三氧化二砷诱导人类风湿性关节炎滑膜细胞凋亡研究

目的：研究三氧化二砷（arsenic trioxide，As2O3）对体外培养的人类风湿性关节炎（rheumatoid arthritis，RA）滑膜细胞凋亡诱导作用。方法：酶消化法进行人关节滑膜细胞原代培养，MTT法研究As2O3对RA滑膜细胞活力影响；用光镜、电镜、DNA凝胶电泳等方法检测As2O3诱导RA滑膜细胞凋亡。结果：成功体外培养人关节滑膜细胞。MTF法显示As2O3可抑制RA滑膜细胞活力；光镜、电镜下发现As2O3作用于RA滑膜细胞可出现凋亡表现；DNA凝胶电泳显示有凋亡梯带出现。结论：As2O3可以诱导RA滑膜细胞凋亡。     

Telmisartan treatment attenuates arsenic-induced hepatotoxicity in mice

The protective effect of telmisartan, the angiotensin II-receptor antagonist, against liver toxicity induced by sodium arsenite (5 mg/kg/day, p.o., for 30 days) was investigated in mice. Telmisartan treatment (10 mg/kg/day, p.o.) was applied for 30 days, starting on the same day of arsenic administration. Telmisartan significantly reduced serum alanine aminotransferase level which was increased by sodium arsenite. Telmisartan significantly suppressed lipid peroxidation, and prevented the reduced glutathione depletion and nitric oxide elevation in the liver tissue resulted from arsenic administration. Also, the increase of arsenic ion, and the reductions of selenium and zinc ions in liver tissue were attenuated by telmisartan. Histopathological examination showed that liver tissue injury mediated by arsenic was ameliorated by telmisartan treatment. Immunohistochemical analysis revealed that telmisartan significantly decreased the arsenic-induced expression of inducible nitric oxide synthase, tumor necrosis factor-α, cyclooxygenase-2, nuclear factor-κB and caspase-3 in liver tissue. It was concluded that telmisartan may represent a potential option to protect the liver tissue from the detrimental effects of arsenic toxicity.     

PC-407 inhibited proliferation and induced apoptosis in human colon cancer SW-1116 cells

AIM: To study whether PC-407 [4-[5-naphthyl-3- (trifluoromethyl)-1H-pyrazol-1-yl] benzenesulfonamide] inhibits cell viability and induces apoptosis in human colon cancer SW-1116 cells. METHODS: Inhibition of SW-1116 proliferation was measured by MTT assay. Morphological assessment of apoptosis was performed with fluorescence microscope and electron microscope. DNA fragmentation was visualized by agarose gel electrophoresis. The amount of apoptotic cells was measured by flow cytometry. RESULTS: PC-407 inhibited SW-1116 cell proliferation in a concentration-dependent manner after 3 d of treatment, and the IC50 for PC-407 inhibition of cell number was 16.67±0.17 μmol/L. After incubation of SW-1116 cells with PC-407 20 μmol/L for 24 h, morphological changes of typical apoptosis were observed by AO/EB staining or transmission electron microscopy. Flow cytometry analysis showed that PC-407 induced apoptosis in SW-1116 cells in a time- and concentration-dependant manner. The agarose gel electrophoresis of DN     

Morphological and functional alterations in human proximal tubular cell line induced by low level inorganic arsenic: evidence for targeting of mitochondria and initiated apoptosis

