ATLA 35, 353–361, 2007
2006 SSCT–EST Conference Proceedings 353
Evaluation of Mitochondrial Function in Isolated Rat
Hepatocytes and Mitochondria during Oxidative Stress
Zuzana C ervinková,1 Halka Lotková,1 Pavla Kr ivaková,1 Tomás Rous ar,1 Otto Kucera,1 Lukas
Tichý,1 Miroslav C ervinka,2 and Zdenek Drahota3
v
v
v
v
v
v
v
1Department of Physiology, Charles University in Prague, Faculty of Medicine in Hradec Králové, Czech
Republic; 2Department of Medical Biology and Genetics, Charles University in Prague, Faculty of Medicine
in Hradec Králové, Czech Republic; 3Institute of Physiology, Academy of Sciences of the Czech Republic,
Prague, Czech Republic
Summary — The majority of toxic agents act either fully or partially via oxidative stress, the liver, specifically the mitochondria in hepatocytes, being the main target. Maintenance of mitochondrial function is
essential for the survival and normal performance of hepatocytes, which have a high energy requirement.
Therefore, greater understanding of the role of mitochondria in hepatocytes is of fundamental importance.
Mitochondrial function can be analysed in several basic models: hepatocytes cultured in vitro; mitochondria in permeabilised hepatocytes; and isolated mitochondria. The aim of our study was to use all of these
approaches to evaluate changes in mitochondria exposed in vitro to a potent non-specific peroxidating
agent, tert-butylhydroperoxide (tBHP), which is known to induce oxidative stress. A decrease in the mitochondrial membrane potential (MMP) was observed in cultured hepatocytes treated with tBHP, as illustrated by a significant reduction in Rhodamine 123 accumulation and by a decrease in the fluorescence of
the JC-1 molecular probe. Respiratory Complex I in the mitochondria of permeabilised hepatocytes showed
high sensitivity to tBHP, as documented by high-resolution respirometry. This could be caused by the oxidation of NADH and NADPH by tBHP, followed by the disruption of mitochondrial calcium homeostasis,
leading to the collapse of the MMP. A substantial decrease in the MMP, as determined by tetraphenylphosphonium ion-selective electrode measurements, also confirmed the dramatic impact of tBHP-induced
oxidative stress on mitochondria. Swelling was observed in isolated mitochondria exposed to tBHP, which
could be prevented by cyclosporin A, which is evidence for the role of mitochondrial permeability transition. Our results demonstrate that all of the above-mentioned models can be used for toxicity assessment,
and the data obtained are complementary.
Key words: hepatocytes, high-resolution respirometry, mitochondria, mitochondrial membrane potential,
oxidative stress, tert-butylhydroperoxide.
v
Address for correspondence: Z. Cervinková, Department of Physiology, Charles University in Prague,
Faculty of Medicine in Hradec Králové, Šimkova 870, 500 38 Hradec Králové, Czech Republic.
E-mail: [email protected]
Introduction
The liver is a multifunctional organ that plays a
crucial role in intermediary and energy metabolism,
biotransformation, secretion and excretion. These
processes are energy demanding, so the liver is a
highly oxygen-dependent tissue, with intensive
metabolic activity. The liver is a particular target
for the toxic effects of various xenobiotics, because
of its roles in metabolising and clearing chemicals.
Toxic liver injury can mimic almost all forms of
acute or chronic hepatobiliary diseases (1). It is now
generally accepted that mitochondria play a crucial
role in many basic biological functions, including
the initiation of both apoptotic and necrotic cell
death. They are increasingly recognised as a frequent target of injury in hypoxia, ischaemia and
chemical toxicity. A major consequence of mitochondrial dysfunction is represented by mitochondrial permeability transition (MPT), which is
dependent on the opening of a voltage-dependent,
high-conductance channel located in the inner
mitochondrial membrane. The opening of this
channel, called the permeability transition pore
(PTP), is controlled by several factors. The most
important inducers of PTP opening are oxidative
stress, elevated matrix Ca2+ concentration, depletion of adenine nucleotides, and a decrease in mitochondrial membrane potential (2, 3). PTP opening
is blocked physiologically by a decrease in pH and
an increased concentration of Mg2+; cyclosporin A
(CsA) is a potent exogenous inhibitor of PTP opening (4, 5).
