Polycyclic aromatic hydrocarbons induce both apoptotic and anti-apoptotic signals in Hepa1c1c7 cells

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Abstract
In this study we show that benzo[ a ]pyrene (B[ a ]P) and the cyclopenta polycyclic aromatic hydrocarbons (CP-PAH) cyclopenta[ c , d ]pyrene (CPP), benz[ j ]aceanthrylene (B[ j ]A) and benz[ l ]aceanthrylene (B[ l ]A) induce apoptosis in Hepa1c1c7 cells, as measured by fluorescence microscopy and flow cytometry. The compounds induced formation of the active form of caspase-3, cleavage of its intracellular substrate, poly(ADP-ribose)polymerase (PARP), and DNA fragmentation. B[ j ]A was found to be the most potent in inducing apoptosis, followed by B[ a ]P, CPP and B[ l ]A. All compounds increased expression of CYP1A1 with relative potencies B[ j ]A > B[ a ]P ≫ CPP > B[ l ]A, corresponding well with their relative apoptotic responses. α-Naphthoflavone (αNF), an inhibitor of CYP1A1, reduced the induced apoptosis. B[ a ]P and CP-PAH exposure also resulted in an accumulation of the tumour suppressor protein p53. No changes were observed in the protein levels of Bax and Bcl-2, whereas the anti-apoptotic Bcl-xl protein was down-regulated, as judged by western blot analysis. Fluorescence microscopic analysis revealed a translocation of p53 to the nucleus and of Bax to the mitochondria. Furthermore, caspase-8 was activated and Bid cleaved. Interestingly, the levels of anti-apoptotic phospho-Bad (Ser155 and Ser112) had a biphasic increase after B[ a ]P or CPP treatment. Whereas αNF markedly reduced the activation of B[ a ]P to reactive metabolites, as measured by covalent binding to macromolecules, it did not inhibit the up-regulation of phospho-Bad. Neither of the compounds triggered apoptosis in primary cultures of rat lung cells (Clara cells, type 2 cells and lung alveolar macrophages), possibly due to a lack of CYP1A1 induction. In conclusion, B[ a ]P and the CP-PAH induced apoptotic as well as anti-apoptotic signals in Hepa1c1c7 cells.

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