Hydroxytyrosol ameliorates oxidative stress and mitochondrial dysfunction in doxorubicin-induced cardiotoxicity in rats with breast cancer.

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BCP-11949; No. of Pages 9 Biochemical Pharmacology xxx (2014) xxx–xxx

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Hydroxytyrosol ameliorates oxidative stress and mitochondrial dysfunction in doxorubicin-induced cardiotoxicity in rats with breast cancer Sergio Granados-Principal a,b, Nuri El-azem a,b, Reinald Pamplona c, Cesar Ramirez-Tortosa d, Mario Pulido-Moran a,b, Laura Vera-Ramirez e, Jose L. Quiles b,f, Pedro Sanchez-Rovira g, Alba Naudı´ c, Manuel Portero-Otin c, Patricia Perez-Lopez b,f, MCarmen Ramirez-Tortosa a,b,* a

Department of Biochemistry and Molecular Biology II, Biomedical Research Center, Granada, Spain ‘‘Jose´ Mataix’’ Institute of Nutrition and Food Technology, Biomedical Research Center, Granada, Spain Department of Experimental Medicine, Faculty of Medicine, University of Lleida-IRB, Lleida, Spain d Pathological Anatomy Service, Jaen City Hospital, Jaen, Spain e GENYO, Granada, Spain f Department of Physiology, Biomedical Research Center, Granada, Spain g Oncology Service, Jaen City Hospital, Jaen, Spain b c

A R T I C L E I N F O

A B S T R A C T

Article history: Received 10 March 2014 Accepted 3 April 2014 Available online xxx

Oxidative stress is involved in several processes including cancer, aging and cardiovascular disease, and has been shown to potentiate the therapeutic effect of drugs such as doxorubicin. Doxorubicin causes significant cardiotoxicity characterized by marked increases in oxidative stress and mitochondrial dysfunction. Herein, we investigate whether doxorubicin-associated chronic cardiac toxicity can be ameliorated with the antioxidant hydroxytyrosol in rats with breast cancer. Thirty-six rats bearing breast tumors induced chemically were divided into 4 groups: control, hydroxytyrosol (0.5 mg/kg, 5 days/week), doxorubicin (1 mg/kg/week), and doxorubicin plus hydroxytyrosol. Cardiac disturbances at the cellular and mitochondrial level, mitochondrial electron transport chain complexes I–IV and apoptosis-inducing factor, and oxidative stress markers have been analyzed. Hydroxytyrosol improved the cardiac disturbances enhanced by doxorubicin by significantly reducing the percentage of altered mitochondria and oxidative damage. These results suggest that hydroxytyrosol improve the mitochondrial electron transport chain. This study demonstrates that hydroxytyrosol protect rat heart damage provoked by doxorubicin decreasing oxidative damage and mitochondrial alterations. ß 2014 Published by Elsevier Inc.

Keywords: Oxidative stress Apoptosis-inducing factor Electron transport chain Protein damage Chemo toxicity Hydroxytyrosol

1. Introduction Oxidative stress is a significant consequence in cardiac injury associated with doxorubicin (ADR) treatment [1]. Cardiac tissue is

Abbreviations: AASA, aminoadipic semialdehyde; ADR, doxorubicin; AIF, Apoptosisinducing factor; ALT, alanine aminotransferase; AST, aspartate aminotransferase; e e CEL, N -(carboxyethyl)-lysine; CK, creatinine kinase; CML, N -(carboxymethyl)lysine; DBI, double bond index; GSA, glutamic semialdehyde; HT, hydroxytyrosol; e i.v., intravenous; LDH, lactate d1ehydrogenase; MDAL, N -malondialdehyde-lysine; METC, mitochondrial electron transport chain; NQO1, NAD(P)H quinine oxidoreductase-1; ROS, reactive oxygen species. * Corresponding author. E-mail address: [email protected] (M. Ramirez-Tortosa).

extremely susceptible to free radical-induced damage because of high aerobic metabolism, lesser amount of antioxidant defenses compared to other tissues [2], and the post-mitotic features of myocytes [3]. Marked hypotension, tachycardia, cardiac dilation ventricular failure, and higher activities of glutamate-oxaloacetic transaminase, lactate dehydrogenase, and creatinine phosphokinase enzymes are characteristics of ADR-caused cardiomyopathy [4]. At the ultrastructural level, myofibril loss, cytoplasmatic vacuolization, increased number of lysosomes, and mitochondrial dysfunction have all been reported [1,5]. These cytotoxic side effects of ADR have been attributed to several events: selective accumulation in the mitochondrial lipid membrane, redox cycling, and subsequent generation of reactive oxygen and nitrogen species (ROS and RNS) [1,6]. It has been shown that ADR redox cycling

http://dx.doi.org/10.1016/j.bcp.2014.04.001 0006-2952/ß 2014 Published by Elsevier Inc.

