Astaxanthin Treatment Confers Protection against Oxidative Stress in U937 Cells Stimulated with Lipopolysaccharide Reducing O2 − Production

Recently, astaxanthin (ASTA) studies have focused on several biological functions such as radical scavenging, singlet oxygen quenching, anti-carcinogenesis, anti-diabetic, anti-obesity, anti-inflammatory, anti-melanogenesis, and immune enhancement activities. In this study, we investigated the potential role protective of ASTA, an antioxidant marine carotenoid, in restoring physiological conditions in U937 cells stimulated with LPS (10 µg/ml). Our results show that pre-treatment with ASTA (10 µM) for 1 h attenuates the LPS-induced toxicity and ROS production. The beneficial effect of ASTA is associated with a reduction intracellular O2 − production by restoring the antioxidant network activity of superoxide dismutase (SOD) and catalase (CAT), which influence HO-1 expression and activity by inhibiting nuclear translocation of Nrf2. We accordingly hypothesize that ASTA has therapeutic properties protecting U937 cells from LPS-induced inflammatory and oxidative stress.


Introduction
Carotenoids are lipid-soluble pigments produced in some plants, algae, fungi and bacterial species which accounts for their red, orange, or yellow hues. Carotenoids are antioxidants due to their ability to quench singlet oxygen, be oxidized, and be isomerized [1]. In addition, carotenoids absorb light, hence providing defense from photo-oxidative damage. Astaxanthin (ASTA) is a red carotenoid pigment belonging to the Xanthophylls class of carotenoids. The physiological functions of ASTA have been studied for more than 20 years. It was found to have beneficial effects supporting human health and well-being and preventing pathologies. ASTA has been reported to be more effective than a representative carotenoid, b-carotene, for prevention of singlet oxygen quenching or generation, as well as lipid peroxidation in biological membranes. Moreover, it has been reported that some ASTA derivatives could scavenge superoxide anion radical [2][3][4].
HO-1 is the inducible isoform of HO that catalyzes the first and rate-limiting step in heme degradation to produce equimolar quantities of biliverdin, carbon monoxide (CO) and free iron. Sequestration of free iron by ferritin lowers the prooxidant state of the cell [7,8].
In addition to its role in heme catabolism, HO-1 has emerged as an important phase II and anti-inflammatory enzyme that is highly up-regulated by oxidative stress. Reactive oxygen species (ROS) are required for normal cellular homeostasis because they serve as critical mediators of cytokine signaling and antimicrobial host defenses. However, excessive ROS levels can induced cellular damage that compromises cellular survival [9,10].
The aim of the present study was to assess the in vitro effect of carotenoid ASTA in a cellular inflammation model through evaluation of the antioxidant enzyme network (superoxide dismutase, SOD; catalase, CAT) and HO-1 expression and activity via Nrf2 on U937 cells stimulated with bacterial lipopolysaccharides (LPS).

Materials and Methods
Cell Culture U937 mononuclear cells were purchased from American Type Culture Collection (Manassas, VA, USA). The cells were cultured in a 5% CO2 atmosphere in RPMI 1640 medium (GIBCO, Invitrogen) containing 10% fetal calf serum, 100 ng/mL streptomycin, 100 U/mL penicillin and 2 mM L-glutamine. Cells derived from the same freeze-down batch were thawed, grown and seeded (at 2610 5 cells per well) onto six-well tissue culture plates and cultured in medium with and without 10 mM astaxanthin (ASTA) (Sigma-Aldrich, St. Louis, MO, USA), LPS (10 mg/mL) and 10 mM of Protoporphyrin IX zinc (ZnPP IX)(Sigma-Aldrich, St. Louis, MO, USA). ASTA was dissolved in dimethyl sulfoxide (DMSO) and diluted with the medium. The final concentration of DMSO in the medium was 0.5%. Control cells did not contain ASTA and/or LPS.

