Mitochondrial Metabolic Activity Research Articles

Evaluation of Cell Cycle Arrest in Estrogen Responsive MCF-7 Breast Cancer Cells: Pitfalls of the MTS Assay

Endocrine resistance is a major problem with anti-estrogen treatments and how to overcome resistance is a major concern in the clinic. Reliable measurement of cell viability, proliferation, growth inhibition and death is important in screening for drug treatment efficacy in vitro. This report describes and compares commonly used proliferation assays for induced estrogen-responsive MCF-7 breast cancer cell cycle arrest including: determination of cell number by direct counting of viable cells; or fluorescence SYBR®Green (SYBR) DNA labeling; determination of mitochondrial metabolic activity by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay; assessment of newly synthesized DNA using 5-ethynyl-2′-deoxyuridine (EdU) nucleoside analog binding and Alexa Fluor® azide visualization by fluorescence microscopy; cell-cycle phase measurement by flow cytometry. Treatment of MCF-7 cells with ICI 182780 (Faslodex), FTY720, serum deprivation or induction of the tumor suppressor p14ARF showed inhibition of cell proliferation determined by the Trypan Blue exclusion assay and SYBR DNA labeling assay. In contrast, the effects of treatment with ICI 182780 or p14ARF-induction were not confirmed using the MTS assay. Cell cycle inhibition by ICI 182780 and p14ARF-induction was further confirmed by flow cytometric analysis and EdU-DNA incorporation. To explore this discrepancy further, we showed that ICI 182780 and p14ARF-induction increased MCF-7 cell mitochondrial activity by MTS assay in individual cells compared to control cells thereby providing a misleading proliferation readout. Interrogation of p14ARF-induction on MCF-7 metabolic activity using TMRE assays and high content image analysis showed that increased mitochondrial activity was concomitant with increased mitochondrial biomass with no loss of mitochondrial membrane potential, or cell death. We conclude that, whilst p14ARF and ICI 182780 stop cell cycle progression, the cells are still viable and potential treatments utilizing these pathways may contribute to drug resistant cells. These experiments demonstrate how the combined measurement of metabolic activity and DNA labeling provides a more reliable interpretation of cancer cell response to treatment regimens.

Evidence for a Role for the Cytoskeleton in Communication Between the L-Type Calcium Channel and the Mitochondria in Isolated Cardiac Myocytes

Cytoskeletal proteins stabilize cell structure but also regulate subcellular distribution of mitochondria and cardiac L-type Ca2+ channel (LTCC) activity. We have previously demonstrated that mitochondrial function can be regulated by alterations in LTCC activity. This effect was attenuated when the cytoskeleton was disrupted with latrunculin A. To further explore this, we determined whether regulation of mitochondrial function by the LTCC is altered in a murine model of Duchenne Muscular Dystrophy(mdx). Mitochondrial membrane potential (Ψm) and metabolic activity was assessed after activation of the LTCC in cardiac myocytes isolated from C57BL/10ScSn-Dmdmdx/Arc (mdx) and C57BL/10ScSnArc (control) mice. Exposure of control myocytes to 10μM BayK(-) (LTCC agonist) caused a 11.4±1.7% increase in Ψm assessed as alterations in JC-1 compared to myocytes exposed to inactive BayK(+) (n=8, p<0.05). The response was attenuated when myocytes were exposed to LTCC antagonist nisoldipine (n=7). However BayK(-) did not induce any significant alteration in JC-1 signal in myocytes from mdx mice (n=6). In control myocytes BayK(-) caused a 105.4±7.4% increase in metabolic activity assessed using an MTT assay (n=8, p<0.05). The response was attenuated when myocytes were exposed to nisoldipine (n=8) or mitochondrial calcium uniporter inhibitor Ru360 (n=8) but unaltered when exposed to ryanodine receptor antagonist dantrolene (n=4). Exposure of mdx myocytes to BayK(-) did not induce any significant alteration in metabolic activity (n=8). These data confirm that alterations in LTCC activity can modulate mitochondrial function and that the cytoskeleton plays an important role in mediating this response. Since the LTCC is the initiator of contraction it has been proposed that a functional coupling between the LTCC and mitochondria may assist in meeting myocardial energy demand on a beat to beat basis.

