Sites of inhibition of mitochondrial electron transport by cadmium

klterod.io~ [ LSI'VII R S( IFNTI~ I< P I : B I ISHI'RS IRI I ANII)

Chemico-Biological Interactions 89 (1993) 159-167

Sites of inhibition of mitochondrial electron transport by cadmium Stefania Miccadei, Aristide Floridi* Laboratory of Cell Metabolism and Pharmacokinetics, Centerfor Experimental Research, Regina Elena Institute for Cancer Research, Via delle Messi D'Oro 156, 00158 Rome, Italy

(Received 16 February 1993; revision received 9 June 1993; accepted 10 June 1993)

Abstract

Cadmium is an extremely toxic environmental contaminant having a long half-life in humans. The greatest accumulation occurs in the liver and kidneys. Since mitochondria are the most sensitive targets, the effect of cadmium on the oxygen consumption and on the redox state of electron carriers of rat liver mitochondria has been evaluated. Cadmium markedly inhibits uncoupler-stimulated oxidation on various NADH-linked substrates as well as that of suceinate. Experiments on specific segments of the respiratory chain showed that cadmium does not inhibit electron flow through cytochrome oxidase, whereas the inhibition of duroquinol oxidation clearly demonstrates an impairment of electron flow through site 2, the ubiquinone-b-cytochrome c~ complex. On the basis of the ability of N,N,N',N' tetramethylp-phenylendiamine and 2,6-dichlorophenolinindophenol bypasses to relieve the cadmium inhibition of succinate oxidation and on the spectroscopic behaviour of the cytochrome b, the inhibition was found to take place before cytochrome b and, more precisely, between ubisemiquinone and cytochrome bx. Furthermore, the finding that cadmium induces a more oxidized state ofcytochrome b in state I demonstrates the existence of a second point in which it inhibits electron transfer. Spectroscopic evidence demonstrates that cadmium induces an oxidation of NAD(P)H in mitochondria in states 1 and 4 and prevents the reduction of mitochondrial NAD(P) ÷ by substrates, thus indicating that the site must be localized between NAD-linked substrates and respiratory chain. Key words: Cadmium; Mitochondria; Electron transport; Inhibition

* Corresponding author. Abbreviations:AA, antimycin A; DHQ, duroquinol; DCIP, 2,6-dichlorophenolindophenol; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; HEPES, 4-(2-hydroxyethyl)l-piperazine ethanesulfonic acid; MX, myxothiazol; TMPD, N,N,N',N' tetramethyl-p-phenylendiamine hydrochloride; WB+, Wuster's blue; Cyt b, cytochrome b; rot, rotenone. 000%2797/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0009-2797(93)03209-D

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1. Introduction

Cadmium is an extremely toxic metal that following ingestion is distributed throughout the soft tissues of the body with major localization in the liver and kidneys [1,2]. The studies on the mechanisms that lead to irreversible cell injury have frequently focused on the role of alterations in energy metabolism [3]. It has been reported that cadmium is a strong uncoupler of the oxidative phosphorylation [4] and inhibits the succinate- and malate/pyruvate-stimulated respiration [5]. This latter effect has been ascribed to a block of electron transfer between substrates and cytochrome b as well as to an inhibition of substrate transport [6]. Nevertheless, since the cadmium sensitive site(s) in the respiratory chain was not well defined, we studied its effect on the rate of FCCP-stimulated respiration, the flow of electrons through specific segments of the respiratory chain and the redox state of NAD(P) and cytochromes in isolated rat liver mitochondria in order to localize in greater detail the inhibitory site(s). 2. Materials and methods

2.1. Preparation of mitochondria Rat liver mitochondria were isolated from adult male Sprague-Dawley rats, according to Pedersen et al. [7]. Animals were fed ad libitum and fasted overnight prior to use. The mitochondria were resuspended in a minimal volume of H-medium (70 mM sucrose, 210 mM mannitol, 2.1 mM Li-HEPES, BSA 0.1 mg%, pH 7.10), at a concentration of 100 mg/ml. Protein content was determined according to Gornall et al. [81.

2.2. Assay of oxygen consumption The rates of oxygen consumption were determined with a Clark oxygen electrode (Yellow Spring Instruments Co., OH, USA) equipped with an ultrathin Teflon membrane. The electrode was inserted horizontally in a thermostated, closed chamber of 2.0 ml and contained final concentrations of 180 mM sucrose, 40 mM KCI, 3 mM Li-HEPES (pH 7.10), without EGTA (because of its ability to bind cadmium), 10 mM substrates and 4 mg of mitochondrial protein. Other additions are given in the figure legends. The temperature was 25°C and the solubility of oxygen was taken as 442 ng atoms m1-1 when the medium was air-equilibrated at 760 Torr [9]. The bypasses with TMPD and DCIP were performed according to Alexandre and Lehniger [ 101.

