A Modified Immuno-Enriched 32P-Postlabeling Methodfor Analyzing the Malondialdehyde-Deoxyguanosine

Adduct, 3-(2-Deoxy-â-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-â-D-erythro-pentofuranosyl)pyrimido-[1,2-R]purin-10(3H)one (M1dG) has been detected in human tissues and is considered to be apromising biomarker for estimating lipid peroxidation-induced DNA damage. With the aim toanalyze the M1dG in small amounts of DNA (<10 µg) and to improve the sensitivity, we havedeveloped an immuno-enriched 32P-postlabeling HPLC method. The main modifications includedthe following steps: (i) an optimization of the immunoenrichment conditions using a monoclonalantibody (MAb D 10A1), (ii) a single labeling step of the purified M1dG 3′-monophosphate toits 5′-monophosphate at pH 6.8, (iii) the addition of O4-ethylthymidine 3′-monophosphate asan internal standard, and (iv) a prepurification of the labeled adduct on a polyethyleneimineminicolumn before HPLC analysis. With this protocol, the percent recovery of M1dG was foundto be ∼70 ( 20; the detection limit in biological samples was ∼200 amol M1dG from 10 µg ofDNA, corresponding to 6 adducts/109 nucleotides. In conclusion, our modified method shows ahigh sensitivity and specificity; when applied to human breast and liver tissue samples,background levels of the M1dG could be reproducibly detected. This ultrasensitive detectionmethod is thus suitable for applications in human biomonitoring and molecular epidemiologystudies.

Introduction

Malondialdehyde (MDA) is one of the major reactionproducts derived from lipid peroxidation. MDA can alsoarise via the cyclo-oxygenase pathway of arachidonic acidmetabolism and C-4′ oxidation of DNA-inducing basepropenals (Figure 1) (1). A major MDA-modified DNAadduct, identified as 3-(2-deoxy-â-D-erythro-pentofura-nosyl)pyrimido[1,2-R]purin-10(3H)one (M1dG), has beendetected in human liver, breast, and total white bloodcells (TWBC) (2-12). It has also been reported that thisadduct is a mutagenic lesion in Escherichia coli (13, 14)and human cells (15, 16). The endogenous formation ofM1dG in human samples and the data on its genotoxicityand mutagenicity suggest that M1dG may play a signifi-cant role in endogenous DNA damage and human car-cinogenesis. Therefore, it is important to estimate cor-rectly background levels of M1dG in different humantissues and monitor any increase in adduct level inrelation to increased oxidative stress and lipid peroxi-dation. For this purpose, development of a sensitive andreliable analytical method to evaluate the backgroundof M1dG levels in small amounts of DNA from humansamples is a prerequisite for risk assessment and mo-lecular epidemiological studies.

Marnett and co-workers (7) have developed a mono-clonal antibody (D 10A1) to the M1dG and recently

reported a development of a gas chromatography/electroncapture-negative chemical ionization/mass spectrometricmethod (GC/EC-NCI/MS) for the analysis of M1dG inhuman leukocyte samples (8). Analyses of M1dG havealso been performed by using 32P-postlabeling, GC-MS,

* To whom correspondence should be addressed. Tel: +49 6221423306. Fax: +49 6221 423359. E-mail: J.Nair@dkfz.de.

Figure 1. Scheme for the formation of M1dG. ROS, reactiveoxygen species; PUFA, polyunsaturated fatty acid; COX-2,cyclooxygenase-2; and PGH2, prostaglandin H2.

268 Chem. Res. Toxicol. 2004, 17, 268-272

10.1021/tx034183p CCC: $27.50 © 2004 American Chemical SocietyPublished on Web 01/30/2004

and LC-MS-MS and immunoslot blot methods (2-12). GCand LC-MS methods used for M1dG detection are specificbut often require relatively large amounts of DNA. 32P-postlabeling methods reported earlier did not considerto use an internal standard for correcting the labelingefficiency. The immunoslot blot method is sensitive anddetects M1dG on whole DNA without adduct resolution.In this paper, we report a modified immunoenriched 32P-postlabeling/HPLC (IEP-HPLC) method that can ef-ficiently separate and accurately quantify M1dG. Tovalidate this IEP-HPLC method, M1dG levels weredetermined in small amounts of DNA (∼10 µg) isolatedfrom human breast, liver, and buffy coat samples andthese results are also presented.

