Synthesis, characterization, and quantitation of a 4-aminobiphenyl-DNA adduct standard.
32P-Postlabeling is a powerful technique for the detection of DNA adducts; however, quantitation of DNA adducts by this method can result in errors due to differences in hydrolysis and labeling efficiencies between adducted and normal nucleotides. We have synthesized a DNA sample modified with 4-aminobiphenyl to serve as a quantitation standard for 32P-postlabeling and other DNA adduct detection methodologies. [2,2'-3H]-N-Hydroxy-4-aminobiphenyl was reacted with calf thymus DNA at pH 5 to give 62 +/- 0.8 adducts/10(8) nucleotides (mean +/- SD) on the basis of 3H content. HPLC analyses following enzymatic hydrolysis to nucleosides indicated one major adduct, N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-4-ABP). The adduct identity was confirmed by HPLC/electrospray ionization mass spectrometry, which indicated a modification level of 19 +/- 1.7 dG-C8-4-ABP/10(8) nucleotides. 32P-Postlabeling analysis gave a value of 0.84 dG-C8-4-ABP/10(8) nucleotides, while a dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) indicated levels of 82 +/- 26 and 63 +/- 20 dG-C8-4-ABP/10(8) nucleotides after enzymatic hydrolysis to nucleotides and nucleosides, respectively. The utility of the DNA adduct standard was determined by assessing the level of dG-C8-4-ABP in liver DNA from mice treated with [2,2'-3H]-4-aminobiphenyl. 32P-Postlabeling analyses, based upon measuring the extent of the 32P incorporation, underestimated the levels of dG-C8-4-ABP, while DELFIA, using a G-C8-4-ABP quantitation standard, overestimated the adduct levels. The adduct levels determined by HPLC/electrospray ionization mass spectrometry best reflected those obtained from 3H incorporation. When the 32P-postlabeling analyses and the DELFIA were conducted using the DNA modified in vitro with dG-C8-4-ABP as a quantitation standard, accurate estimations of the extent of in vivo formation of dG-C8-4-ABP were obtained.
Beland
F A
FA
Division of Biochemical Toxicology, National Center for Toxicological Research, Jefferson, Arkansas 72079, USA. fbeland@nctr.fda.gov
Doerge
D R
DR
Churchwell
M I
MI
Poirier
M C
MC
Schoket
B
B
Marques
M M
MM
D000611:Aminobiphenyl Compounds / Q000737:chemistry* / Q000633:toxicity; D000818:Animals; D002273:Carcinogens / Q000737:chemistry* / Q000633:toxicity; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D018736:DNA Adducts / Q000138:chemical synthesis / Q000737:chemistry*; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000737:chemistry; D006868:Hydrolysis; D008099:Liver / Q000187:drug effects / Q000378:metabolism; D008297:Male; D013058:Mass Spectrometry; D051379:Mice; D008815:Mice, Inbred Strains; D010761:Phosphorus Radioisotopes
D000611:Aminobiphenyl Compounds; D002273:Carcinogens; D018736:DNA Adducts; D010761:Phosphorus Radioisotopes; C006757:4-biphenylamine; C014593:N-hydroxy-4-aminobiphenyl; C053420:N-(deoxyguanosin-8-yl)-4-aminobiphenyl; D004247:DNA; D003849:Deoxyguanosine