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Prioritizing environmental chemicals for obesity and diabetes outcomes research: a screening approach using ToxCast[TM] high-throughput data.

Introduction

The rise in obesity and diabetes rates are major threats to public health in the United States and abroad [Centers for Disease Control and Prevention (CDC) 2011; Dahlquist et al. 2011; DIAMOND Project Group 2006; Ogden and Carroll 2010; Patterson et al. 2009]. Excess caloric consumption and a sedentary lifestyle are well-recognized risk factors for obesity and diabetes. However, there is growing interest in the contribution of "nontraditional" risk factors to these conditions, including environmental chemicals. Research addressing the potential role of environmental chemicals in obesity and diabetes has rapidly expanded in the past several years, and the National Toxicology Program (NTP) has reviewed available information and identified research needs in this area (Behl et al. 2013; Maull et al. 2012; Taylor et al. 2013; Thayer et al. 2012).

One result of the reviews and studies conducted to date is the compilation of a list of molecular pathways whose disruption could increase the risk of obesity or diabetes. A logical step in the search for chemicals that could lead to these diseases is to examine in vitro data that indicate which chemicals may perturb the identified target pathways. To this end, we analyzed high-throughput screening (HTS) data from the U. S. Environmental Protection Agency (EPA) ToxCast[TM] program to identify candidate chemicals for consideration in future research on the environmental causes of obesity and diabetes. It is important not to equate perturbation of one of the diabetes-/obesity-associated pathways with a determination that a chemical causes obesity or diabetes. Bioactivity is one indicator that a chemical has the potential to alter a specific biological process, but whether that altered function produces a phenotypic outcome in an intact animal cannot be determined without further testing. Factors that can modulate the ultimate effects of bioactive chemicals include exposure, pharmacokinetics, diet, and the ability of an intact animal to compensate for the effects of perturbations at the molecular level.

In brief, our strategy was to a) solicit input from experts in the mechanisms of diabetes and obesity who participated in a 2011 NTP workshop, "Role of Environmental Chemicals in the Development of Diabetes and Obesity" (Thayer et al. 2012) to identify assay targets relevant to biological processes related to diabetes and obesity (e.g., insulin sensitivity in peripheral tissue, pancreatic islet and [beta] cell function, adipocyte differentiation, and feeding behavior); and b) identify chemicals that perturb these targets or pathways. These chemicals then become candidates for future research. In this review, we describe the process of identifying pathways, the mapping of pathways to assays, and the identification of chemicals showing significant activity when tested in relevant HTS assays. A major goal of disseminating this information is to encourage the targeted follow-up research that is needed to assess the utility of HTS data for this type of activity.

Methods

An analytical framework to describe the methods described below is presented in Figure 1.

Source In Vitro Data

In this review we analyzed data for an 1,860-compound ToxCast[TM] chemical library. The types of chemicals tested include pesticide actives and inert ingredients, industrial and consumer products, potential "green" chemicals that could be safer alternatives to existing chemicals, in-use and failed pharmaceuticals, and chemicals evaluated in NTP toxicity tests.

ToxCast[TM] currently provides results from [less than or equal to] 821 assay endpoints that make use of numerous technology platforms from 7 vendors (Kavlock et al. 2012) (see Excel File Table S1). These platforms include both cell-free (biochemical) and cell-based measures in multiple human primary cells, human or rodent cell lines, and rat primary hepatocytes (Judson et al. 2010). A wide spectrum of biological targets and effects is covered, including cytotoxicity, cell growth, genotoxicity, enzymatic activity, receptor binding, reporter genes, ion channels, transcription factor activity and downstream consequences, and high-content imaging of cells (Judson et al. 2010). Assays were performed by the individual vendors on uniformly procured chemical samples supplied by the U.S. EPA, and data were provided to the U.S. EPA for normalization and additional processing. In brief, chemicals were tested at 4-15 concentrations depending upon assay complexity, capacity, and cost. The data processing workflow by the U.S. EPA included normalization, curve fitting using Hill equations, visual examination of plots of the concentration-response relationships, and, finally, calculation of the concentration causing half-maximal response (AC50) or, in some platforms, the Lowest Effect Concentration (LEC). The specific criteria for determining the activity of a compound are platform-dependent and are described elsewhere (Kavlock et al. 2012). All analyses utilized the ToxCast[TM] data released in December 2014. In-depth information on the assays, the chemicals, and on ToxCast[TM] data processing can be accessed through the U.S. EPA website (http://actor.epa.gov/dashboard/).

Expert Opinion--Based Approach to Identifying Relevant HTS Gene-Based Assays For Biological Processes

Many of the assays in ToxCast[TM] can be considered "gene-based" because the biochemical activity they assess is linked to a gene or to a set of genes (e.g., peroxisome proliferator-activated receptors [alpha], [delta], and [gamma]; see Excel File Table S1 for a ToxCast[TM] assay list based on annotated gene names). Other assays are related to apical cellular phenotypes (e.g., cell death, mitochondrial damage) and are therefore too complex to map to a single gene or set of genes. In the current analysis, we sought to identify the gene-based assays relevant to the following biological processes related to diabetes or obesity: a) adipocyte differentiation, b) feeding behavior in rodents, c) feeding behavior in Caenorhabditis elegans, d) insulin sensitivity in peripheral tissue, e) pancreatic islet cell function, and f) pancreatic [beta] cell function. With the exception of feeding behavior in rodents, the selected biological processes were considered to be appealing because of the availability of relatively inexpensive and rapid model systems (cell lines, ex vivo, short-term in vivo) that could be used to test hypotheses generated from the HTS results.

We consulted with topic-specific experts to identify relevant ToxCast[TM] gene-based assays for these biological processes (A.H., J.S., S.S., B. Blumberg, D. Clegg, and M. White). In brief, a list of the gene-based assays included in Phase I of ToxCast[TM] with annotated gene names (see Excel File Table S1) was distributed to several participants at the 2011 NTP workshop, "Role of Environmental Chemicals in the Development of Diabetes and Obesity" (Thayer et al. 2012). These experts individually selected the assays that they considered to be the most relevant to the biological processes listed above. The list of gene target assays chosen for each biological process is summarized in Table 1 and is listed by ToxCast[TM] assay names in Excel File Table S2.

ToxPi Analysis of Biological Process Models

We calculated a ToxPi score for each biological process--chemical pair using data from ToxCast[TM].

The ToxPi framework (Filer et al. 2014; Reif et al. 2013) was used to create these scores based on the ToxCast[TM] data for each of the six biological processes selected by the experts. The combination of the genes, assays, and scores for a biological process is called the "biological process model." Each component of the score for a biological process model (a slice in the ToxPi visualization) was equally weighted so that each component/slice had the same potential contribution to the score. It is important to emphasize that this approach only identifies chemicals with predicted absolute effects on these biological pathways and does not necessarily identify the direction of the effect in terms of potentially adverse or therapeutic: for example, pharmaceuticals used to treat diabetes would be expected to affect relevant biological pathways.

The input values for the ToxPi analysis were calculated as follows from the AC50 (concentration at half-maximal activity) and the z-score (the distance from cytotoxicity; higher z-scores indicate increased potency from the chemical-specific cytotoxicity distribution) values provided in the December 2014 ToxCast[TM] release. First, the AC50 values were transformed to negative log molar units. For example, an active chemical-assay pair with an AC50 value of 1 [micro]M would have a negative log-transformed value of 6. Second, inactive chemical-assay pairs or chemical-assay pairs with a z-score [less than or equal to] 2 were assigned values of 0. Third, for active chemical-assay pairs, the z-score was added to the transformed AC50 value. For example, a chemical-assay pair with an [AC.sub.50] value of 1 [micro]M and a z-score of 5.4 would have an input value of 11.4 (transformed [AC.sub.50] value of 6 + z-score value of 5.4).

Exclusion of chemical-assay pairs with z-score values [less than or equal to] 2 accounts for a phenomenon referred to as the "cytotoxic signal burst," which manifests itself as an increase in nonspecific assay activation near concentrations where cell stress and cytotoxicity occur (R. Judson, personal communication). Selecting a cutoff value of 2 eliminates the majority of what appear to be cell stress/cytotoxicity-related false positive activities in the assay data while retaining marginal or ambiguous hits (R. Judson, personal communication). To positively weight more specific responses (higher potency relative to cytotoxicity), the z-scores were added to their respective potency values.

Briefly, the ToxPi scores were calculated by summing the input values across all assays in a component/slice for each chemical. The summed values of the individual assays were then transformed to range from 0 to 1 by subtracting the minimum value and dividing by the range. The values were then multiplied by the proportional weight for that component/slice (1 divided by the number of slices for equally weighted slices, as presented here) to give the component score. The final ToxPi score was calculated by summing each component score, and ranged from 0 to 1, where a ToxPi score of 1 would mean that chemical was the most potent chemical in each component/slice of the model. Note that because some regions of the data matrix are sparse, this approach is only useful for an initial identification of candidate positive chemicals but will miss others for which testing data are not available.

"Signpost" Chemicals for Metabolic Disorders Included in Phase 2 of ToxCast[TM]

To provide context for the HTS data, we compared the screening results for several chemicals included in ToxCast[TM] to findings from the published literature. To identify signpost chemicals, we used a previous summary provided as background material for the 2011 NTP workshop "Role of Environmental Chemicals in the Development of Diabetes and Obesity" (National Toxicology Program, see "Literature Review Documents," Thayer et al. 2012) or as documented in clinical observations of drug effects (Dang et al. 2005; Sheehan 2005). The following chemicals tested in ToxCast[TM] were used as signpost chemicals: troglitazone, tributyltin chemicals, nicotine, haloperidol and chlorpromazine, tolazamide, amitraz, dexamethasone, nicotinic acid (niacin), and chlorinated persistent organic pollutants (POPs). Other environmental chemicals of interest, such as bisphenol A and phthalates, were not considered signpost chemicals because of uncertainties related to the consistency and/or interpretation of findings at the time of the 2011 NTP workshop or in a subsequent systematic review (Kuo et al. 2013; Maull et al. 2012; Taylor et al. 2013). To the best of our knowledge, high-quality reviews (i.e., reviews that adhere to systematic review methodology and reporting standards) have not been published more recently than those mentioned above. However, bisphenol A, phthalates, and other environmental chemicals studied for metabolic effects that were included in ToxCast[TM] (including metabolites and other members of the same chemical class) are highlighted in the ToxPi graphics.

Chemical concordance could not be evaluated using a more systematic comparison because diabetes and obesity-related outcomes are not standard end points in toxicological studies; therefore, these end points are not available for the majority of the environmental chemicals or drugs tested in ToxCast[TM]. In addition, a number of environmental chemicals and drugs associated with diabetes, weight gain, or other metabolic effects have not yet been tested in ToxCast[TM], including atypical antipsychotics (Taylor and McAskill 2000), arsenic (Maull et al. 2012), and certain organochlorine chemicals (Taylor et al. 2013).

Chemical--Chemical Correlation Analysis

To complement the scores for the specific biological processes, a correlation analysis was performed for each chemical--chemical pair across all ToxCast[TM] assays within the subset of ToxCast[TM] chemicals that had the most complete testing coverage (1,061 of the 1,860 chemicals across 685 of the 821 assays). Unlike the biological process analysis, the correlation analysis was limited to the subset of the ToxCast[TM] chemicals with the most complete testing coverage (ToxCast[TM] Phase I and II chemicals) to minimize the impact of missing data in the correlation profiles. Pearson's correlation values for each chemical--chemical pair were calculated on complete pair-wise observations using only transformed z-score values (see below) from each assay. This approach compares the assay-specific profiles of the chemicals across all assays. In addition, we note that this analysis is independent of the genes and pathways that were annotated to assays and used in the above-mentioned metabolic disease biological process models. The z-score values were transformed by binning values into six categories, with the last four indicating increasing specificity of the metabolic bioactivity:

* Chemical-assay pairs not tested = N/A

* Tested, inactive or only tested at single concentration and presumed inactive = 0

* Tested, active, and z-score < 3 = 1

* Tested, active, and 3 < z-score [less than or equal to] 6 = 2

* Tested, active, and 6 < z-score [less than or equal to] 9 = 3

* Tested, active, and z-score > 9 = 4

This procedure provided, for each chemical, a list of chemicals ranked by overall bioassay similarity across the larger ToxCast[TM] assay suite as a way to complement the biological process models. The similarity profiling provided a list of additional candidate chemicals to consider for targeted research and could potentially provide the basis for developing chemotypes for metabolic disorders.

