65, 220 –227 (2002)
Copyright © 2002 by the Society of Toxicology
TOXICOLOGICAL SCIENCES
Multiple Effects of 2,2⬘,5,5⬘-Tetrachlorobiphenyl on Oxidative
Phosphorylation in Rat Liver Mitochondria
Vida Mildaziene,* ,† ,1 Zita Nauciene,* ,† Rasa Baniene,† and Jurgita Grigiene*
*Vytautas Magnus University, Vileikos 8, 3035 Kaunas, Lithuania; and †Institute for Biomedical Research,
Kaunas Medical University, Eiveniu 4, 3009 Kaunas, Lithuania
Received August 13, 2001; accepted October 15, 2001
An experimental investigation of the response of the multicomponent oxidative phosphorylation system to the environmental
pollutant 2,2ⴕ,5,5ⴕ-tetrachlorobiphenyl (2,2ⴕ,5,5ⴕ-TCB) was performed by modular kinetic analysis in rat liver mitochondria
oxidizing succinate (ⴙ rotenone) and glutamate ⴙ malate. This
approach facilitates the analysis of a complex process by dividing
it into a small number of modules, each comprising multiple
enzymatic steps, and allows evaluation of changes in the kinetics
of individual blocks of the complex system induced by multisite
effectors. Kinetic dependencies of the respiratory subsystem, the
phosphorylation subsystem, and the proton permeability of the
inner membrane on the membrane potential ⌬⌿ were determined
in the control and in the presence of 20 ␮M 2,2ⴕ,5,5ⴕ-TCB. The
toxin inhibited the rate of respiration with both substrates to a
similar extent (by 23–26%). We showed that 2,2ⴕ,5,5ⴕ-TCB affected the all three modules of the oxidative phosphorylation
system: it inhibited both the respiratory and the phosphorylation
subsystems, and increased the membrane leak. As a result, the
value of ⌬⌿ in State 3 of mitochondria oxidizing glutamate ⴙ
malate remained the same or slightly increased with succinate,
indicating that in the former case the respiratory subsystem was
more sensitive to 2,2ⴕ,5,5ⴕ-TCB. We explain this by the 2,2ⴕ,5,5ⴕTCB–induced inhibition of Complex I. Moreover, 2,2ⴕ,5,5ⴕ-TCB
decreased the number of oligomycin-binding sites by 20%, caused
a significant drop in the membrane potential generated by ATP
hydrolysis, and inhibited activity of ATP hydrolysis in uncoupled
mitochondria. Thus, we obtained evidence that at least one of the
targets of 2,2ⴕ,5,5ⴕ-TCB action within the phosphorylation module
was ATP synthase.
Key Words: 2,2ⴕ,5,5ⴕ-tetrachlorobiphenyl; liver mitochondria;
oxidative phosphorylation; respiration; membrane potential; ATP
synthase; Complex I; kinetic analysis.
Polychlorinated biphenyls (PCB) are a widespread group of
environmental pollutants consisting of 209 different forms of
chlorinated biphenyl. Their production was halted in most
countries (e.g., in 1977 in the United States, but in Lithuania
only in 2000), but PCB levels persist in the environment
1
To whom correspondence should be addressed. Fax: 370-7-796498.
E-mail: [email protected].
because of their long half-life (Kimbrough, 1987) and further
use in older industrial transformers. Because of their lipophilic
nature, these stable organic compounds accumulate in the lipid
biophase of living cells, such as biomembranes, and remain
there for a long time (Matthews and Anderson, 1975). Toxicity
of PCBs has been proved in humans (Hsu et al., 1984; Sauer et
al., 1994) and experimental animals, and as the results of
numerous studies indicate, the liver is especially susceptible to
deleterious effects of PCBs. It was shown that the administration of PCBs causes change in cristae orientation and an
increase in mitochondrial volume of the liver in animals (Gilroy et al., 1996, 1998). An impairment of mitochondrial functions by different PCBs has been extensively investigated by a
Japanese group (Nishihara et al., 1985, 1986, 1992), who
showed that the inhibitory potency of PCB varied depending
on steric conformation as well as chloro-substituent sites in
PCB. For hexachlorobiphenyls, it was established that orthochlorinated congeners possessing nonplanar conformation and
additional substitution in either meta or para positions were
most effective; moreover, ortho-substitution in both rings determined the highest potency (Nishihara et al., 1992). These
compounds are also strong uncouplers of oxidative phosphorylation (Nishihara et al., 1985). Remarkable differences in the
ability of five different isomers of tetrachlorobiphenyl (TCB)
to inhibit the components of the mitochondrial electron transport chain were indicated (Nishihara et al., 1985) in that
2,2⬘,3,3⬘-, 2,2⬘,4,4⬘- and 2,2⬘,5,5⬘-TCBs were potent inhibitors
of mitochondrial respiration, while 2,2⬘,6,6⬘- and 3,3⬘,4,4⬘TCBs were significantly less effective. It was also suggested
that the TCB-induced inhibition of mitochondrial respiration
was caused by interference with several components of the
electron transport chain (succinate dehydrogenase and cytochrome bc 1), whereas Complex I and cytochrome oxidase were
supposed to be not affected by TCB (Nishihara et al., 1985).
