Use of Early Juvenile Zebrafish Danio Rerio for In-Vivo Assessment of Endocrine Modulation by Xenoestrogens

Abstract

Reliable and cost-effective early-life stage (ELS) bioassays incorporating practical experimentation without compromising scientific relevance are crucial in chemical risk assessment. This study investigated the use of 20 days- post-fertilization life stage ( 20dpfZF ) of zebrafish Danio rerio to screen environmental chemicals known to be estrogenic in adult fish. Firstly, studies with key genes in steroidogenesis were conducted; the brain isoform of aromatase gene ( cyp19a1b ) being the most prominently expressed biomarker. Regulation of mRNA levels of molecular biomarkers, vitellogenin 1 gene ( vtg1) and cyp19a1b were selected to assess the endocrine modulation by xenoestrogens, 17α-ethinylestradiol (EE2), 4- n -nonylphenol (NP), 4- t -octylphenol (OP) and bisphenol A (BPA). Groups of 20dpfZF (n=15) as three replicates were exposed to chemicals over a five-day period in aerated static setups. Exposure of 20dpfZF to sediment spiked with EE2 (nominal 3μg g -1 dw) was also conducted to assess the sensitivity of this life-stage to sediment with estrogenic potency. Whole body homogenates of exposed juveniles showed the estrogenic potential of chemicals in the order: EE2 > OP > BPA > NP. Higher relative expression of cyp19a1b was noticed at lower ambient concentrations of EE2, although vtg1 showed more pronounced expression to it. The 20dpfZF responded in a dose-related way to sediment spiked with EE2, expanding its use as a general aquatic animal model. The suitability of 20dpfZF as an in vivo model, along with stable expression of reference genes was established. In addition to consistent expression pattern of key target genes on xenoestrogenicity, it serves as a practical screening model for the risk assessment of environmental chemicals and samples with estrogenic potential.