Perinatal exposure to oestradiol and bisphenol A alters the prostate epigenome and increases susceptibility to carcinogenesis

An important and controversial health concern is whether low-dose exposures to hormonally active environmental oestrogens such as bisphenol A can promote human diseases including prostate cancer. Our studies in rats have shown that pharmacological doses of oestradiol administered during the critical window of prostate development result in marked prostate pathology in adulthood that progress to neoplastic lesions with ageing. Our recent studies have also demonstrated that transient developmental exposure of rats to low, environmentally relevant doses of bisphenol A or oestradiol increases prostate gland susceptibility to adult-onset precancerous lesions and hormonal carcinogenesis. These findings indicate that a wide range of oestrogenic exposures during development can predispose to prostatic neoplasia that suggests a potential developmental basis for this adult disease. To identify a molecular basis for oestrogen imprinting, we screened for DNA methylation changes over time in the exposed prostate glands. We found permanent alterations in DNA methylation patterns of multiple cell signalling genes suggesting an epigenetic mechanism of action. For phosphodiesterase type 4 variant 4 (PDE4D4), an enzyme responsible for intracellular cyclic adenosine monophosphate breakdown, a specific methylation cluster was identified in the 5'-flanking CpG island that was gradually hypermethylated with ageing in normal prostates resulting in loss of gene expression. However, in prostates exposed to neonatal oestradiol or bisphenol A, this region became hypomethylated with ageing resulting in persistent and elevated PDE4D4 expression. In total, these findings indicate that low-dose exposures to ubiquitous environmental oestrogens impact the prostate epigenome during development and in so doing, promote prostate disease with ageing.     

Neurobehavioral teratogenicity of perfluorinated alkyls in an avian model

Perfluorinated alkyls are widely-used agents that accumulate in ecosystems and organisms because of their slow rate of degradation. There is increasing concern that these agents may be developmental neurotoxicants and the present study was designed to develop an avian model for the neurobehavioral teratogenicity of perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS). Fertilized chicken eggs were injected with 5 or 10 mg/kg of either compound on incubation day 0. On the day of hatching, imprinting behavior was impaired by both compounds. We then explored underlying mechanisms involving the targeting of protein kinase C (PKC) isoforms (α, β, γ) in the intermedial part of the hyperstriatum ventrale, the region most closely associated with imprinting. With PFOA exposure, cytosolic PKC concentrations were significantly elevated for all three isoforms; despite the overall increase in PKC expression, membrane-associated PKC was unaffected, indicating a defect in PKC translocation. In contrast, PFOS exposure evoked a significant decrease in cytosolic PKC, primarily for the β and γ isoforms, but again without a corresponding change in membrane-associated enzyme; this likely partial, compensatory increases in translocation to offset the net PKC deficiency. Our studies indicate that perfluorinated alkyls are indeed developmental neurotoxicants that affect posthatch cognitive performance but that the underlying synaptic mechanisms may differ substantially among the various members of this class of compounds, setting the stage for disparate outcomes later in life.     

Epigenetic and genetic mechanisms of abnormal 11p15 genomic imprinting in Silver-Russell and Beckwith-Wiedemann syndromes

Fetal growth is a complex process depending on the genetics of the fetus, the availability of nutrients to the fetus, maternal nutrition and various growth factors and hormones of maternal, fetal and placental origin. The IGF system, and more particularly IGF2, is one of the most important endocrine and paracrine growth systems regulating fetal and placental growth (reviewed in [1]). The IGF2 gene is regulated by genomic imprinting and is expressed only from the paternally-inherited allele in most tissues during fetal development and after birth. Imprinted genes are tightly regulated and are therefore particularly susceptible to changes, including environmental and nutritional changes. Dysregulation of a cluster of imprinted genes, including the IGF2 gene within the 11p15 region, results in two fetal growth disorders (Silver-Russell and Beckwith-Wiedemann syndromes) with opposite growth phenotypes. Those two syndromes are model imprinting disorders to decipher the regulation of genomic imprinting.     

