title: Comparison of 32P-postlabeling and HPLC-FD analysis of DNA adducts in rats acutely exposed to benzo(a)pyrene.
abstract: DNA adduct analysis is often used for biomonitoring individuals exposed to polycyclic aromatic hydrocarbons (PAH). The 32P-postlabeling assay is routinely applied to study the formation of aromatic bulky adducts, but cannot positively identify individual adduct types. Recently, an HPLC assay with fluorescence detection (HPLC-FD) was developed which was sufficiently sensitive to detect adducts formed by benzo[a]pyrene (B[a]P) diolepoxide isomers [(+/-)anti- and (+/-)syn-BPDE] in occupationally 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 dose was assessed by measuring 3-OH-B[a]P excretion in urine. The detection limit of the HPLC-FD assay varied from 0.5 to 7.4 adducts per 10(8) nucleotides, while the detection limit of the 32P-postlabeling assay 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, which derives from hydrolysis of the major reaction product of DNA and (+)-anti-BPDE. 32P-postlabeling analysis revealed an adduct spot that comigrated with a [3H]BPDE-DNA standard. The putative BPDE-DNA adduct levels were highest in heart followed by lung and liver and correlated 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 detected 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 32P-postlabeling, adducts other than BPDE-DNA were also found, resulting in highest total DNA adduct levels in the liver, heart and lung respectively. Furthermore, mean 24 h urinary excretion of 3-OH-B[a]P was related to BPDE-DNA adduct levels in lung, liver and heart. The 32P-postlabeling assay is sensitive and capable of detecting exposures to complex mixtures, whereas the HPLC-FD assay can be used to identify BPDE-isomers and might therefore be of value in risk assessment of individuals exposed to PAH.
authors.affiliation: Department of Health Risk Analysis and Toxicology, Maastricht University, The Netherlands.
authors.forename: R W
authors.initials: RW
authors.lastname: Godschalk
authors.forename: I T
authors.initials: IT
authors.lastname: Vermeer
authors.forename: E
authors.initials: E
authors.lastname: Kriek
authors.forename: B
authors.initials: B
authors.lastname: Floot
authors.forename: P A
authors.initials: PA
authors.lastname: Schilderman
authors.forename: E J
authors.initials: EJ
authors.lastname: Moonen
authors.forename: J C
authors.initials: JC
authors.lastname: Kleinjans
authors.forename: F J
authors.initials: FJ
authors.lastname: van Schooten
mesh_terms: D000818:Animals; D001564:Benzo(a)pyrene / Q000032:analysis* / Q000378:metabolism*; D001580:Benzopyrenes / Q000378:metabolism; D002273:Carcinogens / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000378:metabolism*; D018736:DNA Adducts / Q000032:analysis*; D004785:Environmental Pollutants / Q000378:metabolism; D005453:Fluorescence; D007700:Kinetics; D008099:Liver / Q000737:chemistry; D008168:Lung / Q000737:chemistry; D008297:Male; D009206:Myocardium / Q000737:chemistry; D010761:Phosphorus Radioisotopes; D011084:Polycyclic Aromatic Hydrocarbons / Q000378:metabolism; D051381:Rats; D011917:Rats, Inbred Lew; D014556:Urine / Q000737:chemistry
chemical_list: D001580:Benzopyrenes; D002273:Carcinogens; D018736:DNA Adducts; D004785:Environmental Pollutants; D010761:Phosphorus Radioisotopes; D011084:Polycyclic Aromatic Hydrocarbons; C056612:benzo(a)pyrene-DNA adduct; D001564:Benzo(a)pyrene; C021338:3-hydroxybenzo(a)pyrene; D004247:DNA