Evaluation of potential sodium-iodide symporter (NIS) inhibitors using a secondary Fischer rat thyroid follicular cell (FRTL-5) radioactive iodide uptake (RAIU) assay.

The Fischer rat thyroid follicular cell line (FRTL-5) endogenously expresses the sodium-iodide symporter (NIS) and has been used to identify environmental chemicals that perturb thyroid hormone homeostasis by disruption of NIS-mediated iodide uptake. Previously, a high-throughput radioactive iodide uptake (RAIU) screening assay incorporating the hNIS-HEK293T-EPA cell line was used to identify potential human NIS (hNIS) inhibitors in 1028 ToxCast Phase I (ph1_v2) and Phase II chemicals. In this study, the FRTL-5 cell line was evaluated and applied as a secondary RAIU assay coupled with cell viability assays to further prioritize highly active NIS inhibitors from the earlier screening. Assay validation with ten reference chemicals and performance assessment by chemical controls suggest the FRTL-5 based assays are robust and highly reproducible. Top-ranked chemicals from the ToxCast screening were then evaluated in both FRTL-5 and hNIS RAIU assays using newly sourced chemicals to strengthen the testing paradigm and to enable a rat vs. human species comparison. Eighteen of 29 test chemicals showed less than 1 order of magnitude difference in IC50 values between the two assays. Notably, two common perfluorinated compounds, perfluorooctanesulfonic acid (PFOS) and perfluorohexane sulfonate (PFHxS), demonstrated strong NIS inhibitory activity [IC50 - 6.45 (PFOS) and - 5.70 (PFHxS) log M in FRTL-5 RAIU assay]. In addition, several chemicals including etoxazole, methoxyfenozide, oxyfluorfen, triclocarban, mepanipyrim, and niclosamide also exhibited NIS inhibition with minimal cytotoxicity in both assays and are proposed for additional testing using short-term in vivo assays to characterize effects on thyroid hormone synthesis.

An in vitro approach for prioritization and evaluation of chemical effects on glucocorticoid receptor mediated adipogenesis.

Rising obesity rates worldwide have socio-economic ramifications. While genetics, diet, and lack of exercise are major contributors to obesity, environmental factors may enhance susceptibility through disruption of hormone homeostasis and metabolic processes. The obesogen hypothesis contends that chemical exposure early in development may enhance adipocyte differentiation, thereby increasing the number of adipocytes and predisposing for obesity and metabolic disease. We previously developed a primary human adipose stem cell (hASC) assay to evaluate the effect of environmental chemicals on PPARG-dependent adipogenesis. Here, the assay was modified to determine the effects of chemicals on the glucocorticoid receptor (GR) pathway. In differentiation cocktail lacking the glucocorticoid agonist dexamethasone (DEX), hASCs do not differentiate into adipocytes. In the presence of GR agonists, adipocyte maturation was observed using phenotypic makers for lipid accumulation, adipokine secretion, and expression of key genes. To evaluate the role of environmental compounds on adipocyte differentiation, progenitor cells were treated with 19 prioritized compounds previously identified by ToxPi as having GR-dependent bioactivity, and multiplexed assays were used to confirm a GR-dependent mode of action. Five chemicals were found to be strong agonists. The assay was also modified to evaluate GR-antagonists, and 8/10 of the hypothesized antagonists inhibited adipogenesis. The in vitro bioactivity data was put into context with extrapolated human steady state concentrations (Css) and clinical exposure data (Cmax). These data support using a human adipose-derived stem cell differentiation assay to test the potential of chemicals to alter human GR-dependent adipogenesis.

Technical evaluation and standardization of the human thyroid microtissue assay.

