| Free radicals such as the hydroxyl radical (·OH) attack DNA to generate multiple modifications including base and sugar lesions, strand breaks and DNA-protein crosslinks. This type of DNA damage, which is also called oxidative DNA damage or free radical-induced DNA damage, is implicated in mutagenesis, carcinogenesis and aging. 8-Oxo-2'-deoxyguanosine (8-oxo-dG) is one of the most critical lesions. The presence of 8-oxo-dG residues in DNA can cause GC to TA transversion, unless repaired prior to DNA replication. Other oxidative lesions such as 8-oxo-2'-deoxyadenosine (8-oxo-dA) also have miscoding properties. The incidence of a variety of cancers, such as breast, liver, lung, prostate and skin cancer appear to be linked at least in part to nucleic acid base oxidation. DNA oxidation products are particularly important biomarkers representing oxidative stress, and these biomarkers may be determined by using specific and sensitive assays.; DNA methylation is an epigenetic modification that occurs in a wide range of eukaryotic organisms. The N-7 and O-6 positions of 2'-deoxyguanosine are the predominant sites of methylation by N-methyl- N-nitrosourea (MNU), which is used to produce a variety of experimental cancers in animal models. Methylation at the N7-position of dG is the most common adduct, accounting for over 70% of all methylation. Methylation at the O6-position is particularly significant since this lesion is involved in G to A transition mutation. This mutation is known to activate certain proto-oncogenes such as members of the ras gene family. The presence of 5-methyl cytosine residues in CpG sites throughout the genome is an important regulator of gene expression, and the alteration of the expression of oncogenes or tumor suppressor genes caused by aberrant cytosine methylation patterns is believed to contribute to carcinogenesis. Therefore, the evaluation of genomic DNA methylation status is critical for understanding the regulation of gene expression and the alteration of expression of oncogenes or tumor suppressor genes.; Accurate measurement of DNA lesions is required for understanding the mechanisms of DNA damage and its cellular repair, and the role of this damage in certain diseases. We have developed a liquid chromatography tandem mass spectrometry (LC-LTV-MS-MS) assay to determine both DNA oxidation and DNA methylation levels. The DNA extraction and hydrolysis procedures were optimized to minimize artifactual oxidation and shorten the sample preparation time. In addition, isotopically labeled internal standards were synthesized and co-eluted with the analysates for peak confirmation and precise quantitative analysis. These LC-UV-MS-MS assays are selective, sensitive and suitable for the analysis of multiple DNA oxidation and DNA methylation products and might be useful for cancer research. |
