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Introduction: The COVID-19 pandemic focused attention on healthcare disparities and inequities faced by individuals within marginalized and structurally disadvantaged groups in the United States. These individuals bore the heaviest burden across this pandemic as they faced increased risk of infection and difficulty in accessing testing and medical care. Individuals experiencing housing insecurity are a particularly vulnerable population given the additional barriers they face. In this scoping review, we identify some of the barriers this high-risk group experienced during the early days of the pandemic and assess novel solutions to overcome these barriers., Methods: A scoping review was performed following PRISMA-Sc guidelines looking for studies focusing on COVID-19 testing among individuals experiencing housing insecurity. Barriers as well as solutions to barriers were identified as applicable and summarized using qualitative methods, highlighting particular ways that proved effective in facilitating access to testing access and delivery., Results: Ultimately, 42 studies were included in the scoping review, with 143 barriers grouped into four categories: lack of cultural understanding, systemic racism, and stigma; medical care cost, insurance, and logistics; immigration policies, language, and fear of deportation; and other. Out of these 42 studies, 30 of these studies also suggested solutions to address them., Conclusion: A paucity of studies have analyzed COVID-19 testing barriers among those experiencing housing insecurity, and this is even more pronounced in terms of solutions to address those barriers. Expanding resources and supporting investigators within this space is necessary to ensure equitable healthcare delivery., Competing Interests: CP was employed by the Healthspring Global Inc. CP reports receiving consulting fees from Abbott Molecular and Rapid Diagnostics. ET has been a consultant for Biomeme, Inc., has a patent pending for Methods to Diagnose and Treat Acute Respiratory Infections (US20180245154A1), and is currently employed by Danaher Corp. MC-W reports receiving support for research from the NIH (1U24-MD016258), National Institute of Allergy and Infectious Diseases (HHSN272201500006I, 1K24-AI143971), U.S. Food and Drug Administration (5U18-FD006298), and industry for drug development in adults and children. CW reports grants or contracts from DARPA, NIH-ARLG/NIAID/VTEU/NIMHO/NIGMS, Sanofi, Najit, CDC, Patient-Centered Outcomes Research Institute, USAMRAA, DOD, Abbott, and Pfizer; consulting fees from Arena Pharmaceuticals, BioFire, FHI Clinical, Giner, Karius, and SeLux Diagnostics; support for attending meetings and/or travel from American Society for Microbiology; patents planned, issued or pending for: biomarkers for the molecular classification of bacterial infection, methods to diagnose and treat acute respiratory infections, gene expression signatures useful to predict or diagnose sepsis and methods of using the same, host based molecular signatures of human infection with SARS-CoV-2 (COVID19), methods of identifying infectious disease and assays for identifying infectious disease, and nasopharyngeal protein biomarkers of acute respiratory virus infection and methods of using same; participation on a Data Safety Monitoring Board or Advisory Board for IDbyDNA, Janssen, Regeneron, Roche Molecular Sciences; leadership or fiduciary role in other board, society, committee or advocacy group, paid or unpaid for American Society for Microbiology and American Society of Tropical Medicine and Hygiene; and other financial or non-financial interests with Biomeme and Predigen, Inc. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Johannesson, Glover, Petti, Veldman, Tsalik, Taylor, Hendren, Neighbors, Tillekeratne, Kennedy, Harper, Kibbe, Corbie, Cohen-Wolkowiez, Woods and Lee.)

Research on the COVID-19 pandemic revealed a disproportionate burden of COVID-19 infection and death among underserved populations and exposed low rates of SARS-CoV-2 testing in these communities. A landmark National Institutes of Health (NIH) funding initiative, the Rapid Acceleration of Diagnostics-Underserved Populations (RADx-UP) program, was developed to address the research gap in understanding the adoption of COVID-19 testing in underserved populations. This program is the single largest investment in health disparities and community-engaged research in the history of the NIH. The RADx-UP Testing Core (TC) provides community-based investigators with essential scientific expertise and guidance on COVID-19 diagnostics. This commentary describes the first 2 years of the TC's experience, highlighting the challenges faced and insights gained to safely and effectively deploy large-scale diagnostics for community-initiated research in underserved populations during a pandemic. The success of RADx-UP shows that community-based research to increase access and uptake of testing among underserved populations can be accomplished during a pandemic with tools, resources, and multidisciplinary expertise provided by a centralized testing-specific coordinating center. We developed adaptive tools to support individual testing strategies and frameworks for these diverse studies and ensured continuous monitoring of testing strategies and use of study data. In a rapidly evolving setting of tremendous uncertainty, the TC provided essential and real-time technical expertise to support safe, effective, and adaptive testing. The lessons learned go beyond this pandemic and can serve as a framework for rapid deployment of testing in response to future crises, especially when populations are affected inequitably., Competing Interests: The authors declare a conflict of interest. Ephraim Tsalik has been a consultant for Biomeme, Inc.; has a patent pending for Methods to Diagnose and Treat Acute Respiratory Infections (US20180245154A1); and is currently employed by Danaher Corp. Michael Cohen-Wolkowiez receives support for research from the NIH [1U24-MD016258]; National Institute of Allergy and Infectious Diseases [HHSN272201500006I, 1K24-AI143971]; U.S. Food and Drug Administration [5U18-FD006298]; and industry for drug development in adults and children. Cathy A Petti receives consulting fees from Abbott Molecular and Rapid Diagnostics. Christopher Woods reports consulting fees from Arena Pharmaceuticals, BioFire, FHI Clinical, Giner, Karius, and SeLux Diagnostics. All other authors report no conflicts of interests.

Exposure to dioxin-like compounds is consistently associated with concentration-dependent induction of cytochrome P4501A (CYP1A) enzymes in primary cultures of avian hepatocytes. We have previously demonstrated that the median effective concentration (EC50) for induction of this response is predictive of in vivo sensitivity to dioxin-like compounds in birds. We investigated sources of interindividual variation in the CYP1A response to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in wild herring gulls and considered how this variation may complicate dioxin sensitivity estimates based on the CYP1A bioassay. Concentration-dependent effects of TCDD on CYP1A mRNA expression were characterized in 55 hepatocyte cultures prepared from individual herring gull embryos. A large degree of variability was observed among the hepatocyte culture preparations. For example, 1) basal CYP1A4 and CYP1A5 mRNA expression varied by 20- and 126-fold, respectively, among individuals, and 2) exposure to TCDD induced CYP1A4 mRNA expression by 57-fold in the most responsive sample but did not significantly induce CYP1A4 mRNA expression above baseline values in 42% of hepatocyte culture preparations. Environmental and genetic factors contributing to the observed variability are discussed. Despite the large amount of interindividual variation, we conclude that reproducible EC50-based estimates of species sensitivity can be obtained from the CYP1A cell culture bioassay when samples are collected from relatively uncontaminated colonies. Environ Toxicol Chem 2019;38:660-670. © 2019 SETAC., (© 2019 SETAC.)

