Screening and testing assays symposium.

Complementary In Vitro and In Vivo Rodent Assays.

Fertuck KC.

Department of Biochemistry & Molecular Biology, National Food Safety & Toxicology Center, and Institute for Environmental Toxicology, Michigan State University, East Lansing, MI.

In attempts to identify pollutants, drugs, dietary constituents, and other compounds possessing hormone agonist or antagonist activity, in vitro assays offer many advantages, including relative simplicity, rapidity, and low cost. However, only in vivo assays provide the pharmacokinetic and pharmacodynamic context necessary to much more fully predict the compound's effects on humans or other organisms of concern. An appealing strategy, therefore, involves an initial in vitro screening to identify a subset of compounds for which in vivo testing is warranted. In a first phase of studies aimed at identifying and characterizing pollutant (anti)estrogens, both cell-free competitive estrogen receptor (ER) binding assays and ER-transactivation reporter gene assays in human breast cancer cells were performed in vitro on a set of related compounds. For parent compounds inducing transactivation but not receptor binding, key metabolites were also examined to verify that the metabolites responsible for activity are those that are formed in vivo by the species of interest. Using this initial data, one compound, benzo[a]pyrene, was selected for in vivo testing using the mouse uterotrophic assay. When both benzo[a]pyrene and major metabolites were unable to induce either uterine weight or expression of lactoferrin, highly estrogen-inducible endpoints in the uterus, a microarray strategy was next adopted. These microarrays, enriched in estrogen-regulated genes, do not depend on increases in organ weight and therefore can be used on any tissue of interest. This ongoing work will be particularly useful in the detection and characterization of compounds that interact with the ER only in a subset of tissues and that therefore may be missed in traditional, solely in vitro and uterine-based testing.