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Lipid peroxidation associated cardiolipin loss and membrane depolarization in rat brain mitochondria
Neurochemistry International 49 (2006) 20–27 www.elsevier.com/locate/neuint
Lipid peroxidation associated cardiolipin loss and membrane depolarization in rat brain mitochondria Tanusree Sen a, Nilkantha Sen b, Gayatri Tripathi b, Uttara Chatterjee c, Sasanka Chakrabarti a,* a
Department of Biochemistry, Dr. B.C. Roy Post-graduate Institute of Basic Medical Sciences & IPGMER, Calcutta, India b Division of Infectious Disease, Indian Institute of Chemical Biology, Calcutta, India c Department of Pathology, Dr. B.C. Roy Post-graduate Institute of Basic Medical Sciences & IPGMER, Calcutta, India Received 20 October 2005; received in revised form 7 December 2005; accepted 20 December 2005 Available online 28 February 2006
Abstract Oxidative stress induced by Fe2+ (50 mM) and ascorbate (2 mM) in isolated rat brain mitochondria incubated in vitro leads to an enhanced lipid peroxidation, cardiolipin loss and an increased formation of protein carbonyls. These changes are associated with a loss of mitochondrial membrane potential (depolarization) and an impaired activity of electron transport chain (ETC) as measured by MTT reduction assay. Butylated hydroxytoluene (0.2 mM), an inhibitor of lipid peroxidation, can prevent significantly the loss of cardiolipin, the increased protein carbonyl formation and the decrease in mitochondrial membrane potential induced by Fe2+ and ascorbate, implying that the changes are secondary to membrane lipid peroxidation. However, iron-ascorbate induced impairment of mitochondrial ETC activity is apparently independent of lipid peroxidation process. The structural and functional derangement of mitochondria induced by oxidative stress as reported here may have implications in neuronal damage associated with brain aging and neurodegenerative disorders. # 2006 Elsevier Ltd. All rights reserved. Keywords: Lipid peroxidation; Mitochondria; Mitochondrial membrane potential; Cardiolipin; Electron transport chain; Reactive oxygen species
Lipid peroxidation is a unique mode of oxidative injury which is triggered and promoted by different radical and nonradical members of reactive oxygen species (ROS) family or by the catalytic decomposition of preformed lipid hydroperoxides in tissues by several agents including most notably the transition metals and microsomal cytochromes (Slater, 1984; Halliwell and Gutteridge, 1989; Niki et al., 2005). The peroxidative injury not only causes structural and functional derangement of phospholipid bilayer of membranes but also produces several deleterious aldehydic end products like malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), acrolein, etc. which may inflict secondary damage to proteins and DNA (Halliwell and Gutteridge, 1989; Uchida, 2003; Luo and Shi, 2005). Thus, lipid peroxidation will further increase any
* Corresponding author at: Department of Biochemistry, Dr. B.C. Roy Postgraduate Institute of Basic Medical Sciences & IPGMER, 244B Acharya J.C. Bose Road, Calcutta 700020, India. Fax: +91 33 2280 1807. E-mail address: [email protected] (S. Chakrabarti). 0197-0186/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2005.12.018
primary oxidant-induced damage through ROS (Niki et al., 2005). The brain is highly vulnerable to lipid peroxidation because of its high rate of oxygen utilization, an abundant supply of polyunsaturated fatty acids, a deficient antioxidant defence and a high content of transition metals like copper and iron in several regions (Halliwell and Gutteridge, 1989; Calabrese et al., 2000). In several neurodegenerative disorders (e.g. Parkinson’s disease, Alzheimer’s disease, amyotropic lateral sclerosis, etc.) and brain aging, an enhanced formation of lipid peroxidation end products or adducts and various forms of mitochondrial dysfunctions have been noticed in post-mortem human brain or in the brains of transgenic or other animal models (Shigenaga et al., 1994; Ferrante et al., 1997; Sayre et al., 1997). However, such observations do not establish a causal relationship between lipid peroxidation and mitochondrial functional alterations in these disease states. On the other hand, the exposure of mitochondria or submitochondrial particles to g-ionizing radiation, xanthine/xanthine oxidase system or exogenously added H2O2 or acrolein has been shown to cause an impairment of electron transport chain (ETC)
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activity, inhibition of a-ketoglutarate dehydrogenase, loss of cardiolipin and some alterations in mitochondrial membrane potential which are largely attributed to the damaging effects of reactive oxygen species but the involvement of lipid peroxidation in causing mitochondrial dysfunction has not been clearly defined in such studies (Zhang et al., 1990; Tretter and Adam-Vizi, 2000; Paradies et al., 2002; Luo and Shi, 2005). On the basis of the results obtained from in vitro experiments and post-mortem studies, it seemed logical to attempt to identify in the present study the damaging consequences of lipid peroxidation on mitochondrial membrane potential and ETC activity and further to suggest the possible implications of our findings in explaining neuronal damage and death in neurodegenerative disorders and brain aging. A combination of iron (FeSO4) and ascorbate has been used to induce lipid peroxidation in brain mitochondria in our study, since this combination is not only a potent source of ROS but can also catalyze lipid peroxidative process in tissue preparation independent of ROS formation (Chakraborty et al., 2001). Our study has indicated that membrane lipid peroxidation is a major contributor to biochemical and functional alterations of mitochondria induced by oxidative stress in vitro and these changes presumably can lead to further deleterious consequences in vivo such as ATP depletion or the release of cytochrome c from the mitochondria. 1. Experimental procedures 1.1. Chemicals Anti-dinitrophenyl (anti-DNP) antibody, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), butylated hydroxytoluene (BHT), 5bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium (NBT) were purchased from Sigma chemical Co. (USA). ‘ApoAlertTM Mitochondria Membrane Sensor Kit’ was purchased from BD Biosciences (USA). JC-1 (5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethylbenzimidazolylcarbocyanine iodide) and nonyl acridine orange were products of Molecular Probes (USA). All other chemicals used were of the highest purity available.
1.2. Animals Healthy adult albino rats of Charles Foster strain were maintained as per the guidelines of the animal ethical committee of the institute. The animals were euthanized by cervical dislocation and the brains dissected out clearly and collected on petri dishes kept over ice.
1.3. Isolation of rat brain mitochondria Rat brain mitochondria were isolated by differential centrifugation as published earlier (Berman and Hastings, 1999). Briefly, the brain from one adult rat was homogenized in 10 ml of buffer containing 225 mM mannitol, 75 mM sucrose, 5 mM HEPES, 1 mM EGTA, 1 mg/ml BSA, pH 7.4. The homogenate was brought to 30 ml with the same buffer and centrifuged at 2000 g for 3 min at 4 8C. The supernatant was divided into two tubes and centrifuged at 12,000 g for 10 min. The pellet containing the mixture of synaptosomes and mitochondria was suspended in 10 ml of homogenization buffer containing 0.02% digitonin to lyse the synaptosomes followed by centrifugation at 12,000 g for 10 min to pellet down both extrasynaptosomal and intrasynaptosomal mitochondria. The mitochondrial pellet was washed twice in the same buffer without EGTA, BSA and digitonin and finally resuspended either in 50 mM phosphate buffer, pH 7.4 or in the isotonic buffer
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A (containing 145 mM KCl, 50 mM sucrose, 5 mM NaCl, 1 mM EGTA, 1 mM MgCl2, 10 mM phosphate buffer, pH 7.4) for further experimentations.
1.4. Assay of citrate synthase for mitochondrial integrity The mitochondria suspended in the isotonic buffer A were checked for membrane integrity by assaying citrate synthase activity before and after treatment with Triton X-100 to obtain the latency value of citrate synthase and ratios exceeding 10 were considered indicative of good membrane integrity (Clark et al., 1997). In this method coenzyme A released during the reaction of oxaloacetate with acetyl coenzyme A catalyzed by citrate synthase present in mitochondrial sample was measured using 5,50 -dithiobis-2-nitrobenzoic acid (DTNB) which reacted with the free-SH groups of coenzyme A.
1.5. Incubation protocol for mitochondria Mitochondria were incubated in 50 mM phosphate buffer, pH 7.4 (200– 300 mg mitochondrial protein for lipid peroxidation and protein carbonyl measurements) or in isotonic buffer A (100–200 mg mitochondrial protein for MTT reduction assay and measurement of mitochondrial membrane potential or 300–500 mg mitochondrial protein for cardiolipin measurement) in the absence (control) or presence of FeSO4 (50 mM) and ascorbate (2 mM) with or without other additions like BHT (0.2 mM) or DMSO (20 mM) or mannitol (20 mM) for 1 h at 37 8C. In some experiments mitochondria were incubated with H2O2 (1 mM) or FeSO4 (50 mM) and H2O2 (1 mM) or KCN (1 mM).
1.6. Assay of lipid peroxides The extent of lipid peroxidation in mitochondrial samples during in vitro incubation was measured by quantitating the amount of malondialdehyde (MDA) formed by 2-thiobarbituric acid (TBA) reaction (Ohkawa et al., 1979). The amount of malondialdehyde (MDA) produced was calculated using the molar extinction co-efficient of MDA–TBA adduct as 1.56 105 cm2 mmol1 (Chakraborty et al., 2001).
