纳米铜和微米铜的毒性比较研究
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摘要
纳米科学与技术在世界范围内均是动态发展、急剧变革的新兴领域；在欧洲、北美及其国内也都是优先开发的科学前沿。故此,纳米技术常常被称之为先驱技术。纳米技术的商业应用也逐步增多,包括电子技术、运动服装、轮胎、耐污染衣料、化妆品以及诊断成像或投送药物的载体。随着纳米技术产品广泛商业化,人类暴露纳米材料及其产品的可能性也急剧增加,评价其毒性和安全性也因而具有极为重要的意义。近年的研究表明,包括纳米铜在内的、应用最广泛的金属纳米粒子在体内外均可诱发不良效应,导致纳米材料因其独特表面活性、催化性质和磁性而引起潜在毒性并受到广泛关注。迄今为止的研究提示,纳米材料的行为和效应并非总能从其它类型纳米材料的现有研究结果预测出来。此外,虽然纳米材料与普通微米材料的组成相同,但在原子和量子水平具有不同于微米材料的性质和行为。故此,纳米毒理学研究的策略、模式、方法和工具均有别于传统的毒理学。纳米铜粒子也是极具代表性的一种金属纳米材料,并且业已作为商业产品如纳米级抗衰老、抗骨质疏松症的治疗药物进入主流市场。虽然.纳米铜材料与我们人类愈来愈关系密切,但是,迄今为止仍仅开展了少量得研究,其结果显示纳米铜材料可能对人类或者其环境可能存在一定的危害。
基于上述考虑,本课题首先采用原子力显微镜、无细胞孵育体系等技术对纳米铜和微米铜的原料本身及其在体外培养体系、给药溶媒和人工胃液中的离子化行为进行全面的鉴定,并以单价铜离子特异性螯合剂BCS为工具药探索离子化过程动力学对纳米铜体外毒性效应的潜在影响,分析离子化过程在纳米铜发挥体外毒性中的作用。其次,以大鼠为模型,研究单次和连续5天给予纳米铜及微米铜引发毒效应的表现、靶器官,通过体重、摄食量、临床体征、临床病理学、解剖病理学等一般毒性检测指标和超微结构分析、生物标志物检测、流式细胞分析等新技术探讨纳米铜诱发肝脏、肾脏、免疫系统、生殖系统毒性的特征和细胞与分子机制,分析毒性表现与铜粒子尺寸、表面积、表面活性等理化性质之间的相关性。最后,采用大鼠全基因组基因芯片技术分析研究纳米铜及微米铜处理大鼠的肾脏组织基因表达水平,结合传统毒理学毒性体征的整合分析和表型锚定探索与纳米铜肾毒性密切相关的靶基因和信号转导通路,进而阐明纳米铜肾毒性发生发展的毒理基因组学机制。本课题的研究目的在于：进一步利用动物模型观察纳米铜的毒理学效应,揭示纳米铜的潜在靶器官和毒效应特征,并利用体外细胞模型和现代组学技术探讨纳米铜的毒性机制,为纳米铜的安全性评价、毒性监测及干预提供理论依据。
在理化鉴定过程中,利用扫描电子显微镜、动态光散射技术、BET比表面积分析仪和X射线荧光光谱法分别对纳米铜的粒径及分布、比表面积和纯度等理化性质进行表征,结果显示：纳米铜的平均粒径及分布为25nm(5-60nm),比表面积为6.93±0.03m2/g,纯度不低于99.9%。受试物纳米铜要明显小于参比物微米铜’(平均粒径171μm,表面积0.2g/m2)。再后再采用MTT法和LDH漏出率测定纳米铜的细胞毒性,并采用一价铜特异性螯合剂、分光光度法、ICP-AES来分析纳米铜粒子在培养体系、给药溶媒中的溶出度。实验结果发现,纳米铜可剂量依赖性地诱导细胞存活力下降,作用24h后对HK-2细胞毒性的IC50值为41.3μg/ml；纳米铜还可剂量依赖性地引发细胞LDH漏出量增加。然而,-价铜离子的特异性螯合剂BCS可相应地抑制纳米铜或氯化铜所诱导的细胞毒性。随着溶液中纳米铜颗粒添加时间的逐步延长,在157.5和315μmol/L浓度下的纳米铜粒子处理体系中,二价铜离子的水平也开始逐渐升高,与此同时培养体系中的纳米铜粒子的溶解率也出现时间依赖性的升高趋势。ICP-AES法定量分析的进一步结果表明：在人工胃液中纳米级的金属铜粒子和微米级的金属铜粒子在溶解速率上存在明显差别；作用5分钟后,纳米级铜粒子在人工胃液中的溶解率达1.2%；其后,铜离子的升高幅度才逐渐变缓,到达2小时时则达到了平台期,其相应的溶解率为2.1%。相同条件下,微米铜的溶解率仅在60-120min之间出现小幅度升高,其溶解率从0.21%上升至0.67%(与纳米铜相比,有明显差异)。在相同质量浓度下,纳米铜对HK-2系肾小管上皮细胞的毒性要明显地高于微米铜。上述结果提示：纳米铜粒子要发挥其生物效应并增强其毒性作用,其中的较小尺寸、较大的表面积、以及较高的表面活性是其中的重要化学结构基础,纳米铜诱导毒性的重要方式为转化为铜离子,单价铜离子螯合剂可明显拮抗纳米铜诱导的毒性,进而可能应用于纳米铜的中毒治疗。
大鼠单次经口急性毒性上下法实验表明,纳米铜的LD50(95%置信区间)为834.3(790～930) mg/kg;限量试验中,2000mg/kg剂量下三只大鼠全部死亡,而微米铜在相同剂量下则仍未导致大鼠死亡。纳米铜的毒性体征与文献报道的可离子化铜化合物的急性毒性体征相近,提示经口给予纳米铜的毒性可能归因于离子化效应。
大鼠连续经口给予纳米铜5天的毒性靶器官为肝脏、肾脏、胃脏、免疫系统和附睾。肝毒性主要表现为肝细胞肿胀、点状或小灶状肝细胞嗜酸性坏死以及中性粒细胞与淋巴细胞等炎细胞浸润。临床生化表现为血清ALT和AST水平升高,TP、Alb、Glo水平的显著下降。肾毒性主要表现为剂量依赖性的肾小管上皮轻度至中度的玻璃样变性。临床生化表现为BUN显著升高和血清中GST-α、KIM-1和β2-MG三种生物标志物的明显改变。纳米铜的胃毒性为本课题首次发现,其主要表现为胃粘膜浅层明显淤血与充血、局部出血、固有层腺体腺上皮胞浆空化、部分胞核溶解消失。免疫毒性主要表现为中性粒细胞数显著升高、淋巴细胞数显著降低、血清补体C3、C4水平升高和IgA抗体水平上升。附睾毒性主要表现为附睾绝对和相对重量剂量依赖性下降以及高剂量下的精子运动性(包括运动精子的百分率)下降。纳米铜染毒5天后,肝肾组织的氧化还原防御酶SOD和GSH-PX水平代偿性升高。
