SVPD-post-labeling detection of oxidative damage negates the problem of adventitious oxidative effects during 32P-labeling
George D.D. Jones3,
Lynda Dickinson,
Joseph Lunec1 and
Michael N. Routledge2
Biomolecular Damage Group and
1 Division of Chemical Pathology, Centre for Mechanisms of Human Toxicity, University of Leicester, Hodgkin Building, PO Box 138, Lancaster Road, Leicester LE1 9HN and
2 Department of Biological Sciences, Hawthorn Building, De Montfort University, The Gateway, Leicester LE1 9BH, UK
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Abstract
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The exploitation of oxidative DNA lesions as biomarkers of oxidative stress in vivo requires techniques that allow for the precise and valid measurement of oxidative damage to DNA. Previously, endogenous levels of the oxidative lesion 8-hydroxy-2'-deoxyguanosine (8-HO-dG) in rat tissues determined by a micrococcal nuclease/calf spleen phosphodiesterase-based 32P-post-labeling protocol were found to be at least 10-fold higher than those determined by HPLC with electrochemical detection. This was attributed to the adventitious oxidation of the normal nucleotides (dGp) occurring during the labeling stage of the post-labeling protocol, which could only be prevented by the introduction of additional chromatographic steps to remove the unmodified species prior to labeling. In the present study we report that an alternative snake venom phosphodiesterase-based 32P-post-labeling procedure (SVPD-post-labeling) negates the problem of adventitious oxidative damage during labeling by virtue of a unique digestion strategy. In SVPD-post-labeling, digestion yields certain lesions (thymine glycols, phosphoglycolates and abasic sites) as damage-containing dimer species which are ready substrates for labeling. In contrast, the undamaged DNA is recovered as mononucleoside species (dN) which are not substrates for labeling and so remain undetected. Furthermore, even if the mononucleosides are oxidized during labeling, they will not contribute to the level of damage detected. Indeed, we demonstrate that neither the external
-irradiation of the digested DNA samples nor increasing the incubation time of the labeling reaction alters the levels of damage detected by SVPD-post-labeling. The negation of adventitious oxidative effects during labeling deems that an optimized SVPD-post-labeling procedure should be well-suited for the biomonitoring of endogenous oxidative stress in vivo.
Abbreviations: 8-HO-dGp, 8-hydroxy-2'-deoxyguanosine-3'-monophosphates; 8-OH-G, 8-hydroxyguanine; 8-OH-dG, 8-hydroxy-2'-deoxyguanosine; CSPD, calf spleen phosphodiesterase; dG, 2'-deoxyguanosine; dGp, 2'-deoxyguanosine-3'-monophosphates; dN, 2'-deoxynucleosides; dNp, 2'-deoxynucleoside-3'-monophosphates; GCMS/SIM, gas chromatographymass spectroscopy with selective ion monitoring; HO-, hydroxyl moieties; HPLCECD, high performance liquid chromatography with electrochemical detection; MN, micrococcal nuclease; OH·, hydroxyl radicals; 32P-HPLC, high performance liquid chromatography with radioactivity detection; PAGE, polyacrylamide gel electrophoresis; pdN, 2'-deoxynucleoside 5'-monophosphates; ROS, reactive oxygen species; SAP, shrimp alkaline phosphatase; SVPD, snake venom phosphodiesterase; T4PNK, T4 polynucleotide kinase; TLC, thin-layer chromatography.
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Introduction
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Evaluating a role for oxidative stress in human disease (and judgement of dietary antioxidants in the prevention of such disorders) necessitates the accurate measure of oxidative effects in vivo. It has been proposed that key mediators of oxidative stress are reactive oxygen species (ROS) (1) and that genomic DNA is an important target for the deleterious effects of ROS in vivo (2). The interaction of ROS with DNA results in a wide variety of lesions (3), and the resulting oxidative damage has been implicated in various deleterious biological end points, including cell death and mutation (4,5), and is thought to contribute to several age-related degenerative pathologies, including rheumatoid arthritis and cancer (6,7). Consequently, the accurate measure of these lesions in vivo would serve as ideal/significant biomarkers of ROS-mediated oxidative stress. To date, the principal assays of oxidative DNA damage have been the analytical techniques of gas chromatographymass spectroscopy (GCMS/SIM) (810), HPLC with electrochemical detection (HPLCECD) (11,12), 32P-post-labeling (1315) and various immuno-assays (16,17). Each of these techniques has its own advantages and disadvantages, depending on the lesions being assayed. Generally, however, 32P-post-labeling techniques have two key advantages over other assays of oxidative DNA damage: (i) they use microgram amounts of DNA, making the assays useful for the study of DNA lesions in tissues; and (ii) the assays are very sensitive, routinely allowing for detection of lesions at the femtomole (1015 mole) level.
The original 32P-post-labeling assay devised by Gupta et al. to detect modified DNA bases (18) utilizes two enzymes in the digestion step: micrococcal nuclease (MN) and calf spleen phosphodiesterase (CSPD). MN mediates the endonucleolytic cleavage of DNA to yield strand breaks possessing 5'-HO termini, whilst CSPD mediates the 5'
3' exonucleolytic digestion of DNA from 5'-HO ends to give nucleoside-3'-monophosphates, with both normal (dNp) and modified bases (dXp) being recovered in this fashion (Figure 1a
). These are subsequently radiolabeled by phosphorylation of the 5'-HO groups with 32P, via incubation with T4 polynucleotide kinase (T4PNK) and [
-32P]ATP, and then separated and quantified by anion-exchange thin-layer chromatography (TLC) or high performance liquid chromatography with radioactivity detection (32P-HPLC) (19).

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Fig. 1. Schemes of (a) CSPD- and (b) SVPD-post-labeling used to detect oxidative damage. DNA containing a modified nucleoside (X) is enzymatically digested by either a combination of micrococcal nuclease (MN) and calf spleen phosphodiesterase (CSPD) or a combination of DNase I, snake venom phosphodiesterase (SVPD) and shrimp alkaline phosphatase (SAP). The products dNp, Xp and dNpX are 32P-end-labeled with T4 polynucleotide kinase (T4PNK) and [ -32P]ATP and analyzed by thin-layer chromatography (TLC) or polyacrylamide gel electrophoresis (PAGE).
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The MN/CSPD-based post-labeling protocol (CSPD-post-labeling) has proved to be very useful for the detection of a wide range of DNA base adducts and modifications, both in vitro and in vivo, at high sensitivity (20). However, as mentioned above, ROS induce a plethora of lesions in DNA, including numerous modifications not only to the DNA base moieties but also to the sugar phosphate backbone (3). Many of these lesions are not detectable by CSPD-post-labeling as they are not substrates for polynucleotide kinase (or are labeled with low efficiency) or they are difficult to separate from unmodified nucleotides.
Devanoboyina and Gupta have described a CSPD-based post-labeling protocol that was purported to allow for the ready detection of basal levels of the principal oxidized purine base lesion 8-hydroxyguanine (8-OH-G) in rat tissue DNA (21). However, the levels of 8-OH-G were far higher than those determined by HPLCECD. Subsequently, it was speculated that the post-labeling protocol may overestimate 8-OH-G levels due to processes specifically associated with the methodology, in particular the ß-radiolytic decay of 32P giving rise to hydroxyl radicals (OH·), and their subsequent reaction with the normal nucleotides (dGp) to give additional 8-OH-G-containing nucleotides (22). Indeed, studies demonstrated that [
-32P]ATP can mediate formation of 8-OH-dGp during the labeling of dGp (22) as well 8-OH-dG on the external and internal irradiation of dG with 32P (22,23). To overcome the problem of adventitious oxidative DNA damage formation during labeling, and so allow for true determinations of basal 8-OH-G levels in DNA, the CSPD-post-labeling protocol has since been further developed to include chromatographic steps which remove dGp prior to 32P-labeling (24).
An alternative post-labeling protocol for the detection of oxidative damage, which overcomes many of the problems described above, has been developed by Weinfeld et al. (25,26). The assay utilizes three enzymes in the digestion step, DNase I, snake venom phosphodiesterase (SVPD) and an alkaline phosphatase [typically shrimp alkaline phosphatase (SAP)] (Figure 1b
). DNase I mediates the endonucleolytic cleavage of DNA to yield strand breaks possessing 3'-HO termini whilst SVPD mediates the 3'
5' exonucleolytic digestion of DNA from 3'-HO ends to produce nucleoside-5'-monophosphates (pdN). However, particular lesions, including certain key oxidative lesions, impede SVPD-mediated hydrolysis of the phosphodiester bond immediately 5' to the lesion (27,28). Having stalled at this position the SVPD cleaves the next phosphodiester bond releasing the damage as dimer species (pdNpdX). Finally, the 5'-phosphates of both the damage-containing dimers and the 5'-mononucleotides are removed by SAP to yield the corresponding damage-containing `dinucleoside'-monophosphate species (dNpX) and nucleosides (dN), respectively.
