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

Accepted Manuscript

Title: Hydroxytyrosol ameliorates oxidative stress andmitochondrial dysfunction in doxorubicin-inducedcardiotoxicity in rats with breast cancer

Author: Sergio Granados-Principal Nuri El-azem ReinaldPamplona Cesar Ramirez-Tortosa Mario Pulido-Moran LauraVera-Ramirez Jose L. Quiles Pedro Sanchez-Rovira AlbaNaudı Manuel Portero-Otin Patricia Perez-Lopez MCarmenRamirez-Tortosa

PII: S0006-2952(14)00225-1DOI: http://dx.doi.org/doi:10.1016/j.bcp.2014.04.001Reference: BCP 11949

To appear in: BCP

Received date: 10-3-2014Revised date: 3-4-2014Accepted date: 3-4-2014

Please cite this article as: Granados-Principal S, El-azem N, Ramirez-Tortosa RP,</sup>Cesar, Pulido-Moran M, Vera-Ramirez L, Quiles JL, Sanchez-Rovira P,Naudi A, Portero-Otin M, Perez-Lopez P, Ramirez-Tortosa MC, Hydroxytyrosolameliorates oxidative stress and mitochondrial dysfunction in doxorubicin-inducedcardiotoxicity in rats with breast cancer, Biochemical Pharmacology (2014),http://dx.doi.org/10.1016/j.bcp.2014.04.001

This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

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Hydroxytyrosol ameliorates oxidative stress and mitochondrial dysfunction in 1

doxorubicin-induced cardiotoxicity in rats with breast cancer 2

Sergio Granados-Principala,b

, Nuri El-azema,b

, Reinald Pamplonac Cesar Ramirez-3

Tortosad, Mario Pulido-Moran

a,b , Laura Vera-Ramirez

e, Jose L. Quiles

b,f, Pedro 4

Sanchez-Rovirag, Alba Naudí

c, Manuel Portero-Otin

c, Patricia Perez-Lopez

b,f, 5

MCarmen Ramirez-Tortosaa,b*

6

aDepartment of Biochemistry and Molecular Biology II, Biomedical Research Center, 7

Granada, Spain. b“José Mataix” Institute of Nutrition and Food Technology, Biomedical 8

Research Center, Granada, Spain. cDepartment of Experimental Medicine, Faculty of 9

Medicine, University of Lleida-IRB, Lleida, Spain. d

Pathological Anatomy Service, 10

Jaen City Hospital, Jaen, Spain. eGENYO Granada, Spain.

fDepartment of Physiology, 11

Biomedical Research Center, Granada, Spain. gOncology Service, Jaen City Hospital, 12

Jaen, Spain.

13

Short title: Hydroxytyrosol in doxorubicin-induced damage. 14

15

*Corresponding author: Dr. MCarmen Ramirez-Tortosa. Instituto de Nutrición y 16

Tecnología de Alimentos “José Mataix Verdú”. Universidad de Granada. Centro de 17

Investigación Biomédica. Parque Tecnológico de Ciencias de la Salud. Avenida del 18

Conocimiento s/n, 18100-Granada (Granada, Spain). Phone: +34 958241000 Ext. 19

20315 Fax: +34 958819132. E-mail: [email protected] 20

21

22

1Abbreviations 23

1AASA, aminoadipic semialdehyde; ADR, doxorubicin; AIF, Apoptosis-inducing

factor; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CEL, Nε-

*Manuscript

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Abstract 1

Oxidative stress is involved in several processes including cancer, aging and 2

cardiovascular disease, and has been shown to potentiate the therapeutic effect of drugs 3

such as doxorubicin. Doxorubicin causes significant cardiotoxicity characterized by 4

marked increases in oxidative stress and mitochondrial dysfunction. Herein, we 5

investigate whether doxorubicin-associated chronic cardiac toxicity can be ameliorated 6

with the antioxidant hydroxytyrosol in rats with breast cancer. Thirty-six rats bearing 7

breast tumors induced chemically were divided into 4 groups: Control, Hydroxytyrosol 8

(0.5mg/kg, 5 days/week), doxorubicin (1mg/kg/week), and doxorubicin plus 9

hydroxytyrosol. Cardiac disturbances at the cellular and mitochondrial level, 10

mitochondrial electron transport chain complexes I-IV and apoptosis-inducing factor, 11

and oxidative stress markers have been analyzed. Hydroxytyrosol improved the cardiac 12

disturbances enhanced by doxorubicin by significantly reducing the percentage of 13

altered mitochondria and oxidative damage. These results suggest that hydroxytyrosol 14

improve the mitochondrial electron transport chain. This study demonstrates that 15

hydroxytyrosol protect rat heart damage provoked by doxorubicin decreasing oxidative 16

damage and mitochondrial alterations. 17

Key words: oxidative stress, apoptosis-inducing factor, electron transport chain, protein 18

damage, chemo toxicity, hydroxytyrosol. 19

20

(carboxyethyl)-lysine; CK, creatinine kinase; CML, Nε-(carboxymethyl)-lysine; DBI,

double bond index; GSA, Glutamic semialdehyde; HT, hydroxytyrosol; i.v.:

intravenous; LDH, lactate d1ehydrogenase; MDAL, N

ε-malondialdehyde-lysine;

METC, mitochondrial electron transport chain; NQO1, NAD(P)H quinine oxido-

reductase-1; ROS, reactive oxygen species.

