玉米赤霉烯酮对MCF-7和T47D细胞凋亡的抑制作用

玉米赤霉烯酮(ZEA)是一种霉菌毒素，在奶制品及霉变谷类食物中含量较高。据报道，ZEA在结构上与内源性雌激素有相似之处，通过与雌激素受体结合而模拟雌激素效应，增加女性体内雌激素负荷，与乳腺癌发病率的升高有关。本实验室前一阶段的研究表明，ZEA可模拟雌激素作用，促进雌激素依赖性乳腺癌细胞MCF-7及T47D细胞增殖，     

玉米赤霉烯酮对MCF-7细胞肿瘤相关基因表达的影响

目的观察真菌类环境雌激素玉米赤霉烯酮(zearalenone,ZEA)对雌激素依赖性乳腺癌细胞MCF-7肿瘤相关基因表达的影响,初步探讨ZEA与乳腺癌发生、发展的关系及其作用机制。方法在同等条件下,溶剂对照组(ethanol,EtOH)和32×10-9mol/L ZEA对MCF-7细胞分别处理72h,收集细胞并提取细胞总mRNA,用Cy5和Cy3两种不同荧光染料通过逆转录反应将mRNA分别标记成两种探针,并与载有一组靶基因的人肿瘤相关基因表达谱芯片进行杂交,经计算机扫描分析得出这些基因在经ZEA处理前后的表达差异。结果筛选出包括与细胞增殖、细胞凋亡、细胞转化、应激反应、肿瘤逃逸等相关表达差异的基因共15条,其中4条基因下调(主要是抑癌基因和调节细胞增殖、凋亡相关的基因等),11条基因上调(主要是热休克蛋白基因、雌激素反应基因、内源性逆转录病毒基因、HPK/GCK激酶基因及与肿瘤发生、发展密切相关的原癌基因等)。结论ZEA上调基因数目多于下调基因数目,且上调基因多数是促进细胞增殖的基因,还有两个是与肿瘤发生有关的基因,提示ZEA对相关肿瘤的发生发展起着促进作用并具有一定的致癌性。     

玉米赤霉烯酮对小鼠胚胎体外发育的影响

目的研究玉米赤霉烯酮(F-2毒素)对小鼠胚胎体外发育的影响。方法将(8只/批)6～8周龄清洁级昆明妊娠4 d小鼠处死,挑选形态良好的8-细胞(8-细胞期是早期胚胎发育过程中便于观察及深入开展胚胎在发育生物学、胚胎移植、转基因动物等领域研究工作的最佳阶段)。胚胎,采用微滴培养法,将形态良好的8-细胞胚胎(10～15个/孔)移入0(溶剂对照)、10-8、10-6和10-4mol/L的F-2毒素培养液(含5%人输卵管液+10%胎牛血清)中,记录囊胚形成数及囊胚脱带数,并计算囊胚形成率和囊胚脱带率。每个剂量设5个平行。结果与溶剂对照组比较,F-2毒素浓度在10-4和10-6mol/L时囊胚形成率较低,差异均有统计学意义(P<0.05);F-2毒素浓度在10-8、10-6和10-4mol/L时,囊胚脱带率较低,差异均有统计学意义(P<0.05)。囊胚形成率和囊胚脱带率均随着F-2毒素浓度的升高呈下降趋势。结论 F-2毒素在小鼠胚胎发育过程中具有毒性作用。     

中药中玉米赤霉烯酮的残留测定

目的建立薏苡仁、麦芽、绿豆中玉米赤霉烯酮的测定方法.方法样品经90%乙腈提取、免疫亲和柱净化后,用高效液相色谱-荧光检测器法进行分析测定,阳性样品采用LC-MS-MS进一步确证.结果玉米赤霉烯酮在125～3 750 pg范围内进样量与峰面积呈良好的线性关系,相关系数为0.999 9,加样回收率在96.6%～114.7%之间.结论本法重复性好、灵敏度高、定量快速准确,可作为薏苡仁、麦芽、绿豆中玉米赤霉烯酮残留的测定方法.     

玉米赤霉烯酮对大鼠子宫内膜细胞增殖作用的影响

目的 研究玉米赤霉烯酮对去卵巢大鼠子宫内膜细胞的增殖作用影响.方法 将成年雌性大鼠去卵巢后,给予不同剂量的玉米赤霉烯酮(0.01mg/kg、0.05mg/kg和0.20mg/kg)连续3d,观察去卵巢大鼠子宫内膜细胞有丝分裂指数和嗜银染色颗粒数.结果 玉米赤霉烯酮低、中、高3个剂量组有丝分裂指数和嗜银染色颗粒数,均高于...     

