Effect of low-dose thalidomide on dopaminergic neuronal differentiation of human neural progenitor cells: a combined study of metabolomics and morphological analysis.

Thalidomide is increasingly used in anticancer and anti-inflammation therapies. However, it is known for its teratogenicity and ability to induce peripheral neuropathy, although the mechanisms underlying its neurological effect in humans are unclear. In this study, we investigated the effect of thalidomide on the metabolism and neuronal differentiation of human neural progenitor cells. We found that levels of tyrosine, phenylalanine, methionine and glutathione, which are involved in dopamine and methionine metabolism, were decreased following thalidomide treatment. Morphological analysis revealed that treatment with 100 nM thalidomide, which is much lower than clinical doses, significantly decreased the number of dopaminergic (tyrosine hydroxylase-positive) neurons, compared with control cells. Our results suggest that these adverse neurological effects of thalidomide should be taken into consideration prior to its use for the treatment of neurodegenerative and other diseases.

Association of variants in genes involved in environmental chemical metabolism and risk of cryptorchidism and hypospadias.

We hypothesized that single-nucleotide polymorphisms (SNPs) of genes involved in environmental endocrine disruptors (EEDs) metabolism might influence the risk of male genital malformations. In this study, we explored for association between 384 SNPs in 15 genes (AHR, AHRR, ARNT, ARNT2, NR1I2, RXRA, RXRB, RXRG, CYP1A1, CYP1A2, CYP1B1, CYP2B6, CYP3A4, CYP17A1 and CYP19A1) and risk of cryptorchidism (CO) and hypospadias (HS) in 334 Japanese (JPN) males (141 controls, 95 CO and 98 HS) and 187 Italian (ITA) males (129 controls and 58 CO). In the JPN study group, five SNPs from ARNT2 (rs2278705 and rs5000770), CYP1A2 (rs2069521), CYP17A1 (rs4919686) and NR1I2 (rs2472680) were significantly associated at both allelic and genotypic levels with risk of at least one genital malformation phenotype. In the ITA study group, two SNPs in AHR (rs3757824) and ARNT2 (rs1020397) were significantly associated with risk of CO. Interaction analysis of the positive SNPs using multifactor dimensionality reduction demonstrated that synergistic interaction between rs2472680, rs4919686 and rs5000770 had 62.81% prediction accuracy for CO (P=0.011) and that between rs2069521 and rs2278705 had 69.98% prediction accuracy for HS (P=0.001) in JPN population. In a combined analysis of JPN and ITA population, the most significant multi-locus association was observed between rs5000770 and rs3757824, which had 65.70% prediction accuracy for CO (P=0.055). Our findings indicate that genetic polymorphisms in genes involved in EED metabolism are associated with risk of CO and HS.

Identification of novel low-dose bisphenol a targets in human foreskin fibroblast cells derived from hypospadias patients.

BACKGROUND/PURPOSE: The effect of low-dose bisphenol A (BPA) exposure on human reproductive health is still controversial. To better understand the molecular basis of the effect of BPA on human reproductive health, a genome-wide screen was performed using human foreskin fibroblast cells (hFFCs) derived from child hypospadias (HS) patients to identify novel targets of low-dose BPA exposure. METHODOLOGY/PRINCIPAL FINDINGS: Gene expression profiles of hFFCs were measured after exposure to 10 nM BPA, 0.01 nM 17ß-estradiol (E2) or 1 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 24 h. Differentially expressed genes were identified using an unpaired Student's t test with P value cut off at 0.05 and fold change of more than 1.2. These genes were selected for network generation and pathway analysis using Ingenuity Pathways Analysis, Pathway Express and KegArray. Seventy-one genes (42 downregulated and 29 upregulated) were identified as significantly differentially expressed in response to BPA, among which 43 genes were found to be affected exclusively by BPA compared with E2 and TCDD. Of particular interest, real-time PCR analysis revealed that the expression of matrix metallopeptidase 11 (MMP11), a well-known effector of development and normal physiology, was found to be inhibited by BPA (0.47-fold and 0.37-fold at 10 nM and 100 nM, respectively). Furthermore, study of hFFCs derived from HS and cryptorchidism (CO) patients (n = 23 and 11, respectively) indicated that MMP11 expression was significantly lower in the HS group than in the CO group (0.25-fold, P = 0.0027). CONCLUSIONS/SIGNIFICANCE: This present study suggests that an involvement of BPA in the etiology of HS might be associated with the downregulation of MMP11. Further study to elucidate the function of the novel target genes identified in this study during genital tubercle development might increase our knowledge of the effects of low-dose BPA exposure on human reproductive health.

siRNA-mediated knockdown of aryl hydrocarbon receptor nuclear translocator 2 affects hypoxia-inducible factor-1 regulatory signaling and metabolism in human breast cancer cells.

