The Effect Of Prenatal Exposure To ZnO Nanoparticles On The Brain Development And Neurobehavior In Rat Offspring

BackgroundNano-materials (NMs) are defined as materials composed of particles in an unbound state, or as an aggregate or agglomerate with at least one external dimensions in the size range from 1nm to 100nm. In recent years, the nano-technology has been closely integrated with medical science in various respects, such as the fundamental research, diagnosis and treatment, and the development of drugs and biomaterials. However, it has to be noted that NMs are not inherently benign, and they may affect biological behaviors at the cellular, subcellular, protein and genetic levels. Many current researches have pointed that nanoparticles can travel throughout the body, deposit in target organs, penetrate cell membranes, lodge in mitochondria, and may trigger injurious responses. Consequently, the study of nano-related biosecurity has received much attention around the world.Nowadays many new types of nano-materials have been applied in dental fields, which contribute to the modification and innovation of traditional dental materials. Zinc oxide nanoparticles are among the nanomaterials most widely incorporated into market goods. Due to their unique antibacterial capability and high bioavailability, ZnO NPs have been utilized in dental composite resin, root canal filling materials, and surface coating materials of dental implants, etc. After exposure, nano ZnO could transfer throughout the body and deposit in target organs, such as the lung, liver and kidney. Different from other organs, the brain is well protected by the blood-brain barrier (BBB), which can prevent the majority of substances from entering the cerebral parenchyma. However, current in vivo studies have demonstrated that ZnO NPs may transport across the BBB into the brain. Moreover, the BBB structure and function are perfect stage by stage, which accompany the animal ontogenesis. Thus, fetal brains are easily affected by blood-borne substances, including nano-sized materials, to a much greater extent than adult brains because the development of the blood-brain barrier in the fetal brains is incomplete.To date, the knowledge of potential neurotoxicity of prenatal exposure of nano-ZnO to the offspring is still limited. For this reason, this research exposed pregnant SD rats to fully characterized commercial ZnO NPs by oral administration for consecutive 18 days. The potential neurotoxic effects on the offspring of the female rats were assessed, Furthermore, a neurobehavioral test, Morris water maze was performed in 8-week-old pups to determine the memory and learning ability in their adulthood. We hope the present study will contribute to a better understanding of the nano-related risks to the CNS and raise public concerns on the applications of ZnO NPs, especially in pregnant females.Objection1. To observe the ability of ZnO NPs to cross the placental barrier of the pregnant rats, and the blood-brain barrier of the fetus to accumulate in their body organs.2. To identify the adverse effect of prenatal exposure of ZnO NPs on brain development of the offspring.3. To determine the major toxicity mechanism of neurotoxicity of ZnO NPs.4. To determine the impact of prenatal exposure of ZnO NPs on the learning and memory of the offspring in their adulthood.Materials and MethodsPart 1:Characterization of ZnO NPs1) Transmission electron microscopy (TEM) was used to observe the primary particle size and morphology of NPs.2) Dynamic Light Scattering was used to determine the hydrodynamic size and zeta potential of NPs in distilled water (DW).3) Energy dispersive spectrometer (EDS) was applied to analyze element content of NPs.4) X-ray-diffraction (XRD) was used to observe the crystal structure of NPs.5) N2 adsorption-desorption Brunauer-Emmett-Teller (BET) analysis was used to measure the specific surface area of NPs.6) Limulus reagent gel method was used to test the level of endotoxin contamination of NPs.7) Inductively coupled plasma-mass spectrometry (ICP-MS) was used to determine solubility of NPs in acidic gastric fluid (AGF).Part 2:Animal treatment and neurotoxic effect of ZnO NPs1) Animal treatmentThe suspension of ZnO NPs was prepared using saline containing 0.05% v/v Tween-80. The pregnant SD rats were divided into two groups, experimental group (treated with 500 mg/kg BW ZnO NPs) and control group (treated with saline containing 0.05%v/v Tween-80). ZnO NPs suspension and the vehicle were respectively administered to the female rats by intragastric gavage daily from gestation day (GD) 2 to 19.2) Coefficients of body organsOn postnatal day (PND) 2, the pups were firstly weighed and sacrificed by decapitation. The hearts, livers, spleens, lungs, kidneys and brains were dissected and weighed immediately. The coefficients of heart, liver, spleen, lung, kidneys and brain to body weight were calculated as the ratio of tissues (wet weight, mg) to body weight (g).3) Biodistribution of zinc in tissues and bloodTissue samples from the heart, liver, spleen, lung, kidney and brain were obtained from 2-day-old neonates. The blood samples were collected from