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Dissociation of DNA damage and mitochondrial injury caused by hydrogen peroxide in SV-40 transformed lung epithelial cells.

Fujii Y, Tomita K, Sano H, Yamasaki A, Hitsuda Y, Adcock IM, Shimizu E - Cancer Cell Int. (2002)

Bottom Line: A 1 h pulse of H2O2 induced small amounts of apoptosis (3%). 8-oxo-dG-positive cells and the length of the comet tail increased within 1 h of exposure to H2O2.The number of cells with reduced DeltaPsim increased after the addition of H2O2 in a concentration-dependent manner.In spite of a continual loss of DeltaPsim, DNA fragmentation was reduced 2 h after exposure to H2O2.

View Article: PubMed Central - HTML - PubMed

Affiliation: Third Department of Internal Medicine, Faculty of Medicine, Tottori University, 36-1 Nishi-machi, Yonago-shi, Tottori-ken 683-8504, JAPAN. tom0223@grape.med.tottori-u.ac.jp

ABSTRACT
BACKGROUND: Since lung epithelial cells are constantly being exposed to reactive oxygen intermediates (ROIs), the alveolar surface is a major site of oxidative stress, and each cell type may respond differently to oxidative stress. We compared the extent of oxidative DNA damage with that of mitochondrial injury in lung epithelial cells at the single cell level. RESULT: DNA damage and mitochondrial injury were measured after oxidative stress in the SV-40 transformed lung epithelial cell line challenged with hydrogen peroxide (H2O2). Single cell analysis of DNA damage was determined by assessing the number of 8-oxo-2-deoxyguanosine (8-oxo-dG) positive cells, a marker of DNA modification, and the length of a comet tail. Mitochondrial membrane potential, DeltaPsim, was determined using JC-1. A 1 h pulse of H2O2 induced small amounts of apoptosis (3%). 8-oxo-dG-positive cells and the length of the comet tail increased within 1 h of exposure to H2O2. The number of cells with reduced DeltaPsim increased after the addition of H2O2 in a concentration-dependent manner. In spite of a continual loss of DeltaPsim, DNA fragmentation was reduced 2 h after exposure to H2O2. CONCLUSION: The data suggest that SV-40 transformed lung epithelial cells are resistant to oxidative stress, showing that DNA damage can be dissociated from mitochondrial injury.

No MeSH data available.


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Indirect immunofluorescence of nitrotyrosine expression in BEAS-2B cells. A, cells without stimulation, B, cells exposed to 500 μM H2O2, C, cells exposed to 1 mM H2O2, D, cells exposed to 1 mM SIN-1, and E, cells exposed to 500 μM H2O2 and 1 mM SIN-1 for 1 h were stained with anti-nitrotyrosine antibody. Each image was captured by laser confocal microscope. Results are representative of 3 individual experiments.
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Figure 5: Indirect immunofluorescence of nitrotyrosine expression in BEAS-2B cells. A, cells without stimulation, B, cells exposed to 500 μM H2O2, C, cells exposed to 1 mM H2O2, D, cells exposed to 1 mM SIN-1, and E, cells exposed to 500 μM H2O2 and 1 mM SIN-1 for 1 h were stained with anti-nitrotyrosine antibody. Each image was captured by laser confocal microscope. Results are representative of 3 individual experiments.

Mentions: An important pathway that enhances NO cytotoxicity is the rapid reaction with superoxide anion radicals (O2·-) to yield peroxynitrite (ONOO-), which has a wide range of biologic activities that includes oxidation of biomolecules and protein tyrosine nitration [18]. It is widely accepted that peroxynitrite is a key mediator of tissue injury in oxidative stress. To determine whether H2O2-induced peroxynitrite formation occurred in BEAS-2B cells, cells were stained with an anti-nitrotyrosine antibody. H2O2 (60 min, 500–1000 μM) induced clear perinuclear nitrotyrosine staining (Fig. 5B and 5C). The peroxynitrite donor, SIN-1, induced a predominantly nuclear membrane distribution of nitrotyrosine staining (Fig. 5D).


