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Chem. Res. Toxicol. 1997, 10, 172-180
Development of Monoclonal Antibodies to the Malondialdehyde-Deoxyguanosine Adduct, Pyrimidopurinone1 Cynthia L. Sevilla,†,2 Norma H. Mahle,†,3 Naomi Eliezer,† Adam Uzieblo,†,4 Shawn M. O’Hara,‡,5 Munetaka Nokubo,§,| Ryan Miller,§ Carol A. Rouzer,§,6 and Lawrence J. Marnett*,‡ Proteins International, 1858 Star Batt Drive, Rochester Hills, Michigan 48309, Department of Chemistry, Wayne State University, Detroit, Michigan 48202, and Department of Biochemistry, Center in Molecular Toxicology, The Vanderbilt Cancer Center, School of Medicine, Vanderbilt University, Nashville, Tennessee 37232 Received July 17, 1996X
Malondialdehyde (MDA), an endogenous product of lipid peroxidation and prostaglandin biosynthesis, is mutagenic in bacterial and mammalian cells and carcinogenic in rats. In order to determine whether MDA-modified bases are formed in nucleic acids in vivo, sensitive immunoassays to detect MDA-DNA and MDA-RNA adducts are being developed in our laboratory. Murine monoclonal antibodies reactive with the MDA-deoxyguanosine adduct 3-β-D-erythro-pentofuranosylpyrimido[1,2-a]purin-10(3H)-one (M1G-R) were prepared and characterized. Several MDA-modified nucleosides and deoxynucleosides and structural analogs were synthesized and characterized and were compared as competitive inhibitors in enzymelinked immunosorbent assays (ELISAs). Less than 5 fmol of M1G in MDA-modified DNA was detected in a direct ELISA, and antibody binding to the modified DNA was competitively inhibited by free M1G-dR. DNA from Salmonella typhimurium treated with concentrations of MDA that induce reversion to histidine prototrophy was enzymatically digested, and M1G-dR was quantitated by competitive ELISA. Over a range of MDA concentrations from 10 to 40 mM, the level of M1G residues in bacterial DNA increased from 0.2 to 2.5/106 base pairs.
Introduction Difunctional aldehydes constitute an important class of carcinogens of widespread natural and industrial occurrence (1-3). Malondialdehyde (MDA),7 a member of this class of compounds, is a product of prostaglandin endoperoxide metabolism via enzymatic and nonenzy* Address correspondence to this author. FAX: (615) 343-7534. E-mail:
[email protected]. † Proteins International. ‡ Wayne State University. § Vanderbilt University. | Deceased 24 April 1996. X Abstract published in Advance ACS Abstracts, January 1, 1997. 1 This article and the accompanying article are dedicated to the memory of Dr. Munetaka Nokubo, our valued colleague who died far too young. He was a hard worker, an imaginative scientist, and a good right fielder. We miss him. 2 Present address: Pharmaceutical Research, Warner-Lambert/ Parke-Davis, 2800 Plymouth Rd., Ann Arbor, MI 48105. 3 Present address: GM Advanced Technology Vehicles, 1996 Technology Drive, Troy MI 48083-7083. 4 Present address: Cayman Chemical, 609 KMS Place, Ann Arbor MI 48103. 5 Present address: Urocore Diagnostics, Oklahoma City, OK, 23104. 6 On sabbatical leave 1 June 1995-31 May 1996 from the Chemistry Department, Western Maryland College, Westminster MD 21157. 7 Abbreviations: malondialdehyde (MDA); pyrimido[1,2-a]purin-10(3H)-one (M1G); 3-β-D-erythro-pentofuranosylpyrimido[1,2-a]purin-10(3H)-one (M1G-R); 3-(2-deoxy-β-D-erythro-pentofuranosylpyrimido[1,2a]purin-10(3H)-one (M1G-dR); 7-methylpyrimido[1,2-a]purin-10(3H)one (7-Me-M1G-R); 1,N2-ethenodeoxyguanosine (-dG); 1,1,3,3-tetramethoxypropane (TMP); 3-β-D-erythro-pentofuranosyl-5,6,7,8-tetrahydropyrimido[1,2-a]purin-10(3H)-one (H4-M1G-R); 3-β-D-erythro-pentofuranosyl-5,6-dihydropyrimido[1,2-a]purin-10(3H)-one (H2-M1G-R); 3-(2deoxy-β-D-erythro-pentofuranosyl)-6,7-dihydroxyimidazo[1,2-a]purin9(3H)-one (glyoxal-dG); β-benzoylaxyacrolein (BBA); keyhole limpet hemocyanin (KLH); keyhole limpet hemocyanin coupled to M1G-R (M1G-R-KLH); bovine serum albumin (BSA); bovine serum albumin coupled to M1G-R (M1G-R-BSA); phosphate-buffered saline (PBS); phosphate-buffered saline containing 0.05% Tween 20 (PBS-T); mediumpressure liquid chromatography (MPLC).
