Volume 36, Issue 1 , Pages 23-35, January 2003
Effect of age on the induction of 8-oxo-2′-deoxyguanosine-releasing enzyme in rat liver by γ-ray irradiation
Article Outline
Abstract
Aged (27 months of age) and young (6 months of age) Fischer 344/DuCrj rats were exposed to γ-ray irradiation, and their livers were compared for levels of oxidative DNA modifications and repair enzyme activities. The amounts of 8-oxo-2′-deoxyguanosine (8-oxodG) in the nuclear DNA of the livers of both young and aged rats increased immediately after irradiation, by 1.7-fold in the livers of young rats and 2.7-fold in the livers of the aged rats. Also, the rate of 8-oxodG decay was slower in the livers of the aged rats than in young rat liver, and remained above the baseline level even 1 week after irradiation. The activities of 8-oxodG-releasing enzymes peaked 2 and 6 h after irradiation in the livers of young and aged rats, respectively. The repair activity in the livers of the young rats was increased by sevenfold 2 h after irradiation, while the livers of the aged rats showed a twofold increase 6 h after irradiation. These results suggest that the ability to repair damaged DNA is lower in aged rats, and that the accumulation of oxidative DNA damage that takes place during aging may be related to this decline in repair activity.
Keywords: Oxidative damage, 8-Oxo-2′-deoxyguanosine, Repair enzyme, γ-Ray irradiation, Aging
1. Introduction
Reactive oxygen species (ROS) impair organisms by causing oxidative damage to proteins, lipids, and DNA, and are thought to be involved in the etiology of diseases such as atherosclerosis and cancer, as well as in the aging process (Ames, 1983). ROS are generated during normal processes that occur in endogenous pathways such as energy metabolism and phagocytosis (Chance et al., 1979), and, furthermore, they are also produced by exposure to exogenous factors such as radiation, ultraviolet (UV) light, carcinogens (Dreher and Junod, 1996), and anticancer agents (Burger et al., 1980, Praet et al., 1988). The ubiquitous presence of antioxidant enzymes, such as superoxide dismutase (SOD) and catalase, in the body is evidence supporting the continuous formation of ROS under usual conditions. Among the types of damage induced by ROS, damage to DNA plays an important role in the process of carcinogenesis (Totter, 1980). Indeed, it is generally accepted that point mutations in oncogenes and tumor suppressor genes often occur because tumor cells contain genetic alterations (Cross et al., 1987, Shen and Ong, 1996). ROS induce several types of DNA damage, including strand breaks, base modifications, and DNA–protein cross-links (Halliwell and Aruoma, 1993).
Oxidatively damaged components in DNA bases have been identified in more than 20 species (Aruoma et al., 1989). Oxidized deoxyguanosine, 8-oxo-2′-deoxyguanosine (8-oxodG), is the main DNA base modification product generated by such ROS as hydroxyl radicals (Kasai et al., 1984) and singlet oxygen (Kohda et al., 1990), and has been recognized as a good marker for oxidative damage to the body. Kuchino et al. (1987) have reported that 8-oxodG residues cause the DNA template to be misread during DNA synthesis, and that misreplication occurs not only at the position of the 8-oxodG residue itself, but also at adjacent positions. It is also reported that transfection into Escherichia coli of a single-stranded viral genome containing 8-oxoguanine (8-oxoGua) at a unique position causes an overwhelming predominance of targeted G to T transversion mutations (Wood et al., 1990). Fidelity is also reduced in DNA synthesis carried out by mammalian DNA polymerase β and an oxidatively damaged template DNA (Feig and Loeb, 1994).
It is reported that 8-oxodG is formed by X-ray (Kasai et al., 1984) or γ-ray (Dizdaroglu, 1985) irradiation in aqueous solutions of calf thymus DNA. Furthermore, 8-oxodG has been detected in DNA isolated from mouse liver irradiated with γ-rays, and the 8-oxodG content produced in DNA of mouse liver irradiated with X-rays decreases with time (Kasai et al., 1986). Thus, the presence of an enzyme(s) that repairs 8-oxodG in mouse liver has been suggested. On the other hand, 8-oxodG (Shigenaga et al., 1989), thymine glycol, and thymidine glycol (Cathcart et al., 1984), oxidized products of thymine residues, have been found in the urine of rodents and humans under normal conditions. Some specific repair enzymes for 8-oxodG have been found in mammalian organs (Mo et al., 1992, Yamamoto et al., 1992, McGoldrick et al., 1995, Rosenquist et al., 1997). In this study, we examined the time-dependent changes in 8-oxodG content and the activities of 8-oxodG-releasing enzymes in the livers of young and aged rats exposed to γ-ray irradiation.
