http://informahealthcare.com/dct ISSN: 0148-0545 (print), 1525-6014 (electronic) Drug Chem Toxicol, Early Online: 1–7 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/01480545.2013.878950
Antioxidant role of hydroxytyrosol on oxidative stress in cadmium-intoxicated rats: different effect in spleen and testes Elisabetta Merra1, Giovanna Calzaretti1, Antonella Bobba2, Maria M. Storelli3, and Elisabetta Casalino1
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1
Department of Veterinary Medicine, University of Bari ‘‘Aldo Moro’’, Str. Prov. per Casamassima km 3, Valenzano, Italy, 2Institute of Biomembranes and Bioenergetics (IBBE), Italian Research Council, (CNR), Bari, Italy, and 3Department of Bioscience, Biotechnologies and Biopharmaceutics, University of Bari ‘‘Aldo Moro’’, Str. Prov. per Casamassima km 3, Valenzano, Italy Abstract
Keywords
Hydroxytyrosol (2-(3,4dihydroxyphenyl)ethanol, (DPE), a phenolic compound present in olive oil, is known to have antioxidant properties. The aim of this study was to investigate the effect of DPE on oxidative stress induced by cadmium injections (CdCl2 2.5 mg/kg body weight) in spleen and testes of adult male rats. Oxidative stress was evaluated by measuring lipid peroxidation by thiobarbituric acid reactive substances (TBARS) as well as superoxide dismutase (SOD) and catalase (CAT) activities in cytosol and mitochondria. We found that in spleen no TBARS formation was detected following CdCl2 injections; however, DPE induces decrease in TBARS level in treated and untreated rats. On the contrary, we observed that DPE showed no effect on cadmium-induced lipid peroxidation in testes. Cytosolic activities of SOD and CAT decreased significantly only in spleen, where DPE restores the values to the control levels. Noteworthy, mitochondrial activities of SOD and CAT were strongly reduced by cadmium treatment both in spleen and testes, and DPE was not be able to restore their activity. Overall, the results from this study indicated that the DPE has different antioxidant efficiency in spleen and testis of cadmium intoxicated rats.
Catalase, food antioxidant, lipid peroxidation, mitochondria, superoxide dismutase
Introduction Cadmium is one of the most common and widespread heavy metals in the environment. Although the intake of contaminated food and water is the common source of contamination for the general population, occupational exposure is considered the most important. Occupational exposure to cadmium occurs when working with pigments containing cadmium, plastic, glass, metal alloys, and the electrode material in batteries. Further, intensive non-occupational exposure is mostly caused by smoking (Waisberg et al., 2003). Acute and chronic cadmium toxicity is associated with toxic and carcinogenic effects in both humans and animals (Lauwerys, 1979; Valko et al., 2005). Depending on the route of administration Cd accumulates in various tissues and is concentrated mainly in the lungs, liver, kidneys, brain, heart, testes and spleen (Wang et al., 2004). In the cell Cd mainly accumulates in the cytosol (70%), followed by the nucleus (15%), and mitochondria and the endoplasmic reticulum (Goering & Klaassen, 1983).
Address for correspondence: Elisabetta Casalino, Department of Veterinary Medicine, Division of Pharmacology and Toxicology, University of Bari ‘‘Aldo Moro’’, Str. Prov. per Casamassima km 3, 70010 Valenzano, Italy. Tel: +39-080-5443864. Fax: +39-080-5443864. E-mail: [email protected]
History Received 6 June 2013 Revised 16 September 2013 Accepted 21 December 2013 Published online 15 January 2014
Mitochondria seem to be highly sensitive target of cadmium in the cell (Zhang et al., 2011). Cd toxicity has been associated with several diseases including osteoporosis, nephrotoxicity, pulmonary emphysema, liver dysfunction, and others. In addition, cadmium is weakly mutagenic and carcinogenic (Nawrot et al., 2010). At cellular level Cd has multiple effects: interference with enzymatic proteins, alterations in thiol proteins, inhibition of energy metabolism, alteration in DNA structure, altered membrane structure/function. It also affects cell cycle progression, proliferation, differentiation, DNA replication and repair, as well as apoptotic pathways (Bertin & Averbeck, 2006). Further, Cd enhances oxidative stress and, although Cd has not redox properties, increased levels of reactive oxygen species (ROS) have been observed both in vitro and in vivo (Valko et al., 2005). As a consequence of oxidative stress induced by Cd, lipid peroxidation has long been considered the primary mechanism for Cd toxicity in the liver, kidney, heart, brain, spleen, lung, erythrocytes and testes (Cuypers et al., 2010; Santos et al., 2005; Yiin et al., 2000). Testes are important targets of Cd, and peroxidative damage is currently considered the most important cause of impaired testicular function underlying the pathological consequences of a wide range of conditions from testicular torsion, cryptorchidism, varicocele and infertility. Cd is known to impair spermatogenesis destroying male fertility
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Table 1. Protocol of rats treatment. Treatments Groups Group Group Group Group
1 2 3 4
(n ¼ 5) (n ¼ 5) (n ¼ 5) (n ¼ 5)
Time 0
Time 1 h
Time 8 h
Saline (i.p.) CdCl2 (2.5 mg/kg, i.p.) Saline (i.p.) CdCl2 (2.5 mg/kg, i.p.)
Saline (i.p.) Saline (i.p.) DPE (9 mg/kg, i.p.) DPE (9 mg/kg, i.p.)
Saline (i.p.) Saline (i.p.) DPE (9 mg/kg, i.p.) DPE (9 mg/kg, i.p.)