1.7. Detection of protein carbonyls by immunoblotting At the end of the incubation, the samples of mitochondria were solubilised in 6% SDS and the protein carbonyls derivatized to hydrazones by treatment with 20 mM dinitrophenylhydrazine (DNPH) in 10% TFA for 10 min (Shacter et al., 1996). The samples were then neutralized with 2 M Tris/30% glycerol/ 19% b-mercapto ethanol and subjected to sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) following the method described previously (Laemmli, 1970). The proteins were then transferred to a nitrocellulose membrane by electroblotting. The dinitrophenylhydrazone derivatives of protein carbonyls were probed with polyclonal anti-DNP antibodies and detected by goat anti-rabbit IgG conjugated to alkaline phosphatase using BCIP/NBT as the substrates.
1.8. MTT reduction assay The incubation of mitochondrial samples (control and experimental) was terminated by the addition of an excess of ice-cold isotonic buffer A followed by centrifugation at 15,000 g for 15 min at 4 8C to pellet the mitochondria. The pellet was resuspended in isotonic buffer A and an aliquot added to a reaction mixture in the same buffer A containing 10 mM succinate (or 10 mM pyruvate or 10 mM a-ketoglutarate for some experiments) and MTT (0.42 mg/ml) and kept at 37 8C for 15 min. The samples were quenched with 500 ml of lysis buffer (45% dimethyl formamide in 10% SDS, pH 4.7) and the difference in absorbance values at 550 and 620 nm noted (Cohen et al., 1997).
1.9. Assay of mitochondrial cardiolipin content Cardiolipin content of mitochondria was measured using the cardiolipin specific dye nonyl-acridine orange (Petit et al., 1992). After 1 h incubation at 37 8C in buffer A with or without other additions, mitochondria were washed
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with an excess of ice-cold buffer A and pelleted at 4 8C by centrifugation at 15,000 g for 15 min. The pellet was resuspended in the same buffer and incubated at 37 8C for further 45 min in the presence of 5 mM nonyl-acridine orange. The excess dye was washed out by centrifugation and the mitochondrial pellet appropriately diluted in buffer A for fluorescence measurements (lex 485 nm, lem 535 nm) in a Hitachi spectrofluorometer (model F-4010). The fluorescence values normalized to 1 mg protein/ml were converted to relative fluorescence units using quinine (1 mg/ml in 0.1N H2SO4, lex 360 nm, lem 457 nm) as the reference.
1.10. Measurement of mitochondrial membrane potential Mitochondrial transmembrane potential was assessed by confocal microscopy using ‘mitosensor dye’ from a commercial kit (mitochondrial membrane sensor kit) as adopted from Sen et al. (2004). ‘Mitosensor’, a cationic dye, was taken up by the mitochondria in proportion to mitochondrial transmembrane potential and the accumulated dye underwent aggregation in the interior of the mitochondria. The monomeric form of the dye emitted green fluorescence while the aggregated form exhibited intense red fluorescence. In our experiments with isolated mitochondria, the excess dye (monomer) remaining outside was washed off thoroughly and, therefore, only the intense red fluorescence emitted from within the mitochondria was visible. For a quantitative assessment of mitochondrial membrane potential in control and experimental samples, a spectrofluorometric measurement was adopted using a mitochondrial membrane potential sensitive carbocyanine dye JC-1 (Reers et al., 1991). When excited at 490 nm, the monomeric form of JC-1 had an emission maximum at 527 nm, but the aggregated form (J-aggregates) showed an emission maximum at 590 nm. The negative transmembrane potential of mitochondria caused a directional uptake of the cationic dye into the matrix with subsequent formation of concentration-dependent J-aggregates and the fluorescence intensity of J-aggregate at 590 nm, therefore, reflected changes in mitochondrial membrane potential (Reers et al., 1991). For both confocal microscopic and spectrofluorometric experiments, isolated mitochondria were incubated in buffer A at 37 8C for 1 h with or without FeSO4 (50 mM) and ascorbate (2 mM) and/or other additions, followed by pelleting of the mitochondria at 4 8C by centrifugation with an excess of buffer A. The mitochondrial pellet was subsequently suspended in appropriate dilution in buffer A containing 10 mM succinate and reincubated at 37 8C for 30 min either in the presence of ‘mitosensor dye’ (5 mg/ml) or JC-1 (10 mg/ml). After 30 min, the dye loaded mitochondria were collected by centrifugation at 4 8C with an excess of buffer A, washed once more with the same buffer to remove thoroughly the dye remaining outside and finally resuspended in appropriate dilution in buffer A for confocal microscopy (TCS-SP Leica Confocal Microscope) or spectrofluorometric measurements (Hitachi F-4010). The spectrofluorometric readings normalized to 1 mg/ml protein were expressed as relative fluorescence units as described earlier in the text.
1.11. Measurement of protein content The protein content was determined by the method of Lowry after solubilizing the protein in 1% SDS and using bovine serum albumin as the standard (Lowry et al., 1951).
1.12. Statistical analysis All experiments were repeated at least five times using mitochondria from separate animals. The statistical significance was calculated by paired Student’s t-test. The immunoblotting and confocal microscopic experiments were repeated at least four times and a representative figure from a set of experiments producing similar results was presented in each case.
2. Results 2.1. Iron-ascorbate induced peroxidative damage to mitochondrial cardiolipin and protein The results presented in Table 1 show that an exposure of mitochondria to a mixture of Fe2+ (50 mM) and ascorbate (2 mM) led to a striking increase in MDA production and a marked loss in cardiolipin content. The lipid-soluble antioxidant BHT (0.2 mM) prevented both the phenomena (Table 1). When brain mitochondria were incubated in the presence of H2O2 (1 mM) or FeSO4 (50 mM) and H2O2 (1 mM) for 1 h the formation of MDA was negligible in comparison to that induced by iron-ascorbate (data not shown). Under similar conditions of incubation with iron-ascorbate an enhanced formation of protein carbonyls was noticed in mitochondrial fraction and this again was abolished by BHT (Fig. 1). 2.2. Effect of iron-ascorbate on mitochondrial MTT reduction The mitochondrial electron transport chain (ETC) activity was monitored by MTT reduction assay with isolated mitochondria using NAD+-linked (pyruvate and a-ketoglutarate) or FAD-linked (succinate) substrates. On incubation at 37 8C in buffer A for 1 h in the absence of any added iron or ascorbate, a striking impairment of mitochondrial MTT reduction in the presence of NAD+-linked substrates (e.g. aketoglutarate or pyruvate) was observed, while succinatesupported MTT reduction remained nearly unaltered (Fig. 2A). All subsequent experiments on the effects of Fe2+ (50 mM) and ascorbate (2 mM) on mitochondrial MTT reduction were, therefore, conducted in the presence of 10 mM succinate (FAD-linked substrate) only. The data shown in Fig. 2B demonstrate that oxidative stress induced by Fe2+-ascorbate led to a dramatic decrease (up to 40%) in succinate-supported mitochondrial respiration as measured
Table 1 Lipid peroxidation and cardiolipin loss in mitochondrial fraction exposed to iron-ascorbate Incubation mixture
Cardiolipin content (relative fluorescence unit/mg protein) (5)
Lipid peroxides (nmol MDA/mg protein) (5)
Mitochondria alone (control) Mitochondria + Fe2+ + Asc Mitochondria + Fe2+ + Asc + BHT
0.150 0.008 0.110 0.006* 0.146 0.007c
0.25 0.04 25.25 0.34* 1.94 0.10c
Incubation of mitochondria was performed in vitro without (control) or with Fe2+ (50 mM) and ascorbate (Asc, 2 mM) in the absence or presence of BHT (0.2 mM) followed by the estimation of cardiolipin and malondialdehyde (MDA) as described in the text. The values presented are the means S.E.M. The figures in parentheses represent the number of observations. * Statistically significant p < 0.001 vs. control. c Significantly different p < 0.001 vs. mitochondria + Fe2+ + ascorbate.