Materials and Methods

Caution: MDA has been determined to be highly cytotoxic,mutagenic, and carcinogenic in animal bioassays. Therefore,appropriate safety procedures should be followed when workingwith this compound. [γ-32P]ATP is a hazardous and radioactivecompound and should be handled with sufficient protection andshielding.

MDA-bis-diethyl acetal (1,1,3,3-tetraethoxypropane, TEP)was obtained from Sigma Chemical Co. (St. Louis, MO). Calfthymus DNA (sodium salt), 2′-deoxyguanosine-3′-monophos-phate (ammonium salt), 2′-deoxyguanosine-5′-monophosphate(ammonium salt), micrococcal nuclease, and Hank’s balancedsalt solution (HBSS) were purchased from Sigma-Aldrich(Taufkirchen, Germany). Collagenase (type IV) and hyalu-ronidase (type I-S) were obtained from Sigma-Aldrich (Stein-heim, Germany). Spleen phosphodiesterase (SPD) was obtainedfrom Worthington Biochemical Corp. (Lakewood, NJ), andcloned T4 polynucleotide kinase (PNK) was obtained from MBI/Fermentas (St. Leon-Rot, Germany). [γ-32P]ATP with a specificactivity of >220 TBq was purchased from Hartman Analytic(Braunschweig, Germany). Polyethyleneimine (PEI) was ob-tained from J. T. Backer, Inc. (Phillipsburg, NJ). The monoclonalantibody 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 withmodification (17). The DNA was extracted from human liver andTWBC derived from buffy coat directly; human breast tissueDNA was extracted from isolated epithelial cells. The tissue wascut into small pieces and was incubated with methylene bluesolution in PBS. The epithelial components that were stainedblue were cut out with a sharp scalpel and a pair of scissorsand were incubated in the mixture of collagenase (2 mg/mL inHBSS) and hyaluronidase (2 mg/mL in HBSS) at 37 °C for 3 h.The resulting suspension was passed through a nylon mesh (100µm) and centrifuged. The pellet was stored at -20 °C until theextraction of DNA.

All solvents used were of HPLC grade. HPLC analysis of the[32P]M1dG-5′-MP was performed with a reverse phase HPLCsystem, model 1090 from Hewlett-Packard (Waldbronn, Ger-many) with a Prodigy ODS column (Phenomenex, 250 mm ×4.6 mm, 5 µm), which was connected to an EG&G Berthold (BadWildbad, Germany) radioactivity detector with cell model Z200-4. Normal nucleotides were quantitated on HPLC diode arraydetector (HP 1050) equipped with a ODS column (Bischoffchromatography, Leonberg, Germany).

Synthesis of the 3′- and 5′-Monophosphate (MP) ofM1dG Standards. The 3′- and 5′-MP of M1dG standards weresynthesized following the procedures described previously withmodification (3, 5) and characterized by mass spectrometry. Theabsolute amount of M1dG 3′- and 5′-MP was determined fromits reported molar extinction coefficient (λ254 nm ) 7.81 × 104

M-1 cm-1) (5).

Chemical Reaction of MDA with Calf Thymus DNA. Toobtain different levels of MDA-modified DNA, 1 mg of purifiedcalf thymus DNA in distilled water was reacted with differentamounts of MDA from hydrolysis of TEP at 37 °C for 10 days(3, 5). After incubation, the DNA was precipitated by adding1/10 vol of 5.0 M NaCl and 2.0 vol of cold ethanol. The levels ofM1dG present in MDA modified calf thymus DNA were analyzedby IEP-HPLC.

Preparation of Immunoaffinity Columns for M1dGEnrichment. The immunoaffinity columns were preparedbased on the manufacturer’s instructions (Pharmacia, Uppsala,Sweden) with minor modification. The monoclonal antibody D10A1, which was a high affinity and specificity for the M1dG(developed in Vanderbilt University), was used. One milligramof purified antibody protein was coupled with 1.0 g of the CNBr-activated Sepharose 4B (Pharmacia). The coupled gel waswashed thoroughly with 0.1 M Tris-HCl buffer (pH 8.0) and thenwashed with 0.1 M acetate buffer (pH 4.0) containing 0.5 MNaCl 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 5.0mL of PBS buffer containing 0.02% (w/v) sodium azide andstored at 4 °C. One milliliter of the gel suspension was placedinto a minisorp polythylene immunotube (Nunc), and theimmunoaffinity columns were washed extensively with PBScontaining 0.02% (w/v) sodium azide and stored at 4 °C beforeuse.

