Serial Review J. Clin. Biochem.Nutr., 34, 51-60,2003 Aging and Oxidative Stress Guest Editor: Mitsuyoshi Matsuo Aging and the Accumulation of Oxidative Damage to DNA Takao Kaneko* RedoxRegulation ResearchGroup, Tokyo Metropolitan Institute of Gerontology,35-2 Sakaecho,Itabashi-ku, Tokyo173-0015, Japan Received 1 September, 2003; Accepted 14 October, 2003 Summary The oxidative stress hypothesis of aging is currently one of the most popular explanations for the cause of aging. Oxidative stress, including that due to reactive oxygen species, causes lesions to biomolecules such as DNA, protein and lipids. 8-Oxo-2'-deox- yguanosine (8-oxodG) is a major oxidative product of DNA and can be detected using a variety of assay methods. For an accurate measurement of 8-oxodG, it is important to mini- mize as much as possible the oxidation of intact DNA during its isolation from cells or tis- sues and during the hydrolysis of DNA to nucleosides. The most popular analytical technique is high-performance liquid chromatography with electrochemical detection. The levels of oxidative damage to DNA are likely to increase in mammalian tissues during aging, although it is not clear whether the levels increase linearly during aging or exponen- tially in aged animals. The accumulation of oxidative damage to DNA is recognized as inducing mutations and dysfunction during aging. The steady state levels of oxidative dam- age to DNA are proposed to depend on the balance between the formation and repair of damage. The repair activity appears to be weak in aged animals compared with young ani- mals, but antioxidative activity has not been shown to undergo a consistent changing pat- tern. Thus, it is assumed that the decline in repair activity in aged animals leads to the accumulation of oxidative damage in aged tissues. Key Words: aging, oxidative stress, DNA damage, 8-oxo-2'-deoxyguanosine cess. The oxidative stress hypothesis of aging, which Introduction was first proposed as the free radical hypothesis of aging [1], is currently one of the most popular expla- During the aging process, the functional capacities nations for the cause of aging. In this hypothesis, the of a variety of physiological systems undergo pro- progressive and irreversible accumulation of molecu- gressive impairment after the maturational phase, lar oxidative damage, induced by oxidative stress, is a decaying the ability to maintain homeostasis and main causal factor underlying the age-related losses resulting in death. Many hypotheses have been of physiological functions. This hypothesis has been offered to explain the mechanism of the aging pro- widely accepted because numerous studies have shown a correlation between increasing in age and * To whom correspondenceshould be addressed. the accumulation of oxidative damage to cellular bio- Tel: +81-3-3964-3241 Fax: +81-3-3579-4776 molecules [2,3]. Since not only free radicals but also E-mail:[email protected] non-radical species attack biomolecules oxidatively, 51 52 T. Kaneko "reactive oxygen species (ROS)" is a preferred term more, it has been shown that certain types of patho- to characterize the oxidants involved in such biomo- logical lesions arisen with age are associated with lecular modifications.The most frequently generated increased levels of oxidative damage to cellular bio- ROS are the superoxideanion radical, hydrogen per- molecules [5,6]. Thus, the rate of ROS formation oxide, and hydroxylradicals, and the main biological appears to be a determining factor in the aging pro- targets of ROS are proteins, lipids, and DNA. Oxi- cess. dative damage is observed as products derived from the oxidative modification of these biomolecules Oxidative DNA Damage such as proteins and protein-associated carbonyls, advanced glycation end products (AGEs), lipid per- Oxidative damage to DNA is also implicated in oxidation products, lipofuscin, 8-oxo-2'-deoxygua- mutagenesis and carcinogenesis as well as in aging nosine (8-oxodG), etc. ROS are formed continuously [7,8]. Numerous lesions have been characterized as in cells during normal cellular metabolism and by arising from oxidative damage to DNA. Hydroxyl exogenous agents such as carcinogenic compounds radicals cause many types of DNA damage including and ionizing radiation [4]. Thus, such damage is the oxidative modification of bases and sugars, strand detectable under normal physiological conditions, breaks, and cross-linking to proteins [9,10]. The even in healthy young animals. Although mammali- structures of main modified DNA bases are shown in an cells have numerous antioxidant defenses to limit Fig. 1. The 8-oxodG lesion has been studied most the generation and attack of ROS, such as catalase, extensivelybecause it is the major oxidative lesion superoxide dismutase and glutathione peroxidase, and because it can be detected relatively easily using ROS can escape these defense systems and injure a variety of assay methods [11]. 