Development Of Recombinant Yeast Screen Systems For Detecting Environmental Endocrine Disruptors

Over the last decade, numerous epidemiologic studies have found that a wide range of chemicals derived from natural and synthetic origins including environmental contaminants, phytoestrogens, fungal products, pharmaceuticals, industrially relevant compounds and pesticides possess hormone activities. These chemicals which are defined as endocrine disrupting chemicals(EDCs) or endocrine disruptors(EDs) have the potential to impact health by disrupting the production, release, transport, metabolism, binding, action or elimination of natural hormones in the body responsible for the maintenance of homeostasis and the regulation of developmental processes. Reports describing the adverse effects of EDs in human or wildlife such as compromised reproductive fitness, functional or morphological birth defects, behavioral abnormalities, cancers as well as altered immune functions, among others, have resulted in considerable public attention and a worldwide intensification of research efforts to characterize endocrine disrupting chemicals. During the past century, tens of thousands of chemicals have been synthesized and released into the general environmental and this number is still mounting every year. As a developing country, China is also facing a serious situation of aggravating environmental pollution as a by-product of the speedy progress in agriculture and industry. More reliable, sensitive and simple in vitro assays are required to screen ubiquitous endocrine disruptors in environment.A large portion of the endocrine disruptors have the potential to interfere with natural estrogens or androgens. Numerous assays have reproducibly shown that most of the endocrine disruptors can directly bind to, or block, hormone receptors, thereby initiating or blocking receptor-activated gene transcription. Hormone receptors including estrogen receptor and androgen receptor are the members of the nuclear receptor superfamily. Binding of an estrogenic compound to the ER causes the dissociation of heat shock protein 90, enabling occupied ERs to homodimerize. Theresulting homodimer complex exhibits high affinity for specific DNA sequences referred to as estrogen response elements (ERE) located in the regulatory region of estrogen-inducible genes. Once bound to the ERE, the homodimer complex recruits transcription factors to the target gene promoter, which leads to increased gene expression. The increased levels of mRNA that are transcribed are then translated into proteins that are the ultimate effectors of the observed response. It has been proposed that exoestrogens act as ER or AR ligand mimics that bind to the receptor, thus modulating endocrine pathways via a receptor-mediated process. Bioassays have been developed exploiting this receptor-mediated mechanism of action to provide a rational strategy for the identification and assessment of chemical interactions with the estrogen and androgen receptors that may adversely affect human health and environmental quality. Two recombinant yeast-based screening systems for detecting chemicals that interfers with androgens and estrogens, respectively, were developed in my study and the former system had never been reported before in domestic literatures. The study comprises three parts as follows:Firstly, a yeast expression plasmid which contained the human estrogen receptor cDNA fused to the ADH1 promoter was constructed. A yeast reporter plasmid carrying two tandem estrogen response elements upstream of the reporter gene LacZ that encodes for the enzyme p-galactosidase was also constructed. The Saccharomyces cerevisae strain RFY206 was stably transfected with two separate plasmids as mentioned above by lithium acetate method. The yeast transformants were selected and recombinant yeast screen system RFYpER/ERE was established for identifying environmental estrogens.Secondly, the human androgen receptor cDNA was amplified by PCR and the BamHI and Sail sites were introduced in 5' and 3' terminus of amplified fragments and subcloned into BamHI-Sall site of a yeast expression plasmid pGl.