DNA Digestion and Immunoaffinity Enrichment ofM1dG. The dried DNA (∼10 µg) from liver and breast tissuesand human buffy coat DNA samples were enzymatically hy-drolyzed to the corresponding 2′-deoxyribonucleoside 3′-MPs at37 °C for 4 h with 100 µL of Tris buffer (100 mM Tris and 20mM CaCl2, pH 6.8) by the addition of 4.0 µL of micrococcalendonuclease (0.2 units/µL) and 4.0 µL of SPD (0.025 units/µL)(18, 19).

The immunoaffinity columns were sequentially washed with10 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 (280µL) from each DNA hydrolysate were loaded onto immunoaf-finity columns. Ten femtomoles of M1dG-5′-MP standard wasadded to each sample for the purpose to improve the recoveryof the M1dG. The columns were washed with 20 mL of cold PBSand 10 mL of cold water at 4 °C in order to remove the bulk ofthe normal nucleotides. The columns were brought to roomtemperature (20 ( 2 °C), and the M1dG was eluted with 2.5mL of methanol/water (1:1; v/v) twice. The eluate from immu-noaffinity columns was pooled and concentrated by vacuumcentrifugation.

32P-Postlabeling and HPLC Analysis of M1dG. Internalstandard (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, themixture was incubated at 37 °C for 60 min. During thispostlabeling step, the dual enzymatic properties of PNK wereutilized (20), where the 3′-MP has been converted to 3′,5′-bisphosphate by the kinase activity of PNK, and subsequentlyconverted to the labeled 5′-MP by the phosphatase activity.PEI minicolumns were prepared by filling 5.0 mg of PEI into a200 µL pipet tip closed with a small fritt (17). The PEIminicolumns were washed with 50 µL of 20 mM NaH2PO4 buffer(pH 4.5) containing 30% methanol before the samples wereloaded, the labeled samples were pipetted onto the top of thePEI minicolumns, and the [32P]M1dG-5′-MP was eluted withsame buffer by centrifugation at 10 000 rpm for 5 min at 4 °C,whereas excess ATP remained bound to PEI. The eluate wasinjected into a reverse HPLC with a Prodigy ODS column(Phenomenex, 4.6 mm × 250 mm, 5 µm), 100 pmol of thenonradioactive M1dG-5′-MP standard was added to each sampleas a UV marker to verify that radioactive chromatographic peak(retention time) contained the [32P] M1dG-5′-MP, and themixture was eluted with using a linear gradient between 20 mMNaH2PO4 buffer (pH 4.5) (A) and methanol (B) as follows: 0-30

Malondialdehyde-Deoxyguanosine Adduct in Human Tissue Chem. Res. Toxicol., Vol. 17, No. 2, 2004 269

min, 100% A; 30-60 min, 0-20% of B in A; 60-70 min, 20-45% of B in A; 70-80 min, 45-55% of B in A; 80-85 min, 55-100% of B in A; 85-90 min, 100% of B isocratically; and 90-92min, 100% A. The HPLC flow rate was 0.8 mL/min. Theradioactive peaks containing [32P]M1dG-5′-MP and internalstandard ([32P]O4-etT-5′-MP) were collected, and the radioactiv-ity was determined by liquid scintillation counting. An externalstandard, a known amount of M1dG-3′-MP standard (1 fmol),was also labeled in parallel. The recovery of M1dG was deter-mined using different amounts of M1dG-3′-MP standards.Background counts were determined from appropriate blankarea on the HPLC and collected. The amount of M1dG wascalculated as (F2/F1), whereby F1 ) [cpm of the peak of [32P]M1-dG-5′-MP in standard]/[cpm of the peak of [32P]O4-etT-5′-MP instandard] and F2 ) [cpm of the peak of [32P]M1dG-5′-MP insample]/[cpm of the peak of [32P]O4-etT-5′-MP in sample]; 1 fmolof O4-etT-3′-MP was added to each sample as an internalstandard.

