Biochimica et Biophysica Acta 1505 (2001) 226^237 www.bba-direct.com
Mitochondrial membrane potential in density-separated trout erythrocytes exposed to oxidative stress in vitro Luca Tiano b
a;
*, Donatella Fedeli a , Patrizia Ballarini a , Giorgio Santoni b , Giancarlo Falcioni a
a Department of Biology MCA, University of Camerino, I-62032 Camerino (MC), Italy Department of Pharmacology and Experimental Medicine, University of Camerino, Camerino, Italy
Received 20 October 2000; received in revised form 22 February 2001; accepted 1 March 2001
Abstract Previous literature reports have demonstrated that nucleated trout erythrocytes in condition of oxidative stress are subjected to DNA and membrane damage, and inactivation of glutathione peroxidase. The present study was undertaken to investigate if mitochondrial membrane potential in stressed conditions was also influenced. Density-separated trout erythrocyte fractions, obtained using a discontinuous Percoll gradient, were submitted to stress conditions and the mitochondrial membrane potential was determined by means of cytofluorimetric analysis after incubation of each subfraction with JC-1, a mitochondrial specific fluorescent probe. The results clearly show that the mitochondrial membrane potential decreased significantly in all erythrocyte fractions, also if the oxidative effect on mitochondria is more severe with increased density (age) of the cell. Ebselen was very effective in preventing mitochondrial depolarization in young as well as in old erythrocytes. ß 2001 Elsevier Science B.V. All rights reserved. Keywords: Trout erythrocyte; Mitochondria; Ageing; Oxidative stress; Membrane potential; Antioxidant
1. Introduction Oxygen free radicals are thought to be the most important factor in determining biochemical and physical changes during the senescence process. The erythrocyte may prove a good model to advance our understanding of the ageing process in other tissues. However, information obtained by studying the ageing process in human erythrocytes is limited by the fact that these cells are structurally simple, devoid of nucleus and mitochondria. More
* Corresponding author. Fax: +39-737-636-216; E-mail:
[email protected]
information can be obtained by studying the ageing process in erythrocytes having these organelles (for instance ¢sh erythrocytes). Hence, the nucleated trout erythrocyte represents a stimulating cellular model to study oxidative damage associated with senescence processes [1^3]. In addition, this model permits to determine in vitro the antioxidant e¤cacy of natural or synthetic compounds and hence to attenuate oxygen radical-induced damage. Trout red blood cells (RBCs) may be separated into three distinct populations by discontinuous density gradient (45^65% Percoll) where older cells are characterized by increasing density. Previously, we reported on the physico-chemical characterization of the plasma membrane [1], and
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the extent of DNA damage [2] on these separated trout erythrocyte fractions. A novel interest in mitochondrial research derives from the unexpected role of mitochondria in the mechanism of apoptotic cell death [4^10], due to the possibility of mitochondria to be both the source and the ¢nal target of free radicals. Recently, we assessed the mitochondrial membrane potential of each density subfraction by means of cyto£uorimetric analysis using JC-1 as £uorescent probe. The analysis revealed a decrease in v8m with the density of the fraction [3]. Here, we reinvestigated the system to evaluate the involvement of oxygen reactive species on mitochondrial membrane potential. For this, we performed a £ow cytometric analysis of intracellular levels of reactive oxygen species (ROS) and mitochondrial membrane potential on the three trout erythrocyte subpopulations submitted to oxidative stress. As previously reported [11,12], in this experimental model it is possible to induce endogenous oxidative stress, both thermally and by varying the pH. The intracellular level of free radicals was evaluated by means of £ow cytometry and confocal microscopy using the non-£uorescent dye carboxy-H2 DCFDA (an analogue of H2 -DCFDA), which is a reporter of ROS generation at the single cell level. To examine mitochondrial membrane potential at the single cell level, cells from each fraction were incubated with the mitochondria speci¢c £uorescent