Please cite this article in press as: Granados-Principal S, et al. Hydroxytyrosol ameliorates oxidative stress and mitochondrial dysfunction in doxorubicin-induced cardiotoxicity in rats with breast cancer. Biochem Pharmacol (2014), http://dx.doi.org/10.1016/ j.bcp.2014.04.001

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takes place in the mitochondrial electron transport chain (METC) [7], more specifically at complex-I, which, alongside complex-III, are more substantial ROS generators in heart [8]. High mitochondrial ROS production after ADR administration results in molecular oxidative damage that affects membrane-bound proteins and enzymes, lipids, the mitochondrial genome, as well as significant other biomolecules [7,9]. The secoiridoid oleuropein, a phenolic compound found in virgin olive oil, has demonstrated protective effects against ADR toxicity, mainly due to its high antioxidant capacity [1] as other biomolecules [10]. Phenolic alcohol hydroxytyrosol (HT), another bioactive molecule found in olive oil, has highly similar antioxidant properties [11]. HT has other features such as iron chelative, antiatherogenic, hypolipidemic, anti-inflammatory, anti-thrombotic, anti-microbial, and anti-tumor properties as well [11]. Moreover, HT is a good hypoglucemic [12] and anti-viral agent [13,14] that has demonstrated protective effects in rat cardiomyocytes in an ischemia–reperfusion model [15]. Our group has recently reported that HT (0.5 mg/kg) is able to suppress breast tumor growth in female Sprague-Dawley rats by modifying the expression of several tumor-related genes [16]. The present study addresses, for the first time, not only the potential protective role of HT against chronic cardiotoxicity generated by ADR in rats with breast cancer, but also the effects of sustained HT on oxidative stress at the cardiac level. To demonstrate this, authors measured cardiac abnormalities at the cellular and mitochondrial level through histopathology and electron microscopy. Total electron flow and the number of ROS-generating-sites into METC can potentially affect the rates of mitochondrial ROS production. Therefore, the content and activity of METC complexes I–IV were also examined. The steady-state levels of five markers of oxidative, glycoxidative, and lipoxidative damage to proteins were measured by gas chromatography/mass spectrometry. Since protein oxidation is secondarily influenced by the membrane’ sensitivity to lipid peroxidation [17], the full fatty acid composition was also measured.

collected and plasma isolated. Hearts were immediately removed and weighed. One half of the heart was snap frozen in liquid nitrogen, while the other half was fixed in 4% buffered formalin (Sigma, Saint Louis, MO, USA). A small fragment was sectioned for electron microscopy.

2. Materials and methods

2.5. Biochemical parameters in plasma

2.1. Animals and reagents

Plasma levels of creatinine kinase (CK), lactate dehydrogenase (LDH), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured with enzymatic kits (Spinreact, Girona, Spain).

Thirty-six female Sprague-Dawley rats (170 20 g), were purchased from Harlan Interfauna Ibe´rica S.L (Barcelona, Spain) at 7 weeks of age. All animals were housed four per cage in an environmentally controlled room at 22 2 8C with a 12:12 h (light/ dark) cycle. They were given free access to rodent chow and deionized water. All experiments were performed in accordance with the principles of the Helsinki Declaration, Spanish animal welfare legislation and Ethical Committee of the University of Granada (CEEA 264-2008) (Spain). Unless otherwise specified, all reagents were from Sigma (Saint Louis, MO, USA). Hydroxytyrosol was purchased from Cayman Chemical (Ann Arbor, MI, USA). Doxorubicin was purchased from Pharmacia-Upjohn Laboratories, Bridgewater, NJ, USA. 2.2. Experimental protocol Mammary tumors were induced as previously described [16]. Mammary tumor-bearing rats were randomized into four groups: (1) Control (n = 10): i.v. (intravenous) saline for 6 weeks; (2) HT (n = 10): HT (0.5 mg/kg, 5 days/week for 6 weeks); (3) ADR (n = 8): doxorubicin (i.v. 1 mg/kg/week for 6 weeks) for a total cumulative dose of 6 mg/kg; and (4) ADR + HT (n = 8): combo group treated with the same doses and timing than HT and ADR groups. One week after the last injection, animals were weighed, anaesthetized with intraperitoneally administered ketamine (Sigma, Saint Louis, MO, USA), and sacrificed by aortic bleeding. Whole blood was

