Silver nanoparticles induce oxidative cell damage in human liver cells through inhibition of reduced glutathione and induction of mitochondria-involved apoptosis

Silver nanoparticles (AgNPs), which have well-known antimicrobial properties, are extensively used in various medical and general applications. Despite the widespread use of AgNPs, relatively few studies have been undertaken to determine the cytotoxic effects of AgNPs exposure. This study investigates possible molecular mechanisms underlying the cytotoxic effects of AgNPs. Here, we show that AgNPs-induced cytotoxicity was higher compared than that observed when AgNO₃ was used as a silver ion source. AgNPs induced reactive oxygen species (ROS) generation and suppression of reduced glutathione (GSH) in human Chang liver cells. ROS generated by AgNPs resulted in damage to various cellular components, DNA breaks, lipid membrane peroxidation, and protein carbonylation. Upon AgNPs exposure, cell viability decreased due to apoptosis, as demonstrated by the formation of apoptotic bodies, sub-G₁ hypodiploid cells, and DNA fragmentation. AgNPs induced a mitochondria-dependent apoptotic pathway via modulation of Bax and Bcl-2 expressions, resulting in the disruption of mitochondrial membrane potential (Δψ_m). Loss of Δψ_m was followed by cytochrome c release from the mitochondria, resulting in the activation of caspases 9 and 3. The apoptotic effect of AgNPs was exerted via the activation of c-Jun NH₂-terminal kinase (JNK) and was abrogated by the JNK-specific inhibitor, SP600125 and siRNA targeting JNK. In summary, the results suggest that AgNPs cause cytotoxicity by oxidative stress-induced apoptosis and damage to cellular components.     

氟伐他汀调控 Sirt3对过氧化氢诱导的骨髓间充质干细胞增殖凋亡的影响

目的 探讨氟伐他汀对过氧化氢诱导的骨髓间充质干细胞(BMSCs)增殖凋亡的影响及作用机制.方法 用400μmol/L的过氧化氢处理BMSCs作为模型组;BMSCs用400μmol/L的过氧化氢培养的同时分别加入0.01、0.1、1μmol/L氟伐他汀作为干预组;未添加任何物质正常培养的细胞作为正常组;将si-NC、si-Sirt3转染至BMSCs中再用400μmol/L的过氧化氢、0.1μmol/L氟伐他汀处理作为干预组2+si-NC组、干预组2+si-Sirt3组.蛋白质印迹(Western Blotting)法检测Sirt3蛋白表达水平;四甲基偶氮唑盐比色法(MTT)检测细胞存活率;流式细胞术检测细胞凋亡.结果 与正常组相比,模型组BMSCs细胞存活率降低[(41.57±4.32)％比(100.40±10.23)％],细胞凋亡率升高[(38.57±4.04)％比(8.28±0.92)％],Sirt3表达水平降低[(0.31±0.03)比(1.01±0.10)],均差异有统计学意义(P<0.05);与模型组相比,氟伐他汀干预组细胞存活率明显升高细胞凋亡率降低,Sirt3表达水平升高,均差异有统计学意义(P<0.05).提示抑制Sirt3能逆转氟伐他汀对过氧化氢诱导的BMSCs细胞增殖抑制和凋亡促进作用.结论 氟伐他汀能抑制过氧化氢诱导的BMSCs细胞凋亡,其机制可能与上调Sirt3有关.     

The effect of lovastatin on oxidative stress and antioxidant enzymes in hydrogen peroxide intoxicated rat

Oxidative stress has been linked to the development of many diseases and hastens the progression of cardiovascular diseases. Since lovastatin is used worldwide as a cholesterol lowering drug, the present study was undertaken to evaluate the antioxidant property of lovastatin against H₂O₂ induced oxidative stress in rats. Four study groups of rats of four animals each were treated with DMSO (control), H₂O₂ (OS), lovastatin (L) and H₂O₂ + lovastatin (OSL). On the 15th day the animals were sacrificed, and the liver and heart tissues were analyzed for oxidative stress biomarkers and anti-oxidant enzymes. Results of the OSL-group showed a reduction in thiobarbituric acid reactive substances in liver (42.7%) and heart tissue (8%) compared with the control group. An increase was observed in the activity of the antioxidant enzymes, catalase (34.6% in liver and 33.3% in heart) and glutathione peroxidase (50.5% in liver and 34.7% in heart). A commensurate increase in the activity of G6PDH was observed indicating an enhanced requirement of NADPH. The ratio GSH:GSSG in liver (1.05) and heart (0.84) was satisfactorily regulated compared to the control group (1.01 in liver and 0.93 in heart). These results suggest that lovastatin possesses antioxidant activity and reduces oxidative stress.     

