Vitellogenin, a Marker of Estrogen Mimicking Contaminants in Fishes: Characterization, Quantification and Interference by Anti-Estrogens

Vitellogenin (Vg), the estrogen inducible protein precursor to egg yolk, serves as an indicator of exposure to estrogen mimicking environmental contaminants. Vg was isolated by size exclusion and ion exchange chromatography from plasma of California halibut (Paralichthys californicus) treated with estrogen. MALDI TOF mass spectrometry (MS) analysis resulted in a molecular mass of 188 kDa. MS/MS de novo sequencing provided evidence that California halibut has more than one form of Vg. Similar analysis on white sturgeon (Acipenser transmontamus) Vg did not reveal adequate evidence to suggest that sturgeon has more than one Vg. The potential of using other MS methods to understand the structure and function of Vg are discussed.An ELISA for measurement of California halibut plasma Vg was optimized and validated using a commercially available antibody developed for another flatfish species, turbot. Inclusion of overnight preincubation was critical for low detection limits. Increasing the amount of Tween-20 to 0.05% in buffers was most effective for improving recoveries of spiked plasma samples. At the IC50, the average recovery of spiked plasma samples was 104% and the interplate CV was 12%. The working range of the assay was 33-1000 ng/mL, while the detection limit in a plasma sample is 2.2 µg/mL. The response to the model compounds 17beta-estradiol and/j-nonylphenol show that this is a suitable model for further studies of estrogen mimicking contaminants.White sturgeon are native to the Sacramento River and subject to agricultural, municipal and industrial waste water effluents that likely contain different classes of endocrine-disrupting contaminants. Reductions in 17beta-estradiol-induced vitellogenin levels were observed in white sturgeon co-injected with beta-naphthoflavone (BNF, 50 mg/kg), an Ah receptor agonist. The inhibition was maximal when the compounds were injected simultaneously versus prior treatment offish with BNF. This timing of the effect compared to increases in ethoxyresorufin-O-deethylase (EROD) activity suggests that the effect is not directly due to enhanced estrogen metabolism by the Ah receptor-induced enzymes. Results of this study will be relevant for those with monitoring programs who measure vitellogenin, as it is important to understand how Ah (dioxin) receptor active environmental contaminants can influence this endpoint