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Carcinogenesis, Vol. 20, No. 7, 1241-1245, July 1999
© 1999 Oxford University Press

Carcinogenesis

32P-postlabeling high-performance liquid chromatography (32P-HPLC) adapted for analysis of 8-hydroxy-2'-deoxyguanosine

Magnus Zeisig, Tim Hofer, Jean Cadet1 and Lennart Möller2

Karolinska Institutet, Department of Biosciences at Novum, Unit for Analytical Toxicology, SE-141 57 Huddinge, Stockholm, Sweden and
1 Département de Recherche, Fordamensole sur la Matière Condensée,SCIB/LAN, CEA/Grenoble, FR-38054 Grenoble Cedex 9, France


   Abstract
Top
 Abstract
Introduction
 Material and methods
 Results
 Discussion
 References
 
8-Hydroxy-2'-deoxyguanosine (8-OH-dG) is a promutagenic lesion in DNA caused by reactive oxygen species. It normally exists at a level of 0.1–1 per 105 2'-deoxyguanosines (dG). To analyze the lesion in easily obtainable biological samples, a very sensitive analytical method is required. The method should also handle the problem with potential oxidation of dG to 8-OH-dG during workup and analysis. 32P-postlabeling high-performance liquid chromatography (32P-HPLC) is an analytical method previously used to analyze lipophilic DNA adducts at levels as low as 1 per 109 normal nucleotides when analyzing microgram amounts of DNA. This method was adapted for analysis of 8-OH-dG. The aim was to develop an analytical method that provided a high sensitivity and good reproducibility, prevented oxidation of dG present in samples to 8-OH-dG, was capable of analyzing DNA from very small samples and still offered high sample throughput and ease of use. In analysis of calf thymus DNA, the method had a detection limit of 0.1 8-OH-dG per 105 dG when 1 µg of DNA was used. The standard deviation of repeated analyses of the same sample was ±10% and the result corresponded well with the established analytical method using HPLC with electrochemical detection. 32P-HPLC is sensitive enough to enable analysis of low levels of 8-OH-dG in biological samples such as small volumes of blood, needle biopsies and tissue swabs. It also substantially reduces oxidation of dG to 8-OH-dG during sample workup and analysis.

Abbreviations: 32P-TLC, 32P-postlabeling thin-layer chromatography and autoradiography; 8-OH-dG, 8-hydroxy-2'-deoxyguanosine; dG, 2'-deoxyguanosine; EDTA, ethylenediaminetetraacetic acid; ELISA, enzyme-linked immunosorbent assay; GC–MS, gas chromatography with on-line mass spectrometry; HPLC, high-performance liquid chromatography; HPLC–EC, high-performance liquid chromatography with on-line electrochemical detection; ISA, immunoslot blot assay; MN, micrococcal nuclease; PNK, T4 polynucleotide kinase; SDS, sodium dodecyl sulphate; SPD, spleen phosphodiesterase; TLC, thin-layer chromatography; Tris, tris[hydroxymethyl] aminoethane.


   Introduction
Top
 Abstract
 Introduction
Material and methods
 Results
 Discussion
 References
 
8-Hydroxy-2'-deoxyguanosine (8-OH-dG) is a chemical modification of DNA caused by reactive oxygen species in the cell (1,2). This modification seems to exist normally in the nuclear DNA at a level of ~0.1–1 per 105 bases or nucleotides (3). To be able to analyze and quantitate 8-OH-dG, an analytical method with a sensitivity of 0.01–0.1 per 105 nucleotides is therefore required. Approximately 30 µg of DNA can be obtained from a blood sample (3 ml), requiring an absolute sensitivity for 8-OH-dG of 3 pg or 10 fmol. A fine needle biopsy or tissue swab (1 mg) produces ~1 µg of DNA, requiring an absolute sensitivity for 8-OH-dG of 0.1 pg or 0.3 fmol. Consequently, analysis of 8-OH-dG in this type of samples requires a very sensitive analytical method.

