ADDUCT DETECTION BY ACYLATION WITH [S-35] METHIONINE - ANALYSIS OF DNA-ADDUCTS OF 4-AMINOBIPHENYL

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