Drugs Induced Liver Injury

doi: 10.1111/j.1472-8206.2008.00608.
x
REVI EW
ART I CL E
Drug-induced liver injury through
mitochondrial dysfunction: mechanisms and
detection during preclinical safety studies
Gilles Labbe
a
, Dominique Pessayre
b
, Bernard Fromenty
b
*
a
Sano-aventis recherche & developpement, Drug Safety Evaluation, Alfortville, France
b
INSERM, U773, Centre de Recherche Biomedicale Bichat Beaujon CRB3, Paris, France
I NTRODUCTI ON
Drug-induced liver injury (DILI) is a major issue for
pharmaceutical companies, because DILI is a frequent
cause for the failure of a drug to get approved, or for
the withdrawal of an already marketed medicine. The
nancial, legal and image-tarnishing consequences are
at their worst when toxicity is only discovered after
marketing, requiring the recall of an unsafe medicine,
which has caused lethal liver injury or has required liver
transplantation in many patients.
Although drugs can cause hepatotoxicity through
different ways [1], mitochondrial dysfunction is one
major mechanisms underlying DILI [2]. By hampering
mitochondrial energy production and/or releasing mito-
chondrial pro-apoptotic proteins into the cytoplasm,
drugs can trigger the necrosis or apoptosis of hepato-
cytes, thus causing cytolytic hepatitis, which can evolve
towards liver failure. Drug-induced mitochondrial dys-
function can also lead to steatosis and steatohepatitis,
which can sometimes progress towards cirrhosis [24].
In addition, drug-induced mitochondrial dysfunction
can also trigger diverse extrahepatic or general mani-
festations, such as hyperlactataemia, lactic acidosis,
myopathy, rhabdomyolysis, pancreatitis, peripheral
neuropathy or lipoatrophy [58]. Thus, because of
its potential severity for patients and its dire conse-
quences for the pharmaceutical industry, drug-induced
Keywords
cell death,
drug,
hepatotoxicity,
mitochondria,
steatohepatitis,
steatosis
Received 14 January 2008;
Revised 11 March 2008;
Accepted 27 March 2008
*Correspondence and reprints:
bernard.fromenty@inserm.fr
This review is the summary of
an oral communication made by
Dr B. Fromenty at the 20th
Annual ADPC Meeting, held in
Paris on 24 October 2007, and
devoted to Predictive Toxicol-
ogy and Drug Safety.
ABSTRACT
Mitochondrial dysfunction is a major mechanism whereby drugs can induce liver
injury and other serious side effects such as lactic acidosis and rhabdomyolysis in
some patients. By severely altering mitochondrial function in the liver, drugs can
induce microvesicular steatosis, a potentially severe lesion that can be associated
with profound hypoglycaemia and encephalopathy. They can also trigger hepatic
necrosis and/or apoptosis, causing cytolytic hepatitis, which can evolve into liver
failure. Milder mitochondrial dysfunction, sometimes combined with an inhibition of
triglyceride egress from the liver, can induce macrovacuolar steatosis, a benign lesion
in the short term. However, in the long term this lesion can evolve in some
individuals towards steatohepatitis, which itself can progress to extensive brosis and
cirrhosis. As liver injury caused by mitochondrial dysfunction can induce the
premature end of clinical trials, or drug withdrawal after marketing, it should
be detected during the preclinical safety studies. Several in vitro and in vivo
investigations can be performed to determine if newly developed drugs disturb
mitochondrial fatty acid oxidation (FAO) and the oxidative phosphorylation
(OXPHOS) process, deplete hepatic mitochondrial DNA (mtDNA), or trigger the
opening of the mitochondrial permeability transition (MPT) pore. As drugs can be
deleterious for hepatic mitochondria in some individuals but not in others, it may also
be important to use novel animal models with underlying mitochondrial and/or
metabolic abnormalities. This could help us to better predict idiosyncratic liver injury
caused by drug-induced mitochondrial dysfunction.
2008 The Authors Journal compilation 2008 Blackwell Publishing Ltd. Fundamental & Clinical Pharmacology 22 (2008) 335353 335
mitochondrial dysfunction should be detected during the
course of preclinical safety studies.
In the present review, we rst briey recall some key
features of mitochondria and the main molecular mech-
anisms whereby drugs can impair mitochondrial func-
tion and lead to liver injury. We then cite a few drugs,
the propensity of which to cause mitochondrial dysfunc-
tion and DILI has led to the premature termination of
clinical trials or to the withdrawal of the drug from the
market. We also indicate which in vitro and in vivo
investigations can be performed during preclinical safety
studies to detect DILI linked to mitochondrial dysfunc-
tion, or later in drug development to explain hepatotoxic
events in some individuals. Finally, we discuss the
possible means to improve the prediction of idiosyncratic
liver injury in the future, such as the use of animal
models with underlying mitochondrial and/or metabolic
abnormalities. It is noteworthy that several excellent
reviews are available for those who wish to gain insight
into preclinical testing strategies independently of the
mechanism of DILI [911].
MI TOCHONDRI AL FUNCTI ONS AND
GENOME
The mitochondria: powerhouses of the cell and
crossroads of metabolic pathways
Mitochondria oxidizes many metabolites including fatty
acids, pyruvate (generated from glycolysis) and several
amino acids such as valine, leucine and methionine
336 G. Labbe et al.
2008 The Authors Journal compilation 2008 Blackwell Publishing Ltd. Fundamental & Clinical Pharmacology 22 (2008) 335353
[3,12,13]. During the fasting state, fatty acid oxidation
(FAO) in the liver is incomplete; the acetyl-CoA that is
generated is not oxidized in the liver, but condenses into
ketone bodies (mainly acetoacetate and b-hydroxybuty-
rate) (Figure 1), which are released into the plasma for
subsequent oxidation in extrahepatic tissues. In kidney,
heart and brain, the ketone bodies are then cleaved into
acetyl-CoA, which is oxidized through the tricarboxylic
acid cycle [3,12].
The intra-mitochondrial oxidation of fatty acids,
pyruvate and amino acids generates reducing equiva-
lents (FADH
2
, NADH), which must be reoxidized by
the mitochondrial respiratory chain (MRC) to regenerate
the FAD and NAD
+
that are needed for the oxidative
processes such as FAO, pyruvate dehydrogenation and
the activity of the tricarboxylic acid cycle (Figure 1). In
addition to regenerating FAD and NAD
+
, the MRC also
creates a large electrochemical gradient across the inner
mitochondrial membrane, the so-called mitochondrial
transmembrane potential or Dw
m
(Figure 1). Indeed,
the transfer of electrons along the different components
of the MRC (for instance, complexes I, III and IV when
electrons come from NADH) is associated with the
ejection of protons from the mitochondrial matrix to
the intermembrane space, thus building up the Dw
m
(Figure 1). The Dw
m
serves as a reservoir of potential
energy. When cells need energy, the rise in ADP levels
within the mitochondria induces the re-entry of protons
into the mitochondrial matrix through the membrane-
spanning F
0
portion of the ATP synthase complex (also
referred to as the complex V of the MRC). This re-entry
of protons partially dissipates the Dw
m
, thus liberating
energy, which is used by the F
1
portion of ATP synthase
to phosphorylate ADP into ATP (Figure 1). Concom-
itantly, the transient decrease in Dw
m
promotes electron
transfer in the MRC and the coupled ejection of
protons towards the intermembrane space, which
restores the Dw
m
. Hence, there is a tight coupling within
the mitochondria between electron transfer within the
MRC (i.e. NADH and FADH
2
oxidation) and ATP
synthesis (i.e. ADP phosphorylation) [3,6,13]. A severe
dysfunction of this oxidative phosphorylation (OXPHOS)
Figure 1 Metabolism and energy production in liver mitochondria, and main drug targets. The entry of long-chain (C
14
C
18
) fatty acids
(LCFAs) within mitochondria requires a specic shuttle system involving four steps: (a) LCFAs are activated into LCFA-coenzyme A (acyl-
CoA) thioesters by long-chain acyl-CoA synthetases (ACS) located in the outer mitochondrial membrane, microsomes and peroxisomes.
