C a r c i n o g e n e s i s v o l . i l n o . l l p p . 1 9 0 3 - 1 9 0 7 . 1990
Serological characterization of polycyclic aromatic diolepoxide—BNA adduncts using monocloiniall antibodies
M.J.Newman 1 , A.Weston2, O.C.Carver, D.L.Mann 3 and C.C.Harris 2 Veterinary Microbiology, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, 2Laboratory of Human Carcinogenesis, NCI, NIH, Bethesda, MA 20892 and 'Laboratory of Viral Carcinogenesis, NCI - Frederick Cancer Research Facility, Frederick, MD 21701, USA
Polycyclic aromatic hydrocarbons (PAHs) are a group of structurally related compounds that are present in the environment in complex mixtures as common pollutants. These compounds have been studied extensively because of their carcinogenic and toxic properties to humans. We reported previously that humans exposed to certain PAHs produce antibodies that bind to different PAH diolepoxide -DNA (PAH-DNA) adducts. The ability to detect and measure antibodies to PAH-DNA adducts in human blood samples could prove useful as a biological dosimeter for identifying persons that have been exposed to high levels of PAHs, i.e. persons who may be at high cancer risk. In our initial studies we found that it was common for persons who were exposed to PAH to produce antibodies against PAH-DNA adducts. However, we were unable to identify the actual chemical types of PAH - DNA adducts that were recognized by the serum antibodies because many serum samples contained antibody activity to more than one adduct. These data indicate that different PAH—DNA adducts may be serologically similar or that humans actually produce immune responses against more than a single PAH-DNA adduct. We have used monoclonal antibody technology to determine the extent to which different PAH-DNA adducts share serologically recognized epitopes. Monoclonal antibodies were produced against two different PAH-DNA adducts, benzo[a]pyrene diolepoxide-DNA (BPDE-DNA) and benz[a]anthracene diolepoxide-DNA (BADE-DNA). The binding of these antibodies to five PAH-DNA adduct preparations and to soluble PAHs was assessed. We found that most monoclonal antibodies bound to more than a single type of PAH-DNA adduct, documenting the serological relatedness of different PAH-DNA adducts. However, two monoclonal antibodies were produced that bound only to BPDE-DNA. Soluble non-metabolized PAHs and PAH tetraols were not recognized by these antibodies, thus
•Abbreviations: PAH, polycyclic aromatic hydrocarbon; BPDE-DNA, benzo[a]pyrene diolepoxide-DNA adduct; ChDE-DNA, chrysene diolepoxide DNA adduct; BADE-DNA, benz[a]anthracene diolepoxide-DNA adduct; BPDE, r-7,(-8-dihydroxy-»-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene; ChDE, r-l,r-2-dihydroxy-/-3,4-oxy-l,2,3,4-tetrahydrochrysene; BADE, r-8,J-9-dihydroxyj-10,1 l-oxy-8,9,10,ll-tetrahydrobenz[a]anthracene; DBADE, r-10,/-ll-dihydroxy-/-12,13-oxy-10,ll,12,13-tetrahydrodibenz[a,c]anthracene; BFDE, r-8,/-9-dihydroxy-r-10,1 l-oxy-8,9,10,11 -tetrahydrobenzo[(fc]fluoranthene; PBS-NGS, PBS containing 10% (v/v) normal goat serum; DBADE-DNA, dibenz[o,c]anthracene diolepoxide — DNA adduct; BFDE —DNA, benzo[&]fluoramhene diolepoxide - DNA adduct. Oxford University Press
Introduction Polycyclic aromatic hydrocarbons (PAHs*) are a group of structurally related compounds that are formed from the incomplete combustion of organic matter. PAHs are found in the environment as complex mixtures and are commonly encountered environmental pollutants (1). Human exposure to PAHs is through inhalation (2-5), ingestion (6,7) and skin contact (8). Exposure to PAHs represents a major health concern because many of these compounds can be converted to their carcinogenic forms (diolepoxides) by metabolic activation through the actions of mixed-function oxidases, primarily the cytochrome P450 enzymes, and subsequent epoxidation by epoxide hydrolases. Carcinogenic diolepoxides of PAHs form covalent addition products (adducts) in DNA. The actual covalent binding of the PAH diolepoxide occurs primarily to the exocyclic amino group of deoxyguanosine (9—13). The presence of benzo[a]pyrene diolepoxide—DNA adducts (BPDE—DNA) in human tissues