S0893-228x(96)00120-8 CCC: $14.00
matic pathways and is generated during lipid peroxidation (4-7). Numerous reports document the production of MDA in animals and humans (5, 8), and MDA has been shown to be mutagenic in Salmonella typhimurium and mouse lymphoma cells and carcinogenic in rats (1, 9-12). A likely explanation for these genotoxic effects is that MDA reacts with the bases of DNA to form adducts, the predominant one at physiological pH being pyrimido[1,2a]purin-10(3H)-one (M1G) (13, 14). This adduct, formed by the reaction of MDA with 2-deoxyguanosine residues, has been implicated as a premutagenic lesion, leading to frame shift as well as base pair substitution mutations in S. typhimurium (15). Despite the extensive toxicology literature on MDA, its contribution to human cancer is uncertain. Part of the reason for this uncertainty is the absence of data on the occurrence of MDA-DNA adducts, such as M1G, in a range of human tissues. Recently, our laboratory reported the measurement of M1G levels in human liver DNA by gas chromatography/mass spectrometry (16), and Vaca et al. have used 32P-postlabeling techniques to measure M1G levels in human blood and breast tissue samples (17). However, neither of these methods has yet been applied to large numbers of subjects or to a wide variety of tissues. To this end, the availability of highly specific antibodies directed against the M1G adduct could be of great value, allowing the use of immunochemical techniques for the purification and/or quantitation of M1G from biological samples. We describe herein the production of a monoclonal antibody directed against M1G-ribose (M1G-R), coupled to bovine keyhole limpet hemocyanin (KLH). Several hybridomas producing monoclonal an© 1997 American Chemical Society
Pyrimidopurinone Antibody
tibodies that recognize M1G were obtained from fusions of murine myeloma cells with splenocytes from immunized mice. One of these antibodies (D10A1) exhibited binding properties that make it a useful reagent for the detection of this important class of MDA-DNA adducts. In this paper, we describe its use in a competitive ELISA assay for the quantitation of M1G levels in DNA from S. typhimurium treated with mutagenic concentrations of MDA. The accompanying paper describes the use of this antibody to purify M1G from human leukocyte DNA for quantitation by gas chromatography/electron capture/ negative chemical ionization/mass spectrometry.
Experimental Procedures Materials. Tetramethoxypropane (TMP) and all organic solvents (analytical grade) were obtained from Aldrich (Milwaukee WI). Guanosine, 2′-deoxyguanosine, 2′-deoxyguanosine 5′-monophosphate, guanine, calf thymus DNA, polylysine, DNase I, snake venom phosphodiesterase, phsophodiesterase I, microccal nuclease, alkaline phosphatase, ribonuclease A, ribonuclease T1, ribonuclease from Aspergillus clavatus, adenosine deaminase, lysozyme, KLH, bovine serum albumin (BSA), antimurine IgM-agarose, alkaline phosphatase, o-phenylenediamine, Tween-20, Trizma base, phosphate/citrate buffer, phosphate-buffered saline, pH 7.4 (PBS), and aminopterin were obtained from Sigma (St. Louis MO). Goat anti-mouse IgG coupled to horseradish peroxidase was obtained from Chemicon (Temecula CA). Phenol/water/chloroform reagent was from Applied Biosystems (Foster City CA). Sodium malondialdehyde (NaMDA) was prepared from TMP as described previously (18). Fetal calf serum was obtained from HyClone (Logan, UT). S. typhimurium hisD3052 was obtained from Bruce Ames, University of California, Berkeley, CA. M1G, M1G-R, and M1G-deoxyribose (M1G-dR) were synthesized and characterized as described by Basu et al. (14). They were purified by medium-pressure liquid chromatography (MPLC) on a column of octadecyl-C18 (40 m, J. T. Baker) eluted with 5% acetonitrile in water at a flow rate of 4 mL/min. The adduct of guanosine and R-methylmalondialdehyde, 7-methylpyrimido[1,2]purin-10(3H)-one (7-Me-M1G-R), was prepared as described by Moschel and Leonard (19). M1G-R-5′-monophosphate was prepared by reacting guanosine 5′-monophosphate (GMP, 500 mg, 1.3 mmol) with TMP (800 mg, 4.9 mmol) at pH 1.5. After 24 h at room temperature, the reaction mixture was evaporated to a volume of 4 mL, applied to a column of octadecyl-C18, and eluted with water at a flow rate of 4 mL/min. Fractions exhibiting green fluorescence were combined, concentrated, and subjected to ion-exchange chromatography on a DE 52 column, which was washed with water and then eluted with 0.01 M potassium phosphate buffer, (pH 4.2). The fluorescent fractions were combined, evaporated to a volume of 4 mL, and desalted by MPLC on the octadecylC18 column eluted with water. The fluorescent fractions were combined and lyophilized. M1G-dR-5′-monophosphate was prepared by dissolving deoxyguanosine 5′-monophosphate (dGMP, 400 mg, 1.1 mmol) in water (pH 2.3), and adding TMP (800 mg, 4.9 mmol). After 24 h at room temperature, the precipitated solid was filtered off; the filtrate was concentrated and subjected to MPLC followed by DE 52 chromatography and desalting as described above for the M1G-R-5′-monophosphate. All of the M1G adducts were homogeneous as judged by chromatography on reversed phase HPLC columns eluted with acetonitrile/water mixtures. They were quantitated by absorption spectroscopy at neutral pH using the molar absorptivity at 251 nm of 13 200 as reported by Seto et al. (13). Synthesis of 3-β-D-erythro-pentofuranosyl-5,6,7,8-tetrahydropyrimido[1,2-a]purin-10(3H)-one (H4-M1G-R) was as described by Chung and Hecht (20). Adduct 3-β-D-erythro-pentofuranosyl5,6-dihydropyrimido[1,2-a]purin-10(3H)-one (H2-M1G-R) was prepared by sodium borohydride reduction of M1G-R (21). 1,N2ethenodeoxyguanosine (-dG) was prepared as described by
