Immunohistochemical detection of carcinogen-DNA adducts and DNA repair in mouse skin.

4-Hydroxyaminoquinoline 1-oxide (4HAQO) and (+/-)-trans-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BP-DE)-DNA adducts were immunohistochemically demonstrated in the nuclei of mouse skin using antibodies directed against carcinogen (4HAQO or BP) modified DNA. The specificity of the immunostaining was confirmed by several tests, including preincubation of the antibody with carcinogen modified DNA or related molecules, and digestion of the sections with DNase. Subcutaneous injection of 4HAQO dissolved in isotonic solution into an isolated portion of the mouse skin clamped off with ring-shaped forceps resulted in dose-dependent generation of DNA adducts in the nuclei of epithelial cells, fibroblasts, and panniculus carnosus cells. BP-DNA adducts could also be similarly detected dose-dependently in the nuclei of skin cells after local application of BP-DE. Nuclear staining was absent in animals injected with isotonic solution alone, and the intensity of staining correlated well with the level of unscheduled DNA synthesis (UDS) demonstrated autoradiographically after treatment with 4HAQO. Killing of mice at different time points after a single injection of 4HAQO revealed a gradual decrease in the intensity of the staining. Thus the postulated generation and repair of DNA adducts can be followed at the cellular level using the presently described method.

Detection of benzo[a]pyrene-DNA adducts in cultured cells treated with benzo[a]pyrene diol-epoxide by quantitative immunofluorescence microscopy and 32P-postlabelling; immunofluorescence analysis of benzo[a]pyrene-DNA adducts in bronchial cells from smoking individuals.

Monoclonal antibodies were raised against the reaction product of benzo[a]pyrene diol-epoxide (BPDE) and deoxyguanosine-5'-monophosphate. The antibodies were used for detection of DNA adducts in situ in BPDE-treated cultured human fibroblasts by immunofluorescence microscopy. Analogue-digital conversion of the fluorescence signal and further image processing allowed measurement of the immunospecific fluorescence in the nuclei of these cells. The results are compared with the adduct levels measured in isolated DNA by 32P-postlabelling. Preliminary results are shown of the application of the immunofluorescence method to the analysis of DNA adducts in bronchial cells obtained from smoking individuals.

Dosimeters of human exposure to carcinogens: polycyclic aromatic hydrocarbon-macromolecular adducts.

The metabolic activation of polycyclic aromatic hydrocarbons (PAH), for example benzo[a]pyrene, leads to the formation of carcinogen-macromolecular adducts. Methods that make it possible to detect low levels of these adducts in human peripheral blood samples should be useful in the dosimetry of human exposure to carcinogens. We demonstrated previously the usefulness of enzyme immunoassays and of synchronous fluorescence spectroscopy (SFS) for detecting and characterizing low levels of PAH-macromolecular adducts present in synthetic adduct mixtures. These methods have now been refined and applied to the analysis of samples of peripheral blood collected from occupationally exposed individuals (coke-oven workers) and from people attending smoking cessation clinics. The results of both immunoassays and SFS show the presence of benzo[a]pyrene diol epoxide (BPDE)-DNA, BPDE-haemoglobin and other putative PAH-macromolecular adducts in peripheral blood samples from certain individuals.

Aromatic DNA adducts in white blood cells of foundry workers.

Blood samples were obtained from volunteers working in a Finnish iron foundry who were occupationally exposed to polycyclic aromatic hydrocarbons (PAH) and from control subjects not known to be occupationally exposed to this class of chemical carcinogens. Foundry workers were classified as belonging to high, medium or low exposure groups according to their exposure to airborne benzo[a]pyrene: high, greater than 0.2: medium, 0.05-0.2: low, less than 0.05 micrograms benzo[a]pyrene/m3 air). Aromatic adducts were found to be present in white blood cell DNA from most of the exposed workers using the enzyme-linked immunosorbent assay (ELISA) to detect aromatic DNA adducts and the 32P-postlabelling technique. There was a dose-response relationship between the estimated exposure and adduct levels by both methods, and a reasonable correlation between the results of the immunoassay and postlabelling carried out in two laboratories. The levels of adducts found in the samples from the high and medium exposure groups by ELISA ranged up to five adducts in 10(7) nucleotides: the aromatic adducts detected by the postlabeling assay were at a level of two adducts/10(8) nucleotides in the high and medium exposure categories. No effect due to age, sex or the smoking habits of the subjects was observed. The results indicate that DNA extracted from white blood cells of highly exposed workers is more likely to contain aromatic DNA adducts than that from workers without occupational exposure to PAH, but large interindividual variations were evident. This study suggests that the antibody and 32P-postlabelling assays may be useful in monitoring human exposure to known and previously unidentified environmental genotoxic agents.

