"Ex vivo" and "in vivo" studies to assess chemical effects on steroidogenesis in fish: development and application of methods

The aquatic environment receives many chemical substances of natural or anthropogenic origin, which can influence the endocrine functions and health of wildlife. Various examples of endocrine disruption in wildlife were documented in aquatic organisms, for which associations between reproductive and developmental effects and exposure to endocrine-disrupting chemicals (EDCs) have been demonstrated. Since most of the endocrine-disrupting effects reported appear to be a consequence of feminization of males, most ecotoxicological research has been directed to identify estrogenic chemicals. However, the endocrine-disrupting effects exerted by EDCs can result from different mechanisms such as agonism or antagonism of endogenous steroid hormones via interaction with steroid hormone-receptors, or interference with the sex steroid hormone synthesis. Given the potential threat of these EDCs for wildlife, effective testing methods are required by regulatory agencies and industry to identify and assess the different mechanisms of action by which the EDCs exert their adverse effects. Testing strategies for endocrine disruption are being developed, in particular with fish test assays. These strategies are based on tiered approach, starting with fish in vitro and in vivo screening assays that identify and inform on potential endocrine mechanisms and effects. The results of the screening assays have then to be confirmed by higher tiered fish in vivo assays that characterize any apical adverse effect resulting from endocrine mode of action. Although the assays to screen for chemicals interacting with sex steroid receptor are widely available, tests to identify and inform on effects of chemicals that act via disrupting sex steroid biosynthesis still need to be developed. The aim of this thesis was therefore to develop and evaluate the potential of different fish test methods focused on chemicals that may interfere with the sex steroid biosynthesis.
In a first step, an ex vivo gonad assay from juvenile brown trout (Salmo trutta fario) was developed to specifically identify substances that disrupt the activity of enzymes involved in the sex steroid biosynthesis. The ex vivo gonad assay was applied to test model chemicals, known or suspected to inhibit sex steroid biosynthesis: 1,4,6-androstatriene-3,17-dione (ATD), an aromatase inhibitor pharmaceutical; prochloraz, an imidazole fungicide; tributyltin (TBT), an organotin compound and persistent organic pollutant. Their effects in the ex vivo gonad assay were assessed by measuring 17ß-estradiol and testosterone
concentrations from the culture medium. The different profile of sex steroid concentrations obtained for each chemical exposure showed that the ex vivo gonad assay cannot only identify the chemicals disrupting the steroidogenesis, but has also the potential to inform on their specific mechanism of action.
To further evaluate the ex vivo gonad assay and its potential to inform on in vivo effects, the responses to prochloraz and TBT exposure were compared in the ex vivo and in vivo exposure assays of juvenile brown trout. The effects were again assessed by measuring 17ß-estradiol and testosterone concentrations, and also by analyzing somatic indices and histopathology of gonads from fish exposed in vivo to the test chemicals. The results of this study demonstrated that the ex vivo gonad assay has the potential to inform on in vivo effects of chemicals disrupting the steroidogenesis and accordingly on their potential to affect sexual development of fish. This study highlights the potential of the ex vivo gonad assay to be a sensitive and informative tool for such EDCs.
The ex vivo gonad assay was then used to further analyze the potential of the steroidogenic inhibitors to impair the regulation of early sexual development of fish. This was investigated by comparing cellular and molecular effects of ex vivo and in vivo exposures to ATD, prochloraz and TBT. The ex vivo 17ß-estradiol and testosterone concentrations were measured and ex vivo/in vivo gene expression of the aromatase and insulin-like growth factors (IGFs), involved in the regulation of sexual development, were compared. It was shown that the test chemicals could interfere with both the sex steroid and IGF systems and potentially lead to altered sexual development.
Finally, to confirm the potential of steroidogenic inhibitors to impair sex differentiation and development, a higher tier fish in vivo test, a Fish Sexual Development Test (FSDT), was applied. Two model fish species, zebrafish (Danio rerio) and fathead minnow (Pimephales promelas) were exposed, from embryo to sexual maturity, to prochloraz and the effects on their sexual differentiation were compared by assessing the sex ratios, the histology of gonads, and the vitellogenin concentration. The results of this last study demonstrated that, although the different strategies of sexual differentiation of zebrafish and fathead minnow influence the response of their gonad morphology and their sensitivity to prochloraz exposure, the exposure to steroidogenic inhibitors has the potential to alter their sexual development and subsequently the reproductive success and population structure of fish.
To conclude, we suggest that the evaluation of the ex vivo and in vivo methods in our different studies are sensitive and valuable tools for application in environmental risk assessment of chemicals interfering with the sex steroid biosynthesis. Although further characterization and validation studies of the ex vivo gonad and FSDT assays are still required, the combination of both ex vivo and in vivo assays represents a good testing approach.