利用基因组学技术研究纳米铜染毒5天大鼠肾脏的基因表达谱发现：在纳米铜100mg/kg和200mg/kg处理组大鼠肾脏中均发生改变的差异基因,其涉及的生物学过程主要包括：生物学调节、应激反应、对损伤的反应、防御反应、脂质代谢、急性炎症反应和损伤修复等过程。涉及的分子功能主要集中在酶调节剂的活性、转运体活性、酶抑制剂活性、蛋白酶抑制剂活性、内肽酶抑制剂活性和脂质结合等方面。
综合以上研究结果,我们初步得出以下结论：纳米铜粒子因其相对较小的粒径、较大的比表面积和高表面活性而较同等剂量的微米铜粒子更易于在培养基体系和人工胃液内发生离子化反应并转化成为铜离子,从而造成较微米铜粒子更强烈的体外细胞毒性、单次体内给药毒性和反复给药5天的亚急性毒性。雄性大鼠经口染毒5天的毒性作用靶器官主要为肝脏、肾脏、胃脏、免疫系统和附睾。同等条件下,纳米铜的毒性明显高于微米铜。纳米铜的毒性主要机制之一与活性氧与抗氧化物质的动态平衡,引起氧化应激有关。肾组织毒理基因组学分析结果发现：氧化应激反应可能是纳米铜粒子引起大鼠肾损伤的一条重要途径。三羧酸循环和氧化磷酸化等能量代谢途径受到抑制,则有可能成为纳米铜粒子引起大鼠肾脏毒性的一种作用机制。纳米铜所引发的信号功能通路中差异表达的相关基因和/或蛋白可作为研究纳米铜诱发肾损伤的生物标志物。
Nanoscience and nanotechnology are dynamically developing scientific fields throughout the world and have already become key research and development priorities in Europe and North America. Nanotechnology is often regarded as an'enabling technology'. The use of nanotechnology in commercial applications is increasing in many scientific disciplines, including electronics, sporting goods, tires, stain-resistant clothing, cosmetics, and medicines for diagnosis, imaging, and drug delivery. With the ongoing commercialization of nanotechnology products, human exposure to nanomaterials will dramatically increase, and evaluation of their potential toxicity is essential. Several manufactured nanomaterials have recently been shown to cause adverse effects in vitro and in vivo, including nanocopper. The concerns about the potential toxicity of nanomaterials are based on their unique surface, catalytic and magnetic properties, and how these properties may be expressed in biological systems and in the environment to produce adverse effects. Results to date suggest that the behavior and effects of nanomaterials are not always directly predictable from the results of previous studies with other types of nanoscale materials. It is becoming increasingly apparent that although they are composed of the same basic elements, at the atomic or quantum level, nanomaterials have different properties and behave differently from their bulk counterparts. Nanocopper has shown great promise as an osteoporosis treatment drug, antibacterial material, additive in livestock and poultry feed, and intrauterine contraceptive device. Furthermore, nanocopper has been widely used in industry, e.g., as an additive in lubricants, for metallic coating, and as a highly reactive catalyst in organic hydrogen reactions. Although there is a gradually increased correlation between the manufacture and use of nanocopper and human health risks, there are only some investigative studies which shown that there are potential hazards to the human and the environments.