2. Materials and methods
2.1. Chemicals
Nuclease P1 and alkaline phosphatase (type III from E. coli) were purchased from Sigma Chemical Co. (St. Louis, MO). Ribonucleases T1 and A were obtained from Boehringer Mannheim (Indianapolis, IN), and proteinase K was from Merck KGaA (Darmstadt, Germany). Phosphodiesterase II (from bovine spleen) and alkaline phosphatase (from calf intestine) were from Worthington Biochemical Co. (Freehold, NJ), and exonuclease III (from E. coli) was from Toyobo Co. (Osaka, Japan). Polyethylene glycol (PEG) (molecular weight: 7300–9000) was from Nacalai Tesque Inc. (Kyoto, Japan) and 8-oxodG was from Wako Pure Chemical Industries (Osaka, Japan). The water used in the isolation and hydrolysis of DNA was treated with Chelex 100 resin (Bio-Rad Lab., Hercules, CA) to remove trace amounts of transient metal ions, in particular, iron ions.
2.2. Animals
Specific pathogen-free male Fischer 344/DuCrj rats were obtained at 6 and 27 months of age from the Animal Facility of the Tokyo Metropolitan Institute of Gerontology. Rats were fed ad libitum a commercial diet, CRF-1 (Oriental Yeast Co., Tokyo, Japan), and water. All experimental procedures involving animals were approved by the Animal Care and Use Committee of the Tokyo Metropolitan Institute of Gerontology.
2.3. Irradiation with γ-rays
Rats were placed in a plastic cage and irradiated with 5 Gy of γ-rays from a 60Co source (Toshiba Electric Co., Tokyo, Japan) at a dose rate of 1 Gy/min. At different time intervals after irradiation, rats were killed by decapitation under anesthesia and their livers were quickly isolated and frozen in liquid nitrogen.
2.4. Isolation of nuclear DNA
Nuclear DNA in the liver (ca. 150 mg) was isolated by the method using sodium iodide and PEG (Kaneko and Tahara, 2000). Tissue homogenates in 0.3 M sucrose solution were centrifuged at 3000 rpm for 20 min to remove the cytosolic fraction containing mitochondria. Pellets were incubated for 1 h at 37
°C in a mixture of 50 μl of proteinase K solution (25 mg/ml) and 350 μl of 1% SDS/1 mM EDTA (pH 8.0) under an argon atmosphere to remove proteins. Then, 300 μl of 7 M NaI and 600 μl of isopropyl alcohol were added to the resulting solution, and the mixture was kept at −20°C for 10 min and centrifuged at 14,000 rpm for 20 min at 4°C. DNA pellets were dissolved in 200 μl of 0.01×SSC/1 mM EDTA and incubated at 37°C for 1 h with a mixture of ribonucleases T1 (20 units) and A (40 μg) under an argon atmosphere. The samples were then extracted with a mixture (300 μl) of chloroform and isoamyl alcohol (24:1, v/v). The aqueous phase was transferred to another tube and mixed with 200 μl of 13% PEG solution containing 1.6 M NaCl, and the mixture was left at −20°C for 1 h. The mixture was then centrifuged at 14,000 rpm for 20 min and the DNA pellet was rinsed twice with 70% ethanol and dissolved in 50 μl of water treated with the Chelex 100 resin. The amount and purity of DNA were determined by UV absorption as described previously (Kaneko et al., 1996).
2.5. Quantitation of 8-oxodG in DNA
DNA (25 μg) was digested with nuclease P1 (2 μg) in 20 mM sodium acetate buffer (pH 4.8) at 37°C for 30 min under an argon atmosphere, and then with alkaline phosphatase (0.65 units) in 100 mM Tris–HCl buffer (pH 7.5) under an argon atmosphere. The resulting mixture was filtered through an Ultrafree MC filter (Millipore Co., Bedford, MA), and the filtrate was applied to a high-performance liquid chromatography (HPLC) system with a Symmetry C18 column (4.6×75 mm; Waters Co., Milford, MA) and an ESA Coulochem II 5200 electrochemical detector (ESA, Bedford, MA) with a guard cell 5020 and an analytical cell 5011. The mobile phase consisted of citrate buffer (12.5 mM, pH 5.1) and methanol (94:6, v/v) at a flow rate of 0.8 ml/min. 8-oxodG was measured by electrochemical detection (ECD) at an oxidation potential of 350 mV. The 8-oxodG content is expressed as the molar ratio of 8-oxodG to 105 2′-deoxyguanosine (dG). The amount of dG was calculated from the absorption at 260 nm in the same measurement.