Results

Synthesis of the 3′- and 5′-MP of M1dG Standards.The 3′- and 5′-MP of M1dG standards were synthesizedand used to characterize unequivocally the 32P-postla-beling reaction products.

Analysis of M1dG. The monoclonal antibody D 10A1was used for the immunoenrichment of M1dG from DNAhydrolysates. The reproducible recovery of M1dG wasfound to be 70 ( 20% when 1 fmol of M1dG-3′-MPstandard was loaded onto the immunoaffinity columns.

O4-etT-3′-MP was added to all of the samples as aninternal standard for quantitation purposes. For verifica-tion of the HPLC retention time, 100 pmol of cold M1-dG-5′-MP standard was added to the reaction mixtureas 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 labelingefficiency of M1dG was found to be 80 ( 20%. A linearcorrelation between substrate (amount of M1dG-3′-MP)and response (radioactivity of [32P]M1dG-5′-MP) wasobtained by 32P-postlabeling of 100, 200, 1000, and 5000amol of the M1dG-3′-MP standard (Figure 2). Similarly,a linear relationship between amounts of M1dG-3′-MPstandard and ratio of the M1dG-3′-MP and a fixed amountof O4-etT-3′-MP (I.S.) was observed (Figure 3). The typicalreverse phase HPLC profiles derived from M1dG-3′-MPstandard, untreated calf thymus DNA, MDA-modifiedcalf thymus DNA, and human buffy coat DNA samplesare shown in Figure 4A-D, respectively; the HPLC run

of the nonradioactive M1dG-5′-MP standard, detected at330 nm, is shown in Figure 4E. The HPLC retention timeand appearance of the radioactive chromatographic peaksare depicted in Figure 4A-D. The M1dG was purifiedusing a specific monoclonal antibody and coeluted witha nonradioactive M1dG-5′-MP standard (Figure 4E), ascompared with the retention time of an internal standard,thus [32P]M1dG-5′-MP could be unequivocally determined.The unmodified calf thymus DNA yielded only back-ground radioactivity (Figure 4B).

Figure 2. Linear relationship of 32P-labeling efficiency withdifferent amounts of M1dG-3′-MP. Figure 3. Linear relationship of internal standard for the

quantification of M1dG using a fixed amount of O4-etT-3′-MP(1 fmol).

Figure 4. Representative HPLC profiles of M1dG analyses: (A)M1dG-3′-MP and O4-etT-3′-MP standards; (B) unmodified calfthymus DNA; (C) MDA-modified calf thymus DNA; (D) DNAsample of human TWBC; and (E) unlabeled M1dG-5′-MPstandard. The retention times of normal nucleotides under thechromatographic conditions for dC, dG, dT, and dA were 9.5,22.0, 32.5, and 46.5 min, respectively.

270 Chem. Res. Toxicol., Vol. 17, No. 2, 2004 Sun et al.

IEP-HPLC Analysis of the M1dG in DNA of Hu-man Samples. To validate the modified IEP-HPLCmethod, the M1dG levels in DNA of human liver (n ) 2),breast tissues (n ) 4), and buffy coat (n ) 26) wereanalyzed. The results are summarized in Table 1 [M1dGper 108 nucleotides {mean ( SD (range)} determined inhuman 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 (0.3-30.1), respectively]. For each analysis, an external stan-dard 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 levelexpected in each DNA sample. The profiles obtained fromIEP-HPLC (Figure 4A-E) showed that the chromato-graphic peak of [32P]M1dG-5′-MP can be efficiently sepa-rated from interfering peaks. These studies clearlydemonstrate that M1dG can be quantitated from samplesin amounts as low as 5-10 µg in DNA.