Recent human studies found that the mRNA expression level of aryl-hydrocarbon receptor nuclear translocator 2 (ARNT2) was positively associated with the prognosis of breast cancer. In this study, we used small interfering RNA techniques to knockdown ARNT2 expression in MCF7 human breast cancer cells, and found that an almost 40% downregulation of ARNT2 mRNA expression increased the expression of sensitive to apoptosis gene (3.36-fold), and decreased the expression of von Hippel-Lindau (0.27-fold) and matrix metalloproteinase-1 (0.35-fold). The metabolite analysis revealed the contents of glucose, glycine, betaine, phosphocholine, pyruvate and lactate involved in the hypoxia-inducible factor (HIF)-1-dependent glycolytic pathway were significantly lower in cells treated with siARNT2. Our results suggested that ARNT2 might play an important role in the modulation of HIF-1-regulated signaling and metabolism.

Xenoestrogens down-regulate aryl-hydrocarbon receptor nuclear translocator 2 mRNA expression in human breast cancer cells via an estrogen receptor alpha-dependent mechanism.

Environmental chemicals with estrogenic activity, known as xenoestrogens, may cause impaired reproductive development and endocrine-related cancers in humans by disrupting endocrine functions. Aryl-hydrocarbon receptor nuclear translocator 2 (ARNT2) is believed to play important roles in a variety of physiological processes, including estrogen signaling pathways, that may be involved in the pathogenesis and therapeutic responses of endocrine-related cancers. However, much of the underlying mechanism remains unknown. In this study, we investigated whether ARNT2 expression is regulated by a range of representative xenoestrogens in human cancer cell lines. Bisphenol A (BPA), benzyl butyl phthalate (BBP), and 1,1,1-trichloro-2,2-bis(2-chlorophenyl-4-chlorophenyl)ethane (o,p'-DDT) were found to be estrogenic toward BG1Luc4E2 cells by an E-CALUX bioassay. ARNT2 expression was downregulated by BPA, BBP, and o,p'-DDT in a dose-dependent manner in estrogen receptor 1 (ESR1)-positive MCF-7 and BG1Luc4E2 cells, but not in estrogen receptor-negative LNCaP cells. The reduction in ARNT2 expression in cells treated with the xenoestrogens was fully recovered by the addition of a specific ESR1 antagonist, MPP. In conclusion, we have shown for the first time that ARNT2 expression is modulated by xenoestrogens by an ESR1-dependent mechanism in MCF-7 breast cancer cells.

Profiles of Chemical Effects on Cells (pCEC): a toxicogenomics database with a toxicoinformatics system for risk evaluation and toxicity prediction of environmental chemicals.

Profiles of Chemical Effects on Cells (pCEC) is a toxicogenomics database with a system of classifying chemicals that have effects on human health. This database stores and handles gene expression profiling information and categories of toxicity data. Chemicals are classified according to the specific tissues and cells they affect, the gene expression changes they induce, their toxicity and biological functions in this database system. The pCEC system also analyzes relationships between chemicals and the genes they affect in specific tissues and cells. The reason why we developed pCEC is to support decision-making within the context of environmental regulation. Especially, exposure to environmental chemicals during fetal and newborn development may result in a predisposition to various disorders such as cancer, learning disabilities and allergies later in life. The identification and prediction of hazardous chemicals using limited information are important issues in human health risk management. Therefore, various toxicity information including lethal dose 50 (LD50), toxicity pathways and pathological data were loaded into pCEC. pCEC is also a facility for query, analysis and prediction of unknown toxicochemical reaction pathways and biomarkers which are based on toxicoinformatical data mining approaches. This database is available online at http://project.nies.go.jp/eCA/cgi-bin/index.cgi. The current version of the database has information on the hepatotoxicity, reproductive toxicity and embryotoxicity of chemicals.

