| Background and objective:Parkinson’s disease (PD) is one of the common neurodegenerative disorder inmiddle an daged population. Its etiology includes genetic and environmental fcetor.Pathologieal features are degeneration and death of dopaminergic neurons in thesubstantia nigra pars compacta (SNe) of the brain, the accumulation of Lewy’s bodyin the neurons, which leads to reduce of dopamine secretion and low level ofdopamine. The sporadic cases accounts for about95%of the PD patients, and they aresusceptible to environmental toxins. With the rapid development of nanotechnology,the manufacture and application of nanomaterials are increasing and humans are morelikely to be exposed occupationally or via consumer products and the environment.Nanoparticles have unique physical and chemical properties, such small size effect,surface effect, and resulting the huge surface, which may its surface producemore active sites to participate in different biochemical reaction. Therefore,nanoparticles have strong ability to penetrate the tissue and cells, the strongoxidation ability and catalytic ability. Recent research shows that, the central nervoussystem is potentially toxic target of nanoparticles. Existing experimental studiesindicate that nanoparticles can cross the blood-brain barrier (BBB) and enter thecentral nervous system (CNS) of exposed animals where they could accumulate anddamage brains. After inhalation exposure, nanoparticles adsorbed and deposited in thenasal mucosa of rats can be transported along the olfactory nerve pathways into thebrain tissue. The environmental health and safety issues of nanomaterials are highlyconcerned recently. However, the research on neurotoxicity of nanomaterials is still atan elementary stage; especially, dopaminergic neuron (DN) damage pathways andmechanisms induced by nanoparticles remain ill-defined. The companion study ofin vivo and in vitro has few reports. In the first part of present study, the male wistarrats were exposed to different kinds of nanomaterials by intratracheal instillation,including single-wall carbon nanotubes (SWCNTs), silica oxide nanoparticles(SiO2-NPs) and zinc oxide (ZnO-NPs), and the oxidative stress level and neurotransmitter in brain homogenate were detected. In the second part of presentstudy, we utilized inductively coupled plasma mass spectrometry (ICP-MS) to detectthe distribution and accumulation of aluminum oxide nanoparticles (Al2O3-NPs),copper oxide nanoparticles (CuO-NPs) and zinc oxide nanoparticles (ZnO-NPs) in thebrain after intranasal instillation and consequently to estimate the potentialparticle-induced injuries and function alterations in the brain. And the preliminarystudy on dopaminergic neuron (PC12cells) toxic effects induced by three kinds ofnano metal oxide were detected. The study results will help to offer scientific basis forthe further mechanism research and estimation of toxic effects after nanoparticlesenter the body through the inhalation exposure; to provide experimental reference forthe study on etiology of neurodegeneration, such as Parkinson’s disease (PD); and toprovide the basic data for establishing a guideline for environmental protection andsafety for normal and susceptible populations.Research:(1)Neurotoxicity on rats induced by three different kinds of nanomaterials afterintratracheal instillation;(2) Distribution and accumulation of three kinds of nano metal oxide in thecrucial functional sub-brain regions of rats after intranasal instillation, andconsequently to estimate the potential particle-induced injuries and functionalterations in the brain.(3) The toxic effects on crucial functional sub-brain regions of rats induced bythree kinds of nano metal oxide.(4) The PC12cells toxic effects induced by three kinds of nano metal oxide.(5) The initial study on toxic effects mechanism of PC12cells induced by threekinds of nano metal oxide.Methods: SWCNTs, SiO2-NPs, Al2O3-NPs, CuO-NPs and ZnO-NPs wererespectively dispersed in physiological saline and their size, shape and chemicalcomposition of the basic parameters and the dispersion of nanoparticles werecharacterized in the transmission electron microscope.Part one:Male wistar rats were divided into the physiological saline control group, highdose group (15mg/kg) and low-dose group (3mg/kg) of three nanomaterials. The ratswere exposed by intratracheal instillation once two days for4weeks, and then killedby abdominal aorta bloodletting. The pathological examinations of rat brain was performed to reflects the substantial changes. The malondialdehyde (MDA),superoxide dismutase (SOD) and reduced glutathione（GSH）in brain homogenatewere detected. The concentrations of monoamine neurotranmitters includingepinephrine (E), norepinephrine (NE), Dopamine (DA),5-hydroxytryptamine (5-HT)and its metabolite5-hydroxyindole acetic acid (5-HIAA) were examined; and thecontent of amino acid neurotransmitter including glutamate (Glu), aspartic acid (Asp),glycine(Gly) and gamma aminobutyric acid (GABA) were also deteced.Part two:In vivo, two separate animal experiments were carried out. We first evaluated thetranslocation and distribution of intranasal instillation with three nanomaterials in therat brain; second we evaluated the antioxidative level and immune