Electrically wired mitochondrial electrodes for measuring mitochondrial function for drug screening

In the continual search of new therapeutics, many possible drug candidates are excluded, because they are found to negatively affect mitochondrial function. We have developed an approach for directly, electrochemically assaying mitochondrial metabolic activity as a function of metabolic substrate to determine drug toxicity. By wiring mouse mitochondria to a carbon electrode surface, electrons can be intercepted before they reach Complex IV, the terminal step of electron transport chain. The electrons are rerouted, to a separate electrode of the electrochemical cell, the cathode. This allows for the direct measurement of electrical current and potential of the mitochondria during their oxidation of substrates such as pyruvate and fatty acids when there are different concentrations of drug present. This analytical technique has been shown to reliably assay several classical mitochondrial toxins and exhibits potential for the further development of a drug candidate screening technique, as well as other applications where the quantitative study of mitochondrial dysfunction is important.

Therapeutic Approaches to Delay the Onset of Alzheimer's Disease

The key cytopathologies in the brains of Alzheimer's disease (AD) patients include mitochondrial dysfunction and energy hypometabolism, which are likely caused by the accumulation of small aggregates of amyloid-β (Aβ) peptides. Thus, targeting these two abnormalities of the AD brain may hold promising therapeutic value for delaying the onset of AD. In his paper, we discuss two potential approaches to delay the onset of AD. The first is the use of low dose of diaminophenothiazins (redox active agents) to prevent mitochondrial dysfunction and to attenuate energy hypometabolism. Diaminophenothiazines enhance mitochondrial metabolic activity and heme synthesis, both key factors in intermediary metabolism of the AD brain.The second is to use the naturally occurring osmolytes to prevent the formation of toxic forms of Aβ and prevent oxidative stress. Scientific evidence suggests that both approaches may change course of the basic mechanism of neurodegeneration in AD. Osmolytes are brain metabolites which accumulate in tissues at relatively high concentrations following stress conditions. Osmolytes enhance thermodynamic stability of proteins by stabilizing natively-folded protein conformation, thus preventing aggregation without perturbing other cellular processes. Osmolytes may inhibit the formation of Aβ oligomers in vivo, thus preventing the formation of soluble oligomers. The potential significance of combining diaminophenothiazins and osmolytes to treat AD is discussed.

Factors which determine the duration of follower potentials in longitudinal smooth muscle isolated from the guinea-pig stomach antrum

In isolated longitudinal muscle tissues of the guinea-pig stomach antrum, recording electrical responses from smooth muscle cells revealed a periodical generation of follower potentials with variable durations. The I-D relationship, made by plotting the duration as a function of the interval before generating follower potential, was linear. Experiments were carried out to investigate the effects of chemicals which had been known to modulate the release of Ca(2+) from the internal stores (2-aminoethoxy-diphenyl-borate, cyclopiazonic acid, caffeine), inhibit mitochondrial metabolic activity (m-chlorophenyl hydrazone, 2-deoxy-D-glucose, potassium cyanide, rotenone), inhibit ATP-sensitive K-channels distributed in mitochondria (glibenclamide, 5-hydroxydecanoic acid) and inhibit the activity of proteinkinase C (chelerythrine), on the I-D relationship of follower potentials. The effects of depolarization on follower potentials were assessed by stimulating tissues with high potassium solution. Experiments were carried out mainly in the presence of nifedipine which minimized the movements of muscles with no modulation of follower potentials. Cycropiazonic acid and caffeine reduced the slope of I-D relationship, with associated reduction of the duration and frequency of follower potentials. 2-Aminoethoxydiphenyl borate reduced the duration and amplitude and increased the frequency of follower potentials, with depolarization of the membrane, and the effects were simulated by high potassium solution. m-Chlorophenyl hydrazone, potassium cyanide, 2-deoxy-D-glucose, rotenone, 5-hydroxydecanoic acid and glibenclamide reduced the slope of I-D relationship, with associated reduction of the frequency of follower potentials. Chelerythrine did not modulate the slope of I-D relationship, with reduced frequency of follower potentials. It seemed likely that the amount of Ca(2+) released from the internal stores and also mitochondrial function had causal relationship to the duration of pacemaker potentials, suggesting that internal Ca-stores and mitochondria are taking the central role for determining the duration of the pacemaker activity. Proteinkinase C did not seem to participate to the function of mitochondria and internal Ca(2+) stores.

