Mechanisms Of Blood-milk Barrier Damage Induced By Lactation Exposure To Zinc Oxide Nanoparticles

BackgroundDue to their antimicrobial,ultraviolet and visible absorption,and photocatalytic and semiconductor properties,zinc oxide nanoparticles(ZnO-NPs)are one of the most widely used nanomaterials and are mostly incorporated into market goods associated with the food industry,daily care and biomedical products.Their applications in dental materials are also extensive.Thus,ZnO-NPs have a high possibility of exposure to humans and inducing potential side effects to human health,which should not be neglected.Overwhelming evidence has shown that breastfeeding is beneficial for the health of the child and the mother and for society in general.Thus,breastfeeding has been encouraged worldwide.However,lactation exposure to nanomaterials has not received enough attention.Whether lactation exposure to ZnO-NPs will affect the mammary gland,the blood-milk barrier(BMB),the breast milk quality and the development of the offspring is undefined.ObjectiveTo uncover whether ZnO-NPs will damage and then cross the endothelial barrier;to uncover whether ZnO-NPs will damage the BMB and enter into the breast milk;to explore the possible mechanism underlying the interactions between the barrier and ZnO-NPs.Materials and methodsZnO-NPs were purchased and were characterized using TEM,DLS,ICP-MS.A model of C57 mice exposed to ZnO-NPs via intravenous administration were conducted.ICP-MS,HE staining and IHC staining were used to examine the zinc level and the pathological changes of the liver,kidney and spleen from the C57 mice.Junction protein levels of the thoracic aorta were also examined to uncover whether ZnO-NPs will damage and then cross the endothelial barrier.Furthermore,the lactating Sprague-Dawley(SD)rats exposed to ZnO-NPs via intravenous administration over a total of 21 days of lactation were used as the in vivo model,and EpH4-Ev cells(mouse mammary epithelial cells;a cell line that can be used to study normal mammary epithelial cell function)were used as the in vitro BMB model.Methods including ICP-MS,IHC staining,Western blot,Transwell sets,cell immunofluorescent staining and co-immunoprecipitation were used to uncover whether ZnO-NPs will damage the BMB and enter into the breast milk.In the in vitro experiment,inhibitors including NAC,Anisomycin and SP600125 were exposed to EpH4-Ev cells together with ZnO-NPs.Methods including flow cytometry,Western blot,cell immunofluorescent staining,co-immunoprecipitation were used to explore the possible mechanism underlying the interactions between the barrier and ZnO-NPs.Results1.The primary diameter of the ZnO-NPs were about 50 nm.The TEM image shows that the NPs were rod-shaped.The ZnO-NPs were larger in solution,especially in saline and culture medium.The dissolution rate of the ZnO-NPs was approximately 33%in saline and approximately 25%in culture medium and milk.2.C57 mice were exposed to ZnO-NPs(1.2 mg/kg and 6 mg/kg body weight)and ZnSO4(3.6 mg/kg).A significantly increased concentration of zinc was detected in the liver,kidney and spleen from the ZnO-NP exposed groups.Inflammatory infiltrates were detected in the vessel walls of liver and kidney from the ZnO-NPs exposed mice in HE staining images.Positive rate was significantly increased from the ZnO-NPs exposed mice in TUNEL and 8-OHdG IHC staining images.The amount of positive staining of occludin,ZO-1 and VE-cadherin,which presented mostly on the luminal side of the thoracic aorta,was significantly reduced in the ZnO-NP exposed groups.Exposure to ZnSO4 did not have such effects.3.The 15 mg/kg group presented with a significant increase in the Zn level of the breast milk and mammary gland tissue.Ultrastructure images detected by TEM showed that the junction border between the epithelial mammary cells in the 15 mg/kg group was wider and looser.Results of IHC staining and Western blot assays showed that TJ protein,such as occludin,ZO-1 and claudin-3,were significantly reduced in the ZnO-NP exposed groups;as for claudin-4,the protein level was increased;the amount of AJ proteins,such as E-cadherin,?-catenin and ?-catenin,were not significantly different in any of the experimental groups.Using the in vitro model represented by EpH4-Ev cells,we found that the ZnO-NP-treated groups presented with dose-and time-dependent decreases in TEER and so did the protein level of occludin,ZO-1 and claudin-3,while the claudin-4 levels did not show significant changes.For AJs,E-cadherin,?