Chem. Res. Toxicol., Vol. 10, No. 2, 1997 173 Sattsangi et al. (22). The 1:1 cyclic adduct of glyoxal and 2′deoxyguanosine (3-(2-deoxy-β-D-erythro-pentofuranosyl)-6,7-dihydroxyimidazo[1,2-a]purin-9(3H)-one) (glyoxal-dG) was synthesized as described by Chung and Hecht (23). Modification of DNA by MDA. Calf thymus DNA was purified by treatment with ribonuclease and proteinase K followed by chloroform/isoamyl alcohol extraction and ethanol precipitation. For some experiments, the DNA was denatured by heating to 100 °C and rapidly cooling to 0 °C. For reaction with MDA, a solution of NaMDA and DNA (3.45 mM each) in 50 mM citrate buffer (pH 4.9) was incubated for 4 days. The modified polynucleotide was precipitated four times with ethanol, extracted with 1-butanol, and dialyzed. The DNA was hydrolyzed by treatment with DNase I, snake venom phosphodiesterase, and alkaline phosphatase as described by Muller and Rajewsky (24). The enzyme digest was passed through a 1 mL Baker C18 solid phase extraction column, and the adducts were eluted with 2 mL of methanol. The methanol was evaporated, and the residue was dissolved in 50 mL of water. The amount of M1G-dR was quantitated by HPLC with fluorescence detection. The nucleotides were chromatographed on an Ultrasphere ODS column eluted with 7% aqueous acetonitrile at 1.5 mL/min. A Kratos 980 fluorescence detector was employed with 342 nm excitation. Quantitation was by comparison to known amounts of authentic M1G-dR. For experiments in which a more highly modified DNA was desired, β-benzoyloxyacrolein (BBA) was used in place of MDA for the modification reaction. Freshly prepared BBA, synthesized by the method of Protopopova and Skoldinov (25), was added at concentrations of 10-50 mM to 1 mL aliquots of a solution (0.5 g/mL) of calf thymus DNA in phosphate buffer (pH 6.5). The reaction mixture was stirred for 8 h at 37 °C, and the DNA was then precipitated with ethanol, dried, and dissolved in 0.5 mL of water. MDA-modified RNA samples were prepared by reacting calf liver RNA in 0.05 M citrate buffer, pH (4.5, 5.5, or 6.5), containing 0.15 M NaCl with 0.01 M NaMDA at 37 °C in the dark for 2 days. The modified RNA was purified by ethanol precipitation, butanol extraction, and finally dialysis against citrate/NaCl buffer, pH (6.5). Preparation of M1G-R Conjugates with KLH (M1G-RKLH) and with BSA (M1G-R-BSA). Conjugation of M1G-R to KLH or BSA was done by a modification of the periodate oxidation procedure of Erlanger and Beiser (26). M1G-R was reacted with 0.1 M NaIO4 for 15 min and then added to a KLH or BSA solution (20 mg/mL) at pH 8.5. The mixture was allowed to react at room temperature for 45 min and then for an additional 3 h at 4 °C. After overnight dialysis against PBS (pH 7.4), the mixture was further purified by gel filtration on Sephadex G-75 to ensure removal of any noncoupled adduct from the conjugate. The ratio of bound adducts to protein was determined spectrally as described by Inouye et al. (27). Preparation of Hybridomas. BALB/c mice (Charles River Labs, Inc., Wilmington MA) were injected intraperitoneally with 50 µg of M1G-R-KLH and Freund’s complete adjuvant. After a 3-week interval, the mice were boosted with the same dose of conjugate but with incomplete adjuvant. Seven days after the second boost, the mice were tail bled, and the anti-M1G titers were determined in an enzyme-linked immunosorbent assay (ELISA). Four weeks after the second boost, mice with the highest titers were again boosted intraperitoneally with conjugate in saline. Three days after the final boost, the mice were sacrificed, the spleens were removed, and the resulting splenocytes were fused with either myeloma cell line P3/NS1/1-Ag4-1 or Pc-X63-Ag8.653 using poly(ethylene glycol) as the fusogen (28). After the fusion, the cells were suspended in RPMI 1640 containing 15% fetal calf serum, glutamine, hypoxanthine (0.1 mM), thymidine (16 mM), and aminopterin (0.4 mM). The cells were plated at a concentration of 105 splenocytes/0.1 mL per well in 96-well tissue culture plates and maintained at 37 °C in a 5% CO2 humidified incubator. The hybridomas were fed after 6 days, and when sufficient growth of cells had occurred (usually 12-18 days),
174 Chem. Res. Toxicol., Vol. 10, No. 2, 1997 aliquots of hybridoma supernatants were removed and tested for the presence of anti-M1G activity in an ELISA. Hybridoma cells from selected positive wells were cloned by limiting dilution, and selected clones were recloned and used for further study. Ascites were produced in pristane primed BALB/c mice. The ascites fluid was centrifuged to remove cells, and the clear supernatant, with added azide and aprotinin, was stored at 4 °C. Isotype Determination and Antibody Purification. Antibody isotype was determined using the mouse monoclonal antibody isotyping kit (MonoAb ID EIA) according to the procedure supplied by Zymed (South San Francisco, CA). IgG antibodies were purified by affinity chromatography using antimurine IgG-agarose (Sigma) as described by Sigma. The IgM antibody was purified by the same method but with anti-murine IgM-agarose. Briefly, ascites fluid was dialyzed against 0.01 M sodium phosphate buffer (pH 7.2), containing 