The kidney is a known target organ for arsenic and is critical for both arsenic biotransformation and elimination. Previous studies have demonstrated that at high doses (ppm) inorganic arsenic is toxic to mitochondria primarily by affecting cellular respiration. However, the effect of inorganic arsenic on mitochondria after low level exposures is not known, particularly in the kidney. Thus the functional and morphological effects of low level inorganic arsenic were investigated in a human proximal tubular cell line, HK-2. Mitochondrial function was assessed at subcytotoxic concentrations of arsenite (< or = 10 microm) by examining the alteration of the mitochondrial membrane potential using MitoTracker Red, a mitochondrion selective dye. In a subset of cells, subcytotoxic arsenite led to mitochondrial membrane depolarization, which could subsequently lead to permeability transition and apoptosis. Subcytotoxic arsenite also induced translocation of phosphatidylserine, indicative of early-stage apoptosis. To confirm whether subcytotoxic arsenite induces cellular and/or mitochondrial morphological alterations consistent with initiated apoptosis, HK-2 cells were evaluated with transmission electron microscopy. Classic morphology of apoptosis was not observed with subcytotoxic arsenite exposures; however, evidence of necrotic changes in the cytoplasmic structure and mitochondrial morphology were apparent. Therefore, based on depolarization of mitochondria and the externalization of phosphatidylserine, HK-2 cells appear to initiate apoptosis following subcytotoxic arsenite insult, but morphological changes indicate that HK-2 cells fail to complete apoptosis and ultimately undergo necrosis. Therefore, subcytotoxic arsenite can be sufficiently toxic to mitochondria that they lose their ability to keep the cell on course for apoptotic cell death.     

分枝石蕊多糖诱导人白血病K562细胞凋亡

目的：研究分枝石蕊多糖（CFP-1）是否能诱导K562细胞凋亡。方法：抑制细胞增殖的测定采用MTT法;用荧光显微镜和透射电镜观察细胞的形态学变化;采用琼脂糖凝胶电泳法观测DNA碎片;用流式细胞仪检测凋亡细胞数。结果：CFP-1（50-800mg/L）明显抑制K562细胞增殖,并且呈浓度依赖性,K562细胞与CFP-1300mg/L共同培养5d后,观察到典型的凋亡形态变化,电泳呈现梯形条带。结论：CFP-1诱导人白血病K562细胞凋亡。     

氧化苦参碱诱导人乳腺癌细胞MCF-7凋亡的实验研究

目的　研究氧化苦参碱对MCF 7细胞的诱导凋亡作用机制。方法　用光学显微镜、电子显微镜、激光共聚焦显微镜、流式细胞仪和DNA凝胶电泳等技术观察细胞凋亡。结果　实验显示氧化苦参碱作用于体外培养的MCF 7细胞可诱导发生凋亡 ,凋亡细胞表现为细胞固缩 ,核染色质聚集或碎裂、胞质空泡化等 ;DNA电泳可见DNA梯形条带 ;激光共聚焦显微镜示DNA含量下降 ,流式细胞仪检测sub G1峰在G1期前出现 ,S期细胞比例增高。结论　氧化苦参碱对体外培养MCF 7细胞生长有抑制作用 ,机制与通过阻止细胞周期的进程 ,启动细胞自身调控程序 ,诱导肿瘤细胞凋亡有关     

Limitations of the single-cell gel electrophoresis assay to monitor apoptosis in U937 and HepG2 cells exposed to 7beta-hydroxycholesterol

The single-cell gel electrophoresis (comet) assay is a method which allows the detection of DNA strand breaks in individual cells. It has been suggested that the single cell gel electrophoresis assay, as an index of DNA fragmentation during cell death, may be applied to monitor apoptosis. The aim of the present study was to determine if the pattern of DNA fragmentation determined by the single cell gel electrophoresis assay can be used to discriminate between the mode of cell death in two cell lines (U937, a human monocytic blood cell line and HepG2, a human hepatocarcinoma cell line) which were treated with 30 microM 7beta-hydroxycholesterol (7betaOHC) over a 48 hr period. The single cell gel electrophoresis assay was compared with more established methods for the determination of apoptosis such as morphological examination, flow cytometry and DNA laddering. The percentage of maximally damaged nuclei as measured by the single cell gel electrophoresis assay was found to be similar at 48 hr in both U937 and HepG2 cells when treated with 7betaOHC. However, morphological examination, flow cytometry and DNA laddering techniques showed that 7betaOHC induced apoptosis in U937 cells but not in HepG2 cells. Thus, although the alkaline single cell gel electrophoresis assay detected DNA strand breaks occurring during cell death, these breaks were observed only when the process was fairly well advanced and a major part of the cells had lost membrane permeability. Therefore the present report demonstrates that the single cell gel electrophoresis assay, used in isolation, cannot accurately be used to distinguish between the mode of cell death induced by 7betaOHC in U937 cells (apoptosis), or HepG2 cells (cell lysis).     

