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A Modified Immuno-Enriched 32P-Postlabeling Method for Analyzing the Malondialdehyde-Deoxyguanosine Adduct, 3-(2-Deoxy-β-D-erythro-pentofuranosyl)pyrimido[1,2-r]purin-10(3H)one in Human Tissue Samples Xin Sun, Jagadeesan Nair,* and Helmut Bartsch Division of Toxicology and Cancer Risk Factors, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany Received September 9, 2003
The malondialdehyde-modified DNA adduct, 3-(2-deoxy-β-D-erythro-pentofuranosyl)pyrimido[1,2-R]purin-10(3H)one (M1dG) has been detected in human tissues and is considered to be a promising biomarker for estimating lipid peroxidation-induced DNA damage. With the aim to analyze the M1dG in small amounts of DNA (220 TBq was purchased from Hartman Analytic (Braunschweig, Germany). Polyethyleneimine (PEI) was obtained from J. T. Backer, Inc. (Phillipsburg, NJ). The monoclonal antibody D 10A1 developed at Vanderbilt University (Nashville, TN) was kindly provided by Dr. L. J. Marnett. O4-etT-3′-MP (I.S.) was synthesized as reported earlier (17). DNA was isolated using Qiagen genomic-tips (no. 13343, Hilden, Germany) according to the manufacturer’s protocol with modification (17). The DNA was extracted from human liver and TWBC derived from buffy coat directly; human breast tissue DNA was extracted from isolated epithelial cells. The tissue was cut into small pieces and was incubated with methylene blue solution in PBS. The epithelial components that were stained blue were cut out with a sharp scalpel and a pair of scissors and were incubated in the mixture of collagenase (2 mg/mL in HBSS) and hyaluronidase (2 mg/mL in HBSS) at 37 °C for 3 h. The resulting suspension was passed through a nylon mesh (100 µm) and centrifuged. The pellet was stored at -20 °C until the extraction of DNA. All solvents used were of HPLC grade. HPLC analysis of the [32P]M1dG-5′-MP was performed with a reverse phase HPLC system, model 1090 from Hewlett-Packard (Waldbronn, Germany) with a Prodigy ODS column (Phenomenex, 250 mm × 4.6 mm, 5 µm), which was connected to an EG&G Berthold (Bad Wildbad, Germany) radioactivity detector with cell model Z2004. Normal nucleotides were quantitated on HPLC diode array detector (HP 1050) equipped with a ODS column (Bischoff chromatography, Leonberg, Germany). Synthesis of the 3′- and 5′-Monophosphate (MP) of M1dG Standards. The 3′- and 5′-MP of M1dG standards were synthesized following the procedures described previously with modification (3, 5) and characterized by mass spectrometry. The absolute amount of M1dG 3′- and 5′-MP was determined from its reported molar extinction coefficient (λ254 nm ) 7.81 × 104 M-1 cm-1) (5).
Chem. Res. Toxicol., Vol. 17, No. 2, 2004 269 Chemical Reaction of MDA with Calf Thymus DNA. To obtain different levels of MDA-modified DNA, 1 mg of purified calf thymus DNA in distilled water was reacted with different amounts of MDA from hydrolysis of TEP at 37 °C for 10 days (3, 5). After incubation, the DNA was precipitated by adding 1/10 vol of 5.0 M NaCl and 2.0 vol of cold ethanol. The levels of M1dG present in MDA modified calf thymus DNA were analyzed by IEP-HPLC. Preparation of Immunoaffinity Columns for M1dG Enrichment. The immunoaffinity columns were prepared based on the manufacturer’s instructions (Pharmacia, Uppsala, Sweden) with minor modification. The monoclonal antibody D 10A1, which was a high affinity and specificity for the M1dG (developed in Vanderbilt University), was used. One milligram of purified antibody protein was coupled with 1.0 g of the CNBractivated Sepharose 4B (Pharmacia). The coupled gel was washed thoroughly with 0.1 M Tris-HCl buffer (pH 8.0) and then washed with 0.1 M acetate buffer (pH 4.0) containing 0.5 M NaCl followed by a wash with 0.1 M Tris-HCl buffer (pH 8.0) containing 0.5 M NaCl. The gel was then suspended into 5.0 mL of PBS buffer containing 0.02% (w/v) sodium azide and stored at 4 °C. One milliliter of the gel