Dissociation of DNA damage and mitochondrial injury caused by hydrogen peroxide in SV-40 transformed lung epithelial cells.

Fujii Y, Tomita K, Sano H, Yamasaki A, Hitsuda Y, Adcock IM, Shimizu E - Cancer Cell Int. (2002)

Indirect immunofluorescence of nitrotyrosine expression in BEAS-2B cells. A, cells without stimulation, B, cells exposed to 500 μM H2O2, C, cells exposed to 1 mM H2O2, D, cells exposed to 1 mM SIN-1, and E, cells exposed to 500 μM H2O2 and 1 mM SIN-1 for 1 h were stained with anti-nitrotyrosine antibody. Each image was captured by laser confocal microscope. Results are representative of 3 individual experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC149434&req=5

Figure 5: Indirect immunofluorescence of nitrotyrosine expression in BEAS-2B cells. A, cells without stimulation, B, cells exposed to 500 μM H2O2, C, cells exposed to 1 mM H2O2, D, cells exposed to 1 mM SIN-1, and E, cells exposed to 500 μM H2O2 and 1 mM SIN-1 for 1 h were stained with anti-nitrotyrosine antibody. Each image was captured by laser confocal microscope. Results are representative of 3 individual experiments.
Mentions: An important pathway that enhances NO cytotoxicity is the rapid reaction with superoxide anion radicals (O2·-) to yield peroxynitrite (ONOO-), which has a wide range of biologic activities that includes oxidation of biomolecules and protein tyrosine nitration [18]. It is widely accepted that peroxynitrite is a key mediator of tissue injury in oxidative stress. To determine whether H2O2-induced peroxynitrite formation occurred in BEAS-2B cells, cells were stained with an anti-nitrotyrosine antibody. H2O2 (60 min, 500–1000 μM) induced clear perinuclear nitrotyrosine staining (Fig. 5B and 5C). The peroxynitrite donor, SIN-1, induced a predominantly nuclear membrane distribution of nitrotyrosine staining (Fig. 5D).

Bottom Line: A 1 h pulse of H2O2 induced small amounts of apoptosis (3%). 8-oxo-dG-positive cells and the length of the comet tail increased within 1 h of exposure to H2O2.The number of cells with reduced DeltaPsim increased after the addition of H2O2 in a concentration-dependent manner.In spite of a continual loss of DeltaPsim, DNA fragmentation was reduced 2 h after exposure to H2O2.

View Article: PubMed Central - HTML - PubMed

Affiliation: Third Department of Internal Medicine, Faculty of Medicine, Tottori University, 36-1 Nishi-machi, Yonago-shi, Tottori-ken 683-8504, JAPAN. tom0223@grape.med.tottori-u.ac.jp

ABSTRACT
BACKGROUND: Since lung epithelial cells are constantly being exposed to reactive oxygen intermediates (ROIs), the alveolar surface is a major site of oxidative stress, and each cell type may respond differently to oxidative stress. We compared the extent of oxidative DNA damage with that of mitochondrial injury in lung epithelial cells at the single cell level. RESULT: DNA damage and mitochondrial injury were measured after oxidative stress in the SV-40 transformed lung epithelial cell line challenged with hydrogen peroxide (H2O2). Single cell analysis of DNA damage was determined by assessing the number of 8-oxo-2-deoxyguanosine (8-oxo-dG) positive cells, a marker of DNA modification, and the length of a comet tail. Mitochondrial membrane potential, DeltaPsim, was determined using JC-1. A 1 h pulse of H2O2 induced small amounts of apoptosis (3%). 8-oxo-dG-positive cells and the length of the comet tail increased within 1 h of exposure to H2O2. The number of cells with reduced DeltaPsim increased after the addition of H2O2 in a concentration-dependent manner. In spite of a continual loss of DeltaPsim, DNA fragmentation was reduced 2 h after exposure to H2O2. CONCLUSION: The data suggest that SV-40 transformed lung epithelial cells are resistant to oxidative stress, showing that DNA damage can be dissociated from mitochondrial injury.

No MeSH data available.


Related in: MedlinePlus