Neurotoxicity Induced By Dibutyl Phthalate Adsorbed To Multi-walled Carbon Nanotubes In Mice

Dibutyl phthalate(DBP)and multi-walled carbon nanotubes(MWCNTs)exist extensively in the environment.DBP has endocrine-disrupting effects and can cause neurotoxicity through neurohumoral regulation,and MWCNTs can cross the blood-brain barrier into the central nervous system.Analysis of the molecular structure of CNTs shows that the carbon atoms in the tubular structure of CNTs form highly delocalized large π-bonds,which can bind to the aromatic benzene ring of DBP through π-π interactions,causing DBP to adsorb on CNTs and forming a complex pollutant in the environment.Based on this,this study aimed to investigate(1)the characteristic differences in the neurological effects of combined exposure to MWCNTs and DBP versus exposure alone;(2)whether MWCNTs can mediate the passage of DBP across the blood-brain barrier to reach the central nervous system,slow down the degradation rate of DBP and thus increase its endocrine disrupting effects,and then affect the expression of many genes in neuronal cells and cause metabolic disorders in brain tissue cells.In this study,40 healthy male Kunming mice were randomly divided into 4 groups:(1)the control group;(2)the MWCNTs group(10 mg/kg/d);(3)the DBP group(2.15mg/kg/d);(4)the MWCNTs and DBP combined group.The mice received a tail vein injection,according to their body weight.After day 14,the Morris water maze and open field test were conducted for one week to evaluate the spatial learning and memory levels and the anxiety-like response of the animals.After 21 days,histopathological studies were carried out to detect the histopathology of various regions of the hippocampus by H&E staining,Nissl staining,and immunohistochemistry,and to assess the effects of the combined exposure on the structure and function of various regions of the hippocampus in mouse brain tissue.The functional enrichment of differentially expressed proteins in the brain tissue of each group of mice was analyzed by proteomics and other techniques to investigate the molecular mechanisms of the neurological effects of combined exposure of MWCNTs and DBP at the molecular biological level.The experimental results showed that mice in the combined exposure group of MWCNTs and DBP showed significantly longer escape latency,shorter retention time in the target quadrant,and fewer crossings of the target platform in the Morris Water Maze experiment,which showed severe spatial learning and memory impairment.In the openfield test,mice in the combined exposure group spent a long time in the limbic region and ran longer distances,which showed worse anxiety-like behavior.The histopathological results showed that neuronal cell nuclei were solidified,cytosolic was atrophied and cell number was reduced in hippocampal CA1,CA2,and DG regions of the brain tissue of mice in the combined group.Combined exposure caused oxidative damage in brain tissues,as evidenced by increased levels of reactive oxygen species and decreased levels of superoxide dismutase and glutathione.Combined exposure disrupted neurotransmitter levels such as acetylcholine,noradrenaline,dopamine,and 5-hydroxytryptamine in mice serum.Proteomic analysis showed that differentially expressed proteins between groups were mainly enriched in a variety of neurodegenerative disease pathways.Combined exposure activated PI3K/Akt signaling pathway,elevated the level of BDNF,and upregulated the expression levels of apoptosis-related genes such as p53,caspase3,and bax in mouse brain tissue.Combined exposure activated microglia associated with central nervous system injury and elevated the levels of inflammatory factors in mouse brain tissues.Combined exposure significantly increased the expression levels of synaptic plasticity-related genes such as Nr4a3,Egr1,Arc,Psd95,and Synapsin1 in the brain tissues of mice in the combined group.The experimental results showed that DBP carried by MWCNTs was easy across the blood-brain barrier into brain tissue cells,which enhanced the endocrine-disrupting effect of DBP,caused metabolic disorders in brain tissue cells,and exacerbating learning and memory impairment,and anxiety-like behavior in mice.This intrinsic mechanism is likely to cause neurotoxic effects by activating the PI3K/Akt signaling cascade,inducing the release of BDNF,promoting neuronal apoptosis,activating microglia,disrupting synaptic plasticity,and interfering with neurotransmitter systems.