RESUMO
@#Abstract: Objective ， To explore the effects of lead exposure on copper level copper transporter protein expression and Methods oxidative stress in mouse cerebral cortex. The specific pathogen free adult male C57BL/6 mice were randomly ， - - - divided into control group low lead exposure group and high lead exposure group with 10 mice in each group. The mice in low - ， and high lead exposure groups were respectively given 250 and 500 mg/L lead acetate in drinking water every day and the mice - ， in the control group were given double distilled water for 12 weeks. Twenty four hours after exposure Morris water maze and ， elevated cross maze were used to test the neurobehavioral function of mice. The cerebral cortex of mice was isolated and the levels of lead and copper were detected by inductively coupled plasma mass spectrometry. The activities of glutathione （ - ）， （ ） （ ） peroxidase GSH Px catalase CAT and malondialdehyde MDA were detected by histochemical method. The relative （ ） ， ， expression levels of copper transporter such as synthesis of cytochrome C oxidase SCO 1 SCO 2 and cytochrome C oxidase （ ） Results - - assembly protein 11 COX11 were detected by western blot. The escape latencies of mice in the low and high lead （ P ）， ， - exposure groups were prolonged all <0.05 while the number of crossing the platform the percentage of open arm entry - （ P ） times and the percentage of open arm retention time decreased all <0.05 compared with the control group. Mice in both the - - （ P ）， - low and high lead exposure groups increased levels of lead and copper in the cerebral cortex all <0.05 decreased GSH Px （ P ）， （ P ） and CAT activity all <0.05 and increased SCO1 relative expression all <0.05 compared with the control group. Mice in - （P ）， - the high lead exposure group showed prolonged escape latency <0.05 reduced GSH Px and CAT activities in the cerebral （ P ）， （ P ） - cortex all <0.05 increased MDA level and relative expression of SCO1 and SCO2 all <0.05 compared to mice in the low Conclusion - lead exposure group. Lead exposure increased the expression of copper and copper transport related proteins in mouse cerebral cortex and induced oxidative stress leading to central nervous system damage resulting in neurobehavioral abnormalities in mice.
RESUMO
OBJECTIVE: To investigate the role of copper transporter protein and copper chaperones in copper accumulation in glioma cell line C6 cells induced by lead acetate exposure. METHODS: i) CCK-8 assay was used to determine the proper lead acetate dose by treating the cells with lead acetate at the final concentration of 0-50 μmol / L for 24. 0 hours. ii) C6 cells were divided into control group and lead-exposure group,treated with 0 and 10 μmol / L lead acetate respectively for24. 0 hours,and then cultured in 2 μmol / L copper chloride for 0. 0,0. 5,1. 0,2. 0,4. 0 and 8. 0 hours; inductively coupled plasma mass spectrometry was used to detect the levels of copper and lead in the cells. Real-time polymerase chain reaction was used to detect the mRNA expression of copper transporter 1( CTR1),divalent metal transporter 1( DMT1),copper-transporting ATPase α polypeptide / β polypeptide( ATP7 A and ATP7B), antioxidant 1 copper chaperone( ATOX1),cytochrome c oxidase copper chaperone( COX17),and copper-chaperone-for-superoxide dismutase( CCS).Laser con-focal microscopy was applied to detect the protein expression of CTR1 and ATP7 A in cells. RESULTS: i) CCK-8assay proved that the 10 μmol / L lead acetate treatment did not affect C6 cells proliferation( P > 0. 05). Thus the final concentration of 10 μmol / L lead acetate was chosen as the treatment dose in later experiments. ii) After 10 μmol / L lead acetate exposure for 24. 0 hours,the lead and copper levels of C6 cells in lead-exposure group were higher than those in the control group( P < 0. 01),but there was no statistical significant difference in the C6 cell survival rate between these two groups( P > 0. 05). After cells were treated with copper,the C6 cell survival rate of lead-exposure group was lower than that in the control group( P < 0. 01). The interactive effect of copper level showed statistical significance between lead exposure and cooper treatment time( P < 0. 01). At the 5 time points from 0. 5-8. 0 hours after exposure to copper,the copper levels in lead-exposure group were higher than those of control group( P < 0. 05). The copper levels in the control group reached a peak after exposure to copper for 2. 0 hours,and maintained at a stable level till the time point of 8. 0hours. The copper levels of lead-exposed groups increased with the increasing time of copper exposure and there was a time-effect relationship,and they reached to the peak at the time point of 8. 0 hours. After 10 μmol / L lead acetate exposure for 24. 0 hours,compared with control group,the CTR1 and DMT1 mRNA relative expression levels in leadexposed group increased by 113. 00% and 36. 00% respectively( P < 0. 01),and the ATP7 A mRNA relative expression level decreased by 25. 00%( P < 0. 01). The protein expression of CTR1 increased by 76. 04%( P < 0. 01),and the protein expression of ATP7 A decreased by 16. 0%( P < 0. 01). There was no significant difference in the mRNA relative expression levels of ATP7 B,ATOX1,COX17 and CCS between the two groups( P > 0. 05). CONCLUSION: Lead acetate exposure can lead to increase accumulation of copper in C6 cells with increasing exposure time showing a time-effect relationship. The increased protein expression of CTR1 and decreased protein expression of ATP7 A might be one of the mechanisms of inducing copper accumulation in cells after the lead acetate exposure.