Study of the cholestatic potential of relevant concentrations of cyclosporine A in primary human hepatocyte spheroids

  • Tiago Antão ((PhD) Student)
  • Vânia Vilas-Boas (Promotor)
  • Mathieu Vinken (Promotor)
  • Rosário Lobato (Co-promotor)

Student thesis: Master's Thesis

Abstract

The liver is the major organ responsible for the metabolism and excretion of endogenous and exogenous compounds, being more exposed to toxic compounds than any organ. Drug-induced cholestasis is attributed to the toxic accumulation of bile acids inside the hepatocytes and its manifestation can be delayed in onset. Cholestasis is a pathology where bile secretion to duodenum is reduced, whether by reduced functionality of the hepatocytes or obstruction of the excretory pathway of bile.
The main objective of this research was to study the long-term effects of cyclosporine A in primary human hepatocytes spheroid cultures.
A dose-response assay was conducted for 28 days, where the cells were exposed to several drug concentrations. Cell viability was measured by quantification of the total ATP content. It was assessed that cyclosporine A was toxic in a dose and time dependent manner.
Furthermore, it was studied the synergistic effects between cyclosporine A and bile acids. Spheroids were exposed to cyclosporine A in the presence and in the absence of bile acids for 28 days. Findings suggested that spheroids were sensitized to cyclosporine A in the presence of bile acids, therefore confirming their synergism.
Cholestatic index was calculated to determine which cyclosporine A concentrations had the potential to cause cholestasis. Cholestatic index was the ratio between the ATP content of the hepatocytes in the presence of bile acids and the ATP content of the hepatocytes in the absence of bile acids. Values lower than or equal to 0.8 indicated that the compound had the potential to cause cholestasis. Results showed cholestatic index values lower than 0.8, when spheroids were exposed to 5 µM cyclosporine A after 14 days of drug exposure. According to the results, cyclosporine A was suggested to demonstrate potential to develop cholestasis.
More studies should be done in this area to discover by which mechanisms cyclosporine A induces cholestasis, since it is not yet fully understood. This can be achieved by performing RT-qPCR and Western Blot assays to study gene and protein expression, respectively.
Date of Award27 Nov 2020
Original languageEnglish
Awarding Institution
  • University of Lisbon

Keywords

  • cholestasis
  • primary human hepatocyte spheroids
  • cyclosporine A
  • synergistic effects of bile acids
  • cholestatic index

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