鱼藤酮诱导线粒体轻度损伤细胞氧化应激时硫氧还蛋白转录水平降低

观察鱼藤酮诱导的线粒体轻度损伤细胞氧化应激时硫氧还蛋白转录水平的变化,探讨细胞氧化损伤的可能机制。通过荧光素发光法检测ATP生成、细胞内活性氧(ROS)水平的变化,流式细胞术检测线粒体膜电位,了解低剂量鱼藤酮对线粒体功能的影响;继而用H2O2诱导细胞氧化损伤,MTT法检测细胞活性,观察正常及线粒体缺陷细胞氧化应激时,胞内硫氧还蛋白(Trx)mRNA水平的变化。结果表明,鱼藤酮以剂量依赖方式抑制线粒体ATP的产生、降低线粒体膜电位,而细胞内ROS水平增高;当线粒体损伤细胞氧化应激时胞内Trx mRNA水平降低,提示鱼藤酮诱导线粒体轻度损伤细胞抗氧化能力降低与Trx转录受到抑制有关。     

Evaluation of mitochondrial function in isolated rat hepatocytes and mitochondria during oxidative stress

The majority of toxic agents act either fully or partially via oxidative stress, the liver, specifically the mitochondria in hepatocytes, being the main target. Maintenance of mitochondrial function is essential for the survival and normal performance of hepatocytes, which have a high energy requirement. Therefore, greater understanding of the role of mitochondria in hepatocytes is of fundamental importance. Mitochondrial function can be analysed in several basic models: hepatocytes cultured in vitro; mitochondria in permeabilised hepatocytes; and isolated mitochondria. The aim of our study was to use all of these approaches to evaluate changes in mitochondria exposed in vitro to a potent non-specific peroxidating agent, tert-butylhydroperoxide (tBHP), which is known to induce oxidative stress. A decrease in the mitochondrial membrane potential (MMP) was observed in cultured hepatocytes treated with tBHP, as illustrated by a significant reduction in Rhodamine 123 accumulation and by a decrease in the fluorescence of the JC-1 molecular probe. Respiratory Complex I in the mitochondria of permeabilised hepatocytes showed high sensitivity to tBHP, as documented by high-resolution respirometry. This could be caused by the oxidation of NADH and NADPH by tBHP, followed by the disruption of mitochondrial calcium homeostasis, leading to the collapse of the MMP. A substantial decrease in the MMP, as determined by tetraphenylphosphonium ion-selective electrode measurements, also confirmed the dramatic impact of tBHP-induced oxidative stress on mitochondria. Swelling was observed in isolated mitochondria exposed to tBHP, which could be prevented by cyclosporin A, which is evidence for the role of mitochondrial permeability transition. Our results demonstrate that all of the above-mentioned models can be used for toxicity assessment, and the data obtained are complementary.     

Apical phosphatidylserine externalization in auditory hair cells

In hair cells of the inner ear, phosphatidylserine (PS), detected with fluorescent annexin V labeling, was rapidly exposed on the external leaflet of apical plasma membranes upon dissection of the organ of Corti. PS externalization was unchanged by caspase inhibition, suggesting that externalization did not portend apoptosis or necrosis. Consistent with that conclusion, mitochondrial membrane potential and hair-cell nuclear structure remained normal during externalization. PS externalization was triggered by forskolin, which raises cAMP, and blocked by inhibitors of adenylyl cyclase. Blocking Na(+) influx by inhibiting the mechanoelectrical transduction channels and P2X ATP channels also inhibited external PS externalization. Diminished PS externalization was also seen in cells exposed to LY 294002, which blocks membrane recycling in hair cells by inhibiting phosphatidylinositol 3-kinase. These results indicate that PS exposure on the external leaflet, presumably requiring vesicular transport, results from elevation of intracellular cAMP, which can be triggered by Na(+) entry into hair cells.     

