- Email: [email protected]
Ebselen prevents mitochondrial ageing due to oxidative stress: in vitro study of fish erythrocytes
Mitochondrion 2 (2003) 428–436 www.elsevier.com/locate/mito
Ebselen prevents mitochondrial ageing due to oxidative stress: in vitro study of fish erythrocytes Luca Tianoa,*, Donatella Fedelia, Giorgio Santonib, Ian Daviesc, Takashi Wakabayashid, Giancarlo Falcionia a
Dipartimento di Biologia Molecolare, Cellulare e Animale, Universita` degli Studi di Camerino, Via Camerini 2, I-62032, Camerino (MC), Italy b Department of Pharmacology and Experimental Medicine, University of Camerino, Via Camerini 2, I-62032, Camerino (MC), Italy c Fisheries Research Services, Marine Laboratory, P.O. Box 101, 375 Victoria Road, Aberdeen AB11 9DB, UK d Department of Medical Chemistry, Medical University of Gdansk, 1 Debinki Str., Gdansk, Poland Received 4 December 2002; received in revised form 10 February 2003; accepted 21 February 2003 Dedicated to the memory of Eraldo Antonini, eminent biochemist, prematurely deceased 20 years ago, on March 19th, 1983.
Abstract Nucleated trout erythrocytes under oxidative stress suffer DNA membrane damage and inactivation of glutathione peroxidase. In addition, oxidative damage increases with the age of the cell. In the present paper, we evaluate the effects of oxidative stress and ageing on mitochondrial functionality by means of transmission electron microscopy and cytofluorimetric determination of mitochondrial membrane potential and intracellular levels of reactive oxygen species. The protective activity of the antioxidant organoselenium compound ebselen, a mimic of glutathione peroxidase, is also evaluated. Ebselen prevents the drastic structural and functional changes in mitochondria in aged RBCs induced by oxidative stress. However, the antioxidant does not prevent swelling of the mitochondria. q 2003 Mitochondria Research Society. Published by Elsevier Science B.V. All rights reserved. Keywords: Trout erythrocytes; Mitochondria; Ageing; Oxidative stress; Ebselen; Membrane potential
1. Introduction Cellular sources, notably mitochondria, can generate oxygen radicals via oxidative phosphorylation in which oxygen is reduced to water through a four-step addition of electrons (Moser et al., 1992; Davies, 1993). The reactive oxygen species (ROS) generated * Corresponding author. Tel.: þ 39-0737-403213; fax: þ 39-0737-636216. E-mail address: [email protected] (L. Tiano).
by incomplete reduction of oxygen are extremely reactive and can interact with cellular lipids, proteins and nucleic acids, impairing their structure and function. Cells have defence systems to prevent injury by these substances. An imbalance between free radical-generating and -scavenging systems is commonly referred to as oxidative stress. Oxidative stress may be a cause or consequence of various diseases such as diabetes, atherosclerosis, cancer, Parkinson’s disease or Alzheimer’s disease (Halliwell et al., 1992; Ames et al., 1993; Swart et al., 1972; Guyton and
1567-7249/03/$20.00 q 2003 Mitochondria Research Society. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S1567-7249(03)00032-1
L. Tiano et al. / Mitochondrion 2 (2003) 428–436
Kensler, 1993; Cerrutti et al., 1994; Harman, 1992). Environmental factors such as a poor diet, tobacco smoke, ozone and various types of xenobiotics may be closely linked to these disease conditions via oxidative stress. Finally, oxidative stress can be a result of senescence processes that are commonly accompanied by a progressive impairment of antioxidant systems (Harman, 1956; Linnane et al., 1989; Agarwal and Sohal, 1994; Lenaz, 1998; Diplock, 1994). Thus, according to the ‘free radical theory of ageing’ proposed by Harman (1956), oxidative stress has been postulated as one of the main factors leading to ageing. Mitochondria play a key role in the oxidative stress processes and ageing, due to their functions in cellular respiration processes that make them simultaneously a source and the final target of free radicals. It has been calculated that 90% of the oxygen taken up by the cell is consumed by mitochondria, and that 2% of the oxygen consumed by mitochondria is converted into the superoxide anion (Lee et al., 1997). Recent advances in molecular biology have disclosed a new function of mitochondria. They are endowed with several important factors controlling the processes of programmed cell death (Mignotte and Vayssiere, 1998; Skulachev, 1996; Petit et al., 1995; Zamzami et al., 1996; Camilleri-Broet et al., 1998; Yakes and Van Houten, 1997; Holt et al., 1988; Susin et al., 1998). Moreover, it is well known that mitochondrial DNA (mtDNA) is more susceptible than genomic DNA (Tritschler and Medori, 1993) to the mutations that are responsible for several diseases (Schapira, 1997). Thus, any mitochondrial lesion, primary or secondary, may trigger a damaging cycle of oxidative stress and mtDNA mutations: a defect in the mitochondrial respiratory chain is followed by the release of mutagenic oxidants, somatic mutations and defects in mitochondrial proteins, thereby amplifying the original error. Nucleated trout red blood cells (RBCs) are a very useful cellular model for the study of oxidative damage associated with oxidative stress and senescence processes (Falcioni et al., 1987; Tiano et al., 2000). RBCs of lower vertebrates differ from those in mammals in that they retain both nucleus and mitochondria after the reticulocyte stage. Also, it is possible to induce a condition of endogenous oxidative stress
429
by promoting haemoglobin autoxidation, leading to met-Hb and formation of ROS. An imbalance in oxidant species achieved in this way is likely to simulate pathophysiological conditions of oxidative stress observed in vivo in response to exogenous oxidative insult. Finally, trout RBCs increase in density with age, and can be separated into three distinct populations (sub-fractions) along a discontinuous density gradient (45 – 65% Percoll). Recently, we assessed the mitochondrial membrane potential (DCm) of each RBC sub-fraction by flow cytometry using JC-1 as a fluorescent probe. We found that the DCm of trout RBCs decreases in parallel with increases in their density (Tiano et al., 2000). The present study is concerned with morphological and functional changes of mitochondria in trout RBCs exposed to oxidative stress states, focusing on possible differences in their behaviour depending on the age of the cells. The possible susceptibility of trout RBCs of different ages to oxidative injury related to senescence processes was assessed. The protective effects of the organoselenium compound ebselen (2-phenyl-1,2-benzisoselenazol-3-(2H)-on) against oxidative damage in mitochondria was also evaluated. Ebselen can minimise the deleterious effects of ROS by mimicking the antioxidant enzyme glutathione peroxidase (Muller et al., 1984; Wendel et al., 1984).