Evidence of crocin against endothelial injury induced by hydrogen peroxide in vitro

Crocin, the digentiobiosyl ester of crocetin, was investigated for its cytoprotective effect on hydrogen peroxide-induced injury in bovine aortic endothelial cells (BAECs). The morphology of BAECs was observed by inverted phase contrast and electron microscopy. The MTT assay was used to measure cell viability. Cell apoptosis was evaluated by DNA argarose gel electrophoresis. The cells treated with H₂O₂ (200 μM) showed apoptotic changes as revealed by cell shrinkage, condensation of nuclei, membrane blebbing and formation of apoptotic body. A concentration-dependent inhibition of cell injury was seen in cultures treated with crocin at dosages ranging from 1 to 10 μM. Furthermore, in the H₂O₂-treated group, agarose gel electrophoresis displayed a “DNA ladder”. Whereas in the 10 μM crocin-pretreated group, cells remained intact and no “DNA ladder” was observed in agarose gel electrophoresis. Only very little DNA debris appeared on DNA-fragmentation analysis in the 1 μM crocin-pretreated group. Our data demonstrated that crocin has preventive effects on the cell apoptosis induced by H₂O₂, which may contribute to its utilisation for cardiovascular diseases (e.g., atherosclerosis and hypertension).     

Evidence of crocin against endothelial injury induced by hydrogen peroxide in vitro

Crocin, the digentiobiosyl ester of crocetin, was investigated for its cytoprotective effect on hydrogen peroxide-induced injury in bovine aortic endothelial cells (BAECs). The morphology of BAECs was observed by inverted phase contrast and electron microscopy. The MTT assay was used to measure cell viability. Cell apoptosis was evaluated by DNA argarose gel electrophoresis. The cells treated with H₂O₂ (200 μM) showed apoptotic changes as revealed by cell shrinkage, condensation of nuclei, membrane blebbing and formation of apoptotic body. A concentration-dependent inhibition of cell injury was seen in cultures treated with crocin at dosages ranging from 1 to 10 μM. Furthermore, in the H₂O₂-treated group, agarose gel electrophoresis displayed a “DNA ladder”. Whereas in the 10 μM crocin-pretreated group, cells remained intact and no “DNA ladder” was observed in agarose gel electrophoresis. Only very little DNA debris appeared on DNA-fragmentation analysis in the 1 μM crocin-pretreated group. Our data demonstrated that crocin has preventive effects on the cell apoptosis induced by H₂O₂, which may contribute to its utilisation for cardiovascular diseases (e.g., atherosclerosis and hypertension).     

普罗布考对过氧化氢诱导心肌细胞凋亡的影响

目的探讨普罗布考对过氧化氢(H2O2)诱导的心肌细胞凋亡的影响,探讨其可能的机制。方法培养的新生鼠心肌细胞随机分组并给予0.2mmol.L-1H2O2或0.2mmol.L-1 H2O2及不同浓度的普罗布考(100μmol.L-1、10μmol.L-1和1μmol.L-1)。采用琼脂糖凝聚电泳检测细胞凋亡;逆转录聚合酶链式反应(RT-PCR)法检测细胞Bcl-2 mRNA及Bax mRNA表达;黄嘌呤氧化酶法测定细胞内超氧化物歧化酶(SOD)活性,硫代巴比妥酸法检测细胞内丙二醛(MDA)含量。结果普罗布考明显抑制H2O2诱导的心肌细胞凋亡,降低心肌细胞BaxmRNA表达,升高Bcl-2mRNA表达。经不同浓度普罗布考干预后,心肌细胞SOD活性显著升高,MDA含量显著降低。以上这些效应均呈剂量依赖性。结论普罗布考抑制H2O2诱导的心肌细胞凋亡,其作用机制与改善心肌细胞氧化应激水平有关。     