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1. Introduction 1

Oxidative stress is a significant consequence in cardiac injury associated with 2

doxorubicin (ADR) treatment [1]. Cardiac tissue is extremely susceptible to free radical-3

induced damage because of high aerobic metabolism, lesser amount of antioxidant 4

defenses compared to other tissues [2], and the post-mitotic features of myocytes [3]. 5

Marked hypotension, tachycardia, cardiac dilation ventricular failure, and higher 6

activities of glutamate-oxalacetic transaminase, lactate dehydrogenase, and creatinine 7

phosphokinase enzymes are characteristics of ADR-caused cardiomyopathy [4]. At the 8

ultrastructural level, myofibril loss, cytoplasmatic vacuolization, increased number of 9

lysosomes, and mitochondrial dysfunction have all been reported [1,5]. These cytotoxic 10

side effects of ADR have been attributed to several events: selective accumulation in the 11

mitochondrial lipid membrane, redox cycling, and subsequent generation of reactive 12

oxygen and nitrogen species (ROS and RNS) [1,6]. It has been shown that ADR redox 13

cycling takes place in the mitochondrial electron transport chain (METC) [7], more 14

specifically at complex-I, which, alongside complex-III, are more substantial ROS 15

generators in heart [8]. High mitochondrial ROS production after ADR administration 16

results in molecular oxidative damage that affects membrane-bound proteins and 17

enzymes, lipids, the mitochondrial genome, as well as significant other biomolecules 18

[7,9]. 19

The secoiridoid oleuropein, a phenolic compound found in virgin olive oil, has 20

demonstrated protective effects against ADR toxicity, mainly due to its high antioxidant 21

capacity [1] as other biomolecules [10]. Phenolic alcohol hydroxytyrosol (HT), another 22

bioactive molecule found in olive oil, has highly similar antioxidant properties [11]. HT 23

has other features such as iron chelative, anti-atherogenic, hypolipidemic, anti-24

inflammatory, anti-thrombotic, anti-microbial, and anti-tumor properties as well [11]. 25

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Moreover, HT is a good hypoglucemic [12] and anti-viral agent [13,14] that has 1

demonstrated protective effects in rat cardiomyocytes in an ischemia-reperfusion model 2

[15]. Our group has recently reported that HT (0.5 mg/kg) is able to suppress breast 3

tumor growth in female Sprague-Dawley rats by modifying the expression of several 4

tumor-related genes [16]. 5

The present study addresses, for the first time, not only the potential protective 6

role of HT against chronic cardiotoxicity generated by ADR in rats with breast cancer, 7

but also the effects of sustained HT on oxidative stress at the cardiac level. To 8

demonstrate this, authors measured cardiac abnormalities at the cellular and 9

mitochondrial level through histopathology and electron microscopy. Total electron 10

flow and the number of ROS-generating-sites into METC can potentially affect the rates 11

of mitochondrial ROS production. Therefore, the content and activity of METC 12

complexes I-IV were also examined. The steady-state levels of five markers of 13

oxidative, glycoxidative, and lipoxidative damage to proteins were measured by gas 14

chromatography/mass spectrometry. Since protein oxidation is secondarily influenced 15

by the membrane´s sensitivity to lipid peroxidation [17], the full fatty acid composition 16

was also measured. 17

18

2. Materials and methods 19

2.1.Animals and reagents 20

Thirty-six female Sprague-Dawley rats (170 ± 20 g), were purchased from 21

Harlan Interfauna Ibérica S.L (Barcelona, Spain) at 7 weeks of age. All animals were 22

housed four per cage in an environmentally-controlled room at 22 ± 2ºC with a 12:12 h 23

(light/dark) cycle. They were given free access to rodent chow and deionized water. All 24

experiments were performed in accordance with the principles of the Helsinki 25

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Declaration, Spanish animal welfare legislation and Ethical Committee of the 1

University of Granada (CEEA 264-2008) (Spain). Unless otherwise specified, all 2

reagents were from Sigma (Saint Louis, MO, USA). Hydroxytyrosol was purchased 3

from Cayman Chemical (Ann Arbor, MI, USA). Doxorubicin was purchased from 4

Pharmacia-Upjohn Laboratories, Bridgewater, NJ, USA. 5

2.2.Experimental protocol 6

Mammary tumors were induced as previously described [16]. Mammary tumor-7

bearing rats were randomized into four groups: 1) Control (n=10): i.v. (intravenous) 8

saline for 6 weeks; 2) HT (n=10): HT (0.5mg/kg, 5 days/week for 6 weeks); 3) ADR 9

(n=8): Doxorubicin (i.v. 1mg/kg/week for 6 weeks) for a total cumulative dose of 10

6mg/kg; and 4) ADR+HT (n=8): combo group treated with the same doses and timing 11

than HT and ADR groups. One week after the last injection, animals were weighed, 12

anaesthetized with intraperitoneally administered ketamine (Sigma, Saint Louis, MO, 13

