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Yoke W. Kow, Ph.D. Division of Cancer Biology, Department of Radiation Oncology, Emory University School of Medicine, 145 Edgewood Avenue, Atlanta, GA 30335 |
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Yoke W. Kow, Ph.D. Division of Cancer Biology, Department of Radiation Oncology, Emory University School of Medicine, 145 Edgewood Avenue, Atlanta, GA 30335 |
Reactive oxygen species (ROS), such as hydrogen peroxide, superoxide and hydroxyl radical are products of oxygen metabolism in all aerobic organisms. ROS are generated as a result of energy production from mitochondria (from the electron transport chain), as part of an antimicrobial1) or antiviral2) response, as well as detoxification reactions carried out by the cytochrome P-450 system3, 4). Environmental agents such as ultraviolet light, ionizing radiation, redox chemicals and cigarette smoke also readily generate ROS. The antioxidant defense system in most cells is composed of two components, the antioxidant enzymes component which includes enzymes such as superoxide dismutase, catalase and glutathione peroxidase, and the low molecular weight antioxidants component that includes vitamins A and E, ascorbate, glutathione and thioredoxin. These substances are the body,s natural defense against endogenous generated ROS and other free radicals, as well as ROS generated by external environmental factors. Oxidative stress occurs when the production of ROS exceeds the body,s natural antioxidant defense mechanisms, causing damage to biomolecules such as lipids, proteins and DNA.
Oxidative damage to DNA is a result of interaction of DNA with ROS, in particular the hydroxyl radical. Superoxide and hydrogen peroxide are normally not reactive towards DNA. However, in the presence of ferrous or cuprous ion (the Fenton reaction), both superoxide and hydrogen peroxide are converted to the highly reactive hydroxyl radical. Hydroxyl radical produces a multiplicity of modifications in DNA. Oxidative attack by OH radical on the deoxyribose moiety will lead to the release of free bases from DNA, generating strand breaks with various sugar modifications and simple abasic (AP) sites. In fact, one of the major types of damages generated by ROS is AP site, a site where a DNA base is lost.
AP sites are also formed at an appreciable rate from spontaneous depurination. It is estimated that at least 10,000 depurination events occur per cell per day under physiological conditions. A similar amount of AP site is thought to be generated by normal aerobic respiration. In addition to AP site, a wide spectrum of oxidative base modification occurs with ROS (Figure 1). The C4-C5 double bond of pyrimidine is particularly sensitive to attack by OH radical, generating a spectrum of oxidative pyrimidine damage including thymine glycol, uracil glycol, urea residue, 5-OHdU, 5-OHdC, hydantoin and others. Similarly, interaction of OH radical with purines will generate 8-OHdG, 8-OHdA, formamidopyrimidines and other less characterized purine oxidative products. It has been estimated that endogenous ROS can result in about 200,000 base lesions per cell per day. The biological consequences of many of the oxidative products are known. For example, unrepaired thymine glycol is a block to DNA replication and is thus potentially lethal to cells. On the contrary, 8-oxoG, an abundant oxidative damage to dG, is readily bypassed by the DNA polymerase and is highly mutagenic. Unrepaired 8-oxoG will mispair with dA, leading to an increase in G to T transition mutations.
