Primary hepatocytes and their cultures are a simple but versatile, well-controlled, and relatively easy to handle in vitro system that is well-accepted for investigating xenobiotic biotransformation, enzyme induction and inhibition, and (biotransformation-mediated) hepatotoxicity. In addition, hepatocyte cultures have proven to be valuable tools in the study of liver physiology, viral hepatitis, and liver regeneration and are proposed as an alternative to orthotopic liver transplantation. It has been observed, however, that a number of liver-specific functions are progressively lost with time when hepatocytes are isolated and cultivated. These phenotypic changes are primarily the result of fundamental changes in gene expression concomitant with a diminished transcription of the relevant liver-specific genes, and can be interpreted as a 'dedifferentiation' of the isolated hepatocytes. Ischemia-reperfusion stress induced during the isolation process, disruption of the normal tissue architecture, as well as an adaptation to the in vitro environment are underlying factors and will be extensively discussed. A detailed description of the regulation of the hepatocyte phenotype in vivo in the first section of this review will help to understand the effect of these factors on hepatocyte gene expression. Although different approaches, mainly mimicking the in vivo hepatocyte environment, have been succesfully used to prevent or slow down the dedifferentiation of primary hepatocytes in monolayer culture, the ideal hepatocyte-based culture model, characterized by a long-term expression of hepatocyte-specific functions comparable to the in vivo level, does not exist at the moment. Consequently, alternative strategies should focus on the isolation procedure, during which dedifferentiation is already initiated. In addition, identification of the conditions needed for the full in vitro maturation of hepatic progenitor cells to quiescent, functional hepatocyte-like cells opens promising perspectives.

Modulation of chromatin structure through histone acetylation/deacetylation is known to be one of the major mechanisms involved in the regulation of gene expression. Two opposing enzyme activities determine the acetylation state of histones: histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively acetylating or deacetylating the epsilon-amino groups of lysine residues located in the amino-terminal tails of the histones. In general, transcriptionally active chromatin is associated with hyperacetylated histones, whilst silenced chromatin is linked to hypoacetylated histones. A number of structurally divergent classes of HDAC inhibitors have been identified. They have been shown to induce cell cycle arrest, terminal differentiation and/or apoptosis in various cancer cell lines and inhibit tumor growth in animals. In particular, the reversible HDAC inhibitor Trichostatin A (TSA) and its hydroxamate analogues can effectively and selectively induce tumor growth arrest at very low concentrations (nano- to micromolar range). They form a group of so-called promising antitumor agents of which some are currently under clinical trial. Since the selection of a molecule for further drug development requires a balance of biological potency, safety and pharmacokinetics, it is of paramount importance to elucidate the pharmacokinetic and toxicological properties of these HDAC inhibitors before they can be considered as potential new drugs. Primary hepatocytes and their cultures are well-differentiated in vitro models and can be used to study simultaneously the biological effects of HDAC inhibitors and their biotransformation. The present review provides a state-of-the-art of our current knowledge of the pharmacological and toxicological effects on proliferating cells of TSA and its hydroxamate-based structural analogues. Besides a theoretical basis, an overview of the experimental results, obtained by the authors using primary rat hepatocytes as an in vitro model, is given.

This is the 54th report of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM). The main objective of ECVAM, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences, and which reduce, refine or replace the use of laboratory animals. One of the first priorities set by ECVAM was the implementation of procedures that would enable it to become well informed about the state-of-the-art of non-animal test development and validation, and of opportunities for the possible incorporation of alternative methods into regulatory procedures. It was decided that this would be best achieved through a programme of ECVAM workshops, each addressing a specific topic, and at which selected groups of independent international experts would review the current status of various types of in vitro tests and their potential uses, and make recommendations about the best ways forward.A workshop on Metabolism: a bottleneck in in vitro toxicological test development, was held at

In vitro models, based on liver cells or tissues, are indispensable in the early preclinical phase of drug development. An important breakthrough in establishing cell models has been the successful high-yield preparation of intact hepatocytes. In this chapter, the practical aspects of the two-step collagenase perfusion method, modified from the original procedure of Seglen, are outlined. Although applicable to the liver of various species, including human, the practical aspects of the method are explained here for rat liver. Critical parameters for the successful isolation of primary rat hepatocytes are highlighted and a troubleshooting guide is provided. In addition, a new development based on the inhibition of histone deacetylase activity is presented. This approach allows inhibition of cell-cycle reentry during hepatocyte isolation, a process known to underlie the dedifferentiation process of cultured hepatocytes.

ABSTRACT:Phase I biotransformation of Trichostatin A (TSA), a histone deacetylase inhibitor with promising antifibrotic and antitumoral properties, was investigated in rat and human liver microsomes and in suspensions of rat hepatocytes. TSA (50 M) was readily and completely metabolized by rat hepatocytes in suspension (2 ؋