Multiple Effects of 2,2`,5,5`-Tetrachlorobiphenyl on Oxidative Phosphorylation in Rat Liver Mitochondria

Vida Mildaziene*,{dagger},1, Zita Nauciene*,{dagger}, Rasa Baniene{dagger} and Jurgita Grigiene*

* Vytautas Magnus University, Vileikos 8, 3035 Kaunas, Lithuania; and {dagger} Institute for Biomedical Research, Kaunas Medical University, Eiveniu 4, 3009 Kaunas, Lithuania

Received August 13, 2001; accepted October 15, 2001


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
An experimental investigation of the response of the multicomponent oxidative phosphorylation system to the environmental pollutant 2,2`,5,5`-tetrachlorobiphenyl (2,2`,5,5`-TCB) was performed by modular kinetic analysis in rat liver mitochondria oxidizing succinate (+ rotenone) and glutamate + 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 {Delta}{Psi} were determined in the control and in the presence of 20 µM 2,2`,5,5`-TCB. The toxin inhibited the rate of respiration with both substrates to a similar extent (by 23–26%). We showed that 2,2`,5,5`-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 {Delta}{Psi} in State 3 of mitochondria oxidizing glutamate + malate remained the same or slightly increased with succinate, indicating that in the former case the respiratory subsystem was more sensitive to 2,2`,5,5`-TCB. We explain this by the 2,2`,5,5`-TCB–induced inhibition of Complex I. Moreover, 2,2`,5,5`-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 2,2`,5,5`-TCB action within the phosphorylation module was ATP synthase.

Key Words: 2,2`,5,5`-tetrachlorobiphenyl; liver mitochondria; oxidative phosphorylation; respiration; membrane potential; ATP synthase; Complex I; kinetic analysis.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 because of their long half-life (Kimbrough, 1987Go) 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, 1975Go). Toxicity of PCBs has been proved in humans (Hsu et al., 1984Go; Sauer et al., 1994Go) 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., 1996Go, 1998Go). An impairment of mitochondrial functions by different PCBs has been extensively investigated by a Japanese group (Nishihara et al., 1985Go, 1986Go, 1992Go), 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 ortho-chlorinated 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., 1992Go). These compounds are also strong uncouplers of oxidative phosphorylation (Nishihara et al., 1985Go). 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., 1985Go) 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 bc1), whereas Complex I and cytochrome oxidase were supposed to be not affected by TCB (Nishihara et al., 1985Go).

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 1Go. Electrons from oxidizable substrates enter the respiratory chain via different routes: electrons to Complex I (NADH 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 bc1 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 ({Delta}pH) and electrical ({Delta}{Psi}) 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 ({Delta}{Psi}).



View larger version (120K):
[in this window]
[in a new window]
 
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.

 
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., 1990Go) for the analysis of kinetics of individual blocks of the oxidative phosphorylation system. In their approach, the system of oxidative phosphorylation is conceptually divided into three subsystems connected by the common intermediate proton motive force ({Delta}p) or, with some restrictions, by the membrane potential ({Delta}{Psi}); {Delta}p is generated by the respiratory subsystem and consumed by the phosphorylation subsystem and the membrane leak subsystem (Fig. 2Go). We estimated activities of the subsystems from dependencies of the fluxes through the three subsystems on the membrane potential ({Delta}{Psi}). The shift in kinetic dependencies induced by the effector indicated which of the three subsystems was directly affected.



View larger version (26K):
[in this window]
[in a new window]
 
FIG. 2. Division of the oxidative phosphorylation system into 3 subsystems connected by the membrane potential {Delta}{Psi}. JR, flux through the respiratory subsystem; JL, flux through the membrane leak subsystem; JP, flux through the phosphorylation subsystem.

 
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
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 x g for 5 min, and the supernatant was centrifuged at 7000 x 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 x 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 biuret method (Gornal et al., 1949Go). 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 (V3/V2) of mitochondria in State 3 and in State 2, according to the common terminology (Chance and Williams, 1955Go).