Critical role of cyclooxygenase-2 activation in pathogenesis of hydronephrosis caused by lactational exposure of mice to dioxin.

Congenital hydronephrosis is a serious disease occurring among infants and children. Besides the intrinsic genetic factors, in utero exposure to a xenobiotic, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), has been suggested to induce hydronephrosis in rodents owing to anatomical obstruction in the ureter. Here, we report that hydronephrosis induced in mouse pups exposed lactationally to TCDD is not associated with anatomical obstruction, but with abnormal alterations in the subepithelial mesenchyma of the ureter. In the kidneys of these pups, the expressions of a battery of inflammatory cytokines including monocyte chemoattractant protein (MCP)-1, tumor necrosis factor alpha (TNFalpha) and interleukin (IL)-1beta were up-regulated as early as postnatal day (PND) 7. The amounts of cyclooxygenase (COX)-2 mRNA and protein as well as prostaglandin E2 (PGE(2)) were conspicuously up-regulated in an arylhydrocarbon-receptor-dependent manner in the TCDD-induced hydronephrotic kidney, with a subsequent down-regulation of the gene expressions of Na+ and K+ transporters, NKCC2 and ROMK. Daily administration of a COX-2 selective inhibitor to newborns until PND 7 completely abrogated the TCDD-induced PGE(2) synthesis and gene expressions of inflammatory cytokines and electrolyte transporters, and eventually prevented the onset of hydronephrosis. These findings suggest an essential role of COX-2 in mediating the TCDD action of inducing hydronephrosis through the functional impairment rather than the anatomical blockade of the ureter.

Estrogen-responsive genes newly found to be modified by TCDD exposure in human cell lines and mouse systems.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) can induce estrogenic action or inhibit estrogen-induced effects in various tissues because of aryl hydrocarbon receptor (AhR)-estrogen receptor (ER) cross-talk. In order to identify the biomarkers of TCDD endocrine disruption, we screened estrogen-responsive genes modified by TCDD exposure using specific cDNA microarrays spotted with estrogen-responsive genes. MCF-7 human breast carcinoma cells and RL95-2 human endometrial carcinoma cells were exposed to TCDD, and an analysis of their gene expression revealed 32 genes exhibiting a significant change. The mRNA expression levels of 27 genes were subsequently verified using real-time RT-PCR. Among these genes, bioinformatic analyses indicated that insulin-like growth factor-binding protein 5 (IGFBP5) gene expression might be influenced by estrogen status. In our animal experiments, IGFBP5 was also shown to be responsive to TCDD exposure in mouse fetuses in utero. These results suggest that TCDD affects the expression levels of a series of estrogen-responsive genes, and follow-up fetal studies in mice indicated that IGFBP5 is useful as a biomarker of TCDD activity.

Activation of telomerase in BeWo cells by estrogen and 2,3,7,8-tetrachlorodibenzo-p-dioxin in co-operation with c-Myc.

Telomerase activation, known to be stimulated by estrogen, is essential for cellular immortalization and trans-formation, both of which play a role in tumorigenesis. Dioxin and dioxin-like compounds have been shown to induce endometriosis and promote estrogen-dependent tumors. In this study, we show that either 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or a combination of TCDD and 17-beta estradiol (E2) increase telomerase activity and the expression of the human telomerase catalytic subunit (hTERT) in human choriocarcinoma (BeWo) cells. Compared with estrogen or TCDD alone, the combination treatment did not show an additive effect. Likewise, treatment with either E2 or TCDD increased DNA synthesis and the cell population in S-phase, as detected by FACS analysis. However, following treatment with the E2 and TCDD combination, the proportion of cells in S-phase was actually lower than in cells treated with TCDD alone. These results suggest that TCDD alone mimics estrogenic action in telomerase activation and cell proliferation but, in the presence of estrogen, TCDD-induced actions were partially counteracted. E2 and TCDD also induced c-Myc, which is a transcriptional activator of hTERT in Bewo, but neither of these agents induced telomerase activity in HO15.19 c-myc-null cells. In contrast, only TCDD upregulated telomerase in TGR-1 cells, which are c-Myc expressing but lacking ER expression. The findings suggest that TCDD induces telomerase activity mediated through AhR signaling and/or ER-independent c-Myc signaling. The present study provides insight into the mechanism of promoter activity of TCDD in estrogen-related tumors.