The effect of a single neonatal treatment (hormonal imprinting) with the antihormones, tamoxifen and mifepristone on the sexual behavior of adult rats

Hormonal imprinting takes place perinatally during the first encounter between the hormone and its developing receptor. The presence of an excess of related molecules in that time provokes faulty (pathological) imprinting. In the present experiments single-neonatal treatment with 100 microg of tamoxifen completely abolished the adult male and female rats' sexual activity. Similar treatment with 100 microg of mifepristone (RU486) significantly enhanced the males sexual activity and non-significantly increased that of the females. The results demonstrate the importance of pathological imprinting during the perinatal development of sexual behavior. There are clear differences between the molecules having steroid (mifepristone) or non-steroid (tamoxifen) character, mediated through a ligand-receptor complex, and its effect in activating particular genes.     

Effect of neonatal allylestrenol treatment and adult benzpyrene treatment on rat thymus glucocorticoid receptors

• 1.1. Neonatal treatment with allylestrenol caused an increase in the number of glucocorticoid receptors in the thymus in the adult.
• 2.2. Benzpyrene treatment of adult animals persistently reduced the glucocorticoid receptor number in rats that were not submitted to neonatal treatment. This effect was not observed in adult animals following neonatal treatment with allylestrenol.
• 3.3. The experiment calls attention to the permanent effect of steroid imprinting and to the developmental stage dependence of the direction of imprinting.
• 4.4. The effect of neonatal steroid treatment on a later steroid influence is also demonstrated.

     

分子印迹技术原理及其在药物分析检测中的应用

分子印迹技束是20世纪90年代快速发展起来的。制备空间结构和结合位点与模板分子完全匹配的聚合物的技术,它具有构效预定性、特异识别性和广泛实用性的特点。本文主要介绍分子印迹技术的产生、原理及其在药物分析检测中的应用．并对此项新技术的未来作了展望。     

Developmental estrogen exposures predispose to prostate carcinogenesis with aging

Prostate morphogenesis occurs in utero in humans and during the perinatal period in rodents. While largely driven by androgens, there is compelling evidence for a permanent influence of estrogens on prostatic development. If estrogenic exposures are abnormally high during the critical developmental period, permanent alterations in prostate morphology and function are observed, a process referred to as developmental estrogenization. Using the neonatal rodent as an animal model, it has been shown that early exposure to high doses of estradiol results in an increased incidence of prostatic lesions with aging which include hyperplasia, inflammatory cell infiltration and prostatic intraepithelial neoplasia or PIN, believed to be the precursor lesion for prostatic adenocarcinoma. The present review summarizes research performed in our laboratory to characterize developmental estrogenization and identify the molecular pathways involved in mediating this response. Furthermore, recent studies performed with low-dose estradiol exposures during development as well as exposures to environmentally relevant doses of the endocrine disruptor bisphenol A show increased susceptibility to PIN lesions with aging following additional adult exposure to estradiol. Gene methylation analysis revealed a potential epigenetic basis for the estrogen imprinting of the prostate gland. Taken together, our results suggest that a full range of estrogenic exposures during the postnatal critical period - from environmentally relevant bisphenol A exposure to low-dose and pharmacologic estradiol exposures - results in an increased incidence and susceptibility to neoplastic transformation of the prostate gland in the aging male which may provide a fetal basis for this adult disease.     

IGF2在小鼠胚胎干细胞定向分化为胰岛样细胞过程中的表达

【摘要】目的观察印迹基因胰岛素样生长因子（IGF）2在小鼠胚胎干细胞定向诱导分化为胰岛样细胞过程中的表达情况。方法体外诱导胚胎干细胞向胰岛样细胞分化,逆转录-PCR（RT-PCR）和细胞免疫荧光检测胰岛细胞标志基因表达;聚合酶链式反应-限制性内切酶片段长度多态性(PCR-RFLP)检测印迹基因IGF2在诱导分化前后细胞中的亲本表达情况。结果诱导分化终末细胞能表达胰岛素、胰高糖素及C肽等胰岛细胞特异性标志物,PCRRFLP检测分析显示,体外诱导分化的细胞的印迹基因IGF2呈双等位基因表达,印迹丢失。结论体外诱导培养可导致胚胎干细胞印迹基因IGF2表达的改变。     

Gene set assembly for quantitative prediction of developmental toxicity in the embryonic stem cell test