A comparison of 32P-postlabelling and immunological methods to examine human lung DNA for benzo[a]pyrene adducts.

Human lung DNA isolated from surgical specimens has been examined for the presence of polycyclic aromatic hydrocarbon-DNA adducts using both 32P-postlabelling and immunological methods. Of 12 samples examined to date, five had detectable amounts of benzo[a]pyrene diol epoxide-DNA adducts (BPDE-DNA) as determined by the enzyme-linked immunosorbent assay (ELISA), after immunoaffinity concentration. Values ranged from 3.5 to 11.5 fmol/mg DNA. When the same group of samples was analysed using the 32P-postlabelling technique, adducts could be detected in all the samples examined. There was generally not a good correspondence between the two methods. The number of adducts measured by 32P-postlabelling ranged from 1-100 per 10(8) nucleotides, which is some two orders of magnitude higher than with the immunological method, indicating that the BPDE-DNA adduct is probably not the major adduct present in these samples.

Binding efficiency of antibodies to DNA modified with aromatic amines and polycyclic aromatic hydrocarbons: implications for quantification of carcinogen-DNA adducts in vivo.

Antibodies raised against the bovine serum albumin conjugates of N-(guanosin-8-yl)-N-2-acetylaminofluorene (Guo-8-AAF), the imidazole ring-opened form of N-(guanosin-8-yl)-2-aminofluorene (roGuo-8-AF) and the methylated bovine serum albumin complex of DNA modified with (+/-)trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyre ne (BPDE) have been employed in a highly sensitive enzyme-linked immunosorbent assay (ELISA) to determine their affinity for DNA modified with the corresponding carcinogens at various levels of modification. All antibodies recognized highly modified DNA more efficiently than DNA of low modification. This property, which may be common to all antibodies raised against carcinogen-DNA adducts, has to be taken into account when these antibodies are used to quantify carcinogen-DNA adducts in biological samples. Appropriate DNA preparations of low modification have to be used as reference compounds in immunoassays to allow reliable quantification of adduct levels in DNA from animals and human cells.

The binding efficiency of polyclonal and monoclonal antibodies to DNA modified with benzo[a]pyrene diol epoxide is dependent on the level of modification. Implications for quantitation of benzo[a]pyrene-DNA adducts in vivo.

A number of polyclonal antibodies specific for DNA modified with (+/-)trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyre ne (BPDE) were obtained from the sera of New Zealand white rabbits immunized with BPDE-DNA, complexed with methylated bovine serum albumin (mBSA). Monoclonal antibodies were developed by fusion of mouse myeloma cells with spleen cells isolated from BALB/c mice immunized with the same complex of BPDE-DNA and mBSA. These antibodies have been characterized for specificity in a highly sensitive, enzyme-linked immunosorbent assay (ELISA). All antibodies showed a very high affinity for single-stranded BPDE-DNA, but had lower affinity towards native BPDE-DNA. The affinity for the free mononucleoside BPDE-dG was at least 100-fold lower than that for BPDE-DNA, and no affinity was detected for BP tetrols or DNA modified with N-acetoxy-N-acetyl-2-aminofluorene. A high cross reactivity was observed with DNA modified with (+/-)-trans-1,2-dihydroxy-anti-3,4-epoxy-1,2,3,4-tetrahydrochrysene++ +. Using five different antibodies, monoclonal or polyclonal, we observed that the antibody affinity for BPDE-DNA was dependent on the level of modification; in the competitive ELISA as little as 4 fmol BPDE-DNA (50 pmol/micrograms) was sufficient for 50% inhibition with our best antisera, but 17 fmol of the adduct was required when [3H]BPDE-DNA of low modification (1-10 fmol/micrograms) was used as inhibitor. When samples of [3H]BP-DNA isolated from the livers of mice, treated i.p. with different doses of [3H]BP were examined by competitive ELISA and calibrated with [3H]BPDE-DNA of low modification (1-10 fmol/micrograms), binding values calculated from the immunoassay were in good agreement with those obtained from radioactivity measurements. In contrast, when this DNA was quantitated in competitive ELISA using highly modified BPDE-DNA as standards, values by ELISA were 20-40% of those obtained by radioactivity. These results indicate that the use of serially diluted BPDE-DNA of high modification as standard competitor in the ELISA will lead to erroneous results in the measurement of adducts in DNAs modified to a low extent (biological samples). The property of antisera specific for BP-DNA, recognizing highly modified DNA more efficiently than DNA modified to a low extent, may be common to all antisera elicited against highly modified DNA immunogens. Therefore we conclude that antibody affinity must be tested also with DNA samples of low modification, obtained either in vitro or in vivo.