Effects Of N-acetyl Cysteine And Sodium Selenite On Hepatotoxicity And Nephrotoxicity Caused By Cadmium And The Apoptosis Of 293 Cells Induced By Cadmium

IntroductionAs one of the most important environmental and occupational metallic toxicants , cadmium is widely dispersed in the environment. Chronic effects of cadmium exposure include kidney and liver damage, anemia, pulmonary enphysema and the bone disease. Despite being one of the most important and widespread pollutant, the toxic mechanism of Cd is still poorly understood. Oxidative stress may be one of the toxic mechanisms of Cd and Cd can trigger apoptosis both in vitro and in vivo. The potential roles of reactive oxygen species and free radicals have been highlighted as mediators for apoptosis. N-acetylcysteine and sodium selenite are important antioxidant agents and may have effects on liver and kidney damage and apoptosis induced by cadmium. Caspase is a mammalian homo-log of CED-3 that play an essential role in apoptosis. The p53 tumor suppressor protein plays a central role in the control of cell cycle progression or apoptosis. Both caspase-3 and p53 gene are correlated with apoptosis signaling pathways.In this study, the effects of N-acetyl cysteine and sodium selenite on rat' liver and kidney damage exposure to cadmium were determined;the occurrence of apoptosis and the expression of Caspase-3 and p53 gene in transformed human embryonic kidney 293 cells after treatment with cadmium were determined.It is of great importance to study the relationship between cadmium-induced oxidative stress, apoptosis and the expression of apoptosis-correlated gene. Such studies may help us understand molecular biological mechanism of cadmium toxicology.Materials and Methods1. Experimental Study on the Effects of N-acetylcysteine and Sodium Sele-nite on Hepatotoxicity and Nephrotoxicity with Acute Exposure to Cadmium32 Wistar rats were randomly divided into four groups. Control group was injected sc with 0. 9% NaCl;Cadmium chloride group was injected sc with 35 fxmol/kg CdCl2;Others were pretreated by administering ip injection of lmmol/kg NAC(N-acetyl cysteine)and lOjxmol/kg NajSeC^ respectively 2 hour before the injection of CdCl2. Liver, kidney cortex and blood samples were collected 24 hours after CdCl2 administration. LDH( lactic dehydrogenase ) , GPT (glutamic pyruvic transaminase) activities in serum were determined. Cd( cadmium ) , MDA ( malondial dehyde ) and GSH ( glutathion) contents in liver and renal cortex as well as GSH-Px( glutathion peroxidase) activities were mensurat-ed.2. Experimental Studuy on The Effects of N-acetyl Cysteine and Sodium Selenite on Hepatotoxicity and Nephrotoxicity Induced by Chronic Exposure to Cadmium32 adult Wistar rats were randomly divided into four groups. Control group was injected sc with 0. 9 % NaCl;Cadmium chloride group, NAC + cadmium chloride group and Na2 SeO3 + cadmium chloride group were injected sc with 7|xmol CdCl2/kg, 5 times a week, for up to 6 weeks;Then NAC + cadmium chloride group and Na-2Se03 + cadmium chloride group were injected ip with lmmol/kg NAC and 10jxmol/kg Na2SeO3 respectively for 2 weeks;Cadmium chloride group and control group were injected with 0. 9 % NaCl at the same time. Urine, liver and kidney cortex samples were collected 24 hours after CdCl2 administration. Cd, protein contents and ALP, LDH, NAG activities in urine were determined. Cd, MDA and GSH contents in liver and renal cortex as well as GSH-Px activities were mensurated.3. The Effects of NAC and Z-DEVD-fmk on Apoptosis of Transformed Human Embryonic Kidney 293 Cells Induced by CadmiumTransformed human embryonic kidney 293 cells were incubated with0fimol/L,20jimol/L, 40|xmol/L, 80|xmol/L, 120|unol/L, 200fjunol/L Cdcl2 for Oh, 3h, 6h, 12h and 24h. The cells in groups of NAC and Z-DEVD-fink pretreatment were treated with 2. 5mmol/L, 5. Ommol/L, 10. Ommol/L NAC and 0. ljxmol/L, 1. 