NF-κB and AP-1 as molecular targets for chemoprevention with EGCG, a review

There are multiple lines of compelling evidence, mostly from population-based studies, supporting that green tea consumption is associated with reduced risk of several human malignancies. Epigallocatechin gallate (EGCG) is a major antioxidant present in green tea. Chemopreventive effects of EGCG have been extensively investigated and well documented. These include inhibition of metabolic activation of carcinogens and/or stimulation of their detoxification, scavenging of reactive oxygen species, induction of apoptosis or differentiation of malignant or transformed cells, suppression of tumor promotion, inhibition of angiogenesis or metastasis, etc. EGCG targets many intracellular signaling molecules and events responsible for malignant transformation or abnormal cell proliferation, and NF-κB and AP-1 appear to be two of most potential targets of EGCG in its exerting chemopreventive activities.     

Regulation of autophagy in rat hepatocytes treated in vitro with low concentration of mercury

Several proteins are implicated in the regulation of autophagy in cells under various physiological and pathological conditions. Recently it was found that in vitro initiation of autophagy in 5 μM mercuric chloride (HgCl₂) treated rat hepatocytes occurred within 30 min of incubation. The aim of this study was to monitor the autophagy–ubiquitination link to determine the regulatory mechanisms underlying autophagy at a concentration of 5 μM HgCl₂. Autophagic cell death was evidenced by the presence of LC3B positive hepatocytes throughout the incubation period of 4 hr. Autophagosome maturation occurred following 1 hr of metal treatment accompanied by an enhanced expression of several regulatory proteins such as p62, Keap1, and caspase-8, which drive the hepatocytes toward autophagy. In addition, the rate of increase in down-regulation in a p38 expression was significantly higher than that of extracellular signal-regulated kinase (ERK) at 1 hr incubation, followed by a significant decline in the p38 level at 2 and 4 hr following metal treatment. In contrast, the expression of ERK remained higher than that of p38 at 2 and 4 hr. Data indicate that intracellular activation of the major regulatory proteins such as LC3B, caspase-8, Keap1, p62, ERK, and p38 modulates autophagy through ubiquitination in rat hepatocytes exposed to low concentrations of Hg.     

单壁纳米碳管对大鼠主动脉内皮细胞损伤作用的研究

为了探索单壁纳米碳管(SWCNT)能否引起血管内皮细胞损伤及其可能的损伤机制,将大鼠主动脉血管内皮细胞暴露于不同浓度的SWCNT水溶液中(0.8、1.6、3.12、6.25、12.5、25、50、100、200μg·mL-1)中染毒,染毒不同时间后测定细胞存活率、细胞内LDH和GSH含量并进行综合分析.结果表明,随着SWCNT染毒浓度和染毒时间的上升,大鼠主动脉血管内皮细胞存活率逐渐下降,死亡率逐渐上升;细胞内LDH在SWCNT染毒浓度为0￣100μg·mL-1范围内其释放随染毒剂量的增加而逐渐上升,在200μg·mL-1剂量组,其释放有所减弱;而细胞内GSH在SWCNT染毒浓度为0￣200μg·mL-1范围内其含量随染毒剂量的增加而逐渐下降.以上结果说明,SWCNT可致血管内皮细胞损伤,其机制可能为氧化损伤途径.     

Effects of nonylphenol on ovarian cancer cells PEO4 c-myc and p53 mRNA and protein expression

The molecular mechanisms underlying proliferation and apoptosis induced by p-nonylphenol (NP) through regulation of mRNA and protein expression of c-myc, p53 mRNA and the protein was investigated using ovarian cancer PEO4 cells. PEO4 cells were grown in the Dulbecco's modified Eagle medium (DMEM), 3(4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT) assay was employed to investigate cell proliferation. RT-PCR and immunohistochemistry was performed to determine mRNA and protein expression of PEO4 c-myc and p53. Within the range of approximately 8–9×10^?7 mol L^?1, NP stimulated proliferation in PEO4 cells in a concentration-and time-dependent manner. RT-PCR and immunohistochemistry analyses revealed that treatment with 32 × 10^?7 mol L^?1 NP for 72 hr resulted in increased mRNA and protein expression of PEO4 c-myc and p53. Evidence indicates that NP exerted estrogenic actions and stimulated proliferation in estrogen responsive ovarian cancer PEO4 cells by up-regulation of c-myc mRNA and protein expression.     