weaning pups (PND 21) and their mothers. The determination of zinc levels in the blood and different tissues were quantified by ICP-MS.4) Histopathological examination of the brainOn PND2, brain tissues were collected from male pups and immediately fixed in 10% neutral buffered formalin overnight. The samples were then dehydrated and embedded in paraffin blocks, sectioned and finally stained with hematoxylin & eosin (HE). Furthermore, Ki-67 and terminal transferase-mediated dUTP nick end-labelling (TUNEL) immunohistochemistry were carried out to identify the specific neurotoxicity effect.5) TEM evaluation of the brainBrains were quickly removed from 2-day-old pups and fixed in 3% glutaraldehyde+2% paraformaldehyde for 2 h. Small pieces of tissuecollected from these samples, were then dehydrated and embedded in resin. Finally, an H-7500 TEM was used to visualize the sections.Part 3:Major toxicity mechanism of neurotoxicity effect1) Homogenate preparation and protein concentration determinationBrain tissues were collected from 2-day-old pups in both experimental and control group. Samples were homogenized in 10:1 (vol/wt) ice-cold PBS, and then diluted. Protein concentrations were determined according to Coomassie brilliant blue assay.2) Determination of ROS levelSuperoxide ion (O2-) in the brain tissue was measured according to the ROS test kit. Propor concentration of homogenate was selected, and added into the 96-well plate with other regent. After complete reaction, a fluorescence microplate was used to read OD value of each well.3) Alteration of enzyme activity related with oxidative stressSOD and GSH-PX in the brain tissue was measured according to the comercial test kit. Proper concentration of homogenate was selected, and added into the 96-well plate with other regent. After complete reaction, a microplate was used to read OD value of each well.4) Level of lipid peroxidation MDA in the brain tissue was measured according to the MDA test kit. Proper concentration of homogenate was selected, and added into the 96-well plate with other regent. After complete reaction, a microplate was used to read OD value of each well.5) Expression of genes related with oxidative stressBrain tissues were collected from 2-day-old and weaned pups in both groups. Homogenate were prepared and then total RNA was extracted. Complementary DNA (cDNA) was prepared from RNA samples. qRT-PCR was carried out to analyze the expression of genes related with oxidative stress, such as SOD、Gsr、 Gsttl-Aloxl2b. The expression level of each target gene was normalized to its β-actin mRNA content.Part 4:Learning and memory ability of the offspring in the adulthood1) Selection of experimental animalNewborns in both groups were weaned at PND 21, and housed according different genders until adulthood (PND 60). Then the learning and memory behavior in the offspring of each group was evaluated using Morris water maze test.2) The acquisition phaseThe pool was divided into four quadrants, and a circular platform was placed 1.5 cm below the surface of the water in the middle of the NE quadrant. Rats received four trials a day from four starting positions. During each trial, the rats were required to find the hidden platform and spend 15 s on the platform. If they failed to reach the platform within 90 s, the rats were guided and placed on the platform by the experimenter and stayed on it for 15 s. Rats received the same training during the first 5 days.3) The probe testOn the 6th day, the platform was removed from the pool. In the probe test, one particular starting position was chosen. Two parameters including the number of crossing of exact place where the platform had been located, and the swimming time in the quadrant of the platform position were measured.4) The reacquisition phase On days 7 and 8 (reacquisition phase), reverse platform training was instituted, and the hidden platform was moved to the center of the SW (opposite) quadrant without changing any distal visual cues. Reversal trials were performed as described in the acquisition phase.Statistical analysisSPSS 19.0 software was used for statistical analysis. All data are expressed as mean±standard deviation. The data of the Morris water maze test were analyzed by repeated measures data of One-way ANOVA. Other comparisons between two groups were tested by one-way analysis of variance (ANOVA). All differences were considered significant when P< 0.05.Results1. TEM micrographs demonstrated that ZnO nanoparticles were hexagonal with a diameter of approximately 50 nm. EDS indicated the element content of NPs belonged to ZnO. XRD image showed that the nanoparticles had the same crystal structure as that of bulk zincite. The intensity-weighted average hydrodynamic diameter of ZnO NPs in distilled water (DW) was 500.8 nm. And the zeta potential was 32.9 mV at pH 7.0. The specific surface area was 32.17 m2/g. The level of endotoxin contamination was not detected, which was higher than the detection limit.2. ICP-MS analysis observed that more than 70% of nanoparticles dissolved in AGF after incubation for 12 h. But thereafter, the dissolution rate slowed down quickly and the solubility of NPs reached 