Combined astaxanthin and fish oil supplementation improves
glutathione-based redox balance in rat plasma and neutrophils
Marcelo Paes Barrosa
, Douglas Popp Marinb
, Anaysa Paola Bolinb
, Rita de Cássia Santos Macedob
Thais Regina Campoiob
, Claudio Fineto Jr.b
, Beatriz Alves Guerrab
, Tatiana Geraldo Polotowa
, Rita Matteic
, Rosemari Ottonb,⇑
aPostgraduate Program, Human Movement Sciences, Institute of Physical Activity and Sport Sciences (ICAFE), Universidade Cruzeiro do Sul, 01506000 São Paulo, SP, Brazil bPostgraduate Program, Health Sciences, CBS, Universidade Cruzeiro do Sul, 03342000 São Paulo, SP, Brazil
cDepartment of Psychobiology, Universidade Federal de São Paulo (UNIFESP), 04023062 São Paulo, SP, Brazil
a r t i c l e
i n f o
Received 3 November 2011
Received in revised form 9 March 2012 Accepted 10 March 2012
Available online 21 March 2012
DHA EPA Omega-3 Carotenoid Anti-inflammatory Leukocyte
a b s t r a c t
The present study aimed to investigate the effects of daily (45 days) intake of fish oil (FO; 10 mg EPA/kg body weight (BW) and 7 mg DHA/kg BW) and/or natural ASTA (1 mg ASTA/kg BW) on oxidative stress and functional indexes of neutrophils isolated from Wistar rats by monitoring superoxide (O
2), hydrogen peroxide (H2O2), and nitric oxide (NO) production compared to the progression of auto-induced lipid 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-phils were compared to the redox status. Our results show evidence of the beneficial effects of FO + ASTA supplementation for immune competence based on the redox balance in plasma (significant increase in GSH-dependent reducing power), non-activated neutrophils (increased activity of the glutathione-recy-cling enzymes GPxand GR) and PMA-activated neutrophils (lower O
2, H2O2, and NOgeneration, reduced membrane oxidation, but higher phagocytic activity). Combined application of ASTA and FO promoted 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.
Ó2012 Elsevier Ireland Ltd.
Habitual intake of marine fish and seafood has been strongly associated with human health. It is particularly considered to enhance immune competence and prevent infectious diseases
. Fish oil (FO) components eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) – well-known as n-3 polyunsaturated fatty acids (n-3/PUFA) – have been studied in immune cells and proved to increase anti-inflammatory responses by stimulating interleukin production. As central players in inflammatory pro-cesses, neutrophils are main protagonists of innate immune re-sponses necessary for early host defense . Regarding nutraceuticals, dietary long-chain (>C20) n-3/PUFAs are considered antioxidant compounds, which significantly diminish superoxide (O
2) and hydrogen peroxide (H2O2) production in activated neutrophils[4,5]. On the other hand, fish oil also showed adverse
effects on immune function due to the influence of PUFAs on mem-brane fluidity, which could result in higher sensitivity to oxidation
[6,7]. Depending on the concentration, DHA and EPA can also affect the microstructure and composition of lipid rafts in the neutrophil membrane, resulting in severe changes of cell–cell molecular com-munication.
Although anti-inflammatory mechanisms in humans have been well discussed, there are still unanswered questions with respect to redox imbalances provoked in neutrophils during the pathogen-activation process. As a consensus, a wide range of reactive oxy-gen/nitrogen species ROS/RNS (including O
2, H2O2, and NO) are continuously produced by neutrophil oxidative burst, and pathogen cells are undoubtedly not the only targets at the inflammatory site. In fact, ROS/RNS can also (auto)-oxidize neutrophil membrane, trig-gering lipid peroxidation and producing lipid-derived cytotoxic aldehydes, e.g. malondialdehyde and 4-hydroxynonenal. Thus, long-living/persistent neutrophils as part of an efficient immune system have to cope with huge oxidative challenges imposed by pathogen activation. Several authors have, therefore, proposed that
0009-2797Ó2012 Elsevier Ireland Ltd.
⇑Corresponding author. Tel./fax: +55 11 26726200.
E-mail address:firstname.lastname@example.org(R. Otton).
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dietary programs based on antioxidant supplementation could of-fer enhancement of immune functions, especially in terms of life-span and slower turnover of damaged immune cells [10,11]. Accordingly, many antioxidants present in human diet have also positively affected immune functions in patients of ROS-related dis-eases, such as diabetes[12,13].
The marine carotenoid astaxanthin (ASTA;Fig. 1) is naturally found in a wide variety of aquatic organisms, such as microalgae, crustaceans (crabs, lobsters, and shrimp), and fishes (salmon and trout). ASTA is renowned as a powerful antioxidant[14,15], and also reported to afford benefits to human health, including photo-protection against UV radiation (skin and eyes), prophylaxis/ remediation of gastric ulcer induced byHelicobacter pylori , and ROS-mediated neurodegenerative processes, as in Parkinson’s disease. Thus, it is reasonable to assume that combined FO and ASTA supplementation could better balance pro- and antioxi-dative events in activated neutrophils to enhance anti-inflamma-tory responses and, thus, provide substantial health benefits to humans.