地昔帕明对皮质酮诱导培养的PC12细胞凋亡的拮抗作用

AIM: To study possible action mechanism of a tricyclic antidepressant, desipramine (DIM). METHODS: Cultured PC12 cells were exposed to corticosterone in the absence or presence of DIM for 5 d. Agarose gel electrophoresis, flow cytometry, and electron microscopy were used to detect the apoptosis of PC12 cells. RESULTS: Corticosterone 10 micromol/L treatment for 5 d elicited typical apoptotic biochemical and morphological changes including condensed chromatin shaped like crescent moon, nuclear fragmentation, and DNA degradation. The highest percentage of apoptotic cells accumulated to 28 % +/- 9 %. Agarose gel electrophoresis showed typical DNA ladders pattern. While in the presence of DIM 1 or 5 micromol/L, apoptosis percentage was markedly decreased with lightened DNA ladder and ultrastructure of the cells was improved. CONCLUSION: DIM could antagonize the apoptosis in PC12 cells induced by corticosterone, which may be one of the cellular mechanisms of its antidepressant effect.     

As2O3诱导K562细胞凋亡过程中细胞表面电荷变化的研究

目的 :研究三氧化二砷 (As2 O3 )诱导K562 细胞凋亡时细胞表面电荷的变化 ,阐明As2 O3 诱导K562 细胞凋亡的可能机制 ,为As2 O3 在临床上的应用提供理论依据。方法 :应用细胞电泳仪检测As2 O3 诱导K562 细胞凋亡中细胞电泳率的变化。通过细胞增殖、活力检测 ,形态学观察 ,亚G1期细胞含量和DNA凝胶电泳等鉴定细胞凋亡。结果 :1.0～ 2 0 μmol·L-1As2 O3 作用于K562 细胞 ,在细胞形态、DNA凝胶电泳、FCM显示出典型的细胞凋亡特征之前 ,试验组细胞表面电荷已在 1.6h左右开始下降 ,6h内降低最明显 ,6h后虽有下降 ,但幅度明显减弱。与对照组比较 ,低于 1.0 μmol·L-1的As2 O3 对细胞表面电荷影响不大。结论 :细胞表面电荷的下降是K562 细胞凋亡的早期事件。细胞表面电荷的下降有一定的时间范围 ,超过此范围 ,细胞表面电荷下降趋向无限大。     

Dimethylarsinic acid causes apoptosis in HL-60 cells via interaction with glutathione

 Inducibility of apoptosis in cultured human HL-60 cells by arsenic compounds, such as arsenite, arsenate, methylarsonic acid (MAA), and dimethylarisinic acid (DMAA), was investigated, together with the role of glutathione (GSH) in the induction. Among the arsenic compounds DMAA was the most potent in terms of the ability to cause the morphological changes (formation of nuclear fragmentation and apoptotic bodies) characteristic of apoptosis. Furthermore, fragmentation of internucleosomal DNA was also induced by DMAA. Depletion of cell GSH by L-buthionine-SR-sulfoximine, a selective inhibitor of γ-glutamylcysteine synthetase, enhanced the cytotoxicity of arsenite, arsenate, and MAA, while such depletion suppressed the cytotoxicity of DMAA. The depletion of GSH also suppressed the morphological changes and the fragmentation of internucleosomal DNA caused by DMAA, both of which are characteristic features of apoptosis. The results suggest that the death of cells caused by DMAA is due to apoptosis and that GSH is involved in the induction of apoptosis by this arsenic compound. Received: 17 January 1996/Accepted: 12 March 1996     

Autophagy is the predominant process induced by arsenite in human lymphoblastoid cell lines