The success and sustainability of U.S. EPA efforts to reduce, refine, and replace in vivo animal testing depends on the ability to translate toxicokinetic and toxicodynamic data from in vitro and in silico new approach methods (NAMs) to human-relevant exposures and health outcomes. Organotypic culture models employing primary human cells enable consideration of human health effects and inter-individual variability but present significant challenges for test method standardization, transferability, and validation. Increasing confidence in the information provided by these in vitro NAMs requires setting appropriate performance standards and benchmarks, defined by the context of use, to consider human biology and mechanistic relevance without animal data. The human thyroid microtissue (hTMT) assay utilizes primary human thyrocytes to reproduce structural and functional features of the thyroid gland that enable testing for potential thyroid-disrupting chemicals. As a variable-donor assay platform, conventional principles for assay performance standardization need to be balanced with the ability to predict a range of human responses. The objectives of this study were to (1) define the technical parameters for optimal donor procurement, primary thyrocyte qualification, and performance in the hTMT assay, and (2) set benchmark ranges for reference chemical responses. Thyrocytes derived from a cohort of 32 demographically diverse euthyroid donors were characterized across a battery of endpoints to evaluate morphological and functional variability. Reference chemical responses were profiled to evaluate the range and chemical-specific variability of donor-dependent effects within the cohort. The data-informed minimum acceptance criteria for donor qualification and set benchmark parameters for method transfer proficiency testing and validation of assay performance.

Development of a bioprinter-based method for incorporating metabolic competence into high-throughput in vitro assays.

The acceptance and use of in vitro data for hazard identification, prioritization, and risk evaluation is partly limited by uncertainties associated with xenobiotic metabolism. The lack of biotransformation capabilities of many in vitro systems may under- or overestimate the hazard of compounds that are metabolized to more or less active metabolites in vivo. One approach to retrofitting existing bioassays with metabolic competence is the lid-based Alginate Immobilization of Metabolic Enzymes (AIME) method, which adds hepatic metabolism to conventional high-throughput screening platforms. Here, limitations of the lid-based AIME method were addressed by incorporating bioprinting, which involved depositing S9-encapsulated microspheres into standard 384-well plates with requisite cofactors for phase I and II hepatic metabolism. Objectives of this study included: 1) compare the lid-based and AIME bioprinting methods by assessing the enzymatic activity of a common cytochrome P450 (CYP) enzyme, 2) use biochemical assays with the bioprinting method to characterize additional measures of phase I and II metabolic activity, and 3) evaluate the bioprinting method by screening 25 chemicals of known metabolism-dependent bioactivity in the VM7Luc estrogen receptor transactivation (ERTA) assay. A comparison of the two methods revealed comparable precision and dynamic range. Activity of additional CYP enzymes and glucuronidation was observed using the AIME bioprinting method. The ERTA experiment identified 19/21 ER-active test chemicals, 14 of which were concordant with expected biotransformation effects (73.7%). Additional refinement of the AIME bioprinting method has the potential to expand high-throughput screening capabilities in a robust, accessible manner to incorporate in vitro metabolic competence.

Evaluation of a high-throughput H295R homogenous time resolved fluorescence assay for androgen and estrogen steroidogenesis screening.

The H295R test guideline assay evaluates the effect of test substances on synthesis of 17ß-estradiol (E2) and testosterone (T). The objective of this study was to leverage commercial immunoassay technology to develop a more efficient H295R assay to measure E2 and T levels in 384-well format. The resulting Homogenous Time Resolved Fluorescence assay platform (H295R-HTRF) was evaluated against a training set of 36 chemicals derived from the OECD inter-laboratory validation study, EPA guideline 890.1200 aromatase assay, and azole fungicides active in the HT-H295R assay. Quality control performance criteria were met for all conditions except E2 synthesis inhibition where low basal hormone synthesis was observed. Five proficiency chemicals were active for both the E2 and T endpoints, consistent with guideline classifications. Of the nine OECD core reference chemicals, 9/9 were concordant with outcomes for E2 and 7/9 for T. Likewise, 9/13 and 11/13 OECD supplemental chemicals were concordant with anticipated effects for E2 and T, respectively. Of the 10 azole fungicides screened, 7/10 for E2 and 8/10 for T exhibited concordant outcomes for inhibition. Generally, all active chemicals in the training set demonstrated equivalent or greater potency in the H295R-HTRF assay, supporting the sensitivity of the platform. The adaptation of HTRF technology to the H295R model provides an efficient way to evaluate E2 and T modulators in accordance with guideline specifications.

