rat hepatocytes

/Tag:rat hepatocytes

Pyruvate-induced Long-term Maintenance of Glutathione S-Transferase in Rat Hepatocyte Cultures

Tamara Vanhaecke, André Foriers, Albert Geerts, Elizabeth A. Shephard, Antoine Vercruysse and Vera Rogiers

The addition of pyruvate to the culture medium has been reported to improve the maintenance of P450-dependent enzyme expression in primary rat hepatocyte cultures. In this study, the effects of 30mM pyruvate on cell morphology, albumin secretion and glutathione Stransferase (GST) expression were investigated as a function of the time in culture. The effect of triiodothyronine (T3) exposure on GST expression was also measured in pyruvate-treated cultures. Transmission electron microscopy showed that untreated hepatocytes deteriorated after culture for 7 days, whereas the morphology of the pyruvate-treated cells was similar to that observed in intact liver tissue. The albumin secretion rate was significantly higher in rat hepatocytes exposed to pyruvate than in control cells. In the presence of pyruvate, μ and α class GST activities were well maintained, whereas GST π activity was increased over the entire culture period. HPLC analysis revealed that the complement of GST subunits present in hepatocytes is altered during culture with pyruvate: μ class proteins remained relatively constant, whereas a decrease in the α class content was accompanied by a strong increase in GST subunit P1 (GSTP1). The induction of GSTP1 was confirmed at the mRNA level. In control cultures, π class GST activity was increased, but total, μ, and α class GST activities continuously declined as a function of culture time and became undetectable beyond 7 days in culture. At the protein and mRNA levels, a much smaller increase in GSTP1 was observed than in the pyruvate cultures. When the pyruvate-treated cell cultures were exposed to T3, an inhibitory effect on GST activities and proteins was found. These results indicate that this simple culture model could be useful for studying the expression and regulation of GST.
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Evaluation of a Flat Membrane Hepatocyte Bioreactor for Pharmacotoxicological Applications: Evidence that Inhibition of Spontaneously Produced Nitric Oxide Improves Cell Functionality

Nikolina Canová, Eva Kmonícková, Dagmar Lincová, Libor Vítek and Hassan Farghali

A laboratory-scale bioreactor was re-evaluated, with the aim of improving its use for the perfused culture of rat hepatocytes. In contrast to conventional culture systems, the flat membrane bioreactor (FMB) showed good functionality and biochemical competence during 2–3 days. Hepatocytes cultured in the FMB, specifically in a “sandwich” configuration, were functionally stable, as shown by a high rate of urea biosynthesis after challenge with NH4Cl, a low alanine-aminotransferase leakage and suppressed spontaneous nitric oxide (NO) production. Moreover, the time-course of the disappearance of cyclosporin A (CsA) from the perfusate demonstrated the high biotransformation capacity of cells in the FMB. The effect of CsA on the modulation of urea and spontaneous NO production demonstrated flexibility, in that
minor changes could be observed at diverse time intervals and in a non-destructive way. The monitoring of nitrite levels during various steps of isolation and culture suggested that spontaneously produced NO has a negative impact on hepatocyte metabolic and functional integrity. In spite of the sophisticated techniques that are being used for the preparation of bioreactors, with hepatocytes surviving for longer periods, our data have shed light on some factors that could be important for the successful use of similar models for pharmacotoxicological and other biomedical applications.
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