Toxicogenomic Assessment of Complex Chemical Signatures in Double-Crested Cormorant Embryos from Variably Contaminated Great Lakes Sites.

Using omics approaches to monitor complex environmental mixtures is challenging. Previously, we evaluated in vitro transcriptomic effects of complex organic extracts derived from avian eggs. However, there is a lack of studies using wild species that are naturally exposed to contaminant mixtures. Here, we examined polychlorinated biphenyl (PCB) and polybrominated diphenyl ether (PBDE) residues and gene expression in embryonic liver tissue of double-crested cormorants (Phalacrocorax auritus) collected from six variably contaminated colonies. Colonies near industrialized areas were distinguished from less contaminated sites based on their PCB and PBDE concentrations. The most variably expressed genes between sites were involved in pathways including, xenobiotic metabolism (e.g., Cyp1a4), lipid/bile acid homeostasis (e.g., Lbfabp), and oxidative stress (e.g., Mt4). Hierarchical clustering, based on relative gene expression, revealed a grouping pattern similar to chemical residue concentrations. Further, partial least squares regression analysis was used to estimate chemical concentrations from transcriptomics data. PCB 155 and BDE 47 showed the highest slopes (0.77 and 0.69, respectively) fitted by linear regression of measured and estimated chemical concentrations. The application of transcriptomics to a wild avian species, naturally exposed to complex chemical mixtures and other stressors, represents a promising means to distinguish and prioritize variably contaminated sites.

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.

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.

Use of a Novel Double-Crested Cormorant ToxChip PCR Array and the EROD Assay to Determine Effects of Environmental Contaminants in Primary Hepatocytes.

In vitro screening tools and 'omics methods are increasingly being incorporated into toxicity studies to determine mechanistic effects of chemicals and mixtures. To date, the majority of these studies have been conducted with well-characterized laboratory animal models. In the present study, well-established methods developed for chicken embryonic hepatocyte (CEH) studies were extended to a wild avian species, the double-crested cormorant (DCCO; Phalacrocorax auritus), in order to compare the effects of several environmental contaminants on cytotoxicity, ethoxyresorufin O-deethylase (EROD) activity, and mRNA expression. Five organic flame retardants and one plasticizer decreased cormorant hepatocyte viability in a similar manner to that observed in previous studies with CEH. EROD activity was induced in a concentration-dependent manner following exposure to two dioxin-like chemicals and the calculated EC50 values were concordant with domestic avian species from similar species sensitivity categories. Transcriptomic effects were determined using a novel DCCO PCR array, which was designed, constructed and validated in our laboratory based on a commercially available chicken PCR array. The DCCO array has 27 target genes covering a wide range of toxicity pathways. Gene profiles were variable among the 10 chemicals screened; however, good directional concordance was observed with regard to results previously obtained in CEH. Overall, the application of well-established methods (i.e., CEH and chicken PCR array) to the double-crested cormorant demonstrated the portability of the techniques to an indicator species of ecological relevance.

Biochemical and Transcriptomic Effects of Herring Gull Egg Extracts from Variably Contaminated Colonies of the Laurentian Great Lakes in Chicken Hepatocytes.

Determining the effects of complex mixtures of environmental contaminants poses many challenges within the field of ecotoxicology. In this study, graded concentrations of herring gull egg extracts, collected from five Great Lakes breeding colonies with variable burdens of organohalogen contaminants (OHCs), were administered to chicken embryonic hepatocytes to determine effects on 7-ethoxyresorufin-O-deethylase (EROD) activity, porphyrin accumulation, and mRNA expression. EROD activity and porphyrin accumulation permitted the ranking of colonies based on the efficacy of eliciting an aryl hydrocarbon receptor-mediated response. An avian ToxChip polymerase chain reaction (PCR) array provided more exhaustive coverage in terms of potential toxicity pathways being affected, including xenobiotic and lipid metabolism and the thyroid hormone pathway. Herring gull eggs from Channel Shelter Island (CHSH, Lake Huron) and Gull Island (GULL, Lake Michigan) had among the highest OHC burdens, and extracts elicited a biochemical and transcriptomic response greater than that of extracts from the other three, less polluted colonies. For example, EROD EC50 values and porphyrin ECthreshold values were lower for CHSH and GULL extracts than for the other colonies. Extracts from CHSH and GULL altered 15 and 13 of 27 genes on the PCR array compared to no more than eight genes for the less contaminated sites. The combination of a well-established avian in vitro assay, two well-characterized biochemical assays, and the avian ToxChip PCR array permitted the geographical discrimination of variably contaminated herring gull eggs from the Great Lakes. Such high-throughput assays show potential promise as cost-effective tools for determining toxic potencies of complex mixtures in the environment.

