Mutagenesis, Vol. 14, No. 2, 239-242,
March 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
A new modification of the 32P-post-labeling method to recover IQ–DNA adducts as mononucleotides
1 Biochemistry and 2 Carcinogenesis Divisions, National Cancer Center Research Institute, 1-1 Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan and 3 Nestec Ltd, Research Centre, Vers-chez-les-Blanc, 1000-Lausanne 26, Switzerland
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Abstract |
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To obtain accurate estimates of DNA adduct levels yielded by genotoxic compounds, it is essential to completely digest adducted nucleotides to mononucleotides. We previously developed a suitable method, called modified method I, to obtain DNA adducts of heterocyclic amines as 32P-labeled-mononucleoside adduct 5'-phosphate forms, by use of nuclease P1 (NP1) and phosphodiesterase I (PDEI) to digest adducted oligonucleotides. In this study, we applied method I to 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)–DNA adduct analysis and found that one of the IQ–DNA adducts, 5-(deoxyguanosin-N2-yl)-2-amino-3-methylimidazo[4,5-f]quinoline 3',5'-diphosphate (pdGp-N2-IQ), was resistant to the 3'-phosphatase activity of NP1, but sensitive to that of T4 polynucleotide kinase (PNK). DNA obtained from the liver of rats fed IQ was 32P-labeled by the standard method and the 32P-labeled nucleotides obtained were incubated with PNK and NP1 to remove 3'-phosphate groups and then digested with PDEI. Three spots were obtained. One major spot was identified as N-(deoxyguanosin-8-yl)-2-amino-3-methylimidazo[4,5-f]quinoline 5'-phosphate (pdG-C8-IQ) and a second abundant adduct as pdG-N2-IQ. The third spot, of which the structure is unknown, was minor. The new method is called modified method II. Modified method II could be applicable to a wide variety of chemicals.
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Introduction |
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TopAbstractIntroductionMaterial and methodsResults and discussionReferences |
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The 32P-post-labeling method, which was developed originally as the standard method (Gupta et al., 1982
) and modified successively as the intensification method (Randerath et al., 1985), butanol extraction method (Gupta, 1985) and nuclease P1 method (Reddy and Randerath, 1986), is very useful and convenient for evaluating DNA base adduct levels yielded by administration of genotoxic compounds. However, further improvement has been sought because, in most cases, many spots are obtained due to incomplete digestion of adducted oligonucleotides. For instance, the 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)–DNA base adduct was detected as multiple spots on thin layer chromatography (TLC) by the standard method. However, our improved method, modified method I, in which 32P-labeled mono- and oligonucleotides obtained by the standard method are subsequently digested with nuclease P1 (NP1) and phosphodiesterase I (PDEI), gives a single spot of N-(deoxyguanosin-8-yl)-PhIP 5'-phosphate (pdG-C8-PhIP) on TLC, with the same recovery as the standard method (Fukutome et al., 1994). This is the same as the 2-amino-3,4-dimethylimidazo[4,5-f]quinoline(MeIQ)–DNA base adduct, as reported previously (Tada et al., 1994). Only a single spot was detected and identified as N-(deoxyguanosin-8-yl)-2-amino-3,4-dimethylimidazo[4,5-f]quinoline 5'-phosphate (pdG-C8-MeIQ) by modified method I, with the same recovery as the standard method, which gave four spots.
As for 2-amino-3-methylimidazo[4,5-f]quinoline(IQ)–DNA base adducts, the standard and the intensification methods are widely used because the recovery of IQ–DNA adducts is higher by these methods than by other methods, such as the nuclease P1 or butanol extraction methods (Hall et al., 1990). However, five spots can be detected by the standard method (Turesky and Markovic, 1994). Two of them have been structurally determined as N-(deoxyguanosin-8-yl)-2-amino-3-methylimidazo[4,5-f]quinoline 3',5'-diphosphate (pdGp-C8-IQ) (Snyderwine et al., 1988b) and 5-(deoxyguanosin-N2-yl)-2-amino-3-methylimidazo[4,5-f]quinoline 3',5'-diphosphate (pdGp-N2-IQ) (Turesky and Markovic, 1994), while the structures of the remaining three spots have yet to be elucidated. The structures of IQ and IQ–DNA base adducts (dG-C8-IQ and dG-N2-IQ) are shown in Figure 1. In an analogy to MeIQ– and PhIP–DNA base adducts, we applied modified method I to digest IQ-adducted oligonucleotides with PDEI after removal of their 3'-phosphate group. However, the 3'-phosphate group of pdGp-N2-IQ was resistant to NP1, so we utilized the phosphatase activity of T4 polynucleotide kinase (PNK) (Cameron and Uhlenbeck, 1977). Using this method, modified method II, it was revealed that IQ induces three different forms of adducts.
