Recoveries of DNA adducts of polycyclic aromatic hydrocarbons in the 32P-postlabelling assay.

Abstract

The 32P-postlabelling assay for analysis of DNA adducts of chemical carcinogens has been applied in a large number of experimental animal and human studies. Most human studies have dealt with occupational and environmental exposures to polycyclic aromatic hydrocarbons (PAHs). The postlabelling assay does not allow direct chemical identification, and most studies with this method have not been performed in a quantitative way. Very little is therefore known about the identity and absolute levels of adducts, which are important contributors to the process of risk identification and quantitation. In the present study it was, therefore, decided to test some parameters suspected to affect recoveries of adducts in the phosphorylation step of the assay. For this purpose 12 different PAHs were reacted individually and in a mixture with DNA in the presence of a rat liver S9 metabolizing system. Different concentrations of ATP, calcium chloride and polynucleotide kinase were tested using the nuclease P1 enhancement. We found that each factor contributed to adduct recovery and that optimal conditions could be defined. Diluting the modified DNA samples up to 1000 times had little influence on the recoveries of adducts. Comparing the nuclease P1 and the butanol extraction procedures for adduct purification showed that both methods gave similar patterns and levels of major adducts. The absolute recoveries in postlabelling, based on 3H-binding of radiolabelled compounds, were for most of the tested compounds relatively low. The fact that the nuclease P1 and the butanol extraction procedures gave similar recoveries points towards common factor(s) involved in the reduction of the recovered adduct levels. Based on the observed recoveries the conclusion can be drawn that when postlabelling related adducts in human samples the true total adduct levels can be considerably underestimated, even if optimal conditions are used.