doi:10.1016/j.brainres.2005.06.030 
Copyright © 2005 Elsevier B.V. All rights reserved.
Research Report
Mitochondrially targeted vitamin E and vitamin E mitigate ethanol-mediated effects on cerebellar granule cell antioxidant defense systems
Kendra I. Siler-Marsiglio
, 
, Qun Pan, Michael Paiva, Irina Madorsky, Nila C. Khurana and Marieta B. Heaton
Department of Neuroscience, McKnight Brain Institute, University of Florida, 100 S Newell Drive, Room L3-151, Gainesville, FL 32611, USA
Accepted 10 June 2005.
Available online 18 July 2005.
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Abstract
Ethanol (EtOH) disrupts the structure and function of the developing nervous system, sometimes leading to birth defects associated with fetal alcohol syndrome (FAS). Animal FAS models indicate that cellular membrane peroxidation, intracellular oxidant accumulation, and suppression of endogenous antioxidant enzymes contribute to the toxic effects of EtOH. Mitochondrially targeted vitamin E (MitoVit E), a chemically engineered form of vitamin E (VE) designed to accumulate in the mitochondria, has been shown to inhibit intracellular oxidant accumulation and cell death more effectively than VE. In previous investigations, we have shown that, in vivo, VE reduces neuronal death in the developing cerebellum of EtOH-exposed animals, and, in vitro, VE prevents apoptotic and necrotic death of EtOH-exposed cerebellar granule cells (CGCs). The present investigation shows that, in a FAS CGC model, 1 nM MitoVit E renders significant neuroprotection against EtOH concentrations as high as 1600 mg/dL. The present study also demonstrates that, in this same model, MitoVit E mitigates EtOH-induced accumulation of intracellular oxidants and counteracts suppression of glutathione peroxidase/glutathione reductase (GSH-Px/GSSG-R) functions, protein expression of gamma-glutamylcysteine synthetase (γ-GCS), and total cellular glutathione (GSH) levels. In the presence and absence of EtOH, VE amplifies the protein expression levels of γ-GCS, an enzyme that performs the rate-limiting step for GSH synthesis, and total GSH levels. These results suggest that MitoVit E and VE ameliorate EtOH toxicity through non-oxidant mechanisms–modulations of endogenous cellular proteins–and antioxidant means.
Keywords: Mitochondrially targeted vitamin E; Vitamin E; Ethanol; Glutathione; Superoxide dismutase; Gamma-glutamylcysteine synthetase
Neuroscience classification codes: Development and regeneration, Neuronal death
Article Outline
- 1. Introduction
- 2. Materials and methods
- 2.1. Primary cerebellar granule cell (CGC) culture
- 2.2. EtOH treatment
- 2.3. Assessment of cellular viability
- 2.4. Assessment of oxidant accumulation
- 2.5. Assessment of GSH-Px activity
- 2.6. Assessment of GSSG-R assay
- 2.7. Assessment of total intracellular GSH levels
- 2.8. Western blot analysis of γ-GCS
- 2.9. Statistical analyses
- 3. Results
- 3.1. Dose-dependent effects of MitoVit E against EtOH-induced cell death
- 3.2. Effects of MitoVit E, VE, and EtOH on intracellular oxidant accumulation
- 3.3. Effects of MitoVit E, VE, and EtOH on GSH-Px activity
- 3.4. Effects of MitoVit E, VE, and EtOH on GSSG-R activity
- 3.5. Effects of MitoVit E, VE, and EtOH on γ-GCS protein expression
- 3.6. Effects of MitoVit E, VE, and EtOH on total cellular GSH
- 4. Discussion
- Acknowledgements
- References
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Fig. 1. The dose-dependent effects of MitoVit E on the neurotoxicity of EtOH in P9 CGCs. Experimental cells were established for 24 h with 0, 1, 10, 25, and 50 nM MitoVit E. All cells were subsequently exposed to 0, 400, 800, and 1600 mg/dL EtOH for 24 h. Results are presented as the mean percentage ± SE of MTT reduced relative to that of control cells (0 nm MitoVit E/0 mg/dL EtOH). Two-factor ANOVA showed that MitoVit E and EtOH had significant main effects on CGC viability, and these two variables also had an interaction effect (P < 0.01). Post-hoc Fischer's LSD comparisons showed that the following MitoVit E or EtOH-only treatment groups were different from the control group and the following MitoVit E + EtOH-treated groups were different from EtOH-only groups: aMitoVit E [10 nM]-only increased compared to controls, P = 0.0002; bMitoVit E [25 nM]-only increased compared to controls, P = 0.0007; cMitoVit E [50 nM]-only increased compared to controls, P < 0.0001; dEtOH [400]-only decreased compared to controls, P = 0.0028; eMitoVit E [10 nM] + EtOH [400] increased compared to EtOH [400], P = 0.0001; fMitoVit E [25 nM] + EtOH [400] increased compared to EtOH [400], P < 0.0001; gMitoVit E [50 nM] + EtOH [400] increased compared to EtOH [400], P < 0.0001; hEtOH [800]-only decreased compared to controls, P = 0.0001; iMitoVit E [1 nM] + EtOH [800] increased compared to EtOH [800], P = 0.0102; jMitoVit E [10 nM] + EtOH [800] increased compared to EtOH [800], P = 0.0008; kMitoVit E [25 nM] + EtOH [800] increased compared to EtOH [800], P < 0.0001; lMitoVit E [50 nM] + EtOH [800] increased compared to EtOH [800], P < 0.0001; mEtOH [1600 mg/dL]-only decreased compared to controls, P < 0.0001; nMitoVit E [1 nM] + EtOH [1600] increased compared to EtOH [1600], P = 0.0001; oMitoVit E [10 nM] + EtOH [1600] increased compared to EtOH [1600], P < 0.0001; pMitoVit E [25 nM] + EtOH [1600] increased compared to EtOH [1600], P < 0.0001; qMitoVit E [50 nM] + EtOH [1600] increased compared to EtOH [1600], P < 0.0001.
 |
Fig. 2. Effects of MitoVit E on EtOH-induced intracellular oxidant accumulation in P9 CGCs. Experimental cells were established for 24 h with MitoVit E (0 or 50 nM). All cells were subsequently exposed to 0, 400, 800, or 1600 mg/dL of EtOH for 24 h. Results are presented as the mean percentage ± SE of fluorescent probe DCFH-DA emissions relative to that of controls. Two-factor ANOVA showed that MitoVit E and EtOH had significant main effects on oxidant accumulation, and these two variables also had an interaction effect (P < 0.01). Post-hoc Fischer's LSD comparisons showed that the following MitoVit E or EtOH-only treatment groups were different from the control group, and the following MitoVit E + EtOH-treated groups were different from EtOH-only groups: aEtOH [400]-only increased compared to controls, P = 0.0355; bMitoVit E [50 nM] + EtOH [400] decreased compared to EtOH [400], P = 0.0245; cEtOH [800]-only increased compared to controls, P = 0.0291; dMitoVit E [50 nM] + EtOH [800] decreased compared to EtOH [800], P = 0.0299; eEtOH [1600]-only increased compared to controls, P = 0.0391; fMitoVit E [50 nM] + EtOH [1600] decreased compared to EtOH [1600], P = 0.0407.
 |
Fig. 3. Effects of MitoVit E, VE, and EtOH on GSH-Px activity in P9 CGCs. Experimental cells were established for 24 h with MitoVit E (0 or 50 nM; MVE) or VE (0 or 50 μM). All cells were subsequently exposed to 0, 400, or 1600 mg/dL of EtOH (Et) for 24 h. Controls (C) were not exposed to either antioxidant or EtOH. Results are presented as the mean percentage ± SE oxidation of NADPH to NADP+ by GSH-Px over time measured at 340 nm relative to that of C. Two-factor ANOVA showed that MitoVit E and EtOH had significant main effects on GSH-Px activity (P < 0.01). Additionally, MitoVit E and EtOH had an interaction effect (P = 0.006). VE did not have a significant main effect on GSH-Px activity. VE and EtOH did not have an interaction effect. Post-hoc Fischer's LSD comparisons showed that the following MitoVit E or EtOH-only treatment groups were different from the control group, and the following MitoVit E + EtOH-treated groups were different from EtOH-only groups: aEtOH [400]-only decreased compared to controls, P = 0.0037; bEtOH [1600]-only decreased compared to C, P = 0.0113; cMVE-only decreased compared to C, P = 0.0136; dMVE + EtOH [400] increased compared to EtOH [400], P = 0.049; eMVE + EtOH [1600] increased compared to EtOH [1600], P = 0.0466.