probe JC-1, which is a delocalized lipophilic cation. JC-1 is more advantageous over other potential-sensitive probes, such as rhodamines and other carbocyanines, since it changes color from green to orange as the membrane potential increases (over values of about 80^100 mV). This property is due to the reversible formation of JC-1 aggregates upon membrane polarization that causes shifts in emitted light from 530 nm (i.e. emission of JC-1 monomeric form) to 590 nm (i.e. emission of J-aggregate). Subsequently, we explored the ability of di¡erent types of compounds (which are known to minimize the deleterious e¡ects produced by ROS) to avoid or reduce the level of intracellular ROS and its eventual linkage with v8m . In particular, we used Ebselen (2 phenyl-1,2-benzisoselenazol-3(2H)-one), a novel seleno-organic compound that has been established to act as a mimic of the antioxidant enzyme glutathione
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peroxidase [13]. Moreover, we used the piperidinic nitroxide TEMPO which is a radical species itself, possessing superoxide-like activity [14], and Trolox, the well known short chain analogue of vitamin E. 2. Materials and methods All reagents were of analytical grade. Percoll, 5-amino-2,3-dihydro-1,4-phthalazinedione (Ebselen), TEMPO and Trolox were purchased from Sigma (St. Louis, MO, USA), stored at 320³C and dissolved in ethanol as a 2 mM stock solution before use. Carboxy-H2 -DCFDA and JC-1 were purchased from Molecular Probes (Eugene, OR, USA) and stored at 320³C as a 1 mM stock solution in DMSO. 2.1. Samples The cells used in this study were obtained from Salmo irideus, an inbred strain of rainbow trout. The ¢sh were kept in tanks containing water from the Scarsito River and fed with commercial ¢sh food. Experiments were performed on ¢sh of the same age (approx. 24 months old), weighing between 180 and 300 g. Blood was withdrawn with a syringe from the lateral tail vein into an isotonic medium (0.1 M phosphate bu¡er, 0.1 M NaCl, 0.2% citrate, 1 mM EDTA, pH 7.8) and further treated within 2 h at 4³C. After removal of the plasma and bu¡y coat, the erythrocytes were washed three times with the same isotonic phosphate bu¡er. The erythrocytes were separated into three subpopulations on Percoll/BSA density gradient according to Rennie [15]. Suitable gradients lay in the range from 45 to 65% Percoll. The three fractions were collected, washed three times and ¢nally resuspended with phosphate bu¡er at pH 6.3 containing concentrations ranging from 5 to 30 WM of Ebselen, TEMPO and Trolox, and incubated at 35³C for 1 h. Control experiments were performed by incubating the erythrocytes with bu¡er at pH 6.3 and pH 7.8 containing only ethanol. 2.2. Flow cytometric analysis of intracellular levels of free radicals Trout erythrocytes were suspended at a concentra-
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tion of 5U106 cells/ml in isotonic phosphate bu¡er, either at pH 7.8 for non-stressed or pH 6.3 for stressed erythrocytes, and stained with 5 WM carboxy-H2 -DCFDA. The former were kept at room temperature while the latter at 35³C, for 60 min. Cells from each subfraction were then washed and resuspended to 1U106 cells/ml with isotonic phosphate bu¡er at pH 7.8 and submitted immediately to analysis using a FACScan £ow cytometer (Becton Dickinson, Mountain View, CA, USA) equipped with a single 488 argon laser. Fluorescence emission was collected at 525 nm. The values of PMT were logarithmically set. Green £uorescence (FL1) represents the oxidized form of carboxy-H2 -DCFDA, proportional to ROS formation. A minimum of 13 000 cells per sample were acquired and analyzed using WINMDI software on an IBM compatible computer. 2.3. Visualization of intracellular levels of free radicals by confocal laser microscopy A suspension of unseparated erythrocytes was prepared as reported above for £ow cytometric analysis of intracellular levels of free radicals. After incubation, the cell suspension was washed in fresh bu¡er and a drop of suspension was spread onto a coverslip glass treated with L-polylysine prior to sealing. Samples were immediately analyzed with a Bio-Rad M600 laser scanning confocal imaging system equipped with a krypton-argon laser and interfaced with a Nikon DIAPHOT-TMD inverted microscope mounting an objective apoplan 60U N.A.1.4. A FITC barrier ¢lter was used and the laser intensities and photodetector gains were held constant to allow comparison of relative £uorescence intensities of cells between the control and experimental cells. 2.4. Flow cytometric analysis of mitochondrial membrane potential After induction of oxidative stress, the erythrocyte suspension was adjusted to a density of 1.5U105 cells/ml and incubated with 10 Wg/ml of JC-1 for 10 min at room temperature in the dark for v8m determination. A suspension of 1U106 cells/ml from each subfraction was analyzed for relative £uorescence intensity using a FACScan £ow cytometer (Becton
