- Email: [email protected]
Copper-induced oxidative damage on astrocytes: protective effect exerted by human high density lipoproteins
Biochimica et Biophysica Acta 1635 (2003) 48 – 54 www.bba-direct.com
Copper-induced oxidative damage on astrocytes: protective effect exerted by human high density lipoproteins Gianna Ferretti, Tiziana Bacchetti *, Cinzia Moroni, Arianna Vignini, Giovanna Curatola Istituto di Biochimica, Facolta` of Medicina, Universita` Politecnica delle Marche, Via Ranieri, I-60131 Ancona, Italy Received 5 June 2003; received in revised form 7 October 2003; accepted 15 October 2003
Abstract In the present study, we confirmed that copper ions induce oxidative damage in human astrocytes in culture, as demonstrated by the significant increase in the levels of hydroperoxides and in the fluorescence intensity of the fluorescent probe dichloro-dihydrofluorescein diacetate (H2DCFDA). The compositional changes were associated with a significant decrease in cell viability in astrocytes treated with 10 AM Cu+ + with respect to control cells. Astrocytes incubated with copper ions in the presence of high density lipoproteins (HDL) isolated from plasma of normolipemic subjects showed lower levels of hydroperoxides and a higher cell viability with respect to cells oxidized alone. Moreover, a significant decrease in the levels of hydroperoxides was observed in oxidized astrocytes treated with HDL. These results demonstrate that HDL exert a protective role against lipid peroxidation. The protective effect could be related to the ability of HDL to bind metal ions at the lipoprotein surface and/or to a stimulation of the efflux of lipid hydroperoxides from cell membranes as demonstrated in other cell types. Oxidative damage of astrocytes was induced at a copper concentration similar to that observed in cerebrospinal fluid (CSF) of patients affected by neurodegenerative diseases such as Alzheimer’s (AD) and Parkinson’s diseases (PD). Lipoprotein particles similar for density and chemical composition to plasma HDL were recently isolated in human CSF, therefore, the protective role exerted by HDL against Cu+ +induced oxidative damage of astrocytes could be of physiological relevance. D 2003 Elsevier B.V. All rights reserved. Keywords: Astrocyte; Copper; High density lipoprotein; Lipid peroxidation; 2V,7V-dichloro-dihydrofluorescein diacetate (H2DCFDA); Neurodegenerative disease
1. Introduction Brain is particularly susceptible to oxidative stress and several studies have demonstrated that copper-induced oxidative damage is involved in the pathogenesis of many neurodegenerative diseases such as Alzheimer’s disease (AD), Creutzfeldt –Jakob disease and amyotrophic lateral sclerosis [1,2] and other oxidative stress-related conditions [3 –5]. Recent studies have demonstrated that astrocytes are chiefly responsible for the antioxidant defence of brain [6,7]. Therefore, the sensitivity of astrocytes to oxidative damage triggered in different experimental conditions has been previously investigated [8 –10]. Changes in antioxidant
* Corresponding author. Tel.: +39-71-220-4673; fax: +39-71-220-4398. E-mail address: [email protected] (T. Bacchetti). 1388-1981/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2003.10.005
enzyme expression in response to hydrogen peroxide have been previously observed in astrocytes [7,10] and it has been suggested that these modifications may contribute to protect cells against oxidative insults. However, the increase in antioxidant enzymes was not sufficient to avoid oxidative damage and lipid peroxidation in cortical astrocyte cultures [7]. Therefore, defence mechanisms in the extracellular space could play a key role in protecting brain cells against oxidative damage and cytotoxic molecules in conditions in which intracellular defences fail. Previous studies have demonstrated that high density lipoproteins (HDL) exert a protective role against lipid peroxidation induced by different agents on plasma lipoproteins [11,12] and cells in culture [13,14]. Lipoprotein particles similar for density and chemical composition to plasma HDL have been isolated in human cerebrospinal fluid (CSF) [15,16] and recent studies demonstrated that astrocytes are involved in their synthesis [17].
G. Ferretti et al. / Biochimica et Biophysica Acta 1635 (2003) 48–54
The lipid and apoprotein composition of human CSF-lipoproteins has been well characterized, but their functions have not been clearly elucidated. It was suggested that they could be involved in the regulation of lipid homeostasis in the central nervous system (CNS) [16] and/or exert a role in the development of neurodegenerative diseases [18]. In our previous study [13], we demonstrated that astrocytes, preincubated with HDL isolated from human plasma, were less susceptible to oxidative damage induced by copper ions at concentrations similar to those observed in CSF of patients affected by neurodegenerative diseases [19,20]. The aim of this study was to further investigate the protective effect exerted by HDL against oxidative damage of brain cells and the molecular mechanisms involved. Therefore, using two different experimental models of astrocytes in culture, we studied the effect of HDL isolated from plasma of normolipemic subjects against astrocyte lipid peroxidation triggered by copper ions.
2. Materials and methods 2.1. Chemicals Dulbecco’s modified Eagle’s medium (DMEM), phenol red-free-DMEM, fetal bovine serum, penicillin/streptomycin solution (10,000 units and 10 mg streptomycin/ml in 0.9% NaCl), L-glutamine (200 mM), ethylenediamine tetraacetic acid (EDTA), trypsin –EDTA solution (10 ), phosphate buffer solution (PBS) and trypan blue (0.4%) were purchased from Sigma Chemical Company (St. Louis, MO). The fluorogenic 2,7-dichloro-dihydrofluorescein diacetate (H2DCFDA) probe was obtained from Molecular Probes, Inc. (Eugene, OR). 2.2. Preparation of HDL Blood from healthy normolipidemic volunteers was obtained by venipuncture. Plasma was prepared by centrifugation at 3000 rpm for 20 min and thereafter used for the preparation of HDL (1.063 –1.21 g/ml) by single vertical spin ultracentrifugation for 1.30 h as described by Chung et al. [21]. After dialysis at 4 jC for 24 h against 5 mM PBS (pH 7.4), the protein concentration of HDL was determined by the method of Lowry et al. [22]. HDL were sterilized on a 0.2 Am Millipore membrane before being incubated with astrocytes. 2.3. Cell culture U 373 MG Human astrocytomas (a gift from Prof. L. Mazzanti-Ancona, Italy) were grown in phenol red-freecomplete-DMEM (containing 10% fetal bovine serum, 1% L-glutamine (200 mM) and 1% penicillin/streptomycin), at 37 jC in a humidified atmosphere of 5% CO2. After 2 or 3 days, cells reached 90 –95% confluence as observed by
49
a light microscope (10 ) and were used for the experiments [23]. 2.4. Oxidative treatment of astrocytes Oxidative treatment of astrocytes was carried out using CuSO4 as oxidizing agent. Briefly, astrocytes were incubated at 37 jC in phenol red-free-complete-DMEM containing 10 AM CuSO4, for different times (4 and 17 h) [13] (CuOX-astrocytes). At the end of the incubation, cells and culture media were used for determination of cell viability and evaluation of oxidative damage. 2.5. Incubation of astrocytes with HDL The protective effect exerted by HDL against Cu+ +induced oxidation of cultured astrocytes was investigated using two different experimental models. 2.5.1. Experimental model A Astrocytes were incubated in phenol-free-completeDMEM (at 37 jC and 5% CO2) for 4 h with 10 AM CuSO4 in the presence or in the absence of different concentrations of HDL (50 – 200 Ag/ml). At the end of the incubation, levels of lipid hydroperoxides were evaluated in culture media and cells. In some experiments, HDL were incubated in the absence or in the presence of 10 AM Cu+ + in the same experimental conditions used for cells and were used for determination of the levels of lipid hydroperoxides. 2.5.2. Experimental model B Astrocytes were incubated in phenol-free-completeDMEM (at 37 jC and 5% CO2) with 10 AM CuSO4 for 17 h. After oxidative treatment, media were removed and cells were washed for three times with 5 mM PBS pH 7.4. Thereafter, cells were resuspended in phenol-free-complete-DMEM (at 37 jC and 5% CO2) and incubated for 4 h in the absence or in the presence of different concentrations of HDL (50 – 200 Ag/ml). At the end of the incubation, HDL were removed from astrocytes and used to evaluate the levels of lipid hydroperoxides. In some experiments, HDL were incubated for 4 h in the same conditions used for cells and were used for determination of the levels of lipid hydroperoxides. 2.6. Evaluation of lipid peroxidation in astrocytes and in HDL The extent of lipid peroxidation in astrocytes, culture media and in HDL treated in different experimental conditions, was evaluated by measuring the levels of hydroperoxides using the FOX-assay as previously described [13]. The levels of hydroperoxides in cells were evaluated by adding the FOX reagent (100 AM xylenol orange, 250 AM Fe+ +, 25 mM H2SO4 and 4 mM BHT in 90% methanol) (1350 Al) to astrocytes previously pelleted and resuspended
50
G. Ferretti et al. / Biochimica et Biophysica Acta 1635 (2003) 48–54
in 650 Al of 5 mM PBS. After 20 min of incubation at 37 jC, cells were centrifuged (3500 rpm) for 15 min and the absorbance of supernatant was evaluated at 560 nm. The levels of hydroperoxides in culture media and in HDL were determined by adding 1350 Al FOX reagent to 650 Al of sample following the experimental procedure previously described for cells. T-butyl hydroperoxide solution was used as standard. The results are presented as nmol of hydroperoxides for 106 cells and as nmol of hydroperoxides per ml of sample (culture media or HDL). 2.7. Determination of oxidative stress by the fluorescent probe H2DCFDA Determination of oxidative stress in control and Cu-OXastrocytes was studied also using the probe H2DCFDA. The molecule H2DCFDA is freely permeable to cells and after incorporation into cells is converted into the fluorescent 2V,7V-dichlorofluorescein (DCF) by oxidative substances. Therefore, DCF fluorescence indicates the intracellular production of redox-active substances and it has been widely used to investigate oxidative damage in intact cells [9,13,24 –26]. At the end of the oxidation treatment, cells were incubated for 1 h at 37 jC in the presence of 10 Al H2DCFDA (stock solution 2 mM). DCF fluorescence was measured at room temperature in a Perkin Elmer LS50B spectrofluorometer (490 nm excitation and 526 nm emission wavelengths). Results of DCF fluorescence intensity were expressed in arbitrary units.
