B), xanthine Figure 2. A B oxidaseScavenging inhibition ( effectsC), and ofquenching astaxanthin of singlet on DPPH oxygen radical (D). (A ( ) ),The galvinoxyl reaction mixture radical contained ( ), xanthine oxidase500 inhibitionμM DPPH (Cand), andthe given quenching concentration of singlet of oxygenastaxant (hin.D). (AllA) values The reaction are means mixture + SD contained(experiments 500 µM DPPHperformed and the givenin triplicate); concentration (B) Various of astaxanthin.astaxanthin concentr All valuesations are were means mixed + SDwith (experiments 500 μM galvinoxyl. performed in triplicate);Changes in (B the) Various radical astaxanthinsignal intensity concentrations are shown on werethe right mixed side withof the 500figure.µM All galvinoxyl. values are Changesmeans in the radical+ SD (three signal independent intensity are experiments shown on performed the right sidein triplicate); of the figure. (C) The All reaction values mixture are means contained + SD (three independent5 U/mL experimentsxanthine oxidase performed in 50 inmM triplicate); potassium (C )phosphate The reaction buffer mixture and containedthe given5 astaxanthin U/mL xanthine oxidaseconcentrations. in 50 mM potassium Allopurinol phosphate was usedbuffer as a positive and the control. given astaxanthin All values concentrations.are means + SD Allopurinol(three was usedindependent as a positive experiments control. performed All values in are triplicate); means + SD(D) (three Singlet independent oxygen quenching experiments activity performed of astaxanthin as a function of concentration (5.4 × 10−7, 1.1 × 10−6, 2.1 × 10−6, 4.2 × 10−6, and 8.1 × 10−6 M in triplicate); (D) Singlet oxygen quenching activity of astaxanthin as a function of concentration astaxanthin), wavelength (left), and time (right). Photo emission was determined by a photon (5.4 ˆ 10´7, 1.1 ˆ 10´6, 2.1 ˆ 10´6, 4.2 ˆ 10´6, and 8.1 ˆ 10´6 M astaxanthin), wavelength (left), and complex after laser irradiation at 532 nm in the presence of the test sample. Two independent time (right). Photo emission was determined by a photon complex after laser irradiation at 532 nm in experiments performed in duplicate. the presence of the test sample. Two independent experiments performed in duplicate. In our cell culture studies, astaxanthin did not exhibit any significant cytotoxicity in Inconcentrations our cell culture of up studies, to 20 astaxanthinμM in Huh7, did PON1-Huh7, not exhibit anyand significantHepG2 as cytotoxicitysummarized inin concentrationsFigure 3. of upTritonX to 20 µ wasM in used Huh7, as a PON1-Huh7,control to induce and cytotoxi HepG2city as as summarized determined by in the Figure neutral3. TritonX red assay. was used as a control to induce cytotoxicity as determined by the neutral red assay. Int. J. Mol. Sci. 2016 17 Int. J. Mol. Sci. 2016,, 17,, 103 103 44 ofof 1413

120 140 120 120 100 120 100 100 80 80 80 60 60 60 40 40 (% of control) (% of(% control)

40 of(% control) 40 (% of control) 40 (% of control) (% of(% control) (% of(% control) (% of control) 40 Huh7 cell viability viability cell Huh7 Huh7 cell viability viability cell Huh7 HepG2 cellviability 20 20 HepG2 cellviability 20 PON1-Huh7 cell viability viability cell PON1-Huh7 20 PON1-Huh7 cell viability viability cell PON1-Huh7 PON1-Huh7 cell viability viability cell PON1-Huh7 PON1-Huh7 cell viability viability cell PON1-Huh7 0 0 0 THF Triton X 20 µM THF Triton X 20 µM THF Triton X 20 µM

Figure 3. Effects of astaxanthin on cell viability in Huh7, PON1-Huh7, and HepG2 cells after 24 h incubation. Data are means + SD SD of of at leas leastt two experiments performed in triplicate.

Supplementation of of cultured cultured PON1-Huh7 PON1-Huh7 cells cells with with 20 20 μµMM synthetic synthetic astaxanthin astaxanthin resulted resulted in in a amoderate moderate induction induction of of paraoxoanse-1 paraoxoanse-1 (PON1) (PON1) (F (Figureigure 44A).A). Furthermore,Furthermore, syntheticsynthetic astaxanthinastaxanthin dose-dependently increasedincreased cellular cellular glutathione glutathione (GSH) (GSH) levels levels in HepG2 in HepG2 cells (Figure cells (Figure4B). The 4B). increase The inincrease cellular in GSH cellular was GSH not accompaniedwas not accompanied by an increase by an in increase Nrf2 transactivation in Nrf2 transactivation as shown inas Figureshown4 C.in CurcuminFigure 4C. andCurcumin resveratrol and resveratrol were used aswere positive used controls,as positive respectively. controls, respectively.

