Harries H M, Fletcher S T, Duggan C M, Baker V A
SEAC Toxicology Unit, Unilever Research, Colworth House, Sharnbrook, Bedfordshire MK44 1LQ, UK.
Toxicol In Vitro. 2001 Aug-Oct;15(4-5):399-405. doi: 10.1016/s0887-2333(01)00043-1.
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.
基因组学领域在毒理学方面具有巨大潜力；然而，该技术仍处于起步阶段，有许多问题需要解决。在本研究中，我们专注于利用毒理基因组学来确定与肝毒性相关的基因表达变化。使用人肝癌细胞系HepG2来评估两种已深入研究的肝毒素——四氯化碳（CCl₄）和乙醇（EtOH）的毒性作用。将HepG2细胞的重复培养皿暴露于两种浓度的CCl₄和EtOH——选择导致20%和50%细胞死亡的剂量（通过MTT法测定）[0.18%和0.4%（v/v）CCl₄；2.5%和5%（v/v）EtOH]，细胞暴露2小时和24小时。提取mRNA并用于探测Atlas Human Toxicology II阵列（Clontech）。初步数据显示，在两种化合物的低剂量下暴露2小时后，未检测到基因表达有明显变化。然而，在细胞暴露于相同低浓度的两种化合物24小时后，观察到基因表达有多种变化，其中许多变化是每种肝毒素特有的，大概反映了它们不同的作用机制。用CCl₄处理HepG2细胞导致参与细胞外转运和细胞信号传导的基因出现处理特异性上调，而用EtOH处理主要导致参与应激反应和代谢的基因下调。此外，两种处理都出现了某些基因（参与应激反应和细胞周期）调控的变化。将HepG2细胞暴露于更高剂量的肝毒素会在更短的暴露时间内导致基因表达出现更多变化。这些结果有力地表明，不同的肝毒性机制可能与特定的基因表达模式相关，而一些与常见细胞反应相关的基因可能作为毒性的早期标志物很有用。