Dickinson) equipped with a single 488 argon laser. The ¢lter in front of the £uorescence 1 (FL1) photomultiplier transmits at 530 nm, and the ¢lter used in the FL2 channel transmits at 617 nm. The values of PMT were logarithmically set. Red £uorescence (FL2) corresponds to the J-aggregate form of JC-1 and is proportional to v8m . Compensation FL1-FL2 was 4% and compensation FL2-FL1 9.5%. A minimum of 13 000 cells per sample were acquired and analyzed using WINMDI 2.8 on an IBM compatible computer. 2.5. Determination of the oxidation index Trout erythrocytes were incubated in stress conditions in isotonic phosphate bu¡er pH 6.3 at 35³C for 1 h in the presence and absence of 10 WM Ebselen, TEMPO and Trolox. Control samples were incubated in isotonic phosphate bu¡er at pH 7.8 at room temperature. Nucleus-free erythrocyte membranes were prepared according to Steer and Levitzki [16] using a discontinuous sucrose gradient. All samples were normalized by Lowry's method [17]. Lipids were extracted according to the method of Folch [18]. The absorbance ratio A233nm /A215nm de¢ned as oxidation index was used as a relative measurement for conjugated dienes which are formed during the early stage of lipid peroxidation, according to the method introduced by Konings [19]. UV measurements were carried out using a Cary1 Varian spectrophotometer. 3. Results Detection of the overall intracellular generation of free radicals was done essentially by the dichloro£uorescein diacetate (H2 -DCFDA) staining method [20] as described by Garland and Halewstrap [21]. In the present study, H2 -DCFDA was replaced with carboxy-H2 -DCFDA because the latter was expected to have enhanced retention inside the cell due to negative charges at physiological pH. Carboxy-H2 DCFDA added to the cells di¡uses across the cell membrane and is hydrolyzed by intracellular esterases to carboxy-H2 -DCF which, upon oxidation, is transformed into highly £uorescent DCF. A £ow cytometric distribution of unseparated cells
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in relation to carboxy-DCF £uorescence is reported in Fig. 1. It is evident from the ¢gure that the amount of ROS, directly proportional to the £uorescence intensity, generated in cells exposed to stress conditions (the stress was induced by suspending the erythrocytes in isotonic bu¡er at pH 6.3 and incubating for 1 h at 35³C), is increased compared to that of the control cells kept for 1 h at room temperature in isotonic bu¡er at pH 7.8. In the same ¢gure, control cells present a unimodal distribution centered on low values of £uorescence whereas stressed erythrocytes present a bimodal distribution with a major population centered on higher £uorescence intensity and a smaller population in correspondence with the control one. In order to allow an easier quanti¢cation of £ow cytometric data, we considered three arbitrary de¢ned regions that indicate levels of £uorescence relative to low, mid and high levels of oxidizing species. A similar experiment was conducted on densityseparated cells. Three di¡erent density-separated trout erythrocyte fractions were obtained using a discontinuous Percoll gradient in the range of 45^65%: a fraction of light cells (top), an intermediate cell fraction (middle) and a third fraction of densest cells (bottom). The cells in each subpopulation showed almost the
Fig. 1. Levels of ROS in unseparated trout erythrocytes as indicated by carboxy-H2 -DCFDA £uorescence. A, control cells incubated for 1 h at room temperature in isotonic bu¡er pH 7.8; B, cells submitted to oxidative stress after incubation for 1 h at 35³C in isotonic bu¡er pH 6.3. Fluorescence intensity on the abscissa and relative cell number on the ordinates. Fluorescence is proportional to ROS formation. Markers of £uorescence relative to low oxidation (R1) mid oxidation (R2) and high oxidation (R3) are presented.
