BILE SALT-INDUCED CYTOTOXICITY AND URSODEOXYCHOLATE CYTOPROTECTION - IN-VITRO STUDY IN PERIFUSED RAT HEPATOCYTES

Objectives: Membrane toxicity induced by hydrophobic bile salts may be important in liver diseases. Administration of ursodeoxycholate reduc es serum liver enzymes in chronic liver diseases, but the nature of th is effect is still unclear. We aimed at establishing a convenient in-v itro system for investigating the hepatotoxic properties of hydrophobi c bile salts and the putative hepatoprotective effect of ursodeoxychol ate. Methods: About 100 mg of freshly isolated rat hepatocytes were su spended on a resin column (Bio-Gel P4 fine) and perifused with differe nt concentrations of bile salts. The effluent was collected at 5-min i ntervals and assayed for lactate dehydrogenase (LDH), aspartate aminot ransferase (AST) and alanine aminotransferase (ALT) activity. Enzyme l eakage induced by bile salts was compared with that induced by Triton X-100 (Union Carbide, Danbury, CT, USA) at different concentrations. A fter perifusion, hepatocytes were collected for electron microscopic o bservation. Results: Cytotoxicity of individual bile salts, assessed b y enzyme release, was time and concentration dependent and corresponde d to their hydrophilic-hydrophobic balance. Perifusion with hydrophili c bile salts, cholate and ursodeoxycholate, did not result in a signif icant enzyme release in concentrations up to 5 mmol/l, whereas hydroph obic bile salts, chenodeoxycholate and deoxycholate, induced significa nt enzyme leakage even in low concentrations, 0.5 and 0.1 mmol/l, resp ectively. Addition of ursodeoxycholate significantly reduced the hepat otoxic effect of deoxychotate. This protective effect was evident with in minutes. The ultrastructural appearance of hepatocytes exposed to h ydrophobic bile salts was very similar to the nonspecific cellular lys is observed after exposition to Triton X-100, suggesting that they act mainly in a detergent-like fashion. Conclusion: Perifused rat hepatoc ytes seem a convenient in-vitro system for investigating the hepatotox ic properties of bile sails and hepatoprotective effect of ursodeoxych olate, offering the opportunity to investigate the effects of bile sal ts under dynamic conditions, mimicking the in-vivo situation, and allo wing continuous enzyme release monitoring. Hydrophobic bile salts seem to act mainly in a detergent-like fashion; ursodeoxycholate-related h epatoprotection could be due not only to a dilution effect of toxic bi le salts, but also to a direct cytoprotective effect.