2.6. Extraction of 8-oxodG-releasing enzyme
Rat liver (1 g) was homogenized with a Teflon–glass homogenizer in 8 ml of 5 mM Tris–HCl buffer (pH 8.0) containing 0.34 M sucrose, 20 mM KCl, and 5 mM MgCl2. The homogenate was centrifuged at 100,000×g for 2 h, and the resulting supernatants were used as crude enzyme extracts.
2.7. Phosphocellulose column chromatography
The enzyme extracts (4 ml each) were dialyzed against 10 mM Tris–maleate buffer (pH 6.5) containing 50 mM KCl and 1 mM 2-mercaptoethanol (buffer A) for 5 h. The dialyzed solutions were applied to a phosphocellulose column (0.5×3 cm) equilibrated with buffer A. The column was washed with 2 ml of buffer A and developed with a 30 ml linear gradient of 50 mM to 1 M KCl in the same buffer. The eluate was collected in 1 ml fractions.
2.8. Assay of 8-oxodG-releasing activity
An aqueous solution of calf thymus DNA (1 mg/ml) was irradiated with 600 Gy of γ-rays from 60Co. The oxidatively damaged DNA was used as a substrate for 8-oxodG-releasing enzymes. Fractions (50 μl) eluted from the phosphocellulose column were mixed with 1.0 nmol MgCl2, 0.5 nmol dithiothreitol, 2.5 nmol Tris–HCl buffer (pH 8.3), and 50 μg of irradiated calf thymus DNA in a total volume of 125 μl. The mixtures were incubated for 1 h at 37°C and divided into four portions. The 8-oxodG-releasing activity in the four portions was measured by the following four separate procedures. In Method 1, for the detection of endonucleases excising 8-oxodG directly in DNA, the amount of 8-oxodG was determined immediately after incubation of the crude extract eluates with substrate. In Method 2, for the detection of endonucleases excising nucleotides containing 8-oxoGua directly in DNA, 0.5 unit of alkaline phosphatase was added to the mixture after incubation of the crude extract eluates with substrate, and the resulting mixture was incubated for an additional 10 min at 37°C. In Method 3, for the detection of endonucleases cleaving on the 3′-side of 8-oxodG in DNA, crude extract eluates were incubated with 1 unit of exonuclease III for 5 min at 37°C after the reaction with substrate, and further dephosphorylated by treatment with alkaline phosphatase as described in Method 2. In Method 4, for the detection of endonucleases cleaving on the 5′-side of 8-oxodG in DNA, chromatographed fractions of crude extract were treated by the same procedure as for Method 3 except that 0.05 unit of phosphodiesterase II was used instead of exonuclease III. After these treatments, the reaction mixtures were centrifuged at 12,000 rpm for 20 min using an Ultrafree MC filter to remove enzymes and high molecular weight DNA. The amount of 8-oxodG in each eluate was measured by the HPLC/ECD method described above.
2.9. Statistical analysis
Analysis of variance with Fisher's PLSD test was used for the statistical analysis of the data concerning 8-oxodG content. Each value represents the mean±SD. Differences were considered significant when the probability (P) values were less than 0.05.
3. Results
3.1. Oxidative DNA damage in rat liver irradiated with γ-rays
The effect of γ-ray irradiation on the 8-oxodG content of nuclear DNA was examined in the livers of rats 6 and 27 months of age. In control rats not exposed to irradiation, the 8-oxodG content at 27 months of age (0.803±0.089 8-oxodG/105 dG) was significantly (P<0.001) higher than at 6 months of age (0.430±0.069 8-oxodG/105 dG). The 8-oxodG contents at both 6 and 27 months of age (0.714±0.042 and 2.140±0.547 8-oxodG/105 dG, respectively) increased immediately and significantly (P<0.001) after irradiation as shown at 0 h in Fig. 1. The 8-oxodG contents in the nuclear DNA of liver at 6 and 27 months of age were 1.7- and 2.7-fold higher than those before irradiation, respectively. The levels of 8-oxodG decreased towards the background levels after irradiation. The 8-oxodG content in the livers of rats 6 months of age fell to the background level 24 h after irradiation (0.410±0.194 8-oxodG/105 dG), whereas the level in the livers of rats 27 months of age was not completely reduced to the background level even after 1 week (1.178±0.346 8-oxodG/105 dG).