(b) The long-chain acyl-CoA is converted into an acyl-carnitine by carnitine palmitoyltransferase-I (CPT-I) in the outer mitochondrial
membrane. (c) The acyl-carnitine is translocated across the inner mitochondrial membrane into the mitochondrial matrix by carnitine-
acylcarnitine translocase. (d) Finally, carnitine palmitoyltransferase-II (CPT-II), located on the inner side of the inner mitochondrial
membrane, transfers the acyl moiety from carnitine back to coenzyme A. LCFA-CoA thioesters are then oxidized into acetyl-CoA moieties via
the b-oxidation process. Acetyl-CoA moieties can then generate ketone bodies (mainly acetoacetate and b-hydroxybutyrate) via ketogenesis,
which is stimulated during fasting. Ketone bodies are liberated into the plasma to be used by extrahepatic tissues for energy production. After
a meal, however, the acetyl-CoA moieties are mainly generated in the liver by the glycolysis of glucose into pyruvate and the oxidative
decarboxylation of pyruvate. The formed acetyl-CoA enters the tricarboxylic acid (TCA) cycle (not shown), to be fully oxidized or to form
citrate, which can leave mitochondria and regenerate acetyl-CoA in the cytosol for subsequent de novo lipogenesis. Mitochondrial fatty acid
b-oxidation (FAO) and the TCA cycle generate NADH and FADH
2
, which transfer their electrons (e
)
) to the mitochondrial respiratory chain
(MRC), regenerating NAD
+
and FAD used for other b-oxidation (or TCA) cycles. Within the MRC, electrons are sequentially transferred to
different polypeptide complexes (numbered from I to IV) embedded within the inner membrane. The nal transfer of the electrons to oxygen
takes place at the level of complex IV, also referred to as cytochrome c oxidase. The mitochondrial DNA (mtDNA) encodes 13 polypeptides,
which are embedded within complexes I, III, IV and V. The ow of electrons within the MRC is coupled with the extrusion of protons (H
+
)
from the mitochondrial matrix to the intermembrane space, which creates the mitochondrial transmembrane potential, Dw
m
. When energy is
needed (i.e. when ATP levels are low), these protons re-enter the matrix through ATP synthase (also referred to as complex V), thus liberating
energy that is used to phosphorylate ADP into ATP. The adenine nucleotide translocator (ANT) then extrudes ATP from mitochondria, in
exchange for cytosolic ADP. Importantly, ANT is one of the components of the mitochondrial permeability transition (MPT) pore, which also
includes the voltage-dependent anion channel (VDAC) in the outer membrane and cyclophilin D in the matrix. The opening of the MPT pore
by exogenous or endogenous compounds leads to the outer membrane rupture and the release into the cytosol of cytochrome c (Cyt. c), a
component of the MRC loosely associated with the inner membrane. Cytochrome c release induces caspase activation and cell death via
apoptosis or necrosis. Drugs can impair mitochondrial function through different mechanisms. They can act (i) by inducing MPT, (ii) by
causing mtDNA depletion, which can reduce the synthesis of mtDNA-encoded MRC polypeptides, impair MRC activity and increase the
generation of the superoxide anion and other reactive oxygen species (ROS), (iii) by directly impairing MRC activity, which can secondarily
inhibit mitochondrial FAO and enhance ROS formation (not shown), (iv) by dissipating Dw
m
, (v) by directly altering FAO via the inhibition of
b-oxidation enzyme(s), and/or (vi) by indirectly impairing FAO through the sequestration of the FAO cofactors, L-carnitine and coenzyme A.
NAPQI and NRTIs signify N-acetyl-p-benzoquinone imine and nucleoside reverse transcriptase inhibitors, respectively.
Drug-induced mitochondrial dysfunction in liver: mechanisms and detection 337
process can jeopardize cell metabolism and cell life in the
liver and elsewhere [2,14].
Double genetic origin of the OXPHOS proteins
One unique feature of mitochondria is the double
genetic origin of the proteins (approximately 100) that
play a role in the OXPHOS process. Indeed, whereas
the majority of these polypeptides are encoded by the
nuclear genome and subsequently imported within the
mitochondria, 13 MRC polypeptides are instead
encoded by the mitochondrial genome, a small piece
of circular doubled-stranded DNA (approximately
16.6 kb in mammals) located within the mitochondrial
matrix (Figure 1). Importantly, there are several hun-
dreds (or thousands) of copies of mitochondrial DNA
(mtDNA) in a single cell, and the replication of these
mtDNA molecules occurs continuously, even in cells
that do not divide [6]. Permanent mtDNA replication by
the DNA polymerase c thus allows mtDNA levels to
remain constant in the cell, despite the continuous
degradation of some mitochondria, particularly dam-
aged and dysfunctional mitochondria [15,16]. It is
important to bear in mind that most cells (including
hepatocytes) have a surplus of mtDNA copies, and
can therefore tolerate a substantial depletion of mtDNA.
Classically, it is considered that the number of normal
mtDNA copies must fall below 2040% of basal
levels to induce mitochondrial dysfunction and adverse
events [6]. Below this threshold, hepatic mtDNA
depletion can impair the MRC. This impairment can
hamper the regeneration of NAD
+
and FAD, thus
leading to a secondary inhibition of the oxidation of
fatty acids and pyruvate. Yet another consequence
of the MRC impairment is to increase the production
of reactive oxygen species (ROS), which can damage
diverse mitochondrial constituents, including mtDNA
[6,1719]. Indeed, a key feature of the mitochondrial
genome is its high sensitivity to oxidative damage,
because of its proximity to the inner membrane (the
main cellular source of ROS, as discussed in the next
section), the absence of protective histone and an
incomplete repertoire of DNA repair enzymes within the
mitochondria [6,20,21].
The respiratory chain as a source of ROS
As pointed out previously, most of the electrons provided
by FADH
2
or NADH to the MRC migrate all the way
along the different MRC components, to nally reach
cytochrome c oxidase (COX, also referred to as complex
IV), where they safely combine with oxygen and protons
to form water (Figure 1). This leads to oxygen consump-
tion, which can be measured by a Clark-type electrode or
oxygen-sensitive probes, as mentioned later on. How-
ever, at several upstream sites of the MRC (most probably
complexes I and III), a small fraction of these electrons
can directly react with oxygen, to generate the super-
oxide anion radical. This radical is then dismutated
by the mitochondrial manganese superoxide dismutase
(MnSOD) into hydrogen peroxide (H
2
O
2
), which is
detoxied into water by the mitochondrial glutathione
peroxidase. Hence, in the normal (non-diseased) state,
most of the ROS generated by the MRC is detoxied by
the mitochondrial antioxidant defences.
However, this detoxication process can be over-
whelmed in some circumstances. One such circumstance
is the depletion of mitochondrial, reduced glutathione.
Normally, glutathione peroxidase plays a key role in
H
2
O
2
detoxication, as liver mitochondria do not
have catalase. However, glutathione peroxidase needs
adequate amounts of reduced glutathione in the mito-
chondrial matrix to detoxify H
2
O
2
. The depletion of
mitochondrial glutathione below a critical threshold can
impair mitochondrial H
2
O
2
detoxication, which can
trigger mitochondrial dysfunction, mitochondrial perme-
ability transition (MPT) and cell death [22].
Mitochondrial antioxidant enzymes can also be
overwhelmed when electron transfer within the MRC is
chronically hindered. A partial block in the ow of
electrons within the MRC leads to their accumulation
within MRC complexes. At some sites of complex I and
complex III, the accumulated electrons directly react
with oxygen to form the superoxide anion radical [23].