2. Materials and methods 2.1. Preparation of samples Specimens of Salmo irideus (, 24 months old, 180– 300 g weight), an inbred strain of rainbow trout, were kept in tanks containing water from Scarsito River, Italy and fed with a commercial fish diet. Blood was withdrawn by syringe from the lateral tail vein into an isotonic medium (0.1 M phosphate buffer, 0.1 M NaCl, 0.2% citrate, 1 mM ethylenediaminetetraacetic acid (EDTA), pH 7.8) and was treated further within 2 h at 48C. After the removal of the plasma and buffy coat, erythrocytes were washed three times with the same isotonic buffer. The RBCs were separated into three sub-populations on a Percoll/BSA density gradient (45 – 65% Percoll, Rennie et al., 1979). The three RBC fractions obtained were washed
430
L. Tiano et al. / Mitochondrion 2 (2003) 428–436
three times and finally suspended in phosphate buffer (pH 6.3) and incubated at 358C for 1 h. Under these conditions, oxidative stress is promoted by inactivation of glutathione peroxidase and acceleration of met-Hb formation, with subsequent formation of ROS. Parallel experiments were conducted by incubating the cells at the same temperature and pH in the presence of ebselen at concentrations ranging from 5 to 30 mM. Control experiments were performed by incubating RBCs with phosphate buffer pH 7.8 at room temperature. 2.2. Flow cytometric analysis of intracellular levels of free radicals Trout RBCs were suspended in isotonic phosphate buffer for 60 min at pH 6.3 at a concentration of 5 £ 106 cells/ml (stressed RBCs). Control RBCs were suspended in the same buffer at pH 7.8. They were then stained with 5 mM carboxy-H2-DCFDA to detect intracellular levels of ROS, essentially, according to the method of Garland and Halestrap (1997). Cells from each sub-fraction were then washed and resuspended in the isotonic phosphate buffer at pH 7.4 at a concentration of 1 £ 106 cells/ml, and immediately submitted to analysis using a fluorescence-activated cell sorter scan (FACSCAN) flow cytometer, Becton, Dickinson, Mountain View, CA equipped with a single 488 nm argon laser. Fluorescence emission (FL1) is proportional to the oxidised form of carboxy-H2-DCFDA, and is proportional to intracellular levels of ROS. A minimum of 13,000 cells per sample was analysed using WINMDI software on an IBM compatible computer. 2.3. Flow cytometric analysis of DCm After being exposed to oxidative stress, suspensions of RBCs were adjusted to a concentration of 1.5 £ 105 cells/ml, and incubated with JC-1 at a concentration of 10 mg/ml for 10 min at room temperature in the dark to detect DCm. JC-1 is a mitochondria-specific fluorescent probe that incorporates into the charged membrane according to Nernst’s Law (Poot et al., 1996; Salvioli et al., 1997; Smiley et al., 1991; Reers et al., 1991; Cossarizza et al., 1993). JC-1 has advantages over other potentialsensitive probes, such as rhodamine and other
carbocyanines, in that, it changes colour from green to orange as the membrane potential increases over values of about 80 – 100 mV. This property arises from the reversible formation of JC-1 aggregates upon membrane polarisation that causes shifts in the wavelength of the emitted light from 520 (emission by monomeric JC-1) to 590 nm (emission by aggregated JC-1). A suspension of 1 £ 105 cells/ml from each subfraction was also analysed for relative fluorescence intensity. The filter in front of the fluorescence 1 (FL1) photomultiplier transmits (PMT) at 530 nm, and the filter used in the FL2 channel transmits at 617 nm. The values of PMT were set logarithmically. Red fluorescence (FL2) corresponds to the aggregated form of JC-1 and is proportional to DCm. Compensation FL1–FL2 was 4% and compensation FL2–FL1 was 9.5%. A minimum of 13,000 cells per sample was acquired and analysed using WINMD1 2.8 on an IBM compatible computer. Mean fluorescence intensities were statistically analysed for significant differences using Student’s t-test. 2.4. Transmission electron microscopy Aliquots of RBC sub-fractions submitted to oxidative stress in the presence and absence of 10 mM ebselen were fixed for 4 h at 48C in a fixative containing 2.5% glutaraldehyde and 0.1 M sodium cacodylate, pH 7.4. They were postfixed for 1 h at room temperature in 1% osmium tetroxide dissolved in distilled water. Fixed cells were then dehydrated in a graded series of ethanol solutions and embedded in TAAB-embedding resin (medium grade). Ultrathin sections were cut on a Reichert – Jung Ultracut ultramicrotome and stained with uranyl acetate and lead citrate. Stained sections were examined in a Philips 301 transmission electron microscope operated at 80 kV. 2.5. Chemicals All reagents were of analytical grade: Percoll, 5amino-2,3-dihydro-1,4-phthalazinedione (Ebselen) were purchased from Sigma, St. Louis, MO. Ebselen was stored at 2 208C and dissolved in ethanol as 2 mM stock solution just before use. Carboxy-H2DCFDA and JC-1 were purchased from Molecular
L. Tiano et al. / Mitochondrion 2 (2003) 428–436
Probes, Eugene, OR and stored at 2 208C as 1 mM stock solution in dimethyl sulfoxide (DMSO).
3. Results Intracellular levels of ROS in RBCs incubated in isotonic phosphate buffer at pH 6.2/358C (Fig. 1 and Table 1) were strongly elevated compared to those in the control. On the contrary, after incubation at physiological pH and 358C, intracellular levels of ROS were comparable with the control cells incubated at room temperature. These data are in accordance with previous literature reports describing the induction of oxidative stress in trout erythrocytes by variation of pH and temperature that involve inhibition of glutathione peroxidase activity and acceleration of the autoxidation of haemoglobin (Falcioni et al., 1992). Autoxidation is the spontaneous oxidation of Hb (haeme iron present as FeII) to met-Hb (haeme iron as FeIII). Superoxide is a reaction product. ROS formation was revealed by the specific oxidation of the fluorochrome carboxy-H2DCFDA. Positive control experiments were performed in order to assess the validity of the test in this experimental model by exposing DCFDA-stained
431
erythrocyte to hydrogen peroxide 5 mM. FACSCAN analysis revealed a time-dependent increase in the fluorescence signal (data not shown). Fig. 1 shows the mean fluorescence intensities of 10,000 measurements, indicating a highly significant rise in fluorescence intensities in erythrocytes of the three age sub-fractions submitted to oxidative stress by incubation at pH 6.3 at 358C ðP , 0:0001Þ. The data showed a bimodal distribution, and, therefore, the 1023 fluorescence channels, proportional to intracellular levels of ROS, were arbitrarily classified into three groups according to low (0 – 295), medium (296 –550) and high levels (551 – 1023) of ROS. This made it possible to estimate the percentage distribution of cells with different fluorescent intensity of carboxy-DCF in each sub-fraction of both control and experimental groups. As shown in Table 1, the percentage of the cells with high intracellular level of ROS was remarkably greater, over 2500-fold in comparison to unstressed controls, in all three subfractions of RBCs exposed to oxidative stress. By contrast, the percentage of the cells with high intracellular level of ROS was lower in all three sub-fractions exposed to oxidative stress in the presence of ebeselen, and was similar to those to those in controls. The lowest effective concentration
Fig. 1. Analysis of DCF fluorescence, proportional to intracellular ROS levels, in flow cytometry of density-separated trout erythrocytes in untreated controls incubated at pH 7.8 both at room temperature and 358C, in conditions of oxidative stress (pH 6.3, 358C) and in the same conditions in the presence of ebselen 10 mM. All three, age sub-fractions are reported: top (white), middle (grey) and bottom (black). Data are expressed as median fluorescence intensities ^ standard deviation *P , 0:0001.
432
L. Tiano et al. / Mitochondrion 2 (2003) 428–436
Table 1 Effects of ebselen on intracellular levels of ROS in sub-fractions of trout erythrocytes exposed to oxidative stress Fraction (ROS level)
Control pH 7.8/room temperature (%)
pH 7.8/358C (%)
pH 6.3/358C (%)
pH 6.3/358C þ ebselen (%)
Top fraction Low Middle High
91.96 7.5 0.03
90.52 8.8 0.6
15.86 8.86 75.4
99.0 0.05 0.05
Middle fraction Low Middle High
95.96 4.72 0.02
92.43 6.82 0.75
13.32 10.72 75.96
99.92 0.04 0.04
Bottom fraction Low Middle High
97.18 1.41 0.03
95.46 3.92 0.62
11.86 10.98 77.16
99.15 0.18 0.67
Percentages of cells presenting low, mid and high intracellular levels of ROS. Markers were arbitrarily set from the analysis of flow cytometry histograms. Data are presented from top, middle and bottom fraction trout erythrocytes, suspended in isotonic buffer pH 7.8 incubated at room temperature, 358C for 1 h and 358C for 1 h in presence of ebselen 10 mM.
of ebselen was 10 mM. No significant differences were detected between 10 and 30 mM (data not shown). The mean intensities of fluorescence proportional to DCm are reported in Fig. 2. Incubation at pH 7.8 and 358C resulted in a non-significant mitochondrial depolarisation, nevertheless, incubation under conditions of oxidative stress induced a dramatic
mitochondrial depolarisation in all three age subfractions ðP , 0:0001Þ and incubation in presence of ebselen 10 mM significantly reduced the degree of depolarisation ðP , 0:0001Þ. Mean fluorescence intensities of ebselen treated erythrocytes are significantly higher than control cells in all three subpopulation. This is due to a broader distribution of the fluorescence intensities in control cells around the
Fig. 2. Analysis of JC-1 red fluorescence, proportional to DCm, in flow cytometry of density-separated trout erythrocytes in untreated controls incubated at pH 7.8 both at room temperature and 358C, in conditions of oxidative stress (pH 6.3, 358C) and in the same conditions in the presence of ebselen 10 mM. All three, age sub-fractions are reported: top (white), middle (grey) and bottom (black). Data are expressed as median fluorescence intensities ^ standard deviation *P , 0:0001.
