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The effects of tetrachlorobiphenyls on the electron transfer reaction of isolated rat liver mitochondria
Life Sciences, Vol. 38, pp. 627-635 Printed in the U.S.A.
Pergamon Press
THE EFFECTSOF TETRACHLOROBIPHENYLSON THE ELECTRONTRANSFER REACTION OF ISOLATED RAT LIVER MITOCHONDRIA Y. Nishihara 1.
L. W. Robertsonz F. Oesch2 and K. Utsumi1
1Department of Medical Biology• Kochi Medical School, Kohasu, Okocho, Nangoku-shi, Kochi 781-51, Japan and 21nstitut f~r Toxikology, Universit~t Mainz, Mainz, Obere Zahlbacher Str. 67, D-6500, Mainz, West Germany (Received in final form December 2, 1985)
Summary A comparative study was made of the effects of several sMnmetrical tetrachlorobiphenyls (TCBs) on the electron transfer from succinate to oxygen of rat liver mitochondria, and some differences in effects caused by the different chlorine positions of the biphenyl ring were clarified. TCBsused in this study included 2 , 3 , 2 ' , 3 ' - , 2 , 4 , 2 ' , 4 ' - , 2 , 5 , 2 ' , 5 ' - , 2 , 6 , 2 ' , 6 ' - , and 3,4,3',4'-TCBs. The inhibitory actions of 2 , 3 , 2 ' , 3 ' - , 2 , 4 , 2 ' , 4 ' - , and 2,5,2',5'-TCBs on succinate oxidase were potent• while those caused by 2,6,2',6'- and 3,4,3',4'-TCBs were significantly weak. The inhibition sites of 2 , 3 , 2 ' , 3 ' - , 2 , 4 , 2 ' , 4 ' - , and 2,5,2',5'-TCBs in succinate oxidase were succinate dehydrogenase and cytochrome b-c segment of the electron transport chain. In the cy'cochrome b-~ segment, these TCBs acted on myxothiazol-sensitive site rather than antimycin-sensitive site. Cytochrome~ oxidase was hardly affected by TCBs. These results indicate that 2 , 3 , 2 ' , 3 ' - , 2 , 4 , 2 ' , 4 ' - , and 2,5,2',5'-TCBs severely depress the electron transfer with succinate as the substrate, which secondarily reduces the s~thesis of ATP. The relationship between the activity and chemical structure of TCBs is also discussed. Polychlorinated biphenyls (PCBs) are industrial chemicals that have been detected in various species of wildlife as well as in human tissues and milk (I-3). The administration of PCBs to experimental animals produces characteristic toxic syndromes and variety of biochemical responses. Among the toxic syndromes reported are skin disorders such as chloracne, th)mic involution, edema, and hepatotoxic events (4,5). Some of the biochemical responses include porphyria (6) and induction of hepatic microsomal drug-metabolizing enz~es (7-9). The relationships between actions on these enz~es and chemical structures of PCBs have been well characterized. We reported previously the biochemical effects of PCBs on the functions of rat liver mitochondria; commercial PCB mixtures (Kanechlors) inhibited mitochondrial ATP synthesis in vitro (10). However, very l i t t l e is known about the structure-activity relationship of PCBs on the energy-transducing functions of mitochondria. We have shown in f a i r l y detail the effects of several sM~metrical tetrachlorobiphenyls (TCBs) on succinate-supported respirations of rat liver mitochondria (11). That is, 2,3•2',3'-, 2 , 4 , 2 ' , 4 ' - , and 2,5,2',5'-TCBs had potent * -- To whom correspondence should be addressed. 0024-3205/86 $3.00 + .00 Copyright (c) 1986 Pergamon Press Ltd.
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inhibitory actions on state 3 respiration (active respiration in the presence of ADP), while those produced by 2 , 6 , 2 ' , 6 ' - and 3,4,3',4'-TCBs were weak. It was also suggested that TCB-induced inhibition of state 3 respiration was mainly caused by the interference with electron transport chain. Here we present a report on the effects of TCBs on the succinate-oxidizing enz~es of rat l i ver mitochonria, and show the sites of inhibition in the electron transport chain. Materials and Methods Chemicals 2 , 3 , 2 ' , 3 ' - , 2 , 4 , 2 ' , 4 ' - , 2 , 5 , 2 ' , 5 ' - , 2 , 6 , 2 ' , 6 ' - , and 3,4,3',4'-Tetrachlorobiphenyls (TCBs) were synthesized by the Ullman condensation of the corresponding dichloroiodobenzenes (12). The purities of these isomers were found to be more than 99% by gas liquid chromatography. The stock solutions ef these isomers except 3,4,3',4'-TCB were prepared in ethanol, and that of 3,4,3',4'-TCB was prepared in dimethylformamide. Antimycin A, and bovine serum albumin were purchased from Sigma Chemical Co. (St Louis, MO). Phenazine methosulfate (PMS), N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD), and 2,4-dinitrophenol (DNP) were purchased fronl Nakarai Chemicals (Kyoto, Japan). Other reagents were commercial products of the highest purity. Preparation of mitochondria Liver mitochondria were isolated from male Wistar rats (200-300 g) by the standard method in a medium containing 0.25 M sucrose, 5 mM Tris-HCI (pH 7.4), and 0.1 n~4 EDTA. EDTAwas omitted in a final wash and resuspension (13). Mitochondrial protein was determined by the biuret method using bovine serum albumin as a standard (14). Enz,vme assays Succinate oxidase, cytochrome ~ oxidase, and succinate dehyOrogenase activities were measured polarographically using a Clark-type oxygen electrode in a 2 ml water-thermostated glass reaction cell maintained at 25°C. The assay medium consisted of 0.2 M sucrose, 20 mM KCI, 3 mM MgCl2, and 5 n~4 potassium phosphate (pH 7.4). Succinate oxidase and cytochrome ~ oxidase a c t i v i t i e s were assayed in assay medium containing 25uM DNP and i mg/ml of mitochondria. Mitochondria were interacted with TCB for 3 min, then respiration was started by the addition of 5 mM succinate (succinate oxidase) or 5 mM ascorbate/O.! mM TMPD (cytochrome £ oxidase), respectively. Succinate dehydrogenase a c t i v i t y was determined using PMS as an electron acceptor in an assay medium containing 2ug/mg protein of antimycin A, 2 n~M NAN3, 5 mM succinate, and ! mg/ml of mitochondria. Mitochondria were interacted with TCB for 3 min, then respiration was initiated by the addition of 0.5 mM PMS (15). Optical spectroscopy The steady-state reductions of b (563-575 nm), and c (550-540 m~)-type cytochro~nes were measured at 25°C using Shimadzu UV-300 duaT wavelength spectrophotometer with a 2 nm band pass. In all experiments, control contained the same volume of solvent (ethanol or dimethylformamide), and the final concentration of solvent was less than 1% (v/v); the concentration of solvent did not affect the cellular a c t i v i t i e s assayed. Results The effects of TCBs on succinate oxidase are shown in Fig. 1. 