Elsevier

Toxicology in Vitro

Volume 22, Issue 1, February 2008, Pages 171-181
Toxicology in Vitro

Genomics and proteomics analysis of cultured primary rat hepatocytes

https://doi.org/10.1016/j.tiv.2007.06.019Get rights and content

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-transferases, sulfotransferases) involved in xenobiotic metabolism, and antioxidative enzymes (e.g., catalase, superoxide dismutase, glutathione peroxidase) was observed. Acute-phase-response enzymes were frequently up-regulated (e.g., LPS binding protein, α-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., β-actin, α-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 successfully 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.

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 development 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 animal studies are urgently required (Harris et al., 2004). However, at the moment, only a limited number of studies on the validation of these in vitro techniques for toxicological risk evaluation have been published (Kienhuis et al., 2007, Boess et al., 2003). Since liver toxicity is a major hurdle in drug development many groups are focusing on hepatotoxicants that are well characterized in vivo to aid in the validation of these in vitro test systems. The main advantage of in vitro test systems is that they can be used at a relatively early time-point in drug development, when only small quantities of the new substance are available (Waring et al., 2001b). In addition, the use of genomics and proteomics technologies enables the discovery of early molecular changes prior to changes that would be visible by traditional pathological analysis.

In the study presented here, we have combined transcriptomic analysis (quantitative real-time-PCR, a low-density oligo chip (MWG-Biotech, Ebersberg, Germany) containing 250 toxicological relevant genes and a high-density chip (RAE 230A, Affymetrix)) and proteomics (2D-PAGE in combination with MS and SELDI-TOF) approaches to generate a wide range of data to characterize rat primary hepatocytes which may have the potential to substitute for animal studies. Because it is well known that hepatocytes lose many of their specific functions during isolation and culture, a time course of 120 h has been carried out to achieve an overview of which genes and proteins are affected by the culturing procedure.

These data, taken together will prove invaluable when these ‘Omics technologies are used for screening compounds early in the drug discovery and development process. A detailed knowledge of how genes and proteins behave in culture, under specific conditions is essential before treating these cells with developmental drugs or novel chemical candidates.

Section snippets

Chemicals

DMEM/Ham F12 medium was purchased from Invitrogen Corporation (Karlsruhe, Germany). Serum, supplements, enzymes, and biochemicals were purchased from Sigma (Deisenhofen, Germany) unless otherwise stated.

Isolation of primary hepatocytes from rat liver

Cells were isolated from adult rats by a standard perfusion technique (Seglen, 1976) and the cell culture of primary rat hepatocytes was performed as previously published, with minor modifications (Ammerschlaeger et al., 2004). The medium was changed every 24 h during the culture and the cells

Results

In order to assess the genome wide transcriptional changes associated with time and conditions in culture, we carried out microarray analysis over a 120 h period. To fully characterize the behavior of primary rat hepatocytes we analyzed cells using low and high-density microarrays for gene expression and 2D-PAGE and SELDI-TOF combined with MS for protein expression profiling. Cells were harvested after 2, 6, 24, 48, 72, 96 and 120 h of culture. Because one of the goals of these experiments was to

Discussion

Before in vitro systems can be used to detect gene and protein expression changes after treatment with drugs or model toxicants, evaluation of culturing conditions related to molecular changes in the cells is essential. In this study, we detected a total of 1262 up and 993 down-regulated genes with the high density and 38 up-regulated and 48 down-regulated genes with the low-density arrays in a time series of rat primary hepatocytes over 120 h. In parallel proteomics experiments revealed 38 up

Acknowledgements

The authors thank Claudia Klement, Yvonne Walter (Institute of Toxicology, Merck KGaA) and Melanie Kuehnl (Target Research Group, Merck KGaA) for technical support.

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