VALIDATION OF A NEW FLUOROMETRIC ASSAY FOR BENZO[A]PYRENE DIOLEPOXIDEDNA-ADDUCTS IN HUMAN WHITE BLOOD-CELLS - COMPARISONS WITH P-32 POSTLABELING AND ELISA

A new fluorometric assay was validated for quantification of benzo[a]p yrene diolepoxide (BPDE)-DNA adducts in white blood cells (WBC) from h umans exposed to polycyclic aromatic hydrocarbons (PAH). This assay ha s a detection limit of 2 pg of r-7,c-10,t-8,t-9-tetrahydroxy-7,8,9,10- hydrobenzo[a]pyrene derived from acid hydrolysis of BPDE-DNA, and can measure 1 BPDE adduct per 10(8) unmodified nucleotides. The quantity of WBC DNA required depends on the modification level and varies betwe en 5 and 500 mu g. The assay was applied to seven WBC DNA samples from lung cancer patients, six of whom were heavy smokers, and to three WB C DNA samples from healthy subjects employed in an aluminum production plant. High levels of BPDE-DNA adducts, ranging from 62 to 533 adduct s/10(8) nucleotides were found in six out of seven DNA samples from th e lung cancer patients. In WBC DNA from healthy persons BPDE-DNA adduc ts were detected only in two non-smokers, but at a much lower level th an in lung cancer patients (4-10 adducts/10(8) nucleotides). Using cod ed WBC DNA samples, BPDE-DNA adduct levels measured by fluorometry of the B[a]P-tetrols, were compared with the results obtained by P-32-pos tlabeling (nuclease P1 enrichment) and ELISA measurements. A good corr elation and proportionality was found between the levels of BPDE-DNA a dducts measured by fluorometry and P-32-postlabeling (r = 0.95, P < 0. 001, n = 8). The correlation between fluorometry and ELISA was much lo wer and not significant (r = 0.61, P = 0.1, n = 6). Moreover, the ELIS A grossly overestimated BPDE-DNA adduct levels measured by the other t wo methods. The results demonstrate that the highly sensitive and spec ific fluorometric assay is suitable for measuring BPDE-DNA adducts in WBC from humans exposed to benzo[a]pyrene.