6-Formylindolo[3,2-b]carbazole (FICZ) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are ligands of the aryl hydrocarbon receptor (AHR) and bind to the AHR with high affinity. Until recently, TCDD was considered to be the most potent AHR agonist, but several recent studies indicate that FICZ binds with greater affinity to the AHR than TCDD. To advance our understanding of the similarities and differences of the effects of FICZ and TCDD exposure in chicken embryo hepatocyte (CEH) cultures, we compared relative expression changes of 27 dioxin-responsive genes by the use of a chicken PCR array, porphyrin accumulation and ethoxyresorufin-O-deethylase (EROD) activity at different time points. In addition, an egg injection study was performed to assess the effects of FICZ on the developing chicken embryo. The results of the current study showed: (1) mean EROD-derived relative potency values for FICZ compared to TCDD changed as a function of time (i.e. 9, 0.004, 0.0008 and 0.00008 at 3, 8, 24, and 48h, respectively) in CEH cultures; (2) FICZ exposure did not result in porphyrin accumulation in CEH cultures; (3) concordance between gene expression profiles for FICZ and TCDD was time- and concentration-dependent, and (4) no mortality or morphological abnormalities were observed in chicken embryos injected with 0.87ng FICZ/g egg into the air cell. The results presented herein suggest that while FICZ and TCDD share similar molecular targets, transient versus sustained AHR activation by FICZ and TCDD result in differential transcriptomic responses. Moreover, rapid metabolism of FICZ in hepatocytes resulted in a significant decrease in the induction of EROD activity., (Crown Copyright © 2016. Published by Elsevier Inc. All rights reserved.)

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.

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.

Systematic consideration of scientific support is a critical element in developing and, ultimately, using adverse outcome pathways (AOPs) for various regulatory applications. Though weight of evidence (WoE) analysis has been proposed as a basis for assessment of the maturity and level of confidence in an AOP, methodologies and tools are still being formalized. The Organization for Economic Co-operation and Development (OECD) Users' Handbook Supplement to the Guidance Document for Developing and Assessing AOPs (OECD 2014a; hereafter referred to as the OECD AOP Handbook) provides tailored Bradford-Hill (BH) considerations for systematic assessment of confidence in a given AOP. These considerations include (1) biological plausibility and (2) empirical support (dose-response, temporality, and incidence) for Key Event Relationships (KERs), and (3) essentiality of key events (KEs). Here, we test the application of these tailored BH considerations and the guidance outlined in the OECD AOP Handbook using a number of case examples to increase experience in more transparently documenting rationales for assigned levels of confidence to KEs and KERs, and to promote consistency in evaluation within and across AOPs. The major lessons learned from experience are documented, and taken together with the case examples, should contribute to better common understanding of the nature and form of documentation required to increase confidence in the application of AOPs for specific uses. Based on the tailored BH considerations and defining questions, a prototype quantitative model for assessing the WoE of an AOP using tools of multi-criteria decision analysis (MCDA) is described. The applicability of the approach is also demonstrated using the case example aromatase inhibition leading to reproductive dysfunction in fish. Following the acquisition of additional experience in the development and assessment of AOPs, further refinement of parameterization of the model through expert elicitation is recommended. Overall, the application of quantitative WoE approaches hold promise to enhance the rigor, transparency and reproducibility for AOP WoE determinations and may play an important role in delineating areas where research would have the greatest impact on improving the overall confidence in the AOP., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Dioxin-like compounds (DLCs) are pollutants of global environmental concern. DLCs elicit their adverse outcomes through activation of the aryl hydrocarbon receptor (AhR). However, there is limited understanding of the mechanisms that result in differences in sensitivity to DLCs among different species of fishes. Understanding these mechanisms is critical for protection of the diversity of fishes exposed to DLCs, including endangered species. This study investigated specific mechanisms that drive responses of two endangered fishes, white sturgeon (Acipenser transmontanus) and lake sturgeon (Acipenser fulvescens) to DLCs. It determined whether differences in sensitivity to activation of AhRs (AhR1 and AhR2) can be predicted based on identities of key amino acids in the ligand binding domain (LBD). White sturgeon were 3- to 30-fold more sensitive than lake sturgeon to exposure to 5 different DLCs based on activation of AhR2. There were no differences in sensitivity between white sturgeon and lake sturgeon based on activation of AhR1. Adverse outcomes as a result of exposure to DLCs have been shown to be mediated through activation of AhR2, but not AhR1, in all fishes studied to date. This indicates that white sturgeon are likely to have greater sensitivity in vivo relative to lake sturgeon. Homology modeling and in silico mutagenesis suggests that differences in sensitivity to activation of AhR2 result from differences in key amino acids at position 388 in the LBD of AhR2 of white sturgeon (Ala-388) and lake sturgeon (Thr-388). This indicates that identities of key amino acids in the LBD of AhR2 could be predictive of both in vitro activation by DLCs and in vivo sensitivity to DLCs in these, and potentially other, fishes.

The potency of tetrachlorodibenzo-p-dioxin (TCDD) and 18 polycyclic aromatic hydrocarbons (PAHs) for induction of ethoxyresorufin-O-deethylase (EROD) activity was assessed in primary hepatocyte cultures prepared from chicken (Gallus domesticus), Pekin duck (Anas platyrhynchos domesticus), and greater scaup (Aythya marila). TCDD and 8 of the PAHs induced EROD activity in a concentration-dependent manner. Seven of these were previously shown to be acutely toxic to avian embryos, while the 10 congeners that did not produce an EROD response caused limited mortality. The rank order potency of the EROD-active congeners in all three species was as follows: TCDD>dibenz[ah]anthracene>benzo[k]fluoranthene>indeno[1,2,3-cd]pyrene>benzo[a]pyrene>chrysene≈benz[a]anthracene≈benz[ghi]perylene>benzo[b]naphtho[2,3-d]thiophene. Chicken hepatoctyes were more sensitive than duck hepatocytes to EROD induction by all test compounds, but the gap in species sensitivity was 100-fold for TCDD, and generally ≤10-fold for PAHs. This study is the first to use in vitro methods to rank the AHR-mediated potency of PAHs in birds. These data may be useful for assessing risks associated with exposure to PAHs in the environment.

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., (© 2014 SETAC.)

Dioxins and dioxin-like chemicals (DLCs) cause a suite of adverse effects in terrestrial species. Most of the adverse effects occur subsequent to binding to the aryl hydrocarbon receptor. Avian species vary in their sensitivity to the effects of DLCs and current research indicates that this is mediated by variations in the amino acid sequence within the ligand binding domain (LBD) of the aryl hydrocarbon receptor 1 (AHR1). Eighty-eight avian species have been classified into three broad categories of sensitivity, based on the amino acid variations within the AHR1 LBD: sensitive type 1 (Ile324_Ser380), moderately sensitive type 2 (Ile324_Ala380), and relatively insensitive type 3 (Val324_Ala380). Risk assessment of avian species can be complicated due to the variability in sensitivity among species. A predictive tool for selecting the priority species at a given site would have broad implications for the risk assessment community. We present a method for AHR1 genotyping using plucked feathers as a source of RNA. The method is extremely robust, requires minimal sample processing and handling, and eliminates the need for blood sampling or tissue collection from the species of interest. Using this method we were able to determine the amino acid sequence of the AHR LBD of three avian species: the chicken, the herring gull, and the zebra finch, and to categorize them based on the identity of amino acids at key sites within the LBD.