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, 1997Go; Hafner et al., 1990Go), we used the creatine phosphokinase ADP-regenerating system, which was developed in our laboratory earlier (Kholodenko et al., 1987Go) 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., 1989Go, 1995Go; Marcinkeviciute et al., 2000Go, Mildaziene et al., 1995Go, 1996Go).

The experiments were performed at 37°C using 5 mM succinate (+ 2 µM 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 KH2PO4, 50 mM creatine, excess of creatine kinase, 1 mM MgCl2, 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 ({Delta}p), which consists of two components: the mitochondrial membrane potential ({Delta}{Psi}) and the proton concentration gradient ({Delta}pH). {Delta}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 {Delta}p as changes in {Delta}{Psi} without introducing a significant error, provided the {Delta}pH value was small and the changes in {Delta}pH were negligible. Direct measurement of {Delta}pH in liver mitochondria (Mildaziene et al., 2000Go) 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 {Delta}{Psi} (Fig. 2Go). We determined the following kinetic dependencies on {Delta}{Psi}:

• The dependence of the flux through the respiratory subsystem (JR) on {Delta}{Psi} 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 (JL) on {Delta}{Psi} is determined under the conditions of complete inhibition of phosphorylation by excess oligomycin (1 µg/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 (JP) on {Delta}{Psi} 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). JP at any given {Delta}{Psi} is calculated as JP = JR – JL at the same {Delta}{Psi}.

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 µM 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 µg/mg protein). In all assays, the functional parameters of mitochondria were monitored after a 3-min preincubation of mitochondria with 20 µM 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 KH2PO4, 5 mM MgCl2, 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, 1987Go) 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 KH2PO4, 1 mM MgCl2, antimycin A (1 µg/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 µg/ml coenzyme Q10. 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 µM 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
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 µM. Therefore, we compared kinetic dependencies of the fluxes through the subsystems of oxidative phosphorylation on {Delta}{Psi} in liver mitochondria oxidizing different substrates (succinate or glutamate + malate) in the absence and in the presence of 20 µM (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 3CGo. 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 {Delta}{Psi} from 143 ± 3 to 146 ± 3 mV. The shift of kinetic curves (the change in respiratory rates at the same value of {Delta}{Psi}) indicated that 20 µM of 2,2`,5,5`-TCB affected the all three subsystems of oxidative phosphorylation: it increased the membrane leak of the inner membrane (Fig. 3AGo) and inhibited the respiratory (Fig. 3BGo) and phosphorylation subsystems (Fig. 3CGo). Although the shift in kinetics of the membrane leak caused by 2,2`,5,5`-TCB was not large (Fig. 3AGo), the flux through this subsystem JL in State 3 increased by more than twice (114%). In addition, 2,2`,5,5`-TCB inhibited the fluxes through the respiratory subsystem JR (by 26%; Fig. 3BGo) and phosphorylation subsystem JP (by 32%; Fig. 3CGo).



View larger version (14K):
[in this window]
[in a new window]
 
FIG. 3. Kinetic dependencies of mitochondrial respiration with succinate (+ rotenone) on the membrane potential {Delta}{Psi}. (A) Kinetics of the membrane leak. (B) Kinetics of the respiratory subsystem. (C) Kinetics of the phosphorylation subsystem. Filled circle, control; open circle, + 20 µM 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.

 
A very important parameter in this type of analysis is the change in {Delta}{Psi} induced by an effector. Whether the {Delta}{Psi} level increases, remains the same, or decreases will depend on which of the subsystems ({Delta}{Psi}-producing or {Delta}{Psi}-consuming reactions) is inhibited or stimulated more. A decrease in {Delta}{Psi} indicates that the {Delta}{Psi}-producing block is inhibited more than the {Delta}{Psi}-consuming block, whereas an increase in {Delta}{Psi} indicates the opposite, and {Delta}{Psi} does not change if the {Delta}{Psi}-producing and the {Delta}{Psi}-consuming blocks are inhibited to the same extent. Thus, the resulting increase in {Delta}{Psi} 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 {Delta}{Psi}.

