Quantitative measurement of genetic and‭ ‬epigenetic‭ ‬biomarkers in molecular epidemiology of cancer

Dr. Fayad Z. Sheabar

The chemical foundation for new method to detect carcinogen-DNA adducts, designated as Adduct Detection by Acylation with Methionine (ADAM) and a method to detect aflatoxin-albumin adducts are reported. The ADAM method is based on reaction of DNA adducts with a protected methionine derivative, (tert-butoxycarbonyl)-L-methionine N-hydroxysuccinimidyl ester (TBM-NHS). Acylation of 2׳-deoxyguanosine (dGuo), used as a prototypical deoxynucleoside, and N-(deoxyguanosine-8-yl)-4-aminobiphenyl (dGuo-8-ABP), the major DNA adduct formed after in vivo exposure to 4-aminobiphenyl, a known human carcinogen, with TBM-NHS was optimized, and products were characterized by ³H radioactivity, UV absorbance, mass spectrometry, and ¹H and ¹³C NMR. Broad applicability of the ADAM procedure for the acylation of various carcinogens was demonstrated. Reaction of synthetic dGuo-8-ABP with ³⁵S-labeled TBM-NHS, under optimized conditions produced mono-, bis, and tris-TBM-acylated nucleosides that were separated by RP-HPLC. The ADAM procedure was successfully applied for the quantification of carcinogen DNA adducts from animal and various human tissues.