Gold Nanoparticles Attenuates Antimycin A-Induced Mitochondrial Dysfunction in MC3T3-E1 Osteoblastic Cells

Gold nanoparticles have shown promising biological applications due to their unique properties. Understanding the interaction mechanisms between nanomaterials and biological cells is important for the control and manipulation of these interactions for biomedical applications. In the present study, we investigated the effects of gold nanoparticles on the differentiation of osteoblastic MC3T3-E1 cells and antimycin A-induced mitochondrial dysfunction. The results showed that gold nanoparticles (5, 10, and 20 nm) caused a significant elevation of cell growth, alkaline phosphatase activity, collagen synthesis, and osteocalcin content in the cells (P?<?0.05). Moreover, pretreatment with gold nanoparticles prior to antimycin A exposure significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, cytochrome c release, cardiolipin peroxidation, and reactive oxygen species generation. Taken together, our study indicated that gold nanoparticles may improve the differentiation and have protective effects on mitochondrial dysfunction of osteoblastic cells.     

Inhibitory effect of paeoniflorin on methylglyoxal-mediated oxidative stress in osteoblastic MC3T3-E1 cells

PurposeMethylglyoxal (MG) has been suggested to be one major source of intracellular reactive carbonyl compounds. In the present study, the effect of paeoniflorin on MG-induced cytotoxicity was investigated using osteoblastic MC3T3-E1 cells.MethodsOsteoblastic MC3T3-E1 cells were pre-incubated with paeoniflorin before treatment with MG, and markers of oxidative damage and mitochondrial function were examined.ResultsPretreatment of MC3T3-E1 cells with paeoniflorin prevented the MG-induced cell death and formation of intracellular reactive oxygen species, cardiolipin peroxidation, and protein adduct in osteoblastic MC3T3-E1 cells. In addition, paeoniflorin increased glutathione level and restored the activity of glyoxalase I to almost the control level. These findings suggest that paeoniflorin provide a protective action against MG-induced cell damage by reducing oxidative stress and by increasing MG detoxification system. Pretreatment with paeoniflorin prior to MG exposure reduced MG-induced mitochondrial dysfunction by preventing mitochondrial membrane potential dissipation and adenosine triphosphate loss. Additionally, the nitric oxide and nuclear respiratory factor 1 levels were significantly increased by paeoniflorin, suggesting that paeoniflorin may induce mitochondrial biogenesis. Paeoniflorin treatment decreased the levels of proinflammatory cytokines such as TNF-α and IL-6.ConclusionsThese findings indicate that paeoniflorin might exert its therapeutic effects via upregulation of glyoxalase system and mitochondrial function. Taken together, paeoniflorin may prove to be an effective treatment for diabeteic osteopathy.     

The protective effects of Achyranthes bidentata root extract on the antimycin A induced damage of osteoblastic MC3T3-E1 cells

Achyranthes bidentata (A. bidentata) Blume is a medicinal herb with the property of strengthening bones and muscles and ensuring proper downward flow of blood in terms of the therapeutic theory of traditional medicine. In the present study, the effect of A. bidentata root extract (AE) on osteoblast function was investigated in osteoblastic MC3T3-E1 cells. AE caused a significant elevation of alkaline phosphatase activity, collagen synthesis, osteocalcin production, and mineralization in the cells (P < 0.05). AE also decreased the production of TNF-α, IL-6, and RANKL induced by antimycin A, mitochondrial electron transport inhibitor. Exposure of MC3T3-E1 cells to antimycin A caused significant reduction of cell viability and mineralization. However, pretreatment with AE prior to antimycin A exposure significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, ATP loss, ROS release, and nitrotyrosine increase, suggesting that AE may be useful for protecting mitochondria against a burst of oxidative stress. Moreover, AE increased the phosphorylation of cAMP-response element-binding protein inhibited by antimycin A. Our study demonstrates that A. bidentata could significantly prevent osteoblast damage in aged patients.     

