Membrane structural changes support the involvement of mitochondria in the bile salt-induced apoptosis of rat hepatocytes.
The accumulation of toxic bile salts within the hepatocyte plays a key role in organ injury during liver disease. Deoxycholate (DC) and glycochenodeoxycholate (GCDC) induce apoptosis in vitro and in vivo, perhaps through direct perturbation of mitochondrial membrane structure and function. In contrast, ursodeoxycholate (UDC) and its taurine-conjugated form (TUDC) appear to be protective. We show here that hydrophobic bile salts induced apoptosis in cultured rat hepatocytes, without modulating the expression of pro-apoptotic Bax protein, and caused cytochrome c release in isolated mitochondria. Co-incubation with UDC and TUDC prevented cell death and efflux of mitochondrial factors. Using spin-labelling techniques and EPR spectroscopy analysis of isolated rat liver mitochondria, we found significant structural changes at the membrane-water surface in mitochondria exposed to hydrophobic bile salts, including modified lipid polarity and fluidity, altered protein order and increased oxidative injury. UDC, TUDC and cyclosporin A almost completely abrogated DC- and GCDC-induced membrane perturbations. We conclude that the toxicity of hydrophobic bile salts to hepatocytes is mediated by cytochrome c release, through a mechanism associated with marked direct effects on mitochondrial membrane lipid polarity and fluidity, protein order and redox status, without modulation of pro-apoptotic Bax expression. UDC and TUDC can directly suppress disruption of mitochondrial membrane structure, which may represent an important mechanism of hepatoprotection by these bile salts.
Solá
Susana
S
Centro de Patogénese Molecular, Faculdade de Farmácia, University of Lisbon, Lisbon, Av. das Forcas Armadas, Portugal.
Brito
Maria A
MA
Brites
Dora
D
Moura
José J G
JJ
Rodrigues
Cecília M P
CM
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001647:Bile Acids and Salts / Q000494:pharmacology*; D003574:Cytochrome c Group / Q000378:metabolism; D022781:Hepatocytes / Q000187:drug effects*; D007425:Intracellular Membranes / Q000187:drug effects*; D008297:Male; D008563:Membrane Lipids / Q000378:metabolism; D008930:Mitochondria, Liver / Q000187:drug effects* / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000187:drug effects / Q000378:metabolism; D010084:Oxidation-Reduction / Q000187:drug effects; D018384:Oxidative Stress; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2; D051381:Rats; D017207:Rats, Sprague-Dawley; D051028:bcl-2-Associated X Protein
C494828:Bax protein, rat; D001647:Bile Acids and Salts; D003574:Cytochrome c Group; D008563:Membrane Lipids; D024101:Mitochondrial Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein