doi:10.1006phrs.1999.0604, available online at http:www.idealibrary.com on HYDROGEN PEROXIDE-INDUCED OXIDATIVE DAMAGE AND APOPTOSIS IN CEREBELLAR GRANULE CELLS: PROTECTION BY GINKGO BILOBA EXTRACT TAOTAO WEI, YUCHENG NI, JINGWU HOU, CHANG CHEN, BAOLU ZHAO and WENJUAN XIN Institute of Biophysics, Academia Sinica, 15 Datun Road, Chaoyang District, Beijing 100101, PR China Accepted 23 September 1999 The ability of oxidative stress to induce apoptosis and the protective effects of Ginkgo biloba extract EGb761. against this induction were studied in cultures of rat cerebellar granule cells. Cells were exposed to oxidative stress by treatment with 50 M hydrogen peroxide100 M ferrous sulphate which generates hydroxyl radicals by Fenton reaction. Both morphological observation and biochemical analysis revealed that H 2O2FeSO4 treatment induced apoptotic cell death in cerebellar granule cells, which was characterized by chromatin condensation and DNA fragmentation. During this process, the fluidity of the cell membrane decreased markedly, and the conformation of membrane proteins altered significantly. Pretreating cerebellar granule cells with the antioxidant EGb761 Ginkgo biloba extract. effectively attenuated oxidative damage induced by H 2O2FeSO 4, and prevented cells from apoptotic cell death. The results suggested that EGb761 might be used as a potential drug for neuronal diseases associated with the excessive production of reactive oxygen species. 2000 Academic Press KEY WORDS: apoptosis, cerebellar granule cells, EGb761, lipid peroxidation, ESR spin labelling. INTRODUCTION In multicellular organisms homeostasis is maintained by the dynamic balance between cell proliferation and cell death. Two types of cell death, apoptosis and necrosis, may occur under certain physiological and pathological conditions. Physiological apoptosis plays important roles in the development of the central nervous system CNS. 1, while inappropriate apoptosis of neuronal cells leads to some neurodegenerative diseases including Alzheimer s disease AD., Parkinson s disease PD., amyotrophic lateral sclerosis ALS., and various forms of cerebellar degeneration 2. In the last decade more and more evidence has suggested that reactive oxygen species ROS. play an important role in the cascade of events leading to neuronal apoptosis associated with these neuronal diseases 3. Accordingly, antioxidants, which protect neuronal cells from ROSinduced apoptosis, may be used as potential therapeutic agents for these diseases. Corresponding author. 104366180004042707$35.000 In this paper, a primary culture of rat cerebellar granule cells, a relatively homogenous and wellcharacterized culture system of neurons 4, was used as the experimental model. The cytotoxic effect of oxidative stress and the protective effect of EGb761 on neuronal cells were examined. The results will help us to understand the process of neuronal apoptosis and neurodegeneration more thoroughly, which will lead to selective methods to control them. MATERIALS AND METHODS Materials Wistar rats were purchased from Beijing Medical University Beijing, China.. Cell culture plastic ware was purchased from Corning Costar Acton, MA, USA.. Dulbecco s modified Eagle medium DMEM., foetal bovine serum FBS., cell culture supplements, proteinase K, RNase A, and trypsin 1:250. were products of Gibco BRL Grand Island, NY, USA.. Agarose, poly-l-lysine, thiazolyl blue MTT., sodium dodecyl sulphate SDS., sodium N-lauroylsarcosine, 2000 Academic Press
428 Triton X-100, 1,1,3,3-tetraethoxypropane TEP., 5- doxyl stearic acid 5-doxyl., 16-doxyl stearic acid 16-doxyl., and 3-maleimido-proxyl 3-mal. were purchased from Sigma St. Louis, MO, USA.. EGb761 was a generous gift from IPSEN Paris, France.. Other reagents made in China were of analytical grade. Cell culture Primary cultures of rat cerebellar granule cells were prepared following procedures described previously 5. Briefly, cerebella from 7-day-old Wistar rats were dissected out, rinsed with HBSS, and dissociated by mild trypsinization. Cells were plated on 6 1 1 six-well multidishes 210 cells ml, 2 ml well. 6 1 or 24-well multidishes 2.510 cells ml, 0.4 ml 1 well. previously coated with poly-l-lysine. Culture medium consisted of DMEM supplemented with KCl 19.6 mm., glutamine 2 mm., HEPES 10 mm. and foetal bovine serum 10%, vv.. Cells were maintained at 37C in a humidified 5% CO295% air atmosphere. Experiments were carried out 48 h after plating. Treatment of cells with H2O2FeSO4 Cell culture medium was changed into fresh DMEM containing a low concentration of FBS 1%. and cells were cultured for 8 h. Then, 50 M HO 2 2 and 100 M FeSO4 were added into cells from freshly prepared stock solutions and cells were cultured for an indicated time. The antioxidant EGb761 was added to cells from freshly prepared stock solu- 1 tion 1 mg ml in DMEM, sterilized upon filtration. 15 min before the treatment with H 2O2 FeSO 4. Assessment of neurotoxicity Cell viability was assessed by the MTT assay 6. 