Cell Viability Assay
MTT, absorbed into the cell and eventually the mitochondria, is broken down into formazan by mitochondria succinate dehydrogenase. Accumulation of formazan reflects the activity of mitochondria directly and cell viability indirectly. Cell viability was measured by the MTT assay. U937 cells were seeded on 96well plates at a density of 8610 3 cells/well, cultured and treated according to the method described above. A total of 20 ml of MTT was added at a concentration of 0.5 mg/ml after medium (200 ml) was added to each well. The plates were incubated at 37uC for 4 h to dissolve the formazan that had formed. The solution (220 ml) was removed from each well and 150 ml of DMSO was added. Reduced MTT was measured on an ELISA reader (Bio-Rad, Hercules, CA, USA) at a wavelength of 570 nm. Values are expressed as a percentage of the control value.

Western Blot Analysis
Total protein extracts were prepared by treating cells with lysis buffer (RIPA). Nuclear extracts were prepared as previously described [11].
Nitrocellulose was then washed in TBS, and incubated with secondary antibody HRP-conjugated (dilution 1:10000, Pierce) for 1 h, washed again and developed. The nitrocellulose was scanned using a computerized densitometric system (Bio-Rad Gel Doc 1000, Milan, Italy). Protein levels were normalized to the housekeeping protein actin and tubulin (Sigma-Aldrich) to adjust for variability of protein loading, and expressed as a percentage of vehicle control.

Quantization of Intracellular Superoxide Production using a Colorimetric NBT Assay
Nitro blue tetrazolium (NBT) (Sigma-Aldrich) assay, performed according to the method described by ROOK et al. (1985), was used to evaluate O 2 2 production. Briefly, after each treatment, cells were incubated with 0.1 mg/ml NBT in pre-filtered culture medium for 3 hours at 37uC; they were then washed three times with methanol. The amount of NBT-formazan produced is an indicator of intracellular production of superoxide anion and can be determined spectrophotometrically (SpectraMaxH 190, Molecular Devices) at 630 nm after solubilization of crystals in 200 ml of KOH 2M/DMSO solution. Cu, Zn-Superoxide Dismutase Activity SOD activity was determined as described previously [13]. The assay mixture contained 50 mM sodium carbonate buffer, pH 10, epinephrine 0.1 mM (Sigma-Aldrich), and tissue fraction (containing about 1-50 mg of protein) in a final volume of 2.5 mL. The inhibitory effect of SOD on the autoxidation of epinephrine, by use of 1.25 mM KCN to discriminate CN-insensitive MnSOD from CN-sensitive CuZnSOD, was assayed spectrophotometrically at 480 nm at 25uC. Percentage inhibition values were converted into activities by using a purified Cu, Zn bovine SOD as standard (Sigma-Aldrich). One unit of SOD is the amount of enzyme required to halve the rate of substrate auto-oxidation.

Catalase Activity
CAT activity was measured spectrophotometrically as described previously [14]. The decomposition of H 2 O 2 was monitored continuously at 240 nm. The assay mixture in a final volume of 3 mL contained 10 mM potassium phosphate buffer, 10 mM H 2 O 2 and 1.5-11 mg of protein of enzymatic extract. CAT units were defined as 1 mmole H 2 O 2 decomposed/min at 25uC.

Assay for HO Activity
An HO activity assay was performed as described previously [7]. Briefly, microsomes from harvested cells were added to a reaction mixture containing 0.8 mM NADPH (Sigma-Aldrich), 2 mM glucose 6-phosphate (Sigma-Aldrich), 0.2 U glucose-6phosphate dehydrogenase (Sigma-Aldrich), 2 mg rat liver cytosol prepared a 105000 g from supernatant fraction as a source of biliverdin reductase, potassium phosphate buffer (100 mM, pH 7.4) and 20 mM hemin (Sigma-Aldrich).
The reaction was conducted at 37uC in the dark for 1 h and then placed on ice for 2 min to terminate the reaction. Bilirubin was determined by calculation from the difference in absorbance between 464 and 530 nm (extinction coefficient, 40 mM 21 cm 21 for bilirubin). HO activity was expressed as nanomoles of bilirubin formed per milligram of microsomal  protein per hour. The total protein content of confluent cells was determined using a Bio-Rad DC protein assay (Bio-Rad, Herts, UK) by comparison with a standard curve obtained with bovine serum albumin.

