Hideko Sone

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.

Effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on T cell-derived cytokine production in ovalbumin (OVA)-immunized C57Bl/6 mice.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is known to suppress both cellular and humoral immunity. Effector T cell-derived type-2 cytokines, including IL-4 and IL-5, play pivotal roles in humoral immunity. Herein, we studied whether TCDD affects type-2 cytokine productions during the immune response. C57Bl/6 mice were intraperitoneally immunized with ovalbumin (OVA) and orally administered 5 or 20 microg TCDD/kg on Day 0, and then challenged with OVA on Day 21. Seven days later (Day 28), antigen-specific antibodies in plasma, and T cell-derived cytokines produced by splenocytes and proliferation of splenocytes upon ex vivo re-stimulation with OVA were investigated. The quantities of IgM class and IgG1 class OVA-specific antibodies in plasma were reduced by 5 or 20 microg TCDD/kg and by 20 microg TCDD/kg, respectively. While thymus weight and cellularity were reduced by 20 microg TCDD/kg, spleen weight and cellularity were not changed by either 5 or 20 microg TCDD/kg. The proportions of B and T cells in the spleen were not affected by TCDD exposure. On the other hand, splenocytes from mice treated with 5 or 20 microg TCDD/kg were shown to produce less IL-4 or IL-5 upon ex vivo re-stimulation with OVA. Production of the T cell growth factor IL-2 was also decreased in splenocytes from TCDD-treated mice. In contrast, the type-1 cytokine IFN-gamma was increased by TCDD. Twenty micrograms of TCDD/kg suppressed OVA- or T cell mitogen (Con A)-stimulated proliferation of splenocytes, but did not affect B cell mitogen (LPS)-stimulated proliferation. These results suggested compromised T cell activation and suppressed type-2 cytokine production by T cells to be involved in the impaired humoral immunity associated with TCDD exposure.

Induction of spermatogenic cell apoptosis in prepubertal rat testes irrespective of testicular steroidogenesis: A possible estrogenic effect of di(n-butyl) phthalate

Although di(n-butyl) phthalate (DBP), a suspected endocrine disruptor, induces testicular atrophy in prepubertal male rats, whether it exerts estrogenic activity in vivo remains a matter of debate. In the present study, we explored the estrogenic potency of DBP using 3-week-old male rats, and then examined the relationship between estrogen-induced spermatogenic cell apoptosis and testicular steroidogenesis. Daily exposure to DBP for 7 days caused testicular atrophy due to loss of spermatogenic cells, whereas testicular steroidogenesis was almost the same with the control values. A single exposure of DBP decreased testicular steroidogenesis in addition to decreasing the level of serum LH at 3 h after DBP treatment, with an extremely high incidence of apoptotic spermatogenic cells at 6 h after administration. To elucidate the estrogenic activity of DBP, we carried out an inhibition study using pure antiestrogen ICI 182,780 (ICI) in a model of spermatogenic cell apoptosis induced by DBP or estradial-3-benzoate (EB). Although both the DBP- and EB-treated groups showed a significant increase in spermatogenic cell apoptosis, ICI pretreatment significantly decreased the number of apoptotic spermatogenic cells in these two groups. In contrast, testicular steroidogenesis and serum FSH were significantly reduced in all the treated groups, even in the DBP+ICI and EB+ICI groups. Taken together, these findings led us to conclude that estrogenic compounds such as DBP and EB induce spermatogenic cell apoptosis in prepubertal rats, probably by activating estrogen receptors in testis, and that reduction in testicular steroidogenic function induced by estrogenic compounds is not associated with spermatogenic cell apoptosis.