suspension was placed into a minisorp polythylene immunotube (Nunc), and the immunoaffinity columns were washed extensively with PBS containing 0.02% (w/v) sodium azide and stored at 4 °C before use. DNA Digestion and Immunoaffinity Enrichment of M1dG. The dried DNA (∼10 µg) from liver and breast tissues and human buffy coat DNA samples were enzymatically hydrolyzed to the corresponding 2′-deoxyribonucleoside 3′-MPs at 37 °C for 4 h with 100 µL of Tris buffer (100 mM Tris and 20 mM CaCl2, pH 6.8) by the addition of 4.0 µL of micrococcal endonuclease (0.2 units/µL) and 4.0 µL of SPD (0.025 units/µL) (18, 19). The immunoaffinity columns were sequentially washed with 10 mL of water, 25 mL of methanol/water (1:1), 10 mL of water, and 10 mL of PBS before the samples were loaded. Aliquots (280 µL) from each DNA hydrolysate were loaded onto immunoaffinity columns. Ten femtomoles of M1dG-5′-MP standard was added to each sample for the purpose to improve the recovery of the M1dG. The columns were washed with 20 mL of cold PBS and 10 mL of cold water at 4 °C in order to remove the bulk of the normal nucleotides. The columns were brought to room temperature (20 ( 2 °C), and the M1dG was eluted with 2.5 mL of methanol/water (1:1; v/v) twice. The eluate from immunoaffinity columns was pooled and concentrated by vacuum centrifugation. 32P-Postlabeling and HPLC Analysis of M dG. Internal 1 standard (1 fmol of O4-etT-3′-MP) and 4 µL of a kinase buffer (125 mM Tris-HCl, 25 mM MgCl2, and 25 mM DTT, pH 6.8) were added to the dried DNA samples. After 2 µL of [γ-32P]ATP (10 µCi) and 2 µL of T4 PNK (10 units/µL) were added, the mixture was incubated at 37 °C for 60 min. During this postlabeling step, the dual enzymatic properties of PNK were utilized (20), where the 3′-MP has been converted to 3′,5′bisphosphate by the kinase activity of PNK, and subsequently converted to the labeled 5′-MP by the phosphatase activity. PEI minicolumns were prepared by filling 5.0 mg of PEI into a 200 µL pipet tip closed with a small fritt (17). The PEI minicolumns were washed with 50 µL of 20 mM NaH2PO4 buffer (pH 4.5) containing 30% methanol before the samples were loaded, the labeled samples were pipetted onto the top of the PEI minicolumns, and the [32P]M1dG-5′-MP was eluted with same buffer by centrifugation at 10 000 rpm for 5 min at 4 °C, whereas excess ATP remained bound to PEI. The eluate was injected into a reverse HPLC with a Prodigy ODS column (Phenomenex, 4.6 mm × 250 mm, 5 µm), 100 pmol of the nonradioactive M1dG-5′-MP standard was added to each sample as a UV marker to verify that radioactive chromatographic peak (retention time) contained the [32P] M1dG-5′-MP, and the mixture was eluted with using a linear gradient between 20 mM NaH2PO4 buffer (pH 4.5) (A) and methanol (B) as follows: 0-30
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Figure 2. Linear relationship of different amounts of M1dG-3′-MP.
32P-labeling
efficiency with
min, 100% A; 30-60 min, 0-20% of B in A; 60-70 min, 2045% of B in A; 70-80 min, 45-55% of B in A; 80-85 min, 55100% of B in A; 85-90 min, 100% of B isocratically; and 90-92 min, 100% A. The HPLC flow rate was 0.8 mL/min. The radioactive peaks containing [32P]M1dG-5′-MP and internal standard ([32P]O4-etT-5′-MP) were collected, and the radioactivity was determined by liquid scintillation counting. An external standard, a known amount of M1dG-3′-MP standard (1 fmol), was also labeled in parallel. The recovery of M1dG was determined using different amounts of M1dG-3′-MP standards. Background counts were determined from appropriate blank area on the HPLC and collected. The amount of M1dG was calculated as (F2/F1), whereby F1 ) [cpm of the peak of [32P]M1dG-5′-MP in standard]/[cpm of the peak of [32P]O4-etT-5′-MP in standard] and F2 ) [cpm of the peak of [32P]M1dG-5′-MP in sample]/[cpm of the peak of [32P]O4-etT-5′-MP in sample]; 1 fmol of O4-etT-3′-MP was added to each sample as an internal standard.