Chemical Clustering Based Upon ToxPi Similarity

Principal components analysis (PCA) was performed on the feeding behavior (C. elegans) ToxPi output matrix to illustrate an approach for identifying similar clusters of compounds. First, we selected all principal components (PCs) that explained [greater than or equal to] 5% of the overall variance. Second, we performed k-means clustering on the reduced PCs matrix using 10,000 iterations and a maximum number of clusters (k) equal to the dimensions of the reduced PCs matrix. Third, we plotted the PCs of each chemical as points colored by cluster, plus the mean ToxPi profiles of each cluster.

Calculations

All calculations and analyses were performed using R (R Core Team 2014). Source data are available at http://www.epa.gov/chemicalresearch/toxicity-forecaster-toxcasttm-data, and R-code is available as supplemental material (R-scripts folder).

Results

Overview of Relative Biological Process Model Results

The top 30 chemicals for each biological process model are listed in Table 2 (also shown as ToxPi graphics in Figures S1-S6). The biological process model scores for all 1,860 chemicals are available in Excel File Tables S3-S8, where chemicals can be sorted by overall score for a given biological process model or for individual components/slices: for example, peroxisome proliferator-activated receptor gamma (PPAR[gamma]) or glucocorticoid receptor (GR) activity. These tables also contain information on chemical properties (i.e., logP, estimated percent human oral absorption), which can be used to further prioritize targeted follow-up research efforts. In Figures S1-S6, we also indicate how other chemicals of high research interest for metabolic effects, such as bisphenol A, phthalate metabolites, perfluorooctane sulfonate (PFOS), perfluorooctanoic acid (PFOA), and several organophosphates and their metabolites, ranked in our analysis. In many cases, heavily studied chemicals (or metabolites) were not included in the top 30 chemicals for the biological processes. The chemical structures represented in the top 30 lists for each biological process are diverse (see Excel File Table S18, Figures S9-S12).

Pharmaceuticals were among the highest-scoring chemicals, and their known mechanistic target(s) were often identified in ToxCast[TM]. For example, the dopaminergic activities of haloperidol and chlorpromazine hydrochloride (both antipsychotic medications) and the PPAR[gamma] activity of farglitazar (a PPAR[gamma] agonist developed for treatment of hepatic fibrosis) were detected.

Of the top 200 ranked chemicals in the adipocyte differentiation process, 138 were identified as having retinoic acid receptor (RAR) agonist activity by the same methods as those described for other assays included in the adipocyte model. These chemicals may not stimulate adipocyte differentiation because activation of RAR can block downstream signaling (Bonet et al. 2012; Frey and Vogel 2011). Researchers interested in using the prioritization results from the adipocyte differentiation prediction process (see Figure S1) should also review the RAR ToxCast[TM] activity data presented in Excel File Table S3 (see column T, "RAR_score").

Chemical Clustering Based upon a Model of Feeding Behavior in C. Elegans

PCA followed by k-means clustering was used to illustrate how ToxPi output can be translated into multidimensional similarity scores of activity across slices. Figure S13 shows the mean ToxPi profiles of feeding behavior in C. elegans for the three clusters with the highest overall ToxPi scores (see Table 1 for an explanation of the component assays in each slice). The 24 chemicals with the highest average ToxPi scores ("Cluster 3") were characterized by activity on slices representing Other, OtherHTR, HTr1, DRD2, and HTR2C, such as chlorpromazine hydrochloride. The 11 chemicals with the second-highest average ToxPi scores ("Cluster 2") were characterized by activity on slices representing Other and NPY, such as the pharmaceutical AVE6324. The 15 chemicals with the third-highest average ToxPi scores ("Cluster 7") were characterized by activity on slices representing Other, PPARd, and INSR, such as the pharmaceutical PharmaGSID_47315. The remaining clusters further partition the variation within ToxPi scores into clusters of similar activity, including a large cluster of 1,200 chemicals representing negligible (or no) activity in this model.

Signpost Chemicals

Most of the signpost chemicals (10 of 12 chemicals or classes of chemicals) would have been prioritized as chemicals of interest using a criterion of being in the top 10% most highly ranked in one or more biological process models. Most organochlorine chemicals included in ToxCast[TM] would not have been prioritized because they were not ranked highly in any biological process (DDT isomers, heptachlor expoxide, mirex, dieldrin, lindane), and nicotinic acid and [beta]-hexachlorocyclohexane ranked in the top 15% of only one biological process; therefore, they likely would not be flagged as chemicals of high interest. In the following sections, we discuss the findings for each signpost chemical (or chemical class) in detail.

Signpost chemicals prioritized in the prediction models. Troglitazone. Troglitazone is an antidiabetic drug that decreases insulin resistance by increasing adipocyte differentiation via activation of PPAR[gamma] (Sheehan 2005). Its use has been associated with weight gain in humans, and it is used as a positive control compound in cellular models of adipogenesis (another widely used positive control compound, rosiglitazone, is not currently included in the ToxCast[TM] library). The PPAR[gamma] activity of troglizazone was identified in ToxCast[TM], and it was ranked highly (in the top 5-10%) for adipocyte differentiation, feeding behavior (C. elegans), insulin sensitivity, and in the biological process models for [beta] cell function (Table 3). Chemicals that have similar activity to troglitazone across the ToxCast[TM] assay set are shown in Table 4, and the full correlation analysis set is available in Excel File Table S9.

Tributyltin chemicals. Trisubstituted organotins, such as tributyltin (TBT), were previously used as biocides for antifouling paints to slow the growth of aquatic organisms, but they are now extremely restricted for use in inland waterways. TBT has been shown to stimulate adipocyte differentiation (in vitro and in vivo) and to increase the amount of fat tissue in adult animals exposed to TBT during fetal life or weaning (Grun and Blumberg 2006; Kirchner et al. 2010) and transgenerationally in the F3 generation following direct treatment to the F0 generation (Chamorro-Garcia et al. 2013). TBT is a potent agonist for both PPAR[gamma] and retinoid X receptor alpha (RXR[alpha]), two receptors that heterodimerize and are known to promote adipocyte differentiation in vitro when activated (Grun et al. 2006). It should be noted that the in vitro profiles of the tin compounds are among the most complex of any of the compounds tested, with hundreds of assays being activated.

The biological process models identified tributyltin compounds, in the form of tributyltin benzoate, tributyltin methacrylate, and tributyltin chloride, as chemicals of interest (Table 3). ToxCast[TM] also detected interactions with dopaminergic, adrenergic, and serotonin receptors at relatively low concentrations ([AC.sub.50] [less than or equal to] 10 [micro]M) for tributyltin chloride and tributyltin methacrylate (data not shown). Chemicals exhibiting similar patterns of activity to those of tributylin chloride are shown in Table 4. The full correlation analysis sets for tributyltin chloride and tributyltin methacrylate are available in Excel File Tables S10 and S11 (tributyltin benzoate was not included in the chemical set used for correlation analyses).

The adipogenic effects of TBT associated with PPAR[gamma] and RXR[alpha] activation have been documented, but its effects on insulin sensitivity have not been throughly explored. It should be noted that a diabetic phenotype for triphenyltin (TPT) has been reported in the literature (see "Organotins and Phthalates Literature Review Documents," Thayer et al. 2012). Studies suggest that rats and mice may be relatively insensitive models for studying the effects of organotins on glucose regulation (Zuo et al. 2011) and that rabbits and hamsters may be more sensitive (Matsui et al. 1984; Ohhira et al. 1999). The diabetic phenotype appears to be transient (Ogino et al. 1996), with no histological abnormalities noted in the islet cells (Matsui et al. 1984; Miura et al. 1997). Implicated mechanisms include reduction of [Ca(2+)](i) and insulin secretion in response to K(ATP) channel-dependent depolarization, and related decreases of NAD(P)H and ATP production during glucose metabolism in pancreatic islet cells (Miura et al. 1997, 2012; Miura and Matsui 2001, 2006; Watanabe et al. 2002).

Nicotine. Nicotine is a parasympathomimetic agent that is present in the nightshade family of plants. It acts as a pharmacological stimulant through the activation of nicotinic acetylcholine receptors. Inhaling tobacco smoke from either active or passive (e.g., second-hand smoke) smoking is the main source of nicotine exposure for the general population (CDC 2013). Epidemiological data support a positive association between maternal smoking and increased risk of obesity or overweight in children after infancy (Behl et al. 2013; Ino 2010; Oken et al. 2008). These data were considered strongly suggestive of a causal relationship by participants in the 2011 NTP workshop and are supported by findings from animal studies (Behl et al. 2013). The association with obesity or overweight following exposure during development is different from effects that occur with exposure later in life, where smoking is known to suppress appetite, and adult smokers tend to gain weight after smoking cessation (Yang et al. 2013; Zoli and Picciotto 2012). Rats exposed to nicotine during perinatal development tended to have higher body weight and more fat mass compared with controls; typically, the effect first became apparent at weaning and persisted through adulthood (Behl et al. 2013). The mechanism(s) by which nicotine might be acting are not well established, but studies have suggested that nicotine alters brain circuitry by affecting leptin signalling in the hypothalamus; a role is also implicated for central hypothyroidism induced by a hypothalamic--pituitary dysfunction (Behl et al. 2013; see "Maternal Smoking During Pregnancy/Nicotine Literature Review Documents," Thayer et al. 2012). The feeding behavior in rodent models identified nicotine as a chemical of interest (ranked 26, in the top 5% of chemicals) (Table 3). The nicotine metabolite cotinine did not rank highly in any biological process model.

Nicotine was considered active on three assay targets at an [AC.sub.50] of < 10 [micro]M, binding to human nicotinic cholinergic receptor, alpha 2 (CHRNA2) and rodent cholinergic receptor, nicotinic, alpha 7 (Chrna7) at [AC.sub.50] values of 0.62 and 1.69 [micro]M, respectively, and up-regulating estrogen-related receptor alpha (ERR[alpha], or gene symbol ESRRA) at an [AC.sub.50] value of 3.39 [micro]M. The high rank of nicotine for feeding behavior in rodents was based mostly on interactions with ERR[alpha]. Although the binding interactions with nicotinic cholinergic receptors were expected, the interaction with ERR[alpha] has not been previously identified and is of interest given the apparent role of ERRs in regulating adipogenesis, energy homeostasis, diabetes, and heart disease (Bonnelye and Aubin 2013; Deblois and Giguere 2011; Ju et al. 2012; Ranhotra 2010; Villena and Kralli 2008). Chemicals exhibiting the greatest similarity in activity across the ToxCast[TM] assays are shown in Table 4, and the full correlation analysis set is available in Excel File Table S12.

Haloperidol and chlorpromazine. Haloperidol and chlorpromazine are primarily used for the treatment of schizophrenia and have been associated with weight gain in patients (Musil et al. 2015; Sheehan 2005). The effects of these drugs on both schizophrenia and weight gain appear to be mediated through a blockade of a number of G-protein coupled receptors that mediate the effects of serotonin, histamine, and dopamine. Individuals taking haloperidol experience increased appetite and sedation along with a decrease in basal metabolic rate. Both haloperidol and chlorpromazine were ranked in the top 5% most active chemicals in biological process models for feeding behavior in C. elegans and in models for islet and [beta] cell function (Table 3), suggesting that these chemicals would have been effectively prioritized for potential effects on metabolic function. Chemicals with activity profiles similar to that of haloperidol are shown in Table 4. The full correlation analysis sets for both haloperidol and chlorpromazine are available in Excel File Tables S13 and S14, respectively.