An essential feature unifying many noxious effectors is their
simultaneous interaction with many different sites within the
oxidative phosphorylation system. The scheme of the oxidative
phosphorylation system in mitochondria is presented in Figure
1. Electrons from oxidizable substrates enter the respiratory
chain via different routes: electrons to Complex I (NADH
220
TCB EFFECTS ON OXIDATIVE PHOSPHORYLATION
FIG. 1. Components of the oxidative phosphorylation system. Complexes
of the respiratory chain are denoted by Roman numerals I, II, III, IV, respectively. The natural membrane permeability to protons (the membrane leak) is
indicated by the dotted arrow. The components responsible for phosphorylation by numbers: 1, phosphate carrier; 2, ATP synthase; 3, ATP/ADP carrier.
dehydrogenase) are donated by the substrates reducing NAD ⫹,
while electrons originating from succinate are passed to ubiquinone (UQ) via Complex II (succinate dehydrogenase). Further,
the electrons are transferred to oxygen via Complex III (cytochrome bc 1 complex), cytochrome c, and Complex IV (cytochrome oxidase). Electron flow through Complexes I, III, and
IV is coupled with the outward pumping of protons, producing
both chemical (⌬pH) and electrical (⌬⌿) gradient because the
inner mitochondrial membrane has very low natural permeability to protons (the proton leak). The ATP synthase makes use
of the electrochemical gradient by coupling the inward proton
flux with the ADP phosphorylation reaction. In addition, transport of substrates for ATP synthesis (phosphate and ADP) into
mitochondrial matrix is also driven by the membrane potential
(⌬⌿).
In this work we investigate the response of this complex
system to one of the most toxic TCB isomers, 2,2⬘,5,5⬘-TCB,
by means of modular kinetic analysis, which is particularly
informative when analyzing the response of multicomponent
systems to multisite effectors. The rationale of the modular
kinetic approach is as follows: the complex metabolic system
is simplified by conceptually dividing it into several modules
centered around the common intermediate. In order to examine
which components of the system are influenced by an effector,
the effector-induced shift in kinetic dependencies of each module on the concentration of the intermediate are determined.
This method (referred to as the “top-down” approach, or the
kinetic elasticity analysis) was first experimentally applied by
a group of researchers in Cambridge (Hafner et al., 1990) for
the analysis of kinetics of individual blocks of the oxidative
221
phosphorylation system. In their approach, the system of oxidative phosphorylation is conceptually divided into three subsystems connected by the common intermediate proton motive
force (⌬p) or, with some restrictions, by the membrane potential (⌬⌿); ⌬p is generated by the respiratory subsystem and
consumed by the phosphorylation subsystem and the membrane leak subsystem (Fig. 2). We estimated activities of the
subsystems from dependencies of the fluxes through the three
subsystems on the membrane potential (⌬⌿). The shift in
kinetic dependencies induced by the effector indicated which
of the three subsystems was directly affected.
The primary goal of the present study is to elucidate possible
sites of action of 2,2⬘,5,5⬘-TCB on the oxidative phosphorylation system by means of modular kinetic analysis. We show
how this method combined with standard biochemical assays
can be used for the identification of molecular targets of toxins
in a multicomponent metabolic system. This approach has
helped us to reveal new sites sensitive to 2,2⬘,5,5⬘-TCB action
in the oxidative phosphorylation system of liver mitochondria,
Complex I, and ATP synthase.
MATERIALS AND METHODS
Mitochondria were isolated by differential centrifugation from the liver of
male Wistar rats weighing 275–300 g. The animals were killed according to the
rules defined by the European Convention for the Protection of Vertebrate
Animals Used for Experimental and Other Scientific Purposes (License No.