Oxidative stress caused by reactive oxygen
species (ROS) damages DNA, proteins and lipids,
and triggers the mitochondrial pathways leading to
cell death. This plays a crucial role in the pathogenesis of a number of diseases, including ischaemicreperfusion injury, toxic injury and the development
of chronic injury, as well as ageing (6).
354
Z. Cervinkova et al.
The aim of our study was to evaluate mitochondrial changes in isolated rat hepatocytes, and the
swelling of isolated rat liver mitochondria, after in
vitro exposure to the potent peroxidating agent,
tert-butylhydroperoxide (tBHP). tBHP is a shortchain lipid hydroperoxide analogue, which is widely
used to mimic the effects of oxidative stress in
short-term cell culture experiments (7). Most of the
experiments focused on the study of PTP opening
were performed on isolated mitochondria. However,
there is growing evidence that mitochondria
require contacts with other structures (e.g. the
endoplasmic reticulum and other mitochondria) for
the maintenance of normal functional activity (8,
9). To evaluate the effects of tBHP-induced oxidative stress on mitochondria, we used rat hepatocytes, permeabilised by digitonin. Permeabilised
hepatocytes provide a useful model, in which the
intracellular cytoskeletal network is maintained
and internalised mitochondria are accessible to
endogenous substrates and cofactors (10).
mitochondrial membrane potential by using a
tetraphenylphosphonium (TPP+)-selective electrode, and for the measurement of oxygen consumption. Control cell suspensions were compared
with cells exposed to tBHP, at concentrations ranging from 0.1–3mM, for 15 minutes. The second part
of the cell suspension was plated in collagen-coated
Petri dishes (2 × 106 cells per 60mm diameter dish),
William’s Medium E (Sigma–Aldrich) was added,
and the dishes were placed in an incubator (5% CO2
in air) for 2 hours to eliminate potential stress
caused by the cell isolation procedure. After this
period, the control cells were incubated in William’s
Medium E for 30 minutes, and the tBHP-treated
hepatocytes were exposed for 15 minutes to different concentrations of tBHP, in William’s E culture
medium.
Materials and Methods
Oxygen consumption was measured by the HighResolution Oxygraph 2K (Oroboros, Innsbruck,
Austria). Measurements were performed in 2ml of
incubation medium at 30°C. Digitonin-permeabilised hepatocytes were incubated in K-medium,
which contained 100mM potassium chloride, 10mM
Tris HCl, 4mM potassium phosphate (dibasic),
3mM magnesium chloride, and 1mM EDTA (at pH
7.4). Oroboros software (DatLab3.1) was used for
the evaluation of oxygen uptake. Oxygen uptake
curves are presented as the first derivation of oxygen tension changes.
Chemicals
Fatty acid-free bovine serum albumin, tBHP,
rotenone, antimycin A, respiratory substrates, ADP,
digitonin, type I collagen, trypan blue, Rhodamine
123, and the commercially-available kit for lactate
dehydrogenase determination were purchased from
Sigma–Aldrich (Prague, Czech Republic). Collagenase (Collagenase cruda) was obtained from SEVAC
(Prague, Czech Republic), and the JC-1 fluorescent
molecular probe from Molecular Probes (New
Brunswick, NJ, USA). All other chemicals of analytical grade were obtained from standard sources.
Isolation of rat hepatocytes
Male Wistar rats (BioTest, Konarovice, Czech Republic), with a body weight of 230–250g, were used.
The rats were housed at 23 ± 1°C, with 55 ± 10% relative humidity, air exchange 12–14 times/ hour, and
12-hour light–dark cycle periods (06.00 to 18.00
hours). The animals had free access to a standard laboratory diet (DOS 2B, Velaz, Czech Republic) and tap
water. All the animals received care according to the
guidelines set by the Institutional Animal Use and
Care Committee of Charles University, Prague,
Czech Republic.
Hepatocytes were isolated by two-step collagenase perfusion (11), and their viability (which was
always > 90%) was assessed by trypan blue exclusion. Isolated cells were suspended in Krebs–
Henseleit buffer (Sigma–Aldrich). One part of the
cell suspension was used for the direct evaluation of
Measurement of oxygen uptake by
permeabilised hepatocytes (high-resolution
respirometry)
Mitochondrial membrane potential
The mitochondrial membrane potential (MMP) was
assessed by:
1. Accumulation of Rhodamine 123 (Rho 123), a
probe that distributes across the mitochondrial
membrane, according to the transmembrane
potential (12). Cultures of hepatocytes were incubated with 60nM Rho 123 at 37°C. After 15 minutes, the medium was removed and centrifuged
(50g; 5 minutes, 4°C). The Rho 123 concentration
was determined in this medium by spectrofluorometry (Perkin–Elmer LS50B, Perkin–Elmer
s.r.o., Prague, Czech Republic; excitation, 498nm;
emission, 525nm).
