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Immunochemical Quantitation of DNA Adducts Derived from the Human Bladder Carcinogen 4-Aminobiphenyl

Division of Biochemical Toxicology, National Cancer for Toxicological Research, Jefferson, Arkansas 72079 [D. W. R., R. W. B., T. J. F., F. F. K.]; Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences [D. W. R., F. F. K.] and Research Service (151), McClellan Memorial Veterans Administration Hospital [W. A. N., A. J. M.] Little Rock, Arkansas 72205; and Boston University School of Public Health, Environmental Health Section, Boston, Massachusetts 02118 [J. D. G.]

To assess target-tissue exposure to the human urinary bladder carcinogen 4-aminobiphenyl (ABP), we have developed a sensitive immunochemical method for measuring the major arylamine-DNA adduct formed, N-(guan-8-yl)-ABP (Gua-C8-ABP). High-affinity polyclonal antisera from rabbits immunized with N-(guanosin-8-yl)-ABP coupled to keyhole limpet hemocyanin were characterized and shown to have high specificity for antigenic determinants on the purine and biphenyl rings of Gua-C8-ABP and minimal cross-reactivity with ABP, deoxyguanosine, or hydrolyzed DNA. Assay standards containing ABP-modified DNA were prepared by reacting [³H]N-hydroxy-ABP with calf thymus DNA. DNA samples were hydrolyzed with trifluoroacetic acid and dried under vacuum, and the residues were dissolved in dimethyl sulfoxide under argon. Using a streptavidin-biotin amplified enzyme-linked immunosorbent assay, DNA hydrolysates competed at 25 µg DNA/microtiter well for a limiting amount of anti-keyhole limpet hemocyanin-(Gua-C8-ABP) in the presence of excess solid-phase bovine serum albumin-(Gua-C8-ABP) coating antigen. The limit of sensitivity for this assay using 25 µg DNA was 2 adducts/10⁸ nucleotides. Gua-C8-ABP adducts in liver and bladder epithelial DNAs were readily quantified after p.o. administration of 5 mg/kg ABP to dogs. This methodology is capable of detecting adducts at levels of biological significance and should be applicable to human target-tissue dosimetry.

¹ To whom requests for reprints should be addressed, at HFT-110, NCTR, Jefferson, AR 72079.

Received 2/26/88. Revised 7/18/88. Accepted 8/10/88.

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