IMPROVED HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY ANALYSIS OF P-32-POSTLABELED 2-AMINO-1-METHYL-6-PHENYLIMIDAZO[4,5-B]PYRIDINE ADDUCTS USING IN-LINE PRECOLUMN PURIFICATION

An improved HPLC-based P-32-postlabeling assay has been developed for the analysis of DNA modified with the food carcinogen 2-amino-1-methyl -6-phenylimidazo[4,5-b]pyridine (PhIP). Postlabeled samples are loaded onto a C-18 precolumn and adducted bases are retained while excess ra dioactivity and unmodified DNA bases are eluted directly to waste thro ugh a switching valve. The use of this HPLC in-line precolumn purifica tion (HIPP) technique allows entire postlabeled samples to be analyzed without prior removal of inorganic phosphate and unmodified DNA bases . The method has a sample to sample precision of 15% and accuracy of 2 0%, at adduct levels of 2 adducts/10(7) bases and shows a linear relat ionship between signal and adduction levels from 1 adduct per 10(4) to approximate to 2 +/- 1 adducts per 10(9) bases. Individual postlabele d DNA samples can be analyzed by HPLC in less than 1 h, allowing high throughput. The use of calf-thymus DNA (CT-DNA), highly modified with PhIP, or DNA isolated from mice chronically fed a PhIP-modified diet s hows two major PhIP-DNA adduct peaks and three additional minor adduct peaks when labeled under ATP-limiting conditions. Isolation of the HP LC purified peaks and analysis by thin layer chromatography (TLC) matc hes the five HPLC peaks to the spots typically seen by TLC, including osin-8-yl)-2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (dG-C8-PhI P). Variations in digestion techniques indicate a potential resistance of the PhIP-DNA adducts to the standard enzymatic digestion methods. Attempts at adduct intensification by solid phase extraction, nuclease P1 enrichment or 1-butanol extraction decreased PhIP-DNA adduct peaks and introduced a large early eluting peak. Removal of the 3'-phosphat e with nuclease P1 following the kinase labeling reaction simplifies t he HPLC profile to one major peak (dG-C8-PhIP monophosphate) with seve ral minor peaks. In addition to the high resolution provided by HPLC s eparation of the PhIP-DNA adducts, this method can be adjusted for ana lysis of other DNA adducts and is readily automated for high throughpu t.