From the Departments of Neurology and ¶ Cell
Biology and Anatomy, University of Miami, School of Medicine,
Miami, Florida 33136
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ABSTRACT |
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The mitochondrial oxidative phosphorylation
system consists of five multimeric enzymes (complexes I-V). NADH
dehydrogenase or complex I (CI) is affected in most of the
mitochondrial diseases and in some neurodegenerative disorders. We have
studied the physiological consequences of a partial CI inhibition at
the cellular level. We used a genetic model (40% CI-inhibited
human-ape xenomitochondrial cybrids) and a drug-induced model (0-100%
CI-inhibited cells using different concentrations of rotenone). We
observed a quantitative correlation between the level of CI impairment
and cell respiration, cell growth, free radical production, lipid
peroxidation, mitochondrial membrane potential, and apoptosis. We
showed that cell death was quantitatively associated with free radical
production rather than with a decrease in respiratory chain function.
The results obtained with human xenomitochondrial cybrid cells were
compatible with those observed in rotenone-induced 40% CI-inhibited
cells. At high concentrations (5-6-fold higher than the concentration necessary for 100% CI inhibition), rotenone showed a second toxic effect at the level of microtubule assembly, which also led to apoptosis. The correlation found among all the parameters studied helped clarify the physiological consequences of partial CI inhibitions at the cellular level.
Electron transport and oxidative phosphorylation are mediated by
five multimeric complexes (complexes I-V) that are embedded in the
mitochondrial inner membrane. Mammalian complex I
(CI)1 or NADH:ubiquinone
oxidoreductase has at least 40 subunits (1), seven of which are coded
by the mitochondrial DNA (mtDNA; Ref. 2), and catalyzes electron
transport from NADH to ubiquinone, which is coupled to vectorial proton movements.
A CI deficiency is characteristic of mitochondrial diseases, as an
isolated defect or, in most of the cases, as a part of multiple
respiratory chain deficiencies. Isolated CI deficiency has been
associated with a wide spectrum of symptomatic phenotypes, varying from
fatal infantile lactic acidosis to some cases of Leigh's disease,
adult onset exercise intolerance, and some neurodegenerative diseases
as Leber's hereditary optic neuropathy (LHON), focal dystonia, and
Parkinson's disease (3-6). The study of transmitochondrial cell lines
harboring mtDNA from LHON patients helped explain the role of some
mtDNA-coded CI subunits. Cybrids carrying the mtDNA G11778A mutation in
the ND4 gene associated with LHON showed a decrease in cell
respiration, but no enzymatic deficiency was detected by
spectrophotometry (7). Jun et al. (8) found that the mtDNA
G14459A transition in the ND6 gene associated with LHON caused a 60%
CI deficiency but a mild respiratory deficiency on polarographic
analysis. The function of the ND4 and ND5 subunits was studied by the
characterization of rotenone-resistant mutants of a human cell line
that in addition to nuclear mutations were also defective in these
mtDNA-encoded CI subunits (9, 10). In addition, cybrid lines created
using mtDNA from Parkinson's disease patients showed a CI deficiency
and increased free radical production (11). We recently created human
xenomitochondrial cybrids (HXC) (12, 13) harboring a 40% CI
deficiency. The CI impairment was caused by a limited number of amino
acid differences in mtDNA-coded subunits (between human and the three
apes used in the creation of HXC). All these genetic models of CI
deficiency have helped us assess the physiological effects of a
particular level of complex I inhibition.
Some specific complex I inhibitors have been classically used to model
CI deficiencies. Rotenone is an inhibitor of the NADH dehydrogenase,
which shuts off the supply of electrons to the quinol
(QH2)-cytochrome c oxidoreductase (14).
1-Methyl-4-phenylpyridinium, the bioactivated product of
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, binds to the
rotenone-binding site, thereby limiting CI activity (15). Inhibition of
CI by rotenone or 1-methyl-4-phenylpyridinium not only leads to a
decline in mitochondrial ATP production but also enhances the
generation of free radical by the mitochondrial respiratory chain and
initiates lipid peroxidation reaction in isolated bovine heart
mitochondria or submitochondrial particles (16-19). Because
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine causes Parkinson's
disease in humans (20) and rats (21), these data suggested a role for
CI deficiency and increased oxidative stress in Parkinson's disease
substantia nigra and in the dopaminergic cell death in this disorder
(22).
The underlying physiological consequences of a partial complex I
inhibition at the cellular level remain mostly unknown. Here we report
the effect of different levels of CI impairment on the cell physiology.
We used a genetic model (40% CI inhibited HXC lines; Ref. 13), and a
drug-induced model (partial CI inhibition in the human
osteosarcoma-derived cell line 143B) with rotenone to quantitatively
correlate CI inhibition and its effect on cell respiration, cell
growth, free radical production, lipid peroxidation, mitochondrial
membrane potential, and apoptosis.