Protection of cell therapeutics from antibody-mediated killing by CD64 overexpression.

Allogeneic cell therapeutics for cancer therapy or regenerative medicine are susceptible to antibody-mediated killing, which diminishes their efficacy. Here we report a strategy to protect cells from antibody-mediated killing that relies on engineered overexpression of the IgG receptor CD64. We show that human and mouse iPSC-derived endothelial cells (iECs) overexpressing CD64 escape antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity from IgG antibodies in vitro and in ADCC-enabled mice. When CD64 expression was combined with hypoimmune genetic modifications known to protect against cellular immunity, B2M-/-CIITA-/- CD47/CD64-transgenic iECs were resistant to both IgG antibody-mediated and cellular immune killing in vitro and in humanized mice. Mechanistic studies demonstrated that CD64 or its intracellularly truncated analog CD64t effectively capture monomeric IgG and occupy their Fc, and the IgG bind and occupy their target antigens. In three applications of the approach, human CD64t-engineered thyroid epithelial cells, pancreatic beta cells and CAR T cells withstood clinically relevant levels of graft-directed antibodies and fully evaded antibody-mediated killing.

p53-Inducible DHRS3 is an endoplasmic reticulum protein associated with lipid droplet accumulation.

The transcription factor p53 plays a critical role in maintaining homeostasis as it relates to cellular growth, proliferation, and metabolism. In an effort to identify novel p53 target genes, a microarray approach was utilized to identify DHRS3 (also known as retSDR1) as a robust candidate gene. DHRS3 is a highly conserved member of the short chain alcohol dehydrogenase/reductase superfamily with a reported role in lipid and retinoid metabolism. Here, we demonstrate that DHRS3 is an endoplasmic reticulum (ER) protein that is shuttled to the ER via an N-terminal endoplasmic reticulum targeting signal. One important function of the ER is synthesis of neutral lipids that are packaged into lipid droplets whose biogenesis occurs from ER-derived membranes. DHRS3 is enriched at focal points of lipid droplet budding where it also localizes to the phospholipid monolayer of ER-derived lipid droplets. p53 promotes lipid droplet accumulation in a manner consistent with DHRS3 enrichment in the ER. As a p53 target gene, the observations of Dhrs3 location and potential function provide novel insight into an unexpected role for p53 in lipid droplet dynamics with implications in cancer cell metabolism and obesity.

The DevTox Germ Layer Reporter Platform: An Assay Adaptation of the Human Pluripotent Stem Cell Test.

Environmental chemical exposures are a contributing factor to birth defects affecting infant morbidity and mortality. The USA EPA is committed to developing new approach methods (NAMs) to detect chemical risks to susceptible populations, including pregnant women. NAM-based coverage for cellular mechanisms associated with early human development could enhance identification of potential developmental toxicants (DevTox) for new and existing data-poor chemicals. The human pluripotent stem cell test (hPST) is an in vitro test method for rapidly identifying potential human developmental toxicants that employs directed differentiation of embryonic stem cells to measure reductions in SOX17 biomarker expression and nuclear localization. The objective of this study was to expand on the hPST principles to develop a model platform (DevTox GLR) that utilizes the transgenic RUES2-GLR cell line expressing fluorescent reporter fusion protein biomarkers for SOX17 (endoderm marker), BRA (mesoderm marker), and SOX2 (ectoderm and pluripotency marker). Initial assay adaption to definitive endoderm (DevTox GLR-Endo) was performed to emulate the hPST SOX17 endpoint and enable comparative evaluation of concordant chemical effects. Assay duration was reduced to two days and screening throughput scaled to 384-well format for enhanced speed and efficiency. Assay performance for 66 chemicals derived from reference and training set data resulted in a balanced accuracy of 72% (79% sensitivity and 65% specificity). The DevTox GLR-Endo assay demonstrates successful adaptation of the hPST concept with increased throughput, shorter assay duration, and minimal endpoint processing. The DevTox GLR model platform expands the in vitro NAM toolbox to rapidly identify potential developmental hazards and mechanistically characterize toxicant effects on pathways and processes associated with early human development.

