HPLC enrichment of hydrophobic DNA--carcinogen adducts for enhanced sensitivity of 32P-postlabeling analysis.

Abstract

The sensitivity of 32P-postlabeling analysis for the quantitation of DNA-carcinogen adducts can be sharply increased by enriching adducted nucleotides relative to normal nucleotides in digested DNA samples prior to postlabeling. Normal and adducted nucleotides from benzo[a]pyrene-adducted DNA were injected onto a reverse phase HPLC column. Normal nucleotides were eluted with 5% methanol, 95% 1 M ammonium formate, pH 3.5. Hydrophobic adducted nucleotides were then recovered from the column using either a linear gradient of methanol (to fractionate adducts) or a step gradient of methanol to elute all hydrophobic adducts in one fraction. Recovered chromatographic fractions were dried, postlabeled with 32P, and aromatic DNA--carcinogen adducts were detected and quantitated using TLC on polyethyleneimine cellulose. Results from the HPLC adduct enrichment procedure were generally similar to those from an alternative enrichment scheme utilizing nuclease P1 to selectively dephosphorylate normal nucleotides. HPLC and nuclease P1 procedures are easily combined for the dual analysis of DNA samples from organisms exposed to environmental carcinogens.