European Journal of Clinical Investigation

European Journal of Clinical Investigation

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


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


Official journal of the European Society for Clinical Investigation

ECI_42_s1_oc.indd 1

2/27/2012 12:36:06 PM

80 Workshop 4: Mitochondria in health and disease

Results: No differences in drug uptake/accumulation capacity

between StemTCs and DiftTCs were observed. Despite equal

amounts of etoposide-induced DNA damage, StemTCs showed

increased histone cH2AX levels, which denotes enhanced

DNA repair response. The higher efficiency of StemTCs in

DNA damage sensing could also be related with higher basal

p53 content in StemTCs. Also, hydrogen peroxide caused more

extensive DNA damage in DiffTCs than StemTCs.

Conclusion: P19 StemTCs were not only better able to

recognize DNA damage, but also suffer less extensive oxidative

DNA damage. The data may suggest that cell death signaling

triggered by nuclear DNA damage is impaired in StemTCs.

Funding: FCT (PTDC/QUI-BIQ/101052/2008), FCT to KAM

(SFRH/BD/66138/2009), Marie-Curie IEF to IVN (#251850).


Habitual physical activity and liver

mitochondrial function in patients with

non-alcoholic fatty liver disease (NAFLD).

A pilot study

I.O. Gonçalves*, P. Portincasa , I. Grattagliano ,

A.N. Pizarro*, A. Ascensão* & J. Magalhães*

*Research Centre in Physical Activity, Health and Leisure,

Faculty of Sport Sciences (CIAFEL), University of Porto,

Porto, Portugal;

Department of Interdisciplinary Medicine, University

Medical School, Bari, Italy

Background: Sedentary behaviors can play a role in the pathogenesis

of NAFLD, being physical activity one first line therapy.

The effect of physical activity on peripheral factors of NAFLD

has been widely reported; however, its role in hepatic mitochondrial

function remains poorly understood. We aim to analyze

the relationship between habitual physical activity (HPA) and

liver mitochondrial function in patients with NAFLD.

Materials and methods: Sixteen NAFLD patients diagnosed

through history and abdominal ultrasonography (steatosis

score) underwent blood analysis (alanine aminotransferase –

ALT, aspartate aminotransferase – AST, gamma-glutamyl

transferase – GGT, triglyceride – TG and total cholesterol –

TC), anthropometric measurements (weight, height, body mass

index), HPA assessment by triaxial accelerometry (5 days

including weekend), and liver mitochondrial function in vivo

evaluation by stable isotope breath testing ( 13 C ketoisocaproate,

2 h sampling with infrared mass spectrometry measurement)

(Portincasa et al., Clin Sci, 2006).

Results: Liver transaminases and GGT, lipid profile and

anthropometric analysis matched reference values for the adult

population. However, mitochondrial decarboxylation capacity,

a marker of the organelle function, was reduced in these

patients due to the (lower cumulative recovery of exhaled

13 CO 2 :18Æ3 ±1Æ2% cumulative dose). The daily time spent in

moderate/vigorous physical activity fulfilled the international

recommendations for health benefits (48Æ1 ±3Æ1 min per day).

Moreover, 13 CO 2 cumulation (60 min) and HPA at moderate/

vigorous intensity are positively correlated (r =0Æ73, P =0Æ09).

Conclusions: The results of this pilot study suggest that the

levels of habitual moderate/vigorous physical activity are

inversely related with severity of mitochondrial dysfunction in

patients with NAFLD.

Acknowledgements: IJUP-70, 2009 and FCT – (PTDC/DES/

113589/2009). The authors are supported by FCT: I.O. Gonçalves

(SFRH/BD/62352/2009), A.N. Pizarro (SFRH/BD/70513/

2010), A. Ascensão (SFRH/BPD/4225/2007) and J. Magalhães



Interstrain differences in the bioenergetic

profile of embryonic fibroblasts from four

strains of mice

C. Pereira*, S. Nadanaciva , P.J. Oliveira* & Y. Will

*Center for Neurosciences and Cell Biology, Department of

Life Sciences, Faculty of Sciences and Technology of the

University of Coimbra, Coimbra, Portugal;

Compound Safety Prediction, Pfizer Global Research &

Development, Groton, CT, USA

Background: The bioenergetic profile of fibroblasts from four

strains of mice containing single nucleotide polymorphisms in

their mitochondrial DNA (mtDNA) was investigated.

Materials and methods: Mouse embryonic fibroblasts were

grown at 37°C, 5% of C0 2 in Knockout DMEM, 2 mM L-Glutamine,

1· Non-essential aminoacid, 0Æ25 mg mL -1 gentamycin

and 0Æ1 mM b-mercaptoethanol. Oxygen consumption rates

(OCR) were measured using the Seahorse Bioscience XF96

Extracellular Flux Analyzer.

Results: PERA/EiJ MEFs have 106 distinct SNPs in mt DNA

in comparison with the reference strain, C57BL/6J. The strains

CZECHII/EiJ and MOLF/EiJ have 390 and 393 SNPs when

compared with C57BL/6J. We found that both PERA/EiJ and

CZECH/EiJ had a much lower maximum respiratory capacity

when compared with the other strains at passage 3, due to a

lower spare respiratory capacity. At passage 10, all the strains

presented lower maximum respiratory capacity when compared

with the reference strain. Overall, no significant differences

were found in the ATP-linked respiration, proton-leak

and in the non-mitochondrial respiration.

Conclusions: This is the first study which shows that there

are differences in the bioenergetic profiles of fibroblasts from

different strains of mice even though a direct link between

those differences and single nucleotide polymorphisms is difficult

to assess.

Acknowledgements: Claudia V. Pereira and Paulo J. Oliveira

are supported by the Portuguese Foundation for Science and

Technology (SFRH/BD/48029/2008 and PTDC/SAU-TOX/

110952/2009, respectively).


Effects of TNF on expression membrane

molecules in tumor K-562 cell

V. Jurisic* & T. Srdic-Rajic

*University of Kragujevac, School of Medicine, Kragujevac,


Institute of Oncology, Belgrade, Serbia

Background: Expression of several molecules on cell surface

in tumor cells is very important for immunotherapy. However,

during cell proliferation, cell growth or immunotherapy, previous

study indicated that expression of cell membrane molecules

on tumor cells can be modulated.

Materials and methods: We studies in vitro effects of TNFalpha

on cell surface expression of several receptors involved

in cell death process in K-562 cells. Here we compared LDH

release as early events for cell death, determined by sensitive

enzyme assay, with changes in cell surface membrane molecule

expression by Flow cytometry during cultures of K-562

cells in presence of diverse dose of TNF-alpha in a time and

dose dependent manner (2, 4, 6, 18, 24 h). The cell death was

also estimated by Flow cytometry using annnexin-V and propidium

iodide. The appearance of the soluble molecules in

supernates of cultures cells wee estimated by western blotting


2012 Wiley-Blackwell

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


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1. Redefinition of oxidative

stress: the good and the bad

2. Mitochondria as a major

source of ROS production

3. The antioxidant guardians of


4. Drug-induced organ increased

cellular OS

5. In vitro detection of ROS and

platforms available

6. Animal models suitable to

study OS

7. In vivo noninvasive ROS


8. Conclusions

9. Expert opinion

The contribution of oxidative

stress to drug-induced organ

toxicity and its detection in vitro

and in vivo

Claudia V Pereira, Sashi Nadanaciva, Paulo J Oliveira & Yvonne Will †

Pfizer R&D, Compound Safety Prediction- WWMC, Cell Based Assays and Mitochondrial Biology,

Groton, CT, USA

Introduction: Nowadays the ‘redox hypothesis’ is based on the fact that thiol/

disulfide couples such as glutathione (GSH/GSSG), cysteine (Cys/CySS) and

thioredoxin ((Trx-(SH)2/Trx-SS)) are functionally organized in redox circuits

controlled by glutathione pools, thioredoxins and other control nodes, and

they are not in equilibrium relative to each other. Although ROS can be

important intermediates of cellular signaling pathways, disturbances in the

normal cellular redox can result in widespread damage to several cell components.

Moreover, oxidative stress has been linked to a variety of agerelated

diseases. In recent years, oxidative stress has also been identified to

contribute to drug-induced liver, heart, renal and brain toxicity.

Areas covered: This review provides an overview of current in vitro and in vivo

methods that can be deployed throughout the drug discovery process. In

addition, animal models and noninvasive biomarkers are described.

Expert opinion: Reducing post-market drug withdrawals is essential for all

pharmaceutical companies in a time of increased patient welfare and tight

budgets. Predictive screens positioned early in the drug discovery process

will help to reduce such liabilities. Although new and more efficient assays

and models are being developed, the hunt for biomarkers and noninvasive

techniques is still in progress.

Keywords: in vitro and in vivo approaches, organ toxicity, oxidative stress; free radicals

Expert Opin. Drug Metab. Toxicol. [Early Online]

1. Redefinition of oxidative stress: the good and the bad

The term oxidative stress (OS) originated from a concept that a significant increase

in reactive oxygen species (ROS) and reactive nitrogen species (RNS) would become

toxic to biomolecules, DNA, proteins and lipids due to the inability of antioxidant

defenses to act. In a healthy cell, ROS/RNS are degraded by the protective antioxidant

machinery and, consequently, only excessive ROS/RNS production and/or a

decrease in the detoxification mechanisms can lead to ‘OS’ and pathological conditions

[1]. For instance, alterations in glutathione (GSH), the most abundant intracellular

nonprotein thiol, indicate a possible global antioxidant or redox change in

cells [2]. Therefore, OS is still often defined as an imbalance of pro-oxidants and

antioxidants. However, the finding that thiol/disulfide couples such as glutathione

(GSH/GSSG), cysteine (Cys/CySS) and thioredoxin ((Trx-(SH)2/Trx-SS)) vary little

among healthy individuals and are maintained in disequilibrium relative to each

other has significantly altered the concept of OS [3]. The theory of redox compartmentalization

and cellular stress is now seen as an alternative to the free radical

hypothesis [4]. More than 50 years ago, Harman postulated the ‘free radical theory’

of aging. The theory is based on the principle that gradual accumulation of damage

10.1517/17425255.2012.645536 © 2012 Informa UK, Ltd. ISSN 1742-5255 1

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Article highlights.

. Oxidative stress is no longer defined as an imbalance of

pro-oxidants and antioxidants since the finding that

glutathione pools, thioredoxins and other control nodes

are maintained in nonequilibrium relative to each other.

. Drug-induced oxidative stress contributes to

cardiovascular, liver and renal toxicity.

. Mitochondria are the main producers of reactive oxygen

species (ROS) within the cell.

. Cells contain important antioxidant systems for reducing

excessive ROS production.

. The combination of different methods and approaches

allows a better estimation of ROS-induced toxicity both

in vitro and in vivo.

. Redox proteomics and new noninvasive methods for

ROS detection will lead the future in this field.

This box summarizes key points contained in the article.

within intracellular macromolecules during aging is caused by

the harmful effects of free radical production during aerobic

metabolism. Later, Harman renamed his initial proposal as

the ‘mitochondrial theory of aging’ [5]. More recently, the

so-called redox hypothesis postulates that oxidizable thiols

are common control elements for biologic processes and are

functionally organized in redox circuits that are controlled

by GSH pools, thioredoxins and other control nodes [4].

‘OS’ under the vision of this new concept can be defined as

the disruption of those redox circuits. The reducing force of

these nodes in different subcellular compartments can be

quantitatively expressed in terms of redox potentials for the

respective couples and calculated using the Nernst equation [6].

Interestingly enough, intracellular organelles differ in terms of

their internal redox potential. Mitochondria present the most

reducing values; because of their mostly alkaline matrix, the

nucleus has a relatively more reducing environment than the

cytoplasm, with the latter by its turn having a more reducing

moiety than the extracellular space [6].

It is known that regulated alterations in the production of

oxidizing species are important in the prevention of consequences

associated with the aging process [5]. Although ROS

are not only toxic cellular metabolic products that are frequently

associated with many diseases but they can also be

critical intermediates of cellular signaling pathways. For

example, it was been shown that an oxidative burst is necessary

for insulin/IGF-1 pathway modulation and vascular

endothelial growth factor (VEGF) signaling. A recent review

from Hamanaka et al. [7] underlines a new insight into modulation

of local OS and its advantages to the cell, which

should also be taken into consideration, for example, when

studying the effects of antioxidants. It has now become clear

that ROS can function as second messengers in signal transduction

[6] and that low levels of ROS are required for cellular

processes such as proliferation and differentiation, pointing a

new direction for the modulation of ROS production as a

means to achieve a beneficial therapeutic outcome.

From a pathological point of view, OS has been associated

with distinct conditions that lead to organ degeneration.

In the cardiovascular system, excessive OS has been

associated with heart failure [8] ischemia and reperfusion

[9,10] and atherosclerosis [11]. Distinct liver pathologies

including Wilson’s disease [12], ischemia and reperfusion [13]

and nonalcoholic fatty liver disease [14] have also been frequently

associated with excessive oxidative damage to

cellular structures, which in most cases originates from

mitochondrial alterations. Overproduction of ROS generated

from mitochondria has been implicated in acute brain

injuries such as stroke and in neurodegenerative diseases

such as Parkinson’s [9,15] and Alzheimer’s disease [9,16],

among others. Furthermore, experimental evidence supports

the role of mitochondrial dysfunction and OS as

determinants of neuronal death as well as endogenous protective

mechanisms after stroke. In addition, renal failure

induced by drug-induced OS can lead to apoptosis, tissue

loss and dysfunction [17,18].

Of importance for the present review, OS has been shown

to contribute to organ-specific toxicity associated with several

drugs [9]. To avoid such liabilities, assays need to be developed

and used throughout the drug discovery process to accurately

detect OS and correlate it with a particular aspect of organ

toxicity, with the ultimate objective of eliminating that

compound or family of compounds from the drug

development process.

This review provides an overview on OS, antioxidant

defense mechanisms, methods currently used to investigate

oxidant production and elimination, animal models that

display OS and some in vivo approaches that have been

developed to assess possible ROS formation [19].

2. Mitochondria as a major source of ROS


The mitochondrial electron transport chain (ETC) is the

main source of ROS production in the form of superoxide

anion, O 2

-. [7,20]. The ETC consists of four enzymatic complexes

that contribute to electron transport according to their

redox potential. Electron transport is coupled with proton

pumping to the mitochondrial intermembrane space and generation

of a protonmotive gradient that is used by the ATP

synthase to synthesize ATP. Although most of electrons are

transferred along the ETC in an orderly manner down their

redox potential, a small percentage of electrons are trapped

directly by oxygen, which is then reduced to superoxide

anion [21]. Damage in the ETC or in the inner mitochondrial

membrane can increase superoxide production. Although

some controversy still exists, it appears that two of the ETC

complexes are responsible for most of ROS production,

namely complex I, the NADH-ubiquinone oxidoreductase

[22,23] and complex III, the ubiquinol-cytochrome c oxidoreductase

[24,25]. Superoxide production can be enhanced

when the ETC decreases its flux due to the formation of a

2 Expert Opin. Drug Metab. Toxicol. [Early Online]

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high membrane potential under non-phosphorylating and

maximum coupled conditions [26]. It has also been demonstrated

that superoxide can escape from the intermembrane

space through porin, a voltage-dependent anion channel

located in the mitochondrial outer membrane [22]. Besides

oxidation of proteins, nucleic acids and membranes, one

important consequence of OS is the induction of the mitochondrial

permeability transition pore (MPTP). Induction

of the MPTP, once irreversible, can lead to mitochondrial

depolarization, loss of calcium control, mitochondrial swelling,

secondary OS and inhibition of ATP synthesis, besides

leading to cell death [27]. Membrane alterations can lead to

the release of cytochrome c once the mitochondrial outer

membrane is disrupted. Thus, MPTP induction can trigger

cell death, either through apoptosis, if enough ATP is present

to maintain caspase activity or through necrosis after complete

bioenergetic failure of the cell. Despite extensive studies, the

composition and mechanism of the MPTP are still under

debate [27].

2.1 Other sources of ROS

In addition to mitochondria, ROS can also be produced during

biotransformation of drugs and xenobiotics, during

inflammatory processes, UV and ionic radiation and also in

conjunction with RNS through the activity of nitric oxide

synthase [2,28].

Non-mitochondrial sources of superoxide anion production

include NADPH oxidases that are located in the plasma

membrane, xanthine oxidase, which is distributed throughout

the cytoplasm and in the endothelial lining cells of the heart,

kidney and brain, aldehyde oxidase, which is localized in the

cytoplasm, and cytochrome P450, which is primarily a

membrane-associated protein that is located either in the

inner mitochondrial membrane or in the endoplasmic reticulum

of cells. Hydrogen peroxide (H 2 O 2 ), a product of superoxide

anion dismutation, can be enzymatically produced by

oxygenases and monoamine oxidase [22].

3. The antioxidant guardians of cells

Cellular antioxidant defenses have evolved to provide an

important network system for prevention, interception and

repair of oxidative damage. They consist of nonenzymatic

scavengers and quenchers, known as antioxidants, as well as

enzymatic systems including superoxide dismutases (SODs)

and hydroxyperoxidases, such as GSH peroxidase, catalase

and other hemoprotein peroxidases [28,29].

SOD exists as three different isoforms: SOD1 is a Cu/Zn

SOD located in the cytoplasm and in the intermembrane

space [30-32], SOD2 is a manganese SOD that is exclusively

located in the mitochondrial matrix (MnSOD) [33] and

SOD3 is a Cu/Zn SOD that has an extracellular localization

[34,35]. Mice lacking mitochondrial MnSOD (SOD2)

cannot survive more than a few days after birth suggesting

that control of a tight level of mitochondrial superoxide anion

is critical for cell survival [36,37]. SOD accelerates the elimination

of superoxide by increasing the rate constant for spontaneous

dismutation. Acting like a scavenger of superoxide,

SOD catalyzes its dismutation to H 2 O 2 in the matrix. Superoxide

anion that is released to the intermembrane space is

partly controlled by the intermembrane space Cu/Zn SOD.

GSH is the most abundant intracellular nonprotein thiol,

which reduces cysteine disulfide bonds formed within cytoplasmic

proteins by serving as an electron donor. GSH-based

systems, including GSH-S transferase and the thioredoxin system,

constitute the major redox buffer in the cytosol [19]. Since

the concentration of the two GSH forms (GSSG/GSH) is so

much higher than that of any other system, the GSH/GSSG

pool dominates the principal redox buffer of the cell [6,38].Mitochondrial

matrix GSH represents 10 -- 15% of the total GSH in

the liver as well as in renal proximal tubule. Since the enzymes

for GSH synthesis are not present in the matrix, GSH has to

be imported to mitochondria from the cytosol. The fact that

the mitochondrial GSH (10 mM) content is higher than that

in cytosol (7 mM) suggests its importance in the cellular GSH

content [39]. However, in the mitochondria, GSH is found

mainly in its reduced [40].

GSH peroxidase (GPx) exists in two forms in the

mitochondria: GPX1 and phospholipid hydroperoxide GPX

(PHGPx) [1,41]. The phospholipid hydroperoxide GSH peroxidase

is known to be inducible under various stress conditions.

This enzyme catalyzes the regeneration of phospholipid hydroperoxides

using the reducing power of GSH, being present in

the cytosol and in the inner mitochondrial membrane of animal

cells, while GPX1 occurs mainly in the mitochondrial matrix.

Both enzymes use reduced GSH in order to reduce hydrogen

peroxide into water, generating GSH disulfide (GSSG) in the

process, which can form protein-mixed disulfides and thus

inhibit protein function [42,43]. The enzyme GSH reductase

can regenerate GSSG to GSH by using NADPH as a cofactor.

Another system capable of degrading H 2 O 2 involves proteins

of the thioredoxin family. These proteins work jointly

with thioredoxin-dependent peroxidase reductases (peroxiredoxins,

PXRs) and a family of selenium-independent GSH-

S-transferases (GST). GSTs catalyze GSH-dependent reduction

of both phospholipid hydroperoxides (PLOOH) and

fatty acid hydroperoxides (FAOOH) and their respective aldehydes.

Thioredoxin, which is especially concentrated in the

endoplasmic reticulum, is a thiol-specific antioxidant that

reduces disulfide bridges of proteins under OS conditions.

Thioredoxin produces mostly intramolecular disulfides and

has different substrate preferences [44,45]. NADPH is a source

of reducing equivalents for both thioredoxin and GSH systems.

NADPH-dependent thioredoxin reductase activity

reduces 12 kDa proteins of the thioredoxin family together

with GSH or PXRs, maintaining protein thiols in the reduced

state [22]. PXR also converts H 2 O 2 into H 2 O using thioredoxin

as a reducing agent. There are two forms of PXR that

are found in the mitochondrial matrix, PXRIII and

PXRV [46,47].

Expert Opin. Drug Metab. Toxicol. [Early Online] 3

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Although the activity of catalase, which converts H 2 O 2 to

water, is restricted in most tissues to peroxisomes, that antioxidant

enzyme represents 0.025% of total heart mitochondrial

protein and it is important for detoxifying mitochondrialderived

H 2 O 2 . Hence, catalase represents a key antioxidant

defense mechanism for myocardial tissue [48,49]. Catalase is

also present inside liver mitochondria where it takes part in

the OS defense system (Figure 1). It should also be emphasized

that if liver and heart mitochondria catalase share similar

detoxification functions, enzyme activity may control the

amount of hydrogen peroxide available to produce hydroxyl

radical and trigger the MPTP. Also it is known that electron

chain complexes and Krebs cycle enzymes in heart mitochondria

can be inactivated by hydrogen peroxide. Target enzymes

include succinate dehydrogenase, alpha-ketoglutarate dehydrogenase

and aconitase [50]. Due to its high Km value, catalase

functions at high H 2 O 2 concentrations (e.g., in skeletal

muscle, red blood cells); on the other hand, at low H 2 O 2 concentrations,

the enzyme exhibits a nonspecific peroxidase

activity. In the liver, catalase removes endogenous H 2 O 2

formed in the peroxisomes and it has been shown to protect

hepatocytes more efficiently from H 2 O 2 formed by peroxisomal

oxidases than that formed by the outer mitochondrial

membrane-bound monoamine oxidase [51]. The most economical

way of removing H 2 O 2 is through catalase activity,

since no reducing equivalents are consumed in the process.

4. Drug-induced organ increased cellular OS

4.1 Liver

Drug attrition due to liver toxicity remains a major problem

for the pharmaceutical industry. There is a growing body of

evidence suggesting that idiosyncratic drug-induced hepatotoxicity

may be mediated, at least in part, by OS [52,53]. The

following examples will focus on mitochondrial-induced

intracellular ROS formation, since several drugs such as acetaminophen

(APAP), nimesulide and valproic acid (VPA),

among others, are associated with drug-induced liver injury

through causing mitochondrial dysfunction, depletion of

cellular ATP and induction of the MPTP [54].

APAP (Figure 2A) toxicity is initiated by the formation of a

reactive metabolite (NAPQI), which depletes GSH and binds

to proteins, especially in mitochondria. The result is mitochondrial

OS and peroxynitrite formation, in part through

amplification by c-Jun-N-terminal kinase activation, leading

to mitochondrial DNA damage and opening of the mitochondrial

transition pore, which subsequently leads to cell

injury/death [55,56]. GSH traps NAPQI first and the GSH

adduct is excreted. However, after GSH is depleted, NAPQI

reacts with cellular proteins and forms an APAP adduct.

Although the original hypothesis that the general protein

binding of NAPQI causes toxicity has been questioned, it

remains undisputed that the metabolic activation of APAP is

the critical initiating event of cell death. After the initial

concerns, the protein binding hypothesis was modified.

With the recognition of a mitochondrial OS and the fact

that formation of mitochondrial protein adducts correlated

with liver injury, the current concept emerged. In this hypothesis,

ROS and protein binding, especially to mitochondrial

proteins, is an important initiating event that by itself is not

sufficient to cause cell death. By contrast, this protein binding

induces mitochondrial OS with the formation of superoxide

anion and peroxynitrite, which in turn amplifies the original

stress, eventually leading to necrotic cell death.

Another example of hepatotoxicity is the nonsteroidal

anti-inflammatory drug (NSAID) nimesulide, a selective

cyclo-oxygenase-2 inhibitor widely used for the treatment of

inflammatory and pain conditions. Nimesulide is almost

exclusively metabolized and cleared by the liver. Metabolic

biotransformation of nimesulide in the liver can occur at

both the phenoxy ring moiety and the aromatic nitro group.

The major metabolite is 4’-hydroxy nimesulide and it is normally

conjugated with sulfate or glucuronic acid. Nitroreduction

is also an important pathway of biotransformation and of

great importance in toxicological terms since it results in the

formation of an aromatic amine. This amine metabolite is

further metabolized by N-acetylation, catalyzed by

N-acetyltransferases (NAT). These intermediates can also

undergo sulfo- or glucurono-conjugation and excreted in

urine. In vitro systems suggested that these intermediates

have the potential to cause oxidoreductive stress and undergo

covalent binding to selective target proteins. Mitochondria are

particularly sensitive organelles. Nimesulide is prone to cause

OS since nitroaromatic compounds can be enzymatically

reduced by nitroreductase-catalyzed one or two electron transfer

pathways. The one electron transfer pathway produces the

nitro anion radical, a highly reactive species that, under aerobic

conditions, is able to transfer the electron to molecular

oxygen, thereby producing superoxide anion radicals.

In vivo, however, this intermediate has not been demonstrated.

Redox cycling is potentially hazardous because one

molecule of nitro-derived molecule can theoretically give rise

to a large number of superoxide anion molecules. Also recent

studies indicated that nimesulide is able to uncouple mitochondrial

oxidative phosphorylation in isolated mitochondria,

induce mitochondrial swelling and NAD(P)H oxidation and

MPTP pore opening [57]. In addition, bioreductive metabolism

of nimesulide in the cytosol and endoplasmic reticulum

causes both OS and covalent modification of several proteins,

which can lead to the MPTP opening. In fact, high concentrations

of nimesulide can be accumulated in mitochondria and

undergo bioreductive metabolism producing redox cycling

and OS and this leads to the MPTP opening. In addition,

the bioreductive metabolism of nimesulide in the cytosol

and/or endoplasmic reticulum causes both OS and covalent

modification of target proteins, which can also lead directly

or indirectly (via calcium mobilization) to the activation of

the MPTP. It is likely that covalent modification of intracellular

proteins and OS-induced depletion of GSH may activate

signaling pathways that can lead to hepatic necrosis [52].

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‘Lifeguards’ of the cell






Cytochrome c







O 2


Healthy cell




‘Lifeguards’ of the cell


O 2


O 2

O 2


O 2

O 2


H + H +

H + H +

H2 O








H2 O




ROS beneficial effets:

- Cell migration

- Cell signaling pathways

- Cell proliferation and


- Cell differentiation





H +










O 2


O 2



O 2


O 2


Cell damage

and disease

ROS negative effects:









- Thiol oxidation

- Lipid peroxidation

- mtDNA and nuDNA damage


- Mitochondrial swelling

- Apoptosis


Other cell sources of ROS:

NADPH oxidases; Cytochrome peroxidases; Xanthine

oxidase; Lysosomes;peroxisomes.

Figure 1. Mitochondria: the main source of reactive oxygen species (ROS) production of the cell. Superoxide anion is mainly produced at mitochondrial complexes I and

III. The excessive ROS accumulation can lead to an imbalance between the antioxidant mechanisms (enzymes such as SOD, catalase, GPx, PXR) and the ROS being

produced in the cell. When ROS accumulate, mitochondrial cell damage might occur. Increased ROS can damage mtDNA and nuDNA, and can cause thiol oxidation and

lipid peroxidation, which can ultimately lead to cytochrome c release from the mitochondria, mitochondrial permeability transition pore opening and apoptosis.

ANT: Adenine nucleotide translocator; CoQ: Coenzyme Q; CycD: Cyclophilin D; Cytc: Cytochrome c; GPx: Glutathione peroxidase; HKII: Hexokinase II; MPTP: Mitochondrial permeability transition pore; PXR: Peroxiredoxin;

SOD: Superoxide dismutase; VDAC: Voltage-dependent anion channel.

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The contribution of oxidative stress to drug-induced organ toxicity and its detection in vitro and in vivo




NAPQI formation

GSH depletion

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- Kupffer cell activation

- Reactive oxygen peroxynitrite

-Endothelial cells


-Necrosis/liver failure

- Oxidative stress

- Mitochondrial dysfunction

- Increased NADH oxidation

- Decreased ATP

- Loss of calcium handling

- Apoptosis



- mtDNA damage and MPTP

- Loss of ATP

- Loss of mtΔΨ

- Reactive oxygen peroxynitrite (Hepatocyte)


Complex I



H +

O 2

O 2


DOX semiquinone




C-Jun N-terminal

kinase activation


Figure 2. Possible mechanisms leading to oxidative stress. A. Acetaminophen (APAP) metabolism causes the formation of a

highly toxic metabolite NAPQ1, which binds to mitochondrial proteins and initiates an oxidative stress cascade due to

glutathione depletion. B. Doxorubicin (DOX) causes mitochondrial impairment through redox cycling in mitochondrial

complex I, resulting in induction of the mitochondrial permeability transition pore, besides other mitochondrial

consequences. DOX-induced cardiomyopathy is a serious consequence of treatment.

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VPA is a broad-spectrum antiepileptic drug and is generally

a safe drug, although it has been associated with idiosyncratic

hepatotoxicity due to microvesicular steatosis. The involvement

of OS in the pathogenesis of VPA hepatotoxicity was

previously investigated [58]. It was found that VPA showed

numerous alterations in OS markers. For example, isoprostanes,

which are a family of compounds formed by free

radical-induced peroxidation of membrane-bound phospholipid-associated

arachidonic acid, were increased and so were

aldehydes, another indicator of lipid peroxidation. In addition,

imaging of hepatocytes treated with VPA showed

increases in ROS formation. Oxidative DNA damage, as measured

by 8-hydroxy-2¢-deoxyguanosine (8-OHdG), was

found in the blood of children with seizure disorders who

were undergoing VPA treatment. Although measurement of

the antioxidant status in rats and humans has been contradictory,

it is thought that a predisposition toward diminished

antioxidant capacity might be predictive for developing

hepatotoxicity on VPA treatment [58].

4.2 Heart

The accumulation of free radicals in peripheral blood has negative

effects on the cardiovascular system, similarly to aging or

other pathophysiological conditions [9]. Cardiotoxicity involving

OS has been associated with the side effects of several

drugs in a similar way to what we reported for hepatotoxicants,

leading to increased mitochondrial ROS generation.

For example, cisplatin is an anticancer drug whose use is

limited because of cardiotoxicity. OS and apoptosis have

been postulated to contribute to this toxicity [59]. Indeed,

cisplatin-treated rats exhibit increased lipid peroxidation as

measured by malondialdehyde (MDA) formation and display

decreased GSH content and decreased SOD activity. Moreover,

both mitochondrial DNA and nuclear DNA of

cisplatin-treated rats showed free radical-induced damage [59].

AZT (zidovudine) is a potent inhibitor of HIV replication

and a major antiretroviral drug used for AIDS treatment.

A major limitation in the use of AZT is the occurrence of

severe side effects, in particular cardiotoxicity. In a study conducted

by de la Asunción (2004), mice were treated with AZT

for 35 days with and without coadministration of the antioxidants

vitamin C and E [60]. Cardiac mitochondrial DNA

(mtDNA) of mice treated with AZT had more than 120%

more oxo-dG (8-oxo-7, 8-dihydro-2’-deoxyguanosine, a

biomarker of oxidative damage to DNA) in their mitochondrial

DNA than untreated controls. AZT treatment also

caused an increase in mitochondrial lipid peroxidation and

oxidation of mitochondrial GSH [61]. Dietary supplementation

with vitamins C and E protected against these signs of

mitochondrial OS.

Compounds such as anthracyclines (doxorubicin (DOX),

daunorubicin) are very effective antineoplastic drugs [17],

although their clinical use is limited by cumulative, doserelated,

progressive myocardial damage that potentially can

lead to congestive heart failure [62]. DOX causes OS through

a redox cycling mechanism in mitochondrial complex I, causing

mitochondrial damage and disruption of mitochondrial

and cellular calcium homeostasis [63,64]. DOX-induced OS

can also be the origin of decreased calcium loading capacity,

which is mediated by increased MPTP, as observed in animal

models (Figure 2B) [65].

4.3 Kidney

Compared with other organs, the kidney is uniquely susceptible

to chemical toxicity, partially because of its disproportionately

high blood flow and due to its anatomically and

functionally complexity. Also liver cells have a tremendous

capacity to repair and regenerate, whereas kidney cells possess

a very limited repair capability [66]. Drugs cause approximately

20% of community- and hospital-acquired episodes

of acute renal failure. Among older adults, the incidence of

drug-induced nephrotoxicity may be as high as 66% [67]. Several

different drug classes such as anticancer drugs, antibiotics,

NSAIDs and immunosuppressants are known to exhibit

kidney injury, OS being a contributing factor to toxicity.