When mitochondrial dysfunction is prolonged, the
enhanced formation of the superoxide anion can over-
whelm the mitochondrial antioxidant defences. High
steady-state levels of ROS then damage OXPHOS pro-
teins, cardiolipin and mtDNA. This oxidative damage
aggravates mitochondrial dysfunction to further aug-
ment electron leakage and ROS formation, thus leading
to a vicious circle [2,6].
The MPT pore
The MPT pore is located at contact sites between
the outer and inner mitochondrial membranes. The pore
may be composed of several proteins, including the
voltage-dependent anion channel (VDAC) located in the
outer membrane, the adenine nucleotide translocator
(ANT) (also called ADP/ATP translocase) in the inner
mitochondrial membrane and cyclophilin D in the
mitochondrial matrix (Figure 1).
338 G. Labbe et al.
In the normal state, MPT pores are kept closed, but in
some circumstances they rapidly open, thus increasing
the permeability of the mitochondrial membranes to
compounds, the molecular weight of which is <1.5 kDa.
As a result, the transmembrane potential (Dw
m
) collapses
because of massive proton re-entry, and the mitochon-
drial matrix expands because of water accumulation,
leading to the rupture of the outer membrane and the
release of pro-apoptotic proteins from the intermembrane
space into the cytosol. The release of apoptosis-inducing
factor (AIF) triggers the fragmentation of large-
sized nuclear DNA. The release of a protein called
second mitochondria-derived activator of caspase/
direct inhibitor of apoptosis-binding protein with low pI
(Smac/DIABLO) inactivates the inhibitor of apoptosis
proteins (IAPs). Finally, the released cytochrome c binds
in the cytosol to Apaf-1 in an ATP-dependent manner to
activate caspase-9. Importantly, cytochrome c, which is
loosely bound to the outer part of the inner membrane, is
a key component of the MRC (Figure 1). Therefore, its
leakage from the mitochondria eventually impairs
electron transfer within the MRC and enhances ROS
generation.
Mitochondrial permeability transition can cause either
apoptosis or necrosis. When MPT occurs in some
mitochondria only, the unaffected organelles synthesize
ATP, thus avoiding necrosis, while the permeabilized
mitochondria release pro-apoptotic substances that trig-
ger apoptosis. In contrast, when MPT occurs in most
mitochondria in a single cell, ATP generation is com-
promised because of the collapse of the Dw
m
, and the
resulting ATP depletion triggers cell necrosis [2,24].
Mitochondrial permeability transition pore opening,
which is calcium-dependent, can be triggered by a
variety of endogenous compounds such as iron, ROS,
nitric oxide, free fatty acids (and acyl-CoA derivatives),
ceramide and bile salts [25,26]. MPT can also be
triggered by extracellular cytokines such as tumour
necrosis factor-a (TNF-a) and Fas ligand (Fas-L) acting
through their plasma membrane receptors [27,28].
Finally, MPT can be triggered by drugs, as discussed in
the next section.
MAI N MECHANI SMS AND
CONSEQUENCES OF DRUG- I NDUCED
HEPATI C MI TOCHONDRI AL
DYSFUNCTI ON
Drugs (or their metabolites) can induce mitochondrial
dysfunction in hepatocytes through several mechanisms.
Induction of MPT and cytochrome c release
Several drugs can trigger MPT in liver mitochondria,
leading to apoptosis and/or necrosis. These drugs cause
cytolytic hepatitis, leading occasionally to fulminant
hepatic failure. Some drugs (or metabolites) listed in
Table I can directly trigger MPT in isolated mitochondria
[2,2931]. The molecular mechanisms whereby these
drugs induce MPT are often poorly understood.
Other drugs (or metabolites) can induce the release
of pro-apoptotic proteins from mitochondria through
indirect mechanisms. For instance, troglitazone and
acetaminophen activate c-Jun N-terminal protein kinase
(JNK), thus inducing the cleavage of Bid, the transloca-
tion of this pro-apoptotic protein to the outer mitochon-
drial membranes and the release of cytochrome c from
mitochondria [32,33]. Another mechanism is seen
during the extensive formation of reactive metabolites,
which increases cytosolic calcium that then enters into
the mitochondria to trigger MPT and apoptosis [34].
FAO impairment
Drugs can also impair the mitochondrial oxidation of
fatty acids, leading to their accretion as triglycerides in
the cytoplasm of the hepatocytes. Although free fatty
acids can also accumulate, this seems to happen
preferentially when FAO is severely inhibited [3]. FAO
can be impaired in different ways. Some drugs directly
impair mitochondrial FAO by inhibiting FAO enzymes
and/or by sequestering the FAO cofactors, L-carnitine
and coenzyme A (Figure 1) [24,35]. Other drugs rst
inhibit MRC activity either directly or as a consequence
of detrimental effects on mtDNA replication and/
or integrity (Figure 1) [24]. Severe MRC inhibition
decreases the regeneration of the oxidized cofactors
(NAD
+
and FAD), which are required for b-oxidation.
Table I Examples of drugs able to directly induce the opening of the
MPT pore.
Drug
a
Therapeutic class
Acetaminophen-derived NAPQI
b
Antalgic drug
Alpidem Anxiolytic drug
Diclofenac Nonsteroidal anti-inammatory
drug (NSAID)
Nimesulide NSAID
Salicylic acid NSAID
Troglitazone Antidiabetic drug (PPARc agonist)
Valproic acid Antiepileptic drug
a
Drugs are listed by alphabetic order.
b
N-Acetyl-p-benzoquinone imine.
Thus, severe MRC inhibition secondarily inhibits mito-
chondrial b-oxidation.
Severe inhibition of mitochondrial b-oxidation can
lead to microvesicular steatosis characterized by the
presence of many tiny lipid droplets within the cytoplasm
of hepatocytes. This potentially severe liver lesion can be
associated with liver failure, profound hypoglycaemia
and encephalopathy [24]. Microvesicular steatosis can
also lead to the death of the patient. Examples of drugs
capable of inducing microvesicular steatosis are given in
Table II [24].
However, a less severe (and more prolonged) inhibi-
tion of mitochondrial b-oxidation can cause macrovac-
uolar steatosis, which is characterized by the presence
of a single, large lipid vacuole within the cytoplasm of
the hepatocytes. Although macrovacuolar steatosis is a
benign liver lesion in the short term, it can evolve in
some patients towards steatohepatitis after several years.
In this more severe condition, steatosis is associated with
necrosis, apoptosis, inammation and brosis, which can
lead to cirrhosis in some patients [2,4]. Examples of
drugs capable of inducing macrovacuolar steatosis and
steatohepatitis are listed in Table III [2,4,3639]. Inter-
estingly, drugs inducing steatohepatitis are usually com-
pounds that impair the MRC (Figure 1), thus causing not
only FAO impairment and steatosis, but also enhanced
ROS production. ROS overproduction may be a key event
in the pathogenesis of drug-induced steatohepatitis by
inducing lipid peroxidation (favoured by lipid accumu-
lation), and possibly by triggering the generation of pro-
apoptotic (TNF-a) and pro-brotic (TGF-b) cytokines by
Kupffer cells and other inammatory cells [2,4,40].
Regarding drug-induced steatosis and steatohepatitis,
several important remarks must be made.
1 Some drugs can acutely induce microvesicular steato-
sis in some patients, and can also cause subacute
or chronic macrovacuolar steatosis or steatohepatitis
in other patients (Tables II and III). It is tempting to
speculate that such drugs may be able to severely impair
mitochondrial FAO in a few patients (possibly those who
may have an underlying mitochondrial dysfunction,
as discussed later on), thus leading to microvesicular
steatosis. In other patients, however, a less severe and
more prolonged impairment of FAO may instead cause
macrovacuolar steatosis.
