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))

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

Effects on Tree Swallows Exposed to Dioxin-like Compounds Associated With: the Tittabawassee River and Floodplain near

Abstract-Concentrations of dioxin-like compounds, primarily polychlorinated dibenzofurans (PCDFs) and polychlorinated dibenzo-pdioxins (PCDDs), in soils and sediments downstream of Midland, Michigan (USA) were greater than upstream sites and prompted a sitespecific hazard assessment of tree swallows breeding in the associated floodplains. Potential for adverse population-level effects from site-specific contaminant exposures were evaluated at study areas (SAs) along the Tittabawassee and Saginaw rivers downstream of Midland. The site-specific multiple lines of evidence approach to hazard assessment included endpoints for dietary-and tissue-based exposures, and population productivity measurements for tree swallows ([TS]; Tachycineta bicolor) measured during the 2005, 2006, and 2007 breeding seasons. Exposure to dioxin-like compounds in TS eggs were some of the greatest recorded and were similar among all upstream and downstream study sites. Conversely, concentrations in nestlings from SAs were significantly greater compared to reference areas (RAs). The pattern of relative concentrations of PCDD/DFs in eggs and nestlings at RAs was dominated by dioxin congeners, whereas at SAs it was dominated by furan congeners. No statistically significant differences were noted in exposure to PCDD/DFs or in population-level responses when compared among locations, and total clutch failures were rare. Hatching success and fledging success were weakly negatively correlated with concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TEQs) in individual eggs and nestlings, respectively. On-site concentrations of TEQs in floodplain soils were some of the greatest ever reported in the environment, and several lines of evidence indicate potential population-level effects on TS overall reproductive productivity

Effects on Tree Swallows Exposed to Dioxin-like Compounds Associated With: the Tittabawassee River and Floodplain near

Abstract-Concentrations of dioxin-like compounds, primarily polychlorinated dibenzofurans (PCDFs) and polychlorinated dibenzo-pdioxins (PCDDs), in soils and sediments downstream of Midland, Michigan (USA) were greater than upstream sites and prompted a sitespecific hazard assessment of tree swallows breeding in the associated floodplains. Potential for adverse population-level effects from site-specific contaminant exposures were evaluated at study areas (SAs) along the Tittabawassee and Saginaw rivers downstream of Midland. The site-specific multiple lines of evidence approach to hazard assessment included endpoints for dietary-and tissue-based exposures, and population productivity measurements for tree swallows ([TS]; Tachycineta bicolor) measured during the 2005, 2006, and 2007 breeding seasons. Exposure to dioxin-like compounds in TS eggs were some of the greatest recorded and were similar among all upstream and downstream study sites. Conversely, concentrations in nestlings from SAs were significantly greater compared to reference areas (RAs). The pattern of relative concentrations of PCDD/DFs in eggs and nestlings at RAs was dominated by dioxin congeners, whereas at SAs it was dominated by furan congeners. No statistically significant differences were noted in exposure to PCDD/DFs or in population-level responses when compared among locations, and total clutch failures were rare. Hatching success and fledging success were weakly negatively correlated with concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TEQs) in individual eggs and nestlings, respectively. On-site concentrations of TEQs in floodplain soils were some of the greatest ever reported in the environment, and several lines of evidence indicate potential population-level effects on TS overall reproductive productivity

Multiple Lines of Evidence Risk Assessment of Terrestrial Passerines Exposed to PCDFs and PCDDs in the Tittabawassee River Floodplain, Midland, Michigan, USA

A site-specific multiple lines of evidence risk assessment was conducted for house wrens (Troglodytes aedon) and eastern bluebirds (Sialia sialis) along the Tittabawassee River downstream of Midland, Michigan, where concentrations of polychlorinated dibenzofurans (PCDFs) and polychlorinated dibenzo-p-dioxins (PCDDs) in flood-plain soils and sediments are greater compared to upstream areas and some of the greatest anywhere in the world. Lines of evidence supporting the population-level assessment endpoints included site-specific dietary- and tissue-based exposure assessments and population productivity measurements during breeding seasons 2005–2007. While a hazard assessment based on site-specific diets suggested that populations residing in the downstream floodplain had the potential to be affected, concentrations in eggs compared to appropriate toxicity reference values (TRVs) did not predict a potential for population-level effects. There were no significant effects on reproductive success of either species. The most probable cause of the apparent difference between the dietary- and tissue-based exposure assessments was that the dietary-based TRVs were overly conservative based on intraperitoneal injections in the ring-necked pheasant. Agreement between the risk assessment based on concentrations of PCDFs and PCDDs in eggs and reproductive performance in both species supports the conclusion of a small potential for population-level effects at this site

