Vascular endothelial effects of dibutyl phthalate: In vitro and in vivo evidence.

Dibutyl phthalate (DBP) is widely used in many consumer and personal care products. Here, we report vascular endothelial response to DBP in three different exposure scenarios: after short-term exposure (24 h) of human endothelial cells (ECs) EA.hy926 to 10-6, 10-5, and 10-4 M DBP, long-term exposure (12 weeks) of EA.hy926 cells to 10-9, 10-8, and 10-7 M DBP, and exposure of rats (28 and 90 days) to 100, 500, and 5000 mg DBP/kg food. We examined different vascular functions such as migration of ECs, adhesion of ECs to the extracellular matrix, tube formation, the morphology of rat aorta, as well as several signaling pathways involved in controlling endothelial function. Short-term in vitro exposure to DBP increased migration of ECs through G protein-coupled estrogen receptor, extracellular signal-regulated kinase 1/2, and nitric oxide (NO) signaling and decreased adhesion to gelatin. Long-term in vitro exposure to DBP transiently increased EC migration and had a bidirectional effect on EC adhesion to gelatin and tube formation. These effects were accompanied by a sustained increase in NO production and endothelial NO synthase (eNOS) and Akt activity. In vivo, exposure to DBP for 90 days decreased the aortic wall-to-lumen ratio and increased eNOS and Akt phosphorylation in ECs of rat aorta. This comparative investigation has shown that exposure to DBP may affect vascular function by altering EC migration, adhesion to gelatin, and tube formation after short- and long-term in vitro exposure and by decreasing the aortic wall-to-lumen ratio in vivo. The eNOS-NO and Akt signaling could be important in mediating the effects of DBP in long-term exposure scenarios.

In vitro and in vivo exposure of endothelial cells to dibutyl phthalate promotes monocyte adhesion.

The effect of endothelial cells' exposure to dibutyl phthalate (DBP) on monocyte adhesion is largely unknown. We evaluated monocyte adhesion to DBP-exposed endothelial cells by combining three approaches: short-term exposure (24 h) of EA.hy926 cells to 10-6, 10-5, and 10-4 M DBP, long-term exposure (12 weeks) of EA.hy926 cells to 10-9, 10-8, and 10-7 M DBP, and exposure of rats (28 and 90 days) to 100, 500, and 5000 mg DBP/kg food. Monocyte adhesion to human EA.hy926 and rat aortic endothelial cells, expression of selected cellular adhesion molecules and chemokines, and the involvement of extracellular signal-regulated kinase 1/2 (ERK1/2) were analyzed. We observed increased monocyte adhesion to DBP-exposed EA.hy926 cells in vitro and to rat aortic endothelium ex vivo. ERK1/2 inhibitor prevented monocyte adhesion to DBP-exposed EA.hy926 cells in short-term exposure experiments. Increased ERK1/2 phosphorylation in rat aortic endothelium and transient decrease in ERK1/2 activation following long-term exposure of EA.hy926 cells to DBP were also observed. In summary, exposure of endothelial cells to DBP promotes monocyte adhesion, thus suggesting a possible role for this phthalate in the development of atherosclerosis. ERK1/2 signaling could be the mediator of monocyte adhesion to DBP-exposed endothelial cells, but only after short-term high-level exposure.

Differential eigengene network analysis reveals benzo[a]pyrene and 2,3,7,8-tetrachlorodibenzo-p-dioxin consensus regulatory network in human liver cell line HepG2.

Aryl hydrocarbon receptor (AHR) is one of the main mediators of the toxic effects of benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). However, a vast number of BaP- and TCDD-affected genes may suggest a more complex transcriptional regulatory network driving common adverse effects of these two chemicals. Unlike TCDD, BaP is rapidly metabolized in the liver, yielding products with a questionable ability to bind and activate AHR. In this study, we used transcriptomics data from the BaP- and TCCD-exposed human liver cell line HepG2, and performed differential eigengene network analysis to understand the correlation among genes and to untangle the common regulatory mechanism in the action of BaP and TCDD. The genes were grouped into 11 meta-modules with an overall preservation of 0.72 and were also segregated into three consensus time clusters: 12, 24, and 48 h. The analysis showed that the consensus genes in each time cluster were either directly regulated by the AHR or the AHR-TF interactions. Some TFs form a direct physical interaction with AHR such as ESR1, FOXA1, and E2F1, whereas others, including CTCF, RXRA, FOXO1, CEBPA, CEBPB, and TP53 show an indirect interaction with AHR. The analysis of biological processes (BPs) identified unique and common BPs in BaP and TCDD samples, with DNA damage response detected in all three time points. In summary, we identified a consensus transcriptional regulatory network common for BaP and TCDD consisting of direct AHR targets and AHR-TF targets. This analysis sheds new light on the common mechanism of action of a genotoxic (BaP) and non-genotoxic (TCDD) chemical in liver cells.

Predicting chemicals' toxicity pathway of female reproductive disorders using AOP7 and deep neural networks.

