Study On Toxicity Of Liver Cells Induced By Methylmercury

In this study, human hepatic cell line HL-7702 was used as model, the proliferative inhibitory effect of MeHg was observed by MTT assay; the lipid peroxidation effect was examined by biochemical method;the changes of cell cycle and mitochondrial membrane potential was detected by flow cytometry (FCM); the cell apoptosis was observed by AO/EB double fluorescent staining and FCM;and the expression of proteins related to apoptosis was detected by immunocytochemical method. Results showed that MeHg could inhibit proliferation of HL-7702, induce S phage arrest, lipid peroxidation and apoptosis. The mechanisms of apoptosis could be related to mitochondrial membrane potential changes,lipid peroxidation and expression of apoptosis related proteins, such as Fas, FasL, Bax, Bcl-2, CytC, Caspase-3 and AIF.1. Proliferative inhibitory effect on HL-7702 induced by MeHg24 and 48 h after exposure to MeHg (10, 20, 30, 40 and 50μmol/L), the OD value decreased with the increase of MeHg, after 24 h there were significant difference between 40,50μmol/L groups and control group respectively (P<0.05), after 48h there were significant difference between all exposure group and negative control group respectively (P<0.05), and the proliferative inhibitory rate increased with the increace of exposure time and dosage, suggesting MeHg had the proliferative inhibitory effect on HL-7702 cell line in time and dose dependent way. 2. Activities influence of Superoxide Dismutase (SOD) and Glutathion Peroxidase (GSH-Px) on HL-7702 induced by MeHg24 h after exposure to MeHg (10, 20, 30, 40 and 50μmol/L), SOD activity decreased with the increace of MeHg, and there were significant differences between 20, 30, 40, 50μmol/L groups and negative control group respectively (P<0.05).24 h after exposure to MeHg (10, 20, 30, 40 and 50μmol/L), the activity of GSH-Px descended with the increace of MeHg, and there were significant differences between 30, 40, 50μmol/L groups and negative control group respectively (P<0.05).The changes of both two enzymes suggested MeHg could induce lipid peroxidation on HL-7702 cells.3. Cell cycle changes of HL-7702 induced by MeHgFCM with PI staining method was performed to detect the cell cycle changes. 24 h after exposure to MeHg (10, 20 and 30μmol/L), percentage of cells in G0/G1 phage decreased while S phage increased with the increasing of MeHg (P<0.05); and compared with 30μmol/L group, percentage of cells in G0/G1 phage increased, S phage and G2/M decreased in 40, 50μmol/L groups. All suggested MeHg could induce S phage arrest.4. Apoptosis of HL-7702 induced by MeHgFCM with PI staining and AO/EB double fluorescent staining methods were performed to examine apoptosis. After exposure to MeHg (10, 20 and 30μmol/L) for 24 h, apoptotic rate ascended with the increasing of MeHg and got to highest at 30μmol/L group, then the rate appeared a descend trend. There were significant differences between exposure and negative control groups (P<0.05) suggesting MeHg could induce apoptosis of HL-7702.5. Changes of mitochondrial membrane potential of HL-7702 induced by MeHgRhodamine-123 staining and FCM were used to detect the changes of mitochondrial membrane potential. 24 h after exposure to MeHg (10, 20, 30, 40 and 50μmol/L), mitochondrial membrane potential descended with the increace of MeHg, and there was significant differences between exposure and negative control group (P<0.05).6. Expression of apoptosis related proteins in HL-7702 induced by MeHg6.1 Expression of Fas,FasL24 h after exposure to MeHg(10, 20, 30 and 40μmol/L), the expression of Fas,FasL enhanced with the increace of MeHg, and there were significant differences in expression between 20, 30, 40μmol/L and negative control group (P<0.05).6.2 Expression of Bax,Bcl-224 h after exposure to MeHg (10, 20 and 30μmol/L), the expression of Bax,Bcl-2 enhanced with the increace of MeHg, and there were significant differences between exposure and negative control group (P<0.05), and Bax/Bcl-2 ratio also appeared an increase trend.6.3 Expression of CytC and Caspase-324 h after exposure to MeHg (10, 20, 30 and 40μmol/L), the expression of CytC and Caspase-3 enhanced with the increace of MeHg, and there were significant differences in CytC,Caspase-3 expression between exposure and negative control group (P<0.05). 6.4 Expression of AIF24 h after exposure to MeHg, the expression of AIF enhanced with the increace of MeHg, and there were significant differences in AIF expression between 20, 30 and 40μmol/L and negative control group (P<0.05).