Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB21 9SB, UK
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Abstract |
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Abbreviations: DMSO, dimethyl sulphoxide; FPG, formamidopyrimidine DNA glycosylase; GMEM, Glasgow-modified Eagle's Minimal Essential Medium; 8-oxoGua, 8-oxo-7,8-dihydroguanine; 8-oxodGuo, 8-oxo-7,8-dihydro-2'-deoxyguanosine; PABA, 4-aminobenzoic acid; PBS, phosphate-buffered saline
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Introduction |
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An alternative approach to the measurement of oxidative DNA damage is based on the incubation of DNA with repair endonucleases that remove oxidized bases. The strand breaks that they introduce are measured by a variety of techniques, including alkaline elution (7), alkaline unwinding (8) and the comet assay (9). Endonuclease III recognizes oxidized pyrimidines (10), while formamidopyrimidine DNA glycosylase (FPG) recognizes 8-oxoGua as well as formamidopyrimidines (fapy-adenine and fapy-guanine) (11). These methods are calibrated against the DNA breaks introduced in cells by ionizing radiation, and so estimates of oxidized bases are indirect. However, there is close agreement between the three different approaches, giving an estimated background level of damage in normal human cells of around 0.5 FPG sites per 106 dGuo (7,12,13).
There is thus a several-fold discrepancy between the lowest HPLC-based estimates and the values reached using FPG with the DNA-breakage assays. This may indicate that the additional oxidation of guanine during preparation of samples for HPLC has still not been eradicated; or there may be a systematic underestimation of 8-oxoGua by the enzyme-based methods. For example, if several bases are oxidized in close proximity in the DNA, the cluster of lesions recognized by FPG will behave as a single DNA strand break in any of the assays based on DNA unwinding. It is also possible that some damage sites in the DNA are inaccessible to the enzyme.
It should be possible to identify the source of the discrepancy by carrying out doseresponse experiments with a suitable DNA-oxidizing system to induce 8-oxoGua. Figure 1 shows the predicted consequences of the two proposed explanations. If additional oxidation occurs during HPLC sample preparation, the levels of 8-oxoGua in the absence of experimental damaging agent will differ, but the slopes of the lines representing induced damage should be equal (b = c). If enzymic methods underestimate damage, then the doseresponse slopes will differ in gradient (a, c) and, in theory, the ratio of slopes (a:c) should equal the ratio of the damage in untreated cells (p:q). To test this (perhaps simplistic) view, we have performed the doseresponse experiments using visible light in combination with the photosensitizing compound Ro 19-8022, which induces few strand breaks relative to oxidized guanine residues (14). As the comet assay detects a range of damage (FPG-sensitive sites) below the level of detection by HPLC, it was not feasible to use the same doses for the two approaches. However, the gradients of the doseresponse curves can validly be compared.
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Here we report the results of a small trial in which we measured urinary 8-oxodGuo, 8-oxodGuo in lymphocyte DNA by HPLC, and FPG sites in lymphocyte DNA by the comet assay. The correlations that we have found indicate that all three biomarkers are reliable indicators of oxidative stress.
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Materials and methods |
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Isolation and hydrolysis of nuclear DNA from human lymphocytes and HPLC analysis of 8-oxodGuo
Lymphocyte samples were thawed, diluted to 50 ml with ice-cold phosphate-buffered saline (PBS) and centrifuged at 875 g for 7 min at 4°C. The cells were washed in 10 mM TrisHCl, 0.4 M NaCl, 5 mM deferoxamine mesylate, pH 8.0 and centrifuged again. Nuclear DNA was isolated as described (21), dissolved in 40 mM TrisHCl, pH 8.5 and stored under nitrogen at 80°C until use; it was then hydrolysed with deoxyribonuclease I, phosphodiesterases I and II and alkaline phosphatase (all from Roche Diagnostics Ltd, Lewes) in the presence of Mg2+ for 2 h at 37°C (22).
The DNA hydrolysate was analysed on a 15x0.46 cm ODS-Apex C18 3 µm column (Capital HPLC Ltd, London) with a 2x0.4 cm guard column containing Perisorb RP18 (Supelco, Poole). The mobile phase was 50 mM potassium phosphate, pH 5.5, containing 8% methanol, and the flow rate was 0.5 ml/min. 8-oxodGuo was measured by an electrochemical detector (ESA Coulochem II) with a 5021 conditioning cell and a 5011 analytical cell. 2'-Deoxyguanosine was measured with a Gilson Holochrome UV detector at 254 nm. Data collection and analysis were done with Gynkotek Chromeleon software.