Chemical Screening in an Estrogen Receptor Transactivation Assay With Metabolic Competence.

The U.S. EPA continues to utilize high-throughput screening data to evaluate potential biological effects of endocrine active substances without the use of animal testing. Determining the scope and need for in vitro metabolism in high-throughput assays requires the generation of larger data sets that assess the impact of xenobiotic transformations on toxicity-related endpoints. The objective of the current study was to screen a set of 768 ToxCast chemicals in the VM7Luc estrogen receptor transactivation assay (ERTA) using the Alginate Immobilization of Metabolic Enzymes hepatic metabolism method. Chemicals were screened with or without metabolism to identify estrogenic effects and metabolism-dependent changes in bioactivity. Based on estrogenic hit calls, 85 chemicals were active in both assay modes, 16 chemicals were only active without metabolism, and 27 chemicals were only active with metabolism. Using a novel metabolism curve shift method that evaluates the shift in concentration-response curves, 29 of these estrogenic chemicals were identified as bioactivated and 59 were bioinactivated. Human biotransformation routes and associated metabolites were predicted in silico across the chemicals to mechanistically characterize possible transformation-related ERTA effects. Overall, the study profiled novel chemicals associated with metabolism-dependent changes in ERTA bioactivity, and suggested routes of biotransformation and putative metabolites responsible for the observed estrogenic effects. The data demonstrate a range of metabolism-dependent effects across a diverse chemical library and highlight the need to evaluate the role of intrinsic xenobiotic metabolism for endocrine and other toxicity-related health effects.

Characterization of Novel Human Immortalized Thyroid Follicular Epithelial Cell Lines.

Introduction: Investigation of normal human thyroid function using in vitro culture systems is dependent on cells that recapitulate physiology of differentiated thyrocytes. Primary thyrocytes retain features of the native organ but have limited lifespan in culture. Immortalized thyrocytes offer an alternative if challenges maintaining phenotypic stability can be overcome to retain functional features of primary cells. Materials and Methods: CI-SCREEN immortalization technology was applied to normal human thyroid tissue to generate four cell line variants. The lines were characterized for transgene integration, biomarker expression, genomic stability, and proliferation rates. Thyroid Stimulating Hormone (TSH)-dependent morphology, thyroglobulin production, thyroxine hormone synthesis, and viability were assessed using conventional 2D monolayer and 3D microtissue culture formats in huThyrEC or h7H medium. Results: Despite differential transgene profiles, the lines had similar biomarker expression patterns and proliferation rates. In 2D culture there was no thyroxine synthesis or changes in viability, but TSH-dependent thyroglobulin production was more significant for several lines in h7H than huThyrEC medium. Comparatively, in 3D microtissues, TSH-dependent thyroglobulin induction was greater for cell lines in h7H medium. Synthesis of thyroxine in one cell line was higher than background with TSH exposure, but not significantly different than control. Discussion: Immortalization of primary human thyrocytes yielded transgenic lines of epithelial origin. When evaluated in 2D or 3D culture formats, h7H medium supported thyroglobulin production to a greater magnitude than huThyrEC medium. One cell line cultured in 3D microtissue format marginally recapitulated T4 synthesis under continuous TSH exposure. Conclusion: Select human thyroid cell lines exhibited morphological and functional features of primary thyrocytes and are a novel resource for in vitro disease modeling and toxicity testing that will enable reproducible culture models more representative of normal human thyroid function.

Cancer-associated mutations in the MDM2 zinc finger domain disrupt ribosomal protein interaction and attenuate MDM2-induced p53 degradation.

