Michael W. Hornung

Adverse outcome pathways: A conceptual framework to support ecotoxicology research and risk assessment

Ecological risk assessors face increasing demands to assess more chemicals, with greater speed and accuracy, and to do so using fewer resources and experimental animals. New approaches in biological and computational sciences may be able to generate mechanistic information that could help in meeting these challenges. However, to use mechanistic data to support chemical assessments, there is a need for effective translation of this information into endpoints meaningful to ecological risk-effects on survival, development, and reproduction in individual organisms and, by extension, impacts on populations. Here we discuss a framework designed for this purpose, the adverse outcome pathway (AOP). An AOP is a conceptual construct that portrays existing knowledge concerning the linkage between a direct molecular initiating event and an adverse outcome at a biological level of organization relevant to risk assessment. The practical utility of AOPs for ecological risk assessment of chemicals is illustrated using five case examples. The examples demonstrate how the AOP concept can focus toxicity testing in terms of species and endpoint selection, enhance across-chemical extrapolation, and support prediction of mixture effects. The examples also show how AOPs facilitate use of molecular or biochemical endpoints (sometimes referred to as biomarkers) for forecasting chemical impacts on individuals and populations. In the concluding sections of the paper, we discuss how AOPs can help to guide research that supports chemical risk assessments and advocate for the incorporation of this approach into a broader systems biology framework.

Comparison of relative binding affinities of endocrine active compounds to fathead minnow and rainbow trout estrogen receptors

Twelve chemicals were tested for binding affinity to rainbow trout liver estrogen receptor (rbtER) and fathead minnow liver ER (fhmER). The chemicals included estradiol (E2), diethylstilbestrol (DES), ethinylestradiol (EE2), estrone (El), estriol, tamoxifen (TAM), genistein (GEN), p-nonylphenol (PNP), p-tert-octylphenol (PTOP), methoxychlor (MXC), testosterone, and methyltestosterone (MT). Relative binding affinity (RBA) was calculated for each chemical as a function of E2 binding to the receptor. The estrogens DES, EE2, and E1 bound with high affinity to both receptors, with respective RBAs of 583, 166, and 28% (fathead minnow) and 179, 89, and 5% (rainbow trout). Relative binding affinity of E3, TAM, and GEN for both fhmER and rbtER were moderate, with values between 0.3 and 5%. The alkylphenols had weak affinity for the ERs with RBAs for the fhmER of 0.1 and 0.01 for PNP and PTOP, respectively. Corresponding values for the rbtER are 0.027 and 0.009. Estradiol ([3H]E2) only partially was displaced from both the fhmER and the rbtER by MXC, T, and MT. Comparison of RBAs of the chemicals tested for fhmER and rbtER indicates that the rank order of RBAs essentially are the same for both species.

Early temporal effects of three thyroid hormone synthesis inhibitors in Xenopus laevis

Thyroid axis disruption is an important consideration when evaluating risks associated with chemicals. Bioassay methods that include thyroid-related endpoints have been developed in a variety of species, including amphibians, whose metamorphic development is thyroid hormone (TH)-dependent. Inhibition of TH synthesis in these species leads to developmental delay, and assays designed to capture these effects take several weeks to complete. In an effort to develop a shorter term approach, the early responses of various endpoints were evaluated in Xenopus laevis throughout 8d of exposure to three TH synthesis inhibitors: methimazole (100mg/L), 6-propylthiouracil (6-PTU) (20mg/L), and perchlorate (4 mg/L). Endpoints included thyroid gland histology and cell numbers, circulating TH concentrations, and thyroidal TH and associated iodo-compounds. Thyroidal 3,5-diodo-L-tyrosine (DIT) and thyroxine (T4) were significantly reduced from day 2 onward by all three chemicals, while 3-monoiodo-L-tyrosine (MIT) was significantly reduced by methimazole and perchlorate, but not by 6-PTU. These reductions were the earliest indicators of TH synthesis inhibition. Histological effects were apparent on day 4 and became more exaggerated through day 8. However, reductions in circulating T4 and increases in thyroid gland cell numbers were not apparent until day 6. Reductions of thyroidal MIT, DIT, and T4 and circulating T4 are indicative of inhibitory effects of the chemicals on TH synthesis. Changes in thyroid histology and cell number represent compensatory effects modulated by circulating TSH. These observations establish a basis for the development of short term amphibian-based methods to evaluate thyroid axis effects using a suite of diagnostic endpoints.