The flame retardant, tris(1,3-dichloro-2-propyl) phosphate (TDCPP), was previously shown to affect chicken embryo growth, gallbladder size, and lipid homeostasis. A microarray study, however, revealed only modest transcriptional alterations in liver tissue of pipping embryos (days 20-21), which was attributed to the rapid metabolism of TDCPP throughout incubation. To identify the most appropriate sampling time for rapidly metabolized compounds, the present study assessed the time-dependent effects of TDCPP on 27 genes, in ovo (50 µg [116 nmol] TDCPP/g egg) and in vitro (10 µM), using a chicken ToxChip polymerase chain reaction array. The greatest magnitude in dysregulation (up to 362-fold) occurred on day 8 of incubation (in ovo) with alterations of genes involved in phase I, II, and III metabolism, among others. Gallbladder hypotrophy was observed by embryonic day 12, corroborating the finding in pipping embryos from our previous study. From days 12 to 19, genes involved in lipid homeostasis, steroid hormone metabolism, and oxidative stress were affected. In chicken embryonic hepatoctyes (CEHs), TDCPP was completely metabolized to bis(1,3-dichloro-2-propyl) phosphate (BDCPP) within 36 h, but transcriptional changes remained significant up to 36 h. These changes were not attributed to BDCPP exposure as it only altered 1 gene (CYP1A4). An 18-h exposure in CEHs altered the greatest number of genes, making it an appropriate time point for high-throughput chemical screening; however, depending on the biological pathways of interest, shorter or longer incubation times may be more informative. Overall, TDCPP elicits the transcriptional and phenotypic alterations observed in vitro and in ovo, whereas its major metabolite, BDCPP, is far less biologically active., (© 2014 SETAC.)

Research has demonstrated that the sensitivity of avian species to the embyrotoxic effects of dioxin-like compounds can be predicted by the amino acid identities at two key sites within the ligand-binding domain of the aryl hydrocarbon receptor 1 (AhR1). The domestic chicken (Gallus gallus domesticus) has been established as a highly sensitive species to the toxic effects of dioxin-like compounds. Results from genotyping and in vitro assays predict that the European starling (Sturnus vulgaris) is also highly sensitive to dioxin-like compound toxicity. The objective of the present study was to test that prediction in vivo. To do this, we used egg injections in field nesting starlings with 3,3',4,4',5-pentachlorobiphenyl (PCB-126), a dioxin-like polychlorinated biphenyl. Eggs were dosed with either the vehicle control or 1 of 5 doses (1.4, 7.1, 15.9, 32.1, and 52.9 ng PCB-126/g egg). A dose-dependent increase in embryo mortality occurred, and the median lethal dose (LD50; 95% confidence interval [CI]) was 5.61 (2.33-9.08) ng/g. Hepatic CYP1A4/5 messenger RNA (mRNA) expression in hatchlings also increased in a dose-dependent manner, with CYP1A4 being more induced than CYP1A5. No effect of dose on morphological measures was seen, and we did not observe any overt malformations. These results indicate that, other than the chicken, the European starling is the most sensitive species to the effects of PCB-126 on avian embryo mortality reported to date, which supports the prediction of relative sensitivity to dioxin-like compounds based on amino acid sequence of the AhR1., (© 2014 SETAC.)

The organophosphate flame retardant, triphenyl phosphate (TPHP), has been detected with increasing frequency in environmental samples and its primary metabolite is considered to be diphenyl phosphate (DPHP). Information on the adverse effects of these compounds in avian species is limited. Here, we investigate the effects of TPHP and DPHP on cytotoxicity and mRNA expression, as well as in vitro metabolism of TPHP, by use of a chicken embryonic hepatocyte (CEH) screening assay. After 36 h of exposure, CEH cytotoxicity was observed following exposure to >10 μM TPHP (LC50 = 47 ± 8 μM), whereas no significant cytotoxic effects were observed for DPHP concentrations up to 1000 μM. Using a custom chicken ToxChip polymerase chain reaction (PCR) array, the number of genes altered by 10 μM DPHP (9 out of 27) was greater than that by 10 μM TPHP (4 out of 27). Importantly, 4 of 6 genes associated with lipid/cholesterol metabolism were significantly dysregulated by DPHP, suggesting a potential pathway of importance for DPHP toxicity. Rapid degradation of TPHP was observed in CEH exposed to 10 μM, but the resulting concentration of DPHP accounted for only 17% of the initial TPHP dosing concentration. Monohydroxylated-TPHP (OH-TPHP) and two (OH)2-TPHP isomers were identified in TPHP-exposed CEH, and concentrations of these metabolites increased over 0 to 36 h. Overall, this is the first reported evidence that across 27 toxicologically relevant genes, DPHP altered more transcripts than its precursor, and that TPHP is also metabolized via a hydroxylation pathway in CEH.

Tetradecabromo-1,4-diphenoxybenzene (TeDB-DiPhOBz) and 2,2',3,3',4,4',5,5',6,6'-decabromodiphenyl ether (BDE-209) are photolytically unstable flame retarding chemicals. Here, photocatalyzed byproducts of TeDB-DiPhOBz and BDE-209 (i.e Br(8)- to Br(11)-PB-DiPhOBz congeners from TeDB-DiPhOBz, and Br(6)- to Br(8)-BDE congeners from BDE-209), formed after 21 days of natural sunlight irradiation (SI), were assessed for exposure effects on cytotoxicity and mRNA expression levels of selected genes in chicken embryonic hepatocytes (CEH). CEHs were exposed for 36 h to concentrations of SI- and nonirradiated (NI)-TeDB-DiPhOBz and BDE-209. Cytotoxic effects were observed only in CEH exposed to 50 μM SI-BDE-209. Results from a custom-designed Avian ToxChip polymerase chain reaction array showed that NI-TeDB-DiPhOBz and NI-BDE-209, up to maximum concentrations of 1.9 and 9 μM, respectively, caused limited changes in mRNA levels of 27 genes from toxicologically relevant pathways, including phase I/II metabolism, the thyroid hormone pathway, lipid/cholesterol metabolism, oxidative stress, immune response, and cell death. In contrast, 12 and 14 of the 27 genes were altered after exposure to 25 μM SI-TeDB-DiPhOBz or 10 μM SI-BDE-209, respectively. Aryl hydrocarbon receptor (AhR)-related CYP1A4 mRNA levels were the most altered on the PCR array with an induction of 560- and 5200-fold after exposure to 1 or 25 μM SI-TeDB-DiPhOBz, respectively, and 2500- and 2300-fold after exposure to 1 or 10 μM SI-BDE-209, respectively. A dioxin-responsive luciferase reporter gene assay confirmed that the CYP1A4 inductions were independent of the dissolution solvents used (tetrahydrofuran/n-hexane, n-hexane, or methanol) during photolysis. Overall, degradation of TeDB-DiPhOBz and BDE-209 by natural sunlight generates byproducts that affect in vitro expression of genes, especially the AhR-mediated CYP1A4.