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.

Cytochrome P4501A induction in avian hepatocyte cultures exposed to polychlorinated biphenyls: comparisons with AHR1-mediated reporter gene activity and in ovo toxicity.

Avian-specific toxic equivalency factors (TEFs) were developed by the World Health Organization to simplify environmental risk assessments of dioxin-like compounds (DLCs), but TEFs do not account for differences in the toxic and biochemical potencies of DLCs among species of birds. Such variability may be due to differences in species sensitivity to individual DLCs. The sensitivity of avian species to DLCs was recently associated with the identity of amino acids 324 and 380 in the aryl hydrocarbon receptor 1 (AHR1) ligand binding domain. A luciferase reporter gene (LRG) assay, measuring AHR1-mediated induction of a cytochrome P450 1A5 (CYP1A5) reporter gene, in combination with a species' AHR1 ligand binding domain sequence, were also shown to predict avian species sensitivity to polychlorinated biphenyls (PCBs) and PCB relative potency in a given species. The goals of the present study were to (1) characterize the concentration-dependent effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin and PCBs 126, 77, 105 and 118 on induction of ethoxyresorufin O-deethylase (EROD) activity and CYP1A4/5 mRNA in chicken, ring-necked pheasant and Japanese quail embryo hepatocytes and (2) compare these in vitro results to those previously generated by the LRG assay and in ovo toxicity studies. EROD activity and CYP1A4/5 mRNA expression data support and complement the findings of the LRG assay. CYP1A enzyme activity and mRNA expression were significantly correlated both with luciferase activity and in ovo toxicity induced by PCBs. Relative potency values were generally similar between the LRG and EROD assays and indicate that the relative potency of some PCBs may differ among species.

In Vitro Screening of 21 Bisphenol A Replacement Alternatives: Compared with Bisphenol A, the Majority of Alternatives Are More Cytotoxic and Dysregulate More Genes in Avian Hepatocytes.

An avian in vitro screening approach was used to determine the effects of 21 bisphenol A (BPA) alternatives. Cytotoxicity and dysregulation of genes associated with estrogen response and other toxicologically relevant pathways evoked by these alternatives were compared with BPA. Most of the BPA alternatives (15/21) were equally or more cytotoxic than BPA in chicken embryonic hepatocytes; variability in cell viability was associated with chemical structure and the log octanol-water partition coefficient (logP) values. A negative linear relationship (r 2 = 0.745; p = 0.49-07 ; n = 18) was observed between logP and the log median lethal concentration (logLC50) values. The least cytotoxic BPA alternatives elicited the greatest gene dysregulation and, overall, most of the alternatives altered more genes than BPA (measured with a custom polymerase chain reaction array). This overall approach shows promise for use as a screen for hazard-based prioritization of BPA replacement alternatives and to ideally identify those that may be less harmful and/or require additional toxicity testing. Environ Toxicol Chem 2021;40:2026-2033. © 2021 Her Majesty the Queen in Right of Canada Environmental Toxicology and Chemistry © 2021 SETAC. Reproduced with the permission of the Minister of Environment and Climate Change Canada.

Toxicity Screening of Bisphenol A Replacement Compounds: Cytotoxicity and mRNA Expression in Primary Hepatocytes of Chicken and Double-Crested Cormorant.