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same level of ROS as in non-stress conditions, although it slightly decreases with the density of the fraction. After submission of cells to stress conditions, ROS levels increased in all the cell populations to the same extent (Fig. 2). In order to evaluate in greater detail the chemical species responsible for the increased £uorescence observed in stressed erythrocytes, we added di¡erent synthetic antioxidant compounds that are able to act as natural ones (Scheme 1). As shown in Figs. 2 and 3, after incubation for 1 h at 35³C in isotonic phosphate bu¡er at pH 6.3 supplemented with Ebselen at a 20 WM concentration, a remarkable reduction in the level of ROS is observed, while Trolox and TEMPO in the same experimental conditions were ine¡ective. Cells in the same conditions were further analyzed by confocal microscopy as reported in Fig. 4. Confocal micrographs con¢rm the data shown by £ow cytometry which indicate that although the overall cellular morphology is identical before and after submission to oxidative stress, only the control and Ebselen treated samples resulted non-£uorescent, thus reporting low levels of ROS. Confocal microscopy allowed to localize the highest £uorescence in correspondence with the nucleus and intracellular compartments, whereas the cytoplasm was faintly £uorescent. JC-1 red £uorescence distribution indicated that before incubation at 35³ for 1 h, variations in pH of the bu¡er had no e¡ect on mitochondrial membrane potential (data not shown). On the contrary, after incubation, the mitochondrial membrane potential decreased signi¢cantly in all the erythrocyte fractions. In particular, cells incubated at pH 6.3 always show mitochondrial depolarization in comparison to cells of the same age incubated at pH 7.8 (Fig. 5). In addition, these data were further analyzed by quanti¢cation of cells in arbitrarily de¢ned regions as shown in Fig. 5, indicating levels of £uorescence relative to low, mid and high levels of membrane potential. From Table 1 it is evident that the oxidative e¡ect on mitochondria is more severe with increased age of the cell: after incubation in stress conditions, the mitochondria of erythrocytes in the top fraction were in a low energetic state (78%), this increased in the middle fraction erythrocytes to 85%, and ¢nally in the bottom fraction, they constituted 95% of the total population.
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Fig. 2. Flow cytometry analysis using carboxy-H2 -DCFDA £uorescence in density-separated trout erythrocytes. Cell distribution chart (£uorescence is proportional to ROS formation). Percentages of cells presenting low (gray), mid (white) and high (black) levels of oxidizing species are presented. (a) Cells suspended in isotonic bu¡er at pH 7.8, (b) cells submitted to oxidative stress by incubation in isotonic bu¡er at pH 6.3 at 35³C for 1 h, (c) cells submitted to oxidative stress supplemented with 20 WM TEMPO, (d) Trolox, (e) Ebselen.
Furthermore, we wanted to evaluate the ability of ROS scavengers to counteract oxidative damage to mitochondria by co-incubating them with erythrocytes. Ebselen was very e¡ective in preventing mitochondria depolarization in young as well as in old erythrocytes. By incubation in bu¡er at pH 6.3 sup-
plemented with 10 WM organoselenium compound, v8m measured as JC-1 £uorescence intensity was higher than in control samples incubated at pH 7.8 and almost comparable with non-incubated samples (Fig. 6, Table 1). Also in this case the e¡ect was, to di¡erent extents, in relation to cell age. In particular,
Scheme 1. Chemical structure of Trolox: 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, TEMPO: 2,2,6,6-tetramethyl-1-piperidinyloxy, and Ebselen: 5-amino-2,3-dihydro-1,4-phthalazinedione.
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Fig. 3. Flow cytometry analysis using carboxy-H2 -DCFDA £uorescence in density-separated trout erythrocytes. (A) Top fraction; (B) middle fraction; (C) bottom fraction. Fluorescence intensity on the abscissa and relative cell number on the ordinates (£uorescence is proportional to ROS formation). RBCs not submitted to oxidative stress (pH 7.8), and submitted to oxidative stress (pH 6.3/35³C) with and without antioxidant supplementation (20 WM) are reported.
6
Fig. 4. Confocal micrographs of unseparated trout erythrocytes incubated with carboxy-H2 -DCFDA to visualize ROS formation. (a) Unstressed cells suspended in isotonic bu¡er at pH 7.8; (b) cells submitted to oxidative stress by incubation in isotonic bu¡er at pH 6.3 at 35³C for 1 h; (c) cells submitted to oxidative stress supplemented with 20 WM TEMPO, (d) Trolox, (e) Ebselen. (f) Control experiment in stress conditions in the presence of EtOH.
