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Bioelectrochemistry and Bioenergetics, 23 (1990) 325-336

A section of J. Electroanal. Chem., and constituting Vol. 298 (1990) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands

Possible involvement of mitochondrial calcium transport in causing cell injury in experimental hepatic chronic iron overload

Daniela Ceccarelli *, Tommaso Trenti, Umberto Muscatello and Albert0 Masini

Istituto di Paiologia Generale, Universitd! di Modena, via Campi 287, I-41100 Modena (Italy)

(Received 30 November 1989; in revised form 5 March 1990)

ABSTRACT

The functional state of isolated mitochondria and, specifically, the integrity of the inner membrane

were investigated in the livers of rats made siderotic by dietary supplementation with carbonyl iron. The

concentration of iron in the mitochondrial fraction increased progressively up to nearly 40 days and it

reached a steady-state level. When the iron content reached a threshold value (higher than 30 nmol/mg

protein) the occurrence of in viva lipid peroxidation in the mitochondrial membrane was detected. This

process did not result in gross alterations in the rnitochondrial membrane, as indicated by phosphoryla-

tive capability and membrane potential measurements. On the contrary, the induction of the lipoper-

oxidative reaction appeared to be associated with the activation of Ca*+ release from mitochondria. This

was shown to occur as a consequence of rather subtle modifications in the inner membrane structure via

a specific efflux route, which appeared to be linked to the oxidation level of mitochondrial pyridine

nucleotides. The induction of this Ca*+ release from iron treated mitochondria resulted in enhancement

of Ca*+ cycling, a process which dissipates energy to reaccumulate the releaased Ca*+ into mitochondria.

The perturbation in mitochondrial Ca *+ homeostasis reported here may be a factor in the onset of cell

damage in this experimental model of hepatic chronic iron overload.

ABBREVIATIONS

EDTA EGTA FCCP FeNTA GSH GSSG Hepes NADH

ethylenediamine tetraacetate ethyleneglycol-bis( P-aminoethylether)-N,N,N’,N ‘-tetraacetic acid carbonyl cyanide-p-trifluoromethoxyphenylhydrazone ferric nitrilotriacetate glutathione, reduced glutathione, oxidized N-2-hydroxyethylpiperazine-n’-2-ethane sulphonic acid nicotinamide adenine dinucleotide, reduced

l To whom correspondence should be addressed.

0302-4598/90/$03.50 0 1990 - Elsevier Sequoia S.A.

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NADPH nicotinamide adenine dinucleotide phosphate, reduced RR ruthenium red

TCA trichloroacetic acid TPP+ tetraphenylphosphonium cation AE electrode potential

A+ mitochondrial transmembrane electrical potential, negative inside

INTRODUCTION

It is firmly established that iron overload in the liver is associated with hepatic injury, fibrosis and ultimately cirrhosis. However, the biochemical mechanism of

iron toxicity is still poorly understood [1,2]. One of the most favoured explanations is that pathological accumulation of iron

initiates membrane lipid peroxidation in cellular organelles, a process which is implicated in tissue damage in a wide variety of disorders [3,4]. Related to this point, experimental evidence is accumulating that chronic iron overload may result in in vivo lipid peroxidation of mitochondrial membranes [5-71. Iron could promote the production of free radicals such as superoxide and hydroxyl radicals and/or

hydrogen peroxide [8,9]. Iron-induced peroxidative damage to liver mitochondria may occur via a number of possible mechanisms. This process may modify the

fluidity state of the mitochondrial membrane with alterations in the transport of solutes and ions [lo]. Swelling and lysis of isolated mitochondria associated with the enhancement of lipid peroxidation has also been reported [11,12]. Furthermore, hydroperoxidic products, derived from the free radical oxidation of mitochondrial polyunsaturated fatty acids (PUFAs) and hydrogen peroxide, may result in more subtle and earlier modifications in the functional properties of liver mitochondria [13-151.

The present study was aimed at investigating the membrane functional integrity of liver mitochondria isolated from rats made siderotic by dietary supplementation with carbonyl iron. Specifically, the susceptibility of the mitochondrial membrane to lipid peroxidation and the mitochondrial ion transport capability were analyzed.

EXPERIMENTAL

Female Wistar albino rats (loo-120 g body weight) were purchased from Nossan (Corezzano, Milan, Italy). They were made siderotic by feeding them a standard diet purchased from Dr. Piccioni (Brescia, Italy) supplemented with 2.5% (w/w) carbonyl iron.

The animals were killed by decapitation after an overnight starvation period. Liver mitochondria were prepared in 0.25 M sucrose according to a standard procedure [16]. The protein content of the final mitochondrial suspension was determined by the biuret method with bovine serum albumin as the standard.

