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The role of desferrioxamine chelatable iron in rat liver mitochondrial dysfunction in chronic dietary iron overload
ELSEVIER
Bioelectrochemistryand Bioenergetics42 (1997) 169-174
The role of desferrioxamine chelatable iron in rat liver mitochondrial dysfunction in chronic dietary iron overload Daniela Ceccarelli a.*, A.V. Kozlov b, D. Gallesi a, A. Tomasi a, F. Giovannini ", A. Masini ~ Department of BioraedicalSciences. Sectionof General Pathology, Universi~'o~Medena. 41100 Modena, Italy b Departmentof Biophysics. Russian Medical Un&ersir).',Ostrovitianm'aStr. 1, i 17347Moscow, Russia
Abstract
Lipid peroxidation and organdie dysfunction are important factors in hepatic iron toxicity. The form of the intracelkdar iron responsible for these abnormalities is still unknown. In order to investigate the iron species inducing cell injury, the level of ct~heab~ iron in the liver roitnchondfia isolated from rats fed a 2.5% carbonyl iron diet for 12 weeks was measured by EPR spectroscopy. The presence of lipid peroxidation products and the energy transducing capability of the mitochondrial inner membrane was e,~aluated in parallel. The total iron concemration in the liver mitochondria from iron fed rats progressively increased up to 6 weeks, almost re~hing a steady-state. By contrast the level of chelatable iron in mitechondrial fraction transiently increased at about 3-6 weeks of treatment. The induction of lipid peroxidation and a large decrease of ATP occurred at the same time. The enhancement of the energy dissipating calcium cycling was in parallel rt:vealed by studying the mitochondria merobrane potential. These results gave experimental evidence to the proposal that the chelatable iron level plays a critical role in initiating organelle dysfunction, at least in this experimental model. © t997 Elsevier Science S.A.
Keywords: Iron toxicity:Iron(dietaryove~'load);Mitochondfialfunction(lipid peroxidation);Mitochondria(membranepotential)
1. Introduction
In iron storage diseases (e.g. idiot'~athic haemochromatosis, thalassemic syndromes, transfusional iron overload, alcoholic cirrhosis, porphyria cutanea tarda), the liver is the main target of injury with hepatic fibrosis and c~rrhosis being the major hallmarks of the disease [I,2]. Although the toxicity of excess iron has been well established clinically [3-6], the specific cytopathol~gical mechanisms whereby hepatocytes are damaged in chronic iron overload remain to be fully explained [7-9]. The most favoured hypothesis is that the pathological accumulation of iron in the liver initiates lipid peroxidation of various cellular organelles that in turn results in the alterations of
Abbreviations: AIP = adenosine triphosphate;BHT = butylated by_ droxytoluene; DFO= desferrio~amine; Ag' = transmembrane electrical potential; EGTA= ethylene glycol-bis(l~-aminoethylether)-N,N.N',N'. tetraacetic acid; FCCP--c;;rbonylcyanide-p-trifluoromethoxyphenylhydrazone;MDA = malondi:ddehyde;RR = ruthenium red- TBA = thiobarbituricacid;TPP+ = tetrap,henylphosphoniumchloride ' Correspondingauthor,Tel: + 59 360044; Fax: + 59 362206: Email: [email protected]. I Presented at the 13th InternationalSymposiumon Bioelectrnchemistry and Bioenergetics,Ein Gedi. Israel,'7-12 January 1996. 0302-4598/97/$17.00 © 1997 Elsevier Science S.A. All rights reserved. Pll $0g02-4598(96)05109-4
tile physical and biochemical properties of the membrane components and finally in cell damage [9,10]. An almost exclusive parenchymal iron overload model, resembling liver pathological appearance in human primary haemochromatosis, is achieved by feeding rats a carbonyl iron-enriched diet [11]. Many reports dealing with this experimental model correlate the induction of lipid peroxidation associated with organclle func~/onal derangement with the level of total iron in the liver [11-14], but the intracellular form of iron that actually is able to init/am the reported anomalies is still unknown. In physiological conditions, iron enters ferritin, the m a ~ iron storage protein in virtually all cells, but under conditions of largely excessive deposition the capacity of hepatocyte to maintain iron in storage form could be exceeded. Recent work has indicated the existence of a transient cellular pool of ionic iron of low molecular weight, chelatable by desferrioxamine (DFO), that is in redox active form capable of initiating oxidative reactions in cellular membranes [15-18]. In the present research, an EPR assay was performed to quantitate the low molecular weight iron (LMW-iron or cbelatable iron) pool in m~tochondria isolated from liw:rs of rats fed an iron enriched diet during a period of 12 weeks. The functional efficiency of the inner mitochondrial membrane
17o
D. Ceccarelliet al./ Bioelectrochemist~"and Bioenergetics42 (1997)169-174
was in pzra]lel evaluated by membrane potential and ATP illeasurelilents.
LC-18 Hypersil 5ODS 4.6 cm x 25 cm (Lab Service, BO, Italy). The separation was accomplished according to Stocchi et al. [24]. ATP was revealed at 26Onto aga:,nst known quantities of external standards (75-200pmol).