Fig. 1.
Changes in the 8-oxodG content of nuclear DNA in Fischer 344/DuCrj rat liver after γ-ray irradiation. DNA was extracted from the livers of rats 6 months (●) and 27 months (▴) of age exposed to 5 Gy of γ-rays. Each value represents the mean±SD (n=5). The contents of 8-oxodG in the livers of young and aged rats without γ-irradiation are shown by the dotted line (⋯) and the broken line (- - -), respectively. *Significantly different from the 8-oxodG level in control rats at P<0.001. **Significantly different from the 8-oxodG level in control rats at P<0.01. ***Significantly different from the 8-oxodG level in control rats at P<0.05.
3.2. 8-oxodG-releasing enzyme activity in the livers of rats irradiated with γ-rays
To clarify the cause for the delay in the decrease in the 8-oxodG levels in the livers of γ-ray-irradiated rats 27 months of age, we compared the 8-oxodG-releasing enzyme activities in the livers of young and aged rats. Since calf thymus DNA irradiated with 600 Gy of γ-rays contains large amounts of 8-oxodG (ca. 690 8-oxodG/105 dG), the irradiated DNA was used as a substrate for 8-oxodG-releasing enzymes. Crude liver extracts should contain a variety of repair enzymes. We separated these enzymes roughly from liver homogenates of rats 6 months of age after 2 h of irradiation by chromatography on a phosphocellulose column, and the enzyme activity in each fraction was examined by the four different procedures described below. In Method 1, the substrate DNA was simply incubated with each fraction eluted from the phosphocellulose column, and the high molecular weight materials (more than about 10,000) were removed by filtration through an Ultrafree MC filter. The filtrates were then analyzed by the HPLC/ECD system. Only one peak was detected in fractions 1–5 (Peak 1) from the phosphocellulose column (Fig. 2). In Method 2, after the incubation in Method 1, the reaction mixture was treated with alkaline phosphatase. Two peaks were observed in fractions 1–5 (Peak 1) and fractions 9–17 (Peak 2) as shown in Fig. 3. In Methods 3 and 4, the eluates incubated with the substrate DNA were further treated with exonuclease III from E. coli or phosphodiesterase II from bovine spleen to detect endonucleases excising on the 3′- and 5′-sides of 8-oxodG residues, respectively, after which the reaction mixture was treated with alkaline phosphatase. The chromatographic profiles following Methods 3 and 4 (Fig. 4, Fig. 5) were almost the same as seen in Method 2.
Fig. 2.
Elution profiles of 8-oxodG-releasing enzymes in the fractions eluted from phosphocellulose column chromatography of homogenates of livers from rats 6 months of age 2 h after γ-ray irradiation. Chromatography was carried out as described in Section 2. Fractions were treated with substrate (γ-ray-irradiated calf thymus) DNA, and enzyme activities were estimated from the amount of free 8-oxodG detected by the HPLC/ECD system. The concentration of KCl in the elution buffer is shown by the broken line.
Fig. 3.
Elution profiles of enzymes that release 8-oxodG mono- and/or di-phosphate in fractions of γ-ray-irradiated rat liver eluted from phosphocellulose column chromatography of homogenates of livers from rats 6 months of age 2 h after γ-ray irradiation. Chromatography was carried out as described in Section 2. Fractions of rat liver homogenates were treated with substrate DNA and then with alkaline phosphatase. Enzyme activities were estimated from the amount of free 8-oxodG detected by the HPLC/ECD system. The concentration of KCl in the elution buffer is shown by the broken line.
Fig. 4.
Elution profiles of enzymes that cleave on the 3′-side of 8-oxodG residues in the fractions eluted from phosphocellulose column chromatography of liver homogenates from rats 6 months of age 2 h after γ-ray irradiation. Chromatography was carried out as described in Section 2. Fractions of rat liver homogenates were treated with the substrate DNA and then with exonuclease III and alkaline phosphatase. Enzyme activities were estimated from the amount of free 8-oxodG detected by the HPLC/ECD system. The concentration of KCl in the elution buffer is shown by the broken line.
Fig. 5.
