Glutathione status of isolated rat hepatocytes affects bile acid-induced cellular necrosis but not apoptosis.
An accumulation of hydrophobic bile acids is implicated in the pathogenesis of cholestatic liver diseases. In the present study, we determined if hydrophobic bile acid-induced cellular injury compromised hepatocyte glutathione (GSH) status, and if modulating intracellular GSH levels prevented or facilitated bile acid-induced cellular cytotoxicities. Freshly isolated rat hepatocytes incubated with >/=125 microM of the hydrophobic bile acid, glycochenodeoxycholic acid (GCDC), underwent a time- and dose-dependent decrease of intracellular GSH levels by 4-h incubation. This loss of intracellular GSH was not associated with an increase of intracellular GSH disulfide (GSSG). Rather, GCDC stimulated the dose-dependent accumulation of extracellular GSSG. The mechanism for extracellular GSSG accumulation by GCDC was through increased efflux of reduced GSH from hepatocytes into the media, where it subsequently oxidized to GSSG. Treatment of hepatocytes with GCDC (0-750 microM) did not directly alter GSH-dependent enzyme activities. The reduction of intracellular GSH with 125 microM GCDC correlated with extensive apoptosis at this concentration as determined by fluorescence microscopy of DAPI (4, 6-diamindino-2-phenylindole hydrochloride)-stained nuclei. Higher concentrations of GCDC (>/=500 microM) favored cellular necrosis and lipid peroxidation. Depleting GSH by treating hepatocytes with 1-bromoheptane increased their sensitivity toward GCDC-induced cellular necrosis, but not apoptosis. However, enhancing the hepatocyte GSH content by supplementation with GSH-ethylester (GSH-EE) failed to protect hepatocytes against either mode of cellular death. In conclusion, while GCDC-induced cytotoxicities were associated with an increased efflux of GSH from rat hepatocytes, GSH status modulated GCDC-induced necrosis, but not apoptosis.
Gumpricht
E
E
Center for Human Nutrition, University of Colorado School of Medicine and The Children's Hospital, Denver, Colorado, 80218, USA.
Devereaux
M W
MW
Dahl
R H
RH
Sokol
R J
RJ
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D005978:Glutathione / Q000378:metabolism*; D019803:Glutathione Disulfide / Q000378:metabolism; D005999:Glycochenodeoxycholic Acid / Q000633:toxicity*; D008099:Liver / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D008297:Male; D009336:Necrosis; D051381:Rats; D017207:Rats, Sprague-Dawley
D005999:Glycochenodeoxycholic Acid; D005978:Glutathione; D019803:Glutathione Disulfide