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. 4Go). The rate of glutamate + malate oxidation (respiratory flux, JR) 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. 4BGo) and the phosphorylation (Fig. 4CGo) subsystems were inhibited (the phosphorylation flux JP decreased from 134 ± 5 to 98 ± 7 ngatO/min per mg), but an increase in the membrane leak (Fig. 4AGo) was smaller (JL 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. 4Go), the value of {Delta}{Psi} 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 {Delta}{Psi}-producing reactions (the respiratory subsystem) is completely compensated by a diminution in the activity of {Delta}{Psi}-consumers (the membrane leak + the phosphorylation subsystem).



View larger version (13K):
[in this window]
[in a new window]
 
FIG. 4. Kinetic dependencies of mitochondrial respiration with glutamate + malate on the membrane potential {Delta}{Psi}. (A) Kinetics of the membrane leak. (B) Kinetics of the respiratory subsystem. (C) Kinetics of the phosphorylation subsystem. Filled circle, control; open circle, + 20 µM 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.

 
Thus, the balance of the 2,2`,5,5`-TCB–induced changes between {Delta}{Psi}-producers and {Delta}{Psi}-consumers differs from that of succinate oxidation (when {Delta}{Psi} 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. 1Go). 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. 5Go) that 2,2`,5,5`-TCB inhibited complex I (the determined value [I50] was 82 ± 10 µM).



View larger version (14K):
[in this window]
[in a new window]
 
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.

 
In the experiments described above (Figs. 3 and 4GoGo), 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. 6Go). As became clear from these experiments (Fig. 6Go), 20 µM 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 finding implied the possible interaction of 2,2`,5,5`-TCB with ATP synthase.



View larger version (17K):
[in this window]
[in a new window]
 
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 µM 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.

 
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. 3BGo) has a major part of the control over phosphorylation flux (Hafner et al., 1990Go; Mildaziene et al., 1996Go, 2000Go). Therefore, evidence of the direct 2,2`,5,5`-TCB action on ATP synthase was obtained by assessing ATP hydrolysis. Under the conditions when {Delta}{Psi} is collapsed, ATPase generates {Delta}{Psi} 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 µM 2,2`,5,5`-TCB caused the drop in ATP hydrolysis-generated {Delta}{Psi} 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, 3AGo), 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 µM 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 {Delta}{Psi} 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
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 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, 1973Go, 1979Go; Heinrich and Rapoport, 1974Go) 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 "top-down" method (or modular kinetic analysis), which was first experimentally applied for the analysis of the fluxes in oxidative phosphorylation (Hafner et al., 1990Go). 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 {Delta}{Psi} 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, 1999Go; Brown et al., 1990Go; Borutaite et al., 1995Go; Kavanagh et al., 2000Go, Mildaziene et al., 1996Go; Marcinkeviciute et al., 2000Go). It has been successfully used for the study of toxic effect of cadmium ions on oxidative phosphorylation in potato mitochondria (Kesseler and Brand, 1994Go).

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 {Delta}{Psi}. Because the resulting change in {Delta}{Psi} reflects the balance of changes in the kinetics of {Delta}{Psi}-producers and {Delta}{Psi}-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., 1986Go) may be deemed responsible for the inhibition of the respiratory subsystem in case of succinate oxidation. However, the analysis of {Delta}{Psi} 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., 1985Go) that Complex I is not sensitive to 2,2`,5,5`-TCB, such assumption was not based on direct estimation of enzymatic activity. Contrary to this assumption, the results obtained by standard biochemical assay (Fig. 5Go) 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, 1986Go), whereas at a physiologically more relevant temperature (37°C), oxidation of the both substrates was inhibited by 20 µM 2,2`,5,5`-TCB to the same extent (Figs. 3 and 4GoGo).

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., 1985Go, Nishihara et al., 1986Go). Although nonplanar TCBs were reported to show strong uncoupling action (Nishihara et al., 1985Go), 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., 1990Go; Mildaziene et al., 1996Go, 2000Go). Therefore, any factor disturbing the activity of the respiratory subsystem (or the membrane leak) via induced changes in {Delta}{Psi} 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 JL from the flux through the respiratory subsystem JR at the certain value of {Delta}{Psi} (i.e., JP = JR – JL). Thus, this approach provides a valuable tool for the detection of changes in JP 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.