1 MTT 0.5 mg ml, final concentration. was added to cells cultured in 24-well multidishes and incubated at 37C for 30 min. Then 1 ml of lysis solution 10% SDS, 25% DMF, ph 3.5. was added and the optical density at 570 nm was measured. Morphological studies The ultrastructure of cells was observed by transmission electron microscopy 7. Briefly, cells were fixed with 2.5% glutaraldehyde at 4C for 1 h and post-fixed with 1% OsO4 at 4C for 1 h, dehydrated through a series of graded ethanol solutions, and embedded in resin. Ultrathin sections of samples were stained with uranyl acetatelead citrate and observed with a transmission electron microscope. Detection of DNA fragmentation The laddering pattern of DNA fragmentation was detected by agarose gel electrophoresis 8. Briefly, 1.210 7 cells were harvested by centrifugation at 200 g for 10 min, washed twice with PBS, and lysed in 500 l lysis buffer 10 mm Tris, 10 mm EDTA, 0.5% sodium N-lauroylsarcosine, ph 8.0.. After treatment with 0.5 mg ml 1 proteinase K0.5 mg ml 1 RNase A at 50C for 3 h, the lysate was extracted with phenol, and the DNA was precipitated with ethanol and ammonium acetate. After being dissolved in TrisEDTA buffer 10 mm Tris, 1 mm EDTA, ph 8.0., DNA was analysed by 1.5% agarose gel electrophoresis. The cytosolic DNA fragments were quantified by 7 the diphenylamine method 9. Briefly, 1.210 cells were lysed in 500 l lysis buffer 10 mm Tris, 10 mm EDTA, 0.5% Triton X-100, ph 7.4. on ice for 20 min. The lysate was centrifuged at 12,000 g for 20 min to separate the intact chromatin the pellet. and the cytosolic DNA fragments the supernatant.. Both the pellet and the supernatant were treated with 100 l of 6% perchloric acid at 4C for 30 min. The precipitates were sendimented at 12,000 g for 20 min. The DNA precipitates were heated at 70C for 20 min in 100 l 6% perchloric acid, and were mixed with 200 l of diphenylamine solution 1.5% diphenylamine, 1.5% sulphuric acid, and 0.01% acetaldehyde in glacial acetic acid.. After overnight incubation at 30C in the dark, the optical density at 600 nm was measured, and the percentage of DNA fragmentation was calculated as the ratio of DNA in the supernatant to the total DNA. Measurement of lipid peroxidation Lipid peroxidation in cells was measured by thiobarbituric acid TBA. assay as described previ- 7 ously 10. Briefly, 1.210 cells were mixed with 0.4 ml of 2.8% trichloroacetic acid and 1 ml of 0.67% thiobarbituric acid, heated at 95C for 1 h, extracted with butanol, and the optical density of the organic layer was determined spectrophotometrically. TEP was used as a standard. The protein concentration was determined by the Bradford method. Spin labelling The fluidity of the cell membrane was determined by ESR using 5-doxyl and 16-doxyl as spin labels. The conformation alteration of cell membrane protein sulphydryl groups was determined by ESR using 3-mal as spin label 11. Briefly, cells were washed three times with PBS, mixed with 10 M spin label 5-doxyl, 16-doxyl. or 100 M spin label 3-mal. and incubated at 37C for 30 min 5-doxyl, 16-doxyl. or 3 h 3-mal.. The labelled cells were washed four times with PBS, and then transferred into quartz capillaries for ESR measurement. The ESR spectra were recorded at room temperature 298 K. by a Varian E-109 spectrometer with measurement conditions as: X-band, central magnetic field 325 mt, sweep width 20 mt, microwave power 20 mw, frequency 100 khz, modulation amplitude 0.2 mt, time constant 0.128 s. The methods of calculation for the order parameter S., the rotational correlation time
429 The data were analysed by one-way analysis of variance ANOVA.. A level of P- 0.05 was considered significant. RESULTS H2 O2 r FeSO4-induced cell death Fig. 1. Time-course of H 2 O 2rFeSO4-induced cell death in cerebellar granule cells. Cells were exposed to 50 M H 2 O 2 q 100 M FeSO4 for indicated time and cell viability was assessed by MTT assay. Data were mean " SD of six experiments. c., and the ratio of strongly immobilized component to the weakly immobilized component SrW. were the same as previously described w12, 13x. Statistical analysis Each experiment was performed at least three times and the results are presented as mean " SD. Exposure to 50 M H 2 O 2 q 100 M FeSO4 induced time-dependent cell death in cultures of cerebellar granule cells as indicated by the decrease of MTT reduction Fig. 1.. Pre-treatment with EGb761 100 g mly1. effectively attenuated cell death induced by H 2 O 2rFeSO4. Characteristics of apoptosis After exposure to 50 M H 2 O 2 q 100 M FeSO4 for 8 h, cerebellar granule cells underwent apoptotic cell death, which was characterized morphologically by chromatin condensation and nuclei fragmentation wfig. 2b.x. Quantitative detection of DNA fragmentation by diphenylamine reagent indicated that exposure to 50 M H 2 O 2 q 100 M FeSO4 caused a time-dependant DNA fragmentation wfig. 3b.x. Agarose gel electrophoresis of DNA extracted from H 2 O 2rFeSO4 treated cells showed a ladder pattern Fig. 2. Ultrastructure of cerebellar granule cells. a. normal cell; b. cell exposed to 50 M H 2 O 2 q 100 M FeSO4 for 8 h; c. cell pre-treated with 100 g mly1 EGb761 and then exposed to 50 M H 2 O 2 q 100 M FeSO4 for 8 h. Magnification: 8000 =.