薏苡仁新标准中玉米赤霉烯酮测定法的方法确认及验证过程

目的 以薏苡仁项下新增的玉米赤霉烯酮测定法为例,阐述实验室首次使用法定标准时应如何进行方法确认以及验证。方法 通过考察玉米赤霉烯酮测定法的专属性、准确度、精密度、检出限、定量限、线性等指标进行方法验证。结果 玉米赤霉烯酮在范围内线性关系良好,r=0.9991,空白溶液无干扰峰、加样回收率83.2%～90.2%,RSD为3.1%,方法检出限为0.1μg/kg,定量限为3.8μg/kg,结果均符合《中华人民共和国药典》2020年版四部“9101分析方法验证指导原则”的要求。结论 方法验证内容应根据分析方法的复杂程度和被测样品的特点进行有针对性的考察,不同的检验方法应考察其关键性的指标。     

细胞间隙连接通讯与肿瘤关系的研究进展

细胞间隙连接通讯 (ｇａｐｊｕｎｃｔｉｏｎａｌｉｎｔｅｒｃｅｌｌｕｌａｒｃｏｍ ｍｕｎｉｃａｔｉｏｎ ,ＧＪＩＣ)是细胞间一种最普遍的通讯方式 ,传递生长抑制和 (或 )增殖信号 ,在调节细胞的生长、分化及凋亡中起重要作用。肿瘤是一种增殖失控和分化紊乱的疾病 ,自从发现肿瘤细胞间的电偶联丧失以来 ,许多学者相继发现肿瘤和转化细胞普遍存在细胞间隙连接通讯缺陷、间隙连接蛋白基因表达异常的现象 ,说明ＧＪＩＣ与肿瘤的发生和发展过程有密切关系。1　细胞间隙连接通讯细胞间隙连接通讯的结构基础是间隙连接 (ｇａｐｊｕｎｃｔｉｏｎ …     

玉米赤酶烯酮对大鼠子宫雌激素受体影响的探讨

目的探讨玉米赤霉烯酮对子宫雌激素受体的影响及其雌激素样作用机制。方法 30只大鼠行双侧去卵巢手术后随机分为阴性对照组（蒸馏水）、低,中、高剂量玉米赤霉烯酮组（0.01mg/kg、0.05mg/kg、0.20mg/kg）和阳性对照组（0.08mkg雌二醇）,连续用药3d后取子宫,采用放射性配基结合分析法和雌激素受体结合试验检测雌激素受体数量。结果玉米赤霉烯酮各浓度组放射活性与阴性对照组比较,差异有统计学意义（P≤0.05）。玉米赤霉烯酮各浓度组雌激素受体数量与阴性对照组比较,差异无统计学意义（P≥0.05）。结论玉米赤霉烯酮可能是通过影响雌二醇与雌激素受体结合和雌激素受体的数量显示雌激素样作用。     

玉米赤霉原生质体融合的研究

应用0.8％纤维素酶(来自Trichoderma.Pseudokoningii)及1.0％蜗牛酶(来自Achatina fulica Ferussac)的混合酶液制备玉米赤霉(Gibberella zeae)原生质体,以30％聚乙二醇(分子量4000)0.01mol/L CaCl_2溶液(pH7.0)作融合剂,进行了原生质体的营养互补融合及双亲紫外灭活融合。在四个菌株组成的两组配对体系中(701×801,7~(AT)×8~(BT))获得了稳定的二倍体及一株四倍体。融合体HL-5及Gl-1较其直接亲本具有良好的生理及遗传性状,二者的赤霉烯酮合成能力均接近高产亲本。     

