Bile salt-induced cytotoxicity and ursodeoxycholate cytoprotection: in-vitro study in perifused rat hepatocytes.
OBJECTIVE
Membrane toxicity induced by hydrophobic bile salts may be important in liver diseases. Administration of ursodeoxycholate reduces serum liver enzymes in chronic liver diseases, but the nature of this effect is still unclear. We aimed at establishing a convenient in-vitro system for investigating the hepatotoxic properties of hydrophobic bile salts and the putative hepatoprotective effect of ursodeoxycholate.


METHODS
About 100 mg of freshly isolated rat hepatocytes were suspended on a resin column (Bio-Gel P4 fine) and perifused with different concentrations of bile salts. The effluent was collected at 5-min intervals and assayed for lactate dehydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activity. Enzyme leakage induced by bile salts was compared with that induced by Triton X-100 (Union Carbide, Danbury, CT, USA) at different concentrations. After perifusion, hepatocytes were collected for electron microscopic observation.


RESULTS
Cytotoxicity of individual bile salts, assessed by enzyme release, was time and concentration dependent and corresponded to their hydrophilic-hydrophobic balance. Perifusion with hydrophilic bile salts, cholate and ursodeoxycholate, did not result in a significant enzyme release in concentrations up to 5 mmol/l, whereas hydrophobic bile salts, chenodeoxycholate and deoxycholate, induced significant enzyme leakage even in low concentrations, 0.5 and 0.1 mmol/l, respectively. Addition of ursodeoxycholate significantly reduced the hepatotoxic effect of deoxycholate. This protective effect was evident within minutes. The ultrastructural appearance of hepatocytes exposed to hydrophobic bile salts was very similar to the non-specific cellular lysis observed after exposition to Triton X-100, suggesting that they act mainly in a detergent-like fashion.


CONCLUSIONS
Perifused rat hepatocytes seem a convenient in-vitro system for investigating the hepatotoxic properties of bile salts and hepatoprotective effect of ursodeoxycholate, offering the opportunity to investigate the effects of bile salts under dynamic conditions, mimicking the in-vivo situation, and allowing continuous enzyme release monitoring. Hydrophobic bile salts seem to act mainly in a detergent-like fashion; ursodeoxycholate-related hepatoprotection could be due not only to a dilution effect of toxic bile salts, but also to a direct cytoprotective effect.
Pazzi
P
P
Department of Gastroenterology, St Anna Hospital, Ferrara, Italy.
Puviani
A C
AC
Dalla Libera
M
M
Guerra
G
G
Ricci
D
D
Gullini
S
S
Ottolenghi
C
C
D000818:Animals; D001647:Bile Acids and Salts / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D066298:In Vitro Techniques; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D008099:Liver / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D008297:Male; D008854:Microscopy, Electron; D011092:Polyethylene Glycols / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors; D014580:Ursodeoxycholic Acid / Q000494:pharmacology*
D001647:Bile Acids and Salts; D011092:Polyethylene Glycols; D014580:Ursodeoxycholic Acid; D007770:L-Lactate Dehydrogenase