inflammatoryresponses. In the first experiment, the weight matched animals were divided into fourgroup: control group and20mg/kg (body weight) dosage groups for threenanomaterials. The rats were exposed to nanomaterials by intranasal instillation1time per day for15days and the control groups were exposed to physiological saline;the sub-brain regions of interest including olfactory bulb, cerebral cortex,hippocampus, striatum and cerebellum were divided from three rats brains in eachgroup for determining Al, Cu and Zn contents using ICP-MS, respectively; theremaining olfactory bulb, hippocampus and striatum of two rats were used for TEMobservations. In the second experiment (10rats per group; intranasal instillation1time per day for15days and30days, respectively), at post-instillation time points of15and30days, above sub-brain regions of five rats were homogenized to assayoxidative damage and cytokines, respectively. The malondialdehyde (MDA) andreduced glutathione（GSH）were detected to indicate the tissues oxidative damage andreact on the oxidative stress. The interleukin-1β (IL-1β) and tumor necrosis factor-α(TNF-α) were detected to react on immune inflammatory responses of the sub-brainregions. In vitro, Al2O3-NPs, CuO-NPs and ZnO-NPs were respectively dispersed infetal bovine serum (FBS) to prepare the suspension of different dosage:2,4,6,8,10,12,14,16,18and20μg/mL ZnO-NPs suspension;5,10,15,20,25,30,35,40,45and50μg/mL CuO-NPs suspension;3.125,6.25,12.5,25,50,100and200μg/mLAl2O3-NPs suspension. PC12cells were respectively exposed to three kinds ofnanopaticles suspension. The cytotoxicity induced by nanomaterials was measured bycellular morphology observation, cck-8assay and lactate dehydrogenase (LDH) assay.PI assay plus FACS technology were employed to quantitatively analyze the cell cycle. Annexin V-FITC/PI assay plus FACS technology were employed to quantitativelyanalyze apoptotic and necrosis PC12cells. The MDA, nitrogen oxide(NO), GSH andsuperoxide dismutase (SOD) were detected to research whether CuO-NPs inducePC12cells damage through oxidative stress mechanism.Results:1. Basic parameters of nanoparticlesThe diameters of metal oxide nanoparticles are less than40nm with more than99.7%incomposition. Al2O3-NPs and CuO-NPs are regular round shape; ZnO-NPsand SiO2-NPs are hexagonal crystal structure. SWCNTs is rope shape, the diameter isless than8nm, and the length is less than5μm.2. In vivo experiment(1) Neurotoxicity on rats induced by three different kinds of nanomaterials:After intratracheal instillation for4weeks, significant decreases in SOD activity andincreases in MDA content of high dosage SWCNTs group were found. The MDAcontent of low&high dosage ZnO-NPs groups were significantly increased and SODactivity in high dosage ZnO-NPs group were significantly decreased. The DA,5-HIAA and5-HT contents of low&high dosage ZnO-NPs groups were significantlydecreased, and DA content in high dosage SWCNTs group were significantlydecreased. The Glu contents of high dosage ZnO-NPs and SWCNTs group weresignificantly decreased. The Asp content of low&high dosage SiO2-NPs groups andthe Glu contents of low dosage SiO2-NPs group were significantly increased. Theinflammation of brain appeared in the high dosage ZnO-NPs group.(2) Sub-brain regions distribution of three kinds of metal oxidenanoparticles: The Al and Cu concentrations in all sub-brain regions were higherthan any of the controls for postexposure time periods, respectively. The increase ofAl and Cu contents in olfactory bulb, hippocampus and striatum were significantlydifferent, respectively. The Zn concentrations in all sub-brain regions were higherthan any of the controls, however, there were no significantly different. Meanwhile,TEM assay showed that exposure of olfactory bulb, hippocampus and striatum tothree nanoparticles result in segmental defection in nuclear membrane, loosearrangement of cells, formation of vacuoles in some region, obvious condensation ofchromatin, different degrees of damage in mitochondria and deposition ofnanoparticles within the cytoplasm and nucleus were observed. (3) Sub-brain regions damage induced by three kinds of metal oxidenanoparticles: The boby weight of rats in three kinds of nanoparticles groupssignificantly decreased. At the time point of15d, significant decreases in GSHactivity and increases in MDA concentration were found in the olfactory bulb,hippocampus and striatum of all exposure groups. Moreover, the GSH activity incerebral cortex was significantly decreased after15d exposure to ZnO-NPs.Significant decreases in GSH activity were found in the olfactory bulb, hippocampus,striatum and cerebral cortex of all exposure groups at the time point of30d.Futhermore, the GSH activity in cerebellum was significantly decreased after30dexposure to ZnO-NPs. Significant increases in MDA concentration were also found inthe olfactory bulb, hippocampus and striatum of all exposure groups at the time pointof30d. Moreover, MDA concentration in cerebral cortex was significantly increasedafter30d exposure to CuO-NPs, and MDA concentration in cerebral cortex andcerebellum was also significantly increased after30d exposure to ZnO-NPs. Inaddition, there are the significantly increased TNF-α and IL-1β levels in the