2.3. Histopathological analysis Formalin-fixed hearts were paraffin-embedded, sectioned (3 mm thickness) and placed onto glass slides. Briefly, paraffinembedded tissue sections were deparaffinized with Neoclear (Panreac Quimica, Barcelona, Spain), rehydrated with graded alcohol, and stained with Harris’ hematoxylin and eosin (Dako, Glostrup, Denmark) in a Leica Autostainer (Wetzlar, Germany). Billingham’s grade [18] was established according to the following criteria: 0.0 = no lesions; 0.5 = abnormal heart but without typical hurt due to ADR-associated toxicity; 1.0 = 5%, 1.5 = 6–15%, 2.0 = 16–25%, 2.5 = 26–35%, 3.0 = >35% of the cells with proper lesions caused by ADR. 2.4. Electron microscopy analysis of cardiac mitochondria Cardiac muscle samples were prefixed in 1.5% formaldehyde in 1% cacodylate buffer, pH 7.4 for 2 h at 4 8C and fixed in 1% osmium tetroxide for 60 min at 0–4 8C (Sigma, Saint Louis, MO, USA). Samples were dehydrated in graded ethanol and embedded in Epon resin and incubated overnight at 65 8C. Ultrathin sections (70 nm) were cut with a diamond knife using an ultrakut S ultramicrotome and placed on 200-mesh copper grids. All sections were stained with uranyl acetate, counterstained with lead citrate, and viewed using a Carl Zeiss (Oberkochen, Germany) EM10C electron microscope at 40,000 magnification in the Scientific Instrument Service, University of Granada. Negatives were digitally transformed into positive images. Mitochondrial area and percentage of altered mitochondria were assessed. Software Image J [19] was used for the quantification of mitochondrial parameters.

2.6. Immunoblot analysis of METC complexes I–IV and AIF Mitochondrial complexes-I to -IV and AIF were estimated using Western-blot analysis as described previously [20]. Immunodetection was performed using specific antibodies (Molecular Probes, Invitrogen Ltd., UK): complex-I (39 kDa-NDUFA9 and 30 kDaNDUFS3 subunits, 1:1000), complex-II (70 kDa-Flavoprotein subunit, 1:500), complex-III (48 kDa-CoreII and 29 kDa-Rieske iron– sulfur-protein subunits, 1:1000), complex-IV (57 kDa-COXI subunit, 1:1000), and AIF polyclonal antibody (1:1000). Relative intensity was determined using porin (1:5000, Molecular Probes, Invitrogen Ltd., UK) as control. Appropriate peroxidase-coupled secondary antibodies and chemiluminescence HRP (horse radish peroxidase) substrate (Millipore, MA, USA) were used for primary antibody detection. Signal quantification and recording was performed with ChemiDoc BioRad equipment (Bio-Rad Laboratories, Inc., Barcelona, Spain). Protein concentration was determined by the Bradford method. 2.7. Mitochondrial complexes-I and -IV activities Complexes-I and -IV activities were determined using enzymeactivity-dipstick-assay kit from MitoSciences (MitoSciences Inc.


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Oregon, USA). Signal intensities were measured with a dipstick reader (MS-1000 Dipstick Reader, MitoSciences Inc. Oregon, USA). Results were analyzed with the MS-1000 measurement software version 1.0.1.2 (MitoSciences Inc., Oregon, USA). 2.8. Oxidation-derived protein damage markers by GC/MS Glutamic semialdehyde (GSA), aminoadipic semialdehyde e e (AASA), N -(carboxyethyl)-lysine (CEL), N -(carboxymethyl)-lye sine (CML) and N -malondialdehyde-lysine (MDAL) were determined as trifluoroacetic acid-methyl-ester derivatives in acidhydrolyzed-delipidated and -reduced protein samples by GC/MS, using an isotope-dilution method as previously described [21].

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2.10. Statistical analysis Results are shown as mean and SEM. Statistically significant differences (p < 0.05) were assessed by ANOVA and Bonferroni pot-hoc test. Histopathological variables and complexes-I and -IV activities were analyzed by Kruskall–Wallis and Mann–Whitney tests. Statistics were conducted with SPSS 15.0 software package for Windows (Chicago, IL, USA).

3. Results 3.1. Assessment of cardiac response by histopathologic, ultrastructural and biochemical analyses

2.9. Fatty acid analysis by GC/MS Fatty acid groups of heart lipids were analyzed as methyl-ester derivatives by GC/MS as previously described [22]. The following fatty acid indices were also calculated: saturated fatty acids (SFA); unsaturated fatty acids (UFA); monounsaturated fatty acids (MUFA); polyunsaturated fatty acids from n-3 and n-6 series (PUFAn-3 and PUFAn-6); average chain length (ACL), double bond index (DBI), and peroxidizability index (PI) (Fig. 1).

