Synthesis, characterization, and quantitation of a 4-aminobiphenyl-DNA adduct standard

P-32-Postlabeling is a powerful technique for the detection of DNA adducts; however, quantitation of DNA adducts by this method can result in errors d ue to differences in hydrolysis and labeling efficiencies between adducted and normal nucleotides. We have synthesized a DNA sample modified with 4-am inobiphenyl to serve as a quantitation standard for P-32- postlabeling and other DNA adduct detection methodologies. [2,2'-H-3]-N-Hydroxy-4-aminobiphe nyl was reacted with calf thymus DNA at pH 5 to give 62 +/- 0.8 adducts/10( 8) nucleotides (mean +/- SD) on the basis of H-3 content. HPLC analyses fol lowing enzymatic hydrolysis to nucleosides indicated one major adduct, N-(d eoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-4-ABP). The adduct identity was c onfirmed by HPLC/electrospray ionization mass spectrometry, which indicated a modification level of 19 +/- 1.7 dG-C8-4-ABP/10(8) nucleotides. P-32-Pos tlabeling analysis gave a value of 0.84 dG-C8-4-ABP/10(8) nucleotides, whil e a dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) indicated l evels of 82 +/- 26 and 63 +/- 20 dG-C8-4ABP/10(8) nucleotides after enzymat ic hydrolysis to nucleotides and nucleosides, respectively. The utility of the DNA adduct standard was determined by assessing the level of dG-C8-4-AB P in liver DNA from mice treated with [2,2'-H-3]-4-aminobiphenyl. P-32-Post labeling analyses, based upon measuring the extent of the P-32 incorporatio n, underestimated the levels of dG-C8-4-ABP, while DELFIA, using a G-C8-4-A BP quantitation standard, overestimated the adduct levels. The adduct level s determined by HPLC/electrospray ionization mass spectrometry best; reflec ted those obtained from H-3 incorporation. When the P-32-postlabeling analy ses and the DELFIA were conducted using the DNA modified in vitro with dG-C 8-4-ABP as a quantitation standard, accurate estimations of the extent of i n vivo formation of dG-C8-4-ABP were obtained.