Avian species differ in sensitivity to the toxic effects of dioxin-like compounds (DLCs) and recent reports have provided insight into the molecular mechanisms underlying this variability. The sensitivity of avian species to DLCs is associated with the identity of amino acids at positions 324 and 380 within the ligand-binding domain (LBD) of the aryl hydrocarbon receptor 1 (AHR1). 6-formylindolo [3,2-b] carbazole (FICZ), a naturally produced photo-oxidation product of tryptophan, is a highly potent AHR ligand. Few studies have attempted to determine if there are species differences in AHR activation by FICZ in a systematic manner. Here we describe results from an in vitro assay that measures AHR1-mediated luciferase reporter gene activity to determine concentration-dependent effects of FICZ and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in COS-7 cells transfected with AHR1 constructs from chicken (Gallus gallus domesticus), ring-necked pheasant (Phasianus colchicus), Japanese quail (Coturnix japonica) and common tern (Sterna hirundo), and three mutant AHR1 constructs. Data were used to (a) compare the potency of FICZ and TCDD for each AHR1 construct (relative potency; ReP) and (b) the sensitivity of each construct to AHR1 activation by FICZ and TCDD (relative sensitivity; ReS). The results show that (1) FICZ was considerably more potent than TCDD in cells transfected with chicken AHR1 (RePavg=41), ring-necked pheasant AHR1 (RePavg=93), Japanese quail AHR1 (RePavg=1392) and common tern AHR1 (RePavg=1534), (2) there were no significant differences in sensitivity to FICZ in cells expressing chicken, pheasant, quail and tern AHR1, but there were significant differences in sensitivity to TCDD, (3) alteration of amino acids at positions 324 and 380 had no effect on avian AHR1 activity in response to FICZ, (4) there was no time-dependent change in the relative potency of FICZ in COS-7 cells, and (5) neither FICZ nor TCDD induced ethoxyresorufin O-deethylase (EROD activity) in COS-7 cells. Our results suggest that FICZ and TCDD activate avian AHR1 by different modes of interaction with AHR1., (Crown Copyright © 2014. Published by Elsevier Ireland Ltd. All rights reserved.)

The organophosphate flame retardants tris(2-butoxyethyl) phosphate (TBOEP) and triethyl phosphate (TEP) are used in a wide range of applications to suppress or delay the ignition and spread of fire. Both compounds have been detected in the environment and TBOEP was recently measured in free-living avian species. In this study, TBOEP and TEP were injected into the air cell of chicken embryos at concentrations ranging from 0 to 45,400 ng/g and 0 to 241,500 ng/g egg, respectively. Pipping success, development, hepatic mRNA expression of 9 target genes, thyroid hormone levels, and circulating bile acid concentrations were determined. Exposure to the highest doses of TBOEP and TEP resulted in negligible detection of the parent compounds in embryonic contents at pipping indicating their complete metabolic degradation. TBOEP exposure had limited effects on chicken embryos, with the exception of hepatic CYP3A37 mRNA induction. TEP exposure decreased pipping success to 68%, altered growth, increased liver somatic index (LSI) and plasma bile acids, and modulated genes associated with xenobiotic and lipid metabolism and the thyroid hormone pathway. Plasma thyroxine levels were decreased at all TEP doses, including an environmentally-relevant concentration (8 ng/g), and gallbladder hypotrophy was evident at ≥ 43,200 ng/g. Tarsus length and circulating thyroxine concentration emerged as potential phenotypic anchors for the modulation of transthyretin mRNA. The increase in plasma bile acids and LSI, gallbladder hypotrophy, and discoloration of liver tissue represented potential phenotypic outcomes associated with modulation of hepatic genes involved with xenobiotic and lipid metabolism., (Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.)

Worldwide, populations of sturgeons are endangered, and it is hypothesized that anthropogenic chemicals, including dioxin-like compounds (DLCs), might be contributing to the observed declines in populations. DLCs elicit their toxic action through activation of the aryl hydrocarbon receptor (AhR), which is believed to regulate most, if not all, adverse effects associated with exposure to these chemicals. Currently, risk assessment of DLCs in fishes uses toxic equivalency factors (TEFs) developed for the World Health Organization (WHO) that are based on studies of embryo-lethality with salmonids. However, there is a lack of knowledge of the sensitivity of sturgeons to DLCs, and it is uncertain whether TEFs developed by the WHO are protective of these fishes. Sturgeons are evolutionarily distinct from salmonids, and the AhRs of sturgeons differ from those of salmonids. Therefore, this study investigated the sensitivity of white sturgeon (Acipenser transmontanus) to DLCs in vitro via the use of luciferase reporter gene assays using COS-7 cells transfected with AhR1 or AhR2 of white sturgeon. Specifically, activation and relative potencies (RePs) of 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD), 2,3,4,7,8-pentachloro-dibenzofuran, 2,3,7,8-tetrachloro-dibenzofuran, 3,3',4,4',5-pentachlorobiphenyl, 3,3',4,4'-tetrachlorobiphenyl, and 2,3,3',4,4'-pentachlorobiphenyl were determined for each AhR. It was demonstrated that white sturgeon expresses AhR1s and AhR2s that are both activated by DLCs with EC50 values for 2,3,7,8-TCDD that are lower than those of any other AhR of vertebrates tested to date. Both AhRs of white sturgeon had RePs for polychlorinated dibenzofurans more similar to TEFs for birds, while RePs for polychlorinated biphenyls were most similar to TEFs for fishes. Measured concentrations of select DLCs in tissues of white sturgeon from British Columbia, Canada, were used to calculate toxic equivalents (TEQs) by use of TEFs for fishes used by the WHO and TCDD equivalents (TCDD-EQs) via the use of RePs for AhR2 of white sturgeon as determined by transfected COS-7 cells. TCDD-EQs calculated for endangered populations of white sturgeon were approximately 10-fold greater than TEQs and were within ranges known to cause adverse effects in other fishes, including other species of sturgeons. Therefore, TEFs used by the WHO might not adequately protect white sturgeon, illuminating the need for additional investigation into the sensitivity of these fish to DLCs.

World Health Organization (WHO) toxic equivalency factors are used to calculate toxic equivalent (TEQ) concentrations of complex mixtures of dioxin-like compounds (DLCs), such as polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans and polychlorinated biphenyls (PCBs), for mammals, fish and birds. The TEQ concept assumes that all species of a taxa respond with similar sensitivity to individual DLCs, but several reports do not support this assumption for birds. Our laboratory is conducting research to attempt to uncover the fundamental mechanism(s) underlying the reasons why avian species differ in sensitivity to DLCs. The present study determined concentration-dependent effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) and 3,3',4,4',5-pentachlorobiphenyl (PCB 126) on ethoxyresorufin-O-deethylase (EROD) activity in primary cultures of northern bobwhite quail (Colinus virginianus) hepatocytes. Bobwhite quail were studied because (1) this species is used in the laboratory for toxicity testing and (2) the amino acids at all locations within the ligand binding domain (LBD) of aryl hydrocarbon receptor 1 (AHR1) in bobwhite quail and ring necked pheasant (Phasianus colchicus) are identical. Because earlier work indicated the importance of the identity of amino acids at key sites within the AHR1 LBD, we hypothesized that bobwhite quail and ring necked pheasant hepatocytes should have similar sensitivity to EROD induction by DLCs. ECthreshold-based relative sensitivity of the bobwhite quail compared to chicken for TCDD, PeCDF and PCB 126 was 0.11, 0.17 and 0.02, respectively. The rank order of potency was PeCDF > TCDD > PCB 126. The results confirm that bobwhite quail and ring-necked pheasant hepatocytes have similar sensitivity to EROD induction by TCDD, PeCDF and PCB 126.

