RESUMEN
Arsenite is an important carcinogen and toxic compound, causing various deleterious effects through multiple mechanisms. In this review, we focused on mitochondrial ROS (mitoROS) and discussed on the mechanisms mediating their formation. The metalloid promotes direct effects in mitochondria, resulting in superoxide formation only under conditions of increased mitochondrial Ca2+ concentration ([Ca2+]m). In this perspective, the time of exposure and concentration requirements for arsenite were largely conditioned by other effects of the metalloid in specific sites of the endoplasmic reticulum (ER). Arsenite induced a slow and limited mobilization of Ca2+ from IP3R via a saturable mechanism, failing to increase the [Ca2+]m. This effect was however associated with the triggering of an intraluminal crosstalk between the IP3R and the ryanodine receptor (RyR), causing a large and concentration dependent release of Ca2+ from RyR and a parallel increase in [Ca2+]m. Thus, the Ca2+-dependent mitoO2-. formation appears to be conditioned by the spatial/functional organization of the ER/mitochondria network and RyR expression. We also speculate on the possibility that the ER stress response might regulate the above effects on the intraluminal crosstalk between the IP3R and the RyR via oxidation of critical thiols mediated by the H2O2 locally released by oxidoreductin 1α.
Asunto(s)
Arsenitos/toxicidad , Retículo Endoplásmico/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Animales , Retículo Endoplásmico/metabolismo , Humanos , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Fluorescent silica nanoparticles (SiNPs) appear to be a promising imaging platform, showing a specific subcellular localization. In the present study, we first investigated their preferential mitochondrial targeting in myeloid cells, by flow cytometry, confocal microscopy and TEM on both cells and isolated mitochondria, to acquire knowledge in imaging combined with therapeutic applications. Then, we conjugated SiNPs to one of the most used anticancer drugs, doxorubicin (DOX). As an anticancer agent, DOX has high efficacy but also an elevated systemic toxicity, causing multiple side effects. Nanostructures are usually employed to increase the drug circulation time and accumulation in target tissues, reducing undesired cytotoxicity. We tested these functionalized SiNPs (DOX-NPs) on breast cancer cell line MCF-7. We evaluated DOX-NP cytotoxicity, the effect on the cell cycle and on the expression of CD44 antigen, a molecule involved in adhesion and in tumor invasion, comparing DOX-NP to free DOX and stand-alone SiNPs. We found a specific ability to release a minor amount of CD44+ extracellular vesicles (EVs), from both CD81 negative and CD81 positive pools. Modulating the levels of CD44 at the cell surface in cancer cells is thus of great importance for disrupting the signaling pathways that favor tumor progression.
Asunto(s)
Antineoplásicos , Neoplasias de la Mama , Nanopartículas , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Doxorrubicina/química , Portadores de Fármacos/química , Sistemas de Liberación de Medicamentos/métodos , Femenino , Humanos , Mitocondrias , Células Mieloides , Nanopartículas/química , Dióxido de Silicio/químicaRESUMEN
The present study used human myeloid leukemia U937 cells, a versatile promonocytic cellular system that, based on its endoplasmic reticulum (ER)/mitochondria functional relationships, responds to low micromolar concentrations of arsenite with a single, defined mechanism of superoxide (O2 -.) formation. Under these conditions, we observe an initial Ca2+ mobilization from the ER associated with the mitochondrial accumulation of the cation, which is followed by Ca2+-dependent mitochondrial O2 -. (mitoO2 -.) formation. These events, which were barely detectable after 3 hours, were better appreciated at 6 hours. We found that markedly shorter exposure to arsenite and lower concentrations of arsenite are required to induce extensive O2 - formation in cells supplemented with inositol-1,4,5-trisphosphate receptor (IP3R) or ryanodine receptor (RyR) agonists. Indeed, nanomolar arsenite induced maximal O2 -. formation after only 10 minutes of exposure, and this response was uniquely dependent on the enforced mitochondrial Ca2+ accumulation. The dramatic anticipation of and sensitization to the effects of arsenite caused by the IP3R or RyR agonists were accompanied by a parallel significant genotoxic response in the absence of detectable mitochondrial dysfunction and cytotoxicity. We conclude that the prolonged, low-micromolar arsenite exposure paradigm resulting in mitoO2 -. formation is necessary to affect Ca2+ homeostasis and accumulate the cation in mitochondria. The arsenite requirements to promote mitoO2 -. formation in the presence of sufficient mitochondrial Ca2+ were instead remarkably lower in terms of both concentration and time of exposure. These conditions were associated with the induction of extensive DNA strand scission in the absence of detectable signs of toxicity. SIGNIFICANCE STATEMENT: In respiration-proficient cells, arsenite causes mitochondrial Ca2+ accumulation and Ca2+-dependent mitochondrial superoxide formation. We now report that the second event requires remarkably lower concentrations of and time of exposure to the metalloid than the former. Indeed, a brief exposure to nanomolar levels of arsenite produced maximal effects under conditions in which the mitochondrial Ca2+ concentration ([Ca2+]m) was increased by inositol-1,4,5-trisphosphate receptor or ryanodine receptor agonists. Hence, specific substances or conditions enhancing the [Ca2+]m may potentiate the deleterious effects of arsenite by selectively increasing mitochondrial superoxide formation.
