The Role And Related Mechanism Of Copper-induced Oxidative Toxicity In Apolipoprotein E-knockout Mice

Wilson’s disease(hepatolenticular degeneration) is an autosomal recessive disorder of copper metabolism. Patients with Wilson’s disease exhibit various clinical manifestations such as different degrees of hepatic damage, neuropsychiatric symptoms, Kayser-Flescher rings, and damage to the kidney and skeletal muscle. Furthermore, liver damage is generally the major clinical manifestation at disease onset in children, whereas neurological symptoms tend to occur in early stage, even as initial symptom, which not conformed to Deiss’ clinical stages. Wilson’s disease caused by mutations in the ATP7 B gene. In European populations, the most common mutation in patients with Wilson’s disease is the H1069 Q mutation. However, the R778 L mutation most often occurs in Asian populations. The diversity of symptoms and differences in age at disease onset suggest that the features of Wilson’s disease may not be determined exclusively by mutations in the ATP7 B gene and that other genetic factors may be involved in the pathogenesis of the disease.Some studies have shown that apolipoprotein E(Apo E) genotypes are associated with clinical presentations and age at onset of symptoms in patients with Wilson’s disease. The Apo E gene has three alleles named as 2, 3, and 4, which encode three different Apo E isoforms with different biological functions. Several lines of evidence have shown that Apo E genotypes are associated with Alzheimer’s disease, vascular diseases, and brain injury; and the outcome is Apo E isoform-dependent. It is believed that the Apo E 3 protein is neuroprotective, whereas the Apo E 4 protein increases neuronal damage in Alzheimer’s disease. However, the mechanisms by which Apo E proteins are involved in neuroprotection remain unknown. Because Apo E proteins can exert an antioxidant effect and high copper concentrations in Wilson’s disease induce tissue damage via generation of free oxygen radicals, it is speculated that Apo E may play a neuroprotective role in patients with Wilson’s disease.In the present study, we established an animal model of Wilson’s disease by feeding the wild-type and Apo E-KO mice with copper for 12 weeks. Apo E-KO mice were used to determine the role of Apo E in copper-induced oxidative damage in the liver and brain. The purpose of this study was to explore the effect of Apo E on copper-induced oxidative toxicity in the liver and brain.The successful animal model was confirmed by measurement of liver copper content. Serous ALT, AST activity, serous and tissular MDA and SOD activities were detected to evaluate the change of oxidative stress in liver, brain and kidney. Immunohistochemistry, confocal, Western blot and RT-PCR techniques were used to detect NQO1 and HO-1 protein or m RNA level in liver and brain. Part Ⅰ Copper distribution and morphological observation in Apo Eknocked-out mice treated with dietary copperObjective: To establish an animal model of Apo E knocked-out mice with dietary copper; to investigate serous ALT, AST, serous copper concentration, tissular copper content, and morphological observation in Apo E knocked-out mice treated with dietary copper.Methods: Male wild-type and Apo E-knockout C57BL/6 mice(15.1±1.45 g) were used in this study. The mice were randomly assigned to four groups(n =12per group): wild-type mice treated with saline, Apo E-knockout mice treated with saline, wild-type mice treated with copper, and Apo E-knockout mice treated with copper. Wild-type or Apo E- knockout mice were intragastrically administered with 0.2 ml of copper sulfate pentahydrate amount to 4mg/d(200mg/kg) or the same volume of saline daily for 12 weeks, respectively. Animals were sacrificed, and blood samples were collected from the tails and eyes at the end of 12 weeks. The brain and liver were removed and stored at-80°C for the following experiments. Behavioral test was conducted by YLS-4C fatigue tester to detect the extrapyramidal damage. Measurement of copper levels: To measure blood copper concentrations, serum samples were obtained as described above. To measure copper concentrations in the liver and brain, liver or brain samples(150–200 mg) were digested with concentrated nitric acid(5 ml) and 30% H2O2(1 ml) at 120°C for 2 min, 160°C for 2 min, and 180°C for 5 min, using a microwave digestion system(CEM, USA). After the samples were cooled down, they were heated to 140°C until the volume was 2 ml. Each sample was then washed with 1% nitric acid three times. Copper concentrations in the serum, liver, and brain were measured using an atomic absorption spectrometer. Serum ALT and AST activities were analyzed by an automatic biochemical analyzer. Morphological observation included HE, Masson, Nissl’s amd Timm’s staining, and electron microscope observation. Timm’s staining was a copper specific staining method.Results: There were no significant differences in the liver copper concentrations between the wild-type and Apo E-knockout mice treated with saline. Compared with the wild-type mice treated with saline, the copper concentration in the liver was significantly higher in the wild-type mice treated