Manufactured silver and gold nanoparticles-induced apoptosis by caspase-pathway in human cell lines

Abstract

Metallic nanoparticles have emerged as an important class of nanomaterials for a wide range of industrial and medical applications. Because of the intensive commercial applications, risk assessment of these nanoparticles is of great importance. In the present study, the human hepatoma and leukemia cells were used to characterize the apoptotic effects of silver nanoparticles (4.7 and 42?nm) and gold nanoparticles (30?nm). Apoptotic cells were identified by chromatin condensation and flow cytometry analysis, using Annexin V/PI, TUNEL and caspase activation assays. Flow cytometry analysis showed that the three metallic nanoparticles induced apoptotic cell death in a concentration and time dependent-manner. Moreover, the three nanoparticles induced activation of caspase-3 and -7 in hepatoma and leukemia cells. Apoptotic effects were stronger after exposure of both cell lines with 4.7?nm silver nanoparticles than those obtained with 42?nm silver and 30?nm gold nanoparticles. In conclusion, silver (4.7 and 42?nm) and gold (30?nm) nanoparticles induced apoptosis in hepatoma and leukemia cells via the caspase dependent pathway. The smaller silver nanoparticles (4.7?nm) had a greater ability to induce apoptosis in both cell lines.     

Single walled carbon nanotubes induce cytotoxicity and oxidative stress in HEK293 cells

The aim of the present study was to evaluate the potential toxicity and general mechanisms involved in single walled carbon nanotubes (SWCNTs)-induced cytotoxicity using human embryonic kidney cell line (HEK293) cells. Carbon nanotubes (coded as CNT) used in this study were synthesized by the chemical vapor deposition method. To elucidate the possible mechanisms underlying SWCNT-induced cytotoxicity, cell viability, cell membrane damage (lactate dehydrogenase activity (LDH) assay), reduced glutathione (GSH), interleukin-8 (IL-8) and lipid peroxidation products levels were quantitatively assessed following SWCNT exposure for 48 hr using HEK293 cells. Exposure of cells to SWCNT at 3–300 μg/ml produced significant reduction in cell viability in a concentration-dependent manner. The IC₅₀ value of SWCNT was found to be 87.58 μg/ml. Exposure of HEK cells to SWCNT at 10–100 μg/ml resulted in concentration-dependent cell membrane damage, increased production of IL-8, elevated levels of thiobarbituric acid reactive substances like malondialdehyde and decreased intracellular GSH levels. In summary, exposure to SWCNT resulted in a concentration-dependent cytotoxicity in cultured HEK293 cells that was associated with increased oxidative stress.     

Low concentration of HgCl2 drives rat hepatocytes to autophagy/apoptosis/necroptosis in a time-dependent manner

Mercury (Hg) is known to produce hepatotoxicity driving cells towards apoptosis. It was recently reported that low concentrations of Hg (5 μM) initiate autophagy in vitro within 30 min of incubation modulated by several autophagy-related gene proteins, and co-regulators through ubiquitination. The present study aimed to elucidate in vitro mode of cytotoxic responses including programed cell death in 5-μM Hg-treated rat hepatocytes. Autophagy proceeded from 30 min to 4 hr mediated by crosstalk between specific regulating factors of cell-death-signaling mechanisms. It was noted that after 4-hr incubation with 5 μM HgCl₂, cells were driven towards apoptosis followed by necroptosis within 6.5 hr. Receptor-interacting serine-threonine protein 3 (RIP3) and caspase-8 played a significant role in interlinking function. The positive role of caspase-8 with RIP3 significantly triggered caspase-3 via extrinsic apoptotic pathway. A shift from apoptosis to necroptosis occurred after 6 hr via tumor necrosis factor α-RIP3-caspase-8 pathway. No alteration in caspase-3 expression and presence of high-mobility group box 1 protein in nucleus indicated absence of apoptosis and necrosis in rat hepatocytes between 6.5 and 8 hr. Data indicated that cellular homeostasis is regulated by modulating different proteins and driving hepatocytes through autophagy to apoptosis to necroptosis in a time-dependent manner.     