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Fig. 1. Immuno-detection of mitochondrial protein carbonyls after oxidative stress. Rat brain mitochondria were incubated with or without Fe2+ (50 mM) and ascorbate (2 mM) in vitro for 1 h at 37 8C in the presence or absence of BHT (0.2 mM) followed by the detection of protein carbonyls by DNPH-derivatization and immunoblotting using anti-DNP antibodies as described in Section 1. Lane a: mitochondria incubated alone; lane b: mitochondria + Fe2+ + ascorbate + BHT; lane c: mitochondria + Fe2+ + ascorbate.
by MTT reduction which was not prevented by BHT or by the scavengers of hydroxyl radicals. Further, when mitochondria were incubated with H2O2 (1 mM) or Fe2+ (50 mM) and dH2O2 (1 mM) for 1 h at 37 8C, a significant decrease in mitochondrial MTT reduction ability could be noticed which however, was less than that caused by Fe2+-ascorbate (Fig. 2B). 2.3. Evaluation of mitochondrial membrane potential by confocal microscopy The mitochondrial transmembrane potential was monitored with confocal microscopy using ‘mitosensor dye’ from a commercial kit. In succinate containing respiratory buffer, the cationic ‘mitosensor dye’ accumulated inside control mitochondria with normal transmembrane potential and formed aggregates which emitted an intense red fluorescence (Fig. 3). The intensity of red fluorescence was diminished markedly when mitochondria were pre-exposed to Fe2+ (50 mM) and ascorbate (2 mM) for 1 h, indicating depolarization of mitochondrial membranes and the phenomenon was significantly prevented by BHT added in the incubation mixture during Fe2+-ascorbate exposure (Fig. 3). The treatment of mitochondria with a strong respiratory poison like KCN led to a complete abolition of red fluorescence indicating a collapse of mitochondrial transmembrane potential (Fig. 3).
Fig. 2. Effect of oxidative stress on mitochondrial MTT reduction. Mitochondria were kept at 0 8C (unincubated control) or incubated at 37 8C in isotonic buffer in the absence (incubated control) or presence of Fe2+ (50 mM) plus ascorbate (Asc, 2 mM) or Fe2+ (50 mM) plus H2O2 (1 mM) or H2O2 (1 mM) with or without other additions like BHT (0.2 mM) or DMSO (20 mM) or mannitol (Man, 20 mM) followed by the measurement of MTT reduction as described in Section 1. (A) MTT-reduction by intact mitochondria (unincubated control and incubated control) was measured in the presence of NAD+-linked substrates (e.g. a-ketoglutarate, 10 mM or pyruvate, 10 mM) or FAD-linked substrate (e.g. succinate, 10 mM). Values from unincubated controls (data not shown) were taken as 100%. *: Statistically different p < 0.001 vs. unincubated control. (B) MTT reduction assay was performed in the presence of 10 mM succinate with control (incubated control) and oxidatively damaged mitochondria. Values from incubated controls (data not shown) were taken as 100%. &: Statistically significant p < 0.001 vs. incubated control.
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Fig. 3. Effect of iron-ascorbate on mitochondrial membrane potential in vitro incubation. Rat brain mitochondria were incubated alone (control) or with KCN (1 mM) or with Fe2+ (50 mM) and ascorbate (2 mM) in the presence or absence of BHT (0.2 mM) for 1 h at 37 8C. At the end of the incubation mitochondrial membrane potential was monitored by confocal microscopy using the cationic ‘mitosensor dye’ as described in Section 1. (a) Control mitochondria, (b) mitochondria + Fe2+ + ascorbate, (c) mitochondria + Fe2+ + ascorbate + BHT and (d) mitochondria + KCN.
2.4. Spectrofluorometric measurement of membrane potential in control and iron-ascorbate treated mitochondria A quantitative assessment of mitochondrial transmembrane potential was made by the measurement of fluorescence emission of J-aggregate at 595 nm within control as well as oxidatively damaged mitochondria. The results (Fig. 4) indicate that in mitochondria treated with Fe2+ (50 mM) and ascorbate (2 mM) for 1 h, J-aggregate fluorescence was diminished remarkably indicating a loss of mitochondrial transmembrane potential by oxidative injury. However, the lipid soluble antioxidant BHT (0.2 mM) prevented the mitochondrial membrane depolarization induced by Fe2+-ascorbate very significantly (Fig. 4). As in the experiments with ‘mitosensor dye’, J-aggregate fluorescence at 595 nm was found to be almost completely abolished by exposure of the mitochondria to a strong respiratory chain inhibitor like KCN (Fig. 4). 3. Discussion 3.1. Iron-ascorbate as a model system for in vitro oxidative stress
Fig. 4. Spectrofluorometric measurement of mitochondrial membrane potential after in vitro oxidative stress. The incubation mixtures containing mitochondria in isotonic buffer A in the absence (control) or with KCN (1 mM) or with Fe2+ (50 mM) plus ascorbate (Asc, 2 mM) with or without BHT (0.2 mM) were kept at 37 8C for 1 h. Mitochondrial membrane potential was measured spectrofluorometrically using JC-1 as described in the text. Values (relative fluorescence intensity in percentage) normalized to 1 mg protein are the means S.E.M. of 6 observations. *: Statistically significant p < 0.001 vs. control, &: statistically different p < 0.001 vs. iron + ascorbate.
In the present study a combination of iron and ascorbate has been used to induce lipid peroxidation in vitro in rat brain mitochondria (Table 1). This combination represents a physiologically relevant pro-oxidant system in the context of brain, since ascorbate content of brain is very high and several regions of the brain are enriched in iron (Spector and Eells, 1984; Halliwell and Gutteridge, 1989). In earlier studies we have shown that iron ascorbate causes extensive membrane lipid peroxidation and consequent secondary protein damage in rat brain crude synaptosomal fraction during in vitro incubation
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and further lipid peroxidation under such conditions is initiated by catalytic breakdown of preformed lipid hydroperoxides present in tissue preparation rather than by reactive oxyradicals (Chakraborty et al., 2001, 2003). Thus, while iron-ascorbate combination is a potent source of oxyradicals, its ability to enhance lipid peroxidation by decomposition of performed tissue lipid hydroperoxides makes this combination an ideal system to examine the effects of peroxidative process vis-a`-vis the direct ROS damage on subcellular components. 3.2. Lipid peroxidation, cardiolipin loss and protein carbonyl formation Cardiolipin is a major phospholipid of mitochondrial membrane and expectedly a significant loss of cardiolipin occurs consequent to lipid peroxidation in incubated mitochondria in the presence of iron-ascorbate and BHT, a chain-breaking inhibitor of lipid peroxidative process, can prevent the cardiolipin loss almost completely (Table 1). The formation of protein carbonyls is considered as a hallmark of oxidative protein damage and may result from a primary damage inflicted by ROS or secondary modifications due to formation of stable adduction compounds between reactive aldehydic end products of lipid peroxidation (e.g. 4-HNE) and amino acid side chains (Halliwell and Gutteridge, 1989; Shacter et al., 1996). Our results indicate that iron-ascorbate mediated protein carbonyl formation in incubated mitochondria represents secondary modifications caused by lipid peroxidation end products since the antioxidant BHT prevents both peroxidation and protein carbonyl formation (Table 1 and Fig. 1). These results are significant since the peroxidative damage to cardiolipin and mitochondrial membrane proteins may affect mitochondrial ETC activity and mitochondrial transmembrane potential which, in turn, could trigger a cascade of damaging events in brain cells. This possibility is supported by earlier studies where a peroxidative cardiolipin loss has been linked to diminished activities of complexes I, III and IV and other mitochondrial proteins like ADP/ATP translocator, phosphate translocator, ATP synthase, etc. (Tretter and Adam-Vizi, 2000; Calabrese et al., 2000; Paradies et al., 2000; Luo and Shi, 2005). Further, a catalytic requirement of cardiolipin has been demonstrated for several respiratory chain enzymes including complex I and complex IV (Fry and Green, 1981). On the other hand several end products of lipid peroxidation, e.g. 4hydroxynonenal (4-HNE) and acrolein added exogenously can inhibit mitochondrial respiration or ETC activity in isolated mitochondria at various levels presumably through formation of stable adducts with proteins (Chen et al., 1998; Picolo et al., 1999; Picklo and Montine, 2001). 3.3. Inhibition of mitochondrial ETC activity is independent of lipid peroxidation The activity of electron transport chain in isolated mitochondria can be assessed at complexes I–III by measuring mitochondrial MTT reduction in the presence of NAD+-linked (e.g. pyruvate, a-ketoglutarate, etc.) and FAD-linked (e.g.