1,2-Dibromo-4-(1,2-dibromoethyl)-cyclohexane (DBE-DBCH; formerly abbreviated as TBECH) and tris(methylphenyl) phosphate (TMPP; formerly abbreviated as TCP) are additive flame retardants that are detected in the environment and biota. A recent avian in vitro screening study of 16 flame retardants identified DBE-DBCH and TMPP as important chemicals for follow-up in ovo evaluation based on their effects on cytotoxicity and mRNA expression in avian hepatocytes. In this study, technical mixtures of DBE-DBCH and TMPP were injected into the air cell of chicken embryos at concentrations ranging from 0 to 54,900ng/g and from 0 to 261,400ng/g, respectively, to determine effects on pipping success, development, hepatic mRNA expression, thyroid hormone levels, and circulating bile acid concentrations. Both compounds were detectable in embryos at pipping and the β-DBE-DBCH isomer was depleted more rapidly than the α-isomer in tissue samples. DBE-DBCH had limited effects on the endpoints measured, with the exception of the up-regulation of two phase I metabolizing enzymes, CYP3A37 and CYP2H1. TMPP exposure caused embryonic deformities, altered growth, increased liver somatic index (LSI) and plasma bile acid concentrations, and altered mRNA expression levels of genes associated with xenobiotic and lipid metabolism and the thyroid hormone pathway. Overall, TMPP elicited more adverse molecular and phenotypic effects than DBE-DBCH albeit at concentrations several orders of magnitude greater than those detected in the environment. The increase in plasma bile acid concentrations was a useful phenotypic anchor as it was associated with a concomitant increase in LSI, discoloration of the liver tissue, and modulation of hepatic genes involved with xenobiotic and lipid metabolism., (Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.)

Results of recent studies showed that 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are equipotent in domestic chicken (Gallus gallus domesticus) while PeCDF is more potent than TCDD in ring-necked pheasant (Phasianus colchicus) and Japanese quail (Coturnix japonica). To elucidate the mechanism(s) underlying these differences in relative potency of PeCDF among avian species, we tested the hypothesis that this is due to species-specific differential binding affinity of PeCDF to the aryl hydrocarbon receptor 1 (AHR1). Here, we modified a cell-based binding assay that allowed us to measure the binding affinity of dioxin-like compounds (DLCs) to avian AHR1 expressed in COS-7 (fibroblast-like cells). The results of the binding assay show that PeCDF and TCDD bind with equal affinity to chicken AHR1, but PeCDF binds with greater affinity than TCDD to pheasant (3-fold) and Japanese quail (5-fold) AHR1. The current report introduces a COS-7 whole-cell binding assay and provides a mechanistic explanation for differential relative potencies of PeCDF among species of birds., (Crown Copyright © 2014. Published by Elsevier Inc. All rights reserved.)

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., (Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.)

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., (© 2013 SETAC.)

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., (Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.)

The World Health Organization toxic equivalency factors (WHO-TEFs) for birds were developed to simplify risk assessments of environmental mixtures of dioxin-like compounds (DLCs). Under this framework, toxic equivalents (TEQs) are used to represent the toxic potency of DLC mixtures as an equivalent concentration of 2,3,7,8-tetrachlorodibenzo-p-dioxin. Recently, a luciferase reporter gene (LRG) assay, measuring aryl hydrocarbon receptor 1 (AHR1)-mediated gene expression, accurately predicted the relative potency of individual polychlorinated biphenyl (PCB) congeners in different avian species. The study presented here used the LRG assay to predict the relative potency of Aroclors 1016, 1221, 1242, 1248, 1254, and 1260 on induction of LRG activity in cells transfected with chicken, ring-necked pheasant, or Japanese quail AHR1 constructs. LRG assay results were compared to (1) results of ethoxyresorufin-O-deethylase (EROD) assays conducted in chicken hepatocytes and (2) calculated TEQs from the literature. The relative potencies of Aroclors were similar between the LRG and EROD assays, and bioassay-derived TEQs for the chicken closely resembled calculated TEQs. However, LRG assay-derived TEQs for the Japanese quail construct were 1-2 orders of magnitude higher than calculated TEQs for Aroclors 1254 and 1016. These results suggest that the WHO-TEFs are not representative of relative PCB potency for all avian species.

Tris(1-chloro-2-propyl) phosphate (TCPP) and tris(1,3-dichloro-2-propyl) phosphate (TDCPP) are organic flame retardants detected in the environment and biota for which avian toxicological data are limited. In this study, domestic chicken eggs were injected with TCPP or TDCPP (maximum dose = 51,600 and 45,000ng/g egg, respectively) to determine dose-dependent effects on pipping success, development, hepatic messenger RNA (mRNA) expression levels of genes associated with xenobiotic metabolism and the thyroid hormone (TH) pathway, and TH levels following 20-22 days of incubation. Neither compound reduced pipping success; however, TCPP significantly delayed pipping at 9240 and 51,600ng/g and reduced tarsus length at 51,600ng/g. TDCPP exposure resulted in significant decreases in head plus bill length, embryo mass, and gallbladder size at 45,000ng/g and reduced plasma free T4 levels at 7640ng/g. Type I deiodinase, liver fatty acid-binding protein, and cytochrome P450 (CYP) 3A37 mRNA levels were significantly induced by TCPP, whereas TDCPP induced CYP3A37 and CYP2H1. Chemical analysis of egg contents at incubation days 0, 5, 11, 18, and 19 revealed that > 92% of the injected TCPP or TDCPP concentration was detectable up to day 5; however, < 1% was detected by day 19. The observed phenotypic responses to TCPP and TDCPP exposure may be associated with disruption of the TH axis, which is critical for normal growth and development in birds. The effects of TDCPP on the gallbladder indicate that the disturbance of lipid metabolism is a likely mechanism of toxicity.

Novel methods that predict the sensitivity of avian embryos to the toxic effects of dioxin-like compounds (DLCs) using either (1) knowledge of the identity of amino acids at key sites within the ligand binding domain of aryl hydrocarbon receptor 1 (AHR1) or (2) a luciferase reporter gene assay that measures AHR1 activation were recently reported. Results from both methods predict that European starling (Sturnus vulgaris) and domestic chicken (Gallus gallus domesticus) embryos have similar sensitivity to the biochemical and toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) and 2,3,7,8-tetrachlorodibenzofuran (TCDF). Chicken embryos are highly sensitive to DLC toxicity, and the prediction that starlings are equally sensitive is surprising given their widespread distribution and large population size. In an attempt to learn more about starling sensitivity to DLCs, we determined concentration-dependent effects of TCDD, PeCDF and TCDF on cytochrome P4501A4 and 1A5 (CYP1A4 and 1A5) mRNA levels in primary cultures of hepatocytes prepared from embryonic European starlings. It has been demonstrated that the sensitivity of avian hepatocytes to CYP1A4/5 induction is well correlated with LD50 values of DLCs for several avian species. The results of the present study indicate that European starling hepatocytes are indeed as sensitive as chicken hepatocytes to CYP1A4/5 induction after exposure to TCDD. However, starling hepatocytes are less sensitive than chicken hepatocytes to CYP1A4/5 induction by PeCDF and TCDF.

The sensitivity of avian species to the toxic effects of dioxin-like compounds (DLCs) varies up to 1000-fold among species, and this variability has been associated with interspecies differences in aryl hydrocarbon receptor 1 ligand-binding domain (AHR1 LBD) sequence. We previously showed that LD(50) values, based on in ovo exposures to DLCs, were significantly correlated with in vitro EC(50) values obtained with a luciferase reporter gene (LRG) assay that measures AHR1-mediated induction of cytochrome P4501A in COS-7 cells transfected with avian AHR1 constructs. Those findings suggest that the AHR1 LBD sequence and the LRG assay can be used to predict avian species sensitivity to DLCs. In the present study, the AHR1 LBD sequences of 86 avian species were studied, and differences at amino acid sites 256, 257, 297, 324, 337, and 380 were identified. Site-directed mutagenesis, the LRG assay, and homology modeling highlighted the importance of each amino acid site in AHR1 sensitivity to 2,3,7,8-tetrachlorodibenzo-p-dioxin and other DLCs. The results of the study revealed that (1) only amino acids at sites 324 and 380 affect the sensitivity of AHR1 expression constructs of the 86 avian species to DLCs and (2) in vitro luciferase activity of AHR1 constructs containing only the LBD of the species of interest is significantly correlated (r (2) = 0.93, p < 0.0001) with in ovo toxicity data for those species. These results indicate promise for the use of AHR1 LBD amino acid sequences independently, or combined with the LRG assay, to predict avian species sensitivity to DLCs.

