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Chem. Res. Toxicol. 1997, 10, 948-952
Development of a Monoclonal Antibody Recognizing Benzo[c]phenanthrenediol Epoxide-DNA Adducts: Application to Immunohistochemical Detection of DNA Damage Yu Jing Zhang,† Nicholas E. Geacintov,‡ and Regina M. Santella*,† Division of Environmental Health Sciences/Cancer Center, Columbia University, New York, New York 10032, and Chemistry Department and Radiation and Solid State Laboratory, New York University, New York, New York 10003 Received April 4, 1997X
A monoclonal antibody was developed against (()-anti-benzo[c]phenanthrenediol epoxidemodified DNA, and sensitivity and specificity were determined by competitive enzyme-linked immunosorbent assay (ELISA). Antibody 10F9 has 50% inhibition in the ELISA at 50 fmol of B[c]PhDE-DNA adducts. There was weak cross-reactivity with DNA modified by (()-antibenzo[a]pyrenediol epoxide (50% inhibition at 150 pmol). Testing of oligonucleotides containing either (+)- or (-)-trans-anti-B[c]PhDE-adenine adducts indicated similar recognition of both stereoisomers. A quantitative immunoperoxidase technique with antibody 10F9 was developed using 10T1/2 cells treated with B[c]PhDE then piloted on exfoliated oral cells from five smokers and five nonsmokers. Mean staining in smokers (184 ( 11) was 1.64-fold higher than in nonsmokers (112 ( 9, p < 0.0001). This antibody should be useful for the detection and quantitation of B[c]PhDE-DNA adducts in cell culture and animal studies and in humans with environmental or occupational exposure to polycyclic aromatic hydrocarbons.
Introduction Polycyclic aromatic hydrocarbons (PAHs) are a major class of chemicals ubiquitous in the environment and causally associated with the development of cancer (1). Benzo[c]phenanthrene is normally present in all PAH mixtures including cigarette smoke (2), and air concentrations are comparable to those of benzo[a]pyrene (BP) (3). The bay region diol epoxides (B[c]PhDE)1 are among the most potent mammalian cell mutagens and tumor initiators of all PAH diol epoxides tested (4, 5). B[c]PhDE reacts extensively with deoxyadenosine resulting in about 65% of the adducts at this base with the remainder at deoxyguanosine (6-8). These deoxyadenosine adducts may be important in tumor induction in animals (6, 9). For example, mutations in codon 61 of the Ha-ras oncogene family of mouse skin tumors induced by (()anti-B[c]PhDE were predominantly A f T (10). This is in contrast to tumors resulting from treatment with BP or 3-methycholanthrene which contains a higher percentage of G f T mutations in codons 12 and 13 than A f T mutations in codon 61 (5, 11, 12). * To whom reprint requests should be addressed at 701 West 168th St., New York, NY 10032. Phone/Fax: 212 305-1996. E-mail: rps1@ columbia.edu. † Columbia University. ‡ New York University. X Abstract published in Advance ACS Abstracts, August 1, 1997. 1 Abbreviations: B[c]PhDE, (()-anti-3,4-dihydroxy-1,2-epoxy-1,2,3,4tetrahydrobenzo[c]phenanthrene; BPDE, (()-anti-7,8-dihydroxy-9,10epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; B[k]FDE, (()-anti-8,9-dihydroxy-10,11-epoxy-8,9,10,11-tetrahydrobenzo[k]fluoranthene; 1,2,3,4BADE, (()-anti-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydrobenz[a]anthracene; 8,9,10,11-BADE, (()-anti-8,9-dihydroxy-10,11-epoxy-8,9,10,11tetrahydrobenz[a]anthracene; CHDE, (()-anti-1,2-dihyroxy-3,4-epoxy1,2,3,4-tetrahydrochrysene; DBADE, (()-anti-10,11-dihyroxy-12,13epoxy-10,11,12,13-tetrahydrodibenz[a,c]anthracene; ELISA, enzymelinked immunosorbent assay; FCS, fetal calf serum; mBSA, methylated bovine serum albumin; PBS, phosphate-buffered saline.