Lack of CYP1A1 expression is involved in unresponsiveness of the human hepatoma cell line SK-HEP-1 to dioxin.

The aryl hydrocarbon receptor (AhR) mediates a wide variety of toxic effects due to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The human hepatoma cell line SK-HEP-1 expresses AhR and ARNT. However, TCDD failed to induce CYP1A1 and XRE-dependent reporter genes in these cells. Although CYP1A1 was not induced by TCDD exposure, both CYP1B1 and AhR repressor (AhRR) were constitutively expressed. The AhR antagonist alpha-naphthoflavone altered the basal level of XRE-dependent reporter gene expression dose-dependently. As our results suggested the activation of AhR signals by putative endogenous ligands, we established SK-HEP-1-derived cell lines that stably expressed CYP1A1. The inducibility of XRE-dependent reporter genes and CYP1B1 by TCDD was restored in these cells. Our findings demonstrated the presence of endogenous ligands in SK-HEP-1 cells due to the absence of the metabolizing enzyme CYP1A1, but not CYP1B1, which allowed the constitutive expression of AhR target genes.

Altered thyroxin and retinoid metabolic response to 2,3,7,8-tetrachlorodibenzo-p-dioxin in aryl hydrocarbon receptor-null mice.

To determine whether the disruption of thyroid hormone and retinoid homeostasis that occurs after exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) can be mediated by the arylhydrocarbon receptor (AhR), pregnant AhR-heterozygous (AhR+/-) mice were administered a single oral dose of 10 microg kg(-1) TCDD at gestation day 12.5. Serum and liver were collected on postnatal day 21 from vehicle-treated control or TCDD-treated AhR+/- and AhR-null (AhR-/-) mouse pups. Whereas TCDD exposure resulted in a marked reduction of total thyroxin (TT4) and free T4 (FT4) levels in the serum of AhR+/- mice, TCDD had no effects on AhR-/- mice. Gene expression of UDP-glucuronosyltransferase (UGT)1A6, cytochrome P450 (CYP)1A1, and CYP1A2 in the liver was induced markedly by TCDD in AhR+/- but not AhR-/- mice. Induction of CYP1A1 in response to TCDD was confirmed by immunohistochemical evidence in that CYP1A1 protein was conspicuously localized in the cytoplasm of hepatocytes in the centrilobular region. Levels of retinyl palmitate were greatly reduced in the liver of TCDD-exposed AhR+/- mice, but not in vehicle-treated AhR+/- mice. No effects of TCDD on retinoid levels in the liver were found in AhR-/- mice. We conclude that disruption of thyroid hormone and retinoid homeostasis is mediated entirely via AhR. Induction of UGT1A6 is thought to be responsible at least partly for reduced serum thyroid hormone levels in TCDD-exposed mice.

Disruption of thyroid hormone homeostasis at weaning of Holtzman rats by lactational but not in utero exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The purpose of this study is to clarify whether lactational exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is entirely responsible for the perturbation in thyroid hormone homeostasis during the neonatal period. Pregnant Holtzman rats were given a single oral dose of 1.0 mug TCDD/kg body weight on gestational day 15. Half of the litters were cross-fostered with the half of the dams treated with vehicle on postnatal day (PND) 1 to make four groups of rats, control (C/C), prenatal TCDD exposure only (T/C), postnatal TCDD exposure only (C/T), and both prenatal and postnatal TCDD exposure (T/T). On PND 21, the C/T and T/T groups, but not the T/C and C/C groups, showed a significant decrease in serum total thyroxin (TT4) and free thyroxin (FT4) concentrations in both sexes and a significant increase in serum thyroid-stimulating hormone (TSH) levels, particularly male pups. These two groups of male and female pups had significantly higher concentrations of TCDD in the liver, with marked induction of cytochrome P450 (CYP) 1A1 mRNA and intense immunostaining of CYP1A1 in the liver. UDP glycosyltransferase 1 family, polypeptide A6 (UGT1A6) and UGT1A7 mRNAs were induced in their livers, with marked immunostaining of UGT1A6. The transfer of TCDD from dams to the pups was confirmed by the detection of TCDD in mother's milk remaining in the stomachs of lactationally exposed pups on PND 1. The present results demonstrate that lactational, but not in utero, exposure to TCDD was responsible for the disruption of thyroid hormone homeostasis.