Influence of renal compensatory hypertrophy on mitochondrial energetics and redox status

A reduction in functional renal mass is common in numerous renal diseases and aging. The remaining functional renal tissue undergoes compensatory growth primarily due to hypertrophy. This is associated with a series of physiological, morphological and biochemical changes similar to those observed after uninephrectomy. Previous work showed that compensatory renal cellular hypertrophy resulted in an increase in susceptibility to several drugs and environmental chemicals and appeared to be associated with oxidative stress. Compensatory renal cellular hypertrophy was also associated with increases in mitochondrial metabolic activity, uptake of glutathione (GSH) across renal plasma and mitochondrial inner membranes, and intracellular GSH concentrations. Based on these observations, we hypothesize that the morphological, physiological and biochemical changes in the hypertrophied kidney are associated with marked alterations in renal cellular energetics, redox status and renal function in vivo. In this study, we used a uninephrectomized (NPX) rat model to induce compensatory renal growth. Our results show alterations in renal physiological parameters consistent with modest renal injury, altered renal cellular energetics, upregulation of certain renal plasma membrane transporters, including some that have been observed to transport GSH, and evidence of increased oxidative stress in mitochondria from the remnant kidney of NPX rats. These studies provide additional insight into the molecular changes that occur in compensatory renal hypertrophy and should help in the development of novel therapeutic approaches for patients with reduced renal mass.

Role of surface charge and oxidative stress in cytotoxicity of organic monolayer-coated silicon nanoparticles towards macrophage NR8383 cells

BackgroundSurface charge and oxidative stress are often hypothesized to be important factors in cytotoxicity of nanoparticles. However, the role of these factors is not well understood. Hence, the aim of this study was to systematically investigate the role of surface charge, oxidative stress and possible involvement of mitochondria in the production of intracellular reactive oxygen species (ROS) upon exposure of rat macrophage NR8383 cells to silicon nanoparticles. For this aim highly monodisperse (size 1.6 ± 0.2 nm) and well-characterized Si core nanoparticles (Si NP) were used with a surface charge that depends on the specific covalently bound organic monolayers: positively charged Si NP-NH2, neutral Si NP-N3 and negatively charged Si NP-COOH.ResultsPositively charged Si NP-NH2 proved to be more cytotoxic in terms of reducing mitochondrial metabolic activity and effects on phagocytosis than neutral Si NP-N3, while negatively charged Si NP-COOH showed very little or no cytotoxicity. Si NP-NH2 produced the highest level of intracellular ROS, followed by Si NP-N3 and Si NP-COOH; the latter did not induce any intracellular ROS production. A similar trend in ROS production was observed in incubations with an isolated mitochondrial fraction from rat liver tissue in the presence of Si NP. Finally, vitamin E and vitamin C induced protection against the cytotoxicity of the Si NP-NH2 and Si NP-N3, corroborating the role of oxidative stress in the mechanism underlying the cytotoxicity of these Si NP.ConclusionSurface charge of Si-core nanoparticles plays an important role in determining their cytotoxicity. Production of intracellular ROS, with probable involvement of mitochondria, is an important mechanism for this cytotoxicity.

