The responses of HT22 cells to the blockade of mitochondrial complexes and potential protective effect of selenium supplementation

Abstract

Mitochondria are the major reactive oxygen species (ROS)--generating sites in mammalian cells. Blockade of complexes in the electron transport chain (ETC) increases the leakage of single electrons to O(2) and therefore increases ROS levels. Complexes I and III have been reported to be the major ROS-generating sites in mitochondria. In this study, using mouse hippocampal HT22 cells as in vitro model, we monitored the change of intracellular ROS level in response to the blockade of ETC at different complex, and measured changes of gene expression of antioxidant enzymes and phase II enzymes, also evaluated potential protective effect of selenium (Se) supplementation to the cells under this oxidative stress. In summary, our results showed that complex I was the major ROS-generating site in HT22 cells. Complex I blockade upregulated the mRNA levels of glutamylcysteine synthetase heavy and light chains, glutathione-S-transferases omega1 and alpha 2, hemoxygenase 1, thioredoxin reductase 1, and selenoprotein H. Unexpectedly, the expression of the enzymes that directly scavenge ROS decreased, including superoxide dismutases 1 and 2, glutathione peroxidase 1, and catalase. Se supplementation increased glutathione levels and glutathione peroxidase activity, indicating a potential protective role in oxidative stress caused by ETC blockade.

Figure 1

The increase of intracellular superoxide level induced by inhibitors of the electron transport chain. The concentration HT22 cells were treated for 30 min with rotenone (0-2.5 µM), malonate (0-15 mM), antimycin A (0-40 µM) and stigmatellin (0-30 µM). DMSO treated cells were used as control. The intracellular superoxide level was indicated by the fluorescent intensity of oxidized hydroethidine. Average values and SD are shown, N=3.

Figure 2

The change of intracellular superoxide level after withdrawal of the inhibitors of electron transport chain. HT22 cells were treated with rotenone (0.5 µM), malonate (5 mM), antimycin A (20 µM) and stigmatellin (20 µM) for 30 min, then washed and recovered in normal medium for up to 8 h. Average values and SD are shown, N=3.

Figure 3

Effects of rotenone treatment on gene expression in HT22 cells. The gene expression level of superoxide dismutase 1 and 2 (SOD1, SOD2), glutathione peroxide 1 (GPx1), catalase (Cat), glutamylcysteine synthetase heavy chain and light chain (GCS-HC, GCS-LC), glutathione s-transferase omega 1 and alpha 2 (GSTo1, GSTa2), heme oxygenase 1 (HO-1), thioredoxin reductase 1 (TR1), and selenoprotein H (SelH) in HT22 cells after treatment with 0.5 µM rotenone for 16 h. Average values and SD are shown.

Figure 4

Effects of selenium supplementation and rotenone treatment on (A) total glutathione level and (B) glutathione peroxidase activity in HT22 cells. HT22 cells were incubated with medium containing 100 nM sodium selenite for 24 h, then treated by 1.25 µM rotenone for 30 min in normal medium. Average values and SD are shown, N=3. In Panel B, the statistical comparisons are between Se Supplementation and normal medium after DMSO or Rot treatment.

Figure 4

Effects of selenium supplementation and rotenone treatment on (A) total glutathione level and (B) glutathione peroxidase activity in HT22 cells. HT22 cells were incubated with medium containing 100 nM sodium selenite for 24 h, then treated by 1.25 µM rotenone for 30 min in normal medium. Average values and SD are shown, N=3. In Panel B, the statistical comparisons are between Se Supplementation and normal medium after DMSO or Rot treatment.