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.

Comparing Transcriptomic Responses to Chemicals Across Six Species Using the EcoToxChip RNASeq Database.

The EcoToxChip project includes RNA-sequencing data from experiments involving model (Japanese quail, fathead minnow, African clawed frog) and ecological (double-crested cormorant, rainbow trout, northern leopard frog) species at multiple life stages (whole embryo and adult) exposed to eight chemicals of environmental concern known to perturb a wide range of biological systems (ethinyl estradiol, hexabromocyclododecane, lead, selenomethionine, 17ß trenbolone, chlorpyrifos, fluoxetine, and benzo[a]pyrene). The objectives of this short communication were to (1) present and make available this RNA-sequencing database (i.e., 724 samples from 49 experiments) under the FAIR principles (FAIR data are data which meet principles of findability, accessibility, interoperability, and reusability), while also summarizing key meta-data attributes and (2) use ExpressAnalyst (including the Seq2Fun algorithm and EcoOmicsDB) to perform a comparative transcriptomics analysis of this database focusing on baseline and differential transcriptomic changes across species-life stage-chemical combinations. The database is available in NCBI GEO under accession number GSE239776. Across all species, the number of raw reads per sample ranged between 13 and 58 million, with 30% to 79% of clean reads mapped to the "vertebrate" subgroup database in EcoOmicsDB. Principal component analyses of the reads illustrated separation across the three taxonomic groups as well as some between tissue types. The most common differentially expressed gene was CYP1A1 followed by CTSE, FAM20CL, MYC, ST1S3, RIPK4, VTG1, and VIT2. The most common enriched pathways were metabolic pathways, biosynthesis of cofactors and biosynthesis of secondary metabolites, and chemical carcinogenesis, drug metabolism, and metabolism of xenobiotics by cytochrome P450. The RNA-sequencing database in the present study may be used by the research community for multiple purposes, including, for example, cross-species investigations, in-depth analyses of a particular test compound, and transcriptomic meta-analyses. Environ Toxicol Chem 2024;00:1-6. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Metabolomic profiles in relation to benchmark polycyclic aromatic compounds (PACs) and trace elements in two seabird species from Arctic Canada.

While exposure of birds to oil-related contaminants has been documented, the related adverse effects this exposure has on Arctic marine birds remain unexplored. Metabolomics can play an important role to explore biologically relevant metabolite biomarkers in relation to different stressors, even at benchmark levels of contamination. The aim of this study was to characterize the metabolomics profiles in relation to polycyclic aromatic compounds (PACs) and trace elements in the liver of two seabird species in the Canadian Arctic. In July 2018, black guillemots (Cepphus grylle) and thick-billed murres (Uria lomvia) were collected by hunters from a region where natural oil seeps occur in the seabed near Qikiqtarjuaq, Nunavut, Canada. A total of 121 metabolites were identified in liver tissue samples using reversed phase and hydrophilic interaction liquid chromatography coupled to high resolution mass spectrometry platforms to detect non-polar and polar metabolites, respectively. Sixty-nine metabolites showed excellent repeatability and linearity and were used to examine possible effects of oil-related contaminants exposure (PACs and trace elements). Metabolites including 3-hydroxy anthranilic acid, adenine, adenosine, adenosine mono-phosphate, ascorbic acid, butyrylcarnitine, cholic acid, guanosine, guanosine mono-phosphate, inosine, norepinephrine and threonine showed significant differences (more than two fold) between the two species. Elevated adenine and adenosine, along with decreased reduced/oxidized glutathione ratio, highlighted the potential for oxidative stress in murres. Lipid peroxidation and superoxide dismutase activity assays also confirmed these metabolomic findings. These results will help to characterize the baseline metabolomic profiles of Arctic seabird species with different foraging behaviour and trace element burden.

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.

Extracts from Dated Lake Sediment Cores in the Athabasca Oil Sands Region Alter Ethoxyresorufin-O-deethylase Activity and Gene Expression in Avian Hepatocytes.