Myofibillar loss, dilation of sarcoplasmic reticulum, and swollen mitochondria were described as major morphological changes in human myocardium following ADR treatment. The severity of these changes can be assessed semiquantitatively according to the score by Billingham et al. [18]. In addition, according to Herman and Ferrans [23], the use of smaller-repeated-doses (an individual dose 20% lower than LD50) in animals adequately mimics clinicalchronic-myocardial-alterations in patients. Typical cumulative

Fig. 1. Representative traces of GC/MS analyses for specific protein oxidative damage markers. Analyses were carried out by selected ion-monitoring GC/MS (SIM-GC/MS). The amounts of product were expressed as mmoles of damage markers per mol of lysine. (Upper figure) Detection of the specific oxidation-derived protein carbonyl glutamic semialdehyde (GSA) by SIM-GC/MS. Quantification was performed by internal and external standardization using standard curves constructed from mixtures of deuterated and non-deuterated standards. The ions used were: lysine and [2H8]lysine, m/z 180 and 187, respectively; 5-hydroxy-2-aminovaleric acid and [2H5]5-hydroxy-2aminovaleric acid (stable derivative of GSA), m/z 280 and 285, respectively. (A) Selected ion chromatograms for rat heart proteins showing the m/z = 280 ion for the different experimental conditions. (B) Selected ion chromatograms for rat heart proteins showing the m/z = 180 ion for the different experimental conditions. (B1) Selected ion chromatograms for rat heart proteins showing the m/z = 180 and 187 ions (for protein lysine content quantification). (C) Selected ion chromatograms for rat heart proteins e showing the m/z = 280 and 285 ions (for protein GSA content quantification). (Lower figure) Detection of the specific glyco- and lipoxidation-derived protein marker N carboxymethyl-lysine (CML) by SIM-GC/MS. Quantification was performed by internal and external standardization using standard curves constructed from mixtures of deuterated and non-deuterated standards. The ions used were: lysine and [2H8]lysine, m/z 180 and 187, respectively; CML and [2H4]CML, m/z 392 and 396, respectively. (A) Selected ion chromatograms for rat heart proteins showing the m/z = 392 ion for the different experimental conditions. (B) Selected ion chromatograms for rat heart proteins showing the m/z = 180 ion for the different experimental conditions. (B1) Selected ion chromatograms for rat heart proteins showing the m/z = 180 and 187 ions (for protein lysine content quantification). (C) Selected ion chromatograms for rat heart proteins showing the m/z = 392 and 396 ions (for protein CML content quantification).


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Table 1 Effects of doxorubicin, hydroxytyrosol and their combination on histopathology, body and heart weights, and plasma parameters of cardiac injury in rat hearts. Group Billingham’s grade

Control 00

HT a

ADR a

00

0.75 0.163

ADR + HT b

0.938 0.147b

Body weight (g) Heart weight (g) Heart/body weight ratio (mg/g)

271.4 8.9 1.04 0.034 3.88 0.2

266.6 7.4 1.03 0.032 3.88 0.1

256.0 3.3 0.93 0.037 3.63 0.13

253.8 6.0 0.97 0.03 3.82 0.17

CK (U/L) LDH (U/L) ALT (U/L) AST (U/L)

103.0 17 376.0 66 21.9 8.5 33.6 8.9

64.0 19 323.0 68 10.7 3.5 18.9 2.8

164.0 45 304.0 33 11.3 3.9 14.9 2.7

151.0 77 327.0 91 15.0 3.8 17.7 2.9

Values are expressed as mean SEM. Letters, when different, represent statistically significant differences (p < 0.05). Control: control group; HT: group treated with hydroxytyrosol; ADR: group treated with doxorubicin; ADR + HT: group treated with hydroxytyrosol and doxorubicin. CK: creatinine kinase; LDH: lactate dehydrogenase; ALT: alanine aminotranferase; AST: aspartate aminotransferase.

cardiotoxic doses of ADR cited by these authors are 15 mg/kg for rats. In this work, cumulative dose of ADR was 6 mg/kg. In this work, ADR induced significant histopathological changes in rat hearts based in the Billingham’s grade (Table 1). However, in accordance with sublethal cumulative doses used in this study, results show that ADR did not cause extensive damage in hearts with clinical manifestations based on either of the following: (i) no differences in body weight, heart weight, and heart/body weight ratio among groups (Table 1); (ii) a slight vacuolization and inflammatory infiltrate in myocytes (Fig. 2); (iii) hearts with a histopathology between abnormal without typical injury due to ADR and an effect on less than 5% of the myocytes with proper lesions caused by ADR, and (iv) no statistical difference following treatment with ADR, HT and their combination on plasmatic markers of cardiac injury (CK, LDH, ALT and AST) (Table 1). In this scenario, there was not an improvement on heart histopathology after using HT in combination with ADR (Table 1).

