Production of a High-Affinity Monoclonal Antibody Specific for 7

George P. Casale*, Eleanor G. Rogan, Douglas Stack, Prabu Devanesan, and ... Regulan RamaNathan, Kenneth P. Roberts, Damon C. Barbacci, John Zhao, ...
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Chem. Res. Toxicol. 1996, 9, 1037-1043

1037

Production of a High-Affinity Monoclonal Antibody Specific for 7-(Benzo[a]pyren-6-yl)guanine and Its Application in a Competitive Enzyme-Linked Immunosorbent Assay George P. Casale,* Eleanor G. Rogan, Douglas Stack, Prabu Devanesan, and Ercole L. Cavalieri Eppley Institute for Research in Cancer, University of Nebraska Medical Center, 600 South 42nd Street, Omaha, Nebraska 68198-6805 Received December 8, 1995X

Molecular dosimetry of depurinating DNA adducts of benzo[a]pyrene (BP) is a promising new approach to measurement of cancer risk associated with exposure to polycyclic aromatic hydrocarbons (PAH). Depurinating adducts of BP are spontaneously released from DNA and can be detected in urine. As a first step toward developing a monoclonal antibody (MAb)based molecular dosimetry for depurinating DNA adducts of BP, a MAb (MAb CB53) has been produced with high specific affinity for 7-(benzo[a]pyren-6-yl)guanine (BP-6-N7Gua), a major depurinating adduct of BP. Production of this MAb was dependent on the successful synthesis of an effective immunogen consisting of the hydrophobic BP-6-N7Gua coupled to carrier protein via a rigid spacer arm. A competitive enzyme-linked immunosorbent assay (ELISA) for BP6-N7Gua has been developed with MAb CB53 and has been applied to evaluation of MAb binding and to quantitation of BP-6-N7Gua in a biological sample. The MAb binds with high affinity to BP-6-N7Gua (Ka ) 1.4 × 108 M-1) and to BP-6-N7Ade (Ka ) 0.7 × 108 M-1), another major depurinating DNA adduct of BP, but discriminates well between BP and BP-6-N7Gua. BP-6-N7Gua produces 50% inhibition at 750 fmol in the competitive ELISA, whereas BP produces 50% inhibition at 960 000 fmol. Binding affinities to selected PAH, BP-DNA adducts, and BP metabolites indicate significant contributions of the hydrophobic region C-3, C-4, and C-5 of BP and the polar oxygen of guanine to MAb/adduct binding. In a preliminary test of the utility of the competitive ELISA for quantitation of BP-6-N7Gua in urine samples, the assay (sensitivity: 200 fmol per well) produced an accurate determination of the adduct added to normal human urine.

Introduction Polycyclic aromatic hydrocarbons (PAH)1 are environmental contaminants produced by incomplete combustion of organic matter. Many of the chemicals in this group are proven carcinogens in animal models and suspected carcinogens in humans (1). Benzo[a]pyrene (BP), one of the most potent carcinogens of the PAH family (2), is a ubiquitous component of PAH mixtures (2) and correlates well with total PAH in contaminated environments (1, 3). Based on these features, BP is considered an appropriate indicator of exposure to carcinogenic PAH (1). Significant exposure to BP may occur in the general population as a consequence of industrial pollution and the use of coal as a home heating fuel. In Gliwice, Poland, for example, residents are exposed to ambient * To whom correspondence should be addressed. X Abstract published in Advance ACS Abstracts, August 1, 1996. 1 Abbreviations: AFB-N7Gua, aflatoxin-N7-guanine; BP, benzo[a]pyrene; BP-6-N7Ade, 7-(benzo[a]pyren-6-yl)adenine; BP-6-N7Gua, 7-(benzo[a]pyren-6-yl)guanine; BP-6-C8Gua, 8-(benzo[a]pyren-6-yl)guanine; BPDE, benzo[a]pyrene 7,8-diol 9,10-epoxide; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; I-BSA, 2-iminothiolane-modified BSA; I50, quantity producing 50% inhibition of MAb binding in the competitive ELISA; HAT, hypoxanthineaminopterin-thymidine; MAb, monoclonal antibody; MCC, N-[(4carboxycyclohexyl)methyl]maleimide; OA, hen egg albumin; PAH, polycyclic aromatic hydrocarbon(s); PBL, peripheral blood leukocytes; PBS, sodium phosphate buffer (pH 7.5), 0.1 M NaCl; SMCC, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate; TBS, Tris buffer (pH 7.5), 0.15 M NaCl; TBST, 50 mM Tris buffer (pH 7.5), 200 mM NaCl, 0.05% (v/v) Tween 20.

