Molecular Responses of Mouse Macrophages to Copper and Copper Oxide Nanoparticles Inferred from Proteomic Analyses

The molecular responses of macrophages to copper-based nanoparticles have been investigated via a combination of proteomic and biochemical approaches, using the RAW264.7 cell line as a model. Both metallic copper and copper oxide nanoparticles have been tested, with copper ion and zirconium oxide nanoparticles used as controls. Proteomic analysis highlighted changes in proteins implicated in oxidative stress responses (superoxide dismutases and peroxiredoxins), glutathione biosynthesis, the actomyosin cytoskeleton, and mitochondrial proteins (especially oxidative phosphorylation complex subunits). Validation studies employing functional analyses showed that the increases in glutathione biosynthesis and in mitochondrial complexes observed in the proteomic screen were critical to cell survival upon stress with copper-based nanoparticles; pharmacological inhibition of these two pathways enhanced cell vulnerability to copper-based nanoparticles, but not to copper ions. Furthermore, functional analyses using primary macrophages derived from bone marrow showed a decrease in reduced glutathione levels, a decrease in the mitochondrial transmembrane potential, and inhibition of phagocytosis and of lipopolysaccharide-induced nitric oxide production. However, only a fraction of these effects could be obtained with copper ions. In conclusion, this study showed that macrophage functions are significantly altered by copper-based nanoparticles. Also highlighted are the cellular pathways modulated by cells for survival and the exemplified cross-toxicities that can occur between copper-based nanoparticles and pharmacological agents.Manufactured nanoparticles are more and more widely used in more and more consumer products, ranging from personal care products to tires and concrete. Among the nanoparticles, metals and metal oxides represent an important part of the total production and are used in water treatment, as antibacterials, in antifouling paints, and in microelectronics. These varied uses in turn pose the problem of the toxicological evaluation of these nanoparticles (1, 2), and especially of the long-term effects that often come not from simple cell mortality but from altered cellular functions.Macrophages are one of the cell types that deserve special attention in toxicology, because of the variety of their functions. Altered cytokine production can lead to adverse long-term effects, as documented, for example, in the case of asbestos (3). Other dysfunctions of the innate immune system can lead to deregulation of the immune responses and to severe adverse effects, such as a higher incidence of tumors (4).It is therefore not surprising that the immunotoxicology of nanoparticles is a developing field (5–7), and several studies have been devoted to macrophages'' response to nanoparticles. However, most of these studies have been limited to the effect of nanoparticles on cell viability and on cytokine production (e.g. 8–11), although some also studied oxidative stress (12–14) and sometimes other functional parameters (15–17). Very few studies have used the analytical power of proteomics to go deeper into the mechanisms of the response to nanoparticles or metals (reviewed in Ref. 18). A few exceptions are studies on, for example, carbon-based nanoparticles (19) and titanium dioxide (20, 21).Most of the toxicological studies in this field have been focused on a few nanoparticles used either as health products, such as iron oxide (15, 17, 22), or in a variety of consumer products, such as silver (13, 14), silica (9, 12), and titanium dioxide (11, 16, 20, 21).However, many other nanoparticles are being used more and more in industrial applications without extensive toxicological testing. Good examples are indium-tin oxide, used in electronic screens, which appears to be toxic (23), and the copper-based nanoparticles used in high-performance batteries (24), in water depollution (25), and as bactericides as a replacement for nano-silver. Copper and copper oxide induce a strong toxicity (26, 27), coupled with inflammation (28), oxidative stress (29), and genotoxicity (30), at least in epithelial cells.In light of these various effects, we decided to use a combination of a proteomics approach and targeted approaches to address in more molecular detail the responses of macrophages to copper-based nanoparticles (i.e. both metallic copper and copper II oxide).     

