European Journal of Clinical Investigation

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European Journal of Clinical Investigation

European Journal of Clinical Investigation Volume 42 Supplement 1 April 2012 1–98

www.ejci-online.com

COPYRIGHT WILEY-BLACKWELL

April 2012 Vol 42 Supplement 1

European Journal of

Clinical Investigation

46th Annual Scientific Meeting of the European

Society for Clinical Investigation

Budapest Hungary

22–24 March 2012

ABSTRACT BOOK

Official journal of the European Society for Clinical Investigation

ECI_42_s1_oc.indd 1

2/27/2012 12:36:06 PM


72 Workshop 4: Mitochondria in health and disease

hamper its implementation in vivo. TCA cycle kinetics was

evaluated under well-defined metabolic constraints and its

sensitivity to them analyzed. Rat hearts were perfused in the

Langendorff mode under altered cytosolic redox sates and

exogenous Ca 2+ concentrations, to alter contractility. High resolution

13 C-NMR spectra of tissue extracts were analyzed and

the time-dependent multiplets observed in the 13 C-NMR resonances

of glutamate, in all hearts, and in malate and aspartate,

in hearts perfused with high-pyruvate/low-lactate concentrations,

were analyzed using a kinetic model of the TCA cycle.

The analysis showed that TCA cycle flux (V TCA ) and exchange

fluxes (V X ) that involve cycle intermediates were both sensitive

to cell redox and altered Ca 2+ concentrations. In particular the

ratio of the two fluxes (V X /V TCA ) proved to be particularly

sensitive, varying approximately 10-fold. This renders this

parameter particularly valuable in analysis of cardiac malfunction

and makes it a preferential target towards clinical implementation

of methodologies based in analysis of carbon

tracers.

411

Organ and treatment protocol-dependent

doxorubicin-induced in vivo

mitochondrionopathy

G.C. Pereira*, S.P. Pereira*, J.A. Lumini , C.V. Pereira*,

J. Magalhães , A. Ascensão , J.A. Bjork à , A.J. Moreno § ,

M.S. Santos*, K.B. Wallace à & P.J. Oliveira*

*Center for Neuroscience and Cell Biology, University of

Coimbra, Coimbra, Portugal;

Research Center in Physical Activity, Health and Leisure

(CIAFEL), University of Porto, Portugal;

à Department of Biochemistry and Molecular Biology,

University of Minnesota Medical School, Duluth, MN, USA;

§ Institute for Marine Research, University of Coimbra,

Coimbra, Portugal

Background: Doxorubicin (DOX) therapeutic usage is limited

due to dose-dependent cardiotoxicity. This work explores in

vivo mitochondrial DOX-induced toxicity associated with two

distinct treatment protocols in order to understand both selectivity

and toxicity caused by DOX.

Materials and methods: Crl:WI(Han) rats were sub-chronically

(7 weeks, 2 mg kg -1 ) or acutely (20 mg kg -1 ) treated with DOX.

Results: Although no echocardiographic alterations were

detected, the majority of differences occurred during subchronic

treatment, notably a decrease in state 3 respiration.

Increased mitochondrial permeability transition was also measured

in cardiac preparations from sub-chronic treatment.

However, gene expression analyses showed no alterations in

sub-chronic model but six genes were altered in the acute

model. Moreover, protein levels remained similar to control in

both treatment protocols. Toxicity was clearly lower in hepatic

and renal mitochondria.

Conclusions: The results shed more light on the role of mitochondria

in DOX-induced cardiotoxicity and demonstrate that

even in the absence of transcriptional alterations of relevant mitochondrial

proteins there is still an alteration in mitochondrial

function without simultaneous echocardiographic abnormalities.

Acknowledgements: The work is supported by the Portuguese

FCT (SFRH/BD/36938/2007 to GCP SFRH/BPD/66935/

2009 to JM, SFRH/BPD/4225/2007 to AA, PTDC/SAU-OSM/

64084/2006, PTDC/QUI-QUI/101409/2008 and PTDC/SAU-

OSM/104731/2008).

412

Targeting mitochondria to induce cancer cell

death

C. Brenner

Labex LERMIT, INSERM U769, University of Paris Sud,

Orsay, France

For many years, medical drug discovery has extensively

exploited peptides as lead compounds. Currently, novel structures

of therapeutic are derived from active pre-existing peptides

or from high-throughput screening, and optimized

following a rational drug design approach. Molecules of interest

may prove their ability to influence the disease outcome in

animal models and must respond to a set of criteria based on

toxicity studies, ease of administration, the cost of their synthesis,

and logistic for clinical use to validate it as a good candidate

in a therapeutic perspective. This applies to the potential

use of peptides to target one central intracellular organelle, the

mitochondrion, to modulate (i.e. activate or prevent) apoptosis.

Putative mitochondrial protein targets and the strategies

already elaborated to correct the defects linked to these proteins

(overexpression, inactivation, mutation…, etc.) will be

described, and recent advances that led or may lead to the

conception of therapeutic peptides via a specific action on

these mitochondrial targets in the future will be discussed.

413

Mitochondrial remodeling during cancer stem

cell differentiation

I. Vega-Naredo*, L.C. Tavares*, J.R. Erickson , R. Loureiro*,

A.C. Burgeiro*, A.F. Branco*, J. Holy , E.L. Perkins à &

P.J. Oliveira*

*Center for Neurosciences and Cell Biology, University of

Coimbra, Coimbra, Portugal;

University of Minnesota-Medical School, Duluth, MN,

USA;

à School of Medicine, Mercer University, Savannah, GA,

USA

Background: The ability of cancer stem cells to evade chemotherapy

results in tumor maintenance. Since mitochondria are

essential in cell death signaling, the objective of his work is to

identify critical mitochondrial alterations in undifferentiated

embryonal carcinoma cells vs. more differentiated cells.

Materials and methods: P19 embryonal carcinoma stem cells

(StemTCs) and retinoic acid-P19 differentiated cells (DiffTCs)

were used. Mitochondrial morphology, oxygen consumption,

oxidative stress, ATP/ADP levels, mtDNA copy number as

well as regulators of mitochondrial biogenesis and dynamic

were evaluated. Apoptotic and autophagic markers were also

measured.

Results: Our results did not show differences in mitochondrial

mass between both groups however, StemTC-DiffTC transition

was accompanied by a conversion from polarized small-round

mitochondria to longer filaments. Accordingly, DiffTCs presented

higher mitochondrial function including higher respiration

rate, oxidative stress and ATP production. Mitochondrial

fusion was also favored in DiffTCs. StemTCs showed the highest

levels of p53 and some of his target proteins, as well as

active caspase-3 but without cleavage of traditional substrates.

At the same time, StemTCs showed a block in lysosomalautophagic

pathway. The higher calpain and lysosomal activities

found in DiffTCs may render these cells susceptible to cell

death.

2012 Wiley-Blackwell

72 ª 2012 The Authors. European Journal of Clinical Investigation ª 2012 Stichting European Society for Clinical Investigation Journal Foundation


International Journal of Cardiology 156 (2012) 4–10

Contents lists available at ScienceDirect

International Journal of Cardiology

journal homepage: www.elsevier.com/locate/ijcard

Review

Exercise as a beneficial adjunct therapy during Doxorubicin treatment—Role of

mitochondria in cardioprotection

António Ascensão a, ⁎, Paulo J. Oliveira b , José Magalhães a

a Research Centre in Physical Activity, Health and Leisure, Faculty of Sport, University of Porto, Portugal

b Centre for Neuroscience and Cell Biology, Department of Life Sciences, University of Coimbra, Portugal

article

info

abstract

Article history:

Received 15 December 2010

Received in revised form 14 March 2011

Accepted 13 May 2011

Available online 1 June 2011

Keywords:

Cardiac injury

Exercise

Doxorubicin

Mitochondria

Apoptosis

One of the mostly used chemotherapeutic drugs is the highly effective anthracycline Doxorubicin. However,

its clinical use is limited by the dose-related and cumulative cardiotoxicity and consequent dysfunction. It has

been proposed that the etiology of this toxicity is related to mitochondrial dysfunction. The present review

aimed to analyze the promising results regarding the effect of several types of physical exercise in cardiac

tolerance of animals treated with acute and sub-chronic doses of Doxorubicin (DOX), highlighting the

importance of cardiac mitochondrial-related mechanisms in the process.

Physical exercise positively modulates some important cardiac defense systems to antagonize the toxic effects

caused by DOX treatment, including antioxidant capacity, the overexpression of heat shock proteins and other

anti-apoptotic proteins. An important role in this protective phenotype afforded by exercise should be

attributed to mitochondrial plasticity, as related adaptations could be translated into improved cardiac

function in the setting of the DOX cardiomyopathy.

© 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

It is well established that some potent and highly effective

chemotherapeutic agents, of which DOX is a clear example, cause a

variety of side effects that limit a more effective use in the reduction or

elimination of tumor cell growth. Specifically, the clinical use of the

broadly successful antineoplastic DOX is limited by the occurrence of a

dose-related cardiac toxicity that results in life-threatening cardiomyopathy.

In an attempt to counteract DOX limiting cardiac-specific

side effect, researchers have investigated efficient adjunct therapies

and the related mechanisms of protective action using animal models.

Among the several pharmacological and non-pharmacological strategies,

physical exercise of different types and characteristics,

including acute bouts of treadmill running, short and long term

forced endurance training in the form of treadmill running or

swimming as well as voluntary physical activity, has been studied.

The present review aims at briefly analyzing possible crosstolerance

effects of physical exercise against the cardiotoxicity

associated to DOX treatments. As both DOX and exercise interfere

with mitochondrial function and plasticity by different mechanisms

and targets, it is reasonable to think that these organelles seem to be

central to explain, at least in some extent, the physiological outcome

of the combination. Moreover, both exercise [1] and DOX toxicity [2]

⁎ Corresponding author at: Research Centre in Physical Activity, Health and Leisure,

Faculty of Sport Sciences, University of Porto, Rua Dr. Plácido Costa, 91, 4200-450 Porto,

Portugal. Tel.: +351 225074774; fax: +351 225500689.

E-mail address: aascensao@fade.up.pt (A. Ascensão).

are known to modulate apoptotic pathways in the cell, in which

mitochondria are central to, which justifies the increased interest in

that organelle. The present review thus highlights the role of

mitochondria in the protective role of exercise against DOX-induced

cardiotoxicity and the mechanisms subjacent to such protective effect.

2. DOX-induced cardiac dysfunction—is there a role for

mitochondrial toxicity

DOX is an anthracycline prescribed alone or in combination with

other chemotherapeutics for the treatment of various neoplasms such

as leukemias, lymphomas, thyroid and lung carcinomas, several

sarcomas, stomach, breast, bone and ovarian cancers. The antitumor

activity of DOX is attributed to its ability to intercalate into both

nuclear and mitochondrial DNA double helix and/or to covalently

bind to proteins involved in DNA replication and transcription,

leading ultimately to cellular death through the inhibition of DNA,

RNA and protein synthesis [2–4]. Unfortunately, a broader clinical use

of this anticancer agent is restrained due to its cardiotoxicity and

consequent development of cardiac dysfunction manifested as

electrophysiological abnormalities and congestive heart failure [5,6].

In addition, hemodynamic impairments are also reported as consequence

of DOX treatment [7–12].