The mitochondrial and hepatic iron concentration were determined with an atomic absorption Perkin Elmer spectrophotometer (model 306).

321

Determination of the conjugated diene signal was performed according to Corongiu et al. [17]. The lipid chloroform extracts were placed in glass test tubes with glass stoppers. The solvent was removed under vacuum at 40” C. The lipid extract was dissolved in cyclohexane in order to obtain a lipid concentration of 100 pg/ml and scanned from 300 to 220 nm and the absorbance and the second-deriva- tive spectrum were recorded. The presence of conjugated dienes was detected directly in the second-derivative spectrum by the presence of minimum peaks that absorb at 233 and 242 nm. These absorbances have been characterized elsewhere as resulting from truns-tram and cis-tram hydroperoxydienes [18]. Before the sample scanning operation, a background correction-memorized scan between Suprasil cells containing cyclohexane was performed in order to avoid spectral differences be- tween sample and reference cell. Because artefactual peroxidation may occur during tissue sampling and processing, experiments were considered acceptable only when lipid extracts, prepared from control animals (at least 4) and studied at the same time as the treated group, did not show any significant second-derivative signal attributable to lipid peroxidative damage. In the figure based on second-derivative spectroscopy, an absorption peak in the conventional sense appears as an absorp-

tion minimum. The transmembrane potential (A$) was measured at 25 o C in a final volume of

1.5 ml by monitoring, with a tetraphenylphosphonium (TPP+)-selective electrode, the movements of tetraphenylphosphonium across the mitochondrial membrane as in ref. 19. An inner rnitochondrial volume of 1.1 pl/mg protein was assumed. The metabolic medium for assaying the electrochemical parameters had the following composition: 210 mM mannitol, 70 mM sucrose; 5 mM Hepes pH 7.4 (MSH buffer); 20 I_LM TPP+. Mitochondria (3 mg protein/ml) were incubated at 25 “C and then energized with 2.5 mM Na-succinate.

Ca2+ movements across the inner mitochondrial membrane were followed by a CaZf-selective electrode as described in detail in refs. 20 and 21. Mitochondria (3

mg protein/ml) were incubated at 25 o C in MSH buffer containing 5 PM rotenone and 150 PM CaCl,. After a pre-incubation period of 5 min, the initial rates of Ca2+

uptake were measured following the addition of 5 mM K-succinate as respiratory substrate.

The endogenous content of K+ and Mg2+ was determined as follows: liver mitochondria (3 mg protein/ml) were incubated at 25 o C in MSH buffer containing 5 mM MgCl,; 2 mM Na, K-phosphate (pH 7.4) and 1.6 mM Na-pyruvate plus 0.4 mM L-malate as the substrate. At timed intervals, 0.5 ml of mitochondrial suspen- sion was removed and centrifuged for 1 min in an Eppendorf bench centrifuge (Model 54148). The pellet was washed twice with cold standard medium which did not contain K+ or Mg2+, then dispersed by extensive vortexing in 1% Na-cholate containing 1 mM EDTA. The K+ and Mg2+ content was determined by atomic absorption.

The mitochondrial GSH content was measured as follows: mitochondria (6.5 mg protein/ml) were incubated at 25” C in MSH buffer in the presence of 2 FM rotenone with succinate as the substrate. After 15 s of incubation, samples of 2 ml

328

were taken and the reaction was stopped by rapid mixing with 1 ml of 5% TCA (w/v) and 5 mM EDTA. After centrifugation, aliquots of supernatant were reacted with Ellman’s reagent as described in ref. 22.

The mitochondrial NADH and NADPH content was determined by an en- zymatic method as in ref. 23. Mitochondria (3 mg protein/ml) were incubated at 25 o C in MSH buffer in the presence of 2 PM rotenone and 2 mM succinate. After 15 s incubation, samples of 3 ml were collected and added to 0.6 ml of ethanol containing 1 M KOH. The mixture was kept at room temperature for 30 min and then cooled on ice for 10 min. Then 1 ml of a solution containing 0.5 M triethanolamine, 0.4 M KH,PO, and 0.1 M K,HPO, was added and the solution, kept at room temperature for an additional 10 min, was centrifuged. Reduced

pyridine nucleotides were determined fluorimetrically (340 nm excitation, 465 nm emission wavelength), on an aliquot of the supematant by the sequential addition of 50 mM Na-pyruvate plus lactate dehydrogenase (NADH) followed by 5 mM glutathione disulphide plus glutathione reductase (NADPH).