Protective effect of α-lipoic acid against antimycin A cytotoxicity in MC3T3-E1 osteoblastic cells

Oxidative stress represents a major cause of cellular damage and death in the process of osteoporosis. Antimycin A (AMA) has been shown to stimulate mitochondrial superoxide anions and reactive oxygen species (ROS). α-Lipoic acid (α-LA) is a naturally occurring essential coenzyme in mitochondrial multienzyme complexes and acts as a key player in mitochondrial energy production. However, whether α-LA affects the cytotoxicity of AMA in osteoblastic cells is unknown. In this study, we investigated the protective effects of α-LA against AMA-induced cytotoxicity using the MC3T3-E1 osteoblast-like cell line. Our results indicated that α-LA treatment attenuated AMA-induced cytotoxicity and LDH release in a dose-dependent manner. Notably, a significant recovery effect of α-LA on mineralization inhibited by AMA was found. Our results also demonstrated that treatment with 50 μM AMA leads to a reduction of mitochondrial membrane potential (MMP) and the complex IV dysfunction, which was inhibited by pretreatment with α-LA in a dose-dependent manner. In addition, treatment with α-LA significantly reduced the generation of ROS and mitochondrial superoxide production induced by AMA. In addition, our result suggests that PI3K/Akt and CREB pathways are related to the protective effect of α-LA. Importantly, Hoechst 33258 staining results indicated that pretreatment with α-LA prevented AMA-induced apoptosis. Mechanistically, we found that α-LA prevents MC3T3-E1 cells from apoptosis through attenuating cytochrome C release and reducing the level of cleaved caspase-3.     

Methylglyoxal induces oxidative stress and mitochondrial dysfunction in osteoblastic MC3T3-E1 cells

Abstract

Methylglyoxal is a reactive dicarbonyl compound produced by glycolytic processing and identified as a precursor of advanced glycation end products. The elevated methylglyoxal levels in patients with diabetes are believed to contribute to diabetic complications, including bone defects. The objective of this study was to evaluate the effect of methylglyoxal on the function of osteoblastic MC3T3-E1 cells. The data indicated that methylglyoxal decreased osteoblast differentiation and induced osteoblast cytotoxicity. Pretreatment of MC3T3-E1 cells with aminoguanidine (a carbonyl scavenger), Trolox (an antioxidant), and cyclosporin A (a blocker of the mitochondrial permeability transition pore) prevented methylglyoxal-induced cytotoxicity in MC3T3-E1 cells. However, BAPTA/AM (an intracellular Ca²⁺ chelator) and dantrolene (an inhibitor of endoplasmic reticulum Ca²⁺ release) did not reverse the cytotoxic effect of methylglyoxal. Methylglyoxal increased the formation of intracellular reactive oxygen species, mitochondrial superoxide, and cardiolipin peroxidation in osteoblastic MC3T3-E1 cells. Methylglyoxal also decreased the mitochondrial membrane potential and intracellular ATP and nitric oxide levels, suggesting that carbonyl stress-induced loss of mitochondrial integrity contributes to the cytotoxicity of methylglyoxal. Furthermore, the results demonstrated that methylglyoxal induced protein adduct formation, inactivation of glyoxalase I, and activation of glyoxalase II. Aminoguanidine reversed all aforementioned effects of methylglyoxal. Taken together, these data support the notion that high methylglyoxal concentrations have detrimental effects on osteoblasts through a mechanism involving oxidative stress and mitochondrial dysfunction.     

Effect of Zinc Oxide Nanoparticles on the Function of MC3T3-E1 Osteoblastic Cells

Zinc oxide nanoparticles (ZnO NPs) can be ingested directly when used in food, food packaging, drug delivery, and cosmetics. This study evaluated the cellular effects of ZnO NPs (50 and 100 nm diameter particle sizes) on the function of osteoblastic MC3T3-E1 cells. ZnO NPs showed cytotoxicity at concentrations of above 50 μg/ml, and there was no significant effect of the size on the cytotoxicity of ZnO NPs. Within the testing concentrations of 0.01～1 μg/ml, which did not cause a marked drop in cell viability, ZnO NPs (0.1 μg/ml) caused a significant elevation of alkaline phosphatase activity, collagen synthesis, mineralization, and osteocalcin content in the cells (P?<?0.05). Moreover, pretreatment with ZnO NPs (0.01～1 μg/ml) significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, complex IV inactivation, and ATP loss. Measurement of reactive oxygen species (ROS) indicated decrease in ROS level upon exposure to ZnO nanoparticles (0.01 μg/ml). Hence, our study indicated that ZnO nanoparticles can have protective effects on osteoblasts at low concentrations where there are little or no observable cytotoxic effects.     

