Increasing evidence suggests that oxidative stress is associated with normal aging and several neurodegenerative diseases, including Alzheimer's disease (AD). Here we quantified multiple oxidized bases in nuclear and mitochondrial DNA of frontal, parietal, and temporal lobes and cerebellum from short postmortem interval AD brain and age-matched control subjects using gas chromatography/mass spectrometry with selective ion monitoring (GC/MS-SIM) and stable labeled internal standards. Nuclear and mitochondrial DNA were extracted from eight AD and eight age-matched control subjects. We found that levels of multiple oxidized bases in AD brain specimens were significantly (p < 0.05) higher in frontal, parietal, and temporal lobes compared to control subjects and that mitochondrial DNA had approximately 10-fold higher levels of oxidized bases than nuclear DNA. These data are consistent with higher levels of oxidative stress in mitochondria. Eight-hydroxyguanine, a widely studied biomarker of DNA damage, was approximately 10-fold higher than other oxidized base adducts in both AD and control subjects. DNA from temporal lobe showed the most oxidative damage, whereas cerebellum was only slightly affected in AD brains. These results suggest that oxidative damage to mitochondrial DNA may contribute to the neurodegeneration of AD. Keywords: Alzheimer's disease, gas chromatography/ mass spectrometry, 8-hydroxyguanine, mitochondrial DNA, nuclear DNA, oxidative stress. Alzheimer's disease (AD) is a progressive, irreversible, neurodegenerative disorder. The key to understanding AD is to elucidate the pathogenesis of neuron degeneration in specific brain regions. AD is associated with several risk factors, including age, genetic factors, presence of apolipoprotein E-4 alleles, educational attainment, head injury, hyperhomocysteinemia and diet (Schofield and Mayeux 1998;Seshadri et al. 2002).Increasing evidence suggests that oxidative stress and damage are associated with aging. The free radical theory of aging suggests that aging is due to the cumulative damage from free radical mediated reactions (Harman 1973; Smith et al. 2000a,b;Harman 2003). Mitochondria are primary sites of free radical production and their DNA and protein may be more easily oxidized than in other organelles (Wallace 1992(Wallace , 1999. Mitochondria produce ATP at the inner membrane through coupling of oxidative phosphorylation with respiration (Eckert et al. 2003), which is the major endogenous source of reactive oxygen species (ROS) (Sampson et al. 1998;Smith et al. 1998). During respiration, 2% of total oxygen consumed by the cell is converted into superoxide radicals by leaked electrons (Halliwell and Gutteridge 1989).Because of the proximity to ROS, lack of protective histones, and limited repair mechanisms (Wallace 1992;Ames et al. 1993), mitochondrial DNA (mtDNA) is susceptible to oxidative damage. These alterations could theoretically cause functional consequences because it has no noncoding sequences (Wallace 1992). As a consequence of ox...

Reactive oxygen species (ROS) are generated after exposure to harmful environmental factors and during normal cellular metabolic processes. The balance of the generating and scavenging of ROS plays a significant role in living cells. The accumulation of ROS will lead to oxidative damage to biomolecules including nucleic acid. Although many types of oxidative nucleic acid damage products have been identified, 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoG) has been commonly chosen as the biomarkers of oxidative damage to DNA and RNA, respectively. It has been demonstrated that oxidative damage to nucleic acid is an initiator in pathogenesis of numerous diseases. Thus, oxidative nucleic acid damage biomarkers have the potential to be utilized for detection of diseases. Herein, we reviewed the relationship of oxidative nucleic acid damage and development of various diseases including cancers (colorectal cancer, gastrointestinal cancer, breast cancer, lung cancer, epithelial ovarian carcinoma, esophageal squamous cell carcinoma), neurodegenerative disorders and chronic diseases (diabetes and its complications, cardiovascular diseases). The potential of oxidative nucleic acid damage biomarkers for detection of diseases and drug development were described. Moreover, the approaches for detection of these biomarkers were also summarized.

5-Methyl-2'-deoxycytidine (5-mdC), 5-hydroxymethyl-2'-deoxycytidine (5-hmdC), 5-methylcytidine (5-mrC) and 5-hydroxymethylcytidine (5-hmrC) are epigenetic marks of DNA and RNA, and aberrant levels of these modified nucleosides were found to be associated with various cancers. Urine is a preferred source of biological fluid for biomarker discovery because the sample collection process is not invasive to patients. Herein, we developed a novel malic acid-enhanced hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method for sensitive and simultaneous quantification of the modified cytosine nucleosides in human urine. Malic acid markedly increased the detection sensitivities of all four cytosine nucleosides, with the limits of detection (LODs) for 5-mdC, 5-hmdC, 5-mrC and 5-hmrC being 0.025, 0.025, 0.025 and 0.050 fmol, respectively. By using this method, we demonstrated, for the first time, the presence of 5-hmrC in human urine, and we successfully quantified 5-mdC, 5-hmdC, 5-mrC and 5-hmrC in urine samples collected from 90 patients with colorectal cancer (CRC) and 90 healthy controls. We found that the levels of 5-mdC, 5-hmdC, 5-mrC and 5-hmrC in urine were all substantially decreased in CRC patients, suggesting that these modified nucleosides might have great potential to be noninvasive biomarkers for early detection and prognosis of CRC. Together, we established a novel and sensitive method for detecting 5-methylated and 5-hydroxymethylated cytosine nucleosides in human urine and the results from this study may stimulate future investigations about the regulatory roles of these cytosine derivatives in the initiation and development of CRC.