Measurement of FPG- and endonuclease III-sensitive sites in human lymphocyte DNA by the comet assay
The lymphocytes were thawed with gentle agitation in a 37°C water bath, and centrifuged at 200 g for 3 min at 4°C. The pelleted cells were dispersed in 0.4 ml RPMI medium with 10% heat-inactivated fetal calf serum, and aliquots of 65 µl were added to 1 ml cold PBS. The cells were centrifuged again, dispersed in 85 µl 1% low melting point agarose (Life Technologies, Paisley) at 37°C, placed on a microscope slide and processed for the comet assay as described (23). For each sample, four gels were incubated with FPG, four with endonuclease III and four with buffer alone. The measures of oxidized purines and pyrimidines (in arbitrary units) were obtained by subtraction of the mean comet assay score with buffer alone from that with FPG or endonuclease III respectively.
Culture of HeLa cells for HPLC and comet assay analysis of 8-oxodGuo/8-oxoGua
HeLa (human transformed epithelial) cells were grown in Glasgow-modified Eagle's Minimal Essential Medium (GMEM) with 5% fetal calf serum, 5% calf serum, supplemented with glutamine and non-essential amino acids.
For measurement of background levels of 8-oxoGua, cells grown to confluence in polystyrene roller bottles (Corning Inc., New York) were harvested by trypsinization, combined and then divided into aliquots for centrifugation at 700 g for 7 min at 20°C. The cell pellets were dispersed in freezing mix (GMEM containing 20% fetal calf serum, 10% DMSO) to a cell density of 5x106/ml, and aliquots of 55x106 cells for HPLC and 1.5x106 cells for comet assay analysis were cooled slowly to 80°C and stored at 80°C until use.
For measurement of induced 8-oxoGua, HeLa cells were grown to confluence in 21x50 ml flasks. They were harvested by trypsinization, combined and divided into 16x140 mm and 12x60 mm dishes (in 25 ml and 4.6 ml medium respectively). The ratio of cell number:surface area of dish was the same for the two types of dishes.
Treatment of HeLa cells with Ro 19-8022
For HPLC analysis, confluent monolayers of HeLa cells in the 140 mm dishes were rinsed twice with cold PBS. 19.5 ml of PBS-G (140 mM NaCl, 3 mM KCl, 8 mM Na2HPO4, 1 mM KH2PO4, 1 mM CaCl2, 0.5 mM MgCl2, 0.1% glucose, pH 7.4) was added to four of these dishes. Other groups of four dishes received 1, 2 or 5 µM Ro 19-8022 (a generous gift from Hoffman-La Roche, Basel) in 19.5 ml PBS-G. All dishes were placed on ice (two dishes at a time) and irradiated 33 cm from a 1000 W halogen lamp for 5 min. Monolayers were rinsed twice with cold PBS and the cells gently scraped into suspension in PBS. The cells from each pair of dishes were combined and centrifuged at 700 g, for 7 min at 4°C. The cells were dispersed gently in freezing mix to a density of 5x106/ml. The cell suspensions were cooled slowly to 80°C as above and were stored at 80°C until use. The four dishes for each dose of Ro 19-8022 were treated with photosensitizer and light and the cells placed at 80°C before treatment of subsequent groups of dishes.
For analysis with the comet assay, confluent monolayers of cells in 60 mm dishes were treated (two dishes at a time) with Ro 19-8022 as described for HPLC analysis with the exceptions that (i) Ro 19-8022 was added at 0, 0.1, 0.2, 0.4 and 0.6 µM in 3.6 ml PBSG and the cells were irradiated for only 2 min and (ii) the cells were collected from each dish separately. The cells were frozen as described above and stored at 80°C until use.
Isolation and hydrolysis of nuclear DNA from HeLa cells and HPLC analysis of 8-oxodGuo
For measurement of background 8-oxodGuo, HeLa cells in freezing mix were thawed in a 37°C water bath. Six aliquots of 55x106 cells were processed in parallel. PBS was added to 50 ml and the cells were centrifuged at 700 g for 7 min at 4°C. The cells were washed again in 50 ml PBS. Nuclear DNA was isolated as described (21), dissolved in 10 mM TrisHCl pH 7.3 and stored at 80°C under nitrogen until hydrolysis with P1 nuclease and alkaline phosphatase (22). The hydrolysate was analysed by HPLC as described above.
Preparation of DNA for measurement of induced 8-oxodGuo was as described above, but was performed on the two samples for each dose of Ro 19-8022.
Authentic 8-oxodGuo (in a mix of dGuo and 8-oxodGuo), and putative 8-oxodGuo in hydrolysed DNA from untreated and Ro 19-8022/light-treated HeLa cells, were analysed over a range of voltages, in order to verify the peak identification by comparison of voltammograms of samples with standard. For ease of comparison, the peak areas of 8-oxodGuo in the standard, untreated and Ro 19-8022/light-treated HeLa cells were normalized to dGuo concentrations of 100, 250 and 150 µM respectively.