L. Tiano et al. / Mitochondrion 2 (2003) 428–436
433
ebselen in maintaining elevated DCm was slightly reduced at 30 mM (data not shown). Structural changes in the mitochondria of RBCs exposed to oxidative stress, and the protective effects of ebselen, were investigated by electron microscopy. Top fraction RBCs in the control contained a few mitochondria elongated to various degrees scattered in the cytoplasm (Fig. 3A). The cristae of these mitochondria were aligned perpendicular to their long axes. On the other hand, mitochondria in the middle and bottom subfractions were round or oval, with cristae confined to the periphery (Fig. 3B, C, respectively). This may suggest that they were functionally impaired to some extent. Previous observations have shown that mitochondria in trout RBCs decline in number and functionality with ageing (Tiano et al., 2000). When RBCs in each sub-fraction were exposed to oxidative stress, marked structural changes were always observed in the mitochondria. Mitochondria in RBCs in the top fraction became distinctly swollen and the cristae were distorted (Fig. 3D). Mitochondria of RBCs in both the middle (Fig. 3E) and bottom (Fig. 3F) sub-fractions were also swollen. Myelin figures were very frequent in mitochondria from the bottom sub-fraction, whereas, they were rarely detected in the middle sub-fraction erythrocytes.
mean values, nevertheless, most of the control cells present higher membrane potential as seen from clustering of fluorescence channels in Table 2: fluorescence intensities proportional to DCm were also arbitrarily classified into three groups, corresponding to low (0 –695) medium (696 – 780) and high values (781 –1023). The percentages of RBCs in each density sub-fraction of the control and experimental treatments are reported according to their DCm. A large proportion of untreated RBCs in all density sub-fractions showed high levels of DCm. Strong mitochondrial depolarisation was evident after incubation under conditions of oxidative stress. Age-related differences were present: the percentage of cells with mitochondria in a low energetic state increased from 3.6 to 77.6% in young erythrocytes, 1.9 to 84.8% in the middle density fraction and exceeded 95% of the population of older cells. Ebselen at 10 mM distinctly reduced the impact of the oxidative stress. However, the degree of mitigation differed between sub-fractions. The percentage of stressed middle fraction erythrocytes, with mitochondria in a high energetic state, was 30 times higher in samples treated with ebselen. It was only doubled in the bottom fraction. The effect of ebselen was dosedependent in the range 5 –10 mM. The efficacy of
Table 2 Effects of ebselen on DCm in sub-fractions of trout erythrocytes exposed to oxidative stress Fraction (DCm level)
Control pH 7.8/room temperature (%)
pH 7.8/358C (%)
pH 6.3/358C (%)
pH 6.3/358C þ ebselen (%)
Top fraction Low Middle High
3.6 11.9 85.4
23.8 68.2 9.7
77.6 19.8 3.9
4.3 75.3 23.6
Middle fraction Low Middle High
1.9 12.4 86.8
28.8 67.6 5.3
84.8 13.8 2.3
2.2 32.5 69.2
Bottom fraction Low Middle High
6.9 29.9 65.1
23.7 62.1 15.7
95.3 4.2 0.9
19.9 79.8 1.7
Percentages of cells showing low, mid and high membrane potential. Markers were arbitrarily set from the analysis of flow cytometry histograms. Data are presented from top, middle and bottom fraction trout erythrocytes suspended in isotonic buffer pH 7.8 incubated at room temperature, 358C for 1 h, and 358C for 1 h in presence of Ebselen 10 mM.
434
L. Tiano et al. / Mitochondrion 2 (2003) 428–436
Fig. 3. Transmission electronic micrographs of mitochondria from density-separated trout erythrocytes: top (A,D,G), middle (B,E,H) and bottom fractions (C,F,I). Erythrocytes were exposed to oxidative stress: pH 6.3/358C/1 h (D,E,F); the same conditions in the presence of ebselen 10 mM (G,H,I) and unstressed controls: pH 7.8/RT/1 h (A,B,C). N, nucleus; m, mitochondria; pm, plasma membrane, mf, myelin figure. Bar: (B,D,G,C), 4 mm; (A,E,H,F,I), 2,3 mm.
Ebselen did not prevent the swelling of mitochondria of RBCs exposed to oxidative stress (Fig. 3G, I), but myelin figures were not seen in samples treated with ebselen.
4. Discussion According to the ‘free radical theory of ageing’, ageing is caused by the accumulation in the cell of macromolecules, such as DNA, proteins and lipids, that have been damaged by free radicals (Harman, 1956). The superoxide anion ðO2 2 Þ, hydrogen peroxide (H2O2) and hydroxy radical ðOHzÞ are commonly referred to as ROS. They are mainly generated by mitochondria, which consume 90% of the oxygen taken up by the cell (Lee et al., 1997). Although various enzymatic and non-enzymatic systems have been developed by the cell to give protection against oxidative damage caused by ROS, it is generally accepted that the defensive capacity of the cell,
especially of mitochondria against ROS, declines with increasing age (Lenaz et al., 1998). It is known that the density of erythrocyte subpopulations is closely related to ageing (Rennie et al., 1979). Older RBCs have higher density. Under the present experimental conditions, oxidative stress arises in part from the formation of superoxide radicals and, at the same time, from the inactivation of glutathione peroxidase that metabolises H2O2 and lipid peroxides formed by the oxidation of Hb (Grelloni et al., 1991). The main targets of oxidative damage in nucleated trout RBCs are membranes and DNA, as in the case of mammalian cells. The data presented here have demonstrated that oxidative stress increases the intracellular levels of ROS in all sub-fractions of trout RBCs. Ebselen, which acts as a mimic of the antioxidant glutathione peroxidase (Wendel et al., 1984), strongly reduced the intracellular level of ROS in all three RBC subfractions. We have shown previously that the DCm decreases with ageing of RBCs (Tiano et al., 2000),
L. Tiano et al. / Mitochondrion 2 (2003) 428–436
and this has been confirmed in the present study. Our new flow cytometric data indicate that the bottom subfraction (older cells) contained the smallest proportion of RBCs with high levels of DCm, suggesting that oxidative damage to mitochondria is accelerated by ageing. This suggestion was supported by ultrastructural analysis, which revealed the formation of large myelin figures. Myelin figures are unusual structures, normally spheroidal, made up of concentric membranes. They originate from nuclear or mitochondrial membranes, and are believed to have a role in the isolation of impaired cellular components. They are, therefore, an expression of intracytosis and reported to occur under conditions of oxidative stress. Ebselen seems to be very effective in preventing the drastic structural changes induced by oxidative stress in mitochondria of RBCs of the top and middle subfractions. However, its protective effect is reduced in older cells. This may arise from a synergy of antioxidant effects resulting from the addition of the glutathione peroxidase mimic ebselen, and the natural increase in antioxidant defences that is characteristic of the ageing process in this cell type. Previous studies have demonstrated marked differences between human (anucleated) and trout (nucleated) densityseparated erythrocytes. The activity of the primary antioxidant defence system in fish erythrocytes, made up of the enzymes superoxide dismutase, catalase and glutathione peroxidase, increased with the density of the cell fraction (Falcioni et al., 1992). By contrast, it decreased with increasing density in human erythrocytes. This possible synergistic antioxidant effect may fail in older cells, where mitochondria may already be compromised by ageing processes (Tiano et al., 2000), as shown in transmission electronic micrographs. In conclusion, the decrease in the DCm in all sub-fractions RBCs exposed to oxidative stress was markedly mitigated by ebselen. However, ebselen did not prevent the swelling of mitochondria. Further studies are required to resolve the significance of these observations.