丹皮酚对过氧化氢诱导的PC12细胞凋亡的抑制作用(英文)

目的探讨丹皮酚对过氧化氢(H2O2)诱导的PC12细胞凋亡的抑制作用及其机制。方法建立H2O2致PC12细胞损伤模型,采用MTT法测定细胞存活率,流式细胞术测定细胞凋亡率及细胞内活性氧含量,化学比色法测定乳酸脱氢酶(LDH)释放量及细胞内丙二醛(MDA)含量。结果PC12细胞经H2O2100μmol.L-1处理10 h可致细胞存活率下降,并能诱导细胞凋亡,LDH释放量及细胞内活性氧和MDA含量明显增加;丹皮酚(12,25和50μmol.L-1)预处理1 h可提高细胞存活率,减少细胞凋亡、LDH释放量及细胞内活性氧和MDA含量。结论丹皮酚对H2O2诱导的PC12细胞凋亡具有抑制作用,该作用可能与其抗氧化作用有关。     

5-HMF对过氧化氢诱导的人脐静脉血管内皮细胞损伤的保护作用（英文）

本文主要探讨5-羟甲基糠醛(5-HMF)对过氧化氢(H2O2)诱导人脐静脉血管内皮细胞(HUVECs)损伤及凋亡的保护作用及机制。研究结果表明,5-HMF通过降低与凋亡过程直接相关的Caspase-3 and NF-κB表达,增加Bcl-2表达,从而抑制H2O2诱导人脐静脉血管内皮细胞凋亡,并降低了NO含量,减轻H2O2对内皮细胞的损伤作用。5-HMF可以缓解血管内皮细胞功能障碍和相关并发症,对血管内皮细胞具有保护作用。     

Role of glutathione in dimethylarsinic acid-induced apoptosis

Inorganic arsenicals are clearly toxicants and carcinogens in humans. In mammals, including humans, inorganic arsenicals often undergo methylation, forming compounds such as dimethylarsinic acid (DMAs(V)). Recent evidence indicates that DMAs(V) is a complete carcinogen in rodents although evidence for inorganic arsenicals as carcinogens in rodents remains equivocal. Thus, we studied the molecular mechanisms of in vitro cytolethality of DMAs(V) using a rat liver epithelial cell line (TRL 1215). DMAs(V) selectively induced apoptosis in TRL 1215 cells; its LC(50) value after 48 h exposure was 4.5 mM. The addition of a glutathione synthase inhibitor, L-buthionine-[S,R]-sulfoximine (BSO), actually decreased DMAs(V)-induced apoptosis. DMAs(V) exposure temporarily decreased cellular reduced glutathione (GSH) levels and enhanced cellular glutathione S-transferase (GST) activity from 6 h after the exposure when the cells were still alive. Also, DMAs(V) exposure activated cellular caspase 3 activity with a peak at 18 h after the exposure when apoptosis began, and BSO treatment completely inhibited this enzyme activity. The additions of inhibitors of caspase 3, caspase 8, and caspase 9 significantly reduced DMAs(V)-induced apoptosis. Taken together, these data indicate that cellular GSH was required for DMAs(V)-induced apoptosis to occur, and activation of cellular caspases after conjugation of DMAs(V) with cellular GSH appears to be of mechanistic significance. Further research will be required to determine the role of intracellular GSH and methylation in the toxicity of arsenicals in chronic arsenic poisoning or in cases where arsenicals are used as chemotherapeutics.     

