Quantitation of benzo[ a]pyrene-DNA adducts by postlabeling with 14C-acetic anhydride and accelerator mass spectrometry

• Published in: Chemico-biological interactions Vol. 126; no. 3; pp. 171 - 183
• Main Authors: Goldman, Radoslav, Day, Billy W, Carver, Tonya A, Mauthe, Robert J, Turteltaub, Kenneth W, Shields, Peter G
• Format: Journal Article
• Language: English
• Published: Ireland Elsevier Ireland Ltd 01.06.2000
• Subjects: 14C-postlabeling; Acetic Anhydrides - chemistry; Acetylation; Animals; Benzo(a)pyrene - analysis; Benzo[ a]pyrene; Carbon Radioisotopes; Carcinogenesis; Carcinogens, Environmental - analysis; Cattle; Chromatography, High Pressure Liquid; DNA adduct; DNA Adducts - analysis; Isotope Labeling - methods; Mass Spectrometry - methods; Molecular epidemiology; Spectrophotometry, Atomic; TFE, trifluoroethanol; THF, tetrahydrofuran; MeIm, 1-methylimidazole; RP-HPLC, reverse phase-high performance liquid chromatography; DNA adduct; TLC, thin layer chromatography; TEA, triethylamine; Carcinogenesis; LSC, liquid scintillation counting; 14C-postlabeling; DMSO, dimethylsulfoxide; PB, phosphate buffer; AMS, accelerator mass spectrometry; BPDE, benzo[ a]pyrene- r-7, t-8-dihydrodiol- t-9,10-epoxide; Molecular epidemiology; Benzo[ a]pyrene; LC–MS, liquid chromatography–mass spectrometry; NP1, nuclease P1; dG, deoxyguanosine; BPdG (RSRS), 7 R,8 S,9 R-trihydroxy-10 S-(N 2-deoxyguanosyl)-7,8,9,10-tetrahydrobenzo[ a]pyrene; SVPD, snake venom phosphodiesterase; AP, alkaline phosphatase; CD, circular dichroism; TFA, trifluoroacetic acid
• ISSN: 0009-2797; 1872-7786
• DOI: 10.1016/S0009-2797(00)00160-5

Quantitation of carcinogen-DNA adducts provides an estimate of the biologically effective dose of a chemical carcinogen reaching the target tissue. In order to improve exposure-assessment and cancer risk estimates, we are developing an ultrasensitive procedure for the detection of carcinogen-DNA adducts. The method is based upon postlabeling of carcinogen-DNA adducts by acetylation with 14C-acetic anhydride combined with quantitation of 14C by accelerator mass spectrometry (AMS). For this purpose, adducts of benzo[ a]pyrene-r-7, t-8-dihydrodiol- t-9,10-epoxide (BPDE) with DNA and deoxyguanosine (dG) were synthesized. The most promutagenic adduct of BPDE, 7 R,8 S,9 R-trihydroxy-10S-(N 2-deoxyguanosyl)-7,8,9,10-tetrahydrobenzo[ a]pyrene (BPdG), was HPLC purified and structurally characterized. Postlabeling of the BPdG adduct with acetic anhydride yielded a major product with a greater than 60% yield. The postlabeled adduct was identified by liquid chromatography–mass spectrometry as pentakis(acetyl) BPdG (AcBPdG). Postlabeling of the BPdG adduct with 14C-acetic anhydride yielded a major product coeluting with an AcBPdG standard. Quantitation of the 14C-postlabeled adduct by AMS promises to allow detection of attomolar amounts of adducts. The method is now being optimized and validated for use in human samples.