ADR showed toxicity at an ultrastructural level, translating into a mitochondrial structure alteration [1]. Mitochondrial swelling and small vacuolization were found in ADR treated rats, while HT partially protected mitochondria when administered alone or in combination with ADR (Fig. 3A). With regards to mitochondrial area (Fig. 3B), the highest values were found in ADR group, and a significant decrease was seen in ADR + HT. In terms of altered mitochondria percentage (Fig. 3C), the ADR group had the highest significant percentage. Both HT and ADR + HT groups exerted less mitochondrial disturbance levels (Fig. 3C). 3.2. METC complexes and AIF ADR-associated cardiotoxicity involves an impairment of METC complexes and an inhibition in mitochondrial function [24–26]. METC complex content is presented in Fig. 4. Lesser levels of complex-I-NDUFA9 subunit were found in HT group, while the highest were found in ADR + HT group (Fig. 4A and G).

Fig. 2. Hematoxylin and eosin staining of rat cardiac tissue after chronic treatment with doxorubicin. Arrows indicate cardiomyocyte cytoplasmic vacuolization (A), inflammatory infiltrate (B), and atrial neuron vacuolization. Magnification: 40.


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Fig. 3. Effects of doxorubicin, hydroxytyrosol, and their combination on mitochondria. Mitochondrial ultrastructure in heart (A), mitochondrial area (B), altered mitochondria percentage (C). Magnification: 10,000. Values are expressed as mean SEM. Letters, when different, represent statistically significant differences (p < 0.05). Control: control group; HT: group treated with hydroxytyrosol; ADR: group treated with doxorubicin; ADR + HT: group treated with hydroxytyrosol and doxorubicin.

ADR + HT group had a significant increase of complex-I-NDUFS3 subunit (Fig. 4B and G). Higher amounts of complex-II were found in ADR + HT and ADR, whereas control and HT groups reported the lowest amounts of flavoprotein (Fig. 4C and G). When complex-III (CORE II subunit) was tested, the highest

quantity was seen in the control group, whereas HT exhibited the lowest, closely followed by ADR and ADR + HT (Fig. 4D and G). Similar results were found upon examination for complex-IIIRieske iron–sulfur protein (Fig. 4E and G). Complex-IV was found in lesser quantities in all groups compared to control groups

Fig. 4. Effects of doxorubicin, hydroxytyrosol, and their combination on protein levels. Complex-I-NDUFA9 (39 kDa) (A) and -NDUFS3 (30 kDa) subunits (B), complex-II70 kDa (Flavoprotein) subunit (C), complex-III-COREII (48 kDa) (D) and -Rieske iron–sulfur protein (29 kDa) subunits (E), and complex-IV-COXI (57 kDa) subunit (F) in rat heart tissue. Values are expressed as means SEM (n = 5). Bars with different letters significantly differ among the groups (p < 0.05). Control: control group; HT: group treated with hydroxytyrosol; ADR: group treated with doxorubicin; ADR + HT: group treated with hydroxytyrosol and doxorubicin. (G) Representative Western blots images of the heart lysates from untreated animals (control group) and effects of doxorubicin (ADR), hydroxytyrosol (HT), and their combination (ADR + HT) on mitochondrial respiratory chain complexes levels (from CI to CIV).


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Fig. 5. Effects of doxorubicin, hydroxytyrosol and their combination on the protein levels of AIF in heart. Values are expressed as means SEM (n = 5). Bars with different letters significantly differ among groups (p < 0.05). Control: control group; HT: group treated with hydroxytyrosol; ADR: group treated with doxorubicin; ADR + HT: group treated with hydroxytyrosol and doxorubicin. Representative Western blots images of the heart lysates from untreated animals (control group) and effects of doxorubicin (ADR), hydroxytyrosol (HT), and their combination (ADR + HT) on AIF protein levels.

(Fig. 4F and G). Finally, AIF significantly decreased in HT compared to other groups (Fig. 5). HT, ADR, and ADR + HT had significantly lower complex-I activity than control (Fig. 6A). Trends demonstrate that HT group had similar complex-IV activity than controls, while the lowest activities were seen in ADR and ADR + HT treated groups (Fig. 6B).

Fig. 6. Activities of mitochondrial complexes-I (A) and -IV (B) in rats treated with hydroxytyrosol, doxorubicin, and their combination compared to controls. Values are expressed as means SEM (n = 5). Bars with different letters significantly differ among groups (p < 0.05). Control: control group; HT: group treated with hydroxytyrosol; ADR: group treated with doxorubicin; ADR + HT: group treated with hydroxytyrosol and doxorubicin.