3. Results 3.1. Effect of Cu++-induced oxidative damage on cultured astrocytes The levels of lipid hydroperoxides in untreated cells were 0.22 F 0.17 nmol/106 cells and were not significantly modified during the incubation at 37 jC (0.25 F 0.14 and 0.30 F 0.12 nmol/106 cells in astrocytes incubated for 4 and 17 h, respectively). A significant increase in the levels of hydroperoxides was observed in cells incubated in the presence of 10 AM Cu+ + with respect to control astrocytes either at 4 and at 17 h (Fig. 1A) ( P < 0.001 vs. control cells). Moreover, a significant time-dependent increase in the levels of hydroperoxides was observed in the culture media of cells incubated in the presence of Cu+ + (Fig. 1B) with respect to culture media of control cells either after 4 and 17 h of incubation ( P < 0.001 vs. culture media of control cells).
2.8. Assay of cell viability The viability of control or oxidized astrocytes was evaluated using Trypan Blue assay [23]. The loss of membrane integrity in dead and dying cells allows the preferential uptake of labels like trypan blue. Briefly, control and treated cells were pelleted and resuspended in 5 mM PBS; 10 Al of the cell suspension were added to 10 Al of 0.4% trypan blue in 1 5 mM PBS (pH 7.4). After 5 min of incubation at room temperature, cell suspension was transferred to the hemocytometer chamber and counted using a light microscope (10 ), the blue-stained cells were considered non-viable. The results were expressed as cell viability percentage. 2.9. Statistics All experiments were carried out in duplicate and were repeated for almost three times. Results were presented as the mean F S.D. Linear regression analysis was used to calculate correlation coefficients. Student’s t-test was used to analyse the difference of the results obtained in different experimental conditions. Values were considered to be significant at the P values less then 0.05 (Microcal Origin 5.0).
Fig. 1. (A) Levels of hydroperoxides (5) and DCF fluorescence (n) in astrocytes incubated at 37 jC with 10 AM Cu+ + for different times (0 – 17 h).*P < 0.001 vs. levels of hydroperoxides in untreated astrocytes; **P < 0.001 vs. DCF fluorescence in untreated astrocytes. (B) Levels of hydroperoxides in culture media of astrocytes incubated at 37 jC for different times (0 – 17 h) in the absence (5) or in the presence of 10 AM Cu+ + (n). *P < 0.001 vs. levels of hydroperoxides in culture media of untreated astrocytes.
G. Ferretti et al. / Biochimica et Biophysica Acta 1635 (2003) 48–54
The fluorescence intensity of DCF in untreated astrocytes was 18 F 9 arbitrary units and were not significantly modified during the incubation at 37 jC for 4 and 17 h, (23 F 9 and 28 F 16 arbitrary units, respectively). As shown in Fig. 1A, the fluorescence intensity was significantly higher in astrocytes incubated with copper ions with respect to control cells ( P < 0.001). The fluorogenic H2DCFDA probe represents a useful and a sensitive approach for measuring the production of reactive oxygen species (ROS) [24]; therefore, the increase in fluorescence observed in our experimental conditions confirms the oxidant effect exerted by copper on astrocytes [13]. A time-dependent decrease of cell viability was observed during incubation of astrocytes with copper ions. As shown in Fig. 2, after 4 h, the viability was about 40% in Cu+ +-treated with respect to control cells and decreased to about 12% after 17 h. The differences were significant ( P < 0.001). The levels of lipid hydroperoxides in astrocytes were positively correlated (r = 0.75, P < 0.001) with the percentage decrease of cell viability, suggesting that the compositional changes observed in Cu+ +-treated astrocytes are associated with modifications in cell viability. 3.2. Effect of HDL on Cu-induced oxidative damage of astrocytes 3.2.1. Experimental model A The protective effect exerted by HDL on Cu+ +-induced oxidative damage was investigated using astrocytes incubated with copper 10 AM for 4 h at 37 jC in the absence (Cu-OX-astrocytes) or in the presence of HDL. As shown in Fig. 3, the increase in the levels of hydroperoxides was significantly lower in astrocytes oxidized with copper ions in the presence of HDL (50 – 200 Ag/ml) with respect to astrocytes incubated alone. At the highest concentration of HDL (200 Ag/ml), the level of hydroperoxides in Cu-OX-
Fig. 2. Cell viability in astrocytes incubated at 37 jC with 10 AM Cu+ + for different times (0 – 17 h). *P < 0.001 vs. cell viability in untreated astrocytes.
51
Fig. 3. Levels of hydroperoxides (n) and cell viability (5) in astrocytes incubated for 4 h alone or with 10 AM Cu+ + (Cu-OX-HDL) in the absence or in the presence of HDL (50 and 200 Ag/ml) (experimental model A). jP < 0.001 vs. levels of hydroperoxides in untreated cells; jjP < 0.001 vs. cell viability in untreated cell; *P < 0.001 vs. levels of hydroperoxides in astrocytes oxidized in the absence of HDL; **P < 0.001 vs. cell viability in astrocytes oxidized in the absence of HDL.
astrocytes was not significantly different with respect to control cells (Fig. 3). As shown in Fig. 3, the astrocyte viability was higher in Cu+ +-treated astrocytes incubated in the presence of HDL with respect to cells oxidized alone and at the higher HDL concentration (200 Ag/ml), cell viability was similar to untreated cells (78 F 5% vs. 87 F 7%). These results suggest that the protective effect exerted by HDL on Cu+ +treated astrocytes was dependent on HDL concentration. 3.3. Effect of HDL on the levels of lipid hydroperoxides in Cu-OX-astrocytes 3.3.1. Experimental model B To investigate whether HDL are able to repair the Cu+ +induced oxidative damage in astrocytes, we investigated the
Fig. 4. Levels of hydroperoxides in Cu-OX-astrocytes before (n) and after incubation for 4 h at 37 jC in the absence or in the presence of different concentration of HDL (50 – 200 Ag/ml) (5) (experimental model B). *P < 0.001 vs. levels of hydroperoxides in astrocytes incubated for 4 h in the absence of HDL.
52
G. Ferretti et al. / Biochimica et Biophysica Acta 1635 (2003) 48–54
effect of incubation of HDL (50 –200 Ag/ml) with astrocytes previously oxidized for 17 h at 37 jC with 10 AM Cu+ + (CuOX-astrocytes). As shown in Fig. 4, the basal level of hydroperoxides in Cu-OX-astrocytes was 3.9nmol/106 cells and was not significantly modified during the subsequent incubation at 37 jC without lipoproteins (4.0nmol/106 cells). A significant decrease in the levels of hydroperoxides was observed in Cu-OX-astrocytes treated with HDL (Fig. 3). Using 50 Ag/ml of HDL the level of hydroperoxides was about one-half with respect to Cu-OX-astrocytes incubated alone and a further decrease was observed at 200 Ag/ml HDL, suggesting that the protective effect is related to HDL concentration. 3.4. Modifications of lipid hydroperoxides in HDL incubated with astrocytes The levels of lipid hydroperoxides in untreated HDL were 2.2 F 0.9 and 6.8 F 1.1 nmol/ml (in 50 and 200 Ag/ml, respectively) and were not significantly modified during incubation at 37 jC for 4 h (2.5 F 0.8 and 7.5 F 1.1 nmol/ ml, in 50 and 200 Ag/ml HDL, respectively). A significant increase of hydroperoxides was observed in HDL incubated for 4 h in the presence of copper 10 AM (4.2 F 0.9 vs. 2.5 F 0.8 nmol/ml, and 14.7 F 1.2 vs. 7.5 F 1.1 nmol/ml in 50 and 200 Ag/ml of HDL, respectively; P < 0.001) (Fig. 5). A higher and significant increase of lipid hydroperoxides has been observed in HDL co-incubated with copper and astrocytes with respect to HDL incubated with copper without cells (experimental model A). The increase of lipid hydroperoxides was three-fold higher at 50 Ag/ml of HDL, a four-fold increase in the levels of lipid hydroperoxides has been observed at the higher HDL concentration (200 Ag/ml) with respect to untreated HDL (Fig. 5). As shown in Fig. 5, a significant increase in the levels of hydroperoxides was observed also in HDL incubated for 4 h with Cu-OX-astrocytes with respect to HDL incubated alone (Fig. 5). The increase of lipid hydroperoxides was
Fig. 5. Levels of hydroperoxides in HDL (5 50 Ag/ml; n 200 Ag/ml) incubated for 4 h at 37 jC in different experimental conditions (for details see Materials and methods). *P < 0.001 vs. 50 Ag/ml HDL; jP < 0.001 vs. 200 Ag/ml HDL; **P < 0.001 vs. 50 Ag/ml Cu+ +-treated HDL; jjP < 0.001 vs. 200 Ag/ml Cu+ +-treated HDL.
dependent on HDL concentration (Fig. 5). At 200 Ag/ml, the percentage increase of lipid hydroperoxides was higher with respect to that observed at 50 Ag/ml.