Arsenic is a widespread environmental toxicant with a diverse array of molecular targets and associated diseases, making the identification of the critical mechanisms and pathways of arsenic-induced cytotoxicity a challenge. In a variety of experimental models, over a range of arsenic exposure levels, apoptosis is a commonly identified arsenic-induced cytotoxic pathway. Human lymphoblastoid cell lines (LCL) have been used as a model system in arsenic toxicology for many years, but the exact mechanism of arsenic-induced cytotoxicity in LCL is still unknown. We investigated the cytotoxicity of sodium arsenite in LCL 18564 using a set of complementary markers for cell death pathways. Markers indicative of apoptosis (phosphatidylserine externalization, PARP cleavage, and sensitivity to caspase inhibition) were uniformly negative in arsenite exposed cells. Interestingly, electron microscopy, acidic vesicle fluorescence, and expression of LC3 in LCL 18564 identified autophagy as an arsenite-induced process that was associated with cytotoxicity. Autophagy, a cellular programmed response that is associated with both cellular stress adaptation as well as cell death appears to be the predominant process in LCL cytotoxicity induced by arsenite. It is unclear, however, whether LCL autophagy is an effector mechanism of arsenite cytotoxicity or alternatively a cellular compensatory mechanism. The ability of arsenite to induce autophagy in lymphoblastoid cell lines introduces a potentially novel mechanistic explanation of the well-characterized in vitro and in vivo toxicity of arsenic to lymphoid cells.     

太白楤木皂苷对砷诱导的肝细胞线粒体途径凋亡的保护作用

目的探讨太白楤木皂苷对砷暴露损伤的保护作用及分子机制。方法人正常肝细胞HL-7702进行砷暴露及太白楤木皂苷保护处理。砷暴露组用10μmol L-1终浓度的NaAsO2处理,太白楤木皂苷保护组用不同浓度太白楤木皂苷预处理12 h后,再进行砷暴露处理。24 h后MTT法检测细胞活力;12 h后收集细胞样品,分别检测细胞的凋亡、活性氧（ROS）、还原型谷胱甘肽（GSH）,氧化损伤产物丙二醛（MDA）水平以及线粒体凋亡途径的重要因子细胞色素C（Cyto C）释放和Caspase-3的激活情况。结果砷暴露可以引起肝细胞活力显著降低,给予不同浓度的太白楤木皂苷具有一定的保护作用,其中5μg mL-1太白楤木皂苷保护效果最佳。砷暴露可以引起细胞凋亡比例显著增加,细胞ROS水平升高,GSH水平降低,氧化损伤产物MDA水平增加,线粒体凋亡途径因子Cyto C和Caspase-3的激活。太白楤木皂苷（5μg mL-1）预处理能够显著拮抗砷暴露引起的细胞上述指标变化。结论砷暴露可以通过诱导细胞氧化应激,激活线粒体途径细胞凋亡,发挥其损伤毒性作用。太白楤木皂苷可以通过抑制细胞氧化应激,抑制线粒体途径的细胞凋亡,从而发挥其抗砷暴露损伤的保护作用。     

Coenzyme Q10 counteracts testicular injury induced by sodium arsenite in rats

The protective effect of coenzyme Q10 against testicular toxicity induced by sodium arsenite (10mg/kg/day, orally for two consecutive days) was investigated in rats. Coenzyme Q10 treatment (10mg/kg/day, i.p.) was applied for five consecutive days, starting three days before arsenite administration. Coenzyme Q10 significantly increased serum testosterone level which was reduced by sodium arsenite. Coenzyme Q10 significantly suppressed lipid peroxidation, restored the depleted antioxidant defenses, and attenuated the increases of tumor necrosis factor-α and nitric oxide resulted from arsenic administration. Also, the elevation of arsenic ion, and the reductions of selenium and zinc ions in testicular tissue were mitigated by coenzyme Q10. Histopathological examination showed that testicular injury mediated by arsenic was ameliorated by coenzyme Q10 treatment. Immunohistochemical analysis revealed that coenzyme Q10 significantly decreased the arsenic-induced expression of inducible nitric oxide synthase, nuclear factor-κB, Fas ligand and caspase-3 in testicular tissue. It was concluded that coenzyme Q10 represents a potential therapeutic option to protect the testicular tissue from the detrimental effects of arsenic intoxication.     

Arsenite-induced apoptosis is prevented by antioxidants in zebrafish liver cell line.

