* Neurotoxicology Laboratory, Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana 47907-1333; and
Department of Pharmacology and Toxicology, Charles University, Faculty of Medicine, Karlovarska 48, CZ 301 66 Pilsen, Czech Republic
Received May 16, 2001; accepted July 23, 2001
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ABSTRACT |
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Key Words: trimethyltin; protein kinase C; oxidative stress; excitotoxicity; cerebellar granule cell death.
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INTRODUCTION |
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Findings in our laboratory show that exposure of differentiated PC12 cells to TMT causes translocation and activation of protein kinase C (PKC) which contributes to TMT-induced neurotoxicity (Pavlakovic et al., 1995). The sustained activation of PKC was mediated by a G-protein coupled receptor and blocked by MCPG, an inhibitor of metabotropic glutamate receptors (Kane et al., 1998
). Mechanisms by which TMT is neurotoxic remain unclear (Costa, 1998
). Oxidative stress is recognized as a common factor in the toxicity of many compounds (Stohs and Bagchi, 1995
), and generation of oxidative species in mouse brain after TMT treatment has been reported (Ali et al., 1992
). Also, neurotoxic effects of TMT can be inhibited by antioxidants (Clerici, 1996
). The present study evaluates oxidative stress, PKC, and glutamate excitotoxicity as factors in the apoptotic and necrotic modes of neuronal cell death produced by TMT.
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MATERIALS AND METHODS |
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Cell culture.
Primary cerebellar granule cells were cultured from 78-day-old rat pups as described previously (Gunasekar et al., 1995a). Briefly, cerebellums were dissected and cells were dissociated and cultured for 10 days in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum, 22 mM glucose, 25 mM KCl, and 1 ml penicillin/streptomycin (5000 U/ml/L) at pH 7.4. Cells were plated at a density of 2 x 106 cells/ml on a 35 mM culture dish containing a sterile cover glass previously coated with 10 µg/ml of poly-L-lysine (mol. wt. = 30,00070,000) and used for experiments in 810 days. Cytosine arabinofuranoside (10 µM) was added 1824 h after plating to prevent proliferation of non-neuronal cells. At 9 days, in vitro cells are considered mature and
95% of the surviving cells are granule cells.
Detection of DNA fragmentation by TUNEL staining.
Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling of fragmented DNA (TUNEL) was performed on paraformaldehyde (4% in phosphate-buffered saline)-fixed cerebellar granule cells (Apotag in situ apoptosis detection kit; Oncor, Gaithersburgh, MD). Briefly, cells were preincubated in equilibration buffer containing 0.1 M potassium cacodylate, pH 7.2, 2 mM CaCl2, and 0.2 mM dithiothreitol for 10 min at room temperature. The tissue was then incubated in the TUNEL reaction mixture (containing 200 mM potassium cocdylate (pH 7.2), 4 mM MgCl2, 2 mM 2-mercaptoethanol, 30 µM biotin-16-dUTP, and 300 U/ml TdT) in a humidified chamber at 37°C for 1 h. After incubating in stop/wash buffer for 10 min, the elongated digoxigenin-labeled DNA fragments were visualized, using antidigoxigenin peroxidase antibody solution followed by staining with DAB/H2O2 in PBS (pH 7.4). Cells were then counterstained with hematoxylin. The selectivity of the assay is based on the presence of 3-OH DNA fragmented ends in apoptotic cells.
Extraction and electrophoresis of DNA.
Intracellular DNA was extracted according to the modified method described previously (Herget et al., 1998; Herrmann et al., 1994
). Untreated or TMT-treated granule cells (2 x 107) were washed with PBS (pH 7.4) twice and collected by centrifugation. Cell pellets were lysed with 1 ml buffer (10 mM TrisHCl, pH 7.4, 10 mM EDTA, 0.5% sodium dodecyl sulfate) for 10 min on ice. After treatment with RNase A (final concentration 100 µg/ml) for 1 h at 37°C, the samples were treated with proteinase K (final concentration 100 µg/ml) and incubated for 4 h at 50°C. DNA was precipitated by adding 0.1 volumes of 3 M sodium acetate (pH 5.2) and 2.5 volumes of cool absolute ethanol. Pellets were dissolved in Tris-EDTA buffer. For analysis, 1020 µg of DNA was loaded on a 1.2% agarose gel containing ethidium bromide (0.5 µg/ml) and electrophoresed at 70 V for 1.5 h. DNA was visualized under ultraviolet light and photographed.
Quantitation of necrotic cell death.
Cytotoxicity was estimated by measurement of LDH efflux from damaged cells into the medium over a 24-h exposure. Cerebellar granule cells grown in 24-well culture dishes (10 days in vitro) were used for the assays. All stock solutions of drugs were sterilized by filtration. Pretreatments were added 10 min before TMT unless otherwise indicated. After incubation, medium was removed and cells lysed for 10 min in 0.5% (v/v) Triton X-100 in 0.1 M potassium phosphate buffer, pH 7.4. The supernatant was removed after centrifugation at 10,000 x g for 5 min and LDH activity was determined by the spectrophotometric method of Vassault (1983) in both the medium and lysis buffer. LDH released into extracellular medium was calculated as LDH release = LDH in medium/LDH in medium + LDH in lysis buffer.