4. Discussion In the present study, we confirmed that copper ions, at a concentration similar to that observed in the CSF of patients affected by neurodegenerative diseases [19,20], induce a time-dependent increase in the levels of lipid hydroperoxides. Moreover, using the fluorescence of H2DCFDA, a probe sensitive to the intracellular formation of oxidative substances, we demonstrated that copper triggers ROS formation in astrocytes, as previously observed in different cellular models [24 –26]. The compositional changes were associated with a decrease in cell viability. These results confirm that copper ions induce ROS formation and oxidative damage in astrocytes [13]. Copper ions have been widely used to induce cell oxidative damage [9,13,25,26] and their cytotoxic effect has been related to the ability to trigger formation of free radicals which in turn cause cell membrane lipid peroxidation, modifications of physico-chemical properties and alterations in cell permeability [27]. Other mechanisms suggested include a toxic effect of copper to other cell constituents such as proteins, nucleic acids or mitochondria. In fact, using isolated mitochondria, it has been demonstrated that copper induces lipid peroxidation and the collapse of the membrane potential [28]. Mitochondrial ROS formation and a decrease in membrane potential has also been observed in intact cells treated with five-fold higher concentrations of copper ions with respect to that used in our experimental conditions [26]. Mitochondrial dysfunction and copper-induced ROS activation of transcription signaling, play a critical role in inducing alterations of cell functions and cell death [29]. Using two different experimental models of astrocytes in culture, we demonstrated that human HDL exert a protective effect against copper-induced oxidative damage of astrocytes. Lower levels of hydroperoxides and a lower decrease in cell viability have been observed in astrocytes oxidized in the presence of HDL with respect to cells oxidized alone. These results suggest that HDL, during the co-incubation with cells, exert a protective effect against oxidative damage, in good agreement with previous studies on LDL oxidized in the presence of HDL [11,12]. The lower susceptibility of astrocytes to copper-induced oxidative damage in the presence of HDL confirms an antioxidant role that could be related to the ability of HDL to bind metal ions in sites at the lipoprotein surface [30]. The antioxidant ability of HDL against lipid peroxidation has also been related to the effect of apoproteins and enzymes associated to the HDL surface. In fact, the enzymes paraoxonase (PON) and platelet activating factor acetylhydrolase (PAF-HA), are able to hydrolyse oxidized
G. Ferretti et al. / Biochimica et Biophysica Acta 1635 (2003) 48–54
fatty acid from phospholipids and prevent their accumulation on LDL, blocking the atherogenic and inflammatory response induced by OX-LDL [31]. In the present study, we also demonstrated a significant decrease in the levels of hydroperoxides associated with CuOX-astrocytes incubated with HDL for 4 h (experimental model B). These results suggest that HDL could protect astrocytes from accumulating lipid hydroperoxides. In our experimental conditions, the levels of lipid hydroperoxides associated with HDL co-incubated with Cu+ + and astrocytes were significantly higher with respect to the values observed in culture media of copper-oxidized astrocytes. We suggest that lipid peroxidation products could be released from oxidized cells to culture media and HDL could stimulate the efflux from OX-astrocytes with some mechanism(s) that deserve further studies. This hypothesis is supported by previous studies using HDL incubated with OX-LDL or OX-cells [11,14]. Klimov et al. [14] demonstrated that the incubation of HDL in the presence of Cu+ +-oxidized erythrocyte membranes increased the content of lipid hydroperoxides in HDL and suggested a transport of phospholipids containing hydroperoxides from oxidized erythrocyte membranes to lipoproteins. A transfer of hydroperoxides from oxidized LDL to HDL and an exchange of lipid peroxidation products between the lipoproteins were previously demonstrated [11]. The high ability of HDL to accumulate lipid hydroperoxides in human plasma has been demonstrated by Bowry et al. [32], therefore it was hypothesised that HDL could play an important role in the transport and detoxification of oxidation products in human plasma [14,32]. In conclusion, using different experimental models, we demonstrated that HDL exert a protective effect against copper-triggered oxidative damage on astrocytes in agreement with previous studies using different cellular models [13,14,33]. Copper-induced cytotoxicity is a multifactorial phenomenon underlying actions due to the generation of ROS and RNS that may alter cell membranes and essential biomolecules with loss of biological functions [25 – 27]. Moreover, modulation of mitochondrial functions, gene expression and production of cytotoxic mediators have been demonstrated in copper-treated cells [26]. The results obtained in the present study suggest that the protective effect of HDL against cell peroxidation could be exerted by different mechanisms: metal binding properties and/or inhibition of the accumulation of lipid hydroperoxides in oxidized cells could be involved. Previous studies have demonstrated that cells preincubated with HDL are less sensitive to the oxidizing effect exerted by copper ions and it has been suggested that HDL could increase cell resistance against the cytotoxicity of oxidant agents by modifying the signaling of intracellular chemical mediators and the intracellular metabolism [13,33]. This hypothesis is supported by several recent studies which have demonstrated that plasma lipoproteins regulate signal transduction and modulate the effects of
53
several molecules (cytokine, free radicals) on cell functions [34]. In particular, HDL modulate the phosphoinositidespecific phospholipase C pathway and protein kinase C (PKC), the sphingosine kinase signaling pathway and endothelial NO production [34]. Moreover, HDL regulate the activation of extracellular-regulated kinases and NF-kappaB signaling cascade in cells in culture [35]. Concerning the patho-physiological relevance of our results, it has to be stressed that in our experimental conditions, the effect of copper on lipid peroxidation of astrocytes was observed at a copper concentration similar to that observed in human CSF of patients affected by neurodegenerative diseases [19,20]. Oxidative stress, defined as the imbalance between biochemical processes leading to production of ROS and the cellular antioxidant cascade, causes molecular damage that can lead to critical failure of biological functions and ultimately to cell death. The role of oxidative damage in the pathogenesis of neurological disease such as Alzheimer’s (AD) and Parkinson’s diseases (PD) was widely demonstrated [3 –5]. Recent studies have demonstrated that astrocytes play a key role in the antioxidant defence of brain [6,7]. The increase in antioxidant enzymes in astrocytes exposed to oxidative damage was not sufficient to avoid lipid peroxidation in cortical astrocyte cultures [7]. Therefore, defence mechanisms in the extracellular space could play a key role in protecting brain cells against oxidative damage and cytotoxic molecules. Moreover, lipoprotein particles similar for density and chemical composition to plasma HDL were isolated in human CSF [15,16]. A higher susceptibility of CSF lipoproteins of AD patients and lower levels of antioxidants have been demonstrated in the CSF of AD patients [18,36]. Therefore, the protective role exerted by HDL against Cu+ +induced oxidative damage to astrocytes could be of physiological relevance. Recent studies have demonstrated that paraoxonase is expressed in brain tissue [37]. Studies are in progress in our laboratory to investigate the activity of paraoxonase in the lipoproteins of human CSF and to study whether the lipoprotein particles isolated from human CSF of controls or AD patients protect astrocytes efficiently as HDL isolated from human plasma.
Acknowledgements This work was supported by a grant from MURST 60% (GF).