Coenzyme Q cytoprotective mechanisms for mitochondrial complex I cytopathies involves NAD(P)H: quinone oxidoreductase 1(NQO1)

The commonest mitochondrial diseases are probably those impairing the function of complex I of the respiratory electron transport chain. Such complex I impairment may contribute to various neurodegenerative disorders e.g. Parkinson's disease. In the following, using hepatocytes as a model cell, we have shown for the first time that the cytotoxicity caused by complex I inhibition by rotenone but not that caused by complex III inhibition by antimycin can be prevented by coenzyme Q (CoQ1) or menadione. Furthermore, complex I inhibitor cytotoxicity was associated with the collapse of the mitochondrial membrane potential and reactive oxygen species (ROS) formation. ROS scavengers or inhibitors of the mitochondrial permeability transition prevented cytotoxicity. The CoQ1 cytoprotective mechanism required CoQ1 reduction by DT-diaphorase (NQO1). Furthermore, the mitochondrial membrane potential and ATP levels were restored at low CoQ1 concentrations (5 microM). This suggests that the CoQ1H2 formed by NQO1 reduced complex III and acted as an electron bypass of the rotenone block. However cytoprotection still occurred at higher CoQ1 concentrations (>10 microM), which were less effective at restoring ATP levels but readily restored the cellular cytosolic redox potential (i.e. lactate: pyruvate ratio) and prevented ROS formation. This suggests that CoQ1 or menadione cytoprotection also involves the NQO1 catalysed reoxidation of NADH that accumulates as a result of complex I inhibition. The CoQ1H2 formed would then also act as a ROS scavenger.     

Exogenous zinc protects cardiac cells from reperfusion injury by targeting mitochondrial permeability transition pore through inactivation of glycogen synthase kinase-3beta

The purpose of this study was to determine whether exogenous zinc prevents cardiac reperfusion injury by targeting the mitochondrial permeability transition pore (mPTP) via glycogen synthase kinase-3beta (GSK-3beta). The treatment of cardiac H9c2 cells with ZnCl2 (10 microM) in the presence of zinc ionophore pyrithione for 20 min significantly enhanced GSK-3beta phosphorylation at Ser9, indicating that exogenous zinc can inactivate GSK-3beta in H9c2 cells. The effect of zinc on GSK-3beta activity was blocked by the phosphatidylinositol 3-kinase (PI3K) inhibitor LY-294002 but not by the mammalian target of rapamycin (mTOR) inhibitor rapamycin or the PKC inhibitor chelerythrine, implying that PI3K but not mTOR or PKC accounts for the action of zinc. In support of this interpretation, zinc induced a significant increase in Akt but not mTOR phosphorylation. Further experiments found that zinc also increased mitochondrial GSK-3beta phosphorylation. This may indicate an involvement of the mitochondria in the action of zinc. The effect of zinc on mitochondrial GSK-3beta phosphorylation was not altered by the mitochondrial ATP-sensitive K+ channel blocker 5-hydroxydecanoic acid. Zinc applied at reperfusion reduced cell death in cells subjected to simulated ischemia/reperfusion, indicating that zinc can prevent reperfusion injury. However, zinc was not able to exert protection in cells transfected with the constitutively active GSK-3beta (GSK-3beta-S9A-HA) mutant, suggesting that zinc prevents reperfusion injury by inactivating GSK-3beta. Cells transfected with the catalytically inactive GSK-3beta (GSK-3beta-KM-HA) also revealed a significant decrease in cell death, strongly supporting the essential role of GSK-3beta inactivation in cardioprotection. Moreover, zinc prevented oxidant-induced mPTP opening through the inhibition of GSK-3beta. Taken together, these data suggest that zinc prevents reperfusion injury by modulating the mPTP opening through the inactivation of GSK-3beta. The PI3K/Akt signaling pathway is responsible for the inactivation of GSK-3beta by zinc.     

Protective effects of piceatannol on methylglyoxal-induced cytotoxicity in MC3T3-E1 osteoblastic cells

Methylglyoxal (MG) is a reactive α-oxoaldehyde that increases under diabetic conditions and subsequently contributes to the complications associated with this disease. Piceatannol is a naturally occurring analogue of resveratrol that possesses multiple biological functions. The present study investigated the effects of piceatannol on MG-induced cytotoxicity in MC3T3-E1 osteoblastic cells. Piceatannol significantly restored MG-induced reductions in cell viability and reduced lactate dehydrogenase release in MG-treated MC3T3-E1 osteoblastic cells, which suggests that it suppressed MG-induced cytotoxicity. Piceatannol also increased glyoxalase I activity and glutathione levels in MG-treated cells, which indicates that it enhanced the glyoxalase system and thus cellular protection. The present study also showed that piceatannol inhibited the generation of inflammatory cytokines and reactive oxygen species and ameliorated mitochondrial dysfunction induced by MG. Furthermore, piceatannol treatment significantly reduced the levels of endoplasmic reticulum stress and autophagy induced by MG. Therefore, piceatannol could be a potent option for the development of antiglycating agents for the treatment of diabetic osteopathy.     