A market for bisphenol A (BPA) replacement compounds has emerged as a result of restrictions on the use of BPA. Some of these compounds have been detected in the environment; however, little is known about their toxicological properties. In the present study, an avian in vitro toxicogenomic approach was used to compare the effects of 5 BPA alternatives. Cell viability and mRNA expression were compared in primary embryonic hepatocytes of chicken (CEH) and double-crested cormorant (DCEH) exposed to 4,4'-(propane-2,2-diyl) diphenol (BPA), bis (4-hydroxyphenyl) methane (BPF), bis (3-allyl-4-hydroxyphenyl) sulfone (TGSH), 7-bis (4-hydroxyphenylthio)-3,5-dioxaheptane (DD-70), 2,2-bis (4-hydroxyphenyl) hexafluoropropane (BPAF), and 4-hydroxyphenyl 4-isoprooxyphenylsulfone (BPSIP). Changes in gene expression were determined using 2 polymerase chain reaction (PCR) arrays: 1) species-specific ToxChips that contain genes representing toxicologically relevant pathways, and 2) chicken-specific AestroChip that measures estrogen responsive genes. In CEH and DCEH, BPA alternatives TGSH, DD-70, and BPAF were most cytotoxic. Some of the replacement compounds changed the expression of genes related to xenobiotic metabolism, bile acid, and cholesterol regulation. The rank order based on the number of genes altered on the chicken ToxChip array was TGSH > DD-70 > BPAF = BPF > 17ß estradiol (E2) > BPSIP > BPA. On the cormorant ToxChip array, BPSIP altered the greatest number of genes. Based on the chicken AestroChip data, BPSIP and BPF were slightly estrogenic. These results suggest that the replacement compounds have comparable or even greater toxicity than BPA and act via different mechanisms. Environ Toxicol Chem 2021;40:1368-1378. © 2021 Her Majesty the Queen in Right of Canada. Reproduced with the permission of the Minister of Environment and Climate Change Canada.

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.

A rapid method of preparing complex organohalogen extracts from avian eggs: Applications to in vitro toxicogenomics screening.

Double-crested cormorants are piscivorous birds that breed in variably contaminated colonies across the Laurentian Great Lakes of North America. Collection and preparation of environmentally relevant extracts from eggs that contain variable concentrations of organohalogen contaminants represents a minimally invasive approach to characterize potential effects of exposure using in vitro bioassays. In the present study, a rapid, efficient lipid freeze-filtration extraction method was used to prepare extracts from double-crested cormorant eggs collected from 5 breeding colonies that had variable organohalogen contaminant burdens. Extracts, solubilized in dimethyl sulfoxide, were administered to chicken embryonic hepatocytes (CEHs) to determine effects on cell viability, 7-ethoxyresorufin-O-deethylase (EROD) activity, and messenger RNA expression using a chicken ToxChip polymerase chain reaction (PCR) array. The EROD median effect concentration (EC50) values were lower for extracts with greater organohalogen contaminant burdens and thus permitted an initial ranking of colonies based on the efficacy of eliciting an aryl hydrocarbon receptor-mediated response. The ToxChip PCR array data provided a more exhaustive, pathway-based evaluation of extract effects; variability in the transcriptomic profiles was associated with organohalogen contaminant burdens. For example, extracts from Mud Island (Detroit River, MI, USA) had among the highest organohalogen contaminant burdens and elicited a greater biochemical (EROD EC50 = 0.005) and transcriptomic response (22/43 genes altered on the array) in CEHs compared with the least contaminated site, which was Mandarte Island (BC, Canada; EROD EC50 = 0.172; 8/43 genes altered). Avian eggs represent a useful biomonitoring tool for determining complex mixture effects, and the combination of a rapid extraction method, an in vitro bioassay, and targeted endpoint evaluation (biochemical and transcriptomic) shows great promise as an environmental effects monitoring approach. Environ Toxicol Chem 2019;38:811-819. © 2019 Crown in the right of Canada. Published by Wiley Periodicals Inc. on behalf of SETAC.

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.

Fluorescent RNA arbitrarily primed polymerase chain reaction. A new differential display approach to detect contaminant-induced alterations of gene expression in wildlife species.

Differential display polymerase chain reaction (PCR) can facilitate the identification of novel molecular end points related to contaminant exposure in a wide range of species. To date, various differential display methodologies have been described in detail. Herein, we describe a modification of the RNA arbitrarily primed PCR (RAP-PCR) method that involves the fluorescent labeling of cDNA transcripts via 5' rhodamine-labeled 18-mer arbitrary primers. These arbitrary primers typically bind to the coding regions of cDNA, which simplifies the downstream identification of contaminant-responsive genes. The technique has been aptly named fluorescent RNA arbitrarily primed PCR, FRAP-PCR, and has been successfully utilized with several avian species and RNA sources (e.g., cultured cells, tissue). This straightforward, safe, and cost-effective approach represents a useful alternative to the radiometric-based RAP-PCR method.