大豆异黄酮和玉米赤酶烯酮对乳腺癌细胞株T47D细胞增殖的影响

环境中天然存在或污染的各类化学物质能够模拟或干扰人和动物内分泌功能 ,称为环境内分泌干扰物 ,环境雌激素是其中的一大类。外源性的天然或合成的雌激素一旦摄入体内 ,在达到一定浓度时 ,都能够与靶细胞上的ＥＲ结合 ,模拟Ｅ2 或拮抗Ｅ2 的生理作用 ,改变靶器官的生长、发育及生理功能[1 3 ] 。大豆异黄酮(ＧＳ)是豆类食品中的主要异黄酮类物质之一 ,流行病学调查表明ＧＳ有预防乳腺癌和前列腺癌作用[4] 。玉米赤霉烯酮 (ＺＥＡ)是一种真菌毒素 ,常见于霉变的玉米、小麦、燕麦等农作物中 ,1～ 10ｎｍｏｌ Ｌ浓度即可刺激雌激素受体转录激…     

亚砷酸钠和氧苯胂对人皮肤成纤维细胞缝隙连接通讯的动态影响及作用特征

目的　了解和比较不同砷化合物对细胞缝隙连接通讯的动态影响及作用特征 ,进一步阐明砷的致癌机理。方法　用激光共聚焦显微镜 ,以细胞漂白后荧光强度的恢复作为评价缝隙连接通讯的指标 ,按激光漂白后荧光恢复实验技术测定亚砷酸钠和氧苯胂对人皮肤成纤维细胞缝隙连接通讯的影响。结果　亚砷酸钠和氧苯胂均可明显抑制激光漂白后细胞荧光强度的恢复 ,其作用有明显的剂量 反应关系 ;进一步的研究发现 ,在去除亚砷酸钠作用后的不同时间 ,实验组细胞荧光强度恢复增加值显著低于对照组 ,表明细胞缝隙连接通讯功能没有恢复 ;而在去除氧苯胂作用 4h后 ,实验组荧光强度恢复增加值与对照组无显著性差异 ,表明细胞缝隙连接通讯有所恢复。结论　无机砷和有机砷均可明显抑制细胞缝隙连接通讯 ,但二者的作用特征有所不同。     

氧苯胂和二甲基胂酸对细胞间通讯的影响

近年来的研究认为抑制细胞缝隙连接间通讯（ＧＪＩＣ）是促癌物的一种重要特性。代谢协同试验是测定ＧＪＩＣ的方法之一。本研究用此法探讨了氧苯胂和二甲基胂酸对Ｖ７９细胞的ＧＪＩＣ的影响。该２种有机砷化物均为无机砷化物在哺乳动物体内的代谢产物。结果发现,氧苯胂能显著抑制Ｖ７９细胞的代谢协同作用。在０．００１μｍｏｌ／Ｌ浓度下,其抑制作用最强。二甲基胂酸在０．０１～１ｍｍｏｌ／Ｌ浓度下对Ｖ７９细胞也有一定的抑制作用。本研究首次提出氧苯胂可能有促癌活性,并进一步提示二甲基胂酸有促癌作用     

Inhibitory effect of Bisphenol A on gap junctional intercellular communication in an epithelial cell line of rat mammary tissue

An endocrine disruptor, bisphenol-A (BPA), has been reported to have several short-term actions in various cells and tissues. However, the mechanisms of these actions have not been fully elucidated. In order to assess the effect of BPA on the intercellular communication mediated by gap junctions, we conducted the present study in the rat epithelium-derived BICR-M1Rk cell-line, in which connexin 43 (Cx43) is a major gap junction channel-forming protein. The cytotoxicity of BPA toward the cultured cells was evaluated by using both MTT reduction and LDH leakage assay systems. The results showed no appreciable loss in cell viability in the presence of increasing concentrations of BPA (from 0.1 to 3.2 microM) for 1 h incubation. However, most of cell viability was lost when cells were incubated for 24 hr with the same concentrations of BPA. The BPA acted as an antagonist on gap junction-mediated intercellular communication (GJIC), and the phenomenon was dose-dependent and irreversible. According to the data obtained from scrape-loading dye-transfer experiments, three quarters of normal GJIC was reduced by concentration of 0.4 microM BPA for 1 h incubation. To identify the relevance of this retardation upon BPA treatment, the GJIC to Cx43 synthesis, the mRNA and protein levels of Cx43 were assessed with RT-PCR and Western-blotting, respectively. The total protein level of Cx43 was almost constant in a wide range of BPA concentrations, as well as in Cx43 mRNA level. These results suggest that BPA inhibits GJIC through a modulation of the gating of gap junction channels, not through a genomic modulation of Cx43.     

Cadmium decreases gap junctional intercellular communication in mouse liver.