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BP at concentrations of 15-57 ng/m3 of air (4). These exposures are associated with high cancer mortality. In Xuan Wei County, China, residents are exposed to indoor BP at concentrations as high as 2485 ng/m3 air, due to combustion of smoky coal (5). Lung cancer mortality rates in Xuan Wei are among the highest in China. By far, the most intensive exposures to BP occur among workers directly participating in certain industrial processes, including aluminum production, iron and steel founding, coke production, and coal gasification (1). A study of coke-oven workers, for example, found BP concentrations of 100-7800 ng/m3 of ambient air, in the work environment (6). In a study of aluminum workers, ambient BP in an anode factory was found to be as high as 11 600 ng/m3 (3). These occupational exposures are correlated with high incidences of lung cancer (1). Lastly, cigarette smoking continues to be a significant source of BP exposure. Mainstream smoke produced by a single cigarette contains 20-40 ng of BP, whereas the sidestream smoke contains 68-136 ng (7). Epidemiological monitoring of BP exposure is fundamental to determining risk of PAH-induced cancers and implementing preventive intervention (1). Conventional studies base risk estimates on measurements of BP concentrations in the environment (8). The precision of these estimates is very low due to large individual variations in absorption and metabolism of BP and the long delay between measured exposure and measured end point (e.g., cancer frequency). In contrast, biologi© 1996 American Chemical Society

1038 Chem. Res. Toxicol., Vol. 9, No. 6, 1996

Casale et al.

Figure 2. Synthesis of the MCC-BP-6-N7Gua conjugate.

DNA adducts of BP will provide a direct measure of DNA damage central to cancer initiation. This report describes the production of a monoclonal antibody (MAb) specific for BP-6-N7Gua, as the first step in developing a MAb-based molecular dosimetry for depurinating DNA adducts of BP. A competitive enzymelinked immunosorbent assay (ELISA) has been developed with this MAb and has been applied to (1) characterization of MAb/ligand binding and (2) detection and quantitation of BP-6-N7Gua.

Experimental Procedures

Figure 1. Structures of selected compounds evaluated in the competitive ELISA for BP-6-N7Gua.

cally effective dose (i.e., the amount of disease-causing damage to biological targets) circumvents variations in absorption and metabolism and is more closely related to disease outcome (8, 9). The use of BP-DNA adducts as biomarkers of biologically effective dose offers the potential for identification of individuals at risk for PAH-induced cancers and for timely preventive measures (8-10). PAH are activated to their ultimate carcinogenic species by two principal pathways: one-electron oxidation to yield radical cations (11) and monooxygenation to yield bay-region diol epoxides (12). These intermediates can bind to DNA and form “stable” adducts that remain in the DNA if not repaired or “depurinating” adducts that are released spontaneously from the DNA by hydrolysis of the N-glycosidic bond. Adducts bound to the N-7 of guanine (BP-6-N7Gua, Figure 1) or adenine (BP-6N7Ade) or the C-8 of guanine (BP-6-C8Gua) are depurinating adducts that account for 71% of all BP-DNA adducts (13-15). Adducts of BP-7,8-diol-9,10-epoxide (BPDE) bound to the 2-amino group of guanine are stable and account for 23% of BP-DNA adducts (13-15). Depurinating adducts of BP are promising biomarkers of PAH-induced DNA damage and risk for PAH-induced cancers. These adducts are the predominant form of BPinduced DNA damage and are excreted in the urine (16). Importantly, recent studies comparing the pattern of DNA adducts for a series of PAH (including BP) with mutations in the Harvey-ras oncogene support an essential contribution of depurination to oncogenic mutation (17). Consequently, quantitation of depurinating