Gene interaction network suggests dioxin induces a significant linkage between aryl hydrocarbon receptor and retinoic acid receptor beta

Gene expression arrays (gene chips) have enabled researchers to roughly quantify the level of mRNA expression for a large number of genes in a single sample. Several methods have been developed for the analysis of gene array data including clustering, outlier detection, and correlation studies. Most of these analyses are aimed at a qualitative identification of what is different between two samples and/or the relationship between two genes. We propose a quantitative, statistically sound methodology for the analysis of gene regulatory networks using gene expression data sets. The method is based on Bayesian networks for direct quantification of gene expression networks. Using the gene expression changes in HPL1A lung airway epithelial cells after exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin at levels of 0.1, 1.0, and 10.0 nM for 24 hr, a gene expression network was hypothesized and analyzed. The method clearly demonstrates support for the assumed network and the hypothesis linking the usual dioxin expression changes to the retinoic acid receptor system. Simulation studies demonstrated the method works well, even for small samples.

Changes in expression of NMDA receptor subunit mRNA by perinatal exposure to dioxin

Since dioxin and related compounds are suspected of affecting permanently the brain function of offspring of human and experimental animals, effects of perinatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on the expression of rat NMDA receptor NR2A and NR2B subunit mRNA were examined. The mRNA quantification by competitive RT-PCR clearly revealed that TCDD inhibited NR2B mRNA expression and enhanced NR2A mRNA expression in the neocortex and hippocampus on postnatal day (PND) 49, whereas these changes in mRNA expression were not found on PND 5. The results demonstrate for the first time that the perinatal exposure to TCDD can alter the molecular basis of brain of offspring in adulthood.

Perinatal exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin alters activity-dependent expression of BDNF mRNA in the neocortex and male rat sexual behavior in adulthood

Dioxin and its related compounds are suspected to cause neurological and nueroendocrinological disruption in human and laboratory animal offspring upon in utero and lactational exposure during growth and development. We tested the hypothesis by utilizing Long-Evans Hooded rats that perinatal exposure to dioxins affects the neocortical function and expression of sexual behavior in adulthood. In the sexual behavior test, perinatal exposure to TCDD significantly reduced the number of mounts and intromissions. The mRNA semi-quantification in in situ hybridization showed that the mating stimulus in control males induced c-fos mRNA expression in the preoptic area (POA) and the brain derived neurotrophic factor (BDNF) mRNA upregulation in the frontal cortex. In contrast, perinatal exposure to TCDD lowered the upregulation of BDNF mRNA in the frontal cortex but not that of c-fos mRNA in the POA. The volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA) was not affected. The results suggest that perinatal TCDD affects the neocortical function independently from the brain sexual differentiation and alters the expression of sexual behavior.

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.

The TAO-Gen Algorithm for Identifying Gene Interaction Networks with Application to SOS Repair in E. coli

One major unresolved issue in the analysis of gene expression data is the identification and quantification of gene regulatory networks. Several methods have been proposed for identifying gene regulatory networks, but these methods predominantly focus on the use of multiple pairwise comparisons to identify the network structure. In this article, we describe a method for analyzing gene expression data to determine a regulatory structure consistent with an observed set of expression profiles. Unlike other methods this method goes beyond pairwise evaluations by using likelihood-based statistical methods to obtain the network that is most consistent with the complete data set. The proposed algorithm performs accurately for moderate-sized networks with most errors being minor additions of linkages. However, the analysis also indicates that sample sizes may need to be increased to uniquely identify even moderate-sized networks. The method is used to evaluate interactions between genes in the SOS signaling pathway in Escherichia coli using gene expression data where each gene in the network is over-expressed using plasmids inserts.

The potential for chemical mixtures from the environment to enable the cancer hallmark of sustained proliferative signalling.

The aim of this work is to review current knowledge relating the established cancer hallmark, sustained cell proliferation to the existence of chemicals present as low dose mixtures in the environment. Normal cell proliferation is under tight control, i.e. cells respond to a signal to proliferate, and although most cells continue to proliferate into adult life, the multiplication ceases once the stimulatory signal disappears or if the cells are exposed to growth inhibitory signals. Under such circumstances, normal cells remain quiescent until they are stimulated to resume further proliferation. In contrast, tumour cells are unable to halt proliferation, either when subjected to growth inhibitory signals or in the absence of growth stimulatory signals. Environmental chemicals with carcinogenic potential may cause sustained cell proliferation by interfering with some cell proliferation control mechanisms committing cells to an indefinite proliferative span.

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.