Asunto(s)
Arsenitos/toxicidad , Retículo Endoplásmico/efectos de los fármacos , Metaloides/toxicidad , Mitocondrias/efectos de los fármacos , Superóxidos , Teratógenos/toxicidad , Relación Dosis-Respuesta a Droga , Retículo Endoplásmico/metabolismo , Humanos , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Superóxidos/metabolismo , Factores de Tiempo , Células U937RESUMEN
The Na+-dependent Vitamin C transporter 2 (SVCT2) is expressed in the plasma and mitochondrial membranes of various cell types. This notion was also established in proliferating C2C12 myoblasts (Mb), in which the transporter was characterised by a high and low affinity in the plasma and mitochondrial membranes, respectively. In addition, the mitochondrial expression of SVCT2 appeared particularly elevated and, consistently, a brief pre-exposure to low concentrations of Ascorbic Acid (AA) abolished mitochondrial superoxide formation selectively induced by the cocktail arsenite/ATP. Early myotubes (Mt) derived from these cells after 4 days of differentiation presented evidence of slightly increased SVCT2 expression, and were characterised by kinetic parameters for plasma membrane transport of AA in line with those detected in Mb. Confocal microscopy studies indicated that the mitochondrial expression of SVCT2 is well preserved in Mt with one or two nuclei, but progressively reduced in Mt with three or more nuclei. Cellular and mitochondrial expression of SVCT2 was found reduced in day 7 Mt. While the uptake studies were compromised by the poor purity of the mitochondrial preparations obtained from day 4 Mt, we nevertheless obtained evidence of poor transport of the vitamin using the same functional studies successfully employed with Mb. Indeed, even greater concentrations of/longer pre-exposure to AA failed to induce scavenging of mitochondrial superoxide in Mt. These results are therefore indicative of a severely reduced mitochondrial uptake of the vitamin in early Mt, attributable to decreased expression as well as impaired activity of mitochondrial SVCT2.
Asunto(s)
Ácido Ascórbico/metabolismo , Diferenciación Celular , Membrana Celular/metabolismo , Membranas Mitocondriales/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Mioblastos Esqueléticos/metabolismo , Transportadores de Sodio Acoplados a la Vitamina C/metabolismo , Adenosina Trifosfato/farmacología , Animales , Arsenitos/farmacología , Ácido Ascórbico/farmacología , Transporte Biológico , Diferenciación Celular/efectos de los fármacos , Línea Celular , Membrana Celular/efectos de los fármacos , Cinética , Ratones , Membranas Mitocondriales/efectos de los fármacos , Fibras Musculares Esqueléticas/efectos de los fármacos , Mioblastos Esqueléticos/efectos de los fármacos , Compuestos de Sodio/farmacología , Transportadores de Sodio Acoplados a la Vitamina C/genéticaRESUMEN
In this study, respiration-proficient (RP) and -deficient (RD) cells were exposed to 2.5 or 10⯵M arsenite to generate superoxide (O2-.) respectively in the mitochondrial respiratory chain or via NADPH oxidase activation. These treatments, while causing similar, although mitochondrial permeability transition-dependent (RP-cells) or independent (RD-cells), delayed apoptosis, surprisingly generated identical kinetics and levels of dihydrorhodamine oxidation, indicative of O2-. formation. These similarities were attributable to the involvement of a common upstream event resulting in activation of the two O2-.-generating systems, and to intrinsic features of the cells. Both mechanisms required an initial and sequential mobilization of Ca2+ from the inositol-1,4,5-trisphosphate receptor and the ryanodine receptor (RyR), with however different implications. The close contacts existing between the RyR and the mitochondria created optimal conditions for the Ca2+ clearance, and the ensuing formation of O2-. in RP-cell mitochondria. Exposure to low concentrations of l-ascorbic acid (AA) transported by high affinity mechanisms in cells and mitochondria, suppressed O2-. formation. Much more Ca2+, and hence more arsenite, was necessary to promote NADPH oxidase activation in RD-cells, as a consequence of the cytosolic dilution and mitochondrial clearance of Ca2+. For the same reasons, an exposure to high concentrations of AA was required to suppress O2-. formation under these conditions.
Asunto(s)
Arsenitos/toxicidad , Contaminantes Ambientales/toxicidad , Mitocondrias/efectos de los fármacos , Superóxidos/metabolismo , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Ácido Ascórbico/farmacología , Calcio/análisis , Calcio/metabolismo , Hipoxia de la Célula , Humanos , Microscopía Intravital/métodos , Microscopía Fluorescente/métodos , Mitocondrias/química , Mitocondrias/metabolismo , Membranas Mitocondriales/efectos de los fármacos , Membranas Mitocondriales/metabolismo , NADPH Oxidasas/metabolismo , Imagen Óptica/métodos , Permeabilidad/efectos de los fármacos , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Células U937RESUMEN
Arsenite is an established human carcinogen that induces cytotoxic and genotoxic effects through poorly defined mechanisms involving the formation of reactive oxygen species (ROS) and deregulated Ca2+ homeostasis. We used variants of the U937 cell line to address the central issue of the mechanism whereby arsenite affects Ca2+ homeostasis. We found that 6-hour exposure to the metalloid (2.5 µM), although not associated with an immediate or delayed toxicity, causes a significant increase in the intracellular Ca2+ concentration ([Ca2+]i) through a mechanism characterized by the following components: 1) it was not affected by ROS produced under the same conditions; 2) a small amount of Ca2+ was mobilized from the inositol-1,4,5-trisphosphate receptor (IP3R), and this response was not augmented by greater concentrations of the metalloid; 3) large amounts of Ca2+ were instead dose dependently mobilized from the ryanodine receptor (RyR) in response to IP3R stimulation; 4) the cells maintained an intact responsiveness to agonist-stimulated Ca2+ mobilization from both channels; 5) arsenite, even at 5-10 µM, failed to directly mobilize Ca2+ from the RyR; and 6) arsenite failed to enhance Ca2+ release from the RyR under conditions in which the [Ca2+]i was increased by either RyR agonists or ionophore-stimulated Ca2+ uptake. We therefore conclude that arsenite elevates the [Ca2+]i by directly targeting the IP3R and its intraluminal crosstalk with the RyR. This mechanism likely mediates mitochondrial superoxide formation, downstream damage on various biomolecules (including genomic DNA), and mitochondrial dysfunction/apoptosis eventually occurring after longer incubation to, or exposure to greater concentrations of, arsenite.