Ketamine induces apoptosis via the mitochondrial pathway in human lymphocytes and neuronal cells

BackgroundKetamine has been shown to have neurotoxic properties, when administered neuraxially. The mechanism of this local toxicity is still unknown. Therefore, we investigated the mechanism of cytotoxicity in different human cell lines in vitro. MethodsWe incubated the following cell types for 24 h with increasing concentrations of S(+)-ketamine and racemic ketamine: (i) human Jurkat T-lymphoma cells overexpressing the antiapoptotic B-cell lymphoma 2 protein, (ii) cells deficient of caspase-9, caspase-8, or Fas-associated protein with death domain and parental cells, and (iii) neuroblastoma cells (SHEP). N-Methyl-d-aspartate (NMDA) receptors and caspase-3 cleavage were identified by immunoblotting. Cell viability and apoptotic cell death were evaluated flowcytometrically by Annexin V and 7-aminoactinomycin D double staining. Mitochondrial metabolic activity and caspase-3 activation were measured. ResultsKetamine, in a concentration-dependent manner, induced apoptosis in lymphocytes and neuroblastoma cell lines. Cell lines with alterations of the mitochondrial pathway of apoptosis were protected against ketamine-induced apoptosis, whereas alterations of the death receptor pathway did not reduce apoptosis. S(+)-Ketamine and racemic ketamine induced the same percentage of cell death in Jurkat cells, whereas in neuroblastoma cells, S(+)-ketamine was slightly less toxic. ConclusionsKetamine at millimolar concentrations induces apoptosis via the mitochondrial pathway, independent of death receptor signalling. At higher concentrations necrosis is the predominant mechanism. Less toxicity of S(+)-ketamine was observed in neuroblastoma cells, but this difference was minor and therefore unlikely to be mediated via the NMDA receptor.

Comparative cytotoxicity evaluation of lanthanide nanomaterials on mouse and human cell lines with metabolic and DNA-quantification assays

Lanthanide nanomaterials are considered a less toxic alternative to quantum dots for bioimaging applications. This study evaluated the cytotoxicity of terbium (Tb)-doped gadolinium oxide (Gd(2)O(3)) and dysprosium oxide (Dy(2)O(3)) nanoparticles exposed to human (BEAS-2B) and mouse (L929) cell lines at a concentration range of 200-2000 μg/ml for 48 h. Two assay methods were utilized-WST-8 assay (colorimetric) based on mitochondrial metabolic activity and Pico-Green assay (fluorescence), which measures total DNA content. The authors' data showed that Tb-doped Gd(2)O(3) nanoparticles were consistently more toxic than Tb-doped Dy(2)O(3) nanoparticles. However, exposure to these nanomaterials caused a decrease in proliferation rate for both cell lines rather than a net loss of viable cells after 48 h of exposure. Additionally, there was some degree of discrepancy observed with the two assay methods. For the mouse L929 cell line, the WST-8 assay yielded consistently lower proliferation rates compared to the Pico-Green assay, whereas the opposite trend was observed for the human BEAS-2B cell line. This could arise because of the differential effects of these nanoparticles on the metabolism of L929 and BEAS-2B cells, which in turn may translate to differences in their postexposure proliferation rates. Hence, the Pico-Green assay could have an advantage over the WST-8 assay because it is not skewed by the differential effects of nanomaterials on cellular metabolism.

Mitochondria in the offspring of old mice exhibit alterations similar to those seen in their mothers

OBJECTIVE: We have shown previously that increased maternal age is associated with decreased mitochondrial metabolic activity in oocytes. As mitochondria are exclusively maternally inherited, any alterations in their performance may be passed on to the offspring, leading to compromised cellular bioenergetics and abnormal redox state. The objective of this study was to determine the effects of maternal age on oocyte mitochondrial function in the offspring.