Regarding antineoplastic renal toxicity, two compounds are

considered classical examples: DOX and cisplatin. Cisplatin

toxicity appears to involve inhibition of protein synthesis,

mitochondrial injury and DNA damage causing blockage

of DNA replication and gene transcription due to the formation

of single- and double-strand DNA breaks [68,69].

Hydroxyl radicals were found responsible for cisplatininduced

apoptosis as hydroxyl radical scavengers inhibited

cytochrome c release and caspase activation [68,69]. When renal

collecting duct-derived cells were incubated with inhibitors of

the mitochondrial respiratory chain or ATP synthase,

cisplatin-induced apoptosis was strongly enhanced showing

that intact mitochondria are essential to prevent cisplatininduced

apoptosis [70]. Furthermore, in vivo studies show

cisplatin-induced oxidative damage to mitochondrial lipids,

including cardiolipin, oxidation of mitochondrial proteins

and lower aconitase activity [69]. Another study showed

that cisplatin depleted the GSH and increased lipid

peroxidation [71].

Aminoglycoside antibiotics including gentamicin can also

cause renal toxicity [72,73]. It is known that gentamicin leads

to the formation of hydrogen peroxide by renal cortical mitochondria

[74]; also radical scavengers and iron chelators provided

functional and histological protection against renal

failure in gentamicin-treated rats. Gentamicin has been shown

to release iron from renal cortical mitochondria and this was

supported by in vitro studies [75]. The results strongly support

the importance of hydroxyl radicals or a similar oxidant in

gentamicin-induced renal failure.

Cyclosporine A has played a major role in the improvement

of solid-organ transplantation, immunosuppression, enhancing

patient survival [66,76,77]. However, its clinical use has

been hampered by frequent reports of nephrotoxicity. The

renal toxicity is attributed to reduced renal blood flow, which

can lead to hypoxia-reoxygenation injury accompanied by

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excessive generation of ROS [77]. It was also previously documented

that cyclosporine A-induced renal toxicity due to an

alteration of intracellular calcium homeostasis, including

increased calcium accumulation in the matrix as well as

increased ROS production [78]. Although many studies

have been conducted, the mechanism of cyclosporine

A-induced nephrotoxicity is far from clear.

Diclofenac (DCLF) is an NSAID that is widely used for the

treatment of various diseases such as osteoarthritis, rheumatoid

arthritis, and acute muscle pain conditions, among

others, although DCLF can cause nephrotoxicity in humans

and experimental animals [79]. Studies showed that DCLF is

a strong inducer of OS through increased levels of MDA

and kidney toxicity. OS induced by DCLF was also coupled

with massive kidney DNA fragmentation [79]. These twofree

radical-mediated events may ultimately translate into

apoptotic cell death of kidney cells in vivo.

Methotrexate (MTX)-induced renal toxicity is thought to

be due to OS since administration to rats increased MDA

levels (lipid peroxidation) in the kidney and decreased

SOD, catalase and GSH peroxidase activities in renal tissue,

resulting in a decrease in the antioxidant capacity [80].

4.4 Brain

The brain is very vulnerable to free radical damage because it

has a high rate of oxygen consumption, abundant supply of

the transition metals, high content of unsaturated lipids and

relatively lower regenerative capacity in comparison with

other tissues [81]. Several chemotherapy drugs are known to

cause significant clinical neurotoxicity, which can lead to early

termination of the treatment [82-84]. Neurotoxicity resulting

from anticancer drugs including taxol, cisplatin and MTX

can involve acute alterations in consciousness, seizures,

cerebral infarctions, paralysis and neuropathy, among

other complications.

Taxol is an effective chemotherapeutic agent that is widely

used for the treatment and management of breast carcinomas.

However, it has also been suggested that taxol can increase

hydroperoxide production and increase OS. Taxol is a

microtubule-stabilizing anticancer drug, which has been

reported to induce neuronal cell death in mouse cortical cultures

and increased cellular OS, assessed by 2,7-dichlorofluorescein

diacetate (2,7-DCFDA) fluorescence. Treatment with

apocynin and trolox markedly inhibited the taxol-induced

oxidative cytotoxicity, demonstrating the importance of free

radicals in the mechanism. Also the treatment with NADPH

oxidase inhibitors or suppression of gp91 (phox), one of the

membrane heme binding subunits of the superoxidegenerating

NADPH oxidase, by siRNA significantly attenuated

the taxol-neuronal death. The results of this study

indicated that taxol induces cellular oxidative neuronal

apoptosis by increasing the activity of NADPH oxidase, a

non-mitochondrial source of ROS [83].

Platinum-based anticancer drugs, such as cisplatin, have

limited usage due to the onset of peripheral nervous system

dysfunction, which can be severe and persistent over a

long period of time [82]. By using the antioxidant lignin

(Schisandrin B) against cisplatin (cDDP), it was demonstrated

that cisplatin deleterious effects in the brain, such as

DNA damage and lipid peroxidation, were inhibited [85]. Histological

analysis performed on peripheral nerves revealed that

the antioxidant alpha-tocopherol also protected the mice from

severe neurologic damage induced by cisplatin treatment [86].

Finally, another example is the folic acid antagonist MTX,

which is widely used as a cytotoxic chemotherapeutic agent.

However, MTX-associated neurotoxicity is an important clinical

problem. Uzar et al. demonstrated that the antioxidant

caffeic acid phenethyl ester (CAPE) prevented cerebellar lipid

peroxidation induced by MTX in rats [84]. Another similar

study demonstrated that chlorogenic acid (CLG) prevented

the increased lipid peroxidation and lower levels of antioxidant

enzymes observed after MTX treatment. Moreover,

MTX caused severe loss of Purkinje cells and apoptotic cell

death in the cerebellum and the previous administration of

CLG markedly attenuated the adverse effects of MTX [87].

5. In vitro detection of ROS and platforms


What makes a good OS marker There are two main prerequisites:

firstly, the marker must be augmented when exposed

to increased OS inducers and, secondly, the marker must

remain unchanged when OS is not present.

Fluorimetric assays are among the techniques with the

highest sensitivity for OS detection in living cells [88,89]. Cells

can be pretreated or loaded with a nonfluorescent compound

that becomes fluorescent on reacting with ROS. In addition

to high sensitivity and specificity, other factors should be considered

in the design of probes for biological systems, such as

low auto-oxidizability, high photostability, good solubility in

aqueous and lipid environments, good cell permeability and

low interference from biological environments [90].

5.1 The classical and the new dyes for OS


Here, we discuss some examples of the most currently used

dyes. Dye specifications can be found in Table 1.H 2 -DCFDA

(2¢,7¢-dichlorodihydrofluorescein diacetate) is a nonpolar

compound that is converted into a nonfluorescent polar derivative,

H 2 DCF, by cellular esterases that remove the acetate

groups (DA). The membrane-impermeable H 2 DCF is rapidly

oxidized to the highly fluorescent 2¢,7¢-dichlorofluorescein

(DCF) in the presence of intracellular ROS. This probe is

largely localized in the cytoplasm and reacts predominantly

with hydroxyl radicals (OH) and peroxynitrite (HNO 3 ). In

spite of the fact that the auto-oxidation of this dye can make

it difficult to quantify the levels of ROS present, the

DCF assay is commonly used for both mitochondrial and

nonmitochondrial ROS detection [89]. A fundamental

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Table 1. Properties of fluorescence probes commonly used for reactive oxygen species (ROS) detection.

Fluorescent probes Localization Advantages/limitations Excitation/

emission wavelengths


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H 2 DCF-DA (dichloro




Almost exclusively


Detects intracellular ROS/reactive

nitrogen species

Unsuitable probe for intracellular

H 2 O 2 and other ROS

Long-term retention in cells

Less susceptible to oxidation

limitation of DCF is that on oxidation to its fluorescent form,

the compound leaks out of the cells. The intracellular halflife

of DCF is in the range of only minutes and this aspect

limits its use in confocal microscopy or fluorescenceactivated

cell sorting (FACS), as well as for microplate reader

assays in intact cells [91]. Also redox-active metals such as Fe 2+

promote DCFH oxidation in the presence of oxygen and

hydrogen peroxide [92]. Many studies reported that products

of the reaction between hydrogen peroxide and various cellular

peroxidases as well as other oxidant species, including

peroxynitrite, are capable of oxidizing DCFH 2 to DCF.

Thus, several studies have raised concerns about the reliability

of using DCF fluorescence as a quantitative measurement of

ROS formation, as DCFH 2 itself can lead to the formation

of ROS [93]. For instance, O’Malley et al. reported that

some redox active compounds such as pyocyanin, phenazine

methosulfate, mitoxantrone and ametantrone directly oxidize

DCFH 2 to DCF [94]. This work provided an additional concern

since the generation of fluorescent oxidation products

of DCFH 2 and other probes can occur through direct oxidation

of the probes by redox-active compounds independent of

ROS production. Furthermore, interference with media composition

has also been reported [91]. To surpass the limitations

of DCF, derivatives of this dye with a thiol-reactive chloromethyl

group (among others) such as 5,6-chloromethyl-

H 2 -DCF have been developed and allow for covalent binding

to intracellular components, allowing for a longer retention

time within the cells [95].

Another dye, dihydrocalcein-AM, is freely permeant to cell

membranes and is oxidized to green-fluorescent calcein,

which has superior retention properties in cells that have

intact membranes. The oxidation product of calcein is known

to be retained in the cells for several days. In comparison

Ex. 485 nm

Em. 530 nm

Ex. 488 nm

Em. 515 nm

MitoTracker red


High mitochondrial



123 (DHR123)

Mitochondria Peroxide and peroxynitrite detection Ex. 488 nm

Em. 520 nm

DHE (dihydroethidium) Nucleic acids Detects intracellular superoxide Ex. 480 or 396 nm

Allows real-time monitoring of its (recently proposed)


Em. 567 or 400 nm

(recently proposed)


Nuclear and Readily oxidized by superoxide but Ex. 510 nm

mitochondrial not by other ROS- or RNS-

Em. 580 nm

generating systems

Suitable for mitochondrial ROS

detection of H 2 O 2 and lipid

hydroperoxides (possible)

Ex. 578 nm

Em. 599 nm




Microplate reader



Microplate reader


Microplate reader





with DCFDA, imaging with calcein appears to be less susceptible

to photo-oxidation [91], which is helpful when

using microscopy.

Dihydrorhodamine 123 (DHR123) can passively diffuse

across membranes where it is oxidized to the cationic rhodamine

123, which can accumulate in mitochondria and exhibit

green fluorescence. DHR123 can detect ROS including

hydrogen peroxide and peroxynitrite anion (ONOO -- ) [96].

The superoxide indicator dihydroethidium, also called

hydroethidine, has become a probe of choice for the detection

of intracellular superoxide. Over the past 20 years, hydroethidine

has been used in diverse biological systems, ranging

from intracellular organelles to whole cells and organs in live

animals [97]. Dihydroethidium exhibits blue-fluorescence in

the cytosol until oxidized, when it intercalates within the cell’s

DNA, staining its nucleus bright fluorescent red.

MitoSox is a fluorogenic HE derivative dye, which contains

a positive charge on the phosphonium allowing this

compound to be specifically targeted to mitochondria and

producing red fluorescence on direct oxidation by superoxide

anion [89,97]. MitoSox accumulates in the mitochondria

as a function of the mitochondrial membrane potential and

exhibits fluorescence on oxidation and subsequent binding

to mitochondrial DNA. In numerous recent studies, Mito-

Sox was validated by fluorescent/confocal microscopy and

flow cytometry for selective detection of mitochondrial

superoxide production in endothelial cells, cardiomyocytes,

keratinocytes, fibroblasts, epithelial and lymphoid cells and

neuronal cells, as well as in isolated blood vessels [98].

The delivery of ROS-detecting systems to mitochondria

and the development of highly specific and selective

probes aim at understanding mitochondrial redox biology in

particular. In general, small molecular probes that target

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mitochondria possess an overall positive charge that is delocalized,

allowing their passage through plasma membranes and

their accumulation within mitochondria in a Nernstian manner,

such as the rhodamine derivatives. In a similar way, TPP

(triphenylphosphonium) can be used to deliver a variety of

cargoes to the mitochondria. Previous work from

Chang et al. (2004) established that H 2 O 2 -mediated conversion

of aryl boronates to phenols can provide a chemospecific,

biologically compatible reaction method for detecting endogenous

H 2 O 2 production. Appending a TPP moiety onto a

boronate-masked hybrid fluorescein/rhodamine reporter originates

MitoPY1, which shows specific mitochondrial localization

and responds selectively to an increase in H 2 O 2 levels by

a turn-on fluorescence increase [99]. Mitochondriapenetrating

peptides (MPPs) can also be used to detect ROS

generation by balancing charge and lipophilicity through a

combination of natural and synthetic amino acids [100]. Various

cargoes can be delivered to the mitochondria, including a

singlet-oxygen generating fluorophore that allows the local

generation of ROS. Rhodamine-like fluorophores capped

with either a 4-amino-phenyl aryl ether (MitoAR) or a

4-hydroxy aryl ether group (MitoHR) have been also

exploited [101]. The ether capping groups quench the fluorophore

emission by photoinduced electron transfer (PET).

Fluorescent imaging experiments have shown that MitoAR

can accumulate selectively within the mitochondria of

live HeLa cells and respond with increased fluorescence to

exogenously added HOCl [90].

Recently, a Coumarin-Neutral Red (CONER) nanoprobe

was designed and was shown to be suitable for the detection

of the hydroxyl radical, but presented less sensitivity to other

ROS such as superoxide anion, hydrogen peroxide, singlet

oxygen and hypochlorite [102].

OS can not only be assessed though measurement of ROS

levels, but also by monitoring the level of cellular damage,

such as through the measurement of lipid peroxidation (isoprostanes,

MDA formation) [103], DNA oxidative damage

peroxidation (through 8-oxo-d guanosine detection) [61,104],

protein oxidation (thiol oxidation, carbonylation, nitration

of tyrosine) [105]. Established protocols are available and

many of the assays are commercially available [106-108].

5.2 Available platforms for ROS detection

What are the types of platforms available to use the plethora

of ROS detection dyes that we have discussed Microplate

readers equipped with UV, fluorimetric or

luminescence detection can offer high-throughput sample

analysis in a 96-, 384- or 1536-well format. The great

advantage of using microplate readers is that several compounds

and/or conditions can be performed in the same

plate, thereby decreasing the variability encountered when

separate cell culture dishes are analyzed. In addition, dose

response curves can be easily generated. However, an

inherent problem with cell culture in the microplate

format is that loss of cells can occur as a consequence of

physical stressors such as medium addition or removal. In

order to reduce this fundamental problem, Hoechst

nuclear or propidium iodide staining can be used to normalize

the results of the cell density of each well. Many

ready-to-go assay kits have been developed in recent years

that make the detection of ROS a user-friendly process.

5.2.1 Imaging approaches

The imaging/microscopy approach has the clear advantage of

not only detecting but also localizing intracellular ROS for the

study of cell death mechanisms under various conditions [89].

Confocal microscopy allows visualization of intracellular

ROS and the detection of their heterogeneity. Furthermore,

it provides the possibility of fast ROS monitoring in different

regions of the cell and detection of ROS flashes. However, the

rather complex chemistry of the reactions involved in the

transformation from the reduced to oxidized, fluorescent

form, and several processes and side effects associated with

laser irradiation may lead to difficulties in data analysis

and interpretation.

5.2.2 Electron spin resonance spectroscopy

Electron spin resonance (ESR) spectroscopy is a technique

used for studying chemical species that have one or more

unpaired electrons, such as organic and inorganic free radicals

or inorganic complexes possessing a transition metal ion.

Transitions can be induced between spin states by applying

a magnetic field and then supplying electromagnetic energy,

usually in the microwave range of frequencies. The resulting

absorption spectra are described as ESR or electron paramagnetic

resonance (EPR). Electron spin should be used with a

spin trapping technique allowing the detection of the shortlived

free radicals resonance and is the most effective and

direct method for detecting free radicals [89,109]. ROS levels

can be estimated from the magnitude of spectral peaks and

are expressed in arbitrary units. Although superoxide radicals

and peroxynitrite are detected by ESR, hydrogen peroxide is

not [89]. Samples can be incubated with different spin probes

(spin trapping technique) such as 1-hydroxy-3-carboxypyrrolidine

(CPH), which is a cell-membrane permeable probe,


(PPH), which is a non-permeable probe. Furthermore, the

application of different spin probes, permeable versus nonpermeable,

allows one to localize and distinguish mitochondrial

versus extramitochondrial ROS and intracellular versus

extracellular ROS. The latter cannot always be detected

by confocal fluorescent microscopy, and in this case, ESR

analysis would serve as a necessary complementary approach.

Another advantage of the ESR approach is that cells or tissue

samples can be frozen, stored, collected and later analyzed

later. However, the sensitivity of ESR is lower when compared

with fluorimetric methods. For instance, ESR cannot

be applied for ROS detection and determination of intracellular

distribution in a single cell, but it can be used for

analysis of suspensions of cells and isolated [89,110,111].

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5.2.3 Redox proteomics

Besides the more traditional detection methods, redox

proteomics is an emerging tool for OS detection. One

can identify proteins that have undergone oxidative

modification, together with the molecular and site-specific

details of these oxidative events. Until now, most

of the redox proteomic studies have used two-dimensional

gel electrophoresis coupled with mass spectrometry (MS).

The quantitative determination of the redox state of

a protein is informative but represents a challenge. The

2D-PAGE techniques effectively separate membrane

proteins having one and two transmembrane segments

but not those with more than four. Also poor separation

of hydrophobic, acidic and alkaline proteins is a strong

limitation of this technique. An improvement of 2DEbased

analysis has been obtained by the application of

two-dimensional differential gel electrophoresis (DIGE)

technique [112]. This approach uses a set of fluorophores

of similar molecular weights and chemical structures

that differ in the absorption and emission wavelengths.

Redox-DIGE has been performed with the use of derivatives

of cyanine, NEM (N-ethylmaleimide) or IAM

(iodoacetamide) and DY--dyes (maleimide-conjugated

infrared dyes). A limitation of all the methods described

above is that only abundant proteins are detected and

transcription factors or other regulatory proteins are

often not detected. A comparative study on the three

quantitative methods frequently used in proteomics, 2D

DIGE, cICAT (cleavable isotope-coded affinity tags)

and iTRAQ (isobaric tags for relative and absolute

quantification) was carried out. iTRAQ is a new LC (liquid

chromatographic)-based technique. In this study,

DIGE was sometimes compromised due to comigration

or partial comigration of proteins while iTRAQ

was more susceptible to errors in precursor ion isolation.

The global-tagging iTRAQ technique revealed to be

more sensitive than cysteine-specific cICAT method,

which was in turn as or more sensitive than DIGE

technique [113].

Post-translational modifications, such as protein tyrosine

nitration, are also considered a valid biomarker of protein

RNS insult. It can be analyzed by immunochemical techniques

using anti-3-nitrotyrosine antibodies. High-throughput

electrospray ionization high-performance liquid chromatography

(HPLC)--MS/MS analysis combined with immunological

techniques can be also useful to identify nitrated tyrosine

modifications among several mitochondrial proteins [114].

Another method used for OS assessment includes PCR

(polymerase chain reaction) arrays that contain an assembly

of genes involved in OS and antioxidant defense mechanisms.

Another technology involves the use of reporter cell

lines and hopefully, in the near future, the development of

reporter animal models. One such reporter cell line is the

Nrf2 reporter, which is activated by compounds that induce

OS [115].

5.3 Methods for the determination of the

antioxidant status

What methods are used for detecting the cellular antioxidant

status The cellular antioxidant status can be assessed by measuring

reduced GSH, GSH peroxidase activity [116], GSH

reductase activity, catalase activity and SOD activity. The traditional

methods for GSH measurement involved biochemical

assays dependent on the reaction of compounds such as

5,5’-dithio-bis-2-nitrobenzoic acid or o-phthalaldehyde that

immediately form chromophoric or fluorescent compounds

measurable by UV/visible or fluorescent spectroscopy, respectively.

This method is reliable and highly quantitative;

however, it is time consuming and not suitable for highthroughput

studies. GSH can also be detected through

HPLC and is considered the most quantitative method.

Despite its advantages, HPLC is also time consuming and of

high cost [117]. Labeling intracellular thiols with fluorescent

bimane compounds and FACS (fluorescent-activated cell

sorting) analysis is an accepted alternative. Recently, the application

of microchip electrophoresis with laser-induced fluorescence

(MCE-LIF) was used to detect simultaneously

GSH and hydrogen peroxide in mitochondria. Organoselenium

probe Rh-Se-2 and bis(p-methylbenzenesulfonate)

dichlorofluorescein (FS) were used as fluorescent probes for

GSH and H 2 O 2 , respectively [118].

The activity of catalase can be determined using assays

based on changes in absorbance of hydrogen peroxide at

240 nm. Commercial kits are widely available for catalase

as well as for GSH reductase, GSH peroxidase and

SOD [119-121].

Table 2 shows a summary of OS measurement approaches.

6. Animal models suitable to study OS

In order to investigate the consequences of OS in vivo, one

important tool originates from genetic manipulation of

mice. Animal models for OS toxicity assessment are being

developed through knockout of important antioxidant

enzymes (Table 3). It is clear that one can carry out studies

using exogenously administered toxicants that induce OS,

observe and characterize the response of the animal model

to this exogenous insult. An alternative is to use genetic models

that have a constitutive pro-oxidative environment within

certain cells or tissues and ideally elicit pathology as a

result [122]. One such model is the heterozygous Sod2 knockout

mouse (Sod2 +/- ). These mice have approximately 50%

reduction in SOD activity and present a phenotype that is

indicative of decreased mitochondrial function and an

increase in oxidative damage such as a 30 -- 80% increase in

nuclear and mitochondrial DNA oxidation (8-oxo-dG).

This model has been used in studying idiosyncratic liver toxicity

[123] and for the investigation of toxicity caused by

AZT [124].

Homozygous Cat (catalase) knockout mice (Cat -/- ) are

completely deficient in catalase and exhibit a retarded rate in

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Table 2. In vitro and ex vivo assessment of oxidative


Type of damage

DNA/RNA damage

Lipid peroxidation

Protein oxidation/nitration

Reactive oxygen species



8-Hydroxyguanosine (8-OHG)



Abasic (AP) sites


Double-strand DNA breaks

Comet Assay (general DNA


UV DNA damage

4-Hydroxynonenal (4-HNE)

8-iso-Prostaglandin F2alpha


Malondialdehyde (MDA)


Protein Carbonyl Content (PCC)


Advanced Glycation End

Products (AGE)

Advanced Oxidation Protein

Products (AOPP)

In vitro ROS

In vivo ROS



Superoxide Dismutase

Oxygen Radical Antioxidant

Capacity (ORAC)

Hydroxyl Radical Antioxidant

Capacity (HORAC)

Total Antioxidant Capacity (TAC)

decomposing extracellular hydrogen peroxide in comparison

with wild-type mice [125]. However, heterozygous mice

(Cat +/- ) do not show any significant increase in OS caused

by photochemical reaction nor do they exhibit a higher susceptibility

to hyperoxia-induced lung injury, suggesting that

the antioxidant function of catalase is negligible in these two

models of oxidant injury. However, brain mitochondria of

these mice are more susceptible to trauma-induced

impairment in OXPHOS than wild-type mice. This acatalasemic

mouse model can be important as a model to further

understand the mechanism of various drugs and associated

diseases related to elevated OS, such as neurodegenerative

diseases, cancer and aging [126]. This mouse model has also

been utilized to examine the toxicity observed by AZT [124].

Another mouse model for OS is the Gpx1-knockout mice

(Gpx -/- ). It has been demonstrated that these mice are highly

susceptible to exogenous ROS generators such as paraquat

and diquat, although they exhibit little or no elevation in basal

levels of oxidative damage, and compensatory antioxidant

upregulation is often observed [127].

The g-glutamyltranspeptidase (GGT)-deficient mouse

excretes large amounts of GSH in urine and exhibits tissuespecific

GSH depletion [128]. The condition causes growth

retardation, cataract development, alteration in the coat color

and premature death. The addition of N-acetylcysteine to the

drinking water of GGT-deficient mice results in reversal of

the phenotype [128].

g-Glutamyl transpeptidase-deficient knockout mice have

been utilized to study cisplatin toxicity to the proximal

tubules in the kidney. Cisplatin was not toxic to GGTdeficient

mice suggesting that nephrotoxicity of cisplatin is

the result of the metabolism of the drug through a GGTdependent

pathway [129]. Other compounds similar to

cisplatin can be evaluated using this mouse model and the

oxidative damage can be determined.

The homozygous knockout Trx2 mouse is embryonic

lethal and partial deficiency of Trx2 results in a decrease in

mitochondrial function by the decrease of ATP levels, decline

in the OXPHOS activity and increased H 2 O 2 formation [130].

Heterozygous Txr2 +/- mice have higher oxidative damage to

protein (protein carbonyl), lipid (F2-isoprostane) and DNA

(8-oxo-dG). These mice present mitochondrial dysfunction

and oxidative damage; however, their life span is not

reduced. Txr2 +/- mice have been shown to be more sensitive

to diquat-induced oxidative damage and also apoptosis [130].

A very recent study on APAP-induced OS and liver injury

used the mouse model of cyclophilin-D-deficient mice.

Cyclophilin-D is a regulatory component of the mitochondrial

membrane transition pore [131]. The treatment of wildtype

mice with APAP resulted in necrosis, nuclear DNA

fragmentation and formation of ROS and peroxynitrite while

the CypD-deficient (Ppif( -/- )) mice were completely protected

against APAP-induced liver injury and DNA damage [132].

This mouse model, in contrast to the other presented models,

is resistant to OS and can, therefore, potentially be used as a

negative control.

The reporter mice have been developed for the rapid, mechanistic

assessment of potential toxic effects in vivo [133,134].

A transcriptionally regulated ‘stress’ promoter from a gene

involved in toxicity pathway is used to drive the expression of

two reporter proteins: an in situ LacZ reporter and an excreted

human chorionic gonadotropin (hCG) beta chain reporter.

Any compound that causes induction of the stress promoter

induces expression of the described reporters. The excreted

hCG biomarker reporter enables real-time and noninvasive

tracking of the stress response via blood and urine analysis.

The LacZ reporter, via immunohistochemistry, enables identification

of the precise target organ and cellular location of the

stress response [133]. The ToxReporter Mice for OS (Hmox

promoter) provides a means of studying the relationship

between chemical exposure and toxic response in a time- and

dose-dependent manner in a single mouse [134].

Animal models shed light on potential cellular targets and

consequences of in vivo ROS and allow the evaluation of

potential therapies against endogenous OS. Furthermore,

even though these animal models are not widely used to evaluate

drug-induced OS, one might hope that the use of

these models will allow us to better understand the overall

consequences of augmented ROS levels in organisms [122].

12 Expert Opin. Drug Metab. Toxicol. [Early Online]

Pereira, Nadanaciva, Oliveira & Will

Table 3. Mouse models potentially useful to study the contribution of oxidative stress to drug-induced organ


Animal models Phenotype/dysfunction Study examples

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Sod2 +/-

Decreased mitochondrial function, increased oxidative damage,

increased nuclear and mitochondrial DNA oxidation

AZT, methamphetamine (METH),


Cat -/- Retarded rate decomposing extracellular hydrogen peroxide AZT, ethanol

Cat +/-

Any significant increase in oxidative stress caused by

photochemical reaction or more susceptibility to hyperoxiainduced

lung injury

Gpx1 -/- mouse

Cyclophilin-D deficient

(Ppif( -/- ))




Trx2 -/- mouse

Trx2 +/- mouse

Highly sensitive to exogenous ROS generators

No elevation in basal levels of oxidative damage

Do not show any compensatory antioxidant mechanisms

Protection against APAP-induced liver injury and DNA damage

Glutathione excretion in the urine

Tissue-specific glutathione depletion

Growth retardation/cysteine deficiency, cataract formation,

alteration in coat color and premature death

Embryonic lethality, apoptosis

Decrease in mitochondrial function by the decrease in ATP levels,

decline in the OXPHOS activity and increased H 2 O 2 formation

These novel discoveries may be useful not only to find

new targets but also to understand the mechanisms behind

drug-induced OS and, consequently, augment drug safety


7. In vivo noninvasive ROS detection

The strategies to assess ROS in vivo remain limited. Samples

can be collected from treated animals and analyzed for various

markers, such as GSH status and antioxidant enzymes as well

as downstream events such as lipid and protein and DNA oxidation.

Samples can be analyzed using commercial kits and

plate reader platforms. PCR arrays, ESR and redox proteomics

can also be applied ex vivo to collected samples. However,

none of these methods are suitable for measuring OS

in a noninvasive manner in patients.

Considerable challenges remain in designing specific

probes that can be utilized noninvasively for patient monitoring.

In 2005, a noninvasive way of measuring OS damage in

humans through breath analysis of exhaled aldehydes, which

are indicators of free radical damage in the body [135], was

developed. This is the only device in use in humans and is

used to monitor effectiveness of antioxidant supplements

and exercise in reducing free radical burden. It would be

interesting to utilize this technology for toxicity monitoring

(available at [136]).

Recently, Dongwon et al. (2007) developed highly specific

and sensitive peroxalate-based nanoparticles. These nanoparticles

are attractive for in vivo imaging because they have tunable

wavelength emission (460 -- 630 nm), nanomolar

sensitivity for hydrogen peroxide and exhibit specificity [137].

Another study showed a chemoselective bioluminescent

reporter for H 2 O 2 and mice. The ability of Peroxy Caged

Luciferin-1 (PCL-1) to detect H 2 O 2 selectively and in a

concentration-dependent manner was established. Moreover,

subsequent studies with exogenous H 2 O 2 and antioxidant

treatment showed that PCL-1 can visualize basal H 2 O 2 basal

levels and H 2 O 2 fluctuations in luciferase-expressing mice

(FVB-luc+ mice) without removal of fur or skin [138].

8. Conclusions

This review discussed the role of OS as a contributor to a variety

of organ toxicities, such as liver, heart, kidney and brain.

Mitochondria are still considered not only the major source

of ROS production but also an important target for ROS deleterious

effects caused by different drugs. Even though several

methods to measure OS both in vitro and in vivo are being

developed, one must be careful when interpreting the results

due to interference, cross talk or nonspecificity of dyes. Assays

can be performed using a variety of platforms with different

throughput capabilities and resolutions, and measurements

of antioxidant status can be performed using biochemical

assays or commercial available assay kits. However, the lack

of noninvasive methods for in vivo ROS detection in suitable

animal studies is still a subject of concern.

9. Expert opinion

Paraquat, diquat, ethanol, doxorubicin,


Acetaminophen, diclofenac (DCLF)

Cisplatin, dexamethasone (DEX),


Cat: Catalase; GPx: Glutathione peroxidase; Gpx1: Glutathione peroxidase; MnSOD/SOD2: Manganese superoxide dismutase 2; Ppif: Peptidylprolyl isomerase; Trx2:



The complexity of the cellular redox system makes the detection

of ROS in vitro and in vivo challenging. This implies that

combinations of different approaches will likely lead to more

Expert Opin. Drug Metab. Toxicol. [Early Online] 13

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accurate results. In fact, it is difficult to study a system without

altering it, since most probes result in increased OS by

themselves or disturb several cell function related to oxidant

and antioxidant metabolism. This is a major concern and

a driving force for the development of better markers and

techniques to follow OS in several biological systems.

Knockout animal models suitable for studying OS have

been developed, but have been underutilized for the study of

drug-induced organ toxicity. The same is true for reporter

mice. In addition, we need to understand the impact of lowlevel

long-term OS to the development of organ toxicity.

Even though many drugs can cause OS and subsequent organ

toxicity, not all drugs within a class do so. By implementing

OS assessment early in the drug discovery process, one can

choose drugs that have the desired efficacy but lack the

OS liability.

The development and validation of new and more complete

methods and tools to assess OS, such as redox proteomics

approaches and the use of animal models, are critical

and should be our priority in the next years. Even though

many studies focus on the deleterious effects of ROS, an

emerging line of thought focuses on the possible beneficial

aspects of ROS in the living systems. Since increased ROS levels

are associated with cancer, diabetes, inflammatory diseases,

ischemia-related diseases and neurodegeneration, multiple

clinical trials have evaluated the efficacy of antioxidants

against the above-mentioned pathologies. Yet most of these

trials were not well succeeded [4]. In addition, genetic experiments

in mouse models have cast doubt on the idea that less

is better when it comes to ROS. For instance, SOD2 -/- mice

die before birth, as mentioned before, and their heterozygous

littermates exhibit mitochondrial damage and oxidative

modifications. However, SOD2 +/- mice have a normal life

span. Recently, new publications drew a correlation between

high OS and life span extension [139]. Mice heterozygous for

mitochondrial GSH peroxidase 4 or the ubiquinone biosynthesis

protein CLK1 display high levels of mitochondrial

OS, yet live longer than their wild-type counterparts [7]. Furthermore,

life span extension in Caenorhabditis elegans mediated

by glucose restriction is associated with increased

mitochondrial metabolism and ROS production [139]. Thus,

it seems that certain levels of ROS might be essential to

some vital functions and for life span.

