Standardization of the 32P-postlabeling assay for polycyclic aromatic hydrocarbon-DNA adducts.

Abstract

A modified method for the 32P-postlabeling assay that permits standardized quantitation for specific polycyclic aromatic hydrocarbon (PAH)-DNA adducts is described. This method has been designed to test the components of the 32P-postlabeling assay for use in the chemically specific detection of individual adducts with the ultimate goal of testing the biological significance of PAH-DNA adducts in humans. The approach relies upon high performance liquid chromatography (HPLC), concomitant labeling of 2'-deoxyguanosine (dGp) as an internal standard and thin layer chromatography (TLC), which identifies unmodified nucleotides along with PAH-DNA adducts on the same TLC plate. This method assesses labeling efficiency, detects the presence of unknown inhibitors, assesses the adequacy of digestion (when combined with HPLC) and allows for the development of calibration curves for directly determined molar ratios (adduct:internal standard). Chemically synthesized adduct standards and quantitative 32P-postlabeling data have been corroborated by UV spectroscopy, fluorescence spectroscopy and liquid scintillation counting where radiolabeled materials were available. Labeling efficiencies of the PAH-DNA adducts were found to be up to 100-fold less than expected and depended upon both the adduct and adduct levels (lower levels being less efficiently detected). The presence of unmodified nucleotides resulted in a 1.5-fold lower labeling efficiency. Mixtures of selected PAH-DNA adducts did not affect the labeling efficiencies of each other. The data suggest that previous 32P-postlabeling assay studies for PAH-DNA adducts may have underestimated adduct levels due to variations in labeling efficiency.