Statistical Analysis
The results are expressed as means 6 SD or mean 6 SEM. Statistical analysis was performed using analysis of variance (ANOVA). A null hypothesis probability of ,5% (p,0.05) was considered statistically significant. The comparison LPS vs. LPS+ ASTA was performed using a post hoc test with the alpha level at 0.05.

Effects of LPS and Astaxanthin on Cell Viability
Macrophage activation by bacterial LPS is an important event involved in inflammation and inflammation-related diseases. LPSinduced macrophage activation is characterized by the induction or up-regulation of genes such as nuclear factor-kappaB (NF-kB), activator protein-1 (AP-1) and cytokines, which play a crucial role in the inflammatory process [14][15][16].
To evaluate the viability of U937 cells after exposure to inflammatory injury, cells were treated with different concentrations of LPS (0.1-100 mg/mL) for 24 h. After washing, and following 24 h incubation, cell viability was measured using the MTT assay. LPS significantly decreased MTT levels, indicating a lower cell count, in a concentration-dependent manner (Figure 1). Treatment with a concentration of 10 mg/mL of LPS for 24 h was selected for subsequent experiments as it reduced cell viability to approximately 4369.2% of control (*p,0.05, Figure 1).
In a previous work we evaluated the effect of ASTA (Figure 2) on cell viability in the U937 cell line. An MTT reduction experiment showed that ASTA did not exhibit any cytotoxic effects on U937 cells at concentrations of 10 mM [16]. Hence, for all experiments this concentration of ASTA was employed.
In the U937 cell line, the viability of LPS-treated cells decreased significantly, but that of cells that were pre-treated for 1 h with ASTA at a concentration of 10 mM, prior to exposure to LPS for 24 h, increased significantly to 82.365.9% ( 1 p,0.01) (Figure 3). Consequently ASTA exerted a protective effect against LPSinduced cytotoxicity.
To assess the antioxidant property of ASTA against LPSinduced inflammation in U937 cells, ROS generation and oxidative stress, an NBT assay was carried out. This method is usually used to detect possible involvement of reactive oxygen species (ROS), primarily superoxide anion radical O 2 2 , in the process of toxicity and cell death [18].
The NBT reduction assay is shown in Figure 4. The experiments were performed in triplicate. The results were expressed as an NBT reduction stimulation index (SI) which was calculated as the optical density (OD) ratio of treated and control cells. The stimulation index (SI) for the control was taken to be 1.
Treatment of U937 cells with LPS (positive control) showed significant (*p,0.05) stimulation of NBT reduction compared to control cells. Free radicals are potentially toxic; they are usually inactivated or scavenged by antioxidants before they can inflict damage on lipids, proteins or nucleic acids. To ameliorate and cope with injury from oxidative damage and maintain redox homeostasis, aerobic organisms have a complex antioxidant defense system that includes antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT). These block the initiation of free radical chain reactions [19,20]. showing that under similar conditions of elevated levels of oxidative stress the dismutase reaction of SOD is reduced ( Figure 5). This decrease in SOD activity is associated with a parallel decline in CAT activity, with consequent disregulation of the antioxidant enzymatic system and accumulation of O 2 2 which is potentially harmful to the cell. Excessive redox-active species may repress the activity of cellular enzymes and activate transcriptional factors involved in resolution of the inflammatory process [21,22].
Treatment with ASTA 10 mM shows no significant ( 1 p,0.05) stimulation of NBT reduction, due to a lower production of O 2 2 which, as shown in Figure 4, appears to be comparable to baseline values. This marked decrease in oxidative stress is combines with reactivation of the detoxification mechanism attributable to CAT and SOD activity being restored by ASTA-treatment ( Figure 5).
Our finding is thus that ASTA exerted a powerful antioxidant effect on O 2 2 produced by the LPS-stimulated U937 cell line.

