Is vitamin E toxic to neuron cells?

Abstract

Besides acting as potent free radical scavengers, tocopherols and tocotrienols have been known to have non-antioxidant properties such as the involvement of alpha-tocopherol (alphaT) in PKC pathway and the anti-cancer properties of gamma-tocotrienol (gammaT3). This study aims to elucidate whether protective effects shown by alphaT and gammaT3 in H(2)O(2)-induced neuron cultures have anti-apoptotic or pro-apoptotic tendency toward the initiation of neuronal apoptosis. H(2)O(2) is used to induce apoptosis in primary cerebellar neuron cultures which is attenuated by pretreatment of alphaT or gammaT3 at concentrations < or =10 microM. Similar to our previous work, gammaT3 was found to be neurotoxic at concentrations > or =100 microM, whereas alphaT showed no neurotoxicity. Cellular uptake of gammaT3 was higher than that of alphaT. Treating cells simultaneously with either gammaT3 or alphaT and with then H(2)O(2) led to higher expression of Bax and Bcl-2 than in neurons exposed to H(2)O(2) alone. Analysis of Bcl-2/Bax ratio as 'survival index' showed that both pretreatment of gammaT3 and alphaT followed by H(2)O(2) increase the 'survival index' of Bcl-2/Bax ratio compared to H(2)O(2)-treated cells, while treatment of gammaT3 alone decrease the ratio compared to unchanged Bcl2/Bax ratio of similar treatment with alphaT alone. Similar treatment of gammaT3 decreased p53 expression and activates p38 MAPK phosphorylation, whereas alphaT did not alter its expression compared to H(2)O(2)-treated cells. Treating neurons with only gammaT3 or alphaT increased the expression of Bax, Bcl-2, p53, and p38 MAPK compared to control with gammaT3 exerting stronger expression for proteins involved than alphaT. In conclusion, low doses of gammaT3 and alphaT confer neuroprotection to H(2)O(2)-treated neurons via their antioxidant mechanism but gammaT3 has stronger pro-apoptosis tendency than alphaT by activating molecules involved in the neuronal apoptotic pathway in the absence of H(2)O(2).

Fig. 1

Cytotoxic effects of α-tocopherol (αT) and γ-tocotrienol (γT3) on cerebellar granule neurons as assessed by a in MTS assay and b in LDH release assays. Percent of MTS reduction corresponds to the viable cell number. Cerebellar neurons were incubated with increasing concentrations of either αT or γT3 for 24 h at 37°C. There was no significant reduction in viable cell number when incubated with αT. However, the viable cell number reduced significantly to 80% when cells were incubated with γT3 at 100 μM and cell viability were reduced further as concentration of γT3 increased. Similar results were obtained with the LDH release assay. *P < 0.05 compared to control. Data are presented as means ± SD, n = 9. Protective effects of α-tocopherol (αT) and γ-tocotrienol (γT3) against H₂O₂-induced cell loss in cerebellar granule neuron cultures, cell viability was determined using MTS assay (c) and apoptosis assay using ELISA kits for ssDNA (d). Neurons were pretreated with different concentrations of αT and γT3 for 1 h before exposure to 100 μM H₂O₂ for 24 h at 37°C. αT and γT3 increased cell viability and decreased apoptosis in H₂O₂-treated cells when used in the concentration range of 1 to 10 μM. At concentration above 10 μM, γT3 was found to increase apoptosis in H₂O₂-treated cells. *P < 0.05 compared to controls. Data are presented as mean ± SD, from 3 independent experiments of triplicate wells (n = 9)

Fig. 2

Fluorescence cell death staining of neurons exposed to different treatments: a control, b 50 μM H₂O₂ treatment, c pre-incubation with γT3 (10 μM) for 1 h followed by H₂O₂ treatment, d pre-incubation with αT (10 μM) for 1 h followed by H₂O₂ treatment. Live cells were stained with 30 μM calcein-AM (green) and dead cells were stained with propidium iodide (PI, red). Neurons undergo early apoptosis as membrane permeability increases, allowing PI to stain cell nuclei. Nuclei show condensation (i) and formation of apoptotic bodies (ii) when exposed to 50 μM H₂O₂. When neurons were pre-incubated with either c γT3 or d αT, cells remained viable and membrane integrity was retained to a greater extent than in cells treated with H₂O₂ alone. Scale bar: 40 μm (refer online version for color figures)

Fig. 3

Uptake of α-tocopherol (αT) and γ-tocotrienol (γT3) in cerebellar granule neurons. Neuron cultures were incubated with 10 μM of either αT or γT3 for 24 hrs at 37°C. Cells were harvested and prepared for HPLC as described in text. Cellular uptake of γT3 and αT is significantly higher than control. Uptake of γT3 is significantly higher than αT uptake into neurons. Data are presented as mean ± SD, n = 3