    ACKNOWLEDGMENTS
 
This study was supported by the EC program Copernicus (Contract No. ERBIC15 CT960307) and the Lithuanian Foundation of Science and Education.


    NOTES
 
1 To whom correspondence should be addressed. Fax: 370-7-796498. E-mail: bioch{at}kmu.lt. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ainscow, E. K., and Brand, M. D. (1999). Internal regulation of ATP turnover, glycolysis and oxidative phosphorylation in rat hepatocytes. Eur. J. Biochem. 266, 737–749.[Abstract/Free Full Text]

Brown, G. C., Lakin-Thomas, P. L., and Brand, M. D. (1990). Control of respiration and oxidative phosphorylation in isolated rat liver cells. Eur. J. Biochem. 192, 355–362.[Abstract]

Borutaite, V., Mildaziene, V., Brown, G. C., and Brand, M. D. (1995). Control and kinetic analysis of ischemia-damaged heart mitochondria: which parts of the oxidative phosphorylation system are affected by ischemia? Biochim. Biophys. Acta 1272, 154–158.[ISI][Medline]

Borutaite, V., Mildaziene, V., Ivanoviene, L., Kholodenko, B. N., Toleikis, A., and Praskevicius, A. (1989). The role of long-chain acyl-CoA in the damage of oxidative phosphorylation in heart mitochondria. FEBS Lett. 243, 264–266.[ISI][Medline]

Chance, B., and Williams, R. B. (1955). Respiratory enzymes in oxidative phosphorylation. Kinetics of oxygen utilization. J. Biol. Chem. 217, 385–393.

Fell, D. A. (1997). Understanding the Control of Metabolism. Portland Press, London and Miami.

Gilroy, C., Connell, B. J., Singh, A., Suidgeest, P., and Chu, I. (1998). PCB congener 77-induced ultrastructural alterations in the rat liver: A quantitative study. Toxicology 127, 179–85.[ISI][Medline]

Gilroy, C., Singh, A., Chu, I., and Villeneuve, D. C. (1996). Toxicity of PCB 156 in the rat liver: an ultrastructural and biochemical study. J. Submicrosc. Cytol. Pathol. 28, 27–32.[ISI][Medline]

Gornal, A. G., Bardavill, G. J., and David, M. M. (1949). Determination of serum proteins by means of the biuret reaction. J. Biol. Chem. 177, 751–766.[Free Full Text]

Hafner, R. P., Brown, G. C., and Brand, M. D. (1990). Analysis of the control of respiration rate, phosphorylation rate, proton leak rate and protonmotive force in isolated mitochondria using the `top-down' approach of metabolic control theory. Eur. J. Biochem. 188, 313–319.[Abstract]

Heinrich, R., and Rapoport, T. A. (1974). A linear steady-state treatment of enzymatic chains. General properties, control and effector strength. Eur. J. Biochem. 42, 89–95.[ISI][Medline]

Hsu, S. T., Ma, C. I., Hsu, S. K., Wu, S. S., Hsu, N. H., and Yeh, C. C. (1984). Discovery and epidemiology of PCB poisoning in Taiwan. Am. J. Ind. Med. 5, 71–79.[ISI][Medline]

Kacser, H., and Burns, J. (1973). The control of flux. In Rate Control of Biological Processes (D. D. Davies, Ed.), pp. 65–104. Cambridge University Press, Cambridge.

Kacser, H., and Burns, J. A. (1979). Molecular democracy: who shares the controls? Biochem. Soc. Trans. 7, 1149–1160.[Medline]

Kavanagh, N. I., Ainscow, E. K., and Brand, M. D. (2000). Calcium regulation of oxidative phosphorylation in rat skeletal muscle mitochondria. Biochim. Biophys. Acta 1457, 57–70.[ISI][Medline]

Kesseler, A., and Brand, M. D. (1994). Quantitative determination of the regulation of oxidative phosphorylation by cadmium in potato tuber mitochondria. Eur. J. Biochem. 225, 923–935.[Abstract]

Kholodenko, B., Zilinskiene, V., Borutaite, V., Ivanoviene, L., Toleikis, A., and Praskevicius, A. (1987). The role of adenine nucleotide translocators in regulation of oxidative phosphorylation in heart mitochondria. FEBS Lett. 233, 247–250.

