Hepatic Transcriptomic Responses to Ethinylestradiol in Embryonic Japanese Quail and Double-Crested Cormorant.

Understanding species differences in sensitivity to toxicants is a critical issue in ecotoxicology. We recently established that double-crested cormorant (DCCO) embryos are more sensitive than Japanese quail (JQ) to the developmental effects of ethinylestradiol (EE2). We explored how this difference in sensitivity between species is reflected at a transcriptomic level. The EE2 was dissolved in dimethyl sulfoxide and injected into the air cell of eggs prior to incubation at nominal concentrations of 0, 3.33, and 33.3 µg/g egg weight. At midincubation (JQ 9 days; DCCO 16 days), livers were collected from five embryos/treatment group for RNA sequencing. Data were processed and analyzed using EcoOmicsAnalyst and ExpressAnalyst. The EE2 exposure dysregulated 238 and 1,987 genes in JQ and DCCO, respectively, with 78 genes in common between the two species. These included classic biomarkers of estrogen exposure such as vitellogenin and apovitellenin. We also report DCCO-specific dysregulation of Phase I/II enzyme-coding genes and species-specific transcriptional ontogeny of vitellogenin-2. Twelve Kyoto Encyclopedia of Genes and Genomes pathways and two EcoToxModules were dysregulated in common in both species including the peroxisome proliferator-activated receptor (PPAR) signaling pathway and fatty acid metabolism. Similar to previously reported differences at the organismal level, DCCO were more responsive to EE2 exposure than JQ at the gene expression level. Our description of differences in transcriptional responses to EE2 in early life stage birds may contribute to a better understanding of the molecular basis for species differences. Environ Toxicol Chem 2024;43:772-783. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Development and Initial Testing of EcoToxChip, a Novel Toxicogenomics Tool for Environmental Management and Chemical Risk Assessment.

New approach methods (NAMs) are increasingly important to help accelerate the pace of ecological risk assessment and offer more ethical, affordable, and efficient alternatives to traditional toxicity tests. In the present study, we describe the development, technical characterization, and initial testing of a toxicogenomics tool, EcoToxChip (384-well quantitative polymerase chain reaction [qPCR] array), to support chemical management and environmental monitoring for three laboratory model species-fathead minnow (Pimephales promelas), African clawed frog (Xenopus laevis), and Japanese quail (Coturnix japonica). Chip design, including gene selection, was informed by a diverse end-user group and quality control metrics (e.g., primer assay, reverse transcription, and PCR efficiency) performed well based on a priori established criteria. Correlation with RNA sequencing (seq) data provided additional confidence in this novel toxicogenomics tool. Although the present study represents an initial testing of only 24 EcoToxChips for each of the model species, the results provide increased confidence in the robustness/reproducibility of EcoToxChips for evaluating perturbations in gene expression associated with chemical exposure and thus, this NAM, combined with early-life stage toxicity testing, could augment current efforts for chemical prioritization and environmental management. Environ Toxicol Chem 2023;42:1763-1771. © 2023 SETAC.

Characterizing Variability and Uncertainty Associated with Transcriptomic Dose-Response Modeling.

Transcriptomics dose-response analysis (TDRA) has emerged as a promising approach for integrating toxicogenomics data into a risk assessment context; however, variability and uncertainty associated with experimental design are not well understood. Here, we evaluated n = 55 RNA-seq profiles derived from Japanese quail liver tissue following exposure to chlorpyrifos (0, 0.04, 0.1, 0.2, 0.4, 1, 2, 4, 10, 20, and 40 µg/g; n = 5 replicates per group) via egg injection. The full dataset was subsampled 637 times to generate smaller datasets with different dose ranges and spacing (designs A-E) and number of replicates (n = 2-5). TDRA of the 637 datasets revealed substantial variability in the gene and pathway benchmark doses, but relative stability in overall transcriptomic point-of-departure (tPOD) values when tPODs were calculated with the "pathway" and "mode" methods. Further, we found that tPOD values were more dependent on the dose range and spacing than on the number of replicates, suggesting that optimal experimental designs should use fewer replicates (n = 2 or 3) and more dose groups to reduce uncertainty in the results. Finally, tPOD values ranged by over ten times for all surveyed experimental designs and tPOD types, suggesting that tPODs should be interpreted as order-of-magnitude estimates.

Hepatic Transcriptomic Responses to Ethinylestradiol in Two Life Stages of Japanese Quail.

