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The protective role of olive oil hydroxytyrosol against oxidative alterations induced by mercury in human erythrocytes Q1 Lidia Tagliafierro a, Arbace Officioso a, Sergio Sorbo b, Adriana Basile c, Caterina Manna a,* a b c
Department of Biochemistry, Biophysics and General Pathology, School of Medicine, Second University of Naples, Naples, Italy Microscopy Section of CeSMA, University of Naples “Federico II”, Naples, Italy Department of Biology, University of Naples “Federico II”, Naples, Italy
A R T I C L E
I N F O
Article history: Received 19 December 2014 Accepted 28 April 2015 Available online Keywords: Mercury Cardiovascular diseases Erythrocyte Hydroxytyrosol Olive oil Oxidative stress
A B S T R A C T
Hydroxytyrosol (HT) is a phenolic antioxidant naturally occurring in virgin olive oil. In this study, we investigated the possible protective effects of HT on the oxidative and morphological alterations induced by mercury (Hg) in intact human erythrocytes. These cells preferentially accumulate this toxic heavy metal. More importantly, Hg-induced echinocyte formation correlates with increased coagulability of these cells. Our results indicate that HT treatment (10–50 μM) prevents the increase in hemolysis and Reactive Oxygen Species (ROS) generation induced by exposure of cells to micromolar HgCl2 concentrations as well as the decrease in GSH intracellular levels. Moreover, as indicated by scanning electron microscopy, the morphological alterations are also significantly reduced by HT co-treatment. Taken together our data provide the first experimental evidence that HT has the potential to counteract mercury toxicity. The reported effect may be regarded as an additional mechanism underlying the beneficial cardio-protective effects of this dietary antioxidant, also endowed with significant anti-atherogenic and anti-inflammatory properties. © 2015 Published by Elsevier Ltd.
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1. Introduction Hg is a highly toxic heavy metal and is one of the main agents which is responsible for environmental pollution (Rice et al., 2014). In the last century, water and soil contamination by both elementary and organic mercury has increased dramatically through anthropogenic sources, including fuel combustion and incinerators. Human exposure to mercury occurs primarily via medical preparations as well as nutritional sources. In particular, contaminated fish products seem to be the major source of methylmercury in food and represent an increasing public health concern (Booth and Zeller, 2005). The health consequences of human exposure to mercury include immune-toxicity, kidney damage and neuronal disorders (Hong et al., 2012). Moreover, the reported anemia-inducing effect of mercury suggests that RBC may be an important target of mercury toxicity (Rooney, 2013). In the last few years, concerns about the negative effects of chronic exposure to mercury on cardiovascular health are rapidly increasing and mercury toxicity is now regarded as a potential new risk factor for cardiovascular diseases (CVD) (Fernandes Azevedo et al., 2012, Q2 Houston, 2011; Virtanen et al., 2007). Mechanisms underlying Hg-
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* Corresponding author. Dipartimento di Biochimica e Biofisica e Patologia Generale, Scuola di Medicina, Seconda Università di Napoli, Via De Crecchio n.7, 80138 Napoli, Italy. Tel.: +39 081 5667523; fax: +39 081 5667608. E-mail address: [email protected] (C. Manna).
64 related endothelial dysfunction include a decrease in nitric oxide 65 bioavailability and Hg-induced increase in oxidative stress (Virtanen 66 et al., 2007). Increased formation of reactive oxygen species (ROS), 67 indeed, is thought to be one of the key mechanisms responsible 68 for Hg-induced toxicity (Ercal et al., 2001). Because mercury is 69 endowed with a high affinity for sulfhydryl groups, it is able to 70 impair the antioxidant defense system by reacting with cellular 71 thiols including glutathione (Rooney, 2007; Velyka et al., 2014). In 72 addition, according to the oxidative stress hypothesis of mercury 73 toxicity, antioxidant compounds are protective (Barcelos et al., 2011; Q3 74 Kaivalya et al., 2011). 75 The aim of this paper is to further explore the involvement of 76 oxidative stress as an underlying mechanism in metabolic changes 77 related to mercury toxicity and the possible protective role played 78 by dietary antioxidants, using intact human RBC incubated in vitro 79 in the presence of mercuric chloride (HgCl2). These cells are a unique 80 cellular model for studies that investigate oxidative stress-related 81 alterations (Manna et al., 1999; Yang et al., 2006) as well as Hg tox82 icity (Harisa et al., 2012, 2013). This metal, indeed, preferentially 83 accumulates in RBC and induces morphological changes (Pal and 84 Ghosh, 2012) which increase the pro-coagulant activity of these cells 85 (Lim et al., 2010). Among the different antioxidants, our attention 86 has been devoted to hydroxytyrosol (3,4-diidroxyphenylethanol, HT), 87 a simple phenol recalling the structure of cathecol, naturally oc88 curring in olive oil (Napolitano et al., 2010; Zappia et al., 2010). This 89 compound is endowed with a variety of biological activities, in90 cluding anti-inflammatory and anti-atherogenic properties (Burattini
http://dx.doi.org/10.1016/j.fct.2015.04.029 0278-6915/© 2015 Published by Elsevier Ltd.
Please cite this article in press as: Lidia Tagliafierro, Arbace Officioso, Sergio Sorbo, Adriana Basile, Caterina Manna, The protective role of olive oil hydroxytyrosol against oxidative alterations induced by mercury in human erythrocytes, Food and Chemical Toxicology (2015), doi: 10.1016/j.fct.2015.04.029
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et al., 2013; Granados-Principal et al., 2010). Mechanisms underlying the biological effects of HT include both radical scavenging properties and metal chelator activity (Manna et al., 2012).
