Detection of 8-hydroxyguanine in small amounts of DNA by 32P postlabeling

ANALYTICAL

BIOCHEMISTRY

201,127-133

(1992)

Detection of 8-Hydroxyguanine of DNA by 32P Postlabeling

in Small Amounts

Jan T. Lutgerink,*+l*’ Ed de Graaf,*T3 Barbara Hoebee,$p4 Hans F. C. Stavenuitez,$ J. Gerard Westra,* and Erik Kriek* *Division of Chemical Carcinogenesis,The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands; tIn.stitute of Human Genetics,Free University, P.O. Box 7161, 1007 MC Amsterdam, The Netherlands; SDepartment of Biophysics, Physics Laboratory of the Free University, De Boelelaan1081, 1081HV Amsterdam, The Netherlands; and #National Institute of Public Health and Environmental Protection, P.O. Box 1, 3720 BA, Bilthoven, The Netherlands Received

September

9, 1991

A method for the sensitive detection of S-hydroxyguanine residues in small amounts of DNA (0.2-2 rg) was developed. It comprises (i) the enzymatic hydrolysis of DNA to 2’-deoxyribonucleotide 3’-monophosphates, (ii) degradation of the bulk amount of normal purine and pyrimidine deoxyribonucleotides in the DNA digest by treatment with trifluoroacetic acid and hydrazine, respectively, under conditions retaining the structure of d(8-OH-G)p necessary for 5’ phosphorylation by T4 polynucleotide kinase (PNK), (iii) 5’ phosphorylation of d(8-OH-G)p by T4 PNK-catalyzed transfer of 3aP from [y-“P]ATP, and (iv) 2D thin-layer chromatography on polyethyleneimine-cellulose sheets to purify and resolve “P-postlabeled d(8-OH-G)p. Model experiments with mixtures composed of synthesized d(8-OH-G)p and DNA hydrolysate indicate that it is possible to detect one 8-hydroxyguanine residue out of 2 x lo6 normal bases starting with 1 Hg DNA. The methodology, which allows for a further decrease of this detection limit, might be very useful for the sensitive detection of DNA damage induced by activated oxygen species in small amounts of DNA. We demonstrate the formation of S-OH-G in DNA in vitro by low doses of 6oCo y-rays. 0 1992 Academic Press, Inc.

Highly reactive oxygen species, known to cause DNA damage, are generated by ionizing radiation (l-4), a number of carcinogenic chemicals (5,6), mutagenic and i Present address: Department of Human Biology, University of Limburg, Beeldsnijdersdreef 101,620O MD Maastricht, The Netherlands. ’ To whom correspondence should be addressed. 3 Present address: Radiation Protection Service, The Netherlands Cancer Institute, Plesmanlaan 121,1066 CX Amsterdam, The Netherlands. ’ Present address: National Institute of Public Health and Environmental Protection, P.O. Box 1,372O BA, Bilthoven, The Netherlands. 0003-2697/92 Copyright All rights

$3.00 0 1992 by Academic Press, of reproduction in any form

carcinogenic cytostatics (7), and tumor promoters (8,9). They are also produced by partial reduction of oxygen during normal cellular oxygen metabolism (10-14). There is increasing evidence implicating them as possible etiological agents in normal aging (15-17), radiation injury (18), carcinogenesis (15), toxic action of certain drugs (19), and the development of a number of pathological states such as ischemic damage to tissues (20) and inflammatory stress (21). To investigate whether interaction with intracellular DNA plays an important role, sensitive methods for the detection of oxidative DNA damage are required. In this paper we describe a simple procedure for the sensitive detection of 8-hydroxyguanine, a potential mutagenic lesion (22) that can be induced in DNA with the hydroxyl radical as ultimate reactive agent (23). The method is partly based on procedures employed in the 32P-postlabeling technique developed during the past decade by Randerath et al. (24-29). This technique is superior for the detection of adducts induced by aromatic carcinogens (25-29) because it combines the sensitivity of 32P with a chromatographic method suitable for separating small amounts of aromatic adduct nucleotides from an excess of unmodified nucleotides (25). For other modified nucleotides, with methylated or oxidized bases, the chromatographic properties do not change radically, so that purification before postlabeling is necessary to obtain the desired sensitivity (30,31). Only in particular cases it is possible to apply the more sensitive nuclease Pl version of the 32P-postlabeling technique (28,32), i.e., for those adducts that retain the 3’ phosphate group necessary for 5’ phosphorylation by T4 polynucleotide kinase when incubated with nuclease Pl (28,32,33). The latter type of adduct enrichment cannot be applied for 8OH-G5 detection, because 8-hydroxydeoxyguanosine 5 Abbreviations phates; pdNp,

