Study On Neurotoxicity And Reproductive Toxicity Of Manganese Chloride In Mice

Objectives:The long-term contact of chronic manganese may cause manganese poisoning.During the process of manufacturing in industrial and mining enterprises, miners,steel and iron smelting workers and welding workers in factory workshops are main occupational contacting peoples.In order to find more sensitive and specific diagnosis index,to detect and treat the subclinical patients at the early stage,and to provide scientific evidences for make preventive measures,and studied the neurotoxicity and reproductive toxicity of manganese chloride in mice by measuring the content of the monoamine in the brain,detecting some indices related to oxidative damages and protein expressions related to apoptosis in the brain and the testicle and using cell cultures.Methods:90 Kun-ming male mice were divided into 3 groups:the control group,the 15mg/kg group and the 30mg/kg group respectively.Male mice were given manganese chloride by intraperitoneal-injection everyday and were then sacrificed by vertebra disjoint 60 days later.Then we carded on the experiment, which included three parts:1 To study the neurotoxicity of manganese chloride in mice:the content of the monoamine,some indices related to oxidative damages and protein expressions related to apoptosis was measured in the brain.2 To study the reproductive toxicity of manganese chloride in mice:some indices related to oxidative damages and protein expressions related to apoptosis was detected in the testicle.Meanwhile the sperm abnormality and kinematical parameters was also measured.3 After that,high differentiated PC12 cells were bought to carry out Dimethylthiahiazo diphenytetrazoliumromide(MTT)test,and the cell growth curve was drew up at the same time.Flow cytometry(FCM)was used to assay the cell cycle,the rate of PC12 cells apoptosis and the mitochondrial trans-membrane potential.Results:1 The neurotoxicity of manganese chloride in mice(1)Compared with the control group,contents of DA,NE and5-HT in the brain were all decreased in the experimental groups.Along with the increase of the dose of manganese chloride,contents of the three monoamines reduced gradually. (2)GSH-PX and T-AOC of whole brains were decreased,and there was a significantly different in T-AOC between each experiment group and the control group(P＜0.05).Along with the increase of the dose of manganese chloride,contents of MDA increased gradually.(3)In the brain we found that the average light density of the P53 protein presented the decline tendency and its gray level presented the incline tendency,and there were significantly different between each experiment group and the control group(P＜0.05).The average light density of Bcl-2 protein and its grey level had the opposite tendency,and there was a significant difference only between the 30mg/kg group and the control group(P＜0.01).2 The reproductive toxicity of manganese chloride in mice:(1)Compared with the control group,GSH-PX and T-AOC in the testicle also decreased in the experiment groups.There was a significant difference in T-AOC between the 30 mg/kg group and the control group(P＜0.05).MDA was increased and there was a remarkable differences in it between each experiment group and the control group(P＜0.05).The sperm abnormality in the different dose groups were analyzed and we found that sperm abnormality rates in experiment groups were all low,and the main abnormality sperm were amorphous and no hook.Compared with the control group,VCL and VSL decreased in the 30 mg/kg group(P＜0.05).(2)In the testicle,the P53 protein mainly expressed in all levels of cells,but the Bcl-2 Protein mainly expressed in the testicle intersititial cells.The average light density of the P53 protein presented the incline tendency,and its gray level presented the decline tendency,and there was a significantly different only between the 30 mg/kg group and the control group(P＜0.01).The average light density of the Bcl-2 protein and its grey level had the same tendency with the P53 protein,and there were significant difference between the 30 mg/kg group and the control group(P＜0.01).3 The toxicity of manganese chloride to dopaminergic PC12 neurocyte cells:MnCl₂ had the inhibition function to the PC12 cells in100-900μmol/L,but the degrees were different.The inhibition rates to the PC12 cells in different dose groups were all lower than 50%and increased along with the extension of the response time and the rising of MnCl₂ concentration.The assay of Flow cytometry(FCM)indicated that compared with the control group,when the MnCl₂ response time achieved 72 hour time,the cell cycle had the obvious change.The G1 stage in the control group, 500,700,and 900μmol/L groups had the increasing tendency,and the stage had the decreasing tendency.This demonstrated that it was more possible to retard the cell cycle at the DNA synthesis stage,along with the increase of MnCl₂ concentration. Additionally,when the MnCl₂ response time achieved 72 hours,there was obvious apoptosis.This indicated that the force to cause apoptosis was greater along with the increase of the response time.To some degree,the Rh123 fluorescence intensity dropped in the PC12 cells of the experiment groups.This showed that mitochondrial trans-membrane potential decreased and indicated that MnCl₂ caused the damage to the membrane of PC12 cells,and may induce apoptosis.Conclusion:The long-term contact of manganese may reduce the contents of the monoamines in mice brains.It may also have effects on the activities of indices related to oxidative damage and change the protein expressions related to apoptosis in the nerve and reproductive systems.The further results in vitro cell culture experiments showed that it had certain poisonous function to the nerve cell and suggested that further study was needed.