Combined Astaxanthin and Fish Oil Supplementation Improves

View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Repositório Institucional UNIFESP

Chemico-Biological Interactions 197 (2012) 58–67

Contents lists available at SciVerse ScienceDirect

Chemico-Biological Interactions

journal homepage: www.elsevier.com/locate/chembioint

Combined astaxanthin and ﬁsh oil supplementation improves glutathione-based redox balance in rat plasma and neutrophils

Marcelo Paes Barros a, Douglas Popp Marin b, Anaysa Paola Bolin b, Rita de Cássia Santos Macedo b, Thais Regina Campoio b, Claudio Fineto Jr. b, Beatriz Alves Guerra b, Tatiana Geraldo Polotow a, ⇑ Cristina Vardaris a, Rita Mattei c, Rosemari Otton b,

a Postgraduate Program, Human Movement Sciences, Institute of Physical Activity and Sport Sciences (ICAFE), Universidade Cruzeiro do Sul, 01506000 São Paulo, SP, Brazil b Postgraduate Program, Health Sciences, CBS, Universidade Cruzeiro do Sul, 03342000 São Paulo, SP, Brazil c Department of Psychobiology, Universidade Federal de São Paulo (UNIFESP), 04023062 São Paulo, SP, Brazil

article info abstract

Article history: The present study aimed to investigate the effects of daily (45 days) intake of fish oil (FO; 10 mg EPA/kg Received 3 November 2011 body weight (BW) and 7 mg DHA/kg BW) and/or natural ASTA (1 mg ASTA/kg BW) on oxidative stress and Received in revised form 9 March 2012 Å functional indexes of neutrophils isolated from Wistar rats by monitoring superoxide (O2 ), hydrogen Accepted 10 March 2012 Å peroxide (H O ), and nitric oxide (NO ) production compared to the progression of auto-induced lipid Available online 21 March 2012 2 2 peroxidation and Ca2+ release in activated neutrophils. Furthermore, phagocytic capacity, antioxidant enzyme activities, glutathione-recycling system, and biomarkers of lipid and protein oxidation in neutro- Keywords: phils were compared to the redox status. Our results show evidence of the beneficial effects of FO + ASTA DHA supplementation for immune competence based on the redox balance in plasma (significant increase in EPA Omega-3 GSH-dependent reducing power), non-activated neutrophils (increased activity of the glutathione-recy- Å Å Carotenoid cling enzymes GPx and GR) and PMA-activated neutrophils (lower O2 ,H2O2, and NO generation, reduced Anti-inflammatory membrane oxidation, but higher phagocytic activity). Combined application of ASTA and FO promoted Leukocyte hypolipidemic/hypocholesterolemic effects in plasma and resulted in increased phagocytic activity of activated neutrophils when compared with ASTA or FO applied alone. In PMA-activated neutrophils, ASTA was superior to FO in exerting antioxidant effects. The bulk of data reinforces the hypothesis that habitual consumption of marine fish (e.g. salmon, which is a natural source of both astaxanthin and fish oil) is beneficial to human health, in particular by improving immune response and lowering the risk of vascular and infectious diseases. Ó 2012 Elsevier Ireland Ltd. Open access under the Elsevier OA license.

1. Introduction effects on immune function due to the influence of PUFAs on membrane fluidity, which could result in higher sensitivity to oxidation Habitual intake of marine fish and seafood has been strongly [6,7]. Depending on the concentration, DHA and EPA can also affect associated with human health. It is particularly considered to the microstructure and composition of lipid rafts in the neutrophil enhance immune competence and prevent infectious diseases membrane, resulting in severe changes of cell–cell molecular com- [1]. Fish oil (FO) components eicosapentaenoic acid (EPA) and munication [8]. docosahexaenoic acid (DHA) – well-known as n-3 polyunsaturated Although anti-inflammatory mechanisms in humans have been fatty acids (n-3/PUFA) – have been studied in immune cells and well discussed, there are still unanswered questions with respect to proved to increase anti-inflammatory responses by stimulating redox imbalances provoked in neutrophils during the pathogen- interleukin production [2]. As central players in inflammatory pro- activation process. As a consensus, a wide range of reactive oxy- Å Å cesses, neutrophils are main protagonists of innate immune re- gen/nitrogen species ROS/RNS (including O2 ,H2O2, and NO ) are sponses necessary for early host defense [3]. Regarding continuously produced by neutrophil oxidative burst, and pathogen

nutraceuticals, dietary long-chain (>C20) n-3/PUFAs are considered cells are undoubtedly not the only targets at the inflammatory site. antioxidant compounds, which significantly diminish superoxide In fact, ROS/RNS can also (auto)-oxidize neutrophil membrane, trig- Å (O2 ) and hydrogen peroxide (H2O2) production in activated gering lipid peroxidation and producing lipid-derived cytotoxic neutrophils [4,5]. On the other hand, fish oil also showed adverse aldehydes, e.g. malondialdehyde and 4-hydroxynonenal [9]. Thus, long-living/persistent neutrophils as part of an efficient immune system have to cope with huge oxidative challenges imposed by ⇑ Corresponding author. Tel./fax: +55 11 26726200. E-mail address: [email protected] (R. Otton). pathogen activation. Several authors have, therefore, proposed that

0009-2797 Ó 2012 Elsevier Ireland Ltd. Open access under the Elsevier OA license. http://dx.doi.org/10.1016/j.cbi.2012.03.005 M.P. Barros et al. / Chemico-Biological Interactions 197 (2012) 58–67 59 dietary programs based on antioxidant supplementation could of- (mostly palmitoleic and oleic acids), and 50% with polyunsaturated fer enhancement of immune functions, especially in terms of life- fatty acids (180 mg EPA and 120 mg DHA). Natural ASTA supple- span and slower turnover of damaged immune cells [10,11]. ments (AstaREAL A1010) were obtained as a donation from the Accordingly, many antioxidants present in human diet have also Swedish company BioReal AB (Gustavsberg, Sweden), a subsidiary positively affected immune functions in patients of ROS-related dis- of BioReal Inc., (Hawaii, USA) and part of the pharmaceutical Group eases, such as diabetes [12,13]. Fuji Chemical Industry CO. AstaREAL A1010 is an astaxanthin-rich The marine carotenoid astaxanthin (ASTA; Fig. 1) is naturally natural microalgal product, consisting of crushed and spray-dried found in a wide variety of aquatic organisms, such as microalgae, aplanospores of the green microalgae Haematococcus pluvialis. crustaceans (crabs, lobsters, and shrimp), and fishes (salmon and AstaREAL A1010 contains 42% of crude fat, 10% of crude protein, trout). ASTA is renowned as a powerful antioxidant [14,15], and 40% of carbohydrates, and 4% of ashes. Regarding carotenoid com- also reported to afford benefits to human health, including photo- position, AstaREAL A1010 contains 5.2–5.8% of total carotenoids, protection against UV radiation (skin and eyes) [16], prophylaxis/ 5.0–5.6% of which are being pure astaxanthin (3.9% as monoesters, remediation of gastric ulcer induced by Helicobacter pylori [17], 0.9% as diesters, and 0.1% in free form) among others. and ROS-mediated neurodegenerative processes, as in Parkinson’s disease [18]. Thus, it is reasonable to assume that combined FO 2.3. Animals and supplementation protocols and ASTA supplementation could better balance pro- and antioxi- dative events in activated neutrophils to enhance anti-inflamma- Adult Wistar male rats, weighing 191 ± 35 g at the beginning of tory responses and, thus, provide substantial health benefits to the study, were provided by the Department of Psychobiology, Uni- humans. versidade Federal de São Paulo (UNIFESP), São Paulo, Brazil. All ani- The present study aimed to investigate the effects of daily in- mals were housed in plexiglas cage (four rats/cage), under take of natural sources of n-3/PUFAs (here provided as fish oil) standard laboratory conditions: 12 h light/dark cycle; lights on at and/or natural ASTA (obtained from the biomass of the green mic- 7:00 a.m.; 22 ± 1 °C; water and Purina rat chow ad libitum. The ani- roalgae Haematococcus pluvialis) [19] on oxidative stress and func- mals used in this study were handled in accordance with the tional indexes of neutrophils isolated from Wistar rats. This study guidelines of the committee on care and use of laboratory animal Å was performed by monitoring superoxide (O2 ), hydrogen peroxide resources of the Ethics Committee on Care and Use of Laboratory Å (H2O2), and nitric oxide (NO ) production compared to the progres- Animal Resources from Universidade Federal de Sao Paulo, which sion of auto-induced lipid peroxidation and Ca2+ release in acti- approved the study protocol (CEP/UNIFESP n° 1938/09). The ani- vated neutrophils. Furthermore, our study evaluated antioxidant mals were treated with ASTA and/or FO by gavage, 5 days a week, enzyme activities, the glutathione-recycling system and biomark- for 45 days. A maximum volume of 400 lL was established for the ers of lipid and protein oxidation as related to the FO and/or ASTA gavage treatment in order to prevent regurgitation or stomach dis- diet, and examined the redox status in plasma as well as in isolated comfort of the animals. Fish oil (FO) content of capsules was di- resting and stimulated neutrophils from experimental animals. luted in 10% Tween 80 aqueous solution (v/v) to reach final n-3/ PUFAs concentrations of 10 mg EPA/kg body weight (BW) and 2. Materials and methods 7 mg DHA/kg BW. An identical procedure was conducted for animal supplementation with 1 mg ASTA/kg BW using AstaREAL 2.1. Chemicals A1010 as the carotenoid source (5.3% of which is pure ASTA). For combined FO and ASTA treatments, both components were diluted All chemicals were of analytical grade and purchased from Sig- in the same stock 10% Tween-80 aqueous solution (v/v) to reach ma–Aldrich Chemical Company (St. Louis, MO, USA), except those previously described concentrations. Although additional antioxi- used in the preparation of common buffers (Labsynth, Diadema, dants as ascorbate, tocopherols and other carotenoids were present SP, Brazil). Commercial biochemical test kits were obtained from in both manufactured natural products, their contribution in the Bioclin-Quibasa Química Básica (Belo Horizonte, Brazil) for mea- total antioxidant capacity of gavage solutions is minor compared surements of glucose (prod. code K082), triacylglycerol (prod. code to the prevalent ASTA or n-3/PUFA components. Thus, four exper- K055) and cholesterol (prod. code k083) concentrations in plasma. imental groups of 16 animals each were formed: (i) control, fed The fluorescent probes acetoxymethyl ester (Fura 2-AM) and 4, with 400 lL of 10% Tween 80 aqueous solution (v/v); (ii) ASTA, 4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-inda fed with 1 mg ASTA/kg; (iii) FO, fed with 10 mg EPA/kg and 7 mg 581/591 DHA/kg; and (iv) ASTA + FO, fed with 1 mg ASTA/kg, 10 mg EPA/ cene-3-undecanoic acid (C11-BODIPY ) were purchased from Invitrogen (Grand Island, NY, USA). kg and 7 mg DHA/kg.