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2.1. Chemicals
2. Materials and methods
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The materials used, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), mercuric chloride (HgCl2), and hydroxytyrosol, were from Sigma Chemical Co. All other chemicals used were of the purest grade available.
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The RBC fraction was obtained from whole blood deprived of leukocytes and platelets by filtration on a nylon net, washed twice with isotonic saline solution (NaCl 0.9%) and finally resuspended to 10% hematocrit with Buffer A (5 mM Tris-HCl containing 0.9% NaCl, 1 mM MgCl2 and 2.8 mM glucose, pH 7.4). Mercury treatment was performed by incubation of intact RBC at 37 °C with increasing concentrations of HgCl2.
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The extent of hemolysis was determined spectophotometrically, according to Manna et al. (1999). At the end of incubation, the reaction mixture was centrifuged at 1100 g for 5 min and the absorption (A) of the supernatant at 540 nm was measured. Packed RBC were hemolyzed with 40 volumes of ice-cold distilled water and the lysate centrifuged at 1500 g for 10 minutes and the absorption of the supernatant (B) was recorded at 540 nm. The hemolysis percentage was calculated from the ratio of the reading (A/B) × 100.
2.2. Preparation of RBC and mercury treatment
2.3. Assay system for hemolysis
2.4. Determination of ROS
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The dichlorofluorescein (DCF) assay was performed to quantify ROS generation, according to Manna et al. (2012). Intact RBC were incubated with the nonpolar, non-fluorescent 2′,7′-dichlorodihydrofluorescin diacetate (DCFH-DA), at final concentration of 10 μM for 15 min at 37°. After centrifugation at 1200 g for 5 min, the supernatant was removed and the hematocrit value was adjusted to 10% with buffer A and RBC were then treated with HgCl2 in the dark. At the end of incubation, 20 μl of RBC was diluted in 2 mL of water and the fluorescence intensity of the oxidative derivative DCF was recorded (λexc 502; λem 520). The results were expressed as fluorescent intensity/mg hemoglobin (Hb).
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The intracellular GSH content was determined spectrophotometrically by reaction with 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) reagent, according to Van den Berg et al. ( 1992). After centrifugation of the samples (0.25 ml) treated as above reported, supernatants were removed and RBC were lysed by addition of 0.6 ml of ice-cold water; proteins were than precipitated by the addition of 0.6 ml ice-cold metaphosphoric acid solution [1.67 g metaphosphoric acid, 0.2 g EDTA and 30 g NaCl in 100 ml of water]. After 5′ incubation at 4 °C, the protein precipitate was removed by centrifugation at 18,000 g for 10 min and 0.45 ml of the supernatant was mixed with an equal volume of a 0.3 M Na2HPO4 . For the reduced GSH determinations, 100 μl of DTNB solution (20 mg DTNB plus 1% of sodium citrate in 100 ml of water) was added to the sample. After 10′ incubation at room temperature, the absorbance of the sample was read against the blank at 412 nm.
2.5. Assay for reduced GSH
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3. Results In order to evaluate the toxic effects of Hg and the possible protection exerted by HT, intact RBC were exposed in vitro to increasing Q6 HgCl2 concentrations. As far as the chemical form of mercury utilized is concerned, methylmercury is considered the more toxic form in that, because of its lipophilicity, it is almost completely absorbed and it easily crosses the cellular membrane and the blood– brain barrier. However, methylmercury is rapidly transformed in the body into its mercuric form, the toxic species in human tissue after conversion (Houston, 2011). Accordingly, several papers are present in the literature that utilized HgCl2 to test the toxic effect of this heavy metal in in vitro systems (Harisa et al., 2013; Kaivalya et al., 2011; Lim et al., 2010). 3.1. HT prevents human RBC from Hg-induced hemolysis To evaluate the ability of HT to prevent Hg-mediated cytotoxicity, the effect of the phenolic antioxidant on hemolysis was measured. As shown in Fig. 1, exposure of cells to HgCl2 results in a significant cytotoxicity in a dose and time-dependent manner. By comparison of the data at 4 and 24 h, we demonstrated that in our experimental conditions Hg toxicity is a relatively late event: no hemolysis is detectable up to 20 μM upon 4 h treatment. A low value but significant hemolysis (2.6%) is present at 40 μM but only at 80 μM HgCl2 does meaningful hemolysis occur (10.3%). Conversely, prolonged exposure to the heavy metal leads to a dramatic decrease in cell viability: after 24 h the increase in the hemolytic process is significant starting from the 10 μM concentration (2.7%) with about 50% of hemolysis at 80 μM (48.3%). On the basis of these results, we used 40 and 80 μM HgCl2 to evaluate the effect of HT at the two different time points utilized. The dose-dependency of the protective effect of HT on Hg-induced cytotoxicity is shown in Fig. 2. HT is able to prevent the toxic effect of the metal by reducing cell death. In fact, hemolysis is significantly decreased in the presence of HT concentrations as low as 10 μM at all time points analyzed. 3.2. HT prevents Hg-induced ROS generation in human RBC In order to investigate the role of oxidative stress in Hg-induced cytotoxicity and the possible protective effects of HT, a DCF assay was performed to measure ROS formation. Fig. 3 shows that incu-
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2.6. Scanning electron microscopy (SEM) analysis
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2.7. Statistical analysis
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Data were expressed as means ± SEM. The significance of differences was determined by one-way ANOVA followed by a post hoc Dunnett’s multiple comparisons test with significance set at p < 0.01. GraphPad Prism 5 was utilized for statistical analysis.