2. Visualisation of the MMP. A JC-1 molecular
probe was used, which is a dye that changes
emission wavelength in response to decreasing
membrane potential, shifting from orange to
green. Hepatocytes were cultured in collagencoated Petri dishes; tBHP-treated cells were
Evaluation of mitochondrial function during oxidative stress
exposed to a dose of 1.5mM for 15 minutes. The
hepatocytes were subsequently cultured for 15
minutes with JC-1 (10mg/ml), in the dark. A fluorescence microscope (Nikon Eclipse E-400,
Nikon s.r.o., Prague, Czech Republic), with a
B2A filter block (excitation 450–490nm; emission 520+nm) and a digital camera (Nikon
COOL 13000 CVDS; Nikon), were used for the
evaluation.
3. Assessment of the MMP in permeabilised hepatocytes with a tetraphenylphosphonium ion
(TTP+)-selective electrode. A decrease of TTP+
concentration in culture medium correlates with
TTP+-uptake by energised mitochondria.
During the measurement, the addition of succinate (a substrate of respiratory Complex II) is
important for the stabilisation of the MMP. We
used FCCP (carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone; Sigma–Aldrich,
Prague, Czech Republic) as a strong uncoupler
of oxidative phosphorylation, to obtain a minimal value of MMP. The measuring device consists of a TTP+-selective electrode (produced in
the Institute of Pathophysiology, the First
Medical Faculty of Charles University in
Prague; see 13), a reference Ag/AgCl electrode,
and a PC with a high impedance measuring card
355
PCI-6036 (National Instruments, Austin, TX,
USA). Data acquisition and handling was performed using MATLAB/Simulink software
(Math Works, Inc., Natick, MA, USA).
4. Swelling of rat liver mitochondria prepared from
liver homogenates by conventional differential
centrifugation (14) in 210mM mannitol, 65mM
sucrose, 5mM HEPES, 1.5mM EGTA and 0.5%
fatty acid-free BSA, at pH 7.35. Mitochondrial
swelling was determined from the decrease in
the turbidity of the mitochondrial suspension,
measured at 520nm in a spectrophotometer
(Shimadzu UV-1601, Shimadzu Handels GmbH,
Vienna, Austria; 15).
Data analysis and statistical analysis
All measurements were made at least six times, but
only representative traces are shown in the figures.
Values in Figure 3 are expressed as the mean ±
S.E.M. The statistical significance was analysed by
using one-way ANOVA. Tukey-Kramer’s post-hoc
test was used for multiple comparisons between
groups (GraphPad InStat 3.06, GraphPad Software,
San Diego, CA, USA), and values of p < 0.05 were
considered to be significant.
Figure 1: Photomicrographs of hepatocytes
a)
b)
a) control hepatocytes, incubated in primary culture for 30 minutes; b) hepatocytes exposed to 1.5mM tBHP, for 15
minutes. Scale bar = 15µm.
Z. Cervinkova et al.
356
Results
Hepatocyte morphology
In our previous experiments, we observed that
freshly isolated hepatocytes are spherical in shape,
and are separated from each other in the cell suspension. Two hours after seeding into a collagencoated dish, the hepatocytes become polygonal and
begin to form contacts with neighbouring cells
(Figure 1). Incubation with tBHP was associated
with remarkable changes in hepatocyte morphology. The treated hepatocytes were more spherical in
shape, the cytoplasm was highly granulated in comparison to that of control cells, and the cells lost
contact with the collagen coating on the dish
(Figure 1b).