-catenin and ?-catenin levels did not change at all,while the level phosphorylated ?-catenin was increased significantly.Cell immunofluorescent staining and co-immunoprecipitation results showed that the combination of ?-catenin with E-cadherin and ?-catenin was significantly reduced.Additionally,extracellular zinc ions were likely not the main factor to induce these effects.4.The nutrient and immune components of the breast milk did not change significantly.From PND 1 to 21,the pup body weights in each exposure group were not significantly different from the weights in the control group.Pup hair growth time was a little bit slower in the 15 mg/kg group than in the control group,and no other significant differences were observed.Although the Zn level was not significantly different among the four organs from each group,the oxidative stress markers,such as T-AOC and MDA,in the brain from the ZnO-NP groups were significantly increased.However,behaviors of the offspring from all groups in the Morris maze test showed no significant different.5.Several oxidative stress marker changes were found in the mammary gland in response to the ZnO-NPs and the 15 mg/kg group presented with a significantly higher percentage of 8-OHdG-stained cells.In vitro,the ROS generation was significantly increased in cells exposed to 20 and 30 ?g/mL ZnO-NPs.In the in vivo experiments,both the p-MKK4 and p-JNK IHC staining and the Western blot results showed that the activation of MKK4 and JNK was significantly enhanced in the ZnO-NP treatment groups.The in vitro experiments further confirmed that MKK4 and JNK could be activated by ZnO-NPs in a dose-and time-dependent manner.With the elimination of ROS using NAC,the activation of MKK4 was significantly inhibited,and so did the decrease in occludin,ZO-1 and claudin-3 and the increase in p-?-catenin induced by ZnO-NPs.Inhibiting the activation of MKK4 using NAC and activating JNK using Anisomycin at the same time had similar effects.In addition,the activation of c-Jun was not necessary for JNK to induce BMB damage.6.Adding JNK inhibitor,SP600125,markedly reduced the activation of JNK and TEER decrease of EpH4-Ev monolayer induced by ZnO-NPs.The level of phosphorylated ?-catenin was significantly reduced by adding SP600125 together with ZnO-NPs,and subsequently the combination of ?-catenin with E-cadherin and ?-catenin recovered.The expression level of SUMO-1 was markedly elevated in a time-dependent manner in EpH4-Ev cells exposed to ZnO-NPs and the positive staining of SUMO-1 was mostly distributed near the cell membrane.ZnO-NPs induced the significant increase in the association of E3 ligase PIAS1 with activated JNK and the combination of Ubc9 with ZO-1,thus caused the disassembly of TJ complex by inducing the SUMOylation of ZO-1.ConclusionIn this study,we found that ZnO-NPs via intravenous administration disrupted the endothelial barrier,accumulated in the mammary gland,disrupted the integrity of BMB by inducing changes in the TJ and AJ components,and let the NPs and zinc ions entering the breast milk.The results were verified both in dams and in the BMB in vitro model composed of EpH4-Ev cells.Other milk components and the development of the offspring were not affected.Furthermore,we verified that ZnO-NP-triggered ROS generation and MKK4/JNK activation are the main mechanism in the process of cell-cell junction damage.Most importantly,JNK activation plays different roles in inducing changes in the TJ and AJ,and these effects do not need to activate the downstream c-Jun and transcription.For the AJs,JNK activation could directly phosphorylate ?-catenin,thus damaging the bonding of ?-catenin with E-cadherin and?-catenin.For the TJs,JNK first activates PIAS1 and induces SUMOylation of ZO-1,by which ZO-1 disassembles from the TJ complex and may further cause the TJ components to degrade.In general,our study focused on an important but seldom studied internal barrier,the BMB,and we conducted a comprehensive evaluation of the biological effects of ZnO-NPs on the barrier's integrity.These data provide new information for understanding how nanomaterials interact with biological internal barriers.Furthermore,the intravenous use of ZnO-NPs or any other kind of nanomaterial in lactating mothers should be carried out with caution.