| In recent years,the trend of studies on environmental chemical toxic substances is from the previous single factor to the present combined ones.Lead(Pb) and cadmium(Cd) are known as toxicants which are accumulative,large-contact frequent,extensive exposure in daily life.Lead and cadmium have deep influences upon the developmenting central nervous system(CNS).The studies on single toxicity of cadmium or lead are reported in many documents.However,systemic studies of toxic damage on combination of lead and cadmium are seldom seen,especially in developmenting CNS.This study aims to investigate the toxic effects and mechanism of lead or/and cadmium on CNS of neonatal rats(in 24h) in vitro and in vivo,which will probably offer some theoretic evidence of further acquiring neurotoxicity mechanism of lead or/and cadmium.1.Experiments in vitro To study lead and/or cadmium neurotoxicity mechanism on CNS,the experimental neurons are obtained from the neonatal Sprague-Dawley rats in 24h.The model of neurons in vitro is successfully established,which is proved by adopting the Nissl's staining,CCK-8 assay methods.The toxic tests of lead and/or cadmium are divided into 9 groups:the control group,P1 group(Pb 100μmol/L),P2 group (Pb 200μmol/L),C1 group(Cd 5μmol/L),C2 group(Cd 10μmol/L),P1C1 group(Pb 100μmol/L+Cd 5μmol/L),P1C2 group(Pb 100μmol/L+Cd 10μmol/L),P2C1 group(Pb 200μmol/L+Cd 5μmol/L),P2C2 group(Pb 200μmol/L+Cd 10μmol/L).The toxic effect of lead and/or cadmium on neuron's morphology,viability,apoptosis rate,the enzymatic activity changes of glutathione peroxidase(GSH-Px),superoxide dismutase(SOD), catalase(CAT) and True choline esterase(TChE) and the contents of Maleic dialdehyde (MDA) are evaluated by related kits.Meanwhile,N-Acetylcysteine(NAC) is added in each experimental group to study the protective effects from the damages of lead and/or cadmium.The results are as follows:①The results of Nissl's staining shows that the purity of neurons is above 92%,and the cortical neuron's viability(OD)is highest on the sixth day of post-beginning of culturing.So neurons are treated the with experimental factors on the sixth day;②In comparison with the control group,the neurons are exposed to different concentrations of lead or/and cadmium,which led to the decrease in the number of neurons and neural network.The results indicated a significant decrease in pericaryons and processes(p＜0.05),and survival rate of neurons(p＜0.05).And the inhibitory effect is gradually enhanced with the prolonging of the culturing time and increasing concentrations of lead or/and cadmium.The nucleus crimpled,crescent liked and chromatin condensed,even nucleus disintegrated by the fluorescence microscope. And apoptosis rate is higher than the control group(P＜0.05);In comparison with the single factor treats groups respectively,the combined factors groups have more significant changes,such as between P1C2 and P2C1,especially in P2C2 group(P＜0.05);③In comparison with the control group,the activities of GSH-Px,SOD and TChE in neurons which are treated by lead or/and cadmium of different concentrations decrease significantly(P＜0.05).However,the activity of CAT and the content of MDA in neurons are more significantly increased than that of the control group(P＜0.05);④To compare the NAC protective groups with the poisoning groups,the neuron's viability was increased,and the apoptosis rate of neurons was degraded relative to the poisoning groups. Meanwhile,the activities of GSH-Px,SOD,TChE and CAT are increased accordingly, but the content of MDA is decreased compared with the poisoning groups.However, there are no significant changes among the relative groups(P＞0.05).From what has been discussed above,it might be concluded that:①The cortical neuron differentiation can be inhibited by lead or/and cadmium,which led to the pericaryon diminutus and process decurtation,lower survival rate and higher apoptosis rate and displays a dose and time effect in comparison with the control group.It is clear that lead and cadmium combined treatment exerts an synergistic effect,and cadmium played a great role in combining lead with cadmium;②The antioxidative enzyme activity of GSH-Px,SOD,CAT,TChE and content of MDA are significantly changed in each of experimental groups.Therefore, Nervous system toxicity of lead and/or cadmium is connected with lipid peroxidation;③The toxicity of lead