Oxidative damage to DNA can occur at the purine/pyrimidine base or on the sugar phosphate backbone. Using technologies that are capable of detecting low level of oxidative DNA lesions (e.g., nmole to fmole), investigators have now identified, over 100 different types of oxidative DNA lesions ( Dizdaroglu, 1992; Poulsen et al., 1998). Of the 100 different types of oxidative damage that have been identified, 8-oxo-2’-deoxyguanosine (oxo8dG) is the most extensively studied oxidative modification of DNA because it represents approximately 5% of the total oxidized bases that are known to occur in the DNA (Helbock et al., 1999). Oxo8dG was discovered in 1984; however, it was not until 1986 that a detection methods were developed that were capable of detecting and quantifying oxo8dG levels.
Because levels of oxidative DNA damage are small (in the nmole to fmole range / 30-50g DNA), it is difficult to detect and quantify these lesions. Advances in the development of sensitive analytical methods that detect modified nucleotides, e.g., high performance liquid chromatography with electrochemical detection (HPLC-EC) and gas chromatography- chromatography-mass spectroscopy (GC-MS) have made it possible to measure specific DNA modifications that arise from oxidative damage in vivo. Although the GC-MS can detect a large number of DNA lesions, the derivatization procedure used to make the nucleosides volatile and stable at high temperatures results in DNA oxidation. The HPLC-EC system developed by Floyd in 1986 allows the separation of nucleosides under mild conditions and is a sensitive assay that can detect compounds in the fmole range (Floyd et al., 1990; Helbock et al., 1998, 1999).
Although much progress in the field of DNA oxidation has occurred since the initial observation of the presence of oxidative damage in the genome, one of the most troubling problems is the contradictory values reported in the levels of various oxidative lesions present in the nDNA and mtDNA. For example, oxo8dG levels measured in nDNA from various cells and tissues have shown an almost 5000-fold difference, and oxo8dG levels in mtDNA have shown a staggering 60,000-fold variation (Hamilton et al., 2001). Such variation has raised important questions concerning the accuracy of various assays used to measure DNA oxidation that occurs in vivo because a significant amount of oxidative damage (e.g., oxo8dG) can occur during the isolation of the DNA, especially in the presence of phenol. Phenol, which is a known reducing agent, is believed to reduce metal ions (e.g., iron) present in biological extracts. After reduction, these ions can enter the Haber-Wiess/Fenton reaction and generate hydroxyl free radicals, which can oxidize the DNA. Because various tissues as well as DNA contain relatively high amounts of iron (2-5 nmoles/mg protein), this reaction could be very important in generating oxidative damage during DNA isolation. In 1995, Nakae et al. reported very low oxo8dG levels in nDNA isolated from liver by a method using sodium iodide (NaI). In this method, NaI, a chaotropic salt, was used to precipitate protein from DNA rather than using an organic solvent, such as phenol. In 2001, our group conducted a comprehensive investigation comparing levels of oxo8dG in DNA isolated using the classic phenol method with the newly developed NaI method (Hamilton et al., 2001a). We showed that the NaI method minimized, if not eliminated, the oxidative damage to DNA that occurs during isolation and that the NaI method was more sensitive than the phenol method. Using the NaI method to isolate mtDNA and nDNA from mouse tissues, we found that the levels of oxo8dG were 6- to 23-fold higher in mtDNA compared to nDNA.
In 1990, Ames’ laboratory reported the first data on the effect of aging on DNA oxidation (Fraga et al., 1990). They observed a significant (approximately 2-fold) increase in oxo8dG levels in nuclear DNA (nDNA) isolated from liver, kidney and intestine of male rats between 2 and 24 months of age. Later, Ames et al. (1993) reported that the levels of oxo8dG in mitochondrial DNA (mtDNA) isolated from male rat liver increased 2- to 3-fold with age. Since 1990, a number of research groups have observed an age-related increase in the level of oxo8dG in both nDNA and mtDNA in a variety of tissues of rats and mice (Hamilton et al., 2001b). In addition, the level of oxo8dG in tissues has been shown to be inversely correlated to the maximum lifespan of species (Barja and Herrero, 2000). However, many investigators have been unable to detect a significant increase in DNA oxidation in rodent tissues with increasing age. The most likely explanation for the contradictory results is artifactual DNA oxidation, which arises during the isolation and analysis of the DNA samples. Our group conducted a comprehensive study in both rats and mice to determine the effect of age and dietary restriction on the levels of oxo8dG in DNA isolated from rodent tissues using the NaI method to eliminate/minimize the artifactual generation of oxo8dG during DNA isolation (Hamilton et al. 2001b). We measured the levels of oxo8dG in DNA isolated from a wide variety of tissues in both rats and various mouse strains to determine the universality of the age-related changes in DNA oxidation, i.e., were the changes tissue- or strain-specific. We observed an age-related increase in oxo8dG levels in nDNA isolated from all tissues studied from both rats and mice and an increase in oxo8dG levels in mtDNA isolated from the livers of both rats and mice. Dietary restriction reduced the levels of oxo8dG in nDNA from most tissues studied as well as in liver mtDNA of both rats and mice. The data in the figure (from Hamilton et al. 2001b) show the levels of oxo8dG in tissues from 6-, 16-, and 24-month-old rats fed ad libitum (o) and 24-month-old rats fed a caloric restricted diet (●).
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