As of 1998, more than 80,000 chemicals were registered by the EPA as being in commerce in the United States. Of the nearly 3000 chemicals that were imported or manufactured at a rate of more than one million pounds per year, 43% had no available toxicology data, and only 7% had full information on basic health and environmental effects. Some of these compounds are now classified as endocrine disruptors, and have been associated with mammary and other tumors, as well as decreases in fertility in human and animal populations. Despite these findings, only a small fraction of the close to 100,000 chemicals listed in the NIH TOXNET database have been fully characterized for endocrine disruptive effects, primarily due to the complexity of the assays required for accurate classification. Thus a critical goal is to develop quick and reliable methods to identify and characterize unknown compounds, so that risks and appropriate regulations can be determined. This proposal seeks to aid in this goal through the development of a bacterial biosensor for the rapid and reliable detection of endocrine disrupting effects in isolated chemicals and complex mixtures. This sensor relies on an engineered, four-domain fusion protein, which includes the ligand-binding domain of a given nuclear hormone receptor linked to a reporter enzyme through a small stabilization domain. When expressed in an appropriate Escherichia coli host strain, this protein generates a hormone-sensitive phenotype, which can be used to detect ligands that interact with the included nuclear hormone receptor domain. This system has been shown to be capable of detecting a wide variety of estrogenic compounds, including phytoestrogens, xenoestrogens, and estrogenic steroids, and it can reliably differentiate agonistic from antagonistic behaviors. Further, the modular design of the sensor has facilitated the construction of additional sensors for thyroid hormone-like compounds, and even insect-hormone like compounds. In our proposed work, we will develop this sensor into a fully validated and functional first-line tool for use in identifying potential endocrine disruptors.
Specific aims i nclude its formal validation for estrogenic compound identification, along with the generation and validation of additional sensors through ligand-binding domain swapping. Enhancement of the system readout is also suggested to facilitate future applications based on high-throughput technologies. It is anticipated that this system will ultimately hold significant utility as part of a multi-tiered program for their systematic discovery and classification of endocrine disrupting compounds.
Over the last several decades, several distressing health trends, including increases in breast cancer, autism rates, and fertility problems have been linked to hormone-mimicking compounds known as endocrine disruptors. The work described in this proposal seeks to develop a unique biosensor system wherein bacterial cells indicate the presence of endocrine disrupting compounds through a simple, inexpensive and reliable growth/no growth assay. This system has many advantages over conventional approaches, and may provide an effective first-line evaluation of natural and artificial compounds, alone and in mixtures, for activity against specific hormone receptor targets. ? ? ?
