Chem. Res. Toxicol. 1996,8,747-752
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Quantitative Immunohistochemical Analysis of 4-Aminobiphenyl-DNA in Cultured Cells and Mice: Comparison to Gas ChromatographyLMass Spectroscopy Analysis Jenan Al-Atrash,’ Yu-Jing Zhang,+Dongxin Lin,; Fred F. Kadlubar,$and Regina M. Santella*s‘ Cancer Center /Division of Environmental Science, School of Public Health, Columbia University, New York, New York 10032, and National Center for Toxicological Research, Jefferson, Arkansas 72079 Received January 25, 1995@
Two monoclonal antisera, 4C11 and 3C8, recognizing 4-aminobiphenyl(4-ABP)-DNAadducts were developed and characterized by competitive enzyme-linked immunosorbent assay (ELISA). Both antisera are highly specific for 4-ABP-DNA and, a t the highest concentration tested, do not recognize the DNA adducts of several other aromatic amines tested including l-aminopyrene, 8-nitro-l-aminopyrene, and 6-nitro-l-aminopyrene. An immunohistochemical method for detecting adducts was developed in R52 cells, a mouse NIH3T3 cell line expressing high levels of cytochrome P450 1A2. Quantitation of fluorescence labeling indicated a dose-related increase in staining in cells treated with 0,6,30,60, and 300 pM 4-ABP. To apply the method to tissue samples, Balb/c mice were treated with 0, 4, 10, 20, 40, and 80 m&g 4-ABP and liver, bladder, and lung tissue analyzed by immunohistochemical staining of tissue sections. There was a dose-related increase in specific nuclear staining in liver and bladder tissues with no detectable staining in lung tissue. DNA from liver tissue was also analyzed by alkaline hydrolysis of 4-ABP, derivatization with pentafluoropropionic anhydride, and gas chromatography/mass spectroscopy analysis. A good correlation (r = 0.98, p 0.0001) was found between DNA damage levels determined by the two methods. Based on adduct levels determined by GC/MS in both R52 cells and liver tissue, the immunohistochemical method has a limit of sensitivity of approximately 1 a d d ~ c t / l O ~nucleotides. -~ Immunohistochemistry should be useful for analysis of 4-ABP-DNA adducts in human tissue biopsies as well as exfoliated cells from the oral mucosa and urinary bladder.
Introduction 4-Amin0bipheny1(4-ABP),~ an aromatic amine present in cigarette smoke, is an established animal carcinogen and in humans has been associated with urinary bladder cancer (I). 4-ABP is metabolized in vivo to a reactive N-hydroxy arylamine which binds covalently to DNA; the major adduct results from binding at the C8 position of guanine ( 2 ) . Several methods have been developed to quantitate 4-ABP-DNA adducts in humans including [32P]postlabelingand immunoassays. Using the postlabeling method, 4-ABP-DNA adducts have been identified both in biopsies of urinary bladder and in exfoliated urothelial cells and found to be elevated in smokers compared to nonsmokers (3-5). A polyclonal antiserum to N-(guanosin-8-yl)-ABP coupled to keyhole limpet hemocyanin has also been developed (6)and used to monitor adduct levels in human lung and urinary bladder tissues (7,8). Monoclonal antibodies were also prepared against 4-ABP coupled to protein and shown to have high * To whom correspondence and reprint requests should be addressed at the Division of Environmental Science, School of Public Health, Copmbia University, 701 W. 168th St., New York, hY 10032. Columbia University. National Center for ToxicologicalResearch. Abstract published in Advance ACS Abstracts, June 1, 1995. Abbreviations: 4-ABP, 4-aminobiphenyl; DAPI, 4’,6-diamidino-2phenylindole dihydrochloride; ELISA, enzyme-linked immunosorbent assay: FCS, fetal calf serum; FITC, fluorescein isothiocyanate; GC/ MS, gas chromatography/mass spectroscopy;Isom,, 5 0 8 inhibition; PBS, phosphate-buffered saline (0.17 M NaC1,3.4 mM KCl, 10 mM sodium phosphate, pH 7.0): PFPA, pentafluoropropionic anhydride.
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affinity for both 4-ABP adducts and metabolites (9). Recently, a gas chromatography/mass spectrometry (GC/ MS) method was developed and used to corroborate the presence of 4-ABP-DNA adducts in human lung and bladder (10). Antisera recognizing carcinogen-damaged DNA can also be applied to quantitative immunohistochemical analysis of DNA adducts. A major advantage is the requirement for small amounts of material, making the method ideal for studies of biopsy or exfoliated cell samples. To develop a sensitive quantitative immunohistochemical method applicable for analysis of 4-ABPDNA adducts in human samples, monoclonal antisera were generated from animals immunized with 4-ABPDNA. An immunohistochemical assay was developed in R52 cells expressing high levels of cytochrome P450 1A2 and then applied to the detection of adducts in liver, bladder, and lung of treated mice.
Materials and Methods Antibody Development. 4-AT3P modified calf thymus DNA (0.18% and 3% modified) was supplied by Dr. Fred Beland (National Center for Toxicological Research, Jefferson, AR). Due to its heat instability, 4-ABP-DNA was denatured with formamide as described before immunization ( 2 1 ) . The denatured DNA was complexed to a n equal weight ratio of methylated bovine serum albumin before emulsification with Freund‘s adjuvant. Female Balb/c mice (6-8 weeks old) were immunized ip with 200 ,ug of the complex in complete Freund’s adjuvant during week 1,and in incomplete adjuvant during weeks 3 and
0893-228X/95/2708-0747$09.o0/0 0 1995 American Chemical Society
748 Chem. Res. Toxicol., Vol. 8, No. 5, 1995 5. During week 7, sera were assayed by ELISA as described below. Three days before fusion, 100 pg of complex without adjuvant was administered iv. Spleen cells were fused with the myeloma cell line P3 X 63-AG.8.653 and subcloned on agarose as described previously (12). Enzyme-Linked Immunosorbent Assay (ELISA). The hybridoma supernatants were screened by a noncompetitive ELISA as described (12). Briefly, polystyrene U-bottomed microwell plates (Corning no. 25805, Corning, NY) were coated with 5 ng of native 4-ABP-DNA (0.18% modified) in phosphatebuffered saline (PBS) by drying at 37 "C overnight. Plates were washed with PBS containing 0.05% Tween with an automatic plate washer (Titertek-Microplate washer, ICN-Flow, Alexandria VA) set for three 200 pL washes. This wash step was repeated after each incubation. Nonspecific binding was blocked with 200 pL of 1%FCS in PBS-Tween for 1 h at 37OC. Hybridoma supernatants (100 pL) were transferred to the microtiter plates and incubated for 1.5 h at 37 "C. Plates were washed and incubated with 100 pL of goat anti-mouse IgGalkaline phosphatase (1:750 dilution, Boehringer Mannheim, Indianapolis, IN) for another 1.5 h. A final wash with PBSTween and two manual washes with 0.01 M diethanolamine were followed by addition of 100 ,uL ofp-nitrophenyl phosphate (1 mg/mL) in 1 M diethanolamine (pH 8.6, Sigma, St. Louis, MO). Absorbance at 405 nm was recorded using a Dynatech MR 5000 microplate reader (Dynatech Laboratories, Chantilly, VA). Supernatants from positive wells were rescreened on plates coated with 5 ng of 4-ABP-DNA, DNA and blank (PBS). Sera from immunized mice were also titered by this method. A competitive ELISA was used to determine antibody sensitivity and specificity. Plates were coated with 5 ng of 4-ABPDNA. After blocking, 50 ,uL of competitor in PBS was added followed by 50 ,uL of diluted supernatant (1:15000 for antibody 4Cl1, 1:500 for 3C8). Goat anti-mouse IgG-alkaline phosphatase was used at 1:750 dilution. Specificity was determined by testing for cross-reactivity with formamide-denatured l-aminopyrene-DNA, 8-nitro-l-aminopyrene-DNA, and 6-nitro-laminopyrene-DNA prepared as reported previously (13). Other carcinogen-modified DNAs available in the laboratory, including heat-denatured aflatoxin BI-DNA and benzo[alpyrene diol epoxide-DNA, were also tested. Treatment of Rs2 (P450IA2) Cells with 4-ABP. To develop the quantitative immunohistochemical protocol, R52 cells, a mouse NIH3T3 cell line expressing high levels of cytochrome P450 1A2 (from Drs. S. Thorgeirsson and K. Nouso, National Cancer Institute), were cultured in Ham's F12 (GIBCO, Grand Island, N Y ) supplemented with 146 mg/L L-glutamine and 400 pg/mL geneticine (G418). Cells were cultured to 70% confluence in 8-chambered slides (Nunc, Napperville, IL) then treated with 0, 6, 30, 60, and 300 p M 4-ABP. 4-ABP is a n established carcinogen and should be handled with appropriate precautions. After 24 h, media was aspirated, and the wells were washed with PBS before fixing in 70% ethanol at room temperature. Animal Experiments. Balb/c female mice (3 miceldose) were treated with a single ip dose of 0, 4, 10, 20, 40, and 80 m g k g of 4-ABP in corn oil plus 0.1% ethanol and maintained on a normal diet. After 24 h animals were sacrificed by cervical dislocation and liver, lung, and bladder tissues removed and frozen at -80 "C. DNA was extracted by standard proteinase K and RNase treatments and phenol and chlorofodisoamyl alcohol extraction. Concentration was determined from the absorbance at 260 nm (1A260nm = 50 pg/mL).