Apelin stimulates proliferation and suppresses apoptosis of mouse osteoblastic cell line MC3T3-E1 via JNK and PI3-K/Akt signaling pathways

The aim of this study was to investigate the effects of apelin on proliferation and apoptosis of mouse osteoblastic MC3T3-E1 cells. APJ was expressed in MC3T3-E1 cells. Apelin did not affect Runx2 expression, alkaline phosphatase (ALP) activity, osteocalcin and type I collagen secretion, suggesting that it has no effect on osteoblastic differentiation of MC3T3-E1 cells. However, apelin stimulated MC3T3-E1 cell proliferation and inhibited cell apoptosis induced by serum deprivation. Our study also shows that apelin decreased cytochrome c release and caspase-3, capase-8 and caspase-9 activation in serum-deprived MC3T3-E1 cells. Apelin activated c-Jun N-terminal kinase (JNK) and Akt (phosphatidylinositol 3-kinase downstream effector), and the JNK inhibitor SP600125, the phosphatidylinositol 3-kinase (PI3-K) inhibitor LY294002 or the Akt inhibitor 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO) inhibited its effects on proliferation and serum deprivation-induced apoptosis. Furthermore, apelin protected against apoptosis induced by the glucocorticoid dexamethasone or TNF-alpha. Apelin stimulates proliferation and suppresses serum deprivation-induced apoptosis of MC3T3-E1 cells and these actions are mediated via JNK and PI3-K/Akt signaling pathways.     

Endoplasmic reticulum Ca(2+) signaling and calpains mediate renal cell death

The goal of the current study was to determine the roles of ATP content, endoplasmic reticulum (ER) Ca(2+) stores, cytosolic free Ca(2+) (Ca(2+)(f)) and calpain activity in the signaling of rabbit renal proximal tubular (RPT) cell death (oncosis). Increasing concentrations (0.3-10 microM) of the mitochondrial inhibitor antimycin A produced rapid ATP depletion that correlated to a rapid and sustained increase in Ca(2+)(f), but not phospholipase C activation. The ER Ca(2+)-ATPase inhibitors thapsigargin (5 microM) or cyclopiazonic acid (100 microM) alone produced similar but transient increases in Ca(2+)(f). Pretreatment with thapsigargin prevented antimycin A-induced increases in Ca(2+)(f) and antimycin A pretreatment prevented thapsigargin-induced increases in Ca(2+)(f). Calpain activity increased in conjunction with ER Ca(2+) release. Pretreatment, but not post-treatment, with thapsigargin or cyclopiazonic acid prevented antimycin A-induced cell death. These data demonstrate that extensive ATP depletion signals oncosis through ER Ca(2+) release, a sustained increase in Ca(2+)(f) and calpain activation. Depletion of ER Ca(2+) stores prior to toxicant exposure prevents increases in Ca(2+)(f) and oncosis.     

Coenzyme Q Cytoprotective Mechanisms for Mitochondrial Complex I Cytopathies Involves NAD(P)H: Quinone Oxidoreductase 1(NQO1)

The commonest mitochondrial diseases are probably those impairing the function of complex I of the respiratory electron transport chain. Such complex I impairment may contribute to various neurodegenerative disorders e.g. Parkinson's disease. In the following, using hepatocytes as a model cell, we have shown for the first time that the cytotoxicity caused by complex I inhibition by rotenone but not that caused by complex III inhibition by antimycin can be prevented by coenzyme Q (CoQ 1 ) or menadione. Furthermore, complex I inhibitor cytotoxicity was associated with the collapse of the mitochondrial membrane potential and reactive oxygen species (ROS) formation. ROS scavengers or inhibitors of the mitochondrial permeability transition prevented cytotoxicity. The CoQ 1 cytoprotective mechanism required CoQ 1 reduction by DT-diaphorase (NQO 1 ). Furthermore, the mitochondrial membrane potential and ATP levels were restored at low CoQ 1 concentrations (5 &#119 M). This suggests that the CoQ 1 H 2 formed by NQO 1 reduced complex III and acted as an electron bypass of the rotenone block. However cytoprotection still occurred at higher CoQ 1 concentrations (>10 &#119 M), which were less effective at restoring ATP levels but readily restored the cellular cytosolic redox potential (i.e. lactate: pyruvate ratio) and prevented ROS formation. This suggests that CoQ 1 or menadione cytoprotection also involves the NQO 1 catalysed reoxidation of NADH that accumulates as a result of complex I inhibition. The CoQ 1 H 2 formed would then also act as a ROS scavenger.     