Based on the above considerations, the nanocopper and micro copper raw materials and its ionization behaviors in the in vitro culture system, in the dosing administration vehicles, and in the artificial gastric juice, were first comprehensively characterized, in present study, with such techniques as atomic force microscope, cell incubation system; and the potential effects of the ionization process kinetics on their in vitro toxicities were also explored with the tool drug monovalent nanocopper chelator BCS, and roles played by ionization process in the in vitro toxicity of nanometer copper, was analyzed too. Secondly, the toxicity manifestations and target organs induced by single dosing and five daily repeated dosing of nanocopper and microcopper were studied in rat model, and the characteristics and cellular or molecular mechanisms for the toxicities induced in liver, kidneys, immune system, reproductive system by the nanocopper were explored by the general toxicity testing indicators such as body weight, food consumption, clinical signs, clinical pathology, anatomical pathology and by the new technologies such as ultrastructural analysis, biomarker assays, and flow cytometry analysis, and the correlation between toxicity manifestations and the physicochemical properties, such as copper particle size, surface area, and surface activities were also analyzed. Finally, gene expression levels in kidney tissues in nanocopper and microcopper treated rats were analyzed using the rat genome-wide gene chip technology, the target genes and signal transduction pathways closely related with renal toxicity of nanocopper were explored by the combined analysis with traditional toxicology toxicity signs and phenotypic anchoring, and toxicogenomic mechanisms for the occurrence and development of renal toxicity of nanocopper were illustrated. The purpose of this topic are as follows:to further examine toxicological effects of nanocopper in animal models, to reveal potential target organs and toxic effects of nanocopper, to investigate the toxicity mechanism of nanocopper using the in vitro cell models and modern genomics technology, and to provide a theoretical basis for safety evaluation, monitoring and intervention of nanocopper toxicity.