2 , 3 , 2 ' , 3 ' - , 2 , 4 , 2 ' , 4 ' - , and 2,5,2',5'-TCBs inhibited succinate oxidase strongly. The concentrations of 2 , 3 , 2 ' , 3 ' - , 2 , 4 , 2 ' , 4 ' - , and 2,5,2',5'-TCBs that gave 50~ inhi-
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bition (150) were 48.4 * 4.1, 53.1 ± 3.6, and 56.2 ± 5.3 pM, respectively(calculated from linear regression analysis, n=3). However, percentage inhibitions of 2,6,2',6'- and 3,4,3', 4'-TCBs never reached 50%, only 19 and 25% inhibitions even at 200pM. Two way analysis of variance and Duncan's multiple range test (p< 0.05) revealed that treatment with 2,3,2',3'-, 2,4,2',4'-, and 2,5,2',5'-TCBs resulted in similar rates of inhibition, while those produced by 2,6,2',6'- and 3,4,3',4'-TCBs were significantly reduced. Cytochrome ~ oxidase was far less inhibited by TCBs (data not shown). This fact indicates that TCBs act at sites before complex IV in the electron transport chain. Fig. 2 shows the effects of TCBs on succinate dehydrogenase. Two way analysis of variance and Duncan'smultiple range test (p< 0.05) revealed that treatments with 2,3,2',3'-, 2,4,2',4'-, and 2,5,2',5'-TCBs resulted in similar rates of inhibition, while those caused by 2,6,2',6'- and 3,4,3',4'-TCBs were significantly reduced. Thus, succinate dehydrogenase was inhibited by TCBs, although the extent of inhibition varied among TCBs, indicating that the inhibitory reaction for succinate oxidase occurs p a r t i a l l y in succinate dehydrogenase. Table I shows the effects of 2,5,2',5'- and 3,4,3',4'-TCBs on the aerobic steady-state reductions of cytochromes b and c of mitochondria during the oxi-
100
3
2
2'
3'
t-
.o_
"~50 C N
,:p
,.~
50 100 Concentration (uM)
200
FIG. I Comparison of the i n h i b i t o r y e f f e c t s of TCBs on succinate oxtdase of rat l t v e r mitochondrta. Succtnate oxidase a c t i v i t y was measured polarographically with a Clark-t~q~e oxygen electrode at 25~C. The mttochondrtal protein was I rag/m1. Control rate was 153.3 ~ 4.8 nat(ms O/man/ mg protein. Each point is a mean of 3 separate experiments. S. O. bars are omitted since they are always less than 51[ of the mean. Sj~bols are as follows: 2 , 3 , 2 ' , 3 ' - , 0 ; 2,4,2',4'-,&; 2,5,2',5'- rl;2,6,2,,6,_ • ; 3,4,3',4'-TCB,~ .
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Effects of TCBs on Mitochondria
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dation of succinate or glutamate/malate. With succinate, 2,5,2',5'-TCB inhibited reduction of cytochromes b and C by 43 and 80%, respectively. The inhibition of cytochrome b is caused by the inhibition of succinate dehydrogenase. When glutamate/malate was used as the substrate, the reduction of cytochrome was inhibited by 65% with 2,5,2',5'-TCB, which had no apparent effect on the reduction of cytochrome b ( see trace B in Fig. 3), suggesting that 2,5,2',5'TCB had almost no effect on the components before cytochrome b of mitochondrial electron transport chain (complex I). The inhibition of cytochrome c reduction by 2,5,2',5'-TCB with both succinate and glutamate/malate indicated that cytochrome b-~ segment of the electron transport chain was blocked by 2,5,2',5'-TCB. Identical results were obtained for 2 , 3 , 2 ' , 3 ' - and 2,4,2',4'-TCBs (data not shown). On the other hand, 3,4,3',4'-TCB had very l i t t l e effect on the cytochrome b-~ segment. 2,6,2',6'-TCB gave the identical result (data not shown). Following experiments were designed to determine the interacting site of 2,5,2',5'-TCB within the cytochrome ~-~ segment. These experin;ents were done using glutamate/malate as the substrate; the results obtained with this substrate directly reflect the information from cytochron,e b-~ segment (complex Ill), since both complex I and IV in the electron transport chain are hardly inhibited by 2,5,2',5'-TCB.
ioo L
j 0
10
50
100
2OO
Conceqntration (uM) FIG. 2
Comparison of the inhibitory effects of TCBs on succinate dehydrogenase of rat liver mitochondria. Succinate dehydrogenase activity was measured polarographicaIIy with a Clark-type oxygen electrode at 25°C. The mitochondrial protein was I mg/m]. Control activity was ]61.1± 5.9 natoms O/min/mg protein. Fach point is a mean of 3 separate experiments. S. D. bars are omitted since they are always less than 5% of the mean. Symbols are as follows: 2 , 3 , 2 ' , 3 ' - , 0 ; 2 , 4 , 2 ' , 4 ' - , • ; 2,5,2',5'0 ; 2 , 6 , 2 ' , 6 ' - , 0 ; 3,4,3',4'-TCB,~.
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Effects
o f TCBs on M i t o c h o n d r t a
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TABLE ] Effects of 2,5,2',5 °- and 3,4,3°,4'-TCBs on the aerobic steady-state reduction of cytochro~es b and ~ in rat liver mitochondria a
Additions
Percentage inhibition of aerobic steady-state reductions of cytochro~es Succinate Glutamate/malate
Control 2,5,2',5'-TCB (150 nmol/mg prot.) 3,4,3',4'-TCB (150 nmol/mg prof.)