The present study aimed to investigate the effects of daily in-take of natural sources of n-3/PUFAs (here provided as fish oil) and/or natural ASTA (obtained from the biomass of the green mic-roalgaeHaematococcus pluvialis)on oxidative stress and func-tional indexes of neutrophils isolated from Wistar rats. This study was performed by monitoring superoxide (O
2), hydrogen peroxide (H2O2), and nitric oxide (NO) production compared to the progres-sion of auto-induced lipid peroxidation and Ca2+ release in acti-vated neutrophils. Furthermore, our study evaluated antioxidant enzyme activities, the glutathione-recycling system and biomark-ers of lipid and protein oxidation as related to the FO and/or ASTA diet, and examined the redox status in plasma as well as in isolated resting and stimulated neutrophils from experimental animals.
2. Materials and methods
All chemicals were of analytical grade and purchased from Sig-ma–Aldrich Chemical Company (St. Louis, MO, USA), except those used in the preparation of common buffers (Labsynth, Diadema, SP, Brazil). Commercial biochemical test kits were obtained from Bioclin-Quibasa Química Básica (Belo Horizonte, Brazil) for mea-surements of glucose (prod. code K082), triacylglycerol (prod. code K055) and cholesterol (prod. code k083) concentrations in plasma. The fluorescent probes acetoxymethyl ester (Fura 2-AM) and 4, 4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-inda cene-3-undecanoic acid (C11-BODIPY581/591) were purchased from Invitrogen (Grand Island, NY, USA).
2.2. Natural products
Fish oil capsules were purchased from Pharmanostra (Sao Paulo, SP, Brazil). Each fish oil (FO) capsule of 500
lL (corresponding to 9 kcal/38 kJ), contains 2.0 mg tocopherols, and 1.0 g of total fat, out of 30% which saturated fats, 20% with monounsaturated
(mostly palmitoleic and oleic acids), and 50% with polyunsaturated fatty acids (180 mg EPA and 120 mg DHA). Natural ASTA supple-ments (AstaREAL A1010) were obtained as a donation from the Swedish company BioReal AB (Gustavsberg, Sweden), a subsidiary of BioReal Inc., (Hawaii, USA) and part of the pharmaceutical Group Fuji Chemical Industry CO. AstaREAL A1010 is an astaxanthin-rich natural microalgal product, consisting of crushed and spray-dried aplanospores of the green microalgae Haematococcus pluvialis. AstaREAL A1010 contains 42% of crude fat, 10% of crude protein, 40% of carbohydrates, and 4% of ashes. Regarding carotenoid com-position, AstaREAL A1010 contains 5.2–5.8% of total carotenoids, 5.0–5.6% of which are being pure astaxanthin (3.9% as monoesters, 0.9% as diesters, and 0.1% in free form) among others.
2.3. Animals and supplementation protocols
Adult Wistar male rats, weighing 191 ± 35 g at the beginning of the study, were provided by the Department of Psychobiology, Uni-versidade Federal de São Paulo (UNIFESP), São Paulo, Brazil. All ani-mals were housed in plexiglas cage (four rats/cage), under standard laboratory conditions: 12 h light/dark cycle; lights on at 7:00 a.m.; 22 ± 1°C; water and Purina rat chowad libitum. The ani-mals used in this study were handled in accordance with the guidelines of the committee on care and use of laboratory animal resources of the Ethics Committee on Care and Use of Laboratory Animal Resources from Universidade Federal de Sao Paulo, which approved the study protocol (CEP/UNIFESP n°1938/09). The ani-mals were treated with ASTA and/or FO by gavage, 5 days a week, for 45 days. A maximum volume of 400
lL was established for the gavage treatment in order to prevent regurgitation or stomach dis-comfort of the animals. Fish oil (FO) content of capsules was di-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 7 mg DHA/kg BW. An identical procedure was conducted for ani-mal supplementation with 1 mg ASTA/kg BW using AstaREAL A1010 as the carotenoid source (5.3% of which is pure ASTA). For combined FO and ASTA treatments, both components were diluted in the same stock 10% Tween-80 aqueous solution (v/v) to reach previously described concentrations. Although additional antioxi-dants as ascorbate, tocopherols and other carotenoids were present in both manufactured natural products, their contribution in the total antioxidant capacity of gavage solutions is minor compared to the prevalent ASTA or n-3/PUFA components. Thus, four exper-imental groups of 16 animals each were formed: (i) control, fed with 400
lL of 10% Tween 80 aqueous solution (v/v); (ii) ASTA, fed with 1 mg ASTA/kg; (iii) FO, fed with 10 mg EPA/kg and 7 mg DHA/kg; and (iv) ASTA + FO, fed with 1 mg ASTA/kg, 10 mg EPA/ kg and 7 mg DHA/kg.
2.4. Neutrophil isolation
After 45 days of treatment, the rats were euthanized by decapi-tation between 11:00 a.m. and 01:00 p.m. to prevent circadian dif-ferences. Prior to decapitation (4 h), rats were intraperitoneally (i.p.) injected with 10 mL 1% (w/v) glycogen solution (Sigma type II, from oyster) in PBS to induce cell migration. Neutrophils were obtained by i.p. lavage with 20 mL PBS, followed by centrifugation in a density gradient of Histopaque 1077. The total number of leukocytes in exudate was determined in a haemocytometer. 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 present in the exudate 4 h after injection of oyster glycogen. Neutrophils (1106cell/mL) were resuspended in RPMI-1640 medium supplemented with 10% foetal calf serum, 20 mM HEPES, 2 mM glutamine, and antibiotics (streptomycin 100 U/mL and
H3C CH3 CH3 CH3
Fig. 1.Chemical structure of the marine carotenoid astaxanthin (ASTA).
penicillin 200 U/mL). Freshly obtained cells were used to measure phagocytic activity, ROS/RNS production, calcium release and membrane oxidation. All other experiments were performed with cells after they were frozen at80°C.