One of the challenges that we face is that OS can be low in

the young-aged biological entity but can accumulate over

time. This is difficult to mimic in a short-term in vitro assay.

Moreover, OS biomarkers are not part of routine evaluation

in regulatory animal studies. Hence, OS and the absence of

frank organ toxicity could be initially missed and may manifest

itself only in chronic treatment of patients. An added

complication is that patient susceptibility (idiosyncratic

responses) to OS could be due to differences in the antioxidant

status of the individual. With human longevity increasing

as science and medicine improves, it becomes essential

that noninvasive methods to measure drug-induced OS in

patients are developed.

Declaration of interest

Y Will and S Nadanaciva are both employees of Pfizer.

CV Pereira research scholarship SFRH/BD/48029/2008

while PJ Oliveira is supported through a research grant

PTDC/SAU-TOX/110952/2009 from the Foundation for

Science and Technology, Portugal (FCT). The authors

declare they have no conflicts and have received no payment

for the preparation of this manuscript.

14 Expert Opin. Drug Metab. Toxicol. [Early Online]

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Claudia V Pereira, Sashi Nadanaciva,

Paulo J Oliveira & Yvonne Will † PhD

† Author for correspondence

Pfizer R&D,

Compound Safety Prediction- WWMC,

Cell Based Assays and Mitochondrial Biology,

Eastern Point Rd, Groton,

CT 06340, USA

Tel: +1 860 686 2832;


Expert Opin. Drug Metab. Toxicol. [Early Online] 19


Mitochondria as a Biosensor for Drug-Induced

Toxicity – Is It Really Relevant

Ana C. Moreira 1,2 , Nuno G. Machado 2 , Telma C. Bernardo 2 ,

Vilma A. Sardão 2 and Paulo J. Oliveira 2

1 Doctoral Programme in Experimental Biology and Biomedicine, University of Coimbra

2 Center for Neuroscience and Cell Biology, Department of Life Sciences,

University of Coimbra


1. Introduction

Mitochondria, from the Greek mito (thread) and chondros (grains) are small organelles that

exist as a network in the cytoplasm of eukaryotic cells, performing a variety of important

functions including energy production, calcium homeostasis, fatty acid metabolism or heme

and pyrimidine biosynthesis (Pereira, Moreira et al., 2009). Moreover, mitochondria play a

critical role in programmed cell death (apoptosis) (Jeong & Seol, 2008; Wang & Youle, 2009).

Mitochondrial structure comprises two different membranes, the outer (OMM) and the

inner membrane (IMM) that functionally separate two distinct compartments, the intermembrane

space (IMS) and the matrix (Jezek & Plecita-Hlavata, 2009) (Fig. 1). The outer

membrane encloses mitochondria and it is somewhat identical to other cell membranes,

including cholesterol in its composition, and is permeable to a large variety of ions and

metabolites. The inner membrane lacks cholesterol, is rich in the tetra fatty acid-containing

phospholipid cardiolipin, and basically controls the entry of metabolites and ions into

mitochondria, through the action of specific transport proteins (Scatena, Bottoni et al., 2007).

Inner membrane invaginations and membrane enclosed structures which can exist

connected to the IMM or freely in the mitochondrial matrix are called cristae. It is in these

latter structures that most of the membrane-bound metabolic proteins and energyproducing

respiratory complexes (complexes I–V) exist (Fig. 1) (Zick, Rabl et al., 2009).

1.1 Organization and genomics

Also considered as a reticulum, the mitochondrial network continuously moves, fuses and

divides in a process tightly regulated by cellular stimuli and disturbances inside this

organelle (Detmer & Chan, 2007). The shape greatly varies depending on the tissue,

developmental and physiological state. Within a cell, the distribution of mitochondria is

unequal depending on the cellular energetic or metabolic demands (Grandemange,

Herzig et al., 2009). The overall shape of mitochondrial network results from an

equilibrium between fusion and fission events (Wallace & Fan, 2010). These events allow

the exchange of organelle contents such as membrane lipids, proteins, solutes, metabolites

and even mitochondrial DNA (Detmer & Chan, 2007), as well as to provide a balance of


Biosensors for Health, Environment and Biosecurity

the electrochemical gradient (Twig, Graf et al., 2006). Balanced mitochondrial fusion and

fission is crucial to preserve mitochondrial integrity and functionality (Wallace & Fan,

2010). Three distinct proteins seem to be involved in mitochondrial fusion: Mitofusins 1

and 2 (Mfn1 and Mfn2) and Optic Atrophy-1 (OPA-1). Mfn 1 and 2 are GTPases proteins

that are localized in the OMM and form homo- and hetero-oligomeric complexes between

themselves and with counterparts in adjacent mitochondria, which mediate their tethering

(Arnoult, 2007). OPA-1 is a dynamin-related protein that can be found in a soluble form in

the IMS or tightly associated with the IMM, being a key protein for the fusion of this

mitochondrial membrane (Arnoult, Grodet et al., 2005). Evidence also suggests that OPA-

1 controls cristae morphology and is implicated in the complete release of cytochrome c

during apoptosis (Jourdain & Martinou, 2009; Perkins, Bossy-Wetzel et al., 2009). Fusion

of both membranes is a two-step process that occurs in a coordinate fashion, although the

precise mechanism remains unclear (Malka, Guillery et al., 2005). Mitochondrial fission

requires the recruitment of dynamin-related protein 1 (Drp1) from the cytosol to the

OMM where it forms multimeric rings and spiral-like structures that surround and

constrict the organelle in a GTP-dependent manner (Sheridan & Martin, 2010). The

mechanism that triggers this recruitment is still unknown, however, Fis1, a small

mitochondrial transmembrane protein, seems to be responsible for this mobilization

(Sheridan & Martin, 2010).

Mitochondria are the only organelles outside of the nucleus that contain their own genome

and replicate itself in an independent manner from the nuclear genome. A single DNA

polymerase (polymerase-gamma), with base excision repair activity, ensures the replication

of the mitochondrial DNA (mtDNA). Moreover, mtDNA has a particular feature since it is

exclusively maternally inherited. Each mitochondrion contains approximately 10-15 copies

of a small circular chromosome that are organized into one or more structures called

nucleoids. Mitochondrial DNA encodes for 13 proteins that are essential for the electron

transport and ATP generation by oxidative phosphorylation (OXPHOS) and 2 rRNA and 22

tRNA (Van Houten, Woshner et al., 2006). The remaining proteins required for

mitochondrial activity are encoded by the nucleus, synthesized in the cytosol and

translocated to mitochondria (Wallace, 2008). Mitochondrial DNA undergoes a mutation

rate that seems to be between 5- to 20- fold higher than what occurs in nuclear DNA

mutations, although this is not consensual (Malka, Lombes et al., 2006; Scatena, Bottoni et

al., 2007). The high rate of mutations, if indeed real, can be explained for both the lack of

mtDNA protective proteins and its proximity to the electron transport chain, where the

majority of, free-radical production occurs (Fruehauf & Meyskens, 2007). Furthermore, the

repair mechanism of mitochondrial DNA is less efficient than of nuclear DNA (Berneburg,

Kamenisch et al., 2006).

1.2 Oxidative phosphorylation and energy production

Production of energy within a living cell is performed by the conversion of dietary fats and

carbohydrates into reducing equivalents. Mitochondria are considered the powerhouses of

the cell, due to a variety of important energy-producing metabolic pathways in their

interior. Pyruvate is formed in the cytosol as an end-product of glucose metabolism

(glycolysis) and can undergo lactic acid or alcoholic fermentation in the absence of oxygen

(anaerobic conditions). Under aerobic conditions, pyruvate is converted into acetyl

coenzyme A (acetyl-CoA) by pyruvate dehydrogenase (PDH) in the mitochondrial matrix

(Pereira, Moreira et al., 2009).

Mitochondria as a Biosensor for Drug-Induced Toxicity – Is It Really Relevant 413

Fig. 1. Mitochondria play a critical role in ATP production, biosynthesis, calcium homeostasis and cell death. The figure represents

some of the functions referred to in the text: (A) The Krebs cycle occurs in the matrix and supplies reducing equivalents for oxidative

phosphorylation, besides participating as intermediate in several biosynthetic pathways. (B) Overall view of mitochondria

morphology: The outer mitochondrial membrane (OMM) encloses the organelle within the cell; the inner mitochondrial membrane

(IMM) separates functionally the matrix from the mitochondrial inter-membrane space (IMS). (C) Oxidative phosphorylation

(OXPHOS): electrons from the Krebs cycle are transferred along the respiratory chain. The energy derived from electron transfer is

used to pump out protons across the inner membrane at complexes I, III and IV, creating a proton electrochemical gradient between

both sides of inner membrane. This electrochemical gradient forms a proton-motive force, which is use to drive the re-entry of

protons to the matrix through complex V (ATP synthase). A small amount of electrons can leak towards the matrix through complex I

and complex III due to an one electron transfer reduction of molecular oxygen forming superoxide anion (O2 ●- ). Figure adapted from

(Pereira, Moreira et al., 2009), with permission.


Biosensors for Health, Environment and Biosecurity

Acetyl-CoA enters the Krebs cycle, being oxidized to generate several intermediates

including NADH and succinate (Fig. 1). Other intermediates of the Krebs cycle are also

important in several metabolic pathways, including biosynthesis of heme and amino acids

(Shadel, 2005). Mitochondria can be involved in the -oxidation of fatty acids (Vockley &

Whiteman, 2002). The end product of this pathway is, once again, Acetyl-CoA, which is

used in the Krebs cycle. NADH and succinate, among other intermediates that are produced

by different pathways are oxidized by the electron transport chain, ultimately leading to the

production of adenosine triphosphate (ATP) in a process known as OXPHOS (Zick, Rabl et

al., 2009; Hebert, Lanza et al., 2010) (Fig. 1). Electrons derived from reduced substrates are

transferred through several multi-protein complexes (mitochondrial complexes I to IV),

down their redox potentials and the energy derived from electron transfer is used to pump

out protons across the IMM at complexes I, III and IV which creates an electrochemical

gradient between both sides of the IMM. This electrochemical gradient is a proton-motive

force driving the re-entry of protons towards the matrix through complex V (ATP synthase),

which is coupled to ATP synthesis (Hebert, Lanza et al., 2010). ATP that is produced is

exported from the mitochondria by the mitochondrial ADP/ATP translocator (ANT).

Molecular oxygen is the final electron acceptor in the mitochondrial respiratory chain, which

is reduced via a sequential four-electron transfer into water by complex IV (cytochrome c

oxidase, COX). However, some of the electrons that are transferred across the mitochondrial

electron transport chain can escape and perform a single electron reduction of molecular

oxygen. This phenomenon occurs continuously even in normal conditions leading to

formation of superoxide anion (O 2

●-) and it will be discussed in the next section of this


1.3 Generation of free radicals

Among the reactive species that are produced within a living cell, reactive oxygen species

(ROS) are the most significant. Mitochondrial complexes I and III account for a significant

proportion of intracellular ROS formation, although complex I is considered the major

contributor (Adam-Vizi & Chinopoulos, 2006; Soubannier & McBride, 2009). The

mitochondrial electron transport chain contains several redox centers, which can react with

molecular oxygen. As a result, a small amount of electrons leaks from complex I (NADH

dehydrogenase) and complex III (CoQ cycle), performing a one-electron reduction of

molecular oxygen that gives rise to superoxide anion (O 2

●-). Approximately 1-2% of the

oxygen consumed during OXPHOS under physiological conditions is converted into this

product (Solaini, Baracca et al., 2010). Superoxide anion produced by respiratory complex I

is released in the mitochondrial matrix and transformed into hydrogen peroxide (H 2 O 2 )

spontaneously or via manganese superoxide dismutase (MnSOD). In turn, O 2

●- generated by

complex III can be released in both sides of the IMM but in the IMS, the dismutation into

H 2 O 2 is achieved via Cu/Zn-dependent SOD (Cu/ZnSOD). Hydrogen peroxide can be

converted into water in the mitochondrial matrix by catalase or glutathione peroxidase

(GSH). Mitochondrial thioredoxin, glutaredoxin and even cytochrome c are other relevant

ROS scavengers (for a review see (Fruehauf & Meyskens, 2007)). The H 2 O 2 produced can

also diffuse to the cytosol and trigger the activation of some transcription factors and

various enzymatic cascades (Cadenas, 2004). General oxidative stress arises when an

imbalance in the redox steady-state occurs and ROS production exceeds the capacity of the

cell for detoxification. If H 2 O 2 encounters a reduced transition metal (Fe 2+ or Cu 2+ ) or O 2

●- it

Mitochondria as a Biosensor for Drug-Induced Toxicity – Is It Really Relevant 415

can be further reduced in a highly reactive and toxic hydroxyl radical ( ● OH) by a Fenton or

Haber-Weiss reaction, respectively (Brandon, Baldi et al., 2006), which is the most potent

ROS. Although very short-lived, ● OH can damage cellular macromolecules including

proteins, lipids and nucleic acids. The oxidation of proteins can inactivate and target them

for degradation; oxidative damage to DNA causes single and double strand-breaks, crosslink

to other molecules and base modifications, while lipid oxidation can generate

membrane disturbances. As described above, mtDNA represents a critical target of

oxidative damage since it does not contain histones and it is located in proximity to the

production site of ROS (Hebert, Lanza et al., 2010). Once damaged, mtDNA can indirectly

amplify oxidative stress since transcription of critical mitochondrial proteins is defective,

leading to a vicious cycle of ROS production and eventually triggering cell death. Oxidative

stress is largely related with aging (Balaban, Nemoto et al., 2005) and is often associated

with some disorders such as cancer and diabetes (Van Houten, Woshner et al., 2006).

Reactive nitrogen species (RNS), including nitric oxide and peroxynitrite, can also contribute

for a regulation of mitochondrial function (especially the former (Brown & Borutaite, 2007)),

as well for increased mitochondrial damage during pathological conditions (Poderoso,


1.4 Cell death

Unlike what was thought during several years, cell death is not a process only observed

when cell tissues are injured by external factors. Actually, cell death is an evolutionary

conserved and genetically regulated process that is crucial for development, morphogenesis

and homeostasis in tissues (Martin & Baehrecke, 2004). Programmed cell death (PCD) was

the first designation attributed to this regulated process. Later, Kerr et al. introduced the

term apoptosis (Kerr, Wyllie et al., 1972) to designate programmed cell death and these

designations remain synonymous until now. Cell death was classified into two types:

apoptosis (programmed cell death) and necrosis (accidental cell death). Nowadays, other

types of cell death have been identified, including autophagy. Although it has become clear

that autophagy can work as an adaptive response to nutrient starvation, cell death can occur

due to autophagy over-stimulation (Rami, 2009). Autophagy is a spatially restricted

phenomenon characterized by the absence of chromatin condensation and in which parts of

the cytoplasm are engulfed by specialized double membrane vesicles, so-called

autophagosomes, and digested by lysosomal hydrolases (Ulivieri, 2010). Mitophagy is a

specific autophagic elimination of mitochondria, identified in yeast and mammals and

regulated by PINK-1, among others (Youle & Narendra, 2011). However, if for some reason

the clearing of old/damaged mitochondria is insufficient, a malignant transformation may

occur (Morselli, Galluzzi et al., 2009). Necrotic cell death is characterized by a moderate or

null chromatin condensation and by an increase in cell volume that culminates in loss of

plasma membrane integrity and swelling of cytoplasmic organelles (Galluzzi, Maiuri et al.,

2007). The disruption of cell membranes leads to the release of cell contents usually resulting

in local inflammatory reactions and damage to contiguous cells. Several studies have

already demonstrated that mitochondria can be involved in this type of cell death due to a

phenomenon called mitochondrial permeability transition (MPT), which results from the

opening of unspecific protein pores in the IMM. The MPT results in dissipation of

mitochondrial membrane potential (∆) and leads to an uncoupling of OXPHOS and


Biosensors for Health, Environment and Biosecurity

decreased ATP, leading cells to necrosis (Sharaf El Dein, Gallerne et al., 2009; Zorov,

Juhaszova et al., 2009). Apoptosis is the best-studied modality of cell death and plays an

essential role in the maintenance of homeostasis by eliminating damaged, infected or

superfluous cells in a regulated form that minimizes inflammatory reactions and damage to

neighboring cells (Jeong & Seol, 2008; Schug & Gottlieb, 2009; Sheridan & Martin, 2010).

Apoptotic imbalance may contribute to the development of neurodegenerative disorders,

autoimmune disorders, cancer or even viral infections (Arnoult, 2007; Jourdain & Martinou,

2009). Apoptotic cells exhibit specific changes, including chromatin condensation, nuclear

fragmentation, and plasma membrane blebbing. The late stages of apoptosis are

characterized by fragmentation of the cell-membrane into vesicles called apoptotic bodies

which contain intact cytoplasmatic organelles or nuclear fragments. These vesicles are

recognized by the immune system macrophages, preventing inflammatory responses

(Martin & Baehrecke, 2004; Jeong & Seol, 2008; Tait & Green, 2010). There are two main

pathways by which a cell can engage apoptosis: the extrinsic (or cell death receptormediated)

apoptotic pathway and intrinsic (or mitochondrial-mediated) apoptotic pathway

(Tait & Green, 2010) (Fig. 2). In both pathways, the apoptotic process is driven by a family of

cysteine proteases that are expressed as pro-enzymes and are activated by proteolysis.

These proteases, known as caspases, specifically cleave their substrates at aspartic residues

and are categorized into initiators (such as caspases -8 and -9) and effectors or executioners

(such as caspases -3 and -7) (Arnoult, 2007; Jeong & Seol, 2008). Mitochondria are central

players in the intrinsic apoptotic pathway; in fact, mitochondria retain a pool of proapoptotic

factors in the IMS. During the development of the intrinsic pathway, pores are

formed in the OMM in a process called outer mitochondrial membrane permeabilization

(OMMP, different from the mitochondrial permeability transition). The OMMP results in the

release of pro-apoptotic factors, such as cytochrome c and the apoptotic-inducing factor,

AIF, to the cytosol (Saelens, Festjens et al., 2004; Sheridan & Martin, 2010). Although the

effects of pro-apoptotic factors that are released in the cytosol are well characterized, the

mechanisms underlying the OMMP remains controversial (Martinou & Green, 2001) and

there are currently several mechanisms that have been proposed. One of these mechanisms

involves members of Bcl-2 proteins family, which comprises three subgroups; the antiapoptotic

family members such as Bcl-2 and Bcl-xL, the pro-apoptotic Bax/Bak sub-family

and the pro-apoptotic BH3-only proteins such as Bim, Bad, Bid, Puma and Noxa. BH3-only

proteins links cell death signals to mitochondria, where the interplay between various

members of the Bcl-2 family determines the fate of the cell (Martinou & Green, 2001; Wong

& Puthalakath, 2008). A mild change in the dynamic balance of these proteins may result

either in inhibition or exacerbation of cell death. The intrinsic and extrinsic pathways can

interact with each other at the mitochondrial level where signal amplification occurs (Fig. 2)

(Saelens, Festjens et al., 2004).

2. Mitochondria and disease

As it was discussed in the previous sections, mitochondria are organelles with crucial

importance in cell bioenergetics, signaling and survival, among others. Mitochondrial

dysfunction is associated with several diseases, as it will be discussed in the present


Mitochondria as a Biosensor for Drug-Induced Toxicity – Is It Really Relevant 417

Fig. 2. An overview of the extrinsic and intrinsic pathways of apoptosis. The intrinsic and

extrinsic pathways can crossroad in mitochondria, which leads to signal amplification. IMM,

inner mitochondrial membrane; IMS, intermembrane space; OMM, outer mitochondrial

membrane. Figure adapted from (Pereira, Moreira et al., 2009), with permission.

2.1 Cancer

As described above, the impact of mitochondria on cellular physiology is not limited to ATP

production. Due to the importance of mitochondria for cellular functions and cell fate, the

role of these small organelles in cancer cell biology is becoming increasingly recognized.

The first suggestion about the role of mitochondria in tumor metabolism appeared in 1920’s,

when Otto Warburg observed increased glycolysis in tumor cells, even in the presence of

abundant oxygen. Following this observation, Warburg hypothesized that tumor cells tend

to obtain most of their energy through aerobic glycolysis (Warburg, 1930). This

phenomenon, known as the Warburg effect, is considered one of the major metabolic

alterations observed during cancer development (Warburg, 1956). Since then, several

hypotheses have been suggested in order to explain the aerobic glycolysis observed in some

(but not all) cancer cells. An irreversible respiratory impairment was first proposed by

Warburg (Warburg, 1956). In fact, the author suggested that the origin of cancer cells was in

an irreversible damage to the respiration apparatus (Warburg, 1956). However, Warburg

results were questioned when Boyland observed an increase in respiration after addition of

succinate or fumarate to tumor slices (Boyland & Boyland, 1936). Also, it was described that

neoplasias can have a normal oxidative phosphorylation capacity when supplemented with

NAD + (Wenner & Weinhouse, 1953). More recently, it was demonstrated that oxidative

phosphorylation can be improved in cancer cells by changing substrate availability

(Rossignol, Gilkerson et al., 2004). Despite all the arguments against the hypotheses raised


Biosensors for Health, Environment and Biosecurity

by Warburg, the truth is the Warburg effect was an important discovery that allowed for an

important progress in cancer research and prognosis (Ak, Stokkel et al., 2000). Being

mitochondria the organelle where several cellular metabolic reactions occur and where the

majority of cellular energy is produced, the role of mitochondria in cancer development is

indubitable. For example, mutations in mitochondrial and nuclear genes encoding proteins

involved in oxidative phosphorylation have been observed in several cancers, suggesting a

role for defective mitochondrial oxidative phosphorylation in tumorigenesis (for a review

see (Chandra & Singh, 2010)). Mutations can be acquired during or after oncogenesis and

result in an inhibition of oxidative phosphorylation, increased ROS production, tumor cells

proliferation and adaptation to tumor microenvironments (Hung, Wu et al., 2010; Lee,

Chang et al., 2010). Also, decreased mtDNA copy number has been associated with

resistance to apoptosis and increased invasiveness (Chandra & Singh, 2010). The loss of

function of mitochondrial-specific enzymes, such as succinate dehydrogenase and

fumarate dehydrogenase, results in the accumulation of specific metabolites in the

cytosol, that can favor the activation of transcription factors (eg. hypoxia-inducible factor,

HIF), directing the metabolism to aerobic glycolysis (Yeung, Pan et al., 2008; Bellance,

Lestienne et al., 2009; Marin-Hernandez, Gallardo-Perez et al., 2009) establishing a

possible correlation between mitochondrial alterations and the Warburg effect observed

in cancer cells.

2.2 Mitochondrial DNA diseases

Besides the nucleus, mitochondria have their own functional genome (Reich & Luck, 1966).

Mutations in mtDNA are associated with the development of different pathologies.

Although the mtDNA of an individual is usually identical in all cell types (homoplasmy),

variations may occur, causing dissimilarities between wild type and mutant mtDNA

(heteroplasmy). Progressive accumulation of mutant mtDNA in affected tissue will increase

the severity of the phenotype associated with those mutations. Besides the rate of

heteroplasmy, the age, gender and environment clearly contribute for the high diversity of

phenotypes (McFarland, Taylor et al., 2002). The so-called mitochondrial diseases are caused

by mutations in mtDNA or in nuclear genes that codify for proteins involved in the

mitochondrial respiratory chain or in overall mitochondrial biology. For the sake of

simplicity, we will focus now in diseases that are the result from mtDNA mutations. The

degree of severity of mtDNA alterations and the impact on organ phenotype is determined

by the threshold effect, or in other words, the dependency of the organ on the mutated

protein, or on the mitochondrial function itself (Dimauro & Davidzon, 2005). Simplifying,

this means that organs that are more dependent on energy will be first affected by

alterations of mitochondrial function caused by mtDNA mutations (Rossignol, Faustin et al.,

2003). Mitochondrial DNA diseases can be divided in two main categories based on the

genomic origin of the disorder: 1) syndromes due to mtDNA rearrangements or 2)

syndromes based on mtDNA point mutations. Kearns-Sayre (KSS) and Person Marrow-

Pancreas Syndromes are classical examples of disorders associated with mtDNA

rearrangements. KSS is characterized by external progressive opthalmoplegia and

pigmentary retinopathy and is associated with heteroplasmatic mtDNA deletions. Pearson

Marrow-Pancreas Syndrome is commonly diagnosed during infancy or postmortem and is

caused by deletions or duplications in mtDNA. It is rarely diagnosed during pregnancy, but

Mitochondria as a Biosensor for Drug-Induced Toxicity – Is It Really Relevant 419

should be suspected in the presence of severe anemia or lactic acidosis (Morel, Joris et al.,

2009). Mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes

(MELAS) is a multisystem mitochondrial maternally inherited disease. It is caused by a

point mutation characterized by a A to G transition at the position 3260 of the mitochondrial

genome. It is normally associated with frequent episodes of migraine and intraventricular

conduction disturbances and syncopal episodes based on paroxysmal atrioventricular block

have been found already (Connolly, Feigenbaum et al., 2010). Leigh Syndrome is a maternalinherited

point mutation in polypeptide-encoding genes based disorder. Although still

largely unknown, it is suggested that the Leigh Syndrome is caused by defects in genes

coding for the pyruvate dehydrogenase complex, cytochrome c oxidase, ATP synthase

subunit 6 or complex I subunits (Quintana, Kruse et al.; Naess, Freyer et al., 2009; Quintana,

Mayr et al., 2009).

2.3 Diabetes

Diabetes mellitus (DM) is a metabolic disease characterized by hyperglycemia and

alterations in carbohydrate, lipid and protein metabolism due to disturbances in insulin

secretion, having as a long-term consequence, the failure in several organs. As in previous

cases, mitochondrial multi-tasking suggests an important role of this organelle not only in

the pathogenesis of this condition, but also in the development of long-term complications.

Several mitochondrial alterations have been described during the progress of diabetes

mellitus, including respiratory alterations and altered induction of the MPT (reviewed in

(Oliveira, 2005)). Besides the heart (Oliveira, Rolo et al., 2001; Oliveira, Seica et al., 2003;

Santos, Palmeira et al., 2003; Bugger & Abel, 2011), alterations of mitochondrial function

have been recorded in liver (Ferreira, Seica et al., 2003), kidney (Oliveira, Esteves et al.,

2004), brain (Moreira, Santos et al., 2004) and testis mitochondria (Palmeira, Santos et al.,

2001; Amaral, Oliveira et al., 2008; Amaral, Mota et al., 2009), which show a multi-organ

scope of hyperglycaemia-induced mitochondrial alterations. Oliveira et al. demonstrated

that streptozotocin (STZ)-induced diabetes results in inhibition of cardiac mitochondrial

respiration and increased susceptibility to calcium-induced MPT (Oliveira, Seica et al., 2003).

In theory, this means that heart mitochondria from diabetic animals are less able to

withstand a metabolic stress, mimicked in this work by the addition of ADP and calcium.

Interestingly, heart mitochondria from Goto-Kakizaki (GK) rats have decreased

susceptibility to the MPT (Oliveira, Rolo et al., 2001). GK rats are an animal model for nonobese

type 2 diabetes, developing hyperglycaemia earlier in life, suggesting that the

severity/duration of the hyperglycaemic period is important for cardiac mitochondrial

alterations. Interestingly, different alterations in terms of hepatic mitochondrial respiratory

activity were found in both STZ-treated and GK rats, such alterations being modulated by

the age of the animals (Ferreira, Palmeira et al., 2003; Ferreira, Seica et al., 2003). Alterations

in MPT induction are also widespread to other tissues. Lumini-Oliveira et al. reported that

18 weeks of STZ treatment lead to a decrease in gastrocnemius mitochondrial respiratory

control ratio and to decreased calcium-dependent MPT, which may counteract the negative

effects of hyperglycaemia. It is still unclear what may cause mitochondrial alterations

during the course of diabetes and why such alterations appear to be organ and age-specific.

Increased oxidative stress due to increased mitochondrial generation of ROS and/or

depression of mitochondrial antioxidant defenses may be an attractive mechanism


Biosensors for Health, Environment and Biosecurity

(Kucharska, Braunova et al., 2000; Turko, Li et al., 2003; Kowluru, Atasi et al., 2006; Ren, Li

et al., 2008; Munusamy & MacMillan-Crow, 2009). A growing body of evidence also

suggests that mitochondrial dysfunction in pancreatic beta-cells may be also one of the

initiation factors responsible for depressed insulin release (Mulder & Ling, 2009). In fact,

mitochondria in beta-cells have a critical role in the release of insulin. Beta cell mitochondria

play a key role in this process, not only by providing ATP to support insulin secretion when

required, but also by synthesizing metabolites that can couple glucose sensing to insulin

exocytosis. ATP alone or possibly modulated by several coupling factors, triggers closure of

the ATP-sensitive potassium channel, resulting in membrane depolarization that increases

intracellular calcium and insulin secretion (Liu, Okada et al., 2009; Jitrapakdee,

Wutthisathapornchai et al., 2011). In several models for diabetes, mitochondrial defects in

beta-cells have been found (reviewed in (Maechler, Li et al., 2011)), including altered

expression of the voltage-dependent anion-channel (Ahmed, Muhammed et al., 2011) and

altered respiratory activity and oxidative stress (Lu, Koshkin et al., 2011). In beta-cell

mitochondria, increased oxidative stress may be critically important in the pathogenesis of

the disease (Nishikawa & Araki, 2007), although what exactly leads to that is still a matter of

debate. What is interesting is that some forms of diabetes are originated by defects on

mitochondrial DNA, present in pancreatic beta-cells (de Andrade, Rubi et al., 2006;

Mezghani, Mkaouar-Rebai et al., 2011). Other mitochondrial-relevant alterations in beta-cells

include enhanced apoptosis in some forms of auto-immune type I and type II diabetes

(Johnson & Luciani, 2011).

3. Drug-induced mitochondrial toxicity

Toxic compounds can interfere and modify physiological mechanisms, leading to cell

alterations and ultimately damage. In many cases of drug-induced toxicity, mitochondria

are the preferential target for toxic compounds and one important initiator of cell damage.

In this section, we will focus on the present knowledge regarding the mechanism of action

of some selected drugs, whose mechanism of toxicity has a clear mitochondrial component.

3.1 Anti-cancer drugs

For five decades, anthracycline antibiotics have played an important role in the treatment of

a variety of cancer types, due to their efficacy and broad spectrum of activity (Sawyer, Peng

et al., 2010). The anti-tumor activity of anthracyclines is based on their ability to intercalate

DNA and to inhibit enzymes involved in DNA replication and transcription such as

topoisomerase II and RNA polymerases, respectively (Sawyer, Peng et al., 2010).

