Effect of methylmercury on glutamate-cysteine ligase expression in the placenta and yolk sac during mouse development

The placenta and the yolk sac play critical roles in fetal development, including protection from oxidative stress through the presence of detoxifying enzymes. Glutathione (GSH; gamma-glutamylcysteinylglycine), a crucial molecule in the maintenance of cellular redox status, plays a critical role in development, and it is also protective against methylmercury toxicity. Glutamate-cysteine ligase (GCL), the enzyme that catalyzes the rate-limiting step in GSH synthesis, is widely expressed in the mouse embryo and extraembryonic membranes throughout development. The aim of this study was to investigate the effect of low-level subchronic methylmercury exposure on GCL expression in the mouse placenta and yolk sac, after describing the basal developmental expression of the enzyme in these tissues. We found that basal mRNA expression levels increased dramatically in the placenta and the yolk sac at gd 18, whereas protein levels did not increase in parallel with the mRNA. We also found that methylmercury induced GCLc mRNA expression in the placenta at gd 18 in a dose-dependent manner, suggesting an important role for this enzyme in the response of the placenta to toxicants. These changes in expression may be useful as a biomarker of MeHg exposure during development.     

The effects of caffeine and bisphenol A singularly or in combination on cultured mouse embryos and yolk sac placenta

Pregnant women drink caffeinated beverages using bisphenol A (BPA)-coated cans without knowing the potential risks. In this study, mouse embryos (embryonic day 8.5) surrounded by yolk sac placenta were cultured with caffeine (30, 60, and 120 μg/ml) and/or BPA (35 μg/ml) for 48 h. In response to a single administration of BPA or caffeine dose, embryonic development was similar to normal control embryos. However, the combined exposure to caffeine and BPA dose-dependently increased embryonic anomalies, and thinner ventricular wall and trabeculae disorders of heart were observed. The mRNA levels of various anti-oxidative, apoptotic, and hypoxic genes were significantly altered in the treated embryos. Furthermore, abnormal vasculogenesis, reduced vasculogenic growth factor expressions, and apoptotic cell death were detected in yolk sac placentas. These findings indicate that the combined exposure to caffeine and BPA induces embryonic anomalies and injuries of the yolk sac placentas through oxidative stress, apoptosis, hypoxia, and vasculogenic defects.     

Role of p53-dependent placental apoptosis in the reproductive and developmental toxicities of caffeine in rodents

相似文献   

1H-异吲哚-1,3(2H)-二酮新衍生物对鸡胚绒毛尿囊膜血管新生的抑制作用研究

目的　研究 1H 异吲哚 1,3(2H) 二酮新衍生物对血管新生的抑制作用。方法　应用鸡胚绒毛尿囊膜血管生成模型 ,通过计数、比较载体周围 0 5cm内血管的分支点数 ,观察不同化合物对于在体血管新生的抑制作用。结果　通过对 18种 1H 异吲哚 1,3(2H) 二酮新衍生物的研究 ,发现化合物 1,2 ,3,4 ,11和 13对鸡胚绒毛尿囊膜的血管新生有明显的抑制作用。结论　筛选出来的化合物 1,2 ,3,4 ,11和13有望成为新的抑制血管新生的药物。     

Breast cancer resistance protein (Bcrp/Abcg2) is selectively modulated by lipopolysaccharide (LPS) in the mouse yolk sac

