Mechanism of cell death induced by aflatoxin B1

描述：(改编自申请人？S摘要)：本研究的目的是 目的：探讨黄曲霉毒素B1(AFB1)对肝细胞的杀伤作用。首先，我将调查 导致肝细胞死亡的一系列生化事件。第二，因为 黄曲霉毒素被认为通过造成DNA损伤来发挥其生物效应， 我将确定黄曲霉毒素引起的几种DNA损伤中的哪一种 诱导死亡前的基因表达模式。第三，我会 筛选在黄曲霉毒素B_1诱导的细胞死亡过程中其信使核糖核酸水平变化的新基因 采用DNA微阵列方法。细胞凋亡是一种机制，通过它 肝脏消除肿瘤前细胞，并已知由黄曲霉毒素B_1诱导 转化的肝细胞。初步实验将检验这一假设 AFB1的细胞毒作用与细胞凋亡有关。基因的表达已知 AFB1的细胞毒作用与其参与细胞凋亡有关。 已知参与细胞凋亡的基因的表达将通过 定量逆转录聚合酶链式反应，以确定参与的特定途径 诱导细胞凋亡，从而确定哪些基因容易感染 致癌突变。将使用原代大鼠肝细胞来建立 在活体内真实暴露AFB1。为了精确定位分子途径 从DNA损伤到细胞死亡，含AFB1的DNA诱导的损伤 AFB1-N7-GUA、AFB1-Fapy和AP位点将被导入肝细胞。这个 每种损伤对细胞死亡和凋亡基因表达的影响将是 测量以确定哪种特定的DNA加合物(S)诱导细胞死亡。最后， 一个大鼠基因微阵列将被构建并用于搜索新的 在黄曲霉毒素B_1诱导细胞死亡过程中诱导或抑制的基因。其基因 AFB1暴露会改变表达，尤其是那些看起来 与已知的凋亡基因共同调控的基因，可能参与 细胞对毒素的反应。

DESCRIPTION: (Adapted from the Applicant?s Abstract): The goal of this study is to determine how aflatoxin B1 (AFB1) kills hepatocytes. First, I shall probe the sequence of biochemical events that leads to liver cell death. Seond, since aflatoxin is believed to exert its biological effects by inflicting DNA damage, I shall determine which of the several types of DNA damage induced by aflatoxin induces the pattern of gene expression that precedes death. Third, I shall screen for novel genes whose mRNA levels change during AFB1-induced cell death, employing a DNA microarray approach. Apoptosis is the mechanism by which the liver eliminates pre-neoplastic cells, and is known to be induced by AFB1 in transformed liver cells. Initial experiments will test the hypothesis that apoptosis is responsible for the cytotoxicity of AFB1. Expression of genes know to participate in apoptosis is responsible for the cytotoxicity of AFB1. Expression of genes known to participate in apoptosis will be examined by quantitative RT-PCR in order to identify the specific pathways involved in the induction of apoptosis, thereby establishing which genes are vulnerable to oncogenic mutations. Primary rat hepatocytes will be employed in order to model in vivo AFB1 exposure realistically. In order to pinpoint the molecular pathway from DNA damage to cell death, DNA containing the AFB1-induced lesions AFB1-N7-Gua, AFB1-FAPY, and AP sites will be introduced into hepatocytes. The impact of each lesion on cell death, and apoptosis gene expression, will be measured to determine which specific DNA adduct(s) induce cell death. Finally, a microarray of rat genes will be constructed and used to search for novel genes that are induced or repressed during AFB1-induced cell death. Genes whose expression is altered by AFB1 exposure, and especially those that appear to be co-regulated with known apoptosis genes, are likely to participate in the cellular response to the toxin.