Caution. The following chemicals are hazardous and should be handled carefully: PAH and metabolites; SOCl2 should be handled only in a fume hood. Experimental Animals. Male BALB/c mice, 7-8 weeks of age, were purchased from Sasco (Omaha, NE) and acclimated in the animal facility at Eppley Institute for 1-2 weeks. Chemicals. The depurinating DNA adducts BP-6-N7Gua, BP-6-C8Gua, BP-6-N7Ade, and BP-6-N3Ade were synthesized by anodic oxidation of BP in the presence of 2′-deoxyguanosine, 2′-deoxyadenosine, or adenine (15, 18, 19). BP, 3-hydroxyBP, 3-methylBP, benzo[e]pyrene, BP-trans-4,5-dihydrodiol, BP-trans7,8-dihydrodiol, BP-trans-9,10-dihydrodiol, 7-methylguanine, deoxyguanosine, benz[a]anthracene, and chrysene were available in our laboratory. Coupling of BP-6-N7Gua with Protein Carriers. Depurinating DNA adducts of BP are excreted in the urine, where they are accessible for individual analysis (16). These adducts can serve as haptens for the production of specific antibodies that may be used for immunochemical detection and quantitation of the adducts in biological fluids. To enhance presentation of BP-6-N7Gua to receptors of the immune system, the adduct was coupled to protein via the rigid heterobifunctional linker N-[(4-carboxycyclohexyl)methyl]maleimide (MCC; Pierce Chemical Co., Rockford, IL). A conjugate of BP-6-N7Gua and MCC was prepared as follows (see Figure 2). MCC (50 mg, 0.21 mmol) was converted to the corresponding acid chloride by treatment with SOCl2, neat, at room temperature for 3 h. Excess SOCl2 was removed under a stream of nitrogen, leaving N-[[(4-(chloroformyl)cyclohexyl]methyl]maleimide sufficiently pure for further use. The acid chloride (32 mg, 0.125 mmol) was dissolved in 1 mL of dry chloroform. Concurrently, BP-6-N7Gua (10 mg, 0.025 mmol) was placed in 5 mL of anhydrous pyridine, producing a yellow suspension. The acid chloride was then added dropwise (for 5 min) to the BP-6-N7Gua suspension at room temperature. Immediately after addition of the acid chloride, the suspension became a clear yellow solution. The mixture was stirred an additional 15 min, and the solvents were then removed under reduced pressure. The crude mixture was dissolved in 1 mL of chloroform and separated by normal-phase silica gel chromatography (19 mm × 100 mm) with gradient mixtures of chloroform and acetone. After an initial wash with 100% chloroform (100 mL), the adduct was eluted with 100 mL of chloroform-acetone (9:1). This fraction, after removal of solvents, was purified by reverse-phase HPLC with a methanol-

MAb-Based ELISA for BP-6-N7Gua water gradient to yield the MCC-BP-6-N7Gua conjugate (10.8 mg, 70% isolated yield). 1H NMR (500 MHz): δ 10.13 (bs, 1H, 1-NH [Gua], exchanged with D2O), 9.13 (d, 1H, 11-H [BP]), 8.44 (d, 1H, 12-H [BP]), 8.32 (d, 1H, 1-H [BP]), 8.17 (s, 1H, 8-H [Gua]), 8.13 (d, 1H, 3-H [BP]), 8.02 (t, 1H, 2-H [BP]), 7.94 (d, 1H, 4-H [BP]), 7.87 (t, 1H, 4-H [BP]), 7.75 (t, 1H, 8-H [BP]), 7.55 (d, 1H, 7-H [BP]), 7.34 (d, 1H, 5-H [BP]), 6.70 (s, 2H, [maleimine group]), 3.35 (d, 2H, 1-methylene [MCC]), 2.46 (m, 1H, 4-H [MCC]), 2.00 (m, 2H, 3-H(eq) [MCC]), 1.74 (m, 2H, 2-H(eq) [MCC]), 1.72 (m, 1H, 1-H [MCC]), 1.46 (d, 2H, 3-H(ax) [MCC]), 0.97 (d, 2H, 2-H(ax) [MCC]). FTIR (cm-1): 3308, 3131, 2924, 1690, 1680, 1117. An immunogen was prepared by coupling MCC-BP-6-N7Gua to hen egg albumin (OA). Lysyl amino groups of OA were converted to free sulfhydryl groups by reaction of the protein (10 mg/mL) with 30 mM 2-iminothiolane (20) in triethanolamine buffer (pH 8.2) containing 0.10 M NaCl, 0.025 M potassium EDTA, and 1% (v/v) 2-mercaptoethanol. The reaction was allowed to proceed on ice, under argon, for 60 min. The modified protein was dialyzed against the same buffer without 2-mercaptoethanol and then purified with a Sephadex G-25 column equilibrated with 0.10 M sodium phosphate buffer (pH 7.4) containing 0.10 M NaCl, 0.05 M potassium EDTA, and 30% (v/ v) DMSO. A volume of 1.5-2.0 mL column eluate, containing ca. 4 mg of protein/mL, was adjusted to 5% (v/v) Tween 20; then 500 µg of MCC-BP-6-N7Gua dissolved in 50 µL of DMSO was added with stirring. The reaction, producing a thioether bond between the free sulfhydryl groups of the modified protein and one of the carbon atoms of the maleimido double bond, was allowed to proceed for 90 min in an argon atmosphere at room temperature. As a control, 50 µL of DMSO was added to 1.52.0 mL column eluate adjusted to 5% Tween 20. The conjugated protein was separated from unreacted MCC-BP-6-N7Gua by acetone precipitation at -10 °C and dried with a stream of argon. The dried protein conjugate was suspended in sodium phosphate buffer (pH 7.5) containing 0.10 M NaCl (PBS), reduced with 2-mercaptoethanol, alkylated with iodoacetamide, and then dialyzed against PBS. The protein concentration of the dialyzed material was determined with bicinchoninic acid (Pierce Chemical Co.). The concentration of MCC-BP-6-N7Gua was determined from the absorbance (405 nm) of duplicate samples of conjugate suspension solubilized with 5 M methylurea and 0.5 M NaOH. The extinction coefficient of MCC-BP6-N7Gua in the same solution was 3.1 × 10-3 mL/µg. The molar ratio of BP-6-N7Gua to OA was 8.5:1 or, when normalized per 100 000 Da protein, 19:1. The immunogen, present as a fine suspension in PBS, was stored at 2-4 °C. The same chemistry was applied to synthesis of a coating complex consisting of MCC-BP-6-N7Gua coupled to bovine serum albumin (BSA). When added to PBS, the coating complex produced a clear solution which was stored at 2-4 °C. The molar ratio of adduct to BSA was 4.8:1 or, when normalized per 100 000 Da protein, 6.8:1. The coating complex served as the target for specific antibody binding in the competitive ELISA. Production and Screening of Mouse Hybridomas. Hybridomas were prepared according to procedures described by Zola (21). Male BALB/c mice were injected into each hind foot with 50 µg of immunogen in 20 µL of a 1:1 mixture of Freund’s adjuvant (Sigma Chemical Co., St. Louis, MO) and immunogen suspension. Two weeks later, the enlarged popliteal lymph nodes were excised, pooled, and then dissociated to a single-cell suspension. The lymph node cells were fused with P3X63-Ag.653 myeloma cells (American Type Culture Collection, Rockville, MD), and the fusion products were cultured in hypoxanthine-aminopterin-thymidine (HAT; Sigma Chemical Co.) selection medium containing Origen hybridoma cloning factor (IGEN, Rockville, MD). Approximately 2 weeks later, cultures were screened for adduct-specific MAbs by an indirect ELISA. The microwells of Nunc Maxisorp plates (Fisher Scientific Co., St. Louis, MO) were coated with 200 ng of coating complex in 100 µL of 0.1 M sodium carbonate buffer (pH 9.6) and then blocked with “blocking