2.3. Spectrophotometric determinations The effect of cadmium on the oxido-reduction state of mitochondrial NAD(P) ÷, cytochrome b was evaluated by dual-wavelength spectrophotometry (Aminco DW2a). The cuvette thermostated at 25°C was provided with magnetic stirrer. The reaction medium (2.5 ml) contained 180 mM sucrose, 40 mM KCI, 3 mM Li-HEPES (pH 7.10) and 3 mg of mitochondrial protein. The addition of substrates and other compounds, as indicated in the figure legends, was made by rapid injection from microsyringes in such a way as to achieve the shortest possible mixing time.

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161

2.4. Chemicals The following chemicals were purchased from the indicated sources: fatty acidfree BSA, TMPD, DCIP, FCCP and ascorbic acid, cadmium chloride from Sigma Chemical Co. (St Louis, MO); pyruvate, malate, glutamate, succinate, ADP, antimycin A, myxothiazol from Boehringer, Mannheim, GmbH (Germany); rotenone and duroquinol from K & K Laboratories (Plainview, NY). All other reagents were analytical grade and were purchased from BDH Italia (Milan, Italy). 3. Results

3.1. Effect of cadmium on the oxidation of various substrates by liver mitochondria Fig. 1 shows the effect of cadmium concentration on the oxidation of succinate by rat liver mitochondria after stimulation by FCCP. The mitochondria were preincubated for 1 min with different cadmium concentrations and then 5 mM succinate was added. When 20% of available oxygen was utilized, 0.3/~M FCCP was injected and the rate of subsequent oxygen consumption recorded and compared to the stimulated rate in the absence of cadmium. Fig. 1 shows that the rate of FCCPstimulated respiration decreased linearly as cadmium concentration increased up to

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JiM Cd CI2 Fig. 1. Effect of cadmium concentration on FCCP-stimulated succinate oxidation. The final volume was 2.0 ml and the temperature was 25°C. The mitochondria (4 mg protein) were preincubated for I min with the indicated concentrations of cadmium in buffered medium, (see Methods); then 5 mM succinate was added and the rate of oxygen consumption was recorded. After 20% of the disssolved oxygen had been utilized, 0.3 ~M FCCP was added and the rate of subsequent oxygen consumption compared to that prior FCCP addition. Each point was averaged from seven different mitoehondrial preparations yielding reproducible results ( ± 5%).

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Table 1 Inhibition of FCCP-uncoupled respiration on various substrates by cadmium chloride Substrate

FCCP

Controlrate (ng atoms 0/min/mg)

~M CdCI2 giving half maximalinhibition

Pyruvate + malate a-Ketoglutarate + malonate Glutamate Succinate Duroquinol Ascorbate + TMPD

+ + + + + +

21 ± 43 ± 20 ± 105 ± 218 ± 94 ±

2.0 2.5 1.5 4.7 9.5 No inhibition at 50 ttM

3 5 2 9 12 6

Each value ± S.D. was averaged from seven different experiments.

10/~M. Half-maximal inhibition was given by 4.7/~M cadmium and an almost complete inhibition of oxygen consumption was achieved by 15 t~M cadmium. Table 1 shows the effect of cadmium on oxidation of other substrates which donate electrons to the energy-conserving site 1 of the respiratory chain. Cadmium strongly inhibited FCCP-stimulated oxidation of pyruvate + malate and glutamate with half-maximal inhibitions attained at 2 and 1.5 #M, respectively. Cadmium also affected the oxygen consumption in mitochondria respiring on a-ketoglutarate, in the presence of malonate in order to inhibit succinate dehydrogenase, with an 'apparent' Ki of 2.5/~M. The effect of cadmium on electron flow through site 3, the cytochrome oxidase reaction, was investigated using ascorbate + T M P D as electron donor in the presence of antimycin A (0.2/zmol mg protein -l) and 4/~M rotenone to block electron flow from endogenous site 1 and 2 substrates. The data of Table 1 indicate that cadmium, up to 50 t~M, did not affect the rate of cytochrome oxidase reaction at all. 3.2. Effect o f cadmium on electron f l o w through site 2