Asunto(s)
Arsenitos/farmacología , Señalización del Calcio/efectos de los fármacos , Calcio/metabolismo , Inositol/metabolismo , Receptor Cross-Talk/efectos de los fármacos , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Transducción de Señal/efectos de los fármacos , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Homeostasis/efectos de los fármacos , Humanos , Especies Reactivas de Oxígeno/metabolismo , Superóxidos/metabolismo , Células U937RESUMEN
Arsenite toxicity is in numerous cellular systems dependent on the formation of reactive oxygen and or nitrogen species. This is also true in U937 cells in which the metalloid selectively promotes the formation of mitochondrial superoxide (mitoO2-) rapidly converted to diffusible H2O2. We tested the hypothesis that, under the same conditions, mitoO2- also mediates the triggering of a parallel survival signaling. We found that a low concentration of the metalloid causes an early activation of nuclear factor erythroid 2 p45-related factor 2 (Nrf2), and a downstream signaling leading to enhanced GSH biosynthesis, via a mechanism sensitive to various treatments/strategies selectively preventing mitoO2- formation. Under the same conditions, the toxic effects mediated by arsenite, leading to delayed mitochondrial permeability transition (MPT)-dependent apoptosis, were also prevented. Additional studies revealed remarkable similarities in the kinetics of mitoO2- formation, MPT induction, Nrf2 activation and GSH biosynthesis, prior to the onset of apoptosis in a small portion of the cells. Importantly, mitoO2- formation, as well as the ensuing toxic events, were significantly potentiated and anticipated under conditions associated with inhibition of de novo GSH biosynthesis triggered by the metalloid through Nrf2 activation. We conclude that, in the arsenite toxicity paradigm under investigation, mitoO2- represents the only trigger of two opposite pathways leading to activation of the Nrf2 signaling and/or to a MPT-dependent apoptotic death. The first pathway, through enhanced GSH biosynthesis, mitigates the extent of further mitoO2- formation, thereby limiting and delaying an otherwise rapid and massive apoptotic death.
Asunto(s)
Apoptosis/fisiología , Arsenitos/toxicidad , Citoprotección/fisiología , Mitocondrias/fisiología , Superóxidos/metabolismo , Apoptosis/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Citoprotección/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Mitocondrias/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Células U937RESUMEN
Exposure of U937 cells to peroxynitrite promotes mitochondrial superoxide formation via a mechanism dependent on both inhibition of complex III and increased mitochondrial Ca2+ accumulation. Otherwise inactive concentrations of the oxidant produced the same maximal effects in the presence of either complex III inhibitors or agents mobilizing Ca2+ from the ryanodine receptor and enforcing its mitochondrial accumulation. l-Ascorbic acid (AA) produced similar enhancing effects in terms of superoxide formation, DNA strand scission and cytotoxicity. However, AA failed to enhance the intra-mitochondrial concentration of Ca2+ and the effects observed in cells supplemented with peroxinitrite, while insensitive to manipulations preventing the mobilization of Ca2+, or the mitochondrial accumulation of the cation, were also detected in human monocytes and macrophages, which do not express the ryanodine receptor. In all these cell types, mitochondrial permeability transition-dependent toxicity was detected in cells exposed to AA/peroxynitrite and, based on the above criteria, these responses also appeared Ca2+-independent. The enhancing effects of AA are therefore similar to those mediated by bona fide complex III inhibitors, although the vitamin failed to directly inhibit complex III, and in fact enhanced its sensitivity to the inhibitory effects of peroxynitrite.
Asunto(s)
Ácido Ascórbico/metabolismo , Macrófagos/metabolismo , Mitocondrias/metabolismo , Monocitos/metabolismo , Ácido Peroxinitroso/farmacología , Superóxidos/metabolismo , Calcio/metabolismo , Humanos , Células U937RESUMEN
We recently reported that U937 cell mitochondria express a functional Na+-dependent ascorbic acid (AA) transporter recognised by anti-SVCT2 antibodies. The present study confirms and extends these observations by showing that this transporter is characterised by a Km and a pH-dependence comparable with that reported for the plasma membrane SVCT2. In isolated mitochondria, Na+ increased AA transport rate in a cooperative manner, revealed by a sigmoid curve and a Hill coefficient of 2, as also observed in intact Raw 264.7 cells (uniquely expressing SVCT2). There was however a striking difference on the Na+ concentrations necessary to reach saturation, i.e., 1 or 100 mM for the mitochondrial and plasma membrane transporters, respectively. Furthermore the mitochondrial, unlike the plasma membrane, transporter was fully active also in the absence of added Ca++ and/or Mg++. Taken together, the results presented in this study indicate that the U937 cell mitochondrial transporter of AA, because of its very low requirement for Na+ and independence for Ca++ and Mg++, displays kinetic characteristics surprisingly similar with those of the plasma membrane SVCT2.