Heart histological observations (Fig. 1C and D) indicate loss of

myofibrils, distension of the sarcoplasmic reticulum, and vacuolization

of the cytoplasm, signs of mitochondrial damage with cristae

degeneration, intramitochondrial vacuoles as well as myelin figures

0167-5273/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.ijcard.2011.05.060


A. Ascensão et al. / International Journal of Cardiology 156 (2012) 4–10

5

Fig. 1. Heart tissue electron micrographs from (A) sedentary saline (magnification: ×16,000); (B) endurance trained saline (magnification: ×10,000); (C and D) sedentary treated

with DOX (magnifications: ×10,000 and ×12,500, respectively); (E and F) endurance trained treated with DOX (magnifications: ×6500 and ×10,000, respectively); Note the

protection afforded by training (E and F) against cytoplasmatic vacuolation, myofibrillar disorganization, and particularly several signs of mitochondrial damage with extensive

degeneration or even loss of cristae, intramitochondrial vacuoles and notorious myelin figures, which characterize heart tissue from sedentary DOX-treated group (C and D).

From Ascensão et al. [13] with permission.

that probably resulted in the formation of secondary lysosomes and

mitochondrial swelling [13–17].

Several mechanisms, particularly related to mitochondria, are

proposed as being responsible for the increased sensitivity of the

heart to DOX-induced toxicity including: (i) higher mitochondrial

density per unit volume in cardiomyocytes when compared to other

tissues, (ii) the elevated affinity of DOX by cardiolipin, a major

phospholipid component of the inner mitochondrial membrane in the

heart, (iii) the possible existence of a specific, although controversial,

NADH dehydrogenase in the heart, which also contributed for DOX

redox cycling and increased formation of reactive oxygen species

(ROS) and (iv) the relative lower antioxidant defense capacity of the

highly oxidative heart tissue [2–4,18].

At a molecular level, despite the fact that the basis for DOX

cardiotoxicity remains a matter of debate, it has been suggested that

cardiomyocyte dysfunction induced by DOX treatment is related to

enhanced levels of ROS-induced damage and apoptotic cellular death,

involving mitochondria in the process [2–4,18]. In fact, it is recognized

that DOX is converted into a semi-quinone reactive by mitochondrial

complex I, leading to formation of superoxide anion [19,20]. The

increased cardiac oxidative stress associated with DOX toxicity leads

to the depletion of reducing equivalents, impairment in oxidative

phosphorylation with consequent decline in ATP, and interference

with cellular calcium homeostasis [2,3]. The latter has been attributed

to increased susceptibility to the mitochondrial permeability transition

(MPT) [15,16,21–24], which is characterized by the loss of the

impermeability that characterizes the inner mitochondrial membrane,

being mediated by the formation and opening of protein

complex-like pores, the MPT pores [25–28]. The increased susceptibility

for MPTP opening can originate from increased oxidative stress,

calcium and phosphate overload, leading to loss of mitochondrial

membrane potential (ΔΨ), increased mitochondrial osmotic swelling

and rupture of the outer mitochondrial membrane [26,29,30]. MPT

pore inhibition or attenuation through pharmacologic and nonpharmacological

strategies has been successfully translated into

amelioration of mitochondrial function during and after DOX

treatment [1,14,15,23,31].

The link between MPTP opening and mitochondrial involvement

in apoptotic cell death is based on the release of pro-apoptotic

proteins housed in mitochondria such as cytochrome C, SMAC/DIABLO

and the apoptosis inducing factor (AIF), which can be regulated by

MPT induction. Although some reports suggest that the voltagedependent

anion channel (VDAC) and the adenine nucleotide

translocase (ANT) are dispensable for the MPT [32,33], a considerable

evidence supports that at least some forms of the MPT pores are

composed by the outer membrane VDAC as well as the inner

membrane ANT and matrix chaperon cyclophilin D (Cyp D) [27].

Increased sensitivity to MPT pore opening occurs in heart

mitochondria from DOX-treated rats, which is in fact considered a

sensitive indicator of DOX-induced mitochondrionopathy [2,23,34].

The activation of apoptosis in animal hearts or cells receiving DOX

treatments irrespective and/or involving mitochondria has been

largely reported, with studies evidencing increased activation of

several caspases such as 3, 9, 12 [14,35–39], an increase in TUNEL

(terminal (TdT)-mediated dUTP-biotin nick end labeling)-positive

nuclei [38,40–42], mitochondrial cytochrome C release [36–38,42],

increased Bax/Bcl-2 ratio [14,35,37,42] and Bad levels [42], and

overexpression of p53 followed by its translocation to mitochondria

[43].

As molecules activated in response to cellular stress, chaperones

and chaperonins such as heat shock proteins (HSP) 60 and 70 kDa

increased in hearts and mitochondria from DOX treated animals and

cells [14,44–47]. Their important roles in protein folding, unfolding,

refolding and transport were demonstrated in cells and transgenic

animals overexpressing HSPs, with consequent increased cardiac

function and viability, decreased mitochondrial morphological alterations,

oxidative damage and cell death indexes after DOX treatment

[48,49].

Despite increasing evidence suggesting mechanistic crosstalk

among autophagic, apoptotic and necrotic pathways involved in


6 A. Ascensão et al. / International Journal of Cardiology 156 (2012) 4–10

cellular death, at least distinct biochemical and morphological

features are classically attributed to each form. Hence, it is important

to note that DOX-induced signs of necrosis have also been reported,

such as heart pro-inflammatory cytokine expression and inflammatory

cell infiltration [50–52]. In these studies, increased oxidative

stress was closely implicated as pre-treatment with antioxidants and

free radical scavengers protected from DOX-induced cardiomyocyte

death. Proposed explanations may include the fact that increased ROS

resulted in mitochondrial calcium overload, MPTP opening and

swelling leading to ATP depletion, thus resulting in necrotic cell

death [3]. In addition, recent studies reported the activation of the

cardiac autophagy in DOX treatment [53,54], being this process also

closely related to mitochondrial dysfunction, including decreased ΔΨ

and increased MPTP opening vulnerability [53]. Although causal

relationship between mitochondrial function and autophagy remains

unclear, it is suggested that MPTP opening may serve as an upstream

signal, triggering autophagy of damaged and depolarized mitochondria

[55].

In summary, the data so far indicates that mitochondria are in fact

important components of DOX-induced cardiotoxicity, contributing to

cardiac degeneration that characterizes patients treated with higher

dosages of DOX. Several heart mitochondrial-related alterations

caused by DOX treatments and the respective tendency are summarized

in Table 1. These include markers of pro-oxidant challenge,

apoptotic signaling, and mitochondrial respiratory function, MPTP

vulnerability, antioxidant and other defense molecules as well as

mitochondrial electron transport chain complex activity.

3. Exercise-induced cardioprotection against Doxorubicin effects

Physical exercise in its various forms has been shown to be an

effective intervention that can provide a safeguard against acute and

chronic deleterious insults for the myocardium. These include ischemia–reperfusion

injury [1,56–58], diabetes [59,60], aging [61,62],

chemical gonadectomy [63] and DOX treatment [7–11,64,65]. The

effects of exercise on mitochondrial consequences of DOX are also

highlighted in Table 1.

3.1. Chronic exercise

There is now considerable number of studies providing biochemical,

morphological and functional data suggesting that daily exercise

antagonizes the harmful consequences of in vivo and in vitro DOX

treatment on rodent heart, either by preventing, attenuating or

reverting the toxicity [7–10,65,79]. Data suggests that mitochondria

may have a pivotal role in this protection [13,14,44,46].

Since the early investigation by Combs et al. [80], in which DOXtreated

rats submitted to an acute bout of forced swimming had no

increased DOX toxicity and in fact, showed increased survival rates,

several works have been performed, particularly to analyze the

chronic effect of exercise against cardiac injury caused by both acute

and sub-chronic DOX treatment. Another early study using endurance

training as an adjunct strategy to diminish DOX cardiac side effects

aimed to correlate exercise-induced alterations in enzymatic antioxidant

activity with the attenuation of DOX-induced oxidative damage

and histological alterations [79]. The degree of morphological damage

observed in sedentary animals treated with DOX was higher than in

trained animals also treated with DOX. This cardioprotection was

attributed to the up-regulation of antioxidant enzymes.

The protective role of swimming training against enhanced cardiac

oxidative stress and damage induced by acute DOX administration

was later confirmed [45]. The authors reported that DOX-induced

increased plasma levels of cardiac troponin I, tissue lipid peroxidation,

protein, including glutathione oxidation were limited by previous

training. The proposed mechanisms for the protection afforded by

swimming training were increased amounts of cardiac reduced

glutathione and heat shock proteins of the 60 kDa family (HSP 60).

An interesting finding was that no variations in cardiac HSP70

Table 1

Summary of some described mitochondrial-related alterations associated with DOX-induced cardiotoxicity and the modulation effect afforded by physical exercise against DOX.

DOX effect Reference Exercise effect against DOX Reference

RONS production ↑ [19,20,38,44,66] ↓ [44]

Oxidative damage markers

Lipid peroxidation ↑ [14,44,46,66–68] ↓ [14,44,46]

Protein oxidation ↑ [14,44,46] ↓ [14,44,46]

DNA oxidation ↑ [67,69,70]

Aconitase activity ↓ or = [14,37] ↑ [14]

Apoptotic signaling

Bax-Bcl-2 ratio ↑ [14,37] ↓ [14]

Cytochrome c release ↑ [36–38]

Caspase 9 activation ↑ [14,31] ↓ [14]

Respiratory endpoints

State 3 ↓ [14,15,31,37,46,68,71–74] ↑or = [14,31,46]

State 4 ↑ or = or ↓ [14,15,31,44,46,68,71,73] =or↓ [14,31,44,46]

RCR ↓ [14,15,31,44,46,72–74] =or↑ [14,31,44,46]

ADP–O ratio = or ↓ [14,15,31,46,72,73]

Uncoupled respiration ↓ [14,74] ↑ [14]

Creatine-stimulated respiration ↓ [75]

Maximal ΔΨ ↓ or = [23,31,74,76] ↑ [31]

Calcium-induced MPTP ↑ [15,16,22–24,31,68,71,77] ↓ [31]

ANT content and functioning ↓ [16,24]

Mitochondrial chaperones ↑ [14,45,46] ↑ [14,46]

Mitochondrial antioxidants

Thiols ↓ [14,68] ↑ [14]

Vitamin E ↓ or = [68,71]

Enzymes = or ↑ [14,44,46] ↑ [14,44,46]

Coenzyme Q isoenzymes = [68]

ETC complex activity

Complex I ↓ [15,31,73] ↑ [31]

Complex II ↓ [73]

Complex II = [73]

Complex IV = or ↓ [15,66,78]

Complex V ↓ [31] ↑ [31]


A. Ascensão et al. / International Journal of Cardiology 156 (2012) 4–10

7

expression after swimming training were observed, despite a large

amount of studies reporting that endurance treadmill training

increased the expression of HSP70 [45]. Up-regulation of HSP 70

was reported to contribute to resistant phenotype against several

cardiac insults, including ischemia–reperfusion, hyperglycemia, ovariectomization,

and spontaneous hypertension, in vitro mitochondrial

anoxia-reoxygenation, hydrogen peroxide-induced oxidative stress

and DOX-induced injury in cardiomyocytes [for refs see 1,57,58].