2.5. Phagocytic activity
Neutrophils (1106cell/mL) were incubated for 60 min at 37°C in 1 mL RPMI 1640 medium with zymosan. Particles were opsonized by incubation in the presence of control serum for 60 min at 37°C. Cells were incubated with particles and counted after cytocentrifugation. Scores of phagocytosis were expressed by the number of cells that had one, two, three, four, or more zymosan particles phagocyted.
2.6. Analyses in plasma
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 centrifuged for 10 min, 300gat room temperature. The clean plas-ma fraction was then removed and stocked in Eppendorf 2 mL-microtubes at80°C for further analyses.
2.6.2. Glucose, triacylglycerol and cholesterol determinations in plasma
Glucose, triacylglycerol (TAG), and cholesterol levels in plasma were all quantified by commercial test kits obtained from Bioclin-Quibasa Química Básica (Belo Horizonte, Brazil). All these assays were based on the peroxidase-catalysed oxidation of 4-ami-noantipyrine by hydrogen peroxide (H2O2) to generate a pink color
2.6.3. Glutathione redox status in plasma
Reduced glutathione content in plasma (GSH) was measured as described by Rahman et al.. The method is based on the reac-tion of reduced thiol groups (such as in GSH) with 5,50 -dithiobis-2-nitrobenzoic acid (DTNB) to form 5-thio-2--dithiobis-2-nitrobenzoic acid (TNB), which is stoichiometrically detected by absorbance at 412 nm. Purified GSH and GSSG were used as standards. The Reducing Power index reveals the ratio between GSH and GSSG – thus, the reducing capacity of a biological system – and is calculated by the equation (Eq.(1)):
Reducing Power¼ ½GSH=ð½GSH þ2½GSSGÞ ð1Þ
2.6.4. Lipid oxidation in plasma
The extent of lipid peroxidation in plasma was determined as the concentration of thiobarbituric acid-reactive substances (TBARS). Butylated hydroxytoluene (4% BHT, in ethanol) was added to stop progressing oxidation reactions in 500
lL diluted samples. For the detection of the colored adducts, 500
lL of each sample was incubated at 100°C for 15 min with 0.375% thiobarbi-turic acid in 0.25 M HCl and 1% Triton X-100. After reaching room temperature, absorbance of the reaction system was measured at 535 nm (blanks lack thiobarbituric acid) using malondialdehyde (MDA) as a standard.
2.7. Biochemical measurements in neutrophils
2.7.1. Superoxide radical (O
The lipophilic and freely diffusible probe dihydroethidium (DHE) was used for the fluorimetric measurement of intracellular production of O
2 . Once inside cells, DHE is rapidly oxidized to ethidium (a red fluorescent compound) by O
2 and H2O2 (in the presence of peroxidase). Ethidium is trapped in the nucleus
by intercalating into DNA, leading to an increase of ethidium fluo-rescence. The cells (5105/well) were preloaded with 5
lM DHE for 15 min at room temperature in the dark. The assay was carried out in the presence and the absence of 20 ng/well phorbol myris-tate acemyris-tate (20 ng PMA/well), here applied as a chemical inducer of ROS production in neutrophils. Fluorescence was measured at 396 nm of excitation wavelength and at 590 nm of emission wave-length in a microplate fluorescence analyzer (Tecan, Salzburg, Austria).
2.7.2. Hydrogen peroxide production
The total production of H2O2was evaluated before/after chem-ical stimulation (with 20 ng PMA) of a 5105cell/well suspension using the horseradish peroxidase-dependent oxidation of phenol red (by H2O2) to form a vivid red color, which was spectrophoto-metrically quantified at 620 nm, using a H2O2concentration curve as a standard.
2.7.3. Nitric oxide production
Nitric oxide production was determined by spectrophotometric analysis of total nitrite, using Griess reagent (1% sulfanilic acid, 0.1%N-(1-naphthyl)ethylenediamine hydrochloride), according to Ding et al.. Supernatant of neutrophils (5105cell/well) with or without lipopolysaccharide (LPS-10
l/mL) was added to 100
lL of Griess reagent, the absorbance measured in 550 nm, and nitrite levels determined by means of a standard curve generated using NaNO2.
2.7.4. Intracellular Ca2+concentration
Intracellular changes of calcium levels (Ca2+) were monitored in neutrophils using the calcium-sensitive fluorescent probe Fura 2-AM. The 1106 neutrophil suspension was pre-loaded with 5
lM Fura 2-AM for 1 h at 37°C in Tyrode’s solution. Afterwards, neutrophils were washed and intracellular Ca2+release was moni-tored for 60 min at a wavelength emission of 510 nm (excitation wavelengths alternating between 340 and 380 nm) in a microplate reader (Tecan, Salzburg, Austria). Transformation of the fluores-cence signal to Ca2+ concentration was performed by calibration with ionomycin (100
lM, maximum concentration) and EGTA (60
lM, minimum concentration) according to the Grynkiewicz equation andKdissof 224 nM.
