| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 8, 2004 as doi:10.1096/fj.04-1767fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
,2
* Rowett Research Institute, Aberdeen, Scotland, UK; and
Department of Nutrition, University of Oslo, Oslo, Norway
2 Correspondence: Department of Nutrition, University of Oslo, PO Box 1046 Blindern, Oslo 0316 Norway. E-mail: a.r.collins{at}medisin.uio.no
SPECIFIC AIMS
The purposes of this study were 1)validation of methods for measuring background levels of 8-oxo-7,8-dihydroguanine (8-oxoGua) or 8-oxo-7,8-dihydro-2’deoxyguanosine (8-oxodGuo) in cellular DNA, for use in human population studies; 2) determination of the range of 8-oxoGua/8-oxodGuo levels found in normal human lymphocyte DNA; and 3) investigation of differences between countries in mean background levels of 8-oxoGua/8-oxodGuo in lymphocytes from healthy volunteers.
Oxidative damage within the body arises through the attack of highly reactive forms of oxygen released during normal respiration and as part of the inflammatory response. This kind of damage to biomolecules such as lipids, proteins, and DNA has been implicated in many chronic and degenerative human diseases and in aging. As oxidative DNA damage may be a cause of cancer, it is important to be able to measure this damage reliably. The most commonly used biomarker of DNA oxidation in epidemiological and experimental studies is 8-oxoGua or the corresponding nucleoside, 8-oxodGuo. Estimates of the concentration of 8-oxoGua in DNA from normal human cells vary by as much as 1000-fold depending on which procedure is used to measure it.
The European Standards Committee on Oxidative DNA Damage (ESCODD), with 25 member laboratories, has coordinated the distribution and analysis of standard samples of 8-oxodGuo, oligonucleotides with defined amounts of 8-oxoGua, calf thymus DNA, calf thymus DNA with experimentally induced 8-oxoGua, pig liver DNA, HeLa cells, and HeLa cells with experimentally induced 8-oxoGua. In the latest trial aimed at validating methods for use in human population studies, laboratories recruited volunteers in their respective countries to provide lymphocytes for analysis of background levels of oxidative DNA damage.
Various methods are used to measure 8-oxoGua or 8-oxodGuo in DNA (Fig. 1
). The chromatographic approach is represented by HPLC with electrochemical detection (HPLC-ECD), either amperometric or coulometric, and HPLC with tandem mass spectrometry (HPLC-MS/MS). The alternative approach we refer to as the "enzymic approach" because it depends on the bacterial DNA repair enzyme formamidopyrimidine DNA glycosylase (FPG) to convert oxidized purines (principally 8-oxoGua) to DNA breaks subsequently measured using the comet assay, alkaline unwinding, or alkaline elution. In recent rounds of ESCODD, the enzymic approach has given estimates of 8-oxoGua that are several times lower (comparing median results from different laboratories) than those obtained using chromatography and fall within a narrower range.
|
There is evidence that substantial oxidation of guanine occurs during the protracted treatment of samples in preparation for chromatographic analysis, and in ESCODD we have designed protocols for DNA extraction and hydrolysis to protect against this oxidation. The enzymic methods involve minimal processing of the sample (e.g., in the comet assay cells are simply centrifuged, embedded in agarose, incubated with FPG, then with alkali, and electrophoresed); samples are therefore less likely to suffer from spurious oxidation. The present trial serves as a test of our success in controlling the oxidation artifact, compares the merits of the two approaches, and affords a realistic estimate of background DNA oxidation in normal human cells.
PRINCIPAL FINDINGS
1. Levels of 8-oxodGuo in HeLa cells measured by HPLC
Identical samples of HeLa (human transformed epithelial) cells were supplied to all participating laboratories to act as a reference standard for analysis of 8-oxodGuo. The median value from all laboratories using HPLC (with ECD or MS-MS) was 2.78 8-oxodGuo per 106 dGuo when using their own method for extraction and hydrolysing DNA (or 2.73 per 106 when using the recommended ESCODD method). The range of mean values from laboratories using their own methods was 32x compared with a range of 13x with the ESCODD method. The latter may be somewhat more reliable; but it is clear that all the sources of variation or of spurious oxidation have not yet been identified and controlled. About half of the laboratories report values of 
4–5 8-oxodGuo per 106 dGuo, and it is tempting to assume that the few results that are far below that level must be the result of an experimental anomaly. But it is equally possible that these low values represent laboratories that have managed to minimize the spurious oxidation. To account for the discrepant results there must be a divergence of experimental conditions among laboratories in steps not covered by the standard protocol (i.e., in the earliest stages of preparation or during HPLC itself).
