Assessment of the effects of estetrol and 17α-ethinylestradiol on zebrafish (Danio rerio) metamorphosis: a morphological and transcriptomic approach

Abstract

17α-ethinylestradiol (EE2) is a synthetic estrogen widely used in combined oral contraceptives (COCs) for years. Estetrol (E4), a natural estrogen synthesized by the fetal liver during pregnancy, is the estrogenic component of a new COC. While E4 is thought to have a limited environmental impact in comparison to EE2, its effects on non-reproductive functions in aquatic species remain underexplored. This study compared the impact of EE2 and E4 on the metamorphosis of zebrafish (Danio rerio), a vital life cycle process. Larvae were exposed to concentrations ranging from 10 to 10,000 times the measured (0.1 ng/L) or predicted (32 ng/L) environmental concentrations of EE2 and E4 respectively. Samples were collected at 14, 22, and 30 days post-fertilization (dpf) to assess morphological traits and perform transcriptomic analysis. EE2 exposure at 1,000 ng/L exhibited developmental delays from the onset of metamorphosis, with most traits affected at 22 dpf. The effects intensified at 30 dpf, with notable impacts at both EE2 100 and 1,000 ng/L. At 100 ng/L, modulations in the expression of genes involved in macronutrient metabolism, crucial to the developmental process, were observed throughout metamorphosis. Additionally, an upregulation of estrogen-specific responses and drug metabolism was noted. In contrast, no notable changes in traits were detected for the concentrations of E4 tested. Although gene expression related to cell structure and adhesion, peptidase activity, and muscle structure and contraction was altered at E4 32,000 ng/L, no metamorphosis-related pathways were affected. These results suggest that E4 presents a greater safety margin and may therefore be more environmentally friendly than EE2.

Keywords: Endocrine disruptor; Estetrol; Ethinylestradiol; Metamorphosis; Zebrafish.