Chem. Res. Toxicol., Vol. 9, No. 6, 1996 1039 buffer” consisting of 50 mM Tris buffer (pH 7.5) containing 200 mM NaCl, 0.05% (v/v) Tween 20, and 1% (w/v) IgG- and protease-free BSA (Bayer Inc., Kankakee, IL). Control wells were coated with nonadducted, 2-iminothiolane-modified BSA (I-BSA). Samples of the medium from each hybridoma culture were diluted 1:1 with “blocking buffer”; then 100 µL was added in duplicate to wells treated with coating complex or with I-BSA. Plates were incubated for 1 h at 37 °C and then washed with 50 mM Tris buffer (pH 7.5), containing 200 mM NaCl and 0.05% (v/v) Tween 20 (TBST). Bound MAbs were detected by addition of a mixture of goat antibodies conjugated with horseradish peroxidase and having specificity for mouse IgA, IgG, and IgM (Life Technologies Inc., Grand Island, NY). Plates were incubated for 1 h at 37 °C; then the substrate, o-phenylenediamine (Life Technologies Inc.), was added to the wells and the plate was incubated an additional 30 min at room temperature. Absorbances were read at 450 nm with a microkinetics reader (Bio-Tek Instruments Inc., Winooski, VT). Hybridoma cultures associated with an absorbance of 2.000 or more in the wells treated with coating complex and 0.100 (background) or less in the I-BSA-coated wells were expanded and cryopreserved (21). Selected cultures were cloned by a combination of limiting dilution (21) and microscopic selection of single-colony cultures. These clones were cryopreserved. Competitive ELISA for BP-6-N7Gua. The competitive ELISA was applied to both immunochemical characterization of selected MAbs and to detection and quantitation of BP-6N7Gua added to normal urine samples. Microwells of the ELISA plates were coated with 20 ng of coating complex in 100 µL of carbonate buffer. The plates were washed with TBST and then blocked for 4 h with “blocking buffer” at 37 °C. Reaction mixtures containing a BP-6-N7Gua-specific MAb and adduct (or other compound) were prepared during this period. Typically, medium from a 3-day hybridoma culture was diluted up to 1:2000 in Tris buffer (pH 7.5) containing 0.15 M NaCl (TBS) and then distributed in 500-µL aliquots to a series of 2-mL Eppendorf tubes. The tubes were placed in an Eppendorf manifold vortexer (Fisher Scientific Co.); then 5 µL of adduct (or other compound) dissolved in DMSO was added to duplicate tubes, with vortexing. DMSO alone was added to the control tubes. The tubes were sealed and incubated at 37 °C for 2 h. The blocked ELISA plates were washed with TBST, 100 µL of each reaction mixture was added to triplicate wells, and then the plates were incubated at 37 °C for 1 h. The plates were again washed with TBST, and then 100 µL of a secondary antibody preparation was added to each well. This preparation consisted of a mixture of polyclonal antibodies conjugated with alkaline phosphatase and having specificity for mouse IgA, IgG, and IgM (Life Technologies Inc.). After 1 h of incubation at 37 °C, the plates were washed with TBS and MAb binding was determined by a two-stage ELISA amplification system (Life Technologies Inc.). In the first stage, antibody-conjugated alkaline phosphatase converts NADPH to NADH. Subsequently, NADH is redox cycled to produce an accumulation of formazan dye. MAb binding was measured as absorbance at 490 nm. The I50 (quantity producing 50% inhibition of MAb binding to the coating complex) of selected compounds was determined by competitive ELISA. Inhibition of MAb binding, expressed as percent reduction of absorbance at 490 nm in comparison to the DMSO control, was plotted as a function of the logarithm of the quantity of compound per well in the ELISA plate. The 50% point was determined from the regression line of the linear portion of the inhibition curve. Determination of MAb Isotype and Quantitation by Immunoassay. MAb CB53, secreted by a cloned hybridoma, exhibited a high degree of discrimination between BP and BP6-N7Gua and a high affinity for BP-6-N7Gua, as indicated by an I50 of 750 fmol for BP-6-N7Gua. Consequently, this MAb was more fully characterized than other MAbs exhibiting lower specificity and affinity. The isotype of MAb CB53 was determined by a competitive ELISA incorporating three different alkaline phosphatase-conjugated secondary antibodies specific

