Abstract
The fish short-term reproduction assay (FSTRA) is a common in vivo screening assay for assessing endocrine effects of chemicals on reproduction in fish. However, the current reliance on measures such as egg number, plasma vitellogenin concentration and morphological changes to determine endocrine effects can lead to false labelling of chemicals with non-endocrine modes- of-action. Here, we integrated quantitative liver and gonad shotgun proteomics into the FSTRA in order to investigate the causal link between an endocrine mode-of-action and adverse effects assigned to the endocrine axis. Therefore, we analyzed the molecular effects of fadrozole-induced aromatase inhibition in zebrafish (Danio rerio). We observed a concentration-dependent decrease in fecundity, a reduction in plasma vitellogenin concentrations and a mild oocyte atresia with oocyte membrane folding in females. Consistent with these apical measures, proteomics revealed a significant dysregulation of proteins involved in steroid hormone secretion and estrogen stimulus in the female liver. In the ovary, the deregulation of estrogen synthesis and binding of sperm to zona pellucida were among the most significantly perturbed pathways. A significant deregulation of proteins targeting the transcriptional activity of estrogen receptor (esr1) was observed in male liver and testis. Our results support that organ- and sex-specific quantitative proteomics represent a promising tool for identifying early gene expression changes preceding chemical-induced adverse outcomes. These data can help to establish consistency in chemical classification and labelling.
Highlights
Substances that interfere with the endocrine system, and exert an adverse outcome on organism development and reproductive capability, are generally referred to as endocrine disrupting chemicals (EDCs)
This study evaluates the efficiency of shotgun proteomics for the identification of organ- and sex-specific molecular responses in zebrafish following aromatase inhibition
No lethality was observed during the 21 days treatment, indicating the exposure concentrations used to be below the threshold for systemic toxicity
Summary
Substances that interfere with the endocrine system, and exert an adverse outcome on organism development and reproductive capability, are generally referred to as endocrine disrupting chemicals (EDCs). Investigating differentially expressed proteins by proteomics can provide in-depth understanding of cellular or tissue expression profiles and networks that could not be achieved by evaluating the expression of individual proteins To this end, a FSTRA according to OECD TG 22915 was performed with fadrozole, a known aromatase inhibitor, in combination with the identification of organ-and sex-specific molecular responses in zebrafish. Its endocrine disrupting effects on fish are reasonably well defined[18,19,20,21,25,26,27,28,29,30] It provides a proof-of-principle model substance to evaluate and validate a shotgun proteomics approach as suitable tool for the integration of molecular methods into standard test guidelines. The obtained molecular signatures could serve as initiating information for the development of a discrimination tool in endocrine disruptor testing
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