Sun et al.
Figure 3. Linear relationship of internal standard for the quantification of M1dG using a fixed amount of O4-etT-3′-MP (1 fmol).
Results Synthesis of the 3′- and 5′-MP of M1dG Standards. The 3′- and 5′-MP of M1dG standards were synthesized and used to characterize unequivocally the 32P-postlabeling reaction products. Analysis of M1dG. The monoclonal antibody D 10A1 was used for the immunoenrichment of M1dG from DNA hydrolysates. The reproducible recovery of M1dG was found to be 70 ( 20% when 1 fmol of M1dG-3′-MP standard was loaded onto the immunoaffinity columns. O4-etT-3′-MP was added to all of the samples as an internal standard for quantitation purposes. For verification of the HPLC retention time, 100 pmol of cold M1dG-5′-MP standard was added to the reaction mixture as a UV marker. The [32P]M1dG-5′-MP eluted at 55.5 min, and [32P]O4-etT-5′-MP eluted at 68.5 min. The labeling efficiency of M1dG was found to be 80 ( 20%. A linear correlation between substrate (amount of M1dG-3′-MP) and response (radioactivity of [32P]M1dG-5′-MP) was obtained by 32P-postlabeling of 100, 200, 1000, and 5000 amol of the M1dG-3′-MP standard (Figure 2). Similarly, a linear relationship between amounts of M1dG-3′-MP standard and ratio of the M1dG-3′-MP and a fixed amount of O4-etT-3′-MP (I.S.) was observed (Figure 3). The typical reverse phase HPLC profiles derived from M1dG-3′-MP standard, untreated calf thymus DNA, MDA-modified calf thymus DNA, and human buffy coat DNA samples are shown in Figure 4A-D, respectively; the HPLC run
Figure 4. Representative HPLC profiles of M1dG analyses: (A) M1dG-3′-MP and O4-etT-3′-MP standards; (B) unmodified calf thymus DNA; (C) MDA-modified calf thymus DNA; (D) DNA sample of human TWBC; and (E) unlabeled M1dG-5′-MP standard. The retention times of normal nucleotides under the chromatographic conditions for dC, dG, dT, and dA were 9.5, 22.0, 32.5, and 46.5 min, respectively.
of the nonradioactive M1dG-5′-MP standard, detected at 330 nm, is shown in Figure 4E. The HPLC retention time and appearance of the radioactive chromatographic peaks are depicted in Figure 4A-D. The M1dG was purified using a specific monoclonal antibody and coeluted with a nonradioactive M1dG-5′-MP standard (Figure 4E), as compared with the retention time of an internal standard, thus [32P]M1dG-5′-MP could be unequivocally determined. The unmodified calf thymus DNA yielded only background radioactivity (Figure 4B).
Malondialdehyde-Deoxyguanosine Adduct in Human Tissue Table 1. Levels of M1dG Detected in DNA of Asymptomatic Human Tissue Samples tissue analyzed
M1dG/108 dG mean ( SD (range)
liver (n ) 2) 25.1 ( 12.9 (15.9-32.4) breast (n ) 4) 7.3 ( 6.3 (1.6-15.2) TWBC (n ) 26) 52.5 ( 53.5 (1.4-144.2)
M1dG/108 nucleotides mean ( SD (range) 5.2 ( 2.8 (3.2-7.2) 1.7 ( 1.3 (0.3-3.2) 9.5 ( 8.6 (0.3-30.1)
IEP-HPLC Analysis of the M1dG in DNA of Human Samples. To validate the modified IEP-HPLC method, the M1dG levels in DNA of human liver (n ) 2), breast tissues (n ) 4), and buffy coat (n ) 26) were analyzed. The results are summarized in Table 1 [M1dG per 108 nucleotides {mean ( SD (range)} determined in human liver, breast tissues, and buffy coat were 5.2 ( 2.8 (3.2-7.2); 1.7 ( 1.3 (0.3-3.2); and 9.5 ( 8.6 (0.330.1), respectively]. For each analysis, an external standard was measured in parallel with the biological samples; the external standard was a fixed amount of M1dG-3′MP standard that closely matched the modification level expected in each DNA sample. The profiles obtained from IEP-HPLC (Figure 4A-E) showed that the chromatographic peak of [32P]M1dG-5′-MP can be efficiently separated from interfering peaks. These studies clearly demonstrate that M1dG can be quantitated from samples in amounts as low as 5-10 µg in DNA.