A B C 550 3500 160 550 3500 *** 140 *** 450 3000 *** 450 120 120 350 2500 100 2000 40 2000 HepG2 cells] 40 HepG2 cells] 6 80

6 80 1500 30 1500 60 30 * Glutathione 60 * Glutathione

[f-luc/] 1000 20 [f-luc/protein] [firefly/renilla ratio] [firefly/renilla 20

1000 40 transactivation Nrf2 [firefly/renilla ratio] [firefly/renilla

40 transactivation Nrf2 PON1 transactivation [µmoL/1*10 PON1 transactivation 500 20 10 500 [µmoL/1*10 20 10 0 0 0 0 0 THF 120 THF 1525 THF 12020 THF 12020 THF 1525 THF 12020 Astaxanthin Res Astaxanthin Curc Astaxanthin Curc Astaxanthin Res Astaxanthin Curc [µM] [µM] [µM] [µM] [µM] [µM] [µM] [µM] [µM] [µM]

Figure 4. Effects of astaxanthin on transactivation of paraoxonase-1 (PON1) (A), cellularcellular glutathioneglutathione (GSH) levels (B) andand transactivationtransactivation of Nrf2Nrf2 ((C)) inin culturedcultured hepatocytes.hepatocytes. (A) PON1-Huh7 cells were 6 6 ˝ seeded atat aa density density of of 0.15 0.15ˆ ×10 10cells/well6 cells/well into into 24 24 well well plates plates and an incubatedd incubated for 24for h 24 at 37h atC. 37 Cells °C. Cells were treatedwere treated with 1 andwith 20 1µ Mand astaxanthin. 20 μM astaxanthin. PON1 transactivation PON1 transactivation was measured was after measured 48 h incubation after of48 the h cellsincubation with synthetic of the cells astaxanthin. with synthe Curcumintic astaxanthin. (Curc; 20CurcuminµM) was (Curc; usedas 20 a μ positiveM) was control;used as ( Ba )positive HepG2 6 6 cellscontrol; were (B seeded) HepG2 at acells density were of seeded 0.15 ˆ 10at acells/well density of into 0.15 24 × well 106 platescells/well and incubatedinto 24-well for plates 24 h.Cells and wereincubated treated for with24 h.1 Cells and were 5 µM treated astaxanthin with 1 and and incubated 5 μM astaxanthin for an additional and incubated 24 h. Resveratrolfor an additional (Res; 6 2524 µh.M) Resveratrol was used (Res; as a positive 25 μM) control;was used (C as) Huh7 a positive cells werecontrol; seeded (C) Huh7 at a density cells were of 0.15 seededˆ 10 atcells/well a density 6 ˝ intoof 0.15 24 well× 106 plates cells/well and incubatedinto 24 well for plates 24 h at and 37 incubatedC. Cells were for 24 transfected h at 37 °C. for Cells 24 h. were Nrf2 transfected transactivation for µ was24 h. measured Nrf2 transactivation after 24 h incubation was measured of the afte cellsr with24 h 1incubation and 20 Mof astaxanthin.the cells with Curcumin 1 and 20 (Curc; μM µ 20astaxanthin.M) was usedCurcumin as a positive (Curc; 20 control. μM) was All valuesused as are a positive means + control. SEM (two All independent values are means experiments + SEM performed(two independent in triplicate), experiments statistical performed significant in differences triplicate), betweenstatistical THF-control significant cellsdifferences and astaxanthin between p p supplementedTHF-control cells cells and are astaxanthin indicated assupp * lemented< 0.05, *** cells< are 0.001; indicated one-way as * ANOVA p < 0.05, LSD*** p (Figure< 0.001; 4one-wayA) and Dunnett-T (Figure4B). ANOVA LSD (Figure 4A) and Dunnett-T (Figure 4B). Additionally, mRNA levels of Gclc (glutamate-cysteine , catalytic subunit) and other Additionally, mRNA levels of Gclc (glutamate-cysteine ligase, catalytic subunit) and other Nrf2-target , including Gsta (glutathione S- alpha 1), Gpx (glutathione peroxidase), Nrf2-target genes, including Gsta (glutathione S-transferase alpha 1), Gpx (glutathione peroxidase), Hmxo1 (heme oxygenase 1), Nqo1 (NAD(P)H dehydrogenase [quinone] 1), Sod1 (), Hmxo1 (heme oxygenase 1), Nqo1 (NAD(P)H dehydrogenase [quinone] 1), Sod1 (superoxide and Sod2, were not significantly changed due to the astaxanthin treatment (Table1). dismutase), and Sod2, were not significantly changed due to the astaxanthin treatment (Table 1). Int. J. Mol. Sci. 2016, 17, 103 5 of 14 Int. J. Mol. Sci. 2016, 17, 103 5 of 13

