The overall long-term objective of these studies is to develop transgenic stains of mice to serve as tools for understanding the toxicology of environmental pollutants. Specifically, these studies will focus on the metallothioneins (MTs), and will demonstrate; a) mechanisms regulating expression of these genes in response to toxicologically important chemicals, and b) the functions of these proteins. The MTs are small cysteine-rich heavy metal binding proteins. They represent the best documented intracellular heavy metal (zinc, copper, cadmium) binding proteins, and are considered to play a pivotal role in protection from metal toxicity. In addition, MTs may play a role in protection from alkylating agents, chemotherapeutic agents, and irradiation. MTs can scavenge free radicals, and may thus provide protection from toxic chemicals that induce oxidative stress. Most, if not all, of the aforementioned chemicals also enhance expression of the MT genes. However, except for the metal ion response, little is known about the mechanisms by which induction of the MT genes occurs. Although in vitro studies suggest that MTs are important components of the cell's responses to a variety of toxins, our ability to precisely define their functional roles in vivo is hampered by a lack of strains of mice that exhibit defects (mutations) effecting MT gene or protein structure-function.
The specific aims of this proposal are, therefore, to establish strains of transgenic mice: 1) in order to determine how inducers, such as oxidative stress inducing agents, enhance expression of the mouse MT genes, 2) that display constitutively heightened or altered tissue-specific expression of the MT genes, and 3) that have targeted ablations of the MT genes. Cis-acting promoter sequences will be mapped in transgenic mouse strains created by microinjection into fertilized mouse eggs of MT promoter deletion mutants linked to a fire fly luciferase reporter gene. Transgenic mice that constitutively over-express the MT gene in many tissues will be created using viral enhancer-driven expression of MT, and transgenic mice that constitutively over-express the MT gene in a tissue-specific manner will be created using albumin (liver specific) enhancer-driven expression of MT. The chicken MT gene will be utilized in studies involving overexpression because the chicken MT mRNA and protein are readily distinguishable from the mouse MT mRNAs and proteins. Embryonic stem (ES) cells with reduced or eliminated MT gene expression will be created by targeted integration of transfected DNA into the MT gene locus. Targeted ablation of the MT-1 and/or MT-11 genes will be performed. Stable transgenic lines will be obtained from germ line chimeras created by microinjection of these ES cells into blastocysts. Gene expression will be determined by analysis of RNA using Northern blotting, solution hybridization, and the reverse transcriptase-polymerase chain reaction, and by analysis of protein synthesis and accumulation using pulse-labeling, western blotting, immunocytochemistry and anion exchange chromatography. Transgenic strains will be analyzed for altered resistance to the hepatotoxic effects of several agents, including cadmium and inducers of oxidative stress, by histopathology and quantitation of serum enzymes.