The p53-inhibitory function of the oncoprotein MDM2 is regulated by a number of MDM2-binding proteins, including ARF and ribosomal proteins L5, L11, and L23, which bind the central acidic domain of MDM2 and inhibit its E3 ubiquitin ligase activity. Various human cancer-associated MDM2 alterations targeting the central acidic domain have been reported, yet the functional significance of these mutations in tumor development has remained unclear. Here, we show that cancer-associated missense mutations targeting MDM2's central zinc finger disrupt the interaction of MDM2 with L5 and L11. We found that the zinc finger mutant MDM2 is impaired in undergoing nuclear export and proteasomal degradation as well as in promoting p53 degradation, yet retains the function of suppressing p53 transcriptional activity. Unlike the wild-type MDM2, whose p53-suppressive activity can be inhibited by L11, the MDM2 zinc finger mutant escapes L11 inhibition. Hence, the MDM2 central zinc finger plays a critical role in mediating MDM2's interaction with ribosomal proteins and its ability to degrade p53, and these roles are disrupted by human cancer-associated MDM2 mutations.

The Alginate Immobilization of Metabolic Enzymes Platform Retrofits an Estrogen Receptor Transactivation Assay With Metabolic Competence.

The U.S. EPA Endocrine Disruptor Screening Program utilizes data across the ToxCast/Tox21 high-throughput screening (HTS) programs to evaluate the biological effects of potential endocrine active substances. A potential limitation to the use of in vitro assay data in regulatory decision-making is the lack of coverage for xenobiotic metabolic processes. Both hepatic- and peripheral-tissue metabolism can yield metabolites that exhibit greater activity than the parent compound (bioactivation) or are inactive (bioinactivation) for a given biological target. Interpretation of biological effect data for both putative endocrine active substances, as well as other chemicals, screened in HTS assays may benefit from the addition of xenobiotic metabolic capabilities to decrease the uncertainty in predicting potential hazards to human health. The objective of this study was to develop an approach to retrofit existing HTS assays with hepatic metabolism. The Alginate Immobilization of Metabolic Enzymes (AIME) platform encapsulates hepatic S9 fractions in alginate microspheres attached to 96-well peg lids. Functional characterization across a panel of reference substrates for phase I cytochrome P450 enzymes revealed substrate depletion with expected metabolite accumulation. Performance of the AIME method in the VM7Luc estrogen receptor transactivation assay was evaluated across 15 reference chemicals and 48 test chemicals that yield metabolites previously identified as estrogen receptor active or inactive. The results demonstrate the utility of applying the AIME method for identification of false-positive and false-negative target assay effects, reprioritization of hazard based on metabolism-dependent bioactivity, and enhanced in vivo concordance with the rodent uterotrophic bioassay. Integration of the AIME metabolism method may prove useful for future biochemical and cell-based HTS applications.

Development of an In Vitro Human Thyroid Microtissue Model for Chemical Screening.

Thyroid hormones (TH) are essential for regulating a number of diverse physiological processes required for normal growth, development, and metabolism. The US EPA Endocrine Disruptor Screening Program (EDSP) has identified several molecular thyroid targets relevant to hormone synthesis dynamics that have been adapted to high-throughput screening (HTS) assays to rapidly evaluate the ToxCast/Tox21 chemical inventories for potential thyroid disrupting chemicals (TDCs). The uncertainty surrounding the specificity of active chemicals identified in these screens and the relevance to phenotypic effects on in vivo human TH synthesis are notable data gaps for hazard identification of TDCs. The objective of this study was to develop a medium-throughput organotypic screening assay comprised of reconstructed human thyroid microtissues to quantitatively evaluate the disruptive effects of chemicals on TH production and secretion. Primary human thyroid cells procured from qualified euthyroid donors were analyzed for retention of NK2 homeobox 1 (NKX2-1), Keratin 7 (KRT7), and Thyroglobulin (TG) protein expression by high-content image analysis to verify enrichment of follicular epithelial cells. A direct comparison of 2-dimensional (2D) and 3-dimensional (3D) 96-well culture formats was employed to characterize the morphology, differential gene expression, TG production, and TH synthesis over the course of 20 days. The results indicate that modeling human thyroid cells in the 3D format was sufficient to restore TH synthesis not observed in the 2D culture format. Inhibition of TH synthesis in an optimized 3D culture format was demonstrated with reference chemicals for key molecular targets within the thyroid gland. Implementation of the assay may prove useful for interpreting phenotypic effects of candidate TDCs identified by HTS efforts currently underway in the EDSP.

