Abstract
BACKGROUND:Reports have demonstrated that cytotoxicity produced by ferric oxide (Fe2O3) nanoparticles is associated with cellular lipid peroxidation. Whether Fe2O3 nanoparticles have toxicity to macrophages, and what is the association of toxic mechanism and oxidization?

OBJECTIVE: To observe the effects of different concentrations of Fe2O3 nanoparticles on the oxidative damage of macrophages.

DESIGN: A controlled observation experiment.

SETTING: School of Public Health, Southeast University.

MATERIALS: RAW264.7 cells were peritoneal macrophages of mouse and purchased from Shanghai Institute of cells, Chinese Academy of Sciences. Fe2O3 nanoparticles (30 nm) suspension was provided by Department of Biomedical Engineering, Southeast University). Fe2O3 nanoparticle suspension was placed in 60 ℃ water for 10 hours, then in 37 ℃ water overnight. This procedure was repeated 3 times for germicidal treatment. Then, the suspension was packed into small bottles and stored at 4 ℃ for later use. DMEM high glucose culture fluid (Gibco Company, USA); trypsinase (Difco Company, USA, imported); new-born calf serum(Sijiqing Company, Hangzhou); hydrogen dioxide （H2O2, Gibco Company）; Kits for measuring hydrogen dioxide(H2O2), hydroxy radical （·OH）, superoxide anion radical （O2·－）, lactic acid dehydrogenase, ultramicro ATP enzyme and Coomassie brilliant blue protein levels (Jiancheng Biotechnique Co., Ltd., Nanjing).

METHODS: This experiment was carried out in the laboratory of Department of Labor and Environmental Health, School of Public Health, Dongnan University between March 2006 and July 2006. RAW264.7 cells (Abelson murine leukemia virus-induced tumor) were cultured in DMEM (Gibco Company) containing 100 g/L fetal bovine serum, 100 000 U/L penicillin and 100 mg/L streptomycin in the environment of 5% CO2. Cell growth was observed under an inverted microscope. Cells, which were at good exponential phase of growth, were chosen for test. ①Detection of oxygen free radical in the cells: 1.5×108 L－1 macrophages were inoculated to 24-well plate, 1 mL a well. After the macrophages were cultured for 24 hours in incubation at 37 ℃ in a humidified atmosphere containing 5% CO2. 1.070 0, 0.535 0 and 0.267 5 g/L Fe2O3 nanoparticles (30 nm) suspension-intervened macrophages were set as Fe2O3 nanoparticle group, and normal saline group was set as control group. Following the intervention of nanoparticles, macrophages were disrupted with vitreous homogenizer. The levels of hydrogen peroxide (H2O2), hydroxyl radical (·OH) and superoxide anion (O2·－) were determined in cell lysates and supernatants using the resgent kit (Nanjing Jiancheng Bioengineering Co., Ltd). ②Determination of the activities of lactate dehydrogenase (LDH), Na+-K+-ATPase and Ca2+-Mg2+-ATPase: Macrophages in the Fe2O3 nanoparticle group and control group were treated as above. The activities of LDH in culture medium were determined according to the instruction of reagent kit (Nanjing Jiancheng Bioengineering Co., Ltd). And the activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase were also determined according to the instruction of reagent kit (Nanjing Jiancheng Bioengineering Co., Ltd) at low temperature.

MAIN OUTCOME MEASURES: ①Effects of different concentrations of Fe2O3 nanoparticles on the production of H2O2, ·OH and O2·－ in RAW264.7 cells. ②Effects of different concentrations of Fe2O3 nanoparticles on the activities of LDH , Na+-K+-ATPase and Ca2+-Mg2+-ATPase in RAW264.7 cell culture fluid.

RESULTS: ① Level of ·OH free radical in Fe2O3 nanoparticle 0.267 5, 0.535 0, 1.070 0 g/L groups was higher than that in control group, respectively [（0.605±0.066），（0.410±0.080），（0.764±0.051），（0.285±0.057）mkat/g, P < 0.05]; Level of O2·－ free radical in Fe2O3 nanoparticle 0.267 5, 0.535 0, 1.070 0 g/L groups was higher than that in control group, respectively[（9.935±1.159），（8.912±0.131），（13.479±0.752），（5.635±0.475）μkat/g，P < 0.05]; Level of H2O2 in Fe2O3 nanoparticle 1.070 0 g/L group was higher than that in the control group [（14.695±2.815），（2.397±0.399） mmol/L，P < 0.05]. ②Fe2O3 nanoparticles within the range of experimental concentration could cause LDH activity significantly increased (P < 0.05). Fe2O3 nanoparticles had effects on the activities of Na+,K+-ATPase and Ca2+,Mg2+-ATPase. With the increase of dose of Fe2O3 nanoparticles, the activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase were gradually decreased. There were significant differences as compared with control group (P < 0.05)

CONCLUSION: Increasing dose of Fe2O3 nanoparticles would cause more H2O2, ·OH and O2·－ free radicals in the cells, increase cell membrane permeability and inhibit the activities of LDH , Na+-K+-ATPase and Ca2+-Mg2+-ATPase.