USA), and sacrificed by aortic bleeding. Whole blood was collected and plasma 14

isolated. Hearts were immediately removed and weighed. One half of the heart was snap 15

frozen in liquid nitrogen, while the other half was fixed in 4% buffered formalin 16

(Sigma, Saint Louis, MO, USA). A small fragment was sectioned for electron 17

microscopy. 18

2.3.Histopathological analysis 19

Formalin-fixed hearts were paraffin-embedded, sectioned (3 µm thickness) and 20

placed onto glass slides. Briefly, paraffin-embedded tissue sections were deparaffinized 21

with Neoclear (Panreac Quimica, Barcelona, Spain), rehydrated with graded alcohol, 22

and stained with Harris’ hematoxylin and eosin (Dako, Glostrup, Denmark) in a Leica 23

Autostainer (Wetzlar, Germany). Billingham’s grade [18] was established according to 24

the following criteria: 0.0= no lesions; 0.5= abnormal heart but without typical hurt due 25

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to ADR-associated toxicity; 1.0= ≤5%, 1.5= 6-15%, 2.0= 16-25%, 2.5= 26-35%, 3.0= 1

>35% of the cells with proper lesions caused by ADR. 2

2.4.Electron microscopy analysis of cardiac mitochondria 3

Cardiac muscle samples were prefixed in 1.5% formaldehyde in 1% cacodylate 4

buffer, pH 7.4 for 2 h at 4 ºC and fixed in 1% osmium tetroxide for 60 min at 0-4ºC 5

(Sigma, Saint Louis, MO, USA). Samples were dehydrated in graded ethanol and 6

embedded in Epon resin and incubated overnight at 65 ºC. Ultrathin sections (70 nm) 7

were cut with a diamond knife using an ultrakut S ultramicrotome and placed on 200-8

mesh copper grids. All sections were stained with uranyl acetate, counterstained with 9

lead citrate, and viewed using a Carl Zeiss (Oberkochen, Germany) EM10C electron 10

microscope at 40,000X magnification in the Scientific Instrument Service, University of 11

Granada. Negatives were digitally transformed into positive images. Mitochondrial area 12

and percentage of altered mitochondria were assessed. Software Image J [19] was used 13

for the quantification of mitochondrial parameters. 14

2.5.Biochemical parameters in plasma 15

Plasma levels of creatinine kinase (CK), lactate dehydrogenase (LDH), alanine 16

aminotransferase (ALT), and aspartate aminotransferase (AST) were measured with 17

enzymatic kits (Spinreact, Girona, Spain). 18

2.6.Immunoblot analysis of METC complexes I-IV and AIF 19

Mitochondrial complexes-I to -IV and AIF were estimated using Western-blot 20

analysis as described previously [20]. Immunodetection was performed using specific 21

antibodies (Molecular Probes, Invitrogen Ltd, UK): complex-I (39 kDa-NDUFA9 and 22

30 kDa-NDUFS3 subunits, 1:1000), complex-II (70 kDa-Flavoprotein subunit, 1:500), 23

complex-III (48 kDa-CoreII and 29 kDa-Rieske iron-sulfur-protein subunits, 1:1000), 24

complex-IV (57 kDa-COXI subunit, 1:1000), and AIF polyclonal antibody (1:1000). 25

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Relative intensity was determined using porin (1:5000, Molecular Probes, Invitrogen 1

Ltd, UK) as control. Appropriate peroxidase-coupled secondary antibodies and 2

chemiluminescence HRP (horse radish peroxidase) substrate (Millipore, MA, USA) 3

were used for primary antibody detection. Signal quantification and recording was 4

performed with ChemiDoc BioRad equipment (Bio-Rad Laboratories, Inc., Barcelona, 5

Spain). Protein concentration was determined by the Bradford method. 6

2.7.Mitochondrial complexes -I and -IV activities 7

Complexes -I and -IV activities were determined using enzyme-activity-8

dipstick-assay kit from MitoSciences (MitoSciences Inc. Oregon, USA). Signal 9

intensities were measured with a dipstick reader (MS-1000 Dipstick Reader, 10

MitoSciences Inc. Oregon, USA). Results were analyzed with the MS-1000 11

measurement software version 1.0.1.2 (MitoSciences Inc. Oregon, USA). 12

2.8.Oxidation-derived protein damage markers by GC/MS 13

Glutamic semialdehyde (GSA), aminoadipic semialdehyde (AASA), Nε-14

(carboxyethyl)-lysine (CEL), Nε-(carboxymethyl)-lysine (CML) and N

ε-15

malondialdehyde-lysine (MDAL) were determined as trifluoroacetic acid-methyl-ester 16

derivatives in acid-hydrolyzed-delipidated and -reduced protein samples by GC/MS, 17

using an isotope-dilution method as previously described [21]. 18

2.9.Fatty acid analysis by GC/MS 19

Fatty acid groups of heart lipids were analyzed as methyl-ester derivatives by 20

GC/MS as previously described [22]. The following fatty acid indices were also 21

calculated: saturated fatty acids (SFA); unsaturated fatty acids (UFA); monounsaturated 22

fatty acids (MUFA); polyunsaturated fatty acids from n-3 and n-6 series (PUFAn-3 and 23

