Global gene expression analysis reveals a subtle effect of DEHP in human granulosa cell line HGrC1.

Di(2-ethylhexyl) phthalate (DEHP) is an endocrine disruptor that exerts anti-steroidogenic effects in human granulosa cells; however, the extent of this effect depends on the concentration of DEHP and granulosa cell models used for exposure. The objective of this study was to identify the effects of low- and high-dose DEHP exposure in human granulosa cells. We exposed human granulosa cell line HGrC1 to 3 nM and 25 µM DEHP for 48 h. The whole genome transcriptome was analyzed using the DNBSEQ sequencing platform and bioinformatics tools. The results revealed that 3 nM DEHP did not affect global gene expression, whereas 25 µM DEHP affected the expression of only nine genes in HGrC1 cells: ABCA1, SREBF1, MYLIP, TUBB3, CENPT, NUPR1, ASS1, PCK2, and CTSD. We confirmed the downregulation of ABCA1 mRNA and SREBP-1 protein (encoded by the SREBF1 gene), both involved in cholesterol homeostasis. Despite these changes, progesterone production remained unaffected in low- and high-dose DEHP-exposed HGrC1 cells. The high concentration of DEHP decreased the levels of ABC1A mRNA and SREBP-1 protein and prevented the upregulation of STAR, a protein involved in progesterone synthesis, in forskolin-stimulated HGrC1 cells; however, the observed changes were not sufficient to alter progesterone production in forskolin-stimulated HGrC1 cells. Overall, this study suggests that acute exposure to low concentration of DEHP does not compromise the function of HGrC1 cells, whereas high concentration causes only subtle effects. The identified nine novel targets of high-dose DEHP require further investigation to determine their role and importance in DEHP-exposed human granulosa cells.

Global gene expression analysis reveals novel transcription factors associated with long-term low-level exposure of EA.hy926 human endothelial cells to bisphenol A.

Bisphenol A (BPA) is an endocrine disruptor that binds to estrogen receptors (ER); however, studies have shown that the ER pathway was not always the primary molecular mechanism of BPA's action in cells and that gene transcription could be altered by different exposure times and doses. Here, we sought to understand the correlation between the BPA-responsive genes that have associated biological functions and the transcription factors (TFs) involved in their regulation by repeatedly exposing human endothelial cells EA.hy926 to three nanomolar concentrations of BPA (10-9 M, 10-8 M, and 10-7 M) for 14 weeks, after which changes in global gene expression were determined by RNA sequencing. Cytoscape plug-in iRegulon was used to infer TFs involved in the control of BPA-deregulated genes. The results show a minimal overlap in deregulated genes between three concentrations of BPA, with 10-9 M BPA having the highest number of deregulated genes. TF analysis suggests that all three concentrations of BPA were active in the absence of an ER-mediated pathway. A unique set of TFs (NES≥4) has been identified for each BPA concentration, including the NFκB family and CEBPB for 10-9 M BPA, MEF family, AHR/ARNT, and ZBTB33 for 10-8 M BPA, and IRF1-7 and OVOL1/OVOL2 for 10-7 M BPA, whereas STAT1/STAT2 were common TFs for 10-9 M and 10-7 M BPA. Overall, our data suggest that long-term low-level exposure of EA.hy926 cells to BPA leads to concentration-specific changes in gene expression that are not controlled by the ER-mediated signaling but rather by other mechanisms.

An in silico toxicogenomic approach in constructing the aflatoxin B1-mediated regulatory network of hub genes in hepatocellular carcinoma.

