Multiple kinetics of mitochondrial cytochrome c release in drug-induced apoptosis

We investigated cytochrome c release kinetics in response to three apoptosis-inducing agents (tumor necrosis factor-alpha, staurosporine, and valinomycin) in MCF-7/Casp-3 cells stably transfected with enhanced green fluorescent protein (EGFP)-tagged cytochrome c. All three agents induced significant caspase activation in the cultures determined by monitoring the cleavage of fluorigenic caspase substrates in extracts from drug-treated MCF-7/Casp-3 cells, albeit the valinomycin-induced activation was less pronounced. Time-lapse confocal microscopy showed that tumor necrosis factor-alpha and staurosporine caused rapid, one- or multiple-step release of cytochrome c-EGFP from mitochondria. In contrast, valinomycin-induced cytochrome c-EGFP release occurred slowly over several hours. Unlike staurosporine, the valinomycin-induced cytochrome c release was not associated with translocation of the proapoptotic Bax protein to the mitochondria, and was not accompanied by co-release of the proapoptotic Smac protein. Immunoprecipitation experiments revealed that cytochrome c was also released out of the cell into the extracellular space before loss of plasma membrane integrity. Our data indicate the existence of multiple kinetics of cytochrome c release in drug-induced apoptosis.     

Reactive oxygen species and p38 mitogen-activated protein kinase activate Bax to induce mitochondrial cytochrome c release and apoptosis in response to malonate

Malonate, an inhibitor of mitochondrial complex II, is a widely used toxin to study neurodegeneration in Huntington's disease and ischemic stroke. We have shown previously that malonate increased reactive oxygen species (ROS) production in human SH-SY5Y neuroblastoma cells, leading to oxidative stress, cytochrome c release, and apoptotic cell death. Expression of a green fluorescent protein-Bax fusion protein in SH-SY5Y neuroblastoma cells demonstrated a Bax redistribution from the cytosol to mitochondria after 12 to 24 h of malonate treatment that coincided with mitochondrial potential collapse and chromatin condensation. Inhibition of Bax translocation using furosemide, as well as Bax gene deletion, afforded significant protection against malonate-induced apoptosis. Further experiments revealed that malonate induced a prominent increase in the level of activated p38 mitogen-activated protein (MAP) kinase and that treatment with the p38 MAP kinase inhibitor SKF86002 potently blocked malonate-induced Bax translocation and apoptosis. Treatment with vitamin E diminished ROS production, reduced the activation status of p38 MAP kinase, inhibited Bax translocation, and protected against malonate-induced apoptosis. Our data suggest that malonate-induced ROS production and subsequent p38 MAP kinase activation mediates the activation of the pro-apoptotic Bax protein to induce mitochondrial membrane permeabilization and neuronal apoptosis.     

AIF多克隆抗体的制备纯化和鉴定

目的制备抗AIF多克隆抗体。方法用人工合成的AIF抗原肽免疫实验动物制备多克隆抗体并纯化。利用ELISA和Western blot方法检测多克隆抗体的效价和特异性。结果实验获得的血清抗AIF抗体经间接ELISA法检测,效价达到1∶128 000。Western blot检测表明抗体效价高、特异性强。结论实验获得了高效价的特异性的抗AIF抗体,为进一步研究肿瘤的抗凋亡机制提供新的方法。     

Smac/DIABLO对胰腺癌SW1990细胞凋亡及化疗敏感性的影响

目的研究Smac/DIABLO与胰腺癌对TRAIL和吉西他滨化疗敏感性的关系。方法构建Flagpc3.1-Smac外源表达质粒,在SW1990细胞中过表达SMAC,采用实时定量荧光PCR方法检测Smac mRNA水平;应用Western免疫印迹法检测caspase-3、caspase-9和Bcl-2蛋白水平;利用MTT法检测转染Flag-pc3.1-Smac和TRAIL、吉西他滨处理后SW1990细胞增殖能力的改变;利用流式细胞术检测转染Flag-pc3.1-Smac后SW1990细胞的凋亡情况。结果转染Flag-pc3.1-Smac后,SW1990细胞中Smac mRNA和蛋白质表达水平显著高于对照组;MTT试验显示,过表达Flag-pc3.1-Smac的SW1990细胞在1~5 d的培养期中490 nm处吸光值均低于对照组。结论 Smac/DIABLO促进胰腺癌SW1990细胞凋亡,并增加对TRAIL和吉西他滨的化疗敏感性。     