Immunofluorescence Quantitation of 4-ABP-DNAAdducts. Prior to tissue mounting, slides were coated with poly(D-lysine) (2 pg/mL deionized water) and dried at 37 "C for 2 h. Frozen tissues were sectioned (6 pm) on a cryostat (ReichertJ u n g 1800, Germany) and fixed in cold (-20 "C) 70% ethanol for 20 min. Both cells and tissue sections were washed for 5-10 min with PBS then incubated at 37 "C for 1 h with 100 pg/mL RNase. After washing, slides were treated with 10 pg/mL proteinase K for 10 min at room temperature to remove histone and non-histone proteins from DNA to increase antibody accessibility. After washing, DNA was denatured with 4 N HC1 for 5 min a t room temperature, neutralized with 50 mM Trizma
Al-Atrash et al. base at room temperature for 5 min, and washed with PBS. Nonspecific binding was blocked with 10% normal goat serum for 1h at 37 "C. After removing blocking buffer, antibody 4 C l l [1:70 dilution in 20% fetal calf serum (FCS)-PBS-Tween 20 for liver, lung, and bladder tissues and 1:600 for R52 cells] was added for 45 min at 37 "C. f i r incubation, slides were washed, and anti-mouse IgG conjugated with fluorescein isothiocyanate (1:40 dilution in 20% FCS-PBS, Boehringer Mannheim, Indianapolis, IN) was incubated for 45 min at 37 "C. Following washing, slides were incubated for 1 h a t 37 "C with 3 pg/mL DAPI (4',6-diamidino-2-phenylindoledihydrochloride, Polysciences, Warrington, PA) to localize nuclei. Slides were mounted using a solution of 50 mM Tris (pH 8.5)/glycerol (1: 10) containing 1 mg/mL p-phenylenediamine. Combined fluorescein isothiocyanate (F1TC)-DAPI staining was observed with a Nikon Optiphot microscope (Nikon, Tokyo). For measurement of FITC, a 450-490 nm excitation filter, with a 510 nm dichroic mirror and 520-560 nm barrier filter, was used. For DAPI fluorescence, a 330-380 nm excitation filter, 400 nm dichroic mirror, and 420 nm barrier filter were used. Photographs were taken with Ektachrome 400 slide film, and fluorescence intensity of nuclei was measured from color slides using a Molecular Dynamics 300A densitometer (Molecular Dynamics, Sunnyvale, CA) using the Image Quant program essentially as described (14). To minimize variability, all samples of the same tissue type were stained on the same day and photographed with the same role of film. Considerable cell variation was observed in FITC staining. To ensure that representative cells were counted, slides of DAPI staining were examined, and approximately 30 cells with good morphology and DAPI staining were randomly selected from at least three regions of the slide for quantitation. Background (cytoplasmic) fluorescence intensity was subtracted from that in the nucleus. G C N S Quantitation of 4-ABP-DNAAdducts. To validate the immunohistochemical method, DNA extracted from both R52 cells and liver tissues were analyzed by alkaline hydrolysis and gas chromatography with negative ion chemical ionization mass spectrometry (GC-NICI-MS) essentially as described previously (10). Briefly, the aliquots of DNA solution (typically 100 pg/mL) in screw-capped tubes were brought to 0.05 N with respect t o NaOH and extracted three times with 2 mL of hexane before spiking with internal standard, 4-ABP-d9. The solution was then heated at 130 "C for 18 h. The samples were cooled t o room temperature, and the released 4-ABP was extracted into hexane (2 mL, twice). 4-ABP and the internal standard were derivatized by the addition of 10 ,uLof trimethylamine in hexane and 10 pL of pentafluoropropionic anhydride (PFPA) to the hexane extracts, followed by reaction at room temperature for 30 min. After removing the solvent and excess reagents by vacuum centrifugation, the PFPA derivates of 4-ABP were redissolved in an appropriate amount of hexane for GC-NICIMS analysis. GC-NICI-MS analyses were carried out on a Varian 3400 gas chromatograph with a SPI injector interfaced (225 "C) to a Finnigan 4050 mass spectrometer system (Finnigan MAT, San Jose, CA). Chromatographic separation was achieved using a DB5 fused silica capillary column (32 m x 0.25 mm i.d.1. Helium was used as carrier gas at a head pressure of 20 psi. The column oven was heated from 60 to 180 "C at 20 W m i n and then at 10 W m i n to 250 "C. Under these conditions, the PFPA derivatives of 4-ABP and 4-ABP-d9 were eluted a t about 7 