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RBC were treated with HgCl2 in the presence or in the absence of HT, as above described. After the treatment, the cells were fixed with a solution of 2.5% glutaraldehyde and 4% paraformaldehyde for 2 h at 4 °C. After fixation, RBC were washed three times with PBS and then post-fixed with 1% osmium tetroxide for 30 min at 4 °C. Cells were washed with PBS several times and then RBC were dehydrated with 50%, 75%, 90%, and 100% ethanol. After drying and coating with gold, the images were observed on a Scanning Electron Microscope (SEM) (FEI QUANTA 200). Echinocytes were quantified by counting ≥200 cells (50 RBC for each different SEM field at a magnification of 1750×) for each experimental condition.
Fig. 1. Effect of Hg treatment on hemolysis in RBC. Cells were treated for 4 and 24 h in the presence of increasing concentrations of HgCl2 as described in Materials and methods. Data are the means ± SEM (n = 10). Statistical analysis was performed with one-way ANOVA followed by Dunnett’s test (p < 0.01). Means with different letters are significantly different.
Please cite this article in press as: Lidia Tagliafierro, Arbace Officioso, Sergio Sorbo, Adriana Basile, Caterina Manna, The protective role of olive oil hydroxytyrosol against oxidative alterations induced by mercury in human erythrocytes, Food and Chemical Toxicology (2015), doi: 10.1016/j.fct.2015.04.029
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Fig. 2. Effect of HT on Hg-induced hemolysis. Cells were treated with HgCl2 at 40 and 80 μM for 4 and 24 h in the presence of increasing concentrations of HT. Data are the means ± SEM (n = 10). Statistical analysis was performed with one-way ANOVA followed by Dunnett’s test (p < 0.01). Means with different letters are significantly different.
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bation with HgCl2 results in an increase in DCF fluorescent signal, which is indicative of ROS formation at both 4 and 24 h, providing confirmation that Hg treatment exposes cells to an oxidative microenvironment. According to the hemolysis data, Hg-induced ROS formation appears as a relatively late event: after 4 h the treatment results in a significant increase in ROS generation in the presence of HgCl2 concentration starting from 40 μM (2.2 fold). In addition, after 24 h, Hg-induced ROS formation is significantly increased in a dose dependent manner, with a significant effect observed at a concentration as low as 10 μM. To test the efficiency of HT in reducing ROS generation, intact RBC were subjected to metal exposure at 40 and 80 μM in the presence of increasing antioxidant concentrations for 4 and 24 h. As shown in Fig. 4, HT decreases the fluorescent signal in metal exposed RBC, with a significant reduction observed at all tested concentrations, starting from a concentration as low as 10 μM. 3.3. HT prevents Hg-induced decrease in GSH levels in human RBC Because of the pivotal role played by glutathione depletion in mercury toxicity, particularly related to the impairment of the antioxidant defense system, we evaluated the possible HT protective effect on this specific Hg-induced metabolic alteration.
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Fig. 4. Effect of HT on Hg-induced ROS production in RBC. Cells were treated with HgCl2 at 40 and 80 μM for 4 and 24 h in the presence of increasing concentrations of HT. ROS production was evaluated by means of the fluorescent probe DCF. Data are the means ± SEM (n = 10). Statistical analysis was performed with one-way ANOVA followed by Dunnett’s test (p < 0.01). Means with different letters are significantly different.
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As shown in Fig. 5, RBC treatment for 4 h in the presence of 20 μM HgCl2 significantly decreases GSH levels by about 15%. The coincubation of the heavy metal with 10 μM HT significantly prevents GSH depletion and starting from 25 μM a complete protection is observable. 3.4. HT prevents Hg-induced cell shape modification in human RBC Membrane alterations are reported to be a major target of Hg damage in RBC (Lim et al., 2010). We therefore examined the effect of HT on echinocyte formation induced by metal treatment. SEM observations reveal that untreated cells show the presence of very few echinocytes (5%) (Fig. 6A), while incubation in the presence of HgCl2 induces a significant increase in altered cells (60%) (Fig. 6A). This cell shape alteration is positively affected by the olive oil antioxidant. In fact, SEM analysis indicates that HT almost fully prevents Hg-induced echinocyte formation (Fig. 6C and 6D). 4. Discussion Mercury is known to pose serious threats to human health owing to its toxic and hazardous nature and a positive link between mercury exposure and CVD has been firmly established (Houston, 2011; Virtanen et al., 2007). Among the molecular mechanisms
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Fig. 3. Effect of Hg treatment on ROS production in RBC. Cells were treated for 4 and 24 h in the presence of increasing concentrations of HgCl2, as described in Materials and methods. ROS production was evaluated by means of the fluorescent probe DCF. Data are the means ± SEM (n = 10). Statistical analysis was performed with oneway ANOVA followed by Dunnett’s test (p < 0.01). Means with different letters are significantly different.
Fig. 5. Effect of HT on Hg-induced GSH decrease in RBC. Cells were treated with 20 μM HgCl 2 for 4 in the presence of increasing concentrations of HT. Data are the means ± SEM (n = 10). Statistical analysis was performed with one-way ANOVA followed by Dunnett’s test (p < 0.01). Means with different letters are significantly different.
Please cite this article in press as: Lidia Tagliafierro, Arbace Officioso, Sergio Sorbo, Adriana Basile, Caterina Manna, The protective role of olive oil hydroxytyrosol against oxidative alterations induced by mercury in human erythrocytes, Food and Chemical Toxicology (2015), doi: 10.1016/j.fct.2015.04.029
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Fig. 6. Effect of HT on Hg-induced morphological alterations in RBC. Cells were exposed for 2 h to 20 μM HgCl2. At the end of incubation, cells were prepared for SEM observation as described in Materials and methods. (A) Untreated RBC; (B) HgCl2treated RBC; (C) HgCl2-treated RBC in the presence of 50 μM HT; (D) Echinocyte percentage quantified under the different conditions, as described in Materials and methods (n = 3). Statistical analysis was performed with one-way ANOVA followed by Dunnett’s test (p < 0.01). Means with different letters are significantly different.
