?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.
