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Environmental pollution is a threat to humans and wildlife species. Of particular concern are endocrine disrupting chemicals (EDCs). An important target of EDCs is nuclear receptors (NRs) that control endocrine and metabolic responses through transcriptional regulation. Owing in part to structural differences of NRs, adverse effects of EDCs vary significantly among species. Here, we describe a multiplexed reporter assay (the Ecotox FACTORIAL) enabling parallel assessment of compounds' effects on estrogen, androgen, thyroid, and PPARγ receptors of representative mammals, birds, reptiles, amphibians, and fish. The Ecotox FACTORIAL is a single-well assay comprising a set of species-specific, one-hybrid GAL4-NR reporter constructs transiently transfected into test cells. To harmonize cross-species assessments, we used a combination of two approaches. First, we used the same type of test cells for all reporters; second, we implemented a parallel detection of reporter RNAs. The assay demonstrated excellent quality, reproducibility, and insignificant intra-assay variability. Importantly, the EC50 values for NR ligands were consistent with those reported for conventional assays. Using the assay allowed ranking the hazard potential of environmental pollutants (e.g., bisphenols, polycyclic aromatic hydrocarbons, and synthetic progestins) across species. Furthermore, the assay permitted detecting taxa-specific effects of surface water samples. Therefore, the Ecotox FACTORIAL enables harmonized assessment of the endocrine and metabolic disrupting activity of chemicals and surface water in humans as well as in wildlife species.

Background: Extensive clinical and experimental research documents the potential for chemical disruption of thyroid hormone (TH) signaling through multiple molecular targets. Perturbation of TH signaling can lead to abnormal brain development, cognitive impairments, and other adverse outcomes in humans and wildlife. To increase chemical safety screening efficiency and reduce vertebrate animal testing, in vitro assays that identify chemical interactions with molecular targets of the thyroid system have been developed and implemented., Objectives: We present an adverse outcome pathway (AOP) network to link data derived from in vitro assays that measure chemical interactions with thyroid molecular targets to downstream events and adverse outcomes traditionally derived from in vivo testing. We examine the role of new in vitro technologies, in the context of the AOP network, in facilitating consideration of several important regulatory and biological challenges in characterizing chemicals that exert effects through a thyroid mechanism., Discussion: There is a substantial body of knowledge describing chemical effects on molecular and physiological regulation of TH signaling and associated adverse outcomes. Until recently, few alternative nonanimal assays were available to interrogate chemical effects on TH signaling. With the development of these new tools, screening large libraries of chemicals for interactions with molecular targets of the thyroid is now possible. Measuring early chemical interactions with targets in the thyroid pathway provides a means of linking adverse outcomes, which may be influenced by many biological processes, to a thyroid mechanism. However, the use of in vitro assays beyond chemical screening is complicated by continuing limits in our knowledge of TH signaling in important life stages and tissues, such as during fetal brain development. Nonetheless, the thyroid AOP network provides an ideal tool for defining causal linkages of a chemical exerting thyroid-dependent effects and identifying research needs to quantify these effects in support of regulatory decision making. https://doi.org/10.1289/EHP5297.

Background: High-content imaging (HCI) allows simultaneous measurement of multiple cellular phenotypic changes and is an important tool for evaluating the biological activity of chemicals., Objectives: Our goal was to analyze dynamic cellular changes using HCI to identify the "tipping point" at which the cells did not show recovery towards a normal phenotypic state., Methods: HCI was used to evaluate the effects of 967 chemicals (in concentrations ranging from 0.4 to 200 μM) on HepG2 cells over a 72-hr exposure period. The HCI end points included p53, c-Jun, histone H2A.x, α-tubulin, histone H3, alpha tubulin, mitochondrial membrane potential, mitochondrial mass, cell cycle arrest, nuclear size, and cell number. A computational model was developed to interpret HCI responses as cell-state trajectories., Results: Analysis of cell-state trajectories showed that 336 chemicals produced tipping points and that HepG2 cells were resilient to the effects of 334 chemicals up to the highest concentration (200 μM) and duration (72 hr) tested. Tipping points were identified as concentration-dependent transitions in system recovery, and the corresponding critical concentrations were generally between 5 and 15 times (25th and 75th percentiles, respectively) lower than the concentration that produced any significant effect on HepG2 cells. The remaining 297 chemicals require more data before they can be placed in either of these categories., Conclusions: These findings show the utility of HCI data for reconstructing cell state trajectories and provide insight into the adaptation and resilience of in vitro cellular systems based on tipping points. Cellular tipping points could be used to define a point of departure for risk-based prioritization of environmental chemicals., Citation: Shah I, Setzer RW, Jack J, Houck KA, Judson RS, Knudsen TB, Liu J, Martin MT, Reif DM, Richard AM, Thomas RS, Crofton KM, Dix DJ, Kavlock RJ. 2016. Using ToxCast™ data to reconstruct dynamic cell state trajectories and estimate toxicological points of departure. Environ Health Perspect 124:910-919; http://dx.doi.org/10.1289/ehp.1409029.

