The Mechanism Of Cadmium-induced Cytotoxicity In Rat Hepatocytes

Cadmium (Cd), an extremely important environment pollutant, was widely used in many industries. In human and other mammals, Cd affects adversely a number of organs and tissues, including the liver, kidney, lung, pancreas, testis, placenta and bone. Cd induced injury is very complex and unclear in the body. Cd induced cellular oxidative stress or apoptosis/death in vitro and in vivo. Apoptosis has relationship with mitochondria, intracellular calcium homeostasis, mitogen-activated protein kinase (MAPK) activitied and so on. Cd mainly accumulate in the liver initialy through rodent studies, so exposure to toxic doses of Cd will cause liver damage first. Therefore, we choose rat hepatocytes to study the mechanism of Cd induced cytotoxicity with cell biology and molecular biology method.1. Cd-induced cytotoxicity in rat hepatocytesRat hepatocytes were isolated by a two-step perfusion technique. After 24h planting, hepatocytes were treated with cadmium acetate (Cd(AC)2) (2.5,5 and 10μmol/L) for 12 h or 24 h. Cell viability, the leakage of lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT) in cell-free medium were measured with MTT assay and spectro photometric assay at the end of incubation. Hepatocytes morphology and ultrastructure were evaluated.The results showed that with the dose of Cd increased and long expored, hepatocytes viability was decreased very significantly (P＜0.01) after 12 h or 24 h treatment, hepatocytes showed sharp deformation and necrosis, mitochondria swelling and degeneration, mitochondrial cristae blurred, deformed or final collapse.The leakage of LDH, ALT and AST increased with doses increased, there was significant difference in partial groups than the control group (P＜0.05 or P<0.01). 2. Oxidative stress induced by Cd triggers apoptosis in rat hepatocytes24 h rat hepatocytes were treated with Cd(AC)2. The effects of Cd on oxidative stress markers, apoptosis, reactive oxygen species (ROS) generation, mitochondrial membrane potential (ΔΨm) collapse were measured.The results showed that the GSH content decreased with the dose of Cd increased, there was very significant difference in partial groups than the control group (P<0.01) at 12 h, but GSH content increased with the dose of Cd increased at 24 h. GSH-PX activities was in opposition with GSH content. The activities of CAT and SOD and MDA level increased, there was significant difference in partial groups than the control group (P＜0.05 or P＜0.01). The GST and GR activities were decreased but have no significant change. Cd induced apoptosis rate increased,ΔΨm collapse and ROS generation. There was significant difference in partial groups than the control group (P<0.05 or P<0.01).These results showed that Cd induces ROS generation and oxidative stress. Cd induces apoptosis through mitochondrial pathway.3. The effect of NAC on Cd induced cytotoxicityRat hepatocytes were treated with Cd in the presence or absence of NAC. The effects of NAC on Cd-induced cell morphology, apoptosis, reactive oxygen species (ROS) generation, mitochondrial membrane potential (ΔΨm) collapse were measured.These results showed that NAC significantly prevented hepatocytes from Cd-induced death (P＜0.05 or P＜0.01). NAC effectively inhibited Cd-induced alterations in the morphology of hepatocytes. NAC insult effectively protected hepatocytes against apoptosis from Cd. Cd induce ROS generation andΔΨm collapse were blocked by NAC. It showed that NAC can protect Cd-induced cytotoxicity.4. Cd-induced apoptosis in rat hepatocytes through caspase-independent way24 h rat hepatocytes were treated with Cd. Caspase-3 activities was measured. The effects of Z-VAD-fmk on Cd-induced cell viability, morphology and apoptosis in hepatocytes were evaluated. The results showed that caspase-3 activity does not increase, Z-VAD-fmk has no effect on cell survival, morphology and apoptosis. It showed that Cd induced hepatocytes apoptosis involvement of caspase-independent pathway.S.Involvement of calcium in Cd-induced apoptosis in rat hepatocytesHepatocytes were treated with Cd for certain time, then intracellular calcium concentration were determinated. And calcium chelator Bapta-AM on the impact of intracellular calcium concentration, cell survival, morphology and apoptosis, ROS generation andΔΨm collapse were evaluated.The results showed the level of intracellular calcium was very significantly increased induced by Cd at 1.5 h (P<0.01). However, with time increasing, the level of intracellular calcium levels rapidly decreased to the level of control group. Bapta-AM can attenuate the level of calcium and significant increase the cell viability induced by Cd (P＜0.05). Bapta-AM effectively inhibited Cd-induced alterations in the morphology of hepatocytes. Bapta-AM insult effectively protected hepatocytes against apoptosis from Cd. Cd induce ROS generation andΔΨm collapse were blocked by Bapta-AM. It showed that Cd can elevate intracellular calcium levels, then induce apoptosis through mitochondrial pathway.6.Cd induced apoptosis throuth p38 MAPK in rat hepatocytesHepatocytes were treated with Cd in the presence or absence of MAPK signaling inhibitors (p38 MAPK inhibitor SB202190, JNK inhibitor SP600125, ERK inhibitor U0126), then their effect on cell viability, morphology and apoptosis were investigated. Immunohistochemistry analysis was performed to recognize the activated phosphorylated forms of p38 MAPK kinases in Cd-treated hepatocytes in the presence or absence SB202190 and NAC. The impact of SB202190 on Cd-inducedΔΨm collapse was resurched.The results showed that SB202190 reversed significantly Cd-induced cell death. SB202190 effectively inhibited Cd-induced alterations in the morphology of hepatocytes. SB202190 protected hepatocytes against apoptosis from Cd. While SP600125 and U0126 increased Cd-induced cell death significantly. The phosphorylation of p38 MAPK increased after Cd treatment and these activations were inhibated by the treatment with its inhibitor SB202190 or NAC. SB202190 blocked disruption ofΔΨm. The findings suggested that Cd induction of ROS activated the p38 MAPK pathway, triggering apoptosis through mitochondrial pathway.