with copper(P<0.05). In addition, the liver copper concentrations were significantly higher in the Apo E-knockout mice treated with copper compared with the wild-type mice treated with copper(P<0.05). Compared with the wild-type mice treated with saline, the brain copper concentrations were significantly higher in the wild-type mice treated with copper and the Apo E-knockout mice treated with saline or copper(P<0.05). In addition, there was no significant difference in the brain copper concentrations between the Apo E-knockout mice treated with saline and those treated with copper(P>0.05). The brain copper concentrations were significantly higher in the Apo E-knockout mice treated with copper compared with the wild-type mice treated with copper(P<0.05).Compared with the wild-type mice treated with saline, the kidney copper concentrations were significantly higher in the wild-type mice treated with copper and the Apo E-knockout mice treated with saline or copper(P<0.05). In addition, there was no significant difference in the kidney copper concentrations among the wild-type mice treated with copper and the Apo E-knockout mice treated with saline or copper(P>0.05). There were no significant differences in the serum copper concentration among the wild-type mice treated with saline or copper and the Apo E-knockout mice treated with saline or copper(P>0.05).The serum ALT activities were not significantly different among the wild-type mice treated with saline or copper and the Apo E-knockout mice treated with saline or copper(P>0.05). However, compared with the wild-type mice treated with saline, the serum AST activities were significantly higher in the wild-type mice treated with copper and the Apo E-knockout mice treated with saline or copper(P<0.05). In addition, the serum AST activities were significantly higher in the Apo E-knockout mice treated with saline or copper compared with the wild-type mice treated with copper(P<0.05).Compared with the wild-type and Apo E-knockout mice treated with saline, characteristic pathological changes occured, and ultrastructure observation showed that cellular edema, high-density electronic increased, mitochondria damage, lysosome increased and fibroplasia generally.Conclusion: Apo E-knockout mice treated with copper, exhibiting increased liver copper content, exhibiting liver copper content increased, could be used as an animal model of Wilson’s disease. Apo E knocked-out mice treated with dietary copper, copper distribution in tissue followed the discipline of dietary intake of copper. Compared with wild-type mice, Apo E knocked-out mice showed that tissue copper content increased obviously. Morphological observation in Apo E knocked-out mice was characteristic of oxidative injure. Part Ⅱ The change of serum and tissue oxidative stress and therelationship between copper and oxidation in Apo E knocked-out mice treated with dietary copperObjective: To investigate the changes of serous and tissular MDA and SOD in wild-type and Apo E knocked-out mice treated with copper, and to discuss the relationship between copper distribution and oxidative stress.Methods: MDA activities were determined using the thiobarbituric acid(TBA) method using commercial kits. Briefly, serum samples, liver and brain omogenates were mixed with 1 ml of TBA. The mixture was incubated at 95°C for 40 min. The samples were then centrifuged at 4000 rpm for 10 min. The absorbance of the supernatant was measured at 532 nm. The concentration of MDA was expressed as nanomoles of MDA per milligram of protein. SOD activities were determined using the xanthine oxidase method. Briefly, SOD activities were examined using commercial kits according to the manufacturer’s instructions. Serum samples or tissue homogenates were mixed with reaction solution, and the mixture was incubated at 37°C for 30 min. The absorbance was measured at 450 nm. SOD activities were expressed as units per milligram of protein.Results: The serum ALT activities were not significantly different among the wild-type mice treated with saline or copper and the Apo E-knockout mice treated with saline or copper(P>0.05). However, compared with the wild-type mice treated with saline, the serum AST activities were significantly higher in the wild-type mice treated with copper and the Apo E-knockout mice treated with saline or copper(P<0.05). In addition, the serum AST activities were significantly higher in the Apo E-knockout mice treated with saline or copper compared with the wild-type mice treated with copper(P<0.05). Compared with the wild-type mice treated with saline, the serum MDA activities were both significantly higher in the wild-type mice treated with copper and the Apo E-knockout mice treated with saline or copper(P<0.05). In addition, the serum MDA activities were significantly higher in the Apo E-knockout mice treated with copper as compared to the wild-type mice treated with copper(P <0.05). Compared with the wild-type mice treated with saline, the serum SOD activities were significantly lower in the wild-type mice treated with copper and in the Apo E-knockout mice treated with saline or copper(P<0.05). In addition, the serum SOD activities were significantly lower