77.4% at 48 h.3. Compared with the control group (7.72 g±0.33 g), rats in the ZnO NPs-treated group had lower body weights (6.83 g±0.65 g). Additionally, the coefficients of the brain, heart and liver in ZnO NPs-treated group were significantly higher than those in the control group. In contrast, the coefficients of the kidney and spleen decreased remarkably. With respect to the coefficients of the lung, no significant change was found in different groups.4. For the two-day-old neonates, Zn accumulated significantly in the heart, liver, kidneys and brain in the rats treated with ZnO-NPs. No obvious changes in the zinc level were observed in the lung and spleen between the two groups. At weaning (PND 21), however, zinc content in total blood was similar in control and nanoparticle exposed dams and offspring.5. The brain slices of rats prenatally treated with ZnO NPs showed slight abnormality in bran structure with sparser tissue in the prefrontal cortex and hippocampus. The immunohistochemistry results found that the number of Ki-67 positive-staining (brown) cells in both areas declined sharply. However, brain cells of nuclei positively stained by TUNEL were significantly higher in NPs treated group.6. TEM analysis revealed that the ultrastructure of the neuron from ZnO NPs-exposed rats presented irregularity of the cell membrane, obvious mitochondrial swelling and autophagosome. Furthermore, evidence of cellular localization of nanoparticles was found in the neural synapse.7. In the NPs treated group, the level of ROS was 12485.56±1435.13 FI/mg protein, and the level of MDA was 16.62±2.75 nmol/mg protein. Compared with the control group, these results were significantly elevated. Furthermore, an obvious decline in activities of SOD and GSH-PX were also observed in the brains from the NPs treated group.8. The qRT-PCR result found that prenatal exposure to ZnO NPs caused significant changes genes related with oxidative stress in the brain from newborns and weaned offspring. Of the eight genes expressed differentially in the nanoparticle exposure group,5 genes were downregulated in response to the treatment at PND 2. In comparison,5 were genes upregulated, and 3 genes were downregulated in the brain of weaning pups.9. ZnO NPs treatment increased the latency of female offspring to reach the platform in the fourth and fifth training day. In the first day of reacquisition training, female rats in NPs group also presented longer latency to reach the platform compared with control rats. Moreover, NPs female offspring spend less time in the quadrant of the former platform position in the probe test. Although male offspring in NPs treated group showed inferior behavioral performance in Morris water maze than control group, but this difference was not statistically significant.Conclusions1. Characterization of NPs has confirmed the particle size and structure, and chemical composition of the comercial material belongs to ZnO NPs. Furthermore, role of endotoxin contamination in the toxic effect was excluded using limulus reagent gel method.2. Prenatal exposure of ZnO NPs may lead to growth restriction of the fetus and the alteration of coefficient of tissues in the offspring. The changing ratios of tissues to body weight indicated that pathological changes, such as inflammation might occur in the offspring following prenatal exposure to nano-ZnO.3. Pregnant SD rats were treated with ZnO NPs by intragastric administration. Considering the acid condition (pH= 1.5) of the gastric fluid, a large proportion of fed ZnO-NPs may dissolve in the stomach, and thus nanoparticle together with Zn ions were absorbed into maternal systemic circulation and further into the fetus, and accumulate in the body organs of the fetus. However, the elevated zinc levels in the blood might gradually decline to a baseline as a large proportion of nanoparticles were distributed to organs or eliminated from the body.4. Prenatal exposure of NPs may have an adverse effect on the brain development of the fetus. H&E staining revealed slight structural abnormalities, with more sparse tissues in the prefrontal cortex and hippocampus compared to control pups. Further Ki-67 and TUNEL immunohistochemistry respectively indicated that ZnO NPs might reduce proliferation and induce apoptosis of brain cells.5. The neurotoxicity of NPs may be closely associated with the production of oxidative stress. The biochemical results indicate a generation of oxidative stress in the offspring brain tissues owning to imbalance in the ROS formation (significant elevation) and antioxidant defense systems.6. Prenatal exposure of ZnO NPs may lead to differential expressions of genes related to oxidative stress in brain tissues. We also suggest that more genes will present response to oxidative stress in the later stage of brain development. Additionally, the expression level of the same gene may differ in different developmental stages.7. The neurotoxic effect of ZnO NPs on the newborn pups may further affect normal brain function in their adulthood, such as the learning and memory behavior. Furthermore, female offspring may possess higher susceptibility to this adverse effect.