Tolazamide. Tolazamide is a sulfonylurea drug used to treat diabetes. Sulfonylureas have been associated with hypoglycemia and weight gain in patients (Dang et al. 2005; Sheehan 2005). As pharmaceuticals, sulfonylurea derivatives help control diabetes by increasing insulin secretion from [beta] cells, which results in a lowering of blood glucose. More specifically, sulfonylureas bind with high affinity to the sulfonylurea receptor-1 subunit (SUR1) of the ATP-sensitive potassium channel [K(ATP)] in pancreatic [beta] cells (Thevenod 2002). Sulfonylurea binding causes K(ATP) channels to close, reducing potassium conductance and leading to membrane depolarization. Membrane depolarization leads to the opening of calcium channels and the entry of [Ca.sup.+2] ions into the [beta] cell, which then triggers insulin secretion and a subsequent decrease in blood glucose levels (NLM 2014). The stimulation of insulin secretion by sulfonylureas, though beneficial in the short term, may cause pancreatic damage because of overstimulation, which may in turn cause an increase in reactive oxygen species, endoplasmic reticulum stress, mitochondrial dysfunction, and [beta] cell death (Remedi and Nichols 2008). Although SUR1 is not included in ToxCast[TM], tolazamide was ranked in the top 5-10% in the islet and [beta] cell function models, in the top 10% for insulin sensitivity, and in the top 15% for feeding behavior in rodents (Table 3). The high ranking of tolazamide in the insulin sensitivity, islet cell, and [beta] cell models was based on binding assay results for the ATP-sensitive potassium inwardly rectifying channel (KCNJ11) gene, which is commonly associated with diabetes of genetic origin (Greeley et al. 2011). K(ATP) channels are found in the cell membranes of pancreatic [beta] cells and open and close in response to blood glucose levels. At least 30 mutations in the KCNJ11 gene have been identified in people with permanent neonatal diabetes mellitus (Greeley et al. 2011; Karges et al. 2011; NLM 2014), and mutations are also associated with gestational diabetes mellitus (Zhang et al. 2013). Mutations prevent K(ATP) channels from closing, which leads to reduced insulin secretion from [beta] cells and impaired blood-sugar control. Several environmental chemicals also ranked highly for insulin sensitivity, islet cell, or [beta] cell function based on the KCNJ11 binding assay, including 2,4,6-trichlorophenol, 4-nitrotoluene, ethofumesate and fluometuron. Chemicals with activity profiles to similar to that of tolazamide are shown in Table 4, and the full correlation analysis set is available in Excel File Table S15.

Sulfonylurea herbicides have been used since the early 1980s for the control of nuisance broadleaf weeds and grasses; these herbicides are taken up by the roots and foliage and act by disrupting protein synthesis. They have high toxicity toward plant growth, low application rates, and they are considered to have low toxicity in mammalian studies (Fletcher et al. 1994). General population exposure to sulfonylurea herbicides is not expected to be high because of the low application rates for these herbicides. This assumption is supported by NHANES data showing median urinary levels below the limit of detection for the 17 sulfonylureas included in the biomonitoring program (CDC 2013). Five sulfonylurea herbicides are included in ToxCast[TM] (under the chemical class "metsulfuron-like") with generally low rankings except for being in the top ~10% for adipocyte differentiation (thifensulfuron-methyl) and feeding behavior in rodents (flucarbazone-sodium).

Amitraz. Amitraz is a formamidine insecticide that has been reported to cause hyperglycemia in children and adults following accidental or deliberate poisoning ("Pesticides Literature Review Documents," Thayer et al. 2012). The mechanism of action for amitraz as an insecticide is not completely clear but appears to involve alpha-adrenergic agonism, interference with octopamine (the invertebrate equivalent of norepinephrine) action in the central nervous system, uncoupling of oxidative phosphorylation, and inhibition of monoamine oxidases and prostaglandin synthesis [Bonsall and Turnbull 1983; California Environmental Protection Agency (CalEPA) 1995]. Amitraz has also been shown to cause hyperglycemia in dogs (Hsu and Schaffer 1988; Hugnet et al. 1996) and worker honeybees (Cascino et al. 1989) and impaired glucose tolerance in rats (Smith et al. 1990). The hyperglycemia in dogs and the impaired glucose tolerance in rats are accompanied by hypoinsulinemia (Hsu and Schaffer 1988; Hugnet et al. 1996; Smith et al. 1990). The effects of amitraz on glucose are attributed to the activation of [alpha]-2 adrenoreceptors, which suppress insulin secretion when activated, presumably through cellular responses that ultimately lead to lower [Ca.sup.2+] concentrations in the cytosol of islet cells (Abu-Basha et al. 1999; Chen and Hsu 1994).

The [alpha]-2 adrenergic receptor interactions of amitraz were identified in ToxCast[TM]. The [AC.sub.50] values for amitraz for [alpha]-2A and [alpha]-2b adrenergic receptors were 0.05-1.6 [micro]M (ADRA2A, Adra2a, Adra2b), but this was not the case for other adrenergic receptor subtypes [[alpha]-2C (ADRA2C), [beta]-1 (ADRBI), [beta]-2 (ADRB2), P-3 (ADRB3)]. Amitraz was ranked in the top 10% in both models for feeding behavior and in the model for [beta] cell function (Table 3). Chemicals exhibiting patterns of activity similar to those of amitraz are shown in Table 4, and the full correlation analysis set is available in Excel File Table S16.

Dexamethasone. Dexamethasone is a synthetic glucocorticoid that is commonly used to treat inflammatory conditions such as allergic disorders, skin conditions, ulcerative colitis, arthritis, lupus, psoriasis, and breathing disorders. Glucocorticoids cause hyperglycemia, and long-term glucocorticoid therapy has been associated with significant weight gain (Dang et al. 2005; Sheehan 2005). Glucocorticoid receptors (GRs) play a role in committing preadipocytes to the adipocyte lineage and in stimulating adipogenesis (Farmer 2006; Janesick and Blumberg 2011). Dexamethasone was ranked in the top 5% of chemicals in the adipocyte differentiation model but was not ranked highly in any other model (Table 3). Dexamethasone was identified as one of the most potent GR agonists in ToxCast[TM], and its GR activity was the only factor contributing to its ranking in the adipocyte differentiation model. Chemicals exhibiting patterns of activity similar to that of dexamethasone are shown in Table 4, and the full correlation analysis set is available in Excel File Table S17.

Signpost chemicals not prioritized in prediction models. In some cases, signpost chemicals derived from the peer-reviewed literature were not ranked highly in our analysis, perhaps because the assay targets underlying the response were not selected by our experts, because assays relevant to the mechanism by which the chemical caused the effects were not included in ToxCast[TM], and/or because potential false negative results in the screening level data were provided by the high-throughput techniques. Understanding the basis for not identifying signpost chemicals is a highly important issue from a public health perspective, where missing active chemicals in a screening strategy is often considered of greater concern than identifying false positives.

Nicotinic acid (niacin). Nicotinic acid, or niacin, is a water-soluble B vitamin. At therapeutic doses, it has been associated with hyperglycemia, and at high doses, it can produce hypolipidemia (Dang et al. 2005). This effect appears to be related to increased insulin resistance and to an increase in hepatic gluconeogenesis. None of the models identified niacin as a chemical of concern for metabolic effects (Table 3), perhaps because the relevant assay targets are not included in ToxCast[TM]. The therapeutic effects of niacin are primarily mediated through G protein--coupled receptors not screened in ToxCast[TM], niacin receptor 1 (NIACR1) and niacin receptor 2 (NIACR2). The niacin receptors have roles in energy regulation (Gille et al. 2008; Hernandez et al. 2010; Mandrika et al. 2010). NIACR1 inhibits cyclic adenosine monophosphate (cAMP) production, which limits fat breakdown in adipose tissue, reducing the amount of free fatty acids available for the liver to produce triglycerides and very-low-density lipoproteins (VLDL) and, consequently, low-density lipoprotein (LDL) or "bad" cholesterol.

Chlorinated persistent organic pollutants. A number of chlorinated persistent organic pollutants (POPs) associated with diabetes in humans were tested in ToxCast[TM] but did not rank highly in our models, including several dichlorodiphenyltrichloroethane (DDT) or dichlorodiphenyldichloroethylene (DDE) isomers (p,p'-DDE, pp'-DDT, o,p'-DDT), heptachlor epoxide, mirex, dieldrin, [beta]-hexachlorocyclohexane ([beta]-HCH), and lindane ([gamma]-HCH) (Taylor et al. 2013). Of these chemicals, the highest ranked were o,p'-DDT and [beta]-HCH, which was ranked in the top 15% for rodent feeding behavior based solely on ESRI activity. Similarly, other chlorinated POPs that have not been as well studied for diabetes outcomes in humans were included in ToxCast[TM] and generally did not rank highly (kepone, endosulfan, endosulfan sulfate, endosulfan I, chlordane, endrin, aldrin, heptachlor, chlorendic acid, o,p -DDD, p,p -DDD). These chemicals can be identified under the Chemical_Super_Category field in Excel File Tables S4-S8 as "phenol chloro," "polychloro-bicycle," and "alkane cyclo chloro." Our models were not designed to assess many aspects of carbohydrate and lipid metabolism, and additional analysis focusing on these chemicals is an area worthy of future consideration.

Signpost chemicals not included in the ToxCast[TM] chemical library. A number of chemicals (and their metabolites) that have been most strongly associated with type 2 diabetes in humans have not been tested in the ToxCast[TM] platforms, including inorganic arsenic species and a number of chlorinated POPs (hexachlorobenzene, oxychlordane, trans-nonachlor, PCBs, and dioxins/dioxin-like chemicals) (Kuo et al. 2013; Maull et al. 2012; Taylor et al. 2013).

Another signpost chemical of interest not tested in ToxCast[TM] is pyrinuron (trade name Vacor), a banned rodenticide associated with type 1 diabetes in humans following acute poisoning episodes (Gallanosa et al. 1981; Karam et al. 1980; Miller et al. 1978; Mindel 1986; Peters et al. 1981; Pont et al. 1979; Prosser and Karam 1978; Yoon 1990). Animal and in vitro studies showed that Vacor damaged pancreatic [beta] cells, which led to impaired glucose tolerance in rats (Lee et al. 1988) and to decreased insulin release in isolated rat pancreatic islet cells and hamster insulinoma HIT-T15 cells (Esposti et al. 1996; Taniguchi et al. 1989; Wilson and Gaines 1983). Vacor is a substituted urea compound containing ~ 2% N-(3-pyridylmethyl)-N'-(p-nitrophenyl) urea (PNU, CASRN 5355825-1) that has been described as causing pancreatic effects similar to those caused by alloxan and streptozotocin (Esposti et al. 1996), two experimental diabetogenic agents that also contain a urea group and were also not included in ToxCast[TM].

Discussion

Overall, our analysis suggests that ToxCast[TM] data can serve as a useful resource for prioritizing chemicals with respect to their potential to alter metabolic function. With the exception of several organotins, the most highly ranked environmental chemicals in the biological process models are not, to our knowledge, being studied for potential metabolic effects. Instead, the research community is focusing on a relatively narrow set of chemicals (or chemical classes) such as bisphenol A, phthalates, perfluorinated chemicals, and certain types of pesticides (Filer et al. 2014).

These results do not demonstrate that the chemicals ranked highest in the models, or considered most similar to signpost chemicals based on correlation analysis, will cause adverse metabolic effects at the organismal level. However, the shortened list of candidates for further testing may increase the feasibility of more time-consuming and expensive follow-up testing to confirm novel metabolic toxicants.

The next steps in considering results from this analysis should include confirming the results presented here with follow-up testing. Additional testing could focus on specific activities (e.g., PPAR[gamma] activation) utilizing different technology platforms, or on phenotypic responses using in vitro or alternative model systems that align with the biological processes modeled in our analyses (e.g., lipid accumulation in adipocytes, body fat in C. elegans, islet cell culture). Follow-up testing is especially important for glycemic control and adipogenic end points because they are understudied in toxicology, making it difficult to systematically evaluate the models presented here with existing data.

Several factors will need to be considered when evaluating the results from follow-up testing. First, binding assays comprise approximately half of the assays used to build the biological process models, in particular for the dopamine, serotonin, and GABA receptor assays used in feeding behavior for C. elegans and the [beta] cell function model (see Excel File Table S2). These assays will not provide information about the directionality of activity (i.e., agonist or antagonist), limiting their utility for developing hypotheses about whether a chemical activates or inhibits a biological pathway. For example, several anti-diabetogenic drugs were identified as active for islet or [beta] cell function and insulin sensitivity in peripheral tissue. Similarly, RAR agonist activity needs to be considered when evaluating the results from the adipocyte differentiation model. Activation of RAR is associated with an antiadiposity phenotype (Bonet et al. 2012; Frey and Vogel 2011), although it should be noted that impaired adipogenesis can itself be metabolically deleterious because failure to expand adipose depots (e.g., in clinical states of lipodystrophy) promotes insulin resistance and diabetes.