To verify that LDH release reflects necrotic cell death in the cerebellar granule cells, 2 DNA fluorescent dyes were used: SYTO-13 and propidium iodide (Ankarcrona et al., 1995). Both dyes stain DNA but only SYTO-13 is membrane permeable. Thus, SYTO-13 stains normal cells with a green fluorescence, whereas only cells with disrupted plasma membranes stain red with propidium iodide. The percentage of cells stained positive for propidium iodide was determined and used along with LDH release as an estimate of necrotic cell death.
Monitoring reactive oxygen species (ROS).
2,7-Dichlorofluorescein (DCF), the fluorescent, oxidized product of DCF-DA, was assayed to monitor generation of ROS and NO (Gunasekar et al., 1995b). Cerebellar granule cells were loaded with DCF-DA and fluorescence was monitored with an SLM-8000 spectrofluorometer attached via fiberoptics to a Nikon diaphot TMD microscope. To load cells with DCF-DA, culture medium was replaced with 1 ml pre-warmed Kreb's Ringer solution and 10 µl of 30 mM DCF-DA was added and incubated for 15 min at room temperature in the dark. Coverslips containing granule cells loaded with DCF were placed in a cell chamber (Medical System, Inc., Greenvale, NY) mounted on a heated (37°C) microscope stage. Fluorescence of single cells was monitored over a 10-min period after addition of TMT (0.110.0 µM) in the presence and absence of L-NAME (nitric oxide synthase inhibitor, 300 µM), catalase (200 U/ml), CHEL (PKC-inhibitor, 1 µM), MK-801 (1 µM) or MCPG (1 mM) at excitation and emission wavelengths of 475 and 525 nm. All the drugs were added 10 min before TMT and fluorescence intensity was recorded over a 10-min period.
Measurement of NOS activity.
As an index of NOS activity, nitrite formation (NO breakdown product) was studied by the method of Ignarro et al. (1987). Nitrite was quantitated after exposure to TMT at different doses (0.110.0 µM) in the presence and absence of L-NAME, CHEL, MK-801 or MCPG for 24 h. Granule cells grown in 6-well culture dishes were used for these experiments. Nitrite levels were estimated at 548 nm, after adding 500 µl of N-(1-napthyl)-ethylenediamine to 1 ml of incubation medium of treated cells.
Thiobarbituric acid assay.
Thiobarbituric acid reactive substances (TBARS), an index of lipid peroxidation, was quantified in cerebellar granule cells using the method of Ohkawa et al. (1979) with minor modifications. Cultured granule cells (6 x 106 cells) were treated with TMT (1.0 µM) in the presence and absence of L-NAME or CHEL for 14 h and then the cells were suspended in media (PMA was used as a positive control). This was followed by addition of 100 µl of 10% (W/V) sodium dodecyl sulfate for solubilization, and then 0.65 ml of 0.5% (W/V) thiobarbituric acid in 20% (V/V) glacial acetic acid (pH 3.5) was added. The mixtures were then incubated at 80°C for 30 min, cooled, and absorbance was recorded at 532 nm against a blank (without cells). TBARS formation was estimated using an extinction coefficient of 1.56 x 105 M1 cm1. To determine if TBARS were generated under control conditions, assays were run in which the respective pretreatment compounds were added individually, The pretreatments did not generate an increase in TBARS above basal conditions. Control cells had a low level of TBARS (1.03 ± 0.03 µM TBARS) as detected by this assay. TBARS levels of the treatment groups were expressed as µM TBARS formation per 6 x 106 cells over the control groups.
Statistics.
Data were expressed as mean ± SE and statistical significance was assessed by 1-way analysis of variance (ANOVA) followed by the Tukey-Kramer multiple range test or Dunnett's comparison test. Differences were considered significant at p < 0.05.
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RESULTS |
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TMT-Induced Oxidant Species Generation
Previous data implicated oxidative stress in the apoptotic and necrotic cell damage caused by TMT. To confirm that TMT can increase formation of oxidative species in cerebellar granule cells, cells were loaded with DCF and were fluorescence monitored after the TMT addition. In 6 experiments, the level of oxidative radicals averaged 123 ± 54% of control following addition of 0.1 µM TMT but the increase was not significant. After 1.0 µM TMT, levels were 354 ± 83% of control and the difference was significant at p < 0.02. Since the data were collected 10 min after TMT treatment, they reveal only the initial effect of TMT. Oxidative stress most likely continues through the entire 24-h-exposure period. It is concluded that cerebellar granule cells exposed to TMT generate oxidative radicals intracellularly. These data are supportive of previous data showing blockade of TMT-induced cell death by antioxidants.