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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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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.

Birds differ in sensitivity to the embryotoxic effects of polychlorinated biphenyls (PCBs), which complicates environmental risk assessments for these chemicals. Recent research has shown that the identities of amino acid residues 324 and 380 in the avian aryl hydrocarbon receptor 1 (AHR1) ligand binding domain (LBD) are primarily responsible for differences in avian species sensitivity to selected dibenzo-p-dioxins and furans. A luciferase reporter gene (LRG) assay was developed in our laboratory to measure AHR1-mediated induction of a cytochrome P450 1A5 reporter gene in COS-7 cells transfected with different avian AHR1 constructs. In the present study, the LRG assay was used to measure the concentration-dependent effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), and PCBs 126, 77, 105 and 118 on luciferase activity in COS-7 cells transfected with AHR1 constructs representative of 86 avian species in order to predict their sensitivity to PCB-induced embryolethality and the relative potency of PCBs in these species. The results of the LRG assay indicate that the identity of amino acid residues 324 and 380 in the AHR1 LBD are the major determinants of avian species sensitivity to PCBs. The relative potency of PCBs did not differ greatly among AHR1 constructs. Luciferase activity was significantly correlated with embryolethality data obtained from the literature (R(2)≥0.87, p<0.0001). Thus, the LRG assay in combination with the knowledge of a species' AHR1 LBD sequence can be used to predict PCB-induced embryolethality in potentially any avian species of interest without the use of lethal methods on a large number of individuals., (Copyright © 2012 Elsevier Inc. All rights reserved.)

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Perfluoroalkyl acids (PFAAs), specifically perfluorinated sulfonates and carboxylates, are synthetic substances known for their chemical stability, resistance to degradation, and potential to biomagnify in food chains. The toxicological and biological effects of PFAAs in avian species are not well characterized, although there is some evidence to suggest that they can impact neurodevelopment and hatching success. Our laboratory recently reported significant effects of perfluorohexane sulfonate (PFHxS) and perfluorohexanoate (PFHxA) on messenger RNA (mRNA) levels of thyroid hormone (TH)-responsive genes in chicken embryonic neuronal cells. In this study, we determined in ovo effects of PFHxS and PFHxA exposure (maximum dose = 38,000 and 9700 ng/g egg, respectively) on embryonic death, developmental endpoints, tissue accumulation, mRNA expression in liver and cerebral cortex, and plasma TH levels. Pipping success was reduced to 63% at the highest dose of PFHxS; no effects were observed for PFHxA. PFHxS exposure (38,000 ng/g) decreased tarsus length and embryo mass. PFHxS and PFHxA accumulated in the three tissue compartments analyzed as follows: yolk sac > liver > cerebral cortex. Type II and type III 5'-deiodinases (D2 and D3) and cytochrome P450 3A37 mRNA levels were induced in liver tissue of chicken embryos exposed to PFHxS, whereas D2, neurogranin (RC3), and octamer motif binding factor 1 mRNA levels were upregulated in cerebral cortex. Plasma TH levels were reduced in a concentration-dependent manner following PFHxS exposure; PFHxA had no effect. This in ovo study successfully validated previous in vitro results concerning the modulation of TH-responsive genes and identified adverse effects associated with TH homeostasis in response to PFHxS treatment.

Primary cultures of ring-necked pheasant (Phasianus colchicus) and Japanese quail (Coturnix japonica) embryo hepatocytes were used to compare the potencies of highly purified hexachlorobenzne (HCB-P), reagent-grade HCB (RG-HCB) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as inducers of ethoxyresorufin O-deethylase (EROD) activity, cytochrome P4501A (CYP1A4) messenger ribonucleic acid (mRNA) and CYP1A5 mRNA. HCB-P, RG-HCB and TCDD all induced EROD activity and up-regulated CYP1A4 and CYP1A5 mRNA. Induction was not caused by contamination of HCB with polychlorinated dibenzo-p-dioxins, dibenzofurans or biphenyls. Based upon a comparison of the EC(50) and EC(threshold) values for EROD and CYP1A4/5 concentration-response curves, the potency of HCB relative to TCDD was 0.001 in ring-necked pheasant and 0.01 in Japanese quail embryo hepatocytes. Differences in species sensitivity to HCB were found to be mainly dictated by differences in species sensitivity to TCDD rather than differences in the absolute potency of HCB. Consequently, ring-necked pheasant and Japanese quail embryo hepatocytes were found to be equally sensitive to HCB exposure. Species sensitivity comparisons were also made with chicken (Gallus gallus domesticus) and revealed that chicken embryo hepatocytes were less responsive to EROD induction (lower maximal response) by HCB compared to the embryo hepatocytes of pheasant and quail., (Copyright © 2011 Elsevier Inc. All rights reserved.)

There are large differences in sensitivity to the toxic and biochemical effects of dioxins and dioxin-like compounds (DLCs) among vertebrates. Previously, we demonstrated that the difference in sensitivity between domestic chicken (Gallus gallus domesticus) and common tern (Sterna hirundo) to aryl hydrocarbon receptor 1 (AHR1)-dependent changes in gene expression following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is based upon the identities of the amino acids at two sites within the ligand binding domain of AHR1 (chicken--highly sensitive; Ile324&#95;Ser380 vs common tern--250-fold less sensitive than chicken; Val325&#95;Ala381). Here, we tested the hypotheses that (i) the sensitivity of other avian species to TCDD, 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), and 2,3,7,8-tetrachlorodibenzofuran (TCDF) is also determined by the amino acids at sites that are equivalent to sites 324 and 380 in chicken, and (ii) Ile324&#95;Ala380 and Val324&#95;Ser380 genotypes confer intermediate sensitivity to DLCs in birds. We compared ligand-induced transactivation function of full-length AHR1s from chicken, common tern, ring-necked pheasant (Phasianus colchicus; Ile324&#95;Ala380) and Japanese quail (Coturnix japonica; Val324&#95;Ala380), and three Japanese quail AHR1 mutants. The results support our hypothesis that avian species can be grouped into three general classes of sensitivity to DLCs. Both AHR1 genotype and in vitro transactivation assays predict in vivo sensitivity. Contrary to the assumption that TCDD is the most potent DLC, PeCDF was more potent than TCDD at activating Japanese quail (13- to 26-fold) and common tern (23- to 30-fold) AHR1. Our results support and expand previous in vitro and in vivo work that demonstrated ligand-dependent species differences in AHR1 affinity. The findings and methods will be of use for DLC risk assessments.

