氧化应激诱导HepG2肝癌细胞凋亡的研究(英)

目的：直接暴露细胞于活性氧能诱导发生凋亡，本文研究氧化应激诱导HepG2肝癌细胞的死亡及其机制。方法：暴露细胞于2 mmol/L过氧化氢产生氧化应激，用DNA凝胶电泳检测细胞凋亡，用荧光染色法检测细胞线粒体膜电位变化，Western blotting检测细胞浆中细胞色素c变化，fluorometric assay kit检测caspase活性变化。结果：氧化应激作用于HepG2细胞后12 h开始发生凋亡；氧化应激作用后4 h，细胞线粒体膜电位明显下降；胞浆中细胞色素c浓度呈时间依赖性增高；氧化应激作用8 h、12 h后细胞内caspase-3、caspase-9活性分别升高6.7及3.6倍，但caspase-8活性无变化。结论：氧化应激能诱导HepG2肝癌细胞发生凋亡，其途径与线粒体通路及caspase激活有关。     

第三丁基过氧化氢损伤皮层神经元线粒体并诱导凋亡

目的：探讨第三丁基过氧化氢（t-BHP）诱导大鼠皮层神经元凋亡的可能机制。 方法： 体外培养大鼠皮层神经元，MTT法测定细胞存活率，DNA断裂评价细胞凋亡，流式细胞术测定线粒体跨膜电位（ΔΨm），分光光度计法测定细胞内谷胱甘肽（GSH）浓度，Western blot法测定Bcl-2和Bax蛋白和胞浆细胞色素c以及活化型半胱氨酸天冬氨酸蛋白酶3（caspase-3）和多聚（ADP-核糖）聚合酶（PARP）水平。 结果： tBHP（25－400 μmol/L）可明显抑制皮层神经元的生长，引起ΔΨm下降和线粒体内细胞色素c向胞浆释放，同时细胞内GSH浓度以及Bcl-2蛋白水平下降，Bax蛋白水平增加，caspase-3和PARP得以激活并最终导致神经细胞凋亡。 结论： tBHP引起的氧化应激可通过损伤线粒体诱导皮层神经元凋亡。     

知母皂苷AⅢ对急性B淋巴细胞白血病细胞的抑制作用及其号转导机制

目的研究知母皂苷AⅢ(TA-Ⅲ)对急性B淋巴细胞白血病(B-ALL)细胞的影响和潜在的作用机制。方法采用不同浓度的TA-Ⅲ对B-ALL细胞进行处理,MTT法检测细胞活力,流式细胞术检测细胞凋亡。Western blot方法检测线粒体途径凋亡信号通路相关蛋白caspase-8、BID、Bax、Bcl-2、细胞色素c、caspase-9、caspase-3、PARP等表达水平和裂解激活水平,并检测PI3K/AKT信号通路相关蛋白AKT,GSK3、FOXO1的磷酸化水平。结果 TA-Ⅲ以剂量依赖方式显著抑制B-ALL细胞活力,促进其凋亡;TA-Ⅲ能剂量依赖性的使caspase-8,caspase-9,caspase-3,PARP裂解激活,上调Bax/Bcl-2比值,并升高细胞质中的细胞色素c表达水平。而caspase抑制剂能显著的抑制这种作用。此外,不同浓度的TA-Ⅲ能提高AKT,GSK3、FOXO1的磷酸化水平。结论 TA-Ⅲ能够抑制B-ALL细胞活力,可能是通过线粒体途径激活caspase级联反应而诱导B-ALL细胞凋亡来完成。这些作用可能与其抑制PI3k/AKT信号通路的激活有关。     

线粒体与细胞凋亡机制

细胞凋亡是生理性的细胞死亡过程,受到多种基因的精确调节。一类被统称为caspase的半胱氨酸蛋白酶是细胞凋亡程序的执行者,它们被激活后作用于细胞内的一些蛋白质,引起细胞凋亡。线粒体中含有许多凋亡相关因子,在凋亡信号转导中起着重要作用。细胞受到凋亡刺激 后,细胞色素c、AIF、caspase-9等凋亡相关因子从线粒体中释放出来。细胞色素c通过和Apaf-1、caspase-9相互作用,激活caspase-9,caspase-9激活下游的caspase蛋白酶。Bcl-2家族成员在细胞凋亡中起着重要的调控作用。     