Acknowledgements This work was supported by a CNR grant to G.F.
435
References Agarwal, S., Sohal, R.S., 1994. DNA oxidative damage and life expectancy in houseflies. Proc. Natl Acad. Sci. USA 91, 12332–12335. Ames, B.N., Shigenaga, M.K., Hagen, T.M., 1993. Oxidants, antioxidants, and the degenerative diseases of ageing. Proc. Natl Acad. Sci. USA 90, 7915–7922. Camilleri-Broet, S., Vanderwerff, H., Caldwell, E., Hockenbery, D., 1998. Distinct alterations in mitochondrial mass and function characterize different models of apoptosis. Exp. Cell Res. 239, 277 –292. Cerutti, P.A., 1994. Oxy-radicals and cancer. Lancet 344, 862–863. Cossarizza, A., Baccarani-Contri, M., Kalashnikova, G., Franceschi, C., 1993. A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethylbenzimidazolcarbocyanine iodide (JC-1). Biochem. Biophys. Res. Commun. 197, 40–45. Davies, K.J., 1993. Protein modification by oxidants and the role of proteolytic enzymes. Biochem. Soc. Trans. 21, 346–353. Diplock, A.T., 1994. Antioxidants and disease prevention. Mol. Aspects Med. 15, 293– 376. Falcioni, G., Cincola, G., Brunori, M., 1987. Glutathione peroxidase and oxidative hemolysis in trout red blood cells. FEBS Lett. 221, 355–358. Falcioni, G., Grelloni, F., Bonfigli, A.R., Bertoli, E., 1992. Biochemical characterization of density-separated trout erythrocytes. Biochem. Int. 28, 379–384. Garland, J.M., Halestrap, A., 1997. Energy metabolism during apoptosis. Bcl-2 promotes survival in hematopoietic cells induced to apoptose by growth factor withdrawal by stabilizing a form of metabolic arrest. J. Biol. Chem. 272, 4680–4688. Grelloni, F., Gabbianelli, R., Falcioni, G., 1991. Inactivation of glutathione peroxidase following hemoglobin oxidation. Biochem. Int. 25, 789–795. Guyton, K.Z., Kensler, T.W., 1993. Oxidative mechanisms in carcinogenesis. Br. Med. Bull. 49, 523 –544. Halliwell, B., Gutteridge, J.M., Cross, C.E., 1992. Free radicals, antioxidants, and human disease: where are we now? J. Lab. Clin. Med. 119, 598–620. Harman, D., 1956. Ageing, a theory based on free radical and radiation chemistry. J. Gerontol. 11, 298–300. Harman, D., 1992. Free radical theory of ageing. Mutat. Res. 275, 257 –266. Holt, I.J., Harding, A.E., Morgan-Hughes, J.A., 1988. Deletions of muscle mitochondrial DNA in patients with mitochondrial myopathies. Nature 331, 717 –719. Lee, C.M., Weindruch, R., Aiken, J.M., 1997. Age-associated alterations of the mitochondrial genome. Free Radic. Biol. Med. 22, 1259–1269. Lenaz, G., Cavazzoni, M., Genova, M.L., et al., 1998. Oxidative stress, antioxidant defences and ageing. Biofactors 8, 195–204. Lenaz, G., 1998. Role of mitochondria in oxidative stress and ageing. Biochim. Biophys. Acta 1366, 53–67. Linnane, A.W., Marzuki, S., Ozawa, T., Tanaka, M., 1989.
436
L. Tiano et al. / Mitochondrion 2 (2003) 428–436
Mitochondrial DNA mutations as an important contributor to ageing and degenerative diseases. Lancet 1, 642 –645. Mignotte, B., Vayssiere, J.L., 1998. Mitochondria and apoptosis. Eur. J. Biochem. 252, 1–15. Moser, C.C., Keske, J.M., Warncke, K., Farid, R.S., Dutton, P.L., 1992. Nature of biological electron transfer. Nature 355, 796–802. Muller, A., Cadenas, E., Graf, P., Sies, H., 1984. A novel biologically active seleno-organic compound – I. Glutathione peroxidase-like activity in vitro and antioxidant capacity of PZ 51 (Ebselen). Biochem. Pharmacol. 33, 3235–3239. Petit, P.X., Lecoeur, H., Zorn, E., Dauguet, C., Mignotte, B., Gougeon, M.L., 1995. Alterations in mitochondrial structure and function are early events of dexamethasone-induced thymocyte apoptosis. J. Cell Biol. 130, 157 –167. Poot, M., Zhang, Y.Z., Kramer, J.A., et al., 1996. Analysis of mitochondrial morphology and function with novel fixable fluorescent stains. J. Histochem. Cytochem. 44, 1363–1372. Reers, M., Smith, T.W., Chen, L.B., 1991. J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential. Biochemistry 30, 4480–4486. Rennie, C.M., Thompson, S., Parker, A.C., Maddy, A., 1979. Human erythrocyte fraction in ‘Percoll’ density gradients. Clin. Chim. Acta 98, 119–125. Salvioli, S., Ardizzoni, A., Franceschi, C., Cossarizza, A., 1997. JC-1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess DC changes in intact cells: implications for studies on mitochondrial functionality during apoptosis. FEBS Lett. 411, 77– 82.
Schapira, A.H., 1997. Mitochondrial disorders: an overview. J. Bioenerg. Biomembr. 29, 105–107. Skulachev, V.P., 1996. Why are mitochondria involved in apoptosis? Permeability transition pores and apoptosis as selective mechanisms to eliminate superoxide-producing mitochondria and cell. FEBS Lett. 397, 7–10. Smiley, S.T., Reers, M., Mottola-Hartshorn, C., et al., 1991. Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1. Proc. Natl Acad. Sci. USA 88, 3671–3675. Susin, S.A., Zamzami, N., Kroemer, G., 1998. Mitochondria as regulators of apoptosis: doubt no more. Biochim. Biophys. Acta 1366, 151–165. Tiano, L., Ballarini, P., Santoni, G., Wozniak, M., Falcioni, G., 2000. Morphological and functional changes of mitochondria from density separated trout erythrocytes. Biochim. Biophys. Acta 1457, 118–128. Tritschler, H.J., Medori, R., 1993. Mitochondrial DNA alterations as a source of human disorders. Neurology 43, 280–288. Wendel, A., Fausel, M., Safayhi, H., Tiegs, G., Otter, R., 1984. A novel biologically active seleno-organic compound – II. Activity of PZ 51 in relation to glutathione peroxidase. Biochem. Pharmacol. 33, 3241– 3245. Yakes, F.M., Van Houten, B., 1997. Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress. Proc. Natl Acad. Sci. USA 94, 514–519. Zamzami, N., Susin, S.A., Marchetti, P., et al., 1996. Mitochondrial control of nuclear apoptosis. J. Exp. Med. 183, 1533–1544.