2. Experimental details 2.5. Total mitochondrial iron 2.1. Animal treatment
Female Wistar albino rats (100-120g) (Morini, Reggio Emilia, Italy) were made siderotic by feeding a standard diet purchased from Dotto~ Piccioni (Brescia, Italy) supplemented with 2.5% (w/w -t) carbonyl iron for increasing periods of up to 12 weeks. Experimental and control rats were handled identically. The National Research Council criteria for the care and use of the laboratory animals in research were carefully followed. 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 [19]. The cytosolic contamination in mitochondrial preparation did not exceed 2% of the total protein content, as assessed by the recovery of marker enzymes [20]. The protein content of the final mitocbondrial suspension was determined by the biuret method with bovine serum albumin as the standard. 2.2. Mitochondrial membrane lipid peroxidation
Mitocbonddai membrane lipid peroxidat~on was asse,~sed by measuring the thyobarbituric acid reactive ;ubstances as end products and the amount of matondialdehyde (MDA) in the mitochondrial fraction was measured by a thiobarbituric acid method modification [21]. 2.3. Transmembrane potential ( ArIt)
Transmembrane potential (A~) was measured at 25°C by a tetraphenylphosphonium chloride ('i"PP+) selective eleclrode [22]. Mitochondfia (2 mg protein ml-t) were incubated in I ml of standard incubation medium of the following composition: t00mM NaCI; 5ram sodiumpotassium phosphate buffer (pH 7.4); lOmlVl Tris-HCl buffer (pH 7.4); IOmM MgCI 2 plus 5tLM rotenone and 20p.M TPP +. An inner mitochoncirial volume of 1.1 p.lmg -t protein was assumed. The respiratory states were those defined by Chance and Williams [23] on the basis of the factors limiting the respiralion. 2.4. Adenosine triphosphate (ATP)
Adenosine triphosphate (ATP) was determined as follows: I ml of mitochondrial suspension (about 8mgprotml -~) was treated with 0.2M of HCIO4 and centrifuged. 20 p.i of the neutralized acid-soluble fraction were used for the HPLC analysis by a liquid chromatografic systein (Hewlett-Packard HP 1090) equipped with a photo-diode array detecter. The column was a
The iron concentration was determined in acid extracts by an atomic absorption Perkin-Elmer spectrophotometer model 31)6 [13]. 2.6. Mitochondrial chelatable iron
The iron concentration was measured by EPR spectroscopy as described in [25]. Samples of 0.5 mi of mitocbondrial suspension (60-90rag ml-I ) were incubated at 20°C for 10rain with I mM desferrioxamine (DFO). The incubation t~me was previously assessed [25] as suitable for DFO to completely chelate all low molecular weight iron present as Fe(IlI) but not sufficient to remove iron from depot proteins. The samples were then placed in cylindrical tubes, frozen in liquid nitrogen and stored until the measurements were made. The DFO-iron complex formed with endogenous ionic iron is characterized by an EPR absorption at g = 4.3. The EPR spectra were recorded on a Brucker 200 spectrometer, at liquid nitrogen temperature, under the following conditions: oscillator Klystron frequency 9.12 GHz, power 20 roW, modulation a;nplitude 2.0mT. The intensity of the signal at g =4.3 was measured as a peak to peak amplitude to estimate the concentration of the DFO-iron complexes because the shape of signals obtained after blank subtraction, (mitochondria suspension not incubated with DFO) was the same in all samples and the ratio amplitude-double integral was constant. Calibration procedures: 'Free' iron concentration was calculated on the basis of a calibration curve, linear in the p.M range, obtained by measuring EPR signals of incremental amounts of FeSO4 • 7H20 added to identical volumes cf mitochondrial suspension, in the presence of I mM DFO. The Fe z+ content in the stock solution was conlrolled using the o-phenanthroline assay.
3. Results Data presented in Fig. 1 clearly demonstrate the significant accumulation of TBA-reactive substances, measured as MDA concentrations, in mitochondrial fraction isolated from the livers of 3-6 weeks iron fed rats (1300+ 180pmolmg -t protein vs. 260+ 33 of control). The extent of the peroxidation progressively decreases toward normal values after a longer treatment time. It has to be pointed out that the very same data resulted when an antioxidant as butylated hydroxytoluene (BHT) was added during the fractionation procedure, ruling out the possibil-
D. Ceccarelliet al. / Bioelectrochemistryand Bioenergetics42 ~1997)169-i74
1,400
• Control~ Iron i
3.s • Con~'o~ [ ] Iron
1,200
3
171
i
~
~.Z5
T ,
T
II
,,m!
m:
600 <~ 400
~; 200 0
i
i-~ 6 9 limeoftreatment(weeks)
3
~
0.5 12
3
6 9 Ttmeoftre~rnent(weeks)
12
~g. 3. Effect of iron ~ = m e n t on ATP content of n~tocl,~l fr~fi~.
Fig. 1. Effect of iron Ireatment of rats on MDA content of the mitochondrial fraction. MDA measurements were performed as described in Methods. The dala are representative of four separate experiments performed on a pool of 3 animals and are expressed as mean+SD, • P < 0.001 in comparison with control.