Bacterial infection alters placental ABC transporters expression. These transporters provide fetal protection against circulating xenobiotics and environmental toxins present in maternal blood. We hypothesized that lipopolysaccharide (LPS-bacterial mimic) alters the yolk sac morphology and expression of key ABC transporters in a gestational-age dependent manner. Yolk sac samples from C57BL/6 mice were obtained at gestational ages (GD) 15.5 and GD18.5, 4 or 24 h after LPS exposure (150ug/kg; n = 8/group). Samples underwent morphometrical, qPCR and immunohistochemistry analysis. The volumetric proportions of the histological components of the yolk sac did not change in response to LPS. LPS increased Abcg2 expression at GD15.5, after 4 h of treatment (p < 0.05). No changes in Abca1, Abcb1a/b, Abcg1, Glut1, Snat1, Il-1β, Ccl2 and Mif were observed. Il-6 and Cxcl1 were undetectable in the yolk sac throughout pregnancy. Abca1, breast cancer resistance protein (Bcrp, encoded by Abcg2) and P-glycoprotein (P-gp/ Abcb1a/b) were localized in the endodermal (uterine-facing) epithelium and to a lesser extent in the mesothelium (amnion-facing), whereas Abca1 was also localized to the endothelium of the yolk sac blood vessels. LPS increased the labeling area and intensity of Bcrp in the yolk sac’s mesothelial cells at GD15.5 (4 h), whereas at GD18.5, the area of Bcrp labeling in the mesothelium (4 and 24 h) was decreased (p < 0.05). Bacterial infection has the potential to change yolk sac barrier function by affecting Bcrp and Abcg2 expression in a gestational-age dependent-manner. These changes may alter fetal exposure to xenobiotics and toxic substances present in the maternal circulation and in the uterine cavity.     

The chick chorioallantoic membrane imaging method as a platform to evaluate vasoactivity and assess irritancy of compounds

Objective To determine if the chick chorioallantoic membrane (CAM) is a potential alternative that is capable of screening test substances for vasoactivity in terms of vessel diameter changes. The CAM was also evaluated as a tool for irritancy screening. Methods Visual assessment of the CAM for irritancy after the application of the test substance or solvent to its surface was made. An imaging based‐in‐vivo CAM model was developed by imaging CAM blood vessels in a pre‐defined area using a semi‐automatic image processing and analysis technique to measure blood vessel diameters. Solvents and drugs such as 70% v/v ethanol, normal saline, 5% w/v glucose monohydrate, glycerin, glucagon, N‐methylpyrrolidone, nicotine, glyceryl trinitrate, glucagon, propranolol and caffeine were tested on the CAM. Key findings Propranolol, nicotine and glycerin were irritants on CAM. Changes in the diameters of fine blood vessels were accurately measured by high resolution image analysis. Vasoconstriction was seen with 70% v/v ethanol while vasodilation was displayed with glucagon and caffeine. The results reflected expected trends with evidence of feedback mechanisms ensuring homeostasis. Conclusion The CAM model can be applied to assess pharmaceutical and cosmetic formulations in early development work to gain useful insights to potential irritancy and biological effects of components and formulations.     

四种抗肝癌中药对鸡胚绒毛尿囊膜血管生成的影响

目的通过测定四种中草药对鸡胚绒毛尿囊膜（CAM）模型血管生成的作用,并与阳性药物索拉菲尼对比,探究药物作用与血管生成的关系,探究药物可能存在的靶向治疗作用。方法醇提取四种广西特色中草药．建立鸡胚绒毛尿囊膜模型测定不同药物醇提物与极性组段抗血管生成的抑制作用。结果索拉菲尼、破骨风、土半夏、血党均有不同程度的抑制鸡胚尿囊血管生长作用,与空白对照组相比,索拉菲尼与破骨风对二、三级血管均有明显抑制作用（P〈0．01）;土半夏、血党对三级血管有抑制作用（P〈0．01）,其高剂量组对二级血管也有明显抑制作用（P〈0．01）。结论索拉菲尼、破骨风、土半夏、血党均可不同程度抑制血管的生长,可能是通过与索拉菲尼相同的作用机制实现;天胡荽对血管生长的抑制作用较弱。     

Daphnetin inhibits TNF‐α and VEGF‐induced angiogenesis through inhibition of the IKKs/IκBα/NF‐κB,Src/FAK/ERK1/2 and Akt signalling pathways