Apical phosphatidylserine externalization in auditory hair cells

In hair cells of the inner ear, phosphatidylserine (PS), detected with fluorescent annexin V labeling, was rapidly exposed on the external leaflet of apical plasma membranes upon dissection of the organ of Corti. PS externalization was unchanged by caspase inhibition, suggesting that externalization did not portend apoptosis or necrosis. Consistent with that conclusion, mitochondrial membrane potential and hair-cell nuclear structure remained normal during externalization. PS externalization was triggered by forskolin, which raises cAMP, and blocked by inhibitors of adenylyl cyclase. Blocking Na⁺ influx by inhibiting the mechanoelectrical transduction channels and P2X ATP channels also inhibited external PS externalization. Diminished PS externalization was also seen in cells exposed to LY 294002, which blocks membrane recycling in hair cells by inhibiting phosphatidylinositol 3-kinase. These results indicate that PS exposure on the external leaflet, presumably requiring vesicular transport, results from elevation of intracellular cAMP, which can be triggered by Na⁺ entry into hair cells.     

Intracellular localization of Herpes simplex virus type 1 thymidine kinase fused to different fluorescent proteins depends on choice of fluorescent tag

Recognition signals are displayed on the cell surface during apoptosis that enable macrophages to engulf and dispose of the dying cell. A common signal is the externalization of phosphatidylserine (PS). Studies in erythrocytes and platelets have suggested that PS exposure requires the concomitant activation of a phospholipid scramblase (PLS) and inhibition of an adenosine triphosphate (ATP)-dependent aminophospholipid translocase. However, the molecular mechanism underlying PS exposure during apoptosis remains poorly understood. In this study, we provide evidence that expression of PLS is neither necessary nor sufficient for PS exposure during Fas-triggered apoptosis. On the other hand, egress of PS is shown to correlate with a decline in intracellular ATP and inhibition of aminophospholipid translocase activity upon Fas stimulation. Moreover, suppression of intracellular ATP levels by the glucose anti-metabolite, 2-deoxyglucose, alone or in combination with glucose-free medium, potentiates Fas-induced PS exposure in the PLS-expressing Jurkat cell line and enables PLS-defective Raji cells to externalize PS in response to Fas ligation. These studies suggest that intracellular ATP levels can modulate the externalization of PS during apoptosis, and implicate the ATP-dependent aminophospholipid translocase in this process.     

Bax translocation to mitochondria subsequent to a rapid loss of mitochondrial membrane potential

Bax, a pro-apoptotic member of the Bcl-2 family, is a cytosolic protein that inserts into mitochondrial membranes upon induction of cell death. Using the green fluorescent protein fused to Bax (GFP-Bax) to quantitate mitochondrial binding in living cells we have investigated the cause of Bax association with mitochondria and the time course relative to endogenous and induced changes in mitochondrial membrane potential (DeltaPsi(m)). We have found that staurosporine (STS) induces a loss in DeltaPsi(m) before GFP-Bax translocation can be measured. The onset of the DeltaPsi(m) loss is followed by a rapid and complete collapse of DeltaPsi(m) which is followed by Bax association with mitochondria. The mitochondria uncoupler FCCP, in the presence of the F(1)-F(0) ATPase inhibitor oligomycin, can trigger Bax translocation to mitochondria suggesting that when ATP levels are maintained a collapse of DeltaPsi(m) induces Bax translocation. Neither FCCP nor oligomycin alone alters Bax location. Bax association with mitochondria is also triggered by inhibitors of the electron transport chain, antimycin and rotenone, compounds that collapse DeltaPsi(m) without inducing rapid ATP hydrolysis that typically occurs with uncouplers such as FCCP. Taken together, our results suggest that alterations in mitochondrial energization associated with apoptosis can initiate Bax docking to mitochondria.     

Mitochondrial effects with ceramide-induced cardiac apoptosis are different from those of palmitate

The objective of this study was to evaluate whether ceramide, palmitate, and inhibitors of mitochondrial electron transport chain shared similar effects on the mitochondria of intact cardiomyocytes in order to determine the likelihood that ceramide and palmitate utilize similar mitochondrial mechanisms or pathways to apoptosis. In embryonic chick cardiomyocytes, ceramide, 100 microM for 24h, induced a 42.9+/-5.8% increase in cell death assessed by the MTT assay, and a significant (P<0.01) 3.9+/-0.6-fold increase in apoptosis assessed by propidium iodide staining of permeabilized cells. Mitochondrial potential (delta psi (m)), as demonstrated microscopically and by flow cytometry of cardiomyocytes stained with a J-aggregate dye, was markedly and significantly reduced by ceramide, palmitate, and two different inhibitors of the mitochondrial electron transport chain-rotenone and antimycin A. In contrast, the effect on mitochondria as assessed by CMX-Ros oxidation was dramatically different, as palmitate, rotenone, and antimycin A each produced a reduction, while ceramide increased CMX-Ros fluorescence. Further ceramide-induced cardiomyocyte apoptosis and loss of delta psi (m) operated through a cyclosporine-insensitive pathway similar to rotenone and antimycin A but distinct from palmitate which induced apoptosis though a cyclosporine-sensitive mechanism in these cells. These data suggest that ceramide acts on the mitochondria of intact cells through a cyclosporine-insensitive mechanism likely from a combination of actions including production of mitochondrial oxidants. The discordant findings between ceramide and palmitate suggest that palmitate-induced cell death is not primarily mediated by de novo ceramide synthesis.     