2.4. Neutrophil isolation 2.2. Natural products After 45 days of treatment, the rats were euthanized by decapi- Fish oil capsules were purchased from Pharmanostra (Sao Paulo, tation between 11:00 a.m. and 01:00 p.m. to prevent circadian dif- SP, Brazil). Each fish oil (FO) capsule of 500 lL (corresponding to ferences. Prior to decapitation (4 h), rats were intraperitoneally 9 kcal/38 kJ), contains 2.0 mg tocopherols, and 1.0 g of total fat, (i.p.) injected with 10 mL 1% (w/v) glycogen solution (Sigma type out of 30% which saturated fats, 20% with monounsaturated II, from oyster) in PBS to induce cell migration. Neutrophils were obtained by i.p. lavage with 20 mL PBS, followed by centrifugation O in a density gradient of Histopaque 1077 [20]. The total number of leukocytes in exudate was determined in a haemocytometer. H3C OH H3CCH3 CH3 CH3 The differential cell count was obtained by optical microscopy (100) (Nikon YS2-H) of a cytospin cell layer stained with Giemsa. The staining allowed the identification of about 98% of neutrophils CH3 CH3 H3C CH3 present in the exudate 4 h after injection of oyster glycogen. HO CH3 Neutrophils (1 106 cell/mL) were resuspended in RPMI-1640 O medium supplemented with 10% foetal calf serum, 20 mM HEPES, Fig. 1. Chemical structure of the marine carotenoid astaxanthin (ASTA). 2 mM glutamine, and antibiotics (streptomycin 100 U/mL and 60 M.P. Barros et al. / Chemico-Biological Interactions 197 (2012) 58–67 penicillin 200 U/mL). Freshly obtained cells were used to measure by intercalating into DNA, leading to an increase of ethidium fluo- phagocytic activity, ROS/RNS production, calcium release and rescence. The cells (5 105/well) were preloaded with 5 lM DHE membrane oxidation. All other experiments were performed with for 15 min at room temperature in the dark. The assay was carried cells after they were frozen at 80 °C. out in the presence and the absence of 20 ng/well phorbol myris- tate acetate (20 ng PMA/well), here applied as a chemical inducer 2.5. Phagocytic activity of ROS production in neutrophils. Fluorescence was measured at 396 nm of excitation wavelength and at 590 nm of emission wave- Neutrophils (1 106 cell/mL) were incubated for 60 min at length in a microplate fluorescence analyzer (Tecan, Salzburg, 37 °C in 1 mL RPMI 1640 medium with zymosan. Particles were Austria). opsonized by incubation in the presence of control serum for 60 min at 37 °C. Cells were incubated with particles and counted 2.7.2. Hydrogen peroxide production after cytocentrifugation. Scores of phagocytosis were expressed The total production of H O was evaluated before/after chem- by the number of cells that had one, two, three, four, or more 2 2 ical stimulation (with 20 ng PMA) of a 5 105 cell/well suspension zymosan particles phagocyted [21]. using the horseradish peroxidase-dependent oxidation of phenol red (by H O ) to form a vivid red color, which was spectrophoto- 2.6. Analyses in plasma 2 2 metrically quantified at 620 nm, using a H2O2 concentration curve as a standard [27]. 2.6.1. Plasma preparation Briefly, total blood volume (5 mL) was collected in EDTA-Vac- utainer tubes (5.4 mg K2EDTA spray-dried, cod. 367835) and 2.7.3. Nitric oxide production centrifuged for 10 min, 300g at room temperature. The clean plas- Nitric oxide production was determined by spectrophotometric ma fraction was then removed and stocked in Eppendorf 2 mL- analysis of total nitrite, using Griess reagent (1% sulfanilic acid, microtubes at 80 °C for further analyses. 0.1% N-(1-naphthyl)ethylenediamine hydrochloride), according to Ding et al. [28]. Supernatant of neutrophils (5 105 cell/well) with 2.6.2. Glucose, triacylglycerol and cholesterol determinations in or without lipopolysaccharide (LPS-10 l/mL) was added to 100 lL plasma of Griess reagent, the absorbance measured in 550 nm, and nitrite Glucose, triacylglycerol (TAG), and cholesterol levels in plasma levels determined by means of a standard curve generated using were all quantified by commercial test kits obtained from NaNO2. Bioclin-Quibasa Química Básica (Belo Horizonte, Brazil). All these assays were based on the peroxidase-catalysed oxidation of 4-ami- 2.7.4. Intracellular Ca2+ concentration noantipyrine by hydrogen peroxide (H2O2) to generate a pink color 2+ [22,23]. Intracellular changes of calcium levels (Ca ) were monitored in neutrophils using the calcium-sensitive fluorescent probe Fura 2- 6 2.6.3. Glutathione redox status in plasma AM. The 1 10 neutrophil suspension was pre-loaded with 5 lM Fura 2-AM for 1 h at 37 °C in Tyrode’s solution. Afterwards, Reduced glutathione content in plasma (GSH) was measured as 2+ described by Rahman et al. [24]. The method is based on the reac- neutrophils were washed and intracellular Ca release was moni- tion of reduced thiol groups (such as in GSH) with 5,50-dithiobis-2- tored for 60 min at a wavelength emission of 510 nm (excitation nitrobenzoic acid (DTNB) to form 5-thio-2-nitrobenzoic acid (TNB), wavelengths alternating between 340 and 380 nm) in a microplate reader (Tecan, Salzburg, Austria). Transformation of the fluores- which is stoichiometrically detected by absorbance at 412 nm. 2+ Purified GSH and GSSG were used as standards. The Reducing cence signal to Ca concentration was performed by calibration Power index reveals the ratio between GSH and GSSG – thus, the with ionomycin (100 lM, maximum concentration) and EGTA reducing capacity of a biological system – and is calculated by (60 lM, minimum concentration) according to the Grynkiewicz the equation (Eq. (1)): equation and Kdiss of 224 nM [29]. Reducing Power ¼½GSH=ð½GSHþ2½GSSGÞ ð1Þ 2.7.5. Membrane oxidation The progression of membrane oxidation in PMA-activated neu- 2.6.4. Lipid oxidation in plasma trophils was monitored by fluorescence kinetics of the C11-BODI- The extent of lipid peroxidation in plasma was determined as PY581/591 probe, which is oxidized by alkoxyl and peroxyl radicals the concentration of thiobarbituric acid-reactive substances (strongly involved in lipid peroxidation progress) and loses thereby (TBARS) [25]. Butylated hydroxytoluene (4% BHT, in ethanol) was fluorescence emission in the red region [30]. Briefly, 5 105 neu- added to stop progressing oxidation reactions in 500 L diluted 581/591 l trophils were pre-incubated with 3.2 lMC11-BODIPY for samples. For the detection of the colored adducts, 500 lL of each 60 min at room temperature, under mechanical agitation and in sample was incubated at 100 C for 15 min with 0.375% thiobarbi- 581/591 ° the dark. The C11-BODIPY -loaded neutrophils were placed turic acid in 0.25 M HCl and 1% Triton X-100. After reaching room in a microplate, and background fluorescence was adjusted to 1.0 temperature, absorbance of the reaction system was measured at relative fluorescence unit (RFU) and measured for 10 min (kexcita- 535 nm (blanks lack thiobarbituric acid) using malondialdehyde tion = 545 nm, kemission = 591 nm) in a microplate reader (Tecan, (MDA) as a standard. Salzburg, Austria). Afterwards, 100 ng PMA/5 105 cells were added to trigger ROS production and start auto-induced membrane 2.7. Biochemical measurements in neutrophils oxidation, which was followed for additional 15 min. A first-order exponential decay function (Eq. (2)) was applied to obtain kinetic Å 2.7.1. Superoxide radical (O2 ) production parameters of lipid peroxidation in the neutrophil membrane. The lipophilic and freely diffusible probe dihydroethidium eðt=kÞ (DHE) was used for the fluorimetric measurement of intracellular yðRFUÞ¼y0 þ A1 1 ð2Þ Å production of O2 [26]. Once inside cells, DHE is rapidly oxidized Å to ethidium (a red fluorescent compound) by O2 and H2O2 (in RFU = relative fluorescence unit; y0 = initial calculated relative fluo- the presence of peroxidase). Ethidium is trapped in the nucleus rescence unit; A1 = intensity factor; t = time (s); k1 = rate constant. M.P. Barros et al. / Chemico-Biological Interactions 197 (2012) 58–67 61

