Vol. 62, Issue 2, 257-264, August 2002

Apoptosis in Murine Hepatoma Hepa 1c1c7 Wild-Type, C12, and C4 Cells Mediated by Bilirubin

John M. Seubert, Alison J. Darmon, Ayman O. S. El-Kadi, Sudhir J. A. D'Souza, and John R. Bend

Department of Pharmacology and Toxicology, University of Western Ontario (J.M.S., A.J.D., J.R.B.); Departments of Pediatrics, Pharmacology and Toxicology, Child Health Research Institute, London, Ontario, Canada (S.J.A.D.); and Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada (A.O.S.E.-K.)

Elevated serum and tissue bilirubin concentrations that occur in pathological conditions such as cholestasis, jaundice, and other liver diseases are known to stimulate cytotoxic responses. In preliminary studies, we noted that bilirubin seemed to cause apoptosis in murine hepatoma Hepa 1c1c7 wild-type (WT) cells. Consequently, we investigated apoptosis caused by bilirubin in WT, mutant C12 [aryl hydrocarbon receptor (AHR)-deficient], and C4 (AHR nuclear translocator-deficient) Hepa 1c1c7 cells. Three independent measures of apoptosis were used to quantify the effects of exogenous bilirubin (0, 1, 10, 25, 50, or 100 µM). Caspase-3 activity and cytochrome c release from mitochondria increased at 3 h post-treatment, before increased caspase-8 activity at 6 h, and nuclear condensation by 24 h after treatment with bilirubin. No differences in whole-cell lipid peroxidation were observed between the cell types; however, intracellular reactive oxygen species (ROS) production was greater in WT cells than C12 or C4 cells 3 h after bilirubin exposure. Pretreatment of cells for 1 h with 1 or 10 µM -naphthoflavone, an AHR antagonist, before bilirubin exposure resulted in decreased caspase-3 activity at 6 h and nuclear condensation at 24 h in WT cells. These results indicate that bilirubin, a potential AHR ligand, causes apoptosis in murine Hepa 1c1c7 WT cells by a mechanism(s) partially involving the AHR, disruption of membrane integrity, and increased intracellular ROS production.