Kimbrough, R. D. (1987). Human health effects of polychlorinated biphenyls (PCBs) and polybrominated biphenyls (PBBs). Annu. Rev. Pharmacol. Toxicol. 27, 87–111.[ISI][Medline]

Matthews, H. B., and Anderson, M. W. (1975). The distribution and excretion of 2,4,5,2`,5`-pentachlorobiphenyl in the rat. Drug. Metab. Dispos. 3, 211–219.[Abstract]

Marcinkeviciute, A., Mildaziene, V., Crumm, S., Demin, O., Hoek, J. B., and Kholodenko, B. (2000). Kinetics and control of oxidative phosphorylation in rat liver mitochondria after chronic ethanol feeding. Biochem. J. 349, 519–526.[ISI][Medline]

Mildaziene, V., Baniene, R., Nauciene, Z., Bakker, B. M., Brown, G. C., Westerhoff, H. V., and Kholodenko, B. N. (1995). Calcium indirectly increases the control exerted by the adenine nucleotide translocator over 2-oxoglutarate oxidation in rat heart mitochondria. Arch. Biochem. Biophys. 324, 130–134.[ISI][Medline]

Mildaziene, V., Baniene, R., Nauciene, Z., and Ciapaite, J. (2000). Impairment of oxidative phosphorylation in rat liver mitochondria by both cadmium and copper ions is caused by inhibition of the respiratory subsystem. In Animating the Cellular Map (J.-H.S. Hofmeyr, J. M. Rowher, and J. L. Snoep, Eds.), pp.143–150. Stellenbosch University Press, Stellenbosch, South Africa.

Mildaziene, V., Baniene, R., Nauciene, Z., Marcinkeviciute, A., Morkuniene, R., Borutaite, V., Kholodenko, B., and Brown, G. C. (1996). Ca2+ stimulates both the respiratory and phosphorylation subsystems in rat heart mitochondria. Biochem. J. 320, 329–334.[ISI][Medline]

Nishihara, Y., Iwata, M., Ikawa, K., Puttmann, M., Robertson, L. W., Miyahara, M., Terada, H., and Utsumi, K. (1992). The influence of chloro-substituent sites of hexachlorobiphenyl on the respiration of rat liver mitochondria. Chem. Pharm. Bull. (Tokyo) 40, 2769–2774.[ISI][Medline]

Nishihara, Y., Robertson, L. W., Oesch, F., and Utsumi, K. (1985). Interaction of tetrachlorobiphenyls with isolated rat liver mitochondria. J. Pharmacobiodyn. 8, 726–732.[Medline]

Nishihara, Y., Robertson, L. W., Oesch, F., and Utsumi, K. (1986). The effects of tetrachlorobiphenyls on the electron transfer reaction of isolated rat liver mitochondria. Life Sci. 38, 627–635.[ISI][Medline]

Nishihara, Y., and Utsumi, K. (1986). 2,2`5,5`-tetrachlorbiphenyl impairs the bioenergetic functions of isolated rat liver mitochondria. Biochem. Pharmacol. 35, 3335–3339.[ISI][Medline]

Ragan, C. I., Wilson, M. T., Darley-Usmar, V. M., and Lowe, P. N. (1987). Sub-fractionation of mitochondria and isolation of the proteins of oxidative phosphorylation. In MitochondriaA Practical Approach (G. C. Brown and C. E. Cooper, Eds.) pp. 39–62. Oxford University Press, Oxford.

Sauer, P. J., Huisman, M., Koopman-Esseboom, C., Morse, D. C., Smits-van Prooije, A. E., van de Berg, K. J., Tuinstra, L. G., van der Paauw, C. G., Boersma, E. R., Weisglas-Kuperus, N., et al. (1994). Effects of polychlorinated biphenyls (PCBs) and dioxins on growth and development. Hum. Exp. Toxicol. 13, 900–906.[ISI][Medline]