has been documented following experimental in vitro exposure to benzo[a]pyrene (11,14-16). Similar adducts have been detected in the DNA of peripheral blood leukocytes (17-21), in the lung tissues of persons exposed to high levels of benzo[a]pyrene through the environment (22,23) and in placenta] tissues (24,25). We have reported recently that persons exposed to PAHs are capable of producing antibodies to the resultant PAH—DNA adducts. Antibodies to BPDE—DNA adducts were detected, using ELISA, in the serum of coke oven workers (18,19). This particular group of coke workers was known to be exposed to high levels of benzo[a]pyrene through the work environment. Antibody binding activity to BPDE-DNA, chrysene diolepoxide —DNA (ChDE —DNA) and benz[a]anthracene diolepoxide-DNA (BADE-DNA) adducts has been detected in sera from individuals with undefined environmental exposure to PAHs (26). The apparent specificity of these serum antibodies was of particular interest. A small number of sera were identified that contained antibodies to only a single PAH-DNA adduct, such as BPDE-DNA or BADE-DNA. However, many sera contained antibodies against more than a single adduct; sera that contained antibodies against all three of the test PAH-DNA 1903
Downloaded from http://carcin.oxfordjournals.org/ at University of California, Santa Barbara on June 29, 2015
'Current address and to whom all correspondence should be sent: Cambridge BioScience, 365 Plantation Street, Worcester, MA 01605, USA
demonstrating their specificity for PAH—DNA adducts and not the PAHs alone. Monoclonal antibodies produced against BADE-DNA also bound to chrysene diolepoxide-DNA but not to BPDE-DNA or to two other PAH-DNA adducts. These data demonstrate that significant serological crossreactivity exists between different PAH—DNA adducts and support our contention that human antibodies to PAH—DNA can often bind to more than a single type of PAH—DNA adduct. However, our ability to produce monoclonal antibodies with restricted specificity suggests that the production of highly monoclonal antibody reagents for use in assays designed to measure PAH-DNA adducts in biological samples is feasible.
M.J.Newman el al.
Materials and methods Preparation of PAH-DNA adducts Vicinal diolepoxides of five selected PAHs were purchased from the NCI Chemical Carcinogen Reference Standard Repository (Division of Cancer Etiology, NCI, NIH, Bethesda, MD). The compounds used were r-7,l-8-dihydroxy(-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), r-\,r-2-dihydroxy-(-3,4oxy-l,2,3,4-tetrahydrochrysene (ChDE), r-8,r-9-dihydroxy-/-10,l 1-oxy8,9,10,ll-tetrahydrobenz]a]anthracene (BADE), /--10,(-ll-dihydroxy-rl2,13-oxy10,11,12,13-tetrahydrodibenz[a,c]anthracene (DBADE) and r-8,r-9-dihydroxyf-10,1 l-oxy-8,9,10,1 l-tetrahydrobenzo[£]fluoranthene (BFDE). These chemicals were chosen to be representative of the PAH class of carcinogens because the parent compounds are widely distributed in the environment (1,4 -6) and because binding to DNA is mediated through similar diolepoxide metabolites. Additionally, these different compounds provided us with a variation of chemical structures needed to compare antibody binding to different PAH - DNA adducts The preparation of PAH-DNA adducts using these diolepoxides and purified calf thymus DNA was done using techniques that have been described previously (26). The chemical structures of the adducts produced and used in this study are shown in Figure 1. The level of modification for the diolepoxide-adducted DNA was estimated by measuring the absorbance spectra of the modified DNA samples (27); epsilon values for each of the chromophores studied were obtained from published sources (28). Modification levels for these adducts were estimated to be between 2.8 and 5.6 pmol/^g DNA, except for BPDE which was adducted at higher levels, 28.1 pmol/jjg DNA. Confirmatory evidence for these modification levels was obtained by analytical 32P-postlabelling (Dr R.C.Gupta, personal communication and 29). In vitro prepared PAH-DNA adducts routinely consisted of double-strand DNA. These preparations were used for immunization and most assay