8 underlying Hg toxicity, increased ROS formation seems to play a 9 major role in endothelial dysfunction (Furieri et al., 2011). However, 10 Gatti et al. (2004) report that Hg toxicity in PC12 cells is indepen11 dent of ROS generation. 12 The data presented in this paper offer experimental evidence that 13 Hg-induced ROS generation is a late event in RBC, and it probably 14 occurs subsequently to a significant decrease of essential antioxi15 dant thiols, which could render cells more susceptible to ROS16 mediated oxidative damage. In fact, after 4 h Hg treatment (20 μM), 17 a significant decrease in GSH concentration is observable, while no 18 ROS generation is detectable, according to data reported in similar 19 experimental conditions (Harisa et al., 2012). 20 Finally, an interesting consideration is that redox-active transi21 tion metal can exacerbate ROS generation acting as a catalyst of OH 22 radical formation: the hypothesis that considers the possible direct 23 formation of ROS by the Fenton type reaction as a contributing factor 24 in building up a pro-oxidative microenvironment in Hg-treated cells 25 can be ruled out in our model system. 26 Based on our observations and in agreement with the litera27 ture data, it is possible to conclude that Hg-induced oxidative 28 alterations may be regarded as contributing factors to the in29 creased rate of human pathologies, including anemia, particularly 30 related to chronic exposure to this heavy metal. In fact, although a 31 particularly high GSH concentration may partially protect RBC from 32 Hg toxic effects, chronic exposure could affect RBC viability and 33 induce shape changes, also affecting CVD. As pointed out before, Hg 34 exposure enhances pro-coagulant activity of these cells, resulting 35 in a contributing factor for Hg-related thrombotic disease (Lim et al., 36 2010). Further studies could validate the fascinating hypothesis that 37 metabolic and shape modification in RBC may be regarded as a clin38 ical biomarker, indicating increased cardiovascular risk in Hg39 exposed individuals. Interestingly, in this respect, Harisa and 40 colleagues (Harisa et al., 2013) utilized erythrocyte nitric oxide syn41 thase as a surrogate marker for Hg-induced vascular damage. 42 Here we also report for the first time that HT has the potential 43 to modulate cytotoxicity and to counteract GSH decrease and the 44 oxidative stress induced in RBC by Hg treatment. Also of clinical im45 portance is the finding that HT prevents Hg-induced procoagulant 46 RBC morphological alteration (echinocyte formation) in RBC. 47 Antioxidant polyphenols are believed to play a major role in the 48 positive correlation between adherence to the Mediterranean Diet 49 (MD) and a low incidence of CVD (Khurana et al., 2013; Tresserra-Rimbau et al. 2014). Our data strengthen the nutritional Q7 Q8 50 51 relevance of the phenolic fraction in olive oil in the aforemen52 tioned claims for the health promoting effects of the MD. Virgin olive 53 oil, an excellent source of oleic acid, vitamin E and nonessential nu54 trients, greatly contributes to the low incidence of CVD associated 55 with an adherence to this dietary habit in that almost all cardio56 vascular risk factors can be positively modulated by olive oil 57 constituents (Carluccio et al., 2007). In this respect, there is a general 58 agreement that the health promoting effects of olive oil intake result 59 from the combined properties of all its constituents, including poly60 phenols (López-Miranda et al., 2010). An increasing amount of data, Q9 61 indeed, clearly indicate that HT can improve cardiovascular health, 62 due to its strong antioxidant activity, presumably counteracting the 63 oxidative stress-induced endothelial dysfunction and modulating 64 key mechanisms implicated in the development of atherosclero65 sis, including expression of adhesion molecules (Manna et al., 2009). 66 Indeed, HT is endowed with significant anti-thrombotic, anti67 atherogenic and anti-inflammatory activities (Granados-Principal 68 et al., 2010). The data reported in this paper may usefully enlarge 69 on HT beneficial effects, indicating that prevention of Hg toxicity 70 could represent an additional mechanism responsible for cardio71 protection in vivo. In this respect, an interesting observation is that 72 HT active concentrations utilized in our study could be approached 73 in vivo upon strict adherence to the MD dietary habit. According to
Please cite this article in press as: Lidia Tagliafierro, Arbace Officioso, Sergio Sorbo, Adriana Basile, Caterina Manna, The protective role of olive oil hydroxytyrosol against oxidative alterations induced by mercury in human erythrocytes, Food and Chemical Toxicology (2015), doi: 10.1016/j.fct.2015.04.029
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the recent EFSA claim on the positive effect of the phenolic fraction of olive oil in the prevention of LDL oxidation, 5 mg of HT and its derivatives should be consumed daily. This amount, if provided by moderate quantities of olive oil, can easily be consumed in the context of a balanced diet. The use of phytochemicals able to significantly counteract oxidative alterations associated with Hg exposure as an attractive tool for the reduction of mercury toxicity has gained a lot of attention recently (Harisa et al., 2013; Kaivalya et al., 2011). Taken together, the reported findings indicate HT as an ideal candidate for nutritional/nutraceutical strategies to prevent mercury toxicity in humans. Particularly important, in this respect, is that HT has been proved by our group to be devoid of toxicity up to 2 g/kg in an animal model (D’Angelo et al., 2001) and a more recent study, investigating HT in terms of its safety profile, indicates the dose of 500 mg/ k/day as “No Observed Adverse Effects Level” (NOAEL). (Auñon-Calles et al., 2013). Conflict of interest The authors declare that there are no conflicts of interest. Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgements
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Food and Chemical Toxicology j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f o o d c h e m t o x
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The protective role of olive oil hydroxytyrosol against oxidative alterations induced by mercury in human erythrocytes Q1 Lidia Tagliafierro a, Arbace Officioso a, Sergio Sorbo b, Adriana Basile c, Caterina Manna a,* a b c
Department of Biochemistry, Biophysics and General Pathology, School of Medicine, Second University of Naples, Naples, Italy Microscopy Section of CeSMA, University of Naples “Federico II”, Naples, Italy Department of Biology, University of Naples “Federico II”, Naples, Italy
A R T I C L E
I N F O
Article history: Received 19 December 2014 Accepted 28 April 2015 Available online Keywords: Mercury Cardiovascular diseases Erythrocyte Hydroxytyrosol Olive oil Oxidative stress
A B S T R A C T
Hydroxytyrosol (HT) is a phenolic antioxidant naturally occurring in virgin olive oil. In this study, we investigated the possible protective effects of HT on the oxidative and morphological alterations induced by mercury (Hg) in intact human erythrocytes. These cells preferentially accumulate this toxic heavy metal. More importantly, Hg-induced echinocyte formation correlates with increased coagulability of these cells. Our results indicate that HT treatment (10–50 μM) prevents the increase in hemolysis and Reactive Oxygen Species (ROS) generation induced by exposure of cells to micromolar HgCl2 concentrations as well as the decrease in GSH intracellular levels. Moreover, as indicated by scanning electron microscopy, the morphological alterations are also significantly reduced by HT co-treatment. Taken together our data provide the first experimental evidence that HT has the potential to counteract mercury toxicity. The reported effect may be regarded as an additional mechanism underlying the beneficial cardio-protective effects of this dietary antioxidant, also endowed with significant anti-atherogenic and anti-inflammatory properties. © 2015 Published by Elsevier Ltd.
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1. Introduction Hg is a highly toxic heavy metal and is one of the main agents which is responsible for environmental pollution (Rice et al., 2014). In the last century, water and soil contamination by both elementary and organic mercury has increased dramatically through anthropogenic sources, including fuel combustion and incinerators. Human exposure to mercury occurs primarily via medical preparations as well as nutritional sources. In particular, contaminated fish products seem to be the major source of methylmercury in food and represent an increasing public health concern (Booth and Zeller, 2005). The health consequences of human exposure to mercury include immune-toxicity, kidney damage and neuronal disorders (Hong et al., 2012). Moreover, the reported anemia-inducing effect of mercury suggests that RBC may be an important target of mercury toxicity (Rooney, 2013). In the last few years, concerns about the negative effects of chronic exposure to mercury on cardiovascular health are rapidly increasing and mercury toxicity is now regarded as a potential new risk factor for cardiovascular diseases (CVD) (Fernandes Azevedo et al., 2012, Q2 Houston, 2011; Virtanen et al., 2007). Mechanisms underlying Hg-
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* Corresponding author. Dipartimento di Biochimica e Biofisica e Patologia Generale, Scuola di Medicina, Seconda Università di Napoli, Via De Crecchio n.7, 80138 Napoli, Italy. Tel.: +39 081 5667523; fax: +39 081 5667608. E-mail address: [email protected] (C. Manna).
64 related endothelial dysfunction include a decrease in nitric oxide 65 bioavailability and Hg-induced increase in oxidative stress (Virtanen 66 et al., 2007). Increased formation of reactive oxygen species (ROS), 67 indeed, is thought to be one of the key mechanisms responsible 68 for Hg-induced toxicity (Ercal et al., 2001). Because mercury is 69 endowed with a high affinity for sulfhydryl groups, it is able to 70 impair the antioxidant defense system by reacting with cellular 71 thiols including glutathione (Rooney, 2007; Velyka et al., 2014). In 72 addition, according to the oxidative stress hypothesis of mercury 73 toxicity, antioxidant compounds are protective (Barcelos et al., 2011; Q3 74 Kaivalya et al., 2011). 75 The aim of this paper is to further explore the involvement of 76 oxidative stress as an underlying mechanism in metabolic changes 77 related to mercury toxicity and the possible protective role played 78 by dietary antioxidants, using intact human RBC incubated in vitro 79 in the presence of mercuric chloride (HgCl2). These cells are a unique 80 cellular model for studies that investigate oxidative stress-related 81 alterations (Manna et al., 1999; Yang et al., 2006) as well as Hg tox82 icity (Harisa et al., 2012, 2013). This metal, indeed, preferentially 83 accumulates in RBC and induces morphological changes (Pal and 84 Ghosh, 2012) which increase the pro-coagulant activity of these cells 85 (Lim et al., 2010). Among the different antioxidants, our attention 86 has been devoted to hydroxytyrosol (3,4-diidroxyphenylethanol, HT), 87 a simple phenol recalling the structure of cathecol, naturally oc88 curring in olive oil (Napolitano et al., 2010; Zappia et al., 2010). This 89 compound is endowed with a variety of biological activities, in90 cluding anti-inflammatory and anti-atherogenic properties (Burattini
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et al., 2013; Granados-Principal et al., 2010). Mechanisms underlying the biological effects of HT include both radical scavenging properties and metal chelator activity (Manna et al., 2012).
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2.1. Chemicals
2. Materials and methods
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The materials used, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), mercuric chloride (HgCl2), and hydroxytyrosol, were from Sigma Chemical Co. All other chemicals used were of the purest grade available.