Table 1.1. Nrf2-targetNrf2-target in astaxanthin treated Huh7 cells (THF control cells were set to be 1.0).

AstaxanthinAstaxanthin [µM] [µM] GeneGene 1201 20 Gclc 0.91 ± 0.08 1.03 ± 0.26 Gclc 0.91 ˘ 0.08 1.03 ˘ 0.26 GstaGsta 1.011.01 ˘± 0.150.15 0.99 0.99 ±˘ 0.180.18 Gpx1Gpx1 1.341.34 ˘± 0.240.24 1.12 1.12 ±˘ 0.300.30 Hmxo1Hmxo1 0.400.40 ˘± 0.040.04 0.85 0.85 ±˘ 0.320.32 Nqo1Nqo1 1.211.21 ˘± 0.180.18 1.32 1.32 ±˘ 0.210.21 Sod1 1.22 ˘ 0.21 0.99 ˘ 0.18 Sod1 1.22 ± 0.21 0.99 ± 0.18 Sod2 1.35 ˘ 0.18 1.39 ˘ 0.54 Sod2 1.35 ± 0.18 1.39 ± 0.54

HepG2 cells were stressed with cumenecumene hydroperoxide/heminhydroperoxide/hemin to to provoke provoke lipid lipid peroxidation. peroxidation. We appliedapplied thethe BODIPYBODIPY assayassay inin orderorder toto determinedetermine lipidlipid peroxidationperoxidation in our HepG2 hepatocytes following the astaxanthin treatment. The The BODIPY BODIPY probe probe exhibits exhibits a a change in fluorescence fluorescence after interaction with peroxyl groups. Under the conditions investigated, supplementation of HepG2 cells with 10 and 20 µμM astaxanthin significantlysignificantly decreaseddecreased lipidlipid peroxidationperoxidation inin HepG2HepG2 cellscells (Figure(Figure5 ).5).

0.7 0.6 ** 0.5 0.4 0.3

Ox/(Ox+Red) 0.2 0.1

0.0 Control 10 20 Astaxanthin [µM] Figure 5. Impact of astaxanthin on the ability of human HepG2 cells to prevent cumene Figure 5. Impact of astaxanthin on the ability of human HepG2 cells to prevent cumene hydroperoxide/hemin stimulated lipid peroxidation. HepG2 cells were seeded at a density of hydroperoxide/hemin stimulated lipid peroxidation. HepG2 cells were seeded at a density of 0.1 ˆ 106 0.1 × 106 cells/well into a 96-well plate and incubated for 24 h. Cells were treated with two cells/well into a 96-well plate and incubated for 24 h. Cells were treated with two concentrations of concentrationsastaxanthin (10 of and astaxanthin 20 µM) for (10 an and additional 20 μM) 24for h.an Cells additional were rinsed 24 h. Cells and loadedwere rinsed with C11-BODIPYand loaded with for C11-BODIPY30 min and then for exposed30 min and to cumene then exposed hydroperoxide to cumene (8 mM)/heminhydroperoxide (8 µ(8M) mM)/hemin for 1 h. Control (8 μM) cells for were 1 h. Controltreated withcells cell were culture treated medium with only.cell Simultaneously,culture medium basal only. levels Simultaneously, of lipid peroxidation basal levels were assessedof lipid peroxidationfor each treatment were concentrationassessed for (dataeach treatment not shown) co andncentration control. Lipid(data peroxidationnot shown) wasand determinedcontrol. Lipid by peroxidationexamining the was ratio determined of green by (oxidized) examining and the theratio sum of green of green (oxidized) and red and ( oxidizedthe sum of + non-oxidizedgreen and red) (oxidizedemissions + usingnon-oxidized) a fluorometric emissions plate using reader. a fluorometric Data are plate means reader. + SD Data of atare least means two + SD independent of at least twoexperiments independent performed experiments in duplicate. performed ** p in< 0.01,duplicate. one-way ** p ANOVA< 0.01, one-way Games–Howell. ANOVA Games–Howell.