Elution profiles of enzymes that cleave on the 5′-side of 8-oxodG residues in the fractions eluted from phosphocellulose column chromatography of liver homogenates from rats 6 months of age 2 h after γ-ray irradiation. Chromatography was carried out as described in Section 2. Fractions of rat liver homogenates eluted from the chromatography of liver homogenates of rats were treated with the substrate DNA and then with phosphodiesterase II and alkaline phosphatase. Enzyme activities were estimated from the amount of free 8-oxodG detected by the HPLC/ECD system. The concentration of KCl in the elution buffer is shown by the broken line.
We, next, examined the time-dependent changes in the activities of Peak 2 in the γ-ray-irradiated livers of rats 6 and 27 months of age. Small amounts of 8-oxodG-releasing activity in Peak 2 were observed in the livers of both young and aged rats not exposed to γ-ray irradiation. After irradiation, the activity increased in the livers of both young and aged rats, but the increase was more rapid in the livers of rats 6 months of age. The 8-oxodG-releasing enzyme activity in the livers of rats 6 months of age increased sevenfold 2 h after irradiation as shown in Fig. 6; the activity subsequently decreased rapidly and returned to the steady-state level at 24 h after irradiation. On the other hand, the activity in the livers of rats 27 months of age reaches a maximum 6 h after irradiation and the increase was twofold. Further recovery to the steady-state activity level required 3 days following irradiation.
Fig. 6.
Changes in the 8-oxodG-releasing enzyme activities in the livers of rats 6 months (●) and 27 months (▴) of age exposed to γ-ray irradiation. The 8-oxodG-releasing activity was estimated from the amounts of free 8-oxodG in Peak 2 using Method 2. Each value represents the average of two separate experiments.
4. Discussion
Genetic instability is profoundly related to carcinogenesis and the aging process. It is recognized that point mutations in oncogenes and tumor suppressor genes are associated with carcinogenesis in organs (Shen and Ong, 1996). Oxidative DNA damage has been reported to induce mismatch mutations (Kuchino et al., 1987, Feig and Loeb, 1994), and has to be repaired since such damage can be mutagenic or carcinogenic, and contribute to aging processes if not removed. Most of the DNA damage produced by ROS is thought to be quenched by defense systems or eliminated by repair systems. Oxidative DNA damage is, however, detected in most animal organs. The accumulation of oxidative DNA damage should be influenced by the balance between the generation and removal of ROS and the balance between the formation and elimination of damage. Probably, oxidative DNA damage occurs continuously in the body, and protective systems cannot completely remove harmful DNA damage. It is very important to clarify changes in antioxidant capacity or repair capacity that occurs during aging. We have previously reported that the 8-oxodG content in nuclear DNA from the heart, liver, and kidneys of Fischer 344/DuCrj rats remains steady up to 24 months of age and then increases progressively (Kaneko et al., 1996). The increase in oxidative DNA damage during aging must be related to an increase in the production of ROS or a decrease in repair capacity. Oxidized nucleosides, such as 8-oxodG and thymidine glycol, in the urine are recognized to be derived from excision repair of oxidized DNA and hydrolysis of 8-oxodGTP in the nucleotide pool (Cooke et al., 2000). In addition, an age-dependent decrease in the 8-oxodG content of rat urine has been observed (Fraga et al., 1990). These results suggest that the capacity to repair oxidative DNA damage decreases with age.
The HPLC/ECD assay can detect trace amounts of 8-oxodG in DNA isolated from control rat liver without additional oxidative stress. Using this method, the effect of age on the formation and removal of 8-oxodG in nuclear DNA from the livers of γ-ray-irradiated rats 6 and 27 months of age was examined. The 8-oxodG contents of both young and aged rats were found to increase immediately and significantly after γ-ray irradiation (Fig. 1). The maximum 8-oxodG content was observed immediately after irradiation, and the degree and quantity of the increase was larger in aged rat liver than in young rat liver. The 8-oxodG content in the livers of young rats returned to the background level 24 h after irradiation, whereas the level in the livers of aged rats required 72 h to return to the background level (Fig. 1). These results suggest that the relative activities of the defense and repair systems protecting against damage are less efficient in the livers of aged rats.
