Experimental Study On Cd-induced Oxidative Damage Of Mitochondria Isolated From Rat Liver And Protective Effect By NAC

IntroductionCadmium(Cd)is one of the most important toxic chemicals due to its increasing level in the environment as a result of industrial and agricultural practices.Cd has a very long biological half-life(10-30 years)in humans and the toxicity of which is dependent on the route,dose and duration of exposure is a nonessential heavy metal with no known biological function in animals.Acute cadmium intoxication induced primarily hepatic and testicular damage,whereas the most severe form of chronic Cd intoxication is the"itai-itai"disease,principal clinical characteristics of which are renal dysfunctions and painful bone injuries in human.While the mechanism of Cd toxicity is not known,it has been hypothesized that it is associated with mitochondrial dysfunction.Evidence has also been accumulated that mitochondria are likely to be one of the primary targets of the noxious action of Cd within the cell.Previous studies have indicated that treatment of cells with Cd results in specific mitochondrial alterations.However,whether cadmium intoxication is associated with oxidative damage and mitochondrial dysfunction are still unclear.The antioxidant and free-radical scavenger N-acetyl-Lcysteine(NAC)is used extensively as a conditional nutrient.NAC acts as a cysteine donor and maintains or even increases the intracellular levels of glutathione,a tripeptide which protects cells from toxins such as free-radicals.It is currently the dietary supplement of choice for building up or conserving the body's stores of glutathione,cysteine,and other sulfhydryl antioxidant resources.The objective of this stoxidative tress in Cd-induced mitochondria dysfunction,we investigated the effects of CdCl₂ on malondialdehyde(MDA),glutathione(GSH)and Cytochrome c(Cyt C) contents,Mn²⁺-superoxide dismutase(Mn-SOD)and mitochondrial complexâ… andâ…¢activities in vitro and on vivo.Studies were also made on the assessment of the preventive effect of NAC.MethodsIn vitro experimental part:Mitochondria were prepared from Wistar rats' liver using differential centrifugation.In vitro,Mitochondrial fractions were incubated in the assay buffer containing different concentrations of CdCl₂(10,100,1000,10000μmol/L) at 37℃for 1h.Effects of the antioxidants NAC(500μmol/L)was studied at a CdCl₂ concentration of 1000μmol/L.The level of MDA,GSH and Cyt C contents;the activities of Mn-SOD and mitochondrial complexâ… andâ…¢were onvestigated.In vivo experimental part:24 Wister rats were randomly divided onto control group,CdCl₂, group,NAC pretreated group by body weight.The control group and CdCl₂ group was given peritoneal injection of 0.9%NaCl,the NAC pretreated group was given peritoneal injection of 1mmol/kg NAC.After 2h,the control group was given subcutaneous injection of NaCl,the CdCl₂.All administration was given at the dose of 5ml/kg for 7d.The animals in all groups were decapitated 24h after the last dose application,and the samples of striatum and cortex were collected,mitochondria were prepared using differential centrifugation.The level of MDA,GSH and Cyt C contents; the activities of Mn-SOD and mitochondrial complexâ… andâ…¢were investigated.ResultsIn vitro experimental part:At 100,1000,10000μmol/L CdCl₂ concentration,the mitochondrial complexâ… andâ…¢activity was reduced significantly(P＜0.05).At 100, 1000,10000μmol/L CdCl₂ concentration the Mn-SOD activity was reduced significantly compared to that of control(P＜0.05).At 100,1000,10000μmol/L CdCl₂ concentration,the MDA contents were increased significantly compared to that of control(P＜0.05).At 100,1000μmol/L CdCl₂ concentration,GSH contents reduced significantly compared to that of control(P＜0.05).At 1000,10000μmol/L CdCl₂ concentration,Cyt C was reduced significantly compared to that of control(P＜0.05).In addition,dose-dependent decrease in mitochondrial complexâ… andâ…¢and Mn-SOD activities,increase in MDA contents and Cyt C released and decrease of GSH contents were observed.the activities of mitochondrial complexâ… andâ…¢and Mn-SOD,the MDA and GSH contents,Cytochrome c released from mitochondria are affected by NAC pre-treatment in vitro.As compared to control group,the Mn-SOD and mitochondrial complexâ… andâ…¢activities were reduced significantly,the MDA contents and Cyt C were increased significantly,GSH contents was reduced significantly in 1000μmol/L CdCl₂ group(P＜0.05).As compared to the 1000μmol/L CdCl₂ exposed group,the mitochondrial complexâ… andâ…¢and Mn-SOD activities were increased significantly,MDA contents were decreased significantly and GSH contents increased significantly,Cyt C released form mitochondria was reduced significantly in NAC pre-treatment group(P＜0.05).Pre-treatment of NAC protected against the CdCl₂ mediated reduction in mitochondrial complexâ… andâ…¢and Mn-SOD activities, increase in MDA contents and Cyt C released form mitochondria,reduction in GSH contents.In vivo experimental part:As compared with the control group,the level of MDA on CdCl₂ group was signigicantly increased,the level of GSH and Cyt C on CdCl₂ group were significantly decreased;the activities of Mn-SOD and mitochondrial complexâ… andâ…¢in CdCl₂ group were gignigicantly decreased(P＜0.05).As compared with CdCl₂ group,the level of MDA in NAC pretreated group was significantly decreased the level of GSH and Cyt C were significanty increased(P＜0.05),the activities of Mn-SOD and mitochondrial complexâ… andâ…¢on NAC pretreated group were signigicantly increased(P＜0.05).Conclusions1.Cadmium could increase of the level of MDA and decrease the level of GSH and Cyt C.Moreover,in vitro experimental part cadmium could dose-dependently induced increase of the level of MDA and decrease of the level of GSH suggested that Cd led to oxidative damage.Cadmium could induce the decline of the activity of Mn-SOD and complexâ… andâ…¢.Moreover,in vitro experimental part cadmium could dose-dependently induced decline of the activity of Mn-SOD and complexâ… andâ…¢suggested that Cd led to oxidative damage.2.Cadmium could cause oxidative damage of mitochondria on rat liver.On vitro experimental part cadmium could dose-dependently induced the decline of oxidative damage of mitochondria isolated from rat liver.And this change could be prevented by pre-treating with NAC.