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succinate) substrates (Khan et al., 2005). The tetrazolium dye, MTT, is reduced by accepting electrons, predominantly between cyrochrome c and cytochrome a and partially at a location between one Fe–S center of complex II and the site of inhibition by antimycin A (Berridge and Tan, 1993). In isolated control brain mitochondria incubated at 37 8C in isotonic medium without added Fe2+ or ascorbate for 1 h, a striking loss of mitochondrial MTT reduction ability is noted in the presence of NAD+-linked substrates, while succinatesupported MTT reduction is not affected to any significant degree (Fig. 2A). The results are surprising but the finding has not been explored any further in this study and in all our experiments related to iron-ascorbate effect on mitochondrial MTT reduction succinate has been used as the respiratory substrate. Our results demonstrate that mitochondrial electron transport chain (ETC) activity as measured by MTT reduction by intact mitochondria in the presence of a FAD-linked substrate, e.g. succinate is remarkably impaired when mitochondria are preincubated with Fe2+ (50 mM) and ascorbate (2 mM) for 1 h (Fig. 2B). The considerable impairment of succinate-supported MTT reduction by mitochondria, however, cannot be attributed to mitochondrial membrane lipid peroxidation or cardiolipin loss or protein carbonyl formation since BHT which nearly completely abolishes the latter phenomena has no effect on the former process (Fig. 2). It is possible that under our incubation condition the effect of iron-ascorbate on mitochondrial MTT reduction is not secondary to lipid peroxidative damage but results from some direct damage to ETC components by reactive oxygen species like OH radicals. The possibility, however, has not been confirmed since OH radical scavengers like mannitol or DMSO have failed to prevent the effect of iron and ascorbate on mitochondrial MTT reduction, although it is entirely possible that the radical scavengers have not reached the site of damage in adequate concentration through intact mitochondrial membrane. On the other hand oxidative stress in the form of H2O2 (1 mM) or a combination of Fe2+ (50 mM) and H2O2 (1 mM) also leads to a significant inhibition of mitochondrial MTT reduction (Fig. 2B), although the former agents induce very little MDA production in comparison to that by iron and ascorbate (data not shown). All these results implicate albeit indirectly a damage to mitochondrial ETC activity by active oxygen species independent of lipid peroxidation. It has been reported earlier by others in different systems that active oxygen species especially OH radicals and singlet oxygen can inactivate mitochondrial respiratory chain complexes by a mechanism independent of lipid peroxidation (Giulivi et al., 1990; Zhang et al., 1990). 3.4. Lipid peroxidation and mitochondrial membrane depolarization Mitochondrial transmembrane potential maintained by the respiratory activity of electron transport chain complexes is instrumental in coupling ATP synthesis with respiration. Various inhibitors of respiratory chain can lead to a loss of mitochondrial transmembrane potential (depolarization) and it
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is likely that oxidative inactivation of electron transport chain components will lead to mitochondrial membrane depolarization (Chinopoulos et al., 1999). Further, disrupted or damaged mitochondrial membranes as a result of lipid peroxidative process may also lead to dissipation of H+-ion gradient and consequent membrane depolarization. In different in vitro systems membrane peroxidation is known to alter permeability properties, transmembrane potential and ionic gradients that often can be prevented by antioxidants (Halliwell and Gutteridge, 1989; Tretter and Adam-Vizi, 1996; Lehotsky et al., 1999). Under our experimental conditions an incubation of isolated mitochondria with Fe2+-ascorbate results in a dramatic decrease of mitochondrial membrane potential (depolarization) as demonstrated by both confocal microscopic and spectrofluorometric experiments (Figs. 3 and 4). The fact that BHT (0.2 mM) significantly (up to 75%) prevents Fe2+ascorbate mediated membrane depolarization and lipid peroxidation (Figs. 3 and 4 and Table 1) but not the impairment of succinate-supported mitochondrial respiration (Fig. 2B) implies that the peroxidative membrane damage rather than the inhibition of mitochondrial ETC activity is predominantly responsible for the loss of mitochondrial transmembrane potential under our experimental conditions. However, since BHT protection against mitochondrial membrane depolarization is not complete, it is likely that the latter in part (nearly 25%) may result from impaired ETC activity of mitochondria induced by iron-ascorbate through ROS mediated damage. 3.5. Consequences of lipid peroxidative damage to mitochondria It is tempting to speculate from the present data that lipid peroxidation in a large measure will uncouple oxidative phosphorylation by disrupting mitochondrial membrane and dissipating the proton gradient with consequent ATP depletion. On the other hand the peroxidative loss of cardiolipin may trigger release of cytochrome c from mitochondria (Shidoji et al., 1999; Petrosillo et al., 2003). It is difficult to extrapolate such in vitro findings to in vivo scenario and to speculate on the consequences of lipid peroxidation associated mitochondrial dysfunctions in brain cells before obtaining further data in animal or cell culture models. In summary, this work has clearly shown that lipid peroxidation per se is an important contributor to mitochondrial dysfunction and that ROS mediated direct damage (e.g. inhibition of ETC activity) and lipid peroxidation mediated secondary damage (e.g. membrane depolarization and cardiolipin loss) may be distinguished from each other by the use of chain-breaking antioxidant like BHT. The damage caused by lipid peroxidation is particularly important since the lipid peroxidative chain reaction may be promoted and propagated by transition metals and other agents independent of ROS. Metal-catalyzed lipid peroxidation is of special significance in the brain, which has high content of polyunsaturated fatty acids and reducing substances like ascorbate along with significant level of transition metals in several areas (Halliwell and Gutteridge, 1989; Calabrese et al., 2000). An increased
accumulation of transition metals and enhanced basal lipid peroxidation have been reported in brain during aging as well as in patients of Parkinson’s disease, Alzheimer’s disease, and Wilson’s disease, etc. (Jener, 1991; Calabrese et al., 2000; Honda et al., 2004). It is, therefore, not unlikely that peroxidation mediated mitochondrial dysfunction will contribute to neuronal degeneration in such conditions. Acknowledgements This work is supported by a research grant from CSIR (37/ 1116/02-EMR-II), Government of India, New Delhi. We acknowledge the kind help and suggestions of Dr. Hemanta Kumar Majumdar, Division of Infectious Disease, Indian Institute of Chemical Biology, Calcutta and Dr. Mitali Chatterjee, Department of Pharmacology, IPGMER, Calcutta. References Berman, S.B., Hastings, T.G., 1999. Dopamine oxidation alters mitochondrial respiration and induces permeability transition in brain mitochondria: implications for Parkinson’s disease. J. Neurochem. 73, 1127–1137. Berridge, M.V., Tan, A.S., 1993. Characterization of the cellular reduction of 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT): subcellular localization, substrate dependence and involvement of mitochondrial electron transport in MTT reduction. Arch. Biochem. Biophys. 303, 474–482. Calabrese, V., Bates, T.E., Stella, A.M.G., 2000. NO synthase and NO-dependent signal pathways in brain aging and neurodegenerative disorders: the role of oxidant/antioxidant balance. Neurochem. Res. 25, 1315– 1341. Chakraborty, H., Ray, S.N., Chakrabarti, S., 2001. Lipid peroxidation associated protein damage in rat brain crude synaptosomal fraction mediated by iron and ascorbate. Neurochem. Int. 39, 311–317. Chakraborty, H., Sen, P., Sur, A., Chatterjee, U., Chakrabarti, S., 2003. Agerelated oxidative inactivation of Na+, K+-ATPase in rat brain crude synaptosomes. Exp. Gerontol. 38, 705–710. Chen, J., Schenker, S., Frosto, T.A., Henderson, G.I., 1998. Inhibition of cytochrome c oxidase activity by 4-hydroxynonenal (HNE). Role of HNE adduct formation with the enzyme subunits. Biochem. Biophys. Acta 1380, 336–344. Chinopoulos, C., Tretter, L., Adam-Vizi, V., 1999. Depolarization of in situ mitochondria due to hydrogen peroxide-induced oxidative stress in nerve terminals: inhibition of a-ketoglutarate dehydrogenase. J. Neurochem. 73, 220–228. Clark, J.B., Bates, T.E., Boakye, P., Kuimov, A., Land, J.M., 1997. Investigation of mitochondrial defects in brain and skeletal muscle. In: Turner, A.J., Bachelard, H.S. (Eds.), Neurochemistry: A Practical Approach. Oxford University Press Inc., New York, pp. 151–174. Cohen, G., Farooqui, R., Kesler, N., 1997. Parkinson disease: a new link between monoamine oxidase and mitochondrial electron flow. Proc. Natl. Acad. Sci. U.S.A. 94, 4890–4894. Ferrante, R.J., Browne, S.E., Shinobu, L.A., Bowling, A.C., Baik, M.J., Mac Marvey, U., Kowall, N.W., Brown Jr., R.H., Beal, M.F., 1997. Evidence of increased oxidative damage in both sporadic and familial amyotrophic lateral sclerosis. J. Neurochem. 69, 2064–2074. Fry, M., Green, D.E., 1981. Cardiolipin requirement for electron transfer in complex I and complex III of the mitochondrial respiratory chain. J. Biol. Chem. 256, 1874–1880. Giulivi, C., Sarcansky, M., Rosenfeld, E., Boveris, A., 1990. The photodynamic effect of rose bengal on proteins of the mitochondrial inner membrane. Photochem. Photobiol. 52, 745–751. Halliwell, B., Gutteridge, J.M.C., 1989. Free Radical in Biology and Medicine. Clarendon Press, Oxford.