0 43 12
0 80 22
0 0 0
0 65 II
aThe reductions of cytochrome b (563-575 nm) and5~c ( 5 5 0 - 5 4 0 2 nm) were monitored by the dual-wavelength spe~trophotometer at . The reaction medium contained 0.2 M sucrose, 25 mM KCI, 3 mMMgCI2, and 5 mM potassium phosphate (pH 7.4). The reduction of the cytochro~es was initiated by the addition of either 5 mM succinate or 5 mM glutamate/5 mMmalate into the mitochondrial suspension 3 min after the addition of TCB. Rotenone (2~M) and malonate (5 mM) were also added to the mitochondrial suspension prior to succinate and glutamate/malate, respectively. Protein concentration was 2 mg/ml. Volume, 3 ml.
A
B
C
11min A
Nuta.
A
Gluta.
I
A
FIG. 3 Effects of 2,5,2',5'-TCB on the reduction of cytochrome ~ by glutamate/malate in rat liver mitochondria. The traces show reduction of cytochrome b monitored at 563 nm versus 575 nm with a 2 nm band pass. The reaction medium consisted of 0.2 M sucrose, 20 mM KCI, 3 mM MgCI2, and 5 mM potassium phosphate (pH 7.4). Mitochondria were interacted with TCB for 3 min, then the reduction of cytochrome b was initiated by the addition of 5 mM glutamate/5 mMmalate (Gluta.).-The protein concentration was 2 mg/~]. CA) control, (B) 2,5,2',5'-TCB,]50 nmol/mg protein, (C) 2,5,2',5'-TCB, 150 nmol/mg protein plus antimycin A, 2~g/mg protein.
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Fig. 3 shows the effect of 2,5,2',5'-TCB on reduction of cytochrome b by glutamate/malate. The trace A shows the reduction of cytochrome b, when glutamate/malate was added in the absence of inhibitor (control). Under these conditions, the cytochrome b which is reduced consists primarily of b-562 (_bK) and a small portion of the low potential b-566 (_bT), residing within one cytuchrome b. When 2,5,2',5'-TCB was added before substrate, i t did not block reduction of cytochrome b (trace B). However, when 2,5,2',5'-TCB and antimycin A were applied together, almost complete inhibition of cytochrome b reduction was seen (trace C). The effect of TCB on cytochrome b reduction By substrate is very similar to that produced by myxothiazol (an antibiotic inhibitor) which has been introduced recently to the study of mitochondrial electron transfer (16,17); myxothiazol does not i n h i b i t reduction of cytochrome b by substrate when applied alone, but does i n h i b i t in the presence of antimycin A (18,19). Therefore, 2,5,2',5'-TCB may interact with myxothiazol-sensitive site in the b-~ segment. To further confirm this, the effect of TCB on the cytochrome b-£ segment was studied by creating a bypass of the antimycin block of electron flow using a r t i f i c i a l electron carrier, TMPD. Alexandreet al. (20) has showed that Wurster's blue (oxidized form of TMPD) promotes the electron flow which is inhib-
A
B
RLM f TMPD ' L ~ . . . . Anu myc, n
!i
RLM I TMPD ~ Antimycin ".5' -TCB
"--r,'°"
"
FIG. 4 The TMPD-promoted b~l)ass of the antimycin block and its sensitivity to 2,5,2',5'-TCB. Oxygenuptake was measured at 2~C in a medium of 0.2 M sucrose, 20 mM KCI, 3 m#~MgC12, 5 mM potassium phosphate (pH 7.4), and 50~M DNP. Where indicated, rat l i v e r mitochondria (RLM), 2 mg/mI;TMPD, 0.1 mM; antimycin A, 2pg/mg protein; glutamate, 5 mM glutamate/5 mMmalate; 2,5,2',5'-TCB, 150 nmol/mg protein. Volume, 2 ml.
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ited by antlmycin, by accepting electrons from some reduced intermediate site within the antimycin-inhibited complex I l l , and transferring electrons to cytochrome ~, and that this bypass is inhibited by myxothiazol. Fig. 4 shows the llqPD-promoted bypass of the antimycin block and i t s sensitivity to 2,5,2',5'TCB. Whenglutamate/malate was added to incubation medium, antimycin-blocked respiration was accelerated greatly (trace A). Thus, llqPD/Wurster's blue couple provides a bypass of the electron flow which was blocked by antimycln. The TMPD-bypass of the antimycin block was highly sensitive to 2,5,2',5'-TCB, simi l a r to myxothiazol (trace B). This fact indicates that 2,5,2',5'-TCB inhibits myxothiazol-sensitive site rather than antimycin-sensitive site in the b-~ segment of the electron transport chain. Although 2,5,2',5'-TCB was shown as the representative compound in Figs. 3 and 4, identical results were observed for 2,3,2',3'- and 2,4,2',4'-TCB (data not shown). Discussion The results presented here clearly indicate that there exist marked differences in the effect of TCBs on electron transport system of rat liver mltochondria. 2,3,2',3'-, 2,4,2',4'-, and 2,5,2',5'-TCBs showed potent inhibitory actions on succinate oxldase, while those produced by 2,6,2',6'- and 3,4,3',4'-TCBs were weak. It is clear that TCBs which show potent inhibitory actions on succinate oxidase (i. e., 2,3,2',3'-, 2,4,2',4'-, and 2,5,2',5'-TCB) have a common structure with chlorine atoms equally attached to both inside (ortho,
