Effects of hydrogen peroxide on mitochondrial gene expression of intestinal epithelial cells

Abstract

Aim: To study the effects of hydrogen peroxide on mitochondrial gene expression of intestinal epithelial cells in in vitro model of hydrogen peroxide-stimulated SW-480 cells.

Methods: RNA of hydrogen peroxide-induced SW-480 cells was isolated, and reverse-transcriptional polymerase chain reaction was performed to study gene expression of ATPase subunit 6, ATPase subunit 8, cytochrome c oxidase subunit I (COI), cytochrome coxidase subuit II (COII) and cytochrome c oxidase subunit III (COIII). Mitochondria were isolated and activities of mitochondrial cytochrome c oxidase and ATPase were also measured simultaneously.

Results: Hydrogen peroxide led to differential expression of mitochondrial genes with some genes up-regulated or down-regulated in a dose dependent manner. Differences were very obvious in expressions of mitochondrial genes of cells treated with hydrogen peroxide in a concentration of 400 micromol/L or 4 mmol/L. In general, differential expression of mitochondrial genes was characterized by up-regulation of mitochondrial genes in the concentration of 400 micromol/L and down-regulation in the concentration of 4 mmol/L. In consistence with changes in mitochondrial gene expressions, hydrogen peroxide resulted in decreased activities of cytochrome c oxidase and ATPase.

Conclusions: The differential expression of mitochondrial genes encoding cytochrome c oxidase and ATPase is involved in apoptosis of intestinal epithelial cells by affecting activities of cytochorme c oxidase and ATPase.

Figure 1

Effects of hydrogen peroxide on expression of hom-ing gene β-actin mRNA. C: control cells; A: 4 mM H₂O_2-treated cells (3 h); B: 400 mM H₂O_2-treated cells (3 h).

Figure 2

Effects of hydrogen peroxide on expression of COI mRNA (^aP < 0.05, vs. normal control). 1 DNA marker: lDNA/Hind III, 2 positive control, 3 normal control, 4400 μmol/L 3 h, 5 4 mmol/L 3 h.

Figure 3

Effects of hydrogen peroxide on expression of COII mRNA (^aP < 0.05, vs normal control). 1 DNA marker; lDNA/Hind III, 2 positive control, 3 normal control, 4400 μmol/L 3 h, 5 4 mmol/L 3 h.

Figure 4

Effects of hydrogen peroxide on expression of COIII mRNA (^aP < 0.05, vs normal control). 1 DNA marker: lDNA/Hind III, 2 positive control, 3 normal control, 4400 μmol/L 3 h, 5 4 mmol/L 3 h.

Figure 5

Effects of hydrogen peroxide on expression of AT-Pase6 mRNA (^aP < 0.05, vs normal control). 1 DNA marker: lDNA/Hind III, 2 positive control, 3 normal control, 4400 μmol/L 3 h, 5 4 mmol/L 3 h.

Figure 6

Effects of hydrogen peroxide on expression of AT-Pase8 mRNA (^aP < 0.05, vs. normal control). 1 DNA marker: lDNA/Hind III, 2 positive control, 3 normal control, 4400 μmol/L 3 h, 5 4 mmol/L 3 h.

Figure 7

Activity of cytochrome c oxidase in hydrogen peroxide-stimulated SW-480 cells (^aP < 0.05, vs. normal control).

Figure 8

Activity of ATPase in hydrogen peroxide-stimulated SW-480 cells (^aP < 0.05, vs. normal control).