We demonstrate a computational network model that integrates 18 in vitro, high-throughput screening assays measuring estrogen receptor (ER) binding, dimerization, chromatin binding, transcriptional activation, and ER-dependent cell proliferation. The network model uses activity patterns across the in vitro assays to predict whether a chemical is an ER agonist or antagonist, or is otherwise influencing the assays through a manner dependent on the physics and chemistry of the technology platform ("assay interference"). The method is applied to a library of 1812 commercial and environmental chemicals, including 45 ER positive and negative reference chemicals. Among the reference chemicals, the network model correctly identified the agonists and antagonists with the exception of very weak compounds whose activity was outside the concentration range tested. The model agonist score also correlated with the expected potency class of the active reference chemicals. Of the 1812 chemicals evaluated, 111 (6.1%) were predicted to be strongly ER active in agonist or antagonist mode. This dataset and model were also used to begin a systematic investigation of assay interference. The most prominent cause of false-positive activity (activity in an assay that is likely not due to interaction of the chemical with ER) is cytotoxicity. The model provides the ability to prioritize a large set of important environmental chemicals with human exposure potential for additional in vivo endocrine testing. Finally, this model is generalizable to any molecular pathway for which there are multiple upstream and downstream assays available., (Published by Oxford University Press on behalf of the Society of Toxicology 2015. This work is written by US Government employees and is in the public domain in the US.)

When assessing risks posed by environmental chemical mixtures, whole mixture approaches are preferred to component approaches. When toxicological data on whole mixtures as they occur in the environment are not available, Environmental Protection Agency guidance states that toxicity data from a mixture considered "sufficiently similar" to the environmental mixture can serve as a surrogate. We propose a novel method to examine whether mixtures are sufficiently similar, when exposure data and mixture toxicity study data from at least one representative mixture are available. We define sufficient similarity using equivalence testing methodology comparing the distance between benchmark dose estimates for mixtures in both data-rich and data-poor cases. We construct a "similar mixtures risk indicator"(SMRI) (analogous to the hazard index) on sufficiently similar mixtures linking exposure data with mixtures toxicology data. The methods are illustrated using pyrethroid mixtures occurrence data collected in child care centers (CCC) and dose-response data examining acute neurobehavioral effects of pyrethroid mixtures in rats. Our method shows that the mixtures from 90% of the CCCs were sufficiently similar to the dose-response study mixture. Using exposure estimates for a hypothetical child, the 95th percentile of the (weighted) SMRI for these sufficiently similar mixtures was 0.20 (i.e., where SMRI <1, less concern; >1, more concern)., (© 2013 Society for Risk Analysis.)

Due to extensive use, human exposure to multiple pyrethroid insecticides occurs frequently. Studies of pyrethroid neurotoxicity suggest a common mode of toxicity and that pyrethroids should be considered cumulatively to model risk. The objective of this work was to use a pyrethroid mixture that reflects human exposure to common pyrethroids to develop comparative toxicokinetic profiles in rats, and then model the relationship between brain concentration and motor activity. Data from a national survey of child care centers were used to make a mixture reflecting proportions of the most prevalent pyrethroids: permethrin, cypermethrin, β-cyfluthrin, deltamethrin, and esfenvalerate. The mixture was administered orally at one of two concentrations (11.2 and 27.4 mg/kg) to adult male rats. At intervals from 1 to 24h, motor activity was assessed and the animals were sacrificed. Pyrethroid concentrations were measured in the blood, liver, fat, and brain. After controlling for dose, there were no differences in any tissue concentrations, except blood at the initial time point. Elimination half-lives for all pyrethroids in all tissues were < 7h. Brain concentrations of all pyrethroids (when cis- and trans-permethrin were pooled) at the initial time point were proportional to their relative doses. Decreases in motor activity indicated dose additivity, and the relationship between pyrethroid brain concentration and motor activity was described by a four-parameter sigmoidal E(max) model. This study links environmental data with toxicokinetic and neurobehavioral assays to support cumulative risk assessments of pyrethroid pesticides. The results support the additive model of pyrethroid effect on motor activity and suggest that variation in the neurotoxicity of individual pyrethroids is related to toxicodynamic rather than toxicokinetic differences.