Selective toxicity of chrysin on mitochondria isolated from liver of a HCC rat model

Flavonoids are natural compounds that show various biological effects, such as the anti-cancer effect. Chrysin is a flavonoid compound found in honey and propolis. Studies have shown that chrysin has anti-cancer activity due to induction of apoptosis signaling. In the present study, we examined the cytotoxic effect of chrysin against liver mitochondria obtained from the hepatocellular carcinoma (HCC) rat model. Diethylnitrosamine (DEN) and 2-acetylaminofluorene (2-AAF) was used for induction of HCC. Mitochondria were isolated from liver hepatocytes using differential centrifugation. Then, hepatocytes and mitochondria markers related to apoptosis signaling were investigated. Our finding indicated an increase in mitochondrial reactive oxygen species (ROS) generation, collapse in the mitochondrial membrane potential (MMP), swelling in mitochondria, and cytochrome c release (about 1.6 fold) after exposure of mitochondria obtained from the HCC rats group with chrysin (10, 20, and 40 µM) compared to the normal rats group. Furthermore, Chrysin was able to increase caspase-3 activity in the HCC rats group (about 2.4 fold) compared to the normal rats group. According to the results, we proposed that chrysin could be considered as a promising complementary therapeutic candidate for the treatment of HCC, but it requires a further in vivo and clinical studies.     

Cyclosporin A Rescues Thymocytes from Apoptosis Induced by Very Low Concentrations of Thapsigargin: Effects on Mitochondrial Function

Raising intracellular calcium levels can induce apoptosis or programmed cell death in many cells. While early rises in intracellular calcium are not universally associated with apoptotic cell death, calcium clearly plays a key role in many of the biochemical events which occur during apoptosis. In this paper we have determined intracellular calcium rises induced by 2, 10, and 100 nMthapsigargin in mouse thymocytes. These concentrations cause increases in cytosolic calcium of 100–250, 400–600, and >1000 nM,respectively. These rises are sustained for at least 85 min and the ratio between the maximum rise caused by 10 nMcompared to 2 nMthapsigargin is 2.1 ± 0.4 (n= 6). Both 2 and 10 nMthapsigargin cause apoptosis at 24 h as shown by DNA fragmentation and morphology when examined by electron microscopy. Cyclosporin A (CsA) inhibits apoptosis caused by 2 nMthapsigargin but not that caused by 10 nMthapsigargin. Electron microscopy of thymocytes treated with 2 nMthapsigargin at 24 h shows intact mitochondria although with altered morphology. There is no loss of ATP or decrease in the ATP/ADP ratio in these cells over 12 h. Mitochondria in cells treated with 10 nMthapsigargin, however, are swollen by 6 h and many are lost by 24 h. These cells show greatly diminished ATP content by 12 h and a decrease in ATP/ADP ratio. Examination of the effects of PMA, an activator of the plasma membrane calcium ATPase pump, on cells treated with 10 nMthapsigargin suggests that two pools of calcium may be responsible for the differential effects of the two calcium levels in the cells. Probing of the mitochondrial membrane potential (MMP) by rhodamine 123 staining of live cells shows that the collapse of the MMP caused by 10 nMthapsigargin is unaffected by CsA. The MMP is also reduced in cells treated with 2 nMthapsigargin but this is restored by CsA. Cells are also rescued from apoptosis caused by 2 nMthapsigargin by incubation with FK506. This immunosuppressive agent has no effect on the membrane permeability transition induced in isolated mitochondria. These results suggest that very low rises in intracellular calcium in thymocytes cause activation-induced cell death inhibited by CsA and FK506 and are without effect on ATP levels and therefore do not involve irreversible mitochondrial damage. Exceeding these calcium levels by only twofold results in apoptosis accompanied by reduced ATP levels and mitochondrial damage, although apoptotic cell death in this instance is unaffected by the classic inhibitor of mitochondrial permeability transition, CsA.     