转染Smac对肝癌细胞凋亡的影响

目的探讨过表达Smac基因对人肝癌HepG2细胞凋亡、细胞周期及凋亡相关蛋白Survivin、半胖氨酸蛋白酶-3（Caspase-3）表达的影响。方法从人K562细胞中扩增Smac cDNA,构建含Smac cDNA的真核表达载体pEGFP-C1-Smac,并转染人肝癌细胞HepG2。流式细胞仪检测分析细胞凋亡百分率,凋亡相关蛋白Survivin、Caspase-3的表达及细胞周期。结果转染Smac基因组、转染空载体组、未转染组细胞凋亡率差异有统计学意义（P〈0.05）。转染Smac基因组细胞凋亡率高于未转染组,也高于转染空载体组细胞,差异均有统计学意义（P〈0.05）。转染Smac基因组细胞Survivin的表达显著高于未转染组,也显著高于转染空载体组细胞,差异均有统计学意义（P〈0.05）。未转染组、转染空载体组、转染Smac基因组中Caspase-3蛋白表达差异无统计学意义（P〉0.05）。未转染组、转染空载体组、转染Smac基因组HepG2细胞的G0/G1期、S期、G2/M各期细胞比例差异无统计学意义（P〉0.05）。结论过表达Smac可诱导人肝癌HepG2细胞凋亡率增加,促进细胞凋亡。     

Proteasome inhibitors prevent cisplatin-induced mitochondrial release of apoptosis-inducing factor and markedly ameliorate cisplatin nephrotoxicity

We demonstrate the effect of proteasome inhibitors in mitochondrial release of apoptosis-inducing factor (AIF) in cisplatin-exposed renal tubular epithelial cells (LLC-PK₁ cells) and in a model of cisplatin nephrotoxicity. Immunofluorescence and subcellular fractionation studies revealed cisplatin-induced translocation of AIF from the mitochondria to nucleus. Mcl-1, a pro-survival member of the Bcl-2 family, is rapidly eliminated on exposure of renal cells to cisplatin. Proteasome inhibitors PS-341 and MG-132 blocked cisplatin-induced Mcl-1 depletion and markedly prevented mitochondrial release of AIF. PS-341 and MG132 also blocked cisplatin-induced activation of executioner caspases and apoptosis. These studies suggest that proteasome inhibitors prevent cisplatin-induced caspase-dependent and -independent pathways. Overexpression of Mcl-1 was effective in blocking cisplatin-induced cytochrome c and AIF release from the mitochondria. Downregulation of Mcl-1 by small interfering RNA promoted Bax activation and cytochrome c and AIF release, suggesting that cisplatin-induced Mcl-1 depletion and associated Bax activation are involved in the release of AIF. Expression of AIF protein in the mouse was highest in the kidney compared to the heart, brain, intestine, liver, lung, muscle, and spleen. In an in vivo model of cisplatin nephrotoxicity, proteasome inhibitor MG-132 prevented mitochondrial release of AIF and markedly attenuated acute kidney injury as assessed by renal function and histology. These studies provide evidence for the first time that the proteasome inhibitors prevent cisplatin-induced mitochondrial release of AIF, provide cellular protection, and markedly ameliorate cisplatin-induced acute kidney injury. Thus, AIF is an important therapeutic target in cisplatin nephrotoxicity and cisplatin-induced depletion of Mcl-1 is an important pathway involved in AIF release.     