Cardiotoxin III suppresses the invasiveness of MDA-MB-231 cells by targeting proto-oncogene tyrosine-protein kinase Src and reversing mesenchymal-to-epithelial transition

The epithelial-to-mesenchymal transition is the first step required for breast cancer to initiate metastasis. The aberrant activation of proto-oncogene tyrosine-protein kinase Src regulates multiple functions during tumor progression. Cardiotoxin III, a basic polypeptide isolated from Naja naja atra venom, has been shown to exhibit anticancer activity; however, the effect of cardiotoxin III on the epithelial-to-mesenchymal transition of cancer cells remains elusive. Exposure of MDA-MB-231 cells to cardiotoxin III resulted in morphological changes and upregulation of E-cadherin with a concomitant decrease in N-cadherin and vimentin protein levels, resulting in the inhibition of cell migration and invasion. Cardiotoxin III induced downregulation of snail and slug expression. Simultaneously, cardiotoxin III suppressed Src phosphorylation and downstream activation of focal adhesion kinase, of the docking protein p130^cas, and of paxillin. In addition, cardiotoxin III inhibited the phosphorylation of extracellular signal-regulated kinase and phosphatidylinositol 3-kinase. The Src specific inhibitor 4-amino-5-(4-chlorophenyl)-7-(dimethylethyl)pyrazolo[3,4-d]pyrimidine decreased in the phosphorylation and the expression changes of epithelial-to-mesenchymal transition markers in a similar way. Thus, cardiotoxin III has a novel anticancer effect by suppressing proto-oncogene tyrosine-protein kinase activity, reversing epithelial-to-mesenchymal transition.     

AzaC预处理增加TCDD对特殊细胞P450基因的诱导

利用RT-PCR检测不同物质诱导细胞的CYP基因 mRNA的表达水平.在HepG2细胞,TCDD能诱导CYP1A1、CYP1B1及CYP1A2基因表达,CYP1A1、CYP1B1基因比CYP1A2基因更容易被诱导,用AzaC处理后CYP1B1基因表达无改变;在A549细胞和SPC-A1细胞,Azac预处理后增加了TCDD对CYP1家族的诱导.也就是AzaC增加了CYP1A1、CYP1A2和CYP1B1基因表达水平.图1表1参10     

Thallium(I) and thallium(III) induce apoptosis in isolated rat hepatocytes by alterations in mitochondrial function and generation of ROS

Environmental metal toxins, generated through diverse anthropogenic activities, constitute one of the major contaminants that have led to global dispersion of these toxic metals in the ecosystem. Thallium is one of these widely dispersed metals that produce severe adverse effects on human and biological systems. The influence of thallium(I) and thallium(III) on the early events that trigger apoptosis signaling were examined in freshly isolated rat hepatocytes. In addition, the role of oxidative stress, and mitochondria in the induction of apoptosis were also investigated. Incubation of thallium(I) and thallium(III) with isolated rat hepatocytes generated reactive oxygen species (ROS), collapse of mitochondrial membrane potential, activation of caspases cascade, and appearance of apoptosis phenotype. Mitochondrial permeability transition (MPT) pore sealing agents (cyclosporine A and carnitine) and ATP generators (L-glutamine, fructose, and xylitol) inhibited the activation of caspase-3 and apoptosis, indicating that both the cations activated apoptosis signaling via mitochondrial pathway. Pretreatment of hepatocytes with antioxidants (α-tocopherol or deferoxamine) also blocked caspase-3 activation induced by these cations, suggesting that oxidative stress may be directly involved in a mitochondrial MPT pore opening and activation of caspases cascade. These findings contribute to a better understanding of the mechanisms that mediate thallium-induced apoptosis in isolated rat hepatocytes.     

