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Connectivity mapping is a method used in the pharmaceutical industry to find connections between small molecules, disease states, and genes. The concept can be applied to a predictive toxicology paradigm to find connections between chemicals, adverse events, and genes. In order to assess the applicability of the technique for predictive toxicology purposes, we performed gene array experiments on 34 different chemicals: bisphenol A, genistein, ethinyl-estradiol, tamoxifen, clofibrate, dehydorepiandrosterone, troglitazone, diethylhexyl phthalate, flutamide, trenbolone, phenobarbital, retinoic acid, thyroxine, 1α,25-dihydroxyvitamin D3, clobetasol, farnesol, chenodeoxycholic acid, progesterone, RU486, ketoconazole, valproic acid, desferrioxamine, amoxicillin, 6-aminonicotinamide, metformin, phenformin, methotrexate, vinblastine, ANIT (1-naphthyl isothiocyanate), griseofulvin, nicotine, imidacloprid, vorinostat, 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD) at the 6-, 24-, and 48-hour time points for 3 different concentrations in the 4 cell lines: MCF7, Ishikawa, HepaRG, and HepG2 GEO (super series accession no.: GSE69851). The 34 chemicals were grouped in to predefined mode of action (MOA)-based chemical classes based on current literature. Connectivity mapping was used to find linkages between each chemical and between chemical classes. Cell line-specific linkages were compared with each other and to test whether the method was platform and user independent, a similar analysis was performed against publicly available data. The study showed that the method can group chemicals based on MOAs and the inter-chemical class comparison alluded to connections between MOAs that were not predefined. Comparison to the publicly available data showed that the method is user and platform independent. The results provide an example of an alternate data analysis process for high-content data, beneficial for predictive toxicology, especially when grouping chemicals for read across purposes., (© The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

To further define the utility of the Ishikawa cells as a reliable in vitro model to determine the potential estrogenic activity of chemicals of interest, transcriptional changes induced by genistein (GES) in Ishikawa cells at various doses (10 pM, 1 nM, 100 nM, and 10 μM) and time points (8, 24, and 48 h) were identified using a comprehensive microarray approach. Trend analysis indicated that the expression of 5342 unique genes was modified by GES in a dose- and time-dependent manner (P ≤ 0.0001). However, the majority of gene expression changes induced in Ishikawa cells were elicited by the highest dose of GES evaluated (10 μM). The GES' estrogenic activity was identified by comparing the Ishikawa cells' response to GES versus 17 α-ethynyl estradiol (EE, at equipotent doses, ie, 10 μM vs 1 μM, respectively) and was defined by changes in the expression of 284 unique genes elicited by GES and EE in the same direction, although the magnitude of the change for some genes was different. Further, comparing the response of the Ishikawa cells exposed to high doses of GES and EE versus the response of the juvenile rat uterus exposed to EE, we identified 66 unique genes which were up- or down regulated in a similar manner in vivo as well as in vitro Genistein elicits changes in multiple molecular pathways affecting various biological processes particularly associated with cell organization and biogenesis, regulation of translation, cell proliferation, and intracellular transport; processes also affected by estrogen exposure in the uterus of the rat. These results indicate that Ishikawa cells are capable of generating a biologically relevant estrogenic response and offer an in vitro model to assess this mode of action., (© The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

We have determined the gene expression profile induced by 17 alpha-ethynyl estradiol (EE) in Ishikawa cells, a human uterine-derived estrogen-sensitive cell line, at various doses (1 pM, 100 pM, 10 nM, and 1 microM) and time points (8, 24, and 48 h). The transcript profiles were compared between treatment groups and controls (vehicle-treated) using high-density oligonucleotide arrays to determine the expression level of approximately 38,500 human genes. By trend analysis, we determined that the expression of 2560 genes was modified by exposure to EE in a dose- and time-dependent manner (p

The rat uterus responds to acute estrogen treatment with a series of well-characterized physiological responses; however, the gene expression changes required to elicit these responses have not been fully characterized. In order to understand early events induced by estrogen exposure in vivo, we evaluated the temporal gene expression in the uterus of the immature rat after a single dose of 17 alpha-ethinyl estradiol (EE) by microarray analysis, evaluating the expression of 15,923 genes. Immature 20-day-old rats were exposed to a single dose of EE (10 microg/kg), and the effects on uterine histology, weight, and gene expression were determined after 1, 2, 8, 24, 48, 72, and 96 h. EE induced changes in the expression of 3867 genes, at least at one time point (p < or = 0.0001), and at least 1.5-fold (up- or downregulated). Specifically, the expression of 8, 116, 3030, 2076, 381, 445, and 125 genes was modified at 1, 2, 8, 24, 48, 72, or 96 h after exposure to EE, respectively (p < or = 0.0001, t-test). At the tissue and organ level, a clear uterotrophic response was elicited by EE after only 8 h, reaching a maximum after 24 h and remaining detectable even after 96 h of exposure. The uterine phenotypic changes were induced by sequential changes in the transcriptional status of a large number of genes, in a program that involves multiple molecular pathways. Using the Gene Ontology to better understand the temporal response to estrogen exposure, we determined that the earliest changes were in the expression of genes whose products are involved in transcriptional regulation and signal transduction, followed by genes implicated in protein synthesis, energy utilization, solute transport, cell proliferation and differentiation, tissue remodeling, and immunological responses among other pathways. The compendium of genes here presented represents a comprehensive compilation of estrogen-responsive genes involved in the uterotrophic response.

