| 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. |
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