Differential regulation of doxorubicin-induced mitochondrial dysfunction and apoptosis by Bcl-2 in mammary adenocarcinoma (MTLn3) cells

Various anticancer drugs cause mitochondrial perturbations in association with apoptosis. Here we investigated the involvement of caspase- and Bcl-2-dependent pathways in doxorubicin-induced mitochondrial perturbations and apoptosis. For this purpose, we set up a novel three-color flow cytometric assay using rhodamine 123, annexin V-allophycocyanin, and propidium iodide to assess the involvement of the mitochondria in apoptosis caused by doxorubicin in the breast cancer cell line MTLn3. Doxorubicin-induced apoptosis was preceded by up-regulation of CD95 and CD95L and a collapse of mitochondrial membrane potential (Deltapsi) occurring prior to phosphatidylserine externalization. This drop in Deltapsi was independent of caspase activity, since benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone did not inhibit it. Benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone also blocked activation of caspase-8, thus excluding an involvement of the death receptor pathway in Deltapsi dissipation. Furthermore, although overexpression of Bcl-2 in MTLn3 cells inhibited apoptosis, dissipation of Deltapsi was still observed. No decrease in Deltapsi was observed in cells undergoing etoposide-induced apoptosis. Immunofluorescent analysis of Deltapsi and cytochrome c localization on a cell-to-cell basis indicates that the collapse of Deltapsi and cytochrome c release are mutually independent in both normal and Bcl-2-overexpressing cells. Together, these data indicate that doxorubicin-induced dissipation of the mitochondrial membrane potential precedes phosphatidylserine externalization and is independent of a caspase- or Bcl-2-controlled checkpoint.     

Cytoprotective and anticancer properties of coenzyme Q versus capsaicin

Coenzyme Q (CoQ) is an essential component of the mitochondrial electron transport chain and serves as an electron donor and acceptor in mitochondrial energy-linked respiration. CoQ1 was shown to prevent ROS formation and cell death in complex 1 inhibited cells. Low concentrations of capsaicin like CoQ1 inhibited ROS formation but CoQ1 was more effective at restoring the mitochondrial membrane potential collapse caused by complex 1 inhibitors such as rotenone. At low concentrations, capsaicin acts as a CoQ mimic by protecting against rotenone induced ROS formation and mitochondrial membrane potential collapse. Lipid peroxidation in isolated rat hepatocytes induced by cumene hydroperoxide and chloroacetaldehyde was also prevented. At higher concentrations, capsaicin and CoQ1 became cytotoxic. Hep G2 cells were more susceptible than hepatocytes. The cytotoxic mechanism for both capsaicin and CoQ1 was shown to involve a collapse of the mitochondrial membrane potential, however, only capsaicin caused ROS formation. The capsaicin side chain was required for capsaicin induced cytotoxicity. The anticancer properties of CoQ1 and capsaicin should prove useful for inducing tumor cell apoptosis.     

低温诱导淡水白鲳尾鳍细胞系早期凋亡

CBT cell line developed from a warm-water fish Colossoma Brachypomum,was used to evaluate the effects of cold stress on fish cells. Cell viability was measured by Thiazolyl blue (MTT) method and cellular ultrastructure was examined under transmission electron microscope. Externalization of phosphatidylserine(PS) and mitochondrial membrane potential(MMP) were monitored with confocal laser scanning microscope. Results showed that cell viability and MMP decreased in a time-dependent manner, and the early events of apoptosis such as chromatin condensation, margination and PS externalization occurred, when the cells were exposed to 10 degrees C. It suggested that low temperature (10 degrees C) could induce apoptosis in Colossoma Brachypomum CBT cells.     

