Comparative analysis of transcriptomic points-of-departure (tPODs) and apical responses in embryo-larval fathead minnows exposed to fluoxetine.

Current approaches in chemical hazard assessment face significant challenges because they rely on live animal testing, which is time-consuming, expensive, and ethically questionable. These concerns serve as an impetus to develop new approach methodologies (NAMs) that do not rely on live animal tests. This study explored a molecular benchmark dose (BMD) approach using a 7-day embryo-larval fathead minnow (FHM) assay to derive transcriptomic points-of-departure (tPODs) to predict apical BMDs of fluoxetine (FLX), a highly prescribed and potent selective serotonin reuptake inhibitor frequently detected in surface waters. Fertilized FHM embryos were exposed to graded concentrations of FLX (confirmed at < LOD, 0.19, 0.74, 3.38, 10.2, 47.5 µg/L) for 32 days. Subsets of fish were subjected to omics and locomotor analyses at 7 days post-fertilization (dpf) and to histological and biometric measurements at 32 dpf. Enrichment analyses of transcriptomics and proteomics data revealed significant perturbations in gene sets associated with serotonergic and axonal functions. BMD analysis resulted in tPOD values of 0.56 µg/L (median of the 20 most sensitive gene-level BMDs), 5.0 µg/L (tenth percentile of all gene-level BMDs), 7.51 µg/L (mode of the first peak of all gene-level BMDs), and 5.66 µg/L (pathway-level BMD). These tPODs were protective of locomotor and reduced body weight effects (LOEC of 10.2 µg/L) observed in this study and were reflective of chronic apical BMDs of FLX reported in the literature. Furthermore, the distribution of gene-level BMDs followed a bimodal pattern, revealing disruption of sensitive neurotoxic pathways at low concentrations and metabolic pathway perturbations at higher concentrations. This is one of the first studies to derive protective tPODs for FLX using a short-term embryo assay at a life stage not considered to be a live animal under current legislations.

The brominated flame retardant, TBCO, impairs oocyte maturation in zebrafish (Danio rerio).

The brominated flame retardant, 1,2,5,6-tetrabromocyclooctane (TBCO), has been shown to decrease fecundity in Japanese medaka (Oryzias latipes) and there is indirect evidence from analysis of the transcriptome and proteome that this effect might be due to impaired oogenesis. An assay for disruption of oocyte maturation by chemical stressors has not been developed in Japanese medaka. Thus, using zebrafish (Danio rerio) as a model, objectives of the present study were to determine whether exposure to TBCO has effects on maturation of oocytes and to investigate potential mechanisms. Sexually mature female zebrafish were given a diet of 35.3 or 628.8 µg TBCO / g food for 14 days after which, stage IV oocytes were isolated to assess maturation in response to maturation inducing hormone. To explore potential molecular mechanisms, abundances of mRNAs of a suite of genes that regulate oocyte maturation were quantified by use of quantitative real-time PCR, and abundances of microRNAs were determined by use of miRNAseq. Ex vivo maturation of oocytes from fish exposed to TBCO was significantly less than maturation of oocytes from control fish. The percentage of oocytes which matured from control fish and those exposed to low and high TBCO were 89, 71, and 67%, respectively. Among the suite of genes known to regulate oocyte maturation, mRNA abundance of insulin like growth factor-3 was decreased by 1.64- and 3.44-fold in stage IV oocytes from females given the low and high concentrations of TBCO, respectively, compared to the control group. Abundances of microRNAs regulating the expression of proteins that regulate oocyte maturation, including processes related to insulin-like growth factor, were significantly different in stage IV oocytes from fish exposed to TBCO. Overall, results of this study indicated that impaired oocyte maturation might be a mechanism of reduced reproductive performance in TBCO-exposed fish. Results also suggested that effects of TBCO on oocyte maturation might be due to molecular perturbations on insulin-like growth factor signaling and expression of microRNAs.

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

There is an urgent demand for more efficient and ethical approaches in ecological risk assessment. Using 17α-ethinylestradiol (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.

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

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.