2 Some drugs can inhibit mitochondrial respiration
through both direct and indirect mechanisms. For
instance, tamoxifen directly inhibits the transfer of
electrons within the MRC, and also progressively depletes
hepatic mtDNA through a mechanism that may involve
the inhibition of mitochondrial topoisomerases and the
impairment of mtDNA replication by this DNA-inter-
calating drug [41,42].
3 By severely inhibiting FAO (either directly or indirectly
through an impairment of the MRC), drugs can deplete
ATP stores within the hepatocytes and lead them to cell
death.
4 Although drug-induced FAO impairment is a key
mechanism leading to steatosis, some drugs (e.g. amio-
darone, amineptine, pirprofen) also inhibit microsomal
triglyceride transfer protein (MTP), an enzyme playing a
major role in the formation of very-low-density-lipopro-
tein (VLDL) particles [43]. Such drugs therefore inhibit
the two main pathways removing fat from the liver,
namely FAO and hepatic VLDL secretion.
Table III Examples of drugs capable of inducing macrovacuolar
steatosis and steatohepatitis.
Drug
a
Therapeutic class
Amiodarone Anti-anginal, anti-arrhythmic drug
Irinotecan Antineoplastic drug (colorectal cancer)
Methotrexate Antipsoriatic, anti-rheumatoid drug
NRTIs
b
(AZT, ddI, d4T) Antiretroviral (anti-HIV) drug
Perhexiline Anti-anginal drug
Tamoxifen Antineoplastic drug (breast cancer)
Toremifene Antineoplastic drug (breast cancer)
a
b
Nucleoside reverse transcriptase inhibitors.
Table II Examples of drugs capable of inducing microvesicular
steatosis.
Drug
a
Therapeutic class
Amineptine Antidepressant drug
2-Arylpropionic acids
(pirprofen, ibuprofen)
Nonsteroidal anti-inammatory
drug (NSAID)
Aspirin (and salicylic acid) NSAID
Fialuridine (FIAU) Antiviral (anti-HBV) drug
NRTIs
b
(AZT, ddI, d4T) Antiretroviral (ant-HIV) drug
Panadiplon Anxiolytic drug
Tetracycline and its derivatives
(high doses)
Antibiotics
Tianeptine Antidepressant drug
a
b
340 G. Labbe et al.
OXPHOS uncoupling
Cationic amphiphilic drugs such as amiodarone, per-
hexiline, diethylaminoethoxyhexestrol (DEAEH) and
tamoxifen can be rst protonated in the intermembrane
space of mitochondria, and then electrophoretically
transported into the mitochondrial matrix thanks to
the Dw
m
[3,4,40,42], as discussed in greater detail
below. This transfer across the inner mitochondrial
membrane dissipates Dw
m
(Figure 1) and increases the
basal (state 4) mitochondrial respiration. This phe-
nomenon, which is called OXPHOS uncoupling, can
also be observed in the case of some mitochondrial
poisons such as the protonophores 2,4-dinitrophenol
(DNP) and carbonylcyanide p-triuoromethoxy-
phenylhydrazone (FCCP). Importantly, the long-lasting
increase in mitochondrial respiration induced by ami-
odarone, perhexiline or tamoxifen can be observed in
isolated liver mitochondria incubated with relatively
low concentrations (20100 lM) of these drugs
[3,40,42]. However, for higher concentrations, their
intramitochondrial accumulation induces a rapid inhi-
bition of electron transfer within the MRC, so that the
transient increase in state 4 is followed by a progres-
sive decrease in mitochondrial respiration [3,40,42].
Finally, it must be pointed out that another important
consequence of drug-induced OXPHOS uncoupling is
the reduction of ATP synthesis, as the re-entry of
protons within the mitochondrial matrix bypasses ATP
synthase [3,40].
Multiple mechanisms
Several drugs impair mitochondrial function via several
of the above-mentioned pathways. For instance,
valproic acid inhibits mitochondrial FAO through the
sequestration of coenzyme A (a cofactor mandatory for
FAO), and possibly also through the inactivation
of b-oxidation enzymes by an electrophilic metabolite
[2,3]. In addition, this antiepileptic drug can induce
MPT (Figure 1) [29], which may explain why valproate-
induced microvesicular steatosis can be associated with
liver cell death [2].
DRUG WI THDRAWAL BECAUSE OF
MI TOCHONDRI AL DYSFUNCTI ON AND
LI VER I NJ URY
In the past 20 years, the development of several drugs
was halted during clinical trials or they were withdrawn
denitively or temporarily from the market because of
mitochondrial dysfunction-associated DILI (Table IV). For
example, aluridine (FIAU) induced unmanageable lactic
acidosis, microvesicular steatosis and severe liver failure
requiring liver transplantation or leading to death, in
several patients receiving this anti-hepatitis B virus
(HBV) nucleoside analogue during clinical trials [3,44].
After interruption of these trials, new in vivo and in vitro
investigations showed that aluridine strongly inhibits
DNA polymerase c and triggers mtDNA depletion by an
unusual mechanism [44,45]. Unlike nucleoside reverse
transcriptase inhibitors (NRTIs) such as stavudine (d4T),
zidovudine (AZT) or didanosine (ddI), the incorporation
of which into a growing chain of mtDNA interrupts
mtDNA replication, aluridine can be incorporated into
mtDNA without immediately terminating mtDNA repli-
cation. Indeed, this drug carries a hydroxyl group on the
3 carbon of the sugar moiety, allowing the subsequent
incorporation of other nucleotides [46]. However, when
several adjacent molecules of aluridine are successively
incorporated into a growing chain of mtDNA, DNA
polymerase c is strongly inhibited, blocking further
mtDNA replication (Figure 1) [45].
Panadiplon is a non-benzodiazepine anxiolytic drug,
the development of which was interrupted during phase I
clinical trials because of increased serum transaminase
activity [2,47]. Subsequent studies showed that pana-
diplon can be converted into cyclopropane carboxylic
acid, which sequesters coenzyme A and L-carnitine, two
cofactors required for mitochondrial FAO (Figure 1).
Hence, panadiplon inhibits mitochondrial FAO and
causes microvesicular steatosis (as well as necrosis) in
Dutch-belted rabbits [47]. As discussed later on, pre-
clinical safety investigations in rats, dogs and monkeys
treated with panadiplon revealed good tolerance,
although microvesicular steatosis was observed in mon-
keys [47].
Table IV Examples of drugs, the potential of which to cause
mitochondrial dysfunction and DILI has led to the interruption of
clinical trials, or their withdrawal after marketing.
Drug
a
Therapeutic class
Drug withdrawn during clinical trials
Fialuridine Antiviral (anti-HBV) drug
Panadiplon Anxiolytic drug
Drug withdrawn after marketing
Alpidem Anxiolytic drug
Pirprofen Nonsteroidal anti-inammatory drug (NSAID)
Troglitazone Antidiabetic drug (PPARc agonist)
a
Drugs are listed by alphabetic order within each subgroup.
The anxiolytic drug, alpidem, has been removed from
the market after it led to several cases of severe,
sometimes fatal, hepatitis [48]. Investigations sub-
sequently showed that this peripheral benzodiazepine
receptor (PBR) ligand can accelerate MPT in rat liver
mitochondria and increase the toxicity of TNF-a in rat
hepatocytes [2,49].