2.7.6. Preparation of cell homogenates 2.8. Statistical analyses Neutrophils (5 106/mL) were isolated and ruptured by ultra- sonication in a Vibra Cell apparatus (Connecticut, USA) in an All data are presented as the mean values of, at least, triplicates ice-water bath using 500 lL of each assay-specific extraction solu- with their standard errors (MEAN ± SE). Data were analyzed by one- tion/buffer. Afterwards, ruptured neutrophils were centrifuged for way ANOVA followed by Tukey’s post-test, considering p < 0.05, 10 min, 10.000g at 4 °C, and the supernatant was removed and p < 0.01, or p < 0.005. The software Origin 6.1 (v6.1052/B232; Orig- kept on ice for further analysis (debris was discarded). inLab Corporation, Northampton, MA, USA) was used for statistical analyses and graph preparation. 2.7.7. Assay of superoxide dismutase activity (SOD) The activity of total superoxide dismutase (SOD) was measured 3. Results according to Ewing & Janero [31], with modifications. The reaction system for total SOD determination included 50 mM sodium phos- 3.1. Plasma indexes phate buffer (pH 7.4), 0.1 mM EDTA, 50 lM nitroblue tetrazolium (NBT), 78 lM NADH, and 3.3 lM phenazine methosulphate (PMS), Å All the experimental groups showed similar growth rates (BW) here used as a chemical O source. Analysis of mitochondrial (man- 2 during the 45-day supplementation period (data not shown), ganese-dependent) MnSOD activity was performed similarly as for although ASTA supplementation alone distinctively increased lipi- total/SOD, but under strong inhibition (88–100%) of the cytosolic demia (55%) and cholesterolemia (35%, compared to control; Table CuZnSOD isoform by addition of 3 lM KCN. As internal controls of 1). This effect was not observed in animals from the FO + ASTA the experiment, all reagents without sample were used for both group. Regarding redox balance, combined FO + ASTA clearly en- totalSOD and MnSOD determination. Absorbance at 550 nm was Å hanced the antioxidant capacity of plasma, especially when com- continuously monitored for over 2 min in order to evaluate O - Å 2 paring GSH/GSSG ratios (6-fold higher). Basal NO concentration mediated reduction of NBT. One SOD unit is defined here as the in plasma was also increased in FO + ASTA animals (65%, Table enzyme concentration required for 50% inhibition of NBT reduction 1), whereas no significant difference was observed in terms of lipid at 25 °C. oxidation.

2.7.8. Assay of catalase activity 3.2. Non-stimulated neutrophils The decomposition of H2O2 was directly followed for 2 min by measuring the decrease in absorbance at 240 nm using a molar 1 1 The redox balance was also studied in neutrophils isolated from extinction of e240 = (0.0394 ± 0.0002) LmM cm , as described by Aebi [32]. One catalase (CAT) unit is defined as the enzyme con- the experimental animals, as shown in Table 2. Concerning the frontline antioxidant enzymes (Table 2), ASTA alone provided the centration required for the decomposition of 1 lmol of H2O2 per min at 25 °C. As internal control, 50 mM (pH 7.4) phosphate buffer most significant effects, especially on the GSH-recycling enzymes GPx and GR (increased by 92% and 52%, respectively). However, was added to H2O2 without sample and monitored for 2 min. GSH content and GSH-related Reducing Power (see Section 2) were 2.7.9. Assay of the glutathione-recycling enzyme system abruptly depleted in neutrophils of ASTA-fed animals (both by The activity of glutathione peroxidase (GPx) was measured approximately 70%; Table 2). FO treatment caused a different effect according to Mannervik [33] using 2.5 U/mL glutathione reductase on neutrophils, since massive decreases in GPx (50%) and GR activ- (GR), 10 mM reduced glutathione (GSH), 250 lM sodium azide (as ities (65%) were accompanied by proportionally augmented levels a catalase inhibitor), and 1.2 mM NADPH. The reaction was trig- of GSH (80%) and Reducing Power (89%). Combined FO + ASTA sup- gered by addition of 4.8 mM tert-butyl hydroperoxide (a GPx sub- plementation resulted in increased activities of total SOD (20%), strate). The other GSH-recycling component enzyme, glutathione mitochondrial MnSOD (22%), GPx (62%), and GR (36%). Unexpect- reductase (GR), was, in turn, measured by a direct reaction be- edly, all supplementation treatments resulted in lower activities tween 3.6 mM NADPH and 10 mM oxidized glutathione (GSSG). of CAT: 35% in ASTA group, and 45% in both FO and FO + ASTA ani- Absorbance of NADPH oxidation (in 0.2 M phosphate buffer, pH mals (compared to control). Despite all changes observed in GSH 7.4) was monitored in both cases at 340 nm for 2 min in an Ultro- content, none of the experimental groups showed statistically dif- spec 3000 spectrophotometer (Pharmacia Biotech). ferent indexes of oxidative modification in lipids (TBARS content) or proteins (carbonyls) compared to control neutrophils, except 2.7.10. Oxidative stress parameters protein carbonyls of ASTA-fed animals (33% higher than control). Carbonyl groups were evaluated as parameters of amino acid oxidation in neutrophils. After pre-treatment with 10% streptomy- 3.3. Activated neutrophils cin to remove interfering nucleic acids, the protein fraction was isolated from homogenate by precipitation with 20% trichloroacetic In order to evaluate one of the most important neutrophil-func- acid solution on ice, followed by the subsequent processes: (i) tional parameters, after 45-day of treatment with FO and/or ASTA, 0 washing once with 0.3 M HClO4, 5 mM EDTA, and 0.06% 2,2 -bipyr- neutrophils were challenged in vitro with opsonized zymosan par- idine solution; (ii) washing twice with the mixture 1:1 ethyl ace- ticles (phagocytic capacity). The effect of ASTA and/or FO supple- tate:ethanol (v/v); and (iii) removing residual organic solvent in mentation on neutrophil was also evaluated by measuring ROS/ vacuum for, at least, 30 min. Afterwards, the protein pellets were RNS production and auto-induced lipid peroxidation after chemical fully dissolved in 6 M guanidine hydrochloride and used for detec- (PMA-) activation of neutrophils. As shown in Fig. 2, the phagocytic tion of the carbonyl groups after reaction with 10 mM dinitrophen- capacity of neutrophils was especially improved by FO supplemen- ylhydrazine (DNPH) in 0.25 M HCl (blanks lack DNPH). Absorbance tation (approximately 2.4-fold higher), whereas ASTA only was recorded at 380 nm, and the carbonyl group concentration was displayed marginal effects (not significant, compared to control; calculated using an extinction coefficient of k = 2.2 104 M1cm1 p = 0.180). [34]. Reduced and oxidized glutathione (GSH and GSSG, respec- In order to investigate whether FO and/ASTA can exert effects on Å tively) and TBARS (biomarker of lipid oxidation) were also O2 production, freshly isolated neutrophils were incubated with measured in homogenates of neutrophils, following the same DHE for 15 min. All supplementation protocols resulted in higher Å procedures as described for plasma samples. basal O2 production in non-stimulated neutrophils. This effect 62 M.P. Barros et al. / Chemico-Biological Interactions 197 (2012) 58–67