雷公藤红素诱导多发性骨髓瘤H929细胞凋亡

目的:研究雷公藤红素对人多发性骨髓瘤H929细胞凋亡的诱导作用及分子机制。方法:体外培养H929细胞,加入不同浓度(0.5、1、5和10 mg/L)的雷公藤红素处理细胞,CCK8法分析药物对细胞活力的抑制率;annexin V-PE/7-AAD双染法检测雷公藤红素对H929细胞凋亡的影响;流式细胞术分析雷公藤红素处理的H929细胞中线粒体膜电位水平;彗星电泳实验分析雷公藤红素对细胞DNA损伤的诱导作用;Western blot分析不同浓度的雷公藤红素对H929细胞中凋亡相关分子P53、XIAP、cleaved PARP-1和cleaved caspase-3的蛋白水平以及线粒体细胞色素C释放的影响。结果:不同浓度雷公藤红素处理H929细胞后明显抑制细胞的活力,并呈浓度依赖和时间依赖性。雷公藤红素呈浓度依赖地诱导H929细胞凋亡,并且降低线粒体膜电位水平(P<0.05)。彗星电泳实验结果显示雷公藤红素可以诱导H929细胞的DNA损伤。Western blot实验结果显示雷公藤红素处理的H929细胞中,促凋亡蛋白P53、cleaved PARP-1和cleaved caspase-3的蛋白水平明显升高,而抗凋亡蛋白XIAP的表达水平明显降低(P<0.05)。雷公藤红素可以促进线粒体中细胞色素C的释放,并依赖于caspase-9而激活caspase-3。结论:雷公藤红素对多发性骨髓瘤H929细胞具有凋亡诱导作用,其机制可能与诱导DNA损伤、激活线粒体凋亡途径有关。     

抗人DR5抗体mDRA- 6细胞毒作用机制分析

目的:探讨鼠抗人DR5单克隆抗体(mAb)mDRA-6对Jurkat细胞的细胞毒作用及其机制。方法:以流式细胞术测定mAbmDRA-6对Jurkat细胞的细胞毒作用和细胞凋亡作用,以及caspase8、9的抑制剂对mAbmDRA-6诱导的Jurkat细胞凋亡的影响。在荧光显微镜下,观察mAbmDRA-6对Jurkat细胞形态的影响。以琼脂糖凝胶电泳检测Jurkat细胞中的DNA片段化。结果:mAbmDRA-6对Jurkat细胞具有显著的细胞毒作用,并呈剂量和时间依赖性。经mAbmDRA-6处理后,Jurkat细胞可出现典型的细胞凋亡的形态特征:细胞膜皱缩,出泡,染色质浓缩,形成凋亡小体等。经mAbmDRA-6处理后,Jurkat细胞膜表面高表达丝氨酸磷脂,并可导致Jurkat细胞中的DNA片段化。caspase8的抑制剂可明显抑制mAbmDRA-6诱导的Jurkat细胞凋亡,caspase9的抑制剂的影响很小。结论:mAbmDRA-6可通过死亡受体信号传导途径诱导Jurakt细胞凋亡,对Jurkat细胞产生细胞毒作用,其在以TRAIL/DR5系统进行的肿瘤治疗和探讨DR5功能结构域方面具有广阔的应用前景。     

TRAIL诱导Hela细胞凋亡的线粒体信号通路

 目的 探讨 TRAIL 诱导人宫颈癌 Hela 细胞凋亡的线粒体通路。 方法 琼脂糖凝胶电泳判断细胞凋亡;激光共聚焦、Western blot、荧光免疫和 caspase-3 活性检测测定细胞线粒体膜电位 (∆Ψm) 、Bcl-2 蛋白、细胞色素 c (Cyt c) 和凋亡诱导因子 (AIF) 蛋白在细胞中的定位以及 caspase-3 活性。结果 TRAIL 能诱导 Hela 细胞凋亡,有凋亡细胞特有的 DNA 梯状条带。同时,TRAIL具有时间依赖性致 ∆Ψm 和 Bcl-2 蛋白含量明显下降,线粒体 Cyt c 蛋白释放,AIF 蛋白向细胞质、细胞核转移,caspase-3 活性增强。结论 TRAIL 诱导 Hela 细胞凋亡途径之一是通过线粒体信号通路进行的。     