During physicochemical characterization process, the physicochemical properties of nanocopper, such as particle size and distribution, surface area and purity, were first respectively characterized by scanning electron microscopy, dynamic light scattering, BET specific surface area analyzer, and X-ray fluorescence spectroscopy, the analysis results shown that the average particle size and distribution of the nanocopper is25nm (5～60nm), the specific surface area of it is6.93±0.03m2/g, and a purity of it is not less than99.9%. The test substance nanometer copper is significantly less than the reference compound microcopper (average particle size of17μm, specific surface area of0.2g/m2). The cytotoxicity of nanoscale copper was bioassayed by MTT tests and LDH leakage rate measurements, and the dissolution of the copper nano-particles in the culture system was determined by monovalent copper-specific chelator, spectrophotometry, and inductively coupled plasma-atomic emission spectrometry analysis. It was found that nanocopper induced a dose-dependent cell viability decreases, with a cytotoxicity IC50value for HK-2cell treatment for24h of41.3μg/ml. Nanocopper induced a dose-dependent LDH leakage increase in the treated cells, and the monovalent copper ion specific chelator BCS can inhibit the cytotoxicity induced by nano-copper or copper chloride. With the elongation of nanocopper particle addtion time, divalent copper ion levels in the157.5and315μmol/L copper nano-particle processing system were gradually increased, and the dissolution rate of nanocopper particles in the culture system is also time-dependent increased. ICP-AES quantification analysis results also confirmed that there are an obvious differences in the dissolution rate in artificial gastric juice between nanocopper particles and the microcopper particles, i.e. when the nanocopper particles were treated with artificial gastric juice for5minutes, the concentration of copper ions in the suspension were significantly increased, with a dissolution rate of1.2%. Subsequently, increase rate is slowing down, with a plateau time at2hours and plateau dissolution rate of2.1%. Under the same conditions, there was only a small increase in the dissolution rate for microcopper between60to120minutes (from0.21%to0.67%), and significant different from those for nano-copper. With the same mass concentration, the toxicity of nanocopper particles to HK-2tubular epithelial cells was significantly higher than that for microcopper particles, therefore, the small size, large surface area and high surface activity of nanocopper are the important basis for biological effects and enhanced toxicity exhibited by nanocopper, the conversion to copper ions is one of the important way to which nanocopper induced toxicity. The monovalent copper chelator antagonizes nanocopper-induced toxicity and thus may be applied to the treatment of nano-copper poisoning.
The single dose oral acute toxicity up-down procedure experiments in rats have shown that nanocopper LD50(95%confidence interval) is834.3(790～930) mg/kg. With the limit test, all three rats dosed with2000mg/kg nanocopper particles were died, however, all three rats dosed with the same dose of microcopper are not died. Toxicity signs of nanocopper were similar to those acute toxicity signs of ionized copper compound reported in the literature, suggesting that toxicity for orally administered nanocopper may be attributed to the ionization effect.
Toxicity target organs in the rats orally administered nanocopper5consecutive days are the liver, kidney, stomach, immune system and epididymis. The main manifestations for hepatotoxicity were swelling of the liver cells, punctate or small focal eosinophilic necrosis of liver cells, and inflammatory cell infiltration of neutrophils and lymphocytes. The clinical biochemical manifestations for hepatotoxicity were elevated serum ALT and AST levels, and significantly lower levels of TP, Alb, and Glo. The main manifestations for renal toxicity were dose-dependent, and mild to moderate hyaline degeneration of the tubular epithelia. Clinical biochemical manifestations for renal toxicity were significantly elevations of BUN, and significantly changes for three biological markers (GST-a, KIM-1and (β2-MG) in sera. Gastric toxicity of nanocopper was first reported in present study, mainly manifested as obvious congestions of shallow gastric mucosa, local bleeding, the cytoplasm cavitation of glandular epithelium in lamina propria of gastric glands, and the dissolution and disappears of partly nucleus. The main manifestations for immunotoxicity were significantly elevated neutrophil count, a significant reduction in the number of lymphocytes, and elevations in serum complement C3and C4levels and IgA antibody levels. The main manifestations for epididymal toxicity were dose-dependent decreases in absolute and relative epididymal weights and the decline in sperm motility (including the percentage of motile sperm) in high dose rats. When treated with nanocopper for5days, the redox defense enzyme SOD and GSH-Px levels in liver and kidney tissues were increased.
Male rats were given nanocopper (50,100,200mg/kg) and microcopper (200mg/kg) at different doses for5days. Whole genome transcriptome profiling of rat kidneys revealed significant alterations in the expression of many genes involved in valine, leucine, and isoleucine degradation, complement and coagulation cascades, oxidative phosphorylation, cell cycle, mitogen-activated protein kinase signaling pathway, glutathione metabolism, and others may be involved in the development of these phenotypes. Results from this study provide new insights into the nephrotoxicity of copper nano-particles and illustrate how toxicogenomic approaches are providing an unprecedented amount of mechanistic information on molecular responses to nanocopper and how they are likely to impact hazard and risk assessment.