分光光度法检测清洁验证中过氧化氢残留

目的建立清洁验证中过氧化氢残留物限度检查的含量测定方法。方法利用过氧化氢与高锰酸钾在硫酸介质中能够发生定量氧化还原反应,并引起高锰酸钾溶液吸光度值降低的原理(反应方程式如下:2KMnO4+3H2SO4+5H2O2=K2SO4+2MnSO4+5O2↑+8H2O),采用分光光度法,以纯化水作参比在525nm处测定高锰酸钾溶液吸光度值的变化,并根据其变化值与过氧化氢含量成正比的关系,计算过氧化氢的含量。结果过氧化氢检测浓度在0～19.48μg·mL-1范围内线性良好,回归方程为:Y=0.0063X+0.0008,r=0.9989;最低检出限及最低定量限分别为0.162μg·mL-1、0.540μg.mL-1;平均回收率为102.1%,RSD为8.24%(n=6)。结论本法简便、准确、灵敏度高、重现性好,可用于清洁验证中过氧化氢残留的含量测定。     

EGFR-dependent ERK activation triggers hydrogen peroxide-induced apoptosis in OK renal epithelial cells

Oxidative stress induces activation of extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein kinase families. However, it is unclear in renal epithelial cells whether the ERK activation is involved in cell survival or cell death in H₂O₂-treated cells. The present study was undertaken to determine the role of the ERK activation in H₂O₂-induced apoptosis of renal epithelial cells using opossum kidney (OK) cells, an established proximal tubular epithelial cell line. H₂O₂ resulted in a time- and dose-dependent apoptosis of OK cells. H₂O₂ treatment caused marked sustained activation of ERK. The ERK activation was prevented by PD98059 and U0126, inhibitors of ERK1/2 upstream kinase MEK1/2. Apoptosis caused by H₂O₂ was prevented by U0126. Transient transfection with constitutive active MEK1 increased the H₂O₂-induced apoptosis, whereas transfection with dominant-negative mutants of MEK1 decreased the apoptosis. H₂O₂ produced hyperpolarization of mitochondrial membrane potential and activation of caspases-3. H₂O₂-induced ERK activation was inhibited by the Src family selective inhibitor PP2 and the epidermal growth factor receptor inhibitor AG1478. The presence of AG1478, but not PP2, prevented H₂O₂-induced cell death. Taken together, our findings suggest that the ERK activation mediated by epidermal growth factor receptor plays an active role in inducing H₂O₂-induced apoptosis of OK cells and functions upstream of mitochondria-dependent pathway to initiate the apoptotic signal.     

Uptake of gold nanoparticles in murine macrophage cells without cytotoxicity or production of pro-inflammatory mediators

Abstract

More information characterizing the biological responses to nanoparticles is needed to allow the U.S. Food and Drug Administration to evaluate the safety and effectiveness of products with nano-scale components. The potential cytotoxicity and inflammatory responses of Au NPs (60 nm, NIST standard reference materials) were investigated in murine macrophages. Cytotoxicity was evaluated by MTT and LDH assays. Cytokines (IL-6, TNF-α), nitric oxide, and ROS were assayed to assess inflammatory responses. Morphological appearance and localization of particles were examined by high resolution illumination microscopy, transmission electron microscopy (TEM), and scanning TEM coupled with EDX spectroscopy. Results showed no cytotoxicity and no elevated production of proinflammatory mediators; however, imaging analyses demonstrated cellular uptake of Au NPs and localization within intracellular vacuoles. These results suggest that 60 nm Au NPs, under the exposure conditions tested, are not cytotoxic, nor elicit pro-inflammatory responses. The localization of Au NPs in intracellular vacuoles suggests endosomal containment and an uptake mechanism involving endocytosis.     

Dimethylarsinic acid causes apoptosis in HL-60 cells via interaction with glutathione

 Inducibility of apoptosis in cultured human HL-60 cells by arsenic compounds, such as arsenite, arsenate, methylarsonic acid (MAA), and dimethylarisinic acid (DMAA), was investigated, together with the role of glutathione (GSH) in the induction. Among the arsenic compounds DMAA was the most potent in terms of the ability to cause the morphological changes (formation of nuclear fragmentation and apoptotic bodies) characteristic of apoptosis. Furthermore, fragmentation of internucleosomal DNA was also induced by DMAA. Depletion of cell GSH by L-buthionine-SR-sulfoximine, a selective inhibitor of γ-glutamylcysteine synthetase, enhanced the cytotoxicity of arsenite, arsenate, and MAA, while such depletion suppressed the cytotoxicity of DMAA. The depletion of GSH also suppressed the morphological changes and the fragmentation of internucleosomal DNA caused by DMAA, both of which are characteristic features of apoptosis. The results suggest that the death of cells caused by DMAA is due to apoptosis and that GSH is involved in the induction of apoptosis by this arsenic compound. Received: 17 January 1996/Accepted: 12 March 1996     