0006). The liver was quickly removed and placed into ice-cold isotonic (0.9%)
KCl solution. The tissue was cut into small pieces and homogenized in a
glass-Teflon homogenizer with a medium containing 10 mM Tris-HCl, 250
mM sucrose, 3 mM EGTA, and 4 mg/ml bovine serum albumin (BSA), pH 7.7
(at 2°C). The homogenate was centrifuged at 750 ⫻ g for 5 min, and the
supernatant was centrifuged at 7000 ⫻ g for 10 min. The obtained pellet was
washed in a buffer containing 250 mM sucrose, 5 mM Tris-HCl, pH 7.3 (at
2°C). The final centrifugation was done at 7000 ⫻ g for 10 min. The
mitochondrial pellet was resuspended in the buffer to an approximate protein
concentration of 50 mg/ml. The protein concentration was determined by the
FIG. 2. Division of the oxidative phosphorylation system into 3 subsystems connected by the membrane potential ⌬⌿. J R, flux through the
respiratory subsystem; J L, flux through the membrane leak subsystem; J P, flux
through the phosphorylation subsystem.
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MILDAZIENE ET AL.
biuret method (Gornal et al., 1949). The BSA standard solution was used for
quality control.
The quality of mitochondrial preparations was determined by the respiratory
control index (RCI), equal to the ratio of the respiratory rates (V 3/V 2) of
mitochondria in State 3 and in State 2, according to the common terminology
(Chance and Williams, 1955).
The respiration and membrane potential of mitochondria were measured in
a closed, stirred, and thermostated 1.5-ml vessel fitted both with a Clark-type
oxygen electrode (Rank Brothers Ltd., Cambridge, UK) and a tetraphenylphosphonium (TPP ⫹)-selective electrode (A. Zimkus, Vilnius University,
Lithuania).
To maintain mitochondrial respiration in the stationary state, one of the
requirements of modular kinetic, or “top-down,” analysis (Fell, 1997; Hafner
et al., 1990), we used the creatine phosphokinase ADP-regenerating system,
which was developed in our laboratory earlier (Kholodenko et al., 1987) for
the analysis of action of different agents affecting the processes involved in
oxidative phosphorylation. The peculiarity of this system is that it does not
exert control over respiration, and therefore the oxygen consumption rate is
determined only by mitochondrial processes. This advantage allows optimum
manifestation of changes in kinetics of oxidative phosphorylation induced by
any effector. This experimental system has been successfully used for the
investigation of effects of various effectors or conditions on the functional
activity of mitochondria (Borutaite et al., 1989, 1995; Marcinkeviciute et al.,
2000, Mildaziene et al., 1995, 1996).
The experiments were performed at 37°C using 5 mM succinate (⫹ 2 ␮M
rotenone) or 5 mM glutamate ⫹ 5 mM malate as a substrate. Mitochondrial
concentration in the probe was 1.0 mg/ml. The rate of mitochondrial respiration corresponding to the rate in State 3 was registered after addition of 1 mM
ATP to the incubation medium containing 110 mM KCl, 20 mM Tris-HCl, 5
mM KH 2PO 4, 50 mM creatine, excess of creatine kinase, 1 mM MgCl 2, pH
7.2. TPP ⫹ (133–266 nM) was added for the membrane potential measurements. Mitochondrial swelling was monitored spectrophotometrically at 520
nm wavelength in the same medium.
Modular kinetic analysis was applied to determine the kinetic changes
induced by 2,2⬘,5,5⬘-TCB on the level of the oxidative phosphorylation system. In this approach, we split the system of oxidative phosphorylation into
three subsystems, or modules, connected by the proton motive force (⌬p),
which consists of two components: the mitochondrial membrane potential
(⌬⌿) and the proton concentration gradient (⌬pH). ⌬p is generated by the
respiratory subsystem (R) and consumed by the phosphorylation subsystem (P)
and the membrane leak subsystem (L). We could measure changes in ⌬p as
changes in ⌬⌿ without introducing a significant error, provided the ⌬pH value
was small and the changes in ⌬pH were negligible. Direct measurement of
⌬pH in liver mitochondria (Mildaziene et al., 2000) confirmed that this was so
in the case of our experiments. Therefore, we estimated activities of the
subsystems from dependencies of the fluxes through these subsystems on ⌬⌿
(Fig. 2). We determined the following kinetic dependencies on ⌬⌿:
● The dependence of the flux through the respiratory subsystem (J R ) on ⌬⌿
is determined by titrating mitochondrial respiration in State 3 with an inhibitor
of the phosphorylation system (carboxyatractyloside 0 – 0.15 nmol/mg mitochondrial protein).
● The dependence of the flux through the membrane leak subsystem (J L ) on
⌬⌿ is determined under the conditions of complete inhibition of phosphorylation by excess oligomycin (1 ␮g/mg). The rate of succinate oxidation is
titrated with malonate (0 –3 mM), and the rate of glutamate ⫹ malate oxidation
with rotenone (0 – 0.06 nmol/mg mitochondrial protein).