Fig. 4

Activity of caspases (caspase-3, caspase-8, and caspase-9) in H₂O₂-treated granule neurons pretreated with α-tocopherol (αT) or γ-tocotrienol (γT3) for 1 h. Exposure to H₂O₂ led to much higher activity of caspase-3 and caspase-8 than in control cultures of cerebellar neurons. In contrast, there were no significant differences in caspase-9 activity in cultures exposed to H₂O₂ alone, cultures pre-treated with γT3 or αT followed by H₂O₂, or control untreated cultures. Pre-incubation with γT3 lowered the activity of both caspase-3 and caspase-8 relative to cultures treated with H₂O₂ alone. Pre-incubation of αT reduced the activity of caspase-8 to the levels in control cultures, but it did not reduce the activity of caspase-3. *P < 0.05 compared to controls, and ^# P < 0.05 compared to cells treated only with H₂O₂. Data are presented as mean ± SD (n = 9)

Fig. 5

H₂O₂ activates apoptosis pathway proteins, Bax, Bcl-2, and p53 protein levels and phosphorylation of p38 MAPK in granule neurons. a Protein levels in primary neurons treated with H₂O₂ for different incubation periods (1, 6, 12, 24 h). During treatment with H₂O₂, the expression of both Bcl-2 and Bax increased and peaked at 6 h, then decreased at 12 and 24 h. H₂O₂ increased expression of p53, with a peak occurring at 1 h, above the levels in control cultures, while H₂O₂ activated phosphorylation of p38 MAPK at 6 h which peaked at 12 h. b Densitometric analysis of Bcl-2 and Bax as Bcl-2/Bax ratio in (i), p53 in (ii), and p38 MAPK (iii). Bcl-2 and Bax bands were quantified using Total Image software and the Bcl-2/Bax ratio was determined as ‘survival index.’ During exposure to H₂O₂, the ratio of Bcl-2/Bax increased at 6 h but progressively decreased thereafter while expression of p53 and activation of p38 MAPK increased. *P < 0.05 compared to control. Data are presented as mean ± SD (n = 3)

Fig. 6

Various treatment of cells in the following fashion: untreated control, incubation of neurons with H₂O₂ for 24 h (H₂O₂), incubation of neurons with γT3 for 24 h (γT3), 1 h of γT3 pretreatment in neurons followed by H₂O₂ incubation for 24 h (γT3 + H₂O₂), incubation of neurons with αT for 24 h (αT), and 1 h of αT pretreatment in neurons followed by H₂O₂ incubation for 24 h (αT + H₂O₂). a Protein levels in primary neurons with various treatments as mentioned above. Expression levels of Bax were higher in neurons pre-treated with γT3 or αT followed by H₂O₂ and also in neurons treated only with γT3 or αT than they were in untreated control neurons or neurons treated only with H₂O₂. Similarly, pre-incubation with γT3 or αT followed by H₂O₂ treatment and cultures treated with only γT3 or αT increased Bcl-2 levels compared to control cultures and cultures treated only with H₂O₂. Pre-incubation with γT3 or αT increased the Bcl-2/Bax ratio back to control levels. Incubation with γT3 only decreased the Bcl-2/Bax ratio relative to control cultures, whereas αT did not change the ratio. Pre-incubation of cells with γT3 or αT followed by H₂O₂ reduced p53 levels to below those in control cultures and cultures treated only with H₂O₂. Incubation with only γT3 and αT induced higher p53 expression compared to the control. Cultures treated with αT alone had significantly higher p53 levels than cultures treated with αT followed by H₂O₂. Pre-incubation with γT3 did not alter p38 MAPK activation, whereas pre-incubation with αT decreased phosphorylation of p38 MAPK. Cultures treated only with γT3 or αT showed increased activation of p38 compared to controls. Activation of p38 in cultures treated with γT3 alone was less than in cultures treated with γT3 followed by H₂O₂. In contrast, cultures treated with αT alone showed greater phosphorylation of p38 than did cultures treated with αT followed by H₂O₂. b Densitometric analysis of Bcl-2 and Bax as Bcl-2/Bax ratio in (i), p53 in (ii), and p38 MAPK in (iii). Cultures treated with γT3 alone had lower Bcl-2/Bax ratios than did cultures treated first with γT3 and then with H₂O₂. The opposite results were seen with αT. *P < 0.05 compared to control, ^# P < 0.05 compared to cells treated only with H₂O₂, and **P < 0.05 compared to cells pre-incubated with γT3 or αT followed by H₂O₂ treatment. Data are presented as mean ± SD (n = 3)