Severe Motor Neuron Degeneration in the Spinal Cord of the Tg2576 Mouse Model of Alzheimer Disease

The transgenic mouse Tg2576 is widely used as a murine model of Alzheimer's disease (AD) and exhibits plaque pathogenesis in the brain and progressive memory impairments. Here we report that Tg2576 mice also have severe spinal cord deficits. At 10 months of age, Tg2576 mice showed a severe defect in the hindlimb extension reflex test and abnormal body trembling and hindlimb tremors when suspended by the tail. The frequency and severity of these abnormalities were overt at 10 months of age and became gradually worsened. On the foot-printing analysis, Tg2576 mice had shorter and narrower strides than the non-transgenic control. Histological analyses showed that neuronal cells including cholinergic neurons in the lumbar cord of Tg2576 mice were severely reduced in number. At 16 months of age, Tg2576 mice showed high levels of amyloid-beta accumulation in the spinal cord. Consistent with this, Tg2576 mice showed that lipid peroxidation levels were increased and mitochondrial metabolic activity were significantly reduced in the spinal cord. Administration of curcumin, a natural compound that has antioxidant properties, notably reversed motor function deficits of Tg2576 mice. The enhanced lipid peroxidation and neuronal loss in the lumbar cord was also partially suppressed by curcumin. Electron microscopic analysis revealed that the sciatic nerve fibers were severely reduced in number and were demyelinated in Tg2576 mice, which were partially rescued by curcumin. These results showed that Tg2576 mice display severe degeneration of motor neurons in the spinal cord and associated motor function deficits.

Evaluation of the biocompatibility and mechanical properties of naturally derived and synthetic scaffolds for urethral reconstruction

The aim of this study was to evaluate the mechanical properties and biocompatibility of biomaterials, including bladder submucosa (BAMG), small intestinal submucosa (SIS), acellular corpus spongiosum matrix (ACSM), and polyglycolic acid (PGA), to identify the optimal scaffold for urethral tissue engineering. Tensile mechanical testing was conducted to evaluate mechanical properties of each scaffold. Rabbit corporal smooth muscle cells were cultured with the extracts of biomaterials and mitochondrial metabolic activity assay was used to determine the cytotoxicity of scaffold. The pore sizes of each scaffold were measured. Additionally, smooth muscle cells were seeded on biomaterials. Cell infiltration was evaluated. Mechanical evaluation showed that Young modulus, stress at break in ACSM were prior to those in other biomaterials (p < 0.05). MTT assay confirmed that all scaffolds supported normal cellular mitochondrial metabolic without inducing cytotoxic events. SEM demonstrated that PGA has the largest pore size (>200 microm). The ACSM has different pore sizes in urethral (<5 microm) and cavernosal surfaces (>10 microm). Widespread distribution of cells could be observed in PGA 14 days after seeding. Multilayer cellular coverage developed in BAMG and urethral surface of ACSM without any sign of cellular invasion. Moderated cellular penetration could be found in SIS and cavernosal surface of ACSM. Although each scaffold demonstrated suitable mechanical properties, which is similar to normal urethra, ACSM showed better response in some parameters than those in other biomaterials. It suggested that this scaffold may be an alternative for urethral reconstruction in the future. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.

The critical role of intracellular zinc in adenosine A2 receptor activation induced cardioprotection against reperfusion injury

Exogenous zinc can protect cardiac cells from reperfusion injury, but the exact roles of endogenous zinc in the pathogenesis of reperfusion injury and in adenosine A2 receptor activation-induced cardioprotection against reperfusion injury remain unknown. Adenosine A1/A2 receptor agonist 5′-(N-ethylcarboxamido) adenosine (NECA) given at reperfusion reduced infarct size in isolated rat hearts subjected to 30min ischemia followed by 2h of reperfusion. This effect of NECA was partially but significantly blocked by the zinc chelator N,N,N′,N′-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN), and ZnCl2 given at reperfusion mimicked the effect of NECA by reducing infarct size. Total tissue zinc concentrations measured with inductively coupled plasma optical emission spectroscopy (ICPOES) were decreased upon reperfusion in rat hearts and this was reversed by NECA. NECA increased intracellular free zinc during reperfusion in the heart. Confocal imaging study showed a rapid increase in intracellular free zinc in isolated rat cardiomyocytes treated with NECA. Further experiments revealed that NECA increased total zinc levels upon reperfusion in mitochondria isolated from isolated hearts. NECA attenuated mitochondrial swelling upon reperfusion in isolated hearts and this was inhibited by TPEN. Similarly, NECA prevented the loss of mitochondrial membrane potential (ΔΨm) caused by oxidant stress in cardiomyocytes. Finally, both NECA and ZnCl2 inhibited the mitochondrial metabolic activity. NECA-induced cardioprotection against reperfusion injury is mediated by intracellular zinc. NECA prevents reperfusion-induced zinc loss and relocates zinc to mitochondria. The inhibitory effects of zinc on both the mPTP opening and the mitochondrial metabolic activity may account for the cardioprotective effect of NECA.