Activation of plasma membrane reduced glutathione transport in death receptor apoptosis of HepG2 cells

Cells undergoing apoptosis release reduced glutathione (GSH) into the extracellular space; however, the physiological significance and the mechanism behind the GSH export remain unclear. The present study demonstrates that GSH is released by HepG2 cells undergoing Fas, tumor necrosis factor alpha (TNFalpha), or tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-stimulated cell death. GSH release was observed at times when extracellular lactate dehydrogenase (LDH) activity and propidium iodide (PI) incorporation were low, suggesting that the GSH release does not occur because of nonspecific cell damage, but is occurring through a specific transport system. Caspase 3-like proteases were activated before GSH was released, indicating that protease may be involved in signaling GSH release. To investigate the mechanism of GSH release, studies were performed in the presence of GSH transport inhibitors, as well as 25 mM GSH in the media. Two organic anion transporter inhibitors, probenecid and dibromosulfophthalein (DBSP), were effective in inhibiting Fas-stimulated GSH release. The addition of 25 mM GSH to the extracellular media also prevented the loss of intracellular GSH and delayed cell death. These findings suggest that an organic anion transporter is involved in GSH release during apoptosis, and that maintenance of intracellular GSH levels during apoptosis provides protection for the cell.     

Effect of hydrogen peroxide on VIP-induced relaxation of the cat lower esophageal sphincter

We investigated the effects of hydrogen peroxide (H2O2) on relaxation of the cat lower esophageal sphincter (LES). Vasoactive intestinal peptide (VIP) caused dose-dependent relaxation of LES, and H2O2 reduced VIP-induced relaxation. Relaxation was also attenuated by pertussis toxin (PTX), indicating a Gi/o component. VIP treatment increased [35S]GTPgammaS binding to Gs and Gi3 protein, but not to Go, Gq, Gil or Gi2. This increase in Gs or Gi3 binding was reduced by H2O2. However, the relaxation induced by sodium nitroprusside (SNP), 3-morpholino sydnomine (SIN-1), 8-br cGMP (cGMP analog), forskolin (adenylate cyclase activator), and dibutyryl-cAMP (a stable cAMP analog) was not reduced by H2O2. These data suggest that H202 inhibits VIP-induced relaxation via a Gi-dependent pathway, perhaps by inhibiting the activation of G(i3) or Gs downstream of the VIP receptor and independent of cAMP or NO-cGMP signaling.     

灯盏乙素对过氧化氢致PC12细胞损伤的抗氧化作用

目的研究灯盏乙素对过氧化氢(H2O2)诱导PC12细胞损伤的保护作用。方法建立H2O2致PC12细胞损伤模型,倒置相差显微镜下进行一般形态学观察,化学比色法测定乳酸脱氢酶(LDH)释放量及细胞培养液和细胞内丙二醛(MDA)含量和超氧化物歧化酶(SOD)活性。结果100μmol/L H2O2诱导PC12细胞4 h,细胞呈现明显损伤形态,LDH释放量增加,细胞培养液和细胞内MDA含量升高,SOD活性降低。灯盏乙素可明显改善形态学损伤,显著降低LDH释放量、细胞培养液及细胞内MDA含量,提高SOD活性。结论灯盏乙素对H2O2诱导PC12细胞损伤具有保护作用,其作用机制可能与提高PC12细胞的抗氧化能力有关。     

Influence of PEG coating on the oral bioavailability of gold nanoparticles in rats

Metallic nanoparticles can be produced in a variety of shapes, sizes, and surface chemistries, making them promising potential tools for drug delivery. Most studies to date have evaluated uptake of metallic nanoparticles from the GI tract with methods that are at best semi-quantitative. This study used the classical method of comparing blood concentration area under the curve (AUC) following intravenous and oral doses to determine the oral bioavailability of 1, 2 and 5?kDa PEG-coated 5?nm gold nanoparticles (AuNPs). Male rats were given a single intravenous dose (0.8?mg/kg) or oral (gavage) dose (8?mg/kg) of a PEG-coated AuNP, and the concentration of gold was measured in blood over time and in tissues (liver, spleen and kidney) at sacrifice. Blood concentrations following oral administration were inversely related to PEG size, and the AUC in blood was significantly greater for the 1?kDa PEG-coated AuNPs than particles coated with 2 or 5?kDa PEG. However, bioavailabilities of all of the particles were very low (相似文献   