Perhexiline is an anti-anginal drug, the administration
of which could cause steatohepatitis that was often
severe, leading to cirrhosis and death of some patients
[3,4,37]. Consequently, perhexiline has been withdrawn
from the market, although it has recently been suggested
that therapeutic plasma monitoring could perhaps
rehabilitate the drug [50]. Similar to amiodarone,
perhexiline accumulates in mitochondria, where it
inhibits both FAO (thus causing steatosis) and mito-
chondrial respiration (thus increasing the mitochondrial
formation of ROS and favouring lipid peroxidation)
[40,51]. Perhexiline and amiodarone accumulate within
mitochondria thanks to Dw
m
[3,52]. Indeed, these
amphiphilic drugs are protonated within the acidic
intermembrane space and pulled into the mitochondrial
matrix, which is basic and negatively charged. In the
more alkaline matrix, the protonated perhexiline or
amiodarone molecule frees its proton, so that the net
result of the import of one drug molecule into the matrix
is to transfer one proton from the intermembrane space
into the matrix. This abnormal entry of protons bypasses
the F
0
portion of ATP synthase, thus uncoupling
OXPHOS and decreasing ATP production. However, as
perhexiline and amiodarone progressively accumulate
within the mitochondria, they soon reach high concen-
trations, which inhibit FAO enzymes and also electron
transfer within the MRC [3,5154]. Hence, after a
transient stimulation caused by uncoupling, mitochon-
drial respiration is progressively inhibited in mito-
chondria incubated with perhexiline or amiodarone
[3,51,52].
Pirprofen, a nonsteroidal anti-inammatory drug
(NSAID), was removed from the market after it had
caused several instances of liver lesions combining both
severe cytolytic hepatitis and microvesicular steatosis
[55]. This aryl-propionate drug was later shown to
inhibit mitochondrial b-oxidation [56] and MTP activity
[43], decreasing both the oxidation of fat within
hepatocytes and the secretion of fat from the liver,
respectively.
Troglitazone, a peroxisome proliferator-associated
receptor-c agonist, has been removed from the market
after the occurrence of several cases of acute liver failure,
which were fatal or required liver transplantation in
some patients [2,57]. In vitro investigations sub-
sequently showed that this antidiabetic drug induces
MPT in mouse liver mitochondria and inhibits ADP-
driven respiration (with glutamate and malate as sub-
strates) in rat liver mitochondria [31,58]. In addition,
troglitazone is a potent inhibitor of complex IV and V
activities in bovine heart mitochondria [58]. As men-
tioned previously, investigations also showed that trog-
litazone-induced liver cell death could also involve JNK
activation [32].
Several other drugs can induce liver toxicity
through mitochondrial dysfunction, but still have a
favourable benetrisk ratio, at least in some circum-
stances. Although these drugs have not been withdrawn
from the market, their use has been restricted by diverse
Black Box warnings added at the request of drug
agencies (Table V).
PREDI SPOSI TI ONS TO DRUG- I NDUCED
MI TOCHONDRI AL DYSFUNCTI ON AND
I DI OSYNCRATI C DI LI
Drugs are only considered for marketing when they do
not cause DILI, or rarely cause DILI. This selection
ensures that DILI only affects a minority of the treated
patients. Otherwise, the compound would not have been
marketed. At the moment, we still poorly understand
which susceptibility factor(s) may explain the higher
susceptibility of these few subjects. However, it is already
clear that genetic, metabolic and environmental factors
that impair mitochondrial function can add their effects
to those of drugs capable of causing mitochondrial
dysfunction. The added effects of the pre-existing con-
dition and the mitochondria-targeting drug can then
Table V Examples of marketed drugs capable of inducing hepato-
toxicity caused by mitochondrial dysfunction, which have received
Black Box warnings from drug agencies.
Drug
a
Therapeutic class
Benzbromarone Uricosuric drug
Buprenorphine Opioid agonist/antagonist
NRTIs
b
(AZT, d4T, ddI) Antiretroviral (anti-HIV) drug
Tamoxifen Antineoplastic drug (breast cancer)
Tolcapone Anti-Parkinson drug
a
b
342 G. Labbe et al.
alter mitochondrial function to reach the low threshold,
under which hepatic manifestations start to occur.
Genetic predisposition
Several studies reported fatal valproate hepatotoxicity in
patients suffering from an underlying genetic mitochon-
drial disease, such as cytochrome c oxidase deciency
or medium-chain acyl-CoA dehydrogenase (MCAD)
deciency [5961]. MCAD is a key FAD-dependent
dehydrogenase involved in the oxidation of medium-
chain (C
6
C
12
) fatty acids within the mitochondria [3].
MCAD deciency is a frequent metabolic disorder of
genetic origin among Caucasians. Indeed, the carrier
frequency of the A985G mutation (which is the most
frequent mutation leading to MCAD deciency) ranges
from approximately one in 55 in northern Europe to one
in 140 in southern Europe [6264]. Because of this high
frequency, it may be worth determining whether the
MCAD carrier state is sufcient or not to increase the risk
of DILI in patients receiving drugs that impair mito-
chondrial function.
Although the report did not mention liver injury, a
mutation in the catalytic subunit of DNA polymerase c
was found in a patient with stavudine-induced hyper-
lactataemia [65]. Interestingly, the mutation markedly
decreased DNA polymerase activity, and rendered lym-
phoblastoid cells more sensitive to stavudine-induced
mtDNA depletion [65]. Thus, it would be interesting to
look for this mutation in patients with NRTI-induced
liver injury.
It must be also pointed out that there is a large
variation of mtDNA copy number in liver or muscle
among different individuals [66,67]. Although the origin
of this variation is currently unknown, low basal mtDNA
levels could perhaps act as a predisposing factor to the
toxicity of NRTIs or other mtDNA-altering drugs.
A recent study showed that individuals with the
C allele (T/C or C/C genotype) of MnSOD had a higher
risk of experiencing DILI with anti-tuberculosis drugs
[68]. Carrying the C/C genotype (i.e. being homo-
zygous for alanine in the mitochondrial targeting
sequence of MnSOD) was also found to greatly enhance
the risk of microvesicular steatosis or cirrhosis in French
alcoholics [69]. Furthermore, the alanine-encoding
allele also increased the risk of hepatocellular carci-
noma development in alcoholic cirrhotic patients [70].
The Ala-MnSOD precursor variant is more efciently
imported into the mitochondria than the Val-MnSOD
precursor variant, resulting in a higher mitochondrial
activity of the MnSOD homotetramer derived from the
Ala-MnSOD precursor [71,72]. MnSOD transforms the
superoxide anion into H
2
O
2
, while alcohol intoxication
can decrease hepatic mitochondrial glutathione, which
is required for the detoxication of H
2
O
2
by glutathione
peroxidase. Therefore, in alcoholics with the Ala-
MnSOD precursor variant, a high MnSOD activity
combined with a low glutathione peroxidase activity
could increase the cellular steady-state levels of
H
2
O
2
, leading to an increased formation of the highly
reactive hydroxyl radical and to liver injury and cancer
[73].
Environmental factors
Environmental factors such as alcohol, microsomal
enzyme inducers or viral infections can enhance the
risk of drug-induced mitochondrial dysfunction and
DILI. Ethanol overconsumption causes numerous dele-
terious effects on liver mitochondria, in particular on
the mitochondrial genome [3,74,75]. Alcohol abuse
may therefore induce an underlying mitochondrial
dysfunction, which may render the liver more vulner-
able to drug-induced mitochondrial dysfunction and
DILI. Surprisingly, this susceptibility factor has been
insufciently addressed in the literature, due in part to
the difculty of disentangling alcohol- and drug-induced
injury in some circumstances. Nevertheless, it is known
that the hepatotoxicity of methotrexate and that of
paracetamol is increased in alcoholics [76]. In addition
to causing mitochondrial alterations, ethanol intoxica-
tion also increases cytochrome P450 2E1 (CYP2E1)
levels, not only in the endoplasmic reticulum but
also in mitochondria [77]. The ectopic localization of
CYP2E1 may enhance the mitochondrial generation of
N-acetyl-p-benzoquinone imine (NAPQI), the toxic
metabolite of acetaminophen. As the reactive NAPQI
has a direct toxicity effect on mitochondria, in partic-
ular by triggering MPT [78,79], the generation of
NAPQI directly within the mitochondria may favour
acetaminophen-induced mitochondrial dysfunction and
liver injury.