ATP was determined by an HPLC tecnique as decried in the Metimds. The data represent mean values for 4 separate expering~±SD. * P < 0.01 in comparison with control.
dria. When the calcium pulse has been completely taken up, Aqt returns to the pre-Ca: ~ level. The a~tidon of an uncoupler such as FCCP immediately makes A~ collapse. By contrast, mitochondria from 3-6 weeks iron fed rats (Fig. 2(b)), upon addition of the same pulse of Ca 2+, display a drop of A~ similar in the extent to tha~ for the control, but A~ trace does not return to the pre-Ca2+ level and quickly (about 2 min) collapses. The addition of a specific Ca:+ chelator, such as EGTA, or ruthenium red (RR), a specitic inhibitor of the elcctrophoreti¢ cag'/um uptake route (uniport) [26] or oligomycin, an inhibitor of ATP synthase [27], largely restores the A~ trace. On the contrary, an antioxidant such as BHT or an iron chel~ng agent such as desferrioxamine, ~ d to the incubating mixture, did not appreciably modify the A~ trace (not
ity that the MDA increase was due to an in vitro formation (not shown). Fig. 2 shows the effect of iron treatment on the mitochondrial functional efficiency as evaluated by measuring the mitochondfial transmembrane electrical potential (A~), by following the movements of the lipid-soluble TPP + cation. This parameter gives a direct indication of the energy transducing capability of the inner mitochondrial membrane. It can he seen that, on addition of an oxidizable substrate such as succinate (succ), the control mitocbondria (Fig. 2(a)) develop a normal A6 of about 200inV. When a pulse of Ca 2+ (150 IxM) is ad~d into the reaction vessel, a decrease of up to 172mV ap~ars which corresponds to the energy used for the Ca 2+ uptake inside the mitochon-
A Ca
FCCP
200
,,'"
OLIGO
180 •
150 •
100 10 =
2 mitt
\ succ
Fig. 2. Effect of iron treatment of rats on mitochondriai internal membcan¢ potential (A~'). The measurements were performed by means of a TFP + sensitive microelectrode, as described iv. the Experimental Details. The arrows indicate the following additions: 2 ram Na-succinate (succ); 150 IJ.M CaLl, (Ca); 1 p,M FCCP; 0.5 mM EGTA; I ~M ruthenium red (RR); 2 I~g rag- i protein oligomycin (oligo), ,3E. electro~ potential, (a) Control mitochoachia: (b) mitochondria from 3-6 week u'eated rats. The traces are representative of at least 3 different experiments performed on a pool of 3 animals.
172
t9. Ceccarelliet aL /Bioelectrochemism.' and Bioenergetics42 (/9971169-/74 [] Chel~l.ab~re ~ Tolat Fe
oi
2
oLf 0
m I 3 6 ,Q 1"=meof ~atrrtent (weeks)
,0o_:
. mm o * 12
Fig. 4. Effectof iron treatmenton total iron and chelatablc iron of mitochondrialfracti,m.Determinationswereperlbrmedby atomicabsorption .q~ectmphotometryand elect,onspin resonancespectrometrytechniquesas dcscrihedin the Methods.The data,'epresentmeanvaluesfor 4 separateexperiments_+SD.• P < 0,01 in comparisonwithcontrol.
shown). It must be pointed out that mitochondria isolated from iron fed rots for a time shorter or longer than 3-6 weeks did not display this A~ ~havinur (results not shown). Fig. 3 illustrates the endogenous adenosine triphosphate (ATP) content of mitochondria isolated from rat livers at various times of iron overloading in comparison with the control mitochondria. A maximal decrease of ATP of about 45% is observed at 6 weeks of iron feeding (!.58 + O.18nmolmg -~ protein vs 2.9+0.4 of control) and an average decrease, although not statistically significant, is also observed at 3 weeks of treatment. The atomic absorption measurement of the total iron in mitochondria from iron treated rats is shown in Fig. 4. It clearly appears that the content of the total iron progressively increases up to 6 weeks of treatment, then maintaining an almost steady value (from 2.8 +_0.8 nmol mgprotein of control to 25 _+3.8). The distribation pattern for the chelatable iron level is presented in the same figure. EP.q measurements indicate a sharp increase in the chelatable iron content for the time interval included between 3-6 weeks of treatment followed by the retum to control values at longcr times.
