The Mechanism Of Cadmium-induced Cytotoxicity In SMMC7721

Cadmium (Cd) is a toxic metal, targeting the lung, liver, kidney, and testes following acute intoxication, and causing nephrotoxicity, immunotoxicity, osteotoxicity and tumors after prolonged exposures. Liver plays a central role in toxicity of cadmium (Cd). Recently, there are lots of Studies reported on cadmium hepatotoxicity, but the specific mechanisms by which it produces its adverse effects have yet to be fully elucidated. In this study, hepatoma SMMC7721cells in the logarithmic phase were treated with cadmium by serum deprivation with different time to study the mechanism of cytotoxicity induced by Cd. The methods of cytobiology, molecular biology and comparative proteome methods were used to evaluate the mechanism of cytotoxicity in hepatocyte induced by cadmium. A series of tests were carried out:1. Cadmium induced cytotoxicity in SMMC7721SMMC7721cells were exposed to cadmium acetate (Cd(AC)2) atl,2.5,5,10,20,40and80μmol/L for a range of6,12and24h. Cell survival rates were measured with MTT assay. At the end of incubation, the leakage of lactate dehydrogenase (LDH) in cell-free medium was measured with spectro photometric assay; apoptosis rate were detectd with flow cytometry. The morphology, nucleus damage and ultrastructure of SMMC7721were also evaluated. The results showed that the inhibitory rate of SMMC7721cells increased significantly with the increasing dosage and time of exposure compared with control group (P<0.01). After24h treatment, hepatocytes showed sharp deformation and more dead cells in culture medium. Ultrastructurally, nucleoli damaged with crush and Chromatin margination and lots of vacuoles in cytoplasm, eventually collapse. After treated with Cd for12h or24h, the activity of LDH in cell free culture increased significantly(P<0.05or P<0.01)compared with control group. The apoptosis rate increased significantly compared with conrol group after24h exposed to Cd. Hoechst33258staining displayed nuclear fragmentation into2-3parts. In short, the injury induced by Cd shows does time effect.5-20μmol/L Cd could induce both necrosis and apoptosis. When treated with10μmol/LCd, we can receive relatively high apoptosis rate.2. The role of oxidative stress in injury induced by cadmium in SMMC7721The content of ROS, MDA and GSH in SMMC7721were detected after treated with Cd atl,2.5,5,10,20μmol/L. SMMC7721cells were treated with10μmol/L Cd in the presence or absence of NAC. The antagonistic effects of NAC on toxicity of Cd were evaluated. The results showed that the concentration of ROS increased significantly only after2h exposure, it decreased to nomal level after6h or longer. When exposed to Cd for12h, the concentration of GSH decreased significantly after treated with10μmol/L and20μmol/LCd compared with control group, while the concentration of MDA increased significantly with group20μmol/LCd. When exposed to Cd for24h, the concentration of GSH decreased significantly in all groups treated with Cd compared with control group, the concentration of MDA was on the contrary. Both apoptosis and necrosis induced by cadmium can be efficiently prevented by NAC. In conclusion, oxidative stress plays an important role in cytotoxicity induced by cadmium on SMMC7721.3. Effects of cadmium on mitochondria of SMMC7721The mitochondrial membrane potential (ΔΨm) was detected and ultramicrostructure of mitochondrial was observed after treated with Cd at5,10,20μmol/L for12h and (or)24h. At the same time the relative expression levels and/or protein expression level of gene Bcl-2, Bax, Cyt-c, Caspase-9and Caspse-3in SMMC7721were detected. The results showed that, after exposed to Cd for12h there was no significant difference on ΔΨm between control and groups (P>0.05); after expoused to Cd for24h, the ΔΨm of all groups exposed to Cd decreased significantly compared with control group(P<0.05or P<0.01). Ultrastructurally, mitochondria swelling and degeneration, mitochondrial cristae blurred, deformed or final collapse. The relative expression levels of Bcl-2decreased significantly in the groups treated with Cd at5,10μmol/L compared with control group, but increased significantly in (20μmol/LCd) group. The reverse change was detected with gene Bax. Western blot analysis showed the same tendency as above. The relative expression levels of Caspase-9increased significantly in group (Cd2.5,5,10,20μmol/L) compared to control group. Cd at10μmol/L decreased the relative expression levels of Caspase-3obviously compared with control, no significant difference was observed between the other groups and control. We can conclude that cytotoxicity induced by cadmium may through the mitochondrial path way in SMMC7721.4. Proteomic study on SMMC7721induced by cadmiumWe chose Cd at10μmol/L for24h to establish the damage model. The total protein of SMMC7721was extracted and separated with2-D gel electrophoresis. The results showed that24protein spots in total expressed differently during exposed to Cd including7new spots,11up-regulated spots and6down-regulated. With MALDI-TOF-MS,24protein spots representing23proteins were successfully identified, including peptidyl-prolyl cis-trans isomerase FKBP4, KRT8protein, heat shock protein27, proteasome activator complex subunit2,60S acidic ribosomal protein P0, heat shock70kDa protein6, glycyl-tRNA synthetase, isoformCRA_, calumenin isoform5, tyrosine3-monooxygenasea/tryptophan5-monooxygenase activation protein zeta, thioredoxin, Galectin-1, rho GDP-dissociation inhibitor1isoform a, ANNEXIN A5, Tubulin beta-2C chain keratin, type I cytoskeletal17, Eukaryotic translation initiation factor5A-1, Ran-specific GTPase-activating protein, KRT18protein, perilipin-3isoform3, heat shock60kDa protein1, Protein S100-A4, cytochrome b-c1complex subunit1mitochondrial, Keratin10. In addition, As confirmed by database query, these differential proteins were mainly associated with cell skeleton protein(21.7%), stress protein(21.7%), proliferation and apoptosis(8.7%), signal transduction(21.7%), nucleric acid binding protein(8.7%) and other proteins(21.7%). Based on the forthcoming research, we analyzed the function of these differential proteins as well as their relationship with mechanism of cytotoxicity induced by treated with Cd. In conclution, cytoskeleton was destroyed and oxidative stress was induced by Cd. Thioredoxin and60S acidic ribosomal protein PO may also participate in regulating apoptosis induced by Cd.