Cadmium (Cd) is an environmental pollutant of increasing importance, due to industrialization, smoking, and the lack of effective therapy for Cd poisoning. The general population is exposed to Cd principally through food and water. The metal accumulates slowly in the liver and kidney, the target organs of acute and chronic Cd toxicity, respectively. We showed recently that liver is also a target organ for chronic Cd toxicity. Gap junctional intercellular communication (GJIC) is a means of maintaining cellular homeostasis in multicellular organisms. It involves the transfer of small, water-soluble molecules through intercellular channels (gap junctions), composed of proteins called connexins. The major connexins of liver (hepatocytes) are connexin 32 (Cx32) and connexin 26 (Cx26). Cd disrupts cellular homeostasis in the liver through its induction of necrosis, apoptosis, and cellular proliferation. It is to be expected, therefore, that Cd must exert some effect on GJIC. This study investigates Cd-induced alterations in GJIC, Cx32, and Cx26 expression, and in cytoskeletal actin, and relates the changes to apoptosis and cell proliferation induced by Cd in vivo. Mice were injected ip with 30 micromol Cd/kg, and were observed for up to 48 h. Other groups of mice were injected with 5-60 micromol Cd/kg and observed for 9 h. Blood and liver were harvested and used for analysis of GJIC, connexin expression, cytoskeletal actin, serum enzymes, and liver pathology. Cd produced a time- and dose-dependent inhibition of GJIC in liver, along with parallel decreases in the expression of Cx32 and Cx26. Cd also produced disruption and loss of cytoskeletal actin in liver in a time- and dose-dependent manner. These observations are discussed in relation to the toxicity of Cd, and possible mechanisms of induction of the GJIC-related alterations are presented.     

Airborne PAHs inhibit gap junctional intercellular communication and activate MAPKs in human bronchial epithelial cell line

Inhalation exposures to polycyclic aromatic hydrocarbons (PAHs) have been associated with various adverse health effects, including chronic lung diseases and cancer. Using human bronchial epithelial cell line HBE1, we investigated the effects of structurally different PAHs on tissue homeostatic processes, namely gap junctional intercellular communication (GJIC) and MAPKs activity. Rapid (<1 h) and sustained (up to 24 h) inhibition of GJIC was induced by low/middle molecular weight (MW) PAHs, particularly by those with a bay- or bay-like region (1- and 9-methylanthracene, fluoranthene), but also by fluorene and pyrene. In contrast, linear low MW (anthracene, 2-methylanthracene) or higher MW (chrysene) PAHs did not affect GJIC. Fluoranthene, 1- and 9-methylanthracene induced strong and sustained activation of MAPK ERK1/2, whereas MAPK p38 was activated rather nonspecifically by all tested PAHs. Low/middle MW PAHs can disrupt tissue homeostasis in human airway epithelium via structure-dependent nongenotoxic mechanisms, which can contribute to their human health hazards.     

热疗上调细胞间隙连接通讯对胶质瘤侵袭性的影响及其机制

目的探讨热疗降低胶质瘤侵袭性的作用与细胞间隙连接通讯(gap junctional intercellular communication,GJIC)的关系。方法热疗处理C6胶质瘤细胞后,免疫组织化学法和Western blot法动态检测HSP70和Cx43的表达水平;划痕标记染料示踪技术检测胶质瘤GJIC功能变化;结晶紫染色法检测胶质瘤侵袭性的改变。结果 C6细胞经热疗后,HSP70表达增加,于30 min时含量最多。C6细胞内Cx43的表达水平也在热疗后明显增加,并于热疗后的120 min达高峰,后逐渐减少。热疗后GJIC功能的恢复与C6细胞内Cx43的表达相一致,且GJIC功能越强,胶质瘤侵袭性越低。结论胶质瘤细胞经热疗后HSP70表达增加,增加的HSP70可能是通过其分子伴侣作用提高Cx43的表达水平,进而上调GJIC功能而引起胶质瘤侵袭性的下降。     

Regulation of gap junctional intercellular communication by TCDD in HMEC and MCF-7 breast cancer cells

