Development of a Comprehensive Toxicity Pathway Model for 17α-Ethinylestradiol in Early Life Stage Fathead Minnows (Pimephales promelas)

Abstract

There is increasing pressure to develop alternative ecotoxicological risk assessment approaches that do not rely on expensive, time-consuming, and ethically questionable live animal testing. This study aimed to develop a comprehensive early life stage toxicity pathway model for the exposure of fish to estrogenic chemicals that is rooted in mechanistic toxicology. Embryo-larval fathead minnows (FHM; Pimephales promelas) were exposed to graded concentrations of 17α-ethinylestradiol (water control, 0.01% DMSO, 4, 20, and 100 ng/L) for 32 days. Fish were assessed for transcriptomic and proteomic responses at 4 days post-hatch (dph), and for histological and apical end points at 28 dph. Molecular analyses revealed core responses that were indicative of observed apical outcomes, including biological processes resulting in overproduction of vitellogenin and impairment of visual development. Histological observations indicated accumulation of proteinaceous fluid in liver and kidney tissues, energy depletion, and delayed or suppressed gonad development. Additionally, fish in the 100 ng/L treatment group were smaller than controls. Integration of omics data improved the interpretation of perturbations in early life stage FHM, providing evidence of conservation of toxicity pathways across levels of biological organization. Overall, the mechanism-based embryo-larval FHM model showed promise as a replacement for standard adult live animal tests.

Assessing the Toxicity of 17α-Ethinylestradiol in Rainbow Trout Using a 4-Day Transcriptomics Benchmark Dose (BMD) Embryo Assay

“…This observation revealed a varied degree but consistent occurrence of proteinaceous fluid that leaked into the coelom. Similar histological observations have been previously reported in early-life stage fathead minnows (FHM) and Xenopus laevis exposed to the same or greater concentration of EE2, respectively. In fact, Tompsett et al confirmed that this proteinaceous fluid contained Vtg protein.…”

“…On the other hand, fish size and the condition factor at 4 dph and 21 dph were not significantly affected at any of the concentrations tested (Figure S4). These observations were in contrast to previously reported stunted growth in earlylife stage FHM 28 and tilapia. 44 The difference in these observations may be due to species-specific responses, particularly with regard to the length of exposure relative to the life cycle of a given species of fish.…”

“…While many protein-coding transcripts were highly dysregulated, particularly those associated with estrogenic responses, similar dysregulations were not reflected at the proteome level. Limited concordance between transcriptomic and proteomic responses was previously observed in EE2-exposed zebrafish and FHM . These observations were expected due to many post-translational factors influencing protein–product synthesis .…”

“…Alevins from the solvent control, 30, and 100 ng/L treatment groups were used for nontargeted proteomics analysis, as described by Alcaraz et al, with minor modifications (SM 4). Briefly, whole-body tissues were processed by filter-aided sample preparation , using 30 kDa Microcon filters (Merck Millipore, MA).…”

The Role of Estrogen and Thyroid Hormones in Zebrafish Visual System Function

“…Similar 7-day EE2 exposures using pM to low nM concentrations caused significant overexpression of brain AroB and ERα/β transcripts in juvenile Atlantic salmon ( Lyssimachou et al, 2006 ) and 7 dpf zebrafish ( Cano-Nicolau et al, 2016 ; Nasri et al, 2021 ). Specific EE2 effects have also been observed in retina, where a 32-day exposure to low nanomolar concentrations (4–100 ng/L) decreased the outer and inner plexiform layers and total retinal thickness of minnows assessed at 28 days post-hatch ( Alcaraz et al, 2021 ), an effect likely attributed to disrupted and/or heightened estrogen signaling.…”