Disturbance of DNA function is thought to be the main responsible for tumor cell death, a

typical behavior shared by other anti-cancer drugs (Singal, Iliskovic et al., 1997). However,

anthracycline therapy is associated with significant side effects, including cardiotoxicity

(Chen, Peng et al., 2007; Sawyer, Peng et al., 2010). A particular leading drug of this group,

Doxorubicin (DOX), has been intensively studied and rapidly stood out from other analog

molecules due to its efficacy. Unfortunately, its cardiotoxicity also stood out, although the

molecular mechanisms are still far of being completely understood (Arola, Saraste et al.,

2000; Horenstein, Vander Heide et al., 2000). The onset of DOX-induced cardiomyopathy is

characterized by several forms of tachycardia (Bristow, Minobe et al., 1981), altered left

ventricular function (Hrdina, Gersl et al., 2000), and severe histological changes such as

Mitochondria as a Biosensor for Drug-Induced Toxicity – Is It Really Relevant 421

vacuolization of the cytoplasm, loss of myofibrils, altered sarcoplasmic reticulum,

deposition of lipid droplets, and mitochondrial swelling (Lefrak, Pitha et al., 1973; Olson &

Capen, 1978; Iwasaki & Suzuki, 1991; Sardao, Oliveira et al., 2009). More evidence suggests

that mitochondria are a critical target in the development of DOX-induced cardiomyopathy

(Yoon, Kajiyama et al., 1983; Praet & Ruysschaert, 1993; Jung & Reszka, 2001; Wallace, 2003;

Berthiaume & Wallace, 2007). Numerous mechanisms for the toxicity of DOX on cardiac

mitochondrial function have been proposed, such as generation of free radicals (Muraoka &

Miura, 2003), interaction with mitochondrial DNA (L'Ecuyer, Sanjeev et al., 2006),

disruption of cardiac gene expression (Berthiaume & Wallace, 2007), alteration of calcium

homeostasis (Lebrecht, Kirschner et al.), lipid peroxidation mediating disturbance of

mitochondrial membranes (Mimnaugh, Trush et al., 1985), and inhibition of mitochondrial

respiration chain, decreasing both intracellular ATP and phosphocreatine (PCr) (Tokarska-

Schlattner, Zaugg et al., 2006). DOX can also interfere with mitochondrial function in other

targets, including by inhibiting phosphorylation steps (Marcillat, Zhang et al., 1989) or by

exerting partial uncoupling (Bugger, Guzman et al.). Although several hypotheses have

been proposed to explain cardiac DOX toxicity, oxidative stress is the most widely accepted;

in fact, data from the literature indicate that the cardiac tissue is particularly susceptible to

free radicals due to reduced levels of enzymatic antioxidants defenses when compared with

other tissues (Hrdina, Gersl et al., 2000). DOX is able to increase ROS through both an

enzymatic mechanism involving a redox cycle and cellular oxidoreductases such as NADH

dehydrogenase of complex I or cytochrome P-450 reductase, and through a non-enzymatic

pathway involving complexes with iron (Fe 3+ ) (Davies & Doroshow, 1986; Doroshow &

Davies, 1986; Jung & Reszka, 2001; Minotti, Recalcati et al., 2004). DOX-induced oxidative

stress can also be related with induction of the MPT (Ascensao, Lumini-Oliveira et al.; Zhou,

Starkov et al., 2001; Oliveira, Santos et al., 2006; Oliveira & Wallace, 2006), which is observed

in both in vivo and in vitro studies (Pereira & Oliveira, 2010). In vitro, DOX-induced MPT

pore opening results in mitochondrial depolarization, respiratory inhibition, matrix

swelling, pyridine nucleotides depletion and release of intermembrane proteins, including

cytochrome c (Oliveira, Bjork et al., 2004; Berthiaume, Oliveira et al., 2005; Oliveira, Santos et

al., 2006).

3.2 Nucleoside-analog reverse transcriptase inhibitors

Nucleoside reverse transcriptase inhibitors (NRTIs), a class of anti-retroviral drugs, are

specifically prescribed as a therapy to Acquired Immune Deficiency Syndrome (AIDS).

Several studies indicate that these drugs induce mitochondrial toxicity by interfering with

mitochondrial DNA (mtDNA) synthesis (Lund & Wallace, 2004; Lewis, Kohler et al., 2006).

The targets of NRTIs are reverse transcriptase enzymes but due to the similarities with

substrates for the mitochondrial enzyme DNA polymerase-gamma, NRTIs also inhibit this

mitochondrial enzyme, affecting mtDNA copy number (Lewis, Simpson et al., 1994). As

described above, mitochondrial DNA depletion may be clinically manifested in one or

several main targets tissues, depending on the energy requirements of that same tissue

(Rossignol, Faustin et al., 2003). Liver mitochondrial complications as hepatomegaly and

increased lipid deposits have been primarily observed with dideoxynucleosidesdidanosine,

stavudine, and zalcitabin. mtDNA depletion has been demonstrated in the liver of HIV

patients, with each of dideoxynucleosides inducing a time- and concentration-dependent

mtDNA depletion (Walker, Bauerle et al., 2004). Several NRTIs were shown to directly

interfere with cardiac mitochondrial respiratory chain decreasing membrane potential and


Biosensors for Health, Environment and Biosecurity

decreasing mitochondrial calcium buffer capacity (Lund & Wallace, 2004). Zidovudine

(AZT) is the most well-known antiviral and its side effects have been subject of several

studies focused on studying mitochondrial interactions (Lewis, Simpson et al., 1994).

Competitive inhibition of thymidine phosphorylation (Lynx, Bentley et al., 2006; Lynx &

McKee, 2006), induction of superoxide anion formation (Szabados, Fischer et al., 1999; de la

Asuncion, Del Olmo et al., 2004), inhibition of adenylate kinase activity (Barile, Valenti et al.,

1994), and inhibition of the ANT both in heart (Valenti, Barile et al., 2000) and liver (Barile,

Valenti et al., 1997) are some of the effects observed in isolated mitochondria incubated with

AZT and other NRTIs. Inhibition of phosphate transport in rat heart mitochondria by AZT

was found to be related with increased superoxide anion production, as shown by the

protective effects of several ROS scavengers (Valenti, Atlante et al., 2002). Oxidative stress

probably plays the most important role in AZT-induced mitochondrial dysfunction. Indeed,

a 2-week treatment of rats with AZT leads to increased ROS and peroxynitrite production

and induced single-strand DNA breaks (Szabados, Fischer et al., 1999). Lipid peroxidation

and oxidation of cell proteins, determined from protein carbonyl content, increased as a

consequence of AZT treatment (Szabados, Fischer et al., 1999). Depletion of mitochondrial

glutathione was also found in mitochondria isolated from the hearts of AZT-treated rats (de

la Asuncion, Del Olmo et al., 2004). Furthermore, NRTIs are able to indirectly inhibit the

regulation of mitochondrial complex I by cyclic adenosine monophosphate (cAMP). This

type of inhibition may explain disturbances observed in many patients regarding ROS

production, NADH/NAD + ratio, and high lactate levels (Lund & Wallace, 2008).

3.3 Anti-diabetic agents

Treatment of hyperglycemia during diabetes involves the use of hypoglycemic drugs.

Initially, biguanide agents such as metformin, phenformin and buformin were used for the

management of hyperglycemia in type 2 diabetes mellitus (T2D). However, these antidiabetic

drugs rapidly resulted into a number of serious adverse effects, which made the

pharmacological management of hyperglycemia still a challenge to the clinic.

Both buformin and phenformin were withdrawn from the market in the 1970´s due to high

incidence of lactic-acidosis-associated mortality and gastrointestinal symptoms, although

phenformin is still available in some countries. Metformin is now believed to be the most

widely prescribed anti-diabetic drug in the world (Correia, Carvalho et al., 2008). The antidiabetic

effect of metformin and phenformin and increased lactic acidosis observed during

treatment are suggested to result from a single mechanism, the inhibition of mitochondrial

complex I (El-Mir, Nogueira et al., 2000; Correia, Carvalho et al., 2008). Other investigators

described that inhibition of hepatocyte complex I not only caused not only a reduction of

blood glucose levels in human subjects but also a complete inhibition of hepatic

gluconeogenesis, a metabolic process that is significantly increased in T2D contributing to

the observed fasting hyperglycemia (Hundal, Krssak et al., 2000). In intact cells, metformin

increases AMP-activated protein kinase (AMPK) activity, resulting in increased fatty acid

oxidation, down-regulation of lipogenic genes, decreased hepatic glucose production and

stimulation of glucose uptake (Zhou, Myers et al., 2001). Beyond biguanides,

thiazolidinediones (TZD) is a class of oral antihyperglycemic drugs also known as

glitazones that have been used as an auxiliary therapy for diabetes mellitus (Petersen,

Krssak et al., 2000; Mudaliar & Henry, 2001). Glitazones includes troglitazone, rosiglitazone,

and pioglitazone, which are used to ameliorate hyperglycemia by increasing insulin-

Mitochondria as a Biosensor for Drug-Induced Toxicity – Is It Really Relevant 423

stimulated glucose removal by skeletal muscle (Petersen, Krssak et al., 2000; Mudaliar &

Henry, 2001). Indeed, TZDs can also be considered insulin sensitizers because they are able

to lower glucose levels in models of insulin resistance without increasing pancreatic insulin

production (Kliewer, Xu et al., 2001). The ability of TZD to lower serum glucose levels and

promote an increase in glucose utilization by accelerating glycolytic flux, can lead to

excessive lactic acid production. Although lowering glucose efficiently is considered a

desired effect of TZD, lactic acidosis seems to be a compensatory mechanism to a decrease in

mitochondrial generated ATP, something that is often observed in diabetic individuals.

These drugs are known to bind and activate the nuclear peroxisome proliferation receptor γ

(PPARγ), and interestingly to also inhibit mitochondrial complex I. The efficacy of TZD to

inhibit complex I or to cause lactate release in skeletal muscle or rat liver homogenates

follows the sequence troglitazone, rosiglitazone, and metformin, being the latter less

efficient (Brunmair, Staniek et al., 2004). Several studies reveal that TZDs may increase the

risk of heart failure (Delea, Edelsberg et al., 2003; Karter, Ahmed et al., 2004), which limits

their clinical application. The risk for heart failure may lie on mitochondrial impairment as

consequence of TZD toxicity. In this case, disruption of NADH oxidation by mitochondrial

complex I tends to occur, although the toxicity effect may also be the mechanism for the

pharmacological benefits observed (Scatena, Martorana et al., 2004). This means the border

line between a desired pharmacological effect and a toxic consequence is very blurred, and

in fact, long-term and/or large-scale inhibition of complex I activity can lead to ATP

depletion, oxidative burst and ultimately cell death (Li, Ragheb et al., 2003). An example of

TZD which had high impact in the clinic is troglitazone (TRO), introduced in 1997 but soon

withdrawn from the market because of reports of serious hepatotoxicity, receiving a black

box warning from the U.S. Food and Drug Administration (FDA). In fact, TRO, when

incubated with HepG2 cells, decreased cellular ATP and (Tirmenstein, Hu et al., 2002;

Bova, Tam et al., 2005). Lim et al. also demonstrated that TRO increases intramitochondrial

oxidative stress that activates the Trx2/Ask1 pathway, leading to mitochondrial

permeabilization (Lim, Liu et al., 2008). More recently, data indicate that significant mtDNA

damage caused by TRO is a prime initiator of the hepatoxicity caused by this drug (Rachek,

Yuzefovych et al., 2009). Overall, the data suggest that the reported mitochondrial effects of

anti-diabetic drugs, especially complex I inhibition are worth of further attention, not only

to explain some of its pharmacological effects but also to predict safety during drug


3.4 Anti-depressant agents

Tricyclic antidepressants (TCAs) are heterocyclic chemicals discovered in the early 1950s

and which have been primarily used to relieve depressive symptoms. Fluoxetine (Prozac),

an antidepressant of the selective serotonin reuptake inhibitor (SSRI) class, presents some

cardiovascular side effects and drug-drug interactions. Interestingly, some studies show that

fluoxetine indirectly affects electron transport and F 1 F o -ATPase activity inhibiting OXPHOS

in isolated rat brain and liver mitochondria (Souza, Polizello et al., 1994; Curti, Mingatto et

al., 1999). The results obtained by Curti et al., suggested that these effects are mediated by

the drug interference with the physical state of lipid bilayer of the IMM (Curti, Mingatto et

al., 1999). In turn, nefazodone is a TCA with a more favorable side effect profile when

compared to fluoxetine and even with other drugs commonly used to mitigate depressive


Biosensors for Health, Environment and Biosecurity

conditions. Nefazodone was initially considered very advantageous among several other

TCAs (Davis, Whittington et al., 1997). Initially, the incidence of specific organ toxicity was

considered very low, and related fatalities by severe toxicity were non-existent on several

hundred of patients during long periods of treatment (Lader, 1996; Robinson, Roberts et al.,

1996; Davis, Whittington et al., 1997). Among other physiological advantages, nefazodone

had the ability to treat some patients who did not respond to other TCAs (Ellingrod & Perry,

1995; Robinson, Roberts et al., 1996). However, some cardiovascular complications such as

asymptomatic reduced systolic blood pressure and asymptomatic sinus bradycardia, started

to be detected and considered as markers for cardiotoxicity (Robinson, Roberts et al., 1996).

Despite the possible therapeutic advantages, the drug was withdrawn from the U.S. market

in 2004, based on cardiotoxicity and later on some severe cases of adverse hepatoxicity as

well. Indeed, more recent data show that when compared to buspirone, nefazodone is more

toxic to hepatic mitochondrial function (Dykens, Jamieson et al., 2008). Dykens et al.

demonstrated that nefazodone promoted inhibition of mitochondrial respiration and

increased glycolysis in isolated rat liver mitochondria and in intact HepG2 cells, respectively

(Dykens, Jamieson et al., 2008). Two other anti-depressant drugs, amineptine and tianeptine,

can also lead to hepatitis associated with microvesicular steatosis, in fact, their heptanoic

acid side chain may be responsible for reversibly inhibiting mitochondrial fatty acid

oxidation by a competitive mechanism (Fromenty, Freneaux et al., 1989).

3.5 Statins and fibrates

Statins (or HMG-CoA reductase inhibitors) are a class of drugs used to decrease cholesterol

levels by inhibiting the enzyme HMG-CoA reductase, which plays a central role in the

production of cholesterol in the liver. Statins are generally safe and well tolerated, but the

major side effect, which occurs in about 1% of patients, is skeletal myopathy (Davidson,

2001). Interestingly, many congenital myopathies are associated with defects in

mitochondrial enzymes (Cornelio & Di Donato, 1985; Wallace, 2000) and bio-accumulation

of statins by fast twitch skeletal muscle cells can increase the risk of mitochondriallyinduced

rhabdomyolysis (Westwood, Bigley et al., 2005). Several reports describe acute

effects of statins on skeletal muscle mitochondria. Lovastatin and simvastatin were reported

to induce the MPT in vitro and decrease the content of total membrane thiol groups in

mitochondria isolated from mouse hind limb (Velho, Okanobo et al., 2006). Mitochondrial

degeneration was observed on rat skeletal muscle fibers treated with cerivastatin (Seachrist,

Loi et al., 2005). A variety of other statins are known to induce the MPT leading to

irreversible collapse of the transmembrane potential and release of pro-apoptotic factors

(Cafforio, Dammacco et al., 2005; Kaufmann, Torok et al., 2006), in a Bcl-xL-preventable

manner (Blanco-Colio, Justo et al., 2003). Kaufman and colleagues also reported inhibition of

-oxidation and swelling of isolated skeletal muscle mitochondria by statins (Kaufmann,

Torok et al., 2006). Ubiquinone coenzyme Q10 (CoQ 10 ) depletion is another hypothetic

contributor to statin-induced myopathy (Folkers, Langsjoen et al., 1990). Thus, CoQ 10

depletion can contribute to mitochondrial dysfunction leading to statin-induced myopathy

since CoQ 10 acts as an electron carrier in the mitochondrial respiratory chain (Schaars &

Stalenhoef, 2008). Besides the effects on skeletal muscle, lovastatin and simvastatin inhibit

mitochondrial respiration of isolated liver mitochondria by a direct effect on complexes II,

III, IV and V (Nadanaciva, Dykens et al., 2007). Fibrates, in turn, are used as accessory

therapy in many forms of hypercholesterolemia, usually in combination with statins

Mitochondria as a Biosensor for Drug-Induced Toxicity – Is It Really Relevant 425

(Steiner, 2007). Fibrates are structurally related to the thiazolidinediones, and

pharmacologically act on PPARγ, impairing mitochondrial function (Barter & Rye, 2006).

In an ex vivo experiment with isolated mitochondria, fenofibrate inhibits complex I

activity and disturbs rat mitochondrial function (Brunmair, Lest et al., 2004). The fibrates

ciglitizone, bezafibrate, gemfibrozil, and clofibric acid were reported to increase lactate

and acetate levels due to increase anaerobic glycolysis and fatty acid beta-oxidation, to

inhibit NADH-cytochrome c reductase activity, and show a correlation between

mitochondrial toxicity and inhibition of HL-60 cell growth (Scatena, Martorana et al.,

2004). In opposition, Scatena et al. argued that fibrates induce toxicity by disrupting

mitochondrial function through a mechanism partly independent on PPARs (Scatena,

Bottoni et al., 2004).

4. Environmental pollutants

Humans are daily exposed to a variety of molecules, which can be present in food,

beverages and even in the atmosphere. Although most are harmless, either due to their

intrinsic safety or to the decreased exposure levels, the truth is that some of those molecules

disturb several biological systems, including mitochondria, leading to short or long-term

organ toxicity (Wallenborn, Schladweiler et al., 2009).

Heavy metal toxicity is widespread in the world due to the very large amount of industrial

activities that release these compounds in nature. Heavy metal toxicity can have different

aspects and result into different pathologies, including carcinogenesis and vascular diseases

(Nash, 2005). As expected, the toxicity of heavy metals also impacts mitochondria.

Cadmium, for example, which has been associated with learning impairments and

neurological disorders, has been described to cause mitochondrial-dependent apoptosis in

oligodendrocytes (Hossain, Liu et al., 2009) and in a skin cell line (Son, Lee et al., 2011).

Cadmium accumulation in the kidney involves alteration of mitochondrial function, which

results into increased generation of mitochondrial free radicals (Gobe & Crane, 2011),

similarly to what occurs in other target organs (Cannino, Ferruggia et al., 2009). As

expected, cadmium, similarly to as mercury and copper, induces the MPT, resulting in

mitochondrial swelling and activation of basal respiration, as well as in membrane

depolarization (Belyaeva, Glazunov et al., 2004). Mercury also caused apoptosis in several

biological models by interfering with mitochondrial function (Shenker, Guo et al., 1998). In

fact, low concentrations of methylmercury cause inhibition of mitochondrial function, which

progresses to apoptotic cell death (Carranza-Rosales, Said-Fernandez et al., 2005).

Mitochondrial respiration in hepatoma AS-30D cells is initially uncoupled for lower

concentrations and progressively inhibited for higher concentrations, resulting also in

increased generation of ROS (Belyaeva, Dymkowska et al., 2008). Although in this same

model, copper (Cu 2+ ) was not as toxic (Belyaeva, Dymkowska et al., 2008), other works have

shown that copper causes toxicity in astrocytes, due to increased MPT induction and

oxidative stress (Reddy, Rao et al., 2008). Also, copper decreased , followed by apoptosis

in MES23.5 dopaminergic cells (Shi, Jiang et al., 2008). Interestingly, at least a significant part

of copper toxicity in non-human species can also be explained by inhibition of

mitochondrial function, including activation of the MPT, as observed in trout hepatocytes

(Krumschnabel, Manzl et al., 2005). Iron has been considered a significant pro-oxidant metal

due to its role in the formation of hydroxyl radical via Fenton reactions (Stohs & Bagchi,

1995). Although iron is essential for life, it can pose serious health risks with the liver being


Biosensors for Health, Environment and Biosecurity

the most relevant target. Heavy iron overload, as described during primary (hereditary) or

secondary forms of hemochromatosis, may cause cirrhosis, liver failure, and hepatocellular

carcinoma (Bonkovsky & Lambrecht, 2000). In addition, iron can contribute to the

development or progression of alcoholic liver disease, nonalcoholic liver steatohepatitis,

chronic viral hepatitis and prophyria cutanea tarda, among other diseases (Bonkovsky &

Lambrecht, 2000). In thalassemia major, one of the clinical end-points is an iron overload

resulting from diverse factors. The excess of iron results in ROS formation, damaging

several intracellular organelles, including mitochondria (Hershko, 2011). The observed

effects are very close to what has been observed in rats subjected to a single injection of a

massive dose of iron-dextran. In this case, mitochondria from treated rats showed decreased

respiratory control ratio (Pardo Andreu, Inada et al., 2009). In another different model, rats

diet-supplemented with iron lactate showed decreased ATP content in the liver and spleen,

which was suggested to occur due to mitochondrial alterations (Fujimori, Ozaki et al., 2004).

An interesting hypothesis is drawn from the work of Liang et al. The authors suggest that

mitochondrial aconitase may be an important early source of mitochondrial iron

accumulation in a model for experimental Parkinson's disease, with an oxidative

inactivation of that enzyme occurring due to iron-mediated oxidative stress (Liang & Patel,

2004). The role of iron in exacerbating the toxic effects of clinically used drugs is

demonstrated, among other examples, by the fact that the iron chelator dexrazoxane

protects cardiac myocytes against the toxicity of DOX (see above), via a mitochondrial

mechanism (Hasinoff, Schnabl et al., 2003).

Dioxins are environmental pollutants with a large impact on human health, being byproducts

of incineration processes and of production of several chloro-organic chemicals

(Sweeney & Mocarelli, 2000; Parzefall, 2002). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is

the best studied and the most toxic dioxin and data are vast describing clear direct effects of

this compound on mitochondria. Several works identified the inhibition of the

mitochondrial electron chain and increased generation of ROS as one mechanism by which

TCDD exerts its toxicity in the heart (Nohl, de Silva et al., 1989) and liver (Stohs, Alsharif et

al., 1991; Latchoumycandane, Chitra et al., 2002; Senft, Dalton et al., 2002). Senft et al.

demonstrated that mitochondria are the source of TCDD-induced ROS, although the exact

mechanism was still not clearly identified (Senft, Dalton et al., 2002). TCDD treatment

resulted in an increased hydrogen peroxide release by the respiratory chain, although no

alterations in mitochondrial superoxide dismutase or glutathione peroxidase were observed

(Senft, Dalton et al., 2002). Interestingly, one week after treating mice with TCDD, coenzyme

Q levels in the liver decreased, while activities of some of the mitochondrial complexes were

increased (Shertzer, Genter et al., 2006). These and other results, led to the proposal that

TCDD causes a defect on the ATP synthase in the liver, resulting in decreased ATP levels in

the liver (Shertzer, Genter et al., 2006). Results in isolated rat hepatocytes confirmed the

mitochondrial role on oxidative stress caused by TCDD (Aly & Domenech, 2009). It was also

demonstrated by using a knock-out model that mitochondrial reactive oxygen production is

dependent on the aromatic hydrocarbon receptor (Senft, Dalton et al., 2002) and causes

direct damage to mtDNA (Shen, Dalton et al., 2005). Interestingly, TCDD induces apoptosis

of human lymphoblastic T-cells, which do not express the aromatic hydrocarbon receptor;

the mechanism being the triggering of mitochondrial-mediated intrinsic apoptotic pathway,

mediated by calcium/calmodulin (Kobayashi, Ahmed et al., 2009). Another interesting

possibility regarding the linkage between mitochondria and TCDD toxicity is the

Mitochondria as a Biosensor for Drug-Induced Toxicity – Is It Really Relevant 427

perturbation of reproductive function by that dioxin (Wu, Li et al., 2001). Reported data

indicate that low doses of TCDD cause increased oxidative stress, including depletion of

antioxidant enzymes, in mitochondria and microsomal fractions from rat testis, which can

alter the mitochondrial ability to supply energy to male germ cells (Latchoumycandane,

Chitra et al., 2002). Mitochondrial interactions of TCDD and the possible carcinogenesis

associated with dioxin exposure (Knerr & Schrenk, 2006; Jenkins, Rowell et al., 2007)

(although others disagree, (Cole, Trichopoulos et al., 2003)) were also demonstrated to be

related since TCDD causes mitochondrial depolarization, stress signaling and tumor

invasion, besides altering calcium homeostasis (Biswas, Srinivasan et al., 2008). Besides,

TCDD directly targets mitochondrial transcription and causes a mitochondrial phenotype

which is similar to what is observed in rho0 cells (Biswas, Srinivasan et al., 2008).

5. Mitochondrial liability in drug development and safety assessment

Mitochondria are indeed, the crossroad for many cellular pathways, which explains the

growing number of publications dealing with the mitochondrial role in cell life and death

(Pereira, Moreira et al., 2009). As a result of the increased efforts focused on the role of

mitochondria on a variety of human disorders as cancer, neurodegenerative, cardiovascular

diseases, obesity, and diabetes, “mitochondrial medicine” emerged as a whole new field of

biomedical research. Based on the recent developments in this field, a large effort is

underway to understand how different molecules regulate or damage mitochondrial

function, with the ultimate goal to improve human health. Two distinct and important

mechanisms/endpoints by which drugs may inhibit mitochondrial function, can be

considered (Fig. 3): a) direct interference with mitochondrial respiration/ATP synthesis

(inhibition of respiratory complex activity, damage by ROS production, uncoupling activity,

MPT induction) and b) inhibition of mtDNA synthesis. Regardless of the initial trigger,

inhibition of ATP synthesis and bioenergetic failure of the tissue are severe manifestations of

mitochondrial impairment. Several drugs or other xenobiotics can drive mitochondrial to an

irreversible collapse via formation of the MPT pore leading to release of pro-apoptotic

factors such as cytochrome c. Drugs that alter the normal equilibrium between pro-apoptotic

and anti-apoptotic proteins, such as Bak/Bax and Bcl-2, can also induce mitochondrial

failure and eventually cell death. Additional information for drug development and safety,

as well for toxicity assessments may be achieved by the use of targeted approaches, affinity

for overexpressed/subexpressed mitochondrial proteins during different diseases types, or

selective mitochondrial accumulation of delocalized lipophilic molecules with positive

charge and with different redox actions. Nevertheless, further investigation in these

endpoints or guidelines of the molecular mechanisms of mitochondria-drug interaction will

be needed for a better understanding of the mechanism of action involved in mitochondrial

toxicity, allowing an improvement in the design of safer drugs or hazard assessment of

xenobiotics with relevant human exposure. Notwithstanding these concerns, until now,

several high-throughput techniques have been used to test and screen drug safety on

mitochondrial function and could easily be studied to improve basic knowledge in drug

development and associated toxicity.

6. High throughput methods – the faster the better

High throughput methods have been developed with the ultimate objective of allowing

company and research laboratories to perform large-scale screening or biochemical


Biosensors for Health, Environment and Biosecurity

analyses for a certain research or commercial objective. During many decades, low

throughput methods were used in most research laboratories, including the Clark-type

electrode or the tetraphenylphosphonium electrode to measure mitochondrial membrane

potential or , respectively (Pereira, Moreira et al., 2009; Pereira, Pereira et al., 2009).

Other low-throughput methods to investigate mitochondrial toxicity of several agents

involved the measurement of activities of components of the mitochondrial respiratory

chain by using polarographic, spectrophotometric or blue-native gel techniques

(Barrientos, Fontanesi et al., 2009; Diaz, Barrientos et al., 2009). Although such methods

are still in use in many laboratories worldwide (and in our own as well), profit-thirsty

pharmaceutical companies require faster and cheaper methods to screen thousands of

compounds per month in an attempt to uncover mitochondrial liabilities. For example, in

the context of mitochondrial toxicity screening in drug development and safety, a

fluorescence-based oxygen consumption assay was developed to analyze the ability of

certain compounds to cause mitochondrial dysfunction. This approach provides detailed

and specific information about the possible mechanisms of toxicity based on

measurements of respiratory states 3 and 4 by means of oxygen-sensitive probes. The

advantages of this particular fluorescence method are the simplicity and large-scale of

measurement, since it can be adapted to a plate reader system. The results can be

visualized in real time and quantified in plate reader software (Hynes, Marroquin et al.,

2006). A later development included a combination of five high-throughput assays adding

important information by identifying enzymes which can be target of the test compounds

(Nadanaciva, Bernal et al., 2007). A set of immunocapture-based assays to identify

compounds that directly inhibit oxidative phosphorylation can be used in the early

evaluation of compound for clinical trials (Nadanaciva, Bernal et al., 2007). The same

research group improved a method based on fluorescent probes for the study of oxygen

consumption. The advantage is the possibility of screening several compounds

simultaneously, being further up-scaled, automated and adapted for other enzyme- and

cell-based screening applications (Will, Hynes et al., 2006). To test compounds that

interfere with the synthesis of mitochondrial DNA or mtDNA-encoded proteins, a 96-well

plate format method, that measures complex IV subunit 1, a protein encoded by mtDNA

and complex V subunit 1, an nuclear DNA- encoded protein was developed (Nadanaciva,

Dillman et al., 2010).

The literature is getting richer in terms of new methods for high-throughput methods to

evaluate mitochondrial function in different applications. When comparing fibroblasts

from patients with mtDNA diseases with control subjects, a decrease in ATP production

rate in muscle with normal OXPHOS enzyme activities was observed (Jonckheere,

Huigsloot et al., 2010). This and other types of assays allow finding primary and

secondary mitochondrial dysfunction, which can facilitate the search for genetic defects

that can lead to mitochondrial diseases (Jonckheere, Huigsloot et al., 2010). Although in a

smaller scale, the Seahorse Bioscience analyzer can be used for a multi-end point of cell

and mitochondrial metabolism. In one particular study, the authors measured the

mitochondrial function of renal proximal tubular cells observing that several

nephrotoxicants alter mitochondria function before altering the basal respiration (Beeson,

Beeson et al., 2010). The future will no doubt yield new fast and cost-effective highthroughput

methods to quickly investigate mitochondrial toxicity of xenobiotics in order

not only to produce safer drugs but also to perform safety screenings on many

compounds that humans are daily exposed to.

Mitochondria as a Biosensor for Drug-Induced Toxicity – Is It Really Relevant 429

Fig. 3. Drugs or environmental xenobiotics can impair mitochondrial function through

affecting different targets, including mitochondrial oxidative phosphorylation and ATP

production. Oxidative stress and calcium overload increase the probability of irreversible

mitochondrial failure via MPT pore, leading to release of pro-apoptotic factors such as

cytochrome c. In addition, drugs that alter the ratio pro-apoptotic and anti-apoptotic

proteins, such as Bak/Bax and Bcl-2, can also induce mitochondrial failure. OMM, Outer

mitochondrial membrane; IMM, Inner mitochondrial membrane; NADH, Nicotinamide

adenine dinucleotide reduced form; NAD + , Nicotinamide adenine dinucleotide oxidized


7. Concluding remarks

The question in the title suggests that doubts would still exist regarding the use of

mitochondria as a biosensor for drug-induced toxicity. Hopefully, the present chapter

provides enough evidence that mitochondria are a critical target in the toxicity of a wide

variety of agents, ranging from clinically-relevant drugs, to environmental poisons.

Moreover, it has been here demonstrated that failure of mitochondrial function originates

several pathologies, which by its turn, contribute to amplify mitochondrial damage.

Idiosyncratic drug reactions have also been proposed to involve mitochondria as well

(Lucena, Garcia-Martin et al.). In fact, an individual who has a lower mitochondrial power

may succumb first to the toxicity of mitochondrial-directed toxicants, even if the original

mild mitochondrial alterations are asymptomatic. This is extremely critical for patients with

diagnosed mitochondrial DNA diseases, who are in a high risk of suffering mitochondrial

failure upon a second hit with a toxicant, either a clinically used drug or an environmental


The large number of mitochondrial targets, some of which were not even explored in this

chapter, and the growing list of compounds presenting mitochondrial liabilities, clearly


Biosensors for Health, Environment and Biosecurity

answers our initial question. The field of mitochondrial pharmacotoxicology (Scatena,

Bottoni et al., 2007) is now critical for many pharmaceutical companies and for a large

number of research laboratories which work on basic toxicology (Chan, Truong et al., 2005;

Dykens, Marroquin et al., 2007; Wallace, 2008; Nadanaciva & Will, 2009; Pereira, Moreira et

al., 2009; Pereira, Pereira et al., 2009). Some for the sake of profit, others for the good of

science itself, but all focusing on that little organelle that is in the spotlight right now.

8. Acknowledgements

Mitochondrial research at the authors’ laboratory is funded by the Foundation for Science

and Technology (FCT), Portugal (research grants PTDC/SAU-OSM/104731/2008,

PTDC/QUI-BIQ/101052/2008, PTDC/QUI-QUI/101409/2008, PTDC/AGR-

ALI/108326/2008 and PTDC/SAU-FCF/101835/2008). Ana C. Moreira and Nuno G.

Machado are funded by FCT (Ph.D. fellowships SFRH/BD/33892/2009 and

SFRH/BD/66178/2009, respectively). Vilma A. Sardão is recipient of a Pos-Doc fellowship

from the FCT (SFRH/BPD/31549/2006).

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


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

Email address –

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-


Mitochondrion 12 (2012) 66–76

Contents lists available at ScienceDirect


journal homepage:


Regulation and protection of mitochondrial physiology by sirtuins

Claudia V. Pereira a , Magda Lebiedzinska b , Mariusz R. Wieckowski b , Paulo J. Oliveira a, ⁎

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

b Nencki Institute of Experimental Biology, Warsaw, Poland




Article history:

Received 3 November 2010

Received in revised form 16 February 2011

Accepted 7 July 2011

Available online 20 July 2011





Oxidative stress


The link between sirtuin activity and mitochondrial biology has recently emerged as an important field. This

conserved family of NAD + -dependent deacetylase proteins has been described to be particularly involved in

metabolism and longevity. Recent studies on protein acetylation have uncovered a high number of acetylated

mitochondrial proteins indicating that acetylation/deacetylation processes may be important not only for

the regulation of mitochondrial homeostasis but also for metabolic dysfunction in the context of various

diseases such as metabolic syndrome/diabetes and cancer. The functional involvement of sirtuins as sensors

of the redox/nutritional state of mitochondria and their role in mitochondrial protection against stress

are hereby described, suggesting that pharmacological manipulation of sirtuins is a viable strategy against

several pathologies.