ortho') and outside (meta or para, meta' or para') positions of the blpe-~l ring, and that TCBs of which all chlorine atoms are localized in either inside (2,6,2',6'-TCB) or outside (3,4,3',4'-TCB) positions of the biphenyl ring are poor inhibitors. 2,3,2',3'-, 2,4,2',4'-, 2,5,2',5'-, and 2,6,2',6'-TCBs possess non-planar configuration becauseof the diortho chlorination, while 3,4,3',4'-TCB is planar configuration because of t ~ e a s e d double-bond character of the C~I~ - C(1,1 bond due to chlorine atoms in para, para' position (6). Therefore,'(Ee ned~s~aryconditions for TCBs to be e~Tec'tive'-e--'Tnhibitors of mitochondrial electron transport chain are that TCBs possess primarily nonplanar configuration and that in addition to non-planarity, position of chlorine substitution as discussed above is important ( because 2,6,2',6'-TCB possesses non-planar configuration, but is a weak inhibitor). A large body of evidence supports the view that the pathway of electrons in the cytochrome b-~ segment of the mitochondrial electron transport chain is not described by a single linear sequence of electron transfer reaction but by a Q cycle mechanism, in which the f i r s t electron from ubiquinol (QH2) passes to the Rieske FeS center and thence to cytochrome £1; the seco~ electron passes from ubisemiquinone (Q~) to cytochrome bT (21,~2). Antimycin A and myxothiazol are known to block different site ~f the Q cycle; antimycin blocks electron transfer fro~ cytochrome bK to ubiquinone in center i (23), whereas myxothiazol suppresses cooperative oxidation of ubiquinol in center o (17). Antimycin A and myxothiazol, when applied alone, do not block the reduction of cytochrome b, but when these agents are applied together, they almost completely block the reduction of cytochrome b (19). Therefore, we tested whether a mixture of antimycin and TCB (2,3,2',~'-, 2,4,2',4'-, or 2,5,2',5'-) had a similar effect. As is shown in Fig. 3, the reduction of cytochrome b by substrate was completely blocked, men antimycin and TCB are applied together. This fact indicates that TCB acts on the myxothiazol-sensitive site.This was further confirmed by use of the bypass of the antimycin block of mitochondrial electron transport (20). TCB blocked ll(PD-pron~oted bypass Of t~e antimycin block (Fig. 4), similar to myxothiazol (see Fig. 4 in Ref. 20). These facts suggest that TCB acts on myxothiazol-sensitive site rather than antimycin-sensitive site of the Q cycle mechanism. This study shows that 2,3,2',3'-, 2,4,2',4'-, and 2,5,2',5'-TCBs had marked inhibitory actions on the electron transport chain of isolated rat l i v e r mito~hondria. These TCBs are classified as phenobarbital (PB)-type inducers of
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microsomal monooxygenase, and are relatively less toxic TCBs (8). Therefore, some of the TCBs classified as PB-type inducers are also potent inhibitors of mitochondrial electron transport chain. The mitochondrial damage may be one of the suitable candidate for toxic mechanism of TCBs of PB-type inducers. A publication has presented evidence indicating mitochondrial damages caused by in vivo treatment with one of these TCBs, 2,5,2',5'-TCB. That is, Lin et al. (24-T reported that conformationally altered mitochondria which contained electron dense matrical material and swollen lucent cristae were observed in rat hepatocytes during the acute 2,5,2',5'-TCB intoxication. These phenomena suggest a damage in electron transport system of mitochondria, which secondarily reduces the synthesis of ATP, as seen in isolated mitochondria. However, mitochondria] damage is not applied to the toxic mechanism of 3,4,3',4'-TCB which categorized as 3-methylcholanthrene (MC)-type inducers and toxic TCB. Poland et aI. (25) suggested that 3,4,3',4'-TCB-induced toxicity is mediated by cytosolic binding protein, referred to as the 2,3,7,8-tetrachlorodibenz(;-~-dioxin (TCDD) receptor. The planarity of 3,4,3',4'-TCB is optimum configuration for binding to this receptor, but is not optimum for binding to the enzymes which constitute the mitochondria] eletron transport chain. Recently, Rifkind et al. (26) suggested a new mechanism. Namely, products of arachidonic acid m e t ~ s m are one class of mediators of toxicity due to 3,4,3',4'-TCB. References I. R. W. RISEBROUGH, P. REICHE, D. B. PEAKALL, S. G. HERMANand M. N. KIRVEN, Nature 22_._00, 1098-1102 (1968). 2. H. BRUNand D. MANZ, Bull. Environ. Contam. Toxicol. 28, 599-604 (1982). 3. R. W. RISEBROUGHand V. BRODINE, Fnvironment 12, 16-77 (]970). 4. D. F. FLICK, R. G. O'DELL and V. A. CHILDS, Po--~'It. Sci. 44, 1460-1465 (1965). 5. J. G. VOS, Environ. Health Perspect. i , 105-117 (1972). 6. S. KAWANISHI, T. MIZUTANI and S. SANO, Biochim. Biophys. Acta 54___0.0, 83-93 (1978). 7. A. PARKINSON, R. COCKERKINEand S. SAFE, Chem. Biol. Interact. 299, 277-289 (1980). 8. A. PARKINSON, L. ROBERISON, L. SAFE and S. SAFE, Chem. Biol. Interact. 30, 271-285 (1980). 9. ~ AHOTUPA, Biochem. Pharmacol. 30, ]866-1869 (1981). I0. Y. NISHIHARA and K. UTSUMI, Arch. Fnviron. Contam. Toxicol. I__44,65-71 (1985). 11. Y. NISHIHARA, L. W. ROBERTSON, F. OESCH and K. UTSUMI, J. Pharmacobio-Dyn. 8, 726-732 (1985). ]2. O. HUTZINGER, S. SAFE and V. ZIIKO, B u l l . Environ. Contam. Toxicol. 6, 209-219 (]971). ]3. G. H. HOGEBOOM,Methods Enzymo]. ~, ]6-19 (]955). 14. A. G. GORNALL, C. J. BARDAWILL and M. M. DAVID, J. Biol. Chem. |7.._~7, 751-766 (1949). ]5. K. S. CHEAHand J. C. WARING, Biochim. Biophys. Acta 723, 45-51 (1983). ]6. G. THIERBACHand H. REICHENBACH, Biochim. Biophys. Acta 638, 282-289 (1981). 17. G. VON JAGOW, PER O. LJUNGDAHL, P. GRAF, T. OHNISHI and B. k. TRUMPOWER, J. Biol. Chem. 259, 6318-6326 (1984). ]8. W. BECKER, G. VON JAGOW, T. ANKE and W. STEGLICH, FEBS Left. 132, 329-333 (1981). 19. G. YON JAGOWand W. D. ~NG[L, FEBS Lett. 136, 19-24 (198]). 20. A. ALEXANDREand A. L. LEHNINGER, Bicchim. Biophys. Acta 76__77, 120-129 (1984). 21. B. [. TRUMPOWER, Biochim. Bioph~s. Acta 639, 129-155 (1981). 22. P. R. RICH, Biochim. Biophys. Acta 768, 53-79 (1984).
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Effects of TCBs on Mitochondria
23. Q. S. ZHU, J. A. BERDEN, S. DE VRIES and E. C. SLATER, Biochim. Bioph2s. Acta 680, 69-79 (1982). 24. F. S. LIN, M. T. HSIA and J. R. ALLEN, Arch. Environ. Contam. Toxicol. 8, 321-333 (1979). 25. A. POLAND, E. GLOVERand A. S. KENDE, J. Biol. Chem. 251, 4936-4946 (1976).