References [1] G. Perry, L.M. Sayre, C.S. Atwood, R.J. Castellani, A.D. Cash, C.A. Rottkamp, M.A. Smith, The role of iron and copper in the aetiology of
54
[2]
[3] [4]
[5]
[6] [7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
G. Ferretti et al. / Biochimica et Biophysica Acta 1635 (2003) 48–54 neurodegenerative disorders: therapeutic implications, CNS Drugs 16 (5) (2002) 339 – 352. G. Rotilio, M.T. Carri, L. Rossi, M.R. Circolo, Copper-dependent oxidative stress and neurodegeneration, IUBMB Life 50 (4 – 5) (2000) 309 – 314. N.A. Simonian, J.T. Coyle, Oxidative stress in neurodegenerative diseases, Annu. Rev. Pharmacol. Toxicol. 36 (1996) 83 – 106. D.L. Marcus, C. Thomas, C. Rodriguez, K. Simberkoff, J.S. Tsai, J.A. Strafaci, M.L. Freedman, Increased peroxidation and reduced antioxidant enzyme activity in Alzheimer’s disease, Exp. Neurol. 150 (1) (1998) 40 – 44. R. Dei, A. Takeda, H. Niwa, M. Li, Y. Nakagomi, M. Watanabe, T. Inagaki, Y. Washimi, Y. Yasuda, K. Horie, T. Miyata, G. Sobue, Lipid peroxidation and advanced glycation end products in the brain in normal aging and in Alzheimer’s disease, Acta Neuropathol. (Berl.) 104 (2) (2002) 113 – 122. S. Desagher, J. Glowinski, J. Premont, Astrocytes protect neurons from hydrogen peroxide toxicity, J. Neurosci. 16 (8) (1996) 2553 – 2562. F. Klamt, C. Gottfried, F. Tramontina, F. Dal-Pizzol, M.L. Da Frota, J.C. Moreira, R.D. Dias, E. Moriguchi, S. Wofchuk, D.O. Souza, Time-related increase in mitochondrial superoxide production, biomolecule damage and antioxidant enzyme activities in cortical astrocyte cultures, NeuroReport 13 (12) (2002) 1515 – 1518. J.M. Servitja, R. Masgrau, R. Pardo, E. Sarri, F. Picatoste, Effects of oxidative stress on phospholipid signalling in rat cultured astrocytes and brain slices, J. Neurochem. 75 (2) (2000) 788 – 794. S.H. Chen, S.H. Liu, Y.C. Liang, J.K. Lin, S.Y. Lin-Shiau, Oxidative stress and c-Jun-amino-terminal kinase activation involved in apoptosis of primary astrocytes induced by disulfiram-Cu (2+) complex, Eur. J. Pharmacol. 414 (2 – 3) (2001) 177 – 188. E. Rohrdanz, G. Schmuck, S. Ohler, Q.H. Tran-Thi, R. Kahl, Changes in antioxidant enzyme expression in response to hydrogen peroxide in rat astroglial cells, Arch. Toxicol. 75 (3) (2001) 150 – 158. S. Parthasarathy, J. Barnett, L.G. Fong, High-density lipoprotein inhibits the oxidative modification of low-density lipoproteins, Biochim. Biophys. Acta 1044 (2) (1990) 275 – 283. M.I. Mackness, P.N. Durrington, B. Mackness, How high-density lipoprotein protects against the effects of lipid peroxidation, Curr. Opin. Lipidol. 11 (4) (2000) 383 – 388. G. Ferretti, T. Bacchetti, C. Moroni, A. Vignini, G. Curatola, Protective effect of human HDL against Cu2 +-induced oxidation of astrocytes, J. Trace Elem. Med. Biol. 17 (3) (2003) 25 – 30. A.N. Klimov, K.A. Kozhevnikova, A.A. Kuzmin, A.S. Kuznetsov, E.V. Belova, On the ability of high density lipoproteins to remove phospholipid peroxidation products from erythrocyte membranes, Biochemistry (Mosc.) 66 (3) (2001) 300 – 304. S. Koch, N. Donarski, K. Goetze, M. Kreckel, H.J. Stuerenburg, C. Buhmann, U. Beisiegel, Characterization of four lipoprotein classes in human cerebrospinal fluid, J. Lipid Res. 42 (7) (2001) 1143 – 1151. M.J. Ladu, C. Reardon, L. Van Eldik, A.M. Fagan, G. Bu, D. Holtzman, G.S. Getz, Lipoproteins in the central nervous system, Ann. N.Y. Acad. Sci. 903 (2000) 167 – 175. R.E. Pitas, J.K. Boyles, S.H. Lee, D. Foss, R.W. Mahley, Astrocytes synthesize apolipoprotein E and metabolize apolipoprotein-E containing lipoproteins, Biochim. Biophys. Acta 917 (1) (1987) 148 – 161. C.N. Bassett, K.S. Montine, M.D. Neely, L.L. Swift, T.J. Montine, Cerebrospinal fluid lipoproteins in Alzheimer’s disease, Microsc. Res. Tech. 50 (4) (2000) 282 – 286. H.S. Pall, A.C. Williams, D.R. Blake, J. Lunec, J.M. Gutteridge, M. Hall, A. Taylor, Raised cerebrospinal-fluid copper concentration in Parkinson’s disease, Lancet 2 (8553) (1987) 238 – 241.
[20] R. Squitti, D. Lupoi, P. Pasqualetti, G. Dal Forno, F. Vernieri, P. Chiovenda, L. Rossi, M. Cortesi, E. Cassetta, P.M. Rossigni, Elevation of serum copper levels in Alzheimer’s disease, Neurology 59 (8) (2002) 1153 – 1161. [21] B.H. Chung, J.P. Segrest, M.J. Ray, J.D. Brunzell, J.E. Hokanson, R.M. Krauss, K. Beaudrie, J.T. Cone, Single vertical spin density gradient ultracentrifugation, Methods Enzymol. 128 (1986) 181 – 209. [22] O.H. Lowry, N.J. Rosebrough, A.L. Farr, P.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193 (1951) 265 – 275. [23] R.I. Freshney, Culture of Animal Cells: A Manual of Basic Technique, Wiley, New York, 2000. [24] Y. Oyama, A. Hayashi, T. Ueha, K. Maekawa, Characterization of 2V,7V-dichlorofluorescin fluorescence in dissociated mammalian brain neurons: estimation on intracellular content of hydrogen peroxide, Brain Res. 635 (1 – 2) (1994) 113 – 117. [25] N.T. Watt, N.M. Hooper, The response of neurones and glial cells to elevated copper, Brain Res. Bull. 55 (2) (2001) 219 – 224. [26] J. Pourahmad, P.J. O’Brien, A comparison of hepatocyte cytotoxic mechanisms for Cu2 + and Cd2 +, Toxicology 143 (3) (2000) 263 – 273. [27] S.C. Luza, H.C. Speisky, Liver copper storage and transport during development: implications for cytotoxicity, Am. J. Clin. Nutr. 63 (5) (1996) 812S – 820S. [28] N.E. Saris, I.A. Skulskii, Interaction of Cu+ with mitochondria, Acta Chem. Scand. 45 (10) (1991) 1042 – 1046. [29] N. Zamzami, P. Marchetti, M. Castedo, C. Zanin, J.L. Vayssiere, P.X. Petit, G. Kroemer, Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo, J. Exp. Med. 181 (5) (1995) 1661 – 1672. [30] S.T. Kunitake, M.R. Jarvis, R.L. Hamilton, J.P. Kane, Binding of transition metals by apolipoprotein A-I-containing plasma lipoproteins: inhibition of oxidation of low density lipoproteins, Proc. Natl. Acad. Sci. U. S. A. 89 (15) (1992) 6993 – 6997. [31] M.I. Mackness, P.N. Durrington, HDL, its enzymes and its potential to influence lipid peroxidation, Atherosclerosis 115 (2) (1995) 243 – 253. [32] V.W. Bowry, K.K. Stanley, R. Stocker, High density lipoprotein is the major carrier of lipid hydroperoxides in human blood plasma from fasting donors, Proc. Natl. Acad. Sci. 89 (21) (1992) 10316 – 10320. [33] I. Suc, I. Escargueil-Blanc, M. Troly, R. Salvayre, A. Negre-Salvayre, HDL and ApoA prevent cell death of endothelial cells induced by oxidized LDL, Arterioscler. Thromb. Vasc. Biol. 17 (1997) 2158 – 2166. [34] J.R. Nofer, B. Kehrel, M. Fobker, B. Levkau, G. Assmann, A. von Eckardstein, HDL and arteriosclerosis: beyond reverse cholesterol transport, Atherosclerosis 161 (1) (2002) 1 – 16. [35] S.H. Park, J.H. Park, J.S. Kang, Y.H. Kang, Involvement of transcription factors in plasma HDL protection against TNF-alpha-induced vascular cell adhesion molecule-1 expression, Int. J. Biochem. Cell Biol. 35 (2) (2003) 168 – 182. [36] C.N. Bassett, M.D. Neely, K.R. Sidell, W.R. Markesbery, L.L. Swift, T.J. Montine, Cerebrospinal fluid lipoproteins are more vulnerable to oxidation in Alzheimer’s disease and are neurotoxic when oxidized ex vivo, Lipids 34 (12) (1999) 1273 – 1280. [37] L. Rodrigo, A.F. Hernandez, J.J. Lopez-Caballero, F. Gil, A.R. Pla, Immunohistochemical evidence for the expression and induction of paraoxonase in rat liver, kidney, lung and brain tissue. Implications for its physiological role, Chem. Biol. Interact. 137 (2) (2001) 123 – 137.