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Animals were sacrificed 24 after the first i.p. injection. ‘‘n’’ represent the number of rats tested in each groups.
through mechanisms involving the induction of lipid peroxidation, depletion of ROS scavengers and disruptions testicular antioxidant enzymes (Aitken & Roman, 2008). The spleen, a major antibody-producing organ, is an important crossroads for antigenic message transported by blood and immune system. On the other hand, Cd is well known as immunotoxic metal and reports on Cd induced abnormal humoral and/or cellular responses are available (Pathak & Khandelwal, 2007). In light of the above, there is an urgent need to identify antioxidants able to integrate the antioxidant strategies own tissue for saving the above organs from the consequences of ROS attack. Several studies have investigated the antioxidant properties of different natural substances among those, carotenoids, retinoids, tocopherols, ascorbic acid, phenolic acids, flavonoids and polyphenols showing the possible protective effect in cellular damage induced by cadmium (Eybl et al., 2006). Olive oil contains several phenolic compounds and hydroxytyrosol (2-(3,4dihydroxyphenyl)ethanol, (DPE), is the most potent and it is more active than antioxidant vitamins (Manna et al., 2002) as well as the synthetic antioxidants (Ranalli et al., 2003). It has been shown to lower thiobarbituric acid reactive substances (TBARS) content in rat liver, heart, kidney, and to increase the serum antioxidant potential and the hepatic catalase (CAT) and superoxide dismutase (SOD) activities in rat (Casalino et al., 2002; Jemai et al., 2008a). In addition, hydroxytyrosol improves mitochondrial function in retinal pigment epithelial cells (Liu et al., 2007). In the present study we assessed the possible effectiveness of hydroxytyrosol against oxidative stress induced in the spleen and testes of rats after cadmium intoxication, with special consideration to the effect on some antioxidant mitochondrial enzymes.
art. 5 D.L. 116/92). Male Wistar rats weighing 180–200 g were used in this study. The rats were housed in polypropylene cages and kept under standard laboratory conditions (temperature 25 ± 2 C natural light-dark cycle). The rats had free access to drinking water and commercial standard pellet diet. Experimental design Rats were randomly divided into four groups (n ¼ 5 in each group) housed in different cages. The animals were injected i.p. with CdCl2 (2.5 mg/kg b.w.), and/or DPE (9.0 mg/kg b.w.) in 0.1 ml NaCl 0.9% and sacrificed 24 h after injection. When administered together, CdCl2 i.p. administration at 0 time was followed by two DPE i.p. administrations (see Table 1). The DPE dose was chosen based on results of previous studies (Casalino et al., 2002). Control animals received the equivalent volume of NaCl 0.9% solution (0.1 ml/kg body weight). The animals were killed by decapitation under ether anesthesia. Analytical procedures Testes and spleen were quickly excised, blotted and then rinsed in ice-cold saline (KCl 0.175 M/Tris-HCl 25 mM pH 7.4) to clear them of blood, weighed, finely minced in the same solution, and homogenized (10% w/v) in a Potter Elvehjem homogenizer with a Teflon pestle. Tissue homogenates from both control and treated rats was used for TBARS determination, while mitochondria and cytosol, obtained by differential centrifugation (Landriscina et al., 1976), were used for enzyme assay. Biochemical analysis Lipid peroxidation
Methods Reagents All chemicals biochemicals used were of the highest quality and purchased from Sigma Chemical Co. Chelex 100 ion exchange resin (Bio-Rad Laboratories, Hercules, CA) was used to remove contaminating metals from all reagents. Organic solvents of analytical grade were used. All solutions were prepared in double-distilled water. Animals and treatment All experiments were performed in accordance with the Italian Guidelines for the use of laboratory animals (art. 4 and
The TBARS assay was used to quantify the oxidative damage (lipid peroxidation). Briefly, aliquot of tissue homogenate was added to the reaction mixture containing TCA–HCl–TBA as reported by Buege & Aust (1987). Butylated hydroxytoluene (0.03 %, final concentration) was added prior to heating the mixture in 80 C water bath for 15 min to avoid any artifactual oxidation due to heating. After cooling, reaction mixture was centrifuged to precipitate the denaturated proteins and the supernatant was used to measure the absorbance at 535 nm. The concentration of lipid peroxides was expressed as nmoles TBARS per mg protein, using tetramethoxypropane as an external standard.
DOI: 10.3109/01480545.2013.878950
Hydroxytyrosol effect on spleen and testes of Cd-intoxicated rats
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Enzyme activity assay Superoxide dismutase (SOD, EC 1.15.1.1) was examined by method of Misra (1985). The activity was determined from its ability to inhibit the autoxidation of epinephrine. Stimulation of epinephrine autoxidation by traces of heavy metals present as contaminants in the reagents or by the other metals under investigation was prevented by adding 104 M EDTA in the buffer to chelate these ions. The two isoforms of SOD, cytosolic (CuZnSOD) and mitochondrial (MnSOD), were distinguished by cyanide activity inactivation, CuZnSOD being strongly inactivated unlike MnSOD whose activity was slightly reduced by cyanide. One unit of CuZnSOD and MnSOD is defined as the amount of enzyme required to inhibit the rate of epinephrine autoxidation by 50%. Catalase (CAT, EC 1.11.1.6) activity was assayed by the method of Aebi (1972), by following the decrease in absorbance of H2O2 at 240 nm (e 40 M1 cm1). One unit of enzyme activity is defined as the amount of enzyme required to degrade 1 micromole of H2O2 in 1 min and is expressed as U/mg protein. Enzyme activities were measured using a temperaturecontrolled Beckman DU 640 spectrophotometer. The assays were run in triplicate. All mitochondrial enzyme activities were determined after 4 freeze–thaw cycles to burst mitochondrial membranes and guarantee access to enzymes.
Figure 1. Effect of 2-(3,4-diydroxyphenyl)ethanol (DPE) on TBARS production in spleen and testes of cadmium-intoxicated rats. Gr I: control; Gr II: Cd treatment; Gr III: DPE treatment; Gr IV: Cd + DPE treatment. Data are the means ± S.D (n ¼ 5). *p50.05; **p50.01.
