Apoptotic as well as anti-apoptotic signals induced by polycyclic aromatic hydrocarbons
Division of Environmental Medicine, Norwegian Institute of Public Health, Oslo, Norway
AIM: Polycyclic aromatic hydrocarbons (PAH) and cyclopenta-PAH (CP-PAH) are major pollutants in the environment formed during incomplete combustion of organic material. Several PAH and CP-PAH have been shown to be mutagenic and to induce tumours. In the present study we have characterised the apoptotic and anti-apoptotic effects of some of these compounds, in order to explore mechanisms and elucidate the relevance of such effects with regard to the development of cancer. METHODS: Hepa1c1c7 cells were exposed to testsubstance and cell death was determined by staining with propidium iodide and Hoechst 33342, and analyses by fluorescence microscopy. The amount of apoptotic cells was also determined by flow cytometry after staining DNA with Hoechst 33258. DNA fragmentation was resolved by agarose gel electrophoresis. Caspase-3, -8, poly(ADP-ribose)polymerase (PARP), CYP1A1, p53, p38 MAPK, ERK1/2, Bid and Bcl-2 proteins, were analysed by immunoblotting using specific antibodies. The amount of covalently [3H]-B[a]P bound to macromolecules was determined by scintillation counting. Intracellular localisation of p53 and Bax was analysed by fluorescence microscopy after immunostaining, while the mitochondria were labelled with mitotracker red. RESULTS: In this study we show that benzo[a]pyrene (B[a]P) and several CP-PAH caused apoptosis in Hepa1c1c7 cells as measured by fluorescence microscopy, flow cytometry, Western blotting of caspase-3 and PARP as well as DNA gelelectrophoresis. All the compounds tested increased expression of CYP1A1 with relative potencies corresponding well with their apoptotic responses. ï¡-Naphthoflavone (ï¡NF), an inhibitor of CYP1A1, reduced covalent binding of B[a]P and the induced apoptosis. Furthermore, B[a]P- and CP-PAH- exposure resulted in an accumulation of p53, and the inhibitor pifithrin-ï¡ reduced the apoptosis. The compounds also resulted in phosphorylation of p38 MAPK, and an inhibitor against p38 MAPK activity (SB202190) markedly weakened the apoptotic effects of the compounds. 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 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 treatment and addition of ï¡NF did not inhibit the up-regulation. Following exposure to B[a]P ERK1/2 was somewhat increased, and the apoptotic effect was increased by the inhibitor U0126. CONCLUSIONS: The reactive B[a]P and the CP-PAH metabolites seem to result in apoptotic signals, while the parent compounds appear to give anti-apoptotic signals. The final result may be an increased probability for the cells to survive with DNA damage. Such properties may be of importance when explaining the carcinogenic effects of PAH and CP-PAH compounds.
Paper presented at the International Symposium on Predictive Oncology and Intervention Strategies; Nice, France; February 7 - 10, 2004; in oral session 795 (Apoptosis mechanisms).