As the damage-containing dNpX species possess an unmodified 5'-HO-bearing nucleoside-3'-monophosphate moiety, these are ready substrates for 5' 32P-end-labeling by incubation with T4PNK plus [
-32P]ATP. However, the recovered mononucleosides (reflecting mostly undamaged DNA) are not substrates for the kinase and so remain unlabeled. This is in direct contrast to CSPD-post-labeling in which the undamaged DNA is recovered as dNp, which, if not removed, will compete with the dXp as efficient substrates for 32P-labeling. After transferring most of the excess 32P to a large oligonucleotide [(dT)16] via a second T4PNK-mediated reaction, the labeled damage-containing `dinucleotide' products (32pdNpX) are then separated by denaturing polyacrylamide gel electrophoresis (PAGE) and detected/quantified by autoradiography/phosphoimager analysis. Although the DNase I/SVPD/SAP-based post-labeling protocol (SVPD-post-labeling) cannot measure 8-OH-dG, it does allow for both the exclusive and efficient labeling of several other oxidative DNA lesions that are not readily measured by other post-labeling techniques, but which can also serve as good biomarkers for oxidative damage. Such lesions include thymine glycols and phosphoglycolates (deoxyribose fragments present at the 3' termini of oxidative strand breaks), induced by the action of ionizing radiation and oxidative stress (25,29), and also abasic sites (30). SVPD-post-labeling has also been exploited in the analysis of UV-induced DNA photoproducts (3133).
As mentioned above, the digestion step of SVPD-post-labeling ultimately releases unmodified DNA as mononucleosides which are present in the labeling reaction at approximately millimolar concentrations. Since these are not substrates for T4PNK, any adventitious oxidation of the nucleosides during the labeling reaction will not contribute to the levels of damage being detected by SVPD-post-labeling. Furthermore, the presence of such high concentrations of nucleosides in the labeling reaction mixture should serve to protect the damage-containing dimer species from any adventitious oxidative effects by acting as scavengers. To demonstrate that this is indeed the case we have used external
-irradiation of digested DNA samples to mimic the effects of adventitious oxidative action during labeling and we have increased the incubation time of the 32P-labeling stage of the SVPD-protocol (cf. ref. 22).
Figure 2a and b
shows the SVPD-post-labeling doseresponse for aerobic aqueous solutions of calf thymus DNA (1 mg/ml in 10 mM phosphate buffer, pH 7.4) following exposure with up to 50 Gy of 60Co
-rays (4 Gy/min). After irradiation, the DNA was precipitated (34), redissolved in water (1 mg/ml) and post-labeled. Full details of the SVPD-post-labeling protocol are provided elsewhere (25,26); briefly, 10 µg samples of irradiated and undamaged DNA were incubated with DNase I (6 U; Sigma, Dorset, UK), SVPD (0.04 U; Sigma), SAP (0.4 U; USB, Cleveland, OH) and in 30 µl of digestion buffer (10 mM Tris, 1 mM EDTA, 4 mM MgCl2, pH 7.5) at 37°C overnight (~16 h). Following removal of the proteins by ethanol precipitation, isolation and drying of the supernatants by lyophilization, resupension of the residue in distilled water (0.1 µg digested DNA/µl) and heating (100°C, 10 min) to inactivate residual nuclease and phosphatase activity, the damage-containing species in 0.10.5 µg of digested DNA were phosphorylated with 32P by incubation with T4PNK (7.5 U; USB) and [
-32P]ATP (3 kCi/mM, 1.65 pmol; Amersham, Buckinghamshire, UK) in 10 µl of the supplied kinase reaction buffer (used at 1x strength) for 1 h at 37°C. To consume excess [
-32P]ATP, each sample is treated with 0.005 A260 units of (dT)16 (5 A260 units/ml; Pharmacia, St Albans, UK) plus a further 3.75 U of T4PNK for 30 min at 37°C. After the addition of an equal volume of formamide loading buffer (34), the radiolabeled products in 50250 ng of digested DNA were separated by polyacrylamide gel electrophoresis (PAGE) using 1.6 or 0.8 mm thick 20% polyacrylamide/7 M urea gels, and visualized/quantified by phosphoimagery (Model 425E; Molecular Dynamics, Sunnyvale, CA, with ImageQuant software version 3.2). The profile of lesions observed (Figure 2a
) is indicative of radiogenic hydroxyl radical action in dilute aqueous solutions of DNA (25). Thymine glycol and phosphoglycolate lesions, in bands 13 and 68, respectively, were observed to be induced linearly with dose (Figure 2b
).