Detection of PBDE effects on mRNA expression in chicken (Gallus domesticus) neuronal cells using real-time RT-PCR and a new differential display method.

Polybrominated diphenyl ethers (PBDEs) are used as flame retardants in a wide range of consumer products. Previous studies have suggested that PBDEs can disrupt thyroid hormone homeostasis and the developing central nervous system in rodents, but few studies have determined whether PBDEs cause similar effects in birds. An in vitro method was used to determine effects of a commercial PBDE flame retardant (DE-71) on mRNA expression in primary chicken neuronal cells derived from the cerebral hemisphere. Real-time RT-PCR assays were developed to quantify changes in mRNA abundance of genes associated with the thyroid hormone pathway; thyroid hormone receptors (TRalpha and TRbeta) and transthyretin (TTR). We also used a new differential display PCR methodology, fluorescent RNA arbitrarily primed PCR (FRAP-PCR), to determine additional effects of DE-71 on mRNA expression. Neither of the TRs responded to DE-71 exposure, but TTR mRNA decreased approximately 2-fold following exposure to 0.1, 1 and 3 microM DE-71. Candidate transcripts associated with signal transduction, neurosteroidogenesis, and neurite and axonal growth were up-regulated by DE-71 exposure. Taken together, the findings from this study indicate that this in vitro cell culture method can be used to characterize the effects of PBDEs in the avian brain.

Bis-(3-allyl-4-hydroxyphenyl) sulfone decreases embryonic viability and alters hepatic mRNA expression at two distinct developmental stages in chicken embryos exposed via egg injection.

Concerns surrounding the toxicological effects and environmental prevalence of bisphenol A (BPA) have increased efforts to identify suitable safer replacement alternatives. Bis-(3-allyl-4-hydroxyphenyl) sulfone (TGSH) represents a potential BPA alternative; however, exposure and ecotoxicological data are scarce. To determine effects on embryonic viability, development, and hepatic mRNA expression at 2 distinct developmental periods (midincubation [day 11] and pipping [days 20-21]), TGSH was injected into the air cell of unincubated, fertilized chicken embryos at 4 concentrations ranging from 0 to 180 µg/g egg. Concentrations of TGSH increased in a dose-dependent manner in whole-embryo homogenates, and the estimated median lethal dose (LD50) based on embryonic viability at midincubation was 66 µg/g (95% confidence interval = 31-142 µg/g), which is similar to the BPA LD50 (∼ 67 µg/g) reported in a previous study. Modulation of hepatic gene targets from a chicken ToxChip polymerase chain reaction (PCR) array was observed at both developmental stages. At midincubation, 21/43 genes on the PCR array were significantly altered (by >1.5-fold) in the 180 µg/g dose group, whereas 9 and 6/43 were altered at pipping in the 9.2 and 48 µg/g groups, respectively. Predominant toxicity pathways included xenobiotic metabolism, lipid homeostasis, bile acid synthesis, and cell cycle regulation. The estrogen-responsive gene apolipoprotein II was significantly up-regulated in liver tissue of midincubation embryos at 180 µg/g; however, neither apolipoprotein II nor vitellogenin II were altered at the other concentrations or developmental time points. Given the importance of identifying suitable BPA replacement alternatives, the present study provides novel, whole-animal toxicological data for a BPA replacement alternative that has an effect on embryonic viability similar to that of the compound it could replace. Environ Toxicol Chem 2018;37:530-537. © 2017 SETAC.

Prioritization of 10 organic flame retardants using an avian hepatocyte toxicogenomic assay.