Copper-induced oxidative damage on astrocytes: protective effect exerted by human high density lipoproteins Gianna Ferretti, Tiziana Bacchetti *, Cinzia Moroni, Arianna Vignini, Giovanna Curatola Istituto di Biochimica, Facolta` of Medicina, Universita` Politecnica delle Marche, Via Ranieri, I-60131 Ancona, Italy Received 5 June 2003; received in revised form 7 October 2003; accepted 15 October 2003
Abstract In the present study, we confirmed that copper ions induce oxidative damage in human astrocytes in culture, as demonstrated by the significant increase in the levels of hydroperoxides and in the fluorescence intensity of the fluorescent probe dichloro-dihydrofluorescein diacetate (H2DCFDA). The compositional changes were associated with a significant decrease in cell viability in astrocytes treated with 10 AM Cu+ + with respect to control cells. Astrocytes incubated with copper ions in the presence of high density lipoproteins (HDL) isolated from plasma of normolipemic subjects showed lower levels of hydroperoxides and a higher cell viability with respect to cells oxidized alone. Moreover, a significant decrease in the levels of hydroperoxides was observed in oxidized astrocytes treated with HDL. These results demonstrate that HDL exert a protective role against lipid peroxidation. The protective effect could be related to the ability of HDL to bind metal ions at the lipoprotein surface and/or to a stimulation of the efflux of lipid hydroperoxides from cell membranes as demonstrated in other cell types. Oxidative damage of astrocytes was induced at a copper concentration similar to that observed in cerebrospinal fluid (CSF) of patients affected by neurodegenerative diseases such as Alzheimer’s (AD) and Parkinson’s diseases (PD). Lipoprotein particles similar for density and chemical composition to plasma HDL were recently isolated in human CSF, therefore, the protective role exerted by HDL against Cu+ +induced oxidative damage of astrocytes could be of physiological relevance. D 2003 Elsevier B.V. All rights reserved. Keywords: Astrocyte; Copper; High density lipoprotein; Lipid peroxidation; 2V,7V-dichloro-dihydrofluorescein diacetate (H2DCFDA); Neurodegenerative disease
1. Introduction Brain is particularly susceptible to oxidative stress and several studies have demonstrated that copper-induced oxidative damage is involved in the pathogenesis of many neurodegenerative diseases such as Alzheimer’s disease (AD), Creutzfeldt –Jakob disease and amyotrophic lateral sclerosis [1,2] and other oxidative stress-related conditions [3 –5]. Recent studies have demonstrated that astrocytes are chiefly responsible for the antioxidant defence of brain [6,7]. Therefore, the sensitivity of astrocytes to oxidative damage triggered in different experimental conditions has been previously investigated [8 –10]. Changes in antioxidant
* Corresponding author. Tel.: +39-71-220-4673; fax: +39-71-220-4398. E-mail address: [email protected] (T. Bacchetti). 1388-1981/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2003.10.005
enzyme expression in response to hydrogen peroxide have been previously observed in astrocytes [7,10] and it has been suggested that these modifications may contribute to protect cells against oxidative insults. However, the increase in antioxidant enzymes was not sufficient to avoid oxidative damage and lipid peroxidation in cortical astrocyte cultures [7]. Therefore, defence mechanisms in the extracellular space could play a key role in protecting brain cells against oxidative damage and cytotoxic molecules in conditions in which intracellular defences fail. Previous studies have demonstrated that high density lipoproteins (HDL) exert a protective role against lipid peroxidation induced by different agents on plasma lipoproteins [11,12] and cells in culture [13,14]. Lipoprotein particles similar for density and chemical composition to plasma HDL have been isolated in human cerebrospinal fluid (CSF) [15,16] and recent studies demonstrated that astrocytes are involved in their synthesis [17].
G. Ferretti et al. / Biochimica et Biophysica Acta 1635 (2003) 48–54
The lipid and apoprotein composition of human CSF-lipoproteins has been well characterized, but their functions have not been clearly elucidated. It was suggested that they could be involved in the regulation of lipid homeostasis in the central nervous system (CNS) [16] and/or exert a role in the development of neurodegenerative diseases [18]. In our previous study [13], we demonstrated that astrocytes, preincubated with HDL isolated from human plasma, were less susceptible to oxidative damage induced by copper ions at concentrations similar to those observed in CSF of patients affected by neurodegenerative diseases [19,20]. The aim of this study was to further investigate the protective effect exerted by HDL against oxidative damage of brain cells and the molecular mechanisms involved. Therefore, using two different experimental models of astrocytes in culture, we studied the effect of HDL isolated from plasma of normolipemic subjects against astrocyte lipid peroxidation triggered by copper ions.
2. Materials and methods 2.1. Chemicals Dulbecco’s modified Eagle’s medium (DMEM), phenol red-free-DMEM, fetal bovine serum, penicillin/streptomycin solution (10,000 units and 10 mg streptomycin/ml in 0.9% NaCl), L-glutamine (200 mM), ethylenediamine tetraacetic acid (EDTA), trypsin –EDTA solution (10 ), phosphate buffer solution (PBS) and trypan blue (0.4%) were purchased from Sigma Chemical Company (St. Louis, MO). The fluorogenic 2,7-dichloro-dihydrofluorescein diacetate (H2DCFDA) probe was obtained from Molecular Probes, Inc. (Eugene, OR). 2.2. Preparation of HDL Blood from healthy normolipidemic volunteers was obtained by venipuncture. Plasma was prepared by centrifugation at 3000 rpm for 20 min and thereafter used for the preparation of HDL (1.063 –1.21 g/ml) by single vertical spin ultracentrifugation for 1.30 h as described by Chung et al. [21]. After dialysis at 4 jC for 24 h against 5 mM PBS (pH 7.4), the protein concentration of HDL was determined by the method of Lowry et al. [22]. HDL were sterilized on a 0.2 Am Millipore membrane before being incubated with astrocytes. 2.3. Cell culture U 373 MG Human astrocytomas (a gift from Prof. L. Mazzanti-Ancona, Italy) were grown in phenol red-freecomplete-DMEM (containing 10% fetal bovine serum, 1% L-glutamine (200 mM) and 1% penicillin/streptomycin), at 37 jC in a humidified atmosphere of 5% CO2. After 2 or 3 days, cells reached 90 –95% confluence as observed by
49
a light microscope (10 ) and were used for the experiments [23]. 2.4. Oxidative treatment of astrocytes Oxidative treatment of astrocytes was carried out using CuSO4 as oxidizing agent. Briefly, astrocytes were incubated at 37 jC in phenol red-free-complete-DMEM containing 10 AM CuSO4, for different times (4 and 17 h) [13] (CuOX-astrocytes). At the end of the incubation, cells and culture media were used for determination of cell viability and evaluation of oxidative damage. 2.5. Incubation of astrocytes with HDL The protective effect exerted by HDL against Cu+ +induced oxidation of cultured astrocytes was investigated using two different experimental models. 2.5.1. Experimental model A Astrocytes were incubated in phenol-free-completeDMEM (at 37 jC and 5% CO2) for 4 h with 10 AM CuSO4 in the presence or in the absence of different concentrations of HDL (50 – 200 Ag/ml). At the end of the incubation, levels of lipid hydroperoxides were evaluated in culture media and cells. In some experiments, HDL were incubated in the absence or in the presence of 10 AM Cu+ + in the same experimental conditions used for cells and were used for determination of the levels of lipid hydroperoxides. 2.5.2. Experimental model B Astrocytes were incubated in phenol-free-completeDMEM (at 37 jC and 5% CO2) with 10 AM CuSO4 for 17 h. After oxidative treatment, media were removed and cells were washed for three times with 5 mM PBS pH 7.4. Thereafter, cells were resuspended in phenol-free-complete-DMEM (at 37 jC and 5% CO2) and incubated for 4 h in the absence or in the presence of different concentrations of HDL (50 – 200 Ag/ml). At the end of the incubation, HDL were removed from astrocytes and used to evaluate the levels of lipid hydroperoxides. In some experiments, HDL were incubated for 4 h in the same conditions used for cells and were used for determination of the levels of lipid hydroperoxides. 2.6. Evaluation of lipid peroxidation in astrocytes and in HDL The extent of lipid peroxidation in astrocytes, culture media and in HDL treated in different experimental conditions, was evaluated by measuring the levels of hydroperoxides using the FOX-assay as previously described [13]. The levels of hydroperoxides in cells were evaluated by adding the FOX reagent (100 AM xylenol orange, 250 AM Fe+ +, 25 mM H2SO4 and 4 mM BHT in 90% methanol) (1350 Al) to astrocytes previously pelleted and resuspended
50
G. Ferretti et al. / Biochimica et Biophysica Acta 1635 (2003) 48–54
in 650 Al of 5 mM PBS. After 20 min of incubation at 37 jC, cells were centrifuged (3500 rpm) for 15 min and the absorbance of supernatant was evaluated at 560 nm. The levels of hydroperoxides in culture media and in HDL were determined by adding 1350 Al FOX reagent to 650 Al of sample following the experimental procedure previously described for cells. T-butyl hydroperoxide solution was used as standard. The results are presented as nmol of hydroperoxides for 106 cells and as nmol of hydroperoxides per ml of sample (culture media or HDL). 2.7. Determination of oxidative stress by the fluorescent probe H2DCFDA Determination of oxidative stress in control and Cu-OXastrocytes was studied also using the probe H2DCFDA. The molecule H2DCFDA is freely permeable to cells and after incorporation into cells is converted into the fluorescent 2V,7V-dichlorofluorescein (DCF) by oxidative substances. Therefore, DCF fluorescence indicates the intracellular production of redox-active substances and it has been widely used to investigate oxidative damage in intact cells [9,13,24 –26]. At the end of the oxidation treatment, cells were incubated for 1 h at 37 jC in the presence of 10 Al H2DCFDA (stock solution 2 mM). DCF fluorescence was measured at room temperature in a Perkin Elmer LS50B spectrofluorometer (490 nm excitation and 526 nm emission wavelengths). Results of DCF fluorescence intensity were expressed in arbitrary units.