0.5 M sodium chloride. The affinity resins were equilibrated with this buffer, and after applying the ascites, the columns were washed with this buffer until the absorbance at 280 nm of the flow through reached background levels. The antibody was then eluted from the resin with 0.1 M glycine (pH 2.4), containing 0.15 M NaCl. The fractions were neutralized immediately with 1 M Tris base. Fractions containing antibody activity were concentrated with an Amicon stirred cell and dialyzed against PBS containing 0.02% azide. ELISA Procedure. ELISA-quality 96-well microtiter plates (Gibco, Beverly, MA) were coated with varying amounts of M1G-R-BSA (1 pg to 1 mg/100 µL of PBS per well) for 90 min (or overnight) at room temperature. Plates were washed twice with 100 µL of PBS containing 0.05% Tween 20 (PBS-T), incubated for 30 min with 300 µL of PBS containing 0.1% BSA, and then washed twice again with PBS-T. Aliquots of murine sera dilutions, hybridoma supernatants, or purified antibody were incubated in the coated wells for 60 min with PBS alone or with PBS containing varying concentrations of free M1G-R, other inhibitors, or test samples in a total final volume of 100 µL. After the wells were washed four times with PBS-T, they were incubated for 1 h with aliquots of diluted peroxidase-labeled goat anti-mouse immunoglobulin (Chemicon). After washing, peroxidase activity remaining in the wells was determined by adding 0.05 M sodium phosphate/0.024 M citrate buffer (pH 5.0), containing 0.04% o-phenylenediamine and 0.012% hydrogen peroxide (200 µL/well) (29). The reaction was stopped after 5 min with 8 N sulfuric acid (50 µL/well), and the absorbance was measured at 490 nm using a Titertek Multiscan microtiter plate reader. For the direct ELISA with MDA-modified RNA or MDAmodified DNA, microtiter plates were coated with either MDARNA, unmodified RNA, MDA-DNA, or unmodified DNA. In order to maximize RNA binding, microtiter plates were first coated with polylysine by incubating plates for 2 h with a solution of 10 mg of polylysine/mL in PBS (100 µL/well). After washing with PBS-T, dilutions of RNA samples were incubated in the wells for 90 min. While DNA could also be bound to the polylysine-coated wells, enhanced binding was observed by coating wells with polynucleotide diluted in PBS containing 0.1 M MgCl2 as described by Nagata and associates (30). After incubation with polynucleotide, plates were washed and blocked with PBS containing 0.1% BSA. The remainder of the assay was carried out as described above. Isolation of DNA from S. typhimurium Treated with MDA. S. typhimurium (TA 100) was grown in 200 mL of Oxoid nutrient broth No. 2 containing the desired concentrations of NaMDA with vigorous shaking at 37 °C for 24 h. The cells were harvested by centrifugation at 3000g for 20 min and washed twice by resuspension and centrifugation in a solution of 50 mM Tris-HCl containing 25% (w/v) sucrose, pH 8.0 (Tris-sucrose). The cells were suspended in 5 mL of Tris-sucrose and disrupted by incubation with lysozyme (4 mg/mL). The resulting lysate was then mixed with an equal volume of 0.5% (w/v) Triton X-100 in 50 mM Tris (pH 8.0), and solid NaCl was then added to give a final concentration of 1 M. RNase A and RNase T1 (400 units
Sevilla et al. each) were added, and the sample was incubated for 1 h at 37 °C, at which time proteinase K (0.5 mg/mL) was added, and the sample was incubated for an additional 3 h. DNA was purified from the resulting mixture by extracting twice with an equal volume of phenol/water/chloroform reagent, and twice with an equal volume of chloroform/isoamyl alcohol (24:1, v/v). Following the addition of one-tenth volume of 3 M sodium acetate, three volumes of ice cold ethanol were added, and the mixture was allowed to stand overnight at -20 °C. The resulting DNA precipitate was collected by centrifugation, dried, and dissolved in 2 mL of water. To the resulting solution was added 0.6 volume of 20% (w/v) poly(ethylene glycol) in 2.5 M NaCl. Following an incubation at 0 °C for 1 h, the precipitates were collected by centrifugation, washed once with 75% ethanol, and dissolved in 2 mL of water. DNA Hydrolysis. To BBA-modified calf thymus DNA or bacterial DNA solutions in 2 mL of water were added 50 µL each of 1 M Tris-HCl (pH 7.4), 0.2 M CaCl2, and 1 M MgCl2, plus 10 µL each of micrococcal nuclease (1 unit), phosphodiesterase I (0.003 unit), alkaline phosphatase (0.25 unit), and adenosine deaminase (0.35 unit). The resulting mixture was incubated overnight at 37 °C and then applied to a C-18 solid phase extraction column (100 mg, 1 mL, Varian Bond Elut). After the column was washed with 10 mL of 1% methanol (v/v) in water, the M1G-dR was eluted with 0.6 mL of 25% (v/v) methanol in water. The eluate was evaporated to dryness under reduced pressure, and the resulting residue was dissolved in 0.5 mL of PBS for quantitation in the ELISA assay. To verify that hydrolysis of DNA samples was complete, and that samples were not contaminated with RNA, aliquots of the hydrolysates were subjected to deoxynucleoside and nucleoside analysis by HPLC on a column (4.6 mm × 25 cm) of Ultrasphere ODS (Beckman). The column was eluted at a flow rate of 1 mL/ min with a solvent system consisting of solvent A (50 mM ammonium acetate) and solvent B (acetonitrile) using the following gradient: 0-5 min, 6% solvent B; 5-10 min, linear gradient to 10% solvent B; 10-13 min, linear gradient to 20% solvent B; 13-16 min, 20% solvent B.