4. Discussion The present results strengthen z recent original observation [28], that a small pool of intracellular chelatable iron (3-10% of total cell iron) is primarily involved in thc induction of oxidant stress, inciuding lipid peroxidation, in the experimemal hepatic iron overload. Specifically, by the use of a new EPR technique, a strict correlation between a transient increase in chelatable iron and mitochondrial functional anomalies has here been confirmed, although at an earlier period of treatment (3-6 weeks vs. 4-7 weeks). In fact, the ultimate oxidizing form of iron responsible for the initiation of lipid peroxidation in iron overload in vivo
is not yet known but only hypothesised [12-14]. Iron dependent lipid peroxidation of hepatic organelles coincide with the impairment of the mitochodnria energy transducing capability has been up to now related with the max:mal concentration of total iron in the hepatic tissue that widely ranged from t000-5 000 ggg-i (wet weight) [I 1-14]. The w.,nsient raise in mitochondria chelatable iron level, here detected, is strictly associated with the induction of lipid peroxidation and the enhancement of the energy dissipating mitocholadriai calcium cycling revealed by membrane potential measurements. Specifically, the study of A~ behaviour of mitochondria from 3-6 weeks iron treated rats showed a clear difference compared with control during the accumulation of a low pulse of Ca2+(see Fig. 2). The observation that, when the A~ trace reaches its lowest value, the addition of EGTA and RR which inhibit Ca~+ cycling, or oligomycin which blocks energy dissipation, promptly reverses the membrane potential almost to its normal value, indicates that the inner membrane is not irreversibly depolarized and the A~ decrease is not caused by direct membrane damage but rather to a continuous energy draining Ca2÷ cycling (i.e., energy is dissipated to reaccumulate the released Ca2÷ into mitochondria). TLe time length of the A~" derangement depended on the amount of the Ca 2+ added; when no calcium was added the A~ decrease was very slow [28] while an immediate drop of A~t resulted after the addition of a high pulse of Ca2+ (results not shown). The metabolism of hydrogen peroxide and fatty acid hydroperoxides through the glutathione enzyme system (GSH peroxid~se-GSH reductase) shifts~,e redox state of mitochondrial i',yridine nucleotides toward an oxidized form, a condition which induces the activation of a specific passive calcium release from mitochondria, via a route distinc~ from the electrophoretical Ca2÷ uptake (uniport) [29-32]. Such an increase in the oxidized form of pyridine nucleotides has already been well documented under these experimental conditions [33]. The chelatable iron here detected may reasonably be involved in the formation of these oxygen reactive species [34]. The observation that the combined treatment of rats with iron and an antioxidant such as silybin, a main constituent of natural flavonoids, completely prevented lipid peroxidation and mitochondfial dysfunctions without appreciably modifying the total iron content of the liver is in agreement with the above proposal [35]. A consideration may be derived by observing the time course of total iron and chelatable iron concentrations in mitochondria (Fig. 4). Iron overload results in a significant transient increase of the mitochondrial chelatable iron pool, which enhances the generation of oxygen free radicals, at the time of intoxication that precedes or is nearly coincident with the reaching of the maximal accumulation of total iron. A comments should be addressed about the lower level of chelatable iron obtained by EPR in the present set of experiments compared with the one previ-
D. Ceccarelliet al. / Bioelectrochemistry and Bioenergetics 42 (1997) 169-174
ously reported by the use of an I-IPLC method [28], In the cas,: of HPLC, proteic samples had to be frozen and thawed, centrifuged and ultrafiltered to give clear extracts before the final reading, while in the case of EPR the measurements were peffo,."med directly on frozen samples. The additional step in the mitochondria treatment leads to a further release of chelatable iron (unpublished observations). Furthermore, it is conceivable that in the present series of animals the amount of preformed or neoformed ferritin was slightly higher to significantly reduce the level of chelatable iron (that is at maximum at 3-10% of the total cell iron) without appreciably affect the total iron pool. Although chelatable iron revealed here is much lower, it apparently constitutes an already critical amount to induce lipoperoxidation. It is well known that iron metabolism is tightly controlled and regulated [36,37] and that ferritin serves to sequester iron in a relatively inert form until it is needed for critical iron-dependent metabolic processes such as heine synthesis [38]. it may be speculated that the existing ferritin could not be able to completely accommodate the large excess of total iron which progressively accumulates and the capacity of the hepatocyte to maintain iron in storage forms is exceeded. After ferritin synthesis becomes stimulated [39] the capability of iron withholding by the hepatocyte may be restored and the level of chelatable iron returns to normal values. The present results add more information to the open problem of biochemical mechanism by which iron, a major catalyst of free radical reactions in the cell, may initiate, propagate and/or amplify the (auto)oxidative phenomena. Specifically they show that: 1) in viva iipoperoxidative reactions in mitochondrial membranes is directly correlated with the presence of a transient increase of redox active low molecular weight iron, or chelatable iron, consequently capable to give radicalic reactions; 2) the lipoperoxidatitn intermediate products bring about an increase in energy dissipation by the induction of an energy draining calciam cycling into mitochondria; 3) the energy wasting as a result of Ca z+ efflux-reuptake found here was active also in the absence of external calcium loading [28] and, in both cases, may be responsible for the significant decrease in ATP content (see Fig. 3). It is conceivable that under conditions of severe hepatic iron overload, when the cell is not able to maintain a steady state in iron storage machinery, the resulting mitochondrial functional derangement may become irreversible thus leading to cell injury. The present results may thus be of interest to give a clue to the therapeutic intervention in various disorders associated with excess tissue iron deposition.
Acknowledgements This work has been supported by a grant of the Italian Ministry of University and Scientific and Technological
173
Research (M.U,R.S.T,) and National Research Council of Italy (C,N.R.) special project 'Ageing'. A.V. KozIov has been supported by the European Science Foundation, grant N-° RF/93/35(T).
