title: Development and validation of a direct sandwich chemiluminescence immunoassay for measuring DNA adducts of benzo[a]pyrene and other polycyclic aromatic hydrocarbons.
abstract: We have developed and validated a sandwich chemiluminescence immunoassay (SCIA) which measures polycyclic aromatic hydrocarbon (PAH)-DNA adducts combining high throughput and adequate sensitivity, appropriate for evaluation of adduct levels in human population studies. Fragmented DNA is incubated with rabbit antiserum elicited against DNA modified with r7,t8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and subsequently trapped by goat anti-rabbit IgG bound to a solid surface. Anti-single-stranded (ss) DNA antibodies binds in a quantity proportional to the adduct levels and is detected by chemiluminescence. The BPDE-DNA SCIA has a limit of detection of 3 adducts per 10(9) nucleotides with 5 μg DNA per well. We have validated the BPDE-DNA SCIA using DNA modified in vitro, DNA from benzo[a]pyrene (BP)-exposed cultured cells and mice. The levels of adduct measured by SCIA were lower (30-60%) than levels of bulky DNA adducts measured in the same samples by (32)P-postlabelling. The BPDE-DNA SCIA also detected adducts produced in vivo by PAHs other than BP. When blood DNA samples from maternal/infant pairs were assayed by BPDE-DNA SCIA, the adduct levels obtained were significantly correlated. However, there was no correlation between (32)P-postlabelling and SCIA values for the same samples. The SCIA can be extended to any DNA adduct and is expected to provide, when fully automated, a valuable high-throughput approach in large-scale population studies.
authors.affiliation: Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, 48 Vassileos Constantinou Avenue, Athens 11635, Greece. panosg@eie.gr
authors.forename: Panagiotis
authors.initials: P
authors.lastname: Georgiadis
authors.forename: Katalin
authors.initials: K
authors.lastname: Kovács
authors.forename: Stella
authors.initials: S
authors.lastname: Kaila
authors.forename: Paraskevi
authors.initials: P
authors.lastname: Makedonopoulou
authors.forename: Livia
authors.initials: L
authors.lastname: Anna
authors.forename: Miriam C
authors.initials: MC
authors.lastname: Poirier
authors.forename: Lisbeth E
authors.initials: LE
authors.lastname: Knudsen
authors.forename: Bernadette
authors.initials: B
authors.lastname: Schoket
authors.forename: Soterios A
authors.initials: SA
authors.lastname: Kyrtopoulos
mesh_terms: D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000737:chemistry*; D000328:Adult; D000818:Animals; D018736:DNA Adducts / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D056945:Hep G2 Cells; D006801:Humans; D007231:Infant, Newborn; D007963:Leukocytes, Mononuclear; D008163:Luminescent Measurements / Q000379:methods*; D061986:MCF-7 Cells; D008297:Male; D051379:Mice; D011084:Polycyclic Aromatic Hydrocarbons / Q000737:chemistry*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods
chemical_list: D018736:DNA Adducts; D011084:Polycyclic Aromatic Hydrocarbons; C043931:benzo(a)pyrene-7,8-dihydrodiol-9,10-epoxide-DNA; C095911:polycyclic aromatic hydrocarbons-DNA adduct; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide