Effects Of TCDD On Mitochondria Of Human Astrocytes And SH-SY5Y Cells

Objective:The aim of this study was to investigate the effects of endocrine disruptors 2,3,7,8-tetrachlorodibenzo-p-dioxins(TCDD)on mitochondria of human astrocytes and SHSY5 Y cells.The effects of TCDD on mitochondrial morphology,content of intracellular ATP,the expression of mitochondrial related genes and proteins were focused.The effects of TCDD on the mitochondria of human nerve cells was studied to explore the toxicity mechanisms of TCDD on human nervous system.Methods:1.The astrocytes and SH-SY5 Y cells were treated with 0-1000 nM TCDD for 24 h,then the activity of astrocytes and SH-SY5 Y cells was detected by MTS.2.The effects of 0-100 nM TCDD exposure for 24 h on the mitochondrial ultrastructure of astrocytes and SH-SY5 Y cells were observed by transmission electron microscopy.3.The intracellular ATP content in astrocytes and SH-SY5 Y cells was detected after exposured to 0-100 nM TCDD for 24 h.4.The mRNA expression levels of mitochondrial related genes in astrocytes and SH-SY5 Y cells was detected by PCR Array after exposured to 0-50 nM TCDD for 24 h.5.After TCDD treated cells for 24 h,the expression of TSPO,SH3GLB1,TOMM22,TOMM40 L,SLC25A10,SLC25A27,MFN1 and MFN2 in astrocytes and SH-SY5 Y cells was detected by RT-PCR.6.The effect of 0-100 n M TCDD exposure on the expression levels of PBR and MFN2 in astrocytes and SH-SY5 Y cells was detected by Western blot after exposured to 0-100 nM TCDD for 24 h.Results:1.MTS test showed that 0-1000 nM TCDD exposure for 24 h had no significantly inhibiting effect on the activity of astrocytes and SH-SY5 Y cells.2.The mitochondrial ultrastructural changes of astrocytes and SH-SY5 Y cells were observed by transmission electron microscopy after the cells exposured to TCDD for 24 h.The mitochondrial ridge number of the two cells was reduced and the mitochondria were swollen and vacuolar in the TCDD treated cells.Indicating that TCDD can affect the normal mitochondrial morphology of astrocytes and SH-SY5 Y cells.3.Compared to the control group,the ATP content in astrocytes was increasing after exposed to 0-100 n M TCDD for 24 h,100 nM TCDD-treated groups were higher significantly(P < 0.05).In SH-SY5 Y cells,the intracellular ATP content increased firstly and then decreased after 100 nM TCDD exposure(P < 0.05).4.The expression of mitochondrial related genes was explored by PCR Array.In astrocytes,1nM TCDD could down-regulate mRNA levels of 12 genes,50 nM TCDD could down-regulate mRNA levels of 11 genes.In SH-SY5 Y cells,1nM TCDD could down-regulate mRNA levels of 12 genes,and 50 nM TCDD could down-regulate mRNA levels of 2 genes.5.In this study,8 genes were selected as target genes,the mRNA expression levels of target genes was detected by RT-PCR.Compared with the control group,in astrocytes,the mRNA expression level of TSPO was increased with 100 nM TCDD treated(P < 0.05).The mRNA expression level of SH3GLB1 was decreased with 50 nM and 100 nM TCDD treated(P < 0.05).The mRNA expression level of TOMM22 was decreased with 10 nM,50nM and 100 nM TCDD treated(P < 0.05).The mRNA expression level of TOMM40 L was decreased in astrocytes with 1nM,10 nM and 100 nM TCDD treated(P < 0.05).The mRNA expression level of SLC25A10 was decreased with 1nM,10 n M,50 nM and 100 nM TCDD treated(P < 0.05).The mRNA expression level of SLC25A27 was increased with 50 nM and 100 nM TCDD treated(P < 0.05).The mRNA expression level of MFN1 was decreased with 10 nM,50nM and 100 nM TCDD treated(P < 0.05).The mRNA expression level of MFN2 was decreased with 1nM,10 nM,50nM and 100 nM TCDD treated(P < 0.05).In SH-SY5 Y cells,compared with the control group,the mRNA expression level of TSPO was increased with 1nM,10 nM,50nM and 100 nM TCDD treated(P < 0.05).The mRNA expression level of SH3GLB1 was decreased with 1nM,50 nM and 100 nM TCDD treated(P < 0.05).The mRNA expression level of TOMM22 was decreased with 1nM and 10 nM TCDD treated(P < 0.05).The mRNA expression level of TOMM40 L was decreased with 10 nM and 100 nM TCDD treated(P < 0.05).The mRNA expression level of SLC25A10 was decreased with 50 nM and 100 nM TCDD treated(P < 0.05).The mRNA expression level of SLC25A27 was increased with 100 nM TCDD treated(P < 0.05).The mRNA expression level of MFN1 was decreased with 10 nM,50nM,100 nM TCDD treated(P < 0.05).The mRNA expression level of MFN2 was decreased with 10 nM,50nM,100 nM TCDD treated(P < 0.05).6.Western blot results showed that compared with the control group,the expression of PBR in astrocytes was decreased with 1nM TCDD treated(P < 0.05),and the expression of PBR in 10 nM and 100 nM TCDD-treated groups was increased with statistically significant(P < 0.05).The expression of MFN2 in astrocytes was decreased with 10 nM and 100 nM TCDD treated(P < 0.05).Compared with the control group,the expression of PBR protein in SH-SY5 Y cells was decreased in 1nM,10 nM and 100 nM TCDD-treated groups,but the difference was not statistically significant.The expression of MFN2 protein in SH-SY5 Y cells was decreased with 1nM,10 nM and 100 nM TCDD treated(P < 0.05).Conclusions:1.TCDD in the experimental concentrations did not inhibit the viability of astrocytes and SH-SY5 Y cells,but had an effect on the normal morphology of mitochondria in both kinds of cells.2.The ATP content in astrocytes were increased,because TCDD can induce the release of intercellular signaling molecules to regulate the activity of astrocytes and neurons.In SH-SY5 Y cells,TCDD can reduce the intracellular ATP content,affecting the normal oxidative phosphorylation of mitochondria.3.TCDD can inhibit the mRNA expression levels of SH3GLB1,TOMM22,TOMM40 L,SLC25A10,MFN1 and MFN2 in astrocytes and SH-SY5 Y cells,inhibit the protein expression level of MFN2,promote the mRNA expression levels of TSPO and SLC25A27 and the protein expression level of PBR in astrocytes,indicating that TCDD has the effect of damage on both kinds of cells,and may activate the self protective mechanism of mitochondria.TCDD can affect the mitochondrial morphology,protein transport and mitochondria dynamic process.