Protein concentration Protein concentrations were determined by the Bradford assay procedure using bovine serum albumin as standard (Bradford, 1976). Statistics Data were expressed as mean ± S.D. and analyzed statistically using one-way analysis of variance (ANOVA) followed by Tukey’s test. Differences from controls were considered significant at *p50.05 and **p50.01.
Results Effect of DPE treatment on lipid peroxidation The effect of DPE treatment on splenic and testicular lipid peroxidation in Cd-intoxicated rat is presented in Figure 1. Twenty-four hours after Cd administration no increase in TBARS levels was observed in spleen. However, treatment with DPE, either in presence and absence of Cd, significantly reduced (p50.01) TBARS levels by about 53%, in both cases, compared to control values. On the other hand, in the same figure it is possible to see that rats receiving only Cd showed a significant increase (p50.05) in TBARS production in testes (49% higher than the control value). Administration of two doses of DPE, one hour and eight hours after intoxication with Cd, was ineffective in restoring TBARS status towards the control level. Effect of DPE treatment on antioxidant enzyme activity The effect of Cd treatment on SOD and CAT activities in cytosol and mitochondria of rat spleen is reported in Figures 2 and 3 respectively.
Figure 2. Effect of 2-(3,4-diydroxyphenyl)ethanol (DPE) on CuZnSOD and catalase activities in cytosol of cadmium-intoxicated rat spleen. Gr I: control; Gr II: Cd treatment; Gr III: DPE treatment; Gr IV: Cd + DPE treatment. Data are the means ± S.D (n ¼ 5). *p50.05; **p50.01.
CuZnSOD was inhibited by 22.8% following Cd intoxication, whereas a decrease by 31.8% was observed in cytosolic CAT. DPE exerted its antioxidant effect by restoring the values of both enzyme activities near control values (Figure 2). Stronger decrease of SOD and CAT activities was observed in spleen mitochondria of rats intoxicated with Cd. In this case DPE has not been able to restore the activity of enzymes (Figure 3). Figure 4 shows that Cd treatment in testes did not alter in any way the activity of CuZnSOD, while CAT activity in the cytosol was strongly enhanced. On both enzymes, DPE did not change the effects induced by Cd. As it is possible to see from Figure 5, activities of the same enzymes in mitochondria were significantly reduced by 31.8 and 23.5% respectively. Once again, DPE did not restore the activity of SOD and CAT in mitochondria.
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Figure 3. Effect of 2-(3,4-diydroxyphenyl)ethanol (DPE) on MnSOD and catalase activities on mitochondria of cadmium-intoxicated rat spleen. Gr I: control; Gr II: Cd treatment; Gr III: DPE treatment; Gr IV: Cd + DPE treatment. Data are the means ± S.D (n ¼ 5). *p50.05; **p50.01.
Figure 4. Effect of 2-(3,4-diydroxyphenyl)ethanol (DPE) on CuZnSOD and catalase activities in cytosol of cadmium-intoxicated rat testes. Gr I: control; Gr II: Cd treatment; Gr III: DPE treatment; Gr IV: Cd + DPE treatment. Data are the means ± S.D (n ¼ 5). *p50.01.
Discussion Testes and spleen are affected by acute cadmium (Cd) intoxication (Santos et al., 2004; Yiin et al., 2000) and mitochondria are one of the targets for Cd injury (Amara et al., 2008; Pathak & Khandelwal, 2007). Oxidative stress and consequent lipid peroxidation seem to be responsible for harm induced by Cd in these organs, so that treatment with antioxidants was effective against deleterious effects of reactive oxygen species (ROS) induced by Cd (Gupta et al., 2004; Koyuturk et al., 2006; Pathak & Khandelwal, 2007; Santos et al., 2005; Yiin et al., 2000; Yiin et al., 1999). Nowadays there is a growing interest in the use of antioxidant nutrients to the modulation of pathological consequences of free radical in biological systems.
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Figure 5. Effect of 2-(3,4-diydroxyphenyl)ethanol (DPE) on MnSOD and catalase activities on mitochondria of cadmium-intoxicated rat testes. Gr I: control; Gr II: Cd treatment; Gr III: DPE treatment; Gr IV: Cd + DPE treatment. Data are the means ± S.D (n ¼ 5). *p50.05; **p50.01.
In recent years, active substances with different chemical properties possessing antioxidant effects have been isolated from plants. Sudharsan and co-workers have shown that lupeol, a triterpene isolated from the medicinal plant Crater nurvala Buch-Ham (Capparidaceae) exhibits antitumor and antioxidant activity in rats (Sudharsan et al., 2005). Silymarin, a purified extract of Silybum marianum Gaertn, composed mainly of flavonolignans, is frequently used in the treatment of liver diseases where inhibits lipid peroxidation and prevents liver glutathione depletion (Gazak et al., 2004). A wide class of compounds of plant origin with antioxidant activity is represented by phenols that occur in fruits and vegetables, wine and tea, chocolate and other cocoa products (Manach et al., 2004). They operate both as scavengers radicals or as chain breakers, depending on their chemical structures (Rice-Evans, 1995). DPE is the main polyphenol present in the olive oil. Many findings have been reported on the benefits, and especially on antioxidant action of DPE exerted in ‘‘in vitro’’ system (Hamden et al., 2009; Casalino et al., 2002; Gutierrez et al., 2001) such as on plasma and isolated cells (Castan˜er et al., 2011; Manna et al., 2000; Pereira-Caro et al., 2012; Rodrı´guez-Ramiro et al., 2011; Schaffer & Halliwell, 2011; Va´zquez-Velasco et al., 2011; Zhang et al., 2009). The effectiveness of DPE on different organs was also proved. In a previous paper we reported findings on antioxidant effect of DPE against damage induced by Cd. Our results showed that DPE was able to lower the levels of TBARS in rat liver intoxicated with an acute dose of cadmium, as well as to reduce TBARS produced in vitro after incubation of rat liver microsomes with Cd (Casalino et al., 2002). Jemai et al. (2008a,b) reported that DPE reduces levels of TBARS in liver, heart, kidney and aorta in cholesterol-fed rats, and hepatic activities of SOD and CAT were stimulated. Positive effects of DPE are also reported in the brain under hypoxic conditions (Gonza´lez-Correa et al., 2008).