1040 Chem. Res. Toxicol., Vol. 9, No. 6, 1996 for IgG, IgA, and IgM, respectively (Life Technologies Inc.). In addition, the concentration of MAb CB53 in the MAb-adduct reaction mixtures of the competitive ELISA was determined by an enzyme immunoassay for quantitation of mouse immunoglobulins in hybridoma culture media and ascites fluid (Boehringer Mannheim Biochemica, Indianapolis, IN). Preparation of a Human Urine Sample. A urine sample was obtained from one healthy volunteer with no history of smoking. The sample was stored in aliquots at -80 °C (16). Twenty-five milliliters of urine was brought to room temperature and then combined with 300 µL of DMSO containing 1680 pmol of BP-6-N7Gua. The urine sample was diluted to 50 mL with H2O and extracted five times with 50 mL volumes of CHCl3 (16). The precipitate was collected on filter paper and washed with 125 mL of CH3OH. The organic extracts were combined and evaporated to dryness under vacuum. The residue was dissolved in 300 µL of DMSO, and the undissolved material was removed by centrifugation. One-half of the DMSO solution was analyzed by competitive ELISA, and the remainder was analyzed by HPLC, as described below. HPLC Analysis of a Urine Extract Containing BP-6N7Gua. As previously described (16), the DMSO solution containing the urine extract was diluted with an equal volume of CH3OH and then sonicated to maximize solubilization of the adduct. A 75-µL sample was analyzed with a YMC, 5-µm ODSAQ reverse-phase analytical column (YMC, Overland Park, KS) coupled to a Waters 600E multisolvent delivery system and a Waters 700 satellite WISP autoinjector (Millipore Corp., Wood Dale, IL). The column was eluted at a flow rate of 0.8 mL/min, for 5 min with 30% CH3OH in water, followed by a 70-min linear gradient to 100% CH3OH. Peaks were detected by UV absorbance at 302 nm with a Waters 990 photodiode array detector, and elution times were compared with the peak produced by pure BP-6-N7Gua.

Results Coupling of BP-6-N7Gua to the Heterobifunctional Linker N-[(4-Carboxycyclohexyl)methyl]maleimide (MCC). Synthesis of an effective immunogen and a coating complex required development of a linker chemistry for coupling the hydrophobic BP-6-N7Gua via its 2-amino group to protein carrier (Figure 2). The commercially available succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) is highly reactive with weakly basic primary amines (e.g., protein lysyl amino groups) at pH 7.0-7.5 (22). However, the strongly basic character of the primary amine in the guanidino group of BP-6-N7Gua requires a pH >9.0 for the analogous reaction. At this pH, the activated succinimide group of SMCC is hydrolyzed to MCC, a product that does not react with primary amines. Consequently, the MCC linker was converted to the acid chloride, which was expected to be highly reactive with the guanidino amine of BP-6-N7Gua. The formation of this new heterobifunctional linker was initiated with the carboxylic acid MCC. When reacted with an excess of SOCl2, MCC was converted quantitatively to its acid chloride without destruction of the maleimide functional group (Figure 2). Reaction of the acid chloride with BP-6-N7Gua produced, in high yield, a conjugate consisting of MCC coupled with BP-6-N7Gua via an amide bond between the carboxyl chloride group of MCC and the 2-amino group of the adduct (Figure 2). Hybridoma Production and Preliminary Evaluation. Fifty-five of 602 hybridoma cultures, produced from two cycles of cell fusion, secreted antibodies highly selective for the coating complex (BP-6-N7Gua coupled to BSA via MCC). In the indirect ELISA, these antibodies produced a strong absorbance signal in wells treated

Casale et al.