Hepatic P450 Enzyme Activity, Tissue Morphology and Histology of Mink (Mustela vison) Exposed to Polychlorinated Dibenzofurans

Dose- and time-dependent effects of environmentally relevant concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TEQ) of 2,3,7,8-tetrachlorodibenzofuran (TCDF), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), or a mixture of these two congeners on hepatic P450 enzyme activity and tissue morphology, including jaw histology, of adult ranch mink were determined under controlled conditions. Adult female ranch mink were fed either TCDF (0.98, 3.8, or 20 ng TEQTCDF/kg bw/day) or PeCDF (0.62, 2.2, or 9.5 ng TEQPeCDF/kg bw/day), or a mixture of TCDF and PeCDF (4.1 ng TEQTCDF/kg bw/day and 2.8 ng TEQPeCDF/kg bw/day, respectively) for 180 days. Doses used in this study were approximately eight times greater than those reported in a parallel field study. Activities of the cytochrome P450 1A enzymes, ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-deethylase (MROD) were significantly greater in livers of mink exposed to TCDF, PeCDF, and a mixture of the two congeners; however, there were no significant histological or morphological effects observed. It was determined that EROD and MROD activity can be used as sensitive biomarkers of exposure to PeCDF and TCDF in adult female mink; however, under the conditions of this study, the response of EROD/MROD induction occurred at doses that were less than those required to cause histological or morphological changes

Environmental Toxicology RISK ASSESSMENT OF GREAT HORNED OWLS (BUBO VIRGINIANUS) EXPOSED TO POLYCHLORINATED BIPHENYLS AND DDT ALONG THE KALAMAZOO RIVER, MICHIGAN, USA

Abstract-The great horned owl (GHO; Bubo virginianus) was used in a multiple lines of evidence study of polychlorinated biphenyls (PCBs) and p,pЈ-dichlorodiphenyltrichloroethane (DDT) exposures at the Kalamazoo River Superfund Site (KRSS), Kalamazoo, Michigan, USA. The study examined risks from total PCBs, including 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TEQ World Health Organization [WHO]-Avian Toxicity Equivalency Factor [TEF] ), and total DDTs (sum of DDT, dichlorodiphenyldichloroethylene [DDE], and dichlorodiphenyldichloroethane [DDD]; ⌺DDT) by measuring concentrations in eggs and nestling blood plasma in two regions of the KRSS (upper, lower) and an upstream reference area (RA). An ecological risk assessment compared concentrations of the contaminants of concern (COCs) in eggs or plasma to toxicity reference values. Productivity and relative abundance measures for KRSS GHOs were compared with other GHO populations. Egg shell thickness was measured to assess effects of p,pЈ-DDE. The concentrations of PCBs in eggs were as great as 4.7 ϫ 10 2 and 4.0 ϫ 10 4 ng PCB/g, wet weight at the RA and combined KRSS sites, respectively. Egg TEQ WHO-Avian calculated from aryl hydrocarbon receptor-active PCB congeners and WHO TEFs ranged to 8.0 and 1.9 ϫ 10 2 pg TEQ WHO-Avian /g, (wet wt) at the RA and combined KRSS, respectively. Egg ⌺DDT concentrations were as great as 4.2 ϫ 10 2 and 5.0 ϫ 10 3 ng ⌺DDT/g (wet wt) at the RA and combined KRSS, respectively. Hazard quotients (HQs) for the upper 95% confidence interval (UCI) (geometric mean) and least observable adverse effect concentration (LOAEC) for COCs in eggs were Յ1.0 for all sites. Hazard quotient values based on the no observable adverse effect concentration (NOAEC) 95% UCI in eggs were Յ1.0, except at the LKRSS (PCB HQ ϭ 3.1; TEQ WHO-Avian HQ ϭ 1.3). Productivity and relative abundance measures indicated no population level effects in the UKRSS

Environmental Toxicology RISK ASSESSMENT OF GREAT HORNED OWLS (BUBO VIRGINIANUS) EXPOSED TO POLYCHLORINATED BIPHENYLS AND DDT ALONG THE KALAMAZOO RIVER, MICHIGAN, USA