Asunto(s)
Ácido Ascórbico/metabolismo , Calcio/farmacología , Magnesio/farmacología , Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , Sodio/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Humanos , Cinética , Ratones , Mitocondrias/efectos de los fármacos , Transportadores de Sodio Acoplados a la Vitamina C/metabolismo , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Células U937RESUMEN
We herein report the results from a comparative study of arsenite toxicity in respiration-proficient (RP) and -deficient (RD) U937 cells. An initial characterization of these cells led to the demonstration that the respiration-deficient phenotype is not associated with apparent changes in mitochondrial mass and membrane potential. In addition, similar levels of superoxide (O2(.-)) were generated by RP and RD cells in response to stimuli specifically triggering respiratory chain-independent mitochondrial mechanisms or extramitochondrial, NADPH-oxidase dependent, mechanisms. At the concentration of 2.5µM, arsenite elicited selective formation of O2(.-) in the respiratory chain of RP cells, with hardly any contribution of the above mechanisms. Under these conditions, O2(.-) triggered downstream events leading to endoplasmic reticulum (ER) stress, autophagy and apoptosis. RD cells challenged with similar levels of arsenite failed to generate O2(.-) because of the lack of a functional respiratory chain and were therefore resistant to the toxic effects mediated by the metalloid. Their resistance, however, was lost after exposure to four fold greater concentrations of arsenite, coincidentally with the release of O2(.-) mediated by NADPH oxidase. Interestingly, extramitochondrial O2(.-) triggered the same downstream events and an identical mode of death previously observed in RP cells. Taken together, the results obtained in this study indicate that arsenite toxicity is strictly dependent on O2(.-) availability that, regardless of whether generated in the mitochondrial or extramitochondrial compartments, triggers similar downstream events leading to ER stress, autophagy and apoptosis.
Asunto(s)
Arsenitos/toxicidad , Respiración de la Célula , NADPH Oxidasas/metabolismo , Superóxidos/metabolismo , Apoptosis/efectos de los fármacos , Autofagia , Supervivencia Celular/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Consumo de Oxígeno , Células U937RESUMEN
Exposure of U937 cells to low concentrations of L-ascorbic acid (AA) is associated with a prompt cellular uptake and a further mitochondrial accumulation of the vitamin. Under the same conditions, dehydroascorbic acid (DHA) uptake was followed by rapid reduction and accumulation of identical intracellular levels of AA, however, in the absence of significant mitochondrial uptake. This event was instead observed after exposure to remarkably greater concentrations of DHA. Furthermore, experiments performed in isolated mitochondria revealed that DHA transport through hexose transporters and Na(+) -dependent transport of AA were very similar. These results suggest that the different subcellular compartmentalization of the vitamin is mediated by events promoting inhibition of mitochondrial AA transport, possibly triggered by low levels of DHA. We obtained results in line with this notion in intact cells, and more direct evidence in isolated mitochondria. This inhibitory effect was promptly reversible after DHA removal and comparable with that mediated by established inhibitors, as quercetin. The results presented collectively indicate that low intracellular concentrations of DHA, because of its rapid reduction back to AA, are a poor substrate for direct mitochondrial uptake. DHA concentrations, however, appear sufficiently high to mediate inhibition of mitochondrial transport of AA/DHA-derived AA.
Asunto(s)
Ácido Ascórbico/metabolismo , Transporte Biológico/efectos de los fármacos , Ácido Deshidroascórbico/farmacología , Mitocondrias/efectos de los fármacos , Transportadores de Sodio Acoplados a la Vitamina C/metabolismo , Línea Celular Tumoral , Humanos , Mitocondrias/metabolismo , Sodio/metabolismo , Células U937RESUMEN
A U937 cell clone, in which low micromolar concentrations of ascorbic acid (AA) and dehydroascorbic acid (DHA) are taken up at identical rates, was used to investigate possible interactions between transport systems mediating cellular uptake of the two forms of the vitamin. Results obtained with different experimental approaches showed that DHA potently and reversibly inhibits AA uptake through Na(+)-AA cotransporters. Hence, a progressive increase in extracellular DHA concentrations in the presence of a fixed amount of AA caused an initial decrease in the net amount of vitamin C accumulated, and eventually, at higher levels, it caused an accumulation of the vitamin solely based on DHA uptake through hexose transporters. DHA-dependent inhibition of AA uptake was also detected in various other cell types. Taken together, our results provide evidence of a novel biological effect mediated by concentrations of DHA compatible with those produced at inflammatory sites.