Ebselen prevents mitochondrial ageing due to oxidative stress: in vitro study of fish erythrocytes Luca Tianoa,*, Donatella Fedelia, Giorgio Santonib, Ian Daviesc, Takashi Wakabayashid, Giancarlo Falcionia a
Dipartimento di Biologia Molecolare, Cellulare e Animale, Universita` degli Studi di Camerino, Via Camerini 2, I-62032, Camerino (MC), Italy b Department of Pharmacology and Experimental Medicine, University of Camerino, Via Camerini 2, I-62032, Camerino (MC), Italy c Fisheries Research Services, Marine Laboratory, P.O. Box 101, 375 Victoria Road, Aberdeen AB11 9DB, UK d Department of Medical Chemistry, Medical University of Gdansk, 1 Debinki Str., Gdansk, Poland Received 4 December 2002; received in revised form 10 February 2003; accepted 21 February 2003 Dedicated to the memory of Eraldo Antonini, eminent biochemist, prematurely deceased 20 years ago, on March 19th, 1983.
Abstract Nucleated trout erythrocytes under oxidative stress suffer DNA membrane damage and inactivation of glutathione peroxidase. In addition, oxidative damage increases with the age of the cell. In the present paper, we evaluate the effects of oxidative stress and ageing on mitochondrial functionality by means of transmission electron microscopy and cytofluorimetric determination of mitochondrial membrane potential and intracellular levels of reactive oxygen species. The protective activity of the antioxidant organoselenium compound ebselen, a mimic of glutathione peroxidase, is also evaluated. Ebselen prevents the drastic structural and functional changes in mitochondria in aged RBCs induced by oxidative stress. However, the antioxidant does not prevent swelling of the mitochondria. q 2003 Mitochondria Research Society. Published by Elsevier Science B.V. All rights reserved. Keywords: Trout erythrocytes; Mitochondria; Ageing; Oxidative stress; Ebselen; Membrane potential
1. Introduction Cellular sources, notably mitochondria, can generate oxygen radicals via oxidative phosphorylation in which oxygen is reduced to water through a four-step addition of electrons (Moser et al., 1992; Davies, 1993). The reactive oxygen species (ROS) generated * Corresponding author. Tel.: þ 39-0737-403213; fax: þ 39-0737-636216. E-mail address: [email protected] (L. Tiano).
by incomplete reduction of oxygen are extremely reactive and can interact with cellular lipids, proteins and nucleic acids, impairing their structure and function. Cells have defence systems to prevent injury by these substances. An imbalance between free radical-generating and -scavenging systems is commonly referred to as oxidative stress. Oxidative stress may be a cause or consequence of various diseases such as diabetes, atherosclerosis, cancer, Parkinson’s disease or Alzheimer’s disease (Halliwell et al., 1992; Ames et al., 1993; Swart et al., 1972; Guyton and
1567-7249/03/$20.00 q 2003 Mitochondria Research Society. Published by Elsevier Science B.V. All rights reserved. doi:10.1016/S1567-7249(03)00032-1
L. Tiano et al. / Mitochondrion 2 (2003) 428–436
Kensler, 1993; Cerrutti et al., 1994; Harman, 1992). Environmental factors such as a poor diet, tobacco smoke, ozone and various types of xenobiotics may be closely linked to these disease conditions via oxidative stress. Finally, oxidative stress can be a result of senescence processes that are commonly accompanied by a progressive impairment of antioxidant systems (Harman, 1956; Linnane et al., 1989; Agarwal and Sohal, 1994; Lenaz, 1998; Diplock, 1994). Thus, according to the ‘free radical theory of ageing’ proposed by Harman (1956), oxidative stress has been postulated as one of the main factors leading to ageing. Mitochondria play a key role in the oxidative stress processes and ageing, due to their functions in cellular respiration processes that make them simultaneously a source and the final target of free radicals. It has been calculated that 90% of the oxygen taken up by the cell is consumed by mitochondria, and that 2% of the oxygen consumed by mitochondria is converted into the superoxide anion (Lee et al., 1997). Recent advances in molecular biology have disclosed a new function of mitochondria. They are endowed with several important factors controlling the processes of programmed cell death (Mignotte and Vayssiere, 1998; Skulachev, 1996; Petit et al., 1995; Zamzami et al., 1996; Camilleri-Broet et al., 1998; Yakes and Van Houten, 1997; Holt et al., 1988; Susin et al., 1998). Moreover, it is well known that mitochondrial DNA (mtDNA) is more susceptible than genomic DNA (Tritschler and Medori, 1993) to the mutations that are responsible for several diseases (Schapira, 1997). Thus, any mitochondrial lesion, primary or secondary, may trigger a damaging cycle of oxidative stress and mtDNA mutations: a defect in the mitochondrial respiratory chain is followed by the release of mutagenic oxidants, somatic mutations and defects in mitochondrial proteins, thereby amplifying the original error. Nucleated trout red blood cells (RBCs) are a very useful cellular model for the study of oxidative damage associated with oxidative stress and senescence processes (Falcioni et al., 1987; Tiano et al., 2000). RBCs of lower vertebrates differ from those in mammals in that they retain both nucleus and mitochondria after the reticulocyte stage. Also, it is possible to induce a condition of endogenous oxidative stress
429
by promoting haemoglobin autoxidation, leading to met-Hb and formation of ROS. An imbalance in oxidant species achieved in this way is likely to simulate pathophysiological conditions of oxidative stress observed in vivo in response to exogenous oxidative insult. Finally, trout RBCs increase in density with age, and can be separated into three distinct populations (sub-fractions) along a discontinuous density gradient (45 – 65% Percoll). Recently, we assessed the mitochondrial membrane potential (DCm) of each RBC sub-fraction by flow cytometry using JC-1 as a fluorescent probe. We found that the DCm of trout RBCs decreases in parallel with increases in their density (Tiano et al., 2000). The present study is concerned with morphological and functional changes of mitochondria in trout RBCs exposed to oxidative stress states, focusing on possible differences in their behaviour depending on the age of the cells. The possible susceptibility of trout RBCs of different ages to oxidative injury related to senescence processes was assessed. The protective effects of the organoselenium compound ebselen (2-phenyl-1,2-benzisoselenazol-3-(2H)-on) against oxidative damage in mitochondria was also evaluated. Ebselen can minimise the deleterious effects of ROS by mimicking the antioxidant enzyme glutathione peroxidase (Muller et al., 1984; Wendel et al., 1984).