in the Apo E-knockout mice treated with copper as compared to the Apo E-knockout mice treated with saline and the wild-type mice treated with copper, respectively(P<0.05). There was no significant difference in liver MDA activities between the wild-type and Apo E-knockout mice treated with saline. Compared with the wild-type mice treated with saline, the liver MDA activities were significantly higher in the wild-type mice treated with copper(P<0.05). Similarly, compared with the Apo E-knockout mice treated with saline, the liver MDA activities were significantly higher in the Apo E-knockout mice treated with copper(P<0.05). Compared with the wild-type mice treated with saline, the liver SOD activities were significantly lower both in the wild-type mice treated with copper and in the Apo E-knockout mice treated with saline or copper(P<0.05). Similarly, compared with the wild-type mice treated with copper, the liver SOD activities were significantly lower in the Apo E-knockout mice treated with copper(P<0.05). There were no significant differences in brain MDA and SOD activities among the wild-type mice treated with saline or copper and the Apo E-knockout mice treated with saline or copper(P>0.05). There was a positive correlation in liver and brain copper content(r=0.707 P=0.033), and a negative correlation in serum MDA and SOD(r=-0.775 P=0.014) activities in wild-type C57B/L6 mice. There was a negaitive correlation between brain copper and kidney copper content(r=-0.78 P=0.022) in Apo E-KO mice. There was no correlation in copper content, MDA, and SOD among Apo E-knockout mice treated with copper.Conclusion: Increased serous MDA and decreased SOD were found in wild type and Apo E knocked-out mice treated with dietary copper, suggesting that copper induced oxidative stress. Compared with wild-type mice, Apo E knocked-out mice treated with copper, exhibited higher MDA activities, lower SOD activities, suggesting that oxidative stress was induced in the liver, and Apo E may protect the liver from copper-induced oxidative injury. Compared with wild-type mice, there were no significant difference in brain MDA and SOD activities in Apo E knocked-out mice treated with copper, suggesting that Apo E may not have neuroprotective effects on copper-induced oxidative injury in brain. In Apo E knocked-out mice treated with copper, there were no relationship among MDA, SOD activities and copper distribution in tissue. Part Ⅲ The mechanism of oxidative stress in liver or brain in wild-type and Apo E knocked-out mice treated with dietary copperObjective: To evaluate the expressions of NQO1 and HO-1 in the liver and brain in wild-type and Apo E knocked-out mice treated with dietary copper, to investigate the machanism of oxidative stress, and to explore whether Apo E has neuroprotective effects in Wilson’s disease.Methods: After copper or saline administration for 12 weeks, immunohistochemistry confocal technique was used to detect NQO1 and HO-1 in the liver and brain tissues, Western blot and RT-PCR were used to determinate the protein or m RNA expressions of NQO1 and HO-1.Results: Western blot analysis showed that the expressions of NQO1 in the liver were both significantly higher in the wild-type mice treated with copper and in the Apo E-knockout mice treated with saline or copper, as compared to the wild-type mice treated with saline, respectively(P<0.05). Compared with the Apo E-knockout mice treated with saline, the expression of NQO1 in the liver was significantly higher in the Apo E-knockout mice treated with copper(P<0.05). Compared with the wild-type mice treated with copper, the expression of NQO1 in the liver was significantly higher in the Apo E-knockout mice treated with copper(P<0.05). Similarly, compared with the wild-type mice treated with saline, the expressions of HO1 in the liver were both significantly higher in the wild-type mice treated with copper and in the Apo E-knockout mice treated with saline or copper(P<0.05). Compared with the Apo E-knockout mice treated with saline, the expression of HO1 was significantly higher in the Apo E-knockout mice treated with copper(P <0.05). Consistent with the western blot results, the immunohistochemical studies showed that the expressions of NQO1 and HO1 were up-regulated in the Apo E-knockout mice treated with copper. The expressions of NQO1 and HO1 in the brain were not significantly different among the wild-type mice treated with saline or copper and the Apo E-knockout mice treated with saline or copper(P>0.05). The relative value of NQO1/ GAPDH in the brain was not significantly different between the Apo E-knockout mice treated with saline or copper(P>0.05).Conclusion: Compared with wild-type mice, Apo E-knockout mice treated with copper exhibited higher expression levels of NQO1 and HO1 in the liver, suggesting that Apo E may protect the liver from copper-induced oxidative injury. However, Apo E-knockout mice treated with copper do not exhibit significant expressions of NQO1 and HO1 in the brain, suggesting that Apo E may not have protective effects on the brain against copper-induced oxidative injury, and the relative value of NQO1/β-actin in the brain further supported the viewpoint.