Coumarins, identified as plant secondary metabolites possess diverse biological activities including anti‐angiogenic properties. Daphnetin (DAP), a plant derived dihydroxylated derivative of coumarin has shown significant pharmacological properties such as anticancer, anti‐arthritic and anti‐inflammatory. The present study was performed to investigate the anti‐angiogenic potential of DAP, focusing on the mechanism of action. The in vivo anti‐angiogenic potential of DAP was evaluated by vascular endothelial growth factor (VEGF)‐induced rat aortic ring (RAR) assay and chick chorioallantoic membrane (CAM) assay. For in vitro evaluation, wounding migration, transwell invasion, tube formation and apoptosis assays were performed on VEGF (8 ng/mL)‐induced human umbilical vein endothelial cells (HUVECs). The cellular mechanism of DAP was examined on TNFα (10 ng/mL) and VEGF‐induced HUVECs by extracting the mRNA and protein levels using RT‐qPCR and western blotting. Our data demonstrated that DAP inhibited the in vivo angiogenesis in the RAR and CAM assay. DAP also inhibited the different steps of angiogenesis, such as migration, invasion, and tube formation in HUVECs. DAP inhibited nuclear factor‐κB signalling together including TNF‐α induced IκBα degradation; phosphorylation of IκB kinase (IKKα/β) and translocation of the NF‐κB‐p65 protein. Furthermore, western blotting revealed that DAP significantly down‐regulated the VEGF‐induced signalling such as c‐Src, FAK, ERK1/2 and the related phosphorylation of protein kinase B (Akt) and VEGFR2 expressions. DAP reduced the elevated mRNA expression of iNOS, MMP2 and also, induced apoptosis in VEGF‐stimulated HUVECs by the caspase‐3 dependent pathway. Taken together, this study reveals that DAP may have novel prospective as a new multi‐targeted medication for the anti‐angiogenesis and cancer therapy.     

Activity of aldrinepoxidase, 7-ethoxycoumarin-O-deethylase and 7-ethoxyresorufin-O-deethylase during the development of chick embryos in ovo

Since the chick embryoin ovo is susceptible to the action of some agents needing metabolic activation we studied the development of the activity of cytochrome P450-dependent monooxygenases in embryo/fetal tissue. The activities of aldrinepoxidase (AE), 7-ethoxycoumarin-O-deethylase (ECOD) and 7-ethoxyresorufin-O-deethylase (EROD) were measured in whole embryos, liver and yolk sac tissue of the chick embryo during developmentin ovo from day 4 to day 15 of incubation (DI). In yolk sac tissue enzyme activities could be detected from DI 4 on. While EROD activity was only marginally developed, AE and ECOD activities were more pronounced in the earlier developmental period and showed a clear decrease by the time the liver activities rose. With the methods used AE activity could be measured in the homogenate of the whole embryo proper from DI 4 on while EROD and ECOD activity was not detectable before DI 6 or DI 7, respectively. In liver tissue enzyme activities of the three monooxygenases studied developed to a considerable degree from DI 9 on and tended to exhibit maximum values around DI 11–13. Studies on monooxygenase activities in extra-embryonically located tissues have not been published until now. The importance of the yolk sac as a metabolically relevant organ during embryogenesis is pointed out in this study.     

The effects of amphotericin B on angiogenesis in chick chorioallantoic membrane

Objective: Amphotericin B (AmB) is widely used as a mainstay in the treatment of sight-threatening fungal endophthalmitis. From the time that itraconazole was discovered to have a previously unknown anti-angiogenic activity, we have suspected that AmB may have possible effects on ocular angiogenesis. The purpose of this study was to evaluate the in vivo anti-angiogenic effect of AmB in the chick chorioallantoic membrane (CAM) model.

Materials and methods: Atak-S type fertilized eggs obtained from the Poultry Institution were used. The eggs were kept under 37?°C at 85–90% relative humidity throughout the experiment. Amphotericin B was prepared in two different concentrations (AmB 125?μg/1?mL and 0.125?μg/1?mL). The CAMs treated with sterile distilled water was specified as controls. About 0.1?mL of each containing 12.5 and 0.0125?µg of AmB, respectively, were dropped to CAM surface. Thirteen eggs were used for each group. The results were evaluated at the 48th hour of the administration of the drugs and recorded by digital camera.