Abstract-The great horned owl (GHO; Bubo virginianus) was used in a multiple lines of evidence study of polychlorinated biphenyls (PCBs) and p,pЈ-dichlorodiphenyltrichloroethane (DDT) exposures at the Kalamazoo River Superfund Site (KRSS), Kalamazoo, Michigan, USA. The study examined risks from total PCBs, including 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TEQ World Health Organization [WHO]-Avian Toxicity Equivalency Factor [TEF] ), and total DDTs (sum of DDT, dichlorodiphenyldichloroethylene [DDE], and dichlorodiphenyldichloroethane [DDD]; ⌺DDT) by measuring concentrations in eggs and nestling blood plasma in two regions of the KRSS (upper, lower) and an upstream reference area (RA). An ecological risk assessment compared concentrations of the contaminants of concern (COCs) in eggs or plasma to toxicity reference values. Productivity and relative abundance measures for KRSS GHOs were compared with other GHO populations. Egg shell thickness was measured to assess effects of p,pЈ-DDE. The concentrations of PCBs in eggs were as great as 4.7 ϫ 10 2 and 4.0 ϫ 10 4 ng PCB/g, wet weight at the RA and combined KRSS sites, respectively. Egg TEQ WHO-Avian calculated from aryl hydrocarbon receptor-active PCB congeners and WHO TEFs ranged to 8.0 and 1.9 ϫ 10 2 pg TEQ WHO-Avian /g, (wet wt) at the RA and combined KRSS, respectively. Egg ⌺DDT concentrations were as great as 4.2 ϫ 10 2 and 5.0 ϫ 10 3 ng ⌺DDT/g (wet wt) at the RA and combined KRSS, respectively. Hazard quotients (HQs) for the upper 95% confidence interval (UCI) (geometric mean) and least observable adverse effect concentration (LOAEC) for COCs in eggs were Յ1.0 for all sites. Hazard quotient values based on the no observable adverse effect concentration (NOAEC) 95% UCI in eggs were Յ1.0, except at the LKRSS (PCB HQ ϭ 3.1; TEQ WHO-Avian HQ ϭ 1.3). Productivity and relative abundance measures indicated no population level effects in the UKRSS

Passerine Exposure to Primarily PCDFs and PCDDs in the River Floodplains Near Midland, Michigan, USA

House wren (Troglodytes aedon), tree swallow (Tachycineta bicolor), and eastern bluebird (Sialia sialis) tissues collected in study areas (SAs) downstream of Midland, Michigan (USA) contained concentrations of polychlorinated dibenzofurans (PCDFs) and polychlorinated dibenzo-p-dioxins (PCDDs) greater than in upstream reference areas (RAs) in the region. The sum of concentrations of PCDD/DFs (ΣPCDD/DFs) in eggs of house wrens and eastern bluebirds from SAs were 4- to 22-fold greater compared to those from RAs, whereas concentrations in tree swallow eggs were similar among areas. Mean concentrations of ΣPCDD/DFs and sum 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (ΣTEQsWHO-Avian), based on 1998 WHO avian toxic equivalency factors, in house wren and eastern bluebird eggs ranged from 860 (430) to 1500 (910) ng/kg wet weight (ww) and 470 (150) to 1100 (510) ng/kg ww, respectively, at the most contaminated study areas along the Tittabawassee River, whereas mean concentrations in tree swallow eggs ranged from 280 (100) to 760 (280) ng/kg ww among all locations. Concentrations of ΣPCDD/DFs in nestlings of all studied species at SAs were 3- to 50-fold greater compared to RAs. Mean house wren, tree swallow, and eastern bluebird nestling concentrations of ΣPCDD/DFs and ΣTEQsWHO-Avian ranged from 350 (140) to 610 (300) ng/kg ww, 360 (240) to 1100 (860) ng/kg ww, and 330 (100) to 1200 (690) ng/kg ww, respectively, at SAs along the Tittabawassee River. Concentrations of ΣTEQsWHO-Avian were positively correlated with ΣPCDD/DF concentrations in both eggs and nestlings of all species studied. Profiles of relative concentrations of individual congeners were dominated by furan congeners (69–84%), primarily 2,3,7,8-tetrachlorodibenzofuran and 2,3,4,7,8-pentachlorodibenzofuran, for all species at SAs on the Tittabawassee and Saginaw rivers but were dominated by dioxin congeners at upstream RAs