Validation of a new fluorometric assay for benzo[a]pyrene diolepoxide-DNA adducts in human white blood cells: comparisons with 32P-postlabeling and ELISA

A new fluorometric assay was validated for quantification of benzo[a]pyrene diolepoxide (BPDE)-DNA adducts in white blood cells (WBC) from humans exposed to polycyclic aromatic hydrocarbons (PAH). This assay has a detection limit of 2 pg of r-7,c-10,t-8,t-9-tetrahydroxy-7,8,9,10-tetrahydrobenzo[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 between 5 and 500 micrograms. The assay was applied to seven WBC DNA samples from lung cancer patients, six of whom were heavy smokers, and to three WBC DNA samples from healthy subjects employed in an aluminum production plant. High levels of BPDE-DNA adducts, ranging from 62 to 533 adducts/10(8) nucleotides were found in six out of seven DNA samples from the lung cancer patients. In WBC DNA from healthy persons BPDE-DNA adducts were detected only in two non-smokers, but at a much lower level than in lung cancer patients (4-10 adducts/10(8) nucleotides). Using coded WBC DNA samples, BPDE-DNA adduct levels measured by fluorometry of the B[a]P-tetrols, were compared with the results obtained by 32P-postlabeling (nuclease P1 enrichment) and ELISA measurements. A good correlation and proportionality was found between the levels of BPDE-DNA adducts measured by fluorometry and 32P-postlabeling (r = 0.95, P < 0.001, n = 8). The correlation between fluorometry and ELISA was much lower and not significant (r = 0.61, P = 0.1, n = 6). Moreover, the ELISA grossly overestimated BPDE-DNA adduct levels measured by the other two methods. The results demonstrate that the highly sensitive and specific fluorometric assay is suitable for measuring BPDE-DNA adducts in WBC from humans exposed to benzo[a]pyrene.