We investigated whether oxidative stress, which contributes to aging, accelerates the telomere shortening in human cultured cells. The terminal restriction fragment (TRF) from WI-38 fibroblasts irradiated with UVA (365-nm light) decreased with increasing of the irradiation dose. Furthermore, UVA irradiation dose-dependently increased the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in both WI-38 fibroblasts and HL-60 cells. In order to clarify the mechanism of the acceleration of telomere shortening, we investigated site-specific DNA damage induced by UVA irradiation in the presence of endogenous photosensitizers using (32)P 5' end-labeled DNA fragments containing telomeric oligonucleotide (TTAGGG)(4). UVA irradiation with riboflavin induced 8-oxodG formation in the DNA fragments containing telomeric sequence, and Fpg protein treatment led to chain cleavages at the central guanine of 5'-GGG-3' in telomere sequence. Human 8-oxodG-DNA glycosylase introduces a chain break in a double-stranded oligonucleotide specifically at an 8-oxodG residue. The amount of 8-oxodG formation in DNA fragment containing telomere sequence [5'-CGC(TTAGGG)(7)CGC-3'] was approximately five times more than that in the DNA fragment containing nontelomere sequence [5'-CGC(TGTGAG)(7)CGC-3']. Furthermore, H(2)O(2) plus Cu(II) caused DNA damage, including 8-oxodG formation, specifically at the GGG sequence in the telomere sequence (5'-TTAGGG-3'). It is concluded that the formation of 8-oxodG at the GGG triplet in telomere sequence induced by oxidative stress could participate in acceleration of telomere shortening.

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Telomere shortening during human aging has been reported to be accelerated by oxidative stress. We investigated the mechanism of telomere shortening by oxidative stress. H 2 O 2 plus Cu(II) caused predominant DNA damage at the 5P P site of 5P P-GGG-3P P in the telomere sequence. Furthermore, H 2 O 2 plus Cu(II) induced 8-oxo-7,8-dihydro-2P P-deoxyguanosine (8-oxodG) formation in telomere sequences more efficiently than that in non-telomere sequences. NO plus O 3 2 efficiently caused base alteration at the 5P P site of 5P P-GGG-3P P in the telomere sequence. It is concluded that the site-specific DNA damage at the GGG sequence by oxidative stress may play an important role in increasing the rate of telomere shortening with aging.z 1999 Federation of European Biochemical Societies.

Reactive oxygen and nitrogen species have been implicated in diverse pathophysiological conditions, including inflammation, neurodegenerative diseases and cancer. Accumulating evidence indicates that oxidative damage to biomolecules including lipids, proteins and DNA, contributes to these diseases. Previous studies suggest roles of lipid peroxidation and oxysterols in the development of neurodegenerative diseases and inflammation-related cancer. Our recent studies identifying and characterizing carbonylated proteins reveal oxidative damage to heat shock proteins in neurodegenerative disease models and inflammation-related cancer, suggesting dysfunction in their antioxidative properties. In neurodegenerative diseases, DNA damage may not only play a role in the induction of apoptosis, but also may inhibit cellular division via telomere shortening. Immunohistochemical analyses showed co-localization of oxidative/nitrative DNA lesions and stemness markers in the cells of inflammation-related cancers. Here, we review oxidative stress and its significant roles in neurodegenerative diseases and cancer.

Telomere shortening is associated with cellular senescence. We investigated whether UVA, which contributes to photoaging, accelerates telomere shortening in human cultured cells. The terminal restriction fragment (TRF) from WI-38 fibroblasts irradiated with UVA (365-nm light) decreased with increasing irradiation dose. Furthermore, UVA irradiation dose-dependently increased the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in both WI-38 fibroblasts and HL-60 cells. To clarify the mechanism of the acceleration of telomere shortening, we investigated site-specific DNA damage induced by UVA irradiation in the presence of endogenous photosensitizers using (32)P 5'-end-labeled DNA fragments containing the telomeric oligonucleotide (TTAGGG)(4). UVA irradiation with riboflavin induced 8-oxodG formation in the DNA fragments containing telomeric sequence, and Fpg protein treatment led to chain cleavages at the central guanine of 5'-GGG-3' in telomere sequence. The amount of 8-oxodG formation in DNA fragment containing telomere sequence [5'-CGC(TTAGGG)(7)CGC-3'] was approximately 5 times more than that in DNA fragment containing nontelomere sequence [5'-CGC(TGTGAG)(7)CGC-3']. Catalase did not inhibit this oxidative DNA damage, indicating no or little participation of H(2)O(2) in DNA damage. These results indicate that the photoexcited endogenous photosensitizer specifically oxidizes the central guanine of 5'-GGG-3' in telomere sequence to produce 8-oxodG probably through an electron-transfer reaction. It is concluded that the site-specific damage in telomere sequence induced by UVA irradiation may participate in the increase of telomere shortening rate.

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Titanium dioxide (TiO2) is a potential photosensitizer for photodynamic therapy. In this study, the mechanism of DNA damage catalyzed by photo-irradiated TiO2 was examined using [32P]-5'-end-labeled DNA fragments obtained from human genes. Photo-irradiated TiO2 (anatase and rutile) caused DNA cleavage frequently at the guanine residue in the presence of Cu(II) after E. coli formamidopyrimidine-DNA glycosylase treatment, and the thymine residue was also cleaved after piperidine treatment. Catalase, SOD and bathocuproine, a chelator of Cu(I), inhibited the DNA damage, suggesting the involvement of hydrogen peroxide, superoxide and Cu(I). The photocatalytic generation of Cu(I) from Cu(II) was decreased by the addition of SOD. These findings suggest that the inhibitory effect of SOD on DNA damage is due to the inhibition of the reduction of Cu(II) by superoxide. We also measured the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine, an indicator of oxidative DNA damage, and showed that anatase is more active than rutile. On the other hand, high concentration of anatase caused DNA damage in the absence of Cu(II). Typical free hydroxyl radical scavengers, such as ethanol, mannnitol, sodium formate and DMSO, inhibited the copper-independent DNA photodamage by anatase. In conclusion, photo-irradiated TiO2 particles catalyze the copper-mediated site-specific DNA damage via the formation of hydrogen peroxide rather than that of a free hydroxyl radical. This DNA-damaging mechanism may participate in the phototoxicity of TiO2.

It has been reported that p53 acetylation, which promotes cellular senescence, can be regulated by the NAD⁺-dependent deacetylase SIRT1, the human homolog of yeast Sir2, a protein that modulates lifespan. To clarify the role of SIRT1 in cellular senescence induced by oxidative stress, we treated normal human diploid fibroblast TIG-3 cells with H₂O₂ and examined DNA cleavage, depletion of intracellular NAD⁺, expression of p21, SIRT1, and acetylated p53, cell cycle arrest, and senescence-associated β-galactosidase (SA-β-gal) activity. DNA cleavage was observed immediately in TIG-3 cells treated with H₂O₂, though no cell death was observed. NAD⁺ levels in TIG-3 cells treated with H₂O₂ were also decreased significantly. Pre-incubation with the poly (ADP-ribose) polymerase (PARP) inhibitor resulted in preservation of intracellular NAD⁺ levels. The amount of acetylated p53 was increased in TIG-3 cells at 4h after H₂O₂ treatment, while there was little to no decrease in SIRT1 protein expression. The expression level of p21 was increased at 12h and continued to increase for up to 24h. Additionally, exposure of TIG-3 cells to H₂O₂ induced cell cycle arrest at 24h and increased SA-β-gal activity at 48h. This pathway likely plays an important role in the acceleration of cellular senescence by oxidative stress.