T. Sen et al. / Neurochemistry International 49 (2006) 20–27 Honda, K., Casadesus, G., Petersen, R.B., Perry, G., Smith, M.A., 2004. Oxidative stress and redox-active iron in Alzheimer’s disease. Ann. NY Acad. Sci. 1012, 179–182. Jener, P., 1991. Oxidative stress as a cause of Parkinson’s disease. Acta Neurol. Scand. 136, 6–15. Khan, F.H., Sen, T., Maiti, A.K., Jana, S., Chatterjee, U., Chakrabarti, S., 2005. Inhibition of rat brain mitochondrial electron transport chain activity by dopamine oxidation products during extended in vitro incubation: implication for Parkinson’s disease. Biochim. Biophys. Acta 1741, 65–74. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Lehotsky, J., Kaplan, P., Racay, P., Matejovicova, M., Drgova, A., Mezesova, V., 1999. Membrane ion transport systems during oxidative stress in rodent brain: protective effect of stobadine and other antioxidants. Life Sci. 65, 1951–1958. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with the Folin-phenol reagent. J. Boil. Chem. 193, 265– 275. Luo, J., Shi, R., 2005. Acroline induced oxidative stress in brain mitochondria. Neurochem. Int. 46, 243–252. Niki, E., Yoshida, Y., Saito, Y., Noguchi, N., 2005. Lipid peroxidation: mechanism, inhibition, and biological effects. Biochem. Biophys. Res. Commun. 336, 1–9. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay of lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95, 351–358. Paradies, G., Petrosillo, G., Pistolese, M., Ruggiero, F.M., 2002. Reactive oxygen species affect mitochondrial electron transport complex I activity through oxidative cardiolipin damage. Gene 286, 135–141. Paradies, G., Petrosillo, G., Pistolese, M., Ruggiero, F.M., 2000. The effect of reactive oxygen species generated from the mitochondrial electron transport chain on the cytochrome c oxidase activity and on the cardiolipin content in bovine heart submitochondrial particles. FEBS Lett. 466, 323– 326. Petit, J.M., Maftah, A., Ratinaud, M.H., Julien, R., 1992. 10-N-nonyl acridine orange interacts with cardiolipin and allows the quantification of this phospholipid in isolated mitochondria. Eur. J. Biochem. 209, 267–273. Petrosillo, G., Ruggiero, F.M., Paradies, G., 2003. Role of reactive oxygen species and cardiolipin in the release of cytochrome c from mitochondria. FASEB J. 17, 2202–2208.
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Picolo, M.J., Amarnath, V., McIntyre, J.O., Graham, D.G., Montine, T.J., 1999. 4-Hydroxy-2(E)-nonenal inhibits CNS mitochondrial respiration at multiple site. J. Neurochem. 72, 1617–1624. Picklo, M.J., Montine, T.J., 2001. Acrolein inhibits respiration in isolated brain mitochondria. Biochim. Biophys. Acta 1535, 145–152. Reers, M., Smith, T.W., Chen, L.B., 1991. J-aggregates formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential. Biochemistry 30, 4480–4486. Sayre, L.M., Zelasko, D.A., Harris, P.L., Perry, G., Saloman, R.G., Smith, M.A., 1997. 4-Hydroxynonenal-derived advanced lipid peroxidation end products are increased in Alzheimer’s disease. J. Neurochem. 68, 2092–2097. Sen, N., Das, B.B., Ganguly, A., Mukherjee, T., Bandyopadhyay, S., Majumdar, H.K., 2004. Camptothecin-induced imbalance in intracellular hemoflagellate Leishmania donovani. J. Biol. Chem. 279, 52366–52375. Shacter, E., Williams, J.A., Stadtman, E.R., Levine, R.L., 1996. Determination of carbonyl groups in oxidized protein. In: Punchard, N.A., Kelly, K.J. (Eds.), Free Radicals—A Practical Approach. Oxford University Press, Oxford, pp. 159–170. Shidoji, Y., Hayashi, K., Komura, S., Ohishi, N., Yagi, K., 1999. Loss of molecular interaction between cytochrome c and cardiolipin due to lipid peroxidation. Biochim. Biophys. Res. Commun. 264, 343–347. Shigenaga, M.K., Hagen, T.M., Ames, B.N., 1994. Oxidative damage and mitochondrial decay in aging. Proc. Natl. Acad. Sci. U.S.A. 91, 10771– 10778. Slater, T.F., 1984. Free-radical mechanisms in tissue injury. Biochem. J. 222, 1– 15. Spector, R., Eells, J., 1984. Deoxynucleoside and vitamin transport into the central nervous system. Fed. Proc. 43, 196–200. Tretter, L., Adam-Vizi, V., 1996. Early events in free radical-mediated damage of isolated nerve terminals: effects of peroxides on membrane potential and intracellular Na+ and Ca2+ concentrations. J. Neurochem. 66, 2057–2066. Tretter, L., Adam-Vizi, V., 2000. Inhibition of Krebs cycle enzymes by hydrogen peroxide: a key role of a-ketoglutarate dehydrogenase in limiting NADH production under oxidative stress. J. Neurosci. 20, 8972–8979. Uchida, K., 2003. 4-Hydroxy-2-nonenal: a product and mediator of oxidative stress. Prog. Lipid Res. 42, 318–343. Zhang, Y., Marcillat, O., Giulivi, C., Ernster, L., Davies, K.J.A., 1990. The oxidative inactivation of mitochondrial electron transport chain components and ATPase. J. Biol. Chem. 265, 16330–16336.
Lipid peroxidation associated cardiolipin loss and membrane depolarization in rat brain mitochondria Tanusree Sen a, Nilkantha Sen b, Gayatri Tripathi b, Uttara Chatterjee c, Sasanka Chakrabarti a,* a
Department of Biochemistry, Dr. B.C. Roy Post-graduate Institute of Basic Medical Sciences & IPGMER, Calcutta, India b Division of Infectious Disease, Indian Institute of Chemical Biology, Calcutta, India c Department of Pathology, Dr. B.C. Roy Post-graduate Institute of Basic Medical Sciences & IPGMER, Calcutta, India Received 20 October 2005; received in revised form 7 December 2005; accepted 20 December 2005 Available online 28 February 2006
Abstract Oxidative stress induced by Fe2+ (50 mM) and ascorbate (2 mM) in isolated rat brain mitochondria incubated in vitro leads to an enhanced lipid peroxidation, cardiolipin loss and an increased formation of protein carbonyls. These changes are associated with a loss of mitochondrial membrane potential (depolarization) and an impaired activity of electron transport chain (ETC) as measured by MTT reduction assay. Butylated hydroxytoluene (0.2 mM), an inhibitor of lipid peroxidation, can prevent significantly the loss of cardiolipin, the increased protein carbonyl formation and the decrease in mitochondrial membrane potential induced by Fe2+ and ascorbate, implying that the changes are secondary to membrane lipid peroxidation. However, iron-ascorbate induced impairment of mitochondrial ETC activity is apparently independent of lipid peroxidation process. The structural and functional derangement of mitochondria induced by oxidative stress as reported here may have implications in neuronal damage associated with brain aging and neurodegenerative disorders. # 2006 Elsevier Ltd. All rights reserved. Keywords: Lipid peroxidation; Mitochondria; Mitochondrial membrane potential; Cardiolipin; Electron transport chain; Reactive oxygen species
Lipid peroxidation is a unique mode of oxidative injury which is triggered and promoted by different radical and nonradical members of reactive oxygen species (ROS) family or by the catalytic decomposition of preformed lipid hydroperoxides in tissues by several agents including most notably the transition metals and microsomal cytochromes (Slater, 1984; Halliwell and Gutteridge, 1989; Niki et al., 2005). The peroxidative injury not only causes structural and functional derangement of phospholipid bilayer of membranes but also produces several deleterious aldehydic end products like malondialdehyde (MDA), 4-hydroxynonenal (4-HNE), acrolein, etc. which may inflict secondary damage to proteins and DNA (Halliwell and Gutteridge, 1989; Uchida, 2003; Luo and Shi, 2005). Thus, lipid peroxidation will further increase any
* Corresponding author at: Department of Biochemistry, Dr. B.C. Roy Postgraduate Institute of Basic Medical Sciences & IPGMER, 244B Acharya J.C. Bose Road, Calcutta 700020, India. Fax: +91 33 2280 1807. E-mail address: [email protected] (S. Chakrabarti). 0197-0186/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2005.12.018
primary oxidant-induced damage through ROS (Niki et al., 2005). The brain is highly vulnerable to lipid peroxidation because of its high rate of oxygen utilization, an abundant supply of polyunsaturated fatty acids, a deficient antioxidant defence and a high content of transition metals like copper and iron in several regions (Halliwell and Gutteridge, 1989; Calabrese et al., 2000). In several neurodegenerative disorders (e.g. Parkinson’s disease, Alzheimer’s disease, amyotropic lateral sclerosis, etc.) and brain aging, an enhanced formation of lipid peroxidation end products or adducts and various forms of mitochondrial dysfunctions have been noticed in post-mortem human brain or in the brains of transgenic or other animal models (Shigenaga et al., 1994; Ferrante et al., 1997; Sayre et al., 1997). However, such observations do not establish a causal relationship between lipid peroxidation and mitochondrial functional alterations in these disease states. On the other hand, the exposure of mitochondria or submitochondrial particles to g-ionizing radiation, xanthine/xanthine oxidase system or exogenously added H2O2 or acrolein has been shown to cause an impairment of electron transport chain (ETC)
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activity, inhibition of a-ketoglutarate dehydrogenase, loss of cardiolipin and some alterations in mitochondrial membrane potential which are largely attributed to the damaging effects of reactive oxygen species but the involvement of lipid peroxidation in causing mitochondrial dysfunction has not been clearly defined in such studies (Zhang et al., 1990; Tretter and Adam-Vizi, 2000; Paradies et al., 2002; Luo and Shi, 2005). On the basis of the results obtained from in vitro experiments and post-mortem studies, it seemed logical to attempt to identify in the present study the damaging consequences of lipid peroxidation on mitochondrial membrane potential and ETC activity and further to suggest the possible implications of our findings in explaining neuronal damage and death in neurodegenerative disorders and brain aging. A combination of iron (FeSO4) and ascorbate has been used to induce lipid peroxidation in brain mitochondria in our study, since this combination is not only a potent source of ROS but can also catalyze lipid peroxidative process in tissue preparation independent of ROS formation (Chakraborty et al., 2001). Our study has indicated that membrane lipid peroxidation is a major contributor to biochemical and functional alterations of mitochondria induced by oxidative stress in vitro and these changes presumably can lead to further deleterious consequences in vivo such as ATP depletion or the release of cytochrome c from the mitochondria. 1. Experimental procedures 1.1. Chemicals Anti-dinitrophenyl (anti-DNP) antibody, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT), butylated hydroxytoluene (BHT), 5bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium (NBT) were purchased from Sigma chemical Co. (USA). ‘ApoAlertTM Mitochondria Membrane Sensor Kit’ was purchased from BD Biosciences (USA). JC-1 (5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethylbenzimidazolylcarbocyanine iodide) and nonyl acridine orange were products of Molecular Probes (USA). All other chemicals used were of the highest purity available.
