RECOVERIES OF DNA-ADDUCTS OF POLYCYCLIC AROMATIC-HYDROCARBONS IN THE P-32 POSTLABELING ASSAY

The P-32-postlabelling assay for analysis of DNA adducts of chemical c arcinogens has been applied in a large number of experimental animal a nd human studies. Most human studies have dealt with occupational and enviromnental exposures to polycyclic aromatic hydrocarbons (PAHs). Th e postlabelling assay does not allow direct chemical identification, a nd most studies with this method have not been performed in a quantita tive way. Very little is therefore known about the identity and absolu te 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 recover ies of adducts in the phosphorylation step of the assay. For this purp ose 12 different PAHs were reacted individually and in a mixture with DNA in the presence of a rat liver S9 metabolizing system. Different c oncentrations of ATP, calcium chloride and polynucleotide kinase were tested using the nuclease P1 enhancement. We found that each factor co ntributed to adduct recovery and that optimal conditions could be defi ned. Diluting the modified DNA samples up to 1000 times had little inf luence on the recoveries of adducts. Comparing the nuclease P1 and th: butanol extraction procedures for adduct purification showed that bot h methods gave similar patterns and levels of major adducts. The absol ute recoveries in postlabelling, based on H-3-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 s imilar recoveries points towards common factor(s) involved in the redu ction of the recovered adduct levels. Based on the observed recoveries the conclusion can be drawn that when postlabelling related adducts i n human samples the true total adduct levels can be considerably under estimated, even if optimal conditions are used.