The targets of 2,2’,5,5’-tetrachlorobiphenyl in the respiratory chain of rat
liver mitochondria revealed by modular kinetic analysis
V. Mildaziene1,2, Z. Nauciene1,2 & K. Krab3
1
Vytautas Magnus University, Vileikos 8, 3035 Kaunas, Lithuania; 2Institute for Biomedical
Research, Kaunas Medical University, Eiveniu 4, 3009 Kaunas, Lithuania; 3Free University,
De Boelelaan 1087, 1081 HV Amsterdam, the Netherlands
Correspondence to Vida Mildaziene, tel. 370-7-733678, fax. 370-7-796498; E-mail:
[email protected]
Key words: 2,2’,5,5’- tetrachlorbiphenyl, liver mitochondria, oxidative phosphorylation,
respiration, kinetic analysis
Abstract
The response of the respiratory subsystem of oxidative phosphorylation to the
environmental pollutant, 2,2’,5,5’-tetrachlorobiphenyl (2,2’,5,5’-TCB) was investigated by
modular kinetic approach. The effects of 20 mM 2,2’,5,5’-TCB on the activity of the
respiratory chain modules in rat liver mitochondria oxidizing succinate (+ rotenone) in state 3
were assessed. The toxin inhibited the rate of respiration by 23%. Analysis around
cytochrome c revealed that 2,2’,5,5’-TCB inhibited both cytochrome c-oxidizing and reducing modules. The toxin inhibited also CoQ-oxidizing module, however it did not affect
the kinetics of CoQ-reducing module. Taken together, these data indicated that 2,2’,5,5’-TCB
inhibited cytochrome bc1 but had no effect on succinate dehydrogenase.
Introduction
Polychlorinated biphenyls (PCB) are a widespread group of enviromental pollutants
inducing many toxic effects in humans and experimental animals. It was shown (Nishihara et
al., 1985, 1986, 1992), that among various PCB congeners, 2,2’,5,5’-tetrachlorobiphenyl
(2,2’,5,5’-TCB) is one of the most potent inhibitors of mitochondrial respiration that
interferes with several components of the electron transport chain (namely, succinate
dehydrogenase and cytochrome bc1, whereas Complex I and cytochrome oxidase were not
affected by 2,2’,5,5’- TCB). Also, 2,2’,5,5’-TCB uncouples oxidative phosphorylation
(Nishihara et al, 1985).
We have recently applied modular kinetic analysis to investigate the effects of 2,2’,5,5’TCB in rat liver mitochondria respiring on succinate (+ rotenone) and glutamate + malate in
state 3 (Mildaziene et al., 2002). The results showed that 2,2’,5,5’-TCB affected all three
modules of the oxidative phosphorylation system: it inhibited both the respiratory and the
phosphorylation subsystems, and increased the membrane leak. We obtained evidence that at
least one of the targets of 2,2’,5,5’-TCB action within the phosphorylation module was ATP
synthase, and that in the respiratory subsystem Complex I was inhibited.
In this work we investigate the response of the oxidative phosphorylation in isolated rat
liver mitochondria to one of the most toxic TCB isomers, 2,2’,5,5’-TCB, by means of
modular kinetic analysis applied at the level of the respiratory subsystem.
Materials and methods
Mitochondria were isolated by differential centrifugation from the liver of male Wistar
rats weighing 275 - 300 g (Mildaziene et. al., 2002). The mitochondrial pellet was
resuspended to an approximate protein concentration 50 mg/ml. The protein concentration
was determined by the Pierce BCA method (Smith et al., 1985).
The experiments were performed at 25oC using 5 mM succinate (+ 2 mM rotenone) as a
substrate. Mitochondrial concentration in experiments was 1.0 mg/ml. The rate of
mitochondrial respiration was measured using the Clark electrode after preincubation of
mitochondria for 3 min in the absence of 2,2’,5,5’-TCB or in the presence of 20 mM 2,2’,5,5’TCB in 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. The reaction was started by
addition of 1 mM of ATP. We used 2,2’,5,5’-TCB (99.8 % purity) manufactured by «Chem
Service», USA.
Results
Modular kinetic analysis was performed with the aim of localizing effects of 2,2’,5,5’TCB on the respiratory chain of the rat liver mitochondria respiring with succinate
(+rotenone) in state 3. The respiratory chain is conceptually divided into two modules around
two different intermediates – coenzyme Q (CoQ) (CoQ-reducing and CoQ-oxidizing
modules) and cytochrome c (cytochrome c-reducing and cytochrome c-oxidizing modules).