Increases in oil sands mining operations in the Athabasca oil sands region have resulted in increased concentrations of polycyclic aromatic compounds (PACs) and heavy metals in aquatic systems located near surface mining operations. In the present study, sediment cores were collected from 3 lakes with varying proximity to surface mining operations to determine the differences in PAC concentrations. Sediment cores were separated into 2 sections-current mining (top; 2000-2017) and premining (bottom; pre-1945)-and extracts were prepared for in vitro screening using a well-established chicken embryonic hepatocyte (CEH) assay. Concentrations and composition of PACs varied between sites, with the highest ∑PACs in Saline Lake, 5 km from an active oil sands mine site. The proportion of alkylated PACs was greater than that of parent PACs in the top sediment sections compared with the bottom. Ethoxyresorufin-O-deethylase activity in CEH permitted the ranking of lake sites/core sections based on an aryl hydrocarbon receptor-mediated end point; mean median effect concentration values were lowest for the top cores from Saline Lake and another near-mining operations lake, referred to as WF1. A ToxChip polymerase chain reaction (PCR) array was used to evaluate gene expression changes across 43 target genes associated with numerous toxicological pathways following exposure to top and bottom sediment core extracts. The 2 study sites with the greatest ∑PAC concentrations (Saline Lake and WF1) had the highest gene expression alterations on the ToxChip PCR array (19 [top] and 17 [bottom]/43), compared with a reference site (13 [top] and 7 [bottom]/43). The avian in vitro bioassay was useful for identifying the toxicity of complex PAC extracts associated with variably contaminated sediment cores, supporting its potential use for hotspot identification and complex mixture screening. EnvironToxicol Chem 2021;40:1883-1893. © 2021 SETAC.

Effects of two Bisphenol A replacement compounds, 1,7-bis (4-hydroxyphenylthio)-3,5-dioxaheptane and Bisphenol AF, on development and mRNA expression in chicken embryos.

Concerns about the estrogenic properties of Bisphenol A (BPA) have led to increased efforts to find BPA replacements. 1,7-bis(4-Hydroxyphenylthio)-3,5-dioxaheptane (DD-70) and 4,4'-(hexafluoroisopropylidene) diphenol (bisphenol AF, BPAF) are two potential chemical substitutes for BPA; however, toxicity data for these chemicals in avian species are limited. To determine effects on avian embryonic viability, development, and hepatic mRNA expression at two distinct developmental periods (mid-incubation [day 11] and term [day 20]), two egg injection studies were performed. Test chemicals were injected into the air cell of unincubated, fertilized chicken eggs at concentrations ranging from 0-88.2 µg/g for DD-70 and 0-114 µg/g egg for BPAF. Embryonic concentrations of DD-70 and BPAF decreased at mid-incubation and term compared to injected concentrations suggesting embryonic metabolism. Exposure to DD-70 (40.9 and 88.2 µg/g) and BPAF (114 µg/g) significantly decreased embryonic viability at mid-incubation. Exposure to DD-70 (88.2 µg/g) decreased embryo mass and increased gallbladder mass, while 114 µg/g BPAF resulted in increased gallbladder mass in term embryos. Expression of hepatic genes related to xenobiotic metabolism, lipid homeostasis, and response to estrogen were altered at both developmental stages. Given the importance of identifying suitable BPA replacements, the present study provides novel, whole animal avian toxicological data for two replacement compounds, DD-70 and BPAF. DATA AVAILABILITY: Data, associated metadata, and calculation tools are available from the corresponding author (doug.crump@canada.ca).

Polycyclic aromatic compounds (PACs) and trace elements in four marine bird species from northern Canada in a region of natural marine oil and gas seeps.

There is a growing understanding of how oil pollution can affect aquatic ecosystems, including physical and chemical effects. One of the biggest challenges with detecting the effects of oil-related contaminants on biota from resource development is understanding the background levels and potential effects of the exposure of biota to contaminants from various natural and anthropogenic sources prior to large scale oil and gas operations. Seabirds are effective indicators of pollution, and can be useful for tracking oil-related contaminants in the marine environment. We sampled four seabird species (black guillemot, Cepphus grylle; thick-billed murre, Uria lomvia; black-legged kittiwake, Rissa tridactyla; and northern fulmar, Fulmarus glacialis) in the Baffin Bay-Davis Strait region of the Northwest Atlantic and Arctic oceans, an area where natural oil and gas seeps are present but lacking any large-scale oil and gas projects. We found detectable levels of PACs and several trace elements in all species examined. Alkylated PAC levels were higher than parent compounds in all four seabird species examined, with fulmars and murres having the highest levels detected; mean hepatic concentrations of ∑16PAC were 99.05, 46.42, 12.78 and 9.57 ng/g lw, respectively, for guillemots, murres, fulmars and kittiwakes. Overall, PAC concentrations in the seabird species examined were similar to PAC concentrations measured in other bird species in regions with more industrialization. These findings provide data which can be used to assess the current oil-related contaminant exposure of biota in the region. As well, they provide background levels for the region at a time when shipping activity is relatively low, which can used for future comparisons following expected increases in shipping and oil and gas activities in the region.