Figure 3. Diversity of anti-BP-6-N7Gua MAb preparations as indicated by distinct inhibition curves in the competitive ELISA for BP-6-N7Gua. Each MAb preparation (CA15, CA74, CB11, CB16, CB25, and CB53) was diluted to produce an absorbance (490 nm) of 0.5-0.7 in the amplified ELISA and then incubated for 2 h at 37 °C with selected concentrations of BP-6-N7Gua. Subsequently, MAb binding to the coating complex, BP-6-N7Gua coupled to BSA, was determined by the amplified ELISA. Reduced MAb binding was expressed as % inhibition vs MAb binding in the absence of the adduct.

with coating complex and no signal in wells treated with chemically modified BSA lacking BP-6-N7Gua. Supernatants were collected from 3-day cultures of the adductspecific hybridomas and titrated for their potency in producing an absorbance signal in the amplified ELISA that served as the basis for the competitive ELISA. Seven of these supernatants produced an absorbance signal of 1.000 or greater at a dilution of 1:400. Four of these supernatants and two producing significantly weaker absorbance signals were selected for preliminary evaluation by competitive ELISA. Inhibition curves for BP-6-N7Gua were developed with all six MAb preparations (Figure 3). In general, the inhibition profiles were distinct, suggesting that these preparations contained unique antibodies. Though the curves generated with supernatants CB25 and CB16 were nearly superimposed, the inhibition profiles produced with BP were distinct (data not shown). The data suggest a diversity of antibody specificities among the adduct-specific hybridomas. Since supernatant CB53 exhibited the highest antibody-binding activity for BP-6-N7Gua (I50 ) 750 fmol), the corresponding culture was selected for further development and evaluation. Immunochemical Characterization of MAb CB53. Hybridoma CB53 was cloned from the corresponding hybrid selection culture, and the isotype of its MAb (MAb CB53) was determined to be IgG. The concentration of this mouse IgG in the MAb-adduct reaction mixture of the competitive ELISA was determined by quantitative immunoassay and found to be 72 pM. With this information, the I50 for BP-6-N7Gua, and an assumption of equilibrium conditions for the MAb-adduct reaction, an affinity constant of 1.4 × 108 M-1 was calculated for the binding of MAb CB53 to BP-6-N7Gua (23). In addition to binding with high affinity to BP-6-N7Gua, MAb CB53 discriminated very well between the DNA adduct and BP in the competitive ELISA (Figure 4). The I50 of BP (960 000 fmol) was ca. 1000 times the I50 of the adduct. The binding specificity of MAb CB53 was more fully characterized by competitive ELISA. Inhibition curves were developed for selected structures including BP metabolites, BP-DNA adducts (in addition to BP-6N7Gua), deoxyguanosine, and PAH (in addition to BP). I50s (Table 1) were determined by regression analysis of

MAb-Based ELISA for BP-6-N7Gua

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DMSO. A sample of this preparation was analyzed by HPLC, and a duplicate sample was diluted (serial 5-fold) with DMSO and the dilutions were assayed for BP-6N7Gua, by competitive ELISA. A plot of percent inhibition vs log dilution produced a straight line with 50% inhibition at a dilution of 1:5. Multiplying the I50 of BP6-N7Gua (750 fmol) by 5, we obtained a value of 3750 fmol of BP-6-N7Gua/µL of the extract or 1100 pmol of the adduct in the total extract (dissolved in 300 µL of DMSO). This value was in agreement with a total of 1100 pmol of the adduct determined by HPLC (Table 2). An undiluted extract of the same urine treated with DMSO alone produced no inhibition in the ELISA. Figure 4. Inhibition profiles of selected depurinating BP-DNA adducts and BP in the competitive ELISA for BP-6-N7Gua. Various concentrations of the competitors BP, BP-6-N7Gua, BP6-N7Ade, and BP-6-C8Gua were incubated with MAb CB53 for 2 h at 37 °C. MAb binding to the coating complex, BP-6-N7Gua coupled to BSA, was determined by an amplified ELISA. Competitor-mediated reduction of MAb binding was expressed as % inhibition vs untreated MAb and then plotted as a function of log quantity of competitor per well of the ELISA plate.

