Journal Article

Highly sensitive chemiluminescence immunoassay for benzo[<i>a</i>]pyrene-DNA adducts: validation by comparison with other methods, and use in human biomonitoring

Rao L. Divi, Frederick A. Beland, Peter P. Fu, Linda S. Von Tungeln, Bernadette Schoket, Johanna Eltz Camara, Monica Ghei, Nathaniel Rothman, Rashmi Sinha and Miriam C. Poirier

in Carcinogenesis

Volume 23, issue 12, pages 2043-2049
Published in print December 2002 | ISSN: 0143-3334
Published online December 2002 | e-ISSN: 1460-2180 | DOI:
Highly sensitive chemiluminescence immunoassay for benzo[a]pyrene-DNA adducts: validation by comparison with other methods, and use in human biomonitoring

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A chemiluminescence immunoassay (CIA) utilizing antiserum elicited against DNA modified with (±)-7β, 8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]- pyrene (BPDE) has been developed and validated to study the formation of polycyclic aromatic hydrocarbon (PAH)–DNA adducts in human tissues. Advantages include a low limit of detection for 10b-(deoxyguanosin-N2-yl)-7β,8α,9α-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPdG, ~1.5 adducts/109 nucleotides using 20 μg DNA) and a high signal-to-noise ratio (≥100). The CIA BPDE–DNA standard curve gave 50% inhibition at 0.60 ± 0.08 fmol BPdG (mean ± SE, n = 30), which was a 10-fold increase in sensitivity compared with the dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA). Calf thymus DNA modified with [1,3-3H]BPDE was assayed by radiolabeling, 32P-postlabeling, DELFIA and CIA, and all assays gave similar values. Liver DNAs from mice exposed to 0.5 and 1.0 mg [7,8-3H]benzo[a]pyrene (BP) were assayed by the same four assays and a dose–response was obtained with all assays. The BPDE–DNA CIA was further validated in MCL-5 cells exposed to 4 μM BP for 24 h, where nuclear and mitochondrial DNA adduct levels were associated with an increase in DNA tail length measured by the Comet assay. Human peripheral blood cell (buffy coat) DNA samples (n = 43) obtained from 25 individuals who were either colorectal adenocarcinoma patients or controls were assayed by BPDE–DNA CIA. Three samples (7%) were non-detectable, and the remaining 40 samples had values between 0.71 and 2.21 PAH–DNA adducts/108 nucleotides. The intra-assay coefficient of variation (CV), for four wells on the same microtiter plate, was 1.85%. Sufficient DNA for two assays, on separate plates, was available for 38 of the 43 samples, and the PAH–DNA adduct values obtained were highly correlated (r2 = 0.95). Coded duplicate DNA samples from 15 individuals were assayed four times gave an inter-assay CV of 13.8%.

Keywords: BP, benzo[a]pyrene; BPDE, (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; BPDE–DNA, DNA modified with BPDE having a single major adduct, BPdG; BPdG, 10b-(deoxyguanosin- N2-yl)-7β,8α,9α-trihydroxy 7,8,9,10-tetrahydrobenzo[a]pyrene; CIA, chemiluminescence immunoassay; CV, coefficient of variation; DELFIA, dissociation-enhanced lanthanide immunoassay; MCL-5, multi competent human lymphoblastoid cell line expressing the cytochrome P450s 1A1, 1A2, 2A6, 3A4, and 2E1, and epoxide hydrolase; PAH, polycyclic aromatic hydrocarbon; PBS, phosphate buffered saline; PBST, phosphate buffered saline with 0.05% Tween 20

Journal Article.  5875 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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