An in vivo testing system for endocrine disruptors in fish early life stages using induction of vitellogenin

An enzyme-linked immunosorbent assay (ELISA) based on carp (Cyprinus carpio ) vitellogenin (c-VTG, an estrogen-dependent precursor of egg yolk) was dev eloped to quantify VTG in the fathead minnow (Pimephales promelas) and was employed to establish an in vivo testing system for estrogens in fish early life stages. The working range of the c-VTG ELISA was between 1 and 50 ng/ ml (20%-80% binding). The inter- and intraassay variations were 9.7 +/- 3% and 8.9 +/- 3.4% (n = 15), respectively. Plasma and whole body homogenates from females and from estrogenized male fathead minnow diluted parallel wit h the c-VTG standard. Measurements of plasma VTG fractionated on sodium dod ecyl sulphate gels and quantified using densitometry were compared with mea surements with the same plasma samples using the c-VTG ELISA and they were similar for the carp and fathead minnow. The plasma concentration of VTG in the male fathead minnow was 25 +/- 6 ng/ml, whereas in females, plasma con centrations of VTG ranged from 5.3 +/- 1 mu g/ml to 182 +/- 78 mu g/ml, dep ending on the state of maturity. Exposure of fathead minnows to estradiol-1 7 beta in the water (nominal concentrations of 25 ng/L, 50 ng/L, and 100 ng /L) from 24 h postfertilization to 30 d posthatch induced VTG synthesis in a dose-dependent manner. Shorter periods of exposure to 100 ng/L estradiol- 17 beta (24 h postfertilization to 10 or 20 d posthatch) similarly induced a vitellogenic response. This work demonstrates that fathead minnow are sen sitive to estrogen and are able to synthesize VTG very early in development ; this provides the basis for the adaptation of existing fish early-life-st age tests for the in vivo testing of estrogenic chemicals.