Genomics and proteomics analysis of cultured primary rat hepatocytes.
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 24h, 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, alpha-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., beta-actin, alpha-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 24h. 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.
Beigel
Juergen
J
Molecular Toxicology, Institute of Toxicology, Merck KGaA, 64271 Darmstadt, Germany.
Fella
Kerstin
K
Kramer
Peter-Juergen
PJ
Kroeger
Michaela
M
Hewitt
Philip
P
D000826:Animal Testing Alternatives / Q000379:methods*; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D015180:Electrophoresis, Gel, Two-Dimensional; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D023281:Genomics / Q000379:methods*; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D040901:Proteomics / Q000379:methods*; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors; D018675:Toxicity Tests / Q000379:methods*; D014116:Toxicology / Q000379:methods