Table 1 Plasma levels of physiological and oxidative stress biomarkers of Wistar rats supplemented with 1 mg/kg/BW ASTA and/or FO (equivalent to 10 mg EPA/kg and 7 mg DHA/kg) for 45 days (5 days/week).

Control ASTA FO FO + ASTA Glucose 156.1 (±20.7) 163.1 (±5.6) #FOA 173.7 (±47.9) 144.6 (±6.5) #A Triacylglycerol 106.4 (±2.9) 164.3 (±27.2) §C, FOA #FO 92.6 (±13.3) #A 106.7 (±13.0) §A Cholesterol 74.9 (±4.1) 100.8 (±12.7) *C, FO 72.5 (±10.1) *A 78.7 (±16.3) GSH 0.081 (±0.041) 0.108 (±0.058) *FOA 0.110 (±0.046) *FOA 0.158 (±0.056) #C, *A, FO GSSG 0.097(±0.026) 0.093 (±0.029) #FOA 0.087 (±0.028) #FOA 0.029 (±0.006) #A, FO Reducing power 0.294(±0.169) 0.367 (±0.228) 0.387 (±0.204) 0.731 (±0.300) *C, A, FO Nitric oxide 6.68(±2.17) 6.57 (±1.44) #FOA 8.88 (±1.63) 11.12 (±1.01) *C, #A TBARS 1.34 (±0.31) 0.85 (±0.39) 1.36 (±0.52) 1.11 (±0.38)

Related to C (Control), A (ASTA), FO (Fish oil), FOA (Fish oil + ASTA). * p < 0.05 § p < 0.01 # p < 0.005.

Table 2 Redox indexes in rested (non-activated) neutrophils isolated from Wistar rats supplemented with 1 mg/kg/BW ASTA and/or FO (equivalent to 10 mg EPA/kg and 7 mg DHA/kg) for 45 days (5 day/week).

Parameter Control ASTA FO FO + ASTA Total SOD 27.10 (±0.92) 28.26 (±1.98) 29.97 (±4.53) 32.29 (±1.44) §C MnSOD 17.66 (±0.04) 17.85 (±2.09) 20.49 (±3.61) 21.68 (±1.16) #C CAT 4.73 (±0.54) 3.06 (±0.54) #C 2.67 (±0.77) #C 2.69 (±0.54) #C GPx 0.87 (±0.04) 1.67 (±0.20) #C, FO 0.43 (±0.01) #C, A, FOA 1.41 (±0.15)#C, FO GR 0.102 (±0.014) 0.155 (±0.018) §C, #FO 0.035(±0.002) #C, A, FOA 0.139 (±0.012) *C, #FO TBARS 3.20 (±0.37) 3.67 (±0.55) 3.65(±1.14) 2.95(±0.51) Protein carbonyls 15.0 (±1.0) 19.9 (±1.2) *C, FO, FOA 14.0 (±0.8) *A 14.4 (±0.2) *A GSH 3.67 (±0.32) 1.01 (±0.17) #C, F, §FOA 6.58 (±0.82) §C, #A, FOA 1.47 (±0.06) #C, FO, §A GSSG 4.05(±0.46) 4.33 (±0.29) 1.84(±0.18) #C, A 1.32 (±0.05) #C, A Reducing Power 0.312 (±0.045) 0.104 (±0.019) #C, FO, FOA 0.641 (±0.102) #C, A, FOA 0.358 (±0.020) #A, FO

Related to C (Control), A (ASTA), FO (Fish oil), FOA (Fish oil + ASTA). * p < 0.05. § p < 0.01. # p < 0.005.

+PMA §C 60000 -PMA 300 #C

45000 200

30000

100

15000 Scores of phagocytic activity phagocytic of Scores

0 Superoxide production (RFU) Control ASTA FO FO+ASTA 0 Control ASTA FO FO+ASTA Fig. 2. Scores of phagocytic capacity of rat neutrophils. The assay was carried out for 60 min, using opsonized zymosan particles. Results are expressed as mean ± - Fig. 3. Superoxide anion production was measured in neutrophils (5 105 per § SEM of three different experiments (n = 8 rats) performed in triplicate. p < 0.01 or well) under baseline and PMA-stimulated conditions. Cells were stimulated with # p < 0.005; related to C (Control). PMA (20 ng/well) for 30 min and superoxide anion generation was measured by dihydroethidium (5 lM) assay. Results are expressed as mean ± SEM of three different experiments (n = 8 rats) performed in triplicate. ⁄p < 0.05; or #p < 0.005, was strongly related to ASTA presence, since ASTA-, FO-, and related to C (Control), A (ASTA), FO (Fish oil), FOA (Fish oil + ASTA). Å FO + ASTA-fed animals showed 53%, 17%, and 65% higher O2 basal production in neutrophils, respectively (compared to control). Upon PMA-activation, however, a different trend was observed. On the other hand, NOÅ production (and probably other RNS that FO alone or as a component of FO + ASTA supplementation signiﬁ- generate nitrite as product) was apparently more affected by ASTA Å cantly reduced O2 production by approximately 10% (Fig. 3). supplementation than by FO per se (Fig. 5). Even though no signif- H2O2 production in both rested and activated neutrophils fol- icant difference was observed in rested neutrophils, LPS stimulus Å Å lowed the same trend as observed for O2 production, except for only resulted in 45% lower NO production in neutrophils isolated PMA-activated neutrophils of ASTA-fed group, which showed 25% from ASTA-fed animals (either alone or combined with FO). 2+ lower H2O2 production than control neutrophils (Fig. 4). Concomitantly, the inﬂux of Ca ions in LPS-activated neutrophils M.P. Barros et al. / Chemico-Biological Interactions 197 (2012) 58–67 63

+PMA 50 -PM A PMA 1,4

M) 40 µ 1,2

30 1,00

20 production ( 2

O Control 2 0,95 H 10 ASTA FO FO+ASTA 0 -BODIPY Lipid peroxidation (RFU)

Control ASTA FO FO+ASTA 11 0,90

C 0 400 800 1200 1600 Fig. 4. Measurement of hydrogen peroxide production in neutrophils (5 105 per Time (s) well) under baseline and PMA-stimulated conditions. Hydrogen peroxide production was induced with PMA (20 ng/well) for 1 h, and measured as absorbance Fig. 6. The progression of auto-induced lipid peroxidation after PMA-stimulation in 5 increase at 620 nm due to horseradish peroxidase-dependent oxidation of phenol neutrophils monitored by C11-BODIPY ﬂuorescence decay. Cells (5 10 /well) were 581/591 red. Results are expressed as mean ± SEM of three different experiments (n = 8 rats) pre-loaded with 3.2 lMC11-BODIPY (60 min). PMA (100 ng) was added to performed in triplicate. ⁄p < 0.05; §p < 0.01; or #p < 0.005, related to C (Control). trigger ROS production and membrane oxidation. Results are expressed as #signiﬁcantly different (#p < 0.005) between C + PMA group and ASTA + PMA or mean ± SEM of three different experiment (n = 8 rats) performed in triplicate. FO + PMA groups.

carotenogenesis pathway [35]. The hypercholesterolemic effect of +PMA these xanthophylls is not related to reported mechanisms of non- 50 -PMA polar carotenoids in mammalians, because beta-carotene does not induce changes in plasma cholesterol. 40 Higher lipid concentration in plasma of ASTA-fed rats was not followed by hyperglycemic conditions or increased antioxidant cell) 5 capacity, as demonstrated by GSH/GSSG scores and oxidative