© 2011 Elsevier B.V. and Mitochondria Research Society. All rights reserved.


1. Sirtuins: function and families . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

1.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

1.2. Classification of sirtuins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

1.3. Sirtuin enzymatic activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

1.4. Sirtuin protein targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

1.5. Tissue specificity and intracellular localization of mammalian sirtuins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

1.6. Cytosolic and nuclear sirtuins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

1.7. Mitochondrial sirtuins: characterization and activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

1.7.1. Localization and processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

1.7.2. Regulation of mitochondrial metabolism by sirtuins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

1.7.3. Protection of mitochondrial function by sirtuins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

1.8. Future perspectives: are sirtuins good therapeutic targets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

Abbreviations: Acetyl-CoA, acetyl-coenzyme A; AceCS2, acetyl-coenzyme A synthethase 2; ACC, Acetyl-CoA carboxylase; ADP, adenosine diphosphate; AIF, apoptosis-inducing

factor; AMPK, 5′ adenosine monophosphate-activated protein kinase; ANT, adenine nucleotide translocator; ATP, adenosine triphophate; BER, base excision repair; CPS1, Carbamoyl

phosphate synthase; CR, calorie restriction; CREB, cAMP response element-binding; CypD, cyclophilin D; DNA-PK, DNA-dependent protein kinase; DSB, double-strand break; ERR α,

Estrogen-related receptor alpha; FAO, fatty acid oxidation; FOXO, Forkhead box O; GDH, glutamate dehydrogenase; H4, histone 4; HDL, high-density lipoprotein; HFD, high fat diet;

HIF1α, Hypoxia-inducible factor 1, alpha subunit; HMGCS2, 3-hydroxy-3-methylglutaryl CoA synthase 2 Km, Michaelis-Menten constant; LCAD, long chain acyl coenzyme A

dehydrogenase; MEFs, mouse embryonic fibroblasts; MPP, mitochondrial processing peptidase; mPTP, mitochondrial permeability transition pore; MnSOD, manganese superoxide

dismutase; NAD + , nicotinamide adenine dinucleotide; NDUFA9, NADH dehydrogenase [ubiquinone]-1 alpha subcomplex subunit; OSCC, oral squamous cell carcinoma; OTC,

ornithine transcarbamoylase; PPARγ, peroxisome proliferator-activated receptor; PARP-1, Poly [ADP-ribose] polymerase 1; PGC-1α, peroxisome proliferator-activated receptor

gamma coactivator 1-alpha; Pol1, polimerase1; ROS, reactive oxygen species; SIRT, sirtuin; Sir2, Silent Information Regulator Two (Sir2) protein; SOD2, superoxide dismutase 2; TNF,

Tumor necrosis factor; UCP2, uncoupling protein 2.

⁎ Corresponding author at: Center for Neuroscience and Cell Biology, Largo Marques de Pombal, University of Coimbra, 3004-517 Coimbra, Portugal. Tel.: +351 239 855760; fax:

+351 239 855789.

E-mail address: (P.J. Oliveira).

1567-7249/$ – see front matter © 2011 Elsevier B.V. and Mitochondria Research Society. All rights reserved.


C.V. Pereira et al. / Mitochondrion 12 (2012) 66–76


1. Sirtuins: function and families

1.1. Introduction

The involvement of mitochondria in multiple fundamental cellular

processes places these organelles under the highlight of many

researchers. In fact, mitochondria are now considered one major

target for many therapeutic approaches. Oxidative stress has been a

particular fulcrum of interest since mitochondrial oxidative damage

has been recognized as being involved in many diseases and in the

aging process itself. Also, disrupted mitochondrial metabolism may

be one of the critical elements leading to cancer, diabetes, ageassociated

and neurodegenerative disorders (Campisi and Yaswen,

2009; Harman, 1956; Sultana and Butterfield, 2010; Weber and

Reichert, 2010; Zhu and Chu, 2010). In this fascinating area of

mitochondrial disease and protection, sirtuins are being specially

focused, since these proteins are able to regulate stress responses

and cell survival (Canto and Auwerx, 2009; Gan and Mucke, 2008).

One of the most intensively investigated compound, resveratrol, is a

known sirtuin 1 (SIRT1) activator, shown to have a positive effect on

mitochondrial metabolism and to delay the aging process (Alcain and

Villalba, 2009; Finkel et al., 2009). The present review comprises our

recent knowledge in the field of sirtuins with a special focus on the

most recent and breakthrough studies related with the protection

and regulation of mitochondrial physiology by SIRT3, SIRT4 and

SIRT5. Several other reviews on the subject are available, including the

very interesting work by Huang et al.(2010). The present review

expands the previous works by incorporating novel and exciting

results, which allows the reader to get a full picture of how sirtuins in

general, and mitochondrial sirtuins in particular, contribute to cellular

and mitochondrial protection.

1.2. Classification of sirtuins

Sirtuins, or Silent information regulator proteins (Sir), and their

homologs are present in a very wide range of organisms, from bacteria

to humans, forming a conserved family of proteins (Denu, 2005). Up

to date, seven sirtuin homologues have been identified in mammalian

cells. These have different intracellular localization as well as various

roles in cell physiology (Blander and Guarente, 2004; Tanny et al.,

1999). Based on the phylogenetic analysis of the core domain,

mammalian sirtuins have been classified into four classes together

with other Sir2-related proteins widely distributed in eukaryotes

and prokaryotes (Frye, 2000; Smith et al., 2000). Mammalian SIRT1

(62.0 kDa), SIRT2 (41.5 kDa) and SIRT3 (43.6 kDa) are considered

Class I sirtuins, while SIRT4 (35.2 kDa) is a Class II sirtuin. Class III

SIRT5 (33.9 kDa) and Class IV SIRT6 (39.1 kDa) and SIRT7 (44.8 kDa)

are other examples. All the described sirtuins contain a conserved 275

amino acid catalytic core domain together with N-terminal and/or C-

terminal domains. Additionally, a novel class (“U”) has been created

to include sirtuins with unique features, such as gram-positive

bacteria and Termoga maritime sirtuins. An appropriate Class

affiliation of the individual Sir2-related proteins has been described

in the comprehensive review article by Michan and Sinclair(2007).

A list of the different sirtuins, plus other details on their physiology/

activity, can be seen in Table 1.

1.3. Sirtuin enzymatic activity

All sirtuins, with only one exception (SIRT4), catalyze protein

deacetylation in which the lysine acetyl group is transferred from

the target protein to the ADP-ribose component of NAD + , which leads

to their full dependence on NAD + availability and indicates that

sirtuins can be sensors of the cellular redox state. As a consequence,

sirtuin activity leads to the generation of deacetylated proteins, 2′-Oacetyl-ADP

ribose and nicotinamide (Sauve, 2010). Moreover, SIRT6

additionally demonstrates ADP-ribosyl transferase activity while

SIRT4 (as mentioned earlier) demonstrates ADP-ribosyl transferase

activity only. As described above, sirtuin activity is regulated by NAD +

availability. On the other hand, nicotinamide noncompetitively

inhibits sirtuins suggesting that the deacetylation reaction product

can also act as an endogenous regulator of sirtuin activity. Interestingly,

isonicotinamide, which binds to the nicotinamide pocket of

yeast sirtuin (Sir2), only inhibits the base exchange, with the

deacetylation activity remaining unaffected or even being increased.

This fact indicates that chemical compounds such as isonicotinamide

can act as potent sirtuins activators in mammalian cells. Additionally,

it has been demonstrated that activity of yeast sirtuin Hst2 (a Class I

member) can be regulated by homo-oligomerization of the enzyme.

Another well known and currently intensively studied sirtuin activator

is resveratrol, a polyphenol present in different sources, including for

example grapes skin and red wine (Lagouge et al., 2006). It has been

demonstrated that polyphenols and especially resveratrol, reduce K m

values for sirtuin substrates, resulting in different biological effects

as increased resistance to apoptosis and to stress stimuli. Apart from

nicotinamide, an inhibitory effect on sirtuins has also been described

for several other compounds. The most well-known are sirtinol,

splitomicin (specific for yeast sirtuins Hst1 and Sir2) and dehydrosplitomicin

(specific mostly to sirtuin Hst1). Human sirtuins are not

inhibited significantly by splitomicin as well as by dehydrosplitomicin.

More about the mechanism of protein deacetylation carried out by

sirtuins as well as detail on their inhibitors and activators can be found

in the review of Sauve et al. (Sauve et al., 2006).

1.4. Sirtuin protein targets

Potential sirtuins substrates are various acetylated proteins

involved in cell metabolism, apoptosis and regulation of gene

transcription. Sirtuins can thus determine the ability of cells to adapt

to different conditions (Finkel et al., 2009; Haigis and Guarente,

2006; Vaquero, 2009). Published data suggest a significant role of

deacetylation in metabolic responses to fasting or caloric restriction as

well as in the response to different stress stimuli, including for

example oxidative stress (Choudhary et al., 2009; Schwer et al., 2009).

Among the several proteins which are deacetylated by sirtuins,

histones and transcription factors such as p53 (Li et al., 2010), FOXO

(Brunet et al., 2004), peroxisome proliferator activated receptor γ

(PPARγ) (Picard et al., 2004), nuclear factor-κB (NFκB) (Kawahara

et al., 2009) and PGC-1α (Sugden et al., 2010) can be found. Sirtuins

are also able to deacetylate α-tubulin (Tang and Chua, 2008) and

acetyl-CoA synthetase (Hallows et al., 2006). Another important

protein for metabolism, glutamate dehydrogenase (GDH) is a wellknown

example of ADP-ribosylation substrates for SIRT4 (Haigis

et al., 2006). The variety of sirtuin substrates, either being crucial

enzymes or gene regulatory elements, indicates the possibility that

multiple sirtuins may modulate cell physiology and metabolism

through interacting with distinct targets regulated under a wide

range of physiological conditions.

1.5. Tissue specificity and intracellular localization of mammalian sirtuins

Molecular analysis revealed that SIRT1, 2, 3, 5 and 6 are

ubiquitously expressed in different tissues and organs. The highest

amount of SIRT6 was detected in muscles, brain and heart, while

SIRT4 is mostly present in muscle, kidney, testis and liver (Michishita

et al., 2005). By its turn, SIRT7 has been found in brain, kidney, liver,

lung and adipose tissue (Michishita et al., 2005). Further detailed

studies determined subcellular localization of several sirtuins. Among

seven mammalian sirtuins, SIRT1 and SIRT2 show both cytoplasmic

and nuclear localization, while SIRT6 and SIRT7 are only located

in the nucleus and nucleoli, respectively (Michishita et al., 2005).

SIRT3, SIRT4 and SIRT5 are mitochondrial proteins, although SIRT3

Table 1

Characterization of seven mammalian sirtuins.

HDACIII Sirtuin Localization Substrates Catalytic activity Function Modulators Knockout outcome References

Class I SIRT1 Cytosol,









p53, MyoD, NF-κB,

histone H3 and H4

α-tubulin, histone


AceCS2, IDH2,


NAD + -dependent

protein deacetylation

NAD + -dependent

protein deacetylation

NAD + -dependent

protein deacetylation

Class II SIRT4 Mitochondria GDH Mono-ADP-ribosyl


Class III SIRT5 Mitochondria Histone H4,

CPS1, cyt c

NAD + -dependent

protein deacetylation

Class IV SIRT6 Nucleus Histone H3K9 NAD + -dependent protein

deacetylation, mono-ADPribosyl


SIRT7 Nucleus RNA pol I, p53 NAD + -dependent

protein deacetylation

Cell survival, insulin signaling,

inflammation, metabolism

regulation, oxidative stress

response, lifespan regulation

Cell cycle regulation, nervous

system development,

Regulation of mitochondrial

energetic metabolism

Regulation of mitochondrial

energetic metabolism, insulin


Urea cycle regulation, apoptosis

Genome stability, DNA repair,

nutrient-dependent metabolism


Regulation of rRNA transcription,

cell cycle regulation


Resveratrol, AROS,

HIC1, splitomicins,




DBC1, NAD + ,



Dihydropyridine Sirtinol, NAD + ,




NAD + , isonicotinamide,


NAD + , isonicotinamide

NAD + , isonicotinamide,


NAD + , isonicotinamide

NAD + , isonicotinamide

High pre-natal mortality,

metabolic abnormalities,

heart and bone defects,

Shortened lifespan

Aberrances during

mitosis, defective


Affected mitochondrial

metabolism, respiratory

chain complex I inhibition

Increased GDH activity

Altered mitochondrial


Chromosomal aberrations,

tumorgenesis, premature

senescence, decreased rate

of mitochondrial respiration

Proliferation arrest, increased

rate of apoptosis, heart

abnormalities, fibrosis,

shortened lifespan

(Alcain and Villalba, 2009; Aquilano

et al., 2010; Bordone et al., 2006,

2007; Borra et al., 2005; Brunet

et al., 2004; Canto et al., 2010;

Feige et al., 2008)

(Garske et al., 2007; Harting and

Knoll, 2010; Hiratsuka et al., 2003;

Nahhas et al., 2007; Southwood

et al., 2007; Wang et al., 2007)

(Ahn et al., 2008; Alhazzazi et al.,

2011; Allison and Milner, 2007;

Cimen et al., 2010; Someya et al.,


(Ahuja et al., 2007; Argmann and

Auwerx, 2006; Haigis et al., 2006)

(Nakagawa et al., 2009; Nakagawa

and Guarente, 2009)

(Kawahara et al., 2009; Mahlknecht

et al., 2006b; McCord et al., 2009;

Michishita et al., 2008; Mostoslavsky

et al., 2006)

(Ford et al., 2006; Michishita et al.,

2005; Vakhrusheva et al., 2008;

Voelter-Mahlknecht et al., 2006)

68 C.V. Pereira et al. / Mitochondrion 12 (2012) 66–76

Abbreviations.: mt′ mitochondrial; n, nuclear; HDACIII, histone deacetylases class III; GDH, glutamate dehydrogenase; PGC1α, Peroxisome proliferator-activated receptor gamma coactivator 1-alpha; e-NOS, epithelial nitric oxide synthase;

FOXO, forkhead transcription factors; NF-κB, nuclear factor κB; Myo D, Myogenic differentiation 1 factor; CPS1, carbamoyl synthetase 1;, cytc, cytochrome c; IDH2, isocitrate dehydrogenase; ShdhA, succinate dehydrogenase flavoprotein;

AceCS2, acetylcoenzyme synthetase2. For more details and references, please refer to the text.

C.V. Pereira et al. / Mitochondrion 12 (2012) 66–76


can be also found in the nucleus.(Schwer and Verdin, 2008; Shoba

et al., 2009).

1.6. Cytosolic and nuclear sirtuins

SIRT1, the closest ortholog of yeast Sirt2, is the most well studied

mammalian sirtuin, harboring both cytosolic and nuclear localization

(although absent from nucleoli). SIRT1 has multiple functions and

its role was confirmed in the regulation of energy metabolism (Yu

and Auwerx, 2010), embryonic development (Mayanil et al., 2006;

Saunders et al., 2010), myocyte differentiation (Fulco et al., 2003), cell

survival and apoptosis (Guo et al., 2010; Yi and Luo, 2010) as well as

in the regulation of gene transcription by histone deacetylation

(Zhang and Kraus, 2010). SIRT1 is also known to mediate the effects of

caloric restriction (Gillum et al., 2011) and to play an important role

in response to different stress stimuli (Leiser and Kaeberlein, 2010).

Additionally, SIRT1 may have an impact on the aging rate (Donmez

and Guarente, 2010). The nuclear fraction of SIRT1 is also localized to

promyelocytic leukemia protein (PML) bodies, where it interacts with

the tumor suppressor p53 which carries several acetylation sites.

Deacetylation of lysine residues in the p53 protein processed by SIRT1

decreases its transactivation activity, which in turn can suppress

apoptosis initiated by DNA damage or oxidative stress (Kume et al.,

2006; Sedding, 2008; Yi and Luo, 2010). The effect of SIRT1 on

cell survival can be also mediated by FOXO transcription factors that

play an important role in cell adaptation to stress conditions including

Foxo1, Foxo3a and Foxo4. Under oxidative stress, deacetylation of

Foxo3a induces its translocation from cytosol to the nucleus where

it forms a complex with SIRT1, regulating the expression of some

antioxidant enzymes, such as mitochondrial superoxide dismutase

(SOD2) and catalase. At the same time, FOXO3a increases the

expression of cell-cycle checkpoint and DNA repair genes, arresting

the cell cycle (Brunet et al., 2004; Jacobs et al., 2008; Motta et al.,

2004; Wang et al., 2007). FOXO1-mediated inhibition of apoptosis

occurs via FOXO4 activity regulation. In transformed epithelial cells,

deacetylated FOXO4 suppresses two proapoptotic caspases, caspase-3

and caspase-7, leading to an arrest of apoptosis (Ford et al., 2005;

Frescas et al., 2005). In turn, deacetylation of FOXO1 results in the

expression of gluconeogenesis genes under control of FOXO1 (Frescas

et al., 2005).

Other SIRT1 targets such as PPARγ and PGC-1α are important

elements controlling energetic balance, especially fatty acid and

glucose metabolism (Michan and Sinclair, 2007; Sugden et al., 2010).

SIRT1 content and activity is increased under starvation and caloric

restriction (CR) (Chen et al., 2008; Nisoli et al., 2005). Deacetylation

of PGC-1α by SIRT1 also stimulates mitochondrial biogenesis.

Experiments by using transgenic animals showed that not all effects

of CR are mediated by SIRT1 but it is noteworthy that both SIRT1

overexpression and caloric restriction are manifested by lower body

weight, reduced cholesterol, glucose and insulin serum level and also

by improved physical performance (Chen et al., 2005; Nisoli et al.,

2005) Lower fat accumulation in SIRT1-overexpressing animals is

associated with PPARγ transcriptional repression which results in

the inhibition of adipogenesis (Picard et al., 2004). In the liver,

SIRT1 regulates glycolysis, gluconeogenesis and fatty acid oxidation

through PGC-1α deacetylation (Rodgers and Puigserver, 2007).

Lipid metabolism (HDL biogenesis) in the liver is also stimulated by

SIRT1-dependent Liver's X receptor deacetylation (Li et al., 2007).

Deacetylation of PGC-1α by SIRT1 also stimulates mitochondrial

biogenesis and induces oxidative phosphorylation (Lagouge et al.,

2006). Similarly, activation of SIRT1 by resveratrol is connected to

enhanced mitochondrial biogenesis and to more efficient metabolism

in the skeletal muscle (Aquilano et al., 2010; Chabi et al., 2009). SIRT1

is also found to be involved in insulin secretion in pancreatic β-cells

(Chen et al., 2010; Liang et al., 2009). The most convincing proof

of concept comes from experiments on SIRT1-overexpressing mice

which present more efficient energy metabolism of pancreatic β-cells

than wild-type individuals (Bordone et al., 2006). This is caused by the

repression of uncoupling protein 2 (UCP2) gene expression by SIRT1,

resulting into decreased content of mitochondrial UCP2 in pancreatic

β-cells, higher mitochondrial coupling and higher rate of ATP

production, which loops to enhanced insulin secretion by pancreatic

β-cells (Bordone et al., 2006). SIRT1 knockout animals showed a lower

level of ATP in pancreatic β-cells as a result of increased UCP2 level

and they exhibited a decrease insulin secretion in the response to

glucose. As indicated above, the feedback between mitochondrial ATP

and insulin secretion is enhanced in SIRT1-overexpressing mice

(Bordone et al., 2007; Chen et al., 2005).

SIRT2 is a mainly cytosolic protein, although a small amount was

also detected in the nucleus, where it carries out histone deacetylation.

In vitro studies demonstrated that SIRT2, similarly to SIRT1, preferentially

deacetylates histone H4 (Vaquero et al., 2007). In the cytosol,

SIRT2 was found to be associated with microtubules, where it

deacetylates α-tubulin (Harting and Knoll, 2010). Based on the

observations that SIRT2 increases during the mitotic phase and colocalizes

with chromatin during transition from G 2 to M phase, it is

thought that this sirtuin can be involved in the regulation of the cell

cycle (Dryden et al., 2003). More detailed studies on sirtuins showed

that SIRT2 plays an important role in glial cells development,

microtubule dynamics in oligodendrocytes and maintenance of

axonal integrity (Michan and Sinclair, 2007; Tang and Chua, 2008),

which makes this isoform an attractive therapeutic target in

neurodegenerative diseases (Wu et al., 2010). SIRT2 was also found

to be involved in Parkinson's and Alzheimer's disease and also in

a Huntington's disease model induced in Drosophila (Pallos et al.,

2008). Additionally, the development and progression of many

brain tumors are connected with a disturbance in SIRT2 deacetylase

activity (Hiratsuka et al., 2003). Thus, many reports describe attempts

to modulate SIRT2 activity with the objective of treating several

malignancies (Harting and Knoll, 2010).

Less published data exists for SIRT6 and SIRT7. Nonetheless, it has

been demonstrated that SIRT6 is similar to SIRT1 regarding the

maintenance of genome integrity, since deletion of this protein leads

to different chromosomal aberrations, impairs cells proliferation and

increases the rate of DNA damage accumulation (Michishita et al.,

2008). There is growing evidence indicating that SIRT6 is involved

in base excision repair (BER) of single-stranded DNA breaks.

Moreover, SIRT6 is directly involved in DNA repair by forming a

macromolecular complex with the DNA double-strand break (DSB)

repair factor, DNA-PK (DNA-dependent protein kinase) promoting

DSB repair. Cells lacking SIRT6 are more sensitive to DNA damaging

agents (McCord et al., 2009). Moreover, SIRT6 can be considered also

as a regulator of metabolism under variable nutrient availability

(Kanfi et al., 2008). Although SIRT6 has weak deacetylase activity, it

can deacetylate histone H3K9, which makes SIRT6 involved in the

stability of telomeric chromatin structure (Michishita et al., 2008).

SIRT6 depletion is associated with premature senescence due to

abnormal telomere structure. It was found that interaction of WRN,

a factor mutated in human premature aging syndrome (Werner

syndrome), with histone H3K9 requires proper SIRT6 activity

(Michishita et al., 2008). By deacetylating histone H3K9, SIRT6 may

additionally regulate expression of glycolytic genes, especially because

it co-represses transcription of Hif1α, which is crucial for the response

to nutrient deprivation. In SIRT6 knockout cells, increased glucose

uptake and higher glycolysis rate is followed by a decrease in

mitochondrial respiration. All these metabolic changes are associated

with a higher Hif1α activity. The regulatory role seems to be important

in several pathologic states associated with diabetes and obesity

(Zhong et al., 2010). Moreover, SIRT6 has been associated with the

production of pro-inflammatory cytokines such as TNF-α by immune

cells (Van Gool et al., 2009). Recently, it was described that SIRT6 plays

a role in the control of somatic growth and in the prevention of obesity

70 C.V. Pereira et al. / Mitochondrion 12 (2012) 66–76

by modulating neural chromatin structure and gene activity (Schwer

et al., 2010).

SIRT7 was found to regulate transcription of ribosomal genes in the

nucleus by interacting and regulating RNA polymerase (Pol1) activity

which means that this sirtuin can have an important impact on multiple

cellular processes (Ford et al., 2006). In fact, it has been shown that

transcription of rRNA directly depends on SIRT7 content (Ford et al.,

2006; Mostoslavsky et al., 2006), with the highest SIRT7 level found in

blood and CD33+ myeloid bone marrow precursor cells. On the other

hand, lower SIRT7 levels were observed in ovaries and mammalian

skeletal muscle (Voelter-Mahlknecht et al., 2006). In case of SIRT7

ablation, cells do not proliferate and enter the apoptotic pathway (Ford

et al., 2006), as opposed to an increased SIRT7 level, which has been

associated with carcinogenesis. In fact SIRT7 overexpression was found

to occur in breast cancer cells (Ashraf et al., 2006). Therefore, the role

of SIRT7 in the regulation of cell cycle is important for cell adaptation

processes under stress conditions. Interestingly, SIRT7 knockout

animals suffer from heart hypertrophy, fibrosis and inflammatory

cardiomyopathy and shortened lifespan (Vakhrusheva et al., 2008).

1.7. Mitochondrial sirtuins: characterization and activity

1.7.1. Localization and processing

Among the seven sirtuins already here described, three (SIRT3,

SIRT4 and SIRT5) are present inside mitochondria, regulating several

metabolic pathways through protein deacetylation (Fig. 1). It has

been shown that about 1/5 of mitochondrial proteins undergo

acetylation, being one of the highest pools in the cell. Proteomic

studies of mice liver mitochondria revealed almost 300 lysine residues

that can be acetylated in 133 proteins involved in different metabolic

pathways, including Krebs and urea cycle, fatty acid beta-oxidation

and oxidative phosphorylation (Kim et al., 2006). Lysine acetylation

profile of mitochondrial proteins demonstrates some similarities

with prokaryotic organisms, which appears to support the symbiotic

origin of mitochondria hypothesis (Huang et al., 2010). All three

mitochondrial sirtuins are located in the mitochondrial matrix (Huang

et al., 2010); however, SIRT5 was also found in the mitochondrial

intermembrane space (Nakamura et al., 2008; Schlicker et al.,

2008). Interestingly, SIRT5 overexpression does not increase the

mitochondrial intermembrane pool of this protein but results

instead into a preferential accumulation in the mitochondrial matrix

(Nakagawa et al., 2009). There are some controversial data showing

that in COS7 cells co-expressing SIRT3 and SIRT5, the 44 kDa isoform

of SIRT3 can be found in the nucleus, although its function still

waits to be elucidated (Nakamura et al., 2008; Scher et al., 2007). It

was speculated that under normal conditions, both a mitochondrial

and nuclear localization of SIRT3 is possible, although under stressful

conditions, most of the nuclear pool moves to mitochondria (Scher

et al., 2007). Mitochondrial sirtuins, especially SIRT3 and SIRT4,

are responsible for sensing NAD + /NADH balance, which by being

disrupted by several factors may affect cellular redox homeostasis

(Yang et al., 2007; Yu and Auwerx, 2009).

All three mitochondrial sirtuins are encoded by nuclear DNA

and contain a mitochondrial targeting sequence located on their

Fig. 1. Mitochondrial sirtuins and their targets. SIRT3 is the major mitochondrial deacetylase which has been shown to physically interact with one of the subunits of complex I

(NDUFA9) and also with ShdhA, one of the flavoprotein subunits of complex II. SIRT3 plays a major role in fatty acid oxidation and in maintaining ATP levels. SIRT3 is also important

to regulate the Krebs cycle since it deacetylates Acetyl-CoA synthase 2, which catalyzes the conversation of acetyl-CoA into acetate. Another target is the enzyme isocitrate

dehydrogenase 2 which catalyzes a key regulation point in the Krebs cycle. SIRT3 can also deacetylate cyclophilin D and induce hexokinase II dissociation from the mitochondrial

outer membrane. SIRT4 is a NAD-dependent mono-ADP-ribosyltransferase that is known to target glutamate dehydrogenase (GDH), which converts glutamate into α-ketoglutarate

and allows it to undergo subsequential steps in the Krebs cycle. GDH can also be deacetylated by SIRT3. SIRT4 also seems to interact with the ANT isoforms 2 and 3 and to act as a

negative regulator of oxidative metabolism in contrast with SIRT3. Finally, SIRT5 is known to play an important role in the urea cycle, deacetylating and activating carbamoyl

phosphate synthetase (CPS1), the enzyme that catalyzes the first step of this cycle. Also, SIRT5 might deacetylate cytochrome c, pointing a pro-apoptotic function for this sirtuin as it

was also pointed to SIRT3.

C.V. Pereira et al. / Mitochondrion 12 (2012) 66–76


N′ terminal end, which is immediately cleaved after reaching the

mitochondrial matrix. Tissue expression profile of SIRT3, SIRT4 and

SIRT5 is similar; however, relatively higher levels of SIRT5 have been

observed in muscles (Hallows et al., 2008; Nakamura et al., 2008).

SIRT3 is a 28 kDa deacytelase, with, as mentioned before, a 44 kDa

isoform found in nucleus, which requires proteolitic processing to

achieve its maximal deacetylase activity. After reaching mitochondria,

premature SIRT3 is cleaved at the N′-terminus targeting peptide by

the mitochondrial processing peptidase (MPP) (Schwer et al., 2002).

SIRT4 is a 35 kDa protein with a ADP-ribosylotransferase activity

present in mitochondrial matrix. SIRT4 transfers ADP-ribose from

NAD + to target proteins. In vitro studies showed no detectable

deacetylase activity of SIRT4 but it cannot be excluded that a very

specific substrate would have to be present in order to observe some

relevant activity (Huang et al., 2010). Similarly to SIRT3, SIRT4 is

also processed in the mitochondrial matrix where a 28 amino acid

sequence is cleaved when pro-SIRT4 reaches its destination (Schwer

et al., 2002).

The 33.0 kDa SIRT5 is present in the mitochondrial matrix, but

when overexpressed, it can also be found in the mitochondrial

intermembrane space. SIRT5 N-terminal 36 amino acids sequence is

immediately cleaved after import into the mitochondrial matrix

(Mahlknecht et al., 2006a; Michishita et al., 2005).

1.7.2. Regulation of mitochondrial metabolism by sirtuins

As described above, reversible protein acetylation allows cells to

adjust to a changing of the environment conditions. A key question

is why and where do mitochondrial proteins become acetylated

This question has not been answered in detail yet but it has been

confirmed that protein acetylation does occur in mitochondria (Guan

and Xiong, 2011) and, in fact, it is also been suggested that the

acetylation mechanism in mitochondrial proteins is different from

cytosolic and nuclear proteins (Kim et al., 2006). Studies in knockout

SIRT4 and SIRT5 mice showed that there was no change in the

acetylation level of mitochondrial proteins, but studies on SIRT3 −/−

showed a significant increase in protein acetylation (Huang et al.,

2010; Lombard et al., 2007). This finding suggests that SIRT3 seems

to play a central role in protein deacetylation in mitochondria. It

is known that sirtuins are directly or indirectly involved in many

different metabolic pathways including lipid metabolism (Lomb et al.,

2010), calorie restriction conditions/insulin uptake (Ahuja et al.,

2007; Guarente, 2008; Qiu et al., 2010b), urea cycle (Nakagawa et al.,

2009; Nakagawa and Guarente, 2009), glycolysis, gluconeogenesis

and Krebs cycle (Huang et al., 2010; Verdin et al., 2010).

Nowadays, major threats to human health are cancer, neurodegenerative

diseases, immune dysfunction and certainly, the metabolic

syndrome (Guarente, 2006). In the US, around 32% of adults and

17% of children and adolescents are obese (Elliott and Jirousek, 2008).

The human body is not adapted to process the excess of calorie

intake per day. Also, in many regards, all of these diseases are also

associated with the aging process. When organisms are calorie

restricted (30–40% decrease in food intake), it was shown to be

possible to increase the lifespan up to 50%, having sirtuins a very

important role in the adaptation process (Guarente, 2007; Qiu

et al., 2010b). When food is scarce, mammals may have to adapt

to the limited resources in order to maintain the basic functions

such as growth, synthesis and reproduction, having to shut down

unnecessary energy expenditures in order to survive. Thus, tissues

that are crucial for survival such as the muscle, the heart and

certain parts of the brain are protected from starvation and go

through mild calorie restriction. On the other hand, tissues that

are normally associated with endocrine and exocrine activity, such

as the liver, pancreas and the reproductive system, are very likely

to experience severe calorie restriction (Qiu et al., 2010b). Upon

abundance of resources, energy is stored as fat. During calorie

restriction, animals up-regulate fatty-acid oxidation and switch

fuel usage from glucose to fatty-acids. At a cellular level, increased

mitochondrial biogenesis in calorie-restricted tissues suggests a

tissue-specific increase in metabolic rate of the affected animals (Qiu

et al., 2010b). Calorie-restricted animals have low levels of blood

insulin in response to limited food intake. Low insulin levels suppress

glycolysis and maintain blood glucose levels. As described above,

SIRT1 suppresses UCP2 in pancreatic β-cells, which allows for a more

efficient ATP production (Bordone et al., 2006). Nevertheless, the

fulcrum of calorie restriction response seems to be mitochondrial

activation which allows a metabolic adaptation to chronic energy

deficit demand (Qiu et al., 2010b). Here is where sirtuins have now

the spotlight. Fig. 1 shows a summary of the interplay between

mitochondrial and in-house sirtuins. SIRT3 and regulation of mitochondrial metabolism. SIRT3 is a

unique mitochondrial sirtuin once it is the only one that was related

with extended lifespan in humans (Rose et al., 2003). SIRT3 may be

critical in sensing NAD + levels in the mitochondria since increased

NAD + would trigger a regulatory pathway that would activate SIRT3

leading to the deacetylation of specific targets. It has been demonstrated

that mice deficient in SIRT3 present hyper protein acetylation

(Lombard et al., 2007), including of the metabolic enzyme glutamate

dehydrogenase (GDH) suggesting that this sirtuin may have an

important impact in metabolic control (Schlicker et al., 2008).

