Original Contribution
DNA damage, repair, and antioxidant systems in brain regions: a correlative study

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Abstract

8-Hydroxy-2′-deoxyguanosine (oxo8dG) has been used as a marker of free radical damage to DNA and has been shown to accumulate during aging. Oxidative stress affects some brain regions more than others as demonstrated by regional differences in steady state oxo8dG levels in mouse brain. In our study, we have shown that regions such as the midbrain, caudate putamen, and hippocampus show high levels of oxo8dG in total DNA, although regions such as the cerebellum, cortex, and pons and medulla have lower levels. These regional differences in basal levels of DNA damage inversely correlate with the regional capacity to remove oxo8dG from DNA. Additionally, the activities of antioxidant enzymes (Cu/Zn superoxide dismutase, mitochondrial superoxide dismutase, and glutathione peroxidase) and the levels of the endogenous antioxidant glutathione are not predictors of the degree of free radical induced damage to DNA in different brain regions. Although each brain region has significant differences in antioxidant defenses, the capacity to excise the oxidized base from DNA seems to be the major determinant of the steady state levels of oxo8dG in each brain region.

Introduction

Free radicals are formed in the central nervous system (CNS) as part of normal metabolic processes [1]. High oxygen uptake and low antioxidant defenses increase the vulnerability of the CNS to oxidative damage [2]. Assessment of free radical–induced damage to biological systems has been carried out by measuring the accumulation of damage to macromolecules. Proteins, lipids, and DNA are major targets for free radical–induced damage. Carbonyl content as an estimate of oxidative damage to proteins has been found to be high in specific brain regions and to be elevated during aging and in some types of neurodegenerative disorders [3], [4]. Levels of malondialdehyde and 4-hydroxynonenal have been used to identify oxidative damage to lipids and its association with aging and disease [5], [6]. The damage to DNA is more variable, and attack by free radicals can produce structural damage (i.e., strand breaks) and/or modification of the bases. 8-Hydroxy-2′-deoxyguanosine (oxo8dG) is one of the most common adducts formed from the reaction of oxyradicals with DNA [7]. A relation between oxidative DNA damage and aging has been postulated based on studies that show inverse associations between steady state tissue levels of oxo8dG and the life spans of different animal species [8], [9]. There are also age-dependent increases in oxo8dG levels in specific rat organs and the human brain [10], [11]. We have recently shown that oxo8dG accumulates differentially across brain regions in the aging mouse [12]. Oxo8dG levels have been found to be high in regions involved in neurodegenerative diseases, which is suggestive of an accelerated aging process in specific populations of neurons [13], [14], [15].

Unrepaired DNA lesions might impair transcription and protein synthesis [16]. This type of damage would be particularly deleterious for terminally differentiated cells.

Cells have a complete set of antioxidant defenses that guard against the damaging effects of free radicals and a DNA repair system that fixes damage done by unscavenged free radicals. Endogenous antioxidants such as glutathione (GSH) and enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase are the first line of defense and act by scavenging potentially damaging free radical moieties. DNA repair systems involve a number of enzymatic processes ranging from base recognition and excision to ligation of DNA strands. The DNA glycosylases recognize a particular damaged base and remove the base by cutting the N-glycosylic bond of the DNA molecule. A bacterial enzyme that recognizes modified purines (including oxo8dG) is formamidopyrimidine DNA glycosylase (Fpg) [17]. A similar activity has been described in mammals, and the enzyme responsible for oxo8dG excision has been cloned and called 8-oxoguanine DNA glycosylase (Ogg1) [18], [19].

Different regional activities in antioxidant systems and variable metabolic rates can lead to a region-specific accumulation of oxidative damage, and such differences can increase the vulnerability of specific brain regions to age-dependent oxidative stress. Several studies have been carried out to establish the age-dependent changes in activities of antioxidant enzymes [20], [21], [22]. It is hypothesized that decreased capacity in DNA repair and/or antioxidant defenses increases brain region vulnerability to oxidative damage and is responsible for age-associated deficits in cellular function, particularly in postmitotic cells such as neurons.

The overall objectives of this study were to measure steady state levels of oxo8dG in specific brain regions as indices of regional oxidative DNA damage and to identify regional differences in oxo8dG glycosylase activities and antioxidant defenses that could render specific brain regions more prone to oxidative DNA damage. This information will be useful in understanding the regional vulnerabilities of the brain during the normal process of aging, after vascular accidents, or elicited with exogenous toxicants.

Section snippets

Measurement of oxo8dG

Black male C57BL/N6 mice 3 months old were obtained from The Jackson Laboratories (Bar Harbor, ME, USA). Animals were killed by decapitation, and brains were place in ice-cold saline. Brain regions were dissected, and the samples were placed at −70°C until the time of assay. Procedures for extraction, purification, and enzymatic hydrolysis of DNA and measurement of oxo8dG levels (detailed elsewhere [23]) were based on published methods [24], [25] with minor modifications. Briefly, approximately

Results

Before presenting a summary of results, which are based on well-known methods for measuring antioxidant enzyme activities and oxo8dG levels, the results on regional differences in DNA repair are detailed, because the method has not been applied previously to measurements in brain regions.

Measurement of oxidized DNA repair in specific brain regions was based on the capacity of nuclear extracts to cleave an oxo8dG-containing oligonucleotide. The repair activity of a specific brain region was

Discussion

This article summarizes the region-specific differences in oxidative DNA damage and antioxidant capacities determined by levels of endogenous antioxidant (GSH), the activities of antioxidant enzymes (Cu/ZnSOD, MnSOD, GPx), and the activity of the glycosylase responsible for oxo8dG removal. Our results demonstrate that low steady state oxo8dG repair activities in the adult mouse brain are inversely correlated with high steady state levels of oxidative DNA damage. In the 3 month old mouse, the

Acknowledgements

This study was supported by a Veterans Affairs Merit Review Grant and the Helen E. Ellis Research Fund at the University of South Florida.

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