Cell Lines and Culture Conditions--
Human xenomitochondrial
cybrids (HXC) (human-chimpanzee clone HC1, human-gorilla HG13, and
human-pigmy chimpanzee clone HP4) were produced and reported previously
(12). The human osteosarcoma-derived cell line 143B(TK Genetic and Experimentally Drug-induced Models of Complex I
Inhibition--
We modeled a partial CI inhibition by treating the
osteosarcoma cell line 143B with different concentrations of the CI
inhibitor rotenone. To define the model and to assess how the CI (NADH
decylubiquinone reductase) inhibition affects the KCN-sensitive
endogenous cell respiration, 143B cells were treated for 24 h with
0-1 µM rotenone. CI was measured spectrophotometrically
in isolated mitochondria, and cell respiration was measured
polarographically in whole cells as described previously (13). In all
the experiments, the culture medium was changed 2 h before
harvesting the cells. To validate the results obtained, a genetic model
of CI deficiency was used. The model consisted in HXC lines harboring
mtDNA from common chimpanzee (Pan troglodytes), pigmy
chimpanzee (Pan paniscus), and gorilla (Gorilla
gorilla; Ref. 12). HXC cells showed an approximately 20% decrease
in the cell respiration and a 40% decrease in the CI activity
(13).
Immunoblotting--
To determine whether the CI inhibition
induced adaptive variations on the expression of the complex I and II
subunits, immunoblotting was performed as described previously. 40 µg
of mitochondrial proteins from 143B cells treated with rotenone during
24 h were used, as well as human succinate dehydrogenase
flavoprotein subunit (SDH (Fp)) monoclonal and human ND1 polyclonal
antibodies. After scanning the autoradiograms, band signals were
quantified using NIH Image 1.62b7 software. The ratio between the
different bands (ND1/SDH ratio) was considered as the division of the
arbitrary densitometric values of the signals using each antibody.
These ratios were used to compare untreated and rotenone-treated 143B cells.
Growth Curves--
Growth measurements were made by platting
5 × 104 cells on 100-cm2 dishes in 10 ml
of the appropriate medium. Cells were grown in Dulbecco's modified
Eagle's medium lacking glucose supplemented with 5 mM
glucose or Dulbecco's modified Eagle's medium lacking glucose
supplemented with 5 mM galactose, both supplemented with 1 mM pyruvate and 10% dialyzed fetal calf serum. Cells were
incubated at 37 °C, and cell counts were performed at daily
intervals. For 143B and 143B/206 Oxidative Stress Analyses--
For each assay, three independent
samples from each cell type (human control lines SUB21 and 143B and the
HXC lines HP4, HG13, and HC1), either intact cells (independently
collected) or isolated mitochondria (independently isolated), were
analyzed, and at least three measurements of each sample were obtained.
To reproduce different degrees of CI inhibition, various concentrations
of rotenone (0-600 nM) were added to 143B cells at
approximately 80% confluence. After 4, 24, 48, and 72 h the
oxidative stress determinations were conducted. Experiments were
performed in triplicate.
Free Radical Production--
To monitor oxidative activity, the
cell-permeant probe 2'-7' dichlorofluorescein diacetate
(H2DCFDA) was used. H2DCFDA passively diffuse
into cells where intracellular esterases cleave the acetates, and the
oxidation of H2DCF by hydrogen peroxide produces a
fluorescent response (25). Cells were collected by trypsinization. 50 µg of cellular protein were resuspended in 500 µl of
phosphate-buffered saline (PBS) medium and labeled with
H2DCFDA (2 µg/ml) (Molecular Probes, Eugene, OR) for
10-60 min in the dark at 37 °C. Fluorometric analyses at 507 nm
excitation and 530 nm emission were performed using a MPF-66
fluorescence spectrophotometer (Perkin-Elmer Corp.). For all the
measurements, the basal fluorescence (time 0, t0) was subtracted. The increase in the
fluorescence was used to measure the chemical process of hydrogen
peroxide production.
Lipid Peroxidation Assays--
Peroxidation of cellular lipids
was measured using 50 µg of cellular protein resuspended in 500 µl
of PBS and labeled with cis-parinaric acid (5 mM)
(Molecular Probes, Eugene, OR) (26) for 10-60 min in the dark at
37 °C. Cells were washed by pelleting to remove excess probe, and
fluorescence at 318 nm excitation and 410 nm emission was detected as
described (27). Loss of parinaric acid fluorescence was used to measure
the chemical process of lipid peroxidation. To measure peroxidation of
mitochondrial membranes lipids, 50 µg of fresh mitochondrial protein
were resuspended in 500 µl of medium A. The experimental conditions
were the same as described above.
Mitochondrial Membrane Potential
( Cell Death Studies--
The apoptotic rotenone-induced cell
death was assessed using a cell death detection enzyme-linked
immunosorbent assay kit (Roche Molecular Biochemicals). Approximately
104 cells were used for the photometric enzyme immunoassay
of cytoplasmic histone-associated DNA fragments after rotenone-induced
cell death. 143B and 143B/206 Immunocytochemical Staining--
143B and 143B/206
Statistical Analysis--
The data were analyzed using the SPSS
software. Results are expressed as the means ± S.D. Comparisons
between cell line groups were carried out using a Levane's test (for
equality of variances) and the Student's t test (for
equality of means) for independent data. When a potential relationship
between variables was of interest, a linear regression analysis was
performed. Values with p < 0.050 were considered
statistically significant.