3. Results 3.1. Effect of Cu++-induced oxidative damage on cultured astrocytes The levels of lipid hydroperoxides in untreated cells were 0.22 F 0.17 nmol/106 cells and were not significantly modified during the incubation at 37 jC (0.25 F 0.14 and 0.30 F 0.12 nmol/106 cells in astrocytes incubated for 4 and 17 h, respectively). A significant increase in the levels of hydroperoxides was observed in cells incubated in the presence of 10 AM Cu+ + with respect to control astrocytes either at 4 and at 17 h (Fig. 1A) ( P < 0.001 vs. control cells). Moreover, a significant time-dependent increase in the levels of hydroperoxides was observed in the culture media of cells incubated in the presence of Cu+ + (Fig. 1B) with respect to culture media of control cells either after 4 and 17 h of incubation ( P < 0.001 vs. culture media of control cells).
2.8. Assay of cell viability The viability of control or oxidized astrocytes was evaluated using Trypan Blue assay [23]. The loss of membrane integrity in dead and dying cells allows the preferential uptake of labels like trypan blue. Briefly, control and treated cells were pelleted and resuspended in 5 mM PBS; 10 Al of the cell suspension were added to 10 Al of 0.4% trypan blue in 1 5 mM PBS (pH 7.4). After 5 min of incubation at room temperature, cell suspension was transferred to the hemocytometer chamber and counted using a light microscope (10 ), the blue-stained cells were considered non-viable. The results were expressed as cell viability percentage. 2.9. Statistics All experiments were carried out in duplicate and were repeated for almost three times. Results were presented as the mean F S.D. Linear regression analysis was used to calculate correlation coefficients. Student’s t-test was used to analyse the difference of the results obtained in different experimental conditions. Values were considered to be significant at the P values less then 0.05 (Microcal Origin 5.0).
Fig. 1. (A) Levels of hydroperoxides (5) and DCF fluorescence (n) in astrocytes incubated at 37 jC with 10 AM Cu+ + for different times (0 – 17 h).*P < 0.001 vs. levels of hydroperoxides in untreated astrocytes; **P < 0.001 vs. DCF fluorescence in untreated astrocytes. (B) Levels of hydroperoxides in culture media of astrocytes incubated at 37 jC for different times (0 – 17 h) in the absence (5) or in the presence of 10 AM Cu+ + (n). *P < 0.001 vs. levels of hydroperoxides in culture media of untreated astrocytes.
G. Ferretti et al. / Biochimica et Biophysica Acta 1635 (2003) 48–54
The fluorescence intensity of DCF in untreated astrocytes was 18 F 9 arbitrary units and were not significantly modified during the incubation at 37 jC for 4 and 17 h, (23 F 9 and 28 F 16 arbitrary units, respectively). As shown in Fig. 1A, the fluorescence intensity was significantly higher in astrocytes incubated with copper ions with respect to control cells ( P < 0.001). The fluorogenic H2DCFDA probe represents a useful and a sensitive approach for measuring the production of reactive oxygen species (ROS) [24]; therefore, the increase in fluorescence observed in our experimental conditions confirms the oxidant effect exerted by copper on astrocytes [13]. A time-dependent decrease of cell viability was observed during incubation of astrocytes with copper ions. As shown in Fig. 2, after 4 h, the viability was about 40% in Cu+ +-treated with respect to control cells and decreased to about 12% after 17 h. The differences were significant ( P < 0.001). The levels of lipid hydroperoxides in astrocytes were positively correlated (r = 0.75, P < 0.001) with the percentage decrease of cell viability, suggesting that the compositional changes observed in Cu+ +-treated astrocytes are associated with modifications in cell viability. 3.2. Effect of HDL on Cu-induced oxidative damage of astrocytes 3.2.1. Experimental model A The protective effect exerted by HDL on Cu+ +-induced oxidative damage was investigated using astrocytes incubated with copper 10 AM for 4 h at 37 jC in the absence (Cu-OX-astrocytes) or in the presence of HDL. As shown in Fig. 3, the increase in the levels of hydroperoxides was significantly lower in astrocytes oxidized with copper ions in the presence of HDL (50 – 200 Ag/ml) with respect to astrocytes incubated alone. At the highest concentration of HDL (200 Ag/ml), the level of hydroperoxides in Cu-OX-
Fig. 2. Cell viability in astrocytes incubated at 37 jC with 10 AM Cu+ + for different times (0 – 17 h). *P < 0.001 vs. cell viability in untreated astrocytes.
51
Fig. 3. Levels of hydroperoxides (n) and cell viability (5) in astrocytes incubated for 4 h alone or with 10 AM Cu+ + (Cu-OX-HDL) in the absence or in the presence of HDL (50 and 200 Ag/ml) (experimental model A). jP < 0.001 vs. levels of hydroperoxides in untreated cells; jjP < 0.001 vs. cell viability in untreated cell; *P < 0.001 vs. levels of hydroperoxides in astrocytes oxidized in the absence of HDL; **P < 0.001 vs. cell viability in astrocytes oxidized in the absence of HDL.
astrocytes was not significantly different with respect to control cells (Fig. 3). As shown in Fig. 3, the astrocyte viability was higher in Cu+ +-treated astrocytes incubated in the presence of HDL with respect to cells oxidized alone and at the higher HDL concentration (200 Ag/ml), cell viability was similar to untreated cells (78 F 5% vs. 87 F 7%). These results suggest that the protective effect exerted by HDL on Cu+ +treated astrocytes was dependent on HDL concentration. 3.3. Effect of HDL on the levels of lipid hydroperoxides in Cu-OX-astrocytes 3.3.1. Experimental model B To investigate whether HDL are able to repair the Cu+ +induced oxidative damage in astrocytes, we investigated the
Fig. 4. Levels of hydroperoxides in Cu-OX-astrocytes before (n) and after incubation for 4 h at 37 jC in the absence or in the presence of different concentration of HDL (50 – 200 Ag/ml) (5) (experimental model B). *P < 0.001 vs. levels of hydroperoxides in astrocytes incubated for 4 h in the absence of HDL.
52
G. Ferretti et al. / Biochimica et Biophysica Acta 1635 (2003) 48–54
effect of incubation of HDL (50 –200 Ag/ml) with astrocytes previously oxidized for 17 h at 37 jC with 10 AM Cu+ + (CuOX-astrocytes). As shown in Fig. 4, the basal level of hydroperoxides in Cu-OX-astrocytes was 3.9nmol/106 cells and was not significantly modified during the subsequent incubation at 37 jC without lipoproteins (4.0nmol/106 cells). A significant decrease in the levels of hydroperoxides was observed in Cu-OX-astrocytes treated with HDL (Fig. 3). Using 50 Ag/ml of HDL the level of hydroperoxides was about one-half with respect to Cu-OX-astrocytes incubated alone and a further decrease was observed at 200 Ag/ml HDL, suggesting that the protective effect is related to HDL concentration. 3.4. Modifications of lipid hydroperoxides in HDL incubated with astrocytes The levels of lipid hydroperoxides in untreated HDL were 2.2 F 0.9 and 6.8 F 1.1 nmol/ml (in 50 and 200 Ag/ml, respectively) and were not significantly modified during incubation at 37 jC for 4 h (2.5 F 0.8 and 7.5 F 1.1 nmol/ ml, in 50 and 200 Ag/ml HDL, respectively). A significant increase of hydroperoxides was observed in HDL incubated for 4 h in the presence of copper 10 AM (4.2 F 0.9 vs. 2.5 F 0.8 nmol/ml, and 14.7 F 1.2 vs. 7.5 F 1.1 nmol/ml in 50 and 200 Ag/ml of HDL, respectively; P < 0.001) (Fig. 5). A higher and significant increase of lipid hydroperoxides has been observed in HDL co-incubated with copper and astrocytes with respect to HDL incubated with copper without cells (experimental model A). The increase of lipid hydroperoxides was three-fold higher at 50 Ag/ml of HDL, a four-fold increase in the levels of lipid hydroperoxides has been observed at the higher HDL concentration (200 Ag/ml) with respect to untreated HDL (Fig. 5). As shown in Fig. 5, a significant increase in the levels of hydroperoxides was observed also in HDL incubated for 4 h with Cu-OX-astrocytes with respect to HDL incubated alone (Fig. 5). The increase of lipid hydroperoxides was
Fig. 5. Levels of hydroperoxides in HDL (5 50 Ag/ml; n 200 Ag/ml) incubated for 4 h at 37 jC in different experimental conditions (for details see Materials and methods). *P < 0.001 vs. 50 Ag/ml HDL; jP < 0.001 vs. 200 Ag/ml HDL; **P < 0.001 vs. 50 Ag/ml Cu+ +-treated HDL; jjP < 0.001 vs. 200 Ag/ml Cu+ +-treated HDL.
dependent on HDL concentration (Fig. 5). At 200 Ag/ml, the percentage increase of lipid hydroperoxides was higher with respect to that observed at 50 Ag/ml.