抗人DR5单克隆抗体诱导Jurkat和U937细胞凋亡的线粒体信号通路

目的:探讨抗人死亡受体5(Death receptor 5,DR5)单克隆抗体mDRA-6诱导Jurkat和U937细胞凋亡的线粒体信号通路。方法:MTT法检测mDRA-6对Jurkat和U937细胞生长增殖的影响,以及caspase-9、3抑制剂对mDRA-6抑制Jur-kat和U937细胞生长增殖的影响;琼脂糖凝胶电泳检测Jurkat和U937细胞DNA片段化降解;Western blot检测mDRA-6对Jurkat和U937细胞bax、bcl-2、bcl-xl、Cyt c及caspase-9、3的激活改变。结果:mDRA-6呈时间、浓度依赖性地抑制Jurkat和U937细胞的生长增殖,10 mg/L的mDRA-6作用6、8和10小时,Jurkat细胞增殖抑制率分别达59.38%、72.56%和76.28%,U937细胞增殖抑制率分别达38.67%、47.54%和50.59%。琼脂糖凝胶电泳显示,10 mg/L的mDRA-6作用6小时,Jurkat和U937细胞均呈现凋亡细胞特有的DNA梯形条带;Western blot检测结果发现,随着mDRA-6作用时间延长,Jurkat和U937细胞内促凋亡分子bax增多,抗凋亡分子bcl-2及bcl-xl减少,Cyt c释放明显增多,同时caspase-9、caspase-3也显示明显的激活表现。预先使用caspase-9抑制剂孵育细胞1小时,mDRA-6所致Jurkat和U937细胞生长抑制率分别降低了24.36%(t=5.44,P﹤0.01)和20.82%(t=4.29,P﹤0.01),mDRA-6所致Jurkat和U937细胞凋亡率分别降低了32.89%和23.97%。结论:线粒体信号通路激活是抗人死亡受体5(Death receptor 5,DR5)单克隆抗体mDRA-6诱导Jurkat和U937细胞凋亡的途径之一。     

创伤弧菌诱导树突状细胞凋亡的过程观察

目的 实验观察创伤弧菌(Vibrio vulnificus,Vv)诱导鼠树突状细胞(dendritic cell,DC)凋亡的过程.方法 建立小鼠树突状细胞(DC2.4株)与创伤弧菌(Vv1.1758株)混合培养模型,DAPI荧光染色分析细胞凋亡的形态特征,DNA Ladder检测凋亡细胞的DNA片段化水平分析,Annexin V FITC/PI染色分析DC2.4细胞凋亡率,分光光度法测定caspase-3、caspase-8活性,JC-1荧光标记检测线粒体膜电位(△Ψm)变化.结果 Vv1.1758株与DC2.4细胞混合培养4h时DAPI荧光染色出现典型的凋亡特征——染色质浓缩及边缘化；DNA琼脂糖凝胶电泳出现凋亡条带；2、4、6h细胞凋亡率分别为(37.8±9.8)％、(54.3±12.7)％和(68.2±14.6)％；1、2、4h线粒体膜电位(△ψm)分别下降了7.1％、16.1％与46.7％；caspase-8活性在1.5h增高,2h达高峰(2.48±0.19) U/μg,而caspase-3活性于3h开始增高,4h达高峰(1.91±0.16) U/μg.结论 创伤弧菌诱导树突状细胞可通过线粒体膜电位下降及激活caspase-8启动子两条途径,最终活化效应因子caspase-3,促使细胞凋亡发生.     

13-甲基十四烷酸诱导膀胱癌细胞株T24 凋亡的机制研究

目的： 研究13-甲基十四烷酸诱导膀胱癌T24细胞凋亡的作用,探讨其可能的机制。方法：将不同浓度的13-甲基十四烷酸作用于膀胱癌T24细胞,用MTT法检测细胞增殖, 流式细胞仪（FCM）检测细胞周期,TUNEL法检测细胞凋亡指数,蛋白免疫印迹法分析参与凋亡的蛋白。结果：药物处理2 h后p38、JNK磷酸化增强,AKT磷酸化减弱,8 h后线粒体中细胞色素C释放,FADD及磷酸化FADD没有明显变化,caspase酶底物 PARP、lamin B、RB在12 h出现明显的裂解片段,且药物诱导T24细胞凋亡的过程有时间浓度依赖效应。结论：在13-MTD诱导T24细胞凋亡的过程中p38MAPK和JNK/SAPK信号通路被激活,PI3K/AKT信号通路被抑制,进而激活线粒体凋亡途径,使线粒体内的细胞色素C释放到胞浆,激活caspase酶,裂解相应的凋亡底物lamin B、Rb、PARP,促进T24细胞凋亡。     