Down-regulation of ceramide production abrogates ionizing radiation-induced cytochrome c release and apoptosis

Previous work has demonstrated that down-regulation of ceramide production after selection of cells with N-oleoylethanolamine (OE), an inhibitor of ceramidase, results in resistance to DNA damage-induced apoptosis. We report here that acute exposure of WEHI-231 cells (murine B-cell lymphoma) to OE activates neutral sphingomyelinase, induces ceramide production and increases intracellular reactive oxygen species. OE exposure also induces mitochondrial permeability, cytochrome c release, and apoptosis. Cells selected for resistance to OE exhibit little if any change in reactive oxygen species and cytochrome c release when exposed either to OE or to toxic doses of ceramide. Importantly, the OE resistant cells are also resistant to ionizing radiation-induced cytochrome c release and apoptosis. These findings demonstrate that down-regulation of neutral sphingomyelinase activity is associated with decreased DNA-damage-induced apoptosis. In addition, the data suggests that agents that modify extranuclear targets responsible for ceramide production select for cells resistant to ionizing radiation-induced apoptosis through alterations in mitochondrial function.     

Inhibition of mitochondrial permeability transition and release of cytochrome c by anti-apoptotic nucleoside analogues

We have investigated whether nucleoside drugs that induce or protect neurones against apoptosis might directly activate or inhibit mitochondrial permeability transition (mPT) since opening of the mPT pore can promote release of cytochrome c and apoptosis, while its closure can prevent these changes. We found that the pro-apoptotic pyrimidine analogues cytosine beta-D-arabinofuranoside and cytosine beta-D-arabinofuranoside 5'-triphosphate, which activated apoptosis in post-mitotic neurones without incorporation into nuclear DNA, induced rapid calcium-dependent mitochondrial swelling of isolated liver mitochondria in a dose-dependent manner. Induction of up to 50 and 80%, respectively, of maximal swelling induced by high calcium was obtained at 1mM concentrations, which also promoted a 17-fold increase in the release of cytochrome c. Both activities were inhibited by cyclosporine A to unstimulated levels; dCTP had no effect. In contrast, the anti-apoptotic adenine analogues, 3-methyladenine (3-MA) and olomoucine (but not iso-olomoucine), inhibited swelling induced by calcium or phenylarsine oxide in a dose-dependent manner at concentrations that protect neurones from apoptosis. Both compounds also inhibited the release of cytochrome c (by 82%, 20 mM 3-MA and 95%, 0.9 mM olomoucine), similar to the inhibition obtained with cyclosporine A, and 5mM ADP or ATP. Similar inhibitory effects with olomoucine and 3-MA were found in isolated heart mitochondria. These studies identify the mPT as an important target for hitherto untested pro- and anti-apoptotic nucleoside-based drugs and suggest that screening for mPT modulation is an important component in the validation of a drug's mechanism of action.     

Combined exposure to DEET (N,N-diethyl-m-toluamide) and permethrin-induced release of rat brain mitochondrial cytochrome c.

The release of cytochrome c from the mitochondrial intermembrane space can induce apoptosis. The levels of mitochondrial cytochrome c in rat brain following a single dermal dose of 400 mg/kg of DEET, and of 1.3 mg/kg of permethrin, alone or in combination were determined. Rats were sacrificed at a time interval of 0.5, 1, 2, 4, 8, 16, 24, 48, or 72 h after dosing. Brain mitochondria were isolated and the levels of cytochrome c were measured using reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet (UV) detection. Average percentage recovery of cytochrome c spiked with control rat brain mitochondria was 83.2 +/- 8.9%. Limits of detection and quantitation were 1 and 5 ng, respectively. The results showed that a single dermal dose of a combination of DEET and permethrin significantly increased the release of brain mitochondrial cytochrome c starting 24 h after treatment. DEET and permethrin alone did not affect the release of cytochrome c. The results indicate that combined exposure to DEET and permethrin might induce the apoptotic processes in rat brain as seen by the release of cytochrome c.     

