Modulation of gene expression in chemical carcinogenesis: Analysis of aflatoxin B(1) induced gene expression in primary human hepatocytes.

摘要

Hepatotoxicity is the most common adverse event of newly marketed drugs. Despite extensive pre-clinical toxicity testing and review by the Food and Drug Administration (FDA), it is difficult to predict human hepatotoxicity from testing in other species. In addition, there are insufficient models to identify drugs and chemicals that are potentially non-genotoxic carcinogens in humans. Therefore, it would be advantageous to develop in vitro human assays to evaluate drug responses in humans.; Any drug or chemically induced endpoint is likely to be associated with some alteration in the gene expression of the exposed cell, therefore an analysis of genes that are responsive to known hepatotoxins or hepatocarcinogens could be used to develop an in vitro screen that is predictive of toxicity or carcinogenicity in new drugs and chemicals.; In this study, primary human hepatocytes and HepG2 cells were evaluated as in vitro models of human hepatoxicity by comparing the variability of basal gene expression in primary hepatocytes from different donors and between human hepatocytes and HepG2 cells. The GDA 1.3 human DNA filter array (GenomeSystems) was used for gene expression analysis. The effects of plating matrix, medium and time in culture on basal levels of stress gene expression in primary human hepatocytes were investigated using the Atlas™ stress filter array. To identify chemically responsive genes, HepG2 cells and primary human hepatocytes from five donors were exposed to sub-toxic doses of two hepatotoxic chemicals, aflatoxin B₁ (AFB₁) or acetaminophen (APAP). RNA was isolated from each treatment group and used to prepare a 33P-radiolabeled cDNA probe that was hybridized to a GDA 1.3 human DNA filter array.; It was found that matrigel was the optimal culturing matrix for primary human hepatocytes. Primary human hepatocytes from different donors had variable basal gene expression patterns and responses to AFB₁. HepG2 cells expressed a subset of genes not expressed in primary human hepatocytes. Fourteen genes were ATB₁ responsive in at least two samples. Six of the genes were also responsive to APAP. These data can be used to develop novel mechanistic studies and to develop gene expression profiles for hepatotoxins and carcinogens.