Results Characteristics of the M1G Conjugated Proteins. The M1G-R-KLH conjugate, used as immunogen, and the M1G-R-BSA conjugate, used as the plate-coating antigen in the assays, fluoresced on excitation at 360 nm. This is a characteristic of the M1G adduct and demonstrated that the adduct was covalently attached to the protein. Sodium borohydride reduction, a step in the original procedure of Erlanger and Beiser (26), was not carried out because borohydride reduces the benzo ring of M1G (21). The unreduced conjugates were stable at neutral pH, and the adduct:protein ratios were calculated from difference spectra by the method of Inouye and associates (27). Ratios of approximately 5:1 M1G:BSA for the BSA conjugate and 40:1 M1G:KLH for the KLH conjugate (assuming a MW of 8 × 105 for the KLH) were calculated. The KLH conjugate was used for immunization of mice. Selection of Hybridomas and Characterization of Antibodies. Ten hybridomas were selected for further cloning on the basis of the ability of their antibodies to bind to M1G-R-BSA-coated plates (100 ng/well) in the absence, but not the presence, of 500 pmol of free M1GR. When ELISA plates were coated with BSA-bound M1G-R at g7 pmol/well (100 ng of M1G-R-BSA), little, if any, inhibition of binding by the natural nucleosides (100 nmol) was observed for antibodies H8C9, G8B3, H5E3, E2A12, H6C3, D4B6, D10A1, and E12F1. In order to further select which hybridomas to reclone, checkerboard ELISAs were performed using decreasing amounts
Pyrimidopurinone Antibody
Chem. Res. Toxicol., Vol. 10, No. 2, 1997 175 Table 1. Dependence of I50 of M1G-R on M1G-BSA Plate Coatinga M1G (BSA bound) (pmol/well)
I50 of M1G-R (free) (pmol/well)
3.4 1.7 0.69 0.34 0.068
3.0 1.5 0.6 0.3 0.15
a Wells were coated with the indicated quantities of M G (BSA 1 bound). The plates were then incubated with D10A1 antibody in the presence of increasing amounts of free M1G-R as described in Experimental Procedures. The I50 is defined as the concentration of M1G-R required for 50% inhibition of D10A1 binding in the competitive ELISA. Assays were performed in triplicate.
Figure 1. Binding of monoclonal antibody D10A1 to M1G (BSA bound) in a direct ELISA. Microtiter plate wells were coated with the indicated amount of M1G (BSA bound) in 0.1 mL of PBS, and the ELISA was performed as described in Experimental Procedures. Each point is the mean and standard deviation of 10 determinations.
of BSA-bound M1G-R for plate coatings, serial dilutions of hybridoma supernatants, and smaller amounts of free M1G-R as the competitive inhibitor. From these experiments, hybridomas E12F1, H5E3, E2A12, H6C3, D10A1, and D4B6 were found to bind to M1G-R-BSA-coated plates (1 ng/well) in the absence but not the presence of 50 pmol/well of free M1G-R; these were further cloned, and ascites was prepared for each. The antibodies produced by these hybridomas all had IgG1 isotypes except for E12F1, which was an IgM. Further analyses of these antibodies resulted in the selection of D10A1 as the antibody having the best overall characteristics, as exemplified by the least inhibition of binding by guanine nucleosides with significant binding inhibition by the smallest amount of M1G-R. Hybridoma D10A1 (653 myeloma parent) was shown to have a λ light chain. The D10A1 antibody protein was purified from the ascites by affinity chromatography and was used in all subsequent experiments. Characteristics of Antibody Binding/Competitive Binding Studies. The dependence of antibody D10A1 binding on the concentration of M1G (BSA bound) used for plate coating is shown in Figure 1. There was no significant inhibition of binding of D10A1 by up to 100 nmol of deoxyadenosine, deoxycytidine, or thymidine regardless of plate coating. However, when the microtiter plates were coated with amounts of M1G (BSA bound) of less than 1 pmol/well, antibody binding inhibition by guanosine and deoxyguanosine was observed. As expected, the concentration of M1G-R required for 50% inhibition of binding for all the antibodies was very dependent on the amount of M1G (BSA bound) used to coat plates (Table 1). Fifty percent inhibition of binding of D10A1 to plates coated with 680 fmol of M1G (BSA bound) was observed with 600 fmol of free M1GR. At this plate coating, 50% inhibition of binding of D10A1 by 30 nmol of guanosine was observed (Figure 2). In addition to the above inhibitors, competitive inhibition studies were performed with a series of analogs (Table 2). The greatest inhibition was observed with M1G-R, which demonstrated somewhat higher inhibition
Figure 2. Competitive inhibition of antibody D10A1 binding to M1G (BSA bound). Microtiter plate wells were coated with 680 fmol of M1G (BSA bound) in 0.1 mL of PBS, and the binding of D10A1 was determined in the presence of the indicated concentrations of each of the five compounds using the competitive ELISA procedure. Each point represents the mean of four determinations.
when it was bound to BSA than when it was free. Several nucleoside derivatives of M1G exhibited I50’s within 1 order of magnitude of M1G-R. The free base M1G was less potent as an inhibitor of binding of D10A1 to M1G-R-BSA. This may indicate some recognition of the sugar backbone, or it may be a function of the poorer solubility of M1G relative to its nucleoside derivatives. Comparison of the inhibitory potency of a variety of M1G analogs suggests that the planarity of the ring is an important determinant of binding. D10A1 binds H2M1G-R and -dG reasonably well, but binds H4-M1G-R much less effectively. The propano ring of H4-M1G-R is completely reduced relative to M1G and is slightly puckered and nonplanar. Interestingly, D10A1 exhibits reasonable binding to 7-Me-M1G-R which contains a methyl group on the benzo ring. The ability to bind 7-MeM1G-R is not shared by all of the other antibodies.8 As expected, the binding of antibody D10A1 to MDAmodified RNA and DNA depends on the extent of modification, which increases with decreased reaction pH. Although no quantitation of adduct in MDA-modified RNA has been done as yet by methods other than immunoassay, antibody D10A1 binds MDA-modified RNA but not unmodified RNA. Binding of D10A1 to these modified RNA samples is competitively inhibited by free M1G-R (Figure 3). 8
C. Sevilla, unpublished results.
176 Chem. Res. Toxicol., Vol. 10, No. 2, 1997
Sevilla et al.
Table 2. Antibody Specificity purine
structure
M1G-R′
I50 (pmol/well)*
structure
e-dG
O
N
R′ ) BSA R′ ) ribose R′ ) deoxyribose R′ ) ribose 5′-PO4 R′ ) deoxyribose 5′-PO4 R′ ) H O
N
0.5 0.6 1-2 4 4 6-20 20
N
H
N
N
N
N
N
R′
H
O
N
ribose O
80-100
R′ ) ribose R′ ) deoxyribose glyoxal-dG
N
N N
30000 70000 >100000
O
HO
N
N
N
H
H
7-MeM1G-R H3C
N N
G-Rl
ribose
H
5000-10000
O N
N
N
deoxyribose
H4-M1G-R
N
N
N
N
H
R′
H2-M1G-R
N
N
N
N
I50 (pmol/well)a 100-200
O
N
N N
purine
HO
N ribose
N H
N
N deoxyribose
deoxyadenosine, deoxycytidine, thymidine
>100000
a Picomoles of inhibitor required for 50% inhibition of D10A1 binding to M G-BSA in an ELISA. Microtiter plate wells were coated 1 with 680 fmol M1G (BSA bound) in 0.1 mL PBS, and the competitive ELISAs were done as described in Methods.
Figure 3. Competitive inhibition of D10A1 binding to MDAmodified RNA. Microtiter plate wells were coated with polylysine and then with 100 ng of control RNA or RNA modified with MDA at pH 4.5, 5.5, or 6.5. The competitive ELISA was performed as described in Experimental Procedures using the indicated concentrations of free M1G-R as the competing compound. In the absence of M1G-R, the corrected absorbance at 490 nm was 1.272 for pH 4.5 MDA-RNA, 0.778 for pH 5.5 MDA-RNA, and 0.333 for pH 6.5 MDA-RNA. D10A1 did not bind to the control RNA. Each point represents the mean and standard deviation of quadruplicate determinations.
Two samples of modified MDA-DNA were prepared, and the number of M1G-dR residues in each was determined by HPLC analysis of a hydrolyzed sample. MDADNA-1 was shown to have 1 M1G-dR/3300 bases. At this level of substitution, the adduct could be detected by the ELISA when DNA containing 15 fmol of adduct was used to coat microtiter plate wells. The content of M1G-dR in MDA-modified DNA-2 was shown to be 1 M1G-dR/400 bases. With this more highly modified DNA, the adduct was detected at levels of 4 fmol of M1G-dR/well. The
Figure 4. Binding of monoclonal antibody D10A1 to M1G (in DNA) in a direct ELISA. Microtiter plate wells were coated with the indicated amount of M1G bound to DNA in 0.1 mL of PBS containing 0.1 M MgCl2, and the direct ELISA was performed as described in Experimental Procedures. Each point represents the mean and standard determination of four determinations.
D10A1 antibody showed no cross-reactivity with unmodified DNA (1 ng-10 mg/well). Figure 4 displays the dependence of absorbance at 490 nm on the amount of modified DNA used for plate coatings. The modified DNA samples contained approximately 8 fmol of M1GdR/ng of MDA-modified DNA-2, and 1 fmol of M1G-dR/ ng of MDA-modified DNA-1. The competitive inhibition of binding of D10A1 to MDA-modified DNA-2 coated plates (32 fmol of M1G-dR/4 ng of DNA per well) is shown in Figure 5. At this plate coating concentration, 50% inhibition of binding was observed with 80 fmol of free M1G-R, 150 fmol of M1G-dR, 5 nmol of guanosine, and 10 nmol of deoxyguanosine. Binding inhibition was also observed using MDA-modified DNA-2 as the competitive
Pyrimidopurinone Antibody
Figure 5. Competitive inhibition of antibody D10A1 binding to M1G (in DNA). Microtiter plate wells were coated with 32 fmol of M1G (in DNA) in 0.1 mL of PBS containing 0.1 M MgCl2, and the competitive ELISA was performed as described in Experimental Procedures, using the indicated quantities of each of the designated compounds as inhibitors. Each point represents the mean of four determinations.
inhibitor, but a higher concentration of M1G (600 fmol of M1G-dR in DNA-2) was required for 50% inhibition. This modified DNA sample was native DNA, and the adducts appear to be less accessible to antibody when the DNA is in solution as compared to when it is bound to a polystyrene plate. DNA binding to the polystyrene may result in partial denaturation and more exposure of the bases to the antibody. Assay of M1G-dR in Hydrolysates of Calf Thymus DNA Modified by BBA. The above results indicated that the D10A1 antibody possessed a high degree of specificity for the binding of M1G nucleosides and that M1G adducts in intact DNA and RNA were also recognized by the antibody. This naturally suggested the application of the competitive ELISA procedure to the detection of M1G-dR in DNA hydrolysates. To test the feasibility of this application, calf thymus DNA samples were reacted with varying concentrations of BBA in order to form M1G adducts for analysis. The samples were hydrolyzed, and HPLC analysis was performed to verify that hydrolysis was complete. Recognizing that the greatest potential problem in using the D10A1 antibody for M1G analyis was the cross reactivity displayed with guanosine nucleosides, a solid phase extraction procedure was used to remove all nonadducted nucleosides from the DNA hydrolysate prior to the ELISA. Figure 6 shows the elution profile of nucleosides from the solid phase extraction column when a hydrolysate of calf thymus DNA sample containing added M1G-dR was chromatographed as described in Experimental Procedures. All of the naturally occurring deoxyribonucleosides in DNA were readily separated from M1G-dR with the exception of deoxyadenosine, which coeluted. Consequently, adenosine deaminase was included in the hydrolysis reaction mixture to convert deoxyadenosine to deoxyinosine, which eluted much earlier than M1G-dR. When 10 nmol of M1G-dR was applied to the solid phase extraction column, eluted with 25% (v/v) methanol in water, and quantitated by HPLC, the recovery of the M1G-dR was found to be 95 ( 3%. Figure 7 displays a representative standard curve
Chem. Res. Toxicol., Vol. 10, No. 2, 1997 177
Figure 6. Elution profile of nucleosides from a C-18 solid phase extraction column. An unmodified calf thymus DNA sample was prepared and hydrolyzed as described in Expermental Procedures. To the hydrolysate was added 10 nmol of M1G-dR, and the sample was subjected to solid phase extraction on a 1 mL column of Bond Elut C-18. The column was eluted as described in Experimental Procedures, except that the solvent was added in 0.75 mL aliquots, and each aliquot was collected as an individual fraction upon elution from the column. The fractions were evaporated to dryness under reduced pressure and subjected to HPLC analysis to detect and quantitate the presence of each nucleoside. Squares, deoxyinosine; diamonds, deoxycytidine; circles, deoxyguanosine; triangles, thymidine; crossed squares, M1G-deoxyribose.