purposes. Singlestrand PAH-DNA adduct preparations were produced when needed by heating native samples to 90°C for 40 min. Samples were diluted immediately to required assay concentrations using cold PBS. The condition of the DNA was documented using the antigen competition ELISA and monoclonal antibodies specific to either single- or double-strand DNA; monoclonal antibody producing cell lines (CH26-1252 and MRSS-1) were obtained from ATCC, Rockville, MD. Preparations of soluble tetrol and non-metabolized forms of the selected test PAHs were prepared by dissolving 1 mg of either the PAH diolepoxides or nonmetabolized PAH crystals (Aldrich Chemical Co., Milwaukee, WI) in 1 ml DMSO and then diluting to 10 ml in PBS. These solutions were incubated in the dark at room temperature for 16 h and used without further purification. Production of monoclonal antibodies Both BALB/c and C57BL mice were used for the production of monoclonal antibodies. The BPDE-DNA and BADE-DNA preparations that were used for immunization were adsorbed to methylated bovine serum albumin (Sigma) at a 1:10 (w/w) ratio for 1 h at room temperature in sterile PBS. The initial immunization consisted of 200 ng PAH - DNA in 1 ml of PBS emulsified with 1 ml of Freund's complete adjuvant (GIBCO, Grand Island, NY) and was administered both i.p. and s.c. into female mice, 12-15 weeks of age. A second set of injections was given after 14 days using 40 /ig/mouse of the same PAH-DNA adsorbed to methylated bovine serum albumin and Freund's incomplete adjuvant (GIBCO). Subsequent booster immunizations were administered at 14 day intervals using 40 ng PAH-DNA adsorbed to methylated bovine serum albumin but without adjuvants. Small samples of blood were collected from the tail after the third and fifth immunizations and tested against
1904
Covalent Binding of Polycyclic Aromatic Hydrocarbon-diol Epoxides with DMA
N 2 -! 10-(7,8,9-tnhydroxy7,8,9.10-tBtrahydrobenzo[a]pyr»ny1)] guanine
N 3 -! 13-O0,11,12-tnhyd rox Y10,11,12,13-tetrahydroxvdibe!ula,c] anthracenyOI guanine
N 2 -M1.
Serological characterization of polycyclic aromatic diolepoxide—BNA adduncts using monocloiniall antibodies
M.J.Newman 1 , A.Weston2, O.C.Carver, D.L.Mann 3 and C.C.Harris 2 Veterinary Microbiology, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, 2Laboratory of Human Carcinogenesis, NCI, NIH, Bethesda, MA 20892 and 'Laboratory of Viral Carcinogenesis, NCI - Frederick Cancer Research Facility, Frederick, MD 21701, USA
Polycyclic aromatic hydrocarbons (PAHs) are a group of structurally related compounds that are present in the environment in complex mixtures as common pollutants. These compounds have been studied extensively because of their carcinogenic and toxic properties to humans. We reported previously that humans exposed to certain PAHs produce antibodies that bind to different PAH diolepoxide -DNA (PAH-DNA) adducts. The ability to detect and measure antibodies to PAH-DNA adducts in human blood samples could prove useful as a biological dosimeter for identifying persons that have been exposed to high levels of PAHs, i.e. persons who may be at high cancer risk. In our initial studies we found that it was common for persons who were exposed to PAH to produce antibodies against PAH-DNA adducts. However, we were unable to identify the actual chemical types of PAH - DNA adducts that were recognized by the serum antibodies because many serum samples contained antibody activity to more than one adduct. These data indicate that different PAH—DNA adducts may be serologically similar or that humans actually produce immune responses against more than a single PAH-DNA adduct. We have used monoclonal antibody technology to determine the extent to which different PAH-DNA adducts share serologically recognized epitopes. Monoclonal antibodies were produced against two different PAH-DNA adducts, benzo[a]pyrene diolepoxide-DNA (BPDE-DNA) and benz[a]anthracene diolepoxide-DNA (BADE-DNA). The binding of these antibodies to five PAH-DNA adduct preparations and to soluble PAHs was assessed. We found that most monoclonal antibodies bound to more than a single type of PAH-DNA adduct, documenting the serological relatedness of different PAH-DNA adducts. However, two monoclonal antibodies were produced that bound only to BPDE-DNA. Soluble non-metabolized PAHs and PAH tetraols were not recognized by these antibodies, thus
