Dna Oxidation (8-OXO-2’-Deoxyguanosine)

Description

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 / 30-50g 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 (●).

References:

Ames,B.N., Shigenaga,M.K. & Hagen,T.M. (1993). Oxidants, antioxidants, and the degenerative disease of aging. Proc.Natl.Acad.Sci.USA  90, 7915-7922. 

Barja, G., Herrero, A. (2000). Oxidative damage to mitochondrial DNA is inversely related to maximum life span in the heart and brain of mammals. FASEB J. 14, 312-318.

Dizdaroglu, M. (1992). Oxidative damage to DNA in mammalian chromatin. Mutat. Res. 275, 331–42.

Floyd, R. A. (1986). The development of a sensitive analysis for 8-hydroxy-2'-deoxyguanosine. Free Radic.Res.Commun. 8, 139-141.

Floyd, R. A., West, M. S., Eneff, K. L., Schneider, J. E., Wong, P. K., Tingey, D. T., Hogsett, W. E. (1990). Conditions influencing yield and analysis of 8-hydroxy-2'-deoxyguanosine in oxidatively damaged DNA. Analyt.Biochem. 155-158.

Fraga, C. G., Shigenaga, M. K., Park, J., Degan, P., Ames, B. N. (1990). Oxidative damage to DNA during aging: 8-hydroxy-2'-deoxyguanosine in rat organ DNA and urine. Proc.Natl.Acad.Sci.USA 87, 4533-4537.

Hamilton, M.L., Guo, Z.M., Fuller, C.D., Van Remmen, H., Ward, W.F., Austad, S.N., Troyer, D.A., Thompson, I., and Richardson,  A. (2001a). A Reliable Assessment of 8-Oxo-3Deoxyguanosine Levels in Nuclear and Mitochondrial DNA using the Sodium Iodide Method to Isolate DNA.  Nucleic Acids Res. 29, 2117-2126.

Hamilton, M.L., Van Remmen, H., Drake, J.A., Yang, H., Guo, Z.M., Kewitt, K., Walter, C.A., and Richardson, A.  (2001b). Does Oxidative Damage to DNA Increase with Age?  Proc. Natl. Acad. Sci., USA. 98, 10469-10474.

Helbock, H., Beckman, K., Shigenaga, M., Walter, P. B., Woodall, A., Yeo, H. C., Ames, B. N. (1998). DNA oxidation matters:  The HPLC-electrochemical detection assay of 8-oxo-deoxyguanosine and 8-oxo-guanine. Proc.National Acad.Science 95, 288-293.

Helbock, H. J., Beckman, K. B., Ames, B. N. (1999). 8-Hydroxydeoxyguanosine and 8-Hydroxyguanine as Biomarkers of Oxidative DNA Damage. Methods Enzymol. 300, 156-165.

Nakae, D., Mizumoto, Y., Kobayashi, E., Noguchi, O., Konishi, Y. (1995) Improved genomic/nuclear DNA extraction for 8-hydroxydeoxyguanosine analysis for small amounts of rat liver tissue. Cancer Lett. 97, 233-239.

Poulsen, H. E., Prieme, H., Loft, S. (1998). Role of oxidative DNA damage in cancer initiation and promotion. Eur.J.Cancer Prev. 7, 9-16.

Experimental Methods

Isolation and Preparation of DNA Hydrolysates.

Nuclear DNA is isolated using a DNA Extractor WB Kit, (Wako Chemicals USA, Inc, Richmond, VA) as described by Hamilton et al. (2001a,b).  Approximately 200 to 500mg of frozen tissue is ground into a powder in liquid nitrogen and then homogenized in ice-cold lysis solution using a Dounce homogenizer and then collecting the nuclear pellets obtained by spinning at 10,000 x g for 20 sec.  The pellets are re-suspended in the enzyme reaction solution and proteinase K (10 µg/ml) and RNAse cocktail (Ambion, Austin, TX) is then added to a final concentration of 20 µg/ml.  