PUFAn-6); average chain length (ACL), double bond index (DBI), and peroxidizability 24

index (PI) (Figure 1) . 25

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2.10. Statistical analysis 1

Results are shown as mean and SEM. Statistically significant differences 2

(p<0.05) were assessed by ANOVA and Bonferroni pot-hoc test. Histopathological 3

variables and complexes -I and -IV activities were analyzed by Kruskall-Wallis and 4

Mann-Whitney tests. Statistics were conducted with SPSS 15.0 software package for 5

Windows (Chicago, IL, USA). 6

7

3. Results 8

3.1.Assessment of cardiac response by histopathologic, ultrastructural and 9

biochemical analyses 10

Myofibillar loss, dilation of sarcoplasmic reticulum, and swollen mitochondria 11

were described as major morphological changes in human myocardium following ADR 12

treatment. The severity of these changes can be assessed semiquantitatively according to 13

the score by Billingham et al. [18]. In addition, according to Herman and Ferrans [23], 14

the use of smaller-repeated-doses (an individual dose 20% lower than LD50) in animals 15

adequately mimics clinical-chronic-myocardial-alterations in patients. Typical 16

cumulative cardiotoxic doses of ADR cited by these authors are 15mg/kg for rats. In 17

this work, cumulative dose of ADR was 6mg/kg. 18

In this work, ADR induced significant histopathological changes in rat hearts 19

based in the Billingham’s grade (Table 1). However, in accordance with sublethal 20

cumulative doses used in this study, results show that ADR did not cause extensive 21

damage in hearts with clinical manifestations based on either of the following: i) no 22

differences in body weight, heart weight, and heart/body weight ratio among groups 23

(Table 1); ii) a slight vacuolization and inflammatory infiltrate in myocytes (Fig. 2); iii) 24

hearts with a histopathology between abnormal without typical injury due to ADR and 25

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an effect on less than 5% of the myocytes with proper lesions caused by ADR, and iv) 1

no statistical difference following treatment with ADR, HT and their combination on 2

plasmatic markers of cardiac injury (CK, LDH, ALT and AST) (Table 1). In this 3

scenario, there was not an improvement on heart histopathology after using HT in 4

combination with ADR (Table 1). 5

ADR showed toxicity at an ultrastructural level, translating into a mitochondrial 6

structure alteration [1]. Mitochondrial swelling and small vacuolization were found in 7

ADR treated rats, while HT partially protected mitochondria when administered alone 8

or in combination with ADR (Fig. 3A). With regards to mitochondrial area (Fig. 3B), 9

the highest values were found in ADR group, and a significant decrease was seen in 10

ADR+HT. In terms of altered mitochondria percentage (Fig. 3C), the ADR group had 11

the highest significant percentage. Both HT and ADR+HT groups exerted less 12

mitochondrial disturbance levels (Fig. 3C). 13

3.2.METC complexes and AIF 14

ADR-associated cardiotoxicity involves an impairment of METC complexes and 15

an inhibition in mitochondrial function [24-26]. METC complex content is presented in 16

Fig. 4. Lesser levels of complex-I-NDUFA9 subunit were found in HT group, while the 17

highest were found in ADR+HT group (Fig. 4A, 4G). ADR+HT group had a significant 18

increase of complex-I-NDUFS3 subunit (Fig. 4B, 4G). Higher amounts of complex-II 19

were found in ADR+HT and ADR, whereas control and HT groups reported the lowest 20

amounts of flavoprotein (Fig. 4C, 4G). When complex-III (CORE II subunit) was 21

tested, the highest quantity was seen in the control group, whereas HT exhibited the 22

lowest, closely followed by ADR and ADR+HT (Fig. 4D, 4G). Similar results were 23

found upon examination for complex-III-Rieske iron-sulfur protein (Fig. 4E, 4G). 24

Complex-IV was found in lesser quantities in all groups compared to control groups 25

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(Fig. 4F, 4G). Finally, AIF significantly decreased in HT compared to other groups 1

(Fig. 5). 2

HT, ADR, and ADR+HT had significantly lower complex-I activity than control 3

(Fig. 6A). Trends demonstrate that HT group had similar complex-IV activity than 4

controls, while the lowest activities were seen in ADR and ADR+HT treated groups 5

(Fig. 6B). 6

3.3.Oxidative molecular damage 7

Amino acid oxidation in proteins was examined, specifically those carbonyl 8

products that suffer metal-catalyzed oxidation [27]. GSA arises from the metal-9

catalyzed oxidation of proline and arginine, while AASA results from lysine oxidation. 10

Moreover, it is known that third-party molecules are also involved in these chemical 11

pathways that link, on one hand, the increased free-radical efflux and, on the other hand, 12

structural modifications in proteins. These pathways may give rise to increased 2,4-13

dinitrophenylhydrazine-reactive carbonyls in proteins [28]. In this study, we 14

investigated the concentration of CEL and CML adducts. Those protein adducts were 15

first described as advanced-glycation-end-products (AGE), later named glycoxidation 16

products and now recognized as mixed AGEs-advanced lipoxidation products [21]. 17

Finally, lipid-peroxidation-derived protein damage was also demonstrated by MDAL. 18

