In vitro systems to predict hepatotoxicity : models based on hepatocarcinoma cell lines

Abstract

The liver has a major role in the metabolism of both endogenous and exogenous compounds. In drug development, reliable and reproducible results regarding the toxicity of a new compound must be obtained. The gold standard is freshly isolated primary human hepatocytes; however, these are costly, difficult to obtain regularly and cannot be cultured extensively. Secondary hepatocyte cell lines are cheaper with a much longer lifespan in culture but do not accurately reflect the expression profile of a hepatocyte. The aim of this work was to promote differentiation of hepatic carcinoma cell lines towards the in vivo hepatic profile and potentially create a well defined and easily accessible model for early stage drug testing. Initial profiling of HepG2 and Huh7 for differentiation markers, transporters and enzymes was carried out by qPCR. Differentiation was attempted by use of growth factors and dimethyl sulfoxide (DMSO) for periods of up to thirty days. Analysis of differentiation markers by qPCR indicated that 1% DMSO treatment for at least 15 days promoted maturation most effectively. Cells treated with 1% DMSO were analysed for mRNA expression of selected transporters and enzymes, followed by treatment with typical inducers, Western blotting and functional assays to assess the presence and function of certain proteins. Although initial results showed potential, further analyses of the 1% DMSO treated cells were less promising. Analysis of transporter and enzyme mRNA expression revealed that many levels did not change favourably towards those observed in liver, or significantly changed from control but remained vastly removed from liver. Results from protein and induction experiments also indicated no benefit of 15-day DMSO treatment in either cell line. In conclusion, 1% DMSO treatment does not promote differentiation towards a more representative hepatic profile in either cell line; alternative methods are needed to develop a more heptocyte-like model using these cells.