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DOI: 10.3109/01480545.2013.878950
Hydroxytyrosol effect on spleen and testes of Cd-intoxicated rats
Nevertheless, no previous study has examined the ability of DPE to reverse oxidative stress induced by Cd in spleen and testes, hence we first provide some indication on the effect of this compound in these organs. The results obtained in the present study show that in spleen DPE is able to lower TBARS content also in rats not exposed to Cd. Furthermore, it also restores the cytoplasmic activity of SOD and CAT decreased by Cd, whereas it is ineffective in recovering the activity of the same enzymes in mitochondria. In testes, in contrast to that observed in spleen, DPE is ineffective in removing Cd-induced TBARS as well as in re-establishing the activities of mitochondrial enzymes SOD and CAT decreased by Cd treatment. On the other hand, Cd treatment in testes did not alter in any way the activity of CuZnSOD, while cytosol CAT activity was strongly enhanced. On both enzymes, DPE did not change the effects induced by Cd. Overall, our results indicate that, despite the different effect of DPE in spleen and testes of rats, the common feature is that in both organs DPE does not appear to exert any effect in mitochondria. Hereby conclusion is in apparent contrast with data reported by other researchers who have shown evident consequences of DPE treatment on mitochondria. Zhu et al. have shown that treatment with DPE protects retinal pigment epithelial cells from acrolein-induced oxidative damage and mitochondrial dysfunction and prevents decrease of activity and expression of MnSOD (Zhu et al., 2010). It has also been proven by others that DPE enhances mitochondrial function and stimulates mitochondrial biogenesis (Hao et al., 2010; Zhu et al., 2010). At first we have hypothesized that the difference between the above and our results could be explained by the fact that these experiments have been conducted on isolated cells and not on the animal model. However a report by Feng shows that DPE, in vivo administered to rats 45 min before beginning the exercise program, enhances mitochondrial complex I and II activities in muscle of rat subjected to strenuous physical exercise (Feng et al., 2011). It is our opinion that the extreme variability of the reported effects of DPE is due to the differences of its availability, and that of its metabolites. Route of administration (orally, intravenously, intraperitoneally) affects the rate and extent of absorption, such as the medium of DPE solution (Tuck et al., 2001). Another important factor is the different distribution of the DPE and its metabolites between different organs of the same animal. Indeed in our laboratory we have shown results on antioxidant effect of DPE on rat liver (Casalino et al., 2002) different than those referred in testes and spleen in this trial, despite having applied the same experimental protocol. The positive effect, observed in the liver, of the same concentration of DPE used here, is probably due to the fact that the compound accumulates primarily in the liver where it is metabolized and converted into sulphated and glucuronidated forms. In testes and spleen of rats, a wide range of metabolites were detected with sulphate conjugates being the main metabolites quantified. Serra and colleagues (Serra et al., 2012) reported that after administration to rats of a
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phenolic extract from olive cake, the testes showed a high concentration of hydroxytyrosol-sulfate, with a maximum accumulation of 1005 nmol/g tissue 2 hours after ingestion extract, decreasing to 200 nmol/g tissue after 4 h of ingestion. On the other hand, hydroxytyrosol-sulfate was detected in the spleen at lower concentrations, such as 21 nmol/g tissue 2 hours after the ingestion of the extract but which persisted even 4 h after ingestion. We can not exclude that, although present in lower doses than testes, the longer time persistence of hydroxytyrosol metabolites in the spleen may account for the different antioxidant effectiveness observed in cytosol of spleen compared to testicle. Apart from these data on the concentration of various metabolites of DPE in the whole spleen and testicles, far as we know there are no data on the concentration of DPE or its metabolites in mitochondria of these organs. It is noteworthy that the results so far reported about the beneficial effect of DPE on mitochondria, show that DPE operates through the modulation of the signaling pathways in the cytoplasm and/or nucleus but not at the mitochondrial level (Hao et al., 2010). Therefore, determination of the different bioavailability of DPE and its metabolites in different tissues and subcellular distribution, may be much more significant than is the knowledge of their plasma concentrations. Additional studies are already under way in our laboratory to further study the distribution and effects of DPE and its metabolites in mitochondria of several organs of rat.
Conclusion In conclusion, in addition to antioxidant effect exerted by DPE on SOD and catalase activity in cytosol of spleen from Cd-intoxicated rat, the outcome emerging from this work is the failure of DPE to restore the enzymatic activities studied in mitochondria of both spleen and testes of Cd-intoxicated rats. Although it may appear at first sight as a negative result, de facto it provides new perspectives of study for a wiser use of the DPE as a potential prophylactic agent against a wide range of disorders, including inflammatory and neurodegenerative diseases, blood disorders, cancer, diabetes and aging. Specifically, more experiments are required to investigate the effect of DPE and its metabolites on the functions of such organs as spleen and testes. Still, the mechanism(s) of DPE in decreasing cadmiuminduced oxidative stress is not well clear, and it would also be interesting to further investigate the effect of DPE against damage induced by cadmium and even by other heavy metals. The extrapolation of in vitro results and their verification in vivo is a critical issue in research on this compound in general and in view of its implementation as a nutraceutical drug.
Acknowledgements Funding for this work was provided by the University of Bari, Fondi di Ateneo 2010. The authors are grateful to Mrs. Arianna Storelli for providing linguistic advice and to Mr. Gaetano Devito for excellent technical assistance in animals treatment.
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Declaration of interest The authors declare that there are no conflicts of interest.
