Concentration-dependent effects of fullerenol on cultured hippocampal neuron viability

Abstract

Background: Recent studies have shown that the biological actions and toxicity of the water-soluble compound, polyhydroxyfullerene (fullerenol), are related to the concentrations present at a particular site of action. This study investigated the effects of different concentrations of fullerenol on cultured rat hippocampal neurons.

Methods and results: Fullerenol at low concentrations significantly enhanced hippocampal neuron viability as tested by MTT assay and Hoechst 33342/propidium iodide double stain detection. At high concentrations, fullerenol induced apoptosis confirmed by Comet assay and assessment of caspase proteins.

Conclusion: These findings suggest that fullerenol promotes cell death and protects against cell damage, depending on the concentration present. The concentration-dependent effects of fullerenol were mainly due to its influence on the reduction-oxidation pathway.

Keywords: fullerenol; hippocampal neuron; nanomaterial; neuroprotection; neurotoxicity.

Figure 1

Transmission electron microscopy image of the physical state of C₆₀(OH)_x(ONa)_y. (A) Image of 1 μM fullerenol in water. (B) Amplified image of 1 μM fullerenol in water. (C) Image of 200 μM fullerenol in water. (D) Amplified image of 200 μM fullerenol in water.

Figure 2

Double staining of neurons and astrocytes. (A) Neurons in the +Ara-C group. (B) Astrocytes in the +Ara-C group. (C) Neurons in the −Ara-C group. (D) Astrocytes in the −Ara-C group. (E) Histogram showing the ratio of astrocyte to cell numbers. Abbreviation: Ara-C, cytosine arabinoside.

Figure 3

Effect of C₆₀(OH)_x(ONa)_y on viability of cultured hippocampal neurons. (A) Hippocampal neurons treated with fullerenol 0, 1, 5, 25, and 100 μM for 24 hours. (B) Hippocampal neurons treated for 48 hours. (C) Hippocampal neurons treated for 72 hours. Notes: The results show mean ± standard error (n = 8) values from a representative experiment that was repeated at least three times. *P < 0.05 versus control group; **P < 0.01, versus control group. One-way analysis of variance with Bonferroni post hoc tests. Abbreviation: PI, propidium iodide.

Figure 4

Protective effect of C₆₀(OH)_x(ONa)_y against lead-induced neurotoxicity. Hippocampal neurons were treated with various concentrations of fullerenol 0, 1, 5, 25, and 100 μM and lead 20 μM for 24 or 48 hours. The Hoechst dye entered into normal cells and emitted blue fluorescence, while the propidium iodide dye entered into necrotic cells and emitted red fluorescence. (A) Three typical images of random fields per treatment group detected with a high-power optical microscope. Left, control; middle, 20 μM lead-exposed for 24 hours; right, 20 μM lead and 1 μM fullerenol exposed for 24 hours. Magnification is 10 × 20. (B) Three typical images detected with fluorescence microscopy. (C) Survival rate of hippocampal neurons after coculturing for 24 hours. (D) Survival rate after coculturing for 48 hours. Notes: *P < 0.05 versus lead-exposed group without fullerenol. **P < 0.01 versus lead-exposed group without fullerenol. ^#P < 0.05 versus control group; ^##P < 0.01 versus control group. One-way analysis of variance with the Bonferroni post hoc tests.

Figure 5

High concentrations of C₆₀(OH)_x(ONa)_y can induce apoptosis. (A) Comet image of a normal neuron detected with CASP software. (B) Comet image of an apoptosis neuron. (C) Mean percentage of tail DNA in hippocampal neurons treated with fullerenol 0, 50, 100, and 200 μM. (D) Olive tail moment distribution (50 observations) in hippocampal neurons treated with fullerenol 0, 50, 100, and 200 μM. Notes: The results are shown as the mean ± standard error (n = 50) of a representative experiment that was repeated at least three times. **P < 0.01 versus control group in 24 hours. ^#P < 0.05 versus control group in 48 hours; ^##P < 0.01 versus control group in 48 hours. One-way analysis of variance with Bonferroni post hoc tests.

Figure 6

Effect of C₆₀(OH)_x(ONa)_y on caspase content of cultured hippocampal neurons. Hippocampal neurons were treated with fullerenol 0, 50, 100, and 200 μM for 24 hours. (A) Caspase-3 activity was detected using a protein assay kit. (B) Caspase-9 activity was detected. Notes: The results are the mean ± standard error (n = 8) of a representative experiment that was repeated at least three times. **P < 0.01 versus control group. One-way analysis of variance with Bonferroni post hoc tests.

Figure 7

Extent of oxidative stress in cultured hippocampal neurons of control and fullerenol-exposed groups (1, 5, 25, and 100 μM). (A) Effects of fullerenol on superoxide dismutase activity in cultured hippocampal neurons. (B) Effects of fullerenol on glutathione level in neurons. (C) Effects of fullerenol on maleic dialdehyde content in neurons. Notes: Values are mean ± standard error (n = 26). *P < 0.05 versus corresponding value of control group; **P < 0.01 versus corresponding value of control group. One-way analysis of variance with the Bonferroni post hoc tests. Abbreviations: GSH, glutathione; MDA, maleic dialdehyde; SOD, superoxide dismutase.