SELCER, Kyle W.; WILLIAMS, Leslie; SKOLODA, Jamie; Duquesne University: Vitellogenin Induction as a Biomarker for Environmental Estrogens in Xenopus laevis
Environmental estrogens pose potential health risks to humans and wildlife by disrupting physiological and developmental processes. Our laboratory has been designing in vivo bioassays for environmental estrogens based on induction of the egg-yolk precursor protein vitellogenin. Vitellogenin bioassays have been developed for numerous fish species but few amphibians. We are using the African clawed frog, Xenopus laevis, as a model species due to the large volume of data on vitellogenesis in this species. Two assays were developed to detect vitellogenin induction: an ELISA for measuring serum vitellogenin and an RT-PCR assay for evaluation of hepatic vitellogenin mRNA. The assays were used to quantify vitellogenin induction in male frogs after injection with, or immersion in, known and suspected estrogenic agents. Time courses and dose response curves were generated for the estrogens ethinyl estradiol and diethylstilbestrol (DES). Using X. laevis vitellogenin-specific primers, RT-PCR generated the expected size cDNA fragment with hepatic RNA from frogs immersed in estrogens. The cDNA fragment from estrogen-treated frogs was 100% identical with the known cDNA sequence for X. laevis vitellogenin. Vitellogenin mRNA was detectable within 4 h after immersion in estrogens while serum vitellogenin was not detectable until 72 h after immersion. The vitellogenin assays were also used to evaluate the response of male frogs to immersion in the environmental estrogens octylphenol, nonylphenol, and bisphenol A (1 mg/L, 11 days). Vitellogenin mRNA was strongly detected in frogs immersed in bisphenol A, weakly detected in nonylphenol-treated frogs, and was not detected in octylphenol-treated frogs. Serum vitellogenin was not detected in any of the xenobiotic-treated frogs. The X. laevis vitellogenin immunoassay and RT-PCR assay could be useful as an in vivo screen for estrogenic chemicals.