Previous studies suggest that many neoplastic tissues exhibit a decrease in gap junctional intercellular communication (GJIC). Many hydrocarbons and organochlorine compounds are environmental pollutants known to be carcinogenic. The effect of an organochlorine compound, TCDD, on GJIC in human breast cell lines has not been established. In the present study, we showed that TCDD causes an inhibition in the gap junctional activity in MCF-7 (breast cancer cells). In MCF-7 cells, an increase in the phosphorylated form of gap junctional protein, connexin 43 (Cx43), and PKC α was seen in the presence of TCDD. Gap junctional plaque formation was significantly decreased in MCF-7 cells in the presence of TCDD. Immunoprecipitation studies of PKC α showed that TCDD caused a significant 40% increase in the phosphorylated Cx43 in MCF-7 cells. TCDD also modulated the translocation of PKC α from the cytosol to the membrane and caused a 2-fold increase in the PKC α activity at 50 nM TCDD in MCF-7 cells. Calphostin C, an inhibitor of PKC α, showed a significant inhibition of PKC α activity in the presence of TCDD. Furthermore, TCDD also caused a decrease in the gap junctional activity and Cx43 protein in human mammary epithelial cells (HMEC). However, we observed a shift in the Cx43 plaques towards the perinuclear membrane in the presence of TCDD by confocal microscopy and Western blot. Overall, these results conclude that TCDD decreases GJIC by phosphorylating Cx43 via PKC α signaling pathway in MCF-7 cells; however, TCDD decreases the GJIC by affecting the localization of Cx43 in HMEC. These new findings elucidate the differential mode of effect of TCDD in the downregulation of GJIC in HMEC and MCF-7 cells.     

Silver nanoparticles up-regulate Connexin43 expression and increase gap junctional intercellular communication in human lung adenocarcinoma cell line A549

Abstract

Silver nanoparticles (Ag NPs) are increasingly being used in wound dressings, medical settings, and various household products due to their unique properties and antimicrobial activity. Despite the widespread use of Ag NP products, the molecular mechanisms underlying the biological effects of Ag NPs remain unclear. Gap junctional intercellular communication (GJIC), formed by the connexin protein family, plays a critical role in the maintenance of tissue and organ homeostasis. This study was undertaken to investigate the effects of well characterized, PVP-coated Ag NPs (69 ± 3 nm) and silver nitrate on GJIC and connexin43 (Cx43) expression in human lung adenocarcinoma cell line A549. Our results showed that Ag NPs increased GJIC in A549 cells as assayed by dye transfer method. Western blot analysis showed that incubation of cells with Ag NPs significantly increased the expression of Cx43 protein. In addition, Ag NPs up-regulated expression of Cx43 mRNA in a dose-dependent manner. Silver nitrate failed to increase GJIC and the expression of Cx43 protein. It, however, increased Cx43 mRNA expression in A549 cells. Taken together, our results provide the first evidence that Ag NPs induced the increase of GJIC activity in A549 cells through up-regulation of Cx43 protein, suggesting that Cx43 and GJIC may be one of the targets for Ag NPs biological effects.     

Effects of organophosphorus pesticides and their ozonation byproducts on gap junctional intercellular communication in rat liver cell line.

The effects of organophosphorus pesticides (OPs), oxons and their ozonation byproducts on gap junctional intercellular communication (GJIC) on cultured BRL cell line were investigated using scrape loading and dye transfer (SL/DT) technique. The neutral red uptake assay was used to identify the non-cytotoxic levels of diazinon, parathion and methyl-parathion applied to GJIC assay. The concentration-dependent inhibition of GJIC was observed over a range of 50-350 mg/l diazinon, parathion and methyl-parathion after 90 min incubation compared with the vehicle control. However, oxons and ozonation byproducts of OPs had no inhibition effect on GJIC at any of the concentrations tested. The inhibition of GJIC by OPs was reversible after removal of the tested pesticides followed by incubation with fresh medium. The present study suggested that the ozonation treatment could be used for the detoxification of drinking water and food crops contaminated with diazinon, parathion and methyl-parathion without formation of GJIC toxicity.     

Targeting gap junctional intercellular communication by hepatocarcinogenic compounds

ABSTRACT

Gap junctions in liver, as in other organs, play a critical role in tissue homeostasis. Inherently, these cellular constituents are major targets for systemic toxicity and diseases, including cancer. This review provides an overview of chemicals that compromise liver gap junctions, in particular biological toxins, organic solvents, pesticides, pharmaceuticals, peroxides, metals and phthalates. The focus in this review is placed upon the mechanistic scenarios that underlie these adverse effects. Further, the potential use of gap junctional activity as an in vitro biomarker to identify non-genotoxic hepatocarcinogenic chemicals is discussed.