1.2. Animals Healthy adult albino rats of Charles Foster strain were maintained as per the guidelines of the animal ethical committee of the institute. The animals were euthanized by cervical dislocation and the brains dissected out clearly and collected on petri dishes kept over ice.
1.3. Isolation of rat brain mitochondria Rat brain mitochondria were isolated by differential centrifugation as published earlier (Berman and Hastings, 1999). Briefly, the brain from one adult rat was homogenized in 10 ml of buffer containing 225 mM mannitol, 75 mM sucrose, 5 mM HEPES, 1 mM EGTA, 1 mg/ml BSA, pH 7.4. The homogenate was brought to 30 ml with the same buffer and centrifuged at 2000 g for 3 min at 4 8C. The supernatant was divided into two tubes and centrifuged at 12,000 g for 10 min. The pellet containing the mixture of synaptosomes and mitochondria was suspended in 10 ml of homogenization buffer containing 0.02% digitonin to lyse the synaptosomes followed by centrifugation at 12,000 g for 10 min to pellet down both extrasynaptosomal and intrasynaptosomal mitochondria. The mitochondrial pellet was washed twice in the same buffer without EGTA, BSA and digitonin and finally resuspended either in 50 mM phosphate buffer, pH 7.4 or in the isotonic buffer
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A (containing 145 mM KCl, 50 mM sucrose, 5 mM NaCl, 1 mM EGTA, 1 mM MgCl2, 10 mM phosphate buffer, pH 7.4) for further experimentations.
1.4. Assay of citrate synthase for mitochondrial integrity The mitochondria suspended in the isotonic buffer A were checked for membrane integrity by assaying citrate synthase activity before and after treatment with Triton X-100 to obtain the latency value of citrate synthase and ratios exceeding 10 were considered indicative of good membrane integrity (Clark et al., 1997). In this method coenzyme A released during the reaction of oxaloacetate with acetyl coenzyme A catalyzed by citrate synthase present in mitochondrial sample was measured using 5,50 -dithiobis-2-nitrobenzoic acid (DTNB) which reacted with the free-SH groups of coenzyme A.
1.5. Incubation protocol for mitochondria Mitochondria were incubated in 50 mM phosphate buffer, pH 7.4 (200– 300 mg mitochondrial protein for lipid peroxidation and protein carbonyl measurements) or in isotonic buffer A (100–200 mg mitochondrial protein for MTT reduction assay and measurement of mitochondrial membrane potential or 300–500 mg mitochondrial protein for cardiolipin measurement) in the absence (control) or presence of FeSO4 (50 mM) and ascorbate (2 mM) with or without other additions like BHT (0.2 mM) or DMSO (20 mM) or mannitol (20 mM) for 1 h at 37 8C. In some experiments mitochondria were incubated with H2O2 (1 mM) or FeSO4 (50 mM) and H2O2 (1 mM) or KCN (1 mM).
1.6. Assay of lipid peroxides The extent of lipid peroxidation in mitochondrial samples during in vitro incubation was measured by quantitating the amount of malondialdehyde (MDA) formed by 2-thiobarbituric acid (TBA) reaction (Ohkawa et al., 1979). The amount of malondialdehyde (MDA) produced was calculated using the molar extinction co-efficient of MDA–TBA adduct as 1.56 105 cm2 mmol1 (Chakraborty et al., 2001).
1.7. Detection of protein carbonyls by immunoblotting At the end of the incubation, the samples of mitochondria were solubilised in 6% SDS and the protein carbonyls derivatized to hydrazones by treatment with 20 mM dinitrophenylhydrazine (DNPH) in 10% TFA for 10 min (Shacter et al., 1996). The samples were then neutralized with 2 M Tris/30% glycerol/ 19% b-mercapto ethanol and subjected to sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) following the method described previously (Laemmli, 1970). The proteins were then transferred to a nitrocellulose membrane by electroblotting. The dinitrophenylhydrazone derivatives of protein carbonyls were probed with polyclonal anti-DNP antibodies and detected by goat anti-rabbit IgG conjugated to alkaline phosphatase using BCIP/NBT as the substrates.
1.8. MTT reduction assay The incubation of mitochondrial samples (control and experimental) was terminated by the addition of an excess of ice-cold isotonic buffer A followed by centrifugation at 15,000 g for 15 min at 4 8C to pellet the mitochondria. The pellet was resuspended in isotonic buffer A and an aliquot added to a reaction mixture in the same buffer A containing 10 mM succinate (or 10 mM pyruvate or 10 mM a-ketoglutarate for some experiments) and MTT (0.42 mg/ml) and kept at 37 8C for 15 min. The samples were quenched with 500 ml of lysis buffer (45% dimethyl formamide in 10% SDS, pH 4.7) and the difference in absorbance values at 550 and 620 nm noted (Cohen et al., 1997).
1.9. Assay of mitochondrial cardiolipin content Cardiolipin content of mitochondria was measured using the cardiolipin specific dye nonyl-acridine orange (Petit et al., 1992). After 1 h incubation at 37 8C in buffer A with or without other additions, mitochondria were washed
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with an excess of ice-cold buffer A and pelleted at 4 8C by centrifugation at 15,000 g for 15 min. The pellet was resuspended in the same buffer and incubated at 37 8C for further 45 min in the presence of 5 mM nonyl-acridine orange. The excess dye was washed out by centrifugation and the mitochondrial pellet appropriately diluted in buffer A for fluorescence measurements (lex 485 nm, lem 535 nm) in a Hitachi spectrofluorometer (model F-4010). The fluorescence values normalized to 1 mg protein/ml were converted to relative fluorescence units using quinine (1 mg/ml in 0.1N H2SO4, lex 360 nm, lem 457 nm) as the reference.
1.10. Measurement of mitochondrial membrane potential Mitochondrial transmembrane potential was assessed by confocal microscopy using ‘mitosensor dye’ from a commercial kit (mitochondrial membrane sensor kit) as adopted from Sen et al. (2004). ‘Mitosensor’, a cationic dye, was taken up by the mitochondria in proportion to mitochondrial transmembrane potential and the accumulated dye underwent aggregation in the interior of the mitochondria. The monomeric form of the dye emitted green fluorescence while the aggregated form exhibited intense red fluorescence. In our experiments with isolated mitochondria, the excess dye (monomer) remaining outside was washed off thoroughly and, therefore, only the intense red fluorescence emitted from within the mitochondria was visible. For a quantitative assessment of mitochondrial membrane potential in control and experimental samples, a spectrofluorometric measurement was adopted using a mitochondrial membrane potential sensitive carbocyanine dye JC-1 (Reers et al., 1991). When excited at 490 nm, the monomeric form of JC-1 had an emission maximum at 527 nm, but the aggregated form (J-aggregates) showed an emission maximum at 590 nm. The negative transmembrane potential of mitochondria caused a directional uptake of the cationic dye into the matrix with subsequent formation of concentration-dependent J-aggregates and the fluorescence intensity of J-aggregate at 590 nm, therefore, reflected changes in mitochondrial membrane potential (Reers et al., 1991). For both confocal microscopic and spectrofluorometric experiments, isolated mitochondria were incubated in buffer A at 37 8C for 1 h with or without FeSO4 (50 mM) and ascorbate (2 mM) and/or other additions, followed by pelleting of the mitochondria at 4 8C by centrifugation with an excess of buffer A. The mitochondrial pellet was subsequently suspended in appropriate dilution in buffer A containing 10 mM succinate and reincubated at 37 8C for 30 min either in the presence of ‘mitosensor dye’ (5 mg/ml) or JC-1 (10 mg/ml). After 30 min, the dye loaded mitochondria were collected by centrifugation at 4 8C with an excess of buffer A, washed once more with the same buffer to remove thoroughly the dye remaining outside and finally resuspended in appropriate dilution in buffer A for confocal microscopy (TCS-SP Leica Confocal Microscope) or spectrofluorometric measurements (Hitachi F-4010). The spectrofluorometric readings normalized to 1 mg/ml protein were expressed as relative fluorescence units as described earlier in the text.
