Time- and dose-dependent differential regulation of copper-zinc superoxide dismutase and manganese superoxide dismutase enzymatic activity and mRNA level by vitamin E in rat blood cells

Abstract

Background: Vitamin E is the most important lipid-soluble antioxidant. Recently, it has been proposed as a gene regulator, and its gene modulation effects have been observed at different levels of gene expression and cell signaling. This study was performed to investigate the effects of vitamin E on the activity and expression of the most important endogenous antioxidant enzyme, superoxide dismutase (SOD), in rat plasma.

Methods: Twenty-eight male Sprauge-Dawley rats were divided into four groups: control group and three dosing groups. The control group received the vehicle (liquid paraffin), and the dosing groups received twice-weekly intraperitoneal injections of 10, 30, and 100 mg/kg of vitamin E ((±)-α-Tocopherol) for 6 weeks. Quantitative real-time reverse transcription-polymerase chain reaction and enzyme assays were used to assess the levels of Cu/Zn-SOD and Mn-SOD mRNA and enzyme activity levels in blood cells at 0, 2, 4, and 6 weeks following vitamin E administration. Catalase enzyme activity and total antioxidant capacity were also assessed in plasma at the same time intervals.

Results: Mn-SOD activity was significantly increased in the 100 and 30 mg/kg dosing groups after 4 and 6 weeks, with corresponding significant increase in their mRNA levels. Cu/Zn-SOD activity was not significantly changed in response to vitamin E administration at any time points, whereas Cu/Zn-SOD mRNA levels were significantly increased after longer time points with high doses (30 and 100 mg/kg) of vitamin E. Catalase enzyme activity was transiently but significantly increased after 4 weeks of vitamin E treatment in 30 and 100 mg/kg dosing groups. Total antioxidant status was significantly increased after 4 and 6 weeks in the 100 mg/kg dosing group.

Conclusion: Only the chronic administration of higher doses of alpha-tocopherol is associated with the increased activity and expression of Mn-SOD in rats. Cu/Zn-SOD activity and expression does not dramatically change in response to vitamin E.

Figure 1.

Dose–response curve of Mn-SOD activity. Mn-SOD activity (U/gHb) was significantly increased by vitamin E treatment in the 100-mg/kg dosing group after 4 weeks (P < 0.001) and 6 weeks (P < 0.0001). In the 30-mg/kg dosing group, vitamin E exerted a weaker, yet eventually enhancing effect on Mn-SOD activity which was statistically significant after 4 weeks (P < 0.01) and 6 weeks (P < 0.001). The lowest dose (10 mg/kg) had a slightly enhancing effect on Mn-SOD activity only after 6 weeks of treatment (P < 0.03). Error bars indicate the standard deviation. *Statistically significant differences compared with control group at the same time point.

Figure 2.

Dose–response curve of Cu/Zn-SOD activity. Cu/Zn-SOD (U/gHb) activity was not significantly changed in the 10-, 30-, or 100-mg/kg dosing groups in response to vitamin E administration. Error bars indicate the standard deviation.

Figure 3.

Effect of different doses of vitamin E on Mn-SOD mRNA compared with the correspondent housekeeping gene in rat blood cells in the control and three dosing groups. Mn-SOD mRNA was significantly increased after 4 weeks (P < 0.0001) and 6 weeks (P < 0.001) in the 30-mg/kg dosing group. Vitamin E had a similar effect in the 100-mg/kg group with statistically significant increase after 4 weeks (P < 0.0001) and 6 weeks (P < 0.0001). Error bars indicate the standard deviation. *Statistically significant differences compared with control group at the same time point.

Figure 4.

Effect of different doses of vitamin E on Cu/Zn-SOD mRNA compared with the correspondent housekeeping gene in rat blood cells in the control and three dosing groups. Cu/Zn-SOD mRNA was weakly but significantly increased after 6 weeks of treatment in the 30-mg/kg dosing group (P < 0.001) and after 4 weeks in the 100-mg/kg dosing group (P < 0.001). Error bars indicate the standard deviation. *Statistically significant differences compared with control group at the same time point.

Figure 5.

Dose–response curve of CAT activity. CAT activity (U/gHb) was significantly and transiently increased by vitamin E treatment in the 30-mg/kg (P < 0.0001) and 100-mg/kg (P < 0.001) dosing groups after 4 weeks of treatment. The lowest dose of vitamin E (10 mg/kg) did not have any effect on CAT activity. Error bars indicate the standard deviation. *Statistically significant differences compared with control group at the same time point.

Figure 6.

Total plasma antioxidant status (TAS) showed a continuous increase from 2 weeks until the end of the study in the 100-mg/kg dosing group, which was statistically significant after 4 weeks (P < 0.0001) and 6 weeks (P < 0.0001) of treatment. Total antioxidant defense was unchanged in the groups receiving 10 and 30 mg/kg of vitamin E. Error bars indicate the standard deviation. *Statistically significant differences compared with control group at the same time point.