the linear portions of the inhibition curves. The presence of a hydroxyl group at position C-3 of BP (3-hydroxyBP, Figure 1) markedly reduced binding to MAb CB53 in comparison with BP, as indicated by an increased I50. When the hydroxyl group was replaced with an equally bulky but nonpolar methyl group (3-methylBP), binding was similar to that of BP. The reduced binding of 3-hydroxyBP was due to the polar nature of the hydroxyl group and was not due to steric hindrance. Substitution of hydroxyl groups at positions C-4 and C-5 of BP (BP4,5-dihydrodiol, Figure 1) also produced a marked reduction of binding, in comparison with BP. These findings suggest significant hydrophobic interaction(s) between the MAb binding site and the region consisting of C-3, C-4, and C-5 of BP in BP-6-N7Gua. The presence of hydroxyl groups at positions 7 and 8 or 9 and 10 of BP (BP-7,8-dihydrodiol and BP-9,10-dihydrodiol, Figure 1) enhanced binding ca. 100- or 50-fold, respectively, in comparison with BP. The magnitude and direction of these changes indicate significant hydrogen bonding to a site(s) in this region of the adduct. The antibody also bound with high affinity to BP-6N7Ade (Table 1), a depurinating BP-DNA adduct exhibiting a chemical configuration very similar to that of BP-6-N7Gua (Figure 1). Binding to BP-6-C8Gua, quantitatively the major depurinating DNA adduct of BP, was ca. 15 times weaker than binding to BP-6-N7Ade and ca. 30 times weaker than binding to BP-6-N7Gua. This result is expected in view of the significantly different chemical configurations of BP-6-C8Gua and BP-6-N7Gua (Figure 1). Quantitation of BP-6-N7Gua Added to Normal Human Urine. Comparison of the I50s of BP-6-N7Gua, BP, benzo[e]pyrene, benz[a]anthracene and chrysene (Table 1) suggests that parent PAH may not interfere with determination of BP-6-N7Gua by competitive ELISA. As a preliminary test of the utility of the competitive ELISA for quantitation of BP-6-N7Gua in biological samples, adduct was added to a sample of normal human urine and then quantified by competitive ELISA. Twentyfive milliliters of urine was warmed to room temperature and then combined with 300 µL of DMSO containing 1700 pmol of BP-6-N7Gua. The urine was extracted with chloroform and methanol (recovery of the adduct was ca. 70%), and the final residue was dissolved in 300 µL of

Discussion This paper describes the production of a monoclonal antibody (MAb CB53) with high specific affinity for the depurinating DNA adduct BP-6-N7Gua. The affinity constant for binding BP-6-N7Gua is 1.4 × 108 M-1, more than 1000 times the affinity constant for the parent compound BP. Both binding affinity and discrimination between the adduct and parent compound significantly exceed these same features of a monoclonal antibody (2B11) applied successfully to molecular dosimetry of aflatoxin-N7-guanine (AFB-N7Gua), a depurinating DNA adduct that serves as an effective biomarker of risk for aflatoxin-induced liver cancer (24, 25). The affinity constant for the binding of MAb 2B11 with AFB-N7Gua is 2 × 107 M-1, nearly 50 times lower than the affinity constant for the parent compound (26). MAb CB53 displays marked cross-reactivity with BP-6-N7Ade, another major depurinating adduct of BP, suggesting the possibility of immunochemical quantitation of both BP6-N7Gua and BP-6-N7Ade in a single assay. Crossreactivity with BP-trans-7,8-dihydrodiol and BP-trans9,10-dihydrodiol poses a challenge to the use of MAb CB53 in the competitive ELISA for direct quantitation of depurinating adducts. However, a preliminary reversephase cleanup of the samples is anticipated to eliminate these metabolites prior to immunochemical analysis. At present, MAb CB53 is under evaluation in both the competitive ELISA and an immunoaffinity/HPLC procedure for molecular dosimetry of adducts in samples of human and rat urine. For the past 15 years, there has been strong interest in developing molecular dosimetry of BP-DNA adducts as a measure of risk for cancers induced by PAH (9, 10, 27). During this period, emphasis has been placed on development of antibody-based molecular dosimetry of stable BPDE-DNA adducts and validation of this dosimetry as an instrument for determining cancer risk (9, 10, 27). The rationale for these efforts has been the view that formation of BPDE-DNA adducts is the primary event in initiation of cancers induced by BP (9, 10, 12). It has been proposed (9, 10) that these adducts would serve as useful biomarkers of biologically effective dose, i.e., the amount of agent-specific biological damage central to cancer production (8). The dosimetry for BPDE bound to DNA makes use of an ELISA that incorporates a polyclonal antiserum prepared against BPDE-DNA. The assay has been applied in many epidemiological studies (3-5, 27-30) aimed at validation of the dosimetry for cancer risk assessment. These studies share common features regardless of the exposed populations, e.g., cigarette smokers, foundry workers, or urban populations. First, the quantity of BPDE-DNA adducts of

1042 Chem. Res. Toxicol., Vol. 9, No. 6, 1996

Casale et al.

