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.

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.

Mapping DEHP to the adverse outcome pathway network for human female reproductive toxicity.

Adverse outcome pathways (AOPs) and AOP networks are tools for mechanistic presentation of toxicological effects across different levels of biological organization. These tools are used to better understand how chemicals impact human health. In this study, a four-step workflow was used to derive the AOP network of human female reproductive toxicity (HFRT-AOP) from five AOPs available in the AOP-Wiki and ten AOPs obtained from the literature. Standard network analysis identified key events (KEs) that are point of convergence and divergence, upstream and downstream KEs, and bottlenecks across the network. To map di-(2-ethylhexyl) phthalate (DEHP) to the HFRT-AOP network, we extracted DEHP target genes and proteins from the Comparative Toxicogenomic and the CompTox Chemicals Dashboard databases. Enriched GO terms analysis was used to identify relevant biological processes in the ovary that are DEHP targets, whereas screening of scientific literature was performed manually and automatically using AOP-helpFinder. We combined this information to map DEHP to HFRT-AOP network to provide insight on the KEs and system-level perturbations caused by this endocrine disruptor and the emergent paths. This approach can enable better understanding of the toxic mechanism of DEHP-induced human female reproductive toxicity and reveal potential novel DEHP female reproductive targets for experimental studies.

Impact of In Vitro Long-Term Low-Level DEHP Exposure on Gene Expression Profile in Human Granulosa Cells.

Here, we applied a model of long-term exposure of human granulosa cells to low environmentally relevant levels of di(2-ethylhexyl) phthalate (DEHP). This approach provides more relevant data regarding the impact of DEHP on the function of human granulosa cells. The immortalized human granulosa cells HGrC1 were exposed to 50 nM and 250 nM DEHP for four weeks. The cells were collected every week to analyze the basal granulosa cells' functions. A portion of the DEHP-exposed cells was stimulated with forskolin (FOR) for 48 h. Steroidogenesis was investigated using ELISA, whereas DNBQ sequencing and RT-qPCR were used to analyze gene expression. The results show that steroidogenesis was not affected by DEHP exposure. RNAsequencing shows that DEHP caused week- and concentration-specific changes in various genes and functions in HGrC1. Sulfotransferase family 1A member 3 (SULT1A3) and 4 (SULT1A4), which are involved in catecholamine metabolism, were the most prominent genes affected by DEHP under both the basal and FOR-stimulated conditions in all four weeks of exposure. This study showed, for the first time, that SULT1A3 and SULT1A4 are expressed in human granulosa cells, are regulated by FOR, and are affected by low-level DEHP exposure. These data provide new insight into the relationship between DEHP, SULT1A3, and SULT1A4 in human granulosa cells.

Integration of data from the in vitro long-term exposure study on human endothelial cells and the in silico analysis: A case of dibutyl phthalate-induced vascular dysfunction.

General population is exposed to dibutyl phthalate (DBP) through continuous use of various consumer products. DBP exhibits its effects mainly on the endocrine and reproductive system but it can also affect the function of the vasculature; however, the underlying mechanisms behind DBP-induced vascular dysfunction are not fully understood. To infer pathways, molecular functions, biological processes, and human diseases associated with DBP exposure, we integrated the toxicogenomic data obtained from the 4-week-long exposure of human vascular endothelial cells (ECs) to three environmentally relevant concentrations of DBP with the in silico analysis. Nine genes were affected by DBP exposure: six of the integrin family, VCAM1, ICAM1, and MMP2. As shown by the in silico analysis, changes in DBP-affected genes could affect extracellular matrix and binding of molecules and cells to ECs, thereby altering cell adhesion and migration. Several pathways, molecular functions, and biological processes were further identified to provide insight into the DBP-vascular disease relationships and the potential mechanism of action. The top three human disease categories associated with DBP exposure and vascular dysfunction include cardiovascular, cerebrovascular, and immune system diseases. Integration of experimental and in silico approaches may offer better understanding of the potential human health risks associated with DBP exposure.

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.

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.