10 30 x modiﬁcations in plasma lipids (Table 1). Based on previous studies

/ 5 from our group, the ASTA effect on plasma glucose, cholesterol or 2 20 triacylglycerol concentrations is supposedly dependent on the carotenoid content in the diet, since those changes were not ob- NO productionNO served when animals were fed with a 20-fold higher dose of ASTA

mol NaNO 10 µ [12]. Interestingly, Aoi et al. (2008) showed that exercising mice ( fed with 0.02% (w/w) ASTA consumed more lipids than glucose as 0 energy source, resulting in an increased running time to exhaustion Control ASTA FO FO+ASTA [36]. Although FO supplementation alone did not alter physiological or redox parameters in plasma (compared to control), its combi- Fig. 5. Measurement of nitric oxide production in neutrophils (5 105 per well) nation with ASTA substantially augmented GSH content, its redox under baseline and LPS-stimulated conditions. Cells were stimulated with LPS turnover (conversion of oxidized GSSG back to its reduced form, (10 lg/well) for 4 h and nitric oxide production was measured by Griess reagent assay. Results are expressed as mean ± SEM of three different experiment (n =8 GSH) and, consequently, thiol-dependent reducing power of plasma rats) performed in triplicate. ⁄p < 0.05; or #p < 0.005, related to C (Control), A (ASTA), (Table 1). Glutathione-based and lipid peroxidation markers in FO (Fish oil), FOA (Fish oil + ASTA). plasma confirm our previous results of the biochemical and putative anxiolytic effects of the ASTA + FO treatment [37]. Important was unaltered regardless of the supplementation protocol (ASTA hypolipidemic and hypocholesterolemic effects were also observed and/or FO, data not shown). in the ASTA + FO group (compared to ASTA-fed animals), suggesting The progression of auto-induced lipid peroxidation after PMA- a specific effect of FO supplementation, since similar results were 581/591 stimulation in neutrophils was monitored by C11-BODIPY also found in the FO-group. Accordingly, experiments in humans fluorescence decay (Fig. 6). Based on a first-order exponential de- indicate a profound hypolipidemic effect of FO, especially by lower- cay function (see Materials and Methods), it is clear that the plas- ing plasma triacylglycerol [38]. In agreement with Engler et al. [39], ma membrane of neutrophils from all supplemented groups was we also observed hypolipidemic/hypocholesterolemic effects asso- Å less exposed to oxidative injury than that from untreated control ciated with 65% higher NO concentration in plasma of FO + ASTA animals. Taking kinetic constants into account (k1), ASTA supple- supplemented Wistar rats (Table 1), although supplementation mentation was proven to limit the progression of auto-oxidation with FO alone demonstrated a non-significant tendency for higher in neutrophil membrane after PMA-stimulus, regardless of FO values (p = 0.175). Reduction in lipidemia and cholesterolemia cou- Å presence (Table 3). All fitting exponential curves were relatively pled to adequate redox rebalance and higher NO bioavailability in well correlated (R2 P 0.600). plasma has been currently suggested as positive events for prevent- ing the progression of early coronary heart disease in high-risk 4. Discussion patients [40].

4.1. Plasma parameters 4.2. Non-activated neutrophils

As observed by other authors, ASTA supplementation alone The long-term FO + ASTA supplementation resulted in substan- (1 mg/kg herein) resulted in hyperlipidemia and hypercholesterol- tial antioxidant responses in rat neutrophils, compared to control. emia in experimental animals (Table 1) [35]. Similar effects were Both total and mitochondrial MnSOD activities were proportion- also observed in rats supplemented with the marine carotenoid ally increased by approximately 20%, whereas CAT showed 43% canthaxanthin, the immediate precursor of ASTA in the lower activities (Table 2). Higher SOD activities in neutrophils from 64 M.P. Barros et al. / Chemico-Biological Interactions 197 (2012) 58–67

Table 3

Kinetic rates of the exponential decay of C11-BODIPY ﬂuorescence induced by PMA activation in neutrophils isolated from Wistar rats supplemented with 1 mg/kg/BW ASTA and/ (t/k ) or FO (equivalent to 10 mg EPA/kg and 7 mg DHA/kg). First-order exponential decay: y(RFU) = y0 + A1.e 1 for 45 days (5 days/week). y = Fluorescence intensity; RFU = relative

ﬂuorescence units; y0 = initial relative ﬂuorescence; A1 = intensity factor; t = time (s); k1 = rate constant.

Experimental groups y0 A1 k1 R2 Control 0.946 (±0.005) 0.828 (±0.581) 260.93 (±70.6) 0.918 ASTA 0.984 (±0.016) 3.10 (±9.33) 131.4 (±72.4)*C 0.600 FO 0.972 (±0.003) 0.839 (±0.968) 217.2 (±78.0) 0.859 FO+ASTA 0.984 (±0.002) 1.29 (±2.83) 157.4 (± 76.0)*C 0.652

* p < 0.05, related to: C (Control); A (ASTA group); FO (Fish oil group); or FOA (Fish oil + ASTA group).