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Fig. 2. Doseresponse for the induction of radiation-induced DNA damage as detected by SVPD-post-labeling. (a) Gel electrophoresis of the end-labeled products obtained after -irradiation of calf thymus DNA samples. Lane A, 0 Gy; lane B, 10 Gy; lane C, 20 Gy; lane D, 30 Gy; lane E, 40 Gy; lane F, 50 Gy. Some of the detectable products have been identified (25). Bands 13 contain the labeled thymine glycol-containing dinucleotides (32pdNpTg); 32pdGpTg is in band 1, 32pdApTg and 32pTpTg are in band 2 and 32pdCpTg is in band 3. Bands 810 are the labeled nucleotide 3'-phosphoglycolate species (32pdNpg); 32pdGpg is in band 6, 32pdApg and 32pTpg are in band 7 and 32pdCpg is in band 8. The lesions in bands 4 and 5 have not been identified but are removed by E.coli endonuclease IV (26). The other prominent unidentified (unnumbered) bands, observed between bands 5 and 6, are seen in the untreated control. Not shown in this figure are the three bands corresponding to inorganic mono-, di- and triphosphates that migrate ahead of the bands 68, and the (dT)16 that remains near the top of the gel. (b) Plots of detectable damage from 1 µg of digested DNA versus dose; open circles, bands 13 (thymine glycols); closed circles, bands 68 (phosphoglycolates). Duplicate determinations were made at each dose. The drawn lines are generated by linear regression of the data points and equate to yields of ~3.7 and ~12.3 fmol/µg DNA digested/Gy for bands 13 and 68, respectively.
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Figure 3a and b
shows the SVPD-post-labeling response following the further exposure of unirradiated and 50 Gy-irradiated DNase I/SVPD/SAP-digested DNA samples with up to 200 Gy of
-rays. After irradiation, the digests were labeled as described above. It is clear that the additional irradiation of the DNA digests with up to four times the amount of radiation as was initially used to irradiate the intact DNA samples does not to lead to any significant alteration in the levels of damage detected by SVPD-post-labeling. Indeed, the levels of damage noted on further irradiation (+200 Gy) of the digested 50 Gy-irradiated and unirradiated DNA samples equates to the levels noted after normal SVPD-post-labeling (Figure 4a and b
). Similarly, in a separate experiment, increasing the incubation time of the labeling reaction for the SVPD-post-labeling of 50 Gy-irradiated and unirradiated DNA samples from 1 to 48 h, does not significantly alter the levels of damage detected (Figure 4c and d
). In contrast, both of the above modifications (external irradiation of the digest and extension of the labeling reaction) were noted to yield increases in the levels of oxidative damage detected by CSPD-post-labeling (see below).


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Fig. 3. SVPD-post-labelling response on the further irradiation of DNase I/SVPD/SAP-digested DNA samples. (a) Gel electrophoresis of the end-labeled products obtained after the further -irradiation of unirradiated digested (lanes AE) and 50 Gy-irradiated digested (lanes FJ) calf thymus DNA samples. Lanes A and F, +0 Gy; lanes B and G, +50 Gy; lanes C and H, +100 Gy; lanes D and I, +150 Gy; lanes E and J, +200 Gy. (b) Plots of detectable damage from 1 µg of DNA versus initial irradiation (050 Gy); open circles, bands 13 (thymine glycols); closed circles, bands 68 (phosphoglycolates); plus plots of detectable damage after the further irradiation (0 to +200 Gy) of unirradiated digested and 50 Gy-irradiated digested samples. Unirradiated digested samples: open triangles, bands 13 (thymine glycols); closed triangles, bands 68 (phosphoglycolates); 50 Gy irradiated digested samples: open squares, bands 13 (thymine glycols); closed squares, bands 68 (phosphoglycolates). The data points at 50, 100, 150 and 200 Gy were from single determinations whilst the points at 250 Gy were from three determinations ± SD. The drawn lines are generated by linear regression of the data points.
