Role of cell signaling in B[a]P-induced apoptosis: characterization of unspecific effects of cell signaling inhibitors and apoptotic effects of B[a]P metabolites.
Here we show that several cell signaling inhibitors have effect on cyp1a1 expression and the metabolism of benzo[a]pyrene (B[a]P) in Hepa1c1c7 cells. The CYP1A1 inhibitor alpha-naphthoflavone (alpha-NF), the p53 inhibitor pifithrin-alpha (PFT-alpha), the ERK inhibitors PD98059 and U0126, and the p38 MAPK inhibitors SB202190 and PD169316 induced the expression and level of cyp1a1 protein. On the other hand, during the first h the inhibitors appeared to reduce the metabolism of B[a]P as measured by the generation of tetrols and by covalent binding of B[a]P to macromolecules. In contrast, the phosphatidylinositol-3 (PI-3) kinase inhibitor wortmannin, had neither an effect on the cyp1a1 expression nor the B[a]P-metabolism. In order to avoid these unspecific effects, we characterized the mechanisms involved in the apoptotic effects of B[a]P-metabolites. B[a]P and the B[a]P-metabolites B[a]P-7,8-DHD and BPDE-I induced apoptosis, whereas B[a]P-4,5-DHD had no effect. B[a]P, B[a]P-7,8-DHD and BPDE-I induced an accumulation and phosphorylation of p53, while the Bcl-2 proteins Bcl-xl, Bad and Bid were down-regulated. Interestingly, the levels of anti-apoptotic phospho-Bad were up-regulated in response to B[a]P as well as to B[a]P-7,8-DHD and BPDE-I. Both p38 MAPK and JNK were activated, but the p38 MAPK inhibitors were not able to inhibit BPDE-I-induced apoptosis. PFT-alpha reduced the BPDE-I-induced apoptosis, while both the PI-3 kinase inhibitor and the ERK inhibitors increased the apoptosis in combination with BPDE-I. BPDE-I also triggered apoptosis in primary cultures of rat lung cells. In conclusion, often used cell signaling inhibitors both enhanced the expression and the level of cyp1a1 and more directly acted as inhibitors of cyp1a1 metabolism of B[a]P. However, studies with the B[a]P-metabolite BPDE-I supported the previous suggestion that p53 has a role in the pro-apoptotic signaling pathway induced by B[a]P. Furthermore, these studies also show that the reactive metabolites of B[a]P induce the anti-apoptotic signals, Akt and ERK. Neither the induction nor the activity of p38 MAPK and JNK seems to be of major importance for the B[a]P-induced apoptosis.
Solhaug
Anita
A
Division of Environmental Medicine, Norwegian Institute of Public Health, PO Box 4404, Nydalen, N-0403 Oslo, Norway.
Øvrebø
Steinar
S
Mollerup
Steen
S
Låg
Marit
M
Schwarze
Per E
PE
Nesnow
Stephen
S
Holme
Jørn A
JA
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001564:Benzo(a)pyrene / Q000037:antagonists & inhibitors / Q000378:metabolism; D001580:Benzopyrenes / Q000378:metabolism; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D019363:Cytochrome P-450 CYP1A1 / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D053938:DNA Fragmentation; D004101:Dihydroxydihydrobenzopyrenes / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors; D015971:Gene Expression Regulation, Enzymologic; D008168:Lung / Q000201:enzymology / Q000378:metabolism; D008297:Male; D051379:Mice; D000081082:Phosphoinositide-3 Kinase Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction / Q000502:physiology*; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors
D001580:Benzopyrenes; D004101:Dihydroxydihydrobenzopyrenes; D004791:Enzyme Inhibitors; D000081082:Phosphoinositide-3 Kinase Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; D001564:Benzo(a)pyrene; C072019:benzo(a)pyrene diolepoxide I; D019363:Cytochrome P-450 CYP1A1; D048049:Extracellular Signal-Regulated MAP Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C505409:Casp3 protein, mouse; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases