Copper Effect On Cognitive Functions And Copper Exposure Levels Of Jiangsu Primary School Students

Background:With the rapid development of industrial and agricultural production,the copper pollution in China is serious.The copper content of soils and water in some areas has exceeded the environmental limits.The domestic and international copper environmental and dietary standards are imperfect at present.The risk of copper exposure in the population is increasing gradually.Disorders of copper metabolism are closely related to various neurodegenerative diseases and cognitive impairment,but the specific mechanism is still unclear.The research on the health risk and mechanism of copper exposure is of great significance.Objective:1.To explore the neurocognitive function of copper exposure,and the mechanism of apoptosis,oxidative stress and synaptic function of hippocampal nerve cells in copper-induced cognitive impairment.2.To determine the copper urinary level of Jiangsu primary school students and analyze the distribution and influencing factors,in order to provide a scientific basis for assessing the health risk of copper exposure and improving environmental limits.Methods and Results:1.Effects of copper exposure on hippocampal neurons and synapsesMice hippocampal neurons（HT22 cells）were divided into 10,20,40,and 80μmol/L copper sulfate treatment groups as well as the control group.The proliferation activity was detected by CCK-8 method.The apoptosis was detected by flow cytometry.The SOD activity,MDA content and GSH-PX activity were measured,respectively.The synaptic morphology was observed by an inverted microscope.The expression of Bcl-2,Bax,SYP,PSD-95,pro BDNF,m BDNF and Trk B proteins were detected by Western blot.1.1 Effects of copper treatment on hippocampal neurons（1）After 24,36 and 48 h treatment,the cell proliferation activity decreased with the higher dose（P<0.05）.After 48 h treatment,the proliferation activity in copper treatment groups were significantly decreased compared with the control group（P<0.05）.（2）The apoptosis rate among groups was significant different.The apoptosis rate of 40 and 80μmol/L copper sulfate treatment groups were significantly increased compared with the control group（P<0.05）.（3）The relative expression of Bax protein in copper sulfate treatment groups were increased compared with the control group（P<0.05）.The relative expression of Bcl-2 protein in 40 and 80μmol/L copper sulfate treatment groups were decreased compared with the control group（P<0.05）.The ratio of Bcl-2/Bax protein was lower than the control group（P<0.05）.（4）No significant differences were found in SOD activity and MDA content（P>0.05）.The GSH-PX activity in copper sulfate treatment groups were decreased compared with the control group（P<0.05）.1.2 Effect of copper exposure on synapses in HT22 cells（1）Compared with the control group,the synapse morphology of copper sulfate treatment groups was changed and the synaptic grid structure was found.Some cells died and the synapse was sparse and disordered in 80μmol/L copper sulfate treatment group.（2）The relative expression of SYP,PSD-95,m BDNF and Trk B proteins in copper sulfate treatment groups were decreased compared with the control group（P<0.05）.The relative expressions of pro BDNF protein in copper sulfate treatment groups were increased compared with the control group（P<0.05）.2.Cognitive effects and mechanisms of copper exposure in miceSixty C57BL/6J mice were randomly divided into control group,low-dose group（100 mg/L Cu SO₄）,and high-dose group（500 mg/L Cu SO₄）,with 20 mice in each group.The mice were given free drinking water and continuously adminstered the conrresponding copper dosages for two months.The cognitive ability of C57BL/6J mice were detected by Morris water maze.The copper content of urine,blood and brain were determined by ICP-MS.The morphology of hippocampus was observed by HE staining.The SOD activity,MDA content and GSH-PX activity of brain were measured respectively.The SYP,PSD-95 and BDNF protein of brain were determined by ELISA.2.1 Effects of copper exposure on behavior in mice（1）The average weight of 100 mg/L Cu SO₄ group was higher than the control group.The spontaneous exercise and water consumption were reduced in 500 mg/L Cu SO₄ group.The brain coefficient of the mice in 500 mg/L Cu SO₄ group were increased compared with 100 mg/L Cu SO₄ group（P<0.05）.