2. Materials and methods 2.1. Preparation of samples Specimens of Salmo irideus (, 24 months old, 180– 300 g weight), an inbred strain of rainbow trout, were kept in tanks containing water from Scarsito River, Italy and fed with a commercial fish diet. Blood was withdrawn by syringe from the lateral tail vein into an isotonic medium (0.1 M phosphate buffer, 0.1 M NaCl, 0.2% citrate, 1 mM ethylenediaminetetraacetic acid (EDTA), pH 7.8) and was treated further within 2 h at 48C. After the removal of the plasma and buffy coat, erythrocytes were washed three times with the same isotonic buffer. The RBCs were separated into three sub-populations on a Percoll/BSA density gradient (45 – 65% Percoll, Rennie et al., 1979). The three RBC fractions obtained were washed
430
L. Tiano et al. / Mitochondrion 2 (2003) 428–436
three times and finally suspended in phosphate buffer (pH 6.3) and incubated at 358C for 1 h. Under these conditions, oxidative stress is promoted by inactivation of glutathione peroxidase and acceleration of met-Hb formation, with subsequent formation of ROS. Parallel experiments were conducted by incubating the cells at the same temperature and pH in the presence of ebselen at concentrations ranging from 5 to 30 mM. Control experiments were performed by incubating RBCs with phosphate buffer pH 7.8 at room temperature. 2.2. Flow cytometric analysis of intracellular levels of free radicals Trout RBCs were suspended in isotonic phosphate buffer for 60 min at pH 6.3 at a concentration of 5 £ 106 cells/ml (stressed RBCs). Control RBCs were suspended in the same buffer at pH 7.8. They were then stained with 5 mM carboxy-H2-DCFDA to detect intracellular levels of ROS, essentially, according to the method of Garland and Halestrap (1997). Cells from each sub-fraction were then washed and resuspended in the isotonic phosphate buffer at pH 7.4 at a concentration of 1 £ 106 cells/ml, and immediately submitted to analysis using a fluorescence-activated cell sorter scan (FACSCAN) flow cytometer, Becton, Dickinson, Mountain View, CA equipped with a single 488 nm argon laser. Fluorescence emission (FL1) is proportional to the oxidised form of carboxy-H2-DCFDA, and is proportional to intracellular levels of ROS. A minimum of 13,000 cells per sample was analysed using WINMDI software on an IBM compatible computer. 2.3. Flow cytometric analysis of DCm After being exposed to oxidative stress, suspensions of RBCs were adjusted to a concentration of 1.5 £ 105 cells/ml, and incubated with JC-1 at a concentration of 10 mg/ml for 10 min at room temperature in the dark to detect DCm. JC-1 is a mitochondria-specific fluorescent probe that incorporates into the charged membrane according to Nernst’s Law (Poot et al., 1996; Salvioli et al., 1997; Smiley et al., 1991; Reers et al., 1991; Cossarizza et al., 1993). JC-1 has advantages over other potentialsensitive probes, such as rhodamine and other
carbocyanines, in that, it changes colour from green to orange as the membrane potential increases over values of about 80 – 100 mV. This property arises from the reversible formation of JC-1 aggregates upon membrane polarisation that causes shifts in the wavelength of the emitted light from 520 (emission by monomeric JC-1) to 590 nm (emission by aggregated JC-1). A suspension of 1 £ 105 cells/ml from each subfraction was also analysed for relative fluorescence intensity. The filter in front of the fluorescence 1 (FL1) photomultiplier transmits (PMT) at 530 nm, and the filter used in the FL2 channel transmits at 617 nm. The values of PMT were set logarithmically. Red fluorescence (FL2) corresponds to the aggregated form of JC-1 and is proportional to DCm. Compensation FL1–FL2 was 4% and compensation FL2–FL1 was 9.5%. A minimum of 13,000 cells per sample was acquired and analysed using WINMD1 2.8 on an IBM compatible computer. Mean fluorescence intensities were statistically analysed for significant differences using Student’s t-test. 2.4. Transmission electron microscopy Aliquots of RBC sub-fractions submitted to oxidative stress in the presence and absence of 10 mM ebselen were fixed for 4 h at 48C in a fixative containing 2.5% glutaraldehyde and 0.1 M sodium cacodylate, pH 7.4. They were postfixed for 1 h at room temperature in 1% osmium tetroxide dissolved in distilled water. Fixed cells were then dehydrated in a graded series of ethanol solutions and embedded in TAAB-embedding resin (medium grade). Ultrathin sections were cut on a Reichert – Jung Ultracut ultramicrotome and stained with uranyl acetate and lead citrate. Stained sections were examined in a Philips 301 transmission electron microscope operated at 80 kV. 2.5. Chemicals All reagents were of analytical grade: Percoll, 5amino-2,3-dihydro-1,4-phthalazinedione (Ebselen) were purchased from Sigma, St. Louis, MO. Ebselen was stored at 2 208C and dissolved in ethanol as 2 mM stock solution just before use. Carboxy-H2DCFDA and JC-1 were purchased from Molecular
L. Tiano et al. / Mitochondrion 2 (2003) 428–436
Probes, Eugene, OR and stored at 2 208C as 1 mM stock solution in dimethyl sulfoxide (DMSO).
3. Results Intracellular levels of ROS in RBCs incubated in isotonic phosphate buffer at pH 6.2/358C (Fig. 1 and Table 1) were strongly elevated compared to those in the control. On the contrary, after incubation at physiological pH and 358C, intracellular levels of ROS were comparable with the control cells incubated at room temperature. These data are in accordance with previous literature reports describing the induction of oxidative stress in trout erythrocytes by variation of pH and temperature that involve inhibition of glutathione peroxidase activity and acceleration of the autoxidation of haemoglobin (Falcioni et al., 1992). Autoxidation is the spontaneous oxidation of Hb (haeme iron present as FeII) to met-Hb (haeme iron as FeIII). Superoxide is a reaction product. ROS formation was revealed by the specific oxidation of the fluorochrome carboxy-H2DCFDA. Positive control experiments were performed in order to assess the validity of the test in this experimental model by exposing DCFDA-stained
431
erythrocyte to hydrogen peroxide 5 mM. FACSCAN analysis revealed a time-dependent increase in the fluorescence signal (data not shown). Fig. 1 shows the mean fluorescence intensities of 10,000 measurements, indicating a highly significant rise in fluorescence intensities in erythrocytes of the three age sub-fractions submitted to oxidative stress by incubation at pH 6.3 at 358C ðP , 0:0001Þ. The data showed a bimodal distribution, and, therefore, the 1023 fluorescence channels, proportional to intracellular levels of ROS, were arbitrarily classified into three groups according to low (0 – 295), medium (296 –550) and high levels (551 – 1023) of ROS. This made it possible to estimate the percentage distribution of cells with different fluorescent intensity of carboxy-DCF in each sub-fraction of both control and experimental groups. As shown in Table 1, the percentage of the cells with high intracellular level of ROS was remarkably greater, over 2500-fold in comparison to unstressed controls, in all three subfractions of RBCs exposed to oxidative stress. By contrast, the percentage of the cells with high intracellular level of ROS was lower in all three sub-fractions exposed to oxidative stress in the presence of ebeselen, and was similar to those to those in controls. The lowest effective concentration
Fig. 1. Analysis of DCF fluorescence, proportional to intracellular ROS levels, in flow cytometry of density-separated trout erythrocytes in untreated controls incubated at pH 7.8 both at room temperature and 358C, in conditions of oxidative stress (pH 6.3, 358C) and in the same conditions in the presence of ebselen 10 mM. All three, age sub-fractions are reported: top (white), middle (grey) and bottom (black). Data are expressed as median fluorescence intensities ^ standard deviation *P , 0:0001.
432
L. Tiano et al. / Mitochondrion 2 (2003) 428–436
Table 1 Effects of ebselen on intracellular levels of ROS in sub-fractions of trout erythrocytes exposed to oxidative stress Fraction (ROS level)
Control pH 7.8/room temperature (%)
pH 7.8/358C (%)
pH 6.3/358C (%)
pH 6.3/358C þ ebselen (%)
Top fraction Low Middle High
91.96 7.5 0.03
90.52 8.8 0.6
15.86 8.86 75.4
99.0 0.05 0.05
Middle fraction Low Middle High
95.96 4.72 0.02
92.43 6.82 0.75
13.32 10.72 75.96
99.92 0.04 0.04
Bottom fraction Low Middle High
97.18 1.41 0.03
95.46 3.92 0.62
11.86 10.98 77.16
99.15 0.18 0.67
Percentages of cells presenting low, mid and high intracellular levels of ROS. Markers were arbitrarily set from the analysis of flow cytometry histograms. Data are presented from top, middle and bottom fraction trout erythrocytes, suspended in isotonic buffer pH 7.8 incubated at room temperature, 358C for 1 h and 358C for 1 h in presence of ebselen 10 mM.
of ebselen was 10 mM. No significant differences were detected between 10 and 30 mM (data not shown). The mean intensities of fluorescence proportional to DCm are reported in Fig. 2. Incubation at pH 7.8 and 358C resulted in a non-significant mitochondrial depolarisation, nevertheless, incubation under conditions of oxidative stress induced a dramatic
mitochondrial depolarisation in all three age subfractions ðP , 0:0001Þ and incubation in presence of ebselen 10 mM significantly reduced the degree of depolarisation ðP , 0:0001Þ. Mean fluorescence intensities of ebselen treated erythrocytes are significantly higher than control cells in all three subpopulation. This is due to a broader distribution of the fluorescence intensities in control cells around the
Fig. 2. Analysis of JC-1 red fluorescence, proportional to DCm, in flow cytometry of density-separated trout erythrocytes in untreated controls incubated at pH 7.8 both at room temperature and 358C, in conditions of oxidative stress (pH 6.3, 358C) and in the same conditions in the presence of ebselen 10 mM. All three, age sub-fractions are reported: top (white), middle (grey) and bottom (black). Data are expressed as median fluorescence intensities ^ standard deviation *P , 0:0001.