The important involvement of mitochondria in cardioprotection

afforded by endurance training against DOX treatment was initially

reported by studies from our group [13,14,46]. It was demonstrated

that endurance training prevented acute DOX-induced mitochondrial

alterations in respiration, oxidative stress and calcium loading

capacity [14]. In addition, training limited the appearance of apoptotic

markers in the hearts of DOX-treated animals, including increased

mitochondrial Bax, Bax-to-Bcl2 ratio and tissue caspase 3 activity.

Changes at the ultrastructural level induced by DOX treatment were

also attenuated in hearts from trained animals [13,14]. As seen in

Fig. 1C and D, sedentary DOX-treated animals showed myocardial

damage when compared to the normal appearance of their saline

sedentary controls (Fig. 1A). The observed morphological alterations

consisted of mitochondrial damage with extensive degeneration and

loss of cristae, swelling and abnormal size and shape, intramitochondrial

vacuoles and notorious myelin figures that probably resulted in

the formation of secondary lysosomes. All these alterations were

attenuated in trained animals treated with DOX (Fig. 1E and F).

Moderate endurance training per se also induced alterations in

myocardial structure suggestive of remodeling, including evidences of

mitochondrial biogenesis.

In contrast to the data shown by Kavazis et al. [44], mitochondrial

protection afforded by training against DOX toxicity was consistent

with overexpression of tissue HSP70 and mitochondrial HSP60 in the

trained groups. Increased HSP levels strongly suggest that upregulation

of these molecular chaperones contributed to the preservation

of the integrity and activity of mitochondrial respiratory

complexes [81–83]. HSP-induced cardioprotection may be accomplished

through facilitation of nuclear-encoded protein import and

assembly in the mitochondrial matrix as well as improved folding of

proteins within mitochondria. Also, it is possible that the upregulation

of mitochondrial superoxide dismutase (MnSOD) activity

accounted for some of the observed protection. As mitochondrial DOX

toxicity has been largely attributed to increased oxidative stress,

increased HSP and MnSOD may be important mechanisms that

explain how endurance training prevents the majority of DOXinduced

myocardial damage. The effect of training on preventing

activation of cardiac apoptotic pathways has been described by others

as well [62,84–86]. However, Chicco et al. [7] did not attribute the

cardioprotective effect of low-intensity exercise against cumulative

DOX treatment to HSP and MnSOD overexpression. Low-intensity

treadmill exercise training-induced inhibition of apoptotic signaling

and the increased activity of glutathione peroxidase were the putative

mechanisms advanced to explain the protection.

It is also suggested that chronic exercise stimulation may also

afford protection against the increased susceptibility to the MPT as the

deleterious effects of calcium on heart mitochondrial respiration of

DOX-treated animals were attenuated in trained group treated with

DOX [14]. As the MPT has a marked oxidative etiology, it is likely that

the possible increased resistance of cardiac mitochondria from trained

animals to the MPT can be related with increased antioxidant

defenses. Therefore, the higher levels of reduced sulfhydryl groups

in trained mitochondria than in sedentary groups may be indicative of

enhanced antioxidant capacity and/or of more elevated sulfhydryldonors,

such as GSH, in mitochondria from trained animals [87].

Further studies are warranted in order to better understand this issue.

Still in the support of the important involvement of mitochondria

in the downstream mechanisms of exercise-induced cardioprotection

in DOX treated rats, a recent study from Kavazis et al. [44] using a

short-term endurance training protocol, confirmed and extended the

existing knowledge. The authors observed that the impaired oxidative

phosphorylation, increased hydrogen peroxide production, lipid

peroxidation and protein oxidation as well as apoptotic levels and

proteolytic degradation induced by acute DOX administration were

limited by a five-day endurance treadmill training regimen prior

treatment. This phenotype was associated with the up-regulation of

antioxidant enzymes and HSP72, although the authors proved in an

independent set up that the prevention of exercise-induced myocardial

HSP72 increased expression did not eliminate the protection

afforded by exercise against DOX-mediated damage. In fact, it has been

demonstrated that HSP expression may not be determinant for

exercise-induced cardioprotection as studies revealed that exercise

in a cold environment, which does not increase HSP content, can also

provide protection against ischemia–reperfusion injury [88,89] or

DOX cardiotoxicity [44]. Exercise in cold vs. normal temperatures may

also display other types of differences regarding alteration of

mitochondrial physiology, besides alteration in the expression of HSPs.

Regarding the putative role of exercise against DOX-induced

cardiac toxicity, it is important to consider the remarkable commentary

made by Emter and Bowles [90] with respect to the work of

Hydock et al. [9] in which it was found out that exercise training in

rats before treatment with DOX attenuated DOX-induced cardiac

dysfunction, through the maintenance of fractional shortening,

developed pressure and contractility. The observation highlights

some unresolved questions regarding the possibility that some

cardiac benefits of exercise training can only be obtained if the

intensity of daily aerobic exercise is high (ranging from 70 to 80% of

maximal oxygen uptake). In fact, being DOX used in patients

undergoing cancer chemotherapy who experience severe fatigue

and display considerable exercise intolerance, the intensity and

duration of exercise training sessions can constitute a limiting factor

for using exercise as co-adjunct tool in cancer patients. This important

comment can easily be applied in other studies, including those from

our laboratory, in which exercise protocols had similar high

intensities and durations. It is however important to note that these

studies aimed to obtain preliminary results regarding the cross

tolerance effect and related mechanisms of how exercise training can

antagonize the side effects of acute and sub-chronic DOX treatment.

The same research group has been using low exercise intensities of

training protocols in their studies with promising results regarding

the protective value of exercise against DOX-induced cardiomyopathy

[7,65].

3.2. Acute exercise

There is evidence that acute exercise can also antagonize cardiac

damage from several deleterious stimuli, since data have revealed that

a single endurance exercise bout preserves ischemia–reperfusioninduced

cardiac oxidative damage [61,88] and decreases infarct size

[91].

With respect to the cross tolerance effects of acute exercise against

DOX-related cardiotoxicity, Wonders et al. [11] reported that the

hemodynamic impairment observed after acute DOX administration

was attenuated when an acute bout of treadmill running was

performed 24 hours prior to DOX injection. In this study, the observed

cardioprotection induced by acute exercise in hearts from DOX

treated rats was associated with inhibition of lipid peroxidation.

In an attempt to understand possible mitochondrial mechanisms

behind the protection triggered by acute exercise, recent data from

our lab showed that an acute bout of treadmill exercise protects

against cardiac mitochondrial dysfunction as well, preserving mitochondrial

phosphorylation capacity and attenuating DOX-induced

decreased tolerance to MPT induction [31]. It was observed that acute

exercise prevented the decreased cardiac mitochondrial function


8 A. Ascensão et al. / International Journal of Cardiology 156 (2012) 4–10

Fig. 2. Scheme summarizing the described adaptations induced by chronic physical exercise against heart mitochondrial dysfunction induced by DOX treatment. The marked

mitochondrial morphological disturbances, increased levels of mitochondrial oxidative capacity, oxidative damage and apoptosis induced by DOX administration are suggested to be

limited by up-regulated antioxidant and anti-apoptotic capacity stimulated by exercise. These adaptations attenuate cardiac histological alterations suggestive of severe damage,

increase oxidative capacity and result in less degree of oxidative damage and apoptosis.

detected as impaired state 3, phosphorylative lag-phase, maximal

transmembrane potential and calcium-induced MPT pore opening

caused by DOX. Exercise also prevented the inhibitory effects of DOX

treatment on the activity of cardiac mitochondrial respiratory chain

complexes I and V, and on increased caspase-3 and -9 activity. Given

the recent results associating variations in the expression of some

known MPT pore component and/or sensitizing proteins such as Cyp

D, ANT and VDAC with the resistance level to MPT pore induction and

apoptosis in several models [24,92–95], these proteins as well as the

Bax/Bcl-2 ratio were determined with no differences between groups

detected. It was hypothetically proposed that the protection observed

was related to possible coordinated up-regulation of stress chaperones

and/or antioxidants. Regardless of the controversy, it is still

recognized that some HSP are up-regulated in the cardiac tissue in

response to endurance exercise [96], possibly contributing to the

preservation of integrity and activity of mitochondrial complexes, as

described above. In addition, it has also been described that acute

endurance exercise increases cardiac uncoupling proteins [97], which

may likely contribute to diminish free radical production by causing a

slight dissipation of membrane potential, a condition termed mild

uncoupling [98] and translocation of fatty acid peroxides from the

inner to the outer membrane leaflet [99]. Further research is

warranted in order to clarify the exact mechanisms by which an

acute exercise bout prior to DOX treatment induces a protective

phenotype in cardiac mitochondria.

4. Concluding remarks

It seems clear that physical exercise positively modulates

physiological mechanisms and elevates some important cardiac

defense systems to antagonize the toxic effects caused by DOX

treatment (Fig. 2). The up-regulation of antioxidant capacity seems to

be the most consensual mechanism for the cardioprotection observed.

Additional beneficial adaptations that promote a cardiac phenotype

which renders the heart more resistant to the deleterious effects of

DOX administration may include increased expression of HSP and

other anti-apoptotic proteins. A central role in this process should be

attributed to mitochondrial plasticity, as mitochondrial adaptations

resulting from physical exercise could potentially be beneficial to the

cardiac tissue in the setting of the DOX cardiomyopathy, a condition in

which acute and chronic exercise were shown to be protective.

Further research should be accomplished in order to better

comprehend the precise mechanisms behind the referred cardioprotection

afforded by exercise. Possible target transcripts and related

proteins associated with increased mitochondrial and tissue defense

systems modulated by physical exercise may be interesting candidates

to counteract DOX cardiac side effects. The interaction of

physical exercise with the reported paths of cell death involved in

DOX toxicity should also be explored.

Acknowledgments

António Ascensão and José Magalhães are supported by Pos-docs

grants from the Portuguese Foundation for Science and Technology

(FCT) (SFRH/BPD/4225/2007 and SFRH/BPD/66935/2009, respectively).

The present work was supported by research grants from the FCT

to António Ascensão (PTDC/DES/113580/2009) and Paulo Oliveira

(PTDC/SAU-OSM/104731/2008).

The authors of this manuscript have certified that they comply

with the Principles of Ethical Publishing in the International Journal of

Cardiology [100].

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Abstract presented in: Jornadas de Bioquímica, University of Coimbra, Portugal, November 25, 2012.

PAULO J. OLIVEIRA

CNC – Center for Neuroscience and Cell Biology, University of Coimbra, Portugal

Email address – pauloliv@ci.uc.pt

From the production of energy to the control of cell death pathways, calcium homeostasis, intracellular

signaling and intermediate metabolism, mitochondria are remarkable dynamic structures. Moreover,

mitochondria are involved in the toxicity of several xenobiotics, which cause adverse reactions in humans.

Drug or disease-induced mitochondrial dysfunction has a larger impact on organs with higher energy

requirements, such as the cardiac and skeletal muscles and the central nervous system. Several clinically

used drugs target mitochondria in the cardiovascular system, which provide the basis for their

pharmacological and/or toxicity effects. We will make the case for Doxorubicin, an anti-cancer agent which

causes a dose-dependent and cumulative cardiac toxicity in which mitochondria is suggested to play a

critical role. We will also show examples of how mitochondrial-directed agents can be useful as

antioxidants and how physical activity can be an effective non-pharmacological strategy to boost

mitochondrial power and afford cross-tolerance against several deleterious insults to the cardiac muscle.