1.11. Measurement of protein content The protein content was determined by the method of Lowry after solubilizing the protein in 1% SDS and using bovine serum albumin as the standard (Lowry et al., 1951).
1.12. Statistical analysis All experiments were repeated at least five times using mitochondria from separate animals. The statistical significance was calculated by paired Student’s t-test. The immunoblotting and confocal microscopic experiments were repeated at least four times and a representative figure from a set of experiments producing similar results was presented in each case.
2. Results 2.1. Iron-ascorbate induced peroxidative damage to mitochondrial cardiolipin and protein The results presented in Table 1 show that an exposure of mitochondria to a mixture of Fe2+ (50 mM) and ascorbate (2 mM) led to a striking increase in MDA production and a marked loss in cardiolipin content. The lipid-soluble antioxidant BHT (0.2 mM) prevented both the phenomena (Table 1). When brain mitochondria were incubated in the presence of H2O2 (1 mM) or FeSO4 (50 mM) and H2O2 (1 mM) for 1 h the formation of MDA was negligible in comparison to that induced by iron-ascorbate (data not shown). Under similar conditions of incubation with iron-ascorbate an enhanced formation of protein carbonyls was noticed in mitochondrial fraction and this again was abolished by BHT (Fig. 1). 2.2. Effect of iron-ascorbate on mitochondrial MTT reduction The mitochondrial electron transport chain (ETC) activity was monitored by MTT reduction assay with isolated mitochondria using NAD+-linked (pyruvate and a-ketoglutarate) or FAD-linked (succinate) substrates. On incubation at 37 8C in buffer A for 1 h in the absence of any added iron or ascorbate, a striking impairment of mitochondrial MTT reduction in the presence of NAD+-linked substrates (e.g. aketoglutarate or pyruvate) was observed, while succinatesupported MTT reduction remained nearly unaltered (Fig. 2A). All subsequent experiments on the effects of Fe2+ (50 mM) and ascorbate (2 mM) on mitochondrial MTT reduction were, therefore, conducted in the presence of 10 mM succinate (FAD-linked substrate) only. The data shown in Fig. 2B demonstrate that oxidative stress induced by Fe2+-ascorbate led to a dramatic decrease (up to 40%) in succinate-supported mitochondrial respiration as measured
Table 1 Lipid peroxidation and cardiolipin loss in mitochondrial fraction exposed to iron-ascorbate Incubation mixture
Cardiolipin content (relative fluorescence unit/mg protein) (5)
Lipid peroxides (nmol MDA/mg protein) (5)
Mitochondria alone (control) Mitochondria + Fe2+ + Asc Mitochondria + Fe2+ + Asc + BHT
0.150 0.008 0.110 0.006* 0.146 0.007c
0.25 0.04 25.25 0.34* 1.94 0.10c
Incubation of mitochondria was performed in vitro without (control) or with Fe2+ (50 mM) and ascorbate (Asc, 2 mM) in the absence or presence of BHT (0.2 mM) followed by the estimation of cardiolipin and malondialdehyde (MDA) as described in the text. The values presented are the means S.E.M. The figures in parentheses represent the number of observations. * Statistically significant p < 0.001 vs. control. c Significantly different p < 0.001 vs. mitochondria + Fe2+ + ascorbate.
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Fig. 1. Immuno-detection of mitochondrial protein carbonyls after oxidative stress. Rat brain mitochondria were incubated with or without Fe2+ (50 mM) and ascorbate (2 mM) in vitro for 1 h at 37 8C in the presence or absence of BHT (0.2 mM) followed by the detection of protein carbonyls by DNPH-derivatization and immunoblotting using anti-DNP antibodies as described in Section 1. Lane a: mitochondria incubated alone; lane b: mitochondria + Fe2+ + ascorbate + BHT; lane c: mitochondria + Fe2+ + ascorbate.
by MTT reduction which was not prevented by BHT or by the scavengers of hydroxyl radicals. Further, when mitochondria were incubated with H2O2 (1 mM) or Fe2+ (50 mM) and dH2O2 (1 mM) for 1 h at 37 8C, a significant decrease in mitochondrial MTT reduction ability could be noticed which however, was less than that caused by Fe2+-ascorbate (Fig. 2B). 2.3. Evaluation of mitochondrial membrane potential by confocal microscopy The mitochondrial transmembrane potential was monitored with confocal microscopy using ‘mitosensor dye’ from a commercial kit. In succinate containing respiratory buffer, the cationic ‘mitosensor dye’ accumulated inside control mitochondria with normal transmembrane potential and formed aggregates which emitted an intense red fluorescence (Fig. 3). The intensity of red fluorescence was diminished markedly when mitochondria were pre-exposed to Fe2+ (50 mM) and ascorbate (2 mM) for 1 h, indicating depolarization of mitochondrial membranes and the phenomenon was significantly prevented by BHT added in the incubation mixture during Fe2+-ascorbate exposure (Fig. 3). The treatment of mitochondria with a strong respiratory poison like KCN led to a complete abolition of red fluorescence indicating a collapse of mitochondrial transmembrane potential (Fig. 3).
Fig. 2. Effect of oxidative stress on mitochondrial MTT reduction. Mitochondria were kept at 0 8C (unincubated control) or incubated at 37 8C in isotonic buffer in the absence (incubated control) or presence of Fe2+ (50 mM) plus ascorbate (Asc, 2 mM) or Fe2+ (50 mM) plus H2O2 (1 mM) or H2O2 (1 mM) with or without other additions like BHT (0.2 mM) or DMSO (20 mM) or mannitol (Man, 20 mM) followed by the measurement of MTT reduction as described in Section 1. (A) MTT-reduction by intact mitochondria (unincubated control and incubated control) was measured in the presence of NAD+-linked substrates (e.g. a-ketoglutarate, 10 mM or pyruvate, 10 mM) or FAD-linked substrate (e.g. succinate, 10 mM). Values from unincubated controls (data not shown) were taken as 100%. *: Statistically different p < 0.001 vs. unincubated control. (B) MTT reduction assay was performed in the presence of 10 mM succinate with control (incubated control) and oxidatively damaged mitochondria. Values from incubated controls (data not shown) were taken as 100%. &: Statistically significant p < 0.001 vs. incubated control.
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Fig. 3. Effect of iron-ascorbate on mitochondrial membrane potential in vitro incubation. Rat brain mitochondria were incubated alone (control) or with KCN (1 mM) or with Fe2+ (50 mM) and ascorbate (2 mM) in the presence or absence of BHT (0.2 mM) for 1 h at 37 8C. At the end of the incubation mitochondrial membrane potential was monitored by confocal microscopy using the cationic ‘mitosensor dye’ as described in Section 1. (a) Control mitochondria, (b) mitochondria + Fe2+ + ascorbate, (c) mitochondria + Fe2+ + ascorbate + BHT and (d) mitochondria + KCN.
2.4. Spectrofluorometric measurement of membrane potential in control and iron-ascorbate treated mitochondria A quantitative assessment of mitochondrial transmembrane potential was made by the measurement of fluorescence emission of J-aggregate at 595 nm within control as well as oxidatively damaged mitochondria. The results (Fig. 4) indicate that in mitochondria treated with Fe2+ (50 mM) and ascorbate (2 mM) for 1 h, J-aggregate fluorescence was diminished remarkably indicating a loss of mitochondrial transmembrane potential by oxidative injury. However, the lipid soluble antioxidant BHT (0.2 mM) prevented the mitochondrial membrane depolarization induced by Fe2+-ascorbate very significantly (Fig. 4). As in the experiments with ‘mitosensor dye’, J-aggregate fluorescence at 595 nm was found to be almost completely abolished by exposure of the mitochondria to a strong respiratory chain inhibitor like KCN (Fig. 4). 3. Discussion 3.1. Iron-ascorbate as a model system for in vitro oxidative stress
Fig. 4. Spectrofluorometric measurement of mitochondrial membrane potential after in vitro oxidative stress. The incubation mixtures containing mitochondria in isotonic buffer A in the absence (control) or with KCN (1 mM) or with Fe2+ (50 mM) plus ascorbate (Asc, 2 mM) with or without BHT (0.2 mM) were kept at 37 8C for 1 h. Mitochondrial membrane potential was measured spectrofluorometrically using JC-1 as described in the text. Values (relative fluorescence intensity in percentage) normalized to 1 mg protein are the means S.E.M. of 6 observations. *: Statistically significant p < 0.001 vs. control, &: statistically different p < 0.001 vs. iron + ascorbate.