Based on the above findings, our preliminary conclusions were as followings. Because of their relatively small particle diameter, larger surface area, and higher surface activities compared with the same dose of microcopper particles, nanocopper particles are easier for ionization reaction and the conversion into copper ion within the medium system and in the artificial gastric juice, thereby causing more strong in vitro cytotoxicity, single dose in vivo toxicity and subacute five repeated daily dosing toxicity, than that for microcopper particles. The major target organs for toxic effects in male rats orally five days exposed to nanocopper were liver, kidney, stomach, immune system and epididymis. Under the same conditions, the toxicity of nanocopper was significantly higher than that for microcopper. One of the major mechanisms of toxicity for nano-copper is the dynamic balance between reactive oxygen species and antioxidants, and the induced oxidative stress. Toxicogenomic analysis of kidney tissues has found that oxidative stress is one of the important ways to cause kidney damage for nanocopper. Energy metabolism, especially the inhibition of the citric acid cycle and oxidative phosphorylation process, may be one of the mechanisms by which nanocopper caused renal toxicity. The related genes and proteins which are differently expressed in these functional pathways can be used as candidate biomarkers for the studies in the nanocopper-induced kidney injuries.
基于上述考虑,本课题首先采用原子力显微镜、无细胞孵育体系等技术对纳米铜和微米铜的原料本身及其在体外培养体系、给药溶媒和人工胃液中的离子化行为进行全面的鉴定,并以单价铜离子特异性螯合剂BCS为工具药探索离子化过程动力学对纳米铜体外毒性效应的潜在影响,分析离子化过程在纳米铜发挥体外毒性中的作用。其次,以大鼠为模型,研究单次和连续5天给予纳米铜及微米铜引发毒效应的表现、靶器官,通过体重、摄食量、临床体征、临床病理学、解剖病理学等一般毒性检测指标和超微结构分析、生物标志物检测、流式细胞分析等新技术探讨纳米铜诱发肝脏、肾脏、免疫系统、生殖系统毒性的特征和细胞与分子机制,分析毒性表现与铜粒子尺寸、表面积、表面活性等理化性质之间的相关性。最后,采用大鼠全基因组基因芯片技术分析研究纳米铜及微米铜处理大鼠的肾脏组织基因表达水平,结合传统毒理学毒性体征的整合分析和表型锚定探索与纳米铜肾毒性密切相关的靶基因和信号转导通路,进而阐明纳米铜肾毒性发生发展的毒理基因组学机制。本课题的研究目的在于：进一步利用动物模型观察纳米铜的毒理学效应,揭示纳米铜的潜在靶器官和毒效应特征,并利用体外细胞模型和现代组学技术探讨纳米铜的毒性机制,为纳米铜的安全性评价、毒性监测及干预提供理论依据。
在理化鉴定过程中,利用扫描电子显微镜、动态光散射技术、BET比表面积分析仪和X射线荧光光谱法分别对纳米铜的粒径及分布、比表面积和纯度等理化性质进行表征,结果显示：纳米铜的平均粒径及分布为25nm(5-60nm),比表面积为6.93±0.03m2/g,纯度不低于99.9%。受试物纳米铜要明显小于参比物微米铜’(平均粒径171μm,表面积0.2g/m2)。再后再采用MTT法和LDH漏出率测定纳米铜的细胞毒性,并采用一价铜特异性螯合剂、分光光度法、ICP-AES来分析纳米铜粒子在培养体系、给药溶媒中的溶出度。实验结果发现,纳米铜可剂量依赖性地诱导细胞存活力下降,作用24h后对HK-2细胞毒性的IC50值为41.3μg/ml；纳米铜还可剂量依赖性地引发细胞LDH漏出量增加。然而,-价铜离子的特异性螯合剂BCS可相应地抑制纳米铜或氯化铜所诱导的细胞毒性。随着溶液中纳米铜颗粒添加时间的逐步延长,在157.5和315μmol/L浓度下的纳米铜粒子处理体系中,二价铜离子的水平也开始逐渐升高,与此同时培养体系中的纳米铜粒子的溶解率也出现时间依赖性的升高趋势。ICP-AES法定量分析的进一步结果表明：在人工胃液中纳米级的金属铜粒子和微米级的金属铜粒子在溶解速率上存在明显差别；作用5分钟后,纳米级铜粒子在人工胃液中的溶解率达1.2%；其后,铜离子的升高幅度才逐渐变缓,到达2小时时则达到了平台期,其相应的溶解率为2.1%。相同条件下,微米铜的溶解率仅在60-120min之间出现小幅度升高,其溶解率从0.21%上升至0.67%(与纳米铜相比,有明显差异)。在相同质量浓度下,纳米铜对HK-2系肾小管上皮细胞的毒性要明显地高于微米铜。上述结果提示：纳米铜粒子要发挥其生物效应并增强其毒性作用,其中的较小尺寸、较大的表面积、以及较高的表面活性是其中的重要化学结构基础,纳米铜诱导毒性的重要方式为转化为铜离子,单价铜离子螯合剂可明显拮抗纳米铜诱导的毒性,进而可能应用于纳米铜的中毒治疗。
大鼠单次经口急性毒性上下法实验表明,纳米铜的LD50(95%置信区间)为834.3(790～930) mg/kg;限量试验中,2000mg/kg剂量下三只大鼠全部死亡,而微米铜在相同剂量下则仍未导致大鼠死亡。纳米铜的毒性体征与文献报道的可离子化铜化合物的急性毒性体征相近,提示经口给予纳米铜的毒性可能归因于离子化效应。
