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BENZO[A]PYRENE AND ITS K-REGION DIOL INDUCE DNA DAMAGE IN C3H10T1/2C18 CELLS AS MEASURED BY THE ALKALINE SINGLE CELL GEL (COMET) ASSAY
Citation:
Tennant, A H., M Pimentel, A D. Kligerman, AND S Nesnow. BENZO[A]PYRENE AND ITS K-REGION DIOL INDUCE DNA DAMAGE IN C3H10T1/2C18 CELLS AS MEASURED BY THE ALKALINE SINGLE CELL GEL (COMET) ASSAY. Presented at ISPAC, Cincinnati, Ohio, 9/9-13/2001.
Description:
160. Benzo[a]pyrene and its K-region diol induce DNA damage in C3HlOTl/2Cl8 cells as measured by the alkaline single cell gel (Comet) assay
In a continuing series of studies on the genotoxicity ofK-region dihydrodiols of polycyclic aromatic hydrocarbons, we have reported the metabolic K-region dihydrodiol of dibenzo[ a, l]pyrene (DB[ a, I]P), trans- DB[ a, I]P-8,9-diol, was a potent morphological cell transforming agent. In addition, we presented evidence that while DB[a,I]P formed stable DNA adducts, no stable DNA adducts were found in C3H10T1/2 cell DNA from cells treated with B[a,I]P-8,9-diol (Nesnow et a!., Carcinogenesis, 21, 1253- 1257,2000). Furthermore with benzo[a]pyrene (B[a]P), and its K-region diol, trans- B[a]P-4,5-diol, we found similar results: both are strong morphological cell transforming agents in C3H10T1/2 cells, B[a]P forms stable DNA adducts, while trans-B[a]P-4,5-diol does not. Since morphological cell transformation is strongly associated with mutation and/or larger scale DNA damage in C3H10T1/2 cells, we sought to identify DNA damage (other than DNA adduction) induced in these cells by rans-B[a]P-4,5-diol.
The comet assay (alkaline single cell gel electrophoresis assay) has been shown to detect 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 (HU) and cytosine arabinoside ( ara-C) to the cells prior to genotoxin treatment provides enhanced responses, as these agents inhibit DNA resynthesis. Using experimental conditions similar to those employed in the morphological cell transformation studies, C3H10Tl/2 cells were pre-treated B[a]P (1,4,10 uM) or trans-B[a]P-4,5-diol (1,4,10 uM) and 18 hr later were treated again with B[a]P (1,4,10 uM) or trans-B[a]P-4,5-diol (1,4,10 uM) in the presence, and absence, ofHU (10 mM) and ara-C (l.8 mM) for 1 and 2 hr. MMS (70 uM) served as a positive control. Using tail DNA length as a measure of activity, and in the presence of HU and ara-C, both B[a]P and trans-B[a]P-4,5-diol exhibited significant activity over background after 1 hr of treatment. B[a]P induced a significant effect after 2 hr of treatment. These results suggest that trans-B[a]P-4,5-diol can induce significant DNA damage in these mouse embyro cells and that this damage might be related to its morphological cell transforming ability.
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Key words: C3HlOT1/2Cl8 cells, morphological cell transformation, K-region diol, comet assay, benzo[a]pyrene