Second, there is concern about the specificity of gene-based assays in the context of cytotoxicity and other secondary mechanisms leading to potential false positive results. To limit the influence of cytotoxicity and other secondary mechanisms in the models, we weighted the input data based on the distribution of cytotoxicity assays, down-weighting and often removing data for lack of specificity. Using this approach biases the analysis toward identifying chemicals that are specific for the assay targets of interest. Consequently, chemicals that exhibit broad-spectrum toxicity at low concentrations, such as tributyltin chloride, will not rank as highly as if less-stringent cytotoxicity filtering were used.

Third, a number of gene targets identified by experts during the 2011 NTP workshop as relevant to the biological processes described in this article are not included in ToxCast[TM], including glucose transporter 2 (GLUT2), insulin receptor substrates 1 and 2 (IRS1, IRS2), the ZFP423 gene and Wnt genes involved in adipogenesis, leptin receptor (LEPR), fatty acid binding protein 4 (FABP4, found in adipocytes), and genes expressed in stem cells that populate white adipose tissue lineage and could be early indicators of commitment to adipocyte lineage (CD24, CD29, CD34, PDGFRb, NG2, Sca1).

Finally, there is limited metabolizing capability in both the Tox21 and ToxCast[TM] platforms. The chemical library contains key metabolites for limited chemicals: for example, metabolites of phthalates and organophosphate pesticides. However, it is likely that many other in vitro screens will have the same limitation.

Despite the limitations in using ToxCast[TM] HTS data, it is encouraging that the models identified the majority of the signpost chemicals for metabolic effects including amitraz, tributyltins, nicotine, and several drugs. In this analysis, we relied exclusively on expert opinion to identify relevant assays for our models. We considered selecting assay targets based on bioinformatics-based biological process/pathway databases, such as Kyoto Encyclopedia of Genes and Genomes (KEGG) (Kanehisa et al. 2014) or CoPub (Frijters et al. 2008), but we decided to utilize expert opinion for several reasons. First, the gene coverage of biological pathways within ToxCast[TM] varies and is limited for pathways related to diabetes and obesity. For example, the KEGG pathway for "Type II diabetes mellitus-Homo sapiens (human)" includes > 50 genes, but approximately half of these are not included in ToxCast[TM] assays. Second, genes identified in text-mining resources such as CoPub do not indicate the directionality of the association with the biological process (i.e., activation/antagonism or up-/downregulation), which is important when trying to identify assay targets associated with potentially adverse health outcomes. Third, the gene targets identified from the pathway databases might not necessarily be applicable to specific medium-throughput methods that could be used to assess the results, including in vitro models of islet/[beta] cell function, adipocyte differentiation, and feeding behavior and body fat in C. elegans. In other words, the relevance of different assays may differ depending on the model system used in more targeted research. We do not consider this a shortcoming of our analysis; instead, it reflects a practical approach to using HTS. Future analyses of this type could perhaps be improved by using a combination of approaches including the use of expert opinion, performing systematic reviews of the literature to identify signpost chemicals for mechanistic insight, and utilization of bioinformatics-based databases such as KEGG and CoPub. ToxCast[TM] data can also be used to complement other databases developed to annotate gene interactions of environmental chemicals such as the Comparative Toxicogenomics Database (CTD, Davis et al. 2013) and the Pesticide Target Interaction Database (PTID, Gong et al. 2013).

Analogous to the biological process scores, we used the z-score values to calculate the chemical-chemical correlations. Anchoring the correlation analysis to z-score values identifies chemicals with similar specific profiles despite shifts in potency, allowing us to identify environmental chemicals similar to the signpost chemicals even though they may often have lower potency values. In this sense, use of the z-scores in similarity profiling can identify potential health outcomes. The dose level at which an effect occurs (i.e., the potency), particularly within the context of potential exposure, also needs to be considered. Using [AC.sub.50] values (concentration at half-maximal activity) in the correlation analysis would better capture the potential potency of an environmental chemical but would likely remove the specificity of any similarity. For completeness, we present the results of the correlation analysis based on [AC.sub.50] data in Excel File Tables S9-S17 because using the [AC.sub.50] data did alter the ranking of chemicals considered most similar.

The clustering presented in Figure S7 illustrates one approach to assessing profile similarity, although ToxPi output data are provided in the supplemental tables to facilitate alternative approaches. Nevertheless, the C. elegans feeding behavior clusters illustrate the notion of chemical "activity" as a multidimensional phenomenon. Across diverse compound and assay sets, different components of activity will come to the fore, which is why ToxPi scores should always be interpreted in context with slice-wise profiles.

Conclusions

The results of this screening-level analysis suggest that the spectrum of environmental chemicals to consider in research related to diabetes and obesity is much broader than indicated from research papers and reviews published in the peer-reviewed literature. Certainly, additional research is required to put these screening-level analyses into context, but our hope is that the information presented in this review facilitates the development of new hypotheses by researchers interested in understanding the potential role of environmental chemicals in the development or progression of disease for diabetes, obesity, and metabolic syndrome.

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Zhang C, Bao W, Rong Y, Yang H, Bowers K, Yeung E, et al. 2013. Genetic variants and the risk of gestational diabetes mellitus: a systematic review. Hum Reprod Update 19(4):376-390.

Zoli M, Picciotto MR. 2012. Nicotinic regulation of energy homeostasis. Nicotine Tob Res 14(11):1270-1290.

Zuo Z, Chen S, Wu T, Zhang J, Su Y, Chen Y, et al. 2011. Tributyltin causes obesity and hepatic steatosis in male mice. Environ Toxicol 26(1):79-85.

Scott Auerbach, (1) Dayne Filer, (2) David Reif, (3) Vickie Walker, (1) Alison C. Holloway, (4) Jennifer Schlezinger, (5) Supriya Srinivasan, (6) Daniel Svoboda, (7) Richard Judson, (2) John R. Bucher, (1) and Kristina A. Thayer (1)

(1) Division of the National Toxicology Program, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina, USA; (2) National Center for Computational Toxicology, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina, USA; (3) Bioinformatics Research Center, Department of Biological Sciences, North Carolina State University, Raleigh, North Carolina, USA; (4) Department of Obstetrics and Gynecology, McMaster University, Hamilton, Ontario, Canada; (5) Department of Environmental Health, Boston University School of Medicine, Boston, Massachusetts, USA; (6) Department of Chemical Physiology, The Scripps Research Institute, La Jolla, California, USA; (7) SciOme, LLC, Research Triangle Park, North Carolina, USA

Address correspondence to K.A. Thayer, NTP Office of Health Assessment and Translation (OHAT), NIEHS/NTP, 530 Davis Dr., Room 2150/Mail Drop K2-04, Morrisville, NC 27560 USA. Telephone: (919) 541-5021. E-mail: thayer@niehs.nih.gov

Supplemental Material is available online (http:// dx.doi.org/10.1289/ehp.1510456).

We gratefully acknowledge the contributions of B. Blumberg (University of California, Irvine), D. Clegg (Cedars-Sinai), and M. White (Children's Hospital Boston) for providing input on ToxCast[TM] assay selection during the 2011 NTP workshop, "Role of Environmental Chemicals in the Development of Diabetes and Obesity," and to J. Heindel and M. Devito for reviewing a draft version of the manuscript.

This research was supported by the National Institute of Environmental Health Sciences/National Institutes of Health and the U.S. Environmental Protection Agency (EPA).

The statements, opinions or conclusions contained in this report do not necessarily represent the statements, policies, opinions or conclusions of the U.S. EPA.

The authors declare they have no actual or competing financial interests.

Received: 9 July 2015; Revised: 9 October 2015; Accepted: 8 February 2016; Published: 15 March 2016.
Table 1. ToxCast[TM] assays included in each of the biological models.

ToxPi model inputs [ToxCast[TM]
assays per input]

Adipocyte differentiation: 5
  slices
PPAR[gamma]                         RXRA
* PPAR[gamma] peroxisome            * RXRA: retinoid X receptor,
  proliferator-activated receptor     alpha [1]
  gamma [4]
* PPRE: peroxisome proliferator-
  activated receptor response
  element [1]
GR (or NR3C1):                      Other

* GR (or NR3C1): nuclear receptor   * CEBPB: CCAAT/enhancer binding
  subfamily 3, group C, member 1      protein (C/EBP), beta [1]
  (or glucocorticoid receptor)      * SREBF1: sterol regulatory
  [4]                                 element binding transcription
                                      factor 1 [1]
LXR
* LXR: NR1H2 (or LXRB)--nuclear
  receptor subfamily 1, group H,
  member 2 (or liver X receptor)
  and NR1H3 (or LXRA)--nuclear
  receptor subfamily 1, group H,
  member 3 [2]
* LXRE: LXR response element [1]
Feeding behavior (rodents): 9
  slices
CCK: cholecystokinin A and B        INSR: insulin receptor [2]
  receptors [2]
ESR1: estrogen receptor a or 1      MAP: mitogen-activated protein
  [4]                                 kinase 3 [3]
ESRRA: estrogen-related receptor    NPY: NPY neuropeptide Y receptors
  alpha [1]                           Y1, Y2, Y5; Bos taurus [3]
FoxO1: forkhead box O1 [1]          STAT3: signal transducer and
                                      activator of transcription 3
                                      (acute-phase response factor)
                                      [1]
IL6: interleukin 6 (interferon,
  beta 2) [1]

Feeding behavior (C. elegans): 12
  slices
DRD2: dopamine receptor D2 [1]      INSR: insulin receptor [2]
GSK3B: glycogen synthase kinase 3   NPY: NPY neuropeptide Y receptors
  beta [1]                            Y1, Y2, Y5; Bos taurus [3]
HTR2C: 5-hydroxytryptamine          PPAR8: peroxisome proliferator-
  (serotonin) receptor 2C [1]         activated receptor delta [1]
HTR3A: 5-hydroxytryptamine          PRKACA: protein kinase, cAMP-
  (serotonin) receptor 3A [1]         dependent, catalytic, alpha [1]
HTR2A: 5-hydroxytryptamine          Sstr1: somatostatin receptor 1
  (serotonin) receptor 2A [1]         [1]
Other HTR                           Other
* Slc6a4: solute carrier family 6   * PPAR[gamma]: peroxisome
  (neurotransmitter transporter,      proliferator-activated receptor
  serotonin), member 4 [2]            gamma [4]
* Htr1a: 5-hydroxytryptamine        * PPRE: peroxisome proliferator-
  (serotonin) receptor 1A [Mus        activated receptor response
  musculus] [1]                       element [1]
* Htr4: 5-hydroxytryptamine         * DRD4: dopamine receptor D4 [1]
  (serotonin) receptor 4 [1]        * DRD1: dopamine receptor D1 [1]
* HTR6: 5-hydroxytryptamine         * NR1I1: vitamin D (1,25-
  (serotonin) receptor 6 [1]          dihydroxyvitamin D3) receptor
* HTR7: 5-hydroxytryptamine           [2]
  (serotonin) receptor 7            * NR1H2 (or LXRB): nuclear
  (adenylate cyclase- coupled)        receptor subfamily 1, group H,
  [1]                                 member 2 (or liver X receptor)
* HTR5A: 5-hydroxytryptamine          and NR1H3 (or LXRA)--nuclear
  (serotonin) receptor 5A [1]         receptor subfamily 1, group H,
                                      member 3 [2]
                                    * NR1H3 (or LXRA): nuclear
                                      receptor subfamily 1, group H,
                                      member 3 (Liver X receptor
                                      alpha) [1]
                                    * CEBPB: CCAAT/enhancer binding
                                      protein (C/EBP), beta [1]