The embryonic stem cell test (EST) is an in vitro method for predicting developmental toxicity based on compound-induced inhibition of embryonic stem cell (ESC) differentiation. We previously described how gene expression analysis in the EST can be used to describe normal ESC differentiation as well as identify compound developmental toxicity, by means of our differentiation track algorithm. In this study, we combined raw data from our three previous studies in a new integrated analysis, to identify a gene set that allows for improved prediction. By evaluating predictions of 100,000 randomly selected gene sets, we identified which genes contribute significantly to the prediction reliability. By additional cross-validation, we identified a set of 52 genes that allows for improved prediction of toxicity. The correlation between the predictions using this gene set and the magnitude of the EST endpoint was 0.85, and the gene set predicted developmental toxicity with 83% accuracy (area under the curve 89%). If compounds with ineffective data because of a too low tested concentration or too much variation between samples were excluded, even 100% accuracy could be reached based on 15 compounds. This novel gene set consists mainly of genes involved in the stem cell differentiation or other developmental processes. We expect that this set can be of use in future studies aimed at improving the EST for risk assessment, thus making a next step towards regulatory implementation of this method.     

Molecular imprinting within hydrogels.

Hydrogels have been used primarily in the pharmaceutical field as carriers for delivery of various drugs, peptides and proteins. These systems have included stimuli-responsive gels that exhibit reversible swelling behavior and hence can show modulated release in response to external stimuli such as pH, temperature, ionic strength, electric field, or specific analyte concentration gradients. The focus of this article is to review molecular imprinting within hydrogels and discuss recent efforts on analyte-responsive intelligent gels, specifically suggesting the possibility of utilizing molecular imprinting strategies to impart analyte specificity and responsiveness within these systems. Molecular imprinting is an emerging field that produces precise chemical architecture that can bind analytes and differentiate between similar molecules with enantiomeric resolution. On the forefront of imprinting gel systems are intelligent, stimuli-sensitive imprinted gels that modify their swelling behavior and in turn modulate their analyte binding abilities. We discuss the challenges creating an imprinting effect in hydrogels and the possibilities of using molecularly imprinted mechanisms within controlled release gels.     

H1-receptor blocker antihistamine, terfenadine durably influences the glucocorticoid receptor, and lymphocyte histamine content of weanling rats.

Hormonal imprinting takes place perinatally when a hormone and its maturing target receptor meet. As a consequence of imprinting the receptor accomplishes its maturation reaching the binding capacity characteristic to the adult age. In this critical period the presence of foreign molecules which are able to bind to the receptor can cause faulty imprinting with life-long consequences. In the recent years it was cleared that not only receptors are influenced by faulty imprinting, however, also the hormone production of the given cell. In addition imprinting was provoked at non-perinatal periods (adolescence and adult age) in cytogenic organs. In the present experiment the prolonged effect of a non-perinatal imprinting by an antihistamine to the histamine content of white blood cells and glucocorticoid receptors of liver and thymus was studied. Two weeks after 3-day terfenadine treatment at weaning, flow cytometry of peritoneal cells and blood lymphocytes for histamine, and receptor kinetic analysis of dexamethasone binding were done. Histamine content of blood lymphocytes and glucocorticoid receptor density of liver cells were significantly decreased. This means that a short treatment with a H(1)-receptor blocker antihistamine durably influences physiological indexes which were not known till now. This means that not only the acute effects, but the prolonged side-effects must be considered.     

All-trans-Retinoic Acid Upregulates the Expression of COUP-TFI in Early-Somite Mouse Embryos Cultured In Vitro

Exposure of embryos to an excess of retinoic acid (RA) modifies the spatio-temporal pattern of expression of developmental genes. RA regulates the expression of target genes through binding of the retinoid nuclear receptors (RARs and RXRs), as heterodimers, to regulatory cis-acting elements. COUP-TF factors, which are able to dimerize with the RXRs and to compete with the retinoid receptors for their DNA binding sites, are suspected to modulate the retinoid signal transduction pathway. Therefore, COUP-TF factors may be involved in the regulation of the expression of developmental genes and/or in the modifications induced by an excess of RA in the expression of these genes. The aim of this work is to assess whether RA-induced modifications in the expression of Krox-20 and Hox genes correlate with alterations of the expression of COUP-TF genes. In addition to spatial modifications in the expression patterns of Krox-20 and Hox genes, we report here an upregulation of the expression level of COUP-TFI after RA exposure. However, this abnormality did not spatially overlap with the modifications observed in the expression of Krox-20 and Hox genes. These data suggest an involvement of COUP-TFI in the generation of RA-induced abnormalities, but do not support the hypothesis of an involvement of this factor in the regulation of the expression of Hox or Krox-20 genes.     

