Heavy metals are extremely toxic environmental pollutants. Cadmium for example, can cause damage to liver and kidney, may increase the risk of cancer, and is embryotoxic and teratogenic. The mechanisms by which cadmium exerts its embryotoxic and teratogenic effects are unknown. However, it is now well established that the cell's first line of defense against heavy metal toxicity is the metal binding protein metallothionein. Cells also react rapidly to toxic levels of metals by induction of the heat shock proteins (ie HSP-70) which may also provide protection from metal induced stress. The overall purpose of these studies, therefore, is to use the developing mouse embryo as a model stystem in which to examine the involvement of metallothionein and HSP-70 in the response of the embryo to cadmium.
The specific aims of this proposal are to: 1) Determine the temporal and spatial patterns of metallothionein and HSP-70 gene expression from fertilization through midgestation in the normal and cadmium treated mouse embryo; 2) Determine whether zinc, which can prevent cadmium induced embryotoxicity and teratogenicity, exerts its protective effect by altering expression of metallothionein and HSP-70 genes in embryonic tissue; 3) Create strains of transgenic mice which will be used to study the regulation and function of these genes in normal and Cd treated embryos. Expression of metallothionein and HSP-70 genes will be determined by detection mRNA using in situ and Northern blot hybridization. Synthesis of these proteins will be examined by electrophoretic analysis of pulse-labelled proteins. Expression of fusion genes, consisting of the metallothionein promoter of the HSP-70 promoter coupled with the chloramphenicol acetyltransferase gene or the luciferase gene, will be assessed in embryos of transfenic animals created by microinjection of cloned DNA. Transgenic animals displaying enhanced or reduced expression of the metallothionein or HSP-70 genes will be created by microinjection of appropriate cloned DNAs and RNAs which result in overexpression or which repress expression of these genes. Levels of metallothionein and HSP-70 as well as development of mouse embryos will be examined following exposure to heavy metals in vivo. Preimplantation embryos will also be exposed to metals in vitro followed by embryo transfer and subsequent analysis of development.