Insulin sensitivity in peripheral
  tissue: 11 slices
AKT: v-akt murine thymoma viral     PPAR[gamma]: peroxisome
  oncogene homolog 1[2]               proliferator-activated receptor
                                      [gamma] [4]
CREB: cAMP responsive element       PPRE: peroxisome proliferator-
  binding protein 3[1]                activated receptor response
                                      element [1]
FOX: forkhead box A2; forkhead      PTPN1: protein tyrosine
  box O1 [2]                          phosphatase, nonreceptor type 1
                                      [1]
INSR: insulin receptor [2]          SREBF1: sterol regulatory element
                                      binding transcription factor 1
                                      [1]
Kcnj11: potassium inwardly          STAT3: signal transducer and
  rectifying channel, subfamily       activator of transcription 3
  J, member 1[1]                      (acute-phase response factor)
                                      [1]
PPAR[alpha]: peroxisome
  proliferator-activated receptor
  alpha [2]

Islet cell function: 11 slices
betaCatenin: TCF/b-catenin          INSR: insulin receptor [2]
  response element [1]
DRD1: DRD dopamine receptors D1,    Kcnj11: potassium inwardly-
  D2, D3, D5 [Bos taurus] [1]         rectifying channel, subfamily
FOXA2: forkhead box A2 [1]            J, member 1 [1]
FOXO1: forkhead box O1 [1]          ONECUT1: one cut homeobox 1 [1]
GSK3B: glycogen synthase kinase 3   PAX6: paired box 6 [1]
  beta [1]                          PTPN1: protein tyrosine
HNF4A: hepatocyte nuclear factor      phosphatase, nonreceptor type 1
  4, alpha [1]                        [1]

[beta] cell function: 14 slices
ACHE: acetylcholinesterase [2]      HRT, solute carrier: solute
                                      carrier family 6
                                      (neurotransmitter transporter,
                                      serotonin), member 4 [2]
BCHE: butyrylcholinesterase [1]     INSR: insulin receptor [2]
DRD: dopamine receptors (multiple   Kcnj11: potassium inwardly-
  subtypes) and opioid receptor,      rectifying channel, subfamily
  delta 1 [5]                         J, member 1 [1]
DRD, solute carrier: [2]            PPAR[alpha]: peroxisome
                                      proliferator-activated receptor
                                      alpha [2]
ESR1: estrogen receptor a or 1      PPAR[delta]: peroxisome
  [4]                                 proliferator-activated receptor
                                      delta [1]
GABA: gamma-aminobutyric acid       PPAR[gamma]: peroxisome
  (GABA) receptor (multiple           proliferator-activated receptor
  subtypes) [5]                       [gamma] [4]
HTR: 5-hydroxytryptamine            PPRE: peroxisome proliferator-
  (serotonin) (multiple subtypes)     activated receptor response
  [8]                                 element [1]

ToxPi model inputs [ToxCast[TM]     References
assays per input]

Adipocyte differentiation: 5
  slices
PPAR[gamma]                         (Farmer 2006;
* PPAR[gamma] peroxisome              Frijters et al. 2008;
  proliferator-activated receptor     Hummasti et al.
  gamma [4]                           2004; Janesick and
* PPRE: peroxisome proliferator-      Blumberg 2011;
  activated receptor response         Mukherjee et al. 1997;
  element [1]                         Tontonoz et al. 1994;
GR (or NR3C1):                        Wang 2010)

* GR (or NR3C1): nuclear receptor
  subfamily 3, group C, member 1
  (or glucocorticoid receptor)
  [4]

LXR
* LXR: NR1H2 (or LXRB)--nuclear
  receptor subfamily 1, group H,
  member 2 (or liver X receptor)
  and NR1H3 (or LXRA)--nuclear
  receptor subfamily 1, group H,
  member 3 [2]
* LXRE: LXR response element [1]
Feeding behavior (rodents): 9
  slices
CCK: cholecystokinin A and B        (Barros and Gustafsson
  receptors [2]                       2011; Deblois and
ESR1: estrogen receptor a or 1        Giguere 2011; Frijters
  [4]                                 et al. 2008; Ranhotra
ESRRA: estrogen-related receptor      2010; Skibicka and
  alpha [1]                           Dickson 2013)
FoxO1: forkhead box O1 [1]

IL6: interleukin 6 (interferon,
  beta 2) [1]

Feeding behavior (C. elegans): 12   (de Bono and Bargmann
  slices                              1998; Frijters et al. 2008;
DRD2: dopamine receptor D2 [1]        Noble et al. 2013; Sawin
GSK3B: glycogen synthase kinase 3     et al. 2000; Srinivasan
  beta [1]                            2015; Srinivasan et al.
HTR2C: 5-hydroxytryptamine            2008)
  (serotonin) receptor 2C [1]
HTR3A: 5-hydroxytryptamine
  (serotonin) receptor 3A [1]
HTR2A: 5-hydroxytryptamine
  (serotonin) receptor 2A [1]
Other HTR
* Slc6a4: solute carrier family 6
  (neurotransmitter transporter,
  serotonin), member 4 [2]
* Htr1a: 5-hydroxytryptamine
  (serotonin) receptor 1A [Mus
  musculus] [1]
* Htr4: 5-hydroxytryptamine
  (serotonin) receptor 4 [1]
* HTR6: 5-hydroxytryptamine
  (serotonin) receptor 6 [1]
* HTR7: 5-hydroxytryptamine
  (serotonin) receptor 7
  (adenylate cyclase- coupled)
  [1]
* HTR5A: 5-hydroxytryptamine
  (serotonin) receptor 5A [1]

Insulin sensitivity in peripheral
  tissue: 11 slices
AKT: v-akt murine thymoma viral     (Frijters et al. 2008;
  oncogene homolog 1[2]               Wang 2010)

CREB: cAMP responsive element
  binding protein 3[1]

FOX: forkhead box A2; forkhead
  box O1 [2]

INSR: insulin receptor [2]

Kcnj11: potassium inwardly
  rectifying channel, subfamily
  J, member 1[1]

PPAR[alpha]: peroxisome
  proliferator-activated receptor
  alpha [2]

Islet cell function: 11 slices
betaCatenin: TCF/b-catenin          (Frijters et al. 2008;
  response element [1]                Greeley et al. 2011)
DRD1: DRD dopamine receptors D1,
  D2, D3, D5 [Bos taurus] [1]
FOXA2: forkhead box A2 [1]
FOXO1: forkhead box O1 [1]
GSK3B: glycogen synthase kinase 3
  beta [1]
HNF4A: hepatocyte nuclear factor
  4, alpha [1]

[beta] cell function: 14 slices
ACHE: acetylcholinesterase [2]      (Amireault et al. 2013;
                                      Barros and Gustafsson
                                      2011; Caicedo 2013;
                                      Eldor et al. 2013; Frijters
BCHE: butyrylcholinesterase [1]       et al. 2008; Greeley
DRD: dopamine receptors (multiple     et al. 2011; Gupta
  subtypes) and opioid receptor,      et al. 2010; Tiano and
  delta 1 [5]                         Mauvais-Jarvis 2012;
DRD, solute carrier: [2]              Ustione et al. 2013;
                                      Wang 2010)

ESR1: estrogen receptor a or 1
  [4]

GABA: gamma-aminobutyric acid
  (GABA) receptor (multiple
  subtypes) [5]
HTR: 5-hydroxytryptamine
  (serotonin) (multiple subtypes)
  [8]

Frijters et al. (2008) was used in a 2010 analysis conducted for the
NTP workshop "Role of Environmental Chemicals in the Development of
Diabetes and Obesity" (Thayer et al. 2012), where CoPub text-mining
tools were used to identify relationships between genes, pathways-
processes, diseases, and drugs. The relationship is summarized in
CoPub with an R-scale score that quantifies the strength of cocitation
between two keywords (e.g., PNPLA3 and fatty liver). In the 2010
analysis, CoPub was searched for genes associated with adipocyte
differentiation, feeding behavior, insulin sensitivity, and islet cell
function, and the results were mapped to the ToxCast[TM] assay targets
listed above. The CoPub analysis was considered to provide support for
an association between the gene and the biological process when the
R-scale score was [greater than or equal to] 25. Many, but not all, of
the gene targets identified by expert opinion were identified in the
CoPub analysis.

Table 2. Top 30 chemicals [CASRN] based on prediction model scores for
adipocyte differentiation, feeding behavior (rodent), feeding behavior
(Caenorhabditis elegans), insulin sensitivity in peripheral tissue,
islet cell function, and beta cell function.

Rank   Adipocyte differentiation        Feeding behavior (rodents)

1      Diallyl phthalate                HMR1171
       [131-17-9]                       [328392-46-7]
       (score = 0.306, *RAR score       (score = 0.192)
       = 0.115)                         Use: pharmaceutical
       Use: plasticizer                 Class: lipid lowering
       Class: phthalate

2      Methyl salicylate                PharmaGSID 48511
       [119-36-8]                       [1062243-51-9]
       (score = 0.293, * RAR score      (score = 0.133)
       = 0.180)                         Use: pharmaceutical
       Use: flavor, antiseptic          Class: polo-like kinase
       Class: salicylate                  inhibitor

3      Melengestrol acetate             4-Hydroxytamoxifen
       [2919-66-6]                      [68392-35-8]
       (score = 0.289)                  (score = 0.125)
       Use: pharmaceutical              Use: pharmaceutical
       Class: steroidal progestin       Class: SERM

4      Rotenone                         Niclosamide
       [83-79-4]                        [50-65-7]
       (score = 0.266, * RAR score      (score = 0.114)
       = 0.222)                         Use: molluscicide
       Use: insecticide                 Class: phenol halide
       Class: botanical

5      Tebufenpyrad                     PharmaGSID 47337
       [119168-77-3]                    [1061517-62-1]
       (score = 0.257, * RAR score      (score = 0.112)
       = 0.120)                         Use: pharmaceutical
       Use: insecticide                 Class: cholecystokinin 1
       Class: pyrazole                    receptor (CCK1R) agonist

6      Frans-retinoic acid              Acetic acid
       [302-79-4]                       C8-10-branched alkyl esters,
       (score = 0.251, * RAR score      C9-rich
       = 1)                             [108419-33-6]
       Use: pharmaceutical              (score = 0.111)
       Class: carboxylic acid           Use: solvent
                                        Class: carboxylate

7      Isazofos                         Methyl parathion
       [42509-80-8]                     [298-00-0]
       (score = 0.248)                  (score = 0.111)
       Use: insecticide                 Use: insecticide
       Class: organophosphate           Class: organophosphate

8      Aspirin                          Isopropyl triethanolamine
       [50-78-2]                        titanate
       (score = 0.246, * RAR score      [36673-16-2]
       = 0.071)                         (score = 0.111)
       Use: pharmaceutical              Use: coupling
       Class: phenyl carboxylic         Class: organometallic
         acid alkoxy

9      GW473178E methyl                 Ilepatril
       benzene sulfonic acid            [473289-62-2]
       [263553-33-9]                    (score = 0.111)
       (score = 0.221)                  Use: pharmaceutical
       Use: pharmaceutical              Class: vasopeptidase
       Class: thrombin inhibitor          inhibitor

10     Bentazone                        Equilin
       [25057-89-0]                     [474-86-2]
       (score = 0.221)                  (score = 0.111)
       Use: herbicide                   Use: pharmaceutical
       Class: carbamate                 Class: steroidal estrogen

11     Sodium abietate                  Triisononyl trimellitate
       [14351-66-7]                     [53894-23-8]
       (score = 0.215)                  (score = 0.111)
       Use: coating                     Use: plasticizer
       Class: abietate                  Class: phthalate

12     2-Ethyl-2-hexenal                Cymoxanil
       [645-62-5]                       [57966-95-7]
       (score = 0.212)                  (score = 0.110)
       Use: intermediate,               Use: fungicide
         insecticide                    Class: acetamide carboxylate
       Class: aldehyde                    amine

13     AVE8923                          AVE6324
       [NOCAS_47381]                    [NOCAS_47377]
       (score = 0.207)                  (score = 0.108)
       Use: pharmaceutical              Use: pharmaceutical
       Class: tryptase inhibitor        Class: factor Xa inhibitor

14     SR271425                         Zearalenone
       [155990-20-8]                    [17924-92-4]
       (score = 0.205, * RAR score      (score = 0.108)
       = 0.084)                         Use: mycotoxin
       Use: pharmaceutical              Class: carboxylic acid
       Class: thioxanthone analog         ketone

