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| Author(s): |
Glaucia Regina Martinez
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | São Paulo. |
| Institution: | Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ) |
| Defense date: | 2003-03-13 |
| Examining board members: |
Paolo Di Mascio;
Ohara Augusto;
Ana Campa;
Luiz Henrique Catalani;
Carlos Frederico Martins Menck
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| Advisor: | Paolo Di Mascio |
| Abstract | |
Singlet oxygen (1O2) exhibits a substantial reactivity towards electron-rich organic molecules. Since DNA damage has been related to aging, cancer and other cytotoxic effects, its reaction with 1O2 have been extensively studied. Although, the mechanism and products of these reactions are not yet completely elucidated. The aim of the present work was to study the mechanism of DNA oxidation by 1O2. Emphasis was placed on the identification of the main products generated by the reaction of 1O2 with DNA. For this purpose, we developed a water-soluble naphthalene endoperoxide, the DHPN18O2, whose thermodecomposition leads to the formation of isotopically labeled singlet oxygen (18[1O2]). Calf thymus DNA and fibroblast cells were incubated with the hydrophilic endoperoxides NDPO2 and DHPNO2, as chemical generators of pure 1O2. It was found that 8-oxodGuo is the major 1O2-mediated DNA damage product. In order to demonstrate that 1O2 is directly involved in the formation of 8-oxodGuo, the DHPN18O2 was used. Incubation of the cells with such a generator of 18[1O2] resulted in the formation of 18O-labeled 8-oxodGuo in the nuclear DNA, clearly demonstrating that 1O2, when released within cells, is able to directly oxidize cellular DNA. The qualitative identification of the 1O2-oxidation products of 8-oxodGuo was achieved using HPLC coupled to electrospray ionization tandem mass spectrometry. Thus, the [18O]-labeled and unlabeled imidazolone, oxazolone, together with the diastereoisomeric spiroiminodihydantoin nucleosides, were detected as the main degradation products. In addition, a modified nucleoside that exhibits similar features than those of the oxidized guanidinohydantoin molecule was also produced. In this way, we contributed in the elucidation of some proposals of great importance to clarify the mechanisms that are involved in the interaction of 1O2 with DNA. (AU) | |