2.7.5. Membrane oxidation
The progression of membrane oxidation in PMA-activated neu-trophils was monitored by fluorescence kinetics of the C11-BODI-PY581/591probe, which is oxidized by alkoxyl and peroxyl radicals (strongly involved in lipid peroxidation progress) and loses thereby fluorescence emission in the red region. Briefly, 5105 neu-trophils were pre-incubated with 3.2
lM C11-BODIPY581/591 for 60 min at room temperature, under mechanical agitation and in the dark. The C11-BODIPY581/591-loaded neutrophils were placed in a microplate, and background fluorescence was adjusted to 1.0 relative fluorescence unit (RFU) and measured for 10 min (k excita-tion= 545 nm, kemission= 591 nm) in a microplate reader (Tecan, Salzburg, Austria). Afterwards, 100 ng PMA/5105 cells were added to trigger ROS production and start auto-induced membrane oxidation, which was followed for additional 15 min. A first-order exponential decay function (Eq.(2)) was applied to obtain kinetic parameters of lipid peroxidation in the neutrophil membrane.
yðRFUÞ ¼y0þA1eðt=kÞ1 ð2Þ
2.7.6. Preparation of cell homogenates
Neutrophils (5106/mL) were isolated and ruptured by ultra-sonication in a Vibra Cell apparatus (Connecticut, USA) in an ice-water bath using 500
lL of each assay-specific extraction solu-tion/buffer. Afterwards, ruptured neutrophils were centrifuged for 10 min, 10.000g at 4°C, and the supernatant was removed and kept on ice for further analysis (debris was discarded).
2.7.7. Assay of superoxide dismutase activity (SOD)
The activity of total superoxide dismutase (SOD) was measured according to Ewing & Janero, with modifications. The reaction system for total SOD determination included 50 mM sodium phos-phate buffer (pH 7.4), 0.1 mM EDTA, 50
lM nitroblue tetrazolium (NBT), 78
lM NADH, and 3.3
lM phenazine methosulphate (PMS), here used as a chemical O
2 source. Analysis of mitochondrial (man-ganese-dependent) MnSOD activity was performed similarly as for total/SOD, but under strong inhibition (88–100%) of the cytosolic CuZnSOD isoform by addition of 3
lM KCN. As internal controls of the experiment, all reagents without sample were used for both totalSOD and MnSOD determination. Absorbance at 550 nm was continuously monitored for over 2 min in order to evaluate O
2 -mediated reduction of NBT. One SOD unit is defined here as the enzyme concentration required for 50% inhibition of NBT reduction at 25°C.
2.7.8. Assay of catalase activity
The decomposition of H2O2was directly followed for 2 min by measuring the decrease in absorbance at 240 nm using a molar extinction of
e240= (0.0394 ± 0.0002) LmM1cm1, as described by Aebi. One catalase (CAT) unit is defined as the enzyme con-centration required for the decomposition of 1
lmol of H2O2per min at 25°C. As internal control, 50 mM (pH 7.4) phosphate buffer was added to H2O2without sample and monitored for 2 min.
2.7.9. Assay of the glutathione-recycling enzyme system
The activity of glutathione peroxidase (GPx) was measured according to Mannervikusing 2.5 U/mL glutathione reductase (GR), 10 mM reduced glutathione (GSH), 250
lM sodium azide (as a catalase inhibitor), and 1.2 mM NADPH. The reaction was trig-gered by addition of 4.8 mM tert-butyl hydroperoxide (a GPx sub-strate). The other GSH-recycling component enzyme, glutathione reductase (GR), was, in turn, measured by a direct reaction be-tween 3.6 mM NADPH and 10 mM oxidized glutathione (GSSG). Absorbance of NADPH oxidation (in 0.2 M phosphate buffer, pH 7.4) was monitored in both cases at 340 nm for 2 min in an Ultro-spec 3000 Ultro-spectrophotometer (Pharmacia Biotech).
2.7.10. Oxidative stress parameters
Carbonyl groups were evaluated as parameters of amino acid oxidation in neutrophils. After pre-treatment with 10% streptomy-cin to remove interfering nucleic acids, the protein fraction was iso-lated from homogenate by precipitation with 20% trichloroacetic acid solution on ice, followed by the subsequent processes: (i) washing once with 0.3 M HClO4, 5 mM EDTA, and 0.06% 2,20 -bipyr-idine solution; (ii) washing twice with the mixture 1:1 ethyl ace-tate:ethanol (v/v); and (iii) removing residual organic solvent in vacuum for, at least, 30 min. Afterwards, the protein pellets were fully dissolved in 6 M guanidine hydrochloride and used for detec-tion of the carbonyl groups after reacdetec-tion with 10 mM dinitrophen-ylhydrazine (DNPH) in 0.25 M HCl (blanks lack DNPH). Absorbance was recorded at 380 nm, and the carbonyl group concentration was calculated using an extinction coefficient ofk= 2.2104M1 cm1
. Reduced and oxidized glutathione (GSH and GSSG, respec-tively) and TBARS (biomarker of lipid oxidation) were also measured in homogenates of neutrophils, following the same procedures as described for plasma samples.
2.8. Statistical analyses
All data are presented as the mean values of, at least, triplicates with their standard errors (MEAN ± SE). Data were analyzed by one-way ANOVA followed by Tukey’s post-test, considering p< 0.05, p< 0.01, orp< 0.005. The software Origin 6.1 (v6.1052/B232; Orig-inLab Corporation, Northampton, MA, USA) was used for statistical analyses and graph preparation.
3.1. Plasma indexes
All the experimental groups showed similar growth rates (BW) during the 45-day supplementation period (data not shown), although ASTA supplementation alone distinctively increased lipi-demia (55%) and cholesterolemia (35%, compared to control;Table 1). This effect was not observed in animals from the FO + ASTA group. Regarding redox balance, combined FO + ASTA clearly en-hanced the antioxidant capacity of plasma, especially when com-paring GSH/GSSG ratios (6-fold higher). Basal NOconcentration in plasma was also increased in FO + ASTA animals (65%, Table 1), whereas no significant difference was observed in terms of lipid oxidation.