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The RBC fraction was obtained from whole blood deprived of leukocytes and platelets by filtration on a nylon net, washed twice with isotonic saline solution (NaCl 0.9%) and finally resuspended to 10% hematocrit with Buffer A (5 mM Tris-HCl containing 0.9% NaCl, 1 mM MgCl2 and 2.8 mM glucose, pH 7.4). Mercury treatment was performed by incubation of intact RBC at 37 °C with increasing concentrations of HgCl2.
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The extent of hemolysis was determined spectophotometrically, according to Manna et al. (1999). At the end of incubation, the reaction mixture was centrifuged at 1100 g for 5 min and the absorption (A) of the supernatant at 540 nm was measured. Packed RBC were hemolyzed with 40 volumes of ice-cold distilled water and the lysate centrifuged at 1500 g for 10 minutes and the absorption of the supernatant (B) was recorded at 540 nm. The hemolysis percentage was calculated from the ratio of the reading (A/B) × 100.
2.2. Preparation of RBC and mercury treatment
2.3. Assay system for hemolysis
2.4. Determination of ROS
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The dichlorofluorescein (DCF) assay was performed to quantify ROS generation, according to Manna et al. (2012). Intact RBC were incubated with the nonpolar, non-fluorescent 2′,7′-dichlorodihydrofluorescin diacetate (DCFH-DA), at final concentration of 10 μM for 15 min at 37°. After centrifugation at 1200 g for 5 min, the supernatant was removed and the hematocrit value was adjusted to 10% with buffer A and RBC were then treated with HgCl2 in the dark. At the end of incubation, 20 μl of RBC was diluted in 2 mL of water and the fluorescence intensity of the oxidative derivative DCF was recorded (λexc 502; λem 520). The results were expressed as fluorescent intensity/mg hemoglobin (Hb).
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The intracellular GSH content was determined spectrophotometrically by reaction with 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) reagent, according to Van den Berg et al. ( 1992). After centrifugation of the samples (0.25 ml) treated as above reported, supernatants were removed and RBC were lysed by addition of 0.6 ml of ice-cold water; proteins were than precipitated by the addition of 0.6 ml ice-cold metaphosphoric acid solution [1.67 g metaphosphoric acid, 0.2 g EDTA and 30 g NaCl in 100 ml of water]. After 5′ incubation at 4 °C, the protein precipitate was removed by centrifugation at 18,000 g for 10 min and 0.45 ml of the supernatant was mixed with an equal volume of a 0.3 M Na2HPO4 . For the reduced GSH determinations, 100 μl of DTNB solution (20 mg DTNB plus 1% of sodium citrate in 100 ml of water) was added to the sample. After 10′ incubation at room temperature, the absorbance of the sample was read against the blank at 412 nm.
2.5. Assay for reduced GSH
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3. Results In order to evaluate the toxic effects of Hg and the possible protection exerted by HT, intact RBC were exposed in vitro to increasing Q6 HgCl2 concentrations. As far as the chemical form of mercury utilized is concerned, methylmercury is considered the more toxic form in that, because of its lipophilicity, it is almost completely absorbed and it easily crosses the cellular membrane and the blood– brain barrier. However, methylmercury is rapidly transformed in the body into its mercuric form, the toxic species in human tissue after conversion (Houston, 2011). Accordingly, several papers are present in the literature that utilized HgCl2 to test the toxic effect of this heavy metal in in vitro systems (Harisa et al., 2013; Kaivalya et al., 2011; Lim et al., 2010). 3.1. HT prevents human RBC from Hg-induced hemolysis To evaluate the ability of HT to prevent Hg-mediated cytotoxicity, the effect of the phenolic antioxidant on hemolysis was measured. As shown in Fig. 1, exposure of cells to HgCl2 results in a significant cytotoxicity in a dose and time-dependent manner. By comparison of the data at 4 and 24 h, we demonstrated that in our experimental conditions Hg toxicity is a relatively late event: no hemolysis is detectable up to 20 μM upon 4 h treatment. A low value but significant hemolysis (2.6%) is present at 40 μM but only at 80 μM HgCl2 does meaningful hemolysis occur (10.3%). Conversely, prolonged exposure to the heavy metal leads to a dramatic decrease in cell viability: after 24 h the increase in the hemolytic process is significant starting from the 10 μM concentration (2.7%) with about 50% of hemolysis at 80 μM (48.3%). On the basis of these results, we used 40 and 80 μM HgCl2 to evaluate the effect of HT at the two different time points utilized. The dose-dependency of the protective effect of HT on Hg-induced cytotoxicity is shown in Fig. 2. HT is able to prevent the toxic effect of the metal by reducing cell death. In fact, hemolysis is significantly decreased in the presence of HT concentrations as low as 10 μM at all time points analyzed. 3.2. HT prevents Hg-induced ROS generation in human RBC In order to investigate the role of oxidative stress in Hg-induced cytotoxicity and the possible protective effects of HT, a DCF assay was performed to measure ROS formation. Fig. 3 shows that incu-
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2.6. Scanning electron microscopy (SEM) analysis
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2.7. Statistical analysis
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Data were expressed as means ± SEM. The significance of differences was determined by one-way ANOVA followed by a post hoc Dunnett’s multiple comparisons test with significance set at p < 0.01. GraphPad Prism 5 was utilized for statistical analysis.
