The Mechanism Of Cadmium-induced Cytotoxicity And The Protection Of NAC In BRL3A Rat Liver Cells

Cadmium (Cd) is a toxic heavy metal, easy to accumulate in the body. Cd causes a mumber of organs and tissues injury and induces dysfunction, including kidney, liver, lung, bone, cardiovascular system, immune system. Some experiments in vitro and in vivo have already proved that Cd induced cellular apoptosis, oxidatived damage and DNA damage, apoptosis was correlated with oxidative stress, mitochondrial damage, the activation of MAPK, death receptor, Caspase and Caspase independent pathway. Studies revealed that liver is the main target organ of Cd injury. Recently, lots of researches had worked on it, but the mechanism is still not clear. Therefore, we choose the BRL3A immortalized rat hepatocytes to study the mechanism of Cd induced cytotocity and the protection of NAC with cytobiological and molecular biological methods. A series of tests were carried out:1. The cytotoxicity induced by Cd and the protection of NAC in BRL3A cellTo explore the cytotoxicity induced by Cd and the protection of NAC, BRL3A cells were treated with0,10,20, and40μmol/L CdAc2for12h (a-d groups). In the other two experiments, the cells were pre-incubated with2mmol/L NAC for30min and then incubated with20μmol/L CdAc2for12h (e-f groups). The morphology damage was observed by a reverse microscope. Cell viability was evaluated by MTT assay and the leakage of lactate dehydrogenase (LDH) was measured with colorimetric method. The results showed with the increasing dosage of Cd, significant morphological changes showing cell shrinkage, rounding, and loss of cell integrity. Cell viability was decreased significantly (p<0.01), the leakage of LDH increased significantly (p<0.01). NAC alone did not affect the cell morphology, cell viability and the leakage of LDH. Cotreatment with NAC and Cd attenuated morphological changes, increased cell viability and decreased the leakage of LDH significantly (p<0.01). NAC can effectively attenuate the damage induced by Cd in BRL3A cells.2. The effect of apoptotic ratio induced by Cd and the protection of NAC in BRL3A cellTo investigate the effect of apoptosis induced by Cd and the protection of NAC, BRL3A cells were treated with0,10,20, and40μmol/L CdAc2for12h (a-d groups). In the other two experiments, the cells were pre-incubated with2mmol/L NAC for30min and then incubated with20μmol/L CdAc2for12h (e-f groups). BRL3A cells were treated with Cd and NAC for12h, nuclear morphology was analyzed by Hoechst33258staining, cell apoptosis was measured by flow cytometry. The results showed with the increasing dosage of Cd, cell chromatin condensation, cell shrinkage and nuclear fragmentation were observed. The apoptotic ratio increased significantly (p<0.01or p<0.05). NAC alone did not affect the cell nuclear morphology and cell apoptosis. Cotreatment with NAC and Cd attenuated nuclear morphological changes, decreased cell apoptotic ratio significantly (p<0.01). NAC can effectively prohibit the apoptosis induced by Cd in BRL3A cells.3. The DNA damage induced by Cd in BRL3A cellTo observe the DNA damage induced by Cd, BRL3A cells were treated with0,10,20, and40μmol/L CdAc2for12h (a-d groups). DNA damage was measured with single cell gel electrophoresis assay. The results showed with the increasing dosage of Cd, cell commet ratio, tail length and tail DNA percentage increased significantly (p<0.01orp<0.05). Cd induced DNA damage in BRL3A cells.4. The oxidative damage induced by Cd and the protection of NAC in BRL3A cellTo measure the oxidative damage induced by Cd and the protection of NAC, BRL3A cells were treated with0,10,20, and40umol/L CdAc2for12h (a-d groups). In the other two experiments, the cells were pre-incubated with2mmol/L NAC for30min and then incubated with20μmol/L CdAc2for12h (e-f groups). Reactive oxygen species(ROS) was detected by flow cytometry, MDA content and SOD、GSH-Px activity were measured with colorimetric method. The results showed with the increasing dosage of Cd, ROS and MDA content increased significantly (p<0.01orp<0.05), SOD