Recently social concerns have been increasing about the effects of environmental factors on children's health, especially on their nervous systems. The U.S. Environmental Protection Agency (EPA) and the Organisation for Economic Co-operation and Development (OECD) have published testing guidelines for developmental neurotoxicity (DNT). Approximately 110 guideline studies have been conducted to date. Importantly, information from these studies has provided data critical for regulatory decisions for a number of chemicals. However, the DNT guidelines do not always satisfy all stakeholders because of some uncertainties in their methodology, evaluation, and regulation. Methodological issues include incomplete harmonization between EPA and OECD guidelines, criticisms of the methodology for learning and memory testing, and unspecified positive control substances. Potential artifacts in morphometric neuropathological measures, criteria for observation measures, uncertainty of postnatal offspring exposure, especially in feeding studies, and extrapolation of data from rats to humans are major evaluation issues. In addition, there is some uncertainty in the use of an additional safety factor for susceptibility of infants and children. Moreover, the DNT guidelines have extensive time and cost requirements, use large numbers of animals, and there is a limited set of laboratories that can conduct the study. This paper reviews some of these issues and summarizes discussions from the symposium 'Developmental neurotoxicity testing: Scientific approaches towards the next generation to protecting the developing nervous system of children' held at the 2011 annual meeting of the Japanese Teratology Society., (© 2012 The Authors. Congenital Anomalies © 2012 Japanese Teratology Society.)

An adverse outcome pathway (AOP) is a sequence of key events from a molecular-level initiating event and an ensuing cascade of steps to an adverse outcome with population-level significance. To implement a predictive strategy for ecotoxicology, the multiscale nature of an AOP requires computational models to link salient processes (e.g., in chemical uptake, toxicokinetics, toxicodynamics, and population dynamics). A case study with domoic acid was used to demonstrate strategies and enable generic recommendations for developing computational models in an effort to move toward a toxicity testing paradigm focused on toxicity pathway perturbations applicable to ecological risk assessment. Domoic acid, an algal toxin with adverse effects on both wildlife and humans, is a potent agonist for kainate receptors (ionotropic glutamate receptors whose activation leads to the influx of Na(+) and Ca²(+)). Increased Ca²(+) concentrations result in neuronal excitotoxicity and cell death, primarily in the hippocampus, which produces seizures, impairs learning and memory, and alters behavior in some species. Altered neuronal Ca²(+) is a key process in domoic acid toxicity, which can be evaluated in vitro. Furthermore, results of these assays would be amenable to mechanistic modeling for identifying domoic acid concentrations and Ca²(+) perturbations that are normal, adaptive, or clearly toxic. In vitro assays with outputs amenable to measurement in exposed populations can link in vitro to in vivo conditions, and toxicokinetic information will aid in linking in vitro results to the individual organism. Development of an AOP required an iterative process with three important outcomes: a critically reviewed, stressor-specific AOP; identification of key processes suitable for evaluation with in vitro assays; and strategies for model development., (© 2010 SETAC.)

Many chemicals released into the environment display estrogenic activity including the oral contraceptive ethinyl estradiol (EE2) and the plastic monomer bisphenol A (BPA). EE2 is present in some aquatic systems at concentrations sufficient to alter reproductive function of fishes. Many concerns have been raised about the potential effects of BPA. The National Toxicology Program rated the potential effects of low doses of BPA on behavior and central nervous system (CNS) as an area of "some concern," whereas most effects were rated as of "negligible" or "minimal" concern. However, the number of robust studies in this area was limited. The current study was designed to determine if maternal exposure to relatively low oral doses of EE2 or BPA in utero and during lactation would alter the expression of well-characterized sexually dimorphic behaviors or alter the age of puberty or reproductive function in the female Long-Evans rat offspring. Pregnant rats were gavaged with vehicle, EE2 (0.05-50 microg/kg/day), or BPA (2, 20, and 200 microg/kg/day) from day 7 of gestation to postnatal day (PND) 18, and the female offspring were studied. EE2 (50 microg/kg/day) increased anogenital distance and reduced pup body weight at PND2, accelerated the age at vaginal opening, reduced F1 fertility and F2 litter sizes, and induced malformations of the external genitalia (5 microg/kg). F1 females exposed to EE2 also displayed a reduced (male-like) saccharin preference (5 microg/kg) and absence of lordosis behavior (15 microg/kg), indications of defeminization of the CNS. BPA had no effect on any of the aforementioned measures. These results demonstrate that developmental exposure to pharmacologically relevant dosage levels of EE2 can permanently disrupt the reproductive morphology and function of the female rat.

