Study On The Toxicity And Its Mechanisms Of Copper Nanoparticles On Liver And Kidneys

Nanocopper, one kind of man-made metal nanoparticles, has been commercially manufactured for many years and shown great promise in the fields of biomedicine and industry. However, its toxicity data are scarce. In the current work, the target organs, toxicity properties and the toxicology mechanisms of nanocopper were explored through in vitro and in vivo studies. The aims were to provide the scientific data for the risk assessment, toxicity monitoring and prevention of the copper nanoparticles. The results are as follow:The primary sizes and their ranges of nano-copper and micro-copper were measured as 25nm (5⁶0nm) and 17μm (0.5³8μm), the purity of copper nanoparticles was not below 99.9%. Compared to the micro-copper, the nano-copper is more readily transformed into ionic copper in solution. As being dispersed in the artificial gastric acid, the dissolution rates of nano-copper and micro-copper were 2.1% and 0.67% after interaction for 2 h, respectively. The dissolution rates of nano-copper in the freshly dispersed in completely culture medium or 1% HPMC suspension were 27.01% and 0.014%. 40 and 100μg/ml nano-copper suspensions of culture medium were incubated under the cell culture condition for 24 h, the copper nanoparticles within them have completely transformed into ionic copper.Copper nanoparticles uptake within the cells was not found through the transmission electron microscopy. The morphological changes of two kinds of cells induced by nanocopper included the vacuolar changes in cytoplasm of HepG2, and gradually shrinkage and detachment from the flask for HK-2 cells after exposure to nanocopper.The damages indicated a dose-dependant fashion. The copper nanoparticles exhibited varying degrees of cytoxicity for both cell lines and the general trends were similar for both HepG2 and HK-2 cell lines. The data of MTT assay showed the IC50 values of copper nanoparticles for HepG2 and HK-2 were 35.2 and 41.3μg/ml for 24 h continuous exposure, respectively. The results of cell viability assay indicated the effects of copper nanoparticles and CuCl2 at the same concentration were similar. BCS, a special chelator for Cu+, can effectively inhibit the effects of cytotoxicity induced by copper nanoparticles or CuCl2. Nano-copper particles can produce increased cells apoptosis of HepG2 in a dose-dependent fashion after 24 h continuous exposure. And the cell apoptosis was accompanied by reduced mitochondria membrane potential and an increase in the level of reactive oxygen species. Increased ROS could further produce lipid peroxidation. The contents of metallothinein markedly increased in the cytoplasm and the mRNA levels of MT1X,MT2A,Ctr1,Atox and Cox17 were up-regulated after cells exposure to copper nanoparticles.The toxicity manifestations of the rats acutely or subacutely treated with copper nanoparticles were similar, and mainly involved diarrhea, loss of appetite and weight. The LD50 value (rat oral) of copper nanoparticles was 834.3 mg/kg. The liver and kidneys were the main target organs. The hepatotoxicity and nephrotoxicity induced by copper nanoparticles indicated in a dose-dependent manner. The characteristics of toxicity are as following: significant changes of the serum clinical biochemistry parameters were restricted to the rats treated with high dose of nano-copper. The serum levels of aspartate aminotransferase, total bilirubin, blood urea nitrogen, and creatinine in the 200 mg/kg group increased significantly (over 5-fold) compared to the control group, and alanine aminotransferase, triglyceride and total bile acid increased to a lesser extent (beyond 2-fold). Additionally, alkaline phosphatase and total cholesterol were slightly reduced while total cholesterol and triglyceride were found to be elevated slightly in the mid- and low-dose groups. Comparison of hematoxylin and eosin liver sections obtained from the control and nano-copper-treated rats indicated scattered dot hepatocytic necrosis in all rats of the 200 mg/kg/d group, and no overt sign of toxicity was found in either the mid- or low-dose nano-copper group. Significant damages were observed in the renal tissue acquired from the rats treated with nano-copper. For all rats dosed with 200 mg/kg/d nano-copper, the changes were characterized by widespread renal proximal tubule necrosis involving most of the nephrons; further, cellular fragments were found in the tubule lumen, where orange crystal matter deposition was commonly observed.Nano-copper (50 or 100 mg/kg/d) only caused the swelling of the proximal tubule epithelia in 2/6 and 6/6 rats, respectively. For the rats treated with copper microparticles, no abnormal changes were found for serum biochemical parameters, only the slightly swelling of the proximal tubule epithelia were found in 3/6 rats.1H NMR-PCA based metabonomics has been applied to investigat the biochemical composition of urine, serum, the aquous extracs and lipidic extracts in the liver and renal obtained from the rats treated copper particles. The results showed 200mg/kg/d nanpcopper can produced overt changes in the biochemical compositions in all test samples. The detailed changes included that the levels of serum lactate, 3-hydroxybutyrate, acetate, creatine and the intensities of CH₃–(CH2)n, (CH2)n, CH₂CH₂*CO, CH₂CO, CH₂OCOR, C=CCH₂C=C, CH=CH and CH₂OPO₂– were elevated. The predominated changes indentified in the liver aqueous extracts included increased creatine and decreased glutamine, and the changes identified in the kidney aqueous extracts included elevated lactate and reduced taurine and glucose. In the liver and renal tissue chloroform/methanol extracts, there was a remarkable increase in many of the lipid signals intensity including CH3–(CH2)n, (CH2)n, and C=CCH2C=C. Increased citrate and 2-oxoglutarate in the urines were identified in rats firstly dosed nano-copper at 200mg/kg/d. Till day 5, elevated glucose, amino acid, citrate, succinate, lactate, acetate, and TMAO together with the decline in creatinine level were obvious in urine spectra obtained from the rats of the high-dose nano-copper group. Mid- and low-dose groups also demonstrated a slight separation compared with the control, and a decrease in the signals of citrate and 2-oxoglutarate was identified. The analysis of the time-course of metabonome changes in urine after nanocopper dosing indicated increased citrate and decreased creatinine were potential biomarkers for the damages in the liver and renal.In summary, nano-copper is moderately toxic material. Liver and kidneys were the main target organs. 50 mg/kg/d approached to the lowest observed adverse effects level for the rats repeatedly treated with nano-copper for 5 days. In the same mass concentration, the toxicity of nano-copper was more than that of micro-copper. The characteristics of small size, huge surface area and ultrahigh surface activity make nanocopper particles more easily transform into ionic copper in the different solutions, and the resulting ionic copper maybe an important factor responsible for their adverse effects either in vivo or in vitro model. Copper chelator can be also used as the treatment of toxicity of copper nanoparticles.The changes in metabolites suggested the failure of the mitochondria, enhanced ketogenesis, fatty acidβ-oxidation, and glycolysis were said to contributed to hepatotoxicity and nephrotoxicity induced by a dose of 200 mg/kg/d nano-copper. 2-oxoglutarate, citrate and serum triglycerides were potential NMR biomarker for the adverse effects of nano-copper. The advantages of metabonomic technique, such as sensitive, time-saving and high-throughput, favor it as a rapid in vivo screening method for nanotoxicity. The biopersistence of nano-copper was different between in vitro and in vivo models. As methods of risk assessment of nanoparticles, in vitro and in vivo test methods should be appropriately applied to ensure the scientific results.