26. A. B. RIFKIND, S. SASSA, J. REYESand H. MUSCHICK, Toxicol. Appl. Pharmacol. /8, 268-279 (1985).
635
Pergamon Press
THE EFFECTSOF TETRACHLOROBIPHENYLSON THE ELECTRONTRANSFER REACTION OF ISOLATED RAT LIVER MITOCHONDRIA Y. Nishihara 1.
L. W. Robertsonz F. Oesch2 and K. Utsumi1
1Department of Medical Biology• Kochi Medical School, Kohasu, Okocho, Nangoku-shi, Kochi 781-51, Japan and 21nstitut f~r Toxikology, Universit~t Mainz, Mainz, Obere Zahlbacher Str. 67, D-6500, Mainz, West Germany (Received in final form December 2, 1985)
Summary A comparative study was made of the effects of several sMnmetrical tetrachlorobiphenyls (TCBs) on the electron transfer from succinate to oxygen of rat liver mitochondria, and some differences in effects caused by the different chlorine positions of the biphenyl ring were clarified. TCBsused in this study included 2 , 3 , 2 ' , 3 ' - , 2 , 4 , 2 ' , 4 ' - , 2 , 5 , 2 ' , 5 ' - , 2 , 6 , 2 ' , 6 ' - , and 3,4,3',4'-TCBs. The inhibitory actions of 2 , 3 , 2 ' , 3 ' - , 2 , 4 , 2 ' , 4 ' - , and 2,5,2',5'-TCBs on succinate oxidase were potent• while those caused by 2,6,2',6'- and 3,4,3',4'-TCBs were significantly weak. The inhibition sites of 2 , 3 , 2 ' , 3 ' - , 2 , 4 , 2 ' , 4 ' - , and 2,5,2',5'-TCBs in succinate oxidase were succinate dehydrogenase and cytochrome b-c segment of the electron transport chain. In the cy'cochrome b-~ segment, these TCBs acted on myxothiazol-sensitive site rather than antimycin-sensitive site. Cytochrome~ oxidase was hardly affected by TCBs. These results indicate that 2 , 3 , 2 ' , 3 ' - , 2 , 4 , 2 ' , 4 ' - , and 2,5,2',5'-TCBs severely depress the electron transfer with succinate as the substrate, which secondarily reduces the s~thesis of ATP. The relationship between the activity and chemical structure of TCBs is also discussed. Polychlorinated biphenyls (PCBs) are industrial chemicals that have been detected in various species of wildlife as well as in human tissues and milk (I-3). The administration of PCBs to experimental animals produces characteristic toxic syndromes and variety of biochemical responses. Among the toxic syndromes reported are skin disorders such as chloracne, th)mic involution, edema, and hepatotoxic events (4,5). Some of the biochemical responses include porphyria (6) and induction of hepatic microsomal drug-metabolizing enz~es (7-9). The relationships between actions on these enz~es and chemical structures of PCBs have been well characterized. We reported previously the biochemical effects of PCBs on the functions of rat liver mitochondria; commercial PCB mixtures (Kanechlors) inhibited mitochondrial ATP synthesis in vitro (10). However, very l i t t l e is known about the structure-activity relationship of PCBs on the energy-transducing functions of mitochondria. We have shown in f a i r l y detail the effects of several sM~metrical tetrachlorobiphenyls (TCBs) on succinate-supported respirations of rat liver mitochondria (11). That is, 2,3•2',3'-, 2 , 4 , 2 ' , 4 ' - , and 2,5,2',5'-TCBs had potent * -- To whom correspondence should be addressed. 0024-3205/86 $3.00 + .00 Copyright (c) 1986 Pergamon Press Ltd.
628
Effects of TCBs on Mitochondria
VoI. 38, No. 7, 1986
inhibitory actions on state 3 respiration (active respiration in the presence of ADP), while those produced by 2 , 6 , 2 ' , 6 ' - and 3,4,3',4'-TCBs were weak. It was also suggested that TCB-induced inhibition of state 3 respiration was mainly caused by the interference with electron transport chain. Here we present a report on the effects of TCBs on the succinate-oxidizing enz~es of rat l i ver mitochonria, and show the sites of inhibition in the electron transport chain. Materials and Methods Chemicals 2 , 3 , 2 ' , 3 ' - , 2 , 4 , 2 ' , 4 ' - , 2 , 5 , 2 ' , 5 ' - , 2 , 6 , 2 ' , 6 ' - , and 3,4,3',4'-Tetrachlorobiphenyls (TCBs) were synthesized by the Ullman condensation of the corresponding dichloroiodobenzenes (12). The purities of these isomers were found to be more than 99% by gas liquid chromatography. The stock solutions ef these isomers except 3,4,3',4'-TCB were prepared in ethanol, and that of 3,4,3',4'-TCB was prepared in dimethylformamide. Antimycin A, and bovine serum albumin were purchased from Sigma Chemical Co. (St Louis, MO). Phenazine methosulfate (PMS), N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD), and 2,4-dinitrophenol (DNP) were purchased fronl Nakarai Chemicals (Kyoto, Japan). Other reagents were commercial products of the highest purity. Preparation of mitochondria Liver mitochondria were isolated from male Wistar rats (200-300 g) by the standard method in a medium containing 0.25 M sucrose, 5 mM Tris-HCI (pH 7.4), and 0.1 n~4 EDTA. EDTAwas omitted in a final wash and resuspension (13). Mitochondrial protein was determined by the biuret method using bovine serum albumin as a standard (14). Enz,vme assays Succinate oxidase, cytochrome ~ oxidase, and succinate dehyOrogenase activities were measured polarographically using a Clark-type oxygen electrode in a 2 ml water-thermostated glass reaction cell maintained at 25°C. The assay medium consisted of 0.2 M sucrose, 20 mM KCI, 3 mM MgCl2, and 5 n~4 potassium phosphate (pH 7.4). Succinate oxidase and cytochrome ~ oxidase a c t i v i t i e s were assayed in assay medium containing 25uM DNP and i mg/ml of mitochondria. Mitochondria were interacted with TCB for 3 min, then respiration was started by the addition of 5 mM succinate (succinate oxidase) or 5 mM ascorbate/O.! mM TMPD (cytochrome £ oxidase), respectively. Succinate dehydrogenase a c t i v i t y was determined using PMS as an electron acceptor in an assay medium containing 2ug/mg protein of antimycin A, 2 n~M NAN3, 5 mM succinate, and ! mg/ml of mitochondria. Mitochondria were interacted with TCB for 3 min, then respiration was initiated by the addition of 0.5 mM PMS (15). Optical spectroscopy The steady-state reductions of b (563-575 nm), and c (550-540 m~)-type cytochro~nes were measured at 25°C using Shimadzu UV-300 duaT wavelength spectrophotometer with a 2 nm band pass. In all experiments, control contained the same volume of solvent (ethanol or dimethylformamide), and the final concentration of solvent was less than 1% (v/v); the concentration of solvent did not affect the cellular a c t i v i t i e s assayed. Results The effects of TCBs on succinate oxidase are shown in Fig. 1. 2 , 3 , 2 ' , 3 ' - , 2 , 4 , 2 ' , 4 ' - , and 2,5,2',5'-TCBs inhibited succinate oxidase strongly. The concentrations of 2 , 3 , 2 ' , 3 ' - , 2 , 4 , 2 ' , 4 ' - , and 2,5,2',5'-TCBs that gave 50~ inhi-