INTRODUCTION

Nanomaterials have shown wide application prospects in many domains because of their unique physical and chemical performance [1,2]. Its wide application also provides more chances for human to contact them (production, application and waste management, etc.) and for their release in the environment. The development and application of nanotechnology has exceeded the analysis and study range of corresponding health and environment risk, meanwhile, people have faced new challenge in comprehending, predicting and managing potential noxious substance [3-5].
Among numerous nanomaterials, ferric oxide (Fe2O3) nanoparticles are a magnetic material with strong magnetism, relatively simple preparation and good biocompatibility. They are commonly used magnetic carrier material in biomedical domain [6]. There are many studies on application of Fe2O3 nanoparticles to magnetic drug delivery and targeted position tumor diagnosis and therapy. Up to now, some progresses have been made in research of cancer therapy with magnetic nanoparticles. Among them, hematite (Fe2O3), magnetite (Fe3O4) and other magnet powder are commonly used.
Some study already indicated that when rats inhaled 92 mg/m3 Fe2O3 nanoparticles 6 hours a day for 3 days, thus, mild respiratory tract reaction could be found[7]. Also, cytological experiment studies demonstrated that cytoxicity produced by Fe2O3 nanoparticles was related to cellular lipid peroxidation[8]. Macrophage is one of the detective cells when body earlily contacted exotic in local [9,10]. When Fe2O3 nanoparticles were injected into bodies, inevitably some of them could enter into tissue, so macrophage would be activated. In order to investigate whether Fe2O3 nanoparticles have cytotoxicity to macrophage and the association of cytotoxicity and oxidative effects.

MATERIALS AND METHODS

Materials
RAW264.7 cells were peritoneal macrophages of mouse and purchased from Shanghai Institute of cells, Chinese Academy of Sciences. Fe2O3 nanoparticles (30 nm) suspension was provided by Department of Biomedical Engineering, Southeast University). Fe2O3 nanoparticle suspension was placed in 60 ℃ water for 10 hours, then placed in 37 ℃ water , overnight. This procedure was repeated 3 times for germicidal treatment. Then, the suspension was packed into small bottles and stored at 4 ℃ for later use. DMEM high glucose culture fluid (Gibco Company, USA); trypsinase (Difco Company, USA, imported); new-born calf serum(Sijiqing Company, Hangzhou); hydrogen dioxide （H2O2, Gibco Company）; Kits for measuring hydrogen dioxide(H2O2), hydroxy radical （·OH）, superoxide anion radical （O2·－）, lactic acid dehydrogenase, ultramicro ATP enzyme and Coomassie brilliant blue protein levels (Jiancheng Biotechnique Co., Ltd., Nanjing).

Methods
This experiment was carried out in the laboratory of Department of Labor and Environmental Health, School of Public Health, Dongnan University between March 2006 and July 2006.

Cells culture
RAW264.7 cells (Abelson murine leukemia virus-induced tumor) were cultured in DMEM (Gibco Company) containing 10% fetal bovine serum, 100 000 U/L penicillin and 100 mg/L streptomycin in the environment of 5% CO2. Cell growth was daily observed under an inverted microscope . Cells, which were at good exponential phase of growth, were chosen for test.

Detection of oxygen free radical in the cells
1.5×105 L－1 macrophages were inoculated to 24-well plate, 1 mL a well. After the macrophages were cultured in incubation at 37 ℃ in a humidified atmosphere containing 5% CO2 for 24 hours.
1.070 0, 0.535 0 and 0.267 5 g/L Fe2O3 nanoparticles (30 nm) suspension-intervened macrophages were set as Fe2O3 nanoparticle group, and normal saline group was set as control group. Following the intervention of nanoparticles, macrophages were disrupted with vitreous homogenizer. The levels of hydrogen peroxide (H2O2), hydroxyl radical (·OH) and superoxide anion (O2·－) were determined in cell lysates and supernatants using the resgent kit (Nanjing Jiancheng Bioengineering Co., Ltd).

Determination of the activities of lactate dehydrogenase (LDH) , Na+-K+-ATPase and Ca2+-Mg2+-ATPase
Macrophages in the Fe2O3 nanoparticle group and control group were treated as above. The activities of LDH in culture medium were determined according to the instruction of reagent kit (Nanjing Jiancheng Bioengineering Co., Ltd). And the activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase were also determined according to the instruction of reagent kit (Nanjing Jiancheng Bioengineering Co., Ltd) at low temperature.