and GSH-Px activity decreased significantly (p<0.01or p<0.05). NAC alone did not affect ROS、MDA content and SOD、GSH-Px activity. Cotreatment with NAC and Cd ROS content decreased significantly (p<0.05), SOD and GSH-Px activity increased significantly (p<0.01orp<0.05). NAC can relieve oxidative damage induced by Cd in BRL3A cell.5. The mitochondrial damage by Cd and the protection of NAC in BRL3A cellTo observe the mitochondrial damage induced by Cd and the protection of NAC, BRL3A cells were treated with0,10,20, and40μmol/L CdAc2for12h (a-d groups). In the other two experiments, the cells were pre-incubated with2mmol/L NAC for30min and then incubated with20μmol/L CdAc2for12h (e-f groups). Mitochondrial ultramicrostructure changes were detected by transmission electron-micriscope and the immunofluorescence of AIF, EndoG were observed, the mitochondrial membrane potential (△Ψm) was detected by flow cytimetry, the activity of Caspase3、Caspse9was detected by colorimetric method, the mRNA and protein level of Bax、Bcl-2, the protein level of Caspse3、Caspase9、PARP were detected by real time fluorescent quantitative PCR and immunoblot. The results showed with the increasing dosage of Cd, mitochondrial swelling and degeneration, mitochondrial cristae blurred, deformed or final collapse, the△Ψm decreased significantly (p<0.01), the activity of Caspase3、Caspse9increased significantly (p<0.01or p<0.05), the mRNA and protein level of Bax increasedand Bcl-2decreased significantly (p<0.01), the protein level of Caspase3、Caspase9、PARP decreased and Cleaved-Caspase3、Cleaved-Caspase9increased. NAC alone did not affect the mitochondrial ultramicrostructure and protein level of Bax、Bcl-2、Caspase3、Caspase9、PARP. Cotreatment with NAC and Cd can inhibit the tendency of the protein level of Bax、Bcl-2、Caspase3、Caspase9、PARP. Cd induced translocation of AIF and EndoG from mitochondria to nucelus. Caspase and Caspase independent pathway toke part in apoptosis induced by Cd in BRL3A cells, NAC can protect the mitochondrial damage induced by Cd in BRL3A cell.6. The effect of Cd on MAPK pathway and the protection of NAC in BRL3A cellTo investigate the effect of MAPK pathway induced by Cd and the protection of NAC, in the first group, BRL3A cells were treated with0,10,20, and40μmol/L CdAc2for12h (a-d groups). In the other two experiments, the cells were pre-incubated with2mmol/L NAC for30min and then incubated with20μmol/L CdAc2for12h (e-f groups). In the second group, BRL3A cells were treated with0,20μmol/L Cd and were pre-incubated with10μmol/L that p38inhibitor (SB203580), JNK inhibitor (SP600125)、ERK inhibitor(U0126) for30min, followed by incubation with20μmol/L Cd for12h, cell apoptotic ratio was detected by flow cytometry and the protein level of MAPK were measured by immunoblot. The results showed the inhibitor of SB203580, SP600125, and U0126decreased cell apoptotic ratio significantly (p<0.01), with the increasing dosage of Cd, the protein level of P-JNK、P-p38increased,10-20μmol/L Cd decreased P-ERK protein level,40μmol/L Cd increased42kD P-ERK protein level,44kD P-ERK protein level didn’t change. MAPK pathway took part in Cd induced BRL3A cell apoptosis and NAC inhibited MAPK pathway.7. The effect of Cd on Fas/FasL pathway and the protection of NAC in BRL3A cellTo investigate the effect of Fas/FasL pathway induced by Cd and the protection of NAC, BRL3A cells were treated with0,10,20, and40μmol/L CdAc2for12h (a-d groups). In the other two experiments, the cells were pre-incubated with2mmol/L NAC for30min and then incubated with20μmol/L CdAc2for12h (e-f groups). The activity of Caspase8was detected by by colorimetric method, the protein level of Caspase8and FasL, the mRNA of Fas、FasL were measured by real time fluorescent quantitative PCR and immunoblot. The results showed with the increasing dosage of Cd, Caspase3activity, the level of Cleaved-Caspase8、FasL protein and Fas mRNA increased significantly (p<0.01), the mRNA level of FasL increased first and then decreased. Fas/FasL pathway is releated with BRL3A cell apoptosis and NAC inhibited Fas/FasL pathway.