The most commonly used methods for 8-OH-dG analysis today are high-performance liquid chromatography with on-line electrochemical detection (HPLC–EC) and gas chromatography followed by on-line mass spectrometry (GC–MS) (4,5). Other methods have also been used, such as fluorescence post-labeling and HPLC with fluorescence detection (6), immunoslot blot assay (ISA) (7), enzyme-linked immunosorbent assay (ELISA) (8), 32P-postlabeling followed by thin-layer chromatography and autoradiography (32P-TLC) (9–11), as well as liquid chromatography with mass spectrometry (12). The absolute sensitivities reported in 8-OH-dG analysis are in the range 1.8–8 fmol (11–14).

A problem encountered with all methods is the risk of auto-oxidation from 2'-deoxyguanosine (dG) to 8-OH-dG during workup procedures (3). This risk is encountered as soon as dG is exposed to oxygen or other oxidants in the atmosphere or in solutions, or when dG is exposed to ionizing radiation, like that from 32P (15). This risk may be reduced by removing potential oxidants or dG. Since dG will be present in biological samples, it is important to keep these samples away from oxidants during storage and sample preparation, at least as long as dG remains.

The most sensitive analytical system for studying DNA damage by covalent binding to DNA (DNA adducts), that is generally available today is the 32P-postlabeling assay (16,17). Sensitivities of 1 adduct per 1010 nucleotides can be reached when microgram amounts of DNA are analyzed. This would give the possibility to detect 10 ag or 0.03 amol of 8-OH-dG. However, owing mainly to poor separation and high backgrounds, the detection limits for 8-OH-dG reported so far are several orders of magnitude higher than 10 ag or 0.03 amol (9–11).

32P-Postlabeling involves the enzymatic digestion of DNA to nucleoside 3'-phosphates and adducts of these, usually some kind of adduct enrichment, 32P-labeling of the adducted and remaining non-adducted nucleoside 3'-phosphates and chromatographic separation of the sample with detection of the radioactivity. The two last steps are usually performed either by thin-layer chromatography (TLC) followed by autoradiography (17) or by HPLC with on-line detection of the labeled compounds (18,19). 32P-Postlabeling assays have mainly been used to study DNA adducts from aromatic compounds (17), but 32P-TLC has also been used to study more polar adducts, such as 8-OH-dG 3'-monophosphate (9,10,20) and 2'-deoxyadenosine-N1-oxide 3'-monophosphate (21). 32P-HPLC is able to separate polar adducts like 7-methyl-2'-deoxyguanosine (22) from normal nucleotides. The 32P-postlabeling assays, however, involve the exposure of the sample to ionizing radiation which may cause oxidation of dG to 8-OH-dG (15,20). It is therefore essential to remove dG before the post-labeling step.

The aim of this study was to adapt the 32P-HPLC method to analysis of 8-OH-dG, thereby developing a method offering a high sensitivity and good reproducibility, preventing oxidation of dG present in samples to 8-OH-dG, being capable of analyzing DNA from very small samples and still offering high sample throughput and ease of use.


   Material and methods
Top
 Abstract
 Introduction
 Material and methods
Results
 Discussion
 References
 
Reagents and chemicals
Solvents and salts were of analytical grade. Reagents were purchased from the indicated sources: micrococcal nuclease (MN), 2'-deoxynucleoside 3'-monophosphates (Sigma, St Louis, MO), T4 polynucleotide kinase (US Biochemical, Cleveland, OH), nuclease P1, spleen phosphodiesterase (Boehringer Mannheim, Mannheim, Germany) and [32P]ATP with an original specific activity of ~3000 Ci/mmol (Amersham, Little Chalfont, UK). Structures and the nomenclature used for nucleotide compounds are shown in Figure 1Go.



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  		Fig. 1. Structure of the compounds discussed in this paper and their abbreviations. The lower half depicts the general pathway for 32P-post-labeling, nuclease P1 treatment and hydrolysis of the postlabeled nucleotides.