FO + ASTA group were possibly a response to the significant raise in massive exposure to higher concentrations of self-generated ROS/ Å basal O2 production of neutrophils (Fig. 3). Accordingly, basal RNS. Furthermore, a crucial mechanism of cell death – apoptosis Å H2O2 production – product of O2 dismutation by SOD isoforms – – is tightly related to redox unbalances in active neutrophils [44]. was also augmented (60%) in non-activated neutrophils of the FO + ASTA group (Fig. 4). Thus, in order to cope with the increase 4.3. Activated neutrophils of cytotoxic H2O2 concentrations and the unresponsiveness of CAT activity (Table 2), the GSH-dependent enzyme system, com- Fish oil supplementation itself was related to the increased prised by coupling enzymes GPx/GR, was upregulated (62% and phagocytic activity of neutrophils (both FO and FO + ASTA groups), 36%, respectively) to stabilize the reducing power of resting since ASTA supplementation alone did not result in higher phago- FO + ASTA neutrophils (Table 2). Interestingly, neutrophils from cytic scores (Fig. 2). Moreover, after PMA activation, neutrophils ASTA-fed animals also showed higher GPx/GR activities, lower from FO-fed groups (both FO- and FO + ASTA, but not from ASTA- CAT activity, but unaltered SOD isoform activities: under these cir- Å group) showed reduced O2 production, whereas all supplemented cumstances, the reducing power was drastically diminished by 65% animals had lower H2O2 generation (Figs. 3 and 4). These data sug- and protein oxidation was exacerbated by 33% (Table 2). Organic gest that phagocytic activity was more closely related to changes in Å peroxides, main products of lipid/membrane oxidation, and H2O2 O2 production of activated neutrophils and/or to pre-acquired po- are regular substrates for GPx in most cells. We assume that spe- sitive redox balance during the FO- and FO-ASTA supplementation cific GSH-dependent enzymes GPx/GR – but not CAT – were upreg- period. Nevertheless, it is plausible to suggest that the improve- ulated as cellular responses to circumvent increased levels of ment of the immune function (phagocytic activity herein) was a di- organic peroxides from exacerbated lipid peroxidation, but not rect effect of FO supplementation in our experimental groups. H2O2 itself. Fig. 6 corroborates this hypothesis. Similar results were Contrasting with this result, FO + ASTA supplementation promoted obtained by our group in an analogous study performed in lymph a reduced proliferative capacity of activated T- and B-lymphocytes nodes lymphocytes. Increased activities of total/SOD, GR and GPx as showed in our previous study [41]. The consensus from histolog- were observed in lymphocytes from rats supplemented with ical studies and tracking fluorescent-targeted cells is that the FO + ASTA [41]. Singular cells in the immune system, such as neu- majority of inflammatory neutrophils never leave an inflamed site trophils and lymphocytes, which exert extremely different func- to return to blood stream, since most activated cells die in situ due tions in the innate and adaptive immune response, respectively, to cellular processes closely related to oxidative stress: apoptosis can present similar responses to supplemention with FO + ASTA. or necrosis [45]. It is well conceived that apoptosis is the preferred Hattori et al. [42] reported the induction of GPx activity by H2O2 injury limiting mechanism for the removal of non-functional neu- exposure in neutrophils, although a key participation of the inflam- trophils from inflamed sites. Moreover, it is notably related to aug- matory cytokine TNF-a was also evidenced. Furthermore, the mented rates of ROS/RNS production (by intrinsic pathway), Ca2+ depletion observed in neutrophil GSH content in FO + ASTA (and influx disruption, and progressive oxidative events in several im- ASTA-fed) animals is consistent with specific percentage changes mune cells, including neutrophils [46]. Therefore, it is possible that of GPx and GR, which favors the consumption of reduced GSH the antioxidant background in neutrophils (obtained during the forms. On the other hand, proportional increases of GSSG content supplementation period) could affect: (i) their life-span as active were not observed (Table 2). One plausible hypothesis for that fact immune cells; (ii) their functionality during inflammatory pro- is the substantial utilization of a fraction of the total GSH content cesses; and (iii) the mechanism by which active neutrophils are de- for S-glutathionylation of proteins, a reversible post-translational graded (with consequences for remaining cells and tissue). As also modification that provides protection of protein thiol groups from observed in lymphocytes [12], ASTA supplementation here offered oxidation and thus proteins from denaturation, concomitantly putative anti-apoptotic effects in neutrophils, based on reduced serving as a redox signal transducer [43]. Reinforcing the afore- production of NOÅ and unaltered intracellular Ca2+ release (Fig. 5). mentioned key participation of SOD isoforms in sustaining redox In addition, the powerful antioxidant activity of ASTA [14] was balance and functionality of neutrophils, no changes in protein oxi- here evidenced in neutrophils by the significantly lower rate con- dation scores were observed in neutrophils of FO + ASTA animals, stant (k1; Table 3) of the exponential progression of membrane oxi- although a significant increase (33%) was found in neutrophils of dation after PMA activation in neutrophils from ASTA-fed animals ASTA-fed animals. Altogether, these data depict the redox scenario (both ASTA- and FO + ASTA groups; Fig. 6). This sensitive assay by of most circulating (rested?) neutrophils from experimental groups the radical-sensitive probe C11-BODIPY depicts the slower pro- and, thus, are possibly related to the life-span of neutrophils under gression of lipid oxidation in neutrophil membrane from both non-inflammatory conditions. On the other hand, neutrophil ASTA- and FO + ASTA-fed animals (see Section 2). Hence, it is rea- (short) life-span is actually more dependent on the occurrence of sonable to assume that ASTA supplementation offered extended inflammatory events, since migration, infiltration, and particularly protection to auto-induced oxidation in neutrophils membrane, the oxidative burst during pathogen (or chemical, herein) activa- with possible influence on cell survival and life-span. tion expose these immune cells to harsher oxidative conditions. Putative anti-apoptotic effects in rat neutrophils are here Thus, it is desirable to investigate the redox balance and progres- suggested from combined FO + ASTA supplementation, since lower sion of oxidative insult in active neutrophils, exactly during their Å Å O2 production was concomitantly monitored with lower NO M.P. Barros et al. / Chemico-Biological Interactions 197 (2012) 58–67 65 concentrations, and both radicals promptly reacted in intracellular The main ASTA + FO effects on neutrophil functions might be asso- compartments to produce the pro-apoptotic factor, peroxynitrite ciated with the GSH-recycling system (with coadjutant collabora- (ONOO) [47]. Interestingly, low doses of NOÅ were shown to pre- tion from total SOD, Table 2), which apparently prioritizes the use vent killing of rat hepatocytes by tert-butylhydroperoxide, whereas of available plasma GSH content to protect neutrophil membrane. further increasing doses resulted in increased killing [48]. This dual The immune system is known to critically depend on accurate pro-/antioxidant role of NOÅ has been also reported in several other cell–cell communication in order to mount an efficient response cell types and could represent an important redox switch for [57]. Consequently, it is tempting to suppose that ASTA + FO might triggering apoptosis in neutrophils. help to maintain fluidity and integrity of neutrophil membranes by reducing the oxidative damage during oxidative bursts, with possi- 4.4. Mechanistic aspects ble positive effects on associated cell receptors, as well as redox- sensitive transcription factors (especially NF-jB), cytokines and A bulk of data has demonstrated that the beneficial action of prostaglandins [58]. Interestingly, ASTA + FO supplementation also Å dietary carotenoids on the immune system is independent of their resulted in significantly lower production of O2 and H2O2 during provitamin A or retinal-derived activities. Most of the immune the oxidative burst of neutrophils (Figs. 3 and 4, respectively). Alto- improving effects provided by nonprovitamin A carotenoids – such gether, the redox rebalance in activated neutrophils resulted in as lycopene, canthaxanthin, lutein and ASTA – on humans and ani- significantly higher phagocytic activities, as shown in Fig. 2. mals has been strongly associated with their antioxidant properties, Regarding physicochemical properties of biological membranes, it even though recent studies also addressed their role in gene regula- is worth mentioning that rigid, rod-like ASTA molecules, anchored tion, apoptosis and angiogenesis [49]. The pharmacodynamics of transversally across lipid bilayers, restrict molecular motion of lip- ASTA plays an essential role in determining its plasma concentra- ids and proteins, thus affecting membrane fluidity and increasing tion, putative direct interaction with circulating neutrophils, and structural stability [59]. On the other hand, n-3/PUFA such as targeting of tissues. Petri & Lundebye (2007) evaluated the tissue DHA and EPA (present in FO) were shown to induce an opposite ef- distribution of ASTA after oral administration to rats for two weeks, fect on biological membranes [60]. Based on the fact that major and found that ASTA accumulated primarily in the spleen, kidneys, ROS production during the oxidative burst of neutrophils also oc- adrenals, liver, skin and eyes [50]. Tested as an anti-carcinogenic curs in membranes (catalyzed by NADPH oxidases), it is reasonable agent, the serum concentration of ASTA was approximately to assume that the proportion of n-3/PUFA and ASTA incorporated 1.2 lM following a 0.02% ASTA supplementation protocol for four in neutrophil membranes could drastically affect physicochemical weeks [51]. Nevertheless, single/multiple oral administrations of properties of lipid bilayers, with crucial consequences in terms of ASTA resulted in short-term accumulation in mice liver, in contrast neutrophil function and whole immune responsiveness. Further to the fast elimination of unmodified ASTA from plasma [52]. ASTA studies are necessary to better understand such molecular-based short-term concentration in mammal liver was shown to propor- – but truly relevant – aspects of ASTA and/or FO supplementation tionally inhibit the activity of hepatic NADPH P450 reductase, lead- in immune enhancement. ing to detrimental toxicological dysfunctions [53]. Although still obscure, the deactivation of hepatic NADPH P450 reductase could 5. Concluding remarks be associated with a hypothetical additional NADPH supply for ASTA-mediated triacylglycerol and cholesterol biosynthesis in the The present study strongly suggests beneficial effects of liver. This hypothesis is in agreement with hyperlipidemia and FO + ASTA supplementation for immune competence, based on hypercholesterolemia observed in ASTA-fed rats, as shown in Table the redox balance in plasma (significant increase in GSH-dependent 1. The specific mechanism of NADPH P450 reductase inhibition by reducing power), non-activated neutrophils (increased glutathi- ASTA is dose-dependent and related to inhibition of cytochrome c one-recycling enzymes GPx and GR) and PMA-activated neutrophils Å Å reduction in microsomes [54]. Therefore, ASTA could directly affect (lower O2 ,H2O2, and NO generation, reduced membrane oxidation, electron transfer on the microsomal membrane of hepatocytes due but higher phagocytic activity) of Wistar rats. The effects of ASTA to its powerful antioxidant properties [14,55]. Furthermore, ASTA and FO combination were summative rather than synergistic, since alone or combined with FO induced higher activities of the antiox- FO strongly contributed to hypolipidemic/hypocholesterolemic ef- idant enzymes GPx and GR belonging to the GSH-recycling system fects on plasma plus increased phagocytic activity in activated neu- that needs constant supply of NADPH from the pentose phosphate trophils, whereas ASTA better offered antioxidant/anti-apoptotic pathway (Table 2). effects to PMA-activated neutrophils. The bulk of data reinforces Although ASTA supplementation alone did not increase GSH the hypothesis that habitual consumption of marine fish (e.g. sal- content of plasma (p = 0.105; Table 1), the combined ASTA + FO mon, which is a natural source of both astaxanthin and fish oil) is supplementation significantly augmented its thiol-based antioxi- strongly associated with human health, in particular with improve- dant capacity (approximately 2-fold higher), with putative protec- ment of immune response, and lower risks for vascular and infec- tive effects on circulating neutrophils. Accordingly, both ASTA- tious pathologies [61]. and ASTA + FO-fed animals showed lower rates of lipid oxidation, 581/591 as monitored by the fluorescent probe C11-BODIPY (k1 in Acknowledgements Table 3). However, it remains to be investigated whether ASTA and/or FO were properly absorbed by neutrophil membrane or The authors are also indebted to the assistance of Marina whether this was simply a result of plasma GSH-neutrophil mem- Santana Gonçalves, and Sebastiana Jeane Nascimento. This research brane interaction. FO treatment alone resulted in unexpected was supported by Fundação de Amparo a Pesquisa do Estado de São downregulation of both GPx and GR by an unknown mechanism. Paulo (FAPESP 2009/12342-8), Coordenação de Aperfeiçoamento de On the other hand, the total conversion of arachidonic acid through Pessoal de Nível Superior (Programa PROSUP/CAPES, Brazil), and the lipoxygenase pathway in platelets was shown to be lowered by Conselho Nacional de Desenvolvimento Científico e Tecnológico GSH depletion, which could be a consequence of diminished GR (Bolsas Produtividade em Pesquisa, Nivel 2, #312404/2009-3 and activities [56]. Interestingly, GPx and GR activities in neutrophils #312190/2009-3, CNPq, Brazil), and Universidade Cruzeiro do Sul. of ASTA + FO-fed animals were significantly augmented (62% and Dr. Marcelo Paes de Barros is also indebted to Dr. Åke Lignell from 36%, respectively), whereas proper intracellular GSH and GSSG con- BioReal AB/Fuji Chemicals (Sweden/Japan) for supplying astaxan- tents both decreased by approximately 60% in neutrophils (Table 2). thin-rich algal biomass for in vivo studies. 66 M.P. Barros et al. / Chemico-Biological Interactions 197 (2012) 58–67