Thiol oxidation enforces phosphatidylserine externalization in apoptosis-sensitive and -resistant cells through a deltapsim/cytochrome C release-dependent mechanism

Previous studies have shown that unlike most apoptotic cells, Raji cells do not externalize phosphatidylserine (PS) upon apoptosis. Here we show that Raji cells are resistant to intrinsic apoptogenic agents, but sensitive to extrinsically triggered Fas-induced apoptosis. Treatment of intrinsic apoptosis-competent Jurkat cells with vitamin E implicated reactive oxygen species in intrinsic apoptosis because, like Raji cells, they became resistant to actinomycin D- but not Fas-triggered apoptosis. Oxidation of sulfhydryls in both cell types with N-ethylmaleimide resulted in rapid disruption of the mitochondrial membrane potential, release of cytochrome c from the mitochondria to the cytoplasm, and externalization of PS by a mechanism that was not inhibited by the pan caspase inhibiter zVAD-fmk. These results suggest that although cell death and PS externalization are both cytochrome c-dependent, they are distinct and separable processes.     

The Role of Heat Shock Protein 90 in the Regulation of Tumor Cell Apoptosis

Programmed death of Jurkat tumor cells was studied under conditions of culturing with 17-AAG selective inhibitor of heat shock protein with a molecular weight of 90 kDa and etoposide. Apoptosis realization was evaluated by fluorescent microscopy with FITC-labeled annexin V and propidium iodide. Activity of caspase-3 was evaluated spectrophotometrically. Inhibition of heat shock protein with a molecular weight of 90 kDa activated the apoptotic program in Jurkat tumor cells and etoposide-induced apoptosis. The heat shock protein with a molecular weight of 90 kDa acted as apoptosis inhibitor in tumor cells.     

Monochloramine enhances Fas (APO-1/CD95)-induced apoptosis in Jurkat T cells

Monochloramine derivatives are physiological oxidants produced by activated neutrophils. We report the effects of chemically prepared monochloramine (NH2Cl) on Fas-induced apoptosis in Jurkat T cells. When the cells were pretreated with NH2Cl (20-70 microM), subsequent addition of apoptosis-inducing anti-Fas antibody resulted in a synergistic enhancement of apoptosis. Treatment of NH2Cl (50-70 microM) alone resulted in a slight but definite apoptosis. Caspase activities, as measured by DEVD and IETD cleavage activities, were also elevated synergistically by NH2Cl + anti-Fas antibody stimulation. Moreover, a broad caspase inhibitor, Z-VAD-fmk, almost completely inhibited the apoptosis induced by NH2Cl and/or anti-Fas antibody. Fas expression on the Jurkat cell surface was not affected by the NH2Cl treatment. After 3 h of NH2Cl treatment, when the apoptosis was beginning to increase, the cells showed cytochrome c release from mitochondria, proteolytic activation of caspase 9, and poly (ADP-ribose) polymerase cleavage, regardless of Fas stimulation. Z-VAD-fmk almost completely inhibited this poly (ADP-ribose) polymerase cleavage, but not cytochrome c release. By contrast, Fas stimulation alone resulted in neither cytochrome c release nor caspase 9 activation at 3 h, and the increase in the DEVD cleavage activity and apoptosis became evident at later time points. These results suggested that NH2Cl enhanced Fas-induced apoptosis through the cytochrome c release and caspase 9 activation at the early stage of apoptosis. Chloramines derived from acute inflammation may modify immune reactions, such as cell-mediated cytotoxicity and some autoimmune diseases, by the enhancement of Fas-induced apoptosis.     

Release of DNA from dead and dying lymphocyte and monocyte cell lines in vitro

DNA is a nuclear macromolecule that circulates in the blood where its levels can reflect the activity of inflammatory and malignant diseases. While dead and dying cells have usually been considered the source of blood DNA, the mechanisms for its release during apoptosis and necrosis are not well defined. To elucidate DNA release, an in vitro model system was used, assessing DNA in the media of living, apoptotic or necrotic Jurkat and U937 cells. Apoptosis was induced by etoposide, camptothecin or staurosporine, while necrosis was induced by heating at 56 degrees C. DNA release was measured by fluorometry with the dye PicoGreen while the extent of death was measured by fluorescence-activated cell sorter analysis with propidium iodide and annexin. Apoptotic Jurkat cells released significantly more DNA in the media than untreated cells while necrotic cells did not show significant DNA release. U937 cells showed similar findings. Pretreatment of Jurkat cells with z-VAD-fmk, a caspase inhibitor, reduced both apoptosis and DNA release. By gel electrophoresis, extracellular DNA from apoptotic cells showed laddering with low molecular weight fragments. These studies suggest that extracellular release of DNA is a consequence of apoptosis and may account for some of the DNA in the blood.     