430 significant increase in TBARS levels. In cells pretreated with EGb761, the formation of TBARS was markedly suppressed Fig. 4.. Fig. 3. a, which indicated the formation of monoand oligonucleosomes. The formation of mono- and oligonucleosomes was a well accepted biochemical characteristic of apoptosis. The above experimental results indicated that exposure to 50 M HO100 2 2 M FeSO4 induced apoptotic cell death in cerebellar granule cells. No apparent morphological alterations occurred in cells pre-treated with antioxidant EGb761 Fig. 2. c ; pre-treating cells with EGb761 also prevented DNA from fragmentation. These suggested that pre-treatment with EGb761 prevented cells from apoptosis effectively. Lipid peroxidation in cells Exposure to H 2O2FeSO4 induced lipid peroxidation in cerebellar granule cells as indicated by the Alteration of cell membrane biophysical characteristics The typical ESR spectra of cell membrane spin labelled with 5-doxyl, 16-doxyl and 3-mal are shown in Fig. 5. The nitroxide groups of 5-doxyl are located in the shallow layers of membrane lipids. The order parameter S. calculated from the ESR spectra reflects the degree of order of the lipid chain around the nitroxide radicals. The high S values correspond to high anisotropic motion and low membrane fluidity; accordingly, the low S values correspond to low anisotropic and high membrane fluidity. The nitroxide groups of 16-doxyl are located in the deep layers of membrane lipids. The correlation rotational time. c reflects the time during which molecules are rotated from one conformation to another. Membrane fluidity and c are inversely correlated. There are two types of sulphydryl groups binding sites on cell membrane proteins. One of them is on the surface of the membrane protein. When 3-mal spin labels are bound to the surface, the nitroxide groups of 3-mal can move freely. Hence, the ESR spectra are weakly immobilized. The other type of binding site is in the deep layers of the protein tertiary structure. When maleimide spin labels are bound to the internal sites, the movement of the nitroxide is limited in space. Hence strongly immobilized spectra are produced. Fig. 3.. a DNA nucleosomal fragmentation induced by H 2O2FeSO 4. Lane 1, DNA extracted from normal cells; lanes 24: cells pre-treated with 100 g ml 1 EGb761 and then exposed to 50 M H2O2100 M FeSO4 for 2, 6, and 8 h, respectively; lanes 57: cells exposed to 50 M H2O2100 M FeSO4 for 2, 6, and 8 h, respectively. Lane 8: DNA marker -DNAEcoR IHind III.. b. Time-course of H 2O2FeSO4 induced DNA fragmentation in cerebellar granule cells. Cells were exposed to 50 M H2O2100 M FeSO4 for indicated time and DNA fragmentation was assessed by diphenylamine method. Data were meansd of four experiments.