Results: A reduction of angiogenesis in CAM area which treated with 125?μg/1?mL of AmB was appreciable macroscopically. The affected areas showed impaired radial arrangement of small vessels with the presence of avascular zone at periphery. The dose of 0.125?μg/1?mL AmB did not show any visible anti-angiogenic effect. Numerous blood vessels with a radially arranged pattern developed toward the periphery after 48?h of treatment. In the CAMs that treated with distilled water, physiological angiogenesis was observed in allantoic vessels. Vessel formation seems to be similar in CAMs treated with 0.125?μg/1?mL AmB with the presence of visibly non-malformed alive embryos.

Conclusions: The present study gives the impression that AmB has the capacity to serve as an anti-angiogenic treatment. As it is a preliminary CAM study only, further studies on both animals and humans are required.     

丹酚酸B对鸡胚绒毛尿囊膜血管新生作用的影响

目的探讨丹酚酸B对鸡胚绒毛尿囊膜血管新生的影响。方法将7日龄鸡胚制备鸡胚绒毛尿囊膜(chick embryo chorioallantoic membrane,CAM)模型,随机分为6个组,分别为PBS(phosphatebuffer solution)对照组,丹酚酸B 4个剂量组(150、50、16.67、5.56 mg.L-1)及VEGF(vascular endo-thelial growth factor)组,培养3 d后时采集数据进行评价。结果丹酚酸B 150、50 mg.L-1组血管新生面积明显高于PBS(phosphate buffer solution)对照组(P<0.01,P<0.05)。结论丹酚酸B可明显促进鸡胚绒毛尿囊膜血管新生。     

The intrauterine renin–angiotensin system: Sex‐specific effects on the prevalence of spontaneous preterm birth

Preterm birth (PTB) is the single largest cause of death in infants and young children. The rate of PTB is significantly higher in male infants, particularly those that are born very preterm. Here we present evidence to suggest that the decidual renin–angiotensin system may play a role in inhibiting inflammation and maintaining the integrity of the fetal membranes during pregnancy, and that sex‐specific alterations in the intrauterine RAS could contribute to the increased risk of PTB in male babies. Women carrying female fetuses have high levels of expression of decidual prorenin at term. Decidua from ‘female’ pregnancies also have greater expression of the anti‐inflammatory angiotensin (Ang)‐(1‐7) pathway, than decidua from ‘male’ pregnancies, and have lower levels of the pro‐inflammatory Ang II pathway. We propose that in ‘female’ pregnancies, the very high levels of decidual prorenin drive the anti‐inflammatory Ang‐(1‐7) pathway, thus reducing the likelihood of PTB. In addition, the high levels of prorenin produced by the decidua in ‘female’ pregnancies are able to diffuse into the amnion and bind to the PRR. We postulate that PRR/prorenin interactions, possibly through both angiotensin dependent and independent pathways, stimulate the production of ECM proteins, inhibit ECM degradation and prevent apoptosis, thus strengthening the amnion. Thus control of the inflammatory signature and the integrity of the fetal membranes prior to parturition may partly depend on the sexually determined activity of the decidual and amniotic renin–angiotensin system pathways.     