This study evaluated oxidative stress-induced apoptosis as a possible mechanism of arsenite toxicity in zebrafish liver cell line (ZFL cells). The heat shock protein 70 (HSP70), a chaperone protein, appears to provide protection against oxidative stress and apoptosis. Using the MTT assay, we demonstrated that survival of ZFL cells treated with arsenite for 24h decreased in a dose-dependent manner. The possible mechanisms that promote the cytotoxicity of arsenite were addressed. Cell viability assays revealed that arsenite caused a dose-dependent increase in cell death, and pretreatment of the ZFL cells with antioxidants blunted these effects. Antioxidants such as N-acetyl-cysteine (NAC, 5 mM) and dithiothreitol (DTT, 80 microM) significantly prevented ZFL cells from arsenite-induced death. Nuclear staining was performed using 1 microg/ml Hoechst, and cells were analyzed with a fluorescent microscope. Arsenite (30 microM) induced massive apoptosis that was identified by morphology and condensation and fragmentation of the nuclei of the ZFL cells. Pretreatment with NAC or DTT before arsenite insult effectively protected the cells against oxidative stress-induced apoptosis from the arsenite. Using a transfected human hsp 70 promoter-enhanced green fluorescent protein (EGFP) reporter, pHhsp70-EGFP, the induction of HSP70 against oxidative stress-induced apoptosis by arsenite was observed. The induction of HSP70 by arsenite increased in a dose-dependent manner, and pretreatment of transfected ZFL cells with NAC or DTT before arsenite insult reduced EGFP expression. Taken together, our results provide evidence that stimulation of the heat shock response is a sensitive biomarker of arsenic exposure and that arsenite causes oxidative stress-induced apoptosis in ZFL cells.     

心血康抗血管内皮细胞凋亡的实验研究

目的采用血管紧张素Ⅱ(AngⅡ)诱导血管内皮细胞凋亡,探讨心血康对其所致内皮细胞凋亡的作用。方法在原代培养的人脐静脉内皮细胞(HUVECs)中,给予不同浓度的心血康作用2h后,再予AngⅡ作用18h诱导内皮细胞凋亡,采用透射电镜技术、琼脂糖凝胶电泳及流式细胞术等方法检测凋亡内皮细胞的超微结构、DNA梯形图和凋亡峰百分率。结果经AngⅡ作用过的血管内皮细胞透射电镜下可见凋亡小体;与AngⅡ诱导组相比,经DNA琼脂糖凝胶电泳有显著变化,DNA的梯状图谱消失,消失程度与心血康的浓度有关,浓度越大消失愈明显;流式细胞术显示DNA直方图上G1峰左侧的凋亡峰明显降低,凋亡百分率与诱导组相比,具有显著性差异(P<0.05)。结论心血康有抑制AngⅡ诱导的血管内皮细胞凋亡的作用。     

Oral administration of Cr(VI) induced oxidative stress, DNA damage and apoptotic cell death in mice

Potassium dichromate (Cr(VI)) was given orally to Swiss mice for 1 and 5 days with the dose of 25, 50 and 100 mg/kg body weight per day, respectively. Oxidative stress including the level of reactive oxygen species (ROS), the extent of lipid peroxidation and the activity of antioxidant enzymes in liver and kidney was determined. DNA damage in peripheral blood lymphocytes was determined by single-cell gel electrophoresis (comet assay). Apoptotic cell death in liver was detected using transmission electron microscopy and TUNEL assay. The results indicated that administration of Cr(VI) had caused a significant increase of ROS level in liver both after 1 and 5 days of exposure, accompanied with a dose-dependent decrease in superoxide dismutase (SOD) and catalase (CAT) activities. The malondialdehyde (MDA) content in liver was not changed as compared to the control animals. In contrast to the liver, no significant changes were observed in kidney on ROS, SOD, CAT and MDA as compared to the control animals. Dose- and time-dependent effects were observed on DNA damage after 1 and 5 days treatment. Significant difference was observed on the number of TUNEL positive liver cells between the control and Cr(VI) treatment groups. The apoptotic cells were also identified by characteristic ultrastructural features. The results obtained from the present study showed that Cr(VI) given orally to mice could induce dose- and time-dependent effects on DNA damage, hepatic oxidative stress and hepatocytes apoptosis. No significant oxidative stress observed in kidney in the study may suggest that the way of Cr(VI) exposure is an important factor affecting its toxicity.     

