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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.)

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.

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.

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.)

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.

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.)

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.)

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.)

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.

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.

Dioxin-like compounds are toxic to most vertebrates, but significant differences in sensitivity exist among species. A recent study suggests that the amino acid residues corresponding to Ile324 and Ser380 in the chicken aryl hydrocarbon receptor 1 (AHR1) are important determinants of differential biochemical responses to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in chickens and common terns. Here, we investigate whether the identity of these amino acid residues can predict embryonic sensitivity to dioxin-like compounds in a wide range of birds. AHR1 sequences were determined in species for which sensitivity data were available. Of all the species surveyed, chickens were unique in having the Ile/Ser genotype and were also the most sensitive to dioxin-like compounds. Turkeys, ring-necked pheasants, and Eastern bluebirds (intermediate Ile/Ala genotype) were less sensitive than chickens but more sensitive than American kestrels, common terns, double-crested cormorants, Japanese quail, herring gulls, or ducks (Val/ Ala genotype). Our work suggests that key amino acids in the AHR1 ligand binding domain are predictive of broad categories of dioxin sensitivity in avian species. Given the large degree of variation in species sensitivity and the paucity of species-specific toxicity data, a genetic screen based on these findings could substantially improve risk assessment for dioxin-like compounds in wild birds.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related halogenated aromatic hydrocarbons (HAHs) are highly toxic to most vertebrate animals, but there are dramatic differences in sensitivity among species and strains. Aquatic birds including the common tern (Sterna hirundo) are highly exposed to HAHs in the environment, but are up to 250-fold less sensitive to these compounds than the typical avian model, the domestic chicken (Gallus gallus). The mechanism of HAH toxicity involves altered gene expression subsequent to activation of the aryl hydrocarbon receptor (AHR), a basic helix-loop-helix-PAS transcription factor. AHR polymorphisms underlie mouse strain differences in sensitivity to HAHs and polynuclear aromatic hydrocarbons, but the role of the AHR in species differences in HAH sensitivity is not well understood. Here, we show that although chicken and tern AHRs both exhibit specific binding of [3H]TCDD, the tern AHR has a lower binding affinity and exhibits a reduced ability to support TCDD-dependent transactivation as compared to AHRs from chicken or mouse. We further show through use of chimeric AHR proteins and site-directed mutagenesis that the difference between the chicken and tern AHRs resides in the ligand-binding domain and that two amino acids (Val-325 and Ala-381) are responsible for the reduced activity of the tern AHR. Other avian species with reduced sensitivity to HAHs also possess these residues. These studies provide a molecular understanding of species differences in sensitivity to dioxin-like compounds and suggest an approach to using the AHR as a marker of dioxin susceptibility in wildlife.

Halogenated dimethyl bipyrroles (HDBPs) recently have been identified as a group of environmentally persistent naturally produced bioaccumulative organohalogens. The ability of these compounds to activate the aryl hydrocarbon receptor (AhR) signaling pathway in vitro was examined. Induction of cytochrome P4501A (CYP1A), measured as ethoxyresorufin-O-deethylase (EROD) activity, and porphyrin accumulation were monitored after exposure of chick embryo hepatocytes to three individual HDBP congeners and two HDBP mixtures. All HDBP congeners and mixtures tested caused induction of EROD activity. Induction equivalency factors, calculated as the ratio of the effective concentration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to induce 10 and 50% of the maximal EROD induction to that of the HDBP congener or mixture ranged from 0.0004 to 0.05. Halogenated dimethyl bipyrroles also caused porphyrin accumulation, which increased with exposure time of the hepatocytes and molar bromine content of the HDBP congener or mixture. Heptacarboxylporphyrin III and corproporphyrinogen III were the most abundant porphyrins observed after an exposure period of 48 h. The individual HDBP congeners and mixtures also inhibited binding of [3H]TCDD to chicken and mouse AhR. Binding of [3H]TCDD to chicken AhR was inhibited approximately 20% by tetra- to hexabrominated congeners at concentrations of 20 to 40 microM; binding to mouse AhR was inhibited 40 to 50% by tri- and tetrabrominated dimethyl bipyrroles (DBPs) at 10 microM. The relative affinity of the hexabrominated DBP congener (vs TCDD) for binding to chicken AhR was estimated to be 0.000017. The results of this work illustrate that HDBPs act as agonist ligands for the AhR in the same manner as many anthropogenic organohalogens.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) and related planar halogenated aromatic hydrocarbons (PHAHs) are highly toxic to most vertebrate animals, but there are dramatic species differences in sensitivity, both within and among vertebrate classes. For example, studies in cultured avian hepatocytes have revealed differential sensitivity of birds to PHAHs [Kennedy et al. (1996). Toxicol. Appl. Pharmacol., 141, 214-230]. Differences in the characteristics or expression of the aryl hydrocarbon receptor (AHR) could contribute to these species differences in PHAH responsiveness. To investigate the molecular mechanism of differential PHAH sensitivity, we have begun to characterize the AHR in white leghorn chicken (Gallus gallus), Pekin duck (Anas platyrhynchos), and common tern (Sterna hirundo), as well as an amphibian, mudpuppy (Necturus maculosus). Partial AHR cDNAs encompassing the helix-loop-helix and PAS domains were cloned and sequenced. Comparison of amino acid sequences in this region indicated a high degree of sequence conservation among the bird species (97% amino acid identity). The percent identity between bird sequences and either mouse or mudpuppy was lower (79%); the mudpuppy AHR was 74% identical to the mouse AHR. Phylogenetic analysis of these and other AHR amino acid sequences showed that the bird and mudpuppy AHRs were more closely related to mammalian and fish AHR1 forms than to fish AHR2. Future studies include the in vitro expression and functional characterization of AHRs from these and other non-mammalian vertebrates.