In the second part of the talk, we will focus on how mitochondrial metabolism is altered in cancer and the

consequences for the pathophysiology of the disease. Mitochondrial remodeling in cancer can also be

used to specifically direct molecules which exploit particular alterations observed in cancer cell

mitochondria. We will focus here on molecules that accumulate in cancer cell mitochondria due to their

higher transmembrane electric potential.

Mitochondrial targeting of pharmaceuticals is thus a growing field, although there is a very fine line

between targeting to heal and targeting to injury.

Acknowledgements: Work in the author´s laboratory is sponsored by the Portuguese Foundation for

Science and Technology, co-funded by COMPETE/FEDER/National Budget (grants # PTDC/QUI-

QUI/101409/2008, PTDC/SAU-OSM/104731/2008, PTDC/SAU-TOX/110952/2009 and PTDC/SAU-

TOX/117912/2010).


Mitochondrionopathy Phenotype in Doxorubicin-

Treated Wistar Rats Depends on Treatment Protocol and

Is Cardiac-Specific

Gonçalo C. Pereira 1 , Susana P. Pereira 1 , Claudia V. Pereira 1 , José A. Lumini 2,3 , José Magalhães 2 ,

António Ascensão 2 , Maria S. Santos 1 , António J. Moreno 4 , Paulo J. Oliveira 1 *

1 Department of Life Sciences, Center for Neuroscience and Cell Biology (CNC), University of Coimbra, Coimbra, Portugal, 2 Faculty of Sport Sciences, Research Centre in

Physical Activity, Health and Leisure, University of Porto, Porto, Portugal, 3 Faculty of Health Sciences, University of Fernando Pessoa, Porto, Portugal, 4 Department of Life

Sciences, Institute for Marine Research, University of Coimbra, Coimbra, Portugal

Abstract

Although doxorubicin (DOX) is a very effective antineoplastic agent, its clinical use is limited by a dose-dependent,

persistent and cumulative cardiotoxicity, whose mechanism remains to be elucidated. Previous works in animal models

have failed to use a multi-organ approach to demonstrate that DOX-associated toxicity is selective to the cardiac tissue. In

this context, the present work aims to investigate in vivo DOX cardiac, hepatic and renal toxicity in the same animal model,

with special relevance on alterations of mitochondrial bioenergetics. To this end, male Wistar rats were sub-chronically

(7 wks, 2 mg/Kg) or acutely (20 mg/Kg) treated with DOX and sacrificed one week or 24 hours after the last injection,

respectively. Alterations of mitochondrial bioenergetics showed treatment-dependent differences between tissues. No

alterations were observed for cardiac mitochondria in the acute model but decreased ADP-stimulated respiration was

detected in the sub-chronic treatment. In the acute treatment model, ADP-stimulated respiration was increased in liver and

decreased in kidney mitochondria. Aconitase activity, a marker of oxidative stress, was decreased in renal mitochondria in

the acute and in heart in the sub-chronic model. Interestingly, alterations of cardiac mitochondrial bioenergetics co-existed

with an absence of echocardiograph, histopathological or ultra-structural alterations. Besides, no plasma markers of cardiac

injury were found in any of the time points studied. The results confirm that alterations of mitochondrial function, which are

more evident in the heart, are an early marker of DOX-induced toxicity, existing even in the absence of cardiac functional

alterations.

Citation: Pereira GC, Pereira SP, Pereira CV, Lumini JA, Magalhães J, et al. (2012) Mitochondrionopathy Phenotype in Doxorubicin-Treated Wistar Rats Depends on

Treatment Protocol and Is Cardiac-Specific. PLoS ONE 7(6): e38867. doi:10.1371/journal.pone.0038867

Editor: Valdur Saks, Université Joseph Fourier, France

Received March 2, 2012; Accepted May 13, 2012; Published June 22, 2012

Copyright: ß 2012 Pereira et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits

unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: The present work was funded by the Foundation for Science and Technology (FCT), Portugal: research grants PTDC/SAU-OSM/64084/2006 and PTDC/

SAU-OSM/104731/2008 (PJO), through FEDER/COMPETE/National funds; Post-Doc fellowships SFRH/BPD/42525/2007 to AA and SFRH/BPD/66935/2009 to JM;

and, Ph.D. fellowships SFRH/BD/30906/2006 to JAL, SFRH/BD/36938/2007 to GCP, SFRH/BD/48029/2008 to CVP, SFRH/BD/64247/2009 to SPP. The funders had no

role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: pauloliv@ci.uc.pt

Introduction

Doxorubicin (DOX) is an anthracycline antibiotic drug which

has been effectively and widely used in the clinic to treat several

types of human and non-human cancers [1]. However, adverse

side effects can be observed during or after treatment cessation,

with a dose dependent and cumulative cardiotoxicity being the

most complex and difficult to manage event [1,2]. In fact, the

associated risk of developing congestive heart failure was one of the

main reasons that lead to limitation of the maximum allowed

dosage during DOX treatment [3].

Acute cardiac toxicity occurs early during the treatment and

usually includes myopericardits, sinus tachycardia, reversible

arrhythmias, prolonged QT interval and flattening of the T wave,

being easily manageable and disappearing once the treatment is

ceased [2]. Alternatively, patients may develop chronic cardiotoxicity

which can appear right after the end of the treatment or even

years later [2,4]. Unlike acute toxicity, the dose-dependence

together with its difficult early detection makes chronic toxicity a

life-threatening and largely uncontrolled condition.

The mechanisms underlying DOX-selective cardiotoxicity have

been the focus of interest in the last four decades, but there is

hardly any consensual conclusion. Nevertheless, it is accepted that

DOX antitumor activity is completely independent from cardiac

toxicity, which may involve disruption of mitochondrial function

[2,5]. Distinctive features of DOX-induced mitochondrial dysfunction

in the cardiac tissue include inhibition of oxidative

phosphorylation, decreased calcium-loading capacity and increased

reactive oxygen species (ROS) production [2,5,6].

Despite the fact that DOX toxicity has been described as being

cardiac selective, none of the hypotheses proposed to date [2] fully

explains the pronounced cardiac effects when compared with

other vital tissues. Although several animal models have been

generated to investigate either sub-chronic [7,8,9,10,11] or acute

[12,13,14] DOX toxicity, one limitation of the majority of studies

is that only one organ was investigated. Thus, comparing DOX

multi-organ effects from different reports becomes non-accurate,

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Mitochondrial Doxorubicin Toxicity in Wistar Rats

confusing and sometimes contradictory since animals species,

strain, age and treatment protocols differ from publication to

publication. By searching PubMed for reports using the keywords

‘‘mitochondria’’ and ‘‘doxorubicin’’ and narrowing the search to

works performed in animal models and in three distinct tissues

(heart, liver and kidney, Table S1), only 16 hits were obtained (Fig.

S1). These studies investigated DOX toxicity in the three tissues

harvested from the same experimental protocol. However, except

for two relatively recent works, most reports originated in the 80 s

and did not compare mitochondrial dysfunction with pathophysiological

state, genetics, metabolomics or proteomics, which we

believe are critical to better understand DOX-induced tissue

toxicity. In an attempt to increase the knowledge regarding DOXinduced

selective cardiotoxicity, the objective of the present work

was to measure alterations of mitochondrial bioenergetics in the

heart, liver and kidney after two distinct treatment protocols (acute

vs. sub-chronic) in male Wistar rats. Mitochondrial function endpoints

were associated with tissue histological alterations (all three

tissues) and function (heart). Our hypothesis is that alterations of

mitochondrial bioenergetics occur predominantly in the heart and

are an early and sensitive marker of DOX-induced toxicity,

occurring even in the absence of histological alterations. A second

tandem hypothesis is that cardiac mitochondrial toxicity is

detectable only in the sub-chronic treatment.

The two distinct treatment protocols used in the present work

were already reported in the literature [13,15] and are accepted

models to investigate DOX-mitochondrionopathy. Note that

accordingly to a reported equation [14], the actual total dosages

of our treatment protocols are slightly below the maximum dosage

allowed in human chemotherapy [16], as our objective was not to

induce substantial tissue damage but to mimic biochemical and

functional alterations that are usually observed in DOX-treated

patients.

Materials and Methods

Reagents

DOX hydrochloride, (7S,9S)-7-[(2R,4S,5S,6S)-4-amino-5-hydroxy-6-methyloxan-2-yl]oxy-6,9,11-trihydroxy-9-(2-hydroxyacetyl)-4-methoxy-8,10-dihydro-7H-tetracene-5,12-dione

hydrochloride,

chemical purity $98%, was obtained from Sigma-Aldrich

Quimica SA (Sintra, Portugal) and prepared in a sterile saline

solution, NaCl 0.9% (pH 3.0, HCl) and stored at 4uC for no

longer than five days upon rehydration. All other chemicals were

of the highest grade of purity commercially available. Aqueous

solutions were prepared in ultrapure (type I) water (Milli-Q Biocel

A10 with pre-treatment via Elix 5, Millipore, Billerica, MA, USA).

For non-aqueous solutions, ethanol (99.5%, Sigma-Aldrich

Quimica SA, Sintra, Portugal) was used as solvent.

Animal Care

Animal handling was performed in accordance with the

European Convention for the Protection of Vertebrate Animals

used for Experimental and Other Scientific Purposes (CETS

no.123) and Portuguese rules (DL 129/92). The procedures were

approved by the CNC Committee for Animal Welfare and

Protection. Animal handlers and the authors GCP, SPP and PJO

are credited by the European Federation for Laboratory Animal

Research (FELASA) category C for animal experimentation

(accreditation no. 020/08). Fourteen weeks of age (acute protocol)

or 6 weeks of age (sub-chronic protocol) male Wistar rats,

Crl:WI(Han), were purchased from Charles River (France),

acclimated for 10–14 days prior to the initiation of experiments

and maintained in the local animal house facility (CNC – School

of Medicine, University of Coimbra, Coimbra, Portugal). Animals

were group-housed in type III-H cages (Tecniplast, Italy) with

irradiated corn cob grit bedding (Scobis Due, Mucedola, Italy) and

environmental enrichment and under controlled environmental

requirements (22uC, 45–65% humidity, 15–20 air changes/hour,

12 h artificial light/dark cycle, noise level ,55 dB) and free access

to standard rodent food (4RF21 GLP certificate, Mucedola, Italy)

and ad libitum acidified water (at pH 2.6 with HCl).

Experimental Design

For the acute study, the experimental protocol was initiated

with 16 weeks old rats (N = 34), randomly allocated in pairs and

administered with either DOX (20 mg Kg 21 of body weight, i.p.,

n = 17) or with an equivalent volume of vehicle solution (NaCl

0.9%, i.p., n = 17) exactly 24 hours before sacrifice, as previously

described [13].

For the sub-chronic study, the experimental protocol was

performed with 8 weeks old rats (n = 40) randomly grouped in

pairs and weekly injected with a subcutaneous injection in the

scruff or flank of either DOX (2 mg kg 21 , n = 20) or equivalent

volume of vehicle solution (NaCl 0.9%, s.c., n = 20) during seven

weeks, and sacrificed one week after the last injection, as

previously described [15].