大鼠连续经口给予纳米铜5天的毒性靶器官为肝脏、肾脏、胃脏、免疫系统和附睾。肝毒性主要表现为肝细胞肿胀、点状或小灶状肝细胞嗜酸性坏死以及中性粒细胞与淋巴细胞等炎细胞浸润。临床生化表现为血清ALT和AST水平升高,TP、Alb、Glo水平的显著下降。肾毒性主要表现为剂量依赖性的肾小管上皮轻度至中度的玻璃样变性。临床生化表现为BUN显著升高和血清中GST-α、KIM-1和β2-MG三种生物标志物的明显改变。纳米铜的胃毒性为本课题首次发现,其主要表现为胃粘膜浅层明显淤血与充血、局部出血、固有层腺体腺上皮胞浆空化、部分胞核溶解消失。免疫毒性主要表现为中性粒细胞数显著升高、淋巴细胞数显著降低、血清补体C3、C4水平升高和IgA抗体水平上升。附睾毒性主要表现为附睾绝对和相对重量剂量依赖性下降以及高剂量下的精子运动性(包括运动精子的百分率)下降。纳米铜染毒5天后,肝肾组织的氧化还原防御酶SOD和GSH-PX水平代偿性升高。
利用基因组学技术研究纳米铜染毒5天大鼠肾脏的基因表达谱发现：在纳米铜100mg/kg和200mg/kg处理组大鼠肾脏中均发生改变的差异基因,其涉及的生物学过程主要包括：生物学调节、应激反应、对损伤的反应、防御反应、脂质代谢、急性炎症反应和损伤修复等过程。涉及的分子功能主要集中在酶调节剂的活性、转运体活性、酶抑制剂活性、蛋白酶抑制剂活性、内肽酶抑制剂活性和脂质结合等方面。
综合以上研究结果,我们初步得出以下结论：纳米铜粒子因其相对较小的粒径、较大的比表面积和高表面活性而较同等剂量的微米铜粒子更易于在培养基体系和人工胃液内发生离子化反应并转化成为铜离子,从而造成较微米铜粒子更强烈的体外细胞毒性、单次体内给药毒性和反复给药5天的亚急性毒性。雄性大鼠经口染毒5天的毒性作用靶器官主要为肝脏、肾脏、胃脏、免疫系统和附睾。同等条件下,纳米铜的毒性明显高于微米铜。纳米铜的毒性主要机制之一与活性氧与抗氧化物质的动态平衡,引起氧化应激有关。肾组织毒理基因组学分析结果发现：氧化应激反应可能是纳米铜粒子引起大鼠肾损伤的一条重要途径。三羧酸循环和氧化磷酸化等能量代谢途径受到抑制,则有可能成为纳米铜粒子引起大鼠肾脏毒性的一种作用机制。纳米铜所引发的信号功能通路中差异表达的相关基因和/或蛋白可作为研究纳米铜诱发肾损伤的生物标志物。
Nanoscience and nanotechnology are dynamically developing scientific fields throughout the world and have already become key research and development priorities in Europe and North America. Nanotechnology is often regarded as an'enabling technology'. The use of nanotechnology in commercial applications is increasing in many scientific disciplines, including electronics, sporting goods, tires, stain-resistant clothing, cosmetics, and medicines for diagnosis, imaging, and drug delivery. With the ongoing commercialization of nanotechnology products, human exposure to nanomaterials will dramatically increase, and evaluation of their potential toxicity is essential. Several manufactured nanomaterials have recently been shown to cause adverse effects in vitro and in vivo, including nanocopper. The concerns about the potential toxicity of nanomaterials are based on their unique surface, catalytic and magnetic properties, and how these properties may be expressed in biological systems and in the environment to produce adverse effects. Results to date suggest that the behavior and effects of nanomaterials are not always directly predictable from the results of previous studies with other types of nanoscale materials. It is becoming increasingly apparent that although they are composed of the same basic elements, at the atomic or quantum level, nanomaterials have different properties and behave differently from their bulk counterparts. Nanocopper has shown great promise as an osteoporosis treatment drug, antibacterial material, additive in livestock and poultry feed, and intrauterine contraceptive device. Furthermore, nanocopper has been widely used in industry, e.g., as an additive in lubricants, for metallic coating, and as a highly reactive catalyst in organic hydrogen reactions. Although there is a gradually increased correlation between the manufacture and use of nanocopper and human health risks, there are only some investigative studies which shown that there are potential hazards to the human and the environments.