Molecular characterization of a toxicological tipping point during human stem cell differentiation.

Endoderm gives rise to the gut tube in the early embryo. We differentiated human induced pluripotent stem cells (hiPSCs) to embryonic endoderm to identify a "tipping point" at which the developing system did not recover from perturbations caused by exposure to all-trans retinoic acid (ATRA). Differentiating hiPSC-derived endoderm exposed to five concentrations of ATRA between 0.001 and 10 µM at 6 h, 96 h, or 192 h was assessed for forkhead box A2 (FOXA2) protein expression and global gene transcript expression. A tipping point of 17 ±â€¯11 nM was identified where patterns of differentially expressed genes supported a developmental trajectory shift indicating a potential teratogenic outcome. This concentration is between women's endogenous ATRA blood plasma levels and teratogenic levels of circulating isotretinoin, an ATRA isomer used to treat acne. Taken together, these data suggest that this approach is a sensitive method to identify a point of departure for chemicals that impact early embryonic development.

Identifying qualitative differences in PPARα signaling networks in human and rat hepatocytes and their significance for next generation chemical risk assessment methods.

In this paper, we evaluate the PPARα signaling network in rats, examining transcriptional responses in primary hepatocytes exposed to a PPARα specific ligand, GW7647. These transcriptomic studies were complemented with ChIP-seq studies of PPARα binding and transcription binding motif identification for PPARα responsive genes. We also conducted a limited study of GW7647 dosing the in intact rat to examine differences in transcriptional responses for primary hepatocytes in vitro and in the intact liver. The rat network has a much larger number of down-regulated genes and pathways than we had found in the human and the PPARα binding motifs in rat differed for upregulated and down regulated genes. Based on these results and comparison with our previous work with the human PPARα signaling network, we identified qualitative differences in the transcriptional networks controlled by PPARα activation in the two species that provide an explanation of the interspecies differences in the responses of humans and rodents to GW7647 and likely to other PPARα agonists. These studies also allow some observations on the manner in which in vitro, fit-for-purpose assays in human hepatocytes could form the basis for risk assessment without recourse to in-life studies in rodents or other test species.

The Next Generation Blueprint of Computational Toxicology at the U.S. Environmental Protection Agency.

The U.S. Environmental Protection Agency (EPA) is faced with the challenge of efficiently and credibly evaluating chemical safety often with limited or no available toxicity data. The expanding number of chemicals found in commerce and the environment, coupled with time and resource requirements for traditional toxicity testing and exposure characterization, continue to underscore the need for new approaches. In 2005, EPA charted a new course to address this challenge by embracing computational toxicology (CompTox) and investing in the technologies and capabilities to push the field forward. The return on this investment has been demonstrated through results and applications across a range of human and environmental health problems, as well as initial application to regulatory decision-making within programs such as the EPA's Endocrine Disruptor Screening Program. The CompTox initiative at EPA is more than a decade old. This manuscript presents a blueprint to guide the strategic and operational direction over the next 5 years. The primary goal is to obtain broader acceptance of the CompTox approaches for application to higher tier regulatory decisions, such as chemical assessments. To achieve this goal, the blueprint expands and refines the use of high-throughput and computational modeling approaches to transform the components in chemical risk assessment, while systematically addressing key challenges that have hindered progress. In addition, the blueprint outlines additional investments in cross-cutting efforts to characterize uncertainty and variability, develop software and information technology tools, provide outreach and training, and establish scientific confidence for application to different public health and environmental regulatory decisions.

p53 upregulates PLCε-IP3-Ca2+ pathway and inhibits autophagy through its target gene Rap2B.