Defining the Complex I-inhibited Models
Modeling Complex I Partial Impairment by Incubating 143B Cells with
Rotenone--
The rotenone-induced model was created by titrating the
effect of different concentrations of the drug on the CI activity, measured as NADH decylubiquinone reductase. Exposure of 143B cells to
increasing concentrations of rotenone produced a progressive inhibition
of CI activity in isolated mitochondria (Fig.
1). A 100% inhibition was achieved with
100 nM rotenone. The endogenous cell respiration was also
inhibited in a dose-dependent manner but showed different
inhibition kinetics (Fig. 1). CI inhibition followed a classical
hyperbolic shape, whereas cell respiration inhibition showed an
S-shaped curve. Only when CI was inhibited by 35-40% (< 5 nM rotenone), cell respiration begun to decrease. Between
40 and 60% of CI inhibition (5-10 nM rotenone), cell
respiration decreased linearly until 30% of the normal rate.
Increasing concentrations of rotenone produced further but slower
decrease in CI activity and cell respiration.
Modeling a Genetically Determined 40% Complex I Inhibition by
Using Human Xenomitochondrial Cybrids--
This cellular model of CI
deficiency is caused by a limited number of amino acid changes in
mtDNA-coded subunits (i.e. those amino acids that differ
between humans and the three apes used in the creation of HXC (13).
Effect of Rotenone Treatment on the Steady-state Levels of mtDNA
and Mitochondrial Respiratory Chain Complexes
To determine whether the inhibition of CI induced adaptative
variations on the expression of the complex I and II subunits, immunoblotting was performed. Using mitochondria isolated from cells
incubated with different concentrations of rotenone for 24 h, no
significant changes in the ND1/SDH(Fp) ratio were observed (Fig.
2). A rotenone-induced CI deficiency did
not produce an adaptive response in the form of an increased expression
of the complex II (CII) in a period of 24 h.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
)
and its mtDNA-less derivative, 143B/206
°, were cultured as described (23). Culture conditions were modified as described below
under "Growth Curves." SUB21 cell line (transmitochondrial cybrid
clone containing wild type mtDNA) was previously characterized (24) and
used in some experiments as a human control cell line.
° cells, medium was supplemented
with different concentrations (0-1 µM) of rotenone. HXC
lines (13) were grown in media with glucose or galactose without
rotenone supplementation.
m)--
m was estimated using
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazole carbocyanide
iodide (JC-1). JC-1 is a fluorescent compound (excitation maximum, 490 nm) that exists as a monomer at low concentrations. At higher
concentrations, JC-1 forms aggregates. Fluorescence of the monomer is
green (emission, 527 nm), whereas that of the J-aggregate is red
(emission, 590 nm). Mitochondria with intact membrane potential (higher
than 100 mV) concentrate JC-1 into aggregates that fluoresce red,
whereas de-energized mitochondria cannot concentrate JC-1 and fluoresce
green (28). Changes in plasma membrane potential do not affect the JC-1
status. JC-1 was chosen because has been described as a reliable probe for analyzing
m changes in intact cells, whereas
other different probes capable of binding mitochondria show a lower
sensitivity (rhodamine 123) or a noncoherent behavior due to a high
sensitivity to changes in plasma membrane potential
(3-3'-dihexilocarbocyanide iodide) (29). Cells were grown in
25-cm2 dishes until 90% confluence and incubated for 45 min with 6.5 µM JC-1. Cells were collected by
trypsinization, washed in PBS, and resuspended in 500 µl of PBS, and
the samples were measured on a MPF-66 fluorescence spectrophotometer
(Perkin-Elmer Corp.). The ratio of the reading at 590 nm to the reading
at 527 nm (590/527 ratio) was considered as a relative
m value.
° were treated with different
concentrations of rotenone and harvested at 24-72 h. To study the role
of the mitochondrial permeability transition (MPT) pore in the
rotenone-induced cell death, cells were pretreated for 30 min with 5 µM of the MPT inhibitor cyclosporin A (CyA) and incubated
for 48 h, changing the medium every 8.5 h. As CyA is only
able to prevent MPT in short term experiments, the assay was repeated
with 15- and 30-h incubation periods and using 5 µM CyA
and 50 µM of the phospholipase A2 inhibitor
aristolochic acid (ArA) (Sigma), which enhances and prolongs the effect
of CyA (30, 31). The culture medium was changed after 15 h when
necessary, and the cells were processed after 30 h.
o cells were seeded on glass coverslips and treated with
different concentrations of rotenone for 24 h. Cells were fixed
with 4% paraformaldehyde in PBS, permeabilized with 0.1% saponin, and
incubated for 4 h with monoclonal antibody anti-
-tubulin
(Sigma). A Texas Red-conjugated AfiniPure donkey anti-mouse IgG
(Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was used as
secondary antibody. Coverslips were incubated for 30 min with 10 µg/ml bis-benzimide Hoechst 3342 (Sigma) to reveal the nuclei and
mounted onto glass slides with ProLongTM Antifade kit
(Molecular Probes). Fluorescence was inspected with a Wild Leitz
photomicroscope (Fluovert. Heerbrugg, Switzerland).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of complex I (NADH decylubiquinone
reductase) inhibition on endogenous cell respiration. Cells were
treated with different concentrations of rotenone for 4 h before
measuring cell respiration in whole cells and CI activity in isolated
mitochondria. Complete CI inhibition was achieved with 100 nM rotenone. The cell respiration was inhibited also in a
dose-dependent manner but showed a different inhibition
kinetics. HXC have an approximately 40% CI inhibition and an
approximately 80% residual cell respiration (13).