Based on the above considerations, the nanocopper and micro copper raw materials and its ionization behaviors in the in vitro culture system, in the dosing administration vehicles, and in the artificial gastric juice, were first comprehensively characterized, in present study, with such techniques as atomic force microscope, cell incubation system; and the potential effects of the ionization process kinetics on their in vitro toxicities were also explored with the tool drug monovalent nanocopper chelator BCS, and roles played by ionization process in the in vitro toxicity of nanometer copper, was analyzed too. Secondly, the toxicity manifestations and target organs induced by single dosing and five daily repeated dosing of nanocopper and microcopper were studied in rat model, and the characteristics and cellular or molecular mechanisms for the toxicities induced in liver, kidneys, immune system, reproductive system by the nanocopper were explored by the general toxicity testing indicators such as body weight, food consumption, clinical signs, clinical pathology, anatomical pathology and by the new technologies such as ultrastructural analysis, biomarker assays, and flow cytometry analysis, and the correlation between toxicity manifestations and the physicochemical properties, such as copper particle size, surface area, and surface activities were also analyzed. Finally, gene expression levels in kidney tissues in nanocopper and microcopper treated rats were analyzed using the rat genome-wide gene chip technology, the target genes and signal transduction pathways closely related with renal toxicity of nanocopper were explored by the combined analysis with traditional toxicology toxicity signs and phenotypic anchoring, and toxicogenomic mechanisms for the occurrence and development of renal toxicity of nanocopper were illustrated. The purpose of this topic are as follows:to further examine toxicological effects of nanocopper in animal models, to reveal potential target organs and toxic effects of nanocopper, to investigate the toxicity mechanism of nanocopper using the in vitro cell models and modern genomics technology, and to provide a theoretical basis for safety evaluation, monitoring and intervention of nanocopper toxicity.
During physicochemical characterization process, the physicochemical properties of nanocopper, such as particle size and distribution, surface area and purity, were first respectively characterized by scanning electron microscopy, dynamic light scattering, BET specific surface area analyzer, and X-ray fluorescence spectroscopy, the analysis results shown that the average particle size and distribution of the nanocopper is25nm (5～60nm), the specific surface area of it is6.93±0.03m2/g, and a purity of it is not less than99.9%. The test substance nanometer copper is significantly less than the reference compound microcopper (average particle size of17μm, specific surface area of0.2g/m2). The cytotoxicity of nanoscale copper was bioassayed by MTT tests and LDH leakage rate measurements, and the dissolution of the copper nano-particles in the culture system was determined by monovalent copper-specific chelator, spectrophotometry, and inductively coupled plasma-atomic emission spectrometry analysis. It was found that nanocopper induced a dose-dependent cell viability decreases, with a cytotoxicity IC50value for HK-2cell treatment for24h of41.3μg/ml. Nanocopper induced a dose-dependent LDH leakage increase in the treated cells, and the monovalent copper ion specific chelator BCS can inhibit the cytotoxicity induced by nano-copper or copper chloride. With the elongation of nanocopper particle addtion time, divalent copper ion levels in the157.5and315μmol/L copper nano-particle processing system were gradually increased, and the dissolution rate of nanocopper particles in the culture system is also time-dependent increased. ICP-AES quantification analysis results also confirmed that there are an obvious differences in the dissolution rate in artificial gastric juice between nanocopper particles and the microcopper particles, i.e. when the nanocopper particles were treated with artificial gastric juice for5minutes, the concentration of copper ions in the suspension were significantly increased, with a dissolution rate of1.2%. Subsequently, increase rate is slowing down, with a plateau time at2hours and plateau dissolution rate of2.1%. Under the same conditions, there was only a small increase in the dissolution rate for microcopper between60to120minutes (from0.21%to0.67%), and significant different from those for nano-copper. With the same mass concentration, the toxicity of nanocopper particles to HK-2tubular epithelial cells was significantly higher than that for microcopper particles, therefore, the small size, large surface area and high surface activity of nanocopper are the important basis for biological effects and enhanced toxicity exhibited by nanocopper, the conversion to copper ions is one of the important way to which nanocopper induced toxicity. The monovalent copper chelator antagonizes nanocopper-induced toxicity and thus may be applied to the treatment of nano-copper poisoning.