 
Instrumentation
The HPLC system for pre-separation and 32P-HPLC consisted of a 600E multisolvent delivery system (Waters Chromatography Division, Millipore, Milford, MA). Radioactivity was measured on-line using a Flo-One\Beta 290 radioactivity detector with a 0.5 ml cell and scintillation fluid Flo-Scint IV (both from Radiomatic Instruments and Chemical, Tampa, FL). The energy window was set at 8–600 keV with a counting efficiency of 60% for 32P. The counting was performed in 12 s cycles. The analytical system had a pre-column NewGuard holder with an RP-18, 7 µm cartridge (both from Brownlee Laboratories, Santa Clara, CA) and two serial Delta Pak 5 µm, C18–100 A, 150x3.9 mm i.d. (Waters Chromatography Division) as main columns. The HPLC system used with EC was prepared as described previously (23).

8-OH-dG 3'-monophosphate synthesis
8-OH-dG 3'-phosphate was prepared and purified according to methods described previously (24,25). dG 3'-phosphate was incubated in sodium phosphate buffer (pH 6.8) with ascorbic acid, ethylenediaminetetraacetic acid (EDTA) and iron (II) sulphate, while leading oxygen through the solution. The resulting 8-OH-dG 3'-monophosphate was purified from the reaction mixture by three subsequent HPLC steps, and stored at –70°C after being evaporated to dryness. The concentration of redissolved aliquots was determined by measuring the absorbance at 245 (12 300/M/cm) and 293 nm (10 300/M/cm).

Hydrolysis, enrichment, 32P-postlabeling and chromatography
DNA (10 µg) for 32P-HPLC analyses was hydrolyzed by dissolving in 2 µl/µg DNA water, adding 1.4 µl/µg DNA of MN (53 mU/µl MN from Staphylococcus aureus Foggis strain in 1.1 mM N,N-bis[2-hydroxyethyl]glycine, 0.18 mM calcium dichloride, pH 9.0) and incubating it for 2 h at 37°C. SPD (1 mU/µl dialyzed phosphodiesterase from calf spleen in 10 mM ammonium acetate, pH 5.0) was added to DNA (1.6 µl/µg DNA) and the mixture was incubated for a further 2 h at 37°C.

Samples consisted of hydrolyzed DNA or 3'-monophosphates of nucleoside and 8-OH-dG standards in water solution. Enrichment of 8-OH-dG prior to postlabeling was performed by HPLC separation. Samples were injected into the HPLC and eluted isocratically with 1 ml/min of a low molar (5–20 mM) ammonium formate buffer (pH 2.2–2.8). Separation was performed at room temperature, 21–22°C. Fractions were collected from the outlet of the column, evaporated to dryness and redissolved in 1 µl of water.

Aliquots of 1 µl of sample were postlabeled by mixing with T4 polynucleotide kinase (PNK) buffer (200 mM bicine, 100 mM DTT, 10 mM spermidine, 100 mM magnesium chloride, pH 9.6) (0.1 µl/µl sample), PNK (0.2 µl/µl sample of 10 U/µl), [32P]ATP (0.7 µl/µl sample of 10 µCi/µl) to a total volume of 2.0 µl/µl sample. The reaction mixture was incubated for 30 min at 37°C. The 3'-phosphate groups were then hydrolyzed by adding nuclease P1 and buffer (0.62 µg nuclease P1, 33 µM zinc dichloride, 15.5 mM sodium acetate, 5.4 mM hydrochloride, pH 4.5) (13 µl/µl original sample) and incubating for 45 min at 37°C.

HPLC analyses of 32P-postlabeled DNA adducts were performed by injection of the total 32P-postlabeling mixture without pre-purification onto the HPLC column, eluting isocratically with 1 ml/min of a low molar (5–50 mM) ammonium formate buffer (pH 2–4). The ammonium formate buffer was prepared by dissolving ammonium formate salt in water to the selected molarity and adjusting pH with formic acid to the indicated value.

DNA for HPLC–EC analyses was hydrolyzed according to methods described previously (23). The hydrolysate was analyzed using 0.8 ml/min of 10% methanol in 50 mM sodium acetate buffer set to pH 5.3 with phosphoric acid.