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Fig. 2.
Effect of rotenone treatment on the
steady-state levels mitochondrial respiratory chain complexes.
Immunoblotting was performed using 40 µg of mitochondrial protein
from 143B cells treated with different concentrations of rotenone for
24 h. Antibodies against SDH (Fp) and the subunit 1 of the
NADH-dehydrogenase (ND1) were used. The ND1/SDH ratio of the
densitometrical values of the correspondent bands did not show
significant differences between untreated and rotenone-treated 143B
cells.
Effect of CI Inhibition on Cell Growth
Respiratory competent 143B cells grew exponentially in medium
containing glucose as carbon source, and they showed a substantial, although slightly reduced, growth rate in galactose-containing medium
(Fig. 3A). The doubling time
in glucose (DGlu) was approximately 17 h,
and in galactose (DGal) it was 20 h (Fig.
3C). When 143B/206 ° cells were cultured in a medium in
which glucose is substituted for galactose, these cells died and
detached from the culture plate in less than 12 h (Fig.
3A), because galactose cannot be used efficiently for
glycolysis. HXC, having a 40% CI deficiency, grew well in
glucose-containing medium, with a DGlu of
23 h, but grew very poorly in galactose-containing medium with a
DGal almost four times higher (Fig. 3). 143B
cells were susceptible to the toxic properties of rotenone so that
increasing concentrations (between 0 and 500 nM) produced
progressively greater inhibition of the growth rate. Fig. 3B
shows that 143B cells can grow in galactose medium in the presence of 5 nM rotenone (concentration inducing 54% of CI inhibition,
Fig. 1) but not in the presence of 7.5 nM (72% of CI
inhibition, Fig. 1). HXC cells grew in galactose medium if supplemented
with less than 1 nM rotenone (concentration that produced
approximately 19% of CI inhibition, Fig. 1). The rotenone-induced
inhibition was additive to the genetically produced 40% deficiency in
these cells. Fig. 3C compares growth parameters of the cell
lines studied in the different culture conditions. The growth of the
143B/206
° in glucose medium was affected by rotenone at
concentrations
500 nM (the growth rate with 500 nM rotenone was 30% slower than untreated cells), and
cells did not grow at a concentration of 600 nM. These data
indicated that rotenone kills cells in a CI inhibition-independent way,
when present at concentrations substantially higher than that producing
complete CI inhibition in 143B cells.
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Effects of CI Inhibition on Oxidative Stress
Studies in HXC Lines--
We studied whether a 40% deficiency in
CI activity resulted in greater reactive oxygen species (ROS)
production in HXC lines. ROS production was estimated using
H2DCF, a dye that is converted to a fluorescent product
upon oxidation by hydrogen peroxide. Fig.
4A shows ROS production as a
function of time. The slope of the regression lines was significantly
higher in the HC (5.64 ± 0.25; p < 0.002), HP
(5.07 ± 0.31; p < 0.016), and HG (5.09 ± 0.57; p < 0.050) xenomitochondrial cell lines compared
with the set of values obtained for the 143B (4.22 ± 0.40) and
SUB21 (4.67 ± 0.32) control cell lines. The electron transport in
HXC may be inefficiently coupled to ATP synthesis, producing an
increase in the rate of ROS formation. Lipid peroxidation is a
deleterious consequence of oxidative stress. To determine whether the
observed increase in the rate of ROS production in the HXC cell lines
produced a higher lipid peroxidation of the membranes in these cells,
the loss of cis-parinaric acid fluorescence was used to measure the chemical process of lipid peroxidation (26). In a whole cell assay, the
time course of the fluorescence decrease showed no significant
differences among cell lines (Fig. 4B). However, when using
isolated mitochondria, an increase in the membrane lipid peroxidation
was observed in HXC cells (Fig. 4C). The fluorescence value
at the start of the reaction (t0) subtracted by
the value measured after 30 min (t30), was
1.36-fold higher in HC (p = 0.015), 1.48-fold higher in
HP (p = 0.044), and 1.51-fold higher in HG (p = 0.036), compared with the mean values obtained for
the control human cell lines.
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Studies in Rotenone-treated 143B Cells--
ROS production and
mitochondrial membrane lipid peroxidation were enhanced by incubation
of cells with rotenone (Fig. 5,
A and B). Both parameters increased roughly
linearly when concentrations of 0-100 nM rotenone were
used (concentration range to produce CI inhibition) but did not
increase significantly at higher concentrations. At the same
concentration of rotenone, both parameters increased with longer
treatment (between 4 and 72 h).