All authors of the present manuscript declare that there is no comparison with homogenates and microsomes, Free Radic. Biol. Med. 4 actual or potential conflict of interest including any financial, (1988) 155–161. [26] H. Zhao, S. Kalivendi, H. Zhang, J. Joseph, K. Nithipatikom, J. Vasquez-Vivar, B. personal or other relationships with other people or organizations Kalyanaraman, Superoxide reacts with hydroethidine but forms a fluorescent that could inappropriately influence, or be perceived to influence product that is distinctly different from ethidium: potential implications in our work. intracellular fluorescence detection of superoxide, Free Radic. Biol. Med. 34 (2003) 1359–1368. [27] E. Pick, D. Mizel, Rapid microassays for the measurement of superoxide and References hydrogen peroxide production by macrophages in culture using an automatic enzyme immunoassay reader, J. Immunol. Methods. 46 (1981) 211–226. [28] A.H. Ding, C.F. Nathan, D.J. Stuehr, Release of reactive nitrogen intermediates [1] P. Singer, H. Shapiro, M. Theilla, R. Anbar, J. Singer, J. Cohen, Anti-inflammatory and reactive oxygen intermediates from mouse peritoneal macrophages. properties of omega-3 fatty acids in critical illness: novel mechanisms and an Comparison of activating cytokines and evidence for independent production, integrative perspective, Intensive Care Med. 34 (2008) 1580–1592. J. Immunol. 141 (1988) 2407–2412. [2] G.E. Caughey, E. Mantzioris, R.A. Gibson, L.G. Cleland, M.J. James, The effect on [29] G. Grynkiewicz, M. Poenie, R.Y. Tsien, A new generation of Ca2+ indicators with human tumor necrosis factor alpha and interleukin 1 beta production of diets greatly improved fluorescence properties, J. Biol. Chem. 260 (1985) 3440–3450. enriched in n-3 fatty acids from vegetable oil or fish oil, Am. J. Clin. Nutr. 63 [30] G.P. Drummen, L.C. van Liebergen, J.A. Op den Kamp, J.A. Post, C11- (1996) 116–122. BODIPY(581/591), an oxidation-sensitive fluorescent lipid peroxidation [3] B.M. Babior, Phagocytes and oxidative stress, Am. J. Med. 109 (2000) 33–44. probe: (micro)spectroscopic characterization and validation of methodology, [4] M.P. Fletcher, V.A. Ziboh, Effects of dietary supplementation with Free Radic. Biol. Med. 33 (2002) 473–490. eicosapentaenoic acid or gamma-linolenic acid on neutrophil phospholipid [31] J.F. Ewing, D.R. Janero, Microplate superoxide dismutase assay employing a fatty acid composition and activation responses, Inflammation 14 (1990) 585– nonenzymatic superoxide generator, Anal. Biochem. 232 (1995) 243–248. 597. [32] H. Aebi, Catalase in vitro, Methods Enzymol. 105 (1984) 121–126. [5] S. Bartelt, M. Timm, C.T. Damsgaard, E.W. Hansen, H.S. Hansen, L. Lauritzen, [33] B. Mannervik, Glutathione peroxidase, Methods Enzymol. 113 (1985) 490– The effect of dietary fish oil-supplementation to healthy young men on 495. oxidative burst measured by whole blood chemiluminescence, Br. J. Nutr. 99 [34] R.L. Levine, J.A. Williams, E.R. Stadtman, E. Shacter, Carbonyl assays for (2008) 1230–1238. determination of oxidatively modified proteins, Methods Enzymol. 233 (1994) [6] M. Tanito, R.S. Brush, M.H. Elliott, L.D. Wicker, K.R. Henry, R.E. Anderson, High 346–357. levels of retinal membrane docosahexaenoic acid increase susceptibility to [35] E. Murillo, Hypercholesterolemic effect of canthaxanthin and astaxanthin in stress-induced degeneration, J. Lipid Res. 50 (2009) 807–819. rats, Arch. Latinoam. Nutr. 42 (1992) 409–413. [7] D.A. Healy, F.A. Wallace, E.A. Miles, P.C. Calder, P. Newsholm, Effect of low-to- [36] W. Aoi, Y. Naito, Y. Takanami, T. Ishii, Y. Kawai, S. Akagiri, Y. Kato, T. Osawa, T. moderate amounts of dietary fish oil on neutrophil lipid composition and Yoshikawa, Astaxanthin improves muscle lipid metabolism in exercise via function, Lipids 35 (2000) 763–768. inhibitory effect of oxidative CPT I modification, Biochem. Biophys. Res. [8] P.C. Calder, Polyunsaturated fatty acids, inflammation and immunity, Lipids 36 Commun. 366 (2008) 892–897. (2001) 1007–1024. [37] R. Mattei, T.G. Polotow, C.V. Vardaris, B.A. Guerra, J.R. Leite, R. Otton, M.P. [9] F. Gueraud, M. Atalay, N. Bresgen, A. Cipak, P.M. Eckl, L. Huc, I. Jouanin, W. Barros, Astaxanthin limits fish oil-related oxidative insult in the anterior Siems, K. Uchida, Chemistry and biochemistry of lipid peroxidation products, forebrain of Wistar rats: putative anxiolytic effects?, Pharmacol Biochem. Free Radic. Res. 44 (2010) 1098–1124. Behav. 99 (2011) 349–355. [10] J.T. Venkatraman, D.R. Pendergast, Effect of dietary intake on immune function [38] S.L. Connor, W.E. Connor, Are fish oils beneficial in the prevention and treatment in athletes, Sports Med. 32 (2002) 323–337. of coronary artery disease?, Am J. Clin. Nutr. 66 (1997) 1020S–1031S. [11] K.P. High, Micronutrient supplementation and immune function in the elderly, [39] M.M. Engler, M.B. Engler, M. Malloy, E. Chiu, D. Besio, S. Paul, M. Stuehlinger, J. Clin. Infect. Dis. 28 (1999) 717–722. Morrow, P. Ridker, N. Rifai, M. Mietus-Snyder, Docosahexaenoic acid restores [12] R. Otton, D.P. Marin, A.P. Bolin, C. Santos Rde, T.G. Polotow, S.C. Sampaio, M.P. endothelial function in children with hyperlipidemia: results from the EARLY de Barros, Astaxanthin ameliorates the redox imbalance in lymphocytes of study, Int. J. Clin. Pharmacol. Ther. 42 (2004) 672–679. experimental diabetic rats, Chem. Biol. Interact. 186 (2010) 306–315. [40] W.E. Connor, C.A. DeFrancesco, S.L. Connor, N-3 fatty acids from fish oil. Effects [13] D.P. Marin, A.P. Bolin, C. Macedo Rde, S.C. Sampaio, R. Otton, ROS production in on plasma lipoproteins and hypertriglyceridemic patients, Ann. N. Y. Acad. Sci. neutrophils from alloxan-induced diabetic rats treated in vivo with 683 (1993) 16–34. astaxanthin, Int. Immunopharmacol. 11 (2011) 103–109. [41] R. Otton, D.P. Marin, A.P. Bolin, R. de Cassia Santos Macedo, T.R. Campoio, C. [14] M.P. Barros, E. Pinto, P. Colepicolo, M. Pedersen, Astaxanthin and peridinin Fineto Jr., B.A. Guerra, J.R. Leite, M.P. Barros, R. Mattei, Combined fish oil and inhibit oxidative damage in Fe(2+)-loaded liposomes: scavenging oxyradicals astaxanthin supplementation modulates rat lymphocyte function, Eur. J. Nutr. or changing membrane permeability?, Biochem Biophys. Res. Commun. 288 (2011) [Epub ahead of print]. (2001) 225–232. [42] H. Hattori, H. Imai, K. Furuhama, O. Sato, Y. Nakagawa, Induction of [15] S. Goto, K. Kogure, K. Abe, Y. Kimata, K. Kitahama, E. Yamashita, H. Terada, phospholipid hydroperoxide glutathione peroxidase in human Efficient radical trapping at the surface and inside the phospholipid membrane polymorphonuclear neutrophils and HL60 cells stimulated with TNF-alpha, is responsible for highly potent antiperoxidative activity of the carotenoid Biochem. Biophys. Res. Commun. 337 (2005) 464–473. astaxanthin, Biochim. Biophys. Acta 1512 (2001) 251–258. [43] M.M. Gallogly, J.J. Mieyal, Mechanisms of reversible protein glutathionylation [16] A. Cort, N. Ozturk, D. Akpinar, M. Unal, G. Yucel, A. Ciftcioglu, P. Yargicoglu, M. in redox signaling and oxidative stress, Curr. Opin. Pharmacol. 7 (2007) 381– Aslan, Suppressive effect of astaxanthin on retinal injury induced by elevated 391. intraocular pressure, Regul. Toxicol. Pharmacol. 58 (2010) 121–130. [44] R.W. Watson, Redox regulation of neutrophil apoptosis, Antioxid. Redox [17] L. Kupcinskas, P. Lafolie, A. Lignell, G. Kiudelis, L. Jonaitis, K. Adamonis, L.P. Signal. 4 (2002) 97–104. Andersen, T. Wadstrom, Efficacy of the natural antioxidant astaxanthin in the [45] J. Savill, C. Haslett, Granulocyte clearance by apoptosis in the resolution of treatment of functional dyspepsia in patients with or without Helicobacter inflammation, Semin. Cell. Biol. 6 (1995) 385–393. pylori infection: a prospective, randomized, double blind, and placebo- [46] S.J. Leuenroth, P.S. Grutkoski, A. Ayala, H.H. Simms, Suppression of PMN controlled study, Phytomedicine 15 (2008) 391–399. apoptosis by hypoxia is dependent on Mcl-1 and MAPK activity, Surgery 128 [18] X. Liu, T. Shibata, S. Hisaka, T. Osawa, Astaxanthin inhibits reactive oxygen (2000) 171–177. species-mediated cellular toxicity in dopaminergic SH-SY5Y cells via [47] C. Ward, T.H. Wong, J. Murray, I. Rahman, C. Haslett, E.R. Chilvers, A.G. Rossi, mitochondria-targeted protective mechanism, Brain Res. 1254 (2009) 18–27. Induction of human neutrophil apoptosis by nitric oxide donors: evidence for a [19] M. Guerin, M.E. Huntley, M. Olaizola, Haematococcus astaxanthin: caspase-dependent, cyclic-GMP-independent, mechanism, Biochem. applications for human health and nutrition, Trends Biotechnol. 21 (2003) Pharmacol. 59 (2000) 305–314. 210–216. [48] M.S. Joshi, J.L. Ponthier, J.R. Lancaster Jr., Cellular antioxidant and pro-oxidant [20] T.J. Rimele, R.J. Sturm, L.M. Adams, D.E. Henry, R.J. Heaslip, B.M. Weichman, D. actions of nitric oxide, Free Radic. Biol. Med. 27 (1999) 1357–1366. Grimes, Interaction of neutrophils with vascular smooth muscle: identification [49] B.P. Chew, J.S. Park, Carotenoid action on the immune response, J. Nutr. 134 of a neutrophil-derived relaxing factor, J. Pharmacol. Exp. Ther. 245 (1988) (2004) 257S–261S. 102–111. [50] D. Petri, A.K. Lundebye, Tissue distribution of astaxanthin in rats following [21] S.C. Sampaio, M.C. Sousa-e-Silva, P. Borelli, R. Curi, Y. Cury, Crotalus durissus exposure to graded levels in the feed, Comp. Biochem. Physiol. C. Toxicol. terrificus snake venom regulates macrophage metabolism and function, J. Pharmacol. 145 (2007) 202–209. Leukoc. Biol. 70 (2001) 551–558. [51] H. Jyonouchi, S. Sun, K. Iijima, M.D. Gross, Antitumor activity of astaxanthin [22] M.W. McGowan, J.D. Artiss, D.R. Strandbergh, B. Zak, A peroxidase-coupled and its mode of action, Nutr. Cancer 36 (2000) 59–65. method for the colorimetric determination of serum triglycerides, Clin. Chem. [52] L.A. Showalter, S.A. Weinman, M. Osterlie, S.F. Lockwood, Plasma appearance 29 (1983) 538–542. and tissue accumulation of non-esterified, free astaxanthin in C57BL/6 mice [23] P. Trinder, Determination of blood glucose using 4-amino phenazone as after oral dosing of a disodium disuccinate diester of astaxanthin (Heptax), oxygen acceptor, J. Clin. Pathol. 22 (1969) 246. Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 137 (2004) 227–236. [24] I. Rahman, A. Kode, S.K. Biswas, Assay for quantitative determination of [53] M. Ohno, W.S. Darwish, Y. Ikenaka, W. Miki, M. Ishizuka, Astaxanthin can alter glutathione and glutathione disulfide levels using enzymatic recycling CYP1A-dependent activities via two different mechanisms: induction of method, Nat. Protoc. 1 (2006) 3159–3165. protein expression and inhibition of NADPH P450 reductase dependent [25] C.G. Fraga, B.E. Leibovitz, A.L. Tappel, Lipid peroxidation measured as electron transfer, Food Chem. Toxicol. 49 (2011) 1285–1291. thiobarbituric acid-reactive substances in tissue slices: characterization and M.P. Barros et al. / Chemico-Biological Interactions 197 (2012) 58–67 67

