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

Abstract

There is an urgent demand for more efficient and ethical approaches in ecological risk assessment. Using 17αethinylestradiol (EE2) as a model compound, this study established an embryo benchmark dose (BMD) assay for rainbow trout (RBT; Oncorhynchus mykiss) to derive transcriptomic points-of-departure (tPODs) as an alternative to live-animal tests. Embryos were exposed to graded concentrations of EE2 (measured: 0, 1. 13, 1.57, 6.22, 16.3, 55.1, and 169 ng/L) from hatch to 4 and up to 60 days post-hatch (dph) to assess molecular and apical responses, respectively. Whole proteome analyses of alevins did not show clear estrogenic effects. In contrast, transcriptomics revealed responses that were in agreement with apical effects, including excessive accumulation of intravascular and hepatic proteinaceous fluid and significant increases in mortality at 55.1 and 169 ng/L EE2 at later time points. Transcriptomic BMD analysis estimated the median of the 20th lowest geneBMD to be 0.18 ng/L, the most sensitive tPOD. Other estimates (0.78, 3.64, and 1.63 ng/L for the 10th percentile geneBMD, first peak geneBMD distribution, and median geneBMD of the most sensitive over-represented pathway, respectively) were within the same order of magnitude as empirically derived apical PODs for EE2 in the literature. This 4-day alternative RBT embryonic assay was effective in deriving tPODs that are protective of chronic effects of EE2.

Transcriptomic Points of Departure Calculated from Rainbow Trout Gill, Liver, and Gut Cell Lines Exposed to Methylmercury and Fluoxetine

“…This divergence in transcriptomic responses is not surprising given that the initial transcriptomic response following methylmercury exposure is likely compensatory and includes genes and pathways that give insight into a chemical's mechanism of action, whereas the later ones (with increasing exposure levels) reflect cellular adversity. Similar patterns are being observed across the tPOD literature including recent data on fish (Alcaraz et al, 2021, 2022; Pagé‐Larivière et al, 2019).…”

“…Studies on fish are drawing similar conclusions. For example, tPODs derived from rainbow trout embryos exposed to ethinylestradiol were in the same order of magnitude as empirically derived BMDs for apical endpoints (Alcaraz et al, 2021). A fluoxetine study derived tPODs from embryo-larval fathead minnows that were similar to apical BMDs in chronically exposed fish (Alcaraz et al, 2022).…”

“…Instead, we used a KEGG pathway library generated by the KEGG Automatic Annotation Server, which is based on KEGG orthologs. As discussed by other researchers, a primary drawback to studying species that lack sufficient functional annotation is the reduced ability to adequately interpret the resulting biological information (Alcaraz et al, 2021; Pagé‐Larivière et al, 2019). Although the investigation of pathway‐level BMDs can yield relevant functional information, the observations should be interpreted judiciously for ecological species with limited functional annotation resources (National Toxicology Program, 2018; Pagé‐Larivière et al, 2019).…”

A Multi–Life Stage Comparison of Silver Nanoparticle Toxicity on the Early Development of Three Canadian Fish Species

“…All methods involving fish were approved by the University of Saskatchewan's Animal Research Ethics Board (protocol 20140079) and adhered to the Canadian Council on Animal Care guidelines for humane animal use. Experimental design and execution followed Alcaraz et al (2021) with minor modifications (Supporting Information, Section SM1). Briefly, AgNPs were diluted with ATRF facility water (dechlorinated and filtered municipal water) to achieve triplicates of six nominal graded concentrations (0.1, 0.3, 1.0, 3.0, 10.0, and 30.0 nM) of AgNPs based on Ag mass.…”

Using the Daphnia magna Transcriptome to Distinguish Water Source: Wetland and Stormwater Case Studies

“…The opportunity to detect chemical‐ or mixture‐specific bioactivities is especially useful when the stressor(s) is unidentified, chemical analysis of all responsible or potentially responsible compounds is too expensive, and a more rapid assay response time is desired compared to observing slower changes in IBI over time. Lastly, it is also possible to generate a “transcriptomic point of departure” (tPOD) by serially diluting wetland waters and evaluating gene expression using a short‐term assay (<20 days, as in the present study or as in Abe et al [2015]), similar to what is currently being done for chemical toxicity (Alcaraz et al, 2021). Such a tPOD can be used to track water quality improvements while providing detailed biological response information potentially traceable to chemicals with known modes of action in a format similar to WET testing.…”