Evaluation of the Aryl Hydrocarbon Receptor Response in LMH 3D Spheroids.

In the present study, we investigated whether the immortalized chicken hepatocellular carcinoma cell line, leghorn male hepatoma (LMH), had a comparable aryl hydrocarbon receptor (AhR) response to primary chicken embryonic hepatocytes (CEHs) when used in a well-established assay for chemical screening and prioritization. The LMH cells were grown as 2-dimensional (2D) confluent cells and 3D spheroids to determine the optimal cell culture states for chemical screening. Cytochrome P450 1A4 and 1A5 (CYP1A) activity and gene expression were compared between CEHs and LMH cells grown in 2 culture states following exposure to the dioxin-like compound 3,3',4,4',5-pentachlorobiphenyl (PCB-126). The CYP1A activity was measured using the ethoxyresorufin-O-deethylase (EROD) assay, and changes in mRNA expression associated with the AhR pathway were determined using a custom-designed polymerase chain reaction array. Among LMH cell culture states (i.e., 2D vs 3D), EROD induction was observed only in 3D LMH spheroids. Similarly, 3D spheroids had the greatest number of changes in AhR-related genes compared with confluent cells. Overall, these results suggest that LMH cells grown as 3D spheroids have a metabolic and gene expression profile that is comparable to that of CEH, and may represent a suitable animal-free alternative for in vitro screening of chemicals. Environ Toxicol Chem 2020;39:1693-1701. © 2020 SETAC.

Computational evaluation of interactions between organophosphate esters and nuclear hormone receptors.

Organophosphate esters (OPEs) have gained considerable interest from many environmental chemists and toxicologists due to their frequent detection in the environment and potential adverse effects on health. Nuclear hormone receptors (NHRs) were found to mediate many of their adverse effects. However, our knowledge regarding the direct binding and interaction between OPEs and NHRs is limited. In this study, Endocrine Disruptome, an online computational tool based on the technique of inverse docking, was used to calculate the binding affinity score of 25 individual OPEs with 12 different human NHRs. Results showed that 20% of potential binding interactions between the OPEs and NHRs had medium-to-high probabilities. The accuracy, sensitivity and specificity of the predictions were 78.8, 60.0 and 80.9%, respectively. OPEs with a benzene ring were more active than those without, among which, tri-o-tolyl phosphate and tri-m-tolyl phosphate displayed the highest activities, suggesting that they might pose the greatest potential risks for interference with endocrine functions. In addition, the antagonistic conformations of androgen receptor and estrogen receptor ß were found to be the two most vulnerable NHR conformations. Our findings can further the understanding about the health risk(s) of OPEs.

A comparative study of 3 alternative avian toxicity testing methods: Effects on hepatic gene expression in the chicken embryo.