Tris(1,3-dichloro-2-propyl) phosphate (TDCPP) and tris(1-chloropropyl) phosphate (TCPP) belong to a group of chemicals collectively known as triester organophosphate flame retardants (OPFRs). OPFRs are used in a wide range of consumer products and have been detected in biota, including free-living avian species; however, data on toxicological and molecular effects of exposure are limited. An in vitro screening approach was used to compare concentration-dependent effects of TDCPP and TCPP on cytotoxicity and messenger RNA (mRNA) expression in cultured hepatocytes and neuronal cells derived from embryonic chickens. TDCPP was toxic to hepatocytes (LC₅₀ = 60.3 ± 45.8μM) and neuronal cells (LC₅₀ = 28.7 ± 19.1μM), whereas TCPP did not affect viability in either cell type up to the highest concentration administered, 300μM. Real-time reverse transcription-PCR revealed alterations in mRNA abundance of genes associated with phase I and II metabolism, the thyroid hormone (TH) pathway, lipid regulation, and growth in hepatocytes. None of the transcripts measured in neuronal cells (D2, D3, RC3, and Oct-1) varied in response to TDCPP or TCPP exposure. Exposure to ≥ 10μM TDCPP and TCPP resulted in significant upregulation of CYP2H1 (4- to 8-fold), CYP3A37 (13- to 127-fold), and UGT1A9 (3.5- to 7-fold) mRNA levels. Transthyretin was significantly downregulated more than twofold by TCPP at 100μM; however, TDCPP did not alter its expression. Liver fatty acid-binding protein, TH-responsive spot 14-α, and insulin-like growth factor-1 were all downregulated (up to 10-fold) in hepatocytes exposed to ≥ 0.01μM TDCPP and TCPP. Taken together, our results indicate that genes associated with xenobiotic metabolism, the TH pathway, lipid regulation, and growth are vulnerable to TDCPP and TCPP administration in cultured avian hepatocytes. The mRNA expression data were similar to those from a previous study with hexabromocyclododecane.

Unlabelled: BACKGROUND/STUDY CONTEXT: The current study examined the relationship between intellectual control and cognition and related the results to everyday problem solving in a mixed ethnicity sample of 35% African American and 65% Caucasian elders., Methods: Participants completed the Personality in Intellectual Aging Contexts Inventory (PIC; Lachman et al., 1982 , Journal of Research in Personality, 16, 485-501), Everyday Cognition Battery (ECB; Allaire & Marsiske, 1999 , Psychology & Aging, 14, 627-644; 2002 , Psychology & Aging, 17, 101-115), and a battery of basic cognitive ability tests assessing memory, inductive reasoning, and verbal meaning., Results: Results indicated that African Americans had significantly lower intellectual control beliefs relative to Caucasian older adults. Regression models suggested that relationship between control beliefs and cognition was moderated by education and race. Decomposing the interactions with simple slope analysis revealed that across cognitive abilities, better cognitive performance was related to higher control beliefs in African Americans with at least 13 years of education. A similar relationship was also found in Caucasian elders with lower education., Conclusion: African American elders' reaching a higher level of education may provide a basis for which individual differences in intellectual control beliefs are activated and thereby more strongly associated with cognitive performance.

Recently it was discovered that the perfluorooctane sulfonate (PFOS) detected in wildlife, such as fish-eating birds, had a greater proportion of linear PFOS (L-PFOS) than the manufactured technical product (T-PFOS), which contains linear and branched isomers. This suggests toxicological studies based on T-PFOS data may inaccurately assess exposure risk to wildlife. To determine whether PFOS effects were influenced by isomer content, we compared the transcriptional profiles of cultured chicken embryonic hepatocytes (CEH) exposed to either L-PFOS or T-PFOS using Agilent microarrays. At equal concentrations (10 µM), T-PFOS altered the expression of more transcripts (340, >1.5-fold change, p < 0.05) compared with L-PFOS (130 transcripts). Higher concentrations of L-PFOS (40 µM) were also less transcriptionally disruptive (217 transcripts) than T-PFOS at 10 µM. Functional analysis showed that L-PFOS and T-PFOS affected genes involved in lipid metabolism, hepatic system development, and cellular growth and proliferation. Pathway and interactome analysis suggested that genes may be affected through the RXR receptor, oxidative stress response, TP53 signaling, MYC signaling, Wnt/β-catenin signaling, and PPARγ and SREBP receptors. In all functional categories and pathways examined, the response elicited by T-PFOS was greater than that of L-PFOS. These data show that T-PFOS elicits a greater transcriptional response in CEH than L-PFOS alone and demonstrates the importance of considering the isomer-specific toxicological properties of PFOS when assessing exposure risk., (Copyright © 2011 SETAC.)

Some currently used brominated flame retardants (BFRs), such as hexachlorocyclopentadienyl-dibromocyclooctane (HCDBCO), bis(2-ethylhexyl)tetrabromophthalate (BEHTBP), 1,2-bis(2,4,6-tribromophenoxy)ethane (BTBPE) and decabromodiphenylethane (DBDPE), are persistent organic contaminants detected in various environmental matrices, including wild birds. Data on potential toxicological and molecular responses to exposure of these BFRs are lacking for avian species. A combined in vitro/in ovo approach was used to determine the concentration-dependent effects of these BFRs on overt toxicity and hepatic messenger RNA (mRNA) expression levels of 11 transcripts in (1) primary cultures of chicken embryonic hepatocytes (CEH; all four BFRs) and (2) chicken embryos (HCDBCO and BTBPE only). Neither hepatocyte viability nor embryonic pipping success were affected by the BFRs at any of the administered concentrations (CEH: 0.001-30 μM, egg injection: 0.1-10 μg/g nominal dose). In CEH, 10 μM HCDBCO induced cytochrome P450 2H1 (CYP2H1) and CYP3A37, while CYP1A4/5 were down-regulated at all tested concentrations. In contrast, only transthyretin was down-regulated by HCDBCO in embryonic liver. There was concordance between the BTBPE-induced transcriptional responses in vitro and in ovo for CYP1A4/5 (up-regulated) and type III iodothyronine 5'-deiodinase (DIO3; down-regulated). DBDPE induced CYP1A4/5 29- and 59-fold at 0.2 μM in CEH and increased DIO1. None of the gene targets were responsive to BEHTBP exposure in CEH. The multi-tiered in vitro/in ovo screening approach was effective for assessing toxicological and molecular biological effects of these BFRs in an avian species., (Crown Copyright © 2011. Published by Elsevier Ireland Ltd. All rights reserved.)

Dechlorane Plus (DP) is an additive chlorinated flame retardant comprising two major isomers, syn- and anti-DP, that is used in a variety of commercial/industrial products. It has been detected in biotic and abiotic matrices including the eggs of herring gulls collected from the Laurentian Great Lakes. However, data on potential toxicological and molecular responses to exposure are lacking, especially for avian species. A combined in vitro/in ovo approach was used to determine concentration-dependent effects of DP in chicken embryonic hepatocytes (CEH) and chicken embryos following injection of DP into the air cell of eggs prior to incubation. Overt toxicity (i.e. cytotoxicity and pipping success) and mRNA expression levels of transcripts previously determined to be responsive to a brominated flame retardant were assessed in CEH and hepatic tissue. DP was not cytotoxic up to a maximum concentration of 3 μM in CEH, and no effects on pipping success were observed up to the highest nominal dose group of 500 ng/g egg. A significant shift in isomeric content of syn- and anti-DP was detected between stock solutions of the commercial mixture and hepatic tissue; the proportion of the syn-DP isomer increased from 0.34 to 0.65 with a concomitant decrease of anti-DP from 0.66 to 0.35. None of the mRNA transcripts changed as a result of in vitro or in ovo exposure to DP indicating that, although there was concordance between the two approaches, DP may evoke its toxicity through other modes of action. At current environmental exposure levels, no adverse effects of DP on embryonic viability or pathways associated with the genes assessed are predicted., (Crown Copyright © 2011. Published by Elsevier Inc. All rights reserved.)