Possible contribution of apoptosis-inducing factor (AIF) and reactive oxygen species (ROS) to UVB-induced caspase-independent cell death in the T cell line Jurkat

We analyzed the mechanism of UVB-induced cell death using the Jurkat T cell line. Apoptosis was assessed by measuring phosphatidylserine (PS) externalization, caspase activity, the decrease in mitochondrial membrane potential (Delta Psi m), nucleosomal DNA fragmentation, and morphological changes such as chromatin condensation. The mitochondrio-nuclear translocation of apoptosis-inducing factor (AIF) was evaluated by confocal laser microscopy. The cell death pattern of UVB-irradiated cells was similar to the Fas-induced cell death pattern. However, zVAD-fmk inhibited the nucleosomal fragmentation of DNA but not the externalization of PS, decrease in Delta Psi m, or mitochondrio-nuclear translocation of AIF. N-acetyl L-cysteine significantly inhibited the translocation of AIF induced by UVB. These results suggested that caspase-dependent and -independent pathways were involved in UVB-induced cell death in Jurkat cells, and the mitochondrio-nuclear translocation of AIF was associated with the latter pathway. In addition, reactive oxygen species generated by UVB might be involved in inducing the mitochondrio-nuclear translocation of AIF.     

Mechanisms Underlying Production And Externalization of Oxidized Phosphatidylserine in Apoptosis: Involvement of Mitochondria

The present study was performed by using selective inhibitors of caspase-8 and caspase-3 functioning upstream and downstream from mitochondria, respectively to determine whether mitochondria are involved in the mechanisms underlying production and externalization of oxidized phosphatidylserine (PSox) during Fas-mediated apoptosis. Treatment with anti-Fas antibody induced caspase-3 activation, chromatin condensation, release of cytochrome c (cyt c) from mitochondria into the cytosol as well as production of PSox and its exposure to the cell surface in Jurkat cells. Inhibition of caspase-8 by pretreatment with Z-IETD-FMK, a membrane permeable selective caspase-8 inhibitor reduced mitochondrial cyt c release, the amount of PSox not only within but also on the surface of Jurkat cells, caspase-3 activation, and apoptotic cell number after treatment with anti-Fas antibody. In contrast, Z-DEVD-FMK, a membrane permeable selective caspase-3 inhibitor was unable to inhibit cyt c release, and the amount of PSox both within and on the surface of the cells after anti-Fas antibody, although it suppressed caspase-3 activation and apoptosis. Thus, these results strongly suggest that mitochondria play an important role in production of PSox and subsequent its externalization during apoptosis.     

Disruption of the cytoskeleton after apoptosis induction with autoantibodies

F-actin cleavage was studied in PBMC after treatment with anti-dsDNA antibodies. Significant changes in F-actin disruption detected by decrease of FITC-phalloidin staining occurred after apoptosis induction with anti-dsDNA antibodies (p < 0.006). Despite of similar F-actin disruption, the switch of phosphatidylserine (PS) to the outer leaflet of the cell membrane as detected by annexin V binding was lower after anti-dsDNA antibody than without antibody treatment (58.4 +/- 11.0% vs. 81.9 +/- 7.7%). F-actin disruption was accompanied by activation of caspase 3 within the cytoplasm (r = -0.92599; p < 8.87446 x 10-(10)) under both conditions with and without autoantibodies. These findings indicate that anti-dsDNA antibody-induced apoptosis is more marked within the cell than upon the cell surface. The diminished externalization of PS might result in a decreased phagocytosis. Thereby, the reduced clearance of apoptotic cells could induce autoantibody production possibly against epitopes which arise due to the apoptotic disruption of cells.     