There is growing interest in developing alternative methods to screen and prioritize chemical hazards, although few studies have compared responses across different methods. The objective of the present study was to compare 3 alternative liver methods derived from white Leghorn chicken (Gallus gallus domesticus): primary hepatocyte culture, liver slices, and liver from in ovo injected embryos. We examined hepatic gene expression changes after exposure to 3 chemicals (17ß-trenbolone [17ßT], 17ß-estradiol [E2], and 2,3,7,8-tetrachlorodibenzo-p-dioxin [TCDD]) using a custom quantitative polymerase chain reaction (qPCR) array with 7 genes (vitellogenin [VTG], apolipoprotein [Apo], cytochrome P450 1A4 [CYP1A4], liver basic fatty acid binding protein [LBFABP], 3ß hydroxysteroid dehydrogenase [HSD3ß1], stearoyl coenzyme A desaturase [SCD], and estrogen sulfotransferase [SULT1E1]). Gene expression across the 3 methods was examined using hierarchical clustering. Up-regulation of CYP1A4 in response to TCDD was consistent across all methods, and the magnitude was higher in hepatocytes (>150-fold) compared with slices (>31-fold) and in ovo liver (>27-fold). In hepatocytes, SCD and VTG up-regulation in response to 17ßT and E2 was >4-fold and 16-fold, respectively. The rank order of cases with significant changes in gene expression among the 3 methods was: hepatocytes (22) > in ovo liver (11) > liver slices (6). Hierarchical clustering grouped liver slices and in ovo liver as more similar, whereas hepatocytes were grouped separately from in ovo liver. More introspective comparisons are needed to understand how and why alternative methods differ and to aid in their integration into toxicity testing. Environ Toxicol Chem 2019;38:2546-2555. © 2019 SETAC.

Extracts of Passive Samplers Deployed in Variably Contaminated Wetlands in the Athabasca Oil Sands Region Elicit Biochemical and Transcriptomic Effects in Avian Hepatocytes.

Recent contaminant monitoring in boreal wetlands situated in Alberta's Athabasca oil sands region revealed increased concentrations of polycyclic aromatic compounds (PACs) in passive sampling devices deployed in wetlands close to bitumen surface mining operations. In this study, graded concentrations of semipermeable membrane device (SPMD) extracts, collected from 4 wetlands with variable burdens of PACs, were administered to chicken and double-crested cormorant (DCCO) embryonic hepatocytes to determine effects on 7-ethoxyresorufin-O-deethylase (EROD) activity and mRNA expression. Concentrations and composition of PACs detected in SPMDs varied among sites, and the proportion of alkyl PACs was greater than parent compounds at all sites. ΣPACs was the highest in SPMDs deployed within 10 km of mining activity (near-site wetland [5930 ng SPMD-1]) compared to those ∼50 km south (far-site wetland [689 ng SPMD-1]). Measures of EROD activity and Cyp1a4 mRNA expression allowed the ranking of wetland sites based on aryl hydrocarbon receptor-mediated end points; EROD activity and Cyp1a4 mRNA induction were the highest at the near-site wetland. ToxChip PCR arrays (one chicken and one DCCO) provided a more exhaustive transcriptomic evaluation across multiple toxicological pathways following exposure to the SPMD extracts. Study sites with the greatest PAC concentrations had the most genes altered on the chicken ToxChip (12-15/43 genes). Exposure of avian hepatocytes to SPMD extracts from variably contaminated wetlands highlighted traditional PAC-related toxicity pathways as well as other novel mechanisms of action. A novel combination of passive sampling techniques and high-throughput toxicity evaluation techniques shows promise in terms of identifying hotspots of chemical concern in the natural environment.

Transcriptomic points-of-departure from short-term exposure studies are protective of chronic effects for fish exposed to estrogenic chemicals.

Resource limitations often require risk assessors to extrapolate chronic toxicity from acute tests using assessment factors. Transcriptomic dose-response analysis following short-term exposures may provide a more reliable and biologically-based alternative for estimating chronic toxicity. Here, we demonstrate that transcriptomic dose-response analysis in fish following short-term exposure to endocrine disrupting chemicals (EDCs) provides estimates of chronic toxicity that may be used as protective points-of-departure (POD) for risk assessment. The benchmark dose (BMD) method was used on publicly available datasets (n = 5) to determine transcriptomic PODs in fish exposed to three EDCs (bisphenol A, ethinylestradiol, and diethylstilbestrol). To test for potential bias related to data processing, our analysis compared the effect of different normalization, filtering, and BMD-grouping methods on the transcriptomic PODs. The resulting PODs were then compared to the empirically-derived chronic LOEC of each substance. Normalization and filtering methods had limited impact on the final PODs. However, we found that PODs derived from ontology- or pathway-based gene grouping methods were highly variable, whereas PODs from grouping methods that focused on the most responsive genes were more stable and provided POD estimates that were most similar to the chronic LOEC. Overall, 72% of transcriptomic PODs were within 1 order of magnitude of the chronic LOEC, regardless of data analysis method. When our recommended analysis approach was applied, the concordance improved to 100%. These results suggest that toxicogenomic dose-response analysis has the potential to be a protective decision-support tool for compounds with chronic toxicity, such as EDCs.

