Genomics and proteomics analysis of cultured primary rat hepatocytes Juergen Beigel, Kerstin Fella, Peter-Juergen Kramer, Michaela Kroeger, Philip Hewitt * Molecular Toxicology, Institute of Toxicology, Merck KGaA, 64271 Darmstadt, Germany Received 22 March 2007; accepted 27 June 2007 Available online 21 July 2007 Abstract The use of animal models in pharmaceutical research is a costly and sometimes misleading method of generating toxicity data and hence predicting human safety. Therefore, in vitro test systems, such as primary rat hepatocytes, and the developing genomics and proteomics technologies, are playing an increasingly important role in toxicological research. Gene and protein expression analysis were investigated in a time series (up to 5 days) of primary rat hepatocytes cultured on collagen coated dishes. Especially after 24 h, a significant down-regulation of many important Phase I and Phase II enzymes (e.g., cytochrome P450’s, glutathione-S-trans- ferases, sulfotransferases) involved in xenobiotic metabolism, and antioxidative enzymes (e.g., catalase, superoxide dismutase, gluta- thione peroxidase) was observed. Acute-phase-response enzymes were frequently up-regulated (e.g., LPS binding protein, a-2-macro- globulin, ferritin, serine proteinase inhibitor B, haptoglobin), which is likely to be a result of cellular stress caused by the cell isolation procedure (perfusion) itself. A parallel observation was the increased expression of several structural genes (e.g., b-actin, a-tubulin, vimentin), possibly caused by other proliferating cell types in the culture, such as fibroblasts or alternatively by hepatocyte dedifferentiation. In conclusion, the careful interpretation of data derived from this in vitro system indicates that primary hepatocytes can be success- fully used for short-term toxicity studies up to 24 h. However, culturing conditions need to be further optimized to reduce the massive changes of gene and protein expression of long-term cultured hepatocytes to allow practical applications as a long-term toxicity test system. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Rat primary hepatocytes; Gene expression; Protein expression; Microarray; 2D-Gel electrophoresis; SELDI-TOF 1. Introduction As the chemistry and biology of drug discovery and development moves towards greater automation and high-throughput-screening procedures, the need for new screens that address potential toxicological issues early in the drug discovery process are essential. In addition, the new EU regulatory framework for chemicals, called REACH (Registration, Evaluation and Authorization of CHemicals), where 1000s of chemicals have to be tested for their toxicological potential, adds to this increasing need. Today, approximately 75% of R&D costs are due to compound failures. A large part of these failures are due to promising compounds being eliminated from devel- opment during preclinical toxicity evaluation. This has increased the pressure on gathering risk evaluation data as early as possible in the drug development process. For this reason, predictive platforms based on in vitro toxicity screening, genomics, proteomics and metabonomics are being established and validated. Several studies based on animal experiments have already been published (Bulera et al., 2001; Hamadeh et al., 2002; Heinloth et al., 2004; Waring et al., 2001a). But the use of animal models in the discovery phase of drug development is a costly and sometimes inefficient method of generating toxicity data. Therefore, in vitro toxicity tests for screening prior to ani- mal studies are urgently required (Harris et al., 2004). However, at the moment, only a limited number of studies 0887-2333/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2007.06.019 * Corresponding author. Tel.: +49 6151 722927; fax: +49 6151 727673. E-mail address: Philip.Hewitt@merck.de (P. Hewitt). www.elsevier.com/locate/toxinvit Available online at www.sciencedirect.com Toxicology in Vitro 22 (2008) 171–181