● The dependence of the flux through the phosphorylation subsystem (J P ) on
⌬⌿ is determined using a similar protocol of titrations (with malonate, 0 –1
mM; rotenone, 0 – 0.013 nmol/mg mitochondrial protein) under the conditions
of active respiration (without oligomycin). J P at any given ⌬⌿ is calculated as
J P ⫽ J R – J L at the same ⌬⌿.
The effect of 2,2⬘,5,5⬘-TCB on oxidative phosphorylation was estimated
from these three titration sets, performed on the same preparation of liver
mitochondria in the absence of 2,2⬘,5,5⬘-TCB and in the presence of 20 ␮M
2,2⬘,5,5⬘-TCB. We used 2,2⬘,5,5⬘-TCB (99.8% purity) manufactured by Chem
Service, West Chester, PA.
The membrane potential generated during ATP hydrolysis was measured
with TPP ⫹-selective electrode in the same medium without a substrate and
creatine kinase, and in the presence of antimycin A (2 ␮g/mg protein). In all
assays, the functional parameters of mitochondria were monitored after a
3-min preincubation of mitochondria with 20 ␮M 2,2⬘,5,5⬘-TCB.
ATPase activity was measured at 37°C by recording the pH changes in the
incubation chamber during the ATP hydrolysis reaction in medium containing
110 mM KCl, 2.5 mM Tris-HCl, 2.5mM KH 2PO 4, 5 mM MgCl 2, antimycin A
(140 ng/mg protein), 3 mM ATP, 70 nM carbonyl cyanid-m-chlorophenylhydrasone (CCCP), pH 8. Concentration of mitochondria in these experiments
was 1.5 mg/ml. Buffering capacity of the reaction mixture was determined
experimentally by recording the pH change induced by addition of the known
quantity of HCl (12 nM H ⫹) to the reaction mixture after each measurement.
ATPase activity was estimated from the initial rate of increase in the H ⫹
concentration during ATP hydrolysis. Control experiments showed that the H ⫹
formation was inhibited by oligomycin by more than 95%.
Activity of Complex I was measured spectrophotometrically by following
the kinetics of NADH reduction at 340 nm (Ragan et al, 1987) in fractured
mitochondria (by rapid freezing-thawing of mitochondria, repeated four
times). Measurements were performed at 37°C in medium containing 110 mM
KCl, 20 mM Tris-HCl, 5 mM KH 2PO 4, 1 mM MgCl 2, antimycin A (1 ␮g/ml),
0.1 mg/ml NADH, fractured mitochondria (0.2 mg mitochondrial protein/ml),
pH 7.2. The reaction was started after a 3-min preincubation by adding 100
␮g/ml coenzyme Q 10. Enzymatic activity was calculated using the extinction
coefficient of NADH 6.81 mM –1cm –1.
Statistical analysis. The mean of each experiment was calculated as average for two or three repetitive runs. The effect of a toxic agent was analyzed
on the same mitochondrial preparation by comparing their functional parameters in the absence of 2,2⬘,5,5⬘-TCB and in the presence of 20 ␮M 2,2⬘,5,5⬘TCB (paired experiments). The data points in figures and text are expressed as
the mean of 3–5 experiments on different preparations of mitochondria ⫾ SE.
Statistical analysis of the data was done by Student t-test. Statistical significance was assumed at p ⬍ 0.05.
RESULTS
The aim of our experiments was to study the effect of
2,2⬘,5,5⬘-TCB at a low concentration that does not compromise
the membrane permeability barrier. Mitochondrial swelling
under our experimental conditions was detectable when the
concentration of 2,2⬘,5,5⬘-TCB in extramitochondrial medium
exceeded 30 ␮M. Therefore, we compared kinetic dependencies of the fluxes through the subsystems of oxidative phosphorylation on ⌬⌿ in liver mitochondria oxidizing different
substrates (succinate or glutamate ⫹ malate) in the absence and
in the presence of 20 ␮M (or 20 nmol/mg) 2,2⬘,5,5⬘-TCB.