Hatching failure and accumulation of organic pollutants through the terrestrial food web of a declining songbird in Western Europe.

Population growth in passerine birds is largely driven by fecundity. If fecundity is affected, for instance by hatching failure, populations may decline. We noted high hatching failure of up to 27% per year in relict populations of the Northern wheatear (Oenanthe oenanthe) in The Netherlands, a strongly declining, migratory passerine in Europe. This hatching failure itself can cause population decline, irrespective of other adverse factors. Additionally, we investigated the cause of hatching failure. Unhatched eggs showed egg yolk infections or embryonic malformations, part of which is associated with the actions of dioxin-like compounds (DLCs). Indeed, DLCs appear to bioaccumulate in the local foodweb, where the soil contained only background concentrations, similar to those found at many other locations. DLC concentrations in Dutch eggs were six-fold higher than those in a reference population in Sweden, where egg failure was only 6%. However, Northern wheatears appear to be only moderately sensitive to the actions of DLCs, because of their specific Ah-receptor type which may moderate the receptor mediated effects of DLCs. This indicates that the concentrations of DLCs, although elevated, may not have caused the embryo malformations or the low hatching rates. We discuss whether other toxins may be important or imbalances in the nutrition and if inbreeding may play a larger role than expected.

Athabasca Oil Sands Petcoke Extract Elicits Biochemical and Transcriptomic Effects in Avian Hepatocytes.

Petroleum coke or "petcoke" is a granular carbonaceous material produced during the upgrading of heavy crude oils, including bitumen. Petcoke dust was recently reported as an environmental contaminant in the Athabasca oil sands region, but the ecotoxicological hazards posed by this complex bitumen-derived material-including those to avian species-have not been characterized. In this study, solvent extracts (x) of delayed and fluid petcoke (xDP and xFP) were prepared and dissolved in dimethyl sulfoxide. A water-accommodated fraction of delayed petcoke (waDP) was also prepared. Graded concentrations of xDP, xFP, and waDP were administered to chicken and double-crested cormorant hepatocytes to determine effects on 7-ethoxyresorufin-O-deethylase (EROD) activity, porphyrin accumulation, and mRNA expression. Polycyclic aromatic compounds (PACs) were characterized, and xDP, xFP, and waDP had total PAC concentrations of 93 000, 270, and 5.3 ng/mL. The rank order of biochemical and transcriptomic responses was xDP > xFP > waDP (e.g., EROD EC50s were lower for xDP compared to xFP and waDP). A total of 22, 18, and 4 genes were altered following exposure to the highest concentrations of xDP, xFP, and waDP, respectively, using a chicken PCR array comprising 27 AhR-related genes. To provide more exhaustive coverage of potential toxicity pathways being impacted, two avian ToxChip PCR arrays-chicken and double-crested cormorant-were utilized, and xDP altered the expression of more genes than xFP. Traditional PAC-related toxicity pathways and novel mechanisms of action were identified in two avian species following petcoke extract exposure. Extrapolation to real-world exposure scenarios must consider the bioavailability of the extracted PACs compared to those in exposed organisms.

Sunlight Irradiation of Highly Brominated Polyphenyl Ethers Generates Polybenzofuran Products That Alter Dioxin-responsive mRNA Expression in Chicken Hepatocytes.