3.3. Oxidative molecular damage

the oxidative status of hearts subjected to ADR treatment (ADR + HT group) by diminishing GSA, AASA, CML, and MDAL. Interestingly, GSA and CML were lower in ADR + HT than controls. When alone, HT caused a marked reduction of oxidative status of AASA, CEL, CML, and MDAL, even below the basal oxidation status occurring in the control group. Finally, this work did not reveal any significant changes in fatty acid composition, double bond, and peroxidizability indices (Table 3).

Amino acid oxidation in proteins was examined, specifically those carbonyl products that suffer metal-catalyzed oxidation [27]. GSA arises from the metal-catalyzed oxidation of proline and arginine, while AASA results from lysine oxidation. Moreover, it is known that third-party molecules are also involved in these chemical pathways that link, on one hand, the increased freeradical efflux and, on the other hand, structural modifications in proteins. These pathways may give rise to increased 2,4-dinitrophenylhydrazine-reactive carbonyls in proteins [28]. In this study, we investigated the concentration of CEL and CML adducts. Those protein adducts were first described as advanced-glycation-endproducts (AGE), later named glycoxidation products and now recognized as mixed AGEs-advanced lipoxidation products [21]. Finally, lipid-peroxidation-derived protein damage was also demonstrated by MDAL. The cardiac content of GSA, AASA, CEL, CML, and MDAL are shown in Table 2. The highest values of those five markers were found in ADR group at significant levels. HT was able to decrease

4. Discussion It has been postulated that adverse cardiac effects resulting from ADR administration is not only dose- but also time-dependent (i.e. during and following treatment) [29,30]. Evident toxicity associated with ADR based on plasma markers (CK, LDH, ALT and AST), body and heart weight changes, and heart/body weight ratio did not find in the experimental animals. These results are in accordance with studies by Injac et al. [31] in rats with colorectal cancer models. In this study, ADR induced significant histopathological changes in rat hearts based in the Billingham’s grade as have

Table 2 Effects of hydroxytyrosol, doxorubicin and their combination on oxidation-derived protein damage markers in rat hearts.

GSA (mmol/mol Lys) AASA (mmol/mol Lys) CEL (mmol/mol Lys) CML (mmol/mol Lys) MDAL (mmol/mol Lys)

Control

HT

ADR

ADR + HT

2424.9 131.1ab 302.9 10.8a 382.8 2.9a 949.4 10.4a 305.1 13.0a

2697.6 141.8ab 260.5 7.8a 354.8 53.0a 747.5 52.1a 269.4 10.6a

3818.6 694.8b 444.5 53.0b 521.3 39.8b 1279.1 109.9b 505.6 39.6b

2180.2 178.8a 325.0 6.5a 424.8 29.1ab 911.8 3.7a 346.4 17.7a

Values are expressed as mean SEM. Letters, when different, represent statistically significant differences (p < 0.05). GSA, glutamic semialdehyde; AASA, aminoadipic semialdehyde; CEL, carboxyethyl-lysine; CML, carboxymethyl-lysine; MDAL, malondialdehyde-lysine. Control: control group; HT: group treated with hydroxytyrosol; ADR: group treated with doxorubicin; ADR + HT: group treated with hydroxytyrosol and doxorubicin.


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BCP-11949; No. of Pages 9 S. Granados-Principal et al. / Biochemical Pharmacology xxx (2014) xxx–xxx Table 3 Effects of hydroxytyrosol, doxorubicin, and their combination on fatty acid profile in rat hearts. Control

HT

ADR

ADR + HT

14:0 16:0

1.18 0.12 21.38 1.18

1.10 0.07 22.08 0.70

1.22 0.13 23.11 1.22

1.12 0.19 23.20 1.55

16:1n-7 18:0

0.86 0.0.07 27.61 1.08

0.74 0.04 29.97 0.36

0.96 0.10 27.25 0.80

0.97 0.0.16 28.96 0.10

18:1n-9

14.34 0.45

14.39 0.60

14.76 0.75

12.79 0.84

18:2n-6 18:3n-3 18:4n-6 20:0 20:3n-6 20:4n-6 20:5n-3 22:0 22:4n-6 22:5n-6 22:5n-3 24:0 22:6n-3 24:5n-3 24:6n-3 ACL SFA UFA MUFA PUFA PUFAn-3 PUFAn-6 DBI PI