An egg injection study was conducted to confirm a proposed model of relative sensitivity of three avian species to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-like chemicals. It was previously reported that the order of species sensitivity to in ovo exposure to TCDD, 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), or 2,3,7,8-tetrachlorodibenzofuran (TCDF) at doses ranging from 0.044 to 37 picomoles (pmol)/g egg was the chicken (Gallus gallus domesticus), common pheasant (Phasianus colchicus), and Japanese quail (Coturnix japonica) based on embryo mortality and hepatic enzyme induction. In the present study, the incidence of developmental deformities, changes in body and relative organ masses, and organ pathology of hatchlings as additional indicators of species sensitivity were assessed; in addition, embryo mortality in the three species was categorized by stage of development. Embryo mortality varied temporally with significant increases generally occurring after organogenesis and just prior to hatching. A significant increase in the percentage of developmental deformities was observed only in Japanese quail exposed to TCDF. Body and relative organ masses of quail, pheasants, and chickens dosed in ovo with TCDD, PeCDF, or TCDF were not consistently affected. Chemical-related pathology occurred only in livers of quail at the greatest doses of each compound. These results indicated that the incidence of developmental deformities, changes in body and relative organ masses and organ pathology could not be used as indicators of species sensitivity or chemical potency., (Copyright © 2011 SETAC.)

There is growing interest in assessing the neurotoxic and endocrine disrupting potential of perfluoroalkyl compounds (PFCs). Several studies have reported in vitro and in vivo effects related to neuronal development, neural cell differentiation, prenatal and postnatal development and behavior. PFC exposure altered hormone levels and the expression of hormone-responsive genes in mammalian and aquatic species. This study is the first to assess the effects of PFCs on messenger RNA (mRNA) expression in primary cultures of neuronal cells in two avian species: the domestic chicken (Gallus domesticus) and herring gull (Larus argentatus). The following thyroid hormone (TH)-responsive genes were examined using real-time reverse transcription-PCR: type II iodothyronine 5'-deiodinase (D2), D3, transthyretin (TTR), neurogranin (RC3), octamer motif-binding factor (Oct-1), and myelin basic protein. Several PFCs altered the mRNA expression levels of genes associated with the TH pathway in avian neuronal cells. Short-chained PFCs (less than eight carbons) altered the expression of TH-responsive genes (D2, D3, TTR, and RC3) in chicken embryonic neuronal cells to a greater extent than long-chained PFCs (more than or equal to eight carbons). Variable transcriptional changes were observed in herring gull embryonic neuronal cells exposed to short-chained PFCs; mRNA levels of Oct-1 and RC3 were upregulated. This is the first study to report that PFC exposure alters mRNA expression in primary cultures of avian neuronal cells and may provide insight into the possible mechanisms of action of PFCs in the avian brain.

Prior to its recent phaseout, perfluorooctane sulfonate (PFOS) was produced by electrochemical fluorination processes, which yielded technical mixtures composed of linear isomer (∼65-79%) and several branched isomers (∼21-35%). Because PFOS can biomagnify in wildlife, birds that occupy higher trophic levels are at increased risk of exposure. We hypothesized that the pharmacokinetic properties of PFOS are isomer-specific in developing chicken (Gallus gallus domesticus) embryos exposed to technical grade PFOS (T-PFOS). In the present study, T-PFOS was composed of 62.7% linear isomer (L-PFOS), and 37.3% branched isomer, including six mono(trifluoromethyl)-branched isomers and four bis(trifluoromethyl)-branched isomers. Concentrations of 0.1, 5, or 100 µg/g of T-PFOS were injected into the air cell of chicken eggs prior to incubation. After pipping, compared with T-PFOS, the PFOS isomer profile in embryonic liver tissue for the 0.1 µg/g dose group showed 21% enrichment in the proportion of L-PFOS with a corresponding decrease in the proportion of branched isomers. Not all branched isomers were discriminated against at equal rates. The proportion of two mono(trifluoromethyl)-branched isomers and three bis(trifluoromethyl)-branched isomers decreased to a greater degree than other branched isomers. In contrast, the mono-branched isomer, P6MHpS, was overrepresented in the low-dose group. In the higher dose groups, L-PFOS was still enriched but only by approximately 10%, which indicated a dose-dependent change in isomer composition relative to T-PFOS. These results show that accumulation of PFOS in chicken embryo livers is dependent on the presence and position of branches on the alkyl backbone. This supports the hypothesis that the pharmacokinetics of PFOS are isomer-specific in biota, and may help explain why wildlife PFOS burdens are dominated by L-PFOS relative to T-PFOS mixtures., (© 2010 SETAC.)

Egg injection studies were performed to confirm a proposed model of relative sensitivity of birds to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In this model, species are classified as belonging to one of three categories of sensitivity based on amino acid substitutions in the ligand-binding domain of the aryl hydrocarbon receptor. Embryo lethality and relative potencies of 2,3,7,8-tetrachlorodibenzofuran (TCDF) and 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) were compared with TCDD for Japanese quail (Coturnix japonica; least sensitive), Common pheasant (Phasianus colchicus; moderately sensitive), and White Leghorn chicken (Gallus gallus domesticus; most sensitive). Doses ranging from 0.044 to 37 pmol/g egg (0.015-12 ng/g egg) were injected into the air cell of eggs prior to incubation. LD(50) (95% confidence intervals) values, based on rate of hatching for TCDD, PeCDF, and TCDF, were 30 (25-36), 4.9 (2.3-9.2), and 15 (11-24) pmol/g egg for the quail, 3.5 (2.3-6.3), 0.61 (0.28-1.2), and 1.2 (0.62-2.2) pmol/g egg for pheasant, and 0.66 (0.47-0.90), 0.75 (0.64-0.87), and 0.33 (0.23-0.45) pmol/g egg for chicken, respectively. LD(50)-based relative potencies of PeCDF and TCDF were 6.1 and 2.0 for quail, 5.7 and 2.9 for pheasant, and 0.88 and 2.0 for chicken, respectively. TCDD was not the most potent compound among the species tested, with PeCDF and TCDF being more potent than TCDD in the quail and pheasant. TCDF was the most potent in chicken. Species sensitivity was as expected for TCDD and TCDF, whereas for PeCDF, the chicken and pheasant were similar in sensitivity and both were more sensitive than the quail. Results from companion in vitro studies are generally similar to those reported here with a few exceptions.

The amino acid sequence of the aryl hydrocarbon receptor 1 ligand binding domain (AHR1 LBD) is an important determinant of sensitivity to dioxin-like compounds in avian species. We are interested in surveying AHR1 LBD sequences in a large number of birds as a means of identifying species that are particularly sensitive to dioxin-like compounds. Our original method for determining AHR1 LBD genotype used liver tissue and required lethal sampling. Here we present two alternate methods for determining AHR1 LBD genotype which use non-lethal sampling and are more appropriate for ecologically sensitive species. First, we establish that AHR1 LBD mRNA is expressed in avian blood and test a variety of blood collection and handling protocols in order to establish a method that is convenient for field collections. Our findings also identify which types of archival blood samples might be appropriate for AHR1 LBD sequence determination. Second, we present a method for obtaining AHR1 LBD coding sequences from DNA. A DNA-based method is advantageous because DNA can be isolated from many tissue types, is more stable than RNA, and requires less specific sample handling and preservation. This work extends applicability of a genetic screen for dioxin sensitivity to a larger number of species and sample types including endangered species and potentially museum specimens.