（2）There was no significant difference among groups in the content of blood copper（P>0.05）.The copper content of 24 h urine and brain were increased with higher dose（P<0.05）.（3）In the place navigation test,the plantform latency and target quadrant latency of 500 mg/L Cu SO₄ group were longer than the control group.In the spatial probe test,the plantform latency of 500 mg/L Cu SO₄ group were longer than the control group（P<0.05）.2.2 The mechanism of SYP,PSD-95 and BDNF proteins in copper-induced cognitive impairment（1）Compared with the control group,the hippocampal nerve cells in 100 mg/L Cu SO₄ group were arranged loosely and disordered.The number of hippocampal nerve cells were decreased.The widened intercellular space and sieve-like structures were observed in 500 mg/L Cu SO₄ group.（2）There was no significant difference among groups in SOD activity in mice brain（P>0.05）.The MDA content in 500 mg/L Cu SO₄ group were increased compared with control group（P<0.05）.The GSH-PX activity in 100 mg/L Cu SO₄ and 500 mg/L Cu SO₄ groups were decreased compared with control group（P<0.05）.（3）The content of SYP protein in mice brain with 500 mg/L Cu SO₄ group were lower than control group（P>0.05）.The content of PSD-95 and BDNF protein with100 mg/L and 500 mg/L Cu SO₄ group were lower than control group（P<0.05）.3.Analysis of copper urinary levels and influcing factors of Jiangsu primary school studentsA method for detecting urinary copper by ICP-MS was established,which were evaluated by standard curve,detection limits accuracy test,precision test and spike recovery test.The basic demographic information of primary school students in Nanjing and Xuyi were surveyed,meanwhile,individual urine samples were collected.The urine copper levels were determined by ICP-MS,and the influencing factors（gender,age,BMI and tobacco exposure）were analyzed.3.1 Establishment of ICP-MS method for detecting urinary copperThe regression equation was y=0.996x（R²=0.9981）.The detection limit was0.0090μg/L.The measured value of the quality control was within the confidence interval.The RSD of the precision test was 4.5%and the recovery rates were80%～120%.3.2 The urinary copper levels and influencing factors in primary school students in Jiangsu province（1）A total of 463 primary school students were collected,including 235 males and228 females.The age was 8～12 years.The BMI of 66.8%respondents were normal,and 50.6%respondents had tobacco exposure.（2）The geometric mean of urinary copper content of Jiangsu primary school students was 14.04μg/g of creatinine.The copper levels of students in Nanjing were higher than Xuyi（P<0.05）.（3）The urinary copper content of males was higher than females in Jiangsu province（P<0.05）.The urinary levels of Nanjing primary school students were higher in younger students（P<0.05）,but there was no significant difference in Xuyi（P>0.05）.The content of urinary copper in obese group and low BMI group were significantly higher than normal BMI group in Nanjing（P<0.05）,but there was no significant difference in Xuyi（P>0.05）.No significant difference among students with different tobacco exposure condition were found（P>0.05）.Conclusion:1.Copper treatment reduced the proliferation activity of HT22 cells,increased apoptosis and oxidative stress.The synapse was sparse and disordered.The expression of synaptic-associated proteins were down-regulated,which was related to pro BDNF/m BDNF/Trk B pathway.2.Copper exposure by drinking water increased the copper content in the urine and brain of C57BL/6J mice.Neurocognitive function was reduced.Hippocampal nerve cells were decreased and oxidative stress of brain was increased.The expression of SYP,PSD-95 and BDNF proteins were decreased.3.The geometric mean of urinary copper in Jiangsu primary school students was14.04μg/g of creatinine.The urinary copper levels in Nanjing primary students were higher than Xuyi students.The urinary copper levels were affected by gender,age and BMI.