4. Discussion In the present study, we confirmed that copper ions, at a concentration similar to that observed in the CSF of patients affected by neurodegenerative diseases [19,20], induce a time-dependent increase in the levels of lipid hydroperoxides. Moreover, using the fluorescence of H2DCFDA, a probe sensitive to the intracellular formation of oxidative substances, we demonstrated that copper triggers ROS formation in astrocytes, as previously observed in different cellular models [24 –26]. The compositional changes were associated with a decrease in cell viability. These results confirm that copper ions induce ROS formation and oxidative damage in astrocytes [13]. Copper ions have been widely used to induce cell oxidative damage [9,13,25,26] and their cytotoxic effect has been related to the ability to trigger formation of free radicals which in turn cause cell membrane lipid peroxidation, modifications of physico-chemical properties and alterations in cell permeability [27]. Other mechanisms suggested include a toxic effect of copper to other cell constituents such as proteins, nucleic acids or mitochondria. In fact, using isolated mitochondria, it has been demonstrated that copper induces lipid peroxidation and the collapse of the membrane potential [28]. Mitochondrial ROS formation and a decrease in membrane potential has also been observed in intact cells treated with five-fold higher concentrations of copper ions with respect to that used in our experimental conditions [26]. Mitochondrial dysfunction and copper-induced ROS activation of transcription signaling, play a critical role in inducing alterations of cell functions and cell death [29]. Using two different experimental models of astrocytes in culture, we demonstrated that human HDL exert a protective effect against copper-induced oxidative damage of astrocytes. Lower levels of hydroperoxides and a lower decrease in cell viability have been observed in astrocytes oxidized in the presence of HDL with respect to cells oxidized alone. These results suggest that HDL, during the co-incubation with cells, exert a protective effect against oxidative damage, in good agreement with previous studies on LDL oxidized in the presence of HDL [11,12]. The lower susceptibility of astrocytes to copper-induced oxidative damage in the presence of HDL confirms an antioxidant role that could be related to the ability of HDL to bind metal ions in sites at the lipoprotein surface [30]. The antioxidant ability of HDL against lipid peroxidation has also been related to the effect of apoproteins and enzymes associated to the HDL surface. In fact, the enzymes paraoxonase (PON) and platelet activating factor acetylhydrolase (PAF-HA), are able to hydrolyse oxidized
G. Ferretti et al. / Biochimica et Biophysica Acta 1635 (2003) 48–54
fatty acid from phospholipids and prevent their accumulation on LDL, blocking the atherogenic and inflammatory response induced by OX-LDL [31]. In the present study, we also demonstrated a significant decrease in the levels of hydroperoxides associated with CuOX-astrocytes incubated with HDL for 4 h (experimental model B). These results suggest that HDL could protect astrocytes from accumulating lipid hydroperoxides. In our experimental conditions, the levels of lipid hydroperoxides associated with HDL co-incubated with Cu+ + and astrocytes were significantly higher with respect to the values observed in culture media of copper-oxidized astrocytes. We suggest that lipid peroxidation products could be released from oxidized cells to culture media and HDL could stimulate the efflux from OX-astrocytes with some mechanism(s) that deserve further studies. This hypothesis is supported by previous studies using HDL incubated with OX-LDL or OX-cells [11,14]. Klimov et al. [14] demonstrated that the incubation of HDL in the presence of Cu+ +-oxidized erythrocyte membranes increased the content of lipid hydroperoxides in HDL and suggested a transport of phospholipids containing hydroperoxides from oxidized erythrocyte membranes to lipoproteins. A transfer of hydroperoxides from oxidized LDL to HDL and an exchange of lipid peroxidation products between the lipoproteins were previously demonstrated [11]. The high ability of HDL to accumulate lipid hydroperoxides in human plasma has been demonstrated by Bowry et al. [32], therefore it was hypothesised that HDL could play an important role in the transport and detoxification of oxidation products in human plasma [14,32]. In conclusion, using different experimental models, we demonstrated that HDL exert a protective effect against copper-triggered oxidative damage on astrocytes in agreement with previous studies using different cellular models [13,14,33]. Copper-induced cytotoxicity is a multifactorial phenomenon underlying actions due to the generation of ROS and RNS that may alter cell membranes and essential biomolecules with loss of biological functions [25 – 27]. Moreover, modulation of mitochondrial functions, gene expression and production of cytotoxic mediators have been demonstrated in copper-treated cells [26]. The results obtained in the present study suggest that the protective effect of HDL against cell peroxidation could be exerted by different mechanisms: metal binding properties and/or inhibition of the accumulation of lipid hydroperoxides in oxidized cells could be involved. Previous studies have demonstrated that cells preincubated with HDL are less sensitive to the oxidizing effect exerted by copper ions and it has been suggested that HDL could increase cell resistance against the cytotoxicity of oxidant agents by modifying the signaling of intracellular chemical mediators and the intracellular metabolism [13,33]. This hypothesis is supported by several recent studies which have demonstrated that plasma lipoproteins regulate signal transduction and modulate the effects of
53
several molecules (cytokine, free radicals) on cell functions [34]. In particular, HDL modulate the phosphoinositidespecific phospholipase C pathway and protein kinase C (PKC), the sphingosine kinase signaling pathway and endothelial NO production [34]. Moreover, HDL regulate the activation of extracellular-regulated kinases and NF-kappaB signaling cascade in cells in culture [35]. Concerning the patho-physiological relevance of our results, it has to be stressed that in our experimental conditions, the effect of copper on lipid peroxidation of astrocytes was observed at a copper concentration similar to that observed in human CSF of patients affected by neurodegenerative diseases [19,20]. Oxidative stress, defined as the imbalance between biochemical processes leading to production of ROS and the cellular antioxidant cascade, causes molecular damage that can lead to critical failure of biological functions and ultimately to cell death. The role of oxidative damage in the pathogenesis of neurological disease such as Alzheimer’s (AD) and Parkinson’s diseases (PD) was widely demonstrated [3 –5]. Recent studies have demonstrated that astrocytes play a key role in the antioxidant defence of brain [6,7]. The increase in antioxidant enzymes in astrocytes exposed to oxidative damage was not sufficient to avoid lipid peroxidation in cortical astrocyte cultures [7]. Therefore, defence mechanisms in the extracellular space could play a key role in protecting brain cells against oxidative damage and cytotoxic molecules. Moreover, lipoprotein particles similar for density and chemical composition to plasma HDL were isolated in human CSF [15,16]. A higher susceptibility of CSF lipoproteins of AD patients and lower levels of antioxidants have been demonstrated in the CSF of AD patients [18,36]. Therefore, the protective role exerted by HDL against Cu+ +induced oxidative damage to astrocytes could be of physiological relevance. Recent studies have demonstrated that paraoxonase is expressed in brain tissue [37]. Studies are in progress in our laboratory to investigate the activity of paraoxonase in the lipoproteins of human CSF and to study whether the lipoprotein particles isolated from human CSF of controls or AD patients protect astrocytes efficiently as HDL isolated from human plasma.
Acknowledgements This work was supported by a grant from MURST 60% (GF).