Aflatoxin B1 (AFB1) can cause hepatocellular carcinoma (HCC) through a mutagenic mode of action but can also lead to global changes in gene expression; however, the AFB1 network of molecular pathways involved in HCC is not known. Here, we used toxicogenomic data from human liver cells exposed to AFB1 to infer the network of AFB1-responsive molecular pathways involved in HCC. The following computational tools: STRING, MCODE, cytoHubba, iRegulon, kinase enrichment tool KEA3, and DAVID were used to identify protein-protein interaction network, hub genes, transcription factors (TFs), upstream kinases, and biological processes (BPs). Predicted molecular events were validated with an external dataset, whereas the hub genes in HCC were validated using the UALCAN database. The results revealed an association between AFB1 and the hub genes involved in the cell cycle. We identified TFs that regulate the hub genes and linked them with upstream kinases including cyclin-dependent kinases, mitogen-activated protein kinase 1, and AKT. This approach enabled the construction of the AFB1-mediated regulatory network consisting of upstream kinases, TFs, hub genes, and BPs, thus revealing the signaling hierarchy and information flow that may contribute to AFB1-induced HCC. This could be a useful tool in predicting the molecular mechanisms involved in chemical-induced diseases when available toxicogenomic data exist.

DEHP Decreases Steroidogenesis through the cAMP and ERK1/2 Signaling Pathways in FSH-Stimulated Human Granulosa Cells.

DEHP is an endocrine disruptor that interferes with the function of the female reproductive system. Several studies suggested that DEHP affects steroidogenesis in human and rodent granulosa cells (GC). Some studies have shown that DEHP can also affect the FSH-stimulated steroidogenesis in GC; however, the mechanism by which DEHP affects hormone-challenged steroidogenesis in human GC is not understood. Here, we analyzed the mechanism by which DEHP affects steroidogenesis in the primary culture of human cumulus granulosa cells (hCGC) stimulated with FSH. Cells were exposed to DEHP and FSH for 48 h, and steroidogenesis and the activation of cAMP and ERK1/2 were analyzed. The results show that DEHP decreases FSH-stimulated STAR and CYP19A1 expression, which is accompanied by a decrease in progesterone and estradiol production. DEHP lowers cAMP production and CREB phosphorylation in FSH but not cholera toxin- and forskolin-challenged hCGC. DEHP was not able to decrease steroidogenesis in cholera toxin- and forskolin-stimulated hCGC. Furthermore, DEHP decreases FSH-induced ERK1/2 phosphorylation. The addition of EGF rescued ERK1/2 phosphorylation in FSH- and DEHP-treated hCGC and prevented a decrease in steroidogenesis in the FSH- and DEHP-treated hCGC. These results suggest that DEHP inhibits the cAMP and ERK1/2 signaling pathways, leading to the inhibition of steroidogenesis in the FSH-stimulated hCGC.

Dibutyl phthalate promotes angiogenesis in EA.hy926 cells through estrogen receptor-dependent activation of ERK1/2, PI3K-Akt, and NO signaling pathways.

Dibutyl phthalate (DBP) is an endocrine disruptor that has been widely used in various products of human use. DBP exposure has been associated with reproductive and cardiovascular diseases and metabolic disorders. Although dysfunction of the vascular endothelium is responsible for many cardiovascular and metabolic diseases, little is known about the effects of DBP on human endothelium. In this study, we investigated the effect of three concentrations of DBP (10-6, 10-5, and 10-4 M) on angiogenesis in human endothelial cell (EC) line EA.hy926 after acute exposure. Tube formation assay was used to investigate in vitro angiogenesis, whereas qRT-PCR was employed to measure mRNA expression. The effect of DBP on extracellular signal-regulated kinase 1/2 (ERK1/2), phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt), and endothelial nitric oxide (NO) synthase (eNOS) activation was examined using Western blotting, whereas the Griess method was used to assess NO production. Results show that the 24-h-long exposure to 10-4 M DBP increased endothelial tube formation, which was prevented by addition of U0126 (ERK1/2 inhibitor), wortmannin (PI3K-Akt inhibitor), and l-NAME (NOS inhibitor). Short exposure to 10-4 M DBP (from 15 to 120 min) phosphorylated ERK1/2, Akt, and eNOS in different time points and increased NO production after 24 and 48 h of exposure. Application of nuclear estrogen receptor (ER) and G protein-coupled ER (GPER) inhibitors ICI 182,780 and G-15, respectively, abolished the DBP-mediated ERK1/2, Akt, and eNOS phosphorylation and increase in NO production. In this study, we report for the first time that DBP exerts a pro-angiogenic effect on human vascular ECs and describe the molecular mechanism involving ER- and GPER-dependent activation of ERK1/2, PI3K-Akt, and NO signaling pathways.