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Effect of CI Inhibition on the Mitochondrial Membrane Potential
A CI deficiency leads to a reduction in proton extrusion to the
intermembrane space, partially depleting the cells from ATP. These
factors, together with the associated increase in oxidative stress,
could produce a significant drop of the mitochondrial membrane
potential (m). The dye JC-1 was used to monitor
m, estimated as the 590/527 nm emission ratio. Used
as low
m controls, 143B/206
o value
was 30-32% of 143B, and 143B cells incubated for 45 min with 10 µM of the uncoupler carbonyl-cyanide
m-chlorophenylhydrazone showed an estimated
m corresponding to 45-48% of the 143B value (Fig.
5C). HXC lines exhibited a
m that was
83-87% of the 143B level. Rotenone-treated 143B cells showed a mild
decrease in their
m (Fig. 5C). Low
concentrations of rotenone (between 1 and 10 nM), produced
a consistent hyperpolarization of the mitochondrial membrane (a
reproducible 15-18% increase compared with the control level) when
cells were incubated with the drug for 4 h. At those concentrations, the
m was maintained above 80% of
the normal value. Only in cells that were incubated for 3 days with
concentrations of rotenone between 100 and 600 nM did their
m decrease to 60-65% of the untreated 143B cell
value. For each concentration of rotenone used,
m
decreased slightly with the time of incubation.
Effect of CI Inhibition on Cell Death
143B and 143B/206 ° were treated with different
concentrations of rotenone and harvested at 24, 48, and 72 h. In
143B cells, cell death was increased with all concentrations of
rotenone up to 10-13 times the level of untreated cells after 3 days
of incubation with 100 nM rotenone (Fig.
6A). Higher concentrations
increased cell death exponentially. Rotenone did not significantly kill 143B/206
° cells until present at concentrations higher than 100 nM. Using 600 nM rotenone, both cell lines died
at indistinguishable rates (Fig. 6A). Experiments were
carried out to test whether cell death in the presence of rotenone
requires MPT. Fig. 6B shows the ability of 5 µM CyA and 50 µM ArA to prevent cell death
from taking place in the presence of different concentrations of
rotenone during 15 and 30 h. CyA inhibits the opening of the MPT
pore (32), and phospholipase A2 inhibitors, such as ArA, enhance and
prolong this effect (30, 31). Because CyA is not able to maintain mitochondrial function, it did not prevent rotenone-induced apoptosis after long periods of rotenone treatment. In cells treated with rotenone for 48 h, CyA essentially did not inhibit apoptosis
(15-30% inhibition), even with changing the medium and adding fresh
CyA every 8.5 h (data not shown). These data indicate that
additional CyA-resistant mechanisms can have a role in the
rotenone-induced apoptosis. ArA prolongs the effect of CyA for at least
16 h (31). We incubated the cells for 15 and 30 h with
different concentrations of rotenone in the presence of CyA and ArA
with a change of the medium in the latter at 15 h. When CyA and
ArA were used in cells treated with < 500 nM
rotenone, the cell death prevention was almost complete in the 15-h
incubation experiments. However, cell death was prevented by 55-75%
in the 30-h incubation experiments. In experiments using 600 nM of rotenone, cell death prevention dropped to under
50%. At these concentrations of rotenone, the mtDNA-less 143B/206
o cells also were killed, suggesting a second mechanism
for rotenone toxicity.
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Exploring the Secondary Effects of Rotenone and Checking the Validity of the CI Inhibition Model
Rotenone seems to have at least two modes of action, because the
mtDNA-less cell line was also killed by relatively high doses of the
drug. To distinguish between these two effects and to define the
effective range of rotenone concentrations that induce only or mostly a
partial mitochondrial defect without major secondary effects, we
studied the effect of rotenone in the microtubular cytoskeleton by
immunocytochemical staining. 143B (data not shown) and 143B/206
o cells (Fig. 7) were
treated with different concentrations of rotenone for 24 h and
stained with a monoclonal antibody against tubulin. The nuclei were
revealed by the specific dye Hoechst 3342. When the cells were
incubated with 0-100 nM rotenone, a normal microtubular
pattern and normal nuclear morphology were observed in all cells (Fig.
7). Concentrations of 500 nM rotenone produced a
disorganization of the microtubular structure and condensation of the
nucleus in approximately 5-10% of the cells (Fig. 7). By incubating
cells with 1000 nM rotenone, more than 90% of the cells that still remained attached showed disruption of the microtubular network and condensation-fragmentation of the nucleus, which are typical apoptotic signs (Fig. 7).
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Correlations between CI Impairment and Physiological Consequences
After 72 h of rotenone treatment, the percentage of CI
inhibition correlated positively, giving an exponential curve, with ROS
production (R2 = 0.973), decay in
m (R2 = 0.985), mitochondrial
membrane lipid peroxidation (R2 = 0.964), and
apoptotic cell death (R2 = 0.924) (Fig.