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[31] C. Richter, D. Frei and F.A. Cerutti. Biochem. 8itphvs. Res. Com,-ntan., 143 (I987) 609-616. [32] D+G.Nicholls and M. Cromptan. FEBS Lett, 111 (1980) 261+268. [33] A. Masini, T. Trenti. D. Caccatelli and U, Muscalello, Biochim. Biophys. Acta, 891 (1987) 150-156. [3~] B. Halliwelt and J.M.C. Gutteridge, Mol. Aspects Med.. 8 (1985) 89-193. [35] A. Pietrangelo, F. Borella, G. Casal~'andi, G. Montosi, D. Cecca-
relli, D. Gallesi, F. Giovannini, A. Gasparetto and A. Masini, Gastroenterology. I09 (199,5) I o41-1949. [36] P. Aisen and !. Listowsky, Ann. Ree+Biochem.. 49 (1980) 357-393. [37] G+ Cairo. L. Tacchini, L. Schiaffonati, E. Rappacciolo, E. Ventura and A. Pietrangelo, Biochem+J+, 264 (1989) 925-928. [38] D.W. Reif. Free Rad. Biol, Med.. 12 (1992)417-427. [39] M.W. Hen~ze, $+ Wright Caughman, J.L. Casey+D,M. KoeUer,T.A. Rouault, J.B. Harford and R.D. Ktausner, Gene. 72 (1988) 201-208.
Bioelectrochemistryand Bioenergetics42 (1997) 169-174
The role of desferrioxamine chelatable iron in rat liver mitochondrial dysfunction in chronic dietary iron overload Daniela Ceccarelli a.*, A.V. Kozlov b, D. Gallesi a, A. Tomasi a, F. Giovannini ", A. Masini ~ Department of BioraedicalSciences. Sectionof General Pathology, Universi~'o~Medena. 41100 Modena, Italy b Departmentof Biophysics. Russian Medical Un&ersir).',Ostrovitianm'aStr. 1, i 17347Moscow, Russia
Abstract
Lipid peroxidation and organdie dysfunction are important factors in hepatic iron toxicity. The form of the intracelkdar iron responsible for these abnormalities is still unknown. In order to investigate the iron species inducing cell injury, the level of ct~heab~ iron in the liver roitnchondfia isolated from rats fed a 2.5% carbonyl iron diet for 12 weeks was measured by EPR spectroscopy. The presence of lipid peroxidation products and the energy transducing capability of the mitochondrial inner membrane was e,~aluated in parallel. The total iron concemration in the liver mitochondria from iron fed rats progressively increased up to 6 weeks, almost re~hing a steady-state. By contrast the level of chelatable iron in mitechondrial fraction transiently increased at about 3-6 weeks of treatment. The induction of lipid peroxidation and a large decrease of ATP occurred at the same time. The enhancement of the energy dissipating calcium cycling was in parallel rt:vealed by studying the mitochondria merobrane potential. These results gave experimental evidence to the proposal that the chelatable iron level plays a critical role in initiating organelle dysfunction, at least in this experimental model. © t997 Elsevier Science S.A.
Keywords: Iron toxicity:Iron(dietaryove~'load);Mitochondfialfunction(lipid peroxidation);Mitochondria(membranepotential)
1. Introduction
In iron storage diseases (e.g. idiot'~athic haemochromatosis, thalassemic syndromes, transfusional iron overload, alcoholic cirrhosis, porphyria cutanea tarda), the liver is the main target of injury with hepatic fibrosis and c~rrhosis being the major hallmarks of the disease [I,2]. Although the toxicity of excess iron has been well established clinically [3-6], the specific cytopathol~gical mechanisms whereby hepatocytes are damaged in chronic iron overload remain to be fully explained [7-9]. The most favoured hypothesis is that the pathological accumulation of iron in the liver initiates lipid peroxidation of various cellular organelles that in turn results in the alterations of
Abbreviations: AIP = adenosine triphosphate;BHT = butylated by_ droxytoluene; DFO= desferrio~amine; Ag' = transmembrane electrical potential; EGTA= ethylene glycol-bis(l~-aminoethylether)-N,N.N',N'. tetraacetic acid; FCCP--c;;rbonylcyanide-p-trifluoromethoxyphenylhydrazone;MDA = malondi:ddehyde;RR = ruthenium red- TBA = thiobarbituricacid;TPP+ = tetrap,henylphosphoniumchloride ' Correspondingauthor,Tel: + 59 360044; Fax: + 59 362206: Email: [email protected]. I Presented at the 13th InternationalSymposiumon Bioelectrnchemistry and Bioenergetics,Ein Gedi. Israel,'7-12 January 1996. 0302-4598/97/$17.00 © 1997 Elsevier Science S.A. All rights reserved. Pll $0g02-4598(96)05109-4
tile physical and biochemical properties of the membrane components and finally in cell damage [9,10]. An almost exclusive parenchymal iron overload model, resembling liver pathological appearance in human primary haemochromatosis, is achieved by feeding rats a carbonyl iron-enriched diet [11]. Many reports dealing with this experimental model correlate the induction of lipid peroxidation associated with organclle func~/onal derangement with the level of total iron in the liver [11-14], but the intracellular form of iron that actually is able to init/am the reported anomalies is still unknown. In physiological conditions, iron enters ferritin, the m a ~ iron storage protein in virtually all cells, but under conditions of largely excessive deposition the capacity of hepatocyte to maintain iron in storage form could be exceeded. Recent work has indicated the existence of a transient cellular pool of ionic iron of low molecular weight, chelatable by desferrioxamine (DFO), that is in redox active form capable of initiating oxidative reactions in cellular membranes [15-18]. In the present research, an EPR assay was performed to quantitate the low molecular weight iron (LMW-iron or cbelatable iron) pool in m~tochondria isolated from liw:rs of rats fed an iron enriched diet during a period of 12 weeks. The functional efficiency of the inner mitochondrial membrane
17o
D. Ceccarelliet al./ Bioelectrochemist~"and Bioenergetics42 (1997)169-174
was in pzra]lel evaluated by membrane potential and ATP illeasurelilents.