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RBC were treated with HgCl2 in the presence or in the absence of HT, as above described. After the treatment, the cells were fixed with a solution of 2.5% glutaraldehyde and 4% paraformaldehyde for 2 h at 4 °C. After fixation, RBC were washed three times with PBS and then post-fixed with 1% osmium tetroxide for 30 min at 4 °C. Cells were washed with PBS several times and then RBC were dehydrated with 50%, 75%, 90%, and 100% ethanol. After drying and coating with gold, the images were observed on a Scanning Electron Microscope (SEM) (FEI QUANTA 200). Echinocytes were quantified by counting ≥200 cells (50 RBC for each different SEM field at a magnification of 1750×) for each experimental condition.
Fig. 1. Effect of Hg treatment on hemolysis in RBC. Cells were treated for 4 and 24 h in the presence of increasing concentrations of HgCl2 as described in Materials and methods. Data are the means ± SEM (n = 10). Statistical analysis was performed with one-way ANOVA followed by Dunnett’s test (p < 0.01). Means with different letters are significantly different.
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Fig. 2. Effect of HT on Hg-induced hemolysis. Cells were treated with HgCl2 at 40 and 80 μM for 4 and 24 h in the presence of increasing concentrations of HT. Data are the means ± SEM (n = 10). Statistical analysis was performed with one-way ANOVA followed by Dunnett’s test (p < 0.01). Means with different letters are significantly different.
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bation with HgCl2 results in an increase in DCF fluorescent signal, which is indicative of ROS formation at both 4 and 24 h, providing confirmation that Hg treatment exposes cells to an oxidative microenvironment. According to the hemolysis data, Hg-induced ROS formation appears as a relatively late event: after 4 h the treatment results in a significant increase in ROS generation in the presence of HgCl2 concentration starting from 40 μM (2.2 fold). In addition, after 24 h, Hg-induced ROS formation is significantly increased in a dose dependent manner, with a significant effect observed at a concentration as low as 10 μM. To test the efficiency of HT in reducing ROS generation, intact RBC were subjected to metal exposure at 40 and 80 μM in the presence of increasing antioxidant concentrations for 4 and 24 h. As shown in Fig. 4, HT decreases the fluorescent signal in metal exposed RBC, with a significant reduction observed at all tested concentrations, starting from a concentration as low as 10 μM. 3.3. HT prevents Hg-induced decrease in GSH levels in human RBC Because of the pivotal role played by glutathione depletion in mercury toxicity, particularly related to the impairment of the antioxidant defense system, we evaluated the possible HT protective effect on this specific Hg-induced metabolic alteration.
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Fig. 4. Effect of HT on Hg-induced ROS production in RBC. Cells were treated with HgCl2 at 40 and 80 μM for 4 and 24 h in the presence of increasing concentrations of HT. ROS production was evaluated by means of the fluorescent probe DCF. Data are the means ± SEM (n = 10). Statistical analysis was performed with one-way ANOVA followed by Dunnett’s test (p < 0.01). Means with different letters are significantly different.
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As shown in Fig. 5, RBC treatment for 4 h in the presence of 20 μM HgCl2 significantly decreases GSH levels by about 15%. The coincubation of the heavy metal with 10 μM HT significantly prevents GSH depletion and starting from 25 μM a complete protection is observable. 3.4. HT prevents Hg-induced cell shape modification in human RBC Membrane alterations are reported to be a major target of Hg damage in RBC (Lim et al., 2010). We therefore examined the effect of HT on echinocyte formation induced by metal treatment. SEM observations reveal that untreated cells show the presence of very few echinocytes (5%) (Fig. 6A), while incubation in the presence of HgCl2 induces a significant increase in altered cells (60%) (Fig. 6A). This cell shape alteration is positively affected by the olive oil antioxidant. In fact, SEM analysis indicates that HT almost fully prevents Hg-induced echinocyte formation (Fig. 6C and 6D). 4. Discussion Mercury is known to pose serious threats to human health owing to its toxic and hazardous nature and a positive link between mercury exposure and CVD has been firmly established (Houston, 2011; Virtanen et al., 2007). Among the molecular mechanisms
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Fig. 3. Effect of Hg treatment on ROS production in RBC. Cells were treated for 4 and 24 h in the presence of increasing concentrations of HgCl2, as described in Materials and methods. ROS production was evaluated by means of the fluorescent probe DCF. Data are the means ± SEM (n = 10). Statistical analysis was performed with oneway ANOVA followed by Dunnett’s test (p < 0.01). Means with different letters are significantly different.
Fig. 5. Effect of HT on Hg-induced GSH decrease in RBC. Cells were treated with 20 μM HgCl 2 for 4 in the presence of increasing concentrations of HT. Data are the means ± SEM (n = 10). Statistical analysis was performed with one-way ANOVA followed by Dunnett’s test (p < 0.01). Means with different letters are significantly different.
Please cite this article in press as: Lidia Tagliafierro, Arbace Officioso, Sergio Sorbo, Adriana Basile, Caterina Manna, The protective role of olive oil hydroxytyrosol against oxidative alterations induced by mercury in human erythrocytes, Food and Chemical Toxicology (2015), doi: 10.1016/j.fct.2015.04.029
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Fig. 6. Effect of HT on Hg-induced morphological alterations in RBC. Cells were exposed for 2 h to 20 μM HgCl2. At the end of incubation, cells were prepared for SEM observation as described in Materials and methods. (A) Untreated RBC; (B) HgCl2treated RBC; (C) HgCl2-treated RBC in the presence of 50 μM HT; (D) Echinocyte percentage quantified under the different conditions, as described in Materials and methods (n = 3). Statistical analysis was performed with one-way ANOVA followed by Dunnett’s test (p < 0.01). Means with different letters are significantly different.