Lombard et al. concluded that SIRT3 deficient mice are metabolically

unremarkable under both fed and fasted conditions, including normal

thermogenesis (Lombard et al., 2007). Another study demonstrated

that SIRT3 was able to deacetylate and activate the mitochondrial

enzyme acetyl-CoA synthase 2 (Hallows et al., 2006), an enzyme that

catalyzes the formation of Acetyl-CoA from acetate (Hallows et al.,

2006; North and Sinclair, 2007). Under ketogenic conditions, such as

during calorie restriction period, the liver releases a large amount of

acetate into the blood. The heart and the muscles express acetyl-CoA

synthase 2 and use acetate in an efficient way as an energy source.

Deacetylation of acetyl-CoA synthetase switches on its activity so it is

relevant that SIRT3 is indeed activated during CR (Hirschey et al.,

2010). A study by Ahn et al. (2008) stressed the importance of NAD + -

dependent deacetylation in the regulation of energy homeostasis and

also provided evidence for SIRT3-dependent regulation of global

mitochondrial function. The authors of this paper showed that SIRT3

is an important regulator of basal ATP levels and observed that SIRT3

can physically interact with at least one of the subunits of complex I,

the 39-KDa protein NDUFA9, although in a reversible manner (Ahn

et al., 2008). Other studies demonstrated that mitochondria from

SIRT3 − / − animals display a selective inhibition of complex I activity

and altered basal ATP content (Ahn et al., 2008). Another target of

SIRT3 is also the enzyme isocitrate dehydrogenase 2 (Schlicker et al.,

2008), which promotes regeneration of antioxidants and catalyzes a

key regulation point of the citric acid cycle. Schilicker C et al. reported

that the N- and C-terminal regions of SIRT3 regulate its activity against

glutamate dehydrogenase and a peptide substrate, indicating roles

of these regions in substrate recognition and sirtuin regulation

(Schlicker et al., 2008). Cimen et al. identified that the succinate

dehydrogenase flavoprotein (ShdhA) subunit is also another SIRT3

target in the mitochondrial respiratory chain (Cimen et al., 2010). It

was demonstrated that SdhA is highly acetylated in SIRT3 knockout

mice and also that the activation of complex II was dependent on

SIRT3 both in wild-type mice and in cells over-expressing SIRT3.

The regulation of complex II by reversible acetylation is actually an

important point of control since is a crossroad between oxidative

phosphorylation and the Krebs cycle, acting on the regulation of

metabolism in mammalian mitochondria.

The skeletal muscle is a metabolically active organ and crucial

for insulin-mediated disposal and lipid catabolism. Palacios et al.

demonstrated that exercise signals can regulate SIRT3 in skeletal

muscle, with a dynamic response of that sirtuin to coordinate

72 C.V. Pereira et al. / Mitochondrion 12 (2012) 66–76

downstream molecular responses (Palacios et al., 2009). The authors

showed that exercise training increases SIRT3 level and CREB

phosporylation and up-regulates PGC-1α. Interestingly, SIRT3 is

more highly expressed in slow oxidative type I soleus muscle in

comparison with fast type II or gastrocnemius muscles (Palacios et al.,

2009). In addition, it was found out that SIRT3 levels in skeletal

muscle are sensitive to diet, with SIRT3 decreasing by a high-fat diet,

and increased by short-term fasting (24-hour) or long term nutrient

deprivation (12-month CR) and exercise training. Since caloric

restriction regiment leads to phospho-activation of AMPK in the

muscle and that SIRT3 −/− mice have low phosphorylation levels of

both AMPK/CREB and low expression of PGC-1α, it was proposed that

SIRT3 levels may respond to various nutritional/energetic and

physiological challenges by regulating muscle energy homeostasis

via AMPK and PGC-1α (Palacios et al., 2009). Another important study

by Hirschey et al. strengths the role of SIRT3 in the regulation of fattyacid

oxidation (Hirschey et al., 2010). It was reported that SIRT3

expression is up-regulated during fasting in liver and brown adipose

tissues. Mass spectrometry results showed that the long chain acyl

coenzyme A dehydrogenase (LCAD) is hyperacetylated at lysine 42 in

the absence of SIRT3, with LCAD hyperacetylation reducing its

enzymatic activity (Hirschey et al., 2010). Mice lacking SIRT3

presented hallmarks of fatty-acid oxidation disorders during fasting,

including reduced ATP levels and intolerance to cold exposure

(Hirschey et al., 2010). This study identified acetylation as a novel

regulatory mechanism for mitochondrial fatty acid oxidation and

demonstrates that SIRT3 modulates mitochondrial intermediary

metabolism and fatty-acid use during fasting. Interestingly, it was

also reported that SIRT3 deacetylates cyclophilin D (Shulga et al.,

2010), diminishing its activity and inducing its dissociation from the

adenine nucleotide traslocator (ANT). In addition, SIRT3-induced

interaction with cyclophilin D also induces the detachment of

hexokinase II from mitochondria. These findings might be important

for the role of SIRT3 in the metabolism of cancer cells and their

susceptibility to toxicity by foreign agents. Recently, Hafner et al.

showed that SIRT3 can regulate the mPTP through the deacetylation

of CypD at lysine 166, suppressing age-related cardiac hypertrophy

(Hafner et al., 2010). In an elegant study, the authors reported that

SIRT3 deacetylates the regulatory component of the mPTP, cyclophilin

D (CypD), at lysine 166, which is adjacent to the binding site of

cyclosporin A, a CypD inhibitor. Cardiac myocytes from SIRT3

knockout mice showed increased mitochondrial swelling due to the

mPTP opening and accelerated signs of cardiac aging including cardiac

hypertrophy and fibrosis at 13 months of age (Hafner et al., 2010). All

together, the data shows that SIRT3 controls the mPTP and that a loss

of SIRT3 promotes mitochondrial alterations resulting in enhanced

ROS production and cell dysfunction. The results are also a clear

evidence that SIRT3 activity is necessary to prevent mitochondrial

dysfunction and cardiac hypertrophy during aging (Hafner et al.,

2010). Although it is not new that under certain conditions SIRT3

mediates the reduction of oxidative damage, a study from Someya et

al. relates age-related hearing loss and calorie restriction with this

topic (Someya et al., 2010). Under caloric restriction conditions, a

reduction of oxidative damage in multiple tissues and a decrease of

age-related hearing loss in WT mice were observed, as opposed to

mice lacking SIRT3. The authors of this study identified SIRT3 as an

essential player in enhancing the mitochondrial glutathione antioxidant

defense system during caloric restriction and concluded that

mitochondrial adaptations and aging retardation in mammals may be

dependent on SIRT3 (Someya et al., 2010). Another paper showed that

the protective effects of CR against oxidative stress and damage are

diminished in mice lacking SIRT3, with the effects of this protein being

mediated by deacetylation of two critical lysine residues in SOD2,

enhancing its antioxidant activity (Qiu et al., 2010a).

Several other SIRT3 targets involved in metabolism have

been recently described. Shimazu et al. identified 3-hydroxy-3-

methylglutaryl CoA synthase 2 (HMGCS2) as another SIRT3 deacetylated

protein (Shimazu et al., 2010). Mice lacking SIRT3 present a

decrease in β-hydroxybutyrate levels during fasting, which can

demonstrate that SIRT3 regulates ketone body production during

fasting (Shimazu et al., 2010). Also, Hallows et al. identified the

urea cycle enzyme ornithine transcarbomoylase (OTC) and other

enzymes involved in β-oxidation as likely SIRT3 targets. Fasted

mice lacking SIRT3 revealed alterations in β-oxidation and in the urea

cycle, demonstrating a direct role of SIRT3 in the regulation of the

two important metabolic pathways during CR (Hallows et al., 2011).

The results also suggested that under low energy input conditions,

SIRT3 modulates mitochondria by promoting amino acid catabolism

and β-oxidation (Hallows et al., 2011). Recently, it has been shown

that livers from mice maintained on a high fat diet (HFD) exhibited

reduced SIRT3 activity, a 3-fold decrease in hepatic NAD + levels, and

increased mitochondrial protein oxidation. In contrast, neither SIRT1

nor histone acetyltransferase activities were altered, suggesting SIRT3

as a crucial factor contributing to the observed phenotype (Kendrick

et al., 2011). In SIRT3 −/− mice, HFD further increased the acetylation

status of liver proteins and reduced the activity of respiratory

complexes III and IV. This study identified acetylation patterns in

liver proteins from HFD-fed mice and the results suggest that SIRT3 is

an integral regulator of mitochondrial function, with its depletion

resulting in hyperacetylation of critical mitochondrial proteins that

protect against liver lipotoxicity under conditions of nutrient excess

(Kendrick et al., 2011). SIRT4 and regulation of mitochondrial metabolism. The mitochondrial

isoform of SIRT4 does not show detectable deacetylase

activity but possesses NAD-dependent mono-ADP-ribosyltransferase

activity (Ahuja et al., 2007; Yamamoto et al., 2007). It is known that

glutamate dehydrogenase (GDH) is a substrate of SIRT4 (Haigis et al.,

2006). By ADP-ribosylating GDH, SIRT4 suppresses its activity and

prevents the usage of amino acids as an energy source. During calorie

restriction, although NAD + levels are increased in mitochondria,

SIRT4 expression is decreased. The decrease in SIRT4 activity results in

the activation of GDH and it induces the usage of glutamate and

glutamine in order to generate ATP (Haigis et al., 2006). This

mechanism is important in pancreatic β-cells of calorie-restricted

mice. Due to low blood glucose levels, glucose-stimulated insulin

secretion is suppressed. However, the activation of GDH allows

amino-acid stimulated insulin secretion and thus maintain basal

levels of insulin (Argmann and Auwerx, 2006). GDH can also be

deacetylated by SIRT3, although the functional link between acetylation

and ADP-ribosylation of this important enzyme is not yet well

understood. Also, a new role for mitochondrial SIRT4 in the regulation

of insulin secretion was identified, with SIRT4 considered as a protein

that negatively regulates insulin secretion (Ahuja et al., 2007). Mass

spectrometry analysis of proteins that co-immunoprecipitate with

SIRT4 identified insulin-degrading enzyme and the adenine nucleotide

translocator isoforms ANT2 and ANT3 (Ahuja et al., 2007). It was

demonstrated that depletion of SIRT4 from insulin-producing INS-1E

cells results in increased insulin secretion in response to glucose

(Ahuja et al., 2007). Nasrin et al. investigated whether the depletion of

SIRT4 would enhance liver and muscle metabolism (Nasrin et al.,

2010). An increase in gene expression of mitochondrial and fatty acid

metabolism enzymes was found in hepatocytes SIRT4 KO cells (Nasrin

et al., 2010). Interestingly, it was also found out that SIRT1 mRNA

levels and protein expression were increased after SIRT4 knockdown

(Nasrin et al., 2010). Interestingly, the loss of activity of SIRT4 in

primary hepatocytes and in whole liver led to an increased expression

of SIRT3. Fatty acid oxidation (FAO) was increased in SIRT4 KO

primary hepatocytes and this effect was dependent on SIRT1 (Nasrin

et al., 2010). In SIRT4 KO primary myotubes, increased fatty acid

oxidation, cellular respiration and pAMPK levels were detected. The

findings demonstrate that SIRT4 inhibition increases fat oxidative

C.V. Pereira et al. / Mitochondrion 12 (2012) 66–76


capacity in liver and mitochondrial function in skeletal muscle. Taken

together, it seems like SIRT4 is a negative regulator of oxidative

metabolism, in contrast with the functions of SIRT1 and SIRT3, which

enhance the oxidative capacity of tissues (Nasrin et al., 2010). SIRT5 and mitochondrial metabolism. As compared with other

sirtuins, there is a lot still to be discovered about SIRT5. For example, it

is known that SIRT5 deacetylates and activates carbomoyl phosphatase

synthetase 1 (CPS1), an enzyme that catalyzes the first step of

the urea cycle (Gertz and Steegborn, 2010; Michishita et al., 2005;

Nakagawa et al., 2009). The expression levels of SIRT5 are unaltered

but NAD + is increased by 50% in mitochondria from calorie restricted

liver (Nakagawa et al., 2009). This increase in the activity of SIRT5

might regulate the urea cycle during caloric restriction conditions by

deacetylation and activation of CPS1. Schilicker et al. reported that

SIRT5 did not deacetylate any of the mitochondrial matrix proteins

tested (Schlicker et al., 2008). The surprising result was that SIRT5

can deacetylate cytochrome c (Schlicker et al., 2008). By using a

mitochondrial import assay, the authors determined that SIRT5 can be

translocated not only to the mitochondrial intermembrane space but

also to the matrix, indicating that localization might contribute to

SIRT5 regulation and substrate selection (Schlicker et al., 2008). In

vivo, SIRT5 appears to co-localize with cytochrome c in the

intermembrane space. The reversible acetylation of cytochrome c

could either affect its function in respiration or in apoptosis, or both. In

cerebellar granule neurons overexpressing SIRT5, a pro-apoptotic

function for this sirtuin was pointed out (Gertz and Steegborn, 2010).

Several other target proteins for SIRT5 are possible due to the intermitochondrial

space localization of the sirtuin. One of them is the

apoptosis inducing factor (AIF), which was already reported to be

acetylated depending on the feeding status (Gertz and Steegborn,

2010). Further and exciting studies are thus needed to understand the

physiological role of SIRT5 as a mitochondrial inter-membrane space


1.7.3. Protection of mitochondrial function by sirtuins

The role of mitochondria in cell physiology and survival, as well as

in drug-induced toxicity is well established (Pereira et al., 2009a,b;

Sardao et al., 2008). Besides control of mitochondrial metabolism,

sirtuins are also involved in the lines of defense of that organelle.

In fact, there is actually evidence that at least SIRT3 can protect

mitochondria from exogenous and endogenous stresses (Kong et al.,

2010; Pillai et al., 2010; Scher et al., 2007; Sundaresan et al., 2008).

Mitochondria are an important site of reactive oxygen species (ROS)

production in a cell, which is why a tight ROS production by that

organelle must be exerted by different mechanisms in order to

prevent structural damage and accelerated aging (Guarente, 2008).

The role of mitochondrial sirtuins in the protection against mitochondrial

damage is now beginning to be clarified. One starting point

was the finding that NAD + levels dictate cell survival (Yang et al.,

2007). It is well known that one of the major causes for cell death

due to genotoxic stress is the hyperactivation of PARP-1 that depletes

nuclear and cytosolic NAD + causing the translocation of the AIF

from the mitochondrial membrane to the nucleus. Yang et al. identified

Nampt as a stress- and nutrient-responsive gene that increases

mitochondrial NAD + levels (Yang et al., 2007). It has been demonstrated

that increased mitochondrial NAD + levels improve cell

survival during genotoxic stress and that this protection is dependent

on SIRT3 and SIRT4, which means that Nampt-mediated cell protection

requires mitochondrial sirtuins (Yang et al., 2007). Nampt increased

SIRT3 activity since Nampt overexpression decreased the acetylation

level of AceCS2. Allison et al. exploited apoptosis trough Bcl-2/p53

regulation and verified a SIRT3 involvement in this process (Allison

and Milner, 2007). Bcl-2 and SIRT3 were silenced separately and

in combination in human epithelial cancer and non-cancer cells. It

was demonstrated that SIRT3 is required for apoptosis under basal

conditions, by selective silencing of Bcl-2 in HCT116 human epithelial

cancer cells. SIRT3 is dispensable for stress-induced apoptosis in

HCT116 human epithelial cancer cells but it is an essential proapoptotic

mediator for both Bcl-2/p53-regulated apoptosis. Interestingly,

SIRT3 functions in JNK2-regulated apoptosis but it is dispensable

for both SIRT-1 regulated and stress-induced apoptosis. It is then

concluded that SIRT3 is a pro-apoptotic protein that participates in

distinct basal apoptotic pathways (Allison and Milner, 2007).

Nevertheless, this paper does not really point to a direct link between

SIRT3 and protection against apoptosis under stress conditions in

contrast to many other papers. For example, it has been shown that

SIRT3 protects the mouse heart by blocking the cardiac hypertrophic

response (Sundaresan et al., 2009). In cardiomyocytes, SIRT3 prevented

cardiac hypertrophy activation by activating the fork head

box O3a-dependent (Foxo3a-dependent), anti-oxidant superoxide

dismutase and catalase genes and decreasing cellular levels of ROS

(Sundaresan et al., 2009). The results demonstrate that SIRT3 is an

endogenous negative regulator of cardiac hypertrophy by the

suppression of ROS levels. Another study showed that SIRT3 levels

are increased under stress not only in mitochondria but also in the

nuclei of cardiomyocytes (Sundaresan et al., 2008). This particular

paper identified Ku70 as a new SIRT3 target, since SIRT3 physically

binds to ku70 and deacetylates the latter, promoting its interaction

with Bax. Under stress, SIRT3 overexpression protects cardiomyocytes

by partially preventing the translocation of Bax to mitochondria.

This study points an essential role of SIRT3 in the survival of

cardiomyocytes under stress conditions. Recently, SIRT3 was pointed

out as a tumor-suppressor mitochondrial-localized protein (Kim et al.,

2010). In this study, it has been shown that the expression of a single

oncogene (Myc or Ras) in SIRT3 knockout MEFs results in in vitro

transformation and altered intracellular metabolism. In addition,

SIRT3 knockout mice developed ER/PR-positive mammary tumors

(Kim et al., 2010). In this work, it is also shown that several human

cancer tissues exhibit reduced SIRT3 levels, thus leading the authors

to propose SIRT3 as a mitochondrial fidelity protein. In this regard,

loss of SIRT3 results in a decrease in the antioxidant defenses, resulting

in an increase of ROS production. It is also speculated that a prooxidant

environment may be permissive for in vivo carcinogenesis.

It is known that PGC-1α is an important inducer of detoxifying

enzymes, even though the molecular mechanism is not clearly

understood. Kong et al. showed that PGC-1α activated the mouse

SIRT3 promoter mediated by an estrogen-related receptor binding

element (ERRE) (Kong et al., 2010). The knockdown of ERR α reduced

the induction of SIRT3 by PGC-1α in C2C12 myotubes. In the same

work, it was found out that SIRT3 was crucial for PGC-1α-dependent

induction of ROS-detoxifying enzymes and other components of the

mitochondrial respiratory chain, such as glutathione peroxidase-1,

superoxidase dismutase-2, ATP synthase 5c and cytochrome c (Kong

et al., 2010). Both overexpression of SIRT3 and PGC-1α decreased basal

ROS levels in myotubes. SIRT3 stimulated mitochondrial biogenesis

and might provide a novel target for treating ROS-related diseases.

Another interesting study focused on SIRT3 capacity of reducing lipid

accumulation via AMKP activation in human hepatic cells (Shi et al.,

2010). The knockdown of SIRT3 downregulated the phosphorylation

of AMPK and acetyl coenzyme A carboxylase (ACC), promoting

increased lipid accumulation. It was concluded that the capacity

of SIRT3 to activate AMPK is dependent on its deacetylase activity

(Shi et al., 2010). A very interesting paper correlates p53, SIRT3 and

protection of in vitro-fertilized mouse against oxidative stress

(Kawamura et al., 2010). During pre-implantation development,

mitochondrial dysfunction or increased levels of oxidative stress

adversely affect the developmental outcome. In this study, it was

shown that SIRT3 inactivation increases mitochondrial ROS production

leading to p53 up-regulation and alterations in downstream gene

expression.The findings indicate that SIRT3 might play a protective

role in pre-implantation of embryos under stress conditions during

74 C.V. Pereira et al. / Mitochondrion 12 (2012) 66–76

in vitro fertilization and culture, pointing out a new and interesting

future clinical application related with manipulation of SIRT3 and

post-implantation success.

Recently, another study involved SIRT3 in oral cancer (Alhazzazi

et al., 2011). The authors demonstrated for the first time that SIRT3

is overexpressed in oral squamous cell carcinoma (OSCC) in vitro and in

vivo, when compared with other sirtuins. Down-regulation of

SIRT3 inhibited cell growth, increased cell sensitivity to chemotherapy

and reduced tumor burden in vivo, demonstrating that SIRT3 can be a

novel potential therapeutic target for oral cancer (Alhazzazi et al., 2011).

Another paper showed that SIRT3-mediated deacetylation of evolutionary

conserved lysine 122 in MnSOD results into increased activity in

response to stress. SIRT3-knockout results in increased mitochondrial

superoxide, formation of a tumor-permissive environment and finally,

enhanced mammary carcinogenesis (Tao et al., 2010).

To the best of our knowledge, there is not much information about

SIRT4 or even SIRT5 but it seems very likely that both sirtuins can also

be important in mitochondrial protection against genotoxic stress

suggesting that they can contribute to apoptosis in tumor-supressive

or stress resistant conditions (Verdin et al., 2010). Interestingly, SIRT4

is apparently involved in mitochondrial oxidative metabolism. SIRT4

KO primary hepatocytes and myotubes were used in order to study

fatty acid oxidation and oxygen consumption under these conditions

(Nasrin et al., 2010). As described above, the findings from this study

suggest that SIRT4 is a negative regulator of oxidative metabolism,

in contrast with SIRT3 and SIRT1, which means that a functional

interplay between different sirtuins must exist in order to coordinate

the flow of energy during a given metabolic state. It seems that SIRT4

inhibition may improve hepatic insulin sensitivity via increased fat

oxidative capacity and hence may be beneficial for the treatment of

type 2 diabetes.

Despite the limited knowledge on in vivo substrates for SIRT5, the

contribution of this sirtuin in disease has been suggested. Repetitive

elements in its gene structure suggested possible genomic instabilities

and malignant diseases (Gertz and Steegborn, 2010). Furthermore,

one particular study indicates that SIRT5 is decreased after alcohol

exposure in rats, which increased hepatic mRNA expression of FoxO1

and p53 (Lieber et al., 2008). Thus, alcohol consumption compromises

nuclear mitochondrial interactions by post-translational modifications

which contribute to alteration of mitochondrial biogenesis

through the newly discovered decrease in SIRT5. It seems that

SIRT5 contributes to liver damage induced through chronic exposure

to alcohol but would also be interesting to understand the role of

this sirtuin under other stress conditions and understand its role in

mitochondrial protection against various stressors.

1.8. Future perspectives: are sirtuins good therapeutic targets

It is plausible that drugs aimed at modulating sirtuins activity

and/or expression may have important consequences in cellular

responses to stress and life span. It is extremely important to fully

identify and characterize targets of these exciting class of proteins and

understand how the manipulation of their enzymatic activity or

protein expression can be involved in the protection, not only of

mitochondria but also of the entire cell. It seems that sirtuins are

strategically positioned along the different organs and cellular

compartments and that they may play distinct roles (Schwer and

Verdin, 2008; Smith et al., 2002; Vaquero, 2009; Verdin et al., 2010;

Yu and Auwerx, 2009). Furthermore, it would be very interesting

to understand how sirtuins communicate within each other and

explore their communication network code that should in theory,

contribute to protect the cell. What do these proteins have in

common How do they interact with each other Is the stoichiometry

of the different sirtuins in mitochondria constant How does it change

there and in other cellular locations upon different stimuli Many

of these questions remain unanswered. Mitochondrial sirtuins might

contribute to the decrease of “unhealthy” mitochondria and some of

them (e.g. SIRT3) might even be protective against drug-induced

toxicity (Scher et al., 2007; Sundaresan et al., 2008, 2009), which is

why it is fundamental to understand the mechanism behind sirtuinmediated

mitochondrial protection and how targeting sirtuins can be

a valid future therapeutic approach for several different diseases.

Although many recent reviews have provided new directions in

this field, there is still much to be elucidated. There is still a gap of

information regarding SIRT4 and SIRT5 functions that warrants

further studies in order to understand the roles of these important

players in the regulation and protection of mitochondrial function.

This review provided an overall view of sirtuin function with a special

relevance on the most recent findings on the function and relevance

of mitochondrial sirtuins. Furthermore, the new and exciting data

opens a completely new road for novel pharmaceutical applications of

inhibitors and inducers of this class of proteins.


Work in the authors' laboratory is funded by the Portuguese

Foundation for Science and Technology (FCT) (research grant

PTDC/SAU-TOX/110952/2009 to Paulo Oliveira). Claudia Pereira is the

recipient of a Ph.D. fellowship from the FCT (SFRH/BD/48029/2008).

The work was also partially supported by the Polish Ministry of

Science and Higher Education under grant NN407 075 137 for Magda

Lebiedzinsk and Mariusz R. Wieckowski.


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



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

Email address –

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-


Toxicology and Applied Pharmacology 264 (2012) 167–181

Contents lists available at SciVerse ScienceDirect

Toxicology and Applied Pharmacology

journal homepage:

Mitochondrial bioenergetics and drug-induced toxicity in a panel of mouse

embryonic fibroblasts with mitochondrial DNA single nucleotide polymorphisms

Claudia V. Pereira a , Paulo J. Oliveira a , Yvonne Will b , Sashi Nadanaciva b, ⁎

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

b Compound Safety Prediction, Pfizer Global Research & Development, Groton, CT, USA




Article history:

Received 14 June 2012

Accepted 29 July 2012

Available online 4 August 2012



Single nucleotide polymorphisms



Drug-induced toxicity

Mitochondrial DNA (mtDNA) variations including single nucleotide polymorphisms (SNPs) have been

proposed to be involved in idiosyncratic drug reactions. However, current in vitro and in vivo models

lack the genetic diversity seen in the human population. Our hypothesis is that different cell strains

with distinct mtDNA SNPs may have different mitochondrial bioenergetic profiles and may therefore

vary in their response to drug-induced toxicity. Therefore, we used an in vitro system composed of four

strains of mouse embryonic fibroblasts (MEFs) with mtDNA polymorphisms. We sequenced mtDNA

from embryonic fibroblasts isolated from four mouse strains, C57BL/6J, MOLF/EiJ, CZECHII/EiJ and PERA/

EiJ, with the latter two being sequenced for the first time. The bioenergetic profile of the four strains of

MEFs was investigated at both passages 3 and 10. Our results showed that there were clear differences

among the four strains of MEFs at both passages, with CZECHII/EiJ having a lower mitochondrial robustness

when compared to C57BL/6J, followed by MOLF/EiJ and PERA/EiJ. Seven drugs known to impair mitochondrial

function were tested for their effect on the ATP content of the four strains of MEFs in both

glucose- and galactose-containing media. Our results showed that there were strain-dependent differences

in the response to some of the drugs. We propose that this model is a useful starting point to

study compounds that may cause mitochondrial off-target toxicity in early stages of drug development,

thus decreasing the number of experimental animals used.

© 2012 Elsevier Inc. All rights reserved.


Mitochondria are pivotal players in the development of drug-induced

toxicity due to their critical role in cellular bioenergetics (Boelsterli and

Lim, 2007). It has been hypothesized that mitochondrial DNA (mtDNA)

alterations which may cause clinically silent abnormalities may predispose

individuals to idiosyncratic drug-responses when exposed to certain

drugs (Boelsterli and Lim, 2007). Episodes of drug-induced injury

have been associated with the concept of a mtDNA threshold effect in

which a phenotype resulting from accumulation of mutated mtDNA is

not evident until a certain degree of heteroplasmy is observed, after

Abbreviations: ATP, adenosine triphosphate; BSA, bovine serum albumin; 2-DG,

2-deoxy-D-glucose; DMSO, dimethyl sulfoxide; DTNB, 5,5′ dithiobis 2 nitrobenzoic acid;

FCCP, Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone; LHON, Leber hereditary

optic neuropathy; MEFs, mouse embryonic fibroblasts; mtDNA, mitochondrial DNA;

NADH, reduced nicotinamide adenine dinucleotide; nDNA, nuclear DNA; OCR, oxygen consumption

rate; rRNA, Ribosomal ribonucleic acid; SNPs, single nucleotide polymorphisms;

tRNA, transfer ribonucleic acid; WY-14, 643, [[4-chloro-6-[(2,3-dimethylphenyl)amino]-

2-pyrimidinyl]thio]-acetic acid.

⁎ Corresponding author at: Compound Safety Prediction, Worldwide Medicinal

Chemistry, Pfizer Global R&D, Groton, CT 06340, USA. Fax: +1 860 686 2353.

E-mail address: (S. Nadanaciva).

which rapid mitochondrial degeneration occurs (Boelsterli and Lim,


Thirteen proteins are encoded by mtDNA, all of them subunits of the

complexes involved in oxidative phosphorylation. In addition, mtDNA

encodes 2 rRNAs and 22 tRNAs which are necessary for the synthesis

of the 13 mtDNA-encoded proteins (Wallace and Fan, 2010). Mutations

within some of the mtDNA-encoded genes cause serious diseases such

as mitochondrial encephalomyopathy with lactic acidosis and strokelike

episodes (MELAS), myoclonic epilepsy with ragged red fibers

(MERRF), and Leber hereditary optic neuropathy (LHON) (Gomez-

Duran et al., 2010; Wallace, 2010). Nevertheless, not all nucleotide

changes in mtDNA are harmful and, in fact, several mtDNA single nucleotide

polymorphisms (SNPs) are known to occur within the human

population (Wallace, 2010). However, accumulation of mtDNA SNPs

could, over time, lead to a decrease in the respiratory capacity of cells

(Czarnecka and Bartnik, 2011). If the accumulation of mtDNA SNPs is

compounded with the effect of drugs that have off-target effects on mitochondrial

function, this could lead to mitochondrial bioenergetic impairment

and hence to idiosyncratic drug reactions (Boelsterli and

Lim, 2007).

Drugs that cause idiosyncratic drug reactions are difficult to identify

in pre-clinical rodent models since these in vivo systems do not display

the diverse genetic variation seen in the patient population. Nor do

0041-008X/$ – see front matter © 2012 Elsevier Inc. All rights reserved.

168 C.V. Pereira et al. / Toxicology and Applied Pharmacology 264 (2012) 167–181

these animals usually have risk factors such as old age or underlying diseases

that are seen in some patients. In an effort to overcome this limitation,

a panel of 36 strains of genetically diverse mice was used to study

the toxicity of acetaminophen (Harrill et al., 2009). In addition, a panel

of inbred mouse strains has been used to study inter-individual variability

in the metabolism of warfarin (Guo et al., 2006). Furthermore, hepatocytes

from 15 genetically different mouse strains have been used to

evaluate strain-specific responses to different toxicants, such as acetaminophen,

WY-14,643 (a peroxisome proliferator activator receptor

(PPAR) agonist) and rifampin (Martinez et al., 2010).

The widespread use of the laboratory mouse in research is mainly

due to the existence of a variety of inbred mouse strains derived by repeated

inter-sibling mating, which also generates an everlasting population

of genetic clones (Szatkiewicz et al., 2008). Since mice within a

single strain share the same genetic background, it is possible to compare

genetic and phenotypic differences among different mouse strains

(Svenson et al., 2003). Much effort has been made to understand and

characterize SNPs in inbred mouse strains ever since the nuclear genomic

sequence of the C57BL/6J strain was reported (Frazer et al., 2004;

Szatkiewicz et al., 2008).

To the best of our knowledge, no bioenergetic characterization has

been performed on murine embryonic fibroblasts cells which vary in

their mtDNA sequence. It is conceivable that cells isolated from mice

which differ in their mtDNA sequence may have different bioenergetic

profiles and, therefore, vary in their response to drugs which cause

mitochondrial impairment. To this end, the first objective of this work

was to compare the bioenergetic profile of embryonic fibroblasts

harvested from four strains of mice, C57BL/6J, PERA/EiJ, CZECHII/EiJ

and MOLF/EiJ. C57BL/6J is a common inbred mouse strain whereas

PERA/EiJ, CZECHII/EiJ and MOLF/EiJ are wild-derived inbred mouse

strains (Goios et al., 2007). The published mtDNA sequence of MOLF/

EiJ shows that it has 393 SNPs in comparison with that of C57BL/6J

(Goios et al., 2007). Since the mtDNA sequences of PERA/EiJ and

CZECHII/EiJ have not been reported before, we sequenced the entire

mtDNA of embryonic fibroblasts from these two strains of mice at passage

3. Furthermore, since cells may accumulate mutations in mtDNA

with increased passage number, we also compared the mtDNA sequence

of each of the four strains used in our study at passage 3 and

passage 10.