8B). Because the effect of
rotenone on CI inhibition and cell respiration was not exactly the same
(Fig. 1), all the curves were modified accordingly, showing hyperbolic
shapes, when cell respiration was plotted against all the parameters
studied. In all cases, R2 was > 0.93 (Fig.
8A). Fig. 1 shows that incubation of 143B cells in medium
supplemented with 1 nM rotenone exhibited a 19% of CI inhibition but maintained 98% of the cell respiration rate.
Percentages of CI inhibition lower than 20% produced mild changes in
all parameters, whereas any change in cell respiration produced
significant increases in all of them (Fig. 8, A and
B). A summary of our findings for four putative levels of CI
deficiency is depicted in Fig. 8C.
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DISCUSSION |
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In the last few years, many studies focused in the causal relationship between physiological or pathological decreases in mitochondrial polarization, ROS generation, the MPT, and subsequent apoptotic cell death (33-35). The main goal of the present work was to define quantitative correlations between partial mitochondrial CI inhibition and the physiological consequences at the cellular level by using a drug-induced model (rotenone dose-dependent CI inhibition in a human osteosarcoma-derived cell line) and a genetic model (40% CI inhibited HXC lines; Ref. 13).
Effect of CI Inhibition on Cell Respiration and
Growth--
Exposure of 143B cells containing normal levels of mtDNA
(+ cells) to increasing concentrations of rotenone
produced a progressive inhibition of CI activity and, subsequently, of
cell respiration. We observed a hyperbolic titration curve of CI
inhibition, whereas the cell respiration inhibition showed a sigmoidal
curve. Our results suggest that there is an excess CI of activity
relative to the maximum rate of electron flow through the respiratory
chain. Consistent with the respiratory control theory (36), a reduction in CI activity up to 40% may be able to limit the flux rate of the
electron transport chain only modestly. In the case of HXC, a 40% CI
inhibition produced a 20% cell respiration reduction, which is in good
agreement with the pharmacological model. Cultured cells, even the ones
defective in oxidative phosphorylation, can grow at essentially normal
rates in medium containing glucose, a sugar that can be well
metabolized glycolytically. Using a medium in which galactose replaced
glucose, cells were forced to derive much of their ATP from oxidative
metabolism. The decrease in respiratory efficiency of the HXC and in
the rotenone-treated cells reduced their rate of ATP production by
oxidative phosphorylation to an extent sufficient to affect their
growth capacity in galactose medium. A 65% CI inhibition, producing
approximately a 35% inhibition in cell respiration, seems to be the
threshold in our pharmacological model that allows cells to grow in
conditions where the respiratory chain is essentially the only source
of ATP. The rotenone-induced CI inhibition was additive to the
genetically determined 40% CI deficiency in HXC cells (13). HXC cells
could grow in galactose medium, although their doubling time was very
high (~80 h). HXC cells were still able to grow in galactose medium
for an additional ~20% CI inhibition (~5% cell respiration
decline), showing that also in this model, the threshold seems to be
~60% CI inhibition (corresponding to ~35% cell respiration
decay). This threshold may change with the cell type, depending on the
control coefficient of their CI. Changes in the threshold can be the
basis for understanding the cell type specificity of CI deficiencies in
some neurodegenerative disorders such as Parkinson's disease. Our
results suggest that if cells are maintained in glucose medium, a
partial CI deficiency is more deleterious than a complete respiratory
chain deficiency for some cellular functions. In glucose-containing
medium,
+ cells experimented a rotenone
dose-dependent (from 0 to 500 nM) reduction in
their growth rate.
° cells did not experience any major change in
their growth rate in glucose medium (<10%) at concentrations lower
than 200 nM rotenone and decreased by only ~35% with 500 nM rotenone, a concentration that seems to be a threshold
for killing these cells. It has been suggested that inhibition of
respiratory chain activity by drugs such as rotenone directly
interferes, as an acute insult, with the normal mitochondrial energy
metabolism, triggering an apoptotic response. The generation of
°
cell lines, on the other hand, undergoes a progressive mtDNA depletion
by treatment with ethidium bromide during at least 30 days, a period of
time that could allow the cells to adapt metabolically (23).
Nevertheless, because cells with complete rotenone-induced CI
inhibition still maintained a cell respiration rate of approximately 20% (because of the electron flow through the CII) and because of the
gradual decline observed on the growth rate of
+ cells
with partial CI inhibition in nonselective glucose medium, it is clear
that factor(s) in addition to the oxidative impairment are implicated
in the CI impairment.