Zinc may increase bone formation through stimulating cell proliferation, alkaline phosphatase activity and collagen synthesis in osteoblastic MC3T3-E1 cells

Zinc is an essential trace element required for bone formation, however not much has been clarified yet for its role in osteoblast. We hypothesized that zinc would increase osteogenetic function in osteoblasts. To test this, we investigated whether zinc treatment enhances bone formation by stimulating osteoblast proliferation, bone marker protein alkaline phosphatase activity and collagen synthesis in osteoblastic MC3T3-E1 cells. MC3T3-E1 cells were cultured and treated with various concentrations of zinc (0, 1, 3, 15, 25 uM) along with a normal osteogenic medium (OSM) as control for 1, 5, 10 days. As measured by MTT assay for mitochondrial metabolic activity, cell proliferation was stimulated even at low zinc treatment (1-3 µM) compared to OSM, and it was stimulated in a zinc concentration-dependent manner during 5 and 10 days, with the most pronounced effect at 15 and 25 uM Zn. Cellular (synthesized) alkaline phosphatase (ALP) activity was increased in a zinc concentration-dependent manner, so did medium (secreted) ALP activity. Cellular collagen concentration was increased by zinc as time went by, therefore with the maximum zinc stimulatory effect in 10 days, and medium collagen concentration showed the same pattern even on 1 and 5 day. This zinc stimulatory effect of collagen synthesis was observed in cell matrix collagen staining. The study results imply that zinc can increase osteogenic effect by stimulating cell proliferation, ALP activity and collagen synthesis in osteoblastic cells.

Modification of endothelial biology by acute and chronic stress hormones

Objective An increasing number of studies have examined the role of emotional stress and coronary heart disease; the underlying pathophysiology is still poorly understood. The present study was designed to evaluate the relationship between acute (epi- and norepinephrine) and chronic stress hormones (dexamethasone, beta-endorphin, corticotropin releasing hormone) and endothelial dysfunction. Methods Human microvascular endothelial cells were incubated with stress hormones for 6 and 24 h. ET-1 release and ADMA were quantified via ELISA, NO release by using cell permeable 4.5-diaminofluorescein diacetate (DAF2-DA), oxidative stress fluometrically by the ROS-sensitive carboxy-H2-DCFDA method, mitochondrial metabolic activity by using the colorimetric assay WST-1, ET-1 receptor type A (ET AR) protein expression by Western blot, and cell proliferation activity was assessed by the colorimetric assay BrdU. Results With respect to analysed acute and chronic stress hormones, ET-1 release was significantly increased. Likewise, protein expression was enhanced after long term incubation (24 h) with norepinephrine and dexamethasone. In contrast, endothelial NO-levels were only influenced by short term stimulation of dexamethasone (upregulation of NO release) and norepinephrine (downregulation of NO release), whereas modified NO concentration mimics altered mitochondrial metabolic activity. Unexpectedly, both oxidative stress and cell proliferation were not modified by stress hormones. Conclusion Results suggest that acute and chronic stress hormones induce a significant ET-1 release whereas NO release remained mainly unchanged. The imbalance of pro- and antiatherosclerotic factors may play a pivotal role in the initiation of stress-related endothelial dysfunction up to myocardial infarction.

