碩士 / 國立臺灣海洋大學 / 生命科學暨生物科技學系 / 104 / Cyclobutane pyrimidine dimer (CPD) and (6-4) photoproduct (6-4PP) are two major types of dipyrimidine photoproducts induced by UV irradiation on DNA. CPDs and 6-4pps are removed from UV-damaged DNA primarily by nucleotide excision repair (NER). Oxidative stress has been proposed as a major factor underlying DNA-damaging effects caused by environmental pollutants. This study explored the effects of oxidative stress on NER in zebrafish (Danio rerio) embryos using cadmium (Cd) and paraquat (PQ) as representation oxidative stressors. Cd at 3 to 5 µM and PQ at 30 to 100 µM induced a weak oxidative stress in zebrafish embryos in which the levels of malondialdehyde (MDA) reached 17 to 36 pmole/ mg protein, but PQ at 200 µM induced a strong oxidative stress with MDA level at 80 pmole/mg protein. A photoproduct-specific band shift assay showed a preferential inhibition of CPD-dependent binding activity following the exposure of embryos at 1 h post fertilization (hpf) to Cd at 3 to 5 μM or to paraquat at 30 to 200 μM for 6 or 9 h and the inhibition was statistically significant in embryos treated with 3 µM Cd and 200 µM PQ. Conversely, 6-4PP binding activity was unaffected unless in embryos exposed to 200 µM PQ . A transcription-based in vitro DNA repair assay revealed a statistically insignificant inhibition of NER activity in embryos exposed to PQ at 30 to 200 µM for 6 h. A statistically insignificant inhibition of NER activity was also found in embryos exposed to 200 µM PQ for 9 h, indicating a close link between 6-4PP-specific damage recognition and NER capacity. Semi-quantitative RT-PCR showed a approximately 1.4-fold increase of UV-DDB2 gene expression in embryos exposed to Cd and PQ at all concentrations, revealing the induction of a DNA damage recognition factor that binds both CPDs and 6-4PPs. In contrast, a constitutive xeroderma pigmentosum C (XPC) gene expression encoding a 6-4PP binding factor was detected. High level of oxidative stress was found to induce apoptosis as analyzed by TUNEL-based in situ, but no induction of the apoptotic factor caspase 3a was detected by western blot. In conclusion, oxidative stress may inhibit NER leading to apoptosis in zebrafish embryos in the absence of a critical level of 6-4PP binding activity.
| Identifer | oai:union.ndltd.org:TW/104NTOU5613013 |
| Date | January 2015 |
| Creators | Ling, Li-Bin, 林里嬪 |
| Contributors | Todd, Hsu, 許濤 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 51 |
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