3.2. Non-stimulated neutrophils
The redox balance was also studied in neutrophils isolated from the experimental animals, as shown in Table 2. Concerning the frontline antioxidant enzymes (Table 2), ASTA alone provided the most significant effects, especially on the GSH-recycling enzymes GPxand GR (increased by 92% and 52%, respectively). However, GSH content and GSH-related Reducing Power (see Section2) were abruptly depleted in neutrophils of ASTA-fed animals (both by approximately 70%;Table 2). FO treatment caused a different effect on neutrophils, since massive decreases in GPx(50%) and GR activ-ities (65%) were accompanied by proportionally augmented levels of GSH (80%) and Reducing Power (89%). Combined FO + ASTA sup-plementation resulted in increased activities of total SOD (20%), mitochondrial MnSOD (22%), GPx(62%), and GR (36%). Unexpect-edly, all supplementation treatments resulted in lower activities of CAT: 35% in ASTA group, and 45% in both FO and FO + ASTA ani-mals (compared to control). Despite all changes observed in GSH content, none of the experimental groups showed statistically dif-ferent indexes of oxidative modification in lipids (TBARS content) or proteins (carbonyls) compared to control neutrophils, except protein carbonyls of ASTA-fed animals (33% higher than control).
3.3. Activated neutrophils
In order to evaluate one of the most important neutrophil-func-tional parameters, after 45-day of treatment with FO and/or ASTA, neutrophils were challengedin vitrowith opsonized zymosan par-ticles (phagocytic capacity). The effect of ASTA and/or FO supple-mentation on neutrophil was also evaluated by measuring ROS/ RNS production and auto-induced lipid peroxidation after chemical (PMA-) activation of neutrophils. As shown inFig. 2, the phagocytic capacity of neutrophils was especially improved by FO supplemen-tation (approximately 2.4-fold higher), whereas ASTA only displayed marginal effects (not significant, compared to control; p= 0.180).
In order to investigate whether FO and/ASTA can exert effects on O
2 production, freshly isolated neutrophils were incubated with DHE for 15 min. All supplementation protocols resulted in higher basal O
2 production in non-stimulated neutrophils. This effect
was strongly related to ASTA presence, since ASTA-, FO-, and FO + ASTA-fed animals showed 53%, 17%, and 65% higher O
2 basal production in neutrophils, respectively (compared to control). Upon PMA-activation, however, a different trend was observed. FO alone or as a component of FO + ASTA supplementation signifi-cantly reduced O
2 production by approximately 10% (Fig. 3). H2O2production in both rested and activated neutrophils fol-lowed the same trend as observed for O
2 production, except for PMA-activated neutrophils of ASTA-fed group, which showed 25% lower H2O2production than control neutrophils (Fig. 4).
On the other hand, NOproduction (and probably other RNS that generate nitrite as product) was apparently more affected by ASTA supplementation than by FOper se(Fig. 5). Even though no signif-icant difference was observed in rested neutrophils, LPS stimulus only resulted in 45% lower NOproduction in neutrophils isolated from ASTA-fed animals (either alone or combined with FO). Concomitantly, the influx of Ca2+ions in LPS-activated neutrophils 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.
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.
0 100 200
300 §C #C
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 ± -SEM of three different experiments (n= 8 rats) performed in triplicate.§p< 0.01 or #p< 0.005; related to C (Control).
0 15000 30000 45000 60000
Control ASTA FO FO+ASTA +PMA -PMA
Fig. 3.Superoxide anion production was measured in neutrophils (5105per
well) under baseline and PMA-stimulated conditions. Cells were stimulated with PMA (20 ng/well) for 30 min and superoxide anion generation was measured by dihydroethidium (5lM) 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 unaltered regardless of the supplementation protocol (ASTA and/or FO, data not shown).
The progression of auto-induced lipid peroxidation after PMA-stimulation in neutrophils was monitored by C11-BODIPY581/591 fluorescence decay (Fig. 6). Based on a first-order exponential de-cay function (see Materials and Methods), it is clear that the plas-ma membrane of neutrophils from all supplemented groups was less exposed to oxidative injury than that from untreated control animals. Taking kinetic constants into account (k1), ASTA supple-mentation was proven to limit the progression of auto-oxidation in neutrophil membrane after PMA-stimulus, regardless of FO presence (Table 3). All fitting exponential curves were relatively well correlated (R2P0.600).
4.1. Plasma parameters
As observed by other authors, ASTA supplementation alone (1 mg/kg herein) resulted in hyperlipidemia and hypercholesterol-emia in experimental animals (Table 1). Similar effects were also observed in rats supplemented with the marine carotenoid canthaxanthin, the immediate precursor of ASTA in the
carotenogenesis pathway. The hypercholesterolemic effect of these xanthophylls is not related to reported mechanisms of non-polar carotenoids in mammalians, because beta-carotene does not induce changes in plasma cholesterol.