Handling of Copper and Copper Oxide Nanoparticles by Astrocytes

Copper is an essential trace element for many important cellular functions. However, excess of copper can impair cellular functions by copper-induced oxidative stress. In brain, astrocytes are considered to play a prominent role in the copper homeostasis. In this short review we summarise the current knowledge on the molecular mechanisms which are involved in the handling of copper by astrocytes. Cultured astrocytes efficiently take up copper ions predominantly by the copper transporter Ctr1 and the divalent metal transporter DMT1. In addition, copper oxide nanoparticles are rapidly accumulated by astrocytes via endocytosis. Cultured astrocytes tolerate moderate increases in intracellular copper contents very well. However, if a given threshold of cellular copper content is exceeded after exposure to copper, accelerated production of reactive oxygen species and compromised cell viability are observed. Upon exposure to sub-toxic concentrations of copper ions or copper oxide nanoparticles, astrocytes increase their copper storage capacity by upregulating the cellular contents of glutathione and metallothioneins. In addition, cultured astrocytes have the capacity to export copper ions which is likely to involve the copper ATPase 7A. The ability of astrocytes to efficiently accumulate, store and export copper ions suggests that astrocytes have a key role in the distribution of copper in brain. Impairment of this astrocytic function may be involved in diseases which are connected with disturbances in brain copper metabolism.     

Iron-Doping of Copper Oxide Nanoparticles Lowers Their Toxic Potential on C6 Glioma Cells

Neurochemical Research - Copper oxide nanoparticles (CuO-NPs) are well known for their cytotoxicity which in part has been attributed to the release of copper ions from CuO-NPs. As iron-doping has...     

Uptake and Toxicity of Copper Oxide Nanoparticles in C6 Glioma Cells

Copper oxide nanoparticles (CuO-NPs) are frequently used for many technical applications, but are also known for their cell toxic potential. In order to investigate a potential use of CuO-NPs as a therapeutic drug for glioma treatment, we have investigated the consequences of an application of CuO-NPs on the cellular copper content and cell viability of C6 glioma cells. CuO-NPs were synthesized by a wet-chemical method and were coated with dimercaptosuccinic acid and bovine serum albumin to improve colloidal stability in physiological media. Application of these protein-coated nanoparticles (pCuO-NPs) to C6 cells caused a strong time-, concentration- and temperature-dependent copper accumulation and severe cell death. The observed loss in cellular MTT-reduction capacity, the loss in cellular LDH activity and the increase in the number of propidium iodide-positive cells correlated well with the specific cellular copper content. C6 glioma cells were less vulnerable to pCuO-NPs compared to primary astrocytes and toxicity of pCuO-NPs to C6 cells was only observed for incubation conditions that increased specific cellular copper contents above 20 nmol copper per mg protein. Both cellular copper accumulation as well as the pCuO-NP-induced toxicity in C6 cells were prevented by application of copper chelators, but not by endocytosis inhibitors, suggesting that liberation of copper ions from the pCuO-NPs is the first step leading to the observed toxicity of pCuO-NP-treated glioma cells.     

Copper Oxide Nanoparticles Greener Synthesis Using Tea and its Antifungal Efficiency on Fusarium solani

Abstract

A great deal of research has been done on various uses of copper oxide. The synthesis of copper oxide nanoparticles was mediated using tea extract. The first sign of the reduction of copper ions to copper oxide was the change in color of extract to dark brown after treating with copper chloride. The resulting nanoparticles were characterized using X-ray diffraction (XRD) and Fourier transform infrared spectrometry (FTIR). Finally, the antimicrobial effects of these nanoparticles on Fusarium solani were studied in vitro by agar dilution method. The TEM images showed the synthesis of copper oxide with size of less than 80?nm. The synthesized copper oxide nanoparticles showed significant inhibitory effects on F. solani cultures so that the concentration of 80?μg/ml prevented approximately 90% of the mycelium growth of the fungus. The results showed that the inhibition zone of silver nanoparticles strongly depends on their concentration and increases by increasing the concentration of copper oxide nanoparticles in the medium.     

