The Center for Alternatives to Animal Testing is an academic center affiliated with the Division of Toxicological Sciences in the Department of Environmental Health Sciences of the Johns Hopkins University Bloomberg School of Public Health.

 

Johns Hopkins School of Public Health

Abstract for TestSmart--Pharmaceuticals: An Efficient and Humane Approach to Predictors of Potential Toxic Effects of Drugs

Genomics: Species Comparison Using Macroarrays

Daniel A. Casciano
National Center for Toxicological Research

The effects of chemical carcinogens on the structure and sequence of DNA are studied extensively by toxicologists to obtain mechanistic information during chemical carcinogenesis. With the advent of commercially available filter arrays and microarrays, it is now also possible to evaluate the effects of compounds with toxicological and carcinogenic properties on the expression of genes. Aflatoxin B1 (AFB1), a genotoxic human and rat carcinogen and hepatotoxin, was used to identify AFB1-responsive genes in primary human hepatocytes and HepG2 cells. Cells were exposed to AFB1 (.001 mM to 1.0 mM) for 16 hours in serum-free hepatocyte maintenance medium at 37oC in a humidified atmosphere of 5% CO2. Total RNA was isolated using TRIzolTM. Radiolabeled cDNA probes were reverse-transcribed from 5.0 mg of oligo dT primed total RNA in the presence of [a-33P]-dATP. Denatured probes were hybridized to a human filter array (GenomeSystems) containing 18,391 non-redundant clones from the I.M.A.G.E. collection spotted in duplicate. There was variability in the basal gene expression of cultured primary human hepatocytes from different donors, and between primary human hepatocytes and HepG2 cells. HepG2 cells expressed a subset of genes that was not detected in cultured primary human hepatocytes. The expression of 14 genes was altered in at least two of the samples tested. AFB1 had effects on gene expression in cultured primary human hepatocytes and HepG2 cells, although the affected genes varied considerably among hepatocytes from different donors.