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

Abstract

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) μg/l range (MT, 10, 50 and 100 μg/l; F, 25, 50, 100 μg/l; MT+F, 10 μg MT per l +50 μg 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.