The cardiac content of GSA, AASA, CEL, CML, and MDAL are shown in Table 2. The 19

highest values of those five markers were found in ADR group at significant levels. HT 20

was able to decrease the oxidative status of hearts subjected to ADR treatment 21

(ADR+HT group) by diminishing GSA, AASA, CML, and MDAL. Interestingly, GSA 22

and CML were lower in ADR+HT than controls. When alone, HT caused a marked 23

reduction of oxidative status of AASA, CEL, CML, and MDAL, even below the basal 24

oxidation status occurring in the control group. Finally, this work did not reveal any 25

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significant changes in fatty acid composition, double bond, and peroxidizability indices 1

(Table 3). 2

4. Discussion 3

It has been postulated that adverse cardiac effects resulting from ADR 4

administration is not only dose- but also time-dependent (i.e. during and following 5

treatment) [29,30]. Evident toxicity associated with ADR based on plasma markers 6

(CK, LDH, ALT and AST), body and heart weight changes, and heart/body weight ratio 7

did not find in the experimental animals. These results are in accordance with studies by 8

Injac et al. [31] in rats with colorectal cancer models. 9

In this study, ADR induced significant histopathological changes in rat hearts 10

based in the Billingham’s grade as have been shown in Table 1. However, in accordance 11

with sublethal cumulative doses used in this study, results show that ADR did not cause 12

extensive damage in hearts with clinical manifestations. As has been shown in Fig. 2 a 13

slight vacuolization and inflammatory infiltrate in myocytes from ADR group were 14

found and hearts with a histopathology between abnormal without typical injury due to 15

ADR and an effect on less than 5% of the myocytes with proper lesions were caused by 16

ADR. Similar results have been previously reported in studies of chronic cardiotoxicity 17

attributed to ADR [30,32]. The low dose and the administration time of the ADR used 18

in this study showed important changes in ultrastructural results but not in 19

histopathological determinations. 20

At ultrastructural level, highly altered mitochondria were observed, as well as 21

swelling and presence of small vacuoles associated with ADR administration. These 22

results are in agreement with previous works at chronic low- [30,31] and high-doses of 23

ADR [32,34]. This is the first study addressing the protective role of HT on 24

mitochondrial disturbances associated with ADR-induced cardiotoxicity in breast 25

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tumor-bearing rats. There is growing evidence supporting the protective role of HT on 1

mitochondria against harmful acrolein [35,36] and ischemia-reperfusion damage [15]. 2

Recently, Hao et al. [37] reported that HT stimulates mitochondrial biogenesis and 3

promotes mitochondrial function in 3T3-L1 adipocytes by increasing: 1) PPARGC1α 4

(peroxisome proliferator-activated receptor coactivator 1 alpha) activation and protein 5

expression, 2) oxygen consumption, 3) mitochondrial DNA quantity, 4) number of 6

mitochondria, and 5) complexes-I, -II, -III, and –IV protein expression and activity 7

levels. A dual effect of HT was observed due to its ability to partially restore the 8

mitochondrial status (ADR+HT group); concomitantly, a slightly damaged in 9

mitochondria was found in HT group. This can be partially explained due to mechanical 10

injury during tissue handling, or even a plausible side-effect arising from HT redox 11

cycling. In this sense, Zhu et al. [36] reported that HT protects ARPE-19 human retinal 12

pigment epithelial cells from acrolein-induced oxidative damage through two 13

mechanisms: 1) induction of phase II detoxifying enzymes like γ-glutamyl cysteine 14

ligase, NAD(P)H quinine oxido-reductase-1 (NQO1) and heme oxygenase-1, and 2) 15

stimulation of mitochondrial biogenesis. 16

ADR did not affect the concentration of mitochondrial complex-I although it 17

affects the activity of this complex. It decreased complexes-III and -IV and increased 18

complex-II, result that agree with previous studies [30,38,39]. Complex-III-Rieske 19

subunit is a nuclear-encoded protein with an iron-sulfur [2Fe-2S] redox center that 20

mainly participates in the Q cycle by transferring an electron from ubiquinol to 21

cytochrome c1. Complex-III-CORE II subunit is also a nuclear-encoded protein 22

important for mitochondrial protein importing, processing and integrity of complex-III. 23

Genetic deletion of this subunit directly affects the complex-III assembling [40]. There 24

is a putative impairment of the integrity and assembling of complex-III by ADR, mainly 25

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associated with low CORE II and unmodified Rieske subunits levels. Lesser activities 1

of both complex-I and –IV could enhance ROS production and subsequently limit ATP 2

production, as previously seen in vitro [30,41]. This is possibly causal, at least in part, 3

to enhanced ROS production by ADR in our study based on high steady-state levels of 4

oxidation-derived protein damage markers (CEL). Precursors of CEL formation are 5

derived from the glycolytic pathway. Thus, the increased levels of CEL described in the 6

present work in the ADR group can be ascribed to both a high oxidative stress status in 7

ADR-treated animals, as well as an increased glycolytic flux in this group in order to 8

compensate the low cellular energy state of the ADR-treated heart [4]. 9

HT did not modify the amounts of complex-I, -III (Rieske subunit) and -IV 10

proteins altered by ADR. Rather, it increased both complex-II and complex-III (CORE 11

