Polycyclic aromatic hydrocarbons induce both apoptotic and anti-apoptotic signals in Hepa1c1c7 cells.
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. alpha-Naphthoflavone (alphaNF), 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 alphaNF 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.
Solhaug
Anita
A
Division of Environmental Medicine, Norwegian Institute of Public Health, PO Box 4404 Nydalen, N-0403 Oslo, Norway.
Refsnes
Magne
M
Låg
Marit
M
Schwarze
Per E
PE
Husøy
Trine
T
Holme
Jørn A
JA
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002352:Carrier Proteins / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism; D005434:Flow Cytometry; D008113:Liver Neoplasms / Q000378:metabolism / Q000473:pathology*; D008168:Lung / Q000187:drug effects / Q000378:metabolism; D008297:Male; D051379:Mice; D008856:Microscopy, Fluorescence; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D011084:Polycyclic Aromatic Hydrocarbons / Q000494:pharmacology*; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051381:Rats; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D051028:bcl-2-Associated X Protein; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein
C494820:Bad protein, mouse; C494821:Bad protein, rat; C494827:Bax protein, mouse; C494828:Bax protein, rat; C494812:Bcl2l1 protein, mouse; C494813:Bcl2l1 protein, rat; D002352:Carrier Proteins; D011084:Polycyclic Aromatic Hydrocarbons; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D051025:bcl-Associated Death Protein; D051020:bcl-X Protein; D019363:Cytochrome P-450 CYP1A1; D011065:Poly(ADP-ribose) Polymerases; C505409:Casp3 protein, mouse; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases