Advancing the Zebrafish embryo test for endocrine disruptor screening using micro-injection: Ethinyl estradiol as a...

Area of Science:

• Environmental Toxicology
• Endocrinology
• Developmental Biology

Background:

• Current fish embryo toxicity tests primarily use aquatic exposures.
• Alternative exposure routes, like micro-injection, may be more practical for certain compounds, especially hydrophobic endocrine disruptors.
• Micro-injection into the yolk can facilitate the delivery of such compounds.

Purpose of the Study:

• To compare micro-injection with continuous aquatic exposure for assessing compound accumulation and biological responses in fish embryos.
• To evaluate the suitability of micro-injection as an alternative or complementary method to aquatic exposure for toxicity testing.
• To identify reliable biomarkers for endocrine disruptor screening across different exposure routes.

Main Methods:

• Optimized micro-injection parameters (solvent, droplet size, needle variation) using 17α-ethinyl estradiol (EE2) as a model compound.
• Compared EE2 accumulation and biological responses between single-dose micro-injection and continuous aquatic exposure.
• Assessed biological endpoints including estrogen receptor activation, and transcription of esr2b, vitellogenin 1 (vtg1), and brain aromatase (cyp19a1b).
• Evaluated the occurrence of malformations in both exposure scenarios.

Main Results:

• Internal dose of EE2 decreased over time in both exposure routes.
• Estrogen receptor activation was dose-dependent and correlated with esr2b transcription.
• Both exposure routes induced vtg1 and cyp19a1b transcription, but cyp19a1b induction timing differed (earlier with injection).
• Specific malformations were observed only after injection, while others occurred in both scenarios.

Conclusions:

• Micro-injection and aquatic exposure elicit different biological responses in fish embryos.
• Micro-injection is not a direct substitute for aquatic exposure but can be a complementary method.
• Vitellogenin 1 and cyp19a1b transcription, along with estrogen receptor activation, are suitable biomarkers for endocrine disruptor screening in both exposure scenarios.