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Profiling cytosine oxidation in DNA by LC-MS/MS
Francois Samson-Thibault1, Guru S Madugundu, Shanshan Gao
1Département de Médecine nucléaire et Radiobiologie, Faculté de Médecine, 3001 12e Avenue Nord, Université de Sherbrooke, Québec, Canada J1H 5N4.
Oxidative damage to cytosine in DNA can cause mutations. This study developed a method to detect 10 oxidized bases, revealing major cytosine oxidation products like 5-hydroxyhydantoin after radiation exposure.
Area of Science:
- Molecular Biology
- Oxidative Stress Research
- Genomic Stability
Background:
- Cytosine oxidation is a primary driver of CG to TA transition mutations in mammalian genomes.
- Previous studies identified numerous cytosine oxidation products using model systems with monomers and oligonucleotides.
- Understanding these oxidative modifications is crucial for comprehending DNA damage and repair mechanisms.
Purpose of the Study:
- To develop and validate an analytical method for detecting multiple oxidized bases in DNA.
- To quantify cytosine oxidation products in DNA subjected to ionizing radiation and Fenton-like reactions.
- To compare the distribution of cytosine oxidation products under different damaging conditions.
Main Methods:
- Development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method.
- Detection of 10 oxidized bases, including 5 specific cytosine oxidation products.
- Analysis of DNA damage in calf thymus DNA exposed to ionizing radiation (0-200 Gy) and Fenton-like reagents.
Main Results:
- Quantified cytosine modifications in DNA exposed to ionizing radiation: 5-hydroxyhydantoin (Hyd-Ura) > 5-hydroxyuracil (5-OHUra) > 5-hydroxycytosine (5-OHCyt) > 5,6-dihydroxy-5,6-dihydrouracil (Ura-Gly) > 1-carbamoyl-4,5-dihydroxy-2-oxoimidazolidine (Imid-Cyt).
- Total cytosine oxidation product yield was comparable to thymine oxidation products and 8-oxo-7,8-dihydroguanine (8-oxoGua).
- Hyd-Ura was the major cytosine oxidation product in DNA, contrasting with Imid-Cyt's dominance in monomer irradiation studies. Fenton-like reactions yielded a different product distribution than ionizing radiation.
Conclusions:
- The developed LC-MS/MS method effectively quantifies various oxidized bases in DNA.
- Ionizing radiation induces distinct cytosine oxidation patterns in DNA compared to Fenton-like reactions.
- Analysis of these products aids in understanding oxidative DNA degradation mechanisms and their biological/medical implications.
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