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bition (150) were 48.4 * 4.1, 53.1 ± 3.6, and 56.2 ± 5.3 pM, respectively(calculated from linear regression analysis, n=3). However, percentage inhibitions of 2,6,2',6'- and 3,4,3', 4'-TCBs never reached 50%, only 19 and 25% inhibitions even at 200pM. Two way analysis of variance and Duncan's multiple range test (p< 0.05) revealed that treatment with 2,3,2',3'-, 2,4,2',4'-, and 2,5,2',5'-TCBs resulted in similar rates of inhibition, while those produced by 2,6,2',6'- and 3,4,3',4'-TCBs were significantly reduced. Cytochrome ~ oxidase was far less inhibited by TCBs (data not shown). This fact indicates that TCBs act at sites before complex IV in the electron transport chain. Fig. 2 shows the effects of TCBs on succinate dehydrogenase. Two way analysis of variance and Duncan'smultiple range test (p< 0.05) revealed that treatments with 2,3,2',3'-, 2,4,2',4'-, and 2,5,2',5'-TCBs resulted in similar rates of inhibition, while those caused by 2,6,2',6'- and 3,4,3',4'-TCBs were significantly reduced. Thus, succinate dehydrogenase was inhibited by TCBs, although the extent of inhibition varied among TCBs, indicating that the inhibitory reaction for succinate oxidase occurs p a r t i a l l y in succinate dehydrogenase. Table I shows the effects of 2,5,2',5'- and 3,4,3',4'-TCBs on the aerobic steady-state reductions of cytochromes b and c of mitochondria during the oxi-
100
3
2
2'
3'
t-
.o_
"~50 C N
,:p
,.~
50 100 Concentration (uM)
200
FIG. I Comparison of the i n h i b i t o r y e f f e c t s of TCBs on succinate oxtdase of rat l t v e r mitochondrta. Succtnate oxidase a c t i v i t y was measured polarographically with a Clark-t~q~e oxygen electrode at 25~C. The mttochondrtal protein was I rag/m1. Control rate was 153.3 ~ 4.8 nat(ms O/man/ mg protein. Each point is a mean of 3 separate experiments. S. O. bars are omitted since they are always less than 51[ of the mean. Sj~bols are as follows: 2 , 3 , 2 ' , 3 ' - , 0 ; 2,4,2',4'-,&; 2,5,2',5'- rl;2,6,2,,6,_ • ; 3,4,3',4'-TCB,~ .
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Effects of TCBs on Mitochondria
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dation of succinate or glutamate/malate. With succinate, 2,5,2',5'-TCB inhibited reduction of cytochromes b and C by 43 and 80%, respectively. The inhibition of cytochrome b is caused by the inhibition of succinate dehydrogenase. When glutamate/malate was used as the substrate, the reduction of cytochrome was inhibited by 65% with 2,5,2',5'-TCB, which had no apparent effect on the reduction of cytochrome b ( see trace B in Fig. 3), suggesting that 2,5,2',5'TCB had almost no effect on the components before cytochrome b of mitochondrial electron transport chain (complex I). The inhibition of cytochrome c reduction by 2,5,2',5'-TCB with both succinate and glutamate/malate indicated that cytochrome b-~ segment of the electron transport chain was blocked by 2,5,2',5'-TCB. Identical results were obtained for 2 , 3 , 2 ' , 3 ' - and 2,4,2',4'-TCBs (data not shown). On the other hand, 3,4,3',4'-TCB had very l i t t l e effect on the cytochrome b-~ segment. 2,6,2',6'-TCB gave the identical result (data not shown). Following experiments were designed to determine the interacting site of 2,5,2',5'-TCB within the cytochrome ~-~ segment. These experin;ents were done using glutamate/malate as the substrate; the results obtained with this substrate directly reflect the information from cytochron,e b-~ segment (complex Ill), since both complex I and IV in the electron transport chain are hardly inhibited by 2,5,2',5'-TCB.
ioo L
j 0
10
50
100
2OO
Conceqntration (uM) FIG. 2
Comparison of the inhibitory effects of TCBs on succinate dehydrogenase of rat liver mitochondria. Succinate dehydrogenase activity was measured polarographicaIIy with a Clark-type oxygen electrode at 25°C. The mitochondrial protein was I mg/m]. Control activity was ]61.1± 5.9 natoms O/min/mg protein. Fach point is a mean of 3 separate experiments. S. D. bars are omitted since they are always less than 5% of the mean. Symbols are as follows: 2 , 3 , 2 ' , 3 ' - , 0 ; 2 , 4 , 2 ' , 4 ' - , • ; 2,5,2',5'0 ; 2 , 6 , 2 ' , 6 ' - , 0 ; 3,4,3',4'-TCB,~.
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Effects
o f TCBs on M i t o c h o n d r t a
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TABLE ] Effects of 2,5,2',5 °- and 3,4,3°,4'-TCBs on the aerobic steady-state reduction of cytochro~es b and ~ in rat liver mitochondria a
Additions
Percentage inhibition of aerobic steady-state reductions of cytochro~es Succinate Glutamate/malate
Control 2,5,2',5'-TCB (150 nmol/mg prot.) 3,4,3',4'-TCB (150 nmol/mg prof.)
0 43 12
0 80 22
0 0 0
0 65 II
aThe reductions of cytochrome b (563-575 nm) and5~c ( 5 5 0 - 5 4 0 2 nm) were monitored by the dual-wavelength spe~trophotometer at . The reaction medium contained 0.2 M sucrose, 25 mM KCI, 3 mMMgCI2, and 5 mM potassium phosphate (pH 7.4). The reduction of the cytochro~es was initiated by the addition of either 5 mM succinate or 5 mM glutamate/5 mMmalate into the mitochondrial suspension 3 min after the addition of TCB. Rotenone (2~M) and malonate (5 mM) were also added to the mitochondrial suspension prior to succinate and glutamate/malate, respectively. Protein concentration was 2 mg/ml. Volume, 3 ml.