8.74 1.27 0.36 0.06 2.68 0.11 1.15 0.07 0.26 0.03 8.18 1.21 0.82 0.10 1.75 0.13 0.71 0.12 0.99 0.02 0.99 0.08 0.31 0.02 3.94 0.0.50 1.25 0.15 2.43 0.22 18.29 0.07 53.39 2.45 46.60 2.45 15.20 0.48 31.40 2.76 9.80 0.57 21.59 2.31 135.40 9.21 132.14 9.44

5.89 0.47 0.44 0.07 2.88 0.22 1.14 0.07 0.22 0.01 6.68 1.09 0.97 0.06 2.23 0.18 0.67 0.08 1.29 0.07 1.09 0.10 0.34 0.05 3.24 0.51 1.59 0.28 3.01 0.31 18.32 0.04 56.86 1.09 43.14 1.09 15.14 0.61 28.00 1.36 10.36 0.43 17.64 1.45 127.98 5.62 128.31 5.46

7.21 1.46 0.45 0.08 2.28 0.17 0.97 0.08 0.20 0.02 7.64 1.12 0.73 0.04 1.64 0.13 0.76 0.11 1.35 0.15 0.98 0.11 0.39 0.06 4.46 0.66 1.23 0.13 2.39 0.26 18.27 0.09 54.58 2.03 45.42 2.03 15.72 0.76 29.69 2.55 10.25 0.91 19.45 2.03 133.72 9.75 132.20 11.71

6.04 0.69 0.34 0.05 2.71 0.26 1.02 0.20 0.23 0.01 7.75 0.19 0.62 0.10 1.71 0.26 0.67 0.06 1.99 0.28 0.98 0.07 0.47 0.08 4.14 0.75 1.54 0.30 2.71 0.54 18.33 0.15 56.48 2.59 43.51 2.60 13.76 0.99 29.75 3.517 10.35 1.50 19.41 2.11 135.85 15.65 137.78 18.85

Values are expressed as mean SEM.

been shown in Table 1. However, in accordance with sublethal cumulative doses used in this study, results show that ADR did not cause extensive damage in hearts with clinical manifestations. As has been shown in Fig. 2 a slight vacuolization and inflammatory infiltrate in myocytes from ADR group were found and hearts with a histopathology between abnormal without typical injury due to ADR and an effect on less than 5% of the myocytes with proper lesions were caused by ADR. Similar results have been previously reported in studies of chronic cardiotoxicity attributed to ADR [30,32]. The low dose and the administration time of the ADR used in this study showed important changes in ultrastructural results but not in histopathological determinations. At ultrastructural level, highly altered mitochondria were observed, as well as swelling and presence of small vacuoles associated with ADR administration. These results are in agreement with previous works at chronic low- [30,31] and high-doses of ADR [32-34]. This is the first study addressing the protective role of HT on mitochondrial disturbances associated with ADR-induced cardiotoxicity in breast tumor-bearing rats. There is growing evidence supporting the protective role of HT on mitochondria against harmful acrolein [35,36] and ischemia-reperfusion damage [15]. Recently, Hao et al. [37] reported that HT stimulates mitochondrial biogenesis and promotes mitochondrial function in 3T3-L1 adipocytes by increasing: (1) PPARGC1a (peroxisome proliferator-activated receptor coactivator 1 alpha) activation and protein expression, (2) oxygen consumption, (3) mitochondrial DNA quantity, (4) number of mitochondria, and (5) complexes-I, II, -III, and -IV protein expression and activity levels. A dual effect of HT was observed due to its ability to partially restore the mitochondrial status (ADR + HT group); concomitantly, a slightly damaged in mitochondria was found in HT group. This can be partially explained due to mechanical injury during tissue handling, or even a plausible side-effect arising from HT redox

