The Role of Bile Acids and Nitric Oxide in Apoptosis of Liver Cells

Introduction: Retention and accumulation of hydrophobic bile acids (BAs) may cause liver toxicity by inducing apoptosis via yet unknown pathways. It was also shown that hydrophobic BAs could induce nitric oxide (NO) production in isolated rat hepatocytes and mitochondria. NO is discussed as one mediator of apoptosis. GOAL: The present study was directed to investigate effects of BAs in liver tissue by means of precision-cut tissue slices (PCTS) in regard to apoptosis and NO production. Methods: PCTS were prepared from porcine livers using Brendel Vitron Tissue Slicer, then incubated submersed in supplemented RPMI medium without or with 1% DMSO and treated with the hydrophobic BAs deoxycholic (DCA) and glycodeoxycholic (GDCA), or with hydrophilic ursodeoxycholic acid (UDCA) for 3, 12, and 24 hours at concentrations of 0,5, 1 and 2mM. Supernatant nitrite and lactate dehydrogenase (LDH) levels were measured. The tissue samples were collected for immunostainings to detect caspase-cleaved cytokeratin–18 fragments as apoptotic marker, proliferation marker Ki–67 and inducible NOS (iNOS) protein. Results: When compared to control, liver slices produced significantly dose- and time-dependent higher amount of NO after BA stimulation with hydrophobic BAs being more potent. All BAs induced expression of iNOS in Kupffer cells. DCA and GDCA increased LDH leakage from PCTS but they slightly decreased number of apoptotic cells throughout the study. Amount of apoptosis in PCTS after stimulation with UDCA were similar to control and increased with time. At the beginning of incubation period, a low proliferation rate was observed in hepatocytes and higher rate was observed in Kupffer cells, both decreasing with time. Conclusion: The use of PCTS revealed cell specific difference in susceptibility of liver cells to undergo BA-induced apoptosis. Hepatocytes exposed to hydrophobic BAs rapidly developed resistance to their toxicity when compared to control and hydrophilic BA. This resistance to apoptosis could be caused by increased iNOS expression and NO production. Thus, NO is among other important signals that regulate process of apoptosis under certain pathological condition related to cholestasis.

Schlüsselwörter

Nitric oxide - bile acids - liver apoptosis