2. Enzymic determination of 8-oxoGua in HeLa cells
The alternative approach to chromatography is the use of enzymic conversion of 8-oxoGua to DNA breaks, which can then be assayed by a variety of essentially physical methods. (FPG has as its main substrate 8-oxoGua, but it also detects formamidopyrimidines FaPyAde and FaPyGua. For convenience, we refer to FPG sites as 8-oxoGua, but the presence of FaPy bases will mean that 8-oxoGua is overestimated.) In the present study, the median concentration of 8-oxoGua in HeLa cell DNA by enzymic analysis was 0.50/106 guanines and the range among laboratories was 10-fold. The overall range reflects extremes; as with HPLC, most individual results agree much more closely than this range suggests.
Enzymic methods tend to give lower estimates of background damage than chromatographic methods. A possible explanation is that the enzymic method in general underestimates 8-oxoGua by several-fold because of a failure of the enzyme FPG to detect all lesions or because lesions occur in clusters so close together they are detected as a single break. On the other hand, since FPG detects oxidized purines in addition to 8-oxoGua, our estimates of 8-oxoGua may be too high. The calibration of these assays, using ionizing radiation to induce known frequencies of breaks, may be inaccurate. Dose/response experiments conducted with alkaline elution and the comet assay vs. HPLC indicate that enzymic methods and HPLC measure experimentally induced 8-oxoGua in cultured cells with similar efficiency (with similar dose-response slopes) and that the main problem is therefore spurious oxidation inflating HPLC measurements.
3. 8-oxodGuo levels in human lymphocyte DNA
Eleven laboratories recruited healthy young adult male volunteers. Six laboratories reported results for HPLC-ECD and the comet assay. One laboratory carried out only the comet assay and four only performed HPLC-ECD. The results are displayed as mean values from each laboratory in Fig. 2
(HPLC-ECD) and Fig. 3
(comet assay). The differences among laboratories may reflect national patterns, but there are too few results from different laboratories in the same country to be sure that the variations are reliable. There is no significant correlation between mean values obtained by the two methods compared across laboratories.
|
|
The "mean of means" (i.e., the mean of all individual laboratory means) for HPLC is 3.73 8-oxodGuo/106 dGuo; the median value is 4.24. With the comet assay, the mean of means and the median are both 0.34 FPG sites/106 guanines. Thus, the chromatographic and enzymic approaches give estimates of background 8-oxodGuo in lymphocytes that differ by factors of 11–12. Looking only at those laboratories that used HPLC and the comet assay, the overall mean (and median) values of 8-oxodGuo are 4.40 and 0.36 per 106 guanines, respectively (again, differing by a factor of 12).
CONCLUSIONS AND SIGNIFICANCE
The difference between the chromatographic and enzymic approaches in median estimates of 8-oxoGua is 6-fold for HeLa cells or 12-fold for lymphocytes compared with the far wider range that we faced at the start of ESCODD. We are able to provide a realistic estimate of the actual background level of damage in lymphocytes, which is likely to lie somewhere between 4.2 and 0.3 8-oxodGuo/106 guanines (median values of the means from different laboratories for HPLC and the comet assay).
The implications of this conclusion are far-reaching. Many trials have been carried out in which human lymphocytes or tissues from normal or tumor tissue have been analyzed for 8-oxodGuo using the techniques tested (or GC-MS, included in previous rounds of ESCODD). One topic under investigation that is affected by this measurement problem is that of dietary antioxidants. In numerous studies, volunteers have taken supplements of antioxidants such as vitamin C, carotenoids, or flavonoids or foods rich in antioxidants, and oxidized bases have been measured in the DNA of white blood cells. It is important to know whether these supplements are able to decrease the level of damage, which would support claims that they may help prevent cancer. It is necessary to reexamine published reports that make claims of much higher values of 8-oxodGuo than 4/106 guanines. With the information we have provided, a more rigorous approach can be followed.
