Quantitation of protein adducts as a marker of genotoxic exposure: immunologic detection of benzo(a)pyrene — globin adducts in mice

Abstract

Immunologic methods have been developed for the determination of benzo(a)pyrene (BP)-protein adducts and validated in animals treated with ( ³ H)BP. A previously developed antibody, 8E11, which recongnizes 7β, 8α-dihydroxy-9α, 10α-epoxy-7, 8, 9, 10tetrahydrobenzo(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 enzymelinked immunosorbent assay (ELISA) with digested BPDE-I-modified bovine serum albumin, 50% inhibition occured at 400 fmol of adduct compared to 1450 fmol for the nondigested albumin. Analysis of globin (Gb) isolated from animals treated in vivo with 0.3–3 mg ( ³ H)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 ≤10% was from Gb while ˜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.