Mitochondrial dysfunction has been implicated in the toxicity of several

drugs including tolcapone, nefazodone and flutamide (Boelsterli

and Lim, 2007; Dykens et al., 2008). It is conceivable that cells which differ

in their mtDNA sequence and likely in their bioenergetic profile may

vary in their response to drugs which impair mitochondrial function.

The second objective of our work was to use the panel of mouse embryonic

fibroblasts to test the effect of rotenone, a mitochondrial inhibitor,

and seven drugs on cellular ATP content, which we considered as an

endpoint for cell viability. The drugs which were tested are known to

cause mitochondrial impairment due to off-target effects. Our results

showed that the four strains of MEFs had differences in their bioenergetic

profiles and, moreover, showed differences in their susceptibility

towards some of the tested drugs.

Materials and methods

Cell culture media and supplements for the mouse embryonic fibroblasts

(MEFs) were purchased from Invitrogen (Carlsbad, CA).

All chemicals were purchased from Sigma Aldrich (St. Louis, MO)

and were of the highest purity available. The CellTiter-Glo® kit was

purchased from Promega (Madison, WI). The 96-well white walled,

opaque bottom plates used for luminescence readings were from

Becton Dickinson (Bedford, MA). XF96 sensor cartridges, XF96-well

plates, calibration buffer and calibration plates were obtained from

Seahorse Bioscience (Billerica, MA). Low-buffered serum-free RPMI

medium was purchased from Molecular Devices (Sunnyvale, CA).

Cell culture conditions. Mouse embryonic fibroblasts (MEFs) were

purchased at passage 0 from the Jackson Laboratory (Bar Harbor, ME)

and were grown in T175 cm 2 cell culture flasks in a humidified atmosphere

at 37 °C, 5% CO 2 in 1× Knockout DMEM (Invitrogen 10829‐018)

containing high glucose (17.5 mM) and sodium pyruvate (110 mg/L)

and supplemented with 2 mM L-glutamine, 15% fetal bovine serum

(FBS), 1× non-essential amino acids (Invitrogen 11140), 30 μg/mL of

gentamicin and 0.1 mM of β-mercaptoethanol. We refer to this medium

as “Cell culture medium” in the rest of Materials and methods section.

Cells were grown until passage 3 or passage 10 before analysis.

mtDNA sequencing. mtDNA sequencing was performed essentially as

described in Goios et al. (2007) with some modifications. Total DNA

from the four strains of MEFs, at passage 3 and passage 10, was

extracted following a standard phenol-chloroform protocol (digested

with proteinase K in TE (10 mM Tris, 1 mM-EDTA, pH 7.5) buffer

containing 0.5% SDS, purified with phenol–chlorophorm–isoamyl alcohol,

and precipitated with ethanol). Thirty‐four overlapping fragments

of approximately 500 bp covering the entire mtDNA genome were

then amplified by PCR (polymerase chain reaction) with the appropriate

oligodeoxynucleotides and annealing temperatures. The primers

used for sequencing the mtDNA of C57BL/6J and PERA/EiJ were as described

in Goios et al. (2007). The primers used for sequencing the

mtDNA of CZECHII/EiJ and MOLF/EiJ are shown in Supplemental Table

1. The PCR products were purified using solid-phase reversible immobilization.

DNA sequencing reactions were carried out using a BigDye Terminator

v3.1 kit (Applied Biosystems, Life Technologies, Carlsbad, CA)

and post-reaction dye terminator removal was done using Agencourt

CleanSEQ (Beckman Coulter Genomics, Danvers, MA). Automated

DNA sequencing was performed on an ABI PRISM 3730xl DNA Analyzer

(Applied Biosystems). All fragments were sequenced from both strands.

mtDNA sequence analysis. The mtDNA sequences of the four strains

of MEFs at passage 3 and passage 10 were compared with the published

mtDNA sequence of the mouse strain, C57BL/6J (NC_005891.1 (Goios et

al., 2007)), using the software, SeqMan Pro (DNAStar Lasergene 8.0,

Madison, WI). The software, Simbiot (, was used to

analyze the synonymous and nonsynonymous substitutions caused by

the single nucleotide polymorphisms (SNPs).

Cell proliferation measurements. MEFs were seeded in cell culture

medium at a density of 160,000 cells/well in 12-well plates for measuring

cell number after 6 h and 18 h. Cells were also seeded at the same

density in 6-well plates for measuring the cell number after 48 h. At

each time point, the cells were trypsinized, stained with trypan blue

and the viable cells counted with a Countess® Automated Cell Counter

(Invitrogen, Life Technologies, Carlsbad, CA).

Cellular oxygen consumption measurements. Oxygen consumption

was measured at 37 °C using a Seahorse XF96 Extracellular Flux Analyzer

(Seahorse Bioscience, Billerica, MA). All four MEF strains were seeded

within the same plate for each experiment and incubated in a 37 °C, 5%

CO 2 humidified atmosphere for 24 h. The cells were seeded at a density

of 16,000 cells/100 μL/well since the absolute rate of oxygen consumption

was linearly related to the cell number between 8000 and 32,000

cells/well. In addition, an XF96 sensor cartridge for each cell plate was

placed in a 96-well calibration plate containing 200 μL/well calibration

buffer and left to hydrate overnight at 37 °C.

The cell culture medium from the cell plates was replaced the following

day with 150 μL/well of pre-warmed low-buffered serum-free RPMI

medium and incubated at 37 °C for 30 min to allow the temperature

and pH of the medium to reach equilibrium before the first rate measurement.

Rotenone, oligomycin, FCCP and antimycin were prepared

as 700× stock solutions in DMSO. 2-Deoxy-D-Glucose (2-DG) was

prepared as a 7× stock solution in serum-free low-buffered RPMI medium

and its pH adjusted to 7.4 with 1 M NaOH. Test compounds which

C.V. Pereira et al. / Toxicology and Applied Pharmacology 264 (2012) 167–181


had to be injected into reagent delivery port A of each well were diluted

7× in low-buffered serum-free RPMI medium and the pH adjusted to 7.4

with 1 M NaOH. Test compounds which had to be injected into port B of

each well following injection of compound in port A were diluted 8× in

low-buffered serum-free RPMI medium. 25 μL ofcompoundorvehicle

(1% (v/v) DMSO) in low-buffered serum-free RPMI medium was then

pre-loaded into the ports of each well in the XF96 sensor cartridge.

Since each well in the cell plate contained 150 μL ofmedium,thefinal

concentration of DMSO in each well after reagent injection was 0.14%


The sensor cartridge and the calibration plate were loaded into the

XF96 Extracellular Flux Analyzer so that the cartridge could be calibrated.

When the calibration was complete, the calibration plate

was replaced with the cell plate. Four baseline rate measurements

of the oxygen consumption rate (OCR) of the MEFs were made

using a 2 min mix, 5 min measure cycle. The compounds were then

injected pneumatically by the XF96 Analyzer into each well, mixed,

and OCR measurements were made using the 2 min mix, 5 min measure

cycle. All oxygen consumption rates were calculated as a percentage

of the fourth baseline rate measurement obtained in the

same well, i.e., the rate obtained just before addition of compound

in port A.

Complex I activity assay. Cell pellets containing approximately 1×10 6

cells were harvested and submitted to 3 freeze-thaw cycles and dissolved

in 100 μL of25mMKH 2 PO 4 ,5mMMgCl 2 ,pH7.4.NADHoxidationwas

assessed spectrophotometrically in a cuvette at 340 nm in a dual beam

JASCO V-660 spectrophotometer at room temperature. 20 μL ofsample

was added to 200 μL of reaction buffer containing 25 mM KH 2 PO 4 ,

5mMMgCl 2 ,pH7.4,1 μM antimycin, 0.2 mM NADH, 0.4% BSA. A baseline

rate in the absence of the substrate, ubiquinone, was first recorded for

2 min. Ubiquinone was then added to a final concentration of 70 μM

and the rate of NADH oxidation recorded for 3 min. NADH oxidation is

due to mitochondrial Complex I as well as other cellular NADH dehydrogenases.

In order to determine Complex I activity, rotenone, an inhibitor

of Complex I, was added to a final concentration of 2 μMandtheratedetermined

for another 3 min. Complex I activity was calculated by

subtracting the rotenone-insensitive NADH oxidation rate in its linear

phase from the total NADH oxidation rate in its linear phase. The protein

concentration of each sample was determined by the BCA method, as described

by the vendor (Pierce, Rockford, IL).

Complex IV activity assay. Cell pellets containing approximately

1×10 6 cells were collected and resuspended in 100 μL of50mM

KH 2 PO 4 , pH 7.2, 1.5% dodecyl maltoside and were kept on ice for

30 min before centrifugation at 14,000g for 15 min at 4 °C. The supernatants

were transferred to new centrifuge tubes and the protein

concentration determined by the BCA method (Pierce, Rockford, IL).

Complex IV activity was determined spectrophotometrically in

96-well format in a Spectramax plate reader for 20 min at 550 nm

in kinetic mode at room temperature. The reaction was started by

adding 5 μg of protein to 200 μL/well of reaction solution containing

20 mM KH 2 PO 4 , pH 7.2, 0.015% dodecyl maltoside, 70 μM reduced cytochrome

c. Complex IV activity was calculated in the linear phase.

Citrate synthase activity assay. Cell pellets containing approximately

1×10 6 cells were collected and resuspended in 100 μL of 50 mM

KH 2 PO 4 , pH 7.2, 1.5% dodecyl maltoside and were kept on ice for

30 min before centrifugation at 14,000g for 15 min at 4 °C. The supernatants

were transferred to new centrifuge tubes and the protein concentration

determined by the BCA method (Pierce, Rockford, IL). Citrate

synthase activity was determined spectrophotometrically in 96-well

format in a Spectramax reader at 412 nm in kinetic mode at room temperature.

The reaction in the absence of the substrate, oxaloacetate, was

first determined for 7 min by adding 5 μg of protein to 200 μL/well of reaction

solution containing 10 mM Tris–HCl, pH 8.1, 200 μM acetyl CoA,

and 500 μM DTNB. Oxaloacetate was then added to a final concentration

of 1 mM and the rate was measured for 30 min. Citrate synthase was

calculated in the linear phase after subtracting the rate obtained in the

absence of oxaloacetate from the rate obtained in the presence of


Measurement of cellular ATP content. MEFs, at passage 10, were

seeded in 96-well white walled opaque bottom plates at 8000 cells/

100 μL/well in the following media: (a) 1× Knockout DMEM

(Invitrogen 10829‐018) containing 17.5 mM glucose (17.5 mM) and

supplemented with 10% FBS and 1 mM sodium pyruvate or (b)

glucose-free DMEM (Invitrogen 11966‐025) supplemented with

10 mM galactose, 2 mM glutamine, 5 mM HEPES, 10% FBS and 1 mM

sodium pyruvate. The following day, compound stock solutions that

were prepared in DMSO were diluted to the appropriate concentration

in either the glucose- or galactose medium described above. The final

DMSO concentration was 0.5%. The medium in each of the cell plates

was aspirated and replaced with 100 μL of medium containing compounds

in either the glucose- or galactose medium. Cellular ATP concentrations

were assessed 24 h after compound addition using a

CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, WI)

according to the manufacturer's instruction.

Statistical analysis. Statistical analysis was performed with GraphPad

Prism 5 (La Jolla, CA) and Microsoft Office Excel 2007. Data are

presented as means±SEM or ±SD. Statistical analysis was performed

using one-way ANOVA followed by the Bonferroni Multiple Comparison

test for inter-strain comparison at the same passage or 2-way ANOVA

when comparing MEFs at passage 3 versus passage 10. For dose

response experiments, a two-tail Student t-test was performed. A p

value of b0.05 was considered significant.


mtDNA single nucleotide polymorphisms in the four strains of mouse

embryonic fibroblasts (MEFs)

Mitochondrial DNA (mtDNA) from embryonic fibroblasts at passage 3

and passage 10 from the mouse strains, C57BL/6J, PERA/EiJ, CZECHII/EiJ

andMOLF/EiJ,wassequencedasdescribedinMaterials and methods.

The mtDNA sequences of PERA/EiJ and CZECHII/EiJ have not been

reported before.

The mtDNA sequence of C57BL/6J from passage 3 embryonic fibroblasts

was identical to that of the published sequence of this strain

(Goios et al., 2007). Single nucleotide polymorphisms (SNPs) were

detected in the mtDNA of fibroblasts from PERA/EiJ (106 SNPs),

CZECHII/EiJ (390 SNPs) and MOLF/EiJ (393 SNPs) at passage 3 when

compared with the published mtDNA sequence of the reference

strain, C57BL/6J (Fig. 1).


•106 SNPs

•9 amino acid



•390 SNPs

•35 amino acid



•393 SNPs

•34 amino acid


Fig. 1. The mtDNA SNPs and amino acid changes in embryonic fibroblasts of the mouse

strains, PERA/EiJ, and CZECHII/EiJ and MOLF/EiJ, when compared with C57BL/6J at passage 3.

170 C.V. Pereira et al. / Toxicology and Applied Pharmacology 264 (2012) 167–181

Table 1

Nonsynonymous substitutions in the mtDNA polypeptide-encoding genes and the corresponding amino acid change in the mouse embryonic fibroblasts (MEFs) at passage 3 from

strains, PERA/EiJ, CZECHII/EiJ and MOLF/EiJ. C57BL/6J is the reference strain.

C.V. Pereira et al. / Toxicology and Applied Pharmacology 264 (2012) 167–181


Table 1 (continued)


Cell Proliferation (% time 0)


Cell Proliferation (% time 0)









0 6 12 18 24 30 36 42 48

Time (hours)


















0 6 12 18 24 30 36 42 48

Time (hours)









Fig. 2. Cell growth curves of the four strains of MEFs at passages 3 and 10. Cells were

seeded at a density of 160,000 cells/mL in cell culture medium. Cells were trypsinized

and counted at 3 time points: 6, 18 and 48 h. The data are expressed as % of time 0.

A) The four strains of MEFs at passage 3. B) The four strains of MEFs at passage 10.

Each data point represents mean±SEM of 3 separate experiments. Statistical analysis

was performed using one‐way ANOVA followed by Bonferroni Multiple Comparison

test and 2‐way ANOVA in order to compare passage 3 vs passage 10. pb0.05 is considered

statistically significant. ⁎ versus reference strain C57BL/6J at the same passage, #

versus same strain for passage 3,

@ versus CZECHII/EiJ,

& versus PERA/EiJ and


The mtDNA sequence of C57BL/6J fibroblasts at passage 10 was

identical to that of C57BL/6J fibroblasts at passage 3. Similarly, the

mtDNA sequence of PERA/EiJ fibroblasts and MOLF/EiJ fibroblasts at

passage 10 was identical to that of PERA/EiJ fibroblasts and MOLF/

EiJ fibroblasts at passage 3, respectively. In contrast, the mtDNA of

CZECHII/EiJ fibroblasts at passage 10 had three more SNPs than the

mtDNA of CZECHII/EiJ fibroblasts at passage 3. These extra SNPs

were located in mt-Nd5, a gene which encodes a subunit of mitochondrial

Complex I. However, these 3 SNPs were synonymous nucleotide

substitutions and hence there were no amino acid changes (data not


We next analyzed the mtDNA SNPs that were nonsynonymous

substitutions (i.e. SNPs that cause amino acid changes) in the 13

polypeptide-encoding genes that code for subunits of the mitochondrial

respiratory chain. PERA/EiJ differed from C57BL/6J by nine

amino acids (Table 1). Seven of these amino acids were in subunits

of Complex I (ND1, ND4L, ND4 and ND5), one amino acid was in a

subunit of Complex IV (COII) and one amino acid was in a subunit

of Complex V (ATP synthase Fo subunit 6). CZECHII/EiJ differed from

C57BL/6J by 35 amino acids (Table 1). Thirty of these amino acids

were in subunits of Complex I (ND1, ND2, ND3, ND4, ND4L, ND5

and ND6), one amino acid was in a subunit of Complex III (Cyt b),

one amino acid was in a subunit of Complex IV (CO1), and three

amino acids were in subunits of Complex V (ATP synthase Fo subunits

6 and 8). MOLF/EiJ differed from C57BL/6J by 34 amino acids

(Table 1). Twenty nine of these amino acids were in subunits of Complex

I (ND1, ND2, ND3, ND4, ND4L, ND5 and ND6), two amino acids

were in a subunit of Complex III (Cyt b), and three amino acids

were in subunits of Complex V (ATP synthase Fo subunits 6 and 8).

CZECHII/EiJ and MOLF/EiJ had 27 amino acid changes in common

when compared with C57BL/6J (Table 1). In contrast, only 3 amino

acid changes found in PERA/EiJ, when compared with C57BL/6J,

were also found in CZECHII/EiJ and MOLF/EiJ. The majority of amino

acid changes that occurred in PERA/EiJ, CZECHII/EiJ and MOLF/EiJ

were in subunits of Complex I.

Nucleotide substitutions in genes encoding tRNAs, rRNAs and the

D-loop are summarized in Supplemental Tables 2–4, respectively. In

the tRNA encoding genes, PERA/EiJ had 7 SNPs, CZECHII/EiJ had 15

172 C.V. Pereira et al. / Toxicology and Applied Pharmacology 264 (2012) 167–181

Table 2

Bioenergetic parameters of the four MEF strains at passage 3 and passage 10.


Bioenergetic parameters

Passage number 3 10 3 10 3 10 3 10

Mitochondrial respiration (% of basal respiration) 84.0±0.7 80.0±1.0 81.0±1.2 85±1.0 @ 77.7±2.8 75±1.0 85±1.3 81±2.0

ATP-linked respiration (% of basal respiration) 63.0±2.0 62.0±1.0 59.0±2.0 70.0±1.0 ⁎#@ 61.0±3.0 58.2±3.0 64.0±2.0 62.0±2.0

Proton-leak (% of basal respiration) 21.0±1.0 18.0±0.2 22.0±2.0 15.0±0.5 # 16.7±2.0 16.8±2.0 21.0±2.0 19.0±1.0 &

Non-mitochondrial respiration (% of basal respiration) 16.0±1.0 20.0±1.0 19.0±1.0 15.0±1.0 @ 22.3±2.0 25.0±2.0 15.0±1.0 19.0±2.0

Data is represented as Mean±SEM, N=5 separate experiments. Statistical analysis was performed using one-way ANOVA followed by Bonferroni Multiple Comparison test to compare

strains within each passage. A 2-way ANOVA was performed to compare strains between passage 3 and passage 10. pb0.05 is considered statistically significant: * vs. reference

strain C57BL/6J at the same passage. & vs. PERA/EiJ (passage 10). @ vs CZECHII/EiJ (passage 10). # vs MOLF (passage 10).

SNPs, and MOLF/EiJ had 17 SNPs when compared with the reference

strain, C57BL/6J (Supplemental Table 2). In the two rRNA encoding

genes, PERA/EiJ had 9 SNPs, CZECHII/EiJ had 29 SNPs and MOLF/EiJ

had 27 SNPs when compared with C57BL/6J (Supplemental Table

3). In the D-loop, PERA/EiJ had 9 SNPs, while CZECHII/EiJ and MOLF/

EiJ each had 34 SNPs when compared with C57BL/6J (Supplemental

Table 4). As in the case with the nonsynonymous substitutions

Spare respiratory

capacity (%)












Passage 3 Passage 10

• C57BL/6J














occurring in the polypeptide-encoding genes, many of the SNPs

found in the genes encoding tRNAs, rRNAs and the D-loop of

CZECHII/EiJ were also found in MOLF/EiJ.

Strain and passage-dependent differences in cell growth rates

In order to characterize the cell growth of the embryonic fibroblasts

from the four mouse strains, cell numbers were determined at three

time-points (6 h, 18 h and 48 h) post-seeding. The data was normalized

to time zero (t0) which corresponds to a seeding density of 160,000

cells/mL. Overall, at both passages 3 and 10, during the first 6 h

Maximum respiratory

capacity (%)













Passage 3 Passage 10

• C57BL/6J













• C57BL/6J







• C57BL/6J






Fig. 3. Spare respiratory capacity of the four strains of MEFs at passages 3 and 10. The

spare respiratory capacity (or reserve capacity) was determined by measuring the maximum

uncoupled respiration upon FCCP injection. A) Spare respiratory capacity at passage

3 and passage 10. Each bar represents the Mean±SEM, N=5 separate experiments. Statistical

analysis was performed using one-way ANOVA followed by Bonferroni Multiple

Comparison test and 2-way ANOVA in order to compare passage 3 vs passage 10.

pb0.05 is considered statistically significant, ⁎ vs. reference strain C57BL/6J at the same

passage, # vs. same strain for passage 3, & vs. CZECHII/EiJ (passage 3 and 10), @ vs.

CZECHII/EiJ (passage 3). Representative recordings from the XF96 platform showing the

percentage change in the oxygen consumption rate upon FCCP addition (at a final concentration

of 1 μM for C57BL/6J, CZECHII/EiJ and MOLF/EiJ, and a final concentration of 0.5 μM

for PERA/EiJ) for the four strains at passage 3 and passage 10 are shown in Figs. 3B and C,

respectively. The basal oxygen consumption rate is denoted as 100% in panels B and C.

Fig. 4. Maximum respiratory capacity of the four strains of MEFs at passages 3 and 10. The

maximum respiratory capacity is the sum of the respiration that results in proton leak and

the respiration that occurs upon sequential addition of oligomycin and FCCP. A) Maximum

respiratory capacity at passage 3 and passage 10. Each bar represents the Mean±SEM,

N=5 separate experiments. Statistical analysis was performed using one-way ANOVA

followed by Bonferroni Multiple Comparison test and 2-way ANOVA in order to compare

passage 3 vs passage 10. pb0.05 is considered statistically significant, ⁎ vs. reference strain

C57BL/6J at the same passage, & vs. PERA/EiJ and CZECHII/EiJ (passage 3 and 10). Representative

recordings of the percentage change in the oxygen consumption rate of the

four strains of MEFs at passage 3 and passage 10 following sequential addition of

oligomycin and FCCP are shown in Figs. 6B) and C), respectively.

C.V. Pereira et al. / Toxicology and Applied Pharmacology 264 (2012) 167–181


% change in

oxygen consumption

rate with 2-DG











Passage 3 Passage 10





Fig. 5. Effect of the glycolysis inhibitor, 2-deoxyglucose (2-DG), on the OCR of the four

strains of MEFs at passages 3 and 10. The data represents the % change in the OCR after

the injection of 2-DG (75 mM final concentration). Each bar represents the Mean±

SEM, N=5 separate experiments. Statistical analysis was performed using one-way

ANOVA followed by Bonferroni Multiple Comparison test and 2-way ANOVA in order to

compare passage 3 vs passage 10. pb0.05 is considered statistically significant, ⁎ vs. reference

strain C57BL/6J at the same passage, # vs. same strain for passage 3, & vs. PERA/EiJ

(passage 3), @ vs. CZECHII/EiJ (passage 3).

post-seeding, cell mass increased for all of the strains, after which cell

growth decreased between t=6 h and t=18 h. After 48 h, the cell

growth showed differences between passages and among strains. At

passage 3, 48 h post-seeding, cell proliferation varied among the strains

in the rank order, CZECHII/EiJ>C57BL/6J, MOLF/EiJ>PERA/EiJ (Fig. 2A),

whereas at passage 10, the rank order was PERA/EiJ>CZECHII/EiJ>

C57BL/6J>MOLF/EiJ (Fig. 2B).



A.U./min/mg of protein


A.U./min/mg of protein




















A.U./min/mg of protein


A.U./min/mg of protein
























Fig. 6. Complex I activity of the four strains of MEFs at passages 3 and 10. Complex I

activity was measured as rotenone-dependent NADH oxidation, and was assessed

spectrophotometrically as described in Methods and materials. The bar graphs represent

Complex I activity at passage 3 (Fig. 6A) and at passage 10 (Fig. 6B). Each bar represents

the Mean±SEM, N=3–5 separate experiments. Statistical analysis was

performed using one-way ANOVA followed by Bonferroni Multiple Comparison test

and 2-way ANOVA in order to compare passage 3 vs passage 10. pb0.05 is considered

statistically significant, * vs. reference strain C57BL/6J at the same passage, # vs. same

strain for passage 3, @ vs. CZECHII/EiJ (passages 3 and 10), & vs. PERA/EiJ (passage 10).

Fig. 7. Complex IV activity of the four strains of MEFs at passages 3 and 10. Complex IV activity

was determined spectrophotometrically as described in Materials and methods.The

data represent Complex IV activity at passage 3 (A) and passage 10 (B). Each bar represents

the Mean±SEM, N=3–4 separateexperiments.

Strain- and passage-dependent differences in mitochondrial bioenergetics

In order to characterize the bioenergetics of the four strains of

MEFs at passages 3 and 10, we measured their ATP-linked respiration,

proton-leak, spare respiratory capacity, maximum respiratory capacity,

and non-mitochondrial respiration using an XF96 extracellular

flux analyzer as described in Materials and methods.

The basal respiration of cells consists of mitochondrial respiration and

non-mitochondrial respiration. Mitochondrial respiration is the component

of basal respiration that is sensitive to the combination of the electron

transport chain inhibitors, rotenone and antimycin, and the ATP

synthesis inhibitor, oligomycin. To assess the component of basal respiration

that is mitochondrial, oligomycin, rotenone and antimycin were

added to the samples at a final concentration of 3 μM, 1 μMand1 μM, respectively.

There was no statistically significant difference (p>0.05) in

mitochondrial respiration among the strains of MEFs at passage 3. At passage

10, the mitochondrial respiration of PERA/EiJ was statistically significantly

higher (pb0.05) than that of CZECHII/EiJ (Table 2).

Mitochondrial respiration is a sum of ATP-linked respiration (i.e. respiration

that is used for ATP synthesis) and proton-leak. To assess the

component of respiration that results from ATP synthesis, oligomycin

was added to inhibit ATP synthesis. There was no statistically significant

difference in ATP-linked respiration among the four strains at passage 3

(Table 2). There was no difference in ATP-linked respiration among the

strains at passage 10 with the exception of PERA/EiJ which showed a

statistically significant (pb0.05) higher ATP-linked respiration than

the other strains (Table 2). There was no difference in ATP-linked respiration

within each strain between cell passages, with the exception of

PERA/EiJ, which showed increased ATP-linked respiration from passage

3 to passage 10 (Table 2).

Mitochondrial respiration that is insensitive to oligomycin results

from proton leak. The proton leak in all the strains was similar at

174 C.V. Pereira et al. / Toxicology and Applied Pharmacology 264 (2012) 167–181


A.U./min/mg of protein


A.U./min/mg of protein


















passage 3. At passage 10, MOLF/EiJ had the highest proton leak whereas

PERA/EiJ had the lowest proton leak (Table 2). There was no difference

in proton-leak within each strain between cell passages with the exception

of PERA/EiJ which had a statistically significant (pb0.05) lower proton

leak at passage 10 than at passage 3 (Table 2).

Non-mitochondrial respiration is insensitive to the combination of

oligomycin, rotenone and antimycin. Non-mitochondrial respiration

was similar in all four strains at passage 3. At passage 10, PERA/EiJ

had the lowest non-mitochondrial respiration whereas CZECHII/EiJ

had the highest non-mitochondrial respiration (Table 2). There was

no statistically significant difference (p>0.05) within each strain between

passage 3 and passage 10.

The spare respiratory capacity (or reserve capacity) was determined

by measuring the maximum uncoupled respiration upon addition of




# &


Fig. 8. Citrate synthase activity of the four strains of MEFs at passages 3 and 10. Citrate

synthase activity was determined spectrophotometrically as described in Materials

and methods. The bar graphs represent the citrate synthase activity at passage 3 (A)

and passage 10 (B). Each bar represents the Mean±SEM, N=3–4 separate experiments.

Statistical analysis was performed using one-way ANOVA followed by

Bonferroni Multiple Comparison test and 2-way ANOVA in order to compare passage

3 vs passage 10. pb0.05 is considered statistically significant, * vs. reference strain

C57BL/6J at the same passage, # vs. same strain for passage 3, & vs. CZECHII/EiJ (passage

3) and PERA/EiJ (passage 10), @ vs. CZECHII/EiJ (passage 10).

FCCP, a protonophore that uncouples electron transport from ATP synthesis

(Figs. 3A, B, and C). Titration with FCCP revealed that C57BL/6J,

CZECHII/EiJ and MOLF/EiJ showed maximum uncoupling with 1 μM

FCCP, whereas PERA/EiJ showed maximal uncoupling with 0.5 μM

FCCP (data not shown). At passage 3, PERA/EiJ and CZECHII/EiJ showed

a significantly lower spare respiratory capacity than C57BL/6J and

MOLF/EiJ. The same trend was seen in passage 10. In addition, PERA/

EiJ showed an even lower spare respiratory capacity at passage 10

(Fig. 3C) than at passage 3 (Fig. 3B), this decrease being statistically significant


The maximum respiratory capacity is the sum of (a) the respiration

that occurs upon sequential addition of oligomycin and FCCP and (b)

the respiration that results in proton leak. At passage 3, PERA/EiJ and

CZECHII/EiJ showed a significantly lower maximum respiratory capacity

than C57BL/6J and MOLF/EiJ (Figs. 4A and B). At passage 10, the maximum

respiratory capacity of the MEFs was in the order, PERA/EiJ,

CZECHII/EiJbMOLF/EiJbC57BL/6J (Figs. 4A and C). No significant differences

between passages within each strain were observed (Fig. 4A).

The Complex I inhibitor, rotenone, reduced the oxygen consumption

rate of the four strains of MEFS in passages 3 and 10 by 70–78% at

1 μM (data not shown). The Complex III inhibitor, antimycin, had a

similar effect on the MEFs at 1 μM (data not shown). No inter-strain

or inter-passage differences in the oxygen consumption rate were observed

in the presence of either 1 μM rotenone or 1 μM antimycin.

The effect of the glycolytic inhibitor, 2-deoxy-D-glucose (2-DG), on

the basal respiration rate of the MEFs was also tested. Inhibition of glycolysis

should increase the respiration of cells since they are forced to

rely on oxidative phosphorylation for their ATP demands. At passage

3, C57BL/6J and PERA/EiJ increased their basal respiration by 54% and

53%, respectively, while CZECHII/EiJ and MOLF/EiJ were significantly

less responsive to 2-DG, showing only a 32% and 33% increase, respectively

(Fig. 5). At passage 10, the response to 2-DG was decreased in

all of the strains although this decrease was not statistically significant

between strains due to data variability (Fig. 5). Furthermore, C57BL/6J

and CZECHII/EiJ showed a significantly decreased response to 2-DG

from passages 3 to 10.

Strain- and passage-dependent differences in Complex I, Complex IV and

citrate synthase activities

Complex I activity in the four MEF strains was determined since

some of the mtDNA SNPs were found in genes encoding subunits of

Complex I. At passage 3, there were no differences between the strains

with the exception of PERA/EiJ which was statistically significantly

higher than both C57BL/6J and CZECHII/EiJ (Fig. 6A). At passage 10,

both CZECHII/EiJ and MOLF/EiJ showed a statistically significant decrease

in Complex I activity when compared to both C57BL/6J and

PERA/EiJ (Fig. 6B). Comparison of each strain between passage 3 and

passage 10 showed that only MOLF/EiJ showed a significant decrease

in the enzymatic activity of Complex I, while the other strains did not

show any significant inter-passage differences.

Since some SNPs were found to exist in the mtDNA genes encoding

some subunits of Complex IV, we also determined the Complex IV

Table 3

Normalization of Complex I and IV activities with citrate synthase activity of 4 strains of MEFs at passages 3 and 10.

Strains/passage C57/6J C57/6J PERA/EiJ PERA/EiJ CZE/EiJ CZE/EiJ MOLF/EiJ MOLF/EiJ

Complex I/Citrate synthase (%) 0.0031±


Complex IV/Citrate synthase (%) 53.4649±


#3 #10 #3 #10 #3 #10 #3 #10






0.0016 ⁎@ 0.0106±

0.0018 @ 0.0054±


75.8174± 67.6519± 92.2838±

2.6345* @ 2.1401







0.0005 ⁎&

61.3162± 68.6087± 66.8948±

2.5886 # 2.3183* @ 8.09059

Data are means±SEM of 3–5 independent experiments. All the presented values were multiplied by 100. Statistical analysis was performed using one-way ANOVA followed by

Bonferroni Multiple Comparison test and 2-way ANOVA in order to compare passage 3 vs passage 10. pb0.05 is considered statistically significant, * vs. reference strain C57BL/

6J at the same passage, & vs. PERA/EiJ (passage 10), @ vs CZECHII/EiJ (passages 3 and 10). Abbreviations: C57—C57BL, CZE—CZECHII, #—number.