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Hydroxytyrosol effect on spleen and testes of Cd-intoxicated rats
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Zhang Li, JH, Liu XR, Jiang FL, et al. (2011). Spectroscopic and microscopic studies on the mechanisms of mitochondrial toxicity induced by different concentrations of cadmium. J Membr Biol 24:39–49. Zhu L, Liu Z, Feng Z, et al. (2010). Hydroxytyrosol protects against oxidative damage by simultaneous activation of mitochondrial biogenesis and phase II detoxifying enzyme systems in retinal pigment epithelial cells. J Nutr Biochem 21:1089–1098.
Antioxidant role of hydroxytyrosol on oxidative stress in cadmium-intoxicated rats: different effect in spleen and testes Elisabetta Merra1, Giovanna Calzaretti1, Antonella Bobba2, Maria M. Storelli3, and Elisabetta Casalino1
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1
Department of Veterinary Medicine, University of Bari ‘‘Aldo Moro’’, Str. Prov. per Casamassima km 3, Valenzano, Italy, 2Institute of Biomembranes and Bioenergetics (IBBE), Italian Research Council, (CNR), Bari, Italy, and 3Department of Bioscience, Biotechnologies and Biopharmaceutics, University of Bari ‘‘Aldo Moro’’, Str. Prov. per Casamassima km 3, Valenzano, Italy Abstract
Keywords
Hydroxytyrosol (2-(3,4dihydroxyphenyl)ethanol, (DPE), a phenolic compound present in olive oil, is known to have antioxidant properties. The aim of this study was to investigate the effect of DPE on oxidative stress induced by cadmium injections (CdCl2 2.5 mg/kg body weight) in spleen and testes of adult male rats. Oxidative stress was evaluated by measuring lipid peroxidation by thiobarbituric acid reactive substances (TBARS) as well as superoxide dismutase (SOD) and catalase (CAT) activities in cytosol and mitochondria. We found that in spleen no TBARS formation was detected following CdCl2 injections; however, DPE induces decrease in TBARS level in treated and untreated rats. On the contrary, we observed that DPE showed no effect on cadmium-induced lipid peroxidation in testes. Cytosolic activities of SOD and CAT decreased significantly only in spleen, where DPE restores the values to the control levels. Noteworthy, mitochondrial activities of SOD and CAT were strongly reduced by cadmium treatment both in spleen and testes, and DPE was not be able to restore their activity. Overall, the results from this study indicated that the DPE has different antioxidant efficiency in spleen and testis of cadmium intoxicated rats.
Catalase, food antioxidant, lipid peroxidation, mitochondria, superoxide dismutase
Introduction Cadmium is one of the most common and widespread heavy metals in the environment. Although the intake of contaminated food and water is the common source of contamination for the general population, occupational exposure is considered the most important. Occupational exposure to cadmium occurs when working with pigments containing cadmium, plastic, glass, metal alloys, and the electrode material in batteries. Further, intensive non-occupational exposure is mostly caused by smoking (Waisberg et al., 2003). Acute and chronic cadmium toxicity is associated with toxic and carcinogenic effects in both humans and animals (Lauwerys, 1979; Valko et al., 2005). Depending on the route of administration Cd accumulates in various tissues and is concentrated mainly in the lungs, liver, kidneys, brain, heart, testes and spleen (Wang et al., 2004). In the cell Cd mainly accumulates in the cytosol (70%), followed by the nucleus (15%), and mitochondria and the endoplasmic reticulum (Goering & Klaassen, 1983).
Address for correspondence: Elisabetta Casalino, Department of Veterinary Medicine, Division of Pharmacology and Toxicology, University of Bari ‘‘Aldo Moro’’, Str. Prov. per Casamassima km 3, 70010 Valenzano, Italy. Tel: +39-080-5443864. Fax: +39-080-5443864. E-mail: [email protected]
History Received 6 June 2013 Revised 16 September 2013 Accepted 21 December 2013 Published online 15 January 2014
Mitochondria seem to be highly sensitive target of cadmium in the cell (Zhang et al., 2011). Cd toxicity has been associated with several diseases including osteoporosis, nephrotoxicity, pulmonary emphysema, liver dysfunction, and others. In addition, cadmium is weakly mutagenic and carcinogenic (Nawrot et al., 2010). At cellular level Cd has multiple effects: interference with enzymatic proteins, alterations in thiol proteins, inhibition of energy metabolism, alteration in DNA structure, altered membrane structure/function. It also affects cell cycle progression, proliferation, differentiation, DNA replication and repair, as well as apoptotic pathways (Bertin & Averbeck, 2006). Further, Cd enhances oxidative stress and, although Cd has not redox properties, increased levels of reactive oxygen species (ROS) have been observed both in vitro and in vivo (Valko et al., 2005). As a consequence of oxidative stress induced by Cd, lipid peroxidation has long been considered the primary mechanism for Cd toxicity in the liver, kidney, heart, brain, spleen, lung, erythrocytes and testes (Cuypers et al., 2010; Santos et al., 2005; Yiin et al., 2000). Testes are important targets of Cd, and peroxidative damage is currently considered the most important cause of impaired testicular function underlying the pathological consequences of a wide range of conditions from testicular torsion, cryptorchidism, varicocele and infertility. Cd is known to impair spermatogenesis destroying male fertility
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Table 1. Protocol of rats treatment. Treatments Groups Group Group Group Group
1 2 3 4
(n ¼ 5) (n ¼ 5) (n ¼ 5) (n ¼ 5)
Time 0
Time 1 h
Time 8 h
Saline (i.p.) CdCl2 (2.5 mg/kg, i.p.) Saline (i.p.) CdCl2 (2.5 mg/kg, i.p.)
Saline (i.p.) Saline (i.p.) DPE (9 mg/kg, i.p.) DPE (9 mg/kg, i.p.)
Saline (i.p.) Saline (i.p.) DPE (9 mg/kg, i.p.) DPE (9 mg/kg, i.p.)