The results obtained in experiments with liver mitochondria
oxidizing succinate ⫹ rotenone are presented in Figures 3A,
3B, and 3C. It can be seen that 2,2⬘,5,5⬘-TCB decreased the
rate of mitochondrial respiration in State 3 from 182 ⫾ 8 to
134 ⫾ 8 ng-atoms of oxygen (ngatomO)/min/mg and induced
a small but statistically significant increase in ⌬⌿ from 143 ⫾
3 to 146 ⫾ 3 mV. The shift of kinetic curves (the change in
respiratory rates at the same value of ⌬⌿) indicated that 20 ␮M
of 2,2⬘,5,5⬘-TCB affected the all three subsystems of oxidative
phosphorylation: it increased the membrane leak of the inner
membrane (Fig. 3A) and inhibited the respiratory (Fig. 3B) and
TCB EFFECTS ON OXIDATIVE PHOSPHORYLATION
223
FIG. 3. Kinetic dependencies of mitochondrial respiration with succinate (⫹ rotenone) on the membrane potential ⌬⌿. (A) Kinetics of the membrane leak.
(B) Kinetics of the respiratory subsystem. (C) Kinetics of the phosphorylation subsystem. Filled circle, control; open circle, ⫹ 20 ␮M TCB. The mean of each
experiment was calculated as average of two or three repetitive runs. Data points in the figure are expressed as the mean of five experiments on different
preparations of mitochondria ⫾ SE.
phosphorylation subsystems (Fig. 3C). Although the shift in
kinetics of the membrane leak caused by 2,2⬘,5,5⬘-TCB was
not large (Fig. 3A), the flux through this subsystem J L in State
3 increased by more than twice (114%). In addition, 2,2⬘,5,5⬘TCB inhibited the fluxes through the respiratory subsystem J R
(by 26%; Fig. 3B) and phosphorylation subsystem J P (by 32%;
Fig. 3C).
A very important parameter in this type of analysis is the
change in ⌬⌿ induced by an effector. Whether the ⌬⌿ level
increases, remains the same, or decreases will depend on which
of the subsystems (⌬⌿-producing or ⌬⌿-consuming reactions)
is inhibited or stimulated more. A decrease in ⌬⌿ indicates
that the ⌬⌿-producing block is inhibited more than the ⌬⌿consuming block, whereas an increase in ⌬⌿ indicates the
opposite, and ⌬⌿ does not change if the ⌬⌿-producing and the
⌬⌿-consuming blocks are inhibited to the same extent. Thus,
the resulting increase in ⌬⌿ by 3 mV in State 3 indicates that,
in the overall response of the system to 2,2⬘,5,5⬘-TCB, inhibition of phosphorylation has dominated over the other two
effects (inhibition of the respiratory subsystem and increase in
the membrane leak), both leading to the decrease in ⌬⌿.
Essentially the same pattern of the 2,2⬘,5,5⬘-TCB–induced
changes was determined when modular kinetic analysis on
mitochondria respiring with glutamate ⫹ malate was performed (Fig. 4). The rate of glutamate ⫹ malate oxidation
(respiratory flux, J R) in State 3 was decreased by 2,2⬘,5,5⬘-TCB
to a similar degree (23%) as in the case of succinate oxidation:
from 141 ⫾ 6 to 109 ⫾ 7 ngatomO/min per mg. The respiratory (Fig. 4B) and the phosphorylation (Fig. 4C) subsystems
were inhibited (the phosphorylation flux J P decreased from
FIG. 4. Kinetic dependencies of mitochondrial respiration with glutamate ⫹ malate on the membrane potential ⌬⌿. (A) Kinetics of the membrane leak. (B)
Kinetics of the respiratory subsystem. (C) Kinetics of the phosphorylation subsystem. Filled circle, control; open circle, ⫹ 20 ␮M TCB. The mean of each
experiment was calculated as the average of two or three repetitive runs. Data points in the figure are expressed as the mean of four experiments on different
preparations of mitochondria ⫾ SE.
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MILDAZIENE ET AL.
FIG. 5. Inhibition of NADH oxidation in freeze-fractured liver mitochondria by TCB. The mean of each experiment was calculated as the average of
two or three repetitive runs. Data points in the figure are expressed as the mean
of five experiments on different preparations of mitochondria ⫾ SE.
134 ⫾ 5 to 98 ⫾ 7 ngatO/min per mg), but an increase in the
membrane leak (Fig. 4A) was smaller (J L in State 3 increased
from 8 ⫾ 1 to 11 ⫾ 1 ngatO/min per mg) as compared with
succinate oxidation. Despite the marked changes in the activities of the three subsystems induced by 2,2⬘,5,5⬘-TCB (Fig. 4),
the value of ⌬⌿ in State 3 of mitochondria oxidizing glutamate ⫹ malate has remained the same (144 ⫾ 2 in control and
143 ⫾ 2 mV with 2,2⬘,5,5⬘-TCB), implying that the decrease in
the activity of ⌬⌿-producing reactions (the respiratory subsystem) is completely compensated by a diminution in the
activity of ⌬⌿-consumers (the membrane leak ⫹ the phosphorylation subsystem).