Cadmium sulfide nanoparticle induces oxidative stress and pro-inflammatory effects in human lung adenocarcinoma epithelial cells

Cadmium sulfide nanoparticles (CdSNP) are increasingly used in biological applications. This study was undertaken to understand the mechanisms underlying adverse effects of CdSNP using human lung adenocarcinoma epithelial (A549) cells. Cellular toxicity was evaluated by using 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide and neutral red assays. Results showed that CdSNP reduced mitochondrial function and induced lysosomal activity in concentration and time-dependent manner. CdSNP produced oxidative stress as evidenced by reduction of glutathione (GSH) levels and increase in reactive oxygen species and lipid peroxidation levels. Induction of caspase-3 enzymes and condensed, fragmented nuclei was observed in CdSNP-treated cells. Furthermore, the levels of interleukin-8, tumor growth factor and DNA fragmentation were significantly higher in CdSNP exposed cells. Data indicated that toxicity of CdSNP noted in A549 cells may be mediated through oxidative stress. This study warrants more comprehensive assessment of CdSNP prior to industrial applications.     

苯并\[a\]芘对马氏珠母贝肝组织抗氧化酶活性的影响

为揭示不同浓度苯并[a]芘(B[a]P)对海洋贝类的生态毒理效应,将马氏珠母贝(Pinctada martensi)暴露于不同浓度(1、4和8μg.L-1)B[a]P中,检测暴露3、7和10d后,B[a]P对马氏珠母贝肝组织抗氧化酶(超氧化物歧化酶SOD、谷胱甘肽硫转移酶GST和过氧化氢酶CAT)活性的影响。结果表明:暴露时间3d时,随着B[a]P浓度的增加,SOD活性无明显变化,GST的活性被激活,在7d和10d受到抑制,GST活性表现为抑制,并表现出一定的剂量-效应关系,而CAT的活性在染毒后第7天受到激活;暴露时间10d时,SOD活性增加,GST活性和CAT活性均受到抑制。B[a]P暴露时间相同,GST活性和CAT活性变化趋势基本相似。B[a]P浓度相同(1和4μg.L-1)时,随着暴露时间的延长,SOD活性无明显变化,GST的活性受到抑制,CAT的活性表现为先激活后抑制的趋势。另外,相对于SOD活性,GST和CAT的活性变化可以更好地反映B[a]P对马氏珠母贝胁迫的毒性效应。     

Induction of apoptosis and cytokine markers in colon cancer cells by magnesium oxide (MgO) nanoparticles

Magnesium oxide nanoparticles (MgONP) are predominantly utilized in industrial products. This study was undertaken to elucidate the mechanisms underlying toxic effect of MgONP in human colon cancer (HT 29) cells over 48 hr period. Cytotoxicity was evaluated by using MTT and neutral red uptake assays. Data demonstrated that MgONP reduced cell viability in concentration- and time-dependent manner. MgONP induced oxidative stress by decreasing glutathione (GSH) concentrations and elevation of reactive oxygen species (ROS) and lipid peroxidation levels. Increased caspase-3 enzyme activity and greater condensed, damaged chromosome was observed following MgONP exposure in HT 29 cells. The level of interleukin-4 (IL-4), tumor necrosis factor (TNF-α), and DNA fragmentation were significantly higher in MgONP incubated cells. The results showed that MgONP-induced toxicity in HT 29 cells may be mediated through oxidative stress.     

五氯酚暴露诱导斑马鱼胚胎细胞凋亡相关基因的变化及caspase-2基因克隆和系统进化分析

应用基因芯片技术研究发现,暴露于五氯酚的斑马鱼胚胎中有14个涉及细胞凋亡行为的基因表达发生了显著改变.利用生物信息学方法构建这些功能基因系统进化树,分析与环境毒物诱导的细胞凋亡行为密切相关的caspase-2基因和其他基因之间的同源关系.斑马鱼caspase-2基因由10个外显子和9个内含子组成,cDNA长1308bp,含一个开放阅读框(ORF),编码435个氨基酸.6种脊椎动物Caspase-2氨基酸序列的保守性及系统进化分析结果表明,在特定功能区结构域中氨基酸序列表现出较高的同源性,Caspase-2在进化上高度保守.利用RT-PCR技术,斑马鱼caspase-2基因cDNA被克隆并确认.研究结果表明斑马鱼caspase-2基因是一个研究细胞凋亡行为的模型分子,可为环境化合物的分子毒性评价及其机制研究提供一种分子标记。     