II subunit) protein concentrations in comparison with the ADR group. These results 12

indicate that HT is able to improve the integrity of complex-III by increasing the CORE 13

II subunit without affecting the Rieske subunit. Since HT was not able to restore 14

complexes-I and -IV activities, it would be expected to maintain higher ROS 15

production. Nonetheless, these findings show that HT dramatically reversed oxidative 16

markers. Therefore, there must be mechanisms, as yet unclarified, by which HT 17

ameliorates oxidative stress and ADR-associated mitochondrial impairment. More 18

studies are needed in order to determine if HT protects electron transfer and decreases 19

ROS generation, without comprising ATP production. 20

In sharp contrast with Hao et al. [37], the present study shows that HT 21

significantly reduced the concentration of complexes-III and –IV. No differences were 22

seen for complexes-I and -II in comparison with controls. With regards to complex-I 23

and -IV activities, HT promotes a marked low activity of complex-I without affecting 24

the complex-IV activity. Taken together, these results show that METC is not affected 25

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by HT. Therefore, a correct flow of electrons may be possible, and lesser ROS 1

generation would be expected. 2

HT did not increase the ROS-derived oxidative damage (GSA, AASA, CEL, 3

CML and MDAL). In its role as a powerful antioxidant, HT was able decrease the 4

oxidative status, even below basal oxidation for certain markers. It can be attributed to 5

its antioxidant effect because the lipids oxidations markers (the degree of membrane 6

unsaturation, fatty acid composition, double bond, and peroxidizability indexes) did not 7

change. 8

AIF is a ubiquitously expressed flavoprotein synthesized as a cytoplasmic 67 9

kDa precursor giving rise to a mature 62 kDa protein at the mitochondrial level. In 10

mitochondria, AIF is involved in oxidoreduction and is considered a potential ROS 11

scavenger [42,43]. This mitochondrial protein has both life and death functions in cells 12

and it is known to be also required for mitochondrial oxidative phosphorylation [13]. 13

AIF-deficient cells exhibit a reduced content of complex-I, suggesting that a certain 14

amount of AIF is required for oxidative phosphorylation [39], all the while pointing 15

towards a role for AIF in the biogenesis of this complex. In this study AIF was only 16

decreased in the HT group, without change of complex-I amounts. This indicates that 17

substantial decreases in AIF are not large enough to become limiting for complex-I 18

biogenesis, and therefore, respiratory chain function is not comprised. Apoptotic or 19

antiapoptotic activity of HT is dependent on the cell type. 20

ADR clearly raised levels of oxidation-derived biomarkers, disrupted 21

mitochondria membrane and inhibited the METC. This could surely lead to an energy 22

imbalance in the cardiomyocytes. This scenario changes when ADR is administrated in 23

combination with HT. This antioxidant protects against ROS generation by ADR, as 24

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15

shown by data from ROS-derived oxidative damage (GSA, ASAA, CEL, CML and 1

MDAL) and data from mitochondria ultrastructure. 2

In summary, the present study reveals, for the first time, that HT can attenuate 3

ADR-associated cardiac toxicity by reducing ROS production enhanced by ADR. 4

Moreover, HT ameliorates mitochondrial dysfunction by partially restoring the 5

respiratory chain altered by ADR chronic treatment in rat models of breast cancer 6

through an as yet undetermined mechanism. Herein, authors hypothesized that 7

hydroxytyrosol scavenges free radicals in mitochondria, and its redox cycling might be 8

responsible, at least in part, for the suitable electron transport from complex-I to the rest 9

of METC proteins. 10

11

Acknowledgements 12

We acknowledge grants from Excelentísima Diputación de Jaén, CEAS 13

Foundation 30.C0.244500 and Junta de Andalucía PI-0210/2007. We thank the Spanish 14

Ministry of Science and Innovation (AP2005-144) and the University of Granada for 15

the personal support of Dr. S. Granados-Principal. Work carried out at the Department 16

of Experimental Medicine was supported in part by R+D grants from the Spanish 17

Ministry of Science and Innovation (BFU2009-11879/BFI), the Spanish Ministry of 18

Health [RD06/0013/0012 and PI081843], the Autonomous Government of Catalonia 19

[2009SGR735] and COST B35 Action of the European Union. We thank Dr. Elvin 20

Blanco for help in editing of the final manuscript. 21

22

Conflict of interest statement 23

The authors have declared no conflict of interest. 24

25

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Figure captions 1

Fig. 1. Representative traces of GC/MS analyses for specific protein oxidative damage 2

markers. Analyses were carried out by selected ion-monitoring GC/MS (SIM-GC/MS). 3

The amounts of product were expressed as µmoles of damage markers per mol of 4

lysine. Upper figure. Detection of the specific oxidation-derived protein carbonyl 5

glutamic semialdehyde (GSA) by SIM-GC/MS. Quantification was performed by 6

internal and external standardization using standard curves constructed from mixtures of 7

deuterated and non-deuterated standards. The ions used were: lysine and [2H8]lysine, 8

m/z 180 and 187, respectively; 5-hydroxy-2-aminovaleric acid and [2H5]5-hydroxy-2-9

aminovaleric acid (stable derivative of GSA), m/z 280 and 285, respectively. (A) 10