Thus, the balance of the 2,2⬘,5,5⬘-TCB–induced changes
between ⌬⌿-producers and ⌬⌿-consumers differs from that of
succinate oxidation (when ⌬⌿ increases). Taking into account
that 2,2⬘,5,5⬘-TCB causes a smaller increase in the membrane
leak with glutamate ⫹ malate, this implies that the sensitivity
of the respiratory subsystem to 2,2⬘,5,5⬘-TCB is higher when
mitochondria oxidize glutamate ⫹ malate as compared with
succinate. Different sensitivity of the respiratory subsystem to
2,2⬘,5,5⬘-TCB in these two cases might be explained by interaction of 2,2⬘,5,5⬘-TCB with Complex I, which is involved in
the electron transport from NADH substrates (e.g., glutamate ⫹ malate), but not from succinate (Fig. 1). To elucidate
this, we determined the activity of Complex I by NADH
oxidation in freeze-fractured liver mitochondria in the absence
of 2,2⬘,5,5⬘-TCB and in the presence of various concentrations
of 2,2⬘,5,5⬘-TCB. Our results showed (Fig. 5) that 2,2⬘,5,5⬘TCB inhibited complex I (the determined value [I 50] was 82 ⫾
10 ␮M).
In the experiments described above (Figs. 3 and 4), we
noticed that, in the presence of 2,2⬘,5,5⬘-TCB, the amount of
oligomycin needed for complete inhibition of phosphorylation
was less than in the control. To quantitate how 2,2⬘,5,5⬘-TCB
changes the binding of specific inhibitors to important components of the phosphorylation subsystem, ATP synthase and
ATP/ADP carrier, we performed titrations of the respiratory
rate in mitochondria oxidizing succinate (⫹ rotenone) in State
3 with oligomycin and with carboxyatractyloside (Fig. 6). As
became clear from these experiments (Fig. 6), 20 ␮M 2,2⬘,5,5⬘TCB significantly decreased (by 20%) the number of the oligomycin-binding sites, whereas the number of the carboxyatractyloside-binding sites remained the same. This
FIG. 6. Oligomycin (A) and carboxyatractyloside (B) titration of the mitochondrial respiration rate in State 3. Substrate, succinate (⫹ rotenone). Filled circle,
control; open circle, ⫹ 20 ␮M TCB. The mean of each experiment was calculated as average of two or three repetitive runs. Data points in figure are expressed
as the mean of three experiments on different preparations of mitochondria ⫾ SE.
TCB EFFECTS ON OXIDATIVE PHOSPHORYLATION
finding implied the possible interaction of 2,2⬘,5,5⬘-TCB with
ATP synthase.
Direct measurement of the ATP synthase activity is complicated because the flux of ADP phosphorylation is under tight
control of part of the oxidative phosphorylation system, i.e., the
respiratory chain that is itself sensitive to 2,2⬘,5,5⬘-TCB (Fig.
3B) has a major part of the control over phosphorylation flux
(Hafner et al., 1990; Mildaziene et al., 1996, 2000). Therefore,
evidence of the direct 2,2⬘,5,5⬘-TCB action on ATP synthase
was obtained by assessing ATP hydrolysis. Under the conditions when ⌬⌿ is collapsed, ATPase generates ⌬⌿ at the
expense of ATP hydrolysis, and therefore the value of the
membrane potential depends only on the activity of ATPase.
Our results showed that 20 ␮M 2,2⬘,5,5⬘-TCB caused the drop
in ATP hydrolysis-generated ⌬⌿ from 107 ⫾ 1 to 94 ⫾ 2 mV
(n ⫽ 3). However this effect may also be caused by the ability
of 2,2⬘,5,5⬘-TCB to increase the membrane leak for protons
(Figs. 3A, 3A), not only by the inhibition of ATPase. Additional evidence of the direct interaction of 2,2⬘,5,5⬘-TCB with
ATPase was obtained by pH-metrically assessing the activity
of ATPase. In the absence of an uncoupler, addition of 20 ␮M
2,2⬘,5,5⬘-TCB slightly stimulated ATP hydrolysis. This finding
is in line with the 2,2⬘,5,5⬘-TCB–induced increase in the membrane leak. However, when the rate of ATP hydrolysis was
maximally stimulated by an uncoupler (continuous elimination
of ⌬⌿ was induced by 70 nM CCCP), the rate of ATP
hydrolysis was inhibited by 2,2⬘,5,5⬘-TCB from 3.0 ⫾ 0.3 to
1.9 ⫾ 0.4 nmol H ⫹/min per mg. Thus, on the whole, our results
show that 2,2⬘,5,5⬘-TCB inhibits ATP hydrolysis. Therefore,
we suggest that at least one of the targets of the 2,2⬘,5,5⬘- TCB
action within the phosphorylation module is ATP synthase.