Comet assay analysis of FPG-sensitive sites in HeLa cells
Untreated cells: seven aliquots of HeLa cells were thawed and prepared for the comet assay as described for lymphocytes above except that 30 µl of cells in RPMI, 10% heat-inactivated fetal calf serum were added to 1 ml PBS. For each of the seven samples, four or six gels were incubated with FPG and the same number with buffer. The measure of FPG-sensitive sites was obtained by subtraction of the mean score for buffer from that with FPG.
Treated cells: the procedure was as above, except that four gels per sample were incubated with FPG and four gels with the enzyme buffer.
Quantitation of the comet assay
Our standard comet scoring method, based on visual examination of 100 randomly selected comets, and classification into categories 04 depending on the relative intensity of tail fluorescence, gives a score for each gel of between 0 and 400 arbitrary units. This method has been validated against computer-based image analysis (24), a score of 400 corresponding to 80% of total DNA fluorescence in comet tails. The assay is calibrated using X-rays which break cellular DNA at a known frequency (25). For comparison with HPLC-derived data, the comet assay results were converted to FPG-sensitive sites per 106 dG. The calibration curve is linear up to a score of 300 arbitrary units; an increase of 10 arbitrary units corresponds to 0.07 breaks per 109 daltons, or 0.11 breaks per 106 dG. For ease of comparison, when presenting results we have assumed that FPG-sensitive sites are equivalent to 8-oxoguanines (see Discussion).
Conversion of double-stranded calf thymus DNA to single-stranded DNA
Highly polymerized calf thymus DNA (Sigma, Poole) at 0.4 mg/ml in 10 mM TrisHCl pH 7.3 was passed six times through a 25Gx1 needle. 0.8 ml aliquots were prepared and nitrogen passed through each for 30 s, followed by 30 s passing over the solution. The aliquots were heated in a boiling water bath for 5 min followed by rapid cooling in an ice/ethanol mix for 4 min. Half of these aliquots were boiled and cooled a second time. The absorbance of the calf thymus solution (at 260 nm) was measured at each step of the treatment.
Hydrolysis of calf thymus DNA
Calf thymus DNA was hydrolysed using P1 nuclease and alkaline phosphatase (22). The hydrolysates were analysed by HPLC as described for nuclear DNA.
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Results |
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Comparative measurement of induced 8-oxodGuo in HeLa cell DNA by HPLC and the comet assay
The photosensitizer Ro 19-8022 is taken up by cells and, in combination with visible light, gives rise predominantly to FPG-sensitive sites in the DNA (14). Because relatively few DNA strand breaks are introduced, the method is particularly suitable for comet assay analysis, in which estimation of FPG-sensitive sites requires subtraction of directly induced breaks from breaks detected in the presence of the enzyme. To establish a doseresponse, we chose to vary the concentration of Ro 19-8022 with a constant time of irradiation.
Figure 2a shows the yield of FPG-sensitive sites, measured by the comet assay, in HeLa cells treated with Ro 19-8022 concentrations up to 0.6 µM with 2 min of light. (The results are expressed against concentration of Ro 19-8022xminutes of light exposure.) The increase in FPG-sites is linear (r = 0.96). HPLC is less sensitive than the comet assay, and higher doses of damage had to be applied; the range of Ro 19-8022 concentrations extended to 5 µM and irradiation was for 5 min rather than 2 min (Figure 2b
). Unpublished results measuring 8-oxodGuo by HPLC have shown a linear response with time of irradiation in the presence of Ro 19-8022. The increase in 8-oxodGuo with dose of Ro 19-8022 is clearly linear (r = 0.997). The comet assay doseresponse curve from Figure 2a
is shown (extrapolated) also in Figure 2b
; the slopes are not significantly different.
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Discussion |
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FPG is not specific to 8-oxoGua, but detects also ring-opened purines (fapy bases) (11). If there are significant numbers of fapy derivatives in the background damage, or in the damage induced by Ro 19-8022 plus light, FPG-sensitive sites as measured with the comet assay would be an over-estimate of 8-oxoGua content. Pflaum et al. (7) found that, in phage DNA treated in vitro with Ro 19-8022 and light, the amount of 8-oxodGuo measured by HPLC accounted for 74% of the lesions detected with FPG (using alkaline elution to measure the resulting breaks). If we were to allow for this over-estimation in expressing the results of the doseresponse experiment (Figure 2), the identity of the slopes would be even more compelling.