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middle fraction erythrocytes had the highest recovery potential, whereas in older cells the ability to revert to high v8m was the lowest. Ebselen presented a dose dependent e¡ect in the range 5^10 WM. The e¤cacy in maintaining v8m was reduced at 30 WM concentration. On the other hand, the piperidinic nitroxide TEMPO, despite its superoxide-like activity, was unable to inhibit ROS-mediated mitochondrial membrane depolarization. Likewise, the antioxidant activity of Trolox, a short chain vitamin E analogue, was insigni¢cant. All compounds were tested in the range 5^30 WM; for TEMPO and Trolox only the highest concentration is reported. The in£uence of oxidative stress and antioxidant supplementation on the rate of hemolysis in these experimental conditions was also investigated. After 1 h of incubation at 35³C/pH 6.3, none of the compounds used enhanced signi¢cantly the rate of hemolysis, although for longer incubation times, Ebselen promoted a hemolytic e¡ect that appeared to have a dose dependent dynamic (Fig. 7). The level of hydroperoxides on plasma membrane in our experimental conditions (1 h of incubation at 35³C pH 6.3 in the presence and absence of antioxidants 10 WM and control samples at pH 7.8) increased after incubation in stress conditions at pH 6.3 and in the presence of Ebselen with respect to the control sample. On the contrary, the presence of Tempo and Trolox reduced the levels of hydroperoxides (Fig. 8). 4. Discussion
Fig. 5. Flow cytometric analysis of JC-1 red £uorescence proportional to v8m in density-separated trout erythrocytes. (1) Top fraction; (2) middle fraction; (3) bottom fraction. A, cells suspended in isotonic bu¡er pH 7.8 not incubated; B, cells suspended in isotonic bu¡er pH 7.8 incubated at 35³C for 1 h; C, cells suspended in isotonic bu¡er pH 6.3 incubated at 35³C for 1 h. Abscissa indicates £uorescence intensity and ordinate the relative cell number. Markers of £uorescence relative to low membrane potential (R1), mid membrane potential (R2) and high membrane potential (R3) are presented.
Mitochondria, through the respiratory chain, are the major producers of ROS, and its components are hence exposed to the hazards of oxygen radicals. A decreased mitochondrial functionality has generally been observed in senescence [22], and this decline could be due to various biochemical processes such as lipid peroxidation, protein oxidation and mtDNA mutations that are a consequence of oxidative stress. Evidence exists, however, that even ROS produced outside the mitochondrion may damage mitochondrial components [23,24], thus impairing mitochondrial functionality. The present work was aimed to investigate mito-
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chondrial membrane potential in density-separated trout erythrocyte subpopulations submitted to endogenous oxidative stress. It is known that there is a correlation between the density of erythrocyte subpopulations and ageing [25,26]; older cells are characterized by an increased density. In our experimental conditions (erythrocytes suspended in isotonic bu¡er pH 6.3 and incubated for 1 h at 35³C), oxidative stress is due in part to both the formation of superoxide radical and to the inactivation of glutathione peroxidase (an enzyme that can metabolize H2 O2 and lipid peroxides) which are a consequence of Hb oxidation as reported previously [12]. Previous literature reports have demonstrated that nucleated trout erythrocytes in these conditions of oxidative stress are subjected to membrane and DNA damage; the latter was signi¢cantly prevented by the presence of some stable nitroxide compounds which have been used recently in biological systems for their capacity to mimic superoxide dismutase [27^30]. Using £ow cytometry and confocal microscopy techniques, we obtained information on intracellular levels of free radicals and membrane potential in individual cells. The data presented here could suggest that the improvement in the primary antioxidant defense system in older cells, as previously reported by us [31], seems to be able to reduce the intracellular level of
