Yoshishige Urata

Several recent studies have suggested that the reactive oxygen species (ROS) generated from mitochondria contribute to genomic instability after exposure of the cells to ionizing radiation, but the mechanism of this process is not yet fully understood. We examined the hypothesis that irradiation induces mitochondrial dysfunction to cause persistent oxidative stress, which contributes to genomic instability. After the exposure of cells to 5 Gy gamma-ray irradiation, we found that the irradiation induced the following changes in a clear pattern of time courses. First, a robust increase of intracellular ROS levels occurred within minutes, but the intracellular ROS disappeared within 30 min. Then the mitochondrial dysfunction was detected at 12 h after irradiation, as indicated by the decreased activity of NADH dehydrogenase (Complex I), the most important enzyme in regulating the release of ROS from the mitochondrial electron transport chain (ETC). Finally, a significant increase of ROS levels in the mitochondria and the oxidation of mitochondrial DNA were observed in cells at 24 h or later after irradiation. Although further experiments are required, results in this study support the hypothesis that mitochondrial dysfunction causes persistent oxidative stress that may contribute to promote radiation-induced genomic instability.

BACKGROUND: Pirfenidone is a novel anti-fibrotic and anti-inflammatory agent that inhibits the progression of fibrosis in animal models and in patients with idiopathic pulmonary fibrosis (IPF). We previously showed that pirfenidone inhibits the over-expression of collagen type I and of heat shock protein (HSP) 47, a collagen-specific molecular chaperone, in human lung fibroblasts stimulated with transforming growth factor (TGF)-β1 in vitro. The increased numbers of HSP47-positive type II pneumocytes as well as fibroblasts were also diminished by pirfenidone in an animal model of pulmonary fibrosis induced by bleomycin. The present study evaluates the effects of pirfenidone on collagen type I and HSP47 expression in the human alveolar epithelial cell line, A549 cells in vitro. METHODS: The expression of collagen type I, HSP47 and E-cadherin mRNAs in A549 cells stimulated with TGF-β1 was evaluated by Northern blotting or real-time PCR. The expression of collagen type I, HSP47 and fibronectin proteins was assessed by immunocytochemical staining. RESULTS: TGF-β1 stimulated collagen type I and HSP47 mRNA and protein expression in A549 cells, and pirfenidone significantly inhibited this process. Pirfenidone also inhibited over-expression of the fibroblast phenotypic marker fibronectin in A549 cells induced by TGF-β1. CONCLUSION: We concluded that the anti-fibrotic effects of pirfenidone might be mediated not only through the direct inhibition of collagen type I expression but also through the inhibition of HSP47 expression in alveolar epithelial cells, which results in reduced collagen synthesis in lung fibrosis. Furthermore, pirfenidone might partially inhibit the epithelial-mesenchymal transition.

To elucidate the pathological metabolism of glutathione synthesis in diabetic endothelial cells, we studied the expression of γ-glutamylcysteine synthetase (γ-GCS) using a mouse vascular endothelial cell line.Exposing normoglycemic endothelial cells to tumor necrosis factor-α (TNF-α) or interleukin-1β (IL-1β) increased the activity and the mRNA expression of γ-GCS. The addition of inhibitors for nuclear factor κB (NF-κB) to the cells caused a loss of the γ-GCS mRNA expression in response to TNF-α.A shift of the concentration of glucose in the medium from 5.5 to 28 mM glucose and a following incubation for 7 days decreased the expression of γ-GCS mRNA. These cells showed no apparent responses of γ-GCS mRNA or the activity of NF-κB to TNF-α or IL-β. Increase in the GSH concentration of the cells treated with 28 mM glucose restored the expression of γ-GCS mRNA and its response to TNF-α or IL-β, suggesting that redox regulation is involved in the expression of γ-GCS.In summary, the expression of γ-GCS is regulated by TNF-α or IL-1β in endothelial cells mediated by NF-κB stimulation, and impairment of the regulation of γ-GCS in hyperglycemic cells may be a cause of medical complications that develop in diabetes mellitus. To elucidate the pathological metabolism of glutathione synthesis in diabetic endothelial cells, we studied the expression of γ-glutamylcysteine synthetase (γ-GCS) using a mouse vascular endothelial cell line. Exposing normoglycemic endothelial cells to tumor necrosis factor-α (TNF-α) or interleukin-1β (IL-1β) increased the activity and the mRNA expression of γ-GCS. The addition of inhibitors for nuclear factor κB (NF-κB) to the cells caused a loss of the γ-GCS mRNA expression in response to TNF-α. A shift of the concentration of glucose in the medium from 5.5 to 28 mM glucose and a following incubation for 7 days decreased the expression of γ-GCS mRNA. These cells showed no apparent responses of γ-GCS mRNA or the activity of NF-κB to TNF-α or IL-β. Increase in the GSH concentration of the cells treated with 28 mM glucose restored the expression of γ-GCS mRNA and its response to TNF-α or IL-β, suggesting that redox regulation is involved in the expression of γ-GCS. In summary, the expression of γ-GCS is regulated by TNF-α or IL-1β in endothelial cells mediated by NF-κB stimulation, and impairment of the regulation of γ-GCS in hyperglycemic cells may be a cause of medical complications that develop in diabetes mellitus.