Several questions remain. Is spurious oxidation under control? Apparently not, to judge by the variable results obtained with identical samples of HeLa cells. If we could eliminate this artifact, would HPLC-ECD be sensitive enough to measure the low levels in real biological samples? There is also the challenge of identifying 8-oxodGuo unequivocally. Although normally a voltammogram can provide a tentative identification, there is insufficient DNA in the lymphocytes from blood samples to do this. The only way to identify the 8-oxodGuo peak is by its retention time. HPLC-MS/MS can provide a definitive identification and measurement not just of 8-oxodGuo, but also of other oxidative DNA lesions. But until now it has proved to be a demanding and complicated method and has not given reliable results in the analysis of 8-oxodGuo in ESCODD trials.
The enzymic approach of using FPG to convert oxidized purines to DNA breaks appears relatively free from the problem of spurious oxidation. To exploit this approach to full effect, it needs to be used quantitatively, so we must address the question of which lesions FPG detects and how efficiently. Calibration of the enzymic methods should not rely solely on a comparison with the effects of ionizing radiation. The use of repair endonucleases is not limited to FPG; endonuclease III can be used in a similar way to detect oxidized pyrimidines, providing another biomarker of oxidative damage.
FOOTNOTES
1 A full list of ESCODD members appears in the full version of this article. 
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-1767fje;
This article has been cited by other articles:
|
|
 |
|
  R. M. Green, N. J. Hodges, J. K. Chipman, M. R. O'Donovan, and M. Graham Reactive oxygen species from the uncoupling of human cytochrome P450 1B1 may contribute to the carcinogenicity of dioxin-like polychlorinated biphenyls Mutagenesis, November 1, 2008; 23(6): 457 - 463. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. G. Sturmey, J. A. Hawkhead, E. A. Barker, and H. J. Leese DNA damage and metabolic activity in the preimplantation embryo Hum. Reprod., October 3, 2008; (2008) den346v1. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-H. Park, D. Mangal, K. A. Tacka, A. M. Quinn, R. G. Harvey, I. A. Blair, and T. M. Penning Evidence for the aldo-keto reductase pathway of polycyclic aromatic trans-dihydrodiol activation in human lung A549 cells PNAS, May 13, 2008; 105(19): 6846 - 6851. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. J. McKenna, S. R. McKeown, and V. J. McKelvey-Martin Potential use of the comet assay in the clinical management of cancer Mutagenesis, May 1, 2008; 23(3): 183 - 190. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. R. Collins, A. A. Oscoz, G. Brunborg, I. Gaivao, L. Giovannelli, M. Kruszewski, C. C. Smith, and R. Stetina The comet assay: topical issues Mutagenesis, May 1, 2008; 23(3): 143 - 151. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Forchhammer, E. V. Brauner, J. K. Folkmann, P. H. Danielsen, C. Nielsen, A. Jensen, S. Loft, G. Friis, and P. Moller Variation in assessment of oxidatively damaged DNA in mononuclear blood cells by the comet assay with visual scoring Mutagenesis, May 1, 2008; 23(3): 223 - 231. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Dusinska and A. R. Collins The comet assay in human biomonitoring: gene-environment interactions Mutagenesis, May 1, 2008; 23(3): 191 - 205. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. Mundt, S. S. Hah, R. A. Sumbad, V. Schramm, and P. T. Henderson Incorporation of extracellular 8-oxodG into DNA and RNA requires purine nucleoside phosphorylase in MCF-7 cells Nucleic Acids Res., January 17, 2008; 36(1): 228 - 236. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. S. Cooke, R. Olinski, S. Loft, and members of the European Standards Committee on Uri Measurement and Meaning of Oxidatively Modified DNA Lesions in Urine Cancer Epidemiol. Biomarkers Prev., January 1, 2008; 17(1): 3 - 14. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-H. Chen, C. N. Hales, and S. E. Ozanne DNA damage, cellular senescence and organismal ageing: causal or correlative? Nucleic Acids Res., December 3, 2007; 35(22): 7417 - 7428. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Cordelli, A. M. Fresegna, A. D'Alessio, P. Eleuteri, M. Spano, F. Pacchierotti, and P. Villani ReProComet: A New In Vitro Method to Assess DNA Damage in Mammalian Sperm Toxicol. Sci., October 1, 2007; 99(2): 545 - 552. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. Humphreys, R. M. Martin, B. Ratcliffe, S. Duthie, S. Wood, D. Gunnell, and A. R. Collins Age-related increases in DNA repair and antioxidant protection: A comparison of the Boyd Orr Cohort of elderly subjects with a younger population sample Age Ageing, September 1, 2007; 36(5): 521 - 526. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. S. Hah, J. M. Mundt, H. M. Kim, R. A. Sumbad, K. W. Turteltaub, and P. T. Henderson From the Cover: Measurement of 7,8-dihydro-8-oxo-2'-deoxyguanosine metabolism in MCF-7 cells at low concentrations using accelerator mass spectrometry PNAS, July 3, 2007; 104(27): 11203 - 11208. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Watters, J. A. Satia, L. L. Kupper, J. A. Swenberg, J. C. Schroeder, and B. R. Switzer Associations of Antioxidant Nutrients and Oxidative DNA Damage in Healthy African-American and White Adults Cancer Epidemiol. Biomarkers Prev., July 1, 2007; 16(7): 1428 - 1436. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. L. Neeley, S. Delaney, Y. O. Alekseyev, D. F. Jarosz, J. C. Delaney, G. C. Walker, and J. M. Essigmann DNA Polymerase V Allows Bypass of Toxic Guanine Oxidation Products in Vivo J. Biol. Chem., April 27, 2007; 282(17): 12741 - 12748. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. J. Thompson Oxidative DNA Damage and Cancer Risk Assessment J. Nutr., October 1, 2006; 136(10): 2693S - 2694S. [Full Text] [PDF]
|
|
|
|
 |
|
 J. P. Thomas, J. Lautermann, B. Liedert, F. Seiler, and J. Thomale High Accumulation of Platinum-DNA Adducts in Strial Marginal Cells of the Cochlea Is an Early Event in Cisplatin but Not Carboplatin Ototoxicity Mol. Pharmacol., July 1, 2006; 70(1): 23 - 29. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-W. Hu, C.-J. Wang, L. W. Chang, and M.-R. Chao Clinical-Scale High-Throughput Analysis of Urinary 8-Oxo-7,8-Dihydro-2'-Deoxyguanosine by Isotope-Dilution Liquid Chromatography-Tandem Mass Spectrometry with On-Line Solid-Phase Extraction Clin. Chem., July 1, 2006; 52(7): 1381 - 1388. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Loft, P. Svoboda, H. Kasai, A. Tjonneland, U. Vogel, P. Moller, K. Overvad, and O. Raaschou-Nielsen Prospective study of 8-oxo-7,8-dihydro-2'-deoxyguanosine excretion and the risk of lung cancer Carcinogenesis, June 1, 2006; 27(6): 1245 - 1250. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. A. Sokhansanj and D. M. Wilson III Estimating the effect of human base excision repair protein variants on the repair of oxidative DNA base damage. Cancer Epidemiol. Biomarkers Prev., May 1, 2006; 15(5): 1000 - 1008. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Ohno, T. Miura, M. Furuichi, Y. Tominaga, D. Tsuchimoto, K. Sakumi, and Y. Nakabeppu A genome-wide distribution of 8-oxoguanine correlates with the preferred regions for recombination and single nucleotide polymorphism in the human genome. Genome Res., May 1, 2006; 16(5): 567 - 575. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. C. Smith, M. R. O'Donovan, and E. A. Martin hOGG1 recognizes oxidative damage using the comet assay with greater specificity than FPG or ENDOIII Mutagenesis, May 1, 2006; 21(3): 185 - 190. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