In order to maintain the fidelity of genetic material, all organisms have evolved many different repair pathways to remove various types of DNA damage, resulting from either endogenous or external DNA reactive agents. Oxidative damage is repaired by an ubiquitous base excision repair pathway5, 6). Base damage is recognized by DNA N-glycosylase. There are two major N-glycosylases for oxidative base damage (a deamination product such as uracil is recognized by uracil N-glycosylase). Endonuclease III from E. coli is the prototype repair enzyme that recognizes many types of oxidative pyrimidine damages7, 8). Homologues of endonuclease III are found in all cells examined, and its gene has been cloned from bacteria, yeast, mouse, and human cells. The substrate specificity of various endonuclease III homologues appear to be similar9, 10). The enzyme has an associated ƒÀ-lyase activity. After the release of the damaged base by endonuclease III, the enzyme cleaves to the phosphodiester bond 3' to the AP site, leaving behind a 3' modified sugar moiety, 4-hydroxypentenal11). On the other hand, oxidative purine damage is recognized by formamidopyrimidine N-glycosylase (fpg)12, 13). Functional homologues of the bacterial fpg protein are present in yeast and human cells. The eukaryotic enzyme that recognizes 8-oxoG is called 8-xooG glycosylase (OGG1 gene product) and shares no amino acid sequence homology with the bacterial fpg protein14). The substrate specificity of bacterial fpg and eukaryotic OGG1 protein is similar, recognizing 8-oxoG and formamidopyrimidines. However, the bacterial fpg protein has an associated ƒÀ,ƒÂ-lyase activity, leaving behind a 3' phosphate terminus15, 16). The eukaryotic OGG1 protein is a ƒÀ-lyase, leaving behind a 3' 4-hydroxypentenal residue. The 3' residue (either the 4-hydroxypentenal or phosphoryl group) left behind by these N-glycosylases are further processed by AP endonucleases, generating the 3' OH that is required for repair synthesis catalyzed by DNA polymerase. There are two major types of AP endonuclease, endonuclease III and the exonuclease IV. In E. coli, both AP endonucleases are present, with exonuclease III being the major AP endonuclease. In human cells, the major AP endonuclease is exonuclease III and in yeast, the major activity is endonuclease IV. After the 3' end of the DNA is processed by AP endonucleases, the repair process is completed following repair synthesis and ligation by DNA polymerase and ligase, respectively.
Oxidative stress has been thought to contribute to the general decline in cellular functions that are associated with many human diseases including Alzheimer,s disease17, 18), amyotrophic lateral sclerosis (ALS)19, 20), Parkinson,s disease21, 22), atherosclerosis23, 24), ischemia/reperfusion neuronal injuries, degenerative disease of the human temporomandibular-joint25), cataract formation26, 27), macular degeneration20, 28), degenerative retinal damage29), rheumatoid arthritis30), multiple sclerosis31), muscular dystrophy32, 33), human cancers34, 35) as well as the aging36, 37) process itself. Increased cellular level of ROS due to oxidative stress can result in an increased steady state level of oxidative DNA damage. There is increasing evidence that an increased level of oxidative damage such as AP site is detected in cells obtained from ALS and Alzheimer patients or after ischemia/reperfusion. Due to the fact that many human diseases might be resulting from chronic oxidative stress and the magnitude of oxygen damage to DNA that is associated with oxidative stress, it this important to have a simple and accurate procedure for estimating the level of oxidative DNA damage in oxidative stressed cells.
ARP reagent (N'-aminooxymethylcarbonylhydrazino-D-biotin, Fig. 2) is a biotinylated hydroxylamine derivative. The chemical reacts specifically with an aldehyde group, thus allowing the detection of DNA modifications that resulted in the formation of an aldehyde group. AP site in DNA exists in equilibrium between the ring closed and the ring opened form (Fig. 3). Approximately 5% of the AP site is in the ring opened form, which has an active aldehyde group. ARP, a biotinylated alkoxylamine (Fig. 2) reacts specifically with the aldehyde group in the ring opened AP site. After treating DNA containing AP sites with ARP reagent, AP sites are thus tagged with a biotin residue. By using an excess amount of ARP reagent, essentially all AP sites can be converted to biotin-tagged AP sites. The amount of biotinylated AP sites can then be easily quantified with an ELISA-like assay, using avidin-biotin complex conjugated with either horseradish peroxidase or alkaline phosphatase as an indicator enzyme (Fig. 4). This procedure has been successfully used by several laboratories for accurate measurement of AP sites in DNA38, 39, 40, 41). A modification of the ELISA-like ARP assay was made by (38), allowing even more sensitivity in the detection of AP site. Instead of binding DNA to a microtiter plate, DNA was bound to a nitrocellulose membrane using a dot blot apparatus. A microtiter plate-based AP site assay kit is currently available from Dojindo Laboratories.