ROS produced compared to control samples (pH 7.8). Besides, it is evident that in our stress conditions, the £uorescence intensity (directly correlated to the level of ROS) increased in all the three subpopulations. The experiments performed in the presence of di¡erent types of antioxidants highlight the fact that only Ebselen is capable of reducing intracellular levels of ROS. Bearing in mind that Ebselen acts as a mimic of the antioxidant enzyme glutathione peroxidase [12], and TEMPO possesses superoxide-like activity [13], one could speculate that the ROS level in our experimental conditions could be mainly due to the production of H2 O2 and not to that of superoxide radical. However, this hypothesis is unlikely because H2 O2 should be eliminated by catalase. Thus, intracellular ROS increase could be due to other reactive species. Our previous results [3] concerning mitochondrial membrane potential on the di¡erent density trout subpopulations, revealed a marked decrease in vim in the bottom layer cell mitochondria compared to the top; similarly, the mitochondrial membrane potential in all three subpopulations decreased in conditions of oxidative stress, although in a di¡erent manner. The link between intracellular ROS production and the decrease in mitochondrial membrane potential in the three erythrocyte subpopulations submitted to oxidative stress is clear, since Ebselen is able to both decrease
Table 1 Cytometric analysis of JC-1 red £uorescence proportional to v8m in density-separated trout erythrocytes Top low potential mid potential high potential Middle low potential mid potential high potential Bottom low potential mid potential high potential
7.8
7.8 inc
6.3 inc
Ebs 5 WM
Ebs 10 WM
Ebs 30 WM
TEMPO 30 WM
Trolox 30 WM
3.6 11.9 85.4
23.8 68.2 9.7
77.6 19.8 3.9
22.1 71.3 8.0
4.3 75.3 23.6
3.7 83.5 15.0
77.5 20.6 3.2
72.8 25.7 2.9
1.9 12.4 86.8
28.8 67.6 5.3
84.8 13.8 2.3
24.3 74.1 3.2
2.2 32.5 69.2
9.4 88.1 3.7
82.7 16.1 1.9
83.5 15.9 1.6
6.9 29.9 65.1
23.7 62.1 15.7
95.3 4.2 0.9
56.9 43.4 1.5
19.9 79.8 1.7
56.6 44.1 1.5
92.7 6.2 1.5
95.7 3.2 1.3
Percentages of cells belonging to areas of low membrane potential, mid membrane potential and high membrane potential are presented. Markers were arbitrarily set from the analysis of £ow cytometry histograms. Data are representative of top, middle and bottom fraction trout erythrocytes suspended in isotonic bu¡er pH 7.8 not incubated (7.8), suspended in isotonic bu¡er pH 7.8 incubated at 35³C for 1 h (7.8 inc), suspended in isotonic bu¡er pH 6.3 incubated at 35³C for 1 h (6.3 inc), and suspended in isotonic bu¡er pH 6.3 incubated at 35³C for 1 h in the presence of antioxidants at di¡erent concentrations (Ebs 5 WM, Ebs 10 WM, Ebs 30 WM, TEMPO 30 WM, Trolox 30 WM).
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Fig. 7. Hemolysis rate of control RBCs and RBCs in the presence of antioxidant 20 WM, after submission to oxidative stress (incubation at 35³C/pH 6.3). 7, control; E, Ebselen; O, TEMPO; a, Trolox.
the intracellular level of ROS and to reverse the effects of oxidative stress on vim . Although it has been established that Ebselen possesses anti-in£ammatory, anti-atherosclerotic and cytoprotective properties [32^34], the fact that it protects mitochondria against free radical damage is of particular interest because of the possible role that this molecule may play as a biological antioxidant in the ageing process, although it is well to point out the early hemolytic event in the presence of Ebselen. It has been reported by Maiorino et al. [35] that Ebselen is capable of reducing membrane hydroperoxides in liposomes; on the contrary in our experimental model Ebselen is not able to reduce the level of hydroperoxides, which in fact is rather increased. That is probably due to its interaction with hemo-
Fig. 6. Flow cytometry analysis using JC-1 red £uorescence in density-separated trout erythrocytes. (A) Top fraction; (B) middle fraction; (C) bottom fraction. Abscissa indicates £uorescence intensity and ordinates relative cell number. Fluorescence is proportional to v8m . RBCs not submitted to oxidative stress (incubated in isotonic bu¡er pH 7.8 at 35³C for 1 h), and submitted to oxidative stress (incubated in isotonic bu¡er pH 6.3 at 35³C for 1 h) with and without antioxidant supplementation (10 WM) are reported.
Fig. 8. Oxidation index obtained by the absorbance ratio A233nm /A215nm measured on lipids extracted from trout erythrocytes incubated in stress conditions in isotonic phosphate bu¡er pH 6.3 at 35³C for 1 h in the presence and absence of 10 WM Ebselen, TEMPO and Trolox. Control samples were incubated in isotonic phosphate bu¡er at pH 7.8 at room temperature.
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globin: the presence of Ebselen increases the met-Hb formation rate (data not shown). Besides, our data are in agreement with the mitochondrial theory of ageing [36,37] that considers senescence as the result of cellular degeneration due to mitochondrial changes induced by long term exposure to free radical attack.
[11]
[12]
[13]
Acknowledgements [14]
The authors wish to thank Prof. G. Lenaz for critical reading and helpful suggestions. This work was supported by a CNR fund to G.F.
[15]
[16]
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