Mitochondria contain not only CYP2E1 but also other
inducible CYPs, such as CYP1A1 and CYP2B1 [80,81].
Thus, the concomitant administration of a CYP-inducer
(such as phenobarbital or phenytoin) may increase the
mitochondrial CYP-mediated generation of reactive
metabolites in subjects co-treated with some drugs, such
as valproic acid [76,82].
Viral infections may also be a predisposing factor
to drug-induced mitochondrial dysfunction in the liver
[76]. Viral infections can release viral proteins, NO,
cytokines and interferon, which can impair mitochon-
drial function and lipid homeostasis, and thus could have
additive effects with drugs inducing mitochondrial dys-
function [5,6,76]. An example is the combined role of
aspirin and viral infections in the occurrence of Reyes
syndrome, a severe post-infectious metabolic disease
characterized by extensive microvesicular steatosis of the
liver, severe hypoglycaemia and a non-inammatory
encephalopathy [3,83]. Although therapeutic doses of
aspirin are well tolerated in uninfected children, they can
trigger Reyes syndrome in a few children sensitized by
varicella or inuenza.
Another example is the hepatic toxicity of some NRTIs
used to prevent the replication of the human immuno-
deciency virus (HIV) (Tables II and III). The hepatic
toxicity of NRTIs is increased in patients co-infected with
either the hepatitis B virus (HBV) or the hepatitis C virus
(HCV) [6,84]. Some HBV or HCV proteins such as the
HCV core protein or the HBV X protein (HBx) can disturb
mitochondrial function [8587]. Together with cyto-
kines, these viral proteins could impair mitochondrial
function and make co-infected subjects more susceptible
to NRTI-induced liver toxicity.
Obesity and NAFLD
A recent surge in the prevalence of obesity threatens the
health and life of millions of individuals, as obesity
increases the risk of type 2 diabetes, hyperlipaemia,
coronary heart disease, stroke, osteoarthritis and cancer.
Furthermore, obesity can cause non-alcoholic fatty liver
disease (NAFLD) [13,88], which can make the liver more
vulnerable to some forms of DILI, and to alcohol-induced
liver injury [38,8993]. This increased vulnerability
could involve several mechanisms, including an under-
lying mitochondrial dysfunction, an oxidative stress, and
an overproduction of cytotoxic cytokines such as tumour
necrosis factor (TNF)-a in NAFLD [13,88,9496]. As the
number of treated overweight or obese patients is
increasing, clinical investigations are urgently needed
to establish which drugs have an increased hepatotoxic
risk in patients with NAFLD.
METHODS TO I NVESTI GATE
DRUG- I NDUCED MI TOCHONDRI AL
DYSFUNCTI ON I N LI VER
Among the battery of in vitro and in vivo investigations
that are presented in this section, some of them can be
performed during the preclinical safety studies to detect
drug-induced hepatotoxicity linked to mitochondrial
dysfunction. These methods allow us to assess the
capacity of a drug to inhibit or uncouple mitochondrial
respiration, to impair FAO, to trigger MPT or to deplete
mtDNA. Importantly, some of these investigations can
also be carried out before preclinical studies in order to
predict mitochondrial dysfunction, thus allowing the
selection of safer compounds for subsequent develop-
ment. Finally, investigational studies can be also per-
formed after clinical trials, or marketing, in order to
explain the occurrence of hepatotoxic events in some
individuals.
The in vitro assays
Several in vitro assays can be used to detect drug-
induced mitochondrial dysfunction either in hepatic cells
or in isolated liver mitochondria (Table VI). Although
technical details are not given in the present review,
some important issues are addressed.
1 When the drug is highly bound to plasma proteins, it is
convenient to perform these in vitro investigations in the
presence of a physiological concentration of albumin
[97]. This allows a direct comparison of the concentra-
tions that impair mitochondrial function in vitro with
the plasma levels in treated patients [97].
2 A very convenient method is to assess the effects of
drugs on FAO, respiration and MPT rst in isolated rat or
mouse liver mitochondria, and then to check the effects
Table VI Examples of in vitro investigations which can be
performed to detect drug-induced mitochondrial dysfunction.
Investigations on isolated liver mitochondria
Assessment of fatty acid oxidation (FAO) with radiolabelled fatty acids
Measurement of oxygen consumption with a Clark-type electrode
Measurement of mitochondrial respiratory chain (MRC) complexes activities
by spectrophotometry
Determination of Dw
m
(mitochondrial transmembrane potential) with the
tetraphenylphosphonium chloride selective electrode, or by ow cytometry
with a uorescent probe (e.g. TMRM
a
), or by spectrouorometry with a
uorescent probe (e.g. safranine)
Assessment of mitochondrial permeability transition (MPT) pore opening by
spectrophotometry (OD recording at 540 nm)
Investigations on hepatic cells
Coloration with oil red O (for the detection of neutral lipids)
Measurement of lactic acid in the incubation medium
Assessment of FAO with radiolabelled fatty acids
Measurement of oxygen consumption with a Clark-type electrode
Determination of Dw
m
(mitochondrial transmembrane potential) by ow
cytometry with a uorescent probe (e.g. MitoTracker Red CMXRos, DiOC6
b
)
Assessment of mtDNA levels by qPCR
a
Tetramethylrhodamine methyl ester.
b
3,3-Dihexyloxacarbocyanine iodide.
344 G. Labbe et al.
of the drug in human hepatoma cell line [97] or in
human lymphocytes. Human lymphocytes can be indeed
used as a useful model to assess the ability of a drug to
inhibit FAO or deplete ATP stores in human cells [98].
3 When using hepatoma cells, it must be remembered
that most hepatic cell lines express very low (or nil) levels
of enzymes involved in drug metabolism such as CYPs
or glutathione S-transferases (GSTs), possibly due to
low expression of the nuclear receptors regulating the
expression of these enzymes such as the constitutive
androstane receptor (CAR) and pregnane X receptor
(PXR) [99,100]. This major limitation of most hepatoma
cells should be taken into consideration during preclin-
ical safety studies, as mitochondrial dysfunction can be
induced by reactive metabolites and not by the parent
drugs [101]. Hence, negative results regarding mito-
chondrial dysfunction in HepG2 cells should be inter-
preted only as indicating a lack of toxicity of the parent
compound. In contrast, the human hepatoma cell line
HepaRG expresses substantial levels of different CYPs,
GSTs and nuclear receptors [99,100], and could there-
fore be preferred for toxicological studies.
4 Hepatoma cell lines cultivated in high-glucose media
(i.e. 25 mM) generate ATP mostly from glycolysis rather
than from mitochondrial OXPHOS. The reliance of
HepG2 cells on glycolysis decreases their vulnerability
to drugs altering mitochondrial function or to OXPHOS
poisons, such as rotenone, antimycin, oligomycin or
FCCP [102]. However, when HepG2 cells are cultivated
with galactose (10 mM) and glutamine (2 mM) for more
than 10 passages, their susceptibility to mitochondria-
targeting compounds increases [102]. Because the
oxidation of galactose to pyruvate does not cause any
net ATP generation, cultivating HepG2 cells with this
substrate progressively forces cells to rely more on
mitochondrial ATP generation [102].
5 Hepatoma cell lines (such as HepG2, H4IIE and Fao)
also present a very poor capacity to oxidize long-chain
fatty acids (LCFAs), such as palmitic acid and oleic acid,
possibly because of high levels of malonyl-CoA, an
endogenous inhibitor of carnitine palmitoyltransferase I
(CPT-I), which catalyses the limiting step for LCFA
oxidation within the mitochondria (Figure 1) [103,104].