L. Tiano et al. / Mitochondrion 2 (2003) 428–436
433
ebselen in maintaining elevated DCm was slightly reduced at 30 mM (data not shown). Structural changes in the mitochondria of RBCs exposed to oxidative stress, and the protective effects of ebselen, were investigated by electron microscopy. Top fraction RBCs in the control contained a few mitochondria elongated to various degrees scattered in the cytoplasm (Fig. 3A). The cristae of these mitochondria were aligned perpendicular to their long axes. On the other hand, mitochondria in the middle and bottom subfractions were round or oval, with cristae confined to the periphery (Fig. 3B, C, respectively). This may suggest that they were functionally impaired to some extent. Previous observations have shown that mitochondria in trout RBCs decline in number and functionality with ageing (Tiano et al., 2000). When RBCs in each sub-fraction were exposed to oxidative stress, marked structural changes were always observed in the mitochondria. Mitochondria in RBCs in the top fraction became distinctly swollen and the cristae were distorted (Fig. 3D). Mitochondria of RBCs in both the middle (Fig. 3E) and bottom (Fig. 3F) sub-fractions were also swollen. Myelin figures were very frequent in mitochondria from the bottom sub-fraction, whereas, they were rarely detected in the middle sub-fraction erythrocytes.
mean values, nevertheless, most of the control cells present higher membrane potential as seen from clustering of fluorescence channels in Table 2: fluorescence intensities proportional to DCm were also arbitrarily classified into three groups, corresponding to low (0 –695) medium (696 – 780) and high values (781 –1023). The percentages of RBCs in each density sub-fraction of the control and experimental treatments are reported according to their DCm. A large proportion of untreated RBCs in all density sub-fractions showed high levels of DCm. Strong mitochondrial depolarisation was evident after incubation under conditions of oxidative stress. Age-related differences were present: the percentage of cells with mitochondria in a low energetic state increased from 3.6 to 77.6% in young erythrocytes, 1.9 to 84.8% in the middle density fraction and exceeded 95% of the population of older cells. Ebselen at 10 mM distinctly reduced the impact of the oxidative stress. However, the degree of mitigation differed between sub-fractions. The percentage of stressed middle fraction erythrocytes, with mitochondria in a high energetic state, was 30 times higher in samples treated with ebselen. It was only doubled in the bottom fraction. The effect of ebselen was dosedependent in the range 5 –10 mM. The efficacy of
Table 2 Effects of ebselen on DCm in sub-fractions of trout erythrocytes exposed to oxidative stress Fraction (DCm level)
Control pH 7.8/room temperature (%)
pH 7.8/358C (%)
pH 6.3/358C (%)
pH 6.3/358C þ ebselen (%)
Top fraction Low Middle High
3.6 11.9 85.4
23.8 68.2 9.7
77.6 19.8 3.9
4.3 75.3 23.6
Middle fraction Low Middle High
1.9 12.4 86.8
28.8 67.6 5.3
84.8 13.8 2.3
2.2 32.5 69.2
Bottom fraction Low Middle High
6.9 29.9 65.1
23.7 62.1 15.7
95.3 4.2 0.9
19.9 79.8 1.7
Percentages of cells showing low, mid and high membrane potential. Markers were arbitrarily set from the analysis of flow cytometry histograms. Data are presented from top, middle and bottom fraction trout erythrocytes suspended in isotonic buffer pH 7.8 incubated at room temperature, 358C for 1 h, and 358C for 1 h in presence of Ebselen 10 mM.
434
L. Tiano et al. / Mitochondrion 2 (2003) 428–436
Fig. 3. Transmission electronic micrographs of mitochondria from density-separated trout erythrocytes: top (A,D,G), middle (B,E,H) and bottom fractions (C,F,I). Erythrocytes were exposed to oxidative stress: pH 6.3/358C/1 h (D,E,F); the same conditions in the presence of ebselen 10 mM (G,H,I) and unstressed controls: pH 7.8/RT/1 h (A,B,C). N, nucleus; m, mitochondria; pm, plasma membrane, mf, myelin figure. Bar: (B,D,G,C), 4 mm; (A,E,H,F,I), 2,3 mm.
Ebselen did not prevent the swelling of mitochondria of RBCs exposed to oxidative stress (Fig. 3G, I), but myelin figures were not seen in samples treated with ebselen.
4. Discussion According to the ‘free radical theory of ageing’, ageing is caused by the accumulation in the cell of macromolecules, such as DNA, proteins and lipids, that have been damaged by free radicals (Harman, 1956). The superoxide anion ðO2 2 Þ, hydrogen peroxide (H2O2) and hydroxy radical ðOHzÞ are commonly referred to as ROS. They are mainly generated by mitochondria, which consume 90% of the oxygen taken up by the cell (Lee et al., 1997). Although various enzymatic and non-enzymatic systems have been developed by the cell to give protection against oxidative damage caused by ROS, it is generally accepted that the defensive capacity of the cell,
especially of mitochondria against ROS, declines with increasing age (Lenaz et al., 1998). It is known that the density of erythrocyte subpopulations is closely related to ageing (Rennie et al., 1979). Older RBCs have higher density. Under the present experimental conditions, oxidative stress arises in part from the formation of superoxide radicals and, at the same time, from the inactivation of glutathione peroxidase that metabolises H2O2 and lipid peroxides formed by the oxidation of Hb (Grelloni et al., 1991). The main targets of oxidative damage in nucleated trout RBCs are membranes and DNA, as in the case of mammalian cells. The data presented here have demonstrated that oxidative stress increases the intracellular levels of ROS in all sub-fractions of trout RBCs. Ebselen, which acts as a mimic of the antioxidant glutathione peroxidase (Wendel et al., 1984), strongly reduced the intracellular level of ROS in all three RBC subfractions. We have shown previously that the DCm decreases with ageing of RBCs (Tiano et al., 2000),
L. Tiano et al. / Mitochondrion 2 (2003) 428–436
and this has been confirmed in the present study. Our new flow cytometric data indicate that the bottom subfraction (older cells) contained the smallest proportion of RBCs with high levels of DCm, suggesting that oxidative damage to mitochondria is accelerated by ageing. This suggestion was supported by ultrastructural analysis, which revealed the formation of large myelin figures. Myelin figures are unusual structures, normally spheroidal, made up of concentric membranes. They originate from nuclear or mitochondrial membranes, and are believed to have a role in the isolation of impaired cellular components. They are, therefore, an expression of intracytosis and reported to occur under conditions of oxidative stress. Ebselen seems to be very effective in preventing the drastic structural changes induced by oxidative stress in mitochondria of RBCs of the top and middle subfractions. However, its protective effect is reduced in older cells. This may arise from a synergy of antioxidant effects resulting from the addition of the glutathione peroxidase mimic ebselen, and the natural increase in antioxidant defences that is characteristic of the ageing process in this cell type. Previous studies have demonstrated marked differences between human (anucleated) and trout (nucleated) densityseparated erythrocytes. The activity of the primary antioxidant defence system in fish erythrocytes, made up of the enzymes superoxide dismutase, catalase and glutathione peroxidase, increased with the density of the cell fraction (Falcioni et al., 1992). By contrast, it decreased with increasing density in human erythrocytes. This possible synergistic antioxidant effect may fail in older cells, where mitochondria may already be compromised by ageing processes (Tiano et al., 2000), as shown in transmission electronic micrographs. In conclusion, the decrease in the DCm in all sub-fractions RBCs exposed to oxidative stress was markedly mitigated by ebselen. However, ebselen did not prevent the swelling of mitochondria. Further studies are required to resolve the significance of these observations.