High-resolution respirometry
The sensitivities of various mitochondrial enzymes
to oxidative damage were tested in isolated rat
hepatocytes, permeabilised by digitonin, by using
high-resolution respirometry. Respiratory rates of
NADH-dependent (glutamate+malate) and flavoprotein-dependent (succinate) substrates were
determined in control hepatocytes (Figure 2a), and
in hepatocytes exposed for 15 minutes to 1.5 mM
tBHP, prior to digitonin permeabilisation (Figure
2b). The results of these experiments are presented
as the outcome of a typical experiment. Nevertheless, we had very reproducible results throughout this study. Specific substrates were added in
sequence into the measuring chamber. Firstly, we
measured the activity of Complex I by addition of
glutamate+malate and ADP, after which Complex I
was inhibited by the addition of rotenone. We then
measured activity of Complex II by the subsequent
addition of succinate. Our data show that oxidation
of NADH-dependent substrates is much more sensitive to tBHP-induced oxidative damage, when
compared with flavoprotein-dependent substrates.
The Rho 123 assay
Rho 123 distributes across mitochondrial membrane with respect to the transmembrane potential.
Figure 3 shows Rho 123 accumulation in control
hepatocytes, and in hepatocytes exposed to tBHP at
various concentrations (from 0.1–3.0mM), for 15
minutes. In the data analysis, accumulation of Rho
123 by control hepatocytes is expressed as 100%.
Exposure of hepatocytes to tBHP significantly
reduced accumulation of Rho 123, in a dose-dependent manner. Even exposure to the lowest dose of
tBHP (0.1mM) results in a statistically-significant
decrease of Rho 123 accumulation (p < 0.05). The
most pronounced decrease was found in the concentration range of 0.5–1.0mM. However, not even the
Figure 2: The effects of tBHP on respiration of glutamate+malate and succinate
a)
1200
1200
rot
800
1000
suc
hepatocytes
dig
600
ADP
glut +
mal
400
200
0
rot
hepatocytes
pmol O2/s/106 cells
pmol O2/s/106 cells
1000
b)
ADP
800
dig
tBHP
600
suc
glut +
mal
400
200
0
200
400
600
800 1000 1200 1400 1600
time (seconds)
0
0
400
800
1200
1600
2000
time (seconds)
a) control hepatocytes in K-medium; b) hepatocytes exposed to 1.5mM tBHP in K-medium, for 15 minutes. The arrows
indicate the successive addition of hepatocytes, tBHP, 20mg/ml digitonin (dig), 10mM glutamate+2.5mM malate
(glut+mal), 1mM ADP, 1µM rotenone (rot) and 10mM succinate (suc).
Evaluation of mitochondrial function during oxidative stress
357
Figure 3: The inhibition of Rho 123
accumulation by tBHP
by using an ion-selective electrode. A marked
advantage of this method is that it is possible to
continuously monitor changes in the MMP. We
used permeabilised hepatocytes, because of the relatively slow passage of TPP+ and substrates into
non-permeabilised cells. Figure 5a depicts tBHPinduced changes in the MMP. In response to the
addition of hepatocytes and digitonin, the TPP+
concentration in the medium decreased, due to its
accumulation in energised mitochondria. Exposure
to tBHP caused a decrease in the MMP, this
decrease being dramatically apparent at the highest
concentration of tBHP (3.0mM). The effect of this
highest dose of tBHP on the MMP was alleviated by
the addition of CsA, an inhibitor of PTP opening
(Figure 5b).
120
100
*
***
% of control
80
60
***
***
40
***
20
0
control
tBHP
0.1mM
tBHP
0.5mM
tBHP
1.0mM
tBHP
1.5mM
tBHP
3.0mM
Hepatocytes in culture were exposed to 0.1, 0.5, 1.0, 1.5
and 3.0mM tBHP, for 15 minutes. Accumulation of Rho
123 by control hepatocytes is expressed as 100%. Each
value represents the mean ± S.E.M. (n = 6); *p < 0.05;
***p < 0.001, as compared to the control.
highest concentration of tBHP (3.0mM) led to complete abolition of mitochondrial membrane potential, the value representing about 20% of control
value (p < 0.001).
The JC-1 assay
Changes of mitochondrial membrane potential can
be visualised by fluorescence microscopy, by using
the JC-1 molecular probe, which changes emission
wavelength in response to decreasing membrane
potential, with a shift from orange to green. Control
cells exert a high mitochondrial membrane potential, demonstrated by strong orange fluorescence
emission (Figure 4a). In tBHP-treated hepatocytes,
the orange fluorescence is almost lost, as a consequence of a dramatic reduction in mitochondrial
membrane potential (Figure 4b).