DISCUSSION
Mitochondria are special organelles in eukaryotic cells that
efficiently convert energy available in the substrate molecules
to the universal fuel for cellular processes, ATP. Oxidative
phosphorylation is a key pathway used by the most aerobic
cells to harvest energy, e.g., about 40 –50% of ATP in the liver
is produced by mitochondria. Therefore, the normal function of
all other processes within these cells is ultimately dependent on
the energy production in mitochondria. Disturbance of the
mitochondrial function underlies many metabolic diseases. For
this reason, the evaluation of dysfunction of oxidative phosphorylation is often given crucial importance in biomedical and
toxicological research.
In numerous mitochondrial studies, changes in separate
functional parameters (e.g., rate of respiration in metabolic
states 4 and 3, and/or uncoupled state, membrane potential,
swelling) are measured with the aim of evaluating the influence
of different effectors or damaging factors on oxidative phosphorylation. However, the established increases or decreases
are only of minor informative value for understanding the
molecular reasons of the observed changes. It appears that
225
standard methods of enzymology are inadequate to deal with
kinetics and control of intricate metabolic pathways. The metabolic control theory was developed (Kacser and Burns, 1973,
1979; Heinrich and Rapoport, 1974) for analyzing the kinetic
behavior of multicomponent enzyme systems. The theory gives
a qualitatively new point of view in understanding the metabolic control, because it considers not only properties related
with individual processes, but also properties determined by
inherent complexity of metabolic systems (systematic properties), reviewed in Fell (1997). Within the framework of this
theory, a group of British researchers developed the “topdown” method (or modular kinetic analysis), which was first
experimentally applied for the analysis of the fluxes in oxidative phosphorylation (Hafner et al., 1990). This approach facilitates the analysis of a complex process by dividing it into a
small number of modules, each comprising multiple enzymatic
steps. By rather moderate experimental efforts (measuring the
uptake of oxygen simultaneously with the membrane potential
⌬⌿ and manipulating the fluxes with specific inhibitors), one
may gain important information about the contribution of each
subsystem to the changes induced by a multisite effector in the
overall flux through the system. This method has been applied
in different ways in different systems: isolated mitochondria,
intact cells, perfused tissues, organs, and entire organisms
(Ainscow and Brand, 1999; Brown et al., 1990; Borutaite et al.,
1995; Kavanagh et al., 2000, Mildaziene et al., 1996; Marcinkeviciute et al., 2000). It has been successfully used for the
study of toxic effect of cadmium ions on oxidative phosphorylation in potato mitochondria (Kesseler and Brand, 1994).
In the present study we demonstrate how the modular kinetic
analysis is used to identify the distribution of a multisite toxic
effect of the organochemical pollutant 2,2⬘,5,5⬘-TCB on the
oxidative phosphorylation system in rat liver mitochondria.
This method allowed us to demonstrate that 2,2⬘,5,5⬘-TCB,
even at low concentration, affects many sites in the machinery
of mitochondrial energy transformation. It increases the membrane leak and inhibits both the respiratory and the phosphorylation subsystems, thus causing a slight (succinate oxidation)
or substantial change (glutamate ⫹ malate oxidation) in ⌬⌿.
Because the resulting change in ⌬⌿ reflects the balance of
changes in the kinetics of ⌬⌿-producers and ⌬⌿-consumers, it
may serve as a sensitive measure to evaluate the degree to
which different blocks are affected. Interaction of 2,2⬘,5,5⬘TCB with the myxothiazole-sensitive site in Complex III and
succinate dehydrogenase (Nishihara et al., 1986) may be
deemed responsible for the inhibition of the respiratory subsystem in case of succinate oxidation. However, the analysis of
⌬⌿ changes indicated stronger inhibition of the respiratory
subsystem with NADH-dependent substrate (glutamate ⫹
malate) than with succinate as a respiratory substrate. This
finding helped us to detect that Complex I in the respiratory
chain was inhibited by 2,2⬘,5,5⬘-TCB. Although it was supposed in earlier studies (Nishihara et al., 1985) that Complex I
is not sensitive to 2,2⬘,5,5⬘-TCB, such assumption was not
226
MILDAZIENE ET AL.
based on direct estimation of enzymatic activity. Contrary to
this assumption, the results obtained by standard biochemical
assay (Fig. 5) clearly show that Complex I is inhibited by the
toxin. Also, it may be possible that the binding of 2,2⬘,5,5⬘TCB to components of oxidative phosphorylation depends on
temperature, as succinate oxidation was inhibited to a much
higher extent compared with glutamate ⫹ malate when the
experiments were performed at 25°C (Nishihara and Utsumi,
1986), whereas at a physiologically more relevant temperature
(37°C), oxidation of the both substrates was inhibited by 20
␮M 2,2⬘,5,5⬘-TCB to the same extent (Figs. 3 and 4).