In thethe present present study, study, we observedwe observed a dose-dependent a dose-dependent free radical free scavenging radical scavenging activity of astaxanthinactivity of astaxanthinin terms of DPPH in terms and of galvinoxyl DPPH and free galvinoxyl radicals free as determined radicals as bydetermined ESR and spin by ESR trapping. and spin Current trapping. ESR Currentdata regarding ESR data the regarding free radical the scavenging free radical activity scavenging of astaxanthin activity of in astaxanthin terms of DPPH in terms free radicalsof DPPH are free in radicalsline with are literature in line data with [24 literat,25], whereure data different [24,25], photometric where different assay systemsphotometric were assay applied, systems e.g., DPPH, were applied,ferric reducing e.g., DPPH, antioxidant ferric reducing power and antioxid hydroxylant power radical and scavenging. hydroxyl radical scavenging. The freefree radicalradical scavenging scavenging activity activity of astaxanthinof astaxant ishin suggested is suggested to be mediatedto be mediated by electron by electron transfer, transfer,radical adduct radical formation adduct formation and hydrogen and atomhydrogen transfer atom [26 transfer]. Singlet [26]. oxygen Singlet quenching oxygen ofquenching carotenoids, of carotenoids,including astaxanthin including isastaxanthin mediated byis mediated energy transfer by energy between transfer the between electrophilic the electrophilic singlet oxygen singlet and oxygenthe polyene and the backbone. polyene Inbackbone. general, In the general, singlet the oxygen singlet quenching oxygen quenching activity of activity carotenoids of carotenoids increases withincreases increasing with increasing length of conjugationlength of conjugation [27]. Furthermore, [27]. Furthermore, our BODIPY our data BODIPY suggest data that suggest astaxanthin that astaxanthinsignificantly significantly prevents lipid prevents peroxidation. lipid peroxidati The preventionon. The of lipidprevention peroxidation of lipid due peroxidation to astaxanthin due may to astaxanthin may improve the sensory quality and shelve life of fish which warrants further investigation. However, it needs to be taken into account that the astaxanthin concentrations, as Int. J. Mol. Sci. 2016, 17, 103 6 of 14 improve the sensory quality and shelve life of fish which warrants further investigation. However, it needs to be taken into account that the astaxanthin concentrations, as administered in our cell culture assays, may be higher than astaxanthin concentrations in fish plasma [28]. We did not observe any inhibition of in response to the astaxanthin treatment. Thus, unlike other antioxidants including flavonoids [29,30], astaxanthin does not seem to mediate antioxidant activity via xanthine oxidase inhibition. For our cell studies, astaxanthin was dissolved in tetrahydrofuran (THF). We used THF since it has been previously shown by our group to be non-cytotoxic to HepG2 cells [31]. Furthermore, astaxanthin dissolved in THF exhibits an appropriate stability in the cell culture medium [31] and is effectively taken up by HepG2 and HT29 cultured cells [31,32]. Current cell culture data clearly suggest that synthetic astaxanthin does not only work as a free radical scavenger, but also as an inductor of cellular antioxidant defense mechanisms, including PON1 and reduced glutathione. PON1 is a hepatic enzyme which prevents and/or delays the oxidation of LDL [33]. Thus, the prevention of plasma lipid peroxidation in humans due to astaxanthin, as reported by Barlic and co-workers [34], may be partly mediated by an induction of paraoxonase-1. Recently, it has been shown that PON1 also counteracts oxidative stress induced by mercury chloride, thereby offering a beneficial strategy against HgCl2 toxicity [35] which may be of particular interest for fish nutrition. We also determined the cellular activity of natural astaxanthin which was isolated from Haematococcus pluvialis (data not shown). Importantly, natural source astaxanthin did not exhibit a superior bioactivity in any of our test systems as also stated by others [36]. In terms of PON1, a transactivation occurred only in response to synthetic astaxanthin. Synthetic astaxanthin dose-dependently increased cellular GSH levels in HepG2 cells which is in line with findings by Saw and co-workers who found an induction of cellular GSH due to both astaxanthin and omega-3 fatty acids [13]. Interestingly, in our study the increase in cellular GSH in HepG2 cells was, however, not accompanied by an increase in Nrf2 transactivation. Furthermore, Gclc, the rate limiting enzyme of GSH synthesis, was not induced by astaxanthin. Additionally, steady state levels of other Nrf2 target genes, including Gsta, Gpx, Hmxo, Nqo1, Sod1, and Sod2 were not induced in response to the astaxanthin treatment. Thus, it is suggested that the increase in cellular GSH due to astaxanthin was not mediated by an Nrf2 dependent signal transduction pathway. It is worth mentioning that GSH synthesis is not only regulated by Nrf2, but also by other transcription factors, including activator protein 1 (AP-1) [37,38], specificity protein 1 (Sp1) [39,40], nuclear respiratory factor 1 (Nrf1) [41,42], and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) [42,43]. Based on the present data, it is suggested that elevated GSH concentration may be a sign of lower oxidative stress levels as a result of the astaxanthin treatment.