Figure 7. Standard curve from the competitive ELISA procedure. Wells in ELISA microtiter plates were coated with 0.1 pmol of M1G (BSA bound) by incubation overnight in 100 mL of PBS. The wells were then washed, blocked with PBS-BSA, and then washed again prior to incubation for 1 h with 0.1 pmol of D10A1 antibody in the presence of the indicated concentrations of M1G-dR. After the wells were washed, they were incubated for 1 h with 0.1 pmol of peroxidase-labeled goat antimouse IgG, and then washed again. Then the peroxidase activity in the wells was determined by incubation with o-phenylenediamine and hydrogen peroxide as described in Experimental Procedures.
generated for the competitive ELISA assay. A linear relationship was observed over the range of M1G-dR concentrations tested. To confirm that the ELISA can detect M1G-dR in a DNA hydrolysate, known quantities of M1G-dR were added to samples of calf thymus DNA; the samples were hydrolyzed and subjected to C-18 solid phase extraction and the competitive ELISA. For samples to which 5 pmol of M1G-dR had been added, 5.3 ( 0.6 pmol of M1G-dR was detected, and for samples to which 10 pmol had been added, 10 (1 pmol was detected (both values are the mean ( SD of five determinations). The ELISA procedure was performed on hydrolysates of calf
178 Chem. Res. Toxicol., Vol. 10, No. 2, 1997
Sevilla et al.
Table 3. Assay of M1G-dR in DNA from MDA-Treated S. typhimuriuma NaMDA (mM)
DNA yield (mg)
M1G-dR (pmol/well)
M1G-dR/DNA (pmol/mg)
M1G-dR/ 106 bases
0 10 20 40 60
6.6 4.5 4.9 3.9 2.4
0.98 2.1 4.6 5.8 3.1
0.73 2.3 4.7 7.5 6.6
0.24 0.78 1.6 2.5 2.2
a S. typhimurium cultures were incubated for 24 h in the presence of the indicated quantities of NaMDA. Cells were harvested, and DNA was isolated and hydrolyzed as described in Experimental Procedures. The DNA was quantitated by HPLC analysis of the hydrolysates. Following partial purification by C-18 solid phase extraction, samples were subjected to the competitive ELISA.
thymus DNA modified by varying concentrations of BBA. A linear relationship was observed between the quantity of M1G-dR detected and the concentration of BBA (e50 mM) used to treat the DNA. The adduct level measured at 50 mM BBA corresponded to 18 M1G-dR’s/106 base pairs. Detection of M1G-dR in the DNA from S. typhimurium Treated with MDA. Cultures of S. typhimurium were incubated for 24 h in the absence or presence of varying concentrations of NaMDA, and the DNA was isolated. Following hydrolysis, HPLC analysis was again performed to verify the purity of the DNA samples and the completeness of the hydrolysis procedure. The samples were then subject to the competitive ELISA. The results are summarized in Table 3. The quantity of DNA isolated from the cultures was relatively constant except for the case of those treated with the two highest concentrations of NaMDA. The decrease in DNA yield from these cultures was likely attributable to toxic effects. Not surprisingly, the quantity of M1G-dR detected in the bacterial DNA samples was directly related to the concentration of NaMDA used to treat the cells, although a decrease was observed at the highest concentration. Note that the quantity of M1G-dR detected in cells not treated with NaMDA is at the lower limit of detection of the assay (0.1 pmol of M1G-dR/well).