Discussion The present method for M1dG was found to be sensitive and specific; the analyses of M1dG could be achieved from ∼10 µg DNA, and the adducts were separated from normal nucleotides using a specific antibody followed by resolution on HPLC. The method has been successfully applied for analyzing small amounts of DNA isolated from human tissues and cells. The immunoaffinity step and the chromatographic resolutions were well-characterized by using synthetic standards. Among several nucleotides tested, O4-etT-3′-MP was found to be the best reliable internal standard in terms of both labeling efficiency and chromatographic resolution from interfering peaks. We have observed that recovery of M1dG-3′MP from the immunoaffinity column was poor when the amount loaded was less than 5 fmol. This problem was resolved by adding cold M1dG-5′-MP along with the DNA hydrolysate. Attempts to resolve M1dG on PEI cellulose plates failed due to decomposition of the adduct during the chromatographic separation. A single labeling step at pH 6.8 to yield M1dG-5′-MP reduced the adduct loss that might have incurred in previous 32P-postlabeling protocols, which converted M1dG into its bisphosphate and then used nuclease P1 to remove 3′-MP (see Materials and Methods). Table 2 compares M1dG levels found by the current method with those detected by other methods reported in the literature. Earlier 32P-postlabeling and GC-MS methods reported 4-18 times higher M1dG levels in human breast, liver, and TWBC (2, 6), whereas a recent LC-MS-MS method failed to detect M1dG in four human livers even when using 100 µg of DNA (10). M1dG levels obtained in TWBC by our current method was comparable with the levels determined by immunoenriched GC-MS, although this method required 1000 µg of DNA (8). M1dG is a biologically important DNA lesion with mutagenic properties (13-16) and was shown to be repaired by nucleotide excision repair and not by base
Chem. Res. Toxicol., Vol. 17, No. 2, 2004 271 Table 2. M1dG Levels Detected by Different Methods in DNA of Human Liver, Breast, and TWBC Samples as Reported in the Literature and Comparison with Our IEP-HPLC Methoda tissue/cells detection amount of analyzed method DNA (µg) TWBC breast liver liver leukocyte WBC liver liver breast TWBC
PL-HPLC LC-MS-MS GC-MS GC-MS ISB PL-HPLC IEP-HPLC IEP-HPLC IEP-HPLC
10 10 100 NR 1000 1.0 10 5-10 5-10 5-10
mean adduct level (M1dG/ 108 nts)
ref
26 (n ) 26) 2 30 (n ) 7) 2 ND (n ) 4) 10 90 (n ) 6) 6 6.2 (n ) 10) 8 5.6-9.5 (n ) 8) 11 14 (n ) 10) 5 5.2 (n ) 2) current study 1.7 (n ) 4) current study 9.5 (n ) 26) current study
a PL-HPLC, 32P-postlabeling/HPLC; ISB, immunoslot; ND, not detected; and NR, not reported.
excision repair (21). Precise determination of steady state levels of this adduct in human samples will facilitate investigations on its formation and repair and its role in human diseases caused by chronic oxidative stress determined. Besides, our method will help in comparing relative adduct levels formed by MDA and by 4-hydroxy2-nonenal that yields etheno-DNA adducts (18, 19). These two are the most abundant DNA reactive aldehydes generated due to oxidative stress and lipid peroxidation. The current method facilitates such simultaneous analysis: M1dG could be serially isolated from DNA, hydrolyzed by micrococcal endonuclease/SPD, and analyzed along with etheno adducts, using immunoaffinity columns in sequence, each carrying an adduct specific antibody. Such analyses are underway in our laboratory.
Acknowledgment. Dr. Xin Sun was a recipient of a visiting scientist fellowship awarded by the DKFZ in 2001. We thank Dr. Lawrence J. Marnett (Vanderbilt University, Nashville, TN) for generously providing the monoclonal antibody D 10A1. This work was supported by EU Contract QLK4-2000-00286.
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