Uptake of Intact Copper Oxide Nanoparticles Causes Acute Toxicity in Cultured Glial Cells

Copper oxide nanoparticles (CuO-NPs) dispersions are known for their high cell toxic potential but contaminating copper ions in such dispersions are a major hurdle in the investigation of specific nanoparticle-mediated toxicity. In order to distinguish between the adverse effects exhibited by CuO-NPs and/or by contaminating ionic copper, the membrane-impermeable copper chelator bathocuproine disulfonate (BCS) was added in a low molar ratio (20% of the total copper applied) in order to chelate the copper ions that had been released extracellularly from the CuO-NPs before or during the incubation. Physicochemical characterization of synthesized CuO-NPs revealed that the presence of this low concentration of BCS did not alter the size or zeta potential of the CuO-NPs. Application of CuO-NPs to C6 glioma cells and primary astrocytes induced a concentration- and temperature-dependent copper accumulation which was accompanied by a severe loss in cell viability. The adverse consequences of the CuO-NP application were not affected by the presence of 20% BCS, while the copper accumulation and cell toxicity observed after application of ionic copper were significantly lowered in the presence of BCS. These results demonstrate that for the experimental conditions applied the adverse consequences of an exposure of cultured glial cells to dispersions of CuO-NPs are mediated by accumulated NPs and not caused by the uptake of contaminating copper ions.

     

Induction of hepatic heme oxygenase activity by bromobenzene

Hepatic heme oxygenase, an enzyme which converts heme to carbon monoxide and bile pigment in vitro, is inducible by heme but also by large “toxic” doses of such nonheme substances as hormones, endotoxin, and heavy metal ions. When we gave rats a single hepatotoxic dose of allyl alcohol, ethionine, acetaminophen, furosemide, or endotoxin, hepatic heme oxygenase activity rose modestly (two- to fivefold) after 20 h. In contrast, administration of bromobenzene (5 mmol/kg) induced heme oxygenase in the liver an average of 15-fold after 20 h but was without effect on the enzyme in the kidney or spleen. The change in heme oxygenase was accompanied by a loss in cytochrome P-450 concentration and, in rats labeled with 5-δ-amino[¹⁴C]levulinic acid, an increased rate of degradation of hepatic [¹⁴C]heme to ¹⁴CO. Induction of heme oxygenase by bromobenzene was blocked by cycloheximide, an inhibitor of protein synthesis, but not by actinomycin D, an inhibitor of RNA synthesis. This suggests that bromobenzene stimulates de novo enzyme synthesis at the step of translation. Subtoxic doses of bromobenzene (less than 1 mmol/kg) gave proportionately greater induction of heme oxygenase. Furthermore, induction of the enzyme remained unaffected when bromobenzene hepatotoxicity was blocked by pretreatment of rats with SKF-525A, 3-methylcholanthrene, or cysteine (which supplements liver sulfhydryl content), or when hepatotoxicity was enhanced by pretreatment with phenobarbital or with diethylmaleate (which depletes hepatic glutathione). These data suggest that with induction of heme oxygenase by bromobenzene, neither liver cell necrosis nor alteration in hepatic sulfhydryl metabolism is indispensible. The latter characteristic differs from induction of the enzyme by metal ions in which depletion of sulfhydryl-containing constituents has been thought to be essential. We conclude that bromobenzene is a novel inducer of heme oxygenase activity in the liver, differing from other nonheme substances in potency and specificity for the liver, and in utilizing mechanism(s) which require neither production of hepatotoxicity, depletion of hepatic glutathione, nor sensitivity to actinomycin D.     

Induction in mouse peritoneal macrophages of 34 kDa stress protein and heme oxygenase by sulfhydryl-reactive agents

The synthesis of 34-kDa stress protein was enhanced, with a simultaneous increase in heme oxygenase activity, when mouse macrophages were exposed to diethylmaleate or sodium arsenite. After 7 h of exposure to the sulfhydryl agents, the 34-kDa protein was the most actively synthesized protein. Immunoblot analysis showed that the induced 34-kDa protein reacted with an antibody raised against bovine heme oxygenase. Cadmium ions or 1-chloro-2,4-dinitrobenzene also induced the 34-kDa protein which reacted with the antibody. Treatments of the cells with buthionine sulfoximine or hydrogen peroxide weakly induced the protein, while diamide treatment or heat shock was without effect. These results are consistent with our previous findings that heavy metal ions including arsenite and cadmium ions induce heme oxygenase (32-kDa stress protein) in human cell lines [Taketani, S., Kohno, H., Yoshinaga, T., & Tokunaga, R. (1989) FEBS Lett. 245, 173-176], and also suggest that the formation of glutathione conjugate with sulfhydryl-reactive agents may mediate the induction of the stress protein in mouse peritoneal macrophages.     