S0893-228x(97)00050-7 CCC: $14.00
Sensitive and specific methods are available to quantitate PAH-DNA damage in biological samples. These methods include immunoassays, gas chromatography/ mass spectroscopy, 32P postlabeling, and fluorescence spectroscopy (reviewed in refs 13 and 14). Several polyclonal antisera and monoclonal antibodies recognizing BP diol epoxide-modified DNA have been developed (15-17) and used in enzyme-linked immunosorbent assays (ELISA) to detect adducts produced in vitro and in blood and tissue samples of humans with occupational, lifestyle, dietary, and environmental exposure (reviewed in ref 13). More recently, a polyclonal antiserum to B[c]PhDE-DNA was also developed and characterized (18). Here, we report the development of a monoclonal antibody specific for B[c]PhDE-DNA and a sensitive ELISA for quantitation of DNA adduct levels. In addition, an immunoperoxidase method was established for detection of adducts in tissue samples and piloted on oral cells of smokers and nonsmokers.
Materials and Methods Chemicals. Goat anti-mouse IgG-alkaline phosphatase, DNase, proteinase K, RNase, p-nitrophenyl phosphate (Sigma 104), and methylated bovine serum albumin (mBSA) were purchased from Sigma Chemical Co., St. Louis, MO. Ham’s F12 and DMEM media were purchased from Grand Island Chemical Co., Grand Island, NY. Fetal calf serum (FCS) was purchased from Sterile System, Logan, Utah. (()-anti-B[c]PhDE used for synthesis of oligonucleotides was synthesized by Dr. S. Amin, American Health Foundation, while that used for treatment of cells was obtained from Dr. R. Harvey, University of Chicago. DNAs modified by BPDE, DBADE, CHDE, 8,9,10,11-BADE, 1,2,3,4-BADE, and B[k]FDE were a generous gift from Dr. Ainsley Weston, Mt. Sinai Medical Center, New York, NY. Oligonucleotides modified by B[c]PhDE were prepared as described (19). Poly(dG-dC) was also modified and the level of adducts estimated from the fluorescence emission at 398 nm
© 1997 American Chemical Society
Monoclonal Antibody Recognizing B[c]PhDE-DNA Adducts following excitation at 346 nm using a SPEX Fluorolog 1902 fluorometer. The modification level was estimated to be 4.6 ( 1.5 adducts/100 nucleotides based on the assumption that the fluorescence yield is the same as in calf thymus DNA. Synthesis of Immunogen and Immunization. B[c]PhDEDNA (2.1 adducts/100 nucleotides) was prepared by reaction of (()-anti-B[c]PhDE with calf thymus DNA for 16 h followed by extraction with water-saturated ethyl acetate and precipitation with ethanol. The modified DNA was denatured by boiling for 3 min and cooling on ice and then complexed with an equal weight ratio of mBSA. Female BALB/c mice (6-8 weeks old) were immunized ip with 200 µg of the complex in complete Freund’s adjuvant during week 1 and in incomplete adjuvant during weeks 3 and 5. During week 7, sera were assayed by ELISA as described below. Three days before fusion 100 µg of complex without adjuvant was administered iv into the tail vein. Cell fusion and screening of hybridomas were carried out essentially as described previously (16), and supernatants were screened by noncompetitive ELISA after 2 weeks as described below. Positive wells were subcloned in agarose with a feeder layer of cloned rat embryo fibroblast cells. Enzyme-Linked Immunosorbent Assay (ELISA). The hybridoma supernatants were screened by a noncompetitive ELISA as described (16). Polystyrene 96-well microwell plates were coated with 5 ng of denatured B[c]PhDE-DNA (2.1 adducts/ 100 nucleotides) in PBS by drying at 37 °C overnight. Plates were washed with PBS containing 0.05% Tween 20 with an automatic plate washer (Titertek-microplate washer, ICN-Flow, Alexandria, VA) set for three 200 µL washes. This wash step was repeated after each incubation. Hybridoma supernatants (100 µL) were transferred to the microtiter plates and incubated for 1.5 h at 37 °C. Plates were washed and incubated with 100 µL of goat anti-mouse IgG-alkaline phosphatase (1:750 dilution; Boehringer-Mannheim, Indianapolis, IN). A final wash with PBS-Tween