used: dNp, P’-deoxynucleoside

2’-deoxyribonucleotide 3’5’-biphosphates;

3’-monophos&OH-G, 8-hy127

Inc. reserved.

128

LUTGERINK

3’-monophosphate [d(8-OH-G)p] is readily dephosphorylated by nuclease Pl (Lutgerink et aZ., unpublished observations). However, we were able to enrich d(8-OH-G)p prior to postlabeling by chemical decomposition of the bulk amount of normal deoxyribonucleotides under conditions that leave d(8-OH-G)p intact, a procedure that increases the sensitivity of 8-hydroxyguanine detection considerably. The present method seems to be a good alternative for an existing assay for the quantification of 8-hydroxydeoxyguanosine by HPLC with electrochemical detection (34,35). The latter procedure, however, requires 50- to 200-fold more DNA for a single analysis to reach the same detection limit (3536). MATERIALS

AND

METHODS

Preparation and Purification of 8-Hydroxy-2’deoxyguanosine 3’-Monophosphute d(8-OH-G)p was prepared from dGp essentially as described by Kasai and Nishimura (37). To a solution of 2 mg dGp in 1560 ~1 of 0.1 M sodium phosphate buffer, pH 6.8, were successively added 280 ~11of 0.1 M ascorbic acid, 130 ~1 of 0.1 M EDTA, and 26 ~1 of 0.1 M FeSO,. Oxygen was led through the solution at 37°C for 3 h in the dark. After incubation the complete reaction mixture was fractionated by semipreparative HPLC on a Partisil RP18 column with 10 mM ammonium acetate, pH 5.4, as eluent. Ultraviolet spectra were monitored every 40 ms using a multiple photodiode array detector. The d(8-OH-G)p was identified by its characteristic uv spectrum: X, = 245 (12,300), 293 (10,300). Fractions containing d(8-OH-G)p were collected and further purification of d(8-OH-G)p was performed by two additional fractionations on a Rosil Cl8 HPLC column. The final preparation was >90% pure: the only contamination appeared to be dGp as determined by both HPLC and 2D PEI-cellulose chromatography after 32P postlabeling. The preparation was stored at -20°C in 10 mM ammonium acetate, pH 5.4. Preparation of Model Deoxynucleotide Postlabeling

Mixtures

for 32P

Model deoxynucleotide mixtures containing known amounts of d(8-OH-G)p were prepared by adding d(8OH-G)p to calf thymus DNA after digestion of the DNA to nucleotide 3’-monophosphates. Digestion of the DNA droxyguanine; d(8-OH-G)p, 8-hydroxy-2’-deoxyguanosine 3’-monophosphate; pd(8-OH-G)p, [32P]d(8-OH-G)p; dG, deoxyguanine; Gp, guamosine 3’-monophosphate; dGp, 2’-deoxyguamine 3’-monophosphate; dAp, 2’-deoxyadenosine 3’-monophosphate; dCp, fl’-deoxycytidine 3’-monophosphate; dm6Cp, 2’-deoxy-5’-methylcytidine 3’-monophosphate: PEI, polyethyleneimine; PNK, polynucleotide kinase; ss, single-stranded; ds, double-stranded, TFA, trifluoroacetic acid, Tp, (a’-deoxy)thymidine 3’-monophosphate.