Molecular glass wool filtration as a new tool for sperm preparation

BACKGROUND: Magnetic-activated cell sorting (MACS) using annexin V-conjugated microbeads in a liquid phase eliminates apoptotic spermatozoa based on the externalization of phosphatidylserine (EPS) residues. The procedure allows the enrichment of a sperm population free of apoptosis markers, giving higher fertilization potential. Our aim was to determine if the annexin V binding principle can be transferred onto a glass wool filter system in order to produce a solid phase filter. METHODS: Semen samples (n = 42) were subjected to a molecular glass wool filter system using glass surfaces coated with annexin V and compared with aliquots separated by conventional glass wool, as well as with annexin V-MACS. The extent of apoptosis was assessed by measuring levels of activated caspase 3 using fluorescein-labelled inhibitors of caspase, alterations in mitochondrial membrane potential (MMP) using a lipophilic cationic dye, and EPS using a fluorescein isothiocyanate-coupled monoclonal antibody. RESULTS: Annexin V-negative sperm filtered out by the newly developed molecular glass wool filtration (GWF) system displayed superior quality in terms of high MMP integrity, as well as, to a small extent, caspase 3 activation and EPS. CONCLUSIONS: The effect of traditional GWF can be further improved by combination with annexin V binding. This newly developed solid phase molecular filter system has been proven to enrich spermatozoa free of apoptosis markers to the same extent as the annexin V magnetic separation technique. The selection of spermatozoa free of apoptosis markers by molecular glass wool filters may enhance the results of IVF.     

喜树碱诱导Jurkat细胞线粒体膜电势和线粒体质量变化的研究

目的： 研究喜树碱（camptothecin，CPT）诱导Jurkat细胞凋亡过程中线粒体膜电势和线粒体质量的变化。 方法： 用喜树碱处理Jurkat细胞，利用Annexin V-FITC/PI双染流式细胞术研究细胞早期凋亡, PI染色流式细胞术测细胞周期, Annexin V-PE/DiOC6(3) 双染流式细胞术检测线粒体膜电势（△ψm），NAO染色流式细胞术检测线粒体质量。 结果： 在10 μmol·L-1 CPT诱导下，6 h 时Jurkat 细胞早期凋亡的细胞比率（22.59±1.04）%显著高于对照组（3.93±0.73）%（P<0.01）。CPT组坏死比率（2.48±0.53）%与对照组（2.78±0.63）%无显著差异（P>0.05）；并可使细胞出现明显的凋亡峰。晚期凋亡的细胞比率为（13.58±0.97）%显著高于对照组（3.18±0.51）%（P<0.01），CPT组G0/G1期细胞比率（48.14±0.96）% ，明显高于对照组（44.09±0.43）%（P<0.01）。CPT组线粒体发生明显去极化现象，AnnexinV+DiOC6(3)-的细胞比率为（19.47±0.69）%，而对照组比率为（4.21±0.40）%，差异显著（P<0.01）。同时，CPT组线粒体质量显著低于对照组：CPT组NAO+细胞比率为（74.77±1.66）%，对照组为（92.24±1.41）%（P<0.01）。 结论： CPT诱导Jurkat细胞凋亡过程中线粒体去极化作用增强并且线粒体质量下降，表明该凋亡过程与线粒体途径密切相关。     

Phosphatidylserine externalization in human sperm induced by calcium ionophore A23187: relationship with apoptosis, membrane scrambling and the acrosome reaction

BACKGROUND: Translocation of phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane is a modification of the lipid architecture occurring in sperm. This is one of the earliest signs of apoptosis that can be monitored by the calcium-dependent binding of annexin V. METHODS AND RESULTS: Flow cytometric analysis of annexin V binding was performed. Calcium ionophore A23187 led to a significant increase in the proportion of living sperm with PS exposure: 7.3 3.2% of cells in the untreated ejaculate versus 47.5 5.6% of cells after 1 h of incubation with A23187. Conversely, diminution of mitochondrial membrane potential [DiOC6(3)/propidium iodide (PI) assay], caspase activation [fluorescein isothiocyanate (FITC)-Val-Ala-Asp-fluoromethylketone (VAD-FMK)/PI assay], increased plasma membrane permeability (Yo-Pro-1/PI assay) and increased DNA fragmentation [TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end labelling assay], which are among the main signs of apoptosis, were not observed in sperm, even after 4 h of incubation with A23187. However, A23187 significantly increased the proportion of sperm with plasma membrane scrambling and with a reacted acrosome, as detected with the merocyanine 540 probe (M540) and the monoclonal anti-human CD46-PE antibody respectively. CONCLUSIONS: Our results suggest that PS exposure in human sperm, as induced by A23187, is mainly related to the acrosome reaction rather than to apoptosis.