15     Tributyltin benzoate             Rifampicin
       [4342-36-3]                      [13292-46-1]
       (score = 0.200, * RAR score      (score = 0.102)
       = 0.145)                         Use: pharmaceutical
       Use: microbicide                 Class: antibiotic
       Class: organotin

16     Farglitazar                      Mestranol
       [196808-45-4]                    [72-33-3]
       (score = 0.200)                  (score = 0.101
       Use: pharmaceutical              Use: pharmaceutical
       Class: PPAR[gamma] agonist       Class: nonsteroidal estrogen

17     Acrylamide                       meso-Hexestrol
       [79-06-1]                        [84-16-2]
       (score = 0.193)                  (score = 0.098)
       Use: reactant                    Use: pharmaceutical
       Class: acrylamide                Class: steroidal estrogen

18     1-(6-fert-Butyl-1,1-dimethyl-    Estriol
       2,3-dihydro-1H-inden-4-yl)       [50-27-1]
       ethanone                         (score = 0.097)
       [13171-00-1]                     Use: pharmaceutical
       (score = 0.180, * RAR score      Class: steroidal estrogen
       = 0.142)
       Use: fragrance
       Class: phenyl ketone

19     Tetrabutyltin                    Pirimiphos-methyl
       [1461-25-2]                      [29232-93-7]
       (score = 0.177, * RAR score      (score = 0.097)
       = 0.157)                         Use: insecticide
       Use: microbicide                 Class: organophosphate
       Class: organotin

20     Triamcinolone                    Diethylstilbestrol
       [124-94-7]                       [56-53-1]
       (score = 0.172)                  (score = 0.095)
       Use: pharmaceutical              Use: pharmaceutical
       Class: corticosteroid            Class: nonsteroidal estrogen

21     Pyridaben                        Raloxifene hydrochloride
       [96489-71-3]                     [82640-04-8]
       (score = 0.171, * RAR score      (score = 0.095)
       = 0.063)                         Use: pharmaceutical
       Use: insecticide                 Class: SERM
       Class: diazine phenyl sulfide
         halide ketone

22     Resorcinol                       17beta-Estradiol
       [108-46-3]                       [50-28-2]
       (score = 0.167, * RAR score      (score = 0.095)
       = 0.162)                         Use: pharmaceutical
       Use: intermediate,               Class: steroidal estrogen
         disinfectant
       Class: phenol

23     Dexamethasone sodium             4,4'-Methylenedianiline
       phosphate                        [101-77-9]
       [2392-39-4]                      (score = 0.093)
       (score = 0.160)                  Use: intermediate
       Use: pharmaceutical              Class: aniline
       Class: corticosteroid

24     Phenobarbital sodium             Pyraflufen-ethyl
       [57-30-7]                        [129630-19-9]
       (score = 0.160)                  (score = 0.092)
       Use: pharmaceutical              Use: herbicide
       Class: barbituate                Class: pyridine alkoxy
                                          carboxylic acid halide

25     CP-457677                        17[alpha]-Ethinylestradiol
       [214535-77-0]                    [57-63-6]
       (score = 0.159)                  (score = 0.092)
       Use: pharmaceutical              Use: pharmaceutical
       Class: not assigned              Class: steroidal estrogen

26     Basic blue 7                     Nicotine
       [2390-60-5]                      [54-11-5]
       (score = 0.156)                  (score = 0.090)
       Use: dye                         Use: pharmaceutical, pesticide
       Class: aniline dye               Class: pyridine amine

27     (Z.f)-Fenpyroximate              Benzal chloride
       [111812-58-9]                    [98-87-3]
       (score = 0.156, * RAR score      (score = 0.088)
       = 0.235)                         Use: dye, reactant
       Use: insecticide                 Class: phenyl halide
       Class: pyrazole

28     CP-612372                        Methyleugenol
       [353280-07-6]                    [93-15-2]
       (score = 0.155)                  (score = 0.088)
       Use: pharmaceutical              Use: fragrance, flavor,
       Class: not assigned                attractant, anesthetic
                                        Class: phenol ethoxylate alkyl

29     2-Methyl-5-nitroaniline          17[alpha]-Estradiol
       [99-55-8]                        [57-91-0]
       (score = 0.155)                  (score = 0.085)
       Use: intermediate                Use: pharmaceutical
       Class: aniline nitro             Class: steroidal estrogen

30     Retinol acetate                  Piperazine
       [127-47-9]                       [110-85-0]
       (score = 0.154)                  (score = 0.085)
       Use: natural; vitamin            Use: insecticide
       Class: carboxylate               Class: amine

       Feeding behavior                 Insulin sensitivity
Rank   (C. elegans)                     peripheral tissue

1      Chlorpromazine                   Farglitazar
       hydrochloride                    [196808-45-4]
       [69-09-0]                        (score = 0.250)
       (score = 0.258)                  Use: pharmaceutical
       Use: pharmaceutical              Class: PPAR[gamma] agonist
       Class: dopamine
         antagonist

2      Trelanserin                      PharmaGSID 47315
       [189003-92-7]                    [444610-91-7]
       (score = 0.241)                  (score = 0.221)
       Use: pharmaceutical              Use: pharmaceutical
       Class: selective serotonin       Class: PPAR[gamma] agonist
         5-HT2A, Antagonist

3      Fabesetron hydrochloride         Basic blue 7
       [129299-90-7]                    [2390-60-5]
       (score = 0.239)                  (score = 0.204)
       Use: pharmaceutical              Use: dye
       Class: serotonin 5-HT3           Class: aniline dye
         receptor antagonist

4      Volinanserin                     Rotenone
       [139290-65-6]                    [83-79-4]
       (score = 0.236)                  (score = 0.203)
       Use: pharmaceutical              Use: insecticide
       Class: serotonin 5-HT2A          Class: botanical
         receptor antagonist

5      Isopropyl triethanolamine        Tebufenpyrad
       titanate                         [119168-77-3]
       [36673-16-2]                     (score = 0.197)
       (score = 0.225)                  Use: insecticide
       Use: coupling                    Class: pyrazole
       Class: organometallic

6      SSR150106                        PharmaGSID 48511
       [NOCAS_47362]                    [1062243-51-9]
       (score = 0.222)                  (score = 0.187)
       Use: pharmaceutical              Use: pharmaceutical
       Class: chemokine receptor        Class: polo-like kinase
         antagonist                       inhibitor

7      PharmaGSID 48511                 Isopropyl triethanolamine
       [1062243-51-9]                   titanate
       (score = 0.174)                  [36673-16-2]
       Use: pharmaceutical              (score = 0.182)
       Class: polo-like kinase          Use: coupling
         inhibitor                      Class: organometallic

8      SB243213A                        Pyridaben
       [200940-23-4]                    [96489-71-3]
       (score = 0.148)                  (score = 0.177)
       Use: pharmaceutical              Use: insecticide
       Class: serotonin 5-HT2C          Class: diazine phenyl
         receptor inverse agonist         sulfide halide ketone

9      Haloperidol                      1,3-Diphenyl-1,3-
       [52-86-8]                        propanedione
       (score = 0.142)                  [120-46-7]
       Use: pharmaceutical              (score = 0.165)
       Class: dopamine inverse          Use: plasticizer
         agonist                        Class: phenyl

10     PharmaGSID 47315                 Fenamiphos
       [444610-91-7]                    [22224-92-6]
       (score = 0.132)                  (score = 0.156)
       Use: pharmaceutical              Use: insecticide
       Class: PPAR[gamma] agonist       Class: organophosphate

11     Elzasonan                        2,4,6-Trichlorophenol
       [361343-19-3]                    [88-06-2]
       (score = 0.130)                  (score = 0.153)
       Use: pharmaceutical              Use: herbicide,
       Class: selective 5-HT1B            fungicide, reactant
         and 5-HT1D receptor            Class: chlorinated
         antagonist                       phenol

12     Raloxifene hydrochloride         (Z,E)-Fenpyroximate
       [82640-04-8]                     [111812-58-9]
       (score = 0.126)                  (score = 0.151)
       Use: pharmaceutical              Use: insecticide
       Class: SERM                      Class: pyrazole

13     Allura red C.I.16035             2-Ethyl-2-hexenal
       [25956-17-6]                     [645-62-5]
       (score = 0.117)                  (score = 0.150)
       Use: dye                         Use: intermediate,
       Class: phenyl sulfuric             insecticide
         acid dye                       Class: aldehyde

14     AVE6324                          Diuron
       [NOCAS_47377]                    [330-54-1]
       (score = 0.108)                  (score = 0.146)
       Use: pharmaceutical              Use: herbicide
       Class: factor Xa inhibitor       Class: phenyl urea

15     SSR241586                        Propargite
       [NOCAS_47353]                    [2312-35-8]
       (score = 0.103)                  (score = 0.137)
       Use: pharmaceutical              Use: insecticide
       Class: 2,2-disubstituted         Class: phenyl ether
         morpholine                       sulfate yne

16     Mercuric chloride                1-(6-ferf-Butyl-1,1-
       [7487-94-7]                      dimethyl-2,3-dihydro-1H-
       (score = 0.103)                  inden-4-yl)ethanone
       Use: bactericide                 [13171-00-1]
       Class: organometallic            (score = 0.130)
                                        Use: fragrance
                                        Class: phenyl ketone

17     Calcium neodecanoate             Isoxaben
       [27253-33-4]                     [82558-50-7]
       (score = 0.099)                  (score = 0.129)
       Use: additive                    Use: herbicide
       Class: carboxylic acid           Class: amide, oxazole

18     FD&C Yellow 6                    1,4-Diaminoanthraquinone
       [2783-94-0]                      [128-95-0]
       (score = 0.09)                   (score = 0.129)
       Use: dye                         Use: dye
       Class: phenyl sulfuric           Class: anthraquinone
         acid dye

19     Aspirin                          Sodium abietate
       [50-78-2]                        [14351-66-7]
       (score = 0.093)                  (score = 0.127)
       Use: pharmaceutical              Use: coating
       Class: nonsteroidal              Class: abietate
         antiinflammatory drugs
         (NSAIDs)

20     Diphenhydramine                  Glyceryl monoricinoleate
       hydrochloride                    [1323-38-2]
       [147-24-0]                       (score = 0.127)
       (score = 0.090)                  Use: intermediate,
       Use: pharmaceutical              emulsifier
       Class: antihistamine             Class: alcohol
         ("Benadryl")                     carboxylate

21     PD 0343701                       Troglitazone
       [676116-04-4]                    [97322-87-7]
       (score = 0.089)                  (score = 0.125)
       Use: pharmaceutical              Use: pharmaceutical
       Class: dopamine D2               Class: thiazolidinediones
         receptor, 5HT2A

22     SSR240612                        Isazofos
       [NOCAS_47351]                    [42509-80-8]
       (score = 0.087)                  (score = 0.123)
       Use: pharmaceutical              Use: insecticide
       Class: kinin B1 receptor         Class: organophosphate
         antagonist

23     Farglitazar                      Famoxadone
       [196808-45-4]                    [131807-57-3]
       (score = 0.084)                  (score = 0.123)
       Use: pharmaceutical              Use: fungicide
       Class: PPAR[gamma] agonist       Class: dicarboximide

24     Fomesafen                        HMR1171
       [72178-02-0]                     [328392-46-7]
       (score = 0.084)                  (score = 0.122)
       Use: herbicide                   Use: pharmaceutical
       Class: diphenyl ether            Class: lipid lowering

25     Diallyl phthalate                Pirinixic acid
       [131-17-9]                       [50892-23-4]
       (score = 0.084)                  (score = 0.121)
       Use: plasticizer                 Use: pharmaceutical
       Class: phthalate                 Class: PPAR[alpha] agonist

26     Methyl parathion                 Dinocap
       [298-00-0]                       [39300-45-3]
       (score = 0.083)                  (score = 0.120)
       Use: insecticide                 Use: fungicide
       Class: organophosphate           Class: dinitrophenol
                                          derivative

27     W-nitrosodipropylamine           Z-tetrachlorvinphos
       [621-64-7]                       [22248-79-9]
       (score = 0.083)                  (score = 0.119)
       Use: breakdown product,          Use: insecticide
         research                       Class: organophosphate
       Class: nitrosoamine