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Material and methods |
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Chemicals
IQ was obtained from the Nard Institute (Osaka, Japan). The dG 3'-phosphate adduct standards of IQ were synthesized and characterized as described previously (Turesky and Markovic, 1994
). All other chemicals and reagents were from the suppliers as previously reported (Fukutome et al., 1994; Tada et al., 1994).
Animal experiments
F344 rats were purchased from Charles River Japan (Atsugi, Japan). Two rats of 8 weeks old were administered the CE-2 diet (CLEA Japan, Tokyo, Japan) containing IQ at a concentration of 300 p.p.m. for 12 weeks and killed. Livers were removed, frozen in liquid nitrogen and stored at –80°C until DNA extraction.
32P-post-labeling method
The standard method and modified method I were performed as reported previously (Fukutome et al., 1994; Tada et al., 1994). Briefly, the DNA (1 µg/µl, 10 µl), isolated by phenol/chloroform extraction, was digested with micrococcal nuclease (3 U) and spleen phosphodiesterase (0.03 U) (Worthington Biochemical Co., Freehold, NJ) at 37°C for 3 h. The DNA digest (0.17 µg) was 32P-labeled by 10 U of PNK (Takara Shuzo Co. Ltd, Kyoto, Japan) with [
-32P]ATP (8.3 TBq/mmol, 45 µM) at 37°C for 1 h in 15 µl of reaction mixture (30 mM Tris–HCl buffer, 10 mM dithiothreitol, 10 mM MgCl2, 1 mM spermidine, pH 9.5). In the standard method, the 32P-labeled samples were treated with 40 mU of potato apyrase (Sigma, St Louis, MO) and then the total amounts of nucleotides and adducted nucleotides were analyzed by TLC.
In modified method I, 2 µl aliquots of the 32P-labeled samples were used for total nucleotide analysis with addition of 5.4 mU of apyrase. The rest was used for adduct analysis. The pH of the sample (13 µl) was adjusted to ~6 by addition of 1.3 µl of 0.3 N HCl and then incubated with 1.6 U of NP1 (Yamasa Shoyu Co. Ltd, Choshi, Japan) and 1 nmol ZnCl2 in 17 mM sodium citrate buffer (pH 5.7) at 37°C for 10 min. The pH was adjusted to 8.0 by adding 3 µl of 0.5 M Trizma base. Aliquots of 30 mU of PDEI (Worthington Biochemical Co.) were added to this reaction mixture and incubated for 30 min.
In modified method II, the amount of the total nucleotides was analyzed as described for modified method I. The pH of the remaining samples (13 µl) was adjusted to ~6 and then incubated with 30 U of PNK (New England Biolabs Inc., Beverly, MA) in 16 mM sodium citrate buffer (pH 5.7) for 30 min and another 10 U of PNK were added and further incubated for 30 min. Then, 1.1 U of NP1 and 1 nmol ZnCl2 were added to this reaction mixture and incubated for 10 min. The pH was adjusted to 8.0 and the mixture treated with PDEI as described for modified method I. These samples were subjected to adduct analysis by TLC. The adducts were resolved in the following TLC solvents: D1, 2.3 M NaH2PO4, pH 6.0; D3, 0.45 M lithium formate, 6.4 M urea, pH 3.5; D4, 0.7 M NaH2PO4, 8.5 M urea, pH 8.0, twice; D5, 1.7 M NaH2PO4, pH 6.0.
Radioactivity was measured with BioImaging Analyzer (BAS2000; Fuji Photo Film Co., Tokyo, Japan).
Adduct standards
The synthesized adduct standards, dGp-C8-IQ and dGp-N2-IQ (Turesky and Markovic, 1994) were labeled with [-32P]ATP and PNK to obtain the 3',5'-diphosphate forms. Adduct spots were extracted from the TLC sheet as described (Turesky and Markovic, 1994) and their 3',5'-diphosphate forms were separated by HPLC under the previously reported conditions (Turesky and Markovic, 1995). The retention times of the 3',5'-diphosphate form of dG-C8-IQ and dG-N2-IQ were 15.2 and 19.2 min, respectively. Aliquots of the extracted samples were digested with NP1 or PNK at pH 6 to obtain the 5'-phosphate form of the IQ–DNA adducts.