The TPP+ assay
TPP+ levels can also be used to evaluate mitochondrial membrane potential. TPP+ is a lipophilic
cation that, similarly to Rho 123, accumulates in
relation to the MMP in mitochondria. It is not a fluorescent probe, so its concentration is determined
Mitochondrial swelling
Mitochondrial swelling was estimated from the
decrease in turbidity (absorbance) of the mitochondrial suspension, measured at 520nm. Isolated
mitochondria exposed to 3.0mM tBHP showed significant swelling (Figure 6). A decrease in
absorbance was also induced by the addition of
400µM Ca2+. A combination of both these factors
multiplied the effect on mitochondrial swelling.
This effect was completely inhibited by addition of
CsA.
Discussion
As the main organ responsible for metabolic and
biotransforming processes, the liver is highly susceptible to various hepatotoxic compounds.
Common final pathways that lead to acute cellular
necrosis may involve ATP depletion after mitochondrial damage. An important mechanism
involved in mitochondrial failure is MPT, a phenomenon first characterised by Hunter and
Haworth in isolated mitochondria, almost 30 years
ago (16).
Oxidative stress is one of the most important
inducers of MPT. Several hypotheses have been
proposed on the mechanisms by which oxidative
stress is linked to MPT. MPT results in osmotic
swelling of the mitochondrial matrix, the uncoupling of oxidative phosphorylation, the rupture of
the mitochondrial outer membrane, and the release
of cytochrome c and other intermembrane space
proteins into the cytosol (17).
The purpose of this work was to evaluate mitochondrial changes in isolated rat hepatocytes, and
the extent of swelling in isolated rat liver mitochondria, after exposure in vitro to the potent peroxidating agent, tBHP. The functional capacity of
the mitochondrial energy provision system was
evaluated by several techniques, by using hepato-
358
Z. Cervinkova et al.
Figure 4: The effects of tBHP on JC-1 accumulation
a)
b)
Hepatocytes were cultured in Krebs–Henseleit medium in collagen-coated Petri dishes. Images show fluorescent
micrographs of a) control hepatocytes; b) hepatocytes incubated with 1.5mM tBHP, for 15 minutes.
Evaluation of mitochondrial function during oxidative stress
359
Figure 5: Changes in MMP, detected by using a TPP+-selective electrode
8
a)
hepatocytes
+ dig
0.5mM
tBHP
1.5mM
tBHP
suc
3.0mM
tBHP
FCCP
TPP+ (µmol/l)
6
4
2
0
0
200
400
600
time (seconds)
800
1000
1200
9 b)
hepatocytes
+ dig
8
TPP+ (µmol/l)
7
0.5mM
tBHP
CsA
suc
1.5mM
tBHP
3.0mM
tBHP
FCCP
6
5
4
3
2
1
0
0
200
400
600
800
1000
time (seconds)
1200
1400
1600
a) changes in MMP, induced by increasing concentrations of tBHP. Arrows indicate the successive addition of
1.85 × 106/ml hepatocytes, 20µg/ml digitonin (dig), 0.5mM tBHP, 10mM succinate (suc), 1.5mM tBHP, 3.0mM tBHP,
and 1µM FCCP; b) the effect of CsA on tBHP-induced changes in MMP. Arrows indicate the successive addition of
1.85 × 106/ml hepatocytes, 20µg/ml digitonin, 2µM CsA, 0.5mM tBHP, 10mM succinate, 1.5mM tBHP, 3.0mM tBHP,
and 1µM FCCP.
cytes cultured as a monolayer in primary culture, or
by using digitonin-permeabilised hepatocytes in
suspension. The advantage of using primary culture
is mainly that it promotes the formation of cell–cell
contacts (see Figure 1a), which are important for
the maintenance of hepatocyte functions.
Morphological changes in tBHP-treated hepato-
cytes (Figure 1b) clearly illustrated a toxic effect,
and are in agreement with our previous results (18).
