Journal Article

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

Bunsyo Shiotani, Yuji Nonaka, Takashi Hashimoto, Kaori Kihara, Kazuki Kanazawa, Gen-ichi Danno and Hitoshi Ashida

in Carcinogenesis

Volume 22, issue 5, pages 693-700
Published in print May 2001 | ISSN: 0143-3334
Published online May 2001 | e-ISSN: 1460-2180 | DOI: http://dx.doi.org/10.1093/carcin/22.5.693
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

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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 μM 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.

Keywords: AMC, 7-amino-4-methyl-coumarin; CYP, cytochrome P 450; DMSO, dimethyl sulfoxide; DTT, dithiothreitol; ECD, electrochemical detector; GDC, glycodeoxycholic acid; HRP, horseradish peroxidase; PARP, poly(ADP-ribose) polymerase; PMSF, phenylmethylsulfonylfluoride; TNF-α, tumor necrosis factor-α; Trp-P-1, 3-amino-1,4-dimethyl-5H-pyrido [4,3-b]indole.

Journal Article.  7506 words.  Illustrated.

Subjects: Clinical Cytogenetics and Molecular Genetics

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