Minimal In Vitro Antimicrobial Efficacy and Ocular Cell Toxicity from Silver Nanoparticles

Silver in various forms has long been recognized for antimicrobial properties, both in biomedical devices and in eyes. However, soluble drugs used on the ocular surface are rapidly cleared through tear ducts and eventually ingested, resulting in decreased efficacy of the drug on its target tissue and potential concern for systemic side effects. Silver nanoparticles were studied as a source of anti-microbial silver for possible controlled-release contact lens controlled delivery formulations. Silver ion release over a period of several weeks from nanoparticle sources of various sizes and doses was evaluated in vitro against Pseudomonas aeruginosa strain PAO1. Mammalian cell viability and cytokine expression in response to silver nanoparticle exposure is evaluated using corneal epithelial cells and eye-associated macrophages cultured in vitro in serum-free media. Minimal microcidal and cell toxic effects were observed for several silver nanoparticle suspensions and aqueous extraction times for bulk total silver concentrations commensurate with comparative silver ion (e.g., ) toxicity. This indicates that (1) silver particles themselves in these size ranges (20–60 nm diameter) are not microcidal under conditions tested, and (2) insufficient silver ion is generated from these particles at these silver ion-equivalent loadings to produce observable biological effects compared to silver ions in these in vitro assays. This is consistent with confounding literature describing both efficacy and lack of microcidal effects for silver nanoparticles, depending on milieu, surface oxide properties, and size. If dosing allows substantially increased silver particle loading in the lens to produce sufficient pathogen-toxic silver ions and/or particle-microbe direct contact, the bactericidal efficacy of silver nanoparticles in vitro could possibly limit bacterial colonization problems associated with extended-wear contact lenses.     

Toxicity Assessment of Iron Oxide Nanoparticles in Zebrafish (Danio rerio) Early Life Stages

Iron oxide nanoparticles have been explored recently for their beneficial applications in many biomedical areas, in environmental remediation, and in various industrial applications. However, potential risks have also been identified with the release of nanoparticles into the environment. To study the ecological effects of iron oxide nanoparticles on aquatic organisms, we used early life stages of the zebrafish (Danio rerio) to examine such effects on embryonic development in this species. The results showed that ≥10 mg/L of iron oxide nanoparticles instigated developmental toxicity in these embryos, causing mortality, hatching delay, and malformation. Moreover, an early life stage test using zebrafish embryos/larvae is also discussed and recommended in this study as an effective protocol for assessing the potential toxicity of nanoparticles. This study is one of the first on developmental toxicity in fish caused by iron oxide nanoparticles in aquatic environments. The results will contribute to the current understanding of the potential ecotoxicological effects of nanoparticles and support the sustainable development of nanotechnology.     

Selenium antagonizes the induction of human heme oxygenase by arsenite and cadmium ions.

Effects of selenium compounds on the induction of heme oxygenase in human cells exposed to sodium arsenite or cadmium chloride have been investigated by an immunoblotting technique. Exposure of HeLa cells to arsenite or cadmium ions caused a marked increase in the synthesis of heme oxygenase, and the presence of sodium selenite suppressed the induction. DL-Selenocystine was an effective suppressor, and sodium selenate was less effective. DL-Selenomethionine had no effect. Northern blot analysis showed that selenite abolished the induction of heme oxygenase mRNA in the cells exposed to arsenite or cadmium ions. These results indicated that selenium antagonizes the induction of heme oxygenase by heavy metals ions.     