Chemical risk assessment for avian species typically depends on information from toxicity tests performed in adult birds. Early-life stage (ELS) toxicity tests have been proposed as an alternative, but incorporation of these data into existing frameworks will require knowledge about the similarities/differences between ELS and adult responses. The present study uses transcriptomics to assess hepatic gene expression in ELS and adult Japanese quail following exposure to ethinylestradiol (EE2). Prior to incubation, ELS quail were dosed with measured EE2 concentrations of 0.54, 6.3, and 54.2 µg/g egg weight via air cell injection. Adult quail were fed a single dose of EE2 at nominal concentrations of 0, 0.5, and 5 mg/kg body weight by gavage. Liver tissue was collected from five to six individuals per dose group at mid-incubation for ELS quail and 4 days after dosing for adults. A total of 283 and 111 differentially expressed genes (DEGs) were detected in ELS and adult quail, respectively, 16 of which were shared across life stages. Shared DEGs included estrogenic biomarkers such as vitellogenin genes and apovitellenin-1. For the dose groups that resulted in the highest number of DEGs (ELS, 6.3 µg/g; adult, 5 mg/kg), 21 and 35 Kyoto Encyclopedia of Genes and Genomes pathways were enriched, respectively. Ten of these pathways were shared between life stages, including pathways involved with signaling molecules and interaction and the endocrine system. Taken together, our results suggest conserved mechanisms of action following estrogenic exposure across two life stages, with evidence from differential expression of key biomarker genes and enriched pathways. The present study contributes to the development and evaluation of ELS tests and toxicogenomic approaches and highlights their combined potential for screening estrogenic chemicals. Environ Toxicol Chem 2022;41:2769-2781. © 2022 SETAC.

Effects on Apical Outcomes of Regulatory Relevance of Early-Life Stage Exposure of Double-Crested Cormorant Embryos to 4 Environmental Chemicals.

Environmental risk assessment is often challenged by a lack of toxicity data for ecological species. The overall goal of the present study was to employ an avian early-life stage toxicity test to determine the effects of 4 chemicals (benzo[a]pyrene [BaP], chlorpyrifos, fluoxetine hydrochloride [FLX], and ethinyl estradiol [EE2]) on an ecologically relevant avian species, the double-crested cormorant (Phalacrocorax auritus), and to compare our results with those we previously reported for a laboratory model species, Japanese quail. Chemicals were dissolved in dimethyl sulfoxide and administered via air cell injection to fertilized, unincubated double-crested cormorant eggs at 3 nominal concentrations, the highest selected to approximate the 20% lethal dose. Of the 4 chemicals, only chlorpyrifos and FLX were detected in liver tissue of embryos at midincubation (day 14) and termination (day 26; 1-2 d prior to hatch); EE2 and BaP were not detectable, suggesting embryonic clearance/metabolism. No apical effects were observed in double-crested cormorant embryos up to the highest concentrations of chlorpyrifos (no-observed-effect level [NOEL] = 25 µg/g) or FLX (NOEL = 18 µg/g). Exposure to EE2 reduced embryonic viability and increased deformities at a concentration of 2.3 µg/g (NOEL = 0.18 µg/g), and BaP decreased embryonic viability (median lethal dose = 0.015 µg/g; NOEL = 0.0027 µg/g). Compared with Japanese quail, double-crested cormorant were more sensitive with regard to embryolethality and deformities for EE2 and embryolethality for BaP, whereas they were less sensitive to embryonic deformities associated with chlorpyrifos exposure. These data reinforce the idea that standardized toxicity tests using a laboratory model species may not always be protective of wild birds, and thus they stress the importance of developing such alternative testing strategies (e.g., the EcoToxChip Project) for ecologically relevant species to augment risk assessment efforts. Environ Toxicol Chem 2021;40:390-401. © 2020 SETAC.

An Early-Life Stage Alternative Testing Strategy for Assessing the Impacts of Environmental Chemicals in Birds.