ET

AL.

was for 4 h at 37°C in 10-/J reaction mixtures containing 1 pg ctDNA, 0.02 U micrococcal endonuclease, 0.001 U spleen phosphodiesterase, 20 mM sodium succinate, and 10 mM CaCl,, pH 6.0, unless otherwise stated. DNA digests [either with or without added d(&OH-G)p] were lyophilized in 50-~1 glass vials with a V-shaped bottom. Decomposition of Normal Purine Deoxynucbotides Pyrimidine Deoxynucleotides

and

Decomposition of dAp and dGp in DNA digests was performed by hydrolysis in trifluoroacetic acid (TFA). For this purpose the lyophilized DNA hydrolysate was dissolved in 90% TFA and incubated for 10 min at room temperature. TFA was evaporated in vacua over NaOH. The pyrimidine deoxynucleotides were decomposed by dissolving the residue in 64% hydrazine or anhydrous hydrazine and allowed to react for 16 h at 60°C. The reaction mixture was lyophilized and the residue was dissolved in an appropriate volume of 20 mM bicine, pH 9.6, for postlabeling. 32P Postlabeling

of DNA Digests

DNA digests (either untreated or treated with TFA and/or hydrazine hydrate) were labeled in lo-p1 reaction mixtures containing an amount of deoxynucleotides corresponding to 0.17-l pg DNA, 60 pM [Y-~~P]ATP (200 Cilmmol), 0.2 U T4 polynucleotide kinase, 10 mM bicine-NaOH, 10 InM MgCl,, 10 mM dithiothreitol, and 1 mM spermidine, pH 9.6. After incubation for 40 min at 38”C, 2.5 ~1 of a solution of 10 U apyrase per milliliter in 10 mM bicine-NaOH, pH 9.6, was added and incubation was continued for 30 min. Thin-Layer Chromatography of 32P-LabeZed Deoxynuckotides Separation of pd(8-OH-G)p from normal deoxynucleotides was performed by two- or three-directional anion-exchange chromatography on polyethyleneimine-cellulose thin layers (PEI-TLC; Machery-Nagel). Sheets were predeveloped with water, soaked in 0.1 M ammonium formate, pH 3.5, and dried. After application of labeled digest (or 12 ~1 of a suitable dilution of the labeled digest) excess ““Pi was removed by development with 0.25 LiCl in direction 1 (Dl, Fig. 3) for 21 h on a paper wick, which had been attached to the top of the sheet by stapling. Under these conditions the labeled deoxynucleotides remain at or stay close to the origin. Sheets were dried, cut at the broken line, and washed in flat trays (30 X 40 cm) with 400 ml of 10 mM Tris (or 400 ml of methanol containing 10 mM Tris) for 10 min, followed by an additional wash in 400 ml of water (or methanol) for 5 min. After drying, the sheets were developed in D2 with 1.5, 2, or 3 M ammonium formate, pH 3.5 (with or without a paper wick), dried, and washed as

=P POSTLABELING

DETECTION

described above. The sheets were then developed in D3 with water and, after drying, with 0.12 or 0.35 M NaH,PO,, pH 6.0, in the same direction. In some cases the first chromatography step (to remove 32Piwith LiCl) was omitted. Separation of all labeled deoxynucleotides was achieved with 1.5 M ammonium formate, pH 3.5 (Dl), and 0.12 M NaH,PO,, pH 6.0 (DZ). Optimal resolution of pdGp and pd(S-OH-G)p was obtained with 3 M ammonium formate, pH 3.5 (Dl), and 0.35 M NaH,PO,, pH 6.0 (D2). Irradiation

129

OF %HYDROXYGUANINE

DNA, modified by reactive oxygen

microccocal endonucleaee spleen phosphodieeferase I

dAp + dCp + dn+Cp + dGp + dTp + ..... + d(&OH-G)p frlfluoroacelic

acid

1 dCp + dmkp + dTp + ..... + d(&OH-G)p

of Single-Stranded Ml3 DNA

Single-stranded M13mplO DNA was irradiated with 6oCo y-rays at a concentration of 6 pg/ml in low3 M sodium phosphate buffer, pH 7.3, and lop4 M MgCl,, with a dose rate of 0.5 Gy min-‘. Before and during irradiation 0, was flushed through the solution.