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Animals were sacrificed 24 after the first i.p. injection. ‘‘n’’ represent the number of rats tested in each groups.
through mechanisms involving the induction of lipid peroxidation, depletion of ROS scavengers and disruptions testicular antioxidant enzymes (Aitken & Roman, 2008). The spleen, a major antibody-producing organ, is an important crossroads for antigenic message transported by blood and immune system. On the other hand, Cd is well known as immunotoxic metal and reports on Cd induced abnormal humoral and/or cellular responses are available (Pathak & Khandelwal, 2007). In light of the above, there is an urgent need to identify antioxidants able to integrate the antioxidant strategies own tissue for saving the above organs from the consequences of ROS attack. Several studies have investigated the antioxidant properties of different natural substances among those, carotenoids, retinoids, tocopherols, ascorbic acid, phenolic acids, flavonoids and polyphenols showing the possible protective effect in cellular damage induced by cadmium (Eybl et al., 2006). Olive oil contains several phenolic compounds and hydroxytyrosol (2-(3,4dihydroxyphenyl)ethanol, (DPE), is the most potent and it is more active than antioxidant vitamins (Manna et al., 2002) as well as the synthetic antioxidants (Ranalli et al., 2003). It has been shown to lower thiobarbituric acid reactive substances (TBARS) content in rat liver, heart, kidney, and to increase the serum antioxidant potential and the hepatic catalase (CAT) and superoxide dismutase (SOD) activities in rat (Casalino et al., 2002; Jemai et al., 2008a). In addition, hydroxytyrosol improves mitochondrial function in retinal pigment epithelial cells (Liu et al., 2007). In the present study we assessed the possible effectiveness of hydroxytyrosol against oxidative stress induced in the spleen and testes of rats after cadmium intoxication, with special consideration to the effect on some antioxidant mitochondrial enzymes.
art. 5 D.L. 116/92). Male Wistar rats weighing 180–200 g were used in this study. The rats were housed in polypropylene cages and kept under standard laboratory conditions (temperature 25 ± 2 C natural light-dark cycle). The rats had free access to drinking water and commercial standard pellet diet. Experimental design Rats were randomly divided into four groups (n ¼ 5 in each group) housed in different cages. The animals were injected i.p. with CdCl2 (2.5 mg/kg b.w.), and/or DPE (9.0 mg/kg b.w.) in 0.1 ml NaCl 0.9% and sacrificed 24 h after injection. When administered together, CdCl2 i.p. administration at 0 time was followed by two DPE i.p. administrations (see Table 1). The DPE dose was chosen based on results of previous studies (Casalino et al., 2002). Control animals received the equivalent volume of NaCl 0.9% solution (0.1 ml/kg body weight). The animals were killed by decapitation under ether anesthesia. Analytical procedures Testes and spleen were quickly excised, blotted and then rinsed in ice-cold saline (KCl 0.175 M/Tris-HCl 25 mM pH 7.4) to clear them of blood, weighed, finely minced in the same solution, and homogenized (10% w/v) in a Potter Elvehjem homogenizer with a Teflon pestle. Tissue homogenates from both control and treated rats was used for TBARS determination, while mitochondria and cytosol, obtained by differential centrifugation (Landriscina et al., 1976), were used for enzyme assay. Biochemical analysis Lipid peroxidation
Methods Reagents All chemicals biochemicals used were of the highest quality and purchased from Sigma Chemical Co. Chelex 100 ion exchange resin (Bio-Rad Laboratories, Hercules, CA) was used to remove contaminating metals from all reagents. Organic solvents of analytical grade were used. All solutions were prepared in double-distilled water. Animals and treatment All experiments were performed in accordance with the Italian Guidelines for the use of laboratory animals (art. 4 and
The TBARS assay was used to quantify the oxidative damage (lipid peroxidation). Briefly, aliquot of tissue homogenate was added to the reaction mixture containing TCA–HCl–TBA as reported by Buege & Aust (1987). Butylated hydroxytoluene (0.03 %, final concentration) was added prior to heating the mixture in 80 C water bath for 15 min to avoid any artifactual oxidation due to heating. After cooling, reaction mixture was centrifuged to precipitate the denaturated proteins and the supernatant was used to measure the absorbance at 535 nm. The concentration of lipid peroxides was expressed as nmoles TBARS per mg protein, using tetramethoxypropane as an external standard.
DOI: 10.3109/01480545.2013.878950
Hydroxytyrosol effect on spleen and testes of Cd-intoxicated rats
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Enzyme activity assay Superoxide dismutase (SOD, EC 1.15.1.1) was examined by method of Misra (1985). The activity was determined from its ability to inhibit the autoxidation of epinephrine. Stimulation of epinephrine autoxidation by traces of heavy metals present as contaminants in the reagents or by the other metals under investigation was prevented by adding 104 M EDTA in the buffer to chelate these ions. The two isoforms of SOD, cytosolic (CuZnSOD) and mitochondrial (MnSOD), were distinguished by cyanide activity inactivation, CuZnSOD being strongly inactivated unlike MnSOD whose activity was slightly reduced by cyanide. One unit of CuZnSOD and MnSOD is defined as the amount of enzyme required to inhibit the rate of epinephrine autoxidation by 50%. Catalase (CAT, EC 1.11.1.6) activity was assayed by the method of Aebi (1972), by following the decrease in absorbance of H2O2 at 240 nm (e 40 M1 cm1). One unit of enzyme activity is defined as the amount of enzyme required to degrade 1 micromole of H2O2 in 1 min and is expressed as U/mg protein. Enzyme activities were measured using a temperaturecontrolled Beckman DU 640 spectrophotometer. The assays were run in triplicate. All mitochondrial enzyme activities were determined after 4 freeze–thaw cycles to burst mitochondrial membranes and guarantee access to enzymes.
Figure 1. Effect of 2-(3,4-diydroxyphenyl)ethanol (DPE) on TBARS production in spleen and testes of cadmium-intoxicated rats. Gr I: control; Gr II: Cd treatment; Gr III: DPE treatment; Gr IV: Cd + DPE treatment. Data are the means ± S.D (n ¼ 5). *p50.05; **p50.01.