Experimental evidence shows that certain chemicals, particularly endocrine disrupting chemicals, may negatively affect the female reproductive system, thereby lowering women's fertility. However, humans are constantly exposed to a number of different chemicals with limited or no experimental data regarding their effect and the mechanism of action in the female reproductive system. To predict chemical hazards to the female reproductive system, we used a previously defined adverse outcome pathway (AOP) that links activation of the peroxisome proliferator-activated receptor γ to the reproductive toxicity in adult females (AOP7) and the Convolutional Deep Neural Network models that produce meaningful predictions when trained on a significant amount of data. The models trained using CompTox assays with intended molecular and biological targets corresponding to AOP7 achieved high performance (over 90% validation accuracy). The integration of AOP7 and Deep Neural Network identified chemicals that could negatively affect female reproduction through the mechanism described in AOP7. We provide a solution to quickly analyze the data and produce machine learning models to identify potentially active chemicals in the female reproductive system. Although we focused on the female reproductive system, this approach could be valid for a number of other chemicals and AOPs if the right data exist.

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.

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.

Characterization of the ERK1/2 phosphorylation profile in human and fish liver cells upon exposure to chemicals of environmental concern.

We developed phospho-ERK1/2 ELISA for human and rainbow trout liver cells, employing HepG2 and RTL-W1 cell lines as models. The assay was applied to detect changes in ERK1/2 activity for nine chemicals, added over a wide concentration range and time points. Cell viability was measured to separate ERK1/2 regulation from cytotoxicity. Perfluorooctane sulfonate and carbendazim did not change ERK1/2 activity; influence on ERK1/2 due to cytotoxicity was indicated for tributyltin and cypermethrin. Mancozeb, benzo[a]pyrene, and bisphenol A stimulated ERK1/2 up to ∼2- (HepG2) and 1.5 (RTL-W1)-fold, though the kinetics differed between chemicals and cell lines. Bisphenol A and benzo[a]pyrene were the most potent concentration-wise, altering ERK1/2 activity in pM (HepG2) to nM (RTL-W1) range. While atrazine and ibuprofen increased ERK1/2 activity by ∼2-fold in HepG2, they did not initiate an appreciable response in RTL-W1. This assay proved to be a sensitive, medium- to high-throughput tool for detecting unrecognized ERK1/2-disrupting chemicals.

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.

Evaluation of cyanobacterial toxicity using different biotests and protein phosphatase inhibition assay.

Cyanobacteria are prolific producers of numerous toxic compounds, among which microcystins (hepatotoxins) are the most frequently found. Cyanobacterial bloom in freshwaters is an increasing problem, and there is still a need for rapid and reliable methods for the detection of toxic cyanobacterial samples. In the present study, the toxicity of crude extracts of 11 cyanobacterial strains from different genera has been assessed on two cell lines (human hepatocellular carcinoma HepG2 and rainbow trout (Oncorhynchus mykiss) liver-derived RTL-W1 cells), crustaceans (Daphnia magna and Artemia salina), and zebrafish (Danio rerio) embryos, as well as by protein phosphatase 1 (PP1) inhibition assay and ELISA test to determine whether the toxicity could be due to the presence of hepatotoxins/microcystins. All the tested strains exhibited toxicity on HepG2 cell line (IC50 from 35 to 702 µg mL-1), including Arthrospira (Spirulina) strains, while toxicity against the RTL-W1 cells was detected only in the positive reference Microcystis PCC 7806 and Nostoc 2S9B. Tested strains expressed higher toxicity to D. magna and zebrafish embryos in comparison to A. salina, whereby Nostoc LC1B and Nostoc S8 belonged to the most toxic strains. The PP1-inhibiting compounds have been detected by PP1 assay only in four strains (Microcystis PCC 7806, Oscillatoria K3, Nostoc LC1B, and Nostoc S8), indicating that their toxic potency can be attributed to these compounds. On the other hand, very low levels of microcystins, as confirmed by ELISA, were insufficient to explain toxicity and different toxic potencies of tested cyanobacteria. Results presented in this study suggested HepG2 cell line as a particularly suitable model for cyanobacterial toxicity assessment. In addition, they highlight terrestrial cyanobacterial strains as potent producers of toxic compounds.

Application of targeted next generation sequencing for the mutational profiling of patients with acute lymphoblastic leukemia.

BACKGROUND: Acute lymphoblastic leukemia (ALL) is the most common cancer in children, whereas it is less common in adults. Identification of cytogenetic aberrations and a small number of molecular abnormalities are still the most important risk and therapy stratification methods in clinical practice today. Next generation sequencing (NGS) technology provides a large amount of data contributing to elucidation of mutational landscape of childhood (cALL) and adult ALL (aALL). METHODS: We analyzed DNA samples from 34 cALL and aALL patients, using NGS targeted sequencing TruSeq Amplicon - Cancer Panel (TSACP) which targets mutational hotspots in 48 cancer related genes. RESULTS: We identified a total of 330 variants in the coding regions, out of which only 95 were potentially protein-changing. Observed in individual patients, detected mutations predominantly disrupted Ras/RTK pathway (STK11, KIT, MET, NRAS, KRAS, PTEN). Additionally, we identified 5 patients with the same mutation in HNF1A gene, disrupting both Wnt and Notch signaling pathway. In two patients we detected variants in NOTCH1 gene. HNF1A and NOTCH1 variants were mutually exclusive, while genes involved in Ras/RTK pathway exhibit a tendency of mutation accumulation. CONCLUSIONS: Our results showed that ALL contains low number of mutations, without significant differences between cALL and aALL (median per patient 2 and 3, respectively). Detected mutations affect few key signaling pathways, primarily Ras/RTK cascade. This study contributes to knowledge of ALL mutational landscape, leading to better understanding of molecular basis of this disease.

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.