28     Trioctyl trimellitate            Apigenin
       [89-04-3]                        [520-36-5]
       (score = 0.083)                  (score = 0.118
       Use: plasticizer                 Use:flavone
       Class: phthalate                 Class: genistein-like

29     Rifampicin                       Sodium dodecyl sulfate
       [13292-46-1]                     [151-21-3]
       (score = 0.083)                  (score = 0.118)
       Use: pharmaceutical              Use: surfactant
       Class: antibiotic                Class: sulfuric acid alkyl

30     Resorcinol                       Sodium 2,4,7-tri(propan-
       [108-46-3]                       2-yl)naphthalene-1-
       (score = 0.082)                  sulfonate
       Use: intermediate,               [1323-19-9]
         disinfectant                   (score = 0.118)
       Class: phenol                    Use: pesticide other,
                                          adjuvant
                                        Class: naphthalene
                                          sulfuric acid

Rank   Islet cell function              Beta cell function

1      Isopropyl                        Raloxifene
       triethanolamine                  hydrochloride
       titanate                         [82640-04-8]
       [36673-16-2]                     (score = 0.240)
       (score = 0.177)                  Use: pharmaceutical
       Use: coupling                    Class: selective
       Class: organometallic              estrogen receptor
                                          modulator (SERM)

2      Basic blue 7                     PharmaGSID 47315
       [2390-60-5]                      [444610-91-7]
       (score = 0.176)                  (score = 0.225)
       Use: dye                         Use: pharmaceutical
       Class: aniline dye               Class: PPAR[gamma] agonist

3      PharmaGSID 48511                 SSR150106
       [1062243-51-9]                   [NOCAS_47362]
       (score = 0.130)                  (score = 0.223)
       Use: pharmaceutical              Use: pharmaceutical
       Class: polo-like kinase          Class: chemokine
         inhibitor                        receptor antagonist

4      Spiromesifen                     PharmaGSID_47259
       [283594-90-1]                    [149062-75-9]
       (score = 0.106)                  (score = 0.221)
       Use: insecticide                 Use: pharmaceutical
       Class: phenyl tetronic           Class: acetylcholin-
         acid                             esterase inhibitor

5      Tris(2,3-dibromopropyl)          Farglitazar
       phosphate                        [196808-45-4]
       [126-72-7]                       (score = 0.203)
       (score = 0.100)                  Use: pharmaceutical
       Use: flame retardant             Class: PPAR[gamma] agonist
       Class: phosphate alkyl
         halide

6      Apigenin                         Chlorpromazine
       [520-36-5]                       hydrochloride
       (score = 0.097)                  [69-09-0]
       Use: flavone                     (score = 0.192)
       Class: genistein-like            Use: pharmaceutical
                                        Class: dopamine
                                          antagonist

7      Resorcinol                       UK-416244
       [108-46-3]                       [402910-27-4]
       (score = 0.091)                  (score = 0.189)
       Use: intermediate,               Use: pharmaceutical
       disinfectant                     Class: selective
       Class: phenol                      serotonin reuptake
                                          inbitor (SSRI)

8      Acetic acid                      Volinanserin
       C8-10-branched alkyl             [139290-65-6]
       esters, C9-rich                  (score = 0.176)
       [108419-33-6]                    Use: pharmaceutical
       (score = 0.091)                  Class: serotonin
       Use: solvent                       5-HT2A receptor
       Class: carboxylate                 antagonist

9      Haloperidol                      Haloperidol
       [52-86-8]                        [52-86-8]
       (score = 0.091)                  (score = 0.163)
       Use: pharmaceutical              Use: pharmaceutical
       Class: dopamine inverse          Class: dopamine
         agonist                          inverse agonist

10     Dibenz[a,fi]anthracene           PharmaGSID 48511
       [53-70-3]                        [1062243-51-9]
       (score = 0.091)                  (score = 0.149)
       Use: research                    Use: pharmaceutical
       Class: polycyclic                Class: polo-like kinase
         aromatic hydrocarbon             inhibitor

11     Caffeine                         meso-Hexestrol
       [58-08-2]                        [84-16-2]
       (score = polycyclic              (score = 0.145)
       aromatic hydrocarbon)            Use: pharmaceutical
       Use: pharmaceutical;             Class: nonsteroidal
         natural                          estrogen
       Class: Not Assigned
         caffeine-like

12     N-nitrosodipropylamine           Trelanserin
       [621-64-7]                       [189003-92-7]
       (score = 0.091)                  (score = 0.135)
       Use: breakdown product,          Use: pharmaceutical
         research                       Class: serotonin
       Class: nitrosoamine                5-HT2A antagonist

13     Dicyclopentadiene                2,2-Bis(4-
       [77-73-6]                        hydroxyphenyl)-1,1,1-
       (score = 0.091)                  trichloroethane
       Use: intermediate                [2971-36-0]
       Class: alkene                    (score = 0.128)
                                        Use: degradate
                                        Class: phenol halide

14     Rotenone                         4-Hydroxytamoxifen
       [83-79-4]                        [68392-35-8]
       (score = 0.091)                  (score = 0.123)
       Use: insecticide                 Use: pharmaceutical
       Class: botanical                 Class: SERM

15     2,4,6-Trichlorophenol            17[alpha]-Ethinylestradiol
       [88-06-2]                        [57-63-6]
       (score = 0.091)                  (score = 0.122)
       Use: herbicide,                  Use: pharmaceutical
         fungicide, reactant            Class: steroidal
       Class: phenol halide               estrogen

16     Silica                           9-Octadecenoic acid,
       [7631-86-9]                      12-hydroxy-, (9Z,12fl)
       (score = 0.087)                  [141-22-0]
       Use: filler                      (score = 0.122)
       Class: silicate                  Use: pharmaceutical,
                                          natural, plasticizer
                                        Class: unsaturated
                                          omega-9 fatty acid

17     Tannic acid                      Calcium neodecanoate
       [1401-55-4]                      [27253-33-4]
       (score = 0.086)                  (score = 0.121)
       Use: natural                     Use: additive
       Class: phenol benzoic            Class: carboxylic acid
         acid

18     Perfluorooctane                  2,4,6-Trichlorophenol
       sulfonate, PFOS                  [88-06-2]
       [1763-23-1]                      (score = 0.120)
       (score = 0.086)                  Use: herbicide,
       Use: fluorosurfactant              fungicide, reactant
       Class: perfluoro sulfuric        Class: chlorinated
         acid                             phenol

19     1-Phenoxy-2-propanol             17[alpha]-Estradiol
       [770-35-4]                       [57-91-0]
       (score = 0.085)                  (score = 0.1120)
       Use: pesticidal inert,           Use: pharmaceutical
         solvent                        Class: steroidal
       Class: phenol ethoxylate           estrogen
         alcohol

20     Dimethyl succinate               Diphenhydramine
       [106-65-0]                       hydrochloride
       (score = 0.084)                  [147-24-0]
       Use: intermediate                (score = 0.119)
       Class: carboxylate               Use: pharmaceutical
                                        Class: antihistamine
                                          ("Benadryl")

21     1,3-Diphenyl-1,3-                Glyceryl
       propanedione                     monoricinoleate
       [120-46-7]                       [1323-38-2]
       (score = 0.083)                  (score = 0.119)
       Use: plasticizer                 Use: intermediate,
       Class: phenyl                      emulsifier
                                        Class: alcohol
                                          carboxylate

22     Auramine hydrochloride           Clomiphene citrate
       [2465-27-2]                      [50-41-9]
       (score = 0.082)                  (score = 0.117)
       Use: dye, disinfectant           Use: pharmaceutical
       Class: aniline                   Class: SERM ("Clomid")

23     Dibenzothiophene                 17[beta]-Estradiol
       [132-65-0]                       [50-28-2]
       (score = 0.081)                  (score = 0.115)
       Use: fragrance, flavor           Use: pharmaceutical
       Class: benzofuran                Class: steroidal
                                          estrogen

24     2,4,6-Tribromophenol             SAR150640
       [118-79-6]                       [NOCAS_47389]
       (score = 0.080)                  (score = 0.114)
       Use: intermediate,               Use: pharmaceutical
         antiseptic                     Class: p3-adrenoceptor
       Class: phenol halide               agonist

25     Sulfasalazine                    N-dodecanoyl-N-
       [599-79-1]                       methylglycine
       (score = 0.080)                  [97-78-9]
       Use: pharmaceutical              (score = 0.111)
       Class: sulfa drug                Use: cosmetic,
                                          surfactant
                                        Class: carboxylic acid
                                          amide

26     1-(6-fert-Butyl-1,1-             Pyrimethamine
       dimethyl-2,3-dihydro-            [58-14-0]
       1H-inden-4-yl)ethanone           (score = 0.111)
       [13171-00-1]                     Use: pharmaceutical
       (score = 0.078)                  Class: protozoal
       Use: fragrance                     infections, antimalarial
       Class: phenyl ketone               drug

27     Chlorpromazine                   2-Naphthalenol
       hydrochloride                    [135-19-3]
       [69-09-0]                        (score = 0.111)
       (score = 0.078)                  Use: antioxidant,
       Use: pharmaceutical                reactant
       Class: dopamine                  Class: naphthalene
         antagonist                       alcohol

28     PharmaGSID_48505                 Propylparaben
       [NOCAS_48505]                    [94-13-3]
       (score = 0.077)                  (score = 0.104)
       Use: pharmaceutical              Use: microbicide
       Class: CDK2 inhibitor            Class: paraben

29     Ethyl butyrate                   SAR377142
       [105-54-4]                       [NOCAS_47385]
       (score = 0.075)                  (score = 0.103)
       Use: flavor                      Use: pharmaceutical
       Class: carboxylate               Class: factor Xa
                                        inhibitor

30     Tolazamide                       Pirinixic acid
       [1156-19-0]                      [50892-23-4]
       (score = 0.074)                  (score = 0.102)
       Use: pharmaceutical              Use: pharmaceutical
       Class: sulfonylurea              Class: PPAR[alpha]
                                          agonist

Table 3. Rank of signpost chemicals out of 1,860 chemicals included in
ToxCast[TM].

                                                          Feeding
                                       Adipocyte         behavior
Compound [CASRN]                    differentiation      (rodent)

Amitraz [33089-61-1]                      --              134 **
  Use: insecticide; Class:
  formamidine
Chlorpromazine hydrochloride              666               --
  [69-09-0]
  Use: pharmaceutical
  (conventional antipsychotic);
  Class: phenyl halide
Haloperidol [52-86-8]                     423               502
  Use: pharmaceutical
  (conventional antipsychotic);
  Class: phenyl-phenyl [COCnN]
  halide alcohol
Nicotine [54-11-5]                        --              26 ***
  Use: pharmaceutical/ pesticide
  other/ natural; Class: pyridine
  amine
Nicotinic acid (niacin) [59-67-6]         --                --
  Use: vitamin; Class: pyridine
  carboxylic acid
Dexamethasone sodium phosphate           23 **              --
  [CASRN2392-39-4]
  Use: pharmaceutical (synthetic
  corticosteroid); Class: steroid
Tributyltin benzoate [4342-36-3]        15 ***              455
  Use: microbicide; Class:
  organometallic
Tributyltin chloride [1461-22-9]        69 ***            81 ***
  Use: microbicide; Class:
  organometallic
Tributyltin methacrylate                112 **              430
  [2155-70-6]
  Use: microbicide; Class:
  organometallic
Tolazamide [1156-19-0]                    --               276 *
  Use: pharmaceutical
  (antidiabetic drug); Class:
  phenyl sulfonamide amine
Troglitazone [97322-87-7]               51 ***              --
  Use: pharmaceutical
  (antidiabetic drug); Class: not
  assigned
Persistant organochlorines (a)
  p,p'-DDE [72-55-9]                      --                --
  p,p'-DDT [50-29-3]                      --                460
  o,p'-DDT [789-02-6]                     --               272 *
  Heptachlor epoxide [1024-57-3]          --                --
  Mirex [2385-85-5]                       --                --
  Dieldrin [60-57-1]                      --                --
  [beta]-Hexachlorocyclohexane            --               206 *
    ([beta]-HCH) [319-85-7]
  Lindane ([gamma]-HCH) [58-89-9]         --                --