The inability of the cadmium to inhibit electron flow through site 3, i.e., cytochrome oxidase clearly indicates that the inhibition of oxygen consumption, therefore, must occur at same point(s) prior to cytochrome c. Because electrons coming from N A D H and succinate must pass via ubiquinone through b-cl complex, experiments were carried out to determine whether cadmium inhibits electron flow in the ubiquinone -- cytochrome c span. Two different experimental approaches were employed. The first and most direct test of the action of the cadmium on site 2 was made using duroquinol. Table 1 shows the 02 uptake data obtained when cadmium was added to rat liver mitochondria with 0.5 mM duroquinol as electron donor in the presence of rotenone to inhibit electron flow from site I and FCCP to yield a maximal rate of duroquinol oxidation. It can be seen that cadmium inhibited duroquinol oxidation and half-maximal inhibition was achieved by 9.5 #M cadmium. The inhibition of the electron flow through site 2 by cadmium was further confirmed by the second experimental approach performed by evaluating its capacity to inhibit

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Fig. 2. Typical traces showing the bypasses of the antimycin (A) or cadmium (B) block by reduced DCIP and TMPD within the ubiquinone - cytochrome c span. When indicated 4 mg of mitochondrial protein, 5 mM succinate, 0.1 mM TMPD, 25 t~M DCIP, 0.3 t~M FCCP, 0.2 nmol antimycin/mg, 0.10 nmol/mg myxothiazol, 30 t~M cadmium were added. Electron transfer from succinate to oxygen. The experiments were repeated with four different mitochondrial preparations yielding reproducible results ( ± 4%). For further experimental details, see Materials and methods.

electron transport via DCIP and TMPD bypasses of the cytochrome b-cytochrome cl complex. Fig. 2 shows that DCIP was not ready oxidized via cytochrome c and cytochrome oxidase in the presence of rotenone, antimycin A and FCCP as indicated by the low rate of oxygen uptake. However, when succinate was also added, a large increase in

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the rate of O2 uptake occurs (Fig. 2A). The non-enzymatic reductant ascorbate was not an absolute requirement in these experiments, since succinate alone can reduce DCIP, in a myxothiazol-insensitive reaction. A similar behaviour has been observed also when antimycin was omitted and the respiration was inhibited by cadmium (Fig. 2B). The oxygen uptake was low, but when succinate was also added to the system there was a large increase in 02 uptake, thus indicating that the reduced DCIP bypassed the cadmium block by transferring electrons to cytochrome c. The DCIPpromoted bypasses of the antimycin and cadmium blocks were almost completely inhibited by myxothiazol (Fig. 2A and B). Fig. 2C shows that TMPD bypasses the antimycin block, with succinate as electron donor, and this reaction was highly sensitive (over 90%) to myxothiazol. Similarly, (Fig. 2D) TMPD relieved the cadmium inhibition of succinate oxidation and, once again, the reaction was myxothiazol-sensitive. 3.3. Effect of cadmium on the oxido-reduction state of mitochondrial electron carriers Fig. 3 shows the effect of cadmium on the oxido-reduction level of cytochrome b. The addition of rotenone, which inhibits electron flow from site 1, induced a rapid and large oxidation of cytochrome b which was promptly reduced by the addition of succinate. The addition of FCCP extensively re-oxidized the cytochrome b as the respiration was stimulated by the uncoupler. Then, about 1 min later, the dissolved oxygen was exhausted and cytochrome b became reduced as indicated by the upward deflection of the 562-575 nm trace (Fig. 3A). The addition of cadmium to mitochon-

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~M Cd C12 Fig. 4. Oxido-reduction state of NAIMP) + of rat liver mitochondria in the absence (A) and in the presence of cadmium added in state 4 (B) or state 1 (C}. Experimental conditions as in Fig. 3 except that the cadmium concentration was 5/~M and 4/~M in B and C, respectively. D, titration curve of the effect of cadmium concentration on NAD(P)H oxidation in state I mitochondria. Each point was averaged from five different mitochondrial preparations and yielded reproducible results (±4%).