C.V. Pereira et al. / Toxicology and Applied Pharmacology 264 (2012) 167–181


Table 4

The effect of a selection of compounds on the ATP content of the four strains of MEFs in (a) cell culture medium containing glucose and (b) glucose-free cell culture medium

supplemented with galactose and glutamine. (Abbreviations: Glu, Glucose; Gal, Galactose).

Compound Strain C57BL/6J PERA/EiJ CZECHII/EiJ MOLF/EiJ

Mean SD Mean SD Mean SD Mean SD

Rotenone IC 50 Glu (μM) >10 ±4.4 >10 ±2.8 >10 ±2.0 >10 ±6.2

IC 50 Gal (μM) 0.005 ±0.001 0.8 ±0.5 0.002 ±0.0001 0.04 ±0.02

IC 50 Glu/Gal >2000 ±7 >12.5 ⁎ ±3.1 >5000 ⁎#& ±5 >250 ±7

Nefazodone IC 50 Glu (μM) 39.2 ±2.4 45.3 ±7.1 117.3 ±1.5 24.5 ±8.3

IC 50 Gal (μM) 12.0 ±9.2 25.0 ±8.5 10.9 ±0.8 14.0 ±0.5

IC 50 Glu/Gal 4.3 ±1.8 1.9 ±0.5 10.9 ⁎&# ±0.9 1.8 ±0.6

Ketoconazole IC50 Glu (μM) 32.9 ±10.1 57.1 ±26.8 111.6 ±12.7 43.0 ±7.5

IC 50 Gal (μM) 24.1 ±7.8 35.3 ±17.9 33.8 ±3.9 42.1 ±4.9

IC50 Glu/Gal 1.5 ±0.8 1.7 ±0.6 3.4 ⁎&# ±0.8 1.0 ±0.3

Tolcapone IC 50 Glu (μM) >300 ±18.2 169.8 ±24.6 235.6 ±6.4 235.6 ±4.1

IC 50 Gal (μM) 140.5 ±29.4 287.4 ±34.9 68.6 ±2.1 112.7 ±1.1

IC50 Glu/Gal >2.1 @&# ±0.2 0.6* @# ±0.2 3.4* &# ±0.1 2.09* @& ±0.04

Flutamide IC 50 Glu (μM) 201.2 ±12.0 >300 ±26.2 >300 ±27.7 125.6 ±15.9

IC 50 Gal (μM) 20.4 ±2.4 45.8 ±21.0 98.8 ±3.2 36.3 ±1.7

IC 50 Glu/Gal 10.0 ±1.5 >6.5 ±2.3 >3.0 ±4.1 3.5 & ±0.5

Tamoxifen IC 50 Glu (μM) 14.5 ±1.6 26.6 ±6.3 40.6 ±4.3 80.8 ±17.4

IC50 Gal (μM) 11.3 ±3.1 16.7 ±4.8 30.6 ±6.5 28.7 ±7.5

IC 50 Glu/Gal 1.3 ±0.2 1.3 ±0.1 1.4 ±0.2 2.9 ⁎&@ ±0.5

Imipramine IC 50 Glu (μM) 27.8 ±3.7 25.0 ±3.3 44.1 ±3.5 101.9 ±8.4

IC 50 Gal (μM) 28.0 ±3.4 19.2 ±2.4 35.9 ±1.1 40.6 ±1.1

IC 50 Glu/Gal 1.0 ±0.2 1.3 ±0.3 1.2 ±0.1 2.5 ⁎&@ ±0.2

Troglitazone IC 50 Glu (μM) 111.1 ±36.2 >300 ±10.1 >300 ±27.1 117.8 ±8.5

IC 50 Gal (μM) 93.0 ±32.6 291.5 ±13.8 103.6 ±19.8 73.6 ±12.1

IC 50 Glu/Gal 1.2 ±0.1 >1.0 ±0.1 >2.9 ⁎&# ±0.9 1.6 ±0.3

Data are Mean±SD, N=3 separate experiments. Statistical analysis was performed using one-way ANOVA followed by Bonferroni Multiple Comparison test. pb0.05 is considered

statistically significant. * vs. reference strain C57BL/6J. & vs. PERA/EiJ. @ vs CZECHII/EiJ. #vs MOLF/EiJ.

activity of the MEFs. Overall, there were no significant differences in the

Complex IV activity among the strains at both passage 3 (Fig. 7A) and

passage 10 (Fig. 7B) and when comparing each strain between passages.

Citrate synthase activity of the MEFs was also determined for all of the

strains at both passages and used as a semi-quantitative marker of mitochondrial

content. The results indicated that CZECHII/EiJ showed significantly

lower citrate synthase activity at passage 3 (Fig. 8A) when

compared to C57BL/6J and MOLF/EiJ. At passage 10 (Fig. 8B), there

were no statistically significant differences in the citrate synthase activity

of PERA/EiJ, CZECHII/EiJ and MOLF/EiJ when compared to C57BL/6J. However,

PERA/EiJ was significantly lower than MOLF/EiJ and CZECHII/EiJ.

Comparison of each strain between passage 3 and passage 10 showed

that C57BL/6J had a statistically significant lower citrate synthase activity

at passage 10 than at passage 3, whereas CZECHII/EiJ showed a significantly

higher activity at passage 10 than at passage 3. After determining

citrate synthase activity, the ratios of Complex I/citrate synthase activities

(CI/CS) and Complex IV/citrate synthase activities (CIV/CS) were also determined

(Table 3). At passage 3, PERA/EiJ showed significantly higher CI/

CS, when compared to C57BL/6J and CZECHII/EiJ. At passage 10, only

MOLF/EiJ showed a statistically significantly lower CI/CS when compared

to C57BL/EiJ and PERA/EiJ, even though CZECHII/EiJ CI/CS was also very

low and significantly different from PERA/EiJ. CIV/CS was significantly

higher for all of the strains at passage 3 while at passage 10, no differences

were observed among the strains. CZECHII/EiJ CIV/CS was significantly

lower at passage 10 when compared to passage 3 (Table 3).

Drug-induced toxicity in the MEF panel: role of primary energy metabolism

In order to test our model of genetically diverse MEFs as a potential

tool to study drug-induced mitochondrial impairment, we tested the effect

of rotenone and seven drugs on the ATP content of the MEFs as an

end-point of cell toxicity. The seven drugs that were chosen are known

to display mitochondrial liabilities and were also reported to induce adverse

idiosyncratic reactions in patients. Rotenone was used as a positive

control since it is a mitochondrial inhibitor. The four strains of MEFs, at

passage 10, were incubated with the compounds over 24 h in (a) cell

culture medium containing glucose and (b) cell culture medium lacking

glucose but supplemented with galactose and glutamine as described in

Materials and methods. This latter approach has been used to detect direct

mitochondrial effects of different compounds since cells, in general,

are almost exclusively reliant on oxidative phosphorylation for their

ATP demands in a glucose-free, galactose/glutamine medium

(Marroquin et al., 2007). Hence, a compound could potentially cause

any of the following four effects when tested in glucose versus galactose

medium: (a) it could be non-toxic at the highest drug concentration tested

in both glucose medium and galactose medium, (b) it could have similar

IC 50 values in both glucose medium and galactose medium,

indicating that it causes multifactorial cytotoxicity, mitochondrial impairment

being one of the many potential factors, (c) it could have an IC 50

value which is at least 3 times lower in galactose medium than in glucose

medium, indicating that the primary mechanism of toxicity is mitochondrial

impairment, or (d) it could have an IC 50 value which is lower in glucose

medium than in galactose medium, indicating that it inhibits


Rotenone had an IC 50 ratio between Glucose:Galactose media that

was greater than 3 in all four strains (Table 4, Fig. 9). The highest ratio

was seen in CZECHII/EiJ and the lowest ratio was seen in PERA/EiJ.

Nefazodone had an IC 50 ratio between Glucose:Galactose media>3

in C57BL/6J and CZECHII/EiJ, indicating that mitochondrial impairment

was the primary mechanism of toxicity in these two strains

(Table 4, Fig. 10). The effect was more pronounced in CZECHII/EiJ

than in C57BL/6J. In contrast, although nefazodone decreased the

ATP content in PERA/EiJ and MOLF/EiJ, both in glucose medium and

galactose medium, the IC 50 ratio between Glucose:Galactose media

wasb3, indicating that multifactorial toxicity occurred in these two

strains. Ketoconazole had an IC 50 ratio between Glucose:Galactose

mediab3 in C57BL/6J, PERA/EiJ and MOLF/EiJ, and decreased the ATP

content in these three strains of MEFs in both glucose and galactose

medium. This indicated that the drug caused multifactorial toxicity

in these strains. In contrast, ketoconazole had an IC 50 ratio between

Glucose:Galactose media>3 in CZECHII/EiJ, suggesting that the drug

caused mitochondrial impairment as a primary mechanism of toxicity

176 C.V. Pereira et al. / Toxicology and Applied Pharmacology 264 (2012) 167–181

ATP content (% control)

ATP content (% control)











* *











0.0001 0.001 0.01 0.1 1 10

Rotenone (µM)







* *

















0.0001 0.001 0.01 0.1 1 10

Rotenone (µM)

ATP content (% control)

ATP content (% control)







* * *



* * *










0.0001 0.001 0.01 0.1 1 10

Rotenone (µM)






* * * *




* *










0.0001 0.001 0.01 0.1 1 10

Rotenone (µM)

Fig. 9. The effect of rotenone on the ATP content of the four strains of MEFs in cell culture medium containing glucose (filled symbol) and galactose (open symbol) 24 h after compound

addition. Each data point represents the mean±SD, N=3 separate experiments. Statistical analysis was performed via Student t-test. pb0.05 is considered statistically significant,

* vs. galactose-grown.

in this strain (Table 4, Fig. 11). Tolcapone had an IC 50 ratio between Glucose:Galactose

mediab3 in C57BL/6J, PERA/EiJ and MOLF/EiJ, indicating

that mitochondrial impairment was not the main mechanism of toxicity

(Table 4). In contrast, the IC 50 ratio between Glucose:Galactose media

was>3 in CZECHII/EiJ, indicating that mitochondrial impairment was

the primary mechanism of toxicity in this strain (Table 4). Flutamide

had an IC 50 ratio between Glucose:Galactose media>3 in all four strains,

indicating that the primary mechanism of toxicity was mitochondrial

impairment. The effect was most pronounced in C57BL/6J where the

ratio was 10 (Table 4). Tamoxifen and imipramine had an IC 50 ratio between

Glucose:Galactose mediab3 in all four strains and decreased the

ATP content of the MEFs in both types of media (Table 4). This indicated

that these two drugs caused multifactorial toxicity in all the strains.

Troglitazone had an IC 50 ratio between Glucose:Galactose mediab3 in

all the strains, indicating that mitochondrial impairment was not the

primary mechanism of toxicity (Table 4).

In summary, CZECHII/EiJ showed the highest IC 50 ratio between

Glucose:Galactose media with rotenone, nefazodone, ketoconazole

and tolcapone when compared with the other strains while C57BL/6J

showed the highest IC 50 ratio between Glucose:Galactose media with



It has been suggested that human genetic diversity should be considered

when predicting and understanding the mechanisms of idiosyncratic

drug-induced toxicity (Boelsterli and Lim, 2007). Pharmacogenomics

aims to identify how gene polymorphisms, such as SNPs, can contribute

to idiosyncratic drug responses (Daly, 2010; Holt and Ju, 2006). Multiple

enzyme polymorphisms can be involved in rare, but sometimes fatal, episodes

of adverse drug reactions. Depending on the activity of the mutated

protein, individuals can have a higher predisposition to the development

of an adverse reaction or, in other cases, respond with lower efficacy to

the treatment (Boelsterli and Lim, 2007).

Recent evidence has shown that mitochondria are off-targets of several

drugs, such as nucleoside reverse transcriptase inhibitors (NRTIs)

(Feng et al., 2001), diclofenac (Deng et al., 2006), troglitazone (Lee et

al., 2008) and doxorubicin (Oliveira and Wallace, 2006). Genetic or acquired

mitochondrial deficiencies have been suggested to be factors

that contribute to increased susceptibility to drug-induced toxicity

that may trigger idiosyncratic responses (Boelsterli and Lim, 2007).

The genetic drift between a mostly normal mtDNA population and an

increased heteroplasmy caused by accumulation of mutated mtDNA

within a cell (DiMauro and Schon, 2003) may lead to the appearance

of a drug toxicity phenotype over time.

So far, there are no validated animal models to study idiosyncratic

drug responses that can be translated to humans. Certain risk factors

such as increasing age, concurrent medications, underlying diseases,

and life-style factors such as smoking and heavy alcohol consumption

are known to predispose patients towards drug adverse reactions,

but ironically, the animals used in toxicology studies are

usually, healthy, young animals. In addition, the animal models

used in regulatory toxicology studies do not represent the genetic diversity

that is seen in the human population. Hence, identifying

C.V. Pereira et al. / Toxicology and Applied Pharmacology 264 (2012) 167–181


drugs that cause idiosyncratic off-target adverse reactions has been

difficult. For this reason, a valid model which could simulate the heterogeneous

genetic background existent in a human population

must be developed to predict drug safety and different drug responses.

An in vitro approach using a suitable biological model mimicking

a genetically diverse population has the advantage of reducing

the costs that in vivo studies entail (O'Shea et al., 2011). Furthermore,

there is still a large gap in our understanding of the role of mtDNA

SNPs and their relationship with mitochondrial bioenergetics and

how this can translate into altered mitochondrial drug liabilities.

In an effort to create a new and suitable in vitro system to investigate

drug-induced mitochondrial toxicity, we characterized the mitochondrial

bioenergetic parameters of embryonic fibroblasts isolated

from four different strains of mice, C57BL/6J, PERA/EiJ, CZECHII/EiJ

and MOLF/EiJ. These strains of mice have distinct mtDNA polymorphisms

that could potentially simulate different sub-populations of

mice that could later be extrapolated for different human genetic

pools. The mtDNA sequences of C57BL/6J and MOLF/EiJ have been

reported previously (Goios et al., 2007). Our results showed that the

mtDNA sequence of the fibroblasts, at passage 3, of the strains,

C57BL/6J and MOLF/EiJ, were identical to that found by Goios et al.

(2007). The mtDNA of PERA/EiJ and CZECHII/EiJ were sequenced for

the first time in our study. We then performed a comparative study of

mtDNA SNPs in PERA/EiJ, CZECHII/EiJ and MOLF/EiJ, using C57BL/6J as

the reference strain. Our results showed that, at passage 3, mtDNA

from fibroblasts of PERA/EiJ had 106 SNPs including 9 nonsynonymous

substitutions, CZECHII/EiJ had 390 SNPs including 35 nonsynonymous

substitutions, and MOLF/EiJ had 393 SNPs including 34 nonsynonymous

substitutions when compared with C57BL/6J (Fig. 1). These results

suggested that PERA/EiJ is more closely related to C57BL/6J than are

either CZECHII/EiJ or MOLF/EiJ. In addition, many of the SNPs found in

CZECHII/EiJ were also found in MOLF/EiJ, suggesting that these two

strains may be phylogenetically related. Overall, the majority of SNPs

which caused amino acid changes occurred in genes encoding subunits

of Complex I, which has been described as the most affected respiratory

chain enzyme in mitochondrial diseases (Wallace, 2010).

The second part of our study was aimed at characterizing the bioenergetic

profile of the four strains of MEFs. We measured the OCR

(oxygen consumption rate) of the MEFs, measured the activity of

Complex I, Complex IV and citrate synthase, and also investigated

alterations in cell proliferation.

When measuring the various components of oxygen consumption

(e.g. ATP-linked respiration, proton leak, etc.) in the different MEF

strains, we did not consider the absolute values of basal respiration of

each strain. In accordance with a recent report (Brand and Nicholls,

2011), comparing absolute values of respiration between cells of different

strains poses challenges since different strains can proliferate at different

rates or may have different morphologies. For instance, if cells

double in size but remain the same number, the respiration rate per

cell will double, but the respiration rate per unit of cell protein or per

ATP content (% control)

ATP content (% control)




















0.01 0.1 1 10 100 1000

Nefazodone (µM)







* *












0.01 0.1 1 10 100 1000

Nefazodone (µM)

ATP content (% control)

ATP content (% control)




















0.01 0.1 1 10 100 1000

Nefazodone (µM)

















0.01 0.1 1 10 100 1000

Nefazodone (µM)

Fig. 10. The effect of nefazodone on the ATP content of the four strains of MEFs in cell culture medium containing glucose (filled symbol) and galactose (open symbol) 24 h after

compound addition. Each data point represents the mean±SD, N=3 separate experiments. Statistical analysis was performed via Student t-test. pb0.05 is considered statistically

significant, * vs. galactose-grown.

178 C.V. Pereira et al. / Toxicology and Applied Pharmacology 264 (2012) 167–181

nucleus or per amount of DNA may remain the same (Brand and

Nicholls, 2011). Our results showed that the different strains of MEFs

proliferated at different rates (Figs. 2A and B). Hence, the endpoints,

ATP-linked respiration, proton leak, non-mitochondrial respiration,

maximum respiratory capacity and spare respiratory capacity, were

measured as percentages of basal respiration for each strain.

The spare respiratory capacity of cells can be defined as the ability

of substrate supply and electron transport to respond to an increase

in energy demand (Brand and Nicholls, 2011; Hill et al., 2009;

Yadava and Nicholls, 2007). The maximum respiratory capacity of

cells indicates the maximal speed at which the respiratory chain can

operate, substrate supply and respiratory chain electron transfer

being the limiting steps. When determining the spare respiratory capacity

and the maximum respiratory capacity, addition of the uncoupler,

FCCP, is required. When using FCCP, one should titrate it

carefully, in order to add the minimum amount necessary to induce

fully uncontrolled respiration, without causing inhibition of respiration

(Brand and Nicholls, 2011). We found that a concentration of

0.5 μM FCCP was enough to achieve maximum uncoupling in MEFs

of PERA/EiJ, whereas 1 μM FCCP was necessary for the other strains.

Our study showed that both PERA/EiJ and CZECHII/EiJ had a decreased

maximum respiratory capacity and a decreased spare respiratory capacity

at passage 3 as well as at passage 10 when compared to the reference

strain, C57BL/6J, indicating that under the conditions of our

experiments, their mitochondria operate closer to their bioenergetic


Surprisingly, although PERA/EiJ MEFs had a lower maximum respiratory

capacity and a lower spare respiratory capacity than the reference

strain, at both passages, this strain also showed higher ATPlinked

respiration and a higher coupling efficiency (coupling efficiency

is the ratio, ATP-linked respiration: ATP-linked respiration plus proton

leak) in comparison with the reference strain, at passage 10. Furthermore,

the proton leak of this strain was lower at passage 10. Using

oligomycin to determine ATP turnover or ATP-linked respiration results

in slight hyperpolarization due to blockage of proton reflux through the

ATP synthase. Since the proton-leak is voltage-dependent and this approach

stimulates proton influx, ATP synthesis is underestimated

(Brand and Nicholls, 2011). Given that oligomycin induces a collapse

of ATP synthesis, cells increase their glycolysis rate by~10-fold in

order to sustain survival (Brand and Nicholls, 2011). Therefore, a

change in the basal rate caused by a change in ATP turnover is most likely

to be a response of mitochondria to altered ATP demand elsewhere in

the cell. This unexpected observation also correlates with the fact that

PERA/EiJ had a higher coupling efficiency and a lower proton leak at

passage 10, as mentioned previously. A modest alteration in the proton

leak might be a possible change in the mitochondrial transmembrane

potential caused by substrate oxidation, although we cannot exclude

that membrane alterations may account for differences in the passive

proton influx. Therefore, these differences in the ATP-linked respiration,

coupling efficiency and proton-leak are likely due to normal cellular

adjustments to substrate oxidation and ATP demands in the cells. Moreover,

we also did not find any differences regarding non-mitochondrial

ATP content (% control)

ATP content (% control)

















0.01 0.1 1 10 100 1000

Ketoconazole (µM)





















0.01 0.1 1 10 100 1000

Ketoconazole (µM)

ATP content (% control)

ATP content (% control)


















0.01 0.1 1 10 100 1000

Ketoconazole (µM)

















0.01 0.1 1 10 100 1000

Ketoconazole (µM)

Fig. 11. The effect of ketoconazole on the ATP content of the four strains of MEFs in cell culture medium containing glucose (filled symbol) and galactose (open symbol) 24 h after

compound addition. Each data point represents the mean±SD, N=3 separate experiments. Statistical analysis was performed via Student t-test. pb0.05 is considered statistically

significant, * vs. galactose-grown.

C.V. Pereira et al. / Toxicology and Applied Pharmacology 264 (2012) 167–181


respiration, which mainly results from cell surface oxygen consumption,

peroxisomes and substrate oxidation, (Abe et al., 2010) with the exception

of PERA/EiJ at passage 10, which showed a statistically significant

lower non-mitochondrial respiration when compared to CZECHII/EiJ.

No differences were found between the tested strains with respect to

the decrease in OCR after addition of either rotenone, a Complex I inhibitor,

or antimycin, a Complex III inhibitor. It is possible that these cells

would reveal more differences if they had been grown in a galactose/

glutamine-containing medium (Pelicano et al., 2006). Other cell types

such as HepG2 cells are more susceptible to drug-induced mitochondrial

dysfunction when grown in a galactose/glutamine-containing medium

since they are forced to use oxidative phosphorylation for making ATP

(Marroquin et al., 2007). Therefore, future studies with these four MEF

strains could be performed in different cell media, such as galactose/

glutamine-containing medium or fatty acid-enriched medium, in order

to explore the underlying mechanisms of their metabolic pathways. In

the present work, we also inhibited glycolysis with the glucose analogue,

2-DG. Our results suggested that C57BL/6J and PERA/EiJ were

more reliant on mitochondria than on glycolysis for their ATP production

in comparison with CZECHII/EiJ and MOLF/EiJ (Fig. 5). Interestingly,

at passage 10, all four strains of MEFs were less able to increase their mitochondrial

respiration. This could be due to decreased substrate delivery

or decreased electron transfer.

Citrate synthase is a crucial pace-making enzyme in the first step

of Krebs cycle and a semi-quantitative marker of mitochondrial content

(Ratkevicius et al., 2010). Only CZECHII/EiJ showed a decreased

citrate synthase activity at passage 3, whereas at passage 10, there

was no statistically significant difference (Fig. 8). Therefore, this information

was important to normalize the data shown in this work,

in order to establish a biologically relevant comparison of the mitochondrial

function of these four murine cell strains.

Since some of the SNPs fell within Complex I and IV mtDNA genes,

we also determined the activities of those two mitochondrial complexes.

Generally, rotenone-sensitive Complex I activity is difficult to

determine in fibroblasts because of the presence of contaminating enzymes

(Janssen et al., 2007). Nevertheless, Complex I deficiencies are

a major cause of mitochondrial dysfunction and consequently a decrease

in Complex I activity is a strong indicator of a possible mitochondrial

dysfunction (Janssen et al., 2007). Our results showed that PERA/

EiJ had increased Complex I activity and CI/CS ratio when compared to

C57BL/6J and CZECHII/EiJ at passage 3. Interestingly, both MOLF/EiJ

and CZECHII/EiJ, which were also the strains with a higher rate of

non-synonymous mutations at subunits of Complex I, showed lower

Complex I activity and CI/CS ratio at passage 10 (Table 3 and Fig. 6)

when compared to the other strains, suggesting a decrease in mitochondrial

capacity. In contrast, Complex IV/citrate synthase activity was significantly

higher for all of the strains when compared to the reference

strain at passage 3, while no differences existed at passage 10 (Table 3

and Fig. 7). This result might be due to a slightly variable citrate synthase

activity/expression among the strains. Despite the fact that the Complex

IV/citrate synthase ratio in C57BL/6J was lower than that for the other

strains at passage 3, the respiratory measurements did not reveal lower

respiratory capacity when compared to the other strains.

Another aim of our study was to investigate the role of cell aging in cell

culture. At passage 10, all four strains of MEFs were less capable

of maintaining respiration when glycolysis was inhibited by 2-DG,

suggesting that the cells became more glycolytic and/or were less capable

of using oxidative phosphorylation to produce ATP at the higher cell passage

(Fig. 5). A decrease in the activity of the respiratory chain or a decrease

in the ability to shuttle substrates into mitochondria may have

been reasons for these observations. We also found that MOLF/EiJ MEFs

had a lower maximum respiratory capacity at passage 10 and lower Complex

I activity per se than at passage 3 while PERA/EiJ MEFs had a lower

spare respiratory capacity at passage 10 than at passage 3, showing a

lower capacity to respond to an increase of energy demand when aged

in culture. The citrate synthase activity decreased for C57BL/6J and

increased for CZECHII/EiJ. Since CS is normally used as a mitochondrial

marker , we can speculate that citrate synthase activity may be a bona

fide indication of mitochondrial mass. The increased CS activity in

CZECHII/EiJ may result from increased mitochondrial proliferation in response

to a mitochondrial bioenergetic deficit found in this strain,

which is suggested by decreased CIV/CS (Table 3) at passage 10 when

compared with passage 3. Despite the differences in some of the bioenergetic

parameters between passage 3 and passage 10, comparison of the

mtDNA sequences of each strain of MEFs between the two passages

showed that there no differences with C57BL/6J, PERA/EiJ and MOLF/EiJ.

Furthermore, the three extra SNPs that we found in CZECHII/EiJ MEFs at

passage 10 did not cause amino acid changes. Overall, our mtDNA SNP

data showed that the number of passages was not sufficient to create

many SNPs. Our data also indicated that mtDNA SNPs cannot be the reason

for the differences seen in the functional endpoints between passage

3 and passage 10. This suggests that other factors such as oxidative damage

to mitochondrial components, SNPs in nuclear DNA-encoded mitochondrial

proteins or decreased nuclear-mitochondrial crosstalk may

have to be taken into account (Aliev et al., 2011; Battersby et al., 2003;

Michikawa et al., 1999). Nevertheless, it is clear that cell aging in culture

shouldbetakeninaccountforbioenergetic or drug safety assessment


One of the challenges in correlating altered mitochondrial bioenergetics

with mtDNA SNPs is that confounding factors such as SNPs

in nuclear DNA-encoded genes that encode subunits of Complexes I,

III, IV and V, are present. For instance, there are 5 SNPs in the nuclear

gene that codes for the Complex I subunit NDUFA1 and 3 SNPs in the

nuclear gene that codes for the Complex I subunit NDUFA6 in MOLF/

EiJ as well as in CZECHII/EiJ when compared to C57BL/6J (http://

Unpredictable drug-induced mitochondrial toxicity is currently a

major reason for post-market drug withdrawals and black box warnings.

Hence, a goal of this study was to determine whether the four

strains of fibroblasts can be used as an in vitro platform to identify

any differences in the susceptibility towards drugs that are known

to cause mitochondrial impairment and idiosyncratic drug responses.

For this reason, we tested the effect of a selection of drugs described

to be involved in mitochondrial impairment, on the ATP content of

the MEFs in glucose- and galactose-medium, at passage 10. Rotenone

showed the expected response in C57BL/6J, CZECHII/EiJ and MOLF/EiJ

in that the cells were significantly more sensitive to rotenone in galactose

medium than in glucose medium, with CZECHII/EiJ displaying

the most significant sensitivity (Table 4, Fig. 9). Surprisingly, PERA/EiJ

was much less sensitive to rotenone in galactose than expected and

did not show complete ATP depletion even at the highest concentration

tested (10 μM). Our results also indicated that, of the four strains,

CZECHII/EiJ showed the highest IC 50 Glucose:Galactose ratio for

nefazodone, tolcapone and ketoconazole. Nefazodone is an antidepressant

drug and it has been reported to cause mitochondrial impairment

by causing Complex I inhibition (Dykens et al., 2008).

Nefazodone had an IC 50 ratio between Glucose:Galactose media which

was>10 in CZECHII/EiJ (Table 3) indicating that it caused strong mitochondrial

impairment in this strain. A slightly less potent mitochondrial

effect was seen in C57BL/6J, where the ratio was 4.3 (Table 3).

Nefazodone has previously been shown to have a mitochondrial effect

on HepG2 cells, with a ratio of 3.6 (Dykens et al., 2008). Thus, C57BL/6J

resembled HepG2 cells in its response to nefazodone, whereas

CZECHII/EiJ was much more sensitive to nefazodone's mitochondrial effects.

In contrast, nefazodone had a ratio which was ~2 in PERA/EiJ and

MOLF/EiJ, suggesting that although mitochondrial impairment may be

occurring in these two strains, nefazodone has other off-targets. This is

in accord with a report (Kostrubsky et al., 2006) which showed that

nefazodone inhibits the bile salt efflux pump.

Ketoconazole is an antifungal agent that is also known to inhibit

Complex I (Rodriguez and Acosta, 1996). Ketoconazole displayed an

IC 50 ratio between Glucose:Galactose media which was>3 in CZECHII/

180 C.V. Pereira et al. / Toxicology and Applied Pharmacology 264 (2012) 167–181

EiJ, indicating that it caused mitochondrial impairment in this strain.

This drug does not cause mitochondrial toxicity in the HepG2 glucose/

galactose model (data not shown). This suggests that CZECHII/EiJ is

more sensitive than HepG2 cells to ketoconazole's mitochondrial effect.

In contrast, no mitochondrial impairment was seen in the other strains

of MEFs. This suggests that ketoconazole may have other mechanisms

of toxicity in these strains in accord with reports that it depletes glutathione

(Rodriguez and Buckholz, 2003), accumulates in lysosomes and

causes phospholipidosis (Rodriguez and Acosta, 1995).

Tolcapone, a catechol-O-methyltransferase inhibitor used in the

treatment of Parkinson's disease, was reported to be a mitochondrial

uncoupler (Haasio et al., 2002). CZECHII/EiJ showed an IC 50 ratio between

Glucose:Galactose media which was>3 (Table 3). This is in contrast

to HepG2 cells which do not show a mitochondrial effect in the

glucose/galactose model (data not shown). Hence, CZECHII/EiJ MEFs

were more sensitive to tolcapone's mitochondrial effect than HepG2


Three of the drugs that we tested, tamoxifen, troglitazone and imipramine,

had an IC 50 ratio between Glucose:Galactose media which

wasb3 in all the strains, indicating that mitochondrial impairment

was not the primary mechanism of toxicity caused by these drugs.

These drugs have been reported to cause multiple mechanisms of toxicity.

For example, tamoxifen, an anticancer drug, and imipramine, an antidepressant,

are both reported to cause phospholipidosis (Kuroda and

Saito, 2010; Nioi and Nguyen, 2007) in addition to mitochondrial impairment.

Troglitazone, a thiazolidinedione removed from the market

due to hepatotoxicity, is reported to inhibit the bile salt efflux pump

(Funk et al., 2001).

In conclusion, four strains of MEFs which had distinct mtDNA polymorphisms

showed strain-dependent and age-dependent differences

in their bioenergetic characteristics. Evaluation of rotenone and seven

drugs on the ATP content of the MEFs in glucose and galactose media

showed that there were strain-dependent differences in the sensitivity

towards the tested compounds. Of the four strains tested, CZECHII/EiJ

showed the largest IC 50 ratio between Glucose:Galactose media with

rotenone, nefazodone, ketoconazole and tolcapone. In addition, this

strain also showed lower mitochondrial robustness when compared

to the other strains, and this might be one of the reasons why it was

easier to identify mitochondrial toxicity in this strain with some of the

drugs that impair mitochondrial function.

Our results indicate that high throughput screening of more drugs

using this panel of MEFs, particularly the strain CZECHII/EiJ, in glucose/galactose

media, may help us predict the toxicity of compounds

which cause off-target mitochondrial effects, often missed in early

stages of drug development. Moreover, our results suggest that, by testing

the effect of rotenone on the ATP content of fibroblasts from many

other strains of genetically diverse mice in glucose/galactose media,

one may identify other strains that are useful for in vitro testing of compounds

that have the potential to cause idiosyncratic drug responses

through mitochondrial impairment.

Despite the open questions, the current investigation can be the

backbone of an in vitro platform to test various drugs and predict toxicity

among different sub-populations of cells with variable number

of SNPs in both mitochondrial and nuclear DNA. The present study

also characterizes, for the first time, the embryonic fibroblast bioenergetic

profile of four distinct mouse strains, showing that they present

differential mitochondrial fitness.

This study conveys a valuable starting-point to the beginning of a new

era where personalized medical care is emerging and individual susceptibilities

should be taken into consideration when developing a new drug.

Funding information

Part of this work was supported by the Foundation for Science and

Technology (FCT). Claudia V. Pereira is the recipient of a PhD fellowship

from the Foundation for Science and Technology, Portugal (FCT),

[SFRH/BD/48029/2008]. Paulo J. Oliveira is supported through a research

grant [PTDC/SAU-TOX/110952/2009], from FCT, co-funded by

COMPETE/FEDER and National Funds.

Conflict of interest statement

The authors declare no conflicts of interest.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://


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