Overexpression of Mitochondrial Ferritin Sensitizes Cells to Oxidative Stress Via an Iron-Mediated Mechanism

Mitochondrial ferritin (MtFt) is a newly identified H-ferritin-like protein expressed only in mitochondria. Previous studies have shown that its overexpression markedly affects intracellular iron homeostasis and rescues defects caused by frataxin deficiency. To assess how MtFt exerts its function under oxidative stress conditions, MtFt overexpressing cells were treated with tert-butyl-hydroperoxide (tBHP), and the effects of MtFt expression on cell survival and iron homeostasis were examined. We found that MtFt expression was associated with decreased mitochondrial metabolic activity and reduced glutathione levels as well as a concomitant increase in reactive oxygen species levels and apoptosis. Moreover, mechanistic studies demonstrated that tBHP treatment led to a prolonged decrease in cytosolic ferritins levels in MtFt-expressing cells, while ferritin levels recovered to basal levels in control counterparts. tBHP treatment also resulted in elevated transferrin receptors, followed by more iron acquisition in MtFt expressing cells. The high molecular weight desferrioxamine, targeting to lysosomes, as well as the hydrophobic iron chelator salicylaldehyde isonicotinoyl hydrazone significantly attenuated tBHP-induced cell damage. In conclusion, the current study indicates that both the newly acquired iron from the extracellular environment and internal iron redistribution from ferritin degradation may be responsible for the increased sensitivity to oxidative stress in MtFt-expressing cells.

Cadmium exposure induces mitochondria-dependent apoptosis in oligodendrocytes

Cadmium toxicity has been associated with learning disabilities and Parkinsonian symptoms in humans. We have previously shown that cultured oligodendrocytes are directly damaged by cadmium exposure. Here, we characterized the molecular mechanisms underlying cadmium-induced cell death in oligodendrocyte progenitors (OLP). Cadmium caused a concentration-dependent decrease in cell viability as assessed by mitochondrial dehydrogenase activity and by the cellular release of lactate dehydrogenase (LDH). A short exposure (1 h) to cadmium (25–100 μM), followed by several hours of recovery, produced a predominant apoptotic mechanism of cell death, involving the mitochondrial intrinsic pathway, as evidenced by nuclear condensation, DNA fragmentation, bax integration into the outer mitochondrial membrane, cytochrome c release, and activation of caspases-9 and -3. Pretreatment of OLPs with the pan-caspase inhibitor, zVAD-fmk, prevented caspase-3 activation but only slightly reduced cell death 11 h after cadmium exposure and failed to prevent cadmium-induced bax insertion into the mitochondrial membrane. In contrast, the anti-oxidant N-acetyl cysteine blocked caspase-3 activation and significantly protected OLPs from cadmium-induced cell death. Continuous exposure (18–48 h) of OLPs to low micromolar concentrations (0.001–25 μM) of cadmium significantly decreased mitochondrial metabolic activity, increased LDH leakage starting at 5 μM and maximally activated caspase-3. These results suggest that cadmium induces OLP cell death mainly by apoptosis, and at higher concentrations or with prolonged exposure to the heavy metal there is an increase in cytoplasmic membrane damage, an index of necrosis. More importantly, transient exposure to cadmium is sufficient to damage OLPs and could in principle impair myelination in the neonate.