Background: There is increasing evidence in humans and in experimental animals for a relationship between exposure to specific environmental chemicals and perturbations in levels of critically important thyroid hormones (THs). Identification and proper interpretation of these relationships are required for accurate assessment of risk to public health., Objectives: We review the role of TH in nervous system development and specific outcomes in adults, the impact of xenobiotics on thyroid signaling, the relationship between adverse outcomes of thyroid disruption and upstream causal biomarkers, and the societal implications of perturbations in thyroid signaling by xenobiotic chemicals., Data Sources: We drew on an extensive body of epidemiologic, toxicologic, and mechanistic studies., Data Synthesis: THs are critical for normal nervous system development, and decreased maternal TH levels are associated with adverse neuropsychological development in children. In adult humans, increased thyroid-stimulating hormone is associated with increased blood pressure and poorer blood lipid profiles, both risk factors for cardiovascular disease and death. These effects of thyroid suppression are observed even within the "normal" range for the population. Environmental chemicals may affect thyroid homeostasis by a number of mechanisms, and multiple chemicals have been identified that interfere with thyroid function by each of the identified mechanisms., Conclusions: Individuals are potentially vulnerable to adverse effects as a consequence of exposure to thyroid-disrupting chemicals. Any degree of thyroid disruption that affects TH levels on a population basis should be considered a biomarker of adverse outcomes, which may have important societal outcomes.

Carbonyl sulfide (COS), a chemical listed by the original Clean Air Act, was tested for neurotoxicity by a National Institute of Environmental Health Sciences/National Toxicology Program and U.S. Environmental Protection Agency collaborative investigation. Previous studies demonstrated that COS produced cortical and brainstem lesions and altered auditory neurophysiological responses to click stimuli. This paper reports the results of expanded neurophysiological examinations that were an integral part of the previously published experiments (Morgan et al., 2004, Toxicol. Appl. Pharmacol. 200, 131-145; Sills et al., 2004, Toxicol. Pathol. 32, 1-10). Fisher 334N rats were exposed to 0, 200, 300, or 400 ppm COS for 6 h/day, 5 days/week for 12 weeks, or to 0, 300, or 400 ppm COS for 2 weeks using whole-body inhalation chambers. After treatment, the animals were studied using neurophysiological tests to examine: peripheral nerve function, somatosensory-evoked potentials (SEPs) (tail/hindlimb and facial cortical regions), brainstem auditory-evoked responses (BAERs), and visual flash-evoked potentials (2-week study). Additionally, the animals exposed for 2 weeks were examined using a functional observational battery (FOB) and response modification audiometry (RMA). Peripheral nerve function was not altered for any exposure scenario. Likewise, amplitudes of SEPs recorded from the cerebellum were not altered by treatment with COS. In contrast, amplitudes and latencies of SEPs recorded from cortical areas were altered after 12-week exposure to 400 ppm COS. The SEP waveforms were changed to a greater extent after forelimb stimulation than tail stimulation in the 2-week study. The most consistent findings were decreased amplitudes of BAER peaks associated with brainstem regions after exposure to 400 ppm COS. Additional BAER peaks were affected after 12 weeks, compared to 2 weeks of treatment, indicating that additional regions of the brainstem were damaged with longer exposures. The changes in BAERs were observed in the absence of altered auditory responsiveness in FOB or RMA. This series of experiments demonstrates that COS produces changes in brainstem auditory and cortical somatosensory neurophysiological responses that correlate with previously described histopathological damage.

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants that are a potential health hazard to human and wildlife populations. Low-frequency auditory impairments have previously been documented in Aroclor 1254 (A 1254)-exposed rats, including elevated behavioral auditory thresholds and decreased amplitude and prolonged latency auditory evoked brain stem responses (ABRs). Furthermore, outer hair-cell loss on the basilar membrane of the cochlea has been documented, demonstrating that the cochlea is a target organ for PCB ototoxicity. The current experiment assessed the effects of A1254 on cochlear function by measuring distortion product otoacoustic emissions (DPOAEs). ABRs were measured to determine the effects of A1254 on the central nervous system auditory pathways. Pregnant Long-Evans rats received either 0 or 6 mg/kg A1254 (po) in corn oil from gestation day 6 to lactational day 21. The auditory function of male and female offspring was assessed at approximately 18 months of age. The rats were anesthetized and a probe-unit, consisting of 2 insert earphones and a microphone, was positioned in the ear canal. DPOAE amplitudes were reduced and thresholds increased in the A1254-exposed rats. The deficits were most pronounced at the lowest frequencies tested (2.1-3.2 kHz), but deficits were also observed at higher frequencies (3.7-8.6 kHz). Males and females were equally affected at the lower frequencies, but females were more impaired at the higher frequencies. In contrast, ABR latencies and amplitudes were not altered by A1254 exposure. These findings provide the first functional evidence supporting a cochlear site of damage in PCB-induced hearing loss.