Eupalinin A isolated from Eupatorium chinense L. induces autophagocytosis in human leukemia HL60 cells

Eupalinin A, a natural phytoalexin included in Eupatorium chinense L., exhibited a marked inhibitory effect on cell growth in HL60 cells. The morphological aspects of eupalinin A-treated cells evaluated by Hoechst 33342 nuclear staining indicated cell death, only a small part of which showed a typical apoptosis with nuclear fragmentation and condensation. To determine what type of cell death is caused by eupalinin A, we examined the contribution of caspases, Bcl-2 family proteins, MAP kinase, and PI3K/Akt, and mitochondrial membrane potential to this cell death. As a result, most part of the cell death was not associated with apoptosis because of caspase independence and no death factor released from mitochondria. Electron microscopic study indicated a characteristic finding of autophagy such as the formation of autophagosomes. Furthermore, the level of microctubule-associated-protein light chain 3 (LC3) II protein and monodancylcanaverin (MDC) incorporation were gradually increased with reduction of mitochondrial membrane potential by the accumulation of intracellular ROS after eupalinin A treatment. From these results, we can conclude that eupalinin A-induced cell death was mainly due to autophagy, which was initiated by increased ROS, resulting in the perturbation of mitochondrial membrane potential. Since the class III PI3K inhibitor such as 3-MA or LY294002 did not inhibit the eupalinin A-induced type II programmed cell death (PCD II), it was suggested that the PCD II was executed by Beclin-1 independent pathway of damage-induced mitochondrial autophagy (mitophagy).     

Inhibition of PI3K/AKT signaling via ROS regulation is involved in Rhein-induced apoptosis and enhancement of oxaliplatin sensitivity in pancreatic cancer cells

Several natural products have been demonstrated to both enhance the anti-tumor efficacy and alleviate the side effects of conventional chemotherapy drugs. Rhein, a main constituent of the Chinese herb rhubarb, has been shown to induce apoptosis in various cancer types. However, the exact pharmacological mechanisms controlling the influence of Rhein on chemotherapy drug effects in pancreatic cancer (PC) remain largely undefined. In this study, we found that Rhein inhibited the growth and proliferation of PC cells through G1 phase cell cycle arrest. Moreover, Rhein induced caspase-dependent mitochondrial apoptosis of PC cells through inactivation of the PI3K/AKT pathway. Combination treatment of Rhein and oxaliplatin synergistically enhanced apoptosis of PC cells through increased generation of intracellular reactive oxygen species (ROS) and inactivation of the PI3K/AKT pathway. Pre-treatment with the ROS scavenger N-acetyl-L-cysteine attenuated the combined treatment-induced apoptosis and restored the level of phosphorylated AKT, indicating that ROS is an upstream regulator of the PI3K/AKT pathway. The combination therapy also exhibited stronger anti-tumor effects compared with single drug treatments in vivo. Taken together, these data demonstrate that Rhein can induce apoptosis and enhance the oxaliplatin sensitivity of PC cells, suggesting that Rhein may be an effective strategy to overcome drug resistance in the chemotherapeutic treatment of PC.     

Reactive oxygen species produced by the mitochondrial respiratory chain are involved in Cd2+-induced injury of rat ascites hepatoma AS-30D cells

Using AS-30D rat ascites hepatoma cells, we studied the modulating action of various antioxidants, inhibitors of mitochondrial permeability transition pore and inhibitors of the respiratory chain on Cd(2+)-produced cytotoxicity. It was found that Cd(2+) induced both necrosis and apoptosis in a time- and dose-dependent way. This cell injury involved dissipation of the mitochondrial transmembrane potential, respiratory dysfunction and initial increase of the generation of reactive oxygen species (ROS), followed by its decrease after prolonged incubation. Inhibitors of the mitochondrial permeability transition pore, cyclosporin A and bongkrekic acid, and inhibitors of respiratory complex III, stigmatellin and antimycin A, but not inhibitor of complex I, rotenone, partly prevented necrosis evoked by exposure of the cells to Cd(2+). Apoptosis of the cells was partly prevented by free radical scavengers and by preincubation with N-acetylcysteine. Stigmatellin, antimycin A and cyclosporin A also abolished Cd(2+)-induced increase in ROS generation. It is concluded that Cd(2+) toxicity in AS-30D rat ascites hepatoma, manifested by cell necrosis and/or apoptosis, involves ROS generation, most likely at the level of respiratory complex III, and is related to opening of the mitochondrial permeability transition pore.     