All animals were injected during the light phase of the cycle,

observed daily and weighed at the beginning and at the end of the

experimental treatment period, being also weekly weighed at the

time of injection. Animals were euthanized in pairs by cervical

dislocation followed by decapitation, to confirm death and

exsanguination. Blood was collected for further biochemical

analysis. Rats were sacrificed between 9:00 and 10:00 AM to

eliminate possible effects due to diurnal variation and were not

fasted before sacrifice. Organs were immediately extracted from

the body and quickly washed in appropriate buffer before being

weighed.

Blood Analysis

Blood was collected after decapitation to sterile tubes without

additives. After blood clot formation, serum was separated by

centrifugation at 1,6006g during 10 minutes at 4uC (Sigma 3–

16 K, 1333 rotor). The supernatant was then transferred to

microtubes and centrifuged at 16,0006g, 20 minutes at 4uC

(Eppendorf 5415 R, FL062 rotor). Serum samples were maintained

for a short time at 4uC for analysis by an external certified

laboratory, or stored frozen at 280uC for troponin I (TnI)

analysis. The latter was performed by external trained personal

one month after collection using the singleplex Rat Cardiovascular

Panel (RCVD1-89K, Millipore, Arium, Portugal). Blood analyses

were performed by blinded operator.

Histological Analysis

Organs were immersion-fixed in Bouin’s solution for 24 h and

then washed with 70% alcohol until the solution became clear and

stored in 70% alcohol until the histological analysis was

performed. At that time, after several incubations with increasing

alcohol percentages (70%, 90%, and 100%) and xylol, tissues were

processed using a normal paraffin procedure and sectioned (3 mm

thick). The sections were then de-paraffinized with xylol and

incubated with solutions containing decreasing alcohol content

(100% and 95%). All slides were stained with hematoxilin and

eosin (HE) by using standard procedures. The samples were

covered with coverslips in Eukitt mounting medium and then

visualized in a Nikon Eclipse 80I microscope coupled with a

camera and computer. Morphological assessment was conducted

in a ‘‘blind’’ fashion by a certified professional.

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Mitochondrial Doxorubicin Toxicity in Wistar Rats

Electron Microscopy

After organ harvesting and washing, a small slice (2–5 mm) was

cut and fixed in 3% glutaraldehyde in phosphate buffer

(100 mM NaH 2 PO 4 , pH 7.3), postfixed in 1% osmium tetroxide

in same buffer and dehydrated in solutions containing increasing

alcohol percentages (70%, 90%, and 100%) before being

embedded in Spurr’s resin. Ultrathin sections were obtained with

an LKB ultra-microtome Ultrotome III (GE Healthcare, Little

Chalfont, Buckinghamshire, UK), stained with methanolic uranyl

acetate followed by lead citrate, and examined with a JEOL Jem-

100SX electron microscope (JEOL, Tokyo, Japan) operated at

80 kV. The operator was blinded to treatment groups and took 5

to 10 micrographs of random fields.

Echocardiogram

Echocardiograms were performed under the same specifications

as previously described [17]. Five days after the last injection, subchronically-treated

animals, free of anesthesia, were examined

lying in the left lateral decubitus position and using a commercial

available echocardiograph system (VIVID i, G.E. Helthcare),

equipped with an 11.5 MHz transducer. Every parameter was

measured accordingly to the American Society for Echocardiography

guidelines (www.asecho.org/guidelines/) and results were

directly obtained from the equipment software by a cardiologist

blinded for treatment groups. Left ventricular mass (LV mass) was

calculated using a standard cube formula which assumes a spherical

left ventricular geometry [18] according to the following equation:

LVmass~1:04|½(LVDdzLVPWzIVS) 3 {LVDd 3 Š. Results

are expressed in mg and 1.04 represents the specific gravity of

muscle. Only four animals of each group were analyzed due to

limitations in the time slot available for the cardiologist/apparatus

used.

Isolation of Mitochondrial Fractions

Mitochondria were isolated by the standard procedure usually

used in our laboratory [19,20,21]. Organs were excised and finely

minced in an ice-cold isolation medium containing 250 mM

sucrose, 10 mM HEPES, 1 mM EGTA and 0.1% defatted BSA

(pH 7.4, KOH). For the isolation of cardiac mitochondrial

fractions, the isolation medium was supplemented with 0.5 mg/

mL of protease (Subtilisin A, Type VIII from Bacillus licheniformis,

Sigma-Aldrich). The mitochondrial protein after isolation was

quantified by the biuret method using bovine serum albumin as a

standard [22] and mitochondrial preparation was kept on ice

during experiments, which were carried out after a 20 min

recovery and within 5 hours post-isolation.

Oxygen Consumption

Oxygen consumption of isolated mitochondria was monitored

polarographically with a Clark oxygen electrode connected to a

Kipp and Zonen recorder in a 1 mL thermostatic, water-jacketed

open chamber with magnetic stirring at 30uC, simultaneously with

mitochondrial membrane potential measurements (see 2.10). The

standard respiratory medium consisted of 130 mM sucrose,

10 mM EGTA, 50 mM KCl, 5 mM H 2 PO 4 , 5 mM HEPES

(pH 7.3, KOH) and supplemented with 2.5 mM MgCl 2 for liver

and kidney. Mitochondria were suspended at a concentration of

0.5 (heart) or 1.0 (liver and kidney) mg protein/mL in the

respiratory medium and mitochondrial state 2 respiration was

initiated with 5 mM glutamate plus malate (mitochondrial

energization through complex I) or 5 mM succinate in the

presence of 2 mM rotenone (mitochondrial energization through

complex II). Adenosine diphosphate (ADP) (225–250 nmol) was

added to initiate state 3 respiration. State 4 respiration was defined

as oxygen consumption after ADP consumption. The respiratory

control ratio (RCR) was calculated as the ratio between state 3

over state 4 and it is an indicator of oxidative phosphorylation

coupling and mitochondrial membrane integrity. ADP/O ratio,

which is expressed as the ratio between the amount of ADP added

and oxygen consumed during state 3 respiration, is an index of

oxidative phosphorylation efficiency. Respiration rates were

calculated considering that the saturation oxygen concentration

was 236 mM at30uC.

Mitochondrial Transmembrane Electric Potential

The mitochondrial transmembrane electric potential (DY max )

was monitored by tetraphenylphosphonium ion (TPP + ) distribution

(see 2.9), by using a TPP-selective electrode in combination

with a Ag/AgCl saturated reference electrode, as previously

described [21]. The difference in potential between the selective

TPP + electrode and the reference electrode was measured with a

potentiometer and continuously recorded in a Kipp and Zonen

recorder (model BD 121; Kipp & Zonen B.V., Delft, Netherlands).

Experimental conditions were the same as for oxygen consumption

assays with the inclusion of 3 mM TPP + in the reaction media.

Absolute DY max values (mV) were determined from the equation

originally proposed by Kamo [23], assuming Nernst distribution of

the ion across the membrane electrode. No correction was made

for the ‘‘passive’’ binding of TPP + to mitochondrial membranes

because the purpose of the experiments was to show relative

changes in potential rather than absolute values. As a consequence,

some overestimation for the DY max values may be

anticipated. A matrix volume of 1.1 mL/mg protein was assumed.

Aconitase Activity

Mitochondrial protein (200 mg) was diluted in 0.6 ml buffer

containing 50 mM Tris–HCl (pH 7.4) and 0.6 mM MnCl 2 and

sonicated for 10–20 seconds, followed by centrifugation at

16,0006g for 5 minutes. Aconitase activity was immediately

spectrophotometrically measured (Jasco V-560, Jasco Europe,

Milan, Italy) by monitoring the formation of cis-aconitate from

isocitrate at 240 nm in 50 mM Tris-HCl (pH 7.4) containing

0.6 mM MnCl 2 and 20 mM isocitrate at 25uC [24]. All assays

were performed in triplicate. Enzyme activity was calculated by

using the mean of the slopes of the three replicates, obtained

before the record reached a plateau. Results were expressed as

percentage of control, which was 174.0627.8 nmol mg protein 21 -

min 21 , 258.3632.4 nmol mg protein 21 min 21 and

245.2631.1 nmol mg protein 21 min 21 for heart, liver and kidney,

respectively, using e 240 = 3.6 mM 21 cm 21 [24].

Statistical Analysis

All data was assessed for normality using the Kolmogorov-

Smirnov test with Dallal-Wilkinson-Lilliefor correction and for

equality of variances using the F test. Since group sample sizes are

equal and the parametric statistical tests applied in this work are

robust for moderate deviations from homoscedasticity [25],

parametric tests were still applied when homoscedasticity was

not observed. However, when data normality was rejected, a

squared-root, logarithmic or reciprocal transformation was applied

in an attempt to achieve normality. If data still rejected normality,

the correspondent non-parametric statistical test was used.

Nevertheless, data is presented to the reader in non-transformed

values for ease of comprehension. In the text, data is expressed as

percentage of the difference of means plus its standard error or as

percentage of the means difference plus its standard error for data

related to isolated mitochondrial fractions. In both situations, the

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Mitochondrial Doxorubicin Toxicity in Wistar Rats

percentage value shown regards the saline group in the same

experimental model. Statistical significance between means was

determined using two-tailed Student’s t test. When homoscedasticity

was not found, Welch’s correction was applied and in the

absence of normality, the Mann-Whitney test was used instead. To

exclude the random effect associated with daily mitochondrial

isolation and electrode variability, a matched pairs Student’s t test

or its non-parametric correspondent Wilcoxon matched pairs test

were performed. Differences were considered significant at 5%

level and p value was categorized accordingly to their interval of

confidence. Statistical analyses were performed using Graph Pad

Prism version 5.0 (GraphPad Software, Inc., San Diego, CA,

USA).

Results

Animal and Organs Mass

Control and DOX-treated animals in the acute model did not

show alterations in their body mass in the 24 hours subsequent to

DOX administration (Table 1). However, when animals were

individually analyzed, i.e. when the mean of the arithmetic

difference between initial and final body mass of every individual

was calculated for the first 24 h, the control group showed a body

mass variation of 20.3561.02 g (n = 17) while variation in DOXtreated

group, 215.1 g 61.7 g (n = 17) representing about 4% of

total body mass, was significantly lower (p,0.001). Surprisingly, a

decrease of 7.662.7% in heart mass was observed 24 h after the

single DOX injection but no alteration was observed in other

tissues analyzed (1.664.4% increase in liver and 3.163.9%

decrease for kidney, Table 1). Therefore, heart mass over body

mass ratio showed a significant decrease of 5.862.3% while no

changes were found for liver or kidney (3.662.6% increase and

1.463.6% decrease, respectively; Table 1).

Treatment of Wistar rats with seven weekly DOX injections

caused a significant reduction in body mass gain of animals, as

seen by the difference of body mass values between groups

(13.862.1%; Table 1). While the body mass variation of the

control group during the treatment showed an increase of

49.162.9%, the DOX-treated group only increased body mass

by 31.263.1%, which can be easily observed in the body mass

gain profile depicted in Fig. 1. However, it was interesting to

observe that the alteration in heart mass detected in the acute

model was not present in the sub-chronic protocol at the end of the

treatment period (Table 1). Since only a non-significant decrease

of all tissues (2.465.6%, 3.264.0% and 5.263.6% for heart, liver

and kidney, respectively) was observed, the ratio of organ mass to

body mass was increased in all analyzed tissues (13.766.6%,

12.463.5% and 10.064.1% for heart, liver and kidney, respectively;

Table 1). The results reflect a change in body mass rather

than alterations in tissue mass.