Early-life stage (ELS) toxicity tests are recognized as an advancement over current testing methodologies in terms of cost, animal use, and biological relevance. However, standardized ELS tests are not presently available for some vertebrate taxa, including birds. The present study describes a Japanese quail (Coturnix japonica) ELS test that is a promising candidate for standardization and applies it to test 8 environmental chemicals (ethinylestradiol, benzo[a]pyrene, chlorpyrifos, fluoxetine, lead(II)nitrate, trenbolone, seleno-L-methionine, hexabromocyclododecane). Individual chemicals were injected into the air cell of unincubated Japanese quail eggs at 3 concentrations, all predicted to cause ≤20% mortality. Survival to embryonic day 16 was consistently high (>90%) among the vehicle-injected controls. All chemicals, except ethinylestradiol, were detected in liver tissue, most at concentrations suggestive of embryonic clearance. Adverse effects were observed for 5 of the 8 chemicals; chlorpyrifos (41.1 µg/g) significantly increased developmental abnormalities and decreased embryo and gallbladder mass. Ethinylestradiol (54.2 µg/g) and hexabromocyclododecane (0.02 µg/g) decreased embryo mass and tarsus length, respectively. Benzo[a]pyrene (0.83 µg/g) and fluoxetine hydrochloride (32.7 µg/g) exceeded the 20% mortality cutoff. No effects were observed following lead(II)nitrate, seleno-L-methionine, or trenbolone exposure up to 10.7, 0.07, and 4.4 µg/g, respectively. Overall, our ELS approach was time- and cost-effective, caused minimal mortality in controls, effectively delivered diverse chemicals to the embryo, and permitted identification of apical outcomes, all of which provide support toward standardization. Environ Toxicol Chem 2019;39:141-154. © 2019 SETAC.

Assessment of environmentally contaminated sediment using a contact assay with early life stage zebrafish (Danio rerio).

Lake Saint-Louis, a shallow fluvial lake near the western tip of the island of Montreal, QC, Canada is an important spawning ground for many species of fish. Sediments in certain areas of the lake are known to be contaminated with high levels of metals and legacy organic chemicals. To improve our understanding of risk to native fish populations, we conducted a study evaluating levels of sediment contamination and potential effects on early life stage fish. Concentrations of PAHs, PCBs, PCDDs and PCDFs were several orders of magnitude higher at two industrial sites (B1 and B2) than at a nearby reference site (IP). Concentrations of 32 metals and metalloids were at least 5-fold higher at B1 and B2 than at IP. Moreover, all available interim sediment quality guidelines (ISQGs) were exceeded at the two contaminated sites, while none were exceeded at the reference site. Biological effects were evaluated using a sediment contact assay. Zebrafish (Danio rerio) embryos were exposed to clean water (control), or to sediment from IP, B1, and B2 until 120 h post fertilization (hpf). Mortality was significantly elevated in fish exposed to the B1, but not the B2 sediment. The frequency of deformities increased with increasing contamination, but this trend was not statistically significant (p > 0.05). Genes that are implicated in the response to PAHs, PCBs, dioxins and furans (cyp1a, cyp1b1, ahr2) were significantly elevated in the 120 hpf larvae exposed to the B1 and B2 sediments. Global DNA methylation, and mRNA expression of genes related to oxidative stress (maft, cat, hmox1, sod2), embryonic development (bmp2b, baf60c), metal exposure (mt2), and DNA repair (gadd45b) were unaffected. Our results suggest that the Beauharnois sector of Lake Saint-Louis is poor quality spawning habitat due to high levels of contamination, and the potential for harmful effects on early life stage fish.

Potency of polycyclic aromatic hydrocarbons in chicken and Japanese quail embryos.

Birds are receptors of concern for polycyclic aromatic hydrocarbons (PAHs), yet limited data describing the relative potency of PAH congeners are available for avian species. In the present study, we determined embryonic median lethal dose (LD50) values for 5 PAH congeners in chicken (Gallus gallus) and one PAH congener in Japanese quail (Coturnix japonica). Graded concentrations of each test compound were injected into the air cell of chicken or quail eggs before incubation. Embryos were monitored through development (quail) or hatching (chicken). All PAHs tested caused dose-dependent increases in embryo mortality, but few other effects (e.g., weight changes, deformities) were observed. In chicken, windows of developmental sensitivity were identified between embryonic days 4 and 9 and between embryonic days 20 and 22. The rank order potency of benzo[k]fluoranthene (BkF; 76 µg/kg) ≈ dibenz[ah]anthracene (83 µg/kg) > indeno[1,2,3-cd]pyrene (325 µg/kg) > benzo[a]pyrene (461 µg/kg) > benz[a]anthracene (529 µg/kg) corresponded well with previous in vitro estimates in birds. Previously published ethoxyresorufin-O-deethylase median effect concentrations from cultured chicken embryo hepatocytes were highly predictive of our LD50s (p < 0.001, r2 = 0.99). To explore differences in sensitivity between species, Japanese quail eggs were injected with BkF, the most potent PAH. We found that chicken and quail were nearly equally sensitive to BkF. The present results contribute to our developing understanding of variability in responses to PAHs among congeners and species. Environ Toxicol Chem 2018;37:1556-1564. © 2018 SETAC.