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Butylated hydroxytoluene (BHT) is a synthetic antioxidant widely used in many industrial sectors. BHT is a well-studied compound for which there are many favorable regulatory decisions. However, a recent opinion by the French Agency for Food, Environmental and Occupational Health and Safety (ANSES) hypothesizes a role for BHT in endocrine disruption (ANSES (2021). This opinion is based on observations in mostly rat studies where changes to thyroid physiology are observed. Enzymatic induction of Cytochrome P450-mediated thyroid hormone catabolism has been proposed as a mechanism for these observations, however, a causal relationship has not been proven. Other evidence proposed in the document includes a read across argument to butylated hydroxyanisole (BHA), another Community Rolling Action Plan (CoRAP)-listed substance with endocrine disruption concerns. We tested the hypothesis that BHT is an endocrine disruptor by using a Next Generation Risk Assessment (NGRA) method. Four different cell lines: A549, HCC1428, HepG2, and MCF7 were treated with BHT and a series of BHT analogs at 5 different concentrations, RNA was isolated from cell extracts and run on the L1000 gene array platform. A toxicogenomics-based assessment was performed by comparing BHT’s unique genomic signature to a large external database containing signatures of other compounds (including many known endocrine disruptors) to identify if any endocrine disruption-related modes of action (MoAs) are prevalent among BHT and other compounds with similar genomic signatures. In addition, we performed a toxicogenomics-based structure activity relationship (SAR) assessment of BHT and a series of structurally similar analogs to understand if endocrine disruption is a relevant MoA for chemicals that are considered suitable analogs to BHT using the P&G read across framework (Wu et al., 2010). Neither BHT nor any of its analogs connected to compounds that had endocrine activity for estrogens, androgens, thyroid, or steroidogenesis.

Mixture toxicity literature is dominated by water pollutants, pesticides, environmental pollutants, insecticides, soil pollutants, and chemicals described as persistent, bioaccumulative, and toxic.

The sodium-iodide symporter (NIS) mediates the uptake of iodide into the thyroid. Inhibition of NIS function by xenobiotics has been demonstrated to suppress circulating thyroid hormones and perturb related physiological functions. Until recently, few environmental chemicals had been screened for NIS inhibition activity. We previously screened over 1000 chemicals from the ToxCast Phase II (ph1v2 and ph2) libraries using an in vitro radioactive iodide uptake (RAIU) with the hNIS-HEK293T cell line to identify NIS inhibitors. Here, we broaden the chemical space by expanding screening to include the ToxCast e1k library (804 unique chemicals) with initial screening for RAIU at 1 × 10–4 M. Then 209 chemicals demonstrating > 20% RAIU inhibition were further tested in multiple-concentration, parallel RAIU and cell viability assays. This identified 55 chemicals as active, noncytotoxic RAIU inhibitors. Further cytotoxicity-adjusted potency scoring (with NaClO4 having a reference score of 200) revealed five chemicals with moderate to strong RAIU inhibition (scored > 100). These data were combined with our previous PhII screening data to produce binary hit-calls for ~ 1800 unique chemicals (PhII + e1k) with and without cytotoxicity filtering. Results were analyzed with a ToxPrint chemotype-enrichment workflow to identify substructural features significantly enriched in the NIS inhibition hit-call space. We assessed the applicability of enriched PhII chemotypes to prospectively predict NIS inhibition in the e1k dataset. Chemotype enrichments derived for the combined ~ 1800 dataset also identified additional enriched features, as well as chemotypes affiliated with cytotoxicity. These enriched chemotypes provide important new information that can support future data interpretation, structure–activity relationship, chemical use, and regulation.

[Image: see text] Since 2009, the Tox21 project has screened ∼8500 chemicals in more than 70 high-throughput assays, generating upward of 100 million data points, with all data publicly available through partner websites at the United States Environmental Protection Agency (EPA), National Center for Advancing Translational Sciences (NCATS), and National Toxicology Program (NTP). Underpinning this public effort is the largest compound library ever constructed specifically for improving understanding of the chemical basis of toxicity across research and regulatory domains. Each Tox21 federal partner brought specialized resources and capabilities to the partnership, including three approximately equal-sized compound libraries. All Tox21 data generated to date have resulted from a confluence of ideas, technologies, and expertise used to design, screen, and analyze the Tox21 10K library. The different programmatic objectives of the partners led to three distinct, overlapping compound libraries that, when combined, not only covered a diversity of chemical structures, use-categories, and properties but also incorporated many types of compound replicates. The history of development of the Tox21 “10K” chemical library and data workflows implemented to ensure quality chemical annotations and allow for various reproducibility assessments are described. Cheminformatics profiling demonstrates how the three partner libraries complement one another to expand the reach of each individual library, as reflected in coverage of regulatory lists, predicted toxicity end points, and physicochemical properties. ToxPrint chemotypes (CTs) and enrichment approaches further demonstrate how the combined partner libraries amplify structure–activity patterns that would otherwise not be detected. Finally, CT enrichments are used to probe global patterns of activity in combined ToxCast and Tox21 activity data sets relative to test-set size and chemical versus biological end point diversity, illustrating the power of CT approaches to discern patterns in chemical–activity data sets. These results support a central premise of the Tox21 program: A collaborative merging of programmatically distinct compound libraries would yield greater rewards than could be achieved separately.

Per- and polyfluoroalkyl substances (PFAS) are a broad class of hundreds of fluorinated chemicals with environmental health concerns due to their widespread presence and persistence in the environment. Several of these chemicals have been comprehensively studied for experimental toxicity, environmental fate and exposure, and human epidemiology; however, most chemicals have limited or no data available. To inform methods for prioritizing these data-poor chemicals for detailed toxicity studies, we evaluated 142 PFAS using an in vitro screening platform consisting of two multiplexed transactivation assays encompassing 81 diverse transcription factor activities and tested in concentration-response format ranging from 137 nM to 300 μM. Results showed activity for various nuclear receptors, including three known PFAS targets--specifically estrogen receptor alpha and peroxisome proliferator receptors alpha and gamma. We also report activity against the retinoid X receptor beta, the key heterodimeric partner of type II, non-steroidal nuclear receptors. Additional activities were found against the pregnane X receptor, nuclear receptor related-1 protein, and nuclear factor erythroid 2-related factor 2, a sensor of oxidative stress. Using orthogonal assay approaches, we confirmed activity of representative PFAS against several of these targets. Finally, we identified key PFAS structural features associated with nuclear receptor activity that can inform future predictive models for use in prioritizing chemicals for risk assessment and in the design of new structures devoid of biological activity.

Sodium absorption in epithelial cells is rate-limited by the epithelial sodium channel (ENaC) activity in lung, kidney, and the distal colon. Pathophysiological conditions, such as cystic fibrosis and Liddle syndrome, result from water-electrolyte imbalance partly due to malfunction of ENaC regulation. Because the quaternary structure of ENaC is yet undetermined, the bases of pathologically linked mutations in ENaC subunits α, β, and γ are largely unknown. Here, we present a structural model of heterotetrameric ENaC α1βα2γ that is consistent with previous cross-linking results and site-directed mutagenesis experiments. By using this model, we show that the disease-causing mutation αW493R rewires structural dynamics of the intersubunit interfaces α1β and α2γ. Changes in dynamics can allosterically propagate to the channel gate. We demonstrate that cleavage of the γ-subunit, which is critical for full channel activation, does not mediate activation of ENaC by αW493R. Our molecular dynamics simulations led us to identify a channel-activating electrostatic interaction between α2Arg-493 and γGlu-348 at the α2γ interface. By neutralizing a sodium-binding acidic patch at the α1β interface, we reduced ENaC activation of αW493R by more than 2-fold. By combining homology modeling, molecular dynamics, cysteine cross-linking, and voltage clamp experiments, we propose a dynamics-driven model for the gain-of-function in ENaC by αW493R. Our integrated computational and experimental approach advances our understanding of structure, dynamics, and function of ENaC in its disease-causing state.