5'-O-tritylated nucleoside derivatives: inhibition of thymidine phosphorylase and angiogenesis

Thymidine phosphorylase (TPase) is one of the key enzymes involved in the pyrimidine nucleoside salvage pathway. However, TPase also stimulates angiogenesis, and its expression correlates well with microvessel density and metastasis in a variety of human tumors. We have shown recently that 5'-O-trityl-inosine (KIN59) allosterically inhibits TPase enzymatic activity. KIN59 also inhibits TPase-induced angiogenesis in the chick chorioallantoic membrane (CAM) assay. The trityl group was found to be instrumental to preserve both the anti-TPase and antiangiogenic effect. We have now synthesized a variety of novel 5'-O-trityl nucleoside derivatives. Enzyme activity studies showed that the anti-TPase activity is significantly improved by replacement of the hypoxanthine base by thymine [3.5-fold; i.e., 5'-O-tritylthymidine (KIN6)] and the introduction of chloride on the trityl group [7-fold; i.e., 5'-O-(4-chlorotrityl)-inosine (TP136)], whereas removal of 2'-hydroxyl in the ribose did not significantly alter the anti-TPase activity. Enzyme kinetic studies also demonstrated that 1-(5'-O-trityl-beta-d-ribofuranosyl)-thymine (TP124), like KIN59, inhibits TPase in a noncompetitive fashion both with respect to phosphate and thymidine. Most KIN59 analogs markedly inhibited TPase-induced angiogenesis in the CAM assay. In vitro studies showed that the antiangiogenic effect of these compounds is not attributed to endothelial cell toxicity. For several compounds, there was no stringent correlation between their anti-TPase and antiangiogenic activity, indicating that these compounds may also act on other angiogenesis mediators. The antiangiogenic 5'-O-trityl nucleoside analogs also caused degradation of pre-existing, immature vessels at the site of drug exposure. Thus, 5'-O-trityl nucleoside derivatives combine antiangiogenic and vascular-targeting activities, which opens perspectives for their potential use as anticancer agents.     

The impact of caffeine on connexin expression in the embryonic chick cardiomyocyte micromass culture system

Cardiomyocytes are electrically coupled by gap junctions, defined as clusters of low‐resistance multisubunit transmembrane channels composed of connexins (Cxs). The expression of Cx40, Cx43 and Cx45, which are present in cardiomyocytes, is known to be developmentally regulated. This study investigates the premise that alterations in gap junction proteins are one of the mechanisms by which teratogens may act. Specifically, those molecules known to be teratogenic in humans could cause their effects via disruption of cell‐to‐cell communication pathways, resulting in an inability to co‐ordinate tissue development. Caffeine significantly inhibited contractile activity at concentrations above and including 1500 μm (P < 0.05), while not affecting cell viability and total protein, in the embryonic chick cardiomyocyte micromass culture system. The effects of caffeine on key cardiac gap junction protein (Cx40, Cx43 and Cx45) expression were analysed using immunocytochemistry and in‐cell Western blotting. The results indicated that caffeine altered the expression pattern of Cx40, Cx43 and Cx45 at non‐cytotoxic concentrations (≥2000 μm ), i.e., at concentrations that did not affect total cell protein and cell viability. In addition the effects of caffeine on cardiomyocyte formation and function (contractile activity score) were correlated with modulation of Cxs (Cx40, Cx43 and Cx45) expression, at above and including 2000 μm caffeine concentrations (P < 0.05). These experiments provide evidence that embryonic chick cardiomyocyte micromass culture may be a useful in vitro method for mechanistic studies of perturbation of embryonic heart development. Copyright © 2015 John Wiley & Sons, Ltd.     

葛根素对AGEs诱导CAM新生血管生成影响的实验研究

目的　探讨葛根素对糖基化终产物(AGEs)诱导的鸡胚绒毛尿囊膜(CAM)新生血管生成的影响。方法　制备AGEs复合物,建立CAM模型,随机分为空白组、AGEs组,不同剂量葛根素组和阳性药对照组,采用单位面积血管百分比法,应用OPTIMAS软件,检测葛根素对AGEs诱导的CAM血管生成的影响。结果　不同剂量的AGEs均可明显促进CAM新生血管的形成(P<0 01),而大、中剂量葛根素对此有明显的抑制作用(P<0 05)。结论　葛根素对AGEs诱导的CAM新生血管的生成具有一定的抑制作用。     

