Immunochemical quantitation of DNA adducts derived from the human bladder carcinogen 4-aminobiphenyl.
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 micrograms 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 micrograms DNA was 2 adducts/10(8) 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.
Roberts
D W
DW
Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, Arkansas 72079.
Benson
R W
RW
Groopman
J D
JD
Flammang
T J
TJ
Nagle
W A
WA
Moss
A J
AJ
Kadlubar
F F
FF
D000611:Aminobiphenyl Compounds / Q000276:immunology / Q000378:metabolism*; D000818:Animals; D002273:Carcinogens / Q000378:metabolism*; D003429:Cross Reactions; D004247:DNA / Q000276:immunology / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D007106:Immune Sera / Q000276:immunology; D011817:Rabbits; D001749:Urinary Bladder Neoplasms / Q000139:chemically induced
D000611:Aminobiphenyl Compounds; D002273:Carcinogens; D007106:Immune Sera; C006757:4-biphenylamine; D004247:DNA