The results represented the mean of 4 to 6 different experiments & S.D. Student’s

t-test was used to compare group means. Carbonyl iron was purchased from Fluka (Fluka AG, Buchs, CH-9470, Switzer-

land). All chemicals and reagents were of the highest purity available.

RESULTS

Figure 1 shows the iron content of liver mitochondria isolated from rats fed a diet supplemented with 2.5% carbonyl iron. The iron level increases progressively from 4 nmol/mg protein up to 40 nmol/mg protein after 60 days of treatment and then reaches a stedy-state level, remaining nearly constant during the period of 90

days tested. No appreciable modifications were observed in the iron concentration of control mitochondria (about 4 nmol/mg protein) during the treatment period (not shown). The iron content of the whole hepatic tissue, from control and iron treated rats, presented a pattern very similar to that of isolated mitochondria (not shown).

The induction of in vivo lipoperoxidative reactions in the mitochondrial mem- branes appears to be associated with the attainment of a threshold value in the concentration of iron in the liver and in the mitochondrial fraction. Figure 2

presents the UV and the second-derivative spectrum of mitochondrial lipids ob- tained from untreated rats (A) and rats treated for 40 days with iron (B). The figure indicates clearly two modifications in the second-derivative spectrum, characteristic of the truns-tram and c&tram conjugated diene signals of iron loaded mitochondria, at 233 and 242 nm, respectively. At shorter treatment times, for example 20 days, no lipid peroxidation was detected. It is worth noting that no modifications were observed in the second-derivative spectrum of microsomal lipids during the entire treatment period.

The determination of the membrane potential on the basis of movements of the lipid-soluble tetraphenylphosphonium cation has proven useful for measuring the

329

Time of Fe treatment/days

Fig. 1. Effect of treatment of rats with iron on the iron content of the mitochondrial fraction. The

mitochondrial iron concentration was determined by atomic absorption spectroscopy. The data represent

mean values for 4 separate experiments f SD.

energy state of mitochondria and for assessing the intactness of mitochondria after hydroperoxide induced calcium release [19]. It appears from Fig. 3 that mitochondria from rats treated with iron for 40 days, on addition of a respiratory substrate, such as succinate, immediately develop a normal A# of about 180 mV. Similar values of the membrane potential were found with mitochondria isolated from rats early in the treatment (not shown). However, the membrane potential pattern during the accumulation of a pulse of Ca2+ revealed a marked difference between control and iron treated mitochondria. Indeed, in these mitochondria the membrane potential trace, after the drop which corresponds to the energy utilized for calcium accumula- tion, does not return to the pre-Ca2+ level, as in the control, but decreases suddenly. When A# has reached its lowest value, addition of the calcium chelator EGTA promptly reverses the membrane potential to its normal value. This observation indicates that the inner membrane is not depolarized irreversibly, thus suggesting that the decrease of the membrane potential is not due to damage to the mitochondria but rather to a continuous and energy draining Ca2+ cycling, a process which dissipates energy due to reaccumulation of the released Ca2+ into the mitochondria.

The suggestion of an enhancement of Ca2+ in iron treated mitochondria is supported experimentally by the results on calcium movements presented in Fig. 4. It is seen that iron treated mitochondria are unable to retain the accumulated pulse of Ca2+. It also appears that the route by which Ca2+ is released by iron treated

330

0 220 250 300 250

Wavelength 1 nm

Fig. 2. UV (a) and second-derivative spectra (b) of lipid extracted from rat liver mitochondria. (A)

Mitochondria isolated from control rats; (B) mitochondria isolated from rats treated with iron for 40

days. The arrows in the second-derivative spectra show the minimum peaks at 233 nm and 242 nm. The

data reported are shown as an example and refer to experiments performed on at least 5 samples

obtained from different animals.

mitochondria can not be inhibited by ruthenium red, a specific inhibitor of the electrophoretic calcium uptake pathway [24]. Indeed, it appears that addition of ruthenium red to control mitochondria results in an immediate and slow release of Ca*+ which is typical of liver mitochondria [20]; in contrast, addition of ruthenium red to iron loaded mitochondria, following this initial release of Ca*+, again induces a significant net Ca*+ efflux. It should be noted that mitochondria isolated from rats treated for 20 days did not exhibit such anomalies in calcium transport (not shown).

331

200

> E 180

\

G-

150

100

10 i

t succ.