It is possible that the HPLC method underestimates 8-oxodGuo if hydrolysis is incomplete because DNA is double-stranded. To test this, we compared yields of 8-oxodGuo from ss DNA and ds DNA. We found an increase in the relative concentration of 8-oxodGuo to dGuo when calf thymus DNA was boiled. This could represent more efficient hydrolysis of ss DNA compared with ds DNA. However, the increase was repeated when the ss DNA was boiled again, strongly implying that the boiling itself is responsible for oxidation of dGuo to 8-oxodGuo, and that ss DNA is no more efficiently hydrolysed than ds DNA.
Further, we recently analysed 8-oxodGuo in ss oligonucleotides (kindly provided by Henrik Poulsen, National University of Copenhagen) constructed to contain a defined number of guanines in the oxidized form (and no unoxidized guanines). Hydrolysis with the 2-enzyme method yielded the expected number of 8-oxodGuo residues (26, C.M.Gedik and S.G.Wood, unpublished).
What is the origin of the discrepancy in background levels of 8-oxoGua?
It has long been suspected that oxidation of DNA occurs during isolation, hydrolysis and storage which could account for the high variability in the levels of 8-oxodGuo reported for chromatographic analyses. Recent improvements in methods by the introduction of antioxidants or nitroxides, reduction of incubation temperatures and times, and substitution of the chelator EDTA with deferoxamine mesylate (desferal) which binds iron in a form unable to catalyse redox reactions, have all been reported to lower the extent of oxidation during preparation of samples (4,5,27,28). We found that treatment of DNA after isolation is important; dialysis, particularly in untreated dialysis tubing, and drying DNA under vacuum or lyophilization lead to an increase in 8-oxodGuo, but we found no evidence of oxidation during hydrolysis (29).
The background levels of 8-oxodGuo for untreated HeLa cells grown in roller bottles were 0.96 and 2.09 per 106 dGuo as determined by the comet assay and HPLC respectively. The closeness of the results suggests that much of the artefactual oxidation during workup has been eliminated, which is very encouraging. However, the comet assay results may be an overestimation if there are significant amounts of fapy-adenine and fapy-guanine present. It is also possible that a systematic error affects the calibration of the comet assay. Support for the accuracy of the comet assay comes from analogous experiments with UV-irradiated cells, in which quantitation was based on the same X-ray calibration curve. T4 endonuclease V was used to detect cyclobutane pyrimidine dimers, and the yield was 30% more than the expected number, which is a relatively small discrepancy (30).
We have found in recent experiments (unpublished) that the background level of damage as measured by HPLC in different batches of HeLa cells varies, between two and five 8-oxodGuo per 106 dGuo. In the doseresponse experiment of Figure 2, the apparent background is about six 8-oxodGuo per 106 dGuo. These cells, although not incubated with Ro 19-8022, were irradiated. However, the amount of light they received is not sufficient to produce a detectable increase in base oxidation (our unpublished results).
Biomarkers of oxidative damage
Both HPLC and the comet assay have been used successfully in human studies to reveal biologically significant differences between groups of individuals. For example, HPLC analysis of 8-oxodGuo in lymphocyte DNA from men and women in five countries showed a striking elevation of damage in men of northern European countries relative to levels in women generally or in men of southern Europe (31). The comet assay, incorporating endonuclease to detect oxidized bases, has revealed a negative correlation between DNA damage and serum levels of carotenoids, supporting a role for dietary antioxidants in protecting against free radical attack on DNA (32); it has also been used to demonstrate the protective effects of antioxidant supplements or antioxidant-rich foods (33,34).
In the present trial, there is no significant correlation between FPG-sensitive sites and 8-oxodGuo (in lymphocytes) measured by HPLC. This is not too surprising, since FPG (as already mentioned) can detect lesions other than 8-oxoGua. However, there are strong correlations between urinary 8-oxodGuo and both measures of 8-oxoGua in DNA. The monoclonal antibody used in the ELISA kit has been tested for cross-reactivity with various normal and modified nucleosides (manufacturer's information). 8-sulphhydrylguanosine and 8-oxoGua were the only ones to be detected by the antibodyat much higher concentrations than required for reaction with 8-oxodGuo. (Fapy nucleosides were apparently not tested.) Our observation of a good correlation with direct measures of oxidized guanine in DNA suggests that cross-reactivity, if it is significant, probably relates only to other oxidation products and not to DNA damage in general. Thus the antibody assay, as well as HPLC and the comet assay, can be used as valid markers of oxidative stress.
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Notes |
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2 To whom correspondence should be addressed Email: a.collins{at}rri.sari.ac.uk
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Acknowledgments |
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References |
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