苏木水提物诱导HL-60细胞凋亡的研究

目的　以HL 6 0细胞为靶细胞 ,观察不同浓度苏木水提物 (AELS)对靶细胞诱导凋亡作用。方法　台盼蓝拒染法检测细胞活性 ,光镜观察细胞形态学变化 ,琼脂糖凝胶电泳法检测凋亡细胞的特征性DNA降解 ,并与阳性对照药Vp16 (etoposide,依托泊苷 )对比。 结果　研究显示 ,40mg/LVp16 ,15 0、30 0mg/L浓度的苏木水提物作用 16h均可诱导靶细胞发生凋亡。光镜下可见到细胞体积缩小 ,染色质浓缩及凋亡小体形成。DNA凝胶电泳显示特征性DNA阶梯图。结论　苏木水提物在一定浓度下可诱导HL 6 0细胞凋亡。提示诱导癌细胞凋亡是其具有抗肿瘤活性的机制之一。     

Chloroacetonitrile induces oxidative stress and apoptosis in mouse fetal liver

Chloroacetonitrile (CAN) is a disinfection by-product of chlorination of drinking water. The present study was designed to investigate the potential adverse effects of maternal exposure to CAN on fetal liver in mice. Based on an initial dose–response experiment, CAN (25 mg/(kg day)) was given orally to pregnant mice at gestation day (GD) 6, till GD 18. Fetuses were collected and fetal livers were used for assessing oxidative status, apoptosis and histopathological changes. Maternal exposure to CAN resulted in observed oxidative stress and redox imbalance in fetal liver tissues as marked by significant decrease in reduced glutathione (GSH) and elevation of oxidized glutathione (GSSG), malondialdehyde (MDA) and 8-hydroxy-2′-deoxyguanosine (8-OHdG) in genomic DNA. Further, CAN induced apoptosis as indicated by a significant increase in binding of Hoechst reagent to damaged DNA fragments of fetal liver and enhancement of the activity of caspase-3 in cytosolic fractions of fetal livers. Histopathological examination of fetal livers of CAN-treated mice showed hepatocytes with vacuolated cytoplasm, karyolysis and karyorrhexis as well as depletion of their glycogen content. In conclusion, maternal exposure to CAN adversely affects mouse fetal livers as evidenced by the induction of oxidative stress, apoptosis and histopathological changes.     

Arsenic-induced cell death in liver and brain of experimental rats

Arsenic is a well established human carcinogen and is ubiquitous in the environment. The present study demonstrates the effect of acute arsenic administration at three different doses in liver and brain of Wistar rats. Sodium arsenite was administered orally at doses of 6.3 mg/kg, 10.5 mg/kg and 12.6 mg/kg of body weight on the basis of a lethal dose 50% (LD50) for 24 hr. After administration of arsenites, liver and brain were analyzed for various parameters of oxidative stress, histopathological changes and caspase-3 activity. Glutathione levels were decreased significantly in the liver at all doses. In liver the following biochemical changes were observed, a significant lipid peroxidation and cytochrome-P450 induction along with significant decrease in catalase and superoxide dismutase was observed at 10.5 mg/kg and 12.6 mg/kg. The activity of glutathione peroxidase was increased significantly at all doses. In brain, no significant change was observed at 6.3 mg/kg. However, a significant increase in lipid peroxidation and glutathione peroxidase activity along with significant decrease in the activity of glutathione, catalase and superoxide dismutase was observed at 10.5 mg/kg and 12.6 mg/kg. The activity of glutathione-S-transferase was decreased significantly in both liver and brain at 10.5 and 12.6 mg/kg. No significant alteration in the activity of glucose-6-phosphate dehydrogenase and glutathione reductase was observed in either liver or brain at any dose. Dose-dependent histopathological changes, observed in both liver and brain are also described. A significant increase in caspase-3 activity was observed at all doses in liver and at 10.5 and 12.6 mg/kg in brain. Sodium arsenite caused DNA cleavage into fragments and manifested as "DNA laddering", a hallmark of apoptosis.