Selected ion chromatograms for rat heart proteins showing the m/z = 280 ion for the 11

different experimental conditions. (B) Selected ion chromatograms for rat heart 12

proteins showing the m/z = 180 ion for the different experimental conditions. (B1) 13

Selected ion chromatograms for rat heart proteins showing the m/z = 180 and 187 ions 14

(for protein lysine content quantification). (C) Selected ion chromatograms for rat heart 15

proteins showing the m/z = 280 and 285 ions (for protein GSA content quantification). 16

Lower figure. Detection of the specific glyco- and lipoxidation-derived protein marker 17

Nε-Carboxymethyl-lysine (CML) by SIM-GC/MS. Quantification was performed by 18

internal and external standardization using standard curves constructed from mixtures of 19

deuterated and non-deuterated standards. The ions used were: lysine and [2H8]lysine, 20

m/z 180 and 187, respectively; CML and [2H4]CML, m/z 392 and 396, respectively. 21

(A) Selected ion chromatograms for rat heart proteins showing the m/z = 392 ion for the 22

different experimental conditions. (B) Selected ion chromatograms for rat heart 23

proteins showing the m/z = 180 ion for the different experimental conditions. (B1) 24

Selected ion chromatograms for rat heart proteins showing the m/z = 180 and 187 ions 25

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(for protein lysine content quantification). (C) Selected ion chromatograms for rat heart 1

proteins showing the m/z = 392 and 396 ions (for protein CML content quantification). 2

3

Fig. 2. Hematoxylin and eosin staining of rat cardiac tissue after chronic treatment with 4

doxorubicin. Arrows indicate cardiomyocyte cytoplasmic vacuolization (A), 5

inflammatory infiltrate (B), and atrial neuron vacuolization. Magnification: 40×. 6

7

Fig. 3. Effects of doxorubicin, hydroxytyrosol, and their combination on mitochondria. 8

Mitochondrial ultrastructure in heart (A), mitochondrial area (B), altered mitochondria 9

percentage (C). Magnification: 10,000×. Values are expressed as mean ± SEM. Letters, 10

when different, represent statistically significant differences (p< 0.05). Control: control 11

group; HT: group treated with hydroxytyrosol; ADR: group treated with doxorubicin; 12

ADR+HT: group treated with hydroxytyrosol and doxorubicin. 13

14

Fig. 4. Effects of doxorubicin, hydroxytyrosol, and their combination on protein levels. 15

Complex-I-NDUFA9 (39 kDa) (A) and -NDUFS3 (30 kDa) subunits (B), complex-II-70 16

kDa (Flavoprotein) subunit (C), complex-III-COREII (48 kDa) (D) and -Rieske iron-17

sulfur protein (29 kDa) subunits (E), and complex-IV-COXI (57 kDa) subunit (F) in rat 18

heart tissue. Values are expressed as means ± SEM (n=5). Bars with different letters 19

significantly differ among the groups (p < 0.05). Control: control group; HT: group 20

treated with hydroxytyrosol; ADR: group treated with doxorubicin; ADR+HT: group 21

treated with hydroxytyrosol and doxorubicin. (G) Representative Western blots images 22

of the heart lysates from untreated animals (control group) and effects of doxorubicin 23

(ADR), hydroxytyrosol (HT), and their combination (ADR+HT) on mitochondrial 24

respiratory chain complexes levels (from CI to CIV). 25

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1

Fig. 5. Effects of doxorubicin, hydroxytyrosol and their combination on the protein 2

levels of AIF in heart. Values are expressed as means ± SEM (n=5). Bars with different 3

letters significantly differ among groups (p< 0.05). Control: control group; HT: group 4

treated with hydroxytyrosol; ADR: group treated with doxorubicin; ADR+HT: group 5

treated with hydroxytyrosol and doxorubicin. Representative Western blots images of 6

the heart lysates from untreated animals (control group) and effects of doxorubicin 7

(ADR), hydroxytyrosol (HT), and their combination (ADR+HT) on AIF protein levels. 8

9

Fig. 6. Activities of mitochondrial complexes-I (A) and -IV (B) in rats treated with 10

hydroxytyrosol, doxorubicin, and their combination compared to controls. Values are 11

expressed as means ± SEM (n=5). Bars with different letters significantly differ among 12

groups (p< 0.05). Control: control group; HT: group treated with hydroxytyrosol; ADR: 13

group treated with doxorubicin; ADR+HT: group treated with hydroxytyrosol and 14

doxorubicin. 15

16

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18

19

20

21

22

23

24

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Table Footnotes

1Abbreviations: AASA, aminoadipic semialdehyde; ADR, doxorubicin; AIF,

Apoptosis-inducing factor; ALT, alanine aminotransferase; AST, aspartate

aminotransferase; CEL, Nε-(carboxyethyl)-lysine; CK, creatinine kinase; CML, N

ε-

(carboxymethyl)-lysine; DBI, double bond index; GSA, Glutamic semialdehyde; HT,

hydroxytyrosol; i.v.: intravenous; LDH, lactate d1ehydrogenase; MDAL, N

ε-

malondialdehyde-lysine; METC, mitochondrial electron transport chain; NQO1,

NAD(P)H quinine oxido-reductase-1; ROS, reactive oxygen species; TUNEL, terminal

deoxynucleotidyl transferase (TdT) dUTP-nick-end labeling

1

2

3

4

5

6

7

8

9

10

11

12

13

14

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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 Control HT ADR ADR+HT