and two manual washes with 0.01 M diethanolamine were followed by addition of 100 µL of p-nitrophenyl phosphate (1 mg/mL) in 1 M diethanolamine (pH 8.6), and absorbance at 405 nm was read on a Dynatech MR5000 microplate reader (Dynatech Laboratories, Chantilly, VA). Positive supernatants were rescreened in three wells: one blank uncoated well, one nonmodified DNA-coated well, and one well coated with B[c]PhDE-DNA. Isotype determination was carried out with a mouse monoclonal isotyping kit from Amersham, Arlington, IL. For the competitive ELISA, plates were coated with 20 ng of denatured B[c]PhDE-DNA (2.1 adducts/100 nucleotides). Diluted hybridoma supernatant (50 µL of 1:50000 for 10F9) was mixed with 50 µL of serially diluted competitor (single-stranded oligonucleotide or heat-denatured DNA) before addition to the wells. All other steps were as in the noncompetitive ELISA. Immunohistochemical Staining. Antibody 10F9 was used for immunoperoxidase staining essentially as described previously (20). Briefly, C3H 10T1/2 cells were cultured in eightchambered slides (Nunc Inc., Naperville, IL) in DMEM medium containing 10% FCS. When the cells were 50-70% confluent, they were treated with 0, 3.6, 18, 36, and 72 µM B[c]PhDE for 3 h at 37 °C. After treatment, cells were washed with PBS and fixed with 70% ethanol. Slides were washed with PBS, treated with RNase (100 µg/ml) at 37° for 1 h, washed with PBS, treated with proteinase K (10 µg/ml) at room temperature for 7 min, and then washed. To denature the DNA, slides were incubated with 4 N HCl for 7 min at room temperature and then with 50 mM Tris base for 5 min at room temperature. After washing with PBS, slides were incubated with 0.3% H2O2 in methyl alcohol at room temperature for 30 min to quench endogenous peroxidase activity. Nonspecific binding was blocked with 1.5% normal horse serum, and then slides were incubated with monoclonal antibody 10F9 (1:50 dilution in 1.5% horse serum) overnight at 4 °C. Elite mouse ABC and DAB kits plus NiCl2 (Vector Laboratories, Burlingame, CA) were used for visualization of bound antibody as directed by the manufacturer. Slides were dehydrated and cleaned in serial ethyl alcohol and xylene and mounted with Permount (Fisher Scientific, Pittsburgh, PA).
Chem. Res. Toxicol., Vol. 10, No. 9, 1997 949 Table 1. Competitive Inhibition of Antibody 10F9 Binding to B[c]PhDE-DNA modification level (adducts/100 nucleotides) (()-anti-B[c]PhDE-DNA (()-anti-B[c]PhDE-poly(dG-dC) CTCTCA(+)-trans-anti-B[c]PhDECTTCC CTCTCA(-)-trans-anti-B[c]PhDECTTCC (()-anti-BPDE-DNA (()-anti-1,2,3,4-BADE-DNA
2.1 4.6 9.1
(()-anti-8,9,10,11-BADE-DNA (()-anti-CHDE-DNA (()-anti-B[k]FDE-DNA (()-anti-DBADE-DNA
2.3 2.2 0.5 1.0
a
9.1 1.1 0.4
50% inhibition (mol of adduct) 50 fmol 260 fmol 680 pmol (43% inhibition) 550 pmol (38% inhibition) 150 pmol 900 fmol (40% inhibition) >3400 fmola >5800 fmola >900 fmola >2050 fmola
No inhibition at the highest concentration tested.
To demonstrate the specificity of the staining, cells were pretreated with DNase (100 µg/mL for 1 h at 37 °C) before staining or stained with a nonspecific antibody (8G1; 1:10 dilution of hybridoma supernatant) recognizing DNA damage produced by the photoactivated drug 8-methoxypsoralen (21) or with antibody preabsorbed with B[c]PhDE-DNA (1 µg/µL) for 20 min at room temperature before use. A Cell Analysis System 200 microscope (Becton Dickinson, San Jose, CA) was used to measure the relative intensity of nuclear staining in 50 randomly selected cells using the Cell Measurement Program software package. Data are presented as the object average optical density multiplied by 1000. To verify that the method has sufficient sensitivity for detection of damage in humans, buccal cell smear slides from five smokers of g20 cigarettes/day and five nonsmokers were stained with 10F9 as for the cells treated in vitro except that a 1:10 dilution of primary antibody was used and the DAB solution was used without NiCl2. DNase pretreatment and preabsorption of antibody with B[c]PhDE-DNA were carried out as above.