Inhibition of the thyroid hormone pathway in Xenopus laevis by 2-mercaptobenzothiazole

Determining the effects of chemicals on the thyroid system is an important aspect of evaluating chemical safety from an endocrine disrupter perspective. Since there are numerous chemicals to test and limited resources, prioritizing chemicals for subsequent in vivo testing is critical. 2-Mercaptobenzothiazole (MBT), a high production volume chemical, was tested and shown to inhibit thyroid peroxidase (TPO) enzyme activity in vitro, a key enzyme necessary for the synthesis of thyroid hormone. To determine the thyroid disrupting activity of MBT in vivo, Xenopus laevis larvae were exposed using 7- and 21-day protocols. The 7-day protocol used 18-357 μg/L MBT concentrations and evaluated: metamorphic development, thyroid histology, circulating T4, circulating thyroid stimulating hormone, thyroidal sodium-iodide symporter gene expression, and thyroidal T4, T3, and related iodo-amino acids. The 21-day protocol used 23-435 μg/L MBT concentrations and evaluated metamorphic development and thyroid histology. Both protocols demonstrated that MBT is a thyroid disrupting chemical at the lowest concentrations tested. These studies complement the in vitro study used to identify MBT as a high priority for in vivo testing, supporting the utility/predictive potential of a tiered approach to testing chemicals for TPO activity inhibition. The 7-day study, with more comprehensive, sensitive, and diagnostic endpoints, provides information at intermediate biological levels that enables linking various endpoints in a robust and integrated pathway for thyroid hormone disruption associated with TPO inhibition.

Inhibition of Thyroid Hormone Release from Cultured Amphibian Thyroid Glands by Methimazole, 6-Propylthiouracil, and Perchlorate

Thyroid gland explant cultures from prometamorphic Xenopus laevis tadpoles were evaluated for their utility in assessing chemicals for thyroid hormone (TH) synthesis disruption. The response of cultured thyroid glands to bovine thyroid stimulating hormone (bTSH) and the TH synthesis inhibitors methimazole, 6-propylthiouracil, and perchlorate was determined. Thyroid glands continuously exposed for 12 days to graded concentrations of bTSH released thyroxine (T4) in a dose-dependent manner. Over time, the glands appeared to reach a constant daily rate of T4 release. This suggested that the T4 stores in the glands were initially depleted but continuous release was maintained by synthesis of new hormone. The potency of methimazole, 6-propylthiouracil, and perchlorate for inhibiting T4 release was determined using glands cotreated with a single maximally effective bTSH concentration and graded concentrations of chemical. Inhibition of T4 release was dose dependent for all three chemicals. Perchlorate was the most potent inhibitor of T4 release. Methimazole and 6-propylthiouracil exhibited lower potency than perchlorate but similar potency to each other. The IC(50) (mean ± SD) for inhibition of T4 release by the thyroid glands was 1.2 ± 0.55, 8.6 ± 1.3, and 13 ± 4.0 μM for perchlorate, 6-propylthiouracil, and methimazole, respectively. This model system shows promise as a tool to evaluate the potency of chemicals that inhibit T4 release from thyroid glands and may be predictive of in vivo T4 synthesis inhibition in prometamorphic tadpoles.