Higher lipid concentration in plasma of ASTA-fed rats was not followed by hyperglycemic conditions or increased antioxidant capacity, as demonstrated by GSH/GSSG scores and oxidative modifications in plasma lipids (Table 1). Based on previous studies from our group, the ASTA effect on plasma glucose, cholesterol or triacylglycerol concentrations is supposedly dependent on the carotenoid content in the diet, since those changes were not ob-served when animals were fed with a 20-fold higher dose of ASTA
. Interestingly, Aoi et al. (2008) showed that exercising mice fed with 0.02% (w/w) ASTA consumed more lipids than glucose as energy source, resulting in an increased running time to exhaustion
. Although FO supplementation alone did not alter physiologi-cal or redox parameters in plasma (compared to control), its combi-nation with ASTA substantially augmented GSH content, its redox turnover (conversion of oxidized GSSG back to its reduced form, GSH) and, consequently, thiol-dependent reducing power of plasma (Table 1). Glutathione-based and lipid peroxidation markers in plasma confirm our previous results of the biochemical and puta-tive anxiolytic effects of the ASTA + FO treatment. Important hypolipidemic and hypocholesterolemic effects were also observed in the ASTA + FO group (compared to ASTA-fed animals), suggesting a specific effect of FO supplementation, since similar results were also found in the FO-group. Accordingly, experiments in humans indicate a profound hypolipidemic effect of FO, especially by lower-ing plasma triacylglycerol. In agreement with Engler et al., we also observed hypolipidemic/hypocholesterolemic effects asso-ciated with 65% higher NOconcentration in plasma of FO + ASTA supplemented Wistar rats (Table 1), although supplementation with FO alone demonstrated a non-significant tendency for higher values (p= 0.175). Reduction in lipidemia and cholesterolemia cou-pled to adequate redox rebalance and higher NObioavailability in plasma has been currently suggested as positive events for prevent-ing the progression of early coronary heart disease in high-risk patients.
4.2. Non-activated neutrophils
The long-term FO + ASTA supplementation resulted in substan-tial antioxidant responses in rat neutrophils, compared to control. Both total and mitochondrial MnSOD activities were proportion-ally increased by approximately 20%, whereas CAT showed 43% lower activities (Table 2). Higher SOD activities in neutrophils from 0
10 20 30 40 50
Control ASTA FO FO+ASTA + P M A - P M A
Fig. 4.Measurement of hydrogen peroxide production in neutrophils (5105per
well) under baseline and PMA-stimulated conditions. Hydrogen peroxide produc-tion was induced with PMA (20 ng/well) for 1 h, and measured as absorbance increase at 620 nm due to horseradish peroxidase-dependent oxidation of phenol red. Results are expressed as mean ± SEM of three different experiments (n= 8 rats) performed in triplicate.⁄p< 0.05;§p< 0.01; or#p< 0.005, related to C (Control). #significantly different (#p< 0.005) between C + PMA group and ASTA + PMA or
FO + PMA groups.
0 10 20 30 40 50
Control ASTA FO FO+ASTA
Fig. 5.Measurement of nitric oxide production in neutrophils (5105per well)
under baseline and LPS-stimulated conditions. Cells were stimulated with LPS (10lg/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 rats) performed in triplicate.⁄p< 0.05; or#p< 0.005, related to C (Control), A (ASTA),
FO (Fish oil), FOA (Fish oil + ASTA).
0 400 800 1200 1600 0,90
0,95 1,00 1,2 1,4
Control ASTA FO FO+ASTA
Fig. 6.The progression of auto-induced lipid peroxidation after PMA-stimulation in neutrophils monitored by C11-BODIPY fluorescence decay. Cells (5105/well) were
pre-loaded with 3.2lM C11-BODIPY581/591(60 min). PMA (100 ng) was added to
trigger ROS production and membrane oxidation. Results are expressed as mean ± SEM of three different experiment (n= 8 rats) performed in triplicate.
FO + ASTA group were possibly a response to the significant raise in basal O
2 production of neutrophils (Fig. 3). Accordingly, basal H2O2production – product of O
2 dismutation by SOD isoforms – was also augmented (60%) in non-activated neutrophils of the FO + ASTA group (Fig. 4). Thus, in order to cope with the increase of cytotoxic H2O2 concentrations and the unresponsiveness of CAT activity (Table 2), the GSH-dependent enzyme system, com-prised by coupling enzymes GPx/GR, was upregulated (62% and 36%, respectively) to stabilize the reducing power of resting FO + ASTA neutrophils (Table 2). Interestingly, neutrophils from ASTA-fed animals also showed higher GPx/GR activities, lower CAT activity, but unaltered SOD isoform activities: under these cir-cumstances, the reducing power was drastically diminished by 65% and protein oxidation was exacerbated by 33% (Table 2). Organic peroxides, main products of lipid/membrane oxidation, and H2O2 are regular substrates for GPxin most cells. We assume that spe-cific GSH-dependent enzymes GPx/GR – but not CAT – were upreg-ulated as cellular responses to circumvent increased levels of organic peroxides from exacerbated lipid peroxidation, but not H2O2itself.Fig. 6corroborates this hypothesis. Similar results were obtained by our group in an analogous study performed in lymph nodes lymphocytes. Increased activities of total/SOD, GR and GPx were observed in lymphocytes from rats supplemented with FO + ASTA. Singular cells in the immune system, such as neu-trophils and lymphocytes, which exert extremely different func-tions in the innate and adaptive immune response, respectively, can present similar responses to supplemention with FO + ASTA. Hattori et al.reported the induction of GPxactivity by H2O2 exposure in neutrophils, although a key participation of the inflam-matory cytokine TNF-
awas also evidenced. Furthermore, the depletion observed in neutrophil GSH content in FO + ASTA (and ASTA-fed) animals is consistent with specific percentage changes of GPx and GR, which favors the consumption of reduced GSH forms. On the other hand, proportional increases of GSSG content were not observed (Table 2). One plausible hypothesis for that fact is the substantial utilization of a fraction of the total GSH content forS-glutathionylation of proteins, a reversible post-translational modification that provides protection of protein thiol groups from oxidation and thus proteins from denaturation, concomitantly serving as a redox signal transducer. Reinforcing the afore-mentioned key participation of SOD isoforms in sustaining redox balance and functionality of neutrophils, no changes in protein oxi-dation scores were observed in neutrophils of FO + ASTA animals, although a significant increase (33%) was found in neutrophils of ASTA-fed animals. Altogether, these data depict the redox scenario of most circulating (rested?) neutrophils from experimental groups and, thus, are possibly related to the life-span of neutrophils under non-inflammatory conditions. On the other hand, neutrophil (short) life-span is actually more dependent on the occurrence of inflammatory events, since migration, infiltration, and particularly the oxidative burst during pathogen (or chemical, herein) activa-tion expose these immune cells to harsher oxidative condiactiva-tions. Thus, it is desirable to investigate the redox balance and progres-sion of oxidative insult in active neutrophils, exactly during their
massive exposure to higher concentrations of self-generated ROS/ RNS. Furthermore, a crucial mechanism of cell death – apoptosis – is tightly related to redox unbalances in active neutrophils.