Handling of Iron Oxide and Silver Nanoparticles by Astrocytes

Metal-containing nanoparticles (NPs) are currently used for various biomedical applications. Since such NPs are able to enter the brain, the cells of this organ have to deal with NPs and with NP-derived metal ions. In brain, astrocytes are considered to play a key function in regulating metal homeostasis and in protecting other brain cells against metal toxicity. Thus, among the different types of brain cells, especially astrocytes are of interest regarding the uptake and the handling of metal-containing NPs. This article summarizes the current knowledge on the consequences of an exposure of astrocytes to NPs. Special focus will be given to magnetic iron oxide nanoparticles (IONPs) and silver nanoparticles (AgNPs), since the biocompatibility of these NPs has been studied for astrocytes in detail. Cultured astrocytes efficiently accumulate IONPs and AgNPs in a time-, concentration- and temperature-dependent manner by endocytotic processes. Astrocytes are neither acutely damaged by the exposure to high concentrations of NPs nor by the prolonged intracellular presence of large amounts of accumulated NPs. Although metal ions are liberated from accumulated NPs, NP-derived iron and silver ions are not exported from astrocytes but are rather stored in proteins such as ferritin and metallothioneins which are synthesized in NP-treated astrocytes. The efficient accumulation of large amounts of metal-containing NPs and the upregulation of proteins that safely store NP-derived metal ions suggest that astrocytes protect the brain against the potential toxicity of metal-containing NPs.     

Human heme oxygenase cDNA and induction of its mRNA by hemin

Hemin treatment increased both activity and mRNA level of heme oxygenase in human macrophages. Using poly(A)-rich RNA prepared from human macrophages treated with hemin, we have constructed a cDNA library in the Okayama-Berg vector. The human heme oxygenase cDNA was isolated by screening this library with a rat cDNA and was subjected to nucleotide sequence analysis. The deduced human heme oxygenase is composed of 288 amino acids with a molecular mass of 32,800 Da. The homology in amino acid sequences between rat and human heme oxygenase is 80%. Like rat heme oxygenase, human enzyme has a putative membrane segment at its carboxyl terminus, which is probably essential for the insertion of heme oxygenase into endoplasmic reticulum. Both rat and human heme oxygenase have no cysteine residues. Recently we have shown that rat heme oxygenase is a heat-shock protein [J. Biol. Chem. 262, 12889-12892 (1987)], and therefore we examined the effects of heat treatment on the induction of heme oxygenase in human macrophages and glioma cells. In contrast to hemin treatment, heat treatment had no apparent effects in either human cell line on the activity of heme oxygenase and its mRNA levels. These results suggest that human heme oxygenase may not be a heat-shock protein.     

Proteome profiling reveals potential toxicity and detoxification pathways following exposure of BEAS-2B cells to engineered nanoparticle titanium dioxide

Oxidative stress is known to play important roles in engineered nanomaterial‐induced cellular toxicity. However, the proteins and signaling pathways associated with the engineered nanomaterial‐mediated oxidative stress and toxicity are largely unknown. To identify these toxicity pathways and networks that are associated with exposure to engineered nanomaterials, an integrated proteomic study was conducted using human bronchial epithelial cells, BEAS‐2B and nanoscale titanium dioxide. Utilizing 2‐DE and MS, we identified 46 proteins that were altered at protein expression levels. The protein changes detected by 2‐DE/MS were verified by functional protein assays. These identified proteins include some key proteins involved in cellular stress response, metabolism, adhesion, cytoskeletal dynamics, cell growth, cell death, and cell signaling. The differentially expressed proteins were mapped using Ingenuity Pathway Analyses^? canonical pathways and Ingenuity Pathway Analyses tox lists to create protein‐interacting networks and proteomic pathways. Twenty protein canonical pathways and tox lists were generated, and these pathways were compared to signaling pathways generated from genomic analyses of BEAS‐2B cells treated with titanium dioxide. There was a significant overlap in the specific pathways and lists generated from the proteomic and the genomic data. In addition, we also analyzed the phosphorylation profiles of protein kinases in titanium dioxide‐treated BEAS‐2B cells for a better understanding of upstream signaling pathways in response to the titanium dioxide treatment and the induced oxidative stress. In summary, the present study provides the first protein‐interacting network maps and novel insights into the biological responses and potential toxicity and detoxification pathways of titanium dioxide.     