It should be noticed that although the objective of the

experimental protocol was to induce a sub-chronic response and

therefore a lower rate of mortality, one animal died during DOX

treatment (marked with an arrow, Fig. 1). This treated animal

received all seven DOX injections and died precisely one week

after the last. Nonetheless, the animal did not show any distinct

sign of distress or illness when compared with its counterparts. A

standard necropsy was performed in the deceased animal but no

particular abnormality was perceived, including heart hypertrophy

or ascites. Liver and kidney tissues appeared normal in size,

morphology and color. The only thing to point out was the fact

that this particular animal showed the larger decrease in weight.

Therefore, the mortality rate associated with the present study is

5% (1 out of 20).

Table 1. Body and organs mass profile of animals subjected to DOX treatment protocols.

Initial body

mass (g) Final body mass (g) Heart mass (g) HM:BM (61000) Liver mass (g) LM:BM (61000) Kidney mass (g) KM:BM (61000)

Model Treatment

Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE

Acute Saline (n = 17) 377.5 7.8 377.2 7.9 0.972 0.020 2.58 0.05 11.6 0.3 30.8 0.05 2.19 0.05 5.84 0.02

DOX (n = 17) 385.1 9.7 370.0 9.6 0.898** 0.018 2.44* 0.04 11.8 0.4 32.9 0.07 2.13 0.07 5.76 0.02

Sub-Chronic Saline (n = 19) 263.0 5.0 391.8 5.9 1.25 0.04 3.20 1.29 12.3 0.4 31.4 0.07 2.50 0.06 6.40 0.02

DOX (n = 19) 255.4 5.3 337.4*** 5.8 1.22 0.05 3.64* 1.65 11.9 0.3 35.3** 0.08 2.36 0.06 7.03* 0.02

Data refers to wet organ mass and its ratio to body mass was obtained dividing the organ mass over the respective total animal mass times 1000. The deceased DOX-treated rat and its matched control in the sub-chronic model

were excluded from this analysis. Differences between treatment groups means within the same model were evaluated by Student’s t test (see Material and Methods for detailed information).

*p#0.05;

**p#0.01;

***p#0.001 vs saline group of the same treatment protocol. HM:BM – heart mass to body mass ratio; LM:BM – liver mass to body mass ratio; KM:BM – kidney mass to body mass ratio; SE – standard error.

doi:10.1371/journal.pone.0038867.t001

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Mitochondrial Doxorubicin Toxicity in Wistar Rats

Figure 1. DOX decreases body mass gain over time in a subchronic

toxicity model. After the fourth injection, the body mass of

DOX-treated animals started to be distinctly different from the salinetreated

and therefore the growth profile is dramatically changed at the

end of treatment. Only one DOX-treated animal died during the

protocol (indicated by the black arrow). Animals in the control group

are depicted by open circles while DOX-treated animals are in full

circles. Lines represent the means of each group at each time point. S –

sacrifice time-point.

doi:10.1371/journal.pone.0038867.g001

Serum Biochemistry

Despite the fact that lactate dehydrogenase (LDH), a general

marker for tissue damage, is not altered (21.8614.0%; Table 2) in

the acute model, the non-specific hepatic marker, aspartate

aminotransferase (AST), is markedly elevated 91.0617.2%. Serum

creatine kinase is unchanged (1.7616.0%) as well as markers of

renal function, namely urea, creatinine, uric acid or blood urea

nitrogen (BUN). The observed high levels of the hepatic-specific

marker, alanine aminotransferase (ALT), 141626%, together with

the lower AST to ALT ratio (15.367.5%) and total protein levels

(TP; 9.861.7%) suggest impaired liver function in DOX-treated

animals. Increased cardiac injury, as evaluated by measuring

troponin I (TnI) was not significantly increased in the acute model

(value increased 15611% vs. control). Regarding circulatingblood

lipids, triglycerides were decreased by 53613% 24 hours

after the acute treatment but no alteration was found in total

cholesterol (4.667%).

Notwithstanding, chronically-DOX-treated rats were hyperlipidemic

with the treated group showing both an increase in

triglycerides and cholesterol (196640% and 127624%, respectively;

Table 2). TnI levels were also increased by 11612%,

although the difference was not statistically significant. Interestingly,

all other parameters analyzed were significantly decreased in

comparison to control animals (average of the parameters

variation of 17.3%, ranging from 8.7% to 31.4%), with the

exception of LDH, uric acid and transaminases ratio, which,

despite being also decreased in the DOX-treated group, did not

reach statistical significance when compared with the saline group.

Echocardiography

Animals from sub-chronic DOX treatment were submitted to

an echocardiogram 5 days after the 7 th injection in order to

evaluate cardiac morphology and function parameters. No

abnormality was found in the four animals analyzed (Table 3).

Thickness of the walls and left ventricular diameter, ejection

fraction and fraction shortening were not different from the

Table 2. Blood plasma profile after DOX treatment.

BUN

(mg/dL)

UA

(mg/dL)

Urea

(mg/dL)

CREA

(mg/dL)

CHOL

(mg/dL) AST (U/L) ALT (U/L) AST/ALT TP (g/dL) LDH (U/L)

TRIG

(mg/dL)

TnI #

(ug/L)

Model Treatment CK (U/L)

Acute Saline (n = 15) 1303961555) 915.9661.5) 114.5614.3) 38.862.0) 178.7615.3) 57.163.0) 3.1160.18) 71.460.9) 25076224) 0.5960.02) 35.061.8) 1.7160.14) 16.360.9)

DOX (n = 15) 1281661564) 1053.0683.7) 54.1*** 64.2) 40.661.9) 341.0*** 626.9) 137.8*** 614.6) 2.64* 60.15) 64.4*** 60.9) 30556271) 0.5760.02) 34.561.3) 1.6660.09) 16.160.6)

Sub-Chronic Saline (n = 18) 913061007) 840.3657.4) 126.968.9) 41.061.2) 183.1610.6) 51.562.5) 3.6360.20) 71.560.6) 42246432) 0.6460.01) 38.860.7) 1.6360.12) 18.160.3)

DOX (n = 18) 6262* 6709) 932.6686.1) 376.3*** 649.6) 93.1*** 69.7) 139.6** 65.0) 43.9* 61.5) 3.2060.10) 62.3*** 60.9) 35616497) 0.58** 60.01) 33.7*** 60.7) 1.3960.09) 15.7*** 60.3)

# For troponin I analysis, only 13 and 16 samples were measured in acute and sub-chronic protocol, respectively due to limitations in the analytical kit used.

Differences between treatment groups means within the same model were evaluated by Student’s t test, when assumptions were not achieved a Welch correction or the non-parametric Mann-Whitney test were applied (see

Material and Methods for detailed information).

*p#0.05;

**p#0.01;

***p#0.001 vs saline group of the same model. List of abbreviations: CK – creatine kinase; TnI – troponin I; TRIG – triglycerides; CHOL – total cholesterol; AST – aspartate aminotransferase; ALT – alanine aminotransferase; TP – total

serum proteins; LDH – lactate dehydrogenase; CREA – creatinine; UA – uric acid; BUN – blood urea nitrogen; SE – standard error.

doi:10.1371/journal.pone.0038867.t002

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Mitochondrial Doxorubicin Toxicity in Wistar Rats

Table 3. Echocardiogram parameters in the sub-chronic protocol.

IVS (mm)

LPWT

(mm) LVDd (mm) LVDs (mm)

LV mass

(mg) LVEF (%) FS (mm) AT s/d (bpm)

Model Treatment Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE

Sub-Chronic Saline (n = 4) 1.49 0.05 1.54 0.05 5.02 0.21 2.44 0.13 416.3 44.7 87.3 0.8 51.2 1.1 520.8 15.1

DOX (n = 4) 1.50 0.03 1.55 0.02 5.00 0.22 2.45 0.16 411.5 23.3 87.0 1.2 51.2 1.3 538.3 16.0

Differences between treatment groups were evaluated by non-parametric Mann-Whitney test due to their lack of normality (see Material and Methods for detailed

information). IVS – interventricular septum; LPWT – left posterior wall thickness; LVDd – left ventricular diastolic dimension; LVDs – left ventricular systolic dimension;

LVEF – left ventricular ejection fraction; FS – fraction shortening; AT s/d – arterial tension systole/diastole.

doi:10.1371/journal.pone.0038867.t003

control group at this time point (average of the parameters

variation of 0.25%, ranging from 0.16 to 0.40%).

Histopathology and Ultrastructure Analyses

When analyzing at least three different samples from each tissue

and from each treatment protocol, no obvious sign of damage or

morphological alterations were found after histological analysis

(Fig. 2). Hearts from treated animals in both treatment protocols

did not present signs of fibrosis or any hallmarks of later stages of

DOX toxicity. However, in the representative pictures of cardiac

slices, minor cytoplasmic vacuolization in the acute model (Fig. 2,

Panels A and B) and also minor increase in cellular volume in the

sub-chronic model (Fig. 2, Panels C and D) can be observed.

Livers were morphologically normal; however, minor centrilobular

dilation was observed, while hepatocytes showed citoplasmic

heterogeneity due to vacuolization. Nevertheless, the vacuolization

was more prominent in slices from the sub-chronic model (Fig. 2,

Panels E–H). When renal slices were observed with hematoxylin

eosin (HE) stain, no differences were found between control and

treated group regardless of the treatment protocol used (Fig. 2,

Panels I–L).

In terms of tissue ultrastructure, electron micrographs of cardiac

slices from acutely treated animals (Fig. 3, Panel B and D) showed

a cellular structure not dissimilar to control (Fig. 3, Panel A and

C). The myofibrillar disorganization, cytoplasm vacuolization and

swollen mitochondria usually observed after DOX treatment in

other rodent models [26,27] were not present in the acute

treatment model (Fig. 3, Panels A–D). In fact, myofibrillar Z-

bands were well defined and with narrow A-bands (Fig. 3, Panels

C and D). Likewise, kidney electron micrographs were similar to

control counterparts (Fig. 3, Panels H–K). However, hepatic slices

presented more heterogeneous cytoplasm with high numbers of

vacuoles and lipid-like droplets (Fig. 3, Panel E). Moreover, liver

mitochondria from DOX acute treated animals appear to be

preferentially in the condensed conformation (Fig. 3, Panel G)

rather than the orthodox one observed in control micrographs

(Fig. 3, Panels D and F).

Regarding the sub-chronic model (Fig. 3, Panels L-X), cardiac

samples from treated animals also showed normal sarcomeres with

well-defined Z-bands and organized myofibrils (Fig. 3, Panels A–

D). Nevertheless, the cytoplasm appeared to present more

vacuolization (Fig. 3, Panel M) although mitochondrial morphology

was not dissimilar from control micrographs (Fig. 3, Panels L

and N). In liver tissue, the striking evidence is the abundance of

small vacuoles in the cytoplasm of hepatocytes from subchronically

treated animals (Fig. 3, Panel S) and, although

mitochondria appear to be greater in volume in some images,

the overall observation is that they are not different in morphology

when compared with saline treatments (Fig. 3, Panels P and R).

Mitochondria from renal slices appear in an intermediate

conformation between orthodox and condensed forms (Fig. 3,

Panel X).