Asunto(s)
Ácido Ascórbico/metabolismo , Ácido Deshidroascórbico/farmacología , Sodio/fisiología , Animales , Transporte Biológico Activo/efectos de los fármacos , Proteínas Portadoras/metabolismo , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Ratones , Monocitos/efectos de los fármacos , Monocitos/metabolismoRESUMEN
U937 cells exposed to physiological concentrations of ascorbic acid (AA) accumulate the reduced form of the vitamin in the cytosol and even further in their mitochondria. In both circumstances, uptake was dependent on Na(+) -AA-cotransport, with hardly any contribution of hexose transporters, which might be recruited to transport the oxidized form of the vitamin. There was an identical linear relationship between the mitochondrial accumulation of the vitamin and the extramitochondrial AA concentration, regardless of whether detected in experiments using intact cells or isolated mitochondria. Western blot experiments revealed expression of both SVCT1 and 2 in plasma membranes, whereas SVCT2 was the only form of the transporter expressed at appreciable amounts in mitochondria. These results therefore provide the novel demonstration of SVCT2-dependent mitochondrial transport of AA and hence challenge the present view that mitochondria only take up the oxidized form of the vitamin.
Asunto(s)
Ácido Ascórbico/metabolismo , Mitocondrias/metabolismo , Cloruro de Sodio/metabolismo , Transportadores de Sodio Acoplados a la Vitamina C/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Humanos , Cinética , Membranas Mitocondriales/metabolismo , Oxidación-Reducción , Células U937RESUMEN
Arsenite is a potent carcinogen and toxic compound inducing an array of deleterious effects via different mechanisms, which include the Ca2+-dependent formation of reactive oxygen species. The mechanism whereby the metalloid affects Ca2+ homeostasis involves an initial stimulation of the inositol 1, 4, 5-triphosphate receptor, an event associated with an endoplasmic reticulum (ER) stress leading to increased ERO1α expression, and ERO1α dependent activation of the ryanodine receptor (RyR). Ca2+ release from the RyR is then critically connected with the mitochondrial accumulation of Ca2+. We now report that the resulting formation of mitochondrial superoxide triggers a second mechanism of ER stress dependent ERO1α expression, which however fails to impact on Ca2+ release from the RyR or, more generally, on Ca2+ homeostasis. Our results therefore demonstrate that arsenite stimulates two different and sequential mechanisms leading to increased ERO1α expression with different functions, possibly due to their different subcellular compartmentalization.
Asunto(s)
Arsenitos , Canal Liberador de Calcio Receptor de Rianodina , Arsenitos/farmacología , Calcio/metabolismo , Homeostasis , Especies Reactivas de Oxígeno/metabolismo , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Oxidorreductasas , Glicoproteínas de MembranaRESUMEN
A 6 h exposure of U937 cells to 2.5 µM arsenite stimulates low Ca2+ release from the inositol 1, 4, 5-triphosphate receptor (IP3R), causing a cascade of causally connected events, i.e., endoplasmic reticulum oxidoreductin-1α (ERO1α) expression, activation of the ryanodine receptor (RyR), mitochondrial Ca2+ accumulation, mitochondrial superoxide formation and further ERO1α expression. At greater arsenite concentrations, the release of the cation from the IP3R and the ensuing ERO1α expression remained unchanged but were nevertheless critical to sequentially promote concentration-dependent increases in Ca2+ release from the RyR, NADPH oxidase activation and a third mechanism of ERO1α expression which, in analogy to the one driven by mitochondrial superoxide, was also mediated by reactive oxygen species (ROS) and devoid of effects on Ca2+ homeostasis. Thus, concentration-independent stimulation of Ca2+ release from the IP3R is of pivotal importance for the effects of arsenite on Ca2+ homeostasis. It stimulates the expression of a fraction of ERO1α that primes the RyR to respond to the metalloid with concentration-dependent Ca2+-release, triggering the formation of superoxide in the mitochondrial respiratory chain and via NADPH oxidase activation. The resulting dose-dependent ROS formation was associated with a progressive increase in ERO1α expression, which however failed to affect Ca2+ homeostasis, thereby suggesting that ROS, unlike IP3R-dependent Ca2+ release, promote ERO1α expression in sites distal from the RyR.
