Journal Article

Effects of the (–)-<i>anti</i>-11<i>R</i>,12<i>S</i>-dihydrodiol 13<i>S</i>,14<i>R</i>-epoxide of dibenzo[<i>a</i>,<i>l</i>]pyrene on DNA adduct formation and cell cycle arrest in human diploid fibroblasts

Brinda Mahadevan, Andreas Luch, Albrecht Seidel, Jill C. Pelling and William M. Baird

in Carcinogenesis

Volume 22, issue 1, pages 161-169
Published in print January 2001 | ISSN: 0143-3334
Published online January 2001 | e-ISSN: 1460-2180 | DOI: http://dx.doi.org/10.1093/carcin/22.1.161
Effects of the (–)-anti-11R,12S-dihydrodiol 13S,14R-epoxide of dibenzo[a,l]pyrene on DNA adduct formation and cell cycle arrest in human diploid fibroblasts

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The tumor suppressor protein p53 plays an important role in recognition of DNA damage and induction of subsequent cell cycle arrest. One of its target genes encodes the protein p21WAF1, which is involved in mediation of growth arrest after DNA damage has occurred. Dibenzo[a,l]pyrene (DB[a,l]P) is a polycyclic aromatic hydrocarbon which is an exceptionally potent carcinogen. A reactive secondary metabolite of DB[a,l]P, the fjord region (–)-anti-11R,12S-dihydrodiol 13R,14S-epoxide [(–)-anti-DB[a,l]PDE] was used to investigate DNA damage via adduct formation and cell cycle arrest in human diploid fibroblast cell cultures (HDF). Synchronous HDF were exposed to increasing concentrations (0.014, 0.028 and 0.07 μM) of (-)-anti-DB[a,l]PDE and at 1, 12, 24 and 42 h after treatment cell pellets were analyzed for DNA adduct formation and cell cycle arrest. Exposure of HDF to 0.07 μM (–)-anti-DB[a,l]PDE caused a total DNA binding level of 113 pmol adducts/mg DNA (42 h after treatment). G1 arrest was induced by this treatment, with 91% of the cells remaining in G1 phase compared with the solvent-treated control cultures (50%) as analyzed by propidium iodide staining and flow cytometry. Further investigation of the percentage of cells in S phase by 5-bromo-2′-deoxyuridine incorporation confirmed the G1 arrest in HDF treated with 0.07 μM (–)-anti-DB[a,l]PDE, with only 1.5% of the cells moving into S phase compared with 39% in the control 42 h after treatment. Induction of p53 and p21WAF1 was demonstrated by western blot analysis.

Keywords: B[a]P, benzo[a]pyrene; BPDE, 7,8-dihydrodiol 9,10-epoxide of B[a]P; BrdU, 5-bromo-2′-deoxyuridine; DB[a,l]P, dibenzo[a,l]pyrene; DB[a,l]PDE, 11,12-dihydrodiol 13,14-epoxide of DB[a,l]P; HDF, human diploid fibroblast cell cultures; NER, nucelotide excision repair; PAH, polycyclic aromatic hydrocarbon; PBS, phosphate-buffered saline; PMSF, phenylmethylsulfonyl fluoride.

Journal Article.  4827 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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