SpeCieS

hydrezine

I ..... + d(&OH-G)p 74 polynucleolide exceee terrier-

kinase free [y?*P]ATP

1 epyrese 1

RESULTS

Chemical Decomposition of Normal Deoxynuckotides

We observed that the N-glycosidic bond of 8-hydroxydeoxyguanosine is much more resistant to hydrolysis with TFA than that of deoxyguanosine or deoxyadenosine. A similar difference in stability toward TFA was observed for the 2’-deoxynucleoside 3’-monophosphates (dNp’s); i.e., 8-hydroxydeoxyguanosine 3’-monophosphate hardly decomposes in trifluoroacetic acid under conditions in which deoxyguanosine 3’-monophosphate and deoxyadenosine 3’-monophosphate are hydrolyzed instantaneously. We also found that d(8OH-G)p is resistant to the action of hydrazine, in contrast to normal pyrimidine deoxynucleotides. The recovery of d(8-OH-G)p after exposure to hydrazine was almost 100% under conditions in which the pyrimidine nucleotides dCp and Tp can be destroyed for at least 99% (see also below). On the basis of these findings we designed a strategy for the sensitive detection of 8-hydroxydeoxyguanosine residues in small amounts of DNA, which is outlined in Scheme 1. It comprises the following steps: (i) digestion of the DNA to deoxyribonucleoside 3’-monophosphates by micrococcal endonuclease and spleen phosphodiesterase, (ii) trifluoroacetic acid hydrolysis of the DNA hydrolysate to decompose dAp and dGp, (iii) decomposition of dCp, dm5Cp, and Tp by hydrazine treatment, (iv) 32P postlabeling of undestroyed d(8-OH-G)p, and (v) 2D chromatography on polyethyleneimine-cellulose sheets. The feasibility of this approach is demonstrated by the results of a model experiment, in which a mixture of a known amount of synthesized d(S-OH-G)p and calf thymus DNA hydrolysate was analyzed by 32P postlabeling after treatment with either TFA, hydrazine, or both TFA and hydrazine (Fig. 1). Excess [T-~‘P]ATP was destroyed by apyrase treatment and a part of a dilu-

.. ...

+ p*d(&OH-G)p Z-or 3-directions1 PEIcellulose chromatography, and autoradiography

1 quantification of p*d(&OH-G)p spot SCHEME 1. Schematic representation of the method developed for the sensitive detection of &hydroxyguanine residues in small amounts of DNA.

tion was applied on PEI-cellulose thin layers. Two modes of 2D chromatography were employed (see Materials and Methods): one to resolve all pdNp’s (Figs. laId) and one to obtain a better resolution of pdGp and pd(B-OH-G)p (Figs. le-lh). Figures la and le show the results obtained from postlabeling analysis of an untreated mixture of digested DNA and synthesized d(8OH-G)p. Treatment of this mixture with 90% trifluoroacetic acid before labeling leads to hydrolysis of the N-glycosidic bonds of dAp and dGp but not of dCp, dm5Cp, Tp, and d(B-OH-G)p. Consequently, only the pyrimidine deoxynucleotides and d(8-OH-G)p are recognized as substrates by the polynucleotide kinase (Figs. lb and If). On the other hand, treatment of the dNp’s with hydrazine prior to postlabeling selectively destroys the pyrimidine rings in dCp, dm’Cp, and Tp (38). In this case only the purine deoxyribonucleotides, including d(8-OH-G)p, are postlabeled (Figs. lc and lg). Figures ld and lh show that after both acid hydrolysis and hydrazine treatment only d(S-OH-G)p retains the structure necessary for 5’ phosphorylation by polynucleotide kinase. The spots shown in Fig. 1 were quantified by Cerenkov counting as described (39). The results are summa-

130

LUTGERINK

ET

AL.