The steady-state rate of oxygen consumption and steady-state reduction level of an
intermediate were measured at the same time. The kinetic dependencies of cytochrome creducing and -oxidizing modules on cytochrome c reduction state were determined by
measuring a number of steady-states in which the rate of cytochrome c oxidation and
reduction was varied by titration with KCN and malonate, respectively. The kinetics of the
CoQ reduction was determined by modulating the activity of the CoQ-oxidizing module by
myxotiazole titration and the kinetics of CoQ oxidation - by malonate titration of the CoQ
reducing module as described earlier (Mildaziene et al., 2002).
The kinetic dependencies of fluxes through each module (with succinate as a respiratory
substrate) were determined in the absence and in the presence of 20 mM (or 20 nmol/mg)
2,2’,5,5’-TCB. The results showed that 2,2’,5,5’-TCB decreased the rate of oxygen
consumption from 132±12 to 101±8 nmol O/min per mg (n = 6). The kinetic curves of
cytochrome c reduction and cytochrome c oxidation were obviously shifted by 2,2’,5,5’-TCB
towards the lower rates at the same level of cytochrome c reduction (Fig. 1,A) implying that
200
Respiratory rate,
nmolO/min per mg protein
160
A
120
B
160
120
80
80
40
40
0
0
0
20
40
60
Fraction of reduced cyt c, %
80
40
50
60
70
Fraction of reduced CoQ,%
Figure 1. The effect of TCB on the kinetics of cytochrome c and CoQ reducing and oxidizing
modules. A - l,¢ - kinetics of cytochrome c-oxidizing module, s,Í - kinetics of cytochrome creducing module; l,s - in the absence of 2,2’,5,5’-TCB, ¢,Í - + 20 mM 2,2’,5,5’-TCB; B - l,¢kinetics of CoQ-oxidizing module; s,Í- kinetics of CoQ-reducing module; l,s- in the absence of
2,2’,5,5’-TCB, ¢,Í -+ 20 mM 2,2’,5,5’-TCB. The results of typical experiment are presented, the
same results were obtained in two other experiments.
80
2,2’,5,5’-TCB inhibited components in both modules. The same type of analysis around
CoQ revealed that 2,2’,5,5’-TCB did not have effect on kinetics of CoQ-reducing module
(dicarboxylate carrier and succinate dehydrogenase), however it inhibited the CoQ-oxidizing
module (Fig. 1, B). From these two sets of results (Fig. 1, A and B) the conclusion was
derived that the inhibition of the respiratory chain by 2,2’,5,5’-TCB is explainable by
inhibition of cytochrome bc1 (since dicarboxylate carrier + succinate dehydrogenase, the other
components of cytochrome c-reducing module, are not affected) and inhibition of the process
catalysed by cytochrome oxidase.
From the kinetic dependencies (Fig. 1) the elasticity of each module towards the
common intermediate was determined and the flux control coefficients were calculated (see
Appendix). The results showed that cytochrome bc1 determines the flux control by 59%
(2,2’,5,5’-TCB increases this value to 69%), cytochrome oxidising module has about 33%
(27% with 2,2’,5,5’-TCB) of the control, however, the contribution of the module comprised
of dicarboxylate carrier and succinate dehydrogenase to the control of flux was negligible.
Discussion
In this study we demonstrate the usefullness of modular kinetic analysis in the decoding
the individual molecular targets of toxic multi-site effects in the multienzyme system. The
results obtained indicate that 2,2’,5,5’-TCB inhibited cytochrome bc1 but did not affect
succinate dehydrogenase in the respiratory chain of liver mitochondria in state 3. Since our
experiments are performed in state 3, the module oxidizing cytochrome c is comprised of
cytochrome oxidase operating together with two subsystems consuming the membrane
potential – the phosphorylation module and the proton leak module. We showed in the
previous study (Mildaziene et al., 2002) that proton leak is stimulated and phosphorylation
subsystem is inhibited by 2,2’,5,5’-TCB, and small increase in the membrane potential in
state 3 was observed in mitochondria respiring with succinate. These two effects through
changes in the membrane potential (Mildaziene et al., 2002) may contribute to the inhibition
in the overall activity of the module oxidizing cytochrome c. Therefore the results obtained in
this study do not provide evidence whether 2,2’,5,5’-TCB has direct effect on cytochrome
oxidase or not.
