One of the most insidious effects of some environmental toxins is their ability to bind to endogenous estrogen receptors and elicit either unnatural responses or block normal physiological processes. These xenobiotic estrogens show little structural similarity, precluding the development of a method to test for estrogenicity based on molecular structure. This grant proposes the development of a sensitive in vivo test for estrogenic activity of toxic compounds utilizing one of the most sensitive and rapid responses to estrogen, the production of serum vitellogenin. Research in my laboratory has demonstrated a rapid response in amphibians to known artificial and xenobiotic estrogens (diethylstilbestrol and o.p.-DDT). The main objective of this proposal is to explore the feasibility of using vitellogenin production in the African clawed frog (Xenopus laevis) as an in vivo biomarker for screening the estrogenicity of environmental toxins.
The specific aims are: 1) to develop specific polyclonal and monoclonal antibodies to Xenopus vitellogenin, 2) to develop and validate a sensitive enzyme-linked immunosorbant assay (ELISA) capable of quantifying plasma vitellogenin, 3) to determine dose-responsiveness of the vitellogenin induction to xenobiotic estrogens in vivo, 4) to determine if there are additive, synergistic or antiestrogenic effects of xenobiotics in combination with each other or with estradiol. The use of an in vivo system can identify potential estrogenic chemicals that would be missed by other methods, e.g. including cell cultures and in vitro induction of vitellogenin. This is partly because some xenobiotics are only estrogenic following metabolic conversions. This model can better represent the dynamics of exposure, absorption, modification, and excretion that occurs in living systems. Further, this assay will readily quantify effects based on exposure to chemicals absorbed from the environment. Xenopus was chosen since it is an accepted model organism, with extensive research on the mechanism of steroid induction of vitellogenin. Further, Xenopus has been shown to be responsive to xenobiotic estrogens. A simple, rapid and sensitive test for xenobiotic estrogens would greatly facilitate screening trials of chemicals that may affect not only reproduction in adults, but also affect the development of human embryos and children, causing permanent damage.
