Health Effects and Life Stage Sensitivities in Zebrafish Exposed to an Estrogenic Wastewater Treatment Works Effluent

Abstract

A wide range of health effects in fish have been reported for exposure to wastewater treatment work (WwTW) effluents including feminized responses in males. Most of these exposure studies, however, have assessed acute health effects and chronic exposure effects are less well established. Using an Estrogen Responsive Element-Green Fluorescent Protein (ERE-GFP)-Casper transgenic zebrafish, we investigated chronic health effects and life stage sensitivities for exposure to an estrogenic WwTW effluent and the synthetic estrogen 17α-ethinylestradiol (EE2). Exposure to the WwTW effluent (at full strength;100%) and to 10 ng/L (nominal) EE2 delayed testis maturation in male fish but accelerated ovary development in females. Exposure to 50% and 100% effluent, and to 10 ng/L EE2, also resulted in skewed sex ratios in favor of females. Differing patterns of green fluorescent protein (GFP) expression, in terms of target tissues and developmental life stages occurred in the ERE-GFP- zebrafish chronically exposed to 100% effluent and reflected the estrogenic content of the effluent. gfp and vitellogenin (vtg) mRNA induction were positively correlated with measured levels of steroidal estrogens in the effluent throughout the study. Our findings illustrate the importance of a fish’s developmental stage for estrogen exposure effects and demonstrate the utility of the ERE-GFP zebrafish for integrative health analysis for exposure to estrogenic chemical mixtures.

Application of Transgenic Zebrafish Models for Studying the Effects of Estrogenic Endocrine Disrupting Chemicals on Embryonic Brain Development

“…the heart valves) have been shown to be especially sensitive to estrogens in both of ERE:GFP lines detecting responses for environmentally relevant exposures ( Gorelick and Halpern, 2011 ; Lee et al, 2012a ) similar to the minimum detection level seen for the radial glia in tg ( cyp19a1b:GFP ) embryo ( Brion et al, 2012 ). Accordingly, these models have been used to assess contamination of surface- and waste-waters with environmental estrogens in the USA ( Gorelick et al, 2014 ), across Europe ( Brion et al, 2019 ) and England ( Cooper et al, 2021a ; Cooper et al, 2021b ).…”

“…They include a line generated through crossing tg ( ERE:Gal4ff ; UAS:GFP ) with a pigment free mutant zebrafish Casper ( Green et al, 2016 ), a line where the UAS:GFP has been substituted with another UAS: reporter gene , Kaede ( Green et al, 2018 ), a line tg [ ERE:Gal4ff ; UAS:nitroreductase (NTR)- mCherry ] (shortened name ERE:mCherry ), that enables NTR-mediated chemical/genetic cell ablation, and a double transgenic line carrying both ERE: mCherry and cyp19a1b:GFP reporter genes, tg ( ERE:mCherry ; cyp19a1b:GFP ) ( Takesono et al, 2022 ) (listed in Table 1 ). The tg ( ERE:Gal4ff ; UAS:GFP ) in a pigment mutant Casper background substantially improves the visual clarity in estrogen responsive GFP expression in vivo as compared with the tg ( ERE:Gal4ff ; UAS:GFP ) in wild type background and has facilitated the detection sensitivity for the study of target tissues and potencies of a wide range of estrogenic chemicals ( Green et al, 2016 ; Green et al, 2018 ) and their EDC mixtures (in surface wastewaters) ( Cooper et al, 2021a ; Cooper et al, 2021b ). In the tg ( ERE:Gal4ff ; UAS:Kaede ) line the reporter protein, Kaede, can be photo-converted from green fluorescent to red fluorescent with a short UV exposure and this allows for time-sequenced and tissue specific ER/ERE-responses to be monitored at a desired timing or location in the live embryo-larvae.…”

Changes in CpG Methylation of the Vitellogenin 1 Promoter in Adult Male Zebrafish after Exposure to 17α-Ethynylestradiol

“…Estrogenic EDCs can elevate Vtg levels unnaturally by mimicking the natural ligand E2 (Bjornstrom & Sjoberg, 2005; Delbes et al, 2022; Wang et al, 2005). Male and juvenile fish normally do not produce Vtg; hence, Vtg induction in male and juvenile fish has been widely used as a molecular marker of exposure to estrogenic EDCs in ecological testing (Cooper et al, 2021; Sumpter & Jobling, 1995). Surprisingly, the mechanism behind Vtg induction is yet to be fully understood.…”

“…Of these, vtg1 mRNA is the most highly expressed (Wang et al, 2005). Further, the vtg1 gene is normally silent in male zebrafish liver and is known to be overexpressed after exposure to estrogenic EDCs (Cooper et al, 2021; Hoffmann et al, 2001; Meng et al, 2010; Versonnen & Janssen, 2004).…”

The use of zebrafish to assess water quality and remediation efforts.

“…Literature also reveals that there is a broad range of synthetic and naturally occurring chemicals regularly detected in aquatic ecosystems as well as wastewater. This includes considerable amounts of pharmaceuticals, heavy metals, emerging contaminants such as endocrine disruptors, antibiotics, pesticides and other such pollutants (Peteffi et al,2018;Patel et al,2019;Kinuthia et al,2020;Baralla et al,2021;Cooper et al,2021;Syafrudin et al,2021).…”