A

Ca

I

2 mln

Ca

FCCP

I

ECTA

Fig. 3. Effect of treatment of rats with iron on the membrane potential of liver mitochondria pre-loaded

with a pulse of Ca*+. Mitochondria in the standard incubation medium, described in the Experimental

section, were energized by the addition of 2.5 mM succinate (Succ.). The arrows indicate the following

additions: 150 pM CaCI, (Ca); 0.5 mM EGTA; 1 gM FCCP. (A) Control mitochondria; (B) mitochondria

from rats treated with iron for 40 days. The mitochondrial membrane potential (A#) was measured by a

tetraphenylphosphonium selective electrode, as described in the Experimental section, in the presence of

20 PM tetraphenylphosphonium chloride. AE is the electrode potential. The traces presented are

representative of at least 4 different experiments performed on a pool of 3 animals.

It was also found that a net release of K+ accompanied the Ca*+ efflux from iron treated mitochondria. After 7.5 min of incubation there was a decrease of

approximately 80% in the mitochondrial potassium content. The presence of either ruthenium red or EGTA in the incubation medium largely prevented K+ release. It is worth noting that under these experimental conditions, no appreciable modifica-

TABLE 1

Effect of chronic dietary iron overload on the reduced pyridine nucleotides and glutathione content of rat

liver mitochondria. Pyridine nucleotides and glutathione analyses were performed according to ref. 11.

Mean values of control (n = 6) and 40 days Fe treated rats (n = 6), respectively, are given + SD.

Treatment Metabolite/nmol/mg-t protein

NADH + NADPH GSH

None 5.12 f 0.4 8.85 + 1.08

Fe 2.18 f 0.68 9.26 + 0.86

332

240 -

5

E \ 2 mo-

m 'i J 150-

+

m; 120-

60-

o-

su-cc. slice. Fig. 4. Effect of treatment of rats with iron on the Ca2+ transport of liver mitochondria. Mitochondria

were incubated for 5 mm in the standard medium, described in the Experimental section, in the presence

of 150 pM Ca’+. Respiration dependent uptake of Ca2+ was started by the addition of 5 mM

K-succinate (Succ.). Further additions were 1 pM ruthenium red (RR). (A) Control mitochondria; (B)

mitochondria from rats treated with iron for 40 days. Ca2+ movements were followed by a Ca2+

selective electrode as described in the Experimental section. All other conditions as in Fig. 2.

tions occurred in the mitochondrial Mg 2+ level, which remained almost constant at a normal value of about 30 nmol/mg protein (not shown).

A quantitative analysis of the factors that conceivably play a regulatory role in the mechanism underlying calcium release from iron treated mitochondria (that is, reduced pyridine nucleotides and reduced glutathione) is presented in Table 1. It appears that the mitochondrial content of reduced pyridine nucleotides, NADH plus NADPH, is decreased by more than 60% compared to the control. In contrast, the content of mitochondrial reduced glutathione is not modified significantly.

DISCUSSION

The present results show that feeding rats a diet supplemented with carbonyl iron results in accumulation of iron in the hepatic tissue and in the mitochondrial fraction. The accumulation of iron is progressive with time up to nearly 60 days, where it reaches a steady-state value. On the basis of the results provided by the technique used in the present research, it cannot be decided whether the excess iron found in the mitochondrial fraction is due to an actual accumulation of iron within

333

the mitochondria or is due, in large part, to accumulation into lysosomes that may be present in the mitochondrial fraction as contaminants [25-281. On the other hand, it has been demonstrated clearly in an analogous experimental model that the presence of contaminant lysosomes and ferritin/hemosiderin aggregates did not affect the functional characteristics of mitochondria appreciably [29].

The increased concentration of iron in the hepatic tissue and in the mitochondrial fraction is associated with an enhancement of in vivo lipid peroxidation of mitochondrial membranes, as shown by the diene spectra (Fig. 2). This process appears to be dependent on the attainment of a threshold iron concentration, since it is observed when the iron concentration is higher than 1000 pg/g of liver tissue and 30 nmol/mg protein.

The biochemical mechanism by which iron may initiate free radical production in vivo has not been established experimentally. It may be speculated that under the condition of excess iron, the capability of the hepatocytes to maintain iron in the non-toxic protein bound ferric state may be exceeded, thus resulting in low amounts of ferrous iron. The latter can initiate membrane peroxidation either directly or indirectly by forming perferril-iron (FeOz’), or a ternary free radical complex arachidonic acid, ferrous iron and oxygen [30]. Moreover, ferrous iron or low molar mass chelates of iron [31] may play a role in the generation of free hydroxyl radicals (OH’) by catalyzing the reaction of superoxide radicals (O;-) with hydrogen peroxide (H,O,) [8,9].

