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This project is a part of the DGRT genomic/proteomics focus area. The goal of this project is to utilize the new gene expression technologies to understand which genes are affected by the exposure of human hepatocytes to a known human carcinogen (aflatoxin B1).
Because this is a new technology, the experimental approach must include research to define the appropriate experimental parameters. Specific goalsare to: 1) Verify aflatoxin B1 effects on steady-state mRNA levels of eight genes previously identified by differential hybridization of a gene filter array to be aflatoxin B1(AFB1)-responsive in human hepatocytes. 2) Use Northern blot, RT-PCR, and/or RNA protection assay to establish AFB1 time- and dose-response curves for maximal gene expression and also determinethe minimum dose at which gene expression can be detected. 3) To identify additional AFB1-induced genes using differential display PCR (DD-PCR) and differential hybridization of a high-density filter array utilizing mRNA from human hepatocytes treated with low, moderate and cytotoxic levels of AFB1. 4) To evaluate selected genes as described for Objective 1.5) To distinguish genes involved in toxicological response to AFB1 exposure from those that contribute to the carcinogenic response by comparing the gene expression profile of human hepatocytes treated with the hepatotoxic non-carcinogenic chemical, acetaminophen. 6) To compare gene expression of selected genes in human hepatocytes treated with known rat liver chemical carcinogens, including 2-acetylaminofluorene, dimethylnitrosamine and methapyrilene.
FY 2000 Accomplishments: 1) Human hepatocytes from four donors were treated with aflatoxin B1 (AFB1) or acetaminophen (APAP). Total RNA, DNA and protein were isolated from all samples. The RNA was used to analyze gene expression on Clontech Tox/Stress Filters (274 genes/filter) and/or Genome Systems GDA filters (18,000genes/filter). 2) Gene expression data were analyzed using Array Vision. 3) Hep2 cells were treated with AFB1. Total RNA, DNA and protein were isolated from all samples. Total RNA was used to analyze gene expression on GenomeSystems GDA filters. The data are currently being analyzed to determine the effects of AFB1 and to compare and contrast with data from normal primary hepatocytes. Northern blot analysis of selected clones in HepG2 RNA is ongoing. 4) Hepatocytes from six rats were isolated and plated on different matrices todetermine the effects of various plating conditions on gene expression.Inaddition, the hepatocytes were treated with methapyrilene and pyrilamine todetermine effects of these two antihistamines on gene expression. Gene expression was analyzed using Research Genetics rat filters (5000 genes/filter). Northern blot analysis of 17 genes in 13 data sets is 75% complete. 5) Presented the data from #3 as a talk at the Seager-Braswell Graduate Student Research Symposium at the UAMS in March 2000. It was also presented as a poster at the Environmental Mutagen Society meeting in New Orleans.
FY 2001 Plans: 1) Write and submit the three manuscripts that are the result of accomplishments #1-3 above. 2) Procure human hepatocyte donors from three females to analyze AFB1-responsive genes and compare to the data from the male donors. 3) Procure human hepatocytes from three additional male donors to analyze the effects of 2-acetylaminofluorene (2-AAF) and dimethylnitrosamine (DMN). Compare to AFB1 and APAP data. 4) HepG2 cells will be treated as in #3 for comparison. 5) Present AFB1 human data at the Society of Toxicology meetings in SanFrancisco, California and at the UAMS.