Protein concentration Protein concentrations were determined by the Bradford assay procedure using bovine serum albumin as standard (Bradford, 1976). Statistics Data were expressed as mean ± S.D. and analyzed statistically using one-way analysis of variance (ANOVA) followed by Tukey’s test. Differences from controls were considered significant at *p50.05 and **p50.01.
Results Effect of DPE treatment on lipid peroxidation The effect of DPE treatment on splenic and testicular lipid peroxidation in Cd-intoxicated rat is presented in Figure 1. Twenty-four hours after Cd administration no increase in TBARS levels was observed in spleen. However, treatment with DPE, either in presence and absence of Cd, significantly reduced (p50.01) TBARS levels by about 53%, in both cases, compared to control values. On the other hand, in the same figure it is possible to see that rats receiving only Cd showed a significant increase (p50.05) in TBARS production in testes (49% higher than the control value). Administration of two doses of DPE, one hour and eight hours after intoxication with Cd, was ineffective in restoring TBARS status towards the control level. Effect of DPE treatment on antioxidant enzyme activity The effect of Cd treatment on SOD and CAT activities in cytosol and mitochondria of rat spleen is reported in Figures 2 and 3 respectively.
Figure 2. Effect of 2-(3,4-diydroxyphenyl)ethanol (DPE) on CuZnSOD and catalase activities in cytosol of cadmium-intoxicated rat spleen. Gr I: control; Gr II: Cd treatment; Gr III: DPE treatment; Gr IV: Cd + DPE treatment. Data are the means ± S.D (n ¼ 5). *p50.05; **p50.01.
CuZnSOD was inhibited by 22.8% following Cd intoxication, whereas a decrease by 31.8% was observed in cytosolic CAT. DPE exerted its antioxidant effect by restoring the values of both enzyme activities near control values (Figure 2). Stronger decrease of SOD and CAT activities was observed in spleen mitochondria of rats intoxicated with Cd. In this case DPE has not been able to restore the activity of enzymes (Figure 3). Figure 4 shows that Cd treatment in testes did not alter in any way the activity of CuZnSOD, while CAT activity in the cytosol was strongly enhanced. On both enzymes, DPE did not change the effects induced by Cd. As it is possible to see from Figure 5, activities of the same enzymes in mitochondria were significantly reduced by 31.8 and 23.5% respectively. Once again, DPE did not restore the activity of SOD and CAT in mitochondria.
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Figure 3. Effect of 2-(3,4-diydroxyphenyl)ethanol (DPE) on MnSOD and catalase activities on mitochondria of cadmium-intoxicated rat spleen. Gr I: control; Gr II: Cd treatment; Gr III: DPE treatment; Gr IV: Cd + DPE treatment. Data are the means ± S.D (n ¼ 5). *p50.05; **p50.01.
Figure 4. Effect of 2-(3,4-diydroxyphenyl)ethanol (DPE) on CuZnSOD and catalase activities in cytosol of cadmium-intoxicated rat testes. Gr I: control; Gr II: Cd treatment; Gr III: DPE treatment; Gr IV: Cd + DPE treatment. Data are the means ± S.D (n ¼ 5). *p50.01.
Discussion Testes and spleen are affected by acute cadmium (Cd) intoxication (Santos et al., 2004; Yiin et al., 2000) and mitochondria are one of the targets for Cd injury (Amara et al., 2008; Pathak & Khandelwal, 2007). Oxidative stress and consequent lipid peroxidation seem to be responsible for harm induced by Cd in these organs, so that treatment with antioxidants was effective against deleterious effects of reactive oxygen species (ROS) induced by Cd (Gupta et al., 2004; Koyuturk et al., 2006; Pathak & Khandelwal, 2007; Santos et al., 2005; Yiin et al., 2000; Yiin et al., 1999). Nowadays there is a growing interest in the use of antioxidant nutrients to the modulation of pathological consequences of free radical in biological systems.
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Figure 5. Effect of 2-(3,4-diydroxyphenyl)ethanol (DPE) on MnSOD and catalase activities on mitochondria of cadmium-intoxicated rat testes. Gr I: control; Gr II: Cd treatment; Gr III: DPE treatment; Gr IV: Cd + DPE treatment. Data are the means ± S.D (n ¼ 5). *p50.05; **p50.01.
In recent years, active substances with different chemical properties possessing antioxidant effects have been isolated from plants. Sudharsan and co-workers have shown that lupeol, a triterpene isolated from the medicinal plant Crater nurvala Buch-Ham (Capparidaceae) exhibits antitumor and antioxidant activity in rats (Sudharsan et al., 2005). Silymarin, a purified extract of Silybum marianum Gaertn, composed mainly of flavonolignans, is frequently used in the treatment of liver diseases where inhibits lipid peroxidation and prevents liver glutathione depletion (Gazak et al., 2004). A wide class of compounds of plant origin with antioxidant activity is represented by phenols that occur in fruits and vegetables, wine and tea, chocolate and other cocoa products (Manach et al., 2004). They operate both as scavengers radicals or as chain breakers, depending on their chemical structures (Rice-Evans, 1995). DPE is the main polyphenol present in the olive oil. Many findings have been reported on the benefits, and especially on antioxidant action of DPE exerted in ‘‘in vitro’’ system (Hamden et al., 2009; Casalino et al., 2002; Gutierrez et al., 2001) such as on plasma and isolated cells (Castan˜er et al., 2011; Manna et al., 2000; Pereira-Caro et al., 2012; Rodrı´guez-Ramiro et al., 2011; Schaffer & Halliwell, 2011; Va´zquez-Velasco et al., 2011; Zhang et al., 2009). The effectiveness of DPE on different organs was also proved. In a previous paper we reported findings on antioxidant effect of DPE against damage induced by Cd. Our results showed that DPE was able to lower the levels of TBARS in rat liver intoxicated with an acute dose of cadmium, as well as to reduce TBARS produced in vitro after incubation of rat liver microsomes with Cd (Casalino et al., 2002). Jemai et al. (2008a,b) reported that DPE reduces levels of TBARS in liver, heart, kidney and aorta in cholesterol-fed rats, and hepatic activities of SOD and CAT were stimulated. Positive effects of DPE are also reported in the brain under hypoxic conditions (Gonza´lez-Correa et al., 2008).