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dria in which the cytochrome b was reduced by succinate (Fig. 3B) induced an extensive and relatively rapid reoxidation of cytochrome b. Fig. 3C shows that when cadmium was added to mitochondria in state 1, (no substrate, no ADP), it induced a cytochrome b oxidation in a manner and to an extent similar to that found in rotenone-treated mitochondria (Fig. 3A and 3C). Nevertheless, the addition of succinate did not reduce the cytochrome b at all. These experiments not only support the conclusion that cadmium inhibited electron flow at some point before cytochrome b, but they also indicate that the electron transport was affected at other point(s) other than at level of b-el complex. However, to better localize the site(s) of action, its effect on the oxido-reduction level of NAD(P) + of mitochondria in different metabolic states has been evaluated. Fig. 4A shows that when glutamate + malate were added to rat liver mitochondria, there was a reduction of NAD(P) ÷ as shown by the upward deflection of 340-370 nm trace. The addition of FCCP determined an immediate and extensive re-oxidation of NAD(P)H. The addition of cadmium to mitochondria in state 4 (Fig. 4B) induced a rapid and large oxidation of mitochondrial NAD(P)H. Cadmium added to mitochondria in state 1 induced an oxidation of NAD(P)H (Fig. 4C). When the trace became stable, the addition of glutamate + malate provided a very small reduction of NAD(P) + which remained unmodified upon FCCP addition. This experiment was repeated in the presence of increasing amounts of cadmium and the rate of NAD(P)H oxidation increased linearly with cadmium concentration (Fig. 4D). 4. Discussion The observations recorded in this paper show that cadmium is a potent inhibitor of the FCCP-stimulated oxidation of various NAD-linked substrates as well as that of succinate. Tests on the segments of the respiratory chain showed a failure to inhibit T M P D + ascorbate oxidation, indicating that energy-conserving site 3, the cyto-

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Fig. 5. Pathways of electron flow in the DCIP and TMPD bypasses of the cadmium block. For the explanation see Discussion. The straight broken lines represent diffusion across the membrane; ( ~ ) , direction of electron transfer, whereas the dotted lines represent the DCIP and TMPD bypasses. Dred and Dox, reduced and oxidized DCIP. This scheme is based on the material from Alexandre and Lehninger [ I 0].

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chrome oxidase reaction, is unaffected. The inhibition of succinate oxidation cannot be ascribed to a block of electron transfer from the substrate to Q because the inhibitory effect on the oxidation of duroquinol, which directly feeds electrons to Q, indicates that the electron flow through the energy-conserving site 2 is impaired by cadmium. More detailed studies on the site(s) of action of cadmium were restricted to an examination of point(s) at which it inhibits mitochondrial respiration. The more oxidized state of cytochrome b upon cadmium addition (Fig. 3B) and the finding that it prevents cytochrome b reduction by succinate (Fig. 3C) indicate that.one site of action should be localized before cytochrome b and, presumably, between ubisemiquinone and cytochrome br (Fig. 5). The validity of such localization is'further confirmed by the failure of the cadmium to inhibit both DCIP and TMPD bypasses. The cadmium-inhibited electron flow from succinate to oxygen can be reactivated by an artificial electron donor (DCIP) as well as by an artificial electron acceptor (WB ÷) according to Q cycle pathway of electron transfer [10]. In this scheme (Fig. 5) the oxidation of QH 2 on the cytosolic side (out) proceeds by two sequential steps. In the first, one electron is released by QH 2 to Rieske FeS center, thus giving ubisemiquinone which, in turn, donates the second electron to b cytochromes. The reduced b cytochromes are reoxidized by donating an electron to ubiquinone in an antimycin-sensitive step. According to this pathway the reduced DCIP can bypass the cadmium block of electron transfer from succinate to cytochrome c (Fig. 2B) by reducing the ubisemiquinone at center out to QH2 as shown in Fig. 5. QH 2 transfers one electron to FeS center and hence to cytochrome cl, thus giving ubisemiquinone that can be reduced to QH2 again by reduced DCIP. This results in a continuous oxidation of reduced DCIP constantly regenerated by enzymatic reduction by succinate, thus providing a bypass around cadmium block. Such a pathway is further supported by the finding that myxothiazol, by inhibiting electron transfer from FeS center to cytochrome cl (Fig. 2B), blocks the cadmiuminsensitive electron flow promoted by reduced DCIP. The TMPD bypass of the cadmium block, in which WB* acts as an oxidant, is also explained on the basis of Q cycle scheme since the WB ÷ must accept electrons from the only reduced species that accumulate in the presence of cadmium, i.e., ubisemiquinone at center out thus providing a continuous escape of electrons from ubisemiquinone. Therefore, the inability of cadmium to inhibit TMPD-promoted bypass confirms once more that its inhibitory site must be localized between cytochrome br and ubisemiquinone. In TMPD bypass one electron from QH2 passes to the Rieske FeS center and the other one from ubisemiquinone to WB ÷ and hence to cytochrome c (Fig. 5). There is no net consumption of WB ÷ which is continuously regenerated by TMPD oxidation by cytochrome c. Once again the inhibition of electron transfer to FeS center by myxothiazol inhibits the overall TMPD bypass reaction (Fig. 2D). QH2 is regenerated again by reduction of Q by succinate. However, the finding that the cadmium, in a similar way to rotenone, induces a rapid and extensive oxidation of cytochrome b in mitochondria in state 1 (Fig. 3) indicates that there must be a second point, other than that just described, at which it inhibits electron flow through the respiratory chain. Spectrophotometric observations on the redox state of NAD(P) ÷ suggest that this second inhibitory site cannot be localized on the respiratory chain itself. In particular, the finding that NAD(P)