The observed effects of 2,2⬘,5,5⬘-TCB on the membrane
leak and the respiratory chain are in full agreement with the
results reported by other authors (Nishihara et al., 1985, Nishihara et al., 1986). Although nonplanar TCBs were reported to
show strong uncoupling action (Nishihara et al., 1985), it is
doubtful that TCB can act as a traditional uncoupler (the
lipophilic proton carrier). The uncoupling of oxidative phosphorylation by TCBs could possibly be explained by changes
in the membrane properties after incorporation of the unusual
lipophilic compound into the membrane structure.
In this study we demonstrate the usefulness of modular
kinetic analysis in the detection of toxic effects on the phosphorylation subsystem. These effects cannot be estimated in a
direct way, because in mitochondria oxidizing different substrates, the flux of ATP synthesis (phosphorylation) is largely
controlled by the other two subsystems, mostly by the respiratory subsystem (Hafner et al., 1990; Mildaziene et al., 1996,
2000). Therefore, any factor disturbing the activity of the
respiratory subsystem (or the membrane leak) via induced
changes in ⌬⌿ elicits secondary effects on the phosphorylation
flux. In modular kinetic analysis, the flux of phosphorylation is
estimated by subtracting the portion of oxygen consumption
determined by the membrane leak J L from the flux through the
respiratory subsystem J R at the certain value of ⌬⌿ (i.e., J P ⫽
J R – J L). Thus, this approach provides a valuable tool for the
detection of changes in J P kinetics per se induced by an
effector. In mitochondria oxidizing both succinate and glutamate ⫹ malate, we obtained an obvious shift toward lower
activity in the kinetics of the phosphorylation subsystem. A
noticeable decrease in the oligomycin-binding sites indicated
interaction of 2,2⬘,5,5⬘-TCB with ATP synthase, and inhibition
of ATPase by 2,2⬘,5,5⬘-TCB was confirmed by the standard
biochemical assay of the ATP hydrolytic activity. Therefore,
we suppose that ATP synthase is at least one of the targets of
2,2⬘,5,5⬘-TCB determining inhibition of the phosphorylation
subsystem and contributing significantly to the overall
2,2⬘,5,5⬘-TCB effect on oxidative phosphorylation.
We conclude that modular kinetic analysis is a useful tool
for the evaluation of multisite effects of toxins, and here we
present an example of how it could be used for the identification of targets of noxious action of 2,2⬘,5,5⬘-TCB on the
oxidative phosphorylation system in rat liver mitochondria.
This approach allowed us to decode the pleiotropic nature of
the impairment of mitochondrial respiration by 2,2⬘,5,5⬘-TCB.
We show that disturbance in the activity of all three subsystems
(or modules) contributes to the overall inhibition of oxidative
phosphorylation, and in further analysis we reveal several
individual components within the system that are sensitive to
the toxin. We conclude that this lipophilic organic pollutant,
which is easily incorporated in the membrane, has a rather
unspecific action as it affects the membrane leak, Complexes I,
II (succinate dehydrogenase), and III (cytochrome bc1) in the
respiratory chain and ATP synthase. We can only speculate on
the extent to which these effects are caused by the change in
composition and properties of the mitochondrial membrane
and the lipid environment that is essential for function of the
membrane proteins. However, at least for ATP synthase, a
more specific interaction with 2,2⬘,5,5⬘-TCB may be supposed
(as judged by the decrease in the oligomycin-binding sites).
Whatever the molecular details of the 2,2⬘,5,5⬘-TCB interaction with components of oxidative phosphorylation, it becomes
clear that by simultaneously acting on many steps, it efficiently
inhibits the physiologically important fluxes of respiration and
ATP synthesis in liver mitochondria. Most probably, other
congeners of 2,2⬘,5,5⬘-TCB share this property to a great
extent. Therefore, modular kinetic analysis will be helpful to
elucidate the relationship between their toxic power and ability
to interact with different components within the oxidative
phosphorylation system.
ACKNOWLEDGMENT
This study was supported by the EC program Copernicus (Contract No.
ERBIC15 CT960307) and the Lithuanian Foundation of Science and Education.
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