3. Experimental Section

3.1. Radical Scavenging Measured by Electron Spin Resonance Spectroscopy (ESR) ESR measurements were performed using a JEOL JES-FR30EX free radical monitor (JEOL Ltd., Akishima, Japan). The measurement conditions were as follows: magnetic field, 337.394 ˘ 7.5 mT; power, 4 mW; sweep time, 1 min; sweep width, 7.5; modulation, 100 kHz, 0.32 mT; amplitude, 400; and time constant, 0.3 s. Signal intensity was compared based on the ratio against the magnetic Mn2+ marker and was represented by relative height ratio. The ESR spectra were measured three times. Preparation of the astaxanthin stock solution. Astaxanthin (DSM, Kaiseraugst, Switzerland) was dissolved in 21.8% tetrahydrofuran (THF) (Wako Chemicals, Osaka, Japan) and 78.2% ethanol (Nacalai Tesque, Kyoto, Japan). The actual concentration of astaxanthin was determined by diluting the stock solution with THF using the molar coefficient of astaxanthin (69,600 M´1¨ cm´1 at 477 nm) [44]. Based on this value, the stock solution of astaxanthin was determined as 30.56 mM. The astaxanthin stock solution was further diluted with tert-butyl alcohol/THF (89.3:10.7, v/v). Int. J. Mol. Sci. 2016, 17, 103 7 of 14

DPPH radical scavenging experiments. To a reaction mixture containing 80 µL distilled water, 20 µL 500 µM DPPH (in methanol; Nacalai Tesque, Kyoto, Japan) and 100 µL astaxanthin (0, 50, 100, 200, 300, 400, 500, 600, 800, 1000, 1200, and 1500 µM) were added and stirred for a few seconds. tert-Butyl alcohol (containing 10.7% THF) was used as a control. After 1 min incubation the ESR spectra were measured. Galvinoxyl radical scavenging experiments. To a reaction mixture containing 80 µL distilled water, 20 µL 500 µM galvinoxyl (in methanol; Nacalai Tesque, Kyoto, Japan) and 100 µL astaxanthin (0, 10, 20, 50, 100, 200, 300, 400, 500 µM) were added and stirred for a few seconds. tert-Butyl alcohol (containing 10.7% THF) was used as a control. After 1 min incubation the ESR spectra were measured. Superoxide radical scavenging experiments. To the reaction mixture of 30 µL of 5 mM hypoxanthine (dissolved in NaOH and then diluted with PBS (pH 7.4)), 40 µL 4 M 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 25 µL ultrapure water, and 10 µL of astaxanthin (0, 1, 7.5, 15 mM) were added to 5 µL of 1 U/mL xanthine oxidase in 200 mM PBS buffer solution (pH 7.4). After 1 min incubation at 25 ˝C the ESR spectra were measured. Three independent experiments were performed in triplicate.

3.2. Inhibition of Xanthine Oxidase The inhibition of xanthine oxidase was measured according to Bräunlich et al. 2013 [45] with some modifications. An enzyme solution consisting of 400 µL xanthine oxidase (5 U/mL) in 7.6 mL 50 mM potassium phosphate buffer (pH = 7.5) was prepared immediately before use. One hundred and fifty microliters of the prepared enzyme solution was added to each well of a 96 well microtiterplate. Astaxanthin (4 g/L in THF) was serially diluted in the wells (dilution factor 2). THF was used as blank sample. Allopurinol (500 µM; Sigma, Darmstadt, Germany) served as positive control and was serially diluted in the wells (dilution factor 2). The hypoxanthine (40 µg/mL; dissolved in NaOH and then in distilled water) was prepared daily and 80 µL were injected at cycle 10. The absorption was measured at 290 nm (microplate reader Tecan infinite 200Pro, Crailsheim, Germany) over 110 cycles («150 s) at 28 ˝C. At least three independent experiments were performed in triplicate.