Asunto(s)
Arsenitos , Especies Reactivas de Oxígeno , Canal Liberador de Calcio Receptor de Rianodina , Arsenitos/toxicidad , Homeostasis , NADPH Oxidasas , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Superóxidos , Calcio/metabolismo , HumanosRESUMEN
Long-term treatment of schizophrenia with clozapine (CLZ), an atypical antipsychotic drug, is associated with an increased incidence of metabolic disorders mediated by poorly understood mechanisms. We herein report that CLZ, while slowing down the morphological changes and lipid accumulation occurring during SW872 cell adipogenesis, also causes an early (day 3) inhibition of the expression/nuclear translocation of CAAT/enhancer-binding protein ß and peroxisome proliferator-activated receptor γ. Under the same conditions, CLZ blunts NADPH oxidase-derived reactive oxygen species (ROS) by a dual mechanism involving enzyme inhibition and ROS scavenging. These effects were accompanied by hampered activation of the nuclear factor (erythroid-derived2)-like 2 (Nrf2)-dependent antioxidant responses compared to controls, and by an aggravated formation of mitochondrial superoxide. CLZ failed to exert ROS scavenging activities in the mitochondrial compartment but appeared to actively scavenge cytosolic H2O2 derived from mitochondrial superoxide. The early formation of mitochondrial ROS promoted by CLZ was also associated with signs of mitochondrial dysfunction. Some of the above findings were recapitulated using mouse embryonic fibroblasts. We conclude that the NADPH oxidase inhibitory and cytosolic ROS scavenging activities of CLZ slow down SW872 cell adipogenesis and suppress their Nrf2 activation, an event apparently connected with increased mitochondrial ROS formation, which is associated with insulin resistance and metabolic syndrome. Thus, the cellular events characterised herein may help to shed light on the more detailed molecular mechanisms explaining some of the adverse metabolic effects of CLZ.
Asunto(s)
Clozapina , Liposarcoma , Humanos , Animales , Ratones , NADPH Oxidasas/metabolismo , Adipogénesis , Especies Reactivas de Oxígeno/metabolismo , Clozapina/farmacología , Clozapina/metabolismo , Peróxido de Hidrógeno/metabolismo , Superóxidos/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Fibroblastos/metabolismo , Mitocondrias/metabolismo , Liposarcoma/metabolismoRESUMEN
We report that a moderate decline in GSH levels causes remarkable changes in Bad sub-cellular localization. An about 30% reduction of the GSH pool, regardless of whether mediated by diamide or DL-buthionine-[S,R]-sulfoximine, indeed promoted loss of the fraction of Bad normally associated with the mitochondria of untreated U937 cells via a phosphatidylinositol 3-kinase (PI3K)-dependent mechanism. Interestingly, inhibition of this pathway was associated with an unexpected delayed lethal response, preceded by the translocation and enforced accumulation of Bad and Bax in the mitochondrial compartment, prevented by inhibitors of mitochondrial permeability transition and characterized by morphological and biochemical features of apoptosis. Collectively, the results herein presented demonstrate that mild redox imbalance associated with a slight reduction of the GSH pool commits U937 cells to apoptosis, however prevented by events leading to PI3K/Akt-dependent mitochondrial loss of Bad.