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Results and discussion |
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TopAbstractIntroductionMaterial and methodsResults and discussionReferences |
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Liver DNA base adducts of F344 rats were analyzed by the standard method, modified method I and modified method II, after feeding a diet containing 300 p.p.m. IQ for 12 weeks. Results were summarized in Table I
. When the standard method was used, IQ–DNA base adducts were detected as five spots (spots 1–5) as reported previously (Turesky and Markovic, 1994; Table I and Figure 2a). Spots 1 and 2 coincided with the 3',5'-diphosphate forms of authentic dG-C8-IQ (pdGp-C8-IQ) and dG-N2-IQ (pdGp-N2-IQ), respectively, shown by co-chromatography on a thin layer (data not shown) as reported previously (Snyderwine et al., 1988b; Turesky and Markovic, 1994). It has been reported that when DNA was reacted with hydroxyIQ in the presence of acetic anhydride and then digested to the mononucleoside with DNase I, PDEI and alkaline phosphatase, the major adduct was identified as dG-C8-IQ after isolation with HPLC analysis and structural determination (Snyderwine et al., 1988a). However, the radioactivity of pdGp-C8-IQ, namely spot 1, was lower compared with those of other spots under the conditions used in this study, as observed previously (Snyderwine et al., 1988b). The discrepancy seemed to be due to incomplete digestion of adducted oligonucleotides. It was previously demonstrated that the dG-N2-IQ oligonucleotide is sensitive to micrococcal nuclease plus spleen phosphodiesterase digestion, but the dG-C8-IQ oligonucleotide is relatively resistant (Turesky and Markovic, 1994).
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When modified method I was applied to the analysis of an IQ–DNA base adduct, four spots (spots 2 and 6–8) were detected (Table I
and Figure 2b). Spot 6, a major spot, coincided with the digestion product of pdGp-C8-IQ by NP1 in co-chromatography analysis (Figure 3a and b). Thus, spot 6 was determined as the 5'-phosphate form of dG-C8-IQ (pdG-C8-IQ). However, no new spots were detected when authentic pdGp-N2-IQ was digested with NP1 (data not shown). In contrast, when pdGp-N2-IQ was digested with PNK, with or without NP1, a new spot, suspected to be the 5'-phosphate form of dG-N2-IQ (pdG-N2-IQ), was detected (data not shown). Thus, it was concluded that the 3'-phosphate group of dG-N2-IQ was resistant to NP1 phosphatase and sensitive to PNK phosphatase.
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In co-chromatography of the spot 7 extract with the PNK digestion product of pdGp-N2-IQ, the relative intensity of spot 7 increased (Figure 3c and d
). Thus, spot 7 was identified as pdG-N2-IQ. Spot 8 in Figure 2 did not coincide with any spots related to dG-C8-IQ or dG-N2-IQ and it is concluded to be a new adduct of unknown structure.
In modified method I, the nucleotide of the dG-N2-IQ adduct was detected as two spots, a major 3',5'-diphosphate form (spot 2) and a lesser 5'-phosphate form (spot 7) (Figure 2b). Spot 7 seems to be derived from oligonucleotides in which dG-N2-IQ was present as the 5'-terminal nucleotide. In modified method II, PNK digestion removed the 3'-phosphate group of pdGp-N2-IQ which has been produced by micrococcal nuclease and spleen phosphodiesterase digestion followed by phosphorylation with PNK at pH 9.5. No signal from spot 2 was detected. Only, three spots, spots 6–8, were detected and these were the 5'-phosphate forms of dG-C8-IQ and dG-N2-IQ and an unknown nucleoside adduct, respectively.
As summarized in Table I, the total levels of IQ–DNA adducts were 88.0, 79.2 and 81.6 mol/107 nucleotides in the standard method, modified method I and modified method II, respectively. Recovery of adducts in modified method II compared with the standard method was ~90% and adduct levels in all three methods were almost the same. Turesky and Markovic (1994) reported that the amount of IQ-modified DNA estimated by 32P-post-labeling was almost the same as that by 14C-based measurement. Therefore, the recovery by modified method II may be almost the same as the recovery by 14C-based measurement. The detection limit of the original standard method was reported as 1 adduct in ~6x107 nucleotides (Gupta et al., 1982). In modified method I, where the background level is very low, the detection limit was 1 adduct in 108 nucleotides(data not shown). Since the background levels of modified method II were almost the same as modified method I, the same detection limit can be expected for method II.
It has been previously reported that the 3'-phosphate group of the dGp-C8 adduct is generally sensitive to NP1 (Beach and Gupta, 1992), but adducts of other forms are resistant. So far, we have analyzed the effectiveness of PNK only on pdGp-N2-IQ. It is possible that method II may be applicable to a wide variety of chemicals.
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Acknowledgments |
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This study was supported by a Grant-in Aid for Cancer Research from the Ministry of Health and Welfare of Japan.
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Notes |
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4 To whom correspondence should be addressed. Tel: +81 3 3542 2511; Fax: +81 3 3542 2530; Email: mnagao{at}ncc.go.jp
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References |
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Received on September 14, 1998; accepted on December 9, 1998.
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