Predicting Early Life Stage Mortality in Birds and Fishes from Exposure to Low‐Potency Agonists of the Aryl Hydrocarbon Receptor: A Cross‐Species Quantitative Adverse Outcome Pathway Approach

Abstract

Dioxin-like compounds (DLCs) cause early life stage mortality of vertebrates through activation of the aryl hydrocarbon receptor (AhR). A prior study developed a cross-species quantitative adverse outcome pathway (qAOP) which can predict full dose-response curves of early life stage mortality for any species of bird or fish exposed to DLCs using the species-and chemical-specific 50% effect concentration (EC50) from an in vitro AhR transactivation assay with COS-7 cells. However, calculating a reliable EC50 for input into this qAOP requires the maximal response of the concentration-response curve to be known, which is not always possible for low-potency agonists, such as some polychlorinated biphenyls (PCBs). To enable predictions for these low-potency agonists, the present study revised this qAOP to use the effect concentration threshold (EC Threshold) from the in vitro AhR transactivation assay as input. Significant linear relationships were demonstrated between EC Threshold and the dose to cause 0, 10, 50, or 100% mortality among early life stages of 3 species of birds and 7 species of fish for 4 DLCs: 2,3,7,8-tetrachlorodibenzo-p-dioxin, PCB 126, PCB 77, and PCB 105. These 4 linear relationships were combined to form the revised qAOP. This qAOP using the EC Threshold enables prediction of experimental dose-response curves for lower-potency agonists to within an order of magnitude on average, but the prior qAOP using EC50 predicts experimental dose-response curves for higher-potency agonists with greater accuracy.

Alkylation of Benz[a]anthracene Affects Toxicity to Early–Life Stage Zebrafish and In Vitro Aryl Hydrocarbon Receptor 2 Transactivation in a Position‐Dependent Manner

“…Further, compared with TCDD, these PAHs are more potent agonists of the zebrafish AhR2 in the standardized in vitro AhR transactivation assay, despite being significantly less potent at causing early-life stage mortality (Supporting Information, Table S2; Elonen et al, 1998;Zhang et al, 2018). As a result, these PAHs do not fit the previously described relationship between sensitivity to activation of the AhR2 and sensitivity to early-life stage mortality contained in the qAOP (Figure 4; Doering et al, 2018Doering et al, , 2020.…”

“…These interspecies differences in sensitivity are explained by differences in the structure of the AhR2 protein that alter its sensitivity to activation by DLCs (Doering et al, 2015(Doering et al, , 2018Karchner et al, 2006;Wirgin et al, 2011). This understanding of interspecies differences in sensitivity has culminated in the generation of a predictive mechanism-based biological model, referred to as a quantitative adverse outcome pathway (qAOP), that can predict doseresponse curves of early-life stage mortality for any DLC in any species of fish, using results from a standardized in vitro AhR transactivation assay with COS-7 cells transfected with the relevant AhR isoform from the species of interest (Doering et al, 2018(Doering et al, , 2020. Some interspecies variation in sensitivity to PAHs has been demonstrated among birds (Franci et al, 2018;Head et al, 2015); but the extent of interspecies variation among fishes is not well characterized, and whether sensitivity to activation of the AhR2 is predictive of early-life stage mortality of fishes is unknown.…”

“…Best-fit line and 95% confidence interval are shown. Linear regressions for polycyclic aromatic hydrocarbons (gray) alongside corresponding linear regressions for dioxin-like compounds (black [Doering et al, 2018[Doering et al, , 2020) for comparison (C, D). Equations for the lines are y = 0.4003x + 1.788 (A) and y = 0.1371x + 1.627 (B).…”

Sensitivity of a Model Reptile, the Common Snapping Turtle (Chelydra serpentina), to In Ovo Exposure to 2,3,7,8‐Tetrachlorodibenzo‐p‐Dioxin and Other Dioxin‐Like Chemicals

“…However, the quantitative relationship between sensitivity to activation of the AhR1 of birds and early-life mortality is not different from the same relationship for AhR2 of fishes (Doering et al, 2018). This mechanistic knowledge from birds and fishes has been used to develop a cross-species, mechanism-based biological model, known as a quantitative adverse outcome pathway (qAOP), which can accurately predict early-life mortality dose curves for any species of bird or fish exposed to any DLC using only results from an in vitro transactivation assay with a transfected avian AhR1 or fish AhR2 expression construct from the species of interest (Doering et al, 2018(Doering et al, , 2020. This in vitro AhR transactivation assay requires only nonlethal samples (e.g., blood, feather, scale, or biopsied tissue) from as few as a single individual from the species of interest to create the AhR expression construct making it amenable to use with species that are not easily used in laboratory-based toxicity testing, such as many reptiles.…”

Reevaluation of 2,3,7,8‐Tetrachlorodibenzo‐p‐Dioxin Equivalency Factors for Dioxin‐Like Polychlorinated Dibenzo‐p‐Dioxins, Polychlorinated Dibenzofurans, and Polychlorinated Biphenyls for Fishes

“…Tissue explant assays are advantageous in that they maintain paracrine interactions and local tissue structure with all representative cell types in the correct proportion, and thus, they can be more representative of in vivo conditions relative to primary or immortalized cells (Eisner et al, 2019). The qAOP model is capable of predicting results of early life mortality assays for any DLC in any species within the range of variability typical of laboratory-based toxicity testing using as input only the chemical-and species-specific median effect concentration (EC50) from a standardized in vitro AhR transactivation assay of COS-7 cells transfected with the AhR2 of the target species of fish (Doering et al, 2018(Doering et al, , 2020. Only relevant AhR-mediated endpoints were considered in the reevaluation, which included early life mortality; increase in CYP1A measured as either transcript abundance using quantitative real-time polymerase chain reaction, protein content using western blot, or enzyme activity using ethoxyresorufin O-deethylase (EROD); induction of aryl hydrocarbon hydrozylase; or specific binding to the AhR measured by velocity sedimentation on sucrose gradients.…”