   Results
Top
 Abstract
 Introduction
 Material and methods
 Results
Discussion
 References
 
Elution of nucleoside 5'-monophosphates, after 32P-post-labeling and nuclease P1 treatment at a low pH, resulted in a good separation when a low molar eluent buffer was used (Figure 2Go). The best separation of 8-OH-dG from the unmodified nucleotides (20 min) was achieved using 5 mM ammonium formate with a pH of 3.0–3.2, while a good resolution between all unmodified nucleotides and 8-OH-dG (3.5–10 min) was achieved using 5 mM ammonium formate with a pH of 3.4–3.6. In both cases, 8-OH-dG was eluted after all normal nucleotides.



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  		Fig. 2. (A) pH dependency of the HPLC resolution of 5'-monophosphates of nucleosides and 8-OH-dG. The curves show the retention times in min at various pH when the compounds were eluted with 10 mM ammonium formate buffer at 1 ml/min. (B) HPLC separation of 5'-monophosphates of nucleosides and 8-OH-dG eluting with 5 mM ammonium formate buffer (pH 4.0) at 1 ml/min. The y-axis shows the detected 32P-radioactivity, measured as 1000 decays per min per 12 s counting cycle (kdpm/12 s). The abbreviations are according to Figure 1Go, with the trailing p indicating 5'-monophosphate; Pi, orthophosphate; x, a so far unidentified compound, possibly 5'-methyl-2'-deoxycytidine 5'-monophosphate.

 
Enrichment of 8-OH-dG prior to postlabeling was best achieved using a low pH with a low molar eluent buffer. A pH of 2.4–2.6 resulted in a good separation of 8-OH-dG from the unmodified nucleotides (4 min) as well as between all unmodified nucleotides (3–5 min) using 5 mM ammonium formate (Figure 3Go). The chromatographic resolution between 8-OH-dG and dG was 7–8 min. 8-OH-dG was eluted after all normal nucleotides. At pH values below 2.0 the detected amount of nucleotides and 8-OH-dG started to decrease. At pH values above 2.6 peaks started to widen.



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  		Fig. 3. (A) HPLC separation of nucleoside and 8-OH-dG 3'-monophosphates eluting prior to 32P-postlabeling with 5 mM ammonium formate buffer (pH 2.5) at 1 ml/min. The compilation shows the fraction of total 32P-radioactivity, P/P(tot), in nucleoside and 8-OH-dG 5'-monophosphates after 32P-postlabeling of all the 1 min fractions collected between 11 and 25 min. 2'-Deoxycytidine 3'-monophosphate eluted before 11 min when the first fraction was collected. (B) 32P-HPLC chromatograms of the corresponding fractions using 5 mM ammonium formate buffer (pH 3.0) at 1 ml/min and the same column system as above. The abbreviations are according to Figure 1Go with the tailing p indicating 3'-monophosphate.

 
Analyses of 8-OH-dG 3'-phosphate standard showed a 32P-postlabeling efficiency of 77 ± 39% (n = 5). The recovery of the standard when dissolved in 1 ml of 5 mM ammonium formate buffer (pH 2.5) and evaporated to dryness (simulating the procedure after pre-separation) was 42 ± 13% (n = 5). The total recovery including 32P-postlabeling of the standard from pre-separation was 32 ± 10% (n = 5).

Five micrograms of calf thymus DNA was hydrolyzed and aliquots of 1 µg were analyzed using pre-separation with 5 mM ammonium formate buffer at pH 2.5, 32P-postlabeling, nuclease P1 hydrolysis of 3'-phosphates and 32P-HPLC using 5 mM ammonium formate buffer at pH 3.5 (Figure 4AGo). Analysis of five samples resulted in an observed modification level of 129 ± 12 8-OH-dG per 108 normal nucleotides or 0.52 ± 0.05 8-OH-dG per 105 dG. This corresponds to an absolute level of 4.2 ± 0.4 fmol of 8-OH-dG.