The DNA is hydrolyzed by incubating 30 to 75 µg of DNA with nuclease P1 at 65oC for 11 min.  Following the digestion, DNA is treated with calf alkaline phosphatase in a Tris-buffer (40 mM Tris-HCl, pH 8.5, containing 10 mM MgCl2) for 60 min at 37oC. Samples are filtered using a Microcon microconcentrator-10 filtration system (Amicon, Beverly,MA) for 45 min at 4oC. Thirty to 75 µg of nDNA or mtDNA are analyzed by HPLC-EC.

Quantification of oxo8dG Levels in DNA.

The oxo8dG and 2-deoxyguanosine (2dG) were resolved by HPLC and quantified using electrochemical detection (EC) developed by Floyd et al. (1990) as we have described previously (Hamilton et al., 2001a).  A CoulArray electrochemical detection system (ESA Model 5500/5600, ESA, Inc. Chelmsford, MA) is employed using a 3 µM, 150 x 4.6 C-18 column (YMC, Wilmington, NC).  The nucleosides are eluted from the column with an isocratic mobile phase consisting of 50 mM sodium acetate (pH 5.2) and 5.0% methanol. The mobile phase is filtered and degassed prior to application using a Sep-pak C-18 cartridge (Waters, Milford, MA) followed by filtration using a 0.02 µM nitrocellulose filter (Millipore, Bedford, MA) and sonication for 30 min. Two coulometric cells (Model 6210-Four Channel, ESA, Chelmsford, MA) are placed in series and were set to the following potentials: 120, 175, 320, 400, 500, 575, 830, and 890.  The HPLC-EC system is calibrated with 500 fmole to 1 nmole of oxo8dG and 5 nmole to 10 mol of 2dG using authentic oxo8dG and 2dG standards obtained from Sigma Chemical Co (St. Louis, MO).  The figure shows typical chromatograms obtained for DNA hydrolysates using a reverse-phase HPLC-EC system where 2dG had a retention time of 6.5 min and oxo8dG had a retention time of 8.7 min. The identity of 2dG and oxo8dG on the chromatograms were determined by co-injection of standards.  Standards were run after every sixth sample for verification, and the data were expressed as the ratio of nmoles of oxo8dG to 105 nmoles of 2dG. 



References:

Floyd, R. A., West, M. S., Eneff, K. L., Schneider, J. E., Wong, P. K., Tingey, D. T., Hogsett, W. E. (1990). Conditions influencing yield and analysis of 8-hydroxy-2'-deoxyguanosine in oxidatively damaged DNA. Analyt.Biochem. 155-158.

Hamilton, M.L., Guo, Z.M., Fuller, C.D., Van Remmen, H., Ward, W.F., Austad, S.N., Troyer, D.A., Thompson, I., and Richardson,  A. (2001). A Reliable Assessment of 8-Oxo-3Deoxyguanosine Levels in Nuclear and Mitochondrial DNA using the Sodium Iodide Method to Isolate DNA.  Nucleic Acids Res. 29, 2117-2126.

Hamilton, M.L., Van Remmen, H., Drake, J.A., Yang, H., Guo, Z.M., Kewitt, K., Walter, C.A., and Richardson, A.  (2001b). Does Oxidative Damage to DNA Increase with Age?  Proc. Natl. Acad. Sci., USA. 98, 10469-10474.

Sample Preparation Guildelines

We require snap frozen tissue stored at -80oC for the oxo8dG assay.  It is best not to store the tissue for more than a few months before conducting the assay to minimize potential oxidation of DNA during storage. For most tissues, 100-200 mg of tissue is optimal for analysis.  However, for skeletal muscle ~500 mg of tissue is required (Hamilton et al. 2001).  

References: 

Hamilton, M.L., Van Remmen, H., Drake, J.A., Yang, H., Guo, Z.M., Kewitt, K., Walter, C.A., and Richardson, A.  (2001). Does Oxidative Damage to DNA Increase with Age?  Proc. Natl. Acad. Sci., USA. 98, 10469-10474.