In the present study a combination of iron and ascorbate has been used to induce lipid peroxidation in vitro in rat brain mitochondria (Table 1). This combination represents a physiologically relevant pro-oxidant system in the context of brain, since ascorbate content of brain is very high and several regions of the brain are enriched in iron (Spector and Eells, 1984; Halliwell and Gutteridge, 1989). In earlier studies we have shown that iron ascorbate causes extensive membrane lipid peroxidation and consequent secondary protein damage in rat brain crude synaptosomal fraction during in vitro incubation
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and further lipid peroxidation under such conditions is initiated by catalytic breakdown of preformed lipid hydroperoxides present in tissue preparation rather than by reactive oxyradicals (Chakraborty et al., 2001, 2003). Thus, while iron-ascorbate combination is a potent source of oxyradicals, its ability to enhance lipid peroxidation by decomposition of performed tissue lipid hydroperoxides makes this combination an ideal system to examine the effects of peroxidative process vis-a`-vis the direct ROS damage on subcellular components. 3.2. Lipid peroxidation, cardiolipin loss and protein carbonyl formation Cardiolipin is a major phospholipid of mitochondrial membrane and expectedly a significant loss of cardiolipin occurs consequent to lipid peroxidation in incubated mitochondria in the presence of iron-ascorbate and BHT, a chain-breaking inhibitor of lipid peroxidative process, can prevent the cardiolipin loss almost completely (Table 1). The formation of protein carbonyls is considered as a hallmark of oxidative protein damage and may result from a primary damage inflicted by ROS or secondary modifications due to formation of stable adduction compounds between reactive aldehydic end products of lipid peroxidation (e.g. 4-HNE) and amino acid side chains (Halliwell and Gutteridge, 1989; Shacter et al., 1996). Our results indicate that iron-ascorbate mediated protein carbonyl formation in incubated mitochondria represents secondary modifications caused by lipid peroxidation end products since the antioxidant BHT prevents both peroxidation and protein carbonyl formation (Table 1 and Fig. 1). These results are significant since the peroxidative damage to cardiolipin and mitochondrial membrane proteins may affect mitochondrial ETC activity and mitochondrial transmembrane potential which, in turn, could trigger a cascade of damaging events in brain cells. This possibility is supported by earlier studies where a peroxidative cardiolipin loss has been linked to diminished activities of complexes I, III and IV and other mitochondrial proteins like ADP/ATP translocator, phosphate translocator, ATP synthase, etc. (Tretter and Adam-Vizi, 2000; Calabrese et al., 2000; Paradies et al., 2000; Luo and Shi, 2005). Further, a catalytic requirement of cardiolipin has been demonstrated for several respiratory chain enzymes including complex I and complex IV (Fry and Green, 1981). On the other hand several end products of lipid peroxidation, e.g. 4hydroxynonenal (4-HNE) and acrolein added exogenously can inhibit mitochondrial respiration or ETC activity in isolated mitochondria at various levels presumably through formation of stable adducts with proteins (Chen et al., 1998; Picolo et al., 1999; Picklo and Montine, 2001). 3.3. Inhibition of mitochondrial ETC activity is independent of lipid peroxidation The activity of electron transport chain in isolated mitochondria can be assessed at complexes I–III by measuring mitochondrial MTT reduction in the presence of NAD+-linked (e.g. pyruvate, a-ketoglutarate, etc.) and FAD-linked (e.g.
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succinate) substrates (Khan et al., 2005). The tetrazolium dye, MTT, is reduced by accepting electrons, predominantly between cyrochrome c and cytochrome a and partially at a location between one Fe–S center of complex II and the site of inhibition by antimycin A (Berridge and Tan, 1993). In isolated control brain mitochondria incubated at 37 8C in isotonic medium without added Fe2+ or ascorbate for 1 h, a striking loss of mitochondrial MTT reduction ability is noted in the presence of NAD+-linked substrates, while succinatesupported MTT reduction is not affected to any significant degree (Fig. 2A). The results are surprising but the finding has not been explored any further in this study and in all our experiments related to iron-ascorbate effect on mitochondrial MTT reduction succinate has been used as the respiratory substrate. Our results demonstrate that mitochondrial electron transport chain (ETC) activity as measured by MTT reduction by intact mitochondria in the presence of a FAD-linked substrate, e.g. succinate is remarkably impaired when mitochondria are preincubated with Fe2+ (50 mM) and ascorbate (2 mM) for 1 h (Fig. 2B). The considerable impairment of succinate-supported MTT reduction by mitochondria, however, cannot be attributed to mitochondrial membrane lipid peroxidation or cardiolipin loss or protein carbonyl formation since BHT which nearly completely abolishes the latter phenomena has no effect on the former process (Fig. 2). It is possible that under our incubation condition the effect of iron-ascorbate on mitochondrial MTT reduction is not secondary to lipid peroxidative damage but results from some direct damage to ETC components by reactive oxygen species like OH radicals. The possibility, however, has not been confirmed since OH radical scavengers like mannitol or DMSO have failed to prevent the effect of iron and ascorbate on mitochondrial MTT reduction, although it is entirely possible that the radical scavengers have not reached the site of damage in adequate concentration through intact mitochondrial membrane. On the other hand oxidative stress in the form of H2O2 (1 mM) or a combination of Fe2+ (50 mM) and H2O2 (1 mM) also leads to a significant inhibition of mitochondrial MTT reduction (Fig. 2B), although the former agents induce very little MDA production in comparison to that by iron and ascorbate (data not shown). All these results implicate albeit indirectly a damage to mitochondrial ETC activity by active oxygen species independent of lipid peroxidation. It has been reported earlier by others in different systems that active oxygen species especially OH radicals and singlet oxygen can inactivate mitochondrial respiratory chain complexes by a mechanism independent of lipid peroxidation (Giulivi et al., 1990; Zhang et al., 1990). 3.4. Lipid peroxidation and mitochondrial membrane depolarization Mitochondrial transmembrane potential maintained by the respiratory activity of electron transport chain complexes is instrumental in coupling ATP synthesis with respiration. Various inhibitors of respiratory chain can lead to a loss of mitochondrial transmembrane potential (depolarization) and it
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is likely that oxidative inactivation of electron transport chain components will lead to mitochondrial membrane depolarization (Chinopoulos et al., 1999). Further, disrupted or damaged mitochondrial membranes as a result of lipid peroxidative process may also lead to dissipation of H+-ion gradient and consequent membrane depolarization. In different in vitro systems membrane peroxidation is known to alter permeability properties, transmembrane potential and ionic gradients that often can be prevented by antioxidants (Halliwell and Gutteridge, 1989; Tretter and Adam-Vizi, 1996; Lehotsky et al., 1999). Under our experimental conditions an incubation of isolated mitochondria with Fe2+-ascorbate results in a dramatic decrease of mitochondrial membrane potential (depolarization) as demonstrated by both confocal microscopic and spectrofluorometric experiments (Figs. 3 and 4). The fact that BHT (0.2 mM) significantly (up to 75%) prevents Fe2+ascorbate mediated membrane depolarization and lipid peroxidation (Figs. 3 and 4 and Table 1) but not the impairment of succinate-supported mitochondrial respiration (Fig. 2B) implies that the peroxidative membrane damage rather than the inhibition of mitochondrial ETC activity is predominantly responsible for the loss of mitochondrial transmembrane potential under our experimental conditions. However, since BHT protection against mitochondrial membrane depolarization is not complete, it is likely that the latter in part (nearly 25%) may result from impaired ETC activity of mitochondria induced by iron-ascorbate through ROS mediated damage. 3.5. Consequences of lipid peroxidative damage to mitochondria It is tempting to speculate from the present data that lipid peroxidation in a large measure will uncouple oxidative phosphorylation by disrupting mitochondrial membrane and dissipating the proton gradient with consequent ATP depletion. On the other hand the peroxidative loss of cardiolipin may trigger release of cytochrome c from mitochondria (Shidoji et al., 1999; Petrosillo et al., 2003). It is difficult to extrapolate such in vitro findings to in vivo scenario and to speculate on the consequences of lipid peroxidation associated mitochondrial dysfunctions in brain cells before obtaining further data in animal or cell culture models. In summary, this work has clearly shown that lipid peroxidation per se is an important contributor to mitochondrial dysfunction and that ROS mediated direct damage (e.g. inhibition of ETC activity) and lipid peroxidation mediated secondary damage (e.g. membrane depolarization and cardiolipin loss) may be distinguished from each other by the use of chain-breaking antioxidant like BHT. The damage caused by lipid peroxidation is particularly important since the lipid peroxidative chain reaction may be promoted and propagated by transition metals and other agents independent of ROS. Metal-catalyzed lipid peroxidation is of special significance in the brain, which has high content of polyunsaturated fatty acids and reducing substances like ascorbate along with significant level of transition metals in several areas (Halliwell and Gutteridge, 1989; Calabrese et al., 2000). An increased
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