Acknowledgements This work was supported by a CNR grant to G.F.
435
References Agarwal, S., Sohal, R.S., 1994. DNA oxidative damage and life expectancy in houseflies. Proc. Natl Acad. Sci. USA 91, 12332–12335. Ames, B.N., Shigenaga, M.K., Hagen, T.M., 1993. Oxidants, antioxidants, and the degenerative diseases of ageing. Proc. Natl Acad. Sci. USA 90, 7915–7922. Camilleri-Broet, S., Vanderwerff, H., Caldwell, E., Hockenbery, D., 1998. Distinct alterations in mitochondrial mass and function characterize different models of apoptosis. Exp. Cell Res. 239, 277 –292. Cerutti, P.A., 1994. Oxy-radicals and cancer. Lancet 344, 862–863. Cossarizza, A., Baccarani-Contri, M., Kalashnikova, G., Franceschi, C., 1993. A new method for the cytofluorimetric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,50 ,6,60 -tetrachloro-1,10 ,3,30 -tetraethylbenzimidazolcarbocyanine iodide (JC-1). Biochem. Biophys. Res. Commun. 197, 40–45. Davies, K.J., 1993. Protein modification by oxidants and the role of proteolytic enzymes. Biochem. Soc. Trans. 21, 346–353. Diplock, A.T., 1994. Antioxidants and disease prevention. Mol. Aspects Med. 15, 293– 376. Falcioni, G., Cincola, G., Brunori, M., 1987. Glutathione peroxidase and oxidative hemolysis in trout red blood cells. FEBS Lett. 221, 355–358. Falcioni, G., Grelloni, F., Bonfigli, A.R., Bertoli, E., 1992. Biochemical characterization of density-separated trout erythrocytes. Biochem. Int. 28, 379–384. Garland, J.M., Halestrap, A., 1997. Energy metabolism during apoptosis. Bcl-2 promotes survival in hematopoietic cells induced to apoptose by growth factor withdrawal by stabilizing a form of metabolic arrest. J. Biol. Chem. 272, 4680–4688. Grelloni, F., Gabbianelli, R., Falcioni, G., 1991. Inactivation of glutathione peroxidase following hemoglobin oxidation. Biochem. Int. 25, 789–795. Guyton, K.Z., Kensler, T.W., 1993. Oxidative mechanisms in carcinogenesis. Br. Med. Bull. 49, 523 –544. Halliwell, B., Gutteridge, J.M., Cross, C.E., 1992. Free radicals, antioxidants, and human disease: where are we now? J. Lab. Clin. Med. 119, 598–620. Harman, D., 1956. Ageing, a theory based on free radical and radiation chemistry. J. Gerontol. 11, 298–300. Harman, D., 1992. Free radical theory of ageing. Mutat. Res. 275, 257 –266. Holt, I.J., Harding, A.E., Morgan-Hughes, J.A., 1988. Deletions of muscle mitochondrial DNA in patients with mitochondrial myopathies. Nature 331, 717 –719. Lee, C.M., Weindruch, R., Aiken, J.M., 1997. Age-associated alterations of the mitochondrial genome. Free Radic. Biol. Med. 22, 1259–1269. Lenaz, G., Cavazzoni, M., Genova, M.L., et al., 1998. Oxidative stress, antioxidant defences and ageing. Biofactors 8, 195–204. Lenaz, G., 1998. Role of mitochondria in oxidative stress and ageing. Biochim. Biophys. Acta 1366, 53–67. Linnane, A.W., Marzuki, S., Ozawa, T., Tanaka, M., 1989.
436
L. Tiano et al. / Mitochondrion 2 (2003) 428–436
Mitochondrial DNA mutations as an important contributor to ageing and degenerative diseases. Lancet 1, 642 –645. Mignotte, B., Vayssiere, J.L., 1998. Mitochondria and apoptosis. Eur. J. Biochem. 252, 1–15. Moser, C.C., Keske, J.M., Warncke, K., Farid, R.S., Dutton, P.L., 1992. Nature of biological electron transfer. Nature 355, 796–802. Muller, A., Cadenas, E., Graf, P., Sies, H., 1984. A novel biologically active seleno-organic compound – I. Glutathione peroxidase-like activity in vitro and antioxidant capacity of PZ 51 (Ebselen). Biochem. Pharmacol. 33, 3235–3239. Petit, P.X., Lecoeur, H., Zorn, E., Dauguet, C., Mignotte, B., Gougeon, M.L., 1995. Alterations in mitochondrial structure and function are early events of dexamethasone-induced thymocyte apoptosis. J. Cell Biol. 130, 157 –167. Poot, M., Zhang, Y.Z., Kramer, J.A., et al., 1996. Analysis of mitochondrial morphology and function with novel fixable fluorescent stains. J. Histochem. Cytochem. 44, 1363–1372. Reers, M., Smith, T.W., Chen, L.B., 1991. J-aggregate formation of a carbocyanine as a quantitative fluorescent indicator of membrane potential. Biochemistry 30, 4480–4486. Rennie, C.M., Thompson, S., Parker, A.C., Maddy, A., 1979. Human erythrocyte fraction in ‘Percoll’ density gradients. Clin. Chim. Acta 98, 119–125. Salvioli, S., Ardizzoni, A., Franceschi, C., Cossarizza, A., 1997. JC-1, but not DiOC6(3) or rhodamine 123, is a reliable fluorescent probe to assess DC changes in intact cells: implications for studies on mitochondrial functionality during apoptosis. FEBS Lett. 411, 77– 82.
Schapira, A.H., 1997. Mitochondrial disorders: an overview. J. Bioenerg. Biomembr. 29, 105–107. Skulachev, V.P., 1996. Why are mitochondria involved in apoptosis? Permeability transition pores and apoptosis as selective mechanisms to eliminate superoxide-producing mitochondria and cell. FEBS Lett. 397, 7–10. Smiley, S.T., Reers, M., Mottola-Hartshorn, C., et al., 1991. Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1. Proc. Natl Acad. Sci. USA 88, 3671–3675. Susin, S.A., Zamzami, N., Kroemer, G., 1998. Mitochondria as regulators of apoptosis: doubt no more. Biochim. Biophys. Acta 1366, 151–165. Tiano, L., Ballarini, P., Santoni, G., Wozniak, M., Falcioni, G., 2000. Morphological and functional changes of mitochondria from density separated trout erythrocytes. Biochim. Biophys. Acta 1457, 118–128. Tritschler, H.J., Medori, R., 1993. Mitochondrial DNA alterations as a source of human disorders. Neurology 43, 280–288. Wendel, A., Fausel, M., Safayhi, H., Tiegs, G., Otter, R., 1984. A novel biologically active seleno-organic compound – II. Activity of PZ 51 in relation to glutathione peroxidase. Biochem. Pharmacol. 33, 3241– 3245. Yakes, F.M., Van Houten, B., 1997. Mitochondrial DNA damage is more extensive and persists longer than nuclear DNA damage in human cells following oxidative stress. Proc. Natl Acad. Sci. USA 94, 514–519. Zamzami, N., Susin, S.A., Marchetti, P., et al., 1996. Mitochondrial control of nuclear apoptosis. J. Exp. Med. 183, 1533–1544.