Adduct detection by acylation with [35S]methionine: analysis of DNA adducts of 4-aminobiphenyl.

Abstract

Reaction of synthetic N-(2'-deoxyguanosin-8-yl)-4-aminobiphenyl (dGuo-8-ABP) with t-butoxycarbonyl-L-[35S]methionine, N-hydroxysuccinimidyl ester (35S-labeled TBM-NHS), under optimized conditions produced mono-, bis-, and tris-TBM-acylated nucleosides that were separable by HPLC. Reaction of different amounts of N-(2'-deoxy-1',2'-[3H]guanosin-8-yl)-4-aminobiphenyl ([3H]dGuo-8-ABP) with 35S-labeled TBM-NHS established that total 35S content of acylated products was linearly related to adduct concentration (r = 0.992) over the range of 10 fmol to 30.6 pmol. Additionally, the N-(deoxyguanosin-8-yl)-4-[3H]aminobiphenyl (dGuo-8-[3H]ABP) adduct was isolated from calf thymus DNA adducted in vitro and from rat liver DNA adducted in vivo and similarly reacted with 35S-labeled TBM-NHS. Acylation products of dGuo-8-ABP from all three sources showed HPLC retention times identical to those of authentic TBM-dGuo-8-ABP, and 35S incorporation into acylated products was linearly related to amount of adduct reacted. These results indicate that the procedure, to which we have referred as adduct detection by acylation with methionine (ADAM), has potential applicability as an analytical procedure for detection and quantification of DNA adducts in human tissues in the molecular epidemiology of cancer.