The effects of tBHP on the MMP in primary cultures of hepatocytes were measured by using Rho
123 accumulation as an indicator of mitochondrial
membrane energisation. The accumulation of Rho
123 was significantly decreased after tBHP expo-
Z. Cervinkova et al.
360
Figure 6: The effect of tBHP on
mitochondrial swelling
2.0
Ca2+
CsA + tBHP + Ca2+
1.8
Ca2+
absorbance (520nm)
1.6
tBHP
1.4
1.2
tBHP + Ca2+
1.0
0.8
0
0
1
2
3
4 5 6 7 8
time (minutes)
9 10 11
Isolated rat liver mitochondria were resuspended in
swelling medium, supplemented with 1µM rotenone and
10mM succinate. Mitochondrial swelling was induced by
3mM tBHP and/or 400µM Ca2+ and was almost blocked
by the addition of 2µM CsA.
sure, in a dose-dependent manner. A statisticallysignificant decrease was observed after exposure to
even the lowest dose of tBHP (0.1mM), which
demonstrates the high sensitivity of MMP to tBHPinduced oxidative action. The decrease in Rho 123
accumulation reached about 30% of the original
value after 15 minutes of incubation with 1.5mM
tBHP. This finding is in good accordance with our
previous results (19).
For the determination of the MMP, we also used
fluorescence microscopy with the JC-1 molecular
probe. Control hepatocytes emit strong orange fluorescence, predominantly in the region close to the
cytoplasmic membrane. Similar functional heterogeneity of mitochondria within single cells, with
respect to the response of the MMP to tBHP, was
observed by Collins et al. (20).
The measurement of oxygen consumption was
performed on permeabilised hepatocytes in suspension culture. This model enabled us to measure
maximal capacity of respiratory enzymes, since the
supply of substrates is non-limiting in the system
used. The other advantage of using permeabilised
hepatocytes is that they mimic the in vivo situation
better than do isolated mitochondria. Various interactions of mitochondria with the cytoskeleton are
essential for maintenance of mitochondrial functional capacity (8).
We confirmed that, in the liver, the activity of
succinate dehydrogenase is significantly higher
than the activity of Complex I (21, 22). Our results
demonstrate that the oxidation of NADH-dependent substrates is much more sensitive to tBHPinduced oxidative damage, as compared with
flavoprotein-dependent substrates (Figure 2b). A
similar decrease in the activity of Complex I was
also observed during ageing (23) — a process
accompanied by the increased production of reactive oxygen species. The high sensitivity of respiratory Complex I to tBHP could be due to oxidation of
NADH and NADPH by tBHP, with concordant disruption of mitochondrial Ca2+ homeostasis. An
increased mitochondrial Ca2+ level stimulates the
mitochondrial formation of reactive oxygen species,
and both of these changes can contribute to the collapse of the MMP, which was shown in our experiments.
Permeabilised hepatocytes were also used for
determination of the MMP with a TPP+-selective
electrode, in cells exposed sequentially to increasing
concentrations of tBHP (final concentration
3.0mM; Figure 5a). Succinate was used for the energisation of the mitochondria. Under these experimental conditions, the dissipation of the MMP was
completely prevented by CsA (Figure 5b), which
indicates the involvement of the CsA-sensitive permeability transition pore (PTP; 24). Nevertheless,
additional mechanisms could be responsible for the
dissipation of the MMP by tBHP, so further studies
are required into this complex process.
PTP opening could be evaluated by the measurement of mitochondrial swelling. A high concentration of calcium, the increased production of ROS,
and other factors, increase the permeability of the
inner mitochondrial membrane to ions and various
substrates. This causes osmotic imbalance and
mitochondrial swelling (25). We estimated the
effects of tBHP on PTP opening from the decrease
in absorbance of isolated mitochondria, measured
at 520nm. Mitochondrial swelling was induced by
the addition of both tBHP and of Ca2+. A dramatic
induction of mitochondrial swelling was observed
when tBHP and Ca2+ were added simultaneously
(Figure 6). CsA was able to abolish the mitochondrial swelling induced by oxidative stress or by the
Evaluation of mitochondrial function during oxidative stress
increased Ca2+ concentration. This demonstrates
that opening of the CsA-sensitive PTP is the mechanism likely to be responsible for mitochondrial
swelling.
Thus, we can conclude that the methods used for
estimation of changes in the MMP and in oxygen
consumption are suitable for the evaluation of the
toxic effects of oxidative stress-inducing drugs.
Further studies with a range of other model drugs
are necessary, to further establish the suitability of
these methods.
Acknowledgements
This work was supported by grant MSM
0021620820 from the Ministry of Education and
Research. We are grateful to Anna Labajova for the
determination of mitochondrial membrane potential with the TPP+-selective electrode.
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Figure 4: The effects of tBHP on JC-1 accumulation
a)
b)
Z. Cervinkova et al.