Combined Exposure Of Low Levels Heavy Metals Disrupted Synaptic Homeostasis And Reversal Effect Of Plant Polyphenols

Objective:Heavy metals lead（Pb）,cadmium（Cd）,arsenic（As）,and mercury（Hg）have widly polluted the environment and food,meanwhile they were the only four metals among the ten chemicals of major public health concern released by the World Health Organization,which were also representative neurotoxicological contaminants detected in the blood of population due to environmental pollution.The health impairments caused by long-term exposure to one of these four metals have been extensively researched,and found the nervous system was obviously damaged which involved the learning and memory function.However,human exposure to environmental chemicals is most correctly characterized as exposure to mixtures.To date,the effects of combined exposure to Pb,Cd,As and Hg on neurological functions have not been reported.Previous studies indicated that neurons processed and retained information through regulating the intensity and frequency of synaptic connections.Thus,synaptic remodeling of neurons was closely associated with neurological functions such as learning and memory.The key of synaptic remodeling was the rearrangement of dendritic spines in the synaptic structure.In addition,plant polyphenols that widely present in plants are secondary metabolites with multiple hydroxyl groups,and have the effects of anti-oxidation,anti-tumor,anti-toxicity and neuroprotection.The common plant polyphenols such as resveratrol and epigallocatechin gallate（EGCG）have good function of neuroprotection and could ameliorate impairments of synaptic remodeling.Our study was purposed to investigate the effects of combined exposure to Pb,Cd,As,and Hg at low levels on synaptic structural homeostatic and the intervention effects of plant polyphenols.Methods:We firstly explored the characteristics of the interactions among these four metal mixtures（MM）and selected the appropriate low dose of the MM in orthogonal and factorial designs in vitro.Then,the low dose MM were exposed to rats to observe the toxic injures in multiple system,especially in nervous system.Furthermore,the effects as well as mechanisms of MM on synaptic remodeling were investigated.At last,resveratrol and EGCG were applied to assess their intervention effects.Results:1.The selection of MM doses.The orthogonal design in vitro was used to explore the interactions among MM,and cell viability was the evaluation indicators which detected by CCK8.Results indicated that when the concentrations of Pb,Cd,As and Hg were not exceed the NOAELs,Pb+Cd+As+Hg combined exposure exhibited synergistic effects to induce cytotoxic.Besides,when the levels of MM was similar to the blood exposure levels of MM detected in general population,the synergistic effect was the strongest.Thus,the levels of the MM in the blood of population（1×MM）,the 5-fold concentrations（5×MM）and the 10-fold concentrations（10×MM）were selected to investigate impact of the interaction among Pb,Cd,As and Hg on cell apoptosis,ROS generation,and[Ca²⁺]i in factorial designs.Results demonstrated that low-level co-exposure to these four metals caused a novel synergistic toxic effect that was different from that induced by two or three of the metals.2.Effect of the MM on multiple systems in rat.The different levels of the MM（Control,1×MM,5×MM,10×MM and 20×MM）were administrated to pregnant rats in drinking water until the pups weaned.ICP-MS was used to determine the Pb,Cd,As and Hg concentrations in the blood and brain tissue of rat pups.ELISA kits were used to detect the biochemical indices in rat tissues.Results showed that the blood concentrations of Pb,Cd,As,and Hg in 1×MM rats was in accordance with the levels of these metals in the blood of population.In addition,the MM levels in blood and brain of rats increased in a dose-dependent manner.Thus,the animal model of low level MM co-exposure were successfully established.The MM exposure caused impairments in biochemical indices as well as multiple systems including brain,liver,testis and kidney,especially severe in nervous system.3.Effect of the MM on function of nervous system and synaptic remodeling.A series of neurobehavioral testing nest were determined to evaluate the learning and memory ability,the sensory perception ability and the athletic ability of the rats.Findings showed that MM exposure caused the dose-dependent disorders of learning and memory ability,and the sensory perception ability,rather than athletic ability.What needs to be emphasized was that the cliff avoidance test reflecting the sensory perception ability had statistical change started at 1×MM level.Furthermore,cells morphology were detected by using TRITC-labelled phalloidin to stain F-actin in cultures of hippocampal neurons and NGF-stimulated differentiation PC12 cells.Results showed that MM decreased the dendritic lengths and branches as well as the spine density and spine maturity in hippocampal neurons,and also shortened the neurite length of NGF-stimulated differentiation PC12 cells.4.Effect of the MM on Snk-SPAR pathway.Immunofiuorescent assay was used to analyze the protein expression levels of serum-induced kinase（Snk）and Spine-associated Rap GTPase-activating protein（SPAR）.The shRNA Snk,Wt-SPAR and Mut-SPAR plasmid were constructed and transferred to hippocampal neurons and NGF-stimulated differentiation PC12 cells.Results showed the SPAR protein levels decreased with the MM exposure levels in a dose-dependent manner.The overexpression of SPAR alleviated the MM induced decrease of the neurite length in NGF-stimulated differentiation PC12 cells,and the Mut-SPAR plasmid presented better than the Wt-SPAR plasmid.Further study found that MM exposure increased the Snk levels in these two nerve cells.In addition,the Snk agonist aggravated the change of the neurite length and Snk-SPAR pathway in NGF-stimulated differentiation PC12 cells.While the low expression of Snk improved the MM exposure induced these changes.5.The intervention effect of resveratrol and EGCG on MM induced impairments of synaptic remodeling.CCK8 was used to detect the cell viability.Flow cytometer was applied to detect the cell apoptosis,ROS generation,and[Ca²⁺]i level.F-actin staining by TRITC-labelled phalloidin was conducted to analyze the cells morphology.Immunofiuorescent assay was used to analyze the protein expression levels of Snk and SPAR.Results showed that 10μM resveratrol and 10μM EGCG both reversed the MM exposure induced nerve cell damage and produced an intervention effect on MM induced changes in Snk-SPAR pathway.Conclusion:The above results indicated that when the single metals was not exceed the NOAEL,MM exposure at the levels in the blood of population may activated the Snk-SPAR pathway to induce synaptic remodeling and further caused the disorders of neurobehaviors.However,resveratrol and ECCG reversed the effects caused by MM.Thus,they maybe the potential drugs to prevent and treat the disorders of the nervous system due to the impairments of synaptic remodeling caused by MM.