Billingham’s grade 0±0a

0±0a

0.75±0.163b

0.938±0.147b

Body weight (g) 271.4±8.9 266.6±7.4 256.0±3.3 253.8±6.0

Heart weight (g) 1.04±0.034 1.03±0.032 0.93±0.037 0.97±0.03

Heart/body weight

ratio (mg/g)

3.88±0.2 3.88±0.1 3.63±0.13 3.82±0.17

CK (U/L) 103.0±17 64.0±19 164.0±45 151.0±77

LDH (U/L) 376.0±66 323.0±68 304.0±33 327.0±91

ALT (U/L) 21.9±8.5 10.7±3.5 11.3±3.9 15.0±3.8

AST (U/L) 33.6±8.9 18.9±2.8 14.9±2.7 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.

Table 1

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Table 2. Effects of hydroxytyrosol, doxorubicin and their combination on oxidation-derived

protein damage markers in rat hearts

Control HT ADR ADR+HT

GSA (µmol/mol Lys) 2424.9±131.1ab

2697.6±141.8ab

3818.6±694.8b

2180.2±178.8a

AASA (µmol/mol Lys) 302.9±10.8a

260.5±7.8a

444.5±53.0b

325.0±6.5a

CEL (µmol/mol Lys) 382.8±2.9a

354.8±53.0a

521.3±39.8b

424.8±29.1ab

CML (µmol/mol Lys) 949.4±10.4a

747.5±52.1a

1279.1±109.9b

911.8±3.7a

MDAL (µmol/mol Lys) 305.1±13.0a

269.4±10.6a

505.6±39.6b

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.

Table 2

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Table 3. Effects of hydroxytyrosol, doxorubicin, and their combination on fatty acid

profile in rat hearts

Control HT ADR ADR+HT

14:0 1.18±0.12 1.10±0.07 1.22±0.13 1.12±0.19

16:0 21.38±1.18 22.08±0.7

0

23.11±1.22 23.20±1.55

16:1n-7 0.86±0.0.07 0.74±0.04 0.96±0.10 0.97±0.0.16

18:0 27.61±1.08 29.97±0.3

6

27.25±0.80 28.96±0.10

18:1n-9 14.34±0.45 14.39±0.6

0

14.76±0.75 12.79±0.84

18:2n-6 8.74±1.27 5.89±0.47 7.21±1.46 6.04±0.69

18:3n-3 0.36±0.06 0.44±0.07 0.45±0.08 0.34±0.05

18:4n-6 2.68±0.11 2.88±0.22 2.28±0.17 2.71±0.26

20:0 1.15±0.07 1.14±0.07 0.97±0.08 1.02±0.20

20:3n-6 0.26±0.03 0.22±0.01 0.20±0.02 0.23±0.01

20:4n-6 8.18±1.21 6.68±1.09 7.64±1.12 7.75±0.19

20:5n-3 0.82±0.10 0.97±0.06 0.73±0.04 0.62±0.10

22:0 1.75±0.13 2.23±0.18 1.64±0.13 1.71±0.26

22:4n-6 0.71±0.12 0.67±0.08 0.76±0.11 0.67±0.06

22:5n-6 0.99±0.02 1.29±0.07 1.35±0.15 1.99±0.28

22:5n-3 0.99±0.08 1.09±0.10 0.98±0.11 0.98±0.07

24:0 0.31±0.02 0.34±0.05 0.39±0.06 0.47±0.08

22:6n-3 3.94±0.0.50 3.24±0.51 4.46±0.66 4.14±0.75

Table 3

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24:5n-3 1.25±0.15 1.59±0.28 1.23±0.13 1.54±0.30

24:6n-3 2.43±0.22 3.01±0.31 2.39±0.26 2.71±0.54

ACL 18.29±0,07 18.32±0,0

4

18.27±0.09 18.33±0.15

SFA 53.39±2.45 56.86±1.0

9

54.58±2.03 56.48±2.59

UFA 46.60±2,45 43.14±1.0

9

45.42±2.03 43.51±2.60

MUFA 15.20±0,48 15.14±0.6

1

15.72±0.76 13.76±0.99

PUFA 31.40±2,76 28.00±1.3

6

29.69±2.55 29.75±3,517

PUFAn-3 9.80±0,57 10.36±0.4

3

10.25±0.91 10.35±1.50

PUFAn-6 21.59±2,31 17.64±1.4

5

19.45±2.03 19.41±2.11

DBI 135.40±9,21 127.98±5.

62

133.72±9.7

5

135.85±15.6

5

PI 132.14±9,44 128.31±5.

46

132.20±11.

71

137.78±18.8

5

Values are expressed as mean ± SEM.

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Figure 1

Figure 1

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Figure B

Figure C

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Figure 2

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Figure 3

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G

Figure 4

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Figure 5

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Figure 6

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*Graphical Abstract (for review)