Tetrahydrocannabinol-induced apoptosis of cultured cortical neurones is associated with cytochrome c release and caspase-3 activation

Delta(9)-tetrahydrocannabinol (THC), the principal psychoactive component of marijuana, is associated with impaired cognition and altered cortical function. THC transduces its central effects via activation of the G-protein linked cannabinoid receptor CB1. In this study we report that THC induces morphological degenerative changes in cultured cortical neurones, such as membrane blebbing and formation of apoptotic bodies, that are consistent with the apoptotic pathway of cell death. The THC-induced apoptosis was blocked by the CB1 receptor antagonist AM251 and pertussis toxin (PTX), suggesting that this effect of THC involves receptor-mediated activation of the G-protein subtypes G(i) or G(o). THC also promoted translocation of mitochondrial cytochrome c to the cytosol and increased the activity of the cysteine protease caspase-3, in a PTX-sensitive manner. The results from this study suggest that coupling of THC to a PTX-sensitive G-protein promotes cytochrome c release, caspase-3 activation and subsequent degeneration of cultured cortical neurones. This apoptotic pathway may underlie the compromised neuronal function that is associated with marijuana usage.     

The course of uncarinic acid E-induced apoptosis of HepG2 cells from damage to DNA and p53 activation to mitochondrial release of cytochrome c

Uncarinic acid E, an active component isolated from Gelsemium elegans BENTH, has been reported to exhibit antitumor effects, but little is known about its molecular mechanisms of action. In this study, the growth-inhibitory activity of uncarinic acid E for HepG2 cells is in time- and dose-dependent manner. HepG2 cells treated with uncarinic acid E exhibited several typical characteristics of apoptosis through photomicroscopical observation, DNA agarose gel electrophoresis. The inhibitory effect of uncarinic acid E on HepG2 cells was partially reversed by the inhibitors of pan-caspase, caspase-3 and caspase-6. The protein expression ratio of Bcl-xL/Bax and Bcl-2/Bax was down-regulated and uncarinic acid E-induced apoptosis involves the initial phase mediated by the balance among Bcl-xL, Bcl-2 and Bax proteins, resulting in cytochrome c release from the mitochondria. Uncarinic acid E significantly increased the expression of p53 proteins indicates that p53 plays a pivotal role in the initiation phase of uncarinic acid E-induced HepG2 cell apoptosis. The phoshatidylinositol 3-kinase (PI3-K) family inhibitor wortmanin and the MEK inhibitor (PD98059) rescued the viability loss induced by uncarinic acid E through the expression of p53. Taken together, uncarinic acid E induces apoptosis in HepG2 cells via accumulation of p53, alters the Bax/Bcl-2 ratio, and activates caspases, resulting in cytochrome c release from the mitochondria.     

Cytotoxicity and apoptosis of benzoquinones: redox cycling,cytochrome c release,and BAD protein expression

The metabolic activation of a variety of quinone-based anticancer agents occurs, in part, as a result of the bioreductive activation by the flavoprotein NAD(P)H:quinone-acceptor oxidoreductase (NQO1) (EC 1.6.99.2). Using the COMPARE algorithm (http://dtp.nci.nih.gov), a significant statistical correlation has been found in the NCI in vitro anticancer drug screen between high endogenous expression of the pro-apoptotic protein BAD, NQO1 enzymatic activity, and the cytotoxicity of certain antitumor quinones. Two statistically correlated groups of quinones can be discerned: positive-correlated compounds, which are more active in cell lines expressing high baseline levels of BAD protein and NQO1 activity (e.g. the MCF-7 breast carcinoma), and negative-correlated compounds, which are more active in cell lines with undetectable levels of BAD and NQO1 activity (e.g. the HL-60 myeloid leukemia). In the present study, the relationship between quinone structure, redox cycling, and cytotoxicity in the MCF-7 and HL-60 cell lines was investigated. A good biological correlation exists between cytotoxicity and NQO1 activity, BAD protein levels and apoptosis, but not always between cytotoxicity and intracellular reactive oxygen species levels. The overall markedly increased cytotoxicity of the aziridinylbenzoquinone compounds used in this study is accompanied by apoptosis, which occurs mostly through a cytochrome c-independent pathway.     

