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.

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_Ser380 vs common tern--250-fold less sensitive than chicken; Val325_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_Ala380 and Val324_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_Ala380) and Japanese quail (Coturnix japonica; Val324_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.

Relative potencies of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF), and 2,3,7,8-tetrachlorodibenzofuran (TCDF) were determined in vitro in primary hepatocyte cultures of chicken (Gallus gallus), ring-necked pheasant (Phasianus colchicus), and Japanese quail (Coturnix japonica) embryos. Concentration-dependent effects on ethoxyresorufin O-deethylase (EROD) activity and expression of cytochrome P4501A4 and cytochrome P4501A5 (CYP1A4 and CYP1A5) messenger RNA (mRNA) were determined in hepatocytes exposed to serial dilutions of TCDD, PeCDF, or TCDF for 24 h. In chicken hepatocytes, the three compounds were equipotent inducers of EROD activity and CYP1A4/CYP1A5 mRNA expression. However, in ring-necked pheasant and Japanese quail hepatocytes, PeCDF was more potent than TCDD (3- to 5-fold in ring-necked pheasant and 13- to 30-fold in Japanese quail). Among species, the rank order of sensitivity (most to least) to EROD and CYP1A4/CYP1A5 mRNA induction for TCDD and TCDF was chicken > ring-necked pheasant > Japanese quail. In contrast, the three species were approximately equisensitive to EROD and CYP1A4/CYP1A5 mRNA induction by PeCDF. It has generally been assumed that TCDD is the most potent "dioxin-like compound" (DLC) and that the chicken is the most sensitive avian species to CYP1A induction by all DLCs. This study indicates that PeCDF is more potent than TCDD in ring-necked pheasant and Japanese quail hepatocytes and that ring-necked pheasant, Japanese quail, and chicken hepatocytes are equally sensitive to CYP1A induction by PeCDF.

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.

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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00244-009-9416-6) contains supplementary material, which is available to authorized users.

In 1990, a portion of the Kalamazoo River in Michigan, USA, was designated a Superfund site because of the presence of polychlorinated biphenyls (PCBs) in the sediment and floodplain soils. During a four-year period from 2000 to 2003, several avian species were monitored for reproductive effects and concentrations of PCBs in tissues attributed to food chain transfer from contaminated sediments. The tree swallow (Tachycineta bicolor) was chosen as a model receptor for contamination of passerine species. A top-down methodology was used to evaluate the bioaccumulation of PCBs, including non-ortho and mono-ortho congeners, in tree swallow eggs, nestlings, and adults at the Kalamazoo River area of concern (KRAOC) and at an upstream reference site. Generally, a sixfold difference in tissue concentrations of total PCBs was observed between the two sites with concentrations in eggs and nestlings at the KRAOC ranging from 0.95 to 15 microg PCB/g wet weight. Concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TEQsWHO Avian) for PCBs, based on bird-specific World Health Organization toxic equivalence factors, were 10- to 30-fold greater in the KRAOC than at the reference location. Egg and nestling TEQsWHO-Avian ranged from 0.21 to 2.4 ng TEQ/g wet weight at the KRAOC. Hazard quotients calculated from literature-derived toxicity reference values were below 1.0 at both the target and the reference site based on the no-observed-adverse-effect level and the lowest-observed-adverse-effect level.

A series of field studies was conducted to determine the bioaccumulation of polychlorinated biphenyl (PCB) congeners in the terrestrial food web of the Kalamazoo River flood plain. Samples included colocated soils, native plants likely to be consumed by wildlife, several taxa of terrestrial invertebrates, small mammals, passerine bird eggs, nestlings, and adults, and great horned owl plasma and eggs. Mean concentrations of total PCBs in samples from the former Trowbridge impoundment were 6.5 mg/kg dry weight for soils and 0.023, 0.13, 1.3, 1.3, 1.6, and 8.2 mg/kg wet weight for plants, small herbivorous mammals, depurated earthworms, shrews, great horned owl eggs, and house wren eggs, respectively. Historical data from the Kalamazoo River have reported Aroclor-equivalent total PCB concentrations in the terrestrial food web; however, the degree of environmental weathering of the parent PCB mixtures was unknown. In this study, earthworms and composite samples of coleoptera exhibited PCB congener patterns that were similar to patterns in colocated soils. However, in plants, less chlorinated PCBs (e.g., mono-, di-, tri-, and tetrachlorinated biphenyls) were predominant, and in small mammals, there was a notable enrichment of PCBs 153, 180, 138, 118, and 99. In general, concentrations of PCBs were lower in most biota than in soil from the Kalamazoo River Area of Concern (KRAOC) although there was a modest biomagnification of PCBs from lower trophic level biota to highertrophic levels. As a consequence of environmental weathering of PCBs in the terrestrial food web of the KRAOC, the relative potency of the PCBs (expressed as mg TEQs/kg PCBs) decreased from soil to most biota. While there was a general trend, as expected, in which concentrations of 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents (TEQs) increased with total PCBs, this relationship was rather poor (R2 = 0.13). Taken together, these data suggest that the differential accumulation of PCB congeners in the terrestrial food web can be explained by congener-specific differences in bioavailability from soil, exposure pathways, and metabolic potential of each of the food web components.

Approximately 125 km of the Kalamazoo River, located in southwestern Michigan (USA), are designated as a Superfund site, with polychlorinated biphenyls (PCBs) as the contaminant of concern. Mink (Mustela vison) are a naturally occurring predator in this area and also a species of concern because of their known sensitivity to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and structurally similar compounds, such as PCBs. Four of nine mink trapped from the Kalamazoo River area of concern (KRAOC) exhibited histological evidence of a jaw lesion previously identified in ranch mink. The jaw lesion, hyperplasia of squamous epithelium in the mandible and maxilla, is known to be caused by 3,3',4,4',5-pentachlorobiphenyl (PCB 126) and TCDD. Mink trapped from an upstream reference area (Fort Custer Recreation Area [FCRA]) did not exhibit the lesion. Mean concentrations of total PCBs were 2.8 and 2.3 mg/kg wet weight in the livers of mink from the KRAOC and FCRA, respectively, and TCDD toxic equivalent (TEQ) concentrations were 0.30 and 0.11 microg/kg wet weight, respectively. Significant correlations were found between the severity of the lesion and the hepatic concentrations of total PCBs and TEQs. To our knowledge, this is the first published report of the lesion occurring in wild mink.