Long-term in vitro exposure of human granulosa cells to the mixture of endocrine disrupting chemicals found in human follicular fluid disrupts steroidogenesis.

Most in vitro studies examine the effects of a single ED or a mixture of EDs on granulosa cells using short-term exposure; however, this approach is unlikely to reflect long-term, real-life exposures that are common in humans. We established an in vitro model that mimics long-term exposure of granulosa cells to real-life ED mixture. Human granulosa cells, HGrC1, were exposed to the mixture consisting of bisphenol A, polychlorinated biphenyl 153, benzo[a]pyrene, and perfluorooctanesulfonate in concentrations found in human follicular fluid (MIX) for 48 h and 4 weeks. Only long-term exposure to MIX decreased estradiol production after 2 and 3 weeks, and CYP19A1 protein after 2 weeks of exposure. By week 4, the cells restored estradiol production and CYP19A1 protein level. MIX increased basal progesterone production after 3 and 4 weeks of exposure but did not affect STAR and CYP11A1 mRNA. Cells that had been exposed to MIX for 4 weeks showed augmentation of forskolin-stimulated progesterone production. These results demonstrate that only long-term exposure to MIX alters steroidogenesis in HGrC1. This study also revealed that adverse effects of MIX on steroidogenesis in HGrC1 occurred a few weeks into MIX exposure and that this effect can be transient.

Integration of data from the cell-based ERK1/2 ELISA and the Comparative Toxicogenomics Database deciphers the potential mode of action of bisphenol A and benzo[a]pyrene in lung neoplasm.

Chemicals can activate a variety of signaling pathways, initiating changes in gene expression and cellular functions. Here, we combined experimental data on the chemical-induced extracellular signal-regulated kinase 1/2 (ERK1/2) activation with the Comparative Toxicogenomics Database (CTD) to connect signaling, genes, and phenotypes to reveal the potential chemical's mode of action (MOA) responsible for the disease state. Experimental data on ERK1/2 activation were derived from the cell-based phospho-ERK1/2 ELISA on human alveolar epithelial cells A549. A549 cells were exposed to bisphenol A (BPA), benzo[a]pyrene (BaP), tributyltin (TBT), and ibuprofen from 10-12 M to 10-5 M. Results show that BPA, BaP, and TBT can activate ERK1/2 in A549 cells. We selected BPA and BaP to elucidate the molecular events connecting chemical exposure, ERK1/2 signaling, phenotypes, and lung neoplasm (LN) using CTD. CTD analysis showed that BPA and BaP share 26 mitogen-activated protein kinase 1/3 (MAPK1/3) signaling genes associated with LN. Phenotype prioritization revealed 37 BPA, 10 BaP, and 11 shared key phenotypes associated with LN. Alignment of MAPK1/3 signaling genes and phenotypes showed that ERK1/2 and oxidative stress, EGFR gene, and positive regulation of cell proliferation and migration could be the shared key events (KE) for BPA and BaP. This analysis also identified protein kinase B and ERK1/2 signaling, FGF9, FGFR1 and FGFR2 genes, positive regulation of cell proliferation and angiogenesis as KE in MOA for BPA, whereas ERK1/2 signaling, IL6 and DAB2IP genes, negative regulation of cell proliferation and inflammatory response were identified as KE in MOA for BaP.

Biological effects of chronic and acute exposure of human endothelial cell line EA.hy926 to bisphenol A: New tricks from an old dog.