Statistical analysis
Data were performed single factor analysis of variance and Dunnett's test by the first author with SPSS 11.5 software.

RESULTS

Effects of different concentrations of Fe2O3 nanoparticles on the production of H2O2, ·OH and O2·－ in RAW 264.7 cells (Table 1)

After interventions of Fe2O3 nanoparticles and RAW264.7 cells, the levels of H2O2, ·OH and O2·－ in the cells increased. As compared with control group, levels of ·OH and O2·－ in the cells of each nanoparticle-treated group significantly increased (P < 0.05), but the production of H2O2 showed no statistically significant increases in the moderate and low-dose groups (P > 0.05).

Effects of different concentrations of Fe2O3 nanoparticles on the activities of LDH , Na+-K+-ATPase and Ca2+-Mg2+-ATPase in RAW264.7 cell culture fluid
Fe2O3 nanoparticles within the range of experimental concentration could cause LDH activity significantly increased (P < 0.05). Fe2O3 nanoparticles had effects on the activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase. With the increase of dose of Fe2O3 nanoparticles, the activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase were gradually decreased. There were significant differences as compared with control group (P < 0.05)(Table 2).

DISCUSSION

Nanomaterial greatly differs from traditional micromaterial with the same composition, and has many excellent performances and entirely new functions. It has shown wide application prospect in many domains. Nanotechnology brings us many advantages, but some studies demonstrate that some nanoparticles also bring some disadvantages due to their small particle diameters and unique surface [11,12]. Macrophages, as the inductors of pathogen, play very important role in eliminating pathogen and regulating proper immune reaction [13]. In vitro experiment has demonstrated that nanoparticles can enter into macrophages [14], and cause oxidative stress and mitochondrial injury [15]. Iron is described as a catalyst which can initiate the form of oxygen free radicals, its function in toxicity is mainly to respond to the catalyst of the iron ion :Fe2+ +H2O2 →Fe3+ + OH·+ OH－[16]. The latter can promote lipid peroxidation of membrane system, which leads to the damage of membrane structure and functions [17,18]. Some study already indicated that the cytotoxicity were related to the oxidative effect in cells exposed to Fe2O3 nanoparticles[19]. In our experiment, Fe2O3 nanoparticles could increase the oxygen free radical in macrophage. Initially, the free radicals were prone to attack the cell membrane structure. The attack of free radicals and following lipid peroxidation would hurt the membrane structure, thus the liquid of membrane, ion transport function and activities of enzyme were changed. After the damage of membrane, LDH was released into culture medium. The activity of extracellular LDH is a sensitive index to reflect the degree of damage. In this study, we found that Fe2O3 nanoparticles could cause the release of LDH. So we can assume that Fe2O3 nanoparticles damage the membrane and lead to the increase of membrane permeability.
Usually, Na+-K+-ATPase can keep relative stabilization of Na+,K+ concentration and balance of osmotic pressure, which are of significance for maintaining normal cell morphology, and normal physiological function of cells and the whole body.
To keep the state of low calcium in cells, Ca2+-Mg2+-ATPase is another ion pump which can actively transfer Ca2+ out of cells. The oxidative stress caused by free radical is an important factor to cause the damage of ATPase. The findings of this experiment demonstrate that with the obvious increase of free radical level, Fe2O3 nanoparticles significantly inhibit the activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase.
To sum up, it can be inferred that Fe2O3 nanoparticles-caused the increase of oxygen free radical is an important factor for increasing cell membrane permeability in macrophages of mouse, inhibiting the activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase, and damaging the function of membrane. Consequently, edema, degeneration and necrosis of macrophages will be resulted in, while the functional damage of macrophages maybe influence the immune system balance of human being.

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磁性三氧化二铁纳米粒子对小鼠腹腔
巨噬细胞的氧化损伤作用**★