As the number of chemicals developed and used by industry increases, the inherent limitations of traditional toxicology approaches become an unavoidable issue. To help meet the demand for toxicity evaluation, new methods, such as high-throughput toxicity screening, are currently being developed to permit rapid determination of toxic, molecular, and/or biochemical effects of a wide range of chemicals. In the present study, we demonstrate the utility of an avian in vitro toxicogenomics screening approach to determine the cytotoxic and transcriptomic effects of 10 organic flame retardants (OFRs) currently of international priority for ecological risk evaluation to prioritize and inform future toxicological studies. Hepatocytes from 2 avian species, chicken and double-crested cormorant, were prepared and exposed for 24 h to various concentrations (0-300 µM) of the following 10 OFRs: Chemical Abstracts Service registration numbers 29761-21-5, 56803-37-3 (p-tert-butylphenyl diphenyl phosphate [BPDP]), 65652-41-7, 68937-41-7 (phenol, isopropylated, phosphate [3:1] [IPPP]), 95906-11-9, 19186-97-1, 26040-51-7, 35948-25-5, 21850-44-2, and 25713-60-4. Cell viability, the 7-ethoxyresorufin-O-deethylase assay, and transcriptomic analysis using species-specific ToxChip polymerase chain reaction arrays were performed to evaluate the in vitro effect of these OFRs. Of the 10 OFRs assessed, BPDP and IPPP elicited the strongest cytotoxic and transcriptomic responses in both chicken and double-crested cormorant hepatocytes and are therefore recommended as priority candidates for further wildlife toxicological investigations. Environ Toxicol Chem 2018;37:3134-3144. © 2018 Crown in the right of Canada. Published by Wiley Periodicals Inc. on behalf of SETAC.

Gene expression profiling in the neuroendocrine brain of male goldfish (Carassius auratus) exposed to 17alpha-ethinylestradiol.

17-alpha ethinylestradiol (EE2), a pharmaceutical estrogen, is detectable in water systems worldwide. Although studies report on the effects of xenoestrogens in tissues such as liver and gonad, few studies to date have investigated the effects of EE2 in the vertebrate brain at a large scale. The purpose of this study was to develop a goldfish brain-enriched cDNA array and use this in conjunction with a mixed tissue carp microarray to study the genomic response to EE2 in the brain. Gonad-intact male goldfish were exposed to nominal concentrations of 0.1 nM (29.6 ng/l) and 1.0 nM (296 ng/l) EE2 for 15 days. Male goldfish treated with the higher dose of EE2 had significantly smaller gonads compared with controls. Males also had a significantly reduced level of circulating testosterone (T) and 17beta-estradiol (E2) in both treatment groups. Candidate genes identified by microarray analysis fall into functional categories that include neuropeptides, cell metabolism, and transcription/translation factors. Differentially expressed genes verified by real-time RT-PCR included brain aromatase, secretogranin-III, and interferon-related developmental regulator 1. Our results suggest that the expression of genes in the sexually mature adult brain appears to be resistant to low EE2 exposure but is affected significantly at higher doses of EE2. This study demonstrates that microarray technology is a useful tool to study the effects of endocrine disrupting chemicals on neuroendocrine function and suggest that exposure to EE2 may have significant effects on localized E2 synthesis in the brain by affecting transcription of brain aromatase.

Bisphenol S alters embryonic viability, development, gallbladder size, and messenger RNA expression in chicken embryos exposed via egg injection.

Amid concerns about the toxicological effects and environmental prevalence of bisphenol A (BPA), efforts to find suitable, safer replacement alternatives are essential. Bisphenol S (BPS) is a potential chemical substitute for BPA; however, few studies are available confirming that it has a more desirable ecotoxicological profile. In the present study, BPS was injected into the air cell of unincubated, fertilized chicken embryos at 6 concentrations ranging from 0 µg/g to 207 µg/g egg to determine effects on pipping success, development, hepatic messenger ribonucleic acid (mRNA) expression, thyroid hormone levels, and circulating bile acid concentrations. Concentrations of BPS increased in a dose-dependent manner in whole-embryo homogenates, and exposure to the highest dose, 207 µg/g, resulted in decreased pipping success (estimated median lethal dose = 279 µg/g; 95% confidence interval = 161-486 µg/g). Exposure to BPS also reduced growth metrics including embryo mass and tarsus length, whereas the most pronounced phenotypic effect was the concentration-dependent, significant increase in gallbladder size at concentrations ≥52.8 µg/g. These adverse phenotypic outcomes were associated with the modulation of gene targets from a chicken ToxChip polymerase chain reaction array, which are involved with xenobiotic metabolism, lipid homeostasis, bile acid synthesis, and the thyroid hormone pathway. Expression levels of 2 estrogen-responsive genes, apolipoprotein II and vitellogenin, were too low at the sampling time point assessed (i.e., pipping embryos) to quantify changes, and no effects were observed on circulating free thyroxine or bile acid concentrations. The present study provides novel, whole-animal toxicological data for a BPA replacement alternative that is not well characterized. Environ Toxicol Chem 2016;35:1541-1549. © 2015 SETAC.