Mitochondrial Bioenergetics

Mitochondrial bioenergetics was evaluated in the three tissues to

detect distinctive DOX-induced alterations. State 3 respiration

mimics an increase in workload which is observed in vitro after

ADP-induced simulation. State 4 respiration is observed after all

ADP is phosphorylated, representing a steady-state of the

respiratory chain, controlled mostly by the passive and unspecific

diffusion of protons through the inner mitochondrial membrane.

Similarly, the phosphorylation lag phase represents the time

needed for the phosphorylative system to convert all added ADP

to ATP, i.e., the time elapsed to mitochondrial repolarization after

ADP depolarization.

In the acute model, complex I-sustained mitochondrial state 3

respiration was increased in hepatic, decreased in renal and not

altered in cardiac fractions (14.765.5%, 5.362.0% and

4.465.3%, respectively; Fig. 3). However, despite equal variation

patterns observed for the lag phase, ranging from 13 to 17%

between tissues, there was no statistical difference between groups

(Table 4). The previous detected differences for complex I-

sustained mitochondrial state 3 respiration were absent when

substrates for complex II were used (4.166.0%, 5.967.9% and

6.766.7% for heart, liver and kidney, respectively). State 4

respiration, RCR and ADP/O remained unchanged in all

fractions regardless of the respiration substrate used (Figs. 3 and

4). The same was observed for all other parameters related to

mitochondrial membrane potential.

Cardiac mitochondria from sub-chronic DOX-treated animals

presented decreased state 3 respiration when using both respiratory

complexes (15.864.9% and 12.863.6% for complex I and II,

respectively; Fig. 3) and lower state 4 respiration, although only

statistically significant when substrates for complex II were used

(12.066.6% and 19.864.2% for complex I and complex II,

respectively). Likewise and in a complementary manner, lag phase

was increased for both complex I- and complex II-sustained

respiration (10.766.0% and 13.165.5%, respectively; Table 4)

and DY max was only slight, yet significantly, decreased when

substrates for complex II were used (1.160.5% and 2.260.6% for

complex I and II, respectively).

Hepatic and renal mitochondrial fractions behaved similarly,

both having decreased DY max with complex I substrates

(1.960.5% and 1.660.4%, respectively; Table 5). Slower state 3

respiration (6.663.8%) in hepatic fractions and kidney

(12.664.2%) with complex I substrates was also measured. Along

with a decreased state 3 respiration, liver mitochondria also

showed a longer phosphorylative lag phase (27.1611.6%). RCR

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Mitochondrial Doxorubicin Toxicity in Wistar Rats

and ADP/O values were similar between control and treated

group in liver and renal mitochondrial.

Aconitase Activity

Aconitase activity was measured in all mitochondrial fractions

as a marker of oxidative stress [24]. Alterations in aconitase

activity were tissue-specific and treatment-dependent. The only

tissue that showed a decreased enzyme activity in the acute model

was the kidney (27.866.7%) while the activity remained unaltered

in the heart and liver (0.3611.8% and 15612.3%, respectively;

Table 5). Cardiac mitochondrial aconitase in the chronic model

was decreased by 21.567.7% in the DOX-treated group while

activity in the two other organs were unaltered (12.9615.6% and

5.469.0% for liver and kidney, respectively).

Figure 2. Histological analysis of organs collected from rats

treated with DOX. No notorious differences or hallmarks of DOX

toxicity were found in the different tissues in both protocols. Panels

represent HE photographs of random chosen tissues: hearts present

minor cytoplasmatic vacuolization (Panel B) and cytoplasmatic dilatation

(Panel D); liver usually show minor cytoplasmatic vacuolization

(Panel F and H); no changes in kidneys (Panel J and L). Organs were

fixed in Bouin’s solution, processed through standard histological

procedures and stained with HE (for more information, see Material and

Methods).

doi:10.1371/journal.pone.0038867.g002

Discussion

The present work demonstrates that DOX treatment induced

different responses depending on the schedule protocol used.

Transaminases and total serum protein levels suggest that the

acute treatment affects the liver. Minor cytoplasmic vacuolization

observed in histological and electron microscopy of thin slices from

hepatic tissue may also suggest metabolic alterations in hepatocytes,

which are supported by decreased triglyceride levels, despite

no alterations in plasma cholesterol (Table 2). Moreover, the slight

increase of state 3 respiration in the presence of complex I-linked

substrates (Fig. 4), which are the most important in a cellular

context, and adoption of the condensed mitochondrial conformation

as seen by electron microscopy (Fig. 3), support the idea that

hepatocyte metabolism and viability are affected after the acute

treatment. Nevertheless, it is however unclear at the moment if

alterations in lipid metabolism can contribute to worsen the

cardiovascular fitness in treated animals. However, hyperlipidemia

was clearly observable in the sub-chronic treatment and liver

histology showed slightly more vacuolization that in the acute

model (Table 2 and Fig. 2), suggesting that altered lipid

metabolism may be a secondary response to drug treatment.

It is intriguing that sub-chronically-treated animals showed only

increase in plasma lipids, while other parameters and markers

were consistently decreased even those which are usually increased

in chronic exposure to DOX [16,28,29] (Table 2). Nevertheless,

because no substantial alterations in histology and ultrastructure

analysis were also detected (Fig. 2 and 3), we believe that the

organism of treated animals reached a new adaptive steady-state

following sub-chronic DOX toxicity. Nevertheless, organ alterations

may probably exist undetected which may lead to a

disrupted response when subjected to metabolic or physiological

stress.

Another interesting difference between treatments relates to

heart mass which was the only organ in both models to show an

alteration in this parameter. However, it was surprising to observe

a difference only in the acute model since hypertrophy is usually

reported along with DOX-induced cardiotoxicity [1,2,30]. Also

surprising was the 7% decrease in heart mass after 24 h of

treatment (Table 1). One hypothesis relates to apoptotic and/or

necrotic events associated with DOX peak dosage in the plasma

[29] and often observed in cardiac cells exposed to DOX [31].

Nevertheless, a previous work showed that a single injection of

10 mg/Kg DOX caused primarily a decrease in heart mass

followed by a restoration to control values [32], which may explain

why no alterations were observed in the sub-chronic model.

However, sample size in the mentioned study was too small for a

good interpretation of results. Nevertheless the authors explained

the weight recovery as an increase in cytoplasm volume and

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Mitochondrial Doxorubicin Toxicity in Wistar Rats

Figure 3. Cellular ultra-structure remains intact after acute or sub-chronic DOX treatment. No notorious differences or hallmarks of DOX

toxicity were found in the different tissues in both protocols. Panels represent electron microphotographs of randomly chosen tissues: hearts present

well-defined Z-bands and organized myofibrils (Panel C,D and L–O) and minor cytoplasmatic dilatation (Panel M and O); livers show cytoplasmatic

vacuolization (Panel E, G and S) and lipid-like droplets structures (Panel Q) and some mitochondria appear in the condensed conformation (Panel G);

renal mitochondria appear in an intermediated conformation between orthodox and condensed form (Panel X). Organs were fixed in 4%

gluteraldehyde and post-fixed in osmium (for more information, see Material and Methods).

doi:10.1371/journal.pone.0038867.g003

dilated ventricles as a sign of hypertrophy, which was not observed

in our two models.

The loss of cardiac structure and deteriorated function usually

observed in long-term treatment with DOX [33,34] was not

present in our model, supporting the idea of unaltered organ

physiology. However, an interesting work performed on young

mice demonstrated that chronic DOX treatment did not result in

any sign of cardiomyopathy until animals were subject to a

stressful swimming protocol [11]. The dissimilarities between

studies, including our own, where no alterations in heart mass in

DOX-treated rats were detected and other reports where those

alterations were measured [8,11,35,36], may be explained by the

different rat strains used or by the fact that the alterations may

only be triggered in the presence of a physiological stress. In fact,

this idea is put in use in the clinical practice where general

diagnostic techniques for cardiac function, such as echocardiograms,

are performed with increased workload and demands for

higher cardiac output increasing therefore the specificity of

screening and decreasing the number of false negatives [37,38].

In fact, the concept of normal organ physiology during resting

conditions but altered when submitted to a stressful event led us to

investigate mitochondrial function, since we can artificially

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Mitochondrial Doxorubicin Toxicity in Wistar Rats

Table 4. Effects of DOX on mitochondrial transmembrane electric potential.

DYmax (-mV) ADP Depolarization (mV) Phosphorylative Lag Phase (sec)

Model Substrate Treatment Heart Liver Kidney Heart Liver Kidney Heart Liver Kidney

Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE Mean SE

Acute n = 10 n = 9 n = 10 n = 10 n = 9 n = 10 n = 10 n = 9 n = 10

Glutamate

Malate

Saline 222.2 3.9 226.3 5.7 219.2 2.6 33.7 2.5 27.7 2.6 27.3 1.4 30.6 5.5 51.5 6.6 22.8 1.8

DOX 224.3 5.4 226.4 4.4 217.8 2.9 38.1 3.2 27.7 2.9 29.0 1.0 25.5 3.2 43.3 4.9 25.8 2.8

Succinate Saline 223.7 2.9 231.8 5.0 222.9 2.7 42.2 1.4 33.3 1.8 31.7 0.8 41.6 6.8 68.0 8.4 27.4 2.8

DOX 227.7 2.2 227.3 2.0 223.1 2.9 44.8 1.4 32.6 1.4 32.2 1.3 48.6 10.4 64.0 10.5 27.3 2.9

Sub-

Chronic

n = 12 n = 11 n = 12 n = 12 n = 11 n = 12 n = 12 n = 11 n = 12

Glutamate

Malate

Saline 210.4 2.6 211.3 1.7 208.4 2.0 24.3 1.3 21.2 1.8 18.1 1.5 29.1 2.5 55.8 6.1 34.9 3.7

DOX 208.1 3.1 207.4** 2.1 205.0** 1.7 24.2 0.9 22.4 1.4 18.9 1.9 32.2* 1.7 70.9* 11.5 38.9 3.5

Succinate Saline 214.9 2.6 217.0 1.6 211.1 3.4 27.0 1.6 26.2 1.7 19.7 2.0 38.4 3.1 64.4 6.2 38.2 3.6

DOX 210.2** 3.0 216.5 2.4 209.2 4.1 27.0 1.7 26.5 1.9 19.2 2.2 43.5* 3.7 76.1 8.1 46.4 6.3

Data was collected with a TPP + -sensitive electrode (for details see Materials and Methods) where 225–250 nmol ADP were added to induce depolarization (phosphorylative cycle). Differences between treatment groups means

within the same model were evaluated by matched pairs Student’s t test to exclude the variability related to mitochondrial isolation and electrode calibration but when assumptions were rejected the non-parametric Wilcoxon

matched pairs test was applied (see Material and Methods for detailed information).

*p#0.05;

**p#0.01 vs saline group of the same model. SE – standard error.

doi:10.1371/journal.pone.0038867.t004

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Mitochondrial Doxorubicin Toxicity in Wistar Rats

Table 5. Effects of DOX on mitochondrial aconitase activity.

Model Treatment Heart Liver Kidney

Mean SE Mean SE Mean SE

%

Acute Saline (n = 6) 100 13.1 100 12.5 100 12.7

DOX (n = 6) 100.3 12.8 85.0 18.9 72.2** 9.0

Sub-Chronic Saline (n = 5) 100 10.4 100 14.7 100 11.7

DOX (n = 5) 78.5* 14.5 112.9 25.9 105.4 12.4

Differences between treatment groups means within the same model were

evaluated by matched pairs Student’s t test to exclude the variability related to

mitochondrial isolation (see Material and Methods for detailed information).