Asunto(s)
Apoptosis , Glutatión/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteína Letal Asociada a bcl/metabolismo , Humanos , Mitocondrias/metabolismo , Oxidación-Reducción , Fosforilación , Transducción de Señal , Células U937RESUMEN
Mammalian cells accumulate vitamin C either as ascorbic acid (AA), via Na+-AA co-transport, or dehydroascorbic acid (DHA, the oxidation product of AA), via facilitative hexose transport. As the latter, unlike the former, is a high-capacity transport mechanism, cultured cells normally accumulate greater levels of vitamin C when exposed to increasing concentrations of DHA as compared with AA. We report herein similar results using the U937 cell clone used in our laboratory only under conditions in which DHA and AA are used at concentrations greater than 50-60 µm. Below 60 µm, i.e. at levels in which AA is normally found in most biological fluids, AA and DHA are in fact taken up with identical rates and kinetics. Consequently, extracellular oxidation of AA switches the mode of uptake with hardly any effect on the net amount of vitamin C accumulated. As a final note, under these conditions, neither AA nor DHA causes detectable toxicity or any change in the redox status of the cells, as assessed by the reduced glutathione/reduced pyridine nucleotide pool. These findings therefore imply that some cell types do not have a preferential route for vitamin C accumulation, and that the uptake mechanism is uniquely dependent on the extracellular availability of AA v. DHA.
Asunto(s)
Ácido Ascórbico/metabolismo , Ácido Deshidroascórbico/metabolismo , Macrófagos/metabolismo , Monocitos/metabolismo , Ácido Ascórbico/efectos adversos , Transporte Biológico , Línea Celular , Supervivencia Celular , Células Clonales , Ácido Deshidroascórbico/efectos adversos , Proteínas Facilitadoras del Transporte de la Glucosa/metabolismo , Glutatión/metabolismo , Humanos , Cinética , Macrófagos/inmunología , Monocitos/inmunología , NAD/metabolismo , NADP/metabolismo , Concentración Osmolar , Oxidación-Reducción , Transportadores de Sodio Acoplados a la Vitamina C/metabolismoRESUMEN
Our recent studies suggest that arsenite stimulates the crosstalk between the inositol 1, 4, 5-triphosphate receptor (IP3R) and the ryanodine receptor (RyR) via a mechanism dependent on endoplasmic reticulum (ER) oxidoreductin1α (ERO1α) up-regulation. Under these conditions, the fraction of Ca2+ released by the RyR via an ERO1α-dependent mechanism was promptly cleared by the mitochondria and critically mediated O2-. formation, responsible for the triggering of time-dependent events associated with strand scission of genomic DNA and delayed mitochondrial apoptosis. We herein report that, in differentiated C2C12 cells, this sequence of events can be intercepted by genetic deletion of ERO1α as well as by EN460, an inhibitor of ERO1α activity. Similar results were obtained for the early effects mediated by arsenite in proliferating U937 cells, in which however the long-term studies were hampered by the intrinsic toxicity of the inhibitor. It was then interesting to observe that ISRIB, an inhibitor of p-eIF2 alpha, was in both cell types devoid of intrinsic toxicity and able to suppress ERO1α expression and the resulting downstream effects leading to arsenite geno- and cyto-toxicity. We therefore conclude that pharmacological inhibition of ERO1α activity, or expression, effectively counteracts the deleterious effects induced by the metalloid via a mechanism associated with prevention of mitochondrial O2-. formation.
Asunto(s)
Arsenitos , Glicoproteínas de Membrana/metabolismo , Metaloides , Oxidorreductasas/metabolismo , Arsenitos/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , Humanos , Inositol , Canal Liberador de Calcio Receptor de Rianodina/genética , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Superóxidos/metabolismoRESUMEN
Long-term ingestion of arsenicals, a heterogeneous group of toxic compounds, has been associated with a wide spectrum of human pathologies, which include various malignancies. Although their mechanism of toxicity remains largely unknown, it is generally believed that arsenicals mainly produce their effects via direct binding to protein thiols and ROS formation in different subcellular compartments. The generality of these mechanisms most probably accounts for the different effects mediated by different forms of the metalloid in a variety of cells and tissues. In order to learn more about the molecular mechanisms of cyto- and genotoxicity, there is a need to focus on specific arsenic compounds under tightly controlled conditions. This review focuses on the mechanisms regulating the mitochondrial formation of ROS after exposure to low concentrations of a specific arsenic compound, NaAsO2, and their crosstalk with the nuclear factor (erythroid-2 related) factor 2 antioxidant signaling and the endoplasmic reticulum stress response.