Effect of CI Inhibition on Oxidative Damage-- In both genetic and pharmacological models, CI impairment was accompanied by an enhancement of ROS production and lipid peroxidation. Under aerobic conditions, the respiratory chain is a potent source of free radical (33, 37, 38). A CI impairment leads to enhanced formation of ROS, as has been shown in submitochondrial particles (16, 18) and in different cell systems (39, 40). In the case of HXC, defects in CI may predispose the respiratory chain to produce excess superoxide because of structural or stoichiometric alterations in the subunits. This alteration may lead to increased interaction of oxygen with an electron carrier, as it has been proposed for patients with genetically determined CI deficiency (39). In 143B cells treated with rotenone, ROS production increased in a dose-dependent manner. At 100% CI inhibition, the rate of ROS generation increased by a maximum of 20-25% in short term incubations (4 h). Similar results were reported on a human skin fibroblasts treated with 1 µM rotenone (39). This pattern is compatible with a scenario where ROS production may result directly from CI inhibition, being enhanced during the subsequent apoptotic process (see below). Activation of apoptosis has been associated with generation of ROS, and it has been shown that superoxide is produced by mitochondria isolated from apoptotic cells due to a switch from the normal four-electron reduction of O2 to a one-electron reduction when cytochrome c is released from mitochondria (41). It is still unclear whether the site of superoxide production in CI inhibited mitochondria is CI itself (16-19) or not (42). In any case, a partial inhibition of CI by rotenone should favor the transfer of electrons by CII and, as a consequence, an increase in the superoxide anion production. Electrons channeled via CII produce greater than four times more mitochondrial superoxide than electrons channeled via CI (43), and in studies in isolated cerebral mitochondria using succinate as substrate, ROS production was increased almost 9-fold (44). This problem will be of greater magnitude if cells try to compensate the CI impairment by overexpressing CII subunits. Although the question of whether CI inhibition up-regulates the mitochondrial CII activity was not directly addressed in this study, the unchanged ND1/SDH ratio observed in our experiments suggest that the compensatory mechanism in the form of CII overexpression, if any, cannot be observed in a period up to 24 h. The possible adaptive mechanisms in the form of modulation of enzymic activities of distal respiratory chain complexes, increase in mitochondrial content of the cell, or other effects in the mitochondrial biogenesis remain unknown and are currently under investigation.
It has been shown that CI inhibition induces increase in the MnSOD
levels (39, 45). Because oxidative stress is considered an imbalance
between oxidants being metabolized or scavenged by antioxidants, our
work cannot discern whether the ROS generation was underestimated by
the action of intramitochondrial scavenging activity. To evaluate the
consequences of increased level of ROS under the circumstance of a
partial CI impairment, we examined the degree of lipid peroxidation.
Lipid peroxidation increased linearly until approximately 75% CI
inhibition, remaining basically constant until total CI blockage. The
increased membrane sensitivity to oxidants could be due to changes in
membrane lipid composition (46) and may contribute to a decreased
fluidity (47), affecting in turn parameters such as m
and cell death. Certainly, ROS have damaging effects on cellular
components triggering defensive responses by the cells and even
apoptosis (48, 49).
Effect of CI Inhibition on m and
Apoptosis--
Although the regulation of apoptosis is complex and not
fully understood, it is known that mitochondria play an important role
in the process. A reduction in
m is an early event in
many cells undergoing apoptosis (50-53); isolated mitochondria induced to undergo MPT can induce nuclear apoptosis (53), CyA inhibits MPT pore
opening, inhibiting apoptosis in some cases, and the subsequent loss of
m (31, 53, 54). It is interesting to consider whether
or not a fall in
m is a signal for the CI
impairment-associated cell death. According to this and previous
studies (55), despite the failure to generate ATP oxidatively, rotenone
only slightly depolarized mitochondria, maintaining more than 75% of
control
m, even when CI was completely inhibited for
long incubation periods (3 days). Glycolysis provides the cells with
sufficient ATP, which can be used via the mitochondrial ADP/ATP
translocator to create a
m across the mitochondrial
inner membrane (56). Such a fall in
m may be
insufficient to trigger apoptosis when cells grow in glucose medium,
and additional biochemical insults (e.g. oxidative stress)
may finally lead to cell death. It is interesting that by using
concentrations of rotenone lower than 10 nM (~75% CI
inhibition), mitochondria actually became hyperpolarized, especially
under incubation times shorter than 48 h. Similar
m increases have been shown before in Jurkat cells
early during the apoptotic response to different stimuli (57). As a
result of a decreased electron transport, the rate of H+
ion delivery to the intermembrane space should be decreased. However,
alterations in ionic homeostasis may produce an increase in that rate
or a decrease in the removal of the H+ ions from the
intermembrane space, inducing the observed hyperpolarization. When
cells lose ion homeostasis, this scenario could be reversed, producing
a fall in the
m. The
° cells used in this study
maintained approximately 30% of the
m exhibited by
the
+ cells (20% in previous studies using the dye
R123; Ref. 56), enough to support a growth rate in glucose medium
comparable with that from
+ cells. In the light of these
results, HXC reduced growth rate in glucose medium cannot be explained
by the 10-15% fall of their
m but probably because
HXC cells suffer the consequences of a higher ROS production rate.