4.3. Activated neutrophils
Fish oil supplementation itself was related to the increased phagocytic activity of neutrophils (both FO and FO + ASTA groups), since ASTA supplementation alone did not result in higher phago-cytic scores (Fig. 2). Moreover, after PMA activation, neutrophils from FO-fed groups (both FO- and FO + ASTA, but not from ASTA-group) showed reduced O
2 production, whereas all supplemented animals had lower H2O2generation (Figs. 3 and 4). These data sug-gest that phagocytic activity was more closely related to changes in O
2 production of activated neutrophils and/or to pre-acquired po-sitive redox balance during the FO- and FO-ASTA supplementation period. Nevertheless, it is plausible to suggest that the improve-ment of the immune function (phagocytic activity herein) was a di-rect effect of FO supplementation in our experimental groups. Contrasting with this result, FO + ASTA supplementation promoted a reduced proliferative capacity of activated T- and B-lymphocytes as showed in our previous study. The consensus from histolog-ical studies and tracking fluorescent-targeted cells is that the majority of inflammatory neutrophils never leave an inflamed site to return to blood stream, since most activated cells diein situdue to cellular processes closely related to oxidative stress: apoptosis or necrosis. It is well conceived that apoptosis is the preferred injury limiting mechanism for the removal of non-functional neu-trophils from inflamed sites. Moreover, it is notably related to aug-mented rates of ROS/RNS production (by intrinsic pathway), Ca2+ influx disruption, and progressive oxidative events in several im-mune cells, including neutrophils. Therefore, it is possible that the antioxidant background in neutrophils (obtained during the supplementation period) could affect: (i) their life-span as active immune cells; (ii) their functionality during inflammatory pro-cesses; and (iii) the mechanism by which active neutrophils are de-graded (with consequences for remaining cells and tissue). As also observed in lymphocytes, ASTA supplementation here offered putative anti-apoptotic effects in neutrophils, based on reduced production of NOand unaltered intracellular Ca2+release (Fig. 5). In addition, the powerful antioxidant activity of ASTA  was here evidenced in neutrophils by the significantly lower rate con-stant (k1;Table 3) of the exponential progression of membrane oxi-dation after PMA activation in neutrophils from ASTA-fed animals (both ASTA- and FO + ASTA groups;Fig. 6). This sensitive assay by the radical-sensitive probe C11-BODIPY depicts the slower pro-gression of lipid oxidation in neutrophil membrane from both ASTA- and FO + ASTA-fed animals (see Section2). Hence, it is rea-sonable to assume that ASTA supplementation offered extended protection to auto-induced oxidation in neutrophils membrane, with possible influence on cell survival and life-span.
Putative anti-apoptotic effects in rat neutrophils are here suggested from combined FO + ASTA supplementation, since lower O
2 production was concomitantly monitored with lower NO 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/
or FO (equivalent to 10 mg EPA/kg and 7 mg DHA/kg). First-order exponential decay:y(RFU) =y0+A1.e(t/k1)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
concentrations, and both radicals promptly reacted in intracellular compartments to produce the pro-apoptotic factor, peroxynitrite (ONOO). Interestingly, low doses of NOwere shown to pre-vent killing of rat hepatocytes bytert-butylhydroperoxide, whereas further increasing doses resulted in increased killing. This dual pro-/antioxidant role of NOhas been also reported in several other cell types and could represent an important redox switch for triggering apoptosis in neutrophils.
4.4. Mechanistic aspects
A bulk of data has demonstrated that the beneficial action of dietary carotenoids on the immune system is independent of their provitamin A or retinal-derived activities. Most of the immune improving effects provided by nonprovitamin A carotenoids – such as lycopene, canthaxanthin, lutein and ASTA – on humans and ani-mals has been strongly associated with their antioxidant properties, even though recent studies also addressed their role in gene regula-tion, apoptosis and angiogenesis. The pharmacodynamics of ASTA plays an essential role in determining its plasma concentra-tion, putative direct interaction with circulating neutrophils, and targeting of tissues. Petri & Lundebye (2007) evaluated the tissue distribution of ASTA after oral administration to rats for two weeks, and found that ASTA accumulated primarily in the spleen, kidneys, adrenals, liver, skin and eyes. Tested as an anti-carcinogenic agent, the serum concentration of ASTA was approximately 1.2
lM following a 0.02% ASTA supplementation protocol for four weeks. Nevertheless, single/multiple oral administrations of ASTA resulted in short-term accumulation in mice liver, in contrast to the fast elimination of unmodified ASTA from plasma. ASTA short-term concentration in mammal liver was shown to propor-tionally inhibit the activity of hepatic NADPH P450 reductase, lead-ing to detrimental toxicological dysfunctions. Although still obscure, the deactivation of hepatic NADPH P450 reductase could be associated with a hypothetical additional NADPH supply for ASTA-mediated triacylglycerol and cholesterol biosynthesis in the liver. This hypothesis is in agreement with hyperlipidemia and hypercholesterolemia observed in ASTA-fed rats, as shown inTable 1. The specific mechanism of NADPH P450 reductase inhibition by ASTA is dose-dependent and related to inhibition of cytochrome c reduction in microsomes. Therefore, ASTA could directly affect electron transfer on the microsomal membrane of hepatocytes due to its powerful antioxidant properties[14,55]. Furthermore, ASTA alone or combined with FO induced higher activities of the antiox-idant enzymes GPxand GR belonging to the GSH-recycling system that needs constant supply of NADPH from the pentose phosphate pathway (Table 2).