The purpose of this study was to determine (1) the transcriptional program elicited by exposure to three estrogen receptor (ER) agonists: 17 alpha-ethynyl estradiol (EE), genistein (Ges), and bisphenol A (BPA) during fetal development of the rat testis and epididymis; and (2) whether very low dosages of estrogens (evaluated over five orders of magnitude of dosage) produce unexpected changes in gene expression (i.e., a non-monotonic dose-response curve). In three independently conducted experiments, Sprague-Dawley rats were dosed (sc) with 0.001-10 microg EE/kg/day, 0.001-100 mg Ges/kg/day, or 0.002-400 mg BPA/kg/day. While morphological changes in the developing reproductive system were not observed, the gene expression profile of target tissues were modified in a dose-responsive manner. Independent dose-response analyses of the three studies identified 59 genes that are significantly modified by EE, 23 genes by Ges, and 15 genes by BPA (out of 8740), by at least 1.5 fold (up- or down-regulated). Even more genes were observed to be significantly changed when only the high dose is compared with all lower doses: 141, 46, and 67 genes, respectively. Global analyses aimed at detecting genes consistently modified by all of the chemicals identified 50 genes whose expression changed in the same direction across the three chemicals. The dose-response curve for gene expression changes was monotonic for each chemical, with both the number of genes significantly changed and the magnitude of change, for each gene, decreasing with decreasing dose. Using the available annotation of the gene expression changes induced by ER-agonist, our data suggest that a variety of cellular pathways are affected by estrogen exposure. These results indicate that gene expression data are diagnostic of mode of action and, if they are evaluated in the context of traditional toxicological end-points, can be used to elucidate dose-response characteristics.

The profound effects of 17beta-estradiol on cell growth, differentiation, and general homeostasis of the reproductive and other systems, are mediated mostly by regulation of temporal and cell type-specific expression of different genes. In order to understand better the molecular events associated with the activation of the estrogen receptor (ER), we have used microarray technology to determine the transcriptional program and dose-response characteristics of exposure to a potent synthetic estrogen, 17 alpha-ethynyl estradiol (EE), during prepubertal development. Changes in patterns of gene expression were determined in the immature uterus and ovaries of Sprague-Dawley rats on postnatal day (PND) 24, 24 h after exposure to EE, at 0.001, 0.01, 0.1, 1 and 10 micro g EE/kg/day (sc), for four days (dosing from PND 20 to 23). The transcript profiles were compared between treatment groups and controls using oligonucleotide arrays to determine the expression level of approximately 7000 annotated rat genes and over 1740 expressed sequence tags (ESTs). Quantification of the number of genes whose expression was modified by the treatment, for each of the various doses of EE tested, showed clear evidence of a dose-dependent treatment effect that follows a monotonic response, concordant with the dose-response pattern of uterine wet-weight gain and luminal epithelial cell height. The number of genes whose expression is affected by EE exposure increases according to dose. At the highest dose tested of EE, we determined that the expression level of over 300 genes was modified significantly (p < or = 0.0001). A dose-dependent analysis of the transcript profile revealed a set of 88 genes whose expression is significantly and reproducibly modified (increased or decreased) by EE exposure (p < or = 0.0001). The results of this study demonstrate that, exposure to a potent estrogenic chemical during prepubertal maturation changes the gene expression profile of estrogen-sensitive tissues. Furthermore, the products of the EE-regulated genes identified in these tissues have a physiological role in different intracellular pathways, information that will be valuable to determine the mechanism of action of estrogens. Moreover, those genes could be used as biomarkers to identify chemicals with estrogenic activity.

Exposure to some compounds with estrogenic activity, during fetal development, has been shown to alter development of reproductive organs, leading to abnormal function and disease either after birth or during adulthood. In order to understand the molecular events associated with the estrogenicity of different chemicals and to determine whether common sets of gene expression changes can be predictive of estrogenic activity, we have used microarray technology to determine the transcriptional program influenced by exposure to this class of compounds during organogenesis and development. Changes in patterns of gene expression were determined in the developing uterus and ovaries of Sprague-Dawley rats on GD 20, exposed to graded dosages (sc) of 17alpha-ethynyl estradiol (EE), genistein, or bisphenol A (BPA) from GD 11 to GD 20. Dose levels were roughly equipotent in estrogenic activity. We compared the transcript profiles between treatment groups and controls, using oligonucleotide arrays to determine the expression level of approximately 7000 rat genes and over 1000 expressed squence tags (ESTs). At the highest tested doses of EE, BPA, or genistein, we determined that less than 2% of the mRNA detected by the array showed a 2-fold or greater change in their expression level (increase or decrease). A dose-dependent analysis of the transcript profile revealed a common set of genes whose expression is significantly and reproducibly modified in the same way by each of the 3 chemicals tested. Additionally, each compound induces changes in the expression of other transcripts that are not in common with the others, which indicated not all compounds with estrogenic activity act alike. The results of this study demonstrate that transplacental exposure to chemicals with estrogenic activity changes the gene expression profile of estrogen-sensitive tissues, and that the analysis of the transcript profile of these tissues could be a valuable approach to determining the estrogenicity of different compounds.