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  		Fig. 4. (A) 32P-HPLC analysis of 1 µg calf thymus DNA using pre-separation with 5 mM ammonium formate buffer (pH 2.5) at 1 ml/min, 32P-postlabeling, nuclease P1 hydrolysis of the 3'-phosphate and 32P-HPLC using 5 mM ammonium formate buffer (pH 3.0) at 1 ml/min. The abbreviations are according to Figure 1Go with the trailing p indicating 5'-monophosphate; Pi, orthophosphate; x, a so far unidentified compound, possibly 5'-methyl-2'-deoxycytidine 5'-monophosphate. (B) Comparison between 8-OH-dG analyses using 32P-HPLC and HPLC–EC. The samples were varying amounts of human lymphocytes.

 
When human lymphocyte DNA was prepared and analyzed for 8-OH-dG by 32P-HPLC and HPLC–EC the results from the two methods corresponded well when 2–10 µg DNA was used (Figure 4BGo). However, with both methods, the levels of 8-OH-dG detected increased when the amount of DNA prepared and analyzed was <5 µg.


   Discussion
Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
References
 
The method used in this study was developed from the 32P-HPLC assay designed at our laboratory (19,22,26) and previously used mainly for analysis of aromatic DNA adducts (27–31). As the method had also shown its capacity to analyze more polar DNA modifications, such as 7-methyl-2'-deoxyguanosine (22), it was considered a possibility for measuring 8-OH-dG as well.

The original 32P-HPLC method was enhanced by adding a second main column in series with the first. This narrowed the peak width to ~50% of that seen when using one column, thereby increasing both resolution and sensitivity.

The nucleoside 3',5'-bisphosphates obtained after 32P-postlabeling were difficult to separate on HPLC. By eliminating the unlabeled 3'-phosphate group using nuclease P1, the compounds were transformed into the less polar nucleoside 5'-monophosphates (Figure 1Go), which proved to be more readily resolved using HPLC.

Owing to the polar nature of the main compounds to be resolved, i.e. unmodified and polarly modified nucleoside 5'-phosphates, an acetonitrile gradient similar to that in the 32P-HPLC method for aromatic compounds proved to be unnecessary. Also, the high molar eluent buffer used could also be diluted from 2 M ammonium formate to 5–10 mM (Figure 2Go). These two modifications allowed for a simpler HPLC setup.

The use of a low molar eluent without any organic solvent has the disadvantage of less polar postlabeled compounds accumulating in the HPLC column, slowly eluting and causing an increased background during analysis. This problem was overcome by washing the chromatographic system once per day with 2 M ammonium formate (pH 4.5) and acetonitrile.

Adjusting the pH of the 32P-HPLC eluent enabled flexibility in the separation of the polar DNA compounds. A high pH caused 8-OH-dG to be eluted between the unmodified nucleotides, while a low pH permitted the elution of 8-OH-dG after the unmodified nucleotides (Figure 2AGo). This may be advantageous when performing preparative HPLC.

The adapted 32P-HPLC system provided a sufficient resolution between unmodified nucleotides and 8-OH-dG to permit analysis of 8-OH-dG without prior enrichment. The huge excess of normal nucleotides (105–106 times) compared with expected 8-OH-dG levels would, however, demand very large amounts of 32P-ATP to be used in the postlabeling step. Therefore, an enrichment of 8-OH-dG prior to 32P-postlabeling was considered necessary. Also, ß radiation from 32P can cause extensive conversion of dG to 8-OH-dG (10,15). It is, therefore, of the utmost importance to separate 8-OH-dG from dG prior to postlabeling (32,33).

The chemical similarities between nucleoside 3'-monophosphates and nucleoside 5'-monophosphates allowed for pre-separation of 8-OH-dG and unmodified nucleotides using an HPLC system identical to that in the adapted 32P-HPLC, except for the eluent. The optimal pre-separation was achieved at a pH of ~2.5 (Figure 3Go). At pH values lower than 2.5, the amount of nucleotides detected after 32P-postlabeling decreased, probably owing to acid hydrolysis of the 3'-phosphate group necessary for postlabeling. At pH values higher than 2.5 the peaks showed tendencies to widen, thereby deteriorating the pre-separation.

