Mechanism Of Mitochondria-mediated Apoptosis Induced By Methylmercury In Human Hepatocytes

Background Mercury is a global pollutant.It is persistent,easy to migrate,highly bioaccumulative,and highly toxic,causing contamination to air,soil,and ocean.Various chemical forms of mercury can be converted to organic mercury,which is more toxic and poses a health hazard to living organisms.More than 100 countries around the world have signed the Minamata Convention on Mercury,working together to reduce the adverse effects of mercury on the environment and humans.Liver is an important detoxifying organ responsible for the metabolism of heavy metals and other poisons.After entering the human body,methylmercury can accumulate in target organs such as the liver,causing toxic effects.At present,there are few studies on hepatotoxicity of methylmercury and non-Caspase-dependent PARP-1/AIF apoptosis pathway in hepatocytes.Therefore,this study aimed to investigate the toxic effects of different concentrations of methylmercury on hepatocytes.The mechanism of apoptosis of mitochondrial pathway cells induced by methylmercury,explores the temporal and spatial relationship between PARP-1/AIF pathway and Cyt C/Caspase 3 pathway,and the hazard of methyl mercury in humans.Provide a theoretical basis for the development of scientific and effective protective measures.Aim To study the toxic effects of methylmercury on human hepatocytes and the mechanism by which methylmercury induces mitochondria-mediated apoptosis in hepatocytes.Method Hep G2 cells were selected as experimental subjects.MTT assay was used to detect the effect of methylmercury on the survival rate of Hep G2 cells.The concentration of methylmercury and the time of administration were determined.The morphology of methylmercury on Hep G2 cells was observed by inverted microscope.Immunofluorescence technique and Western Bloting method was used to detect DNA double-strand breaks induced by methylmercury;fluorescence microscopy and flow cytometry were used to detect the apoptosis of Hep G2 cells induced by methylmercury;flow cytometry was used to detect the effect of methylmercury on Hep G2 cell cycle;Western Bloting assay was used to detect the expression levels of apoptotic proteins(PARP-1,AIF,Pro-Caspase 3,Caspase 3)after treatment with different concentrations of methylmercury for 24 h..Western blotting was used to detect the apoptosis of mitochondria-associated cells in different time.The levels of PARP-1/AIF pathway-related proteins(PARP-1,PAR,AIF,Cy PA)and Cyt C/Caspase 3 pathway-related proteins(Cyt C,Caspase 9,Apaf-1,Pro-Caspase 3,Caspase 3,Hsp70)were detected by Western Bloting assay.Results 1.Methylmercury reduces Hep G2 cell survival in a dose-and time-dependent manner: Hep G2 cells treated with methylmercury for 24 h,and the survival rate of Hep G2 cells decreased with the increase of methylmercury concentration,the difference was statistically significant(P<0.05).The effect of methylmercury on the survival rate of Hep G2 cells was dose dependent.The cells were treated with 1.0 μg/m L methylmercury for 0,1,3,6,12,24,48 h.As the treatment time increased,the survival rate of Hep G2 cells gradually decreased,and the difference was compared with the survival rate of 0 μg/m L.Statistically significant(P<0.05).Methylmercury has a time-dependent survival rate for Hep G2 cells.2.The effect of methylmercury on the morphology of Hep G2 cells: 0 μg/m L Hep G2 cells are polygonal,adherent growth,good refractive index,clear cell boundaries and sharp edges.As the concentration of methylmercury increases,the cell volume becomes smaller,gradually shrinks and rounds,forming a smooth contour,and the refractive index is deteriorated.In the 1.25 μg/m L group,the cell boundaries were unclear,cell detachment increased,and cells were broken and melted.3.Methylmercury caused chromosomal DNA double-strand breaks in Hep G2 cells: After treatment with different concentrations of methylmercury for 24 h,γH2AX fluorescence focus signal was detected and increased with increasing methylmercury concentration.The green fluorescence focus was detected in the 1.0 μg/m L group methylmercury for 10 min.The green fluorescence signal intensity increased significantly at 20 and 30 min,and the fluorescence signal gradually decreased at 40,50,and 60 min.Western Bloting showed that the expression level of γH2AX increased first and then decreased.The level of γH2AX increased in the 20 th and 30 th minutes,and the level of γH2AX decreased in the 60 th group,which was significantly different from 0μg/m L(P<0.05).4.Methylmercury increases the apoptosis rate of Hep G2 cells: Apoptosis occurs in cells,and the apoptotic rate increases with the increase of methylmercury concentration.Compared with 0 μg/m L,the apoptosis in the 0.75,1.00,and 1.25 μg/m L groups was significant,and the apoptosis rate was statistically significant(P<0.05).5.The effect of methylmercury on Hep G2 cell cycle: With the increase of methylmercury concentration,the proportion of cells in G0/G1 phase decreased,and the proportion of S phase cells in 1.25 μg/m L group increased significantly,the difference was statistically significant(P<0.05).The percentage of cells in G2/M phase was significantly increased,and the difference was statistically significant(P<0.05).G2/M phase arrest occurred in Hep G2 cells.6.The effect of methylmercury on the expression of apoptosis protein in Hep G2 cells: The expression levels of the 116 k Da fragment and the 89 k Da fragment of PARP-1 protein were up-regulated and increased with the dose of methylmercury.The 116 k Da fragment of PARP-1 protein in 1.0 and 1.25 μg/m L group was significantly different from the control group(P<0.05),and the 89 k Da fragment of PARP-1 protein in 0.5,0.75,1.0,1.25 μg/m L group and control group (P<0.05).The expression of 67 k Da fragment of AIF protein was decreased,and the difference was statistically significant between the 1.25 μg/m L group and the control group(P<0.05).The expression of AIF protein was up-regulated at 57 k Da(P<0.01).The level of Pro-Caspase 3 protein was up-regulated and increased first and then decreased.The 0.5 and 0.75 μg/m L groups were significantly up-regulated(P<0.05),and the expression levels in 0.75,1.00,1.25 μg/m L group were slightly higher(P< 0.05).The level of Caspase 3 protein was on the rise,and the difference was statistically significant compared with 0 μg/m L(P<0.05).7.The effect of methylmercury on the PARP-1/AIF apoptotic pathway in Hep G2 cells: Compared with the control group,the expression levels of PARP-1 protein 116,89 k Da fragment,PAR polymer,AIF protein 57 k Da fragment and Cy PA increased with the increase of administration time.(P<0.05).The expression of 67 k Da fragment of AIF protein increased first and then decreased,and the difference was statistically significant(P<0.05).8.Effect of methylmercury on Cyt C/Caspase 3 apoptosis pathway in Hep G2 cells: Compared with the control group,the expression levels of Cyt C,Caspase 9,Apaf-1,Pro-Caspase 3 and Caspase 3 increased(P<0.05).The Pro-Caspase 3 protein level was not significantly different from the control group.The expression of Caspase 3 protein in the 3 and 6 h groups was significantly higher than that in the control group,and the difference was statistically significant(P<0.05).Compared with the control group,the Hsp70 protein level increased in the 1 h group,and the protein expression level decreased after 3 h of methylmercury administration.The Hsp70 level in the 6th,12 th,and 24 h groups was significantly different from the control group(P <0.05).9.Inhibition of caspase apoptosis pathway has no effect on PARP-1/AIF protein expression: Z-VAD-fmk can reduce the apoptosis rate of Hep G2 cells induced by methylmercury(P<0.05);compared with Me Hg group,the expression of Pro-Caspase 3 and Caspase 3 in Me Hg+Z-VAD-fmk group decreased(P<0.05).Compared with Control group,the expression of Pro-Caspase 3 and Caspase 3 in Me Hg+Z-VAD-fmk group decreased,the expression of Caspase 3 protein was statistically significant(P<0.05),and the expression of Caspase 3 protein was not statistically significant.There were no significant differences in the expression levels of Pro-Caspase 3 or Caspase 3 between Me Hg group and Me Hg+DMSO group.There was no significant difference in the expression of PARP-1(116 k Da)between Me Hg group,Me Hg+Z-VAD-fmk group and Control group.There was no significant difference in the expression levels of PARP-1(89 k Da)and AIF(67,57 k Da)compared with Me Hg group,Me Hg+DMSO group(solvent control group)and Me Hg+Z-VAD-fmk group.Compared with Control group(blank control group),the expression levels of PARP-1(89 k Da)and AIF(57 k Da)increased in Me Hg group,Me Hg+Z-VAD-fmk group and Me Hg+DMSO group,and the expression of AIF(67 k Da)decreased(P<0.05).Conclusion 1.Methylmercury decrease the survival rate of Hep G2 cells,cause cell morphology changes,DNA double-strand breaks,G2/M phase arrest in cells,and cell apoptosis.2.Methylmercury induce apoptosis in Hep G2 cells via PARP-1/AIF and Cyt C/Caspase 3 pathways 3.Inhibition of caspase pathway reduce apoptosis of Hep G2 cells and does not prevent apoptosis.