Involvement of mitochondria in acetaminophen-induced apoptosis and hepatic injury: roles of cytochrome c,Bax, Bid,and caspases

The role of apoptosis in acetaminophen (AAP)-induced hepatic injury was investigated. Six hours after AAP administration to BALB/c mice, a significant loss of hepatic mitochondrial cytochrome c was observed that was similar in extent to the loss observed after in vivo activation of CD95 by antibody treatment. AAP-induced loss of mitochondrial cytochrome c coincided with the appearance in the cytosol of a fragment corresponding to truncated Bid (tBid). At the same time, tBid became detectable in the mitochondrial fraction, and concomitantly, Bax was found translocated to mitochondria. However, AAP failed to activate the execution caspases 3 and 7 as evidenced by a lack of procaspase processing and the absence of an increase in caspase-3-like activity. In contrast, the administration of the pan-inhibitor of caspases, benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone (but not its analogue benzyloxycarbonyl-Phe-Ala-fluoromethylketone) prevented the development of liver injury by AAP and the appearance of apoptotic parenchymal cells. This correlated with the inhibition of the processing of Bid to tBid. The caspase inhibitor failed to prevent both the redistribution of Bax to the mitochondria and the loss of cytochrome c. In conclusion, apoptosis is an important causal event in the initiation of the hepatic injury inflicted by AAP. However, as suggested by the lack of activation of the main execution caspases, apoptosis is not properly executed and degenerates into necrosis.     

2,3,5-tris(Glutathion-S-yl)hydroquinone (TGHQ)-mediated apoptosis of human promyelocytic leukemia cells is preceded by mitochondrial cytochrome c release in the absence of a decrease in the mitochondrial membrane potential.

2,3,5-tris(Glutathion-S-yl)hydroquinone (TGHQ), a metabolite of benzene, induces apoptosis in human promyelocytic leukemia (HL-60) cells. However, the mechanisms by which TGHQ induces apoptosis are unclear, and they were the focus of the present investigation. TGHQ stimulated the rapid formation (30 min) of reactive oxygen species (ROS) in HL-60 cells, and co-treatment with catalase or the antioxidant N-acetylcysteine (NAC) completely blocked TGHQ-induced apoptosis, implicating a causative role for ROS in HL-60 cell death. Western blot analysis revealed the complete disappearance of pro-caspase 9 between 1 and 2 hours after exposure of HL-60 cells to TGHQ, concomitant with the appearance of cleaved caspase 9 and increases in caspase 9 activity. The appearance of two cleaved forms of caspase 3 occurred subsequent to increases in caspase 9 activity. Levels of the anti-apoptotic Bcl-2 protein remained constant during TGHQ-induced apoptosis of HL-60 cells, but Bcl-2 S70 phosphorylation decreased. In contrast, changes in the subcellular localization of the pro-apoptotic molecule Bax were observed, with a rapid (15-60 min) increase in the ratio of cytosolic to mitochondrial Bax. Cytochrome c release from mitochondria to the cytosol occurred after Bax translocation and the dephosphorylation of pS70 Bcl-2. However the mitochondrial inner transmembrane potential (deltapsi(m)) was maintained, even after cytochrome c was released from the mitochondria. Cyclosporin A, an inhibitor of the mitochondrial membrane permeability transition pore (PTP), did not completely rescue HL-60 cells from apoptosis. Taken together, we conclude that TGHQ facilitates ROS production, alters the post-translational modification of Bcl-2 and subcellular localization of Bax, culminating in the release of cytochrome c and caspase activation.     