One of the early events in apoptosis seems to involve MPT triggered by
a phosphorylation event or by the action of free radical (58). The MTP
pore opening is mediated by collapse of m, ROS, high
levels of Ca2+, or low ATP. When these conditions are
established, mitochondrial solutes leak out, and nuclear apoptosis is
induced. CyA, an inhibitor of MPT, plus ArA, a phospholipase
A2 inhibitor acting synergistically with CyA, prevented
rotenone-induced cell death up to 80% at concentrations lower than 400 nM and up to 60% with higher concentrations. The ability
of CyA to protect cells from death suggests that some of the
physiological consequences of the CI impairment, such as a sudden
reduction in ATP production (59), a decrease (even if small) in the
m, or an increase in the ROS production, can promote
MPT. In agreement with our results, CI inhibitor-induced apoptosis in
dopaminergic PC12 cells was prevented by CyA (40) and also by free
radical scavengers (60), supporting the involvement of ROS in the
pathway leading to apoptosis following CI inhibition.
Cellular Events Associated with CI Inhibition-- After chronic rotenone treatment (72 h) of 143B cells, the percentage of CI inhibition in these cell line correlates exponentially with all the physiological parameters studied. In Fig. 8C, the model of the mechanisms whereby cells incubated with rotenone suffer the physiological consequences of a mitochondrial dysfunction is depicted. Following the values for four putative levels of CI residual activity exemplified in the figure, we suggest that when electron transport is inhibited at the CI level, cells are depleted of ATP, and ROS production is enhanced, damaging mitochondrial membranes, resulting in slight membrane depolarization and MPT induction. These mitochondrial alterations are the starting point of a cascade of events promoting apoptosis. In response to respiratory chain impairment, a calcium efflux from mitochondria probably occurs, which could contribute to perpetuate a vicious cycle.
Our results show that the cytotoxicity of rotenone at high
concentrations does not correlate to its inhibitory effect on CI because: (i) ° cells underwent apoptosis in the presence of high doses of rotenone; the levels of cell killing by concentrations of the
drug higher than 600 nM were indistinguishable from those corresponding to
+ cells and (ii)
+ cells
did not survive, even in high glucose medium, at concentrations of
rotenone five times higher of that producing a total CI inhibition. It
was important to address whether or not side effects of the drug would
affect its use in creating pharmacological models of CI inhibition.
Rotenone, at µM concentrations, is able to arrest mammalian cells in metaphase (61), not by inhibiting a specific energy-requiring step (62) but by binding directly to tubulin and
preventing microtubule assembly (61, 63), similarly to colchicine or
other "c-mitotic agents" (64). Our immunocytochemical studies
demonstrated that by using rotenone concentrations that produce
0-100% of CI inhibition, the microtubule assembly was not affected,
and no nuclear condensation was observed. Therefore, it is evident that
the two modes of action of rotenone occur at nonoverlapping ranges of
drug concentration, validating the rotenone-induced model of CI impairment.
Conclusions--
The goal of this study was to provide a
comprehensive analyses of the cellular consequences of a partial
complex I deficiency. Our chief conclusions were as follows: (i) We
demonstrated a toxic effect of CI inhibition that is different from the
one caused by a decrease in ATP production. This effect is likely to be
mediated by increased free radical production, which triggers an
apoptotic program. This cell death, however, could be prevented by
blocking the mitochondrial permeability transition pore. (ii) Our
studies validated rotenone inhibition as a good model for studying
complex I, provided that the concentrations used do not exceed the ones sufficient to cause a complete inhibition of the enzyme, thus avoiding
secondary toxicity. (iii) Finally, the quantitative correlation found
among all the parameters studied provided a "guide" for the several
physiological consequences of a partial CI inhibition at the cellular
level and underscored the important deleterious effects of even a mild,
chronic impairment in CI activity.
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ACKNOWLEDGEMENTS |
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We thank Dr. Ashok Verma and Dr. Runu Dey for
critically reading the manuscript and all the members of the MitoClub
(University of Miami) for constructive criticism. We are indebted to
Dr. Michael P. King (Thomas Jefferson University, Philadelphia, PA) for
the cell line 143B/206 °, Dr. R. A. Capaldi (Institute of
Molecular Biology, University of Oregon, Eugene, OR) for SDH (Fp)
monoclonal antibody, and Dr. A. Lombes (Groupe Hospitalier
Pitié-Salpêtrière, INSERM U-153, Paris, France) for
the human ND1 polyclonal antibody.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants GM55766 and EY10804.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Recipient of Grant 37289410 from the Spanish Ministerio de Educacion y Ciencia, FPI-Extranjero-1996.
To whom reprint requests should be addressed: Dept. of
Neurology, University of Miami, 1501 NW 9th Ave., Miami, FL 33136. E-mail: cmoraes{at}med.miami.edu.
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ABBREVIATIONS |
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The abbreviations used are: CI, complex I; CII, complex II; ArA, aristolochic acid; CyA, cyclosporin A; Fp, flavoprotein; HXC, human xenomitochondrial cybrids; LHON, Leber's hereditary optic neuropathy; mtDNA, mitochondrial DNA; MPT, mitochondrial permeability transition; SDH, succinate dehydrogenase; H2DCFDA, 2'-7' dichlorofluorescein diacetate; PBS, phosphate-buffered saline; JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazole carbocyanide iodide; ROS, reactive oxygen species.
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REFERENCES |
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