                                    Feeding behavior
                                    (Caenorhabditis        Insulin
Compound [CASRN]                        elegans)         sensitivity

Amitraz [33089-61-1]                     113 **              419
  Use: insecticide; Class:
  formamidine
Chlorpromazine hydrochloride             1 ***               490
  [69-09-0]
  Use: pharmaceutical
  (conventional antipsychotic);
  Class: phenyl halide
Haloperidol [52-86-8]                    9 ***               399
  Use: pharmaceutical
  (conventional antipsychotic);
  Class: phenyl-phenyl [COCnN]
  halide alcohol
Nicotine [54-11-5]                         --                --
  Use: pharmaceutical/ pesticide
  other/ natural; Class: pyridine
  amine
Nicotinic acid (niacin) [59-67-6]          --                --
  Use: vitamin; Class: pyridine
  carboxylic acid
Dexamethasone sodium phosphate             --                --
  [CASRN2392-39-4]
  Use: pharmaceutical (synthetic
  corticosteroid); Class: steroid
Tributyltin benzoate [4342-36-3]         135 **            154 **
  Use: microbicide; Class:
  organometallic
Tributyltin chloride [1461-22-9]          257              114 **
  Use: microbicide; Class:
  organometallic
Tributyltin methacrylate                  449                322
  [2155-70-6]
  Use: microbicide; Class:
  organometallic
Tolazamide [1156-19-0]                     --              112 **
  Use: pharmaceutical
  (antidiabetic drug); Class:
  phenyl sulfonamide amine
Troglitazone [97322-87-7]                108 **            21 ***
  Use: pharmaceutical
  (antidiabetic drug); Class: not
  assigned
Persistant organochlorines (a)
  p,p'-DDE [72-55-9]                       --                --
  p,p'-DDT [50-29-3]                       --                --
  o,p'-DDT [789-02-6]                     689                --
  Heptachlor epoxide [1024-57-3]           --                --
  Mirex [2385-85-5]                        --                --
  Dieldrin [60-57-1]                       --                --
  [beta]-Hexachlorocyclohexane             --                --
    ([beta]-HCH) [319-85-7]
  Lindane ([gamma]-HCH) [58-89-9]         707                635

                                      Islet cell      [beta] cell
Compound [CASRN]                       function         function

Amitraz [33089-61-1]                    149 **            320
  Use: insecticide; Class:
  formamidine
Chlorpromazine hydrochloride            27 ***           6 ***
  [69-09-0]
  Use: pharmaceutical
  (conventional antipsychotic);
  Class: phenyl halide
Haloperidol [52-86-8]                    7 ***           9 ***
  Use: pharmaceutical
  (conventional antipsychotic);
  Class: phenyl-phenyl [COCnN]
  halide alcohol
Nicotine [54-11-5]                        --               --
  Use: pharmaceutical/ pesticide
  other/ natural; Class: pyridine
  amine
Nicotinic acid (niacin) [59-67-6]         --               --
  Use: vitamin; Class: pyridine
  carboxylic acid
Dexamethasone sodium phosphate            --               --
  [CASRN2392-39-4]
  Use: pharmaceutical (synthetic
  corticosteroid); Class: steroid
Tributyltin benzoate [4342-36-3]         197 *           146 **
  Use: microbicide; Class:
  organometallic
Tributyltin chloride [1461-22-9]         218 *           103 **
  Use: microbicide; Class:
  organometallic
Tributyltin methacrylate                 199 *            354
  [2155-70-6]
  Use: microbicide; Class:
  organometallic
Tolazamide [1156-19-0]                  30 ***           93 **
  Use: pharmaceutical
  (antidiabetic drug); Class:
  phenyl sulfonamide amine
Troglitazone [97322-87-7]                 --             69 ***
  Use: pharmaceutical
  (antidiabetic drug); Class: not
  assigned
Persistant organochlorines (a)
  p,p'-DDE [72-55-9]                      --               --
  p,p'-DDT [50-29-3]                      --              917
  o,p'-DDT [789-02-6]                     --              699
  Heptachlor epoxide [1024-57-3]          --               --
  Mirex [2385-85-5]                       --               --
  Dieldrin [60-57-1]                      --               --
  [beta]-Hexachlorocyclohexane            --              614
    ([beta]-HCH) [319-85-7]
  Lindane ([gamma]-HCH) [58-89-9]         --              481

(a) Dichlorodiphenyltrichloroethane (DDT) or
dichlorodiphenyldichloroethylene (DDE). *** (pink squares) In top ~5th
percentile. ** (blue squares) In top 10th percentile. * (green
squares) In top 15th percentile. --Not active, score = 0.

Table 4. Similarity analysis: Top 10 most similar nonpharmaceuticals
in ToxCast[TM] (rank ordered by Pearson correlation of z-score
values).

Rank   Amitraz                 Haloperidol

1      Diquat dibromide        Gentian violet
       monohydrate             0.439
       0.337                   [548-62-9]
       [6385-62-2]             Fungicide
       Herbicide

2      Tralkoxydim             Difenzoquat metilsulfate
       0.307                   0.405
       [87820-88-0]            [43222-48-6]
       Herbicide               Herbicide

3      Pentamidine             1-Benzylquinolinium
       isethionate             chloride
       0.288                   0.4018
       [140-64-7]              [15619-48-4]
       Microbicide             Industrial

4      N-phenyl-1,4-           Didecyldimethylammonium
       benzenediamine          chloride
       0.258                   0.388
       [101-54-2]              [7173-51-5]
       Intermediate            Bactericide

5      FD&C yellow 5           Pentamidine isethionate
       0.256                   0.387
       [1934-21-0]             [140-64-7]
       Dye                     Microbicide

6      Mercuric chloride       Mercuric chloride
       0.252                   0.382
       [7487-94-7]             [7487-94-7]
       Bactericide             Bactericide

7      Difenzoquat             Tributyltin methacrylate
       metilsulfate            0.373
       0.248                   [2155-70-6]
       [43222-48-6]            Microbicide
       Herbicide

8      FD&C yellow 6           Dodecyltrimethylammonium
       0.246                   chloride
       [2783-94-0]             0.369
       Dye                     [112-00-5]
                               Bactericide

9      1,2-Benzisothiazolin-   N-methyldioctylamine
       3-one                   0.354
       0.238                   [4455-26-9]
       [2634-33-5]             Reactant
       Fungicide

10     Forchlorfenuron         Tributyltin chloride
       0.227                   0.334
       [68157-60-8]            [1461-22-9]
       Plant growth            Microbicide
       regulator

Rank   Nicotine             Dexamethasone        Tributyltin chloride

1      Mepiquat chloride    Cyclohexanol         Tributyltin
       0.553                0.292                methacrylate
       [24307-26-4]         [108-93-0]           0.859
       Herbicide            Precursor            [2155-70-6]
                                                 Microbicide

2      Imidacloprid         1,3-Dichloro-5,5-    Triphenyltin hydroxide
       0.430                dimethylhydantoin    0.517
       [138261-41-3]        0.268                [76-87-9]
       Insecticide          [118-52-5]           Fungicide
                            Disinfectant,
                            reactant

3      Triisononyl          Benzoic acid         Gentian violet
       trimellitate         0.265                0.500
       0.428                [65-85-0]            [548-62-9]
       [53894-23-8]         Intermediate,        Fungicide
       Plasticizer          preservative

4      Acetamiprid          2-Phenoxyethanol     Phenylmercuric acetate
       0.328                0.253                0.492
       [135410-20-7]        [122-99-6]           [62-38-4]
       Insecticide          Intermediate,        Fungicide
                            fragrance, solvent

5      Thiacloprid          Pentaerythritol      Didecyldimethyl-
       0.299                0.249                ammonium chloride
       [111988-49-9]        [115-77-5]           0.483
       Insecticide          Explosives/weapons   [7173-51-5]
                                                 Bactericide

6      Clothianidin         Clove leaf oil       Octhilinone
       0.276                0.237                0.444
       [210880-92-5]        [8000-34-8]          [26530-20-1]
       Insecticide          Natural              Fungicide

7      Nitrobenzene         1-Tetradecanol       Mercuric chloride
       0.251                0.229                0.395
       [98-95-3]            [112-72-1]           [7487-94-7]
       Reactant             Intermediate         Bactericide

8      Biphenyl             Sodium saccharin     1,2-Benzisothiazolin-
       0.247                hydrate              3-one
       [92-52-4]            0.228                0.383
       Intermediate,        [82385-42-0]         [2634-33-5]
       fungicide            Additive             Fungicide

9      2,6-Dimethylphenol   4,4'-Bipyridine      2,4-Bis(1-methyl-1-
       0.238                0.216                phenylethyl)phenol
       [576-26-1]           [553-26-4]           0.381
       Intermediate         Degradate            [2772-45-4]
                                                 Intermediate

10     2-Butoxyethanol      Diacetone alcohol    Ziram
       0.238                0.214                0.377
       [111-76-2]           [123-42-2]           [137-30-4]
       Solvent              Solvent              Fungicide

Rank   Tolazamide              Troglitazone

1      Sucrose                 Quinoxyfen
       0.477                   0.511
       [57-50-1]               [124495-18-7]
       Sweetener               Herbicide

2      Butylbenzene            Dichlorprop
       0.477                   0.476
       [104-51-8]              [120-36-5]
       Plasticizer, solvent,   Herbicide
       surfactant

3      4-Aminofolic acid       Dihexyl phthalate
       0.395                   0.464
       [54-62-6]               [84-75-3]
       Rodenticide             Plasticizer

4      4-Nitrotoluene          3,3',5,5'-Tetrabrom
       0.351                   bisphenol A
       [99-99-0]               0.442
       Reactant                [79-94-7]
                               Flame retardant

5      Methenamine             Oxadiazon
       0.348                   0.434
       [100-97-0]              [19666-30-9]
       Intermediate            Herbicide

6      Pyrithiobac-sodium      Clotrimazole
       0.343                   0.412
       [123343-16-8]           [23593-75-1]
       Herbicide               Fungicide

7      4-Vinyl-1-              Spirodiclofen
       cyclohexene dioxide     0.408
       0.335                   [148477-71-8]
       [106-87-6]              Insecticide
       Pesticide, reactant

8      Novaluron               Octrizole
       0.321                   0.408
       [116714-46-6]           [3147-75-9]
       Insecticide             UV absorber

9      Etridiazole             Butralin
       0.316                   0.404
       [2593-15-9]             [33629-47-9]
       Fungicide               Herbicide

10     2,4,6-Trichlorophenol   2,4-Bis(1-methyl-1-
       0.297                   phenylethyl)phenc
       [88-06-2]               0.399
       Herbicide, fungicide,   [2772-45-4]
       reactant                Intermediate

Figure 1. Analytical framework for source data and analyses.

              Prediction models for
            adipocyte differentiation,
            feeding behavior, insulin
             sensitivity, islet cell        Similarity profiling to
                     function                  signpost chemicals

Source     * 1860 chemicals ToxCast       * 1061 chemicals
data         Phase 2                        -? A subset of ToxCast
           * 821 assays (724 gene-             Phase 2 with 90%
             based)                            complete information on
                                               685 assays
                                          * 685 assays

Analysis   * Expert opinion to select     * z-Scores calculated for
             relevant gene based assays     each chemical-assay pair
           * Filter to remove z-score     * Assigned into 1 of 6
             values [less than or equal     categories (chemical-assay
             to] 2 from cytotoxicity        pair not tested; 0, 1, 2,
           * Missing data considered        3, or 4 score for
             inactive                       bioactivity)
           * z-Scores added to
             [AC.sub.50] values
           * Summed values transformed
             to a range of 0 to 1 where
             1 reflects most potent

Output     * Numerical ToxPi scores and   * Pearson's correlation
             ranked list of chemicals       scores
           * Visual displays of top-      * Ranking of chemicals most
             ranked chemicals from          similar to signpost
             ToxPi analysis                 chemicals
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Author:Auerbach, Scott; Filer, Dayne; Reif, David; Walker, Vickie; Holloway, Alison C.; Schlezinger, Jennif
Publication:Environmental Health Perspectives
Geographic Code:1U9CA
Date:Aug 1, 2016
Words:15056
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