Using a customized DNA microarray for expression profiling of the estrogen-responsive genes to evaluate estrogen activity among natural estrogens and industrial chemicals.

We developed a DNA microarray to evaluate the estrogen activity of natural estrogens and industrial chemicals. Using MCF-7 cells, we conducted a comprehensive analysis of estrogen-responsive genes among approximately 20,000 human genes. On the basis of reproducible and reliable responses of the genes to estrogen, we selected 172 genes to be used for developing a customized DNA microarray. Using this DNA microarray, we examined estrogen activity among natural estrogens (17beta-estradiol, estriol, estrone, genistein), industrial chemicals (diethylstilbestrol, bisphenol A, nonylphenol, methoxychlor), and dioxin. We obtained results identical to those for other bioassays that are used for detecting estrogen activity. On the basis of statistical correlations analysis, these bioassays have shown more sensitivity for dioxin and methoxychlor.

BMP7/ActRIIB regulates estrogen-dependent apoptosis: new biomarkers for environmental estrogens.

A ligand-receptor pair, bone morphogenetic protein-7 (BMP7) and activin receptor IIB (actRIIB), was identified from a pool of DNA fragments recovered from MCF7 cells treated with 17beta-estradiol (E2) by chromatin immunoprecipitation with antiestrogen receptor-alphaantibody. The E2 responsiveness of both genes was confirmed in MCF cells and in the mouse uterus. Repeated treatment with E2 resulted in decreased expression of both actRIIB and BMP7 mRNA in the uteri of ovariectomized mice. A single oral administration of bisphenol A (BPA), an environmental estrogen, inhibited actRIIB and BMP7 expression and apoptosis in the luminal epithelium of the mouse uterus at diestrus (or early proestrus). This decrease, due to BPA administration, was restored by an estrogen receptor (ER) antagonist suggesting that it is mediated through ERs. These results suggest that E2 and BPA suppress estrogen-dependent apoptosis of epithelial cells of the endometrium through down-regulation of actRIIB and BMP7. Thus, we propose that BMP7 and actRIIB, a ligand-receptor pair, are involved in regulation of the apoptotic signaling pathway and might therefore be new biomarkers of the effects of environmental estrogens on the female reproductive tract.

Increased glycogen content and glucose transporter 3 mRNA level in the placenta of Holtzman rats after exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Exposure to a low dose of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) produces a variety of toxic manifestations, including fetal death. In order to evaluate the effects of low-dose TCDD on placental function in this study, pregnant Holtzman rats were given a single oral dose of 800 or 1600 ng TCDD/kg body wt or an equivalent volume of vehicle (control) on gestation day (GD) 15 and the results were observed on GD16 and GD20. The number of fetal deaths increased in the animals exposed to TCDD. Although fetal and placental weight did not differ significantly between the control group and the TCDD groups, histological differences from the control rats were clearly observed in the junctional zone (JZ) of the placentas of the TCDD-exposed rats. In the control placenta, glycogen cells occupied the majority of the JZ on GD16, but then decreased in number and almost disappeared by GD20, whereas on GD20 the placenta of the TCDD-exposed rats exhibited a larger area occupied by the glycogen cells and cysts filled with eosinophilic material surrounded by glycogen cells in the JZ than that of the control group. Glycogen assay revealed that the glycogen content of the placentas from the TCDD-exposed rats was higher than in the control rats. Semiquantitative RT-PCR analysis was performed to assess the expression of glucose transporter 1 (GLUT1) and GLUT3, the two major placental glucose transporter isoforms. On GD20 the level of expression of GLUT1 mRNA in the placentas was not different between the control and TCDD groups, whereas the level of expression of GLUT3 mRNA approximately doubled in both the 800 and 1600 ng/kg TCDD groups. GLUT3 mRNA expression was restricted to the labyrinth zone of placenta, where zone-specific expression of mRNA arylhydrocarbon receptor and induction of cytochrome P450 1A1 mRNA by TCDD were observed, and none was detected in the JZ. These results, including the increase of glycogen content and GLUT3 mRNA level in TCDD-exposed placentas, provide the first evidence of alteration of glucose kinetics in the placenta by TCDD.