1 3.3. Quenching Experiments of Near-Infrared Emission Spectra of O2 by Astaxanthin The singlet oxygen scavenging activity of astaxanthin was determined by photon counting according to Shimizu et al. (2010) [46] with some modifications. Ten millimolar astaxanthin was prepared in THF/ethanol (20/80; v/v). After preparing 10 mM astaxanthin, 1 mM astaxanthin was prepared in THF/ethanol (10.7/89.3; v/v). The final concentration of astaxanthin was confirmed by measuring the solution of 2 mL ethanol and 80 µL astaxanthin solution (1 mM) by UV–Vis spectrometer (Hitachi, Tokyo, Japan) using the molar coefficient of astaxanthin (69,600 M´1¨ cm´1 at λ = 477 nm). 1 The O2 formation was directly measured by the near-infrared luminescence at around 1270 nm 1 1 1 3 3 ´ from deactivated O2 which corresponds to the O2 ( ∆g)– O2 ( Σg ) transition. The emission from 1 O2 was measured using an apparatus based on a commercially available apparatus and improved for high-sensitivity detection (NIR-PII System, Hamamatsu Photonics K.K., Hamamatsu, Japan). The excitation pulse was obtained using a dye laser (CL-EGC USHO Optical Systems Co., Ltd., Osaka, Japan) excited by an Nd-YAG Laser (NL 240/TH EKSPLA Lithuania). The excitation wavelength was 530 nm, pulse width and intensity were approximately 7 ns and 17 mW, respectively, and the repetition 1 rate was 500 Hz. Emission of O2 was monitored using an infrared-gated image intensifier (NIR-PII, Hamamatsu Photonics) after passage through a polychromator (250 is, Chromex Inc., Albuquerque, NM, USA). Measurements started at 1 µs after application of the excitation pulse and the exposure time was 50 µs. Signals were accumulated by repeated detection (500 Hz, 10 s). The emission decay was detected by the NIR-PII system (Hamamatsu Photonics) after passing through the bandpass filter (1270 nm) and recorded by the multichannel scalar (NanoHarp 250, PicoQuant GmbH, Berlin, Germany). The measuring time was set at 16 ns/bin for ca. 260 µs and accumulated for 2 min at 500 Hz. The solution studies consist of 2 mL of 5 µM Rose Bengal (in ethanol) as a photosensitizer. The quenching of the singlet oxygen decay was examined by adding 109 µM astaxanthin as follows: Int. J. Mol. Sci. 2016, 17, 103 8 of 14 addition of 10 µL, 10 µL (final 20 µL), 20 µL (final 40 µL), 40 µL (final 80 µL), and 80 µL (final 160 µL). The decay curve and reaction rate of astaxanthin were calculated using the decay curve and quenching ratio of the duplicate experiments.

3.4. Cell Culture Human Huh7 hepatic cellular carcinoma cells (Institute of Applied Cell Culture, Munich, Germany) were cultivated in high glucose (4.5 g/L) Dulbecco’s modified Eagle’s medium (DMEM, with sodium pyruvate, L-glutamine and 3.7 g/L NaHCO3 supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin and 100 µg/mL streptomycin (all PAN-Biotech, Aidenbach, Germany) ˝ at 37 C in a 5% CO2 setting. For the preparation of PON1-Huh7 cells, Huh7 cells were stably transfected with a 1009 bp [´1013, ´4] fragment of the human PON1 promoter (kindly provided by X. Coumoul and R. Barouki, INSERM UMR-S, Paris, France [47]). PON1-Huh7 cells were cultured in high glucose (4.5 g/L) DMEM (without L-glutamine) supplemented with 10% heat-inactivated FBS, 2 mmol/L glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 100 µg/mL G418 sulfate ˝ (PAN-Biotech, Aidenbach, Germany) in a humidified atmosphere of 5% CO2 at 37 C. Human HepG2 hepatocellular carcinoma cells (Institute of Applied Cell Culture, Munich, Germany) were cultured in RPMI 1640 (with L-glutamine and 2.0 g/L NaHCO3; PAN-Biotech, Aidenbach, Germany) containing ˝ 10% FBS, 100 U/mL penicillin and 100 µg/mL streptomycin in an incubator (95% air, 5% CO2) at 37 C. At least two independent experiments were performed in triplicate. All cell culture experiments were conducted at non-cytotoxic concentrations of astaxanthin.