Exposure to polychlorinated biphenyls (PCBs) has been shown to detrimentally affect learning and memory in children as well as schedule-controlled behavior in experimental animals. The objective of the present series of experiments was to extend research into the effects of PCBs on behavior maintained under both short (30 s) and long (5 min) fixed-interval (FI) schedules as well as an FI 3-min with reinforcement omission. Long-Evans rats were exposed to 0 or 6 mg/kg/day Aroclor 1254 (A1254) via oral gavage from Gestation Day 6 (GD 6) through Postnatal Day 21 (PND 21). At approximately PND 90, acquisition and steady-state performance were assessed under a series of FI reinforcement schedules consisting of FI 30-s, FI 5-min, and FI 3-min with 33% of the scheduled reinforcers omitted. Performance measures included index of curvature (IOC), response rate, and postreinforcement pause (PRP). There were no effects of A1254 on the acquisition of behavior under the FI 30-s schedule. Subsequently, there was an initial decrease in response rate and IOC and an increase in PRP following the transition from FI 30-s to the FI 5-min; there were, however, no treatment-related effects on any measure. During the reinforcement-omission procedure, there was an increase in the rate of responding and a decrease in IOC and PRP following omission intervals irrespective of treatment. These data are inconsistent with previous findings and suggest that perinatal A1254 exposure in the rat does not disrupt temporally organized behavior.

Thyroid hormone is well-known to play essential roles in brain development. Therefore, environmental factors that interfere with thyroid function or thyroid hormone action may produce deleterious effects on brain development by interfering with thyroid hormone action in the developing brain. The purpose of this review is to identify in broad terms the gaps in our knowledge of thyroid hormone action in brain development, to relate these gaps to present information on thyroid disruption, and to review briefly our recent research that is germane to these issues. The endocrinology of the thyroid system is first reviewed briefly with an emphasis on the neuroendocrine and extrathyroidal mechanisms controlling circulating levels of thyroid hormones. The second section reviews the evidence that thyroid hormone is important for fetal, as well as neonatal, brain development. We review the mechanism of thyroid hormone action in the third section and briefly relate this information to information about the mechanism of thyroid hormone action on brain development. In the final section, we review the endocrinology of thyroid disruption with an emphasis on disruption of thyroid hormone action.

Maternal exposure to polyhalogenated hydrocarbons results in early postnatal hypothyroxenemia and a low-frequency hearing loss in adult offspring (Goldey et al., 1995a. Toxicol. Appl. Pharmacol. 135, 67-76; Herr et al., 1996. Fundam. Appl. Toxicol. 33, 120-128). The purpose of the present work was to determine whether the site-of-action of this auditory impairment was within the cochlea. Primiparous Long-Evans rats were given daily oral doses of corn oil (control) or 8 mg/kg of the commercial PCB mixture Aroclor 1254 (A1254) from gestation day (GD) 6 through postnatal day (PND) 21. Auditory thresholds for 1-, 4-, 16-, and 40-kHz tones were assessed using reflex modification audiometry in young adult offspring on postnatal days (PND) 92-110. Approximately 6 weeks after auditory assessments, a subset of animals (n=4 per group) were killed for histological assessment of the cochlea. Surface preparations of the organ of Corti were prepared from one cochlea per animal and modiolar sections were prepared from the opposite cochlea. Consistent with previous findings, auditory thresholds for 1-kHz tones were elevated by approximately 25 dB in the A1254-exposed animals. Thresholds for all higher frequencies were not different compared to controls. Surface preparations of the organ of Corti revealed a mild to moderate loss of outer hair cells in the upper-middle and apical turns. Inner hair cells were not affected. Modiolar sections failed to reveal alterations in any other cochlear structures. There was also no apparent loss of ganglion cells. These data clearly link the loss of low-frequency hearing caused by exposure during development to A1254 to a loss of outer hair cells in the organ of Corti. The mechanism that underlies this developmental ototoxicity remains to be determined. These data provide the first evidence of a structural deficit in the nervous system of adult animals exposed to PCBs during development.