FIG. 1. Detection of 8-hydroxydeoxyguanosine residues in DNA hydrolysates after chemical decomposition of the normal deoxynucleotides and subsequent ‘*P-postlabeling analysis. A mixture of a calf thymus DNA hydrolysate and synthesized d(8-OH-G)p was not treated (a and e) or was treated with either trifluoroacetic acid (b and f), hydrazine (c and g), or both (d and h) prior to postlabeling. The labeled 2’-deoxynucleoside 3’,5’-biphosphates (pdNp’s) were separated by two different modes of 2D polyethyleneimine-cellulose thin layer chromatography (see Materials and Methods), one to resolve all pdNp’s (a-d) and one to obtain a better resolution of pdGp and pd(B-OH-G)p (e-h). Pi, inorganic phosphate.

rized in Table 1 and show that, under the conditions employed, d(S-OH-G)p is very resistant to both the trifluoroacetic acid hydrolysis and the hydrazinolysis. Some loss of d(B-OH-G)p might occur upon longer incubation in TFA (Fig. 2). In contrast, no significant reduction of d(8-OH-G)p was observed by treatment with 64% hydrazine at 60°C even after prolonged incubation periods up to 24 h. Because it has been reported that aqueous hydrazine under certain conditions mediates the formation of d(8OH-G)p from dG (37) it was necessary to check whether artifactual d(S-OH-G)p is formed during the hydrazine treatment described above. This was done by incubating commercially available dGp with both 64% hydrazine and anhydrous hydrazine. In both cases we could not

TABLE

detect an increase of d(&OH-G)p. Unfortunately, however, a high amount of background d(S-OH-G)p (0.42%) appeared to be present in the dGp used for this experiment (see also Discussion). Our preliminary conclusion is that the theoretical lower limit of detection by the protocol mentioned above is 1 d(S-OH-G)p out of lo6 dGp’s when at most 0.1% of dGp is left after TFA treatment.

Labeling with Carrier-Free Chromatography

[i-“‘P]ATP

and

Selective chemical decomposition of at least 99% of normal nucleotides in a DNA digest before labeling

1

100.

U-EC

Y

Percentages of 32P-LabeledNucleoside 3’,5’-Biphosphates Found Either

after 32P Postlabeling 90% Trifluoroacetic

ture or 64% Hydrazine for

of a DNA Digest Treated with 10 min at Room Tempera16 h at 60°C or Both

Acid for

Treatment Nucleotide

TFA

PTP

98.5

PdCp

97.8 0.2

PdAP

PdGp

1.5

0.9 95.1 97.1 94.9

0.1 102.1

pd(S-OH-G)p Note.

Hydrazine

One percent

corresponds

to at least

350 cpm.

Both

1.5 0.7 0.2 0.1 96.3

10 CL 5 a s

1

.l

.Ol 0

20

40

•i

pd(B-OH-G)p

A

PdAp

A

PdGP

60

80

100

min. in TFA

FIG. 2. Decomposition of dAp, dGp, or d(8-OH-G)p fluoroacetic acid as measured by 32P-postlabeling analysis rials and Methods).

in 90% tri(see Mate-

=P

POSTLABELING

DETECTION

FIG. 3. Three-directional chromatography of a mixture of 32Ppostlabeled deoxynucleotides and d(B-OH-G)p demonstrating the removal of excess of 32P in the mixture of labeled nucleotides. Removal of 32P was achieved by overnight development with 0.25 M LiCl in direction Dl, i.e., conditions under which pyrimidine deoxynucleotides hardly migrate and purine deoxynucleotides do not migrate at all. After the chromatogram was cut at the broken line, the deoxynucleotide biphosphates were resolved by development in D2 with 3 M ammonium formate, pH 3.5, and in D3 with 0.35 M NaH,PO,, pH 6.1.