Results Hybridoma cell lines from Balb/c mice immunized with (()-trans-B[c]PhDE-modified DNA (about 2 adducts/100 nucleotides) were established, and the most sensitive clone, 10F9, was further characterized. Isotype classification indicated an IgG1 κ isotype. Competitive ELISA was used to determine the specificity and sensitivity of the antibody. Antibody 10F9 binding to B[c]PhDE-DNA was inhibited 50% by 50 fmol of (()-antiB[c]PhDE-DNA adducts in the modified DNA (Table 1). There was weak cross-reactivity with DNA modified by (()-anti-benzo[a]pyrenediol epoxide (50% inhibition at 150 pmol). There was no cross-reactivity with DNAs modified with diol epoxides of chrysene, benzo[k]fluoranthrene, benzo[b]fluoranthrene, and dibenz[a,c]anthracene at the highest concentrations tested (1-6 pmol) in which adduct levels were 20-120-fold higher than the concentration of B[c]PhDE-DNA adduct which resulted in 50% inhibition. Antibody cross-reactivity was also tested with oligonucleotides (CTCTCACTTCC) containing either (-)or (+)-trans-anti-B[c]PhDE adducts on the single adenine. Similar binding was observed with both oligomers [(+)-trans-oligonucleotide, 43% inhibition at 680 pmol of adduct; (-)-trans-anti-oligonucleotide, 38% inhibition at 550 pmol]. Since oligonucleotides containing guanine adducts were not available, recognition of guanine adducts was tested using B[c]PhDE-poly(dG-dC) and indicated 50% inhibition at approximately 260 fmol of adduct. Cross-reactivity with both native and denatured non-
950 Chem. Res. Toxicol., Vol. 10, No. 9, 1997
Zhang et al.
Figure 1. Immunoperoxidase staining of 10T1/2 cells for B[c]PhDE-DNA adducts with monoclonal antibody 10F9. Cells where treated with 36 µM (A), 3.6 µM (B), and 0 µM (C) B[c]PhDE. Cells were treated with 72 µM B[c]PhDE but stained with antibody 10F9 preabsorbed with B[c]PhDE-DNA before use (D) (400×; reproduced at 60% of original).
modified calf thymus DNA was also tested, but there was no reactivity with up to 50 µg of nonmodified DNA/well. To determine whether the antibody could be used for immunohistochemical detection of adducts in biological samples, 10T1/2 cells were treated with 0, 3.6, 18, 36, and 72 µM B[c]PhDE for 3 h at 37 °C. Cells were treated with RNase, proteinase K, and 4 N HCl as detailed in Materials and Methods to increase assay sensitivity and eliminate potential cross-reactivity with RNA adducts. Specific nuclear staining was observed in cells treated with 36 µM B[c]PhDE (Figure 1A), with weaker staining in cells treated with 3.6 µM B[c]PhDE (Figure 1B) and minimal background staining in control cells (Figure 1C). Intensity of nuclear staining was determined for 50 randomly selected cells for each dose. Cells treated with 0, 3.6, 18, 36, and 72 µM B[c]PhDE had staining intensities of 151 ( 42, 189 ( 82, 218 ( 55, 328 ( 101, and 446 ( 128, respectively (Figure 2). Cells treated with 72 µM B[c]PhDE and stained with antibody preabsorbed with B[c]PhDE-DNA before use had minimal background nuclear staining (145 ( 35, Figure 1D). Also showing minimal staining were 72 µM B[c]PhDE-treated cells incubated with DNase before staining (116 ( 32) or stained with a nonspecific primary antibody recognizing 8-methoxypsoralen-DNA (115 ( 34, not shown). To determine whether the assay has sufficient sensitivity to detect exposure in humans, a pilot study was carried out on exfoliated oral cells of five smokers of g 20 cigarettes/day and five nonsmokers. Representative nuclear staining in cells from a smoker and nonsmoker is shown in Figure 3. Mean staining intensity in the five smokers was 184 ( 11 and ranged from 165 ( 74 to 197 ( 44, while in the five nonsmokers it was 112 ( 9 and ranged from 100 ( 64 to 123 ( 94. Thus, smokers had a mean staining intensity 1.64-fold higher than nonsmokers (p < 0.0001). To test the specificity of staining,
Figure 2. Dose-response curve for relative immunoperoxidase staining of 10T1/2 cells for B[c]PhDE-DNA adducts with antibody 10F9 versus dose of B[c]PhDE.
cells from a smoker were stained after pretreatment of slides with DNase or preabsorption of the antibody with B[c]PhDE-DNA. Staining decreased from 167 ( 40 to 122 ( 31 after DNase treatment and to 72 ( 41 after preabsorption.