Discussion

The present method for M1dG was found to be sensitiveand specific; the analyses of M1dG could be achieved from∼10 µg DNA, and the adducts were separated fromnormal nucleotides using a specific antibody followed byresolution on HPLC. The method has been successfullyapplied for analyzing small amounts of DNA isolatedfrom human tissues and cells. The immunoaffinity stepand the chromatographic resolutions were well-charac-terized by using synthetic standards. Among severalnucleotides tested, O4-etT-3′-MP was found to be the bestreliable internal standard in terms of both labelingefficiency and chromatographic resolution from interfer-ing peaks. We have observed that recovery of M1dG-3′-MP from the immunoaffinity column was poor when theamount loaded was less than 5 fmol. This problem wasresolved by adding cold M1dG-5′-MP along with the DNAhydrolysate. Attempts to resolve M1dG on PEI celluloseplates failed due to decomposition of the adduct duringthe chromatographic separation. A single labeling stepat pH 6.8 to yield M1dG-5′-MP reduced the adduct lossthat might have incurred in previous 32P-postlabelingprotocols, which converted M1dG into its bisphosphateand then used nuclease P1 to remove 3′-MP (see Materi-als and Methods). Table 2 compares M1dG levels foundby the current method with those detected by othermethods reported in the literature. Earlier 32P-postla-beling and GC-MS methods reported 4-18 times higherM1dG levels in human breast, liver, and TWBC (2, 6),whereas a recent LC-MS-MS method failed to detect M1-dG in four human livers even when using 100 µg of DNA(10). M1dG levels obtained in TWBC by our currentmethod was comparable with the levels determined byimmunoenriched GC-MS, although this method required1000 µg of DNA (8).

M1dG is a biologically important DNA lesion withmutagenic properties (13-16) and was shown to berepaired by nucleotide excision repair and not by base

excision repair (21). Precise determination of steady statelevels of this adduct in human samples will facilitateinvestigations on its formation and repair and its role inhuman diseases caused by chronic oxidative stress de-termined. Besides, our method will help in comparingrelative adduct levels formed by MDA and by 4-hydroxy-2-nonenal that yields etheno-DNA adducts (18, 19).These two are the most abundant DNA reactive alde-hydes generated due to oxidative stress and lipid per-oxidation. The current method facilitates such simulta-neous analysis: M1dG could be serially isolated fromDNA, hydrolyzed by micrococcal endonuclease/SPD, andanalyzed along with etheno adducts, using immunoaf-finity columns in sequence, each carrying an adductspecific antibody. Such analyses are underway in ourlaboratory.

Acknowledgment. Dr. Xin Sun was a recipient of avisiting scientist fellowship awarded by the DKFZ in2001. We thank Dr. Lawrence J. Marnett (VanderbiltUniversity, Nashville, TN) for generously providing themonoclonal antibody D 10A1. This work was supportedby EU Contract QLK4-2000-00286.

References

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Table 1. Levels of M1dG Detected in DNA ofAsymptomatic Human Tissue Samples

tissue analyzedM1dG/108 dG mean

( SD (range)M1dG/108 nucleotidesmean ( SD (range)

liver (n ) 2) 25.1 ( 12.9 (15.9-32.4) 5.2 ( 2.8 (3.2-7.2)breast (n ) 4) 7.3 ( 6.3 (1.6-15.2) 1.7 ( 1.3 (0.3-3.2)TWBC (n ) 26) 52.5 ( 53.5 (1.4-144.2) 9.5 ( 8.6 (0.3-30.1)

Table 2. M1dG Levels Detected by Different Methods inDNA of Human Liver, Breast, and TWBC Samples as

Reported in the Literature and Comparison with OurIEP-HPLC Methoda

tissue/cellsanalyzed

detectionmethod

amount ofDNA (µg)

mean adductlevel (M1dG/

108 nts) ref

TWBC PL-HPLC 10 26 (n ) 26) 2breast 10 30 (n ) 7) 2liver LC-MS-MS 100 ND (n ) 4) 10liver GC-MS NR 90 (n ) 6) 6leukocyte GC-MS 1000 6.2 (n ) 10) 8WBC ISB 1.0 5.6-9.5 (n ) 8) 11liver PL-HPLC 10 14 (n ) 10) 5liver IEP-HPLC 5-10 5.2 (n ) 2) current studybreast IEP-HPLC 5-10 1.7 (n ) 4) current studyTWBC IEP-HPLC 5-10 9.5 (n ) 26) current study

a PL-HPLC, 32P-postlabeling/HPLC; ISB, immunoslot; ND, notdetected; and NR, not reported.

Malondialdehyde-Deoxyguanosine Adduct in Human Tissue Chem. Res. Toxicol., Vol. 17, No. 2, 2004 271

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