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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 [ 3 H]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 dioxinlike compounds and suggest an approach to using the AHR as a marker of dioxin susceptibility in wildlife.basic helix-loop-helix-PAS ͉ comparative toxicology ͉ mechanisms ͉ risk assessment ͉ susceptibility

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. These considerations include (1) biological plausibility and (2) empirical support (dose-response, temporality, and incidence) for Key Event Relationships (KERs), and (3) essentiality of key events (KEs). Here, we test the application of these tailored BH considerations and the guidance outlined in the OECD AOP Handbook using a number of case examples to increase experience in more transparently documenting rationales for assigned levels of confidence to KEs and KERs, and to promote consistency in evaluation within and across AOPs. The major lessons learned from experience are documented, and taken together with the case examples, should contribute to better common understanding of the nature and form of documentation required to increase confidence in the application of AOPs for specific uses. Based on the tailored BH considerations and defining questions, a prototype quantitative model for assessing the WoE of an AOP using tools of multi-criteria decision analysis (MCDA) is described. The applicability of the approach is also demonstrated using the case example aromatase inhibition leading to reproductive dysfunction in fish. Following the acquisition of additional experience in the development and assessment of AOPs, further refinement of parameterization of the model through expert elicitation is recommended. Overall, the application of quantitative WoE approaches hold promise to enhance the rigor, transparency and reproducibility for AOP WoE determinations and may play an important role in delineating areas where research would have the greatest impact on improving the overall confidence in the AOP.

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.