Effects of a phycotoxin,okadaic acid,on oyster heart cell survival

Okadaic acid (OA) is a dinoflagellate toxin which accumulates in shellfish producing diarrhetic shellfish poisoning (DSP) in humans. It was found that OA is a highly selective inhibitor of protein phosphatase types 1 (PP1) and 2A (PP2A) which produces a marked increase in phosphorylation of several proteins, including p38 mitogen-activated protein (MAP) kinase. The cytotoxicity attributed to OA and the effects on p38 MAP kinase and calcium current were examined in the oyster Crassostrea gigas in this study. Data showed that p38 MAP kinase is strongly expressed in oyster heart and that OA bioaccumulated in cultured heart cells. Hence the effects of OA was tested in vitro and in vivo on oysters. OA was found to (i) exert a positive chronotropic effect on cultured atrial cardiomyocytes which is related to an increase in calcium current via PKC as shown by patch clamp measurements, (ii) produce an activation/phosphorylation of MAP kinase as shown by Westernblot while the non-phosphorylated p38 remained constant during treatment, (iii) did not induce a pro-apoptotic effect. Data suggest that OA may also stimulate the anti-apoptotic pathway by phosphatase inhibition.     

Nanosilica exerts cytotoxicity and apoptotic response via oxidative stress in mouse embryonic fibroblasts

Nanoscale silica is an important industrial material and extensively used in medicines. The objective of this study was to determine potential cytotoxicity and genotoxic effects attributed to nanosilica exposure in mouse embryonic fibroblasts (L929) cells. Nanosilica produced mild cytotoxicity in L929 cells. Results showed that nanosilica increased thiobarbituric acid reactive substance levels and enhanced superoxide dismutase activity but decreased levels of glutathione. This was accompanied by a concomitant generation of reactive oxygen species, loss of mitochondrial membrane potential, and activation of caspase-3 activity. In addition, in the single-cell gel test, nanosilica (50–300 μg/ml) at two treatment times 24 and 48 hr produced concentration- and time-dependent increase of DNA damage. Therefore, the obtained results indicate that nanosilica may induce genotoxic effects in cultured L929 cells associated with induction of oxidative stress.     

微囊藻毒素-LR致小鼠肝细胞DNA-蛋白质交联作用的研究

为探讨微囊藻毒素-LR致小鼠肝细胞的DNA-蛋白质交联作用,将20只昆明雄性小鼠随机分为4组:1个对照组和3个染毒组,采用腹腔注射进行染毒7d,染毒剂量分别为3.0、6.0和12.0μg·kg-1,检测小鼠肝细胞DNA-蛋白质交联程度.结果显示,3.0、6.0和12.0μg·kg-1微囊藻毒素-LR均可导致小鼠肝细胞显著的DNA-蛋白质交联作用(与对照组相比,p<0.01,p<0.01,p<0.05),当微囊藻毒素为6.0μg·kg-1时,这种作用最明显.     

丙烯腈暴露对大鼠脑组织损伤及iNOS/p38 MAPK信号通路关键蛋白表达的影响

通过探究iNOS/p38 MAPK信号通路在丙烯腈(acrylonitrile,ACN)诱导脑组织损伤中的作用,为进一步研究ACN的神经毒性作用提供依据。选取50只SPF级健康成年雄性SD大鼠,随机分为5组,每组10只。适应性饲养一周后,以12.5、25.0、50.0 mg·kg~(-1)ACN对大鼠进行灌胃染毒,对照组给予玉米油,另设NAC组(300.0 mg·kg~(-1)NAC+50.0 mg·kg~(-1)ACN),1次·天~(-1),6天·周~(-1),共染毒13周。次日称重并处死大鼠,测定大鼠脑组织NO含量、总NOS水平及iNOS、p-p38和p38蛋白表达水平。结果显示,ACN各剂量组大鼠脑组织脏器系数与对照组比较均显著降低(P0.05),高剂量组大鼠脑脏器系数与NAC组比较降低(P0.05)。高剂量组NO含量和总NOS水平显著高于对照组,与NAC组比较,高剂量组NO含量降低(P0.05),总NOS水平升高(P0.05)。Western blot结果显示,ACN高剂量组大鼠脑组织iNOS、p-p38蛋白表达水平和p-p38/p38比值显著高于对照组和NAC组(P0.05)。ACN可激活iNOS/p38MAPK信号通路,这可能是ACN致大鼠脑组织损伤的机制之一。