Interindividual variation in the cytochrome P4501A response to 2,3,7,8-tetrachlorodibenzo-p-dioxin in herring gull embryo hepatocytes.

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.

Time-dependent transcriptomic and biochemical responses of 6-formylindolo[3,2-b]carbazole (FICZ) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are explained by AHR activation time.

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.

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

We report on two highly brominated polyphenyl ether flame retardants, tetradecabromo-1,4- diphenoxybenzene (TeDB-DiPhOBz) and 2,2',3,3',4,4',5,5',6,6'-decabromodiphenyl ether (BDE-209), that formed photolytic degradation products in tetrahydrofuran (THF)/hexane solvent after 21 days of natural sunlight irradiation (SI). These degradation products of SI-TeDB-DiPhOBz and SI-BDE-209 included the numerous polybrominated homologue groups of polybenzofurans and dibenzofurans, respectively. Formation of similar polybenzofuran and dibenzofuran products was also observed following a 3 month exposure of the solid powder forms of TeDB-DiPhOBz and BDE-209 to natural SI.

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.

Increasing Scientific Confidence in Adverse Outcome Pathways: Application of Tailored Bradford-Hill Considerations for Evaluating Weight of Evidence.

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.

Differences in activation of aryl hydrocarbon receptors of white sturgeon relative to lake sturgeon are predicted by identities of key amino acids in the ligand binding domain.

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.

Potency of polycyclic aromatic hydrocarbons (PAHs) for induction of ethoxyresorufin-O-deethylase (EROD) activity in hepatocyte cultures from chicken, Pekin duck, and greater scaup.

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.

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

A market for alternative brominated flame retardants (BFRs) has emerged recently due to the phase out of persistent and inherently toxic BFRs. Several of these replacement compounds have been detected in environmental matrices, including wild birds. A chicken embryonic hepatocyte (CEH) assay was utilized to assess the effects of the BFR, tetrabromobisphenol-A (TBBPA), and its replacement alternative, tetrabromobisphenol A bis(2,3-dibromopropyl ether [TBBPA-DBPE]) on cell viability and messenger ribonucleic acid (mRNA) expression.

Rapid in vitro metabolism of the flame retardant triphenyl phosphate and effects on cytotoxicity and mRNA expression in chicken embryonic hepatocytes.

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.

Feathers as a source of RNA for genomic studies in avian species.

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).

Time-dependent effects of the flame retardant tris(1,3-dichloro-2-propyl) phosphate (TDCPP) on mRNA expression, in vitro and in ovo, reveal optimal sampling times for rapidly metabolized compounds.

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.

Photolytic degradation products of two highly brominated flame retardants cause cytotoxicity and mRNA expression alterations in chicken embryonic hepatocytes.

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).

Amino acid sequence of the AhR1 ligand-binding domain predicts avian sensitivity to dioxin like compounds: in vivo verification in European starlings.

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.

Sensitivity of avian species to the aryl hydrocarbon receptor ligand 6-formylindolo [3,2-b] carbazole (FICZ).

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.

Tris(2-butoxyethyl)phosphate and triethyl phosphate alter embryonic development, hepatic mRNA expression, thyroid hormone levels, and circulating bile acid concentrations in chicken embryos.

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.

Functionality of aryl hydrocarbon receptors (AhR1 and AhR2) of white sturgeon (Acipenser transmontanus) and implications for the risk assessment of dioxin-like compounds.

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.

1,2-Dibromo-4-(1,2-dibromoethyl)-cyclohexane and tris(methylphenyl) phosphate cause significant effects on development, mRNA expression, and circulating bile acid concentrations in chicken embryos.

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.

Ethoxyresorufin-O-deethylase (EROD) induction by TCDD, PeCDF and PCB 126 in bobwhite quail hepatocytes.

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.

Species-specific relative AHR1 binding affinities of 2,3,4,7,8-pentachlorodibenzofuran explain avian species differences in its relative potency.

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).

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.

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.

Relative potencies of aroclor mixtures derived from avian in vitro bioassays: comparisons with calculated toxic equivalents.

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.

In Ovo effects of two organophosphate flame retardants--TCPP and TDCPP--on pipping success, development, mRNA expression, and thyroid hormone levels in chicken embryos.

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.

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

Perfluoroundecanoic acid (PFUdA) is one of the most highly detected perfluoroalkyl compounds in wild bird tissues and eggs. Although PFUdA does not affect hatching success, many PFCs are known to impair post-hatch development and survival. Here we use microarrays to survey the transcriptional response of cultured chicken embryonic hepatocytes (CEH) to PFUdA for potential targets of PFUdA action that could lead to developmental deficiencies in exposed birds.

Cytochrome P4501A induction in primary cultures of embryonic European starling hepatocytes exposed to TCDD, PeCDF and TCDF.

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.