Morphological sex reversal upon short-term exposure to endocrine modulators in juvenile fathead minnow (Pimephales promelas).

Indications of effects on fish endocrine system have been noted when exposed to effluents of sewage treatment plants and subsequently in the receiving surface waters. For screening purposes, the concentration of vitellogenin (VTG) in plasma is employed to detect potential exposure of fish, to (anti-)estrogenic substances. However, little is known about the variability of VTG determinations and morphological endpoints (secondary sexual characteristics) in fish under exposure conditions employing compounds with hormonal activity other than estrogens. An in vivo test system was established to study the effects of methyltestosterone (MT, a potential model androgen) and fadrozole (F, an aromatase inhibitor) as well as the combination of MT and F on juvenile, sexually undifferentiated fathead minnows (Pimephales promelas). Fish were exposed to those compounds continuously in the (nominal) microg/l range (MT, 10, 50 and 100 microg/l; F, 25, 50, 100 microg/l; MT+F, 10 microg MT per l +50 microg F per l), for 14 days (MT+F) or 21 days (MT and F) using a flow-through system. The concentration of VTG and the expression of VTG mRNA was determined using whole body homogenates in an enzyme linked immunosorbant assay (ELISA) or reverse transcription-polymerase chain reaction (RT-PCR), respectively. Exposure to MT alone led to de novo mRNA expression as well as up to a four-fold increase of VTG. F had no effect on the VTG mRNA expression and VTG protein synthesis. The combination of MT and F had no effect on VTG concentrations, however, this produced a strong masculinisation of the juvenile fish, e.g. after 13 days of exposure 100% of the fish showed typical male sex characteristics, e.g. formation of nose tubercles and pigmentation of the dorsal fin. The above findings suggest that in fish MT may be aromatised to an estrogen. F, on the other hand, inhibits testosterone aromatisation. Consequently, the combination of MT and F strongly morphologically masculinised the juvenile fathead minnows. VTG detection at the mRNA and protein level is a sensitive parameter, however, it does not provide for any information regarding the baseline "estrogenicity" of a given parent compound.

Utility of a juvenile fathead minnow screening assay for detecting (anti-)estrogenic substances.

The European Chemical Industry's aquatic research program for endocrine disrupters includes the development of an in vivo juvenile fathead minnow (Pimephales promelas) screening assay. Working within the Organization for Economic Cooperation and Development's (OECD, Paris, France) tiered approach to endocrine disrupter evaluation in fish, the juvenile fish screening protocol was adapted from the OECD test guideline 204. Six chemicals, with different (anti-)estrogenic potencies, were used to develop the in vivo juvenile fish screening protocol: diethylstilbestrol, 17alpha-ethynylestradiol, genistein, methoxychlor, 4-tert-pentylphenol, and ZM189,154 (a novel pharmaceutical antiestrogen). Mixed-sex juvenile fathead minnows were exposed to individual chemicals (with chemical analyzes) and sampled after 4, 7, 14, and 21 d of exposure. Wet weight, total length, condition factor, and whole-body homogenate concentrations of vitellogenin (VTG) were determined. Estrogens and antiestrogens were detected in this screen by virtue of the VTG response (an elevation or suppression, respectively) after 14 d. The study showed that the use of VTG concentrations in mixed-sex juvenile fish provides a sensitive and robust assay for the detection of both estrogenic and antiestrogenic chemicals, with widely divergent potencies.