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167

in cadmium-treated mitochondria, either in state 4 (Fig. 4B) or in state 1 (Fig. 4C), is in an oxidized state strongly indicates that the site of inhibition should be localized before NAD(P) ÷, i.e., between NAD-linked substrates and respiratory carriers, probably at level of dehydrogenases, as found with lonidamine and rhein, two antitumor drugs which inhibit electron transport in tumor and liver mitochondria at the dehydrogenase-coenzyme level [ 11-13]. Furthermore, it should be stressed that cadmium concentrations to inhibit electron transfer from substrates to respiratory chain as well as through ubiquinone -c~ span are remarkably lower than those required to exert its uncoupling effect. It is therefore suggested that low intracellular cadmium concentrations, resulting from the metal exposure, exert their toxic effects mainly through a block of electron flow rather than an uncoupling of the oxidative phosphorylation.

5. Acknowledgements The authors thank Mr Luigi Dall'Oco and Mr. Mauro Di Giovanni for their skilful graphic and photographic work. This work was partially supported by AIRC and Ministero della Santi/t.

6. References I 2 3 4 5 6

7

8 9 10 I1

12 13

M.R.S. Fox, Biochemical basis ofcadmium toxicity in human subjects, in: (Ed.), Clinical biochemical and nutritional aspects of trace elements, A.R. Liss, New York, 1982, pp. 537-547. S.M. Andrews, M.S. Johnson and J.A. Cooke, Cadmium in small mammals from grasslands established on metalliferous mine waste, Environ. Pollut., (Series A), 33 (1984) 153-162. E.E. Jacobs, M. Jacob, D.R. Sanadi and L.B. Bradley, Uncoupling of oxidative phosphorylation by cadmium ion, J. Biol. Chem., 223 (1956) 147-156. E.M. Diamond and J.E. Kench, Effects of cadmium on the respiratory of rat liver mitochondria, Environ. Physiol. Biochem., 4 (1974) 280-283. L. Mfiller and F.K. Ohnesorge, Cadmium-induced alteration of the energy level in isolated hepatocytes, Toxicology, 31 (1984) 297-306. N. Sato, T. Kamada, T. Suematsu, H. Abe, F. Furuyama and B. Hagihara, Cadmium toxicity and liver mitochondria II - - Protective effect of hepatic soluble fraction against cadmium-induced mitochondrial dysfunction, J. Biochem., 84 (1978) 127-133. P.L. Pedersen, J.W. Greenwalt, B. Reynafarje, J. Hullihen, G.L. Decker, J.W. Soper and E. Bustamante, Preparation and characterization of mitochondria and submitochondrial particles of rat liver and liver-derived tissue, Methods Cell Biol, 20 (1978) 411-481. A.G. Gornall, C.J. Baldwin and M.M. David, Determination of serum proteins by means of biuret reaction, J. Biol. Chem., 177 (1949) 751-766. B. Reynafarje, L. Costa and A.L. Lehninger, 02 solubility in aqueous media determined by a kinetic method, Anal. Biochem., 145 (1985) 406-418. A. Alexandre and A. Lehninger, Bypasses ofthe antimycin A block ofmitochondrial electron transport in relation to ubisemiquinone function, Biochim. Biophys. Acta, 767 (1984) 120-129. A. Floridi and A.L. Lehninger, Action of the antitumor and antispermatogenic agent Ionidamine on electron transport in Ehrlich ascites tumor mitochondria, Arch. Biochem. Biophys., 226 (1983) 73-83. A. Floridi, S. Castiglione and C. Bianchi, Sites of inhibition of mitochondrial electron transport by rhein, Biochem. Pharmacol., 38 (1989) 743-751. A. Floridi, S. D'Atri, M. Bellocci, M.L. Marcante, M.G. Paggi, B. Silvestrini, A. Caputo and C. De Martino, The effect of gossypol and Ionidamine on electron transport in Ehrlich ascites tumor mitochondria, Exp. Mol. Pathol., 40 (1984) 246-261.