Although ASTA supplementation alone did not increase GSH content of plasma (p= 0.105; Table 1), the combined ASTA + FO supplementation significantly augmented its thiol-based antioxi-dant capacity (approximately 2-fold higher), with putative protec-tive effects on circulating neutrophils. Accordingly, both ASTA-and ASTA + FO-fed animals showed lower rates of lipid oxidation, as monitored by the fluorescent probe C11-BODIPY581/591(k1 in
Table 3). However, it remains to be investigated whether ASTA and/or FO were properly absorbed by neutrophil membrane or whether this was simply a result of plasma GSH-neutrophil mem-brane interaction. FO treatment alone resulted in unexpected downregulation of both GPxand GR by an unknown mechanism. On the other hand, the total conversion of arachidonic acid through the lipoxygenase pathway in platelets was shown to be lowered by GSH depletion, which could be a consequence of diminished GR activities. Interestingly, GPxand GR activities in neutrophils of ASTA + FO-fed animals were significantly augmented (62% and 36%, respectively), whereas proper intracellular GSH and GSSG con-tents both decreased by approximately 60% in neutrophils (Table 2).
The main ASTA + FO effects on neutrophil functions might be asso-ciated with the GSH-recycling system (with coadjutant collabora-tion from total SOD,Table 2), which apparently prioritizes the use of available plasma GSH content to protect neutrophil membrane. The immune system is known to critically depend on accurate cell–cell communication in order to mount an efficient response
. Consequently, it is tempting to suppose that ASTA + FO might help to maintain fluidity and integrity of neutrophil membranes by reducing the oxidative damage during oxidative bursts, with possi-ble positive effects on associated cell receptors, as well as redox-sensitive transcription factors (especially NF-
jB), cytokines and prostaglandins. Interestingly, ASTA + FO supplementation also resulted in significantly lower production of O
2 and H2O2during the oxidative burst of neutrophils (Figs. 3 and 4, respectively). Alto-gether, the redox rebalance in activated neutrophils resulted in significantly higher phagocytic activities, as shown in Fig. 2. Regarding physicochemical properties of biological membranes, it is worth mentioning that rigid, rod-like ASTA molecules, anchored transversally across lipid bilayers, restrict molecular motion of lip-ids and proteins, thus affecting membrane fluidity and increasing structural stability . On the other hand, n-3/PUFA such as DHA and EPA (present in FO) were shown to induce an opposite ef-fect on biological membranes. Based on the fact that major ROS production during the oxidative burst of neutrophils also oc-curs in membranes (catalyzed by NADPH oxidases), it is reasonable to assume that the proportion of n-3/PUFA and ASTA incorporated in neutrophil membranes could drastically affect physicochemical properties of lipid bilayers, with crucial consequences in terms of neutrophil function and whole immune responsiveness. Further studies are necessary to better understand such molecular-based – but truly relevant – aspects of ASTA and/or FO supplementation in immune enhancement.
5. Concluding remarks
The present study strongly suggests beneficial effects of FO + ASTA supplementation for immune competence, based on the redox balance in plasma (significant increase in GSH-dependent reducing power), non-activated neutrophils (increased glutathi-one-recycling enzymes GPxand GR) and PMA-activated neutrophils (lower O
2, H2O2, and NOgeneration, reduced membrane oxidation, but higher phagocytic activity) of Wistar rats. The effects of ASTA and FO combination were summative rather than synergistic, since FO strongly contributed to hypolipidemic/hypocholesterolemic ef-fects on plasma plus increased phagocytic activity in activated neu-trophils, whereas ASTA better offered antioxidant/anti-apoptotic effects to PMA-activated neutrophils. The bulk of data reinforces the hypothesis that habitual consumption of marine fish (e.g. sal-mon, which is a natural source of both astaxanthin and fish oil) is strongly associated with human health, in particular with improve-ment of immune response, and lower risks for vascular and infec-tious pathologies.
The authors are also indebted to the assistance of Marina Santana Gonçalves, and Sebastiana Jeane Nascimento. This research was supported by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP 2009/12342-8), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Programa PROSUP/CAPES, Brazil), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (Bolsas Produtividade em Pesquisa, Nivel 2, #312404/2009-3 and #312190/2009-3, CNPq, Brazil), and Universidade Cruzeiro do Sul. Dr. Marcelo Paes de Barros is also indebted to Dr. Åke Lignell from BioReal AB/Fuji Chemicals (Sweden/Japan) for supplying astaxan-thin-rich algal biomass forin vivostudies.
All authors of the present manuscript declare that there is no actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations that could inappropriately influence, or be perceived to influence our work.
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