Endothelial heme oxygenase-1 induction by hypoxia. Modulation by inducible nitric-oxide synthase and S-nitrosothiols

The stress protein heme oxygenase-1 (HO-1) is induced in endothelial cells exposed to nitric oxide (NO)-releasing agents, and this process is finely modulated by thiols (Foresti, R., Clark, J. E., Green, C. J., and Motterlini R. (1997) J. Biol. Chem. 272, 18411-18417). Here, we report that up-regulation of HO-1 in aortic endothelial cells by severe hypoxic conditions (pO(2) 相似文献   

Rapid Pharmacokinetic and Biodistribution Studies Using Cholorotoxin-Conjugated Iron Oxide Nanoparticles: A Novel Non-Radioactive Method

Background

Recent advances in nanotechnology have led to the development of biocompatible nanoparticles for in vivo molecular imaging and targeted therapy. Many nanoparticles have undesirable tissue distribution or unacceptably low serum half-lives. Pharmacokinetic (PK) and biodistribution studies can help inform decisions determining particle size, coatings, or other features early in nanoparticle development. Unfortunately, these studies are rarely done in a timely fashion because many nanotechnology labs lack the resources and expertise to synthesize radioactive nanoparticles and evaluate them in mice.

Methodology/Principal Findings

To address this problem, we developed an economical, radioactivity-free method for assessing serum half-life and tissue distribution of nanoparticles in mice. Iron oxide nanoparticles coated with chitosan and polyethylene glycol that utilize chlorotoxin as a targeting molecule have a serum half-life of 7–8 hours and the particles remain stable for extended periods of time in physiologic fluids and in vivo. Nanoparticles preferentially distribute to spleen and liver, presumably due to reticuloendothelial uptake. Other organs have very low levels of nanoparticles, which is ideal for imaging most cancers in the future. No acute toxicity was attributed to the nanoparticles.

Conclusions/Significance

We report here a simple near-infrared fluorescence based methodology to assess PK properties of nanoparticles in order to integrate pharmacokinetic data into early nanoparticle design and synthesis. The nanoparticles tested demonstrate properties that are excellent for future clinical imaging strategies and potentially suitable for targeted therapy.     

组蛋白H3第10位丝氨酸的磷酸化：金属纳米材料早期毒性的潜在生物标志物

目的 阐明金属纳米材料（MNPs）对组蛋白H3第10位丝氨酸磷酸化（p-H3S10）修饰变化的影响，探讨典型MNPs暴露后细胞全基因表达的变化，为MNPs早期毒性筛选提供理论基础。方法 通过蛋白质免疫印迹及流式细胞术等方法评价了10种MNPs对p-H3S10修饰变化的影响。此外，利用转录组测序技术在转录水平上探讨了1种典型MNPs——纳米氧化铜对细胞全基因表达的影响。结果 除纳米氧化镍外，其余用于测试的9种MNPs均在不同程度上诱导了p-H3S10。进一步分析发现，MNPs诱导的p-H3S10与MNPs的细胞内蓄积高度相关，且细胞内金属离子的持续释放可能是MNPs诱导 p-H3S10的关键因素之一。另外，转录组测序的结果表明，纳米氧化铜的暴露导致了275个基因的显著差异表达（P<0.05），其中185个基因上调，90个基因下调。基因本体分析表明，在分子功能类别中，排名靠前的术语包括与多种转录因子活性、序列特异性DNA结合及丝裂原活化蛋白激酶活性相关的术语。京都基因和基因组百科全书分析表明，纳米氧化铜暴露后丝裂原活化蛋白激酶的信号级联显著上调。结论 MNPs的细胞内蓄积与其早期诱导的p-H3S10表达高度相关，并且细胞内MNPs持续释放的金属离子可能会在MNPs进入细胞后的很长一段时间内持续诱导p-H3S10的高表达。综上，p-H3S10具有作为评估MNPs毒性的生物标志物的潜力。