431 Fig. 4. Time-course of H 2O2FeSO4-induced lipid peroxidation in cerebellar granule cells. Cells were exposed to 50 M H2O2100 M FeSO4 for an indicated time and lipid peroxidation was assessed by TBA method. Data were meansd of four experiments. After treatment with H 2O2FeSO 4, the order parameter and the rotational correlation time of cells increased markedly, suggesting the decrease in the fluidity of both the surface layer and the deep layer of the cell membrane Table I.. The ratio of strongly immobilized component to the weakly immobilized component also increased markedly, suggesting the alteration of the membrane protein conformation. Pretreatment with EGb761 showed significant protective effects against oxidative stressinduced alteration of cell membrane biophysical characteristics. DISCUSSION It has been well documented that reactive oxygen species are involved in neuronal apoptosis. Troy and co-workers reported that the down-regulation of superoxide dismutase SOD. led to apoptosis in neuronal cells 15, while Greenlund and co-workers reported that the up-regulation of SOD delayed apoptosis in neuronal cells 14. In this paper, both morphological and biochemical evidence indicate that apoptosis is induced by exposure of cerebellar granule cells to H 2O2FeSO 4, which generates hy- Fig. 5. ESR spectra of cell membrane spin labelled with 5-doxyl. a, 16-doxyl. b, and 3-mal. c. Cerebellar granule cells were exposed to 50 M H2O2100 M FeSO4 for 8 h and then spin labelled with 5-doxyl, 16-doxyl, and 3-mal. The ESR spectra were recorded by a Varian E-109 spectrometer at room temperature 298 K.. droxyl radicals by Fenton reaction. Electron microscopy revealed nuclear morphological changes characteristic of apoptosis Fig. 2.. These changes were accompanied by the internucleosomal fragmentation of DNA as qualitatively demonstrated by the DNA ladder pattern detected by agarose gel electrophoresis Fig. 3. a. Quantitative analyses of DNA fragmentation demonstrated an increase in DNA fragmentation. Pre-treating cells with antioxidants EGb761 effectively attenuates H 2O2FeSO4- induced apoptosis. After exposure to hydroxyl radicals, the lipid peroxidation level in cerebellar granule cells increased time-dependently. Lipid peroxidation may cause the damage of cell membrane, which alters the biophysical characteristics of the cell membrane such as membrane fluidity and the conformation of protein sulphydryl groups as confirmed by ESR spin labelling. Table I H O FeSO -induced cell membrane biophysical characteristics alteration in cerebellar granule cells 2 2 4 Normal cells H O FeSO exposure EGb761 pretreatment 2 2 4 S 0.7130.011 0.7420.006 0.7210.006 10 10 s. c 9.150.09 9.680.15 9.120.12 SW 0.2230.009 0.2500.016 0.2180.008 Notes. Cells were exposed to 50 M H2O2100 M FeSO4 for 8 h and then spin labelled with 5-doxyl, 16-doxyl, and 3-mal. The cell membrane biophysical characteristics order parameter S., rotational correlation time. c, and the ratio of strongly immobilized component to the weakly immobilized component SW. were calculated from the ESR spectra. Data were meansd of four experiments. P0.05 in comparison with normal cells; P0.05 in comparison with cells treated with 50 M H2O2100 M FeSO4 for 8 h.
432 Fig. 6. Chemical structures of typical flavonoids in EGb761. Lipid peroxidation may also damage the receptors and ion channels on the cell membrane 16, which may result in more serious consequences including calcium influx, and cause cell death finally. On the other hand, unpublished data of our laboratory demonstrated that H O FeSO treatment could 2 2 4 down-regulate the anti-apoptotic gene bcl-2 and upregulate the apoptotic gene p53. H 2O 2 FeSO might induce apoptosis in neuronal cells by 4 regulating the expression of these apoptosis-related genes. Pre-treating cells with EGb761 attenuates oxidative stress-induced alteration of cell membrane biophysical characteristics and prevented the cells from apoptosis effectively. EGb761 is a complex mixture 17, which contains flavonoid and non-flavonoid components. The main chemical structures that flavonoid components possess are aromatic rings and double bonds Fig. 6., so flavonoid components prefer to react with hydroxyl radicals to form an additional product, and thus scavenge the hydroxyl radicals directly. On the other hand, flavonoid components of EGb761 also possess phenolic hydroxyl groups, which may chelate the ferrous cation and decrease the formation of hydroxyl radicals generated from H 2O2 and FeSO 4. The protective effect of EGb761 on apoptosis may be the effect of both scavenging of hydroxyl radicals directly and chelating iron to inhibit Fenton reaction indirectly. On the other hand, EGb761 could up-regulate the activity of SOD, the key antioxidant enzyme in the brain 18, and thus enhance the antioxidant capacity of the neuronal system. In conclusion, exposure of cerebellar granule cells to oxidative stress triggers apoptosis, and pre-treatment with EGb761 could effectively have prevented cells from apoptosis. REFERENCES 1. Henderson CE. Programmed cell death in the developing nervous system. Neuron 1996; 17: 57985. 2. Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science 1995; 267: 145662. 3. Jacobson MD. Reactive oxygen species and programmed cell death. Trends Biochem Sci 1996; 21: 836. 4. Dudek H, Datta SR, Franke TF, Birnbaum MJ, Yao R, Cooper GM, Segel RA, Kaplan DR, Greenberg ME. Regulation of neuronal survival by the serine-
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