LC-18 Hypersil 5ODS 4.6 cm x 25 cm (Lab Service, BO, Italy). The separation was accomplished according to Stocchi et al. [24]. ATP was revealed at 26Onto aga:,nst known quantities of external standards (75-200pmol).
2. Experimental details 2.5. Total mitochondrial iron 2.1. Animal treatment
Female Wistar albino rats (100-120g) (Morini, Reggio Emilia, Italy) were made siderotic by feeding a standard diet purchased from Dotto~ Piccioni (Brescia, Italy) supplemented with 2.5% (w/w -t) carbonyl iron for increasing periods of up to 12 weeks. Experimental and control rats were handled identically. The National Research Council criteria for the care and use of the laboratory animals in research were carefully followed. 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 [19]. The cytosolic contamination in mitochondrial preparation did not exceed 2% of the total protein content, as assessed by the recovery of marker enzymes [20]. The protein content of the final mitocbondrial suspension was determined by the biuret method with bovine serum albumin as the standard. 2.2. Mitochondrial membrane lipid peroxidation
Mitocbonddai membrane lipid peroxidat~on was asse,~sed by measuring the thyobarbituric acid reactive ;ubstances as end products and the amount of matondialdehyde (MDA) in the mitochondrial fraction was measured by a thiobarbituric acid method modification [21]. 2.3. Transmembrane potential ( ArIt)
Transmembrane potential (A~) was measured at 25°C by a tetraphenylphosphonium chloride ('i"PP+) selective eleclrode [22]. Mitochondfia (2 mg protein ml-t) were incubated in I ml of standard incubation medium of the following composition: t00mM NaCI; 5ram sodiumpotassium phosphate buffer (pH 7.4); lOmlVl Tris-HCl buffer (pH 7.4); IOmM MgCI 2 plus 5tLM rotenone and 20p.M TPP +. An inner mitochoncirial volume of 1.1 p.lmg -t protein was assumed. The respiratory states were those defined by Chance and Williams [23] on the basis of the factors limiting the respiralion. 2.4. Adenosine triphosphate (ATP)
Adenosine triphosphate (ATP) was determined as follows: I ml of mitochondrial suspension (about 8mgprotml -~) was treated with 0.2M of HCIO4 and centrifuged. 20 p.i of the neutralized acid-soluble fraction were used for the HPLC analysis by a liquid chromatografic systein (Hewlett-Packard HP 1090) equipped with a photo-diode array detecter. The column was a
The iron concentration was determined in acid extracts by an atomic absorption Perkin-Elmer spectrophotometer model 31)6 [13]. 2.6. Mitochondrial chelatable iron
The iron concentration was measured by EPR spectroscopy as described in [25]. Samples of 0.5 mi of mitocbondrial suspension (60-90rag ml-I ) were incubated at 20°C for 10rain with I mM desferrioxamine (DFO). The incubation t~me was previously assessed [25] as suitable for DFO to completely chelate all low molecular weight iron present as Fe(IlI) but not sufficient to remove iron from depot proteins. The samples were then placed in cylindrical tubes, frozen in liquid nitrogen and stored until the measurements were made. The DFO-iron complex formed with endogenous ionic iron is characterized by an EPR absorption at g = 4.3. The EPR spectra were recorded on a Brucker 200 spectrometer, at liquid nitrogen temperature, under the following conditions: oscillator Klystron frequency 9.12 GHz, power 20 roW, modulation a;nplitude 2.0mT. The intensity of the signal at g =4.3 was measured as a peak to peak amplitude to estimate the concentration of the DFO-iron complexes because the shape of signals obtained after blank subtraction, (mitochondria suspension not incubated with DFO) was the same in all samples and the ratio amplitude-double integral was constant. Calibration procedures: 'Free' iron concentration was calculated on the basis of a calibration curve, linear in the p.M range, obtained by measuring EPR signals of incremental amounts of FeSO4 • 7H20 added to identical volumes cf mitochondrial suspension, in the presence of I mM DFO. The Fe z+ content in the stock solution was conlrolled using the o-phenanthroline assay.
3. Results Data presented in Fig. 1 clearly demonstrate the significant accumulation of TBA-reactive substances, measured as MDA concentrations, in mitochondrial fraction isolated from the livers of 3-6 weeks iron fed rats (1300+ 180pmolmg -t protein vs. 260+ 33 of control). The extent of the peroxidation progressively decreases toward normal values after a longer treatment time. It has to be pointed out that the very same data resulted when an antioxidant as butylated hydroxytoluene (BHT) was added during the fractionation procedure, ruling out the possibil-
D. Ceccarelliet al. / Bioelectrochemistryand Bioenergetics42 ~1997)169-i74
1,400
• Control~ Iron i
3.s • Con~'o~ [ ] Iron
1,200
3
171
i
~
~.Z5
T ,
T
II
,,m!
m:
600 <~ 400
~; 200 0
i
i-~ 6 9 limeoftreatment(weeks)
3
~
0.5 12
3
6 9 Ttmeoftre~rnent(weeks)
12
~g. 3. Effect of iron ~ = m e n t on ATP content of n~tocl,~l fr~fi~.
