Xian-Yang Qin

Effects of bisphenol A exposure on the proliferation and senescence of normal human mammary epithelial cells

The carcinogenic activity of bisphenol A (BPA) is responsible for stimulating growth in estrogen-dependent breast cancer tissues, cell lines and rodent studies. However, it is not fully understood how this compound promotes mammary carcinogenesis. In our study, we examined the effect of BPA on cellular proliferation and senescence in human mammary epithelial cells (HMEC). Exposure to BPA for 1 week at the early stage at passage 8 increased the proliferation and sphere size of HMEC at the later stage up to passage 16, suggesting that BPA has the capability to modulate cell growth in breast epithelial cells. Interestingly, the number of human heterochromatin protein-1γ positive cells, which is a marker of senescence, was also increased among BPA-treated cells. Consistent with these findings, the protein levels of both p16 and cyclin E, which are known to induce cellular senescence and promote proliferation, respectively, were increased in BPA-exposed HMEC. Furthermore, DNA methylation levels of genes related to development of most or all tumor types, such as BRCA1, CCNA1, CDKN2A (p16), THBS1, TNFRSF10C and TNFRSF10D, were increased in BPA-exposed HMEC. Our findings in the HMEC model suggested that the genetic and epigenetic alterations by BPA might damage HMEC function and result in complex activities related to cell proliferation and senescence, playing a role in mammary carcinogenesis.

Identification of Stage-Specific Gene Expression Signatures in Response to Retinoic Acid during the Neural Differentiation of Mouse Embryonic Stem Cells

We have previously established a protocol for the neural differentiation of mouse embryonic stem cells (mESCs) as an efficient tool to evaluate the neurodevelopmental toxicity of environmental chemicals. Here, we described a multivariate bioinformatic approach to identify the stage-specific gene sets associated with neural differentiation of mESCs. We exposed mESCs (B6G-2 cells) to 10−8 or 10−7 M of retinoic acid (RA) for 4 days during embryoid body formation and then performed morphological analysis on day of differentiation (DoD) 8 and 36, or genomic microarray analysis on DoD 0, 2, 8, and 36. Three gene sets, namely a literature-based gene set (set 1), an analysis-based gene set (set 2) using self-organizing map and principal component analysis, and an enrichment gene set (set 3), were selected by the combined use of knowledge from literatures and gene information selected from the microarray data. A gene network analysis for each gene set was then performed using Bayesian statistics to identify stage-specific gene expression signatures in response to RA during mESC neural differentiation. Our results showed that RA significantly increased the size of neurosphere, neuronal cells, and glial cells on DoD 36. In addition, the gene network analysis showed that glial fibrillary acidic protein, a neural marker, remarkably up-regulates the other genes in gene set 1 and 3, and Gbx2, a neural development marker, significantly up-regulates the other genes in gene set 2 on DoD 36 in the presence of RA. These findings suggest that our protocol for identification of developmental stage-specific gene expression and interaction is a useful method for the screening of environmental chemical toxicity during neurodevelopmental periods.

Effects of methylmercury exposure on neuronal differentiation of mouse and human embryonic stem cells.

The establishment of more efficient in vitro approaches has been widely acknowledged as a critical need for toxicity testing. In this study, we examined the effects of methylmercury (MeHg), which is a well-known developmental neurotoxicant, in two neuronal differentiation systems of mouse and human embryonic stem cells (mESCs and hESCs, respectively). Embryoid bodies were generated from gathering of mESCs and hESCs using a micro-device and seeded onto ornithine-laminin-coated plates to promote proliferation and neuronal differentiation. The cells were exposed to MeHg from the start of neuronal induction until the termination of cultures, and significant reductions of mESCs and hESCs were observed in the cell viability assays at 1,10,100 and 1000nM, respectively. Although the mESC derivatives were more sensitive than the hESC derivatives to MeHg exposure in terms of cell viability, the morphological evaluation demonstrated that the neurite length and branch points of hESC derivatives were more susceptible to a low concentration of MeHg. Then, the mRNA levels of differentiation markers were examined using quantitative RT-PCR analysis and the interactions between MeHg exposure and gene expression levels were visualized using a network model based on a Bayesian algorithm. The Bayesian network analysis showed that a MeHg-node was located on the highest hierarchy in the hESC derivatives, but not in the mESC derivatives, suggesting that MeHg directly affect differentiation marker genes in hESCs. Taken together, effects of MeHg were observed in our neuronal differentiation systems of mESCs and hESCs using a combination of morphological and molecular markers. Our study provided possible, but limited, evidences that human ESC models might be more sensitive in particular endpoints in response to MeHg exposure than that in mouse ESC models. Further investigations that expand on the findings of the present paper may solve problems that occur when the outcomes from laboratory animals are extrapolated for human risk evaluation.