RNA can fold into complex tertiary structures, much like proteins, and exhibits significant conformational dynamics that are a central facet of its functions. Two limiting types of RNA motions occur: equilibrium fluctuations and induced conformational changes. Equilibrium fluctuations are spontaneous transitions between conformers along the RNA free energy landscape and conformational transitions occur when the free energy landscape is perturbed to generate new minima and change the energy barriers between minima. Surprisingly, little experimental data are available for equilibrium fluctuations for even simple RNA, and even less is known about the driving forces for conformational transitions in large RNAs. In part this is due to limiting technologies. One technology, SHAPE, has overcome many of the limitations for studying large RNAs. SHAPE, for selective 2′-hydroxyl acylation analyzed by primer extension, has revolutionized RNA secondary structure prediction and provides a transformative experimental approach to investigate nucleotide specific equilibrium fluctuations and conformations. SHAPE reactivity is governed primarily by nucleotide flexibility, which in turn is governed by secondary and tertiary structure constraints. In practice, SHAPE provides nucleotide level resolution of RNA structure and dynamics in RNA of any size. We hypothesize that the solvent isotope effect on SHAPE chemistry will allow insights into the roles of hydrogen bonding and solvation on RNA structure and dynamics. Here, we describe the initial experiments and theoretic basis for interpreting this solvent isotope effect.

Telomerase is a special reverse transcriptase that adds repetitive DNA sequences, GGGTTG for Tetrahymena thermophila, at the 3’ end of DNA strand in the telomere region to ensure DNA replication completion. This enzyme is a ribonucleoprotein complex with RNA subunit as a template for synthesis of the repetitive telomeres. Telomerase is a key element to understand cellular aging and tumorigenesis due to its direct impact on chromosome length maintenance. The mechanism with which telomerase adds the six nucleotide repeat is not well-understood with current experimental biochemical and biophysical methods. The lack of three-dimensional structure of telomerase further hinders the current effort to fully understand its crucial biological function. Here, we attempt to propose a 3D structural model of the six catalytic steps using computational modeling with experimental constraints. We perform discrete molecular dynamics simulations with experimental constraints derived from SHAPE chemistry, FRET and crystal structure homology modeling. Our preliminary results reveal interesting structural features and dynamic properties of telomerase in action. Further simulations and detailed computational analysis will allow us to generate experimentally testable hypothesis. The synergetic approaches of computational modeling and experimental validation will help us understand the molecular mechanism of telomerase.

Sodium absorption in epithelial cells is rate-limited by the epithelial sodium channel (ENaC) activity in lungs, kidney and the distal colon. Pathophysiological conditions, such as cystic fibrosis and Liddle syndrome, result from water-electrolyte imbalance partly due to malfunction of ENaC's molecular regulation. The molecular mechanism(s) of pathologically implicated mutations in ENaC subunits are largely unknown due to absence of structural models for ENaC's oligomers. Here, we propose a dynamics-driven mechanism of the gain of function mutation αW493R implicated in atypical cystic fibrosis. We utilize a combination of discrete molecular dynamics simulations (DMD) of the extracellular region of ENaC's heterotrimer αβγ and functional data from whole-cell electrophysiology experiments. Structure-function studies suggest that tetramers and trimers are the major characterized functional oligomeric states of ENaC. Using the crystal structure of the acid sensing ion channel, ENaC's structural homolog in the ENaC/degenerin mechanosensitive ion channel family, we have constructed homology models of ENaC subunits alpha, beta and gamma, in addition to the heterotrimers αβγ, αγβ and the heterotetramer αβαγ. Electrophysiology data show constitutive activity of αW493Rβγ in oocytes indicated by higher open probability and elevated basal activity compared to WT. Our DMD simulation provides an allosteric framework, which agrees with experimental data. W493R rewires the electrostatically mediated inter-residue interaction network in close proximity to W493, resulting in widening of the pore geometry in the outer mouth of the pore in the extracellular region. Rewiring effect of inter-residue interactions in 493R mutant pocket allosterically propagates across the channel resulting in a more stabilized global conformational ensemble of the channel. These findings predict a novel mechanism of ENaC's constitutive activity, in which changes in local dynamics can affect the relative population of the channel's active states and its open probability.