•Abbreviations: PAH, polycyclic aromatic hydrocarbon; BPDE-DNA, benzo[a]pyrene diolepoxide-DNA adduct; ChDE-DNA, chrysene diolepoxide DNA adduct; BADE-DNA, benz[a]anthracene diolepoxide-DNA adduct; BPDE, r-7,(-8-dihydroxy-»-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene; ChDE, r-l,r-2-dihydroxy-/-3,4-oxy-l,2,3,4-tetrahydrochrysene; BADE, r-8,J-9-dihydroxyj-10,1 l-oxy-8,9,10,ll-tetrahydrobenz[a]anthracene; DBADE, r-10,/-ll-dihydroxy-/-12,13-oxy-10,ll,12,13-tetrahydrodibenz[a,c]anthracene; BFDE, r-8,/-9-dihydroxy-r-10,1 l-oxy-8,9,10,11 -tetrahydrobenzo[(fc]fluoranthene; PBS-NGS, PBS containing 10% (v/v) normal goat serum; DBADE-DNA, dibenz[o,c]anthracene diolepoxide — DNA adduct; BFDE —DNA, benzo[&]fluoramhene diolepoxide - DNA adduct. Oxford University Press
Introduction Polycyclic aromatic hydrocarbons (PAHs*) are a group of structurally related compounds that are formed from the incomplete combustion of organic matter. PAHs are found in the environment as complex mixtures and are commonly encountered environmental pollutants (1). Human exposure to PAHs is through inhalation (2-5), ingestion (6,7) and skin contact (8). Exposure to PAHs represents a major health concern because many of these compounds can be converted to their carcinogenic forms (diolepoxides) by metabolic activation through the actions of mixed-function oxidases, primarily the cytochrome P450 enzymes, and subsequent epoxidation by epoxide hydrolases. Carcinogenic diolepoxides of PAHs form covalent addition products (adducts) in DNA. The actual covalent binding of the PAH diolepoxide occurs primarily to the exocyclic amino group of deoxyguanosine (9—13). The presence of benzo[a]pyrene diolepoxide—DNA adducts (BPDE—DNA) in human tissues has been documented following experimental in vitro exposure to benzo[a]pyrene (11,14-16). Similar adducts have been detected in the DNA of peripheral blood leukocytes (17-21), in the lung tissues of persons exposed to high levels of benzo[a]pyrene through the environment (22,23) and in placenta] tissues (24,25). We have reported recently that persons exposed to PAHs are capable of producing antibodies to the resultant PAH—DNA adducts. Antibodies to BPDE—DNA adducts were detected, using ELISA, in the serum of coke oven workers (18,19). This particular group of coke workers was known to be exposed to high levels of benzo[a]pyrene through the work environment. Antibody binding activity to BPDE-DNA, chrysene diolepoxide —DNA (ChDE —DNA) and benz[a]anthracene diolepoxide-DNA (BADE-DNA) adducts has been detected in sera from individuals with undefined environmental exposure to PAHs (26). The apparent specificity of these serum antibodies was of particular interest. A small number of sera were identified that contained antibodies to only a single PAH-DNA adduct, such as BPDE-DNA or BADE-DNA. However, many sera contained antibodies against more than a single adduct; sera that contained antibodies against all three of the test PAH-DNA 1903
Downloaded from http://carcin.oxfordjournals.org/ at University of California, Santa Barbara on June 29, 2015
'Current address and to whom all correspondence should be sent: Cambridge BioScience, 365 Plantation Street, Worcester, MA 01605, USA
demonstrating their specificity for PAH—DNA adducts and not the PAHs alone. Monoclonal antibodies produced against BADE-DNA also bound to chrysene diolepoxide-DNA but not to BPDE-DNA or to two other PAH-DNA adducts. These data demonstrate that significant serological crossreactivity exists between different PAH—DNA adducts and support our contention that human antibodies to PAH—DNA can often bind to more than a single type of PAH—DNA adduct. However, our ability to produce monoclonal antibodies with restricted specificity suggests that the production of highly monoclonal antibody reagents for use in assays designed to measure PAH-DNA adducts in biological samples is feasible.
