Application of Biomarkers for Endocrine Disruptor Screening in Zebrafish: A Multi-Endpoint Approach to Assessing the Effects of Diflubenzuron and Pyriproxyfen

• Grant number: 25/07127-3
• Support Opportunities: Scholarships abroad - Research Internship - Doctorate
• Start date: August 01, 2025
• End date: July 31, 2026
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Animal Pathology
• Principal Investigator: Bruno Fiorelini Pereira
• Grantee: Rafaella da Silva Brito
• Supervisor: Lisa Baumann
• Host Institution: Instituto de Ciências Ambientais, Químicas e Farmacêuticas (ICAQF). Universidade Federal de São Paulo (UNIFESP). Campus Diadema. Diadema , SP, Brazil
• Institution abroad: University Amsterdam (VU), Netherlands
• Associated to the scholarship: 23/09884-0 - Analysis of histopathological effects and endocrine disruption in chronic exposure to Diflubenzuron and Piriproxyfen in the experimental model Danio rerio, BP.DR

Abstract

Insect growth regulators are extensively utilized in agriculture, veterinary medicine, aquaculture, and mosquito vector control. The control of mosquito larvae in aquatic environments depends on larvicides such as diflubenzuron (DFB) and pyriproxyfen (PPF), which target insect development by inhibiting chitin synthesis and metamorphosis. While effective for vector control, these compounds may pose significant risks to non-target aquatic organisms, particularly fish, through potential endocrine disruption and neurotoxic effects. This study employs a comprehensive transgenic zebrafish (Danio rerio) platform to systematically evaluate the ecotoxicological impacts of DFB and PPF exposure across multiple biological systems. The experimental design incorporates three transgenic lines: Tg(tg:mCherry) for assessing thyroid follicle development, Tg(vtg1:mCherry) for detecting estrogenic activity, and the novel Tg(eno2:Cerulean, gfap:mCherry, mbp:citrine) triple-transgenic line for simultaneous monitoring of neurons, astrocytes, and oligodendrocytes to detect neurotoxic effects. Methodologies include standardized fish embryo toxicity tests (0.025-10 mg/L), quantitative morphological analyses (swim bladder inflation, thyroid follicle size/density), detailed histopathological examination of retinal layers and brain structures, qPCR quantification of HPT-axis genes and visual system markers, and high-throughput behavioral assays using an automated behavior tracking device (ZebraBox, Viewpoint). These concentrations were chosen based on previous studies from our group and the solubility limits of both substances. They will be used to define baseline toxicity levels for the transgenic lines. Expected outcomes include: (1) characterization of thyroid hormone system disruption potential of DFB and PPF through impaired swim bladder inflation, altered thyroid follicle morphology, and eye development; (2) evaluation of estrogenic activity via vitellogenin expression changes; (3) assessment of neurodevelopmental toxicity by quantifying changes in neuronal, astrocytic, and oligodendrocytic markers; and (4) identification of potential correlations between molecular alterations and behavioral deficits. The integration of these advanced transgenic models with molecular and phenotypic endpoints will provide remarkable resolution in understanding DFB and PPF modes of action. (AU)