Metabolomics reveals the depletion of intracellular metabolites in HepG2 cells after treatment with gold nanoparticles

Studies on the safety of gold nanoparticles (GNPs) are plentiful due to their successful application in drug delivery and treatment of diseases in trials. Cytotoxicity caused by GNPs has been studied on the physiological and biochemical level; yet, the effect of GNPs (particularly gold nano-spheres) on the metabolome of living organisms remains understudied. In this investigation, metabolomics was used to comprehensively study the metabolic alterations in HepG2 cells caused by GNPs; and to investigate the role of representative GNP coatings. GNPs were synthesized, coated and characterized before use on HepG2 cell cultures. Cells were treated for 3?h with citrate-, poly-(sodiumsterene sulfunate)-, and poly-vinylpyrrolidone (PVP)-capped GNPs, respectively. The internalization of the different GNPs and their effect on mitochondrial respiration and the metabolome were studied. Results indicated that the PVP-capped GNPs internalized more and also caused a more observable effect on the metabolome. Conversely, it was the citrate- and poly-(sodiumsterene sulfunate) coated particles that influenced ATP production in addition to the metabolomic changes. A holistic depletion of intracellular metabolites was observed regardless of GNP coating, which hints to the binding of certain metabolites to the particles.     

Competitive oxidation of 6-hydroxydopamine by oxygen and hydrogen peroxide.

Simultaneous oxygen electrode and conventional polarographic measurements show the net concentration of hydrogen peroxide produced by air oxidation of 6-hydroxydopamine is considerably less than that predicted from the known stoichiometry of the reaction. This is due to competitive oxidation of 6-hydroxydopamine by the generated hydrogen peroxide. The presence of ascorbic acid in this reaction also results in significant decreases of hydrogen peroxide under most conditions. The implications of these results to the molecular mechanism of 6-hydroxydopamine neurotoxicity are discussed.     

金雀异黄素对正常的和过氧化氢损伤的内皮细胞的影响(英文)

目的 :观察金雀异黄素 (genistein)对体外培养的血管内皮细胞 (ECV 3 0 4)生长的影响和对过氧化氢引起ECV 3 0 4细胞损伤的保护作用。方法 :将ECV 3 0 4细胞与金雀异黄素 0 .1～ 1 0 0 μmol·L-1共同孵育 2 4～ 96h ,通过MTT法测定细胞存活率。为了观察金雀异黄素对过氧化氢引起ECV 3 0 4细胞损伤的保护作用 ,将细胞分成 3组 ,分别为对照组、过氧化氢损伤模型组和金雀异黄素治疗组 ,治疗组ECV 3 0 4细胞先与金雀异黄素 6.2 5～ 1 0 0 μmol·L-1共同孵育 3 0min ,然后模型组和治疗组ECV3 0 4细胞均加入过氧化氢 1 0mmol·L-1孵育 ,3 0min后以MTT法测定OD值 ,以此反映各组ECV 3 0 4的生存情况。结果 :金雀异黄素对于正常ECV 3 0 4细胞生长的作用与浓度和作用时间有关 ,浓度为1 0 0 μmol·L-1时 ,作用 2 4,48h能明显促进ECV3 0 4生长 (P <0 .0 1 ) ,而作用 96h则表现为明显的抑制作用 (P <0 .0 5 ) ;浓度为 1 0 μmol·L-1时 ,作用48,72h对ECV 3 0 4细胞生长也有促进作用 (P <0 .0 5 ) ;而当金雀异黄素浓度降低为 0 .1 ,1 μmol·L-1时 ,对ECV 3 0 4细胞生长无明显影响。过氧化氢 1 0mmol·L-1可使ECV 3 0 4的存活率明显下降(P <0 .0 5 ) ;预先给予金雀异黄素 2 5 ,5 0 ,1 0 0 μmol·L-1能浓度依赖性地