Experimental Study On Mn-induced Oxidative Damage And Energy Metabolism Dysfunction As Well As Protective Effect By NAC

IntroductionManganese(Mn)is widely employed in industry,in agriculture and it has recently been introduced as antiknock agent in unleaded gasoline.The increasing environmental exposure of large populations to Mn-containing products thus justifies the extensive study for a correct assessment of the potential health risk due to this metal.Exposure to Mn may lead to accumulation of Mn in the basal ganglia of the brain,where it may have its toxic effects lead to neurotoxicity(also known as manganism)often resemble Parkinson's disease.While the mechanism of Mn toxicity is not known,it has been hypothesized that it is associated with mitochondrial dysfunction because Mn accumulates specifically within mitochondria and adversely affects mitochondrial function both in vivo and in vitro.Clinical and experimental evidence suggest that Mn neurotoxicity involves mitochondrial dysfunction leading to impaired cerebral energy metabolism and possibly energy failure.Also oxidative stress had been implicated in Mn toxicity.However,the mechanisms of inhibition of mitochondrial energy metabolism as well as whether manganese intoxication is associated with oxidative damage and mitochondrial dysfunction was still unclear.N-Acetyl-cysteine(NAC)is a thiol which can act as a precursor for GSH synthesis as well as a stimulator of the cytosolic enzymes involved in GSH regeneration,and which when administered systemically had been shown to significantly increase brain GSH levels in rats.The objective of this study was to investigate the mechanisms and possible role of energy metabolism and oxidative stress in Mn-induced mitochondria dysfunction,we investigated the effects of MnCl₂ on malondialdehyde(MDA)and glutathione(GSH) contents,aconitase and mitochondfial complexâ… activities,mitochondrial membrane potential(MMP)in vitro and in vivo.Studies were also made on the assessment of the preventive effect of NAC.MethodsIn vitro experimental part:Mitochondria were prepared from Wistar rats' whole brain using differential centrifugation.In vitro,Mitochondfia were incubated in the assay buffer containing different concentrations of MnCl₂(5,50,500,1000μmol/L) at 30℃for 2h.The effect of NAC(500μmol/L)was studied at a MnCl₂ concentration of 500μmol/L.The level of MDA,GSH;the activities of aconitase, mitochondrial complexâ… and mitochondrion membrane potential were investigated.In vivo experimental part:24 Wistar rats were randomly divided into control group, MnCl₂ group,NAC pretreated group by body weight.The control group and MnCl₂ 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 0.9%NaCl,the MnCl₂ group and NAC pretreated group was given subcutaneous injection of 200μmol/kg MnCl₂.All administration was given at the dose of 5 ml/kg for 21d.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; the activities of aconitase,mitochondrial complexâ… and mitochondrion membrane potential was investigated.ResultsIn vitro experimental part:As compared with the control group,the level of MDA in 50,500,1000μmol/L MnCl₂ groups was significantly increased;the level of GSH in 500,1000μmol/L MnCl₂ groups was significantly decreased;the activity of aconitase in 500,1000μmol/L MnCl₂ groups and the activity of mitochondrial complexâ… in 50, 500,1000μmol/L MnCl₂ groups were significantly decreased(P＜0.05), mitochondrion membrane potential in 500,1000μmol/L MnCl₂ groups was significantly decreased(P＜0.05).As compared with the 500μmol/L MnCl₂ group,the level of MDA in 500μmol/L NAC pretreated group was significantly decreased,the level of GSH was significantly increased(P＜0.05),the activities of aconitase, mitochondrial complexâ… in 500μmol/L NAC pretreated group were significantly increased(P＜0.05),and mitochondrion membrane potential in 500μmol/L NAC pretreated group was significantly increased(P＜0.05).In vivo experimental part:As compared with the control group,the level of MDA in MnCl₂ group was significantly increased,the level of GSH in MnCl₂ group was significantly decreased;the activities of aconitase,mitochondrial complexâ… in MnCl₂ group were significantly decreased; mitochondrion membrane potential in MnCl₂ group was significantly decreased (P＜0.05).As compared with MnCl₂ group,the level of MDA in NAC pretreated group was significantly decreased,the level of GSH was significantly increased(P＜0.05),the activities of aconitase,mitochondrial complexâ… in NAC pretreated group were significantly increased(P＜0.05),and mitochondrion membrane potential in NAC pretreated group was significantly increased(P＜0.05)DiscussionsBrain function is critically