min. The mass spectrometer was operated in the negative ion chemical ionization mode with an electron energy of 100 eV. Methane gas was admitted to an indicated ion source pressure of about 0.25 torr, and the source temperature was maintained at 220 "C. Quantification was acomplished using selected ion monitoring (SIM) of the (M - HF) ions of the PFPA derivatives of 4-ABP (mlz 295) and 4-ABP-dg (mlz 304). Data were processed by an INCOS data system using IDOS 2 software. The limit of detection for GC-NICI-MS is 10 fmol of 4-ABP/mg of DNA (0.32 adduct per lo8 normal nucleotides) due to the amount of about 0.0006 pg of 4-ABP present in the blank. Samples were assayed in duplicate.
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4-ABP-DNA Adducts
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Figure 2. Dose-response curve for relative fluorescence intensity of staining for 4-ABP-DNA adducts with antibody 4 C l l and a n FITC-labeled secondary antiserum as a function of 4-ABP dose in R52 cells treated for 24 h. A total of 30 cells were quantitated for each dose. Data are the mean and SD.
Figure 1. Immunofluorescence staining for 4-ABP-DNA in R52 cells with antibody 4 C l l and goat anti-mouse FITC. For details see Materials and Methods. Cells treated with 60 pM 4-ABP (A) and control cells (B). Magnification is 400 x .
Results Antibody Characterization. Two monoclonal antibodies (4Cll and 3C8) were developed from mice immunized with 4-ABP-DNA and characterized by competitive ELISA. 150% was at 15 and 33 fmol of 4-ABPDNA for antibody 4 C l l and 3C8, respectively. To determine specificity, DNAs containing C8 guanine adducts of several other aromatic amines were tested as competitors. No cross-reactivity was found with the highest concentration tested (2.5 pmol) of 1-aminopyrene -DNA, 8-nitro-1-aminopyrene-DNA, and 6-nitro1-aminopyrene-DNA. Other carcinogen-modified DNAs available in the laboratory were also tested, including aflatoxin B1-DNA and benzo[a]pyrene diol epoxideDNA, but for both antisera 150% was greater than 2.5 pmol, the highest concentration tested. Both antisera showed some cross-reactivity with nonmodified calf thymus DNA (approximately 20% inhibition with 50 pg of denatured DNNwell). Immunohistochemistry. To develop the immunofluorescence technique, R52 cells were treated with 0,6, 30, 60, and 300 pM 4-ABP for 24 h and stained with antiserum 4C 11and an FITC-labeled secondary antiserum. Specific nuclear staining was observed in treated R52 cells (Figure 1A)but not in control cells (Figure 1B). Additional controls, including 4-ABP-treated cells stained with antiserum preabsorbed with 4-ABP-DNA before use or cells treated with DNase before staining, were negative (not shown). Staining with monoclonal antibody 8 E l l (12),which recognizes benzo[alpyrene diol epoxide-modified DNA, was used as an additional control and was also negative (not shown). Relative fluorescence intensity of staining, determined by densitometric scanning of photographic slides, increased with 4-ABP dose (Figure 2). There was a 40-fold increase in intensity between control
cells (11f 3) and cells treated with the highest dose of 300 pM (438 f 29). Modification levels were also determined by alkaline hydrolysis release of 4-ABP from DNA isolated from treated cells followed by derivatization and GC/MS. At the 30 and 60 pM dose adduct levels were 2 and 11.5/108 nucleotides, respectively; levels in control cells were below the limit of sensitivity of the assay (2.5 pmol). However, significant cross-reactivity with nonmodified calf thymus DNA (20% inhibition at 50 &well) limits the competitive ELISA to analysis of 25 ,ug of DNA or less per well. By combining FITC detection of adducts with DAPI staining for localization of nuclei, specific nuclear binding of antibody 4 C l l was demonstrated in R52 cells and in liver and bladder tissues of treated mice. Additionally, a dose-related increase in 4-ABP-DNA adduct formation
