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.

Quantitation of protein adducts as a marker of genotoxic exposure: immunologic detection of benzo[a]pyrene--globin adducts in mice.

Immunologic methods have been developed for the determination of benzo[a]pyrene (BP)-protein adducts and validated in animals treated with [3H]BP. A previously developed antibody, 8E11, which recognizes 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10- tetrahydrobenzo[a]pyrene (BPDE-I)-modified DNA or protein as well as BPDE-I-tetraols, was used. The sensitivity of the assay was increased by enzymatic digestion of the modified protein with insoluble protease into peptides and amino acids before analysis. In a competitive enzyme-linked immunosorbent assay (ELISA) with digested BPDE-I-modified bovine serum albumin, 50% inhibition occurred at 400 fmol of adduct compared to 1450 fmol for the non-digested albumin. Analysis of globin (Gb) isolated from animals treated in vivo with 0.3-3 mg [3H]BP indicated that the ELISA could detect 90-100% of the adducts determined by radioactivity. Levels of adducts in lung and liver DNA and serum albumin were correlated with the levels of Gb adducts. Of the total radioactivity associated with hemoglobin, only less than or equal to 10% was from Gb while approximately 80% was from the heme fraction and the remainder from free BP metabolites. Significant cross-reactivity of antibody 8E11 was found with several BP-diols and phenols, suggesting that the immunoassay will not only be specific for BPDE-I adducts but will also detect adducts of other BP metabolites as well as other aromatic hydrocarbon diol epoxides. An immunoaffinity column of antibody 8E11 coupled to Sepharose 4B was used to isolate modified peptides from the digested Gb. About 65% of the applied radioactivity was retained on the column. Between 1 and 2 mg of non-modified digested Gb could be added to the sample without interfering with binding of adducts. Protein digestion and immunoaffinity chromatography should be useful for the measurement of protein adducts in biomonitoring studies.

[Cytochrome P-450-dependent mechanisms of the biosynthesis of protein-conjugated benzo(a)pyrene antigens and their role in the development of a specific immune response to this carcinogen]. / Izuchenie tsitokhrom-P-450-zavisimykh mekhanizmakh biosinteza kon''iugirovannykh antigenov benzo(a)pirena s belkami i ikh rol' v razvitii spetsificheskogo immunnogo otveta na étot kantserogen.

There was studied the possibility of covalent binding to proteins of a carcinogenic compound benzo(a)pyrene in the system of cytochrome P-450 of the liver and the possibility of the development of the immune reaction to administration of the conjugated antigens obtained in such a way to animals. It was shown that under experimental conditions modelling the processes of microsomal oxidation of benzo(a)pyrene in the organism there occurs irreversible binding of 14C-benzo(a)pyrene both to microsomes and the added albumin. Immunization of rabbits by conjugates benzo(a)pyrene-albumin and benzo(a)pyrene-microsomes leads to the development of the immune response to the original carcinogen. The antibodies and lymphocytes specifically binding 14C-benzo(a)pyrene appear in the blood.

Quantitation by electron microscopy of the binding of highly specific antibodies to benzo[a]pyrene-DNA adducts.

Highly specific antibodies bound to carcinogen adducts in DNA modified with (+/-)7 beta,8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE I) were quantitated by electron microscopy (EM) visualization and these observations were compared with quantitation of adducts by enzyme-linked immunosorbent assay (ELISA). The antiserum, elicited in rabbits following inoculation with BPDE I-modified DNA, has been found to be highly specific in its recognition of BPDE I-deoxyguanosine moieties. Parallel DNA samples prepared for analysis by ELISA and EM quantitation were randomized, encoded, and analyzed to determine extents of carcinogen modification in double-blind studies. After levels of modification were determined by immunoassays, DNA samples were prepared for EM analysis by incubation with amounts of anti-BPdG-DNA serum in excess of that necessary for complete binding of antibody to antigenic sites. At equilibrium, samples were enzymatically digested with papain in order to cleave anti-BPdG-DNA IgG molecules into Fab fragments in situ. Following column exclusion chromatography, BPdG-DNA-Fab complexes were incubated with ferritin-labeled Fab' fragments of goat [anti-rabbit F(ab')2] IgG in amounts in excess of those necessary for complete binding. When DNA samples were modified to between 0 and 40 fmol adduct/micrograms DNA, excellent agreement was obtained between ELISA quantitation and visualization by EM of antibodies bound to adducts.