Caffeine-induced fetal rat over-exposure to maternal glucocorticoid and histone methylation of liver IGF-1 might cause skeletal growth retardation

Several epidemiological investigations, including previous work by our laboratory, indicate that maternal caffeine consumption is associated with intrauterine growth retardation and impaired fetal length growth. Skeletal development is critical for length growth. In the present study, our goals were to determine the effects of prenatal caffeine exposures on fetal skeletal growth and to investigate the mechanisms associated with such effects. Pregnant Wistar rats were injected intragastrically with 120 mg/kg of caffeine intragastrically each day from gestational days 11–20. Maternal prenatal caffeine exposure was associated with decreased fetal femur lengths and inhibited of synthesis of extracellular matrices in fetal growth plates Moreover, caffeine exposure significantly increased the levels of fetal blood corticosterone and decreased IGF-1mRNA expression levels in the liver and growth plate. The expression levels of IGF-1 signaling pathway components (IGF-1R, IRS-1, AKT1/2 and Col2A1) were also reduced. In addition, the results of chromatin immunoprecipitation assays indicated that caffeine exposure down-regulated histone methylation of fetal IGF-1 in the liver. These results suggest that prenatal caffeine exposure may inhibit fetal skeletal growth through a mechanism that is associated with increased fetal exposure to maternal glucocorticoids and results in lower IGF-1 signaling pathway activity. Taken together, these results raise important concerns regarding the skeletal growth toxicity of caffeine and potentially indicate the intrauterine origins of adult osteoporosis and osteoarthritis.     

内源性血管生成抑制因子A rresten在E.coli JM109中的表达及抗新生血管生成的药理学研究

目的构建内源性血管生成抑制因子Arresten基因的原核表达载体,并进行表达,抑制新生血管的药理学实验中发现,该表达产物具有抑制鸡胚绒毛尿囊膜血管生长的功能。方法从健康产妇的胎盘组织中提取总RNA,经逆转录-聚合酶链式反应(RT-PCR)扩增出Arresten基因,构建重组质粒pBV220-Arr转化E.coli JM109进行原核表达,大量表达提取Arresten蛋白,用鸡胚绒毛尿囊膜实验进行活性测定。结果成功构建的重组质粒pBV220-Arr在E.coliJM109菌株中2~8 h均可获得表达,其中诱导4 h表达效率最高,Arresten蛋白可明显抑制鸡胚绒毛尿囊膜血管生长,活性功能明显强于血管抑素。结论成功构建Arresten基因重组质粒pBV220-Arr,并可在E.coli JM109菌株中获得表达,Arresten蛋白具有明显的抑制血管生成的作用。     

从赤魟组织中分离到的福安肽的抗血管生成作用

目的研究从赤魟组织中分离到的福安肽(FAT)对鸡胚绒毛尿囊膜(CAM)血管生成、人低分化鼻咽癌细胞(CNE-2Z)诱导的CAM血管生成和小鼠肝癌组织血管生成的影响。方法用CAM法检测FAT对CAM血管生成和CNE-2Z细胞诱导的CAM血管生成的影响;用免疫组化法检查FAT对小鼠肝癌组织内微血管密度(MVD)的影响。结果FAT显著抑制CAM血管生成,当其用量为30,60,120μg/(胚.d)×3时,其抑制率分别为30.8%,50.1%,64.0%;CNE-2Z细胞明显诱导CAM血管生成,当CNE-2Z细胞的接种量为1.9×106细胞/胚时,其血管生成诱导率为30.1%;FAT明显抑制CNE-2Z细胞诱导的CAM血管生成,当其用量为30,60,120μg/(胚.d)×4时,其抑制率分别为35.8%,43.1%,51.3%。FAT明显降低小鼠肝癌组织的MVD,FAT平均微血管数为(8.2±1.3)与对照组的(21.3±2.8)相比,差异显著(P<0.05)。结论FAT有显著的抗血管生成作用,这可能是其抑制小鼠肿瘤生长的重要原因。