The single dose oral acute toxicity up-down procedure experiments in rats have shown that nanocopper LD50(95%confidence interval) is834.3(790～930) mg/kg. With the limit test, all three rats dosed with2000mg/kg nanocopper particles were died, however, all three rats dosed with the same dose of microcopper are not died. Toxicity signs of nanocopper were similar to those acute toxicity signs of ionized copper compound reported in the literature, suggesting that toxicity for orally administered nanocopper may be attributed to the ionization effect.
Toxicity target organs in the rats orally administered nanocopper5consecutive days are the liver, kidney, stomach, immune system and epididymis. The main manifestations for hepatotoxicity were swelling of the liver cells, punctate or small focal eosinophilic necrosis of liver cells, and inflammatory cell infiltration of neutrophils and lymphocytes. The clinical biochemical manifestations for hepatotoxicity were elevated serum ALT and AST levels, and significantly lower levels of TP, Alb, and Glo. The main manifestations for renal toxicity were dose-dependent, and mild to moderate hyaline degeneration of the tubular epithelia. Clinical biochemical manifestations for renal toxicity were significantly elevations of BUN, and significantly changes for three biological markers (GST-a, KIM-1and (β2-MG) in sera. Gastric toxicity of nanocopper was first reported in present study, mainly manifested as obvious congestions of shallow gastric mucosa, local bleeding, the cytoplasm cavitation of glandular epithelium in lamina propria of gastric glands, and the dissolution and disappears of partly nucleus. The main manifestations for immunotoxicity were significantly elevated neutrophil count, a significant reduction in the number of lymphocytes, and elevations in serum complement C3and C4levels and IgA antibody levels. The main manifestations for epididymal toxicity were dose-dependent decreases in absolute and relative epididymal weights and the decline in sperm motility (including the percentage of motile sperm) in high dose rats. When treated with nanocopper for5days, the redox defense enzyme SOD and GSH-Px levels in liver and kidney tissues were increased.
Male rats were given nanocopper (50,100,200mg/kg) and microcopper (200mg/kg) at different doses for5days. Whole genome transcriptome profiling of rat kidneys revealed significant alterations in the expression of many genes involved in valine, leucine, and isoleucine degradation, complement and coagulation cascades, oxidative phosphorylation, cell cycle, mitogen-activated protein kinase signaling pathway, glutathione metabolism, and others may be involved in the development of these phenotypes. Results from this study provide new insights into the nephrotoxicity of copper nano-particles and illustrate how toxicogenomic approaches are providing an unprecedented amount of mechanistic information on molecular responses to nanocopper and how they are likely to impact hazard and risk assessment.
Based on the above findings, our preliminary conclusions were as followings. Because of their relatively small particle diameter, larger surface area, and higher surface activities compared with the same dose of microcopper particles, nanocopper particles are easier for ionization reaction and the conversion into copper ion within the medium system and in the artificial gastric juice, thereby causing more strong in vitro cytotoxicity, single dose in vivo toxicity and subacute five repeated daily dosing toxicity, than that for microcopper particles. The major target organs for toxic effects in male rats orally five days exposed to nanocopper were liver, kidney, stomach, immune system and epididymis. Under the same conditions, the toxicity of nanocopper was significantly higher than that for microcopper. One of the major mechanisms of toxicity for nano-copper is the dynamic balance between reactive oxygen species and antioxidants, and the induced oxidative stress. Toxicogenomic analysis of kidney tissues has found that oxidative stress is one of the important ways to cause kidney damage for nanocopper. Energy metabolism, especially the inhibition of the citric acid cycle and oxidative phosphorylation process, may be one of the mechanisms by which nanocopper caused renal toxicity. The related genes and proteins which are differently expressed in these functional pathways can be used as candidate biomarkers for the studies in the nanocopper-induced kidney injuries.
引文
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