We report on two highly brominated polyphenyl ether flame retardants, tetradecabromo-1,4- diphenoxybenzene (TeDB-DiPhOBz) and 2,2',3,3',4,4',5,5',6,6'-decabromodiphenyl ether (BDE-209), that formed photolytic degradation products in tetrahydrofuran (THF)/hexane solvent after 21 days of natural sunlight irradiation (SI). These degradation products of SI-TeDB-DiPhOBz and SI-BDE-209 included the numerous polybrominated homologue groups of polybenzofurans and dibenzofurans, respectively. Formation of similar polybenzofuran and dibenzofuran products was also observed following a 3 month exposure of the solid powder forms of TeDB-DiPhOBz and BDE-209 to natural SI. These resulting degradation product mixtures were administered to chicken embryonic hepatocytes (CEH) to determine effects on mRNA expression levels of 27 dioxin-responsive genes. For the solvent-based SI study, equivalent concentrations of 1 or 25 µM of SI-TeDB-DiPhOBz or 1 or 10 µM of SI-BDE-209 resulted in gene expression profiles that were similar to those of the most potent dioxin-like compound, 2,3,7,8-tetrachlorodibenzo-p-dioxin. In addition, a concentration-dependent induction of CYP1A4 and CYP1A5 mRNA was observed following exposure to SI-TeDB-DiPhOBz and SI-BDE-209. Based on ECthreshold values for CYP1A4/5 mRNA expression, relative potency (ReP) values were 1 × 10(-6) and 1 × 10(-5) for SI-TeDB-DiPhOBz and SI-BDE-209, respectively. The SI TeDB-DiPhOBz and BDE-209 powder degradation product mixture also significantly induced CYP1A4 mRNA levels in CEH. Our findings clearly show that the environmental stability of TeDB-DiPhOBz and BDE-209, and possibly other highly brominated polyphenyl ethers, is of great concern from a dioxin-like degradation products and toxicity perspective.

Comparing the effects of tetrabromobisphenol-A, bisphenol A, and their potential replacement alternatives, TBBPA-bis(2,3-dibromopropyl ether) and bisphenol S, on cell viability and messenger ribonucleic acid expression in chicken embryonic hepatocytes.

A market for alternative brominated flame retardants (BFRs) has emerged recently due to the phase out of persistent and inherently toxic BFRs. Several of these replacement compounds have been detected in environmental matrices, including wild birds. A chicken embryonic hepatocyte (CEH) assay was utilized to assess the effects of the BFR, tetrabromobisphenol-A (TBBPA), and its replacement alternative, tetrabromobisphenol A bis(2,3-dibromopropyl ether [TBBPA-DBPE]) on cell viability and messenger ribonucleic acid (mRNA) expression. Bisphenol A (BPA) and 1 of its replacement alternatives, bisphenol S (BPS), were also screened for effects. Both TBBPA and BPA decreased CEH viability with calculated median lethal concentration (LC50) values of 40.6 µM and 61.7 µM, respectively. However, the replacement alternatives, TBBPA-DBPE and BPS, did not affect cell viability (up to 300 µM). Effects on mRNA expression were determined using an Avian ToxChip polymerse chain reaction (PCR) array and a real-time (RT)-PCR assay for the estrogen-responsive genes, apolipoproteinII (ApoII) and vitellogenin (Vtg). A luciferase reporter gene assay was used to assess dioxin-like effects. Tetrabromobisphenol-A altered mRNA levels of 4 genes from multiple toxicity pathways and increased luciferase activity in the luciferase reporter gene assay, whereas its alternative, TBBPA-DBPE, only altered 1 gene on the array, Cyp1a4, and increased luciferase activity. At 300 µM, a concentration that decreased cell viability for TBBPA and BPA, the BPA replacement, BPS, altered the greatest number of transcripts, including both ApoII and Vtg. Bisphenol A exposure did not alter any genes on the array but did up-regulate Vtg at 10 µM. Characterization of the potential toxicological and molecular-level effects of these compounds will ideally be useful to chemical regulators tasked with assessing the risk of new and existing chemicals.

Tris(1,3-dichloro-2-propyl) phosphate perturbs the expression of genes involved in immune response and lipid and steroid metabolism in chicken embryos.