Tiered High-Throughput Screening Approach to Identify Thyroperoxidase Inhibitors Within the ToxCast Phase I and II Chemical Libraries

High-throughput screening for potential thyroid-disrupting chemicals requires a system of assays to capture multiple molecular-initiating events (MIEs) that converge on perturbed thyroid hormone (TH) homeostasis. Screening for MIEs specific to TH-disrupting pathways is limited in the U.S. Environmental Protection Agency ToxCast screening assay portfolio. To fill 1 critical screening gap, the Amplex UltraRed-thyroperoxidase (AUR-TPO) assay was developed to identify chemicals that inhibit TPO, as decreased TPO activity reduces TH synthesis. The ToxCast phase I and II chemical libraries, comprised of 1074 unique chemicals, were initially screened using a single, high concentration to identify potential TPO inhibitors. Chemicals positive in the single-concentration screen were retested in concentration-response. Due to high false-positive rates typically observed with loss-of-signal assays such as AUR-TPO, we also employed 2 additional assays in parallel to identify possible sources of nonspecific assay signal loss, enabling stratification of roughly 300 putative TPO inhibitors based upon selective AUR-TPO activity. A cell-free luciferase inhibition assay was used to identify nonspecific enzyme inhibition among the putative TPO inhibitors, and a cytotoxicity assay using a human cell line was used to estimate the cellular tolerance limit. Additionally, the TPO inhibition activities of 150 chemicals were compared between the AUR-TPO and an orthogonal peroxidase oxidation assay using guaiacol as a substrate to confirm the activity profiles of putative TPO inhibitors. This effort represents the most extensive TPO inhibition screening campaign to date and illustrates a tiered screening approach that focuses resources, maximizes assay throughput, and reduces animal use.

Thyroid-stimulating hormone (TSH): Measurement of intracellular, secreted, and circulating hormone in Xenopus laevis and Xenopus tropicalis

Thyroid-stimulating hormone (TSH) is an important regulator of the hypothalamic-pituitary-thyroid (HPT) axis in Xenopus laevis. To evaluate the role of this hormone on developing tadpoles, immunologically-based Western blots and sandwich ELISAs were developed for measuring intracellular (within pituitaries), secreted (ex vivo pituitary culture), and circulating (serum) amounts. Despite the small size of the tadpoles, these methods were able to easily measure intracellular and secreted TSH, and circulating TSH was measurable in situations where high levels were induced. The method was validated after obtaining a highly purified and enriched TSH sample using anti-TSH-β antibodies conjugated to magnetic beads. Subsequent mass-spectrometric analysis of the bands from SDS-PAGE and Western procedures identified the presence of amino acid sequences corresponding to TSH subunits. The purified sample was also used to prepare standard curves for quantitative analysis. The Western and ELISA methods had limits of detection in the low nanogram range. While the majority of the developmental work for these methods was done with X. laevis, the methods also detected TSH in Xenopus tropicalis. To our knowledge this is the first report of a specific detection method for TSH in these species, and the first to measure circulating TSH in amphibians. Examples of the utility of the methods include measuring a gradual increase in pituitary TSH at key stages of development, peaking at stages 58-62; the suppression of TSH secretion from cultured pituitaries in the presence of thyroid hormone (T4); and increases in serum TSH following thyroidectomy.

Cross-species analysis of thyroperoxidase inhibition by xenobiotics demonstrates conservation of response between pig and rat