Fig. 1. Effect of iron Ireatment of rats on MDA content of the mitochondrial fraction. MDA measurements were performed as described in Methods. The dala are representative of four separate experiments performed on a pool of 3 animals and are expressed as mean+SD, • P < 0.001 in comparison with control.
ATP was determined by an HPLC tecnique as decried in the Metimds. The data represent mean values for 4 separate expering~±SD. * P < 0.01 in comparison with control.
dria. When the calcium pulse has been completely taken up, Aqt returns to the pre-Ca: ~ level. The a~tidon of an uncoupler such as FCCP immediately makes A~ collapse. By contrast, mitochondria from 3-6 weeks iron fed rats (Fig. 2(b)), upon addition of the same pulse of Ca 2+, display a drop of A~ similar in the extent to tha~ for the control, but A~ trace does not return to the pre-Ca2+ level and quickly (about 2 min) collapses. The addition of a specific Ca:+ chelator, such as EGTA, or ruthenium red (RR), a specitic inhibitor of the elcctrophoreti¢ cag'/um uptake route (uniport) [26] or oligomycin, an inhibitor of ATP synthase [27], largely restores the A~ trace. On the contrary, an antioxidant such as BHT or an iron chel~ng agent such as desferrioxamine, ~ d to the incubating mixture, did not appreciably modify the A~ trace (not
ity that the MDA increase was due to an in vitro formation (not shown). Fig. 2 shows the effect of iron treatment on the mitochondrial functional efficiency as evaluated by measuring the mitochondfial transmembrane electrical potential (A~), by following the movements of the lipid-soluble TPP + cation. This parameter gives a direct indication of the energy transducing capability of the inner mitochondrial membrane. It can he seen that, on addition of an oxidizable substrate such as succinate (succ), the control mitocbondria (Fig. 2(a)) develop a normal A6 of about 200inV. When a pulse of Ca 2+ (150 IxM) is ad~d into the reaction vessel, a decrease of up to 172mV ap~ars which corresponds to the energy used for the Ca 2+ uptake inside the mitochon-
A Ca
FCCP
200
,,'"
OLIGO
180 •
150 •
100 10 =
2 mitt
\ succ
Fig. 2. Effect of iron treatment of rats on mitochondriai internal membcan¢ potential (A~'). The measurements were performed by means of a TFP + sensitive microelectrode, as described iv. the Experimental Details. The arrows indicate the following additions: 2 ram Na-succinate (succ); 150 IJ.M CaLl, (Ca); 1 p,M FCCP; 0.5 mM EGTA; I ~M ruthenium red (RR); 2 I~g rag- i protein oligomycin (oligo), ,3E. electro~ potential, (a) Control mitochoachia: (b) mitochondria from 3-6 week u'eated rats. The traces are representative of at least 3 different experiments performed on a pool of 3 animals.
172
t9. Ceccarelliet aL /Bioelectrochemism.' and Bioenergetics42 (/9971169-/74 [] Chel~l.ab~re ~ Tolat Fe
oi
2
oLf 0
m I 3 6 ,Q 1"=meof ~atrrtent (weeks)
,0o_:
. mm o * 12
Fig. 4. Effectof iron treatmenton total iron and chelatablc iron of mitochondrialfracti,m.Determinationswereperlbrmedby atomicabsorption .q~ectmphotometryand elect,onspin resonancespectrometrytechniquesas dcscrihedin the Methods.The data,'epresentmeanvaluesfor 4 separateexperiments_+SD.• P < 0,01 in comparisonwithcontrol.
shown). It must be pointed out that mitochondria isolated from iron fed rots for a time shorter or longer than 3-6 weeks did not display this A~ ~havinur (results not shown). Fig. 3 illustrates the endogenous adenosine triphosphate (ATP) content of mitochondria isolated from rat livers at various times of iron overloading in comparison with the control mitochondria. A maximal decrease of ATP of about 45% is observed at 6 weeks of iron feeding (!.58 + O.18nmolmg -~ protein vs 2.9+0.4 of control) and an average decrease, although not statistically significant, is also observed at 3 weeks of treatment. The atomic absorption measurement of the total iron in mitochondria from iron treated rats is shown in Fig. 4. It clearly appears that the content of the total iron progressively increases up to 6 weeks of treatment, then maintaining an almost steady value (from 2.8 +_0.8 nmol mgprotein of control to 25 _+3.8). The distribation pattern for the chelatable iron level is presented in the same figure. EP.q measurements indicate a sharp increase in the chelatable iron content for the time interval included between 3-6 weeks of treatment followed by the retum to control values at longcr times.