dependent on a continuous supply of O₂..In our study, MnCl₂ cause the significant increase of MDA content suggest an increased lipid peroxidation(LPO),NAC pre-treatment significantly reversed the increase of MDA content.MDA content increase may be linked to an enhanced production of ROS by MnCl₂.MnCl₂-induced LPO associated damage to membrane may therefore trigger/propagate various diseases.In our study,MnCl₂ cause the significant decrease of GSH content suggesting decrease in antioxidant protection leading to a failure to repair oxidative damage,NAC pre-treatment significantly reversed the decrease of GSH content.Decrease of GSH content may be linked to an enhanced production of ROS by MnCl₂,excess production of ROS oxidizes the glutathione pool and may also allow the formation of critical protein dithiols leading to decrease in antioxidant protection.In our study,MnCl₂ cause the significant decline of aconitase activity,NAC pre-treatment significantly reversed the decline of aconitase activity.Mn may insert itself into the fourth Fe site of aconitase,suppress the enzyme's catalytic function. Aconitase is also highly susceptible to oxidative inactivation of superoxide(·O₂^-), oxidative inactivation of aconitase may cause the formation of an inactive[3Fe-4S]⁺ cluster results in the concomitant release of Fe²⁺and H₂O₂.Aconitase plays a key role in cellular energy metabolism,a decrease in aconitase activity is likely to affect the overall turnover efficiency of the citric acid cycle.Therefore,a decrease in aconitase activity may impair energy metabolism and possibly energy failure leading to mitochondrial dysfunction.In our study,MnCl₂ cause the significant decline of mitochondrial complexâ… activity,NAC pre-treatment significantly reversed the decline of mitochondrial complexâ… activity.Since mitochondrial complexâ… is an iron-sulfur [Fe-S]center enzyme and is rich in vital sulfhydryl groups,it is particularly susceptible to oxidative damage,its decreased activity may be linked to an enhanced production of ROS by MnCl₂ and to the consequent damage of the complex.Major consequences of mitochondrial complexâ… inactivation are impairment of the ability of the respiratory chain to oxidize NADH to NAD⁺ and overproduction of ROS.Mitochondrial complexâ… plays a major role in controlling oxidative phosphorylation in mitochondria,therefore, its inactivation may cause impaired energy metabolism leading to mitochondrial dysfunction.In our study,it was demonstrated that MnCl₂ induces a reduction in MMP in isolated mitochondria,this study was also demonstrate that NAC protects against the MMP reduction induced by MnCl₂ to a significant degree.An enhanced ROS formation by MnCl₂ may cause the reduction of MMP and a disturbance of mitochondrial function. MMP plays a key role in cellular energy metabolism,a reduction in MMP is likely to affect the overall efficiency of mitochondrial respiratory chain leading to impaired mitochondrial energy metabolism and possibly energy failure.In conclusion,MnCl₂ accumulates specifically in the mitochondrial matrix,where it inhibits energy metabolism,increases the rate of radical production,and induces a reduction in MMP leading to initiation of the apoptotic processes.These conclusions prompt the hypothesis that MnCl₂ interferes with mitochondrial function in several interdependent ways.In our study,NAC pre-treatment significantly reversed the toxic effects of MnCl₂. Although NAC is widely exerted,there are only few reports available analyzing the molecular events establishing these therapeutic attribute to heavy mentals especially manganese,so our study is of significantly important.Because of the ease of administration and low cost and low toxicity of NAC,it is of potential use in the therapy of manganese toxicity.Conclusions1.Manganese could induce the decline of the activity of aconitase and mitochondrial complexâ… .Moreover,in vitro experimental part manganese could dose-dependently induced the decline of the activity of aconitase and mitochondrial complexâ… suggested that Mn led to energy metabolism compromise.2.Manganese could increase of the level of MDA and decrease the level of GSH. Moreover,in vitro experimental part manganese could dose-dependently induced increase of the level of MDA and decrease of the level of GSH suggested that Mn led to oxidative damage.3.Manganese could cause oxidative damage and induce the decline of energy metabolism of mitochondria in rat brain,in vitro experimental part manganese could dose-dependently induced the decline of energy metabolism and the oxidative damage of mitochondria isolated from rat brain.And this change could be prevented by pre-treating with NAC.