王晓娜1，3，唐 萌1，3，张 婷1,3，杨 磊1，夏 婷1,3，曾垂焕1,3，熊丽林1,3，张 宇2,3，顾 宁2,3
1东南大学公共卫生学院，江苏省南京市 210009； 2东南大学生物科学与医学工程系，江苏省南京市 210096；3江苏省生物材料与器件高技术重点实验室，江苏省南京市 210096
王晓娜★，女，1978年生，广西壮族自治区柳州市人， 2002年东南大学毕业，在读硕士，主要从事纳米毒理学研究。
通讯作者:唐 萌，博士，教授，东南大学公共卫生学院，江苏省南京市210009
国家重点基础研究发展计划973项目资助（2006CB705602）*；国家自然科学基金项目资助（30671782）*
摘要
背景：已有报道表明纳米级的Fe2O3产生的细胞毒性与细胞的脂质过氧化存在一定的联系。氧化铁纳米粒子是否对巨噬细胞产生毒性，其毒性机制与氧化作用有何关联？
（To page 2585）
（From page 2577）
目的：观察不同浓度Fe2O3纳米粒子对巨噬细胞的氧化损伤作用。
设计：观察对比实验。
单位:东南大学公共卫生学院。
材料：RAW264.7细胞为小鼠腹腔巨噬细胞，购自中国科学院上海细胞所。Fe2O3纳米粒子(30 nm)悬浮液由东南大学生物医学工程系制备提供。实验前先进行预处理：将Fe2O3 纳米粒子悬浮液置60 ℃水浴中10 h，然后37 ℃水浴过夜。如此反复3次，灭菌处理。小瓶分装，4 ℃保存。DMEM高糖培养液（美国Gibco公司）；胰蛋白酶（美国Difco公司，进口分装）；新生牛血清（杭州四季青公司）；过氧化氢、羟自由基、超氧阴离子自由基、乳酸脱氢酶、超微量ATP酶和考马斯亮蓝蛋白含量测定试剂盒（南京建成生物技术公司）。
方法：实验于2006－03/07在东南大学公共卫生学院劳动卫生与环境卫生学系实验室完成。RAW264.7细胞用DMEM培养基于37 ℃、体积分数为0.05的CO2培养箱中进行培养。每日用倒置显微镜观察细胞生长情况，取生长状态良好的对数生长期细胞进行试验。① 细胞中氧自由基的检测：将密度为1.5×105 L－1的巨噬细胞接种于24孔培养板，每孔1 mL，37 ℃、体积分数为0.05的CO2培养箱中培养24 h后，以质量浓度为1.0 700、0.5 350和0.2 675 g/L的Fe2O3纳米粒子(30 nm)悬浮液染毒细胞为纳米粒子干预组，并设生理盐水为溶剂对照组，24 h后终止培养，染毒结束后，低温条件下用玻璃匀浆器破碎细胞，按试剂盒说明，分别测定细胞中过氧化氢、羟自由基、超氧阴离子自由基。②培养液中乳酸脱氢酶活性及膜ATP酶活性的测定：纳米粒子干预组及对照组干预方法同上，染毒结束后，检测培养液中乳酸脱氢酶活性及膜ATP酶活性，乳酸脱氢酶活性的检测采用南京建成生物工程研究所提供的试剂盒，严格按照说明书进行操作。膜ATP酶活性的检测采用低温条件下用玻璃匀浆器破碎细胞，按试剂盒说明检测膜Na+-K+-ATP酶和Ca2+-Mg2+-ATP酶活性。
主要观察指标：① 不同质量浓度Fe2O3纳米粒子对细胞过氧化氢、羟自由基和超氧阴离子的影响。②乳酸脱氢酶活性、膜Na+-K+-ATP酶和Ca2+-Mg2+-ATP酶活性检测结果。
结果：①Fe2O3 0.267 5, 0.535 0, 1.070 0 g/L纳米粒子干预组羟自由基水平高于溶剂对照组[（0.605±0.066），（0.410±0.080），（0.764±0.051），（0.285±0.057）mkat/g,P < 0.05]，超氧阴离子自由基高于溶剂对照组[（9.935±1.159），（8.912±0.131），（13.479±0.752），（5.635±0.475）μkat/g，P < 0.05], Fe2O3 1.070 0 g/L过氧化氢水平高于溶剂对照组[（14.695±2.815），（2.397±0.399） mmol/L，P < 0.05] ②Fe2O3纳米粒子在实验浓度范围内，能够引起培养液中乳酸脱氢酶活性显著升高（P < 0.05）。Fe2O3纳米粒子对细胞膜Na+-K+-ATP酶和Ca2+-Mg2+-ATP酶活性均产生影响，且随着Fe2O3纳米粒子染毒剂量增加，Na+-K+-ATP酶和Ca2+-Mg2+-ATP酶活性逐渐降低，与对照组比较，差异均具有显著性（P < 0.05）。
结论：Fe2O3纳米粒子剂量增加导致细胞内过氧化氢、羟自由基、超氧阴离子自由基增多，从而使细胞膜通透性增加，膜Na+-K+-ATP酶和Ca2+-Mg2+-ATP酶活性受到抑制。
关键词： Fe2O3；纳米粒子；巨噬细胞；氧化应激；毒性
中图分类号:R349.5 文献标识码:A 文章编号:1673-8225(2007)13-02575-03
王晓娜，唐萌，张婷，杨磊，夏婷，曾垂焕，熊丽林，张宇，顾宁.磁性三氧化二铁纳米粒子对小鼠腹腔巨噬细胞的氧化损伤作用[J].中国组织工程研究与临床康复，2007，11(13):2575-2577,2585
[www.zglckf.com/zglckf/ejournal/upfiles/07-13/13k-2575(ps).pdf]
(Edited by Sui G/Song LP/Wang L)