M.J.Newman el al.
Materials and methods Preparation of PAH-DNA adducts Vicinal diolepoxides of five selected PAHs were purchased from the NCI Chemical Carcinogen Reference Standard Repository (Division of Cancer Etiology, NCI, NIH, Bethesda, MD). The compounds used were r-7,l-8-dihydroxy(-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE), r-\,r-2-dihydroxy-(-3,4oxy-l,2,3,4-tetrahydrochrysene (ChDE), r-8,r-9-dihydroxy-/-10,l 1-oxy8,9,10,ll-tetrahydrobenz]a]anthracene (BADE), /--10,(-ll-dihydroxy-rl2,13-oxy10,11,12,13-tetrahydrodibenz[a,c]anthracene (DBADE) and r-8,r-9-dihydroxyf-10,1 l-oxy-8,9,10,1 l-tetrahydrobenzo[£]fluoranthene (BFDE). These chemicals were chosen to be representative of the PAH class of carcinogens because the parent compounds are widely distributed in the environment (1,4 -6) and because binding to DNA is mediated through similar diolepoxide metabolites. Additionally, these different compounds provided us with a variation of chemical structures needed to compare antibody binding to different PAH - DNA adducts The preparation of PAH-DNA adducts using these diolepoxides and purified calf thymus DNA was done using techniques that have been described previously (26). The chemical structures of the adducts produced and used in this study are shown in Figure 1. The level of modification for the diolepoxide-adducted DNA was estimated by measuring the absorbance spectra of the modified DNA samples (27); epsilon values for each of the chromophores studied were obtained from published sources (28). Modification levels for these adducts were estimated to be between 2.8 and 5.6 pmol/^g DNA, except for BPDE which was adducted at higher levels, 28.1 pmol/jjg DNA. Confirmatory evidence for these modification levels was obtained by analytical 32P-postlabelling (Dr R.C.Gupta, personal communication and 29). In vitro prepared PAH-DNA adducts routinely consisted of double-strand DNA. These preparations were used for immunization and most assay purposes. Singlestrand PAH-DNA adduct preparations were produced when needed by heating native samples to 90°C for 40 min. Samples were diluted immediately to required assay concentrations using cold PBS. The condition of the DNA was documented using the antigen competition ELISA and monoclonal antibodies specific to either single- or double-strand DNA; monoclonal antibody producing cell lines (CH26-1252 and MRSS-1) were obtained from ATCC, Rockville, MD. Preparations of soluble tetrol and non-metabolized forms of the selected test PAHs were prepared by dissolving 1 mg of either the PAH diolepoxides or nonmetabolized PAH crystals (Aldrich Chemical Co., Milwaukee, WI) in 1 ml DMSO and then diluting to 10 ml in PBS. These solutions were incubated in the dark at room temperature for 16 h and used without further purification. Production of monoclonal antibodies Both BALB/c and C57BL mice were used for the production of monoclonal antibodies. The BPDE-DNA and BADE-DNA preparations that were used for immunization were adsorbed to methylated bovine serum albumin (Sigma) at a 1:10 (w/w) ratio for 1 h at room temperature in sterile PBS. The initial immunization consisted of 200 ng PAH - DNA in 1 ml of PBS emulsified with 1 ml of Freund's complete adjuvant (GIBCO, Grand Island, NY) and was administered both i.p. and s.c. into female mice, 12-15 weeks of age. A second set of injections was given after 14 days using 40 /ig/mouse of the same PAH-DNA adsorbed to methylated bovine serum albumin and Freund's incomplete adjuvant (GIBCO). Subsequent booster immunizations were administered at 14 day intervals using 40 ng PAH-DNA adsorbed to methylated bovine serum albumin but without adjuvants. Small samples of blood were collected from the tail after the third and fifth immunizations and tested against
1904
Covalent Binding of Polycyclic Aromatic Hydrocarbon-diol Epoxides with DMA
N 2 -! 10-(7,8,9-tnhydroxy7,8,9.10-tBtrahydrobenzo[a]pyr»ny1)] guanine
N 3 -! 13-O0,11,12-tnhyd rox Y10,11,12,13-tetrahydroxvdibe!ula,c] anthracenyOI guanine
N 2 -M1.