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cycling. In this sense, Zhu et al. [36] reported that HT protects ARPE-19 human retinal pigment epithelial cells from acroleininduced oxidative damage through two mechanisms: (1) induction of phase II detoxifying enzymes like g-glutamyl cysteine ligase, NAD(P)H quinine oxido-reductase-1 (NQO1) and heme oxygenase1, and (2) stimulation of mitochondrial biogenesis. ADR did not affect the concentration of mitochondrial complexI although it affects the activity of this complex. It decreased complexes-III and -IV and increased complex-II, result that agree with previous studies [30,38,39]. Complex-III-Rieske subunit is a nuclear-encoded protein with an iron–sulfur [2Fe–2S] redox center that mainly participates in the Q cycle by transferring an electron from ubiquinol to cytochrome c1. Complex-III-CORE II subunit is also a nuclear-encoded protein important for mitochondrial protein importing, processing and integrity of complex-III. Genetic deletion of this subunit directly affects the complex-III assembling [40]. There is a putative impairment of the integrity and assembling of complex-III by ADR, mainly associated with low CORE II and unmodified Rieske subunits levels. Lesser activities of both complex-I and -IV could enhance ROS production and subsequently limit ATP production, as previously seen in vitro [30,41]. This is possibly causal, at least in part, to enhanced ROS production by ADR in our study based on high steady-state levels of oxidation-derived protein damage markers (CEL). Precursors of CEL formation are derived from the glycolytic pathway. Thus, the increased levels of CEL described in the present work in the ADR group can be ascribed to both a high oxidative stress status in ADRtreated animals, as well as an increased glycolytic flux in this group in order to compensate the low cellular energy state of the ADRtreated heart [4]. HT did not modify the amounts of complex-I, -III (Rieske subunit) and -IV proteins altered by ADR. Rather, it increased both complex-II and complex-III (CORE II subunit) protein concentrations in comparison with the ADR group. These results indicate that HT is able to improve the integrity of complex-III by increasing the CORE II subunit without affecting the Rieske subunit. Since HT was not able to restore complexes-I and -IV activities, it would be expected to maintain higher ROS production. Nonetheless, these findings show that HT dramatically reversed oxidative markers. Therefore, there must be mechanisms, as yet unclarified, by which HT ameliorates oxidative stress and ADR-associated mitochondrial impairment. More studies are needed in order to determine if HT protects electron transfer and decreases ROS generation, without comprising ATP production. In sharp contrast with Hao et al. [37], the present study shows that HT significantly reduced the concentration of complexes-III and -IV. No differences were seen for complexes-I and -II in comparison with controls. With regards to complex-I and -IV activities, HT promotes a marked low activity of complex-I without affecting the complex-IV activity. Taken together, these results show that METC is not affected by HT. Therefore, a correct flow of electrons may be possible, and lesser ROS generation would be expected. HT did not increase the ROS-derived oxidative damage (GSA, AASA, CEL, CML and MDAL). In its role as a powerful antioxidant, HT was able decrease the oxidative status, even below basal oxidation for certain markers. It can be attributed to its antioxidant effect because the lipids oxidations markers (the degree of membrane unsaturation, fatty acid composition, double bond, and peroxidizability indexes) did not change. AIF is a ubiquitously expressed flavoprotein synthesized as a cytoplasmic 67 kDa precursor giving rise to a mature 62 kDa protein at the mitochondrial level. In mitochondria, AIF is involved in oxidoreduction and is considered a potential ROS scavenger [42,43]. This mitochondrial protein has both life and death functions in cells and it is known to be also required for


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8

mitochondrial oxidative phosphorylation [13]. AIF-deficient cells exhibit a reduced content of complex-I, suggesting that a certain amount of AIF is required for oxidative phosphorylation [39], all the while pointing toward a role for AIF in the biogenesis of this complex. In this study AIF was only decreased in the HT group, without change of complex-I amounts. This indicates that substantial decreases in AIF are not large enough to become limiting for complex-I biogenesis, and therefore, respiratory chain function is not comprised. Apoptotic or antiapoptotic activity of HT is dependent on the cell type. ADR clearly raised levels of oxidation-derived biomarkers, disrupted mitochondria membrane and inhibited the METC. This could surely lead to an energy imbalance in the cardiomyocytes. This scenario changes when ADR is administrated in combination with HT. This antioxidant protects against ROS generation by ADR, as shown by data from ROS-derived oxidative damage (GSA, ASAA, CEL, CML and MDAL) and data from mitochondria ultrastructure. In summary, the present study reveals, for the first time, that HT can attenuate ADR-associated cardiac toxicity by reducing ROS production enhanced by ADR. Moreover, HT ameliorates mitochondrial dysfunction by partially restoring the respiratory chain altered by ADR chronic treatment in rat models of breast cancer through an as yet undetermined mechanism. Herein, authors hypothesized that hydroxytyrosol scavenges free radicals in mitochondria, and its redox cycling might be responsible, at least in part, for the suitable electron transport from complex-I to the rest of METC proteins. Conflict of interest statement The authors have declared no conflict of interest. Acknowledgements We acknowledge grants from Excelentı´sima Diputacioń de Jaeń, CEAS Foundation 30.C0.244500 and Junta de Andalucıá PI-0210/ 2007. We thank the Spanish Ministry of Science and Innovation (AP2005-144) and the University of Granada for the personal support of Dr. S. Granados-Principal. Work carried out at the Department of Experimental Medicine was supported in part by R + D grants from the Spanish Ministry of Science and Innovation (BFU2009-11879/BFI), the Spanish Ministry of Health [RD06/0013/ 0012 and PI081843], the Autonomous Government of Catalonia [2009SGR735] and COST B35 Action of the European Union. We thank Dr. Elvin Blanco for help in editing of the final manuscript.

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