Involvement of the Nrf2/HO-1 System in the Antioxidant Effects of Astaxanthin
We have previously shown that ASTA blocks H 2 O 2 mediated activation of NF-kB and its downstream cytokine secretion through modulating protein tyrosine phosphatase-1 (SHP-1) expression [16].
Activation of NF-kB, a transcription factor, controls the expression of several inducible genes such as HO-1 and iNOS engaged in the inflammatory response and involved in cellular  defense against oxidative stress. A number of proinflammatory agents are potent activators of the HO-1 response. The most notable example is bacterial LPS, which typically produces robust HO-1 activation in cell cultures [23,24]. It has, in fact, been shown that the expression of HO-1 is crucial in inhibiting LPS-induced proinflammmatory responses in RAW 264.7 cells and that ROS mediates the induction of HO-1 in a redox-regulated pathway [23,25].
HO-1 is a well-known Nrf2 target gene with established antioxidative and anti-inflammatory properties. Nrf2 plays an important role in inducing HO-1 in many cells, including macrophages [26].
Under normal homeostatic conditions, Nrf2 is retained in the cytoplasm via its interaction with Kelch-like ECH-associated protein 1 (Keap1) and is degraded by the proteasome. Under oxidative conditions, Nrf2 is released from Keap1, translocates to the nucleus, recognizes and binds to the ARE site, which causes transcription of its target genes [27].
We thus needed to determine whether or not the O 2 2 reduction observed in U937 cells treated with ASTA reduces Nrf2 nuclear translocation. For these experiments we prepared nuclear extracts and cytosolic fractions, and measured Nrf2 levels in each fraction by Western blot analysis. As shown in Figure 6, Nrf2 was retained in the cytosolic fraction in untreated cells, whereas it was translocated to the nuclear fraction when treated with LPS.
In such a condition, nuclear Nrf2 expression is up-regulated, playing an important role in regulating cellular defense through induction of HO-1 expression [28,29].
The induction of HO-1 may therefore be a generalized marker of oxidative stress, for which reason we measured the effects of ASTA treatment on alteration of the cellular redox state and hence on HO-1 protein.
HO-1 analysis (Figure 7) showed that, when the cells were stimulated with LPS, ASTA induced a return a the physiological state, decreasing O 2 2 production, restoring scavenger enzyme activity (SOD and CAT), and hence reducing the expression and activity of HO-1 protein ( Figure 7A,B). To support our hypothesis, Protoporphyrin IX zinc (ZnPPIX), an HO inhibitor, was used. As shown in Figure 8, O 2 2 reduction is associated only with ASTA treatment. This meant that ASTA was able to restore the cellular redox state by reduction of O 2 2 production, mediated by resumption of SOD and CAT activity, which preventsNrf2 translocation in U937 cells, resulting in down-regulation of the expression of inducible genes such as HO-1.

Conclusions
Oxidative stress caused by the imbalance between reactive oxygen species (ROS) and biological antioxidant systems can lead to modification of macromolecules such as DNA, lipids, and proteins. Upon exposure to cellular stress this physiological homeostasis is endangered. It is known that the induction of HO-1 can be a response to and a generalized marker of oxidative stress [30].
We have demonstrated that the marine drug ASTA exerts an antioxidant effect by suppressing the O 2 2 level in an LPSstimulated U937 cell line. This occurs through the ability of ASTA to interact with various different radicals: the pattern of conjugated double bonds in the polyene backbone of ASTA affects its quenching of singlet oxygen [31]. Quenching involves the transfer of excitation energy from O 2 2 to the carotenoid, resulting in ground state oxygen and an excited triplet state carotenoid. The energy is dissipated between the excited carotenoid and the surrounding solvent to yield ground state carotenoid and thermal energy. In the process of quenching the carotenoid remains intact and can undergo further cycles of singlet oxygen quenching [32].
In lowering the O 2 2 levels, treatment with ASTA, therefore, leads to functional recovery of the antioxidant network (SOD and CAT). Consequently, as shown by our results, the nuclear translocation of Nrf2 does not occur, and neither does any parallel up-regulation of the expression and catalytic activity of HO-1.
The interest of our research lies in the current need to find new substances of natural origin which have proven efficacy and a low degree of toxicity in the therapeutic treatment of various diseases characterized by alteration of the cellular redox state.
From the results gathered the conclusion is that the natural compound ASTA may be used for therapeutic purposes to protect cells against oxidative conditions.