Although epidemiological and animal studies suggest a possible correlation between bisphenol A (BPA) exposure and atherosclerosis, very few in vitro mechanistic and functional studies regarding the effect of BPA on vascular cells have been conducted. Here, we applied a "real-life" exposure scenario by continuously exposing human endothelial cell (EC) line EA.hy926 to environmentally relevant concentrations of BPA (10-9, 10-8, and 10-7 M) during 14 weeks. We also exposed EA.hy926 cells to higher concentrations of BPA (10-7, 10-6, and 10-5 M) for up to 48 h to gain mechanistic insight into the BPA's action in ECs. Chronic exposure to BPA produced some unexpected effects in EA.hy926 cells including a transient decrease in the adhesion of monocytes to the EC monolayer and decrease in the expression of cellular adhesion molecules, improvement in endothelial barrier function and elevated expression of tight junction proteins occludin and zonula occludens-1 (ZO-1), increased adhesion of ECs, and increased nitric oxide (NO) production. Some of these effects, such as diminished adhesion of monocytes to the EC monolayer and elevated NO production have also been replicated during acute exposure experiments. Using Western blotting and specific pharmacological inhibitors in the acute study, we have shown that direct BPA's action in EA.hy926 cells involves activation of estrogen receptor (ER), phosphorylation of protein kinase B (PKB/Akt) and endothelial nitric oxide synthase (eNOS)-mediated production of NO. Collectively, these data indicate that BPA induces functional and molecular changes in EA.hy926 cells associated with the promotion of endothelial integrity through activation of the ER/Akt/eNOS pathway.

Rosiglitazone increases expression of steroidogenic acute regulatory protein and progesterone production through PPARγ-EGFR-ERK1/2 in human cumulus granulosa cells.

The mechanism by which rosiglitazone (ROSI: a thiazolidinedione (TZD)) affects steroid production in undifferentiated human granulosa cells is not known. In this study, cultured human cumulus granulosa cells were exposed to ROSI and pharmacological inhibitors of the extracellular signal-regulated kinase 1/2 (ERK1/2), epidermal growth factor receptor (EGFR) and peroxisome proliferator-activated receptor gamma (PPARγ) signalling pathways. Expression of progesterone biosynthetic enzymes, PPARγ and PPARα, progesterone production and ERK1/2 activation were analysed. After 48h, 30µM ROSI increased STAR, 3ßHSD and PPARγ mRNA and elevated progesterone production in human cumulus granulosa cells. Addition of ERK1/2 (U0126), EGFR (AG1478) and PPARγ (GW9662) inhibitors prevented the ROSI-induced STAR mRNA expression and progesterone production after 48h. Inhibition of PPARγ, but not EGFR or ERK1/2, decreased the PPARγ mRNA levels induced by ROSI in human cumulus granulosa cells after 48h. On the other hand, U0126 and GW9662 prevented the ROSI-induced increase in PPARγ transcripts after 6h. Western blot analysis showed that ROSI induced a rapid ERK1/2 activation, which was prevented by inhibition of ERK1/2, EGFR and PPARγ in human cumulus granulosa cells. Overall, these data suggested that PPARγ, EGFR and ERK1/2 were involved in the stimulatory effect of ROSI on STAR expression and progesterone production in undifferentiated human cumulus granulosa cells.

BPA activates EGFR and ERK1/2 through PPARγ to increase expression of steroidogenic acute regulatory protein in human cumulus granulosa cells.

