DNA-damaging carcinogen 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) induces apoptosis via caspase-9 in primary cultured rat hepatocytes.
The mechanism of cytotoxicity induced by the DNA-damaging carcinogen 3-amino-1,4-dimethyl-5H-pyrido[4,3-b] indole (Trp-P-1) was investigated in primary cultured rat hepatocytes. Cytotoxicity was caused by intact Trp-P-1 and not by metabolically activated derivatives prepared using a recombinant yeast strain AH22/pAMR2 expressing rat cytochrome P450 1A1, and not by metabolically activated derivatives. We also found internucleosomal DNA fragmentation 6 h after treatment with 30 microM Trp-P-1, indicating that the cytotoxicity was due to the induction of apoptosis. After treatment with Trp-P-1, c-Myc protein level increased in a time-dependent manner and p53 protein also increased transiently with a subsequent increase in Bax protein level. This apoptotic pathway required the activation of caspase-9 as an initiator after leakage of cytochrome c into the cytosol from mitochondria and the activation of caspase-3 and -7 as executioners, but not caspase-1, -6 or -8 as measured using the corresponding peptide inhibitors and substrates or western blotting. The activated caspases in turn cleaved poly(ADP-ribose) polymerase as an intracellular substrate. Furthermore, we detected NUC18-like endonuclease activity during apoptosis induced by Trp-P-1. These findings suggest that this apoptosis may have a role against heterocyclic amine-type carcinogens in normal cells.
Shiotani
B
B
Division of Life Science, Graduate School of Science and Technology and Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Japan.
Nonaka
Y
Y
Hashimoto
T
T
Kihara
K
K
Kanazawa
K
K
Danno
G
G
Ashida
H
H
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002243:Carbolines / Q000494:pharmacology*; D002273:Carcinogens / Q000494:pharmacology*; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D002478:Cells, Cultured; D003574:Cytochrome c Group / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D022781:Hepatocytes / Q000187:drug effects* / Q000201:enzymology / Q000648:ultrastructure; D006868:Hydrolysis; D008297:Male; D008928:Mitochondria / Q000201:enzymology; D009152:Mutagenicity Tests; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar
D002243:Carbolines; D002273:Carcinogens; D003574:Cytochrome c Group; D011065:Poly(ADP-ribose) Polymerases; C506435:Casp9 protein, rat; D053453:Caspase 9; D020169:Caspases; C019132:3-amino-1,4-dimethyl-5H-pyrido(4,3-b)indole