*p#0.05;

**p#0.01 vs saline group of the same model. SE – standard error.

doi:10.1371/journal.pone.0038867.t005

stimulate this model system, by creating a pseudo-metabolic stress

by the addition of ADP. Furthermore, alterations of cardiac

mitochondrial function were already described [37]. Once again, a

different response to DOX treatment was observed in the two

models. Mitochondrial alterations, as assessed by oxygen consumption

and transmembrane electric potential, were noticeable

in both treatment protocols but the degree of effect and their

targets were distinct. If one assumes that the extension of statistical

significance of the 7 distinct end-points regarding respiration/

transmembrane electric potential (Table 4, Fig. 4 and 5) can

indicate the extension of treatment damage to mitochondria (total

of 14 parameters for tissue specificity, 21 for respiratory complex

specificity and 42 for model specificity) we can make the following

assumptions: a) Mitochondrial dysfunction is clearly more present

in the sub-chronic model with 11/42 parameters altered in

comparison to 2/42 in the acute, b) Cardiac mitochondria in the

acute model are the less affected population with 0/14 altered

parameters while both hepatic and renal mitochondria have at

least one (1/14) altered parameter, c) Contrarily, heart mitochondria

is the most affected group in the sub-chronic model with 6/14

altered parameters compared to the liver (3/14) and kidney (2/14)

and d) DOX-treatment also leads to more alterations in complex-I

sustained respiration with 2/21 and 8/21 altered parameters in

acute and sub-chronic model, respectively, in comparison to

complex-II sustained respiration which had 0/21 and 4/21 altered

parameters in same treatment schedules, respectively. It seems

therefore plausible to consider that mitochondrial bioenergetic

dysfunction, together with harmful effects of DOX on energy

substrate channeling, synthesis and availability [2,39] may be prior

and thus responsible for altered cardiac metabolism and structure

remodeling.

Oxygen consumption and calcium-loading capacity were

previously reported to be accurate markers for DOX-induced

mitochondriopathy [13,26,29,36,39]. Although some argue about

the sensitivity of mitochondrial bioenergetics parameters [15],

state 3 respiration was a good indicator for mitochondrial

dysfunction in this study since it uncovered differences between

tissues and treatment protocols. Importantly, changes in this

parameter were detected before changes in organ structure or

function occurred.

DOX is known for its futile redox cycle on mitochondrial

complex I [40]. DOX enhances the production of ROS which is

closely related to mitochondrial toxicity and further damage to cell

tissue. In the present study, activity of the tricarboxylic acid cycle

enzyme aconitase, an indirect but specific marker of oxidative

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Mitochondrial Doxorubicin Toxicity in Wistar Rats

Figure 5. Respiratory Control Ratio (RCR) and ADP phosphorylated

per consumed oxygen ratio (ADP/O). A – heart; B – liver; C

– kidney. Bars represent means of treatment groups (saline in white

bars; DOX in black bars) with SE. Differences between treatment

groups means within the same model were evaluated by matched pairs

Student’s t test to exclude the variability related to mitochondrial

isolation and electrode calibration but when assumptions were rejected

the non-parametric Wilcoxon matched pairs test was applied (see

Material and Methods for detailed information). *, p#0.05 vs saline

group of the same model. n = 10, 9 and 10 (acute model – heart, liver

and kidney, respectively) or n = 12, 11 and 12 (sub-chronic model –

heart, liver and kidney, respectively). GM - glutamate/malate; SUC -

succinate.

doi:10.1371/journal.pone.0038867.g005

stress, was used. The profile of enzyme inhibition followed the

previous idea that DOX toxicity is treatment schedule and targetspecific;

cardiac aconitase activity was decreased in the subchronic

model while it was unchanged in the acute (Table 5). The

results suggest increased cardiac mitochondrial oxidative stress in

the sub-chronic model but not in the acute; in this latter case,

only kidney mitochondria presented decreased aconitase activity.

This was rather surprising since we were expecting effects in both

models due to the fact that DOX accumulates rapidly in the

tissue and remains at high levels even after the treatment is ended

[41]. In fact, DOX-induced increase in oxidative stress is

exacerbated in long-term treatments since the primary damage

of reactive oxygen species (ROS) on mitochondrial DNA can lead

to a defective respiratory chain, increasing therefore the

productions of ROS. Nevertheless, we believe that oxidative

stress is indeed present in the acute model but perhaps

antioxidant enzymes were also up-regulated, as previously

described [42], although more work is needed in this regard.

Interestingly, most published works done by using animal models

and a single injection with higher DOX concentrations found

evidences of cardiomyopathy several days later [14,43,44,45].

Although this observation is not consistent with the well-known

late-onset DOX-induced cardiomyopathy, we believe that

previous studies combined with our present data suggest a

possible time window where strategies to counteract the drug

toxicity can be effectively applied.

To our knowledge this was the first time that a 7+1 week

treatment protocol was used in Wistar rats. Since different rat

strains differ in their metabolism and susceptibility to toxic agents

[46,47], data interpretation from the present work is not directly

comparable to previous data using other rat strains, including

Sprague-Dawley [8,15,26,35,36,48], which may present different

tolerance to DOX. Interestingly, the results from Sprague-Dawley

[6,9,27] and Wistar rats (our study) suggest that the former are

more susceptible to DOX cardiotoxicity. Although needed to be

confirmed by a new study, the differences between rat strains

corroborate the idea of polymorphism-driven susceptibility to

chemotherapy [49,50]. In fact, the same sub-chronic protocol in

Sprague-Dawley caused extensive ascites (Oliveira, personal

communication), a marker of heart failure, which was absent in

the present work.

Our data confirms once again the idea of a preferential toxicity

targeted to the heart although this was now clearly demonstrated

in a multi-organ experimental model. Moreover, mitochondrial

dysfunction is detected before detection of cardiomyopathy as

assessed by echocardiography, or morphological changes. Animals

appear to be mostly normal although presenting impaired cardiac

mitochondrial function, which may pre-dispose these organelles

for failure during stressful events. The results suggest that DOX

cardiotoxicity is better revealed when animals models or humans

are placed under stress, as referred in this study [11]. Stressful

events can include pregnancy, which has been described to present

a higher risk in survivors of childhood leukemia treated with DOX

[51].

In conclusion, our data confirms that mitochondrial dysfunction

is one major cause of DOX-selective cardiotoxicity and not a

consequence as sometimes is questioned [29,30]. The present work

is also the first to provide a three organ analysis of DOX toxicity

using two different experimental protocols in Wistar rats. DOX

did not cause substantial morphological or echocardiographic

alterations in the heart or any other organs analyzed, although

cardiac mitochondria showed alterations. Therefore, data confirms

that mitochondrial alterations result from DOX treatment,

being more severe in the heart and which are dependent on the

treatment protocol. Thus, mitochondrial dysfunction is an early

marker of DOX toxicity, although it remains to be determined if

mitochondrial alterations in organs such as liver and kidney are a

direct effect of DOX on mitochondria or instead if they result from

secondary effects of DOX on other target tissues.

Supporting Information

Figure S1 PubMed results distribution of research

involving ‘‘doxorubicin’’ and ‘‘mitochondria’’ according

to tissue category. The Venn diagram presented in the figure

was elaborated after collecting data from the PubMed website

(assessment date February 27 th ) using specific #keywords to obtain

the desired output. Briefly, papers in the database that included

works related to the #drug and #mitochondria were retrived,

restricting the output for research performed in the defined #tissue,

excluding #reviews and works performed in #humans as long as

they are not indexed with other animals. Therefore, the base of the

search string was as follow: (((#mitochondria AND #drug) AND

#tissue) NOT #reviews) NOT #humans Further explanation

about each of the keywords is given in supporting Table 1. The

authors recognize that the present search string is not flawless;

however, the idea is to give the reader an overview of report

rankings across the selected tissues. In fact, we acknowledge the

fact that, for example, the keyword #humans will not include

recent reports since they are yet to be indexed to Medline.

(TIF)

Table S1 Description of keywords used in PubMed

search for construction of Venn diagram of Fig.5, as well

as the number of results retrieved with for each

corresponding keyword.

(DOCX)

Acknowledgments

We are thankful to Dr. Lina Carvalho (Services of Pathological Anatomy

and Medical Oncology, Hospitals of University of Coimbra) for histological

analyses, Dr. Cristina Brás (Institute of Experimental Pathology, Faculty of

Medicine, University of Coimbra) for echocardiograms and Dr. Mário

Grãos (Biocant, Cantanhede, Coimbra) for the troponin I multiplex assay.

Author Contributions

Conceived and designed the experiments: GCP PJO AJM MSS. Performed

the experiments: GCP SPP CVP JAL JM AA. Analyzed the data: GCP

SPP AJM PJO JM AA JAL. Contributed reagents/materials/analysis tools:

MSS AJM. Wrote the paper: GCP PJO.

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Mitochondrial Doxorubicin Toxicity in Wistar Rats

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PLoS ONE | www.plosone.org 12 June 2012 | Volume 7 | Issue 6 | e38867


Abstract presented in: Targeting Mitochondria Meeting, Berlin, Germany, November 8, 2012.

MITOCHONDRIA AS A PERFECT TARGET IN HEALTH AND DISEASE

PAULO J. OLIVEIRA

CNC – Center for Neuroscience and Cell Biology, University of Coimbra, Portugal

Email address – pauloliv@ci.uc.pt

From the production of energy to the control of cell death pathways, calcium homeostasis, intracellular

signaling and intermediate metabolism, mitochondria are remarkable dynamic structures, with an

important, yet undesirable, role as mediator of several disease processes. Moreover, mitochondria are

also involved in the toxicity of several xenobiotics, which are known to cause adverse reactions in

humans. In this context, several drugs cause idiosyncratic reactions which are known to be mediated, at

least in part by mitochondrial toxicity caused at different levels. It is of course expected that drug or

disease-induced mitochondrial dysfunction have a larger impact on organs with higher energy

requirements, such as the cardiac and skeletal muscles and the central nervous system. If mitochondrial

dysfunction can contribute to organ degeneration, then aiming at the protection of mitochondrial function

should be a priority to rescue the affected organ. It is thus clear that mitochondria are important drug

targets in both health and disease due to the variety of molecular targets and metabolic processes that

occur in the organelle. Some structural and biophysical properties are also important to target specific

compounds to mitochondria in situ.

Several clinically used drugs target mitochondria, which provide the basis for their pharmacological and/or

toxicity effects. One example mentioned in this presentation will be doxorubicin, an anti-cancer agent

which causes a dose-dependent and cumulative cardiac toxicity, in which mitochondria is suggested to

play a critical role. We will also show examples of how mitochondrial-directed agents can be useful as

anti-cancer drugs or antioxidants and how physical activity can be an effective non-pharmacological

strategy to boost mitochondrial power and afford cross-tolerance against several deleterious insults.

Acknowledgements: Work in the author´s laboratory is sponsored by the Portuguese Foundation for

Science and Technology, co-funded by COMPETE/FEDER/National Budget (grants # PTDC/QUI-

QUI/101409/2008, PTDC/SAU-OSM/104731/2008, PTDC/SAU-TOX/110952/2009 and PTDC/SAU-

TOX/117912/2010).

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