We recently conrmed these results on HepG2 cells and
showed that HuH7 and Chang Liver cells also have a
poor capacity to oxidize LCFAs [105]. Thus, these
hepatoma cell lines are not suitable to explore the effect
of drugs on mitochondrial LCFA oxidation. It would be
interesting to determine whether the mitochondrial
oxidation of LCFAs can be improved if these cells are
maintained in a galactose medium. In contrast, the
mitochondrial oxidation of medium-chain fatty acids,
such as octanoic acid, seems active in hepatoma cells
[104].
6 Caution is needed when mitochondrial function is
investigated in cryopreserved/thawed hepatocytes.
Indeed, a recent study showed profound mitochondrial
dysfunction in cryopreserved/thawed human and mouse
hepatocytes [106]. In particular, ATP levels and mito-
chondrial respiration with glutamate/malate, which give
electrons to complex I, were severely decreased. More-
over, electron microscopy showed swollen mitochondria
and partial loss of cristae [106]. Uncontrolled damage to
mitochondria and other organelles in cryopreserved/
thawed hepatocytes is likely to alter the vulnerability
of cells to drugs. Hence, further studies are needed to
improve the conditions of cryopreservation and thawing,
and determine whether cryopreserved/thawed hepato-
cytes can be valuable tools to assess the effects of drugs
on mitochondrial function.
7 Besides isolated mitochondria, it is also possible to
investigate electron transfer along the MRC thanks to
submitochondrial particules (SMPs). SMPs are prepared
from isolated mitochondria by sonication, and consist of
inverted, vesicular portions of the inner mitochondrial
membrane [107]. With SMPs, NADH can be used as an
electron donor to complex I instead of the glutamate/
malate substrates usually employed. Hence, one can take
advantage of this feature if one suspects that a drug can
inhibit the substrate carriers of the inner mitochondrial
membrane, or enzymes involved in substrate processing
and NADH generation. For instance, studies have shown
that valproic acid inhibits pyruvate and succinate
transport as well as glutamate dehydrogenase activity
[3].
8 Finally, it is important to mention that investigations
are currently being performed in order to set up and
validate high-throughput assays for mitochondrial
toxicity screening. Yvonne Will and colleagues have
developed high-throughput-applicable screens based on
luminescent, oxygen-sensitive probes and immunocap-
ture-based MRC complex assays, which allow the assess-
ment of oxygen consumption in isolated mitochondria,
or the measurement of the activities of different MRC
complexes, respectively [58,108,109].
Plasma metabolites and enzymes
In vivo investigations aimed at detecting the toxicity of a
drug on liver mitochondria may include the measure-
ment of several metabolites and enzymes in plasma
(Table VII). Regarding plasma chemistry, the European
Medicines Agency (EMEA) recommends the determina-
tion of lactate levels, as well as glutamate dehydrogenase
(GLDH) and ornithine carbamoyltransferase (OCT) activ-
ities [110]. GLDH and OCT are mitochondrial enzymes.
An increased plasma activity of these enzymes reects
structural damage to the mitochondria and cell mem-
brane, leading to the leakage of these enzymes into the
plasma [111,112]. OCT is particularly expressed in the
liver [111]. Therefore, a high plasma OCT activity can
occur when mitochondrial damage has specically
caused liver injury.
Elevated lactate (and/or pyruvate) levels indicate an
insufcient oxidation of pyruvate by mitochondria. This
may not only be due to drug-induced mitochondrial
dysfunction, but also to many other causes, including
anoxia and/or shock. It must be also pointed out that
hyperlactatemia is not strictly specic to mitochondrial
dysfunction in liver. We therefore also suggest measur-
ing the two plasma ketone bodies, b-hydroxybutyrate
and acetoacetate. The b-hydroxybutyrate/acetoacetate
ratio mostly reects the NADH/NAD
+
ratio present in
hepatic mitochondria [3,113], and therefore provides
indirect information on the activity of the hepatic MRC.
When the hepatic MRC is inhibited, the insufcient re-
oxidation of NADH into NAD
+
increases the NADH/
NAD
+
ratio in the liver. The increase in NADH, in turn,
favours the reduction of acetoacetate into b-hydroxybu-
tyrate by b-hydroxybutyrate dehydrogenase, a mito-
chondrial enzyme mainly expressed in the liver
[114,115]. The b-hydroxybutyrate/acetoacetate ratio
therefore increases when the MRC is inhibited in the liver
not only at the level of complex I but also downstream
to this site [116].
In addition to the b-hydroxybutyrate/acetoacetate
ratio, one should also consider the sum of b-hydroxybu-
tyrate and acetoacetate levels in plasma. FAO-inhibiting
drugs can diversely affect the plasma levels of ketone
bodies. Some FAO inhibitors such as valproic acid,
ibuprofen and pirprofen decreased, as expected, plasma
ketone bodies in rodents [3,56,117]. However, other
hepatic FAO inhibitors, such as aspirin, amineptine,
tianeptine, tetracycline or amiodarone instead aug-
mented plasma ketone bodies in rodents [3,118120].
Although the mechanism behind this paradoxical
increase has not been determined, it has been suggested
that the latter drugs may inhibit the activity of the
tricarboxylic acid cycle in extra-hepatic tissues, which
massively use ketone bodies as an energy source during
fasting [3].
Acyl-carnitine and acyl-glycine derivatives can be also
assessed in blood or urine, as their augmentation can
reect an inhibition of mitochondrial FAO within the
liver [121]. For instance, rats or mice treated with
valproic acid had increased levels of acyl-carnitine
derivatives in plasma (or serum) and urine [122,123].
However, it must be kept in mind that abnormal acyl-
carnitine proles can be also the secondary consequence
of an impaired MRC activity [124].
Liver histology and electron microscopy
Liver histology can also give precious information
regarding the mitochondrial origin of DILI (Table VII).
For instance, the presence of microvesicular steatosis is
highly suggestive of a strong inhibition of mitochondrial
FAO [3,4,56,117120,123]. It is noteworthy, however,
that pure microvesicular steatosis is rare, and that this
lesion is often accompanied by macrovacuolar steatosis.
The presence of steatohepatitis can also suggest drug-
induced mitochondrial dysfunction. However, investiga-
tions should be performed to test the possibility that this
liver lesion could be due also (or instead) to an increased
hepatic de novo lipogenesis, such as that triggered by
insulin resistance in a context of body fatness [13,88]. It
is important to mention that drugs can indirectly impair
mitochondrial FAO in liver and cause steatosis (or
steatohepatitis) by inducing lipoatrophy (i.e. a reduction
in body fat mass) [125]. Fatty liver (or steatohepatitis)
secondary to lipoatrophy is, at least in part, the con-
sequence of a lower production of leptin by the white
Table VII Examples of in vivo or ex vivo investigations which can
be performed to detect drug-induced mitochondrial dysfunction.
Plasma biochemistry: lactate (and pyruvate), ketone bodies
(b-hydroxybutyrate and acetoacetate), GLDH
a
and OCT
b
activities,
acyl-carnitine derivatives
Urine biochemistry: acyl-carnitine and acyl-glycine derivatives
Histopathology (with oil red O and haematoxylin-eosin staining) and electron
microscopy
Assessment of whole-body fatty acid oxidation (FAO) after administration
of
14
C-labelled fatty acids
Investigations on liver mitochondria isolated from treated animal:
measurement of mitochondrial FAO with
14
C-labelled fatty acids and/or
oxygen consumption with a Clark electrode, assessment of mitochondrial
permeability transition (MPT) pore opening by spectrophotometry
Investigations on liver homogenates prepared from treated animals:
assessment of mtDNA levels and/or activities of different mitochondrial
respiratory chain (MRC) complexes, immunoblot analysis of selected MRC
or FAO polypeptides
a
Glutamate dehydrogenase.
b
Ornithine carbamoyltransferase.