Induction of apoptosis in human hepatoblastoma cells by tetrandrine via caspase-dependent Bid cleavage and cytochrome c release

Tetrandrine, a bis-benzylisoquinoline alkaloid from the root of Stephania tetrandra, induces apoptosis in human T-cell lines, lung carcinoma and hepatoblastoma cells. However, the mechanisms by which tetrandrine inhibits tumor cell growth are poorly understood. The purpose of the present study was to investigate the intracellular signaling mechanism of tetrandrine-induced apoptosis in HepG2 cells. The induction of apoptosis was determined by morphological analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. Treatment of cells with tetrandrine caused the upregulation of p53, downregulation of Bcl-X(L), cleavage of Bid and Bax, and release of cytochrome c, which were accompanied by activation of caspases 9, 3 and 8. The activation of caspases 9 and 3 preceded that of caspase 8. A broad-spectrum caspase inhibitor and a caspase 8-specific inhibitor completely blocked tetrandrine-induced Bid processing, cytochrome c release, activation of caspase 3, and cell death. These findings and data showing the early release of cytochrome c, cleavage of Bid and downregulation of Bcl-X(L) suggest that the mitochondrial pathway is primarily involved in tetrandrine-induced apoptosis. The activation of caspase 8 after early caspases 9 and 3 activation might act as an amplification loop for activation of upstream signals such as Bid cleavage or cytochrome c release. These data suggest that tetrandrine may constitute a plausible therapeutic for hepatocellular carcinoma.     

Acetoxycycloheximide (E-73) rapidly induces apoptosis mediated by the release of cytochrome c via activation of c-Jun N-terminal kinase

Cycloheximide (CHX) is an inhibitor of protein synthesis and commonly used to modulate death receptor-mediated apoptosis or to induce apoptosis in a number of normal and transformed cells. In this study we show that a close structural derivative of CHX, acetoxycycloheximide (E-73) induced rapid processing of procaspases and subsequent apoptosis with much higher efficacy than CHX in human leukemia Jurkat T cells. E-73 induced the release of cytochrome c from mitochondria even in the presence of the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone. The Bcl-2 family protein Bcl-x(L) suppressed cytochrome c release as well as processing of procaspases-3, -8, and -9 in E-73-treated cells. In Jurkat T cells transfected with the caspase-8 modulator FLIP(L), E-73 still induced activation of procaspase-3 and subsequent apoptosis, suggesting that the caspase-8 activity is dispensable for apoptosis. In contrast to CHX, E-73 drastically induced activation of extracellular signal-regulated kinase, c-Jun N-terminal kinase (JNK), and p38 MAP kinase. Inhibitory profiles of small-molecular kinase inhibitors revealed that JNK activation was critical for induction of cytochrome c release in E-73-induced apoptosis. Thus, our present results demonstrate that E-73, unlike CHX, induces strong activation of the JNK pathway and triggers rapid apoptosis mediated by the release of cytochrome c.     

Protection by ascorbic acid from denaturation and release of cytochrome c, alteration of mitochondrial membrane potential and activation of multiple caspases induced by H(2)O(2), in human leukemia cells

We investigated peroxide and superoxide accumulation, cytochrome c nature and release from mitochondria, as well as caspase activation during exposure of HL-60 cells to H(2)O(2) and the protective effect of ascorbic acid. Exposure of the cells to 100 microM H(2)O(2) resulted in intracellular accumulation of peroxides, denaturation of cytochrome c that was identified in the mitochondria and cytosol, release of native cytochrome c to the cytosol and fall in mitochondrial membrane potential (DeltaPsi(m)). Loading of cells with ascorbic acid before exposure to H(2)O(2) resulted in a dose-dependent protective effect against: intracellular accumulation of peroxides, DeltaPsi(m) alteration, cytochrome c denaturation and release. The accumulation of peroxides induced processings and activations of procaspase-8, -9 and -3. Double staining with antiserum which recognizes the p18 subunit of cleaved caspase-3 and with Hoechst had shown that a high percentage of cells exposed to 100 microM H(2)O(2) stained positively with the antibody and showed features of apoptosis. Ascorbic acid loading prevented caspase activation induced by H(2)O(2). We conclude that ascorbic acid protects against activation of apoptotic cascades in HL-60 cells exposed to H(2)O(2) and against apoptosis.     

Catalpol inhibits apoptosis in hydrogen peroxide-induced PC12 cells by preventing cytochrome c release and inactivating of caspase cascade.