ZnT7 can protect MC3T3-E1 cells from oxidative stress-induced apoptosis via PI3K/Akt and MAPK/ERK signaling pathways

The osteoblasts could be lead to the occurrence of apoptosis by oxidative stress. The zinc transporter family SLC30A (ZnTs) plays an important role in the regulation of zinc homeostasis, however, its function in apoptosis of MC3T3-E1 cells remains unknown. This study was aimed to investigate the role of zinc transporters in cell survival, particularly in MC3T3-E1 cells, during oxidative stress, and the molecular mechanism involved. Our study found that hydrogen peroxide can induce zinc-overloaded in the cells. While high concentration of zinc plays an important role in inducing apoptosis of the MC3T3-E1 cells, we demonstrated that ZnT7 can protect MC3T3-E1 cells and reduce the aggregation of intracellular free zinc ions as well as inhibit apoptosis induced by H₂O₂. Moreover, ZnT7 overexpression enhanced the anti-apoptotic effects. Interestingly, suppression of ZnT7 by siRNA could significantly exacerbate apoptosis in MC3T3-E1 cells. We also found that ZnT7 promotes cell survival via two distinct signaling pathways involving activation of the PI3K/Akt-mediated survival pathway and activation of MAPK/ERK pathway. Collectively, these results suggest that ZnT7 overexpression significantly protects osteoblasts cells from apoptosis induced by H₂O₂. This effect is mediated, at least in part, through activation of PI3K/Akt and MAPK/ERK pathways.     

Thiol depletion induces lethal cell injury in cultured cardiomyocytes.

Treatment of cultured neonatal cardiomyocytes with ethacrynic acid (EA) induced a rapid depletion of glutathione (GSH) that preceded a gradual elevation of cytosolic Ca2+ (monitored by phosphorylase a activation), a loss of protein thiols, and a marked inactivation of the thiol-dependent enzyme glyceraldehyde-3-phosphate dehydrogenase (G3PD). A subsequent decline of mitochondrial transmembrane potential (delta psi) and ATP occurred prior to the onset of lipid peroxidation which closely paralleled a loss of cardiomyocyte viability. The antioxidant N,N'-diphenyl-p-phenylenediamine prevented lipid peroxidation and cell death but had no effect on elevated cytosolic Ca2+, delta psi loss, GSH depletion, or G3PD inactivation. Pretreatment with the iron chelator, deferoxamine, decreased both lipid peroxidation and cell death. EA-induced lipid peroxidation and cell damage were also diminished by preincubation with acetoxymethyl esters of the Ca2+ chelators Quin-2 and ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid, even though cytosolic Ca2+ remained elevated. The extent of GSH depletion was unaltered by either chelator; however, Quin-2 did protect G3PD from inactivation by EA. An inhibitor of the mitochondrial respiratory chain, antimycin A, decreased EA-induced lipid peroxidation and cell death but had no effect on thiol depletion or elevated cytosolic Ca2+. These data suggest that cardiomyocyte thiol status may be linked to intracellular Ca2+ homeostasis and that peroxidative damage originating in the mitochondria is a major event in the onset of cell death in this cardiomyocyte model of thiol depletion.     

ZIP1 and Zinc Inhibits Fluoride-Induced Apoptosis in MC3T3-E1 Cells

Excess fluoride intake could induce apoptosis in the cells. As an essential micronutrient and cytoprotectant, zinc is involved in many types of apoptosis. Here, we studied the effects of zinc and ZIP1 on fluoride-induced apoptosis in mouse MC3T3-E1 cells and examined the underlying molecular mechanisms. Our study found that fluoride not only inhibited cell proliferation and increased the intracellular reactive oxygen species (ROS) but also induced cell apoptosis. Whereas pretreatment with zinc significantly attenuated fluoride-induced ROS production and partly protected cells against fluoride-induced apoptosis through MAPK/ERK signaling pathway. Our study also found that fluoride upregulated the expression of ZIP1 in a time-dependent manner. Moreover, overexpression of ZIP1 also inhibited fluoride-induced apoptosis by activation of PI3K/Akt pathway. This cytoprotective effect of zinc and ZIP1 may be new factors that affect the physiological activity of fluoride and need study further.