Table 1. Binding Specificity of MAb CB53 Characterized in the Competitive ELISA for BP-6-N7Gua compd

I50 (fmol)a

compd

I50 (fmol)a

BP-6-N7Gua BP-6-N7Ade BP-6-N3Ade BP-6-C8Gua deoxyguanosine 7-methylguanine

7.5 × 1.5 × 103 1.8 × 103 2.4 × 104 >1.7 × 108 >2.3 × 106

BP 3-methylBP 3-hydroxyBP BP-trans-4,5-dihydrodiol BP-trans-7,8-dihydrodiol BP-trans-9,10-dihydrodiol benzo[e]pyrene benz[a]anthracene chrysene

9.6 × 105 6.1 × 105 >1.5 × 107 >1.5 × 107 9.0 × 103 1.8 × 104 4.9 × 105 9.7 × 105 2.7 × 106

102

a The quantity of compound expressed as fmol per well producing 50% inhibition of the binding of MAb CB53 to the coating complex (BP-6-N7Gua coupled to BSA) in the competitive ELISA.

Table 2. Quantitation of the Depurinating DNA Adduct BP-6-N7Gua Added to a Sample of Human Urine: Evaluation of the Competitive ELISA for BP-6-N7Gua assay procedure

extract dilutiona

fmol of adduct in the assay

pmol of adduct in 25 mL of urine

HPLC ELISA50b ELISA30b

none 1/5 1/22

273 000 750 200

1100 1100 1300

a 1700 pmol of BP-6-N7Gua was added to 25 mL of human urine, and the sample was extracted with chloroform-methanol (ca. 70% recovery). b Adduct concentration was calculated from the extract dilution producing 30% (ELISA30) or 50% (ELISA50) inhibition of MAb binding in the competitive ELISA.

individuals in well-defined exposure groups varies widely from one individual to the next. Second, individual measurements of BPDE-DNA adducts do not correlate with individual exposures to BP. Third, mean BPDEDNA adducts of well-defined exposure groups generally display significant but weak correlations with group exposures. Disagreement between measured BP exposure and biologically effective dose determined as BPDE-DNA adducts may be, in part, a function of certain limitations of the ELISA for BPDE bound to DNA. First, the test DNA must be obtained from peripheral blood leukocytes (PBL). It is well established that cytochrome P450 metabolism in these cells is extremely low and is qualitatively different from cytochrome P450 metabolism in target tissues, e.g., lung (31, 32). Second, profiles of cytochrome P450 metabolism are significantly different among subsets of peripheral blood leucocytes, e.g., monocytes vs lymphocytes or antigen-stimulated vs resting lymphocytes (33, 34). These variables generally are not controlled. Third, the ELISA is not specific for BPDEDNA, detects a broad spectrum of PAH-DNA adducts, and cannot provide differential quantitative information about the adducts (including BPDE-DNA adducts) detected in the DNA matrix (27). The latter is particularly confounding for cancer risk assessment. Human exposures to PAH are highly complex, due to the diversity of carcinogenic compounds in PAH mixtures. The evidence suggests that these exposures produce a diversity of stable PAH-DNA adducts (35). Given the wide range of carcinogenic potencies of the compounds present in PAH mixtures, an undifferentiated measure of PAHDNA adducts provides little information about relative cancer risk and obviates identification of the best biomarkers for risk assessment. It is now clear that PAH produce, in addition to stable DNA adducts, depurinating adducts that are spontaneously released from the DNA (13-15, 36, 37). Depurinating adducts formed by one-electron oxidation are produced exclusively with double-stranded DNA, and not with single-stranded DNA or RNA (14, 36). For those

PAH studied thus far, depurinating adducts are the predominant DNA adducts formed. For example, depurinating adducts represent 71%, 85%, and 99% of all DNA adducts formed by BP, dibenzo[a,l]pyrene, and 7,12dimethylbenz[a]anthracene, respectively (14, 15, 36, 37). Importantly, recent findings indicate a major contribution of depurinating adducts to oncogenic mutations (17). Clearly, depurinating adducts offer unique advantages over BPDE-DNA adducts as biomarkers of PAH-induced DNA damage and risk of PAH-induced cancers. Depurinating adducts are more effective in immunochemical analyses since they can function as classical haptens that (1) induce highly specific antibodies when linked to a protein carrier and (2) bind with high specificity to the corresponding antibodies. Since these adducts are present in urine as free molecules, they obviate the need to probe the DNA matrix and can be quantified individually. Molecular dosimetry of depurinating adducts is a promising new approach to determining risk of PAH-induced cancers.

Acknowledgment. This research was supported by U.S. Public Health Service Grant RO1 CA25176 from the National Cancer Institute and an American Cancer Society Special Institutional Grant on Cancer Cause and Prevention.

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