Vitellogenin as a biomarker for xenobiotic estrogens in an amphibian model system

A number of chemicals released into the environment have the potential to interfere with physiological and developmental processes by disrupting endocrine pathways. Among the best known of these endocrine disruptors are compounds that mimic the action of the steroid hormone 17 beta-estradiol. These xenobiotic estrogens are believed to pose health risks to both humans and wildlife. Our laboratories are designing in vivo bioassays for xenobiotic estrogens based on induction of the egg-yolk precursor protein vitellogenin. Vitellogenin is normally produced by the liver of adult female nonmammalian vertebrates under estrogen stimulation. In immature or male animals, which have low levels of endogenous estrogens, vitellogenin can serve as a reliable biomarker for exposure to xenobiotic estrogens. Our model system used the African clawed frog, Xenopus laevis, an ideal species for laboratory screening of endocrine disruptors. Xenopus laevis vitellogenin was purified by diethylaminoethyl (DEAE) chromatography and used to generate polyclonal antibodies in rabbits. The resulting antiserum was used to develop an enzyme-linked immunosorbent assay (ELISA) for measurement of serum vitellogenin. Frogs were exposed to compounds by immersion in order to mimic environmental exposure to aquatic contaminants. Initially, frogs were immersed in the potent estrogenic agent diethylstilbestrol (DES) at a concentration of 1 ppm for 11 d to test the efficacy of the immersion protocol. Diethylstilbestrol exposed animals showed substantial induction of serum vitellogenin, indicating that the frogs are capable of responding to estrogenic agents present in their aquatic environment. Vitellogenin induction was then investigated for chlordane, dieldrin, endosulfan, and toxaphene, compounds that have been shown through in vitro assays to be weakly estrogenic when administered individually but more strongly estrogenic in combination. Adult male frogs were immersed in water containing the compounds (1 ppm, 11 d), both singly and in paired combinations. Endosulfan proved toxic at this concentration. Toxaphene- and dieldrin-treated frogs showed significant levels of vitellogenin induction, while chlordane-treated animals did not differ from controls. There was no evidence of a synergistic response between any of the combinations. This research demonstrates the utility of vitellogenin as a biomarker for exposure to estrogenic agents. The assays developed could be used to screen chemicals for estrogenic properties, to test waters for the presence of estrogenic agents, or to assess wildlife exposure to environmental estrogens.