Mitochondrial electron transport inhibitors cause lipid peroxidation-dependent and -independent cell death: protective role of antioxidants

Mitochondrial electron transport inhibitors induced two distinct pathways for acute cell death: lipid peroxidation-dependent and -independent in isolated rat hepatocytes. The toxic effects of mitochondrial complex I and II inhibitors, rotenone (ROT) and thenoyltrifluoroacetone (TTFA), respectively, were dependent on oxidative stress and lipid peroxidation, while cell death induced by inhibitors of complexes III and IV, antimycin A (AA) and cyanide (CN), respectively, was caused by MMP collapse and loss of cellular ATP. Accordingly, cellular and mitochondrial antioxidant depletion or supplementation, in general, resulted in a dramatic potentiation or prevention, respectively, of toxic injury induced by complex I and II inhibitors, with little or no effect on complex III and IV inhibitor-induced toxicity. ROT-induced oxidative stress was prevented by the addition of d-alpha-tocopheryl succinate (TS) but surprisingly TS did not afford hepatocytes protection against TTFA-induced oxidative damage. TS treatment prevented ROT-induced mitochondrial lipid hydroperoxide formation but had no effect on the loss of mitochondrial GSH or cellular ATP, suggesting a mitochondrial lipid peroxidation-mediated mechanism for ROT-induced acute cell death. In contrast, only fructose treatment provided excellent cytoprotection against AA- and CN-induced toxicity. Our findings indicate that complex III and IV inhibitors cause a rapid and severe depletion of cellular ATP content resulting in acute cell death that is dependent on cellular energy impairment but not lipid peroxidation. In contrast, inhibitors of mitochondrial complex I or II moderately deplete cellular ATP levels and thus cause acute cell death via a lipid peroxidation pathway.     

Hormonal regulation of mitochondrial function. Description of a system capable of mimicking several effects of glucagon

Isolated rat liver mitochondria were incubated at 0 degrees C in a medium consisting of 225 mM sucrose, 10 mM KCl, 1 mM EDTA, 10 mM KH2PO4, 5 mM MgCl2 and 10 mM Tris-HCl, pH 7.4 (buffer 1) for 10 min, centrifuged and resuspended in 0.3 M sucrose. This treatment resulted in a stimulation of mitochondrial functions, mimicking several of the effects that follow glucagon treatment of the intact rat or isolated hepatocytes. Both phosphate and potassium are required for this effect; the addition of magnesium serves to enhance it. Mitochondrial respiration is essential for the development of the activated state as the stimulation is blocked by increasing concentrations of rotenone in the incubation. The intramitochondrial ATP/ADP ratio is increased, but when this increase was prevented by including low levels of rotenone or oligomycin in buffer 1, the stimulation of mitochondrial function was not diminished, thus demonstrating that an increased ATP/ADP ratio is not essential for activation. The rate of citrulline formation was unaffected by buffer 1 treatment unless glutamate was also included in the medium, indicating that control of this mitochondrial function differs from other functions studied.     

荆皮癣湿酊诱导红色毛癣菌凋亡的机制研究北大核心

【目的】本研究旨在探讨复方中药荆皮癣湿酊(Jingpixian tincture,JPXT)对红色毛癣菌(Trichophyton rubrum)的凋亡诱导作用,以阐明其可能的抗真菌作用机制。【方法】采用细胞计数试剂盒-8(cell counting kit-8,CCK-8)评价荆皮癣湿酊对红色毛癣菌生长活力的影响;流式细胞仪检测红色毛癣菌细胞内活性氧(reactive oxygen species,ROS)水平和线粒体膜电位(mitochondrial membrane potential,MMP)变化;Annexin V-FITC/PI染色荧光显微镜观察红色毛癣菌细胞磷脂酰丝氨酸(phosphatidylserine,PS)外翻情况;流式细胞术检测红色毛癣菌细胞凋亡率;FITC-VAD-FMK染色观察红色毛癣菌偏半胱天冬酶(metacaspase)活性;紫外分光光度计测定红色毛癣菌细胞色素C氧化酶的活性。【结果】荆皮癣湿酊处理后的红色毛癣菌细胞活力与MMP水平均有所降低,ROS水平显著升高,PS外翻与凋亡率明显增加,偏半胱天冬酶活性显著升高,细胞色素C氧化酶活性降低。【结论】荆皮癣湿酊可通过诱导菌体凋亡的方式发挥对红色毛癣菌的抗菌作用。     

A mechanism of sulfite neurotoxicity: direct inhibition of glutamate dehydrogenase

