ABSTRACT
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.