The use of genomics technology to investigate gene expression changes in cultured human liver cells.
The field of genomics has great potential in toxicology; however, the technology is still in its infancy and there are many questions that need to be addressed. In this study we focus on the use of toxicogenomics for the determination of gene expression changes associated with hepatotoxicity. The human hepatoma cell line HepG2 was used to assess the toxic effects of two well-studied hepatotoxins, carbon tetrachloride (CCl(4)) and ethanol (EtOH). Replicate dishes of HepG2 cells were exposed to two concentrations of CCl(4) and EtOH--doses which caused 20% and 50% cell death (as determined by the MTT assay) were chosen [0.18% and 0.4% (v/v) CCl(4); 2.5% and 5% (v/v) EtOH] and the cells exposed for periods of 2 and 24 h. mRNA was extracted and used to probe Atlas Human Toxicology II arrays (Clontech). Preliminary data revealed that following a 2-h exposure at the low doses of both compounds, few changes in gene expression were detected. However, after 24-h exposure of the cells to the same low concentration of both compounds, multiple changes in gene expression were observed, many of which were specific to the individual hepatotoxins, presumably reflecting their different mechanisms of action. CCl(4) treatment of HepG2 cells gave rise to treatment specific up-regulation of genes involved in extracellular transport and cell signalling, whereas EtOH treatment gave rise predominantly to down-regulation of genes involved in stress response and metabolism. In addition, changes in regulation of certain genes (involved in stress response and cell cycle) were common to both treatments. Exposure of HepG2 cells to higher doses of the hepatotoxins gave rise to more changes in gene expression at lower exposure times. These results strongly suggest that different mechanisms of hepatotoxicity may be associated with specific patterns of gene expression, while some genes associated with common cellular responses may be useful as early markers of toxicity.
Harries
H M
HM
SEAC Toxicology Unit, Unilever Research, Colworth House, Sharnbrook, Bedfordshire MK44 1LQ, UK. helen.harries@unilever.com
Fletcher
S T
ST
Duggan
C M
CM
Baker
V A
VA
D000826:Animal Testing Alternatives; D002251:Carbon Tetrachloride / Q000633:toxicity; D002470:Cell Survival / Q000187:drug effects; D004273:DNA, Neoplasm / Q000032:analysis; D004305:Dose-Response Relationship, Drug; D000431:Ethanol / Q000633:toxicity; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects / Q000235:genetics*; D023281:Genomics / Q000379:methods*; D018197:Hepatoblastoma; D022781:Hepatocytes / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D008113:Liver Neoplasms; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000032:analysis / Q000187:drug effects / Q000378:metabolism; D012334:RNA, Neoplasm / Q000032:analysis; D013778:Tetrazolium Salts / Q000378:metabolism; D013844:Thiazoles / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000187:drug effects / Q000378:metabolism
D004273:DNA, Neoplasm; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D013778:Tetrazolium Salts; D013844:Thiazoles; D000431:Ethanol; D002251:Carbon Tetrachloride; C022616:thiazolyl blue