Species-specific relative ahr1 binding affinities of 2,3,4,7,8-pentachlorodibenzofuran explain avian species differences in its relative potency

Author Posting. © The Author(s), 2013. This is the author's version of the work. It is posted here by permission of Elsevier for personal use, not for redistribution. The definitive version was published in Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology 161 (2014): 21-25, doi:10.1016/j.cbpc.2013.12.005.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.This research was supported by an unrestricted grant from the Dow Chemical Company to the University of Ottawa, Environment Canada’s Wildlife Toxicology and Disease and STAGE programs and, in part, by a Discovery Grant from the National Science and Engineering Research Council of Canada (Project # 326415-07). The authors wish to acknowledge the support of an instrumentation grant from the Canada Foundation for Infrastructure. Professor Giesy was supported by the Canada Research Chair program and an at large Chair Professorship at the Department of Biology and Chemistry and State Key Laboratory in Marine Pollution, City University of Hong Kong, and the Einstein Professor Program of the Chinese Academy of Sciences. M. Hahn was supported by NOAA Sea Grant (grant number NA06OAR4170021 (R/B-179))

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

ABSTRACT: 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-pentachlorodibenzofuran, 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 EC 50 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

Amino acid sequence of the ligand-binding domain of the aryl hydrocarbon receptor 1 predicts sensitivity of wild birds to effects of dioxin-like compounds

Author Posting. © The Author(s), 2012. This is the author's version of the work. It is posted here by permission of Oxford University Press for personal use, not for redistribution. The definitive version was published in Toxicological Sciences 131 (2013): 139-152, doi:10.1093/toxsci/kfs259.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 inter-species differences in aryl hydrocarbon receptor 1 ligand binding domain (AHR1 LBD) sequence. We previously showed that LD50 values, based on in ovo exposures to DLCs, were significantly correlated with in vitro EC50 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 2,3,8,8-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 86 avian species to DLCs and (2) in vitro luciferase activity in AHR1 constructs containing only the LBD of the species of interest is significantly correlated (r2 = 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.This research was supported by unrestricted grants from the Dow Chemical Company and Georgia-Pacific LLC to the University of Ottawa, Environment Canada’s STAGE program and, in part, by a Discovery Grant from the National Science and Engineering Research Council of Canada (Project # 326415-07). The authors wish to acknowledge the support of an instrumentation grant from the Canada Foundation for Infrastructure. Professor Giesy was supported by the Canada Research Chair program and an at large Chair Professorship at the Department of Biology and Chemistry and State Key Laboratory in Marine Pollution, City University of Hong Kong, the Einstein Professor Program of the Chinese Academy of Sciences and the Visiting Professor Program of King Saud University. M. Hahn and S. Karchner were supported by NOAA Sea Grant (grant number NA06OAR4170021 (R/B-179)), and by the Walter A. and Hope Noyes Smith endowed chair.2013-08-2

Prediction of the Sensitivity of Avian Species to the Embryotoxic Effects of Dioxin-like Compounds

The main goal of this thesis was to develop new methods and knowledge that will explain and predict species differences in sensitivity to dioxin-like compounds (DLCs) in birds. The important achievements and results obtained from the four experimental chapters of this thesis are summarized as follow: (1) an efficient luciferase reporter gene (LRG) assay was developed for use with 96-well cell culture plates; (2) the results obtained from LRG assay were shown to be highly correlated to available in ovo toxicity data; (3) amino acids at positions 324 and 380 within the aryl hydrocarbon receptor 1 ligand binding domain (AHR1 LBD) were shown to be responsible for reduced Japanese quail (Coturnix japonica) AHR1 activity to induce a dioxin-responsive reporter gene in comparison to chicken (Gallus gallus domesticus), and ring-necked pheasant (Phasianus colchicus) AHR1 in response to different DLCs; (4) AHR1 LBD sequences of 86 avian species were studied and differences at amino acid sites 256, 257, 297, 324, 337 and 380 were identified. It was discovered that only positions 324 and 380 play a role in AHR1 activity to induce a dioxin-responsive gene; (5) in COS-7 cells expressing chicken AHR1, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) are equipotent inducers of the reporter gene and bind with similar affinity to chicken AHR1, however, in the cells expressing pheasant, Japanese quail and common tern (Sterna hirundo) AHR1, PeCDF is a stronger inducer than TCDD. PeCDF also binds with higher affinity to pheasant and quail AHR1 than TCDD. The results of this thesis show that embryo lethal effect of DLCs in avian species can be predicted by use of two new non-lethal methods: (1) the LRG assay and (2) determination of the identity of the amino acids at positions 324 and 380. The findings and methods described in this thesis will be of use for environmental risk assessments of DLCs

Photolysis of highly brominated flame retardants leads to time-dependent dioxin-responsive mRNA expression 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 flame retardant chemicals that can undergo photolytic degradation. The present study compared the time-dependent photolyic degradation of TeDB-DiPhOBz and BDE-209, and dioxin-like product formation as a result of (UV) irradiation (I; irradiation time periods of 0, 1, 4, 15 and 40 days). Photo-degraded product fractions of UV-I-TeDB-DiPhOBz (nominal concentration: 1.9 μM) were administered to chicken embryonic hepatocytes (CEH), and significant induction of CYP1A4/5 mRNA expression was observed for fractions collected at the day 15 and 40 time points (fold change of 7.3/3.6 and 9.1/4.7, respectively). For the UV-I-BDE-209 fractions (nominal concentration: 10 μM), significant CYP1A4/5 up-regulation occurred at all time points, and the fraction collected on day 1 induced the greatest fold change of 510/86, followed by 410/68 (day 4) and 110/26 (day 15), respectively. For the UV-I-BDE-209 fraction collected at day 40, significant CEH cytotoxicity was observed. As a result, CYP1A4/5 expression was determined at a nominal concentration of 1 μM instead of 10 μM and CYP1A4/5 fold changes of 11/8.2 (day 40) were observed. Fractions eliciting the greatest CYP1A4/5 mRNA upregulation were further screened for transcriptomic effects using a PCR array comprising 27 dioxin-responsive genes. A total of 6 and 16 of the 27 target genes were up or down-regulated following UV-I-TeDB-DiPhOBz and UV-I-BDE-209 exposure, respectively. Overall, and regardless of the formation rate, these results raise concerns regarding the potential formation of dioxin-like compounds from flame retardant

Potency of Polycyclic Aromatic Hydrocarbons (PAHs) for Induction of Ethoxyresorufin‑<i>O</i>‑deethylase (EROD) Activity in Hepatocyte Cultures from Chicken, Pekin Duck, And Greater Scaup

The potency of tetrachlorodibenzo-<i>p</i>-dioxin (TCDD) and 18 polycyclic aromatic hydrocarbons (PAHs) for induction of ethoxyresorufin-<i>O</i>-deethylase (EROD) activity was assessed in primary hepatocyte cultures prepared from chicken (<i>Gallus domesticus</i>), Pekin duck (<i>Anas platyrhynchos domesticus</i>), and greater scaup (<i>Aythya marila</i>). 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[<i>a</i><i>h</i>]­anthracene > benzo­[<i>k</i>]­fluoranthene > indeno­[1,2,3-<i>c</i><i>d</i>]­pyrene > benzo­[<i>a</i>]­pyrene > chrysene ≈ benz­[<i>a</i>]­anthracene ≈ benz­[<i>g</i><i>h</i><i>i</i>]­perylene > benzo­[<i>b</i>]­naphtho­[2,3-<i>d</i>]­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