0(xmol/L, 10. Ojxmol/L Z-DEVD-fmk before incubated with 40jxmol/L Cdcl2. Cells viability were mensurated by MTT. The rate of ap-optosis was mensurated by Flowcytometry staining of cells with phosphatidyl-serine (PS)-annexin- V.4. The Relationship Between the Apoptosis of Transformed Human Embryonic Kidney 293 Cells Induced by Cadmium and the Expression of Caspase-3 and p53Transformed human embryonic kidney 293 cells were incubated with 40jxmol/L Cdcl2 for Oh, 3h, 6h, 12h and 24h. The cells in groups of NAC and Z-DEVD-fmk pretreatment were treated with 5. Ommol/L NAC and 10. Ojxmol/L Z-DEVD-fmk before incubated with 40jxmol/L Cdcl2. the expression of Caspase-3 and p53 gene in transformed human embryonic kidney 293 cells after treatment with cadmium were determined by the methods of reverse transcription polymer-ase chain reaction ( RT-PCR) and Western-blot analysis. And the occurrence of apoptosis was determined by Flowcytometry.Results1. Experimental Study on Effects of N-acetyl Cysteine and Sodium Selenite on Hepatotoxicity and Nephrotoxicity with Acute Exposure to CadmiumContrast with control group, LDH and GPT activities in serum and Cd contents in both liver and renal cortex with Cd alone were obviously increased. GSH, MDA contents in liver were increased significantly and GSH-Px activities were obviously decreased with Cd alone. Contrast with the group of given Cd a-lone, LDH and GPT activities in serum and GSH, MDA contents in liver with the administration of NAC were decreased significantly. Cd contents in both liver and renal cortex also decreased significantly;The pretreatment of NajSeC^ had significantly reduced LDH, GPT activities in serum;GSH and Cd contents in both liver and renal cortex were also reduced significantly. It also obviously in-creased GSH-Px activities in liver and MDA contents in renal cortex as compared with those given Cd alone.2. Experimental Studuy on the Effects of N-acetyl Cysteine and Sodium Sel-enite on Hepatotoxicity and Nephrotoxicity Induced by Chronic Exposure to CadmiumContrast with control group, Cd contents in liver, renal cortex and urine with Cd alone were obviously increased;ALP, NAG activities and protein contents in urine were obviously increased;GSH contents were obviously increased while GSH-Px activities obviously decreased in both liver and renal cortex;MDA contents in renal cortex were increased significantly with Cd alone. Contrast with the group of given Cd alone, Cd contents in both renal cortex and urine with the administration of NAC were increased significantly;NAG activities and protein contents in urine and GSH contents in both liver and renal cortex were obviously decreased;MDA contents in renal cortex decreased significantly with the administration of NAC group. The injection of Na2Se03 had significantly increase Cd contents in renal cortex and reduced Cd contents and ALP activities in urine;The injection of Na2Se03 had also significantly reduced GSH contents while increased GSH-Px activities in both liver and renal cortex.3. The Effects of NAC and Z-DEVD-fmk on Apoptosis of Transformed Human Embryonic Kidney 293 Cells Induced by CadmiumCells viability were time and dose dependent decreased with cadmium. Compared with O(xmol/L cadmium,the apoptosis rate increased obviously in the incubating cell with 40,80,120 ^OO^mol/L cadmium ( p < 0. 01). Both NAC and Z-DEVD-fmk, an antioxidant agent and a selective inhibitor of caspase-3, respectively, suppressed significantly cadmium-induced cell apoptosis.4. The Relationship Between the Apoptosis of Transformed Human Embryonic Kidney 293 Cells Induced by Cadmium and the Expression of Caspase-3 and p534. 1 RT-PCR analysis revealed that incubating cell with 40ujmol/L cadmium 6hN12h^24h, the expressin of caspase-3 mRNA were 2.1 fold, 3.8 fold and 2.7 fold respectively of that in control. The pretreatment of NAC and Z-DEVD-fmk increased the expression of caspase-3 mRNA compared with incubating cellwith 40jxmol/L cadmium only. The expressin of p53 mRNA had no change between 0 ~ 24h incubating cell with 40jjumol/L cadmium.4.2 Western-blot analysis revealed that caspase-3 20kDa and p53 protein were increased 6h after incubating ceU with 40funol/L cadmium. Pretreatment with NAC decreased the caspase-3 20kDa while pretreatment with Z-DEVD-fink had no effects on it. Both the NAC and Z-DEVD-fmk had no effects on the expression of p53 protein.Conclusion1. NAC and NajSeOj might have protective effects on Cd induced acute hepatotoxicity, which may related to increase the content of GSH and the activities of GSH-Px.2. NAC and NajSeC^ might have protective effects on Cd induced chronic nephrotoxicity, which may relate to increase the content of GSH and the activities of GSH-Px.3. Antioxidant agent and selective inhibitor of caspase-3 may suppress the apoptosis induced by Cd. Oxidative stress and caspase-3 were involved in Cd-in-duced apoptosis.4. Cadmium induces caspase-mediated cell apoptosis;Caspase play a central role in cadmium-induced cell apoptosis. The role of p53 in apoptosis induced by cadmium should be study thoroughly.