opens the possibility of increasing the sensitivity of 8hydroxyguanine detection considerably, because (i) small amounts of unaffected d(8-OH-G)p residue in this digest can be labeled with excess carrier-free [-Y-~~P]ATP and (ii) radioactive background on the PEI-cellulose thin layers, which otherwise would obscure minor pd(8-OH-G)p spots after 2D chromatography, can be reduced. The sensitivity of the method was further improved by reduction of radioactive background due to free ““Pi in postlabeled digests. This was achieved by employing another mode of chromatography, in which free ““Pi is chromatographically removed prior to 2D separation of the deoxynucleoside diphosphates (Fig. 3) (see Discussion).

Detection

of 8-OH-G in y-Irradiated

DNA

Since it is known that y-irradiation induces 8-OH-G in DNA [see Refs. (l-4,40)], we wanted to validate the detection method with purified single-stranded Ml3 DNA irradiated in aqueous solution with low doses of 6oCo y-rays. Figure 4 shows the results of a 32P-postlabeling analysis of this DNA before and after irradiation with 1 or 5 Gy. The nucleotides were either untreated (Figs. 4a-4c), treated with both trifluoroacetic acid and hydrazine (not shown), or treated with trifluoroacetic acid alone (Figs. 4d-4f) and labeled with an excess of [Y-~~P]ATP with a low specific activity (200 Ci/mmol). The figure illustrates that y-irradiation induces 8-OHG in a dose-dependent way and that reduction of deoxyguanylic acid in DNA digests by TFA treatment before labeling increases the sensitivity of the detection of 8OH-G considerably. Unfortunately, however, induction

OF

8-HYDROXYGUANINE

131

of 8-OH-G residues in DNA by even lower doses of yrays could not be detected in this case because of the presence of a relatively high amount of 8-OH-G in the unirradiated DNA (0.10% versus 0.25 and 0.88% found after irradiation with 1 and 5 Gy, respectively). This makes both chemical decomposition of normal deoxynucleotides and removal of excess 32Pi (see Fig. 3) redundant in this experiment. Figure 4 also shows that not only d(8-OH-G)p but also guanylic acid (Gp) is resistant toward TFA. Because pGp migrates much closer to pd(8-OH-G)p during TLC than pdGp, the sensitivity of the method depends not only on the amount of background 8-OH-G but also on the amount of RNA present in the DNA preparation used for analysis (see also Discussion). DISCUSSION The methodology described in this paper might be very useful for the sensitive detection of 8-OH-G in low amounts of DNA. Using the protocol shown in Scheme 1 and the chromatographical procedure described in the legend to Fig. 3 it should be possible to detect 1 d(8-OHG)p out of 2 X lo6 nucleotides in an amount of 1 pg DNA, provided that the number of Gp’s present in DNA hydrolysates (due to the presence of small amounts of RNA in DNA preparations) does not exceed 0.01% of the total number of dGp’s. Since Gp is the major nucleotide interfering with the detection of d(8-OH-G)p after postlabeling and 2D chromatography (Fig. 4) the sensitivity of the method might be enhanced by prepurification of d(S-OH-G)p in such a way that the amount of Gp is strongly reduced before TFA treatment and postlabeling. In that case also the hydrazinolysis step may be omitted, viz. when the bulk amount of dCp, dm5Cp, and Tp is reduced concomitantly. Another, perhaps more simple, way to reduce interference by 32P-postlabeled Gp is to retard its migration relative to that of pd(8-OHG)p in the final TLC step (Fig. 4, from left to right) by employing solvents containing boric acid. [Borate anions form complexes with the cis-glycols of ribonucleotides and retard their migration (41)]. However, from the results presented in this paper it is evident that these improvements are redundant when DNA, isolated with the intention of examination for the presence of low levels of newly introduced a-OH-G, already contains levels of this lesion far above the detection limit. For instance, because of the presence of a relatively high amount of background 8-OH-G in the ss Ml3 DNA (0.1% of the total number of G’s), a significant increase of this lesion could not be detected at radiation doses much lower than 1 Gy, although this should be possible with the detection procedure described here. It is possible that certain manipulations during DNA isolation may cause the formation of 8-OH-G. For instance, it is known that purification of DNA by extrac-

132

LUTGERINK

ET

AL.