References [1] G. Perry, L.M. Sayre, C.S. Atwood, R.J. Castellani, A.D. Cash, C.A. Rottkamp, M.A. Smith, The role of iron and copper in the aetiology of
54
[2]
[3] [4]
[5]
[6] [7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
G. Ferretti et al. / Biochimica et Biophysica Acta 1635 (2003) 48–54 neurodegenerative disorders: therapeutic implications, CNS Drugs 16 (5) (2002) 339 – 352. G. Rotilio, M.T. Carri, L. Rossi, M.R. Circolo, Copper-dependent oxidative stress and neurodegeneration, IUBMB Life 50 (4 – 5) (2000) 309 – 314. N.A. Simonian, J.T. Coyle, Oxidative stress in neurodegenerative diseases, Annu. Rev. Pharmacol. Toxicol. 36 (1996) 83 – 106. D.L. Marcus, C. Thomas, C. Rodriguez, K. Simberkoff, J.S. Tsai, J.A. Strafaci, M.L. Freedman, Increased peroxidation and reduced antioxidant enzyme activity in Alzheimer’s disease, Exp. Neurol. 150 (1) (1998) 40 – 44. R. Dei, A. Takeda, H. Niwa, M. Li, Y. Nakagomi, M. Watanabe, T. Inagaki, Y. Washimi, Y. Yasuda, K. Horie, T. Miyata, G. Sobue, Lipid peroxidation and advanced glycation end products in the brain in normal aging and in Alzheimer’s disease, Acta Neuropathol. (Berl.) 104 (2) (2002) 113 – 122. S. Desagher, J. Glowinski, J. Premont, Astrocytes protect neurons from hydrogen peroxide toxicity, J. Neurosci. 16 (8) (1996) 2553 – 2562. F. Klamt, C. Gottfried, F. Tramontina, F. Dal-Pizzol, M.L. Da Frota, J.C. Moreira, R.D. Dias, E. Moriguchi, S. Wofchuk, D.O. Souza, Time-related increase in mitochondrial superoxide production, biomolecule damage and antioxidant enzyme activities in cortical astrocyte cultures, NeuroReport 13 (12) (2002) 1515 – 1518. J.M. Servitja, R. Masgrau, R. Pardo, E. Sarri, F. Picatoste, Effects of oxidative stress on phospholipid signalling in rat cultured astrocytes and brain slices, J. Neurochem. 75 (2) (2000) 788 – 794. S.H. Chen, S.H. Liu, Y.C. Liang, J.K. Lin, S.Y. Lin-Shiau, Oxidative stress and c-Jun-amino-terminal kinase activation involved in apoptosis of primary astrocytes induced by disulfiram-Cu (2+) complex, Eur. J. Pharmacol. 414 (2 – 3) (2001) 177 – 188. E. Rohrdanz, G. Schmuck, S. Ohler, Q.H. Tran-Thi, R. Kahl, Changes in antioxidant enzyme expression in response to hydrogen peroxide in rat astroglial cells, Arch. Toxicol. 75 (3) (2001) 150 – 158. S. Parthasarathy, J. Barnett, L.G. Fong, High-density lipoprotein inhibits the oxidative modification of low-density lipoproteins, Biochim. Biophys. Acta 1044 (2) (1990) 275 – 283. M.I. Mackness, P.N. Durrington, B. Mackness, How high-density lipoprotein protects against the effects of lipid peroxidation, Curr. Opin. Lipidol. 11 (4) (2000) 383 – 388. G. Ferretti, T. Bacchetti, C. Moroni, A. Vignini, G. Curatola, Protective effect of human HDL against Cu2 +-induced oxidation of astrocytes, J. Trace Elem. Med. Biol. 17 (3) (2003) 25 – 30. A.N. Klimov, K.A. Kozhevnikova, A.A. Kuzmin, A.S. Kuznetsov, E.V. Belova, On the ability of high density lipoproteins to remove phospholipid peroxidation products from erythrocyte membranes, Biochemistry (Mosc.) 66 (3) (2001) 300 – 304. S. Koch, N. Donarski, K. Goetze, M. Kreckel, H.J. Stuerenburg, C. Buhmann, U. Beisiegel, Characterization of four lipoprotein classes in human cerebrospinal fluid, J. Lipid Res. 42 (7) (2001) 1143 – 1151. M.J. Ladu, C. Reardon, L. Van Eldik, A.M. Fagan, G. Bu, D. Holtzman, G.S. Getz, Lipoproteins in the central nervous system, Ann. N.Y. Acad. Sci. 903 (2000) 167 – 175. R.E. Pitas, J.K. Boyles, S.H. Lee, D. Foss, R.W. Mahley, Astrocytes synthesize apolipoprotein E and metabolize apolipoprotein-E containing lipoproteins, Biochim. Biophys. Acta 917 (1) (1987) 148 – 161. C.N. Bassett, K.S. Montine, M.D. Neely, L.L. Swift, T.J. Montine, Cerebrospinal fluid lipoproteins in Alzheimer’s disease, Microsc. Res. Tech. 50 (4) (2000) 282 – 286. H.S. Pall, A.C. Williams, D.R. Blake, J. Lunec, J.M. Gutteridge, M. Hall, A. Taylor, Raised cerebrospinal-fluid copper concentration in Parkinson’s disease, Lancet 2 (8553) (1987) 238 – 241.
[20] R. Squitti, D. Lupoi, P. Pasqualetti, G. Dal Forno, F. Vernieri, P. Chiovenda, L. Rossi, M. Cortesi, E. Cassetta, P.M. Rossigni, Elevation of serum copper levels in Alzheimer’s disease, Neurology 59 (8) (2002) 1153 – 1161. [21] B.H. Chung, J.P. Segrest, M.J. Ray, J.D. Brunzell, J.E. Hokanson, R.M. Krauss, K. Beaudrie, J.T. Cone, Single vertical spin density gradient ultracentrifugation, Methods Enzymol. 128 (1986) 181 – 209. [22] O.H. Lowry, N.J. Rosebrough, A.L. Farr, P.J. Randall, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193 (1951) 265 – 275. [23] R.I. Freshney, Culture of Animal Cells: A Manual of Basic Technique, Wiley, New York, 2000. [24] Y. Oyama, A. Hayashi, T. Ueha, K. Maekawa, Characterization of 2V,7V-dichlorofluorescin fluorescence in dissociated mammalian brain neurons: estimation on intracellular content of hydrogen peroxide, Brain Res. 635 (1 – 2) (1994) 113 – 117. [25] N.T. Watt, N.M. Hooper, The response of neurones and glial cells to elevated copper, Brain Res. Bull. 55 (2) (2001) 219 – 224. [26] J. Pourahmad, P.J. O’Brien, A comparison of hepatocyte cytotoxic mechanisms for Cu2 + and Cd2 +, Toxicology 143 (3) (2000) 263 – 273. [27] S.C. Luza, H.C. Speisky, Liver copper storage and transport during development: implications for cytotoxicity, Am. J. Clin. Nutr. 63 (5) (1996) 812S – 820S. [28] N.E. Saris, I.A. Skulskii, Interaction of Cu+ with mitochondria, Acta Chem. Scand. 45 (10) (1991) 1042 – 1046. [29] N. Zamzami, P. Marchetti, M. Castedo, C. Zanin, J.L. Vayssiere, P.X. Petit, G. Kroemer, Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo, J. Exp. Med. 181 (5) (1995) 1661 – 1672. [30] S.T. Kunitake, M.R. Jarvis, R.L. Hamilton, J.P. Kane, Binding of transition metals by apolipoprotein A-I-containing plasma lipoproteins: inhibition of oxidation of low density lipoproteins, Proc. Natl. Acad. Sci. U. S. A. 89 (15) (1992) 6993 – 6997. [31] M.I. Mackness, P.N. Durrington, HDL, its enzymes and its potential to influence lipid peroxidation, Atherosclerosis 115 (2) (1995) 243 – 253. [32] V.W. Bowry, K.K. Stanley, R. Stocker, High density lipoprotein is the major carrier of lipid hydroperoxides in human blood plasma from fasting donors, Proc. Natl. Acad. Sci. 89 (21) (1992) 10316 – 10320. [33] I. Suc, I. Escargueil-Blanc, M. Troly, R. Salvayre, A. Negre-Salvayre, HDL and ApoA prevent cell death of endothelial cells induced by oxidized LDL, Arterioscler. Thromb. Vasc. Biol. 17 (1997) 2158 – 2166. [34] J.R. Nofer, B. Kehrel, M. Fobker, B. Levkau, G. Assmann, A. von Eckardstein, HDL and arteriosclerosis: beyond reverse cholesterol transport, Atherosclerosis 161 (1) (2002) 1 – 16. [35] S.H. Park, J.H. Park, J.S. Kang, Y.H. Kang, Involvement of transcription factors in plasma HDL protection against TNF-alpha-induced vascular cell adhesion molecule-1 expression, Int. J. Biochem. Cell Biol. 35 (2) (2003) 168 – 182. [36] C.N. Bassett, M.D. Neely, K.R. Sidell, W.R. Markesbery, L.L. Swift, T.J. Montine, Cerebrospinal fluid lipoproteins are more vulnerable to oxidation in Alzheimer’s disease and are neurotoxic when oxidized ex vivo, Lipids 34 (12) (1999) 1273 – 1280. [37] L. Rodrigo, A.F. Hernandez, J.J. Lopez-Caballero, F. Gil, A.R. Pla, Immunohistochemical evidence for the expression and induction of paraoxonase in rat liver, kidney, lung and brain tissue. Implications for its physiological role, Chem. Biol. Interact. 137 (2) (2001) 123 – 137.