Proinflammatory Cytokines Activate the Intrinsic Apoptotic Pathway in β-Cells

OBJECTIVEProinflammatory cytokines are cytotoxic to β-cells and have been implicated in the pathogenesis of type 1 diabetes and islet graft failure. The importance of the intrinsic mitochondrial apoptotic pathway in cytokine-induced β-cell death is unclear. Here, cytokine activation of the intrinsic apoptotic pathway and the role of the two proapoptotic Bcl-2 proteins, Bad and Bax, were examined in β-cells.RESEARCH DESIGN AND METHODSHuman and rat islets and INS-1 cells were exposed to a combination of proinflammatory cytokines (interleukin-1β, interferon-γ, and/or tumor necrosis factor-α). Activation of Bad was determined by Ser136 dephosphorylation, mitochondrial stress by changes in mitochondrial metabolic activity and cytochrome c release, downstream apoptotic signaling by activation of caspase-9 and -3, and DNA fragmentation. The inhibitors FK506 and V5 were used to investigate the role of Bad and Bax activation, respectively.RESULTSWe found that proinflammatory cytokines induced calcineurin-dependent dephosphorylation of Bad Ser136, mitochondrial stress, cytochrome c release, activation of caspase-9 and -3, and DNA fragmentation. Inhibition of Bad Ser136 dephosphorylation or Bax was found to inhibit cytokine-induced intrinsic proapoptotic signaling.CONCLUSIONSOur findings demonstrate that the intrinsic mitochondrial apoptotic pathway contributes significantly to cytokine-induced β-cell death and suggest a functional role of calcineurin-mediated Bad Ser136 dephosphorylation and Bax activity in cytokine-induced apoptosis.

Trichothecene-induced cytotoxicity on human cell lines

Trichothecene cytotoxicity of type A (T-2 toxin and HT-2 toxin), type B (deoxynivalenol, DON, and nivalenol, NIV), and type D (satratoxins G and H) compounds was determined comparatively by using eight permanent human cell lines (Hep-G2, A549, CaCo-2, HEp-2, A204, U937, RPMI 8226, and Jurkat). Viability of cells was measured by a water-soluble tetrazolium (WST-1) reagent cell proliferation assay assessing mitochondrial metabolic activity. Toxicity was expressed as the toxin concentration inhibiting 50% of cell viability (IC50). Depending on the chemotype of the tested trichothecenes, relative cytotoxic activity differed by a factor of 100-1,000, and the corresponding IC50 values were in the range from 2.2nmol/l (satratoxin H on Jurkat and U937 cells) to 4,900nmol/l (deoxynivalenol on HEp-2 cells). In contrast, the specific toxicity of each individual mycotoxin towards different cell lines was within remarkable close limits, and between-cell line differences were much smaller than previously reported. For the cell lines tested, IC50 values were 4.4-10.8nmol/l for T-2 toxin, 7.5-55.8mol/l for HT-2 toxin, 600-4,900nmol/l for DON, 300-2,600nmol/l for NIV, and 2.2-18.3nmol/l for satratoxins G/H. In addition, for the first time, the toxic activity of trichothecenes on primary cell culture of human endothelial cells (HUVEC) was tested. The susceptibility of this cell line was comparable to the other cell lines tested, with IC50 values ranging from 16.5nmol/l (T-2 toxin) to 4,500nmol/l (DON). The results suggest that the current focus of cytotoxicological studies on trichothecenes on lymphoid cell lines may lead to an underestimate of their potential on other target cell systems.

Enhanced nerve growth factor efficiency in neural cell culture by immobilization on the culture substrate

Nerve growth factor (NGF) immobilization on a culture substrate may dramatically reduce the amount of NGF required for pheochromocytoma (PC12) cell culture. Coverslips on which NGF had been immobilized, or with NGF added to the culture medium daily, were used to culture PC12 cells. We examined the effects of adding 5, 10, or 100ng of NGF to cultures daily, and compared them to the effects of immobilizing 5, 10, or 100ng of NGF on culture substrates in a single dose. Cultures with 10 or 5ng NGF added daily showed dramatically decreased cell viability, mitochondrial metabolic activity, and neuronal differentiation compared to cultures with 100ng NGF added daily, while also exhibiting increased apoptosis. In contrast, a single dose of 100ng immobilized NGF yielded results similar to 100ng NGF added daily (total: 300ng over 3days), and 10 or 5ng immobilized NGF showed far better results than 10 or 5ng NGF added daily. These results demonstrate that NGF immobilization can dramatically reduce the amount of NGF required in neuronal cell culture.