Discussion Cyclic nucleic acid adducts have been previously identified as products of the reaction of RNA, DNA, or nucleosides with acrolein, crotonaldehyde, glyoxal, methylglyoxal, vinyl chloride, substituted acroleins, R-alkylMDA, and MDA itself (31, 32). Highly specific immunoassays have been developed to detect several of these cyclic adducts. For example, Foiles et al. have developed four monoclonal antibodies that can be used in immunoassays for cyclic guanine adducts of crotonaldehyde (33). These antibodies are able to detect picomole amounts of crotonaldehyde adducts and also recognize acrolein adducts (34, 35). Specific immunoassays for ethenoadenosine and ethenocytidine have been described by Young and Santella (36). Under in vitro conditions, MDA has been shown to react with guanine, adenine, and cytidine nucleosides in decreasing order of reactivity (14). Seto and associates isolated the pyrimidopurinone adducts M1G-R and M1GdR from in vitro modified RNA and DNA, respectively (37). Furthermore, Seto detected M1G-R in urine and confirmed its structure by mass spectrometry (38). There is a report of the detection of the base, M1G, in human
urine by HPLC with fluorescence detection, but we were unable to confirm this finding by mass spectrometry (39, 40). In order to aid in the determination of M1G residues in DNA and/or RNA in vivo, we have prepared murine monoclonal antibodies directed against the adduct by fusing murine myeloma cells with splenocytes from mice immunized with periodate oxidized M1G-R coupled to KLH. This conjugate was sufficiently stable under in vivo conditions to elicit an immune response even though the complex was not reduced with sodium borohydride. Six positive hybridomas were obtained, and antibody D10A1, from one of these hybridomas, was characterized in detail. Binding of this antibody was proportional to the concentration of M1G (BSA-bound) or M1G (DNA) used for plate coating, and the antibody detected less that 5 fmol of bound M1G in a direct ELISA. Although D10A1 binds to RNA or DNA that has been reacted with MDA as well as to proteins containing M1G, it does not bind to unmodified BSA, KLH, RNA, or DNA. Although only two DNA samples at different levels of modification were characterized by methods other than immunoassay (HPLC) in this report, the finding that D10A1 binds to DNA-1 suggests that M1G residues can be directly detected in DNA samples with lower levels of modification (1 M1G-dR/3300 bases) by this antibody. For RNA and DNA samples that have not been analyzed by methods other than immunoassay, the extent of binding correlates with the conditions used for modification of the polynucleotide, with no binding observed for unmodified polynucleotide. For example, more antibody binds to RNA samples that have been modified by MDA at pH 4.5 as compared to pH 6.5. Antibody binding to MDA-modified polynucleotide samples as well as to M1G-BSA was inhibited by free M1G-R or M1G-dR in a competitive ELISA. While the actual amount of M1G (BSA-bound) remaining in plate wells after coating was not determined, the concentration of inhibitor required to cause 50% inhibition of binding was dependent on the concentration of bound adduct used for coating. The affinity of D10A1 for the M1G-ribonucleoside was 2-fold higher than its affinity for the deoxyribonucleoside and 10-fold higher than its affinity for the M1G base as determined in the competitive ELISA. Although a number of structurally related, modified nucleosides were shown to bind to D10A1 with varying affinities, the only unmodified bases found in RNA and DNA that showed cross-reactivity with the antibody were guanosine and deoxyguanosine. The high affinity and specificity of the D10A1 antibody suggested that is should be useful for detecting M1G-dR in DNA hydrolysates. This hypothesis was tested using calf thymus DNA treated with BBA, and with DNA isolated from S. typhimurium treated with mutagenic concentrations of NaMDA. Possible interference from nonadducted nucleosides was eliminated by subjecting DNA hydrolysates to solid phase extraction prior to assay. This procedure resulted in efficient separation of M1G from the four naturally occurring deoxyribonucleosides if deoxyadenosine was first converted to deoxyinosine during the hydrolysis step. That the ELISA assay could efficiently detect M1G-dR in DNA hydrolysates was confirmed in experiments in which known quantities of M1G-dR added to calf thymus DNA samples were detected at near 100% recovery. When the assay was applied to modifed DNA samples (either calf thymus DNA or S typhimurium DNA), a correlation was found
Pyrimidopurinone Antibody
between the amount of M1G-dR detected and the concentration of either BBA or NaMDA used. Together, the above results suggest that the D10A1 antibody is a promising tool for the detection of M1G-dR in biological samples. This conclusion is most clearly illustrated by the fact that the assay could be used to determine the level of DNA adduction in bacteria incubated under conditions in which mutagenesis is known to occur. However, it should be noted that, at its present level of sensitivity (approximately 100 fmol of M1G-dR detected per well), the competitive ELISA assay may have limited applicability to other DNA sources, depending on available sample size. For example, we have shown by gas chromatograpy/mass spectrometry that M1G-dR is present in normal human liver samples at an average level of 9 adducts/107 bases (10). At this level of adduction, approximately 30 µg of DNA would be required to detect M1G-dR in the competitive ELISA, assuming 100% recovery of adduct. In contrast, we report in the accompanying article that the average M1G level in human leukocyte DNA is 6 adducts/108 bases (12). At this level of adduction, 500 µg of DNA would be required for the assay if the recovery of adduct throughout the isolation procedure is 100%, which is unrealistic. In addition to concerns about sensitivity, the issue of specificity is important for samples in which M1G levels are near the limit of detection of the assay. Although we have eliminated the known major source of crossreactivity (guanine nucleosides) by incorporating a solid phase extraction step prior to ELISA, we cannot absolutely rule out the presence of other interfering species in biological samples. In particular, the high reactivity of D10A1 to M1G-R means that it is essential to remove all RNA that might contaminate the DNA that is to be analyzed for M1G-dR. In the analysis of DNA from S. typhimurium treated with MDA, we found that the DNA samples from Salmonella treated with greater than 40 mM MDA contained significant RNA contamination and the level of M1G residues in RNA was higher than the levels in DNA.9 It appears that when RNA is highly modified by MDA, it is difficult to completely digest with ribonucleases. High concentrations of ribonucleases A and T1 in combination were more effective than ribonuclease A alone in removing contaminating RNA, but detectable amounts remained even after digestion with both nucleases for 4 h. Complete removal of contaminating RNA was only acheived by treatment of the DNA with ribonuclease from A. clavatus. This example illustrates that if broad applicability of immunochemical techniques for the analysis of M1G residues is to be achieved, both the specificity and the sensitivity of the assay must be improved. In the accompanying paper, we describe the combination of immunoaffinity purification with gas chromatography/mass spectrometry to provide an assay with the same sensitivity as the competitive ELISA but with greatly enhanced specificity.
Acknowledgment. This work was supported by Grants CA45878 and CA47479 from the National Cancer Institute.
Chem. Res. Toxicol., Vol. 10, No. 2, 1997 179
(2) (3)
(4) (5) (6) (7)
(8) (9) (10) (11) (12) (13)
(14)
(15) (16)
(17)
(18) (19) (20) (21)
(22)
(23)
(24)
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TX960120D