A
B
C
11min A
Nuta.
A
Gluta.
I
A
FIG. 3 Effects of 2,5,2',5'-TCB on the reduction of cytochrome ~ by glutamate/malate in rat liver mitochondria. The traces show reduction of cytochrome b monitored at 563 nm versus 575 nm with a 2 nm band pass. The reaction medium consisted of 0.2 M sucrose, 20 mM KCI, 3 mM MgCI2, and 5 mM potassium phosphate (pH 7.4). Mitochondria were interacted with TCB for 3 min, then the reduction of cytochrome b was initiated by the addition of 5 mM glutamate/5 mMmalate (Gluta.).-The protein concentration was 2 mg/~]. CA) control, (B) 2,5,2',5'-TCB,]50 nmol/mg protein, (C) 2,5,2',5'-TCB, 150 nmol/mg protein plus antimycin A, 2~g/mg protein.
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Effects of TCBs on Mltochondrla
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Fig. 3 shows the effect of 2,5,2',5'-TCB on reduction of cytochrome b by glutamate/malate. The trace A shows the reduction of cytochrome b, when glutamate/malate was added in the absence of inhibitor (control). Under these conditions, the cytochrome b which is reduced consists primarily of b-562 (_bK) and a small portion of the low potential b-566 (_bT), residing within one cytuchrome b. When 2,5,2',5'-TCB was added before substrate, i t did not block reduction of cytochrome b (trace B). However, when 2,5,2',5'-TCB and antimycin A were applied together, almost complete inhibition of cytochrome b reduction was seen (trace C). The effect of TCB on cytochrome b reduction By substrate is very similar to that produced by myxothiazol (an antibiotic inhibitor) which has been introduced recently to the study of mitochondrial electron transfer (16,17); myxothiazol does not i n h i b i t reduction of cytochrome b by substrate when applied alone, but does i n h i b i t in the presence of antimycin A (18,19). Therefore, 2,5,2',5'-TCB may interact with myxothiazol-sensitive site in the b-~ segment. To further confirm this, the effect of TCB on the cytochrome b-£ segment was studied by creating a bypass of the antimycin block of electron flow using a r t i f i c i a l electron carrier, TMPD. Alexandreet al. (20) has showed that Wurster's blue (oxidized form of TMPD) promotes the electron flow which is inhib-
A
B
RLM f TMPD ' L ~ . . . . Anu myc, n
!i
RLM I TMPD ~ Antimycin ".5' -TCB
"--r,'°"
"
FIG. 4 The TMPD-promoted b~l)ass of the antimycin block and its sensitivity to 2,5,2',5'-TCB. Oxygenuptake was measured at 2~C in a medium of 0.2 M sucrose, 20 mM KCI, 3 m#~MgC12, 5 mM potassium phosphate (pH 7.4), and 50~M DNP. Where indicated, rat l i v e r mitochondria (RLM), 2 mg/mI;TMPD, 0.1 mM; antimycin A, 2pg/mg protein; glutamate, 5 mM glutamate/5 mMmalate; 2,5,2',5'-TCB, 150 nmol/mg protein. Volume, 2 ml.
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Effects of TCBs on Mitochondrla
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ited by antlmycin, by accepting electrons from some reduced intermediate site within the antimycin-inhibited complex I l l , and transferring electrons to cytochrome ~, and that this bypass is inhibited by myxothiazol. Fig. 4 shows the llqPD-promoted bypass of the antimycin block and i t s sensitivity to 2,5,2',5'TCB. Whenglutamate/malate was added to incubation medium, antimycin-blocked respiration was accelerated greatly (trace A). Thus, llqPD/Wurster's blue couple provides a bypass of the electron flow which was blocked by antimycln. The TMPD-bypass of the antimycin block was highly sensitive to 2,5,2',5'-TCB, simi l a r to myxothiazol (trace B). This fact indicates that 2,5,2',5'-TCB inhibits myxothiazol-sensitive site rather than antimycin-sensitive site in the b-~ segment of the electron transport chain. Although 2,5,2',5'-TCB was shown as the representative compound in Figs. 3 and 4, identical results were observed for 2,3,2',3'- and 2,4,2',4'-TCB (data not shown). Discussion The results presented here clearly indicate that there exist marked differences in the effect of TCBs on electron transport system of rat liver mltochondria. 2,3,2',3'-, 2,4,2',4'-, and 2,5,2',5'-TCBs showed potent inhibitory actions on succinate oxldase, while those produced by 2,6,2',6'- and 3,4,3',4'-TCBs were weak. It is clear that TCBs which show potent inhibitory actions on succinate oxidase (i. e., 2,3,2',3'-, 2,4,2',4'-, and 2,5,2',5'-TCB) have a common structure with chlorine atoms equally attached to both inside (ortho,
ortho') and outside (meta or para, meta' or para') positions of the blpe-~l ring, and that TCBs of which all chlorine atoms are localized in either inside (2,6,2',6'-TCB) or outside (3,4,3',4'-TCB) positions of the biphenyl ring are poor inhibitors. 2,3,2',3'-, 2,4,2',4'-, 2,5,2',5'-, and 2,6,2',6'-TCBs possess non-planar configuration becauseof the diortho chlorination, while 3,4,3',4'-TCB is planar configuration because of t ~ e a s e d double-bond character of the C~I~ - C(1,1 bond due to chlorine atoms in para, para' position (6). Therefore,'(Ee ned~s~aryconditions for TCBs to be e~Tec'tive'-e--'Tnhibitors of mitochondrial electron transport chain are that TCBs possess primarily nonplanar configuration and that in addition to non-planarity, position of chlorine substitution as discussed above is important ( because 2,6,2',6'-TCB possesses non-planar configuration, but is a weak inhibitor). A large body of evidence supports the view that the pathway of electrons in the cytochrome b-~ segment of the mitochondrial electron transport chain is not described by a single linear