[54] H.D. Choi, H.E. Kang, S.H. Yang, M.G. Lee, W.G. Shin, Pharmacokinetics and [59] W.I. Gruszecki, J. Sielewiesiuk, Orientation of xanthophylls in first-pass metabolism of astaxanthin in rats, Br. J. Nutr. 105 (2011) 220–227. phosphatidylcholine multibilayers, Biochim. Biophys. Acta 1023 (1990) 405– [55] K. Nakagawa, S.D. Kang, D.K. Park, G.J. Handelman, T. Miyazawa, Inhibition of 412. beta-carotene and astaxanthin of NADPH-dependent microsomal [60] X. Yang, W. Sheng, G.Y. Sun, J.C. Lee, Effects of fatty acid unsaturation numbers phospholipid peroxidation, J. Nutr. Sci. Vitaminol. (Tokyo) 43 (1997) 345–355. on membrane fluidity and alpha-secretase-dependent amyloid precursor [56] T.D. Hill, J.G. White, G.H. Rao, Role of glutathione and glutathione peroxidase in protein processing, Neurochem. Int. 58 (2011) 321–329. human platelet arachidonic acid metabolism, Prostaglandins 38 (1989) 21–32. [61] J.J. Lara, M. Economou, A.M. Wallace, A. Rumley, G. Lowe, C. Slater, M. Caslake, [57] D.A. Hughes, Carotenoids and Immune Responses, in: N.I. Krinsky, S.T. Mayne, N. Sattar, M.E. Lean, Benefits of salmon eating on traditional and novel vascular H. Sies (Eds.), Carotenoids in Health and Disease, Marcel Dekker Inc., New risk factors in young, non-obese healthy subjects, Atherosclerosis 193 (2007) York, 2004, pp. 503–517. 213–221. [58] G. Leonarduzzi, B. Sottero, G. Poli, Targeting tissue oxidative damage by means of cell signaling modulators: the antioxidant concept revisited, Pharmacol. Ther. 128 (2010) 336–374.