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DOI: 10.3109/01480545.2013.878950
Hydroxytyrosol effect on spleen and testes of Cd-intoxicated rats
Nevertheless, no previous study has examined the ability of DPE to reverse oxidative stress induced by Cd in spleen and testes, hence we first provide some indication on the effect of this compound in these organs. The results obtained in the present study show that in spleen DPE is able to lower TBARS content also in rats not exposed to Cd. Furthermore, it also restores the cytoplasmic activity of SOD and CAT decreased by Cd, whereas it is ineffective in recovering the activity of the same enzymes in mitochondria. In testes, in contrast to that observed in spleen, DPE is ineffective in removing Cd-induced TBARS as well as in re-establishing the activities of mitochondrial enzymes SOD and CAT decreased by Cd treatment. On the other hand, Cd treatment in testes did not alter in any way the activity of CuZnSOD, while cytosol CAT activity was strongly enhanced. On both enzymes, DPE did not change the effects induced by Cd. Overall, our results indicate that, despite the different effect of DPE in spleen and testes of rats, the common feature is that in both organs DPE does not appear to exert any effect in mitochondria. Hereby conclusion is in apparent contrast with data reported by other researchers who have shown evident consequences of DPE treatment on mitochondria. Zhu et al. have shown that treatment with DPE protects retinal pigment epithelial cells from acrolein-induced oxidative damage and mitochondrial dysfunction and prevents decrease of activity and expression of MnSOD (Zhu et al., 2010). It has also been proven by others that DPE enhances mitochondrial function and stimulates mitochondrial biogenesis (Hao et al., 2010; Zhu et al., 2010). At first we have hypothesized that the difference between the above and our results could be explained by the fact that these experiments have been conducted on isolated cells and not on the animal model. However a report by Feng shows that DPE, in vivo administered to rats 45 min before beginning the exercise program, enhances mitochondrial complex I and II activities in muscle of rat subjected to strenuous physical exercise (Feng et al., 2011). It is our opinion that the extreme variability of the reported effects of DPE is due to the differences of its availability, and that of its metabolites. Route of administration (orally, intravenously, intraperitoneally) affects the rate and extent of absorption, such as the medium of DPE solution (Tuck et al., 2001). Another important factor is the different distribution of the DPE and its metabolites between different organs of the same animal. Indeed in our laboratory we have shown results on antioxidant effect of DPE on rat liver (Casalino et al., 2002) different than those referred in testes and spleen in this trial, despite having applied the same experimental protocol. The positive effect, observed in the liver, of the same concentration of DPE used here, is probably due to the fact that the compound accumulates primarily in the liver where it is metabolized and converted into sulphated and glucuronidated forms. In testes and spleen of rats, a wide range of metabolites were detected with sulphate conjugates being the main metabolites quantified. Serra and colleagues (Serra et al., 2012) reported that after administration to rats of a
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phenolic extract from olive cake, the testes showed a high concentration of hydroxytyrosol-sulfate, with a maximum accumulation of 1005 nmol/g tissue 2 hours after ingestion extract, decreasing to 200 nmol/g tissue after 4 h of ingestion. On the other hand, hydroxytyrosol-sulfate was detected in the spleen at lower concentrations, such as 21 nmol/g tissue 2 hours after the ingestion of the extract but which persisted even 4 h after ingestion. We can not exclude that, although present in lower doses than testes, the longer time persistence of hydroxytyrosol metabolites in the spleen may account for the different antioxidant effectiveness observed in cytosol of spleen compared to testicle. Apart from these data on the concentration of various metabolites of DPE in the whole spleen and testicles, far as we know there are no data on the concentration of DPE or its metabolites in mitochondria of these organs. It is noteworthy that the results so far reported about the beneficial effect of DPE on mitochondria, show that DPE operates through the modulation of the signaling pathways in the cytoplasm and/or nucleus but not at the mitochondrial level (Hao et al., 2010). Therefore, determination of the different bioavailability of DPE and its metabolites in different tissues and subcellular distribution, may be much more significant than is the knowledge of their plasma concentrations. Additional studies are already under way in our laboratory to further study the distribution and effects of DPE and its metabolites in mitochondria of several organs of rat.
Conclusion In conclusion, in addition to antioxidant effect exerted by DPE on SOD and catalase activity in cytosol of spleen from Cd-intoxicated rat, the outcome emerging from this work is the failure of DPE to restore the enzymatic activities studied in mitochondria of both spleen and testes of Cd-intoxicated rats. Although it may appear at first sight as a negative result, de facto it provides new perspectives of study for a wiser use of the DPE as a potential prophylactic agent against a wide range of disorders, including inflammatory and neurodegenerative diseases, blood disorders, cancer, diabetes and aging. Specifically, more experiments are required to investigate the effect of DPE and its metabolites on the functions of such organs as spleen and testes. Still, the mechanism(s) of DPE in decreasing cadmiuminduced oxidative stress is not well clear, and it would also be interesting to further investigate the effect of DPE against damage induced by cadmium and even by other heavy metals. The extrapolation of in vitro results and their verification in vivo is a critical issue in research on this compound in general and in view of its implementation as a nutraceutical drug.
Acknowledgements Funding for this work was provided by the University of Bari, Fondi di Ateneo 2010. The authors are grateful to Mrs. Arianna Storelli for providing linguistic advice and to Mr. Gaetano Devito for excellent technical assistance in animals treatment.
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Declaration of interest The authors declare that there are no conflicts of interest.
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DOI: 10.3109/01480545.2013.878950
Hydroxytyrosol effect on spleen and testes of Cd-intoxicated rats
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