We previously demonstrated that in ovo exposure to the flame retardant tris(1,3-dichloro-2-propyl) phosphate (TDCPP) decreased plasma thyroxine levels, reduced growth parameters, and decreased gallbladder size in chicken embryos. In the current study DNA microarrays were used to evaluate global mRNA expression in liver tissue of male chicken embryos that exhibited the above mentioned effects. Injected doses were dimethyl sulfoxide vehicle control, 7.6 or 45 µg TDCPP/g egg. TDCPP caused significant changes in the expression of five genes at the low dose and 47 genes at the high dose (False Discovery Rate p ≤ 0.1, fold change ≥ 1.5). The gene expression analysis suggested a compromised immune function, a state of cholestatic liver/biliary fibrosis, and disrupted lipid and steroid metabolism. Circulating bile acid levels were elevated, which is an indication of liver dysfunction, and plasma cholesterol levels were reduced; however, hepatic bile acid and cholesterol levels were unaltered. Interactome analyses identified apolipoprotein E, hepatocyte nuclear factor 4 alpha, and peroxisome proliferator-activated receptor alpha as key regulatory molecules involved in the effects of TDCPP. Our results demonstrate a targeted effect of TDCPP toxicity on lipid metabolism, including cholesterol, that helps explain the aforementioned phenotypic effects, as chicken embryos are highly dependent on yolk lipids for growth and maintenance throughout development. Finally, our results are in concordance with the literature that describes TDCPP as a cancer-causing agent, since the majority of dysregulated genes were involved in cancer pathways.

In vitro microarray analysis identifies genes in acute-phase response pathways that are down-regulated in the liver of chicken embryos exposed in ovo to PFUdA.

Perfluoroundecanoic acid (PFUdA) is one of the most highly detected perfluoroalkyl compounds in wild bird tissues and eggs. Although PFUdA does not affect hatching success, many PFCs are known to impair post-hatch development and survival. Here we use microarrays to survey the transcriptional response of cultured chicken embryonic hepatocytes (CEH) to PFUdA for potential targets of PFUdA action that could lead to developmental deficiencies in exposed birds. At 1 µM and 10 µM PFUdA significantly altered the expression of 346 and 676 transcripts, respectively (fold-change>1.5, p<0.05, false discovery rate-corrected). Using functional, pathway and interactome analysis we identified several potentially important targets of PFUdA exposure, including the suppression of the acute-phase response (APR). We then measured the expression of five APR genes, fibrinogen alpha (fga), fibrinogen gamma (fgg), thrombin (f2), plasminogen (plg), and protein C (proC), in the liver of chicken embryos exposed in ovo to PFUdA. The expression of fga, f2, and proC were down-regulated in embryo livers (100 or 1000 ng/g, p<0.1) as predicted from microarray analysis, whereas fibrinogen gamma (fgg) was up-regulated and plg was not significantly affected. Our results demonstrate the utility of CEH coupled with transcriptome analysis as an in vitro screening tool for identifying novel effects of toxicant exposure. Additionally, we identified APR suppression as a potentially important and environmentally relevant target of PFUdA. These findings suggest in ovo exposure of birds to PFUdA may lead to post-hatch developmental deficiencies, such as impaired inflammatory response.

Transcriptional profiles in the cerebral hemisphere of chicken embryos following in ovo perfluorohexane sulfonate exposure.

In a recent egg injection study, we showed that in ovo exposure to perfluorohexane sulfonate (PFHxS) affects the pipping success of developing chicken (Gallus gallus domesticus) embryos. We also found evidence of thyroid hormone (TH) pathway interference at multiple levels of biological organization (i.e., somatic growth, messenger RNA expression, and circulating free thyroxine levels). Based on these findings, we hypothesize that PFHxS exposure interferes with TH-dependent neurodevelopmental pathways. This study investigates global transcriptional profiles in cerebral hemispheres of chicken embryos following exposure to a solvent control, 890 or 38,000 ng PFHxS/g egg (n = 4-5 per group); doses that lead to the adverse effects indicated above. PFHxS significantly alters the expression (≥ 1.5-fold, p ≤ 0.001) of 11 transcripts at the low dose (890 ng/g) and 101 transcripts at the high dose (38,000 ng/g). Functional enrichment analysis shows that PFHxS affects genes involved in tissue development and morphology, cellular assembly and organization, and cell-to-cell signaling. Pathway and interactome analyses suggest that genes may be affected through several potential regulatory molecules, including integrin receptors, myelocytomatosis viral oncogene, and CCAAT/enhancer-binding protein. This study identifies key functional and regulatory modes of PFHxS action involving TH-dependent and -independent neurodevelopmental pathways. Some of these TH-dependent mechanisms that occur during embryonic development include tight junction formation, signal transduction, and integrin signaling, whereas TH-independent mechanisms include gap junction intercellular communication.