COMPARISON OF P-32 POSTLABELING AND HPLC-FD ANALYSIS OF DNA-ADDUCTS IN RATS ACUTELY EXPOSED TO BENZO(A)PYRENE

DNA adduct analysis is often used for biomonitoring individuals expose d to polycyclic aromatic hydrocarbons (PAH). The P-32-postlabeling ass ay is routinely applied to study the formation of aromatic bulky adduc ts, but cannot positively identify individual adduct types. Recently, an HPLC assay with fluorescence detection (HPLC-FD) was developed whic h was sufficiently sensitive to detect adducts formed by benzo[a]pyren e (B[a]P) diolepoxide isomers [(+/-)anti- and(+/-)syn-BPDE] in occupat ionally exposed subjects (Rojas et al. Carcinogenesis, 16 (1995) 1373- 1376). In this study, we compared both techniques using DNA samples of rats which were treated i.p. with B[a]P (10 mg/kg bw). The internal d ose was assessed by measuring 3-OH-B[a]P excretion in urine. The detec tion limit of the HPLC-FD assay varied from 0.5 to 7.4 adducts per 10( 8) nucleotides, while the detection limit of the P-32-postlabeling ass ay was around 1 adduct per 10(9) nucleotides. HPLC-FD analysis showed that BPDE-DNA adduct levels were highest in the heart, lung and liver respectively. The most predominant B[a]P-tetrol was the I-1 isomer, wh ich derives from hydrolysis of the major reaction product of DNA and ( +)-anti-BPDE. P-32-postlabeling analysis revealed an adduct spot that comigrated with a [H-3]BPDE-DNA standard. The putative BPDE-DNA adduct levels were highest in heart followed by lung and liver and correlate d significantly with tetrol I-1 levels determined by HPLC-FD (r = 0.72 , P = 0.006). In samples in which both tetrol I-1 and II-2 were detect ed by means of HPLC-FD, this correlation was even better (r = 0.95, P = 0.01). Estimated half-lives of BPDE-DNA adducts were in the ranking order; heart, lung and liver for both techniques. By P-32-postlabeling , adducts other than BPDE-DNA were also found, resulting in highest to tal DNA adduct levels in the liver, heart and lung respectively. Furth ermore, mean 24 h urinary excretion of 3-OH-B[a]P was related to BPDE- DNA adduct levels in lung, liver and heart. The P-32-postlabeling assa y is sensitive and capable of detecting exposures to complex mixtures, whereas the HPLC-FD assay can be used to identify BPDE-isomers and mi ght therefore be of value in risk assessment of individuals exposed to PAH. (C) 1997 Elsevier Science Ireland Ltd.