The analysis of flux control distribution implies that cytochrome bc1 carried the largest
part of the control over respiration flux among all of the complexes of the respiratory chain,
followed by cytochrome c oxidizing module. Therefore, inhibition of these two modules by
2,2’,5,5’-TCB had a clear effect on the overall flux. The response analysis (Fell, 1997)
showed that inhibition of cytochrome bc1 determined 67% and inhibition of cytochrome
oxidizing module – 33% of the 2,2’,5,5’-TCB-induced inhibition of the overall flux through
the respiratory chain.
Interaction of 2,2’,5,5’-TCB with the myxothiazole-sensitive site in Complex III was
reported by other authors (Nishihara et al.,1986) and our study confirms their finding.
However, in contrast to their suggestions, our results indicated that succinate dehydrogenase
is not affected, but instead, the process catalysed by cytochrome oxidase is inhibited by
2,2’,5,5’-TCB. This contradiction may be explained by the fact that 2,2’,5,5’-TCB effects on
activity of succinate dehydrogenase cytochrome oxidase were directly estimated by
Nishihara’s group under the conditions when mitochondria cannot maintain the
transmembrane proton gradient (Nishihara et al.,1986). Possibly, interaction of lypophylic
toxin with membrane protein complexes might depend on their conformational changes
induced by membrane energisation.
Acknowledgement : This study was supported by the EC program Copernicus (contract No.
ERBIC15 CT960307) and the Lithuanian Foundation of Science and Education.
References
Fell D.A., In: Understanding the control of metabolism. Snell K. (Ed.) London and Miami: Portland
Press, 1997, p. 121.
Mildaziene, V., Nauciene, Z., Baniene, R., and Grigiene, J., Toxicol. Sci., 65 (2002) 220.
Mildaziene, V., Nauciene, Z., Baniene, R., Ciapaite, J., Krab K., BioComplexity, 1 (2002) in press.
Nishihara, Y., Iwata, M., Ikawa, K., Puttmann, M., Robertson, L. W., Miyahara, M., Terada, H., and
Utsumi, K., Chem Pharm Bull (Tokyo), 40 (1992) 2769.
Nishihara, Y., Robertson, L. W., Oesch, F., and Utsumi, K. J., Pharmacobiodyn., 8 (1985) 726.
Nishihara, Y., Robertson, L. W., Oesch, F., Utsumi, K., Life Sci., 38 (1986) 627.
Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto,
E.K., Goeke, N.M., Olson, B.J. and Klenk, D.C., Anal. Biochem., 150 (1985) 76.
Appendix
The contribution of CoQ-reducing module (dicarboxylate carrier and succinate
dehydrogenase) to the control of respiration was negligible, most of the flux control (92%)
resided in the CoQ-oxidizing module (cytochrome bc1 + cytochrome c oxidizing module), and
2,2’,5,5’-TCB even increased contribution of this module to the control (to 96%). The flux
control between the two modules around cytochrome c was distributed to a larger degree,
however, the dominating role belonged to cytochrome c-reducing module (dicarboxylate
carrier + succinate dehydrogenase + cytochrome bc1). The flux control of cytochrome bc1
calculated by substracting the value of either CCoQ-red from Ccyt c-red, or Ccyt c-ox from CCoQ-ox
equaled to 0.59. This value increased to 0.69 upon exposure to 2,2’,5,5’-TCB. The shift of the
control from cytochrome c-oxidizing to –reducing module was not statistically significant,
possibly because in this case 2,2’,5,5’-TCB inhibited one component in each of the modules.
Table 1. The flux control coefficients of the CoQ-reducing (CCoQ-red), CoQ-oxidizing
(CCoQ-ox), cytochrome c-reducing (Ccyt c-red) and cytochrome c-oxidizing (Ccyt c-ox) modules
over the rate of respiration under state 3 conditions in liver mitochondria in the absence of
2,2’,5,5-TCB and in the presence of 20 mM 2,2’,5,5-TCB.
Ci
Without 2,2’,5,5-TCB
20 mM 2,2’,5,5-TCB
CCoQ-red
0.08±0.01
0.04±0.01*
CCoQ-ox
0.92±0.01
0.96±0.01*
Ccyt c-red
0.67±0.07
0.73±0.06
Ccyt c-ox
0.33±0.07
0.27±0.06
Substrate - succinate (+rotenone). * - indicates statistically significant difference (p<0.05),
n = 3.