Many kinds of base damages are recognized by damage specific DNA glycosylases. The substrate spectrum of DNA glycosylases varies depending on the enzymes; some have very narrow substrate specificity, such as uracil DNA N-glycosylase and T4 endonuclease V, while some can recognize a variety of base modifications such as endonuclease III, 8-oxoguanine N-glycosylase and alkA protein. These glycosylases remove modified bases, leaving behind either intact AP sites or modified sugar moieties (4-hydroxy-pentenal) still attached to the 3' termini of nicked DNA. Both products of N-glycosylases still retain the active aldehyde that can easily be quantified by the use of ARP assay. Therefore, treating damaged DNA with a specific repair enzyme will permit the determination of a class of base damages that is normally recognized by the repair enzyme. The advantage of the enzyme-coupled ARP assay is that it allows the investigator to assess the contribution of a whole spectrum of base damage that is normally recognized by the repair enzymes. Furthermore, if one would like to assess the amount of oxidative DNA damage due to increased oxidative stress, treatment of the damaged DNA with both endonuclease III and yeast OGG1 will provide a relatively good assessment of the total amount of oxidative base damage that has occurred on the DNA. Endonuclease III has been shown to recognize many different types of pyrimidine oxidative damages. Therefore DNA samples treated with excess endonuclease III will leave behind a 3' modified sugar moiety (4-hydroxypentenal) that can be tagged with the ARP reagent. The amount of ARP tag can then be determined and be used as a measurement for endonuclease III sensitive site or an oxidative pyrimidine lesion. In fact, the enzyme coupled-ARP assay has been used for the quantification of thymine glycols and alkylation damage in DNA39, 41). Similarly, oxidative purine damage can be detected using either the yeast 8-oxoguanine glycosylase (yOGG1) or the human 8-oxoguanine glycosylase (hOGG1). In the latter case, the bacterial fpg protein cannot be used since the enzyme generates a phosphoryl group. OGG1 has been shown to recognize mostly 8-oxoG and formamidopyrimidines (OGG does not recognize 8-oxoA), but the amount of damage determined by the use of OGG might underestimate the total oxidative purine damage. However, it should provide a good assessment of the level of biologically important purine damage present in the cells. A kit for estimating the amount of oxidative pyrimidine and purine damage is currently under development by Dojindo Laboratories, and should be available soon.
References
1. Weiss, S. J., and LoBuglio, A. F., Laboratory Investigation, 47, 5-18(1982).
2. Griot, C., Burge, T., Vandevelde, M., and Peterhans, E., Acta Neuropathol., 78, 396-403(1989).
3. Scholz, W., Schutze, K., Kunz, W., and Schwarz, M., Cancer Res., 50, 7015-22(1990).
4. Cederbaum, A. I., Free Radical Biology & Medicine, 7, 559-67(1989).
5. Kow, Y. W., Faundez, G., Melamede, R. J., and Wallace, S. S., Radiation Res., 126, 357-66(1991).
6. Kow, Y. W., Annals of the New York Academy of Sciences, 726, 178-80(1994).
7. Kow, Y. W., and Wallace, S. S., Biochemistry, 26, 8200-6(1987).
8. Purmal, A. A., Rabow, L. E., Lampman, G. W., Cunningham, R. P., and Kow, Y. W., Mutation Res., 364, 193-207(1996).
9. Aspinwall, R., Rothwell, D. G., Roldan-Arjona, T., Anselmino, C., Ward, C. J., Cheadle, J. P., Sampson, J. R., Lindahl, T., Harris, P. C., and Hickson, I. D., Proc. Natl. Acad. Sci. USA, 94, 109-14 (1997).
10. Ikeda, S., Biswas, T., Roy, R., Izumi, T., Boldogh, I., Kurosky, A., Sarker, A. H., Seki, S., and Mitra, S., J. Biol. Chem., 273, 21585-93 (1998).
11. Mazumder, A., Gerlt, J. A., Absalon, M. J., Stubbe, J., Cunningham, R. P., Withka, J., and Bolton, P. H., Biochemistry, 30, 1119-26 (1991).
12. Chetsanga, C. J., Lozon, M., Makaroff, C., and Savage, L., Biochemistry, 20, 5201-7 (1981).
13. Tchou, J., Bodepudi, V., Shibutani, S., Antoshechkin, I., Miller, J., Grollman, A. P., and Johnson, F., J. Biol. Chem., 269, 15318-24 (1994).
14. Radicella, J. P., Dherin, C., Desmaze, C., Fox, M. S., and Boiteux, S., Proc. Natl. Acad. Sci. USA, 94, 8010-5 (1997).
15. Tchou, J., and Grollman, A. P., J. Biol. Chem., 270, 11671-7 (1995).
16. Rabow, L. E., and Kow, Y. W., Biochemistry, 36, 5084-96 (1997).
17. Multhaup, G., Ruppert, T., Schlicksupp, A., Hesse, L., Beher, D., Masters, C. L., and Beyreuther, K., Biochem. Pharm., 54, 533-9 (1997).