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Fig. 4. Determined yields of damage with (+200 Gy) and without (none, normal protocol) further irradiation of the digests of 50 Gy-irradiated (a) and unirradiated (b) DNA samples; plus determined yields of damage on extending the labeling incubation time for SVPD-post-labelling from 1 h (normal protocol) to 48 h for both 50 Gy-irradiated (c) and unirradiated (d) DNA samples. The differences between the levels of damage noted for the two unirradiated DNA samples (shown in b and d) reflect differences in the background measurements for the two different commercial lots of calf thymus DNA used in the two separate experiments.
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The identical levels of damage noted both before and after the further irradiation of the digests, and on extension of the labeling reaction, are a direct consequence of the unique strategy of the SVPD-post-labeling protocol. On DNase I/SVPD/SAP-digestion of the damaged DNA, the lesions that impede SVPD-digestion are revealed as damage-containing dimer species, dNpX, whilst the rest of the DNA (mostly undamaged DNA) is reduced to the level of mononucleosides (dN), with the latter being present in an overwhelming extent (~103104 M) in comparison with dNpX (~10121015 M). Whilst the unmodified mononucleosides are undoubtedly damaged by the further irradiation of the digests and extended labeling reaction time, the resulting modified products (dX) are not substrates for the labeling kinase and are not detected by SVPD-post-labeling. Consequently, SVPD-post-labeling only detects/registers damage which is present in the DNA prior to digestion. After digestion, any additional adventitious damage induced during the labeling stage is not detected by SVPD-post-labeling. Furthermore, the presence of high amounts of normal nucleosides in the digest and labeling reaction serve to protect the damage-containing dimer species from further oxidative action by acting to scavenge any adventitious oxidative species. Consequently, we observed no loss of dNpX, even at the highest doses used in the further irradiation of the digests, nor after the longer labeling incubation times.
The above observations of SVPD-post-labeling negating the problem of adventitious oxidative damage formation during labeling contrasts the observations for CSPD-post-labeling reported by Schuler et al. (22). In a comparative analysis of 8-HO-dG in DNA by CSPD-post-labeling and HPLCECD, the yields of basal 8-HO-dG in commercial calf thymus DNA as determined by CSPD-post-labeling were ~16-fold greater than the values determined by HPLCECD. To study the possibility that ionizing radiation from the radiolytic decay of 32P during labeling was causing the higher levels of 8-HO-dG observed by CSPD-post-labeling, both the incubation time and the level of radioactivity in phosphorylation reactions with commercial dGp were increased, and the external irradiation of commercial dG with 32P was also investigated. All modifications resulted in higher levels of 8-HO-dG being measured. However, the effect was insufficient to fully account for the difference between CSPD-post-labeling and HPLCECD suggesting that processes other than the 32P radiogenic action, such as metal catalyzed in vitro formation of OH·, may also contribute to the artefactual formation of oxidative lesions. Nevertheless, whatever the source of adventitious damage during the labeling stage, the SVPD-post-labeling, by virtue of its unique molecular strategy, acts to negate this problem.
In conclusion, we have demonstrated that the SVPD-post-labeling method negates the problem of adventitious oxidative effects during labeling without the need to include additional chromatographic (24) or chemical (35) steps to separate/remove the unmodified species from the damage-containing species prior to labeling. This advantage suggests that an optimized SVPD-post-labeling assay (36) should be well-suited for the biomonitoring of endogenous oxidative stress in human studies.
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Acknowledgments
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This work is supported by research contracts (ANO4/26 and ANO4/32) from the UK Ministry of Agriculture Fisheries and Food (Antioxidants in Food Programme, ANO4) awarded to G.D.D.J., with further support from a project grant (G9527655MA) from the UK Medical Research Council awarded to G.D.D.J.
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Notes
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3 To whom correspondence should be addressed Email: gdj2{at}leicester.ac.uk 
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Received September 22, 1998;
revised November 11, 1998;
accepted November 12, 1998.