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 17β-estradiol (E2) or 1 nM 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for 24 h. Differentially expressed genes were identified using an unpaired Students 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.

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.

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.

The Effect of Acyclic Retinoid on the Metabolomic Profiles of Hepatocytes and Hepatocellular Carcinoma Cells

Background/Purpose Acyclic retinoid (ACR) is a promising chemopreventive agent for hepatocellular carcinoma (HCC) that selectively inhibits the growth of HCC cells (JHH7) but not normal hepatic cells (Hc). To better understand the molecular basis of the selective anti-cancer effect of ACR, we performed nuclear magnetic resonance (NMR)-based and capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS)-based metabolome analyses in JHH7 and Hc cells after treatment with ACR. Methodology/Principal Findings NMR-based metabolomics revealed a distinct metabolomic profile of JHH7 cells at 18 h after ACR treatment but not at 4 h after ACR treatment. CE-TOFMS analysis identified 88 principal metabolites in JHH7 and Hc cells after 24 h of treatment with ethanol (EtOH) or ACR. The abundance of 71 of these metabolites was significantly different between EtOH-treated control JHH7 and Hc cells, and 49 of these metabolites were significantly down-regulated in the ACR-treated JHH7 cells compared to the EtOH-treated JHH7 cells. Of particular interest, the increase in adenosine-5′-triphosphate (ATP), the main cellular energy source, that was observed in the EtOH-treated control JHH7 cells was almost completely suppressed in the ACR-treated JHH7 cells; treatment with ACR restored ATP to the basal levels observed in both EtOH-control and ACR-treated Hc cells (0.72-fold compared to the EtOH control-treated JHH7 cells). Moreover, real-time PCR analyses revealed that ACR significantly increased the expression of pyruvate dehydrogenase kinases 4 (PDK4), a key regulator of ATP production, in JHH7 cells but not in Hc cells (3.06-fold and 1.20-fold compared to the EtOH control, respectively). Conclusions/Significance The results of the present study suggest that ACR may suppress the enhanced energy metabolism of JHH7 cells but not Hc cells; this occurs at least in part via the cancer-selective enhancement of PDK4 expression. The cancer-selective metabolic pathways identified in this study will be important targets of the anti-cancer activity of ACR.

Multi-Parametric Profiling Network Based on Gene Expression and Phenotype Data: A Novel Approach to Developmental Neurotoxicity Testing

The establishment of more efficient approaches for developmental neurotoxicity testing (DNT) has been an emerging issue for children’s environmental health. Here we describe a systematic approach for DNT using the neuronal differentiation of mouse embryonic stem cells (mESCs) as a model of fetal programming. During embryoid body (EB) formation, mESCs were exposed to 12 chemicals for 24 h and then global gene expression profiling was performed using whole genome microarray analysis. Gene expression signatures for seven kinds of gene sets related to neuronal development and neuronal diseases were selected for further analysis. At the later stages of neuronal cell differentiation from EBs, neuronal phenotypic parameters were determined using a high-content image analyzer. Bayesian network analysis was then performed based on global gene expression and neuronal phenotypic data to generate comprehensive networks with a linkage between early events and later effects. Furthermore, the probability distribution values for the strength of the linkage between parameters in each network was calculated and then used in principal component analysis. The characterization of chemicals according to their neurotoxic potential reveals that the multi-parametric analysis based on phenotype and gene expression profiling during neuronal differentiation of mESCs can provide a useful tool to monitor fetal programming and to predict developmentally neurotoxic compounds.

Peroxisome proliferator-activated receptor α mediates di-(2-ethylhexyl) phthalate transgenerational repression of ovarian Esr1 expression in female mice.

Di-(2-ethylhexyl)-phthalate (DEHP) is a phthalate ester that binds peroxisome proliferator-activated receptor α (PPARα) to induce proliferation of peroxisomes and regulate the expression of specific target genes. The question of whether the effect of DEHP on female reproductive processes is mediated via PPARα-dependent signaling is controversial. In this study, we investigated the effect of exposure to DEHP on ovarian expression of estrogen receptor α (Esr1) and aromatase (Cyp19a1) in three generations of Sv/129 wild-type (WT, +/+) and PPARα (-/-) knockout mice. Compared with untreated controls, ovarian expression of Esr1 decreased in response to DEHP treatment in the F0 (0.56-fold, P=0.19), F1 (0.45-fold, P=0.023), and F2 (0.35-fold, P=0.014) generations of WT mice, but not PPARα-null mice. Our data indicate that transgenerational repression by DEHP of ovarian Esr1 gene expression is mediated by PPARα-dependent pathways. Further studies are required to elucidate the mechanisms underlying crosstalk between PPARα and Esr1 signaling in reproductive processes.

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.