Bisphenol A (BPA) negatively affects steroid production in human luteinized granulosa cells (GC). This study was designed to address two important questions: (1) whether BPA exerts the same disruptive effect in human cumulus granulosa cells (hCGC) and (2) to reveal the molecular mechanism underlying the BPA's action on steroidogenesis. We used cultured hCGC since these cells exert the properties of GC from early antral follicles. Results showed that BPA at 100 µM decreased estradiol level and CYP19A1 mRNA, but increased progesterone production, steroidogenic acute regulatory protein (STAR) and peroxisome proliferator-activated receptor gamma (PPARγ) mRNA expression after 48 h. Shorter (6 h) exposure to BPA elevated PPARγ mRNA level in hCGC. Addition of ERK1/2 (U0126), EGFR (AG1478) and PPARγ (GW9662) inhibitors prevented the BPA-induced STAR and PPARγ mRNA expression. Western blot analysis showed that BPA induced a rapid EGFR and ERK1/2 activation. The BPA-induced EGFR phosphorylation was prevented by addition of the PPARγ inhibitor, whereas the BPA-induced ERK1/2 activation was prevented by addition of the EGFR or PPARγ inhibitor. These data show that BPA increases the progesterone and decreases the estradiol biosynthetic pathway in hCGC. Augmentation of the progesterone biosynthetic pathway is mediated through the PPARγ-dependent activation of EGFR and ERK1/2, leading to increased expression of STAR mRNA.

T-2 toxin downregulates LHCGR expression, steroidogenesis, and cAMP level in human cumulus granulosa cells.

Our goals were to investigate whether environmentally relevant doses of T-2 toxin can affect human ovarian granulosa cells' function and to reveal the potential mechanism of T-2 toxin's action. Results showed that T-2 toxin strongly attenuated luteinizing hormone/choriogonadotropin receptor (LHCGR) mRNA expression in follicle-stimulating hormone (FSH)-stimulated human cumulus granulosa cells. Addition of human chorionic gonadotropin was not able to elicit maximal response of ovulatory genes amphiregulin, epiregulin, and progesterone receptor. T-2 toxin reduced mRNA levels of CYP19A1 and steroidogenic acute regulatory protein (STAR) and lowered FSH-stimulated estradiol and progesterone production. Mechanistic experiments demonstrated that T-2 toxin decreased FSH-stimulated cyclic adenosine monophosphate (cAMP) production. Addition of total PDE inhibitor 3-isobutyl-1-methylxanthine prevented T-2 toxin's action on LHCGR, STAR, and CYP19A1 mRNA expression in FSH-stimulated human cumulus granulosa cells. Furthermore, T-2 toxin partially decreased 8-bromoadenosine 3'5'-cyclic monophosphate (8-Br-cAMP)-stimulated LHCGR and STAR, but did not affect 8-Br-cAMP-stimulated CYP19A1 mRNA expression in human cumulus granulosa cells. Overall, our data indicate that environmentally relevant dose of T-2 toxin decreases steroidogenesis and ovulatory potency in human cumulus granulosa cells probably through activation of PDE, thus posing a significant risk for female fertility.

Environmental mixture with estrogenic activity increases Hsd3b1 expression through estrogen receptors in immature rat granulosa cells.

Humans are exposed not only to single endocrine disruptors, but also to chemical mixtures that can adversely affect their reproductive health. Steroidogenesis in reproductive tissues is emerging as the key target of endocrine disruptor action. Here, we analyzed the effect of environmental chemical mixtures with estrogenic activity on steroidogenic processes in immature rat granulosa cells and whether the observed steroidogenic effects were mediated through estrogen receptors. Extracts from untreated wastewater were prepared by solid-phase extraction and silica gel fractionation. ER-CALUX assay showed that the polar fractions of wastewater exerted different levels of estrogenic activity. Exposure of immature granulosa cells to the polar fraction exerting 9 ng of 17ß-estradiol equivalents per liter of water of estrogenic activity increased mRNA expression of the key enzymes of progesterone biosynthetic pathway Star and Hsd3b1, but did not alter the level of Cyp19a1 and Lhr. Addition of estrogen receptor inhibitor ICI 182 780 prevented the estrogenic mixture-induced increase in Hsd3b1, but not Star mRNA level in immature granulosa cells. These results indicate that the environmental chemical mixtures with estrogenic activity exert endocrine disrupting effects by augmenting the progesterone biosynthetic pathway in immature rat granulosa cells, which is an effect achieved in part through activation of the estrogen receptors.