In the present study, using a rat pheochromocytoma (PC12) cell line, the effect of catalpol on H2O2-induced apoptosis was studied. The apoptosis in H2O2-induced PC12 cells was accompanied by down-regulation of Bcl-2, up-regulation of Bax, the release of mitochondrial cytochrome c to cytosol and sequential activation of caspase-1 and caspase-3 then leading to cleavage of poly-ADP-ribose polymerase (PARP). Catalpol not only suppressed the down-regulation of Bcl-2, up-regulation of Bax and the release of mitochondrial cytochrome c to cytosol, but also attenuated caspase-3 activation, PARP cleavage, and eventually protected against H2O2-induced apoptosis. Taken together, these results suggest that treatment of PC12 cells with catalpol can block H2O2-induced apoptosis by the regulation of Bcl-2 family members, as well as suppression of cytochrome c release and caspase cascade activation.     

Aroclor 1254 inhibits the mitochondrial permeability transition and release of cytochrome c: a possible mechanism for its in vivo toxicity

The mitochondrial permeability transition (MPT) occurs in several forms of necrotic cell death induced by various insults, including oxidative stress, ischemia/reperfusion injury Ca(2+)-ionophore toxicity, and apoptosis. In fact, the release of an apoptogenic factor such as cytochrome c is often associated with the opening of the transition pore. The present study shows that Aroclor 1254, a mixture of polychlorinated biphenyls that was banned in the U.S. in 1977 but is still present in the environment, inhibits the MPT in a dose-dependent manner in a concentration range of 1 to 25 nmol/mg protein. The compound prevents key phenomena associated with the MPT, including colloid-osmotic swelling, the collapse of membrane potential, nonspecific bidirectional traffic of solutes through the transition pore, and the oxidation of pyridine nucleotides. In contrast, Aroclor 1254 does not inhibit uptake of Ca(2+) or P(i). The effects of Aroclor 1254 are evident both in sucrose-based media and in saline and are observed when the compound is added before the opening of the pore. Aroclor 1254 prevents MPT induction provoked by a variety of agents, including phosphate, menadione, tert-butylhydroperoxide, and atractyloside. Aroclor 1254 also inhibits the specific release of cytochrome c, a correlate of MPT induction. These effects reveal a possible toxicological mechanism of action of this compound. The possibility that its effect on mitochondrial function is linked to its action as a tumor promoter is discussed.     

Bisphenol A induced apoptosis via oxidative stress generation involved Nrf2/HO-1 pathway and mitochondrial dependent pathways in human retinal pigment epithelium (ARPE-19) cells

Bisphenol A (BPA) is an estrogen-like compound, and an environmental hormone, that is commonly used in daily life. Therefore, it may enter the human body through food or direct contact, causing BPA residues in blood and urine. Because most studies focused on the analysis of BPA in reproductive cells or tissues, regarding evidence the effect of BPA on human retinal pigment epithelium (ARPE-19) cells unavailable. Accordingly, the present study explored the cytotoxicity of BPA on ARPE-19 cells. After BPA treatment, the expression of Bcl-XL an antiapoptotic protein, in the mitochondria decreased, and the expression of Bax, a proapoptotic protein increased. Then the mitochondrial membrane potential was affected. BPA changed in mitochondrial membrane potential led to the release of cytochrome C, which activated caspase-9 to promote downstream caspase-3 leading to cytotoxicity. The nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and heme oxygenase 1 (HO-1) pathway play a major role in age-related macular degeneration. Our results showed that expression of HO-1 and Nrf2 suppressed by BPA. Superoxide dismutase and catalase, which Nrf2 downstream antioxidants, were degraded by BPA. AMP-activated kinase (AMPK), which can regulate the phosphorylation of Nrf2, and the phosphorylation of AMPK expression was reduced by BPA. Finally, BPA-induced ROS generation and cytotoxicity were reduced by N-acetyl-l -cysteine. Taken together, these results suggest that BPA induced ARPE-19 cells via oxidative stress, which was associated with down regulated Nrf2/HO-1 pathway, and the mitochondria dependent apoptotic signaling pathway.