8-OxodG has been biomolecules, suggesting that the efficiency of the reported to be induced by the treatment of DNA antioxidativeand repair mechanisms is not complete. with Fenton-type reagents [12], carcinogens [13] The amount of oxidative damage to various biomol- and X ray-irradiation [14]. These treatments are ecules has been shown to increase during aging in a known to generate ROS during the reaction. Metal variety of tissues from different species [3]. Further- ions, such as iron and copper ions, both of which are thymine thymine glycol 5-hydroxymethyluracil 5 ,6-dihydrothymine 5-hydroxy-5-methylhydantoin cytosine cytosine glycol 5-hydroxycytosine 5,6-di hydroxycytosine 5-hydroxyhydantoin adenine 2-hydroxyadenine 8-oxoadeninel 4,6-diamino-5-formamidopyrimidine guanine 8-oxoguanine 2,6-diamino-4-hydroxy-5-formamidopyrimidine Fig. 1. Molecular structures of the main modified bases produced from the reaction of DNA with hydroxyl radicals. J. Clin. Biochem. Nutr. Aging and Oxidative DNA Damage 53 levels of 8-oxodG in mtDNA than in nDNA [24- 26]. However, Higuchi and Linn reported that the levels of 8-oxodG measured in mtDNA isolated by cesium chloride density gradient centrifugation were lower than those reported for nDNA [27]. Anson et al. measured the levels of oxidative damage in mtDNA from rat liver using an Fpg/EndoIII/south- em blot assay and concluded that endogenous oxida- tive damage to mtDNA had been overestimated [28]. Measurement of Oxidative Damage to DNA Because the levels of oxidative damage to DNA are low(on the order of fmole range per μg DNA), it is not easy to detect and quantify these lesions with accuracy. Techniques used to measure oxidative dam- age to DNA include high-performance liquid chro- matography with electrochemical detection (HPLC/ ECD), gas chromatography-mass spectroscopy (GC/ MS), immunochemical techniques, post-labeling as- Fig. 2. Mispairing of 8-oxoG in the syn-conformation says, and the comet assay for DNA break measure- (keto form) with adenine. ment, not for oxidative damage. The most frequently used technique is HPLC/ECD among these analyt- ical techniques. By the HPLC/ECD following the known to bind to DNA because of its overall nega- mild enzymic hydrolysis of DNA, modified nucleo- tive charge, are believed to produce 8-oxodG sides such as 8-oxodG can be detected in a fmole through metal-catalyzed oxidation reactions. The C- range [29]. Because of the availability of this tech- 8 hydroxyl group of 8-oxodG tautomerizes to the C- nique and of the mutagenic properties of 8-oxodG 8 keto form (syn-conformation) in neutral aqueous [11, most studies using HPLC/ECD have concen- solutions [15]. Since the keto form is more stable trated on this compound. On the other hand, GC/ than the enol form (8-hydroxyguanine) [16], 8- MS in combination with a selective-ion monitoring oxodG adopts the syn-conformation. Because 8- (SIM) technique can detect a large number of DNA oxodG pairs preferentially with adenine rather than lesions simultaneously. Recently, liquid chromatogra- cytosine, its presence can generate G—T transver- phy/tandem mass spectrometry (LC/MS/MS) [30] sions as shown in Fig. 2 [17,18], which are likely to and liquid chromatography/mass spectrometry (LC/ be involved in carcinogenesis [7,8]. Indeed, 8- MS) [31] have emerged as new techniques for the oxodG has been detected at higher levels in cancer measurement of modified nucleosides in DNA. cells [19,20], and, further, the formation of 8- It has become apparent that a significant amount oxodG has been demonstrated in target organ DNA of DNA oxidation can occur during the DNA isola- in a wide variety of in vivo experimental carcinogen- tion and sample preparation for assay. The oxidation esis models [21,22]. of intact bases during DNA isolation and/or under Mitochondrial DNA (mtDNA) is not protected subsequent procedures is recognized to depend on by nucleosomes, being different from nuclear DNA analytical techniques used for the measurement of (nDNA), and it lies in close proximity to the free DNA damage. Indeed, it has been shown that DNA radical producing processes of oxidative phosphory- is oxidized during isolation, and that precautions lation. Therefore, mtDNA is thought to be highly must be taken to prevent the oxidation of guanine prone to oxidative damage. Richter et al. first report- [32]. The oxidation of DNA has been discussed ed that the levels of 8-oxodG were 16-fold higher in extensively in recent reviews [33, 34]. Such oxidation mtDNA than nDNA in rat liver [23]. Subsequently, has raised important questions about the accuracy of many investigators have reported significantly higher various assays used to measure in vivo levels of DNA Vol.34, No.2, 2003 54 T. Kaneko oxidation. For example, 8-oxodG levels obtained by GC/MS are considerablyhigher than those obtained using HPLC/EC [35]. Artifacts may occur during the derivatization of DNA acid-hydrolysatesat high temperature prior to GC/MS analysis [36]. Guanine is the most prone to oxidation among DNA bases [37], and is oxidized to yield 8-oxoguanine (8- oxoG).