was found both in R52 cells and in liver and bladder tissues. A significant correlation between quantitative immunofluorescent intensity and level of DNA adducts, determined by GCMS, was seen in liver tissue ( r = 0.98, p < 0.0001) (Figure 5). However, the slope of this line was 0.25, indicating a proportionally smaller increase with 4-ABP dose in fluorescence staining than in adduct levels determined by GCMS. This may be due to inefficient detection of adducts in fixed tissues related to incomplete exposure of adducts by the treatments utilized to increase antibody acessibility or clustering of adducts resulting in steric hindrance of antibody binding. In R52 cells, immunohistochemical analysis can clearly distinguish cells treated with 6 pM 4-ABP (relative intensity 31 f 2.2) from untreated control cells (11f 3). Unfortunately, adduct levels for this dose could not be analyzed using GCMS due to insufficient sample. Adduct levels for the 30 ,uM dose were near the limit of sensitivity of the GCMS method of 0.32/108. These results suggest the sensitivity of the immunohistochemical method for cells treated in culture is approximately l/107-8. Studies of liver tissue indicated comparable background staining in controls (8.3 f 4.6) compared to the control R52 cells (11 f 3). However, there was more variability in staining of control tissue (CV = 57-122%) than control cultured cells (CV = 27%). Average staining in the lowest dose (4 mgkg) treated animal (19 f 5 ) was not significantly elevated compared to that in control animals (8 f 5). However, there was a significant ( p < 0.05, Mann Whitney test) increase in staining in animals treated with 10 mgkg 4-ABP (22 f 2) compared to control animals. GCMS data indicated an average DNA adduct level of 4.72/108 in liver tissues of mice treated with 10 mgkg. Thus, these data also suggest that the
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limit of sensitivity of the immunohistochemical method is in the range of l/107-8. Two monoclonal antisera (3D6 and 2 E l l ) against N-acetyl-4-aminobiphenyl-modifiedbovine y-globulin and a polyclonal antiserum (BH2) against 4-ABP-deoxyguanosine-keyhole limpet hemocyanin were developed previously (6, 15). With 4-ABP-dG, 150% was at 2 and 65 pmol for antisera 3D6 and 2El1, respectively. The polyclonal antiserum, BH2, has 160% at 148 fmollwell with a detection limit of 2/108 when assaying 25 pg of DNA per well (6). The monoclonal antisera have been used for immunoaffinity chromatographic isolation of metabolites and DNA adducts from biological samples while the polyclonal serum has been used for quantitation of adducts in lung and bladder tissue (7, 8). The antisera reported here are approximately 130-fold more sensitive than the monoclonal and 10-fold more sensitive than the polyclonal antisera previously developed. Using [32Plpostlabeling,mean 4-ABP-DNA adducts in human bladder biopsy samples were 1.5/108in smokers and 0.5/108 in nonsmokers ( 3 ) . Also by postlabeling, relative adduct labeling for 4-ABP-DNA adducts in exfoliated urothelial cells ranged from nondetectable to 4/108 (4,5 ) . Although mean values were 0.81/108 in smokers and 0.48/108 in nonsmokers, these differences were not significant. In peripheral lung and bladder tissues, using an ELISA with polyclonal antiserum BH2, 4-ABP-DNA ranged between 2 and 85/108in smokers and nonsmokers (7, 8). These results suggest that the immunohistochemical method may have sufficient sensitivity to detect adducts in some human samples. Quantitative immunohistochemical analysis of DNA damage allows biomonitoring of small biopsy samples as well as exfoliated cells such as oral mucosa and urinary bladder cells. We have used this method to monitor aflatoxin B1-DNA adducts in liver tissue obtained at the time of surgery (16)as well as in biopsy samples obtained for diagnosis of liver cancer (17). Antisera t o benzoblpyrene diol epoxide-DNA have been used to detect adducts in skin biopsies of coal tar-treated psoriasis patients (18) and, more recently, for the detection of adducts in oral mucosa cells (19)and lymphocytes of coke oven workers and controls (20). The methodology developed in this work is capable of detecting adducts at levels of biological significance and should be applicable to human target-tissue dosimetry. Additionally, provided that antisera of appropriate specificities are available, the simultaneous quantitation of multiple DNA adducts in the same cell should be feasible.
Acknowledgment. This work was supported by NIH Grant ES05294, American Cancer Society Sig 13, and an award from the Lucille P. Markey Charitable Trust.
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