4. Discussion The present results strengthen z recent original observation [28], that a small pool of intracellular chelatable iron (3-10% of total cell iron) is primarily involved in thc induction of oxidant stress, inciuding lipid peroxidation, in the experimemal hepatic iron overload. Specifically, by the use of a new EPR technique, a strict correlation between a transient increase in chelatable iron and mitochondrial functional anomalies has here been confirmed, although at an earlier period of treatment (3-6 weeks vs. 4-7 weeks). In fact, the ultimate oxidizing form of iron responsible for the initiation of lipid peroxidation in iron overload in vivo
is not yet known but only hypothesised [12-14]. Iron dependent lipid peroxidation of hepatic organelles coincide with the impairment of the mitochodnria energy transducing capability has been up to now related with the max:mal concentration of total iron in the hepatic tissue that widely ranged from t000-5 000 ggg-i (wet weight) [I 1-14]. The w.,nsient raise in mitochondria chelatable iron level, here detected, is strictly associated with the induction of lipid peroxidation and the enhancement of the energy dissipating mitocholadriai calcium cycling revealed by membrane potential measurements. Specifically, the study of A~ behaviour of mitochondria from 3-6 weeks iron treated rats showed a clear difference compared with control during the accumulation of a low pulse of Ca2+(see Fig. 2). The observation that, when the A~ trace reaches its lowest value, the addition of EGTA and RR which inhibit Ca~+ cycling, or oligomycin which blocks energy dissipation, promptly reverses the membrane potential almost to its normal value, indicates that the inner membrane is not irreversibly depolarized and the A~ decrease is not caused by direct membrane damage but rather to a continuous energy draining Ca2÷ cycling (i.e., energy is dissipated to reaccumulate the released Ca2÷ into mitochondria). TLe time length of the A~" derangement depended on the amount of the Ca 2+ added; when no calcium was added the A~ decrease was very slow [28] while an immediate drop of A~t resulted after the addition of a high pulse of Ca2+ (results not shown). The metabolism of hydrogen peroxide and fatty acid hydroperoxides through the glutathione enzyme system (GSH peroxid~se-GSH reductase) shifts~,e redox state of mitochondrial i',yridine nucleotides toward an oxidized form, a condition which induces the activation of a specific passive calcium release from mitochondria, via a route distinc~ from the electrophoretical Ca2÷ uptake (uniport) [29-32]. Such an increase in the oxidized form of pyridine nucleotides has already been well documented under these experimental conditions [33]. The chelatable iron here detected may reasonably be involved in the formation of these oxygen reactive species [34]. The observation that the combined treatment of rats with iron and an antioxidant such as silybin, a main constituent of natural flavonoids, completely prevented lipid peroxidation and mitochondfial dysfunctions without appreciably modifying the total iron content of the liver is in agreement with the above proposal [35]. A consideration may be derived by observing the time course of total iron and chelatable iron concentrations in mitochondria (Fig. 4). Iron overload results in a significant transient increase of the mitochondrial chelatable iron pool, which enhances the generation of oxygen free radicals, at the time of intoxication that precedes or is nearly coincident with the reaching of the maximal accumulation of total iron. A comments should be addressed about the lower level of chelatable iron obtained by EPR in the present set of experiments compared with the one previ-
D. Ceccarelliet al. / Bioelectrochemistry and Bioenergetics 42 (1997) 169-174
ously reported by the use of an I-IPLC method [28], In the cas,: of HPLC, proteic samples had to be frozen and thawed, centrifuged and ultrafiltered to give clear extracts before the final reading, while in the case of EPR the measurements were peffo,."med directly on frozen samples. The additional step in the mitochondria treatment leads to a further release of chelatable iron (unpublished observations). Furthermore, it is conceivable that in the present series of animals the amount of preformed or neoformed ferritin was slightly higher to significantly reduce the level of chelatable iron (that is at maximum at 3-10% of the total cell iron) without appreciably affect the total iron pool. Although chelatable iron revealed here is much lower, it apparently constitutes an already critical amount to induce lipoperoxidation. It is well known that iron metabolism is tightly controlled and regulated [36,37] and that ferritin serves to sequester iron in a relatively inert form until it is needed for critical iron-dependent metabolic processes such as heine synthesis [38]. it may be speculated that the existing ferritin could not be able to completely accommodate the large excess of total iron which progressively accumulates and the capacity of the hepatocyte to maintain iron in storage forms is exceeded. After ferritin synthesis becomes stimulated [39] the capability of iron withholding by the hepatocyte may be restored and the level of chelatable iron returns to normal values. The present results add more information to the open problem of biochemical mechanism by which iron, a major catalyst of free radical reactions in the cell, may initiate, propagate and/or amplify the (auto)oxidative phenomena. Specifically they show that: 1) in viva iipoperoxidative reactions in mitochondrial membranes is directly correlated with the presence of a transient increase of redox active low molecular weight iron, or chelatable iron, consequently capable to give radicalic reactions; 2) the lipoperoxidatitn intermediate products bring about an increase in energy dissipation by the induction of an energy draining calciam cycling into mitochondria; 3) the energy wasting as a result of Ca z+ efflux-reuptake found here was active also in the absence of external calcium loading [28] and, in both cases, may be responsible for the significant decrease in ATP content (see Fig. 3). It is conceivable that under conditions of severe hepatic iron overload, when the cell is not able to maintain a steady state in iron storage machinery, the resulting mitochondrial functional derangement may become irreversible thus leading to cell injury. The present results may thus be of interest to give a clue to the therapeutic intervention in various disorders associated with excess tissue iron deposition.
Acknowledgements This work has been supported by a grant of the Italian Ministry of University and Scientific and Technological
173
Research (M.U,R.S.T,) and National Research Council of Italy (C,N.R.) special project 'Ageing'. A.V. KozIov has been supported by the European Science Foundation, grant N-° RF/93/35(T).
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