sequence of electron transfer reaction but by a Q cycle mechanism, in which the f i r s t electron from ubiquinol (QH2) passes to the Rieske FeS center and thence to cytochrome £1; the seco~ electron passes from ubisemiquinone (Q~) to cytochrome bT (21,~2). Antimycin A and myxothiazol are known to block different site ~f the Q cycle; antimycin blocks electron transfer fro~ cytochrome bK to ubiquinone in center i (23), whereas myxothiazol suppresses cooperative oxidation of ubiquinol in center o (17). Antimycin A and myxothiazol, when applied alone, do not block the reduction of cytochrome b, but when these agents are applied together, they almost completely block the reduction of cytochrome b (19). Therefore, we tested whether a mixture of antimycin and TCB (2,3,2',~'-, 2,4,2',4'-, or 2,5,2',5'-) had a similar effect. As is shown in Fig. 3, the reduction of cytochrome b by substrate was completely blocked, men antimycin and TCB are applied together. This fact indicates that TCB acts on the myxothiazol-sensitive site.This was further confirmed by use of the bypass of the antimycin block of mitochondrial electron transport (20). TCB blocked ll(PD-pron~oted bypass Of t~e antimycin block (Fig. 4), similar to myxothiazol (see Fig. 4 in Ref. 20). These facts suggest that TCB acts on myxothiazol-sensitive site rather than antimycin-sensitive site of the Q cycle mechanism. This study shows that 2,3,2',3'-, 2,4,2',4'-, and 2,5,2',5'-TCBs had marked inhibitory actions on the electron transport chain of isolated rat l i v e r mito~hondria. These TCBs are classified as phenobarbital (PB)-type inducers of
634
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microsomal monooxygenase, and are relatively less toxic TCBs (8). Therefore, some of the TCBs classified as PB-type inducers are also potent inhibitors of mitochondrial electron transport chain. The mitochondrial damage may be one of the suitable candidate for toxic mechanism of TCBs of PB-type inducers. A publication has presented evidence indicating mitochondrial damages caused by in vivo treatment with one of these TCBs, 2,5,2',5'-TCB. That is, Lin et al. (24-T reported that conformationally altered mitochondria which contained electron dense matrical material and swollen lucent cristae were observed in rat hepatocytes during the acute 2,5,2',5'-TCB intoxication. These phenomena suggest a damage in electron transport system of mitochondria, which secondarily reduces the synthesis of ATP, as seen in isolated mitochondria. However, mitochondria] damage is not applied to the toxic mechanism of 3,4,3',4'-TCB which categorized as 3-methylcholanthrene (MC)-type inducers and toxic TCB. Poland et aI. (25) suggested that 3,4,3',4'-TCB-induced toxicity is mediated by cytosolic binding protein, referred to as the 2,3,7,8-tetrachlorodibenz(;-~-dioxin (TCDD) receptor. The planarity of 3,4,3',4'-TCB is optimum configuration for binding to this receptor, but is not optimum for binding to the enzymes which constitute the mitochondria] eletron transport chain. Recently, Rifkind et al. (26) suggested a new mechanism. Namely, products of arachidonic acid m e t ~ s m are one class of mediators of toxicity due to 3,4,3',4'-TCB. References I. R. W. RISEBROUGH, P. REICHE, D. B. PEAKALL, S. G. HERMANand M. N. KIRVEN, Nature 22_._00, 1098-1102 (1968). 2. H. BRUNand D. MANZ, Bull. Environ. Contam. Toxicol. 28, 599-604 (1982). 3. R. W. RISEBROUGHand V. BRODINE, Fnvironment 12, 16-77 (]970). 4. D. F. FLICK, R. G. O'DELL and V. A. CHILDS, Po--~'It. Sci. 44, 1460-1465 (1965). 5. J. G. VOS, Environ. Health Perspect. i , 105-117 (1972). 6. S. KAWANISHI, T. MIZUTANI and S. SANO, Biochim. Biophys. Acta 54___0.0, 83-93 (1978). 7. A. PARKINSON, R. COCKERKINEand S. SAFE, Chem. Biol. Interact. 299, 277-289 (1980). 8. A. PARKINSON, L. ROBERISON, L. SAFE and S. SAFE, Chem. Biol. Interact. 30, 271-285 (1980). 9. ~ AHOTUPA, Biochem. Pharmacol. 30, ]866-1869 (1981). I0. Y. NISHIHARA and K. UTSUMI, Arch. Fnviron. Contam. Toxicol. I__44,65-71 (1985). 11. Y. NISHIHARA, L. W. ROBERTSON, F. OESCH and K. UTSUMI, J. Pharmacobio-Dyn. 8, 726-732 (1985). ]2. O. HUTZINGER, S. SAFE and V. ZIIKO, B u l l . Environ. Contam. Toxicol. 6, 209-219 (]971). ]3. G. H. HOGEBOOM,Methods Enzymo]. ~, ]6-19 (]955). 14. A. G. GORNALL, C. J. BARDAWILL and M. M. DAVID, J. Biol. Chem. |7.._~7, 751-766 (1949). ]5. K. S. CHEAHand J. C. WARING, Biochim. Biophys. Acta 723, 45-51 (1983). ]6. G. THIERBACHand H. REICHENBACH, Biochim. Biophys. Acta 638, 282-289 (1981). 17. G. VON JAGOW, PER O. LJUNGDAHL, P. GRAF, T. OHNISHI and B. k. TRUMPOWER, J. Biol. Chem. 259, 6318-6326 (1984). ]8. W. BECKER, G. VON JAGOW, T. ANKE and W. STEGLICH, FEBS Left. 132, 329-333 (1981). 19. G. YON JAGOWand W. D. ~NG[L, FEBS Lett. 136, 19-24 (198]). 20. A. ALEXANDREand A. L. LEHNINGER, Bicchim. Biophys. Acta 76__77, 120-129 (1984). 21. B. [. TRUMPOWER, Biochim. Bioph~s. Acta 639, 129-155 (1981). 22. P. R. RICH, Biochim. Biophys. Acta 768, 53-79 (1984).
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Effects of TCBs on Mitochondria
23. Q. S. ZHU, J. A. BERDEN, S. DE VRIES and E. C. SLATER, Biochim. Bioph2s. Acta 680, 69-79 (1982). 24. F. S. LIN, M. T. HSIA and J. R. ALLEN, Arch. Environ. Contam. Toxicol. 8, 321-333 (1979). 25. A. POLAND, E. GLOVERand A. S. KENDE, J. Biol. Chem. 251, 4936-4946 (1976).
26. A. B. RIFKIND, S. SASSA, J. REYESand H. MUSCHICK, Toxicol. Appl. Pharmacol. /8, 268-279 (1985).
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