Octylphenol (OP) alters the expression of members of the amyloid protein family in the hypothalamus of the snapping turtle, Chelydra serpentina serpentina.

The gonadal estrogen estradiol-17beta (E(2)) is important for developing and regulating hypothalamic function and many aspects of reproduction in vertebrates. Pollutants such as octylphenol (OP) that mimic the actions of estrogens are therefore candidate endocrine-disrupting chemicals. We used a differential display strategy (RNA-arbitrarily primed polymerase chain reaction) to isolate partial cDNA sequences of neurotransmitter, developmental, and disease-related genes that may be regulated by OP or E(2) in the snapping turtle Chelydra serpentina serpentina hypothalamus. Hatchling and year-old male snapping turtles were exposed to a 10 ng/mL nominal concentration of waterborne OP or E(2) for 17 days. One transcript [421 base pairs (bp)] regulated by OP and E(2) was 93% identical to human APLP-2. APLP-2 and the amyloid precursor protein (APP) regulate neuronal differentiation and are also implicated in the genesis of Alzheimer disease in humans. Northern blot analysis determined that the turtle hypothalamus contains a single APLP-2 transcript of 3.75 kb in length. Exposure to OP upregulated hypothalamic APLP-2 mRNA levels 2-fold (p < 0.05) in month-old and yearling turtles. E(2) did not affect APLP-2 mRNA levels in hatchlings but stimulated a 2-fold increase (p < 0.05) in APLP-2 mRNA levels in yearling males. The protein beta-amyloid, a selectively processed peptide derived from APP, is also involved in neuronal differentiation, and accumulation of this neurotoxic peptide causes neuronal degeneration in the brains of patients with Alzheimer disease. Therefore, we also sought to determine the effects of estrogens on the expression of beta-amyloid. Using homology cloning based on known sequences, we isolated a cDNA fragment (474 bp) from turtle brain with 88% identity to human APP. Northern blot analysis determined that a single 3.5-kb transcript was expressed in the turtle hypothalamus. Waterborne OP also increased the expression of hypothalamic APP after 35 days of exposure. Our results indicate that low levels of OP are bioactive and can alter the expression of APLP-2 and APP. Because members of the APP gene family are involved in neuronal development, we hypothesize that OP exposure may disrupt hypothalamic development in young turtles.

Use of an avian hepatocyte assay and the avian Toxchip Polymerse chain reaction array for testing prioritization of 16 organic flame retardants.

Risk assessors are challenged with the task of providing data for an increasing number of priority chemicals. High-throughput toxicity screening methods--which permit rapid determination of toxic, molecular, and/or biochemical effects of a wide range of chemicals--are essential to help meet this demand. The avian embryonic hepatocyte in vitro screening method has been utilized in the authors' laboratory to assess the effects of a wide range of environmental contaminants on cytotoxicity and mRNA expression of genes associated with xenobiotic metabolism, the thyroid hormone pathway, lipid metabolism, and growth. Sixteen structurally variable organic flame retardants (OFRs)--including tetrabromoethylcyclohexane (TBECH), tris(2-butoxyethyl) phosphate (TBEP), tricresyl phosphate (TCP), and tris(1,3-dichloro-2-propyl) phosphate (TDCPP)--were screened using the in vitro method in the present study. Hepatocytes from 2 avian species, chicken and herring gull, were prepared, and species differences in hepatocyte viability were observed for several OFRs. For example, TCP was not cytotoxic in chicken hepatocytes up to the highest concentration tested (300 µM), whereas the median lethal concentration (LC50) was 31.2 µM in herring gull hepatocytes. Effects on mRNA expression in chicken embryonic hepatocytes were determined using a 3 × 32 custom-made Avian ToxChip polymerse chain reaction array and were variable among OFRs; TCP, TDCPP, and tris(2,3-dibromopropyl) isocyanurate showed the most significant alterations among the target genes assessed. Overall, this rapid screening method helped prioritize OFRs for further assessment. For example, OFRs that elicited significant effects on cytoxicity or mRNA expression represent prime candidates for egg injection studies that determine adverse effects on the whole animal but are more costly in terms of time, money, and embryo utilization.