8 underlying Hg toxicity, increased ROS formation seems to play a 9 major role in endothelial dysfunction (Furieri et al., 2011). However, 10 Gatti et al. (2004) report that Hg toxicity in PC12 cells is indepen11 dent of ROS generation. 12 The data presented in this paper offer experimental evidence that 13 Hg-induced ROS generation is a late event in RBC, and it probably 14 occurs subsequently to a significant decrease of essential antioxi15 dant thiols, which could render cells more susceptible to ROS16 mediated oxidative damage. In fact, after 4 h Hg treatment (20 μM), 17 a significant decrease in GSH concentration is observable, while no 18 ROS generation is detectable, according to data reported in similar 19 experimental conditions (Harisa et al., 2012). 20 Finally, an interesting consideration is that redox-active transi21 tion metal can exacerbate ROS generation acting as a catalyst of OH 22 radical formation: the hypothesis that considers the possible direct 23 formation of ROS by the Fenton type reaction as a contributing factor 24 in building up a pro-oxidative microenvironment in Hg-treated cells 25 can be ruled out in our model system. 26 Based on our observations and in agreement with the litera27 ture data, it is possible to conclude that Hg-induced oxidative 28 alterations may be regarded as contributing factors to the in29 creased rate of human pathologies, including anemia, particularly 30 related to chronic exposure to this heavy metal. In fact, although a 31 particularly high GSH concentration may partially protect RBC from 32 Hg toxic effects, chronic exposure could affect RBC viability and 33 induce shape changes, also affecting CVD. As pointed out before, Hg 34 exposure enhances pro-coagulant activity of these cells, resulting 35 in a contributing factor for Hg-related thrombotic disease (Lim et al., 36 2010). Further studies could validate the fascinating hypothesis that 37 metabolic and shape modification in RBC may be regarded as a clin38 ical biomarker, indicating increased cardiovascular risk in Hg39 exposed individuals. Interestingly, in this respect, Harisa and 40 colleagues (Harisa et al., 2013) utilized erythrocyte nitric oxide syn41 thase as a surrogate marker for Hg-induced vascular damage. 42 Here we also report for the first time that HT has the potential 43 to modulate cytotoxicity and to counteract GSH decrease and the 44 oxidative stress induced in RBC by Hg treatment. Also of clinical im45 portance is the finding that HT prevents Hg-induced procoagulant 46 RBC morphological alteration (echinocyte formation) in RBC. 47 Antioxidant polyphenols are believed to play a major role in the 48 positive correlation between adherence to the Mediterranean Diet 49 (MD) and a low incidence of CVD (Khurana et al., 2013; Tresserra-Rimbau et al. 2014). Our data strengthen the nutritional Q7 Q8 50 51 relevance of the phenolic fraction in olive oil in the aforemen52 tioned claims for the health promoting effects of the MD. Virgin olive 53 oil, an excellent source of oleic acid, vitamin E and nonessential nu54 trients, greatly contributes to the low incidence of CVD associated 55 with an adherence to this dietary habit in that almost all cardio56 vascular risk factors can be positively modulated by olive oil 57 constituents (Carluccio et al., 2007). In this respect, there is a general 58 agreement that the health promoting effects of olive oil intake result 59 from the combined properties of all its constituents, including poly60 phenols (López-Miranda et al., 2010). An increasing amount of data, Q9 61 indeed, clearly indicate that HT can improve cardiovascular health, 62 due to its strong antioxidant activity, presumably counteracting the 63 oxidative stress-induced endothelial dysfunction and modulating 64 key mechanisms implicated in the development of atherosclero65 sis, including expression of adhesion molecules (Manna et al., 2009). 66 Indeed, HT is endowed with significant anti-thrombotic, anti67 atherogenic and anti-inflammatory activities (Granados-Principal 68 et al., 2010). The data reported in this paper may usefully enlarge 69 on HT beneficial effects, indicating that prevention of Hg toxicity 70 could represent an additional mechanism responsible for cardio71 protection in vivo. In this respect, an interesting observation is that 72 HT active concentrations utilized in our study could be approached 73 in vivo upon strict adherence to the MD dietary habit. According to
Please cite this article in press as: Lidia Tagliafierro, Arbace Officioso, Sergio Sorbo, Adriana Basile, Caterina Manna, The protective role of olive oil hydroxytyrosol against oxidative alterations induced by mercury in human erythrocytes, Food and Chemical Toxicology (2015), doi: 10.1016/j.fct.2015.04.029
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the recent EFSA claim on the positive effect of the phenolic fraction of olive oil in the prevention of LDL oxidation, 5 mg of HT and its derivatives should be consumed daily. This amount, if provided by moderate quantities of olive oil, can easily be consumed in the context of a balanced diet. The use of phytochemicals able to significantly counteract oxidative alterations associated with Hg exposure as an attractive tool for the reduction of mercury toxicity has gained a lot of attention recently (Harisa et al., 2013; Kaivalya et al., 2011). Taken together, the reported findings indicate HT as an ideal candidate for nutritional/nutraceutical strategies to prevent mercury toxicity in humans. Particularly important, in this respect, is that HT has been proved by our group to be devoid of toxicity up to 2 g/kg in an animal model (D’Angelo et al., 2001) and a more recent study, investigating HT in terms of its safety profile, indicates the dose of 500 mg/ k/day as “No Observed Adverse Effects Level” (NOAEL). (Auñon-Calles et al., 2013). Conflict of interest The authors declare that there are no conflicts of interest. Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgements
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Please cite this article in press as: Lidia Tagliafierro, Arbace Officioso, Sergio Sorbo, Adriana Basile, Caterina Manna, The protective role of olive oil hydroxytyrosol against oxidative alterations induced by mercury in human erythrocytes, Food and Chemical Toxicology (2015), doi: 10.1016/j.fct.2015.04.029
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