The in vivo induction of lipoperoxidative reactions in the mitochondrial mem- branes seems to be associated with the activation of calcium release from these organelles. This proposal derives from the findings that no calcium release is observable in mitochondria isolated from rats treated for 20 days, where no lipid peroxidation is detected. This calcium efflw did not result from gross alterations in the structure of the mitochondria. In fact, a clear indication of the functional integrity of the inner membrane is given by the fact that a normal membrane potential of about 180 mV [16] is developed by these mitochondria.

Direct measurement of calcium movements shows that iron treated mitochondria release the accumulated calcium rapidly by a pathway which can not be inhibited by ruthenium red, a specific inhibitor of the electrophoretic calcium uptake route (uniport) [24], thus indicating that this may occur via a distinct electroneutral “antiport” [32]. Should calcium be released by the reversal of the electrogenic uptake pump, the calcium efflux would be inhibited by ruthenium red.

An aspect which merits consideration is that the enhancement of the calcium cycling process seems to elicit a net efflux of K+ from mitochondria. Indeed, when calcium cycling is blocked by either ruthenium red or by EGTA, the efflux of K+ is largely prevented also. This finding and the observation that no release of mitochondrial Mg*+, an ion involved in the maintenance of the functional integrity of the mitochondria [33], took place under these conditions, confirms further that the K+ efflux is not due to a non-specific increase of the inner membrane permeability [34,35], due, for instance, to generalized damage to mitochondrial membranes.

334

BIOCHEMICAL MECHANBM OF IRON INDUCED Mll’DCHONDRIAL Ca2* RELEASE

\ R-ooH~*G~*N~~~~~~~d~ed substrate, H24

I increased permeability, -Ca%slease

lncrkased extramitochondrlal

Can

Fig. 5. A proposed biochemical mechanism for iron induced Ca*+ release from isolated mitochondria in

the experimental model of hepatic chronic iron overload.

Experimental evidence is accumulating that two factors may be primarily in- volved in the regulation of calcium release from the mitochondria: a shift in the redox state of pyridine nucleotides [13,15,36,37] and/or of mitochondrial glutath- ione [34,35,38]. The present results show that no appreciable modifications are observable in the level of reduced glutathione (GSH), whereas a decrease of approx. 60% is observed in the content of NADH plus NADPH in mitochondria isolated from iron treated rats. This finding suggests that the latter factor regulates the activation of the specific calcium release pathway reported here. In this vein it may be speculated that hydroperoxidic products and/or hydrogen peroxide, which most probably are formed in this experimental model, may lead to enzymatic pyridine nuclcotide oxidation through glutathione peroxidase-glutathione reductase and the energy linked transhydrogenase system [39]. Such a biochemical pathway is depicted in Fig. 5. Another possible explanation for the anomalies in calcium transport observed here may be derived from the observation that hydroperoxides and Ca2+ may lead to Ca2+ pore opening in the mitochondrial inner membrane [40]. The finding that, under conditions of oxidative stress, the oxidation of some polyun- saturated fatty acids may give rise to products acting as specific Ca2+ ionophores, may represent an additional mechanism for explaining the results reported here [41].

335

The finding that no significant decreases is observable in the mitochondrial GSH

content makes untenable the proposal that the mitochondrial GSH redox state may be involved primarily in the induction of the process of calcium release from iron treated mitochondria, although it cannot be excluded that in vivo mitochondrial GSH may in some way be replenished continuously from the cytosolic compart-

ment. As to the causal events of this calcium transport perturbation, it has to be

stressed that lipid peroxidation and changes in the reduced pyridine nucleotide level was measured in mitochondria immediately after the isolation; therefore they are somewhat representative of the in vivo situation in the liver cell.

In the light of the present results and the above considerations, the perturbation in mitochondrial calcium homeostasis described here for a mitochondrial fraction isolated from iron treated rats, which results from an original in vivo situation, may be of importance for the understanding of the biochemical mechanism of in vivo iron hepatotoxicity. In fact, an activation of calcium release leads to an increase in the process of calcium reaccumulation, thus resulting in an enhancement of calcium cycling with a continuous energy dissipation. Given the role played by rnitochondria in the cell metabolism, this situation might be one of the causal events of cell damage. This derangement in mitochondrial calcium transport may also result in a decreased capability of mitochondria to contribute to cellular calcium homeostasis, with excess work for the endoplasmic reticulum and plasma membrane calcium pump [42,43]. An alteration of cellular calcium homeostasis is a proposed factor in liver cell injury [44]. To test this hypothesis directly, further studies are needed and at the moment its direct extrapolation to intact cells is a matter of speculation.

ACKNOWLEDGEMENTS

This work was supported by a grant from Minister0 Pubblica Istruzione of Italy (40%) project “Patologia da Radicali Liberi e degli Equilibri Redox”.

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