3.5. Neutral Red Cell Viability Assay PON1-Huh7, Huh7, and HepG2 cells were seeded at a density of 1.5 ˆ 105 cells per well in 24-well plates. Cells were treated with 1–20 µM astaxanthin for 48 (PON1-Huh7) or 24 h (Huh7, HepG2). Afterwards, the culture medium was replaced with neutral red solution (50 µg/mL, prepared in cell culture medium) and incubated for 2 h at 37 ˝C. Cells were extracted with ethanol:water:glacial acetic acid (50:49:1, v/v/v; glacial acetic acid purchased from Carl Roth, Karlsruhe, Germany) and incubated at room temperature for 15 min under continuous shaking. The absorbance was measured at 540 nm in a plate reader (Labsystems iEMS Reader, Helsinki, Finland). At least two independent experiments were performed in triplicate.

3.6. Paraoxonase Activity PON1-Huh7 cells were seeded at a density of 0.15 ˆ 106 cells per well in 24-well plates and incubated for 24 h. Subsequently, cells were treated with 1 and 20 µM synthetic (DSM, Kaiseraugst, Switzerland) versus algae-based (BioAstin, Cyanotech Corporation, Kailua-Kona, HI, USA) astaxanthin. Curcumin (20 µM) served as positive control. After 48 h incubation, cells were washed with PBS and lysed with cell culture lysis reagent (1:5, v/v; Promega, Mannheim, Germany). Luciferase activity (Luciferase Assay System; Promega, Mannheim, Germany) was measured by luminescence reading (Infinite 200 microplate reader, Tecan, Crailsheim, Germany). Results were normalized to total protein content, determined by the BCA assay (Pierce, Bonn, Germany) according to the manufacturer’s instructions. At least two independent experiments were performed in triplicate.

3.7. Glutathione Assay HepG2 cells (0.15 ˆ 106 cells/well) were incubated for 24 h at 37 ˝C. Cells were treated with 1 and 5 µM astaxanthin, respectively and incubated for an additional 24 h. Glutathione measurements were performed according to Esatbeyoglu et al. (2014) [48] and Vandeputte et al. (1994) [49]. Int. J. Mol. Sci. 2016, 17, 103 9 of 14

3.8. Nrf2 Transactivation Huh7 cells (0.15 ˆ 106 cells per well) were incubated in 24-well plates for 24 h. Cells were transiently transfected with pARE_GIGPx_Luc according to Wagner et al. (2010) [50]. After 24 h-transfection, cells were incubated with 1 and 20 µM astaxanthin for further 24 h. Curcumin (20 µM) served as positive control. Cell lysis and measurement of Nrf2 transactivation were conducted as reported previously [50]. At least two independent experiments were performed in triplicate.

3.9. RNA Isolation and qRT-PCR Cells were harvested with TriFAST (VWR International GmbH, Erlangen, Germany) and RNA was isolated according to the manufacturer’s protocol. RNA concentration and quality were determined using the NanoDrop ND-2000 spectrophotometer (VWR International GmbH, Erlangen, Germany). Aliquots of RNA samples were stored at ´80 ˝C until PCR analyses. mRNA expression levels of glutathione peroxidase 1 (Gpx1), superoxide dismutase 1, soluble (Sod1), superoxide dismutase 2, mitochondrial (Sod2), heme oxygenase 1 (Ho-1), NAD(P)H dehydrogenase [quinone] 1, (Nqo1), glutamate-cysteine ligase, catalytic subunit (Gclc), glutathione S-transferase alpha 1 (Gsta), and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) were determined using target-specific primers. Primer3 Input software version 0.4.0 was used for primer design (http://bioinfo.ut.ee/primer3-0.4.0/primer3/). Primer sequences are depicted in Table2. Primers were purchased from Eurofins MWG (Ebersberg, Germany). qRT-PCR was carried out as a one-step procedure using the SensiFAST™ SYBR No-ROX One-Step Kit (Bioline, Luckenwalde, Germany) with SYBR Green detection on a Rotorgene 6000 cycler (Corbett Life Science, Sydney, Australia). Calculation of results was done by an external standard curve. Transcription levels of target genes were normalized by the transcription of Gapdh which served as housekeeping gene in qRT-PCR analyses.