346 G. Labbe et al.
adipose tissue. Leptin insufciency in turn reduces
hepatic FAO and ketogenesis and favours de novo
lipogenesis [125,126].
The presence of apoptosis and necrosis in liver does
not specically imply direct toxicity of the drug on
mitochondria. Indeed, both kinds of cell death can be
induced by other mechanisms such as oxidative stress,
reticulum endoplasmic stress, nitric oxide overproduc-
tion, activation of kinases such as JNK, or alteration of
calcium homeostasis [127130].
Electron microscopy can also provide valuable pieces
of information (Table VII) as it can reveal not only
ultrastructural changes in liver mitochondria (enlarge-
ment or swelling, disruption of cristae), but also the
presence of small lipid droplets within the cytoplasm
suggesting microvesicular steatosis [21,131133]. How-
ever, ultrastructural alterations of mitochondria are not
necessarily a direct consequence of drug toxicity. Indeed,
these alterations can represent collateral effects linked
to the generation of free radicals (and/or lipid peroxida-
tion products) in extra-mitochondrial compartments
[134,135].
Other in vivo or ex vivo investigations
Other investigations can be performed in vivo or ex vivo
(Table VII). For instance, whole-body FAO can be
assessed thanks to the measurement of [
14
C]CO
2
exha-
lation after the administration of
14
C-labelled fatty acids
[3,42,119,120,123]. Interestingly,
14
C-labelled fatty
acids of different lengths can be used to determine
whether FAO inhibition affects the whole oxidative
process or only some chain length-specic processes
[117,119].
Alternatively, mitochondria can be isolated from the
liver of treated animals and immediately used to measure
mitochondrial FAO ex vivo, thanks to
14
C-labelled fatty
acids [3,117,119,120]. Finally, a fraction of the liver can
be frozen and subsequently used to assess mtDNA levels,
the activities of different MRC complexes, and the
expression of selected MRC and FAO proteins by immuno-
blotting [42,125,132,133,136].
Uncommon animal species
Rats and dogs (and sometimes monkeys) are the regu-
lation species commonly used by pharmaceutical com-
panies in preclinical studies. Thus, investigations in any
other animals are not required by drug agencies. Despite
this, uncommon animal species could be valuable tools
for the screenings carried out prior to the preclinical
studies, as they could help select safer drugs. Alterna-
tively, these animal species can help understand a
posteriori the complex ways whereby a marketed drug
induced hepatotoxicity in some patients, in particular,
through mitochondrial dysfunction.
Uncommon animal species such as marmots and
rabbits have been used to test aluridine and panadiplon
(Table IV) [11]. Indeed, aluridine did not induce hepa-
totoxicity in rats, dogs and nonhuman primates, but
liver toxicity similar to that in humans was revealed
when this antiviral drug was administered to wood-
chucks (Marmota monax) [44,132]. Panadiplon was
found to be safe to rats and dogs during preclinical
safety studies. However, this anxiolytic drug induced
microvesicular steatosis and centrilobular necrosis when
Dutch-belted rabbits were used for toxicological investi-
gations performed after the interruption of the clinical
trials [47,137]. Interestingly, preclinical safety investi-
gations in monkeys showed microvesicular steatosis, but
as this lesion was not accompanied with overt signs of
hepatotoxicity the clinical development of panadiplon
was continued [47].
Unfortunately, it is unknown why aluridine and
panadiplon are particularly toxic to woodchucks
and Dutch-belted rabbits, respectively. Owing to their
mechanisms of mitochondrial toxicity (Figure 1), aluri-
dine could be incorporated at a very high rate within
woodchuck hepatic mtDNA and panadiplon could
severely deplete coenzyme A and/or L-carnitine stores
in Dutch-belted rabbit liver. Obviously, these uncommon
animal species cannot be used on a regular basis in
toxicological studies, although they could be used, for
example, for investigational compounds that share
structural or pharmacological analogies with aluridine
or panadiplon. Finally, as discussed in the next section,
the use of genetically modied mice could also improve
our understanding of the complex ways whereby
a marketed drug can induce hepatotoxicity in some
patients, in particular when DILI is mediated by
mitochondrial dysfunction [101,123,138,139]. More
generally, signicant efforts should be devoted to assess-
ing the potential of different animal models in detecting
mitochondrial toxicity-mediated DILI.
FUTURE DI RECTI ONS
A major challenge for pharmaceutical companies in the
future will be to develop drugs, which do not induce
mitochondrial dysfunction in liver and in other tissues
too. Improvement of in vitro screenings by using high-
throughput technologies during lead selection would
certainly help select safer compounds for subsequent
in vivo preclinical safety investigations [7]. Recent
investigations also suggest that better predictions could
be possible thanks to the use of novel animal models
such as the heterozygous MnSOD
+/)
(sod2
+/)
) knockout
mouse [138]. Although sod2
+/)
mice are apparently
normal, they present underlying mitochondrial altera-
tions in liver and other tissues, such as decreased Dw
m
,
reduced MRC activity, and an enhanced propensity to
undergo MPT [140,141]. Interestingly, sod2
+/)
mice
treated with troglitazone developed increased serum
alanine aminotransferase activity and hepatic necrosis
whereas wild-type mice did not [136]. Using the sod2
+/)
mice model can also unmask the mitochondrial toxicity
of nimesulide and stavudine [138,142].
Another murine model with underlying mitochon-
drial/metabolic dysfunction is the juvenile visceral
steatosis (jvs) mouse. Heterozygous jvs
+/)
mice have
decreased L-carnitine stores because of impaired renal re-
absorption of this mitochondrial FAO cofactor, and thus
should be expected to be more susceptible to FAO-
impairing drugs [123]. It would be also helpful to use
MCAD
+/)
mice [143], as partial MCAD deciency is one
of the most frequent metabolic abnormalities of genetic
origin among Caucasians, as previously mentioned.
Finally, because of the increasing prevalence of obesity
and NAFLD, and the propensity of NAFLD, and partic-
ularly non-alcoholic steatohepatitis (NASH), to alter
mitochondrial function [13,88], it could be also valuable
to use rodent models of obesity and pre-existing NAFLD
such as ob/ob mice (which are leptin decient), or db/db
mice and fa/fa rats (which both are decient in leptin
receptor) [9496,144]. However, further studies are
needed to assess the interest if any of these different
animal models.
CONCLUSI ONS
Together with the metabolic activation of drugs into
reactive metabolites, mitochondrial dysfunction is yet
another frequent cause of DILI. Severe side effects such
as microvesicular steatosis, lactic acidosis or rhabdo-
myolysis can threaten the life of patients, and can lead to
the premature interruption of clinical trials, or the recall
of an unsafe medicine. The enormous investments made
for drug development are not recovered, which can
jeopardize the nancial viability and the image of a drug
company. A major challenge for the pharmaceutical
industry is therefore to be able to detect drug-induced
mitochondrial dysfunction during preclinical studies. We
therefore strongly recommend performing mechanism-
based investigations during preclinical safety studies in
order to assess whether a selected drug is able to impair
mitochondrial function, in particular in the liver. The
development and improvement of high-throughput
in vitro screening techniques should also allow pharma-
ceutical companies to assess mitochondrial effects during
lead selection, before subsequent preclinical safety inves-
tigations [7]. Finally, further investigations should be
carried out in mice with pre-existing mitochondrial and/
or metabolic abnormalities, such as sod
+/)
, jvs
+/)
or
ob/ob mice. Indeed, data collected from such investiga-
tions could help to determine if these animal models can
prove useful in the future to detect mitochondrial
dysfunction-mediated DILI, which could selectively occur
in patients with pre-existing mitochondrial and/or met-
abolic abnormalities.
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Drugs Induced Liver Injury

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Drugs Induced Liver Injury

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doi: 10.1111/j.1472-8206.2008.00608.

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