分子印迹技术高效分离中药活性成分的应用

分子印迹技术是一门分子识别技术,由于其制备的分子印迹聚合物对目标分子具有特异性识别的功能,在中药领域受到了广泛关注。本文综述了分子印迹技术的原理、特点、聚合物的制备方法及其在中药现代化研究中的应用。     

A mechanism-based complementary screening approach for the amelioration and reversal of neurobehavioral teratogenicity

The identification of mechanisms and outcomes for neurobehavioral teratogenesis is critical to our ability to develop therapies to ameliorate or reverse the deleterious effects of exposure to developmental neurotoxicants. We established mechanistically-based complementary models for the study of cholinergic systems in the mouse and the chick, using both environmental neurotoxicants (chlorpyrifos, perfluoroalkyls) and drugs of abuse (heroin, nicotine, PCP). Behavioral evaluations were made using the Morris maze in the mouse, evaluating visuospatial memory related to hippocampal cholinergic systems, and imprinting in the chick, examining behavior dependent on cholinergic innervation of the IMHV. In both models we demonstrated the dependence of neurobehavioral deficits on impairment of cholinergic receptor-induced expression, and translocation of specific PKC isoforms. Understanding this mechanism, we were able to reverse both the synaptic and behavioral deficits with administration of neural progenitors. We discuss the prospects for clinical application of neural progenitor therapy, emphasizing protocols for reducing or eliminating immunologic rejection, as well as minimizing invasiveness of procedures through development of intravenous administration protocols.     

Right Hemisphere Involvement in Imprinting Memory Revealed by Glutamate Treatment

The lateralized use of the forebrain hemispheres during recall of imprinting memory was investigated using unilateral intrahemispheric injections of glutamate. Administration of glutamate to the right hemisphere 1, 3, or 6 h after exposure to the imprinting stimulus disrupted recall 8 h after the end of training, whereas the same treatment of the left hemisphere had no effect. Imprinted chicks treated with glutamate injected into the right hemisphere did not approach the imprinting stimulus in preference to an alternative, unfamiliar stimulus during a simultaneous choice test, whereas imprinted chicks treated with glutamate injected into the left hemisphere showed a preference for the imprinting stimulus. Thus, the left and right hemispheres are involved differentially in the recall of imprinting memory. Fear behavior or activity levels were not altered by glutamate treatment of either the right or left hemisphere, indicating that the effects of glutamate were specific to recall of imprinting memory. However, the amnestic effect of treatment of the right hemisphere with glutamate was transient: it was no longer evident by 48 h after the end of training. Also, glutamate had no effect when the chicks were treated 9 h after the end of training. These results suggest that regions in right hemisphere of the chick brain are involved in early (0–8 h after training) recall of imprinting memory.     

分子印记技术在毛细管电色谱手性分离中的应用

分子印记技术是制备具有预定选择性分离介质的有效途径，其应用于毛细管电色谱(CEC)手性分离具高效及独特功用。综述分子印记技术的基本原理及其在CEC中的应用模式和实例。     

Pathway modeling of microarray data: A case study of pathway activity changes in the testis following in utero exposure to dibutyl phthalate (DBP)

Pathway activity level analysis, the approach pursued in this study, focuses on all genes that are known to be members of metabolic and signaling pathways as defined by the KEGG database. The pathway activity level analysis entails singular value decomposition (SVD) of the expression data of the genes constituting a given pathway. We explore an extension of the pathway activity methodology for application to time-course microarray data. We show that pathway analysis enhances our ability to detect biologically relevant changes in pathway activity using synthetic data. As a case study, we apply the pathway activity level formulation coupled with significance analysis to microarray data from two different rat testes exposed in utero to Dibutyl Phthalate (DBP). In utero DBP exposure in the rat results in developmental toxicity of a number of male reproductive organs, including the testes. One well-characterized mode of action for DBP and the male reproductive developmental effects is the repression of expression of genes involved in cholesterol transport, steroid biosynthesis and testosterone synthesis that lead to a decreased fetal testicular testosterone. Previous analyses of DBP testes microarray data focused on either individual gene expression changes or changes in the expression of specific genes that are hypothesized, or known, to be important in testicular development and testosterone synthesis. However, a pathway analysis may inform whether there are additional affected pathways that could inform additional modes of action linked to DBP developmental toxicity. We show that Pathway activity analysis may be considered for a more comprehensive analysis of microarray data.