3.10. Lipid Peroxidation Determined by BODIPY Assay C11-BODIPY (581/591) (Life technologies, Darmstadt, Germany) fluorescent dye served as a lipid peroxidation sensor. C11-BODIPY is a lipophilic fatty acid analog which incorporates into biomembranes and shifts its fluorescent properties from reduced (λex = 540 nm/λem = 595 nm) to oxidized (λex = 480 nm/λem = 520 nm) status. Adjusted index of BODIPY oxidation was calculated as follows: Adjusted Index “ EmOx{pEmOx ` EmRedq (1) HepG2 cells (density of 0.15 ˆ 106 cells per well in 24-well plates and incubated for 24 h) were incubated with 10 and 20 µM astaxanthin for 24 h. Cells were treated with 10 µM C11-BODIPY (581/591) in medium without FBS for 30 min. BODIPY was removed and cells were stressed with 80 µM cumene hydroperoxide/80 nM hemin in PBS for 1 h. Afterwards, PBS was refreshed and fluorescence was measured in adherent cells.

3.11. Statistical Analysis Statistical analysis was conducted using SPSS software version 23.0 (SPSS Inc., Munich, Germany). All cell culture data were tested for normal distribution (Kolmogorow–Smirnow). One-way ANOVA was applied. In case of homogeneous variances, LSD and Dunnett-T post hoc test and, in case of heterogeneous variances, Games–Howell post hoc test was conducted to test significant differences between the control group and the test groups. All data are expressed as the mean + SD or SEM. Significance was accepted at p < 0.05. Int. J. Mol. Sci. 2016, 17, 103 10 of 14

Table 2. Nucleotide sequences and annealing temperatures of primers used in qRT-PCR analyses in cultured Huh7 cells.

Gene Gene-ID Description Primer, Forward (51–31) Primer, Reverse (51–31) Annealing (˝C) Gpx1 2876 glutathione peroxidase 1 ACACCCAGATGAACGAGCTG CCGGACGTACTTGAGGGAAT 58 Sod1 6647 superoxide dismutase 1, soluble GGGGAAGCATTAAAGGACTG CAACATGCCTCTCTTCATCC 55 Sod2 6648 superoxide dismutase 2, mitochondrial GCACTAGCAGCATGTTGAGC GATCTGCGCGTTGATGTG 55 Hmox1 3162 heme oxygenase 1 CCA GGC AGA GAA TGC TGA GT GTA GAC AGG GGC GAA GAC TG 59 Nqo1 1728 NAD(P)H dehydrogenase [quinone] 1 CTG ATC GTA CTG GCT CAC TC GAA CAG ACT CGG CAG GAT AC 58 Gclc 2729 glutamate-cysteine ligase, catalytic subunit TTT GGT CAG GGA GTT TCC AG TGA ACA GGC CAT GTC AAC TG 59 Gst 2938 glutathione S-transferase alpha 1 CGTATGTCCACCTGAGGAAA GCCAACAAGGTAGTCTTGTCC 60 Gapdh 2597 glyceraldehyde-3-phosphate dehydrogenase CAATGACCCCTTCATTGACC GATCTCGCTCCTGGAAGATG 58 Int. J. Mol. Sci. 2016, 17, 103 11 of 14

4. Conclusions Collectively, our data suggest that, beyond its coloring properties, synthetic astaxanthin exhibits important free radical scavenging, oxygen quenching, and antioxidant activities. The direct free radical scavenging activity of astaxanthin was determined by ESR and spin trapping and its oxygen quenching activity by photon counting. The cellular antioxidant activity of astaxanthin was examined in various bioassays (e.g., transactivation of PON1, cellular GSH levels, transactivation of Nrf2 and expression of its target genes, lipid peroxidation) in cultured cell lines. Since we worked with cell lines, our findings can not be generalized to primary cells and animal models, respectively, and should therefore be verified in appropriate in vivo models in the future. Additionally, it would be interesting to test whether there are synergistic interactions between astaxanthin and other lipid (e.g., vitamin E) and water soluble antioxidants (e.g., ascorbic acid, flavonoids) in terms of their free radical scavenging, antioxidant and gene-regulatory activity.

Acknowledgments: We thank Christoph Plieth (Center of Biochemistry and Molecular Biology, University of Kiel) for providing the microplate reader (Tecan infinite 200Pro, Crailsheim, Germany) and for the fruitful discussions. We also thank Vivien Schmuck for excellent technical assistance. Author Contributions: Janina Dose, Gerald Rimbach, and Tuba Esatbeyoglu designed the study and wrote the manuscript. Seiichi Matsugo, Haruka Yokokawa, and Yutaro Koshida conducted the electron spin resonance spectroscopy experiments. Shigetoshi Okazaki conducted the singlet oxygen quenching activity experiments. Ulrike Seidel performed the BODIPY assay. All other experiments were conducted by Janina Dose and Tuba Esatbeyoglu. All authors read, edited and gave feedback for the final manuscript. Conflicts of Interest: Manfred Eggersdorfer is an employee of DSM.

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