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COMPARISON OF THE GENOTOXIC ACTIVITIES OF THE K-REGION DIHYDRODIOL OF BENZO[A]PYRENE WITH BENZO[A]PYRENE IN MAMMALIAN CELLS: MORPHOLOGICAL CELL TRANSFORMATION; DNA DAMAGE; AND STABLE COVALENT DNA ADDUCTS
Citation:
Nesnow, S, C. R. Davis, G B. Nelson, G. Lambert, W. Padgett, M Pimentel, A H. Tennant, A D. Kligerman, AND J A. Ross. COMPARISON OF THE GENOTOXIC ACTIVITIES OF THE K-REGION DIHYDRODIOL OF BENZO[A]PYRENE WITH BENZO[A]PYRENE IN MAMMALIAN CELLS: MORPHOLOGICAL CELL TRANSFORMATION; DNA DAMAGE; AND STABLE COVALENT DNA ADDUCTS. MUTATION RESEARCH 521(1-2):91-102, (2001).
Description:
Benzo[a]pyrene (B[a]P) has been the most thoroughly studied polycyclic aromatic hydrocarbon (PAH) with regard to its occurrence, metabolism, toxicological and carcinogenic activities. Many mechanisms have been suggested to explain its carcinogenic activity, yet many questions still remain. K-region dihydrodiols of PAHs are common metabolic intermediates and have been thought to be detoxification products. However, recently K-region dihydrodiols of several PAHs have been shown to morphologically transform mammalian cells in culture. Here, we present evidence that the K-region dihydrodiol of B[a]P, trans-B[a]P-4,5-diol, is a strong morphological cell transforming agent in C3H10T?CL8 mouse embryo fibroblasts (C3H10T? cells). This dihydrodiol also induced DNA damage in these cells, but did not form stable covalent DNA adducts. trans-B[a]P-4,5-diol and B[a]P morphologically transformed C3H10T? cells by producing significant numbers of Type II and Type III foci over a concentration range of 1.8-10.5 M, for trans-B[a]P-4,5-diol, and 2.1-8.1 M for B[a]P. The dose response curves for trans-B[a]P-4,5-diol and B[a]P were indistinguishable. Since morphological cell transformation is strongly associated with mutation and/or larger scale DNA damage in C3H10T? cells, the identification of DNA damage induced in these cells by trans-B[a]P-4,5-diol was sought. The comet assay (single cell gel electrophoresis assay) under alkaline conditions has been shown to identify a wide variety of classes of genotoxins as it detects single strand breaks, alkali-labile lesions and excision repair-induced strand breaks. The addition of hydroxyurea and cytosine arabinoside to the cells prior to genotoxin treatment provides enhanced responses, as these agents inhibit DNA resynthesis. Using experimental conditions and concentrations similar to those employed in the morphological cell transformation studies, C3H10T? cells were pre-treated with trans-B[a]P-4,5-diol or B[a]P to induce the predominant cytochrome P450 1B1, and 18 hr later were treated again with B[a]P or trans-B[a]P-4,5-diol. Both trans-B[a]P-4,5-diol (8 and 10 M) and B[a]P (1,4,8,10 M) exhibited significant DNA damaging activity (determined as comet tail content) compared to the control after 1 hr of treatment without significant concurrent cytotoxicity. Furthermore, the distributions of comet tail contents were altered in the trans-B[a]P-4,5-diol and B[a]P treatment groups compared to the controls. DNA adduct patterns from C3H10T? cells were examined after trans-B[a]P-4,5-diol or B[a]P treatment using 32P-postlabeling techniques and improved TLC elution systems designed to separate polar DNA adducts. While B[a]P treatment produced one major DNA adduct identified as anti-trans-B[a]P-7,8-diol-9,10-epoxide-deoxyguanosine, no stable covalent DNA adducts were detected in the DNA of trans-B[a]P-4,5-diol-treated cells. In summary, this study provides evidence for the DNA damaging and morphological cell transforming activities of the K-region dihydrodiol of B[a]P, in the absence of covalent stable DNA adducts. In concert with the morphological cell transformation activities of other K-region dihydrodiols of PAHs, these data suggest a new mechanism/pathway for the bioactivation of PAHs.