• Tatyana, Yordanova Doktorova
• Athersuch, Toby
• Vinken, Mathieu
• Cavill, Rachel
• Ellis, James K.
• Chan, Pui Hei
• Rogiers, Vera
• Keun, Hector
• Vanhaecke, Tamara
• Nicholson, Jeremy K.
• Ebbels, Timothy

Abstract

The use of cultured primary hepatocytes is of great importance in the pharmaco-toxicological testing of chemical compounds, and represents an alternative to dosing regimes conducted in immortalised cell lines or in vivo testing. By stabilising the epigenome, the histone deacetylase inhibitor TSA (trichostatin A) has been shown to preserve hepatocyte xenobiotic metabolic capacity after isolation, however the effects of TSA on endogenous metabolism are to date unknown.Using an NMR-based metabonomics approach we have investigated the impact of TSA treatment on the global profile and individual metabolite levels in hepatocytes to evaluate if the metabolome was stabilised and the normal metabolic phenotype retained in this model. Aqueous soluble metabolites were extracted from isolated rat hepatocytes after 24 and 72 hours exposure to TSA (25?M) together with time-matched controls and measured by 1H NMR spectroscopy. Multivariate analysis of the global profiles revealed a clear effect of TSA-treatment and discrimination between different time points. Metabolic differences between time points in the TSA treated group were less than those observed in the control, suggesting that TSA reduced the time-related effect on metabolism. Considering individual metabolites, TSA treatment was associated with decreased amounts of glycerophosphocholine (GPC), 3-hydroxybutyric acid (3HBA) and adenosine, an increase in glycogen and slowed the decrease of glutathione (GSH) over time. The effect on intracellular glycogen (glucose store) and the ketone body 3HBA (ketosis product) may indicate a change in energy metabolism.Effects on adenosine metabolism are consistent with other reported events following TSA exposure, specifically down-regulation of ecto-5'-nucleotidase. The effect on GSH suggests that bioavailability is higher in TSA treated cells: this would have implications in many metabolic processes and specifically phase II conjugation of xenobiotics. Levels of choline metabolites are frequently altered as cells are transformed to an immortalised or tumourigenic phenotype, and so the effect of TSA on GPC may reflect cell cycle arrest and preservation of the differentiated metabolic state. Collectively our data suggest that TSA treatment reduces the loss of a normal metabolic phenotype in cultured primary hepatocytes, improving the model as a tool to study endogenous liver metabolism and potentially affecting the accuracy of all biological assays in this system.

Topics

• impedance spectroscopy
• compound
• phase
• laser emission spectroscopy
• Nuclear Magnetic Resonance spectroscopy
• ketone