18. Bozner, P., Grishko, V., LeDoux, S. P., Wilson, G. L., Chyan, Y. C., and Pappolla, M. A., J. Neuropathol. Experimental Neurol., 56, 1356-62 (1997).
19. Jackson, C. E., and Bryan, W. W., Seminars in Neurology, 18, 27-39 (1998).
20. Christen, W. G., Glynn, R. J., and Hennekens, C. H., Ann. Epidemiol., 6, 60-6 (1996).
21. Radunovic, A., Porto, W. G., Zeman, S., and Leigh, P. N., Neurosci. Lett., 239, 105-8 (1997).
22. Mukherjee, S. K., and Adams, J. D., Jr., Molecular & Chemical Neuropathol., 32, 59-74 (1997).
23. Alexander, R. W., Transactions of the American Clinical & Climatological Association, 109, 129-45 (1998); discussion, 109, 145-6 (1998).
24. Fiorillo, C., Oliviero, C., Rizzuti, G., Nediani, C., Pacini, A., and Nassi, P., Clinical Chemistry & Laboratory Medicine, 36, 149-53 (1998).
25. Milam, S. B., Zardeneta, G., and Schmitz, J. P., Journal of Oral & Maxillofacial Surgery, 56, 214-23 (1998).
26. Tissie, G., Guillermet, V., Latour, E., Coquelet, C., and Bonne, C., Ophthalmic Res., 20, 27-30 (1988).
27. Varma, S. D., Devamanoharan, P. S., and Morris, S. M., Critical Reviews in Food Science & Nutrition, 35, 111-29 (1995).
28. Nicolas, M. G., Fujiki, K., Murayama, K., Suzuki, M. T., Shindo, N., Hotta, Y., Iwata, F., Fujimura, T., Yoshikawa, Y., Cho, F., and Kanai, A., Experimental Eye Research, 62, 399-408 (1996).
29. Andersson, M., Sjostrand, J., Petersen, A., and Karlsson, J. O., Ophthalmic Research, 30, 157-67 (1998).
30. Miyata, T., Ishiguro, N., Yasuda, Y., Ito, T., Nangaku, M., Iwata, H., and Kurokawa, K., Biochem. Biophys. Res. Commun., 244, 45-9 (1998).
31. Calabrese, V., Raffaele, R., Cosentino, E., and Rizza, V., International Journal of Clinical Pharmacology Research, 14, 119-23 (1994).
32. Rando, T. A., Disatnik, M. H., Yu, Y., and Franco, A., Neuromuscular Disorders, 8, 14-21 (1998).
33. Murphy, M. E., and Kehrer, J. P., Biochem. J., 260, 359-64 (1989).
34. DeWeese, T. L., Shipman, J. M., Larrier, N. A., Buckley, N. M., Kidd, L. R., Groopman, J. D., Cutler, R. G., te Riele, H., and Nelson, W. G., Proc. Natl. Acad. Sci., 95, 11915-20 (1998).
35. Meyer, T. E., Liang, H. Q., Buckley, A. R., Buckley, D. J., Gout, P. W., Green, E. H., and Bode, A. M., International Journal of Cancer, 77, 55-63 (1998).
36. Stadtman, E. R., and Berlett, B. S., Drug Metabolism Reviews, 30, 225-43 (1998).
37. Beckman, K. B., and Ames, B. N., Physiol. Reviews, 78, 547-81 (1998).
38. Nakamura, J., Walker, V. E., Upton, P. B., Chiang, S. Y., Kow, Y. W., and Swenberg, J. A., Cancer Research, 58, 222-5 (1998).
39. Kubo, K., Ide, H., Wallace, S. S., and Kow, Y. W., Biochemistry, 31, 3703-8 (1992).
40. Ide, H., Akamatsu, K., Kimura, Y., Michiue, K., Makino, K., Asaeda, A., Takamori, Y., and Kubo, K., Biochemistry, 32, 8276-83 (1993).
41. Asaeda, A., Ide, H., Tano, K., Takamori, Y., and Kubo, K., Nucleosides & Nucleotides, 17, 503-13 (1998).