When analyzing calf thymus DNA with the above adapted 32P-HPLC method, 8-OH-dG was detected at a level of 0.5 per 105 unmodified 2'-deoxyguanosines (Figure 4AGo). This rather high value likely depends on the exposure of the commercial calf thymus DNA to reactive oxygen during preparation, storage and handling. Methods to prevent the oxidation of dG to 8-OH-dG during sample preparation, storage and handling are being developed at our laboratory. These methods indicate that artifact levels of 8-OH-dG could be reduced by a factor of 2.5–25 (23).

32P-HPLC was performed using 1 µg of DNA. The 8-OH-dG peak would still be detectable above the background noise even if its height was reduced by a factor of five. This means a sensitivity for the method of 26 8-OH-dG per 108 normal nucleotides or 0.1 8-OH-dG per 105 dG when 1 µg of DNA is analyzed, and an absolute sensitivity of 1 fmol of 8-OH-dG. Alternatively, a sensitivity of 0.5 8-OH-dG per 105 dG can be reached when 0.2 µg DNA is analyzed. The absolute sensitivity is about eight times the highest sensitivity reported for GC–MS, five times that of 32P-TLC and about twice that of HPLC–EC. Also, the sensitivity of the 32P-HPLC method reported in this paper is based on analysis of DNA and not an 8-OH-dG standard (Table IGo). GC–MS and HPLC–EC normally require 20–50 µg DNA for analyses (13,14).


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  		Table I. Best sensitivity in analysis of 8-OH-dG by different methods according to literature
 
When human lymphocyte DNA was prepared and analyzed, the levels of 8-OH-dG detected with 32P-HPLC and HPLC–EC were very similar when 2–10 µg of DNA was used (Figure 4BGo). Above 10 µg, the levels of 8-OH-dG detected with 32P-HPLC dropped owing to the presence of increasing amounts of normal nucleotides competing for the available [32P]ATP. This phenomenon can be circumvented by increasing the amount of [32P]ATP used. When <5 µg was used, the levels of 8-OH-dG detected increased with both methods, probably owing to auto-oxidation during preparation of the samples. This is also in accordance with previous studies (15,20,23).

The sensitivity of 32P-HPLC analysis could be improved if the radioactive background of the 32P-chromatograms could be decreased. This could probably be achieved either by an improved pre-separation or by modification of the chromatographic conditions during the 32P-HPLC step. However, to take full advantage of this sensitivity, auto-oxidation during sample preparation needs to be reduced even further.

The possibility of analyzing microgram amounts of DNA for 8-OH-dG allows for the application of the 32P-HPLC method to a wide variety of biological samples. Ordinary blood samples, needle biopsies and tissue swabs all produce only small amounts of DNA and have, thus far, not been usable for routine 8-OH-dG analysis, but may become samples of choice for 32P-HPLC analysis. This means that studies of 8-OH-dG in humans that have previously required tissue amounts achievable only through autopsies or surgical biopsies could be performed more easily.

In conclusion, 32P-HPLC was adapted to 8-OH-dG analysis. A pre-separation utilizing the same HPLC system was used to prevent conversion of dG to 8-OH-dG during 32P-postlabeling, which had most probably been caused by ß radiation from 32P. The method had a sensitivity of 0.1 8-OH-dG per 105 dG when 1 µg of DNA was analyzed, or 0.5 8-OH-dG per 105 dG if 0.2 µg DNA was analyzed, and an absolute sensitivity of 1 fmol 8-OH-dG. This would enable the analysis of very small biological samples for 8-OH-dG by 32P-HPLC. Further experiments are ongoing in our laboratory to further optimize the 32P-HPLC method for analyses of 8-OH-dG.


   Acknowledgments
 
The authors wish to express their gratitude to Mary-Ann Zetterqvist for her skilful technical assistance. The contribution of Dr Jean-Luc Ravanat for the synthesis of 8-OH-dG 3'-monophosphate and 8-OH-dG 5'-monophosphate is also gratefully acknowledged. This work was supported by the Swedish Environmental Protection Agency and the Swedish Medical Research Council (contract 11567).


   Notes
 
2 To whom correspondence should be addressed Email: lennart.moller{at}cnt.ki.se Back


   References
Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 

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Received July 22, 1998; revised March 23, 1999; accepted March 25, 1999.


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