The tumor suppressor p53 plays a pivotal role in numerous cellular responses as it regulates cell proliferation, metabolism, cellular growth, and autophagy. In order to identify novel p53 target genes, we utilized an unbiased microarray approach and identified Rap2B as a robust candidate, which belongs to the Ras-related GTP-binding protein superfamily and exhibits increased expression in various human cancers. We demonstrated that p53 increases the intracellular IP3 and Ca2+ levels and decreases the LC3 protein levels through its target gene Rap2B, suggesting that p53 can inhibit the autophagic response triggered by starvation via upregulation of the Rap2B-PLCε-IP3-Ca2+ pathway. As a confirmed target gene of p53, we believe that further investigating potential functions of Rap2B in autophagy and tumorigenesis will provide a novel strategy for cancer therapy.

Editor's Highlight: Screening ToxCast Prioritized Chemicals for PPARG Function in a Human Adipose-Derived Stem Cell Model of Adipogenesis.

The developmental origins of obesity hypothesis posits a multifaceted contribution of factors to the fetal origins of obesity and metabolic disease. Adipocyte hyperplasia in gestation and early childhood may result in predisposition for obesity later in life. Rodent in vitro and in vivo studies indicate that some chemicals may directly affect adipose progenitor cell differentiation, but the human relevance of these findings is unclear. The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARG) is the master regulator of adipogenesis. Human adipose-derived stem cells (hASC) isolated from adipose tissue express endogenous isoforms of PPARG and represent a biologically relevant cell-type for evaluating activity of PPARG ligands. Here, a multi-endpoint approach based on a phenotypic adipogenesis assay was applied to screen a set of 60 chemical compounds identified in ToxCast Phase I as PPARG active (49) or inactive (11). Chemicals showing activity in the adipogenesis screen were further evaluated in a series of 4 orthogonal assays representing 7 different key events in PPARG-dependent adipogenesis, including gene transcription, protein expression, and adipokine secretion. An siRNA screen was also used to evaluate PPARG-dependence of the adipogenesis phenotype. A universal concentration-response design enabled inter-assay comparability and implementation of a weight-of-evidence approach for bioactivity classification. Collectively, a total of 14/49 (29%) prioritized chemicals were identified with moderate-to-strong activity for human adipogenesis. These results provide the first integrated screening approach of prioritized ToxCast chemicals in a human stem cell model of adipogenesis and provide insight into the capacity of PPARG-activating chemicals to modulate early life programming of adipose tissue.

RP-MDM2-p53 Pathway: Linking Ribosomal Biogenesis and Tumor Surveillance.

Ribosomal biogenesis is tightly associated with cellular activities, such as growth, proliferation, and cell cycle progression. Perturbations in ribosomal biogenesis can initiate so-called nucleolar stress. The process through which ribosomal proteins (RPs) transduce nucleolar stress signals via MDM2 to p53 has been described as a crucial tumor-suppression mechanism. In this review we focus on recent progress pertaining to the function and mechanism of RPs in association with the MDM2-p53 tumor-suppression network, and the potential implications this surveillance network has for cancer development.

The Evolution of the Ribosomal Protein-MDM2-p53 Pathway.

The progression of our understanding of ribosomal proteins as static building blocks of the ribosome to highly integrated sensors of p53 surveillance and function has achieved a tremendous rate of growth over the past several decades. As the workhorse of the cell, ribosomes are responsible for translating the genetic code into the functional units that drive cell growth and proliferation. The seminal identification of ribosomal protein binding to MDM2, the negative regulator of p53, has evolved into a paradigm for ribosomal protein-MDM2-p53 signaling that extends into processes as diverse as energy metabolism to proliferation. The central core of signaling occurs when perturbations to rRNA synthesis, processing, and assembly modulate the rate of ribosome biogenesis, signaling a nucleolar stress response to p53. This has led to identification of a number of disease pathologies related to ribosomal protein dysfunction that are manifested as developmental disorders or cancer. Advancing research into the basic mechanics of ribosomal protein-MDM2-p53 signaling is paving the way for novel translational research into biomarker identification and therapeutic strategies for ribosome-related diseases.