Pretreatment of cells with L-NAME or catalase before addition of TMT, significantly blocked oxidative species generation, suggesting that both nitric oxide and other ROS are involved in the action of TMT (Fig. 6). Furthermore, metabotropic glutamate receptor antagonist MCPG (but not NMDA receptor antagonist MK-801) prevented TMT-induced ROS accumulation, indicating significant involvement of metabotropic glutamate receptor activation in TMT-induced oxidative stress (Fig. 6
).
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DISCUSSION |
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In neurons, NO is generated after phosphorylation of NOS by PKC (Bredt and Snyder, 1992). Activation of PKC is important in granule cell apoptosis and necrosis since inhibition of PKC prevented cell death in both instances. PKC activation may contribute to oxidative stress in cerebellar granule cells after TMT exposure by increasing NOS activity. This would explain the decrease in NO formation following pretreatment with chelerythrine (PKC inhibitor) or L-NAME. In differentiated PC12 cells TMT treatment translocates and activates PKC, and chelerythrine protects against TMT-mediated cytotoxicity (Kane et al., 1998
; Pavlakovic et al., 1995
). However mediation of TMT toxicity by PKC is blunted in the presence of glial cells (Gunasekar et al, 2001). It is concluded that PKC mechanisms are part of the TMT-induced apoptotic and necrotic cell death cascade in cerebellar granule cells, even though hydrogen peroxide is involved only in the apoptotic effect.
Oxidative stress is an initiating event in apoptosis (Payne et al., 1995) and is associated with a number of neurodegenerative conditions (Martin et al., 1998
; Mattson and Furukawa, 1996
). In our study, substances that prevented TMT-induced apoptosis (chelerythrine, catalase, and L-NAME) also prevented accumulation of oxidative species supporting the hypothesis that TMT-induced toxicity is due to reactive oxygen radicals. Other reports show that oxidative radicals can be contributing factors in either apoptotic or necrotic cell death (Kass et al., 1996
; Mason et al., 1999
; Zeevalk et al., 1998
).
Oxidative stress may mediate cell death in vivo and in vitro following glutamate receptor activation (Gunasekar et al., 1995a; Martin et al., 1998
). Blockade of NMDA receptors protected against cyanide toxicity in hippocampal cultures (Patel et al., 1990
, 1992
) and cerebellar granule cells (Gunasekar et al., 1996
). However, in the present study, MK-801 blocked neither oxidative species formation nor the apoptosis induced by TMT. On the other hand, MCPG blocked oxidative species and NOS activation, while it did not prevent DNA fragmentation or apoptosis. Oxidative stress appears to be an initiating event in TMT-induced granule cell apoptosis and is independent of NMDA glutamate-receptor activation.
Glutamate excitotoxicity can play a significant role in the signaling cascade associated with apoptosis (Chang, 1986; Martin et al., 1998
), and some suggest TMT neurotoxicity is mediated by glutamate (Koczyk, 1996
, Patel et al., 1990
). Yet in the present study, glutamate excitotoxicity plays little role in TMT-induced apoptosis. In contrast, TMT-induced necrosis of granule cells involves both NMDA and non-NMDA glutamate receptors. Cerebellar granule cells that undergo necrosis due to TMT may be reacting to 2 different mechanisms, one an oxidative stress mechanism (due to non NMDA glutamate receptors) and another related to NMDA glutamate receptor activation. The glutamate NMDA response may potentiate the oxidative effect and accelerate TMT-induced neuronal cell death.
Cerebellar dysfunction (ataxia and nystagmus) has been reported in humans after acute TMT intoxication (Besser et al., 1987), and vacuolization and dense granules were noted in cells of rat cerebellum 24 h after treatment (Brown et al., 1984
). Also permanent, dose-related weight deficits were seen in rat cerebellum after administration of TMT to developing animals (Miller et al., 1984). Cerebellar destruction by TMT has also been reported in gerbils (Nolan et al., 1990
). Present results indicate mechanisms by which these TMT-induced changes in the cerebellum occur.
In conclusion, our results demonstrate that low-level exposure to TMT damages cerebellar granule cells through an apoptotic pathway. Higher concentrations of TMT cause necrosis in which NMDA and non-NMDA glutamate receptor activation are involved. Generation of oxidative species occurs in response to both low and high concentrations of TMT. Antioxidants can diminish either the apoptosis or necrosis caused by TMT. The nature of the oxidative species generated by TMT varies with the dose employed, since hydrogen peroxide plays little role in TMT-induced necrosis but is important in the apoptotic response. PKC and NO, however, are involved in both the apoptotic and necrotic actions of TMT. Thus, the neurotoxic actions of TMT are complex and vary with the concentration of TMT employed.
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ACKNOWLEDGMENTS |
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NOTES |
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