Dissertação apresentada para a obtenção do Grau de Mestre em Genética Molecular e Biomedicina, pela Universidade Nova de Lisboa, Faculdade de Ciências e TecnologiaCholestasis is the reduction or stoppage of bile flow. When bile flow is interrupted, the bile compounds, namely bile acids and bilirubin, accumulate into the hepatocyte causing cellular injury and cell death. The hepatocyte function under cholestasis must be studied using liver cell lines closely resembling human primary hepatocytes. However, most of the cell lines used is derived from hepatic tumors which have altered gene expression. In this project, we used the novel non-neoplasic cell line, HHL-5, which retain primary adult hepatocyte phenotype.
In addition, we used the fetal hepatocyte cell line WRL-68, which was shown to present
similar morphological properties and antigenic profile of human fetal hepatocytes in situ.
Using these two cell lines, we evaluated whether fetal and adult hepatocytes respond
differently to conditions that mimic cholestasis with associated jaundice, never evaluated in vitro before. The hepatocytes were exposed to 100 μM glycochenodeoxycholic acid(GCDCA), 100 μM conjugated bilirubin (CB), 100 μM unconjugated bilirubin (UCB), 100 μM GCDCA + 100 μM CB + 100 μM UCB or vehicle alone, in the presence of 100 μM
human serum albumin (HSA), at various time points. After, we assessed cellular toxicity
analyzing cytolysis by lactate dehydrogenase (LDH) release and apoptosis by caspase-3
activity and nuclear fragmentation. There was a significant increase of all these parameters in hepatocytes stimulated with the GCDCA+CB+UCB mixture. LDH release significantly increased after 48 h incubation for GCDCA+CB+UCB treatment (~40%, P<0.01) in the adult cell line. This increase was also statistically significant when compared to GCDCA, CB or UCB incubation alone (P<0.05). Relatively to WRL-68 cells, the release of LDH also increased significantly (~22%) after 48 h incubation with GCDCA+CB+UCB when compared to control (P<0.05), GCDCA (P<0.05) or UCB (P<0.01) incubations. Regarding caspase-3 activity, the increase was evident for GCDCA+CB+UCB treatment. In the adult cell line, it
increased ~1.5-fold after 6 h (P<0.01 vs. control, P<0.01 vs. GCDCA, P<0.05 vs. UCB),
peaking at 12 h (~3.5-fold, P<0.05 vs. control, P<0.05 vs. GCDCA) and remaining elevated after 24 h (P<0.01 vs. control, P<0.01 vs. GCDCA, P<0.05 vs. CB, P<0.01 vs. UCB). In the fetal cell line, the peak of caspase-3 activity with the same treatment occurred earlier and in a higher magnitude. Indeed, caspase-3 activity increased 4-fold after GCDCA+CB+UCB treatment at 6 h incubation (P<0.05) and this activation was sustained until 12 h incubation(P<0.01). Concerning nuclear fragmentation, in both cell lines, hepatocytes incubated with GCDCA+CB+UCB exhibited profound changes in nuclear morphology, consistent with apoptosis, in a more marked way than when incubated with GCDCA, CB or UCB alone. The
results showed similar data to those previously obtained for caspase-3 activity