 |
Fig. 4. Effects of MitoVit E, VE, and EtOH on GSSG-R activity in P9 CGCs. Experimental cells were established for 24 h with MitoVit E (0 or 50 nM; MVE) or VE (0 or 50 μM). All cells were subsequently exposed to 0, 400, or 1600 mg/dL of EtOH (Et) for 24 h. Controls (C) were not exposed to either antioxidant or EtOH. Results are presented as the mean percentage ± SE reduction of DNTB by GSSG-R over time measured at 412 nm relative to that of C. Two-factor ANOVA showed that MitoVit E, VE, and EtOH had significant main effects on GSSG-R activity (P < 0.01). Additionally, MitoVit E and EtOH had an interaction effect (P = 0.011). VE and EtOH did not have an interaction effect. Post-hoc Fischer's LSD comparisons showed that the following MitoVit E, VE, or EtOH-only treatment groups were different from the control group and the following MitoVit E + EtOH-treated groups were different from EtOH-only groups: aEtOH [400]-only decreased compared to C, P = 0.0006; bEtOH [1600]-only decreased compared to C, P = 0.0126; cMVE-only increased compared to C, P = 0.0392; dMVE + EtOH [400] increased compared to EtOH [400], P = 0.0004; eMVE + EtOH [1600] increased compared to EtOH [1600], P = 0.0077; fVE-only increased compared to C, P = 0.0282.
 |
Fig. 5. Effects of MitoVit E, VE, and EtOH on γ-GCS protein expression in P9 CGCs. Experimental cells were established for 24 h with MitoVit E (0 nM or 50 nM; MVE) or VE (0 or 50 μM). All cells were subsequently exposed to 0, 400, or 1600 mg/dL of EtOH (Et) for 24 h. Controls (C) were not exposed to either antioxidant or EtOH. Cell lysates (25 μg/lane) were fractionated using SDS-PAGE, transferred to nitrocellulose membranes, and probed with γ-GCS antibody, as described in Materials and methods. Results are presented as the mean percentage ± SE of the densitometric analysis of the signal strength of γ-GCS (73 kDa) relative to that of C. Two-factor ANOVA showed that MitoVit E, VE, and EtOH had significant main effects on γ-GCS protein expression (P < 0.01). Additionally, MitoVit E and EtOH had an interaction effect (P < 0.0001). VE and EtOH did not have an interaction effect. Post-hoc Fischer's LSD comparisons showed that the following MitoVit E, VE, or EtOH-only treatment groups were different from the control group and the following MitoVit E + EtOH-treated groups were different from EtOH-only groups: aEtOH [1600]-only decreased compared to C, P = 0.0009; bMVE + EtOH [1600] increased compared to EtOH [1600], P = 0.0009.
 |
Fig. 6. Effects of MitoVit E, VE, and EtOH total cellular GSH levels in P9 CGCs. Experimental cells were established for 24 h with MitoVit E (0 or 50 nM; MVE) or VE (0 or 50 μM). All cells were subsequently exposed to 0, 400, or 1600 mg/dL of EtOH (Et) for 24 h. Controls (C) were not exposed to either antioxidant or EtOH. Results are presented as the mean percentage ± SE of reduction of DNTB over time measured at 405 nm relative to that of C. Two-factor ANOVA showed that MitoVit E and EtOH had significant main effects on total cellular GSH levels (P < 0.01). Additionally, VE and EtOH had an interaction effect (P = 0.02). MitoVit E and EtOH did not have an interaction effect. Post-hoc Fischer's LSD comparisons showed that the following MitoVit E or EtOH-only treatment groups were different from the control group and the following VE+ EtOH-treated groups were different from EtOH-only groups: aEtOH [1600]-only decreased compared to C, P = 0.0022; bMVE-only increased compared to C, P = 0.047; cVE-only increased compared to C, P = 0.0356; dVE + EtOH [400] increased compared to EtOH [400], P = 0.0209.
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