Identification of genes involved in drug-induced hepatotoxicity

在本研究中，我们对与药物肝毒性相关的基因进行了研究。我们这次研究的重点药物是扑热息痛、异烟肼、二甲双胍和布福明。为了鉴定药物在人肝细胞株HepG2中表达变化的基因，构建了消减文库。通过对文库的差异筛选，有几个克隆被鉴定为药物诱导或还原基因。例如，扑热息痛和异烟肼增加了赖氨酰羟基酶2(LH2)的表达，而对乙酰氨基酚降低了胸苷合成酶的表达。二甲双胍和布福明降低了3-磷酸甘油醛脱氢酶的表达，增加了β-肌动蛋白的表达。赖氨酰羟基酶催化胶原中赖氨酸残基的翻译后羟基化。胶原分子中赖氨酸残基的羟基化对于胶原交联物的形成和稳定是必不可少的，被认为在肝纤维化过程中起着重要作用。因此，我们把重点放在药物增加LH2的表达上。为了探讨药物促进LH2基因表达的机制，我们制备了含有LH2上游区的报告载体，发现LH2基因的表达上调是由于药物对LH2基因转录的激活。此外，我们还发现两种药物均可诱导转录因子c-myb的表达，c-myb的过表达可诱导LH2基因的表达和LH2的转录激活。上述结果提示，对乙酰氨基酚和异烟肼的肝毒性可能与LH2的表达增加有关。

In this study, we investigated the genes involved in drug-induced hepatotoxicity. The drugs we focused in this study were acetaminophen, isoniazid, metformin and buformin. To identify the genes changes their expression by the drugs in human hepatic HepG2 cells, a subtractive cDNA library was constructed. Through differential screening of the library, several clones were identified as drug induced or reduced gene. For example, acetaminophen and isoniazid increased lysyl hydroxylase, 2 (LH2) expression, and acetaminophen decreased thymidine synthetase expression. Metformin and buformin decreased glyceraldehyde 3-phosphate dehydrogenase expression and increased beta actin expression.Lysyl hydroxylase catalyzes the posttranslational hydroxylation of lysine residues in collagen. Hydroxylation of lysine residues in collagen molecules is essential for the formation and stabilization of collagen crosslinks, and is believed to act as an important role in the process of hepatic fibrosis. We therefore focused in the increased LH2 expression by the drugs. To examine the mechanism of LH2 expression increased by the drugs, we prepared the reporter plasmid containing the LH2 upstream region, and found that the increased mRNA expression was due to the transcriptional activation of LH2 by the drugs. In addition, we found that the two drugs induce the expression of transcriptional factor c-myb and the overexpression of c-myb induces LH2 mRNA expression and the transcriptional activation of LH2. From these results, increased LH2 expression may involve in the hepatotoxicity by acetaminophen and isoniazid.