Exposure of Neuro-2a and PC12 cells to micromolar concentrations of sulfite caused an increase in reactive oxygen species and a decrease in ATP. Likewise, the biosynthesis of ATP in intact rat brain mitochondria from the oxidation of glutamate was inhibited by micromolar sulfite. Glutamate-driven respiration increased the mitochondrial membrane potential (MMP), and this was abolished by sulfite but the MMP generated by oxidation of malate and succinate was not affected. The increased rate of production of NADH from exogenous NAD+ and glutamate added to rat brain mitochondrial extracts was inhibited by sulfite, and mitochondria preincubated with sulfite failed to reduce NAD+. Glutamate dehydrogenase (GDH) in rat brain mitochondrial extract was inhibited dose-dependently by sulfite as was the activity of a purified enzyme. An increase in the Km (glutamate) and a decrease in Vmax resulting in an attenuation in Vmax/Km (glutamate) at 100 microm sulfite suggest a mixed type of inhibition. However, uncompetitive inhibition was noted with decreases in both Km (NAD+) and Vmax, whereas Vmax/Km (NAD+) remained relatively constant. We propose that GDH is one target of action of sulfite, leading to a decrease in alpha-ketoglutarate and a diminished flux through the tricarboxylic acid cycle accompanied by a decrease in NADH through the mitochondrial electron transport chain, a decreased MMP, and a decrease in ATP synthesis. Because glutamate is a major metabolite in the brain, inhibition of GDH by sulfite could contribute to the severe phenotype of sulfite oxidase deficiency in human infants.     

Caspase-independent phosphatidylserine exposure during apoptosis of primary T lymphocytes

Exposure of phosphatidylserine (PS) on the outer leaflet of the plasma membrane is a key feature of apoptosis. As the signals underlying these phenomena are unknown, it is generally assumed that PS exposure is a consequence of caspase activation, another hallmark of apoptosis. In this study we investigated the role of caspases in PS externalization during apoptosis of activated PBL triggered by drugs (etoposide, staurosporine), CD95 engagement, or IL-2 withdrawal. Anti-CD95 mAb induces a rapid activation of caspases, followed by PS exposure and mitochondrial transmembrane potential (DeltaPsim) disruption. In contrast, etoposide (ETO), staurosporine (STS), or IL-2 withdrawal triggers concomitant caspase activation, PS exposure, and DeltaPsim disruption. Such kinetics suggest that PS exposure could be independent of caspase activation. As expected, in activated PBL treated by anti-CD95 mAb, the pan-caspase inhibitor Cbz-Val-Ala-Asp(OMe)-fluoromethylketone and the caspase-8 inhibitor Cbz-Leu-Glu-Thr-Asp(OMe)-fluoromethylketone, but not the caspase-9 inhibitor Cbz-Leu-Glu-His-Asp(OMe)-fluoromethylketone, inhibit PS externalization and DeltaPsim disruption. Surprisingly, during apoptosis induced by ETO, STS, or IL-2 withdrawal, none of those caspase inhibitors prevents PS externalization or DeltaPsim disruption, whereas they all inhibit DNA fragmentation as well as the morphological features of nuclear apoptosis. In Jurkat and H9 T cell lines, as opposed to activated PBL, PS exposure is inhibited by Cbz-Val-Ala-Asp(OMe)-fluoromethylketone during apoptosis induced by CD95 engagement, ETO, or STS. Thus, caspase-independent PS exposure occurs in primary T cells during apoptosis induced by stimuli that do not trigger death receptors.     

The abnormal-shaped mitochondria in thymus lymphocytes treated with inhibitors of mitochondrial energetics

The effects of uncouplers (DNP, FCCP), oligomycin, and rotenone on the energetics and mitochondrial ultrastructure in lymphocytes have been studied. We confirmed the previous observations done on Ehrlich ascites and cardiomyocyte culture cells that uncouplers and respiratory inhibitors cause the appearance of ringlike and dumbbell-like mitochondria. It is shown that this effect does not correlate with decrease in ATP concentration, changes in oxygen consumption, or condensation of the mitochondrial matrix. FCCP (2 µM) is more effective in the induction of abnormal-form mitochondria than 240 µM DNP, oligomycin, or rotenone. Combined treatment with DNP, oligomycin, and rotenone or with DNP and rotenone produces an effect as strong as 2 µm FCCP. DNP (240 µM) and FCCP (2 µM) have a similar effect on respiration and intracellular ATP, but only the latter induces condensation of the mitochondrial matrix.