Many repair enzymes against modified DNA have so far been reported, and can be classified roughly into two types, glycosylases and endonucleases. It is generally recognized that modified DNA bases are mainly released as bases by digestion with glycosylases. Some glycosylases that remove 8-oxoGua have been identified in the mammalian cells (McGoldrick et al., 1995, Arai et al., 1997). However, oxidized nucleosides, such as 8-oxodG (Shigenaga et al., 1989) and thymidine glycol (Cathcart et al., 1984), have been detected in mammalian urine. Cooke et al. (2000) have suggested that the nucleotide pool represents a greater source of oxidized nucleosides. MTH (MutT (8-oxodG triphosphatase) mammalian homologue), which hydrolyses 8-oxodGTP to 8-oxodGMP, has been found to exist (Mo et al., 1992). On the other hand, an endonuclease excising 8-oxodG has been reported in human cells (Bessho et al., 1993). Furthermore, the action of nucleases and phosphatases upon DNA released from dead cells has also been suggested as the origin of 8-oxodG in the urine (Lindhal, 1993). However, little is known so far about endonucleases that excise 8-oxodG. In this study, we attempted to detect enzymes involved in 8-oxodG release, and compare the enzyme capacities in the livers of young and aged rats.
Crude protein extracts obtained from rat liver homogenates are presumed to contain several kinds of DNA glycosylases and endonucleases. The 8-oxodG-releasing endonuclease activity was examined in the fractions of crude protein extracts eluted from the phosphocellulose column. Calf thymus DNA irradiated with γ-rays was used as a substrate to detect 8-oxodG-releasing enzymes. When endonucleases act on modified DNA, in general, the modified portions are first excised by the endonucleases, and then removed as deoxynucleotide monophosphates by exonucleases. The fractions obtained by the four different methods described in Section 2 were assayed for 8-oxodG by the HPLC/ECD system. Although 8-oxodG was not detected in the simple incubation (Method 1) of each fraction eluted from the phosphocellulose column with substrate (Fig. 2), 8-oxodG was observed by Method 2 as Peaks 1 and 2 by additional treatment with alkaline phosphate (Fig. 3). The enzymes in Peak 1 were not examined further in this study because this fraction is often not sufficiently separated. No increase in 8-oxodG nucleotides was observed by additional treatment with exonuclease III (Method 3) or phosphodiesterase II (Method 4) (Fig. 4, Fig. 5). Therefore, the endonuclease activities detected by Methods 3 and 4 are thought to be based on the endonuclease activity detected in Method 2. These results indicate that endonucleases excising at positions adjacent to nucleotides containing 8-oxoGua exist in rat liver irradiated with γ-rays, but that enzymes that remove 8-oxodG directly or break on either side of nucleotides containing 8-oxoGua are not observed in rat liver. Since the detected activity releases 8-oxodG-3′- or 5′-monophosphate or 8-oxodG-3′ ,5′-diphosphate from DNA strands, it appears to be the 8-oxodG endonuclease reported previously (Bessho et al., 1993).
Time-dependent changes in the activity of the 8-oxodG-releasing enzyme after γ-ray irradiation were examined in Peak 2 using the livers of rats 6 and 27 months of age. The activity in young and aged rats reached their maximum levels 2 and 6 h after γ-ray irradiation, respectively (Fig. 6). The maximum enzyme activity in aged rats was lower than that in young rats. The level of 8-oxodG-releasing enzyme in young rats decreased rapidly 2 h after irradiation, while the level in aged rats did not show a quick decrease. The higher activity of 8-oxodG-releasing enzyme was maintained in aged rats from 2 h until at least 24 h after irradiation. These results indicate that the repair ability induced in aged rats irradiated with γ-rays is insufficient to repair all the damage. The decline in repair capacity with age may lead to an accumulation of oxidative damage and DNA mutations in somatic cells. The random mutations originating from oxidative DNA damage could lead to a decrease in cellular functions or to cell death by producing alterations in essential proteins. Information related to DNA repair is very valuable for studies designed to maintain health and physical strength. The detection of endonucleases cleaving phosphodiester bonds on the sides of nucleotides containing 8-oxoGua residues has been reported in electrophoretic studies using synthetic oligonucleotides containing a single 8-oxoGua at a defined position in the substrate (Bessho et al., 1993, Cardozo-Pelaez et al., 2000, de Souza-Pinto et al., 2001). However, this method is unable to distinguish between endonuclease activity and combination of glycosylase and AP lyase activity. Since the method used in this study detects 8-oxodG released from DNA directly, it is thought that the endonuclease activity alone is detected. Thus our method is useful in studies to identify the endonuclease that excise 8-oxodG from DNA.
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Volume 36, Issue 1 , Pages 23-35, January 2003