olfactorybulb, hippocampus and striatum of all exposure groups at15d and30d postexposure.TNF-α level in cerebellum was significantly increased after exposure to ZnO-NPs,and TNF-α level in cerebral cortex was also significantly increased after30d exposureto Al2O3-NPs.3. In vitro experiment(1) Morphological changes: The control PC12cells had normal morphologieswith long neuritis, many intercellular processes and transparent cytoplasms.However, after treating with nanoparticles, PC12cells demonstrated differentdegrees of deformation: rounding and floating, full nuclear condensation andformation of several intracellular vacuoles. Additionally, the number and length ofcellular neurites was significantly reduced or shortened. The increased number ofintracellular particles visibly reduced cellular transparency. The distribution ofnanoparticles within and on the surface of cells had an appreciable impact on thegrowth and metabolism of cells. This resulted in a significantly reduced number ofviable cells.(2) Cytotoxicity of PC12cells: The cell activity in significantly decreased ascompared with control, and the significantly decreases in cell activity were inducedby three kinds of nanoparticles on a time-dependent and dose-dependent manner.Within the exposure time of current study, low dosage (<10μg/mL) ZnO-NPs had no toxicity on PC12cells, However, when the dasage of ZnO-NPs was more than12μg/mL, abundant death PC12cells were observed. Three kinds of nanoparticlesexposure significantly increased the LDH levels in the cell media compared withcontrols. Moreover, the result of LDH leakage exhibited a significant cytotoxicity atZnO-NPs concentrations of5~30μg/ml each dose level while the LDH releaseinduced by Al2O3-NPs and ZnO-NPs were relatively feeble.(3) Cell cycle and apoptosis: Three kinds of nanoparticles treatmentssignificantly induced apoptosis and necrosis. Furthermore, the apoptosis playprincipal action in cells death process at low exposure dosage and short exposure time.However, as the exposure dasage was increased and the exposure time was prolonged,the necrosis gradually play principal action in cells death process. After treatmentwith Al2O3-NPs for24h, the G1/G0phase significant accumulation was accompaniedby a corresponding reduction in the percentages of cells in G2/M phase. Afterexposure to low dosage (10μg/mL) CuO-NPs for24h, the G1/G0phase significantaccumulation was accompanied by a corresponding reduction in the percentages ofcells in G2phase. However, as the exposure dosage were increased to20and40μg/mL, a significant increase in the percentage of cells in S phase was observed,with was accompanied by a corresponding reduction in the percentages of cells inG2/M phase.(4) Oxidative stress level induced by CuO-NPs: After treatment with CuO-NPsfor24h, the cytotoxicity was examined in accordance with the intracellular oxidativestress level measure by GSH and total GSH depletion, SOD inhibition and MDA andNO production.Conclusions:(1) After intratracheal instillation exposure to three different kinds ofnanomaterials, ZnO-NPs and SWCNTs groups can cause oxidative stress andoxidative damage to rats brain, and effect the synthesis of monoamineneurotranmitters (DA,5-HIAA and5-HT) and amino acid neurotransmitter (Glu andAsp). SiO2-NPs can effect the synthesis of amino acid neurotransmitter (Glu and Asp).Moreover, the inflammatory reaction to rat brain of high dasage ZnO-NPs group wasobserved. The oxidative stress may be one of the main toxic effects which trigger theimmunotoxicity induced by nanoparticles.(2) After intranasal instillation exposure, three kinds of typical metal oxidenanoparticles can be transported along the olfactory nerve pathways into the brain tissue. The deposition in the brain of nanoparticles can stimulate oxidative stress,inflammatory responses, and ultrastructure changes. Oxidative stress andinflammatory might be important for inducing the neurotoxicity of these nanoparticles,which accorded with the present universal approval pathogenesis of PD, indicatingthese three naniparticles may be one of environmental motivator of PD.(3) Three typical kinds of metal oxide nanomaterials can lead to PC12cellsmorphological changes, decrease activity of PC12cells, affect cell membraneintegrity and permeability to inhibite cell activity, induce apoptosis and accumulatecell cycle to inhibit the cell proliferation.(4) CuO-NPs exposure can increase the PC12cells oxidative stress level.CuO-NPs may induce PC12cells cytotoxicity by oxidative stress mechanism.(5) Three kinds of metal oxide nanomaterials and SWCNTs may increase thesusceptibility to PD and be one of the environmental promotive factors.(6) The toxic effect comparisions amid three different kinds of nanomaterialswere distinct: the toxicity of inorganic metal oxide nanomaterials, ZnO-NPs, wasgreater than organic nanomaterials, SWCNTs; and the inorganic nometal oxidenanomaterials, SiO2-NP, has not apparent toxicity. In addition, the toxic effectcomparisions amid three kinds of metal oxide nanomaterials were distinct: the toxicityof ZnO-NPs and CuO-NPs was greater than Al2O3-NPs, and the difference may beattributed to several parameters including shape and chemical composition. |
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