Thyroperoxidase (TPO), the enzyme that catalyzes the synthesis of thyroid hormone, is a known target for thyroid-disrupting chemicals. In vivo toxicological evidence supporting TPO-inhibition as one molecular-initiating event that leads to thyroid disruption is derived largely from rat models; however, a significant fraction of research on the inhibition of TPO by xenobiotics has been conducted using porcine TPO. The current work tested the hypothesis that porcine and rat thyroid microsomes exposed to TPO-inhibiting chemicals would demonstrate different responses in a guaiacol oxidation assay. A primary objective of this work is to establish the degree of concordance between rat and porcine TPO inhibition data. Microsomes were isolated from both rat and pig thyroid glands, and the guaiacol oxidation assay was performed for a training set of 12 chemicals, including previously reported TPO inhibitors, thyroid-disrupting chemicals thought to perturb other targets, and several previously untested chemicals, to determine the relative TPO inhibition responses across species. Concentration-response curves were derived for methimazole (MMI), dibutylphthalate (DBP), diethylhexylphthalate (DEHP), diethylphthalate (DEP), 3,5-dimethylpyrazole-1-methanol (DPM), iopanoic acid (IOA), 2-mercaptobenzothiazole (MBT), sodium perchlorate (PERC), p-nonylphenol (PNP), 4-propoxyphenol (4POP), 6-propylthiouracil (PTU), and triclosan (TCS). MMI, PTU, MBT, DPM, 4POP, and at extremely high concentrations, PERC, inhibited TPO activity. Results demonstrated a strong qualitative concordance of response between the two species. All chemicals that inhibited TPO in porcine microsomes also inhibited TPO in rat microsomes. Hill model-derived IC50 values revealed approximate 1.5- to 50-fold differences in relative potency to MMI between species for positive chemicals. DPM, MBT, 4POP, and PTU exhibited greater relative potency to MMI using rat TPO versus porcine TPO, but rank order potency for inhibition was similar for the other test chemicals, with: PTU>MBT>DPM>4POP>PERC for rat TPO and MBT>PTU>DPM>4POP>PERC for porcine TPO. These data support the extrapolation of porcine TPO data to potential thyroid-disrupting activity in rodent models to evaluate TPO-inhibiting chemicals.

Evaluation of the scientific underpinnings for identifying estrogenic chemicals in nonmammalian taxa using mammalian test systems

The US Environmental Protection Agency has responsibility for assessing endocrine activity of more than 10 000 chemicals, a task that cannot reasonably be achieved solely through use of available mammalian and nonmammalian in vivo screening assays. Hence, it has been proposed that chemicals be prioritized for in vivo testing using data from in vitro high-throughput assays for specific endocrine system targets. Recent efforts focused on potential estrogenic chemicals-specifically those that activate estrogen receptor-alpha (ERα)-have broadly demonstrated feasibility of the approach. However, a major uncertainty is whether prioritization based on mammalian (primarily human) high-throughput assays accurately reflects potential chemical-ERα interactions in nonmammalian species. The authors conducted a comprehensive analysis of cross-species comparability of chemical-ERα interactions based on information concerning structural attributes of estrogen receptors, in vitro binding and transactivation data for ERα, and the effects of a range of chemicals on estrogen-signaling pathways in vivo. Overall, this integrated analysis suggests that chemicals with moderate to high estrogenic potency in mammalian systems also should be priority chemicals in nonmammalian vertebrates. However, the degree to which the prioritization approach might be applicable to invertebrates is uncertain because of a lack of knowledge of the biological role(s) of possible ERα orthologs found in phyla such as annelids. Further, comparative analysis of in vitro data for fish and reptiles suggests that mammalian-based assays may not effectively capture ERα interactions for low-affinity chemicals in all vertebrate classes. Environ Toxicol Chem 2016;35:2806-2816. Published 2016 Wiley Periodicals Inc. on behalf of SETAC. This article is a US Government work and, as such, is in the public domain in the United States of America.

Effects-based chemical category approach for prioritization of low affinity estrogenic chemicals

Regulatory agencies are charged with addressing the endocrine disrupting potential of large numbers of chemicals for which there is often little or no data on which to make decisions. Prioritizing the chemicals of greatest concern for further screening for potential hazard to humans and wildlife is an initial step in the process. This paper presents the collection of in vitro data using assays optimized to detect low affinity estrogen receptor (ER) binding chemicals and the use of that data to build effects-based chemical categories following QSAR approaches and principles pioneered by Gilman Veith and colleagues for application to environmental regulatory challenges. Effects-based chemical categories were built using these QSAR principles focused on the types of chemicals in the specific regulatory domain of concern, i.e. non-steroidal industrial chemicals, and based upon a mechanistic hypothesis of how these non-steroidal chemicals of seemingly dissimilar structure to 17ß-estradiol (E2) could interact with the ER via two distinct binding types. Chemicals were also tested to solubility thereby minimizing false negatives and providing confidence in determination of chemicals as inactive. The high-quality data collected in this manner were used to build an ER expert system for chemical prioritization described in a companion article in this journal.