or/and cadmium could antagonize by adding NAC,which improve survival rate of neurons and reduce apoptosis rate of neurons and increase activity of antioxidase and reduce lipid peroxidation.It is not obvious that adding NAC has a protective effects on the damage of lead or/and cadmium.2.Experiments in vivo To set up experimental animal mode of the damage Pb or/and Cd exposed in neonatal SD rats,35 SD pregnancy rats are used from the center of experimental animal of Yangzhou University,and are randomly divided into 7 groups:the control group(drinking water),lead acetate group(300mg/L),lead acetate+NAC(300 mg/L+20mmol/L),cadmium acetate group(10mg/L),cadmium acetate+NAC (10mg/L+20mmol/L),group of lead and cadmium(300mg/L+10mg/L),group of lead and cadmium+NAC(300mg/L+10mg/L+20mmol/L),which are given by drinking water during gestational periods.After delivery,neonatal SD rats are used to examine the toxic effects of lead or/and cadmium and appraise protective effect of NAC.Experimental examination index was that①Toxic effect of lead and/or cadmium on physical growth (such as body weight,brain weight) and development of brain;②The studies on the changes in histopathology and ultrastructure of cerebral cortex of the neonatal SD rats;③Toxic effects of lead or/and cadmium on the biochemistry index of neonatal rats brain, such as the activity of GSH-Px,SOD,CAT and TChE and the content of MDA;④Effect of lead or/and cadmium on contents of trace element(Cu,Fe,Zn,Mn and Se) in neonatal rats;⑤The expression of bcl-2,Bax,c-fos in the cerebral cortex by FQ-PCR and MT-3 by FQ-PCR and immunohistochemistry method;⑥The protective effects of NAC on damage of lead or/and cadmium in developing brain of neonatal rats.The results are as follows:①In comparison with the control group,the body weight and brain weight are obviously decreased in lead or/and cadmium group(P＜0.05 or P＞0.05), especially in the combination group;②The changes in histopathology of cerebral cortex is not observed under light microscope.However,the changes in ultrastructure of cerebral cortex is obvious that pyknosis of neuron nucleus,chromatin assemble,mitochondrial swelling and partial cristae under transmission electronic microscope;③In each of experimental groups,the activities of GSH-Px,SOD,CAT and TChE are significantly decreased than those in the normal group(P＜0.05),however,the content of MDA is significantly increased in the experimental group than that in the normal group(P＜0.05);④In the poisoning groups,the contents of trace element(Cu,Fe,Zn,Mn and Se) descend more significantly than the normal group(P＜0.05);⑤In comparison with control group,the expression of bcl-2 in the cerebral cortex decrease(P＜0.05 or P＞0.05),but the expression of Bax,c-fos and MT-3 increase in the experimental groups(P＜0.05 or P＞0.05);⑥In comparison with the poisoning groups respectively,the protective groups of NAC are not only contents of trace element(Cu,Fe,Zn,Mn and Se) are improved,but also the activity of GSH-Px,SOD,CAT and TChE are higher(P＞0.05)and the content of MDA is significantly lower(P＜0.05).The expression levels of bcl-2 in the cerebral cortex is obviously increased(P＜0.05 or P＞0.05),but the expression levels of Bax and c-fos are significantly decreased in the experimental group(P＜0.05).The conclusions drawn by analyzing the test results are as following:①The body weight and brain weight of neonatal rats are significantly decreased by lead or/and cadmium exposed to gestational rats;②There are obvious changes in ultrastructure of the cerebral cortex which displayed as cytomembrane disappearance and neuron nucelus concentration,mitochondrial swelling,disruption and total cristae loss;③The reducing activity of antioxidase led to lead or/and cadmium could be related to the decreasing contents of trace element in neonatal rats;④The apoptosis of neurons induced by lead or/and cadmium are in connection with the exceptional expression of bcl-2,Bax and c-fos genes.It is clear that lead and cadmium combined treatment exerts an synergistic effect;⑤NAC,as a new-style antioxidant,could antagonize toxicity of lead or/and cadmium, which improves activity of antioxidase and reducing lipid peroxidation and increasing content of trace element and increasing gene expression of bcl-2 and decreasing gene expression of Bax,c-fos and MT-3,while its protective effect was limited.
|
PDF Full Text Request