FIG. 4. “P-postlabeling analysis of single-stranded phage Ml3 DNA before (a and d) or after irradiation of the DNA with 1 Gy (b and e) and 5 Gy (c and f). The nucleotides were either untreated (a, b, and c) or treated with trifluoroacetic acid (d, e, and Q. Labeled nucleotides other than pdGp, pGp, and pd(&OH-G)p were removed by chromatography on paper wicks, which were attached to the thin-layer sheets by stapling. Only the relevant parts of the chromatograms are shown.

tion with phenol-containing peroxides may artificially introduce high levels of this type of modification (42). However, other causes for isolation-mediated &OH-G introduction are conceivable because we also found relatively high amounts (20 to 100 &OH-G’s per lo5 G’s) in Ml3 DNA (both ss and ds) and plasmid DNA purified by extraction with phenol that was freshly distilled and freed from peroxides by washing with saturated disodium tetraborate. We also found high amounts of d(S-OH-G)p after postlabeling analysis of both commercially available calf thymus DNA [even up to 570 d(S-OH-G)p per lo5 normal dGp’s] and deoxyguanylic acid [5 to 420 d(8OH-G)p per lo5 normal dGp’s]. From previous reports it can also be deduced that high background levels of 8OH-G are present in commercial calf thymus DNA [ - 140 per lo5 guanines as detected by gas chromatography-mass spectrometry with selected-ion monitoring or by fluorescence postlabeling (43-45)J. All these observations demonstrate the necessity of assessing whether and to what extent 8-OH-G residues are present in commercially available deoxyguanylic acid or DNA when in vitro experiments to detect low levels of newly introduced 8-OH-G are planned. Our results together with those found by others illustrate the need for systematic studies that may help to minimize artificial introduction of this lesion during the isolation of DNA. We included such studies in a recently started project on oxidative base damage induced in DNA in uiuo by radiation, carcinogens, and cytostatics. Preliminary results, obtained by using the detection method described in this paper, show that DNA from normal rat liver contains 1 to 5 8-OH-G per lo5 G’s

when isolated according to Visser and Westra (45) and when freshly distilled phenol that was freed from peroxides is used. These amounts fall within the range found by others in various rat organs, including rat liver [m-0.5-48 ~-OH-G’S per 10’ G’s (47-49)]. Although there has been some argument about the physiological occurrence of 8-OH-G in DNA, recent evidence for the presence of a specific 8-OH-G endonuclease in Escherichia coli and probably also in human tissue culture cells (50) supports the idea that at least part or possibly all of the 8-OH-G found in the isolated DNA is formed in uiuo by reactive oxygen species. Other evidence for the presence of oxidative DNA damage in cells in viuo has been obtained by Richter et al. (47), who found that the level of 8-OH-G in mitochondrial DNA from rat liver was about 16 times higher than that in nuclear DNA, which is not unexpected since mitochondria are a major source of oxygen radicals. A final remark regarding the hydrazinolysis step employed to remove the pyrimidine deoxynucleotides before postlabeling should be made. In contrast with the results of Kasai and Nishimura (37), we did not find significant amounts of d(8-OH-G)p after hydrazine treatment of dGp. We attribute this to the possible presence of contaminating metal ions in the reaction mixtures of these authors, which may allow reducing agents such as hydrazine to participate in a Fenton-like reaction producing hydroxyl free radicals (51,52). REFERENCES 1. Cadet, J., and Berger, M. (1985) Znt. J. Rodiat. Bid. 47,127-143. 2. Schulte-Frohlinde, D., and Von Sonntag, C. (1985) in Oxidative Stress (Sies, H., Ed.), pp. l-40, Academic Press, New York.

=P

3. Hutchinson, 154.

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