Discussion Monoclonal antibody 10F9 has high sensitivity and specificity for B[c]PhDE-DNA (50% inhibition at 50 fmol). Both adenine and guanine adducts are formed in modified DNA, but only oligonucleotides containing adenine adducts were available for testing. For this reason, B[c]PhDE-poly(dG-dC) was also tested and found to crossreact with 50% inhibition at 260 fmol. This value is an estimate since modification levels were based on fluo-
Monoclonal Antibody Recognizing B[c]PhDE-DNA Adducts
Figure 3. Representative immunoperoxidase staining for B[c]PhDE-DNA adducts with monoclonal antibody 10F9 of exfoliated oral cells from a smoker (A) and a nonsmoker (B).
rescence measurements (see Materials and Methods) but indicates that there is significant recognition of guanine adducts. The antibody exhibited poor cross-reactivity with DNA modified by BPDE (50% inhibition at 150 pmol) and did not cross-react with nonmodified DNA. There was weaker or no cross-reactivity with DNAs modified by several other PAH diol epoxides, but adduct concentrations only 20-120-fold higher than the 50% inhibition value for B[c]PhDE-DNA adducts were tested due to limited amounts of sample. Cross-reactivity with oligonucleotides containing either (+)- or (-)-trans-antiB[c]PhDE adduct on adenine indicated equal recognition of both adducts. However, several orders of magnitude higher levels of adduct were required for 50% inhibition with the oligonucleotides than for B[c]PhDE-DNA. Similar results, weaker recognition of damage in oligonucleotides compared to DNA, were observed for antibodies recognizing thymidine dimers and suggest that the antibody recognizes some of the surounding DNA structure (22). Previously, a polyclonal antiserum was developed against B[c]PhDE-DNA (18). While monoclonal antibody 10F9 is slightly more sensitive than the polyclonal antiserum (50% inhibition at 200 fmol of B[c]PhDE-DNA adduct), its major advantage is that large amounts can be produced making it more readily available. For example, 8E11, one of our monoclonal antibodies recognizing BPDE-DNA, has been used by a number of researchers for adduct quantitation by immunoassay or adduct purification by immunoaffinity chromatography (e.g., see refs 23-25). Preliminary data with plasmids modified with B[c]PhDE indicate that antibody 10F9 will also be useful for immunoaffinity purification of modified DNA fragments. An immunoperoxidase method for quantitation of adducts using antibody 10F9 was developed in 10T1/2
Chem. Res. Toxicol., Vol. 10, No. 9, 1997 951
cells treated with B[c]PhDE. A dose-response was observed between relative staining intensity and dose of B[c]PhDE (Figure 2). In addition, the small pilot study indicates that the assay has sufficient sensitivity to detect human exposure to cigarette smoke. Significantly higher levels of damage were seen in smokers compared to nonsmokers, and in this small sample size, there was no overlap in staining intensity between smokers and nonsmokers. The mean staining in smokers was 1.64fold higher than in nonsmokers, similar to the 1.8-2fold difference observed in oral cells using a rabbit antiserum against BPDE-DNA (20, 26). In the competitive ELISA, approximately 3000-fold higher concentrations of BPDE-DNA than B[c]PhDE-DNA adducts are required for 50% inhibition. This suggests that the assay is detecting B[c]PhDE-DNA adducts and not those of BPDE-DNA which are known to be present in human cells (20, 26). However, it is also possible that other PAH diol epoxide adducts that have not been tested give significant cross-reactivity. The quantitative immunohistochemical method allows analysis of DNA modification in small biopsies or single cells such as exfoliated oral or bladder cells. One limitation is that absolute adduct levels cannot be determined and only relative staining intensity is measured. While the ELISA has been used much more extensively to monitor human exposure to chemical carcinogens, there are some reports using immunohistochemical methods. We used an immunofluorescence method to monitor aflatoxin B1-DNA adducts in liver tissue from hepatocellular carcinoma patients obtained at the time of surgery (27) or at the time of needle biopsy, for confirmation of diagnosis (28). Antisera to BPDEDNA were used to detect adducts in lung, cervix, and placental tissue (29), in skin biopsies of coal tar treated psoriasis patients (30), in bronchial cells of smokers (31), in oral mucosa cells of smokers and nonsmokers (20), and in lymphocytes of coke oven workers and controls (32). Chemotherapy-induced adducts of cis-diamminedichloroplatinum(II) (33) and N-ethyl-N-nitrosurea (34) have also been measured. Taken together, these studies demonstrate the utility of the method for molecular epidemiology studies.
Acknowledgment. This research was supported by NIH Grant ES05294 and the Lucille P. Markey Charitable Trust. The B[c]PhDE-modified oligonucleotides were prepared at New York University as part of a project supported by the Department of Energy, Office of Health and Environmental Research, Grant DE-FG0286ER60645. The authors express their appreciation to A. Cartagena for secretarial assistance.
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